key: cord-284694-bk6bnox0 authors: wang, changsong; kang, kai; gao, yan; ye, ming; lan, xiuwen; li, xueting; zhao, mingyan; yu, kaijiang title: cytokine levels in the body fluids of a patient with covid-19 and acute respiratory distress syndrome: a case report date: 2020-05-12 journal: ann intern med doi: 10.7326/l20-0354 sha: doc_id: 284694 cord_uid: bk6bnox0 nan during this patient's illness to see whether they could help us decide how to modify his treatment as the disease progressed. we found high and fluctuating levels of these cytokines in his peripheral blood, bronchoalveolar lavage fluid, and pleural fluid. however, these levels correlated only inconsistently with the treatments we administered, even for plasmapheresis, which was intended to dilute circulating cytokines, and for a dialysis filter that was designed to adsorb cytokines ( figure) . in addition, these cytokine levels correlated inconsistently with his clinical course, except that the levels increased dramatically in the last days before he died. we suspect that this patient's immune system was partially suppressed due to his advanced age and multiple chronic conditions, which might have contributed to the virus's continued replication and the disease's progress. in addition, the time from symptom onset to confirmation of covid-19 diagnosis was relatively long, the patient's hospital course was longer, and we wonder whether this long duration of viral replication contributed to the high cytokine levels we measured. other studies have reported that patients with covid-19 have evidence of local damage, which includes diffuse alveolar injury with cellular fibrous mucus-like exudates (2). we measured il-6 levels in bronchoalveolar lavage fluid that were higher than the corresponding serum levels. on one occasion (7 march), the il-6 level was approximately 10 times higher. this difference is even more remarkable because the process of collecting bronchoalveolar fluid dilutes the specimen. in addition, the level of il-6 in pleural effusion was higher than the corresponding serum levels on the 2 times we measured it. if these observations indicate a cytokine storm, we propose that the local storm may be worse than the systemic storm. interleukin-6 blockers have been used to treat cytokine storm in patients with other causes of cytokine storm (3), and tocilizumab has been suggested for immunotherapy for severe patients with extensive lung lesions and elevated il-6 levels (3). as a result, we wonder whether tocilizumab would have affected the il-6 levels we observed and whether it might have improved this patient's disease course, especially because others have reported that as covid-19 progresses to its middle and late stages, the expression of inflammatory cytokines is related to the severity of the disease (4). on the basis of our experience, we encourage additional research to determine whether inflammatory cytokines in the lungs predict the clinical course of covid-19 and whether these cytokines should be a target for intervention and treatment. in summary, this case suggests an increased inflammatory response in the lung tissues of critically ill patients with covid-19, and it suggests that future research should include examinations of local inflammation in the lungs. continued on the following page epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study pathological findings of covid-19 associated with acute respiratory distress syndrome toxicity management for patients receiving novel t-cell engaging therapies clinical features of patients infected with 2019 novel coronavirus in wuhan key: cord-266034-811lov8f authors: benameur, karima; agarwal, ankita; auld, sara c.; butters, matthew p.; webster, andrew s.; ozturk, tugba; howell, j. christina; bassit, leda c.; velasquez, alvaro; schinazi, raymond f.; mullins, mark e.; hu, william t. title: encephalopathy and encephalitis associated with cerebrospinal fluid cytokine alterations and coronavirus disease, atlanta, georgia, usa, 2020 date: 2020-09-17 journal: emerg infect dis doi: 10.3201/eid2609.202122 sha: doc_id: 266034 cord_uid: 811lov8f there are few detailed investigations of neurologic complications in severe acute respiratory syndrome coronavirus 2 infection. we describe 3 patients with laboratory-confirmed coronavirus disease who had encephalopathy and encephalitis develop. neuroimaging showed nonenhancing unilateral, bilateral, and midline changes not readily attributable to vascular causes. all 3 patients had increased cerebrospinal fluid (csf) levels of anti-s1 igm. one patient who died also had increased levels of anti-envelope protein igm. csf analysis also showed markedly increased levels of interleukin (il)-6, il-8, and il-10, but severe acute respiratory syndrome coronavirus 2 was not identified in any csf sample. these changes provide evidence of csf periinfectious/postinfectious inflammatory changes during coronavirus disease with neurologic complications. there are few detailed investigations of neurologic complications in severe acute respiratory syndrome coronavirus 2 infection. we describe 3 patients with laboratory-confirmed coronavirus disease who had encephalopathy and encephalitis develop. neuroimaging showed nonenhancing unilateral, bilateral, and midline changes not readily attributable to vascular causes. all 3 patients had increased cerebrospinal fluid (csf) levels of anti-s1 igm. one patient who died also had increased levels of anti-envelope protein igm. csf analysis also showed markedly increased levels of interleukin (il)-6, il-8, and il-10, but severe acute respiratory syndrome coronavirus 2 was not identified in any csf sample. these changes provide evidence of csf periinfectious/ postinfectious inflammatory changes during coronavirus disease with neurologic complications. elisa with 90% sensitivity and 89% specificity for confirmed covid-19 against 78 pre-2020 controls. csf was serially diluted from 1:2 to 1:16, and csf from 1 case-patient who had hiv infection (hospitalized during march 2020) and from 3 pre-2020 healthy subjects (9) were included for comparison. we measured levels of plasma igg against the receptor-binding domain of s1 by using a commercial elisa (genscript, https:// www.genscript.com) at a 1:16 dilution. we analyzed csf inflammatory proteins (milli-poresigma, https://www.emdmillipore.com) by using a luminex-200 platform and a modified manufacturer's protocol as described (9) . these proteins include interleukin (il)-1α, il-1β, il-2, il-4, il-6, il-7, il-8, il-9, il-10, il12-p40, il12-p70, interferon-gamma-induced protein 10 (ip-10), monocyte chemoattractant protein 1 (mcp-1/ccl2), macrophage-derived chemokine (mdc/ccl22), fractalkine (cx3cl1), and tumor necrosis factor α (tnf-α). we performed molecular testing for sars-cov-2 by using real-time quantitative reverse transcription pcr (qrt-pcr). we extracted total nucleic acid from 120 µl of csf from each person by using the ez1 virus mini kit version 2.0 and the ez1 advanced xl instrument (qiagen, https://www.qiagen.com) after lysis with avl lysis buffer (qiagen). we performed a 1-step qrt-pcr by using 2019-ncov_n1 or 2019-ncov_n2 combined primer/probe mix (integrated dna technologies, inc., https://www.idtdna.com) in a roche lightcycler 480 ii (https://lifescience. roche.com), an endogenous control, and an in vitro transcribed full-length rna of known titer (integrated dna technologies, inc.) as a positive control. we followed the same procedure for influenza a virus except using a primer/probe mixture (10) and a mitochondrial cytochrome oxidase subunit 2 dna endogenous control (11) . we tested all samples in duplicate. patient 1, a 31-year-old african-american woman who had sickle cell disease (scd) and was receiving dabigatran for a recent pulmonary embolus, came to a community hospital after 5 days of progressive dyspnea. an initial chest radiograph showed a right lower lobe infiltrate, and she was given a blood transfusion and antimicrobial drugs for presumed scd crisis and pneumonia. her breathing became more labored, and a repeat chest radiograph showed worsening bilateral infiltrates. a nasopharyngeal swab specimen was positive for sars-cov-2 and influenza a virus (negative for influenza b virus). she was empirically given hydroxychloroquine (400 mg daily) and peramivir (100 mg daily), but acute kidney injury and progressive hypoxemic respiratory failure developed. she was intubated and transferred to our institution on day 11. her paralysis and sedation were discontinued on day 13 after improved oxygenation, but she remained comatose with absent brainstem reflexes on day 15. brain magnetic resonance imaging (mri) showed nonenhancing cerebral edema and diffusion weighted imaging abnormalities predominantly involving the right cerebral hemisphere, as well as brain herniation ( figure 1 ). an occlusive thrombus was identified in the right internal carotid artery, and edema was also identified in the cervical spinal cord. the overall appearance was most consistent with encephalitis and myelitis, with superimposed hypoxic ischemic changes. csf showed high opening pressure of 30 cm of water, 115 nucleated cells/ml, 7,374 erythrocytes/ml, an increased protein level (>200 mg/dl), and a glucose level within a standard range (table) . her nucleated cell count remained strongly increased even after correction for the traumatic tap (≈1 nucleated cell/700 erythrocytes). given a grave prognosis, the family withdrew life-sustaining care and the patient died on day 16. patient 2, a 34-year-old african-american man who had hypertension, showed development of fever, shortness of breath, and cough. computed tomography of the chest showed bilateral, diffuse ground glass infiltrates. a nasopharyngeal swab specimen obtained on day 1 showed sars-cov-2. he was given a 6-day course of hydroxychloroquine, but hypoxic respiratory failure developed, which required intubation, followed by encephalopathy with myoclonus on day 9. his neurologic examination showed profound encephalopathy, absent corneal and gag reflexes, multifocal myoclonus involving both arms, and absent withdrawal to painful stimuli. electroencephalography showed diffuse slowing with a suggestion that the myoclonus was seizure-related. brain mri on day 15 showed a nonenhancing hyperintense lesion within the splenium of the corpus callosum on fluid-attenuated inversion recovery and diffusion weighted imaging sequences ( figure 1 ). csf showed high opening pressure of 48 cm h 2 o, no pleocytosis, 27 erythrocytes/ml, a mildly increased protein level, and glucose level within the reference range. patient 3, a 64-year-old african-american man who had hypertension, showed development of cough, dyspnea, and fever with multifocal, patchy, ground glass opacities on chest computed tomography and a nasopharyngeal swab specimen positive for sars-cov-2. his symptoms progressed to hypoxic respiratory failure requiring intubation, and his multifocal myoclonus began soon after starting to take hydroxychloroquine. his neurologic examination showed profound encephalopathy, absent oculocephalic reflex, multifocal myoclonus affecting bilateral arms and legs, absent withdrawal to pain, and diminished deep tendon reflexes. the resolution of his myoclonus coincided with fentanyl cessation, but it is not clear that the 2 symptoms were related. a motion-degraded brain mri showed an equivocal nonenhancing area of fluid-attenuated inversion recovery abnormality in the right temporal lobe. csf obtained on hospital day 11 showed a normal opening pressure; levels of nucleated cells, erythrocytes, and protein within reference ranges; and an increased glucose level (table) . his mentation began to improve on day 13, and he was subsequently discharged without major neurologic sequelae. plasma anti-s1 receptor-binding domain igg levels were increased for all 3 patients, consistent with severe covid-19 (t. ozturk et al., unpub. data ). an indirect elisa for plasma showed an increased level of anti-s1 igm for patients 1 (1:512) and 2 (1:256), a highly increased level of anti-s1 igm for patient 3 (1:2,048); an increased level of anti-e igm for patients 1 and 2 (1:128), and a standard level of anti-e igm for patient 3. an indirect elisa for csf showed markedly increased levels of igm for sars-cov-2 s1 (figure 2 , panel a) and e (figure 2, panel b) proteins for the most severely ill patient 1, and mildly elevated levels of igm for s1 only for patients 2 and 3. the number of csf erythrocytes in patient 1 suggested plasma contamination at an approximate dilution of 1:1,000, which still 2018 emerging infectious diseases • www.cdc.gov/eid • vol. 26, no. 9, september 2020 placed these csf igm levels higher than those for patients 2 and 3. csf from patients 1 and 3 underwent detailed inflammatory protein profiling as described (9, 12, 13) . when we compared historical and present control subjects who had normal cognition (no viral illness) (13), we found that patients with covid-19 and neurologic symptoms had increased csf levels of il-6, il-8, il-10, ip-10, and tnf-α ( figure 2 , panel c). levels of il-8, il-10, ip-10, and tnf-α were also available for subjects who had hiv-associated neurocognitive disorders (12) . increased levels of il-8 and il-10 appeared to be unique for neurologic complications of sars-cov-2, and increased levels of ip-10 and tnf-α were common features between neurologic complications of sars-cov-2 and hiv. we used a real-time rt-pcr to test for sars-cov-2 and influenza a virus (tested because patient 1 showed a co-infection). results were negative for all patients. we report 3 patients who had severe covid-19 and showed development of various neurologic symptoms and findings in a us hospital. all patients had more severe symptoms affecting cortical and brainstem functions at the peak of their neurologic illnesses than a recent series of 7 case-patients with milder illness in france (6) . all 3 patients were also co-incidentally given a short course of empiric hydroxychloroquine, although there was no temporal correlation between the medication and their neurologic manifestation. similar to the case-series in france, we did not isolate sars-cov-2 rna from csf, although such viral rna has been inconsistently identified in other cases (14) . however, increased levels of csf anti-s1 igm and altered levels of csf cytokines are consistent with direct cns involvement by sars-cov-2. because mri changes seen in these patients could be caused by hypercoagulability (15) or metabolic encephalopathy (16) , we propose that csf investigation can improve the distinction between neurologic involvement of sars-cov-2 (or neuro-covid) and neurologic symptoms caused by other covid-related causes. in health and many noninflammatory neurologic disorders, the intact blood-brain barrier prevents major central translocation by plasma immunoglobulins or cells that secrete them (17) . increased levels of csf antibodies can thus result from disrupted blood-brain barrier, regulated migration of peripheral antibodysecreting cells into the cns, or de novo antibody synthesis within the cns. the relatively normal protein levels in patients 2 and 3 would argue against an unequivocal blood-brain barrier disruption. the lack of clear correlation between plasma and csf titers provides some support for an active cns process. the failure to detect csf sars-cov-2 rna does not diminish the likelihood of direct cns infection because it is only recovered from blood in 1% of the actively infected cases (18) , and increased levels csf igm are also more commonly found as evidence for cns infection than viral recovery in other encephalitides, including those for infection with japanese encephalitis virus (19) , dengue virus (20) , human parvovirus 4 (21) , and rabies virus (22) . at the same time, undetectable csf rna raises the possibility that mechanisms other than direct brain infection might account for the observed mri and clinical changes. these changes include peri-infectious inflammation (mediated by antibodies, complement, or both) (5,23), vasculopathy, and altered neurotransmission. until definitive neuropathologic studies or effective antiviral therapies are possible, infectious and peri-infectious etiologies need to be examined for neuro-covid. increased levels of csf multiple cytokines in these neuro-covid patients are consistent with earlier reports of cytokine analysis of blood (24; m. woodruff et al., unpub. data). we additionally identified changes shared (and not shared) by sars-cov-2 and hiv. factors associated with increased levels of csf il-10 in patients infected with hiv should be investigated in future neuro-covid studies, and increased levels of csf il-8 might uniquely provide useful information on the pathophysiology of cns. we did not include plasma cytokine levels because their levels are much more influenced by demographic factors than their csf counterparts (w.t. hu et al., unpub. data). a larger cohort is necessary to better distinguish between csf and plasma cytokine alterations, and including patients without confounding disease (e.g., scd in patient 1) or standard mri results can also determine the relative roles of noninfectious/inflammatory causes of encephalopathy, including hypoxia or hypercoagulability (25, 26) . nevertheless, we demonstrated in these case-patients that sars-cov-2 antibodies are detectable in the csf for patients with neurologic complications and are associated with selective csf cytokine alterations. future investigations should align neurologic outcomes with csf infectious and immunologic profiles, such that an evidence-based treatment algorithm can be determined for preventing and treating neuro-covid-19. dr. benameur is an neurologist and associate professor in the department of neurology at emory university school of medicine, atlanta, ga. her primary research interest is in neuroinflammatory changes related to covid-19 asymptomatic patients with novel coronavirus disease (covid-19) clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study possible central nervous system infection by sars coronavirus severe neurologic syndrome associated with middle east respiratory syndrome corona virus (mers-cov) nervous system involvement after infection with covid-19 and other coronaviruses neurologic features in severe sars-cov-2 infection evidence of the covid-19 virus targeting the cns: tissue distribution, host-virus interaction, and proposed neurotropic mechanisms severe acute respiratory syndrome coronavirus infection causes neuronal death in the absence of encephalitis in mice transgenic for human ace2 interleukin 9 alterations linked to alzheimer disease in african americans epidemiology of hospital admissions with influenza during the 2013/2014 northern hemisphere influenza season: results from the global influenza hospital surveillance network antiviral activities and cellular toxicities of modified 2′,3′-dideoxy-2′,3′-didehydrocytidine analogues linked csf reduction of phosphorylated tau and il-8 in hiv associated neurocognitive disorder csf cytokines in aging, multiple sclerosis, and dementia a first case of meningitis/encephalitis associated with sars-coronavirus-2 hematological findings and complications of covid-19 toxic and acquired metabolic encephalopathies: mri appearance immune regulation of antibody access to neuronal tissues detection of sars-cov-2 in different types of clinical specimens how many patients with anti-jev igm in cerebrospinal fluid really have japanese encephalitis? importance of cerebrospinal fluid investigation during dengue infection in brazilian amazonia region detection of human parvovirus 4 dna in the patients with acute encephalitis syndrome during seasonal outbreaks of the disease in gorakhpur long-term follow-up after treatment of rabies by induction of coma human coronaviruses and other respiratory viruses: underestimated opportunistic pathogens of the central nervous system? viruses clinical features of patients infected with 2019 novel coronavirus in wuhan difference of coagulation features between severe pneumonia induced by sars-cov2 and non-sars-cov2 vaso-occlusive crisis and acute chest syndrome in sickle cell disease due to 2019 novel coronavirus disease (covid-19) key: cord-352687-gncmygda authors: science, michelle; maguire, jonathon l.; russell, margaret l.; smieja, marek; walter, stephen d.; loeb, mark title: low serum 25-hydroxyvitamin d level and risk of upper respiratory tract infection in children and adolescents date: 2013-05-15 journal: clinical infectious diseases doi: 10.1093/cid/cit289 sha: doc_id: 352687 cord_uid: gncmygda background. vitamin d may be important for immune function. studies to date have shown an inconsistent association between vitamin d and infection with respiratory viruses. the purpose of this study was to determine if serum 25-hydroxyvitamin d (25(oh)d) was associated with laboratory-confirmed viral respiratory tract infections (rtis) in children. methods. serum 25(oh)d levels were measured at baseline and children from canadian hutterite communities were followed prospectively during the respiratory virus season. nasopharyngeal specimens were obtained if symptoms developed and infections were confirmed using polymerase chain reaction. the association between serum 25(oh)d and time to laboratory-confirmed viral rti was evaluated using a cox proportional hazards model. results. seven hundred forty-three children aged 3–15 years were followed between 22 december 2008 and 23 june 2009. the median serum 25(oh)d level was 62.0 nmol/l (interquartile range, 51.0–74.0). a total of 229 participants (31%) developed at least 1 laboratory-confirmed viral rti. younger age and lower serum 25(oh)d levels were associated with increased risk of viral rti. serum 25(oh)d levels <75 nmol/l increased the risk of viral rti by 50% (hazard ratio [hr], 1.51; 95% confidence interval [ci], 1.10–2.07, p = .011) and levels <50 nmol/l increased the risk by 70% (hr, 1.67; 95% ci, 1.16–2.40, p = .006). conclusions. lower serum 25(oh)d levels were associated with increased risk of laboratory-confirmed viral rti in children from canadian hutterite communities. interventional studies evaluating the role of vitamin d supplementation to reduce the burden of viral rtis are warranted. viral upper respiratory tract infections (rtis) are very common worldwide. despite their perceived benign nature, the burden of disease is significant in terms of morbidity and economic loss [1] . viral respiratory infections may also be associated with mortality in certain patient populations [2, 3] . unfortunately, treatment remains unsatisfactory and is often focused on symptom relief. as a result, prevention remains a key strategy in reducing the burden of these infections. there has been increasing interest in the role of vitamin d in respiratory infections. vitamin d has been shown to have important roles for both innate [4] [5] [6] and adaptive immune responses [7] [8] [9] . in particular, vitamin d has been linked to innate immune responses in lung epithelial cells [10, 11] . in addition, antimicrobial peptides induced by vitamin d may have antiviral effects with activity shown against herpes simplex virus type 1 [12] , adenovirus [13] , human immunodeficiency virus (hiv) [14] , and vaccinia virus [15] . several observational studies have evaluated the role of serum 25-hydroxyvitamin d (25(oh)d) concentration in respiratory tract infections [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] . however, all these studies have important limitations including short follow-up period [16] , small sample size [16-20, 24, 25] , case-control design [18] [19] [20] , or crosssectional design with retrospective ascertainment of symptoms [21, 22] and failure to obtain laboratory confirmation of self-reported illness [16, [21] [22] [23] . the studies in adults have shown an association between lower vitamin d levels and increased respiratory infections (self-reported) [21, 22, 24] and absence from work due to respiratory symptoms [23] . however, pediatric studies have focused predominantly on lower rtis (chest radiography-confirmed pneumonia or bronchiolitis) [17] [18] [19] [20] 25] . the relationship between serum 25(oh)d concentration and upper rtis in children has not been evaluated. the purpose of this study was to determine if serum 25(oh) d levels are associated with subsequent risk of laboratoryconfirmed viral upper rtis in children and adolescents. we conducted a prospective cohort study of children and adolescents participating in a cluster randomized controlled trial evaluating the effect of influenza vaccination of children on viral infection rates in hutterite communities, the results of which have been published elsewhere [26] . hutterite communities (colonies) are rural, self-governing, and self-sufficient communal-living groups of anabaptists. in the rct, children with no underlying chronic medical conditions between 3 and 15 years of age (n = 947) from 46 colonies were randomly assigned by colony to receive either inactivated seasonal influenza vaccine ( children with underlying conditions (n = 65) and other children (n = 174) were followed but not randomized. all participants were followed regularly with twice-weekly assessments by research nurses over the influenza season, defined by the start date (1 laboratory-confirmed influenza case for 2 consecutive weeks) and stop date (no laboratory-confirmed influenza cases for 2 consecutive weeks). this period was from 28 december 2008 to 23 june 2009. covariables of interest were age, sex, presence of asthma, presence of other underlying conditions, influenza vaccination, and 25(oh)d level (nmol/l). serum 25(oh)d levels were collected on children at baseline unless bloodwork was refused. serum from venous blood was frozen at −80°c until batched analysis was performed according to the manufacturer's instructions using the diasorin liaison chemiluminescence assay. serum 25(oh)d levels were taken between 16 october 2008 and 28 april 2009 with most taken between 16 october 2008 and 31 december 2008 (n = 724, 97%). the primary outcome was laboratory-confirmed viral infection defined by a positive nasopharyngeal swab polymerase chain reaction (pcr) result. copan flocked nasopharyngeal swabs (copan italia, brescia, italy) were collected in universal transport medium (copan italia) if 2 or more of the following symptoms were present: fever (≥38°c), cough, nasal congestion, sore throat, headache, sinus problems, muscle aches, fatigue, ear ache or infection, or chills. specimens were first tested for influenza a (including ph1n1) and b using the centers for disease control and prevention human influenza virus real-time reverse transcription pcr detection and characterization panel [27] . negative specimens were then tested for influenza (a, b), coronavirus (229e, nl63, oc43), enterovirus (including rhinovirus), parainfluenza (1-4), respiratory syncytial virus (rsv) (a, b), and human metapneumovirus by xtag respiratory virus panel multiplex pcr (luminex, austin, texas). survival analysis was used to assess the relationship between time to laboratory-confirmed viral respiratory infection and serum 25(oh)d level. univariable analyses were conducted to obtain unadjusted hazard ratios (hrs), and significance was determined using the log-rank test. vitamin d levels were analyzed as both a continuous variable (log-transformed to correct positive skew) and dichotomized based on both the american academy of pediatrics (aap [≥50 nmol/l]) [28] and canadian paediatric society (cps [≥75 nmol/l]) [29] recommendations. age was also analyzed both as a continuous variable and categorized into 3 groups (<5 years, 5-9 years, 10-15 years). a cox proportional hazards model was used to estimate adjusted hazard ratios (ahrs), and the model was adjusted for clustering at the colony level. variables with a p value < .1 were considered for inclusion in the multivariable model, and the final model was determined using a stepwise backwards elimination method. it was decided a priori to adjust the final model for age and sex. the proportional hazards assumption was evaluated using shoënfeld residual test, graphically using shoënfeld residuals and log-log curves and examination of variables for time dependence. overall model fit was assessed using cox-snell residuals and deviance residual plots. a recurrent events analysis was conducted to examine the relationship between 25(oh)d and rate of occurrence of respiratory tract infection adjusted for the biologically plausible covariables specified above. a counting process model was used to treat recurrent events as identical. the model was adjusted for clustering at the colony level. all estimates are presented with 95% confidence intervals (cis). p values <.05 was considered statistically significant. all analyses were conducted using stata statistical software release 12 (statacorp, college station, texas). baseline characteristics are summarized in table 1 . of 1186 children (aged ≤15 years) in the rct, 947 were randomized to influenza or hepatitis a vaccine, and 743 (63%) children from 43 colonies had serum 25(oh)d levels and were included in the study. there were no appreciable differences between participants with serum 25(oh)d levels and those without. the mean age was 9. in univariable analyses, age and serum 25(oh)d level were associated with rti (table 2 ). in multivariable analysis accounting for clustering at the colony level, serum 25(oh)d level was independently associated with viral rti (table 2) . for every 1-unit increase in log serum 25(oh)d level (corresponding to a 2.72-fold increase in serum 25(oh)d level), the hazard of developing a respiratory tract infection decreased by 50% (ahr, 0.52; 95% ci, .35-.79, p = .002). when levels were dichotomized based on aap and cps recommendations, levels <50 nmol/l (ahr, 1.67; 95% ci, 1.16-2.40, p = .006) and <75 nmol/l (ahr, 1.51; 95% ci, 1.10-2.07, p = .011), were both associated with increased risk of infection (figures 1 and 2) . age was also independently associated with viral rti in the multivariable analysis. children aged <5 years were at highest risk of rti compared to those aged 5-9 years (ahr, 1.92; 95% ci, 1.26-2.88, p = .002) and 10-15 years (ahr, 2.40; 95% ci, 1.48-3.87, p < .001). there were no interactions found between serum 25(oh)d level and either age and sex. when multiple viral rti events were taken into account, age and log serum 25(oh)d levels were associated with increased rate of occurrence of respiratory tract infection (table 3) . serum 25(oh)d level was an important predictor both as a continuous variable (ahr, 0.57; 95% ci, .39-.83, p = .003) and when dichotomized based on levels <50 nmol/l (ahr, 1.49; 95% ci, 1.11-2.02, p = .008) and <75 nmol/l (ahr, 1.50; 95% ci, 1.11-2.03, p = .009). younger age conferred an increased risk of viral rti with a hazard ratio of 1.45 for every 5-year decrease in age (ahr, 1.45; 95% ci, 1.09-1.94, p = .011). we found a statistically significant association between serum 25(oh)d and laboratory-confirmed viral rtis in children from hutterite communities in canada. lower serum 25(oh)d levels were associated with increased risk of rti after adjusting for age and sex. younger age was also associated with increased risk. this study provides novel information on the effects of vitamin d status on laboratory-confirmed viral upper rtis in children and adolescents. previous studies have shown that children with rickets are at increased risk of lower respiratory tract disease or pneumonia [30] [31] [32] . more recently, several observational studies have shown an association between low serum 25(oh)d levels and risk of lower respiratory tract infection in children in india [17] , bangladesh [20] , and turkey [18] . our study extends these results, suggesting that vitamin d status may also be important for susceptibility to viral upper rtis in children and adolescents, a finding consistent with the adult literature [21] [22] [23] [24] . if confirmed with intervention trials, our findings may have important public health implications given the frequency of viral upper rtis and their associated morbidity and the prevalence of low vitamin d levels. we found that serum 25(oh)d level was an important predictor of rti both as a continuous variable ( per 1-unit change in log 25(oh)d), and also when dichotomized into levels <50 nmol/l (aap recommendations) and levels <75 nmol/l (cps recommendations). therefore, although maintaining levels >50 nmol/l is important to prevent rickets, [33] higher levels may be needed for prevention of viral rti. this was suggested in a recent study involving healthy adults, where a serum 25(oh)d concentration of >38 ng/ml (approximately 95 nmol/l) was associated with a reduced incidence of acute viral respiratory tract infection [24] . further studies in children are needed to determine the optimal level required for immune function. other canadian studies have failed to show an association between vitamin d status and risk for hospitalization for acute lower respiratory infection in children <5 years of age [19, 25] . although differences in vitamin d levels were not found in these studies, a higher proportion of participants with acute lower respiratory infection were found to have vitamin d receptor polymorphisms associated with reduced receptor expression in one study [34] . we did find an association between serum 25(oh)d level and upper rti in children <5 years of age, which may be related to differences in population (urban vs rural, genetic differences such as vitamin d receptor polymorphisms), study design, serum 25(oh)d measurements, or outcome (upper vs lower rti). the role of serum 25(oh)d level and individual respiratory viruses has not been studied. it has been postulated that lower vitamin d levels may explain the seasonal variation in influenza [35] . in the case of rsv infection, studies have shown that in rsvinfected human airway epithelial cells, vitamin d induces ikβα, an nf-kβ inhibitor, in airway epithelium and decreases rsv induction of inflammatory genes [11] . although we were unable to look at serum 25(oh)d level and the risk of individual respiratory viruses, this area warrants further research as serum 25 (oh)d level may impact viruses differently. our study has several limitations. first, we obtained only 1 vitamin d measurement for each participant and therefore do not have the exact level around the time of rti. however, serum 25(oh)d measurements were taken around the same time in october and november and reflect the vitamin d status at the start of the respiratory infection season prior to the development of viral rti. we believe this estimate is more appropriate than estimates taken at the time of illness because the impact of acute viral illness on serum 25(oh)d levels has not been studied. these levels were also taken at a time just after levels tend to peak in the northern hemisphere [36] . therefore, it is unlikely that vitamin d levels would have rebounded by early spring and had an appreciable impact on infections that occurred later in the follow-up period. in addition, 10 participants had serum for 25(oh)d levels obtained after the follow-up period had started. sensitivity analysis removing these estimates also did not affect the results. a third limitation was that data were not collected on sources of vitamin d intake for each individual participant. however, there are no data demonstrating that vitamin d supplementation or the use of fortified foods is part of routine practice in hutterite communities, and therefore we do not feel that this would affect our analysis. finally, this study was conducted in hutterite children and adolescents and may not be generalizable to other populations. studies evaluating other pediatric populations are warranted. we found that children and adolescents with lower vitamin d levels were at increased risk for laboratory-confirmed viral upper rti. current recommendations regarding target serum 25(oh)d levels may be too low to prevent viral upper rti. this study provides evidence in support of future interventional trials examining the efficacy of vitamin d supplementation on viral rtis in children and adolescents. the economic burden of non-influenza-related viral respiratory tract infection in the united states risk factors associated with severe influenza infections in childhood: implication for vaccine strategy s principles and practice of infectious diseases cutting edge: 1,25-dihydroxyvitamin d3 is a direct inducer of antimicrobial peptide gene expression toll-like receptor triggering of a vitamin d-mediated human antimicrobial response functional antagonism between vitamin d3 and retinoic acid in the regulation of cd14 and cd23 expression during monocytic differentiation of u-937 cells immunoregulation by 1,25-dihydroxyvitamin d3: basic concepts modulatory effects of 1,25-dihydroxyvitamin d3 on human b cell differentiation 25-dihydroxyvitamin d3 has a direct effect on naive cd4(+) t cells to enhance the development of th2 cells hunninghake gw. respiratory epithelial cells convert inactive vitamin d to its active form: potential effects on host defense vitamin d decreases respiratory syncytial virus induction of nf-kappab-linked chemokines and cytokines in airway epithelium while maintaining the antiviral state human cathelicidin (ll-37), a multifunctional peptide, is expressed by ocular surface epithelia and has potent antibacterial and antiviral activity cap37-derived antimicrobial peptides have in vitro antiviral activity against adenovirus and herpes simplex virus type 1 the antimicrobial peptide ll-37 inhibits hiv-1 replication selective killing of vaccinia virus by ll-37: implications for eczema vaccinatum vitamin d status in healthy romanian caregivers and risk of respiratory infections association of subclinical vitamin d deficiency with severe acute lower respiratory infection in indian children under 5 association of subclinical vitamin d deficiency in newborns with acute lower respiratory infection and their mothers vitamin d status is not associated with the risk of hospitalization for acute bronchiolitis in early childhood vitamin d status and acute lower respiratory infection in early childhood in sylhet vitamin d status has a linear association with seasonal infections and lung function in british adults association between serum 25-hydroxyvitamin d level and upper respiratory tract infection in the third national health and nutrition examination survey an association of serum vitamin d concentrations < 40 nmol/l with acute respiratory tract infection in young finnish men serum 25-hydroxyvitamin d and the incidence of acute viral respiratory tract infections in healthy adults rosenberg am. vitamin d deficiency in young children with severe acute lower respiratory infection effect of influenza vaccination of children on infection rates in hutterite communities: a randomized trial emergence of a novel swine-origin influenza a (h1n1) virus in humans prevention of rickets and vitamin d deficiency in infants, children, and adolescents vitamin d supplementation: recommendations for canadian mothers and infants case-control study of the role of nutritional rickets in the risk of developing pneumonia in ethiopian children the frequency of nutritional rickets among hospitalized infants and its relation to respiratory diseases presentation and predisposing factors of nutritional rickets in children of hazara division 25-hydroxyvitamin d: functional outcomes in infants and young children vitamin d receptor polymorphisms and the risk of acute lower respiratory tract infection in early childhood epidemic influenza and vitamin d hypovitaminosis d in british adults at age 45 y: nationwide cohort study of dietary and lifestyle predictors key: cord-267237-wbwlfx7q authors: gómez-rial, jose; currás-tuala, maria josé; rivero-calle, irene; gómez-carballa, alberto; cebey-lópez, miriam; rodríguez-tenreiro, carmen; dacosta-urbieta, ana; rivero-velasco, carmen; rodríguez-núñez, nuria; trastoy-pena, rocio; rodríguez-garcía, javier; salas, antonio; martinón-torres, federico title: increased serum levels of scd14 and scd163 indicate a preponderant role for monocytes in covid-19 immunopathology date: 2020-09-23 journal: front immunol doi: 10.3389/fimmu.2020.560381 sha: doc_id: 267237 cord_uid: wbwlfx7q background: emerging evidence indicates a potential role for monocytes in covid-19 immunopathology. we investigated two soluble markers of monocyte activation, scd14 and scd163, in covid-19 patients, with the aim of characterizing their potential role in monocyte-macrophage disease immunopathology. to the best of our knowledge, this is the first study of its kind. methods: fifty-nine sars-cov-2 positive hospitalized patients, classified according to icu or non-icu admission requirement, were prospectively recruited and analyzed by elisa for levels of scd14 and scd163, along with other laboratory parameters, and compared to a healthy control group. results: scd14 and scd163 levels were significantly higher among covid-19 patients, independently of icu admission requirement, compared to the control group. we found a significant correlation between scd14 levels and other inflammatory markers, particularly interleukin-6, in the non-icu patients group. scd163 showed a moderate positive correlation with the time lapsed from admission to sampling, independently of severity group. treatment with corticoids showed an interference with scd14 levels, whereas hydroxychloroquine and tocilizumab did not. conclusions: monocyte-macrophage activation markers are increased and correlate with other inflammatory markers in sars-cov-2 infection, in association to hospital admission. these data suggest a preponderant role for monocyte-macrophage activation in the development of immunopathology of covid-19 patients. emerging evidence from sars-cov-2 infected patients suggests a key role for monocyte-macrophage in the immunopathology of covid-19 infection, with a predominant monocytederived macrophage infiltration observed in severely damaged lungs (1) , and morphological and inflammation-related changes in peripheral blood monocytes that correlate with the patients' outcome (2) . an overexuberant inflammatory immune response with production of a cytokine storm and t-cell immunosuppression are the main hallmarks of severity in these patients (3) . this clinical course resembles viral-associated hemophagocytic syndrome (vahs), a rare severe complication of various viral infections mediated by proinflammatory cytokines, resulting in multiorgan failure and death (4) . a chronic expansion of inflammatory monocytes and over-activation of macrophages have been extensively described in this syndrome (5) (6) (7) . viral-associated hemophagocytic syndrome has been identified as a major contributor to death of patients in past pandemics caused by coronaviruses (8) , including previous sars and mers outbreaks (9) , and currently suggested for sars-cov-2 outbreak (10) . cd14 and cd163 are both myeloid differentiation markers found primarily on monocytes and macrophages, and detection of soluble release of both in plasma is considered a good biomarker of monocyte-macrophage activation (11, 12) . elevated plasma levels of soluble cd14 (scd14) are associated to poor prognosis in vih-infected patients, are a strong predictor of morbidity and mortality (13, 14) , and associated with diminished cd4+-t cell restoration (15) . in addition, soluble cd163 (scd163) plasma levels are a good proxy for monocyte expansion and disease progression during hiv infection (16) . in measles infection, a leading cause of death associated with increased susceptibility to secondary infections and immunosuppression, scd14 and scd163 levels have been found to be significantly higher, indicating an important and persistent monocyte-macrophage activation (17) . we hypothesized that monocytes/macrophages may be an important component of immunopathology associated to sars-cov-2 infection. in this paper, we analyze serum levels of soluble monocyte activation markers in covid-19 patients and their correlation with severity and other inflammatory markers. we recruited 59 patients with confirmed pcr-positive diagnosis of sars-cov-2 infection, classified according to icu admission requirement (n = 22 patients), or non-icu requirement (n = 37), and age-matched healthy individuals (n = 20) as a control group. demographic data, main medication treatment and routine lab clinical parameters including inflammatory biomarkers were collected for all infected patients. leftover sera samples from routine analytical controls were employed for the analysis, after obtaining the corresponding informed consent. time elapsed from hospital admission to sample extraction was also recorded. to determine levels of soluble monocyte activation markers in serum specimens, appropriate sandwich elisa (quantikine, r&d systems, united kingdom) were used following manufacturer indications. briefly, diluted sera samples were incubated for 3 h at room temperature in the corresponding microplate strips coated with capture antibody. after incubation, strips were washed and incubated with the corresponding human antibody conjugate for 1 h. after washing, reactions were revealed and optical density at 450 nm was determined in a microplate reader. concentration levels were interpolated from the standard curve using a four-parameter logistic (4-pl) curvefit in prism8 graphpad software. final values were corrected applying the corresponding dilution factor employed. data are expressed as median and interquartile range. all statistical analyses were performed using the statistical package r. mann-whitney tests were used for comparison between icu and non-icu groups versus healthy controls. pearson's correlation coefficients were used to quantify the association between scd14 and scd163 concentration and other lab parameters in non-icu patients. data outliers, falling outside the 1.5 interquartile range, were excluded from the statistical analysis. the nominal significance level considered was 0.05. bonferroni adjustment was used to account for multiple testing. patients in the icu group showed significant differences when compared to non-icu group in several clinical laboratory parameters: lymphocytes, ferritin, d-dimer, lactate dehydrogenase (ldh), procalcitonin (pct), and interleukin-6 (il-6). the absolute value for circulating monocytes did not show significant differences between groups. however, these values may have been distorted by the use of tocilizumab, an il-6 blocking drug extensively employed in the icu group which interferes with monocyte function. age and time elapsed from admission to sample extraction did not show differences between groups. values are summarized in table 1 . median levels for scd14 in sera from icu patients were 2444.0 (95%ci: 1914.0-3251.0) ng/ml, compared to 2613.0 (95%ci: 2266.0-2991.0) ng/ml in non-icu patients. the healthy control group median value was 1788.0 (95%ci: 1615.0-1917.0) ng/ml. we observed significant statistical differences when comparing infected patients against controls (p-value < 0.0001), however no significant differences were observed between icu and non-icu groups. median levels for scd163 in sera from icu patients were 911.5 (95%ci: 624.7-1167.0) ng/ml, and 910.4 (95%ci: 733.1-1088.0) ng/ml in non-icu patients. the healthy control group value was 495.6 (95%ci: 332.5-600.7) ng/ml. as with scd14, we observed significant differences for values from infected patients compared to control group (p-value < 00001), but no differences between icu and non-icu infected patients. values are summarized in table 2 and figure 1 . we assessed the correlation between scd14 and scd163 levels and time elapsed from hospital admission to sample extraction (figure 2) . we found a significant positive correlation between scd163 levels and time elapsed (r 2 = 0.3246, p-value = 0.0156) we did not observe a significant correlation between scd14 levels and time elapsed from hospital admission to sample extraction. we found significant correlations between scd14 and scd163 levels and several clinical laboratory parameters in infected patients (in these analysis, adjusted significance under bonferrori correction is 0.01), but only in the non-icu group, possibly reflecting an interference of the use of tocilizumab or corticoids in the icu group. levels of scd14 showed a negative correlation with the absolute value of lymphocytes (r 2 = −0.5501, p-value = 0.0005) and a positive correlation with levels of ldh (r 2 = 0.5906, p-value = 0.0001), crp (r 2 = 0.6275, p-value < 0.0001); pct (r 2 = 0.4608, p-value = 0.0091), and ferritin (r 2 = 0.4414, p-value = 0.0090) (figure 3) . no other significative associations were found with other lab parameters. levels of scd163 did not show significant correlation with clinical laboratory parameters (figure 3) . particularly, il-6 also showed significant positive correlation with scd14 (r 2 = 0.6034, p-value = 0.0003) (figure 4) . we analyzed possible interference of different treatments on scd14 and scd163 serum levels for all patients. we found an interference of corticoid treatment on scd14, levels with median values of 2034 (95%ci: 1319-3159) ng/ml for treated group, and values of 2613 (95%ci: 2466-2913) ng/ml for nontreated group. values were significantly lower in corticoid-treated group (p-value = 0.0069) (figure 5) . no impact was found for corticoids on scd163 levels. likewise, hydroxychloroquine and/or tocilizumab were not found to have an impact on scd14 and scd163 serum levels. levels of scd14 and scd163 did not show association with length of hospital stay in both groups. also, these biomarkers did not show association with the number of days of onset of symptoms. we analyzed for possible age-dependence of scd14 and scd163 levels. values did not show association between these biomarker levels and the age of patients. our results show, for the first time, increased levels of scd14 and scd163 in sera from sars-cov-2 infected patients admitted to hospital. we did not observe statistical differences when comparing icu versus non-icu patients. this is probably due to the interference on monocyte function and scd14 levels produced by the use of corticoid treatment in icu patients, as shown here and previously by others (18, 19) . however, levels of scd14 showed a strong correlation with clinical laboratory parameters, including acute phase reactants (ferritin, ldh, c-reactive protein, procalcitonin) and a strong correlation with il-6 levels in the non-icu patient group, where no corticoids treatments were used. hydroxychloroquine and tocilizumab treatment did not show interferences on scd14 and scd163 levels. furthermore, scd163 levels showed a correlation with the time elapsed from hospital admission to sample extraction, suggesting a potential indicator of disease progression. monocytes and macrophages constitute a key component of immune responses against viruses, acting as bridge between innate and adaptive immunity (20) . activation of macrophages has been demonstrated to be pivotal in the pathogenesis of the immunosuppression associated to several viral infections (such as vih, measles), where expansion of specific subsets of monocytes and macrophages in peripheral blood are observed, and considered to be drivers of immunopathogenesis (21) . our results support the hypothesis of a preponderant role for monocytes in sars-cov-2 immunopathology, associated to an overexuberant immune response. increased levels of monocytemacrophage activation markers, and their correlation with other inflammatory biomarkers (particularly il-6), indicate a close relationship between monocyte activation and immunopathology in these patients. inflammatory markers are closely related to severity in covid-19 pathology (22) and selective blockade of il-6 has been demonstrated to be a good therapeutic strategy in covid-19 pathology (23). our results thus suggest that monocyte-macrophage activation can act as driver cells of the cytokine storm and immunopathology associated to severe clinical course of covid-19 patients. further, monitorization of monocyte activity trough these soluble activation markers and/or follow-up of circulating inflammatory monocytes in peripheral blood, could be useful to assess disease progression in the same way as in other viral infections (16) . in addition, our results identify monocyte-macrophage as a good target for the design of therapeutic intervention using drugs that inhibit monocyte-macrophage activation and differentiation. in this sense, anti-gm csf inhibitor drugs, currently under clinical trials for rheumatic and other auto-inflammatory diseases, might provide satisfactory results in covid-19 patients. other drugs targeting monocyte and/or macrophage could also be useful in covid-19, as in other inflammatory diseases (24) . the strategy of inhibiting monocyte differentiation has proved useful in avoiding cytokine storm syndrome after car-t cell immunotherapy (25), suggesting a possible therapeutic application to covid-19 immunopathology (26, 27) . the present study has several limitations, including a relatively low sample size and the interference of corticoids in icu patients' results. however, these preliminary results are strongly suggestive of an important implication of monocytemacrophage in covid-19 immunopathology, as highlighted by the correlations found between these biomarker levels and inflammatory parameters. further studies using broader series are needed to confirm our findings. in summary, our data underscore the preponderant role of monocyte and macrophage immune response in covid-19 immunopathology and provide pointers for future interventions in drug strategies and monitoring plans for these patients. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the studies involving human participants were reviewed and approved by comité de ética de la investigación con medicamentos de galicia (fast-track approval 18-march-2020). written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. the landscape of lung bronchoalveolar immune cells in covid-19 revealed by single-cell rna sequencing. medrxiv covid-19 infection induces readily detectable morphological and inflammation-related phenotypic changes in peripheral blood monocytes, the severity of wich correlate with patient outcome. medrxiv speciality collaboration, covid-19: consider cytokine storm syndromes and immunosuppression virus associated hemophagocytic syndrome cd14(dim)/cd16(bright) monocytes in hemophagocytic lymphohistiocytosis how viruses contribute to the pathogenesis of hemophagocytic lymphohistiocytosis. front immunol recommendations for the management of hemophagocytic lymphohistiocytosis in adults virus-associated hemophagocytic syndrome as a major contributor to death in patients with 2009 influenza a (h1n1) infection is secondary hemophagocytic lymphohistiocytosis behind the high fatality rate in middle east respiratory syndrome corona virus? the pathogenesis and treatment of the 'cytokine storm' in covid-19 soluble cd14 is a nonspecific marker of monocyte activation differential expression of cd163 on monocyte subsets in healthy and hiv-1 infected individuals plasma levels of soluble cd14 independently predict mortality in hiv infection elevated levels of serum-soluble cd14 in human immunodeficiency virus type 1 (hiv-1) infection: correlation to disease progression and clinical events immunologic failure despite suppressive antiretroviral therapy is related to activation and turnover of memory cd4 cells increased monocyte turnover from bone marrow correlates with severity of siv encephalitis and cd163 levels in plasma persistent high plasma levels of scd163 and scd14 in adult patients with measles virus infection modulation of human monocyte/macrophage activity by tocilizumab, abatacept and etanercept: an in vitro study effects of corticosteroids on human monocyte function co-ordinating innate and adaptive immunity to viral infection: mobility is the key soluble cd163, a novel marker of activated macrophages, is elevated and associated with noncalcified coronary plaque in hiv-infected patients correlation analysis between disease severity and inflammation-related parameters in patients with covid-19 pneumonia. medrxiv the cytokine release syndrome (crs) of severe covid-19 and interleukin-6 receptor (il-6r) antagonist tocilizumab may be the key to reduce the mortality gm-csf inhibition reduces cytokine release syndrome and neuroinflammation but enhances car-t cell function in xenografts a strategy targeting monocyte-macrophage differentiation to avoid pulmonary complications in sars-cov2 infection role of monocytes/ macrophages in covid-19 pathogenesis: implications for therapy the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 gómez-rial, currás-tuala, rivero-calle, gómez-carballa, cebey-lópez, rodríguez-tenreiro, dacosta-urbieta, rivero-velasco, rodríguez-núñez, trastoy-pena, rodríguez-garcía, salas and martinón-torres. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-011360-1n998win authors: zloto, keren; tirosh-wagner, tal; bolkier, yoav; bar-yosef, omer; vardi, amir; mishali, david; paret, gidi; nevo-caspi, yael title: preoperative mirna-208a as a predictor of postoperative complications in children with congenital heart disease undergoing heart surgery date: 2019-11-15 journal: j cardiovasc transl res doi: 10.1007/s12265-019-09921-1 sha: doc_id: 11360 cord_uid: 1n998win major perioperative cardiovascular events are important causes of morbidity in pediatric patients with congenital heart disease who undergo reparative surgery. current preoperative clinical risk assessment strategies have poor accuracy for identifying patients who will sustain adverse events following heart surgery. there is an ongoing need to integrate clinical variables with novel technology and biomarkers to accurately predict outcome following pediatric heart surgery. we tested whether preoperative levels of mirnas-208a can serve as such a biomarker. serum samples were obtained from pediatric patients immediately before heart surgery. mirna-208a was quantified by rq-pcr. correlations between the patient’s clinical variables and mirna levels were tested. lower levels of preoperative mirna-208a correlated with and could predict the appearance of postoperative cardiac and inflammatory complications. mirna-208a may serve as a biomarker for the prediction of patients who are at risk to develop complications following surgery for the repair of congenital heart defects. major perioperative cardiovascular events, such as cardiac arrest, arrhythmia, and pericardial tamponade, are important causes of morbidity in patients undergoing repair of congenital heart disease (chd) [1] . a number of clinical risk indices have been developed, but their predictive power is limited [2] , raising the need for a simple and strongly predictive noninvasive alternative. mirnas are a class of noncoding rnas of approximately 22 nucleotides in length that was shown to be useful biomarkers in various diseases. the posttranslational regulatory roles of mirnas have been demonstrated in almost all physiological processes. unlike other extracellular rna molecules, extracellular mirnas are remarkably stable in serum and, together with their tissue specificity and the ease by which they can be detected and quantified, they have sparked great interest in their use as clinical biomarkers for a wide range of medical states [3] . we had reported that the level of circulating cardiac mirna-208a following surgery can serve as a sensitive biomarker for the postoperative course of pediatric patients with chd undergoing heart surgery for the repair of their defect [4] . in the present study, we hypothesized that preoperative mirna-208a levels in the blood of pediatric patients with chd can predict postoperative complications. all pediatric patients with chd who underwent cardiac surgery at safra children's hospital between 2012 and 2016 and whose legal representative provided informed consent were enrolled. the inclusion criteria were age younger than 21 years and an echocardiographic diagnosis of chd that required cardiac surgery. the laboratory parameters assessed in the study were as follows: troponin at 24 h after surgery, lactate at 0 h, 6 h, 12 h, 24 h, and maximal lactate after surgery, and maximal creatinine, aspartate aminotransferase (ast), and alanine aminotransferase (alt) levels measured during the hospitalization period. the preoperatively measured parameters were aristotle score [5] , invasive or noninvasive ventilator support, oxygen saturation levels, and whether the surgery was elective or nonelective. the surgical parameters were the cardiopulmonary bypass (cpb) and aortic crossclamp (acc) times. the postoperative parameters were invasive and noninvasive ventilator support, length of ventilation, length of hospitalization (loh), need for reintubation, need for extracorporeal membrane oxygenation (ecmo), maximal inotropic support, and number of days during which inotropic support was given. these parameters were divided into two groups in order to examine correlations. the first group contained the parameters that were related to the loh (i.e., invasive and noninvasive ventilation days and the total number of hospitalization days), and they did not account for children who died following surgery. the second group contained all the other parameters and accounted for all the children in the study. the appearance of cardiac and inflammatory complications was the primary outcome. cardiac complications were defined as the need for cardiorespiratory resuscitation, pericardial and/or pleural effusion requiring treatment or drainage, and postoperative arrhythmia (junctional ectopic, supraventricular, and ventricular tachycardias). inflammatory complications were defined as the need for antibiotic treatment. twenty-six patients comprised the group that sustained complications, of whom 19 patients were diagnosed with complications that involved the heart. laboratory parameters, loh, and higher or lower than the median maximal lactate value were the secondary outcomes. all samples were processed within 24 h of surgery. the blood was centrifuged at 4°c for 10 min at 1200g followed by separation of serum. centrifugation was then repeated at 4°c for 10 min at 10000g. rna was extracted from 250 μl serum to which 5.6 × 10 8 copies of cel-mirna39 were added using tri-reagent®-ls (sigma). rna was resuspended in 45 μl h 2 o of which 5 μl was taken for cdna synthesis (taqman® microrna-rt-kit, abi, #4366597) using a specific primer. mirna quantification was performed on a stepone™ pcr (qpcr). reactions (10 μl total) were run in triplicates and consisted of: 5 μl pcr-mix (abi, #ab-4444557), 0.5 μl of either of the taqman mirna assays (abi, #ab-4427975): cel-mirna39 (#000200) or mirna-208a (#000511), and 1.5 μl of the cdna and 3 μl h 2 o. the cycle threshold (ct) values were calculated with the stepone software v2. 3 . δct values are calculated by subtracting the ct of mirna-208a from the ct of cel-mirna39 of the same sample. the δct values are inversely correlated with the amount of template mirna present in the reaction. the rq was analyzed using the δδct method. categorical variables were described as frequency and percentage. continuous variables were evaluated for normal distribution using histograms and q-q plots. the normally distributed continuous variable was described as mean and standard deviation (sd), and non-normally distributed continuous variables as median and interquartile range (iqr). continuous variables were compared between patients with and without complications using an independent sample t test. correlations between continuous variables were evaluated using the spearman correlation coefficient. univariate and multivariate logistic regressions were used to evaluate the association between the mirna expression and the complications. the multivariate models were performed twice. the model included age and aristotle as confounders. the area under the receiver operating characteristic (roc) curve was used to evaluate the discrimination ability of the mirna. odds ratios (or) with 95% confidence intervals (ci) were reported. all statistical tests were 2-tailed, and p < 0.05 was considered statistically significant. all calculations were performed using spss (ver. 25.0). all statistical analyses were performed on three groups of patients: (1) the entire cohort, (2) patients with oxygen saturations below 90%, and (3) patients with oxygen saturations above 90%. the protocol of this study was approved by the institutional review board of the chaim sheba medical center. informed consent was obtained from the legal representatives of all subjects. seventy-nine consecutive children who underwent cardiac surgery for repair of congenital anomalies at safra children's hospital between 2012 and 2016 were enrolled. there were 34 (43%) females and 45 (57%) males. the characteristics of the population are shown in table 1 and the surgical procedures are listed in table 2 . the surgical characteristics, the laboratory parameters, and the preoperative and postoperative characteristics of the study patients are summarized in tables 3, 4 , and 5. the expression of mirna-208a was measured immediately before the beginning of the operation in all the patients. the patients were divided into two groups: those that did not sustain postoperative complications and those that did. the relative levels of expression of mirna-208a were 3.7 times higher in the group of children with an uncomplicated postoperative course (p = 0.03) (fig. 1) . logistic regression analysis showed an association between the preoperative levels of mirna208a and the risk of developing complications: the lower the level of mirna-208a in the patient's blood before the operation, the higher was the risk for developing complications following surgery (crude or 1.16; 95% ci 1.01-1.33; p = 0.03). this association remained significant after adjusting for the child's age and aristotle score (adjusted or 1.14; 95% ci 0.99-1.32; p = 0.05) ( table 6) . preoperative levels of mirna-208a were studied as predictors of postoperative complications using an roc curve and the area under the curve (auc). the result of this analysis reached a level of significance, with an auc of 64% (95% ci 51.1-77.0%; p = 0.04) (fig. 2) . there was a significant (p = 0.03) inverse correlation between the amount of mirna-208a before surgery and the aristotle score. there was also a correlation (p = 0.01) between preoperative low oxygen saturation values with a shorter loh. we hypothesized that mirna-208a may have better prediction performances in a more homogenous group of patients in relation to their oxygen saturation levels. to test that hypothesis, we divided our cohort into two groups: patients with oxygen saturation levels below 90% (hypoxic) and patients with oxygen saturation levels above 90% (normoxic). we analyzed the data on the preoperative levels of mirna-208a in each group separately, looking for correlations between the amount of mirna-208a and postoperative outcome. the relative levels of expression of mirna-208a before surgery among the children with oxygen saturation levels below 90% (n = 38) were 9.0 times higher for those without postoperative cardiac complications compared with the children who did sustain them (p = 0.01) (fig. 3) . logistic regression calculations in the group of patients with oxygen saturation levels below 90% yielded a statistically significant association between the preoperative level of mirna-208a and the risk of developing postoperative complications: the lower the level of mirna-208a in the patient's blood before the operation, the higher was the risk for developing cardiac complications following surgery (crude or 1.28; 95% ci 1.02-1.60; p = 0.02). table 6 ). the ability of preoperative levels of mirna-208a to predict postoperative cardiac complications in the group of patients with oxygen saturation levels below 90% was studied using an roc curve and the auc. this analysis reached a level of significance, with an auc of 71% (95% ci 54.5-88.1%; p = 0.02) (fig. 4) . a δct of 18.0 can serve as a cutoff value for the prediction of the risk of complications following heart surgery in that group of patients. specifically, δct > 18.0 predicted that the child will sustain complications whereas δct < 18.0 predicted that he/she will not. the sensitivity of this value was 81% and its specificity was 46%. the 41 children who had oxygen saturation levels above 90% had significant inverse correlations between the amount of mirna-208a before the operation and the following postoperative parameters: fewer days of postoperative invasive or noninvasive ventilation (p = 0.05) and fewer days of inotropic support (p = 0.00). in addition, higher levels of mirna-208a correlated with lower lactate values (p = 0.01), with lower creatinine levels (p = 0.00), and with lower aristotle scores (p = 0.00). these results also indicated that higher levels of preoperative mirna-208a correlated with a better outcome in this group (table 7) . dividing the patients with oxygen saturation levels above 90% according to their postoperative lactate values revealed a significant (p = 0.01) difference in their preoperative mirna-208a levels (fig. 5) . patients with higher postoperative lactate values had lower preoperative mirna-208a levels. this result supports the ability of preoperative mirna-208a levels to predict the patient's postoperative outcome. it is also worth noting that dividing this group of patients according to their loh (more or less than the median of 8 days) yielded a substantial, although not significant, difference in the amount of preoperative mirna-208a: specifically, children with a shorter loh had 2.7 times more mirna-208a in serum before their operation than those with longer lohs. the findings of our current investigation revealed that preoperative levels of circulating cardiac mirna-208a in the serum are predictive of the pediatric patient's complications following surgery for life-threatening anatomical malformations associated with chd. to the best of our knowledge, this is the first study to identify a mirna which can serve as a single preoperative biomarker effective in predicting the postoperative outcome in this setting. this is a continuation of our previous study in which we identified that circulating mirna-208a can serve as an accurate biomarker for predicting the risk of developing postoperative complications 6 h following surgery: high levels of mirna-208a in the serum several hours after surgery were indicative of complications which increased the risk of sustaining a worse outcome [4] . the potential benefit of being able to preoperatively identify patients at high risk for complications following heart surgery for such life-threatening conditions is enormous. accurate preoperative risk assessment at an individual patient level enhances clinical decision-making of postoperative patient management and the estimation of associated risks. to date, preoperative prediction of the postoperative course involves the use of the aristotle score which takes into account the complexity of the surgical procedure and predicts 30-day mortality and loh during the first postoperative week. in addition, recent papers have shown the potential benefit of preoperative st2 levels to identify children with increased risk of mortality or readmission after pediatric heart surgery [6, 7] . we were intrigued by the possibility that mirna-208a could also serve as a preoperative biomarker for the child's potential sequelae. our results indeed showed that high expression levels of preoperative circulating mirna-208a were predictive of a better postoperative outcome, as reflected by fewer complications following surgery and, accordingly, were correlated with a low aristotle score and a shorter loh. we hypothesized that the elevated mirna-208a found in the sera of patients before the operation was secondary to the presence of a disturbance in normal physiology and that such patients benefited from preconditioning of the heart, thereby leading to a greater likelihood of an uncomplicated postoperative course. the hearts of those patients were "prepared" before surgery by a yet to be determined mechanism, which enabled them to cope optimally with the stress caused during and immediately after the operation, with the result of fewer postoperative complications. our study is not the first to suggest that a beneficial postoperative outcome is seen in patients whose hearts were exposed to preoperative stress. remote ischemic preconditioning (ripc) was found to be clinically effective in a study by wu et al. on tof children undergoing open heart surgery [8] . those authors showed that ripc attenuated myocardial ischemia/reperfusion (i/r) injury and improved the short-term prognosis of those patients [8] . not surprisingly, mirnas have been reported to mediate such protection of the heart in several studies [9, 10] . the involvement of mirnas in protective mechanisms has also been described in preconditioning of the brain [11] . the source of the high levels of circulating mirna-208a before surgery has yet to be determined. in general, extracellular circulating mirnas originate from two main sources: they can be merely byproducts of cellular activity and cell death [12] , or they can be the result of a selective export system which enables the secreted mirna to be transferred to a recipient cell where it can carry out its role [13] . we and others have shown that the presence of mirna-208a in the blood correlates with damage caused to the heart [4, 14, 15] ; however, there are no studies that define the exact mechanism of the export system. notably, however, circulating mirna-208a in the blood could be a trigger for inducing a heartprotective mechanism whose results become evident following surgery in both of the above-mentioned scenarios. a role for mirna-208a in communicating signals from the heart to other parts of the body has been reported by feng et al., who found that cardiac mirna-208a released into the circulation following myocardial i/r is capable of activating innate [16] . we suggest that because the hearts of the children that were subjected to prolonged physiological stress until the date of the operation had the opportunity to be more prepared, and possibly more protected, from the effects of the operation, those children sustained fewer postoperative complications. although the correlation between the levels of mirna-208a and the postoperative course was more significant in the cyanotic patients, results pointing in the same direction were also obtained for the non-cyanotic ones. this observation strengthens our position that exposing the heart to abnormal physiological constraints, as could be expected in children with low oxygen saturation, produces a stronger "signal" to provoke a protective mechanism, whatever it may be. this is a pilot study which is intended to serve as the basis for a larger investigation designed to examine the value of obtaining preoperative mirna levels for the purpose of guiding decisions regarding surgery and the postoperative management that will need to be provided for a specific patient. in addition to demonstrating the benefit of having such information, our results suggest that mirna-208a may serve as a preoperative target for therapy in children with chd. increasing its levels in the blood of such patients may trigger the preconditioning of their heart and thus contribute to a smoother postoperative course. we are aware of a main limitation of our study that bears mention. our cohort comprises patients with diverse chds which necessarily affect their medical parameters. our attempts to divide the cohort into homogenous groups resulted in groups too small to enable us to reach statistically significant results. we are also aware that our small sample size may be a limitation for our multivariate analysis results. we have shown that the level of preoperative mirna-208a in serum is a reliable biomarker for the prediction of patients who are at risk to develop postoperative complications. unlike other extracellular rna molecules, extracellular mirnas are remarkably stable in serum and, together with their tissue specificity and the ease by which they can be detected and quantified, they have sparked great interest in their use as clinical biomarkers for a wide range of medical states. such a biomarker can help in surgical comorbidity assessment, which is an integral part of patient risk stratification. further studies to assess its applicability to specific cardiac surgeries and to other invasive cardiac procedures should be performed in a larger group of patients. funding information this work was funded with institutional departmental funds. all procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 helsinki declaration and its later amendments. ethical approval was obtained from the ethics committee of the sheba medical center. no animal studies were carried out by the authors for this article. the authors declare that they have no conflict of interest. informed consent informed consent was obtained from all individual participants or their parents/legal guardians included in the study. correlations were calculated for δct values fig. 5 relative average mirna-208 expression in children with oxygen saturation more than 90%. expression levels were quantified by qpcr and the average level of the group of patients for whom lactate maximal levels were above the median (i.e., > 42 mg/dl) was set as 1. values are shown ± sem seminal postoperative complications and mode of death after pediatric cardiac surgical procedures utility of clinical risk predictors for preoperative cardiovascular risk prediction micrornas as biomarkers for clinical studies mirna-208a as a sensitive early biomarker for the postoperative course following congenital heart defect surgery the aristotle comprehensive complexity score predicts mortality and morbidity after congenital heart surgery biomarkers associated with 30-day readmission and mortality after pediatric congenital heart surgery novel biomarkers improves prediction of 365-day readmission after pediatric congenital heart surgery cardiac protective effects of remote ischaemic preconditioning in children undergoing tetralogy of fallot repair surgery: a randomized controlled trial microrna-125b protects against myocardial ischaemia/reperfusion injury via targeting p53-mediated apoptotic signaling and traf6 microrna-144 attenuates cardiac ischemia/reperfusion injury by targeting foxo1. experimental and therapeutic medicine role of micrornas in innate neuroprotection mechanisms due to preconditioning of the brain circulating mir-499 as a potential biomarker for acute myocardial infarction delivery of microrna-126 by apoptotic bodies induces cxcl12-dependent vascular protection analysis of plasma mir-208a and mir-370 expression levels for early diagnosis of coronary artery disease correlation between serum exosome derived mir-208a and acute coronary syndrome extracellular micrornas induce potent innate immune responses via tlr7/myd88-dependent mechanisms publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments we thank amisragas for their continuous support of the department of intensive care. key: cord-022070-soqeje4z authors: parry, christopher m.; peacock, sharon j. title: microbiology date: 2019-05-28 journal: hunter's tropical medicine and emerging infectious diseases doi: 10.1016/b978-0-323-55512-8.00021-1 sha: doc_id: 22070 cord_uid: soqeje4z the management and containment of many treatable and preventable infectious diseases in resource-poor countries is limited by the failure to make an accurate diagnosis. most of the world's population lacks access to accurate, affordable, easy-to-use, quality-assured, reliable, and accessible diagnostic tests and misdiagnosis of infectious diseases is common and compromises patient care. laboratory diagnostics are also needed for the detection and surveillance of the increasing levels of antimicrobial resistance. accurate clinical diagnosis in resource-poor settings relies strongly on the laboratory service, and the need to support the development of a quality-assured laboratory service in such settings is increasingly recognized. international organizations are actively working with local and national providers to improve laboratory services. the development of laboratory services will contribute to improved health for the local population, protection against emerging pathogens, and ensure better use of scarce health care resources. christopher m. parry, sharon j. peacock support. diagnostic algorithms have been developed for situations with no laboratory backup, an approach adopted, for example, in the integrated management of childhood illness (imci). unfortunately, for many infections, clinical features lack sufficient specificity to allow them to be used to differentiate the possible diagnoses, and over-treatment to cover the various possibilities is common. in the assessment of the febrile child in the tropics, for example, malaria and systemic bacterial infections often have an indistinguishable clinical picture. malaria may be diagnosed by smear microscopy, but bloodstream infections require a blood culture service. it has become clear in recent years that bloodstream infections represent an underappreciated burden of disease and mortality. this was clearly demonstrated by a study conducted in kenya in which bacterial bloodstream infections diagnosed by blood culture were responsible for 26% of deaths among children admitted to a rural district hospital. 1 without an accurate diagnosis and specific treatment, bloodstream infections such as those due to salmonella enterica, staphylococcus aureus, streptococcus pneumoniae, or burkholderia pseudomallei can carry a high mortality. distinguishing cerebral malaria, bacterial meningitis, and encephalopathic typhoid may be similarly difficult without laboratory support. children in sub-saharan africa with clinical symptoms of pneumonia may have pneumococcal pneumonia but can equally have malaria or invasive salmonellosis. a child with dysentery may be suffering from amebic colitis, shigella infection, or enterohemorrhagic escherichia coli. in adults, syndromic management of sexually transmitted infections is widespread but needs to be informed by periodic surveillance of antimicrobial susceptibility patterns. emerging and potentially epidemic viral infections such as severe acute respiratory syndrome (sars), influenza (h5n1 and h1n1), ebola, and zika require relatively sophisticated tests to confirm the diagnosis. 2 infections by pathogens that are resistant to multiple antimicrobials are common in many tropical countries where there is widespread availability of over-the-counter antimicrobials. appropriate therapy of these infections requires isolation of the causative organism and antimicrobial susceptibility testing. 3 laboratories also have an important public health role within the health care system. the ability to investigate outbreaks of disease as part of epidemic preparedness is a key function. these might include outbreaks of watery or bloody diarrhea, epidemics of meningitis, or clusters of patients with fever of unknown etiology. in addition, laboratories are a critical component of disease control programs such as the national programs for the control of tuberculosis, hiv, and malaria. the lack of laboratory capacity to support the expansion of diagnostic testing and antiretroviral therapy in hiv programs and in disease outbreaks such as ebola has made many international organizations appreciate the desperate plight of the laboratory service for the first time. 2 tuberculosis can be diagnosed in many patients with a ziehl-neelsen-stained smear of sputum, but to extend the diagnosis in those who are acid-fast bacilli (afb) negative or have multi-drug-resistant (mdr) disease requires more developed laboratory support. furthermore, laboratories have an increasing role in infection control in health care settings and congregate facilities and in the prevention of health care-associated infections. accurate disease surveillance requires a laboratory network and is vital to inform public health policy concerning allocation of resources and disease prevention. laboratories can help to • accurate diagnosis in resource-poor settings is severely limited by the absence of good diagnostic laboratory services. • laboratories in resource-restricted settings struggle with poor facilities, lack of reliable water and electricity, inadequate equipment and consumables, insufficient staff, poor training and low morale, absence of standard operating procedures and quality assurance programs, and inadequate levels of biosafety. • a country plan for the development of a laboratory network requires consideration of the needs at primary, district, provincial/regional, and national levels. • at the district hospital level, a quality-assured repertoire of essential laboratory tests can contribute to improved health care. • surveillance by microbiology laboratories provides an understanding of the causes of infection in the local population and the levels of antimicrobial resistance in key pathogens, and informs public health policy on appropriate antimicrobial therapy and preventive strategies. • there is increasing recognition of the need to support the development of a quality-assured laboratory service in resource-restricted settings and develop simple and robust point-of-care diagnostics both for routine clinical care and outbreak response. • point-of-care rapid diagnostic tests are changing our approach to the diagnosis of some infectious diseases, but care needs to be taken about their usage and interpretation of results. the effective management and containment of many treatable and preventable infectious diseases in resource-restricted countries is limited by the failure to make an accurate diagnosis. access to accurate, affordable, easy-to-use, quality-assured, reliable, and accessible diagnostic tests is severely lacking for most of the world's population, and misdiagnosis of infectious diseases is common. disease identification, appropriate treatment choice, and implementing public health measures for the prevention and control of endemic and epidemic infections all require laboratory support. this lack of reliable diagnostics compromises patient care. laboratory diagnosis also highlights the increasing levels of resistance to antimicrobials in many infections and the need for newer, possibly unaffordable, antimicrobials such as broad-spectrum antimicrobials in bacterial sepsis, or second-line combination therapy for aids, malaria, and tuberculosis. this issue is increasingly recognized and being addressed in many regions. in most resource-restricted settings, individual patient diagnosis is based on clinical signs and symptoms with little or no laboratory be rudimentary so that specimens referred to the next level are not transported in a timely manner and results do not return in a time period that will influence clinical management. it is standard practice in tuberculosis programs that patients who fail treatment should have a sample cultured for tuberculosis so that susceptibility tests can be performed. in a study of the transport of such specimens to the central reference laboratory in malawi, only 40% of specimens arrived in the reference laboratory and only 36% of those samples received were successfully cultured for susceptibility testing. 5 the shortage of staff with appropriate education and training is a further problem. many laboratory workers have no formal training and are simply trained at the bench. at the peripheral level, there may be only one laboratory assistant, with no more than secondary school education. at the district level, there may be assistants and technicians (formally educated in laboratory medicine for 3 years). at the central level, technicians may work alongside technologists (with 2 years specialist post-technician training) and scientists (university science graduates). regardless of qualifications, laboratory workers often have a lowly status within the health sector, and the attrition of health care personnel out of government service results in low morale among those who remain. private or research laboratories may attract the best technicians from the government sector. diagnostic laboratories frequently have no representation at the local, provincial, or national level, or, if they do, it is only as part of the support services. in many countries, the voice of the laboratory is rarely heard. these many problems contribute to a poor biosafety situation in laboratories. the lack of equipment, knowledge, and training means that laboratory workers are processing samples with hazardous pathogens in an unsafe manner. in a study of tuberculosis laboratories in korea, before safety conditions had been upgraded, the relative risk of being diagnosed with tuberculosis for the technicians performing drug susceptibility tests was 21.5 (95% ci 4.5-102.5) compared with non-laboratory workers. 6 the true magnitude of this problem in laboratory workers is difficult to gauge because surveillance of infection in laboratory workers is rarely performed or reported. at a national level, the important contribution of laboratories needs to be appreciated within the ministry of health, by national and local health care managers, and by funding organizations. a representative of the laboratory services should be present in the key decision-making committees. support is also needed from clinicians, who often have disproportionate influence within the system. a plan for the laboratory network should become part of the overall health care development plan. there needs to be a priority list of core and essential services provided in a qualityassured manner. the laboratory plan should include the provision for a tiered laboratory network at the primary, district, regional/ provincial, and national levels. the plans should be realistic, affordable, and sustainable. at the level i or primary level, perhaps in a health post or health center serving outpatients, microscopy for malaria and tuberculosis and testing for hiv with a same-day service would be essential. these laboratories can serve as a collection point for samples that need referral to the next level. the level ii facility in the local district hospital would have a dedicated laboratory space and a broader repertoire of tests serving inpatients and outpatients. the tests offered would depend on the spectrum of local diseases and resources available, and may be limited to microscopy, simple biochemistry and serology, and blood transfusion, or may include bacterial culture facilities. laboratories can act as a hub for the primary-level laboratories, providing them with support, supplies of reagents, and qa activities. at the level iii, define clinical problems by sampling surveys. for example, determining the antimicrobial susceptibilities of bacterial pathogens such as s. aureus, s. pneumoniae, or s. enterica for a selection of isolates can inform the appropriate empiric therapy in a particular area. an understanding of the burden of disease in an area-drugresistant typhoid in an urban slum, for example-could lead to public health measures such as a vaccination program. laboratory surveillance programs may produce the clue to the possibility of new organisms emerging, including both bacteria and viruses, most commonly at the animal-human interface. at for many health care staff working in resource-restricted areas, the major problem is simply a lack of laboratory services. hospital laboratories may be absent, or, if they are available, only offer a limited repertoire of tests. in other areas, particularly in asia, a wide range of alternative services is offered by private diagnostic laboratories, typically outside the front gate of the hospital but with uncertain quality. even when the tests are available, they may not be used or the results ignored. lack of use may stem from a poor perception of the laboratory, and tests may not be available because the costs are prohibitive. even when laboratories are present, they face the many challenges that are familiar to all areas of the health care sector. inadequate facilities are common, with laboratories that lack space and a secure supply of electricity and water. appropriate equipment may be unavailable or poorly maintained. even basic equipment required for a functioning laboratory can be in disrepair because of the absence of regular care and servicing. a functioning microscope is a key piece of equipment for a basic microbiology laboratory but is frequently found in poor condition. in a survey of 90 microscopes in laboratories in nine districts in malawi, only 50% were in good condition. 4 there were 1.1 functioning microscopes per 100,000 population, and even microscopes in need of full servicing were still in daily use. the 90 microscopes were from 16 different manufacturers, illustrating the lack of standardization of laboratory equipment so frequently seen. the provision of biological safety cabinets is another area where equipment from multiple manufacturers and lack of spare parts and maintenance are common, and in this case may lead to unsafe and hazardous conditions for laboratory workers. standardization of equipment and consumables with central ordering, maintenance contracts, and supplies of spare parts would seem a sensible response to this issue but is rarely seen. tests may also be unavailable because of an inadequate supply route for consumables. this is another area where standardization of tests and central ordering and supply can lead not only to more reliable supply of quality-assured consumables but also to potential cost savings for the country. the laboratory can generate results, but the quality may be poor. standard operating procedures may be absent and quality control of routine procedures non-existent. the absence of national or regional laboratory guidelines or programs of external quality assurance (qa) by the laboratory network is common. communications between different levels within the laboratory network may laboratories are further categorized into biosafety levels (bsl) so that the facilities available are matched to the pathogens handled. a standard diagnostic laboratory would be at bsl2, and the basic requirements for such a laboratory are outlined in table 21 .2 and box 21.1. more specialized laboratories such as tuberculosis reference laboratories where culture and susceptibility testing are performed require bsl3 facilities. bsl3 laboratories have particular design features to reduce the hazard of airborne transmission and incorporate directional airflows and the use of biological safety cabinets. they are particularly appropriate for laboratories handling pathogens such as tuberculosis and influenza. however, bsl3 facilities are very expensive and difficult to build and maintain. the who has recently indicated that in some circumstances, slightly less rigorous guidelines, so-called bsl2+ as outlined in table 21 .2, may be appropriate for selected laboratories, for example, processing samples for tuberculosis culture. 8 health care staff working at the district hospital level may be asked to advise on what would constitute an appropriate laboratory service for the hospital and district. the provision of an extensive range of tests is likely to be unaffordable and impractical. in a study evaluating the role of the laboratory in a district hospital in malawi, the services considered essential were blood transfusion (including blood grouping and compatibility testing and screening for hiv, hepatitis b, and syphilis), hemoglobin estimation, and the microscopic diagnosis of malaria and tuberculosis. 9 this list will vary in different areas, and the services of the laboratory should be orientated to the requirements of the district and the available resources. other tests that require relatively little investment and can be done where there are limited resources include microscopy of urine and stool samples for ova, cysts, and parasites; gram stain and cell count in cerebrospinal fluid and other sterile fluids; and gram stains of pus samples. the microscopic appearance of some typical bacterial pathogens is shown in fig. 21 .1a-f. guidelines for standard laboratory methods appropriate for resource-restricted areas are available. 10 a checklist of issues that should be considered when evaluating a diagnostic laboratory is in box 21.2. provincial or regional level, laboratories will be located in larger referral hospitals. laboratories at this level should be performing a more sophisticated range of tests with higher throughput. for example, facilities for tuberculosis culture might be available, together with molecular techniques for specific diseases and the ability to investigate disease outbreaks. support for the level ii laboratories would be an important function, including periodic visits and laboratory assessment as part of a qa program. national reference laboratories at level iv are likely to be located in the capital and serve specialized public health functions that may be linked to specific disease control programs such as the central reference laboratory for the national tuberculosis programme. it is important that laboratories at the national level have links to regional supranational reference laboratories for advice and quality assurance. level iii and iv laboratories would conduct surveillance and monitoring of infections using laboratory data collected throughout the network, establish standard operating procedures and protocols, conduct training and quality improvement, and plan for equipment needs and maintenance throughout the network. biosafety is an essential consideration at all levels of the laboratory network and depends on three principles. 7 good laboratory practice and technique are fundamental and require established standard operating procedures and appropriate induction and training of staff. safety equipment provides a primary barrier, and this includes appropriate, properly maintained and used equipment (e.g., centrifuges, biological safety cabinets) and personal protective equipment (e.g., gloves, respirators). finally, facility design and construction are a secondary barrier providing, for example, appropriate workflows (from clean to dirty areas) and directional airflows and containment if required. microorganisms are categorized into four hazard groups according to their risk to individuals and society and the availability of treatment and preventive measures (table 21. the diagnosis of infection depends on detection of the pathogen or the host response to the pathogen. direct pathogen detection is traditionally performed by light microscopy, although antigen detection and nucleic acid amplification tests (such as polymerase chain reaction [pcr]) are increasingly used. pathogen detection may also be carried out by isolation of the microorganism by culture of relevant clinical samples, and this allows susceptibility testing to be performed. methods based on detecting the immune response mainly rely on detecting pathogen-specific igm or igg antibodies. technological advances in the design of testing methods have simplified antigen and antibody detection to the point that simple point-of-care test kits are now widely available. the rapid kits for hiv antibody detection have an established place in the voluntary counseling and testing framework being established in many countries. rapid malaria detection tests have been recommended as a replacement for malaria microscopy in some guidelines and need to be positive before antimalarial treatment is given. in recent years, organizations such as the unicef/united nations development programme/world bank/who special programme for research and training in tropical diseases (tdr), and the foundation for innovative new diagnostics (find) have played an important role in developing and evaluating new diagnostic tests for many tropical diseases. 11 the who sexually transmitted diagnostics initiative has developed an approach to the characteristics of an ideal diagnostic test in the developingcountry context. "assured" tests should be affordable by those resistance surveillance system manual (e. coli, klebsiella pneumoniae, acinetobacter baumannii, s. aureus, s. pneumoniae, salmonella spp., shigella spp., and neisseria gonorrhoeae), as well as other pathogens of local or national importance. there have also been considerable advances in the format and ease of use of molecular tests. this is exemplified by the increasing use in tuberculosis laboratories of nucleic acid amplification tests directly from afb smear-positive sputum, or from culture isolates. line probe assays (lpas) use a multiplex pcr amplification followed by reverse hybridization to identify mycobacterium tuberculosis complex and mutations in the genes associated with at risk of infection, sensitive and specific, user friendly (simple to perform and requiring minimal training), rapid (to enable treatment at the first visit), robust (does not require refrigerated storage), equipment-free, and able to be delivered to those who need it. there has been increased attention on the problem of antimicrobial resistance for many important pathogens and the critical role that the laboratory plays in the management of this. initiatives have focused on methods and systems of surveillance of antimicrobial resistance in bacterial infections that countries can readily implement. 3, 12 the who guideline has recommended a focus on eight priority pathogens as described in the global antimicrobial rifampicin and isoniazid resistance. lpa can be performed with results in 1 to 2 days, which is considerably quicker than the weeks required for traditional culture methods, and the overall agreement for the diagnosis of mdr between these tests and conventional methods is 99%. the format of these tests is being simplified so that the feasibility of their routine use in tuberculosis reference laboratories in developing countries is becoming a reality. these methods are an important component of the roll-out of the programmatic management of mdr tuberculosis globally. quality assurance is defined as "planned and systematic activities to provide adequate confidence that requirements for quality will be met." the qa system is the basis for a guaranteed result. if this system is not followed, patients may get the wrong results, with important consequences for their health-such as receiving inadequate treatment. a program of qa in diagnostic laboratories involves not only internal quality control and external qa but also attention to appropriate staffing, training and supervision, and maintenance of equipment and facilities. international guidelines are now available and increasingly implemented for qa in many areas of laboratory practice such as afb smear microscopy and hiv testing. accurate clinical diagnosis in resource-restricted settings relies strongly on the laboratory service. the increasing recognition of the need to support the development of a quality-assured laboratory service in such settings is therefore welcome. in many regions, international organizations are actively working with local providers to improve laboratory services. the development of laboratory services will contribute to improved health for the local population and ensure better use of scarce health care resources. bacteremia among children admitted to a rural hospital in kenya diagnostic preparedness for infectious disease outbreaks amr surveillance in low and middle-income settings -a roadmap for participation in the global antimicrobial surveillance system (glass) evaluation of microscope condition in malawi using a bus service for transporting sputum specimens to the central reference laboratory: effect on the routine tb culture service in malawi risk of occupational tuberculosis in national tuberculosis programme laboratories in korea world health organization (who) guidance on bio-safety related to tb laboratory diagnostic procedures the operation, quality and costs of a district hospital laboratory service in malawi medical laboratory manual for tropical countries diagnostics for the developing world world health organization: global antimicrobial resistance surveillance system: manual for early implementation key: cord-034746-uxhpufnv authors: nusshag, christian; stütz, alisa; hägele, stefan; speer, claudius; kälble, florian; eckert, christoph; brenner, thorsten; weigand, markus a.; morath, christian; reiser, jochen; zeier, martin; krautkrämer, ellen title: glomerular filtration barrier dysfunction in a self-limiting, rna virus-induced glomerulopathy resembles findings in idiopathic nephrotic syndromes date: 2020-11-05 journal: sci rep doi: 10.1038/s41598-020-76050-0 sha: doc_id: 34746 cord_uid: uxhpufnv podocyte injury has recently been described as unifying feature in idiopathic nephrotic syndromes (ins). puumala hantavirus (puuv) infection represents a unique rna virus-induced renal disease with significant proteinuria. the underlying pathomechanism is unclear. we hypothesized that puuv infection results in podocyte injury, similar to findings in ins. we therefore analyzed standard markers of glomerular proteinuria (e.g. immunoglobulin g [igg]), urinary nephrin excretion (podocyte injury) and serum levels of the soluble urokinase plasminogen activator receptor (supar), a proposed pathomechanically involved molecule in ins, in puuv-infected patients. hantavirus patients showed significantly increased urinary nephrin, igg and serum supar concentrations compared to healthy controls. nephrin and igg levels were significantly higher in patients with severe proteinuria than with mild proteinuria, and nephrin correlated strongly with biomarkers of glomerular proteinuria over time. congruently, electron microcopy analyses showed a focal podocyte foot process effacement. supar correlated significantly with urinary nephrin, igg and albumin levels, suggesting supar as a pathophysiological mediator in podocyte dysfunction. in contrast to ins, proteinuria recovered autonomously in hantavirus patients. this study reveals podocyte injury as main cause of proteinuria in hantavirus patients. a better understanding of the regenerative nature of hantavirus-induced glomerulopathy may generate new therapeutic approaches for ins. . characteristics of 26 patients with acute hantavirus infection. acr = albumin-to-creatinine ratio, crp = c-reactive protein, dpo = days post onset of first symptoms, gcr = gram creatinine, los = length of hospital stay, max = maximum, min = minimum, pcr = protein-to-creatinine ratio, scr = serum creatinine. bold values are statistically significant for p < 0.05. (fig. 1 ), but the characteristic picture of tubular interstitial nephritis in the renal medulla (fig. 2) . electron microscopy of glomeruli revealed enlarged visceral podocytes, a focal foot process effacement together with a mild thickening of the glomerular basement membrane (gbm) and vacuolization of podocytes in hantavirus patients (fig. 1 , figure s1 ). immune deposits or further ultrastructural changes were not present. mean gbm widths were 367.3 nm (± 69.6), 504.9 nm (± 74.1), 482.5 nm (± 92.6) and 685.2 nm (± 133.8) for the control and hantavirus biopsy samples i, ii and iii, respectively. maximum podocyte foot process width was highest in proteinuric patients i and iii with 2064.2 nm and 2301.0 nm and significantly lower in patient ii and control with 1998.3 nm and 1885.5, respectively. however, due to the focal nature of the foot process effacement, mean podocyte width did not differ between patients and control (table s3) . compared to the control, em analysis of proximal tubular cells showed relevant subcellular lesions indicated by severe apical cytoplasmic vacuolization (fig. 2) . interestingly, these changes were predominantly observed in the two patients with severe proteinuria at the time of biopsy (hantavirus patient i and iii). biomarker levels on admission. on admission, patients suffering from hantavirus infection showed significantly increased urinary nephrin, igg, α1-mg and serum supar levels compared to healthy controls (fig. 3a ). when further dividing hantavirus patients according to the severity of pcr on admission, patients with severe pcr showed significantly higher median nephrin and igg levels compared to patients with moderwww.nature.com/scientificreports/ ate pcr. remarkably, an almost dichotomous distribution of urinary biomarkers of a defective gfb (nephrin and igg) was observed when moderate and severe pcr were compared on admission, indicating substantial differences in permeability of the glomerular slit diaphragm at that specific time point. a trend towards higher supar levels was also seen in patients with severe proteinuria. correlation analyses between serum supar levels, maximum scr and scr levels within the first 48 h showed no significant correlations, whereas a significant positive correlation was found for serum supar levels and levels of urinary nephrin, pcr, acr and igg (table s4 ). the normalization of nephrin and igg levels to urinary creatinine excretion led to similar results ( figure s2 ). in contrast, the previous significant difference of absolute α1-mg levels between patients with moderate and severe pcr disappeared after creatinine normalization ( figure s2 ). though, urinary biomarker levels decreased in both groups over time, patients with severe pcr showed significantly higher levels of nephrin, igg, acr and pcr during the first 48 h after admission ( table 2 ). the greatest absolute differences were seen for urinary biomarkers that indicate a defective glomerular barrier such as nephrin, igg, acr. at the same time, only minor absolute differences were observed for the tubular proteinuria marker α1-mg. when analyzing urinary nephrin concentrations over time in individual patients and in relation to the dpo instead of time after admission, urinary nephrin and pcr levels decreased almost in parallel. interestingly, the start of normalization of urinary nephrin and pcr levels preceded the first decline of scr by 48-72 h. furthermore, in patients with an available urine sample at the time of pcr normalization, the normalization of urinary nephrin levels tended to precede the normalization of pcr levels. figure 3b shows two exemplary biomarker courses of patients with acute hantavirus infection. in a next step, we analyzed the course of dpo-synchronized pcr values between patients with moderate and severe pcr in the entire cohort (table 3) . patients with severe pcr showed significantly higher pcr levels up to dpo 8 compared to patients with moderate pcr, indicating a higher renal disease severity between both groups at the same dpo. due to the self-limiting character of the hantavirus disease and subsequent autonomous recovery of the gfb in both groups, no differences in pcr levels were seen beyond dpo 8 ( table 3 ). the morphology and time course of pcr slopes within both groups www.nature.com/scientificreports/ was comparable, but the origin of the slope started at higher pcr levels in patients with severe proteinuria, especially before dpo 8. analyses showed a strong positive correlation between urinary nephrin levels and pcr, acr, igg, α1-mg and c-reactive protein (crp) levels ( table 4 ). the highest correlation coefficients (r) were thereby achieved between nephrin levels on admission and biomarkers of (non-selective) proteinuria. in contrast, weaker correlations where seen for scr, especially on admission and for maximum levels. furthermore, nephrin levels on admission showed a moderate positive association with the length of hospital stay (los) and a moderate negative association with platelet count and the time of admission in terms of dpo. hemoglobin and leukocytes values showed no relevant correlation with urinary nephrin levels. to our knowledge, this is the first comprehensive study to investigate the role of direct podocyte damage in acute puuv infection in vivo. our data show a strong association between urinary nephrin levels and the extent of (non-selective) glomerular proteinuria, suggesting that hantavirus infection causes a pronounced podocyte damage and subsequent impairment of the gfb. the significant findings in electron microscopy analyses were a focal foot process effacement, podocyte vacuolization and apical tubular vacuolization (indicating massive proteinuria) which all are known as typical histopathological features in ins 16, 17, [31] [32] [33] [34] [35] . however, the pathomechanical role and underlying mechanisms of the observed podocyte vacuolization need further clarification. while differences between patients with moderate and severe proteinuria were preserved for urinary nephrin and igg levels after normalization to urinary creatinine excretion, differences in α1-mg levels were no longer present. this further supports the idea that proteinuria in hfrs is predominantly from glomerular origin. furthermore, when individual patients were analyzed, the normalization of urinary nephrin levels tended to precede the normalization of proteinuria. the highest correlation coefficients (r) were achieved between urinary nephrin levels on admission and pcr, acr and igg levels within 48 h. both observations suggest urinary nephrin levels as an early indicator of gfb dysfunction and a direct pathophysiological connection between the current impairment of gfb integrity by hantavirus infection and the subsequent extent and clinical course of proteinuria. as a further similarity to ins 16, 18 , hantavirus patients showed significantly elevated serum supar levels in comparison to healthy controls. supar levels correlated significantly with urinary nephrin, pcr, acr and igg levels, but not with scr. it was generally believed that hantaviruses predominantly infect endothelial cells, leading to capillary permeability due to a loss of cell-to-cell contacts, but without a direct cytopathic effect 6 . the deregulation of systemic angiopoietin levels 9 and a vascular endothelial growth factor a (vegfa)-induced downregulation of ve-cadherins accompanied by a β3 integrin-mediated dysregulation of vegf receptor 2 (vegf2) are suggested mechanisms 19, [36] [37] [38] . though, these findings may explain major symptoms and complications of hfrs such as capillary leakage and pulmonary edema, the exact cause of (nephrotic) proteinuria is still poorly understood. vascular endothelial barrier dysfunction or tubular damage, as discussed by some authors 13,15 , does not adequately explain the extent of proteinuria. we have recently shown in vitro that hantaviruses additionally infect tubular epithelial cells, glomerular endothelial cells and especially podocytes, leading to disruption of cell-to-cell contacts and impaired intra-cellular integrity with rearrangements of the podocyte cytoskeleton 7, 39, 40 . here, we show for the first time in vivo an interdependent relationship between ultrastructural and functional impairments of the gfb and a direct podocyte damage as indicated by increased nephrin excretion and electron microscopy. in addition, urinary nephrin, pcr, acr and igg levels correlated significantly with serum supar values. to date, one other study showed significantly elevated blood supar levels and their association with hantavirus disease severity but did not include nephrinuria and the extent of proteinuria in their analysis 19 . supar is suggested to interfere with the cross-directional signaling between the glomerular basement membrane (gbm) and podocytes, thereby affecting gfb integrity 16 . in addition, alfano et al. recently showed that supar downmodulates nephrin expression in podocytes in vitro and in vivo 41 . together, these mechanisms may disturb table 3 . comparison of proteinuria in relation to the days post onset of first symptoms. dpo = days post onset of first symptoms, gcr = gram creatinine, pcr = protein-to-creatinine ratio. bold values are statistically significant for p < 0.05. www.nature.com/scientificreports/ table 4 . spearman's correlation of urinary nephrin levels on admission with parameters of hantavirus disease activity. acr = albumin to creatinine ratio, α1-mg = α1-microglobulin, ci = confidence interval, crp = c-reactive protein, dpo = days post onset of first symptoms, igg = immunoglobulin g, r = correlation coefficient, s-alb = serum albumin, scr = serum creatinine. bold values are statistically significant for p < 0.05. www.nature.com/scientificreports/ podocyte-gbm-interaction, where rearrangements of the podocytic cytoskeleton may result in podocyte damage, subsequent foot process effacement and gfb dysfunction with release of intercellular gfb components such as nephrin into the urine 7, 16, 18, 39 . further studies are required to clarify whether supar is a directly involved pathomechanically mediator in hantavirus-induced podocyte injury. nevertheless, a very unique feature in puuv infection is that proteinuria and kidney function usually recover autonomously in all patients 8 , while therapeutic outcomes in ins are variable and autonomous recovery is rare 16, 17 . hantavirus induced proteinuria usually peaks around dpo 5 and normalizes within 1-3 weeks 13 . but, due to a varying hantavirus disease severity, the actual extent of proteinuria differs in patients 13 . the same observation applies to our cohort. patients with severe pcr on admission still showed significantly higher pcr levels compared to patients with moderate pcr, when pcr values were matched based on the dpo instead of the time of hospital admission. scr in turn peaks 4-5 days after the peak of proteinuria 13 . this tempts authors to claim that proteinuria predicts the severity of emerging aki, by showing an association between glomerular proteinuria and maximum scr levels 13 . however, it is generally accepted that scr reflects rapid changes in renal function with a latency of at least 24-72 h (time until a new steady state is reached after a single renal insult) 42 . we therefore hypothesize that the extent of maximum renal impairment is at least in part already present at the time of maximum proteinuria but is indicated with a delay by scr. our results support this hypothesis by showing a stronger correlation between pcr values on admission and scr concentrations after 24-48 h (24 h: r = 0.69, p < 0.0001; 48 h: r = 0.81, p < 0.0001) than for maximum scr levels or scr levels on admission (adm: r = 0.27, p = 0.1837; max: r = 0.56, p = 0.029). other accepted markers of hantavirus disease severity such as leukocyte and platelet count, crp and hemoglobin levels, showed weaker correlations with nephrin. this suggest gfb dysfunction once more as an independent, subcellular disease manifestation of hantavirus infection in addition to capillary leakage and aki. both impairment of renal function and proteinuria must probably be seen as two individual disease manifestations, the extent of each depending on the current hantavirus disease activity. there are study limitations, which need to be addressed. first, kidney biopsy material was only available for three hantavirus patients in our database who were initially suspected to have kidney diseases other than hantavirus infection. routine kidney biopsies could not be justified in terms of a risk-benefit analysis, due to the mostly reliable hantavirus diagnostics by serological tests and the self-limiting disease character with exclusively symptomatic therapy. second, the use of a creatinine-normalized description of proteinuria/albuminuria in hantavirus patients may overestimate total protein excretion relative to 24-h volume measurements because urinary creatinine excretion decreases with aki. however, we are confident that this does not affect the results of our study, as our results were extremely consistent when absolute or creatinine-normalized proteinuria parameters were used in the analyses of glomerular proteinuria. third, the reported minimum or maximum biomarker levels in our study may differ from the actual absolute minimum or maximum values that may have occurred prior to hospitalization. in summary, puuv hantavirus infection shows clinical and ultrastructural similarities to ins, but with the unique feature of an autonomous recovery. this special feature highlights the potential of further comparative studies with ins and other rna-virus induced glomerulopathies in order to improve our understanding of regenerative mechanisms in the context of gfb dysfunctions and potential future therapeutic approaches. study design and patient population. this retrospective study was conducted at the department of nephrology at heidelberg university hospital and approved by the local ethics committee of the medical faculty of heidelberg. all patients with acute hantavirus infection hospitalized in 2017 were included for further analyses. in total, 26 patients without pre-existing kidney diseases and with serologically proven, acute puuv infection were analyzed. 18 age and gender matched volunteers served as controls (table s1 ). written informed consent was obtained from all participants. all methods or experiments were performed in accordance with relevant guidelines and regulations. the overall median creatinine-normalized proteinuria (protein-to-creatinine ratio, pcr) at the time of admission was used to categorize patients in two disease severity groups (moderate pcr vs. severe pcr): (a) moderate pcr (≤ 2485 mg/g creatinine, gcr) and (b) severe pcr (> 2485 mg/gcr). baseline serum creatinine (scr) was defined as the lowest value between 6 months before or after an acute hantavirus infection. maximum pcr or scr values were defined by the highest value measured during hospitalization or recorded in medical reports immediately prior to hospitalization. data collection and laboratory methods. patient characteristics and presented laboratory parameters such as scr, urinary albumin-to-creatinine ratio (acr) and pcr were obtained from medical records. urine nephrin, igg, α1-mg and serum supar levels were measured retrospectively. nephrin and supar levels were quantified by enzyme-linked immunosorbent assay (elisa) as instructed by the manufacturer (human nphn antibody elisa kit, elabscience biotech co. ltd, wuhan, china; supar elisa kit, r&d systems, minneapolis, mn, usa). urinary igg and α1-mg concentrations were measured in the accredited central laboratory of the heidelberg university hospital. light and electron microscopy. three biopsy samples of patients with acute hantavirus infection were analyzed. biopsy samples (heidelberg biopsy register) were fixed in glutaraldehyde and embedded in epon-araldite after post-fixation with osmium tetroxide and analyzed by transmission electron microscopy (jem-1400, jeol, freising, germany). biopsy material from a time-zero biopsy of a living kidney allograft served as control. all analyses were performed at the institute of pathology (heidelberg university hospital, germany). the gbm width was evaluated using 20 representative measurements per patient. to account for the focal nature scientific reports | (2020) 10:19117 | https://doi.org/10.1038/s41598-020-76050-0 www.nature.com/scientificreports/ acute kidney injury in critically ill patients with covid-19 acute kidney injury in patients hospitalized with covid-19 multiorgan and renal tropism of sars-cov-2 renal histopathological analysis of 26 postmortem findings of patients with covid-19 in china kidney disease is associated with in-hospital death of patients with covid-19 uncovering the mysteries of hantavirus infections virus-and cell 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activator in renal disease? hantavirus infection with severe proteinuria and podocyte foot-process effacement electron microscopy of nephropathia epidemica. renal tubular basement membrane urine podocyte mrnas, proteinuria, and progression in human glomerular diseases scanning electron microscopy of the nephrotic kidney diagnostic and prognostic significance of glomerular epithelial cell vacuolization and podocyte effacement in children with minimal lesion nephrotic syndrome and focal segmental glomerulosclerosis: an ultrastructural study podocytes undergo phenotypic changes and express macrophagic-associated markers in idiopathic collapsing glomerulopathy podocyte autophagy is associated with foot process effacement and proteinuria in patients with minimal change nephrotic syndrome in nephrotic syndromes podocytes synthesize and excrete proteins into the tubular fluid: an electron and ion microscopic study elevated vegf levels in pulmonary edema fluid and pbmcs from patients with acute hantavirus pulmonary syndrome andes virus disrupts the endothelial cell barrier by induction of vascular endothelial growth factor and downregulation of ve-cadherin hantaviruses direct endothelial cell permeability by sensitizing cells to the vascular permeability factor vegf, while angiopoietin 1 and sphingosine 1-phosphate inhibit hantavirus-directed permeability motility of human renal cells is disturbed by infection with pathogenic hantaviruses pathogenic old world hantaviruses infect renal glomerular and tubular cells and induce disassembling of cell-to-cell contacts full-length soluble urokinase plasminogen activator receptor down-modulates nephrin expression in podocytes issues of acute kidney injury staging and management in sepsis and critical illness: a narrative review christian nusshag was funded by the physician scientist programme of heidelberg faculty of medicine. author contributions n.c. conceived the study design, was responsible for acquisition, analysis and interpretation of data and drafted the article. s.a. assisted with acquisition, analysis and interpretation of data and assisted in drafting the article. h.s., s.c., k.f., e.c., b.t., w.m.a. and m.c. contributed to the acquisition and interpretation of data and revised the article critically. z.m., r.j. and k.e. contributed to the conception of the study, assisted with analysis and interpretation of data and revised the article critically. all authors approved the final version of the article for publication. open access funding enabled and organized by projekt deal. reiser j. is a co-founder and shareholder of trisaq, a biotechnology company developing therapies for renal diseases. the authors declare no competing interests. supplementary information is available for this paper at https ://doi.org/10.1038/s4159 8-020-76050 -0.correspondence and requests for materials should be addressed to c.n. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. key: cord-005633-oyhpwut7 authors: oppert, michael; reinicke, albrecht; gräf, klaus-jürgen; barckow, detlef; frei, ulrich; eckardt, kai-uwe title: plasma cortisol levels before and during "low-dose" hydrocortisone therapy and their relationship to hemodynamic improvement in patients with septic shock date: 2000-11-18 journal: intensive care med doi: 10.1007/s001340000685 sha: doc_id: 5633 cord_uid: oyhpwut7 objectives: to compare cortisol levels during "low-dose" hydrocortisone therapy to basal and acth-stimulated endogenous levels and to assess whether clinical course and the need for catecholamines depend on cortisol levels and/or pretreatment adrenocortical responsiveness. design and setting: prospective observational study in a medical icu of a university hospital. patients: twenty consecutive patients with septic shock and a cardiac index of 3.5 l/min or higher, started on "low-dose" hydrocortisone therapy (100 mg bolus, 10 mg/h for 7 days and subsequent tapering) within 72 h of the onset of shock. measurements and results: basal total and free plasma cortisol levels ranged from 203 to 2169 and from 17 to 372 nmol/l. in 11 patients cortisol production was considered "inadequate" because there was neither a response to acth of at least 200 nmol/l nor a baseline level of at least 1000 nmol/l. following the initiation of hydrocortisone therapy total and free cortisol levels increased 4.2and 8.5-fold to median levels of 3587 (interquartile range 2679–5220) and 1210 (interquartile range 750–1846) nmol/l on day 1, and thereafter declined to median levels of 1310 nmol/l and 345 nmol/l on day 7. patients with "inadequate" steroid production could be weaned from vasopressor therapy significantly faster, although their plasma free cortisol concentrations during the hydrocortisone treatment period did not differ. conclusions: (a) during proposed regimens of "low-dose" hydrocortisone therapy, initially achieved plasma cortisol concentrations considerably exceed basal and acth stimulated levels. (b) cortisol concentrations decline subsequently, despite continuous application of a constant dose. (c) "inadequate" endogenous steroid production appears to sensitize patients to the hemodynamic effects of a "therapeutic rise" in plasma cortisol levels. abstract objectives: to compare cortisol levels during ªlow-doseº hydrocortisone therapy to basal and acth-stimulated endogenous levels and to assess whether clinical course and the need for catecholamines depend on cortisol levels and/or pretreatment adrenocortical responsiveness. design and setting: prospective observational study in a medical icu of a university hospital. patients: twenty consecutive patients with septic shock and a cardiac index of 3.5 l/min or higher, started on ªlow-doseº hydrocortisone therapy (100 mg bolus, 10 mg/h for 7 days and subsequent tapering) within 72 h of the onset of shock. measurements and results: basal total and free plasma cortisol levels ranged from 203 to 2169 and from 17 to 372 nmol/l. in 11 patients cortisol production was considered ªinadequateº because there was neither a response to acth of at least 200 nmol/l nor a baseline level of at least 1000 nmol/l. following the initiation of hydrocortisone therapy total and free cortisol levels in-creased 4.2-and 8.5-fold to median levels of 3587 (interquartile range 2679±5220) and 1210 (interquartile range 750±1846) nmol/l on day 1, and thereafter declined to median levels of 1310 nmol/l and 345 nmol/l on day 7. patients with ªinadequateº steroid production could be weaned from vasopressor therapy significantly faster, although their plasma free cortisol concentrations during the hydrocortisone treatment period did not differ. conclusions: (a) during proposed regimens of ªlow-doseº hydrocortisone therapy, initially achieved plasma cortisol concentrations considerably exceed basal and acth stimulated levels. (b) cortisol concentrations decline subsequently, despite continuous application of a constant dose. (c) ªinadequateº endogenous steroid production appears to sensitize patients to the hemodynamic effects of a ªtherapeutic riseº in plasma cortisol levels. adrenal insufficiency per se can lead to a high output circulatory failure that resembles septic shock [3] . in addition, animal experiments [4] and clinical observations [5, 6, 7, 8] have shown that diminished steroid levels during sepsis are associated with adverse prognosis. in experimental settings in humans the administration of hydrocortisone before or during endotoxin challenge significantly reduces the inflammatory response [9] . on the other hand, some studies have shown that mortality is higher with increased levels of cortisol in patients with sepsis and septic shock [10, 11] . a recent prospective cohort study in 189 patients with septic shock found that patients with high cortisol levels and a reduced response to adrenocorticotropic hormone (acth, corticotropin) have the worst outcome [12] . several attempts to improve the outcome of septic patients with high doses of steroids (approx. 2±8 g methylprednisolone in 24 h) have failed [13, 14, 15, 16, 17] . one meta-analysis concluded that steroids at such doses may even be harmful since they appear to increase the mortality in patients with overwhelming infection [16] . in contrast, two open studies [18, 19] and two recent randomized controlled trials [20, 21] have shown ªlowdoseº steroid therapy to have beneficial effects on hemodynamics and outcome in patients with septic shock, using no more than 300 mg hydrocortisone daily administered either as bolus injections of 100 mg three times daily or as continuous infusion. this work greatly stimulated the interest in using corticosteroids for septic patients, but little is known about the cortisol concentrations achieved by such a ªlow-doseº regimen and their relationship to endogenous basal and acth-induced cortisol levels. in fact, a number of studies have considered similar moderate amounts of hydrocortisone as a ªsupraphysiologicalº [18, 20] , a ªphysiologicalº [19] , a ªreplacementº [22] , or a ªstressº dose [21] . moreover, it remains uncertain whether the response to hydrocortisone depends on endogenous steroid secretion. while some investigators believe that hemodynamic improvement after moderate doses of hydrocortisone detects adrenal insufficiency [18] , a recent randomized trial by bollaert et al. [20] suggested that the effectiveness of hydrocortisone is unrelated to adrenocortical function. to obtain further insight into the pharmacological and pathophysiological basis of the proposed ªlowdoseº steroid treatment in septic patients we performed a prospective observational study in 20 medical intensive care patients with septic shock receiving continuous hydrocortisone therapy (10 mg/h). total and free cortisol levels were determined under basal conditions, after acth stimulation, and during the subsequent course of hydrocortisone therapy to compare the levels under therapy with the endogenous production and to assess whether clinical course and hemodynamic response dif-fer in patients with and without ªinadequateº endogenous production. adult patients with septic shock were included who were being treated in the medical intensive care unit of the virchow klinikum. demographic data, the focus of sepsis, and causative organisms are presented in table 1 . according to the american college of chest physicians/society of critical care medicine consensus committee [1] septic shock was defined as sepsis with hypotension of 90 mmhg or less or a drop of 40 mmhg or more despite adequate fluid resuscitation along with the presence of perfusion abnormalities. furthermore, patients were only included with evidence of a causative organism or focus of infection and a cardiac index 3.5 l min ±1 m ±2 or greater. patients with pancreatitis, infection with human immunodeficiency virus, and those in whom withholding maximal therapy was considered were excluded. informed consent was obtained from next of kin. all patients were monitored hemodynamically with an arterial, a central venous, and a swan-ganz pulmonary artery catheter. after inclusion in the study dopamine, when given in daily doses above 240 mg, was switched to noradrenaline and dobutamine when possible depending on cardiac output and peripheral vascular resistance in order to make the needed doses of vasopressors comparable. tapering of catecholamines and fluid expansion was guided by hemodynamic and pulmonary function to optimize tissue perfusion and gas exchange. vasopressor therapy was usually titrated to achieve a mean arterial pressure of 70 mmhg or higher and a cardiac index of 3 l min ±1 m ±2 . the pulmonary artery occlusion pressure was aimed to be between 15 and 18 mmhg. within 72 h following the onset of septic shock a ªshortº corticotropin test was performed in all patients [23, 24] . for this plasma samples were drawn before and 30 min after the administration of 0.25 mg of 1,24-corticotropin (synacthen; ciba, switzerland; corresponding to 25 iu acth) for measurement of basal and stimulated cortisol levels. cortisol production was defined as ªadequateº when the baseline level was greater than 500 nmol/l (to convert values for cortisol to mg/dl, divide by 27.5) and the increase after acth was greater than 200 nmol/l, or the baseline level was already above 1000 nmol/l (see ªdiscussionº). following the corticotropin test all patients were given a bolus injection of 100 mg hydrocortisone (pharmacia & upjohn, germany), followed by a continuous infusion. this infusion was given at a rate of 10 mg/h for 7 days, was reduced to 6 mg/h on day 8, and then reduced by 2 mg/h per day until it was discontinued on day 10. plasma samples for determination of plasma cortisol and transcortin levels were drawn daily between 6 and 8 a.m. measurements were performed en bloc, and the results were therefore not available to the physicians treating the patients. plasma cortisol was measured by solid-phase radioimmunoassay (biermann, bad nauheim, germany). plasma transcortin was de-termined using a competition radioimmunoassay (medgeniox diagnostics, fleurus, belgium). the concentration of free cortisol was calculated from total cortisol and transcortin concentrations using the following formula: free cortisol (mmol/l) = (z 2 +0.00128 c±z) ±1/2 , with z = 0.0167+0.182 (t±c) [c = total cortisol (mmol/l); t = transcortin (mmol/l)] [25] . for comparison of differences between groups the mann-whitney u test was used. fisher's exact test was used to test for dependencies between groups. a p value less than 0.05 was considered significant. all statistics were computed using spss for windows, version 7.0. unless otherwise indicated values are presented as medians with the interquartile range in parenthesis. basal and stimulated cortisol levels of the 20 patients studied are given in table 1 . both basal and stimulated cortisol levels varied considerably, ranging from 203 to 2169 and from 269 to 2253 nmol/l, respectively. cortisol production was considered ªadequateº in 9 patients because their cortisol level increased by at least 200 nmol/l following synacthen (nos. 8, 13, 14, 17, 20) , which most investigators consider as a normal response [23] , or the pre-acth concentration was already above 1000 nmol/l (nos. 12, 15, 16, 19 ; see ªdis-cussionº). in the remaining 11 patients endogenous cortisol production was defined as ªinadequateº because there was neither a response to acth of at least 200 nmol/l nor a baseline concentration of more than 1000 nmol/l. in one female patient (no. 2) cortisol levels were strikingly low. she had no evidence, however, of preexisting adrenal insufficiency, and after recovery her endogenous basal cortisol level increased to 545 nmol/l (day 14). the median basal levels of total cortisol were 780 nmol/l (601±883) in patients with ªinadequateº and 1068 nmol/ l (706±1769) in patients with ªadequateº cortisol production (p < 0.05; table 2 ). the concentrations of free cortisol, as derived from the determination of transcortin and total cortisol levels, were 263 nmol/l (154±381) and 104 (79±154) in both groups (p < 0.01) and thus amounted to about 24.6 % and 13.3 % of the total cortisol concentration. it should be noted that the formula does not include albumin, which also binds cortisol at a lower affinity. although this may lead to a slight overestimation of the fraction of free cortisol, plasma albumin levels were low (2.85 g/dl, 2.62±3.53) and did not change significantly during the course of the study. following acth administration the increment in median total cortisol level was 16.1 % and 5.1 %, respectively. patients in the two groups did not differ with respect to their demographic data, disease severity, as assessed by apache ii score levels, organ dysfunction indices, the type of causative organisms, hemodynamics, or catecholamine doses. plasma cortisol levels following hydrocortisone therapy the time course of total and free cortisol levels during the hydrocortisone treatment period in patients with and without ªadequateº endogenous cortisol production is illustrated in fig. 1 free cortisol levels that were calculated from total cortisol and transcortin concentrations rose 8.5-fold to a median of 1210 nmol/l (750±1846) following the initiation of hydrocortisone therapy and did not differ be-tween patients with and those without ªadequateº endogenous cortisol production (fig. 1, lower panel) . before the initiation of hydrocortisone therapy patients on renal replacement therapy had lower total and free cortisol levels [620 (545±803) and 95 (79±196) vs. 945 (853±1503) and 262 (129±337) nmol/l; p < 0.04). however, during hydrocortisone therapy cortisol levels did not differ between patients receiving and those not receiving hemodialysis or hemofiltration, with the exception of day 2, when free cortisol was even higher in patients on renal replacement therapy [1057 (802±1481) vs. 430 (290±924) nmol/l; p < 0.01]. thus, as expected from the molecular size of cortisol, blood purification did not seem to contribute to differences or to the reduction in cortisol levels with prolonged therapy. hemodynamics and outcome following hydrocortisone therapy in all but two patients catecholamines could be reduced during the observation period. although systolic and mean arterial blood pressure blood did not differ between patients with and without ªinadequateº cortisol production (table 3) , the rate of reduction in noradrenaline did differ. figure 2 illustrates that patients with ªinadequateº cortisol production required 15 % (5±25 %) of the initial noradrenaline dose on day 2. in contrast, in patients with ªadequateº cortisol production noradrenaline could only be reduced by 50 % (21±88 %; p < 0.01). in addition, patients with ªinadequateº cortisol production were free of vasopressor support significantly earlier than patients with ªadequateº cortisol production (table 3 ). there was, however, no difference in survival or the course of inflammatory parameters between the two groups ( table 3) . to identify patient characteristics other than endogenous cortisol production that determine hemodynamic improvement during hydrocortisone therapy patients were divided into two groups according to whether the catecholamine dose could be reduced by at least 70 % within 48 h. fourteen of the 20 patients investigated ful-filled this criterion. as shown in table 4 , the only detectable difference between patients with and without rapid weaning from catecholamines was their endogenous cortisol production; 11 of 14 patients with rapid hemodynamic improvement, but none of the 6 patients with continued high catecholamine dependence, showed inadequate endogenous cortisol production. accordingly, free and total cortisol levels before the initiation of hydrocortisone therapy in patients with early improvement were significantly lower. during the course of corticosteroid therapy, however, cortisol levels were very similar in the two groups. there was also no difference between the groups with respect to patient demographics, the disease severity score, or the course of inflammatory parameters. the pretreatment cortisol status did not differ between survivors (n = 11) and nonsurvivors (n = 9). respective levels for total cortisol were 800 (560±1662) and 883 nmol/l (700±955). free cortisol levels also did not differ between the two groups [103 (79±376) vs. 187 (115±242) nmol/l]. the endogenous cortisol production was considered ªinadequateº in 6 of 11 survivors and in 5 of 9 nonsurvivors. this study confirms that endogenous plasma cortisol concentrations are increased in patients with septic shock, but that the degree of increase is highly variable. only one of 20 patients had a post-acth cortisol plasma level less than 500 nmol/l, which supports the concept that absolute adrenocortical deficiency is rare in critically ill patients [5, 6, 7, 26, 27, 28, 29, 30, 31] . whether increases in cortisol levels are appropriate for the severity of illness or reflect functional hypoadrenalism is less clear. the rapid acth test is widely used as a simple method to identify adrenocortical hyporesponsiveness, but controversy exists as to how diagnostic criteria should be derived from the basal cortisol level, the stimulated cortisol level or their difference [24] . three [6, 7, 32] of four previous studies [6, 7, 28, 32] using a rapid acth test to investigate adrenocortical function in septic shock used an increment of at least 200±250 nmol/l to define ªadequateº responsiveness. however, in healthy controls, large unselected patient populations and also in patients with septic shock [7, 24] (table 1) cortisol increments are inversely related to basal levels. as discussed previously [24, 29, 31, 33, 34] , it appears likely that disproportionately low increments from high basal cortisol concentrations are due to maximal endogenous acth and therefore not indicative of adrenocorticoid hyporesponsiveness. we have thus arbitrarily defined endogenous cortisol production as ªadequateº in patients not only when they responded significantly to acth, but also when their baseline level was above 1000 nmol/l. using these criteria endogenous cortisol production appeared to be ªinadequateº in approximately one-half of our patients. it should be noted, however, that an elevation in plasma cortisol levels above 1000 nmol/l in septic patients may reflect not only increased production but could also indicate decreased hepatic cortisol clearance [35, 36] . following cortisol therapy with a similar dose as in previous trials (340 mg/day) [18, 19, 20, 21 ], peak concentrations of total cortisol were almost threefold higher than the post-acth level in patients considered to have an ªadequateº endogenous response. these peak concentrations corresponded to a 6.2-fold and 9.1-fold increase in free cortisol concentrations in patients with and without ªadequateº endogenous production. clearly therefore this dose must be considered as supraphysiological. interestingly, although the median total cortisol level remained above 1000 nmol/l thereafter, the levels of both total and free cortisol declined progressively during the course of therapy despite the administration of a constant dose of 10 mg/h. a similar, albeit less pronounced reduction in cortisol levels during ªlow-doseº hydrocortisone therapy has also been reported by briegel et al. [22] . however, in this study the dose was tapered after only 1±5 days. therefore, in contrast to our observations, the decline could be attributed to the reduction in steroid dose. even if one assumes that in the present investigation the administration of acth and the initial bolus injection of 100 mg contributed to the peak concentration on day 1, and that endogenous cortisol production was subsequently suppressed, these two effects cannot entirely explain the reduction in median level from 3587 to 1310 nmol/l between days 1 and 7. moreover, it has been shown that cortisol production in septic shock is in fact not suppressible by steroids [34] . it is possible therefore that the metabolism of hydrocortisone changes during the treatment period. earlier studies have shown that cortisol extraction from blood is decreased, and that its half-life is prolonged during septic shock [35, 36] . the observed decline in cortisol levels during the treatment period could reflect a reversal of these alterations. our intention in this study was not to compare the efficacy of cortisol therapy to untreated controls. it is nevertheless noteworthy that inotropes could be reduced rather quickly after initiation of the hydrocortisone infusion. the reduction in catecholamine dose was comparable [21] or even faster than in previous studies using similar doses of cortisol [18, 20] , which may be related to the fact that we started therapy earlier during the septic course. in contrast to previous reports [6, 7, 28], mortality in patients with ªinadequateº endogenous cortisol was not higher (table 2) , and survivors and nonsurvivors did not have significantly different steroid levels fig. 2 time course of noradrenaline needs in patients with and without adequate endogenous cortisol production (cp). two days after the beginning of ªlow-doseº hydrocortisone therapy patients with inadequate endogenous production needed significantly less vasopressor support before hydrocortisone therapy. this observation is in accordance with the results of a recent randomized controlled trial by bollaert et al. [20] and is compatible with the growing evidence that ªlow-doseº therapy reverses the putative adverse risk factor of inappropriate cortisol production [18, 19, 20, 21, 22] . although patients with ªadequateº and ªinadequateº endogenous production were clinically indistinguishable at the onset of therapy (table 2) , and did not have significantly different levels of free cortisol during hydrocortisone therapy (fig. 1) , those with ªinadequateº endogenous response showed a significantly faster reduction in their vasopressor support (fig. 2) . in addition, the only detectable difference between patients with and without rapid hemodynamic improvement was their basal cortisol status (table 4) . similarly brie-gel et al. [18] found that 6 of 14 patients weaned from catecholamines within 48 h of hydrocortisone therapy had lower cortisol levels before steroid treatment. in addition, annane et al. [32] reported that patients in septic shock with inadequate as compared to adequate cortisol production had lower baseline responsiveness to noradrenaline but showed a more marked improvement in vasopressor sensitivity in response to hydrocortisone bolus. it appears therefore that the greatest benefit of hydrocortisone therapy is achieved in patients with blunted endogenous production, although the exact mechanisms of this sensitization remain to be elucidated. in contrast to the hemodynamic changes the febrile response and the course of c-reactive protein and procalcitonin levels or white blood cell counts did not depend on the pretreatment cortisol production (table 3) . thus, although it has been suggested that hydrocortisone treatment reduces indicators of the acute-phase response [22] , these effects appear not to be directly related to the hemodynamic effects. we also found no association between the hemodynamic response and survival rates (table 4 ), but larger controlled trials are necessary to exclude or verify effects on such outcome parameters. in conclusion, there are three main findings of this study. firstly, during proposed regimes of ªlow-doseº hydrocortisone therapy plasma cortisol concentrations are achieved initially which considerably exceed basal and acth stimulated levels. thus in order to achieve a substitution of deficient endogenous production, doses even lower than those used so far may be sufficient. secondly, during continuous administration of hydrocortisone, cortisol levels decline, suggesting that changes in cortisol metabolism have a significant impact on its plasma concentration. thirdly, ªinadequateº endogenous steroid production appears to sensitize patients to the hemodynamic effects of a rise in plasma cortisol levels. a 3-level prognostic classification in septic shock based on cortisol levels and cortisol response to corticotropin the effects of high-dose corticosteroids in patients with septic shock a controlled clinical trial of high-dose methylprednisolone in the treatment of severe sepsis and septic shock effect of high-dose glucocorticoid therapy on mortality in patients with clinical signs of systemic sepsis corticosteroid treatment for sepsis: a critical appraisal and metaanalysis of the literature steroid controversy in sepsis and septic shock: a metaanalysis contribution of cortisol deficiency to septic shock abrupt hemodynamic improvement in late septic shock with physiological doses of glucocorticoids stress doses of hydrocortisone reverse hyperdynamic septic shock: a prospective, randomized, double blind low-dose hydrocortisone infusion attenuates the systemic inflammatory response syndrome screening for adrenocortical insufficiency with cosyntropin rapid adrenocorticotropic hormone test in practice clinical use of unbound plasma cortisol as calculated from total cortisol and corticosteroid binding globulin adrenocortical function: an indicator of severity of disease and survival in chronic critically ill patients spectrum of serum cortisol response to acth in icu patients adrenal insufficiency occurring during septic shock: incidence, outcome, and relationship to peripheral cytokine levels plasma cortisol levels in patients with septic shock a comparison of the adrenocortical response during septic shock and after complete recovery adrenocortical function during septic shock gajdos p (1998) impaired pressor sensitivity to noradrenaline in septic shock patients with and without impaired adrenal function reserve adrenal insufficiency hypercortisolism in septic shock is not suppressible by dexamethasone infusion comparative studies on adrenal cortical function and cortisol metabolism in healthy adults and in patients with shock due to infection secretion and metabolism of cortisol after injection of endotoxin key: cord-264427-frrq4h39 authors: huang, ling; liu, ziyi; li, hongli; wang, yangjun; li, yumin; zhu, yonghui; ooi, maggie chel gee; an, jing; shang, yu; zhang, dongping; chan, andy; li, li title: the silver lining of covid‐19: estimation of short‐term health impacts due to lockdown in the yangtze river delta region, china date: 2020-07-07 journal: geohealth doi: 10.1029/2020gh000272 sha: doc_id: 264427 cord_uid: frrq4h39 the outbreak of covid‐19 in china has led to massive lockdowns in order to reduce the spread of the epidemic and control human‐to‐human transmission. subsequent reductions in various anthropogenic activities have led to improved air quality during the lockdown. in this study, we apply a widely used exposure‐response function to estimate the short‐term health impacts associated with pm(2.5) changes over the yangtze river delta (yrd) region due to covid‐19 lockdown. concentrations of pm(2.5) during lockdown period reduced by 22.9% to 54.0% compared to pre‐lockdown level. estimated pm(2.5)‐related daily premature mortality during lockdown period is 895 (95% confidential interval: 637‐1081), which is 43.3% lower than pre‐lockdown period and 46.5% lower compared with averages of 2017‐2019. according to our calculation, total number of avoided premature death associated pm(2.5) reduction during the lockdown is estimated to be 42.4 thousand over the yrd region, with shanghai, wenzhou, suzhou (jiangsu province), nanjing, and nantong being the top five cities with largest health benefits. avoided premature mortality is mostly contributed by reduced death associated with stroke (16.9 thousand, accounting for 40.0%), ischemic heart disease (14.0 thousand, 33.2%) and chronic obstructive pulmonary disease (7.6 thousand, 18.0%). our calculations do not support or advocate any idea that pandemics produce a positive note to community health. we simply present health benefits from air pollution improvement due to large emission reductions from lowered human and industrial activities. our results show that continuous efforts to improve air quality are essential to protect public health, especially over city‐clusters with dense population. the outbreak of the tragic coronavirus disease 2019 by the end of 2019 has caused tremendous impacts on people's life around the world. at the time of this writing (may 6 th , 2020), covid-19 has made more than 3.6 million people sick and led to more than 257,301 deaths worldwide (https://www.statista.com/statistics/, last access on may 6 th , 2020). during its peak, the pandemic at one point caused over 15,000 new confirmed cases in china in just one single day back in february, and presently in may very few new local infections are reported in china (http://www.nhc.gov.cn/, last access on may 6 th , 2020). the effective containment of covid-19 within china is mostly attributed to a series of prevention and control measures implemented rapidly by the chinese government. starting from late january 2020, national emergency response policies were launched in china in order to reduce the intensity of the spread of the epidemic to slow down the increase of number of new cases, including but not limited to: schools shut down, traffic strictly restricted, industries and construction activities suspended, mass gatherings and events cancelled or suspended, and social distancing become the new norm. as a result of the massive lockdown, emissions of primary air pollutants from various human and industrial activities decreased substantially and pm2.5 concentrations during covid-19 lockdown in china has been shown to be much better than previous years during the same period (nasa, 2020; wang et al., 2020a) . it is well known that poor air quality, with pm2.5 (particulate matters with aerodynamic diameters less than 2.5 μm) being a key criteria pollutant, could have adverse health impacts (boldo et al., 2011; cao et al., 2012; song et al., 2017) and lead to premature mortality (fang et al., 2016; liu et al., 2016; lu et al., 2015b ). an integrated exposure risk (ier) model for pm2.5 exposure-response function is widely used to estimate the premature mortality attributed to pm2.5 exposure. for example, maji et al. (2018) estimates pm2.5-related long-term premature mortality for 161 cities in china for year 2015 as well as the potential health benefits of air pollution control policies for year 2020. wang et al. (2020b) calculates the number of premature death due to acute and chronic exposure of ambient pm2.5 in china during 2013-2017. with substantial reductions in pm2.5 concentrations due to covid-19 lockdown, a follow-up question is what are the health impacts of the short-term changes in air quality. the yangtze river delta (yrd) region is one of the most economic developed and populated regions in china. in the past, the yrd region has frequently experienced heavy haze pollution (cheng et al., 2013; wang et al., 2015) . with various control strategies continuously being carried out, the overall air quality over the yrd region has greatly improved for the past few years (ministry of ecology and environment of china, 2019). according to the latest report released by the ministry of ecology and environment of china (ministry of ecology and environment of china, 2020, 40 out of 41 cities in the yrd region has successfully met the goals of reducing pm2.5 concentrations during the 2019-2020 fall and winter season. in our most recent study , we investigate the air quality changes over the yrd region due to lowered human activities during covid-19 lockdown using multipollutant observations and photochemical model simulations. in this follow-up study, we attempt to quantify the short-term health impacts associated with pm2.5 changes over the yrd region due to covid-19 lockdown. we estimate the premature mortality associated with pm2.5 exposure before lockdown and during lockdown periods. utilizing simulated results based on an integrated meteorology and air quality modeling system, we estimate the number of avoided premature death due to lowered pm2.5 concentrations during covid-19 lockdown over the yrd region. methods and results from our previous study are partially adopted in this study to support health related estimation. 2.1 quantitative analysis pm2.5 changes due to covid-19 lockdown the yangtze river delta (yrd) region, consisting of shanghai, jiangsu, zhejiang, and anhui province (fig. 1) , is one of the most economic developed and populated regions in china. on january 23 th 2020, being one of three earliest provinces (the other two being hunan and guangdong provinces), zhejiang province (located in south of the yrd region) announced provincial lockdown as "level i" (particularly serious) response, followed by shanghai and anhui province on the next day and jiangsu province two days later. coincided with the chinese spring festival (january 24 th -february 1 st , 2020), all kinds of human activities were greatly reduced during level i response period. with the epidemic gradually controlled, emergency response in anhui and jiangsu province was downgraded to level ii (serious) on february 25 th , followed by zhejiang province on march 2 nd . shanghai announced level ii response on march 24 th due to high numbers of imported infectious cases. same as previous study, we define pre-lockdown period as january 1 st -january 23 rd , level i response period as january 24 th -february 25 th , and level ii response period as february 26 th -march 31 st . fig. 1 location of the yangtze river delta (yrd) region with city-level population to quantify the changes of air quality caused by reduced human activities during covid-19 lockdown, the integrated weather research forecasting model (wrf) -comprehensive air quality model with extensions (camx) modeling system is used ). details of model configurations and input data can be found in and are briefly summarized here. the integrated wrf/camx model is applied to simulate air quality over the yrd region ( fig. 1 ) during pre-lockdown, level i response, and level ii response periods. two parallel simulations are conducted with two sets of anthropogenic emissions while keeping all other inputs and model configurations identical. for the base case simulation, the baseline emissions (i.e. emissions from normal activities assuming no lockdown) are used. for the covid-19 scenario, emissions estimated based on reduced human activities due to lockdown are applied. for emission reductions outside the yrd region during lockdown, we applied the reduction ratio used by wang et al. (2020a) . the relative improvement factor (rf) is defined as the ratio of simulated concentrations between the two scenarios and is applied to the observed concentrations to obtain the theoretical concentrations of air pollutants that would be if there is no lockdown. results of model performance evaluation of the covid-19 scenario show acceptable agreement between simulated and observed results ). 2.2 premature mortality due to short-term pm2.5 exposure we estimate the premature mortality due to ambient pm2.5 exposure based on a widelyused log-linear exposure-response function below (fang et al., 2016; gao et al., 2016) : where y is the number of premature deaths caused by ambient pm2.5 exposure due to five leading causes (k=5): cerebrovascular disease (stroke), ischemic heart disease (ihd), chronic obstructive pulmonary disease (copd), lung cancer (lc) for adults (≥ 25 years), and acute lower respiratory infection (alri) for infants (< 5 years). β is the cause-specific exposureresponse coefficients and values reported from a meta-analysis study (lu et al., 2015a) are utilized in this study. for an increase of 10 μg/m 3 pm2.5, β is 0.63% [95% confidential interval (ci): 0.35% -0.9%] for cardiovascular disease (i.e. stoke、ihd) and 0.75% (95% ci: 0.39% -1.11%) for respiratory disease (i.e. copd, alri, lc). the baseline incidence rate (r) at provincial level is obtained from the sixth national population census (http://www.stats.gov.cn/tjsj/pcsj/rkpc/6rp/indexch.htm, last access on 20 th april, 2020) and the contribution of individual disease to total mortality are based on the national estimates from the global burden of diseases (gbd) project of institute for health metrics and evaluation (ihme) and health effects institute (hei) for year 2017 (https://vizhub.healthdata.org/gbd-compare/, last access on 23 rd april, 2020). according to gbd study, stroke, ihd, copd, lc, and alri contribute 20.2%, 16.7%, 9.2%, 6.4% and 1.7% of total deaths in china for year 2017. p is the exposed population for each city in the yrd region and is obtained from statistical yearbooks for year 2018. the exposed pm2.5 concentration in eq. (4) 3.2 premature mortality attributable to short-term pm2.5 exposure ambient pm2.5 exposure leads to higher mortality in infants (<5 years) from alri and in adults (≥25 years) due to stroke, ihd, copd and lc. we calculate the premature mortality due to above-mentioned causes based on the health impact function (eq. 1) over the yrd region during pre-lockdown, level i, and level ii period of 2017-2020 (fig. 4) . during the pre-lockdown period, the total premature mortality attributed to pm2.5 exposure are relatively consistent during 2017-2020 and the number in 2020 is 36.4 thousand (95% ci: 30.4-38.8 thousand) for the whole yrd region. stroke and ihd contribute to 14.4 thousand (95% ci: 12.0-15.4 thousand) and 11.9 thousand (95% ci: 10.0-12.7 thousand) premature death, together accounting for 72.2% of total pm2.5-related premature death. copd, lc, and alri contribute the rest 27.8% of pm2.5-related death, each causing 6.8 thousand (95% ci: 5.7-7.2 thousand), 3.2 thousand (95% ci: 2.7-3.4 thousand), and 0.05 thousand (95% ci: 0.04-0.06 thousand) premature death during the pre-lockdown period. during level i and level ii response periods, while the premature morality due to pm2.5 exposure during 2017-2019 fluctuated a little bit, a sharp decrease is observed for year 2020. the total pm2.5-related premature mortality during 2020 level i and level ii period is 33.2 thousand (95% ci: 23.9-38.5 thousand) and 27.7 thousand (95% ci: 18.6-33.8 thousand), which dropped by 32.3% and 47.7% compared to the 2017-2019 average values. the relative contributions from different diseases remain unchanged since the only changing variable here is the pm2.5 concentration. in order to directly compare the premature mortality before and during lockdown (level i plus level ii period), we present the daily premature mortality in figure s1 . during 2017-2019, the daily premature morality dropped by 3.2% to 12.7% before and during lockdown. for 2020, the daily premature mortality across the yrd region during pre-lockdown is 1.6 thousand (95% ci: 1.3-1.6 thousand) and 0.9 thousand (95% ci: 0.6-1.1 thousand) during lockdown (level i + level ii), representing a sharp decrease by 43.3%. the significant reduction in premature mortality during lockdown periods, whether it is compared to the prelockdown periods of the same year, or the same periods of historical years, indicate substantial health benefits associated with lowered pm2.5 concentrations due to covid-19 lockdown. fig. 4 premature mortality due to lc, stroke, ihd, copd during pre-lockdown, level i and level ii periods of 2017-2020 (data for alri is not shown due to low numbers). estimated premature mortality with the assumption of no-lockdown is also shown for level i and level ii. table s1-s4 shows the city-level premature mortality by period and year. with the same base incidence rate (r) being applied for all cities, city-level premature mortality depends on the exposed population and the pm2.5 concentration for that city. with the largest population (2418.3 thousand in 2018) of all cities in the yrd region (fig. 5) , shanghai has the highest pm2.5-related premature mortality during 2017-2019, even though its averaged pm2.5 concentration is ranked the 6 th , 9 th , and 9 th from the bottom during january to march of 2017, 2018 and 2019 (fig. 5) . on the other hand, high premature mortality for cities like bozhou and suqian in anhui province (for example, each is ranked 3 rd and 7 th during level ⅱ period in 2020 in terms of premature mortality) is more associated with the high pm2.5 concentrations, which is ranked 3 rd and 7 th in terms of average pm2.5 concentration and only 13 rd and 19 th in terms of population. cities with small population and low pm2.5 concentrations, for example, huangshan (in anhui province), has the lowest premature mortality. during 2020 pre-lockdown, daily premature mortality due to pm2. an integrated wrf/camx model is used to estimate pm2.5 concentrations that would be during level i and level ii period if no lockdown occurred. the potential health benefits due to lockdown are estimated as the differences of premature mortality calculated based on the observed pm2.5 concentrations and the simulated 'no lockdown' concentrations during the same periods. if no lockdown occurred, the total premature death due to pm2.5 exposure over the yrd region would be 50.2 thousand (95% ci: 42.0-53.3 thousand) during level i period and 52.9 thousand (95% ci: 40.6-58.5 thousand) during level ii period. these two numbers are lower than the corresponding values of 2017-2019 (except for level i in 2017 and 2019). the total avoided premature mortality over the yrd region is 17.1 thousand during level i and 25.1 thousand during level ii (table s5) , each representing 51.5% and 90.6% of current premature mortality rate due to pm2.5 exposure. in terms of diseases, avoided premature morality due to ihd and stroke are 14.0 thousand and 16.9 thousand, each accounting for 33.2% and 40.1% of current base incident mortality rate due to pm2.5. avoided premature mortality due to copd, lc, and alri are 7.6 thousand, 3.6 thousand, and 0.06 thousand. figure 6 shows the city-level total avoided premature mortality during level i and level ii (avoided premature mortality associated with different diseases are shown in fig. s2 ). the number of avoided premature death for different cities depends on the base population and the changes in pm2.5 concentrations due to lockdown ( , and suzhou (anhui province, 1449, 95% ci: 1027-1672). a few of uncertainties exist with our estimation of health impacts, which are also recognized in a couple of similar studies maji et al., 2018; wang et al., 2020) . first and foremost, there exists uncertainties with the parameters (e.g. concentrationresponse coefficient, threshold concentration) used in the integrated exposure-response function. although limitation exists, we use values reported by lu et al.(2015a) , which is developed based on meta-analysis of 59 studies covering 22 cities in mainland china (3 cities in the yrd region), hong kong and taiwan to reduce uncertainties with the concentrationresponse coefficient. other simple assumptions when using the exposure-response function include that the exposure-response coefficient does not vary by age and a provincial-level base incidence rate is used for all cities within the province. secondly, we only calculate the premature mortality associated with pm2.5 exposure while realizing that synergistic effects exist when exposed to other or multiple pollutants (apte et al., 2015; billionnet et al., 2012) and our estimation of premature mortality perhaps represents an underestimation. on top of that, the influences of pm2.5 chemical composition, size distribution and sources on health impacts (ostro et al., 2015) are ignored in this study. when we calculate city-level population exposure, we ignore the heterogeneities of ambient pm2.5 concentrations and population within the city. we simply use the observed pm2.5 concentrations averaged over all monitoring sites within the city as the exposed concentrations. this introduces underestimation when more monitoring sites are located in the rural and less populated areas and overestimation when monitoring sites are more likely to be located in urban and populous areas for a city. ways to improve the spatial accuracy of health impact estimation include utilizing population distribution with high spatial resolution and interpolated pm2.5 concentrations based on networks of low-cost sensors (cavaliere et al., 2018; holstius et al., 2014) or satellite based data (e.g. the aerosol optical depth, chen et al., 2019; . finally, estimations of avoided premature death assuming no-lockdown are associated with uncertainties with the meteorology and air quality model. this may all be explored in the near-future when more data are available. in this study, we attempt to quantify the health impacts associated pm2.5 improvement due to reduced human and industrial activities during covid-19 lockdown in the yangtze river delta region, a region with heavy air pollution during the past years. as a result of reduced human activities, concentrations of pm2.5 during lockdown periods reduced by 22.9% to 54.0% over the yrd region compared to pre-lockdown level. avoided premature death due to lowered pm2.5 concentrations are estimated to be 42.2 thousand over the yrd region, representing 69.3% of the total present premature mortality due to pm2.5 exposure. the avoided premature mortality is mostly contributed by reduced death associated with stroke (16.9 thousand, accounting for 40.0%), ischemic heart disease (14.0 thousand, 33.2%) and chronic obstructive pulmonary disease (7.6 thousand, 18.0%). top five cities with highest avoided premature mortality are shanghai (5433 persons), wenzhou (2751), suzhou (in jiangsu province, 2660), nanjing (2000) and nantong (1969) . the outbreak of covid-19 is disastrous. this study is by no means to suggest that pandemics are bringing a positive effect for health. the passive emission reductions during covid-19 lockdown provide a good opportunity to show the health impacts related to reduction in pm2.5. it merely reinforces our knowledge that pm2.5 has detrimental health effects. results from our study suggested that substantial health benefits could be achieved with reduced pm2.5 concentrations by emission reductions, which confirms the importance of recent efforts to mitigate the haze pollution nationwide. however, although pm2.5 concentration decreased substantially during lockdown period, the residual pm2.5 concentrations are still much higher than the recommended 24-hr standard by who. continuous efforts are needed to reduce emissions in the long term and through the most cost-effective ways in order to protect public health. addressing global mortality from ambient estimating the health effects of exposure to multi-pollutant 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analysis of a severe prolonged regional haze episode in the yangtze river delta severe air pollution events not avoided by reduced anthropogenic activities during covid-19 outbreak. resources, conservation and recycling acute and chronic health impacts of pm2.5 in china and the influence of interannual meteorological variability this study is financially sponsored by the shanghai science and technology innovation plan monitoring service). the baseline incidence rate (r) at provincial level is obtained from ©2020 american geophysical union. all rights reserved.http://www.stats.gov.cn/tjsj/pcsj/rkpc/6rp/indexch.htm (the sixth national population census). the contribution of individual disease to total mortality are based on the national estimates from https://vizhub.healthdata.org/gbd-compare/ (the global burden of diseases (gbd) project of institute for health metrics and evaluation (ihme) and health effects institute (hei) for year 2017). key: cord-003520-f3jz59pt authors: arabi, yaseen m.; tamimi, waleed; jones, gwynne; jawdat, dunia; tamim, hani; al-dorzi, hasan m.; sadat, musharaf; afesh, lara; sakhija, maram; al-dawood, abdulaziz title: free fatty acids’ level and nutrition in critically ill patients and association with outcomes: a prospective sub-study of permit trial date: 2019-02-13 journal: nutrients doi: 10.3390/nu11020384 sha: doc_id: 3520 cord_uid: f3jz59pt objectives: the objectives of this study were to evaluate the clinical and nutritional correlates of high free fatty acids (ffas) level in critically ill patients and the association with outcomes, and to study the effect of short-term caloric restriction (permissive underfeeding) on ffas level during critical illness. patients/method: in this pre-planned sub-study of the permit (permissive underfeeding vs. target enteral feeding in adult critically ill patients) trial, we included critically ill patients who were expected to stay for ≥14 days in the intensive care unit. we measured ffas level on day 1, 3, 5, 7, and 14 of enrollment. of 70 enrolled patients, 23 (32.8%) patients had high ffas level (baseline ffas level >0.45 mmol/l in females and >0.6 mmol/l in males). results: patients with high ffas level were significantly older and more likely to be females and diabetics and they had lower ratio of partial pressure of oxygen to the fraction of inspired oxygen, higher creatinine, and higher total cholesterol levels than those with normal ffas level. during the study period, patients with high ffas level had higher blood glucose and required more insulin. on multivariable logistic regression analysis, the predictors of high baseline ffas level were diabetes (adjusted odds ratio (aor): 5.36; 95% confidence interval (ci): 1.56, 18.43, p = 0.008) and baseline cholesterol level (aor, 4.29; 95% ci: 11.64, 11.19, p = 0.003). serial levels of ffas did not differ with time between permissive underfeeding and standard feeding groups. ffas level was not associated with 90-day mortality (aor: 0.49; 95% ci: 0.09, 2.60, p = 0.40). conclusion: we conclude that high ffas level in critically ill patients is associated with features of metabolic syndrome and is not affected by short-term permissive underfeeding. fatty acids are a major source of fuel in the body and play an important role in cell signaling [1, 2] . free fatty acids (ffas) are nonesterified fatty acids that are released by the hydrolysis of triglycerides (triglyceride molecule is composed of three fatty acid molecules bound to glycerol) within the adipose tissue by lipoprotein lipase. they circulate in the blood protein-bound, serving as an energy source for tissues [1, 3] . chronically elevated ffas level has been observed in obese people and in diabetic patients and is associated with insulin resistance and with sudden death in middle-aged men without known ischemic heart disease [1, 3, 4] . acutely, ffas level is often increased during critical illness and may contribute to organ dysfunction. critical illness is characterized by hypercatabolic state and by a change in the contribution of the endogenous protein, fat, and carbohydrate sources to oxidative fuel [5] . lipolysis is accelerated by the high catecholamine and other stress hormone milieu leading to increased release of ffas from adipocytes, thus, increasing ffas level [6] . insulin resistance during critical illness impairs the use of ffas for energy, and, thus, contributes to increased ffas level [7] . heparin given during critical illness may also increase ffas level by activating lipoprotein lipase [8] . ffas may have toxic effects by increasing reactive oxygen species leading to cell death and necrosis [9] and by depressing the immune cell function [10] . in addition, ffas potentiate insulin resistance and impair glucose metabolism by inhibiting glucose oxidation and by stimulating protein kinase c [1, 10] . in an acute setting, elevated ffas level has been associated with the development of acute lung injury in at-risk patients with sepsis, trauma, and pancreatitis and after on-pump coronary artery bypass grafting [8, 11, 12] . can ffas level in critically ill patients be modulated by short-term caloric restriction (permissive underfeeding)? normally, serum ffas level increases during fasting and exercise and after a fatty meal. ffas level goes down postprandially due to the anti-lipolytic effect of insulin that is released after carbohydrate intake [13] . on the other hand, caloric restriction and weight loss lead to a lowering of ffas level and can attenuate ffas-induced hepatic insulin resistance in obese healthy patients [14] [15] [16] . however, the effect of caloric restriction on serum ffas level has not been investigated in critically ill patients. the aims of this study were (1) to evaluate the clinical and nutritional correlates of high ffas level in critically ill patients and the association with outcomes, and (2) study the effect of short-term caloric restriction (permissive underfeeding) on ffas level during critical illness. this is a pre-planned sub-study of the permit [17] (permissive underfeeding vs. target enteral feeding in adult critically ill patients-isrctn68144998) trial, in which critically ill patients were randomized to permissive underfeeding (40-60% of calculated caloric requirements) or standard feeding (70-100%) for up to 14 days while maintaining similar protein intake in both groups. the trial found no difference in the primary endpoint of 90-day mortality. in this sub-study which was separately funded by king abdulaziz city for science and technology (kacst), riyadh, saudi arabia (grant number-at 32-25 kacst), we enrolled consecutive patients from the permit trial at king abdulaziz medical city, riyadh, saudi arabia between september 2012 and september 2014 who were expected to stay ≥14 days in the intensive care unit as judged by their primary team. a separate informed consent was obtained for participation in this sub-study. the study was approved by the institutional board review of the ministry of the national guard health affairs, riyadh, saudi arabia. blood was collected at the time of enrollment (baseline or study day 1) within 48 h of icu admission and on days 3, 5, 7, and 14. serum was prepared from the blood samples by centrifugation at 4 • c at 1600 g for 20 min and divided into aliquots. these aliquots were stored immediately in a designated freezing area at −70 • c to be analyzed once the sample size was completed. the samples were analyzed blindly, and then the sample codes were broken. the measurement of ffas was performed in bioscientia reference laboratory in germany using an in-vitro enzymatic calorimetric assay with wako-nefa-hr (2) reagent (wako-chemicals, neuss, germany) [18] . in this method, ffas with the coexistence coenzyme a (coa) and adenosine-5'-triphosphate (atp) disodium salt were converted to acyl-coa, adenosine monophosphate (amp) and pyrophosphoric acid by the action of acyl-coa synthetase (acs). the acyl-coa was oxidized yielding 2,3-trans-enoyl-coa and hydrogen peroxide (h 2 o 2 ) by the action of acyl-coa oxidase. in the presence of peroxidase, h 2 o 2 yielded a blue-purple pigment by quantitative oxidation condensation with 3-methyl-n-ethyl-n-(β-hydroxyethyl)-aniline (meha) and 4-aminoantipyrine (4aa). ffas level was obtained by measuring absorbance at the blue and purple color at wavelengths of 546 nm and 660 nm. the normal fasting serum ffas level is 0.1 to 0.45 mmol/l for females and 0.1 to 0.6 mmol/l for males. however, ffas level in the critically ill is poorly studied. in the current study, patients with ffas more than 0.45 mmol/l in females and 0.6 mmol/l in males were considered to have high ffas level; otherwise ffas level was considered normal. baseline data included demographics, acute physiology and chronic health evaluation scores (apache) ii [19] , presence of sepsis upon admission, sequential organ failure assessment (sofa) score [20] , the ratio of partial pressure of arterial oxygen to the fraction of inspired oxygen (pao 2 :fio 2 ), glasgow coma scale and various laboratory results (baseline blood glucose, hemoglobin, international normalized ratio(inr), platelets, bilirubin, creatinine, c-reactive protein, albumin, pre-albumin, transferrin, 24-h urinary urea nitrogen excretion, and nitrogen balance). for the intervention period, which lasted for up to 14 days, we collected daily nutritional data (feeding formula and calories from enteral feeds, propofol, intravenous dextrose, and parenteral nutrition), insulin dose for hyperglycemia management, daily blood glucose, and use of certain medications, such as aspirin, beta-blockers and statins. we noted the daily carbohydrate, fat, and protein calories from enteral and parenteral sources and then calculated the total fat-to-carbohydrate ratio by dividing fat calories by carbohydrate calories. the outcomes evaluated in this study were 28-, 90-, and 180-day all-cause mortality. other outcomes included hospital and icu mortality, incident renal replacement therapy, icu-associated infections [21] , icu and hospital length of stay (los), and mechanical ventilation duration. in addition, icu-free days, renal replacement therapy-free days, and ventilator-free days were also calculated. we reported categorical variables as frequencies with percentages and continuous variables as medians with quartile 1 and 3 (q1, q3). we compared categorical variables using chi-square or fisher's exact test and continuous variables using mann-whitney u test. we examined pearson correlation among the following baseline variables ffas level, age, body mass index, total cholesterol, high-density lipoprotein (hdl) cholesterol, low-density lipoprotein (ldl) cholesterol, non-hdl cholesterol, triglycerides, glucose, and hemoglobin a1c. in addition, multivariable logistic regression analysis was performed to assess the predictors of high ffas level. we entered in the model a priori decided baseline variables that were of clinical interest and/or had significant association with high ffas level by univariable analysis (p ≤ 0.05) which included age, gender, body mass index (bmi), apache ii, diabetes, triglycerides, ldl cholesterol, hdl cholesterol, medical admissions (vs. non-medical admissions), and randomization (permissive vs. standard feeding). we also carried out a linear mixed model to test whether ffas level is affected over time with permissive underfeeding compared to standard feeding. we carried out logistic and linear regression models to examine the association between ffas level and outcomes adjusting for age, gender, bmi, apache ii, diabetes, triglycerides, ldl cholesterol, hdl cholesterol, non-hdl cholesterol, and medical admissions (vs. non-medical admissions). a two-tailed p value < 0.05 was considered statistically significant. the results were expressed as adjusted odds ratio (aor) or parameter estimate with 95% confidence intervals (95%ci). all statistical analyses were performed using sas version 9.2 (sas institute, cary, nc, usa). of the 70 patients included in the study ( figure s1 ), 23 (32.8%) had high ffas level (median 0.74 mmol/l (q1, q3: 0.63, 1.06) and 47 (67.1%) had normal ffas level (0.34 mmol/l (0.22, 0.45))). patients with high ffas level were significantly older, more likely to be females and diabetic, had higher hgba1c, creatinine and non-hdl and ldl cholesterol levels, and had lower pao 2 :fio 2 ratio compared with patients with normal ffas (table 1 ). there were significant correlations between ffas level and total cholesterol (r = 0.45, p = 0.0001), non-hdl cholesterol (r = 0.38, p = 0.002), hdl cholesterol (r = 0.30, p = 0.01) and ldl cholesterol (r = 0.43, p = 0.0003), and age (r = 0.40, p = 0.0006) ( table s1 ). table 2 shows the nutritional data during the study period and the trial co-interventions. the total daily caloric intake was 1065. ffas level (p = 0.37). the baseline blood glucose was similar in the two groups; however, during the study period, the glucose level was significantly higher in patients with high ffas level compared to patients with normal ffas level (9.5 mmol/l (q1, q3: 7.6, 11.9) compared to 7.7 mmol/l (q1, q3: 6.5, 10.3) p= 0.05) with higher use of insulin (18.9 units per day (q1, q3: 3.8, 37.5) compared to 0.0 units per day (q1, q3: 0.0, 14.9) p = 0.003). additionally, more patients in the high ffas level received disease-specific formulae, renal replacement therapy, aspirin and statins during icu stay. on multivariable logistic regression analysis, the independent predictors of high ffas level were diabetes (aor, 5.36; 95% ci, 1.56, 18.43; p = 0.008), and baseline cholesterol (aor, 4.29; 95% ci, 1.64, 11.19; p = 0.003). figure 1 , panel a shows the serial levels of ffas for patients with high ffas and normal ffas. figure 1, panel b shows ffas level in patients who received permissive underfeeding and standard feeding. the ffas level was not different between the two feeding strategies. on multivariable logistic regression analysis, the independent predictors of high ffas level were diabetes (aor, 5.36; 95% ci, 1.56, 18.43; p = 0.008), and baseline cholesterol (aor, 4.29; 95% ci, 1.64, 11.19; p = 0.003). figure 1 , panel a shows the serial levels of ffas for patients with high ffas and normal ffas. figure 1, panel b shows ffas level in patients who received permissive underfeeding and standard feeding. the ffas level was not different between the two feeding strategies. a) and in patients who received permissive underfeeding and standard feeding (panel b). p values for between-group differences and between-group differences over time are provided using mixed linear model. there was no significant difference in crude mortality between patients with high and normal ffas level ( table 3) . incident of renal replacement therapy was more frequent in patients with high ffas level (6/23 (27.3%) compared to 2/47 (4.7%), p = 0.009). multiple variable analyses adjusting for age, gender, bmi, apache ii, diabetes, triglycerides, ldl cholesterol, hdl cholesterol, non-hdl cholesterol, and medical admissions (vs. non-medical admissions) showed no significant association between high ffas level and 90-day mortality (aor 0.49, 95% ci 0.09, 2.60, p = 0.40) or any other study outcomes (table 3) . . p values for between-group differences and between-group differences over time are provided using mixed linear model. there was no significant difference in crude mortality between patients with high and normal ffas level ( table 3) . incident of renal replacement therapy was more frequent in patients with high ffas level (6/23 (27.3%) compared to 2/47 (4.7%), p = 0.009). multiple variable analyses adjusting for age, gender, bmi, apache ii, diabetes, triglycerides, ldl cholesterol, hdl cholesterol, non-hdl cholesterol, and medical admissions (vs. non-medical admissions) showed no significant association between high ffas level and 90-day mortality (aor 0.49, 95% ci 0.09, 2.60, p = 0.40) or any other study outcomes (table 3) . in this study, we evaluated serum ffas level in critically ill patients. we found that ffas level was elevated at baseline in 32% of patients, and that it was associated with features of the metabolic syndrome. ffas level was not affected by permissive underfeeding versus standard feeding. high ffas level appear to be largely a reflection of the underlying metabolic condition of the patient rather than the critical illness itself. our study provides a characterization of critically ill patients with high ffas level. we found that high ffas level correlated highly with other lipid profile parameters (total, hdl and ldl cholesterol, but not triglycerides) and with age. compared to those with normal ffas level, patients with high ffas level were at baseline significantly older, more likely to be diabetic, had higher hgba1c, blood glucose, creatinine and non-hdl and ldl cholesterol concentrations, and had more hypoxemia (as reflected lower po2: fio2 ratio) despite lack of differences in apache ii scores, sofa scores, and vasopressor demands. during icu stay, patients with high ffas level had increased demand for insulin, disease-specific nutrition therapy, rrt, aspirin, and statins. the differences suggest the association of high ffas level with metabolic syndrome. there was no difference in bmi, between the two groups; however, bmi is known to have its limitations in predicting obesity [22] . because of these differences, we carried out multivariable analyses to account for the confounding effect of some of these variables on clinical outcomes. these analyses show that high ffas level is not associated independently with clinical outcomes. interestingly, patients with high ffas level had less 24-h urinary nitrogen excretion and less negative nitrogen balance. this may be related to lower muscle mass in this older population and more frequent insulin therapy. normally, ffas are elevated during fasting and exercise, and their level drops postprandially after carbohydrate-rich meals. ffas level are elevated in obesity and diabetes [1, 3] . in acute critical illness, where lipolysis increases, serum ffas level increases [6] . our study demonstrated that one-third of critically ill patients had high ffas level; most (63.6%) of these patients were diabetics. we found that baseline cholesterol level and diabetes were independent risk factors for high ffas level on multivariable logistic regression analysis. the effect of propofol on ffas level is uncertain. in an experiment on dogs undergoing general anesthesia, high concentration of propofol (200 and 400 mcg/kg/min) were associated with increased ffas level, although a study in humans undergoing general anesthesia for cardiopulmonary bypass showed that propofol (50 mcg/kg/min) compared to midazolam did not alter serum ffas level [23, 24] . in our study, categorization of patients into high and low ffas level was based on baseline serum specimens, and doses of propofol preceding enrolment were not collected. our study was not designed to specifically address the effect of propofol on ffas level. nevertheless, the doses of propofol that were used during the icu stay were on average much lower than what was used in these studies, and, therefore, the effect of propofol on ffas level in our cohort is likely to be small. the slightly lower dose of propofol given to patients with high-ffas level was likely to be related to being older and more susceptible to sedation. therefore, these patients would normally receive smaller doses of propofol. in addition to being a fuel source, ffas have multiple other physiologic effects. ffas are associated with insulin resistance and impaired glucose metabolism by inhibiting glucose oxidation and by stimulating protein kinase c [1, 10, 25] . they may also stimulate the autophagy of pancreatic beta cells [26] . in our study, patients with high ffas level had higher blood glucose and required more insulin therapy during the icu stay even though the baseline blood glucose level was similar in patients with high and normal ffas, suggesting an association of ffas and insulin resistance in icu patients. ffas may also affect the course of acute critical illness. ffas were found to exacerbate hyperglycemia-induced toll-like receptor expression and activity in monocytic cells, increase superoxide release, enhance nuclear factor-κb activity, and induce the release of proinflammatory factors in diabetics [27] . whether ffas affect inflammation in critically ill patients is less clear. in a porcine endotoxemia model, infusing lipids at two different concentrations was associated with no differences in plasma tumor necrosis factor-α, interleukin6, and leucocytes between animals with low and high ffas suggesting that ffas does not play a significant pro-inflammatory mediator effect [28] . however, ffas have been implicated in the pathogenesis of acute respiratory distress syndrome and has been identified as a prognostic factor for this syndrome. in a lipopolysaccharide-induced acute lung injury model, a 15-fold increase in free oleic acid was observed in bronchoalveolar lavage fluid from mice 8 h after lipopolysaccharide application [20] . the ffa, oleic acid has been demonstrated to be elevated in patients with ards (acute respiratory distress syndrome) and in patients at-risk for ards [11, 29] . patients with sepsis demonstrated a six-fold increase in plasma oleic acid levels compared to healthy volunteers [19] . in addition, ffas are elevated in the blood of patients with sepsis who are at increased risk for ards [30] . the exact mechanism of ffas-associated lung injury is unclear; however, ffas have been shown to increase permeability and to impair transepithelial active sodium transport mechanisms in the lung, and could, thus, promote alveolar edema formation and prevent edema resolution [31] . in our study, where almost all patients were on mechanical ventilation at baseline, hypoxemia was more significant in patients with high ffas compared with patients with normal ffas (median pao2: fio2 ratio was 115 vs. 200, p = 0.02), a finding that may be in line with the association of ffas and lung injury. whether ffas are toxic to the kidneys is unclear. in an animal study, ffas led to severe tubulointerstitial damage [32] . ffas and their metabolites have been implicated in renal cell injury and development of chronic kidney in patients with the metabolic syndrome [33] . in our study, patients with high ffas had a higher rate of new renal replacement therapy, although this association became not significant in multivariable analysis. this finding suggests that the observed crude association may be related to other confounders that put these patients at a higher risk for acute kidney injury; for example, patients with high ffas level were more likely to be diabetics and had higher baseline creatinine compared to patients with normal ffas level. weight loss leads to a lowering of ffas level in the long run and can attenuate ffas-induced hepatic insulin resistance in obese healthy patients [14] [15] [16] . however, the effects of short-term caloric restriction are different. in one study, 11 subjects were fed for two periods of 6 days with hypo-and eucaloric diet with the same macronutrient composition in random order [34] . at 6 days, fasting ffas significantly increased with the hypocaloric diet compared with the eucaloric diet [34] . whether the macronutrient composition affects ffas was investigated in an animal model, and the study found that energy-restricted high-fat versus low-fat diet did not result in different ffa levels [35] . in the current study, serial levels of ffas did not differ with time between patients receiving permissive underfeeding and standard feeding. the study results should be interpreted taking into considerations its strengths and limitations. strength include that data came from a randomized controlled trial, and that serial measurements of ffas were obtained. the limitations include the sample size, which makes the study underpowered to detect a mortality difference. we measured total ffas but not individual levels of each ffas. in addition, the study included patients who had an expected duration of icu stay ≥14 days and, thus, the results may not be generalizable to patients who have a shorter stay. in conclusion, we found that serum ffas level was elevated in almost one-third of critically ill patients. high ffas level was associated with features of the metabolic syndrome and was not affected by short-term moderate caloric restriction. supplementary materials: the following are available online at http://www.mdpi.com/2072-6643/11/2/384/s1, table s1 : pearson correlations among baseline free fatty acids (ffas) level and other related measures of lipid metabolism, figure s1 : flow diagram for patients enrolled in the sub-study of free fatty acids (ffas) level. obesity, insulin resistance and free fatty acids elevation of free fatty acids induces inflammation and impairs vascular reactivity in healthy subjects lipid metabolism, metabolic diseases, and peroxisome proliferator-activated receptors circulating nonesterified fatty acid level as a predictive risk factor for sudden death in the population metabolic response to the stress of critical illness the acute splanchnic and peripheral tissue metabolic response to endotoxin in humans insulin stimulates lipoprotein lipase activity and synthesis in adipocytes from septic rats elevated free fatty acid level is a risk factor for early postoperative hypoxemia after on-pump coronary artery bypass grafting: association with endothelial activation fatty acids trigger mitochondrion-dependent necrosis short-chain fatty acids produced by anaerobic bacteria alter the physiological responses of human neutrophils to chemotactic peptide an increase in serum c18 unsaturated free fatty acids as a predictor of the development of acute respiratory distress syndrome serum free fatty acid concentration in patients with acute pancreatitis fatty acids, obesity, and insulin resistance: time for a reevaluation effect of weight loss and ketosis on postprandial cholecystokinin and free fatty acid concentrations effects of exercise training and diet on lipid kinetics during free fatty acid-induced insulin resistance in older obese humans with impaired glucose tolerance free fatty acid-induced hepatic insulin resistance is attenuated following lifestyle intervention in obese individuals with impaired glucose tolerance permissive underfeeding or standard enteral feeding in critically ill adults a severity of disease classification system use of the sofa score to assess the incidence of organ dysfunction/failure in intensive care units: results of a multicenter, prospective study. working group on "sepsis-related problems" of the european society of intensive care medicine middle east respiratory syndrome coronavirus infection: a short note on cases with renal failure problem is bmi the best measure of obesity? effect of propofol continuous-rate infusion on intravenous glucose tolerance test in dogs propofol as a continuous infusion during cardiopulmonary bypass does not affect changes in serum free fatty acids elevated plasma nonesterified fatty acids are associated with deterioration of acute insulin response in igt but not ngt free fatty acids stimulate autophagy in pancreatic β-cells via jnk pathway free fatty acids in the presence of high glucose amplify monocyte inflammation via toll-like receptors circulating free fatty acids do not contribute to the acute systemic inflammatory response. an experimental study in porcine endotoxaemia plasma fatty acid changes and increased lipid peroxidation in patients with adult respiratory distress syndrome parenteral nutrition with fish oil modulates cytokine response in patients with sepsis oleic acid inhibits alveolar fluid reabsorption: a role in acute respiratory distress syndrome? urinary free fatty acids bound to albumin aggravate tubulointerstitial damage mechanisms of tubulointerstitial injury in the kidney: final common pathways to end-stage renal failure the effects of underfeeding on whole-body carbohydrate partitioning, thermogenesis and uncoupling protein 3 expression in human skeletal muscle energy-restricted high-fat diets only partially improve markers of systemic and adipose tissue inflammation we wish to thank the following those who made valuable suggestions or who have otherwise contributed to the preparation of the manuscript: maram sakhija, turki almoammar, muhammad rafique sohail, shihab mundekkadan and aeron toledo. key: cord-254646-psolkrom authors: matsui, mary s. title: vitamin d update date: 2020-10-14 journal: curr dermatol rep doi: 10.1007/s13671-020-00315-0 sha: doc_id: 254646 cord_uid: psolkrom purpose: the goal of this review is to provide an update in the field of vitamin d, in particular, the role of vitamin d in non-skeletal health, the complexity of providing patient guidance regarding obtaining sufficient vitamin d, and the possible involvement of vitamin d in morbidity and mortality due to sars-cov-2 (covid-19). recent findings: in addition to bone health, vitamin d may play a role in innate immunity, cardiovascular disease, and asthma. although rickets is often regarded as an historical disease of the early twentieth century, it appears to be making a comeback worldwide, including “first-world” countries. broad-spectrum sunscreens (with high uva filters) that prevent erythema are unlikely to compromise vitamin d status in healthy populations. summary: new attention is now focused on the role of vitamin d in a variety of diseases, and more individualized patient recommendation schemes are being considered that take into account more realistic and achievable goals for achieving sufficient vitamin d through diet, supplements, and sun behavior. in the last 10 years, over 41,000 peer-reviewed research studies have been published on vitamin d, and since its original discovery as a sunlight-generated factor important to bone health, a more complex story of vitamin d has continued to evolve. vitamin d, technically not a vitamin, has been linked not just to rickets in children and osteomalacia in adults, but also has been suggested to play a role in diabetes, celiac disease, asthma, atopic dermatitis, tuberculosis, and, most recently, covid-19. this review will briefly summarize fundamental, well-established aspects of vitamin d and human health and then will also discuss (a) some of the most recent work related to vitamin d and non-skeletal-associated health issues; (b) the complexity of establishing meaningful vitamin d measurement metrics and assessing vitamin d status; c)decisionmaking for obtaining vitamin d through diet, supplements, or sun exposure; (d) the impact of skin type, pigmentation, and sunscreen on vitamin d levels; and (e) evidence for a potential influence of vitamin d on the mortality and morbidity of covid-19 through modulation of the pro-inflammatory cytokine response and respiratory response to the virus. some would urge us to move away from the nomenclature of vitamin d as a vitamin and, instead, acknowledge that vitamin d3 is a prohormone produced in skin through ultraviolet irradiation of 7-dehydrocholesterol (7dhc or provitamin d3) [1•] . this previtamin d3 is biologically inert and must undergo thermal isomerization and two hydroxylations. the first hydroxylation occurs in the liver and converts vitamin d to 25-hydroxyvitamin d [25(oh)d], also known as calcidiol. physiologically active 1,25-dihydroxyvitamin d [1, 25(oh) 2d], or calcitriol, is then synthesized primarily in the kidney. the importance of vitamin d for calcium absorption and bone health is undisputed [2, 3] . the classic function of vitamin d is to promote calcium absorption in the gut and maintain adequate serum calcium and phosphate concentrations necessary for normal mineralization of bone. rickets in children and osteomalacia in adults results from insufficient vitamin d [1•] . together with calcium, vitamin d helps protect this article is part of the topical collection on photodermatology older adults from osteoporosis. there is strong, consistent evidence supporting the role of vitamin d in childhood bone health, although most studies have been done in a limited range of skin types, as exemplified in a very recent report showing that high-dose vitamin d supplementation during pregnancy improved bone health in children [4] . in this case, all participants were white and danish. globally, rickets and vitamin d deficiency remain a significant public health problem even in highly developed countries [5••] , despite much research and many government and nongovernmentally funded programs. in europe, little vitamin d is made endogenously in the skin of individuals during the winter months [1•] , and yet vitamin d fortification of foods is largely absent. in the view of many scientists in the vitamin d field, the recommended dietary allowance is too low. recommendations for vitamin d3 at 2000 iu/day are being considered, an intake which should be safe and remain below toxic levels [1•] . factors contributing to a modern "third wave" of rickets have been reviewed recently [5••] from prospective surveillance studies of vitamin d deficiency rickets in australia, canada, and new zealand, as well as multiple retrospective studies from across the globe. in part, this third wave is believed to be caused by reduced uvb exposure due to sun avoidance (sunscreen, clothing, cultural behavior) and a shift toward indoor work. malabsorption syndromes such as celiac disease, short bowel syndrome, gastric bypass, and cystic fibrosis can trigger vitamin d deficiency [6] . medications such as phenobarbital, carbamazepine, dexamethasone, nifedipine, spironolactone, clotrimazole, and rifampin induce hepatic p450 enzymes which can accelerate the degradation of vitamin d and thereby lead to vitamin d deficiency. chronic liver disease and chronic kidney disease increase the risk for vitamin d deficiency due to the dependence on these organs for vitamin d activation. the prevalence of patients with vitamin d deficiency is highest in the elderly, obese, nursing home residents, and hospitalized patients [6] . the issue of vitamin d deficiency in populations who have higher melanin content and/or use extensive skin coverage will be discussed separately. according to the nih, serum concentration of 25(oh)d is the best indicator of vitamin d status. it reflects total vitamin d produced cutaneously as well as obtained through food and supplements and has a fairly long circulating half-life of 15 days. 25(oh)d functions as a biomarker of exposure, but it is not clear to what extent 25(oh)d levels also serve as a biomarker of physiological disease or more subtle conditions or indicate the amount of vitamin d stored in body tissues (primarily adipose tissue). in contrast to 25(oh)d, circulating 1,25(oh)2d is not a good indicator of vitamin d status. levels of 1,25(oh)2d do not typically decrease until vitamin d deficiency is severe. table 1 shows the current serum concentrations and interpretations from the institute of medicine [7• ]. in addition, because the two test protocols used to measure 25(oh)d show some variability among laboratories that conduct the analyses, a standard reference material for 25(oh)d was developed to improve confidence in the result [8] . although from table 1 it would appear that there exists one set of ideal values for serum vitamin d, the situation is somewhat more complex. a slightly alternate rule of thumb is the following [6] : levels of 25(oh)d less than 20 ng/ml indicate vitamin d deficiency, while levels below 30 ng/ml indicate vitamin d insufficiency. levels of 25(oh)d between 30 and 50 ng/ml are generally regarded to be optimal levels; however, a number of variables, including race and age, add complexity [6] . a more thorough discussion of vitamin d assay standardization and recommendation guidelines is provided by sempos and binkley [9] . in any case, the categories of vitamin d sufficiency or deficiency are based on the classic function of vitamin d, bone health. again, based on bone health parameters, vitamin d levels can also be referred to as deficient, insufficient, sufficient, or optimal vitamin d status. the severity of vitamin d deficiency is divided into mild, moderate, and severe. but what about other conditions for which vitamin d may have some modifying actions? there are numerous and conflicting studies that suggest that there may be an association between vitamin d deficiency and cancer, cardiovascular disease, diabetes, autoimmune diseases, asthma, atopic dermatitis, and depression. if indeed, vitamin d status is a modifying factor for these health issues, and it cannot be assumed that the ideal 25(oh)d serum values for prevention of rickets and osteomalacia would prevent or modify these other diseases. it is now recognized that many cells in the body express vitamin d receptors (vdr) which modulate cell proliferation, differentiation, and apoptosis and also regulate gene expression associated with modulation of cell growth, neuromuscular and immune function, and inflammation and may help regulate antimicrobial peptides [10••-14] . several cells involved in immune function express vdr and cyp27b1, which suggests that the active form of vitamin d, 1,25(oh)2d, may control immune function at different levels [10••] . vitamin d is known to regulate parathyroid growth and parathyroid hormone production; it plays a role in the islet cells of the pancreas and may help in suppression of certain autoimmune diseases and some cancers. there are many suggestions that deficiencies in vitamin d levels are linked to conditions such as rheumatoid arthritis, multiple sclerosis, alzheimer's disease, and schizophrenia (for reviews, see [10••, 13••]). prospective studies have reported inverse correlations between 25(oh)d serum levels and cardiovascular disease, serum lipids, inflammation, disorders of glucose metabolism, weight gain, mood disorders, declining cognitive function, and alzheimer's disease. however, no effect of vitamin d supplementation has been demonstrated for these outcomes, so the proximal relationship could be genetic, cultural behavior, or other confounding. first suggested over 30 years ago, vitamin d is now often referred to as a "wellknown" regulator of innate immunity [10••] . the apparent mechanism for this is vitamin d enhancement of defensin β2 and cathelicidin antimicrobial peptide (camp) production, increasing their antimicrobial activity [15] . a thorough review of vitamin d's regulatory function in both innate and acquired immunity can be found in wei and christakos [15] . the biologically active form of vitamin d, 1,25(oh)2d, modulates innate and adaptive immunity via regulation of at least 15 genes by the vdr [16] . 1,25(oh)2d upregulates camp not only in monocytes/ macrophages but also in other cells participating in the innate immune system. that said, there are insufficient data at this time to recommend any specific vitamin d dosage or treatment regimen for asthma, autoimmune diseases, multiple sclerosis, or other conditions. there is confusing and sometimes contradictory evidence for the therapeutic use of vitamin d to treat nonskeletal conditions. for example, although treatment with 1,25(oh)2d was shown to be effective in reducing respiratory infections in asthma patients, and suboptimal serum 25(oh)d in childhood may have adverse effects on tuberculosis and asthma, vitamin d supplementation in pregnancy does not appear to prevent school-age asthma [17] . in a study on vitamin d levels and susceptibility to asthma and atopic dermatitis, no evidence was found for genetically determined low 25(oh)d levels to be linked to an increased risk of either conditions [18] . initial reports indicate that vitamin d and omega-3 supplements failed to reduce risk of cancer or heart events in a 5-year trial of nearly 26,000 healthy us adults [19] . this is, in part, why there is currently active discussion over both the recommended levels of vitamin d as well as the testing methods used to assess adequacy for health and prevention of diseases. recommendations for optimum vitamin d levels, for obtaining a healthy level of vitamin d and even for assessment methods, are currently not uniform across the globe. to some extent, this is because relevant factors vary: vitamin d food fortification regulations, the strength of ambient ultraviolet radiation (uvr), levels of smog, culture and ethnicity, skin phototype, chronological age, and ease and accuracy of specific clinical laboratory measurements. for example, some geographic regions with strong sun have a very high prevalence of rickets [20] . the american academy of dermatology (aad) is regarded as a primary resource for good practice in the field of skin health and related medical specialties. the aad position statement on vitamin d can be accessed online [21] and lists the recommended dietary allowance (rda) and the upper limit values for both calcium and vitamin d intake that should cover "97.5% of the normal healthy population." the optimum vitamin d intake varies by age: for children under 1 year, the rda is 400 iu/day; between 1 year and 70 years, the rda is 600 iu/day; and for people over 70, the rda is 800 iu/day. these rda guidelines are based on aad guidelines that promote minimal or no sun exposure primarily due to the risk of skin cancer associated with sun exposure. the american cancer society also does not support increasing vitamin d levels through sun exposure. the cancer council of australia has slightly eased its sun protection message concerning sunscreen, outdoor clothing, and hats. protection from the sun is currently recommended by the world health organization when the uvr index is ≥ 3 [22] . in general, the paradigm accepted is that an initial linear dose-response relationship exists between exposure to uv radiation and change in concentration of 25(oh)d, followed by a plateau with continuing exposures over a longer period of time. because uvr from the sun and tanning beds can lead to the development of skin cancer, sun exposure (natural) or indoor tanning (artificial) is strongly discouraged. instead, it is recommended that vitamin d be supplied from a "healthy diet," which includes naturally enriched vitamin d foods, fortified foods and beverages, and/or vitamin supplements while practicing sun protection. unfortunately, it is almost impossible to achieve a satisfactory vitamin d intake from diet alone, since it is found at significant levels in only a few commonly consumed foods. fat-soluble molecule is found almost exclusively in oily fish such as salmon, sardines, herring and mackerel, liver, egg yolks, and fortified foods. since dietary sources are unlikely to be sufficient, especially for vegetarians and vegans, supplements are necessary to obtain the rda. the us department of agriculture's (usda's) websites (https:// fdc.nal.usda.gov/index.html.) can be searched for recommended healthy eating guidelines, the nutrient content of many foods, and a list of foods containing vitamin d. in contrast, one comprehensive review [6] concluded that 30 min of sunshine daily with over 40% of skin surface area is required to prevent vitamin d deficiency (although surface area numbers seem to vary greatly). the difficulty of promulgating simple blanket recommendations as pathways to optimal vitamin d status is well described by macdonald et al. [23] . this same reference describes a survey of multiple ethnic groups which demonstrated the difficulty (if not impossibility) of reaching optimal vitamin d levels if no sun exposure is considered safe. most women would find it almost impossible to achieve satisfactory vitamin d status if they had to rely on their current diets. other measures including fortification of additional foods may have to be considered. several different vitamin d fortification programs have been initiated across the globe [24] . in contrast to the aad guidelines that advise against any sun exposure, recommendations from the national health service of the uk [25] states this: "from about late march/early april to the end of september, most people should be able to get all the vitamin d we need from sunlight." clearly, one issue that is immediately apparent is the seasonality of vitamin d levels in certain latitudes. this brings up other key questions in the world of vitamin d. is there such a thing as "sensible" sun exposure? are vitamin d guidelines a "one size fits all" or should they be more nuanced to respect the range of human pigmentation and culture? for example, the recommendations and sufficiency numbers do not take into account an almost complete lack of data from africa and south america [13••] . one recent editorial goes so far as to say that "vitamin d guidelines development is in a state of paralysis" due to a lack of agreement on the parameters for the terms insufficiency, sufficiency, and toxicity based on 25(oh)d concentrations [9] . further, the commentary urges that a set of recommendations based on work of the vitamin d standardization program (vdsp) paves the way for development of rational universal vitamin d status guidelines. although it is believed that most vitamin d (about 80%) is acquired from solar exposure, sunscreen use is an important tool in prevention of skin cancer and is strongly recommended by the dermatology community. uvb wavelengths (280-320 nm) are absorbed by dna and result in direct damage in the form of cyclobutane pyrimidine dimers (cpds) and other mutagenic events that ultimately lead to nonmelanoma skin cancer and melanoma. uva wavelengths (320-400 nm) generate oxidative stress which is associated primarily with photoaging but can also promote skin cancer. unfortunately, the same uvb wavelengths absorbed by dna and that cause dna mutations are also those that induce photoconversion of 7-dehydrocholesterol, to previtamin d3. one of the most important questions to ask then, with regard to the issue of sun avoidance and vitamin d, is directed toward the balance between (a) sunscreen use and uvr generation of vitamin d and (b) the efficacy of sunscreen to prevent uvr-induced dna damage. a consensus review published in 2019 [26] concluded that broad-spectrum sunscreens (with high uva filters) that prevent erythema are unlikely to compromise vitamin d status in healthy populations and that daily and recreational photoprotection does not compromise vitamin d synthesis. vitamin d screening should be used to monitor patients with photosensitivity disorders, who require the most rigorous photoprotection combined with vitamin d supplements [27••] . improved vitamin d status by uvr is always associated with the possibility of higher dna damage and skin cancers; however, production of vitamin d may be optimized and skin dna damage minimized, by increasing the body surface area exposed and decreasing the uvb dose per unit area [13••] . one group has examined the relationship between sunscreen use and vitamin d production in some detail [26, 28••, 29] . a study of sunscreen use in polish volunteers on vacation in tenerife had both interventional and observational groups and was able to monitor the effect of two spf 15 sunscreens over 1 week [26, 29] . one sunscreen had a high uva protection factor (uva-pf), while the second had low uva protection. the authors were able to make the important conclusion that sunscreens may be used to prevent sunburn yet allow vitamin d synthesis, although with the caveat that the absence of sunburn does not necessarily mean the absence of dna mutations [30] . however, concern about unrepaired mutagenic dna lesions should be tempered by evidence that efficient repair occurs within 12 to 48 h, although there is significant individual variation in this ability [30] . in the tenerife study, both high uva and low uva sunscreens prevented sunburn, but subjects using the high uva sunscreen had higher levels of serum 25(oh)d at the conclusion of the study. these findings were attributed to the fact that high uva sunscreen allows transmission of more uvb than low uva sunscreen. another very important study on sun exposure and vitamin d levels followed polish children over 12 days at a baltic sea summer camp [31] . relatively low daily uv radiation doses resulted in a modest but significant improvement in 25(oh)d (24%) but a very much greater increase in cpd (1162%). dna damage was worse in skin types i/ii. this conundrum is reflected in a recent global consensus on rickets prevention that was unable to recommend a safe uvr exposure level to enhance vitamin d status [32] . it has been argued, following in vitro assays, that vitamin d itself reduces the risk of skin cancer by several mechanisms [33] [34] [35] . under laboratory conditions, dna photolesions can be reduced in irradiated skin cells treated with 1,25(oh)2d. one mechanism for this may relate to the increases in p53 and nucleotide excision repair observed within hours after uvr exposure in keratinocytes and melanocytes treated with 1,25(oh)2d (1 or 10 nm) or vitamin d analogs. there was a corresponding reduction in cpds in uv-irradiated skin cells treated with vitamin d compounds. the indirect dna damage and the reduction in dna repair that is normally caused by nitric oxide products may also be reduced by vitamin d compounds. a group in cleveland reported data from an exploratory study in which high oral doses of vitamin d3 resulted in a sustained reduction in skin redness after experimental sunburn, as well as less epidermal structural damage, reduced expression of pro-inflammatory markers in the skin, and a gene expression profile characterized by upregulation of skin barrier repair genes [11] . one barrier to establishing recommendations for sensible sun exposure is the impact of human skin pigmentation and genetic polymorphisms on the response to uvr. these are still largely unknown variables. a careful literature review of vitamin d and the impact of pigmentation was published in 2015 [36•] and noted that a better understanding of how and how much melanin influences vitamin d photosynthesis is critical to meaningful public health messages. the research is difficult to integrate due to variations in study methodology, including the source, dose and frequency of uv irradiation, phototype classification, measurement methods for vitamin d, and the lack of information on relevant genetic polymorphisms. however, on balance, the review team concluded that m o r e h i g hl y p i g m e n t e d s ki n h as l es s e f f e ct i v e photoproduction of vitamin d and 25-hydroxyvitamin d. the ratio of sun exposure to pigmentation level to achieve vitamin d sufficiency remains uncertain. a recent 4-week study of 71 school children [37] controlled for time spent outdoors and assessed the contribution of clothing coverage, initial vitamin d levels, skin color, and other variables to the serum vitamin d levels at the conclusion of the study. the subjects were fitzpatrick skin phototypes iv and v with a range of melanin levels. for all subjects, there was a significant increase in serum 25(oh)d at the conclusion of the study compared with baseline, and a greater increase in serum vitamin d was seen in children with the lowest initial values. melanin levels were inversely correlated with the increase in vitamin d levels. in a study of racially diverse children in boston, most of the variability in 25(oh)d was correlated with constitutive skin color [38] . the correlation between skin color and serum levels of 25(oh)d is usually attributed to the photoprotective properties of melanin. however, there may be additional variables in play which may influence the ultimate health consequences. in a recent study, black americans (n = 2085) had lower concentrations of 25 (oh)d, but also lower concentrations of vitamin d binding protein, than white americans. the consequence of this was similar (calculated) levels of bioavailable 25(oh)d. this may explain why people of color with low total 25(oh)d had a higher average bone mineral density than the white group with similar 25(oh)d concentrations. furthermore, it may imply that bone health is more accurately reflected by the concentration of bioavailable, rather than total, 25(oh)d. taken together, the limited data available suggests that there should be some caution about attributing the observed lower levels of vitamin d in skin of color (soc) simply to a blocking effect of melanin. a very recent rigorously performed and analyzed study was just published that indicates melanin to have less impact on vitamin d levels than previously thought, possibly due to the spatial positions of melanin and 7-dehydrocholesterol in human skin [39••] . a brief review of confounding related to the question of pigment, uvrrelated skin cancers, and serum vitamin d levels can also be found in a 2015 review [13••] . the role of vitamin d as a possible modulator of susceptibility, morbidity, and mortality in sars-cov-2 (covid19) that vitamin d should play a role in covid-19 is not unexpected, since there has been believed to be a link between vitamin d deficiency and respiratory disease for over 100 years. observational studies have reported independent associations between low serum concentration of 25hydroxyvitamin d and susceptibility to acute respiratory tract infections [40] . vitamin d deficiency is associated with an increased risk of the acute respiratory distress syndrome (ards), intensive care admission, and mortality in patients with pneumonia [41] . in a systematic review and metaanalysis of 25 randomized controlled studies, martineau et al. found that vitamin d protected against acute respiratory tract infection overall [42] . patients who were severely vitamin d deficient experienced the most benefit. subgroup analysis revealed that daily or weekly vitamin d supplementation without additional bolus doses protected against acute respiratory tract infection, whereas regimens containing large bolus doses did not. among those receiving daily or weekly vitamin d, protective effects were strongest in those with profound vitamin d deficiency at baseline, although those with higher baseline 25-hydroxyvitamin d concentrations also experienced benefit. the interaction of vitamin d status and susceptibility to sars-cov-2 is currently being investigated [43-45••] . most immune cells express the vdr and actively convert 25(oh)d into 1,25(oh)2d, its active form. vdr signaling has a suppressive role on autoimmunity and an antiinflammatory effect, promoting dendritic cell and regulatory t cell differentiation and reducing th 17 cell response and inflammatory cytokine secretion (with relevance to the covid-19 induced cytokine storm). also mentioned earlier, vitamin d is thought to have a regulatory effect on innate immunity. there is no general consensus on the desired level of 25(oh)d to achieve immunomodulatory effects; thus, there is no current indication for vitamin d supplementation in specific infections and/or autoimmune diseases. however, dr. anthony fauci, director of the national institute of allergy and infectious diseases, in a recent interview by jama, agreed that "if you are vitamin d deficient, you might have a poor outcome or a greater chance of getting into trouble with an infection" (jama medical news & perspectives, june 8, 2020). the most vulnerable group with respect to covid-19, the aging population, also has a high proportion of individuals with deficient vitamin d levels. further studies have been both proposed (at least one clinical trial is registered with the nih) and initiated to clarify the contribution of vitamin d levels to susceptibility and the severity of response to covid-19 as well as the use of vitamin d supplementation as a possible therapeutic agent [43••-45••]. vitamin d deficiency is a major global public health issue with vitamin d deficiency rickets at significant levels even in highly developed countries. about 1 billion people worldwide have vitamin d deficiency, while 50% of the population has vitamin d insufficiency. the fact that exposure of the skin to uvb radiation, wavelengths that cause mutations in the dna of epidermal cells, is also the source of vitamin d is problematic. can the average person finely calibrate 0.2 minimal erythema dose, the point at which vitamin d synthesis occurs without perceptible dna damage? for relevant nonskeletal conditions or diseases such as multiple sclerosis, alzheimer's disease, or even ards, it is unknown whether low vitamin d status causes the disease or the disease causes the low vitamin d status. communication with patients about vitamin d calls for judgment and individualization in patient care, including more nuanced advice about sun exposure. human and animal rights and informed consent this article does not contain any studies with human or animal subjects performed by any of the authors. this is a generally helpful primer on the classic functions and understanding of vitamin d vitamin d and health: the need for more randomized controlled trials serum cholecalciferol may be a better marker of vitamin d status than 25-hydroxyvitamin d effect of high-dose vs standard-dose vitamin d supplementation in pregnancy on bone mineralization in offspring until age 6 years: a prespecified secondary analysis of a double-blinded, randomized clinical trial a brief history of nutritional rickets vitamin d deficiency medicine committee to review dietary reference intakes for vitamin d, calcium. the national academies collection: reports funded by national institutes of health nist releases vitamin d standard reference material. nist 25-hydroxyvitamin d assay standardisation and vitamin d guidelines paralysis vitamin d: nutrient, hormone, and immunomodulator oral vitamin d rapidly attenuates inflammation from sunburn: an interventional study control of cutaneous antimicrobial peptides by vitamin d3 the consequences for human health of stratospheric ozone depletion in association with other environmental factors cyp11a1 in skin: an alternative route to photoprotection by vitamin d compounds mechanisms underlying the regulation of innate and adaptive immunity by vitamin d key vitamin d target genes with functions in the immune system vitamin d supplementation in pregnancy does not prevent school-age asthma vitamin d levels and susceptibility to asthma, elevated immunoglobulin e levels, and atopic dermatitis: a mendelian randomization study study questions the benefits of vitamin d and omega 3 supplements. cardiosmart news american college of cardiology the effect of high-dose postpartum maternal vitamin d supplementation alone compared with maternal plus infant vitamin d supplementation in breastfeeding infants in a high-risk population. a randomized controlled trial o / dfff5f47b0dddfb82676270505afd09f/ps-vitamin_d_postition_ statement.pdf 22. ultraviolet (uv) index. world health organization sunlight and dietary contributions to the seasonal vitamin d status of cohorts of healthy postmenopausal women living at northerly latitudes: a major cause for concern? vitamin d microencapsulation and fortification: trends and technologies how to get vitamin d from sunlight sunscreen photoprotection and vitamin d status an important discussion of study protocol variables and their influence on policy sub-optimal application of a high spf sunscreen prevents epidermal dna damage in vivo optimal sunscreen use, during a sun holiday with a very high ultraviolet index, allows vitamin d synthesis without sunburn kinetics of uv light-induced cyclobutane pyrimidine dimers in human skin in vivo: an immunohistochemical analysis of both epidermis and dermis children sustain high levels of skin dna photodamage, with a modest increase of serum 25-hydroxyvitamin d3, after a summer holiday in northern europe global consensus recommendations on prevention and management of nutritional rickets vitamin d and death by sunshine protective effects of 1,25 dihydroxyvitamin d3 and its analogs on ultraviolet radiation-induced oxidative stress: a review photoprotective properties of vitamin d and lumisterol hydroxyderivatives a systematic review of the influence of skin pigmentation on changes in the concentrations of vitamin d and 25-hydroxyvitamin d in plasma/serum following experimental uv irradiation impact of solar ultraviolet b radiation (290-320 nm) on vitamin d synthesis in children with type iv and v skin association of serum 25-hydroxyvitamin d with race/ethnicity and constitutive skin color in urban schoolchildren melanin has a small inhibitory effect on cutaneous vitamin d synthesis: a comparison of extreme phenotypes vitamin d(3) supplementation reduces the symptoms of upper respiratory tract infection during winter training in vitamin d-insufficient taekwondo athletes: a randomized controlled trial vitamin d deficiency contributes directly to the acute respiratory distress syndrome (ards) vitamin d supplementation to prevent acute respiratory tract infections: systematic review and meta-analysis of individual participant data evidence that vitamin d supplementation could reduce risk of influenza and covid-19 infections and deaths covid-19 and vitamin d-is there a link and an opportunity for intervention? letter: covid-19, and vitamin d publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-016300-vw11c2wt authors: jain, kewal k. title: biomarkers of pulmonary diseases date: 2017-09-18 journal: the handbook of biomarkers doi: 10.1007/978-1-4939-7431-3_16 sha: doc_id: 16300 cord_uid: vw11c2wt lungs and airways are affected by several pathologies, the most important of which are inflammation, infection and cancer. some of the biomarkers of these pathologies are similar to those found in involvement of other organs. this chapter will briefly discuss general issues of biomarkers of pulmonary disorders listed in table 16.1. biomarkers of lung cancer are described in chapter 13. lungs and airways are affected by several pathologies, the most important of which are inflammation, infection and cancer. some of the biomarkers of these pathologies are similar to those found in involvement of other organs. this chapter will briefly discuss general issues of biomarkers of pulmonary disorders listed in table 16 .1. biomarkers of lung cancer are described in chapter 13. low lung function is associated with increased morbidity and mortality. it is therefore of interest to identify biomarkers that are associated with impaired lung function. lung function (fev1 and fvc) and a panel of 15 inflammatory biomarkers (including cytokines, chemokines, adhesion molecules, crp and wbc count) from blood samples were analysed subjects aged 70 years (kuhlmann et al. 2013) . wbc count, crp and vcam-1 were found to relate to poorer lung function. a doserelated association was found for the combination wbc count and crp towards fev1 and wbc and vcam-1 towards fvc. this indicates that combination of two biomarkers yielded more information than assessing them one by one when analysing the association between systemic inflammation and lung function. oxidative stress is the hallmark of various chronic inflammatory lung diseases. increased concentrations of ros in the lungs of such patients are reflected by elevated concentrations of oxidative stress markers in the breath, airways, lung tissue and blood. traditionally, the measurement of these biomarkers has involved invasive procedures to procure the samples or to examine the affected compartments, to the patient's discomfort. non-invasive approaches to measure oxidative stress have been investigated. the collection of exhaled breath condensate (ebc) is a noninvasive sampling method for real-time analysis and evaluation of oxidative stress biomarkers in the lower respiratory tract airways. the biomarkers of oxidative stress such as h 2 o 2 , f2-isoprostanes, malondialdehyde, 4-hydroxy-2-nonenal, antioxidants, glutathione and nitrosative stress such as nitrate/nitrite and nitrosated species can be measured in ebc. oxidative stress biomarkers also have been measured for various antioxidants in disease prognosis. ebc is currently used as a research and diagnostic tool in free radical research, yielding information on redox disturbance and the degree and type of inflammation in the lung. it is expected that ebc can be exploited to detect specific levels of biomarkers and monitor disease severity in response to treatment. community-acquired pneumonia (cap) is one of the most common reasons for emergency department. despite its prevalence, there are many challenges to proper diagnosis and management of pneumonia. there is no accurate and timely gold standard to differentiate bacterial from viral disease, and there are limitations in precise risk stratification of patients to ensure appropriate site-of-care decisions. clinical risk scores such as pneumonia severity index (psi) and curb-65 (confusion, urea, respiratory rate, blood pressure, age > 65 years), and blood biomarkers of different physiopathological pathways are used in predicting longterm survival in patients with cap. in a prospective study, patients admitted with cap were followed for 6 years and cox regression models as well as area under the receiver operating characteristics curve (auc) were used to investigate associations between initial risk assessment and all-cause mortality (alan et al. 2015) . initial psi and curb-65 scores both had excellent long-term prognostic accuracy, with a step-wise increase in mortality per risk class. the addition of inflammatory (pro-adrenomedullin) and cardiac (pro-atrial natriuretic peptide) blood biomarkers measured upon hospital admission further improved the prognostic capabilities of the psi. pathological changes in severe acute respiratory syndrome (sars) suggest that sars sequelae are associated with dysregulation of cytokine and chemokine production. a study from taiwan showed that cytokine or chemokine profiles in patients with sars differ markedly from those in patients with community-acquired pneumonia (cap) and control groups (chien et al. 2006) . serum levels of three cytokines were significantly elevated in sars patients versus the cap: interferon-γ-inducible protein-10 (ip-10), interleukin (il)-2, and il-6. cytokine levels began to rise before the development of chest involvement and peaked earlier than did lung injury assessed by chest x-ray. conversely, in cap patients but not sars patients or controls, levels of interferon-γ, il-10, and il-8 were elevated, and rose in tandem with radiographic changes. a further difference between groups was the ratio of il-6 to il-10, at 4.84 in sars patients versus 2.95 in cap patients. however, in both sets of patients, levels of il-6 correlated strongly with the severity of lung injury. the early induction of ip-10 and il-2, as well as the subsequent overproduction of il-6 and lack of il-10, probably contribute to the main immunopathological processes involved in sars lung injury and may be early biomarkers of lung injury. these findings differ from those observed in subjects with cap. plasma biomarkers related to inflammation − il-8 and enhanced neutrophil recruitment to the lung (icam-1) − are independently associated with increased mortality in patients with ali. higher levels of il-8 and icam-1 independently predicted death (mcclintock et al. 2008 ). in addition, lower levels of the coagulation marker protein c were independently associated with an increased risk of death. the association of lower protein c levels with non-survivors continues to support the role for disordered coagulation in ali/ards. these associations exist despite consistent use of lung protective ventilation and persist even when controlling for clinical factors that also impact upon outcomes. the two biomarkers with an independent association with mortality, il-8 and icam-1, need to be be studied further for their potential value in stratifying patients in clinical trials. acute respiratory distress syndrome (ards) is the rapid onset of respiratory failure − the inability to adequately oxygenate the blood − that often occurs in the critically ill. acute lung injury (ali) precedes ards as severe respiratory illnesses progress. both conditions can be life-threatening. in a large-scale, multicenter trial of patients with ards or ali, higher levels of nitric oxide (no) in urine were strongly associated with improved survival, more ventilator-free days, and decreased rates of organ failure (mcclintock et al. 2007 ). the authors speculated that no has a beneficial effect on ali since it scavenges oxygen free radicals that are generated during oxidative stress. since no increases microcirculation, it helps to better perfuse tissue beds in the lungs. the investigators offered an alternative hypothesis to explain their findings: no created inside the body may have a beneficial effect on organs other than the lung during ali. it might help prevent further tissue damage by improving oxygen and nutrient delivery to the tissues, while helping to decrease the amount of toxic oxygen species. the authors also speculated that no might have antibacterial effects that could be important in infectious conditions that predispose patients to ali. pulmonary surfactant, a complex of lipids and proteins, functions to keep alveoli from collapsing at expiration. surfactant proteins a (sp-a) and d (sp-d) belong to the collectin family and play pivotal roles in the innate immunity of the lung. pulmonary collectins directly bind with broad specificities to a variety of microorganism and possess antimicrobial effects. these proteins also exhibit both inflammatory and antiinflammatory functions. the collectins enhance phagocytosis of microbes by macrophages through opsonic and/or non-opsonic activities. the proteins stimulate cell surface expression of phagocytic receptors including scavenger receptor a and mannose receptor. since the expression of sp-a and sp-d is abundant and restricted within the lung, the proteins are now clinically used as biomarkers for lung diseases. the levels of sp-a and sp-d in bronchoalveolar lavage fluids, amniotic fluids, tracheal aspirates and pleural effusions reflect alterations in alveolar compartments and epithelium, and lung maturity. the determination of sp-a and sp-d in sera is a noninvasive and useful tool for understanding some pathological changes of the lung in the diseases, including pulmonary fibrosis, collagen vascular diseases complicated with interstitial lung disease, pulmonary alveolar proteinosis, acute respiratory distress syndrome and radiation pneumonitis (takahashi et al. 2006) . interstitial lung disease (ild) is defined as restrictive lung function impairment with radiographic signs of ild. kl-6, a mucinous high-molecular weight glycoprotein, is expressed on type ii pneumonocytes and is a potential biomarker of ild. a retrospective, cross-sectional analysis caucasian patients with polymyositis (pm) or dermatomyositis (dm) and ild were shown to have elevated serum levels of kl-6 compared to patients without ild (fathi et al. 2012) . at a cut-off level of 549 u/ml, the sensitivity and specificity for diagnosis of ild was 83% and 100%, respectively. the level of serum kl-6 may serve as measure of ild in patients with pm/dm, and is a promising biomarker for use in clinical practice to assess response to treatment. chronic obstructive pulmonary disease (copd) consists of two main forms − chronic bronchitis and emphysema − and sufferers usually have a combination of these conditions. there has been increasing interest in using pulmonary biomarkers to understand and monitor the inflammation in the respiratory tract of patients with copd. bronchial biopsies and bronchoalveolar lavage provide valuable information about inflammatory cells and mediators, but these procedures are invasive, so that repeated measurements are limited. sputum provides considerable information about the inflammatory process, including mediators and proteinases in copd, but samples usually represent proximal airways and may not reflect inflammatory processes in distal bronchi. analysis of exhaled breath is a noninvasive procedure so that repeated measurements are possible, but the variability is high for some assays. there is relatively little information about how any of these biomarkers relate to other clinical outcomes, such as progression of the disease, severity of disease, clinical subtypes or response to therapy. more information is also needed about the variability in these measurements. in the future pulmonary biomarkers may be useful in predicting disease progression, indicating disease instability and in predicting response to current therapies and novel therapies, many of which are now in development. the copd foundation biomarker qualification consortium (cbqc) is a unique public-private partnership established in 2010 between the copd foundation, the pharmaceutical industry, and academic copd experts with advisors from the us national heart lung & blood institute and fda (miller et al. 2016) . the initial intent of the cbqc was to integrate data collected in 2009 and submit a dossier for the qualification. this led to the fda qualification of plasma fibrinogen as a prognostic or enrichment biomarker for all-cause mortality and copd exacerbations in 2015. it is the first biomarker drug development tool qualified for use in copd under the fda's drug development tool qualification program. alpha1-antitrypsin (aat) is a plasma glycoprotein that inhibits neutrophil elastase, and individuals who inherit altered aat genes resulting in deficiency of the protein are at high risk for copd and liver cirrhosis. this deficiency can be detected by serum protein pattern studies. in the past, testing for the deficiency has been done retrospectively in patients with copd or liver disease, but the introduction of a home-administered finger-stick blood spot test for aat genotype enables affected families to construct pedigrees to enable them to identify children who are at risk for developing copd in later life and should avoid exposure to dust and smoke. extracellular matrix (ecm) remodeling of the lung tissue releases protein fragments into the blood, where they may be detected as serologic surrogate biomarkers of disease activity in copd. association of ecm turnover with severity and outcome of copd has been assessed in a prospective, observational, multicenter study, global initiative for chronic obstructive lung disease grades ii to iv, and serum samples were analyzed at stable state, during exacerbation as well as 4 weeks after exacerbation (stolz et al. 2017) . results showed that patients with the lowest levels of pro-forms of collagen type iii (pro-c3) and type vi (pro-c6) had more severe airflow limitation, hyperinflation, air trapping, and emphysema. collagen type iii (c3m) and collagen type vi (c6m) were associated with dyspnea. in conclusion, serum biomarkers of ecm turnover were significantly associated with disease severity and clinically relevant outcomes in patients with copd. lung ecm remodeling in healthy controls and copd patients was investigated in the copdgene study. the data suggest that type vi collagen turnover and elastin degradation by neutrophil elastase are associated with copd-induced inflammation (eosinophil-bronchitis) and emphysema (bihlet et al. 2017) . serological assessment of type vi collagen and elastin turnover may assist in identification of phenotypes likely to be associated with progression and amenable to precision medicine for clinical trials. lung failure, also termed "lung attack", is the most common organ failure seen in the intensive care unit. lung attacks, which effect individuals with copd are among the leading cause of visits to emergency rooms among chronic disease sufferers. other causes are neuromuscular impairment, pulmonary edema, pneumonia, and vascular diseases such as acute or chronic pulmonary embolism. when a patient is admitted into the hospital with a severe lung failure, it usually takes >3 months to get to 80% of his or her baseline health. if the patient's health is poor to start with, the new attack can be devastating or even fatal. a test that could more accurately present a patient's disease could make it easier to predict and treat copd progression to lung failure. there is need for a test that could be performed in any clinical lab and could be used far more widely than the current lung function tests, which are performed in certain centers by specially trained personnel. in 2012, canada's prevention of organ failure (proof) center of excellence in vancouver received funding from genome british columbia to develop a biomarkerbased test for determining a copd patient's risk for having a lung attack. genes and protein biomarker sets that have been discovered at proof center could have the ability to predict copd-caused lung attacks and need to be validated. circulating bnp levels were evaluated as a parameter for the presence and severity of pulmonary hypertension (ph) in patients with chronic lung disease (leuchte et al. 2006) . during a follow-up time of approximately 1 year, significant pulmonary hypertension (mean pulmonary artery pressure > 35 mm hg) was diagnosed in more than one-fourth of patients and led to decreased exercise tolerance and life expectancy. elevated bnp concentrations identified significant pulmonary hypertension with a sensitivity of 0.85 and specificity of 0.88 and predicted mortality. moreover, bnp served as a risk factor of death independent of lung functional impairment or hypoxemia. it is concluded that plasma bnp facilitates noninvasive detection of significant ph with high accuracy and can be used as a screening test for the presence of ph. in addition, bnp enables an assessment of the relevance of ph and could serve as a useful prognostic parameter in chronic lung disease. a study has revealed that serum levels of the neuroendocrine activity biomarker chromagranin a (cga) are increased in male smokers with impaired lung function, and are associated with both respiratory symptoms and the degree of airway obstruction (sorhaug et al. 2006) . the subgroup of airway epithelial cells belonging to the diffuse neuroendocrine system, termed pulmonary neuroendocrine cells, may represent a putative regulatory function of cga as a prohormone. they are considered to control growth and development of the fetal lung and regulation of ventilation and circulation, but may also have a role in the pathogenesis of smoking-induced airway disease. the findings indicate that neuroendocrine activation may be important in smoking-related airway inflammation and remodeling, and raise the possibility that cga could be of predictive value as a biomarker of prognosis in smoking-associated diseases. measurements of c-reactive protein (crp), a biomarker of inflammation, provide incremental prognostic information beyond that achieved by traditional biomarkers in patients with mild to moderate copd, and may enable more accurate detection of patients at a high risk of mortality. lung function decline is significantly related to crp levels, with an average predicted change in fev1 of −0.93% in the highest and 0.43% in the lowest quintile. however, respiratory causes of mortality are not significantly related to crp levels. genome-wide expression profiling of peripheral blood samples from subjects with significant airflow obstruction was performed to find non-invasive gene expression biomarkers for copd (bhattacharya et al. 2011) . correlation of gene expression with lung function measurements identified a set of 86 genes. a total of 16 biomarkers showed evidence of significant correlation with quantitative traits and differential expression between cases and controls. further comparison of these peripheral gene expression biomarkers with those previously identified from lung tissue of the same cohort revealed that two genes, rp9 and nape-pld, were decreased in copd cases compared to controls in both lung tissue and blood. these results contribute to our understanding of gene expression changes in the peripheral blood of patients with copd and may provide insight into potential mechanisms involved in the disease. patients with copd are often at high risk of early death and identification of prognostic biomarkers may aid in improving their survival by providing early intensive therapy for high-risk patients. a study has investigated the prognostic role of hyperuricemia at baseline on the prognosis of patients with copd by retrospective evaluation of data . hyperuricemia was found to be not associated with other baseline characteristics in patients with copd. kaplan-meier survival curve showed that patients with copd with hyperuricemia had higher risk of mortality compared with patients with normouricemia. thus, hyperuricemia is a promising biomarker of early mortality in patients with copd. decreased expression of vascular endothelial growth factor (vegf) and its receptor has been implicated in the pathogenesis of copd. levels of placenta growth factor (plgf), another angiogenic factor, are increased in the serum and bronchoalveolar lavage (bal) fluid of patients with copd and are inversely correlated with fev1 (cheng et al. 2008) . serum levels of plgf in patients with copd were more than double those in smokers and nonsmokers without copd. these findings suggest that bronchial epithelial cells can express plgf, which may contribute to the pathogenesis of copd. both plgf and vegf expression levels were increased in cultured bronchial epithelial cells exposed to pro-inflammatory cytokines such as tnfα and il-8. although the mechanisms underlying the observed detrimental effects of plgf remain to be clarified, persistent plgf expression might have adverse effects on lung parenchyma by down-regulating angiogenesis. although the aim of management of patients with asthma is to control their symptoms and prevent exacerbations and morbidity of the disease, optimal management may require assessment and monitoring of biomarkers, i.e., objective measures of lung dysfunction and inflammation. clinical observations suggest that rhinovirus infection induces a specific inflammatory response in predisposed individuals that results in worsened asthmatic symptoms and increased airway inflammation. a study has shown that ifn-γinduced protein (ip)-10 is specifically released in acute virus-induced asthma, and can be measured in the serum to predict a viral trigger of acute exacerbations (wark et al. 2007) . primary bronchial epithelial cell models of rhinovirus infection were used to identify mediators of rhinovirus infection and responded to infection with rhinovirus-16 by releasing high levels of ip-10, rantes, and il-16, as well as smaller amounts of il-8 and tnf-α. ip-10, perhaps in combination with tnf-α, might be a useful clinical marker to identify rhinovirus and other virus-induced acute asthma. additional findings suggest that ip-10 or cxcr3 (an ip-10 receptor that is highly expressed in activated t cells) might have a role in worsening of airflow obstruction and airway inflammation, and may therefore be potential therapeutic targets. international guidelines on the management of asthma support the early introduction of corticosteroids to control symptoms and to improve lung function by reducing airway inflammation. however, not all individuals respond to corticosteroids to the same extent and it would be an desirable to be able to predict the response to corticosteroid treatment. several biomarkers have been assessed following treatment with corticosteroids including measures of lung function, peripheral blood and sputum indices of inflammation, exhaled gases and breath condensates. the most widely examined measures in predicting a response to corticosteroids are airway hyperresponsiveness, exhaled no (eno) and induced sputum. of these, sputum eosinophilia has been demonstrated to be the best predictor of a short-term response to corticosteroids. more importantly, directing treatment at normalizing the sputum eosinophil count can substantially reduce severe exacerbations. the widespread utilization of sputum induction is hampered because the procedure is relatively labor intensive. the measurement of eno is simpler, but incorporating the assessment of no in an asthma management strategy has not led to a reduction in exacerbation rates. the challenge now is to either simplify the measurement of a sputum eosinophilia or to identify another inflammatory marker with a similar efficacy as the sputum eosinophil count in predicting both the short-and long-term responses to corticosteroids. airway inflammation is associated with an increased expression and release of inflammatory reactants that regulate processes of cell migration, activation and degranulation. one study was done to quantify bronchial lavage (bal) fluid and serum levels of il-8, secretory leukocyte protease inhibitor (slpi), soluble intracellular adhesion molecules-1 (sicam-1) and scd14, as surrogate markers of inflammatory and immune response in asthma and copd patients with similar disease duration time (hollander et al. 2007 ). biomarkers were measured using commercially available elisa kits. the findings show that of four measured biomarkers, only the bal il-8 was higher in copd patients when compared to asthma. severe asthma is characterized by elevated levels of proinflammatory cytokines and neutrophilic inflammation in the airways. blood cytokines, biomarkers of systemic inflammation, may be a feature of increased inflammation in severe asthma. one study found that il-8 and tnf-α levels were higher in severe asthmatics than in mild-moderate asthmatics or in controls and, in conjunction with augmented circulating neutrophils, suggest the involvement of neutrophil-derived cytokine pattern (silvestri et al. 2006) . furthermore, in patients with severe asthma, tnf-α levels were positively correlated with both exhaled nitric oxide and circulating neutrophil counts. cytokine levels were elevated even though the patients were on high-dose inhaled steroids. this finding might reflect the inability of these drugs to significantly suppress production of this cytokine by airway cellular sources including epithelial cells and inflammatory cells. in patients with severe asthma there may be an imbalance between il-8 production and the blocking capacity of il-8 autoantibodies. the findings of this study may be clinically relevant and suggest that drugs that block tnf-α release or activity might represent a new treatment option in severe asthma. airway hyperresponsiveness is the main feature of asthma and is defined as an increase in the ease and degree of airway narrowing in response to brochoconstrictor stimuli. inflammation plays a central role in the pathogenesis of asthma and much of it can be attributed to helper t cell type 2 cytokine activation, the degree of which strongly correlates to disease severity. one of the inflammatory mediators in asthma is nitric oxide (no). the exhaled no level is elevated in asthma, particularly allergic asthma during the pollen season, and can predict asthma exacerbation. it may be clinically more useful to compare exhaled no values with a subject's previous values than to compare them with a population based normal range. cough variant asthma (cva) and atopic cough both present with bronchodilator-resistant non-productive cough but may be differentiated from and other causes of chronic non-productive cough by measuring exhaled no. exhaled no levels in patients with atopic cough are significantly lower than those in patients with cva and bronchial asthma (fujimura et al. 2008 ). there are no significant difference in the exhaled no levels between patients with cva and bronchial asthma. a uk study findings show that it is feasible to measure bronchial flux no concentration ( j no) and alveolar no concentration (c alv ) in 70% of children, with c alv levels potentially reflecting alveolar inflammation in asthma (paraskakis et al. 2006) . c alv and j no were measured from the fractional exhaled no (feno 50 ) at multiple exhalation flow rates in asthmatic children. although feno 50 and jno give essentially the same information, c alv is higher in asthmatic children than in normal children. this study also highlights the relationship between poor control of asthma and c alv (a biomarker of alveolar inflammation) but further work is needed to confirm the relevance of this. a novel nanosensor can detect a possible asthma attack before it begins. the minute sensor can be fitted into a hand-held device, and when a person blows into the device, it measures the no content of their breath. use of this device would provide asthma sufferers with a simple and cost effective way to monitor their asthma inflammation. an explanation for increased levels of exhaled no is nonenzymatic generation of no from nitrite due to airway acidification in asthmatics. reduced arginine availability may also contribute to lung injury by promoting formation of cytotoxic radicals such as peroxynitrite. as arginine levels decline, nitric oxide synthase (nos) itself can begin to generate superoxide in lieu of no, thereby favoring no consumption via the generation of peroxynitrite that could induce lung injury. this reduction in bioavailability of no via formation of species such as peroxynitrite could be further amplified by the rapid loss of sod activity during the asthmatic response. plasma arginase activity declines significantly with treatment and improvement of symptoms. additional studies are needed to determine whether measurements of plasma arginase activity will provide a useful biomarker for underlying metabolic disorder and efficacy of treatment for this disease. the arginase activity present in serum probably does not accurately reflect whole body arginase activity or that compartmentalized in the lungs, since the arginases are intracellular enzymes. because arginase is induced in monocytes in response to helper t cell type 2 cytokines, it is speculated that these cells are one likely source of the elevated arginase in serum, consistent with the localization of arginase expression within macrophages in the lungs. athough exhaled no is a clinically useful biomarker of eosinophilic airway inflammation in asthma, significant validation and investigation are required before exhaled breath condensate could be utilized for making decisions in clinical practice (simpson and wark 2008) . endothelins are proinflammatory, profibrotic, broncho-and vasoconstrictive peptides, which play an important role in the development of airway inflammation and remodeling in asthma. a study has evaluated the endothelin-1 (et-1) levels in exhaled breath condensate (ebc) of asthmatics with different degree in asthma severity (zietkowski et al. 2008) . et-1 concentrations in ebc of all asthmatic patients were significantly higher than in healthy volunteers. et-1 levels were significantly higher in patients with unstable asthma than in the two groups with stable disease. thus, measurements of et-1 in ebc may provide another useful diagnostic tool for detecting and monitoring inflammation in patients with asthma. the release of et-1 from bronchial epithelium through the influence of many inflammatory cells essential in asthma and interactions with other cytokines, may play an important role in increase of airway inflammation, which is observed after postexercise bronchoconstriction in asthmatic patients. ige plays a central role in the pathophysiology of asthma. the two essential phases in this pathophysiology are sensitization to allergen and clinical expression of symptoms on reexposure to the sensitizing allergen. omalizumab (xolair, genentech) is a recombinant humanized igg1 monoclonal anti-ige antibody that binds to circulating ige, regardless of allergen specificity, forming small, biologically inert ige-anti-ige complexes without activating the complement cascade. an 89-99% reduction in free serum ige (i.e., ige not bound to omalizumab) occurs soon after the administration of omalizumab, and low levels persist throughout treatment with appropriate doses. a total serum ige level should be measured in all patients who are being considered for treatment with omalizumab, because the dose of omalizumab is determined on the basis of the ige level and body weight. the dose is based on the estimated amount of the drug that is required to reduce circulating free ige levels to less than 10 iu per milliliter. lebrikizumab (roche) is an injectable humanized mab designed to block il-13, which contributes to key features of asthma. lebrikizumab improves lung function in adult asthma patients who are unable to control their disease on inhaled corticosteroids. il-13 induces bronchial epithelial cells to secrete periostin, a matricellular protein. increased levels of periostin, a biomarker of asthma, can be measured in the blood. in the milly phase ii trial, patients with high pretreatment periostin levels had greater improvement in lung function when treated with lebrikizumab, compared to patients with low periostin levels (corren et al. 2011) .the primary endpoint of the trial showed that at week 12, lebrikizumab-treated patients had a 5.5% greater increase in lung function from the baseline compared to placebo. lebrikizumabtreated patients in the high-periostin subgroup experienced an 8.2% relative increase from baseline forced expiratory volume in 1 second (fev1), compared with placebo. in the low-periostin subgroup, those patients on the drug experienced a 1.6% relative increase in fev1, compared with placebo. these results support further investigation of lebrikizumab as a personalized medicine for patients who suffer from moderate to severe uncontrolled asthma periostin enables selection of patients who will benefit most from the drug. cystic fibrosis (cf) is the most common serious genetic disease among caucasians in the us. the disease results from a defective gene that affects multiple aspects of cellular function. its most serious symptom is a build-up of thick, sticky mucus in the airways, which can lead to fatal lung infections. the usual method for screening and diagnosis is genotyping of cystic fibrosis transmembrane conductance regulator (cftr) gene mutations. antibody microarrays have been developed as a platform for identifying a cf-specific serum proteomic signature. serum samples from cf patients have been pooled and compared with equivalent pools of control sera in order to identify patterns of protein expression unique to cf. the set of significantly differentially expressed proteins is enriched in protein mediators of inflammation from the nfkappab signaling pathway, and in proteins that may be selectively expressed in cf-affected tissues such as lung and intestine. in several instances, the data from the antibody microarrays can be validated by quantitative analysis with reverse capture protein microarrays. in conclusion, antibody microarray technology is sensitive, quantitative, and robust, and can be useful as a proteomic platform to discriminate between sera from cf and control patients. saliva, because of the noninvasive collection process, shows great potential as a biological fluid for cf monitoring. extensive protein degradation and differentially expressed proteins have been identified in sputum as biomarkers of inflammation relating to pulmonary exacerbations of cf. use of fiber microarrays for measuring significant variations of the levels of six proteins in saliva supernatants -vegf, mmp-9, ip-10, il-8, il-1β and egf -as well as the correlations of these levels with clinical assessments, has demonstrated the value of saliva for cf research and monitoring (nie et al. 2015) . the prohosp study group. clinical risk scores and blood biomarkers as predictors of long-term outcome in patients with community-acquired pneumonia: a 6-year prospective follow-up study peripheral blood gene expression profiles in copd subjects biomarkers of extracellular matrix turnover are associated with emphysema and eosinophilic-bronchitis in copd increased expression of placenta growth factor in chronic obstructive pulmonary disease temporal changes in cytokine/chemokine profiles and pulmonary involvement in severe acute respiratory syndrome lebrikizumab treatment in adults with asthma kl-6: a serological biomarker for interstitial lung disease in patients with polymyositis and dermatomyositis exhaled nitric oxide levels in patients with atopic cough and cough variant asthma serum and bronchial lavage fluid concentrations of il-8, slpi, scd14 and sicam-1 in patients with copd and asthma association of biomarkers of inflammation and cell adhesion with lung function in the elderly: a population-based study brain natriuretic peptide is a prognostic parameter in chronic lung disease higher urine nitric oxide is associated with improved outcomes in patients with acute lung injury biomarkers of inflammation, coagulation and fibrinolysis predict mortality in acute lung injury plasma fibrinogen qualification as a drug development tool in chronic obstructive pulmonary disease. perspective of the chronic obstructive pulmonary disease biomarker qualification consortium correlations of salivary biomarkers with clinical assessments in patients with cystic fibrosis measurement of bronchial and alveolar nitric oxide production in normal children and children with asthma parathyroid hormone as a novel biomarker for chronic obstructive pulmonary disease: korean national health and nutrition examination survey high serum levels of tumour necrosis factor-α and interleukin-8 in severe asthma: markers of systemic inflammation? the role of exhaled nitric oxide and exhaled breath condensates in evaluating airway inflammation in asthma increased serum levels of chromogranin a in male smokers with airway obstruction systemic biomarkers of collagen and elastin turnover are associated with clinically relevant outcomes in copd pulmonary surfactant proteins a and d: innate immune functions and biomarkers for lung diseases ifn-gamma-induced protein 10 is a novel biomarker of rhinovirus-induced asthma exacerbations hyperuricemia is a biomarker of early mortality in patients with chronic obstructive pulmonary disease endothelin-1 in exhaled breath condensate of stable and unstable asthma patients key: cord-270184-bq5p2gs6 authors: alrubaiee, gamil ghaleb; al-qalah, talal ali hussein; al-aawar, mohammed sadeg a. title: knowledge, attitudes, anxiety, and preventive behaviours towards covid-19 among health care providers in yemen: an online cross-sectional survey date: 2020-10-13 journal: bmc public health doi: 10.1186/s12889-020-09644-y sha: doc_id: 270184 cord_uid: bq5p2gs6 background: the growing incidence of coronavirus (covid-19) continues to cause fear, anxiety, and panic amongst the community, especially for healthcare providers (hcps), as the most vulnerable group at risk of contracting this new sars-cov-2 infection. to protect and enhance the ability of hcps to perform their role in responding to covid-19, healthcare authorities must help to alleviate the level of stress and anxiety amongst hcps and the community. this will improve the knowledge, attitude and practice towards covid-19, especially for hcps. in addition, authorities need to comply in treating this virus by implementing control measures and other precautions. this study explores the knowledge, attitude, anxiety, and preventive behaviours among yemeni hcps towards covid-19. methods: a descriptive, web-based-cross-sectional study was conducted among 1231 yemeni hcps. the covid-19 related questionnaire was designed using google forms where the responses were coded and analysed using the statistical package for the social sciences software package (ibm spss), version 22.0. descriptive statistics and pearson’s correlation coefficient test were also employed in this study. a p-value of < 0.05 with a 95% confidence interval was considered as statistically significant. the data collection phase commenced on 22nd april 2020, at 6 pm and finished on 26th april 2020 at 11 am. results: the results indicated that from the 1231 hcps participating in this study, 61.6% were male, and 67% were aged between 20 and 30 years with a mean age of 29.29 ± 6.75. most (86%) held a bachelor’s degree or above having at least 10 years of work experience or less (88.1%). however, while 57.1% of the respondents obtained their information via social networks and news media, a further 60.0% had never attended lectures/discussions about covid-19. the results further revealed that the majority of respondents had adequate knowledge, optimistic attitude, moderate level of anxiety, and high-performance in preventive behaviours, 69.8, 85.10%, 51.0 and 87.70%, respectively, towards covid-19. conclusion: although the yemeni hcps exhibited an adequate level of knowledge, optimistic attitude, moderate level of anxiety, and high-performance in preventive behaviours toward covid-19, the results highlighted gaps, particularly in their knowledge and attitude towards covid-19. a cluster of pneumonia cases of unknown origin or causes was reported in wuhan, china, on 12th december 2019 [1] . among the initial 41 cases reported, most originated from vendors and dealers working in the huanan seafood market in wuhan [2] . the world health organisation (who) and the chinese authorities identified the causative agent as a new strain of coronavirus (sars-cov-2), named at that time as a coronavirus disease 2019, commonly referred to after that as covid-19 [3] . initially, sars-cov-2 quickly spread within china before dramatically spreading to other countries on a global scale [4] . on 11th march 2020, who declared the outbreak of covid-19 as a global pandemic [5] . since 12th september 2020, the virus has infected over 28,329,790 people, causing 911,877 deaths in 216 countries worldwide [6] . in yemen, the fight against covid-19 began on 10th april 2020 resulting from the initial case confirmed in ash shihr, the hadramout province, southern yemen. on 29th april 2020, five more cases of covid-19 were confirmed and registered in aden city, the temporary capital of yemen. after that, the cases started to increase in other cities daily. since 12th september 2020, 2011 cases of covid-19 have been reported in the republic of yemen, of which 1211 cases have since recovered, resulting in 583 deaths. however, the number of covid-19 cases is anticipated to be much higher than these figures, particularly given the transparency and the inability to effectively track and control the spread and number of cases reported in north yemen [6] . at present, the exact dynamics and transmission of the virus have not been determined. however, according to who, the virus can be transmitted via air-droplets and fomites during close and unprotected contact between an infected person and a healthy person [7] . according to the centre for disease control and prevention (cdc) sars-cov-2 is transmitted from person to person through close contact (within 6 ft); from an infected person via respiratory droplets during coughing or sneezing or when touching a surface or an object that is contaminated with the virus, including touching one's eyes, nose or mouth [8] . in most cases, those infected with covid-19 experience none or mild to moderate symptoms that are alleviated within several weeks of isolation. however, in contrast, it can cause severe respiratory syndrome or death, particularly in older people or patients with chronic health diseases [9] . similarly, healthcare providers (hcps) as the front line defence in treating patients with covid-19 are more susceptible to this spreading infection [10] . the who on 27th july 2020, estimated that close to 10% of all covid-19 cases globally, which accounts for nearly 1.5 million cases, were related to hcps. however, this figure is possibly underestimated, as, at that time, no systematic reporting or other measures were in place [11] . indeed, information released by the international council of nurses (icn), reported that until june 2020, nearly 230,000 hcps worldwide had acquired covid-19, with over 600 nurses dying [12] . in the context of yemen, at present, the ongoing war and civil unrest over the past six years within the country has severely impacted or destroyed the much of the country's infrastructure, with only 51% of the country's health facilities remaining in operation [13] . this consists of two testing centres and 500 ventilators for a population of nearly 30 million people. further, the country continues to suffer from limited testing capacity, critical shortage in health care supplies, including basic personal protective equipment (ppe) and other measures, limited by the ability to track the spread of the virus, especially, given the similarity covid-19 symptoms with other diseases that already prevail in the country [14] . all these factors place the country sadly in a unique if not, an uncompromising and dangerous position should covid-19 spread uncontrollably within the community, adding further burdening hcps' in the country. however, viewing this situation from a wider perspective, the rapid spread of covid-19 globally has caused considerable level anxiety, fear and panic among the population in countries worldwide, especially given that fact that hcps and the elderly are most vulnerable to the risk of infection [15] . according to who, the shortage of appropriate ppe and other preventive measures directly endangers hcps and represents a major cause of concern for countries [16] . likewise, the availability and correct use of ppe is critical in order to protect and safeguard frontline workers such as hcps in coping with though, what is of prime importance at this stage, is for hcps to adhere to applying these preventive measures, which largely depend on their knowledge, attitude, and practice in addressing this highly contagious virus [2] . nevertheless, yemeni hcps have been facing a double battle even before this pandemic eventuated given that yemen, according to who, is more than 50% below the basic health services global benchmark concerning the coverage of health care services. furthermore, while there are a limited number of skilled hcps in the country, they have not received salaries for nearly five years. surprisingly, the proportion of medics in yemen has been calculated as 10 medics to every 10,000 of the population, notwithstanding that the number of nurses and midwives that are available remains inadequate to fill this shortage. these issues are further compounded by the 'brain drain' in the country of people seeking better opportunities offshore and weakening medical health education [17] . therefore, to ensure the protection of hcps and safeguard yemen from covid-19, there is an urgent need to upskill and enhance the understanding and awareness of covid-19 among hcps. this study aims to assess the knowledge, attitude, fear, and anxiety, as well as the preventive behaviours of hcps towards covid-19. study area, study design, and study period a descriptive, web-based cross-sectional survey was conducted among yemeni hcps between 22nd april 2020, 6 pm and 26th april 2020, 11 am. all hcps who provided direct healthcare services to patients were invited to participate in the study. the questionnaire developed and used in this study was adapted from previously published studies based on the authors' permission [2, 18] . the questionnaire consisted of 58 items that sought to collect information on the respondents' knowledge, attitude, anxiety, and preventive behaviours toward covid-19. the questionnaire comprised of five parts. part (1) the socio-demographic characteristics such as age, sex, occupation, education level, years of working experience, and sources of covid-19 related knowledge. part (2) the respondents' knowledge (21-items). part (3) the respondents' attitude (10-items) and part (4) the respondents' anxiety (17-items). part (5) included questions on the respondents' preventive behaviours (10-items). scoring of knowledge, attitude, anxiety, and preventive behaviours the scoring system that was used in this study was adapted from the work of taghrir et al. [18] and roy et al. [2] . the 21-items related to knowledge were assessed with either a "yes" or "no" response in which each correct response was awarded a score of one (1), while a zero (0) score was assigned to an incorrect response. the scores ranged between 0 (no correct answers) and 21 (all answers are correct). a score of less than 11 was considered as having inadequate knowledge, and between 11 and 16, the scores were considered as having moderate knowledge, while a score of 17 and above was considered as having adequate knowledge. similarly, the 10-items signifying the respondents' attitudes were evaluated with a "correct" or "incorrect" response. the scores ranging between zero (0) and seven (7) were considered as acquiring a negative attitude, while the scores between eight (8) and ten (10) were considered as having a positive attitude. the 17-items related to anxiety were assessed via a 5-point likert scale, in which a score between 1 = "never" to 5 = "always". the total cumulative score ranged between 17 and 85. here, scores between 17 and 50 were considered as "low anxiety", and those scores ranging between 51 and 67 were considered as "moderate anxiety", while those ranging between 68 and 85 were considered as "high anxiety". the 10-items related to preventive behaviours were assessed with a "yes" or "no" response. a score between zero (0) and seven (7) was considered as "low performance", while a score between eight (8) and ten (10) was considered as "high-performance". three experts with a background in infectious disease and epidemiology (one specialist in infectious disease and two epidemiologists) were invited to participate in assessing the content validity of the questionnaire items. the reliability of the questionnaire items was based on a pilot study that included 40 participants, and the reliability was tested using a cronbach's alpha test with the results showing 0.79 for the knowledge part, 0.77 for the attitude part, 0.80 for the anxiety part, and 0.75 for the preventive behaviours part. at present, due to the outbreak of covid-19 and the specific preventive precautions and measures recommended by the ministry of health and population in yemen, an electronic web-based self-reported questionnaire was designed to comply with the recommendations. the internet link was distributed to the hcps via email, whatsapp, telegram, and other forms of social media. the criteria of the hcps to participated in the study needed to be living in the republic of yemen, regardless of gender, aged 20 years or older, was aware of the covid-19 outbreak, and who had signed a consent form to participate in the study. although participation in the study was voluntary, personal details of the participants were not recorded on the questionnaire. the respondents in receipt of the questionnaire were encouraged to forward the survey to other colleagues who may be interested in participating in the study as well. approval of the ethics committee of al-razi university was obtained before conducting the study. the respondents needed to confirm their willingness to participate on a voluntary basis by answering a "yes or no" question on a written informed consent form before being allowed to complete the online self-reporting questionnaire. the statistical package for social sciences (ibm spss), version 22.0 was used in the administration and analysis of the collected data. descriptive analyses using mean values and standard deviations for continuous variables and the count and percentages for the dichotomous or categorical variables were used in describing the data. the relationship between the study variables was assessed using pearson's correlation coefficient test. a pvalue of < 0.05 (two-tailed) with a 95% confidence interval was reported as significant for the correlation analysis. the respondents' socio-demographic data are presented in table 1 below. as shown in the table, over half (61.6%) of the hcps were male, with more than (67%) of respondents were aged between 20 and 30 years with a mean of 29.29 ± 6.75. regarding the occupation of respondents, 22.5% were physicians, followed by pharmacists (17.8%), laboratory technicians/workers (16.5%), and nurses (16.0%). regarding their education and working experience, 4.5% of respondents held a phd, 1.8% held a board position with 88.1% of all respondents having 10 years or less of working experience. concerning covid-19 related information sources, social media was highlighted as the main source (31.0%) followed by news media (26.1%). around 99.0% of respondents were aware of covid-19, with a further 60.0% having never attended lectures or discussions on covid-19. the level of knowledge among healthcare providers regarding the covid-19 pandemic is presented in fig. 1 below. twenty-one items within the questionnaire instrument having a "true" or "false" response choice was used to assess the extent of the respondents' knowledge regarding covid-19. as shown in fig. 1 , the majority of hcps (69.80%) were believed to have acquired an adequate level of knowledge regarding covid-19, while 29.70% had moderate knowledge, and only 0.60% were considered to have inadequate knowledge. the lower percentages were attributed to four (4) statements that discussed the importance of wearing face masks, the need to wear n95 face masks only during intubation, suction, bronchoscopy, and cardiopulmonary resuscitation, in treating the disease by usual antiviral drugs and antibiotics as the first-line (of defence) treatment, that scored 69.9, 68.8, 28.47, and 27.3%) respectively. the level of attitude of yemeni hcps towards the covid-19 pandemic is shown in fig. 2 below. the respondents' attitude towards the covid-19 pandemic was assessed using ten (10) items with a "yes" or "no" response choice. as shown in fig. 2 , the findings indicate that the majority of respondents (85.10%) had a positive attitude, while 14.90% had a negative attitude towards the covid-19 pandemic. however, although the vast majority of respondents exhibited a high degree of optimism and attitude towards the pandemic, 75.1% still believed that they would not contract the disease, and almost 29.4% were willing to move to other locations within the country to be safe and secure during the pandemic. the level of anxiety among yemeni hcps toward the covid-19 pandemic is illustrated in fig. 3 below. the level of anxiety among hcps was assessed using 17-items, with the answers rated against a 5-point likert ranging between 0 = "never" to 5 = "always". as shown in fig. 3 , the findings indicate that just of half of the respondents had a moderate level of anxiety towards the pandemic, 27.70% had a high level of anxiety, and 21.30% had a low level of anxiety towards the covid-19 pandemic. healthcare providers' self-reported preventive behaviours toward the covid-19 pandemic ten-items each requiring a "yes" or "no" response was used to assess the respondents' level of self-reported preventive behaviours towards covid-19. five (5) items were to avoid or reduce visiting public places in their daily life. one item was related to preventive behaviour such as coughing/sneezing, two items were related to hand washing and frequently disinfecting surface areas on a frequent basis, and one item was related to talking with family and friends about preventive measures associated with of covid-19. as can be seen in fig. 4 , the vast majority (87.70%) of respondents exhibited sufficient preventive behaviours, while only 12.30% demonstrated low preventive behaviours. the lowest score (84.8%) was related to cancelled or postponed activities and events such as eating out, sporting activities, and meeting with colleagues. association between the respondents' socio-demographic characteristics and their knowledge, attitude, anxiety, and preventive behaviours the association between the respondents' sociodemographic characteristics and their knowledge, attitude, anxiety, and preventive behaviours towards the covid-19 pandemic are reflected in table 2 below. as the correlation between hcps knowledge, attitude, anxiety, and preventive behaviour scores is shown in table 3 below. the correlations were divided into four (4) levels based on the following criteria: weak = 0-0.25, fair = 0.25-0.5, good = 0.5-0.75, and excellent = 0.75 or greater [19] . as shown in table 3 , there was a significant positive linear correlation between knowledge-attitude (r = 0.176, p < 0.001), knowledge-anxiety (r = 0.136, p < 0.001), knowledge-preventive behaviours (r = 0.320, p < 0.001), attitude-anxiety (r = 0.078, p < 0.006), attitude-preventive behaviours (r = 0.293, p < 0.001) and anxiety-preventive behaviours (r = 0.284, p < 0.001). accordingly, the results indicate the relationship between knowledge, attitude, anxiety, and preventive behaviours towards the covid-19 pandemic. since the first confirmed case announced in yemen on 10th april 2020, in ash shihr, (a port city in the hadhramaut province of southern yemen), rising fear and anxiety extended to other provinces from the possibility of contracting covid-19 and its outbreak. the hcps as the front line of defence and older people were the most vulnerable in contracting covid-19 that the majority of other people. during this time, there was also a critical shortage of ppe given the current conflict in the region, and civil unrest in the country [14] . equally important was the need during this period to understand the level of preparedness of hcps' in order to cope with the outbreak of covid-19 in the country. this fact motivated the need to undertake the current study aiming to explore the level of knowledge, attitude, anxiety, and preventive behaviours among hcps towards the outbreak of covid-19 in the country. the findings have shown that while the majority of respondents (60.0%) had never attended covid-19 training courses with respect to covid-19, most (69.80%) had acquired an adequate level of knowledge about the outbreak of the virus. on the other hand, the four (4) statements reflecting the importance of wearing face masks in the community, having to wear n95 face masks only during intubation, suction, bronchoscopy, and cardiopulmonary resuscitation, the possibility to treat the disease using antiviral drugs and antibiotics as first-line treatment scored the lowest at 69.9, 68.8 28.47 and 27.3%, respectively. this result possibly highlights the need to direct more attention toward developing educational courses and programmes related to covid-19. likewise, the adequate level of knowledge among the respondents could be attributed to their educational level since most (73.0%) of respondents held a bachelor's degree or higher, (i.e. a master's degree). accordingly, an educated professional group such as this could help to collect knowledge of covid-19 from a variety of 1% of hcps seemed to use social media and news media as the main source of information, which is a significant concern given the reliability of this information. this is because utilising such media can mislead hcps by spreading fabricated and unverified information. it is also worth highlighting that the respondents' level of knowledge was only statistically significantly different according to their age, occupation, and educational level. furthermore, these results are consistent with the results of a previous study [20] which reported that the level of knowledge towards covid-19 differs significantly across different age groups, educational levels, and levels of different professions. the results are also in line with the results of giao et al. [9] and saqlain et al. [21] regarding the difference in the level of respondents' anxiety based on their profession. concerning the level of respondents' attitude, it was found to differ based on the participants' occupations significantly. this corroborates with a study by giao et al. [9] , which reported a significant association between respondents' attitude and their occupation. however, in contrast, the result seems in differ from the results of saqlain et al. [21] and rahman and sathi [20] , who stated that a positive attitude toward covid-19 did not significantly vary nor differ across different occupations. equally, the results revealed that the respondents' level of anxiety was significantly different based on their gender and educational levels. these results support the findings reported by al-hanawi et al. [22] that respondents' level of worry or concern attributed to covid-19 differs significantly across gender and educational level. this result is also in line with previous studies [23, 24] carried out in china, indicating that females have higher levels of anxiety compared to males. similarly, the respondents' level of self-reported preventive behaviour significantly differed according to their gender, occupation, years of working experience, and educational level. these results are in agreement with the results by rahman and sathi [20] on the variation of respondents' preventive behaviour according to different age groups, al-hanawi et al. [25] regarding the gender of respondents, saqlain et al. [21] regarding the respondents' years of working experience and khasawneh et al. [26] about the respondents' educational level. with respect to the attitude of the respondents', the result showed that 85.10% of respondents had an optimistic attitude towards covid-19, though unfortunately, the findings also revealed that 75.1% believe that they avoid infection, and close to 29.4% of respondents were willing to relocate to protect themselves from covid-19. this result suggests that most of the respondents were either confident of protecting themself from the virus or unaware about the nature of covid-19 how contagious it is. similarly, one-third of respondents would look to leave their work and relocate for fear of infection, which contributed to the shortage of hcps if the situation was to become more serious, i.e. rising infections. accordingly, based on the results and the information presented above, it is imperative given the seriousness of the issue that training courses and awareness programmes be created on covid-19 and disseminating such information via official websites. regarding the high level of optimism and attitude of respondents in the current study, this could also be explained, at this stage, by the limited number of cases reported in yemen, and the adequate level of knowledge they had gained since the outbreak of the virus, and until this research study was conducted. according to roy et al. [2] , adequate awareness often leads to optimistic attitudes, which could positively affect the preparedness of hcps to address pandemic issues. furthermore, the results of the current study showed a positive correlation between the respondents' knowledge and their attitude, which could support this conjecture. moreover, the findings of the current study are consistent with a study by giao et al. [9] , that healthcare workers had a high level of knowledge and a positive attitude towards covid-19. these findings are also in line with the results of a cross-sectional study conducted among saudi health college students [27] , which revealed that more than half of the students had a positive attitude towards mers-cov. concerning the respondents' level of anxiety, the results indicated that nearly half (51%) of the respondents had a moderate level of anxiety and 27.70% had a high level of anxiety regarding the covid-19 outbreak. according to roy et al. [2] , fear and anxiety within a population are usually expected given the significant impact of the pandemic on the community, which could also affect the mental well-being of people and influence their behaviour in the wider community. in this study, only 27.7% of the respondents exhibited a high level of anxiety concerning covid-19, which could possibly be attributed to their level of knowledge given they were still experiencing the first wave of the virus covid-19. interesting, the current study indicated lower anxiety level results compared to other studies that were carried out during the outbreak as reported by huang and zhao [28] on chinese healthcare workers and nemati et al. [29] on iranian nurses. in these studies, the results showed that the level of anxiety among healthcare workers was higher compared to other people. the high anxiety level among the hcps could be attributed to the uncontrolled nature of the pandemic and concerns of becoming infected, particularly given the shortage of healthcare institutions and ppe. concerning the self-reported preventive behaviours, it was found that the majority (87.70%) of respondents had a high-performance level of preventive behaviours towards covid-19, which could be attributed to the having an adequate level of knowledge and awareness among the respondents towards covid-19. as reported in a previous study, those who had acquired adequate knowledge exhibited optimistic attitudes and appropriate, it not proactive practices toward covid-19 [30] . another study revealed that the level of good or sound knowledge in a given population about covid-19 is significantly reflected in their behaviour and attitude [2] . however, the findings from the current study were seemingly lower than a study conducted during covid-19 by taghrir et al. [18] on medical students in iran finding that 94.2% of the respondents showed relatively high-performance in preventive behaviours toward covid-19. according to the results of this study, females were found to exhibit a higher-performance-level in preventive behaviours compared to their male counterparts, possibly due to their better compliance in preventive measures towards covid-19. this result is consistent with the result by taghrir et al. [18] that females demonstrated more precautionary behaviours compared to males. notwithstanding, another key result in this study was of the positive linear correlation between knowledge-attitude, knowledge-anxiety, knowledgepreventive behaviours, attitude-anxiety, attitudepreventive behaviours, and anxiety-preventive behaviours. this result confirms the relationship between the respondents' level of knowledge and their level of anxiety, attitude, and preventive measures towards covid-19. such a correlation could be explained by the theory of reasoned action (tra) [31] , which states that a person's intention to carry out a specific behaviour is determined by their attitude towards this behaviour. in the current study, the findings are in line with the results of other studies [20, 30, 32] showing that acquiring a good level of knowledge of covid-19 is correlated with optimistic attitudes and proper practices towards covid-19. however, in contrast, the results of this study disagree with the results by nemati et al. [29] in which they found that most iranian nurses displayed their anxiety and that of their families as a result of covid-19 though the knowledge they had acquired about covid-19 to be sufficient. lin et al. [24] found that the level of knowledge of covid-19 did not influence anxiety levels. however, they found that higher levels of attitude were highly associated with high levels of anxiety. furthermore, in a study carried out in hong kong by leung et al. [33] , the results revealed that the level of anxiety during the sars outbreak was highly associated with behavioural responses such as wearing face masks. in a separate study by roy et al. [2] , they revealed that people's level of anxiety correlated with their behaviour. the results showed that under the effect of rumours, people tend to modify their behaviour positively compared to an undesirable one. reuben et al. [20] also reported the relationship between respondents' attitudes and their preventive behaviours. regarding the relationship between the respondents' attitudes and their preventive behaviour, rubin et al. [34] conducted a study during the swine flu outbreak, reporting a significant association between the respondents' attitude and their behavioural change (e.g. performing one or more avoidance behaviours). nevertheless, several limitations were inherent in this study which should be addressed for future research. the first limitation concerns the nature of collecting the data. the data in this study were collected via a webbased survey since it was not possible to conduct a faceto-face survey among yemeni hcps during given the uncertainty surrounding the outbreak of the virus and level of contagious. therefore, the data may be seen as being less reliable having less accountability compared to face-to-face interviews and the lack of a trained interviewer. secondly, collecting the data was challenging, given the availability of respondents and cooperation. thirdly, the exclusiveness of the study to hcps. therefore, future research should involve a more diverse community or population, employing a community-based study design. the results of this study have demonstrated that the majority of hcps in yemen had acquired an adequate level of knowledge of covid-19. however, their level of knowledge concerning situations that require wearing n95 masks and the possibility of using current antiviral drugs and antibiotics as the first-line of treatment for covid-19 could be improved through training and other programmes. the moderate anxiety level, as revealed in this study, would undoubtedly increase, particularly if the prevalence curve of the outbreak of covid-19 elevated, and the situation became much worse. therefore, implementing preventive measures and regulation strategies to control the emotional status among hcps is recommended. in addition, organisations such as who and the ministry of public health and population in yemen must continue to provide updated information regarding covid-19 to warrant better control concerning covid-19. a novel coronavirus from patients 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public in china attitude and practice toward covid-19 among the public in the kingdom of saudi arabia: a cross-sectional study medical students and covid-19: knowledge, attitudes, and precautionary measures. a descriptive study from jordan. front public health knowledge and attitude toward middle east respiratory syndrome coronavirus among heath colleges' students in najran, saudi arabia generalised anxiety disorder, depressive symptoms, and sleep quality during covid-19 epidemic in china: a web-based crosssectional survey. medrxiv preprint assessment of iranian nurses' knowledge and anxiety toward covid-19 during the current outbreak in iran knowledge, attitudes, and practices towards covid-19 among chinese residents during the rapid rise period of the covid-19 outbreak: a quick online crosssectional survey understanding and promoting aids-preventive behavior: insights from the theory of reasoned action attitudes and practices towards covid-19: an epidemiological survey in north-central nigeria longitudinal assessment of community psychobehavioral responses during and after the 2003 outbreak of severe acute respiratory syndrome in hong kong public perceptions, anxiety, and behaviour change in relation to the swine flu outbreak: cross sectional telephone survey publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank all the healthcare providers who agreed to participate in this study and for their support in distributing the link to the questionnaire to other colleagues to participate.authors' contributions gga, taha, and msaa were involved in the inception of the idea and study design. taha and msaa were responsible for data collection. gga supervised, and taha performed the data analysis. gga drafted and finalised the manuscript. all the authors contributed to the interpretation of the data, reviewing, and drafting the manuscript, and approving the final manuscript. this study did not receive any form of grants or financial support. data are available from the corresponding author on a reasonable request. this study obtained ethical approval from the ethics committee for research of al-razi university. the participants provided their consent to participate voluntarily through answering a "yes or no" question in the online written informed consent form before they were allowed to complete the questionnaire. not applicable. the authors declare they have no competing interests. key: cord-285151-zynor0b2 authors: eisenhut, michael title: neopterin in diagnosis and monitoring of infectious diseases date: 2013-12-08 journal: j biomark doi: 10.1155/2013/196432 sha: doc_id: 285151 cord_uid: zynor0b2 neopterin is produced by activated monocytes, macrophages, and dendritic cells upon stimulation by interferon gamma produced by t-lymphocytes. quantification of neopterin in body fluids has been achieved by standard high-performance liquid chromatography, radioimmunoassays, and enzyme-linked immunosorbent assays. neopterin levels predict hiv-related mortality more efficiently than clinical manifestations. successful highly active antiretroviral therapy is associated with a decrease in neopterin levels. elevated neopterin levels were associated with hepatitis by hepatitis a, b, and c viruses. serum neopterin levels were found to be a predictor of response to treatment of chronic hcv infection with pegylated interferon combined with ribavirin. neopterin levels of patients with pulmonary tuberculosis were found to be higher in patients with more extensive radiological changes. elimination of blood donors with elevated neopterin levels to reduce risk of transmission of infections with known and unknown viral pathogens has been undertaken. neopterin measurement is hereby more cost effective but less sensitive than screening using polymerase chain reaction based assays. in conclusion neopterin is a nonspecific marker of activated t-helper cell 1 dominated immune response. it may be a useful marker for monitoring of infectious disease activity during treatment and for more accurate estimation of extent of disease and prognosis. neopterin was first isolated from larvae of bees, in worker bees and in royal jelly in 1963, and subsequently from human urine by sakurai and goto in 1967 [1] . neopterin or 2-amino-4-hydroxy-6-(d-erythro-1 ,2 ,3trihydroxypropyl)-pteridine is produced from guanosine triphosphate via guanosine triphosphate cyclohydrolase i (gtpch i) by activated monocytes, macrophages, dendritic cells, and endothelial cells and to a lesser extent in renal epithelial cells, fibroblasts, and vascular smooth muscle cells upon stimulation mainly by interferon gamma and to a lesser extent by interferon alpha and beta with its release being enhanced by tumor necrosis factor [2, 3] . gtpch i mrna expression is synergistically and independently induced by interferon gamma through the jak2/stat pathway of nuclear transcription regulation and through tnf by the nf-kappab pathway (see figure 1 ) [4] . release in response to cytokines released by t-lymphocytes and natural killer cells make neopterin an indicator of activation of cell mediated immunity including release by infections associated with activation of t-lymphocytes and natural killer cells, malignancies, autoimmune diseases, rejection of transplanted organs, and atherosclerosis. at its first isolation in the 1960s neopterin was detected in the pupae of bees by anion exchange chromatography followed by paper chromatography [1] . in the seventies gas chromatographic-massfragmentographic methods were described allowing measurement in urine. subsequently detection and quantification of neopterin succeeded in serum, urine, and other body fluids using standard high pressure and by reverse-phase high-performance liquid chromatography with fluorescence detection. later simpler radioimmunoassays and more recently enzymelinked immunosorbent assays have been developed which are suitable for large numbers of samples [1] . semiquantitative measurement with a dipstick system using polyclonal antineopterin antibodies has been validated and may be suitable for bedside testing and in the setting of developing countries [5] . levels have been observed, which correlate with the activity of disease. this was first described in 1979 [6] and subsequently neopterin elevations were noted in infections with hepatitis viruses, epstein-barr, cytomegalo, measles, mumps, varicella zoster, rubella, and influenza viruses [1, [7] [8] [9] [10] [11] . elevated neopterin levels in body fluids were found at the end of the incubation period before the onset of clinical symptoms. the highest neopterin levels occur just before specific antibodies against the virus become detectable, which is about two to four weeks after onset of increased neopterin production. in acute varicella zoster virus infection peak neopterin levels were observed at the end of the appearance of the rash and in measles virus infection one to three days after appearance of the rash [12, 13] . immunisation with live viruses, for example, measles, mumps and rubella and virus vaccine, resulted in a significant increase of neopterin independent of presence of any symptoms. in measles vaccination neopterin levels were observed to rise at a median of 5 days after vaccination about 7 days before the appearance of antibodies [13] . these investigations point to a future application of measurements neopterin as a correlate of a successful vaccination. neopterin should be investigated as a marker to evaluate protective efficacy of vaccines stimulating cell mediated immunity against mycobacterial, parasitic, or viral diseases. the magnitude of the elicited neopterin levels could be put into relationship to incidence of the disease immunised against the population of immunised children. serum neopterin levels were also found to be significantly elevated in symptomatic dengue virus infections with levels higher than in measles and influenza virus disease [14] . levels correlated with duration of fever and severity of disease [14, 15] . investigations into the physiological functions of neopterin in viral infections revealed that it is able to delay the development of the cytopathic effect of coxsackie b5 virus in hep-2 cells [16] . a proposed mechanism is the stimulation of inducible nitric oxide synthase expression leading to an increase in nitric oxide production. other mechanisms include the induction of the translocation of the nuclear factor-kappa b to the nucleus. infection. testing of 328 samples of 29 hiv infected individuals found that 44/68 (64.7%) of samples, which were hiv-1 rna and p24 antigen positive had elevated neopterin levels (>10 nmol/l). 6/216 (2.8%) samples, which were both hiv-1 and p24 antigen negative had elevated neopterin levels [17] . neopterin levels were also found to be significantly elevated in hiv-2 infection compared to controls [18] . studies investigated markers of immune activation for their usefulness as prognostic markers in hiv infection and showed an increase of neopterin levels in people with hiv infection compared to patients without hiv infection [19] [20] [21] [22] . neopterin levels hereby were found to increase early in the course of hiv infection preceeding cd4+-t-cell decline and clinical manifestations of aids [23, 24] . plasma neopterin levels were found to correlate with plasma hiv viral load [25] . neopterin levels were found to predict hiv related mortality [26, 27] . a retrospective study compared 2 microglobulin, immunoglobulin a, g, and m, adenosine deaminase, and neopterin levels above normal range as predictors of clinical or immunological deterioration in 256 patients with hiv infection. changes in 2 microglobulin levels showed the greatest sensitivity to detect worsening (43%) with neopterin slightly less sensitive (41.9%) followed by immunoglobulin levels (26.8-35 .2%) and adenosine deaminase levels with 21.8% having the lowest sensitivity [28] . marker for viral load to monitor response to antiretroviral treatment. in a land mark study the effects of dual reverse transcriptase inhibitor (rt) therapy and highly active antiretroviral therapy (haart) on neopterin levels in patients with hiv infection were compared to hiv uninfected controls, hiv infected patients not on treatment, and patients who had stopped treatment [22] . rt inhibitor treatment decreased circulating levels of neopterin (mean of 15.6 for treated versus a mean of 22.3 ng/ml for untreated hiv patients, < 0.04). haart decreased neopterin levels significantly further. this confirmed results of a previous study on the effects of haart on neopterin levels [29] . neopterin levels in patients who discontinued haart became similar to untreated hiv patients. neopterin may be a particularly useful surrogate marker for monitoring of control of hiv replication in settings in developing countries where hiv rna viral load measurement is not available and may be a cheaper alternative particularly if semiquantitative dip stick tests are used for urine samples [5] . longitudinal serial measurements in the same individual could overcome difficulties with interpretation in settings where chronic parasitic (malaria) or bacterial schistosoma mansoni praziquantel blood serum levels normalized on treatment [43, 44] (tuberculosis) infections may elevate the baseline neopterin level and could allow monitoring of response to antiretroviral treatment in the absence of resistance testing and provide means to monitor compliance in the outpatient setting (see table 1 for list of diseases in which neopterin levels have been used to monitor treatment response). hepatitis. the first study investigating the role of neopterin in specific forms of viral hepatitis tested urinary levels in patients with hepatitis a, hepatitis b, and non-a, non-b hepatitis virus infection [9] . the authors noted that in 51 patients with acute viral hepatitis 49 patients had elevated urinary neopterin levels with the highest levels found in patients with acute hepatitis a. while all patients with active hepatitis b had elevated neopterin levels, 49/62 hbsag carriers (77%) had normal urinary neopterin levels. the authors noted that neopterin levels were not a reflection of hepatocellular damage as 3 patients with alcoholic hepatitis had normal urinary neopterin levels. in order to address the question whether neopterin is a useful marker for early detection of viral infection in donated blood products before seroconversion, one study investigated neopterin levels in anti-hcv-negative specimens, which were hcv rna and hcv core antigen positive. the investigators found that 8/217 (3.7%) had elevated neopterin levels (>10 nmol/l). 4/115 (3.5%) specimens positive for hbv dna had elevated neopterin levels [17] . in 106 patients with thalassemia major receiving multiple blood transfusion significantly more patients with histologically proven chronic hepatitis (19/21 were anti hcv antibody positive) had elevated blood neopterin levels compared to patients with siderosis of the liver [45] . alanine aminotransferase levels in hcv infected persons correlated significantly with neopterin levels. serum neopterin levels were found to be a useful predictor of response to treatment of chronic hcv infection with pegylated interferon combined with ribavirin. neopterin concentrations were evaluated in 260 hcv patients treated by pegylated interferon combined with ribavirin. mean and median pretreatment neopterin concentrations were lower in patients with sustained virological response than in nonresponders. the rate of response was twofold higher among patients with pretreatment neopterin levels <16 nmol/l than in patients with neopterin levels ≥16 nmol/l, even after controlling for hcv genotype status [38] . a recent study investigated specifically whether serum neopterin levels can discriminate between patients with replicative ( = 30) and nonreplicative ( = 25) hbv carriage [46] . replicative hbv carriage was defined as hbv dna >5 pg/ml by hybrid capture system. neopterin levels had a mean of 14.5 nmol/l in replicative versus 8.8 nmol/l in nonreplicative hbv carriers ( < 0.05). this result was not reproducible in another study, which found that in patients with replicative hbv infection ( = 30) mean serum neopterin level was 24.73 nmol/l and in nonreplicative hbv ( = 30) 14.8 nmol/l a difference, which was not statistically significant [47] . this may have been due to large standard deviations and small numbers in groups. a more recent investigation found that in chronic hepatitis the mean ± sd serum neopterin levels were 14.2 ± 5.6 nmol/l, 20.3 ± 7.9 nmol/l in patients with liver cirrhosis and 5.2 ± 1.4 nmol/l in the control group. serum neopterin levels were significantly higher in patients with chronic hepatitis ( = 0.005) and cirrhosis patients ( = 0.008) than in control subjects. cirrhotic patients had significantly higher serum neopterin levels than patients with chronic hepatitis ( = 0.004). there was a positive correlation between serum neopterin levels and alanine aminotransferase levels in patients with chronic hepatitis ( = 0.41, = 0.004) and cirrhotic patients ( = 0.39, = 0.005). positive correlations were detected between serum neopterin levels and inflammatory score in patients with chronic hepatitis ( = 0.51, = 0.003) and cirrhotic patients ( = 0.49, = 0.001) [48] . infections. neopterin has been investigated as a marker to distinguish viral from bacterial lower respiratory tract infections. the investigators found that serum neopterin levels were elevated (>10 nmol/l) in 96% of patients with viral lrti. the median serum neopterin concentration was almost 2-fold higher in the viral lrti group than bacterial lrti patients (30.5 versus 18.7 nmol/l) and 5-fold higher than those in healthy controls. the specificity for correct identification of viral lrti was 69.5% for a cut-off of >15 nmol/l [49] . serial monitoring of serum neopterin levels in patients with severe acute respiratory syndrome (sars) associated virus revealed that all ( = 129) investigated patients had elevated neopterin levels by day 9 [50] . duration of pyrexia in sars patients correlated positively with neopterin levels. patients on steroid therapy had significantly lower neopterin levels. measurement of neopterin in isolation and in relationship to other inflammatory markers like procalcitonin and c-reactive protein were investigated for discriminatory power between viral and bacterial lower respiratory tract infections. investigators used the crp/neopterin ratio (c/n ratio) to discriminate viral and bacterial etiology of respiratory tract infections. in a study conducted in hong kong sera obtained on the day of hospitalization for lrti from 139 patients with confirmed bacterial etiology and 128 patients with viral etiology were examined. a further 146 sera from healthy chinese subjects with no infection were included as controls. the area under the receiver operating characteristic (roc) curve (area under curve [auc]) for distinguishing bacterial from viral infections was 0.838 for crp and 0.770 for pct. the auc for distinguishing viral from bacterial infections was 0.832 for neopterin. when the markers were used in combination, auc of roc of the c/n ratio was 0.857, whereas (crp × pct)/neopterin was 0.856 [49] . in a subsequently reported study the median of the c/n ratio was 10 times higher in patients with bacterial aetiology than with viral aetiology (12.5 versus 1.2 mg/nmol; < 0.0001) and 42 times higher than those in healthy subjects (12.5 versus 0.3 mg/nmol; < 0.0001). the area under the receiver operator characteristic curve for the c/n ratio was 0.840. a cut-off value of "c/n ratio >3" for ruling in/out bacterial/viral infection yielded optimal sensitivity and specificity of 79.5% and 81.5%, respectively [51] . early studies showed elevated neopterin levels in cerebrospinal fluid (csf) of patients with aseptic meningitis and herpes simplex and measles virus encephalomyelitis [52] [53] [54] . csf levels of neopterin seem to reflect intrathecal production by microglia as pterins have a low permeability across the blood brain barrier with a serum-to-csf distribution at a quotient of 1/40 [55] . it has recently been established that normal csf neopterin is brain-derived. the interindividual variation of csf neopterin in healthy adults was found not to depend on serum neopterin concentration variation (coefficient of variation, cv-csf = 9.7% < cv-serum = 24.5%). additionally individual normal csf neopterin concentrations were found to be invariant to the variation of the albumin quotient, qalb; that is, csf neopterin does not derive from leptomeninges [56] . patients with viral meningitis had elevated csf neopterin levels compared to healthy controls but normal serum levels [54] . csf neopterin levels correlated hereby with csf monocytic cell count. patients with various forms of encephalitis including those caused by herpes simplex virus, varicella zoster virus, and tick borne encephalitis virus had significantly elevated csf neopterin levels compared to controls and higher levels than in patients with viral meningitis without overlap of levels in the two conditions. in hiv infection there was a clear relationship between the severity of aids-related dementia and csf neopterin levels [12, [30] [31] [32] . higher csf hiv viral loads were associated with higher csf neopterin levels [33] . after commencement of combination antiretroviral therapy (art), csf neopterin decreased markedly but remained slightly above normal levels in a substantial number of patients despite several years of receiving art [32, [34] [35] [36] . even patients with systemic virological failure exhibit a substantial reduction of csf neopterin concentrations, though above that of virologically suppressed patients [37] . in patients on combination art, the lowest csf neopterin levels have been found in patients with the lowest csf viral loads (<2.5 copies/ml) [32] . no significant difference in csf neopterin concentrations was found between those treated with protease inhibitor-and nonnucleoside reverse transcriptase based regimens in combination with 2 nucleoside analogues [57] . this would support the idea that viral replication within or close to the csf, at least to some extent, is partly driving the inflammatory response. it has also been suggested that an inflammatory response, once triggered, may lead to a self-sustaining state of cellular activation as has been seen in patients with herpes simplex virus type-1 encephalitis [58] . findings in this study are consistent with these reports. hiv rna levels measured in csf or plasma were not significantly associated with csf neopterin trajectories. in addition, all study participants had experienced virologic control to the limit of standard detection as a result of their treatment and csf neopterin levels were the only factor strongly associated with subsequent decay rates and the ultimate set-point levels [32] . patients with bacterial infections with species other than mycobacteria showed significantly lower urinary neopterin levels compared to patients with viral infections in one study [59] but no statistically significant difference in a more recent study [60] . within the group of bacterial infections it was shown that patients with symptoms for at least 5 days had significantly higher neopterin concentrations than patients with acute illness. this applied particularly to bacterial pneumonia. patients with urinary tract infections were found to have similar levels to patients with viral infections with data on urinary neopterin concentrations but not serum concentrations. thus it remains unclear whether local production of neopterin takes place in urinary tract infections and serum neopterin would stay low. there was no significant difference in neopterin levels between patients with febrile neutropenia and underlying haematological and oncological conditions and gram-negative versus gram-positive infections [61] . in patients on an intensive care unit with sepsis journal of biomarkers 5 and septic shock urinary neopterin/creatinine ratios were found to be significantly higher compared to patients with other forms of systemic inflammatory responses syndromes [62] and serum neopterin levels were higher in nonsurvivors compared to survivors of sepsis and multiorgan failure scores correlated with neopterin levels [63] [64] [65] [66] . in this context it was however noted that neopterin levels correlated negatively with reduced renal function reflecting renal failure causing a reduced excretion of neopterin. future studies could correct for reduced excretion due to reduced renal function by calculation of the serum neopterin/creatinine ratio. investigations on critically ill patients on intensive care units evaluated neopterin levels as tool to discriminate patients with systemic inflammatory response syndrome with and without infectious etiology. neopterin levels were found to have a specificity of 78% for discriminating infectious and noninfectious etiology of critical illness [66] . bacterial meningitis was associated with both elevated serum and csf neopterin levels compared to controls [55] . in lyme neuroborreliosis-a late complication of infection by the tick-born spirochete borrelia burgdorferi-high neopterin concentrations were found in csf of patients, whereas serum neopterin levels were not markedly increased, confirming intrathecal neopterin production [67] . infection with treponema pallidum subsp. pallidum (syphilis) was not associated with elevated neopterin levels [18] . in melioidosis by pseudomonas pseudomallei neopterin concentrations were found to be significantly higher than controls [12] . in brucellosis neopterin levels were with a mean 52.5 mmol/ml significantly higher than healthy controls and patients with tuberculosis [68] . in leprosy caused by mycobacterium leprae 75% of patients with tuberculoid and lepromatous leprosy presented with elevated urinary neopterin excretion [69] . on the basis of in vitro and in vivo data showing that macrophages release neopterin in response to stimulation by t-lymphocytes [70, 71] fuchs et al. [39] investigated urinary neopterin levels by hplc in 55 patients with culture confirmed pulmonary tuberculosis and compared them with 417 normal controls. 83% of patients had levels above the upper tolerance limit (containing with 95% probability 97.5% of healthy controls). neopterin levels were higher than age and gender matched controls for every extent of pulmonary disease and correlated with its extent. the correlation with extent of pulmonary tuberculosis was also demonstrated for serum levels and levels found in bronchioalveolar lavage fluid [72] . subsequent studies showed higher levels of neopterin in serum and pleural effusions of patients with pulmonary tuberculosis compared to controls [73, 74] . peripheral blood mononuclear cells (pbmnc) from tuberculosis patients showed a significantly higher spontaneous production of neopterin. stimulation with phytohaemagglutinin or purified protein derivative did not yield higher neopterin production in pbmnc of patients with tuberculosis showing that it is not the production capacity for neopterin which is different [74] . elevated serum neopterin levels were also found in hiv infected patients with tuberculosis and decreased significantly on antituberculous treatment [40] . a relapse of tuberculosis was in 2 cases characterized by increase in neopterin levels. further studies compared neopterin levels in urine, serum, and bronchoalveolar lavage fluid and found that they correlate significantly in patients with tuberculosis [72, 75] . the elevation of neopterin in patients with tuberculosis was more pronounced in urine than in serum or bronchoalveolar lavage [75] . diseases. patients with pulmonary tuberculosis had significantly higher urinary neopterin levels compared to patients with lung cancer or pneumonia with more than twice the concentration reported in adults [74] . pleural fluid neopterin levels were investigated for its ability to differentiate between tuberculous and malignant pleural effusion and were found to be significantly higher in patients with pleural tuberculosis but performance characteristics including receiver-operating characteristics curve analysis was inferior to adenosine deaminase [76] . waiting for the results of susceptibility tests to select an effective antituberculosis drug regimen often causes a delay in effective treatment which can be disastrous, especially in children. because the x-ray changes tend to resolve very slowly and may get even worse after starting therapy because of a paradoxical reaction due to immune-reconstitution even in otherwise immune-competent patients, other than clinical status there is no reliable parameter to reflect success or failure of the drug regimen [77] . urinary neopterin levels declined on twice weekly measurements in all monitored patients with pulmonary tuberculosis on treatment and fell to below tolerance limits within 10 weeks of treatment in 6/10 patients [39] . measurement of serum neopterin levels in patients on treatment for microbiologically confirmed pulmonary tuberculosis confirmed this observation and showed a significant decline of levels to near normal levels within 6 months of treatment [41] . in the context of emerging multiple drug resistance and difficulties in monitoring compliance and drug absorption neopterin needs to be explored as a tool for monitoring of success in treatment of mycobacterium tuberculosis infection. it may also help to distinguish active from latent disease. people with hiv-m. tuberculosis coinfection with active tuberculosis responded with a reduction of plasma neopterin to antituberculotic treatment but neopterin levels remained above the baseline levels of hiv negative tuberculosis patients and levels were higher in patients with lower cd4 count [78] . the first study of neopterin levels in parasitic infections included measurements of urinary neopterin by hplc in patients with plasmodium falciparum and vivax infections including patients with low grade parasitemia [7] . all patients had elevated urinary neopterin levels compared to uninfected controls to a level of 664 to 5189 micromol neopterin/mol creatinine. levels in patients treated with quinine sulphate and levels in untreated patients were not significantly different. a subsequent detailed interventional study provided data on urinary neopterin levels in volunteers experimentally infected with plasmodium falciparum [79] . serial monitoring revealed that urinary neopterin levels were not elevated until peripheral blood parasite densities had increased through 3 to 4 cycles of intraerythrocytic schizogony. a sharp rise in urinary neopterin was detectable at the beginning of day 14 after infection. there was an increase one day after onset of fever. in one patient a urinary neopterin increase was noted without the occurrence of fever. neopterin production in falciparum malaria seems to be a direct effect of plasmodial antigens on monocytes/macrophages. in vitro studies showed that the monocytic cell line u937 could be stimulated to produce neopterin with lysates of plasmodium falciparum parasitized human erythrocytes and recombinant p. falciparum proteins [80] . at a cut-off point of 10.0 ng/ml, neopterin had a positive and negative predictive value of 0.38 and 0.98 for detection of severe falciparum malaria [81] . chloroquine treatment was followed by a reduction of urinary neopterin levels. when clinical disease resolved within 3-7 days of treatment, neopterin levels normalized rapidly [42] . neopterin levels in nonimmune patients and young children were higher than were those of semiimmune individuals. csf (csf) neopterin levels were investigated for ability to discriminate between different stages of cerebral trypanosoma (t.) brucei (b.) gambiense infection. in an investigation of 512 t. b. gambiense patients originating from angola, chad, and the democratic republic of the congo csf igm and neopterin were the best in discriminating between the two stages (s1 and s2) of disease with 86.4% and 84.1% specificity, respectively, at 100% sensitivity. when a validation cohort (412 patients) was tested, neopterin (14.3 nmol/l) correctly classified 88% of s1 and s2 patients, confirming its high staging power [82] . serum neopterin was also assessed as a disease marker in human schistosoma mansoni infection and levels were found to reflect the extent of hepatic involvement with higher levels found in patients with hepatomegaly. treatment with praziquantel led to a normalisation of serum neopterin levels as a result of a reduction of egg induced immunopathology [43, 44, 83] . the detection of new blood borne viruses including hiv and non-a non-b hepatitis viruses led to investigations into new ways of excluding transmission of blood borne viruses by transfusion of blood products. the government of tirol in austria introduced routine measurement of neopterin levels in all donated blood in 1986. a cut-off of 10 nmol/l was used and led to the exclusion of 1.6% of donors (total number of donors = 76587). the most common cause of elevated levels was in 123 (67%) of cases a viral respiratory tract infection. 6 donors with elevated neopterin had an acute toxoplasmosis and 4 had hiv infection or non-a-non b-hepatitis [84] . in another study 5.26% of 1767 donations with increased neopterin levels were positive for cmv igm indicating acute infection. 0.3% of patients with low neopterin levels had cmv igm. seroconversion was detected in 10 patients with initially elevated neopterin levels on a second serum sample indicating that neopterin may precede the appearance of cmv antibodies by 2-4 weeks [85] . a further study of austrian blood donors showed that neopterin levels were significantly higher in early compared to late infection or carrier state. all early infections (seroconversions) had elevated neopterin levels while only 17% of late and carrier states [86] . a recent study found that using a neopterin elisa 61% of cmv dna-positive samples had elevated neopterin levels [87] . with regard to other viruses 5.5% of donors with above normal neopterin had epstein-barr virus igm generating an almost threefold greater chance of acute ebv infection in donors with increased neopterin (odds ratio: 2.85 (95% confidence interval, 1.5-5.6). with regard to parvovirus b19 infection 73/1060 (6.9%) donors were found to be seropositive for parvovirus b19 igm [88] . a later study by the same group found no hpv dna positive results amongst 1200 patients with normal neopterin levels [89] . an investigation of the association of neopterin levels with chronic hepatitis c virus infection revealed that significantly more patients with elevated neopterin levels and hcv antibodies were hcv pcr positive for hcv rna (odds ratio: 3.76 = 0.002) [90] . neopterin is a nonspecific marker of activated cell mediated immunity involving release of interferon gamma. neopterin may be a useful marker for more accurate estimation of extent of disease and hence prognosis. knowledge of all potential causes of its elevation can overcome problems with reduced specificity in a patient known to have a specific infectious disease. longitudinal serial measurements in the same individual could overcome difficulties with interpretation in settings where chronic parasitic (malaria) or bacterial (tuberculosis) infections may elevate the baseline neopterin level and could allow monitoring of response to antiretroviral, antituberculous, and antiparasitic treatment in the absence of resistance testing and provide means to monitor compliance in the outpatient setting (see table 1 ). this is particularly important in the current context of emerging multiple drug resistance of hiv and mycobacterium tuberculosis. neopterin for which high quality elisa systems to measure urine and blood levels are commercially available is an underused marker in clinical practice and is suitable for introduction into the routine clinical laboratory practice. the author declares that there is no conflict of interests regarding the publication of this paper. neopterin measurement in clinical diagnosis potential role of immune system activation-associated production of neopterin derivatives in humans cytokine-stimulated gtp cyclohydrolase i expression in endothelial cells requires coordinated activation of nuclear factor-b and stat1/stat3 simple dipstick assay for semi-quantitative detection of neopterin in sera erhoehte ausscheidung von neopterin im harn von patienten mit malignen tumoren und mit viruserkrankungen urinary neopterin is elevated in patients with malaria clinical presentation of cmv infection in solid organ transplant recipients and its impact on graft rejection and neopterin excretion urinary neopterin levels in acute viral hepatitis immune activation during measles: beta 2-microglobulin in plasma and cerebrospinal fluid in complicated and uncomplicated disease neopterin levels during acute rubella in children the 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represented by cell surface antigen and plasma activation marker changes highly active antiretroviral therapy (haart) and circulating markers of immune activation: specific effect of haart on neopterin serum neopterin changes in hiv-infected subjects: indicator of significant pathology, cd4 t cell changes, and the development of aids increased immune activation precedes the inflection point of cd4 t cells and the increased serum virus load in human immunodeficiency virus infection predicting clinical progression or death in subjects with early-stage human immunodeficiency virus (hiv) infection: a comparative analysis of quantification of hiv rna, soluble tumor necrosis factor type ii receptors, neopterin, and 2 -microglobulin serum neopterin level predicts hiv-related mortality but not progression to aids or development of neurological disease in gay men and parenteral drug users are plasma biomarkers of immune activation predictive of hiv progression: a longitudinal comparison and analyses in hiv-1 and hiv-2 infections? 2 -microglobulin and immunoglobulins are more useful markers of disease progression in hiv than neopterin and adenosine deaminase reduction of viral load and immune complex load on cd4+ lymphocytes as a consequence of highly active antiretroviral treatment (haart) in hivinfected hemophilia patients neopterin concentrations in cerebrospinal fluid and serum of individuals infected with hiv-1 levels of human immunodeficiency virus type 1 rna in cerebrospinal fluid correlate with aids dementia stage csf neopterin decay characteristics after initiation of antiretroviral therapy central nervous system immune activation characterizes primary human immunodeficiency virus 1 infection even in participants with minimal cerebrospinal fluid viral burden continuing intrathecal immunoactivation despite two years of effective antiretroviral therapy against hiv-1 infection cerebrospinal fluid neopterin: an informative biomarker of central nervous system immune activation in hiv-1 infection immune activation of the central nervous system is still present after >4 years of effective highly active antiretroviral therapy treatment benefit on cerebrospinal fluid hiv-1 levels in the setting of systemic virological suppression and failure neopterin as a marker of response to antiviral therapy in hepatitis c virus patients neopterin as an index of immune response in patients with tuberculosis neopterin, 2 -microglobulin, and acute phase proteins in hiv-1-seropositive and -seronegative zambian patients with tuberculosis serum interleukin-2 and neopterin levels as useful markers for treatment of active pulmonary tuberculosis neopterin as marker for activation of cellular immunity: immunologic basis and clinical application liver involvement in human schistosomiasis mansoni. regression of immunological and biochemical disease markers after specific treatment praziquantel in the treatment of hepatosplenic schistosomiasis: biochemical disease markers indicate deceleration of fibrogenesis and diminution of portal flow obstruction neopterin as a marker of c hepatitis in thalassaemia major serum neopterin levels in patients with replicative and nonreplicative hbv carriers serum neopterin levels in patients with hbv infection at various stages serum neopterin levels in children with hepatitis-b-related chronic liver disease and its relationship to disease severity value of serum procalcitonin, neopterin, and c-reactive protein in differentiating bacterial from viral etiologies in patients presenting with lower respiratory tract infections serum neopterin for early assessment of severity of severe acute respiratory syndrome diagnostic utility of crp to neopterin ratio in patients with acute respiratory tract infections intrathecal production of neopterin in aseptic meningo-encephalitis and multiple sclerosis immune activation during measles: interferon-and neopterin in plasma and cerebrospinal fluid in complicated and uncomplicated disease role of il-6 and neopterin in the pathogenesis of herpetic encephalitis cerebrospinal fluid neopterin concentrations in central nervous system infection cerebrospinal fluid neopterin is brain-derived and not associated with blood-csf barrier dysfunction in noninflammatory affective and schizophrenic spectrum disorders persistent intrathecal immune activation in hiv-1-infected individuals on antiretroviral therapy persistent intrathecal immune activation in patients with herpes simplex encephalitis value of urinary neopterin in the differential diagnosis of bacterial and viral infections evaluation of procalcitonin and neopterin level in serum of patients with acute bacterial infection evaluation of procalcitonin, neopterin, c-reactive protein, il-6 and il-8 as a diagnostic marker of infection in patients with febrile neutropenia neopterin as a prognostic biomarker in intensive care unit patients the value of neopterin and procalcitonin in patients with sepsis d-erythroneopterin plasma levels in intensive care patients with and without septic complications course of immune activation markers in patients after severe multiple trauma procalcitonin and neopterin as indicators of infection in critically ill patients neopterin production and tryptophan degradation in acute lyme neuroborreliosis versus late lyme encephalopathy assessment of diagnostic enzyme-linked immunosorbent assay kit and serological markers in human brucellosis is neopterin-a marker of cell mediated immune response, helpful in classifying leprosy immune responseassociated production of neopterin. release from macrophages primarily under control of interferon neopterin as a new biochemical marker for diagnosis of allograft rejection. experience based upon evaluation of 100 consecutive cases bal neopterin. a novel marker for cell-mediated immunity in patients with pulmonary tuberculosis and lung cancer neopterin in tuberculous and neoplastic pleural fluids neopterin as a marker for cell-mediated immunity in patients with pulmonary tuberculosis urinary neopterin measurement as a non-invasive diagnostic method in pulmonary tuberculosis pleural fluid neopterin levels in tuberculous pleurisy neopterin levels and pulmonary tuberculosis in infants incomplete immunological recovery following anti-tuberculosis treatment in hiv-infected individuals with active tuberculosis urinary neopterin in volunteers experimentally infected with plasmodium falciparum malaria antigene stimulate neopterin secretion by pbmc and u937 celts neopterin and procalcitonin are suitable biomarkers for exclusion of severe plasmodium falciparum disease at the initial clinical assessment of travellers with imported malaria csf neopterin as marker of the meningo-encephalitic stage of trypanosoma brucei gambiense sleeping sickness liver involvement in human schistosomiasis mansoni. assessment by immunological and biochemical markers serum-neopterinbestimmung zur zusaetzlichen sicherung der bluttransfusion neopterin screening and acute cytomegalovirus infections in blood donors acute cytomegalovirus infections in blood donors are indicated by increased serum neopterin concentrations high prevalence of cytomegalovirus dna in plasma samples of blood donors in connection with seroconversion increased prevalence of igm antibodies to epstein-barr virus and parvovirus b19 in blood donations with above-normal neopterin concentration human parvovirus b19 detection in asymptomatic blood donors: association with increased neopterin concentrations association between chronic hepatitis c virus infection and increased neopterin concentrations in blood donations key: cord-312418-e4g5u1nz authors: melillo, alessandro title: rabbit clinical pathology date: 2007-09-18 journal: j exot pet med doi: 10.1053/j.jepm.2007.06.002 sha: doc_id: 312418 cord_uid: e4g5u1nz with rabbit patients, as in other species, analyzing blood and urine samples can be useful and informative, although interpretation of the results is sometimes challenging. this article summarizes the interpretation of laboratory results from rabbits. hematological parameters can yield information about the red blood cell population and leukocyte response to stress and pathogens. biochemistry evaluation can be used to investigate liver, kidney, and other organ function, and urinalysis results may yield additional information about kidney function and electrolyte imbalances. serological tests are available for several pathogens of rabbits, including encephalitozoon cuniculi, although the significance of positive results and antibody titers is not clear. serum protein electrophoresis aids the understanding of protein disorders and the immune response to acute and chronic inflammation. r abbits can mask signs of illness or show few or confusing clinical signs. additional information may be gained from laboratory tests, and in-house analyzers can provide a complete profile with a small volume of the patient's blood. unfortunately, most of the published data on rabbit hematology and biochemistry values are descriptions of the effects of toxins on hematological and biochemical parameters of laboratory rabbits. there is little information available that describes the effect of clinical disease on the blood parameters of companion rabbits, or on the use of blood tests as diagnostic and prognostic indicators. the lack of biochemical data for pet rabbits is changing as practitioners collect information and researchers are more cognizant of diagnostic and prognostic hematologic indicators. the blood volume of a healthy rabbit is approximately 55 to 65 ml/kg, and 6% to 10% of the blood volume may be safely collected. many sites are described for blood collection in rabbits. cardiac punc-ture is used in laboratory rabbits but is not recommended for pet animals. both the marginal ear vein and central ear artery are easily accessible, but they may be difficult to access in some patients. collecting a sufficient volume of blood from these sites to perform all of the desired clinical tests may also be difficult, especially from dwarf breeds with small ears. sampling from ear vessels can occasionally result in thrombosis and subsequent avascular necrosis of parts of affected pinna tissue. blood may be collected from the cephalic vein, which is straight and easily accessible, but, because of the short antebrachium, occlusion of the vessel by encircling the limb at the elbow is difficult. the cephalic vein is also small and easily collapses. the jugular veins are large and allow for ample-sized blood volumes to be collected, but jugular phlebotomy can be stressful for rabbits and may require chemical restraint. the dewlap may interfere with jugular vein visualization, especially in obese does. an accessible and efficient site for blood sampling in rabbits is the lateral saphenous vein (fig 1) . most biochemical parameters of rabbits can be measured from serum or plasma. rabbit blood clots easily at room temperature and will coagulate quickly if not mixed with anticoagulant during collection. heparin is a suitable anticoagulant because it does not alter biochemical parameters, even though the anticoagulant/blood ratio is not always optimal. hemolysis can be prevented by letting the blood drop from the needle into the tube, but often, only a few drops can be collected before the blood clots. the technique can be improved by heparinizing syringes and needles through aspiration of a few drops of heparin into the syringe, then removing the excess with injection pressure. the small amount of heparin remaining in the needle prevents clotting without significant alterations in biochemical parameters. it is important to make several air-dried blood smears at the time of venipuncture before the anticoagulant in the tube and transport can modify red and white cell morphology. most of the standard blood stains work well. automated flow cytometry is reliable for analyzing most hematological parameters for rabbit patients. age, sex, breed, and circadian rhythms all affect hematological and biochemical parameters in rabbits. rabbits under 12 weeks of age have lower red blood cell (rbc) and white blood cell (wbc) counts. the total wbc and lymphocyte counts are lowest in the late afternoon and evening, when the heterophils and eosinophil counts rise. urea and cholesterol levels tend to increase at the end of the day. stress can alter many different hematological parameters (e.g., blood glucose). prolonged stress, such as transportation, unfamiliar noises, smell, chronic pain, and poor environment, can induce heterophilia, lymphopenia, and leukocytosis. simple handling does not induce this response, but several muscle enzymes including lactate dehydrogenase (ldh), aspartate aminotransferase (ast), and creatine kinase elevate after physical restraint of the rabbits, especially if they are fractious or unfamiliar with handling. sedation or general anesthesia can be helpful. isoflurane anesthesia does not appear to affect blood parameters in rabbits. hemolysis can induce several artifacts, such as decreased rbc and amylase values and increased ldh, ast, creatine kinase, total protein, and potassium levels. in-house analyzers can be very sensitive to hemolysis, thereby altering the true blood parameters of the patient. hematology results of rabbits can be difficult to interpret. most reference ranges are from experimental laboratory studies that are run on homogeneous groups of rabbits belonging to the same breed, strain, age, and environmental conditions. this can be very different from the clinician's situation of dealing with a heterogeneous population of pet rabbits. many texts amalgamate reference ranges from different sources to create ranges so wide that they include almost any result. another problem is that healthy pet rabbits are very hard to find, so samples from rabbits with an apparently acute condition may also show changes due to an underlying chronic problem, such as malnutrition, improper husbandry, or subclinical disease. for example, harcourt-brown and baker 1 showed that rabbits that were caged, fed on commercial mixes, and suffered from dental disease had consistently lower packed cell volumes (pcv), rbc counts, hemoglobin values, and lymphocyte counts in comparison with rabbits kept outside with a more natural diet and exercise. rabbit erythrocytes are typical mammalian anucleate biconcave discs with an average diameter of 6.8 m, which is midway between cats and dogs. 2 erythrocyte size varies between 5.0 and 7.8 m, which is often reported as a marked anisocytosis (fig 2) . the short life span (57 days) and high turnover of erythrocytes is reflected as polychromasia, which is not clinically significant. the presence of a few nucleated rbcs (1-2 ϫ 100 leukocytes) and the occasional howell-jolly bodies (fig 2) should be considered within the normal reference range for rabbits and not an indicator of cellular regeneration. the published reference range for pcv in the rabbit is 30% to 50%, but pet rabbits often have lower values of 30% to 40%. 2 values higher than 45% may indicate dehydration, which is usually linked to gastrointestinal (gi) stasis. combined pcv and total protein (tp) is useful to differentiate acute conditions from subclinical chronic diseases that have suddenly deteriorated. a pcv of less than 30% indicates anemia, especially if the rbc and hemoglobin levels are low as well. nonregenerative anemia associated with chronic disease is common in pet rabbits. otitis media, dental disease with or without abscesses, pneumonia, pododermatitis, mastitis, endometritis and pyometra, renal disease, and osteomyelitis are all examples of chronic infections that can be associated with nonregenerative anemia in pet rabbits. regenerative anemia is manifested by significant and rapid reticulocyte production and usually indicates blood loss. causes of external hemorrhage in rabbits include trauma and severe flea infestation. common internal causes include hematuria due to kidney or bladder stones or bleeding uterine adenocarcinomas or endometrial aneurysms in does. intravascular hemolysis is an unusual cause of regenerative anemia after ingestion of leaves and stems of potato plants and possibly other solanaceae. alliums (onion, garlic, and chives) may also cause heinz body anemia. 3 autoimmune hemolytic anemia has been reported in laboratory rabbits in association with lymphosarcoma, and isolated cases have been treated in private practice (harcourt-brown, personal communication, november, 2006). 4 lead toxicosis is a cause of regenerative anemia, characterized by many nucleated erythrocytes, hypochromasia, poikilocytosis, and basophilic cytoplasmatic stippling. 2 nucleated red cells of more than 1% to 2% of the rbc can be linked with the acute, septicemic phase of an infectious disease, although this is unusual because of the presence of a nonregenerative anemia from underlying chronic disease. a well-prepared air-dried smear is required for an accurate differential white cell count and evaluation of the cytological appearance of each cell type (figs 2-6). differential white cell counts and cell morphology can be used to develop a differential diagnoses list and to determine the general condition of the patient. interpretation of wbc data from rabbits is different from other domestic species including dogs, cats, and birds, in which a leukocytosis is the response to inflammation. with rabbits, although leukocytosis can be identified in cases that have been diagnosed with lymphosarcoma, it is not the usual response to inflammation. laboratory investigations have shown no increase in the total number of circulating leukocytes in rabbits injected with bacteria or yeast, in domestic carnivores, anisocytosis usually reflects the presence of reticulocytes and indicates regenerative anemia. this is not true in rabbits in which 1% to 4% of the circulating erythrocytes may be reticulocytes. the occasional howell-jolly body is not clinically significant either. rabbit clinical pathology although fever, increased plasma cortisol concentrations, neutrophilia, and lymphopenia were observed. 5, 6 in clinical practice, rabbits with sepsis can show a variety of responses including a neutropenia, normal neutrophilic count, or a mature neutrophilia, accounting for more than 90% of wbcs. band neutrophils appear to be a rare finding in clinical infection, and the absence of a left shift does not rule out an infectious problem. an alteration of the neutrophil/lymphocyte ratio showing a relative neutrophilia coupled with lymphopenia may indicate a response to infection. this ratio is approximately 1:1 in an adult healthy rabbit, but stress can alter the neutrophil/lymphocyte ratio. transport, waiting in a room full of unfamiliar sounds and smells, or even restraint for clinical examination can change the ratio. gentle clinical examination and blood collection do not seem to affect the differential white cell count, whereas more prolonged stress like travel or exposure to barking dogs in the waiting room can induce a lymphopenia and relative neutrophilia, that may persist for up to 24 to 48 hours. 7 the total wbc can be used to further characterize acute stress from chronic stress (e.g., malnutrition, improper husbandry, prolonged social stress, dental disease), as both a leukopenia and lymphopenia are eosinophil. rabbit eosinophils measure 10 to 16 m in diameter and have a purple bilobed or horseshoe-shaped nucleus. the cytoplasm is obscured by so many granules that the cell looks orange-pink and foamy. the abundance of granules is the main difference between the eosinophils and the neutrophils. removal of histamine and histaminelike toxins is the most important function of the eosinophils, suggesting that they therefore play an important role in controlling allergic reactions. more common with chronic stress. chronic stress is often reported as general leukopenia and lymphopenia in a rabbit's differential wbc evaluation. the primary role of the lymphocytes is to respond to those activities that stimulate the immune system. in rabbits, lymphocytes are primarily found in the blood, spleen, bone marrow, lymph nodes and the lymphatic tissues in the gi tract. the number of circulating lymphocytes is a balance between the cells entering and leaving the bloodstream and does not necessarily reflect a change in lymphopoiesis. in rabbits, it has been shown that an increase in adrenaline levels (acute stress) induces lymphocytosis, whereas raised cortisol levels (chronic stress) leads to lymphopenia. viral diseases may result in a normal or higher lymphocyte count. other causes of lymphocytosis are lymphoma and lead poisoning. 8 eosinophilia in rabbits can occur when tissues rich in mast cells, such as the skin, lungs, gi tract, or uterus, are involved in disease. eosinophilia can indicate the presence of an abscess and may be found during wound healing. in other species, eosinophilia is linked to parasitic diseases, especially when larvae are moving through tissues, but this is rare in domestic rabbits. encephalitozoon cuniculi does not stimulate an eosinophilic response. in clinically healthy rabbits, a very low eosinophilic count, even 0, is a common finding. high levels of cortisol (chronic stress) can induce eosinopenia. monocytosis is linked with chronic inflammation (e.g., abscesses, mastitis, tympanic bulla empyema). however, the absence of a monocytosis does not rule out inflammation. monocyte counts within the normal range are a common finding in rabbits with osteomyelitis due to dental disease. the diameter of rabbit basophil measures 8 to 12 m. its nucleus is less segmented than the eosinophil or heterophil and difficult to see because of the many deep purple granules that obscure the light gray cytoplasm. as in other species, the function of the rabbit basophil is not fully understood, but these cells are often present in large numbers of rabbit blood smears. basophilia with concurrent eosinophilia has been described in rabbits with chronic skin problems (e.g., atopy, pyoderma). 3 in rabbits, ast is widely distributed in many tissues. it is present in cardiac tissue and muscle, as well as the liver, and has a short half-life (5 hours). although higher ast levels may be found in patients diagnosed with liver damage, struggling during collection or hemolysis of the sample also raises ast levels. creatine phosphokinase levels also increase after restraint and are purely muscular in origin. ldh is produced by muscle and liver cells in rabbits and therefore is not beneficial as a diagnostic tool for many disease evaluations. reference levels for ast, ck, and ldh can be found in table 1 . in many mammalian species, alanine aminotransferase (alt) is a useful indicator of hepatocyte damage because of its specificity for liver tissue and its long half-life (45-60 hours in dogs). in rabbits, alt is not as useful as an indicator of liver damage as with other species because, like other herbivores (e.g., horses, cattle, guinea pigs), alt is not liver specific and has a shorter half-life (around 5 hours). however, alt concentrations are not affected by restraint and therefore can be used as a diagnostic tool. slightly increased alt levels are a common finding in apparently healthy rabbits. mildly increased alt levels in healthy rabbits have been attributed to exposure to low concentration of toxic substances, such as resins in wood-based litter or aflatoxins in food. 9 raised alt levels (with alkaline phosphatase (alp), bilirubin and glutamyltransferase [ggt]) can be associated with hepatic lipidosis or may be found in patients with hepatic coccidiosis (eimeria steidae) or torsion of a liver lobe. alp is a widely distributed enzyme; liver and bone contain the highest concentrations, but it is also found in bowel epithelium, kidney tubules, and placenta. a physiological cause of high-serum alp concentrations is osteoblastic activity in growing animals. animals with bone lesions will show raised alp levels. as a liver enzyme, alp does not increase because of hepatocellular damage but is indicative of bile stasis (e.g., hepatic coccidiosis, liver abscesses, neoplasia, lipidosis). extrahepatic causes, such as abscesses or neoplasia, can cause bile stasis by occluding the bile ducts. rabbits produce 2 alp isoenzymes in the liver. an intestinal isoenzyme is quite abundant, so serum alp concentrations are actually the sum of these 3 isoenzymes, which may explain why many reference ranges are vague and wide and why raised alp levels in clinically healthy animals are a common finding. alp does have a diagnostic value because it is not altered by restraint and thus is considered a good indicator of real tissue damage. reference levels for alt and alp can be found in table 1 . ggt is a useful indicator of chronic liver disease with bile stasis in horses, cattle, and domestic carnivores; however, in rabbits, the activity of ggt is low. activity of this enzyme is high in the kidney, yet renal ggt does not reach the circulation because it is eliminated with the urine. therefore, elevated ggt levels in the rabbit are most often linked to obstructive lesions of the bile ducts, but with a lower sensitivity than that found in other species. reference levels for ggt can be found in table 1 . bile pigments are produced during the breakdown of the heme molecule by hepatocytes and are excreted into the bowel. bilirubin levels reflect either hepatocyte or bile tract function. rabbits produce a large amount of bile for their weight, and the main compound is biliverdin, for which there is no commercial laboratory test. about 30% of biliverdin is converted to bilirubin, which is found in the blood in measurable amounts. the main cause of hyperbilirubinemia is bile flow obstruction. in young rabbits, hepatic coccidiosis is the most common cause of biliary obstruction, while in adult rabbits it is biliary neoplasia. cellular causes of hyperbilirubinemia, and rarely icterus, are aflatoxicosis (e.g., eating moldy food), which induces hepatic fibrosis (alt is usually raised too), and viral hemorrhagic disease, which causes acute hepatic necrosis with concurrent high levels of all hepatocellular enzymes. if the rabbit survives long enough, icterus may be seen. bilirubin may also be increased in diseases that cause hemolysis (e.g., immune-mediated hemolytic anemia). in other species, a comparison of preprandial and postprandial serum bile acid concentrations is used as an indicator of liver function. in rabbits, cecotrophy makes it almost impossible to fast a rabbit for the preprandial sample, so bile acid measurement is not a routine procedure in clinical practice, although persistently raised bile acids have been reported in association with hepatic disease. 10 cholesterol is synthesized in the liver or obtained from the diet and is a precursor of steroids. it is metabolized by the liver and excreted in bile. in carnivores, hypercholesterolemia is linked with several metabolic diseases, such as hypothyroidism, hyperadrenocorticism, diabetes, and hepatopathies. hypocholesterolemia indicates liver failure. cholesterol and triglycerides levels peak after a meal and fasting is needed for accurate measurement, which limits their diagnostic value in rabbits because of cecotrophy. abnormal levels of cholesterol and tri-glycerides can be related to a diet rich in fats, obese patients, or hepatic disease. in anorexic patients, hypercholesterolemia carries a poor prognosis because it indicates end-stage hepatic lipidosis. hypercholesterolemia has also been linked with pancreatitis, diabetes mellitus, nephrotic syndrome, and chronic renal failure. 3 decreased cholesterol levels in rabbits might be found in cases of liver failure, chronic malnutrition, and even pregnancy (up to 30% below the range). unlike other species, amylase is an almost pure pancreatic enzyme in rabbits, with little or no content in salivary glands, intestinal tissue, or liver. therefore, raised amylase levels in rabbits reflects pancreatic damage from pancreatitis, pancreatic duct obstruction, peritonitis, or abdominal trauma. renal failure can also cause hyperamylasemia because this enzyme is cleared by renal filtration. corticosteroids (exogenous or endogenous) can raise amylase values in serum, whereas hemolysis lowers it. there is little information on the function and diagnostic value of lipase in rabbits. increased lipase values may indicate cellular damage to the pancreas as it does in other species. as with amylase, lipase is artifactually elevated by corticosteroids. urea is a by-product of protein catabolism and is excreted by the kidneys into the urine. urea levels in rabbits depend on the circadian rhythm (peak in late afternoon and early evening), quantity and quality of proteins in the diet, nutritional status, liver function, intestinal absorption, urease activity of the caecal flora, and hydration status. often, small changes in urea levels are difficult to interpret. reference ranges have been determined from laboratory rabbits fed on a standardized diet and bled at the same time of the day, whereas clinicians see pet rabbits fed on a variety of foods and samples are taken at random. slight elevations in blood urea are a common finding. reference levels for urea can be found in table 1 . creatinine is a protein catabolite that is produced from the muscle creatine and excreted by glomerular filtration at a constant rate. creatinine is a more reliable test of renal function than blood urea. in rabbits, prerenal azotemia can be caused by dehydration because rabbits have a limited ability to concentrate urine. only a few hours without drinking or losing fluids, as in cases of ileus or diarrhea, may cause an increase of urea and creatinine to levels compatible with renal failure. urea and creatinine levels rapidly return to normal once the dehydration deficit is corrected. stress can also induce shock and cardiac disease, classified as prerenal disease, causing a decrease in renal perfusion. another potential cause of prerenal azotemia is gi hemorrhage, which results in increased protein digestion. azotemia is also indicative of renal disease, usually affecting the rabbit patient in association with hyperkalemia or hypokalemia, hypercalcemia and coexisting hyperphosphatemia, nonregenerative anemia, and isostenuric urine. the most common cause of renal failure in rabbit patients is e. cuniculi, which causes granulomatous and then fibrotic lesions in the renal parenchyma. other possible causes of renal failure are chronic interstitial nephritis, glomerulonephritis, pyelonephritis, nephrolithiasis, renal cysts, and lymphosarcoma. postrenal azotemia can occur because of obstruction to urine flow as a complication of bladder sludge or urolithiasis. an abdominal radiograph is mandatory in any azotemic rabbit. blood urea levels below the reference range indicate hepatic insufficiency or muscle mass loss (e.g., dental disease). reference levels for creatinine can be found in table 1 . glucose metabolism in rabbits is different from dogs or cats. not only do rabbits eat continuously during the day, but they also use volatile fatty acids produced by cecal flora as a primary energy source. a fasting blood sample is impossible to obtain because rabbits ingest fecal pellets. a rabbit that is not given food can continue to ingest cecotrophs. it has been shown that 4 days of starvation does not reduce blood glucose levels in rabbits. 11 diabetes mellitus is rare in rabbits, although hyperglycemia is a common finding and may be associated with glucosuria. reports of confirmed diabetes mellitus are from laboratory strains bred as a model for human diabetes. clinical signs commonly observed in pet rabbits with diabetes mellitus are polyphagia, polyuria, polydipsia, very high blood glucose levels (ͼ500 mg/dl), and glycosuria with significantly elevated glycosylated hemoglobin and raised triglycerides; however, obesity and ketoacidosis are not observed. 12 in clinical practice, most cases of hyperglycemia are due to stress (e.g., transport, handling, venipuncture, underlying disease). a marked hyperglycemia (around 350 mg/dl) is reported in cases of acute intestinal blockage by a foreign body. 10 early mucoid enteropathy may be associated with hyperglycemia. in rabbits with gi stasis, hyperglycemia carries a bad prognosis because it may indicate hepatic lipidosis. other causes of raised serum glucose levels are traumatic or hypovolemic shock and hyperthermia. acute pancreatitis could cause blood glucose abnormalities, even though the role of the pancreas in glucose metabolism in rabbits is less important than in other species. glucocorticoids and other drugs can raise blood glucose. hyperadrenocorticism has not been described in rabbits. hypoglycemia is an important finding. in anorexic patients, it indicates that the rabbit is using adipose tissue and is at risk of developing hepatic lipidosis. hypoglycemia may occur in terminal mucoid enteropathy, liver failure, or other chronic diseases. rabbits with acute sepsis may be hypoglycemic too. 3 insulinoma has not been described in rabbits. reference levels for glucose can be found in table 1 . the complex gi physiology and the limited ability of the kidneys to correct acid-base alterations make rabbits susceptible to electrolyte imbalances. any disturbance of serum electrolytes should alarm the clinician to possible pathology of the digestive or excretory system. anorexia can rapidly lead to metabolic acidosis. the diagnostic value of sodium for rabbit patients is low. hypernatremia can be due to dehydration or loss of fluids (e.g., diarrhea, peritonitis, burns, myiasis). hyponatremia is usually associated with polyuric renal failure (acute or more commonly chronic), when urine flow in the renal tubules is too fast to impede the sodium-potassium exchange. lipemia or hyperproteinemia can artifactually decrease sodium levels in serum. reference levels for sodium can be found in table 1 . potassium is homeostatically important because it is essential for maintenance of membrane potential. changes in membrane potential can be lethal (e.g., impaired contractility of myocardial cells due to hyperkalemia can cause arrhythmias and cardiac arrest). intracellular and extracellular potassium levels are maintained by a complex mechanism of exchange between cells and microenvironment, and regulated by several hormones such as aldosterone, insulin, and catecholamines. hypoadrenocorticism has not been described in rabbits. instead, raised potassium levels are more often due to acute renal failure or urine flow obstruction. severe tissue damage can also cause hyperkalemia by dispersing potassium into the extracellular space. for the same reason, hemolysis (e.g., intravascular, bad sampling technique, letting the sample wait too long before separating the serum) can artifactually raise potassium levels. another indirect cause of higher potassium levels in serum is metabolic acidosis, which increases the exchange of potassium ions across the cell membrane. reference levels for potassium can be found in table 1 . causes of hypokalemia in rabbits include dietary insufficiency and loss of fluids from the gi system (e.g., saliva, mucoid diarrhea) or the kidneys (e.g., renal failure, diuretic drugs). stress-induced increases in catecolamine levels can also cause hypokalemia. alkalosis, although rare in rabbits, decreases the blood potassium concentrations by stimulating the cellular uptake of potassium ions. hyperproteinemia and lipemia can artifactually decrease blood potassium levels that may present clinically as sensory depression and muscle weakness. a correlation between low potassium levels in blood and "floppy rabbit syndrome" has been reported. 10 in other herbivorous species (e.g., horses), blood potassium levels fluctuate widely, depending on physical activity or even on the quantity of saliva produced during the meals. 13 these examples of serum potassium level fluctuation are purely physiological and may occur in rabbits. calcium is found in blood either bound to serum proteins or ionized free in the serum. most laboratories report a total serum calcium value, which is the sum of bound and ionized calcium. ionized calcium is a more precise measurement but is a more difficult and expensive parameter to test. total serum calcium is influenced by dietary intake, serum protein levels, and other metabolic conditions. calcium metabolism in rabbits is different from that in other animals. blood calcium levels are influenced more by the calcium content of the diet than in the dog or the cat. rabbits absorb calcium in proportion to the concentration of the ion in the gut, and the kidney eliminates the excess. vitamin d is not important in calcium absorption if dietary levels are high, yet it does play an important role if dietary levels are low. vitamin d is also important in calcium distribution within the body. as with other species, parathyroid hormone regulates blood calcium levels, but the level at which calcium is moved from the blood to the bones is high in rabbits. consequently, blood calcium levels are higher, and the normal range is broader than that in other species. growing youngsters and pregnant does use more calcium, resulting in lower blood calcium concentrations. in these rabbits blood calcium concentrations rarely rise above 14 mg/dl, even when fed calcium-rich diets, whereas adult rabbits on a varied diet can show calcium levels up to 16 to 17 mg/dl ( table 1) . the urinary excretion rate of calcium is around 45% to 60% for rabbits, whereas most mammals excrete no more than 2% of their calcium through renal filtration. this predisposes rabbits to the formation of sludge and stones in the rabbit urinary system. although the constant excretion of calcium could be a cause of renal failure in rabbits fed unbalanced diets, hypercalcemia is also a consequence of renal disease in rabbits because of the inability of the kidney to eliminate excess calcium. measurement of blood calcium is essential to diagnose and treat renal disease in rabbits. hypocalcemia is rare but is reported in rabbits. the most frequent cause of low blood calcium levels is hypoalbuminemia due to poor nutrition. hypocalcemic seizures have been described in late-pregnant and lactating does. 13 phosphorus is involved in many enzymatic systems in rabbits, but its main function is contributing to the proper formation of bones and teeth. because phosphorus is present inside cells, blood phosphate concentrations are easily increased by hemolysis (e.g., spontaneous or sampling problems). blood phosphate values should always be evaluated with blood calcium levels to determine the mineral balance in patients diagnosed with urinary tract stones, dental disease, or other signs of nutritional secondary hyperparathyroidism. because the kidney is the main organ involved in phosphorus balance by regulation of glomerular filtration and tubular reabsorption, blood phosphate levels can be an indirect measurement of kidney function and, in general, parallel azotemia. serum phosphorus levels can be elevated as a result of prerenal, renal, and postrenal effects. hyperphosphatemia usually indicates chronic kidney failure (a loss of more than 80% of nephrons) given that serum phosphorus levels are normalized by compensatory mechanisms in early-onset renal disease. hyperphosphatemia may also be an indicator of soft tissue trauma. reference levels for phosphorus can be found in table 1 . hypophosphatemia is not rare, but its clinical significance is unknown at this time, with dietary deficiencies or reduced intestinal absorption possibly involved. total protein, consisting of the sum of albumin and globulin, is an important parameter in any species of animal. many factors (e.g., age of the animal, reproductive status, pregnancy) can affect tp levels. total serum protein levels can be artifactually raised by hemostasis at the sample site through fluid loss because of digital pressure on the vessel from which the blood is collected. reference levels for tp, albumin and globulins can be found in table 1 . the main cause of hyperproteinemia in rabbits is dehydration. raised tp levels may also indicate a chronic infectious or metabolic process. measuring albumin and globulin fractions helps to differentiate the causes of hyperproteinemia. hypoproteinemia is usually due to chronic malnutrition or protein loss. if both albumin and globulin are low, hemorrhage or protein loss through exudative skin lesions such as burns or flystrike should be considered. other possible causes must be examined in cases of hypoalbuminemia with normal or raised globulins. because the liver is the only site of albumin synthesis, a lowered albumin level may indicate an advanced hepatic disease, such as hepatic coccidiosis (e stiedae) or scarring and necrosis due to the migrations of cysticercus (taenia) pisiformis larvae. protein-losing nephropathies (glomerulonephropathy) and enteropathies are rarely diagnosed in rabbits. a common cause of hypoalbuminemia in pet rabbits is chronic malnutrition either from poor diet or advanced dental disease. all causes of reduced cecotrophy (e.g., dental disease, obesity, back pain) can be reflected as low protein, especially albumin levels. a method of evaluating serum proteins is by electrophoresis (eph), which divides the globulins into distinct fractions. in acute disease, alphaglobulins are elevated; consequently, a rabbit with an alpha-globulin peak may have a bacterial infection or a developing abscess, and/or present febrile. the beta portion of globulins consists of several proteins classified as "acute-phase" proteins including fibrinogen. fibrinogen correlates with inflammation in rabbits, although the correlation is not as evident as in other species. plasma is preferable to serum for eph because it does include fibrinogen. gamma-globulins are mainly antibodies, and a peak of this fraction indicates a subacute to chronic inflammation, especially when associated with a bacterial infection. coronavirus infections lead to an impressive increase of rabbit globulins as feline coronavirus does in cats, but this disease is probably limited to laboratory set-tings. the correlation between eph curves and different rabbit pathologies is unknown and currently under investigation. there are serological tests for antibodies against e cuniculi, toxoplasma gondii, treponema cuniculi, myxomatosis, viral hemorragic disease, and pasteurella multocida, although their availability varies in different parts of the world. the most common serological assay used in private practice is for the antibodies to e cuniculi. laboratory studies have shown that infected rabbits start to develop a measurable serological immune response 4 weeks after infection, 2 weeks before e cuniculi is found in the kidney or in the urine, and at least 8 weeks before any brain lesion. 14, 15 if a serologic test for e cuniculi is negative for a neurological rabbit patient, one could assume, with confidence, that this patient does not have the disease. if the test is positive, neurological signs may be due to e cuniculi or a different disease process. the seropositive result may be due to past exposure to the pathogen, which is common within most rabbit populations. further laboratory tests may be helpful to determine a definitive diagnosis. a correlation between antibody titers and neurological signs is hard to prove. a rising titer after 2 weeks is often considered diagnostic in suspect cases. the kidney is a target organ for e cuniculi, so evaluation of renal function and structure by biochemistry, urinalysis, and ultrasound may aid one's diagnostic overview. inflammatory changes in the cerebrospinal fluid are suggestive of e cuniculi but are nonspecific. at the present time, the definitive diagnosis of rabbit encephalitozoonosis requires histopathology or isolation of the spores from the urine by microscopy or polymerase chain reaction assay. although serology for t cuniculi can be used to screen a breeding facility, it has little value in clinical practice. at least 3 months are needed for antibody levels to become measurable in the blood even with clinically evident dermatological lesions. this substantial lag in the development of antibody titers can lead to false-negative test results. positive titers without skin lesions can indicate subclinical disease or previous infection in which the skin lesions are missed. antibody titers fall rapidly once the rabbit is treated for t cunicoli. 2 serological testing for p multocida is available in some countries, but its use in clinical practice is limited. a single positive response has no clinical significance because many rabbits harbor the bacteria in the nasal cavities without illness. a positive titer can also indicate previous exposure to the bacteria rather than clinical infection. repeat testing after 2 weeks, while looking for a rising titer, can be helpful in obtaining a correct diagnostic evaluation. results from rabbits younger than 2 months can be difficult to interpret because maternal antibodies are still present. a high antibody titer is not protective and may indicate subclinical disease. urine samples can be collected from spontaneous micturition, bladder expression, catheterization (quite difficult because sedation/anesthesia is required), or cystocentesis, with the latter being the preferred method for obtaining a sample for bacterial culture. care should be taken when performing a cystocentsis, because microtrauma to the bladder wall can stimulate local mineralization and it is possible to puncture the cecum, an enlarged uterus, or even an abscess. if bacteriology is not required, free catch from a clean litter tray is the easiest way to obtain a urine sample. manual expression of the bladder should be done carefully because the wall is thin and ruptures easily, especially if an obstruction is present or the rabbit struggles. in cases of chronic cystitis, the risk for bladder rupture is lower because the bladder is thickened. it is preferable to collect the first urine of the morning and to run the analysis as soon as possible. normal rabbit urine is dense and rich in minerals, so it should be centrifuged or filtered for biochemical analysis or examinations. clear urine indicates low calcium excretion, which may be pathological because of renal failure or physiological in the case of growing or lactating rabbits. the color may range from light yellow to reddish brown. in most cases, dark urine is caused by dietary pigments; however, the urine should be checked for hematuria, which may be caused by uroliths, urinary tract inflammation/infection, uterine problems, or anticoagulants. test dipsticks work well to evaluate blood, glucose, ketone, and ph levels in rabbit urine but are not accurate for other parameters. glucose may be found as a consequence of stress hyperglycemia; checking urine collected at home can help to rule out stress-related problems. true glucosuria indicates altered energy metabolism, such as hepatic lipidosis, or, very rarely, diabetes mellitus. ketones are always abnormal and indicate anorexia (even short duration), hepatic lipidosis, pregnancy toxemia, or diabetes. the ph of rabbit urine tends to be high (7.5 to 9) in rabbits fed a correct diet. acidic urine indicates acidosis due to anorexia, fever, pregnancy toxemia, or hepatic lipidosis. it is possible that, because a normal kidney would eliminate acidic urine in cases of acidosis, the finding of alkaline urine in an anorexic rabbit could indicate compromised renal function. specific gravity (sg) indicates the ability to concentrate urine. a refractometer is a more reliable tool to measure sg than dipsticks. most normal rabbit urine is quite dilute, with an average sg of 1.015 (range, 1.003-1.036). prerenal azotemia is associated with raised sg (ͼ1.030), whereas true azotemia linked to renal failure is associated with dilute urine (sg ͻ1.013). urine-specific gravity is useful if it is examined alongside urine protein concentrations. protein traces in clinically normal rabbits, especially the young, are not significant, whereas a dilute urine (ͻ1.020) with proteins is very significant. proteinuria appears earlier than biochemical changes in renal disease, making this test useful in clinical practice. measuring urine proteins/urine creatinine ratio (ͻ0.6 is suggested as normal) may further improve the sensitivity of urine-specific gravity testing. sediment examination can differentiate normal urine that is rich in crystals from sludge. after centrifugation, normal crystals should resuspend when shaken, while sludge remains as a solid mass. cytology is not very different from that of other mammals; a small number of leukocytes are considered normal in rabbits. gram or trichrome stains can reveal e cuniculi spores. 3 parathyroid hormone, hematological and biochemical parameters in relation to dental disease and husbandry in pet rabbits laboratory medicine: avian and exotic pets notes on rabbit internal medicine the biology of laboratory rabbit alteration of sleep in rabbits by staphylococcus aureus infection hematological effects of exposure to three infective agents in rabbits physiological stabilization of rabbits after shipping rabbit surgical pathology effects of t-2 toxin on ovarian activity and some metabolic variables of rabbits textbook of rabbit medicine the anatomy, physiology and the biochemistry of the rabbit spontaneous diabetes mellitus in the new zealand white rabbit veterinary laboratory medicine serological and histological studies on adult rabbits with recent naturally acquired encephalitozoonosis ferrets, rabbits and rodents clinical medicine and surgery key: cord-018623-of9vx7og authors: saghazadeh, amene; rezaei, nima title: the physical burden of immunoperception date: 2019-04-27 journal: biophysics and neurophysiology of the sixth sense doi: 10.1007/978-3-030-10620-1_10 sha: doc_id: 18623 cord_uid: of9vx7og the previous chapter introduced the immunoemotional regulatory system (immers). also, there was a brief discussion about psychological states/psychiatric disorders that so far have been linked to the immers. the present chapter considers another aspect of the immers in which physiological states/physical diseases can be fit to the immers. such as pemphigus [10, 11] . further, human studies provided evidence pointing to the increased development of emotional problems and edr-related disorders in patients with various types of aids, such as sle and multiple sclerosis (ms), in a disease state/severity-dependent manner [12] [13] [14] [15] [16] [17] . for example, among patients with childhood-onset sle, 95% manifest neuropsychiatric sle (nsle). mood and anxiety disorders were the most common psychiatric conditions with the prevalence rate of 60% and 20% [13] . even about 40% of patients with sle without cns manifestations suffer from psychological distress compared with 6% in controls. it is, thus, not surprising that both emotional coping and depressive symptoms were correlated with non-nsle [14, 15] . interestingly, there was an increased activation of the brain regions related to emotion regulation/processing (e.g., the amygdala and superior temporal) in sle patients. however further analyses led to identifying this increased activity of emotional circuit as a consequence of cns involvement by sle [18] . among patients with ms, emotional troubles were more than twofold more likely to occur in patients who had an exacerbation or progressive nonremitting ms compared to stable patients. this was reflected by an increased rate of using emotion-focused coping styles in patients with relapsingremitting multiple sclerosis (rrms) compared to stable patients [16, 17] . mood disturbance was correlated negatively with sil-2r levels and positively with joint pain in patients with ra [19] . consistent with data from human studies, animal experiments have also supported the link between emotionality-related behaviors and aids. clearly, aids result from immdr. interestingly, the aids-related immdr has been observed in the specific brain regions associated with emotional behaviors, particularly anxiety-and depressive-like behaviors [20] . in this manner, the link between aids and immers is strengthened. a high rate of increased emotionality and emotional-like behaviors in aids led to propose the term autoimmune-associated behavioral syndrome (aabs). studies emphasize the pivotal role of cytokines and neuroendocrine factors in the pathogenesis of aabs [21] . b-cell-activating factor (baff) transgenic mice model, which is used as an experimental model of systemic lupus erythematosus (sle), rheumatoid arthritis (ra), and sjögren syndrome, exhibited an anxious phenotype along with the following changes in immune brain signaling, such as increased igg titers in the hippocampus, hypothalamus, and cortex and increased cd68 (as a maker of activated microglia/macrophages) and gfap (as a maker of activated astrocytes) immunoreactivity in the hippocampus in mice at 4.5-5 months of age, but not in young (2 months of age) mice [22] . eae models of ms showed an increase in levels of il-1β and tnf-α in the hypothalamus. this indicates an inflammatory central basis behind anxiety-and depressive-like behaviors [20] . these emotional deficits were shown to display before the onset of ms [20, 23] . consistently, behavioral problems usually manifest before symptoms of impaired cognitive and motor performance in dementia. moreover, this model showed an early (at day 4) and a meaningful increase in circulating cytokine levels and cd3 + t cell counts. of note, these inflammatory markers began to decrease in the periphery (at day 8) almost when their infiltration in the cns (at day 10) started [23] . in the mrl-lpr model of aid, which is a well-documented model of emotional deficits [24, 25] , a reduced preference to glucose, and as an index of emotionality, was detected in 5-to 6-week-old mice [26] . this deficit could be diminished by immunosuppressive treatment with cyclophosphamide and was pronounced by means of chronic administration of il-6 [26] . along with psychosocial stressors, either chronic and acute, and social networkrelated factors (i.e., social ties, social conflict, and social support), the experience of unpleasant emotions, including anger, depression, sadness, and stress, or, in general, extremely exciting emotions, often promptly, pulls susceptible individuals into a steep road leading to cardiovascular events, particularly acs (for review see references [27] [28] [29] ). for example, emotional stress is ranked as the second most common neuropsychological cause of acute myocardial infarction (ami) owing to its record in approximately 40-50% of these patients [30, 31] . at the molecular levels, these patients have shown increased levels of the proinflammatory cytokines (e.g., tnf-α, il-1, il-2, il-6, and il-18) and decreased levels of the anti-inflammatory cytokine (il-10). thus, it is not surprising that the inflammatory response and respective cytokines are supposed as one of the possible mechanisms linking the experience of negative emotions or er-related disorders and the progression of cardiovascular diseases, of course along with the neuroendocrine system and apoptosis signaling pathways [27, 30, [32] [33] [34] [35] . it is to be noted that when patients with cardiovascular diseases are stratified according to their emotional background, some cytokines are more highlighted than others, for example, tnf-α and il-10, but not il-6, considering depressive symptoms in chf patients [35] . cognitive reappraisal is found to correlate positively with the engagement of the lateral and prefrontal regions and inversely with the engagement of the amygdala and medial orbitofrontal cortex [36] . the role of the inflammatory cytokine il-6 in the progression of cardiovascular diseases is widely appreciated [37] . studies show that il-6 is significantly involved in efforts to arbitrate between the sides of the relationship between the reappraisal-related activation of the dorsal anterior cingulate cortex and preclinical atherosclerosis (evaluated by carotid artery intima-media thickness and inter-adventitial diameter) in healthy individuals [38] . the normative aging study carried out a 3-year follow-up study in older men (mean 60.3 ± 7.9 years). there was a dose-response relationship between negative emotions, evaluated by the minnesota multiphasic personality inventory (mmpi), and incidence of coronary heart disease (chd) within the duration of the study (p = 0.005) [39] . meanwhile, the circulating levels of il-6 were positively associated with the reappraisal-related activation of the dorsal anterior cingulate cortex in healthy subjects [38] . however, higher reappraisals and suppressions were positively and inversely associated with the serum levels of crp, respectively [40] . on the other hand, the reflection of watching the ice hockey match, as a real-life emotional excitement, on serum levels of endothelin-1 (et-1) and il-6 was more pronounced in spectators with coronary artery disease compared with healthy spectators [41] . patients with type d personality display concurrently two absolute opposite, positive and negative, tendencies towards the experience and the expression of negative emotions by themselves and in front of other people, correspondingly. meta-analysis studies and comprehensive literature reviews have revealed that this type of personality is positively associated with contracting cardiovascular conditions and their consequent mortality and morbidity, as well as with a constellation of non-cardiovascular complaints (for details, see [42] [43] [44] ). also, individual studies, either cross-sectional or follow-up, have providence evidence of increased levels of proinflammatory cytokine tnf-α and its receptors, stnfr1 and stnfr2 [45] [46] [47] [48] , an enhanced il-6/il-10 ratio, and decreased levels of anti-inflammatory cytokine, il-10, in chf patients with type d personality compared to those without type d personality [45] . interestingly, in chf patients, the inflammatory effect of type d personality appears to resemble closely the effect of aging. there was a similar increased pattern of stnfr1 and stnfr2 in younger chf patients with type d personality and older patients without this personality trait [46] . plasminogen activator inhibitor-1 (pai-1) is a factor contributing to thrombosisrelated cardiovascular diseases in elderly people. both cytokines and hormones take part in the regulation of the gene expression of pai-1 (for review, see reference [49] ). in a model of premature immunosenescence, mice were assigned to either fast or slow group if the amount of time taken to explore the first arm of the maze was ≤20 s or >20 s, correspondingly [50] . when compared to fast mice, slow mice expressed high emotional response to stress and had lower life span [50] . at the immunological levels, slow mice showed a reduction in proliferative response to concanavalin a (con a) and related release of il-2 and il-1β and nk cell activity, while increasing the production of tnf-α [50] . an investigation on women who had to undergo breast biopsy indicated that this procedure should be considered as an emotional stressor if the final diagnosis is determined benign. in parallel with this emotional stress, the immune system prepares itself before the procedure and seeks for ways to prolong this preparation even 4 months after the procedure. this is a reflection of the joint regulation of our body by both the immune system and the emotional brain [51] . the immune system responds to this challenge by decreasing nk cell activity, decreasing production of ifn-γ, and increasing production of il-4, il-6, and il-10 [51] . further, there was a significantly positive relationship between mothers with breast cancer and their adult daughters on distress levels. this persuaded scientists to investigate the immune profile and its association with distress in daughters' group. daughters' distress levels were inversely associated with il-2, il-12, and ifn-γ production and also with il-2-induced natural cytotoxic activity (nca) [52, 53] . further, nca activity and the production of th1 cytokines were both negatively related to the emotional distress degree [53] . antoni and his colleagues accomplished a genome-wide transcriptional analysis on leukocyte samples taken from women subject to the treatment of stage 0-iii breast cancer and demonstrated that the negative affect, evaluated by the affects balance scale (abs), was significantly associated with greater than 50% increased expression of leukocyte transcripts such as proinflammatory marker-related genes [54] . another multiplex analysis on circulating concentration of 27 cytokines identified the il-6 profile as the predictor of physical and cognitive functioning and also the vascular endothelial growth factor (vegf) profile as the predictor of emotional functioning [55] . further, the experience of childhood emotional neglect/abuse was associated with lower levels of nca at the first evaluation after breast cancer surgery [56] . emotional processing and expression (evaluated by emotional approach coping scale), respectively, tended to be inversely and positively correlated with plasma levels of il-6, soluble tnf-receptor type 2 (stnf-rii), and crp in male patients with prostate cancer [57] . however, among those correlations, two of them, the correlations of emotional expression with il-6 and crp, were not found significant (p < 0.10) [57] . in vivo model of ultraviolet-b light-induced squamous cell carcinoma concluded that the high stress and anxiety levels can leave mice prone to the more considerably progression of the tumor through increasing the expression of immunosuppressive (ccl2 and t regulatory cells) and angiogenic (vegf: vascular endothelial growth factor) markers and decreasing the expression of antitumor immune markers (ctack/ccl27, il-12, and ifn-γ) [58] . on the other side, a peripheral tumor, by itself, could lead to a reduction in the hippocampal function, as reflected in increased depressive-like behaviors and memory impairment. this was, at least in part, underpinned by triggering an inflammatory process both in the hippocampus (↑il-1β, ↑il-6, ↑il-10, and ↑tnf-α) and in the circulation (↑il-6, ↑il-1β, and ↑il-10) [59, 60] . this process was found to be significantly strengthened in infection models compared to peripheral tumor models, explaining the presence and absence of the sickness state in these models, respectively [60] . using hospital anxiety and depression scale (hads) test, it was estimated that nearly half of patients with hemodialysis (hd) were in a depressive mood, which was significantly higher than what was reported for control group [61] . also, there was a higher production of il-6 in hd patients with anxiety (hads≥8) than those without anxiety (hads≤8) [61] . at least one major psychiatric illness, particularly depression, affects greater than 60% of hiv patients [62] . by virtue of the fact that specific substances of abuse aggregate further the situation of hiv patients at the neuropathological level [62] , the inverse correlation between affect regulation and regular substance motivates us to utilize er as a therapeutic intervention in this population [63] . hiv patients encounter commonly with situations where the social self is threatened. this threat causes shame feelings, which have been associated with increased proinflammatory cytokines [64] . both emotional and environmental factors affect the gut [65] . similar to that mentioned for asthma, this affect is mediated by crf and similar neuropeptides and also by mucosal mast cells [65] . immunoregulatory factors along with genetic and environmental factors contribute to the pathogenesis of inflammatory bowel disease (ibd). patients with ibd confront various er-related problems in their social life in a disease severity-dependent manner, such as higher sensitivity to negative emotions, fewer dropping into bar/disco and delayed falling in love, and experiencing more depressive and anxious symptoms, not only compared to controls, but also compared to patients with other chronic conditions [66] [67] [68] [69] [70] . neuroimaging studies have indicated a reduction in both the volume and the activation of brain regions related to emotional processing [71, 72] . the gray matter (gm) volume of both the frontal cortex and the anterior midcingulate cortex was reduced in patients with crohn's disease (a type of ibd) compared to controls. more interestingly, disease duration was found to correlate with the gm volumes of some brain regions, importantly, limbic areas [71] . also, patients with ulcerative colitis (another type of ibd) showed reduced activity, evaluated by bold signal, within the amygdala, thalamic regions, and cerebellar areas during the emotional visual task, compared to the control group [72] . for the first time in 1991, cohen and his colleagues demonstrated that higher psychological stress is associated with lower resistance to respiratory viruses (rhinovirus type 2, 9, or 14, respiratory syncytial virus, or coronavirus type 229e) in a dose-response manner [73] , while positive emotional style (pes), but not negative emotional style (nes), was found to correlate inversely with susceptibility to common cold and upper respiratory infections following exposure to rhinoviruses and influenza a virus in a dose-response manner [74, 75] . various regression analyses showed that this correlation is independent of prechallenge virus-specific antibody, virus type, age, sex, education, race, body mass, season, and nes, optimism, extraversion, mastery, self-esteem, purpose, and selfreported health [74, 75] . by contrast, childhood socioeconomic status, as assessed by "the number of childhood years during which their parents owned their home," was found to correspond negatively with both the risk of illness and infection and, in a word, with vulnerability to common colds [76] . this finding along with approximately the similar increased risk of common colds in "those whose parents did not own their home during their early life but did during adolescence" in "those whose parents never owned their home" [76] indicate that (a) the childhood period takes more impression of socioeconomic status of their family than other lifetime periods (e.g., adolescence) such that (b) it would influence the mind-body background of future life. meanwhile, pes and nes were negatively and positively related to the subjective report of unfounded symptoms of common cold, respectively [74, 75] . however, the basal protein levels of all the investigated proinflammatory cytokines, e.g., il-1β, il-6, and il-8, were associated with illness symptoms/ signs after exposure to rhinoviruses. however, il-6 was the best cytokine which could predict nasal symptoms/signs [77] . further, daily evaluation of emotional style and cytokine production in infected individuals on each one of 5 days after exposure to rhinoviruses and influenza virus showed that the production of inflammatory cytokines including il-6, il-1β, and tnf-α was negatively related to positive affect (pa) on that day or on the next day [78] . neurological diseases including parkinson's disease (pd) and alzheimer's disease (ad) are accompanied by serious shortfalls in emotional processing in a severity-dependent manner. for example, patients with frontotemporal dementia (ftd) represent a poor recognition of several basic emotions, e.g., anger, sadness, disgust, fear, and contempt. also, patients with the probable ad are more likely to fail to recognize fear and contempt compared with controls [79] . in patients with mild ad, the recognition of more basic emotions are missed, and they are less able to differentiate between some emotions, e.g., happiness and sadness [80] . apathy in patients with ad was found to correlate positively with dysfunction in the prefrontal and anterior temporal regions [81] . regarding memory recall, individuals with ad presented no preference to recall better emotional memories other than nonemotional ones, standing in stark contrast to healthy subjects, either young or older [82] . experimental models provided evidence that there are deficits in the emotional memory performance in ad, which can be diminished by treatment with cytotoxic necrotizing factor 1 (cnf1) [83] . this pleasant effect of cnf1 was accompanied by a reduced il-1β expression in the hippocampus, along with other encouraging events, especially enhancing the energy amount evaluated by the atp (adenosine triphosphate) levels [83] . there is a spectrum of behavioral problems in patients with ad [84] . agitation is the second most common behavior in ad, after apathy. it has been associated with cognitive impairment [84] . inflammatory changes appear to pave the way for agitation. there were higher il-1β levels and decreased nk cell activity in both the morning and evening periods corresponding with preagitation and agitation phases of ad [85] . even, esterling and his colleagues demonstrated changes in the immune profile of ad patients' spousal caregivers, either former or current. there was a reduced response of enriched nk cells to either ril-2 or rifn-γ cytokines in patients' caregivers than controls [86] . interestingly, this response was related positively to the emotional and tangible social support levels [86] . women with severe or morbid obesity had significantly increased levels of proinflammatory markers il-6 and hscrp in a bmi-dependent manner, which were closely related to anxiety and depression subscales of neuroticism, even after the bmi adjustment [87] . since these patients had to undergo gastric surgery, these markers were measured again after surgery. interestingly, decreased levels of il-6 and hscrp were correlated with lower anxiety and depressive behaviors postoperation [87] . the long-term maternal exposure (4 weeks before mating and during pregnancy and lactation) to a high-fat diet (hfd) led to a decrease in the basal serum levels of cort in offspring [88] . in addition, the steps toward normalizing the stress-induced cort levels were made with the lower speed in long-term hfd-exposed offspring than standard chow diet (sd)-exposed offspring at the end of stress challenge [88] . regarding inflammation-related markers, the increased expression of il-6 and il-1ra in long-term hfd-exposed offspring than chow-exposed ones in the amygdala was found in both females and males [88] , while changes in the expression of nf-kb and i-kappa-b-alpha (ikba) were observed only in female, but not in male, offspring [88] . mice subjected to short-term (1-3 weeks) hfd also exhibited anxiety-like behaviors in addition to learning and memory impairments and had significantly higher levels of homovanillic acid-a metabolite of dopamine-in their hippocampus and cortex but without any alteration in the gene expression of inflammatory markers [89] . further, chronic western diet (wd) intake led to the increased responsiveness to lps, which was represented in higher and more prolonged protein/mrna measures of il-6 in both plasma and hypothalamus, while there was no significant difference in the plasma levels of other proinflammatory cytokines such as tnf-α, il-1β, and ifn-γ between wd and sd groups [90] . in parallel with the increased expression of il-6, there was significantly increased mrna expression of socs-3, which belonged to the suppressors-of-cytokinesignaling (socs) family of proteins, in the hypothalamus in wd than sd mice [90] . however, the lps-induced mrna expressions of tnf-α and ifn-γ in the hippocampus were significantly higher in wd than sd mice. also, lps augmented the levels of adipokines, e.g., cst, leptin, and resistin, more significantly in wd mice when compared with mice exposed to sd [90] . altogether, both short-term and long-term obesity in either young adult or maternally can lead to display disturbed anxiety-like behaviors and impaired learning/memory, and brain inflammation might be one of the reasons behind these hfd-related events [88] [89] [90] [91] . chronologically, at first, learning/memory and then anxiety-like behaviors are impaired, and disturbance in depressive-like behaviors is subject to exposure to an immune challenge, such as lps [90] . a high age-adjusted prevalence rate of ~24% is estimated for metabolic syndrome (visceral obesity, dyslipidemia, hyperglycemia, and hypertension) in the united states [92] . both er-and edr-related subscales have been associated with the metabolic syndrome factor [93] . even, a disease pathway involving edr can be proposed, which is triggered by low socioeconomic status (ses), followed with low reserve capacity for high negative emotions and eventuated in the metabolic syndrome factor [94] . inflammation plays a major role in metabolic syndrome [95] . it has also been indicated that this role is not performed in the periphery merely, but a mice model of metabolic syndrome proved the presence of central inflammation (↑tnf-α, ↑il-1β, and ↑il-6) in the hippocampus, explaining the anxiety-like behavior in this model [96] . a possible pathogenic pathway for metabolic syndrome is initiated by emotional stress and ensuing enhancement in the levels of proinflammatory cytokines, e.g., il-1, il-6, and tnf-α. then, these cytokines lead to increased levels of ngf, which in turn stimulates a series of cascades toward insulin resistance and finally resulting in diabetes mellitus (for review, see [97] ). skin diseases are frequently associated with troubled er, reflecting in problematic emotional expression. for instance, patients with psoriasis, who are more likely to be alexithymic, employ more control over negative emotions and more avoidance of emotional closeness and intimacy compared with controls [98] . it may explain the negative relationship between psoriasis symptoms and affective expression in a severity-dependent manner [99] . when patients with atopic dermatitis were compared to healthy controls, the effect of psychological stress on the various immune parameters, such as ↑ eosinophil count, ↑ cd8 + /cd11 + and cla + t cells, and ↑ cytokines (il-5 and ifn-γ), was significantly strengthened [100] . the route from edr to immdr in acnegenesis has been explained elsewhere [101] . in summary, emotional distress would make sebocytes prone to the increased expression of receptors for crh, melanocortins, b-endorphin, vasoactive intestinal polypeptide, neuropeptide y, and calcitonin gene-related peptide, and then the production of proinflammatory cytokines are stimulated by means of these receptors and binding to their ligands. this notion that sleep disturbances are, irrespective of their cause, seen as chronic stressors is supported by evidence at different levels, e.g., immunological, neuropathological, and neuroimaging studies (for review, see [102] ). to assess the effects of sleep and its efficiency on susceptibility to respiratory infections, cohen and his colleagues conducted an investigation on healthy individuals and recorded at first their sleep duration and its efficiency within 14 consecutive days and then made them exposed to rhinoviruses, and after 5 days postchallenge, calculated the rate of clinical cold development [103] . this investigation indicated that those who had average sleep duration (asd) ≤7 hours were three times more susceptible to clinical cold than those with asd≥8 hours. further, there was a 5.5-fold increased risk of clinical cold in individuals with sleep efficiency <92% compared to those with an efficiency of ≥98%. therefore, it is well expected that circadian arrhythmia has been associated with edr-related parameters, e.g., decreased social motivation/functioning, decreased exploratory anxiety, and decreased emotional functioning [104, 105] . in a cancer population, the presence of circadian arrhythmia was associated with decreased levels of all the investigated cytokines, e.g., tnf-α, tgf-α, and il-6 [105] . alexithymic patients were more likely to suffer from severe forms of stroke when compared with non-alexithymic patients. this alexithymia trait appears to contain an inflammatory component either as a cause or an effect [106] . study of patients with a first-ever symptomatic ischemic stroke revealed that (a) the circulating levels of il-18 were correlated positively with the severity of alexithymia, (b) stratification of patients made this correlation more statistically significant in those who had right hemisphere lesions, and (c) these increased il-18 levels were pronounced in alexithymic (tas-20 score 61) than non-alexithymic patients [106] . in patients hospitalized for orthopedic injuries, the use of emotion-focused coping was found to correlate positively with the levels of proinflammatory cytokines, il-6 and il-8, whereas it was negatively correlated with tgf-β levels [107] . compared with controls who received placebo, circulating levels of cytokines, in particular, il-6, were increased at 3 hours after the first-ever typhoid vaccination. it coincided with significant mood impairment [108, 109] . it has been elucidated that when subjects perform psychological tasks (i.e., stress condition) after injection, the il-6 response is inversely related to optimism in either the typhim vi typhoid vaccine or saline placebo group [110] . in response to the implicit emotional face perception task, there was an increased activity in the subgenual anterior cingulate cortex (sacc) along with reduced functional connectivity between sacc and reduced activity within the anterior rostral medial prefrontal cortex (armpfc), mni coordinates, nucleus accumbens, right amygdala, sts, and ffas. these changes were observed in inflammation-associated mood change compared to the placebo group [108] . chapter 10 presented evidence supporting the notion that there are a variety of psychological states/psychiatric diseases where the immune responses, as well as the emotion regulation, are impaired. this chapter provided evidence linking physiological states/physical diseases to the impairment of both the immune system and emotion regulation. altogether, the immunoemotional regulatory system (immers) covers both psychological states/psychiatric diseases and physiological states/physical 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cord-255575-640v00jg authors: binny, r. n.; baker, m. g.; hendy, s. c.; james, a.; lustig, a.; plank, m. j.; ridings, k. m.; steyn, n. title: early intervention is the key to success in covid-19 control date: 2020-10-23 journal: nan doi: 10.1101/2020.10.20.20216457 sha: doc_id: 255575 cord_uid: 640v00jg new zealand responded to the covid-19 pandemic with a combination of border restrictions and an alert level system that included strict stay-at-home orders. these interventions were successful in containing the outbreak and ultimately eliminating community transmission of covid-19. the timing of interventions is crucial to their success. delaying interventions for too long may both reduce their effectiveness and mean that they need to be maintained for a longer period of time. here, we use a stochastic branching process model of covid-19 transmission and control to simulate the epidemic trajectory in new zealand and the effect of its interventions during its covid-19 outbreak in march-april 2020. we use the model to calculate key outcomes, including the peak load on the contact tracing system, the total number of reported covid-19 cases and deaths, and the probability of elimination within a specified time frame. we investigate the sensitivity of these outcomes to variations in the timing of the interventions. we find that a delay to the introduction of alert level 4 controls results in considerably worse outcomes. changes in the timing of border measures have a smaller effect. we conclude that the rapid response in introducing stay-at-home orders was crucial in reducing the number of cases and deaths and increasing the probability of elimination. an outbreak of covid-19, a novel zoonotic disease caused by the sars-cov-2 virus, was first detected in wuhan, china in november 2019. the virus spread rapidly to other countries resulting in a pandemic being declared by the world health organisation in march 2020. governmental policy responses to covid-19 outbreaks have varied widely among countries, in terms of the nature and stringency of policy interventions, how quickly these interventions were implemented (table 1 ) (desvars-larrive et al., 2020) and their effectiveness at reducing spread of the virus (flaxman et al., 2020; hsiang et al., 2020; binny et al., 2020a) . while it is tempting to judge the success of interventions by comparison across jurisdictions, this assessment may be confounded by local context that may influence success, as well as by the fact that policy choices can be driven by the severity of initial outbreaks. models of disease spread played an important role in the design and timing of interventions, but they can also be used post hoc, to evaluate the effectiveness of those interventions. for example, flaxman et al. (2020) and brauner et al. (2020) fitted models of disease dynamics to case count and death data in different countries to estimate the effect of specific non-pharmaceutical interventions on the transmission rate of covid-19. in response to the escalating covid-19 pandemic and the outbreak that was establishing in new zealand in march 2020, a number of policy interventions were implemented to mitigate risk at the border and risk of community transmission. from 15 march 2020 (11.59pm), border restrictions were put in place requiring all international arrivals to 'self-isolate' (home quarantine) for 14 days. on 19 march 2020, the border was closed to everyone except returning citizens and residents. a system of four alert levels was introduced on 21 march with the alert level initially set at level 2. on 23 march, it was announced that the alert level was increasing to level 3 , and that the country would move to alert level 4 as of 11.59pm on 25 march, signalling that nz was taking a decisive covid-19 response that would become known as an elimination strategy (baker et al., 2020a) . at the time alert level 4 came into effect, there had been 315 reported (confirmed and probable) cases. alert level 4 stayed in place until 27 april when restrictions were eased to alert level 3. on 13 may, after 16 days at alert level 3, daily new cases had dropped to 3 and there was a phased easing into alert level 2 ( table 2) . the seven weeks spent under stringent alert level 3 or 4 restrictions, which included stay-at-home orders (see appendix table s2 for full list of measures) alongside systems for widespread testing, contact tracing and case isolation, were effective at reducing transmission (reff = 1.8 prior to alert level 4; reff = 0.35 during alert level 4; binny et al., 2020b) . daily numbers of new cases declined to between zero and one by mid may and the last case of covid-19 associated with the march outbreak was reported on 22 may. on 8 june, it was estimated that new zealand had very probably eliminated community transmission of covid-19 after 17 consecutive days with no new reported cases . between 22 may and 11 august, the only new cases detected were associated with international arrivals and during this period these arrivals were required to spend 14 days in government-managed isolation or quarantine facilities (baker et al, 2020b) . on 9 august 2020, new zealand reached a milestone of 100 days with no community transmission. for each hypothetical scenario, we simulate a model of covid-19 spread and compare key outcomes, including the peak load on the contact tracing system, the cumulative numbers of cases and deaths, and the probability of elimination predicted by the model. in particular, we assess how important new zealand's decision to move 'hard and early' was for the successful elimination of community transmission during the march-april outbreak. to this end, we compare scenarios with different timings until the start of alert level 4 to see how these choices could have affected the size of the outbreak. while alert level 4 was successful in achieving elimination, the benefits of elimination had to be weighed against the negative impacts of stringent stay-at-home measures, for example job losses, increased rates of domestic violence, disruption to education, and impacts on mental health. if careful border management could have avoided the need for a lockdown or reduced its intensity, this approach may have been preferable. for instance, taiwan's early border closure, travel restrictions and 14-day quarantine for those entering the country have meant that, to date, taiwan has avoided a mass lockdown (summers et al., 2020) . we explore whether introducing border restrictions earlier in new zealand might have been sufficient to eliminate or reduce transmission from international arrivals to the extent where stringent alert level 4 restrictions could have been avoided or less restrictive measures been sufficient. compared to other countries, new zealand was very quick to close its border to all except returning citizens and residents (table 1) . we explore a scenario where border closure is delayed by 5 days to assess how much larger the outbreak might have been had new zealand been slower to act. finally, we consider a scenario with no al4/3 restrictions to compare the size of outbreak that new zealand could have experienced if border restrictions and closure had been the only control measures. in this study, we focus on the timing of interventions; we do not explicitly consider the duration of interventions, although this will be investigated in future work. indeed, the likelihood of elimination was one of the factors taken into account in new zealand government decision-making concerning the duration of alert levels (dpmc, 2020). we simulated a stochastic model of covid-19 spread in new zealand under alternative scenarios in which implementation of border restrictions, border closure and alert level 4 were either delayed or started earlier. in each scenario, we kept the duration of each alert level the same as actually occurred, i.e. 33 days at alert level 4 followed by 16 days at alert level 3. we explored the following scenarios (see table 2 a full description of the model is provided in james et al. (2020a) . we obtained case data from esr, containing arrival dates, symptom onset dates, isolation dates and reporting dates for all international cases arriving in new zealand between february and june. border restrictions, border closure and start of al4 were all implemented at 11.59pm so we start simulating their effects on the day after their implementation date. for scenarios 0, 1, 2, 3 and 6, the model was seeded with the same number of international cases as were actually reported. in scenarios where border restrictions were implemented on the actual start date (15 march; scenarios 0, 1, 2, 4 and 6), the self-isolation dates of international cases were set to the same isolation dates as were actually reported. in all scenarios, prior to 9 april the modelled effect of self-isolation is to reduce an individual's infectiousness to 65% of their infectiousness when not isolated. this reflects some risk of onward transmission for cases self-isolating at home. after 9 april, the model assumes that all international cases are placed in government-managed isolation and quarantine (miq) facilities and do not contribute to local transmission. we also simulated a poisson-distributed random number of international subclinical cases in proportion to the number of international clinical cases (assuming 1/3 of all cases are subclinical), with arrival and symptom onset dates that were randomly sampled with replacement from the international case data. we assume that these international subclinical cases are not detected and therefore do not self-isolate, but those arriving after 9 april are placed in miq. to simulate border restrictions starting 5 days early (scenarios 3 and 5a), international cases arriving between the earlier start date and the actual start date (11 -15 march, inclusive) were assumed to be . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october 23, 2020. ; self-isolated on their date of arrival. to simulate a 5-day delay to border closure (scenarios 4 and 5b), we delayed the arrival dates (and associated symptom onset, reporting and isolation dates) of international seed cases arriving after 19 march by 5 days. we then allowed for new international cases arriving over these 5 days (e.g. additional non-residents that may have chosen to travel had the border remained open for longer) by seeding an additional poisson-distributed random number of international cases from 20 march to 24 march, with an average daily number of seeded cases equal to the actual average daily number of international cases arriving during the week prior to 19 march (33 international cases per day). these additional seeded cases were assumed to self-isolate on arrival and their delays from arrival-to-symptom onset and arrival-to-reporting were randomly sampled with replacement from the corresponding delays in the actual international case data. we did not attempt to simulate scenarios with delayed border restrictions or earlier border closure because these would have required additional modelling assumptions about isolation dates of international arrivals and about the reduction in the volume of international arrivals resulting from border closure. model predictions would have been highly sensitive to these assumptions and, without data available to validate them, this would introduce additional model uncertainty. for each scenario, we assessed the following key measures describing the dynamics of a covid-19 outbreak: 1. the maximum contact tracing load, by calculating the maximum number of daily new reported cases and the date on which this occurred. 2. the number of daily new reported cases at the end of alert level 4. 3. cumulative number of reported cases and the cumulative number of deaths at the end of the seven week period of alert level 3-4 restrictions. 4. probability of elimination, p(elim), 5 weeks after the end of al3. we performed 5000 realisations of the model and report the average value of each key measure as well as the interval range within which 90% of simulation results were contained (in square brackets throughout). here, we define elimination as there being no active cases (we assume a case remains 'active' for 30 days after date of exposure) that could contribute to future community transmission. this definition excludes cases in miq, that is it excludes international arrivals after 9 april 2020. in the model, p(elim) was calculated as the proportion of all model realisations that resulted in elimination. simulations were run using estimates of reproduction number reff that provided the best fit to actual data : for the period prior to lockdown reff = 1.8; during alert level 4 reff = 0.35. under the alert levels 3, 2 and 1, which followed the lockdown, the daily numbers of new cases were too low to obtain reliable estimates of the effective reproduction number reff. instead we simulated the model for assumed values of reff = 0.95, 1.7 and 2.4, respectively. reff = 2.4 is in line with estimates reported in plank et al (2020b) for the pre-lockdown period of new zealand's august-september outbreak, when al1 restrictions were in place. al2's reff would likely be lower than al1 but greater than one due to relatively high activity levels and contact rates as stay-at-home orders are lifted and public venues, businesses and schools re-open; reff = 1.7 is in the range of estimated values for the prelockdown period of the march-april outbreak given in plank et al (2020b) . the reff for al3 was chosen to be less than 1, however we tested the sensitivity of our results to using a value greater than one. for the scenario with no stringent alert level restrictions (scenario 6), we simulated the model using reff = 1.8 for the entire period (i.e. the same value as was used in all scenarios for the period prior to al4). . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october 23, 2020. ; we investigated the sensitivity of our results to varying the length of delay for the start of al4, to introducing border restrictions 10 days early (cf. 5 days early in scenario 3), and to other choices of reff under al3. to check that the model could accurately replicate the outbreak, we first simulated our model with border restrictions, border closure and al4 implemented on the dates they actually occurred. the predicted dynamics of daily new reported cases were a very good visual match to observed daily case data ( fig. 1) and predicted key measures showed good agreement with the values that were actually observed ( table 3 , bold text). after moving into al4, the model prediction and the actual number of daily new reported cases both levelled off at 70-80 for around one week before case numbers started to decline (fig. 1 ). in actual case data, the maximum of 84 new cases per day was observed at the start of this flat-topped peak, while our model predicted a similar maximum (80 [67, 99] new cases per day) occurring 6 days later. by the end of al3, the model predicted similar cumulative totals to the 1502 cases and 21 deaths actually reported. five weeks after al3 restrictions were relaxed, elimination of community transmission of covid-19 was achieved in 66% of model simulations, giving p(elim)=0.66 (table 3 ). in the following scenarios with alternative timings of interventions, we use scenario 0 as a baseline for comparing key measures. under a scenario where al4 was implemented 5 days earlier (only one day after border closure), the model predicts slightly lower values for most key measures than were actually observed: daily new cases peaked at a lower level of 69 [61, 79] cases around 26 march and at the end of al4 had dropped to a similar level of 4 new cases per day as was actually observed (fig. 1) . by the end of the 7 weeks of al4/3 it predicts approximately 500 fewer cases in total and 10 fewer deaths (table 3 ; scenario 1 cf. scenario 0). however, this estimate should be taken with caution because of the small numbers of daily cases and fine-scale variations involved: for instance, whether an outbreak occurred in an aged care facility or not. five weeks after al3, the probability of elimination was 63%, slightly lower than in scenario 0. this counter-intuitive result is due to the presence of an international case in the data that had an arrival date prior to the start of al4 (25 march) but a much later symptom onset date near the end of al4. in scenario 0, when international cases are seeded in the model, this individual's peak infectiousness occurs during al4. however, in scenario 1, the earlier start to al4 means that the individual is instead most infectious during al3. with a lower reff in al3, this individual infects more people, on average, in this scenario than in scenario 0. similarly, any simulated subclinical cases with the same arrival and symptom onset dates (drawn from the international case data) will also be most infectiousness during al3. these subclinicals do not appear in the numbers of reported cases but will reduce the probability of elimination. if this international case outlier is excluded from the data, the model predicts a very similar probability of elimination in both scenarios. delaying the move into alert level 4 would have led to a higher peak in daily new cases, and greater cumulative totals of cases and deaths. for a delay of 20 days (scenario 2c), the outbreak would have reached a considerably higher maximum of close to 500 daily new cases (cf. 80 cases in scenario 0; table 3 , fig. 1 and fig. 2 ). this number would certainly have overwhelmed the contact tracing system, which was already pushed close to capacity in places by the 70-80 daily new cases in late march is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october 23, 2020. ; https://doi.org/10.1101/2020.10.20.20216457 doi: medrxiv preprint (verrall, 2020) . after a week in al4, case numbers would start to decline and by the end of the 4 weeks in al4 daily new cases would still have been as high as 34 [22, 49] (close to the actual number of domestic daily reported cases when new zealand went into al4 on 25 march). by the end of the 7 week period of stringent restrictions (i.e. end of al3) the incidence would have dropped to approximately 4 new cases per day (fig. 1) , but the cumulative total could have climbed to 11,534 [8854, 15048] reported cases and 200 [147, 266] deaths, substantially more than scenario 0 and the 1,502 cases and 21 deaths actually reported on 13 may. additionally, the probability of elimination 5 weeks after the end of al3 was only 7%, much lower than scenario 0. we next investigated a scenario where border restrictions were put in place 5 days earlier, but border closure and al4 were started on their actual dates. border restrictions would therefore have been in place for 9 days (cf. actual 4 days) before the border was closed. our model predicted this would have had very little impact for the initial trajectory ( fig. 1) or eventual size of the outbreak, with values for all key measures very similar to those in scenario 0 ( table 3 ). this finding suggests that key measures are more sensitive to varying the timing of al4 than to the timing of border restrictions. in reality, out of the 563 international cases who arrived prior to the start of miq and could have contributed to local transmission, only 78 (14%) arrived before border restrictions were implemented on 15 march and were not required to self-isolate. furthermore, out of these 78 cases, 52 arrived between 10 and 15 march and 19 of these were reported to have voluntarily self-isolated immediately on arrival (the model simulates these 19 cases as being self-isolated on arrival in all scenarios). therefore, under this scenario, only an additional 33 international cases have their infectiousness reduced by early self-isolation requirements. this reduction is not sufficient to prevent an outbreak, nor does it reduce transmission to an extent where al4/3 restrictions would not have been necessary to control the outbreak. under a scenario where closure of the border (to all except returning residents and citizens) was delayed by 5 days (24 march; 9 days after border restrictions and 1 day before al4), our model predicted slightly worse outcomes, on average, for key measures than were predicted in scenario 0. however, due to the stochasticity of individual simulations, the range of key measures always had overlap with the scenario 0 values and actual values, suggesting that a 5 day delay to border closure alone would not have made a significant difference. a delayed border closure did, however, have a greater impact for the probability of elimination 5 weeks after al3 restrictions were relaxed, which was only 55%, compared to 66% chance in scenario 0. this reduced probability of elimination is partly due to the additional international clinical cases (captured in the key measures of reported cases) and international subclinicals (not captured in reported cases) arriving prior to the delayed border closure. it is also likely affected by the international case outlier with the pre-miq arrival date and late onset date, discussed above. if border restrictions were implemented 5 days early and al4 came into effect 5 days early (scenario 5a), this would have led to outcomes very similar to those predicted in scenario 1 (where only al4 started early) ( table 3 ). this again suggests that results are more sensitive to changes in timing for the start of al4 than to an earlier start to border restrictions. in contrast, if border closure and the start of al4 had both been delayed by 5 days (scenario 5b), outcomes would have been worse than a delay in only one of these interventions ( is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october 23, 2020. ; https://doi.org/10.1101/2020.10.20.20216457 doi: medrxiv preprint the end of the 7 week period in al4/3, there would have been close to 1,050 more cases in total and nearly 20 more deaths than in scenario 0 ( table 3 ). the probability of elimination 5 weeks after al3 would have also been reduced to 53%. finally, we explored the impact of only having border restrictions and border closure in place, but without implementing al4/3. under this scenario, the international cases who arrived prior to 9 april and were either in self-isolation or were not isolated have a chance of seeding an outbreak which, without al4/3 measures to reduce reff below one, leads to community transmission and a large uncontrolled outbreak. new zealand would have seen close to 1127 [841, 1492] new cases per day by 27 april, the date on which new zealand moved from al4 to al3 in reality. by 13 may (the date on which new zealand moved from al3 to al2), there could have been over 60,000 cumulative reported cases and over 1100 deaths. new cases would have continued to increase, reaching a peak of 47,592 [47, 240, 47, 962] daily new cases on 14 june (table 3 ). by the end of the outbreak, around october 2020 on average, there could have been over 1.81 million reported cases in total and 31,905 [31, 606, 32, 204] deaths. no simulations resulted in elimination by 18 june (5 weeks after end of actual al3), indicating a 0% chance of covid-19 having been eliminated by this time, compared to the 66% chance on this date in scenario 0. we assessed the effect that different lengths of delay (in days) until the start of al4 (fig. 2) had on key measures: maximum load on contact tracing system; cumulative total reported cases; total infected cases (including both clinical and subclinical); total deaths at end of al3; and probability of elimination 5 weeks after the end of al3. measures of numbers of cases and deaths increased exponentially with increasing delay to al4, emphasising the importance of acting quickly to reduce the risk of large outbreaks arising. probability of elimination decreased linearly with increasing delays to al4. counterintuitively, earlier starts to al4 slightly reduced the probability of elimination; again, this is caused by the international case outlier discussed previously. if the outlier is excluded from the international case data, the predicted probability of elimination is insensitive to al4 starting 1 to 5 days early. introducing border restrictions ten days earlier still results in an outbreak and gives very similar results to scenario 3 (5 days early), with a maximum of 77 [65, 94] new daily cases, 1385 [1166, 1706] cumulative reported cases at the end of al3, p(elim) = 0.68, and other measures the same as in scenario 3. with border restrictions ten days earlier, the only difference compared to scenario 3 is that an additional 16 cases who arrived between 5 and 10 march have their infectiousness reduced (a further 2 cases arriving in this 5-day period were voluntarily self-isolated on arrival in reality, so are simulated with self-isolation on arrival in all scenarios). this restriction has little impact on the overall contribution to local transmission by all 563 international cases who arrive prior to the start of miq. we also tested the sensitivity of all key measures to using different values of reff under al3 (table s1 ; reff = 1.1, 0.95 and 0.7). different choices of al3 reff had very little effect on predicted cumulative totals of cases at the end of al3 and no effect on total deaths at end of al3. however, the predicted probability of elimination was sensitive to varying al3 reff ; for all scenarios, assuming a lower reff = 0.7 (more effective al3) gave a p(elim) that was approximately 0.14 higher than with reff = 0.95, while a higher reff = 1.1 (less effective al3) reduced p(elim) by approximately 0.07. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october 23, 2020. ; level 4 (i.e. end of alert level 3) , and the total number of deaths 7 weeks after the start of alert level 4. for each measure, except p(elim) , the mean value from 5000 simulations is reported alongside the interval range, in parentheses, in which 90% of simulations results are contained. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october 23, 2020. ; https://doi.org/10. 1101 is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october 23, 2020. ; https://doi.org/10.1101/2020.10.20.20216457 doi: medrxiv preprint new zealand's decision to act quickly and to implement stringent restrictions to reduce sars-cov-2 transmission meant that, to date, new zealand has experienced amongst the lowest mortality rates reported worldwide (kontis et al., 2020) . on 8 june 2020, nearly 11 weeks after al4 was initiated, new zealand declared elimination of covid-19. over the course of the march-april outbreak, a total of 1504 cases and 22 deaths were reported before elimination was achieved. our results suggest that the timing of alert level 4 is a much stronger driver of reductions in daily new cases than timings of border restrictions and closure. this finding makes sense because the effect of al4 in the model is to greatly reduce reff for all cases, domestic and international arrivals, to 0.35, while border restrictions reduce the delay until case isolation of international cases only (i.e. international cases have their infectiousness reduced earlier) and border closure reduces the daily numbers of international cases only. out of the scenarios we considered, an earlier start to al4 by 5 days resulted in the greatest reduction in numbers of cases and deaths, with approximately 500 fewer cases in total and 10 fewer deaths. however, in reality, the rapid escalation of the covid-19 situation in mid-march may have made an earlier start to al4 impractical and would have allowed less time to prepare for ongoing provision of essential services under al4. introducing border restrictions requiring 14-day self-isolation for international arrivals earlier than 15 march would have been unlikely to have much impact on the trajectory of new zealand's march-april outbreak, unless such measures were started prior to the first case on 26 february and used methods that were particularly effective (notably full miq). the 563 international cases arriving between 15 march and 9 april were already required to self-isolate; had border restrictions been in place prior to the arrival of new zealand's first case, this would have required self-isolation for, at most, an additional 56 international cases (22 cases who arrived prior to 15 march self-isolated voluntarily immediately on their arrival). in mid-march, there was a lower global prevalence of covid-19 and between 2 and 12 cases arrived at the border each day in the week prior to 15 march. with a higher global prevalence and correspondingly higher numbers of international cases arriving per day, earlier implementation of border restrictions may have had a greater impact than our model predicted for this outbreak. self-isolation is less stringent than miq and relies heavily on public compliance. without additional safety nets, such as official monitoring and support for people who are self-isolating, there is a greater risk of the virus spreading into the community than in miq facilities. for example, risk of non-compliance may be higher for individuals who are concerned about loss of income (bodas & peleg, 2020) . without alert level restrictions in place to require strong communitywide social distancing, any infected individuals who do not self-isolate effectively are more likely to spark an outbreak. self-isolation restrictions for international arrivals can therefore reduce the frequency of cases leaking into the community but are unlikely to be sufficient to prevent an outbreak entirely, unless additional measures are also put in place. delaying border closure by 5 days could have led to a slightly larger outbreak, but not as large as if al4 had been delayed by 5 days. the full effect on local transmission potential of the additional international cases expected under a delayed border closure was partially dampened because international cases arriving after 9 april were still placed in miq and assumed not to contribute to community transmission. if the timing of this miq policy was also delayed, a larger outbreak may have occurred, but we did not model such a scenario here. if the start of al4 had been delayed by 20 days, our results suggest new zealand could have experienced over 11,500 reported cases and 200 deaths, reducing the chance of elimination to only 7%. as with other severe viral disease, the infection fatality risk for covid-19 is greater for mä�ori and pacific peoples (close to 50% higher for mä�ori than for non-mä�ori) wilson et al., 2012; verrall et al., 2010) . therefore, in scenarios resulting in significantly higher numbers of covid-19-related deaths (e.g. scenario 2c), mä�ori and pacific communities would likely have been disproportionately affected, however a population-structured model would be required to assess this consequence in detail. delaying al4 would have also increased the chance of a longer lockdown period being required to reduce daily new case numbers to low levels. with a 20 day delay to al4, new zealand could still have been experiencing close to 35 new reported cases per day at the end of al4. while in reality, a 33-day period in al4 was sufficient to reduce daily new cases to below 10, and the government announced an easing to al3, these higher case numbers predicted for a delayed start to al4 may have motivated an extension to the lockdown to allow more time for cases to drop below a safe threshold. in terms of the key measures we considered, the counterfactual scenario with no al4/3 restrictions (scenario 6) had disastrous outcomes, including close to 2 million reported cases and tens of thousands of deaths. our model uses a value of reff =0.35 during al4, which was estimated by binny et al (2020b) by fitting the model to data, and is consistent with a later estimate of reff from reconstructions of the epidemiological tree . this is a relatively low value of reff compared to other countries who implemented interventions roughly equivalent to al4 (flaxman et al, 2020; binny et al, 2020a) . a combination of a highly effective social distancing in al4, fast contact tracing, effective case isolation, and the fact that the outbreak occurred at the end of the southern hemisphere summer, likely contributed to this low reff . for scenarios where the load on contact tracing exceeded system capacity (e.g. scenario 2c with a maximum 500 daily new cases), this effect would have likely resulted in longer delays to isolation of cases and a higher reff. we did not attempt to model this potential feedback effect and so our results for scenarios where contact tracing system capacity is exceeded may underestimate the size of the outbreak. we also did not attempt to directly model the burden of covid-19 on the healthcare system (e.g. numbers of cases requiring hospitalisation or intensive care), or the effects of an overwhelmed healthcare system. once numbers of daily new cases requiring hospitalisation or icu admission exceed new zealand's healthcare system capacity, this could result in increased fatality rates and considerably more deaths . these effects would have been most pronounced under the scenario with no al4/3 restrictions. it is important to note that, while we report average values for outbreak dynamics, each individual realisation of the stochastic model can deviate (sometimes widely) from the average behaviour. when case numbers are small, as they were in new zealand, the predicted dynamics are particularly sensitive to fine-scale variations. while reff < 1 means that an outbreak will eventually die out, on average, it is still possible for a small number of cases to spark an outbreak in a particular stochastic realisation if interventions are relaxed too soon. conversely, when case numbers are small, an outbreak can still die out by chance even when reff > 1. it is therefore important to account for this stochasticity when weighing the effectiveness and risks of different intervention strategies, for example by considering the probability of elimination. on 18 june, five weeks after al3 restrictions were relaxed, the probability that community transmission of covid-19 had been eliminated in model simulations was estimated to be 66% in scenario 0. this estimate is calculated by finding the proportion of stochastic realisations that resulted in elimination. in reality, as the outbreak died out and more days with zero new cases were observed, this provided additional information about which trajectory new zealand was most likely experiencing. each additional consecutive day with no new reported cases reduced the likelihood of . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october 23, 2020. ; being on an upward trajectory. making use of this information meant that the actual probability of elimination on 18 june was estimated to be 95% higher than in scenario 0 . however, this estimate required up-to-date information about recent case numbers. the results reported in this paper compare average outcomes under different scenarios, which is appropriate for evaluating the effect of alternative actions and guiding future decision making. in general, for the other scenarios we explored, bringing in earlier interventions had very little impact on probability of elimination, while delaying border closure or al4 reduced the chance of elimination. our results are important for reflecting on the effectiveness of intervention timing in new zealand's covid-19 response, relative to alternative scenarios, to help guide future response strategies. early intervention was critical to the successful control of new zealand's march-april outbreak. for modelling future disease outbreaks, epidemiological parameters should be updated to reflect changes in national pandemic preparedness (e.g. improved policy and response plans) and behavioural changes influencing the dynamics of future outbreaks. for instance, the degree of compliance with alert level restrictions in future may differ dramatically from the march-april outbreak, resulting in different values of reff. further work is needed to explore the social dynamics affecting transmission and the effectiveness of interventions, for instance whether wearing masks in public spaces becomes more common, or whether more people will choose to work from home or avoid travel if a suspected new outbreak is reported. the key measures of outbreak dynamics assessed here should be considered alongside other measures of economic, social and health impacts (e.g. job losses, consumer spending, impacts for mental health, rates of domestic violence or disrupted education). particular attention needs to be given to identifying vulnerable groups who may experience inequitable impacts so that future policies can be tailored to support these groups. at the end of al3, health benefits (e.g. number of cases and deaths avoided) differed between scenarios. for cost-benefit analyses, age-dependent morbidity and mortality rates of covid-19 (kang & jung, 2020) allow numbers of cases and deaths to be quantified in terms of disability-adjusted life years (dalys) avoided (or quality-adjusted life years gained), which can (with obvious issues) be converted to monetary units to facilitate comparison with economic costs. because the duration spent under al4/al3 was fixed at 7 weeks for scenarios 0-5, the short-term economic costs of the different scenarios would have been similar, so we did not convert health benefits into dalys here. after the end of al3, benefits and costs would differ between scenarios depending on the value of reff for al1-2 and on whether or not elimination was achieved. increased levels of activity and contact rates under al1-2 mean that reff is very likely to have been greater than one. in scenarios with lower probabilities of elimination, it is more likely that new zealand would continue to experience new cases while under al1-2 which, with reff > 1, would likely lead to another outbreak and require a second lockdown (with its associated costs). conversely, scenarios with higher probabilities of elimination mean there is a greater chance of the outbreak dying out entirely and less risk of a second lockdown being required. future work could consider the costs and benefits of alternative scenarios where the duration of time spent in al4 and al3 is dictated by the need to achieve a certain threshold probability of elimination. . cc-by-nc-nd 4.0 international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted october 23, 2020. ; https://doi.org/10. 1101 new zealand's elimination strategy for the covid-19 pandemic and what is required to make it work successful elimination of covid-19 transmission in new zealand effect of alert level 4 on effective reproduction number: review of international covid-19 cases effective reproduction number for covid-19 in aotearoa new zealand. medrxiv preprint probability of elimination for covid-19 in aotearoa new zealand self-isolation compliance in the covid-19 era influenced by compensation: findings from a recent survey in israel the effectiveness of eight nonpharmaceutical interventions against covid-19 in 41 countries cab-20-sub-0270 review of covid-19 alert level 2 cccsl: complexity science hub covid-19 version 2.0 estimating the effects of non-pharmaceutical interventions on covid-19 in europe the effect of large-scale anti-contagion policies on the coronavirus (covid-19) pandemic. medrxiv preprint a structured model for covid-19 spread: modelling age and healthcare inequities successful contact tracing systems for covid-19 rely on effective quarantine and isolation model-free estimation of covid-19 transmission dynamics from a complete outbreak age-related morbidity and mortality among patients with covid-19 magnitude, demographics and dynamics of the effect of the first wave of the covid-19 pandemic on all-cause mortality in 21 industrialized countries a stochastic model for covid-19 spread and the effects of alert level 4 in aotearoa new zealand. medrxiv preprint effective reproduction number and likelihood of cases outside auckland estimated inequities in covid-19 infection fatality rates by ethnicity for aotearoa new zealand. medrxiv preprint potential lessons from the taiwan and new zealand health responses to the covid-19 pandemic. the lancet regional health -western pacific rapid audit of contact tracing for covid-19 in new zealand. ministry of health, new zealand. 0.95 80 evaluated at end of outbreak, approx weeks after end of actual al3) the authors acknowledge the support of statsnz, esr, and the ministry of health in supplying data in support of this work. we are grateful to samik datta, nigel french, markus luczak-roesch, melissa mcleod, anja mizdrak, fraser morgan and matt parry for comments on an earlier version of this manuscript. this work was funded by the ministry of business, innovation and employment and te på«naha matatini, new zealand's centre of research excellence in complex systems. table s1 : key measures from alternative scenarios of early or delayed implementation of policy interventions: the maximum number of daily new cases, date on which the peak occurs, the number of daily new cases at the end of the alert level 4 period, the cumulative number of cases 7 weeks after the start of alert level 4, and the total number of deaths 7 weeks after the start of alert level 4. for each measure, except p(elim) , the mean value from 5000 simulations is reported alongside the interval range, in parentheses, in which 90% of simulations results are contained. â�¢ stay at home except for essential personal movement â�¢ safe recreational activity allowed in local area.â�¢ travel is severely limited.â�¢ all gatherings cancelled and public venues closed.â�¢ businesses closed, except essential services and lifeline utilities.â�¢ educational facilities closed.â�¢ rationing of supplies and requisitioning of facilities possible.â�¢ reprioritisation of healthcare services. â�¢ stay at home except for essential personal movement -including going to work, school or for local recreation.â�¢ work from home if possible.â�¢ low risk local recreation activities allowed.â�¢ inter-regional travel highly limited (e.g. allowed for essential workers, with limited exemptions for others).â�¢ public venues closed (e.g. libraries, museums, cinemas, food courts, gyms, pools, playgrounds, markets).â�¢ gatherings of up to 10 people allowed but only for wedding services, funerals and tangihanga, with physical distancing and public health measures maintained. â�¢ two-metre physical distancing outside home. one metre in controlled environments, e.g. schools and workplaces.â�¢ people must stay within their immediate household bubble, but can expand this to reconnet with close family/whä�nau, or bring in caregivers, or support isolated people. this extended bubble should remain exclusive.â�¢ businesses can open premises, but cannot physically interact with customers.â�¢ schools (years 1 to 10) and early childhood education centres can safely open but with limited capacity. children should learn from home if possible. â�¢ healthcare services use virtual, non-contact consultations where possible.â�¢ people at high risk of severe illness (older people or those with pre-existing medical conditions) are encouraged to stay at home and take extra precautions when leaving home. they may choose to work. key: cord-121777-3zrnz9nc authors: qian, xuelin; fu, huazhu; shi, weiya; chen, tao; fu, yanwei; shan, fei; xue, xiangyang title: m3lung-sys: a deep learning system for multi-class lung pneumonia screening from ct imaging date: 2020-10-07 journal: nan doi: nan sha: doc_id: 121777 cord_uid: 3zrnz9nc to counter the outbreak of covid-19, the accurate diagnosis of suspected cases plays a crucial role in timely quarantine, medical treatment, and preventing the spread of the pandemic. considering the limited training cases and resources (e.g, time and budget), we propose a multi-task multi-slice deep learning system (m3lung-sys) for multi-class lung pneumonia screening from ct imaging, which only consists of two 2d cnn networks, i.e., sliceand patient-level classification networks. the former aims to seek the feature representations from abundant ct slices instead of limited ct volumes, and for the overall pneumonia screening, the latter one could recover the temporal information by feature refinement and aggregation between different slices. in addition to distinguish covid-19 from healthy, h1n1, and cap cases, our m 3 lung-sys also be able to locate the areas of relevant lesions, without any pixel-level annotation. to further demonstrate the effectiveness of our model, we conduct extensive experiments on a chest ct imaging dataset with a total of 734 patients (251 healthy people, 245 covid-19 patients, 105 h1n1 patients, and 133 cap patients). the quantitative results with plenty of metrics indicate the superiority of our proposed model on both sliceand patient-level classification tasks. more importantly, the generated lesion location maps make our system interpretable and more valuable to clinicians. coronavirus disease 2019 , caused by a novel coronavirus (sars-cov-2, previously known as 2019-ncov), is highly contagious and has become increasingly prevalent worldwide. the disease may lead to acute respiratory distress or multiple organ failure in severe cases [1] , [2] . as of june 28th, 2020, 495, 760 of 9, 843, 073 confirmed cases across countries have led to death, according to who statistics. thus, how to accurately and efficiently diagnose covid-19 is of vital importance not only for the timely treatment of patients, but also for the distribution and management of hospital resources during the outbreak. the standard diagnostic method being used is real-time polymerase chain reaction (rt-pcr), which detects viral nucleotides from specimens obtained by oropharyngeal swab, nasopharyngeal swab, bronchoalveolar lavage, or tracheal aspirate [3] . early reports of rt-pcr sensitivity vary considerably, ranging from 42% to 71%, and an initially negative rt-pcr result may convert into covid-19 after up to four days [4] . recent studies have shown that typical computed tomography (ct) findings of covid-19 include bilateral pulmonary parenchymal groundglass and consolidative pulmonary opacities, with a peripheral lung distribution [5] , [6] . in contrast to rt-pcr, chest ct scans have demonstrated about 56∼98% sensitivity in detecting covid-19 at initial manifestation and can be helpful in rectifying false negatives obtained from rt-pcr during early stages of disease development [7] , [8] . however, ct scans also share several similar visual manifestations between covid-19 and other types of pneumonia, thus making it difficult and time-consuming for doctors to differentiate among a mass of cases, resulting in about 25∼53% specificity [4] , [7] . among them, cap (community-acquired pneumonia) and influenza pneumonia are the most common types of pneumonia, as shown in figure 1 ; therefore, it is essential to differentiate covid-19 pneumonia from these. recently, liu et al. compared the chest ct characteristics of covid-19 pneumonia with influenza pneumonia, and found that covid-19 pneumonia was more likely to have a peripheral distribution, with the absence of nodules and tree-in-bud signs [9] . lobar or segmental consolidation with or without cavitation is common in cap [10] . although it is easy to identify these typical lesions, the ct features of covid-19, h1n1 and cap pneumonia are very diverse. in the past few decades, artificial intelligence using deep learning (dl) technology has achieved remarkable progress in various computer vision tasks [11] [15] . recently, the superiority of dl has made it widely favored in medical image analysis. specifically, several studies focus on classifying different diseases, such as autism spectrum disorder [16] , [17] or alzheimer's disease in the brain [18] [20] ; breast cancers [21] [23] ; diabetic retinopathy and glaucoma in the eye [24] [26] ; and lung cancer [27] , [28] or pneumonia [29] , [30] in the chest. some efforts have also been made to partition images, from different modalities (e.g., ct, x-ray, mri) into different meaningful segments [31] [33] , including pathology, organs or other biological structures. existing studies [30] , [34] , [35] have demonstrated the promising performance of applying deep learning technology for covid-19 diagnosis. however, as initial studies, several limitations have emerged from these works. first of all, [36] [39] utilized pixel-wise annotations for segmentation, which require taxing manual labeling. this is unrealistic in practice, especially in the event of an infectious disease pandemic. second, performing diagnosis or risk assessment on only slice-level ct images [34] , [40] [45] is of limited value to clinicians. since a volumetric ct exam normally includes hundreds of slices, it is still inconvenient for clinicians to go through the predicted result of each slice one by one. although, 3d convolutional neural networks (3d cnns) are one option for tackling these limitations, their high hardware requirements, computational costs (e.g., gpus) and training time, make them inflexible for applications [43] , [46] , [47] . to this end, we propose a multi-task multi-slice deep learning system (m 3 lung-sys) for multi-class lung pneumonia screening, which can jointly diagnose and locate covid-19 from chest ct images. using the only category labeled information, our system can successfully distinguish covid-19 from h1n1, cap and healthy cases, and automatically locate relevant lesions on ct images (e.g., ggo) for better interpretability, which is more important for assisting clinicians in practice. to facilitate the above objective, two networks using a 2d cnn are devised in our system. the first one is a slice-level classification network, which acts like a radiologist to diagnose from coarse (normal or abnormal) to fine (disease categories) for every single ct slice. as the name suggests, it can ignore the temporal information among ct volumes and focus on the spatial information among pixels in each slice. meanwhile, the learned spatial features can be further leveraged to locate the abnormalities without any annotation. to recover the temporal information and provide more value to clinicians, we introduce a novel patient-level classification network, using specifically designed refinement and aggregation modules, for diagnosis from ct volumes. taking advantage of the learned spatial features, the patient-level classification network can be trained easily and efficiently. in summary, the contributions of this paper are four-fold: 1) we propose an m 3 lung-sys for multi-class lung pneumonia screening from ct images. specifically, it can distinguish covid-19 from healthy, h1n1 and cap cases on either a single ct slice or ct volumes of patients. 2) in addition to predicting the probability of pneumonia assessment, our m 3 lung-sys is able to simultaneously output the lesion localization maps for each ct slice, which is valuable to clinicians for diagnosis, allowing them to understand why our system gives a particular prediction, rather than simply being fed a statistic. 3) compared with 3d cnn based approaches [47] , [48] , our proposed system can achieve competitive performance with a cheaper cost and without requiring large-scale training data 1 . 4) extensive experiments are conducted on a multi-class pneumonia dataset with 251 healthy people, 245 covid-19 patients, 105 h1n1 patients and 133 cap patients. we achieve high accuracy of 95.21% for correctly screening the multiclass pneumonia testing cases. the quantitative and qualitative results demonstrate that our system has great potential to be applied in clinical application. the ethics committee of shanghai public health clinical center, fudan university approved the protocol of this study and waived the requirement for patient-informed consent (yj-2020-s035-01). a search through the medical records in our hospital information system was conducted, with the final dataset consisting of 245 patients with covid-19 pneumonia, 105 patients with h1n1 pneumonia, 133 patients with cap and 251 healthy subjects with non-pneumonia. of the 734 enrolled people with 415 (56.5%) men, the mean age was 41.8±15.9 years (range, 2 ∼ 96 years). the patient demographic statistics are summarized in table i . the available ct scans were directly downloaded from the hospital picture archiving and communications systems (pacs) and non-chest cts were excluded. consequently, 734 threedimensional (3d) volumetric chest ct exams are acquired for our algorithm study. all the covid-19 cases (mean age, 51.5 ± 15.9 years; range, 16 ∼ 83 years) and h1n1 cases (mean age, 28.5 ± 14.6 years; range, 4 ∼ 78 years) were acquired from january 20 to february 24, 2020 and from may 24, 2009 to january 18, 2010, respectively. all patients were diagnosed according to the diagnostic criteria of the national health commission of china and confirmed by rt-pcr detection of viral nucleic acids. patients with normal ct imaging were excluded. the patients with cap subjects (mean age, 48.5 ± 17.4 years; range, 8 ∼ 96 years) and healthy subjects (mean age, 32.4 ± 11.8 years; range, 2 ∼ 73 years) with non-pneumonia were randomly selected between january 3, 2019 and january 30, 2020. all the cap cases were confirmed positive by bacterial culture, and healthy subjects with non-pneumonia undergoing physical examination had normal ct imaging. to better improve the algorithm framework and fairly demonstrate the performance, we do not use any ct volumes from re-examination, that is, only one 3d volumetric ct exam per patient is enrolled in our dataset. as shown in figure 2 , all eligible patients were then randomized into a training set and testing set, respectively, using random computer-generated numbers. unlike other studies [45] , [47] which employ a small number of cases (10%∼15%) for testing, we utilize around 40% of each category to evaluate the effectiveness and practicability of our system. the annotation was performed at a patient and slice level. first of all, each ct volume was automatically labeled with a one-hot category vector based on ct reports and clinical diagnosis (i.e., 0: healthy; 1: covid-19; 2: h1n1; 3: cap). considering that each volumetric exam contains 512 × 512 images with a varying number of slices from 24∼495, for training, five experts subsequently annotated each ct slice following four principles: (1) the quality of annotation is supervised by a senior clinician; (2) if a slice is determined to have any lesion, label it with the corresponding ct volume's category; (3) except for healthy cases, all slices from other cases considered as normal are discarded; (4) all slices from healthy people are annotated as healthy. note that we evaluate our model with the whole ct volume (i.e., realistic and arbitrary-length data), the discarded slices are only removed for training. eventually, the number of slices annotated for the four categories is listed in table i figure 3 shows the schematic of our proposed multitask multi-slice deep learning system (m 3 lung-sys), which consists of two components, the image preprocessing and classification network. specifically, the image preprocessing receives raw ct exams, and prepare them for model training or inference (in section iii-a). for the classification network, we divide it into two subnets (stages), i.e., slice-level and patient-level classification networks, with the purpose of jointly covid-19 screening and location. concretely, slicelevel classification network is trained with multiple ct slice images and predicts the corresponding slice-level categories (in section iii-b), i.e., healthy, covid-19, h1n1 or cap. besides, patient-level classification network only has four layers, and takes a volume of ct slice features, instead of images as input, which are extracted by the former network, to output the patient-level labels (in section iii-c). both classification networks are trained separately due to different tasks, but can be used concurrently in an end-to-end manner for the efficiency. more importantly, for cases classified as positive (i.e., covid-19, h1n1 or cap), our system can locate suspected area of abnormality without any pixel-level annotations (in section iii-b). the pixel value of ct images reflects the absorption rate of different human tissues (e.g., bone, lung, kidney) to xrays, which is measured by hounsfield unit (hu) [49] . if we directly apply raw images for classification, this will inevitably introduce noise or irrelevant information, such as the characteristics of equipment, making the performance of the model inaccurate and unreliable. consequently, according to the priors from radiologist, here, we introduce two effective yet straightforward approaches for preprocessing. 1) lung crop: given chest ct images, lungs are one of the most important organs observed by radiologists to check whether there exist abnormalities. considering the extreme cost and time-consumption of manual labeling, instead of training an extra deep learning network for lung segmentation [47] , [48] , [50] , we propose a hand-crafted algorithm to automatically subdivide/segment the image into 'lungs' and 'other', and then crop the area of lungs using the minimum bounding rectangle within a given margin. as illustrated in figure 4 , the details of the algorithm involved in lung segmentation and cropping are as following: • step 1: load the raw ct scan image (figure 4 (a) ). • step 2: set a threshold to separate the lung area from others, such as bone and fat ( figure 4 (b)). in this paper, we set the threshold of hu as t hu = −300. • step 3: to alleviate the effect of 'trays', which the patient lays on during ct scanning, we apply a morphological opening operation [51] (figure 4 (c)). specifically, we set the kernel size as 1 × 8. • step 4: remove the background (e.g., trays, bone and fat) based on 8-connected components labeling [52] ( figure 4 (d)). • step 5: apply the morphological opening operation again to eliminate the noise caused by step 3 (figure 4 (e)). • step 6: compute the minimum bounding rectangle with a margin of 10 pixels for lung cropping and then resize the cropped image ( figure 4 (f)). 2) multi-value window-leveling: in order to simulate the process of window-leveling when a radiologist is looking at ct scans, we further apply multi-value window-leveling to all images. more concretely, the value of the window center is assigned randomly from −700 to −500, and the window width is assigned with a constant of 1200. this preprocessing provides at least two benefits: (1) generating much more ct samples for training, i.e., data augmentation; (2) during inference, the assessment based on multi-value window-leveling ct images will be more accurate and reliable. after the above-mentioned image preprocessing, slices from ct volumes are first fed into the slice-level classification network. considering the outstanding performance achieved by the residual networks (resnets) [53] on the 1000-category image classification task, we utilize resnet-50 [53] as our backbone and initialize it with the imagenet [54] pretrained weights. this network consists of four blocks (a.k.a, resblock1∼4) with a total of 50 layers, including convolutional layers and fully connected layers. each block has a similar structure, but different number of layers. the skip connection and identity mapping functions in the blocks make it more possible to apply deeper layers to learn stronger representations. for the purpose of pneumonia classification and alleviating the limitations discussed in section i, we improve the network from three aspects, i.e., multi-task learning for radiologist-like diagnosis, weakly-supervised learning for slice-level lesion localization (attention) and coordinate maps for learning location information, as shown in figure 5 . 1) multi-task learning: usually, given a ct slice, a radiologist will gradually check for abnormalities and make a decision according to these. to act like an experienced radiologist, we introduce a multi-task learning scheme [55] by dividing the network into two stages. specifically, image features obtained from the first three resblocks are fed into an extra classifier to determine whether they have any lesion characteristics. then, the features are further passed through resblock4 to determine fine-grained category, i.e., healthy, covid-19, h1n1 or cap. 2) weakly-supervised learning for lesion localization: instead of using pixel-wise annotations or bounding box labels for learning to locate infection areas, we devise a weaklysupervised learning approach, that is, employ only the category labels. specifically, the weights of the extra classifier described in sec. iii-b.1 have a dimension of 2 × d, where d is the dimension of the feature and '2' denotes the number of classes (i.e., 'with lesion' and 'without lesion'). these learned weights can be regarded as two prototypical features of the corresponding two classes. similar to the class activation the details of our proposed slice-level classification network. we improve the network from three aspects: (1) multi-task learning to diagnose like a radiologist; (2) weakly-supervised learning for slice-level lesion localization (attention); (3) coordinate maps for location information. the symbol '©' indicates a concatenation operation. map [56] , we first select one prototypical feature according to the predicted class, and then calculate the distance between it and each point of the image feature extracted from the first three resblocks. intuitively, a closer distance between a point and the prototypical feature of 'with lesion' indicates that the area of this point mapping to the input ct slice has a higher probability of being an infection region, e.g., ggo. as one output of our m 3 lung-sys, such generated location maps are complementary to the final predicted diagnosis and provide interpretability for our network, making the assistance to clinicians more comprehensive and flexible. more visualization samples are demonstrated in figure 10 , 11 and 12. furthermore, we regard the lesion location map as an attention map and take full advantage of it to help the slicelevel differential diagnosis, as shown in figure 5 . 3) coordinate maps: from the literature [57] , it is known that infections of covid-19 have several spatial characteristics. for example, they frequently distribute bilaterally on lungs, and predominantly in peripheral lower zone. nevertheless, convolutional neural networks primarily extract the features of textures. to explicitly capture the spatial information, and inspired by [58] , we integrate our slice-level classification network with the coordinate maps (h × w × 3) containing three channels, where h and w are the height and width of the image feature extracted from the first three resblocks, to facilitate the distinction among covid-19, h1n1 and cap. the first two channels of the coordinate maps are instantiated and filled with the coordinates of x ∈ [0, w ) and y ∈ [0, h) respectively. and we further apply a normalization to make them fall in the range of [−1, 1]. the last channel encodes the distance d from the point (x, y) to the center (0, 0), i.e, d = x 2 + y 2 . specifically, these three additional channels are fed into the resblock4 together with the image feature and attention map to learn representations with spatial information. as mentioned in section i, performing diagnosis or risk assessment on only slice-level ct images is of limited value to clinicians. although several studies [47] , [48] have been proposed to take advantage of temporal information with 3d cnns for patient-level differential diagnosis, they require thousands of patient-level data for deep model training, which makes the cost particularly high. to overcome these limitations, we further propose a patient-level classification network. it takes a volume of ct slice-level features as input rather than 3d images, and only comprises four layers, allowing it to be trained with lower hardware, time and data cost. details will be described below. note that we concatenate the features from resblock3 and resblock4 in section iii-b as the input. 1) feature refinement and aggregation head.: inspired by [12] , [59] , we introduce a three-layer head to conduct feature refinement and aggregation, so that the image features from different slices can be correlated with each other and aggregated into one final feature for patient-level classification. the key intuition behind this is to utilize the attention mechanism to exploit the correlation between different ct slices, and accomplish the refinement and aggregation based on the explored correlation. as shown in figure 6 , the head includes a feature refinement module with two layers and a feature aggregation module with one layer, the structures of which are similar. formally, for the feature refinement module, given a volume of ct image features with the feature dimension of d, we first utilize three parallel fc layers to map the input to three different feature spaces for dimension reduction (d < d) and self-attention [12] . then, we calculate the distance, as attention, between each pixel in different slices using features from the first two spaces, and refine the features from the last space based on the attention. finally, another fc layer is employed to expand the feature dimension back to d, so that the skip-connection operation [53] can be applied. similar to [59] , we also apply a multi-head mechanism 2 to strengthen the refinement ability. without loss of generality, we define the input volume feature as f ∈ r n×d , where n is the number of slices, and the overall formulation of our refinement module can be expressed as, where h indicates the correlation between each pixel in different slices, f θi indicates the i-th fc layer with parameter θ i , and we omit the reshape operation for simplicity. with regard to the feature aggregation module, its structure, as well as the equations, is similar to the refinement module, except that we remove the multi-head mechanism and the last fc layer, and replace the first fc layer with a learnable parameter k ∈ r 1×d , so that the total number of n ct image features can be aggregated into one. details can be found in figure 6 (c). 2) multi-scale learning: if the number of slices with lesions in the early stage is relatively small (i.e., < n ), this may result in key information being leaked when performing feature aggregation from n × d to 1 × d. therefore, we introduce a multi-scale learning mechanism to aggregate features from different scales. as illustrated in figure 6 (a), given a set of scales s = [s 1 , s 2 , . . . , s k ], for each scale s j , we first divide the input feature f ∈ r n ×d evenly into s j parts, then, a shared feature refinement and aggregation head is applied to each part. in the end, we concatenate a set of aggregated features from all parts of different scales, and feed it into one fc layer to reduce the dimension from k j=1 s k d to d as the final patient-level feature for classification. we implement our framework with pytorch [60] . all ct slices are resized to 512×512. we set the hyper parameters of d and h as 512 and 12 respectively, and use four scales in the feature refinement and aggregation head, i.e., s = [1, 2, 3, 4]. for training, the slice-level/patient-level classification network is trained with two/one nvidia 1080ti gpus for a total of 110/90 epochs, the initial learning rate is 0.01/0.001 and gradually reduces by a factor of 0.1 every 40/30 epochs. both classification networks are trained separately with the standard cross-entropy loss function. random flipping is adopted as data argumentation. during inference, our system is an endto-end framework since the input of the patient-level classification network is the output of the slice-level one, so that it can be applied effectively. we additionally set the window center as [−700, −600, −500] for multi-scale window-leveling and average the final predicted features/scores for assessment. for the statistical analysis, we apply lots of metrics to thoroughly evaluate the performance of the model, following standard protocol. concretely, 'sensitivity', known as true positive rate (tpr), indicates the percentage of positive patients with correct discrimination. referred as true negative rate (tnr), 'specificity' represents the percentage of negative persons who are correctly classified. 'accuracy' is the percentage of the number of true positive (tp) and true negative (tn) subjects. 'false positive/negative error' (fpe/fne) measures the percentage of negative/positive persons who are misclassified as positive/negative. 'false disease prediction error' (fdpe) calculates the percentage of positive persons whose disease types (i.e., covid-19, h1n1 or cap) are predicted incorrectly. receiver operating characteristic curves (roc) and area under curves (auc) are used to show the performance of classifier. we also report the p-values compared our model with other competitors to demonstrate the significance level 3 . the main purpose of our system is to assist the diagnosis of covid-19 at a patient level rather than slice level [40] [44] , which is more significant and practical in the real-world applications. therefore, we first evaluate our system on the patient-level testing set with 102 healthy, 96 covid-19, 41 h1n1 and 53 cap cases. the competitors include one 2d cnn based method of covnet [30] and three 3d cnn based models as med3d-50 [61] , med3d-18 [61] and decovnet [62] . the results are shown in table ii and figure 7 . for the overall performance, our system achieves 95.21% on accuracy, with only 2.83% and 4.15% in false positive error and false negative error, respectively. although it may be difficult for clinicians to differentiate covid-19 from other kinds of viral pneumonia or cap pneumonia according to ct features, our system, as expected, only gets confused on a small number of cases, i.e., 1.57% in false disease prediction error, which beats the second best model by a margin of 3.05%. similarly, for h1n1, it obtains approximately 99.6% in both sensitivity and specificity, which is definitely a promising performance. moreover, our system significantly improves the sensitive of covid-19 from 95% to 99% comparing with med3d-18 and decovnet. however, we observe that the sensitivity or specificity of healthy is relatively inferior to med3d-18 and decovnet by approximately 2∼4 points, it seems our model is a little oversensitive to noise. on the other hand, med3d-50 achieves much worse performance at most of metrics unexpectedly, especially in sharp contrast to med3d-18. our explanation is that it may be difficult to train a 3d cnn with such large parameters and limited dataset, which is consist with our motivation of using 2d cnn based network. in addition to performance, we also compare the computation cost between our system and other competitors. as shown in table iii , our m 3 lung-sys takes full advantage of training data (ct slices and volumes) and has the lowest computation cost, including training time and gpu requirement. combining with the results in table ii and table iii , our method can achieve better performance with less computing resources, which is more practical for assisting diagnosis. 2) slice-level performance: another advantage of our proposed m 3 lung-sys is that we can flexibly switch whether the input is ct slices or volumes, i.e., slice-level or patientlevel diagnosis. naturally, we further evaluate our model on slices, using the total of 48,818 ct slices from the four categories (i.e., healthy, covid-19, h1n1 and cap) for testing. as shown in figure 8 , our model achieves 98.40%, 98.99%, 100.00% and 94.58% in auc for the four categories, respectively. this strongly demonstrates the superiority of our proposed m 3 lung-sys on slice-level diagnosis. d. ablation study 1) improvements in patient-level classification network: it is worth mentioning that our proposed slice-level classification network is strong and the extracted features are very discriminative. even without parameters, simple mathematic operations can obtain competitive results on patient-level diagnosis. meanwhile, the proposed multi-scale mechanism and refinement and aggregation head are able to further boost performance. to verify this, as shown in table iv, we conduct experiments to demonstrate improvements with different variants of the patient-level classification network. more specifically, 'non-parametric assessment' denotes a simple variant without parameters for differential diagnosis (we refer readers to i for details). 'max pooling' indicates that the input features are directly aggregated by a max pooling operation. 'single-scale + a. head' refers to a variant without the multi-scale mechanism (i.e., s=[3]) and feature refinement module. 'multi-scale + a. head' is similar to the previous model but applies the multi-scale strategy (i.e., s= [1, 2, 3, 4] ). from the results in table iv and figure 9 , we highlight the following observations: (1) using only the non-parametric assessment method, we can achieve competitive results of 94.18% in accuracy, which suggests the stronger feature representations acquired by our slice-level classification network. however, a big performance gap between 'false positive error' and 'false negative error' also reflects its inferior robustness, since a higher value of hyper-parameter t may result in more healthy cases being misdiagnosed due to some noise. (2) from the results in the second row to the last, the performance on all metrics improves gradually with more and more specifically designed components, which clearly demonstrates the benefits of our proposed feature refinement and aggregation head and multi-scale mechanism. (3) we notice that the false positive error gets worse when applying the method of 'single-scale + a. head', and it decreases dramatically when involving multi-scale mechanism. we argue that this does not suggest the inferiority of our proposed 'a. head' (since the overall accuracy is improved by 1%), but reflects the importance and rationality of the multi-scale mechanism. 2) improvements in slice-level classification network: to explicitly demonstrate the advantages of our improvements in slice-level classification network, we compare it with several competitors on patient-level diagnosis. without loss of generality, we choose the method of non-parametric assessment with t = 0.99 as the patient-level classification network. concretely, since the backbone of our slice-level classification network is resnet-50, which is widely adopted by other works [30] , [42] , we directly train a vanilla resnet-50 for fourway classification as a baseline. based on this, we conduct further experiments by gradually adding different improvements, including multi-task learning, lesion location (attention) maps and coordinate maps. all results are listed in table v . compared with the baseline, our model achieves significant improvement in all four metrics. for example, the overall accuracy is improved from 89.04% to 94.18% and the proportion of false positive is reduced effectively by 12 points. although we obtain a few more failure cases on false disease predication when utilizing the multi-task mechanism, the number of both false positive and false negative samples is dramatically reduced. the twotask approach acting like a radiologist is expected to better distinguish between healthy people and patients. furthermore, if we introduce the coordinate maps, fewer positive samples are misclassified as the wrong type of disease and some negative cases with noise are correctly diagnosed as positive, resulting in a decrease in both false positive and false disease prediction error. these results clearly suggest that the compo-nents in slice-level classification network play important roles in extracting discriminative features, e.g., attention maps for awareness of small lesions, and coordinate maps for capturing location diversity among different types of pneumonia. as one of its contributions, our m 3 lung-sys can implement lesion localization using only category labels, i.e., weaklysupervised learning. to qualitatively evaluate this, we randomly select several ct slices of three categories from the testing set, and show the visualizations of lesion location maps in figure 10 . for each group, the left image is the raw ct slice after lung cropping, and the right image depicts the detected area of abnormality. note that a warmer color indicates that the corresponding region has a higher probability of being infected. several observations can be made from figure 10 : 1) first of all, the quality of location maps are competitive. all highlighted areas are concentrated on the left or right lung region, and most abnormal manifestations, such as ground-glass capacities (ggo), are completely captured by our model, which is trained without any pixel-level or bounding box labels. in addition, there is no eccentric area being mistaken as a lesion, such as vertebra, skin or other tissues. our system can even precisely detect small lesions with relatively high response, as shown in the top-right image of figure 10 (a). above all, our system can achieve visual localization of abnormal areas with good interpretability, which is crucial for assisting clinicians in diagnosis and improving the efficiency of medical systems. 2) second, we found that the location map results are consist with the experience or conclusions of radiologists. several studies have found that covid-19 typically presents ggo with or without consolidation in a predominantly peripheral distribution [63] [65] , which has already been used as guidance for covid-19 diagnosis endorsed by the society of thoracic radiology, the american college of radiology, and rsna [66] . in contrast, h1n1 pneumonia most commonly presents a predominantly peribronchovascular distribution [67] , [68] . lobar or segmented consolidation and cavitation suggest a bacterial etiology [10] . therefore, the visualizations of location maps can reflect the characteristics of lesion distributions in some ways, which may be a valuable indicator for clinicians in analyzing or differentiating these three diseases. to fully demonstrate the practicability and effectiveness of our system, we further simulate its real-world application and present the outputs, i.e., the diagnosis assessment of diseases and the localization of lesions. concretely, we randomly select two covid-19 patients, and feed their ct exams into our system. the full outputs are illustrated in figure 11 and 12. due to page limitation, we show a ct sequence by sampling every five slices. the lesion location maps, with the predicted slice-level diagnosis on the upper right, are attached at the bottom of the corresponding raw ct slices. at the end of the sequence, the probability of each category predicted by the patient-level classification network is provided (i.e., 0: healthy; 1: covid-19; 2: h1n1; 3: cap). as can be seen, our system can accurately locate the lesion areas in each slice, 4 and these areas also have good continuity in sequence, which are very important characteristics to assist the clinician in diagnosing covid-19. in this paper, we proposed a multi-task multi-slice deep learning system (m 3 lung-sys) to assist the work of clinicians by simultaneously screening multi-class lung pneumonia, i.e., healthy, covid-19, h1n1 and cap, and locating lesions from both slice-level and patient-level ct exams. different from previous studies, which incur high hardware, time and data costs to train 3d cnns, our system divides this procedure into two stages: slice-level classification and patientlevel classification. we first utilize the slice-level classification network to classify each ct slice. an introduced multitask learning mechanism makes our model diagnose like a radiologist, first checking whether each ct slice contains any abnormality, and then determining what kind of disease it is. both attention maps and coordinate maps are further applied to provide awareness of small lesions and capture location diversity among different types of pneumonia. with the improvements provided by these components, our system achieves a remarkable performance of 94.18% accuracy, 10.66% false positive error and 0.0% false negative error. then, to recover the temporal information in ct sequence slices and enable ct volumes screening, we propose a patientlevel classification network, taking as input a volume of ct slice features extracted from the slice-level classification network. such a network can achieve the feature interaction and aggregation between different slices for patient-level diagnosis. consequently, it further promotes our system by dramatically reducing the cases of false positive and false disease predication and improving the accuracy by 1.1%. above all, from a clinical perspective, our system can perform differential diagnosis with not only a single ct slice, but also a ct volume. the combined outputs of risk assessment (predicted diagnosis) and lesion location maps make it more flexible and valuable to clinicians. for example, they can easily estimate the percentage of infected lung areas or quickly check the lesions at any time before making a decision. as illustrated in figure 11 and 12 , with these lesion location maps, clinicians are able to understand why the deep learning model gives such prediction, not just face a statistic. even with the outstanding performance, there are three limitations remaining in our system that need to be improved. first of all, it is very easy for clinicians to distinguish covid-19 from healthy cases. however, from table 2 , we find that our system may still misclassify some healthy people. we examine failure cases and find that the main challenge lies in the pulsatile artifacts in pulmonary ct imaging. second, our framework, which contains slice-level and patient-level classification networks, is not end-to-end trainable yet. although it just increases the negligible training and testing time, we hope the end-to-end training manner would be more conducive to the learning and combination of spatial and temporal information. third, our proposed localization maps accurately show the location of abnormal regions, which are valuable to clinicians in assisting diagnosis. however, they still lack the ability to automatically visualize the unique lesions' distributions for each disease. in the future, we will attempt to tackle the first and third limitation by improving the attention mechanism to enhance the feature representations. besides, developing the technology of coordinate maps may be an optimal option. frankly speaking, the third obstacle is a very challenging and ideal objective, but we will continuously promote research along this line. as for the second limitation, we are going to polish our method into a unified framework, through such training mechanism, both spatial and temporal information can complement each other. in conclusion, we proposed a novel multi-task multi-slice deep learning system (m 3 lung-sys) for multi-class lung pneumonia screening from chest ct images. different from previous 3d cnn approaches, which incur a substantial training cost, our system utilizes two 2d cnn networks, i.e., slicelevel and patient-level classification networks, to handle the discriminative feature learning from the spatial and temporal domain, respectively. with these special designs, our system can not only be trained with much less cost, including time, data and gpu, but can also perform differential diagnosis with either a single ct slice, or a ct volume. more importantly, without any pixel-level annotation for training, our system is able to simultaneously output the lesion localization for each ct slice, which is valuable to clinicians for diagnosis, allowing them to understand why our system gives a particular prediction, rather than just being faced with a statistic. according to the remarkable experimental results on 292 testing cases with multi-class lung pneumonia (102 healthy people, 96 covid-19 patients, 41 h1n1 patients and 53 cap patients), our system has great potential for clinical application. given a volume of ct exam with n slices in sequence, we feed them into our slice-level classification network to obtain two kinds of probabilities for each slice, p lesion = (p 0 li , p 1 li ) n i=1 and p multi−class = (p 0 mi , p 1 mi , p 2 mi , p 3 mi ) n i=1 , where p lesion means the probability whether a ct slice contains any lesion or not, and p multi−class denotes the predicted probability of multi-class pneumonia assessment (i.e., 0: healthy; 1: covid-19; 2: h1n1; 3: cap). then, we derive the final probabilities of four classes for each slice from p lesion and p multi−class , which can be expressed as follows, intuitively, if all or most of slices are predicted as health, the patient has a very high chance of being healthy. otherwise, he will be diagnosed as either covid-10, h1n1 or cap, according to ct imaging manifestation. to simulate this process, our proposed non-parametric holistic assessment on patient-level can be formulated as follows, where n k = n i=1 δ(max p i − p k i ) and δ( * ) = 1 if * = 0, otherwise, δ( * ) = 0. t is a hyper-parameter to control the degree of 'most of', that is, the proportion of healthy (normal) slices. normally, the chest ct slices of healthy people should be nearly or completely all normal. therefore, without loss of generality, in this paper, we set it as a reasonable and acceptable value, i.e., t = 0.99. epidemiology, causes, clinical manifestation and diagnosis, prevention and control 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automated methods for glaucoma assessment from fundus photographs age challenge: angle closure glaucoma evaluation in anterior segment optical coherence tomography computer aided lung cancer diagnosis with deep learning algorithms end-to-end lung cancer screening with three-dimensional deep learning on low-dose chest computed tomography variable generalization performance of a deep learning model to detect pneumonia in chest radiographs: a cross-sectional study artificial intelligence distinguishes covid-19 from community acquired pneumonia on chest ct automatic segmentation of liver tumor in ct images with deep convolutional neural networks deep learning-based image segmentation on multimodal medical imaging hi-net: hybrid-fusion network for multi-modal mr image synthesis a deep learning algorithm using ct images to screen for corona virus disease (covid-19) a fully automatic deep learning system for covid-19 diagnostic and prognostic analysis inf-net: automatic covid-19 lung 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learning ct image analysis a weakly-supervised framework for covid-19 classification and lesion localization from chest ct a quantitative theory of the hounsfield unit and its application to dual energy scanning large-scale screening of covid-19 from community acquired pneumonia using infection size-aware classification method and device for automatic segmentation of a digital image using a plurality of morphological opening operation a simple and efficient connected components labeling algorithm deep residual learning for image recognition imagenet: a large-scale hierarchical image database multitask learning learning deep features for discriminative localization chest ct findings in coronavirus disease-19 (covid-19): relationship to duration of infection an intriguing failing of convolutional neural networks and the coordconv solution attention is all you need automatic differentiation in pytorch med3d: transfer learning for 3d medical image analysis a weakly-supervised framework for covid-19 classification and lesion localization from chest ct radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study emerging 2019 novel coronavirus (2019-ncov) pneumonia comparison of hospitalized patients with acute respiratory distress syndrome caused by covid-19 and h1n1 radiological society of north america expert consensus statement on reporting chest ct findings related to covid-19. endorsed by the society of thoracic radiology, the american college of radiology, and rsna computed tomography findings of influenza a (h1n1) pneumonia in adults: pattern analysis and prognostic comparisons pneumonia in novel swine-origin influenza a (h1n1) virus infection: high-resolution ct findings the authors would like to thank all of clinicians, patients and researchers who gave valuable time effort and support for this project, especially in data collection and annotation additionally, we appreciate the contribution to this paper by wenxuan wang (for the help of paper revision), junlin hou for her suggestion in 3d baselines) and longquan jiang (for the assistance of data processing). key: cord-252343-a85wz2hs authors: skoda, eva-maria; teufel, martin; stang, andreas; jöckel, karl-heinz; junne, florian; weismüller, benjamin; hetkamp, madeleine; musche, venja; kohler, hannah; dörrie, nora; schweda, adam; bäuerle, alexander title: psychological burden of healthcare professionals in germany during the acute phase of the covid-19 pandemic: differences and similarities in the international context date: 2020-08-07 journal: j public health (oxf) doi: 10.1093/pubmed/fdaa124 sha: doc_id: 252343 cord_uid: a85wz2hs background: healthcare professionals (hps) are the key figures to keep up the healthcare system during the covid-19 pandemic and thus are one of the most vulnerable groups in this. to this point, the extent of this psychological burden, especially in europe and germany, remains unclear. this is the first study investigating german hps after the covid-19 outbreak. methods: we performed an online-based cross-sectional study after the covid-19 outbreak in germany (10–31 march 2020). in total, 2224 hps (physicians n = 492, nursing staff n = 1511, paramedics n = 221) and 10 639 non-healthcare professionals (nhps) were assessed including generalized anxiety (generalized anxiety disorder-7), depression (patient health questionnaire-2), current health status (eq-5d-3l), covid-19-related fear, subjective level of information regarding covid-19. results: hps showed less generalized anxiety, depression and covid-19-related fear and higher health status and subjective level of information regarding covid-19 than the nhps. within the hp groups, nursing staff were the most psychologically burdened. subjective levels of information regarding covid-19 correlated negatively with generalized anxiety levels across all groups. among hps, nursing staff showed the highest and paramedics the lowest generalized anxiety levels. conclusions: in the context of covid-19, german hps seem to be less psychological burdened than nhps, and also less burdened compared with existing international data. the covid 19 pandemic reached germany in late february 2020. it brought not only objective medical challenges for healthcare professionals (hps), but also reports and findings from other more affected countries. due to exponentially increasing case numbers and large numbers of patients requiring intensive care, those more affected countries are facing unexpected challenges. countries such as china, italy, spain, brasil and the usa were and are currently reaching the limits of their healthcare systems in the context of this pandemic: something that was previously unimaginable in industrialized countries. 1 such a development seems to have been avoided in germany but is not completely ruled out for the future. in the face of an ever-renewing european and a further worldwide escalation, there is no shortage of uncertainty and concern among hps. it is already known from countries other than germany that hps are under elevated psychological stress during the covid-19 pandemic and show increased levels of various psychometric values, including anxiety and depression. 2-5 existing evidence, e.g. from china, already shows the extent of the psychological burden on hps. front-line healthcare workers were identified as bearing a particularly heavy psychological burden. 2,6 however, these studies were conducted during the extreme stress phase of the covid-19 epidemic in china. only few data in the context of other studies suggest that, e.g. in the uk, a heightened psychological burden for the hps may exist. 7 there is, as yet no comparable data, especially from a time when the health system is still mainly coping normally, alongside already population-wide uncertainty, particularly in europe. the german situation to this point is 2-fold: continuing and past restrictions in public life, contact restrictions, empty supermarket shelves and daily updated increasing case numbers are still coupled with a hospital system that is and was largely still able to cope normally. this is combined with mortality rates, which are, for the moment, low when compared internationally. 8, 9 though, the german population shows itself already burdened in terms of generalized anxiety, depression and distress, which is in line with evidence from other countries, 10,11 customized low-threshold interventions, offline as well as online, are needed and already implemented. [12] [13] [14] the aim of this study was to close the research gap and provide initial findings on psychological burden of german hps after the covid-19 outbreak. it is hypothesized that the group of hp in germany will mirror the existing, population-wide elevated psychological burden 15 to an even greater extend by being in the 'front line', as already could be observed in previous studies in other countries. 2,3 a nationwide, online-supported cross-sectional survey was conducted. participants were recruited via online channels and official channels e.g. websites of clinics. the survey period was from the 10-31 march 2020. it was during this period that the first increased numbers of covid-19 cases in germany, increasingly restrictive government regulations, the closure of european borders and the restriction of individual freedoms occurred. in total, 12 863 people completed the questionnaire, of which we identified 2224 people in the medical sector as hps and 10 639 as non-healthcare professionals (nhps). hps were from three different groups: physicians, nursing staff and paramedics. the sample description can be seen in table 1 . all participants gave their written consent to participate in the survey and the evaluation of the collected data. the study was conducted in accordance with the ethical guidelines from the declaration of helsinki and was approved by the local ethics committee of the faculty of medicine. details of general socio-demographic variables were asked. validated psychometric instruments were used to assess psychological burden. the generalized anxiety disorder-7 (gad-7) to measure generalized anxiety symptoms over the course of the last 2 weeks (gad-7, 7 items, 4-point likert scale meaning 0 = never to 3 = nearly every day), 16 the patient health questionnaire-2 (phq-2) to screen for depression symptoms over the course of the last 4 weeks (phq-2, 2 items, 4-point likert scale meaning 0 = never to 3 = nearly every day) 17 and the visual analogous scale of the euroqol eq-5d-3l scale to assess current health status (ranging from 0 [worst imaginable health status] to 100 [best imaginable health status]). 18 additionally, based on scientific and media reports, multiple items and item scales were formed in expert consensus with regard to 'covid-19-related fear' (one item, 7-point likert scale meaning 1 = very low to 7 = extremely high), 'the subjective level of information regarding covid-19' (3 items: i feel informed about covid-19; i feel informed about measures to avoid an infection with covid-19; i understand the health authorities' advice regarding covid-19. seven-point likert scale, meaning 1 = complete disagreement to 7 = complete agreement). scale reliability for was tested using cronbach's α for internal consistency. 'the subjective level of information regarding covid-19' showed high internal consistency (cronbach's α = 0.801). the descriptive and inferential statistics were performed with r3.6.1 (r core team, 2019). sum scores for the gad-7 and phq-2 and mean scores for all other scales were calculated. to assess the hypotheses, the 95% confidence of the association measures are reported; for each difference between the groups after having assessed the global mean difference in the respective scale. hence, the assumptions were assessed based on their precision. [19] [20] [21] generally, test statistics and p values are not reported given that at this sample size even the slightest deviation from equivalence results in extremely low p values. when the confidence interval (ci) of the effect size covers 0, we assume there is no effect. as soon as this is the case, we use the guidelines by sawilowsky 22 to evaluate the importance of the effect; a cohen's d ∼0.2 is considered a small, a d ∼0.5 is considered medium-sized and d ∼0.8 is regarded as large effects. due to the large sample size and the intuitive and common interpretation of the effect sizes, parametric methods were also used for violation of the normality assumption. 23 for mean comparisons welch's t-test with the cohen's d association measure was used, for multiple mean comparisons and between-subject analysis of variance with the association measure η 2 with subsequent t-tests for post hoc comparisons with tukey error correction. a complete summary of all post hoc group comparisons after calculation of the variance analyses and post hoc tests can be assessed in the supplementary materials. to clear the association of subjective level of information regarding covid-19 and other variables, spearman correlations between variables were performed. to subsequently test the interdependence of variables a robust linear mestimator regression was performed (rlm from the r package mass, 2002). all spearman correlations including confidence between the measures are provided in the supplemental material. following the results of the correlation analyses, prevalence ratios for the amount of participants with moderate generalized anxiety in relation to the subjective level of information regarding covid-19 were explored. levels of generalized anxiety were divided by using the gad-7 sum score of ≥10 24 as a split into low levels of generalized anxiety (<10) and moderate to high levels of generalized anxiety (≥10). this was compared with a pre-covid-19 standard population, where 5.9% of the population scored above ≥10. 25 the subjective level of information regarding covid-19 was split by the median into high (≥median) and low (<median) levels. prevalence ratios were calculated using unconditional maximum likelihood. cis were calculated using normal approximation (via the r-packages epitools, 2020). concerning generalized anxiety measured by the gad-7, differences between the different hp groups and between the hps and the nhps could be found (η 2 = 0.009, ci = subjective level of information regarding covid-19 correlates with generalized anxiety. the association of high or low subjective levels of information regarding covid-19 and low or moderate to high generalized anxiety (cutoff of ≥10) is shown in table 2 . the proportion of moderately anxious participants regardless of level of information is 15.99% in the nhps, 4.25% in physicians, 11.41% in nursing staff and 4.55% in paramedics. the probability of reaching the pathological gad-7 cutoff score of ≥10 thus diminishes across almost all hp groups and the nhps with the degree of subjective level of information regarding covid-19 (fig. 3) . for physicians, nursing staff and others, the prevalence of having generalized anxiety when sufficiently informed is critically reduced (with cis of the prevalence ratio deviating significantly from 1). however, reduced moderate generalized anxiety in participants with high subjective levels of information can also observed when the sample is analyzed as a whole. assessing the psychological burden and covid-19-related concerns of people working in the healthcare system is of high relevance in the light of the covid-19 pandemic events. to this point, this study is the first to assess those points in the german healthcare system. in this study, nhps reported overall higher levels of psychological burden than the hps, which is particularly pronounced in generalized anxiety and depression scores. within the hp groups there was, in relative terms, less psychological burden on physicians and paramedics than on nursing staff. health status was better in the hp groups and within those, physicians reported the best health status, followed by paramedics. hps also showed less covid-19-related fear than nhps, paramedics showing the least fear. subjective levels information regarding covid-19 was high overall hps and nhps. high levels of subjective levels of information regarding covid-19 correlated negatively with high levels of generalized anxiety symptoms globally. in the group of individuals with low subjective levels of information regarding covid-19, 17.88% of the nhps, 8.60% of the physicians, 14.69% of the nursing staff and 5.00% of the paramedics scored above the cutoff of ≥10, indicating the presence of gad symptoms. even without the division in subjective levels of information regarding covid-19, still 15.99% of the nhps, 5.89% of the physicians, 11.41% of the nursing staff and 4.55% of the paramedics scored above this cutoff. especially concerning the nhps and the nursing staff, this is a massive elevation of prevalence in comparison to a pre-covid-19 sample, where only 5.9% of the population scored above this cutoff. 25 the psychological burden of hps during the covid-19 pandemic has already been investigated in the early hotspots of the pandemic, especially in china and southeast asia. high psychological burden of front-line workers could be found in china 4-6,26 and other countries (e.g. tan et al . 3 ). in detail, at the height of the crisis in china, 50.7% of healthcare workers showed symptoms of depression, 44.7% those of generalized anxiety and 73.4% showed increased stress symptoms. 27 the psychological response of hps during epidemics has already been investigated during other virus outbreaks, e.g. sars and h1n1, drawing similar pictures. 28,29 a clear picture concerning the hps in europe, especially in the beginning of the virus outbreak still does not exist. in the present study, it was possible to show a rare picture of a healthcare system in the early outbreak stages of a pandemic in the western world. during this phase of the outbreak in germany, in which uncertainty and anxiety prevailed but covid-19 cases and death rates were still moderate and low, hps were less psychologically burdened than the nhps and less psychologically burdened than comparable hp groups in other countries. [3] [4] [5] [6] 26 this poses the question why the german hps coped and cope so well in the light of the current pandemic and in comparison to the nhps. the finding that physicians in the current situation are among the least burdened by generalized anxiety and depression can maybe be understood by the fact that generalized anxiety was most correlated with the subjective level of information regarding covid-19. this may explain why physicians and hps show lower overall levels of generalized anxiety and covid-19related fear, which also is in line with findings from tan et al . 3 where nonmedical healthcare workers had higher prevalence of anxiety than medical healthcare workers. this, in a second step, points to the inevitable need for the population to be informed transparently and evidence based in order to experience the covid-19 pandemic with a low level of fear as possible. 15, 30 the present findings of this study also urge especially western countries to conduct still more research concerning the psychological burden of their hps. existing literature is majorly consisting of studies from southeast asia and during the early, dramatic scenes of the pandemic. to meet the needs of the hps, especially as soon as the situation worsens again or a much feared 'second wave' hits europe, the mental health of hps will have to be given high priority in supporting measures. 31 limitations need to be considered. data were obtained via an anonymous online, self-reported questionnaire in a crosssectional study design. a potential selection bias may exist. during the short survey period, a series of various governmental restrictions were put into place, which may represent different manifestations of psychological states at different points in time. data were acquired during a rather early period of the outbreak, which poses the risk of an under-or even overestimation of psychological burden. on the other hand, this exceptional early assessment period might be a major strength of posed study-a thus large sample as the present on the beginning of the current outbreak is scarce and adds a piece to the covid-19 puzzle in order to gain a clearer picture of the unprecedented covid-19 pandemic. in conclusion, hps in germany show less psychological burden compared with hps in other countries and compared with the general population in germany after the outbreak of covid-19. in the investigated sample, nursing staff seems to be the most vulnerable group for mental health burden during the covid-19 pandemic, whereas a high subjective level of information seems to be associated with less psychological burden. supplementary data mentioned in the text are available to subscribers in pubmed online. this study was supported by the essen university medicine foundation. the funder had no role in the design and conduct of the study; management, collection, analysis and interpretation of the data; preparation, review or approval of the manuscript and decision to submit the manuscript for publication. the authors declare that they have no competing interests. covid-19 in europe: the italian lesson psychological support in times of covid-19: the essen community-based cope concept an e-mental health intervention to support burdened people in times of the covid-19 pandemic: cope it techniques, methods, and dissemination of community based psychological support strategies in times of the covid-19-pandemic not all world leaders use twitter in response to the covid-19 pandemic: impact of the way of angela merkel on psychological distress, behaviour and risk perception validation and standardization of the generalized anxiety disorder screener (gad-7) in the general population detecting and monitoring depression with a two-item questionnaire (phq-2) euroqol-a new facility for the measurement of healthrelated quality of life heuristic thinking and inference from observational epidemiology sifting the evidence-what's wrong with significance tests? 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multilevel analysis of neighborhood effects in chicago, illinois date: 2020-10-29 journal: nan doi: nan sha: doc_id: 149748 cord_uid: ucsxbzen mobility resilience refers to the ability of individuals to complete their desired travel despite unplanned disruptions to the transportation system. the potential of new on-demand mobility options, such as ridesourcing services, to fill unpredicted gaps in mobility is an underexplored source of adaptive capacity. applying a natural experiment approach to newly released ridesourcing data, we examine variation in the gap-filling role of on-demand mobility during sudden shocks to a transportation system by analyzing the change in use of ridesourcing during unexpected rail transit service disruptions across the racially and economically diverse city of chicago. using a multilevel mixed model, we control not only for the immediate station attributes where the disruption occurs, but also for the broader context of the community area and city quadrant in a three-level structure. thereby the unobserved variability across neighborhoods can be associated with differences in factors such as transit ridership, or socio-economic status of residents, in addition to controlling for station level effects. our findings reveal that individuals use ridesourcing as a gap-filling mechanism during rail transit disruptions, but there is strong variation across situational and locational contexts. specifically, our results show larger increases in transit disruption responsive ridesourcing during weekdays, nonholidays, and more severe disruptions, as well as in community areas that have higher percentages of white residents and transit commuters, and on the more affluent northside of the city. these findings point to new insights with far-reaching implications on how ridesourcing complements existing transport networks by providing added capacity during disruptions but does not appear to bring equitable gap-filling benefits to low-income communities of color that typically have more limited mobility options. unexpected transit disruptions, service interruptions due to accidents, infrastructure breakdowns, and passenger distress are common occurrences in urban transit systems. the desire of riders is usually to continue their journeys to a timely completion -to be resilient in their travel. the current growth of ridesourcing services offers a novel opportunity for urban mobility resilience by filling unpredicted gaps in transit operations. in this study we examine the role of ridesourcing to enhance adaptive mobility to disruptions by complementing traditional services (reggiani et al., 2015) . specifically, we study the variation in this type of resilience across communities. we analyze the equity in ridesourcing for mobility resilience for the city of chicago. notably, chicago is home to the second largest transit network in the u.s. with the chicago transit authorities (cta) serving 3.5 million riders (cta, 2020a) with nearly 16 million rail rides each month (cta, 2020b) . while unequal access to essential resources is common in many u.s. cities, chicago contends with historically rigid, spatially-defined, social and economic inequality that is frequently linked to race. for example, the income divide between white households and minority households is wider in chicago than it is across the nation as a whole (asante-muhammed, 2017) . urban mobility systems typically grapple with multiple layers of inequitable mobility investments and service-access that determine service quality for different population segments. the city of chicago is subject to urban mobility inequity both at the service level (e.g., poor mobility accessibility coverage), as well as disproportionate impacts (e.g., lack of pedestrian-friendly infrastructure or biased policing) in low-income communities (krapp, 2020) . to understand how well a mobility system serves diverse community members, it is essential to understand the interplay between modes in the transportation system. on the one hand, public transit addresses the transportation needs of those with mobility disadvantages, implying that any disruptions in transit could disproportionately affect under-resourced communities of color. on the other hand, on-demand mobility services can offer expanded flexible mobility, although there is evidence of disparities in the use of ridesourcing related to spatial and sociodemographic diversity (soria et al., 2020) . this has led the local planning agency cmap to highlight the need to study the potential benefits and pitfalls of new mobility technologies, such as ridesourcing, with regard to accessibility, affordable mobility, and local quality of life (cmap, 2018) . our awareness of existing disparities in mobility begs a fundamental question when analyzing the substitution of ridesourcing services during transit disruptions: is this disruption recovery equitable; that is, are under-resourced transit riders benefitting from ridesourcing-based mobility resilience on par with other travelers? in this project, we take a natural experiment approach where we systematically identify major transit disruptions over the course of a year and match them with large scale, spatio-temporal ridesourcing trip data from the city of chicago. we then develop a multilevel model (mlm) to examine the degree to which ridesourcing demand surges during transit disruptions, providing evidence of an adaptive ridership response. the multilevel structure is tailored to account for variation in spontaneous mobility resilience across the city and to explore whether it occurs due to neighborhood differences. to examine this potential adaptation strategy among transit riders, we compare the demand for ridesourcing during unplanned rail transit disruptions to the baseline demand while also controlling for the time of day, day of the week, and location. the main contributions of this study are the insights it provides into: (1) whether ridesourcing is used as a gap-filling transportation mode during transit disruptions in chicago, (2) whether its utilization for this purpose is distributed equitably across the city in terms of racial and economic circumstances, and (3) whether variation in ridesourcing demand during disruptions is attributable to station-level, community-level, or quadrant-level contexts. our findings provide evidence of significant localized station-level effects, such as the timing of the disruption. importantly, we also discover that the community area where the station is located is responsible for the majority of the variation in observed disruption-based ridesourcing substitution. specifically, the racial composition and transit commuting rates show significant interaction with the aforementioned station-level factors. these insights contribute to an improved understanding of how riders cope with disruptions in different communities. in terms of practice, we discuss how our findings can guide more equitable communication strategies for transportation agencies and potential partnerships with private on-demand mobility service operators to treat mobility as a service regardless of transportation mode. transit disruptions may be long-term or unplanned, and their impacts can cause riders to shift to traditional modes (such as cars and buses) or on-demand mobility (such as bikeshare and ridesourcing). the effects of long-term transit disruptions on transportation behavior have been widely studied over the past decade (marsden and docherty, 2013; pnevmatikou et al., 2015; pu et al., 2017; van exel and rietveld, 2001; zhu et al., 2017) . long-term transit line closures may extend for several months, providing riders with time to adjust their behavior, including departure time, route, and mode, temporarily or permanently. often during long-term disruptions, such as transit strikes, the majority of travelers switch to cars (van exel and rietveld, 2001; zhu et al., 2017) . this may be a function of available resources, as those who are less likely to travel by car during transit disruptions include lower income individuals, women, and individuals with more flexible work schedules (pnevmatikou et al., 2015) . mode-shifting behavior during long-term transit disruptions depends not only on sociodemographics but also on the surrounding context, such as the city in which it occurs. longterm rail transit disruptions in washington, d.c. are associated with increased bus ridership (pu et al., 2017) , especially among lower income individuals (zhu et al., 2017) . by contrast, an analysis of smart-card data for chicago rail riders shows that the majority of riders continue to use rail during planned maintenance with a minor share shifting to bus (mojica 2008) . furthermore, the effects of long-term transit disruptions have resulted in a permanent decline in transit ridership across europe and the u.s. (van exel and rietveld, 2001; zhu et al., 2017) . similarly, in chicago, lengthy disruptions from track operations led to an estimated 3.9% of riders abandoning transit (mojica, 2008) . in the few cases of research on long-term transit disruptions that consider on-demand mobility, the focus has been largely on bikeshare. findings show temporary increases in bikesharing demand during transit strikes or maintenance projects, suggesting the ability of on-demand transportation to increase mobility resilience (fuller et al., 2012; kaviti et al., 2018; pu et al., 2017; saberi et al., 2018) . compared to the extensive body of research on planned, long-term transit disruptions, research on unplanned, short-term events has been scant (sun et al., 2016) . very recently, however, research has begun to consider the role of ridesourcing by transit users in response to unplanned service disruptions . one survey-based study, which is among the first to research transit rider behavior during unplanned service disruptions in chicago, examined whether riders would cancel their trip, change destinations, or change modes to shuttle bus, ridesourcing, taxi, personal vehicle, or carpool . the findings reveal that the individuals who would shift to ridesourcing during a transit disruption tend to be millennials, have a higher level of education, have a smartphone, or have prior ridesourcing experience . in the survey-based study, race was not found to be a significant factor in the hypothetical shift to ridesourcing. while earlier work has revealed the role of bikeshare as a gap-filling mechanism during longterm transit disruptions, so far, ridesourcing has only been investigated as a mode-adaptation strategy for hypothetical unplanned transit disruption scenarios. the current study is among the first to use a natural experiment to examine the impacts of unplanned, short-term transit disruptions on the usage of ridesourcing across the city of chicago. we believe that examining unplanned transit disruptions can improve understanding of adaptation strategies that substitute on-demand mobility for fixed transportation services. herein, we develop an mlm model to examine the magnitude of ridesourcing demand-surges due to transit disruptions. more importantly, we analyze the variation, especially related to racial composition, in the gap-filling utilization of ridesourcing and compare the disruption source and broader community-level contexts to explain this observed variation. due to the limited access of on-demand mobility by individuals with disabilities, low income or historically marginalized communities, rural populations, under-banked households, and individuals without smartphones, the ridesourcing business model has been accused of being based on privileged access (daus esq., 2016) . studies show consistently that ridesourcing users are typically young, male, higher income (zhang and zhang, 2018) , highly educated, full-time workers , own fewer vehicles per household, and live closer to transit stations (deka and fei, 2019) . similarly, users of a commute-focused vanpooling service formerly operating in seattle were mainly white, male, highly educated, higher income, and millennials (lewis and mackenzie, 2017) . other works highlight contradictory findings on declared hailingtransit substitution according to income segment and service quality factors (gehrke et al., 2018) . recent work based on large-scale trip data and experiments has revealed more aggregate demand insights. in chicago, greater ridesourcing usage is observed in areas with higher population and employment density, land-use diversity, household incomes, percentages of transit commuters, and percentages of zero vehicle households (ghaffar et al., 2020) . in seattle, areas with higher percentages of racial minorities experience longer wait times for ridesourcing services at night after adjusting for differences in income (hughes and mackenzie, 2016) , and in new york city, ridesourcing pickups are found to be less common in lower income areas (jin et al., 2019) . additionally, evidence of racial and gender discrimination has been identified in ridesourcing practices in boston and seattle (ge et al., 2016) . although a smaller body of research has found evidence of more equitable ridesourcing practices in some cities, such as longer wait times in areas with higher average income in seattle (hughes and mackenzie, 2016) or more frequent ridesourcing in low-income neighborhoods in los angeles by one trip per month while controlling for residential location (brown, 2019b), ridesourcing inequity is still a problem that needs to be addressed in many cities and for many groups. furthermore, findings that racial discrimination against riders may be worse among taxis than ridesourcing (brown, 2019a), still does not constitute evidence that ridesourcing is equitable on the whole. in terms of bikeshare, chicago has an embattled history of socioeconomic segregation leading to starkly different daily use patterns. biehl et al. (2018) shows that divvy uptake is lower in the less affluent southside even after controlling for station tenure and density. an early investigation of newly released chicago ridesourcing data suggests a similar concentration of rides in the more affluent north and central quadrants of the city (soria et al., 2020) . this context of on-demand mobility usage and equity helps to inform our variable selection and interpretation of revealed ridesourcing substitution behavior as it relates to sociodemographics at the station, community area, and city quadrant levels. this perspective offers a valuable contribution to the literature, because equity aspects of transportation resilience have traditionally been understudied (mattsson and jenelius, 2015) . twenty-eight chicago transit authority rail transit disruptions, listed in table 1 , are identified as having occurred during the period of november 2018 through october 2019 using a google news search for the phrase "cta disruption". we filtered cases to only include significant disruptions (i.e., lasting a minimum of one hour). the event-specific variables include the location (in terms of city side, quadrant, community area, and station), number of other stations impacted, disruption cause, disruption duration, deployment of shuttle buses, outside air temperature, weekday status, holiday status, peak hour status, late night status, and the cause being a medical emergency (cta, 2019). the ridesourcing data used in this study were obtained from the city of chicago data portal (chicago data portal, 2019) . the transportation network companies in the dataset include uber, lyft, and via. the entire dataset consists of over 152 million trips spanning the period of november 2018 through october 2019. the variables that we analyze in this study are trip start date and time, trip miles, pickup community area, and pickup census tract. the ridesourcing trip data is matched to the identified transit disruption days and comparable baseline operations. the starting location of each ridesourcing trip is identified by census tract or community area, and if that location is within a 0.25-mile radius of a disrupted transit station, the trip is included in the analysis. this frequently-used walking estimate (younes et al., 2019; zhao et al., 2003) is applied to account for riders who source rides on their way to or from the impacted transit station or who step away from the potential crowd surrounding the impacted station to facilitate pick-up by their ridesourcing driver. to generate a robust four-day ridesourcing demand baseline, trip counts during the disruption time period are averaged across the same day of week and time of day as the disruption for two weeks prior to the event and two weeks following. this assumes mode shifting behavior from transit to ridesourcing would typically not continue beyond two weeks following a single, unplanned transit disruption that lasts on average 2.5 hours. while this may be a conservative estimate, it avoids the risk of confounding changes in station accessibility and seasonality. the city of chicago is divided into 77 community areas, which can be further aggregated into four quadrants or sectors of the city: central, north, west, and south. the community area variables that we include in our analysis are the number of bus stations, number of divvy stations, station ridership, population density, area, airport presence, total population, median household income, percentage of zero vehicle households, percentage of commuters taking transit, and percentage of residents who self-identify as predefined categories of hispanic or latino, white non-hispanic, black non-hispanic, asian non-hispanic, or any other race and ethnicity category, according to five-year estimates from the american community survey (u.s. census bureau, 2017) and as reported by the chicago transit authority (cta, 2019), the chicago metropolitan agency for planning (cmap, 2019), and divvy (divvy, 2020) . sociodemographic data on riders of transit and ridesourcing are not available, so the socioeconomic characteristics of the surrounding geographic areas are used instead. multilevel mixed (mlm) modeling is designed to properly account for the hierarchical nesting of data and effects happening at different levels (goldstein, 2003; julian, 2001; wampold and serlin, 2000) . mlm models provide a mechanism for analyzing datasets where observations (in this case, station disruptions) are nested within higher-order spatial contexts, such as neighborhoods. in the past, mlm or hierarchical models have been used to represent the structure of social relations within personal networks (carrasco and miller, 2009) , temporal changes in bike share trips (el-assi et al., 2017) , and transit demand between origin-destination station pairs (iseki et al., 2018) . we use a multilevel regression analysis to identify the station level, community area level, and city quadrant level factors associated with systematic variations in ridesourcing demand during transit disruptions. we can thereby examine explanatory variables at each level of the data hierarchy, and in doing so, control for community area effects on station ridership variation. the advantage of using the multilevel structure is the ability to estimate the variability in results that can be attributed to neighborhood (e.g., community area) effects rather than only to individual station effects. by carefully controlling variable-inclusion at the appropriate level, the model takes into account correlations between observations within the same group (i.e., a given community area) as distinct from correlations between groups (jones and duncan, 1996) . instead, a standard one-level regression model would ignore group-level distinctions (for example, different commuting patterns in different communities) and group level correlations (for example, similar patterns of use among stations in the same community related to the income-level of riders). a useful way to think of mlm models is as a structure sitting between two modeling extremes when groupings are known: fully pooled and fully unpooled specification (gelman and hill, 2007) . a fully pooled model treats group-level variables as individual variables, thereby ignoring grouplevel distinctions. the opposite extreme, a fully unpooled model, asserts that the groups are so completely different that they cannot be associated in the same model. the mlm model offers a compromise between complete distinction of groups and the complete ignorance of group-level effects by modeling individual-level fixed effects as well as distributional assumptions on the random effects. to address the research question of ridesourcing surges triggered by transit disruptions, we control not only for the immediate station attributes where the disruption occurs (level 1), but also for community area (level 2) and quadrant (level 3) factors in a three-level structure. fig. 2 shows the hierarchical, three-level model framework. the dependent variable is the number of ridesourcing trips compared to the baseline demand two weeks prior and two weeks following the disruption (i.e., individual station observations). covariates related to the disruption cause, context, and timing are included as explanatory variables at this level, in line with mojica (2008) and pu et al. (2017) . we further investigate whether the fact that stations are nested within community areas and major quadrants plays a role in ridesourcing demand shifts. it is likely that a comparable disruption can generate different mode-shifting effects depending on where it is located, owing to the different composition of travelers and availability of alternative modes. specifically, the broader context is controlled for by including sociodemographic and mobility factors measured at the community level that are in turn aggregated to the quadrant level of analysis. it is worth noting that since the disruptions we measure result from a natural experiment, we are unable to control exhaustively for all combinations of factors that are at play within and between community areas. therefore, we include random intercept effects at each of the lower-nested group levels to partition the unexplained variability effects on the dependent variable. conceptually, the model can be articulated as regression equations occurring at different levels where each group-level coefficient has its own regression equation. following gill and womack (2013) , the general three-level structure is defined in eq. 1 as: where i represents the station, j represents the community area, and k represents the quadrant. '#$ is the (random) intercept measuring average ridesourcing use (defined in eq. 2), and )"#$ is a predictor, such as the average daily transit use measured at the station level, while )#$ is the (random) slope depicting the relationship between the station-variables and the change in ridesourcing demand (as defined in eq. 3). the error term "#$ relates to station-level effects. by including level 2 and 3 explanatory variables in the model, we uncover context-level effects. namely, we account for variability in coefficients at the station level owing to community area or city quadrant level factors. level 2 includes variables aggregated to the community area level expected to impact all stations in the area. this can be thought of as being equivalent to the way in which student educational performance is affected by their classroom teacher in a way that is distinct from the effects of their individual factors or from more aggregate school-level effects. the subscript jk denotes the distinct community area impacts. the é£ has numbered subscripts, the first denotes the intercept (0) or slope (1), while the second subscript denotes the independent variable. at level 2, the general regression equations are defined as: where the random intercept '#$ is a function of é£ ''$ , the grand mean of ridesourcing demand surges across stations in the community (defined below in eq. 4). departures from this average intercept represented by -#$ are community-level predictors with é£ ')$ denoting the random slope for community level predictors (eq. 5), and '#$ is the unique effect associated with communities assumed to have a multivariate normal distribution. the random slope )#$ is a function of é£ )'$ representing the average effect of the station-level predictors (i.e. the slope over all stations shown in (eq. 6)). departures from the slope (i.e., random effects) over station predictors are represented by the é£ ))$ coefficient (eq. 7) that would be removed for a random intercept-only model (as in the current case). at level 3, variables vary by quadrant and apply to all individual cases and community areas assigned to this group. therefore, they contain the subscript k as opposed to ijk or jk. at level 3, the separate regression equations for the intercepts and slopes are defined as: é£ )'$ = ) + 4 1$ + )'$ (6) é£ ))$ = 1 + 5 1$ + ))$ where ' is the intercept shared by all individual cases, ) , -, and 1 are the main effects, 0 , 3 , and 4 are two-way interactions, and 5 is a three-way interaction. in our specific modeling, the outcome variable of the three-level hierarchy "#$ is defined as the change in ridership over baseline. after specification testing, the final model takes the forms shown in eq. 8-16. the model includes a random intercept '#$ and two main effects ( _â�� "#$ and _â�� "#$ ) at level 1, shown in eq. 8. level 2 brings in contextual variables used to explain variability in ridesourcing demand via cross-level interactions. that is, now we model the intercept and slopes explicitly, and include level-one and level-two independent variables interacted to describe variation in the intercept. eq. 9-11 shows the random intercept é£ ''$ and the cross-level interaction terms ( _ â�� #$ ã� _â�� "#$ and _ #$ ã� _ "#$ ). level 2 also specifies )#$ and -#$ which represent the parameter slopes with é£ )'$ and é£ -'$ . level 3 includes the random intercept ' and one quadrant level interaction ( â��_ $ ã� â�� "#$ ) that is found to generate variability in ridesourcing (eq. 12), with remaining parameters ) anddenoting the fixed slope coefficients. the disturbance parameters are included at the community '#$ and quadrant levels ''$ (eq. 15-15). it is important to note that the cross-level interactions explain a significant amount of variance of ridesourcing demand changes in addition to that already explained by the station-level equations. level 1 model ''$ ~ (0, s -) the descriptive analysis suggests that a significant surge in the use of ridesourcing does occur following no-notice transit line disruptions, as suggested in fig. 3(a) . this depicts a high-impact northside case in lake view at the belmont station (the source of the disruption) on a monday in december during morning peak hours. the disruption cause was a train striking a person. for reference, the baseline ridesourcing demand for this timespan and location is 807 rides. the disruption is associated with a significant surge in ridesourcing requests, totaling 2,883 and corresponding to an increase of 257% (a t-test of the disruption versus the baseline means has a pvalue of 0.00001). however, ridesourcing adaptation is not uniform across the city. a different case is shown in fig. 3(b) of a similar disruption event, this time occurring in an under-resourced westside neighborhood where there appears to be limited shifting towards on-demand services. this shows a low-impact case in east garfield park at the kedzie station (the source of the disruption). similar to the belmont disruption, it occurred during weekday morning peak hours and was caused by a person on the tracks. the baseline ridesourcing demand for this time and location is a fraction of that at belmont: 89 rides. the number of ridesourcing rides during the disruption event is lower than the baseline at 76 (-15%), which is not a significant change (a t-test of the disruption versus the baseline means has a p-value of 0.3984). given that lake view has a median household income of $86,119 and 79% of its residents are white, while east garfield park has a median household income of $23,116 and 5.6% of its residents are white, this behavioral difference could be tied to racial and economic inequity. to examine the different patterns of ridesourcing demand shifts triggered by transit disruptions systematically, we turn to the model results. table 2 , the mlm model includes both station-level (level 1) fixed effects (nonholiday disruption and peak hours disruption), which are similar to standard regression parameters, and explanatory features that reflect the context surrounding the station: namely, two cross-level random effects (percent white during peak hours and percent transit commuters at the source of disruption), and one quadrant-level effect (shuttle deployment in the north quadrant). all parameters are significant to a 98.9% level of confidence or greater except for the model constants. the model also includes random intercepts for the community area (level 2) and city quadrant (level 3) effects. first, we estimate a null model that partitions the variance at each level without including any explanatory variables. this enables us to calculate the intraclass correlation (icc), also known as the variance partition coefficient, for three levels, following snijders and boskers (1999) . this statistic measures the correlation among data at the lower levels to determine how much of the variation in ridesourcing demand is accounted for by variation among community and quadrant level factors. a smaller variance partition coefficient indicates that the variation in ridesourcing shifts is attributable more to variation among lower-level units (such as stations) than to that among upper-level units (like community areas and quadrants). the empty mlm model (model 1) thereby provides an estimate of baseline variance of ridesourcing demand shifts due to factors beyond the immediate station. the intra-quadrant correlation is small and suggests that 20% of the variance of the dependent variable is due to quadrant-level effects. in contrast, the intra-community correlation reveals that the largest share of total variance (43%) is related to community-level factors, suggesting that the lowest level of analysis (the station level) explains the remaining variance (37%). this indicates the largest share of the variation in ridesourcing demand substitution is related to factors that occur across communities, followed by the station level. the evolution of the variance estimation deserves attention. when adding station-level independent variables in model 2, the quadrant random intercept and thereby icc is shown to be insignificant, while the variance is now partitioned between the station (44%) and community area levels (56%). along the same lines, when adding cross-level effects by including variables measured at the community area level, the variance explained clearly pivots toward the community area variables (model 3). we note that despite the level 3 quadrant random intercept collapsing to zero, removing this variance component from the analysis causes a significant reduction in overall model fit. this analysis of random intercepts points to two important observations. first, the variance partitioning shows the importance of controlling for variables measured at the neighborhood factors that would have been overlooked in a standard regression solely focused on station substitution analysis. moreover, the inclusion of more explanatory factors leads to the absorption of more variability in the ridesourcing demand shift at the neighborhood levels. the main takeaway from the variance controls is the following: the differences between factors occurring across different community areas in the city are the most decisive in shaping the ridesourcing demand shifts following disruptions. we interpret this to mean that there are significant latent neighborhood effects at play in the shift to ridesourcing during transit disruptions. to model systematic variables, we follow the block entry approach consisting of the gradual addition of covariates level by level (cohen et al., 1983) , following the plan previously outlined in fig. 2 . first, each of the hypothesized predictors measured at the station-level are tested independently, then jointly. owing to high variable collinearity, only two fixed-effect explanatory variables related to the timing of the disruption and a constant are included in the resulting model 2. these statistically significant effects result in a significant improvement in model fit as measured by the deviance difference (836.99 -822.10 = 14.89, exceeding the critical ï� 2 of 5.99 with alpha set at .05) and aic reduction. looking at the constant, the model suggests a moderate average increase of 54 ridesourcing trips (or 15.6%) during a transit disruption, compared to baseline. to contextualize this finding, we note that the average baseline ridesourcing ridership is 347 trips across the chicago community areas covered in the disruption analysis. this value can be seen as the ridesourcing demand that would be occurring for the same station and timespan without the disruption. with this baseline in mind, the timing of disruptions is revealed to be highly impactful. on average, when a disruption occurs on a weekday (excluding holidays), ridesourcing rides increase by 541 from baseline (a 156% increase). when a transit disruption occurs during peak hours, ridesourcing demand increases by 408 rides from baseline (a 118% surge). the analysis suggests the existence of some citywide trends related to the timing of no-notice disruptions, likely related to less flexible trips during peak hours and weekdays. this finding is not surprising considering that business and commuting trips are more likely to be shifted to another mode than canceled, which has been shown for pre-planned disruptions (van exel and rietveld, 2009 ) and for unreliable metro services (pnevmatikou and karlaftis, 2011) . the novelty of our findings refers to the degree of shifting towards on-demand ridesourcing, a mode which has not been considered in previous work, which has been dominated by car substitution and transit replacement analysis. for completeness, following the fig. 2 modeling framework, we note that we are able to find no consistently significant impact of temperature, deployment of shuttle buses, number of nearby bus or divvy stations, nor general transit commute ridership. this is somewhat surprising given that the research on consequences of long-term transit disruption shows that the context (e.g., spatial or temporal) results in different rider adaptation strategies. therefore, we had expected specifically that modal alternatives (e.g., buses, bikeshare, etc.) would impact ridesourcing. for example, given previous research findings on the role of bikeshare as an adaptive strategy during long-term transit disruptions in washington, d.c. and london, we had expected to find that the availability of nearby divvy bikeshare stations would significantly decrease the observed shift to ridesourcing during unplanned transit disruptions. however, this was not the case, possibly due in part to trip purpose, travel distance, local bikesharing culture, and/or the inconvenience of requesting a membership in response to a single unplanned disruption. nevertheless, from the icc calculation in the current study, we know that the community area context is the main source of unexplained systematic differences in ridership shifting strategies. these neighborhood effects likely vary as a function of ridership culture (including car, transit, and ridesourcing culture), socioeconomic and political factors, and transportation agency strategies. while it is possible that some of these effects could be measured narrowly around the disruption events (i.e., stations), we note that the mlm enables us to analyze important factors like population density, median household income, and racial composition measured at more aggregated levels. the next section seeks to pinpoint the systematic factors that can explain the observed stochasticity. a fundamental goal of this multilevel analysis is to estimate the variability in ridesourcing use that can be attributed to community area level characteristics rather than to individual station factors and to identify how these components of variation change with the inclusion of predictors that quantify the context. in model 3, we hypothesize that the context surrounding the disruption also plays a role in determining the transfer of ridership from transit to ridesourcing during nonotice events. specifically, we hypothesize that the rate of ridesourcing substitution is associated with sociodemographic privilege, in line with earlier research (deka and fei, 2019) . this guides the inclusion of a number of predictor variables measured at the community area (not station) level in the form of cross-level interactions, including factors such as zero car households and population density. we apply group mean centering for community area variables (enders and tofighi, 2007) to facilitate the interpretation of the cross-level interactions. model 3 reveals a significant impact of two community area level factors; racial composition and percent of transit commuters. the addition of these cross-level factors leads to significant improvements in goodness-of-fit measured by the deviance difference and aic. while the crosslevel effects look comparatively small, they need to be related to the percentage unit of the variable measurements. the positive effect associated with the interaction term for percentage of white residents in the community area with the dummy variable for peak-hour travel (a coefficient of 12.3 additional trips) provides insight into the combined effect of race composition in the local area on the previous peak-hour effect findings. namely, across community areas, the peak-hour impact (353 added trips) is further boosted when disruptions occur in communities with higher shares of white residents. the implied difference is that, other things equal, a disruption occurring in a community area with a 10% higher share of white residents would result in a boost of 120 (or 34.6%) ridesourcing trips compared to the average peak-hour baseline. recalling that communities of color in chicago are more likely to be under-resourced in relation to job accessibility, transit supply, and on-demand mobility, we believe this finding is more likely a reflection of gaps in access to resources in areas with lower shares of white residents than of a lower willingness to use ridesourcing during disruptions. this finding adds to existing evidence that ridesourcing, in this case as a disruption gap-filling resource, gives more benefit to privileged user groups (zhang and zhang, 2018) . additionally, a novel effect is found related to the proportion of transit commuters in the community area and the incident location. on the whole, every percentage unit increase in transit commuting in the community area results in 18 added ridesourcing trips (or a 5.2% increase). however, this effect only occurs at the station where the incident responsible for the disruption is occurring. we speculate that transit commuters more readily shift to hailing services when they experience the disruption and receive information about it directly. in other words, riders at the source of the disruption are likely to have more information regarding the nature of the disruptive event from official sources and other riders, which will likely factor into their adaptation strategy. on the other hand, in areas with less transit commuting, there is presumably less collective experience with disruptions and therefore a higher likelihood of shifting to other private modes. despite there being no unexplained systematic differences related to the quadrant level beyond model 1, we conduct a model search to look for further impactful interactions including quadrant dummies for the four parts of the city. the model suggests a surprising finding. in the north quadrant, when a shuttle is deployed, ridesourcing rides increase by 321 instances (or 92.5%) from baseline. the deployment of replacement bus services during rail disruptions is the most common agency response (pender et al., 2013) . yet, there appears to be an unintended negative effect of this strategy. that is, the deployment of added transit bus capacity to assist riders should not boost ridesourcing requests. we interpret the unexpected increase in ridesourcing to be related to the signaling effect of this action. riders could perceive bus deployment as a strong cue for the severity of the disruption, or they may fear excessive crowding on the buses, both of which justify the decision to use ridesourcing. the north quadrant is home to the biggest share of disruptions in our dataset (16/28 or 57%), as well as heavy transit demand by commuters (fig 1.d) . the fact that replacement bus deployments trigger more ridesourcing substitution in the north quadrant is likely related to the higher income levels among commuters in this corridor. given the situational and locational contexts impacting the use of ridesourcing as an adaptive transportation strategy, it is important to consider the potential mobility resilience inequity across the city. if ridesourcing as a gap-filling mechanism is mainly associated with mandatory travel, disruptions of greater severity (i.e., requiring the deployment of a shuttle bus), and community areas with higher percentages of white residents and transit commuters, its role in mobility resilience is specific, selective, and unlikely to address existing issues of mobility inequity. specifically, our findings show that riders in areas with a higher proportion of non-white residents are less likely to divert to ridesourcing in times of disruption. this result does not in itself suggest supply inequity. however, when taking into account the fact that gaps in mobility services disproportionately impact communities that lack good access to transportation, the finding raises an important issue. under-resourced communities with poorer accessibility options would benefit the most from access to more adaptability options in the face of transit disruptions, and ridesourcing could play a greater role in this adaptation portfolio. taken together, our findings point to an opportunity to increase equitable mobility resilience by addressing the barriers that limit access to on-demand transportation. this highlights the need to consider socioeconomic constraints in disruption response planning and to fill service gaps through collaboration between transit providers and ridesourcing companies. when a major transit disruption occurs, riders may not be well-informed of the reason for the disruption or the expected duration. typically, some information is provided to passengers already on a transit platform, and only a brief description of a service alert may be offered online. however, this information may not be timely or may be generic or incomplete, making it difficult for passengers to develop informed response strategies. in this paper, we show how riders spontaneously respond to disruptions by substituting transit with ridesourcing. in some settings this occurs despite agency efforts to appease their passengers. for example, ridesourcing demand surges more when shuttles are deployed or when the disruption occurs at the traveler's station. through improved communication, transit providers could advise travelers of when and how to seek alternative transportation means and efficiently inform ridesourcing companies of disruptions. given this information, ridesourcing companies and drivers could work in tandem with route-around bus services to meet spikes in demand and avoid exploiting the situation through surge pricing. the flexibility of ridesourcing services offers on-call availability to provide extra capacity, while buses are able to maintain fixed routes for prolonged periods of time. in some cases, it might actually be cheaper for transit carriers to subsidized shared ridesourcing instead of delivering shuttle buses. by communicating the nature of the disruption and anticipated needs, transit agencies could engage ridesourcing drivers in adaptive, gap-filling services to address unplanned disruptions and reduce the adverse impacts experienced by transit riders. while collaborations between transit providers and ridesourcing companies may provide a way to decrease disruption response time and assist a greater number of affected travelers, these partnerships are not without challenges. most notably, transit providers are required to ensure fair service to all individuals in accordance with title vi and the americans with disabilities act, while ridesourcing services are not currently held to the same standards. another crucial challenge is the negotiation of data and information sharing among public transit agencies and private transportation providers. public-private data-sharing partnerships are an important collaborative mechanism through which governmental agencies and ridesourcing companies may develop datasupported policies to fill transportation gaps while protecting user privacy (cohen and shaheen, 2018). our findings suggest that transit riders in areas with a higher proportion of non-white residents and in less affluent quadrants may be left out of the option to shift to on-demand ridesourcing during unplanned transit disruptions. in this section we discuss how the joint goal of providing mobility adaptiveness and achieving transportation equity, can be achieved across three levels of analysis: spatial, economic, and social. first, existing spatial inequities in chicago have resulted in low-income communities and communities of color experiencing long-running transportation challenges. residents in these communities are subject to spatial mismatch, have less accessibility to public transit within walking distance, and experience longer commutes, summarized as having fewer livability opportunities (ferrel et al., 2016) . underlying mobility inequity at the spatial level can be addressed by transportation policymakers through partnerships between public transit agencies and ridesourcing companies and by providing more micro-transit in lower-density areas. second, economic inequity is apparent in the distribution of resources that are needed to use ridesourcing, including direct costs (e.g., proportion of income spent on fares) and indirect costs (e.g., smartphones, credit cards, and internet access). as urban areas become more digitally integrated, offering residents greater mobility resilience to disruptions through on-demand ridesourcing services, those on the underserved side of the digital mobility divide are left further behind. practical measures to combat direct and indirect costs include providing lower income commuters or travelers with subsidies or vouchers for lower cost access to ridesourcing during transit disruptions, access to smartphones, multi-modal hubs with internet access, and unbanked payment options similar to transit smartcards. third, social inequities refer to existing racial, cultural, and language barriers to ridesourcing usage. for example, there is a potential supply equity challenge in light of the evidence that communities of color experience longer ridesourcing wait times than non-white communities in seattle (hughes and mackenzie, 2016) , also suggested for chicago (cnt, 2019) . longer wait times can be especially troublesome for peak period commuters. to address social inequities, such as supply inequity wherein the opportunity to use ridesourcing is not readily available to all communities, there is a need to consider ridesourcing incentives, safety initiatives, and driver training on socially equitable practices to minimize sociodemographic profiling. to combat poorly targeted marketing, transportation agencies could offer information campaigns and outreach programs geared toward historically underserved communities that highlight equitable transportation initiatives to improve adaptability and resilience during travel disruptions. in particular, it is important that under-resourced communities have a say in the decisions that impact them through bottom-up concept generation and participatory policymaking. ultimately, agencies may shift toward the idea of mobility as a service. if transit operators, like the cta, were to adopt a mandate to deliver stop-to-stop service despite unplanned disruptions, they might internalize the responsibility of providing transportation alternatives when a disruption occurs. this lack of separation between agencies and domains may represent the future of transportation operations, and public-private partnerships between transit agencies and ridesourcing companies may provide a first step toward making this future a reality. to meet service quality constraints, the on-demand rapidity of ridesourcing service response could be advantageous. if deployed ridesourcing was moreover provided as a pooled service, it might be more cost efficient for transit agencies than a shuttle service, which requires equipment maintenance by the cta and short-notice driver availability (shared-use mobility center, 2020). furthermore, such partnerships would be beneficial to have in place ahead of long-term transit service disruptions, such as those observed during the covid-19 pandemic. for example, the la metro in the los angeles region was able to leverage a preexisting partnership with the ridesourcing company via during the pandemic by expanding their role from providing first-and last-mile services to private, point-to-point trips to accommodate essential travel (grossman, 2020) . this exemplifies the ability of public-private partnerships to increase mobility resilience to unexpected disruptions. while this study is among the first to examine impacts of unplanned transit disruptions on the usage of on-demand ridesourcing services across the city of chicago, some caveats warrant discussion. first, information on transit disruption and shuttle bus deployment were gathered from local news sources and are subject to source accuracy. for the analysis, assumptions were made that disruptions lasted the same duration at each affected station and that shuttle services (when provided) were deployed to all affected stations. second, determining the spatial band where riders change travel behavior is complicated to determine and depends in part on their location when informed of the disruption, as well as their intended destination. we consider mode-switching behavior within a 0.25-mile radius of each affected station which is likely to underestimate the true degree of shifting. particularly, mode-shifting behaviors may have occurred across a broader time-space domain, including travelers who learned of the disruption prior to departure. third, we use aggregated measurements of community variables as a proxy for individual-level attributes, which masks variation in socioeconomic characteristics of riders. the characteristics of the actual riders aboard the train and the areas in which they reside are unknown. despite these limitations, which are all associated with the use of a natural experiment approach, our research contributes new insights that would be difficult to gage using smaller scale stated data. namely, we capture the actual circumstances of the disruptions leading to adaptive use of ridesourcing. importantly, the findings of this study bring to light which community groups are able to shift to on-demand mobility during a disruption and who is left behind to seek out other alternatives. our findings suggest two main avenues for future research. first, to better gage socioeconomic distributions of mobility resilience, further collaborative research should aim for a more nuanced analysis of the behavioral adaptations practiced by transit riders who do and do not shift to ondemand mobility. for example, access to disaggregated data on ridesourcing pickup locations with rider sociodemographics matched with spatio-temporal bus ridership would reveal more detailed insights into a user-based (in)equitable distribution of multi-modal, context-specific adaptive strategies that are enacted to complete disrupted travel (i.e., our measure of mobility resilience) across disaggregated marginalized populations. second, the latent area effects observed in this multilevel analysis suggest that future work should investigate the impacts of mobility culture, values, and attitudes surrounding the use of ridesourcing as an adaptive mobility strategy. better understanding of latent constraints would enable the tailoring of transportation policies by area to improve equity and adaptability, thereby enhancing mobility resilience. while research on the provision of information to riders and their observable reactions is extensive (leng and corman, 2020; sarker et al., 2019 , ben-elia et al., 2013 , mahmassani & liu 1999 , there is still a need for an empirical understanding of adaptation decision-making to recover from unplanned travel disruptions in the presence of ridesourcing options. in this study we use a natural experiment design to identify the main determinants of ridesourcing substitution as an adaptive response strategy during unplanned transit disruptions. our findings reveal that spikes in ridesourcing demand are strongly tied to peak-hour and weekday travel, suggesting a city-wide effect of mandatory travel. however, the relationship between transit disruptions and ridesourcing substitution behavior is also influenced by the temporal and spatial context in which the disruption occurs. peak-hour disruption shifts to ridesourcing are positively correlated with the percentage of white residents in the surrounding community area, which suggests potential accessibility inequity in terms of options for mobility adaptation (i.e., resilience). that is, riders in areas with higher shares of non-white residents are less likely to turn to ridesourcing for mandatory travel during transit disruptions. this suggests a greater vulnerability of racial minorities to the negative impacts of transportation disruptions. inequitable access to multiple (redundant) and robust mobility options needs to be addressed by transit agencies and transportation policymakers. in this paper we outline and discuss two policy perspectives arising from these findings, namely public-private partnerships and information campaigns, and a three-level framework for agencies to think more broadly about mobility justice initiatives. a comparison of the personal and neighborhood characteristics associated with ridesourcing, transit use, and driving with nhts data centering predictor variables in cross-sectional multilevel models: a new look at an old issue effects of built environment and weather on bike sharing demand: a station level analysis of commercial bike sharing in toronto handbook for building livable transit corridors: methods, metrics and strategies. ( no. tcrp h-45) transit cooperative research program the impact of public transportation strikes on use of a bicycle share program in london: interrupted time series design substitution of ride-hailing services for more sustainable travel options in the greater boston region modeling determinants of ridesourcing usage: a census tract-level analysis of chicago racial and gender discrimination in transportation network companies (no. w22776) data analysis using regression and multilevel/hierarchical models the multilevel model framework. the sage handbook of multilevel modeling multilevel statistical models time machines and virtual portals: the spatialities of the digital divide resiliency of transit agency partnerships with private mobility services in the face of covid-19 transportation network company wait times in greater seattle, and relationship to socioeconomic indicators the determinants of travel demand between rail stations: a direct transit demand model using multilevel analysis for the washington dc metrorail system uber, public transit, and urban transportation equity: a case study people and places: the multilevel model as a general framework for the quantitative analysis of geographical data. spatial analysis: modelling in a gis environment the consequences of ignoring multilevel data structures in nonhierarchical covariance modeling impact of pricing and transit disruptions on bikeshare ridership and revenue. transportation transportation equity project: prioritization criteria the role of information availability to passengers in public transport disruptions: an agent-based simulation approach uberhop in seattle: who, why, and how? transportation research record dynamics of commuting decision behavior under advanced traveler information systems insights on disruptions as opportunities for transport policy change vulnerability and resilience of transport systems-a discussion of recent research examining changes in transit passenger travel behavior through a smart card activity analysis (doctoral dissertation, mit) metro service disruptions: how do people choose to travel? state of transportation in a day without metro in the washington region analysis of transit users' response behavior in case of unplanned service disruptions transport resilience and vulnerability: the role of connectivity understanding the impacts of a public transit disruption on bicycle sharing mobility patterns: a case of tube strike in london applying affective event theory to explain transit users' reactions to service disruptions dynamics of travelers' modality style in the presence of mobility-on-demand services multilevel analysis: an introduction to basic and advanced multilevel modeling 2020) k-prototype segmentation analysis on large-scale ridesourcing trip data estimating the influence of common disruptions on urban rail transit networks public transport strikes and traveller behaviour the consequence of ignoring a nested factor on measures of effect size in analysis of variance how transit service closures influence bikesharing demand; lessons learned from safetrack examining the relationship between household vehicle ownership and ridesharing behaviors in the united states forecasting transit walk accessibility: regression model alternative to buffer method travel behavior reactions to transit service disruptions: study of metro safetrack projects in this research was supported in part by funding from the national defense science and engineering graduate (ndseg) fellowship provided to borowski. stathopoulos was partially supported by the us national science foundation (nsf) career grant no. 1847537. key: cord-035308-996ysogr authors: twining, peter; butler, deirdre; fisser, petra; leahy, margaret; shelton, chris; forget-dubois, nadine; lacasse, michel title: developing a quality curriculum in a technological era date: 2020-11-11 journal: educ technol res dev doi: 10.1007/s11423-020-09857-3 sha: doc_id: 35308 cord_uid: 996ysogr there is considerable rhetoric internationally around the need for national curricula to reflect the changes that are taking place in the world outside school. this raises questions about what a quality curriculum in a technological era should look like, and equally challenging issues about how to achieve the necessary changes in schooling in order for such a curriculum to be realised. this paper summarises the views of 11 experts from seven countries. it introduces a sociocultural framework that highlights the complexity of achieving alignment between policies and practice spanning the national to local school to classroom levels. three key issues that underpin alignment are then explored, each of which link with the issue of trust: stakeholders engagement; teacher professionalism; summative assessment. by exploring and exemplifying these three issues the paper indicates potential ways of addressing them and provides ‘tools to think with’ to enhance future curriculum development initiatives. we live in a rapidly changing world that is struggling "to respond to economic, environmental and social transformations -including technological advances, climate change and migration" (oecd 2019, p. 9 ). these transformations, and in particular the impact of digital technologies, have increased demand for higher order skills, competencies and knowledge. they have also led to uncertainty about the future, for example: the potential impact of artificial intelligence on some workplaces; how to keep up with an accelerating pace of change; or how to manage global competition for skills . within this global context, the school sector is expected not only to meet the demand for new, higher-order skills and competencies but also to prepare students to thrive in a future where the competencies and knowledge they will need to succeed are constantly changing. this is not about learning about digital technology per se, it is about how our digital era impacts on all aspects of schooling. indeed, focussing on one aspect of the system, such as digital technology, without considering the system as a whole is unlikely to bring about sustained change (butler et al. 2018) . in an effort to meet these challenges, many countries are re-designing their national curricula so as to better prepare young people now and in the future (wolfenden et al. 2018 ). this begs the question 'what does a quality curriculum look like in a digital era?' that is the question that is the focus of this paper and that technical working group 12 (twg12) at edusummit 2019 set out to answer. in so doing they needed to consider what educational visions, policies and practices might be most appropriate (butler et al. 2018) . this involved discussion of: (a) what knowledge, competencies, attitudes and values today's school students need to thrive and shape their world (e.g. see erstad and voogt 2018) . (b) how school systems can develop such knowledge, competencies, attitudes and values effectively (e.g. oecd 2018). however key to these discussions was developing an understanding of what is understood when we use the term 'curriculum' and how 'quality' is defined. the starting point for any consideration of 'quality curriculum' for schools must include a discussion of what is understood by the concept 'curriculum'. the word comes from latin, where 'curriculum' (related to the verb currere, i.e. running) refers to a 'course' or 'track' to be followed. in the context of schooling, where learning is the central activity, the most obvious interpretation of the word curriculum is then to view it as a course, trajectory, or 'plan for learning ' (cf. taba 1962) . however, this is a rather narrow view. van den akker (2003) differentiates between different levels of curriculum: • supra level: international debates or agreements on aims and quality of education • macro level: curriculum at the system/society/nation/state level (e.g. national syllabi or core objectives) • meso level: curriculum at the school/institution level (e.g. school-specific curriculum) • micro level: curriculum at the classroom level (e.g. textbooks, instructional materials) • nano level: curriculum at the individual/personal level thus the process of curriculum development can be seen as narrow (developing a specific curricular product) or broad (a long term, ongoing process of curriculum improvement, often including many related aspects of educational change, such as teacher education, school development, and examinations). building on un sustainable develoment goal 4 (united national general assembly 2015) and butler et al. (2018) , a quality curriculum would be a 'plan for learning' which supports young people in acquiring the knowledge, competences and dispositions needed to be successful in the digital era. an important aspect of the digital era is growing global interconnectedness, and thus it makes sense to examine curriculum developments at the international (supra) level. in a review of international trends and practices in curriculum development walsh (2018) identified that one of the key characteristics of international practices (at the supra level) was the preparation of curriculum frameworks rather than prescribed curricula, allowing flexibility in interpretation and enactment at a national (macro) and school (meso) level. such frameworks provide an insight into the key issues underlying curriculum development debates internationally. two such curriculum frameworks which were explicitly developed in the light of understandings of the digital era are outlined below. the oecd learning framework 2030 (oecd 2018), which is summarised in fig. 1 , explicitly addresses the needs of individuals and of society. it is underpinned by a vision of "helping every learner develop as a whole person, fulfil his or her potential and help shape a shared future built on the well-being of individuals, communities and the planet" (p. 3). this framework merges different views of knowledge (including 'knowledge about'/ content) with understanding different ways of making sense of the world and knowing how people act in different disciplines. this is combined with skills ('knowledge of') in the form of the application of knowledge to enable students to act in appropriate/valued ways in particular contexts. attitudes and values are seen as mediating these different forms of knowledge. the combination of these three elements ('knowledge about', 'knowledge of', and values and attitudes) result in what the oecd refer to as competencies. this contrasts with traditional curricula in many school systems, which have tended to focus on 'knowledge about' (biesta 2004) . importantly, the oecd framework recognises that the development of competencies requires changes in pedagogy, with a particular focus on increasing student agency. the eu's key competences for lifelong learning includes eight key competences (see fig. 2 ), which are identified as being "essential to citizens for personal fulfilment, a healthy and sustainable lifestyle, employability, active citizenship and social inclusion" (european commission 2019, p. 4). as with the oecd framework there is a focus on both individual and societal needs, and a recognition that learning needs to go beyond content to include skills and attributes. these supra level frameworks provide a useful starting point for discussions of 'quality curriculum' at the national (macro) level. both the eu key competences framework and the oecd learning framework 2030 are underpinned by views about the purposes of education that are encapsulated in their aims. in both cases these encompass individual performance and collective well being, which may be seen as being in tension with each other. indeed, both frameworks see the purposes of education in terms of capacities, competencies or capabilities, so articulation is not "in terms of what the students should learn but in terms of what they should become" (biesta and priestley 2013, p. 4 ). this provides a stimulus for national curricula to think more broadly about what knowledge, competencies and dispositions school students need to thrive and shape their world (e.g. see erstad and voogt 2018) and thus should be included in a quality curriculum; i.e. a 'plan for learning' which supports young people in acquiring the knowledge, competences and dispositions needed to be successful in the digital era. having outlined the context, this paper will now examine the importance of purpose before exploring issues that arise when creating a quality curriculum in the digital era. the aim being to inform and hence enhance future curriculum development. it has long been recognised that being clear about the purpose of schooling is critical (e.g. goodlad 2006 ). the importance of defining the purpose of schooling however should not be confined to international frameworks but should be central to the development of curriculum at a national level. for example, as part of the ongoing redesign of the irish primary school curriculum, walsh (2018) reviewed international trends and practices in curriculum development and stressed the need for clarity of purpose, aims and principles: central to the revised overview must be an articulation of the purpose, aims and principles of the curriculum in ireland. the inclusion of these elements will distinguish the curriculum from a syllabus, where the primary focus is on subject content. being clear about this central purpose is imperative to ensure that all other elements of the curriculum, including the content, become subservient to this fundamental driver. (walsh 2018, p. 16) there is wide ranging support for the view that curriculum development essentially involves making decisions around what are considered to be the most important values and purposes for inclusion from the culture of a society (lawton 1989) . kelly (2004) advocated that curriculum developers should place their energies on articulating the ideological position and broad principles rather than defining specific content. similarly, biesta and priestley (2013) noted that having explicit aims and purpose in the curriculum provides a rationale for the inclusion or exclusion of particular elements in a transparent manner. furthermore, there is evidence from a cross-national study of nine jurisdictions that countries considered to have high performing education systems had clear and well thought through aims, principles and goals of education (creese et al. 2016) . however, it is clear that there are differences of views about the purposes of schooling across and within educational jurisdications (butler et al. 2018) . thus, the starting point for any discussion of curriculum needs to be an agreement about the purposes of schooling. while this idea is not new (e.g. biesta and priestly 2013; kelly 2004; lawton 1989; tedesco et al. 2013 ) and may seem self-evident, in some instances it has not been the case. for example, the english national curriculum's aims were added a number of years after the curriculum content and, as alexander (2014, p. 160 ) points out, were little more than "a high-sounding statement you attach to the curriculum after you've determined its content and whose function is therefore cosmetic". in contrast, having a clear rationale, which informs your purpose, provides the major orientation point, to which other components of the curriculum should be linked. this applies at each level of the system. a way to present this visually is to arrange them as a spider's web (van den akker 2003) (fig. 3) . this curricular spider web not only illustrates the many interconnections between the components, but also its vulnerability. it illustrates that although the emphasis of curriculum design on specific components may vary over time, eventually some kind of alignment has to occur to maintain coherence, as each element is tied to the rationale at the centre. this coherence and alignment is often not evident within national curricula as outlined in the next section. as a starting point for their discussions members of twg12 individually created blog posts that set out their personal views about what an ideal curriculum for the digital age would look like. they each then compared their 'ideal curriculum' with the reality of the curricula in their own country, which included: australia, canada (quebec), england, ireland, sri lanka, slovakia, and the netherlands. the data were analysed inductively, using emergent theme analysis (wong and blandford 2002) . this analysis foregrounded two issues: • differences in the purposes and thus curriculum elements that group members were focussing on when thinking about their blog posts; • a clear disconnect between the visions of a quality curriculum within the blog posts and the realities of the curricula within the twg12 members' countries. each of these is explored below. analysis of group members' blog posts highlighted differences in the underpinning purposes that group members were focussing on when thinking about their blog posts. for example, whilst some posts looked at the overarching aims of schooling, others focussed more narrowly on aspects of the curriculum explicitly linked to digital technology. in the subsequent group discussions it was agreed that the focus should be on that broader view. building on the work of edusummit 2017 (butler et al. 2018) , members of twg12 adopted the yin-yang vision framework (fig. 4 ). this sets out what the ultimate purpose of schooling should be and key elements that are necessary for the vision to be realised. this framework is underpinned by a sociocultural theoretical perspective (twining et al. 2017) which drew upon sociocultural theory as set out by lave (1988) , within which the concepts of identity, agency and participation are key. the items on the left of the figure relate to individual fulfilment. the items on the right focus on universal wellbeing. the items in the middle are the capabilities that are necessary to achieve the vision as a whole, providing a bridge between the individual (identity) and the collective (participation). all of these dispositions and capabilities are developed through engagement with content. this content should reflect key concepts -powerful ideas that have relevance in multiple contexts and enable one to make sense of the world. for example, the notion of 'systems', which has relevance in biology (environmental systems, nervous system), wellbeing (health systems), business and it (computer systems), and many other areas. within this conception of curriculum the development of knowledge, skills and dispositions is seen as fig. 4 the yin-yang vision framework (twining 2019a) happening through engagement with key concepts which span discipline boundaries. this contrasts with traditional school curricula which tend to compartmentalise 'content' into discrete subjects that are often taught in isolation. underpinning this framework is a recognition that in order to achieve a vision of individual fulfilment and universal wellbeing you need to be able to apply your understandings in appropriate ways. this means that you need knowledge, capabilities and dispositions. we need to move away from a binary debate about content vs skills. using the yin-yang vision framework, we believe could act as a bridge between supra (international) and macro (national) level curriculum debates, highlighting the importance of purpose, and providing a tool to think with at the macro and meso (school) levels. the disconnect between twg12 members' visions and the curricula realities in their school systems seemed to reflect tensions around the purposes of schooling in our rapidly changing world. this is mirrored in the gap between the dominant rhetoric of '21st century skills' and the reality of predominatly knowledge based curricula aligned with high stakes testing (e.g. naplan) and international assessments (e.g. pisa) that cannot assess many of these critical skills and dispositions (etag 2015). butler et al. (2018) explored the issue of alignment, noting that this required alignment of purpose, policy (including curriculum, assessment, accountability and teacher professional learning), and practice at three levels (macro/national, meso/school, and micro/ teacher). twining (2018) extended this analysis using a sociocultural framework (see fig. 5 ). this framework has three levels, • the constitutive order -the broader context, including cultural norms, values and beliefs, as well as more explicit policies, rules and regulations. this includes any nationally specified curriculum. • the arena -the enduring elements of the school context which are taken up from the constitutive order. for example, how policies and expectations at the national level are interpreted and enshrined in the school expectations, policies and facilities. this includes any school specified curriculum (e.g. school schemes of work). the arena provides opportunities for action. • the setting -the local context (e.g. the classroom) in which practice is implemented. at this level of analysis, the actors (e.g. teacher and students) perceive what is possible within the context of the school arena in the light of their identities. as illustrated by the shaded box within the setting in fig. 5 actors 'take up' some of those perceived possibilities in their iterative interactions with the other people in their setting. achieving alignment between and within each of these levels (constitutive order, school arena, and setting) is complex. this reflects a tension in finding a reasonable balance between curriculum freedom and regulation when developing a quality curriculm. having agreed on the overarching yin yang vision framework twg12 focussed on three key issues that need to be addressed in order to achieve alignment within and between levels. these key issues are stakeholder ownership, teacher professionalism, and assessment, each of which is explored below. curriculum development and alignment is a process with many stakeholders. even though the stakeholders might have different values and interests in relation to the curriculum, it is important to involve them in a timely and authentic manner, to ensure commitment and ownership. in relation to broad curriculum development, van den akker (2003) states that this is usually a long and cyclical process with many stakeholders and participants; in which motives and needs for changing the curriculum are formulated; ideas are specified in programmes and materials; and efforts are made to realise the intended changes in practice. members of twg12 agreed that in order to ensure alignment between the specified and enacted curricula it was critical that all stakeholders should be involved in co-constructing the curriculum; including agreeing the purposes that the curriculum is intended to meet. table 1 illustrates the key stakeholders at each level of alignment. at every level there are large numbers of stakeholders. the nature of those stakeholders changes between levels, with their foci becoming more local and immediate as you move from the constitutive order to arena to setting. teachers are a particularly important group because ultimately they enact a curriculum at the setting level. involving them in the curriculum development process can help address the challenge of clarity and hold: • clarity, in the sense of having a shared understanding about what needs to be learnt, and • hold in the sense of ensuring alignment between the specified curriculum and what is enacted in the setting. the consequence of misalignment between curriculum purposes and curriculum practices can be seen in donaldson's (2015) report on the national curriculum in wales. donaldson's suggestions for improving the curriculum were based on an analysis of the problems of the previous curriculum in wales. donaldson notes that a highly prescriptive curriculum combined with powerful legislative and accountability mechanisms led to "much of the curriculum as experienced by children and young people has become detached from its avowed aims and too focused on the short-term" (p. 10). donaldson's approach to addressing these problems and to align curriculum, assessment and accountability was to "involve as many people in wales as possible" (2015, p. 13). however, how to organise involving as many stakeholders as possible is a thorny issue. so it is useful to learn and build on what other countries have tried as outlined in the example below from the netherlands. the netherlands is an example which illustrates a range of typical approaches to tackling the problems and tensions of trying to address the shifting priorities of society particularly in relation to the use of digital technologies and how to accommodate their use in the school curriculum. however, what is unique is how the focus came round to having a national discussion on the future of schooling. since the 1980s/90 s, 'information science' and 'informatics' was part of the national curriculum in the netherlands, this was: (a) particularly the case in secondary schooling and; (b) primarily focused on understanding and being able to work with computers and programming. these subjects eventually proved very difficult to implement and they disappeared in 2000 from the curriculum (voogt and ten brummelhuis 2014) . the discussion about ict in schooling gradually changed from learning about ict to using ict for learning, and more and more attention was paid to the integration of ict in schooling as a "tool" for teachers. however a report from the royal netherlands academy of arts and sciences (knaw 2013) stated that the increasing digitisation of information and communication in society required new skills but that these skills (digital literacy) were not getting sufficient attention in schooling. this report ignited the discussion again with regard to the role and use of digital technologies in schools. this focus, coupled with a range of other issues which were gaining attention (such as twenty-first century skills, equity, and the perceived overload of the current curriculum), led to a broad national discussion about the future of schooling in the netherlands. in november 2014, the state secretary for education, culture and science of the netherlands officially launched an online countrywide consultation about the future of primary and secondary schooling. everyone in the netherlands had the opportunity to take part. the board consultation process outlined in fig. 6 was the start of a process of curriculum design. this consultative process was an effort to garner the views of as wide a range of stakeholders as possible to begin the process of reaching an agreement on a shared common purpose for the curriculum (rationale in terms of the spider web). for example, via social media over 16,000 people contributed their ideas on what the young students of today should learn if they are to be productive members of society in the year 2032. based on the consultation an independent commission, platform onderwijs2032, wrote an advisory report about future-oriented education in the netherlands which they presented in january 2016 to the state secretary, this was meant as an overall rationale for the ongoing curriculum review. next to the importance of specific content domains such as language, science, numeracy and social studies they concluded that citizenship and digital literacy should also be part of the formal curriculum (platform onderwijs2032 2016). conscious of building a shared understanding of a common purpose to underpin the curriculum, it took a year to discuss the report with pupils, teachers, parents, school leaders, administrators, scientists, representatives of social and cultural organizations, business and political parties (platform onderwijs2032 2016). based on these discussions parliament agreed that a new curriculum for the primary and secondary school sector was needed and in 2018 the process to develop this was started. based on the consultative process this new curriculum would consist of nine subjects: dutch, arithmetic/mathematics, english/ modern foreign languages, exercise & sport, art & culture, human & nature, human & society, citizenship, and digital literacy. the next stage of the curriculum design process is particularly difficult in a co-constructive process. there is always a challenge to get a balance between involving everyone and making sure you have a workable situation. what the dutch example illustrates is what a government and the coordination group accomplished by letting the teacher design teams do the work and giving all others the opportunity to react. as a starting point, 125 teachers and 18 school leaders were recruited to work on the new curriculum. for each subject a teacher design team (tdt) was formed to work on a subject. the tdt worked together in six three-day sessions. after each three-day session, the documents they had produced were open for online consultation. everyone with an interest in the various subjects was able to react to the documents. next to the general consultation rounds, all documents were discussed with the specific "subject associations". these associations are groups of experts from a specific subject. the reactions/feedback was not taken for granted. everything was read and either included in the documents or -when decided that it should not be included -arguments were given for why it was omitted. in addition, a special working group on "coherence" with experts from the national expertise centre for curriculum development analysed all documents from all working groups to see if the big ideas and the key stages or learning trajectories were a) in line with the rationale and b) in line with each other. taking digital literacy as an example, a teacher design team consisting of 13 teachers, two school leaders, and two members of the national institute for curriculum development built a vision for the subject, designed big ideas, and corresponding learning trajectories for the big ideas (ontwikkelteam digitale geletterdheid 2019). the tdt defined digital literacy as the skills that relate to using ict effectively, efficiently and responsibly. they involve a combination of ict (basic) skills, computational thinking, media literacy and information literacy (thijs et al. 2014) . big ideas (also known as essential understandings) are broad statements that frame what students will learn (government of alberta 2019). eight big ideas were described by the tdt: (1) data and information, (2) safety and privacy, (3) using and controlling, (4) communication and cooperation, (5) digital citizenship, (6) digital economy, (7) applying and designing, and (8) sustainability. in order to help teachers from primary and secondary schooling to teach the concepts of digital literacy key stages or learning trajectories needed to be designed. learning trajectories were defined as "a reasoned structured set of intermediate objectives and content leading to a certain core objective" (strijker 2010, p. 10). learning trajectories not only describe what students should learn, providing clarity about the core objective, but also afford the opportunity to personalise learning by adjusting learning goals and related learning activities to the needs of the learners. the tdt concluded that digital literacy should be both a separate subject and embedded as a cross-curricular theme across all disciplines. taking all the feedback at each stage into consideration, the tdt for digital literacy published their final version of the vision, big ideas and learning trajectories in june 2019. the products of all tdts were combined into a final report that was sent to parliament in october 2019. after discussions in parliament the decision was made to start with the next stage of curriculum development for primary and lower secondary schools: making the learning trajectories and goals part of the legal and mandatory framework for primary and lower secondary schooling in the netherlands. working with teacher design teams again, teachers, school leaders and teacher trainers will work together, supported by experts from the national expertise centre for curriculum development to complete this next stage of curriculum development. it is expected that the new curriculum will be put into practice in 2022. there are a wide range of groups and individuals who can make a strongly justified claim to be a key stakeholder in schooling and thus have a right and a responsibility to contribute to the curriculum design process. while the approach taken in the netherlands ensures that teacher design teams are at the core of the curriculum reform, we need to pay particular attention to the wider body of teachers who, although given the opportunity to contribute to consultations, may have been less directly involved in the overall curriculum design. these teachers form a particularly significant stakeholder group because of their direct control over how curriculum intentions and expectations are actualised in classroom teaching and learning activities (at the setting level). the second key challenge relates to the role and professional status of the school teacher in the enactment of any new curriculum at the setting level. accepting that teachers are central to the adoption of any curriculum reform (kärkkäinen 2012 ; schoenfeld 2014), does not just require teacher 'buy in' but recognition of what it takes to develop ownership by the teaching profession of any new curriculum or curriculum innovation. in considering the schooling system as a whole (constitutive order, school arena, setting), the challenge is therefore how to capture and build on the expertise of teachers so that they become invested and genuinely participative in curriculum reform, thus ensuring that intended curriculum aims are achieved in practice. the extent to which teachers adhere to the specified curriculum and have control over classroom activities will vary between countries, local school systems, and within individual schools. this relates to the professional status of the teacher in their context and directly impacts on the effectiveness of any attempt at curriculum reform. the talis 2018 (oecd 2020) teacher survey suggests that although teachers in most countries have a large degree of autonomy over their practice in the classroom, they still have significant challenges ahead of them. only 14% of teachers in the 48 countries surveyed say that policy makers in their country/region value their view, and only 24% believe that they can influence education policy. this reflects a lack of trust of teachers, which emerged as active distrust in the later part of the twentieth century followed by a focus on achievement data and top-down measures that emphasised performance targets and public scrutiny (hargreaves and shirley 2009) . the implications according to schleicher (2020, p. 31 ) are that teachers "rarely own their professional standards to the extent other professionals do, and rarely work with the level of autonomy and in the collaborative culture that people in other knowledge-based professions take for granted". decades of research show that curriculum reform does not automatically result in sustainable changes in the everyday practices of schools (fullan and miles 1992) and that how reform is implemented plays a key role in determining whether an intended curriculum achieves its desired outcomes (mclaughlin 1990; tuinamuana 2011) . it is also well documented that teachers have a central role in the successful adoption of any curriculum reform (kärkkäinen 2012; schoenfeld 2014) and that new curricula are often not implemented as planned. this lack of implementation as planned is due in large part to conflicts between the principles of a new curriculum and teachers' existing beliefs and practices about teaching (orafi and borg 2009) . a new curriculum may demand significant shift in thinking and practice which according to prendergast and treacy (2018) leads to concerns among teachers on issues such as the reasoning behind the reform, the implications for classroom practices and students, as well as their ability to implement the changes. thus, educators may implement the reform with fidelity by following the curriculum as prescribed, adapt the curriculum to the local context while adhering to its core principles, comply with the curriculum by only implementing surface-level changes, co-opt the curriculum to fit with existing practices, or not implement the curriculum at all (berman and mcloughlin 1976; tichnor-wagner et al. 2018 ; both cited in taguma and barrera 2019). there are two possible conceptions of the role of the teacher in transacting a curriculum: to view teachers as technicians whose job is to deliver what others have decided, or to treat teachers as professionals who are best placed to ensure that schooling meets the aims that society has agreed on. the first view, a top-down, centralised approach where policymakers or curriculum designers seek compliance from implementers on the ground (e.g. teachers) to carry out their directives without any alterations to the intended curriculum design, can lead to teacher de-professionalisation as the judgement of teachers is seen to no longer be valuable (ball 2008) . it can be enacted through attempts to 'teacher-proof' the curriculum via overly-prescriptive lesson plans or scripts based on a "theoretical assumption that … the behaviour of teachers needs to be controlled and made consistent and predictable across different schools and student populations" (giroux 2002, p. 2) . however, such attempts can have negative consequences, including reducing teachers to 'task managers' rather than 'concept/skill builders' who are concerned with the pupils' deep learning of knowledge and skills (twistleton 2004) . it leads to the erosion of teacher autonomy, along with their personal and professional identities (day 2002) . ultimately teachers' knowledge and professional practices are diminished (giroux 2010; fitzgerald and knipe 2016; mclaren and farahmandpur 2006) . as pollard (2010) suggests, pedagogy is 'impoverished' (p. 5) if teachers are unable to engage in curriculum development. the de-professionalisation of teaching has happened in many countries (rubin 2011; rubtcova et al. 2015) . according to priestley et al. (2012, p. 192) this control over schooling "could be seen as the result of such systems having been subject for at least two decades to the combined influence of prescriptive national curricula and the use of outcomes steering, both backed by rigorous inspection regimes and the quantitative use of attainment data". in the uk for example, in his summary of findings from the cambridge primary review, alexander (2010, p. 7) states that "in many primary (uk) schools a professional culture of excitement, inventiveness and healthy scepticism has been supplanted by one of dependency, compliance and even fear; and the approach may in some cases have depressed both standards of learning and the quality of teaching". in portugal, teodoro and estrela (2010, p. 636 ) report that teacher autonomy was "progressively removed with the emergence of administrative control of the curriculum". while in the usa, au (2011, p. 30 ) claims that 'teachers' power' [is] being increasingly usurped through policy and curriculum structure. this erosion of the teaching profession and deprofessionalising of the teacher now presents as a serious challenge and both reflects and undermines trust in teachers. recent trends in curriculum development have placed greater emphasis on the formulation of curricula in terms of competences and capacities and on the teacher as a central agent in curriculum development (priestley and biesta 2013; sinnema and aitken 2014) . the focus on twenty-first century skills and competences has been identified in various comparative analyses of curriculum (e.g. creese et al. 2016) . trends towards school-based curriculum within a central framework (kärkkäinen 2012) and an outcomes-based educational approach (biesta et al. 2015) have also been idenified. many countries have accordingly prepared curriculum frameworks rather than prescribed curricula. such frameworks allow flexibility in interpretation and enactment at teacher level. for example, priestley and sinnema (2014) found that current curriculum policies in new zealand and scotland are characterised by flexibility at both national and school levels. similarly, in quebec, the ministry of education's digital action plan measures for how digital technology might be used are presented as suggestions, although the use of digital technologies in teaching and learning is required. ireland also has a digital learning framework (department of education and skills 2017a, b) which is comprised of standards and statements of practice for embedding digital technologies in teaching. however, it is up to schools and teachers to decide which statements to focus on. these changes represent a shift in emphasis from teachers as 'curriculum implementers' to 'curriculum developers and co-constructors'. however, this shift not only places the teacher at the centre of curriculum development but accentuates the importance of teacher capacity and autonomy (biesta et al. 2015) . the challenge is how to gain the 'buy-in' of teachers so that they become invested in and develop ownership of the reform. while it is suggested that embracing a culture of trust and autonomy, and promoting teachers' agency over the curriculum reform, increases teachers' engagement in the reform work (mccharen et al. 2011; priestley 2011) ; it is accepted that teachers are often not fully engaged in the consultation process when decisions are made in relation to curriculum (e.g. elliott 1994; williamson 2017) . this is significant when one considers that teachers' expertise and attitudes, including their understanding of the reform, contributes to the way the reform is implemented (tikkanen et al. 2017 ). in addition, tensions continue to exist in this form of curriculum development between the autonomy proffered to teachers as curriculum developers and the control and accountability mechanisms of the wider system (biesta and priestley 2013 ). this raises questions about how to re-professionalise teaching and hence increase trust of teachers. fundamentally this is about ensuring that teachers are treated and act like profesionals. the key characteristics of professionals include: • being knowlegable about the underpinning principles and theories in your field; • having expertise in curriculum and assessment enactment; • engaging in regular professional learning and re-acreditation; • being the creators and curators of knowledge in your field; • working the hours necessary to perform their role rather than a fixed number of hours; • having a high degree of autonomy, within the bounds of agreed professional standards; • having a independent professional body that oversees the professional standards. the first four of these are closely linked to pre-service and continuing professional learning. based on evidence from the literature twining and henry (2014) identitied key characteristics of effective professional learning (see fig. 7 )-it should be: • strategic-involving the senior management team, linking with the school development plan, and being integrated with the school's self-review and/or performance management processes; • impact focussed -in terms of enhancing curriculum, pedagogy, and/or learning outcomes; • context relevant -related to the needs of the staff and students and using resources/ equipment available in the school; • informed by both internal expertise (e.g. school staff) and external expertise (e.g. consultants); • collaborative -supporting sharing and professional dialogue; • experimental and reflective -trying out new ways of working and reflecting collectively on practice; • sustained rather than one-off; • evaluated in relation to planned impact. practitioner research can encapsulate all of those elements, and positions teachers as the creators of their professional knowledge base (twining and henry 2014) . the last three characteristics of professionals relate to the regulation and autonomy of the field. the key challenge here is how to uncouple education policy (at the constitutive order level) from short-term political control. finland, which is widely recognised as being one of the more successful education systems in the world, has a model in which schools have considerable autonomy (world bank 2012, p. 1). they note two key factors that underpin the success of the finnish education system: "the two most important factors explaining the success of the finnish education system are: (i) education has been a national priority for decades, and (ii) the system operates on trust." trust (or lack of it) and summative assessment are closely linked. as stated in the introduction, the word curriculum is related to the latin verb currere, which means course or track to be followed. in schooling contexts summative assessment is sometimes seen as the finish line of a race. indeed, summative assessment generally drives practice in schools irrespective of what the curriculum specifies (e.g. conseil supérieur de l'éducation 2018; etag 2015) and thus, where something is not being assessed it is likely not to be a priority (robinson and aronica 2015) . the pressure to improve student performance on mandated tests diminishes teachers' authority and control over what and fig. 7 characteristics of effective teacher professional learning (twining 2019b) how they teach (au 2011; wills and sandholtz 2009) . fundamentally, there are two main issues with current forms of summative assessment. the first issue is that summative assessments are often used not only to establish what students have learnt, but also as indicators of the quality of the educational provision (e.g. klenowski 2015) . thus, teachers focus on summative assessment because that is what they are held accountable against (etag 2015) . berliner (2011) highlights that this can have a range of negative educational impacts. such impacts include: off-rolling students; holding students back by a year; focussing on students who are on grade boundaries; cheating; narrowing the curriculum; and teaching to the test. this problem could be overcome by separating out the evaluation of the quality of school provision from the assessment of what young people have learnt. the second issue is that traditional forms of summative assessment are unable to capture evidence of many of the competences and dispostions that are valued as being essential today if schools are going to succeed in enhancing individual fulfilment and universal wellbeing. the fact that summative assessments, especially national assessments, are determinant for pupils' access to higher education (e.g. a levels in england; hscs in australia; the leaving certificate in ireland) results in too narrow a focus on academic understanding at the expense of other critical competences and dispositions. high stakes testing replaces trust in teachers' professional judgment. summative assessment is of course important for selection and certification purposes. the challenge is how to assess the competences and dispositions that are seen as being essential, as well as the knowledge that current high stakes assessments focus on. we need to develop new forms of summative assessment that complement existing ones, and align with the purposes of schooling. whilst this would increase the validity of school assessment in the sense of measuring the things that are important, achieving this raises many challenges as well as opportunities (webb and ifenthaler 2018) , including potentially reducing construct validity and reliability. thus, for example, including teacher judgements of practical performance, such as in a science experiment, would increase the validity of a science assessment. however, it would also increase the risk of inter-marker inconsistency, which would need to be mitigated by some form of moderation process. this highlights three key issues that are explored further below: • criteria for evaluating summative assessment • approaches to complementing traditional forms of assessment including emerging possibilities • teachers professional learning to enable them to design assessment tasks twining (2020a) set out criteria for evaluating summative assessment of young people's learning. these include being: • relevant -this is an aspect of validity: summative assessment needs to measure things that are important (i.e. the goals identified in the curriculum) • credible -this combines aspects of validity (measuring what it claims to measure) and reliability (being both accurate and consistent) • practical -it must be feasible to implement, and ideally integral to the learning process • scalable -it must be able to be used with large numbers of learners • formative -assessment should inform future learning as well as providing a summary of current knowledge, competences and dispositions • positive -it should be constructive, and focus on success rather than failure • ethical, including: -it should enable everyone to succeed by being at an appropriate level and/or providing alternative pathways to success. norm based assessment is unethical because it requires that some people fail in order for others to succeed -it should be transparent -both in the sense of not being covert and being clear why particular judgements have been made -it should be unbiased -it should not favour particular individuals • able to be reported concisely -this is important because recruiters and admissions officers typically don't have time to read through copious documentation about student performance a number of approaches to complementing traditional forms of assessment have been developed, including: portfolios, eexams and digital badges. portfolios, can support reflection and self-assessment. at the junior cycle (15 year olds) in ireland there has been a shift towards the use of portfolios in place of traditional examinations. whilst they can provide rich evidence of achievement, that can be mapped to standards, they are time consuming to mark or evaluate. digital badges, and other forms of micro credentials, can provide certification for competencies and dispostions which may have been developed either within or outside formal schooling. they suffer from a lack of standardisation, which potentially undermines their credibility. eexams, which use the power of digital technology, potentially enable students to demonstrate competences and dispositions (etag 2015) , for example through the use of simulations. however, the pragmatics of moving to eexams are considerable. for example, in a school context where all students studying a particular subject need to take their exam at the same time and in 'exam conditions', as is common for high stakes tests, most schools do not have the necessary infrastructure (e.g. hundreds of devices and robust internet). approaches to addressing this challenge have been developed, based on a bring your own (byo) device model (e.g. fluck and hillier 2016), but have not gained traction in schools. there are also a number of emerging possibilities, including: • point of learning (pol) which is a new approach to collecting compelling evidence of competences and dispositions in the form of 'claims' based on observations of practice by multiple people over prolonged periods (twining, rix and sheehy 2016) . • social learning analytics which utilises ai, and in particular data mining techniques to assess learning in online environments (e.g. see twining 2019c). • the use of online 'living cvs' which include endorsements and recommendations from members of the learner's community both inside and outside school (e.g. twining 2020b). this reflects how adults use linkedin to develop their professional profiles and networks. one way to start to develop new approaches to assessment would be to ensure that teachers are better educated to design assessment tasks. whilst in some countries teacher education provision does focus on assessment, this tends to be formative rather than summative assessment. in other jurisdictions, such as quebec, there are very few courses dedicated to assessment in teachers' professional learning programmes, and what there are tend to be disconnected from courses about teaching subjects (conseil supérieur de l'éducation 2018). unless solutions are found to 'the assessment problem', the gap between the rhetoric of national curricula and the reality of practice in schools will remain. there is a growing consensus that current school curricula are not meeting the needs of individuals or society. if we are going to strive for a vision of individual fulfilment and universal wellbeing then the curriculum needs to prepare young people to influence the world outside school and to tackle the wicked challenges that humanity faces. in short, the curriculum needs to follow purpose. this means that we need to change the predominantly content focus of traditional curricula so that they encompass knowledge, competences and dispositions. that of course has knock on implications for many other aspects of educational provision, as highlighted in the spider web of inter-relationships (fig. 1) . we also need to recognise that a curriculum that genuinely focusses on learners having the ability to act outside school will require not just a change in curriculum but also in pedagogy. however a change in pedagogy won't be enough if the teacher is not recognised and respected as a professional who has an active role in determining the curriculum and how learning is to be assessed. furthermore, we need to recognise that it doesn't matter what the curriculum says if it is not aligned with summative assessment and does not have the buy in of key stakeholders. in exploring the development of a quality curriculum in the digital era, this paper has clarified that a quality curriculum is one that supports young people in acquiring the knowledge, competences and dispositions needed to be successful in the digital era. the yin-yang vision framework was introduced, as a bridge between supra (international) and macro (national) level curriculum debates, that highlights the importance of purpose, and provides a tool to think with at the macro and meso (school) levels. however, in tandem with highlighting the importance of purpose as a critical starting point for developing a quality curriculum, understanding the issue of alignment was identified as essential. using a socio-cultural framework (fig. 5) this paper illustrates the complexity of alignment of purpose, policy (including curriculum, assessment, accountability and teacher professional learning) and practice between and within each level (constitutive order, school arena, and setting). three key challenges in achieving alignment were discussed, namely: stakeholder engagement, enhancing the professionalism of teachers, and developing better forms of summative assessment. understanding and addressing these issues is essential in order to achieve alignment between purpose, policy and practice across levels of the school system. the recent covid-19 crisis has opened up debates and started to crystalise these issues. for example, clarifying one key purpose of schooling being to provide child care so that parents (and in particular 'essential workers') can go to work. similarly, the replacement of high stakes tests in many countries (e.g. england, scotland, ireland, australia, slovakia) with alternative approaches to summatively assessing school leavers raises questions about the role of teacher assessments and opens the door for a wider debate about teacher professionalism and how best to judge students' learning. running through all three of the key issues explored in this paper is a tension between flexibility and control of the curriculum, and fundamentally the extent to which teachers are trusted. perhaps the time is right, to move from what hargreaves and shirley (2009) describe as the third way-trust through control and accountability-to what they call the fourth way-trust based on a 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publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.peter twining is professor of education (innovation in schooling and educational technology) at the university of newcastle (australia). he has been professor of education (futures) at the open university (uk), co-editor in chief for computers & education, and the lead on a number of major projects looking at the use of digital technology both inside and outside formal education.deirdre butler is a professor in dcu's institute of education, whose passion in life is exploring what being digital in learning can mean and what skills or competencies are needed to live in today's complex globally connected world, challenging us to examine how we learn and questioning our assumptions about "traditional" models of schooling. she has designed and managed a range of projects / school-based initiatives which focus on sustainable, scalable models of teacher professional learning and creative uses of digital technologies.petra fisser is currently working as educational advisor at the college of health care of roc van twente, a regional training centre for vocational education and training and adult education. projects she is involved in are related to educational innovation of secondary vocational education and training (vet), adult education, and tailor-made courses for sme's. before working at roc van twente, petra worked at the national institute for curriculum development and the university of twente in the netherlands, where she was engaged in projects, knowledge development and professional development in the field of curriculum development and evaluation, internationalization, digital literacy and tpack.margaret leahy is head of the school of stem education, innovation and global studies at the institute of education, dcu university. she has extensive experience in the theory and practice of digital learning, the design and development of professional learning programmes for teachers and has been involved in a range of innovations/research projects, all of which focus on ways digital technologies can be used to create more powerful learning environments.chris shelton is head of education within the institute of education, health and social sciences at the university of chichester where he is responsible for education and teacher education programmes. chris has a range of research interests relating to technology, computing and digital literacy in schools and universities. his recent work has included publications about teacher's thinking about technology in higher education; the pedagogy of teaching computing; and the curriculum.nadine forget-dubois has a ph.d. in anthropology from the université de montréal and is a research agent at the conseil supérieur de l'éducation du québec, attached to the production of the report on the states and needs of education.michel lacasse is a doctoral candidate in education administration and policy at université laval. his thesis subject concerns the technological competence of school directors and, between theory and practice, he participates in various research projects in the field of education, which include school governance, alternative pedagogies, educational technologies and the training of teachers and administrators of education. key: cord-264749-m1awr1rm authors: saad, julian m.; prochaska, james o. title: a philosophy of health: life as reality, health as a universal value date: 2020-03-18 journal: palgrave commun doi: 10.1057/s41599-020-0420-9 sha: doc_id: 264749 cord_uid: m1awr1rm emphases on biomarkers (e.g. when making diagnoses) and pharmaceutical/drug methods (e.g. when researching/disseminating population level interventions) in primary care evidence philosophies of health (and healthcare) that reduce health to the biological level. however, with chronic diseases being responsible for the majority of all cause deaths and being strongly linked to health behavior and lifestyle; predominantly biological views are becoming increasingly insufficient when discussing this health crisis. a philosophy that integrates biological, behavioral, and social determinants of health could benefit multidisciplinary discussions of healthy publics. this manuscript introduces a philosophy of health by presenting its first five principles of health. the philosophy creates parallels among biological immunity, health behavior change, social change by proposing that two general functions—precision and variation—impact population health at biological, behavioral, and social levels. this higher-level of abstraction is used to conclude that integrating functions, rather than separated (biological) structures drive healthy publics. a philosophy of health provides a framework that can integrate existing theories, models, concepts, and constructs. a philosophy of health w hat is health? is it a state of the body or the mind? is health primarily a natural, biological state or a holistic, value-laden state? naturalistic and holistic philosophies of health have provided very important, but very different, perspectives of population health. naturalistic views (e.g. as seen in boorse, 1997) provide insight into physical, natural, biological, or physiological processes that are tangible (in the material sense), observable, and measurable with modern technology. complementarily, holistic views contend that value-laden phenomena (e.g. vital goals, meaning, and purpose) play a central role in population health (nordenfeldt, 2007) . a dialog, or as we see it, an important dialectic among naturalistic and holistic perspectives plays out between the biostatistical theory of health (bst) and the holistic theory of health (hth). the bst posits that a person is healthy if and only if, all natural organs function normally given a statistically normal environment (boorse, 1997) . the hth posits that a person is healthy if and only if (given standard circumstances) he/she has the ability to attain their vital goals (nordenfeldt, 2007) . in addition to defining health, each philosophy defines disease. the bst poses that disease is the internal state of impairment to the normal functioning of organs (boorse, 1997) . in the hth, an organ dysfunction is a disease if and only if the organ's process reduces the person's ability to pursue vital goals or life-purpose (nordenfelt, 2007) . in bst health is the absence of disease; and in hth, health is not the absence of biological disease, but is the whole person's ability to function in relation to vital goals. both naturalistic and holistic perspectives guide important observations of health and disease. when one considers health through the bst one pays close attention to the functions of the internal, biological functioning of the human being. when one considers health through the hth, one pays close attention to the functioning of an individual, in relation to their external, societal/ cultural functions. is there a hybrid model that accounts for both internal and external functioning? wakefield's (2014) harmful dysfunction analysis (hda) creates a hybrid model that integrates natural-and value-laden phenomena when conceptualizing disease. hda asserts that a person suffers from a disorder/disease if (1) the condition causes harm (as judged by the standards of the person's culture); or if (2) the person's internal, natural processes cannot perform normal functioning (as judged by the standards set by evolution). hda creates a hybrid model that can integrate perspectives of the bst (i.e. by considering internal organ functioning); and the hth (i.e. by considering external societal/goal functioning). however, while hda may define health processes in relation to disease, it serves primarily as an integrative model of disease. is there an integrative model of health that can account for natural and value-laden functions? schroeder (2012) identifies a significant, common thread among these competing (or perhaps complementing) philosophies: functionalism. the researcher suggests that each philosophy is concerned with the functioning of organisms. although the bst, hth, and the hda might not agree on which functions inform the first principles of health, schroder (2012) uses higherlevel abstraction to identify one common first principle: the state of functioning in an organism impacts its state of health. when paralleling the three philosophies based upon functioning one might observe that (1) bst declares an individual healthy if its organs function normally; (2) hth declares an individual healthy if he/she can function in relation to vital goals; and (3) hda declares an individual unhealthy if internal mechanisms cannot perform natural, evolutionary functions, and/or when a condition prevents a person from functioning in relation to goals/norms/values. through this higher-level abstraction, an integration of seemingly separate philosophies of health is made possible. learning from leaders in the field. as we attend to these philosophies of health, we too observe how discussions about functions and functioning produce integrative perspectives. although a definition of "function" is not explicitly stated in the above research, it appears that nordenfeldt (2007) , boorse (1997) , wakefield (2014) , and schroeder (2012) are each discussing functions as pre-existent (i.e. either from evolution, personal goalsetting, cultural tradition) processes-with-purposes. whether one is describing a value-laden function (e.g. decision-making in pursuit of a valuable career) or an evolutionary-biological function (e.g. the heart beating for circulation), each process (i.e. decision-making processes or cardiac processes) serves identifiable purposes (e.g. maintained financial stability or maintained blood flow). whether an organ is functioning normally in relation to the body or a human being is functioning in relation to vital goals, it appears that both perspectives consider if an active "process" (i.e. an organ's activity, an individual's activity) can express its "purpose" (i.e. evolutionary-purpose, life-purpose). in the present manuscript we will propose that naturalistic and holistic perspectives can be integrated within a single philosophy of health. we will propose two universal functions-termed precision and variation-that can account for both natural functions and value-laden functions of the existing philosophies. this functional language will support a higher level of abstraction that integrates, rather than separates, biological functions, behavioral functions, and social functions under a philosophy of health. the need for new perspectives in population health. the chronic disease crisis beckons the need for an updated philosophy of health that can account for biological, behavioral, and social functioning. why? chronic diseases, which account for 60% of all-cause deaths worldwide (chartier and cawthorpe, 2016) , do not emerge from naturalistic, biological, or physical contact with an illness. rather, chronic diseases do emerge in biological functions (e.g. tumor proliferation in an organ) after prolonged contact with health risk behaviors and lifestyle factors that active the conditions (mokdad et al., 2018; edington, 2009; li et al., 2018) . chronic diseases are not curable by purely naturalistic or biological means (e.g. pharmaceuticals). rather, some diseases may be effectively prevented or intervened on through healthy behavior (dansinger et al., 2005; daubenmier et al., 2007) . population health risk behaviors are unique determinants of population health because researchers can actively observe how they simultaneously alter biological functioning (e.g. chronic smoking alters cells in lung tissue), behavioral functioning (e.g. chronic smoking alters decision-making and daily habits) and social functioning (e.g. chronic smoking creates an economic, social, and healthcare burden) of the population. these behaviors not only have biological, behavioral, and social implications for the individual doing the behavior, but also have intergenerational and interpersonal effects. the individual who binges on refined sugar not only puts themselves at risk of diabetes, but can put their future offspring at risk. the individual who smokes two packs of cigarettes per day not only puts themselves at risk of lung cancer, but can put their housemates at risk of lung cancer from second-hand smoke. therefore, the chronic disease crisis is neither purely naturalistic, nor purely value-laden; rather it reflects an integration of natural and value-laden phenomena. there remains a real need for principles of health that can integrate existing naturalistic and holistic perspectives of population health. the principles since april 7, 1948, the constitution of the world health organization (2010) has utilized an intuitive definition of health by suggesting that health is "a state of complete physical, mental, and social well-being." while this definition might be intuitive and even accessible to a wide audience; the defininition is not necessarily researchable across health disciplines. integrating principles of health might begin with a common-sense definition of health that can also be upheld across existing naturalistic and holistic perspectives. without operationally defining functions that drive physical, mental, and social well-being, it is a challenge for multidisciplinary collaborators to unite under the who mission. further, without a common definition of health, important communications from patients to doctors, from subjects to researchers, from researchers to collaborators, and from peer-reviewers to peer-reviewees, can become fragmented or lost in translation. in the proceeding sections, a common-sense definition of health is used to present the first principles of a philosophy of health. principle 1: "health" is the state of maintainable-ease of functioning. a "disease" is a state of prolonged-dysfunction that prevents ease. chronic diseases emerge from prolonged exposure to dysfunctional behaviors like smoking, alcohol abuse, unhealthy diet, and inactivity (mokdad et al., 2018) that also create dysfunctional expressions of life functions. smoking creates dysfunctional breathing; alcohol abuse creates dysfunctional drinking; sugar binging creates dysfunctional eating; and sedentary behavior creates dysfunctional moving. when these health risk behaviors lead to chronic disease, they have already prolonged dysfunctional breathing, drinking, eating, and/or moving. the chronic smoker breathes in smoke so frequently that he no longer experiences an ease-of-breathing. rather, his breathing becomes short and shallow. prior to the emergence of lung tumors, the chronic smoker prolongs dysfunctional patterns of breathing. the "couch potato" sits so frequently that he no longer experiences an ease-of-movement. rather his movement becomes rigid and limited. prior to the emergence of cardiovascular dysfunction or obesity, the sedentary person prolongs dysfunctional patterns of movement. if chronic smoking facilitates prolonged-dysfunction in breathing, and sedentary behavior facilitates prolonged-dysfunction in movement, what do functional breathing and moving look like? healthy breathing and moving (as well as eating and drinking) are characteristic of an ease of one's functioning that can be maintained in normal conditions. for example, the chronic smoker and the "couch potato" might report momentary-ease in breathing and posture when engaging in their health risk behaviors; but they do not maintain that ease outside of smoking or sitting. conversely, the yogi might report that their yoga practices expose them to momentary dis-ease in breathing and moving that lead to maintainable-ease in breathing and movement in everyday life. in contrast to disease as a prolongeddysfunction, healthy functioning can be commonly sensed as a maintainable-ease of functioning. when observing a disease, perhaps we are observing a prolonged-dysfunction that prevents ease. rather than define health as the absence of disease (as seen in bst), notice here how we instead define disease in relation to health; and we define health in relation to maintainability, ease, and functioning. consideration of "maintainable-ease of functioning" will allow us to consider how not all "dis-ease" is bad (i.e. exposure to acute dis-ease/stress maintains healthy functioning in the long-term); and not all "ease" is good (i.e. avoidance of stress and prolonged "comfort" creates fragility seen in sedentary behavior). we propose that: 1. dysfunction parallels a state of "dis-ease"; and prolongeddysfunction parallels the state of disease. 2. function parallels a state of "ease"; and maintainable-ease of functioning parallels the state of health. this definition of health will be applied in the proceeding principles to integrate naturalistic and holistic perspectives of population health. principle 2: health emerges from maintainable-ease of functioning at multiple levels. maintainable-ease of functioning in the general population can be observed at the level of the cell, the self, and the society simultaneously. cooperation across multiple levels of functioning is required for the organization and adaptation of living systems (nowak and sigmund, 2005; antonucci and webster, 2014) . when developing an integrative model of health, it is important to consider how biological cells, individuals, and the larger society simultaneously play a role in population health (xavier da silveira dos santos and liberali, 2019; antonucci and webster, 2014) . in this philosophy, we define health from three levels: cells, selves, and societies. what happens when these levels do not function in cooperation? when the functioning of cells disrupts the functioning of the self, a state dis-ease in the self can follow. for example, prolonged dysfunction in autoimmune conditions can lead to prolonged dysfunction for the (individual's sense of) self by triggering depression, decreased motivation, or anxiety (lougee et al., 2000; garud et al., 2009) . the reverse can also be true. when the functioning of the self (i.e. one individual) disrupts the functioning of their cells, a state dis-ease in the cells can also follow. for example, prolonged sugar binging and addictive eating can lead to prolonged high blood sugar and pancreatic dysfunction seen in diabetes (de koning et al., 2011; imamura et al., 2015) . cells and selves are not separate. when the functioning of the self disrupts the functioning of the society we observe a state dis-ease in the society. for example, one person's unprotected sex with multiple partners can also lead to epidemics and social conflicts. the reverse can also be true. when the functioning of the society disrupts the functioning of the individual, a state dis-ease in the self can follow. for example, dysfunctional social conditions (as seen in rutter, 1998) , can lead to prolonged psychological and behavioral dysfunctions of individuals. selves and societies are not separate. when the functioning of society disrupts the functioning of cells, a state of dis-ease in the cells can also follow. for example, prolonged dysfunction in society in the form of misguided values about cleanliness, can lead to over-sanitization practices that create superbugs and antibiotic-resistant bacteria (zaccheo et al., 2017; finkelstein et al., 2014; bower and daeschel, 1999) . the reverse can also be true. when the functioning of cells disrupts the functioning of the society, a state of dis-ease in the society can follow. prolonged dysfunction in cells from naturally occurring parasites (e.g. yersinia pestis [cui et al., 2013] ) can lead to prolonged dysfunctions like the economic collapse following 14th century black death (haensch et al., 2010) . cells and societies are not separate. what does health look like when these levels work together? recent reports on the blue zones (i.e. the areas of the world where populations live significantly longer and healthier than the average) demonstrate that healthy functioning at these levels enhances physical longevity and mental wellbeing in populations (buettner, 2012; poulain et al., 2013) . buettner (2012) reports on how blue-zone populations intentionally and habitually enrich their physical bodies with healthy eating and physical activity. in addition to integrating physical and behavioral practices, these communities also integrate behavioral and social practices, such as, goal-setting, meditations/prayer, social engagement, pursuit of purpose, and community gathering. humor is used by individuals and groups as a means to practice ease when challenges present themselves (buettner, 2012) . blue zone communities place value upon physical/natural, behavioral and social processes, generating them intentionally and habitually. both states of ease and dis-ease can teach us about the contributions of cells, selves, and societies to population health. although it is important to be able to observe the levels separately to describe their contributions, it is also important to consider how the levels integrate to impact healthy publics. we acknowledge that meaningful changes can be observed above and below these levels (e.g. at the level of the biosphere and genome). however, this initial paper will introduce levels that are most proximal and accessible to the experience of a general readership (fig. 1) . principle 3: health emerges from systems whose primary purpose is to generate maintainable-ease of functioning at a respective level. we propose that systems exist at each level with the purpose of generating maintainable-ease of functioning at that level. the biological immune system, an individual's system of health behaviors, and the social system will be observed as systems that generate maintainable-ease of functioning in cells, selves, and societies respectively (fig. 2 ). principle 3a: the biological immune system is directly responsible for maintainable-ease of functioning at the level of the cell. throughout the course of human evolution, the complexity and biodiversity of the human body continued to increase (rodríguez et al., 2012) . what keeps the trillions of cells and microorganisms in cooperation in a human body? the biological immune system maintains functional cells (rodríguez et al., 2012) . although it is documented that the functioning of the biological immune system has implications for behavioral functioning (ader, 1974 (ader, , 2000 johnston et al., 1992; cdc, 2016) and social functioning (cdc, 2016; reidel, 2005; cutler and miller, 2005) the system's primary purpose is supporting functioning in the cellular/biological system. principle 3b: health behavior is directly responsible for maintainable-ease of functioning at the level of the self. throughout the course of time, the complexity of human behavior, has continued to increase (boulding and khalil, 2002) . what keeps an individual in a state of balance during times of rapid change? one's system of health behaviors (e.g. one's practices of breathing, drinking, eating, and moving) maintain a functional self. although it is well documented that the behavior of the individual impacts biological functioning (fadel, 2013 (fadel, , 2015 and social functioning (omer et al., 2009 ), one's system of health behaviors directly impacts one's experience of (or one's 'sense of') their "self". principle 3c: the social system is directly responsible for maintainable-ease of functioning at the level of the society. throughout history, the social diversity of human societies continued to increase. during periods of rapid increases in social diversity and cultural integration, what supported cooperation in the society? social systems (e.g. public governments, private social organizations, religious/spiritual organizations) emerge to maintain a functional society. although it is well documented that a social system can impact biological functioning (cdc, 2016; riedel, 2005; cutler and miller, 2005) and behavioral functioning (buettner, 2012) , the social system's primary role is to maintain functions at the level of the society. principle 3d: by considering health as maintainable-ease of functioning generated by systems, we have the ability generalize health across levels. to observe health at the level of the cell, the self, and the society simultaneously, we consider systems that support maintainable-ease of biological, behavioral, and social functioning. the biological immune system, an individual's system of health behaviors, and the social system make meaningful contributions to the functioning of cells, selves, and societies, respectively. while these systems are not the only systems that impact each level (e.g. one's cardiovascular system impacts cells, one's "personality" impacts the self, the environment impacts society), the biological immune system, health behavior, and the social system have great implications for population health from their respective levels; and they can be operationalized at these levels based upon their functions. by considering health as maintainable-ease of functioning (rather than maintained biological structures) at multiple levels, we set a point of reference from which to integrate important determinants of population health. when taking the structuralist's perspective, the biological immune system, health behavior, and social systems appear as distinctly separated. when taking a functionalist's perspective, the biological immune system (i.e. the integration of host defense functions and microbiota functions), one's (system of) health behaviors (i.e. the integration of decisionmaking/executive functions and habits/habitual life functions), and the social system (i.e. the integration of population values and population behaviors) appear together in a philosophy of health. fig. 1 the levels of functioning. this philosophy of health investigates three levels of health: cell, self, and society. the level of the cell accounts for biological functioning within human beings. the level of the self accounts for first-person functioning of each human being. the level of the society accounts for group functioning of human beings. fig. 2 the systems at each level. each system is responsible for generating maintainable-ease of functioning at a level. the biological immune system is responsible at the level of the cell. a human's system of health behaviors is responsible at the level of the self. the social system is responsible at the level of the society. article palgrave communications | https://doi.org/10.1057/s41599-020-0420-9 principle 4: each system employs two general functions-variation and precision-to generate maintainable-ease of functioning at a level. the functionalist perspective allows us to observe systems based upon their functions. the biological immune system will be observed as an integration of host defense functions and microbiota functions (hooper and littman macpherson, 2012) ; (2) an individual's system of health behaviors will be observed as an integration of decisions/executive functions and habits/habitual life functions (de bruin et al., 2016; verplankern, 2005; norman et al., 1998; prochaska et al., 1994; prochaska et al., 1991) ; and the social system will be observed as an integration of actively functioning values and population-wide behaviors that function in relation to those values (dowling and pfeffer, 1975; cotgrove and duff, 1981) . by researching the role of these functions at each level, we distilled two general functions of each system: variation and precision. variation appears in the functions of each system that generate a range of abilities, the "varied-abilities", that sustain health in presently changing conditions. the microbiota, habits/ habitual life functions and population behaviors will be observed (in principle 4a) as the variation-functions of the biological immune system, health behavior, and the social system, respectively. precision appears in those functions that prioritize and organize the patterns of variation that can sustain health at a level in future, changing conditions. the host-defense functions, decision-making/executive functions, and values systems will be observed (in principle 4b) as the precision-functions in the biological immune system, health behavior, and the social system, respectively. consideration of a complementary relationship among precision and variation is not novel. precision and variation have been discussed as central to the development of neural and biological systems (hiesinger and bassem, 2018) . discussions of precision and variation have also provided important insight into research on the biological immune system (albert-vega et al., 2018; brodin et al., 2015) . through this philosophy, one can go beyond biological systems to observe how precision (in the form of hostdefense functions, decision-making/executive functions, and values) and variation (in the form of microbiota functions, habits/habitual life functions, and population-wide behaviors) integrate to generate to maintainable-ease of functioning in cells, selves, and societies simultaneously (fig. 3) . principle 4a: variation is responsible for generating the range of abilities, the "varied-abilities", that can express ease-of-functioning in presently changing conditions. without functional variation, life is fragile because the present environment is always changing (taleb and blyth, 2011) . fragile systems' inability to experience changing conditions (in part) relates to limited variability. conversely, adaptive system's ability to experience changing conditions (in part) relates to functional variability (taleb, 2012) . when one microorganism in the microbiome takes over, biological fragility reflects a state of infection. when one habit takes over, behavioral fragility reflects a state of an addiction/dependence. when one population behavior takes over (e.g. when economic participation or access to food is restricted to a small percentage of the population) social fragility reflects a state of social/civil unrest. the human microbiota is comprised of trillions of microorganisms, such as bacteria, fungi, and viruses. when variability in the human microbiota exists, an ease of functioning, or "homeostasis" in cells can be expressed in the present biological/ ecological environment bogaert et al., 2011; claesson et al., 2011) . research demonstrates that variation in the microbiota impacts the health of human cells by metabolizing complex carbohydrates, converting proteins to neural signals, and modulating diurnal rhythms that maintain biological homeostasis (clemente et al., 2012; rothe and blaut, 2012; blaut and clavel, 2007; de vadder et al., 2014) . when variation in the microbiota is dramatically limited or changed (e.g. following antibiotic overuse), cellular tissue in the human body is fragile and vulnerable to infections, allergies, and inflammatory outbreaks (francino, 2016) . when one's habitual life functions (e.g. breathing, drinking, eating, and moving) and one's healthy habits (e.g. one's weekly exercise schedule, or weekly meal preparation) can be expressed freely, an ease of functioning is felt by one-self in the present environment. when life functions are no longer expressed with ease (e.g. breathing and movement are compromised due to prolonged sedentary lifestyle), or when a single habit takes over one's lifestyle (e.g. smokes breaks "must" occur every 30 min), an individual is vulnerable to stressful outbreaks and chronic states (al'absi, 2011; conrad et al., 2007; suess et al., 1980; león and sheen, 2003; parrott, 1999; koob, 2008) . when the basic human rights in a society are preserved in the present (e.g. right to life, freedom of speech; right to property), human populations have the ability to freely engage in the population-wide behaviors (e.g. health behaviors, social behaviors, economic behaviors) that support a functioning society. health behaviors drive health and longevity. social behaviors drive communication and cooperation. economic behaviors drive goods and resources. when these population-wide behaviors are chronically restricted in a population (e.g. poor access to health care, oppression of free-speech, economic crash), societies become vulnerable to social/civil unrest [as commented historically by victor frankl (1985) , alexander solzhenitsyn (2003) , franklin d. roosevelt (1941) , and dr. martin luther king (1985) ]. variation is essential so that a system has varied-abilities that can express ease-of-functioning in present environmental conditions. dramatic and prolonged restrictions to variation in the microbiota, habits/habitual life functions, and population-wide behaviors characterize fragile and vulnerable states in cells, selves, and societies. conversely, functional-variation supports resilience, robustness, and antifragility (taleb, 2012) . this does not mean that infinite variation is desirable; however, in this philosophy, precision is responsible for organizing expressions of variation so that the system does not degrade into unpredictably random variation or chaos (see principle 4b). fig. 3 precision and variation in each system. maintainable-ease of functioning is generated by two functions in each system: precision and variation. the human microbiota, habits, and population-wide behaviors evidence variation in cells, selves and societies respectively. the host defense system, decisions, and values evidence precision in cells, selves and societies respectively. palgrave communications | https://doi.org/10.1057/s41599-020-0420-9 article palgrave communications | (2020) 6:45 | https://doi.org/10.1057/s41599-020-0420-9 | www.nature.com/palcomms principle 4b: precision is responsible for prioritizing and organizing the patterns of variation that maintain ease-of-functioning in future, changing conditions. some environmental changes are too challenging for ease to be expressed in the present. however, following an exposure to challenging conditions, some systems adapt and become more functional (taleb, 2012) . without the ability to functionally organize after stressors, a system degrades into disorder or chaos over time. host-defense functions, decision-making/executive functions and values systems prioritize and organize variation in the microbiota, habits/habitual life functions, and population behaviors respectively. when a pathogen invades the biological system, precise responses must occur to organize this potentially chaotic situation. at the level of the cell, a functional host-defense system (comprised of the innate, adaptive and complement immune system branches) organizes the biological system so that functional invaders (i.e. symbionts) and healthy cells are maintained and dysfunctional invaders (i.e. pathogens) and damaged cells are removed (hoeb et al., 2004; janeway, 1992; janeway and medzhitov, 2002; janeway et al., 2014) . when precision is dysfunctional, the host-defense system may (1) fail to prioritize responses to a costly invasion, leading to a state of infection; or (2) the host-defense system might prioritize dysfunctional responses to the cells of body that prolong a state of autoimmunity (naor and tarcic, 1982) . when a bad habit emerges, precise responses must occur to organize this potentially chaotic situation. at the level of the self, functional decision-making (or at smaller scales executive functioning) prioritizes and organizes behavior so that functional expressions of habit (or at smaller scales, habitual life functions) are prioritized regularly, and dysfunctional ones are replaced or minimized (de bruin et al., 2016; prochaska et al., 1994; prochaska and prochaska, 2016; prochaska et al., 1988; redding et al., 2011; weissenborn and duka, 2003; bickel et al., 2012) . when dysfunctional, decisions may (1) fail to prioritize responses that remove a costly expression of habit (e.g. a teen started smoking cigarettes to "be cool" and now has to smoke in the bathroom before each class to get through the day; by not deciding to move at work, one's breathing becomes shallow and movement becomes rigid); or decisions may (2) prioritize habits that prolong dysfunction despite knowing the dangerous consequences (e.g. an adult continues smoking cigarettes despite knowing the family's history of lung cancer; an adolescent continues binge on sugar despite a diabetes diagnosis). when dangerous population-wide behaviors threaten life in a society, precise responses must occur to organize this potentially chaotic situation. at the level of society, the agreed upon values organize the social system so that functional population behaviors are prioritized and dysfunctional population behaviors are minimized. functional values prioritize behaviors that support the society (e.g. as seen when societies mandate that students get certain vaccines before attending university), while also setting standards that remove/replace behaviors that threaten the society (e.g. new laws create legal repercussions for risk behaviors in society). without values that functionally prioritize populationwide behavior, society may (1) fail to prioritize responses to a dysfunctional population behavior (e.g. as seen during aids epidemic of the 1980s due to insufficient public health values around safe sex); or society may (2) prioritize dangerous behaviors that can prolong societal dysfunction (e.g. the antibiotic resistance crisis (ventola, 2015; michael et al., 2014) has been attributed in part to the over-valuing or over-use of antibiotic medications in healthcare practices). precision is essential so that a system can maintain ease-offunctioning in future, changing conditions. when precision does not adequately detect the presence of costly conditions, a response may not be prioritized (e.g. as seen during acute infection, addiction/dependence following a surgery, the aids outbreak in the 1980s). when precision prioritizes responses that prevent ease longitudinally, dysfunction is prolonged (e.g. autoimmunity, continued smoking despite family history of cancer, misguided values that create an antibiotic-resistant bacteria). through dysfunctional-precision, the conditions for life in cells, selves, and societies becomes disordered over time. through functional-precision, a system prioritizes responses that maintain ease-of-functioning in future conditions. prioritizing functional microorganisms (i.e. symbionts) supports the developing life of cells; prioritizing functional habits (e.g. weekly exercise) and habitual life functions (e.g. diaphragmatic breathing and relaxed movement) supports the developing life of the self; and prioritizing functional population behaviors (e.g. access to functional health care, economic resources; access to social support) supports the developing life of the society. principle 5: health is valued by a system when precision-andvariation generate maintainable-ease of functioning. health is de-valued by a system when precision or variation prevent maintainable-ease of functioning. by defining precision-andvariation, we can better understand maintainable-ease of functioning in population health: • functional-variation generates ease-of-functioning in the present (e.g. fluid and variable motion reflects an ease and variability of one's movement); while functional-precision prioritizes expressions that can maintain ease-of-functioning in the future (e.g. prioritizing challenging exercise for 20 min each day may lead to an ease in bodily movement long term). • dysfunctional-variation prevents ease-of-functioning in the present (e.g. prolonged sitting might lead to rigid movement and shallow breathing); while dysfunctional-precision might prioritize expressions that prevent ease in the future (e.g. rather than focus on relaxing breathing and movement on work breaks, one decides to drink alcohol to relax). without functional-variation, life is fragile and vulnerable to changing conditions of the present. without functional-precision, life becomes disorganized from the system's exposure to changing conditions across time. when functional-and-integrated, precision-and-variation value maintainable-ease of functioning in cells, selves, and societies. when dysfunctional or fragmented, precision or variation can de-value maintainable-ease of functioning in cells, selves, or societies. if maintainable-ease of functioning can be valued in cells, selves, and societies, we will likely observe healthy publics. five principles of health are presented: (1) health is the maintainable-ease of functioning; (2) maintainable-ease of functioning emerges from multiple levels; (3) at each level, maintainable-ease of functioning is generated by systems; (4) each system employs two functions, precision-and-variation, that generate maintainable-ease of functioning; and (5) health is valued by a system if precision-and-variation generate maintainable-ease of functioning. through these five principles, both naturalistic and holistic perspectives can be considered simultaneously because maintainable-ease of functioning is relevant to biological functioning (e.g. as described in bst) and personal/social, goal-oriented functioning (e.g. as described in hth). this philosophy can also be used to investigate how naturalistic and holistic phenomena have informed past healthcare interventions. what do vaccine interventions, behavior change interventions, and social change interventions have in common? when successful, these interventions enhance both precision and variation. vaccine interventions can enhance both the precision of the host-defense functions and variation in the microbiome. during a vaccine intervention, the microbiome is exposed to a new variation in the form of a new virus (reidel, 2005) . through this exposure, the precision of host defense functions can adapt to prioritize maintainable-ease of functioning in the microbiome in the future. how? the host-defense system produces antibodies that allow the immune system to respond effectively and efficiently to this virus when exposed to it again in the future (janeyway, 2014) . although the precision of the immune system has been enhanced to handle historical threats through vaccines (e.g. for small pox, chickenpox, measles), new viruses like the coronavirus can still emerge. with this philosophy, vaccine developers and public health officials might not only ask the question, "how do we combat the coronavirus?" researchers, vaccine developers and public health officials may also ask the functional question: "how do we enhance the precision of the host-defense system and the variation of the human microbiome to adapt following an exposure to the coronavirus?" behavior change interventions can enhance both the precision in one's decisions and the variation in one's habits. during a behavior change intervention, a person's existing habits are exposed to a new variation in habit. for example, the beginning of a new exercise intervention exposes the individual's current habits/habitual functioning to changes in movement and breathing (i.e. exercise) that may also change their patterns of eating and hydration. through this exposure, a person's decisionmaking might adapt to prioritize maintainable-ease of functioning in the individual's lifestyle. how? some behavior change interventions train one's decision-making to remove or "countercondition" unhealthy habits, by replacing them with healthy habits (prochaska et al., 1988) . although modern behavior change interventions have shaped the precision of decisionmaking during health behavior change (e.g. of smoking, diet, alcohol use, inactivity), new problems for health behavior still emerge when the individual is exposed to a new, potentially addictive technology. with this philosophy, behavior change interventionists and health officials might not only ask the question, "how do we support good decision-making of individuals?" researchers, behavior change technology developers, and public health officials may also ask the functional question: "how do we enhance the precision of one's decisions and the variation of one's habits following the exposure to a new, potentially addictive technology?" public health campaigns disseminated by social organizations can enhance the precision of the population's health values and variation in population-wide health behaviors. leading up to first surgeon general's advisory committee on smoking and health (1964) , the u.s. department of health had become increasingly aware of (i.e. exposed to) variations in a population health behavior. if populations smoked, then populations were more likely to develop lung cancer, laryngeal cancer, or chronic bronchitis (cdc, 2018) . following this exposure to (the consequences of) population smoking behavior, society's values shifted to prioritize health. how? the federal cigarette labeling and advertising act of 1965 was adopted, and the public health cigarette smoking act of 1969 was adopted to create new health values. this shift in values prioritized new variations in population health behavior by: (1) requiring a health warning on cigarette packages; (2) banning cigarette advertising in the broadcasting media; and (3) calling for an annual report on the health consequences of smoking (cdc, 2018) . since these first initiatives adult smoking rates have fallen from about 43% (in 1965) to about 18% today; and mortality rates from lung cancer, the leading cause of cancer death, are declining (department of health and human services, 2014). although the precision of the population's values has been enhanced to impact population behaviors (e.g. the tobacco laws described above supported healthy change), new chronic states can still emerge following exposure to social changes (e.g. the invention of the juul impacted high school and college aged populations). with this philosophy, public policy officials and public health researchers might not only ask the question, "how do we create new laws to protect population health from nicotine addiction?" they may also ask the functional question: "how do we enhance the precision of the population's values and the variation of the population's behavior following the invention of a new nicotine delivery system technology (e.g. flavored juuls)?" previously we described that without functional variation, life is fragile when exposed to present changing conditions; and without functional precision, life becomes disorganized from exposure to changing conditions across time. when successful, the above interventions upon biological, behavioral, and social functioning have a common theme: each facilitates exposures to biological, behavioral or social conditions that support (1) increasingly complex/diverse variation; and (2) increasingly organizable precision. exposure, not avoidance, has facilitated population health in these interventions. while healthcare systematically prioritizes biological exposures in the form of vaccine interventions, they do not systematically prioritize behavioral or social exposures. however, it is documented that exposure to healthy behaviors in youth prevents risk behaviors in adolescence (velicer et al., 2000) ; and exposure to community-based health initiatives can support population health (dulin et al., 2018; cdc, 2018) . given that systematic biological exposures in the form of vaccination have led to a global control of some acute infectious diseases (tangermann et al., 2007) ; might systematic behavioral and social exposures (especially in youth) be needed to enhance global campaigns toward the control of chronic disease? a functional language of health is central to the success of a philosophy of health. why? the levels are not separate, but rather are continuously connecting with one another. a good philosophy of health should have the ability to discuss assessment, diagnosis, intervention, and prevention across levels, across systems, across cultural populations, and across time. using the common language of precision and variation creates discussions that connect the levels and integrate research disciplines. a case (to) study: mental health as between-level functioning in this philosophy. historically, and still too often, health professionals have an expertise at one level, that limits their prescription of interventions to that level. this can actually create barriers to a complete solution when a health problem is multileveled. while a person's mental health is typically assessed based upon their firstperson experience of thoughts, feelings, and behaviors; symptoms can be triggered by biological, physiological, behavioral, psychological, and/or social dysfunction. most clinicians typically do not have the ability to assess and address all forms functioning. so if one person, john, is meeting with a clinician who specializes in primary care medicine, he may only be prescribed a biological intervention like medication. if john is meeting with a clinician who specializes in behavioral medicine, he may only be prescribed a health behavior change intervention. if john is meeting with a clinician who specializes in a certain theory of psychotherapy, he may only be prescribed a psychotherapy intervention based on the clinician's training. if john is meeting with a clinician who specializes in social work, he may only be prescribed a group, community or social intervention. while the above specializations have been helpful in establishing an empirical bases for mental health interventions, overspecialization can be problematic when a multi-leveled solution is needed. in addition, it can also be problematic when a levelspecific solution is needed that the clinician cannot provide (e.g. when psychotherapy is needed but a clinician only has the ability to prescribe psychiatric medication). technology poses a multileveled issue for population mental health in 2020. selves have more social connection then ever in history, yet societies are characterized by increasing rates of depression and loneliness (sum et al., 2008; hammond, 2020; srivastava and tiwari, 2013; twenge, 2017) . researchers might use this philosophy of health to facilitate between-level conversations that address seemingly paradoxical outcomes that emerge during this new age of rapid technological growth. to do this, a researcher might first begin by asking questions about functioning at each level; second, ask questions about processes between the levels; and third, concurrently ask questions at and between levels. first: begin by asking questions at each level. novel challenges face the igeneration (and their parents) due to technology's novel impacts on the development of individual and social functioning (twenge, 2017) . for example, if john's decisions (self-precision) and habits (self-variation) remain consistent during school hours because his parents do not let him have a phone; but his class' social behaviors around him (society-variation) change dramatically because everyone else at school uses the newest smartphone application to talk during class; will john's mental health suffer? although his parents' intentions are to protect john, the contrast between his behavior (self's precision-and-variation) and the population social behavior (society-variation) can impact john's health. notice here how we have not yet considered functions that connect the self to the society (e.g. john's thoughts and feelings). rather we first consider (or contrast) functioning at the level of the self (i.e. john's decisions-and-habits) and the society (i.e. population social behavior) in accordance with principles 1-5 (see figs 1-3). second: look for functional processes that connect the levels. one person's thoughts and emotions/feelings are processes that help to integrate the functioning of one-self within the functioning of a society. how might john's thoughts and feelings connect his (sense of) self to his society? perhaps john's parents teach him that it is important to feel separate from his classmates during class so he can think clearly in class; and that he can feel connected to his friends by inviting them over to communicate together after school. this parenting may impact john's thoughts and feelings during school. if john's parents do not talk with him about this topic, john may experience different thoughts and feelings during school hours. when kept to one-self, thoughts and emotions are foundational to an internal sense of self as one functions in the larger society; and, when acted upon, thoughts and feelings can become verbal communication (e.g. speech) and non-verbal communication (e.g. body language, facial expressions) that form an external sense of self that is visible to the society. the (internal) experience of and (external) communication of thoughts, feelings and actions form the foundation of all systems of psychotherapy (prochaska and norcross, 2018) . this view can be particularly helpful as researchers begin to investigate how smart technology impacts developmental changes to the self within the society beginning in youth. third: concurrently ask questions at and between levels. perhaps, a clinical researcher is interested in investigating protective mental health factors in the igeneration; and they hypothesize that lower rates of loneliness, anxiety, and depression will be seen in subjects that do not respond to text messages immediately. the researcher might investigate further by using the philosophy to develop questions for the research subjects: "(1) do you use conscious decision-making (self-precision) to prevent yourself from habitually responding to your phone when a text appears (self-variation)? (2) how fast do other's in your social group typically respond to texts (society-variation)? (3) what changes in thoughts and feelings are experienced (internal self-society connection) after you communicate via text (external self-society connection)?" perhaps this researcher also wants to investigate how those who are addicted to the technology perceive nonresponders. the clinical researcher might again apply the philosophy: "(1) how fast do other's in your social group typically respond to your texts (society-variation)? (2) do you experience changes in thought and feeling (internal self-society connection) when others do not respond to you within an hour (societyvariation)? (3) how do you communicate those thoughts and feelings (external self-society connection) with others when they do not respond for a prolonged period of time (society-variation)?" future research might use this method to gather and organize levels of information on mental health factors across different self-and societal-conditions. the processes that form our mental health form a functional connection between self and society. if mental health is a reflection of the self-society connection, what might be a reflection of the self-cell connection? physiological health evidences a functional connection between our sense of self and our cells. for example, aerobic exercise is a health behavior that stimulates changes to variations in breathing and movement. by engaging in this behavior, the biological cells of the body are also stimulated via various physiological processes. breathing will stimulate cellular functioning via the cardiovascular and respiratory systems; and movement will stimulate cellular functioning via the cardiovascular, musculoskeletal, and central nervous systems. while all physiological systems are working in collaboration in the body, certain changes to behavioral and biological functioning will stimulate certain physiological systems. by viewing health through this lens, between-level observations join the philosophy: biological functions emerge at the level of the cell; physiological functioning emerges as the cell-self connection; behavioral functions emerge at the level of the self; psychological/ mental functioning emerges as the self-society connection; and social functions emerge at the level of the society. future papers will explore maintainable-ease of functioning at and between levels. future directions: new images of healthcare integration and new perspectives of healthcare innovation. by considering this integrative philosophy, one can define health based upon a tangible connectedness, rather than separateness, of cells, selves, and societies. we provide image 4 as a way to visualize the common paths to the health of healthy publics. when researchers observe that a host defense system is changing cellular functions following an infection, they may also expect these changes to have an impact [along path 1] on expressions of habitual or physiological functions (e.g. immune function can stimulate the sensation of "achiness" or "pain" altering one's physical movement, breath rate, hydration, and hunger) (kelley, 2003; johnson et al., 1992; danzer, 2009) . when researchers observe an individual deciding to engage in health behavior change following an addiction, they may also expect these changes to have an impact [along path 2] on the group-behavior of their family system or social systems. when researchers observe changes to society's values following a newly detected problem (e.g. laws ban cigarette advertising in broadcasting media; public health standards mandate certain vaccines before attending school), they may also expect that these changes can have an impact on behavioral functions of individuals [along path 2] and biological functions of cells/organs [along path 3]. these levels are continually integrating along these common paths to the health of healthy publics (fig. 4) . when attending to this connectedness new, important questions can have new answers. what function does modern technology serve in population health and healthcare? if technology algorithms prioritize variations in population behaviors, then they fulfill a role as society-level precision. when modern technologies like machine learning (ml) technology and computer tailored interventions (cti) prioritize patterns of population behavior, we can see profound impacts on social change in a society. although one might argue that technologies can be used by individual-level functions, the algorithms that are currently deployed and updated on devices interface with bigdata gathered on population behaviors (manogaran and lopez, 2017; dinov, 2016; mullainathan and spiess, 2017; cheng et al., 2017) . in this paper, we identified that precision can be functional or dysfunctional. similarly, technologies can support or prevent healthy population behavior. some technologies prioritize health behavior in populations by tracking physical activity and providing feedback on activity progress; while others prevent healthy behavior by prioritizing sedentary behavior through video-gaming. some social media technologies facilitate social communication with distant friends and relatives that supports wellbeing; while others facilitate conflictual communication that diminishes wellbeing. given that modern technology can support or hinder health, we believe it is important that healthcare can prioritize technological innovations that value health in cells, selves, and societies. to do this, technology innovators might seek to value a higher order construct (e.g. maintainable-ease of functioning) in their algorithms. medical technology is currently used to titrate the doses of vaccines so that maintainable-ease of biological functioning (i.e. inoculation) is made available to the general population. when biological exposures are not properly titrated, infections can become active in the population and health is no longer valued at the level of the cell. similarly, when behavioral and social exposures are not tailored to the needs of individuals and groups, populations can become resistant to healthy change, and health is no longer valued at the level of the self and the society. behavior change researchers prochaska and prochaska (2016) report that when individuals and populations are not ready for a change, interventions that force individuals or populations to take action can increase resistance and prolong dysfunction. by tailoring (or what they term "staging") behavioral and social level interventions, computer tailored interventions upon behavioral and social functioning are made possible (prochaska et al., 2001; velicer et al., 2000, prochaska and prochaska, 2016) . despite these advances, there remains a need for technological advances that can make maintainable-ease of behavioral and social functioning available to the general population. future healthcare interventions could benefit from ml algorithms that tailor behavioral and social exposures to enhance precision-and-variation. research already demonstrates that tailoring interventions for biological precision (albert-vega et al., 2018) and biological variation (brodin et al., 2015) can impact long-term biological functioning. future innovations might seek to use technology to tailor behavioral and social interventions to generate maintainable-ease of functioning. through the functional language used in this paper we hope readers are inspired to present new questions, new comments, and new perspectives about needed healthcare innovations. behaviorially conditioned immunosuppression on the development of 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change, and is the director of the cancer prevention research center at the university of rhode island. jms, m.ed. is currently a ph.d. student conducting dissertation research on population health and behavior change under jop's supervision at the university of rhode island. correspondence and requests for materials should be addressed to j.m.s.reprints and permission information is available at http://www.nature.com/reprintspublisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. key: cord-104157-rivaoo73 authors: noreika, valdas; kamke, marc r.; canales-johnson, andrés; chennu, srivas; bekinschtein, tristan a.; mattingley, jason b. title: alertness fluctuations during task performance modulate cortical evoked responses to transcranial magnetic stimulation date: 2019-06-28 journal: biorxiv doi: 10.1101/155754 sha: doc_id: 104157 cord_uid: rivaoo73 transcranial magnetic stimulation (tms) has been widely used in human cognitive neuroscience to examine the causal role of distinct cortical areas in perceptual, cognitive and motor functions. however, it is widely acknowledged that the effects of focal cortical stimulation on behaviour can vary substantially between participants and even from trial to trial within individuals. here we asked whether spontaneous fluctuations in alertness can account for the variability in behavioural and neurophysiological responses to tms. we combined single-pulse tms with neural recording via electroencephalography (eeg) to quantify changes in motor and cortical reactivity with fluctuating levels of alertness defined objectively on the basis of ongoing brain activity. we observed rapid, non-linear changes in tms-evoked neural responses – specifically, motor evoked potentials and tms-evoked cortical potentials – as eeg activity indicated decreasing levels of alertness, even while participants remained awake and responsive in the behavioural task. impact statement a substantial proportion of inter-trial variability in neurophysiological responses to tms is due to spontaneous fluctuations in alertness, which should be controlled for during experimental and clinical applications of tms. transcranial magnetic stimulation (tms) is a widely used tool for probing human brain function, with applications ranging from the characterization of inter-hemispheric motor cortex interactions and establishment of causal links between neural oscillations and attention, to the clinical treatment of depression and other diseases (dugué & vanrullen, 2017; valero-cabré et al., 2017; ziemann, 2017) . a number of neurophysiological indices of cortical tms perturbation have been used to contrast experimental conditions of interest, including motor evoked potentials (meps) recorded from peripheral muscles (barker et al., 1985; bestmann & krakauer, 2015) , and tms-evoked potentials (teps) which are thought to reflect the reactivity of underlying cortical circuits (chung et al., 2015; . these and other outcome measures show varying sensitivity to different experimental manipulations, as well as confounding factors. perhaps the largest within-participant variation in motor and cortical responses to tms are observed when contrasting wakefulness and sleep. as healthy adult participants fall into slow wave sleep, mep amplitude diminishes (bergmann et al., 2012; hess et al., 1987; grosse et al., 2002) , whereas tep amplitude increases in association with a breakdown of effective connectivity, reflecting a state shift from a global to a more local or stereotypical mode of processing (massimini et al., 2005) . likewise, sleep pressure has been shown to modulate tms responses during normal waking in daytime hours. for example, mep-derived motor thresholds are elevated following sleep deprivation (de gennaro et al., 2007) , whereas tep amplitude increases throughout the day as a function of a natural build-up of sleep pressure (huber et al., 2013) . to date, however, it is not known whether the effects of tms on neural activity are influenced by fluctuations in the level of alertness that occur during wakefulness. here we combined single-pulse tms with concurrent eeg recording and a simple behavioural task to quantify changes in motor and cortical reactivity with fluctuating levels of alertness defined objectively on the basis of ongoing brain activity. in most studies that have employed tms to perturb or modify brain activity, behavioural or physiological data are typically collected over experimental sessions that last up to an hour or more (e.g. darmani et al., 2018; herring et al., 2015; salminen-vaparanta et al., 2013) . the implicit assumption is that participants' levels or alertness remain relatively consistent for the duration of the testing session. this assumption has recently been challenged by findings from brain imaging studies. for instance, tagliazucchi and laufs (2014) found that 30% of research participants drifted into a drowsy state (n1 sleep) during resting-state functional magnetic imaging (fmri) protocols after only three minutes. these periods of early n1 sleep during passive resting-state scans were accompanied by increased signal variance in sensory and motor cortices, increased temporal-temporal and temporal-frontal connectivity, and decreased thalamic-frontal connectivity patterns (tagliazucchi & laufs, 2014) , highlighting a pervasive source of variance in neuroimaging data due to drowsiness. likewise, using an active decision-making task, de gee et al. (2017) demonstrated that brainstem-controlled inter-trial fluctuations in phasic arousal are accompanied by modulation in the involvement of prefrontal and parietal cortices in choice encoding, again implying that fluctuating levels of alertness are potentially a significant source of variability in neural activity. further evidence for the contribution of fluctuating levels of alertness to variability in neural activity has come from studies of mep amplitudes, which tend to be highly variable from trial to trial even when the intensity of the tms pulses is held constant (ellaway et al., 1998; maeda et al., 2002) . several studies have shown that a significant portion of this variance is related to eeg oscillatory activity in a pre-tms time window (mäki & ilmoniemi, 2010; sauseng et al., 2009; zarkowski et al., 2006) . in particular, trials with higher pre-stimulation alpha power tend to be associated with lower mep amplitude (sauseng et al., 2009; zarkowski et al., 2006) . to the extent that increases in alpha power are associated with decreased levels of alertness in participants who are awake and have their eyes open (gharagozlou et al., 2015; kaida et al., 2006) , spontaneous fluctuations in alertness could be a significant source of inter-trial mep variability. unfortunately, previous tms investigations have not measured or controlled for changes in alertness in their participants, and so it remains unknown whether fluctuations in alertness are systematically associated with changes in tms-evoked neural activity. in the present study, we characterize effects of spontaneous fluctuations in alertness on neurophysiological indices of tms perturbation over the primary motor cortex during a single daytime session. we had four goals: (1) to estimate the latency and stability of fluctuations in alertness over the course of an active, single-pulse tms session; (2) to test whether fluctuations in alertness modulate the occurrence and amplitude meps; (3) to determine whether the amplitude of tep responses within the first 50 ms after a tms pulse changes across different levels of alertness; and (4) to assess whether inter-trial variance of mep and tep amplitudes is altered with decreases in alertness. participants (n=20) relaxed with eyes closed in a reclining chair, and received single pulses of tms over the right motor cortex (targeting the first dorsal interosseous (fdi) muscle of their left hand) at 9 different intensities centred on their individual motor threshold (see materials and methods for a detailed outline of the experimental protocol). participants were asked to press a mouse button with their right hand (which was not targeted) after each tms pulse if they felt a tactile sensation or twitch in their targeted left hand ( fig. 1a-b ). at the commencement of the study, participants were informed that they were free to fall asleep but that they should otherwise continue performing the task. they were gently awakened if they failed to respond after 3 successive tms pulses. to assess the instantaneous level of alertness, a two-fold eeg analysis was applied over the time window immediately preceding each tms pulse: (1) a binary definition of awake and drowsy states following eeg spectral power signatures (θ/α) averaged across all eeg electrodes (bareham et al., 2014) , and (2) a dynamical definition of alertness levels following a detailed sub-staging system for scoring the transition to n1 sleep (hori et al., 1994) (fig. 1c ). both measures were highly correlated (see fig 1f) . temporal structure of an individual trial. two eeg windows preceding single-pulse tms were used to assess alertness: a 4 s window was used for manual scoring of hori stages (5 alertness levels), and a 2 s window was used for automatic calculation of the theta (3-7 hz) to alpha (8-12 hz) spectral power ratio. following each tms pulse delivered over the right motor cortex, motor evoked potentials (meps) were recorded from the first dorsal interosseous (fdi) muscle of the left hand, and tms-evoked potentials (teps) were recorded using high density eeg to characterize cortical reactivity. (b) schematic of experimental set-up, showing emg, eeg, tms and response mouse in situ. (c) brief definitions and eeg examples of 9 hori stages of sleep onset, progressing from relaxed wakefulness (hori stage 1) to nrem stage 2 sleep (hori stage 9) (modified with permission from ogilvie (2001) ). in the current study, alertness levels 1-5 (marked in green) correspond to hori stages 1-5. (d) percentage of trials obtained within each alertness level, shown separately for the 20 participants. datasets are sorted from the most alert participants (lower rows) to the drowsiest participants (upper rows). there were very few epochs of alertness level 6 or above. (e) representative dataset for one participant, showing good agreement between the two eeg measures of alertness across the whole testing session. the upper subplot indicates θ/α ratio; the lower subplot shows fluctuations of alertness levels on the hori scale. (f) cross-validation of eeg measures of alertness: intra-individual correlations between θ/α power and alertness levels across single session trials. bars represent intra-individual spearman's rank order correlation coefficients for the 20 participants, sorted from the most to the least positive coefficients. on average, tms sessions lasted for 92.5 min (sd=7, range=73.5-104.3 min) including time spent switching tms coils and allowing breaks for participants. during this period, all 20 participants reached alertness level 5 or higher, reflecting deep drowsiness with a dominance of eeg theta ripples (see fig 1c) . notably, it took only 9.44 min on average for participants to reach alertness level 5 (sd=8.95, range=2.25-33.35 min), indicating a rapid decrease of alertness despite the fact that participants were receiving tms pulses and generating task-specific motor responses. all participants ceased responding at some point during the testing session, after which they either woke up spontaneously due to tms, or they were awoken by an experimenter after 3 consecutive unresponsive trials. on average, 13.1% of trials were categorised as "unresponsive" (sd=9.7, range=2.83-40.6), suggesting a notable impact of drowsiness on task performance. alertness levels and unresponsive trials tended to be spread across the testing session, i.e., participants tended to "oscillate" between awake and drowsy states (see fig 1e and s1). consequently, there was no systematic increase or decrease in alertness level within a session at the group level. four participants showed a significant but weak positive correlation between alertness level and trial number (mean rho=0.21), 6 participants showed a significant negative association (mean rho=-0.27), while the remaining 10 participants showed no significant correlation between alertness level and trial number (mean rho=-0.008) (see fig s1) . these results suggest that a given participant's level of alertness cannot be assumed to decrease continuously over a testing session. only concurrent eeg measures of alertness can definitively determine a participant's moment-to-moment level of alertness. visualisation of the alertness level (vertical axis) shown for the entire tms testing session (horizontal axis). each subplot represents a different participant (indicated by id number). red vertical lines depict the first trial within a session scored as alertness level 5. blue dots indicate unresponsive trials. results of spearman rank order correlation tests between alertness level and trial number are presented in the top right corner of each subplot (ns=not significant). we first assessed corticospinal excitability as a function of alertness and tms intensity. to this end we calculated the proportion of trials with mep peak-to-peak amplitude above 50 μv for each of the 9 tms intensities, separately for the θ/α-defined awake and drowsy trials. a sigmoid function was then fitted across alertness conditions for each participant. the slope of the mep sigmoid was slightly but significantly shallower in drowsy compared with awake trials (wilcoxon signed-rank test: z-score=2.02, p=0.044, r=0.32), suggesting mildly increased noise and instability in corticospinal processing (see fig. 2a ; individual participant results are shown in fig. s2 ). by contrast, the mep sigmoid threshold did not differ between awake and drowsy trials (t(19)=1.31, p=0.21, d=0.13, bayes factor in favour of the null=2.04). we considered whether the observed difference in mep slopes was specifically related to alertness, as the amplitude of pre-stimulus alpha oscillations has also been implicated in fluctuations in attention and sensory gating. in these cases, eeg alpha effects are typically evident only within a relatively short pre-stimulus time period of a few hundred milliseconds (romei et al., 2008) , and are restricted to sensory or fronto-parietal regions (capotosto et al., 2009; van dijk et al., 2008) . contrary to this, the difference observed here in mep sigmoid slope as a function of eeg θ/α power was temporally and spatially widespread (fig. s3 ). (a) group-averaged frequency of trials with meps above a threshold value of 50μv across 9 tms intensities, centred on individual motor thresholds (0%). sigmoidal functions are fitted separately to the θ/α-defined awake (red) and drowsy (blue) conditions (error bars represent one standard error of mean, sem). insets on the right depict each participant's sigmoid threshold and slope difference (awake minus drowsy), with horizontal bars sorted in ascending order. only responsive trials are included in the analysis shown in this and other subplots. alertness states are distinguished here using the eeg θ/α measure taken from a 2000 ms window immediately prior to the tms pulse. (b, upper panel) group-level dynamics of meps (averaged across three tms intensities centered on motor threshold) across alertness levels 1-5. horizontal green dashed lines delineate peaks at 25 and 29 ms post-tms (0 ms) for alertness level 1. (b, lower panel) change in mep peak-to-peak amplitude across alertness levels 1-5. circles represent individual participants. for each alertness level, the red line depicts the group-level mean of peak-to-peak amplitude. the pink shaded region represents 1 standard deviation (sd), and the blue shaded region represents the 95% confidence interval of the mean. states. percentage of trials with meps above threshold value of 50μv, calculated separately for the awake and drowsy trials across 9 tms conditions centred on individual motor threshold (0%). sigmoidal functions are fitted to the awake (red) and drowsy (blue) conditions separately for each individual (n=20). slopes (lower panel) between θ/α-defined awake and drowsy conditions. alertness states were measured and contrasted separately for each of the 20 time bins in steps of 100 ms across a 2000 to 0 ms pre-tms time window. electroencephalography (eeg) spectral power was averaged over all electrodes. (b) difference in mep sigmoid thresholds (upper row) and slopes (lower row) between θ/α-defined awake and drowsy conditions. alertness states were measured and contrasted separately for each of the 63 eeg electrodes. eeg spectral power is averaged over a -2000 to 0 ms pre-stimulation time window. we next compared mep peak-to-peak amplitudes between alertness level 1, reflecting relaxed wakefulness, and alertness levels 2-5, reflecting increasing levels of drowsiness. as shown in figure 2b , there was a significant increase in mep amplitude between alertness levels 1 and 4 (t(19)=3.5, p=0.0096, d=0.64), as well as an intermediate stepped increase across alertness levels 2 (d=0.15) and 3 (d=0.23), and a small decrease at alertness level 5 (d=0.27). a linear trend of increasing mep amplitude was observed across alertness levels 1-4 (f(1,19)=11.55, p=0.003, partial η2=0.38), but this was no longer significant when level 5 was also included (f(1,19)=2.11, p=0.165, partial η2=0.1). these findings indicate a reliably non-linear reorganization of corticospinal excitability at a time when drowsy participants are still conscious and responsive. the most noticeable change in dynamics occurred with the disappearance of alpha waves, at a point where there was eeg flattening and the first occurrence of eeg theta-range ripples, i.e., alertness levels 4 and 5despite the fact that participants were still responding behaviourally in the task. these observations suggest a much earlier modulation of corticospinal excitability in the initial moments of drowsiness than has been reported previously in studies of mep changes with sleep deprivation or during nrem sleep (de gennaro et al., 2007; grosse et al., 2002; manganotti et al., 2004) . we next assessed post-tms cortical reactivity measured as tms-evoked potentials (teps) within the first 40 ms after each pulse. early tep amplitude is known to increase in response to homeostatic sleep pressure (huber et al., 2013) and during nrem sleep (massimini et al., 2005) , likely reflecting a combination of synaptic strengthening, changes in neuromodulation, and impaired inhibition (huber et al., 2013) . we hypothesized that, as with mep amplitude, teps should be affected by the level of alertness in drowsy but responsive participants. comparing tep amplitudes between θ/α-defined awake and drowsy trials revealed a reliable increase in cortical reactivity in drowsy trials in a time window from 26-36 ms after the tms pulse (t(19)=4.02, p=0.00074, d=0.49) (see fig 3a) . this pattern was evident in 18/20 participants (see fig. s4 ). while displaying a wide fronto-central spread, the peak difference between awake and drowsy states occurred over the right motor region, directly beneath the tms coil (see fig 3b-c) . additional control analyses confirmed that the observed tep increase for drowsy trials was not due to tms-evoked scalp muscle artefacts (see fig s5) . we further compared tep amplitudes at the site of stimulation across alertness levels 1-5. as hypothesized, tep amplitude increased as participants became more drowsy (mann-kendall trend test: z=4.7, p=0.0000025, tau=0.77) (fig 3d-e) . planned comparisons revealed a significant increase in tep amplitude between alertness levels 1 and level 3 (t(19)=4.54, p=0.00088, d=0.5), 1 and 4 (t(19)=4.38, p=0.00099, d=0.68), and 1 and 5 (t(19)=3.43, p=0.0056, d=0.6). these findings provide the first direct evidence for an inverse association between cortical reactivity and alertness, suggesting that sleep-related changes in neural activity may intrude early in the transition between wakefulness and sleep, while participants are still able to respond in an ongoing behavioural task. strikingly, the tep effects emerged at a relatively early alertness level, before the appearance of drowsiness ripples or early slow waves (hori et al., 1994) . shading depicts standard error of the mean (sem). (b) topographical distribution of the early tep mean peak at 26-36 ms post-tms pulse in the θ/α-defined awake (upper left) and drowsy (upper right) states. black dots indicate locations of three eeg electrodes with the maximal amplitude in the map. non-parametric z map (below) reveals region reliably different between awake and drowsy states. (c) 0-100 ms data-driven spatiotemporal clustering of eeg potentials post-tms pulse between θ/α-defined awake (red) and drowsy (blue) states. tep amplitude was significantly higher in drowsy trials in a 5-67 ms time window (cluster peak: 27 ms, t = 4884.47, p = 0.004). the green horizontal line depicts the time window of significant difference. the electrode with the largest difference between awake and drowsy states is marked as a green dot in the topographic voltage map, and its waveforms are plotted below. the black contours within the map show the electrodes with statistically reliable differences (cluster). the topographic voltage map is at the peak difference between awake and drowsy states. (d) individual-level dynamics of tep cortical reactivity peak amplitude across alertness levels 1-5 (tep amplitude averaged over 26-36 ms across 4 electrodes beneath the tms coil). normalized amplitude is shown relative to alertness level 1 (green dashed line). black lines represent participants with higher tep amplitude at alertness level 5 relative to alertness level 1 (n=15); grey lines represent participants with lower tep amplitude at alertness level 5 relative to alertness level 1 (n=5). (e) group-level dynamics of tep waveforms across alertness levels 1-5 (teps averaged over 4 electrodes beneath the tms coil). horizontal green dashed line delineates tep cortical reactivity peak at 31 ms post-tms at alertness level 1. teps averaged across 4 eeg electrodes within a region-of-interest (roi) beneath the tms coil. awake (red) and drowsy (blue) tep waveforms are depicted separately for each individual (n=20). electromyography (emg) waveforms depicting tms-evoked scalp muscular contraction artefact identified during independent component analysis (ica). after identification of this component, trials were split between awake (red) and drowsy (blue) conditions. data shown here are averaged across 16 participants in whom the artefact could be identified. error shading depicts standard error of mean (sem). there was no significant difference between the two conditions. having examined participant-level changes in mep and tep amplitudes with spontaneous fluctuations in alertness, we next carried out single-trial analyses of tms-evoked response variability by pooling data across participants, separately for each alertness level. response variability was quantified as the absolute difference between the tms-evoked response amplitude in each trial and the mean amplitude in a given alertness condition. the variability of single-trial mep amplitude changed significantly between alertness levels figure 4b , there was a consistent increase in tep amplitude variability from alertness level 1 to level 5 (standardized jonckheere-terpstra trend test statistic=14.65, p=0.00000), suggesting increases in cortical reactivity with decreases in alertness. mep peak-to-peak amplitude. (b) tep peak amplitude. jittered dots represent individual trials across participants (n=936 per condition). for each alertness level, the red line depicts mean amplitude. pink shading represents 1 standard deviation (sd), which was very small and is thus difficult to discern in the figure. blue shading represents the 95% confidence intervals for the mean. insets on the right indicate locations of hand and scalp electrodes and time windows used to detect peak amplitude values. in a final step, we asked whether mep and tep amplitudes were correlated at a single trial level, and whether any such association was modulated with changes in alertness. there was no significant correlation between mep and tep amplitudes at alertness level 1 (spearman's rho=0.07, p=0.026; not significant after correction) or level 2 (spearman's rho=0.07, p=0.025; not significant after correction). as shown in figure s6 , however, there was a significant positive correlation between mep and tep amplitudes at the deeper levels of drowsiness, including alertness level 3 (spearman's rho=0.22, p=1.73e-11), alertness level 4 (spearman's rho=0.19, p=3.14e-09), and alertness level 5 (spearman's rho=0.29, p=9.73e-20) (see fig. s6 ). taken together, these findings indicate that while mep and tep responses are independent during wakefulness, they become positively associated with increases in drowsiness, suggesting a gradual shift in state toward a non-differentiated mode of neural processing. individual dots in the scatter plots depict single trial amplitudes of mep and tep responses. z-scored amplitude values are shown, and least-squares lines are plotted to visualize associations that were assessed using a spearman rank order correlation test. most studies that use tms to investigate perceptual, cognitive and motor function in human participants do not consider the possibility that fluctuating levels of alertness across a typical testing session might lead to measurable changes in both behavioural and associated patterns of brain activity. here we used single-pulse tms delivered over the right motor cortex while simultaneously measuring meps and teps across different, objectively defined levels of alertness while participants engaged in a simple tactile perception task. participants exhibited fluctuating levels of alertness across the testing session, as indexed by continuous eeg recordings, but continued to respond behaviourally even in relatively deep states of drowsiness. strikingly, both motor evoked responses and tms-evoked cortical reactivity were altered across different levels of alertness. specifically, we found that mep amplitudes peaked during eeg flattening (alertness level 4), whereas tep cortical reactivity increased earlier and remained stable across alertness levels 4 and 5. these findings highlight that a substantial proportion of inter-trial variability in neurophysiological responses to tms can potentially be attributed to spontaneous fluctuation in alertness. inter-trial and inter-subject variability in mep amplitude is a well-known source of data variance in tms experiments (kiers et al., 1993; ellaway et al., 1998; rösler et al., 2008; schutter et al., 2010; sommer et al., 2002) , and it has been suggested that 30 or more trials are required to provide a reliable estimate of mep amplitude (goldsworthy et al., 2016) . the non-stationarity of mep amplitudes has been attributed to a number of factors, including prestimulus voluntary muscle contraction (kiers et al., 1993) , variation in the number of recruited alpha-motor neurons (rösler et al., 2008) , variation in the synchronization of motor neuron discharges (rösler et al., 2008) and functional hemispheric asymmetries (schutter et al., 2010) . furthermore, a recent series of studies found that the amplitude or phase of pre-tms eeg oscillations can predict mep amplitude, including alpha (iscan et al., 2016; sauseng et al., 2009; schulz et al., 2014; zarkowski et al., 2006) , beta (mäki & ilmoniemi, 2010; keil et al., 2014; schulz et al., 2014) and gamma (zarkowski et al., 2006) frequency bands. extending these studies, our findings suggest that changing levels of alertness could be a key factor in brain state-modulation of corticospinal excitability. for instance, zarkowski et al. (2006) demonstrated that mep amplitude is negatively correlated with pre-tms alpha power (10-13 hz) and positively correlated with pre-tms gamma power (30-60 hz), with an alpha/gamma ratio being the strongest predictor of mep amplitude. while a theta/alpha ratio can index the level of alertness in eyes-closed experiments, such as in the present study, the alertness-indexing frequencies are shifted upward in eyes-open paradigms (eoh et al., 2005; kaida et al., 2006; zhao et al., 2012) . it is therefore likely that the results of zarkowski et al. (2006) and other studies were influenced by changing levels of alertness in the participant sample. our finding of non-linear changes in mep amplitude with decreasing levels of alertness might explain previous contradictory findings regarding sleep deprivation effects on mep amplitude. while several studies have reported an increase in corticospinal motor threshold following sleep deprivation (manganotti et al., 2001; de gennaro et al., 2007) , other studies have failed to find any such effect (civardi et al., 2001; manganotti et al., 2006) . arguably, due to individual differences in instantaneous drowsiness levels and potentially different time of day and duration of testing, the dominant level of alertness varied between these studies, confounding their comparison. for instance, datasets with a relatively high proportion of trials obtained during alertness level 4 would likely indicate higher mep amplitude compared with other datasets. unfortunately, a fine-grained measurement of alertness is seldom undertaken in mep studies, even when eeg is recorded, e.g., "sleepiness" or nrem stage 1 sleep are usually treated as a uniform state (manganotti et al., 2004) , even though a more detailed analysis can reveal at least 6 micro-states within n1 sleep (hori et al., 1994; see fig 1c) . regarding the modulation of teps with sleepiness, huber et al. (2013) observed an increase of tep amplitude as a function of prolonged wakefulness as well as following sleep deprivation. contrary to our results, however, they found no association between tep amplitude and short-lasting episodes of drowsiness. this discrepancy is most likely attributable to the fact that huber et al. (2013) relied solely on a behavioural definition of drowsiness (performance in a visuomotor tracking task), whereas we used fine-grained eeg measures of alertness that could be quantified independently of fluctuations in behaviour. in another recent study, tep amplitude was found to depend on the interaction between sleep pressure and a phase of circadian cycle rather than sleep homeostasis alone (ly et al., 2016) . furthermore, the same study found that an increase in tep amplitude was associated with an increase in eeg theta power across 29 h of sustained wakefulness. unfortunately, the authors did not report whether such an association held over a shorter period of time (e.g., 45 -90 mins), as would be the case in typical tms studies. sleep-related increases in tep amplitudes likely reflect facilitation of a stereotypical, local mode of processing, which has been linked to the loss of consciousness (massimini et al., 2005 (massimini et al., , 2012 tononi & massimini, 2008) . similarly, an increase in tep amplitude is observed during dreamless xenon anaesthesia, whereas dreamful ketamine anaesthesia shows no changes in tep amplitude compared with wakefulness, suggesting a specific link between tep amplitude and (un)consciousness (sarasso et al., 2015) . contrary to these suggestions, however, here we found that the perturbational tep marker of (un)consciousness arose at an early stage of drowsiness, when participants were still able to respond behaviourally and well before they fell asleep. thus, we would suggest that changes in local cortical processing as indexed by teps primarily reflect different levels of alertness rather than the presence or absence of sensory awareness. an increase in stereotypical local processing with increasing drowsiness was also observed in the association between teps and meps. fecchio et al. (2017) showed that trials with a high mep amplitude were also associated with a relatively high tep amplitude compared with trials with a low mep amplitude. in the present study, we found a gradually increasing intertrial variability of tep amplitude across decreasing levels of alertness. by contrast, variability in mep amplitude showed a more complex u-shaped pattern characterised by an initial decrease during early stages of drowsiness and a subsequent increase with higher levels of drowsiness, suggesting potentially independent neural pathways in the transition between wakefulness and sleep. nevertheless, while tep and mep amplitudes showed no correlation at alertness levels 1 and 2, these two neural indices became positively correlated with increasing drowsiness, i.e. at alertness levels 3-5. these findings suggest that withintrial associations between corticospinal excitability (mep) and cortical reactivity (tep) depend on the level of alertness, which should be controlled for when experiments involve hundreds of trials delivered over a prolonged testing session (fecchio et al., 2017; helfrich et al., 2012) . several strategies exist for dealing with changing levels of alertness during behavioural testing. if alertness decreases throughout a session, an additional factor of trial or block number could be added to a statistical model as an alertness regressor or covariate. however, we found that an initial decrease in alertness did not persist throughout the testing session, and participants tended to "oscillate" between awake and drowsy periods (see fig s1) , which precludes any straightforward inference of decreasing alertness over the course of a single testing session. alternatively, reaction times (rts) could be used as a behavioural index of alertness in active tms experiments, as rts typically lengthen with decreases in vigilance (schmidt et al., 2009) , increases in drowsiness (ogilvie & wilkinson, 1984) , and following sleep deprivation (ratcliff & van dongen, 2011) . however, such a strategy would not be possible in passive paradigms in which participants are not required to respond (gordon et al., 2018; massimini et al., 2005) . furthermore, as both trial counts and reaction times are relative measures, participants who maintain high alertness throughout a session could be falsely labelled as drowsy in a proportion of trials. arguably, concurrent eeg recording should be the gold standard for assessment of single-trial alertness, as it provides quantifiable and reliable signatures of instantaneous brain-states, including alpha-and theta-derived hori stages of sleep onset (hori et al., 1994) . as an alternative to the tedious manual scoring of hori stages (alertness levels), an automated eeg method based on wakefulness and sleep grapho-elements is available for the detection of drowsiness from eeg data (jagannathan et al., 2018) . while methods that weight the dominance of eeg theta and alpha oscillations are suitable for eyes-closed paradigms (hori et al., 1994; jagannathan et al., 2018) , such as resting state or phosphene studies (bonnard et al., 2016; de graaf et al., 2017) , the power of higher eeg frequencies should be considered when assessing alertness during active eyesopen experiments (eoh et al., 2005; kaida et al., 2006; zhao et al., 2012) . finally, when eeg measurements are not available or feasible, tms experiments could be carried out in short blocks of just a few minutes each (e.g., 3-5 min) and inter-block intervals could be used to assess instantaneous subjective sleepiness, for example by asking participants to undertake the 9-graded karolinska sleepiness scale (åkerstedt & gillberg, 1990; kaida et al., 2006) . paradigms. it will also be important to investigate whether neurophysiological effects of decreasing alertness are limited to individuals who are likely to fall asleep in a situation of prolonged inactivity, such as our participants. if this turned out to be the case, it might be advisable to selectively recruit individuals who are very unlikely to fall asleep during daytime hours to tms experiments that require a high and stable level of alertness. to conclude, our findings challenge the widely held assumption that the cortex is maintained in a more or less "steady state" when participants undertake experimental investigations of perceptual, cognitive or motor function. our findings demonstrate that spontaneously occurring fluctuations in alertness differentially modulate cortical reactivity over relatively short durations, even when participants are tested during day time hours when they would normally be awake and performing typical activities of daily living. our study highlights the importance of controlling for spontaneous fluctuations in alertness at a single trial level in non-invasive brain stimulation studies. twenty participants (7 male; mean age 23.7 years: age range 21-33 years) took part in the study. all participants were screened for contraindications to tms (rossi et al., 2009) , which included having no history of hearing impairment or injury, and no neurological or psychiatric disorders. all participants were right handed, as assessed using the edinburgh handedness scale (oldfield, 1971) . the mean handedness index was 0.79 (sd=0.19; range 0.3 to 1). potential participants were also screened with the epworth sleepiness scale (ess) (johns, 1991) . the mean ess score was 9.4 (sd=4.3), which indicates that most of the participants had a slight to moderate chance of dozing off in a situation of prolonged inactivity. university of queensland (uq), and the study was carried out in accordance with the declaration of helsinki. all participants gave informed, signed consent. participants were recruited through an electronic volunteer database managed by uq's school of psychology. they received $30 for taking part in the study. there were no adverse reactions to tms. surface emg was recorded from the first dorsal interosseous (fdi) of the left and right hands using disposable 24 mm ag-agcl electrodes (kendall h124sg by covidien; ma, usa). the electrodes were placed in a belly-tendon montage with the reference over the proximal phalanx of the index finger and a common reference on the right elbow. raw emg signals were amplified (×1000) and filtered (20-2000 hz; 50 hz notch filter) using a digitimer neurolog system (digitimer; hertfordshire, uk). the data were digitised at 5000 hz using a power 1401 and signal (v5) software (cambridge electronic design; cambridge, uk) and stored for offline analysis on a pc. throughout the experiment emg activity was monitored on-line using a digital oscilloscope with a high gain. participants were prompted to relax if any unwanted muscle activity was observed. tms was applied to the right primary motor cortex using a magstim 2002 stimulator and a 70 mm figure-of-eight coil (#9925-00; the magstim company; carmarthenshire, uk). the site for stimulation was the point on the scalp over the motor cortex that elicited the largest and most consistent amplitude meps from the left fdi. this stimulation 'hotspot' was found by placing the tms coil tangentially on the scalp with the handle pointing posteriorly and laterally at ~45° to the sagittal plane, and stimulating at an intensity that was assumed to be slightly suprathreshold for most individuals. once the hotspot had been identified it was marked using an infrared neuro-navigation system (visor 2 by ant neuro; enschede, the netherlands). a small piece of foam (~ 5) mm thick was then placed under the centre of the tms coil so that it was not in physical contact with any eeg electrodes. the hotspot was remarked and the location and orientation of the tms coil were maintained throughout the testing session with the aid of the neuro-navigation system. accuracy of coil position and handle orientation were kept within 5 mm and 5 degrees, respectively, but were typically within 3 mm and 3 degrees. resting motor threshold was determined using the relative frequency method with a criterion of ≥50 µv (peak-to-peak) mep amplitude in at least five out of ten consecutive trials rossini et al., 1994; samii et al., 1996) . a two-down, one-up staircase was used, starting at a suprathreshold intensity. mean motor threshold for the group was 53.1% (range 34 -74%) of maximal tms output intensity. continuous eeg data were acquired using a 64 channel brainamp mr plus amplifier, tms braincap and brain vision recorder (v1) software (brain products; gilching, germany). a high chloride abrasive electrolyte gel was used (abralyt hicl by easycap; herrsching, germany) and electrode placement corresponded with the international 10-10 system. data were sampled at 5 khz with a bandpass filter of dc-1000 hz and resolution of 0.5 mv (± 16.384 mv). recordings were referenced online to the left mastoid, and electrode impedance was typically kept below 5 kω. participants were seated in a comfortable reclining chair that included head and leg support (see figure 1b) . after placing the emg and eeg electrodes, participants had their eyes blindfolded and the lights in the lab were dimmed. they were instructed to relax for a few minutes while estimation of individual resting motor threshold was performed. participants' hands were comfortably supported with pillows. after threshold estimation the combined tms-eeg experiment was carried out, and participants were reminded to stay relaxed and keep their eyes closed. they were also instructed to pay attention covertly to their left hand and to respond by clicking one of the two keys on a mouse held in their right hand if they felt a tactile sensation, such as a twitch or a touch, in their left hand at the time of each tms pulse (see figure 1a ). participants were explicitly instructed that they were permitted to fall asleep should they wish to. if no responses were registered after 3-5 consecutive trials (i.e., failure to press a mouse button within 6 seconds of a tms pulse), participants were gently awakened verbally and reminded to continue the task. during stimulation, nine tms intensities centred on the individual resting motor threshold were used (-20%, -15%, -10%, -5%, 0%, +5%, +10%, +15%, +20%). given that tms stimulator output intensity is measured in whole numbers from 1 to 100, the calculated percentage from threshold intensity was rounded. this yielded slightly different sized steps from -20% to +20% for some individuals, and this was taken into consideration when fitting sigmoidal functions at the single participant level. for each individual, 520 trials of single pulse tms were delivered, with an average inter-pulse interval of 9.5 sec and a uniformly distributed random jitter of ±1000 ms. thus, the inter-pulse interval lasted between 8.5-10.5 s. we incorporated a relatively long inter-pulse interval to facilitate the natural development of drowsiness, and to allow sufficient time for a return of tonic emg activity to its baseline level. as our aim was to obtain the maximum number of evoked responses (meps and teps) around the tms threshold intensity, the following number of trials was delivered at each tms intensity: 40 trials (7.7% of a total) at each of the -20%, -15%, -10%, +10%, +15% and +20% intensities; 80 trials (15.4% of a total) at each of the -5% and -5% intensities; 120 trials (23.1% of a total) at 0% (i.e., at the individual resting motor threshold). trial order was randomized throughout the experiment. tms pulses were delivered in 8 blocks of 65 trials. one experimenter held the tms coil and the other monitored ongoing eeg; these individuals switched their places after each block. an extended rest was provided after 4 blocks to allow participants a break from the task, to change the heated tms coil, and to reduce the impedance of any eeg electrodes if required. data collection lasted approximately 90 minutes. in an effort to reduce the potential impact of any circadian fluctuation in cortical excitability (sale et al., 2007) , all testing sessions commenced at 1.00 pm (a time at which participants were more likely to feel drowsy after having had their lunch). peak-to-peak amplitudes of meps evoked by tms pulses delivered over the right motor cortex were calculated for each trial within a 20-45 ms time window using signal (v5) software (cambridge electronic design; cambridge, uk). trials containing phasic muscle activity in the left fdi channel within 100 ms prior to a tms pulse being delivered were discarded from the analyses. we characterized modulations of meps as a function of alertness levels by fitting a sigmoid function to the proportion of trials that evoked meps (constrained from 0 to 1 on the y axis) across the 9 tms intensities (-20%, -15%, -10%, -5%, 0%, +5%, +10%, +15%, +20). we then compared threshold and slope measures in awake and drowsy trials separately for each participant. a 50 μv cut-off threshold in peak-to-peak amplitude rossini et al., 1994; samii et al., 1996) was used to define the presence of an mep. a sigmoid function was fitted to each individual participant's data: where f is the mep ratio, x is the tms intensity, µ is the threshold value (the tms intensity at the inflection point), and s is inversely proportional to the slope at the threshold. the actual slope of the fitted sigmoid was calculated by fitting a straight line between a point 0.1 above the inflection point and a point 0.1 below it (see figure s2 ). to assess dynamics of mep peak-to-peak amplitude across alertness levels 1-5, responsive trials with mep amplitude at least twice as high as the peak-to-peak distance in the -100-0 ms baseline window were averaged separately for each alertness level and each participant. in an effort to control for mep variance as a function of tms intensity, only three tms intensities (-5%, 0%, +5%) around each individual's motor threshold were included in the analysis of mep changes across alertness levels. eeg pre-processing and analysis: pre-tms spectral power eeg data pre-processing was carried out using eeglab toolbox for matlab (delorme & makeig, 2004) , with two separate pre-processing pipelines developed for the analysis of eeg activity before and after tms pulses. to calculate eeg spectral power before tms, the recordings were downsampled to 500 hz, and then epoched in -4000 ms to -10 ms time segments preceding each tms pulse. the noisiest epochs were manually deleted, and the most deviant eeg channels were detected with the 'spectopo' function before running the independent component analysis (ica) for further removal of artefacts such as eye blinks and saccades, heartbeats, and muscle noise. ica was carried out on relatively clean channels only, whereas the noisy channels were recalculated by spherical spline interpolation of surrounding channels after deleting ica components with artefacts. data were again manually inspected and several remaining noisy epochs were deleted. on average, 58 trials (11.2%) were discarded per single participant during emg and eeg pre-processing, leaving on average 462 trials per participant (sd=38; range=348-513 trials) for the subsequent analyses. the spectral power of eeg oscillations over the 4 s time interval immediately preceding each tms pulse was computed using a hilbert transform, set from 1.5 hz to 48.5 hz in steps of 1 hz. given that estimation of spectral power of slow oscillations can be difficult close to the edges of eeg segments (cohen, 2014) , and we were particularly interested in the spectral power just before each tms pulse, a dummy copy of each eeg epoch was created by flipping the left and the right sides of each pre-tms epoch along the time axis. the resulting "mirror image" data were the concatenated with the original pre-tms data; that is, the time axis of the obtained 7.976 s eeg epochs extended from -4000 ms to -12 ms (original) and then back from -8 ms to -4000 ms (mirror). in this manner, an abrupt discontinuity was avoided in the time window just before the tms pulse, thus enabling a more stable estimate of spectral power. after hilbert transformation, the "mirror" part of the eeg epoch was deleted, retaining the original pre-tms window from -4000 ms to -12 ms. to reduce data size, eeg recordings were down-sampled to 250 hz before running the hilbert transform. two complementary eeg measures were used to assess participants' level of alertness before each tms pulse: (1) the hori scoring system of sleep onset eeg (hori et al., 1994) , and (2) a ratio between eeg spectral power of pre-tms theta and alpha oscillations, which we refer to here as the 'θ/α' measure of alertness (bareham et al., 2014; noreika et al., 2017 ). the hori system relies on visual scoring of 4 s segments of continuous eeg data (hori et al., 1994) . it consists of 9 stages reflecting a gradual progression from wakefulness to sleep, from hori stage 1 which refers to alpha-dominated relaxed wakefulness, to hori stage 9 which is defined by the occurrence of complete spindles coinciding with classic stage 2 nrem sleep (see fig 1c) . the hori system has been used to map dynamic wake-sleep changes in erps (nittono et al., 1999) , eeg spectral power (tanaka et al., 1997) , reaction times, and the rate of subjective reports of being asleep (hori et al., 1994) . in the present study, hori stages were visually assessed by an experienced sleep researcher (vn) who was blind to participants' responsiveness and the tms intensity on any trial. for scoring purposes, only 19 eeg channels of the standard 20-10 system were used (fp1, fp2, f7, f3, fz, f4, f8, c3, cz, c4, t7, t8, p7, p3, pz, p4, p8, o1, o2), and eeg recordings were low pass filtered (20 hz). previous research has found that participants are typically unresponsive in hori stages 6 and above (ogilvie, 2001) , so our analysis was restricted to hori stages 1-5, which we refer to here as alertness levels 1-5. hori stages 1 to 4 are marked by decreasing activity in the alpha range, and hori stages 4 to 8 are characterized by an increase in activity in the theta range (hori et al., 1994) . thus, progression of drowsiness can be quantified by a ratio of the spectral power of the alpha and theta eeg frequency bands. specifically, here drowsiness was quantified as a period of time with an increased θ/α ratio of spectral power (bareham et al., 2014) . to apply this measure, theta (4.5-7.5 hz) and alpha (8.5-11.5 hz) power was first averaged in time from -2000 ms to -12 ms, and the θ/α ratio was then calculated for each trial and electrode. next, the θ/α ratio was averaged across all electrodes, resulting in a single "alertness" value per trial. finally, trials were split into the most strongly "awake" (45%) and most strongly "drowsy" (45%) trials, excluding the 10% of trials that were intermediate between the two extremes. importantly, in addition to spontaneous fluctuations in alertness, the spectral power of eeg pre-stimulus oscillations can reflect attentional sampling and/or sensory gating (capotosto et al., 2009; romei et al., 2008; van dijk et al., 2008) . we expected that an alertness-related effect would be spatially and temporally widespread and consistent, so we repeated the mep analysis by splitting the data between awake and drowsy trials separately for each eeg electrode, and in 20 equally sized pre-tms time bins of 100 ms duration, from -2000 ms to 0 ms relative to the tms pulse. all participants completed the experimental task and reached the expected alertness level 5 or higher, marked by the occurrence and dominance of theta waves. at a group level, a comparable proportion of awake and drowsy trials were obtained as per the criteria defined above (alertness levels 1-2: m=45.17%, sd=19.92; alertness levels 4-5: m=35.68%, sd=16.44) (see fig 1d) . thus, even though the hori system provides absolute electrophysiological signatures of the depth of drowsiness, the θ/α ratio was used to identify equal proportions of awake and drowsy trials within each participant. given that the θ/α measure is relative, there was a risk of mislabelling trials for some participants, as it would make a split between "awake" and "drowsy" trials even if all of them happened to be alertness level 1. thus, to verify the use of θ/α data splits, we compared these two measures at an individual level and at the level of the group as a whole. first, we carried out correlation analyses between the two measures of alertness within each participant. second, we compared correlation coefficients against zero to assess the consistency of association between the hori and the θ/α measures. at an individual level, hori and θ/α scores were positively and significantly correlated for all 20 participants (individual rho ranged from 0.66 to 0.9). group analysis confirmed a very strong association between these two electrophysiological measures of alertness (one sample t test: t(19)=51.99, p<0.000005), confirming that the θ/α ratio was well suited to assessing the level of alertness in the sample here (see fig 1f) . analysis of eeg reactivity to tms pulses in the first 50 ms time window requires a perfect alignment of tms markers with the onset of the actual tms pulses. given that there was some delay and jittering between a tms marker and the pulse itself (m=9.6 ms, sd=1.7 ms), eeg markers indicating tms intensity were automatically adjusted to the time point of the actual tms pulse. for this, raw eeg data were segmented ±200 ms around each tms marker, and global field power (gfp) was calculated as a standard deviation of voltage across all electrodes, resulting in a single time waveform for each tms marker. each obtained waveform was baseline corrected to the -200 ms to -50 ms time window, and each time sample was transformed to its absolute value. the remaining time window of -49 ms to +200 ms was scanned, searching for the first time point where a gfp value that exceeded the maximal baseline gfp value by a factor of five, which indicated the onset of a tms artefact. the tms marker was then reallocated to this point in the continuous eeg recording. the eeg data were processed following an ica-based approach of tms-eeg artefact cleaning (rogasch et al., 2014) . first, eeg data were segmented from -1000 ms to +1000 ms around the onset of tms artefact. next, the segments were baseline corrected to the mean of the interval from -500 ms to -100 ms time window. a line was then fitted to the data from -2 ms to 15 ms, thus deleting the initial tms-eeg artefact, and the epochs were down-sampled to 1000 hz. the most deviating eeg channels were then detected with the 'spectopo' function and the first round of independent component analysis (ica) was performed without using noisy channels. after deleting a very distinctive early high amplitude component that reflected tms-evoked contraction of scalp muscles, eeg data were filtered (1-80 hz) and epoched from -400 ms to +600 ms around the onset of the tms marker. once again, any deviating eeg channels were identified and a second round of ica was carried out without using noisy channels. independent components reflecting tms-eeg decay artefact, eye movements, auditory evoked potentials, 50 hz line noise, and other sources of noise, were deleted, after which bad channels were recalculated using spherical spline interpolation. the eeg segments were again baseline corrected (-100 ms to -3 ms), and manually inspected to delete epochs that still contained a residual tms artefact. to account for within-trial variance, raw voltages of each individual trials were transformed to z-scores using the mean and standard deviation of the baseline period (-100 to -3 ms). trials were then split into different levels of alertness. to assess changes in eeg reactivity to tms perturbation as a function of alertness, the four electrodes immediately beneath the tms coil were chosen to contribute their voltage values to a region of interest (roi), and these were then averaged within each participant. the group-level waveform was then plotted, revealing an early tep peak at 31 ms post-tms pulse. the data were then split between alertness levels and the mean amplitude (±5 ms) around the peak (26-36 ms) was calculated for each participant and each level of alertness. erp dynamics were additionally studied using data-driven spatiotemporal clustering analyses similar to what we have described previously (chennu et al., 2013) . awake and drowsy trials were compared in the time windows of interest (15-100 ms) by averaging single-subject data and running group level clustering. using modified functions of fieldtrip toolbox (maris and oostenveld, 2007; oostenveld et al., 2011) , we compared corresponding spatiotemporal points in individual awake and drowsy trials with an independent samples t-test. although this step was parametric, fieldtrip uses a nonparametric clustering method (bullmore et al., 1999) to address the multiple comparisons problem. t values of adjacent spatiotemporal points whose p values were less than 0.05 were clustered together by summating their t values, and the largest such cluster was retained. a minimum of two neighbouring electrodes had to pass this threshold to form a cluster, with the neighbourhood defined as other electrodes within a 4 cm radius. this whole procedure, that is, calculation of t values at each spatiotemporal point followed by clustering of adjacent t values, was repeated 1000 times, with recombination and randomized resampling before each repetition. this monte carlo method generated a nonparametric estimate of the p value representing the statistical significance of the originally identified cluster. the cluster-level t value was calculated as the sum of the individual t values at the points within the cluster. we considered the possibility that a hypothetical alertness-modulation of the contraction of scalp muscles following tms may have contributed to the alertness-modulation of tep amplitude. to address this hypothesis, we compared the amplitude of the ica component of the scalp muscle, which was removed during the first stage of ica cleaning (rogasch et al., 2014) , between θ/α-defined awake and drowsy states. no amplitude difference was observed between awake and drowsy trials (see fig s4) , ruling out the possibility that tep corticalreactivity changes between awake and drowsy states were due to a change in the intensity of scalp muscle contraction. paired samples t tests were used to compare behavioural and neural summary measures between θ/α-defined awake and drowsy states. pooled variance was used to calculate cohen's d, with 0.2 indicating a small effect size, 0.5 a medium effect size, and 0.8 a large effect size (cohen, 1988) . for a similar comparison of summary measures across alertness levels 1-5, a one way repeated measures anova was carried out with linear as well as non-linear contrasts. huynh-feldt correction was used when mauchly's test indicated violation of the assumption of sphericity. partial η 2 was calculated as an effect size in anova tests, with 0.01 indicating a small effect size, 0.06 a medium effect size, and 0.14 a large effect size (cohen, 1988) . shapiro-wilk's test was used to assess normality of the distribution before running parametric tests. square-root or log10 transform were used to normalize skewed data. when transformations failed, non-parametric statistical tests were used, such as wilcoxon's signed-ranks test instead of a paired samples t test, and the mann-kendall trend test instead of a one-way repeated measures anova for linear contrasts across alertness levels 1-5. a jonckheere-terpstra trend test was used to assess a linear change of single trial response variance across alertness levels 1-5, followed up by planned contrasts using a mann-whitney test. for any significant main effects, bonferroni-holm multiplicity correction (holm, 1979) of p values was carried out to 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and electrocardiograph assessment of mental fatigue in a driving simulator the study was supported by the wellcome trust (wt093811ma to tab), the eu key: cord-102738-e5zojanb authors: lieberoth, andreas; pedersen, mads kock; marin, andreea catalina; planke, tilo; sherson, jacob friis title: getting humans to do quantum optimization user acquisition, engagement and early results from the citizen cyberscience game quantum moves date: 2015-06-26 journal: nan doi: 10.15346/hc.v1i2.11 sha: doc_id: 102738 cord_uid: e5zojanb the game quantum moves was designed to pit human players against computer algorithms, combining their solutions into hybrid optimization to control a scalable quantum computer. in this midstream report, we open our design process and describe the series of constitutive building stages going into a quantum physics citizen science game. we present our approach from designing a core gameplay around quantum simulations, to putting extra game elements in place in order to frame, structure, and motivate players' difficult path from curious visitors to competent science contributors. the player base is extremely diverse for instance, two top players are a 40 year old female accountant and a male taxi driver. among statistical predictors for retention and in-game high scores, the data from our first year suggest that people recruited based on real-world physics interest and via real-world events, but only with an intermediate science education, are more likely to become engaged and skilled contributors. interestingly, female players tended to perform better than male players, even though men played more games per day. to understand this relationship, we explore the profiles of our top players in more depth. we discuss in-world and in-game performance factors departing in psychological theories of intrinsic and extrinsic motivation, and the implications for using real live humans to do hybrid optimization via initially simple, but ultimately very cognitively complex games. when online participants are used as workhorses for difficult problems such as eterna's needle-inhaystack-like rna model selection task (lee et al, 2014) or eyewire's formidable challenge of mapping neural connectivity in the mouse retina (marx, 2013) , conclusive results may lie months and years into the future. in the rapidly growing field of human computation the design of new initiatives is often based on intuition rather than proven design hypotheses. the citizen cyberscience community is, however, a remarkably open scientific group, which allows us to capitalize on a unique and generous culture to learn from each other at each step of the journey -not just in the end, when all is securely tested and published. the citizen cyberscience game quantum moves was designed to help build a quantum computer -a computer more powerful than any other in the world based on moving atoms around under the principles governing quantum physics. this delicate process involves a constant risk of losing hold on the volatile atoms, if they are not moved precisely and quickly. the simulations in which our algorithms tried to optimize this process bear a remarkable similarity to side scrolling casual games, and so the notion of human quantum optimization was hatched: what if real humans would do things differently than the logical step-by-step nature of the algorithms? would explicit understandings of the counterintuitive quantum problems help people solve our problem more intelligently? would human physical and cognitive fallibility add an interesting random factor? and even if people in general could not beat the ai, would play trajectories resulting from certain lucky punches or persistent quantum heroes be enough to help the ais learn in new ways? this is the premise of human-computer hybrid optimization: helping ais learn through real people's blooming buzzing mess of solutions, when problems can be represented as engaging game levels. quantum moves places itself with eyewire, galaxy zoo, foldit and eterna in a small category of large-scale resource demanding online citizen cyberscience endeavors where real problem solving is taking place beyond pure data gathering. with this midstream paper, we want to share our experiences from this first year of beta design and player recruitment, and make our reflections and learning curves available. we believe that our findings about the engagement process -although preliminarycontain a series of conclusions pertinent to the design-and engagement processes hidden behind human computation. we first give a brief introduction to the physics behind the game, and the method of turning its core tenets into a playable game. this paper does not focus on the actual game results and the reader with no interest in physics can safely skip the first part. we then turn to the main focus of the paper, the description of the design considerations from the first year, especially relating to user acquisition and the structural gameplay surrounding the game's core loop. finally, we present data about participation for the beta year, noting how different properties like recruitment source and physics interest predict tenacity and performance in the game. we conclude by looking closer at our most dedicated "heroes" and discussing future perspectives for human computation and quantum moves. quantum mechanics originated in the beginning of the 20th century when the physicists of the time realized that the known laws of physics were not capable of describing the structure of atoms. experiments made by ernest rutherford showed that the atom had to consist of a positively charged nucleus orbited by negatively charged particles called electrons (longair, 2003) . in 1913, niels bohr showed that only certain orbits were allowed, and that the electron could only jump from one obit to another by absorbing or emitting a quantum of light with the correct amount of energy. even more remarkably the electron was allowed to be in two different orbits simultaneously until a measurement was performed to determine in which of the orbits it was. from this, quantum mechanics evolved through the work of heisenberg (born, 1926 ), de broglie (1925 , schrödinger (1926) and many others. they showed that atoms should be described by a wave function: a distribution describing the probabilities of measuring the atom in every point in space. computer technology has also progressed rapidly over the past decades. moore's law states that available computer power doubles every 18 months (moore, 1965) , due to the ability to fabricate ever-smaller transistors. however, the miniaturization has a lower limit due to quantum effects. this led to the proposal of the quantum computer (feynman, 1982) , in which the computer bits which are traditionally only allowed to be either 0 or 1 are replaced by quantum bits (qubits) that are allowed to be 0 and 1 simultaneously. a quantum computer holds the potential for huge calculation power since 300 qubits would be capable of representing 2 300 numbers (larger than the total number of atoms in the universe), in contrast to the normal computer where 300 bits can represent just 1 number. quantum computers have already been created in a very small system of 7 qubits (vandersypen, 2001) , but they have yet to be implemented in a scalable system capable of outperforming traditional computers. many proposals for such scalable architectures exist in various systems and in one of them, atoms are contained in an egg-tray like trap made of interfering light beams (weitenberg, 2011a , weitenberg, 2011b . the quantum computation is then performed by concatenating a sequence of individual qubit flip operations (jørgensen, 2014) and two-qubit operations consisting of picking up an atom in one well with a focused tweezer of light and transporting it into contact with another atom somewhere else in the computer (weitenberg, 2011b) . this is non-trivial because any fast movement of the tweezer causes the (probability distribution of the) atom to slosh around and this sloshing will result in an error in the calculation because a sloshing atom contains kinetic energy, which means that it is not in the ground state. the challenge of quantum moves consists in finding algorithms describing how the laser in the physical machine should be controlled to move atoms quickly from one location to another without introducing sloshing at the end. in quantum moves, players help build a powerful quantum computer by finding ways of moving a simulated atom from one location in the game interface to another, without inducing sloshing. the movement of the atom is guided and constrained by a so-called potential landscape spanning the screen (black line in figure 1 ). the probability distribution of the atom (green in figure 1 ) resembles a liquid, but will slosh and distribute itself in smaller waves at the slightest wrong movement according to the rules of quantum physics. we call the collective shape of the atom at any given instance of time its state. each level describes a unique problem represented by the potential landscape's line combined with pre-specified beginning-and target states. success is measured by the degree of overlap between the final state of the atom and that of the target area. a game always consists of controlling the simulated tweezer with your computer mouse for a given amount of time (by moving the bottom of an indentation in the potential landscape). dragging the mouse horizontally changes the position, whereas a vertical move increases or decreases the depth of the trapping indentation (physically realized by turning the power of the laser up or down). a game consists of one complete trajectory of the mouse through a particular level. this can be characterized as the game's "core loop" (fields, 2014) or (fittingly) "game atom" (elias, garfield, & gutschera, 2012) , as it recurs in every game level, variably modified with new obstacles, potential landscapes, goal-states and bonus-points to challenge players or simulate particular physics problems. the levels are organized according to an overall structural gameplay, or metagame, where they get unlocked in stepwise fashion and players receive rewards through different symbolic feedback such as highscores, 1-3 "stars" according to the degree of success, and acquisition of skill-and achievement badges. the entire solution for each game played is stored on our servers for potential future use in our laboratory. computer algorithms used to optimize this type of problems typically exist in two variants. in local algorithms, an initial trajectory is slightly perturbed in a stepwise fashion, and if a change proves beneficial, further in that same direction. this step -although it sounds simple -can be very sophisticated in state-of-the-art algorithms. unfortunately, this deterministic update often causes it to get stuck in what is called a local maximum. in contrast, algorithms with a random component such as genetic optimization algorithms can jump erratically from one solution to the next. this randomness ensures that they will never get stuck and eventually find the best solution -called the global maximum. the disadvantage with this type of optimization is the fact that the steps are random and therefore only beneficial in a small fraction of the times. this makes algorithms with random components exceedingly slow, often in fact so sluggish that in practice they will never converge to the optimal solution. the aim of quantum moves is to combine the best of both worlds in our gamified human quantum optimization: optimization that is rational most of the time, but sometimes makes seemingly random errors or leaps of intuition to rapidly find the sought after solutions. one example of a gameplay trajectory which requires a certain critical breakthrough (iacovides, aczel, scanlon, & woods, 2011 , 2013 is the level bring home water fast (see figure 1 ). the aim is to fetch an atom from the well on the far right of the screen, and ferry it carefully back to your beginning position on the left. here, a good solution consists in utilizing the principle of quantum tunneling by bringing the two wells close together without merging them into one. the atom will the tunnel through the classically forbidden intermediate region, and appear in the well created by the moveable tweezer. specifically, we compare the players to the computer algorithms in two ways. first, for problems which can eventually be solved by the computer, we compare the score after each optimization to the equivalent high score of players after having played an equal amount of times. as long as the player score is higher than the computer score, the human result really represents the fastest way of getting results at that particular junction. of course, since the specialty of the computer is to make minute adjustments and improvements, if the particular problem is of a nature that can be solved by the computers, eventually it will overtake the players. in such cases it is an extremely interesting question, to which extent player results can be used as a starting point for a computer optimization that will yield faster convergence rate than the computer optimization alone. finally, for some of the problems posed the computer fails to find good solutions and it is of course extremely interesting to which extent players can find these solutions. although the analysis of the more than 300,000 unique play trajectories generated so far is not complete yet, a pattern seems to emerge that the human optimization is indeed superior to the computer in many problem spaces investigated in all of the three areas discussed above. even more surprising, it seems that the fraction of the players actually outperforming the computer is quite large. whether the players succeed by fluke, by building in-game skills, or possibly through a simple theoretical understanding of the quantum physics principles represented in the game is still an open question, which will be the topic of future research. a central challenge to achieve the optimization described above is that quantum moves needs to recruit engageable players and hone their in-game skills over time. player acquisition is a central part of online game launch strategies. the industry metric user acquisition cost (uac) is an aggregate of advertisement cost, development cost, back-end expenditures and similar prices used to describe how much money a social game developer will spend, on average, to get a new user (fields, 2014) . developers thus expect to spend quite a bit of time and money to get click-through and installations of their games. this is both an ongoing process of marketing and design, and a focused enterprise to build critical mass at launch. however, while the commercial industry needs to balance this effort and expenditure against each player's average lifetime value (ltv) in dollars and cents, as well as their ability to make the game a social place and recruit friends to join them (lifetime network value, lnv), citizen science games need to consider the time and relative price of a different kind of payoffnamely tangible science contribution from each player and his network. we can label the average direct player contribution user science value (usv). player contributions on our sister-game galaxy zoo have been aggregated into four main clusters, or participation profiles. these reveal that some contribute a lot early, usually on their first login, never to return, while others establish a stable pattern of play over a prolonged period of time (brasiliero, 2014; ponciano, brasiliero, simposon & smith, 2014) . each galaxy pattern recognized is a worthwhile addition of data. in more complex games like eyewire (robinson, personal communication, 2/21. 2014) and quantum moves, however, players need to build a modicum of skill before they can reliably contribute to the core scientific challenges (barring flukes arising from the random factor that is human cognitive and behavioral processing, i.e. bob, 2009) . we could call the average point at which players start doing anything directly useful, the game's user contribution threshold (uct). we have come to call the small percentage of players who tenaciously acquire the necessary skills and persist far beyond the uct "heroes". our notion of heroes mirrors that of whales from commercial games that rely on free-to-play strategies where only a small group of players are ever really monetized through in-game purchases, premium memberships and the like. while citizen science games with low cognitive complexity can benefit from every minute any player spends, games with high cognitive complexity like quantum moves and eyewire only usually gain direct value from a player if he/she becomes a hero. players who just visit out of curiosity and then drop out can be called flâneurs, while average participants explore the structural gameplay deeper, and might contribute by fluke. to that calculation we must then add a player's network value, which means that it seems like a viable strategy to make the game fun and attractive for everyone, even if only a few manage to contribute to the core scientific challenge. generating sustained engagement at less complex levels of game participation may also be a way to gradually build the loyalty and skills needed for a flâneur to transition into a hero. a real concern, which our empirical work aims to address, is, however, if the average hero's preferences and play trajectories diverge substantially from average players. from a psychological standpoint, it is important to mention the difference between intrinsic and extrinsic motivation, as they apply to participant retention in citizen science projects. there are competing schools of thought on the matter (deci, 1980; malone, 1981) , but they agree on core tenets: in extrinsic motivation, action is based on outside rewards like money or socially valued praise, or avoidance of unpleasant states like scolding. this entails a tendency to act half-heartedly and cease the behavior when the outside factors dry out (deci, koestner, & ryan, 1999) . in a state of intrinsic motivation, on the other hand, the user is driven by desires to participate, explore, learn and master the activity in itself. extrinsic design-logics are commonly found in vulgar points-badges-leaderboards (pbl) gamification, where mainstream ideas (e.g. bowser, preece, & hansen, 2013; marczewski, 2012) resemble diluted versions of behaviorist token economy (e.g. kazdin, 1982; skinner, 1973) and 20 th century utility-based economics brought into question by behavioral economists like kahneman (2011) . extrinsically informed motivational design places great importance on the magnitude and temporal distribution of rewards, as well as their psychological framing. intrinsic motivation theories, on the other hand, prescribe design structures that support self-determination via a sense of competence, autonomy and social relatedness (deci & ryan, 2008; rigby & ryan, 2011) , or challenge, fantasy and curiosity (malone, 1981) plus interpersonal factors like recognition, competition and cooperation (malone & lepper, 1987) . for an overview, see below figure 2 . fogg and eckles' (2007) "behavior chain for online participation" can be used to conceptualize 3 phases of user involvement. the discovery phase "learning about the service" and "visiting the site. once they have arrived on the site, users have plenty of opportunities to explore the information available and get the chance "to be educated and influenced". for example, the quantum moves site has videos and photos integrated. also, the website hosts a forum, where visitors can read discussions of registered players and get the chance to be exposed to the game. in the superficial involvement stage, users are influenced to "decide to try" and "get started" with the game. at this stage the structural gameplay will fluently guide them through the tutorial levels and hopefully prompt them to create a profile and validate an email activation link, so they can save their progress. it is only in the final phase, called true commitment, users generate large added value. in quantum moves this translates to users making a valuable contribution to either the science, game development, or through their lifetime network value. such examples range from creation of posts and video materials to creating levels, but contributors' core commitment to quantum moves is measured in play counts and scores. this 3-phase process mirrors the conceptual difference between interest (berlyne, 1954 (berlyne, , 1970 , motivation (grant & dweck, 2003; jensen & buckley, 2012; prestopnik & crowston, 2011; raddick et al., 2013; ryan, rigby, & przybylski, 2006) and sustained engagement (kular, gatenby, rees, soane, & truss, 2008; rigby & ryan, 2011; skinner, seddon, & postlethwaite, 2008) . interest can be understood as the immediate psychological allure of something encountered in the world, usually as a property of perceptual processing (berlyne, 1954 (berlyne, , 1970 ) exacting a motivational pull of curiosity to investigate. engagement (kahn, 1990) includes both motivation, behavioral change, and persistence in an activity. to acquire engaged players across the beta period, we used four separate recruitment strategies. the early parts of the game (see below) were tested in high-school science classrooms and our own university lectures, where large numbers of students were forced to sign up. we also had the opportunity to speak at several high-profile events, such as a couple of public lectures with over 500 people combined, which we used for an a/b-test of certain game elements (see below). the project has additionally garnered a good deal of attention in traditional media and online, which has generated substantial influxes in identifiable jolts. finally, a fourth group of ongoing clickthrough can be attributed to community efforts and general buzz arising online and in-world, making their origin essentially unknown. since many players cease participating quite quickly (a common pattern in games and citizen science projects alike, but especially prevalent for students forced to participate), we also announced several "featured challenges", where existing players were prompted to come back and knuckle down on particular levels. we awarded extra in-game badges for participation, and offered combinations of books, logo mugs, t-shirts, and even a lab visit with all expenses paid (see below) to top contributors. we can thus envision a two-dimensional space for motivational devices, with one axis constituted by in-game (points, progress, good core gameplay, community, etc.) versus in-world (our talks and teaching efforts, prizes, recommendations from friends) (lieberoth & roepstorff, 2014; stevens, satwicz, & mccarthy, 2007) situation, and the other ranging from intrinsic (science participation, challenging gameplay, fun, fascination with physics, etc.) to extrinsic (physical prizes, mandatory high school participation) motivational flavors. game designs based on intrinsic motivation are thought to satisfy the criteria through gameplay processes alone (the core loop and the structural gameplay surrounding it), while our recruitment strategies in educational settings and prizes in featured challenges can be said to be classically extrinsic. extrinsic reward has been found to exact a detrimental effect on intrinsic motivations (deci et al., 1999) , but it is worth noticing that points, leaderboard placements and even physical prizes for top-performance may act as intrinsically motivating feedback, social devices and signs of mastery. but people may also already have interests and preferences in the real world, that makes citizen science participation intrinsically motivating, as for instance seen in player data from galaxy zoo where the most prevalent motivators were not related to the site's superficial gamification devices, but rather a fascination with outer space or a sense of participating in something greater (raddick et al., 2013) . as such, creating engagement is usually a question of balancing design strategies rather than relying on a single approach, such as vulgar pbl-gamification. recruitment activities in-world and online, an engaging in-game core loop, a structural gameplay to frame, structure and motivate the player's continual progression through the levels, as well as an active community where participants get a sense of continually contributing to science, are all central components of the strategy laid out to hopefully realizing the scientific goals of quantum moves. in the remaining sections, we describe the game design process as it has unfolded in a sometimes stumbling but always-creative fashion with its many different ideas and theoretical rationales. we then report statistics about users, recruitment and retention, and the attributes that characterize our small crop of heroes so far. we developed quantum moves as the first game under the scienceathome.org umbrella, one of the first citizen science projects in the quantum physics segment. the initiative is developed within our cross disciplinary centre for community driven research (coder) 1 , where we aim to bridge theoretical and experimental research with online community efforts. the early development of quantum moves, previously known as the quantum computer game, started in december 2011 with the first coding iterations deployed in matlab -an algebra program widely used in physics. the decision was based on the program's plotting flexibility and the programming experience available in the student pool. an early version of the game was ready in february 2012 (see figure a .1 in the appendix) and subsequently tested in several danish high schools. this served as an overall proof of concept, yet we noticed that several challenges hindered the game experience, mainly due to matlab's limited portability and graphic support. over the following months, we improved various aspects of the initial prototype. however, a test session with 100 volunteers in the summer of 2012 made it clear that, if we wanted the game to have a large public appeal, we had to abandon matlab and rewrite the code in a flexible, end-user accessible programming language. the best solution at that time was java, as it addressed most of our concerns of end-user accessibility, robustness, flexibility in programming and variety in graphics. a preliminary java edition came out in early october 2012 (see figure a. 2), and a testing version made available to a small group outside the coder team in dec 2012 (see figure a. 3, a.4, a.5). the first, fully-fledged beta version was publicly launched and advertised on the 18 th of june 2013 (see figure a .6). the game was structured around 4 levels: tutorial, arcade (a series of games where players could practice) scientific (where we included the games we considered most relevant for our lab research) and user space (a sandbox environment, where players could design their own games or try those created by other users in the community). when the 12 tutorial games were successfully completed, a user was allowed to roam around and try any of the other games found in the arcade and scientific levels. the launch of this beta version was covered in several national media outlets, e.g. national geographic and videnskab.dk. the media attention played a definite role in making the game known to an audience beyond the usual high school students. yet, we experienced several glitches with the university server which slowed down our initial success. a brief follow-up survey sent personally to the top 10 players after launch pointed out that the technical issues were doubled by frustrations with the abrupt increase in difficulty, starting with the tutorial. also, looking at the data gathered in the days after the release, we noticed, that once players got past the tutorial, they predominately chose to play in the scientific level with insufficient time spent in the arcade level intended to help them acquire necessary core skills. this became a premise for our discussions and, as the team expanded to include a business school graduate and a psychology researcher, we focused on reevaluating the game design. in august 2013, the current beta version was introduced ( figure a.7) . from this point on, we refer to that version in this paper. early, we were confronted with two pressing aspects that needed to be addressed: one was redesigning the tutorial to create a lower entry barrier, while also ensuring an effective learning curve. the other was to create a game structure that would enable players to hone the skills needed to perform with high effect in the scientific levels, hopefully turning some into heroes. we started by operationalizing the main physics concepts applied in the game and their equivalent in-game operations into a set of core skills that players would need to acquire: deceleration of the atom speed, tunneling the atom into the target state and stabilization of the atom state. these became our guiding references for the structural redesign of both the tutorial and the advanced levels. firstly, we reorganized the tutorial into a set of 7 games, which gradually introduced the main physics elements and core game loop: the atom as a ball, continuing with the atom as a wave, and finishing with the insertion of a static obstacle. to ensure that players had appropriate visual scaffolding and understood the goals of each challenge, we added video animations preceding each level, presenting one possible trajectory along with written hints. the successful completion of the last tutorial level allows players to access the main menu, with the option of playing in separate skill labs: cool, tunneling and control. we decided to create predetermined paths, presented in a tree structure (see figure a.7) . by doing so, we aimed to push players to go through the skill training levels before the scientific levels where the main contributions to our current citizen science problems are made. since individual levels differ in difficulty and require refinement of particular in-game operations, each skill lab was divided into a bachelor and master section. the user has to successfully complete 4-6 levels to acquire skill badges on their profile, which would unlock specific new levels. once the bachelor level was completed, players would gain partial access to both the master section and the scientific labs: qcomp and beat ai. to achieve full scientific access, the master's would have to be completed at some point. beyond this, the structural gameplay tree consisted of a few more nodes: created as a more theoretically grounded, finetune contains a set of tools that makes it easy for a player to manually change points on the path of his previously played levels, adjust the timing, stretch or shrink the total time or smooth the path. since the tool functions are not automated, it is ultimately up to the user to decide which parameters are to be changed in order to optimize the path. the user-defined space is open to all players, regardless of skills. it includes construction yard, a sand box type of environment where users can build their own levels with goals and obstacles and playground, a space where users can play each other's' creations. compared to the predefined games, these are not connected to the main tree structure, and can be played immediately after completing the tutorial. therefore, we decided not to set any skills requirements for creating games. furthermore, since the user games created are primarily supposed to be used as community building drivers, they are neither checked for resolvability, nor included in any of our data analysis processes. based on the number of plays, the user-created games are ranked on the "the most popular games in the community" leaderboard. the last and newest branch in early 2014 combined predefined user-created games in challenges with the intention to enhance competition and the community feeling. for instance the newest game type is called "quantum quests". here we catered to achievement oriented players (heeter, lee, medler, & magerko, 2011; sherry & lucas, 2006) , who crave more traditionally "gamy" elements such as finite super mario-like lives. building on the iron man type of game principles, this game consisted of a series of selected games, where a player has to progress as much as possible through the level with only a limited number of attempts, called "lives". it is currently being used as a base for creating more immersive 3d levels using the unity game development platform. to create a sense of progress in quantum moves with minimal effort, we implemented the simple points-badges-leaderboards (pbl) mechanic recurrent in casual games like candy crush saga and shallow marketing gamification. in quantum moves, the score is calculated on the basis of various parameters, such as time penalty, overlap with target state, points collected while avoiding the obstacles, and is presented to players in the end screen triggered by the finalization of a game sequence. based on the obtained score, a player would move up or down on the leaderboard, presented in the lower right corner of the game window (e.g. fig. a4 ). to mark a player's progress, his score on the leaderboard is shown in relation to similar scores in his range. this is similar to the techniques found on social games, where the scores of a player are presented in relation to others in his/her social network. we made use of two feedback techniques found in gamified applications (groh, 2012) and mobile games to introduce visual cues that could enhance a player' sense of progression (deterding, 2012) . the first is a bar presented at the top of the game interface, which provides players with real time feedback on their performance. the bar changes its color from red to yellow and finally to green to suggest the effectiveness of a player's trajectory. mirroring the wellknown concept of stars found in games like candy crush saga, we also introduced "atoms" as an alternative three-tier grading system reflecting acceptable, good and excellent performance thresholds for each levels, indirectly challenging players to revisit levels and perfecting their collection. the potential downside is that once a game is completed with three stars, players might lose motivation to further optimize his/her score. the last technique to indicate progress is by acknowledging specific achievements in the form of badges on the player profile (antin and churchill, 2011, denny, 2013) . we designed a series of possible badges for two types of achievement: performance and engagement. depending on performance at various junctions of the game, and cumulative time spent logging and interacting with the game universe, a player could receive a recognition for reaching a specific milestone, "bachelor degree" or a particular threshold of play counts like "quantum frenzy 350". to test the effectiveness of the pbl framework and constrained paths of play on player engagement, we also set up a randomized a/b-test surrounding a major event in august 2013, where around 500 people would attend talks about quantum moves and the quantum computer. the literature on gamification is rife with pundits clamoring about the efficacy of badges in motivational design, but there is not much quantitative data to back them up, so we jumped on the opportunity to test whether giving badges to players at various junctions would be a significant motivator for repeated visits and engaged gameplay. at the time, we needed to figure out how to best guide players on their path to the scientific levels, without creating barriers to progress and the feeling of competence. we had conflicting hypotheses about how to build this into structural gameplay: on one hand, the central psychological usability (norman, 2002) and choice architecture (thaler & sunstein, 2009 ) principle of guiding constraints recommends that a system should have a certain amount of openness, but the basic interface for core operations should be limited to the most preferable options. locking levels until the necessary skills are accumulated fits this view. on the other hand, we want players to follow their curiosity, maximizing autonomy, motivation, and access to the science levels. in this view, the structural gameplay would not need stringent locks based on skills, but just the tree-like lab structure laid out as an open map, with friendly hints about where to go, if a level proves too difficult. to this end, an "a/b test" was conceived as a 2x2 factorial design, randomly assigning the 500 expected new players to one of four conditions: locked levels or open levels with or without badges. unfortunately, the test was crippled by both time constraints and technical difficulties. our programmers were working overnight to implement the system to automate a/b-test cell assignment, but also had a lot of more pressing design issues to address before launch. in the end, only a fraction of the badges we had designed were implemented, and players only got weak cues when achieving them. since we did not have time to test it, the skill system unlocking levels seemed also somewhat counterintuitive. some old server issues also came back to haunt us on the day, so many invitees were probably unable to log on to the game after creating their account. with these limitations, the usable data were only a fraction of the people assigned to each condition (down to n=1 player in the "open levels with no badges" cell). the results were statistically insignificant, and will not be reported below. we are awaiting new opportunities to gather this kind of data, which we believe is central to both our own work and adding a much-needed controlled evidence-base to the academic discussion of gamification in general. as these game design choices were being implemented, we collected data on user acquisition, play trajectories, and the few dedicated players we call heroes from beta launch in 2013 to writing this paper in april 2014. the results are reported in the next section. between the10 th of november 2013 and 10 th of april 2014 2 , there were approximately 6000 users visiting the www.scienceathome.org website, 56.6 % of these being new unique visitors. average visits last 5 minutes, where users visit an average of 11 pages on the website. ip addresses came from at least 47 countries (see figure 3 ); yet, the country tally could be even higher, as we could not place a country of origin to approximately 1000 users. at this point, most registrations originate from denmark, followed then by germany and the united states. in the following analysis we present results derived from data collected over a period of 10 months, from the beta version launched on 18 th of june 2013 until 10 th of april 2014. the sample relevant for the demographic and human computation data is equal to a cohort of 2511 people. 60% of all players actually finish the tutorial (see figure 4) , which is an encouraging number. figure 4 illustrates early drop-off, as people move from superficial involvement to commitment via the 7 mandatory tutorial levels. these data were collected from all users registered between 15 th of july 2013 to 10 th of march 2014, which is equivalent to a total sample of 1190 players using the newest version of the tutorial. as it can be noticed on figure 4 , the drop-off mainly happens between play during the first six levels (m=98%, 95% ci[97%,99%]). however, the 7th tutorial level display a large discrepancy between the numbers of tried versus completed levels. a one-way anova was performed, to see if the 7th tutorial level (m=81%) belongs to a different normal distribution of completion ratios than the first six levels (f(2, 4) = 127.12, p < 0.001**). this indicates that the 7th level has a significantly lower completion ratio than the first six tutorial levels, suggesting that the 7th tutorial level, which is the first level that includes the zones of death, which the atom is not allowed to touch, and which constitutes a significant barrier to an otherwise fluid flow of progress, most likely proving too difficult or disrupting competence motivation. an alternative explanation could, however, be that noncommitted visitors simply realize that they have now seen all the game has to offer, and either stop because their flanêurs' curiosity has been satisfied, or because they are not sufficiently attracted by the gameplay to engage further. as detailed at the top of section 4, during the beta period we used four strategies to recruit players. the classifications online/media and voluntary by talks were based on all registration in a ±3 days' time span before and after a registered major online or in-world event. the 3 days before the event was included to account for the users that wanted to checkout the game in anticipation of the event. to avoid overlap with online/media, we attributed the voluntary by talks tag only to the users registered in the same country as where the event took place. the classification of forced by talks was given by a tag added at the point of registration. the results of these recruitment efforts are summarized in figure 5 , which displays increases in unique users attributable to one of the 4 main groups. a large percentage of our registered beta users originate in the online/media group. the steep initial rise of this curve can be attributed to national media coverage of the game launch in june 2013, while the stepwise increases beginning around day 140 is due to ongoing publicity. from mid-september 2013, we increased our communication efforts by setting up a content strategy for the blog and forum on scienceathome.org, where players ask and answer questions related to the game, report bugs or request new game features. these efforts were complemented by a social media presence, with the launch of a facebook page and twitter account, and an increased focus on producing video content for vimeo. also, more attention was directed towards a proactive public relations approach, in order to ensure the recognition of the game on established human computation websites and blogs like scistarter and citizen science centre. following this, we noticed an increase in the number of other sources mentioning the game or giving positive reviews referring back to our website. to analyze if any of our data could indicate a predictor for the number of play counts (see figure 6 ), a one-way anova test for differences in the number of play counts generated per active day for each player was performed between the four registration origin groups (see table 1 ). this showed a significant between-groups difference (f(3,116)=23.93, p=<0.001**). in order to discover which groups are different from each other a tukey-kramer post-hoc comparison was performed and it shows that both forced by talks and voluntary by talks differed significantly from online/media (p=0.04* and p<0.001** respectively), unknown (p=0.002** and p=0.002** respectively) and each other (p<0.001**). online/media and unknown did not differ significantly from each other (p=0.28) suggesting that these two large player groups were qualitatively similar, revealing a pattern where people who actually went out the door in-world on their own accord are highly motivated to play, compared to those who found their way to us online, and especially the poor souls forced by teachers who only played a little. a similar procedure was conducted for physics interest, years of physics education beyond 9 th grade, and finally between male and female (all summarized in table 1 ). the one-way anova shows significant differences for both physics interest (f(2,873)=11.45, p<0.001**), gender (f(1,582)=40.37, p<0.001**) and physics education (f(2,873)=10.87, p<0.001**). tukey-kramer post-hoc comparisons show that high interest in physics leads to significantly more play that low and middle interest (both p<0.001**), while the latter two do not differ significantly (p=0.762). for physics education beyond the 9 th grade the post-hoc comparisons show that the very large group with 0-3 years of schooling played much more than those with 4-6 years (p=0.006**) and 7-10 years (p<0.001**) of additional schooling behind them, which did not differ significantly from each other (p=0.31). the game framing of our citizen science project thus seems to speak to people with a notable casual interest in physics, but not a too professional background. we were also interested in who performed well after having completed the tutorial levels, so we calculated the average score on the tutorial levels (the most stable figure, since players may have chosen many different paths after the 7 th tutorial level) calculated for the four registration groups, interest, physics education beyond 9 th grade, and gender (see table 2 a one-way anova reveals that the registration groups differed significantly on average score reached on the tutorial levels (f(3,819)=6.71, p<0.001**). the tukey-kramer post-hoc comparisons show that forced by talks differed significantly from online/media (p<0.001**) and unknown (p=0.008**), but not from voluntary by talks (p= 0.178). voluntary by talks surprising differ from online/media (p=0.035*) but not unknown (p=0.132), which do not differ from each other (p=0.391). interestingly, the forced by talks group performed best, perhaps because they received better in-world instructions than those tackling the tutorial only with in-game cues as scaffolding. but it is also worth noticing, that these players forced by extrinsic means were much less likely to complete the tutorial levels the first place, so the population registered here is likely to reflect only the most tenacious and intrinsically motivated members of the cohort, who might also have engaged fully in the game on their own time. no significant effect on tutorial scores was found for interest (f(2,671)=0.76, p=0.469), nor education f(2,671)=0.99, p=0.370), but there was a significant difference between the genders, with females significantly outperforming their male competition (f(1,672) =5.55, p=0.019**). to assess how quantum moves retained players recruited by the four different means, we calculated the number of active days, defined as a day with at least one play count, for each user. a score of 6 can thus mean someone who plays intensely for 6 days on end never to return, or someone who returns a day per months for half a year. a look at the drop-off curves in figure 7 indicates that 99% of the users drop out within 7 days and never return to the game. a one-way anova comparing the drop-off rates did not show a significant difference between the four registration groups. however, players that stayed for more than 7 active days were all recruited at public events, and subsequently signed up voluntarily. these 19 players are the ones we refer to in our paper as "heroes". sadly, demographic data is available only for 14 of them. even though the sample is small, we present a comparative analysis of the heroes versus more casual players in quantum moves. in figure 8 , it can be noticed the results derived are based on a sample of 14 "heroes" and 1885 casual users. a considerable part of our work has converged on heroes: that is, identifying players who significantly contribute in the science levels based on existing personal attributes like talent, interests and cognitive surplus, and in turn designing structural gameplay to help more casual players cross the uct through combination of motivation and fluid skill acquisition. a wilcoxon rank sum test for equal medians was conducted to compare heroes to more casual participants interest in physics (reported on a likert scale ranging 1-5) (hero mean=4.214 sd=1.311; casual mean=3.907 sd=1.072) and years of education related to physics (e.g. high school science) (hero mean= 4 years, sd=2.418; casual player mean=4.188 years sd= 2.848), revealing no significant predictive power for who becomes a hero on either of these. firmly believing in the power of mixed methods triangulation, however, we also have some interesting qualitative data to illuminate this. out of the 14 identified heroes, we had direct contact with three, namely sus, shb and meilby. for privacy reasons, we will refer to them by their quantum moves user name. due to their early contributions, sus and shb were invited as special guests to an offline community event and lab visit on the 6 th of november 2013, where they gave brief interviews to help us understand player motivations and promote quantum moves. the semi-structured chat was based on 5 questions related predominantly to their motivation to play quantum moves and their preferred game elements. contact to meilby was established via email. his response was among those we received as a reply to the email sent out after the beta version launch in june 2013. in this email, the questions were formulated around the strategies top players used to obtain above average scores in particular games, as well as to give their impressions with regard to the game structure and the newly launched features. sus (f, 40 years old) was working as an accountant, based in copenhagen, denmark, when we met her. she signed up as a player in quantum moves after a public talk on the topic of information security and quantum computers, held by jacob sherson at the experimentarium on 27 -28 august 2013. since that date, sus had 120 active days and 47570 play counts. this is noteworthy as, according to her own testimony, sus had never played a computer game until she signed up for quantum moves. when we asked sus what motivates her to keep playing the game, she mentioned that what motivated her to play quantum moves was the "knowledge that the results will be used for real, scientific purposes" (recorded interview with sus, 6 th of nov 2013). sus' case helps us to understand, that quantum moves does not necessarily acquire its heroes from a traditional gamer demographic (although women 30+ are the quickest expanding gamer group, software entertainment association (esa), 2013), and should take this into account for both recruitment, structural and core game design, and framing purposes. the drive to help science by playing a computer game was also what shb (f, 17) mentioned as main motivation to sign up for quantum moves. shb is a danish high school student and member of the unf, a youth organization which aims to attract and develop young talents into natural sciences. just as sus, shb signed up as a player after the public talk at the experimentarium on 27 -28 august 2013. she has had 19 active days and 5441 play counts. when asked what motivates her to keep playing quantum moves, she answered "it is fun to play, among others because i know (that by doing so) i help science" (danish-english translation of an excerpt from recorded interview with shb, 6 th of nov. 2013). shb's case suggests that in-world membership in scientific communities of interest may provide a fertile ground for future recruitment. meilby joined the quantum moves community early and was part of testing several previous editions of the game before the beta version launch in august 2013. in the selected time span, he had a total number of 17 active days and a 2656 cumulative level plays. meilby drives a taxi and has no physics education beyond the standard level obtained by attending high school. his case becomes interesting because he reported being able to replicate, through several iterations, the correct sequences of actions needed to perform an operation known in physics as "quantum tunneling" based on theoretical reasoning rather than trial and error (for full description, see appendix 9.1). based on his formal education, he would not have the sufficient knowledge to analytically translate the physics concepts used in the game. meilby thus exemplifies the contrast between several hypotheses we have about how a hero manages to move to the top of the leaderboards: through explicit theoretical knowledge about quantum principles (as expected by the physicists in our group, who advocate focusing on explicit semantic knowledge in player skill acquisition), through implicit familiarity and complex processes of predictive coding in the core handeye coordination used to play (friston, 2012; scott & dienes, 2008) , or some combination of these higher-and lower-order cognitive processes, unique to the human mind/brain/body complex, allowing human players to explore, tinker and strategize their way through difficult and counterintuitive physics problems via more or less conscious trajectories that no computer algorithm would ever fathom. in this paper, we have tried to open up our design process for quantum moves, and reported significant findings about factors predicting play activity and performance, ranging from gender to recruitment. notably, we have devoted time to understand the most tenacious and talented kind of players, who we label heroes. quantum moves gets most of its participants online, but our beta data reveal a much more interesting pattern: people who were very interested in physics but had little formal background in the field, and who actually at some point went out the door to meet us, played much more tenaciously than those who clicked their way to www.scienceathome.org online -or were forced by extrinsic means. thus, genuinely interested amateurs reached in-world seem to be the most fertile ground for recruiting intrinsically motivated citizen cyberscientists to games like quantum moves. having attended talks with real physicists also seems to help people achieve better scores once they start playing. for citizen science, the relationship between in-world and in-game motivation is nontrivial. as for the actual human computation, the first year of quantum moves has been devoted to "calibrating our users" to solve various challenges (first scientific results will be published elsewhere shortly). we purposely abstained from helping the users beyond teaching them the basic game mechanics, even when we knew of efficient solutions to a problem. this choice provided an unbiased ensemble of solutions, which has two advantages: first it contains solutions we would never have thought of, and second, it provides a measure of what the users can realistically contribute. we use this to compare user learning curves with computer algorithms as they both found solutions for the same level. the score of a user after each try is compared with the score the computer would have after the same number of iterations. this has shown that users are fast at obtaining a good score, but they will never obtain the perfect score, whereas the computer algorithms need a lot more iterations to obtain a good score, but they are capable of finding the solution with a level of precision which yields the perfect score that we need. however, for a level like bring home water fast the users try out solution-patterns which the computer algorithms would never find by themselves, and they discover solutions which are faster than the computer algorithms' more linear computational trajectory. getting to know our player-base and especially the heroes reveals that citizen cyberscience games like quantum moves cater to a nontraditional demographic compared to both science and gaming. for instance, female players significantly outscore males after having played the tutorial, even though males play more per day. our data comparing those forced to play via schoolwork to players who came to us via in-world events or online channels also suggest that users are much more inclined to play if the action springs from curiosity or if players are intrinsically motivated to contribute to science, supporting earlier findings by raddick and colleagues (2013) as well as the central tenets of self-determination theory. what is less clear, however, is what game elements help engage and retain players once they have been recruited, and begin to move from superficial to true commitment. while quantum moves is unique compared to other citizen science games in having an engaging and challenging core game loop that by itself lives up to prominent definitions of (casual) games (juul, 2005; salen & zimmerman, 2004) , we also expect that a well-designed structural gameplay (sometimes called metagame) is central to frame, structure and motivate the play experience, both helping and goading players to move from level to level along appropriate learning curves balanced between boredom and anxiety. in the end, we accept that the high level of cognitive complexity in quantum moves compared to citizen science games that rely on players as simple pattern-hunters or "mules" carrying data gathering devices into the real world , means that we will lose players at a higher rate due to the difficulty, but we believe that it is a viable strategy to work towards a game that is fun and learnable for everyone: on one hand to capitalize on each player's lifetime network value, and on the other in the hope of helping players hone the three skills central to succeed at the scientific levels, moving them beyond the user contribution threshold. we were vague about the difference between a hero and a player who crosses the uct. this is because we view hero-status as a significant individual property combining sustained engagement and a high level of skill, while crossing the uct can happen to anyone by a fluke or one good gameplay. in this sense, we conceive of our heroes less as loincloth clad conan-types who individually topple empires and more alike the ww1 trench-heroes who made up the bulge with grit, skill, and intrinsic determination, and together make a difference worth reverence. however, even though many players may be truly engaged and come back for several days, we still need deeper analysis of the scientific results to tell how many actually manage to cross the uct by accident and/or by attaining hero status -that is, who actually manages to contribute to the quantum computer, as our beta population has mainly guided us in designing an effective game interface, and test simple hybrid optimization against only specific algorithms. the limits to the current study are manifold, as we have compiled this contribution as an open midstream report rather than waiting for the best possible data years into the future. centrally, the n of active players, and especially heroes, is limited, and mainly stem from a danish context. as such, new patterns are likely to emerge when we establish an even wider international profile. also, the many anovas run are almost certain to show some kind of statistically significant results, even though they may not be practically significant. mancovas would have been preferable, but since the population for each test differed slightly, this is the right kind of statistical test available at this point. further analysis and data gathering is clearly needed. in this instance, our a/b test was unable to show a statistically significant role for challenging constraints, openness to explore or the infamous. the lesson learned is, that large-scale tests should only be conducted after a satisfactory alpha-test of the gameplay in each cell, and ideally supported by using qualitative measures of player engagement and interaction-patterns to allow triangulation of causal mechanisms shaping gameplay trajectories. the games industry might not have time for these kind of detailed studies with most casual games being developed by small expert teams in less than 6 months, but as scientists, we should. so much the wiser, we still hope to one day lead the citizen cyberscience games community in methodologically sound testing of design hypotheses about recruitment, engagement and different kinds of motivation. the relationship between curiosity on one hand, and intrinsic versus extrinsic motivation on the other, goes some way toward explaining the steep and permanent drop-off in player engagement as they make their way through the tutorial, and is especially seen after quantum moves teaching and recruitment events, where people may have been forced to register and thus driven primarily by external factors. a steep drop-off curve is in no way unusual for online games (fields, 2014) , so a full 60% tutorial playthrough suggests that the levels encountered early in the behavior chain actually keep players interested for a good while. future design perspectives include building better just-intime feedback into the core game loop, making it more 'juicy' and helping players understand exactly what is going well or wrong at any moment. after a year of stalling due to programming pool limitations, we also finally have the capacity to integrate the game with social media such as facebook, which will add an entirely new social dimension to both gameplay and recruitment. finally, we have yet to narratively structure the structural gameplay and implement truly juicy graphics. as our community manager noted, we do not need to become the next candy crush saga in terms of juiciness and engagement -just the candy crush saga of physics. we are now in a position to deploy more advanced psychological methods in mapping player trajectories and trying to predict who might be born heroes and who can become ones, which should significantly inform our future design of the structural gameplay. our centre's unique crossdisciplinary nature allows us access to eye-tracking and other tools that will soon help us test interaction design, and conflicting hypotheses about the usefulness of explicit understanding of the physics issues (as expressed my meilby) versus implicit learning of the hand-eye coordination and establishment of predictive coding (bubic, von cramon, & schubotz, 2010; friston, 2012) to account for the counterintuitive movement of the 'liquid' wave. as another exiting step we will soon be able to report the first comprehensive psychometrics battery collated especially for citizen science gaming, in collaboration with other major players. the revolutionary bit will, however, not be the our data collection, but the analysis of complex relationships between hosts of in-game and in-world variables via our next game: a title both for teaching statistics and engaging our community in analyzing data with advanced techniques like path analysis, that require intelligent human causal propositions and critical evaluations of other user's solutions -something no algorithm can ever do. human optimization is superior to the computer in many instances, but we have yet to understand the exact cognitive nature of these solutions as they apply to the varying problem spaces in quantum moves. this, along with more work on motivation, will be the subject of extensive future research. contrary to commercial casual games, we cannot change or abandon our core game loop, which represents the real quantum optimization processes needed to operate a scalable quantum computer. we can, however, change the look, feel, usability, and structural gameplay surrounding our game to frame, structure and motivate more engaging play trajectories, and ensure a sufficient learning curve to help people cross our high science contribution threshold. we have opened up that messy and experimental design process in this inaugural issue, hoping that others will be able to learn from our experiences. we encourage other researchers to publish future human computation pieces along the same lines. the present study would not have been possible without all the citizen scientists and casual players on http://www.scienceathome.org/. this is your research as much as ours. "merge merge was a pain (…) and since i did not really figured out how to get a single atom to the excited state i gave up kind of fast. i got the best score i think it was 3,4, and i was a little mad about being so bad at the challenge because the score went to 692 as max, and 3.4 is just not even near it. well i had some time off, and i tried again a, or some days after. with no luck. then i said to myself, since i was not doing any progress, and did not know how is should get the first atom to the excited state, i started doing the excited challenge again, and found the only way i could do it. since the background was with lines i started using them, i started raising the curve with the first atom to just above the second atom (according to the lines) and figured i was letting then merge too fast because the first atom was what i would say too excited, and not going to relax. then i had to be patient, when they were merging, and with some patience letting it flow in itself when they got near each other. the first times it did not work out, instead of the 1 st atom splitting into 2 it spilt into 3, and i knew i had it i just needed more tries. then it happened after some cursing it split into two, and i got a score 16 points, which i was chocked about, i was like wtf, i just did it, and i am getting nothing! i think it is too hard to do, and the fact that you cant do it fast is giving me stress, the fact i have to wait all 20 seconds every time to make it happen is a reason why it's not so funny to do. (i guess i played too much, and hate the fact i can't get it done right, pisses me off j) then i started finetune my score 16 point, i knew already it was done right because it spilt into 2 and the 2 nd atom was really relaxed. it took me some time to be familiar with the finetune part, but after spending some time in there, i finally got something, by zoom and keep moving small parts and watching the score i got to 460 points. i used the smooth tool early on, because it was flickering the line in the mittle graph, and after i had dragged the line around and around, it started to flicker (jeg mener linjen bliver zig zagget), i believe it is not good for the score, but i'm not able to remove it, i tried the locking tool to lock the line around the part and the use the smooth tool on the not locked line but it fucked it all up. i've added some pictures to show you what happened." badges in social media: a social psychological perspective a theory of human curiosity novelty, complexity, and hedonic value chaos, cognition and the disordered brain gamifying citizen science : lessons and future directions volunteers engagement profiles in volunteer thinking systems prediction, cognition and the brain the psychology of self-determination a meta-analytic review of experiments examining the effects of extrinsic rewards on intrinsic motivation self-determination theory: a macrotheory of human motivation, development, and health the effect of virtual achievements on student engagement gamification: designing for motivation a universe of consciousness characteristics of games mobile & social game design: monetization methods and mechanics mobile persuasion: 20 perspectives on the future of behavior change predictive coding, precision and synchrony clarifying achievement goals and their impact gamification: state of the art definition and utilization beyond player types: gaming achievement goal what can breakdowns and breakthroughs tell us about learning and involvement experienced during game-play? making sense of game-play : how can we examine learning and involvement ? why people attend science festivals: interests, motivations and self-reported benefits of public engagement with research half-real: video games between real rules and fictional worlds psychological conditions of personal engagement and disengagement at work thinking, fast and slow the token economy: a decade later employee engagement : a literature review rna design rules from a massive open laboratory deep and shallow gamification: the thin evidence for effects in marketing and forgotten powers of good games engaging consumers through branded entertainment and convergent media mixed methods in games research -playing on strengths and countering weaknesses toward a theory of intrinsically motivating instruction* making learning fun -a taxonomy of intrinsic motivations aptitude, learning, and instruction gamification -a simple introduction. tips, advice and thoughts on gamification the design of everyday things volunteers' engagement in human computation astronomy projects gaming for (citizen) science: exploring motivation and data quality in the context of crowdsourced science through the design and evaluation of a social-computational. e-science workshops (esciencew) galaxy zoo: motivations of citizen scientists glued to games -how video games draw us in and hold us spellbound the motivational pull of video games: a self-determination theory approach rules of play: game design fundamentals. leonardo the conscious, the unconscious, and familiarity video game uses and gratifications as predictors of use and game preference playin computer fames: motives, responses and consequences the free and happy student creating a model to examine motivation for sustained engagement in online communities. education and information technologies 2013 essential facts about the computer and video game industry in-game , in-room , in-world : reconnecting video game play to the rest of kids ' lives nudge: improving decisions about health, wealth, and happiness ).scienceathome, home key: cord-344656-xx76w7c0 authors: sarder, md title: logistics customer services date: 2020-10-16 journal: logistics transportation systems doi: 10.1016/b978-0-12-815974-3.00008-3 sha: doc_id: 344656 cord_uid: xx76w7c0 this chapter discusses customer service in logistics in terms of different elements, the relative importance of those elements, and how these elements impact the effectiveness of logistics operations. it also explains the sales–service relation model and how to measure service level. other topics include order cycle time, how to determine optimal service levels, and acceptable service variation in logistics. such as transport planning, scheduling, and modal selection. there are also strategies involving location analysis and the networking planning. all these strategies are critical for an effective logistics customer service ( fig. 8.1 ). logistics planners need to focus on certain approaches and and features to ensure a good customer service experience. such approaches include building up a strategic process to provide highly valued services to the customers, on-time deliveries, ensuring trade-off between costs and services, maintaining a harmonious relationship among all supply chain partners, continuously improving customer loyalty, and customer satisfaction as well as bringing the competitive environment in the market (fig. 8.2 ). logistics customer service is a part of a firm's overall customer service offering, customer service elements that are specific to logistics operations including fulfillment, speed, quality, and cost. the term fulfillment process has been described as the entire process of filling the customer's order. the process includes the receipt of the order, managing the payment, picking and packing the goods, shipping the package, delivering the package, providing customer service for the end-user, and handling the possible return of the goods. the term "customer service" needs clear explanation in order to relate with logistics. for example, manufacturers' first concern always is with how efficiently the cargo reaches its destination without any delay or any sort of complication. this is important because of the reputation of the company, which solely depends on customer perception. businesses flourish based on the manufacturer's capability of meeting these customer expectations. one approach to maintaining good logistical support and cutting costs is to concentrate on communication solutions such as tracking shipment, status update, and accommodating last minute change request. with the advancement of technology, many services are available to the customer by limiting confusion, ambiguity, and inefficiency. as a result, these services such as shipment tracking helps not only pushes away unnecessary expenses out of the manufacturer's existing operational exercises, but also increase the overall customer experience and helps improve financial aspects. some technology driven service goals are described as follows: g automate timing/location updates, rate quotes, pick-up scheduling, current transit times, or proof of delivery with interactive voice response (ivr) self-service. g provide inquiries about updates regarding service and measures the needs of service calls within the system. g generate and deliver notifications, such as weather alerts, changes in schedules, and more with campaign management tools to alert the respective personnel. g provide security of overall customer information and payment transactions and minimize fraud. g empower customers by providing information regarding the purchased products so that they can express and communicate better their expectations. g identify and predict customer interest to make every smooth interaction between the customer service provider and the customer. continuously enhance policies and approaches through gathered customer feedback data and analyze and make reports for executing better business strategies. g ensure customer reliability and a consistent experience for clients by avoiding unnecessary costs and improving workforce development. logistics planners must understand all logistics services offered by the firm so that they can articulate the benefits to the customer. if articulate properly, customer service could add significant value to create demand for the products and improve customer loyalty. customer service starts with order entry of the product from the inventory to the transport of the final product to the desired destination. well-organized customer service logistics focuses on providing technical support as well as required equipment service maintenance. as mentioned earlier that customer satisfaction depends on the speed and efficiency of ensuring the availability of the product ordered and delivered. the following sections describe the different elements of customer service. customer service has several integral parts, which are interconnected with each other, such as price, product quality, and speed of service. for instance, the price goes up with higher speed of service and vice versa. there are four valuable marketing mixes such as product, price, promotion, and place, which are combinedly elaborated as four ps. the "place" is associated with physical distribution, which means it involves customer service. a study on customer service by the national council of physical distribution management identified these elements of customer service according to when the transaction between the supplier and customer take place. these elements are categorized as pretransaction, transection and posttransaction. according to lalonde and zinszer, there are three elements to customer service. the first one is the pretransaction element. this element establishes the business relationship climate. ideally, all terms of customer service policy are identified prior to shipment of goods that establishes an expected level of customer service in the transaction. the pretransaction element consists of returns policies, expected delivery time, and contingency plans for problems that may occur during shipment. the expectations are established during the pretransection stage, but it is important for companies to adhere to established policies. the second element of customer service occurs during the transaction stage. this element is very simple. companies must deliver the right product to the correct location in the prescribed delivery time. also, the product received must be in good condition. lalonde and zinszer identified the third element of customer service as posttransaction activities. these are the services provided to customers following receiving their goods. these activities must be planned in the pretransaction and transaction stages (ballou, 2004) . these elements are shown graphically in fig. 8 .3. in the corporate business climate, all these elements are considered individual components of the larger overall customer service. there have been several studies, such as the works of innis and lalonde or sterlingàlambert, which indicate that while all these individual elements do make up the overall customer service, some elements are considered more important than others. innis and lalonde concluded that as much as 60% of desirable customer service attributes can be directly attributed to logistics (innis & lalonde, 1994) . these include fill rates, frequency of delivery, and supply chain visibility (innis & lalonde, 1994) . researchers have consistently discovered that customer service is highly dependent on logistics. fig. 8 .3 summarizes the most important customer service elements as on-time delivery, order fill rate, product condition, and accurate documentation. pretransaction elements of customer service mean to establish a climate for good customer service. which is basically a nonroutine activity. this element of services deals with the service level and related activities in qualitative and quantitative terms. pretransaction elements provide the roadmap to the operating personnel regarding the tactical and operational aspects of customer service activities of the company. for the reverse logistics process, this phase is essential because it helps to shape the firm to focus on customer such way to create influence the perception of the firm into the customer's mind. transaction elements include everything between a order is received and delivered to the customer. during the transaction phase of customer service, a firm focusses on retrieving, packing, and delivering the order to the customer in a timely and cost effective manner. this phase also includes scheduling of shipment, communication with the customer, delivery tracking, and delivery confirmation. this phase represents the array of services needed to support the product in the field; to protect consumers from defective products; to provide for the return of packages; and to handle claims, complaints, and returns. corporate customer service is the sum of all these elements because customers react to the overall experience. according to studies of sterling and lambert, most of the industries show that buyers, customers, and influencers of purchases of related industries mainly focus on variables including product, price, promotion, physical distribution, and speed of delivery among others. sterling and lambert clearly showed in their research that logistics customer service is the critical factor for the office systems as well as plastic and furniture factories. factors such as high fill rate, frequent delivery, detailed inventory visibility, estimated shipping date, and expected delivery time from the time of order placement and order received are very important to the retail customers. in the surveys of purchasing and distributing suppliers, presented by shycon associates, there are several common service failures including late delivery, faulty products, damaged goods, and discontinued products. late delivery is the most critical issue, as it represents 44% of the entire customer complaints. again, faulty products fall around one-third of the total complaints. fig. 8 .4 shows some of the most common customer service complaints noted by industrial surveys. the following are considered the most important logistics customer service elements: in logistics, it is said that nothing happens until somebody orders something. "order cycle time is defined as the elapsed time between when a customer order, purchase order, or service request is placed and when the product or service is received by the customer" (ballou 2004) . logisticians can affect the overall customer service level through efficient management of operations. the cycle time of each order must be carefully monitored to properly judge the efficiency within each cycle. therefore order cycle time is considered all the processes that must occur prior to the customer receiving their product or service. total order cycle time includes order transmittal time, order processing and receiving time, stock acquisition time, and delivery time. order processing and receiving time includes the bill of lading preparation, credit clearance, and order assembly times. however, the delivery time has three basic components: shipping time from the plant, shipping time from the warehouse, and customer shipment process. fig. 8 .5 shows the various components of a typical customer order cycle. depending on the system used for communicating orders, the transmittal time varies. the transmittal time includes transferring the order request from the origin to the entry of the order for further processing. order entry may be handled manually such as physically carrying the order or electronically via toll-free number, satellite communication or via the internet. the manual processing is slow but inexpensive, while the electronic methods are most reliable, accurate and fast but expensive. the next important element of an order cycle is the steps required for order processing and order assembly. these processes are involved steps like send notifications to the buyer/supplier, updating inventory records, preparing and scheduling shipping details for delivery, and communicating with customers as priorities can affect or change the speed of order processing for delivery. to some extent, order processing and assembly occurs concurrently to save time for both of these operations. unavailability of stock has a significant negative effect on total order cycle time, as it takes searching for the stock items, reconciling missing items, and delays in order assembly. the final primary element in the order cycle over which the logistician has direct control is the delivery time, the time required to move the order from the stocking point to the customer location. order cycle time can be adjusted for various reasons including the changes in customer needs, order priorities, shipping capacities, promotions, among others. a customer may chose to change the order delivery time by paying for an expedited service anytime after placing the order. it is normally assumed that the elements of the order cycle have remain unaffacted, but customer service policies and disruptions may distort the normal order cycle time patterns. such as priorities of order processing, condition of the order, size of the order, natural disaster, etc. priorities of order processing are determined by factors including delivery time and window, premimums paid by the customers, urgency of ontime delivery, consequence of late delivery, customer reputation, and many others. when backlogs in the order cycle occur, it is required to distinguish orders from each other. an individual customer may vary greatly from the company standard, depending on the priority rules, or lack of them, that have been established for processing incoming orders. typical order cycle time may change significantly for the goods delivered in their destinations as damaged or unusable. in that situation order cycle time significantly increase as reorder, replacement, or repair has to happen. depending on the factors for setting standards for the packaged goods including design, returning and replacing processes if needed for the incorrect, damaged goods, the cycle of order time may vary. also, there are specific standards established in any business to monitor the quality of order and check the average order time and keep it steady. order constraints are preset expectations or requirements that prevent flexibility in order processing and delivery. due to the order constraints, the cost of order processing and delivery can increase. the example of order constraints includes minimum order size, fixed days for receiving order, maintained specifications for order, etc. order constraints also help with the order planning as the restrictions are known ahead of time. according to the logistic planners, presetting the delivery schedule, order conditions, packaging, etc. help the business to impose a organized processing of order and improve the delivery to the customer on time in a great extent. presetting specifications also help low volume markets serve reliable and efficiently in a continuous manner. customer service is extremely important in the logistics world because of the highly synchronized and detailed planning and execution that is required when operating on a global scale. multiple factors are critical in delivering high levels of customer service and they include high rates of order fulfillment, speed and frequency of delivery, inventory visibility, on-time delivery, condition of product on delivery, and accurate documentation on po's and bill of ladings. it is a multi-faceted concept of gaining and maintaining differentiation in the market-place. the customer service must meet the needs of different customers. 'perfect order' should form the basis for measuring service performance and to develop new service standards. logistics management plays a vital role in enhancing the customer lifetime value by increasing customer satisfaction and enhanced customer retention. in any business, especially in the transportation business, good customer service is a top priority. this is because customer satisfaction helps the business survive and grow simultaneously. in any sort of logistics operation, providing good customer service for example, monitoring shipments periodically from the warehouse until destination and notifying customers if their orders are facing delay for any circumstances will elevate customer satisfaction. monitoring deliveries at every point and communicating with respective personnel in need and sending notifications to the customer to brief them regarding the issue and arranging adjustments increases the customer's loyalty and thus sets the business in a unique position compared to other competitors in the market. poor customer service will drive customers away from the brand. customers usually shares with others regarding product quality. if the product is good and they are satisfied by the customer care service, they recommend the brand to others but if they feel unsatisfied due to low quality or poor service, they tend to alert others, which negatively affects the reputation of the company or brand. a negative reputation could be very hard to erase and tends to degrade the share value of the company. the relationship between customer service and sales is symbiotic. after having a positive experience with a business, most of the customers are actually willing to refer that company to another person. a positive experience in customer service not only help retain customers, but also help with the acquisition of new customers. retained and loyal customers can help increase incremental growth of a business. when comparing, retaining customers costs 4 to 10 times less than the cost of acquiring new customers. it is obvious that low-quality customer service has tremendous side effects in any sort of business. additionally, a business could lose the loyalty of the valued customers and there are risks of losing the best employees because whenever companies have a customer service problem. the best employees are obliged to fill up the slack for other employees, so they search for better opportunities for their talents. an industry survey revealed many penalties of bad customer service and their significance on businesses. for instance, reduction of the business volume contributed to almost one-third of the entire customer service related failures. other penalties include called in manager/salesman, cut-off of all purchases with suppliers, significant number of items discontinued, deny of purchasing new items and refusal to invest in promotion. fig. 8 .5 shows some significant customer service penalties noted from an industry survey. so how can businesses go about fixing bad customer service experiences? it is very critical that business identify the root causes of bad customer service and address them before it is too late. before doing anything, business need to be more informed about the situation and underlying causes. they can connect with the employees and customers involved to identify the problems. once root causes are identified, business need to focus on addressing them applying various methods including training employees, reviewing business practices and strategic partnership, involving high level leadership, fixing the system, and compensating customer losses. in short, there are several ways to fix a bad customer service situation but arguably the best way is to prevent them from happening altogether. make sure the businesses have the right customer support infrastructure and consistently improve their customer experiences. to look at the importance of customer service is through the costs associated with customer retention. logistics customer service plays a critical role in maintaining customer patronage and must be carefully set and consistently provided if customers are to remain loyal to their supplier. on the average it is approximately six times more expensive to develop a new customer than it is to keep a current customer. thus, from a financial point of view, resources invested in customer service activities provide a substantially higher return than resources invested in promotion and other customer development activities. it is not always clear how important logistics customer service is until we understand how logistics decision making would be enhanced if we knew more precisely how sales change with changes in logistics customer service levels. business sales are related to customer experience and customer satisfaction. the exact relationship between sales and customer service varies by industry and specific business. generally, when customer service is poor, sales decline. as services increase above the level offered by the competition, sales gain can be expected as superior customer service increases the retention of existing customers and attract new customers. when a firm's customer service level reaches this threshold (level offered by the competition), further service improvement relative to competition can show good sales stimulation. it is possible that service improvements can be carried too far, resulting in no substantial increase of sales. efficiency in customer service can result from the combined impact of improving the elements of customer service, which has a quantitative effect on sales for a company. this is referred to as the sales-service relationship. there are several theories that conclude that if price and quality are equal a company must offer customer service to approximately the same degree as their competitors in order to maintain competitive advantage in a given market. the service level offering that is offerd by the competition in a market is considered the threshold service level. this threshold service level assumes that a company cannot sustain themselves in any market it they do not offer a base level of customer service greater than or equal to their competitors. once a company has reached the threshold service level, any improvements above the threshold are expected to stimulate sales. these sales can come from new and unexplored markets or customers converted from other companies. this section discusses varios models that formulate the theoritical relationship between sales/revenues and services. usually, better service generates more sales. in some cases, salesàservice relationship for a given product may deviate from the theoretical relationship. following methods for modeling the actual relationship could be used in those specific cases. the two-point method involves establishing two points on the diminishing return portion of the sales-service relationship through straight lines. the method is based on the notion that multiple data points to accurately define the salesàservice curve would be expensive or unrealistic to obtain, and if data were available, it is not usually possible to describe the relationship with a great deal of accuracy. first, set logistics customer service at a high level for a particular product and observing the sales that can be achieved. then the level is reduced to a low level and sales are again noted. these limitations suggest that a careful selection of the situation to which it is to be applied must be made if reasonable results are to be obtained. fig. 8.6 shows how the two-point method is used to correlate sales-service relations by establishing two points and the area covered based on the relationship of product sales and logistic customer service offered. the impact on sales/revenues to a change in service level may be all that is needed to evaluate the effect on costs. the sales-service relationship over a wide range of service choices may be unnecessary and impractical. sales response is determined either by inducing a service level change and monitoring the change in sales. these experiments are easier to implement because the current service level serves as the before data point. before and after experiments of this type are subject to the same methodological problems as the two points method described earlier. one problem in measuring the sales response to service changes is controlling the business environment so that only the effect of the logistics customer service level is measured. one approach is to set up a laboratory simulation, or gaming situation, where the participants make their decisions within a controlled environment. this environment attempts to replicate the elements of demand uncertainty, competition, logistics strategy, and others that are relevant to the situation. game involves decisions about logistics activity levels and hence service levels. by monitoring the overall time period of game playing, extensive data is obtained to generate a sales-service curve. the artificiality of the gaming environment will always lead to questions about the relevance of the results to a particular firm or product situation. predictive value of the gaming process is established through validation procedures. one of the popular methods for gathering customer service information is surveying buyers or other people who influence purchases. mail questionnaires and personal interviews are frequently used because a large sample of information can be obtained at a relatively low cost. survey methods must be used with caution because biases can occur. the questions must be carefully designed so as not to lead the respondents or to bias their answers and yet capture the essence of service that the buyers find important. the finding of survey can be used to model the relationship between the cost and the customer service level. there is a cost associated with providing the logistics customer services. as the level of customer service goes up, the cost associated with providing that service also goes up. for example, a business has to spend more money to improve order fullfillment rate from 90% to 95%. the most critical question for a logistics manager is where they choose to be in relation to cost and customer service levels. as activity levels are increased to meet higher customer service levels, costs increase at an increasing rate. this is a general phenomenon observed in most economic activities as they are forced beyond their point of maximum efficiency. the diminishing returns in the sales-service relationship and the increasing cost-service curve results in a profit curve. the profit contribution curve results from the difference between revenue and costs at various service levels. because there is a point on the profit contribution curve where profit is maximized, it is this ideal service level that is sought in planning the logistics system. customer service level is defined by vrious factors such as percentage of ontime deliveries, percentage of correct orders, fulfillment rate, etc. optimum service level is a target service level where net profit is maximum while providing acceptable customer service. to maximize the net profit, it is imperative to maximize the revenue while minimize the cost at that particular service level fig. 8.7 . identifying the revenue and cost for each service level will provide the logistics professionals a starting point to make this critical decision. revenue, cycle time, shipment cost, handling costs, and inventory costs are some of the factors to determine the optimum service level. each level of service has an associated cost level. when activity levels are figure 8.7 relationship between revenue and logistics customer service. increased to meet higher customer service levels, costs increase at an increasing rate. profit contribution curve results from the difference between revenue and costs at various service levels. the maximum profit point occurs between the extremes of low and high service levels. net profit is the driving force for businesses that provide logistics services. the optimum service level is found when the net profit is maximized. net profit (np) is the difference between the revenue (r) and the costs (c) associated with all logistics services. the relationship can be expressed as np 5 r 2 c. for each level of customer service, a company can realize a specific revenue and cost. the difference between the revenue and cost varies along the service level. although net profit in a logistics business is essential, determining logistics decisions about transportation has many factors and one key factor is quality. a shipment arriving on time in the condition intended is a key factor in customer service. imagine you have ordered for your child a stereo for christmas over the internet. the package is supposed to arrive on december 22, at your home in plenty of time for wrapping and you are pleasantly pleased with the free shipping offered. the package leaves on time and you are tracking it to your home in anticipation. now it is christmas eve and you do not have your package and your unhappiness is growing with every moment. the package arrives on december 27, and looks like it was dropped from the truck on the way. in this situation, your transportation costs expectations were met but your expected service quality was not met. the mix of the two is the ideal spot for customer service and happiness. another factor in the overall customer service level is the amount of variability present in each service provided. "service variability is a characteristic that differentiates services from goods, and it can be defined as changes in performance from one service encounter to another with the same service provider" (mcquitty et al., 2004) . variability in any service implies additional risks and uncertainty. the larger the uncertainty in a supply chain the larger the costs for safety inventories, time in transit, or cost of expedited deliveries. in the case of customer service, variability is generally considered negative to overall customer experience. variability is a powerful term in the logistics customer service arena. the global economy has contributed greatly to the variability in customer service. instead of depending on a local supplier to deliver a component, companies now relies on suppliers from the other side of the planet. due to the global nature of supply chain, service variability is very high. for instance, ocean shipping causes variability due to various factors including shipping schedule changes, international rules and regulations, customs delay, navigation challanges, and port capacity. how much variability can be tolerated by a customer is the million dollar question? customer service in supply chain operations can be quantified by the percentage of products or services that meet delivery due dates, order filling accuracy, stock-out percentage, and several other service variables. genichi taguchi developed a loss function that is critical to managing the supply chain processes that determine customer service levels. "taguchi proposed that inconsistent quality in product and services results in expense, waste, loss of goodwill, and lost opportunity whenever the quality target value is not met exactly" (ballou, 2004) . service levels are viewed to be satisfactory and without any penalty cost as long as variations in service levels remain within the upper and lower limits of the accepted range. fig. 8 .8 graphically represents this loss function. a loss function defines the potential loss of a business due to not fulfilling the target service level. for example, a service was expected or promised to deliver at a certain location, at a certain date and time, at a certain price, at a certain condition. if there is a deviation of service from the expected or promised targets, there is a potential loss for that service provider, taguchi's loss function determined that cost penalty (losses) occur at an increasing rate as the level of service deviates from the target value. the following formula is used to derive the loss function: l 5 kðy2mþ 2 where: l 5 loss per unit ($) y 5 value of variable m 5 target service variable k 5 constant representing the importance of service variable taguchi's loss function allows a value to be placed on not meeting the expected customer service levels within supply chains. in this way, companies are able to quantify the loss associated with poor customer service performance. additionally, this loss function formula can be utilized to optimize service levels by determining the appropriate amount on variability for service levels. example: target delivery time for an autoparts supplier is 2 hours. parts delivered more than 15 minutes late incur a penalty of $5 off the total bill. delivery costs are estimated at $3 but decline at the rate of $0.25 for each minute of deviation from target. how much variation should be allowed in the delivery service? step1: find k l5 kðy2mþ 2 55 kð1520þ 2 k5 5 15 2 5 $0:022=per minute 2 step2: find varðyþif m is taken as 0 y 2 05 0:25 2ð0:022þ 5 5:68 minutes no more than 5.68 minutes should be allowed from the 2-hour delivery target to minimize cost. supply chain visibility in global outsourcing is the visualization of information related to product or service quality and makes it available to all actors in the supply chain network. actors in supply chain network include retailers, 3pl/4pl providers, manufacturers, sub contractors, suppliers, etc. as global outsourcing continues to become complicated, visibility of quality information is rapidly becoming the fundamental building block for outsourcing supply chain networks. information technology advances now make extended visibility across organizations possible. information visibility of orders, plans, supplies, quality specifications of supplies, inventory, and shipments is key to successfully coordinating events across the network and to monitoring analytics that track the health of the network and allow for proactive action. the greatest benefit comes from leveraging visibility information to identify and eliminate root causes of quality problems, and to rapidly respond to ensure the quality of outsourced products and services. this early identification and correction of quality problems in global outsourcing can help companies reduce the consequences of poor quality of products and services. customer service can be a constraint to a logistics system. service levels set by competitors and often traditional service levels can affect the customer service and cost relationship. sensitivity analysis can help aid a logistics operation to determine the factors that constrain the operation. the ideal solution is still the optimum balance between quality and cost; this should be weighed heavily in all analysis of the constraints. assuring quality in logistics operations such as global outsourcing is very challenging due to the multiple layers involved in the supply chain. supply chain layers include worldwide retailers who outsource products or services globally, intermediaries such as 3pl/ 4pl, freight forwarder, broker, overseas manufacturers and their sub contractors, and various levels of vendors. these layers are sometime loosely integrated and hence hard to maintain quality throughout the chain. some layers have quality assurance, but to truly ensure quality products and services, every member of supply chain layers should be considered quality assurance so that the work is done according to specifications. one could say that creates a culture of quality that is ingrain to every layer of the supply chain including an outsourced vendor. companies may actually decide that in order to meet their quality objectives, some services or products must be outsourced overseas to more skilled laborers. they feel that they do not have the skills in house, and quality is better met by outsourcing the necessary work. a test may be needed on a product and the company may not have the facilities, equipment or the skilled manpower to perform it and therefore they find a company that is more capable and has the facility to perform the test. by that decision, a needed operation is performed and the company's schedule is not interrupted if accurately planned. steps can be taken to help ensure the vendor provides services and products at quality levels that are acceptable to both internal and external customers. as stated before proper integration of the outsourced work into the supply chain is paramount. no work can properly be accomplished and managed with an integration plan to guide and oversee the vendor's work. if outsourcing is a strong option for the company, but yet there is a lack of trained workers, the company should provide training for the vendors to prepare them for the work that need to be accomplished. the company should also work on the cultural differences between them and the outsourced vendor. they should not seek just to completely change the vendor's way of accomplishing work, but they should strive to understand the vendor's cultural. this will assist in making decisions on how to define requirements to the group and how to help them meet the requirements. u.s. companies should understand that there are different ways at arriving to a solution as long as the requirements are met. in realizing the cultural differences, u.s. companies should make sure the vendor clearly understands what is expected of them. words that are used in the u.s. may have a totally different meaning to someone in india or china. the company may feel they clearly defined their requirements and the vendor may feel they clearly accomplished the work according the requirements as they read or understood them. only later, sometimes too late, they find out the product or service did not meet the requirements and the vendor did not clearly understand. a liaison from the parent company should network with a liaison from the vendor who has a clear understanding of the english diction. they will assist in knowing whether the company is effectively providing their requirements to the vendor and the vendor clearly understand what is needed of them. the company should also set up quality metrics that are understood by the vendor and should become a part of the vendor's way of business. in order for quality to become a complete part of the company's supply chain, the outsourced company has to make quality inherit to their business. the company should be able to provide back to the vendor what work is acceptable and what goals are not being met. they should also provide suggestions on how to achieve the required goal. incentives should be provided to the vendors who continuously provide quality products and product non-confirming vendors should be addressed appropriately, including termination of their services if they continue not to meet the expected quality level. most of the time, logistics operation run smoothly and as planned. there are times when disruptions cause havoc to logistics operations. the aftermath of any disaster could be enormous and annihilating for any logistics operations, especially for healthcare industry. in case of an emergency, the healthcare organizations in the affected region may experience out of stock situation for medical supplies which eventually impact their services. healthcare providers need to replenish their supplies from central distribution centers or unaffected regional distribution centers. the most difficult situation that authorities face is the complexity of operating conditions where they had to work in order to supply medical items to the affected region from a central position. some regions may be very difficult to reach under disaster condition. in this scenario it may be required to share medical items from contiguous health care organizations. product recall or system breakdowns also demand contingency plans. a global economy has inherently a very complex logistical system. getting a raw material from china to a us manufacturer and then the final product back to japan can have many factors that can cause a system breakdown. weather, a natural disaster, an economic upheaval, or even political changes can affect the supply chain in many drastic ways. for instance, covid-19 and its associated impacts paralyzed the health system deliveries in many places includinng in the u.s. many hospitals were out of ventilators and other personal protective equipment during this pandemic. inventory is the attribute of a supply chain or logistical system that will allow them to strive in one of these dramatic events. inventory will allow the system the time it needs to recover to prevent performance levels. product recalls are becoming more and more the norm of businesses today. the tremendous growth in returns has enthused new interest in reverse logistics (rl) as firms attempt to meet various challenges. typically, the higher the level of challenge greater is the opportunity for improvement. this is especially true in the case of rl management. engineering a rl network is fraught with daunting challenges due to the sheer uncertainty that surrounds returns quality, quantity and time. transporting returned goods is usually difficult and a cumbersome process. statistically, there are up to 12 times the number of transactions involved in the returns process than to sell the product in the first place, and more require human intervention. for example, an outbound shipment of goods only involves one or two transactions (picking up the goods from a warehouse and delivering them to a small number of locations, or even just one location). however, the process of returning just ten items could mean supply from many locations, plus a different problem resolution per item, and at different times. rl may be an area where companies can gain a sizeable advantage over the competitors. in today's highly competitive economy, high-quality customer services are the tickets to the game. it behooves an organization to differentiate itself from its competitors. in this regard, rl could be one of the major differentiators that organizations can take into account. many companies in the world, including in the us, lack a methodology for designing an efficient reverse logistics that focuses on the industry's salient features: high "marginal value of time", high "value recovery" and high "volume" of returned goods. customer service is a very important measure of the efficiency of a logistical system. many measures and processes allow the logistics professional an opportunity to receive feedback from the customer on their efficiency. the adage that the customer is always right may not always be true but certainly reigns supreme in most companies. the complexity added by a global economy has increased the visibility of customer service in logistics and emphasizes the importance of measuring and examining the process. customer service will influence many decisions in logistics and require much analysis for optimum performance. business logistics/supply chain management (fifth ed) strategic nature of the logistics customer service in the supply chain customer service: thekey to customer satisfaction, customer loyalty, and marketshare service variability and its effect on consumer perceptions and intentions managing a customer-driven supply chain, inbound logistics logistica. editura uranus, bucureşti. importance of customer service in transportation operations key: cord-312580-r57rkrya authors: harcourt-brown, frances title: chapter 6 clinical pathology date: 2002-12-31 journal: textbook of rabbit medicine doi: 10.1016/b978-075064002-2.50009-6 sha: doc_id: 312580 cord_uid: r57rkrya nan as rabbits are used extensively for toxicological and physiological studies, there are many scientific papers about the effects of experimental infections, drugs and toxic substances on haematological and biochemical parameters. there is also information about diseases of commercial rabbits, which are mainly investigated by post-mortem examination. in contrast, there is a dearth of literature about the effects of clinical diseases on the blood picture of rabbits or the use of blood tests as diagnostic or prognostic indicators. it is not always possible to extrapolate from other species, especially carnivores such as dogs and cats to the herbivorous rabbit with its specialized physiology. at the present time, much of the information that is available on the haematology and biochemistry of pet rabbits is anecdotal, although it can be helpful and is better than no information at all. the collection of blood and urine samples is covered in section 3.12. parameters such as glucose, creatine kinase and aspartate aminotransferase (ast) can be altered by stress associated with handling and restraint, or tissue damage that has occurred during sample collection. for example, potassium results appear less reliable in samples taken with a plastic cannula as opposed to a hypodermic needle . rabbit blood haemolyses easily and clots quickly (perry-clark and meunier, 1991). small clots affect haematology results and haemolysis affects certain biochemistry results, especially potassium and serum inorganic phosphorus that are released from erythrocytes. rapid clotting can affect the performance of some analysers and heparinized syringes and needles are required. it is not possible to take a guaranteed fasting sample from a rabbit because they ingest caecotrophs. parameters such as blood glucose are affected by digestion. some parameters such as bile acids, cholesterol and urea follow a diurnal rhythm that also affects the total and differential white cell count (fox and laird, 1970; fekete, 1989; loeb and quimby, 1989) . stress associated with car journeys or a period in unfamiliar surroundings will increase blood glucose and alter haematological parameters such as the distribution of neutrophils and lymphocytes. pregnancy affects parameters such as protein, haematocrit, cholesterol, alkaline phosphatase, triglycerides (viard-drouet et al., 1984) , glucose, sodium, calcium, phosphate and red cell indices (palm, 1997) . serum cholesterol is the parameter that is most affected and can be up to 30% lower in pregnant than in nonpregnant animals (palm, 1997) . anaesthesia affects some blood parameters such as potassium . the effect of anaesthesia on biochemical parameters is minimized by taking samples within 5 minutes of induction. intravenous or intraosseous fluids will also affect haema-clinical pathology tological findings (ros et al., 1991) and blood samples should be taken prior to treatment. there are a number of published reference ranges for haematological and biochemical parameters in rabbits. conversion factors for the variety of units that are used in reference ranges are given in table 6 .1. differences in analytical techniques between laboratories can lead to disparity in results. laboratory data are often derived from populations of rabbits of the same breed, sex and age that are not genetically diverse. in contrast, the pet rabbit population is made up of a variety of breeds, cross breeds and is composed of rabbits of all ages. significant breed and sex differences have been noted for some parameters in laboratory rabbits (kozma et al., 1974) . published reference ranges for pet rabbits are either derived from sets of data from laboratory rabbits or from data collected by the author. some reference ranges are an amalgamation of other references ranges and result in a range so wide that almost any result will fall within it. for example, the reference range for serum albumin concentra-tions of pet rabbits is given as 27-46 g/l (malley, 1996) based on four published sets of data. gillett (1994) gives an even wider range of 27-50 g/l for laboratory rabbits based on five sets of data. for some parameters there are big differences between published reference ranges. an example is blood calcium. gillett (1994) gives a range of 1.5-3.4 mmol/l in comparison with 3.4-4.0 mmol/l by harkness and wagner (1995) . differences in calcium content of the diet between the groups of rabbits or differences in analytical technique could explain these discrepancies. different laboratory methods can result in large differences between reference ranges. an example is alkaline phosphatase. collins (1988) gives a reference range of 4.1-16.2 iu/l in comparison with 10-70 iu/l (gillett, 1994) or 112-350 iu/l (harkness and wagner, 1995) . automated flow cytometers are designed to measure numbers of human blood cells. rabbit erythrocytes are smaller in diameter than human erythrocytes and also vary in diameter. these differences cause problems with some automated analysers. automated differential white cells counts cannot be relied upon for rabbit blood and accurate results can only be obtained using manual counting methods (kabata et al., 1991) . clinical pathology some morphological characteristics of rabbit blood cells are different from other species. the red blood cells vary in diameter within a range of 5.0-7.8 µm (sanderson and philips, 1981) . this variation in diameter (anisocytosis) is a feature of blood smears from rabbits and is not a significant finding (see plate 2). the red cell distribution width (rdw) is a measurement of the variation in size of erythrocytes and is higher in rabbits (11-15, idexx reference range) than in dogs and cats (8-10; bush, 1981) . in dogs and cats, anisocytosis is indicative of the presence of reticulocytes and a regenerative anaemia. in rabbits, 1-4% of circulating erythrocytes may be reticulotytes. polychromasia and reticulocytes in rabbit blood smears have been attributed to the short life span and high turnover of erythrocytes (kraus et al., 1984) . nucleated red cells and howell-jolly bodies can also be found occasionally (mclaughlin and fish, 1994) . rabbit neutrophils have an almost colourless cytoplasm and contain two types of granules. the smaller granules stain pink giving a pinkish colour to the cytoplasm. larger granules stain a deeper pinkish-red. the overall colour of the neutrophils varies according to the proportion of large and small granules. the granular appearance of the cytoplasm has led to different nomenclature. rabbit neutrophils may be called heterophils, pseudoeosinophils, acidophils or amphophils depending on the text (sanderson and philips, 1981; benson and paul-murphy, 1999) . neutrophils measure 10-15 µm in comparison with eosinophils that measure 12-16 µm (sanderson and phillips, 1981) . small lymphocytes are seen more commonly than large lymphocytes. the average cell diameter for small lymphocytes is 7-10 µm (cooke, 2000) . the lymphocytes are round cells with the typical morphology described for other species. an occasional large lymphocyte may have a few azurophilic granules in the cytoplasm (jain, 1986) . monocytes are large nucleated cells measuring 15-18 µm (cooke, 2000) . the nucleus has a diffuse lacey chromatin pattern that lightly stains a purple blue. the vacuolar cytoplasm stains light blue. eosinophils can be distinguished from neutrophils by their greater size and large acidophilic granules. in contrast to other laboratory species, basophils are frequently found in the circulation of rabbits in small to modest numbers (jain, 1986). a reference range for haematological parameters is given in table 6 .2. the haematological picture gives an indication of the general health status of a rabbit. stress and a range of diseases will alter haematological parameters. hinton et al. (1982) analysed the haematolog-• rabbit blood clots quickly and haemolyses easily • food deprivation does not guarantee a fasting blood sample as rabbits ingest caecotrophs • stress associated with transport or handling can affect parameters such as blood glucose and the distribution of neutrophils and lymphocytes • pregnancy, anaesthesia, blood collection techniques and intravenous fluid therapy will influence some blood results • time of day can influence blood results as many parameters follow a duirnal rhythm in common with many physiological processes in rabbits • laboratory reference ranges are often derived from animals of the same breed and strain. pet rabbits come from a more genetically diverse population • reference ranges for pet rabbits are often an amalgamation of laboratory reference ranges that can be so wide that almost any result will fall within them • different analytical techniques can result in disparity between laboratory reference ranges • automated flow cytometry is not suitable for differential white cell counts in rabbits. accurate results can only be obtained by manual counting methods. ical findings in 117 healthy and diseased rabbits and found that blood cellularity was a good indicator of disease especially with regard to erythrocyte and lymphocyte counts. these findings are in agreement with studies of experimental infections in rabbits krueger, 1988, 1989) and in a clinical study by harcourt-brown and baker (2001) . in this study, significantly higher red cell counts, haemoglobin values, haematocrits and lymphocyte counts were found in rabbits kept outside with unlimited access to grazing and exercise. a comparison was made with rabbits kept in hutches and those suffering from dental disease (see figure 6 .1). reference ranges for packed cell volumes (pcv) vary between sources with values between 30% and 50% (malley, 1996) . pet rabbits tend to have pcv values at the lower end of the range, typically between 30 and 40% (harcourt-brown and baker, 2001) . values greater than 45% are indicative of dehydration, especially in rabbits suffering from gut motility problems. values of less than 30% indicate anaemia that can be classi-fied into non-regenerative and regenerative in a similar manner to other species. a regenerative anaemia is associated with chronic blood loss, e.g. due to heavy flea infestation or from a site that intermittently bleeds such as a uterine endometrial aneurysm. uterine adenocarcinomas are a common finding in middle-aged does. lead poisoning can result in a regenerative anaemia with the presence of nucleated erythrocytes, hypochromasia, poikilocytosis and cytoplasmic basophilic stippling (fudge, 2000) . a nonregenerative anaemia is caused by diseases such as lymphoma or chronic renal failure. autoimmune haemolytic disease has not been described as a clinical phenomenon in pet rabbits although there are reports that it occurs. autoimmune haemolytic anaemia has been reported in laboratory rabbits in association with lymphosarcoma (weisbroth, 1994) . chronic debilitating disease such as dental disorders or abscesses often cause a mild anaemia in pet rabbits (harcourt-brown and baker, 2001 ) (see figure 6 .1). nucleated red blood cells can be associated with acute infectious processes although a few may be present in normal blood films. experimentally, infections with escherichia coli and staphylococcus aureus cause an increase in 143 clinical pathology number of circulating nucleated red blood cells during the septicaemic phase of the disease krueger, 1988, 1989 ). there is a natural diurnal variation in total white cell count with lowest counts occurring during the late afternoon and evening (fox and laird, 1970) . the white cell count also varies with age (jain, 1986) . it is higher in young rabbits of approximately 3 months of age and in adults over 1 year old. the first peak in leucocyte count is due to an increase in the number of lymphocytes. the second peak is due to an increase in the number of neutrophils. in other species, an increased white blood cell count is seen in response to bacterial infection or in response to endogenous or exogenous corticosteroids. rabbits do not develop marked leucocytosis after either acute infectious challenge or the intramuscular injection of cortisone acetate (toth and january, 1990) . in two studies by krueger (1988, 1989) controlled experimental infections with staphylococcus aureus, streptococcus pyogenes, escherichia coli and candida albicans resulted in fever, increased plasma cortisol concentrations, neutrophilia and lymphopaenia but no significant increase in total white blood cell count. high white cell counts can be found in rabbits with suffering from lymphosarcoma (mclaughlin and fish, 1994) . low white blood cell counts can be found in association with chronic disease (hinton et al., 1982) . clinical pathology figure 6.1. comparison of some blood parameters of pet rabbits. as part of an investigation into the relationship of metabolic bone disease with dental disease, blood samples were taken from pet rabbits presented for veterinary treatment or for health checks. at the time of sampling the rabbits were assigned to one of four groups: add (advanced dental disease), edd (early dental disease), h (healthy rabbits kept in hutches), fr (rabbits kept in free-range conditions in a large enclosure with unlimited access to exercise and natural daylight all year round). no rabbits in the free-range group (fr) showed signs of dental disease; all the rabbits in the add, edd and h groups were kept in hutches. blood results from rabbits that were found to be suffering from other conditions, such as renal disease or neoplasia, were not included in the statistical analysis. the add group consisted of rabbits that were presented for veterinary treatment for dental disorders such as acquired malocclusion or abscesses. all the other rabbits in the edd, h and fr groups were presented for neutering, health checks, vaccination or euthanasia. rabbits in the edd group had signs of early dental disease, such as horizontal ribs on the incisors, swellings along the ventral border or the mandible or epiphora related to elongation of the roots of the upper primary incisors. the investigation was conducted to compare blood parameters related to calcium metabolism in rabbits with or without dental disease. the healthy free-range group was used as a control. during the course of the study, differences in haematological pictures and albumin values emerged among rabbits kept under the different husbandry regimens. complete blood counts from free-range rabbits were comparable with laboratory reference ranges whereas there were significantly lower red cell and lymphocyte counts in rabbits suffering from advanced dental disease. the low lymphocyte counts of rabbits with dental disease suggest they suffered from chronic stress. serum albumin values were significantly higher in rabbits kept in free-range conditions than in those suffering from advanced dental disease or those unaffected by dental disease but kept in hutches. total serum calcium concentrations were highly correlated with serum albumin levels. rabbits kept in hutches showed trends towards anaemia and lymphopaenia. plasma parathyroid hormone (pth) concentrations were higher and total serum calcium concentrations were lower in hutch-kept rabbits with advanced dental disease in comparison with rabbits kept in free-range conditions. these results indicated that acquired dental disease of pet rabbits is related to husbandry and is associated with alterations in calcium metabolism. reprinted from harcourt-brown and baker (2001) with kind permission from the journal of small animal practice. although the total white cell count of rabbits seldom alters in diseased rabbits, the differential white cell count may show a number of changes due to the redistribution of white blood cells. a feature of many diseases in rabbits is an alteration in the ratio of neutrophils to lymphocytes and a reduction in blood cellularity (hinton et al., 1982) . the neutrophil: lymphocyte ratio has been suggested as a method of predicting whether a rabbit is normal or abnormal (mclaughlin and fish, 1994) . jain (1986) described a physiological variation in the neutrophil: lymphocyte ratio according to the age of the rabbit. the ratio changes from 33:60 in the second month of life to 45:45 in rabbits over 1 year of age. stress and increased cortisol levels can affect this ratio as well as disease. 6.2.4.3 effect of stress on the differential white cell count stress alters the differential white cell count in any species. rabbits are particularly susceptible to the effects of stress. a car journey to the surgery, a period in the waiting room next to a barking dog or the excitement of handling can be reflected in the blood picture. adrenaline and cortisol affect the distribution of lymphocytes throughout the body. administration of exogenous adrenaline to rabbits results in redistribution of lymphocytes from spleen and bone marrow to peripheral blood, lungs and liver (toft et al., 1992a) . conversely, exogenous corticosteroid administration results in a redistribution of lymphocytes from the peripheral blood, bone marrow and spleen to the lymphatic tissue in rabbits (toft et al., 1992b) . prolonged periods of stress cause neutrophilia and lymphopaenia. marked changes in white cell distribution with a relative neutrophilia and lymphopaenia were found in a study of the cortisol levels and haemograms of rabbits after transport, either by air or by lorry. the changes in white cell distribution lasted for 24-48 h and were correlated with increased cortisol levels (toth and january, 1990) . disease is stressful as well as having a direct effect on the production and distribution of white cells. rabbits with experimental infections exhibit a neutrophilia and lymphopaenia in comparison with control rabbits handled and sampled in exactly the same manner but inoculated with heat killed cultures (toth and krueger, 1989) . rabbits inoculated with a heat-killed culture do not experience the same rise in plasma cortisol concentrations as those inoculated with a live culture, indicating that the stress response is initiated by disease rather than by handling. therefore the stressful effects of a long car journey to the surgery or a morning spent in a kennel next to a barking dog is more likely to affect the neutrophil:lymphocyte ratio than the excitement response of taking blood. neutrophils function primarily as phagocytes and are important in infectious conditions and in inflammation. in other species, a neutrophilia occurs in response to inflammation, especially bacterial infection. an increase in the number of circulating neutrophils causes a rise in total white blood cell count. this response is not marked in rabbits. however, a change in the distribution of white cells can occur in response to infection with a relative neutrophilia and lymphopaenia but no alteration in total white cell count (toth and krueger, 1989) . a mature neutrophilia accompanied by an increase in plasma cortisol can also be associated with stress (toth and january, 1990 ). lymphocytes are involved in immunological responses and are distributed throughout the body in various tissues including blood, bone marrow, lymph nodes, spleen and gut-associated lymphoid tissue. the number of lymphocytes in the blood reflects a balance between cells leaving and entering the circulation and does not necessarily reflect a change in lymphopoiesis. increased cortisol levels cause a lymphopaenia and increased adrenaline levels cause lymphocytosis (toft et al., 1992a, b) . in rabbits, lymphopaenia is a feature of a variety of clinical diseases (hinton et al, 1982) . marked lymphopaenia has been reported as a feature of differential white cell counts of pet rabbits especially those suffering from dental disease (see figure 6 .1) (harcourt-brown and baker, 2001) . lymphoma is a relatively common tumour of rabbits and atypical lymphocytes can be found in the peripheral blood of these patients. the main function of eosinophils is detoxification either by inactivation of histamine, or histamine-like toxic materials. eosinophils are important in the allergic response and are capable of phagocytosis (kerr, 1989) . chronic eosinophilia can be seen in diseases of tissues that contain large numbers of mast cells such as the skin, lungs, gastrointestinal tract and uterus. eosinophilia can be associated with parasitism, especially when parasites are migrating through tissue. mild eosinophilia has been associated with experimentally induced chronic ascarid parasitism in rabbits (gupta and trivedi, 1981) . however, heavy worm burdens are rare in pet rabbits. encephalitozoonosis does not appear to cause an eosinophilia. slight to moderate elevations in eosinophil counts can be observed after traumatic wound repair in rabbits (fudge, 2000) . although eosinopaenia can be a significant finding in other species, low eosinophil counts or a zero count are not unusual in rabbits. basophils are similar to neutrophils but have dark blue cytoplasmic granules. although basophils are rare in blood films from species such as the dog, they may be seen commonly in rabbit blood (kerr, 1989) . basophil counts as high as 30% have been reported in clinically normal animals (benson and paul-murphy, 1999 ). in other species, monocytosis is associated with chronic disease, particularly chronic inflammatory conditions. in rabbits, increased monocyte counts can be associated with chronic bacterial infection. hinton et al. (1982) noted increased monocyte counts in rabbits with subcutaneous abscesses, mastitis and 'labyrinthitis'. however, monocyte counts within the laboratory reference do not signify the absence of chronic infection. rabbits with chronic osteomyelitis due to dental disease can have monocyte counts within the laboratory reference range (harcourt-brown, unpublished data). a reference range for biochemical parameters is given in table 6 .3. clinical pathology key points 6.2 • rabbit erythrocytes vary in diameter and anisocytosis can be a normal finding in rabbit blood films • polychromasia and small numbers of reticulocytes and nucleated red cells may be seen on normal blood films • rabbit neutrophils have a granular cytoplasm and may be mistaken for eosinophils • there are several different terms for the rabbit neutrophil. some authors use terms such as heterophil, pseudoeosinophil, acidophil or amphophil instead of neutrophil • basophils are frequently found on blood films from rabbits • low blood cellularity, i.e. anaemia and lymphopaenia, is a non-specific feature of disease in rabbits • high numbers of nucleated red blood cells may be associated with infectious disease • the neutrophil:lymphocyte ratio should be approximately 1:1 in adult rabbits. alterations in the ratio can be associated with stress or disease • adrenaline causes a shift of lymphocytes from the spleen and bone marrow to blood. cortisol causes a shift of lymphocytes away from the bloodstream to the spleen and lymphatic tissue • an increase in total white cell count is unusual in rabbits even in the presence of infection • a neutrophilia with a left shift occurs in response to infection • monocytosis can be seen in association with chronic infection. herbivores, such as rabbits, differ from carnivores in their carbohydrate metabolism. carnivores eat periodically and have sudden large intakes of nutrients that must be stored for utilization during the fast between meals. herbivores graze for long periods of the day and are continually absorbing nutrients from the digestive tract. in rabbits, volatile fatty acids are produced from bacterial fermentation in the caecum and are continually absorbed as an energy source. a fasting sample is difficult to obtain from a rabbit. withholding food does not prevent caecotrophy and the digestion of caecotrophs provides a source of glucose. blood samples taken after 96 h of food deprivation may show no alteration in blood glucose levels (kozma et al., 1974) . hyperglycaemia is a relatively common finding in rabbits and can be accompanied by glycosuria. handling alone can cause an increase in blood glucose to the order of 8.5 mmol/l experimentally (knudtzon, 1988) and 15 mmol/l or more anecdotally. diabetes mellitus has not been described in pet rabbits and there is some difference of opinion about its importance as a clinical disease (hoefer, 2000; jenkins, 2000; rosenthal, 2000) . herbivorous animals withstand the absence of insulin more readily than carnivorous ones (bentley, 1998) and are therefore not so susceptible to diabetes mellitus. diabetes mellitus has been induced in laboratory rabbits by the administration of alloxan. it has also been described as total protein 54-75 g/l e urea 6.14-8.38 mmol/l b vitamin a (plasma) 30-80 µg/ml ± < 10 µg/ml indicates deficiency (liver levels of < 10 µg/g liver denote deficiency) b vitamin e (plasma -tocopherol) > 1 µg/ml (< 0.5 µg/ml indicates deficiency) a hereditary disease in rabbits. a laboratory strain was selectively bred as an animal model of human diabetes mellitus. (roth and conaway, 1982) . affected animals were polydipsic, polyuric and polyphagic with severely impaired insulin release. elevated glycosylated haemoglobin values of 12.2% were observed in the overtly diabetic animals in comparison with 3.9% in normal animals. increased glycosylated haemoglobin levels did not correlate with plasma glucose concentrations (cannon and conaway, 1981) . histologically there was hypergranulation of ␤-cells of the islets of langerhans. obesity and ketoacidosis were not features of diabetes mellitus in the laboratory rabbits. the hyperglycaemia was in the region of 540-590 mg/dl (30-33.4 mmol/l) and there was marked glycosuria. roth and conaway (1982) described the maintenance of one diabetic individual on insulin at a dose of up to 8 units per day for 3 years. ketonuria was not observed. in pet rabbits, a diagnosis of diabetes mellitus cannot be made on a single blood sample and requires serial blood and urine sampling to confirm the diagnosis. in view of the physiological factors that can increase blood glucose levels it is advisable to take repeat blood samples from hyperglycaemic rabbits with time of day, phase of digestion, anaesthesia, influence of handling or car journeys in mind. mild glycosuria is not a significant finding. hyperglycaemia can be seen in the terminal stages of gut stasis and is a poor prognostic sign (harcourt-brown, personal observation). it is associated with fatty degeneration of the liver at post-mortem examination. marked hyperglycaemia is also seen in association with painful conditions such as acute intestinal obstruction. blood glucose levels can rise to 20-25 mmol/l and return to normal once the condition is resolved (harcourt-brown, unpublished observation). experimental haemorrhagic or traumatic shock results in hyperglycaemia proportional to the severity of the condition. hyperthermia also results in hyperglycaemia (mclaughlin and fish, 1994) . diseases that elevate serum glucose levels in other species, such as hyperadrenocorticism or acute pancreatititis have not been reported in pet rabbits, although they could occur. hypoglycaemia is a significant finding in rabbits and is associated with anorexia, starvation or disturbances in the digestion and absorption of carbohydrates. it can be a sign of hepatic dysfunction. a drop in blood glucose leads to mobilization of free fatty acids from adipose tissue and contributes to the development of ketoacidosis and fatty degeneration of the liver. measurement of serum glucose is of value in moribund rabbits as a basis for the selection of appropriate fluid therapy. other causes of hypoglycaemia such as addison's disease or insulinomas have not been reported in pet rabbits although such conditions could occur. interpretation of total protein concentrations is similar to other mammals. artefactual increases in protein concentrations can result from excessive venous stasis during blood collection. fluid and small molecules leave the plasma, resulting in a relative increase in proteins. this situation can occur in rabbits, especially in miniature breeds with small veins. an increase in total protein indicates dehydration, chronic and immune-mediated disease. in rabbits dehydration due to water deprivation or gastrointestinal disturbances commonly occurs. examination of the haematocrit and albumin and globulin fractions can assist differential diagnosis. liver disease, chronic enteropathy, starvation or malnutrition may result in reduced protein levels. glomerulonephropathy or protein-losing enteropathy are uncommon conditions that could cause low total protein levels in rabbits. a decrease in both albumin and globulin may be associated with haemorrhage or exudative skin lesions such as fly strike. the liver is the sole site of albumin synthesis and hypoalbuminaemia is feature of advanced liver disease in all species. in rabbits, heavy parasitism is a cause of liver disease. eimeria steidae causes hepatic coccidiosis (see section 10.10.1.2). cysticercus pisiformis, the larval stage of taenia pisiformis, migrates through the liver and results in the development of fibrous tracks and necrotic foci (see section 16.3.2). severe infestations can result in low albumin levels. non-hepatic causes of low serum albumin include glomerulonephropathy, protein-losing enteropathy, malabsorption and cardiogenic ascites. laboratory reference ranges for serum albumin levels in rabbits can be wide and vary between sources. sex differences have been reported in laboratory rabbits. one study showed that female new zealand white rabbits had higher serum albumin levels than males, although other studies have found no sex differences (kozma et al., 1974) . in rabbits, hypoalbuminaemia is most likely to be associated with nutritional factors such as abnormal caecotrophy, incorrect diet, starvation or malnutrition associated with dental disease. primary or secondary hepatic neoplasia occasionally occurs in pet rabbits. hepatic coccidiosis is a cause of low serum albumin levels, especially in young rabbits that have been kept in colonies. a high serum albumin concentration is not a feature of any specific disease, although an increased albumin level in conjunction with a raised pcv is indicative of dehydration. in a study by harcourt-brown and baker (2001), pet rabbits kept in free-range conditions had significantly higher serum albumin concentrations than rabbits that were suffering from advanced dental disease. they were also significantly higher than rabbits kept in hutches that were not suffering from dental problems (see figure 6 .1). the difference in albumin levels was attributed to differences in diet and husbandry. caecotrophs are a source of amino acids for rabbits and normal caecotrophy is an important element of their protein metabolism. low fibre diets, obesity, dental disease or skeletal abnormalities can prevent rabbits ingesting caecotrophs from the anus and reduce the available amino acids for protein synthesis. rabbits that are kept in hutches are more likely to be eating a low fibre diet and to suffer from obesity and skeletal problems than rabbits living outside with unrestricted access to natural vegetation and exercise. plasma globulins are made up of a range of proteins including carrier proteins and immunoglobulins or antibodies. the types of globulin can be separated into five fractions by electrophoresis. the ␥-globulin fraction is almost entirely composed of immunoglobulins. some globulins can be synthesized in the liver but immunoglobulins are synthesized exclusively in lymphoid tissue. acute inflammation, chronic disease or immune-mediated disease can cause an increase in globulin levels. myeloproliferative disease results in abnormal levels of immunoglobulin production. there are few published data on the significance of globulin concentrations in rabbits. lipaemia can artefactually elevate protein levels with some analytical methods. experimental infections with rabbit coronavirus result in hypergammaglobulinaemia. analogies have been made between coronavirus infection in rabbits and feline infectious peritonitis in cats (digiacomo and mare, 1994) . coronavirus occurs in laboratory rabbits but is an unlikely diagnosis in the pet rabbit. cholesterol is synthesized in the liver or absorbed from the diet. it is a metabolic precursor of steroid hormones. cholesterol is broken down in the liver and excreted in bile. in other species, elevated cholesterol levels are indicative of a variety of metabolic disorders such as hypothyroidism, hepatopathy, diabetes mellitus, and hyperadrenocorticism. low levels can occur in association with impaired hepatic function. changes in serum triglyceride levels reflect a similar range of diseases. blood levels of triglycerides increase after a meal, especially if it is a fatty meal. in rabbits, there are some physiological factors that affect cholesterol levels. male rabbits have lower cholesterol levels than females and there is a diurnal variation with higher levels occurring during the late afternoon (loeb and quimby, 1989) . large variations in blood cholesterol and triglyceride values can occur between individual rabbits (yu et al., 1979) . a fasting blood sample is required for cholesterol and triglyceride assay. in rabbits, it is difficult to obtain a fasting sample because of caecotrophy. abnormal cholesterol or triglyceride levels are most likely to be associated with dietary factors or hepatic impairment. in anorexic rabbits, especially obese ones, a lipaemic sample is a poor prognostic indicator as it signifies impaired fat metabolism and the presence of hepatic lipidosis (see section 10.3). a rise in triglyceride levels has been found in association with experimentally induced chronic renal failure in rabbits (tvedegaard, 1987) . in other species, amylase is found in the pancreas and to a lesser extent in the salivary glands, liver and small intestinal mucosa. amylase has a short half-life and is rapidly removed from the circulation. it is excreted by the kidney. elevated levels indicate pancreatic disease or renal insufficiency. in rabbits, amylase is present in pancreatic tissue in high concentrations. low concentrations are found in the salivary glands and none is produced by the liver (jenkins, 2000) . amylase is also produced by caecal microorganisms and is present in caecotrophs aiding conversion of glucose to lactic acid during digestion in the stomach and small intestine. serum amylase levels are lower in rabbits than other species (mclaughlin and fish, 1994) . pancreatic duct obstruction or pancreatic disease can result in a rise in blood amylase values. rabbits can survive experimental ligation of the pancreatic duct (brewer and cruise, 1994) . the rabbit secretes a large amount of bile, approximately seven times as much as a dog on a weight basis (brewer and cruise, 1994) . the rabbit also differs from other species in the excretion of breakdown products of haemoglobin. the rabbit has low biliverdin reductase activity (fekete, 1989) and only 30% of biliverdin is converted to bilirubin. bilirubin values can be affected by fasting. glucose administration to rabbits lowers serum bilirubin concentrations by modifying hepatic conjugation and increasing biliary secretion (mclaughlin and fish, 1994) . in rabbits, biliary obstruction results in jaundice and raised serum bilirubin values. in young rabbits, hepatic coccidiosis is the most usual cause of jaundice. in older rabbits, bile duct obstruction from neoplasia is more likely. aflatoxicosis from the ingestion of mouldy feed can result in hepatic fibrosis and jaundice (krishna et al., 1991) . viral haemorrhagic disease (vhd) causes acute hepatic necrosis with elevated bilirubin concentrations in association with dramatic increases in ast and alt concentrations. vhd is invariably fatal, although some rabbits may survive long enough to develop jaundice before they die. there is little on information on haemolytic disease as a cause of jaundice in pet rabbits. haemolytic anaemia has been reported in association with lymphosarcoma in laboratory rabbits (weisbroth, 1994 ). in other species, alt is used as an indicator of hepatocellular damage, especially in dogs and cats. alt is also in found in other tissues such as muscle and red blood cells. an increase in alt signifies cell damage, although the degree of the increase does not correlate with the severity of hepatic disease and is not a prognostic indicator (willard et al., 1999) . in rabbits, liver alt activity is lower than in other species and there is less organ specificity (rosenthal, 1997) . alt is also present in cardiac muscle. the half-life of alt in the rabbit is approximately 5 h. in the dog the half-life is 45-60 hours (jenkins, 2000) . hepatic coccidiosis due to eimeria steidae is a cause of increased blood alt concentrations especially in conjunction with an increase in alkaline phosphatase, bilirubin and gamma gt. elevated alt values have been found in asymptomatic house rabbits and have been attributed to the effects of organic solvents in wood shavings used as litter material. other liver diseases such as neoplasia can cause a rise in alt but sometimes not until the condition is advanced (mclaughlin and fish, 1994) . low doses of aflatoxin caused a significant rise in alt concentrations in a group of laboratory rabbits (fekete and huszenicza, 1993) . hepatic lipidosis will elevate alt levels. in other species ast is widely distributed throughout the body. in particular, it is found in skeletal muscle, cardiac muscle, liver and erythrocytes. like alt, ast is an indicator of tissue damage. it is sometimes used as an indicator of liver disease, especially in horses in which alt is not liver specific. in rabbits, ast is found in liver, heart, skeletal muscle, kidney and pancreas with the highest activity in the liver and skeletal muscle (benson and paul-murphy, 1999) . physical exertion or tissue damage during blood collection can elevate results. raised ast levels can be found in association with liver disease. ggt is found in liver and kidney tissue. in other species, ggt is used as an indicator of hepatobiliary disease especially in horses and ruminants where it is associated with longterm liver damage. although ggt is found in high concentrations in renal tubular cells, kidney disease does not lead to elevated blood levels, probably because the enzyme is excreted in the urine (bush, 1991) . in the rabbit ggt is located predominantly in the renal epithelium with low activity in the liver. liver ggt is present primarily in bile duct epithelial cells and is therefore an indicator of hepatobiliary disease rather than hepatocellular damage (mclaughlin and fish, 1994) . in cases where there is renal tissue damage, urine ggt may be increased in addition to plasma concentrations. alkaline phosphatase consists of a group of several isoenzymes that hydrolyse phosphates at an alkaline ph (kerr, 1989) . it is one of the most widely distributed enzymes in the body. alkaline phosphatase is found particularly in bone, liver and intestinal wall. different isoenzymes are produced from each site. increases in plasma activity are usually due to the isoenzymes derived from liver and bone. higher concentrations are found in young animals with high osteoblastic activity. in rabbits, alkaline phosphatase is present in nearly all tissues. it is found in association with cell membranes and especially in intestinal epithelium, renal tubules, osteoblasts, liver and placenta. the rabbit has three ap isoenzymes -the intestinal form as well as two isoenzymes present in both liver and kidney (mclaughlin and fish, 1994) . there is a wide variation between laboratory reference ranges for ap values for rabbits. examples include: 4.1-16.2 iu/l (collins, 1988) ; 10-70 iu/l (gillett, 1994) ; 112-350 iu/l (harkness and wagner, 1995) . different analytical techniques could account for these variations. in a survey of blood parameters relating to calcium metabolism in pet rabbits, serum alkaline phosphatase values varied widely even in apparently healthy individuals (harcourt-brown and baker, 2001) . increased levels of alkaline phosphatase are seen in biliary obstruction, e.g. neoplasia or hepatic coccidiosis. experimental ligation of the common bile duct results in increased levels of alkaline phosphatase up to 600 iu/l (mclaughlin and fish, 1994) . enteric disease can also elevate alkaline phosphatase values (jenkins, 2000). bile acids are derived from cholesterol and are secreted into the intestine to aid fat digestion. from the gut, they are reabsorbed into the circulation and transported to the liver to be resecreted in the bile. impaired hepatic function results in increased concentrations of bile acids in peripheral blood. there are physiological variations in circulatory bile acid concentrations in association with the digestion of food and stimulation of the gall bladder to release bile into the small intestine. in most species, a fasting sample should have a low concentration of bile acids of less than 15 µmol/l (kerr, 1989) . impaired hepatic function can result in marked rises in fasting serum bile acid concentrations. in rabbits, the production of bile acids shows a circadian rhythm (fekete, 1989) . there is a problem in obtaining a fasting sample from rabbits due to the ingestion of caecotrophs. bile acids are not included in published reference ranges for rabbits at the present time. bile acid levels in excess of 100 µmol/l have been found in association with hepatic coccidiosis in comparison with levels that are generally less than 40 µmol/l (harcourt-brown, unpublished data). urea is a nitrogenous waste product that is formed in the liver as the end product of deamination of amino acids. it is transported in the blood to the kidney where it is excreted in the urine. in other species, high blood urea concentrations are indicative of impaired renal function that may be due to renal disease or poor perfusion due to circulatory disorders or cardiac disease. low blood urea levels can reflect hepatic dysfunction. in rabbits, many physiological factors influence the concentration of urea in the blood. dietary protein concentrations and quality, withholding food and natural diurnal rhythms can all affect blood urea concentrations. higher levels occur in the late evening (loeb and quimby, 1989) . the rabbit's urea metabolism is further complicated by urea utilization by caecal microflora during catabolism or during periods of dietary excess. therefore small fluctuations in serum urea concentrations are difficult to interpret. laboratory reference ranges apply to animals that are fed a standard diet and have usually been bled at a specific time of day. pet rabbits are subject to greater fluctuations in blood urea values due to the variation in diet and other factors, and can have values slightly higher than laboratory reference ranges. prerenal azotaemia associated with poor renal perfusion occurs during periods of dehydration. the rabbit has a limited capacity to concentrate urea and a greater volume of urine is required when urea load increases (brewer and cruise, 1994) . increased blood urea values were recorded in a study by licois et al. (1978) of young rabbits with diarrhoea experimentally induced with coccidiosis. the authors suggested that the blood urea values rose as a result of intense nitrogen catabolism during weight loss associated with the disease. water deprivation can lead to high blood urea values as high as 40 mmol/l in association with creatinine values in excess of 200 µmol/l (harcourt-brown, unpublished data). water deprivation can be due to a lack of available drinking water, caused either by an oversight by the owner or by a faulty mechanism on the drinking bottle. in rabbits, dehydration can cause urea and creatinine values that would signify renal disease in the dog and cat. high levels usually return to normal once the animal is rehydrated. therefore, urea and creatinine values should be checked before making an absolute diagnosis of renal failure. as in other species, elevated blood urea values in rabbits are associated with renal insufficiency. nephrolithiasis is a cause of kidney disease in the rabbit (see section 14.5). abdominal radiography is indicated in rabbits with raised urea and creatinine levels. encephalitozoon cuniculi can cause low-grade kidney disease in rabbits with mild elevations in blood urea. most cases are subclinical. e. cuniculi infection causes granulomatous lesions in the kidneys that become pitted and scarred with fibrotic areas. the parasite has been associated with chronic renal failure with blood urea values in the region of 152.7 mg/dl (25.45 mmol/l) and creatinine of 5.8 mg/dl (512.72 µmol/l) in a study by ewringmann and göbel (1999) . affected rabbits were anaemic with low haemoglobin and red cell counts and had elevated serum potassium concentrations. neoplasia, interstitial nephritis, nephrotoxicity also occur in rabbits and cause renal disease. low blood urea values in association with impaired hepatic function and the use of anabolic steroids have been described (benson and paul-murphy, 1999 ). creatinine is a nitrogenous waste product that, like urea, is transported in the blood to the kidney where it is excreted in the urine. creatinine is not the product of amino acid breakdown but of creatine which is a substance present in the muscle and is involved in high energy metabolism (kerr, 1989) . the slow catabolism of creatine results in a slow inflow of creatinine to the plasma at a rate which is directly proportional to the individual's muscle mass but is unaffected by any change in muscular activity or muscle damage. any changes in blood creatinine concentrations are due to changes in excretion and are a reflection of renal function. concentrations rise quickly at the outset of renal disease and decrease when an improvement of renal function takes place. creatinine deteriorates in plasma and readings from old samples (>24 h) cannot be relied upon. there is interference from a variety of other substances such as bilirubin (which decreases creatinine) or cephalosporins (which increase creatinine). the rabbit's complex digestive physiology and the compromised renal capability of correcting acid-base disorders make the rabbit a prime candidate for electrolyte imbalances (see section 1.6). dietary deficiency of electrolytes such as sodium and potassium is unlikely in the herbivorous diet of rabbits. instead, electrolyte problems are more likely to be associated with abnormal losses. although rabbits do not vomit, water and electrolyte absorption and secretion are affected by gastrointestinal disease. if facilities are available, electrolyte assays, especially potassium, can be a valuable part of the diagnostic workup for critically ill rabbits. in general, changes in sodium concentrations reflect the osmolality of extracellular fluid rather than the total body sodium content. increased blood sodium concentrations (hypernatraemia) can be the result of water deprivation or the loss of low sodium fluids. decreased sodium concentrations (hyponatraemia) may occur as a result of chronic renal failure when the kidney cannot concentrate urine and fast urine flow through the renal tubules prevents effective sodium/ potassium exchange. lipaemia or hyperproteinaemia can artefactually reduce affect sodium concentrations if certain laboratory methods are used. at the present time, there are few data available on clinical conditions that affect sodium concentrations in rabbits. about 95% of the total body potassium is intracellular, so measurement of extracellular potassium in blood samples does not give a true reflection of the potassium status of the patient. the balance between intracellular and extracellular potassium is regulated by aldosterone, insulin and catecholamines and is affected by blood ph. aldosterone stimulates renal excretion of potassium. insulin promotes the movement of potassium into cells. the effects of these hormones prevent large diet-induced changes in plasma potassium concentrations. potassium is an important ion in the maintenance of membrane potential. abnormally high or low potassium concentrations can have life-threatening consequences due to impaired electrical activity of cells. high blood potassium concentrations can result in cardiac arrest. alterations in blood potassium levels can be due to alterations in dietary intake and 154 textbook of rabbit medicine key points 6.3 • stress of handling can cause a marked elevation of blood glucose levels in rabbits. levels as high as 15 mmol/l can occur in association with handling. higher levels (> 20 mmol/l) may be seen in association with stressful or painful diseases such as intestinal obstruction • there is debate about the occurrence of diabetes mellitus in domestic rabbits. it has been induced in laboratory rabbits and a genetically susceptible laboratory strain has been bred • large individual variations in blood cholesterol and triglyceride values can occur in rabbits • jaundice is unusual in rabbits but may be seen in association with cholestasis in diseases such as hepatic coccidiosis or neoplasia • there is a wide variation between laboratory reference ranges for alkaline phosphatase values • blood urea and creatinine values can be high in cases of prerenal azotaemia in rabbits and do not always signify renal failure. causes include dehydration or water deprivation. excretion, or redistribution across cell membranes. to maintain electroneutrality, potassium ions shift from intracellular to extracellular fluid in exchange for hydrogen ions. in other species, hypoadrenocorticism (addison's disease) reduces the exchange of sodium and potassium ions across the cell membrane and results in increased serum potassium and decreased serum sodium concentrations. hyperkalaemia can be the result of impaired renal excretion of potassium due to kidney disease or from tissue trauma such as crushing injuries that release large amounts of potassium into the circulation. acidosis causes a redistribution of potassium across the cell membrane. artefactually high levels of potassium can result from leakage from red cells in haemolysed samples or those that have not been separated until several hours after the blood was taken. low blood potassium can cause muscular weakness and depression. hypokalaemia can be the result of dietary potassium deficiency or as a result of potassium loss from the gastrointestinal tract. diuresis or the use of potassium-free intravenous fluids also cause hypokalaemia. alkalosis can cause redistribution of potassium and sodium across the cell membrane and result in hypokalaemia. artefactually low potassium concentrations are uncommon although they can occur secondarily to hyperlipidaemia or hyperproteinaemia (willard et al., 1999) . blood collection through a catheter that contains residual potassium-free fluids can lead to erroneously low results. in rabbits, the effect of blood collection methods on plasma potassium levels has been investigated. discrepancies in results were found between blood collected from the ear and from the carotid artery when the blood was collected with a plastic catheter but not with a 21 g needle . general anaesthesia with pentobarbitone depressed plasma potassium values but sedation with chlorpromazine did not affect results. serum potassium concentrations were found to be higher than plasma and in venous rather than arterial blood . low serum potassium values have been found in unanaesthetized rabbits in conjunction with signs of muscular weakness (harcourt-brown, unpublished data). affected animals can still eat and drink but are unable to move. it is not known whether hypokalaemia is the cause of the muscular weakness. possible causes of hypokalaemia are discussed in section 12.6.1.1. further investigations of serum potassium concentrations of rabbits are required to know the clinical significance of measured values and the influence of various physiological states. in horses, blood potassium concentrations can fall to 2.0 mmol/l after prolonged exercise due to potassium loss in sweat and to 2.5 mmol/l while eating hay due to potassium loss in saliva. during moderate exercise concentrations can rise to 4.0 mmol/l due to potassium release from muscle cells (kerr, 1989) . similar physiological variations could occur in the rabbit. calcium is an essential element that is involved in many body systems. most of the body's calcium is stored in bone in conjunction with phosphate. calcium is an essential part of the structure of bones and teeth. it is an important cation in intracellular and extracellular fluid where it is required for muscle metabolism, enzyme activation, blood coagulation and osmoregulation. calcium is found in the blood in three forms: ionized, bound to other anions (especially phosphate) and bound to protein (especially albumin). because of the protein binding capacity of calcium, total serum calcium concentrations are proportional to albumin concentrations. ionized calcium is the physiologically active component of blood and is involved in the permeability of cell membranes. hypocalcaemia is a life-threatening condition. in many species, a high demand for calcium during late pregnancy and lactation can result in hypocalcaemic tetany. there are also some metabolic disorders that can result in alterations in serum calcium concentrations in other species. examples include renal, pancreatic and neoplastic diseases. the rabbit has a different calcium metabolism from other domestic species (see section 1.6.7). dietary calcium is readily absorbed from the intestine and total plasma values reflect dietary intake. total blood calcium levels are higher and can vary over a wider range than other species. an erroneous diagnosis of hypercalcaemia is often made because of the rabbit's high total serum calcium levels in comparison with other animals. parathyroid hormone (pth) regulates calcium metabolism in a similar manner to other animals, but a reduction in plasma pth level occurs at a higher plasma calcium concentration than in other species . the kidney plays an important role in calcium regulation and has a high fractional excretion for calcium when blood levels are high. calcium is excreted in the urine in which it forms calcium carbonate precipitate. some authors have suggested monitoring blood calcium concentrations as part of the protocol for treating 'sludgy urine'. however, high blood calcium levels are not the sole cause of urinary tract disease in rabbits (see section 14.4.1). there are differences between published reference ranges for total serum calcium values in rabbits. variations in dietary calcium intake could account for some of the discrepancies. the peak blood level that was obtained by increasing dietary calcium intake in laboratory rabbits was 5.42 mmol/l (21.7 mg/dl) in a study by chapin and smith (1967a) . experimental calcium restriction resulted in minimum serum concentrations of 3.22-3.5 mmol/l (13-15 mg/dl) before a rapid decline just before death in a separate study by chapin and smith (1967b) . a reference range of 3.2-3.7 mmol/l taken from eight laboratory reference sources has been made by goad et al. (1989) . values outside this range are encountered in otherwise healthy individuals and a range of 3.0-4.2 mmol/l is acceptable for pet rabbits on a varied diet. total blood calcium levels in rabbits are also affected by age and reproductive status. kamphues et al. (1986) found that increased calcium intake only resulted in higher total plasma calcium concentrations in adult rabbits and not young ones of 5-19 weeks. serum calcium concentrations in growing rabbits are fixed at a value of approximately 3.5 mmol/l (14 mg/dl) (kamphues et al., 1986 , gilsanz et al., 1991 . blood calcium levels decrease during pregnancy (assane et al., 1993) . due to the protein binding properties of calcium, albumin levels can also affect total calcium concentrations. albumin concentra-tions in pet rabbits are variable and appear to be affected by the manner in which they are kept (harcourt-brown and baker, 2001) . in dogs and man, total serum calcium values can be adjusted by using a mathematical formula that takes albumin concentration into account. (adjusted calcium concentration = measured serum total calcium concentration -serum albumin (g/dl) + 3.5). this formula is unreliable in cats (flanders et al., 1989) and has not been investigated in rabbits. at present, most published reference ranges for pet rabbits refer to total serum calcium concentrations. ideally, ionized calcium should be measured for an accurate assessment of calcium status but special sample handling and equipment is required that precludes its measurement in most practice situations. however, affordable equipment is now becoming available that can be used in the practice laboratory. measurements of ionized calcium have been made during experimental investigations using rabbits. warren et al. (1989) found a linear relationship between total serum calcium and ionized calcium values. a group of 29 non-pregnant female and male rabbits were found to have ionized serum calcium levels of 1.71 + 0.11 mmol/l. kamphues et al. (1986) reported ionized calcium values of 6.94 + 0.21 mg/dl (1.73 + 0.05 mmol/l) in adult rabbits on an average dietary calcium intake (0.85%). hypocalcaemic tetany has been reported in lactating does (barlet, 1980) . hypercalcaemia is seen in rabbits with chronic renal failure and impaired calcium excretion (see section 14.5.3.1). in a study by tvedegaard (1987) , experimentally induced chronic renal failure resulted in total serum calcium concentrations of 4.82 + 0.77 mmol/l. levels in excess of 4.25 mmol/l have been recorded in association with neoplasia (voelkel et al., 1978) . inorganic phosphate is involved in many enzyme systems and is important in carbohydrate and muscle metabolism as well as forming a major component of bone. phosphate is obtained from the diet. vitamin d and pth influence intestinal absorption of phosphorus in a similar manner to calcium. pth stimulates renal excretion of phosphate and renal conservation of calcium. the ph of intestinal contents and the presence of cations such as calcium and magnesium can affect the availability of dietary phosphate. abnormalities of phosphate metabolism are complex and interdependent with many other factors. blood phosphate values can be difficult to interpret and need to be examined alongside other parameters. in other species, physiological activities such as feeding and exercise reduce serum phosphorus concentrations (aitken and allen, 1994) . there are drug interactions that alter serum phosphorus values. examples include phosphate binders, anaesthetic agents, bicarbonate, parenteral glucose, anabolic steroids, diuretics and tetracyclines (willard et al., 1999) . phosphorus can shift between the intracellular and extracellular space in response to alterations in acid-base metabolism. in addition to these problems of interpretation, phosphate values are subject to artefactual error caused by poor sample handling. haemolysis releases phosphate from erythrocytes, which results in elevated values. in rabbits, there is little information about the clinical relevance of serum phosphate concentrations. rabbit blood clots easily and good quality non-haemolysed samples can be difficult to obtain. hyperphosphataemia can be due to impaired renal phosphorus excretion due to kidney disease. hypophosphataemia may result from dietary deficiency, impaired intestinal absorption or metabolic disorders. blood lead concentrations are given in different units depending on the source. the conversion factor for µg/dl to µmol/l is 0.0483. a study by roscoe et al. (1975) evaluated three diagnostic tests for lead toxicity in rabbits. whole blood lead concentrations of greater than 0.03 mg/dl (1.45 µmol/l) were considered a reliable indicator of lead ingestion. measurement of urinary delta-aminolevulinic acid (␦ala) was considered unreliable. erythrocytes from rabbits that were given lead fluoresced red when exposed to light rays of 320-400 nm. the fluorescent erythrocyte test (fet) was considered a convenient and reliable test for lead ingestion. swartout and gerken (1987) described two clinical cases of lead poisoning that had blood levels of 70 µg/dl (0.07 mg/dl) and 40 µg/dl (0.04 mg/dl). they gave a range of 2-27 µg/dl (0.002-0.027 mg/dl) as a normal range for laboratory rabbits. pth is released by the parathyroid gland in response to both a fall in blood calcium and low serum 1,25-(oh) 2 d 3 levels. it is responsible for the minute to minute regulation of calcium, due to its quick, short duration response. pth stimulates conversion of (25-oh-d) to the active from of vitamin d (1,25-(oh) 2 d 3 ) that, in turn, stimulates intestinal absorption of calcium. pth also stimulates osteoclastic resorption of bone to release calcium, phosphorous and magnesium into the circulation. pth stimulates renal conservation of calcium but not phosphorous, which results in an increase in blood calcium without an increase in phosphorous concentrations. in animals with disturbances in calcium metabolism that result in bone demineralization, pth levels are high, and pth can be used to investigate metabolic bone disease that is often nutritional in origin. dietary calcium deficiency, or a failure to absorb calcium are reasons for metabolic bone disease. failure to absorb calcium can be due to vitamin d deficiency or unavailability of calcium due to binding with substances such as oxalates, fats or phosphates in the gut. pth assays are available in commercial laboratories that specialize in endocrinological investigations. pth assays have been performed on pet rabbits as part of an investigation of the possibility of metabolic bone disease as a cause of poor tooth and bone quality and the development of dental disease in rabbits (harcourt-brown and baker, 2001) . sample handling is of paramount importance as the hormone is labile and haemolysis interferes with the assay. samples require separating and freezing immediately after collection and must be shipped in the frozen state to a laboratory. sufficient, non-haemolysed blood to harvest 1-2 ml of serum or plasma needs to be collected. as pth is responsible for the minute to minute regulation of blood calcium, values can vary over a wide range making interpretation of a single result difficult. diet, age, pregnancy, lactation, diurnal rhythms cause physiological variations in results. warren et al. (1989) reported pth values of 59.6 + 41.2 pg/ml in a group of 29 nonpregnant farm and laboratory rabbits and harcourt-brown and baker (2001) reported values of 40.3 + 10.7 pg/ml in a group of 12 pet rabbits kept outside under free-range conditions all year round (see figure 6 .1). values as high as 100-200 pg/ml have been recorded in baseline samples from laboratory rabbits . values in excess of 230 pg/ml have been found in pet rabbits, one of which was found to have a liver tumour on subsequent post-mortem examination (harcourt-brown, unpublished data). in the uk, serological tests are available for encephalitozoon cuniculi, toxoplasma gondii, myxomatosis, viral haemorrhagic disease and treponema cuniculi as part of the commercial health screening of laboratory rabbits. commercial laboratories may accept individual samples from pet rabbits for serological screening. it is advisable to consult a veterinary laboratory in the first instance. in the usa, serology and a pcr test are also available to detect pasteurella multocida infection (sanchez et al., 2000) but these tests are not available in the uk at the present time (september, 2000) . serological testing for encephalitozoon cuniculi antibodies can be useful in the differential diagnosis of neurological diseases such as vestibular syndrome or paraplegia (section 12.4) or uveitis (section 11.7.3.1). it is also indicated in animals with mild renal insufficiency (section 14.5.1). serological and histological tests from naturally infected rabbits have demonstrated the presence of antibodies before the organism can be seen in the kidney. lesions were not seen in the brain until at least 8 weeks after the first detectable antibodies, suggesting that serology is a sensitive procedure for early diagnosis (cox and gallichio, 1978) . therefore animals with clinical signs that are seronegative are unlikely to be suffering from encephalitozoonosis, although experimental infections with encephalitozoon cuniculi have shown the presence of granulomas in the brain of animals that had become seronegative (kunstyr et al., 1986) . conversely, asymptomatic rabbits can be seropositive. therefore serology can only be used as a guide in the diagnosis of encephalitozoon cuniculi infection. antibody titres can be helpful in distinguishing between recent and chronic infection. simultaneous detection of igm and igg suggest recent infection. urinanalysis is summarized in table 6 .4. urine collection is described in section 3.12.3. in common with other herbivorous species, rabbits excrete alkaline urine. urinary ph is approximately 8-8.2. rabbit urine is normally turbid due to the presence of calcium carbonate that can be seen as sediment in collected samples. the urine from anorexic, pregnant, lactating or young rabbits is often clear. the colour of normal urine can vary from pale yellow, to orange, brown or red, mimicking haematuria. plant porphyrin pigments are the cause of red coloured urine and can be distinguished from haematuria with urinanalysis dipstick tests. alternatively, a wood's lamp can be used, as urinary pigments fluoresce when exposed to ultraviolet light (benson and paul-murphy, 1999) . in addition to the presence of calcium carbonate crystals, oxalate or ammonium magnesium phosphate crystals can also be found in normal rabbit urine. the specific gravity of rabbit urine is difficult to evaluate accurately due to the presence of mineral deposits but is approximately 1.003-1.036. traces of glucose and protein can be present in normal rabbit urine. as in other species, rabbit urine can be spun and the sediment examined microscopically for the presence of crystals, red cells, inflammatory cells and bacteria. cultures can be taken to confirm bacterial infection and aid antibiotic selection. examination of urine sediment stained with gram stain can reveal encephalitozoon cuniculi spores that are oval, strongly gram-positive with a coiled filament inside (pye and cox, 1977; patton, 2000) . ketones may be detected in the urine of anorexic rabbits and is a poor prognostic sign as it is associated with the development of hepatic lipidosis. rabbits produce two types of faeces: hard dry pellets that are composed of compressed indigestible fibre and soft caecotrophs that are composed of a smooth paste rich in bacteria and other microorganisms. the first step in faeces examination is to determine which type of faeces has been collected. it is often samples of soft faeces or caecotrophs that are collected for examination because their consistency is abnormally loose or the owner has mistaken uningested caecotrophs for diarrhoea. microscopically the two types of faeces are completely different. hard faeces contain indigestible fragments of plant debris and little else. caecotrophic material contains a wide range of microorganisms including large gram-negative bacilli, bacteroides, large metachromic staining bacilli and many other bacteria including oval and fusiform rods. in some types of diarrhoea, a mixture of indigestible fragments of plant material can be seen alongside a range of typical caecal microorganisms. this signifies a failure of the proximal colon to separate the indigestible and digestible fractions of the diet. coccidial oocysts or eggs of the non-pathogenic oxyurid passalurus ambiguus can be found in both hard and soft faeces of infected rabbits. coccidial oocysts can be confused with a nonpathogenic saccharomyces, a budding sporogenous yeast that can be present in large numbers in rabbit faeces (see figure 6 .2). clostridium spiroforme is a large grampositive, semicircular or spiral-shaped bacterium that may be seen in faecal smears from diarrhoeic rabbits or those that have died from enterotoxaemia. centrifuging faecal material at 20 000 rpm for 15 minutes and gram staining the residue after the supernatant has been removed improves the chance of diagnosis (langan and o'rourke schaeffer, 2000) . although the presence of semicircular bacteria in the faeces or caecal material is suggestive of clostridial enterotoxaemia, it is not a reliable diagnostic criterion. clostridia species can be present in the normal caecal flora and proliferate after death. the demonstration of the toxin and anaerobic culture of the organism is required for positive diagnosis (carman and borriello, 1983) . clostridium piliforme, the causative organism of tyzzer's disease, is not detected in faeces. a pcr test is available in the usa for detection of this organism. escherichia coli is not a normal inhabitant of the rabbit gut flora 159 clinical pathology (a) coccidial oocysts and saccharomyces (ϫ 100). shows large numbers of coccidial oocysts interspersed with saccharomyces guttulatus. the faecal sample was from a 14-week-old, thin rabbit that was suffering from diarrhoea. faecal material was emulsified with water before being passed through a fine sieve to remove the coarse debris. the homogenate was then centrifuged and the supernatent discarded. the residue was mixed with saturated salt solution and centrifuged again. after the sample had been spun, more saturated salt solution was added to the test tube until a meniscus formed. a cover slip was placed over the meniscus and the sample left for approximately 30 minutes for the oocysts or worm eggs to float to the top of the tube. at the end of this period, the cover slip and surface film was removed from the test tube, placed on a microscope slide and examined under low power. (b) eimeria steidae (high power) (image kindly supplied by dr sheelagh lloyd, division of animal pathology, university of cambridge). several eimeria spp. affect rabbits and mixed infections occur. the species can be differentiated by the morphological characteristics of the oocysts. the most pathogenic species is eimeria steidae, which causes hepatic coccidiosis (see section 16.4.1). e. steidae invades the epithelial cells of the bile ducts and can cause severe liver damage. oocysts may be seen in faeces from infected rabbits or in smears of bile collected from rabbits during post mortem examination. the relative sizes of coccidial oocysts and saccharomyces guttulatus can be seen in (a,b). worm eggs are larger than coccidial oocysts. the most common helminth infestation of pet rabbits is passalurus ambiguus. adult worms or ova from p. ambiguus may be seen in rabbit faeces. the ova are ovoid, slightly flattened and asymmetrical with a cap at one end. (c) saccharomyces guttulatus (high power) (image kindly supplied by idexx laboratories, wetherby). saccharomyces guttulatus is a budding yeast that is commonly found in the faeces of rabbits, chinchillas and guinea pigs. it is not believed to be pathogenic. some texts call the yeast cyniclomyces guttulatus. although small numbers may be present in some animals. enteropathogenic strains can be found in association with diarrhoea in weanling rabbits. pathogenic salmonella spp. may be isolated although post-mortem material is usually available in these cases. infectious enteritis is rare in the individual pet rabbit. plucked hair samples may be examined visually for the presence of mites that are just visible with the naked eye. under good illumination, cheyletiella mites can be seen in scale and skin debris that has been brushed out of the coat. after a few minutes, individual mites can be seen moving into the warmth of the illuminating light. egg cases and cuticles from developmental stages of the fur mite leporacus gibbus (formerly known as listrophorus gibbus) may also be seen on visual examination of hair brushings. they remain attached to hair shafts after hatching or moulting and give the fur a characteristic 'salt and pepper appearance' in heavy infestations. visual evidence of l. gibbus is seen more easily on white or light coloured areas of fur, especially if it is wet. occasionally lice may be found. microscopic examination of acetate strips applied to the skin of alopecic areas can be used to detect cheyletiella parasitovorax. all stages of the life cycle of c. parasitovorax can be seen by this method. skin brushings can also be examined microscopically. fleas, flea dirt, cheyletiella parasitovorax and leporacus gibbus can be seen in skin brushings examined under low magnification. a trichogram is useful in differentiating between conditions that cause alopecia. for this technique a sample of hair is plucked from as close to skin as possible using forceps. the hair is placed on a microscope slide, taking care to ensure the hairs remain orientated in the same direction. a drop of mineral oil and a cover slip are applied before examining the shafts of hair under the microscope. abrupt, fragmented, distal ends of the hair shaft suggest barbering by a companion. egg cases, cuticles or adult mites, usually leporacus gibbus, may be seen attached to hair shafts. the presence of fungal spores in broken hair shafts plucked from a lesion is diagnostic of dermatophytosis. dermatophyte infection can be demonstrated by the presence of mycelia or ectothrix arthospores in potassium hydroxide preparations of macerated scale. asymptomatic infections can be detected by brushing the entire body with a sterile toothbrush and incubating the brushings at 25°c on dermasel agar (oxoid). plates that do not show fungal growth within 3 weeks can be considered negative (vangeel et al., 2000) . dermatophyte infections are usually due to trichophyton mentagrophytes that does not fluoresce under ultraviolet light. microsporum canis infections can also occur that are evident from the characteristic apple green spores that fluoresce under a wood's lamp. clinical pathology key points 6.5 • rabbit urine may be coloured red due to the presence of plant pigments. dipsticks can be used to differentiate haematuria from red urine • normal rabbit urine contains calcium carbonate sediment. small quantities of triple phosphate and oxalate crystals may be present • traces of glucose and protein can be present in normal rabbit urine • rabbits produce two types of faeces that differ in composition. the first step in faeces' examination is to determine which type of faeces has been collected • hard faeces are composed of compressed strands of indigestible fibre • soft faeces or caecotrophs are composed of a strong smelling paste rich in bacteria • some rabbits with diarrhoea excrete a mixture of hard and soft faeces signifying a failure of the proximal colon to separate the indigestible and digestible fractions of the diet • coccidial oocysts and ova from passalurus ambiguus may be seen in both types of faeces • a non-pathogenic yeast saccharomyces guttulatus may be seen in large numbers and can be mistaken for coccidial oocysts (see figure 6 .2) • clostridium spiroforme can sometimes be seen in large numbers on a gramstained smear of faeces collected from a rabbit suffering from enterotoxaemia. exudate from crusty lesions may be examined for the presence of mites. although psoroptes cuniculi normally inhabits the ear canal, the mite can be found in other areas of the body such as the perineal skin folds and can be seen on microscopic examination of the exudate from affected areas. skin scrapings may be required to demonstrate sarcoptic mange mites in scabies cases that are characterized by intense pruritus and crusty lesions. dark field microscopy can be used to look for treponema cuniculi organisms in crusty lesions suggestive of rabbit syphilis (section 16.5.9). the organism is a motile corkscrew-shaped spirochaete. lesions are found on the mucocutaneous junctions of the anus and genitalia or on the nose, lips and eyelids (see plate 10). the lesion is abraded with a sterile saline-soaked swab. serum from the lesion is then expressed on to a slide and covered with a cover slip before being examined immediately (digiacomo et al., 1984) . the slide can be placed in a moisturized chamber. the differential diagnosis of exudative skin lesions in rabbits can be difficult and histopathological examination of biopsy specimens may be required. cerebrospinal fluid can be collected from the cisterna magna of rabbits in a similar manner to other animals. some normal parameters are summarized in table 6 .5. depressed glucose concentrations (< 56 mg/dl) may be indicative of purulent inflammation (kusumi and plouffe, 1980) . key points 6.6 • cheyletiella parasitovorax, fur mites (leporacus gibbus), lice (haemodipsus ventricosus), fleas or flea dirt can be seen in skin brushings from affected animals • acetate tape strips can be used to detect all stages of the life cycle of cheyletiella parasitovorax • a trichogram can differentiate between dermatophytosis and barbering. mites, cuticles and egg cases may be seen attached to the hair shafts • psoroptes cuniculi, the rabbit ear mite, can sometimes be seen on microscopic examination of smears from skin lesions in other parts of the body such as the perineum • dark field microscopy may be used to detect treponema pallidum organisms. minerals and electrolytes part 1 influence of dietary calcium/phosphorous ratio on blood calcium, phosphate and magnesium during gestation in the rabbit. (article in french, english abstract) plasma calcium, inorganic phosphorus and magnesium levels in pregnant and lactating rabbits clinical pathology of the domestic rabbit comparative vertebrate endocrinology physiology interpretation of laboratory results for small animal clinicians laboratory diagnosis of clostridium spiroforme-mediated diarrhoea (iota enterotoxaemia) of rabbits glycosylated haemoglobin levels in a colony of spontaneously diabetic rabbits (abstract) the calcium tolerance of growing rabbits calcium requirement of growing rabbits common diseases and medical management of rodents and lagomorphs clinical chemistry serological and histological studies on adult rabbits with recent naturally acquired encephalitozoonosis leucocyte subpopulations in cerebrospinal fluid of normal rabbits (abstract) clinical course and treatment of venereal spirochaetosis in new zealand white rabbits viral diseases untersuchungen zur klinik und therapie der encephalitozoonose beim heimtierkaninchen (article in german recent findings and future perspectives of digestive physiology in rabbits: a review effects of t-2 toxin on ovarian activity and some metabolic variables of rabbits adjustment of total serum calcium concentration for binding to albumin and protein in cats: 291 cases (1986-1987) biochemical parameters of clinical significance in rabbits. ii. diurnal variations rabbit hematology selected drug dosages and clinical reference data effect of dietary calcium on bone density in growing rabbits total serum calcium concentrations in rabbits effect of ascaris suum infection on blood picture of rabbits in relation to the production of immunity (abstract) textbook of medical physiology parathyroid hormone, haematological and biochemical parameters in relation to dental disease and husbandry in pet rabbits clinical biochemistry and haematology clinical pathology values in pregnant and non-pregnant rabbits rabbit and ferret parasite testing vascular access ports for chronic serial infusion and blood sampling in new zealand white rabbits isolation of encephalitozoon cuniculi from urine samples evaluation of the effect of pentobarbitone anaesthesia on the plasma potassium concentration of the rabbit and the dog effect of intraosseous saline infusion on hematological parameters chronic plumbism in rabbits: a comparison of three diagnostic tests (abstract) interpretation of selected clinical pathology values in ferrets and rabbits ferret and rabbit endocrine disease spontaneous diabetes mellitus in the new zealand white rabbit rabbits. in an atlas of laboratory animal haematology pasteurellosis in rabbits. compendium on continuing education lead induced toxicosis in two domestic rabbits redistribution of lymphocytes after cortisol administration (abstract) the redistribution of lymphocytes during adrenaline infusion. an in vivo study with radiolabelled cells (abstract) alteration of sleep in rabbits by staphylococcus aureus infection haematological effects of exposure to three infective agents in rabbits physiological stabilization of rabbits after shipping arterial disease in chronic renal failure. an experimental study in the rabbit prevalence of dermatophytes in asymptomatic guinea pigs and rabbits changes in plasma parameters in rabbit does as a function of their physiological state and feed rationing acute phase reactants ceruloplasmin and haptaglobin and their relationship to plasma prostaglandins in rabbits bearing the vx2 carcinoma (abstract) regulation of calciotropic hormones in vivo in the new zealand white rabbit neoplastic diseases small animal clinical diagnosis by laboratory methods biochemical values of normal rabbit serum medicine of rabbits and rodents, 4th edn. williams and wilkins. hinton, m., jones d.r.e., festing m.f.w. (1982). haematological findings in healthy and diseased rabbits, a multivariate analysis. lab anim., 16, 123-129. hoefer, h.l. (2000). rabbit weisbroth s.h., flatt r.e., brewer n. (1984).biology and diseases of rabbits. in laboratory animal medicine. pp 207-237. academic press. krishna l., dawra r.k., vaid j., gupta v.k. (1991). an outbreak of aflatoxicosis in angora rabbits (abstract). key: cord-296717-ay4wcmk3 authors: long, wen; zhao, manyi; tang, yeran title: can the chinese volatility index reflect investor sentiment? date: 2020-10-20 journal: nan doi: 10.1016/j.irfa.2020.101612 sha: doc_id: 296717 cord_uid: ay4wcmk3 the volatility index is the implied volatility calculated inversely from the option prices. this study investigates whether the official chinese volatility index, ivx, can represent investor sentiment. in order to describe investor sentiment comprehensively, we build a three-dimensional investor sentiment measurement system composed of macro, meso and micro level, and decompose ivx into three components to obtain short-term, medium-term fluctuations and long-term trend by eemd method. the relationships between ivx, its components and sentiment indexes at each level have been analyzed separately, and the empirical results reveal all components of ivx can reflect the investor sentiment at the corresponding level but to which extent they can reflect are not the same. further we introduce the mixed-frequency dynamic factor analysis to extract the common sentiment factor, which shows stronger correlation with contemporaneous ivx, compared with the sentiment indexes at each level. the adl model in robustness check also demonstrates the results. our findings confirm ivx can represent the common sentiment and expectations of chinese investors in different time scales. since the stock came into being, its risk characteristics have been the focus of investors. risk refers to the uncertainty of return, which is generally defined as volatility. according to different calibers and methods, volatility can be categorized into historical volatility, predicted volatility, and implied volatility. historical volatility is calculated as the average deviation from the average price of a financial instrument in the given time period. predicted volatility is the volatility estimated based on asset conditions, economic situation, historical experience, etc. when calculating the option price, it is brought into the option pricing formula to obtain the price. implied volatility is the volatility inversely derived by putting the market price of the option and other known parameters except volatility into the black-scholes option pricing model. investor sentiment refers to a belief formed by the expectation of future cash flows and investment risks of assets, which yet does not fully reflect the existing facts (wurgler and baker, 2006) . this kind of belief is not only influenced by the fundamentals of assets and the information transmission of capital market, but also strongly related to the education, personal experience and personal preference of investors. therefore, for the same asset, different investors will hold different beliefs, namely sentiment. herd effect and other phenomena show that sentiment is contagious, which will lead to consistent actions among people. due to this irrational behavior, if limited arbitrage exists, asset prices will have systematic bias. in 1993, chicago board options exchange (cboe) delivered the world's first volatility index vix, which is the implied volatility calculated using the s&p 500 index. in 2016, shanghai stock exchange of china also announced the first official certain advantages in vix futures pricing. kanniainen et al. (2014) price s&p500 index options based on the garch model and the vix index. basher at el. (2016) compare the effects of hedging market risks with indicators such as vix index and oil prices. some researchers also study the volatility index in other stock markets (siriopoulos and fassas, 2012; badshah i. et al., 2018) . due to the late release of the chinese volatility index ivx, there are few studies on ivx. the related researches mainly focus on three aspects: the prediction ability of ivx (qiao et al., 2019) , the relationship between ivx and the yield, including the "leverage effect" yue et al., 2019) , and the inclusion of volatility index as an indicator in the sentiment index construction system (xu and zhou, 2018) . in order to measure the expectation of investor, scholars put forward the concept of investor sentiment index. previous methods for obtaining investor sentiment indicators can be divided into three categories: (1) investor sentiment index composed of questionnaires and interviews. american studies mainly use the university of michigan consumer survey sentiment index, while chinese indexes mainly include "cctv watch index", good and bad index by stock market trend analysis weekly, -long and short polls‖ by huading, consumer confidence index et al. (lemmon, 2006; schmeling, 2009) . although the survey method aims to directly quantify investor sentiment, its operation cost is high, the data frequency of constructing sentiment index is relatively low, and the time span is relatively short. (2) indirect indicators obtained from the historical data of the stock market. this method mainly uses historical data of several proxies that can be observed on the stock market to synthesize the investor sentiment index. at present, the academic community mainly adopts the method proposed by baker and wurgler (2007) . they use principal component analysis to extract a sentiment index from six variables of the stock market, including closed-end fund discount, nyse share turnover, the number and average first-day returns on ipos, the equity share in new issues, and the dividend premium. although the approach of using indirect indicators in the stock market is more time-saving and labor-saving than survey and interview, it is limited by some conditions such as index selection and synthesis methods. furthermore, using market j o u r n a l p r e -p r o o f variables as proxies for investor sentiment may not only reflect investor sentiment, but also reflects the equilibrium results after the interaction of sentiment and other economic factors (da et al., 2014; qiu and welch, 2004) . (3) analysis of information on the internet. many researchers start with the internet information and apply text analysis to analyze the information released by investors on social platforms or searched content to get the investor sentiment. this method avoids the shortcomings of the first two methods, but is susceptible to network noise information. text data mainly includes yahoo finance posts (antweiler and frank, 2004; das and chen, 2007; kim and kim, 2014; tsukioka et al., 2018) , wechat (shi et al. 2018) , weibo platform (checkley et al., 2017; renault, 2017) , twitter (behrendt and schmidt, 2018; li et al., 2017) and google search (da et al., 2014; gao et al. 2019) , etc. the purpose of this paper is to investigate the ability of the chinese volatility index ivx to represent investor sentiment. since ivx is computed given the option prices and the prices reflect the expectations of the stock market by the investors, thus ivx reveals the investors' predictions towards future of the stocks. and the predictions reflect the investors' sentiment. therefore, ivx can simultaneously represent the investors' sentiment in theory. li et al. (2019) find that ivx is negatively associated with the price and return of shanghai 50etf separately, which indirectly proves that ivx could reflect investors' panic sentiment, but did not directly measure the sentiment. regarding the sentiment representation of the vix created by cboe, most of the researches take it as the known facts and conduct research on this basis (pan, 2018; smales, 2017; yang et al., 2016) . however, there is little literature that confirms vix has this effect. some of them indirectly proves it by analyzing the negative correlation between vix's change and the return rate of the underlying index (smales,2016; whaley, 2000; whaley, 2009) . the researches which directly prove it only involve the relationship of volatility index on sentiment index based on news, social media or other single-level sentiment (pineiro et al., 2016; smales, 2014; zhang, 2011) . in order to obtain fluctuations on different time scales, this paper will use ensemble empirical mode decomposition (eemd) to decompose ivx. eemd provides a j o u r n a l p r e -p r o o f feasible way to deal with non-linear and non-stationary data. it consists in a local and fully data-driven separation of a signal in fast and slow oscillations (torres et al., 2011) . it has been widely used in industry and resources (wang et al, 2014; wang et al.,2013; lei and zuo, 2009) . meanwhile, zhang (2008 ) and yu et al. (2008 have successfully applied this idea to the field of social science, and prove that the integrated empirical mode decomposition method is a useful approach for financial time series analysis. the contributions of this paper include the following three aspects: (1) this study seeks to examine systematically on whether ivx has the ability to represent sentiment, and analyze it at the macro, meso and micro levels, while previous studies rarely discuss this issue, especially for the newly released and short-lived chinese volatility index ivx. (2) this paper constructs a three-dimensional comprehensive measurement system of investor sentiment, which is composed of three levels: macro-economy, stock market and individual opinions. the frequency is monthly, weekly and daily, respectively, including rational and irrational components, so our measure system can capture more information about the investor attitudes. however, most of the previous studies only focus on one level of sentiment, such as micro-blog sentiment from the micro perspective. (3) by employing dynamic factor analysis on different sentiment indexes with mixed-frequency to extract the common factor, we investigate whether ivx can comprehensively represent investor sentiments at different time scales. the remaining of the article is organized as follows. section 2 introduces ivx and compiles sentiment indexes at different levels. section 3 decomposes ivx by eemd to get three components and analyzes their representation on investor sentiment at different levels. section 4 applies mixed-frequency dynamic factor analysis to obtain a common sentiment factor, and discuss its relationship with ivx. section 5 is the robustness test. finally, this study concludes with a few remarks. j o u r n a l p r e -p r o o f chinese volatility index ivx, released by the shanghai stock exchange, can be traced back to february 9, 2015, and was suspended on february 22, 2018 for unknown reasons. using model-free methodologies similarly to vix, the ivx is estimated from the bid and ask prices of the underlying options for shanghai 50etf. this paper collects all the data during the ivx release period, with 740 effective observations from the wind database. and 50etf's variation δ50etf in the sample period, respectively. it is clearly visible from the graphs that when the chinese stock market falls sharply in mid-2015, ivx rises to a historical peak of 63.79, which suggests ivx is negatively related to the underlying assets to a certain extent. the left vertical axis depicts δivx and the right depicts δ50etf. (excel format , 1-column fitting image)， table 1 reports the summary statistics for ivx and δivx. the mean value of ivx is 23.879 and δivx is approximately zero. the two are both positively skewed. δivx presents excess kurtosis while ivx does not. the above two points suggest that big positive changes of ivx occur more frequently than large negative changes and vice versa. the reported first-order autocorrelations show that ivx is highly persistent, but δivx is not. the augmented dickey fuller (adf) and pp tests on the levels, reject the null hypothesis of unit root for δivx, while accepts for ivx. hypothesis at the 1%, 5% and 10% significance levels, respectively. investor sentiment reflects the expectation of investors for the future economy and stock market. this paper builds a three-dimensional measurement system of investor sentiment, which depicts sentiment from three levels: macro, meso and micro. among them, macro level quantifies investor sentiment towards the whole economy; meso level measures investor sentiment towards the stock market; and micro level shows investor sentiment towards each individual stock. the three levels of sentiment index are monthly, weekly and daily data separately. in order to reflect the latest situation of economic development in time, taking into account the representativeness of china's macroeconomic cycle variables and the availability of data, this paper uses the purchasing manager index (pmi) and production price index (ppi) on the production side, the consumer satisfaction index (si) , consumer price index (cpi) and total retail sales of consumer goods (rscd) on the consumer side, and the consistent index of business climate index (cbci) indicating the economic boom and bust issued by the china economic prosperity monitoring center, to form a composite macro-level index of sentiment. these data are monthly and from wind database. (excel format , 1-column fitting image)， further, considering the fact that some variables take longer to reveal the same sentiment (baker and wurgler, 2006) , if the variables are synthesized directly in the same period, the composite index will not effectively reflect the market sentiment movement. referring to baker and wurgler (2006) , this paper uses the following two (excel format , 1-column fitting image)， the formation of the investor sentiment index at the meso level is mainly based on the method of wurgler (2006, 2007) . the six proxy variables they selected are: the closed-end fund discount, nyse share turnover, the number and average first-day returns on ipos, the equity share in new issues, and the dividend premium. however, compared with the us stock market, the chinese stock market has its own particularities, mainly containing the following three points: (1) almost every ipo will hit a daily limit on the first day of listing, which means the average value for first day's earnings is almost 44%. among the above variables, in theory, the price of closed-end funds should be consistent with the value of the unit net assets of the stock portfolio. however, in reality, closed-end funds often trade at a discount (lee et al., 1991) . it is generally believed that the higher the absolute value of the discount rate, the less optimistic view investors take about the market outlook. since fddr is negative in normal conditions, it is positively correlated with investor sentiment. to signifies market liquidity. under the restrictions of market short selling and the participation of irrational investor, high liquidity is often accompanied by overvaluation, which has positive correlation with investor sentiment. in addition, pe and nia are also positively associated with sentiment. in order to control the impact of fund size, we adopt the following criteria for fund table 4 reports that there is a certain degree of association between the four sentiment agent variables. j o u r n a l p r e -p r o o f (excel format , 1-column fitting image)， we use the same method as section 2.2.1 to select the proxies or their lags, and then perform principal component analysis to calculate the meso-level sentiment index. the selected terms are to t-1 , pe t , nia t-1 , and fddr t . taking of the sample variance respectively, thus the two factors with cumulative rate of 81.351% capture much of the common variation. therefore, we take weighted average of the first and second principal components as meso sentiment index, without the macro influence. the resulting sentiment index mesi is shown in eq. (2), and its variation throughout the sample period is shown in fig. 6 . the index falls sharply in the mid-2015, after then appears to be less volatile. (excel format , 1-column fitting image)， based on investors' online opinion posts on the internet social media, the investor sentiment at micro level is extracted and analyzed from massive text data. in terms of data acquisition, we use web crawler tools to grab the information on the east money stock forum, which has a considerable number of visits and influence among the stock forums in china. according to ranking of visitor volume by alexa's website and baidu weight rankings, east money stock forum currently ranks first in the major there are usually two types of sentiment classification, based on sentiment dictionary or classifier, respectively (liu et al. 2012) . it is hardly possible to meet the professional needs in the field of finance by using a general sentiment dictionary in natural languages, and empirical evidence has shown that the classification results obtained by the latter are better than the former (wu et al., 2014) . therefore, on the grounds of previous research, we adopt machine learning algorithm and choose svm classifier to identify investor sentiment and calculate the micro sentiment index. considering that online posts contain a great deal of noise, we use two-step classifier for sentiment analysis (shi et al., 2018) . the first step is to get rid of noise by separating the text into noise and non-noise, and the second step is to divide non-noise text into bullish and bearish ones. we use several technologies including data cleaning, text representation, feature extraction and classification, to compute investor sentiment index with individual sentiments, which will be applied into following study. (1) data cleaning the posts on the online stock forum involve a lot of punctuation, noise, etc. which cannot be directly used to sentiment analysis. therefore, in the data cleaning process, we first remove the punctuation and gibberish, and then operate word segment. (2) text representation in order to enable the computer to read text, words must be changed into digital j o u r n a l p r e -p r o o f data for computer processing. in this paper, word2vec, which is commonly used in academia, is applied to represent words. it can calculate word vectors according to the context of words, fully capture the semantic information of the context, and has good performance in text classification (lilleberg et al., 2015; wolf et al., 2014) . then we use tf-idf (term frequency-inverse document frequency) algorithm to compute the weight of words in short text, and provide weighted word2vec text vector. word2vec is a tool based on deep learning and released by google in 2013 . this neural network includes two architectures: continuous bag-of-words model (cbow) and skip-gram. the former predicts the current word based on the context, while the latter predicts surrounding words given the current word (mikolov et al., 2013) . compared with the cbow model, skip-gram has higher semantic accuracy, higher computational complexity, and longer model training time. in this paper, the skip-gram model is used to predict words through context, the mathematical representation (3) is as follows: where the input is a word in the corpus. the main idea of tf-idf is if a word or phrase appears frequently in one article and rarely in other articles, we infer that this word or phrase shows good performance on distinguishing different text information. formula (4) shows tf-idf is the product of tf term frequency and idf reverse document frequency. where , is the number of times that the word appears in the file , while the denominator ∑ , is the sum of number of times that all the words appear in the file , |d| represents the total number of files in the corpus, and |{ : ∈ }| represents the number of files containing . by adding the weighted word2vec vector of words in document, we get the new j o u r n a l p r e -p r o o f vector r( ) of document : where 2 ( ) is the word2vec vector of word . (3) classifiers for sentiment identification we use support vector machine (svm) to identify investor sentiment, which has already become an important approach for classification due to its outstanding performance (cortes and vapnik, 1995; deng et al., 2012) . in the first step of "noise elimination" identification, non-noise data is labeled with +1 and noise data with -1. in the second step of "bullish-bearish" identification, bullish sentiment is labeled with +1 and bearish sentiment with -1. through the procedures of classifying the text, we can identify the investors' attitudes towards individual stock. table 4 reports the classification accuracy rate obtained by the 10-fold cross validation method. it indicates that the classification accuracy and recall rate based on the svm algorithm have reached more than 70%. after identifying individual investor sentiment, we can combine the views of all individual investors into micro investor sentiment index. in accordance with the method in previous literatures (antweiler and frank, 2004; kim and kim, 2014; wu et al., 2014) , we define as total bullish posts in time interval t, and as total bearish posts in time interval t. the calculation for sentiment index is: eq. (6) is used to compose the csi 800 daily micro investor sentiment index, named as micsi, which can reflect the comparison of investors' long or short views. the higher the investor sentiment index, the more investors hold positive expectation on the future stock market. average, investor sentiment is towards bearish, which demonstrates the viewpoint of investors' irrational biases in behavioral finance (odean, 1998) . (excel format , 1-column fitting image)， the u.s. volatility index vix has the ability of reflecting investor sentiment, thus it is known as "the investor fear gauge". in order to study whether the chinese volatility index ivx possess this capability, we conduct the following research. first, we decompose ivx into short-term, medium-term and long-term fluctuations, considering macro-level sentiment is the investor's sentiment towards the market, which lasts for a long time, and the persistence of meso level and micro level sentiment decreases in turn. then we explore the relationship between the decomposed ivx and corresponding sentiment respectively. compared to that, the relationship between the ivx and sentiment is also examined on each level. with the above process, we can verify whether ivx is a proper representation of investor sentiment. previous studies show short-term volatility clustering (gray,1996; cont, 2007) and long-term mean reverting of the volatility (bollerslev and mikkelsen, 1996; arav et al.,2018) , which indicates the characteristics of stock market on different time scales are not consistent. in order to obtain the fluctuations on different time scales, we need to decompose ivx. since the duration of three levels of sentiment is different, if ivx has the ability of representing sentiment, the short-term with high frequency, medium-term fluctuations with low frequency and long-term trend obtained after decomposition should have a relationship with corresponding sentiment at the level of macro, meso and micro. we use eemd to operate decomposition. compared with wavelet analysis, it can avoid the instability of the result caused by manual selection of wavelet function. moreover, compared with fast fourier transform, it can realize the analysis of high-frequency data volatility. therefore, eemd is chosen to decompose ivx. (1) eemd model the eemd algorithm can be summarized as follows: step 1. initialize the number of realizations and amplitudes of added white noise. set m equal 1. step 2. perform the mth emd decomposition. 1)add white noise ( ) with the given signal ( ). ( ) = ( ) + ( ) j o u r n a l p r e -p r o o f where ( ) is the white noise added at the mth time, and ( ) is the signal containing white noise at the mth time. 2)after employing emd to decompose the signal ( ), we get a group of imf , ( = 1,2, ⋯ , ), where , is the nth imf in the mth decomposition. 3)if m<m, then go back to step 1) and let = + 1. repeat steps 1) and 2) until m equals m. step 3. compute the population mean in the mth decomposition. step 4. take n imfs in the mth decomposition, i.e. ( = 1,2, ⋯ , ) as the final imfs. (2) fine-to-coarse reconstruction in practice, fine-to-coarse reconstruction is used to get short-term, medium-term fluctuations and the long-term trend of the original signal (zhang et al., 2008) . the algorithm is as follows: step 1. except for the residue, calculate the mean of the sum of 1 to for each component. step 2. identify for which i the mean departs from zero significantly. step 3. if i is identified as a significant change point, partial reconstruction with imfs from this to the end is identified as the medium-term fluctuations, the partial reconstruction with other imfs is identified as the short-term fluctuations and the residue as the long-term trend. we get five the imfs and residue by decomposing ivx, of which the variations are depicted in fig. 8 . shows the average of imfs as a function of imfs index k. it can be observed that the average of the fine-to-coarse reconstruction departs significantly from zero at imf4. furthermore, we use t-test to demonstrate the result (the significance value is set at 0.05). therefore, we take the partial reconstruction with imf1, imf2 and imf3 as short-term fluctuations and the partial reconstruction with imf4 and imf5 as medium-term fluctuations. short-term fluctuations have the characteristics of small amplitudes, and each steep rise or fall in medium-term fluctuations corresponds to a significant event. the residue retains as long-term trend which is slowly varying around the long-term mean. the variation of three components including hivx, livx, tivx is shown in fig. 10 . in this section, we plot the scatter diagrams of sentiment index and ivx or its components on the level of macro, meso and micro respectively. then we calculate their correlation coefficients, and analyze the sentiment representation of ivx at different levels. j o u r n a l p r e -p r o o f in the above discussion, we decompose ivx into three components with different characteristics, and then study the sentiment representation of different components on each level. it is concluded that only the component at the macro level has a strong ability to reflect sentiments. however, the weekly and daily data account for the majority of the research of financial market, to which the corresponding levels are meso and micro. according to our study, the correlations between ivx or its components and sentiment index are low at the meso and micro levels (the maximum absolute values are about 0.2 and 0.4), which is not enough to prove that ivx can reflect investor sentiment. but meanwhile, it is noted that the correlations between the components of ivx at different levels and the corresponding sentiment index are almost stronger than those between ivx and the sentiment index, which suggests that ivx also reflects the sentiments and expectations of investors at different levels to some degree. therefore, it is necessary to combine the sentiment indexes of different levels together to test how the volatility index ivx represents the investor sentiment. the dynamic factor model is mainly used in the modeling and analysis of macroeconomics. in recent years, it has received more and more attention. its theoretical basis can be traced back to lucas (1977) . he finds that a single macro variable is not enough to reflect all the characteristics of economic fluctuations, and the evolution of fluctuations accompanies with the interaction and coordinated j o u r n a l p r e -p r o o f movement of many macro variables. the main advantage of the dynamic factor model is that it can extract dominating factors from a variety of macro data through metrological methods, that is, assuming that the mutual movement of many macro variables can be explained by an unobservable common random factor, which can better reflect the variation and characteristics of economic (bańbura and rünstler, 2007; koopman and harvey, 2003) . in this paper, we think that all levels of sentiment are influenced by not only specific factors but also common sentiment factors. section 3.2 shows that ivx has different correlations with the sentiment index at three levels, hence it should also have potential relationship with the common sentiment factors. therefore, the dynamic factor model is employed on the extraction of common sentiment factors. considering that the frequencies of sentiment indexes at three levels are different, we further combine the mixed-frequency modeling method (aruoba et al., 2009) , and build the mixed-frequency dynamic factor model to extract the common sentiment factor. the frequencies of sentiment indexes at macro, meso and micro level are monthly, weekly and daily, respectively. in order to construct a high-frequency daily common sentiment factor, denotes the unobservable common sentiment factor at day t, which evolve daily with ar(p) dynamics, where is a white noise innovation with unit variance. interested in tracking and forecasting real activity, we use a single-factor model, i.e. is a scalar. denotes the sentiment index at day t, which depends linearly on and also on lags of , and i represents the level of sentiment index, which equals 1, 2, and 3 in the case of micro, meso and macro, respectively. where coefficient for , reflects the effect of the common sentiment factor on the sentiment index . where 3, is missing for t ∈ a and 2, is missing for t ∈ b. based on eq. (13), eq. (11) is transformed into the following form: where is a 3×3 identity matrix when there is no missing value in observed vector , is a 2×3 matrix deleted ith row when the ith value in is missed, and is a 1×3 matrix when two of values in is missed. in eq. (14), there is no problem with missing data. hence for the state space model composed of eq. (12) and eq. (14), the maximum likelihood estimation based on kalman filter is used to obtain the consistent estimators, and simultaneously, kalman filter and smoothing estimators of state variable can be calculated. since eq. (12) and eq. (14) demand the variables in state space model should be stationary, we first take the adf test on three sentiment indexes of different levels. table 6 reports macsi is non-stationary, whereas the other two are opposite. in an attempt to maintain the data properties, we take first difference of the variables with trend. finally, all variables are standardized eliminating the influence of dimension. j o u r n a l p r e -p r o o f table 6 also gives some descriptive statistics of three sentiment indexes. according to aic、bic, we set p equals 1. hence the measurement and transition equations become on the basis of the optimal estimation of unobserved state vector, table 7 gives the estimation values of related parameters. the estimates are all significant, and those of β1, β2, and β3 are positive, which suggests the effects of the common sentiment factor on the three sentiment at different levels are consistent. ρ is significantly positive, indicating the first-order common sentiment factor has a positive effect on the current one, i.e. investor sentiment is influenced by inertia, which will lead to conservatism bias in behavioral finance: investors' insufficient response to new information, resulting in short-term momentum effect. according to the estimations of the common sentiment factor obtained by kalman smoothing, fig. 12 represents its time series. we calculate the correlation coefficients between the common sentiment factor and ivx as shown in table 8 . in comparison with table 5 , the three correlation coefficients show a significant negative correlation between the common sentiment factor and ivx. the absolute values are larger than the correlation coefficients between the meso and micro level sentiment indexes and the corresponding ivx component, and also significantly larger than the correlation coefficients between the two sentiment indexes and ivx. it suggests that compared with the sentiment index of each level, the common sentiment factor we extract is more closely related to ivx. therefore, we believe that ivx represents more a mutual portion of all levels of sentiments than the diversity. besides, compared with lags ranging from 1 to 3, ivx has the strongest correlation with the contemporaneous common sentiment factor, indicating that ivx can reflect the current mutual sentiment. in the previous section, we use three correlation coefficients to discuss the sentiment representation of ivx. in this section, we will further investigate the relationship of ivx and sentiment based on econometric model. according to the formula of ivx, it is affected by risk-free rate, option prices and strike prices. here we take shibor 1-year rate as the risk-free rate and average strike price of all near-term and next-term options as the strike prices. as for option prices, except for investors' sentiment, it can be influenced by other factors. therefore, the explanatory variables will include lagging ivx or its component. the polynomial order is decided by maximum of residual sum of squares divided by degrees of freedom, recommended by mitchell and speaker (1986) . at the macro level, all monthly variables, including ivx (mivx), average strike price (msp), shibor 1-year rate (msr) and macsi are tested non-stationary, while their first differences (which are denoted as dmivx, dmsp, dmsr and dmacsi, respectively) are the opposite. the results are shown in table 9 . the estimations are presented in table 11 . for equation (21), current sentiment has significantly strongest negative impact on mtivx, compared with other periods. for equation (22), the results suggest current sentiment influence ivx most strongly. these inferences indicate ivx and its low-frequency component can reflect investors' sentiment simultaneously. note: ***, **, and * denote rejection of the null hypothesis at the 1%, 5% and 10% significance levels, respectively. at the meso level, we test weekly ivx (wivx), average strike price (wsp), shibor 1-year rate (wsr) and their first differences (which are denoted as dwivx, dwsp and dwsr, respectively), as well as mesi and weekly livx(wlivx). table 12 reports summary statistics for the variables. table 13 reports the estimations. it shows that current sentiment cannot effectively affect ivx or its component. for both wlivx and ivx, first-order lagging sentiment has the significantly negative impact. thus, ivx and its medium-frequency component represent previous sentiment, consistent with our conclusions. note: ***, **, and * denote rejection of the null hypothesis at the 1%, 5% and 10% significance levels, respectively. we define the regression model as the following way: we estimate the equations, as shown in table 15 . for equation (25), only 0 1 is significantly negative among 1 , which suggests ivx reveals more the current j o u r n a l p r e -p r o o f sentiment. although 2 1 and 3 1 are significantly positive, but the sum of all the coefficients on sentiment is negative, which means ivx can reflect investor sentiment. for equation (26), 0 2 is negatively larger than 1 2 . note: ***, **, and * denote rejection of the null hypothesis at the 1%, 5% and 10% significance levels, respectively. after discussing ivx and investors' sentiment at different levels, we analyze its representation of common sentiment factor (csf). the factor is tested stationary with adf test of -6.625 and pp test of -6.427. the other variables have been tested during the previous investigation. then we run the following regression model: note: ***, **, and * denote rejection of the null hypothesis at the 1%, 5% and 10% significance levels, respectively. table 16 shows that our conclusions hold, and only the current sentiment can significantly negatively impact ivx. therefore, the application of adl confirms our findings on ivx' s representation to investor sentiment, for different levels or the common sentiment factor. in this paper, we investigate whether chinese volatility index ivx can represent investor sentiment. most of the previous researches treat the sentiment representation of the volatility index as a known fact, or only analyze a certain level of sentiment. however, we construct a three-dimensional investor sentiment measurement system, and use eemd method to decompose ivx into three components, i.e. short-term fluctuations, medium-term fluctuations and long-term trend. we study the relationship of ivx and micro, meso and macro sentiments respectively. further, in the framework of dynamic factor model, with mixed-frequency data, a common sentiment factor is constructed to verify the representation of ivx to mutual sentiment. finally, we use adl to test our results. our findings mainly include the following aspects: first, the ivx's components at the macro, meso, and micro levels all have certain ability to represent sentiment. among them, the ivx decomposition component at the macro level has the strongest ability to represent the current sentiment; the one at the meso level reflects the first-order or second-order lagging sentiment; the one at the micro-level can represent the current sentiment, but the effect is weak. overall, ivx tends to more reflect the macro sentiment, yet has a weaker ability to represent sentiment at meso and macro levels. second, this paper finds that compared with the sentiment index at each level, the common sentiment factor has a stronger relationship with ivx. this fact implies that j o u r n a l p r e -p r o o f ivx more represents the mutual composition of sentiment at different levels, and ivx has the strongest correlation with the current common sentiment factor. hence, we believe that ivx can represent mutual sentiment of all levels to a certain extent. the underlying asset of ivx is shanghai 50etf option, and the pricing of option should theoretically reflect the expectations and sentiments of investors. that is confirmed to be effective, for we have verified the ability of ivx to represent sentiment in this paper. since chinese volatility index ivx is still in its infancy, the empirical evidence of this paper would contribute to providing a theoretical basis for the development of ivx. our results show ivx can comprehensively reflect the sentiment and expectations of investors on different time scales. the measurement of investor sentiment is complicated from the macro level to the micro level, while ivx can be calculated at high frequency. thus, we strongly recommend resuming the release of ivx. it will help investors better understand the overall financial market situation, allocate asset portfolios, and perform risk management, and is also beneficial for regulatory authorities to deepen their understanding of changes in the domestic financial market and effectively conduct financial supervision, so as to achieve the healthy and sustainable development of the financial market and ensure the stable operation of the macroeconomic system. hedging emerging market stock prices with oil, 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a new approach for crude oil price analysis based on empirical mode decomposition estimating the impact of extreme events on crude oil price: an emd-based event analysis method sentiment analysis key: cord-006435-4o8uu6d5 authors: kivity, shaye; katz, uriel; daniel, natalie; nussinovitch, udi; papageorgiou, neophytos; shoenfeld, yehuda title: evidence for the use of intravenous immunoglobulins—a review of the literature date: 2009-07-10 journal: clin rev allergy immunol doi: 10.1007/s12016-009-8155-9 sha: doc_id: 6435 cord_uid: 4o8uu6d5 intravenous immunoglobulins (ivig) were first introduced in the middle of the twentieth century for the treatment of primary immunodeficiencies. in 1981, paul imbach noticed an improvement of immune-mediated thrombocytopenia, in patients receiving ivig for immunodeficiencies. this opened a new era for the treatment of autoimmune conditions with ivig. since then, ivig has become an important treatment option in a wide spectrum of diseases, including autoimmune and acute inflammatory conditions, most of them off-label (not included in the us food and drug administration recommendation). a panel of immunologists and internists with experience in ivig therapy reviewed the medical literature for published data concerning treatment with ivig. the quality of evidence was assessed, and a summary of the available relevant literature in each disease was given. to our knowledge, this is the first all-inclusive comprehensive review, developed to assist the clinician when considering the use of ivig in autoimmune diseases, immune deficiencies, and other conditions. abstract intravenous immunoglobulins (ivig) were first introduced in the middle of the twentieth century for the treatment of primary immunodeficiencies. in 1981, paul imbach noticed an improvement of immune-mediated thrombocytopenia, in patients receiving ivig for immunodeficiencies. this opened a new era for the treatment of autoimmune conditions with ivig. since then, ivig has become an important treatment option in a wide spectrum of diseases, including autoimmune and acute inflammatory conditions, most of them off-label (not included in the us food and drug administration recommendation). a panel of immunologists and internists with experience in ivig therapy reviewed the medical literature for published data concerning treatment with ivig. the quality of evidence was assessed, and a summary of the available relevant literature in each disease was given. to our knowledge, this is the first all-inclusive comprehensive review, developed to assist the clinician when considering the use of ivig in autoimmune diseases, immune deficiencies, and other conditions. intravenous immunoglobulins (ivig) are gamma globulins purified from the pooled plasma of thousands of donors, typically containing more than 95% of unmodified immunoglobulin g (igg) and only trace amounts of iga or igm. immune globulin products from human plasma were first used in 1952 to treat immune deficiencies. about 30 years later, paul imbach observed that patients with immune thrombocytopenia and agammaglobulinemia receiving immunoglobulins as immune replacement therapy recovered from their thrombocytopenia [1] . this was the first observation to suggest treatment of autoimmune diseases with ivig. later on, the ability to administer large quantities of immunoglobulin intravenously was gained owing to technological advances, among them the improvement in plasma fractionation. as a result, ivig slowly became an important treatment option in a number of diseases beyond primary immune deficiencies, including autoimmune and acute inflammatory conditions, most of them off-label indications. these indications have crossed over into almost every medical specialty. the us food and drug administration (fda) has approved the use of ivig for the following six conditions: primary immunodeficiencies, immune thrombocytopenic purpura (itp), kawasaki disease, hematopoietic stem cell transplantation, chronic b cell lymphocytic leukemia, and pediatric hiv. many offlabel indications have emerged; some of these new indications for ivig are based on solid clinical evidence; others are based on relatively few data or anecdotal reports (case series, case reports). this lack of firm evidence is due to the difficulty in performing appropriate clinical trials in diseases with low prevalence. there is a need for an evidence-based guidance for the use of ivig to help improve patient care consistency. another issue is the efficacy of different preparations of ivig. the fda has recommended the use of particular preparations of ivig for each labeled indication in accordance to the specific preparation used to demonstrate a beneficial effect. of course, there is selection bias since generally only a few preparations have been tested for a given disease. this is in recognition of the difficulty to reproduce the properties of an ivig preparation, which may vary from one manufacturer to the other due to differences in the donor population, number of donors, period of donation, production methods, virus/bacteria inactivation methods, etc. ivig properties may also vary from batch to batch made by the same manufacturer, complicating homogeneity even more. possible mechanisms of action of ivig in autoimmune and inflammatory diseases are: intact fc-dependent blockage of igg (as in itp), inhibition of membrane attack complexes (c5b-c9) and activated components c3b and c4b (as in kawasaki's disease), and anti-idiotypes against autoantibodies (as in acquired hemophilia due to autoantibodies against factor viii). ivig also contains various cytokines and natural antibodies that may act against pathogens, altered molecules, cells, autoreactive b cell clones, and tumors. ivig therapy reviewed the medical literature indexed in pubmed using specific terms for each specific disease/ condition and ("ivig" or "igiv" or "intravenous immunoglobuli*" or "gamma globuli*"). there was no limitation on language, year of publication, or publication status. we used all clinical data ranging from multicentered randomized controlled trials (rct) and meta-analysis to case reports. from each article, we extracted details of the study design, number of patients, type of intervention including the dose and ivig preparation used (if mentioned), and response to treatment. the relevant data were summarized in a hierarchical manner according to the study design and number of participants. when evidence was based on higher level of evidence studies, such as rcts, lower levels of evidence studies (such as case control studies) were disregarded. specific diseases were classified in tables according to the specialty they belong to and are followed by a short summary of recommendations, including the level of evidence and the strength of recommendation, as assessed by known guidelines (table 1 ) [2] . to our knowledge, this is the first comprehensive review which summarizes all up-to-date published data regarding the usage of ivig in autoimmune diseases, immune deficiencies, and other indications. the tables below summarize the clinical data gathered from studies dealing with ivig treatment for different conditions. each table is followed by our evidence-based recommendations for the usage of ivig. the following conditions refer to ( table 2) . level of evidence b systemic lupus erythematosus (sle) is a multisystemic disease with various manifestations. there is some evidence that ivig, given in patients without an increased risk for thromboembolic events or renal failure, is a safe and beneficial adjunct therapy for sle patients with systemic flare-ups who are resistant to or refuse conventional treatment (strength of recommendation iia). level of evidence c lupus myocarditis is an uncommon but severe complication of sle. there is no consensus on the specific treatment of sle myocarditis. most reports describe treatment with high-dose corticosteroids (cs), followed by either cyclophosphamide or azathioprine, in addition to conventional treatment for heart failure. there is little evidence that ivig is effective when immunosuppressive therapy fails (strength of recommendation iia). level of evidence b as with the treatment of itp, ivig seems to be useful in managing the bleeding complications of patients with lupus-associated thrombocytopenia (strength of recommendation i). level of evidence c standard therapy for subacute cutaneous lupus erythematosus (scle) includes cs (topical, intralesional, systemic), antimalarials, and other immunosuppressive agents. ivig may be considered in refractory cases, but more study should be done (strength of recommendation iia). level of evidence c kikuchi, also called histiocytic necrotizing lymphadenitis, is a rare benign disease. there is little evidence that treatment with ivig is superior to other antiinflammatory treatments (strength of recommendation iia). level of recommendation c giving the fact that adult still disease responds to cs and biological and anti-tumor necrosis factor (tnf) treatment, there is low-level evidence that ivig has an additional benefit for treatment of this disease (strength of recommendation iia). level of recommendation b immunosuppression with css and cytotoxic agents should be used in systemic vasculitis before considering ivig. there is however some evidence that ivig might be useful and therefore in refractory cases it may be worth to try (strength of recommendation iia). level of evidence b css is a rare disease. the initial management of css consists of high doses of cs. for patients with severe disease or those who are unresponsive to cs, treatment with ivig was warranted (strength of recommendation iia). level of evidence b infection with hepatitis c virus (hcv) may be associated with a variety of autoimmune phenomena causing a therapeutic dilemma for treatment with interferon alpha (ifn alpha), which stimulates autoimmune symptoms, or with cs, which may lead to an increase of viral load. treatment with ivig may act synergistically with table 1 recommendation guidelines [2] level of evidence a: more than one randomized controlled trial (rct)/meta-analysis b: a single rct or well-designed nonrandomized trial like prospective observational registries (case-controls, cohorts). c: expert consensus: this includes case reports and retrospective series; here, the expert decides based on his experience strength of recommendation class i: conditions for which there is evidence and/or general agreement that a given procedure/therapy is useful and effective class ii: conditions for which there is conflicting evidence and/or divergence of opinion about the usefulness/efficacy of performing the procedure/therapy class iia: weight of evidence/opinion is in favor of usefulness/efficacy class iib: usefulness/efficacy is less well established by evidence/opinion class iii: conditions for which there is evidence and/or general agreement that a procedure/therapy is not useful/effective and in some cases may be harmful (2) , acquired factor viii inhibitors (2) , acquired von willebrand disease (2) , pure red cell aplasia(3), pancytopenia (1), myelofibrosis (1), pneumonitis (2) , pleural effusion (1), pericarditis (2) , myocarditis (2) , cns improvement of sle-related condition level of evidence c mixed connective tissue disease (mctd) is an autoimmune condition which combines features of polymyositis, systemic lupus erythematosus, scleroderma, and dermatomyositis and is thus considered an overlap syndrome. there is scarce evidence that treatment with ivig helps to improve skin manifestations in mctd (strength of recommendation iia). level of evidence b systemic sclerosis is characterized by hardening and scarring of the skin and inner organs. there is some evidence that treatment with ivig helps to improve skin and systemic symptoms (strength of recommendation i). level of evidence a there is evidence that ivig is useful for the treatment of patients with severe chronic jra; its treatment may help also reduce the need for cs and other immunosuppressive therapy (strength of recommendation i). level of evidence b there is hardly any convincing evidence that ivig benefits patients with ra (strength of recommendation iib). level of evidence c there is some evidence that patients with ataxic sensory neuropathy refractory to immunosuppressive therapy will benefit from ivig (strength of recommendation iia). level of evidence b in patients with dm and pm that are resistant or partially responsive to conventional therapies, ivig was effective (strength of recommendation i). level of evidence a in ibm, ivig showed marginal improvements in muscle strength which were nonsignificant and thus were not recommended (strength of recommendation iii). level of evidence c behcet's disease is a multisystemic disorder presenting with recurrent oral and genital ulcers as well as ocular and central nervous system involvement. the severe cases may respond to systemic css, interferon, or anti-tnf therapy. there is some evidence to support ivig treatment for refractory eye and skin involvement (strength of recommendation i). the following conditions refer to (table 3) . level of evidence b there is weak evidence that ivig is useful in the treatment of acquired hemophilia. ivig may be tried in the case in which cs and cytotoxic agents fail or when facing an emergency situation as an additional therapy ivig (strength of recommendation iia). level of evidence a there is strong evidence that ivig is of benefit in reducing the number and severity of infections in patients with acquired hypogammaglobulinemia in the context of hematological malignancy (strength of recom-mendation i). while this is true, both cost and the potential of adverse effects, most prominently acute renal failure and thromboembolic phenomena [3] [4] [5] , prevent a clear-cut recommendation for the use of ivig in acquired hypogammaglobulinemia. with this in mind, ivig may be recommended at the replacement dose of 400 mg/kg each 3 to 4 weeks in cases in which severe or recurrent infections have occurred. level of evidence c pure red cell aplasia may be immunemediated due to a background neoplasia, most frequently hematologic, or due to an immune disease or as an immune response triggered by drugs. it may also occur secondary to parvovirus b19 infection which may also lead to aplastic anemia. treatment of the causative disease is the most important issue. weak evidence supports the use of ivig in pure red cell aplasia. ivig may however be used in the case in which first-line therapy (i.e., cs and immunosuppressive drugs) fails to achieve a remission (strength of recommendation iia). ivig is first-line therapy in immunosuppressed hosts in which infection with parvovirus b19 results in pure red cell aplasia. level of evidence b and c according to federici and colleagues [6] , 48% of the cases of acquired von willebrand syndrome are associated with lymphoproliferative diseases, 15% with myeloproliferative disorders, and 21% with cardiovascular diseases. in another paper, federici and colleagues [7] claim that 33% of the reported cases are associated with a monoclonal gammopathy of uncertain significance. then, the detection and specific treatment of the underlying disease is as important as the immediate therapy of the coagulation disorder. in accordance to the scant existing evidence of ivig therapy in this disorder, ivig should be used in cases of standard therapy (desmopressin and factor viii/von willebrand factor concentrate) failure and in urgent situations as an addition to such therapy (strength of recommendation iib). ivig may also be used in the preparation for surgery. level of evidence c while idiopathic aplastic anemia can be transient as in the case of parvovirus-b19-induced bone marrow depression, idiopathic aplastic anemia should first be treated with immunosuppressive drugs like cs or cyclosporine or with antithymocyte or antilymphocyte immunoglobulins as well as supportive blood components transfusions. stem cell poems syndrome polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes transplantation is always the alternative in the case of failure of the aforementioned treatments. due to the low level of evidence of ivig for the treatment of idiopathic aplastic anemia, we cannot recommend its liberal use but it may be tried in the case of treatment failure, before stem cell transplantation is performed (strength of recommendation iib). level of evidence b immunosuppression with cs and then cytotoxic agents should be used in autoimmune hemolytic anemia before considering ivig. there is however some evidence that ivig might be useful and therefore in refractory cases before considering splenectomy it may be worth a try (strength of recommendation iib). level of evidence c although immune neutropenia may also be an adverse effect resulting from ivig treatment as have been published elsewhere [8] , ivig has been used to treat immune neutropenia. the evidence is weak and responses are not as good as those with cs and granulocyte colony-stimulating factor (strength of recommendation iib). level of evidence c evans' syndrome is usually a therapyresistant condition that has been treated combining cs, ivig, and immunosuppressive therapy. although convincing evidence is lacking for the use of ivig in view of the severity and the refractory nature of several cases of evans' syndrome, ivig may be considered among the treatment options generally together with cs with or without immunosuppressive therapy (strength of recommendation iib). level of evidence b the only study comparing ivig against other treatment (cs), although nonrandomized, suggests that ivig raises the platelet number and prevents intracranial bleeding in a significant percentage of treated patients. the level of evidence is low, but the alternatives for prenatal treatment of fetuses at risk are not better and the risks involved are huge (strength of recommendation iib). level of evidence a strong evidence denies any benefit from the use of ivig around stem cell/bone marrow transplantation, both from the infectious or from the graftversus-host disease point of views (strength of recommendation iii). level of evidence a there is clear evidence for the use of ivig when facing newborn hemolysis as it reduces the need of plasmapheresis (pp) therapy (strength of recommendation i). level of evidence c there is no reliable evidence to recommend the use of ivig to prevent or to treat hemolytic transfusion reactions (strength of evidence iib). level of evidence c apart from two papers reporting two cases and another case, there is no evidence to recommend the use of ivig either to prevent or to treat hemolytic transfusion reactions in sickle cell patients, specially taking into consideration the additional risk of thrombosis due to a change in the rheologic properties of the blood after ivig infusion (strength of recommendation iib). level of evidence b there is conflicting evidence that ivig benefits patients with hemolytic uremic syndrome (strength of recommendation iib). level of evidence b there is no evidence that ivig benefits patients with thrombotic thrombocytopenic purpura (strength of recommendation iii). level of evidence c there is low-level evidence that ivig is useful for this disease. on the other hand, thromboembolic phenomena, which are part of the heparin-induced thrombocytopenia clinical picture, might be enhanced by the rheologic blood changes after ivig infusion. therefore, ivig should not be generally recommended for this disease (strength of recommendation iib). level of evidence b evidence comes only from one crossover randomized placebo-controlled trial which supports the use of ivig in hiv-associated thrombocytopenia, especially when platelet count is very low or the risk of bleeding is high (strength of recommendation iia). level of evidence a since imbach's observation [1] , acute itp has been the prototype of ivig-responsive immune disease. there is consistent evidence that ivig is beneficial in children with itp (strength of recommendation i). notwithstanding this, cs therapy is still the first-line treatment for itp, and single-dose anti-d immunoglobulin [9, 10] is a [8] good alternative for rh-positive patients. ivig should be considered when there is bleeding or when the bleeding risk is high or upon failure of other treatments. in adult acute itp patients, there is no placebo-controlled rct, but when compared with cs ivig induces a faster response. again, cs is the first-line treatment for adults but ivig may be given in severe or refractory cases. there is weak evidence that ivig can improve mother and fetal prognosis in pregnancy. ivig may be used when a fast correction in platelet number is needed. level of evidence b by the merits of its own research, ivig may be considered to treat itp in the context of hiv. although no hard evidence exists due to the fact that this particular group of patients has not been investigated as well as has non-hiv-related itp, it will be hard to argue that the recommendation of the latter may not be extended to hiv-related itp (strength of recommendation i). in chronic itp, ivig has so far not demonstrated a plausible benefit. only prospective noncontrolled trials are available and we found only one evaluating maintenance ivig for chronic itp, in which the overwhelming majority of patients had a recrudescence of thrombocytopenia in spite of a good initial response to ivig (level of evidence and strength of recommendation b-iib). a small number of patients benefited and therefore ivig might be tried in drug refractory cases of chronic itp in which there is a contraindication for splenectomy. level of evidence c series of patients treated with fast response to ivig suggest a benefit of ivig treatment in posttransfusional purpura, although with low-level of evidence. in the context of a patient with severe risk to bleeding or actual bleeding, ivig can be used if the diagnosis of posttransfusional purpura is made (strength of recommendation iib). level of evidence c hemophagocytic syndrome is a severe reaction leading to high mortality. inconsistent and low-level evidence do not warrant a clear-cut recommendation of ivig for this syndrome. however, due to the lack of effective and standardized treatment in an otherwise highly lethal disease, ivig may be used along with other therapies like monoclonal antibodies, cs, cytotoxic drugs, and support measures (strength of recommendation iib). level of evidence c there are only anecdotal reports, both showing benefit and lack of benefit. there is no evidence that ivig has a beneficial effect on polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (strength of recommendation iib). level of evidence c there is no convincing evidence that ivig has any beneficial effect in this disease (strength of recommendation iib). the following conditions refer to (table 4 ). level of evidence b ivig is not used for the treatment of cmv infection but may be helpful in treatment of hemophagocytic syndrome related to cmv and other viruses (see hematology section); there is some evidence for its effectiveness in preventing seroconversion in transplant patients who are immunosuppressed (strength of recommendation iia). level of evidence b ivig can reduce infections in children with perinatal hiv but has not been proven to reduce mortality (strength of recommendation iia). level of evidence b no evidence for the effectiveness of ivig in the treatment in malaria exists (strength recommendation iii). level of evidence a postpolio syndrome (pps) typically affects survivors of poliovirus infection, 15-20 years after the original infection, with fatigue, muscular weakness, and pain. there are two rcts demonstrating some inconsistent evidence for the effectiveness of ivig treatment on quality of life, pain, and muscle strength in patients with pps (strength of recommendation iia). level of evidence a sepsis is a life-threatening condition, resulting mainly from the patient's immune response to a severe infection. significant but heterogenic reduction in mortality with ivig treatment in septic patients was demonstrated in a meta-analysis of 14 rcts. these results were not confirmed when only high-quality studies were analyzed (strength of recommendation iia). level of evidence c infection with west nile virus can cause fatal encephalitis in immunosuppressed and elderly patients, in which case effective treatment is lacking. the effectiveness of ivig in these situations is supported by a few case reports and may be considered (strength of recommendation iia) level of evidence a treatment with ivig has not proven to reduce infectious-related mortality in postsurgical patients, but there is inconsistent evidence that ivig reduces icurelated infections and hospital stay in these patients (strength of recommendation iia). level of evidence a there is conflicting evidence regarding the usefulness of ivig treatment for these patients. it should be taken into consideration that patients treated with ivig after bone marrow transplantation (bmt) may have a higher incidence of fatal hepatic veno-occlusive disease (strength of recommendation iia). level of evidence b necrotizing fasciitis is caused by deep-skin infection with bacteria, mainly group a streptococcus. the mainstay of nuclear factor treatment is prompt surgical exploration and antibiotic therapy. ivig might have additional benefit to antibiotic therapy for treatment of patients who refuse surgery or are not surgical candidates. (strength of recommendation iia). level of evidence b there is weak evidence that ivig is useful in the treatment of recurrent otitis media (strength of recommendation iib). level of evidence b some evidence that ivig is effective in preventing perinatal transmission of varicella. ivig may also be used for treatment adults with severe respiratory failure due to varicella pneumonia (level of recommendation iia). the following conditions refer to (table 5) . level of evidence b although the best level of evidence is not strong, the improvement shown for treated pregnancies is beyond the natural possibility of a subsequent spared pregnancy which has been estimated to be approximately 40% ( [11] #110). in view of this data and the difficulty for large rcts, ivig may be considered to prevent recurrent perinatal hemochromatosis (strength of recommendation iia). level of evidence a there is evidence that ivig improves the outcome of pregnancy in secondary recurrent miscarriage (in women that had a previous pregnancy that reached at least the second trimester (strength of recommendation iib)). this has so far not been shown for primary recurrent miscarriage (strength of recommendation iii). level of evidence a the evidence is against the use of ivig in pregnancy of women affected by antiphospholipid syndrome (strength of recommendation iii). level of evidence b evidence from a small rct indicates that ivig may reduce the rate of fetal infection after premature rupture of membranes (strength of recommendation iia). level of evidence a the evidence is scarce but the only meta-analysis based on data from three rcts found a benefit in the number of live births in women treated with ivig. after in vitro fertilization (ivf) failure, ivig along with ivf techniques may be weighted (strength of recommendation iib). the following conditions refer to (table 6) . level of evidence b a randomized double-blind study has demonstrated significant increase of ejection fraction (ef) and improved quality of life following ivig administration, regardless of the congestive heart failure (chf) cause. chf is a proinflammatory state due to an increase of cytokines such as tnf-α and interleukin (il)-1. some of the cytokines have been shown to induce myocardial dysfunction due to negative inotropic effect [12] . another possible mechanism in chf pathogenesis is mediated by anti-β1 adrenergic receptor (strength of recommendation iia). level of evidence b dilated cardiomyopathy (dcm) is caused by various triggers or may be idiopathic. immune abnormalities and autoantibodies may play a role in the pathogenesis. nevertheless, no significant effect was noted following ivig administration in recent-onset dcm compared to placebo [13] . a trial on 17 patients with idiopathic dcm showed significant improvement of ef and quality of life compared with placebo [14] . therefore, ivig treatment in dcm remains controversial, and it is possible that ivig treatment is beneficial in specific subpopulation and when treatment is initiated at a certain time window (strength of recommendation iib). level of evidence c peripartum cardiomyopathy (ppcm) is a rare disorder. it is believed that autoimmune mechanisms play a role in the pathogenesis. in a small nonrandomized retrospective trial, there was a significant improvement of ef following ivig administration compared with conventional treatment. although there is partial evidence of ivig effectiveness in ppcm, its use should be considered due to the generally poor prognosis of ppcm patients who show no clinical improvement (strength of recommendation iia). level of evidence b treatment with cs or cytotoxic agents was found ineffective [15, 16] . in an rct trial of 62 patients with new-onset dcm of which some had myocarditis [13] , there was no significant difference in ef improvement between ivig and placebo group. nevertheless, in a prospective nonrandomized trial, there was a significant improvement of fractional shortening compared to control. we conclude that further research is warranted regarding ivig use in myocarditis due to inconclusive results (strength of recommendation iib). chronic idiopathic pericarditis (cip) appears in up to 25% of acute pericarditis cases. it may be associated with viral infections and autoantibodies [21] . there is limited evidence on the role of ivig as an alternative therapy for prolonged steroid or colchicine treatment in cip (strength of recommendation iia). atherosclerosis is believed to be mediated also by humeral and cellular immune mechanisms. there are accumulating data that support a role of ivig in prevention of atherosclerosis. possible mechanisms include decrease of matrix metalloproteinase 9 secretion from mononuclear cells [22], increase of il-10 [12] , and decrease of oxidized lowdensity lipoprotein uptake by macrophages [23] . nevertheless, clinical trials are required in order to establish the relationship between ivig use and atherosclerosis therapy. level of evidence a it is the leading cause of acquired heart disease in the usa. kawasaki disease is an fda-approved absolute indication for ivig therapy. the frequency of ca aneurysm development and associated morbidity and mortality have been dramatically decreased as a result of ivig therapy when given within 10 days following the onset of fever. a single dose of 2 g/kg of ivig over 10 h is usually level of evidence b because there is no efficient treatment for established rheumatic carditis, several agents has been proposed in an attempt to change the natural history. ivig has failed to change clinical outcome and disease progression and therefore is not recommended for treatment of acute rheumatic fever (strength of recommendation iii). level of evidence c neonatal lupus erythematosus is a rare disease that is associated with anti-ro and anti-la autoantibodies [26] . a single case series of eight subjects had level of evidence c ivig has anti-idiotypic properties, as well as human leukocyte antigen molecules that may neutralize high panel reactive antibodies in sensitized patients awaiting cardiac transplantation. there is some evidence for ivig benefit in patients with left ventricular assist device who are awaiting cardiac transplantation (strength of recommendation iia). nevertheless, ivig did not reduce sensitization in previously unsensitized patients who underwent norwood procedure (strength of recommendation iii). the following conditions refer to (table 7) . level of evidence c linear iga bullous disease may affect the eye (in 67% of patients) and may present as chronic cicatrizing conjunctivitis. systemic disease may be treated with systemic cs and dapsone. in poorly responsive patients, ivig treatment may be used, although its use should be further investigated (strength of recommendation iia). level of evidence c mucous membrane pemphigoid (mmp) may involve the eye. there is some evidence for the beneficial effect of ivig in mmp that involves the eye. ivig may provide more rapid control of symptoms and prevents remissions during long-term follow-up [28, 29] . this notion should be supported by larger randomized trials (strength of recommendation iia). level of evidence c there are some but limited data regarding the potential benefit of ivig use in behcet's disease. further study is required. nevertheless, ivig should be carefully considered in patients resistant to conventional immunosuppressive therapy (strength of recommendation iia). level of evidence a the natural history of optic neuritis in multiple sclerosis patients is not altered by ivig transfusion, neither clinically nor radiologically. therefore, ivig is not recommended in that setting (strength of recommendation iii). level of evidence c there is anecdotal evidence for the possible beneficial role of ivig in the treatment of inflammatory pseudotumor of orbit (strength of recommendation iib). level of evidence c birdshot retinochoroiditis is a rare inflammatory disease (bilateral autoimmune posterior uveitis of idiopathic origin). without immunosuppressive treatment, a progressive visual deterioration will occurs in 80% of patients [30] . there is supporting evidence for the use of ivig, especially in patients unresponsive to other therapies (strength of recommendation iia). level of evidence c it is caused by inflammatory process of unknown etiology, which is confined to the orbit. there are very limited data on the effect of ivig in patients with orbital myositis. such treatment might be considered in symptomatic patients, resistant to other therapies (strength of recommendation iia). level of evidence c most cases of refractory uveitis (ru) are associated with autoimmune mechanisms. there are some data supporting the use of ivig in resistant ru, although more research is required in order to establish clinical guidelines (strength of recommendation iia). level of evidence b it seems that ivig is as efficient as cs in the treatment of graves' ophthalmopathy. nevertheless, despite similar clinical response to treatment, ivig was associated with fewer side effects and the study group showed more tolerance towards it. therefore, we conclude that ivig should be considered in cs intolerance (strength of recommendation i). level of evidence c there is limited evidence for the benefit of ivig in cancer-associated retinopathy. nevertheless, since spontaneous recovery usually does not occur, ivig may be used in progressive visual compromise in addition to cs or plasmapheresis (strength of recommendation iib). the following conditions refer to (table 8 ). level of evidence b a few case reports and a single rct clearly indicate the efficacy of ivig in the treatment of lupus nephritis. in all cases, patients had a beneficial response to ivig and a significant improvement of renal function was noted. therefore, ivig is recommended as an alternative treatment in lupus nephritis or in cases that conventional immunosuppressive treatment fails (strength of recommendation i). level of evidence c although evidence for the use of ivig in renal transplant rejection is limited to case reports and case series results showed that ivig may be effective for treating acute or chronic renal transplant rejection. ivig may improve renal function and reverse ab-mediated rejection. ivig may be considered among the treatment options generally together with immunosuppressive therapy (strength of recommendation i). level of evidence c there is weak evidence indicating significant benefit from the use of ivig in antineutrophil cytoplasmic antibody (anca)-associated rpgn. several case series and case reports showed that ivig may improve renal function and therefore is recommended as a potential therapy for anca-associated rpgn (strength of recommendation i). level of evidence c the use of ivig in bk-virus-associated nephropathy in renal allograft recipient is limited only in a few case reports. nevertheless, in these cases, ivig showed significant efficacy and with concomitant reduction of immunosuppression it may be considered as one of the treatment options in this disease (strength of recommendation i). level of evidence b results coming from a single-arm nonrandomized study and a prospective cohort study showed that ivig may be effective in treating severe iga nephropathy and therefore it may be considered as a possible treatment option. however, rcts are needed to confirm this efficacy (strength of recommendation i). the efficacy of ivig in membranous nephropathy was studied in few retrospective trials and a case series. results from these studies indicate that ivig may be effective in induction of remission. although there is no strong evidence, ivig may be considered as an additional option in treatment of membranous nephropathy (strength of level of evidence c; recommendation i). the following conditions refer to (table 9 ). level of evidence b there is some evidence that ivig have steroid-sparing effect and may be effective as monotherapy and/or adjunctive therapy in patients with previously severe unresponsive pemphigus vulgaris (strength of recommendation iia). level of evidence c adjunctive to standard immunotherapy treatment with ivig may cause improvement in clinical course and may have steroid-sparing effect in previously steroid-dependent patients with refractory pemphigus foliaceus (strength of recommendation iia). level of evidence c in some severe cases of bullous pemphigoid, ivig was found to be effective as monotherapy and adjunctive therapy as well; it had steroid-sparing effect and led to improvement of quality of life (strength of recommendation iia). level of evidence c there is some evidence that ivig monotherapy may be at least as effective as standard immunosuppressive treatment and lead to quick therapy response and better quality of life (strength of recommendation iia). level of evidence c the sparse data of positive effect of ivig on severe cases of epidermolysis bullosa acquisita, linear iga disease, and pemphigoid gestationis were published (strength of recommendation iia). summary in autoimmune blistering diseases, the treatment with ivig may be effective and has to be implicated in cases with severe disease, resistant to conventional therapy or those who experienced severe complications of such therapy. level of evidence c the randomized studies have not been performed and the current knowledge about effectiveness of ivig in sjs and ten is based on results of multiple prospective noncontrolled studies, retrospective case series, and case reports. the data are limited and inconclusive (strength of recommendation iib). level of evidence b the current data based on single, randomized, controlled, and evaluator-blinded trial and number of prospective noncontrolled studies suggest that ivig may be effective as monotherapy in pediatric patients and as adjunctive therapy in adults. however, in view of the low-cost effectiveness of ivig, this treatment should be used in cases of severe disabling atopic dermatitis (strength of recommendation iia). level of evidence c a number of case reports and case series describe the beneficial effect of ivig in chronic idiopathic and autoimmune urticaria, but randomized controlled studies are still lacking. the use of ivig has to be limited to severe unresponsive cases of chronic urticaria or in case of severe complications of conventional treatment (strength of recommendation iia). level of evidence c only three cases of severe resistant psoriasis with psoriatic arthritis responsive to treatment with high-dose immunoglobulin were reported. we conclude that there is now evidence of effectiveness of ivig in psoriasis (strength of recommendation iib). level of evidence c ivig was successfully used in few cases of previously unresponsive pyoderma gangrenosum, but its systematic use cannot be recommended (strength of recommendation iia). level of evidence c several case reports and case series showed ivig to be effective for nephrogenic fibrosing dermopathy, pretibial myxedema, and arndt-gottron scleromyxedema, but still well-controlled studies are lacking. we suppose that ivig can be used in severe cases of these rare conditions, unresponsive to conventional treatment (strength of recommendation iia). the following conditions refer to (table 10 ). retrospective series report [299] no difference in length of stay, mechanical ventilation, severity of inflammatory response or incidence of sepsis, wound progression, time to healing, and mortality 16 ten patients with initial rash involving 65±29%, treated with ivig vs. 16 patients with ten initial rash involving 65±27% not treated with ivig ivig (gamimune® n, bayer inc., toronto, ca) 0.2 to 0.7 g/kg per day according to discretion of attending physician on duty, as 5% or 10% solution according to availability, for 4±1 day retrospective [300] no difference of scorad index and in global evaluation of disease severity by patients at day 30 10 adult patients (mean age 28) with sever atopic dermatitis ivig 1 g/kg per day (sandoglobulin®, sandoz, france) immediate or delayed by 1 month (meanwhile intensive therapy with emollients and topical cs (limited to class ii 60 g/month)), for 2 days in 8-h infusion, as monotherapy rc evaluatorblinded trial [301] atopic dermatitis improvement in skin score (measi) was apparent in responders (4/6 patients) from 2 to 3 months and continued to improve over a 6-month period; after 7 months, there was a significant reduction of the overall measi; cd69-expressing t cells decreased to 60% from baseline; no change of tnf-a and ifn-g 6 adult patients with severe stable atopic dermatitis flebogamma® 5% 2 g/kg per month given in 2-5 days for 6 months as adjunctive treatment, followed for 3 months open, single center, prospective [302] 3/3 patients improved skin scores, allowing reduction of steroid dose 3 adult patients with severe atopic dermatitis and steroid-related side effects alphaglobin® (grifols, uk) or sandoglobulin ® (novartis, uk) 2 g/kg per month in 3-5 days adjunctive to cs case report [303] clinic rev allerg immunol (2010) 38:201-269 case report [318] first patient had significant reduction in skin scores (36/60 to 11/60) for 1 year only; second had a dramatic sustained response with continued tx (interval subsequently increased to 10 weeks) 2 patients with scleromyxedema and severe skin disease ivig 0.4 g/kg per day for 5 days, monthly case report [319] improvement after the first treatment, continuous skin softening and reduction of induration, sustained response to treatment after 20 courses a 48-year-old female with long-standing scleromyxedema unresponsive to highdose cs and immunosuppressive (cp, melphalan) treatment ivig 2 g/kg in 2 days (intratect®; biotest, dreieich, germany) every 4 weeks, adjunctive to pd 10 mg/day case report [320] progressive clinical improvement over several months allowing discontinuation of pd but rapid deterioration afterwards with lethal cva despite reinstating pd and cp an 81-year-old female with scleromyxedema and iddm ivig (gamimune n, 10%, bayer) 1.5 g/ kg (dose reduced due to chf) every 4 weeks and oral pd 30 mg/day case report [321] a positive response after 2 cycles an 82-year-old female with a 12-year history of scleromyxedema unresponsive to various preceding therapies venimmun® n (zlb behring, germany) 0.5 g/kg for 5 days at 4-week intervals as monotherapy case report [322] marked clinical improvement, maintained with repeated ivig infusions a 56-year-old woman with scleromyxedema ivig (sandoglobulin®) 2 g/kg over 5 days as monotherapy case report [323] complete clearance of skin lesions, sustained effect after 1 year without treatment case report [324] clinic rev allerg immunol (2010) 38: kaposi's sarcoma level of evidence c a patient with polymyositis and kaposi sarcoma had remission of both conditions on ivig therapy (strength of recommendation iib). level of evidence c stabilization of metastatic melanoma was shown in the small group of patients after addition of ivig to standard therapy (strength of recommendation iib). level of evidence civig was shown to be effective in preventing severe recurrent chemotherapy-induced oral mucositis in two patients treated with methotrexate (strength of recommendation iia). level of evidence c anecdotal case report describes complete remission of metastatic malignant thymoma after four cycles of ivig given to myasthenic patient (strength of recommendation iib). level of evidence c after thymectomy, two patients with good syndrome were maintained with ivig for developed immunodeficiency; one of them developed kaposi sarcoma after 3 years (strength of recommendation iib). the following conditions refer to (table 11) . summary there are still no randomized controlled trials evaluating efficacy of ivig and it cannot be recommended for treatment of epithelial or other solid malignancies. level of evidence c there is some evidence that ivig therapy can improve the neuropathy impairment score in patients with diabetic demyelinating polyneuropathy (strength of recommendation iia). a few case reports describe regression of symptoms of proximal diabetic neuropathy, diabetic amyotrophy, and cranial nerve neuropathy in different diabetic patients treated with ivig (strength of recommendation iia). we conclude that evidence for the use of ivig in diabetic neuropathies is poor and its systematic use cannot be recommended. disease improvement in all but relapse in 1 patient, 2 weeks to 6 months for full response, most needing 6-8 weekly cycles 10 patients, 30-79 years old with scleromyxedema ivig 2 g/kg per month given over 5 days; octagam® (2 patients), sandoglobulin® (3 patients), gammagard ®sd (1 patient), unknown (2patients); adjunctive to pd (2 patients) and thalidomide (1 patient) or as monotherapy (7 patients) review of published case reports [325] regrowth of eyelashes, eyebrows, and body and scalp hair after second dose, significant hair regrowth with 5-6 cm of scalp hair level of evidence b therapeutic efforts including dietary therapy and immunomodulation with ivig fail to improve prognosis in patients with x-linked adrenoleukodystrophy and bmt remains the only treatment that can reverse early neurological manifestations and adrenal impairment of ald (strength of recommendation iii). level of evidence c no evidence supports the use of ivig in waterhouse-friderichsen syndrome due to meningococcal sepsis (strength of recommendation iii). level of evidence c no evidence supports the use of ivig in autoimmune polyendocrine syndrome type 2 (strength of recommendation iib). the following conditions refer to (table 12) . level of evidence c there is weak evidence coming out from several case reports that ivig may be a useful treatment option either as a combination therapy with cs or in cases that steroid treatment fails (strength of recommendation i). level of evidence b taking into consideration the limited evidence (one rct) and the unfavorable effect of ivig treatment, at this point, ivig should not be used as treatment of adrenoleukodystrophy (strength of recommendation iii). level of evidence c no benefit was observed from the use of ivig in two case series. considering the weak evidence and the negative outcome, ivig is not recommended for the treatment of amyotrophic lateral sclerosis (strength of recommendation iii). level of evidence c the use of ivig in autism is only limited to case series and the effect is questionable. ivig is not recommended at the moment for the treatment of autism (strength of recommendation iii). level of evidence a several rcts evaluated the effectiveness of ivig. the results of these studies are strongly pointing to the useful effect of ivig in patients with chronic inflammatory demyelinating polyneuropathy. ivig should be considered as an additional option in combination with other immunosuppressive agents (strength of recommendation i). level of evidence c in this rare syndrome, ivig was reported in several case reports and a retrospective study proved that it induced significant response. although there is no strong evidence, ivig may be considered as an additional option in treatment of opsoclonus myoclonus (strength of recommendation i). level of evidence b results coming from one small rct testing the effect of ivig showed significant improvement in symptoms; therefore, ivig is recommended as an additional option for treatment of pandas (strength of recommendation i). level of evidence a data from several rcts and a metaanalysis showed significant differences between ivig and placebo favoring the use of ivig in relapsing-remitting multiple sclerosis (ms). on the other hand, a recent rct including 127 patients with relapsing-remitting ms treated with ivig or placebo showed no significant difference in the proportion of relapse-free patients among treated groups. another recent rct including 231 patients showed that ivig can delay progression of disease in primary chronic progressive ms but no significant difference in progression was noted for secondary chronic progressive ms. to conclude, ivig may be used as an alternative therapy in relapsingremitting ms and in primary chronic progressive ms when standard immunosuppression therapy fails (strength of recommendation iib). level of evidence a there is strong evidence, several randomized controlled trials, and a meta-analysis that prove that ivig is of benefit in improving the disability grade in patients with guillain-barré syndrome. no significant difference between ivig and pp was found. ivig should be considered as a treatment option in guillain-barré syndrome (strength of recommendation i). level of evidence a there is no reliable evidence to recommend the use of ivig in paraproteinemic neuropathy. a restricted number of rct's and a systematic review showed only modest benefit with ivig in the short term (strength of recommendation iib). level of evidence b a single small rct showed significant improvement in patients suffering from lambert-eaton myasthenic syndrome with the use of ivig compared to placebo. therefore, it is acceptable to consider the use of ivig in this rare and severe neurological syndrome (strength of recommendation i). level of evidence b data from a small randomize control trial and several case reports showed that the use of ivig led to significant improvement in patients with stiff-person syndrome and was superior to placebo. ivig should be considered as a treatment option in this syndrome especially if the first-line treatment fails (strength of recommendation i). level of evidence a two rcts investigated the efficacy of ivig in patients with intractable childhood epilepsy compared with placebo. results from these studies do not support benefit from the use of ivig. therefore, ivig is not recommended for the treatment of intractable childhood epilepsy (strength of recommendation iii). level of evidence c there is no reliable evidence (level of evidence c-iii) to recommend the use of ivig in the treatment of critical illness polyneuropathy (strength of recommendation iii). level of evidence a several rcts and a meta-analysis examined the efficacy of ivig in patient with myasthenia gravis. results are conflicting. a recent metaanalysis concluded that there is insufficient evidence to determine the efficacy of ivig in myasthenia gravis. ivig may be used as treatment for myasthenia gravis severe acute exacerbations (strength of recommendation iia). the following conditions refer to (table 13 ). level of evidence b retrospective analyses revealed that ivig is beneficial in terms of reduction of acute and chronic infections and days of stay in the hospital. intravenous route is preferred over intramuscular administration of immunoglobulin and the number and severity of complications are inversely correlated with the dose of ivig (strength of recommendation i). level of evidence c despite relatively low evidence of effectiveness of ivig, the majority of was patients are treated with ivig, which appears to be effective in reduction of acute and chronic infections in these immunodeficient patients (strength of recommendation iia). level of evidence c there is weak evidence that ivig is useful in the treatment of hyper-ige syndrome. ivig may be tried in cases in which recurrent live-threatening diseases cannot be controlled by antibiotic prophylaxis (strength of recommendation iib). level of evidence c there is scarce evidence that ivig is useful in the treatment of igg subclass deficiencies. most of the experience reported is patients with igg2 deficiency. in our opinion, when antibiotic treatment is not effective, ivig would be a reasonable alternative (strength of recommendation iib). level of evidence b this is a heterogeneous group. specific deficiency of antibodies after vaccination has been found to correlate with infection susceptibility. most of the published experience regarding ivig is in patients with deficiency of antibodies to capsular polysaccharide. ivig has been reported to be beneficial in some cases. this is not surprising as antibodies to bacterial capsular polysaccharide are contained in ivig [31] . the evidence for the use of ivig in specific antibody deficiency is not strong but might be warranted when recurrent infection is present and it is possible to demonstrate low antibody responses to a relevant vaccine (strength of recommendation iia). level of evidence b the hyper-igm syndrome (higm) is a rare hereditary immune deficiency, characterized by a low or nil level of igg and iga and a normal or increased level of igm, predominately affecting boys. its clinical manifestations are dominated by recurrent infection, notably of the digestive tube, the ears, nose, and throat and the lungs. ivig may be considered to treat patients with higm and recurrent infections (strength of recommendation i). level of evidence c transient hypogammaglobulinemia of infancy is characterized by a prolongation and accentuation of the physiologic hypogammaglobulinemia normally occurring during the first 3 to 6 months of life and recovers spontaneously between 18 and 36 months of age. infants with transient hyogammaglobulinemia of infancy (thi) may remain asymptomatic or develop recurrent sinopulmonary infections, but severe or lifethreatening infections are rare. in general, supportive and antimicrobial therapies are sufficient management for the treatment of specific infections in patients with thi. in cases in which infections are severe or refractory to conventional therapy, ivig is sometimes considered although literature reports are lacking (strength of recommendation i). level of evidence b scid is a severe form of heritable immunodeficiency in which both "arms" of the adaptive immune system are defected. these babies, if untreated, usually die within 1 year due to severe recurrent infections. treatment with bmt is successful, and ivig is used as an adjuvant for this therapy (strength of recommendation i). many different genetic diseases will ultimately result in primary phagocytic defect: cyclic neutropenia, severe congenital neutropenia, shwachman-diamond syndrome, leu-kocyte adhesion deficiency, rac2 deficiency, interferon-γ and il-12 defects, chronic granulomatous disease, myeloperoxidase deficiency, chediak-higashi syndrome, and neutrophil-specific granule deficiency [34] . an extensive pubmed-based search revealed little if any information regarding the efficacy of ivig in the above entities. iga deficiency may be associated with preventable anaphylaxis or anaphylactoid reaction following ivig transfusion [35] . this adverse reaction is encountered in patients with selective iga deficiency and high titer of anti-iga antibodies. in those patients, anaphylaxis can be avoided by the use of iga-depleted ivig preparation [36] . this article summarizes most of the studies dealing with ivig administration that were publicized until recently. we have included an array of clinical conditions, some in which the use of ivig is expected according to their pathogenesis, such as primary immunodeficiencies and rheumatic diseases, and others somewhat less obvious indications including cardiac and oncologic diseases. in some diseases, the usage of ivig was found to be highly recommended (with a level of evidence a and strength of recommendation i): kawasaki disease, acquired hypogammaglobulinemia, juvenile rheumatoid arthritis, hemolytic disease of the newborn, acute immune thrombocytopenic purpura, chronic inflammatory demyelinating polyneuropathy, and guillain-barré syndrome. for many other diseases, treatment with ivig was found effective as well, but this was less well established, either due to a lack of well-designed studies or due to conflicting evidence among them. in some diseases, there is a strong recommendation against using ivig (with a level of evidence a and strength of recommendation iii): stem cell/bone marrow transplantation, inclusion body myositis, recurrent pregnancy loss due to antiphospholipid syndrome, optic neuritis, and intractable childhood epilepsy. nevertheless, the effectiveness of ivig in such a large span of diseases fortifies the accumulating evidence that many pathological conditions are autoimmune mediated. high-dose intravenous gamma globulin therapy of refractory, in particular idiopathic thrombocytopenia in childhood acc/ aha guidelines for the evaluation and management of chronic heart failure in the adult: executive summary. a report of the american college of cardiology/american heart association task force on practice guidelines (committee to revise the 1995 guidelines for the evaluation and management of heart failure): developed in collaboration with the international society for heart and lung transplantation review: intravenous immunoglobulin therapy and thromboembolic complications intravenous immunoglobulin: adverse effects and safe administration intravenous immunoglobulin and the kidney-a two-edged sword acquired von willebrand syndrome: data from an international registry treatment of acquired von willebrand syndrome in patients with monoclonal gammopathy of uncertain significance: comparison of three different therapeutic approaches transient neutropenia induced by intravenous immune globulin randomized trial of anti-d immunoglobulin versus low-dose intravenous immunoglobulin in the treatment of childhood chronic idiopathic thrombocytopenic purpura single dose of anti-d immune globulin at 75 microg/kg is as effective as intravenous immune globulin at rapidly raising the platelet count in newly diagnosed immune thrombocytopenic purpura in children new hope for treatment of neonatal haemochromatosis complement activation in patients with congestive heart failure: effect of high-dose intravenous immunoglobulin treatment controlled trial of intravenous immune globulin in recent-onset dilated cardiomyopathy immunomodulating therapy with intravenous immunoglobulin in patients with chronic heart failure successful highdose intravenous immunoglobulin therapy for a patient with fulminant myocarditis the current status of immune modulating therapy for intravenous immunoglobulins for adult still's disease and pregnancy intravenous immunoglobulin for ancaassociated systemic vasculitis with persistent disease activity pooled intravenous immunoglobulin in the management of systemic vasculitis treatment of anti-neutrophil cytoplasmic antibody (anca)-associated systemic vasculitis with high-dose intravenous immunoglobulin serologic and clinical response to treatment of systemic vasculitis and associated autoimmune disease with intravenous immunoglobulin intravenous immunoglobulin as sole therapy for systemic vasculitis intravenous immunoglobulin (ivig) therapy in mpo-anca related polyangiitis with rapidly progressive glomerulonephritis in japan long term effectiveness of intravenous immunoglobulin in churg-strauss syndrome treatment of churg-strauss syndrome with high-dose intravenous immunoglobulin efficacy of intravenous immunoglobulin therapy in a case of autoimmunemediated chronic active hepatitis intravenous immunoglobulin plus interferon-alpha in autoimmune hepatitis c high-dose immunoglobulin therapy for severe iga nephropathy and henoch-schönlein purpura intravenous immunoglobulin therapy for severe digestive manifestations of henoch-schönlein purpura successful treatment of adult-onset henoch-schönlein purpura nephritis with high-dose immunoglobulins henoch-schönlein nephritis. adverse effect of treatment with intravenous immunoglobulin intravenous immunoglobulin in henoch-schönlein purpura fasciitis in mixed connective tissue disease successfully treated with highdose intravenous immunoglobulin efficacy of pulsed intravenous immunoglobulin therapy in mixed connective tissue disease intravenous immunoglobulin modulates cutaneous involvement and reduces skin fibrosis in systemic sclerosis: an open-label study high-dose intravenous immunoglobulin infusion as treatment for diffuse scleroderma intravenous immunoglobulins improve the function and ameliorate joint involvement in systemic sclerosis: a pilot study skin score decrease in systemic sclerosis patients treated with intravenous immunoglobulin-a preliminary report intravenous immunoglobulin in the treatment of polyarticular juvenile rheumatoid arthritis: a phase i/ii study. pediatric rheumatology collaborative study group therapeutical and immunological effects of methylprednisolone pulse therapy in comparison with intravenous immunoglobulin. treatment in patients with juvenile chronic arthritis intravenous immunoglobulin therapy in systemic onset juvenile rheumatoid arthritis: a follow-up study high dose immunoglobulin therapy in severe juvenile chronic arthritis: long-term follow-up in 16 patients failure of low-dose intravenous immunoglobulin therapy to suppress disease activity in patients with treatment-refractory rheumatoid arthritis immunomodulating therapy of rheumatoid arthritis by high-dose intravenous immunoglobulin high dose intravenous immunoglobulin (ivig) in severe refractory rheumatoid arthritis: no evidence for efficacy successful treatment of 'malignant rheumatoid arthritis' in japan with pooled intravenous immunoglobulin benefit of ivig for long-standing ataxic sensory neuronopathy with sjögren's syndrome. iv immunoglobulin intravenous immunoglobulin treatment in painful sensory neuropathy without sensory ataxia associated with sjögren's syndrome intravenous immunoglobulin therapy in sensory neuropathy associated with sjögren's syndrome a controlled trial of high-dose intravenous immune globulin infusions as treatment for dermatomyositis intravenous immunoglobulin treatment in autoimmune diseases efficacy of high-dose intravenous immunoglobulin therapy in japanese patients with steroid-resistant polymyositis and dermatomyositis a controlled study of intravenous immunoglobulin combined with prednisone in the treatment of ibm high-dose immunoglobulin therapy in sporadic inclusion body myositis: a double-blind, placebo-controlled study treatment of inclusion-body myositis with ivig: a double-blind, placebo-controlled study intravenous immunoglobulin therapy for resistant ocular behcet's disease behcet's syndrome treated with highdose intravenous igg and low-dose aspirin a prospective study of treatment of acquired (autoimmune) factor viii inhibitors with high-dose intravenous gamma globulin low response to high-dose intravenous immunoglobulin in the treatment of acquired factor viii inhibitor intravenous immunoglobulin therapy for acquired coagulation inhibitors: a critical review combined prednisolone and intravenous immunoglobulin treatment for acquired factor viii inhibitors: a 2-year review prophylaxis against infections with low-dose intravenous immunoglobulins (ivig) in chronic lymphocytic leukemia. results of a crossover study crossover study of immunoglobulin replacement therapy in patients with low-grade b-cell tumors randomized trial of intravenous immunoglobulin prophylaxis for patients with chronic lymphocytic leukaemia and secondary hypogammaglobulinaemia randomised trial of intravenous immunoglobulin as prophylaxis against infection in plateau-phase multiple myeloma. the uk group for immunoglobulin replacement therapy in multiple myeloma immunoglobulin prophylaxis during intensive treatment of acute lymphoblastic leukemia in children treatment of fever and neutropenia with antibiotics versus antibiotics plus intravenous gamma globulin in childhood leukemia immunoglobulin replacement in chronic lymphocytic leukaemia prophylaxis against infections with intravenous immunoglobulins in multiple myeloma hypogammaglobulinaemia in low grade b cell tumours; significance and therapy pure red cell aplasia: treatment with intravenous immunoglobulin concentrate treatment of pure red cell aplasia by high dose intravenous immunoglobulins dose-by-dose virological and hematological responses to intravenous immunoglobulin in an immunocompromised patient with persistent parvovirus b19 infection pure red cell aplasia due to parvovirus b19 infection after liver transplantation: a case report and review of the literature acute renal failure in a pediatric kidney allograft recipient treated with intravenous immunoglobulin for parvovirus b19 induced pure red cell aplasia pure red cell aplasia due to parvovirus following treatment with chop and rituximab for b-cell lymphoma relapsing pure red cell aplasia associated with b-cell chronic lymphocytic leukemia successfully treated by intravenous immunoglobulin concentrate a case of persistent anemia in a renal transplant recipient: association with parvovirus b19 infection fulminant parvovirus infection following erythropoietin treatment in a patient with acquired immunodeficiency syndrome chronic pure red cell aplasia caused by parvovirus b19 in aids: use of intravenous immunoglobulin-a report of eight patients successful treatment of persistent erythroid aplasia caused by parvovirus b19 infection in a patient with common variable immunodeficiency with lowdose immunoglobulin red cell aplasia caused by parvovirus b19 in aids: use of i.v. immunoglobulin parvovirus b19 in hiv infection: a treatable cause of anemia management of persistent b19 parvovirus infection in aids chronic idiopathic pure red cell aplasia: successful treatment during pregnancy and durable response to intravenous immunoglobulin pure red cell aplasia associated with systemic lupus erythematosus: remission after a single course of intravenous immunoglobulin parvovirus b19-induced red cell aplasia treated with plasmapheresis and immunoglobulin treatment of antibody-mediated pure red-cell aplasia with high-dose intravenous gamma globulin persistent parvovirus b19 infection and pure red cell aplasia in waldenstrom's macroglobulinemia: successful treatment with high-dose intravenous immunoglobulin clinical significance of inhibitors in acquired von willebrand syndrome three cases of acquired von willebrand disease associated with systemic lupus erythematosus intravenous immunoglobulin in the treatment of aplastic anemia transient aplastic anemia caused by parvovirus b19 infection in a heart transplant recipient treatment of hematologic disorders other than immune thrombocytopenic purpura with intravenous immunoglobulin (ivig)-report of seven cases and review of the literature efficacy of intravenous immunoglobulin in the treatment of autoimmune hemolytic anemia: results in 73 patients diagnosis and clinical course of autoimmune neutropenia in infancy: analysis of 240 cases use of intravenous gamma globulin for the treatment of autoimmune neutropenia of childhood and autoimmune hemolytic anemia evans syndrome: results of a national survey evans syndrome. results of a pilot study utilizing a multiagent treatment protocol intravenous gamma globulin for thrombocytopenia in children with evans syndrome induction of remission with intravenous immunoglobulin and cyclophosphamide in steroid-resistant evans' syndrome associated with dermatomyositis fetomaternal alloimmune thrombocytopenia: antenatal therapy with ivigg and steroids-more questions than answers. european working group on fmait should immunoglobulin therapy be used in allogeneic stem-cell transplantation? a randomized, double-blind, dose effect, placebocontrolled, multicenter trial a randomized trial of high dose polyvalent intravenous immunoglobulin (hdigg) vs. cytomegalovirus (cmv) hyperimmune igg in allogeneic hemopoietic stem cell transplants (hsct) a controlled trial of long-term administration of intravenous immunoglobulin to prevent late infection and chronic graft-vs.-host disease after marrow transplantation: clinical outcome and effect on subsequent immune recovery intravenous immunoglobulin and cmv-seronegative blood products for prevention of cmv infection and disease in bone marrow transplant recipients a multicenter, randomized, double-blind comparison of different doses of intravenous immunoglobulin for prevention of graft-versus-host disease and infection after allogeneic bone marrow transplantation high-dose weekly intravenous immunoglobulin to prevent infections in patients undergoing autologous bone marrow transplantation or severe myelosuppressive therapy. a study of the american bone marrow transplant group immunomodulatory and antimicrobial efficacy of intravenous immunoglobulin in bone marrow transplantation immunoglobulin infusion for isoimmune haemolytic jaundice in neonates systematic review of intravenous immunoglobulin in haemolytic disease of the newborn high-dose intravenous immunoglobulin in non-abo transfusion incompatibility haemolytic transfusion reaction-successful attenuation with methylprednisolone and high dose immunoglobulin hyperhemolytic transfusion reaction in sickle cell disease use of intravenous immunoglobulin and intravenous methylprednisolone in hyperhaemolysis syndrome in sickle cell disease post-transfusion hyperhaemolysis in a patient with sickle cell disease: use of steroids and intravenous immunoglobulin to prevent further red cell destruction the use of intravenous gamma globulin in the treatment of typical hemolytic uremic syndrome high-dose intravenous gamma globulin infusions in hemolytic-uremic syndrome: a preliminary report treatment of thrombotic thrombocytopenic purpura with high-dose immunoglobulins. results in 17 patients. italian cooperative group for ttp efficacy of intravenous immunoglobulin in the treatment of thrombotic thrombocytopaenic purpura. a study of 44 cases high-dose intravenous gamma-globulins for heparininduced thrombocytopenia: a prompt response the therapy of the heparin-induced thrombosis-thrombocytopenia syndrome with immunoglobulins an evaluation of intravenous immunoglobulin in the treatment of human immunodeficiency virus-associated thrombocytopenia intravenous immunoglobulin or high-dose methylprednisolone, with or without oral prednisone, for adults with untreated severe autoimmune thrombocytopenic purpura: a randomised, multicentre trial the comparison of gamma globulin to steroids in treating adult immune thrombocytopenia. an interim analysis a prospective, randomized trial of high-dose intravenous immune globulin g therapy, oral prednisone therapy, and no therapy in childhood acute immune thrombocytopenic purpura clinical course of children with immune thrombocytopenic purpura treated with intravenous immunoglobulin g or megadose methylprednisolone or observed without therapy intravenous immunoglobulin in the treatment of human immunodeficiency virus-related thrombocytopenia usefulness of a low-dose intravenous immunoglobulin regimen for the treatment of thrombocytopenia associated with aids clinical efficacy and safety of a novel intravenous immunoglobulin preparation in adult chronic itp treatment of adult chronic autoimmune thrombocytopenic purpura with repeated high-dose intravenous immunoglobulin high-dose intravenous immunoglobulin for posttransfusion purpura high-dose intravenous immunoglobulin for post-transfusion purpura post-transfusion purpura following open heart surgery: management by high dose intravenous immunoglobulin infusion high-dose immunoglobulin therapy in renal transplant recipients with hemophagocytic histiocytic syndrome hemophagocytic syndrome: a review of 18 pediatric cases immunomodulation treatment for childhood virus-associated haemophagocytic lymphohistiocytosis infection associated hemophagocytic syndrome: a report of 50 children intravenously administered immune globulin for the treatment of infection-associated hemophagocytic syndrome successful treatment of cytomegalovirus-associated hemophagocytic syndrome by intravenous immunoglobulins rapidly deteriorating polyneuropathy associated with osteosclerotic myeloma responsive to intravenous immunoglobulin and radiotherapy poor response to intravenous immunoglobulin therapy in patients with castleman's disease and the poems syndrome intravenous immunoglobulin therapy in poems syndrome: a case report successful treatment of post-transplant lymphoproliferative disorder with interferon-alpha and intravenous immunoglobulin cytomegalovirus prophylaxis with intravenous polyvalent immunoglobulin in high-risk renal transplant recipients the national institute of child health and human developments intravenous immunoglobulin study group (1991) intravenous immune globulin for the prevention of bacterial infections in children with symptomatic human immunodeficiency virus infection intravenous immunoglobulin in symptomatic and asymptomatic children with perinatal hiv infection plasmodium falciparum: diversity of isolates from malawi in their cytoadherence to melanoma cells and monocytes in vitro parasitologic and clinical human response to immunoglobulin administration in falciparum malaria intravenous immunoglobulin for post-polio syndrome: a randomised controlled trial post-polio syndrome patients treated with intravenous immunoglobulin: a double-blinded randomized controlled pilot study effect of intravenous immunoglobulin in patients with post-polio syndrome-an uncontrolled pilot study polyclonal intravenous immunoglobulin for the treatment of severe sepsis and septic shock in critically ill adults: a systematic review and meta-analysis possible benefit of intravenous immunoglobulin therapy in a lung transplant recipient with west nile virus encephalitis treatment of west nile virus encephalitis with intravenous immunoglobulin prophylactic intravenous administration of standard immune globulin as compared with core-lipopolysaccharide immune globulin in patients at high risk of postsurgical infection polyvalent immunoglobulins for prophylaxis of bacterial infections in patients following multiple trauma. a randomized, placebo-controlled study prophylactic intravenous immunoglobulin replacement in high-risk burn patients safety and efficacy of intravenous immunoglobulin prophylaxis in pediatric head trauma patients: a double-blind controlled trial prevention of infection in multiple trauma patients by high-dose intravenous immunoglobulins reduced incidence of postoperative infection after intravenous administration of an immunoglobulin a-and immunoglobulin menriched preparation in anergic patients undergoing cardiac surgery immunoglobulin replacement in patients with chronic lymphocytic leukaemia: a comparison of two dose regimes clinical experience with 20 cases of group a streptococcus necrotizing fasciitis and myonecrosis: 1995 to 1997 successful management of severe group a streptococcal soft tissue infections using an aggressive medical regimen including intravenous polyspecific immunoglobulin together with a conservative surgical approach high-dose immunoglobulin-life-saving in invasive group a streptococcal infection group a streptococcal bacteraemia and necrotizing fasciitis in a renal transplant patient: a case for intravenous immunoglobulin therapy the effect of intravenous immunoglobulin treatment in recurrent acute otitis media effect of intravenous gamma globulin therapy in igg2 deficient and igg2 sufficient children with recurrent infections and poor response to immunization with haemophilus influenzae type b capsular polysaccharide antigen prophylaxis of intravenous immunoglobulin and acyclovir in perinatal varicella experience of intravenous immunoglobulin and acyclovir in neonates at risk for severe varicella infection-report of five cases two cases of severe adult varicella pneumonia varicella pneumonia in a healthy adult presenting with severe respiratory failure intravenous immunoglobulin in adult varicella pneumonia complicated by acute respiratory distress syndrome varicella pneumonia complicated by acute respiratory distress syndrome in an adult intravenous gamma globulin therapy for thrombocytopenic purpura associated with active varicella infection an adult patient with varicella preceded by acute thrombocytopenia high-dose immunoglobulin during pregnancy for recurrent neonatal haemochromatosis use of intravenous immunoglobulin for treatment of recurrent miscarriage: a systematic review immunotherapy for recurrent miscarriage intravenous immunoglobulin (ivig) and recurrent spontaneous pregnancy loss a randomized, double-blind, placebo-controlled trial of intravenous immunoglobulin in the prevention of recurrent miscarriage: evidence for a therapeutic effect in women with secondary recurrent miscarriage critical analysis of intravenous immunoglobulin therapy for recurrent miscarriage short-term therapy for recurrent abortion using intravenous immunoglobulins: results of a double-blind placebo-controlled italian study intravenous immunoglobulin for treatment of recurrent pregnancy loss placebocontrolled trial of treatment of unexplained secondary recurrent spontaneous abortions and recurrent late spontaneous abortions with i intravenous immunoglobulin in the prevention of recurrent miscarriage prevention of recurrent miscarriage for women with antiphospholipid antibody or lupus anticoagulant randomized study of subcutaneous low molecular weight heparin plus aspirin versus intravenous immunoglobulin in the treatment of recurrent fetal loss associated with antiphospholipid antibodies a multicenter, placebo-controlled pilot study of intravenous immune globulin treatment of antiphospholipid syndrome during pregnancy. the pregnancy loss study group pregnancy outcome in recurrent spontaneous abortion associated with antiphospholipid antibodies: a comparative study of intravenous immunoglobulin versus prednisone plus low-dose aspirin pregnancy complicated by the antiphospholipid syndrome: outcomes with intravenous immunoglobulin therapy the selective use of heparin/aspirin therapy, alone or in combination with intravenous immunoglobulin g, in the management of antiphospholipid antibody-positive women undergoing in vitro fertilization recurrent first trimester spontaneous abortion associated with antiphospholipid antibodies: a pilot study of treatment with intravenous immunoglobulin normal fetal growth in women with antiphospholipid syndrome treated with high-dose intravenous immunoglobulin (ivig) the effect of antenatal intravenous immunoglobulin on ascending intrauterine infection after preterm premature rupture of the membranes: a pilot study is intravenous immunoglobulins (ivig) efficacious in early pregnancy failure? a critical review and meta-analysis for patients who fail in vitro fertilization and embryo transfer (ivf) treatment of repeated unexplained in vitro fertilization failure with intravenous immunoglobulin: a randomized, placebo-controlled canadian trial intravenous immune globulin in the therapy of peripartum cardiomyopathy treatment of acute inflammatory cardiomyopathy with intravenous immunoglobulin ameliorates left ventricular function associated with suppression of inflammatory cytokines and decreased oxidative stress gamma-globulin treatment of acute myocarditis in the pediatric population the prevention of coronary artery aneurysm in kawasaki disease: a meta-analysis on the efficacy of aspirin and immunoglobulin treatment the treatment of kawasaki syndrome with intravenous gamma globulin treatment of kawasaki disease with a moderate dose (1 g/kg) of intravenous immunoglobulin clinical responses of patients with kawasaki disease to different brands of intravenous immunoglobulin therapeutic effectiveness of intravenous immunoglobulin at 1 g/kg and 2 g/kg on kawasaki disease: a comparative and follow-up study a randomized prospective study on the use of 2 g-ivig or 1 g-ivig as therapy for kawasaki disease a single intravenous infusion of gamma globulin as compared with four infusions in the treatment of acute kawasaki syndrome intravenous immunoglobulin in acute rheumatic fever: a randomized controlled trial intravenous immunoglobulin reduces anti-hla alloreactivity and shortens waiting time to cardiac transplantation in highly sensitized left ventricular assist device recipients low-dose prophylactic intravenous immunoglobulin does not prevent hla sensitization in left ventricular assist device recipients failure of prophylactic intravenous immunoglobulin to prevent sensitization to cryopreserved allograft tissue used in congenital cardiac surgery linear iga bullous disease limited to the eye: a diagnostic dilemma: response to intravenous immunoglobulin therapy a randomized trial of intravenous immunoglobulin in inflammatory demyelinating optic neuritis a double-blind, randomized trial of iv immunoglobulin treatment in acute optic neuritis successful intravenous immunoglobulin therapy for resistant inflammatory pseudotumor of the orbit efficacy of intravenous immunoglobulin in the treatment of birdshot retinochoroiditis successful treatment of orbital myositis with intravenous immunoglobulins the treatment of refractory uveitis with intravenous immunoglobulin efficacy of intravenous immunoglobulin treatment in refractory uveitis intravenous immunoglobulin versus corticosteroid in treatment of graves' ophthalmopathy randomized trial of intravenous immunoglobulins versus prednisolone in graves' ophthalmopathy treatment of paraneoplastic visual loss with intravenous immunoglobulin: report of 3 cases improvement in visual fields in a patient with melanoma-associated retinopathy treated with intravenous immunoglobulin a case-bycase protocol of membranous nephropathy treatment with endovenous infusion of high doses of human immunoglobulins intravenous immunoglobulin therapy of membranous nephropathy: efficacy and safety the short-and long-term outcomes of membranous nephropathy treated with intravenous immune globulin therapy. kanazawa study group for renal diseases and hypertension high-dose intravenous immunoglobulin pulse therapy in patients with progressive immunoglobulin a nephropathy: a long-term follow-up intravenous immunoglobulin as a treatment for bk virus associated nephropathy: one-year follow-up of renal allograft recipients intravenous immunoglobulin as rescue therapy for bk virus nephropathy clinical efficacy of intravenous immunoglobulin for patients with mpo-ancaassociated rapidly progressive glomerulonephritis successful use of high dose intravenous immunoglobulin in rapidly progressive crescentic glomerulonephritis with vasculitis treatment of antineutrophil cytoplasmic autoantibodypositive systemic vasculitis and glomerulonephritis with pooled intravenous gamma globulin successful treatment of chronic antibody-mediated rejection with ivig and rituximab in pediatric renal transplant recipients early diagnosis and successful treatment of acute antibodymediated rejection of a renal transplant plasmapheresis, intravenous cytomegalovirus-specific immunoglobulin and reversal of antibody-mediated rejection in a pediatric renal transplant recipient: a case report posttransplant therapy using high-dose human immunoglobulin (intravenous gamma globulin) to control acute humoral rejection in renal and cardiac allograft recipients and potential mechanism of action intravenous immunoglobulin compared with cyclophosphamide for proliferative lupus nephritis intravenous immunoglobulin treatment of lupus nephritis intravenous immunoglobulin therapy in a patient with lupus serositis and nephritis successful treatment of rapidly progressive lupus nephritis associated with anti-mpo antibodies by intravenous immunoglobulins intravenous immunoglobulin therapy in the treatment of patients with pemphigus vulgaris unresponsive to conventional immunosuppressive treatment the role of ivig treatment in severe pemphigus vulgaris treatment of pemphigus with intravenous immunoglobulin corticosteroid-sparing effect of intravenous immunoglobulin therapy in patients with pemphigus vulgaris effectiveness of intravenous immunoglobulin therapy for skin disease other than toxic epidermal necrolysis: a retrospective review of mayo clinic experience intravenous immunoglobulin therapy for patients with pemphigus foliaceus unresponsive to conventional treatment influence of ivig therapy on autoantibody titers to desmoglein 1 in patients with pemphigus foliaceus steroid sparing effect of intravenous immunoglobulin therapy in patients with pemphigus foliaceus intravenous immunoglobulin therapy for patients with bullous pemphigoid unresponsive to conventional immunosuppressive treatment role of intravenous immunoglobulin in the treatment of bullous pemphigoid: analysis of current data treatment of oral pemphigoid with intravenous immunoglobulin as monotherapy. long-term follow-up: influence of treatment on antibody titers to human alpha6 integrin a severe persistent case of pemphigoid gestationis treated with intravenous immunoglobulin and cyclosporin effect of high-dose intravenous immunoglobulin therapy in stevens-johnson syndrome: a retrospective, multicenter study intravenous immunoglobulin therapy for children with stevens-johnson syndrome prospective, noncomparative open study from kuwait of the role of intravenous immunoglobulin in the treatment of toxic epidermal necrolysis high-dose intravenous immunoglobulin for severe drug reactions: efficacy in toxic epidermal necrolysis intravenous immunoglobulin treatment for stevens-johnson syndrome and toxic epidermal necrolysis: a prospective noncomparative study showing no benefit on mortality or progression treatment of toxic epidermal necrolysis. experience with 9 patients with consideration of intravenous immunoglobulin intravenous immunoglobulin for treatment of toxic epidermal necrolysis use of intravenous immunoglobulin in children with stevens-johnson syndrome and toxic epidermal necrolysis: seven cases and review of the literature treatment of toxic epidermal necrolysis with high dose intravenous immunoglobulins: multicenter retrospective analysis of 48 consecutive cases analysis of intravenous immunoglobulin for the treatment of toxic epidermal necrolysis using scorten: the university of miami experience treatment of toxic epidermal necrolysis with intravenous immunoglobulin in children intravenous immunoglobulin does not improve outcome in toxic epidermal necrolysis a randomized controlled evaluator-blinded trial of intravenous immunoglobulin in adults with severe atopic dermatitis adjunctive high-dose intravenous immunoglobulin treatment for resistant atopic dermatitis: efficacy and effects on intracellular cytokine levels and cd4 counts the treatment of atopic dermatitis with adjunctive high dose intravenous immunoglobulin: a report of three patients and review of the literature high dose immunoglobulin in atopic dermatitis and hyper-ige syndrome a review of high-dose intravenous immunoglobulin treatment for atopic dermatitis intravenous immunoglobulin un autoimmune chronic urticaria the effectiveness of low dose intravenous immunoglobulin in chronic urticaria effect of highdose intravenous immunoglobulin delayed pressure urticaria solar urticaria: one case treated by intravenous immunoglobulin treatment of solar urticaria by intravenous immunoglobulins and puva therapy intravenous immunoglobulin therapy suppresses manifestations of the angioedema with hypereosinophilia syndrome psoriasis: response to high-dose intravenous immunoglobulin in three patients treatment of pyoderma gangrenosum with intravenous immunoglobulin pyoderma gangrenosum treated with high-dose intravenous immunoglobulins: two cases and review of literature nephrogenic fibrosing dermopathy: response to high-dose intravenous immunoglobulin pretibial myxedema and high-dose intravenous immunoglobulin treatment scleromyxedema. a case series highlighting long-term outcomes of treatment with intravenous immunoglobulin (ivig) scleromyxoedema: treatment of cutaneous and systemic manifestations with high-dose intravenous immunoglobulin scleromyxedema: response to highdose intravenous immunoglobulin (hdivig) arndt-gottron scleromyxedema: successful therapy with intravenous immunoglobulins combination oral prednisone and intravenous immunoglobulin in the treatment of scleromyxedema successful therapy of scleromyxedema arndt-gottron with low-dose intravenous immunoglobulin marked improvement in scleromyxedema with high-dose immunoglobulin tumorous variant of scleromyxedema. successful therapy with intravenous immunoglobulins use of igiv in the treatment of atopic dermatitis, urticaria, scleromyxedema, pyoderma gangrenosum, psoriasis, and pretibial myxedema effect of ivig on the hair regrowth in a common variable immune deficiency patient with alopecia universalis regression of kaposi's sarcoma after intravenous immunoglobulin treatment for polymyositis efficacy and safety of intravenous immunoglobulin in patients with metastatic melanoma intravenous immunoglobulin as prophylaxis of chemotherapy-induced oral mucositis total remission of thymus carcinoma after treatment with intravenous immunoglobulin thymoma and immunodeficiency (good syndrome): a report of 2 unusual cases and review of the literature diabetic demyelinating polyneuropathy responsive to intravenous immunoglobulin therapy intravenous immunoglobulin therapy markedly ameliorates muscle weakness and severe pain in proximal diabetic neuropathy simultaneous multiple cranial nerve neuropathies and intravenous immunoglobulin treatment in diabetes mellitus a case of diabetic amyotrophy with severe atrophy and weakness of shoulder girdle muscles showing good response to intravenous immune globulin antibodymediated rejection of a pancreas allograft multifocal motor neuropath, type 1 diabetes and asymptomatic hashimoto's thyroiditis: an unusual association multifocal motor neuropathy and asymptomatic hashimoto's thyroiditis: first report of association thyrotoxic autoimmune encephalopathy in a female patient: only partial response to typical immunosuppressant treatment and remission after thyroidectomy high dose immunoglobulin iv treatment in adrenoleukodystrophy discussion on: high dose immunoglobulin iv treatment in adrenoleukodystrophy waterhouse-friderichsen syndrome and bilateral renal cortical necrosis in meningococcal sepsis autoimmune polyendocrine syndrome. treatment with intravenous immunoglobulins rituximab and intravenous immunoglobulins for relapsing postinfectious opsoclonus-myoclonus syndrome treatment of idiopathic opsoclonus-myoclonus syndrome with intravenous immunoglobulin parainfectious opsoclonus-myoclonus syndrome: high dose intravenous immunoglobulins are effective forty-one year follow-up of childhood-onset opsoclonus-myoclonus-ataxia: cerebellar atrophy, multiphasic relapses, and response to ivig treatment with intravenously administered immunoglobulins of the neuroblastoma-associated opsoclonus-myoclonus treatment of opsoclonus-myoclonus with high-dose intravenous immunoglobulin neuroblastoma-associated opsoclonus-myoclonus treated with intravenously administered immune globulin g high-dose i.v. human immunoglobulin in a case with infantile opsoclonus polymyoclonia syndrome clinical outcome in adult onset idiopathic or paraneoplastic opsoclonusmyoclonus therapeutic plasma exchange and intravenous immunoglobulin for obsessive-compulsive disorder and tic disorders in childhood intravenous immunoglobulin in relapsingremitting multiple sclerosis: a dose-finding trial intravenous immunoglobulin in primary and secondary chronic progressive multiple sclerosis: a randomized placebo controlled multicentre study no effect of intravenous immunoglobulins on cytokine-producing lymphocytes in secondary progressive multiple sclerosis intravenous immunoglobulins in the treatment of relapsing remitting multiple sclerosis-results of a retrospective multicenter observational study over five years intravenous immunoglobulin in secondary progressive multiple sclerosis: randomised placebo-controlled trial intravenous immunoglobulin g for the treatment of relapsing-remitting multiple sclerosis: a meta-analysis intravenous immunoglobulins in therapy of intermittent multiple sclerosis. an update sarova-pinhas i (1998) intravenous immunoglobulin treatment in multiple sclerosis. effect on relapses a double-blind, cross-over trial of intravenous immunoglobulin g in multiple sclerosis: preliminary results immunotherapy for guillain-barré syndrome: a systematic review high-dose immunoglobulin therapy for guillain-barré syndrome in japanese children intravenous immune globulins in patients with guillain-barré syndrome and contraindications to plasma exchange: 3 days versus 6 days intravenous immunoglobulin therapy for guillain-barré syndrome with igg anti-gm1 antibody randomised trial of plasma exchange, intravenous immunoglobulin, and combined treatments in guillain-barré syndrome intravenous immune globulin in the guillain-barré syndrome immunotherapy for igm antimyelin-associated glycoprotein paraprotein-associated peripheral neuropathies a randomised controlled trial of intravenous immunoglobulin in igm paraprotein associated demyelinating neuropathy a controlled study of intravenous immunoglobulin in demyelinating neuropathy with igm gammopathy intravenous immunoglobulin therapy in amyotrophic lateral sclerosis effect of high-dose intravenous immunoglobulin on amyotrophic lateral sclerosis and multifocal motor neuropathy effects of intravenous immunoglobulin on muscle weakness and calcium-channel autoantibodies in the lambert-eaton myasthenic syndrome treatment of stiff-man syndrome with intravenous immune globulin treatment of stiffman syndrome with intravenous immunoglobulin long-term remission of refractory stiff-man syndrome after treatment with intravenous immunoglobulin high-dose intravenous immune globulin for stiff-person syndrome high dose immunoglobulin iv treatment in adrenoleukodystrophy treatment of refractory epilepsy with intravenous immunoglobulins. results of the first double-blind/ dose finding clinical study intravenous immunoglobulin: a single-blind trial in children with lennox-gastaut syndrome effects of early treatment with immunoglobulin on critical illness polyneuropathy following multiple organ failure and gramnegative sepsis failure of high dose intravenous immunoglobulins to alter the clinical course of critical illness polyneuropathy experience with immunomodulatory treatments in rasmussen's encephalitis positive response to immunomodulatory therapy in an adult patient with rasmussen's encephalitis improvement in adult-onset rasmussen's encephalitis with long-term immunomodulatory therapy medical treatment of rasmussen's syndrome (chronic encephalitis and epilepsy): effect of high-dose steroids or immunoglobulins in 19 patients intravenous immune globulin (10% caprylate-chromatography purified) for the treatment of chronic inflammatory demyelinating polyradiculoneuropathy (ice study): a randomised placebocontrolled trial randomized controlled trial of intravenous immunoglobulin versus oral prednisolone in chronic inflammatory demyelinating polyradiculoneuropathy randomized controlled trial of ivig in untreated chronic inflammatory demyelinating polyradiculoneuropathy intravenous immunoglobulin treatment in chronic inflammatory demyelinating polyneuropathy. a double-blind, placebo-controlled, cross-over study intravenous immunoglobulin for myasthenia gravis iv immunoglobulin in patients with myasthenia gravis: a randomized controlled trial randomized, controlled trial of intravenous immunoglobulin in myasthenia gravis immunoglobulin treatment versus plasma exchange in patients with chronic moderate to severe myasthenia gravis immunoglobulin treatment in refractory myasthenia gravis high-dose, rapid-infusion ivig in postvaccination acute disseminated encephalomyelitis outcome of severe encephalomyelitis in children: effect of highdose methylprednisolone and immunoglobulins effectiveness of intravenous immunoglobulin treatment in adult patients with steroid-resistant monophasic or recurrent acute disseminated encephalomyelitis improvement of atypical acute disseminated encephalomyelitis with steroids and intravenous immunoglobulins dramatic improvement of severe acute disseminated encephalomyelitis after treatment with intravenous immunoglobulin in a three-year-old boy treatment of acute disseminated encephalomyelitis with intravenous immunoglobulin brief report: a pilot open clinical trial of intravenous immunoglobulin in childhood autism early and prolonged intravenous immunoglobulin replacement therapy in childhood agammaglobulinemia: a retrospective survey of 31 patients highvs. low-dose immunoglobulin therapy in the long-term treatment of xlinked agammaglobulinemia efficacy of intravenous immunoglobulin on the prevention of pneumonia in patients with agammaglobulinemia intravenous immunoglobulin therapy for antibody deficiency the effect of two different dosages of intravenous immunoglobulin on the incidence of recurrent infections in patients with primary hypogammaglobulinemia intravenous immunoglobulin, splenectomy and antibiotic prophylaxis in wiscott-aldrich syndrome new diagnostic and therapeutic aspects of hyper-ige syndrome hyperimmunoglobulin e syndrome: two cases and a review of the literature high dose intravenous immunoglobulin in atopic dermatitis and hyper-ige syndrome immunodeficiency presenting as hypergammaglobulinaemia with igg2 subclass deficiency deficiency in immunoglobulin subclasses as cause of increased infection susceptibility-a family study chronic rhinosinusitis and recurrent nasal polyps in two children with igg subclass deficiency and review of the literature a case of igg subclass deficiency with the initial presentation of transient hypogamma immunoglobulinemia of infancy and a review of igg subclass deficiencies antibody response in patients with osteomyelitis of the mandible. oral surg oral med oral pathol oral radiol endod clinical and laboratory characteristics of 75 patients with specific polysaccharide antibody deficiency syndrome selective antibody immune deficiency in a patient with smith-lemli-opitz syndrome clinical, immunologic and genetic analysis of 29 patients with autosomal recessive hyper-igm syndrome due to activation-induced cytidine deaminase deficiency the hyper-igm syndrome: 13 observations effects of intravenous immunoglobulin on clinical and immunological findings of patients with humoral immunodeficiency diseases non-x-linked hyperimmunoglobulin m syndrome with chronic interstitial pneumonitis successful management of neutropenia in a patient with cd40 ligand deficiency by immunoglobulin replacement therapy hyper-igm syndrome: report of one case transient hypogammaglobulinemia of infancy presenting as staphylococcus aureus sepsis with deep neck infection transient hypogammaglobulinemia of infancy with severe bacterial infections and persistent iga deficiency hematopoietic stem-cell transplantation for the treatment of severe combined immunodeficiency efficacy of intravenous immunoglobulin in the prevention of pneumonia in patients with common variable immunodeficiency key: cord-332432-q7u943k6 authors: hofkirchner, wolfgang title: a paradigm shift for the great bifurcation date: 2020-06-30 journal: biosystems doi: 10.1016/j.biosystems.2020.104193 sha: doc_id: 332432 cord_uid: q7u943k6 this paper is an attempt to achieve an understanding of the situation the evolution of humanity is confronted with in the age of global challenges. since global challenges are problems of unprecedented complexity, it is argued that a secular paradigm shift is required away from the overemphasis on allegedly neutral standpoints, on a mechanistic picture of the world and on deductive logics towards accounts of emergence, of systemicity, informationality and conviviality, building upon each other and providing together a transdisciplinary edifice of the sciences, in the end, for, and by the inclusion of, citizens. viewed from such a combined perspective, the current social evolution is punctuated by a great bifurcation similar to bifurcations other emergent systems have been facing. on the one hand, humankind is on the brink of extinction. it is the world occurrence of the enclosure of commons that is detrimental to sharing the systemic synergy effects and thus to the cohesion of social systems. on the other hand, humanity is on the threshold of a planetary society. another leap in integration would be the appropriate response to the complexity confronted with. humans and their social systems are informational agents and, as such, they are able to generate requisite information and use it to catch up with the complex challenges. they can establish convivial rules of living together in that they disclose the commons world-wide. by doing so, they would accomplish another evolutionary step in anthroposociogenesis. the concept of the global sustainable information society describes the framework of necessary conditions of conviviality under the new circumstances. the seemingly disruptive advent of the covid-19 pandemic outshone the climate change that has been gaining obvious momentum since the last fifty years to an extent that it threatens with a much more decisive rupture if science, technology and society are not unwilling to learn from the pandemic that there is nonlinear growth with complex challenges be they small or large and that human actors are not completely doomed to helplessness though. for such a lesson to learn, a secular shift in thinking and acting throughout sciences and everyday life is required because human actors need to be capacitated to cope with complex challenges such as the global problems. a shift is already underway though not yet hegemonic. this shift has to overcome three prejudices of conventional science: • the outdated ideology of value-free scientific research. the absence of values would make science distinct from biased everyday thought. but that's not the distinction. any research is driven by societal interests even if mediated by personal curiosity. any research implies particular values, reflected or not if not even camouflaged. of course, these values must not divert the findings of research, quite the contrary, they shall stimulate evidence-based research -and that's the distinction from biased opinion of everyday. science can critique opinions. in the last two decades, several labels have become aspirational for scientists: research shall be responsible, university research shall be aware of its third mission, namely, to serve the common good, applied research shall be replaced by use-inspired basic research, research shall become practically transdisciplinary in that it transcends science towards, and include in science, the values of everyday people that are affected by the results of research, best by letting them participate in research. these are attempts in the right direction: the acceptance that there is a limited controllability of what can be done. as everyday-thinking and -acting, science has limited controllability over its impact on society but within that certain limit it is capable of controlling and thus it must aim at doing so in a precautious way. neither phantasies of omnipotence nor of impotence are called for but a deliberate activism is (hofkirchner, 2013a, 62-71) . not everything that might be feasible is also desirable and not everything that might be desirable is also feasible. the feasible and the desirable need to be made compatible with each other. • the outdated mechanistic picture of the world. cause-effect relationships are fancied as if pertaining to a machine constructed by humans. cause and effect would obey laws of strict determinacy such that the effect would follow necessarily from the cause. but that is true only for a small subdivision of effective causality -causes can have different effects and effects can be brought about by different causes -, let alone final, material and formal causality (hofkirchner, 2013a, 86-95) . laws of nature and other parts of the world are not given for eternity. the late karl raimund popper called those laws propensities -an asymmetrical, contingent behaviour of the universe (1990) . this shows the right direction. strict determinism as well as indeterminism are false alternatives. less-than-strict determinism avoids both fallacies. • the outdated preponderance of methodologies that are based on deductive logics. deductivism is an attempt to deduce that which shall be explained or predicted from premises such that the phenomenon can be subsumed under a proposition of the form of a universal implication that covers the phenomenon. the premises suffice by definition for the conclusion, the phenomenon is thus reduced to a sufficient condition. but, in fact, those conditions are rarely sufficient. there is the search for alternative logics such as the hype for abduction or claims for a trans-classical logic (günther, 1990) . and there is the logic in reality of joseph brenner (2008) who grounds logic in reality, that is, he gives primacy to how reality works in principle when postulating logical principles. anyway, it needs to be accepted that explanation and prediction are incomplete and that they should focus on the adjacent necessary instead, that is, a necessary condition that might rarely be sufficient but should form a basis of understanding as close as possible to the phenomenon that shall be explained or predicted (hofkirchner, 2013a, 131-139; 1998) . neither deductivism nor irrationalism -for which anything would go -can convince but a reflexive rationalism that accepts incomplete deducibility with an ascendance from the abstract to the concrete where by each step a new assumption is introduced without deduction. the build-up of such a specification hierarchy is important for the transdisciplinarity in its theoretical sense -the consideration of as many facets of the phenomena as possible in order to achieve a unified understanding. the removal of those impediments for the progress of science are the milestones that the paradigm shift has to master. only if they will have been achieved, humanity will be ready to confront the global challenges in a way that safeguards mankind against man-made extinction. the next sections substantiate how, on the three pillars of deliberate activism, less-than-strict determinism and reflexive rationalism, a new understanding of the current situation of world society can be erected and how it can make the disciplines of the whole edifice of science responsive to that task. the sections will proceed from a systemic level to an informational level to a convivialist level. 1 emergentist systemism 'emergentist systemism' is a term introduced by poe yu-ze wan (2011) in the context of social theory, after it had been used in the field of social work in switzerland (e.g., obrecht, 2005) to characterise the approach of philosopher of science, mario bunge. bunge himself was rather used to terms such as 'emergentist materialism' to signify, e.g., his position in the field of the mind-body problem. however, he defined systemism in a broader sense, namely as 'ontology: everything is either a system or a component of some system. epistemology: every piece of knowledge is or ought to become a member of a conceptual system, such as a theory. axiology: every value is or ought to become a component of a system of interrelated values ' (2012, 189) . and he defined emergence as '[…] advent of qualitative novelty. a property of systems ' (2012, 185) . thus, emergentism is 'a world view or an approach' that focuses on emergence (2003, 38) . 'systemism, or emergentism', as he said (2003, 38-39) , 'is seem to subsume four general but one-sided approaches: 1. each of these four views holds a grain of truth. in putting them together, systemism (or emergentism) helps avoid four common fallacies.' this is by and large the sense in which emergentist systemism is understood here (hofkirchner, 2017a ) -as weltanschauung, that is, a world view that is not value-free (a german term by which mark davidson 1983 summed up ludwig von bertalanffy's general system theory and which will be subsumed here under the term praxiology), as conception of the world (ontology) and as way of thinking to generate knowledge about the world (epistemology). in a nutshell, emergent systemism means that, practically, humans intervene as a rule for the betterment of social life in real-world systems they conceive of by patterns they have already identified and that these systems are emergent from the co-operation of other systems that become or are their elements. this is called selforganisation. since the idea of emergent systems implies a kind of evolution, these systems are also known by the term evolutionary systems. evolutionary systems theory -a term coined by ervin laszlo (1987) , vilmos csanyi (1989) and susantha goonatilake (1991) but extended here to cover the meaning it received after the seminar held at the konrad lorenz institute for evolution and cognition research in vienna 1995 (van de vijver et al., 1998) -is the proper theory of self-organisation, a merger of systems theory and evolutionary theory by which the first was enabled to include more than ideas of maintaining systems only and the latter could emancipate from mechanistic interpretations of the darwinian model. self-organisation is characterised by evolvability and systemicity. that means that matter, nature, real-world events or entities evolve such that systems materialise as organisation of components (hofkirchner, 2013a, 58-59) . applying emergentist systemism to the edifice of science(s) brings a profound change (hofkirchner, 2017a) . how is the old paradigm's view of that edifice? let's start with philosophy, composed of epistemology, ontology and praxiology (ethics, aesthetics and else) as the most abstract enterprise and put it in the background. before that background, you have the three categories of formal sciences, real world sciences and applied sciences in juxtaposition. the formal sciences include logics and mathematics as disciplines. the real-world sciences comprise disciplines that investigate nature, on the one hand, and were called typically physics, chemistry, biology and else, and disciplines that investigate the social world, on the other, nowadays summarised under the term social and human sciences including sociology, cultural, political, and economic sciences and else. applied sciences assemble engineering, management, arts and else. every discipline is divided by sub-disciplines and sub-sub-disciplines besides all having their own legitimation as basic research, formal sciences are known for providing instruments for gaining knowledge in the real-world sciences, real-world sciences are needed for the provision of evidence for developing technologies, organisation, pieces of art. however, what makes the co-operation of sciences difficult is that they are siloed against each other by impermeable boundaries. connection between those mono-disciplines can be attempted only by heaping some of them together in a multi-disciplinary approach, which is no connection at all, or by peripheral exchanges in an interdisciplinary approach, which does not admit internal changes and keeps the disciplines as alien to each other as they have been before. driven by the confrontation with complex problems, things have begun to change already in the direction of semi-permeability of disciplinary boundaries, which, in the long run, paves the way for the establishment of new stable relations between them. emergentist systemism is not another discipline that just adds to the picture of the old disciplines. it causes rather a paradigm shift that has the potential to transform the whole edifice of science(s). philosophy that was deprived of fruitful relations to the disciplines of science in what had become normal science turns into systems philosophy now; formal sciences turn into formal as well as non-formal systems methodology; realworld sciences turn into sciences of real-world systems, that is, material, living or social systems; and, finally, applied sciences turn into a science that makes artefacts by designing systems and, in doing so, integrates them with social systems. so, at one blow, connectedness is unveiled between all inhabitants of the edifice. transgressions from one scientific endeavour to another can be mediated via jumping forth and back over shared levels of scientific knowledge. those levels form now a specification hierarchy. jumping from one specific level to a more general level allows comparison and adjustment of both levels. it allows the initial level to instigate knowledge adaptations on the target level or adoption of knowledge on re-entry from the target level. in addition, a more general level works as bridge for jumping up the ladder to even higher levels or down to different lower levels so as to help understand that their knowledge is just another specification of the knowledge they share at a higher level. this makes the sciences of evolutionary systems a transdiscipline and its inherent emergentist systemism makes the edifice of disciplines a transdisciplinary, common endeavour of all science. semi-permeability does not lift the boundaries. relative autonomy of disciplines is maintained in the overall transdisciplinary network. paraphrasing bunge, informationism is a term used here to denote a praxiological perspective on, an ontological conception of, and an epistemological way of thinking about, information, which takes centre stage in this tenet. for the sake of consistence, information is set to be based upon, and concretise further, systems, in particular, emergent information shall easily relate to emergent systems. this can be achieved through the assumption of informational agents -agents being emergent systems. thus, the generation of information is enshrined in the self-organisation of systems. any time a system self-organises, its agency brings forth information. an evolutionary system can be defined as 'a collection of (1) elements e that interact such that (2) relations r emerge that -because of providing synergistic effects -dominate their interaction in (3) a dynamics d. this yields a distinction between micro-level (e) and macro-level (r) and a process (d) that links both levels in a feedback loop' (hofkirchner, 2013a, 105) . with reference to, but in modification of, a triadic semiotics after charles sanders peirce (2000) , laying emphasis on the intrinsic connection of self-organisation with negentropy after edgar morin (1992, 350 and 368) and by usage of the term 'perturbation' introduced by humberto maturana and francesco varela (1980), information can be defined as 'relation such that (1) the order o built up spontaneously (signans; the sign) (2) reflects some perturbation p (signandum/signatum; (to-be-)signified) (3) in the negentropic perspective of an evolutionary system s e (signator; the signmaker' (hofkirchner, 2013a, 171) . 'information is generated if self-organising systems relate to some external perturbation by the spontaneous build-up of order they execute when exposed to this perturbation. in the terms of triadic semiotics, the self-organising systems, by doing so, assign a signification to the order and make it a sign which stands for the so signified perturbation' (hofkirchner, 2013a, 172) . this is the approach of a unified theory of information (hofkirchner, 2013) . it is worth noting that those assumptions attribute information generability to emergent systems according to the evolutionary type they represent. not only social systems and their inhabitants are qualified as informational agents (this would import the acceptance of umberto eco's threshold of semiosis applicable to the realm of human culture exclusively), not only biotic systems (this is the threshold of biosemiotics) but also physical systems in so far as they are able to self-organise are qualified to generate information in shades -as far as the respective evolutionary stages allow. emergent systems of any kind produce emergent information. as to the new scientific edifice, informationism is mounting systemism. systems philosophy becomes a systemic philosophy of information; systems methodology becomes a systemic information methodology; the sciences of real-world systems become sciences of information of real-world systems, that is, of material information, living information and social information; and the science of designing artificial systems becomes the science of designing information in artificial systems. all that information is emergent information. convivialism is a term denoting a social perspective (praxiology), a conception of the social world (ontology) and a social-scientific way of thinking (epistemology) for which conviviality is key. conviviality as term was introduced by the austrian-american writer, ivan illich, who published a book with the title tools for conviviality (1973) . it contained a philosophy of technology according to which technology should be socially controlled so as to reclaim personal freedom that is restricted by uncontrolled technological development. conviviality -illich was familiar with the spanish term convivialidad -has latin origins and means the quality of living together in the manner of dining together (convivor) of hosts (convivator) and guests (conviva) at common feasts (convivium). in the last decade, that term gained new attention when mainly about fourty french intellectuals -among them serge latouche, edgar morin or chantal mouffe -opened the discussion on a political manifesto for the redesign of social relations. the first manifesto was followed by a second, up-dated one in 2020 (internationale convivialiste). according to the latter, convivialism 'is the name given to everything that in doctrines and wisdom, existing or past, secular or religious, contributes to the search for principles that allow human beings to compete without massacring each other in order to cooperate better, and to advance us as human beings in a full awareness of the finiteness of natural resources and in a shared concern for the care of the world. philosophy of the art of living together, it is not a new doctrine that would replace others by claiming to cancel them or radically overcome them. it is the movement of their mutual questioning based on a sense of extreme urgency in the face of multiple threats to the future of humanity. it intends to retain the most precious principles enshrined in the doctrines and wisdom which were handed down to us.' convivialism is emergentist if seen in the context of emergent systems and emergent information. social systems are here considered as evolutionary systems, which is in stark contrast to how german sociologist niklas luhmann (1995) considered them (wan, 2011). though luhmann originally claimed to start with general system theory when elaborating his theory of social systems, a revisiting of bertalanffy would lead to different conclusions . such an approach has been pursued not only by bunge but also by members of ervin laszlo's general evolution research group, among them robert artigiani (1991) , by representatives of critical realism, in particular margaret s. archer (1995; 2010; and her project group on social morphogenesis at the centre for social ontology, and workers departing from us sociologist walter f. buckley (1967) , including the economist tony lawson (2013) and the relational sociologist pierpaolo donati (donati, 2011; donati and archer, 2015) . of course, many other sociologists are worth mentioning; even if they do not explicitly share a systems approach, they have nevertheless contributed with important insights to such a framework (giddens, 1984; alexander, 1995; mouzelis, 1995; reckwitz 1997) . social systems are the evolutionary product of living systems but contain living systems that get a social shape. the elements of social systems as conceived here are social agents -humans called actors -and their organisational relations are social relations -called structure. actors inhabit the micro-level of social systems, while the macro-level is where the structure is located. the structure is produced by the actors and it exerts a downward causation on the actors and their agency (hofkirchner, 2013b, 136; lawson, 2013 ). thus, social systems self-organise as living and material systems do, but they differ from living and material systems as to their mode of self-organisation. 'social self-organisation goes beyond biotic self-organisation, which, in turn, goes beyond physicochemical self-organisation' (hofkirchner, 2014, 120) . social self-organisation does so in that it transcends, re-invents, creates the social systems through the action, interaction and co-action of their actors, who -as informational agents -cognise, communicate and co-operate mindfully when reproducing and transforming their social systems, which, in turn, can be considered as higher-level informational agents. social self-organisation is not conceivable without the generation of specific social information. the triple-c model postulates a hierarchy of information processes such that cognition is the necessary condition for the functioning of communication and communication the necessary condition for the functioning of co-operative information, in short, co-operation (hofkirchner, 2013) . psychic functions such as thought and others, the ability to speak and the ability to devise and manufacture artefacts, in particular, tools, are characteristic of humans. all of them are knit together in social information: 'human thought is part of human cognition […]; human language is part of communication […] ; human tools are part of work that belongs to human co-operation' (hofkirchner, 2016, 286) . starting with work at the top (which refers to the structure on the system's macro-level), it is about constituting common goals and instituting common goals. work is consensual. co-operation involves finding and building consensus. what is needed here, are common intentions. common intentionality provides the perspective of the whole we, the perspective of the social system. consensualisation, in turn, presupposes a certain collaboration that designs specific tasks for reaching the shared goals and assigns these tasks to certain actors. that is done on the social information level below, on the level of language (which refers to the network of interactions the actors form on the system's micro-level). communication functions as the means to realise that kind of collaboration that is needed for the upper level. that is, taking the perspective of the other facilitates collaboration. however, taking the perspective of the other is promoted by taking the perspective of the whole in which one's own and the others' roles are included. what is required here, is readiness for a dialogue with sense for consilience. collaboration, in turn, presupposes a certain co-ordination that devises certain operations for fulfilling the tasks and supervises certain actors in performing the operations. that is worked out on the lowermost level, on the level of thought (which refers to the actions of the individual actors who are also located on the system's micro-level). cognition allows the actors to understand what kind of co-ordination is needed by the upper level. it enables the actors to reflect upon the relationship between operations, tasks and goals. what is necessitated here, is reflexivity, the capacity to reflect the social context in which the cognising actor is embedded (archer, 2010) , and conceptuality, the capacity to use concepts, all of which are influenced by verbal language (logan, 2007; . the rationale of every complex system is synergy (corning, 1983; . agents produce synergetic effects when co-operating systemically -effects they could not produce when in isolation. in social systems, synergy 'takes on the form of some social good. actors contribute together to the good and are common beneficiaries of that good -the good is a common good, it is a commons' (hofkirchner, 2014, 121) . the social relations are commoning relations. conviviality then, as a feature of emergent social systems with emergent social information, can be determined as the historical-concrete shape of the commoning relations. it is a social value that is highly esteemed, it is a theoretical conceptualisation of a social practice, and it is a measurand the value of which can be estimated by empirical studies -it is all of them in one because it is an expression of the quality of the commoning relations. it expresses how just those relations are constructed, how equitable, free and solidary, and to which degree they enclose or disclose the commons. conviviality is visionary and longs for actualisation. its actualisation would 'make the social systems inclusive through the disclosing of the enclosed commons and, by doing so, […] warrant eudaimonia, a good life in a good society, the flourishing of happy individuals in convivial social relations' (hofkirchner, 2017b, 286) . having defined the commons as social synergy and conviviality as measure of the actualisation of envisioned commoning relations, the critical theory perspective becomes apparent (hofkirchner, 2015, 97-99) -the perspective of a critical social systems theory as part of the social systems sciences in the new edifice of disciplines. conviviality is emergent. it develops over time and changes its forms in a contingent way. referring to michael tomasello's shared intentionality hypothesis and his interdependence hypothesis (tomasello et al., 2012; tomasello, 2014; , there have been two key steps in anthroposociogenesis (the becoming of humans and society) so far and, following the new systemic, informational and convivialist paradigm, a possible third one is imminent. the next subsections discuss those steps. leaps in quality emerge in systems as novel organisation due to a change in the organisational relations. thus, changes on the top-most levels of information generation and usage are decisive. all of them are shifts in co-operation. if work, language and thought build the human/social hierarchical levels of information from a synchronic point of view, then, from the diachronic point of view, it may well be assumed 'that it is conditions of co-operation that made the difference in evolution. evolutionary pressure unfolded a ratchet effect that yielded ever higher complex co-operation' (hofkirchner, 2016, 287) . the state of co-operation in the ancestors of humans is the origin of anthroposociogenesis. self-reinforcing processes came about. changes in the state of co-operation proliferated down to provoke changes on the level of communication -the development of human language -in order to propel co-operation and changes in the state of communication proliferated down to provoke changes on the cognition level -the development of thinking -in order to propel communication. in the beginning, so tomasello, there was a shift from individual to joint intentionality. individual intentionality of common ancestors of chimpanzees and humans was the point of departure about six million years ago. as living together was driven by self-interest of animal monads, there was no need for taking in consideration common goals, no need for thinking on a level beyond the actual egocentric perspective (tomasello, 2014, 4, 30) . early humans began to speciate only when they took advantage of going beyond individual intentionality and adopted 'more complex forms of cooperative sociality' (31). a first step occurred 'in the context of collaborative foraging' (33), that is, the hunting of large game and gathering of plant foods, around 2 million years ago. this step culminated about 400.000 years ago, when joint intentionality emerged. hunters and gatherers developed dyadic cooperations driven by a 'second-person morality ' (2016, 6) . hence a need for acknowledging a common goal, that is, an understanding that the partner shares the goal and both are committed to act according to its achievement. multiple and vanishing dyadic relationships formed in which early humans shared a joint goal. in order to support the negotiation of joint goals and the coordination of collaboration, human communication originated with 'a commitment to informing others of things honestly and accurately, that is, truthfully ' (2014, 51) . cognitively, 'when early humans began engaging in obligate collaborative foraging, they schematized a cognitive model of the dual-level collaborative structure comprising a joint goal with individual roles and joint attention with individual perspectives' (69). this was a premature state of conviviality. dyadic co-operation guaranteed the common good for the included actors. the shift from individual to joint intentionality was followed by a shift from joint to collective intentionality. collective intentionality emerged with early humans about 150.000 to 100.000 years ago. this shift occurred with the advent of culture, that is, of separate and distinct cultural groups, the interdependence that caused co-operation reigned 'not just at the level of the collaborating dyad, and not just in the domain of foraging, but at the level of the entire cultural group, and in all domains of life' (tomasello, 2016, 85) . this step created objective morality (6). co-operation became triadic. since then a need for group-thinking has become characteristic of humanity, that is, knowing that any person belonging to the same group culture can be expected to share the same values and norms -by constructing a meta-level such that any group member can imagine the whole of the group, the roles taken, her own as well as others' replaceability. in line with that, communication was to start with discourses about 'objective' facts in need of compelling arguments and cognition had to turn into fullblown human reasoning; 'the individual no longer contrasted her own perspective with that of a specific other […]; rather, she contrasted her own perspective with some kind of generic perspective of anyone and everyone about things that were objectively real, true and right from any perspective whatsoever ' (2014, 122) . cognition involved a new feature of generalisation capacity. this was the next step of conviviality. the third of the triad is relations of society that relate individuals to each other with respect to the common good -even if the concrete content of the common good became a matter of disputation and conflict. and today, a third step of anthroposociogenesis can be hypothesised. there might be a shift from collective intentionality to one that is shared universally, that is, on a planetary scale. that would be the transition to another convivial regime -an extension of the triad to the whole of humanity, an omniad. this extension would be necessary because the conflict over the commons has reached an extension that endangers conviviality at all and the curbing of the extension of the conflict over the commons by extending the triad to an omniad can be considered possible, which is discussed in the sub-subsections to follow. another step is necessary, given that it is agreed that there shall be a human future, which is tantamount with a humane future (hofkirchner 2017b ). in the course of evolution, complex systems move on trajectories on which bifurcations occur. they occur if and when the provision of synergy effects becomes problematic. bifurcations force the systems to change their trajectory. the old one cannot be continued any more. it bifurcates into a variety of possible future trajectories. there are two of them that span the whole variety in the possibility space between two extremes: systems might be able to achieve a leap from the previous level of evolution on which they could enjoy a steady state onto a higher level which forms part of a successful mega-evolution (haefner, 1992, 314; oeser, 1992 , 103-104) -a breakthrough to a path that transforms the systems into what is called meta-system (the metasystem transition) or supra-system -or they might even not be in the position to avert devolution -a path that leads to the breakdown of the systems. amplified fluctuations of parameters indicate the crossroads that demand taking one path or another. the nonlinear dynamics of complex systems make the crossroads appear in one as windows of opportunity to spiral upwards and as tipping points that let the systems spiral downwards into oblivion (laszlo 2001; 2006) . complex systems that can be observed today are those that could manage so far to harness synergy. the evolution of social systems is no exception. 'today, enclosures of the commons have been aggravated to such a degree that all of them morphed into global challenges. global challenges drive an accumulation of crises that mark a decisive bifurcation' (hofkirchner, 2017a, 10) . not only do global challenges cause a multi-crisis in the tension between, and among, social systems from the granularity of today's nation states down to the granularity of the smallest units made up by individualised actors, cutting across all social areas such as the cultural, the political, the economic, the ecological (eco-social) and the technological (techno-social) area and affect so humanity as a whole, but they also threaten, for the first time in human evolution, with the ultimate impact for humanity -with extinction. thus, that decisive bifurcation, in which a branch is much sought after to lead out of a dead-end branch, is called here the great bifurcation. that term resembles karl polanyi's term of the great transformation (1944) in that it embeds the conflict of market capitalism with democracy -the point that was of utmost importance to polanyi -in the complex systems context of anthroposociogenesis. 'either the social systems that together constitute mankind undergo a metasystem transition onto a higher level of organisation that allows the continuation of social evolution on earth or they, eventually, fall apart and discontinue anthropogenesis; either they succeed in rising their complexity such that they break through to a new step in the mega-evolution of humanity or there is a decline in complexity, a breakdown and devolution; either their differences can be integrated or they disintegrate themselves' (hofkirchner, 2020a, 1) . another step is not only necessary for a surviving and flourishing humankind. it is also possible and the reason for that is not only that such a step is grounded objectively in the possibility space given by the bifurcation. moreover, humans can be conceded the subjective potency to find the right way out of the crossroads, in particular, since the problems they come across are of anthropogenic origin and can be solved by a proper re-organisation of the social systems. they can view the evolution of humanity from the inside, explore and anticipate the way out and, finally, intervene accordingly (laszlo, 1987; 2001; 2006) . they belong to the first species on earth that can overcome self-made problems in the sharing of synergy. any emergent system can boost emergent information to catch up with the complexity of the challenges. 'if there is a mismatch between the complexity of a system and the complexity of the problems faced by the system, that system can catch up. […] intelligence is the capability of selforganising systems to generate that information which contributes in the best way to solving problems. the better their collective intelligence, that is, the better their problem-solving capacity and the better their capability to generate information, the better their handling of the crisis and the order they can reach. higher complexity not only signifies a higher degree of differentiation. at least as importantly, it signifies a new quality of integration. only a new level of integration can deal with an intensification of differentiation' (hofkirchner, 2017a, 10) . this can be called the law of requisite information (hofkirchner, 2020a, 3) that is elaborated on the basis of w. ross ashby's law of requisite variety (ashby, 1956) . according to the latter, a system is said to be able to steer another system, if the variety it disposes of corresponds, if not surpasses, the variety of the system to be steered. by departing from the narrow cyberneticist view through connecting variety with complexity and complexity with information and extending the reach of that which is to be steered from the outside to the inside, it can be concluded: 'requisite information is that appropriate information a system has about the complexity of the exterior and interior environment. requisite information safeguards the functioning of the system' (hofkirchner, 2020a, 3) . humanity entered the great bifurcation because 'the social relations of any partition of humanity are based on the principle of othering of partitions that are considered outside of them, thus not doing justice to legitimate self-interests of the rest of the partitions. frictions […] are caused by the lack of relations that would be valid for all partitions from a bird's eye view, that is, from a meta-level perspective. the establishment of such relations would mean the abolition of those frictions by a new supra-system in which all existing systems take part and shape according to the new relations on a higher level, following the application of the subsidiary principle as a basis for the preservation of diversity and autonomous agency" (hofkirchner et al., 2019, 455) . despite some literature based on biases due to biologism unable to imagine a transgression of the conceptual framework of the nationstate we, transnational relations have been taking shape. there is empirical evidence of co-operation between culturally homogeneous groups several tens of thousands of years ago, between cities around five thousand years ago, and between modern states since the seventeenth century (messner and weinlich, 2016; neumann, 2016; grimalda, 2016) . 'this co-operation between collective actors like groups, cities and states has already been paving the way for co-operation among the whole of humankind in the same way that dyadic, interpersonal co-operation between individual actors opened up the space of possibilities for triadic, societal co-operation' (hofkirchner et al., 2019, 455) . the term information society is gaining a new meaning. it does not mean a society that is only informatised, that is, penetrated by information technology as a report to france's president originally insinuated (nora and minc, 1978) . it means an informatised society only if that society uses its informatisation for becoming informational in a non-technical sense. becoming informational entails becoming sustainable and becoming sustainable entails, in turn, becoming global. such is information society on the point of obtaining another meaning in the context of critical social systems theory, which crystallises as critical information society theory. it points towards the global sustainable information society as a framework (hofkirchner, 2017c) : • informationality of the global sustainable information society means 'the envisioned state of informedness of informational actors and systems in which they will catch up with the complexity they are challenged by the great bifurcation to such an extent that […] they will dispose of the capacity to recognise the causes of the global challenges in order to accordingly re-organise human life on earth to master those challenges' (hofkirchner, 2020a, 2) . informationalisation signifies the provision of social information for the installation of safeguards against the deprivation of commons world-wide and thus a new step in the evolution of conviviality. • the provision of such safeguards, in turn, is the process of executing sustainability. sustainability in the framework of the global sustainable information society does so receive a new meaning too. it means 'the envisioned state of the world system that will be shaped and shaping the social relationships between all parts, and throughout any part, of humanity pursuant to the commoning relations on the higher level' such that 'dysfunctions in the working of the organisation of the social system are kept below a threshold the transgression of which would discontinue social evolution' (hofkirchner, 2020a , 2). • the higher level on which the commons shall be provided, is 'the envisioned state of world society as an integrated meta-and supra-system in which social relationships will connect all parts of humanity in all fields of human life', which, eventually, conveys a new meaning to globality in the context of the global sustainable information society. 'these commoning relations need to be lifted onto the planetary level, and the emerging superordinate system will nest all actors and systems according to the new expanded commoning relations. by that, global governance is carried out' (hofkirchner, 2020a, 2) . globalisation signifies the provision of the commons worldwide. the notion of the global sustainable information society is far from a blueprint of the future society but describes which necessary conditions need to be met if the great bifurcation shall be successfully passed. there are three imperatives of social information that must be obeyed so as to enable actors to take that next step in the evolution of convivial humanity. on the co-operative level, normative, valueladen information must become hyper-commonist, that is, it must orient the consciousness and conscience of the actors towards the reclaiming of the commons in a universal manner; on the communicative level, dialogical information must become all-inclusive, that is, it must not exclude any actor in a universal conversation about the common good; on the cognitive level, reflexive information must become meta-reflexive, that is, it must be concerned about changes of the meta-level that is a universe for all actors (hofkirchner, 2017c) . in order to accomplish that third step in conviviality, those imperatives, investigated by social sciences and humanities, need to be provided to civil society by translational sciences, all of them integrated and implemented by the new paradigm shift as transdisciplinary basis. scientific thinking as well as everyday thinking need to support each other in the comprehension and tackling of the next step. thus, emergentist systemism, informationism and convivialism, shifting research to a remedy for the global challenges, to a reconciliation of determinacy and indeterminacy, and to a logic of emergence, are no academic exercise of no avail. also, common sense is, in principle, capable of understanding those issues of the paradigm shift as well as becoming activist on that premises. the step will be an unprecedented revolutionary one. revolutionary thinking 'needs to focus on future social relations that are not yet actualised. it needs to anticipate them ideationally on a new meta-level, it needs to anticipate the meta-/suprasystem transition of the social systems.' and, taking up an idea of ernst bloch (1967), it 'does not only need to anticipate what is desirable but needs to explore which desirable is also possible in the here and now. only what is potential can be actualised. thus, it looks in the space of possibilities now for the foreshadowing of something that might become a future third' (hofkirchner, 2020b, 4) . the conclusion is that the current state of human evolution has been reached as emergent response to requirements of co-operation through two steps in anthroposociogenesis, namely, from the living together of individual monads towards a joint interaction in dyads and from that to a collective working together that was mediated by social relations -which are the social system's relations of the organisation of the commons -such that a triad has taken over the co-action of humans: a meta-level was constructed as a third that relates the interaction of the group members as a second and any action of a member as a first. now that global conditions require global co-operation, the third needs to be extended to another level ushering in a new phase. current discourses on whether or not our time shall be called anthropocene or whether or not we can stop the climate change or prevent pandemics like the covid-19 one, are dominated by pejorative connotations and negative imaginaries of the future. they lack a focus on the real potentialities of humanity that is just on the point of going through a possible next step of social evolution. extermination is the risk of the crisis. meta-reflexive global citizens, engaging in a global dialogue can kick off the emergence of global governance and thus solve the crisis. fin de siecle social theory -relativism, reduction, and the problem of reason realist social theory -the morphogenetic approach structure, agency and the internal conversation making our way through the world -human reflexivity and social mobility the reflexive imperative in late modernity social evolution: a nonequilibrium systems model an introduction to cybernetics sociology and modern systems theory the synergism hypothesis -a theory of progressive evolution nature's magic -synergy in evolution and the fate of humankind evolutionary systems and society -a general theory of life, mind and culture uncommon sense. tarcher relational sociology -a new paradigm for the social sciences the possibilities of global we-identities die tradition der logik und das konzept einer transklassischen rationalität emergence and the logic of explanation emergent information -a unified theory of information framework self-organisation as the mechanism of development and evolution on the validity of describing 'morphogenic society' as a system and justifyability of thinking about it as a social formation generative mechanisms transforming the social order ethics from systems -origin, development and current state of normativity transdisciplinarity needs systemism. systems 5, 15 creating common good -the global sustainable information society as the good society information for a global sustainable information society social relations -building on ludwig von bertalanffy intelligence, artificial intelligence and wisdom in the global sustainable information society taking the perspective of the third -a contribution to the origins of systems thinking icts connecting global citizens, global dialogue and global governance -a call for needful designs seconde manifeste convivialiste -pour une monde post-néolibéral macroshift -navigating the transformation to a sustainable world emergence and morphogenesis -causal reduction and downward causation the extended mind. the origin of language and culture what is information? propagating organisation in the biosphere, symbolosphere, technosphere and econosphere social systems the evolution of human cooperation the nature of nature sociological theory: what went wrong? routledge global cooperation and the human factor in international relations l'informatisation de la société mega-evolution of information processing systems semiotische schriften, 3 vols the great transformation -the political and economic origins of our time struktur -zur sozialwissenschaftlichen analyse von regeln und regelmäßigkeiten the direction of evolution -the rise of cooperative organization the metasystem transition a natural history of human thinking two key steps in the evolution of human cooperation -the interdependence hypothesis evolutionary systems -biological and epistemological perspectives on selection and self-organization such an account can be reached by a paradigm shift towards emergentist systemism, on the basis of which emergentist informationism is elaborated, on the basis of which, in turn, emergentist convivialism is elaborated. from that perspective, the great bifurcation can be regarded as a problem of coming-of-age of humanity. by accomplishing that evolutionary step, the rise of co-operative organisation would enable 'the emergence of a coordinated and integrated global entity' (stewart, 2014, 35) not seen before. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. key: cord-336201-fl606l3b authors: daryabor, gholamreza; atashzar, mohamad reza; kabelitz, dieter; meri, seppo; kalantar, kurosh title: the effects of type 2 diabetes mellitus on organ metabolism and the immune system date: 2020-07-22 journal: front immunol doi: 10.3389/fimmu.2020.01582 sha: doc_id: 336201 cord_uid: fl606l3b metabolic abnormalities such as dyslipidemia, hyperinsulinemia, or insulin resistance and obesity play key roles in the induction and progression of type 2 diabetes mellitus (t2dm). the field of immunometabolism implies a bidirectional link between the immune system and metabolism, in which inflammation plays an essential role in the promotion of metabolic abnormalities (e.g., obesity and t2dm), and metabolic factors, in turn, regulate immune cell functions. obesity as the main inducer of a systemic low-level inflammation is a main susceptibility factor for t2dm. obesity-related immune cell infiltration, inflammation, and increased oxidative stress promote metabolic impairments in the insulin-sensitive tissues and finally, insulin resistance, organ failure, and premature aging occur. hyperglycemia and the subsequent inflammation are the main causes of microand macroangiopathies in the circulatory system. they also promote the gut microbiota dysbiosis, increased intestinal permeability, and fatty liver disease. the impaired immune system together with metabolic imbalance also increases the susceptibility of patients to several pathogenic agents such as the severe acute respiratory syndrome coronavirus 2 (sars-cov-2). thus, the need for a proper immunization protocol among such patients is granted. the focus of the current review is to explore metabolic and immunological abnormalities affecting several organs of t2dm patients and explain the mechanisms, whereby diabetic patients become more susceptible to infectious diseases. the metabolic syndrome is defined by the presence of metabolic abnormalities such as obesity, dyslipidemia, insulin resistance, and subsequent hyperinsulinemia in an individual (1) . dyslipidemia, the main characteristic of metabolic syndrome, is defined by decreased serum levels of high-density lipoproteins (hdls) but increased levels of cholesterol, free fatty acids (ffas), triglycerides (tg), vldl, small dense ldl (sdldl), and oxidized ldl (ox-ldl) ( table 1 ) (2) . individuals with the metabolic syndrome are much more likely to develop type 2 diabetes mellitus (t2dm), cardiovascular diseases (cvds), and fatty liver disease (2) (3) (4) . t2dm, the most common form of diabetes (∼90%), is characterized by a systemic inflammatory disease accompanied by insulin resistance (ir) or decreased metabolic response to insulin in several tissues, including the adipose tissue, liver, and skeletal muscle, as well as by reduced insulin synthesis by pancreatic beta cells (4, 5) . studies on immunometabolism have indicated that the metabolic states and immunological processes are inherently interconnected (6) . in this scenario, metabolites derived from the host or microbiota regulate immunological responses during health and disease (6) . accordingly, in obese individuals, expanded adipose tissue at different locations, by initiating and perpetuating the inflammation, induces a chronic low-level inflammatory state that promotes ir (4). every organ system in human body can be affected by diabetes, but the extent of organ involvement depends largely on the severity and duration of the disease (figure 1 and table 1 ). during the progression of diabetes, hyperglycemia promotes mitochondrial dysfunction and induces the formation of reactive oxygen species (ros) that cause oxidative stress in several tissues such as blood vessels and pancreatic beta cells (7) (8) (9) . accumulating damage to the mitochondria, as well as several macromolecules, including proteins, lipids, and nucleic acids by ros promotes the process of aging (10) . as a result, pancreatic β cells that require functional mitochondria to maintain insulin synthesis fail to generate high enough levels of insulin (11, 12) . in the absence of compensatory mechanisms, stress-responsive intracellular signaling molecules are activated and cellular damage occurs. elevated intracellular levels of ros and subsequent oxidative stress play an important role in the pro-atherosclerotic consequences of diabetes and the development vascular complications (9, 13) . moreover, the non-enzymatic covalent attachment of glucose and its toxic derivatives [e.g., glyoxal, methylglyoxal (mgo), and 3deoxyglucosone] to the biological macromolecules such as nucleic acids, lipids, and proteins leads to the formation of advanced glycation end products (ages) (14, 15) . accumulated ages block the insulin signaling pathway and promote inflammation (16, 17) . in addition, the attachment of ages to their receptors [e.g., cd36, galectin-3, scavenger receptors types i (sr-a1), and ii (sr-a2)] on the surfaces of immune cells in the circulation and tissues activates the expression of pro-inflammatory cytokines and increases free radical generation (18) . furthermore, due to the chronic exposure of cells to high glucose levels in untreated t2dm patients, glucose toxicity might occur in several organs. this will figure 1 | effects of t2dm on body organs. t2dm is an inflammatory state that affects circulatory system, gastrointestinal tract, pancreatic beta cells, liver, and skeletal muscles and makes them dysfunctional. nfald, non-alcoholic fatty liver disease; nash, non-alcoholic steatohepatitis; er, endoplasmic reticulum. eventually lead to nephropathy, cardiomyopathy, neuropathy, and retinopathy. gut microbiome dysbiosis is another important factor that can facilitate the induction and progression of metabolic diseases such as t2dm (19) . the gut microbiome dysbiosis, by altering the barrier functions of intestine and the host metabolic status, promotes the insulin resistance in diabetic patients (19) . diabetes also impairs the immune system and increases the susceptibility of patients to serious and prolonged infections (20) . this is likely to be the case with the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), as well (21, 22) . in the current paper we will review recent research to explore the impairment of body organs in t2dm patients and explain how diabetic patients become more susceptible to certain infectious diseases. vascular homeostasis is an important function of the endothelium. under homeostatic conditions, the ecs maintain the integrity of blood vessels, modulate blood flow, deliver nutrients to the underlying tissues, regulate fibrinolysis and coagulation, control platelet adherence and patrol the trafficking of leukocytes (figure 2a ) (23) . normal ecs also internalize high-density lipoproteins (hdls) and its main protein part apolipoprotein a-i (apoa-i) in a receptor-mediated manner to activate endothelial cell nitric oxide (enos) synthase and promote anti-inflammatory and antiapoptotic mechanisms ( figure 2b ) (24) . hdl receptors on the surfaces of ecs include: the atp-binding cassette (abc) transporters a1 and g1, the scavenger receptor (sr)-b1 and the ecto-f1-atpase (24) . according to the epidemiological studies, diabetes mellitus is considered as one of the main risk factors for cvd (figure 1 ) (25) . from the beginning of t2dm, the functions of ecs are impaired, which is the main cause of disease-related side-effects (26) . ecs can initiate and perpetuate the inflammatory milieu during the pathogenesis of diabetes. due to the negative impacts of hyperglycemia and subsequent oxidative stress, cvds are more common among diabetic patients (27) . it has been observed that incubation of human aortal endothelial cells (haecs) with a medium containing high glucose concentrations (hg, 20 mm) increases the intracellular levels of mgo and glycated proteins that in turn activate the unfolded protein response (upr) and trigger inflammatory and prothrombotic pathways (28) . glycated apoa-i, which is formed during hyperglycemia, modifies its structure, decreases its lipid-binding ability, prevents cholesterol efflux from macrophages and impairs its anti-inflammatory function (29, 30) . vaisar et al. have shown that hdls from diabetic patients have a reduced capacity to trigger enos production and suppress tumor necrosis factor-α (tnf-α)mediated inflammatory responses within ecs (31) . diseases such as t2dm that induce high levels of vascular injury are accompanied by an elevated number of circulating endothelial cells (cecs) (32) . t2dm-related risk factors such as dyslipidemia, hyperglycemia, and hyperinsulinemia as well as other conditions (e.g., inadequate physical activity, smoking, and high blood pressure) facilitate the formation of atherosclerotic plaques/lesions (33) . dyslipidemia, due to the elevated flux of ffa from insulin-resistant tissues and spillover from entry (c) blood vessels in t2dm patients. during the progression of the disease, red blood cells become glycated, while activated ecs synthesize elevated levels of adhesion molecules and chemokines that facilitate monocytes recruitment, adhesion, and transmigration across the endothelium toward the subendothelial region. monocytes are then differentiated into macrophages and eventually, by excess lipid uptake, generate foam cells. subsequently, further immune cell infiltration into the atherosclerotic lesion occurs, where their inflammatory cytokines promote platelet activation, ec apoptosis, and increased generation of ros and ox-ldl. (d) interactions between oxldl and its receptor aggravate ros generation, nf-κb activation and inflammation. ec, endothelial cell; rbc, red blood cell; plt, platelet; hdl, high-density lipoprotein; ox-ldl, oxidized low-density lipoprotein; ros, reactive oxygen species; enos, endothelial nitric oxide synthase; no, nitric oxide; lox-1, lectin-type oxidized ldl receptor 1. into adipocytes, is considered as an important risk factor for developing cvd among diabetic patients. this is because dyslipidemia promotes inflammation, endothelial dysfunction, and platelet hyperactivation (34, 35) . during the progression of atherosclerosis, lipids, immune cells, and extracellular matrix accumulate in the arterial intima or subendothelial regions ( figure 2c ) (33) . advanced plaques can impede blood flow and cause tissue ischemia or might become disrupted and generate a thrombus that stops the blood flow of important organs. vascular complications of diabetes engage either tiny or large blood vessels (micro-and macroangiopathy, respectively). microangiopathies, which can be seen in the kidneys, vasa nervorum and eye tissues, cause nephropathy, neuropathy, and retinopathy. macroangiopathies, by inducing atherosclerosis in the coronary, carotid, and peripheral arteries, increase the risk of myocardial infarction (mi), stroke and peripheral artery disease (pad). macrovascular complications due to ec dysfunction are considered as an important cause of mortality and morbidity among diabetic patients (36) . oxidative stress has an essential role in the induction of vascular complications during the course of diabetes (8) . ec dysfunction (e.g., delayed replication, dysregulated cell cycling, and apoptosis), as well as enhanced ox-ldl formation are some consequences of oxidative stress. it has been well-established that sdldl and ox-ldl have an enhanced atherogenic ability and are more useful biomarkers than total ldl for predicting cvd (37, 38) . sdldl particles have a smaller size than other ldl particles. thus, sdldl particles are more easily oxidized, and their atherogenic potential is enhanced. during oxidative stress, levels of ox-ldl increase by the excess action of reactive oxygen species (ros) (13) . subsequently, ox-ldl interaction with scavenger receptors, including cd36, sr-a1/cd204, sr-b1, and lectin-like ox-ldl receptor-1 (lox-1) on the surface of ecs activates the nadph oxidase that in turn increases the expression of ros and activates the transcription factor nf-αb (39) . afterwards, the expression of lox-1, adhesion molecules (e.g., selectins and integrins) and the secretion of pro-inflammatory cytokines and chemokines are increased, while no synthesis is decreased in ecs ( figure 2d ) (39) (40) (41) . ec-derived chemokines bind to their cognate receptors on the surfaces of monocytes and recruit them toward the inflamed endothelium. following this, selectin-based rolling and integrin-based attachment of monocytes to the ecs cause their migration toward the subendothelial region, where they develop into lipid-laden macrophages or foam cells later on (42) . the scavenger receptor lox-1 plays an important role in the uptake of ox-ldl during atherogenesis. it is strongly expressed on the surfaces of ecs, but has an inducible pattern of expression on the surface of macrophages and smooth muscle cells (43) . the accelerated uptake of ox-ldl by macrophages accounts for their transformation into foam cells, the initial hallmark of atherosclerosis (41, 43) . besides, diabetes leads to both quantitative and qualitative defects in circulating angiogenic progenitor cells (capcs) that take part in the repair of injured endothelium (44) . it has been shown that humans or mice with decreased numbers of cd31 + cd34 + cd133 + cd45 dim sca-1 + flk-1 + capcs have an increased prevalence of t2dm, elevated hba1c levels and aggravated cvd risk scores (44, 45) . in diabetic patients, despite elevated serum levels of proangiogenic molecules, like angiopoietin-1/2, epo, and vegf-a, angiogenesis is impaired. this is mainly due to the decreased expression levels of vegfr2 and cxcr4 on the surfaces of capcs, which makes them unresponsive to the angiogenic factors (44, 46) . it has also been shown that circulating proangiogenic granulocytes composed of eosinophils and neutrophils are also impaired in diabetic patients (47) . besides, elevated levels of ages in t2dm cause ec dysfunction and vascular inflammation (48) . ren et al. have shown that incubation of human coronary artery endothelial cells (hcaecs) with ages causes decreased expression (at both mrna and protein levels) and enzymatic activity of enos, increased levels of ros, diminished mitochondrial membrane potential and declined activity of catalase and superoxide dismutase in treated cells (49) . another study by lan et al. has shown that ages in the pancreas decrease ec viability and induce their apoptosis in an nfκb signaling-related manner (50) . however, apigenin (4 ′ ,5,7trihydroxyflavone) can protect ecs against oxidative stress and subsequent inflammatory reactions mediated by ages (51) . apigenin binds to methylglyoxal (mgo) and forms a complex that inhibits age formation. chettab et al. have shown that the expression of icam-1 as well as the production of il-8, are significantly increased in huvecs cultured in hg medium compared to cells cultured in normal glucose (ng, 5.5 mm) conditions (52). bammert et al. found out that incubation of huvecs with hg media promotes the generation of endothelial microparticles (emps) that, when added to normally cultured huvecs, downregulate the expression of anti-apoptotic microrna mir-let7a, but enhance the synthesis of active caspase-3 and cause cell apoptosis (53) . several micrornas, including mir-21, mir-26a, mir-30, mir-92a, mir-126, mir-139, mir-199a, mir-222, and mir-let7d, regulate vascular homeostasis. it has been shown that the expressions of mir-26a and mir-126 are significantly reduced in circulating mps isolated from diabetic patients compared with normal individuals. this could be involved in making diabetic individuals more susceptible to coronary heart disease (54) . moreover, hg media upregulate the expression of nadph oxidase that will induce the generation of ros. this leads to subsequent apoptosis of the huvecs through a ros-dependent caspase-3 pathway (55). su et al. have demonstrated that argirein medication, by inactivating nadph oxidase, can prevent endothelial cell apoptosis in a rat model of t2dm and hence attenuate vascular dysfunction (56) . hg further increases the permeability of the huvecs in a protein kinase c (pkc)-dependent manner (57, 58) . hassanpour et al. showed that incubation of endothelial progenitor cells with the serum of t2dm patients inhibits their migration toward bfgf, increases their expression of vegfr-2, but reduces their expression of vegfr-1 and induces their apoptosis (59) . however, humanin (hn), a mitochondriumderived peptide, is cytoprotective against apoptosis during pathological conditions, such as diabetes mellitus (60) . it has been demonstrated that simultaneous incubation of h9c2 cells, a line of rat cardiac myoblasts, with h 2 o 2 and hn decreases the intracellular levels of ros, preserve mitochondrial function/structure and decline cellular apoptosis (61) . wang et al. have indicated that the treatment of huvecs with hn before their incubation with hg medium increases the expression of enos, while decreasing the expression of endothelin 1 (et-1), vcam-1, tnf-α, il-1β, and e-selectin in a krüppellike factor 2 (klf2)-dependent manner. such changes in the expression of integrins prevent the attachment of monocytes to huvecs (62) . accordingly, hn might be used to prevent the development of hyperglycemia-associated ec dysfunction in t2dm. ec activation and expression of adhesion molecules also facilitate activation and adhesion of platelets. this will increase the risk of thrombosis and promote the development of thrombotic angiopathy, typical for diabetic patients. platelets are tiny anucleated cellular fragments generated from megakaryocytes in the bone marrow. they circulate in the blood for ∼5-9 days and play essential roles in hemostasis and in controlling vascular integrity (63) . circulating inactive platelets move in the proximity of vessel walls (figure 2a ) and rapidly get activated in response to vascular injury. at the end of their life, platelets are cleared from circulation with the action of the liver and spleen-resident macrophages. platelets have an essential role in the initiation and progression of inflammation. platelet hyperactivation that occurs during inflammatory states (e.g., t2dm) facilitates the pathogenesis of cvds ( figure 2c ) (64, 65) . it has been shown that elevated levels of resistin, an adipokine, in diabetic patients enhances oxidative stress, promotes endothelial dysfunction and facilitates platelet activation (66) . activated platelets with an increased mean volume [mean platelet volume (mpv)] secrete microparticles (mps) and soluble adhesion molecules (e.g., sp-selectin and scd40l) that in turn activate endothelial and immune cells (67) (68) (69) . higher levels of platelet-derived mps, which correlate positively with fasting blood sugar and glycated hemoglobin, have been shown in newly diagnosed t2dm patients compared to healthy individuals (70) . in t2dm patients thrombotic microangiopathies can lead to the development of cvds (71) . platelets in the patients adhere to ecs and aggregate more rapidly than in healthy individuals thereby increasing the risk of thrombosis. in a mouse model of t2dm, zhu et al. have shown that ages interact with cd36, a member of the type 2 scavenger receptor family, on the surfaces of murine platelets to activate them and induce a prothrombotic state (72) . elevated levels of the p2y12 receptor on the surface of platelets in t2dm expose diabetic patients to a prothrombotic condition. this receptor has an essential role in platelet activation (73) . zhou et al. have shown that long non-coding rna (lncrna) metallothionein 1 pseudogene 3 (mt1p3), which is markedly upregulated in megakaryocytes of t2dm patients, enhances the expression of p2y12 receptor in platelets (74) . they indicated that this is due to the inhibitory action of mt1p3 on mir-126. virtually all parts of the human digestive system, including the gastrointestinal tract, pancreas, and the liver are affected by diabetes. the git is populated with a myriad of microorganisms, including principally bacteria but also archaea, viruses, fungi, and protozoans that dynamically influence the health status and homeostasis of the host. the physiological functions of the git resident microbes improve gut integrity, protect against microbial pathogens and regulate immune responses (75) . mucosal barriers, such as intestinal epithelial cells (iecs) and the mucus layer, spatially isolate the host immune system and gut microbiota to prevent unnecessary immune activation and intestinal inflammation. they also facilitate the uptake of nutrients through receptors and transporters. however, hyperglycemia, in a glut2-dependent manner, can influence the mucus and alter the integrity of adherence and tight junctions between intestinal epithelial cells of diabetic mice. this will enhance the permeability of the intestinal barrier leading to so called "leaky gut." subsequently, hyperglycemia may facilitate the dispersal of an enteric infection into a systemic infection (figure 1 ) (76) . interestingly, the reversal of hyperglycemia, conditional deletion of glut2 from the iecs and inhibition of glucose metabolism will fix the barrier dysfunction and prevent the spread of bacteria (76). xu et al. have shown that faecalibacterium prausnitzii, one of the most frequent commensal bacteria in normal individuals with essential roles in gut homeostasis, generates anti-inflammatory molecules that enhance the expression of tight junctions and improve intestinal integrity during diabetes (77) . however, in some cases, gut microbiota dysbiosis or altered microbial composition of the intestines could induce t2dm and lead to its progression (78) . of interest, the widely used antidiabetic drug metformin can improve barrier integrity and restore the healthy microbiota composition of the gut in diabetic patients (79) . the intestinal commensal bacterium akkermansia muciniphila can also act as a sentinel to reduce microbial translocation across the gut and prevent the subsequent inflammation in patients with t2dm (80) . hyperglycemia can further decrease the intracellular levels of glutathione (gsh) but increase inos activity and no production in the iecs (81). zhao et al. have found out that hyperglycemia in a pkcα-dependent manner inhibits the ubiquitination, internalization and degradation of the divalent metal transporter 1 (dmt1) present on the microvillar membranes of iecs. subsequently, intestinal iron uptake is enhanced and accumulated iron ions aggravate diabetes-related complications and increase mortality (82, 83) . the pancreas consists of the exocrine and endocrine compartments. the endocrine part is made of different cell types, including α, β, δ, and ε cells that secrete glucagon, insulin, somatostatin, and ghrelin hormones, respectively. these cells are aggregated into specialized structures called islets of langerhans, which play an important role in controlling blood glucose levels through the secretion of insulin and glucagon. in t2dm, despite normal levels of β-cell replication and islet formation, β-cell apoptosis is increased so that the number of cells declines by ∼50% (figure 1 ) (84) . during the progression of t2dm, the insulin-resistant state forces β-cells to compensate for the lack of insulin by elevating its synthesis to restore the normal blood glucose level. however, in severe diabetic patients, β-cell exhaustion, and subsequent persistent hyperglycemia occur (7) . furthermore, chronic elevated serum levels of free fatty acids, seen in obesity and t2dm, induce lipotoxicity in beta-cells and suppress their insulin secretion ability (85) . to alleviate chronic inflammation, overcome insulin resistance (ir) and to prevent β-cell apoptosis, stem cells or stem cell derivatives such as insulin-producing cells (ipcs) and exosomes have been suggested (86) (87) (88) (89) . their effects are believed to be mainly due to their anti-inflammatory activities. secretagogin (scgn) is predominantly expressed by pancreatic β-cells protecting their normal functions. scgn also acts as an insulin binding protein to make it more stable, avoid its aggregation, improve its functions and enhance its secretion (90, 91) . in t2dm patients, due to the islet cell dysfunction and endoplasmic reticulum (er) stress, serum levels of scgn are elevated reflecting stress and dysfunctional islet cells (92) . moreover, in patients with t2dm, islet amyloid polypeptide (iapp or amylin), a peptide hormone and one of the main secretory products of pancreatic β-cells, tends to deposit in the islets of langerhans, form insoluble fibrils and impair secretory functions of β-cells (93) . iapp is costored with insulin in the secretory granules of pancreatic β cells. in steady-state conditions it regulates food intake, insulin secretion, and glucose metabolism (94). ribeiro et al. have noted that pancreatic extracellular vesicles (evs) from healthy individuals, but not from t2dm patients, directly bind to iapps and prevent amyloid formation within the pancreatic islets (95) . the authors showed that the altered protein-lipid composition of the evs is the main reason for this discrepancy (95) . however, chatterjee et al. have shown that β-cells from t2dm patients have a dysfunctional proteasome complex that fails to degrade pancreatic iapp, whereby amyloid formation is induced (96) . furthermore, in t2dm patients, lipids accelerate the formation of fibrillary iapp, which aggravates islet cell damage (97) . dhar et al. have demonstrated that chronic use of mgo in sprague-dawley rats increases the expression of nf-αb, mgo-derived ages and their receptors in pancreatic β cells. mgo can also induce apoptosis of islet β cells, increase fasting plasma glucose levels and impair glucose tolerance (98) . in t2dm patients the plasma level of mgo directly correlates with fasting blood sugar and hba1c levels (99) . bo et al. further showed that mgo in a dose-based manner impairs insulin secretion of pancreatic β-cell lines min6 and ins-1 through increased generation of ros and by induction of mitochondrial dysfunction (100). robertson et al. have found out that elevated levels of ros in pancreatic β-cells inhibit the pancreas duodenum homeobox-1 (pdx-1) transcription factor that is needed for insulin synthesis (7) . it has been shown that chronic use of mgo in animals could induce t2dm, while simultaneous use of alagebrium, which breaks age compounds, attenuates the disease (98) . it has also been reported that during the course of diabetes dedifferentiation and conversion of β-cells into αand δ-"like" cells occurs (101) . in conclusion, the pancreatic β cell function is progressively reduced during the progression of t2dm. the liver is by far the most important metabolic organ with essential roles in regulating homeostasis and mediating glucose and lipid metabolism. metabolic activities of the tissue are precisely controlled by the actions of metabolic substrates, including free fatty acids (ffas) and hormones (102) . t2dm patients usually suffer from a chronic liver condition called non-alcoholic fatty liver disease (nafld). it is characterized by steatosis that means ectopic fat storage in hepatocytes and subsequent insulin resistance (figure 1 ) (103) . lipid accumulation in hepatocytes leads to impaired biogenesis of mir-206 that facilitates insulin signaling and prevents lipogenesis (104) . several factors such as obesity, increased serum levels of fatty acids, and insulin resistance can increase the risk of fatty liver disease. p2y2 receptor, through the induction of the c-jun n-terminal kinase (jnk) and prevention of insulin signaling, can promote insulin resistance in hepatocytes in t2dm (105) . in some cases, nafld may progress into an aggressive form of inflammatory fatty liver disease called non-alcoholic steatohepatitis (nash), which might cause liver cirrhosis and organ failure (106). dang et al. have indicated that exosomes released from the adipose tissues of obese mice due to the smaller mir-141-3p content can promote insulin resistance in the murine hepatocyte cell line aml12 (alpha mouse liver 12) (107) . the adipokine visfatin that is released from the adipose tissue of obese individuals has also been shown to activate the pro-inflammatory stat3 signaling pathway and nf-κb in the human liver cell line hepg2 and promote their insulin resistant state (108) . nevertheless, the hepatocyte growth factor (hgf) can alleviate the insulin resistance of hepatocytes and control their triglyceride and cholesterol contents (109) . skeletal muscle (sm) is the main tissue that releases glucose after insulin stimulation. hence, insulin resistance in sm has a pivotal role in the metabolic dysregulation of t2dm. insulin resistance in sm is the primary defect of t2dm that facilitates the progression of fatty liver disease, deposition of fat in the liver (figure 1 ) (110) . skeletal muscle from diabetic patients expresses less genes related to insulin signaling and metabolic pathways, but more apoptosis and immune-related genes (111) . this inflammatory milieu is mainly due to the proinflammatory actions of obesity-related adipose tissue mediators, which are released into the circulation and promote inflammation within the sm (4). furthermore, obesity causes intermyocellular and perimuscular adipose tissue expansion that acts like adipose tissue depots to enhance sm inflammation (112) . it has been shown that human skeletal muscle cells (hsmc), isolated from diabetic patients, after a 24-h culture generate significantly more tnf-α, il-6, il-8, il-15, monocyte chemotactic protein (mcp)-1, growth-related oncogene (gro)-α, and follistatin compared to non-diabetic individuals (113) . this altered secretion of myokines (e.g., cytokines secreted by sms) is an intrinsic feature of sm during the progression of t2dm. in sm, glut-4, which is quickly translocated to the cell surface, facilitates glucose uptake in response to insulin hormone as well as muscle contraction. pinto-junior et al. have shown that the use of age-albumin in rats increases the expression of the inflammatory molecule nf-κb1 within the sm. nf-κb1 binds to the promoter of the glut-4 gene and suppresses its expression (at both mrna and protein levels) (114) . accordingly, glut-4 levels on the surfaces of sm decrease and subsequently, whole-body ir develops. the immune system is generally classified into two main arms, innate and adaptive (or acquired) immunity. adaptive immunity is mediated by b cells, which produce antibodies and t cells, which are classified into cd4 + helper cells and cytotoxic cd8 + cells. a considerable literature has discussed the dysfunctional immune responses in diabetic patients ( table 2 ) (115) (116) (117) (118) (119) (120) . abnormal immune cell activation and subsequent inflammatory environment has an essential role in the progression of t2dm (121) . in this regard, chronic inflammation due mainly to the activation of the myeloid cell lineage (e.g., macrophages and neutrophils), is directly related to the induction of ir (4, 122). their numbers are elevated, are larger and more granular, express diminished levels of antioxidant genes but elevated levels of pro-apoptotic and pro-inflammatory genes. complement system attachment of c-type lectin proteins to mannose residues is decreased, lectin pathway is impaired, cd59 activity is reduced, mac deposition in vascular walls is increased. dendritic cells (dcs) their numbers and activity are reduced. their cholesterol efflux is decreased, generate foam cells, have dysfunctional efferocytosis. are activated, constitutively release nets, produce high levels of mpo, ros, and calprotectin (s100a8/a9), are more susceptible to apoptosis, their migration, phagocytosis and microbial killing are impaired. nk cells their numbers are increased but are usually dysfunctional, express high levels of glut4 but decreased levels of nkg2d and nkp46, have reduced degranulation capacity, are more susceptible to apoptosis. nkt cells their numbers are increased, produce high levels of ifn-γ, il-4, and il-17, express high levels of nkp30, nkg2d, and nkp44 but low levels of nkg2a and 158b. innate lymphoid cells (ilcs) ilc1s are increased and produce high levels of ifn-γ. humoral immunity (b cells) germinal centers are reduced, ab production and isotype switching is defective, abs become glycated, abs fail to activate complement. functions of osteoclasts, which are bone-resident innate immune cells (126) . this may affect bone structure and delay bone healing. defects in the innate, as well as adaptive immunity, are supposed to be the main cause of diabetic individuals' susceptibility to infections (127) . furthermore, some microorganisms, especially bacteria, in hyperglycemic conditions are better nourished and become more virulent, while also having a better milieu to cause infections. complement system the complement system is a first-line defense mechanism against invading microorganisms. it acts via different but interconnected classical, alternative, and lectin pathways (128) . ilyas et al. have shown that under high glucose conditions, the attachment of ctype lectin proteins to high-mannose containing glycoproteins is substantially decreased in a dose-dependent manner. these carbohydrate-binding proteins include mannose-binding lectin (mbl), surfactant protein d (sp-d), dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (dc-sign, cd209), and dc-sign-related (dc-signr) protein (129) . reduced binding of mbl in the presence of high levels of sugar causes a significant reduction in the lectin pathway activity, but does not influence classical or alternative pathway activity (129) . nevertheless, barkai et al. did not find significant differences in the function of classical or mbl pathways between t2dm and healthy individuals (130) . however, significantly decreased activity of ficolin-3-mediated lectin and alternative pathways, as well as decreased levels of c4d and soluble complement c5b-9 (sc5b-9) were seen in diabetic patients with escherichia coli-mediated urinary tract infections (130) . this may be linked to a reduced ability of diabetics to protect themselves against bacterial infections. the lipopolysaccharides of certain gram-negative bacteria, like salmonella serotype o6,7 as well as the cell walls of fungi, are rich in mannose. possibly, because of this, in addition to additional provision of nutrients, an increased prevalence of fungal infections is seen in t2dm patients (131, 132) . patel et al. found a significantly higher prevalence of oral candida carriage in diabetic patients compared to healthy controls (131) . they found that candida albicans was the most commonly isolated species followed by c. tropicalis, but uncommon species such as c. lusitaniae and c. lipolytica were also isolated (131) . another study by jhugroo et al. showed that c. albicans is the predominant yeast isolated from oral mucosal lesions of diabetic patients, followed by. c. tropicalis and c. krusei dendritic cells (dcs) are a heterogeneous population of specialized and professional antigen-presenting cells (apcs) that create a crucial link between the innate and adaptive immune responses (136, 137) . some studies have shown that the numbers of dcs are reduced in both type 1 and 2 diabetes (138, 139). seifarth et al. have found that t2dm patients with poor metabolic control have decreased numbers of both myeloid and plasmacytoid dcs compared with healthy controls. this could make them more susceptible to opportunistic infections (139) . in the case of good blood glucose control, the reduction in dc numbers was less prominent but still significant, especially for myeloid dc1 (mdc1) cells (139) . another study by blank et al. demonstrated that women with t2dm and poor glycemic control (hba1c ≥ 7%) have fewer numbers of circulating plasmacytoid dcs (pdcs) compared to diabetic women with good glycemic control (hba1c < 7%) or to healthy women (140). montani et al. have recently shown that hyperglycemic medium and hyperglycemic sera derived from t2dm patients prevent the maturation of monocytes into effective dcs and their activation in vitro (141) . interestingly, quercetin, a flavonoid with antiinflammatory and antioxidant characteristics, prevented such effects (141) . macrophages are important immune cells that play critical roles through all stages of the pathogenesis of t2dmrelated atherosclerosis (41). swirski et al. have shown a significantly elevated number of pro-inflammatory monocytes in the circulation of apoe −/− mice, an animal model of atherosclerosis, compared to control mice (142) . modifications of the lipoproteins in the arterial walls of diabetic individuals make them pro-inflammatory and activate the overlying endothelium. in response, monocytes are recruited into the subendothelial region, differentiate into macrophages and internalize the accumulated lipoproteins. finally, cholesterol-laden foam cells are generated. they promote inflammation and progression of the disease through the synthesis and secretion of cytokines, chemokines, ros, and matrix metalloproteinases (mmps) (figure 2c ) (42) . foam cells lose their migratory potential, die by apoptosis and generate a necrotic core within the atherosclerotic plaque (143) . it has been demonstrated that the use of mesenchymal stem cells in apoe −/− mice reduces the numbers of monocytes/macrophages at the site of inflammation, decreases lipid deposition and diminishes plaque size (144). ma et al. have studied the effects of long-term hyperglycemia in diabetic mice and found out that compared to non-diabetic control mice, the numbers of f4/80 + macrophages isolated from spleen (spms), as well as from peritoneal exudates (pems) of diabetic mice are significantly decreased (145) . subsequently, sun et al. showed that stimulation of pems from diabetic mice in vitro with ifn-γ and lipopolysaccharide (lps) significantly decreased the expression of intercellular adhesion molecule 1 (icam-1 or cd54), cd86, tnf-α, and il-6, while it increased the production of nitric oxide (no) (146) . they further showed that stimulation of pems isolated from diabetic mice with il-4 caused an enhanced arginase activity (146) . kousathana et al. have demonstrated that circulating monocytes isolated from diabetic patients produce higher levels il-6, while having an impaired activation of the nlrp3 inflammasome and subsequently reduced il-1β production (147) . however, they showed that proper glycemic control would restore such modifications. poor inflammatory responses in circulating monocytes, as well as in macrophages, are responsible for elevated susceptibility to infections and their severity in patients with t2dm. macrophages play a critical role in tissue repair. early in wound healing, they are pro-inflammatory to clear pathogens and debris but later, they resolve inflammation and promote tissue repair. in pathological conditions, failure to transform from pro-inflammatory to the anti-inflammatory proliferative phase can cause chronic inflammation in the affected tissue (148). have shown that an impaired wound healing process in animals with t2dm is due to high levels of nlrp3 inflammasome activity, which promotes the generation of il-1β and il-18 in macrophages (149, 150) . efficient skin wound healing process is mediated by the up-regulation of the peroxisome proliferatoractivated receptor (ppar)-γ in macrophages that convert their pro-inflammatory phenotype into healing-related. pparγ suppresses cytokine production by macrophages and hence is upregulated in inflamed tissue-resident macrophages. however, in t2dm, pparγ expression is down-regulated in skin-resident macrophages that enhance the activity of nlrp-3 inflammasome and cause chronic inflammation. using myeloid-specific pparγ −/− mice, it has been shown that the absence of ppar-γ in macrophages is sufficient to delay the healing process and extend tissue inflammation (150) . in t2dm patients, chronic hyperglycemia and hyperlipidemia trigger the secretion of a damage-associated s100a8 molecule (calgranulin a) from pancreatic islets that in turn increase macrophage infiltration (151) . westwell-roper et al. have shown that iapp aggregates in t2dm patients polarize islet-resident macrophages toward the m1-like f4/80 + cd11b + cd11c + phenotype that produces pro-inflammatory cytokines, including tnf-α, il-1β, and il-6. furthermore, m1 cells promote islet inflammation, cause β-cell malfunction and apoptosis (152) . in t2dm, excess phagocytosis of apoptotic β-cells by macrophages induces their lysosomal permeabilization, generation of ros, inflammasome activation, and pro-inflammatory cytokines secretion (153) . collectively, these observations reveal that the functions and plasticity of macrophages are compromised during the progression of t2dm. neutrophils are the most prevalent circulating leukocytes and one of the main components of innate immunity. they are recruited to the sites of infection through chemotaxis following complement activation, most importantly by c5a. activated neutrophils bind via their surface receptors to induced ligands on the surfaces of inflamed endothelial cells to migrate to tissues. there they phagocytose and kill invading microbes with lysosomal enzymes, antimicrobial peptides and by the generation of ros (154). neutrophils from patients with t2dm, but not from healthy individuals, are activated and produce elevated levels of ros. so, it could increase the risk of random organ injury (155) . in diabetic patients, the plasma levels of homocysteine are elevated, which is mainly due to its impaired clearance rate (156) . this will induce neutrophils to constitutively release neutrophil extracellular traps (nets) that can cause vascular damage and delays in wound healing (157, 158) . it has been shown that the circulating level of hydrogen sulfide (h 2 s) is significantly reduced in fasting blood of patients with t2dm compared with healthy individuals as well as in streptozotocin-induced diabetic rats compared with controls (159) . h 2 s is produced from cysteine by the action of several enzymes. it acts as a regulator of cell signaling and homeostasis (160) . it is essential to maintain balanced levels of antioxidants and protect tissues from oxidative stress (160) . the use of h 2 s or the endogenous l-cysteine in vitro blocks the production of il-8 and monocyte chemoattractant protein-1 (mcp-1) in the human u937 monocyte cell line incubated in high-glucose medium (159) . yang et al. have shown that h 2 s treatment decreases netosis and enhances the healing process of diabetic wounds by preventing ros-dependent erk1/2 and p38 activation (161) . it has been shown that the levels of net components, including histones, elastase and proteinase-3, are elevated in the sera from patients with diabetic foot ulcers (162) . wang et al. have recently indicated that hg dramatically enhances nadph oxidasedependent net generation in diabetic rats and humans. it was proposed that this could have a role in the induction of diabetic retinopathy (163) . indeed, patients with t2dm have elevated plasma levels of mgo, which can induce the production of proinflammatory cytokines like tnf-α, il-6, and il-8 by neutrophils and make them more susceptible to apoptosis (99) . myeloperoxidase (mpo), which is abundantly produced by neutrophils, but only to a small extent by monocytes and macrophages, might be useful as an early biomarker of inflammation in diabetic individuals (164) . binding of mpo to endothelial cells increases its half-life. thereby, more proinflammatory oxidant hypochloric acid (hclo) is generated that extends the damage to blood vessels (165) . in t2dm patients, neutrophil activities, including migration, phagocytosis and microbial killing are impaired. this makes diabetic individuals more susceptible to infections (166) . it has been welldocumented that neutrophils isolated in animal models of t2dm have an impaired tlr4 signaling pathway. this is reflected as a diminished cytokine and chemokine production, possibly as a consequence of reduced phosphorylation of nfκb and iκbα (167) . the half-life of these neutrophils as well as their in vivo migration and myeloperoxidase activity are decreased. during hyperglycemia, neutrophils produce calprotectin (s100a8/a9), which interacts with the receptor for advanced glycation end products (rage) on the surface of hepatic kupffer cells and promotes the synthesis of il-6 (168). subsequently, il-6 stimulates hepatocytes to increase the generation of thrombopoietin that in turn attaches to its receptor on the surfaces of bone marrow precursor cells and megakaryocytes to enhance their proliferation and expansion. this results in reticulated thrombocytosis, which means elevated megakaryocyte activity and thrombopoiesis. interestingly, diabetes-related thrombocytosis and subsequent atherothrombosis can be reduced by lowering blood glucose, depleting kupffer cells or neutrophils or by preventing the binding of s100a8/a9 to rage using paquinimod (168) . thom et al. have shown that the incubation of human and murine neutrophils with hg medium would cause their cytoskeletal and membrane instability. this will induce the generation of 0.1 to 1 µm diameter microparticles and activate the nlrp3 inflammasome (169) . microparticles, which are potently pro-inflammatory, are found in the circulation of healthy individuals, but their generation is increased during cell activation in several diseases, including t2dm and cardiovascular diseases (170, 171) . furthermore, serum levels of soluble fasl (sfasl) are increased in patients with t2dm thereby activating neutrophils and aggravating the inflammatory milieu (172, 173) . the proinflammatory roles of sfasl are mediated through increased amounts or activity of nfκb, il-1β, caspase-1, cd11b/cd18, and ros (173) . caspase-1 activation prevents the sfasl-dependent apoptosis of neutrophils and inhibits their expression of fas and caspase-3 (173) . accordingly, hyperglycemia disturbs the normal functions of neutrophils and increases the susceptibility to infections by pathogenic microorganisms. the expression level of nkg2d is negatively correlated with hba1c levels implying that chronic hyperglycemia would cause nk cell dysfunction (176) . also, hyperglycemia increases the expression of unfolded protein response (upr) genes in nk cells and induces their apoptosis (176) . nkt cells express simultaneously markers of both t cells (tcr and cd3) and nk cells [cd16, cd56, cd314 (nkg2d), and cd337 (nkp30)]. nkt cell subsets produce a broad range of cytokines, including gm-csf, ifn-γ, tnf-α, il-2, il-4, il-5, il-9, il-10, il-13, il-17, and il-21 (178) . they recognize lipids and glycolipids presented by cd1d molecules. phoksawat et al. have shown that the frequency of cd3 + cd4 + cd28 null cd56 + nkg2d hi nkt cells, which produce high levels of il-17, are increased in diabetic patients and their numbers are directly correlated with hba1c levels (179, 180) . lv et al. have recently shown that the numbers of cd3 + cd56 + nkt cells are higher in diabetic patients compared to healthy individuals (181) . they further showed that such cells are mostly cd4 + , produce elevated levels of ifn-γ and il-4 and express high levels of nkp30, nkg2d, and nkp44 but low levels of inhibitory receptors nkg2a and 158b (181) . the co-culture of these cells with huvecs significantly decreased their proliferation and migration abilities that were mainly il-4dependent (181) . taken together these studies show that diabetic individuals appear to have elevated levels of inflammationpromoting nkt cells. ilcs are critical effectors of innate immunity that produce both regulatory and pro-inflammatory cytokines to promote tissue repair, immunity, and inflammation (182) . mature ilcs lack the tcrs. based on their cell surface markers, cytokine production as well as expression of transcription factors the ilcs are classified into types 1, 2, and 3 (183) . these correspond to the different types of cd4+ t helper cells: th1, th2, and th17, respectively. ifn-γ is the cytokine signature of ilc1s, while type 2 cytokines (e.g., il-5 and il-13) are mainly produced by ilc2s and the main product of ilc3s are il-17 and il-22. regarding transcription factors, t-bet is mainly expressed by ilc1s, gata3 and rorα are mostly expressed by ilc2s and rorγt is predominantly expressed by ilc3 (183) . in t2dm, the numbers of circulating as well as adipose tissue-resident ilc1s are increased compared with normal individuals (184, 185) . the frequency of circulating ilc1s is positively correlated with fasting plasma glucose (fpg), hba1c, homeostasis model assessment for insulin resistance (homa-ir), serum-free fatty acids (ffas) and adipose tissue insulin resistance index (adipo-ir) (184, 185) . it has also been shown that patients with increased numbers of ilc1 have an elevated risk of developing t2dm (184) . a study by wang et al. indicated that adipose tissue-resident ilc1s, via the production of ifn-γ, promote tissue fibrosis and induce diabetes in obese individuals (185) . liu et al. have demonstrated that the numbers of ilc2s as well as serum cytokine levels of il-4, il-5, and il-13 are significantly elevated in diabetic kidney disease patients and have a positive correlation with disease severity (186) . they further demonstrated that ilc2s, through the tgf-β1 signaling pathway, are involved in renal fibrosis seen in diabetic kidney disease (184) . however, galle-treger et al. indicated that the engagement of the glucocorticoid-induced tumor necrosis factor receptor (gitr/or tnfrsf18) on the surface of activated ilc2s promotes their secretion of il-5 and il-13, ameliorates glucose homeostasis, protects against the onset of and improves established insulin resistance (187) . the protective role of ilc2s during acute metabolic stress has also been well-documented by dalmas et al. (188) . humoral immunity (b cells) elevated levels of blood glucose generate covalent sugar adducts with several proteins through non-enzymatic glycation. this can impair humoral immunity in many ways, e.g., by modifying the structure and functions of immunoglobulins (igs) (189) (190) (191) (192) (193) (194) . such modifications in the structure of igs can be determined using matrix-assisted laser desorption ionization (maldi) mass spectrometry (119, 191) . the molecular mass of igs in diabetic patients is higher than in normal subjects (189) . this can lead to reduced efficiency of vaccines that stimulate humoral immunity in these patients. it has been shown that immunization with influenza (flu) vaccines in diabetic patients induces normal or even elevated levels of flu-specific antibodies compared with normal individuals (195) (196) (197) (198) . however, the ability of the dysfunctional glycated antibodies to neutralize viruses is impaired, which will increase the susceptibility to infections. farnsworth et al. have shown that in t2dm, class switch defects in the assembly of antibody genes are also present (199) . in a model system, mice with t2dm have decreased amounts of specific anti-staphylococcus aureus antibodies (total as well as igg), which will increase the risk of infection and morbidity of diabetic mice. however, the levels of igm were elevated, but inefficient in protecting against infection, possibly because of their inability to directly promote phagocytosis. in another study, farnsworth et al. have demonstrated that defects in humoral immunity, as shown by decreased levels of total igg and anti-staphylococcus aureus antibody, aggravate foot infections in a murine model of t2dm (200) . this was due to a reduced germinal center induction and decreased numbers of t and blymphocytes within the germinal centers. this causes failures in antibody generation and class-switch recombination (200) . mathews et al. have shown that the protective levels of antibodies against streptococcus pneumoniae surface protein a are lower in diabetic patients compared to non-diabetic individuals. these antibodies also have a reduced potential to trigger complement activation on the surface of pneumococci, whereby phagocytosis of the bacteria becomes compromised (201) . they showed that hyperglycemia reduces both the antibody titers as well as the ability to deposit complement on the bacteria. the abovementioned changes in the ability to protect against s. aureus and s. pneumoniae are important, because these bacteria belong to the most common infection-causing pathogens in diabetic patients. another major group is constituted by gram-negative bacteria that commonly cause e.g., urinary tract infections. many studies have shown that t-cell functions are impaired in individuals with t2dm (202) (203) (204) (205) . elevated levels of activated cd4 + cd278 + t helper cells, cytotoxic t-cells, and th17 cells have been observed in obese diabetic patients compared to nonobese ones (205, 206) . nevertheless, pbmcs isolated from obese diabetic patients produced smaller amounts of il-2, il-6, and tnf-α after stimulation with phytohemagglutinin (pha) (205) . martinez et al. indicated that diabetic patients have reduced pathogen-specific memory th17 responses as well as decreased numbers of cd4+ t cells in response to stimulation with streptococcus pneumoniae (206) . th17 cells are critical for the recruitment of neutrophils to the infection site and improve the phagocytosis of invading bacteria and yeast (207) . moura et al. have shown that diabetic patients, particularly those with foot ulcers, have reduced levels of naive t-cells, but an elevated number of effector t cells and a reduction in the tcr-vβ repertoire diversity (204) . the observed changes are mainly due to an abnormal amount of inflammatory cytokines (e.g., ifn-γ and tnf-α) produced during infection and to subsequent robust stimulation of t-cells. leung et al. have reported that ischemic tissues of t2dm patients contain elevated numbers of tnf-α and ifn-γ producing th1 cells but diminished numbers of regulatory t cells (tregs), which suppress angiogenesis and decrease vascular density (208) . the high rate of infectious diseases in t2dm patients might also be linked to a reduction in the mitochondrial dna function that causes downstream lymphocyte dysfunction and subsequently increased susceptibility to infection (209) (210) (211) (212) . in support, we have recently shown that the numbers of ifn-γ producing cells against cytomegalovirus (cmv), epstein-barr virus (ebv), and influenza virus are fewer in t2dm patients compared to normal controls (202) . kumar et al. have also investigated the functions of cd8 + t cells and nk cells in the whole blood of t2dm patients infected with mycobacterium tuberculosis (m.tb). compared to controls, the patients exhibited a reduction in cytokine production (ifn-γ, il-2, il-17a/f, and tnf-α) and decreased expression of cytotoxic molecules (perforin, granzyme b, and cd107a) (203, 213) . these studies conclude that the functions of both cd4 + and cd8 + t-cell are defective in t2dm patients. t2dm is usually associated with an elevated risk of asymptomatic bacteriuria, urinary tract infections (utis), pyelonephritis and non-sexually transmitted genital infections, such as balanitis and vulvovaginal infections (213) (214) (215) . the incidence of infections with a complicated course is significantly higher in diabetic patients compared to healthy controls ( table 3) . it seems that it is principally defects in the innate immune responses of diabetic individuals that are responsible for the increased susceptibility and prevalence of infections (4, 225 (199, 201) cd8 + tcells mycobacterium tuberculosis (203, 213) susceptible to the causative pathogen of lyme disease, borrelia burgdorferi (216) . the disease is mainly due to the ability of the bacteria to escape complement opsonization and attack, which leads to an impaired uptake and killing of bacteria by neutrophils (227) . neutrophil dysfunction also increases the susceptibility of diabetic animals to staphylococcus aureus (217) (233) . during the progression of t2dm in human subjects, the basal phenotype of macrophages is altered so their capacity to control mycobacterium tuberculosis is diminished (222) . martinez et al. have indicated that alveolar macrophages isolated from diabetic mice express decreased levels of macrophage receptor with collagenous structure (marco) and cd14 that are engaged in the recognition of trehalose 6,6'-dimycolate, a bacterial cell wall component (223) . diabetes increases the severity of tuberculosis (tb) and enhances the risk of progression to the active form in latent infections (234, 235) . diabetic tb patients have elevated frequencies of th1 and th17 cells as well as increased serum levels of inflammatory cytokines, including ifn-γ, tnf-α, il-1β, il-2, il-6, il-17a, and il-18 but decreased levels of il-22 compared to non-diabetic tb patients. this can contribute to dysfunctional immune responses and poor immune control of a tb infection (236) . a positive correlation between the serum levels of ifn-γ, tnf-α, il-2, and il-17a with hb-a1c levels was also observed. this indicates an association between impaired control of diabetes and the proinflammatory milieu. tripathi et al. have demonstrated that serum levels of il-22 were significantly decreased in tb-infected t2dm mice and humans compared to non-diabetic tb-infected mice and humans (224) . they revealed that the treatment of tb-infected diabetic mice with recombinant il-22 or ilc3s (cellular source of il-22) increased the survival of mice, prevented the accumulation of neutrophils near alveoli, diminished the generation of neutrophil elastase 2 (ela2) and prevented epithelial cell damage (224) . tan et al. have shown that b. pseudomallei and m. tuberculosisinfected pbmcs of diabetic patients fail to produce il-12. this leads to a decreased ifn-γ production, poor bacterial killing and elevated intracellular bacterial loads (237) . an impaired il-12 production is mainly due to decreased intracellular glutathione (gsh) concentrations within the infected cells of diabetic individuals (237) . such a combination of an inflammatory microenvironment and dysfunctional immune responses enhances the bacterial load and can subsequently amplify lung injury and fibrosis in diabetic tb patients. chellan et al. have further shown that infections caused by enterococcus faecalis, staphylococcus aureus, and pseudomonas aeruginosa are more prevalent in the wounds of diabetic patients (238) . t2dm patients are more susceptible to utis caused by antibioticresistant escherichia coli, proteus spp., klebsiella spp., coagulasenegative staphylococci, enterobacter spp., and enterococci (215, 239) . diabetic patients are also more susceptible to helicobacter pylori (h. pylori) infections (240). cui et al. have recently reported that t2dm patients have an increased risk of infection with kaposi's sarcoma-associated herpesvirus (kshv or hhv-8) (241) . they further showed that the viral load and antibody titers are positively correlated with blood glucose levels (241) . diabetic patients also have been shown to have an increased risk of infection with the severe acute respiratory syndrome coronavirus (sars-cov) ( (248) . the influenza virus that usually causes self-limiting infections can induce severe forms of the disease in diabetic patients (249, 250) . following the 2009 h1n1 influenza pandemic, diabetic individuals suffered from more severe infections compared to non-diabetic people (251, 252) . diabetic patients have also a higher prevalence of chronic cytomegalovirus (cmv), herpes simplex virus (especially hsv-1), and varicellazoster virus infections (253) (254) (255) . accordingly, it seems that the immune response against viruses is impaired in diabetics, and these patients need more care during viral infections. coronavirus virions are enveloped positive-strand rna spherical viruses with a diameter of ∼125 nm characterized by spike proteins projecting from their surface and with an unusual large rna genome (256) . the spike (s) protein of the virus binds to its receptor on the surface of cells by which intracellular proteases are induced (257) (258) (259) . subsequently, the s protein priming and cleavage occurs that allow viral fusion to the plasma membrane and entrance of viral genome into the cells (259) . sars-cov and sars-cov-2 use angiotensin-converting enzyme 2 (ace2) as their receptor while mers-cov uses dipeptidyl peptidase-4 (dpp4) to enter the cells (260, 261) . ace2 is strongly expressed in blood vessels, pancreas, intestine, brain, lungs, heart, and testis (262) . interestingly, nasal epithelial cells, especially goblet, and ciliated cells express the highest levels of ace2 and the intracellular protease transmembrane serine protease 2 (tmprss2) that facilitates the entrance of the sars-cov-2 (263) . furthermore, the expression of ace2 is significantly up-regulated in diabetic patients and those treated with ace inhibitors (264) . coronaviruses cause respiratory, enteric and central nervous system (cns) diseases in various animal species except rats and mice (264) . most coronavirus infections are mild, but major outbreaks of deadly pneumonia have been caused by sars-cov, mers-cov, and sars-cov-2 in 2002, 2014, and 2019-2020, respectively (265) . on march 11, 2020, the world health organization (who) announced the pandemic of sars-cov-2, the etiologic agent of coronavirus disease-19 (covid-19) (265) . the novel coronavirus pandemic, which has emanated from wuhan, china, promotes symptoms similar to those caused by the sars-cov outbreak in 2002. the viral pandemic, which has put the world on alert, has caused over 7.9 × 10 6 confirmed human cases and at least 43 × 10 4 deaths throughout the world (https://www.worldometers.info/coronavirus/) by june 14, 2020. most of the infected people experience only mild to moderate respiratory disease and recover soon without the need for special treatment. however, aged individuals and those with health problems, including diabetes, obesity, cardiovascular disease (cvd), hypertension, immune deficiency, and chronic respiratory disease are more likely to develop serious illness (https://www.who.int/health-topics/coronavirus#tab= tab_1). patients death is mainly due to the acute respiratory distress syndrome, disseminated intravascular coagulation, hemorrhage, coagulopathy, acute organ (e.g., kidney, heart, liver) injury, multi-organ failure, and secondary bacterial infections (266) . elevated levels of adipose-tissue derived adipokines, interferon, and tnf-α in diabetic patients may impair immune-responses against sars-cov-2 (267, 268) . it has been shown that diabetic patients have impaired clearance of sars-cov-2 from their circulation (269) . accordingly, diabetic patients due to the diminished viral clearance, impaired t cell function, and accompanied cardiovascular disease are more susceptible to the coronaviruses infection and subsequent cytokine release syndrome (crs) (270, 271) . in support, elevated levels of il-1β, il-2, il-6, il-7, il-8, il-10, ifn-γ, interferon gamma-induced protein 10 (ip-10), granulocyte colony-stimulating factor (g-csf), macrophage inflammatory protein 1α (mip1α), serum ferritin, fibrinogen, plasminogen, c-reactive protein (crp), and d-dimer have been observed in patients with covid-19 (266, 269, 272, 273) . covid-19 patients, especially those requiring intensive care unit (icu) have decreased total lymphocytes (lymphopenia), t cells (both cd4+ and cd8+), b cells, and nk cells (274, 275) . it should be noted that most of the surviving t cells in such patients have an exhausted phenotype (274) . consequently, disease severity is mainly because of the host immune response to viral infection. current evidence about the relationship between pathophysiological mechanisms of diabetes and covid-19 are limited and further research is still needed. patients with t2dm have an elevated risk of infection with plasmodium falciparum (276) , toxoplasma gondii (277), opisthorchis viverrini (278), strongyloides stercoralis (279), cryptosporidium parvum (280), blastocystis hominis (281), ascaris lumbricoides (280, 282, 283) , and giardia lamblia (283) . interestingly, diabetic patients who were treated with metformin had less p. falciparum infections compared to untreated patients (276) . omaña-molina et al. have shown that in a mouse model of t2dm the animals have an increased susceptibility to granulomatous amoebic encephalitis (gae) caused by trophozoites of acanthamoeba culbertsoni (284) . the possible reasons for the increased risk of diabetics for parasitic infections are metabolic abnormalities and immune dysregulation. chellan et al. have shown a higher prevalence of fungal infections in the wounds of diabetic patients (238) . the prevalence correlated with the levels of hba1c. the most widely observed fungal isolates were c. albicans, candida parapsilosis, c. tropicalis, trichosporon asahii, and aspergillus species. some of them were resistant to antifungal medications (238) . al mubarak et al. have also demonstrated that diabetic patients with periodontitis are more susceptible to infection with c. albicans, c. dubliniensis, c. tropicalis, and c. glabrata (285) . the incidence of candidiasis was significantly increased in patients over the age of 40 with hba1c > 9 (285). it has also been shown that diabetic patients are more susceptible to utis caused by c. albicans (239) . hyperglycemia impairs the normal functions of the circulatory system, gastrointestinal tract, pancreatic beta cells, liver as well as of skeletal muscles to boost systemic insulin resistance. a hyperglycemic environment also leads to immune cells dysfunction. it increases 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prognosis and practical considerations diabetes is a risk factor for the progression and prognosis of covid-19 the trinity of covid-19: immunity, inflammation and intervention reduction and functional exhaustion of t cells in patients with coronavirus disease 2019 (covid-19) characteristics of peripheral lymphocyte subset alteration in covid-19 pneumonia type 2 diabetes mellitus and increased risk for malaria infection toxoplasma gondii infection in diabetes mellitus patients in china: seroprevalence, risk factors, and case-control studies association between helminth infections and diabetes mellitus in adults from the lao people's democratic republic: a cross-sectional study is there an association between positive strongyloides stercoralis serology and diabetes mellitus? intestinal parasitosis and associated factors among diabetic patients attending arba minch hospital, southern ethiopia intestinal parasitic infections in patients with diabetes mellitus: a case-control study intestinal parasitic infections among diabetes mellitus patients host-parasite interactions in individuals with type 1 and 2 diabetes result in higher frequency of ascaris lumbricoides and giardia lamblia in type 2 diabetic individuals type 2 diabetes mellitus balb/c mice are more susceptible to granulomatous amoebic encephalitis: immunohistochemical study the prevalence of oral candida infections in periodontitis patients with type 2 diabetes mellitus the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 daryabor, atashzar, kabelitz, meri and kalantar. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-280040-xphxlaat authors: rutala, william a.; weber, david j. title: disinfection and sterilization in health care facilities an overview and current issues date: 2016-09-30 journal: infectious disease clinics of north america doi: 10.1016/j.idc.2016.04.002 sha: doc_id: 280040 cord_uid: xphxlaat when properly used, disinfection and sterilization can ensure the safe use of invasive and noninvasive medical devices. the method of disinfection and sterilization depends on the intended use of the medical device: critical items (contact sterile tissue) must be sterilized before use; semicritical items (contact mucous membranes or nonintact skin) must be high-level disinfected; and noncritical items (contact intact skin) should receive low-level disinfection. cleaning should always precede high-level disinfection and sterilization. current disinfection and sterilization guidelines must be strictly followed. in the united states in 2010 there were approximately 51.4 million inpatient surgical procedures and an even larger number of invasive medical procedures. 1 in 2009, there were more than 6.9 million gastrointestinal (gi) upper, 11.5 million gi lower, and 228,000 biliary endoscopies performed. 2 each of these procedures involves contact by a medical device or surgical instrument with patients' sterile tissue or mucous membranes. a major risk of all such procedures is the introduction of pathogenic microbes, which can lead to infection. failure to properly disinfect or sterilize equipment may lead to transmission via contaminated medical and surgical devices (eg, carbapenem-resistant enterobacteriaceae [cre] ). 3, 4 achieving disinfection and sterilization through the use of disinfectants and sterilization practices is essential for ensuring that medical and surgical instruments do not transmit infectious pathogens to patients. because it is not necessary to sterilize all patient-care items, health care policies must identify whether cleaning, disinfection, or sterilization is indicated based primarily on each item's intended use, manufacturers recommendations, and guidelines. multiple studies in many countries have documented lack of compliance with established guidelines for disinfection and sterilization. 5 failure to comply with scientifically based guidelines has led to numerous outbreaks and patient exposures. [6] [7] [8] because of noncompliance with recommended reprocessing procedures, the centers for disease control and prevention (cdc) and the food and drug administration (fda) issued a health advisory alerting health care providers and facilities about the public health need to properly maintain, clean, and disinfect and sterilize reusable medical devices in september 2015. 9 in this article, which is an updated and modified version of earlier articles, [10] [11] [12] [13] [14] a pragmatic approach to the judicious selection and proper use of disinfection and sterilization processes is presented, based on well-designed studies assessing the efficacy (via laboratory investigations) and effectiveness (via clinical studies) of disinfection and sterilization procedures. almost 50 years ago, earle h. spaulding 15 devised a rational approach to disinfection and sterilization of patient-care items or equipment. this classification scheme is so clear and logical that it has been retained, refined, and successfully used by infection control professionals and others when planning methods for disinfection or sterilization. [10] [11] [12] [13] [14] spaulding thought that the nature of disinfection could be understood more readily if instruments and items for patient care were divided into 3 categories based on the degree of risk of infection involved in the use of the items. the 3 categories he described were critical, semicritical, and noncritical. this terminology is used by the cdc's "guidelines for environmental infection control in healthcare facilities" 16 and the cdc's "guideline for disinfection and sterilization in healthcare facilities." 13 these categories and the methods to achieve sterilization, high-level disinfection, and low-level disinfection are summarized in table 1 . although the scheme remains valid, there are some examples of disinfection studies with prions, viruses, mycobacteria, and protozoa that challenge the current definitions and expectations of high-level disinfection (hld) and low-level disinfection. 22 in may 2015, the fda convened a panel to discuss recent reports and epidemiologic investigations of the transmission of infections associated with the use of duodenoscopes in endoscopic retrograde cholangiopancreatography (ercp) procedures. 23 after presentations from industry, professional societies, and invited speakers, the panel made several recommendations to include reclassifying duodenoscopes based on the spaulding classification from semicritical to critical to support the shift from hld to sterilization. 24 this change could be accomplished by shifting from hld for duodenoscopes to sterilization and modifying the spaulding definition of critical items from "objects which enter sterile tissue or the vascular system or through which blood flows should be sterile" to "objects which directly or secondarily (ie, via a mucous membrane such as duodenoscope) enter normally sterile tissue of the vascular system of through which blood flows should be sterile." 24, 25 implementation of this abbreviations: epa, environmental protection agency; glut, glutaraldehyde; hld, high-level disinfection; hp, hydrogen peroxide; opa, ortho-phthalaldehyde; pa, peracetic acid; ppm, parts per million. a prions (such as creutzfeldt-jakob disease) exhibit an unusual resistance to conventional chemical and physical decontamination methods and are not readily inactivated by conventional sterilization procedures. 17 b consult the fda-cleared package insert for information about the cleared contact time and temperature, and see reference 18 for discussion why greater than 2% glutaraldehyde products are used at a reduced exposure time (2% glutaraldehyde at 20 minutes, 20 c). increasing the temperature using an automated endoscope reprocesser (aer) will reduce the contact time (eg, ortho-phthalaldehyde 12 minutes at 20 c but 5 minutes at 25 c in aer). exposure temperatures for some of the aforementioned high-level disinfectants varies from 20 c to 25 c; check fda-cleared temperature conditions. 19 tubing must be completely filled for highlevel disinfection and liquid chemical sterilization. material compatibility should be investigated when appropriate (eg, hydrogen peroxide [hp] and hp with peracetic acid will cause functional damage to endoscopes). intermediate-level disinfectants destroy vegetative bacteria, mycobacteria, most viruses, and most fungi but not spores and may include chlorine-based products, phenolics, and improved hp. intermediate-level disinfectants are not included in table 1 as there as there is no device or surface for which intermediate-level disinfection is specifically recommended over low-level disinfection. adapted from refs. [11] [12] [13] 20, 21 recommendations requires sterilization technology that achieves a sterility assurance level of 10 à6 of complex medical instruments, such as duodenoscopes. ideally, this shift would eventually involve not only endoscopes that secondarily enter normally sterile tissue (eg, duodenoscopes, bronchoscopes) but also other semicritical devices (eg, gi endoscopes). 24, 25 critical items critical items are so called because of the high risk of infection if such an item is contaminated with any microorganism, including bacterial spores. thus, it is critical that objects that enter sterile tissue or the vascular system be sterile because any microbial contamination could result in disease transmission. this category includes surgical instruments, cardiac and urinary catheters, and implants used in sterile body cavities. the items in this category should be purchased as sterile or be sterilized by steam sterilization if possible. if heat sensitive, the object may be treated with ethylene oxide (eto), hydrogen peroxide (hp) gas plasma, vaporized hp, hp vapor (hpv) plus ozone, or by liquid chemical sterilants if other methods are unsuitable. table 1 and tables 2 and 3 list sterilization processes and liquid chemical sterilants and the advantages and disadvantages of each. with the exception of 0.2% peracetic acid (12 minutes at 50 c-56 c), the indicated exposure times for liquid chemical sterilants range from 3 to 12 hours. 19 liquid chemical sterilants can be relied on to produce sterility only if cleaning, which eliminates organic and inorganic material, precedes treatment and if proper guidelines as to concentration, contact time, temperature, and ph are met. another limitation to sterilization of devices with liquid chemical sterilants is that the devices cannot be wrapped during processing in a liquid chemical sterilant; thus, it is impossible to maintain sterility following processing and during storage. furthermore, devices may require rinsing following exposure to the liquid chemical sterilant with water that, in general, is not sterile. therefore, because of the inherent limitations of using liquid chemical sterilants in a nonautomated (or automated) reprocessor, their use should be restricted to reprocessing critical devices that are heat sensitive and incompatible with other sterilization methods. in contrast to semicritical items that have been associated with greater than 100 outbreaks of infection, 6 critical items have rarely, 26 if ever, been associated with disease transmission. for example, any deviation from proper reprocessing (such as crevices associated with the elevator channel) of an endoscope could lead to failure to eliminate contamination with a possibility of subsequent patient-to-patient transmission due to a low or nonexistent margin of safety. this low (or nonexistent) margin of safety associated with endoscope reprocessing compares with the 17-log 10 margin of safety associated with cleaning and sterilization of surgical instruments (ie, 12-log 10 reduction via sterilization and at least a net 5-log 10 reduction based on the microbial load on surgical instruments [2-logs] 27 and microbial reduction via a washer disinfector [7-logs] ). 18 semicritical items are those that come in contact with mucous membranes or nonintact skin. respiratory therapy and anesthesia equipment, gastrointestinal endoscopes, bronchoscopes, laryngoscopes, endocavitary probes, prostate biopsy probes, 28 cystoscopes, 29 hysteroscopes, infrared coagulation devices, 30 and diaphragm fitting rings are included in this category. these medical devices should be free of all microorganisms (ie, mycobacteria, fungi, viruses, bacteria), although small numbers of bacterial spores may be present. intact mucous membranes, such as those of the lungs or the gastrointestinal tract, are generally resistant to infection by abbreviations: aer, automated endoscope reprocessor; opa, ortho-phthalaldehyde. a all products effective in presence of organic soil, relatively easy to use, and have a broad spectrum of antimicrobial activity (bacteria, fungi, viruses, bacterial spores, and mycobacteria). the aforementioned characteristics are documented in the literature; contact the manufacturer of the instrument and sterilant for additional information. all products listed are cleared by the fda as chemical sterilants except ortho-phthalaldehyde, which is an fda-cleared hld. adapted from refs. [10] [11] [12] [13] 20 (continued on next page) common bacterial spores but susceptible to other organisms, such as bacteria, mycobacteria, and viruses. semicritical items minimally require hld using chemical disinfectants. glutaraldehyde, hp, ortho-phthalaldehyde (opa), peracetic acid with hp, and chlorine (via electrochemical activation) are cleared by the fda 19 and are dependable high-level disinfectants provided the factors influencing germicidal procedures are met (see tables 1 and 2 ). the exposure time for most high-level disinfectants varies from 8 to 45 minutes at 20 c to 25 c. 19 because semicritical equipment has been associated with reprocessing errors that result in patient lookback and patient notifications, it is essential that control measures be instituted to prevent patient exposures. 7 before new equipment (especially semicritical equipment as the margin of safety is less than that for sterilization) 25 is used for patient care on more than one patient, reprocessing procedures for that equipment should be developed. staff should receive training on the safe use and reprocessing of the equipment and be competency tested. at the university of north carolina (unc) hospitals, to ensure patient-safe instruments, all staff that reprocess semicritical instruments (eg, instruments which contact a mucous membrane such as vaginal probes, endoscopes, prostate probes) are required to attend a 3-hour class on hld of semicritical instruments. the class includes the rationale for and importance of high-level disinfection, discussion of high-level disinfectants and exposure times, reprocessing steps, monitoring minimum effective concentration, personal protective equipment, and the reprocessing environment (establish dirty-to-clean flow). infection control rounds or audits should be conducted annually in all clinical areas that reprocess critical and semicritical devices to ensure adherence to the reprocessing standards and policies. results of infection control rounds should be provided to the unit managers, and deficiencies in reprocessing should be corrected and the corrective measures documented to infection control within 2 weeks (immediately correct patient safety issues, such as exposure time to high-level disinfectant). noncritical items are those that come in contact with intact skin but not mucous membranes. intact skin acts as an effective barrier to most microorganisms; therefore, the abbreviations: eto, ethylene oxide; hp, hydrogen peroxide; twa, time-weighted average. adapted from refs. [10] [11] [12] [13] 20 sterility of items coming in contact with intact skin is "not critical." examples of noncritical items are bedpans, blood pressure cuffs, crutches, bed rails, linens, bedside tables, patient furniture, and floors. in contrast to critical and some semicritical items, most noncritical reusable items may be decontaminated where they are used and do not need to be transported to a central processing area. there is virtually no documented risk of transmitting infectious agents to patients via noncritical items 31 when they are used as noncritical items and do not contact nonintact skin and/or mucous membranes. however, these items (eg, bedside tables, bed rails) could potentially contribute to secondary transmission by contaminating hands of healthcare personnel or by contact with medical equipment that will subsequently come in contact with patients. 32 table 1 and table 4 list several low-level disinfectants that may be used for noncritical items. table 4 lists the advantages and disadvantages of the low-level disinfectants that are used on noncritical patient care items (eg, blood pressure cuffs) and noncritical environmental surfaces. the exposure time for low-level disinfection of noncritical items is at least 1 minute. physicians use endoscopes to diagnose and treat numerous medical disorders. although endoscopes represent a valuable diagnostic and therapeutic tool in modern medicine, more health care-associated outbreaks have been linked to contaminated endoscopes than to any other reusable medical device. 6, 8 additionally, endemic transmission of infections associated with gi endoscopes may go unrecognized for several reasons, including inadequate surveillance of outpatient procedures, long lag time between colonization and infection, low frequency of infection, and because pathogens are the usual enteric flora. in addition, the risk of some procedures might be lower than others (eg, colonoscopy vs ercp), whereby normally sterile areas are contaminated in the latter. in order to prevent the spread of health care-associated infections (hais), all heat-sensitive endoscopes (eg, gi endoscopes, bronchoscopes, nasopharyngoscopes) must be properly cleaned and, at a minimum, subjected to hld following each use. hld can be expected to destroy all microorganisms; although when high numbers of bacterial spores are present, a few spores may survive. recommendations for the cleaning and disinfection of endoscopic equipment have been published and should be strictly followed. 13, 35, 36 unfortunately, audits have shown that personnel often do not adhere to guidelines on reprocessing 5 and outbreaks of infection continue to occur. 3, 6, 8, 37 additionally, recent studies have suggested that current reprocessing guidelines are not sufficient to ensure successful decontamination. 38 in order to minimize patient risks and ensure that reprocessing personnel are properly trained, there should be initial and annual competency testing for each individual who is involved in reprocessing endoscopic instruments. 13, 35, 36 in general, endoscope disinfection or sterilization with a liquid chemical sterilant or high-level disinfectant involves 5 steps after leak testing: (1) clean: mechanically clean internal and external surfaces, including brushing internal channels and flushing each internal channel with water and a enzymatic cleaner or detergent; (2) disinfect: immerse endoscope in high-level disinfectant (or chemical sterilant) and perfuse (eliminates air pockets and ensures contact of the germicide with the internal channels) disinfectant into all accessible channels, such as the suction/biopsy channel and air/water channel, and expose for a time recommended for specific products; (3) rinse: rinse the endoscope and all channels with sterile water, filtered water (commonly used with automated endoscope reprocessors), or tap water; (4) dry: rinse the insertion tube and inner channels with alcohol and dry with forced air after disinfection and before storage; and (5) store: store the endoscope in a way that prevents recontamination and promotes drying (eg, hung vertically). in the past 3 years, multiple reports of outbreaks have led the fda, the cdc, and national news to raise awareness among the public and health care professionals that the complex design of duodenoscopes (used primarily for ercp) may impede effective reprocessing. several recent publications have associated multidrug-resistant (mdr) bacterial infections, especially due to cre, in patients who have undergone ercp with reprocessed duodenoscopes. 3, 4, 25, 37, 39 unlike other endoscope outbreaks, 6 these recent outbreaks occurred even when the manufacturer's instructions and professional guidelines were followed correctly. 3, 4 the key concern raised by these outbreaks is that current reprocessing guidelines are not adequate to ensure a patient-safe gi endoscope (one devoid of potential pathogens), as the margin of safety associated with reprocessing endoscopes is minimal or nonexistent. there are at least 2 (and maybe 3) reasons for this reprocessing failure and why outbreaks continue to occur. first, studies have shown that the internal channel of gi endoscopes, including duodenoscopes, may contain 10 7à10 (7-10-log 10 ) enteric microorganisms. 40, 41 investigations have demonstrated that the cleaning step in endoscope reprocessing results in a 2-to 6-log 10 reduction of microbes and the hld step results in another 4-to 6-log 10 reduction of mycobacteria for a total 6to 12-log 10 reduction of microbes. [40] [41] [42] thus, the margin of safety associated with cleaning and hld of gi endoscopes is minimal or nonexistent (level of contamination: 4-log 10 [maximum contamination, minimal cleaning/hld] to à5-log 10 [minimum contamination, maximum cleaning/hld]). therefore, any deviation from proper reprocessing (such as crevices associated with the elevator channel) could lead to failure to eliminate contamination with a possibility of subsequent patient-to-patient transmission. this low (or nonexistent) margin of safety associated with endoscope reprocessing compares with the 17-log 10 margin of safety associated with cleaning and sterilization of surgical instruments. 23 second, gi endoscopes not only have heavy microbial contamination (10 7 -10 10 bacteria) but they are also complex with long, narrow channels, right-angle turns, and difficult-to-clean and disinfect components (eg, elevator channel). the elevator channel in duodenoscopes is unique to side-viewing endoscopes. it has a separate channel and provides orientation of catheters, guidewires, and accessories into the endoscopic visual field. 25 this channel is complex in design and has crevices that are difficult to access with a cleaning brush and may impede effective reprocessing. 43 based on this and other recent studies, it is likely that mdr pathogens are acting as marker or indicator organisms for ineffective reprocessing of the complex design of duodenoscopes, which is an infectious risk to patients. third, biofilms could impact endoscope reprocessing failure and continued endoscope-related outbreaks. 44 biofilms are multilayered bacteria plus exopolysaccharides that cement cells to surfaces. they develop in a wet environment. if reprocessing is performed promptly after use and the endoscope is dry, the opportunity for biofilm formation is minimal. 45, 46 however, the formation of endoscopic biofilm during clinical practice may be related to reuse of reprocessing methods, such as reuse of detergent, manual cleaning, and incomplete drying. 47 ideally, reprocessing should be initiated within an hour of use; however, there are no evidence-based guidelines on delayed endoscope reprocessing. 48 it is unclear if biofilms contribute to failure of endoscope reprocessing. what should we do now? unfortunately, there is currently no single, simple, and proven technology or prevention strategy that hospitals can use to guarantee patient safety. of course, we must continue to emphasize the enforcement of evidenced-based practices, including equipment maintenance, and routine audits with at least yearly competency testing of reprocessing staff. 13, 35, 36 all reprocessing personnel must be knowledgeable and thoroughly trained on the reprocessing instructions for duodenoscopes. this training includes the new recommendations to use a small bristle cleaning brush and for additional flushing and cleaning steps of the duodenoscope elevator channel (http://medical.olympusamerica.com/sites/default/files/ pdf/150326_tjf-q180v_customer_letter.pdf). although these steps were described as validated, no public data are available on the ability of these new cleaning recommendations to yield an ercp scope devoid of bacteria. but we must do more or additional outbreaks will likely continue. for example, all hospitals that reprocess duodenoscopes should select one of the enhanced methods for reprocessing duodenoscopes. these enhanced methods have been priority ranked with the first providing the greatest margin of safety. 25 they include (1) eto sterilization after hld with periodic microbiologic surveillance; (2) double hld with periodic microbiologic surveillance; (3) hld with scope quarantine until negative culture results are returned; (4) liquid chemical sterilant processing system using peracetic acid (rinsed with extensively treated potable water) with periodic microbiologic surveillance; (5) other fdacleared low-temperature sterilization technology (provided material compatibility and sterilization validation testing performed using the sterilizer and endoscope) after hld, with periodic microbiologic surveillance; and (6) hld with periodic microbiologic surveillance. these supplemental measures to enhance duodenoscope reprocessing made in may-june 2015 25 were reinforced by the fda in august 2015. 43 unc hospitals has chosen eto sterilization after hld with periodic microbiologic surveillance as its primary reprocessing method for duodenoscopes and if the eto sterilizer is not available, then double hld with periodic microbiologic surveillance. 49 there is excellent evidence in the scientific literature that environmental contamination plays an important role in the transmission of several key health care-associated pathogens, including methicillin-resistant staphylococcus aureus (mrsa), vancomycinresistant enterococcus (vre), acinetobacter sp, norovirus, and clostridium difficile. [50] [51] [52] [53] all these pathogens have been demonstrated to persist in the environment for days (in some cases months), frequently contaminate the environmental surfaces in rooms of colonized or infected patients, transiently colonize the hands of health care personnel, be transmitted by health care personnel, and cause outbreaks in which environmental transmission was deemed to play a role. importantly, a study by stiefel and colleagues 54 demonstrated that contact with the environment was just as likely to contaminate the hands of health care personnel as was direct contact with patients. further, admission to a room in which the previous patient had been colonized or infected with mrsa, vre, acinetobacter or c difficile has been shown to be a risk factor for newly admitted patients to develop colonization or infection. [55] [56] [57] improving room cleaning and disinfection and demonstrating the effectiveness of surface decontamination in reducing health care-associated infections investigators have reported that intervention programs aimed at improving surface cleaning and disinfection reduced hais. 58 such interventions have generally included multiple activities: disinfectant product substitutions and interventions to improve the effectiveness of cleaning and disinfection (eg, improved housekeeper education, monitoring the thoroughness of cleaning [eg, by use of atp assays or fluorescent dyes] with feedback of performance to the environmental service workers, and/or use of cleaning checklists). [58] [59] [60] [61] health care facilities must also allow adequate time for room processing to ensure adherence to all steps recommended by institutional policies and professional organization guidelines. the authors have found that collaboration between infection prevention and environmental services staff, nursing, and management is critical to an effective environmental cleaning program. this collaboration includes ensuring that environmental services staff recognize the significance and relationship of adhering to proper work procedures to reduction of microbial contamination. the assignment of cleaning responsibility (eg, medical equipment to be cleaned by nursing; environmental surfaces to be cleaned by environmental service) is also important to ensure all objects and surfaces in a patient room are decontaminated, especially the surfaces of medical equipment (eg, cardiac monitors). improved environmental cleaning has been demonstrated to reduce the environmental contamination with vre, 61 mrsa, 62 and c difficile. 63 further, all studies have only focused improvement on a limited number of high-risk objects. thus, a concern of published studies is that they have only demonstrated improved cleaning of a limited number of high-risk objects (or targeted objects) not an improvement in the overall thoroughness of room decontamination, which is the objective. to the authors' knowledge only one study has objectively evaluated what constitutes high-touch objects in a patient room and no study has demonstrated epidemiologically what constitutes a high-risk object. examples of what the literature refers to as high-touch objects includes bed rails, intravenous (iv) poles, call buttons, door knobs, floors, and bathroom facilities 64 ; however, a study demonstrated high-touch objects in the intensive care unit were the bed rail, bed surface, and supply cart, whereas the high-touch surfaces in a patient ward were the bed rail, over-bed table, iv pump, and bed surface. 65 importantly, the level of microbial contamination of room surfaces was not statistically different regardless of how often they were touched before and after cleaning. until research identifies which objects and surfaces pose the greatest risk of pathogen transmission, all noncritical surfaces that are touched must be cleaned/disinfected. 66 no-touch (or mechanical) methods for room decontamination as noted earlier, multiple studies have demonstrated that environmental surfaces and objects in rooms are frequently not properly cleaned and these surfaces may be important in transmission of health care-associated pathogens. further, although interventions aimed at improving cleaning thoroughness have demonstrated effectiveness, many surfaces remain inadequately cleaned and, therefore, potentially contaminated. for this reason, several manufacturers have developed room disinfection units that can decontaminate environmental surfaces and objects. these no-touch systems generally use one of 2 methods: either uv light or hpv/mist. 53 these technologies supplement, but do not replace, standard cleaning and disinfection because surfaces must be physically cleaned of dirt and debris. ultraviolet light for room decontamination uv radiation has been used for the control of pathogenic microorganisms in a variety of applications, such as control of legionellosis, as well as disinfection of air, surfaces, and instruments. 53, 67 at certain wavelengths, uv light will break the molecular bonds in dna, thereby destroying the organism. uv radiation has peak germicidal effectiveness in the wavelength range of 240 to 280 nm. mercury gas bulbs emit uv-c at 254 nm, whereas xenon gas bulbs produce a broad spectrum of radiation that encompasses the uv (100-280 nm) and the visible (380-700 nm) electromagnetic spectra. 68 the efficacy of uv radiation is a function of many different parameters such as dose, distance, direct or shaded exposure, exposure time, lamp placement, pathogen, carrier or surface tested, inoculum method, organic load and orientation of carriers (eg, parallel vs perpendicular). data demonstrate that several uv systems have effectiveness (eg, eliminate >3-log 10 vegetative bacteria [mrsa, vre, acinetobacter baumannii] and >2.4-log 10 c difficile) at relatively short exposure times (eg, 5-25 minutes for bacteria, 10-60 minutes for c difficile spores). [68] [69] [70] the studies also demonstrated reduced effectiveness when surfaces were not in direct line-of-sight. [68] [69] [70] [71] [72] hydrogen peroxide systems for room decontamination several systems that produce hp (eg, hpv, aerosolized dry mist hp) have been studied for their ability to decontaminate environmental surfaces and objects in hospital rooms. hpv has been used for the decontamination of rooms in health care. [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] studies have demonstrated that hp systems are a highly effective method for eradicating various pathogens (eg, mrsa, mycobacterium tuberculosis, serratia, c difficile spores, clostridium botulinum spores) from rooms, furniture, and equipment. comparison of ultraviolet irradiation versus hydrogen peroxide for room decontamination uv devices and hp systems have their own advantages and disadvantages ( table 5) , 53 and there is now ample evidence that these no-touch systems can reduce environmental contamination with health care-associated pathogens and reduce hais. 84 however, each specific marketed system should be studied and its efficacy demonstrated before being introduced into health care facilities. the main advantage of both types of units is their ability to achieve substantial reductions in vegetative bacteria. another advantage is their ability to substantially reduce c difficile spores, as low-level disinfectants (such as quaternary ammonium compounds) have only limited or no measurable activity against spore-forming bacteria. 85 both systems are residual free, and they decontaminate all exposed surfaces and equipment in the room. based on data that demonstrated a reduction of colonizations and/or infections associated with these technologies, the authors recommend they should be used for terminal room decontamination after discharge of patients on contact precautions. because different uv and hp systems vary substantially, infection preventionists should review the peer-reviewed literature and the advantages/disadvantages of each technology (box 1) and choose only devices with demonstrated bactericidal capability as assessed by carrier tests and/or the ability to disinfect actual patient rooms. ultimately, one would select a device that also has demonstrated the ability to reduce hais. 84 disinfection and sterilization are critical components of infection control. unfortunately, breaches of disinfection and sterilization guidelines are not uncommon. patient notifications due to improper reprocessing of semicritical (eg, endoscopes) and critical medical instruments have occurred regularly. 7 this referenced article also provides a method for assessing patient risk for adverse events, especially infection. use of a 14-step algorithm (box 2) can guide an institution in managing potential disinfection and sterilization failures. 7,86 human papilloma virus is an extremely common sexually acquired infection and is the most important cause of cervical cancer. a 2014 article reported that the fda-cleared high-level disinfectants (ie, glutaraldehyde, opa) tested did not inactivate human papilloma virus, a nonenveloped virus. 87 these findings are inconsistent with many there is reliable biocidal activity against a wide range of health care-associated pathogens. room surfaces and equipment are decontaminated. there is rapid room decontamination (w5-25 minutes) for vegetative bacteria, which reduces the downtime of the room before another patient can be admitted. it is demonstrated to reduce hais (eg, c difficile, mrsa). it is effective against c difficile, although requires longer exposure (w10-50 minutes). hvac system does not need to be disabled and the room does not need to be sealed. uv is residual free and does not give rise to health or safety concerns. there are no consumable products, so costs include only capital equipment and staff time. there is good distribution in the room of uv energy via an automated monitoring system. all patients and staff must be removed from the room before decontamination, thus, limiting use to terminal room decontamination. decontamination can only be accomplished at terminal disinfection (ie, cannot be used for daily disinfection) as room must be emptied of people. capital equipment costs are substantial. it does not remove dust and stains, which are important to patients and visitors; hence, cleaning must precede uv decontamination. it is sensitive to use parameters (eg, dose, distance, carrier or surface tested, exposure time, pathogen). it requires that equipment and furniture be moved away from the walls. advantages it has reliable biocidal activity against a wide range of health care-associated pathogens. room surfaces and equipment are decontaminated. it has been demonstrated to reduce hais (eg, c difficile, mrsa, vre). it is useful for disinfecting complex equipment and furniture. it does not require that furniture and equipment be moved away from the walls. hp is residual free and does not give rise to health or safety concerns (aeration unit converts hp into oxygen and water). there is uniform distribution in the room via an automated dispersal system. all patients and staff must be removed from the room before decontamination, thus, limiting use to terminal room decontamination. hvac system must be disabled to prevent unwanted dilution of hp during use, and the doors must be closed with gaps sealed by tape. articles in the peer-reviewed literature, which demonstrates that high-level disinfectants, such as opa and glutaraldehyde, inactivate nonenveloped viruses, such as hepatitis a virus, polio, adenovirus, norovirus, and so forth. because the high-level disinfectants are commonly used to disinfect endocavitary probes (eg, vaginal probes, rectal probes), there is an urgency to corroborating these data. in a conversation with cdc staff regarding this issue, it was determined hospitals should continue to use the fda-cleared high-level disinfectants consistent with the manufacturers' instructions until the data can be corroborated. data have demonstrated the activity of a hp mist device to inactivate human papilloma virus. 88 although the most common way of performing hld of contaminated endocavitary probes is by immersion in an fda-cleared high-level disinfectant (eg, glutaraldehyde), an alternative procedure for disinfecting the endocavitary and surface probes is a hp mist system, which uses 35% hp at 56 c with the probe reaching no more than 40 c (ie, trophon epr, nanosonics, alexandria, australia). in one study, the results demonstrated complete inactivation (>6-log 10 reduction) of vre and a cre-klebsiella pneumoniae strain both in the presence and absence of 5% fetal calf serum (fcs). the trophon epr system showed good, but not complete, inactivation of mycobacterium terrae (5.2-log 10 reduction for m terrae with fcs, a 4.6-log 10 reduction for m terrae without fcs) and c difficile spores (5.1-log 10 reduction for c difficile spores with fcs, 6.2-log 10 reduction for c difficile spores without fcs). 89 to simulate a worse-case condition, cleaning was not done before disinfection in these experiments; but proper cleaning of probes is necessary to ensure the success of high-level disinfection. other data have demonstrated the activity of trophon to inactivate human papilloma virus 88 and other pathogens (eg, bacteria, mycobacteria, viruses) including a greater than 6-log 10 reduction of m terrae and c difficile spores in carrier tests and a greater than 6-log 10 reduction in m terrae on inoculated ultrasound probes. 90 these results differ slightly from those presented earlier, presumably because of the differences in testing methodology. in the authors' study only the probe devices were inoculated (carriers of different materials were not tested); for recovery of bacteria on the probe, the probes were immersed in media (not swabbed, which would likely result in lower recovery). 89 the trophon system processes the portion of the probe that has mucous membrane contact but also the handle of endocavitary probes, which may be contaminated; it is an alternative to high-level chemical disinfection for ultrasound probes. the department of justice and the fda have joined forces in prosecuting health care providers that reuse single-use devices. for example, one physician was criminally prosecuted for reusing needle guides meant for single use during prostate procedures. these prosecutions are based on conspiracy to commit adulteration and medicare fraud. third-party reprocessing is allowed by the fda as the reprocessor is considered the device manufacturer as defined under the code of federal regulations title 21 part 820. in 2011, the joint commission (tjc) recommended that laryngoscope blades be packaged in a way that prevents recontamination. examples of compliant storage include, but are not limited to, a peel pouch or a closed plastic bag. examples of noncompliant storage would include unwrapped blades in an anesthesia drawer as well as an unwrapped blade on top of or within a code cart. the packaging not only prevents recontamination but also distinguishes a processed from a nonprocessed semicritical item, such as a specula, laryngoscope blade, or endoscope. the use of a tagging system, in both inpatient and outpatient facilities, 91 that separates processed from nonprocessed items minimizes the risk that a nondisinfected, semicritical device would be used and potentially lead to cross-transmission of a pathogen. 7 this tagging system could involve a tag (eg, green tag, patient ready; red tag, requires reprocessing) for gi endoscopes or a plastic sheath or plastic-paper peel pouch (eg, endocavitary probes). ideally, hospitals and ambulatory care facilities 91 (as appropriate) should develop a strategy (eg, tagging, storage covers for patient-ready devices) that prevents patient exposures to contaminated devices. in the united states, it is estimated that more than 4 million cystoscopies are performed each year. 29, 92 cystoscopy is a diagnostic procedure that uses an endoscope especially designed to examine the bladder, lower urinary tract, and prostate gland or is used to collect urine samples, perform biopsies, and remove small stones. a flexible or rigid scope can be used to carry out the procedure. because the procedure, and other channeled scopes (eg, hysteroscopes, some nasopharyngoscopes), involves a medical device in contact with the patients' mucous membranes, it is considered a semicritical device that must minimally be high-level disinfected. the authors recently evaluated the disinfection of cystoscopes, and their results demonstrated that disinfection (ie, a reduction in bacterial load of greater than 7-log 10 colony-forming unit [cfu]) did not occur unless the channel was actively perfused with the glutaraldehyde. in fact, failure to perfuse the channel led to only minimal, if any, reduction in bacterial contamination. however, complete inactivation of 10 8 cfu of both vre and cre was achieved when the channel was actively perfused. it seems that no high-level disinfectant entered the channel unless it was actively perfused, as the level of microbial contamination was not reduced by immersion. 29 this failure to perfuse the channel occurs because the air pressure in the channel is stronger than the fluid pressure at the fluid-air interface. recommendations are provided for cystoscope hld and include actively perfusing the device while immersed in the high-level disinfectant. 93 unfortunately, some cystoscope reprocessing recommendations published in the literature are incorrect. for example, investigators have recommended complete immersion of the cystoscope into the high-level disinfectant but did not mention perfusion of the high-level disinfectant into the channel. 94 laryngoscopes laryngoscopes are routinely used to view the vocal cords and larynx and facilitate airway management. it typically consists of a blade that connects to a handle, which usually contains 2 batteries that power the light source. limited guidelines are available for reprocessing laryngoscope blades and handles, and hospital practices vary. [95] [96] [97] for example, some guidelines recommend and hospitals use low-level disinfection of the handle as it does not have direct contact with a mucous membrane, whereas others recommend the handle be high-level disinfected to prevent disease transmission. although blades have been linked to hais, handles have not been directly linked to hais. however, reports of contamination with blood (40% of the handles positive for occult blood) and potentially pathogenic microorganisms (86% of the handles deemed ready for patient use were contaminated with pathogens, such as s aureus, acinetobacter) suggest its potential, 97-100 and the blade and handle function together. for this reason, it is ideal that the blades and handles be high-level disinfected or sterilized even if a protective barrier or sheath is used during the procedure. in 2007, the state of california required that both blades and handles be hld or sterilized. unc hospitals is sterilizing the blades and handles (ie, blades via hp gas plasma, handle [without batteries] by steam). other methods for hld or sterilization are acceptable, but one must ensure the blade and handle are compatible with the hld or sterilization process chosen. after sterilization the blades and handles are checked for function before packaging and then packaged in a ziploc bag. per tjc, the laryngoscope blade and handle must be packaged in a way that prevents recontamination after processing (frequently asked questions, the joint commission, october 24, 2011). examples of compliant storage include, but are not limited to, a peel pack after sterilization (long-term storage) or wrapping in a sterile towel (short-term storage). recent advances in video technology have led to the development of video laryngoscopes, such as the glidescope (verathon medical, bothell, wa) and mcgrath (medtronic, minneapolis, mn) video laryngoscopes. 92 these new intubation devices assist in difficult airway management. for the mcgrath an image is displayed on a liquid-crystal display screen that is contained within a monitor mounted to the handle of the device. a sterile, single-use disposable laryngoscope blade covers the camera and light-emitting diode assembly to prevent direct patient contact. even though a cover is used, hld or sterilization via eto or hp gas plasma (battery removed) is recommended for the mcgrath mac video laryngoscope. 93 the manufacturer states, whenever practical, a hld or sterilization is preferred to a wipebased process. the portable glidescope video laryngoscope system is available in a single-use and a reusable configuration. they should be cleaned and disinfected per the manufacturer's recommendations. the single-use system features a reusable video baton and sterile stats that must be disposed of immediately after use. low-level disinfection is recommended for the video baton after each use using an environmental protection agency-registered disinfectant (eg, antimicrobial disposable wipe per manufacturer's instructions) after each use. the manufacturer recommends hld for the video baton when it is visibly soiled. the manufacturer recommends that the advanced video laryngoscope reusable blade be high-level disinfected and the gliderite rigid stylets be sterilized. 101 emerging pathogens, antibiotic-resistant bacteria, and bioterrorism agents emerging pathogens are of growing concern to the general public and infection control professionals. relevant pathogens include ebola, 102 mdr organisms such as cre, enterovirus d68, mdr pathogens, middle east respiratory syndrome-coronavirus, mdr m tuberculosis, human papilloma virus, norovirus, and nontuberculous mycobacteria (eg, m chelonae). the susceptibility of each of these pathogens to chemical disinfectants/sterilants has been studied; all of these pathogens (or surrogate microbes such as feline-calicivirus for norwalk virus, vaccinia for variola, 103 and bacillus atrophaeus [formerly b subtilis] for b anthracis) are susceptible to currently available chemical disinfectants/sterilants. 19, 104 standard sterilization and disinfection procedures for patient-care equipment (as recommended in this article) are adequate to sterilize or disinfect instruments or devices contaminated with blood or other body fluids from persons infected with blood-borne pathogens, emerging pathogens, and bioterrorism agents, with the exception of prions, hpv, and c difficile spores (see earlier discussion). no changes in current procedures for cleaning, disinfecting, or sterilizing need to be made. 13 in addition, there are no data to show that antibiotic-resistant bacteria (mrsa, vre, mdr m tuberculosis) are less sensitive to the liquid chemical germicides than antibiotic-sensitive bacteria at currently used germicide contact conditions and concentrations. [105] [106] [107] summary when properly used, disinfection and sterilization can ensure the safe use of invasive and noninvasive medical devices. the method of disinfection and sterilization depends on the intended use of the medical device: critical items (contact sterile tissue) must be sterilized before use; semicritical items (contact mucous membranes or nonintact skin) must be high-level disinfected; and noncritical items (contact intact skin) should receive low-level disinfection. cleaning should always precede hld and sterilization. current disinfection and sterilization guidelines must be strictly followed. national hospital discharge survey: 2010 table, procedures by selected patient characteristics-number by procedure category and age burden of gastrointestinal disease in the united states: 2012 update new delhi metallo-beta-lactamaseproducing carbapenem-resistant escherichia coli associated with exposure to duodenoscopes endoscopic retrograde cholangiopancreatography-associated ampc escherichia coli outbreak endoscope reprocessing methods: a prospective study on the impact of human factors and automation transmission of infection by flexible gastrointestinal endoscopy and bronchoscopy how to assess risk of disease transmission to patients when there is a failure to follow recommended disinfection and sterilization guidelines lessons learned from outbreaks and pseudo-outbreaks associated with bronchoscopy immediate need for healthcare facilities to review procedures for cleaning, disinfecting, and sterilizing reusable medical devices disinfection, sterilization and control of hospital waste disinfection, sterilization and antisepsis: an overview association for professionals in infection control and epidemiology guideline for disinfection and sterilization in healthcare facilities disinfection and sterilization in healthcare facilities disinfection, sterilization, and preservation. philadelphia: lea & febiger healthcare infection control practices advisory committee. guidelines for environmental infection control in health-care facilities disinfection and sterilization of prion-contaminated medical instruments, reply to belay efficacy of a washer-disinfector in eliminating healthcare-associated pathogens from surgical instruments fda-cleared sterilants and high level disinfectants with general claims for processing reusable medical and dental devices disinfection and sterilization in healthcare facilities guidelines for infection control in dental health-care settings-2003 disinfection: is it time to reconsider spaulding? brief summary of the gastroenterology and urology devices panel meeting gastrointestinal endoscopes: a need to shift from disinfection to sterilization? ercp scopes: what can we do to prevent infections? outbreak of pseudomonas aeruginosa surgical site infections after arthroscopic procedures: texas microbial contamination on used surgical instruments disinfection of a probe used in ultrasoundguided prostate biopsy effective high-level disinfection of cystoscopes: is perfusion of channels required? disinfection of an infrared coagulation device used to treat hemorrhoids role of environmental contamination in the transmission of vancomycin-resistant enterococci role of hospital surfaces in the transmission of emerging health care-associated pathogens: norovirus, clostridium difficile, and acinetobacter species outbreaks associated with contaminated antiseptics and disinfectants stability and bactericidal activity of chlorine solutions multisociety guideline on reprocessing flexible gi endoscopes society of gastroenterology nurses and associates i. standards of infection control and reprocessing of flexible gastrointestinal endoscopes risk of transmission of carbapenem-resistant enterobacteriaceae and related "superbugs" during gastrointestinal endoscopy persistent contamination on colonoscopes and gastroscopes detected by biologic cultures and rapid indicators despite reprocessing performed in accordance with guidelines control of a multi-hospital outbreak of kpc-producing klebsiella pneumoniae type 2 in france disinfection, sterilization and antisepsis: principles and practices in healthcare facilities worst-case soiling levels for patient-used flexible endoscopes before and after cleaning fda labeling requirements for disinfection of endoscopes: a counterpoint brief summary of the gastroeneterology and urology devices panel meeting is biofilm accumulation on endoscope tubing a contributor to the failure of cleaning and decontamination association for professionals in infection control and epidemiology effectiveness of current disinfection procedures against biofilm on contaminated gi endoscopes correlation between the growth of bacterial biofilm in flexible endoscopes and endoscope reprocessing methods delayed reprocessing of endoscopes outbreaks of carbapenem-resistant enteriobacteriaceae infections associated with duodenoscopes: what can we do to prevent infections? the role played by contaminated surfaces in the transmission of nosocomial pathogens environmental contamination makes an important contribution to hospital infection role of the environment in the transmission of clostridium difficile in health care facilities disinfectants used for environmental disinfection and new room decontamination technology contamination of hands with methicillin-resistant staphylococcus aureus after contact with environmental surfaces and after contact with the skin of colonized patients methods for assessing the adequacy of practice and improving room disinfection risk of acquiring antibiotic-resistant bacteria from prior room occupants evaluation of hospital room assignment and acquisition of clostridium difficile infection does improving surface cleaning and disinfection reduce health care-associated infections? evaluating hygienic cleaning in health care settings: what you do not know can harm your patients monitoring the effectiveness of hospital cleaning practices by use of an adenosine triphosphate bioluminescence assay interventional evaluation of environmental contamination by vancomycin-resistant enterococci: failure of personnel, product or procedure? impact of an environmental cleaning intervention on the presence of methicillin-resistant staphylococcus aureus and vancomycin-resistant enterococci on surfaces in intensive care unit rooms reduction of clostridium difficile and vancomycin-resistant enterococcus contamination of environmental surfaces after an intervention to improve cleaning methods environmental cleaning for the prevention of healthcare-associated infection. technical brief no. 22 (prepared by the ecri institute -penn agency for healthcare research and quality a quantitative approach to defining "high-touch" surfaces in hospitals microbial assessment of high-, medium-, and low-touch hospital room surfaces applications of ultraviolet germicidal irradiation disinfection in health care facilities: effective adjunct, but not standalone technology evaluation of a pulsed xenon ultraviolet disinfection system for reduction of healthcare-associated pathogens in hospital rooms room decontamination with uv radiation room decontamination using an ultraviolet-c device with short ultraviolet exposure time evaluation of an automated ultraviolet radiation device for decontamination of clostridium difficile and other healthcare-associated pathogens in hospital rooms terminal decontamination of patient rooms using an automated mobile uv light unit impact of hydrogen peroxide vapor room decontamination on clostridium difficile environmental contamination and transmission in a healthcare setting tackling contamination of the hospital environment by methicillin-resistant staphylococcus aureus (mrsa): a comparison between conventional terminal cleaning and hydrogen peroxide vapour decontamination environmental methicillinresistant staphylococcus aureus (mrsa) disinfection using dry-mistgenerated hydrogen peroxide use of hydrogen peroxide vapor for deactivation of mycobacterium tuberculosis in a biological safety cabinet and a room rapid recontamination with mrsa of the environment of an intensive care unit after decontamination with hydrogen peroxide vapour evaluation of hydrogen peroxide vapour as a method for the decontamination of surfaces contaminated with clostridium botulinum spores efficacy of vaporized hydrogen peroxide against exotic animal viruses vapor-phase hydrogen peroxide as a surface decontaminant and sterilant use of hydrogen peroxide vapour for environmental control during a serratia outbreak in a neonatal intensive care unit activity of a dry mist hydrogen peroxide system against environmental clostridium difficile contamination in elderly care wards airborne hydrogen peroxide for disinfection of the hospital environment and infection control: a systematic review effectiveness of uv devices and hydrogen peroxide systems for terminal room decontamination: focus on clinical trials selection of the ideal disinfectant assessing the risk of disease transmission to patients when there is a failure to follow recommended disinfection and sterilization guidelines susceptibility of high-risk human papillomavirus type 16 to clinical disinfectants susceptibility of hpv 16 and 18 to high-level disinfectants indicated for semi-critical ultrasound probes effectiveness of a hydrogen peroxide mist (trophonò) system in inactivating healthcare pathogens on surface and endocavitary probes evaluation of an automated high-level disinfection technology for ultrasound transducers disinfection and sterilization in physician practices and specialty clinics reprocessing semicritical items: current issues and new technologies mcgrath mac video laryngoscope operator's manual outbreak of cystoscopy related infections with pseudomonas aeurginosa: new mexico recommendations to resolve inconsistent guidelines for the reprocessing of sheathed and unsheathed rigid laryngoscopes prevention of disease transmission during flexible laryngoscopy reassessment of the risk of healthcare-acquired infection during rigid laryngoscopy nosocomial contamination of laryngoscope handles: challenging current guidelines contamination of laryngoscope handles incidence of visible and occult blood on laryngoscope blades and handles evaluating environmental persistence and disinfection of the ebola virus makona variant the inactivation of viruses by germicides sporicidal activity of a new low-temperature sterilization technology: the sterrad 50 sterilizer susceptibility of antibiotic-susceptible and antibiotic-resistant hospital bacteria to disinfectants susceptibility of vancomycin-resistant enterococci to environmental disinfectants antimicrobial activity of home disinfectants and natural products against potential human pathogens key: cord-349581-o320ogmg authors: robertson, lindsay j. title: the technological 'exposure' of populations; characterisation and future reduction date: 2020-05-25 journal: futures doi: 10.1016/j.futures.2020.102584 sha: doc_id: 349581 cord_uid: o320ogmg the nature and level of individuals' exposure to technological systems has been explored previously and is briefly restated here. this paper demonstrates how the concept of technological exposure can be extended to generic needs of individuals, and further to the needs of populations of individuals and even as far as “existential threats” to humanity. technological categories that incur high levels of population exposure are explored, and categories are described. a theoretical basis for reducing population exposure is developed from the basic concepts of technological exposure. technological developments that potentially enable less centralised societies having lower levels of population exposure, are considered for practicality and effectiveness as are the factors that could allow and cause transition to a less technologically centralised model. some conclusions regarding practicality, triggers, and issues arising from a decentralised society are considered and include the key conclusion that a higher level of decentralisation and exposure reduction is both desirable and possible. the fundamental impossibility of never-ending growth have been recognised for a long time (e.g. daly 1990 , henrique et al. 2013 ) and similarly authors (e.g. rao 1975) have recognised the danger of corporates of transnational size. this paper considers an associated but distinct issue, the growing technological vulnerability of populations. increasingly, essential goods and services are only accessible via technological systems that are both sophisticated, and also centralized. in this situation, end-user technological vulnerability becomes significant, but quantifying the extent and nature of such vulnerabilities have been hindered by the complexity of the analysis (haimes & jiang the supply of essential goods and services will involve heterogeneous systems (i.e. systems where goods/services are created by a progression of steps, rather than a system in which completed goods are simply transported/distributed), that involve an arbitrary number of linked operations each of which requires inputs, executes some transformation process, and produces an output that is received by a subsequent process. four essential principles have been proposed (author 2017) to allow and justify the development of a metric that evaluates the contribution of a heterogeneous technological system, to the vulnerability of an individual (author 2017a). these principles are: 1) the metric is applicable to an individual end-user: when an individual user is considered, not only is the performance of the supply system readily defined, but the relevant system description is clearer. 2) the metric acknowledges that events external to a technology system only threaten the output of the technology system if the external events align with a weakness in the technology system. if a hazard does not align with a weakness then it has no significance. conversely if a weakness exists within a technological system and has not been identified, then hazards that can align with the weakness are also unlikely to be recognised. if the configuration of a particular technology system is changed, weaknesses may be removed while other weaknesses may be added. 3) the metric depends upon the observation that although some hazards occur randomly and can be assessed statistically, over a sufficiently long period the probability of these occurring approaches 1.0. the metric also depends upon the observation that intelligently (mis)guided hazards (i.e. arising when a person j o u r n a l p r e -p r o o f intentionally seeks a weakness in order to create a hazard) do not occur randomly. the effect of a guided hazard upon a risk assessment is qualitatively different from the effect of a random hazard. the guided hazard will occur every time the perpetrator elects to cause the hazard and therefore such a hazard has a probability of 1.0. 4) the metric depends upon the observation that it is possible to not only describe goods or services that are delivered to the individual (end-user), but also to define a service level at which the specified goods or services either are or are-not delivered. this approach allows the output of a technological system to be expressed as a boolean variable (true/false), and allows the effect of the configuration of a technological system to be measured against a single performance criterion. applying these principles, the arbitrary heterogeneous technological system supplying goods/services at a defined level to an individual, can be described by a configured system of notional and/or/not functions. having represented a specific technological system using a boolean algebraic expression, a 'truth table' can be constructed to display all permutations of process and stream availabilities as inputs, and technological system output as a single true or false value. from the truth table, a count of the cases in which a single input failure will cause output failure, and assign that total to the variable "e1". a count of the cases where two input failures (exclusive of inputs whose failure will alone cause output failure) cause output failure, and assign that total value to e2. a further count of the cases in which three input failures cause output failure (and where neither single nor double input failures within that triple combination would alone cause output failure) and assign that total value to the variable "e3" and similarly for further "e" values. the exposure metric, {e1, e2, e3…}, for a defined system supplying specific goods/services, can be shown to measure the level of vulnerability of the individual to that technological system (author 2017). for many practical cases a generic service could be considered -e.g. "basic food" rather than "fresh milk", and vulnerability assessed by nominating multiple specific services as alternative suppliers (an "or" gate) of a nominated generic service. the exposure of the individual to lack of secure access to the necessities of life, can thus be assessed using exactly the same approach used to assess exposure to nonavailability of any specific need. illustrative examples of generic services could include "all needs for maslow's physiological level". conceptually this would include ((food_a or food_b or food_c…) and warmth and water and shelter) or possibly "all needs to occupy apartment on month-by-month basis". (sewage services and water supply and power supply). the concept of an individual's exposure may be yet further modified and extended to consider the quantification of a population's exposure, in regard to supply of either a particular, or a generic service. it is common for some "population" to need the same services/goods, and obtain these via an identical technological system. the "technological system" that supplies such a population may not be identical to that which supplies an individual: consider a case in which four individuals each rely on their cars to commute to work; for the output "arrive at work", each of their individually-owned cars contribute to the e1 exposure of each of the 4 individuals. if they reach a car-pooling agreement, the "car" then contributes to the e4 exposure for j o u r n a l p r e -p r o o f "arrive at work" of each individual, since any of the 4 cars can supply that functionality. a population exposure is developed by modifying an individual exposure evaluation (for a population using a functionally identical service) by considering each functional element used by more than one of the population, and whether each such functional element can be made accessible to more than one. if an exposure level (metric) is then nominated, it is possible in principle to establish the largest population which is subject to this level of exposure. for example, we might identify the largest population using a functionally identical service or supply of goods, that has a non-zero e1 exposure (i.e. has at least on single point of failure): should a city discover that the progressive centralisation of services had resulted in a situation where the whole population of the city had a non-zero e1 value for an essential service, some urgent action would be strongly suggested. conversely, if the supply of generic service to even small suburbs had low exposure values, then a good level of robustness would have been demonstrated for that generic service, at the suburb level. extrapolating these principles, where a "population" is actually the totality of the homo-sapiens species, and the "service" is essential for every individual's survival, then a definition of "existential threat" as proposed by bostrom (2002) , is approached. the analysis of exposure considers loci of failure and hence intentionally avoids consideration of specific hazards or their probabilities. nevertheless, hazards that affect various populations certainly exist, and a short list will serve to illustrate the significance of evaluating population exposure levels. the following list makes no attempt to be comprehensive, but illustrates hazards relevant to large corporates, j o u r n a l p r e -p r o o f apartment-dwellers, members of isolated communities and users of electronic communications.  a large group could be criminally held to ransom by malicious encryption of essential data (pathak 2016) .  a large group could be effectively excluded from access to a service because of conditions imposed by a (single, national) owner (tanczer, et al. 2016 , deibert 2003 , sandywell 2006 .  an isolated group (on antarctica, moon or perhaps mars) could cease to be viable if they cannot import supplies that cannot be generated locally (p?kalski & sznajd the occupiers of an apartment block could encounter a situation in where there was minimal (financial or other) pressure on the provider of sewage removal services to restore service, but where the occupiers faced major consequences (need to de-camp) in the absence of this service. similarly, (author 2017a) has noted that a failure to provide financial transaction services to a small retailer may incur negligible financial cost to a bank, but may cause the retailer's business to fail. these are examples of asymmetric consequences.  the whole of humanity exists on the planet earth: a failure of the natural environment can doom either a regional or possibly a global population (diamond 2005) , and can be certainly be considered to be an "existential threat", as defined by bostrom (2002) .  persons born early in the 20th century were very familiar with the possibility of highly unjust treatments (e.g. loss of employment), if private information became known -and they acted accordingly. for many born early in this century, such concerns seem remote. personal information privacy issues have thus failed to gain a j o u r n a l p r e -p r o o f high visibility, and we have perhaps become blasé about the costs of lack of privacy. nevertheless, the ubiquity of surveillance (miller 2014 , citron & gray 2013 , the increasing frequency of major data breaches (edwards et al. 2016) , and the recent rise in algorithmic decision-making (moses 2018) on such matters as health insurance, credit worthiness, validity of job application and security targeting are bringing this issue to increased prominence.  the recent revelation of hardware vulnerabilities in general processors (eckersley & portnoy 2017 , ermolov & goryachy 2017 ) demonstrated the significance of unappreciated weaknesses, even when overall operation of a system has been reliable. other publications (author 2010 , martin 1996 have noted society's vulnerability to technological systems and considered the relationship between time-todisaster and time-to-repair: that analysis was not an exposure analysis, but did consider the range of services corresponding to a broad category of human needs and effectively considered the significance of the exposure categories noted above. later works (author 2017a) have analysed the individual exposure associated with a broad range of goods and services. those works also noted that the analysed values for these specific examples could be reasonably extrapolated to many other similar goods and services. the general analyses of (author 2010) did attempt to cover the scope of individual needs and as such identified a range of services (or goods-supply needs) that were considered to incur high levels of vulnerability. the more detailed exposure analyses of (author 2017a) were indexed to the exposure of an individual, and although a limited number of examples were studied, these were considered to be representative of the high-exposure items relevant to j o u r n a l p r e -p r o o f individuals living in urbanised settings in the 21st century. the assessment of the population exposure of those exemplar technological systems shows several distinctive changes from the analysis indexed to the individual. primarily, the e1 items that arise close to the final point of use by the individual lose significance and for many cases cease to be significant at the e3 level (arbitrarily chosen as the cut-off point for consideration). the components that remain significant are those that genuinely represent exposure values for the population under consideration. the analysis of population exposure of a broad spectrum of needs showed that it was possible to identify some high exposure technological fields, specifically complex components, complex artificial substances, finance, communications, energy and information, these are considered more fully as follows;  complex components: complex components may be distinguished from large components (such as a building, hydroelectric dam, or road-bridge), and qualitatively described as being beyond the capacity of a skilled individual craftsperson to fashion in reasonable time and to required tolerances. under this category we might consider such items as a carburettor body for an internal combustion engine, food processing equipment and construction machinery (crane). for many consumer items within the "complex component" category, the population exposure is significantly lower than the individual exposure, since numerous examples of item exist (either owned by others, or stockpiled for sale). the level of centralisation may however be very high, with (for example) perhaps only one worldwide source of a carburettor body for a specific vehicle.  complex artificial substances: complex artificial substances includes advanced metallurgical alloys, advanced plastics and composites, drugs, and vaccines: these j o u r n a l p r e -p r o o f are distinguished by the complexity of composition, rather than complexity of form. in many cases the centralisation that causes high exposure levels for the production of complex substances, has resulted primarily from available economies of scale, and only secondarily from the substances' complexity. some complex substances (notably pharmaceuticals) have patent protection, which creates centralisation at up to global level.  finance: as the range of goods and services, and the geographical scope of supply of those goods and services has increased, so has the need for the facility to exchange value in return for goods and services: this has inevitably led to elaborate mechanisms for secure exchange of value. recognising that the exchange of value can also facilitate illegal activities, state actors have also enacted significant surveillance and control of financial transactions. technologies associated with the exchange of value have acquired high large levels of exposure (colloquially, many things that can go wrong) and high levels of centralisation, in the process of meetings these demands.  communications: the ubiquity of the internet has become a remarkable feature of the last 20 years: although there are exceptions, most areas of the earth and a very large proportion of total population can communicate via internet. although cellphone use is common, it is evolving as a mobile access connection, with the internet carrying the data. internet communications has been made possible by wellestablished protocols, however high level systems for routing continue to be centralised, and while problems are rare, nation-states have occasionally decided to discontinue connection to the internet, showing that high levels of centralisation exist (howard et al. 2011) . although the feasibility of internet communications can be attributed to open source protocols, the practicality of current connectivity has been j o u r n a l p r e -p r o o f largely enabled by the very large data rates possible via fibre-optic cables, yet this capacity has also introduced a high level of exposure for both individuals and populations. duplication of undersea fibre-optic cables has somewhat reduced the level of population exposure, yet the trend for dependence on high-data-rates has increased at similar pace (market forces have driven the need for additional cables), and communications via internet carry a high level of population exposure. recent reports (rose 2017 , clark 2016 have noted that the disruption of a very small number of fibre-optic cable would lead to unacceptable slowdowns, and trends to ownership of undersea cables by corporate entities further contributes to e1 values of exposure and high population exposure. the numbers of cables in service show that internet communications is centralised at a small-nation level.  energy: energy has been noted as key to civilisation (smil 2017) . coke allowed the smelting of iron, and oil enabled almost all current transportation. coal, nuclear and geothermal heat and hydro storage allow electricity generation on-demand. national power transmission systems generally have high reliability and many have some level of design redundancy. nevertheless, the generation and distribution of electric power incurs significant individual and population exposure: large power stations are bespoke designs as are large transformers (sobczak & behr 2015) , and transmission networks may face significant "resilience" issues (carvajal et al. 2013 , sidhu 2010 . liquid fuels can be stockpiled at national level, and to a lesser extent locally, however the level of stockpiling is limited -and even the higher levels of stockpiling are likely to be small compared to the time to rebuild a major production plant (terminal, or refinery). at the consumer and industrial user-level, although solar pv and wind can produce power at $/kwh rates close to thermal generation, but currently no economically viable technology that allows storage of megawatt-hours  information: information, whether medical reference information, contractual records or engineering design information, is not a coincidental by-product of a technological society, it is information that fundamentally allows the design and construction of technological systems and to the full operation of society (dartnell 2014 , shapiro 2009 , van den heuvel 2017 ). yet while it has become possible to generate and transmit enormous quantities of information, the information storage remains a particularly high-exposure issue (bergeron 2001) . currently the ascii codes largely standardise information representation, and protocols for transmission are also largely standardised but a gap in the standardisation of information storage (including writing and recovery) contributes to a high exposure for the final delivery of information to users. hard-disk drives are still the most common storage technology, yet these have short lives, and use proprietary data storage formats plus proprietary approaches to the writing and recovery of data. this issue has been wellreported, authors such as (cerf 2015) have predicted a "digital dark age", i.e. a future society that cannot recover/read most of the information generated in the current era. this describes a situation of high individual and population exposure, and since there j o u r n a l p r e -p r o o f are few of manufacturers of hdd's, information storage can also be noted to illustrate centralisation at multi-nation level. several technologies allowing longterm storage have been proposed (kazansky et al 2016, longnow foundation; nanoarchival™ technology) but these are currently expensive and lack the integration to allow them to truly offer changes to population exposure. by contrast, some fields have generally low population exposure: basic building materials, foodstuffs, natural fabrics and clothing are commonly supplied via relatively simple technological systems in which technological design redundancies are commonly large, and for which population exposure is therefore low. technologies such as creation of ceramics and glassblowing may require skill but are also not dependent on high technological sophistication and so contribute low technological exposure. similarly, the collection and storage of rainwater is feasible with low technological exposure for even densely populated urban populations. in addition to identifying high exposure fields, it is also possible to identify categories of exposure contribution that commonly apply across a range of technological fields. these are proposed to include initial resource availability, complex unit operations, lack of buffering, single points of failure (spof), contributory systems, highly centralised processes and "practical unavailability", and are described more fully as follows: initial resource availability: all technological systems producing goods and services for users, ultimately depend upon raw materials and viable environmental conditions. where raw materials cease to be available, or environmental conditions change permanently, services to users will inevitably be affected. raw material supplies and acceptable environmental conditions must therefore be identified (diamond 2005) j o u r n a l p r e -p r o o f as sources of exposure and hence vulnerability to users. complex unit operations: we use the descriptor "complex" as a characteristic of a process whose internal operation is practically unknowable to the user and cannot realistically be repaired by the user. personal computers, routers and related equipment are examples. it is also possible to consider situations where a critical application has been compiled from an outdated programming language and runs on a computer for which no spare hardware is available (hignet 2018). another example might consider critical information held on a very old storage medium (teja 1985) . these examples illustrate three categories of complex processes: in the first case, while the inner workings of a pc may be exceedingly complex, the format of incoming data (tcp/ip packets) and protocols (www, email etc.) are in the public domain (fall & stevens 2011 ) and so it is not only possible but practical for alternative machines to offer the same services. in the second case, assuming the functional specifications of the application processes are known, the application can be re-coded (and fully documented) using a language for which larger numbers of maintenance programmers exist, and on a more common platform. the third case of data encoded on old storage medium, illustrates a subcategory where the internal details of the storage are proprietary (not in public domain), alternative equipment is unavailable, and creation of replica equipment for reading the data is probably impractical, leading some authors e.g. (cerf 2015) to express fears of a "digital dark age". lack of buffering: for the supply of long-life products, it is both possible and practical to provide buffer stocks at various points in the process. by contrast, since ac power (for example) is not readily storable, all processes involving uses of ac power will fail immediately if the power supply fails. single points of failure (spof): all single points-of-failure (spof) contribute to e1 values and so make a primary contribution to users' vulnerability. three subcategories of spof are noted: the first is j o u r n a l p r e -p r o o f where delivery of services to users involves some processes immediately adjacent to the user, known as "last mile" services in the telecommunications field. the second subcategory of spof is illustrated by considering a small rural town whose eftpos, landline phone service, cell-phone service and internet connection have all been progressively migrated to data services, carried by a single fibre-optic cable and thus have inadvertently created a spof. the third is where a particular failure will inevitably cause failure of other components that are not functionally connected -a cascading failure. finally it is noted that contributory systems are a common source of exposure: whenever a system is made dependent upon another, the contributory system's exposures are reflected in the total exposure to the user. a common example is where a simple user service is made dependent upon internet access; the mandatory internet access may add huge levels of exposure to a system that would otherwise incur low levels of vulnerability. some specific technologies including artificial intelligence, nuclear weapons and asteroid strikes have been examined, and authors such as baum (2018) have pondered their potential to incur existential threats by threatening multiple systems. others including baum (2015) and green (2016) have considered approaches to limiting the scope of such hazards. the above categories of exposure may apply to any technological process; there are additionally several categories that are specifically relevant to the study of "population exposure", these include highly centralised processes, for example, the evacuation and treatment of sewage requires a network of pipes and pumps to collect sewage and deliver it to the treatment station. this is an example of a centralised system that is large but technologically simple. other examples of large centralised systems include financial transaction systems (o'mahony et al. 1997) , and the international data transmission systems of undersea fibre-optic cables (clark 2016) . such systems tend to j o u r n a l p r e -p r o o f be monopolies and are commonly controlled by entities that have little if any obligation to provide service or to negotiate terms acceptable to individual users. authors such as li et al. (2019) have shown that highly interconnected systems have similar characteristics to centralised systems, and it is well-recognised that the most "centralised" system is earth's natural environment, because the natural environment (including air and water) are essential to all life. practical unavailability: consider the hypothetical case where a user wishes to communicate sensitive information, but only has access to one data transmission facility that is known to be under surveillance. although technically operational, the inevitable absence of privacy associated with that data transmission facility has made the facility practically unavailable. for technological systems that are highly centralised and near-monopoly, practical unavailability is a significant possibility. the en metric has been explained earlier in terms of its significance for measuring vulnerability, however a wider and more future-oriented applicability of the metric itself can also be demonstrated in several ways. the assumption that there is an average cost of defending any vulnerability could be challenged but across a broad enough spread of examples, it is workable. under that broad assumption, whereas the cost of defence for an e3 exposurecontributor is precisely the same as the cost of defence for a contributor to an e1 vulnerability (it is the cost of protecting one only vulnerability, since the e3 contributor requires successful attack of all three vulnerabilities), the cost of mounting a successful attack on a e3 vulnerability is 3 times greater than the cost of attacking a vulnerability j o u r n a l p r e -p r o o f contributing to the e1 value, since the attacker needs to identify and successfully attack all 3 e3-contributory nodes simultaneously. in addition to its value for measuring vulnerability, the exposure metric is therefore also broadly significant for planning the reduction in vulnerability of a system. considering the generation of an exposure metric, the process itself provides valuable insights into options for reduction of the final values. the process of generating an exposure metric must be started from the delivery point, and follow a systematic redrawing and track-back process until a justifiable end-point is reached. the selection of a justifiable end-point has been addressed elsewhere, and could be associated with "no further contributors to an en level" criterion. the process of achieving a final representation of the system will very likely require a progressive redrawing of stream and process relationships; an example of a common re-drawing is presented in figure 1 . in a practical process of building an exposure metric, subsystems (which may have been analysed separately) commonly contribute goods or services to "higher" systems. if the exposure metric of a subsystem is known then there is little value in recreating a truth table for a higher level super-system, that re-explores all of the inputs to every sub-system. the more-effective approach is to consider the point at which the subsystem output contributes to the input of a gate within the higher level system, and how the subsystem exposure metric is transmitted/accumulated by the upstream gate of the super-system. we may generalise this process by considering that each input to a gate (boolean and or or operation) has an exposure vector, and developing the principles by which the gate output can be calculated from these inputs. this is illustrated in figure 2 . for the and gate, the contributory exposure vectors are simply added. for the or gate, the issue is more complex, but the higher levels of exposure are quickly avoided. the process of generating the metric will itself highlight sources of large exposure, and hence specific options for reduction. the detailed process for analysis of exposure has illustrated how the e values are accumulated and how specific exposure reduction options may be identified. generalised approaches to the reduction of the exposure of a population can also be identified. the process of redrawing shows that even where alternative subsystems can supply higher systems, if there is a locus of weakness that is common to alternative subsystems, the exposure analysis will ensure that such facts are preserved and the e1 contribution will actually appear in the exposure value for the whole target consumer (group). if the "o" ring seal failure had been identified as a contributor to the e1 j o u r n a l p r e -p r o o f exposure of the thiokol solid booster rocket then the elimination of all e1 values for the parent system that was the challenger space shuttle, could not have been achieved unless the "o" ring weakness were addressed. the use of an exposure metric therefore potentially addresses the colloquial saying "it's always the smallest things that get you". one of the learnings from the analysis of the "challenger" tragedy was that even known subsystem weaknesses could become lost as analyses were progressively summarised. when genuinely independent, alternative sources are available, their effect on the next-highest systems (process, consumer or group) is illustrated by the operation of an "or" gate. if 4 genuinely independent/alternative subsystems that each have exposure vectors with non-zero values of e1, are combined via an "or" gate, the higher system does not see any exposure at above the e4 level from that source. this principle might be qualitatively or intuitively perceived, but the effect upon an analysis of exposure provides a starkly quantitative analysis. it may also be observed that while reducing the exposure of each subsystem would require separately addressing each contributor to the subsystems e1, e2 and e3 values (potentially a major task), if 4 genuinely alternative subsystems exist then the combined "or" exposure has no nonzero contributor more serious than e4 and may warrant no further action. whereas many 20th century devices were designed for a single purpose and high throughput (mass production), some recent trends have been to devices that can be repurposed -and the pinnacle of flexibility is the human being! for any case where a piece of equipment contributes to the e1 value (is a single point-of-failure), if the capability of that equipment were able to be undertaken by multiple options (e.g. j o u r n a l p r e -p r o o f alternative human operators), the exposure contribution may be reduced to the en level, where n is the number of alternatives or persons capable of undertaking the equipment's function. an illustration may help to clarify this principle: if a sensor provides input that is required by an upstream function, that upstream function and all higher dependencies are exposed to the capability of the sensor to provide input: if 'n' humans are able to provide their best assessment of the sensor's input, then the exposure of the higher system to that functionality is reduced to the en level. considering the high-exposure technological fields that were identified earlier, generalised approaches to reducing the accrued actual exposure include standardisation of specifications that allow competitive supply of complex components and complex substances, avoiding the large exposure of some contributory systems, retention of genuine alternatives (e.g. cash/gold as well as electronic transactions). there is an intuitive appreciation that it is undesirable to have high vulnerability levels for systems that affect large numbers of persons. some may also intuitively appreciate that trends to centralisation, driven by economies of scale, increases the technological vulnerability of large population groups. the analysis of population exposure and the principles of exposure analysis therefore provide a quantitative approach that can be used not only to assess the level of exposure of current systems, but more importantly to show quite widely applicable principles for exposure reduction. specifically the analysis shows that centralisation of production almost inevitably j o u r n a l p r e -p r o o f creates higher population exposure values, and provides a sound theoretical basis for promoting decentralisation of production as an approach for the reduction of population exposure. it is important to consider the practicality of population exposure reduction by decentralisation; this is reviewed within each of the fields previously identified as incurring high exposure: a) complex components. in order for this functionality to be genuinely available at a significantly decentralised level, we can consider the sub-systems that are required, and the current level of maturity of technology options within each of those subsystems. for a complex component, relevant subsystems include those associated with the supply of materials and the creation of complex shapes from basic material stocks. for many cases, assembly of components is likely to be straightforward but we must also consider cases where assembly itself requires specialised equipment. for the majority of cases, the composition of the complex component can be assumed to be uniform, however cases where composition is not uniform (e.g. multilayer circuit board) must also be acknowledged. equipment for additive and subtractive manufacturing is available; specifically 3d printing equipment is readily available at small scale, and large scale implementations (bassoli et al. 2007 , kraft et al. 2013 ) have been tested. a moderately standardised format for 3d printer design information exists as iso 14649-1:2003, and iso 10303:2014 (many sections) , and instructions for operating additive and subtractive manufacturing equipment are such that high skill levels are un-necessary. a significant body of designs (pinshape™,  complex substances. in order for complex substance synthesis to be genuinely available at a significantly decentralised level, we can consider the types of complex substances that are of interest, the sub-systems that are required for each, and the current level of maturity of technology options within each of those subsystems. broadly, the complex substances could be categorised as: complex alloys, complex inorganic liquids (oils, detergents, etc.), complex organic materials (pharmaceuticals, insecticides, herbicides), complex substances derived from living organisms -yeasts, vaccines, fermentation bacteria, and polymers. for a complex molecular substance, it is currently common for a range of supply chains to bring raw materials to a synthesis plant. a sequence of synthesis steps (including unit operations of heating and cooling, separation, reaction, precipitation and dissolution) are carried out to generate a complex substance. for "organic" compounds, temperatures are generally limited to below 120ºc. for a complex metallic component, it is currently common for granulated pure metals or metal-compounds to be melted together, sometimes in vacuum or inert gas, and then a controlled set of cooling/holding/reheating steps are used to generate the final material. considering whether these syntheses could be practically decentralised, drexler's seminal paper (drexler 1981) considered the options and practicality issues associated with general-purpose synthesis of complex substances. since 2010, the "engineering and physical sciences research council" (epsrc) have been funding a "dial-a-molecule" challenge which runs parallel to the work of others (crow 2014 , peplow 2014 , and commercial ventures such as "mattersift™" (manipulating matter at the molecular scale) have demonstrated the progress towards this goal. even where general-purpose synthesis capabilities are not available, the availability of knowledge does in principle allow relatively complex syntheses e.g. daraprim™, to be undertaken by small groups (hunjan 2016) . for j o u r n a l p r e -p r o o f inorganic materials, progress has been made with solar pyrometallurgy (neelameggham 2008) and although much development is needed, there would seem to be no fundamental reason why the required temperatures and composition constraints could not be achieved on small scale and with limited equipment. published proposals (crow 2014 , peplow 2014 ) have proposed that it is possible to build a standardised facility capable of carrying out an arbitrary sequence of unit operations required to make any organic compound. while the technologically maturity of decentralised synthesis of complex materials is lower than the decentralised production of complex components, many of the processes are actually feasible at present, and others are rapidly maturing.  finance: decentralised tokens of wealth (tokens of exchange) have existed for as long as societies have existed. in order for a token of exchange to continue to avoid the large exposure of current centralised financial systems, a token must retain the qualities of ubiquitous acceptance, transparent and irrevocable transactions; this is currently feasible. blockchain technology (swan 2015) has recently offered another decentralised system for secure and irrevocable transfer of wealth, allowing broad acceptance and thus meets the criteria for acceptability. blockchain-based currencies are however reliant on a communications system that is currently highly centralised, and so fall short of the security expected and exist in numbers of as-yet-incompatible forms. the difficulties with current blockchain technologies seem to be solvable, and this technology offers a promising approach to decentralised exchange of value. current, high-security banknotes and bullion fulfil many of the requirements for a decentralised approach to transactions, and do not incur the exposure that is inherent with blockchain-based currencies, but do incur a high risk of theft and the exposure of a physical transmission system if the transaction is to span significant distance. j o u r n a l p r e -p r o o f  communications: a communications system could be considered decentralised (within the population size envisaged) when it has no e1 value above zero, and is capable of communicating with any other (decentralised) population. secure encryption is currently possible (schneier 2015) , and despite some practical difficulties, mature approaches such as one-time-pads, seem to be proof against even projected (de wolf 2017) technologies. radio transmission on shortwave bands (using ionospheric reflection) of encrypted material can be received globally. assuming a 10 mhz "open" band, and a very narrow bandwidth (and very slow transmission rate), many channels would be available in principle. this is a low level of practicality, but is must be noted that completely decentralised communication is inherently feasible. massively-redundant fibre-optic cable systems with decentralised routing systems are also technically feasible, and it is even feasible to consider significantly higher levels of design redundancy for undersea cables. while both feasible and practical for land-based systems, massive design redundancy appears to be feasible for undersea routes but not practical for the exposure level sought. selfdiscovering radio-based mesh communications for land-based systems are (author 2007) feasible at present and are likely to be more practical and economical than massively redundant fibre-optic systems for land-based communications. hybrid approaches, e.g. using self-discovering mesh for communications within a single land mass, and allowing access to a significant number of undersea cables, could meet the population levels and exposure levels to allow a claim of feasible decentralisation using current technology. suggests that fundamental breakthroughs may not be likely. so-called "ultracapacitor" technology (de rosa et al. 2017 ) may or may-not overtake battery storage for smaller power levels in the future. the decentralised production of biofuel using macro-algae (zev et al. 2017 , chen et al. 2017 ) is also immature, but the capability to treat sewage and create storable hydrocarbon fuel with high energy-density is promising and is perhaps the decentralised heating-energy storage mode that is closest to technical maturity at present. information. the information storage requirements for a community could include significant medical information, all data required for manufacture of complex components and synthesis of complex substances in addition to contractual, financial, genealogical etc. data. human-readable information storage (books, tablets) have very low exposure and high decentralisation currently. a decentralised and low-exposure approach for storage of machine readable information does j o u r n a l p r e -p r o o f however pose a non-trivial technological challenge. existing computer hard-disk drive storage technology is mature, but this approach has a very high technological exposure since the format of storage and the recovery of data is via a complex and proprietary system, and the actual storage medium is not user-accessible. a criterion of e1<1 could in principle be achieved by massive redundancy, but in practice the use of identical operating systems and software make it likely that residual exposure will remain. technologies such as the 5d glass storage approach (kazansky et al. 2016) or proposals by the longnow foundation; or organisations such as "nanoarchival™ technology" avoid the reliance upon proprietary data retrieval systems and provide very long life -but still lack a mature and decentralised datawriting approach, and a mature and decentralised approach for reading stored data back into a machine-readable form. the advances needed in order to achieve a durable, low-exposure storage medium are immature but are technologically achievable. other: it is useful to note many other fields in which significant capabilities can be achieved with simple components: while far short of laboratory quality, the principles of spectrography and chromatography (lichtenegger et al. 2017 , ghobadian et al. 2017 are actually accessible at a domestic level, and microscope (prakash et al. 2017) (jones 1979) and mars explorer spacecraft. organisations such as the longnow corporation have considered the requirements for durable engineering, and plan items such as a "10,000 year clock". in the context of this paper and earlier studies (author 2010), we can consider that it is practical to design at least some classes of components and equipment for a usable timeframe that is demonstrably longer than the time required to re-develop a production facility to recreate them; this criterion is a valid test of low exposure practicality. in summary: low exposure and decentralised options for a range of technologies have been examined: some are mature and some have a low level of technological maturity. it is likely that some could mature rapidly (e.g. additive/subtractive manufacturing, self-discovering communications networks, information storage), while others such as energy storage have absorbed enormous r&d effort already with limited progress. this does not mean that a trend to decentralisation cannot begin, simply that a decentralised society may have more sophisticated capabilities in some fields than others. the ability to form fixed-duration, ad-hoc associations to enable some largescale development are not only possible for a society comprising decentralised groups, but are considered to be essential. this conclusion differs from the assertion by jebari & jebari (2015) that "…isolated, self-sufficient, and continuously manned underground refuges…" are appropriate, rather proposing intentionally independent population units who control their specific interactions with other units, achieving reduced vulnerability but without forfeiting the capability to aggregate selected resources for mutual gain. smaller-scale concepts such a crowdsourcing approaches are already demonstrating somewhat similar options for shorter-duration, ad-hoc design advances. situations such as ships travelling in convoy are common and perhaps form a close analogy, demonstrating both the independence of each and the possibility of cooperation. this paper focussed on broad issues of technological vulnerability, but implementation details (resource usage efficiency, waste creation/treatment, gross greenhouse gas emissions and many other issues are also acknowledged. sociological/anthropological research (dunbar 2010) has indicated that quite small populations provide a sufficient sociological group size for most, and this conclusion seems to remain broadly valid even where high levels of electronic connection are possible (maccarron 2016). the concept of a technological system's exposure provides both a tool and a metric, which can be applied to either individuals or to populations of individuals, and can supply useful data to the forecasting process. population exposure is found to be high for many categories of current technological systems, and the current trend is to increased levels of population exposure. the categories that have been considered as typical, span and affect many goods and services that would be considered essential. items as diverse as internet usage and financial transactions already have multi-national levels of population exposure (and hence high levels of centralisation) and although various authors (diamond 2005) j o u r n a l p r e -p r o o f does not use the term "exposure" nor precisely describe the concept, he explains that environmental damage actually has a global exposure level. population exposure is a topic that does generate intuitive awareness of vulnerability: it can be observed that awareness of technological vulnerability exists at both national level (see j critical infrastructure protection, pub elsevier) and at individual level (slovic 2016 , martin 1996 , huddleston 2017 , kabel & chmidling 2014 , reiderer 2018 . since a measure of exposure correlates closely with the effort required to protect a system, a continued trend to centralisation and increased population exposure is very likely to lead to progressively herculean efforts to ensure that vulnerable loci are not attacked; such efforts are likely to include progressively wider surveillance to identify potential threats, and progressively stronger efforts to control and monitor access -each of which are themselves factors that can serve to make the service practically unavailable to individuals. if no value were attached to high and rising levels of population exposure, then highly centralised options are likely to continue to be preferred -but the consequences of the high and rising population exposure are demonstrated by reference to a metric of exposure, are also intuitively understood and are illustrated by the "continued centralisation" option in figure 3 . if there were indeed no technological options for reducing the level of population exposure without major reductions in the levels of technological sophistication available, then large populations are indeed exposed to the danger of losing access to services. that scenario would indeed result in a catastrophic situation where a major reduction in sophistication of services occurs, but the remnant j o u r n a l p r e -p r o o f exposure is also reduced. that scenario has been labelled as the "apocalyptic survivor" scenario in figure 3 . the third alternative shown in figure 3 , is for progress towards a significantly decentralised society, with a comparatively low population exposure and a level of sophistication (in terms of technological services and goods available) that does not decrease and actually has the real possibility of advancing with time. in this paper, the description of "forecastable options" has been considered under each of the categories of high population exposure, and it has been noted that for each of them technological developments that enable significantly reduced population exposure exist. the current level of technological maturity of these options vary, but none are infeasible and complement the proposals advanced by a number of authors including gillies (2015) and blühdorn (2017) . j o u r n a l p r e -p r o o f in the course of considering decentralisation options, this paper has identified a small number of technological capabilities that would facilitate a more decentralised option, but which are currently at a low level of technological maturity. these include:  further advances on general purpose chemical synthesis  a durable, open-source machine-accessible information storage system that can be created and read in a decentralised context (requiring only minor development)  an self-discovering network using an open-source approach to allow information transmission (requiring some development)  a large-capacity, durable and economically accessible energy storage technology (requiring significant development)  further development of trustworthy and decentralised financial transaction systems (requiring some development).  a non-technological system to allow ad-hoc cooperation between decentralised groups, allowing resource aggregation without long-term centralisation (requiring significant development). despite the inevitable variations in technological maturity across a broad range of technologies, the analyses have concluded firstly that centralisation and high population exposure result in severe and increasing vulnerabilities for large numbers of persons, and secondly that the combination of maturing decentralised technological capabilities and the storage of knowledge allows a transition to a "sophisticated decentralisation" model to be considered as a serious option. while not the primary topic of this paper, it is noted that even in the presence of technological options, change may not occur until some triggering event occurs; events that could trigger a more rapid transition to a "sophisticated decentralisation" could j o u r n a l p r e -p r o o f include a truly major and long-term disruption of some highly centralised technology such as undersea cables or an irremediable malfunction of international financial systems, or a pandemic requiring high levels of population isolation 1 . the analysis of technological exposure and reduced exposure options have concluded that practical options for substantial decentralisation exist, or could be reasonably forecast as possible. it has also been proposed that there are substantive and immediate reasons to consider a qualitatively distinct "fork" from the high population exposure of current centralised society to a more decentralised model. any selection of a decentralised technological model might be triggered by some event which crystallised the exposure of a highly centralised model: the drivers that had produced the centralised model will still remain however, and will arguably tend to cause a re-centralisation without ongoing efforts. this issue is outside the scope of this paper, but is noted as a topic requiring further research. the capability for decentralised and machine-accessible storage of knowledge and the creation of complex components and substances has recently, or will soon, create a cusp at which local equipment is capable of reproducing itself. sufficient computation facilities at local scale are already able to make genuine advances, and with equipment capable of replication, is sufficient to allow fully sustainable, sophisticated and decentralised communities to diverge from current trends. declarations of interest: none consumer preferences for household-level battery energy storage on some recent definitions and analysis frameworks for risk, vulnerability, and resilience 3d printing technique applied to rapid casting confronting future catastrophic threats to humanity dark ages ii: when the digital data die eco-political hopes beyond sustainability. global discourse existential risks: analyzing human extinction scenarios and related hazards calling dunbar's numbers colombian ancillary services and international connections: current weaknesses and policy challenges digital vellum macroalgae for biofuels production: progress and perspectives undersea cables and the future of submarine competition addressing the harm of total surveillance: a reply to professor neil richards mechanics of turn-milling operations the anything factory sustainable growth-an impossibility theorem the knowledge: how to rebuild civilization in the aftermath of a cataclysm black code: censorship, surveillance, and the militarisation of cyberspace collapse: how societies choose to fail or succeed cascading blackout overall structure and some implications for sampling and mitigation molecular engineering: an approach to the development of general capabilities for molecular manipulation how many friends does one person need?: dunbar's number and other evolutionary quirks intel's management engine is a security hazard, and users need a way to disable it hype and heavy tails: a closer look at data breaches how to hack a turned off computer, or running unsigned code in intel management engine the protocols kinematic self-replicating machines an innovative homemade instrument for the determination of doxylamine succinate based on the electrochemiluminescence of ru(bpy)2+ 3 paul mason's postcapitalism emerging technologies, catastrophic risks, and ethics: three strategies for reducing risk a critical review of cascading failure analysis and modelling of power system in: near-net shape manufacturing of miniature spur gears by wire spark erosion machining. materials forming, machining and tribology leontief based model of risk in complex interconnected infrastructures weaving the web: otlet's visualizations of a global information society and his concept of a universal civilization long-lost satellite tech is so old nasa can't read it. tech & science. nasa image. observer. nasa satellite lost for 13 years recovered by amateur astronomer when do states disconnect their digital networks? regime responses to the political uses of social media. the communication review conceptual design and dimensional synthesis for a 3-dof module of the trivariant-a novel 5-dof reconfigurable hybrid robot prepper" as resilient citizen. responses to disasters and climate change daraprim) drug's key ingredient recreated by high school students in sydney industrial automation systems and integration --physical device control --data model for computerized numerical controllers automation systems and integration -product data representation and exchange. it is known informally as "step existential risks: exploring a robust risk reduction strategy automatic fault protection in the voyager spacecraft disaster prepper: health, identity, and american survivalist culture eternal 5d data storage via ultrafast-laser writing in glass nasa tests limits of 3-d printing with powerful rocket engine check. nasa engineers prepare to hot-fire test a 3-d printed rocket part on test stand 115 at the marshall center recent progress on cascading failures and recovery in interdependent networks visible light spectral domain optical coherence microscopy system for ex vivo imaging longnow foundation total surveillance, big data, and predictive crime technology: privacy's perfect storm is your algorithm dangerous? solar pyrometallurgy-an historic review electronic payment systems. artech house organic synthesis: the robo-chemist. the race is on to build a machine that can synthesize any organic compound. it could transform chemistry permanent archival solution: nanoarchival provides ultra-long term archival solutions to enterprise customers, based on its proprietary a dangerous trend of cybercrime: ransomware growing challenge population dynamics with and without selection optical microscope 3d printing based on imaging data: review of medical applications doomsday goes mainstream high performance spiro ammonium electrolyte for electric double layer capacitors emerging threats: outer space, cyberspace, and undersea cables von neumann machine. encyclopedia of computer science. 4th. pages 1841-1842 monsters in cyberspace cyberphobia and cultural panic in the information age. information applied cryptography, second edition: protocols, algorthms, and source code in c, 20th anniversary edition isbn information. online isbn: 9781119183471. print isbn a new rationale for returning to the moon? protecting civilization with a sanctuary a thesis submitted to faculty of graduate studies and research, mcgill university in partial fulfilment of the requirements of the degree of the perception of risk energy and civilization; a history hulking transformers prove a challenge for vulnerable grid. e&e 2015-11-24 and see energy news digest for blockchain: blueprint for a new economy censorship and surveillance in the digital age: the technological challenges for academics the designer's guide to disk drives the potential impact of quantum computers on society. ethics and information technology high-yield bio-oil production from macroalgae (saccharina japonica) in supercritical ethanol and its combustion behaviour key: cord-349831-0u9y35qo authors: ellis, george f. r. title: the causal closure of physics in real world contexts date: 2020-08-18 journal: found phys doi: 10.1007/s10701-020-00366-0 sha: doc_id: 349831 cord_uid: 0u9y35qo the causal closure of physics is usually discussed in a context free way. here i discuss it in the context of engineering systems and biology, where strong emergence takes place due to a combination of upwards emergence and downwards causation (ellis, emergence in solid state physics and biology, 2020, arxiv:2004.13591). firstly, i show that causal closure is strictly limited in terms of spatial interactions because these are cases that are of necessity strongly interacting with the environment. effective spatial closure holds ceteris parabus, and can be violated by black swan events. secondly, i show that causal closure in the hierarchy of emergence is a strictly interlevel affair, and in the cases of engineering and biology encompasses all levels from the social level to the particle physics level. however effective causal closure can usefully be defined for a restricted set of levels, and one can experimentally determine effective theories that hold at each level. this does not however imply those effective theories are causally complete by themselves. in particular, the particle physics level is not causally complete by itself in the contexts of solid state physics (because of interlevel wave–particle duality), digital computers (where algorithms determine outcomes), or biology (because of time dependent constraints). furthermore inextricably intertwined levels occur in all these contexts. it is often supposed that causal closure occurs at the micro level in physical systems, and hence prevents the occurrence of strong emergence because the macrostate supervenes on the microstate [81, 82] . this is discussed in [27, 68, 89, 54] . in contrast, [20] shows by careful philosophical argument that one can have emergence with reduction, as well as without it; and emergence without supervenience, as well as with it. here i want to examine the issue in a different way, by dealing in some detail with the hierarchical nature of emergence in real world contexts: the cases of engineering, based in the underlying solid state physics, and biology, based in the underlying molecular biology, in turn based in the underlying physics. the context is my paper [41] that establishes that strong emergence does indeed take place in both those cases, so that the argument against strong emergence has to be wrong in those contexts. reference [41] examines the issue of strong emergence of properties p of macrodynamics m out of the underlying microdynamics m in the context of condensed matter physics and biology. following anderson's lead [4, 5] that symmetry breaking is at the heart of emergence, its method was to identify five different kinds of symmetry breaking occurring in different contexts ( [41] , sect. 2.3] and then to trace how broken symmetry states at the macro and micro levels interact with each other. • the microscale dynamics m, based in the laws of physics l, obeys symmetries s: s(m)=m. spontaneous symmetry breaking ssb(m) leads to symmetry breaking of the macro scale dynamics m through the emergence process e whereby m emerges from the microscale dynamics m. this is weak emergence ∶ → ∶ ( ) ≠ . • this spontaneously broken macro state m reaches down to create quasiparticles such as phonons at the micro level, which play a key dynamical role at that level. 1 the base microdynamics m is altered to produce an effective microdynamics m ′ which breaks the symmetry of the underlying physical laws l. thus → � ∶ ( � ) ≠ . • to derive correctly the properties of macro dynamics m from the micro dynamical level, you must coarse grain the effective theory m ′ rather than m. • thus strong emergence takes place in this case: you cannot even in principle derive the macrodynamics m from the microdynamics m in a strictly bottom up way, because m satisfies the symmetry s and m does not. 2 as a consequence [41] : sect. 4.4, in the case of solid state physics, the underlying microphysics m cannot be causally complete, because by itself it cannot lead to the emergence of known properties of solids such as electrical conductivity. the lower level physics only gives the correct outcome when modified by inclusion of terms a(m) arising from the higher level state m (so ( ) ≠ ). the same is true for living systems. that is, in both these cases, causal completeness is only attained by considering both the low level properties m and the higher level properties m (which lead to the alteration → ′ ) together. the real causally closed system comprises both those levels. the aim of this paper is to extend that result by investigating causal closure of physics in terms of determining dynamic properties 3 p(d) of entities in engineering and biological contexts (i use the word closure rather than completeness for reasons that become apparent below as the theme develops). a separate very interesting project would extend this to considering the dynamic emergence of entities e(d) over time, see e.g. [24] . section 2 sets the context for the discussion, which is the hierarchies of emergence in the cases of engineering and the life sciences respectively (table 1) . it introduce the idea of an effective theory at each level l, and discusses bottom up and top down causation in the hierarchy of emergence (sect. 2.3). section 3 introduces the idea of a domain of interest (doi), and the concept of effective spatial closure (sect. 3.3) . it is shown that in terms of spatial interactions, causal closure in engineering and biology only holds ceteris parabus. yes of course philosophers know that this is the case; the point is that it has real consequences in real world contexts. section 4 defines levels of interest (loi) and sect. 4.4 introduces the need for restricted domains of interest. section 5 introduces interlevel causal closure in the case of biology (sect. 5.3), sect. 6 extends this to the case of digital computers and physics. section 7 summarizes the main results of this paper, emphasizes that unavoidable unpredictability also undermines causal closure, and comments on ways people ignore the issues raised in this paper. the novel concepts introduced are effective theories the context of the discussion is the hierarchy of emergence. as stated by [4] , at each level of complexity, entirely new properties appear ... at each stage entirely new laws, concepts, and generalizations are necessary. the emergent hierarchy is shown in table 1 for the cases of engineering on the left and life sciences, in particular the case of humanity, on the right. the left hand side represent the sciences of the artificial as discussed by [126] . the right hand side represents the structures and processes of biology, as discussed by [23] . this hierarchy has important aspects. • the bottom three levels l1-l3 are the same on both sides. this is one of the great discoveries science has made: inanimate matter and living matter are made of the same stuff at the bottom. electrons are at level l3, interacting with the nucleus. • the atomic level l3 is where new properties emerge out of the underlying physics, as characterised by the periodic table of the elements, another great discovery. • the components enabling complexity to arise occur at level l4. both semiconductors and metals are crystals, and they are the key components of machines. 4 solid state physics covers levels l3-l4. biomolecules such as dna, rna, and proteins are the foundations of biological emergence. • level l5 is where the basic units of complexity arise, showing functional emergent properties, and being the basis for building complex entities. on the machines side, these are devices such as transistors, light emitting diodes, photodetectors, and lasers. on the life sciences side, they are cells: the basic building block of life, which come in many different types. this is the lowest level where the processes of life occur, entailing metabolism and information processing hartwell et al. [61] . • level l6 is where on the machine side, devices are integrated into functional units, such as the central processing unit in a microprocessor, which is itself a device. on the life sciences side, organs comprising physiological systems occur. • level l7 is where functional units occur that have an integrity of their own: they are effectively causally closed systems imbedded in a larger environment. on the engineering side, they are machines built to carry out some purpose, such as aircraft or digital computers or particle colliders. on the life sciences side, they are individuals with autonomy of action. • level l8 is the same on both sides. both machines and individual human beings exist in the context of a society with social, economic, and political aspects, which sets the stage for their existence and functioning. • finally, level l9 is again the same on both sides. it reflects the fact that each society exists in a natural environment with both ecological and geophysical aspects. note that this table has chosen a particular set of levels to represent causation all the way from the particle physics level l1 to the environmental level l9. however most scientific studies will be interested in a much more restricted sets of levels: the levels of interest (lois) discussed in sect. 4. given such a choice, one will in general use a more fine-grained set of levels than represented in table 1 . thus for example if the loi is (l4-l6), one might divide that range into a finer set of sublevels. it is a common belief that the lower levels are more real than the higher levels, because bottom up causation from the lower to higher levels is the source of higher level properties. arthur eddington in on the nature of the physical world ( [34] , pp. 5-12) muses on the dual (solid macroscopic/atomic microscopic) nature of his writing desk, and concludes (p. 10) that because of the scientific world view, the external world of physics has thus become a world of shadows. in removing our illusions we have removed the substance, for indeed we have seen that substance is one of our great illusions. however this view is subject to dispute. richard feynman in his book the character of physical law (50] , pp. 125-126) considers whether one level or another is more fundamental, and using a religious metaphor, argues that 'the fundamental laws are no nearer to god than emergent laws'. 5 phil anderson arguably had a similar view. sylvan schweber commented as follows [124] : anderson believes in emergent laws. he holds the view that each level has its own "fundamental" laws and its own ontology. translated into the language of particle physicists, anderson would say each level has its effective lagrangian and its set of quasistable particles. in each level the effective lagrangian -the "fundamental" description at that level -is the best we can do. thus this does not recognize any level as more fundamental than any other. recently, denis noble has proposed a "principle of biological relativity" [105] : all levels one deals with in studying emergence in biology are equally valid, there is no privileged level of causation. effective theories a good way to express this is that there is a valid effective theory 6 (et) at each level. elena castellani gives this definition [25] : an effective theory (et) is a theory which 'effectively' captures what is physically relevant in a given domain, where 'theory' is a set of fundamental equations (or simply some lagrangian) for describing some entities, their behaviour and interactions... more precisely, an et is an appropriate description of the important (relevant) physics in a given region of the parameter space of the physical world. in parallel to the way the functioning of the laws of physics was sketched in [41] , one can characterise an effective theory valid at some level l as follows; an effective theory at a level l is a reliable relation between initial conditions described by effective variables v ∈ and outcomes o ∈ : in a reliable way, whether is an exact or statistical law. it is important to note that an effective theory may have a randomisation element r: where r might for example produce a gaussian distribution. equal causal validity in terms of effective theories for emergent properties p(d), noble's principle [105] as extended in [41] can be restated: table 1 ) represents an effective theory , so each level is equally valid in a causal sense. this implies no level is a fundamental level with priority over the others, and particularly there is not a primary one at the bottom level. this is just as well, because there is no well-established bottom-most physical level to which physics can be reduced [102] . every emergent level equally represents an effective theory. 7 equality of validity of effective theories at every level is possible because causation is not just bottom-up. rather higher level properties p(d) are linked to lower levels by a combination of upwards and downwards causation [39, 41, 105] , which enables emergence of effective laws at each level. upwards emergence this has two different aspects ( [41] , §1.1). 7 while luu and meißner are critical of my claims on emergence [87] , they agree on this point. 6 not to be confused with an effective field theory (eft), see [19, 25, 60] , which is a special case of an et. note that efts such as in [87] cannot deal with emergence in solid state physics, as they do not allow for symmetry breaking. first there is the emergence e of a macro system from its components. in terms of levels, this corresponds to creation of a higher level ln from a lower level ln: that is, → , > . this may lead to topological non-trivial states emerging such as networks, or quantum entanglement may take place. the issue of phase transitions is important here. first order phase transitions occur when spontaneous symmetry breaking ssb occurs leading to the emergent level et having lower symmetries than the underlying et. in terms of the associated micro dynamics m and macrodynamics m, if s is the symmetry set of m, then second there is emergence p of properties of the emergent level ln out of properties of the underlying constituent level ln once ln has come into existence. this corresponds to emergence of a higher level out of a lower level one. some form of coarse graining c of properties may suffice if the higher and lower levels have the same symmetries s, but not if their symmetries are different due to ssb (see [41] ). downward causation a classification of different types of downward causation was given in [37, 39] . here i will rather approach the issue from an viewpoint. there are essentially two kinds of downwards effects that can happen: downward alteration of lower level dynamics l via either constraints or effective potentials, and downward alteration of dynamics at level l by altering the set of lower level variables. constraints and effective potentials the way downward causation by constraints works is that the outcomes p(d) at level l depend on constraints c at the level l arising from conditions at a level of influence . thus when interlevel interactions are taken into account, relation (1) is modified (see [41, (30) essentially the same holds if the effect of the level of influence li on the level l is expressed in terms of an effective potential v(v ) at level l (see [41, (9) ]). thus constraints act as causes [76] . the constraints c may be time independent: c ∕ t = 0 in which case they are structural constraints; or they may be time dependent: c = c (t), c ∕ t ≠ 0, in which case they are signalling or controlling constraints. an important case is feedback control (engineering), which is essentially the same as homeostasis (biology). then the constraints c (t) depend on goals g valid at level l but set at the level of influence li. similarly the potential v(v ) may depend on time-dependent variin both cases the level l is no longer causally complete on its own; at a minimum, only the combination {l,{li} of levels can be causally complete. altered variables the causal effect due to the level of influence li may rather be due to changes in the variables v at level l due to variables v at the higher level li: where the new set {v � } of effective variables at level l may be smaller, larger, or altered. they are smaller if they are changed by deleting lower level elements. this occurs when downward causation by adaptive selection takes place, altering or deleting selected lower level elements according to some selection criterion c. this enables alteration of structures and functions at level l so as to meet new challenges at level li. this plays an important role in enabling by organisms to have agency and choice, enabled by stochasticity, as explained in [106] : choice in the behavior of organisms involves novelty, which may be unpredictable. yet in retrospect, we can usually provide a rationale for the choice. a deterministic view of life cannot explain this. the solution to this paradox is that organisms can harness stochasticity through which they can generate many possible solutions to environmental challenges. they must then employ a comparator to find the solution that fits the challenge. what therefore is unpredictable in prospect can become comprehensible in retrospect. harnessing stochastic and/or chaotic processes is essential to the ability of organisms to have agency and to make choices. they are larger if for example one has downward creation of quasiparticles such as phonons via interlevel wave-particle duality ( [41] and sect. 6.3), which underlies the properties of metals and semi-conductors. this is what carl gillett calls a "foundational determinative relation" [55] . the are altered if the number is the same but the properties of an element changes. when they are bound in an emergent complex their own properties may change (for example, neutrons decay in 11 min when free but last for billions of years when bound in a nucleus), or their interactions with external entities may change (for example electrons bound in an atom interact with light quite differently than a free electron does). downward causation is related to aristotle's formal cause, see [130] , but i will not follow that strand here. to give these rather abstract statements flesh, see many examples given in [39, 104] . downward causation in relation to the key physics-chemistry link is discussed in [86] . multiple realisability of higher level variables at lower levels plays a key role in downward causation [93] . any particular higher level state can be realised in a multiplicity of ways in terms of lower level states. in an engineering or biological cases, a high level need determines the high level effective function that needs to be realised and thus the high level structure that fulfills it. this higher structure and function is then realised by suitable lower level structures and functions, but there are billions of ways this can happen it does not matter which of the equivalence class of lower level realisations is used to fulfill the higher level need, as long as it is indeed fulfilled. consequently you cannot even express the dynamics driving what is happening in a sensible way at a lower level. the issue is not just the huge number lower level entities involved in realising a higher level systems, as characterised by avagadro's number it is the huge different numbers of ways combinations of lower level entities can represent a single higher level variable. any one of the entire equivalence class at the lower level will do. thus it is not the individual variables at the lower level that are the key to what is going on: it is the equivalence class to which they belong. but that whole equivalence class can be describer by a single variable at the macro level, so that is the real effective variable in the dynamics that is going on. this is a kind of interlevel duality: where e − (v − ) is the equivalence class of variables v − at level − corresponding to the one variable v at level l. the effective law at level l for the (possibly vectorial or matrix) variables v at that level is equivalent to a law for an entire equivalence class e − (v − ) of variables at level l-1. it does not translate into an effective law for natural variables v − per se at level l-1. it is important to note the following: one establishes the validity of an for some chosen level l by doing experiments or making observations on phenomena occurring at that level. this involves the experimenter intervening at the level l, hence it is an interlevel interaction. for example a particle physics experiment considers effective laws at level l1 but involves scientists at level l7 and organisations at level l8 acting down to affect things at level l1. consequently, one can make the following important observation: existence and functioning of effective theories at level l does not necessarily imply causal closure of level l. the issue is what determines constraints c, potentials v, and effective variables v ′ that may occur at that level. they may be influenced by other levels. that is what sects. 5 and 6 are about. determining an effective law at level l involves other levels then l. looking in more detail at the hierarchy, it is a hierarchy made of modules (this section) which form networks (next section). it is a modular hierarchy for very good reasons. five principles of complex structure ([16] , §1.3) gives five principles of complex structure, developing from [126] , starting from the idea the role of decomposition: the technique of mastering complexity has been known since ancient times: divide et impera (divide and rule). the five principles, applicable to both engineering and biology, are stated by him to be, (1) modules modularity is the property of a system that has been decomposed into a set of cohesive and loosely coupled modules ( [16] , p. 56). they can be represented by abstractions, where "an abstraction denotes the essential characteristics of an object that distinguish it from all other kinds of objects and thus provide crisply defined conceptual boundaries, relative to the perspective of the viewer. an abstraction focuses on the outside view of an object and so serves to separate an object's essential behavior from its implementation" ( [16] , pp. [44] [45] [46] [47] [48] [49] [50] . they involve encapsulation ( [16] , pp. [50] [51] [52] [53] , that is, the internal details of the module's workings are hidden from the external world), and multiple realisability: the required module functioning can be fulfilled in many ways by its internal structure and variables. hierarchy is a ranking or ordering of abstractions ( [16] , p. 58). a feature of particular interest is that emergent systems may give rise to effective theories that involve topological constraints. indeed this happens quite often because emergent complexity in both engineering and biology often involves interaction networks, and a key feature of such networks is their topological connectivity, described by graph theory. thus for example arthur peacocke points out that in electrical circuit theory there are certain topological constraints, the boundary conditions that one element imposes on another ( [112] , p. 74). they obviously have strongly emergent properties: their functioning does not follow from any local characteristics of the elements that make up the circuit. the electric light won't work until you change its open circuit topology (isomorphic to an open interval) when the switch is off, to a closed topology (isomorphic to a circle) when the switch is on. this macro event than reaches done to alter the flow of billions of electrons at the micro level. networks can be physical networks, or interaction networks. physical networks physical networks are embodied in physical links between nodes, which constrain what interactions can take place by dictating what nodes can interact with what other nodes. thus physical networks in fact create interaction networks by constraining interactions between links. this is the key structure-function relationship of engineering and biology. examples in engineering are computer architecture [132] , computer networks [83] , and artificial neural networks [73] . the case of importance in biology is the nervous system [59] and neural networks [63] . interaction networks interaction networks occur due to the presence of a variety of reagents that selectively interact with each other. this requires firstly a container that keeps the reagents within interaction distance of each other, rather than just diffusing away, and second the presence of an appropriate set of reagent that do indeed interact with each other. a key role is then played by selectively letting specific reagents enter or exit the container so as to control their interaction densities. on a large scale, examples of importance in engineering are purification plants, chemical engineering reaction vessels, water treatment plants, sewage treatment plants. in biology, they arguably are the endocrine system, controlling signalling, and the digestive system, controlling metabolism at the systems level [59] , and on a larger scale, ecological networks [75] . on a small scale, there are many interaction networks in cell biology [18] . these are crucially dependent on the existence of cells bounded by cell walls, that serve as the necessary reaction containers. they have ion channels imbedded in those walls that control movement of ions in and out of the cells, and molecular channels controlling movement of molecules in and out. they include • gene regulatory networks [24, 75, 139] , also known as transcription networks [3] , • metabolic networks [18, 75, 139] , • cell signalling networks [11, 18] , • protein interaction networks [18, 75] , • signal transduction networks [18, 75] . these networks are the heart of cell biology [85] and underlie how information flows and logic underlie biological functioning as emphasized by [30, 45, 109] . networks and hierarchy networks may have a hierarchical character in that subnetworks can often be identified within an overall network, and so define levels within the network [111, 121, 142] . this is an interesting topic i will not develop further here except to remark that firstly, subnetworks include network motifs [3, 94] , which are small subnetworks of particular functional significance. for example they include the autoregulation motif, which is nothing other than feedback control ( [3] , pp. [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] and the feed-forward loop motif ( [3] , pp. . they may contain higher-dimensional interactions characterised by clique complexes [118] . networks may also contain hubs, central nodes of importance [75] . their nature is highly dynamic [32] . causation because interaction networks are directed graphs (i.e. the edges between nodes have orientations), they represent causal effects, where causation is defined as [113, 114] . reference [67] shows how such diagrams can be used to exemplify causal entailment in a diverse range of processes: enzyme action, construction of automata, and ribosomal polypeptide synthesis through the genetic code. their causal effects can be tested by experiment, where this is possible (vary conditions at one node and show that, ceteris parabus (i.e. conditions at other links to the node are unchanged) this results in a reliable change at another node. when this is not possible, one can use counterfactual arguments: demonstrating that as a result of the nature of the network links this should indeed be the outcome if one were to make such a change. this is the kind of argument i will use to claim that both upward and downward relations between levels are also causal ( table 3) . networks and strong emergence because of their systemic properties, biochemical networks display strong emergence [17] . in examining the issue of causal closure of properties p, one must have the context clearly in mind. to do so, it is useful to define the domain of interest (doi) of such study. this has three quite different aspects. first, there will a specific topic of interest (toi) one wishes to investigate. for example, it might be physics or engineering or chemistry or biology. in physics, one might have in mind atomic physics or condensed matter physics or plasma physics; in biology, molecular biology or physiology or neuroscience or population evolution. or one might want to investigate relations between various of these topics. in this paper, the interest is the nature of causal closure in the relation between physics, engineering, and biology. second, given a choice of topic of interest, the domain of interest doi of a system of interest s consists firstly of interaction limits for s with its surroundings, and secondly of time limits on the duration when we are interested in the behaviour of s. together these comprise spacetime limitations (sect. 3.1), leading to effectively isolated systems in the case of physics (sect. 3.2) and effective spatial closure in the case of biology and engineering (sect. 3.3). thirdly, there will be a choice of levels of interest (loi). the issue of lois is the focus of this paper, and is discussed in the following sect. 4. to be of physical interest, s must be spatially limited. although they are often talked about, systems of infinite extent do not occur in the real universe [46] . 8 spacetime boundaries define the time and spatial domain we are interested in in relation to s. from a spacetime viewpoint, this is a world tube of finite radius r that surrounds s, large enough to contain s and all the elements strongly interacting with it, bounded by an initial time t i and final time t f defined in a suitable way. this governs the kinds of interactions it can have with other systems. time limitations we may be interested in short or long timescales characterised by the starting time t i and ending time t f , depending on what we wish to study. we may be interested in, • evolutionary processes e whereby the family of systems of similar type to s came into existence over long timescales via reproduction with variation followed by selection; • developmental processes e whereby a specific system s came into existence through developmental or manufacturing processes, or perhaps by self assembly; • functional processes whereby the properties p of the system s considered over short timescales emerge from the underlying physics. this is the focus of this paper. each involves very different choices of the timescale δt ∶= t f − t i relevant to our study. isolated systems causal closure of a system s cannot happen if uncontrolled influences arrive from the surroundings: as these influences vary with time, they will cause changes in the the state of the system with time that cannot be predicted from a knowledge of the properties of the system alone. the system is not causally closed. physics deals with this by introducing the idea of an isolated system. this is usually expressed by giving limits on any incoming influences "at infinity", for example such conditions are imposed in studying electromagnetic and gravitational radiation. however, as just stated, infinity is not a valid physics concept. one should instead refer to finite infinity i [36] : a world tube of finite radius r i ≫ r chosen so that incoming radiation and matter will not seriously interfere with s. 9 the dynamics of the system will then be autonomous except for small perturbations due to incoming matter and radiation crossing i, which can be treated as small effects. the dynamics of s can be treated as an autonomous system, affected by small incoming perturbations over i. however, there are two problems with this idea: one to do with physics, and one to do with engineering and biology. causal domains the first is that famously, in general relativity, causal domains are determined by null cones rather than timelike tubes [62] . why have i not defined the causal limits, which are basic in term of causal closure, in terms of null surfaces rather than a timelike world tube? the answer is that on astronomical scales, effective causal limits are indeed given by timelike world tubes rather than null surfaces. on astronomical scales at recent times, the dynamic effects of radiation are very small compared with those of matter. we get a very usable i by choosing r i to be about 1 megaparsec in comoving coordinates [47] . nothing outside there has had a significant effect on the history of our galaxy or the solar system. yes some radiation and matter is coming in, but it is negligible compared to the energies involved in daily life. the one form of radiation of significance for the world is light from the sun, which comes from well within those limits: 1 astronomical unit might indeed suffice for local physics. the radiative energy coming from greater distances has negligible dynamical effects on earth. a timelike world tube of radius 1 au will do just fine in terms of considering causal closure of the solar system. isolated systems: laboratories however physics practice works in a different way: the key concept is an isolated system in a laboratory. it's a system that is in fact interacting strongly with the environment (sect. 3.3), but that interaction is strictly controlled so that it is highly predictable. the system is shielded from influences outside the laboratory as far as possible. this then enables the results of experiments to also be highly predictable. and that is what enables the determination of the effective theories that hold at a level l. examples are the expensive isolation and cooling systems underlying the success of quantum optics experiments. engineering and biology as open systems the real problem is different. it is that no biological system can be closed: they have by their nature to be open systems. and the same is true for engineering systems. in these cases, the 'isolated system' paradigm is simply wrong. life cannot exist as an isolated system. biological systems are inherently open systems interacting with the environment [65, 112, pp. 10-11] ) states, biological organisations can only maintain themselves in existence if there is a flow of energy and this flow requires that the system not be in equilibrium and therefore spatially inhomogeneous. 10 the effect of the outside world is not negligible. on the contrary, it is essential to biological functioning. it cannot be treated as a perturbation. the biosphere experiences incoming high grade radiation from the sun and radiates outgoing low grade heat to the dark sky, and this is its energy source enabling it to function thermodynamically [115] . organisms need a flow of material in, and, because of the second law of thermodynamics (essentially: as time progresses, matter and energy will be transformed from usable to unusable forms), need to dispose of waste matter and heat resulting from internal non-equilibrium metabolic processes. a living system s must take in materials and energy from the surrounding environment e and dispose of waste matter and energy to e: in summary, living systems are essentially interacting systems. the same is true for engineering systems, because they do work of some kind. reliable interactions they must therefore interact strongly with an environment that is stable enough that the interactions with the environment are reliable and reasonably constant so they do not disrupt the dynamics of the system over time. in physics this is the concept of a heath bath or thermal reservoir. you are in contact with an environment but don't need to take its dynamics into account because it is in a state usually assumed to be static, characterised only by a constant temperature t; and it is so large that the system s has negligible influence on it state. 11 life: interaction limits how then does one limit those interactions to those that will enable life to sustain itself? humberto maturana and francisco varela essentially dealt with this by introducing the idea of autopoiesis [91] , which inter alia expresses the idea of causal closure in terms of system interaction with its environment. the key point is how does one define the boundary of a system in this context. instead of choosing a spacetime tube of some chosen radius r as in the astronomy case, one chooses a system boundary b that characterizes the system as being effectively autonomous. this underlies the meaning of level l8 in the hierarchy of emergence (table 1 , sect. 2.1). a person has a skin that is her physical boundary with the outside world, but still allows interaction with it. energy and matter transfer takes place across the boundary. a machine similarly has a boundary that defines its limits, but will have some form of energy input enabling it to do work, and in many cases complex cooling devices to get waste heat out (paradoxically, they may consume large amounts of energy). but this idea extends down to other levels, for example it holds also at the device level/cellular level l5 and the component level/organ level l6. for example, a cell is the core of biology. it exists as an entity with its own integrity, characterised by the cell wall which allows controlled ingress and egress of materials and energy, yet interacts strongly with the environment. how then does life handle functioning in this context? the environment must be sufficiently stable so as to allow effective predictability. this is the case when the system is not causally closed but has predictable interactions with the environment that makes its own functions predictable. it has an environment that can be treated as predictable up to perturbations. the environment may change with time, but if so, slowly enough to allow adaptation to the changing situation. this is often the case, and is what on the one hand allows living systems to flourish, and on the other allows biology to be a genuine scientific subject [23] . the exceptions but that is not always the case: take covid-19 as an example. that started at the social level (l9) in one house, then spread worldwide via aircraft affecting life world wide and thereby affecting in a downward way all the biomolecules (l4) and electrons (l1) in the bodies of doctors and nurses and patients effected. but additionally it affected the engineering side by closing down thousands of flights across the world, thereby reaching down to affect all the billions of atoms (l3) and particles (l1) that make up those aircraft: a classic example of the interlevel causation i turn to next. as far as predictability is concerned, the system s has reliable interactions with the environment and so is predictable most of the time, except when we this is not the case. predictability holds when all things are equal, but there is no guarantee this will be the situation. and that is the best we can do. 12 elaborating, investopaedia states this concept as follows: 12 and no this could not be predicted in principle in a strictly bottom up way if we knew the detailed positions and momenta of all the particles in our causal past to utmost precision, for multiple reasons [41, 42] . these are strongly emergent phenomena, unpredictable even in principle. ceteris paribus is a latin phrase that generally means 'all other things being equal.' in economics, it acts as a shorthand indication of the effect one economic variable has on another, provided all other variables remain the same. ... ceteris paribus assumptions help transform an otherwise deductive social science into a methodologically positive 'hard' science. it creates an imaginary system of rules and conditions from which economists can pursue a specific end. put another way; it helps the economist circumvent human nature and the problems of limited knowledge'. in other words, effective spatial closure works except when it doesn't. unpredictability happens when black swan events take place, possible when there is a fattailed rather than a gaussian distribution [131] , and with major significance at the macro level. and when it doesn't work, the effects chain all the way down from level l8 to the atomic level l3 and particle level l1 in table 1 , as in the case of the coronavirus pandemic. setting this aside, the key conceptual issue i will deal with in this paper is the relation of the causal closure of emergent properties p to levels in the hierarchy of emergence. effective theories and existence of levels is discussed in sect (sect. 2.2) is a set of variables and equations representing interactions and constraints at a particular level l, such that initial data implies a reliable outcome at that level. it is the possibility of existence of an effective theory (1) at each level l (the dynamics at that level is determined at that level) that underlies the very concept of levels in the first place. as commented by peacocke ([112] , p. 10), following from [126] natural hierarchies are more often than not 'nearly decomposable' -that is, the interactions among the sub-systems (the 'parts') are relatively weak compared withe the interactions among the subsystems, a property which simplifies there behaviour and description. the fact that such levels exist is a consequence of the nature of the underlying physical laws and the values of the constants of nature [136] . it allows the existence of the modular hierarchical structures that are the core foundation of complexity (sects. 2.1, 2.6). reference [126] defines a hierarchical system as follows, quoted in ( [112] , p. 249): 'a hierarchical system: a system of composed of inter-related subsystems, each of the latter being in turn, hierarchical in structure until we reach some lowest level of elementary subsystem (the choice of this lowest level he regarded as arbitrary). this is what i have taken for granted above. while it has all the levels shown in table 1 (sect. 2.1), we usually do not want to consider them all at once. reference [15] says it thus: thus we take it as a given that when a portion of the universe is selected for study, be it a gas or a galaxy, we are allowed to blissfully ignore what is going on at scales that are much larger, or indeed much smaller, than the one we are considering. that is what i am formalizing by defining the concept of levels of interest (loi). they are defined as follows: a loi is defined by its top level tl and its bottom level bl, thus where the union sign " ∪ " means include all levels between tl and bl. some studies are unilevel: tl = bl, and some are explicitly interlevel: > • one can validly define such a loi regardless of what levels you choose, because the levels are equally causally valid (sect. 2.2). • your choice will depend on your topic of interest (toi) (see sect. 3). it is helpful if the choice of levels is made explicit, e.g. loi(3-5) covers levels 3 to 5. • then one can for the purposes of studying the dynamics at those levels legitimately ignore higher and lower levels, in the sense to be explored below. • this is possible because the levels effectively decouple in the sense of allowing valid effective theories at each level l. • however this does not give you the right to deny the validity of levels that lie outside your levels of interest. • there is no guarantee that causal completion will occur by including only those levels characterised by your choice of loi. that is the topic of interlevel causal closure, which i discuss below (sect. 5). are there limits on the lois one can choose? one can choose to investigate any desired lois, not investigating or take for granted the interactions that will inevitably occur from higher and lower levels. this is done to establish s. for example noble [103] "modeling the heart-from genes to cells to the whole organ" chooses to investigate the range of levels stated, namely levels l5 to l8 in the hierarchy of emergence (table 1) , and not for example on the one hand the interactions between protons and electrons that make this possible, and on the other hand the mental and social influences that will inevitably be having an effect on how the heart is functioning. what he does do is investigate the interlevel relations within the levels loi(5-8) he has chosen, because that is the domain of physiology. other examples are shown in table 2 . physics right down to level l1 always underlies what is happening, even if it lies outside the levels of interest to you. what happens in the real world right down to the physics levels l1-l3 is always influenced by what happens at higher levels including l9, even if that is not what interests you in your particular studies. what is a sensible doi depends on conditions. the suitable choice of lois will follow. this is the context within which "causal completeness of physics" must be evaluated. strongly interacting levels there is a basic problem with lois, however. that is the fact that, as mentioned above, every level interacts with every other level! the choices one makes relates inter alia as to whether one wants to answer how questions, why questions, or both (sect. 4.5). there is a practical way out that i discuss in sect. 5.3 by introducing the idea of effective causal closure. in order to examine the emergence of machines on the engineering side and organisms on the biology side, all one needs is newton's laws of motion, maxwell's equations, galilean gravity, and maybe the schrödinger equation ( [84] , §4.1); in [41] . that is, level l3 is an adequate base. all engineering and biology emerges from this level, it is the lowest level engineers and biologists need to study. as explained in [41] and sect. 2.3, conversely engineering and biology reach down to shape outcomes at level l3 via time dependent potentials or constraints. however levels l3 and l2 emerge from the quantum field theory and the standard model of particle physics at level l1 [110] . thus l1 is a deeper foundation of emergence. however it is essentially decoupled from everyday life [4, 84] . nevertheless outcomes at level l1 too must also be shaped in a downward way by engineering and biological variables, via outcomes at level l3. but this is not the bottom. underlying l1 is some theory of fundamental physics at level l0, a "theory of everything" (toe) [141] : maybe string theory/m theory, maybe not. there are variety of competing theories on offer [102] . this is the ultimate source of the emergence of dynamic properties p(d) in engineering and biology (using the notation of ( [41] , §2.2). it affects us every day. these lie outside the lois of engineers and biologists. that is just as well, as we do not know what the answer is at level l0, even though it underlies all physical emergence. as explained above, it suffices to deal with effective theories that hold at higher levels. at the top: cosmology and existence e consider isaac newton seeing an apple drop. this occurs for a variety of reasons: the law of gravity acting on the apple, the light rays that convey this image to his retina, the analysis of the image by his brain, and so on. but there are far deeper underlying issues. why does the apple exist? why does the earth exist? why does the solar system and the galaxy exist? why does the universe exist and have the nature it does? these are all the background reasons why the apple fell, and why isaac newton existed for that matter. these questions of how everything came into being (e) is the domain of cosmology [117] . it deals with issues such as, where do elements such as helium and carbon come from? how did the galaxy, the sun, and the earth arise? the philosophy of cosmology [38] considers issues such as why is physics of such a nature as to allow life to exist? reference [128] . particularly: why are the constants of nature [136] of such a character as to allow the hierarchical structure in table 1 to emerge? thus cosmology affects us every day by underlying our existence. everyday effects of cosmology p there are more immediate issues as well, in the relation of cosmology to everyday life [36] : why is the sky dark at night, serving as a heat sink for the earth's waste energy? this is crucial to the functioning of the biosphere ( [115] , pp. 411-417). why is there an arrow of time? references [29, 44, 115] ? this is crucial to all macro level physics, biology, and chemistry. both are due to the cosmological context. thus cosmology affects the emergence of properties p in engineering and biology today. the point then is that while it does indeed have a major causal effect on daily life [125] , this is a rock solid relation that does not change with time. it is a fixed unchanging background that does not alter effective laws as time passes. it is thus not a case of ceteris parabus 13 (c.f. sect. 3.3) and so can be taken for granted and not considered further when investigating causal closure in engineering, biology, and physics. an issue in choosing lois is if one is interested in how questions or why questions. how questions consider physical interactions on the one hand, and mechanisms on the other. thus they will relate to levels l1-l7 in table 1 , including l7 because that relates to the integration of systems to produce the organism as a who [120, 122] . why questions relate to motivation, meaning, and philosophical issues. thus they will relate to levels l7-l8 in table 1 , including l7 because this is the level where as well as philosophy, psychology and motivation come in [33, 79] and l8 where the causal power of social structures enters [35] . there is of course a trend for some strong reductionists to deny that the why questions are valid or have any real meaning. from the viewpoint of this paper, that simply means that they themselves have a restricted set of lois that excludes those higher levels. because of the equal validity of levels espoused in [41, 105] , and in this paper, that restricted set of interests does not provide a justification for denying the validity of the levels with effective theories outside their particular set of interests. the crucial concept in this paper is that of interlevel causal closure of properties p(d). i first consider causation and causal closure (sect. 5.1) and the nature of biology (sect. 5.2). then i consider interlevel causal closure in the case of biology (sect. 5.3), because this is where it is clearest and has been discussed most. i introduce here the key concept of effective causal closure. a stronger relation is the idea of inextricably intertwined levels(iils) which i discuss in sect. 5.4. 13 unless we live in a false vacuum in which case local physics could suddenly change in a way that might wipe out all life [133] . causation in order to consider causal closure, one must first have a view on how one justifies claims of causation. this has been laid out in depth by pearl [113, 114] . causal inference is based in causal models (directed graphs) validated by experimental intervention, or when that is not possible, by counterfactual arguments. causal models here one consider causal models of the influences at work . in effect, the diagram of the hierarchy of levels table 1 in sect. 2 is such a (very simplified) model, when one introduces the arrows of both upward emergence (left) and downward constraint or control (right). to develop this approach more fully, one needs to expand table 3 to a hierarchical diagram that represents the modular nature of the hierarchy (sect. 2.6). this is a very worthwhile project, but i will not attempt it here. it is roughly indicated in ( [112] , pp. [8] [9] [10] [11] [247] [248] , and examples are in ( [18] , pp. 6, 10-11, 22, 95, 110, 132, 160) . intervention here one actually intervenes at level li and reliably observes a resultant change at level lf. this has been done both for effective theories at each level l, and in many case for both upwards and downwards interlevel effects. one can do this also using digital computer models; for example [51, 105] have done this to show downwards causation occurring in computer models of heart function. counterfactual views [48] here one considers what would happen if one intervened at level li, and plausibly argues that this will cause an actual difference at level lf, when upward causation takes place: > (left column bu) or when downward causation takes place: > (right column td). this has been used to establish that downward causal effects exist, e.g. [22, 43, 45] . causal closureconsider a multilevel system s (which could have only one level). note that this includes the physicalist idea of causal closure where all follows from a single lowest physical level ll, for that is the case = , chosen as l1 in table 1 . causal closure requires effective predictability on the one hand (sect. 3.3), and an effectively causally closed set of levels on the other, which concept i now consider. the issue: two opposing strands • there are no isolated sets of levels, as just discussed (sect. 4.4). causal closure as just defined is an ideal that does not occur in practice unless one takes ll = l1, hl = l9: you are giving data for the whole thing. • however there are in practice preferred restricted sets levels with a special integrity in terms of causal closure (see the comments just after table 1 ). how do we deal with this tension? the clearest domain in which to tackle this is biology (sect. 5.3). the lessons from there carry over to engineering (sect. 6.2), and physics (sect. 6.4). biological organisms have purpose, as stated by nobel prize winning biologist hartwell et al. [61] 14 : although living systems obey the laws of physics and chemistry, the notion of function or purpose differentiates biology from other natural sciences. organisms exist to reproduce, whereas, outside religious belief, rocks and stars have no purpose. selection for function has produced the living cell, with a unique set of properties that distinguish it from inanimate systems of interacting molecules. cells exist far from thermal equilibrium by harvesting energy from their environment. they are composed of thousands of different types of molecule. they contain information for their survival and reproduction, in the form of their dna. their interactions with the environment depend in a byzantine fashion on this information, and the information and the machinery that interprets it are replicated by reproducing the cell. many biological concepts and language are often sui generis and not reducible to physics and chemistry, certainly not in the form to which they apply to simpler and restricted atomic and molecular systems. in the case of biology, unless the concepts considered include purpose and function, it will miss the essence of what is going on, as pointed out by [61] . you also need to introduce the concepts "alive" and "dead", which do not occur at any lower level than the cellular level in biology, and do not occur at any physics level. without this concept you cannot for example discuss the theory of natural selection [92] . upward and downward causation as just stated, all biological entities have purpose or function, and that controls in a top-down way what happens at lower levels [105] reaching down to the underlying physical levels [45] . the physics does not control the higher levels, rather-without any violation of the laws of physics-it does what the biology asks it do. this functioning occurs via a combinations of upwards and downwards causation [104, 105] , for example gene regulation taking place on the basis of the state of the heart [51] or the brain [78] . this dynamic reaches down to the molecular level and then the underlying electron level. the enabling factors are black boxing [7] to get higher level logic out of lower level logic, together with time dependent constraints at the lower level that are regulated by higher level biological variables [45] . together they underlie the emergent effective laws at each level l in biology. preferred levels the cellular level l5 is a key level in biology: cells have an organisation and integrity of their own, and are living integral entities that are the basic units of life. they interact with other cells at the same level, and react to their environment in appropriate ways. in multicellular organisms they depend on higher levels for nutrition, materials, waste disposal, and signals as to what to do. but they can be treated as modules (sect. 2.6) with an integrity of their own that responds to inputs and produces outputs. the associated set of biological levels, taking the underlying physics for granted, is the set of levels l4-l5. similarly the level of individual organisms l7 again represents a level of emergent integrity. individuals are entities that can be treated as autonomous entities that respond to environmental cues (from the levels above) and other individuals (at the same level). the associated set of biological levels, taking the underlying physics for granted, is the set of levels l4-l7. so the issue is, how does this kind of autonomy emerge at these particular levels, given that all levels are interacting? what characterizes these special sets of levels? the key point as to what occurs is organisational closure in biological organisms [100, 101] : the central aim of this paper consists in arguing that biological organisms realize a specific kind of causal regime that we call 'organisational closure'; i.e., a distinct level of causation, operating in addition to physical laws, generated by the action of material structures acting as constraints. we argue that organisational closure constitutes a fundamental property of biological systems since even its minimal instances are likely to possess at least some of the typical features of biological organisation as exhibited by more complex organisms. this is a distinct causal regime, as explained in [98] : in biological systems, closure refers to a holistic feature such that their constitutive processes, operations and transformations (1) depend on each other for their production and maintenance and (2) collectively contribute to determine the conditions at which the whole organization can exist. according to several theoretical biologists, the concept of closure captures one of the central features of biological organization since it constitutes, as well as evolution by natural selection, an emergent and distinctively biological causal regime. this is developed further in [95] , identifying biological organisation as closure of constraints we propose a conceptual and formal characterisation of biological organisation as a closure of constraints. we first establish a distinction between two causal regimes at work in biological systems: processes, which refer to the whole set of changes occurring in non-equilibrium open thermodynamic conditions; and constraints, those entities which, while acting upon the processes, exhibit some form of conservation (symmetry) at the relevant time scales. we then argue that, in biological systems, constraints realise closure, i.e. mutual dependence such that they both depend on and contribute to maintaining each other. thus biological organisation is an interlevel affair, involving downward causation as well as upwards emergence, thus enabling teleology [12, 99] . from the viewpoint of this paper, these authors are identifying specific sets of levels where effective interlevel causal closure occurs: the topmost level links to the bottom-most level to close the dynamic loop that leads to biological emergence. i will quote three more papers that have essentially the same view. reference [66] emphasizes this property in the case of the cell: [the] property of self-fabrication is the most basic expression of biological anticipation and of life itself. self-fabricating systems must be closed to efficient causation... i identify the classes of efficient biochemical causes in the cell and show how they are organized in a hierarchical cycle, the hallmark of a system closed to efficient causation. broadly speaking, the three classes of efficient causes are the enzyme catalysts of covalent metabolic chemistry, the intracellular milieu that drives the supramolecular processes of chaperoneassisted folding and self-assembly of polypeptides and nucleic acids into functional catalysts and transporters, and the membrane transporters that maintain the intracellular milieu, in particular its electrolyte composition. you need all these components and levels for the thing to work. reference [49] emphasizes that multi-level homeostasis is part of the mix: two broad features are jointly necessary for autonomous agency: organisational closure and the embodiment of an objective-function providing a 'goal': so far only organisms demonstrate both. organisational closure has been studied (mostly in abstract), especially as cell autopoiesis and the cybernetic prin-ciples of autonomy, but the role of an internalised 'goal' and how it is instantiated by cell signalling and the functioning of nervous systems has received less attention. here i add some biological 'flesh' to the cybernetic theory and trace the evolutionary development of step-changes in autonomy: (1) homeostasis of organisationally closed systems; (2) perception-action systems; (3) action selection systems; (4) cognitive systems; (5) memory supporting a selfmodel able to anticipate and evaluate actions and consequences. each stage is characterised by the number of nested goal-directed control-loops embodied by the organism, summarised as will-nestedness. finally [107] argue for circular causality: we argue that (1) emergent phenomena are real and important; (2) for many of these, causality in their development and maintenance is necessarily circular; (3) the circularity occurs between levels of organization; (4) although the forms of causation can be different at different levels, there is no privileged level of causation a priori: the forms and roles of causation are open to experimental investigation; (5) the upward and downward forms of causation do not occur in sequence, they occur in parallel (i.e. simultaneously); (6) there is therefore no privileged direction of emergence -the upper levels constrain the events at the lower levels just as much as the lower levels are necessary for those upper-level constraints to exist. modern biology has confirmed [...] that organisms harness stochasticity at low levels to generate their functionality. this example shows in fine detail why higher-level causality can, in many cases, be seen to be more important than lower-level processes. this is closely related to the idea of autonomous systems, characterized by their organizational and operational closure [138] , where [135] organizational closure refers to the self-referential (circular and recursive) network of relations that defines the system as a unity, and operational closure to the re-entrant and recurrent dynamics of such a system which is just the idea above. this discussion is related to the idea of autopoiesisa system capable of reproducing and maintaining itself-mentioned above, and to the idea of autocatalytic sets [69, 70] . however i will not develop those links here. rather my purpose is to claim that exactly the same applies in engineering systems in general, and even in physics in some cases. that is what i develop below. given that it is understood i am considering causal closure in terms of levels and lois, i can summarise as follows 15 : [131] that lie outside the normal operating environment. thus this characterizes the set of levels needed for an entity (a cell or an organism) to function successfully. there is then no preferred level enabling the system to function: they all equally enable this to happen [107] . reference [57] argue that in such cases, while the effective theories are contained in the range {bl-tl}, one needs different models at each level: no single mathematical model can account for behaviors at all spatial and temporal scales, and the modeler must therefore combine different mathematical models relying on different boundary conditions. these are the different s for each level. but note that one then needs data d for each level l too. thus the set of levels where data is given has to be the same as the set of levels where ecc occurs. thus in the definition of effective causal closure just given, one should set {bl-tl} = {ll-hl}. reference [57] give the examples of epithelial sheets and mechanical modeling of gastrulation. to be clear: one is free to work with an effective theory , with appropriate data for that level, at any level chosen l in the range {bl-tl}; but one only gets effective causal closure by including that full set of levels and data. when does this occur in biology? there are two cases where ecc occurs in biology. • cells the cellular level is the lowest level showing all the attributes of life. it is a case of ecc involving levels l4-l5. • individuals the organism level is the major coherent emergent level in life, assuming it is a multicellular organism such as a human being. this is a case of ecc involving levels l4-l7. however there is an interesting different view: that human beings are essentially social beings, so that in fact it is a mistake to view them as being capable of living on their own, as is implied by that categorisation, thus berger and luckmann [10] wrote about the social construction of reality: our worldview-an inescapable part of our nature shaping our actions-is crucially shaped by the society in which we live. merlin donald's book a mind so rare [33] essentially agrees, as does andy clark's book supersizing the mind [26] and [108] . in short, top-down effects from society so crucially shape our being that they are not just perturbations of independent existence: they are essential, and that characterisation is wrong. the correct ecc statement is • social human beings human beings are essentially social, and are in fact a case of ecc involving levels l4-l8. ignoring the lower levelsa further key comment regards the other end of the scale: why is it legitimate to ignore levels l1-l3 here? the answer is the existence of quantum and classical protectorates that are governed by emergent rules and are insensitive to microscopics [84] . this is another way of affirming the causal efficacy of the effective theories at each emergent level l. however the effectively causally closed levels will reach down to determine what happens at those levels via time dependent constraints (4) [45] . in summary: interlevel effective causal closure as identified here is a key feature of biological functioning, emphasized in [8] and [96] . [24] (where it is not identified as such, but is there) and [123] , where the relation is made explicit. setting the data one final issue remains: we don't in practice set data at all the levels ll-hl required in a particular context. there is no way we could in fact set the data at the lower levels. and there is no need for us to do so. this comment applies both in theory and in practice. that is, it is a statement both about epistemology (what we can know) and ontology (what we can do). a higher level may be essential to a lower level an important possibility is that the properties p of two levels {bl,tl} may have an essential relationship with each other: each level cannot function without the other, as in some cases of symbiosis. consider an individual human being. it is no surprise that the level l7 of the individual cannot exist without the level l5 of cells, for human beings are made out of cells and depend on them for their existence and physiological functioning. but the fact is that the converse is also true: the cells cannot exist and function without the existence of the body that they comprise. the reason is that cells have specialised for specific functions, and cannot survive on their own. they are supplied with oxygen-laden blood by the lungs, heart, and indeed the entire circulatory system, without which they die in a matter of minutes (as happens if a heart attack occurs). thus levels l5 and l6 are inextricably intertwined. but organs are part of the individual and won't function without systemic integration at that level. hence levels {l5-l7} are in fact inextricably intertwined. now an interesting issue arises: ecc occurs for levels l5-l7. should i have included l4 in the inextricably intertwined levels? certainly level l4 is required in order that cells exist at level l5, but is the other way round true also? i believe one can claim it is, because the gene regulatory networks that control production of proteins at level l4 are at level l5, and they would not exist if it were not for their functioning. thus the real inextricably intertwined set of levels is {l4-l7}: the same as the ecc set of levels. there is however this difference: the ecc relation is ceteris parabus, as explained above. the iil relation is not, it is essential, whatever happens at other levels, these levels are crucially dependent on each other. the discussion in the last two sections makes clear a set of principles that apply equally to engineering, and that is what i will show in this section. to make the discussion concrete, i will consider the case of digital computers. but it will apply equally to other branches of engineering: automobiles, aircraft, chemical plant, water supply systems, sewerage systems, and so on. i consider the nature of digital computers (sect. 6.1), where interlevel effective causal closure again occurs (sect. 6.2). inextricably intertwined levels again occur in computers (sect. 6.3), and in physics and chemistry (sect. 6.4). the relevant hierarchy [43, 132] 16 is shown in table 4 . upward and downward causation the dynamics of a computer is driven by the algorithms encoded in the programs loaded, together with the data used by those programs. these control the flow of electrons through gates at the transistor level via a combinations of upward and downward causation ( [39] , chap. 2, [43] ). this enables the emergent effective laws at each level l. different algorithms result in different flows of electrons, as can be demonstrated by running different computer programs which produce different patterns of electron flows through transistors at level l2, and cause major effects at social levels l8 and l9 [88] . the bigger picture the fact that the higher levels {l8,l9} reach down to affect what happens at the lower levels {l0-l6} is stated in [43] as follows: causal closure in the case of computers: in the real world, it is only the combination of physics with its logical, social, psychological, and engineering contexts (which includes the values guiding policy) that can be causally complete, because it is this whole that determines what computer programs will be written and what data utilised, hence what electron flows will take place in integrated circuits, as per the discussion in this paper. this is in parallel to the interlevel causal closure that takes place in biology, as discussed in sect. 5.3. reference [43] gives a specific example: as a specific example: the amount of money that can be dispersed to you from an atm will be limited by an agreement you have reached with your bank. the program used to control the atm will take into account the existence of such limits, and the specific amount you are able to take out in a given time period will be limited by a logical and operation linking this agreed amount to the amount of money in your account. thus these abstract variables will control electron flows in both the bank computers and the atm dispenser mechanism. every relevant abstract variable has physical counterparts; in other words, it?s realized by some physical properties on some relevant physical substrate. but crucially there is much more than this: there is the whole issue of the purposes computers are used for in society, from controlling manufacturing to enabling the internet, cell phones, and social media, and they way that this whole enterprise is shaped by the values of those that control the system. the book coders [134] considers "the morality and politics of code, including its implications for civic life and the economy. programmers shape our everyday behavior: when they make something easy to do, we do more of it. when they make it hard or impossible, we do less of it." all this is expressed in the flows of electrons through gates at the digital levels l2-l3. nevertheless, just as in the case of biology, effective causal closure can occur when interlevel causation results in a high degree of autonomy of operation. analogously to the case of biology, one can state to an autonomous system that do not destroy its autonomy, and (ii) it is a ceteris parabus relation, as discussed in §3.3. it can be destroyed by black swan events that lie outside the normal operating environment. the two emergent levels with their own causal integrity emerging through ecc are the integrated circuit level (l4), the equivalent of the cell in biology, with ecc given by levels l2-l4; and the computer level (l5), the equivalent of the individual in biology, with ecc given by levels l2-l5. but just as in the case of biology one can make a case that one should really include the societal level, the same applies here too. the quotes above suggest that ecc for computers really only occurs for levels l2-l9, including the highest level because of the effect of the world wide web. engineering and applied physics the same kind of considerations apply to all branches of engineering, and equally to all branches of applied physics. the applications (socially determined at level l8) determine what physical effects occur (levels l1-l3). inextricably intertwined levels (iils) do these occur in this case too, as they did in biology? here there is a major difference: the transistors do not depend on the computer for their continued existence, whereas cells depend on the organism for their existence. while in biology iils link the individual as a whole to the molecular level, here they also occur, but only at the levels l0-l1 in table 4 . the reason for that relation is that downward emergence of key properties at the electron level l0 takes place, due to properties of the crystal level l1, as explained in detail in [41] . this is called a "foundational determinative relation" (fdr) by carl gillett, see [55] . in more detail, quasiparticles such as phonons exist due to the broken symmetries of the emergent lattice structure. they come into being as effective particles at the lower level l0 because they are dynamically equivalent to collective oscillations of a level l1 structure (the crystal lattice) ( [127] , pp. [82] [83] . this is an essentially quantum theory phenomenon. one can think of it as an interlevel wave(macro)-particle(micro) duality. reference [52] say it this way: phonons [are] quasi-particles that have some claim to be emergent, not least because the way in which they relate to the underlying crystal is almost precisely analogous to the way in which quantum particles relate to the underlying quantum field theory. stephen blundell states the key point thus ( [15] , p. 244): so now we come to the key question: are these emergent particles real? from the perspective of quantum field theory, the answer is a resounding yes. each of these particles emerges from a wave-like description in a manner that is entirely analogous to that of photons. these emergent particles behave like particles: you can scatter other particles off them. electrons will scatter off phonons, an interaction that is involved in superconductivity. neutrons can be used to study the dispersion relation of both phonons and magnons using inelastic scattering techniques. yes, they are the result of a collective excitation of an underlying substrate. but so are 'ordinary' electrons and photons, which are excitations of quantum field modes. as a consequence, the levels {l0, l1} are inextricably intertwined. another way of stating this is the way that bloch's theorem [14] shows how the crystal structure causes the electron wave functions to have a basis consisting of bloch eigenstates with the same periodicity as the crystal. this is a proof that the solid state physics occurring in digital computers is a case where causal closure is impossible at the micro level l0 alone. it also shows that in general the set of iils is not the same as the eccs. while these considerations apply to digital computers, of course they also apply in particular to the solid state physics itself that underlies their operation, due to the nature of crystals. for the reasons just discussed in solid state physics, as a consequence of interlevel wave-particle duality, levels {l1,l4} in table 1 are inextricably intertwined levels. however the iil phenomenon is not confined to this case. the laser is another example ( [119] , §2) the laser involves a kind of 'circular' causality which occurs in the continuous interplay between macrolevel resonances in the cavity guiding, and being reinforced by, self-organization of the molecular behaviour. a quite different example is resonance energy transfer (ret) in the transport of electronic energy from one atom or molecule to another [74] . as described in that paper, the individual electrons do not migrate between molecules during the transfer process, since the molecular orbitals (the wavefunctions) do not overlap, but instead move between individual electronic states within the molecules. ... energy transfer, through dipole coupling between molecules, mostly depends on two important quantities: spectral overlap and intermolecular distance. this lead to a r −6 distance-dependence for the resonance energy transfer rate in the short-distance regime. the behaviour results from interwining between the electron level l1 and the molecular level l4. because of these interactions, second-order perturbation theory is the minimal needed to describe ret. while this paper refers to 'molecules', ret occurs in atoms, chromophores, particles and carbon nanotubes. its applications include nanosensors and photodynamic therapy. this raises the issue that whenever molecular physics [21] is concerned, there is inextricable intertwining between the molecular structure at level l4, bound by electrons, and the motions of the electrons at level l1, controlled by that structure. this is manifest in binding energy ( [21] , pp. 17-19, [90] , p. 569) and the existence of covalent bonds between atoms ( [21] , pp. [17] [18] [19] . molecular physics and quantum chemistry also exhibit iils between levels l1 and l4, which is why chemistry is a classic example of downward causation [86] . the group theory underlying this intertwining is discussed in [13] . it is believed by many that because the bottom-most physics level is causally complete, and only upward causation takes place, higher levels are purely derivative: they have no real causal validity. in this paper and its companion [41] , i argue against that position. it is invalid because it treats physics in a way that ignores context, whereas physics outcomes always depend on context. in fact downward effects imply the opposite: in real world contexts, the bottom-most physics level is not by itself causally complete. in this section, i look at the contextual nature of causal closure of physics (sect. 7.1), the way that unpredictability undermines causal closure of physics per se (sect. 7.2), and comment on ways people ignore the issues discussed in this paper (sect. 7.3). we can consider physics per se, or in relation to the natural world, or in relation to biology, or in relation to engineering. within physics, the issue of causal closure depends on what aspects we are considering: particle physics, nuclear physics, condensed matter physics, cosmology for example. firstly, we have no reliable tested toe at the very bottom level l0. we don't try to reduce to that most fundamental physical level (sect. 4.4). rather we reduce to a level that is convenient. that that can work is due to the existence of quantum protectorates, as explained in [84] . but then it is common to assume that level l1 (particle physics) is causally complete. is that indeed so? i have argued that this is not the case in the contexts of physics and biology (sect. 5.3); physics and engineering, as exemplified by digital computers (sect. 6.2); solid state physics (sect. 6.3); and physics and chemistry (sect. 6.4). in each case the real causal closure that takes place is an interlevel affair, as emphasized in particular in the case of biology by many perceptive writers (sect. 5.3). effective causal closure in real world contexts spans many levels, in the case of biology reaching down from the level of the organism to the underlying physics via time dependent constraints. this implies how it works in terms of physics in relation to society. the causal effects of the coronavirus pandemic at the social level reaches down to cause major changes at the physical levels l1-l3 through a complex interaction between social behaviours, virology, and microbiology that for example has temporarily destroyed international air travel and so the trajectories of the billions of particles that make up aircraft. causal closure only occurs when we take all these factors and levels into account. considering only disembodied physical laws seriously misleads about the nature of causation and causal closure in real world contexts. in summary, this is formalized by the concepts of effective causal closure (sects. 5.3, 6.2), and inextricably intertwined levels (sects. 5.4, 6.3). within solid state physics itself, the lower levels l1-l3 are not causally closed by themselves because of interlevel particle-wave duality between the particle level l1 where electrons and phonons live, and the crystal level l4 where lattice vibrations take place (sect. 6.3). properties of level l4 decouple from the lower physics levels. as stated by [84] , the crystalline state is the simplest known example of a quantum protectorate, a stable state of matter whose generic low-energy properties are determined by a higher organizing principle and nothing else. ignoring interlevel issues, if by causal closure one means that data specified as precisely as possible leads to unique outcomes, then unavoidable unpredictability undermines the possibility of physics per se (whether quantum or classical) being causally closed. quantum physics quantum effects doubly cause uncertainty in outcomes. firstly, the heisenberg uncertainty principle states that the standard deviations of position x and momentum p obeys so one cannot even in principle apply laplace's dream of setting initial data precisely at level l1. consequently, outcomes are also uncertain. secondly, collapse of the wave function introduces an irreducible uncertainty in classical outcomes at this level when interactions take place [53] . this can reach up to macro levels through various amplifiers such as photon multipliers and ccds. in the engineering case it causes predictability issues at macro scales in terms of digital computer reliability because cosmic rays cause errors in computer memories [56, 143] , and the emission of a cosmic ray by an excited atom is a quantum event that is (10) x p ≥ ℏ∕2 unpredictable even in principle. as regards biology, cosmic rays have had a significant effect on evolutionary history by causing genetic mutations [116] . the classical case uncertainty of outcomes occurs in this case too, because one can't set initial data to infinite precision [31] . this is an outcome of the fact that infinity never occurs in physical reality [46] . thus physics is not causally closed in the classical case at higher levels because of chaotic dynamics (the butterfly effect), together with the impossibility of specifying initial data to infinite accuracy. this occurs for instance at the level at which fluid motion is determined. reference [6] puts it this way: a fluid dynamicist when studying the chaotic outcome of convection in a benard cell knows to a gnat's eyelash the equations of motion of his fluid but also knows, through the operation of those equations of motion, that the details of the outcome are fundamentally unpredictable, although he hopes to get to understand the gross behaviour. this aspect is an example of a very general reality: the existence of universal law does not, in general, produce deterministic, cause-and-effect behaviour. this fundamentally undermines the concept of a causally closed physical levels in the case of classical physics. as was already known to poincare, this occurs even in the 3-body gravitational case. microbiology in the case of microbiology, interactions take place in the context of what hoffmann [65] has called "the molecular storm". molecular machines use ratchet-like mechanisms to harness energy from that storm, and organisms use it to provide an ensemble of options from which they can choose preferred lower level states and so attain biological objectives [106] (see the quote in sect. 2.3). one has the opposite of the calm relation between initial data and outcomes supposed by laplace. here are some ways that the nature of causal closure as discussed in this paper is avoided. partial reduction this is very common. francis crick in the astonishing hypothesis [28] states, you, your joys and your sorrows, your memories and your ambitions, your sense of personal identity and free will, are in fact no more than the behavior of a vast assembly of nerve cells and their associated molecules. in other words, he is reducing l7 to {l5-l4}. now my physics colleagues who believe that all that matters is the particle interactions at level l1 will just laugh and say, cells at level l4 and molecules at l5 are nothing but particles interacting with each other. thus crick believes in the reality and effectiveness of causality at levels l4 and l5 that for example [71, 58] , who believe that all causality resides at level l1, clearly must deny. so why did crick emphasize causality at those levels? the answer of course is that those were the levels at which he worked-and experienced the effectiveness of causality in terms of the interactions between entities (molecules, neurons) at those levels. from a strictly reductionist viewpoint, this is an illegitimate move. it is however fine if you accept noble's principle of biological relativity [105] , as extended in [41] and this paper: then causality is real at the levels {l4, l5} he studies. but that removes crick's justification for denying the reality of causation at level l7. ignoring context crucial contextual effects are simply ignored by some writers. for example [58, 71] . the view is "you are nothing but a bag of particles, it's just a matter of particles interacting via a known set of forces". context has nothing to do with it. the physicists holding this view all come from the particle physics/cosmology side, where this is to some extent true. physicists from the solid state physics side (the largest section of the physics community) do not hold this view, see e.g. [5, 127] . this leads to the large divide in the physics community between these two groups, as discussed by schweber [124] . denying top-down causation there is a frequent denial of the possibility of top-down causation, even though it occurs in physics and cosmology. in the latter case it occurs in the context of primordial nucleosynthesis in the early universe ( [117] , §4.3) and structure formation in the 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the ultimate laws of nature. penguin random house the architecture of biological networks effect of cosmic rays on computer memories that paper gives fascinating examples of downward causation from morphogenesis and regenerative medicine.downward causation is obvious in subjects other than particle physics and cosmology, and in particular in the functioning of the brain [39, 40, 77, 79] . for example, our minds are shaped by society, and shape society, including material outcomes in terms of architecture, engineering, art, and so on. society does not exist without mind, and mind does not exist without society [9, 10, 33] . that is the nature of interlevel causal closure. key: cord-018414-6ffhm895 authors: kang, yoogoo; elia, elia title: anesthesia management of liver transplantation date: 2016-07-22 journal: contemporary liver transplantation doi: 10.1007/978-3-319-07209-8_9 sha: doc_id: 18414 cord_uid: 6ffhm895 anesthesia for liver transplantation pertains to a continuum of critical care of patients with end-stage liver disease. hence, anesthesiologists, armed with a comprehensive understanding of pathophysiology and physiologic effects of liver transplantation on recipients, are expected to maintain homeostasis of all organ function. specifically, patients with fulminant hepatic failure develop significant changes in cerebral function, and cerebral perfusion is maintained by monitoring cerebral blood flow and cerebral metabolic rate of oxygen, and intracranial pressure. hyperdynamic circulation is challenged by the postreperfusion syndrome, which may lead to cardiovascular collapse. the goal of circulatory support is to maintain tissue perfusion via optimal preload, contractility, and heart rate using the guidance of right-heart catheterization and transesophageal echocardiography. portopulmonary hypertension and hepatopulmonary syndrome have high morbidity and mortality, and they should be properly evaluated preoperatively. major bleeding is a common occurrence, and euvolemia is maintained using a rapid infusion device. pre-existing coagulopathy is compounded by dilution, fibrinolysis, heparin effect, and excessive activation. it is treated using selective component or pharmacologic therapy based on the viscoelastic properties of whole blood. hypocalcemia and hyperkalemia from massive transfusion, lack of hepatic function, and the postreperfusion syndrome should be aggressively treated. close communication between all parties involved in liver transplantation is also equally valuable in achieving a successful outcome. dr. thomas starzl of denver, colorado, usa, who believed that "liver transplantation is an effective treatment providing exactly what is needed for patients with end-stage liver disease (esld)," performed the first successful orthotopic liver transplantation (olt) in a 3-year-old boy with biliary atresia in 1963 (starzl et al. 1963) . during the first two decades of the procedure's history, liver transplantation led by starzl and sir roy calne of cambridge encountered almost insurmountable challenges, including complexity of surgical technique, primitive anesthesia and intensive care, less-than-adequate immunosuppression and organ preservation, and devastating infection. the number of procedures performed was relatively few, and the success rate was low. however, their keen observations on these early clinical experiences laid the foundation of modern liver transplantation (starzl and putnam 1969; calne 1983) . breakthroughs were made in each decade following the first transplantation. in the 1980s, venovenous bypass was introduced to maintain better hemodynamic stability (shaw et al. 1984) , cyclosporine was found to be a superior immunosuppressant to azathioprine, and anesthesiologists answered important clinical questions, including those relating to the monitoring and treatment of coagulopathy, hemodynamic changes, and the role of the electrolyte imbalance. in the 1990s, fk506 (tacrolimus) became the immunosuppressant of choice ), university of wisconsin solution was introduced to extend the safe cold ischemia time to 24 h (kalayoglu et al. 1988) , and the piggyback technique simplified surgery in select patients (tzakis et al. 1989 ). in the past 15 years, liver transplantation has been performed in most major medical centers with a 1-year survival rate of greater than 85 %, and living donor liver transplantation has become a valuable alternative. liver transplantation requires a true multidisciplinary approach, and anesthesiologists and intensivists have played a major role in the successful outcome of liver transplantation. in support of the important role of anesthesiologists in liver transplantation, the american society of anesthesiologists (asa) developed the guidelines for director of liver transplant anesthesia in 2001. the guidelines specified that the director should have fellowship training in critical care medicine, cardiac anesthesiology, or transplantation anesthesiology that includes the perioperative care of at least ten liver transplant recipients or experience in the perioperative care of at least 20 liver transplant recipients in the operating room. in addition, the director is expected to obtain a minimum of 8 h of accreditation council for continuing medical education (accme) category i continuing medical education (cme) credit in transplantation-related educational activities within the most recent 3-year period. in this chapter, physiology and pathophysiology of liver disease and anesthesia care of liver transplantation are described based on clinical experience at the university of pittsburgh (pittsburgh, pa, usa) and thomas jefferson university (philadelphia, pa, usa). the liver, which weighs 1200-1500 g in adults, is traditionally divided into the right and left lobe in reference to the location of the falciform ligament. couinaud, however, divided the liver into the right and left hemiliver using the cantlie's line, which extends from the inferior vena cava (ivc) to the gall bladder, and each hemiliver is further divided into four segments (couinaud 1954) . the left hemiliver is composed of the traditional left lobe along with the caudate and quadrate lobe. liver resections based on these segmental definitions are right hepatectomy (segments 5-8), right lobectomy (segments 4-8), left hepatectomy (segments 1-4), and left lobectomy (segments 1-3) ( fig. 1) (bismuth 1982) . the liver has a unique dual blood supply: arterial supply from the hepatic artery, a branch of the celiac axis, and venous supply from the portal vein formed by the union of the splenic and superior mesenteric vein. despite liver mass constituting only 2.5 % of the total body weight, the total hepatic blood flow is approximately 100 ml/ 100 g/min, or 25 % of cardiac output. the hepatic artery supplies approximately 25-30 % of hepatic blood flow and 45-50 % of the oxygen requirement, while the portal vein supplies 70-75 % of hepatic blood flow and 50-55 % of oxygen. the venous drainage is through the right, middle, and left hepatic veins, which merge into the ivc. the valveless portal vein is a low pressure/low resistance circuit, while the hepatic artery is a high pressure/high resistance system. hepatic 553-565, copyright (2000) , with permission from elsevier) blood flow is primarily regulated by local metabolic demand with an inverse relationship between portal venous and hepatic arterial flow: an increase in the hepatic adenosine level triggered by a reduced portal venous flow increases hepatic arterial blood flow (gelman and ernst 1977; lautt et al. 1985) . the hepatic artery buffer response appears to be functional even after liver transplantation (payen et al. 1990) , and this response may be responsible for the development of the small-for-size syndrome after living donor or split liver transplantation (kiuchi et al. 1999 ). small-for-size syndrome develops in a patient who received a donor graft that was less than 1 % of the recipient's body weight and is caused by decreased hepatic arterial flow in response to increased portal venous flow and pressure. subsequently, a prolonged postoperative reduction in hepatic arterial flow can lead to centrilobular tissue necrosis, biliary ischemia, and hepatic arterial thrombosis (smyrniotis et al. 2002) . there is no buffer response in the portal system because the portal vein cannot regulate its blood flow. therefore, alterations in the hepatic arterial blood flow do not induce compensatory changes in the portal blood flow (lautt 1983) . the mean pressure in the hepatic artery is similar to that in the aorta, while portal vein pressure ranges between 6 and 10 mmhg. the portal pressure depends primarily on the degree of constriction or dilatation of the splanchnic arterioles and on intrahepatic resistance. both afferent systems merge at the sinusoidal bed, where the pressure is estimated to be 2-4 mmhg higher than that in the ivc. the liver serves as a blood reservoir, and it replenishes blood volume of up to 25 % rapidly in the case of an acute bleeding episode (lautt 2007) . hepatic blood volume may expand considerably in cardiac failure by venous congestion. the liver is innervated by the left and right vagi, the right phrenic nerve, and fibers from the t7-t10 sympathetic ganglia. the hepatic artery is innervated mainly by sympathetic fibers, and hepatocytes, by the unmyelinated sympathetic fibers. the bile ducts are innervated by both sympathetic and parasympathetic fibers. the role of hepatic innervation is unclear, as denervation of the transplanted liver does not affect its function (kjaer et al. 1994) . bile flow begins from the bile canaliculi to the common bile duct. hepatic lymph forms in the space between the sinusoid and the hepatocyte (space of disse) and flows to lymph nodes in the hilum and ivc. the transdiaphragmatic lymphatic flow is the cause of pleural effusions in the presence of large ascites. the liver is made of parenchymal cells (hepatocytes) and non-parenchymal cells (sinusoidal endothelial, kupffer, stellate, dendritic, and lymphocyte) . hepatocytes make up 60-80 % of liver cells and carry out hepatic metabolic, synthetic, and detoxification functions. polyhedral hepatocytes are arranged in one-cell thick plates with endothelium-lined sinusoids on both sides. each hepatocyte cell membrane has three distinct membrane domains. the sinusoidal membrane is adjacent to the sinusoidal endothelium and has numerous microvilli abutting into the space of disse. fenestrae within the sinusoidal endothelium without the basement membrane permit intimate contact between sinusoidal blood and the hepatocytes to allow the passage of big molecules, including lipoproteins. liver sinusoidal endothelial cells make up 15-20 % of liver cells and release nitric oxide to regulate vascular resistance. they are, along with dendritic cells and lymphocytes, part of the innate immune system. the space of disse contains phagocytic kupffer cells that participate in the hepatic inflammatory process. the ito cells, also known as stellate cells, are the major site of vitamin a storage, and their activation results in hepatic fibrosis and cirrhosis. reticulin fibers in the space of disse support the sinusoidal framework, and weakening of these supporting fibers results in rupture of sinusoidal walls and formation of blood-filled cysts known as peliosis hepatis, a forerunner of cirrhosis. the apical membrane circumscribes the canaliculus, the earliest component of the biliary system. the lateral hepatic membrane is found between adjacent hepatocytes. the functional unit of the liver is the acinus. terminal portal veins communicate with terminal hepatic venules, with sinusoids bridging the gap between the two vessels ( fig. 2) . each sinus contains three zones with equal blood pressure and oxygen content. the periportal zone (zone 1) receives blood highest in oxygen content and the pericentral or perivenular zone (zone 3) receives blood lowest in oxygen content. as a result, the hepatocytes in the perivenular zone (zone 3) are more vulnerable to ischemic damage and nutrient depletion. oxidative and reductive functions are predominantly performed by hepatocytes at the periportal zone and glucuronidation is performed by those at the perivenular zone, although hepatocytes of the two different zones are functionally integrated (lamers et al. 1989 ). the unique structure of the liver acinus is well-suited for bidirectional transfer of nutrients. the low pressure in the portal venous system allows blood to flow slowly through the sinusoids. hepatic arterial blood flows mainly to the terminal bile canaliculi, although it augments sinusoidal flow to give a gentle pulsatility. in patients with liver cirrhosis, the sinusoids acquire features of systemic capillaries: the space of disse widens with collagen deposits at the basement membrane, endothelial fenestrations become smaller and fewer, and hepatic microvilli efface. all of these changes reduce transport across the sinusoidal walls and result in hepatic dysfunction. furthermore, widespread fibrosis and scarring reduce the number and size of the small portal and hepatic veins and increase intrahepatic vascular resistance to the development of portal hypertension (popper 1977) . the sluggish blood flow in the altered vascular architecture promotes thrombosis, causing further cell necrosis and fibrosis (wanless et al. 1995) . the liver undergoes rapid regeneration through proliferation of hepatocytes to maintain the critical mass necessary for normal liver function. for example, the newly transplanted hemiliver from a living related donor regenerates to about 85 % of its original whole liver size in 7-14 days. the major hepatic growth factors are epidermal growth factor and hepatocyte growth factor (michalopoulos 1990) . administration of the epidermal or hepatocyte growth factor to normal rats, however, does not cause hepatocyte replication. this negative response suggests that liver regeneration involves a two-step process: the initial signal generated by an acute increase in metabolic demand associated with the loss of hepatocytes triggers a set of early response genes that prime hepatocytes to respond to various growth factors. in apoptosis or programmed cell death, aging hepatocytes are removed and new cells are produced in a continuous manner (ellis et al. 1991 ). the liver has three major functions: metabolism, bile production and secretion, and filtration of harmful substances. the principal role of the liver is to provide the body with normal glucose levels, which are regulated by insulin, glucagon, growth hormone, and catecholamines (pilkis and granner 1992) . the liver converts glucose into glycogen (glycogenesis) and utilizes glucose for the synthesis of fatty acids. cirrhotic patients are frequently hyperglycemic although their insulin level is elevated (petrides and defronzo 1989) . this insulin resistance is caused by multiple mechanisms. cirrhotic patients have an increased basal metabolic rate and use preferentially fatty acids as an energy source. reduced glucose uptake and limited glucose storage in the liver and muscle lead to hyperglycemia. other contributing factors are increased serum fatty acids, which inhibit glucose uptake by muscle; altered second messenger activity after insulin binding to its receptors; an increased concentration of serum cytokines associated with elevated levels of endotoxins; and increased levels of glucagon and catecholamines. the liver is the major organ for protein synthesis, and albumin is the most important protein product. albumin is the major contributor to plasma oncotic pressure and binds and transports bilirubin, hormones, fatty acids, and other substances. hypoalbuminemia is commonly caused by decreased hepatic synthetic function, although it can be secondary to an enlarged volume of distribution, reduced level of amino acid precursors, and losses into the urine, peritoneum and pleural cavity, and leads to peripheral edema, ascites, and pleural effusions. the low serum oncotic pressure stimulates the hepatic albumin synthesis in healthy subjects, but this is impaired in patients with cirrhosis (pierrangelo et al. 1992) . the liver synthesizes all coagulation factors (except von willebrand factor) and protein c and s. factors ii, vii, ix, and x undergo a posttranslational vitamin k-dependent modification involving γ-carboxylation of specific glutamic acid residues in the liver. the liver is the primary site of interconversion of amino acids. anabolic processes synthesize proteins from amino acids, while catabolic processes convert amino acids either to keto acids by transamination or ammonia by oxidative deamination. ammonia, in turn, is converted to urea by the krebs-henseleit cycle. in patients with liver disease, derangement of both anabolic and catabolic processes results in decreased production of blood urea nitrogen (bun) and accumulation of ammonia, a contributing factor in the development of hepatic encephalopathy. the liver produces acute-phase reactants, such as α-fetoprotein, ceruloplasmin, fibrinogen, transferrin, complement, and ferritin. they are expressed during acute and chronic systemic inflammation, and their activation is mediated by interleukin-6, tumor necrosis factor, interferon-γ, and glucocorticoids. the liver takes up fatty acids and cholesterol from diet and peripheral tissues to produce and release lipoprotein complexes into circulation. fatty acids released from adipocytes are bound to serum albumin and transported to the liver for the synthesis of phospholipids and triglycerides. the liver produces fatty acids from small molecular weight precursors, and cholesterol synthesis is regulated by the ratelimiting enzyme 3-hydroxyl-3 methylglutaryl coenzyme a reductase (hmg-coa reductase). lipids are exported out of the liver by very low-density lipoprotein (vldl) particles, which are the major carriers of plasma triglycerides during non-absorptive states. lipids are temporarily stored in the liver as fat droplets, or as cholesteryl esters in the case of cholesterol, and are directly excreted into bile or metabolized into bile acids. the liver is the major site for sterol excretion and production of bile acids. various abnormalities in lipid metabolism are common in liver disease. hypertriglyceridemia (250-500 mg/dl) is the most common presentation and may be caused by decreased synthesis of lipoproteins, decreased hepatic clearance of lipoprotein complexes, or re-entry of biliary content into the serum. alcoholic liver injury results in increased fatty acid synthesis and steatosis (lieber 1993) . paradoxically, an increased high-density lipoprotein (hdl) 3 level has been noted with moderate alcohol consumption, which may explain the reduced risk of atherosclerosis in these patients (chait and brunzell 1990) . patients with cholestatic liver diseases have elevated total serum cholesterol and triglycerides because the bile is rich in cholesterol, phospholipids, and lecithin. the liver eliminates drugs through two types of reactions. the phase 1 reactions include oxidation, reduction, hydroxylation, sulfoxidation, deamination, dealkylation, and methylation of reactive substances. these reactions involve systems such as cytochrome p450 and typically occur in the periportal area of the liver. the phase 2 reactions, which transform lipophilic agents into more water-soluble compounds, take place in the pericentral area. in patients with liver disease, hepatic drug clearance is usually reduced due to the enlarged volume of distribution and decreased hepatic metabolism. as a result, a large initial dose of medications followed by small, titrated maintenance doses are required to achieve the desired pharmacologic effects. several hormones are deactivated or altered in the liver. the deactivated hormones are insulin, glucagon, steroid hormones, aldosterone, thyroxine, and triiodothyronine. the liver converts testosterone into androsterone and estrogen into estrone and estriol. abnormal levels of estrogen and testosterone in patients with liver disease lead to testicular atrophy, loss of pubic and axillary hair, spider angioma, and gynecomastia. the liver removes various substances from the body, and bile formation is one of the most important excretory functions. when membranes of old erythrocytes rupture, the released hemoglobin is taken up by the reticuloendothelial cells and is split into heme and globin. heme converts to biliverdin, which, in turn, is reduced to free bilirubin and released into the plasma. the free bilirubin-albumin complex is taken up by the hepatocytes. bilirubin conjugates primarily with glucuronic acid and is actively transported into the bile. a small portion of conjugated bilirubin returns to the plasma directly from the sinusoids or indirectly by absorption from the bile ducts and lymphatics. bilirubin is converted into urobilinogen by the intestinal bacterial flora. some urobilinogen is reabsorbed through the intestinal mucosa and is re-excreted into the intestine. bile acids, which enhance absorption of vitamin k, are also excreted into the bile by the liver. the liver, located between the splanchnic and systemic venous system, acts as a vascular filter. kupffer cells phagocytose immune complexes, endotoxins, and bacteria in the portal venous blood and process antigens for presentation to immunocompetent cells. the liver also removes activated coagulation elements from circulation to prevent excessive coagulation or fibrinolysis. liver cirrhosis is defined as progressive fibrosis and the formation of regenerative nodules, and is the final common pathway in which hepatocytes are replaced by connective tissue after various, repetitive insults. the amount of remaining functional hepatic mass and the degree of architectural distortion determine the functional state of the liver. portal hypertension is inevitable in advanced cirrhosis and leads to ascites, variceal bleeding, and encephalopathy. the severity of cirrhosis is frequently classified using the child-pugh score (table 1) , and a score of >6 suggests a short life expectancy. hepatic encephalopathy is a reversible neuropsychiatric condition in both acute and chronic liver failure. in chronic liver disease, hepatic encephalopathy develops in 28 % of patients within 10 years of compensated cirrhosis and is associated with spontaneously developed or surgically created portosystemic shunting (butterworth 2001) . the degree of encephalopathy is stratified by a coma scale: grade 1, subtle confusion; grade 2, somnolence; grade 3, unconsciousness with response to pain stimulation; and grade 4, deep coma. clinically, asterixis, flapping tremor, and fetor hepaticus (musty, sweet breath odor) are confirmatory of hepatic encephalopathy. the main cause of hepatic encephalopathy is the altered expression of several genes for various neurotransmitter proteins in the brain (butterworth 2001) . decreased expression of the glutamate transporter (glt-1) increases extracellular brain glutamate. an increased expression occurs in some receptors: monoamine oxidase increases degradation of monoamine transmitters, the peripheral-type benzodiazepine receptor increases inhibitory neurosteroids, and neuronal nitric oxide synthase increases nitric oxide production. although its plasma level is not closely related to the severity of encephalopathy, ammonia is still considered to be a major contributing factor. ammonia and manganese are known to alter the expression of the peripheraltype benzodiazepine receptor and neuronal nitric oxide synthase in exposed cells (warskulat et al. 2001) . magnetic resonance spectroscopy reveals brain edema and increased brain glutamine/glutamate in the frontal and parietal lobes; histologic findings are swelling and glycogen deposition in astrocytes. these changes in the brain coincide with impairment in the visuopractic capacity, visual scanning, and perceptual-motor speed on neuropsychiatric testing (tarter et al. 1984) . subclinical hepatic encephalopathy can be detected by having patients perform a simple timed connect-the-numbers test. treatment of hepatic encephalopathy is based on the ammonia-lowering strategy, such as protein restriction, oral non-absorbable antibiotics, and lactulose. rifaximin reduces the plasma ammonia level by destroying intestinal bacteria that produce urease. metronidazole (800 mg/day) is another antibiotic, although its adverse effects limit its use to 1 week at a time. lactulose is a substrate for gut bacteria and reduces the formation of ammonia by lowering intestinal ph. rifaximin class a = 5-6 points, b = 7-9 points, and c = 10-15 points inr international normalized ratio and lactulose are commonly used together, and the antibiotic is discontinued once eradication of disaccharide-metabolizing intestinal bacteria is indicated by an increase in stool ph. oral or parenteral ornithine aspartate, a substrate for the conversion of ammonia to urea and glutamine, has effects similar to those of lactulose, but with fewer adverse effects. in patients with severe encephalopathy, the molecular-absorbent recycling system (mars) may be utilized to remove small and middle molecular weight water-soluble substances (sorkine et al. 2001) . the system appears to increase blood pressure and systemic vascular resistance, possibly by removing nitric oxide. in fulminant hepatic failure, progressive hepatic coma is accompanied by a gradual increase in cerebral blood flow and intracranial pressure (icp) (see ▶ chap. 12, "fulminant hepatic failure: diagnosis and management"). subsequently, vasogenic cerebral edema and severe intracranial hypertension develop and approximately 30-50 % of patients die of brain herniation. monitoring of icp using a ladd epidural sensor is useful in detecting intracranial hypertension, monitoring the therapeutic effects, and identifying patients who would survive after transplantation without neurologic damage (lidorsky et al. 1992) . non-invasive neurologic assessment includes transcranial doppler (tcd) to measure cerebral bloodflow velocity, determination of the cerebral metabolic rate for oxygen by calculating the oxygen content difference between arterial and jugular bulb venous blood, evoked potentials, and serial computed tomography (ct) scans (aggarwal et al. 1994) . treatment includes osmotic and loop diuretics, barbiturateinduced coma, and hypothermia. the definitive treatment is usually transplantation. the presence of hyperdynamic circulation with a markedly increased cardiac output and decreased systemic vascular resistance was first described by kowalski and abelmann in the early 1950s (kowalski and abelmann 1953) . several hypotheses have been proposed to explain this phenomenon, including an overactive sympathetic nervous system, inadequate clearance of vasoactive substances by the diseased liver, the presence of arteriovenous shunts, nitric oxide-induced vasodilation, and relative hypoxia in peripheral tissues (benoit et al. 1984; yokoyama et al. 1989; kalb et al. 1993; d'souza et al. 1993) . although cardiac output is frequently two to three times normal, impaired systolic and diastolic function together with attenuated cardiac responsiveness to stimuli suggests that cardiomyopathy is present in cirrhotics (cirrhotic cardiomyopathy) (lee 1989) . caramelo et al. noted a 50 % decrease in cardiac output with volume expansion in a ccl 4 -induced cirrhotic rat model (caramelo et al. 1986) . in another rat model, the chronotropic response to isoproterenol was attenuated compared with that in control animals (lee et al. 1990 ). cardiac response to physical exercise is blunted in patients with cirrhosis, indicated by alterations in the pre-ejection period, isometric contraction time, and ratio of the pre-ejection period to left ventricular ejection time. in addition, abnormalities in myocardial diastolic indices suggest non-compliant ventricles. histologically, myocardial fibrosis, mild subendocardial edema, and vacuolation of myocyte nucleus and cytoplasms are observed. the development of cirrhotic cardiomyopathy is multifactorial. it appears that the β-receptor system, the main stimulant of the ventricle, is dysfunctional. in humans, lymphocyte β-receptor density, which reflects cardiac β-receptor status, is reduced in patients with severe ascites (gerbes et al. 1986) , and β-receptor density of the cardiomyocyte sarcolemmal plasma membrane is reduced in cirrhotic rats (liu and lee 1999) . further, the β-receptor signal transduction pathway is impaired at several levels (ma et al. 1996) . although cardiac contractile impairment may result from overactivity of the muscarinic m 2 receptor, the receptor density and binding affinity are unchanged, suggesting normal parasympathetic function (jaue et al. 1997) . high serum catecholamine levels, a result of desensitization and down-regulation of β-receptors, may lead to myocardial dysfunction in the presence of α-mediated coronary vasoconstriction. additionally, overproduction of nitric oxide inhibits β-receptor-stimulated cyclic adenosine monophosphate (camp) release, causing myocardial dysfunction and vasodilation (hare and colucci 1995) . coronary artery disease (cad) was previously believed to be relatively uncommon in patients with cirrhosis as a result of generalized vasodilation and elevated levels of hdl and estrogen. in addition, autopsy findings showed relatively fewer atherosclerotic changes and myocardial infarction. however, studies have shown that cad is not uncommon, and moderate-to-severe cad was found in approximately 27 % of patients who underwent coronary artery catheterization as a part of liver transplantation workup (carey et al. 1995) . in another study of 161 liver transplantation candidates who were at risk for cad and referred for coronary angiography, 25 % of patients had at least one moderate or severe (>50 %) coronary stenosis (tiukinhoy-laing et al. 2006) . endocarditis is three times more common in patients with liver disease (snyder et al. 1977) . this is attributed to translocation of intestinal bacteria through the intestinal wall and portosystemic collaterals, and reduced immune response. the incidence of pericardial effusion in cirrhotic patients is approximately 32-63 % and correlates with the degree of liver failure (shah and variyam 1988) . the effusion is usually small and may require drainage if it affects cardiac function. patients with liver disease exhibit three common cardiac electrophysiological disturbances: electromechanical dissociation, prolongation of ventricular repolarization (the q-t interval), and chronotropic incompetence (milani et al. 2007) . pulmonary hypertension associated with portal hypertension was first described in 1951 (mantz and craige 1951) . pulmonary hypertension defined as a mean pulmonary artery pressure of >25 mmhg and pulmonary vascular resistance of >240 dyn/s/cm à5 (3 wood units) is more common in patients with liver disease, with a prevalence of 0.25-0.73 % (lebrec and capron 1979; mcdonnell et al. 1983) . pulmonary artery pressure is a function of pulmonary venous pressure, pulmonary vascular resistance, and cardiac output [(pulmonary artery pressure = pulmonary venous pressure + (pulmonary vascular resistance â cardiac output)]. therefore, pulmonary hypertension is not uncommon in patients with liver disease because of their poor left ventricular compliance, increased pulmonary vascular resistance, and increased pulmonary blood flow from portosystemic shunting. the pathophysiology and management of pulmonary hypertension are well-described in ▶ chap. 10, "hepatopulmonary syndrome and portopulmonary hypertension". portal hypertension is caused by an increased intrahepatic vascular resistance and increased splanchnic blood flow. endothelin-1, a powerful vasoconstrictor produced by the sinusoidal endothelial cells, is known to increase intrahepatic vascular resistance and activates stellate cells, and its level increases as cirrhosis progresses (kojima et al. 2002; gandhi et al. 1996) . normally, vasodilatory compounds, such as nitric oxide, counterbalance the increased intrahepatic vascular resistance induced by endothelin. in liver cirrhosis, however, nitric oxide production is inhibited by caveolin-1, a hepatic membrane protein that binds with endothelial nitric oxide synthase. hypoxemia of varying severity is present in 45-69 % of patients with significant liver disease (krowka and cortese 1985) . the common causes are pleural effusions, impaired diffusion capacity, arteriovenous shunting, atelectasis caused by ascites or diaphragmatic dysfunction, aspiration secondary to encephalopathy, and deconditioning (hourani et al. 1991) . ventilation-perfusion mismatch, pulmonary vasodilation, and infection also contribute to hypoxemia. mild forms of hypoxemia are most common, although moderate-tosevere hypoxemia may be found in patients with advanced liver disease complicated by adult respiratory distress syndrome (ards), infection, and multiple organ failure. hepatopulmonary syndrome, first described by fluckiger in 1884 (fluckiger 1884) , may cause severe hypoxemia in a subset of patients with liver disease. the syndrome consists of a triad of liver dysfunction, severe hypoxemia (pao 2 < 70 mmhg in room air), and pulmonary vasodilation, and is characterized by dyspnea, cyanosis, clubbing of the digits, exercise desaturation, and orthodeoxia (hypoxemia in upright position). other concomitant clinical signs are a markedly increased alveolar-arterial oxygen gradient, portal hypertension, and vascular abnormality such as spider angioma and pulmonary vasodilation. the pulmonary vascular dilation (from 8-15 μ to 15-100 μ) at the precapillary level is believed to be the main pathology of the hepatopulmonary syndrome, which is caused by decreasing erythrocyte transit time and impairing diffusion of oxygen to the erythrocytes at the center of the bloodstream (genovesi et al. 1976 ). in contrast with other pulmonary diseases, oxygenation improves dramatically with a high inspired oxygen concentration (fio 2 ), because a high alveolar concentration of oxygen overcomes the diffusion barrier and oxygenates the erythrocytes in the center of the bloodstream. the pathophysiology and management of hepatopulmonary syndrome are described in ▶ chap. 10, "hepatopulmonary syndrome and portopulmonary hypertension". non-cardiogenic pulmonary edema occurs in 37-79 % of patients with advanced liver disease, particularly in those with fulminant hepatic failure, and appears to be associated with sepsis and a neurogenic mechanism. the presence of this complication is ominous: matuschak and shaw reported that all 29 patients who developed non-cardiogenic pulmonary edema died before liver transplantation (matuschak and shaw 1987) . in contrast, a rapid reversal of ards after liver transplantation has been reported (doyle et al. 1993) . pulmonary edema caused by fluid overload responds to diuretics and has a relatively benign course. pleural effusions are found on chest x-rays in about 10 % of patients. these are caused by the unidirectional passage of ascites via diaphragmatic defects into the pleural space. diagnostic thoracentesis is necessary to confirm the transudative nature and to exclude infection, malignancy, or embolic disease. optimal control of ascites may prevent symptomatic pleural effusions, and transjugular intrahepatic portosystemic shunt (tips) is effective in treating refractory hydrothorax in 84 % of patients (siegerstetter et al. 2001 ). approximately 10 % of hospitalized cirrhotic patients with ascites develop the hepatorenal syndrome, which is a form of acute pre-renal kidney injury caused by circulatory dysfunction secondary to an imbalance between circulating vasodilatory and vasoconstrictive substances. the primary contributing factor for the hepatorenal syndrome is nitric oxide-induced vasodilation of the splanchnic vascular bed causing systemic arterial underfilling and relative hypovolemia (arroyo et al. 1996) . this relative hypovolemia activates baroreceptor-mediated sympathetic and the renin-angiotensin system to constrict all vascular beds including the renal vasculature (guevara et al. 1998 ). the initial prostaglandin-mediated compensatory renal vasodilation is followed by renal arterial vasoconstriction and renal hypoperfusion. a striking feature of the hepatorenal syndrome is the lack of any histologic change and its reversibility: the affected kidneys resume their function after successful liver transplantation. the renal failure may be rapid (type 1) or insidious (type 2) and results in sodium and water retention and dilutional hyponatremia. since the hepatorenal syndrome is a functional renal failure, the urine is similar to that found in pre-renal azotemia: oliguria, low urinary sodium, and an increased urine osmolality and urine to plasma osmolality ratio. the major criteria for the diagnosis of the hepatorenal syndrome are as follows: (1) advanced hepatic disease and portal hypertension; (2) low glomerular filtration rate (serum creatinine >1.5 mg/dl or creatinine clearance <40 ml/ min); (3) absence of nephrotoxic drug use, shock, systemic infection, or recent fluid losses; (4) lack of sustained improvement after diuretic withdrawal and volume resuscitation with 1.5 l of normal saline; (5) proteinuria (<500 mg/dl); and (6) no ultrasound evidence of urinary obstruction or parenchymal disease. minor criteria include oliguria (<500 ml/day), urinary sodium <10 meq/l, urinary osmolality greater than plasma osmolality, urinary red blood cells (rbcs) <50/hpf, and serum sodium <130 meq/l. it is noteworthy that conventional renal function tests, such as bun and creatinine levels, overestimate renal function in patients with liver failure because malnutrition and muscle wasting contribute to a low creatinine level and liver dysfunction impairs urea synthesis. the hepatorenal syndrome is treated with the administration of vasopressin-1 agonists (i.e., terlipressin), tips, and, most reliably, liver transplantation. one uncontrolled trial using terlipressin with albumin for a median duration of 26 days (range 8-68 days) showed improvement in serum sodium as well as a decrease in the creatinine level below 2 mg/dl (mulkay et al. 2001) . hemodialysis is a temporary measure and its efficacy is not reliable. the only primary preventive measure showing some promise is the administration of albumin along with antibiotics as soon as the presence of spontaneous bacterial peritonitis is diagnosed; this possibly works by preventing hypovolemia and subsequent activation of vasoconstrictor systems. all phases of hemostasis are impaired in patients with liver disease, including clot formation, fibrinolysis, and their inhibitory processes. thrombocytopenia is found in 30-64 % of cirrhotic patients, and platelet count is commonly below 75,000/mm 3 . thrombocytopenia is primarily caused by splenomegaly associated with portal hypertension, which pools up to 90 % of platelets in the spleen. however, the degree of thrombocytopenia does not closely correlate with the size of the spleen. impaired hepatic synthesis of thrombopoietin also leads to thrombocytopenia. thrombopoietin is involved in the maturation and formation of platelets, and its return to a normal level coincides with a gradual increase in platelet count by the fifth day after liver transplantation (kawasaki et al. 1999) . other contributing factors are increased destruction of platelets by immune mechanisms, excessive activation of coagulation, and direct bone marrow suppression by toxins such as ethanol and folate deficiency. additionally, platelet dysfunction is common, as demonstrated by impaired platelet aggregation to adenosine diphosphate (adp), collagen, and thrombin (rubin et al. 1979) . the liver produces all coagulation factors except for von willebrand factor. therefore, plasma levels of clotting factors are directly related to the severity of liver disease, and prothrombin time (pt) is considered to be one of the most sensitive hepatic synthetic function tests. the plasma fibrinogen level, being an acutephase reactant, typically is normal or increased in chronic liver disease. a reduction in the fibrinogen level may indicate either a greatly reduced hepatic reserve or significant extravascular loss to ascites. markedly prolonged thrombin time indicates the presence of dysfibrinogenemia in some patients. dysfibrinogenemia is characterized by an excessive number of sialic acid residues in the fibrinogen molecule and abnormal polymerization of fibrin monomers. its clinical significance is unclear. patients with liver disease have a tendency to develop fibrinolysis due to decreased hepatic clearance of plasminogen activators, especially tissue plasminogen activator (tpa), and reduced production of α 2 -antiplasmin and thrombin activatable fibrinolysis inhibitors (van thiel et al. 2001) . elevated levels of d-dimers, fibrin degradation products, and plasminogen are present in ascitic fluid, indicating that absorption of ascitic fluid may contribute to the hyperfibrinolysis. on the other hand, excessive activation of coagulation is common in liver disease because of inadequate hepatic clearance of activated coagulation factors, reduced level of coagulation inhibitors, and enlarged vascular beds. the hypercoagulable state may lead to localized or disseminated intravascular coagulation (dic), particularly in the presence of sepsis, trauma, or major surgery. the diagnosis of excessive activation of coagulation is based on the presence of a known triggering factor and the progressively worsening of coagulation with thrombocytopenia. an anesthesia consultation is performed once a patient with esld is referred to the liver transplantation center. the type of liver disease is identified because patients with hepatocellular disease may have more pronounced hepatic dysfunction than those with cholestatic disease or hepatocellular cancer, and certain types of liver disease may affect other vital organ function (i.e., hemochromatosis, familiar amyloidosis, etc.). the anesthesia consultation is focused on evaluation of the functional reserve of extrahepatic organs, and various tests or specific consultations may be requested (table 2) . cardiovascular assessment is performed to determine two things: (1) whether a patient can be expected to survive the operation and immediate postoperative period; and (2) whether transplantation in patients with severe cardiopulmonary disease would be futile and an inappropriate use of a scarce donor organ (lentine et al. 2012) . a suggested strategy for cardiac assessment is shown in fig. 3 (raval et al. 2011) . overall cardiac performance is evaluated by transthoracic echocardiography to assess myocardial contractility, abnormality in cardiac anatomy, intracardiac or intrapulmonary shunting, and pulmonary artery pressure. most patients over age 50 years undergo non-invasive stress testing because they may have multiple cad risk factors (i.e., diabetes, hypertension, hyperlipidemia, and pre-existing cardiovascular disease), and limited physical activity masks underlying ischemic heart disease. an exercise stress test may not be feasible in many patients with advanced liver disease, and dobutamine stress echocardiography (dse) is commonly used, although adenosine or dipyridamole may be used when dobutamineinduced tachycardia is not desirable. dse, with its high sensitivity and specificity, appears to be the most reliable screening test (plotkin et al. 1998) , and dobutamine-induced tachycardia may mimic intraoperative stress on the cardiovascular system. on the contrary, dse has been reported to have poor sensitivity (as low as 13 %) and negative predictive value (as low as 75 %) (harinstein et al. 2008) , and its results may not correlate with adverse cardiac events within 30 days after transplantation (safadi et al. 2009 ). cardiac ct scan is a non-invasive technique measuring calcium deposits within the coronary vasculature. the total amount of calcium, adjusted to the age and gender of the patient, is reported as a calcium score. high scores suggest a greater potential for coronary artery stenosis (shaw et al. 2003; o'rourke et al. 2000) , and a calcium score of >400 has a predictive value of cardiac complications within 1 month after transplantation (kemmer et al. 2014 ). this test, however, may have limited predictive value as a single screening study for cad. cardiac ct angiography is an alternative to invasive coronary angiography. it does appear to have negative predicting value of 100 % for clinical coronary events in patients undergoing liver transplantation (cassagneau et al. 2012 ) but may not be suitable for the diagnosis of obstructive lesions at this time. because of the difficulty in diagnosing cad using non-invasive testing methods, coronary angiography is recommended for patients with a positive dse or multiple high-risk factors to identify the degree and type of obstruction. in addition, coronary angiography should be able to detect non-obstructive lesions (coronary artery stenosis <50 %), which are unlikely to be detected by stress tests but can be responsible for acute coronary syndromes (unstable angina, myocardial infarction, or sudden cardiac death) (rubin et al. 1994; gulati et al. 2009 ). cardiac catheterization, however, can be difficult in patients with severe liver disease due to bleeding complications and the increased risk of contrast-induced nephropathy (sharma et al. 2009 ). if significant coronary artery stenosis (>70 % stenosis) is detected, revascularization may be table 2 liver transplantation evaluation at the thomas jefferson university hospital attempted before liver transplantation. bare metal stents are favored over drug-eluting stents to avoid the need for long-term antiplatelet therapy (6 weeks vs. 1 year). when angioplasty is not amenable, coronary artery bypass grafting (cabg) is performed. it is clear that 1-year survival after cabg is greater in patients with child-pugh class a (80 %) than with child-pugh class b (45 %) and c (16 %) (filsoufi et al. 2007 ). therefore, patients with child-pugh class a can undergo cabg relatively safely while waiting for liver transplantation. on the other hand, patients with child-pugh class b and c may require simultaneous cabg and liver transplantation. patients with mild-to-moderate valvular disease undergo liver transplantation without excessive complications. similar to that of cabg, mortality after corrective valvular surgery depends on the severity of liver disease. therefore, child-pugh class c patients with severe aortic or mitral valve stenosis may undergo percutaneous balloon valvuloplasty or simultaneous valve replacement with cardiopulmonary bypass and liver transplantation. myocardial disease is commonly detected by transesophageal echocardiography (tee). patients with chronic cardiomyopathy may have attenuated systolic contraction and diastolic relaxation, altered repolarization, and reduced cardiac response to β stimulation (liu et al. 2002 for pulmonary evaluation, results of chest x-ray, arterial blood oxygen tension in 100 % oxygen, and spirometry are reviewed to identify the degree of pulmonary shunting, obstructive, or restrictive disease. when the hepatopulmonary syndrome is suspected, contrast tee or tc-99 m macro aggregated albumin scintigraphy may be performed for its definitive diagnosis (krowka et al. 2000) . for evaluation of renal function, results of bun, creatinine, glomerular filtration rate, levels of serum and urine electrolytes, urine output, and renal ultrasound are reviewed. the diagnosis criteria of the hepatorenal syndrome have been described earlier. in patients with chronic renal failure, simultaneous liver and kidney transplantation is performed, the criteria for which are end-stage renal disease with dialysis, no dialysis but a glomerular filtration rate <30 ml/min and proteinuria >3 g/day with a 24-h urine protein/creatinine ratio >3, and acute kidney injury requiring dialysis at least twice per week for more than 6 weeks (charlton et al. 2009 ). in patients with fulminant hepatic failure, reversibility of the neurologic function should be investigated using clinical signs, eeg, brain ct scan, the cerebral metabolic rate of oxygen, and tcd. in addition, icp monitoring is recommended when a high icp (>25 mmhg) is suspected, although its benefit should be weighed against potential complications (vaquero et al. 2005) . poor prognostic indicators of fulminant hepatic failure are progressive hepatic failure for 7-14 days, grade 3-4 encephalopathy, intracranial hypertension, cerebral swelling, severe coagulopathy, rapid shrinkage of the liver, metabolic acidosis, hemodynamic instability, and sepsis. for the coagulation system, pt, activated partial thromboplastin time (aptt), and platelet count are reviewed. in general, no specific coagulation therapy is requested because of the potential long waiting period and fluid overloading. abdominal magnetic resonance imaging (mri) is reviewed to assess the degree of portosystemic shunting and anatomy of hepatic vasculature. additional consultation may be requested from various specialists to identify the type and severity of the specific organ dysfunction. after the evaluation, all information of the potential recipient is compiled to stratify whether the patient's condition can be optimized or meet the criteria of contraindications. contraindications are diseases or conditions patients could have that may not improve survival after liver transplantation. they include malignancy with poor prognosis, active bacterial and viral infection, severe cardiopulmonary dysfunction, and technical difficulties. active alcoholism is a contraindication, although demonstrable abstinence for 6 months is considered acceptable. the presence of multiple organ dysfunction is a relative contraindication for liver transplantation as the 2-year survival is approximately 25 %. indications and contraindications of liver transplantation, however, have evolved over the past 50 years, and further modifications are expected to occur. anesthesiologists participate in the transplantation candidate selection committee for discussion of the hepatic disease, its complications, and the extrahepatic organ function of each patient. once the patient is placed on the active candidate list, the united network for organ sharing (unos) is notified and the patient is given a meld (model for end-stage liver disease) or peld (pediatric end-stage liver disease) score for fair distribution of donor livers. although surgical techniques are fully described elsewhere, a brief description of their physiologic effects is warranted here. in olt, after removal of the diseased liver, the donor liver is placed anatomically in the right upper quadrant. for the convenience of description, the procedure is divided into three stages: stage 1 (dissection stage), stage 2 (anhepatic stage), and stage 3 (neohepatic stage). the dissection stage begins with an inverted y-shaped bilateral subcostal skin incision and ends with the skeletonization of the diseased liver. the anhepatic stage begins with the occlusion of the hepatic artery, portal vein, and ivc for hepatectomy. however, the patient is virtually anhepatic once the hepatic artery or portal vein is occluded. three surgical techniques are used for hepatectomy and vascular reconstruction during the anhepatic stage: olt with simple venous crossclamping, olt with venovenous bypass, and the piggyback technique. in olt with simple venous cross-clamping, the diseased liver is removed together with the retrohepatic portion of the ivc after crossclamping of the suprahepatic and infrahepatic ivc, hepatic artery, and portal vein (fig. 4) . after surgical hemostasis of the hepatic bed, the donor liver is placed in the right upper quadrant, and sequential anastomoses of the suprahepatic ivcs, infrahepatic ivcs, portal veins, and hepatic arteries are performed. during the infrahepatic ivc anastomosis, the liver allograft is flushed with 1000 ml of cold lactated ringer's solution or 5 % albumin solution through a cannula in the portal vein. this flush technique allows preservation solution, metabolites, and air in the donor liver to escape through the incompletely anastomosed infrahepatic ivc. a second flush may be used by allowing 300-500 ml of blood to escape through the incompletely anastomosed portal vein by unclamping the infrahepatic ivc (back-bleeding technique). when the portal vein of the recipient is less than optimal, the superior mesenteric vein, collateral vein, or venous graft may be used for portal blood supply. the hepatic artery is reconstructed by end-to-end hepatic arterial anastomosis. however, an arterial graft is placed between the graft hepatic artery and the infrarenal aorta of the recipient with a side clamp on the aorta when the size or anatomy of the recipient hepatic artery is less than optimal. the liver is reperfused by the sequential unclamping of the infrahepatic ivc, portal vein, suprahepatic ivc, and hepatic artery. after hemostasis, choledochocholedochostomy is performed frequently with a t-tube. choledochojejunostomy using a roux-en-y loop is performed when the bile ducts are diseased or mismatched in size. the abdomen is closed once the absence of foreign bodies in the peritoneal cavity is confirmed. in patients with a large graft or swollen intestine, the abdomen may require secondary closure. olt with venovenous bypass was developed in 1983 to minimize reduction of venous return associated with the cross-clamping of the ivc and portal vein by diverting blood from the ivc and portal vein to the axillary vein using a centripetal magnetic pump (shaw et al. 1984) . once the hepatic hilum is dissected, cannulas are inserted into the left superficial femoral vein (7 mm) and portal vein (9 mm) for outflow from the patient and into the left axillary vein (7 mm) for venous inflow. the cannula site and size may vary depending on the preference of the surgical team or anatomic variations. the cannulas and heparin-bonded tubings are flushed with heparin solution (2000 u/l) to avoid thrombosis during preparation. systemic heparinization is not used because of the presence of pre-existing coagulopathy and the use of heparin-bonded tubings. the bypass run begins by unclamping all cannulas while the pump speed is gradually increased to achieve the maximal flow rate. hepatectomy and anastomoses of the suprahepatic and infrahepatic ivc are performed once full bypass is achieved. the removal of the portal cannula for portal venous anastomosis leads to a partial bypass, which reduces venous return. bypass is terminated after the engrafted liver is reperfused, and cannulas are removed. the advantages of venovenous bypass are (1) well-preserved cardiac output by uninterrupted venous return from the viscera and lower extremities; (2) effective decompression of the portal venous system, which decreases bleeding and intestinal congestion; (3) avoidance of renal congestion, oliguria, and hematuria; and (4) simplified anhepatic stage allowing meticulous hepatectomy and vascular anastomoses. long-term complications are neurovascular injury, thrombosis, infection, lymphocele, and seroma at the cannulation sites. as an alternative to the traditional venovenous bypass technique, percutaneous cannulation was introduced. in this technique, inflow to the patient is achieved by percutaneous cannulation of the right internal jugular vein (16-20 french) performed by the anesthesia team using a seldinger technique, and outflow from the patient by percutaneous cannulation of the left femoral vein (16-20 french) and a portal cannula by the surgical team. this technique is generally safe, but the inadvertent extravascular placement of an inflow cannula may cause a massive hemothorax (sakai et al. 2007 ). the piggyback technique was originally designed for patients with significant cardiovascular disease, portacaval shunt, superior vena caval syndrome, or small donor livers (tzakis et al. 1989) . in this technique, the diseased liver is removed without the retrohepatic portion of the ivc by peeling the diseased liver off the ivc after transaction of the hepatic veins, hepatic artery, and portal vein. therefore, systemic venous return can be relatively well preserved via the intact ivc during the anhepatic stage. vascular anastomoses are made between the reconstructed ostia of the recipient by combining hepatic veins and the suprahepatic ivc of the graft for the drainage of the hepatic venous blood. the portal vein of the recipient and the graft are anastomosed for portal blood supply, and the infrahepatic ivc of the graft is ligated. the neohepatic stage begins with reperfusion of the grafted liver by sequential unclamping of the infrahepatic ivc, portal vein, suprahepatic ivc, and hepatic artery, although the sequence of unclamping may vary depending on the surgical technique. reperfusion is followed by hepatic arterial anastomosis (if it has not been performed already), biliary reconstruction, and closure of the abdomen. immediate preoperative consultation is made when a donor organ is identified. the patient is re-evaluated to identify any interval changes during the waiting period. anesthetic and postoperative management and their risks are explained to the patient one more time. in general, pre-medication is withheld in most cases because of potential encephalopathy and hypovolemia, and narcotics (e.g., fentanyl 1-5 μg/kg) are commonly administered intravenously in the operating room. necessary medications and anesthesia equipment are listed in table 3 . a device that delivers fluids and blood rapidly on demand is considered standard equipment (i.e., fms2000 ® fluid warming system, belmont instrument corp., billerica, ma, usa) (elia and kang 2002 ). an autotransfusion system is helpful in minimizing the need for bank blood (dzik and jenkins 1985; kang et al. 1991) . a system that monitors coagulation, either a conventional coagulation profile, thromboelastography r with circle (teg; haemonetics, braintree, ma, usa), or (kang 1986 (kang , 1997 . teg and rotem provide similar physical properties of blood coagulation, although teg monitors shear elasticity and rotem monitors viscoelasticity. in general, 20 units each of cross-matched packed rbcs (prbcs) and fresh frozen plasma (ffp) are available at all times, and 10 units of each are prepared in the operating room. platelets (10-20 units) should be available on demand. two large-bore intravenous (iv) catheters (up to 8.5 or 9 french) are secured, typically in the right antecubital and right or left internal jugular vein. when the antecubital vein is unavailable, two catheters may be placed in the same internal jugular vein. catheter patency is confirmed by noting the line infusion pressure of <300 mmhg during fluid infusion at 500 ml/ min. sterile technique should be followed during catheterization, and antiseptic ointment or antiseptic patch is applied at the skin puncture site. a nasogastric tube is placed with copious lubrication and topical vasoconstrictor to avoid nasal or esophageal variceal bleeding. proper monitoring is prerequisite to a successful outcome because patients undergoing liver transplantation develop clinically significant hemodynamic, hematologic, metabolic, and other homeostatic abnormalities. non-invasive monitoring is similar to that for patients undergoing any major surgery. for invasive monitoring, two intra-arterial catheters (20 gauge in the left radial artery and 16-18 gauge in the right femoral artery) are used at the thomas jefferson university hospital. femoral arterial pressure monitoring is preferred because it reflects central arterial blood pressure more accurately in the presence of low systemic vascular resistance, particularly after reperfusion (lee et al. 2015) . radial arterial pressure monitoring is useful for blood sampling and backup pressure monitoring when the aorta is partially or completely clamped during aorta-tohepatic artery anastomosis. a pulmonary artery catheter (pa catheter) is inserted via the right internal jugular vein to monitor cardiac output, intracardiac pressures, and core temperature. carotid artery puncture should be assiduously avoided because of the presence of coagulopathy. an oximetric-type pa catheter provides additional information on mixed venous hemoglobin oxygen saturation (svo 2 ). the right ventricular ejection fraction-type pa catheter monitors the right ventricular ejection fraction and right ventricular end-diastolic volume. it has been shown that central venous pressure (cvp) and pulmonary capillary wedge pressure (pcwp) are not as sensitive as right ventricular end-diastolic volume in estimating preload, particularly during the anhepatic stage (dewolf et al. 1993b ). recently, non-invasive, continuous cardiac output monitoring was introduced; however, the technique is not reliable in monitoring cardiac output in hyperdynamic patients. in some centers, a cvp catheter is used instead of a pa catheter. this, of course, is justified if hemodynamic derangement is kept minimal during the entire surgical procedure. however, most centers use a pa catheter for three reasons: (1) hemodynamic instability can be unpredictable during liver transplantation; (2) determination of cardiac output and preload is more clinically significant than cvp monitoring; and (3) it is an important educational tool for trainees. tee is used in all patients at the thomas jefferson university hospital to monitor myocardial contractility, ventricular end-diastolic volume, wall motion abnormality, air or thromboembolism, intrapulmonary shunting, and patency of the reconstructed major veins. a tee probe may cause esophageal variceal bleeding (burger-klepp et al. 2012) and it should therefore be placed gently. various laboratory tests are performed, including arterial blood gas tension and acid-base state, and serum level of electrolytes, ionized calcium, glucose, lactate, and ionized magnesium if available. typical test times are before and after induction of anesthesia, every hour during the dissection stage, 5 min after the onset of the anhepatic stage, every 30 min during the anhepatic stage, 15 min before reperfusion, 5 and 30 min after reperfusion, and every hour thereafter. coagulation is monitored by conventional coagulation profile (pt, aptt, fibrinogen level, and platelet count) and teg or rotem at the following times: before induction of anesthesia, every hour during the dissection stage, 15 and 60 min after onset of the anhepatic stage, 15 min before reperfusion, 5 and 30 min after reperfusion, and every hour thereafter. monitoring of teg or rotem and the platelet count is preferable as a conventional coagulation profile has several drawbacks when used during liver transplantation (kang 1995) . pt is a very sensitive hepatic function test and is prolonged in most patients undergoing liver transplantation. administration of ffp to correct the pt may not be possible or desirable in the course of surgery. aptt follows a similar time course to pt, and its correction may not be practical. it is a sensitive test for the heparin effect, and its prolongation indicates the presence of heparin released from the bypass circuit or grafted liver. the fibrinogen level is frequently maintained within the acceptable range, although severe hypofibrinogenemia may indicate either active fibrinolysis or excessive activation of coagulation. the level of fibrin(ogen) degradation products is usually elevated in most patients due to excessive activation of coagulation and reabsorption of defibrinated blood from the abdominal cavity and does not have any immediate clinical significance. further, coagulation profile results may not be available in a timely manner. teg/rotem has several advantages over a conventional coagulation profile and has been accepted as a standard coagulation monitoring tool by the asa (american society of anesthesiologists 2015). it rapidly and reliably measures blood coagulability (quality) instead of the quantity of each coagulation component. an accurate differential diagnosis can be made for replacement therapy and pharmacologic therapy by comparing teg/rotem of untreated blood with that of blood treated with various blood components (ffp, platelets, cryoprecipitate) or pharmacologic agents (protamine sulfate, heparinase, ε-aminocaproic acid [eaca], aprotinin) (fig. 5 ) . lastly, circumferential identification tags around the wrists or ankles are removed to avoid the compartment syndrome. both arms are placed on padded arm boards in an abducted position, and excessive abduction should be avoided to prevent a plexus stretch injury. the extremities are protected with foam padding to avoid pressure injuries. a rapid-sequence induction is preferred because of uncertain gastric emptying. anesthesia is commonly induced with propofol (2-3 mg/kg) or etomidate (0.3 mg/kg), and fentanyl (2-5 μg/kg) is frequently added. succinylcholine (1-2 mg/kg) or rocuronium bromide (1.2 mg/kg) is used to facilitate intratracheal intubation. anesthesia is maintained using volatile inhalation agents and narcotics. isoflurane is the preferred inhalation agent because its effect includes less myocardial depression and biotransformation. nitrous oxide is avoided because it distends the bowel and increases the size of any entrained air. midazolam (1-4 mg) may be added for amnesia. for muscle relaxation, protamine-treated blood untreated blood 5 min after reperfusion fig. 5 effects of pharmacologic agents on pathologic coagulation immediately after reperfusion (from kang yg (1986) monitoring and treatment of coagulation. in: winter pm, kang yg (ed) hepatic transplantation, anesthetic and perioperative management. prager, new york, with the permission of the publisher) rocuronium bromide, vecuronium bromide, or cisatracurium besilate are commonly used. antibiotics and immunosuppressants administered during surgery may vary from center to center. at the thomas jefferson university hospital, unasyn ® (ampicillin/sulbactam 3 g) is given before incision and every 4 h thereafter. for patients allergic to cephalosporin or penicillin, vancomycin is administered within 2 h before skin incision (1 g for patients <80 kg and 1.5 g for those >80 kg). for immunosuppression, methylprednisolone (500 mg iv) and basiliximab (20 mg iv) are given during the anhepatic stage and tacrolimus is given in the postoperative period. liver transplantation imposes a great deal of physiologic stress on patients, and maintenance of physiologic homeostasis is essential to a successful outcome. the goal of hemodynamic management is to optimize tissue perfusion by maintaining the hyperdynamic state characteristic of esld. in general, there are two schools of thought about maintaining hemodynamic stability. the first endorses maintaining the hyperdynamic state to optimize cardiac output and tissue perfusion. patients with esld have generalized vasodilation and are known to have oxygen debt at the tissue level. therefore, maintaining the hyperdynamic state, instead of 'normal blood pressure,' ensures ample oxygen delivery to tissues and avoids tissue acidosis. this, in turn, optimizes tissue metabolism and hepatic blood flow. the second school of thought endorses maintaining arterial blood pressure within the normal range. this may include the use of various vasopressors (phenylephrine, norepinephrine, vasopressin, etc.) with or without hypovolemia. in extreme cases, blood is removed from the patient to induce hypovolemia, and blood pressure is supported by vasopressors (massicotte et al. 2010) ; it has been claimed that blood loss is minimal without increasing perioperative complications, although fluid restriction may lead to tissue ischemia, renal failure, and air embolism (melendez et al. 1998; schroeder et al. 2004 ). further, α-vasopressors (norepinephrine and phenylephrine) decrease hepatic blood flow by reducing portal venous flow dramatically in the presence of a limited hepatic arterial buffer response (mehrabi et al. 2005 ). hemodynamic instability represented by reduced cardiac output and hypotension is typically caused by hypovolemia associated with drainage of ascites, rapid third-space fluid loss, surgical bleeding, and inadvertent compression of major vessels (ivc, portal vein, hepatic veins, and aorta). intravascular volume is usually replenished by administration of a mixture of prbcs and ffp (typically, prbc:ffp: plasmalyte-a ® = 200:300:250 ml) using a rapid-infusion device. this mixture yields hematocrit of 26-28 vol.% and coagulation factor levels of 30-50 % of normal. a low hematocrit is chosen to optimize microcirculation and minimize the rbc wastage. calcium-containing fluid (i.e., lactated ringer's solution) should not be used to prevent clot formation in the reservoir of the rapid-infusion device. continuous administration of ffp is necessary to compensate for the loss of coagulation elements (procoagulants, prolysins, and their inhibitors) by surgical bleeding and excessive activation of coagulation. in patients with minimal blood loss, colloids (albumin or ffp) and crystalloids may be required to compensate for the third-space fluid loss and continuous production of ascites. close communication with the surgical team is essential to identify the cause of hemodynamic instability, as is communication with the blood bank to facilitate adequate supply of blood products. intraoperative autotransfusion has been shown to be effective and safe during liver transplantation, and its use may be considered when the prbc requirement is >5 units. its use is not recommended for patients with peritoneal infection or malignancy (liang et al. 2008 ). when lower cardiac output and/or hypotension persists even with adequate preload, dopamine or epinephrine may be infused for patients with hypotension, while dobutamine can be used when patients are normotensive. high venous pressures (cvp and pcwp) may be seen in patients with volume overload, large ascites, and pleural or pericardial effusion. drainage of ascites and effusion may decrease intrathoracic pressure and central venous pressures and improve cardiac performance. thoracentesis and pericardiocentesis can be performed after the abdomen is opened to minimize the risk of injury to the thoracoabdominal organs. unexpected pulmonary hypertension may be observed in some patients. because of the high perioperative mortality in patients with pulmonary hypertension, it deserves a thorough intraoperative investigation. the pa catheter should be able to differentiate between pulmonary hypertension with high pulmonary vascular resistance and pulmonary hypertension with fluid overloading. pulmonary hypertension caused by fluid overloading may dissipate gradually by intraoperative fluid loss, and phlebotomy may be required in severe hypervolemia. in portopulmonary hypertension with increased pulmonary vascular resistance, the presence of right ventricular function is investigated. a low cardiac output with a high cvp suggests the presence of right ventricular dysfunction. tee findings of right ventricle dysfunction are low fractional area change (fac), tricuspid annular plane excursion (tapse) of <16 mm, flattening of the ventricular septum, apicalization of the right ventricle, and right ventricular dilation. additionally, the pulmonary vascular response to various vasodilators (i.e., diltiazem, nitroglycerin, epoprostenol, and nitric oxide) may be evaluated. liver transplantation may continue when pulmonary hypertension is mild to moderate with normal right ventricular function. in such cases, right ventricular function is supported by maintaining optimal preload and improving myocardial contractility by inotropes. complications of massive blood transfusion (ionic hypocalcemia, ionic hypomagnesemia, hyperkalemia, and acidosis) may develop at this stage and should be treated aggressively. normothermia can be well-maintained even during massive transfusion when a rapid-infusion device is used. hemodynamic changes that occur during the anhepatic stage are caused primarily by interruption of venous return from the ivc and portal vein. in the simple cross-clamping technique, clamping of the ivc and portal vein reduces venous return by up to 40 %, leading to low cardiac output, hypotension, and compensatory tachycardia (pappas et al. 1971) . calculated systemic vascular resistance is frequently elevated, although this is a reflection of the exclusion of the vascular tree of the lower extremities and splanchnic bed. it is noteworthy that cross-clamping of the ivc and the portal vein decreases the central blood volume and pressure (cvp and pcwp) but progressively increases total intravascular blood volume as blood is sequestered in the vascular bed of the gastrointestinal and pelvic organs, kidneys, and lower extremities. a prolonged low output state, portal hypertension, and renal venous congestion may lead to acidosis, intestinal swelling, and hematuria. we, at thomas jefferson university hospital, prefer to treat the low output state by administration of fluid and/or inotropes (dopamine or dobutamine 2-5 μg/kg/min). venovenous bypass is more physiologic technique than a simple cross-clamping technique as it returns venous blood from the portal and ivc system (shaw et al. 1984) . hence, hemodynamic changes that occur during the anhepatic stage with venovenous bypass are minimal when the bypass flow rate is greater than 25 % of the baseline cardiac output and, therefore, the bypass flow should be monitored and adjusted as needed. improper positioning of the cannula tip in the femoral or portal vein, or a kinked bypass circuit, may not drain blood adequately and the surgical team should correct their positions. a low pump speed reduces venous return, while a high pump speed collapses the outflow venous wall and decreases the bypass flow. the perfusionist, therefore, should adjust the pump speed to maximize the bypass flow. in addition, hypovolemia decreases the bypass flow, and it should be corrected by the anesthesia team. the anesthesia and surgical teams should be prepared for potential acute complications of venovenous bypass. bleeding or air entry may result from venous laceration during cannulation or improperly secured cannulas. entry of a small volume of air (up to 50 ml) into the bypass pump may not cause immediate systemic air embolism because it is trapped in the cone-shaped pump head by centripetal force. thromboembolism may be caused by the migration of pre-existing thrombi or those developed during a low bypass flow rate (<1000 ml/min), particularly in hypercoagulable conditions (i.e., budd-chiari syndrome, neoplasms, and congenital protein c deficiency). most importantly, the bypass may have to be terminated unexpectedly when serious complications occur. therefore, the anesthesia team should be prepared for unexpected cross-clamping of the ivc and portal vein at all times. after completion of the ivc anastomosis, the portal cannula is removed to facilitate the portal venous anastomosis, resulting in partial bypass. low bypass flow and low cardiac output during this period can be improved by the administration of fluids or dopamine, but full correction of central hypovolemia, as reflected on cvp, pcwp, or tee, is avoided to prevent fluid overload on reperfusion. in the original description of the piggyback technique, adequate venous return is maintained through the intact ivc and portal vein using portoaxillary venovenous bypass (tzakis et al. 1989 ). currently, many transplantation centers do not incorporate the portoaxillary venovenous bypass in the piggyback technique, which makes patients vulnerable to significant hypovolemia. hepatectomy in the presence of portal hypertension can be difficult, and hypovolemia is not uncommon as a consequence of inadvertent compression of the ivc and portal vein, partial side-clamping of the ivc, and cross-clamping of the portal vein during portal anastomosis. hence, temporary portacaval shunt or portal-axillary venovenous bypass may be instituted in surgically challenging patients to maintain preload. as described earlier, 1000 ml of cold lactated ringer's solution or albumin (5 %) is flushed through the portal vein and drained via the incompletely anastomosed ivc to remove preservative solution, metabolites, and air from the allograft. additionally, approximately 300-500 ml of blood may be allowed to escape through the incompletely anastomosed portal vein to enhance the washout by partial unclamping of the infrahepatic ivc (back-bleeding technique) immediately before reperfusion of the grafted liver. in this case, blood should be administered simultaneously to avoid hypovolemia. other factors that affect circulation during the anhepatic stage are similar to those of the dissection stage, although lactic acidosis, citrate intoxication, hypomagnesemia, hyperkalemia, and coagulopathy are more pronounced. hyperkalemia is treated by dextrose (5-10 g) and insulin (5-10 units) to move potassium intracellularly (dewolf et al. 1993a ). in severe hyperkalemia, prbc or phlebotomized blood can be washed using an autotransfusion system to remove potassium before transfusion (ellis et al. 1987) . at the end of the anhepatic stage, all biochemical variables are normalized to prepare for reperfusion. significant hemodynamic changes occur on reperfusion of the grafted liver (fig. 6) . unclamping of the infrahepatic ivc and portal vein results in transient hypovolemia and hypotension due to acute sequestration of the blood in the engrafted liver. unclamping of the suprahepatic ivc increases preload by mobilizing blood from the low extremities and splanchnic circulation. this is followed by severe hemodynamic changes, the so-called postreperfusion syndrome (aggarwal et al. 1993) . the postreperfusion syndrome, which occurs in approximately 30 % of patients, is defined by abrupt hypotension (below 70 % of the baseline value) that develops within 5 min of reperfusion and lasts for more than 1 min. other associated hemodynamic changes are bradycardia, high cvp and pcwp, low systemic vascular resistance, and conduction defects. acute reduction in myocardial contractility is observed in tee. the postreperfusion syndrome appears to be caused by a combination of several factors. for example, an acute increase in preload may result in right ventricular strain and an acute decrease in blood temperature (2-3 c) by the systemic entry of the cold preservation solution may decrease cardiac conduction and contractility. other physical factors are air embolism and thromboembolism, which may cause right ventricular strain or right ventricular outflow tract obstruction (ellis et al. 1989; suriani et al. 1996) . chemical factors involved are acute hyperkalemia and acidosis. systemic entry of hyperkalemic preservation solution increases serum potassium level to a very high level (up to 12 mmol/l), causing severe bradycardia and conduction defects (martin 1986 ). return of the acidic blood from the viscera and lower extremities increases the base deficit by 5-10 mmol/l. in addition, unknown endogenous vasodilators or myocardial depressants (i.e., vasoactive intestinal polypeptide, nitric oxide, and eicosanoid) released from the allograft or congested viscera may decrease systemic vascular resistance and impair myocardial function. several measures may be taken to prevent the postreperfusion syndrome, although they are not always successful. at the end of the anhepatic stage, blood volume is adjusted to avoid fluid overloading on reperfusion, and ionic hypocalcemia, hyperkalemia, and metabolic acidosis are corrected. prophylactic administration of cac1 2 (15 mg/kg), nahco 3 (0.5-1 mmol/kg), regular insulin (10 units), 50 % dextrose (1 ml/kg), and epinephrine (5-10 μg) are recommended by some centers (ellis et al. 1989) . once the postreperfusion syndrome develops, severe hypotension and bradycardia are treated with small doses of epinephrine (5 μg increments) to support contractility, heart rate, and vasomotor tone, followed by a dopamine or epinephrine infusion, if necessary. symptomatic hyperkalemia (tall, peaked-t wave, and widening qrs complex with bradycardia) is treated by administration of cac1 2 (15 mg/kg) and nahco 3 (0.5-1 mmol/kg). arrhythmias are treated in the standard fashion. when pulmonary edema develops, positive end-expiratory pressure (peep) is applied and inotropes may be given. patients who develop intracardiac or pulmonary embolism are supported by inotropes. when severe fluid overloading is a concern, phlebotomy may be considered. the postreperfusion syndrome dissipates gradually over the next 5-15 min, although low (1987)) systemic vascular resistance and hypotension with a high cardiac output may persist for several hours. when hypotension is suspected to cause tissue and myocardial ischemia, it may be treated with ephedrine, dopamine, or epinephrine. overzealous administration of fluids may result in hepatic congestion, while norepinephrine may interfere with hepatic blood flow by decreasing portal venous flow. octreotide and vasopressin may increase arterial blood pressure by decreasing portal pressure and flow, although its effects on hepatic circulation and metabolism are unclear (fayed et al. 2013; wagener et al. 2008) . hemodynamic changes that occur during hepatic arterial and biliary reconstruction are relatively minor, except for intermittent fluctuation of the preload associated with continuous third-space fluid loss and compression of the liver and great vessels. gas exchange is maintained satisfactorily in most patients. minute volume is gradually decreased during the anhepatic stage to match the reduced oxygen consumption and carbon dioxide production, and is increased during the neohepatic stage. alveolar recruitment maneuvers are performed intermittently to avoid atelectasis caused by pleural effusions, cephalad traction of the rib cage, and compression of diaphragm. intermittent endotracheal suctioning, using a suction catheter or bronchoscope, may be required to remove secretions. drainage of pleural effusions and ascites decreases intrathoracic pressure and improves oxygenation within 2 h. patients with preoperative ards may require a high fio 2 and a high level of peep to ensure adequate gas exchange, and a volume ventilator may be necessary to overcome the high airway pressure. frank pulmonary edema can develop, particularly after reperfusion, from the increased pulmonary capillary permeability or fluid overload. in such cases, patients are ventilated with a high level of fio 2 and peep, while the underlying cause is treated. closure of the abdominal cavity may interfere with ventilation by increasing intrathoracic and airway pressures. primary closure with mesh or secondary closure may be necessary. in patients with fulminant hepatic failure, preoperative cerebral monitoring is continued as cerebral hyperemia and intracranial hypertension persist during surgery, although they are somewhat attenuated by general anesthetics. however, a sudden increase in preload may dramatically exacerbate intracranial hypertension on reperfusion of the grafted liver. hence, optimal preload should be maintained during the entire procedure. after reperfusion, cerebral hyperemia may gradually decrease as the liver begins to function. intraoperative changes in coagulation are summarized in table 4 . surgical bleeding is common due to numerous collateral vessels associated with portal hypertension, difficulty in dissection of the diseased liver, pre-existing coagulopathy, and pathologic changes in coagulation. the average blood loss in adults is 5-15 units each of prbc and ffp, although blood loss may reach more than 100 units each. during the dissection stage, dilutional coagulopathy develops as bleeding reduces the levels of coagulation factors and platelets (fig. 7) . fibrinolysis may develop, particularly in patients with hepatocellular disease, as a result of a low level of inhibitors of fibrinolysis and impaired hepatic clearance of tpa (lewis et al. 1989a) . excessive activation of coagulation, evidenced by a gradual increase in thrombin-antithrombin complex, develops at the end of the dissection stage (kratzer et al. 1991) . management of coagulation begins with normalization of physiologic variables, such as ionic hypocalcemia, hypothermia, and acidosis impair coagulation (rohrer and natale 1992) . this is followed by continuous infusion of coagulation factor-rich blood (rbc:ffp: plasmalyte-a ® or normal saline = 1 unit:1 unit:250 ml) to maintain coagulation factor levels above the critical level (30-50 % of normal). specific blood components may be administered based on teg/rotem. in general, platelets (5-10 units) are administered for a small maximum amplitude (ma) (<40 mm). platelet administration, in addition to increasing ma, improves reaction time (r) and clot formation rate (α), because the coagulation cascade leading to fibrin formation occurs on the surface of platelets. its administration, however, is withheld during the anhepatic stage to avoid potential thrombosis and during massive blood transfusion (>150 ml/min) to minimize wastage. two units of ffp may be administered when the reaction time is prolonged (r> 12 min) even after platelet administration cryoprecipitate (6 units) containing factors i and viii are rarely required unless severe fibrinolysis is left untreated because plasmin selectively destroys factors i, v, and viii. however, cryoprecipitate may be used for patients with severe hypofibrinogenemia (<100 mg/dl). pathologic coagulation superimposes on dilutional coagulopathy during the anhepatic stage. the heparin effect is seen as a prolonged aptt and reaction time on teg/rotem at the onset of the venovenous bypass as a small dose of heparin (2000-5000 units) in the bypass circuit enters systemic circulation. this heparin effect dissipates over the next 30-60 min. the effects of the absence of the hepatic synthetic and clearance function begin to develop during this stage. the absence of hepatic clearance of tpa promotes fibrinolysis in approximately 30 % of patients . similarly, the absence of hepatic clearance of activated coagulation factors results in excessive activation of coagulation evidenced by a progressive increase in thrombin-antithrombin complex and fibrin (ogen) degradation products. severe fibrinolysis (fibrinolysis time <60 min) may be treated by the administration of a single, small dose of eaca (250-500 mg) ). administration of a large or repeated dose of eaca is not recommended in order to avoid potential thromboembolism (gologorsky et al. 2001) . the postreperfusion syndrome occurs in coagulation at the onset of the neohepatic stage. a typical coagulation profile shows prolonged pt, aptt, reptilase time, and thrombin time. a generalized decrease in coagulation factors (i, v, vii, and viii) and platelets is accompanied by a sharp increase in the tpa level, a shortened euglobulin lysis time, and a moderate increase in fibrin(ogen) degradation products and thrombin-antithrombin complex. fibrinolysis is observed in up to 80 % of patients and is severe in about 40 % . fibrinolysis is caused by a 20-fold increase in tpa being released from the allograft and congested viscera, which overwhelms the activity of the plasminogen activator inhibitor (virji et al. 1989; porte et al. 1989 ). there are ample data to support the finding that fibrinolysis is primary in origin: a relatively steady antithrombin level, only moderate levels of fibrin(ogen) degradation products and d-dimers, selective decreases in factors i, v, and viii, and no known microthrombi formation (lewis et al. 1989a, b) . fibrinolysis resolves over the 120 min following reperfusion. the heparin effect occurs in approximately 30 % of patients, as heparin is released from the allograft and dissipates over the next 60-90 min. to identify the presence of fibrinolysis and the heparin effect, teg/rotems of untreated blood (native), blood treated with antifibrinolytic agent (eaca or aprotinin), and blood treated with an agent neutralizing heparin (protamine sulfate or heparinase) are compared 5 min after reperfusion. when fibrinolysis is present, early treatment using a single, small dose of eaca (250-500 mg) is recommended in order to reduce delayed oozing and to minimize the loss of factors i, v, and viii . prophylactic administration of eaca or tranexamic acid (amca) is a common practice in many centers, but has not shown scientific efficacy. fibrinolysis prophylaxis is not recommended by the authors, because the presence of fibrinolysis can easily be detected by teg/rotem and can be treated effectively with a small dose of eaca in most patients. when the heparin effect is present, a small dose of protamine sulfate (25-50 mg) may be given in severe cases. in addition, blood coagulability can be impaired by reperfusion hypothermia, acidosis, and ionic hypocalcemia. in contrast, excessive activation of coagulation leading to fatal intracardiac or pulmonary embolism may occur in some patients (gologorsky et al. 2001; warnaar et al. 2008 ). this complication appears to be associated with a massive transfusion, release of a large quantity of tissue thromboplastin from the less than optimal allograft, impaired tissue perfusion, and possibly antifibrinolytic therapy. intracardiac thrombosis can be treated by infusion of tpa (40-100 mg over 2 h) while observing resolution of thrombi using tee (boone et al. 2011; jackson et al. 2006) . coagulopathy improves gradually after reperfusion. generalized oozing, however, may occur even in the presence of acceptable coagulation profiles and teg/rotem, possibly due to delayed bleeding caused by the loss of a poorly formed clot or by the residual effects of reperfusion fibrinolysis. several other pharmacologic agents are reported to improve coagulation. aprotinin (2,000,000 kiu followed by 500,000 kiu/h), a non-specific inhibitor of plasminogen and serine protease, may reduce blood loss by inhibiting fibrinolysis and excessive activation of coagulation (neuhaus et al. 1989; cottam et al. 1991 ). however, clinical use of aprotinin declined even before the drug was withdrawn by the manufacturer: clinical reports did not show a significant reduction in blood loss (ickx et al. 1993; groh et al. 1993) , and fibrinolysis can be treated with eaca or amca more efficiently with negligible side effects boylan et al. 1996) . recombinant factor viia (rfviia) has been suggested to improve coagulation and reduces bleeding by actively enhancing coagulation and stimulating fibrin formation in the presence of tissue factor. its beneficial effects have been shown in patients with fulminant hepatic failure, "critical bleeding," and a ruptured liver (meadows et al. 2011; merchant et al. 2004; yamaguchi et al. 2015) . however, results of clinical trials are controversial (planinsic et al. 2005; gasperi and baudo 2006; niemann et al. 2006 ) and a european consensus concluded that a paucity of data from clinical trials with rfviia limits both the strength and the scope of clinical recommendations (vincent et al. 2006) . recently, the use of prothrombin complex concentrate has been assessed in a limited number of centers. prothrombin complex concentrate is prepared from ffp and contains clotting factors ii, vii, ix, and x, protein c, and protein s, and its use may improve coagulation without increasing preload (arshad et al. 2013 ). however, it has limited components of coagulation and its clinical advantage requires further investigation. desmopressin acetate (ddavp), a synthetic analog of 8-arginine vasopressin, increases the endothelial release of factor viii, von willebrand factor, and plasminogen. its beneficial effects have been demonstrated in vitro and in patients with liver disease, and it may be used to improve coagulation (0.3 μg/kg) . conjugated estro-gen has been reported to improve coagulation and reduce blood loss (frenette et al. 1998) , although its use has not been accepted widely. calcium metabolism patients with hepatic dysfunction invariably develop ionic hypocalcemia during massive blood transfusion, which is caused by chelation of serum calcium with citrate in the banked blood. ionic hypocalcemia begins to appear during the dissection stage (marquez et al. 1986 ) and becomes severe during the anhepatic stage. the serumionized calcium level is inversely related to the serum citrate level, as the absence of hepatic metabolism of citrate increases the serum citrate level close to that in the banked blood (fig. 8) . significant hypocalcemia (ca 2+ <0.55 mmol/l) is associated with a prolonged q-t interval and decreases in the cardiac index, stroke-work index, and blood citrate level ca ++ level mg ++ level fig. 8 intraoperative changes in serum calcium, magnesium, and citrate level (results of two studies (marquez et al. 1986 and scott et al. 1996) are superimposed, with the permission of the publisher) pressure. therefore, the ionized calcium concentration is monitored hourly or more frequently, and cac1 2 (15 mg/kg) or calcium gluconate (30 mg/kg) is administered to maintain a normal level (martin et al. 1990 ). ionic hypocalcemia improves gradually as the engrafted liver begins to metabolize citrate, unless the speed of the transfusion exceeds the metabolic function of the liver. hypokalemia is not uncommon in patients with liver disease due to poor dietary intake of potassium and its loss from chronic diuretic therapy and diarrhea. severe hypokalemia (<2.5 mmol/l) is treated with potassium chloride to increase its level to 3.0-4.0 mmol/l. moderate hypokalemia (<3.5 mmol/l) is not treated because it is welltolerated by patients and self-corrected by blood transfusion. hyperkalemia is a serious concern because it interferes with myocardial conduction and contractility, particularly in the presence of acidosis and hypocalcemia. progressive hyperkalemia (up to 6-7 mmol/l) may occur in patients with renal dysfunction or those requiring massive blood transfusion. mild hyperkalemia (up to 5.5 mmol/l) is treated with insulin (10 units) and glucose (12.5 g). it has been shown that glucose and insulin therapy is effective in lowering the serum potassium level even in the absence of hepatic function (dewolf et al. 1993a) . for moderate-to-severe hyperkalemia (>5.5 mmol/l), in addition to insulin therapy, prbc or phlebotomized blood can be washed to remove potassium using an autotransfusion system before transfusion (ellis et al. 1987) . reperfusion hyperkalemia is caused by potassium influx from the preservation solution and hepatocytes, and its systemic effects and treatment have been described previously. acute hyperkalemia returns to a normal range within 5-10 min as a result of redistribution. the potassium level gradually returns to the baseline value as the rbcs and the engrafted liver take up excess potassium. hypokalemia (<3.5 mmol/l), which occurs toward the end of procedure, is treated using a kcl infusion (20 mmol increments). hyponatremia (<130 mmol/l) is a common occurrence in patients with liver disease, particularly those with fluid retention, ascites, diuretic therapy, and restricted sodium diet. the serum sodium level gradually increases towards normal during surgery via administration of blood products and a balanced salt solution. a rapid rise in the serum sodium level (>10 mmol/l) is a clinical concern because it may contribute to the development of central pontine myelinolysis, a serious neurological injury caused by the destruction of the myelin sheath in the pons (videira et al. 1991) . therefore, the preoperative serum sodium level should be raised to >130 mmol/l, if possible, and a rapid increase in sodium should be prevented by administration of low sodium-containing crystalloids during surgery. in addition, tromethamine (tham) is the preferred drug for treatment of metabolic acidosis as it does not contain sodium. hypernatremia may be seen in some patients who receive a large dose of nahco 3 preoperatively. this hypernatremia is gradually normalized by administration of blood products and a balanced electrolyte solution. a clinical investigation showed that the serum ionized magnesium level, similar to the ionized calcium level, has an inverse relationship with the serum citrate level as magnesium ion chelates with citrate in banked blood (scott et al. 1996) . although the clinical significance of ionic hypomagnesemia during liver transplantation is unclear, mgso 4 (1-4 g) can be administered to minimize potential cardiac irritability and myocardial depression. metabolic acidosis begins to appear during the dissection and anhepatic stages because of impaired hepatic metabolism of the acid load from the banked blood and the peripheral tissues. the base deficit and lactate level increase further (approximately 5 mmol/l) on reperfusion due to the acid load from the graft and congested viscera and lower extremities. it gradually improves as hepatic function is restored and tissue perfusion improves during the neohepatic stage. persistent lactic acidosis (>15 mmol/l) appears to be associated with graft dysfunction (begliomini et al. 1989) . metabolic acidosis is aggressively corrected by administration of nahco 3 to maintain base deficit levels <5 mmol/l because acidosis is frequently progressive and leads to myocardial depression, inadequate cellular respiration, and decreased sensitivity to catecholamines. as described earlier, tham is preferred in hypo-or hypernatremic conditions to minimize fluctuation of the serum sodium level: 150 ml of 0.3 m tham is equivalent to 50 mmol of nahco 3 . alternatively, dichloroacetate (40 mg/kg every 4 h) appears to reduce lactate production by stimulating pyruvate oxidation (shangraw and robinson 1997) . metabolic alkalosis may develop during the neohepatic stage, and this was believed to be associated with nahco 3 -administered and citrate metabolism-generating bicarbonate. however, it has been shown that the degree of metabolic alkalosis is unrelated to the citrate and nahco 3 load (fortunato et al. 1987) and may be associated with residual hyperaldosteronism. body temperature may gradually decrease to 34 c during the dissection stage as a result of the exposure of the abdominal contents to the cold environment, vasodilatation, and lack of shivering. hypothermia continues during the anhepatic stage as energy production decreases further. an abrupt decrease in core temperature (2-3 c) occurs on reperfusion as cold preservation solution enters systemic circulation. the temperature increases during the neohepatic stage, and the surgery ends with a body temperature of approximately 35-36 c. hypothermia is difficult to avoid, although raising the room temperature, application of forced warm air devices, use of a warming blanket, and a heat exchanger in the venovenous bypass system may be beneficial. the blood glucose level is relatively wellmaintained (100-200 mg/dl) with blood transfusion, as the banked blood contains glucose (approximately 200 mg/dl). a gradual decrease in glycogenolysis reduces the blood glucose level during the dissection and anhepatic stages. in patients with fulminant hepatic failure or severe hepatocellular disease, the blood glucose level may decrease precipitously, making glucose supplementation necessary. hyperglycemia (up to 300 mg/dl) occurs on reperfusion as glucose is released from the engrafted liver (dewolf et al. 1987 ). insulin does not appear to be effective in treating reperfusion hyperglycemia because glucose reuptake requires restoration of hepatic function. the insulin level is relatively steady during surgery, and the glucagon level increases after reperfusion. the blood glucose level usually returns to normal within 12-24 h. persistent hyperglycemia caused by impaired hepatic glucose reuptake and hormonal imbalance is an early sign of poor graft function (mallett et al. 1989 ). urine output is well-preserved in most patients once the intravascular volume is optimized. oliguria or anuria, however, may persist in patients with the hepatorenal syndrome or underlying renal disease. the presence of oliguria and hematuria during the anhepatic stage of the simple cross-clamping technique has been described earlier. urine output increases during the neohepatic stage as a result of the restoration of renal function and circulation. various agents have been tried to protect or improve renal function: the role of dopamine is controversial, dopexamine appears to be beneficial, and triple-drug therapy (dopamine [2-3 μg/kg/min], mannitol [250 mg/kg], and furosemide) improves urine output but not renal function (gray et al. 1991; planinsic et al. 1997 ). when fluid overload or severe electrolyte imbalance is a concern, intraoperative venovenous ultrafiltration or hemodialysis may be utilized. the restoration of hepatic function is evident about 2 h after reperfusion: levels of citrate and lactate decrease, the glucose level returns toward normal, coagulopathy improves, and bile production begins. persistent citrate intoxication, acidosis, hyperglycemia, coagulopathy, and pale-colored bile are poor prognostic signs. recently, tracheal extubation in the operating room has been successful in several centers when the patient meets the liver transplantationspecific extubation criteria, including the severity of pre-existing liver disease, blood loss, and hemodynamic stability (mandell et al. 2002; biancofiore et al. 2005; glanemann et al. 2007 ). however, most patients are still transported to the intensive care unit (icu) while receiving invasive monitoring and ventilatory support. upon arrival to the icu, the ventilator setting is reported to the respiratory therapist, the lungs are auscultated, and vital signs are displayed on the icu monitor. detailed intraoperative information is reported to the icu physician and nursing staff. primary non-function primary non-function is defined as graft failure occurring within 90 days after liver transplantation in the absence of either rejection or technical factors such as hepatic arterial thrombosis (bzeizi et al. 1997) . this complication occurs in up to 10 % of patients and is frequently caused by hepatic dysfunction of the donor liver or prolonged cold ischemia (>18 h). the patient develops progressive multi-organ failure including encephalopathy, coagulopathy, minimal bile production, and oliguria. supportive therapy may be helpful until the liver resumes its function, although urgent retransplantation is the only solution in many patients. worsening liver function without technical complications in the second week after liver transplantation suggests acute cellular rejection. biopsy findings are inflammation of the intrahepatic endothelium and bile duct and a mononuclear cell infiltration with eosinophilia (wiesner 1996) . hepatic arterial stenosis occurs in approximately 5 % of patients, and is four times more common in children. common clinical signs are biliary tract breakdown, recurrent bacteremia, hepatic abscess, and occasionally massive hepatic necrosis (tzakis et al. 1985) . hepatic arterial stenosis is suspected when an ultrasound examination reveals increased focal arterial flow velocities and is confirmed by angiography. in the immediate postoperative period, direct repair or reconstruction using an infrarenal arterial conduit is usually successful. stenosis occurring several weeks after transplantation is treated with percutaneous hepatic arterial angioplasty, which has a success rate of more than 90 % in achieving long-term patency. vena caval stenosis and thrombosis occur in 1-2 % of patients. in traditional liver transplantation, outflow obstruction is managed by balloon angioplasty with or without a metallic stent placement (simo et al. 1993) . in the piggyback technique, it is treated with end-to-side anastomoses between the donor infrahepatic ivc and the recipient retrohepatic ivc (stieber et al. 1997) . portal venous stenosis and thrombosis are relatively uncommon in the adult population and present with graft dysfunction, massive ascites formation, and hemodynamic instability. this complication is corrected by an urgent reconstruction of the portal vein or construction of a superior mesenteric venous graft to the liver, together with a ligation of large collaterals that may reduce the portal flow. biliary complications are more common in children and have an overall incidence of 8-15 %. early recognition is difficult, leading to high morbidity and mortality. bile leaks usually occur at the anastomotic site, although they may be found at the t-tube site or aberrant ducts. most biliary complications occur within the first 3 months and are diagnosed by liver function tests (serum bilirubin, γ-glutamyltransferase, and alkaline phosphatase) and imaging techniques. these complications are treated by percutaneous or endoscopic drainage of bile collections. in cases of roux-en-y choledochojejunostomy, surgical reconstruction is required. intra-abdominal bleeding occurs in about 7-15 % of patients and requires exploration in about half of these cases (ozaki et al. 1994) . gastrointestinal bleeding may develop from ulcers, viral enteritis, varices, and an afferent roux-en-y loop. variceal bleeding is usually associated with portal vein thrombosis and requires an urgent ultrasound or angiographic evaluation. bleeding from the roux-en-y limb occurs 1 week after surgery and is usually self-limited. additionally, bleeding can be caused by persistent thrombocytopenia associated with splenic sequestration, drug toxicity, heparin-induced thrombocytopenia, and immunologic reactions. intestinal perforation is caused by serosal injury to the intestines and usually occurs in patients who have had a technically difficult hepatectomy, prolonged portal venous clamping, or a massive blood transfusion. intestinal perforation or leakage is treated by urgent surgery and antifungal therapy. cardiac complications any type of cardiac complication can develop in the postoperative period. hypotension can occur due to hypovolemia, either from underresuscitation or ongoing bleeding, decrease in contractility secondary to the pre-existing myocardial disease, or new onset of dilated or ischemic cardiomyopathy. other potential causes are acidosis, hypocalcemia, or vasodilation from sepsis or graft failure. management of cardiac complications is based on the underlying cause. hypertension occurs in patients with pre-existing hypertension, inadequate pain control, hypoglycemia, and cerebral edema. restoration of normal liver function may increase systemic vascular resistance, and calcineurin inhibition can increase systemic blood pressure. calcium channel blockers (i.e., diltiazem and verapamil) are avoided because they can increase the levels of the calcineurin inhibitors. myocardial infarction is relatively rare due to thorough preoperative evaluation being undertaken to detect cad. however, when it does develop, a cardiologist should be consulted for possible emergent cardiac catheterization during surgery and revascularization. pulmonary edema is commonly seen postoperatively and may be caused by significant transfusion requirements, increased capillary permeability, prolonged intubation, and reversible dilated cardiomyopathy. reversible dilated cardiomyopathy with pulmonary edema may develop in the first 5 days after transplantation. sampathkumar et al. reported that 1 % of patients who did not have ventricular dysfunction developed dilated cardiomyopathy postoperatively, most of whom recovered completely without any long-term complications (sampathkumar et al. 1998) . the cause of this condition is unknown, although it may be a form of stress-induced cardiomyopathy. atrial fibrillation, ventricular tachycardia, and other arrhythmias may develop as a result of electrolyte abnormalities (i.e., hypomagnesemia, hyperkalemia, and hypocalcemia), cardiac ischemia, or irritation from cvp or pa catheters. in a study by xia et al., atrial fibrillation was observed in 7.4 % of patients and was associated with increased mortality, graft failure, and acute kidney injury (xia et al. 2015) . all arrhythmias are treated following the standard guidelines. thromboembolism is a common cause of sudden postoperative death. deep vein thrombosis should be prevented by early extubation and mobilization, use of compressive stockings, and administration of heparin (subcutaneous or low molecular weight). most patients require mechanical ventilation for only a few hours or days after transplantation. however, prolonged ventilatory support is required in some patients with atelectasis, pleural effusions, and central nervous system (cns) depression. intraoperative cross-clamping of the ivc occasionally results in right phrenic nerve crush injury and diaphragmatic paralysis in the immediate postoperative period (mcalister et al. 1993 ). ards may develop in patients with intra-abdominal infection, pancreatitis, hepatic necrosis, acute cellular rejection, and occasionally with muromonab-cd3 (okt3) treatment. bronchoalveolar lavage and bacterial culture are frequently performed to rule out pulmonary infection from any other pulmonary pathology. pre-existing pulmonary hypertension may persist postoperatively and is controlled by epoprostenol or nitroglycerin. neurological complications occur in 12-20 % of patients, mostly in the first week of transplantation (singh et al. 1994 ). these are more common in adults and present as mental status changes ranging from dysphasia to frank coma. dysfunction of the cns is commonly caused by medications, such as cyclosporine, tacrolimus, histamine h 2 -blockers, acyclovir, and antibiotics such as imipenem. non-convulsive seizures may occur, and an eeg is performed for patients with unexplained mentation changes. intracranial hemorrhage and watershed infarcts are ruled out using ct scans. hyponatremia and hypomagnesemia can also delay awakening. central pontine myelinolysis may develop several days after transplantation, and recovery is often slow and incomplete (winnock et al. 1993) . hepatic encephalopathy may be present for several days after transplantation in patients with persistent portosystemic shunting. meningitis should be ruled out when the mental status change is accompanied by fever. disseminated aspergillosis is a devastating complication in a patient with multiple brain infarcts and fever. peripheral neuropathy presenting as weakness is usually myopathic in nature and is more common in patients with preoperative severe liver disease, poor graft function, high steroid doses, and uremia, and are confirmed by electromyography and muscle biopsy. in patients with fulminant hepatic failure, cerebral hyperemia and hypertension usually decrease gradually, and the patient regains consciousness as the liver begins to function. renal dysfunction is usually transient and is commonly associated with intraoperative hypovolemia and hypotension, allograft dysfunction, and nephrotoxicity of cyclosporine and tacrolimus. oliguria is an early sign of renal dysfunction and is managed by restoring intravascular volume and renal perfusion. the hepatorenal syndrome may persist after transplantation, and its recovery depends on its preoperative severity and allograft function. in some patients, addition of vasoconstrictive immunosuppressants (cyclosporine and tacrolimus) may lead to acute tubular necrosis. in general, renal function returns to the normal range in most patients, and approximately 10 % of patients require temporary dialysis (mccaulley et al. 1990 ). long-term prognosis is fair, although hypertension, diabetes, and chronic nephropathy induced by steroids and the calcineurin inhibitors may result in chronic renal failure. more than half of the postoperative infections following liver transplantation are bacterial in origin. these infections typically occur in the first 2 weeks, when blood levels of immunosuppressants are high. the most common sites of infection are the liver, biliary tract, peritoneal cavity, and pulmonary system. common organisms in the abdomen are aerobic gram-positive organisms (streptococci and staphylococci) and gram-negative bacilli (escherichia coli, enterobacter species, and pseudomonas), while pseudomonas infection is most common in the lungs. approximately 20 % of infections are caused by fungus, with candida species accounting for more than 80 % of all fungal infections. the risk factors are a high steroid dosage, usage of broad-spectrum antibiotics, and prolonged surgical time. candida infection is treated with amphotericin or fluconazole. aspergillus infection accounts for 15 % of all fungal infections and is associated with a very high mortality; high-dose liposomal amphotericin b followed by prolonged itraconazole is the treatment of choice. viral infections are seen 2-3 months after transplantation, with cytomegalovirus and herpes simplex accounting for the bulk of these infections. epstein-barr virus is not usually seen until approximately 6 months after transplantation, but is an important cause of lymphoproliferative disease. pneumocystis pneumonia, an opportunistic infection, responds to trimethoprim-sulfamethoxazole. late metabolic complications following liver transplantation include diabetes, hyperlipidemia, weight gain, and hypertension. diabetes is induced by steroids, cyclosporine, and tacrolimus and may respond to oral hypoglycemic agents or insulin. hyperlipidemia is associated with diabetes, obesity, steroids, and immunosuppressive drugs and is treated by diet and exercise. hypertension is seen in as many as 85 % of patients after transplantation; the use of steroids or tacrolimus is the most likely cause. hypomagnesemia has been implicated as the cause of the hypertension in some cases. approximately 15 % of all patients require retransplantation of the liver. early retransplantation is performed within several days after the primary transplantation to rescue patients from primary non-function (graft factor), acute rejection, and technical failure (vascular thrombosis), or secondary non-function (host factor) associated with poor hepatic perfusion. hepatic necrosis is the common pathway of graft non-function and results in progressive, severe encephalopathy, ards, lactic acidosis, coagulopathy, hypoglycemia, and significant circulatory instability. although infrequent, hepatectomy with a portacaval shunt may be performed to protect the patient from the ill effects of the necrotizing liver on extrahepatic organ functions. in such a case, retransplantation should be performed as soon as the donor organ is available. the surgical procedure itself is relatively simple because surgical dissection has already been made and adhesions have not yet formed. anesthetic management of these patients is similar to that of patients undergoing primary transplantation. late retransplantation is performed in patients with chronic rejection, vascular complications, and recurrence of the original disease. the physical condition of the patient may have improved, but complications of immunosuppression (i.e., hypertension, renal insufficiency) may be present. adhesions and the steroid-induced fragile tissues frequently complicate late retransplantation. anesthetic management is similar to that of primary liver transplantation, but a large amount of blood loss is anticipated. in pediatric liver transplantation, rapid-sequence iv induction is preferred, although mask induction is chosen in patients in whom there is difficulty obtaining iv access (borland et al. 1985) . large-bore iv catheters are placed in the upper extremities after induction of anesthesia. a central venous catheter with cvp monitoring is the usual procedure, and pulmonary arterial catheterization is rarely indicated. blood pressure is monitored using a femoral intra-arterial catheter. it appears that children tolerate cross-clamping of the ivc and portal vein reasonably well without significant hemodynamic changes, possibly by compliant vasomotor tone. therefore, venovenous bypass is rarely used in children under 20 kg. coagulation changes that occur during liver transplantation are not as severe as those of adults, and this may be associated with more prevalent cholestatic diseases in children ). blood loss in children with biliary atresia can be large due to the technical difficulty associated with previous biliary surgery (i.e., ksai procedure). maintenance of body temperature is difficult, as the large surface area promotes heat loss. live-donor hepatectomy is usually a challenging procedure. the young and healthy donors (asa physical status [ps] 1 or 2) undergo a complete evaluation by hepatologists, surgeons, anesthesiologists, and psychologists. the anesthetic goals are minimizing surgical blood loss and allogeneic blood transfusion, maintaining liver blood flow, facilitating early extubation, preventing deep venous thrombosis and infection, and providing adequate postoperative pain control. preoperatively, donors may be given erythropoietin to boost rbc production and they can donate 2 units of autologous whole blood 2-3 weeks before surgery. on the day of surgery, donors may be given heparin (5000 units, subcutaneous injection) to prevent deep venous thrombosis, and 6 units of typed and cross-matched prbcs are prepared. in the holding area, a peripheral iv catheter is secured, anxiolytics are administered, and donors may elect to receive thoracic epidural anesthesia for postoperative analgesia. the need for epidural local anesthetics with or without narcotics is determined by the attending anesthesiologist and pain service. in the operating room, unasyn ® (3 g iv) or vancomycin (if allergic to penicillin) is administered to prevent infection, and the patient is positioned with minimal stress to the brachial plexus to avoid neurologic injury (dulitz et al. 2005) . induction and maintenance of anesthesia follows the standard guidelines of any major surgical procedure. ultra-short-acting narcotics such as remifentanil may be beneficial for early extubation after surgery as it is rapidly metabolized by plasma esterase and does not have a prolonged effect in the presence of hepatic and renal dysfunction. intraoperative monitoring is similar to that of patients undergoing major surgery, and a radial arterial catheter and cvp are placed for hemodynamic monitoring. additional ivaccess is secured to prepare for the potential need for rapid infusion of fluids using a rapid-infusion system. immediately after induction of anesthesia, isovolemic hemodilution may be performed: 2 units of the patient's whole blood is collected in cpda (citrate phosphate dextrose adenine) blood collection bags, agitated to prevent clot formation, stored at room temperature, and returned to the patient within 8 h. intraoperatively, physiologic condition should be maintained at all times to ensure adequate perfusion of all tissues including the liver by monitoring the cardiopulmonary system and stat laboratory. metabolic acidosis should be avoided, and use of a balanced salt solution (lactated ringer's solution or plasmalyte-a ® ) is the preferred choice in order to avoid the acid load from normal saline (waters et al. 2001 ). blood loss is not excessive and pre-and intraoperatively donated autologous blood and intraoperative autotransfusion are sufficient in most patients. the relationship between the cvp level and surgical blood loss is controversial: chhibber et al. reported that intraoperative blood loss did not correlate with cvp (<5 mmhg) in their study (chhibber et al. 2007 ), while jones et al. demonstrated a significant reduction in blood loss with low cvp (jones et al. 1998 ). the authors recommend euvolemia to maintain hepatic blood flow during dissection of the liver. however, fluid overloading should be avoided after hepatectomy because relatively high portal venous flow to the reduced liver mass may lead to liver congestion and small-for-size syndrome (dahm et al. 2005) . at the conclusion of surgery, the patient can be extubated safely in the operating room and transported to the icu. all types of surgical procedures may be necessary in the early postoperative period. within the first 2 months after transplantation, surgical procedures are performed to treat complications of transplantation, such as exploratory laparotomy for abdominal bleeding or reconstruction of the biliary system. some degree of hepatic dysfunction may still be present, and ventilatory and circulatory support and invasive monitoring may be required. regional anesthesia is not recommended because of potential bleeding and infectious complications. anesthesia care of these patients is similar to that of other urgent abdominal procedures. patients may return to the operating room at any time for biliary reconstruction, replacement of a hip joint, or almost any other procedure. liver function and drug metabolism are usually within the normal range, and anesthetic management differs little from that of other patients. side effects of immunosuppressants (hypertension and renal insufficiency) and drug interactions should be considered. the main goal of organ procurement is the maintenance of optimal conditions for all organ systems to promote as normal as possible an environment for the organs prior to harvesting. specifically, integrity of organs should be maintained by optimizing organ perfusion and preventing further damage associated with pre-existing illness or trauma. therefore, donor care during procurement is a continuum of the intensive care provided before brain death. the donor is reviewed and examined by the anesthesia team to evaluate their medical history and vital organ function. the equipment and medications necessary for multiple organ procurement are shown in table 5 . a multiple-channel vital-sign monitor is an essential piece of equipment because of the unavoidable hemodynamic changes associated with the absence of brain stem function, surgical manipulation, and fluid shift. a volume ventilator may be required for donors requiring high levels of peep or airway pressure. a large volume of crystalloids and colloid solutions is prepared, and 5 units of prbcs are frequently required. the transit from the icu to the operating room is a crucial period; the anesthesia care team directs the transportation while the donor is continuously monitored, ventilated, and treated. intraoperatively, blood pressure is monitored by an indwelling radial or brachial arterial catheter as abrupt changes in blood pressure are anticipated. cvp monitoring is essential, and a pa catheter may be used in unstable donors. general anesthesia is provided as donors respond to surgical stimulation by dramatic hemodynamic changes such as tachycardia, hypertension, perspiration, and involuntary movement (wetzel et al. 1985) . this so-called mass reflex is caused by the neurogenic vasoconstriction and stimulation of adrenal medulla by reflex spinal arc. isoflurane is the most commonly used agent, because its myocardial depression is relatively benign, and short-acting narcotics (i.e., fentanyl, up to 5 μg/kg/min) may be used in unstable donors. rocuronium bromide or vecuronium bromide is administered for muscle relaxation. the specific goals of ventilatory care are to maintain normal pao 2 (70-100 mmhg), arterial hemoglobin oxygen saturation (>95 %), and paco 2 (35-45 mmhg) as well as to avoid pulmonary complications. this goal is frequently achieved by ventilating with a tidal volume of 10-15 ml/kg, fio 2 of 30-40 %, respiratory rate of <20/min, and a low level of peep (<5 cmh 2 o). however, in donors with pulmonary complications, adjustments are made in tidal volume (up to 20 ml/kg), respiratory rate (up to 20/min), and peep (up to 10 cmh 2 o). aggressive circulatory care is essential because hemodynamic instability may impair organ perfusion. specifically, hypotension (systolic blood pressure <80 mmhg or mean arterial pressure <40 mmhg) is associated with a high incidence of acute tubular necrosis, non-function of the graft kidneys, and poor hepatic function. it is generally agreed that systolic blood pressure should be within the normal range (100-120 mmhg) and cvp should be <10 cmh 2 o with minimal vasopressor support. maintaining circulatory homeostasis, however, can be challenging. preload is frequently decreased because of blood loss, vasomotor paralysis, diuretic therapy, and diabetes inspidus, although fluid resuscitation may result in overload. the heart rate may vary depending on the degree of brain injury, ranging from tachycardia to bradycardia. arrhythmia is not uncommon, and myocardial contractility is frequently impaired by myocytolysis, myocardial necrosis, coronary spasm, and reduction of myocardial energy storage (novitzky et al. 1988 ). afterload may be high, from excessive sympathetic tone, or low, from vasomotor paralysis. volume deficit is usually corrected with lactated ringer's or colloid solution, and transfusion of prbcs (1-3 units) may be necessary to maintain hematocrit between 25 and 35 % (hardesty and griffith 1986) . once the fluid deficit is corrected, a glucose-containing hypotonic solution (5 % dextrose in 0.45 % nacl 1 ml/kg/h) is administered to replace urine output and insensible loss, guided by cvp and urine output. excessive urine output (>200-250 ml/h) is replaced using a hypotonic electrolyte solution with supplementation of kcl (20 meq/l). tachycardia with hypertension should be avoided as it may cause pulmonary edema, decrease organ perfusion, and increase myocardial oxygen consumption. a β-antagonist (i.e., labetalol hydrochloride or esmolol hydrochloride) or a calcium channel blocker (verapamil hydrochloride) is used to treat tachycardia and arrhythmia (novitzky et al. 1984) . for bradycardia, isoproterenol or epinephrine is used for positive chronotropic effects because donors are unresponsive to centrally acting chronotropic drugs (i.e., atropine). supraventricular or ventricular arrythmia is treated using antiarrhythmic drugs. low afterload is compensated for by increasing preload because α-vasopressors increase the myocardial work load and decrease splanchnic and coronary blood flow. in severely hypertensive donors, an α-blocker (hydralazine or sodium nitroprusside) may be given to reduce the afterload. when cardiac output and organ perfusion are impaired, inotropes (dopamine hydrochloride, dobutamine hydrochloride, and isoproterenol hydrochloride) are recommended to improve cardiac contractility. in brain-dead animal models, serum levels of triiodothyronine, insulin, and cortisol have been found to be low, and the administration of triiodothyronine may improve hemodynamic stability by maintaining myocardial high-energy stores and glycogen (james et al. 2010) . circulatory arrest, which occurs in 10 % of potential donors (emery et al. 1986) , is managed in the standard fashion, except atropine is not effective. adequate diuresis (>0.5 ml/kg/h, preferably 1-1.5 ml/kg/h) is recommended as urine output (>100 ml/h) is the most significant factor that determines the outcome of the kidney and liver graft. oliguria is generally caused by hypovolemia and hypotension and frequently responds to fluid administration. diabetes insipidus leads to polyuria, hypovolemia, and electrolyte imbalance. in addition to the fluid replacement, ddavp (0.5-1 units/h) may be administered (richardson and robinson 1985) , although an excessive dose of ddavp may increase the risk of acute tubular necrosis and reduce hepatic blood flow (burggraaf et al. 1994) . donors are poikilothermic, and hypothermia plays a major role in hemodynamic instability. body temperature should be kept above 35 c by raising the operating room temperature, infusing all fluids through a blood warmer, and using heating lamps, a warming blanket, and a heated humidifier in the ventilation circuit. metabolic acidosis, caused by inadequate tissue perfusion, is corrected by administration of nahco 3 or tham. commonly seen electrolyte imbalances are hypernatremia, hypokalemia, hypocalcemia, hypophosphatemia, and hypomagnesemia, and they are treated in a standard fashion. glucose metabolism is relatively well-maintained, and any abnormality in glucose metabolism is corrected by administration of insulin or glucose on the basis of the serum glucose level. dilutional coagulopathy is common, and consumption coagulopathy may develop secondary to the release of tissue thromboplastin from injured tissues and the ischemic organs (kaufman et al. 1984) . fibrinolysis is not uncommon in donors, possibly as a result of the release of tpa from the necrotic brain. replacement of coagulation factors and platelets or any pharmacologic therapy is rarely indicated as donors are fully heparinized when the aorta is cannulated. once cardiac arrest is induced by cardioplegia, no further supportive care is necessary. liver transplantation is one of the most stressful procedures for patients with multiple organ dysfunction and it is a challenge for anesthesiologists. it is remarkable that anesthesiologists have played a major role in the progress of liver transplantation and its successful outcome. it cannot be overemphasized, however, that a thorough understanding of pathophysiology and close communication and cooperation among hepatologists, surgeons, anesthesiologists, intensivists, and other healthcare workers are vital to successful outcomes and further progress in this field. ▶ hepatopulmonary syndrome and portopulmonary hypertension postreperfusion syndrome: hypotension after reperfusion of the transplanted liver cerebral hemodynamic and metabolic changes in fulminant hepatic failure: a retrospective study guidelines for director of liver transplant anesthesia practice guidelines for 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salvage endotoxemia and human liver transplantation key: cord-015090-n6f4xupw authors: nan title: ps 339-563 date: 2005-09-10 journal: intensive care med doi: 10.1007/s00134-005-2780-4 sha: doc_id: 15090 cord_uid: n6f4xupw nan bronchoscopy bronchoalveolar lavage (bal) may be followed by a systemic inflammatory response which clinical effects in critical patients are unknown. we designed this study to examine the effects of fiberoptic bronchoscopy (fob) with and without bal on body temperature, systemic arterial pressure, heart rate and supportive therapies requirements in mechanically ventilated patients. methods. 46 consecutive mechanically ventilated patients were prospectively enrolled. fob with bal (150 ml of isotonic saline instilled by aliquots of 50 ml) was performed in 29 patients and without bal in 17. heart rate and mean arterial pressure were recorded 4 hrs before (time 1), at the beginning (time 2), and 4 (time 3) and 24 hrs after the procedure (time 4). body temperature, vasopressors, urine output and fluid balance were registered during 24 hrs before and after fob. there were not changes in hemodynamic parameters, body temperature nor supportive therapies (p: n.s.) in fob patients without bal. on the contrary, in patients with bal, there was a significant decrease in mean arterial pressure 4 hrs after the procedure (table). fluid balance and urine output remained unchanged (p: n.s.), but number of patients on vasopressor therapy and mean vasopressors dosage increased after bal (from 0.20 ± 0.51 µg/kg/min to 0.48 ± 0.92 µg/kg/min; p < 0.01); body temperature did not change (p: n.s.). conclusion. global tests may better reflect physiological haemostasis than standard screens or individual factor assays in critically ill patients with sepsis. studies that involve clinically relevant endpoints of bleeding are required. abizanda r 1 , reig-valero r 1 , bisbal-andres e 1 , mas-font s 1 , sánchez-morán f 1 , madero-peréz j 1 , iranzo-velasco j 1 1 intensive care department, hospital universitario asociado general de castello, castello, spain to limit therapeutic effort (let) is a reasonable acceptable medical and ethical attitude that must be offered to those cases where the evolution of patients is no good in spite appropiate treatment. our aim has been to look into the let decisions and in the participation of patients and surrogates in them. retrospective analysis of decision making processes about let attitudes in a multidisciplinary 19 beds icu during 2003 and 2004, as compared to practices registered during 1996 and 1999. since 2003, a consensus attitudes form has been fulfilled between the attending medical team and the surrogates of patients to whom let was proposed (by any part). the agreement offered differentiated possibilities of let (no cpr, no increases in fio2, no use of vasoactive drugs, no use of extracorporeal renal depurative techniques, and so on). existing agreement for each individual patient was reviewed every time that the attending team or the surrogates ask for that. during the analyzed years, 118 let attitudes were performed (60 in 2003 and 58 in 2004) over a whole in icu mortality of 288 patients (141 and 147). in patients in whom let was decided, age, severity of illness, risk of death, nursing workload and icu length of stay, were significantly higher than in patients not subjected to let. agreement on let attitudes, between the attending medical team and the patient's representatives was present in 63 % of cases, and in the 18 % the let was decided only on professional-technical reasons. in those cases, medical decision was also informed to relatives. the number of patients being discharged from the icu in whom let was established was 26 (22 %). withholding approaches were more than three times higher than withdrawing indications. shorten of death process, was performed in 12 patients (10 % of let). these observational data agree with the theoretical approaches established by our group in 1994 1. no claim was presented by relatives or patients against medical attitudes. conclusion. an increasing transparency in decision making about let does not meet refusal from the patient's representatives, and, by the contrary, it favours that the decision becomes a collegiate one and makes more easy the implementation of non futile attitudes. rubulotta f m 1 , gullo a 2 , levy m m 3 , ramsay g 4 1 intensive care, gasthuisberg uz, leuven, belgium, 2 intensive care, cattinara, trieste, italy, 3 intensive care, brown, providence, united states, 4 hospital director, atrium, heerlen, netherlands the objective of this study is to assess the end-of-life decision-making process in different countries. questionnaire administered by the same interviewer to 50 north american physicians after an end-of-life discussion with proxies of patients admitted to a medical intensive care unit (micu). questionnaire administered by the same interviewer to 50 italian icu physicians during a regional meeting. physicians in both countries were asked, using a five point linkert scale, to indicate their percentage of responsibility in the decision making process. . usa physicians reported a 43% of responsibility in the end-of life decision-making process in the micu. italian physicians claimed 80% of responsibility in the end of life decision making process. physicians have different percentage of responsibility in the end-of-life decision making process according to their culture and working environment (1) . decision to withhold or withdraw of life-sustaining treatment for an incapacitated brain-damaged patient raises complex issues regarding legal or medical concerns. it is the main question how a "clear and convincing" evidence based decision can be constituted or who has the right to make decision in end of life for a patient living in persistent vegetative state. here we present the care of a woman who was left in a persistent vegetative state after having a cardiac arrest. in 1992, the case of n.u. had a cardiac arrest triggered by a delivery with severe abruption placentae. hypoxic-ischemic encephalopathy developed after a poor response to resuscitation. after one-year stay in icu, she was discharged from hospital without a ventilatory support but still required tracheotomy cannula, gastrostomy tube and urinary catheter. nursing homes caring for incapable patients are not under the cover of health insurance policy in turkey. so the sole solution for caring ms. u was being nourished at own home. to both improve and supply a constant care for ms. u, her sister and her husband agreed on to 'err on the side of life': the final decision was a marriage between mr. u and sister-in-low. it perhaps seems like a moral disturbing solution at first sight, but we do not have to forget that the families commonly disagree over how best to care for a loved one. those unfortunate circumstances need stronger bond shared by each family member, rather than a familial dispute. participation of sibling into the care of ms. u, of course, did not improve the neurological outcome or any cognitive function, but resulted in nearly uninterrupted care, which could be so hard to ensure in a different manner. furthermore since 1992, artificial nutrition and hydration delivered by family without a nurse support, surprisingly maintain normal levels of hematological and biochemical parameters, measured periodically. we believe that the acceptance of diagnosis by all family members that her condition was irreversible, and then they had submitted to her unfortunate destiny. nevertheless, it is difficult to analyze underlying factors, which cause perhaps a compulsive marriage. this profound decision, whether reflects what the patient would want for herself or what the family would want for their incapacitated loved one, is rather debatable. in order to help relieve conscious patients and family members from the burden of an icu stay, we introduced in our icu non-professional hospice volunteers (hv), trained to intervene in palliative care settings. we performed an evaluation after one year. after an agreement was signed with the hv association, hv's role and hours of presence were defined and adapted between the 4 hv women and the icu staff. an hv was on duty one afternoon every 2 weeks from 09/2003 to 03/2004 and every week from 04 to 09/2004. hv met 25 patients and 60 family members either upon request or suggestion by the staff. a special logbook could be consulted to transmit information. evaluation was performed using questionnaires for staff and hv. these results clearly indicate that introducing hv in an icu is considered by a large majority of the medical and nursing staff as useful and appropriate, though some expressed reluctance to share informations with the hv. satisfaction of patients and family members is being evaluated. a working group has proposed improvements. italy has 472 icus. these include adult and pediatric units. we contacted 50 icus selected at random all over the country. local icu regulations allow family members one visit a day in 89% of cases. only one family member can enter inside the icu after wearing gloves, mask and gown in 73% of cases, two family members wearing the same protection in 20% of cases, and more than two people in 7% of icus. icu physicians meet the family members at admission and once a day in 90% of cases. reasons given for adopting these rules include-in 80% of cases a lack of physical space to receive relatives longer than 30 minutes, in 40% of cases a lack of icu staff, in 10% of cases concern about an increased risk of infection, and in 10% of cases other reasons. all units have special rules for caring for families of terminal patients. the communication between physicians and families in italy could be improved first of all by more space inside the unit, second by dedicating more staff time. families seem to be adequately supported by the icu staff in terminal or difficult situations. masnou n 1 , pont t 1 , gracia r 1 , salamero p 1 1 transplant coordination, hospital vall d'hebron, bcn, spain the aim of the study is to compare tissue donation refusal between organ donors and potential tissue donors. methods. prospective and descriptive study of all family interviews from 2000 to 2004, through specifically designed protocol. this included epidemiological data, the manner of comunication to transplant coordinator (tc), interview features and perceived family attitude. there were 3400 deaths in this period. we evaluated as potential tissue donnors 519 patients. a tc carried out the interviews with these families, 42% (217) refused donation. over 30% were actual tissue donors.we followed 256 patients in brain death in this period. only 142 could be organ donors, 10% of these families didn't consent to tissue donation; so only 68% of patients could give tissue. we did 784 interviews ; 6% of families were familiar with spanish law about transplant and presumed consent, and only 2% of people carry donor cards or have made a living will. regarding the family ties to the deceased, in the case of organ donation the parents refused in 37% of cases, the partner in 21% ,the children in 16%, siblings in 8% and 17% in the case of more distant relatives.in the case of tissue donors the refusal was from the parents in 8% of cases,the partner in 37%, the children in 40%, siblings in 10% and other relatives in 6% of cases.these differences were due, in part, to the advanced age of the tissue donors. if we relate the cultural level of the interviewed people with the refusals we find the following results: mo low cultural level 29%, average 35% and high 1% while in the tissue group , low18%, average 20% and high 5% (ns). the reasons for family refusal were as follows (mo-mt %): deceased had expressed negative attitude 28/33, family opposition 17/23, resentment of the health system 11/7, difficulties with image of the corpse 17/3, deceased's will unknown 5/12 , contrary religious beliefs 6/1,flat refusals 6/12, others 11/19.regarding reversed refusals in mo, consent was obtained in 6% of cases and in 11% of mt. tissue donation is still the war horse for tc.this is due to a general lack of awareness of every aspect of this subject.there remains to carry out the lengthy task of social education in this area. methods. this is a descriptive study on the level of satisfaction of students who received these classes.it was carried out with 4th year students(14y)and 6th(18y).we used a post-course questionnaire, which included:level of interest,clarity and usefulness of explanation, students'environtment.it was agreed by the professionals working on this project not to use audiovisuals.the lesson was structured in six parts: introduction,donation rate, difference between organs and tissues,characteristics of the waiting list,conditions to become an organ donnor(brain death),discussion on transplant law and personal experiences.sometimes,kidney receptors participated and explained their experiences. we considered the experience useful and satisfying.both the institutions and the students supported the presentation in over 95% of cases.the teachers considered the course enjoyable and accessible.we feel that we need to evaluate the level of comprehension and therefore we have designed a new study with two questionnaires, one prior and one a week after the course so as to evaluate the changes in the students' sensivity and perceptions. organ transplantation activities are dependent on legislation, attitudes of the general public and health care professionals, and the organization of transplantation. reports from countries throughout the world have emphasized the importance of positive attitudes in health workers on organ donation and transplantation, yet there is a lack of studies on this subject. even in spain, the leading country in organ donation rates, there is also an existing shortage of these studies. however, donation rates have not kept pace with demand, resulting in a critical deficit of available healthy organs. it has been suggested that the attitudes of medical personnel regarding organ retrieval is a key success factor to improve organ donation. the aim of this study is to examine attitudes towards organ donation in health care workers. we analysed a long survey, which evaluates attitudes, knowledge, roles and experience towards organ, and tissue donation and transplantation. this survey was administered to all participants before and after the post graduate courses (2003 and 2004) in organ donation. we studied this changes in term of prognosis (glasgow outcome scale). we prospectly studied 51 patients the first 24 hours after sah. patients with chronic cardiac disease or brain death were excluded. clinical characteristics (glasgow scale, heart rate, systolic blood pressure), cardiac enzymes (troponin i, total serum creatine kinase and myocardial isoenzyme, myoglobin), ecg changes (st-t changes, prolonged qt and corrected qt intervals), echocardiographic assessment of cardiac function (left ventricular ejection fraction, hypokinesia) were studied on the day of the admission. data are shown in the table. only systolic blood pressure 95 bpm were found to be independent factors of poor outcome. measurements of myocardial specific enzymes and echocardiographic assessment of cardiac function have no prognosis impact in this study. 2)the more the patient sleeps during the day, the more likely to have poor quality sleep at night. 3)sleep disruption in icu is related to the degree of illness-severity and length of stay in the unit. 4)actigraphy is a simple method of assessing sleep that is well tolerated by patients and doesn't interfere with nursing activities. it is well-known that the ischemia-reperfusion injury in patients resuscitated from cardiopulmonary arrest (cpa) severely damages the brain. some recent studies have been reported that neuron-specific enolase (nse) is an useful marker for outcome prediction. the aim of this study was to compare the prognosis of patients resuscitated from cpa with levels of s100 protein and nse in serum and cerebrospinal fluid (csf). twenty four patients resuscitated from cpa were eligible in this study. patients were divided into two groups according to the glasgow outcome scale (gos) at three months after the initiation of therapy. group g had the favorable neurological outcome and group p had the poor outcome, evaluated by gos. the blood and csf samples were taken within 48 hours after resuscitation and the levels of s100 protein and nse were compared between the two groups. jugular bulb oxygen saturation levels were measured when hemodynamics were stabilized. mann-whitney's u test was used for continuous variables. correlations were tested using spearman's rank correlation test. a p-value less than 0.05 was considered statistically significant. six patients in group g had favorable neurological outcome and eighteen patients in group p had poor outcome. the levels of s100 protein in serum and csf (median, 1.55 and 25.4 ng/ml, respectively) in group p were significantly higher than those (median, 0.05 and 4.05 pg/ml, respectively) in group g. the levels of nse in serum and csf (median, 99.4 and 248.1 ng/ml, respectively) in group p were significantly higher than those (median, 32.4 and 39.5 ng/ml, respectively) in group g. jugular bulb oxygen saturation levels in group p were significantly higher than those in group g. the levels of s100 protein in serum correlated well with those of nse in serum (σ=0.76, p<0.005). the outcome and jugular bulb oxygen saturation levels correlated with the levels of s100 protein in serum (σ=0.81, p<0.005, and σ=0.69, p<0.05, respectively). the levels of s100 protein in serum and csf correlated well with nse and their neurological outcome, so that s100 protein would be an excellent biological predictor in patients resuscitated from cpa. grant acknowledgement. this work was supported by the grant 14207060 from the japan society for the promotion of science, japan. wright k 1 , munasinge a 1 1 critical care and emergency medicine, royal surrey county hospital, guildford, united kingdom injury to the cervical spine occurs in 5-10% of blunt multisystem trauma. spinal immobilisation consists of a hard collar, headblocks and tape immobisation.this immobilisation is maintained until the cervical spine can be cleared of injury. often this relies on the patient being able to co-operate with a neurological examination. following head trauma some patients may never regain sufficient neurological ability to co-operate with an examination. others may remain obtunded for some time. until the spine is cleared the patient needs to be log-rolled and turned in accordance with spinal care bundles. complications such as pressure sores can ensue if this is not followed. evidence has also shown that removal of the cervical spine collar in head injured patients improves venous drainage from the head and so is beneficial in managing intracranial pressure1.we therefore need an approach to clearing the spine in obtunded multisystem trauma patients. literature review study leading to institutional protocol. a safe clearence protocol is suggested. a protocol guided approach will allow the rapid removal of cervical spine precautions in multisystem injured patients. patients who have a spinal injury demonstrated are excluded from this approach and are managed in accordance with the spinal service guidance. hypothalamic-pituitary-adrenal (hpa) function has been recently studied in patients with traumatic brain injury (tbi), but few studies have shown its relationship with outcome. the aim of this study was to analyze hpa response and its relationship to intensive care unit (icu) survival in patients with isolated tbi. we studied 38 consecutive patients (33 male) with isolated tbi. norepinephrine (ne) was used to maintain cerebral pressure perfusion over 60 mmhg when necessary. at 24-48 hours following tbi, we recorded values for plasma acth, baseline serum cortisol and stimulated cortisol at 30 and 60 minutes after performing high-dose corticotropin stimulation test (hdcst). mean and sd are reported. chi-square and logistic regression analysis were done. age was 37.5 ± 18.9 years. iss 25.0 ± 8.7; apache ii 17.4 ± 6.5; gcs score after resuscitation 7.7 ± 3.0. plasma acth was 22.2 ± 42.3 ng/ml (normal values 9-52 ng/ml). baseline cortisol was 15.6 ± 8.9 ug/dl, stimulated cortisol at 30 minutes 27.3 ± 7.7 ug/dl and at 60 minutes 30.8 ± 7.3 ug/dl. all patients increased at least 9 ug/dl after hdcst or had a stimulated value greater than 20 ug/dl. overall survival was 73.7% (28 patients). univariate analysis of variables related to icu survival showed: age <45 years (p=0.02),apache ii <15 (p=0.01),acth <9 ng/ml (p=0.001),baseline cortisol < 20 ug/dl (p=0.08),use of ne (p=0.20),second-tier measures to control icp (p=0.02), gcs > 8 (p=0.12). logistic regression analysis revealed that no need of second-tier measures to control icp (or 11.9 ci95% 1.3 to 103.2) and plasma acth lower than 9 ng/ml (or 37.2 ci95% 3.1 to 449.5) were significant independent predictors of icu survival. conclusion. 1)adrenal gland function, assessed by hdcst, is not impaired at early stage of tbi. 2) tbi patients with low levels of plasma acth had a high icu survival. paramythiotou e 1 , katsarelis n 1 , papakonstantinou k 1 , stathopoulos g 1 , varveri m 1 , fousfoukis s 1 , roussos a 1 , karabinis a 1 1 icu, george gennimatas general hospital, athens, greece aspiration of foreign bodies during trauma is a known com-plication. it usually concerns teeth, pieces of food etc and for their removal several procedures, invasive or not -like bronchoscopy or thoracotomy -must be underta-ken. we describe three patients with foreign body aspira-tion in our icu. a 20 year -old male was admitted in our icu with face trauma, a broken mandible and a broken femoral bone. a foreign body was observed in the left lower bronchus on the chest x -ray. an attempt to retrieve it with the flexi-ble bronchoscope failed and the foreign body moved to the right lower bronchus. a rigid bronchoscope was then used with success and the foreign body was removed. it was a part of the broken mandible. the patient was discharged after two weeks. case 2. a 20 year -old male was admit-ted after a road accident suffering from a severe cerebral injury, a pneumothorax and a broken lower mandible. a fo-reign body (piece of a broken tooth) was aspirated in the right upper bronchus. it was retrieved with a flexible bronchoscope. he remained in a "vegetative" situation for a long time and finally died because of a septic shock. case 3. a 47 yearold patient was admitted with cerebral injury and a low glascow coma scale, a pneumothorax and acute respiratory failure. a foreign body was present in his right upper bronchus. the flexible bronchoscope and a basket were used in order to retrieve it. it was a large tooth. his neurological situation never ameliorated and the patient developed a septic shock and a multiorgan fai-lure and died. severe cerebral injury may result in foreign body aspira-tion especially when it is accompanied by facial trauma. for comatose patients, x -ray of the chest and thorax c/t scan are the main diagnostic tools for this situation. retrieval of the foreign bodies is necessary to avoid further complications such as atelectasis, pneumoniae etc. flexible bronchoscope used through the endotracheal tube is very effective in their removal. medical personnel dealing with trauma patients must have a high index of suspicion for the presence of foreign bodies in the tracheobronchial tree. flexible bronchoscopy or use of the rigid bronchoscope in case of failure, are very use-ful and safe techniques for the removal of these foreign bodies. mandila c 1 , koukoulitsios g 1 , stathopoulos g 1 , paramythiotou e 1 , theodoropoulos g 1 , karabinis a 1 1 icu, general hospital of athens ''g gennimatas'', athens, greece we report angiographic detection of vertebral artery dissection (vad) in two sedated patients in the intensive care unit (icu). in both cases vad was suspected solely by the presence of ischemic lesions evident on cervical spine and brain magnetic resonance imaging (mri). two patients were intubated, sedated, and admitted to the icu with glascow coma scores <7 after having suffered blunt head and neck injuries due to motor vehicle accidents. in the first patient computed tomography (ct) of the brain and cervical spine revealed traumatic subarachnoid haemorrhage, anterior atlas arc fracture, axis fracture, and a c3 body fracture. in addition, brain and cervical spine mri depicted a medullar contusion at the c7 level, an increased interarticular space c5-c6, and a left cerebellar hemisphere infarct. based on these findings carotid and vertebral angiography was performed, which showed complete left vertebral artery occlusion at the c6 level with incomplete distal filling due to a hypoplastic right vertebral artery. in the second patient brain ct was normal, while cervical spine ct revealed c4-c5 dislocation with accompanying posterior sliding of c5, and a subdural haematoma at the c2-c3 level on the right. cervical mri showed dislocation with spinal cord dissection at the c4-c5 level, and a large ischemic right occipital brain lobe lesion that was ascribed to putative right vertebral artery thrombosis/dissection. carotid and vertebral angiography revealed bilateral vad at the c3-c5 level with distal reopacification by collateral perfusion. anticoagulant therapy was not administered due to coexisting contraindications (subarachnoid haemorrhage, hemorrhagic contusions, subdural hematoma). the level of consciousness increased step-wise in both patients. while the second patient suffered bilateral vad, his recovery was more complete than that of the first patient. in patients with brain and cervical trauma, the coexistence of cerebral lesions due to accompanying vad is probable. mri can prompt further investigation by depicting ischemia of vertebral artery-dependent areas. the impact of vad largely depends on the efficiency of collateral flow to the affected parenchyma. maintaining of normal cerebral oxygenation is the main goal of intensive care of patients with severe head injury. it can be achieved by different methods. one of them is hyperoxya. in this study we investigated the influence of different fractions of inspired oxygen (fio2) on cerebral oxygenation and intracranial pressure (icp). two patients with traumatic brain injury (tbi) with glasgow coma scale 7 on admission enrolled in the study. patients had one-side lesions and underwent decompressive craniotomy. we compared fio2 with icp (n=30), cerebral oxymetry in non-lesioned hemisphere rso2(nl) (n=15), oxygen partial pressure in cerebral tissue (ptio2) in lesioned (les) (n=29) and nonlesioned (nl) hemisphere (n=29), pao2 (n=27), jugular bulb saturation (sjo2) (n=26), o2 extraction ratio (o2er) (n=26), arterio-venous o2 difference (avdo2) (n=26) and lactate concentration in jugular bulb (lac(v)) (n=26). plasma osmolality, cardiac output, invasive mean arterial blood pressure, paco2 and blood temperature were stable during investigation. fio2 changing from 1 to 0,3 leaded to decrease in pao2 (m±sd) (402,6±67,9 vs 109,8±43,9 torr ((p<0,0001)), sjo2 (76,5±3% vs 70,6±2,8% (p<0,05)), rso2(nl) (77,3±5% vs 66±2,8% (p<0,05)), ptio2(les) (46,8±11,8 vs 19,6±4,4 torr (p<0,01)) and non-significant changes in ptio2(nl) (26,2±17,2 vs 9,9±3,4 torr), lac(v) (1,1±0,4 mmol/l vs 1,27±0,6 mmol/l), icp (15,2±2,7 vs 14,2±4,9 torr), o2er (0,24±0,03 vs 0,28±0,04) and avdo2 (4,7±1,8 vol% vs 1,8±1,8 vol%).we found good correlation between fio2 and pao2 (r=0,89 (p<0,01)), sjo2 (r=0,63 (p<0,01)), rso2(nl) (r=0,65 (p<0,01)), ptio2(nl) (r=0,56 (p<0,01)) and ptio2(les) (r=0,76 (p<0,01)).during comparing of different methods of cerebral oxygenation assessment we found good correlation between sjo2 and ptio2(nl) (r=0,65 (p<0,05)) and no correlation between sjo2 and ptio2(les) (r=0,39 (p=0,09)), rso2(nl) and ptio2(nl) (r=0,41 (p=0,27)), rso2(nl) and ptio2(les) (r=0,39 (p=0,3)). fio2 increasing is effective and quick method of cerebral oxygenation improving.icp is not influenced by fio2 changes.fio2 must be noticed during interpretation of high levels of sjo2 and rso2. jugular oxymetry reflects the oxygenation mostly of the non-lesioned brain hemisphere. cerebral oxygenation monitoring can be improved by combination of sjo2 and ptio2 methods. nijboer j m m 1 , van der horst i c c 2 , hendriks h g d 3 , ten duis h j 1 , nijsten m w n 1 1 surgery, 2 cardiology, 3 anesthesiology, university medical center groningen, groningen, netherlands there is a longstanding belief that in trauma patients hematocrit(ht) is more sensitive than hemoglobin(hb) in detecting blood loss. this association of ht with trauma is reflected by numerous references in medline. we studied the relation between hb and ht in trauma patients. trauma patients with an iss>15 from 1996 to 2004 were included. all blood samples taken during the first week in which hb and ht were both measured, were analysed. in 678 patients (mean age 37 ± 23 yrs; 72% male) 2963 paired hb and ht values were available. the mean hb was 6.39 ± 1.40 mmol/l with a range from 1.3 to 10.7 mmol/l. the mean ht was 0.305 ± 0.07, ranging from 0.061 to 0.505. hb and ht had a pearson r2 of 0.99 (figure) . in a large series of trauma patients hb and ht behaved as identical parameters. the idea that ht is different from or superior to hb is a misconception and there is no reason for determining both hb and ht in trauma patients. paramythiotou e 1 , papakonstantinou k 1 , tsirantonaki m 1 , kalogeromitros a 1 , noulas n 1 , pedonomos m 1 , apostolakou h 1 , karabinis a 1 1 icu, george gennimatas general hospital, athens, greece introduction. propofol is often used as a sedative in icu patients. unfortunately large doses may be needed sometimes causing propofol infusion syndrome (pris). we are presenting a patient with this syndrome followed by manifestations compatible with a catastrophic antiphospholipid syndrome (caps). a 14 year old female was admitted to our icu with a multiple trauma. she had many skull fractures, a subarachnoid hemorrhage and a small acute subdural hematoma. she was put to sedation with propofol. large propofol doses were used to keep her sedated (25 -30 ml/h of propofol infusion 2%) along with noradrenaline and corticosteroids to maintain a normal arter. pressure. three days later she developed high fever, cpk rose to 75.000 µg/l and a multiple organ failure followed including renal and right heart failure. a cvvhd was immediately started. she was also put on broad spectrum antibiotics and the propofol infusion was interrupted. a week later her situation had become stable, she was free from vasoactive agents and her renal and cardiac functions were reestablished. the blood cultures taken did not prove the presence of bacteremia, though the simultaneous presence of an infection could not be excluded. thirty -two days after her admission she presented a status epilepticus. a brain c/t and mri were performed, revealing the presence of multiple hypodense areas not following a vessel distribution. an anticardiolipinantibody titer igg (1st 113 u, 2nd >130 u ) gave us the hint for a probable caps. after a combined therapy with plasma exchange and immunoglobulins she recovered and survived later on. propofol infusion is very popular in icus hospitalizing patients with cerebral injuries permitting physicians to perform regularly a neurological examination. large propo-fol doses and concomitant use of corticosteroids and catecholamines with or without sepsis could precipitate pris as in our case. our patient's condition was complicated by the neurological manifestations attributed to probable catastrophic antiphospholipid syndrome. the question aroused is if pris could have triggered such an autoimmune disorder. conclusion. attention must be paid to propofol doses used for sedation of patients with craniocerebral injuries especially adolescents. alternative sedation or combination with other sedative and/or analgesics must be considered. tsarenko s v 1 , petrikov s s 1 , huseynova k t 1 , krylov v v 2 1 neurosurgical icu, 2 neurosurgery, sklifosovsky scientific research institute of emergency medicine, moscow, russian federation invasive measurement of the intracranial pressure (icp) is known as the best method of intracranial hypertension evaluation. unfortunately it is associated with high equipment costs and risk of infection complications. we compared non-invasive methods of intracranial hypertension assessment with invasive icp measurement. methods. 48 patients enrolled in the study (severe head injury (n=32), arterial aneurism rupture (n= 7), hemorrhagic stroke (n=7), arterio-venous malformation (n=2). average age (m±sd) 44±16. m/f ratio was 35/13. all patients were operated (26 underwent decompressive craniotomy, 13 boneplastic craniotomy and 9 -insertion of icp sensor only). all patients received invasive icp monitoring (average time 4,8 ± 3 days). we used codman intracranial pressure microsensors or ventricular icp monitoring systems (hanni-set, smith medical). average preoperative glasgo coma scale (gcs) was 8,1±2,1. all patients had head ct scan and neurological examination on admission, and dynamically in postoperative period. we compared icp values with ct scan data (volume of zones with high and low density, signs of lateral and axial dislocation), gcs and neurological signs of brain stem dislocation. analyses of all data showed correlation between icp and gcs (r= -0,48; p<0,00001; n=109), neurological signs of brain stem dislocation (r=0,4; p<0,00001; n=109), volume of zones with high and low density (r=0,25; p=0,016; n=95) and lateral dislocation on head ct scan (r=0,24; p=0,016; n=101). then icp values obtained before the mass lesion evacuation were compared with preoperative head ct scan and neurological signs of brain stem dislocation. we found good correlation between icp and signs of axial (r=0,59; p<0,00001; n=42) and lateral dislocation (r=0,43; p=0,004; n=42) on head ct scan. we did not find correlation between icp values and gcs (r=-0,02; p=0,196; n=48), neurological signs of brain stem dislocation (r=0,27; p=0,07; n=42) and volume of zones with high and low density on ct scan (r=0,3; p=0,06; n=30). we found that invasive icp monitoring is the best method of intracranial hypertension assessment. neurological examination or ct scan data can not reflect all cases of icp changes but they can be used as screening methods of intracranial hypertension estimation. markogiannakis h 1 , sanidas e 2 , messaris e 1 , tsiftsis d 2 1 1st department of propaedeutic surgery, hippocration hospital, athens medical school, university of athens, athens, 2 department of surgical oncology, herakleion university hospital, herkleion medical school, university of crete, herakleion, greece nonoperative management (nom) is considered to be the treatment of choice for carefully selected blunt hepatic trauma patients. the objective of this study is to identify and evaluate the factors that can safely predict nom of these patients. our study is a retrospective analysis of trauma registry data of all 55 consecutive adult blunt hepatic trauma patients admitted in a greek level i trauma center over a 4-year period. factors that were included in the analysis were: sex, age, mechanism of injury, initial vital signs, grade of liver injury, concomitant injuries, and injury scoring systems used for total injury severity estimation. nineteen patients (34%) were immediately operated, whereas 36 (66%) were initially selected for nom. concomitant abdominal, pelvic and spinal cord trauma, high injury severity score (iss), low international classification of diseases -9th revision injury severity score (iciss), and low probability of survival (ps) were predictive factors for operative management of these patients. immediately operated patients suffered statistically significantly more frequently concomitant abdominal (84.2% vs 47.2%, p=0.004), pelvic (42.1% vs 16.7%, p=0.03), and spinal cord injuries (36.8% vs 2.8%, p=0.005) than conservatively treated patients. additionally, immediately operated patients with blunt liver injury were significantly more severely totally injured than those treated with nom as expressed by higher iss (27.2±3.2 vs 19±1.5, p=0.01), lower iciss (0.49±0.04 vs 0.74±0.03, p=0.003), and lower ps (0.79±0.04 vs 0.93±0.02, p=0.005). moreover, the percentage of patients that were admitted in the icu and mortality rate were significantly lower in patients treated with nom than those treated with immediate operation (47.2% vs 78.9%, p=0.002 and 5.5% vs 21%, p=0.03, respectively). thirty-three patients that were initially selected for nom were successfully treated conservatively; thus, the rate of success of nom was 92%. conclusion. nom of blunt hepatic trauma patients is safe and efficient resulting in significant reduction of icu admission and mortality. concomitant abdominal, pelvic and spinal cord trauma, iss, iciss, and ps are predictive factors for operative or nonoperative management of these patients. ruler van o 1 , lamme b 1 , reitsma j b 2 , gouma d j 1 , boermeester m a 1 1 surgery, 2 epidemiology and biostatistics, academic medical center, amsterdam, netherlands the decision when to perform a relaparotomy for secondary peritonitis is largely subjective and experience-based. to date there is no reliable scoring system that aids the decisional process by predicting relaparotomy outcome. our aim was to identify variables predictive of a positive outcome of relaparotomy in the acute phase of the disease. the study population was derived from a retrospective cohort of secondary peritonitis patients(n=219). patients with a positive relaparotomy (n=62) were compared to patients undergoing a negative relaparotomy (n=55) and patients undergoing an index laparotomy only (n=102). a prediction model was build from a logistic regression model by the addition of patient, peritonitis, operative and postoperative variables. a stepwise build-up of predictive models incorporating the chronology in which information is achieved in clinical practice was used. variables entered were assessed on clinical judgment and statistical analysis. accounting for chronology of information, postoperative variables are most predictive for positive relaparotomy. this implicates that information on the clinical course after the index laparotomy is required to predict who will need a relaparotomy. further adjustment and external validation of this model and development of a prediction rule is needed in a prospective, cross-sectional series of patients with secondary peritonitis. schöniger-hekele a 1 , klingbacher e 2 , hiesmayr m 2 1 department of cardiac thoracic vascular anaesthesia and intensive care, 2 department of cardiothoracic anaesthesia and intensive care medicine, medical university of vienna, vienna, austria in general icu-patients nasal carriage of staphylococcus aureus was associated with a higher risk of developing staphylococcal infections and death. the aim of this study was to determine the impact of mssa-colonisation on postoperative infections and los in elective cardiac surgery patients. we prospectively collected all data for the analysis. the cohort consisted of 122 patients, that underwent routine preoperative nasal swab one week before surgery. only patients with mrsa were treated topically with mupirocin. before surgery 46 patients (37,7%) were identified as staphylococcal aureus nasal carriers, while 62,3% were free of nasal colonization. compared to the non-carriers the mssa-carriers did not have a significant difference of the total and staphylococcus aureus infection rate. other indicators of infection and inflammation parameters(crp, leukocytes)did not show significant differences. data-mining searched publications from 8 common languages for randomised clinical trials that supplemented with impact® before and/or after major elective surgery. infectious complications, mortality, and hospital stay were primary outcomes. seventeen studies (n=2305) were analyzed, and 14 (n=2083) described patients undergoing elective gi surgery. studies were conducted in 7 countries, however, the clinical effects of impact® treatment were homogenous across the set of trials. impact® use significantly reduced postoperative infections overall (p < 0.0001), and anastomotic leaks in gi surgical patients (p = 0.003). furthermore, impact® use shortened the average hospital stay by 2.5 d (p < 0.0001), and a trend was observed towards reduced risk of mortality. considerable differences in country-specific operation modalities were noted. in a chinese trial, nosocomial infection prevalence was unusually low, 3% when supplemented with a control formula vs. 0% with impact®. in all other trials, nosocomial rates were 18-67% with control feeds vs. 3-38% with impact®. in germany and switzerland, hospital stays were extended for gi surgical patients. there, average stays were 20 d for the impact® group and 30 d for the control group vs. 11 d and 13 d, respectively, in all other countries. conclusion. impact® specialized nutrition support, as a component of infection control during surgery, is valuable for all methods practiced worldwide. to determine the incidence of nosocomial infection in critically ill patients with brain trauma. it is a prospective study performed during 12 months of the patients with brain trauma admitted in a 24-beds medical-surgical icu of a 650-beds university hospital. infections were diagnosed according to cdc criteria. infections were classified bassed on the onset moment as early onset and late onset: early onset (eo) were those developed during the first 4 days of icu-stay; and late onset (lo) were those developed 5 days after icu-admission. the statistical analysis was performed using spss 11.0 program. continuous variables are reported as means and standard deviation, and categoric variables as percentages. turkmen a 1 , turgut n 1 , altan a 1 , medetoglu a 1 , gökkaya s 1 1 department of anaesthesiology and reanimation, okmeydani training hospital, istanbul, turkey airway suctioning is classically performed with disconnection of the patient from the ventilator and the introduction of suction catheter into endotracheal tube. several authors suggest that application of closed suction catheters (csc)in intubated patients for more than 24 h is safe and can reduce the costs associated with mechanical ventilation. therefore, we evaluated the possible role of prolonged application of csc in causing enhanced colonization of the lower respiratory tract. the prospective, randomized study included 39 mechanically ventilated patients. the csc tips, tracheobronchial aspirates of each patient were examined for microbial growth. we analyzed the data with the student's t test for paired samples and fisher exact test. application for 72 h significantly enhanced the microbial growth on the csc tips (table) . to decrease vap incidence in the icu is necessary to implement infection control policies. nevertheless that implementation is not always simple and requires effort by the icu workers. new infection control policies were applied in our icu in 2003. those recommendations were adapted from the published guidelines concerning the prevention of vap and adapted to our icu. particular concern was made on the handwashing and contact isolation precautions. to analysed the impact of these measures the incidence of vap was analysed before and after that implementation. prospective study of all patients admitted in the icu for more than 24 hours, between 2002 and 2004. patients data collected included the number of ventilation days, the date of the vap diagnosis with or without microbiological confirmation. the vap diagnosis was made by new radiographic infiltrate for at least 48 h and at least two of the following criteria: fever > 38.5°c or < 35.0°c; leukocytes > 10,000/µl or < 3,500/µl, purulent sputum, or isolation of pathogenic bacteria from lower respiratory tract. the microbiological samples were collected by proximal or distal bronchial aspirated. the vap diagnosis was made on the patients receiving mechanical ventilation, 113, 164 and 166 patients during the study period. theodorakopoulou m 1 , lignos m 1 , diamantakis a 2 , zoupa p 2 , stelliou a 2 , karabekiou i 2 , armaganidis a 1 1 icu, 2 nursing icu, attiko university hospital, athens, greece hand hygiene is the most important action to control spread of nosocomial infections.hand washing compliance among health care workers remains low.the objective of this study was to assess compliance of hygiene in our icu. a 3 month prospective study on a 5 bed icu of a university hospital.antiseptic solution were placed at the bottom side of each bed and one hand washing facility exists within the unit.3 well instructed observers recorded opportunities of hand washing, and actual performance of hand washing or hand disinfection. observation time was set at 2 hrs. it was performed on morning and afternoon shifts all days of the week.every observer monitored 2 beds.the staff was not informed of the study.staff was classified according to their work status (doctors, nurses etc.). . 360 hrs of observations were recorded in 180 sessions. a total of 7880 opportunities for hand hygiene were observed. see table 1 for hand washing opportunities and actual hand washing compliance among the staff. the average hand washing opportunities were 8.76 opp/pt/hr and the average actual hand washing was 5.10 act.wash/pt/hr.hand washing compliance was similar for doctors,nurses and nursing assistants.medics, physiotherap, and visiting doctors showed significant difference in actual hand washing compliance.the overall compliance rate was 4592/7880 (58.3%). it is a prospective study during 30 months of the patients admitted in icu during 24 hours o more. were taken throat swab, tracheal aspirate and urine on admission and twice weekly. were registered the colonization and infection by pseudomonas. the infections were diagnosed according to cdc criteria. the infections were classified bassed on thorat flora as: primary endogenous (pe) when they were caused by germs that were already colonizing the throat on the icu admission; secondary endogenous (se) when they were caused by germs that were not colonizing the throat on the icu admission but were acquired during the stay in icu; exogenous (ex) when they were caused by germs which were not colonizing the throat. the infections were classified bassed on the onset moment as: early onset (eo) were those developed during the first 4 days of icu-stay; late onset (lo) were those developed 5 days after icu-admission. conclusion. in our serie, the most of infections caused by pseudomonas were pneumonias, had a late onset and were secundary endogenous. prolonged critical illness is characterized by feeding-resistant wasting of lean body mass. this catabolic state is due to an impaired activity of the thyroid and gh axes, since restoration of physiological levels of igf-1 and thyroid hormones by continuous infusion of trh+ghrp-2 is capable to induce anabolism [1] . whereas the cause of hyposomatropism during prolonged critical illness is mainly located within the hypothalamus, concomitant changes in peripheral thyroid hormone metabolism are involved in the low t3 syndrome. the aim of this study was to examine these peripheral changes into more detail in an animal model of prolonged critical [2] . burn-injured, parenterally fed, new zealand white rabbits (4 x n=8) were randomized to receive 4-d treatment with saline, trh (60 \mug/kg/h) ,ghrp-2 (60 \mug/kg/h), or trh+ghrp-2. blood glucose was maintained below 180 mg/dl by continuous insulin infusion. endocrine and biochemical organ system markers were studied. animals were sacrificed for assay of deiodinase activity in snap frozen samples. infusion of trh+ghrp-2 and trh increased hepatic activity of type 1 deiodinase (d1) versus the saline group (p= 0.02 and 0.1 resp.), restoring tt3 levels within physiological range. only combined infusion of trh+ghrp-2 induced a significant increase in igf-1 levels into the range observed in healthy rabbits. administration of trh alone resulted in a further decline of serum igf-1 levels. (1) infusion of ghrp-2+trh is able to restore peripheral thyroid hormone and serum igf-1 levels within the physiological range, mainly due to re-activation of d1. (2) d1 activity during critical illness is regulated via alterations in the thyroid axis. (3) reactivation of the thyroid axis in prolonged critical illness, without concomitant reactivation of the gh-axis, might worsen catabolism. and strong ion difference (sid) approach, to our knowledge is still not available a systematic comparison. the approach to sid may be more or less rigorous: we can calculate the apparent sid (the difference between strong ions, sidapp) or the effective sid (the sum of weak anions, sideff); moreover, when computing the sid variation (äsid) the reference value of sid can be considered fixed (40 meq/l) or variable (the expected sid) as a function of total non volatile weak acids concentration. the aim of this study was to suggest how the computation of äsid should be sophisticated in order to obtain a good correspondence with be in icu patients. conclusion. the rigorous computation of the corrected äsid seems to be necessary in the icu population, because of non-neglectable concentration of unmeasured anions and of diffuse and serious hypoalbuminemia in these patients. moreover, äsid is a measure of plasma buffer base variation, so it should be compared with an equivalent be formulation, that is plasma be. leditschke i a 1 , southcott e 2 , gissane j 1 , enslin a 1 , hickman p e 3 , potter j m 3 1 intensive care unit, 2 act pathology, 3 australian national university medical school, the canberra hospital, canberra, australia recently it has been shown that total plasma cortisol measured by immunoassay may not detect elevations in plasma free cortisol in hypoproteinaemic critically ill patients(1). we investigated the relationship between urinary free cortisol and total serum cortisol in a group of critically ill patients. methods. 5 patients were studied within 24 hours of icu admission. patients with neurotrauma or oliguria were excluded. hourly total plasma cortisol and 4-hourly urinary cortisol were measured for 24 hours using routine immunoassay for the plasma samples and high performance liquid chromatography for the urine samples. statistical analysis was performed using graphpad instat software. summary results for total plasma cortisol at the mid point of the urine collection and urinary free cortisol are shown in table 1 . using a non parametric (spearman r) test of correlation, urinary free cortisol was found to correlate moderately well with total plasma cortisol; spearman r = 0.7063, 95% confidence intervals 0.4533 to 0.8533, p to further investigate this topic, we conducted a prospective study of patients admitted to a general adult icu. morning blood samples were taken within 24 hours of icu admission to measure plasma cortisol, corticotropin (acth), dehydroepiandrosterone sulphate (dheas), free thyroxine (ft4), tri-iodothyronine (t3), thyroid-stimulating hormone (tsh) and prolactin (prl). . 150 critically ill patients (120 males) with diverse underlying diagnoses, having a median age of 50 years (range 17-84 years) were enrolled. their median apache ii and sofa scores were 11 and 6 respectively. there were no differences between survivors and non-survivors in plasma cortisol, acth, ft4, and t3. in contrast, survivors had higher median values for tsh (0.70 mciu/l vs. 0.48 mciu/l, p=0.04), dheas (1638 ng/dl vs. 995 ng/dl, p=0.04) and prl (13 ng/ml vs. 9 ng/ml, p=0.03) compared to non-survivors. our data indicate that hormone concentrations differ between survivors and nonsurvivors acutely ill patients. further studies are required to investigate whether endocrine measurements are helpful in predicting clinical outcome. mekontso-dessap a 1 , lellouche n 2 , brochard l 1 , brun-buisson c 1 , dubois-randé j 2 1 medical intensive care unit, 2 coronary care unit, henri mondor hospital, créteil, france relative adrenal insufficiency has been demonstrated to be associated with increased mortality in septic shock patients. cardiogenic shock (cs) induces a stress response involving the adrenal cortex, but functional hypoadrenalism has never been investigated in this setting. the aim of the present study was to prospectively evaluate adrenal function in patients admitted to intensive and coronary care unit for cardiogenic shock. methods. 40 consecutive patients (35 men) admitted for cs, with a mean age of 66 ± 16 years were included. patients submitted to any steroid therapy or etomidate were excluded. 4 patients needed mechanical ventilation and 4 patients were equipped with an intraaortic balloon pump. causes of cs included acute myocardial infarction (n=14), cardiomyopathy (n=13), arrrythmia (n=3), and others (n=10). patients underwent a high dose short corticotrophin test (sct) and relative adrenocortical insufficiency (nonresponders) was defined by a rise in cortisol less than 90 microg/l after stimulation. . 13 (32.5%) patients were classified as nonresponders and 27 (67.5%) as responders. no significant difference was evidenced between responders and nonresponders concerning clinical characteristics and outcome (table 1) . in contrast to international guidelines, it is common practice in some icu's in the netherlands to treat septic critically ill patients with high dose dexamethason on admission. increase in mortality might be associated with the induction of adrenal failure. we compared adrenal function in patients with high, single dose (100 mg) dexamethason (dexa) with patients receiving no steroids during the study period. we studied ventilated patients with mods admitted for emergency reasons. excluded were patients after elective surgery, with an expected short stay or steroid use. cortisol (co) was measured day 1 and 4 at 6.00 am. at day 4 the co response 30 and 60 minutes after 250 mcgr synthetic acth was determined. the patients did not receive corticosteroids, other than dexa on admission if they were included in the dexa+ group. all 25/25 patients (100%) with dexa had baseline co levels on day 4 below 0.69 mmol/l, compared to 7/8 (88%) in the control group (ns). however, adequate co response (rise in co of more than 0.25 mmol/l, 60 min after 250 mcg synthetic acth iv) was 100% in patients with dexa and 50% (4/8) for patients without dexa. in a case control analysis apache score was not a determinant. neutrophils are believed to occupy a prominent position in the pathogenesis of organ failure that arises from the systemic inflammatory response syndrome (sirs). the epidermal growth factor-like 7-transmembrane (egf-tm7) family of molecules are a group of glycoproteins whose structure suggests a dual role in cell adhesion and intracellular signaling. two members of this family, hcd97 and the egf molecule-containing mucin-like hormone receptor (emr2) are expressed on human monocytes and macrophages. the aim of this study was to examine the expression of hcd97 and emr2 on neutrophils from patients with sirs and ascertain if they were associated with sepsis or the clinical course of disease. we analysed erythropoietin, interleukin-3 (il-3), interleukin-6 (il-6), and interleukin-12p70 (il-12p70) in the blood of 301 patients (controls n=250) with circulating nrbcs. in-hospital mortality of nrbc-negative and nrbc-positive patients was 1.2% (3/250) and 22.6% (68/301; p<0.001), respectively. in-hospital mortality increased with the nrbc concentration ( figure 1 ). 85.7% (12/14) of patients with more than 500 nrbcs/µl in the peripheral blood died. multiple logistic regression revealed a significant association between the appearance of nrbcs in the blood and age (odds ratio 1.019; 1.009-1.030; p<0.001), erythropoietin (odds ratio 1.017; 1.007-1.027; p<0.001), il-3 (odds ratio 1.293; 1.180-1.417; p<0.001), and il-6 (odds ratio 1.138; 1.016-1.275; p<0.05), respectively. gender and il-12p70 were not significantly associated with the appearance of nrbcs in the blood to estimate the red blood cell production in the bone marrow the increase in the reticulocyte concentration in blood was measured. the reticulocyte concentration in nrbc-positive patients was 69±2/nl, being significantly higher than in nrbc-negative patients (60±2/nl; p<0.01). furthermore, in the course of hospitalization the increase in the reticulocyte concentration in nrbc-positive patients was significantly higher (24±2/nl; n=224) than in nrbc-negative patients (11±3/nl; n=126; p<0.01). conclusion. an association of the appearance of nrbcs were found with increased levels of erythropoietin, il-3, and il-6, respectively. therefore, nrbcs in the circulation could be an indicator which summarises hypoxic and inflammatory injuries. thus, generally the appearance of nrbcs in blood is a valid parameter to identify patients at high mortal risk. moreover, the increased number of reticulocytes in the blood of nrbc-positive patients may indicate that the appearance of nrbcs is not associated with disturbed bone marrow function as far as the erythropoiesis is concerned. grant acknowledgement. sysmex europe corp. macrophage migration inhibitory factor (mif) was originally described as a tlymphocyte derived cytokine that inhibits the migration of the macrophages at the site of inflammation(1).subsequently it was also identified as a stress induced hormone released from the anterior lobe of the pituitary in response to some pro-inflammatory stimuli (2) .the glucocorticoid counterbalancing proinflammatory actions of mif have been thoroughly documented. our study compared postoperative changes in serum mif levels of patients undergoing bowel and liver resections. 28 patients were recruited in our descriptive study.patients in the first group (a) underwent only hepatic resection without surgically opening the bowel. the other group (b) comprised of patients who have had bowel resection with surgical bowel opening. mif, il-1β, il-8, prealbumin, albumin, α fibrinogen and c-reactive protein levels were measured before and immediately after the operations and also for three consecutive days. to evaluate organ functions the mods-test was used. statistical analysis was carried out by means of spss for windows, applying the mann-whitney test. a higher level of mif (4505 pg/ml /1489-7148/) was found in group a as compared to that of group b immediately after the operations, that proved to be significant. other parameters monitored in this study were not statistically different between the two sets of patients. higher elevations in mif levels with liver resections compared to bowel resections might be attributable to mif release from damaged liver cells. the presumably minimal endotoxin exposure during the bowel surgery was either insufficient or inefficient to induce relevant mif elevations in our patients. chromogranins are prohormones, precursors of numerous peptides displaying various biological activities. some even have antifungal and antibacterial properties. as catecholamines, they result from secretory granules of the chromaffin cells in adrenal medulla. aims of the study: to analyze the physiological secretion of cgb and its derivatives in healthy subjects; and to compare its characteristics with those of patients undergoing the stress of septic shock. methods. 4 healthy voluntaries and 3 patients with septic shock were included. samples of serums were taken at several times to establish a kinetic of secretion. serum proteins were studied by mono and two-dimensional electrophoresis with anti-cgb specific immunodetection, using polyclonal antibodies; and by chromatography (rp-hplc) with specific immuno-detection of each eluted sample, and then by mass spectrometry (maldi tof) and antimicrobial tests. the healthy subjects' electrophoretic profiles are identical. we did not find fragments of molecular weights (mw) lower than 20 kda. but patients' profiles show a great number of short fragments. there were no qualitative modifications of monodimensional electrophoresis profile over time in healthy subjects, whereas for patients, we observed the disappearance of a 37 kda band and of short fragments of weak mw. this modification occurs 4 hours after the end of the infusion of norepinephrine. rp-hplc chromatograms show strong similarities between controls and patients. however the peaks of albumin (hsa) and transferrin are higher in healthy controls. for the whole population, we observe at the end of the chromatogram, 2 immonreactive peaks: the peak of hsa (immunoreactive zone which corresponds to an association of cgb and hsa); and an isolated peak after hsa peak. conclusion. this is the first study of cgb secretion in human serum. we show noticeable differences between healthy controls and patients with septic shock. the clinical improvement of a patient corresponds to the modifications of the electrophoretic profile (backwards to the profile of a healthy control). for the first time, an association is also shown between the hsa and the cgb. in septic shock, the free cgb seems to be more abundant. patients with septic shock or non infectious sirs within 48 hours of admission were included and allocated to the following groups according to usual criteria : group 1 (surgical patients with septic shock), group 2 (surgical patients with sirs), group 3 (medical patients with septic shock) and group 4 (medical patients with sirs). pct at study entry was compared between groups 1 and 2 and between groups 3 and 4 to determine the diagnostic cutoff value for septic shock in surgical and in medical patients respectively. identifying sepsis in intensive care unit (icu) can be difficult. we assessed the utility of the biphasic aptt waveform (bpw) and procalcitonin (pct) determinations, alone or combined, for the diagnosis of sepsis in icu patients. this prospective observational study included 200 adult patients admitted to a 31-bed university hospital medical-surgical icu during a 3-month period. the presence of sepsis, severe sepsis or septic shock was determined on the day of admission by standard clinical and laboratory criteria, without knowledge of aptt or pct. aptt transmittance waveforms (biomérieux mda system) and pct levels (brahms pct lumitest) were determined on the day of admission. threshold values for the prediction of any form of sepsis were assessed by receiver operating characteristic (roc) curves. the bpw was detected when the slope of the pre-coagulation phase (slope_1) exceeded the threshold value (i.e., became more negative). the combined assessment of aptt transmittance waveforms and pct levels provides a rapid means of identifying septic patients on icu admission. van nuffelen m 1 , abraham a 1 , zakariah a 1 , vincent j l 1 1 intensive care medecine, erasme university hospital, brussels, belgium both c-reactive protein (crp) and procalcitonin (pct) concentrations have been proposed to monitor sepsis in acutely ill patients. the aim of this study was to study their time course in septic icu patients. the study included 97 infectious episodes (mean age: 60 years, ratio m/f: 2/1), as defined by standard cdc criteria. patients were divided into two groups, depending on their evolution: favorable (clinical and white blood cell count) or unfavorable (need for additional procedure and/or change in antibiotic regimen). crp was measured daily by direct immunoturbimetry and pct by immuno luminometric assay. and pct were as follows(median values): where day 0 represents the day where antibiotics were started. conclusion. crp and pct kinetics in septic patients show no significant trend in patients who respond favorably to therapy. however, an increase in these variables indicates a poor response. percutaneous tracheotomy (pt) is frequent in the icu to help wean patients from mv. we compared the effectiveness and airway management of laryngeal mask-airway (lma) vs endotracheal intubation (ei) we included 40 consecutive intubated adult patients in the icu who required pt, randomized into two groups of 20. one group had a proseal lma and the other underwent laryngoscope-assisted partial withdrawal of the endotracheal tube. ventilator settings in both groups were: volume-control ventilation, fio2 1, minute volume 8.8 l, peep 0. arterial blood gas pressure was measured before the start of each pt and before insertion of the tracheotomy tube. data were recorded concerning the duration of the procedure from commencing airway manipulation to insertion of tracheotomy tube and airway complications results. 67% of patients were men (median age 55 years). reasons for tracheotomy were a low level of consciousness(48%), lung disease (25%), neuromuscular disease (20%) and airway obstruction (7%). no significant changes were seen in duration, ph, p02 or pc02. complications included six accidental extubation, four tube cuff tears, four guidewire bends and four difficulty to insert the tracheotomy tube. three patients planned for lma required ei because of impossibility to place correctly the pro-seal laryngeal mask-airway. no other complications arose in this group the differential diagnosis between sepsis and sirs is of considerable importance in burn patients. delay in the initial adequate treatment increases the mortality rate. the aim of this study was to assess whether plasma procalcitonin (pct) level was related to sepsis, burn size and organ failure in severely burned patients over the entire clinical course. methods. forty one patients, mean age 53 ± 23 (sd), (range 18-80 year), mean burn size 43.7 ± 22 (sd) % of body surface area (bsa), (range 18% -95 % bsa) were included in our study. all patients were classified daily in one of the following three categories: negative, sirs, sepsis according to the definitions of the accp/sccm. a total of 227 patient days were evaluated: negative (n: 162), sirs (n: 28), sepsis (n: 37). measurement of pct levels and evaluation of organ function by sofa score were performed daily until discharge from icu. admission pct levels were significantly higher in patients with burn size >60% of bsa than in those with burn of less than 60% of bsa (0.57ng/ml vs 2.6 ng/ml, p=0.01). pct plasma concentrations differed among the three diagnostic classes and were higher in sepsis than in sirs ( table 1) . a statistically significant correlation was observed between pct levels and sofa score (r= 0.49, p<0.001 (pearson' bivariate correlation)). the optimal timing of tracheotomy in critically ill patients requiring prolonged mechanical ventilation (mv) is debated. recent studies suggest that early tracheotomy could substantially reduce both infectious morbidity and mortality. in a prospective, randomized, study we compared early tracheotomy with prolonged endotracheal intubation in icu patients needing prolonged ventilatory support. patients projected to need ventilatory support for > 7 days were prospectively randomized to either early (open or percutaneous) tracheotomy within 4 days (et) or prolonged intubation (pi) with or without delayed tracheostomy. the primary end-points were: 28 days mortality and cumulated incidence of nosocomial pneumonia, and number of ventilatory free days between day 1 and 28. time in the icu and on mv, 60 days mortality, number of septic episodes, accidental extubation and amount of sedation were recorded as secondary end-points. a sample size of 470 patients was determined for a reduction of the 28 days mortality from 45% to 32%¨(two-sided, power=0.8). the study was prematurely closed because of poor accrual, after 123 patients (et=61, pi=62)have been included. no difference was found between the 2 groups for any of the primary (table 1 ) or secondary end-points. in addition, laryngeal or tracheal damage and time for resuming oral nutrition did not differ between the 2 groups. early pdt has several advantages when long-term mechanical ventilation is adamant. however, in patients suffering from tbi, one major concern are increased intracranial pressures (icp´s). during pdt, decrease of venous return and hypercapnia might seriously comprise icp. therefore, changes in icp´s during videobronchoscopic guided pdt were measured. methods. 40 patient with tbi,treated at our neurosurgical intensive care unit, required long-term (> 15 days) mechanical ventilation due to intracranial lesions. indication and feasibility to perform pdt were evaluated in patients treated with severe tbi from the day 1 after admission on a daily routine. icp levels below 20 mmhg (over at least 6 hours) without extended icp treatment and no icp increase > 15 mmhg during neck extension was considered to be a safe timepoint for pdt. videobronchoscopic guided, single-step pdt with modified ciaglia technique (blue rhino, cook, germany) was performed in 38 patients, in two patients pdt had do be aborted for anatomic reasons. as operation time we defined begin of videobronchoscopy until the intra-tracheal position of the tracheostoma was confirmed. icp´s were recorded either through intraparenchymal catheters (n= 14) or by external ventricular catheters (n= 24). methods. an anonymous questionnaire was distributed among croatian anaesthesiologists at three universities (zagreb, split, rieka) and during two anaesthesia meetings (split, dubrovnik) between sept. 2003 and may 2004. . 152 completed forms were returned which was 31% of the 450 anaesthesiologists in croatia. male and female respondents were 37% and 63%, respectively, with a mean age of 42.4 years. they had been practicing anesthesia from 1 to 32 years with 91% practicing in an academic center, and 9% in a community hospital. 51% completed a difficult airway course, receiving training at their hospital or at a meeting such as the european society of anaesthesiology. per respondent per year, an average of 975 anesthetics were performed, with 638 patients having endotracheal intubation. the most frequently preferred laryngoscope blade was macintosh (75%) followed by miller (21%) and mccoy (4%). 83% indicated they rarely failed an intubation using a conventional laryngoscope. in difficult airway situations, following laryngoscopy, the technique of choice was the laryngeal mask airway followed by the gum elastic bougie. for anticipated difficult intubations, 54% performed sedated awake intubation, and 29% used the flexible bronchoscope. while the asa difficult airway algorithm was used by 42% of respondents, 25% stated that they used an internally developed difficult airway protocol. croatia. laryngoscopy and sedated awake intubation are used more frequently than fiberoptic bronchoscopy. the asa difficult airway algorithm was used by 42% of the anaesthesiologists surveyed. in a randomized crossover trial, 25 special forces (sf)-medics of the royal netherlands army and 19 residents in anesthesiology performed cricothyrotomies using two different emergency airway devices on larynges from freshly slaughtered pigs (1) . we compared the quicktrach with the portex emergency cricothyroidotomy kit. all data were analyzed using spps version 11.0 (wilcoxon test for non-normal distributed 2-paired comparison and mcnemar test for nominal values). the quicktrach-technique was done significant faster than the portex-technique in both groups. intratracheal placement of the cannula was achieved by 14 (56%) sf-medics and 12 (63%) residents using the portex-technique and using the quicktrach-technique by 18 (72%) sf-medics and 15 (79%) residents. despite the fact that it was a procedure performed in very critically ill patients, tracheostomy was associated with very few minor complications in this sample. we hipothetized that this low rate of complications is due in part to the very high expertise of the operators involved in the realization of conventional tracheostomies in the two centers. grant acknowledgement. the authors are indebted with dr. ederlon a. c. rezende for his support and suggestions. kiessling a h 1 , isgro f 1 , skuras j 1 , lehmann a 2 , pieper s 2 , saggau w 1 1 klinikum ludwigshafen, cardiac surgery, 2 klinikum ludwigshafen, anaesthesiology, ludwigshafen, germany tracheotomies are routinely performed for severely ill patients with respiratory failure. the procedure facilitates the weaning procedures by reducing dead space and decreasing airway resistance, by improving secretion clearance and by decreasing the risk of aspiration. this intervention is correlated with a poor survival rate. the aim of the investigation was the evaluation of the quality of life scores (qof) and outcome after cardiac surgical procedures. the retrospective, non-randomized follow up study was performed in a single surgical intensive care unit in 49 patients after cardiac procedures and surgical tracheotomy. preoperative data and items were collected and outcomes analyzed after a mean follow up period of 2.9 years. a written questionnaire for the documentation of the sf 36 score and beck depression scale were used. in addition to the test battery, healing outcome and vocal function were components of the questioning. overinflation of the endotracheal tube cuff (> 25 mmhg) may cause tracheal damage and complications such as tracheal stenosis and tracheo-oesophageal fistula. we have surveyed the practice of tracheal cuff pressure measurement in our medical-surgical intensive care unit (icu) and evaluate the impact of a regular cuff pressure monitoring program (cpmp) on reducing cuff overinflation. cuff pressure have been evaluated over three periods (p1= before cpmp, p2 = 3 months after cpmp and p3 = 3 years after cpmp) obtained in 100 measurements in 35-40 patients each period. the cpmp consists of regular cuff pressure monitoring twice a day. comparing to the first period, mean cuff-pressure decreased in the second period from 42±22mmhg to 26±13mmhg (p< 0.001) and the rate of overinflated cuffs from 77% to 24% (p< 0.001). in the third period, mean pressure was in the normal range (21±19 mmhg) but there was a significant increase in underinflated cuffs. however, in these patients, the operator hasn't noticed any leakage around the tube cuffs. a regular cuff pressure monitoring program can reduce significantly the overinflation of tracheal cuffs in icu and this may lead to prevent subsequent complications. icu medical stuff may also maintain this protocol by a regular education of the nurse team in order to always keep endotracheal tube cuff pressures in the normal range preventing over (tracheal damage) and underinflation side effects (nosocomial pneumonia). further studies are needed to evaluate this educational procedure on the outcome of the icu patients. forty-three patients (27 men, 16 women) with a mean age of 54.8 and a mean simplified acute physiologic score (saps) ii of 49.3 were studied. three patients were excluded because of insufficient data. ts was done because of traumatic brain injury with persistent glasgow coma score <8 (nineteen patients), unsuccessful weaning -failure of spontaneous breathing trial in 2 or more occasions (8 patients), hypoxic encephalopathy (6 patients) and prolonged invasive ventilation (6 patients). in the subgroup of 8 patients with unsuccessful weaning, spontaneous breathing could be achieved in seven patients by day 1 to day 7 (mean of 3.0 days) after ts. in 24 patients, ts has been considered an adjunctive intervention for the weaning process, and in these patients, spontaneous breathing was achieved in 17 patients and bipap ventilation in 3 patients. thirty nine patients could be discharged from icu (mean of 8.1 days after ts) in spontaneous breathing (36 patients) or bipap ventilation (3 patients). mortality analysis revealed a total of 18 deaths (four in the icu, 9 during hospital stay and 5 after hospital discharge at six months). in patients with hypoxic encephalopathy (6), five deaths were observed during hospital stay. complication rate was low, with local haemorrhage in seven patients. our study revealed that ts was useful as an adjunctive therapy in the weaning process in the majority of patients and could reduce icu stay; however, the subgroup of patients with hypoxic encephalopathy did not benefit from ts and should be considered for alternative strategies of airway protection. zgoda we report a prospective case series of 10 successful percutaneous tracheostomy procedures in the critically ill without complication. the balloon-tracheostomy tube apparatus (image 1) was placed overwire then inflated to form the stoma, then deflated. the tracheostomy tube followed the deflated balloon into the airway. almost no anterior tracheal compression took place. the average procedure time from puncture to tube placement was 4-6 minutes. ten icu patients underwent bfpt. six of the 10 patients had a successful tube placement after only 1 balloon dilation. the rest had successful tracheostomy placement after a second dilation. one of these 10 patients had a previous tracheostomy and the procedure was successful with 2 balloon dilation attempts at the site of the previous tracheostomy. two were coagulopathic with inr>2 and/or platelet count(s) of less than 50k. the average estimated blood loss was less than 2ml. one patient had an obvious tracheal ring fracture without immediate clinical significance. there was no posterior tracheal wall damage, no pneumothorax, and no obvious damage to the anterior neck. thus far, there have been 10 consecutive tracheostomy tubes placed without bleeding complications, or damage to the posterior tracheal wall but more study is needed. bfpt is an easy and effective means of placing an elective tracheostomy tube at the bedside in the icu. despite surgical percutaneous emphysema is a recognised complication following percutaneous tracheostomy [1] ,it is not usually reported with a fenestrated trachesotomy tube as the direct cause [2] .the rationale to use a fenestrated tube when performing percutaneous tracheostomy is to eliminate the need to change the tube when the patient is weaned from mechanical ventilation. report of a cluster of complications associated with fenestrated tracheostomy tubes placed percutaneously. in our trust (3 hospitals) within a 4 week period 8 patients developed subcutaneous emphysema (one with an associated pneumothorax). the cases were performed by experienced doctors. all using portex blue rhino kits, with the insertion of tracoe-twist fenestrated tracheostomy tubes (using the non-fenestrated inner cannula); bronchoscopic guidance was used in all of the cases.we also have performed a bench top study on the fenestrated tubes to find the source of leak. eight patients developed subcutaneous emphysema (one with an associated pneumothorax).the emphysema was immediate in some, but only becoming apparent several hours after insertion in the majority.in at least two,the emphysema was so extensive that it compromised the patients' airway making exchanging the tracheostomy impossible and oral endotracheal intubation very difficult. fortunately there were no directly attributable deaths or hypoxic injuries. the bench top study revealed air can track between the inner and outer cannulae at quite low pressures. surgical percutaneous emphysema is a complication following percutaneous tracheostomy using fenesterated tubes which can lead to pneumothorax and airway compromise.it seems that the fenestrations can remain in the pre-tracheal fascia with air tracking between the inner and outer cannula leading to the development of subcutaneous emphysema.we have now changed our practice to insert only non-fenestrated tubes for percutaneous tracheostomies. therapeutic hypothermia (th) improves outcome after cardiac arrest (ca) due to ventricular fibrillation (vf). however, due to lack of protocols and to technical difficulties inherent to its practical application, this treatment has not been widely implemented in daily practice. we evaluated whether th could be effectively introduced in icu practice and assessed its impact on patient outcome. we retrospectively analyzed 110 comatose patients resuscitated from out-of-hospital ca due to vf and non-vf rhythms (asystole or pulseless electrical activity in patients with circulatory shock before initiating the treatment, th was also beneficial (4/10 patients had good outcome vs 0/9 patients treated with sr, p=0.03). in contrast, th had no impact on the outcome of survivors of ca due non-vf rhythms (2/12 patients in the th group survived with good neurological outcome vs 1/12 in the sr group). conclusion. therapeutic hypothermia can be safely and effeciently introduced in icu practice for the treatment of all comatose patients resuscitated from cardiac arrest with a major impact on the outcome of patients resuscitated from ca due to vf, independently from their hemodynamic status. in contrast, our data do not support the use of therapeutic hypothermia after cardiac arrest due to asystole or pulseless electrical activity. lavery g g 1 , hickland b 1 , caddell p1, dillon m 1 , northern, ireland intensive care society audit group 1 1 regional intensive care unit, royal hospitals trust, belfast, united kingdom since october 2000, a centralized service has facilitated the interhospital transfer (iht) of over 1400 critically-ill adult patients using a standard ambulance and mobile icu equipment. quality of escort is an important factor in the transport of all potentially unstable patients (1, 2) and so all ihts are performed by an experienced icu team (1 doctor and 1 nurse) . the aim of this project was to assess the use and the quality of this service. information regarding the indications for, and conduct of, iht was recorded prospectively for all patients transferred by the service over 1 yr (03/04-02/05). icus prospectively collected data including admission apache ii score and icu (and hospital) outcomes. all data were entered on a central database (ms access). molnar t 1 , köszegi t 2 , bogar l 1 , szakmany t 1 1 anesthesiology and intensive therapy, 2 institute of laboratory medicine, university of pecs, pecs, hungary it has been proposed, that procalcitonin (pct) might be used as a prognostic factor for outcome after cardiac arrest (1) . to date no studies addressed the question whether pct levels are different after vf and pea induced in-hospital cardiac arrest. methods. 45 consecutive patients were studied following cardiac arrest. pct levels were measured on icu admission (t 0 ), then on the first (t 24 ) and third day (t 72 ) post-arrest. for statistical analysis mann-whitney u test and chi-square test were used with spss 11.5. data are presented as median and interquartile range. out of the 45 patients 37 suffered pea and 8 vf arrest. there was no significant difference between the groups regarding age, male/female ratio and anoxic time and time to rosc. mortality was 77% vs. 12.5% in the pea and vf groups, respectively, p<0.01. serum pct levels were significantly higher in the pea group (table 1) . s100b levels did not differ significantly between the two groups. serum pct: 3.5(0.10-8.95) vs.0.9(0.33-1.86) and s100b:2.99(1.36-5.42) vs.1.19(0.19-2.33) were significantly higher at t 0 among non-survivors in the pea group, p<0.05 respectively, whereas in the vf group no such difference was observed. conclusion. significantly lower inflammatory response was detected in patients initially in vf arrest, with significantly better survival compared to pea arrest, although anoxic time and time to rosc was similar in the two groups as reflected by nearly identical s100b levels. however, patients with pea arrest often have long, undetected hypoxic period, which may trigger the release of inflammatory markers such as pct. the significantly higher pct and s100b values in the nonsurvivor group of pea patients may indicate the potential prognostic value of such measurements. horn j 1 , zandbergen e j g 2 , vos p e 3 , verlooy p 4 , van dijk g w 5 , vroom m b 1 , hijdra a 6 1 ic, amc, amsterdam, 2 neurology, rijnstate, arnhem, 3 neurology, umc, nijmegen, 4 neurology, olvg, amsterdam, 5 neurology, umcu, utrecht, 6 neurology, amc, amsterdam, netherlands after cardiopulmonary resuscitation (cpr) many patients develop post-anoxic encephalopathy (pae) often accompanied by myoclonic seizures or epilepsy.(1,2)treatment is often difficult, several strategies have been advocated. (3, 4) in this study we investigated the medication used in these patients. from the database of the propac study, a prospective cohort study in pae patients, we selected patients with myoclonic or epileptic seizures. medication used to treat these conditions was extracted from the records. in 188 patients, 97 showed myoclonic seizures or epilepsy. records of 76 patients could be retrieved. differentiation between myoclonus and epilepsy was difficult, we used the description as found in the records. eleven patients received no medication. treatment was started in 65 patients (86%): in 58 (89%)a benzodiazepine, in 35 (54%) another antiepileptic drug, in 32 a combination of both. clonazepam was used most often (36 patients, 62%). valprioc acid was used in 19 patients (54%), phenytoin in 16. seventeen patients (out of 65) received propofol and in 14 patients a second benzodiazepine was administered. outcome after 1 month: 66 had died (87%), 7 were in coma, vegetative state or severely handicapped (9%) and 3 were moderately handicapped or completely recovered. conclusion. dutch neurologists prefer benzodiazepines in patients with seizures in post-anoxic encephalopathy, often combined with an antiepileptic drug. myoclonic status reacts poorly to medication, however, treatment is often started because of problems in daily care or mechanical ventilation. in this study we found that the different types of seizures were often not specified in the records, despite the consequences on prognosis. we suggest to use the definition proposed by wijdicks et al for myoclonus status.1 in this study epileptic or myoclonic seizures in patients with post-anoxic encephalopathy seemed to be related to poor outcome, as 96% had a poor outcome. we conducted an etiologic study among parturients presenting a cerebrovascular stroke. the aim was to determine the frequency of the various types of vascular accident and their moment of arisen,to underline the factors of risk and to estimate the prognosis of vascular accidents in this population(p-values <0,05 were considered to be statiscally signifiant). among our 25 patients,17 had an ischemic accident,12 of which had venous origin,5 an arterial origin and 8 had an hemorragic accident .the majority of damage occurs in the 3 rd quarter of pregancy or in the post-partum. five of our patients had no risk factor and 20 had several risk factors.as for the arterial accident , the etiologic inquiry was not decisive for four patients.they had however several risk factors of thrombosis vascular. five patient with an ischemia died and three of the patients having a bleeding died. uni-varieted logistic regression did not find statiscally-significant result concerning mortality in relation with the age,the term gestationnel or the type of accident . conclusion. cerebrovascular strokes complicating the evolution of a pregnancy remain an unknown entity. they can cause sequela and have fatal issues.studies including a larger number of patients are requested in order to decrease the incidence and the important morbi-mortality. they are also meant to find out all risk factors,take them in to consideration and therefore work on their mechanism. bubnova i d 1 , dobrinin i n 1 , astakhov a a 1 1 anaesthesiology and reanimatology, ural postgraduate medical academy, chelyabinsk, russian federation one of the ways for the cerebral perfusion support in severe brain trauma (sbt) patients is the cardiac output optimization. but we must know whether the decreasing of hypovolemia range be better for brain protection or not in each case. this study we tried to reveal if the the topic level of central haemodynamic regulation disturbances (chrd) can determine the response on the volume load (vl). in the previous works we showed that the patients with sbt may have different types of the haemodynamic regulation due to interfere of the humoral and the autonomic nervous stimulus. this study we examined 52 patients with 3 main regulatory types. all patients were under artificial ventilation and had 11 and less gcs. for the estimation of the type and topic level of chrd we compared the absolute data and the variability (spectral power (sp) in 0 -0.5 hz band) of blood pressure (bp), heart rate (hr), peripheral vessels pulse (pvp), and stroke volume (sv), determined by the bioimpedans method. also we determined the variability of eeg amplitude in the alone biparietal channel. all comparisons were made before and after infusion of 500,0 ml of 6% stabisol. especial attention were paid to the p2 (0.05-0.075 hz) and p4 (0.2-0.5hz) bands of sv, which reflect the hormonal, more often adh activity (p2) and predominantly connect with patients breathing (p4). last findings showed it may be used as a marker of hypovolemia. the patients with the worst type of regulation (a result of brain stem dysfunction) responded on the vl by the sv increasing in 87,5% cases. but they showed a decreasing sp of p4 only in 43,7%, and sp of p2 increased in 62,5%. in cases of hypothalamic dysfunction (type 2) the sv grew in 50% patients, sp of p4 decreased in 62,5% and p2 increased in 50%. the patients with the best adaptive type of regulation (type 1) responded on the volume load only in 42,8%, but had sp of p4 decreasing in 78,5% and low growth of p2 (21,4%). surprisingly, in some cases we revealed the great decreasing of variability of hr, bp, pvp and eeg amplitude as a transformation from type 1 or 2 to 3 after infusion. in sbt the vl partly compensates hypovolemia, but creates an exertion in regulatory system, especially in case of significant chrd. so we need to find out the predictive marker of response on the vl in different level of brain damage. engström m 1 , schött u 1 , reinstrup p 1 1 anaesthesia and intensive care, lund university hospital, lund, sweden acidosis has been found to be a predictor of worse outcome in trauma patients suffering from exsanguination. it has, however, not been studied if acidosis may be a causal factor in the development of coagulopathy. rotational thromboelastography (roteg) is a coagulation monitoring tool that is gaining increasing popularity as it seems to be more sensitive and specific than routine coagulation tests in detecting defects of the coagulation system. clot formation time (cft) and alpha angle are roteg parameters primarily dependent on the rate of fibrin formation and the platelet activity. methods. 5 blood samples of 5 ml each were obtained from 6 healthy volunteers. one sample was studied without any additions. three samples were adjusted to ph 7.2, 7.0 and 6.8 by the addition of 25, 50 and 75 µl of 1m hydrochloric acid (hcl). the last sample was first adjusted to a ph of 6.8 by the addition of 75 µl of hcl and then reversed to a ph of 7.4 by addition of 30 µl of tromethamol (tham) 3.3 mmol/ml. after adaptation of the ph to the desired level roteg was performed to study the coagulation system. we found a strong correlation between decreasing ph levels and an impairment of the coagulation (p<0.00001) (figure 1 ). the impairment of the coagulation caused by the acidosis was reversible after addition of the buffer tham. in subarachnoid haemorrhage (sah), old age and high clinical grade at presentation are poor prognostic factors. treatment for these patients has been largely conservative. with endovascular coil embolisation a less invasive treatment option has become available(1). this study focuses on elderly and high grade patients admitted to the nicu. retrospective analysis of 167 patients with aneurysmal sah. demographic features, wfns grade at presentation (low grade: 1&2), fisher grade, data for aneurysm site and mode of intervention were recorded. outcome at three months coded according to the modified rankin score (good outcome: rankin 0-2). conclusion. our data suggest that favourable outcomes (rankin score 0-2) can be achieved in elderly patients with high grade (wfns 3-5) sah. 35% of 20 high grade patients >60 years made a good recovery. this may be due to the less invasive nature of coil embolisation and careful patient selection. gama r x 1 , oller a m 1 , bortoletto t c 1 , almeida c r m 1 , gurgel a p a 1 , henrique l m p 1 , zanini a a 1 , faintuch j 1 1 central pharmacy, hospital alemao oswaldo cruz, sao paulo, brazil lipid-based parenteral nutrition (tpn) mixtures are deemed safer than glucosebased preparations as regards possibility of hyperglycemia, but few comparative studies are available. aiming to determine glucose concentrations during such therapy, a clinical study was done. stable septic patients submitted to tpn (n= 96) during a 12-month period were investigated on the 1st and the 7th day of therapy. both glucose-based (group i, n= 29, 3.3±1.2% of total calories as fat) and lipid-based (group ii, n= 67, 25.8±9.2% of calories as fat) programs were employed. groups were comparable regarding age (55.5±14.7 vs 59.0±14.1 years , ns), gender (55.2% females in both groups) and features of septic problems . energy intake was slightly higher in the lipid-based preparations, but without statistical difference (1120±376 vs 1352±550 kcal/day ). conclusion. 1) glucose-based tpn was associated with moderate hyperglycemia when compared to a lipid-containing prescription in this septic population; 2) no clinically significant hyper or hypoglycemia was registered. guidelines for blood transfusion (bt) are based on plasmatic haemoglobin value (hb) and on clinical state. apart cardiac and septic patients, the threshold value of hb for bt is 7 g/dl. the aim of the study was to evaluate the central venous oxygen saturation (scvo2) as a guide for bt decision. methods. 60 patients of general and urologic surgery for whose a bt was discussed were included. scvo2 (%) and hb (g/dl) were measured before and after bt. the following parameters were registered: age, history of cardiovascular disease (cv), presence of sepsis, number of blood units. patients were retrospectively divided into 2 groups according to scvo2 before bt < or > 70 %. overall, demographic characteristics were similar. bt provided a significant increase of hb for each patient while scvo2 value rose significantly only in patients with scvo2 before bt <70% (table 1) . results are given in median (range). * wilcoxon test for values before vs after bt; # mann-whitney test or chi-2 for scvo2 < vs > 70 %; significance for p< 0.05. 8.05 (6.4-9.8) hb after bt 9.8 * (7.9-11.9) 9.6 * (7.9-11.6) 9.9 * (8.5-11.9) conclusion. among the 60 patients studied, only those with a low scvo2 before bt had a better tissue oxygenation by hb increase. scvo2 might be an interesting parameter to help the clinician in his decision of postoperative bt. szakmany t 1 , dodd m 2 , dempsey g 1 , lowe d 2 , rogers s n 2 1 department of anaesthesia, 2 regional maxillofacial unit, university hospital aintree, liverpool, united kingdom perioperative blood transfusion is reported to be related to cancer recurrence and reduced survival1. to date, little is know about the effects of blood transfusion on outcome in oropharyngeal cancer. we undertook this study to test the hypothesis that perioperative blood transfusion has an adverse effect on survival of patients with oropharyngeal cancer. methods. 557 patients undergoing oropharyngeal cancer resection were evaluated from jan 1992 to december 2002. transfusion rate, units of blood transfused and tumour stage were recorded. the primary outcome measure was oropharyngeal cancer death within two years. cox logistic regression was used to assess the association between cancer death and blood transfusion. data are presented as median (interquartile range). overall transfusion rate was 65% (362/557), units of blood transfused 3 (1) (2) (3) (4) (5) . mortality was 20.1% (112/557). mortality was significantly higher in the transfused group (table 1 .) however, in the cox regression analysis only tumour size, stage and clear resection margins were predictive of survival. after stratification of patients for these predictors, transfusion did not affect disease specific survival. in patients who are supported by mechanical ventilation with tracheostomy and who undergo neck surgery because of the neck trauma or neck infection, there is some risk of dislocation of the tracheostomy tube, contamination of the fixation device during daily surgical wound management and daily nursing care. the object of this study is to clarify the usefulness and safety of our technique of easily detachable fixation of tracheostomy tube with small clip in these patients. methods. 5 patients who underwent this technique were examined. we detach the clip fixing the tracheostomy tube during daily surgical wound management and attached it as soon as finishing wound management. we did not experienced dislocation of the tube during daily surgical management and daily nursing care in all cases. we easily protected contamination of fixation device of the tracheotomy tube during surgical wound management. our technique of fixing the tracheostomy tube using detachable small clip is useful and safe in patients who are supported by mechanical ventilation with tracheostomy and who undergo neck surgery because of the neck trauma or neck infection. schachtrupp a 1 , toens c 2 , afify m 1 , lawong g 1 , schumpelick v 1 1 surgery, rwth aachen, aachen, 2 surgery, marien hospital, dusseldorf, germany in the presence of abdominal compartment syndrome (acs) the increased intraabdominal pressure (iap) leads to organ damage and reduced cardiac output (co). decompression is of utmost importance but occasionally circulatory collapse occurred. moreover, it is unknown whether reperfusion will increase organ damage. aim of the underlying study was to determine the influence of decompression on circulation and organ damage in a porcine model of the acs. we investigated 18 pigs (dl, 50 kg). in two groups (each n=6), iap was increased to 30 mmhg for 18h using co2. in one group a period of decompression lasting for a period of 6h followed. in the control group, iap remained unchanged for 24h. all animals received a basic volume substitution of 2 ml/kg. additionally, 500ml of kristalloids were given whenever the continuously monitored co was lower than the control reading of 70 ml/min x kg. heart rate (hr), mean arterial pressure (map), central venous pressure (cvp) and urine output (uo) were recorded. at the end of the experiment, specimen from the lung, liver, kidney and bowel were taken for histological examination. moreover, liver tissue was examined for the expression of icam-1 displaying leukocyte sticking. statistical analysis was done using analysis of variance as well as paired and unpaired ttesting. a p<0.05 was considered significant. in case of repeated pairwise testing, level of significance was adjusted. results. co did not differ between groups but additional volume was needed in study groups. hr, map and uo did not differ. cvp was significantly increased. after decompression, hemodynamic parameters remained stable, uo increased significantly. medium grade histological was found after 18h of increased iap. reperfusion did not increase organ damage. the highest expression of icam-1 was found after 18h of increased iap without reperfusion. conclusion. in this model, administration of additional volume was sufficient to preserve co despite the presence of an iap of 30 mmhg. decompression did not lead to circulatory collapse. nonetheless, organ damage was present which was not increased by decompression. these results imply, that in the presence of critically increased iap, adequate volume substitution is needed together with an immediate decompression in order to avoid organ damage. there were 8 episodes of ventilator associated pneumonia in 8 patients of total 250 admitted patients (3.2%) and 105 patients who required mechanical ventilation support (7.6%). the mean ventilator associated pneumonia rate was 15.6/1000 ventilator days. leading causative agents detected in our picu patients were pseudomonas aeruginosa (3.8%),enterococcus (2.8%) and staphilococcus aureus (0/9%). all patients with ventilatory associated pneumonia survived. ventilator associated pneumonia occurs at significant rates among mechanically ventilated picu patients. ultrasonic guided pleural aspiration is a safe ed accurate method of obtaining fluid in pleural effusion, caused by several mechanisms (pneumonia, cancer, congestive heart failure etc). drainage could improve pulmonary ventilation and allow the laboratory examination of the fluid, useful for the differential diagnosis. pneumothorax (pnx)is the principal complication of thoracentesis. for this reason, five years ago, the emergency department of this hospital, adopted the plastic catheter (pc)in use for iv infusion in order to perform a pleural drainage. the aim of this study was to evaluate the effectivness of this method compared with the more common metallic needle (mn)contents in the set for thoracentesis. where insert the needle. after a local injection of anesthetic lidocaine, one of the two needles was chosen. in particular, for the pc, after the inserction, the metallic core was removed and only the plastic tube was left in place and connected to the drainage system. pleural aspiration was removed when the patient had thoracic pain, cough or fluid flow ceased. by ultrasonography, at the point of drainage, was measured the space between the two pleural layers and this was considered a parameter of drainage entity: the lower the space, the greater the drainage. results were analised on a statistical manner by t test of student for impaired data. patients who underwent thoracentesis by pc had more complete thoracic drainage (pleuric space 5,8 +/-3,2 cm vs 12,7 +/-4,2 cm; p < 0,03)without case of pnx (0 vs 1). ultrasound is more accurate than plain chest radiography for estimating pleural fluid volume and aids thoracentesis. chest drainage by plastic catheter increases efficacy and safety. prospective randomized study included 40 enterally fed patients with an expected mechanical ventilation period of at least 6 days. the diagnosis of vap was based on clinical, radiological and bacteriological criteria. qualitative and quantitative bacteriological study of microorganisms isolated from gastric content as well as from upper and lower respiratory tract was carried out on the 1st, 4th and 6th day of the therapy. material from lower respiratory tract was taken by protected specimen brush (psb) using bronchoscope. introduction. vap is a frequent nosocomial infection. since delayed appropiate antimicrobial therapy worsens prognosis, broad-spectrum antibiotics are frequently administered. early clues on potential microorganisms involved could help select a more focused antimicrobial therapy. the known relation between vap and upper airways colonization prompted us to determine if uas at the time of icu admission (day 1) could accurately identify microbial agents involved in early vap (within the first 5 days following tracheal intubation). 136 consecutive icu patients who had a clinical pulmonary infection score (cpis) consistent with the diagnosis of vap between 1996 and 2001 were retrospectively analyzed. uas (nose and throat) were obtained at day 1 for all patients, and specific pathogens (other than normal oropharyngeal flora) were cultured. pulmonary plugged specimen (pps) were obtained whenever vap was suspected and were considered positive beyond 10 3 cfu/ml. the concordance between uas at day 1 and the first pps was analyzed. in level i(unit-based),6.8%(95% ci 6.6-6.9)of patients stayin >2d, acquired at least one episode of iapn. the percentage varied strongly according to the country (4.0 to 15.5%),type of icu (7.2% in mixed,5.5% in medical and 4.6% in surgical icus) and percentage of intubation. incidence density (id)per 1000 patient-days was 5.3(5.0-5.6)in icus with <30% of intubation, 9.2 (8.9-9.7) in icu with 30-59% intubation and 10.8(10.4-11.1) in >60% of intubation, p<0.001). the median n of days from admission to iapn were 12.8(10.7-17.7). the most frequently isolated were p aeruginosa (17.9%) and s aureus (16.2%), with large variations between countries.gp cocci were isolated in 28.7%, gnb-enterobacteriaceae in 27.4%,gnb non-enterobacteriaceae 31.8% and fungi/parasites 10.2%. table 1 shows the distribution of micro-organism according to the time of onset of iapn. in level ii surveillance (patient-based) intubation utilisation ratio was 0.49 (0.38-0.71) and deviceadjusted indicator: 19.9 iapn* 1000/ intubation days (4.0-22.0). 18% of all general critical care patients were transferred for their care. these patients accounted for 25% of all bed days in the network. transferred patients had a mean icu stay of 10 days, 3 days longer than non-transferred patients (p=0.014) together with a slightly longer hospital stay. there was also a small (1.6%) increase in hospital mortality associated with transfer which was not statistically significant. there is a large number of level 1 patients who are at risk of deterioration or have stepped down from higher levels of care.these patients can be looked after on an acute ward with additional support from the critical care/outreach team the critical care network has 73 level 2 beds and the audit identified 232 level 2 patients.159(68.53%)of these patients(at the time of the audit) could not access a level 2/hdu bed despite their condition warranting care in an hdu area. the care delivered to this group of patients is therefore by staff trained for a level 0/1 area.with 82 level 3 beds, capacity for level 3 patients was appropriate on this day with 5 beds available. the networks patient transfer activity for 2004 was 500 non clinical transfers and 300 clinical transfers. sepsis is one of the leading causes of death in intensive care medicine (icm). the rapid diagnosis and management of sepsis is critical to successful treatment. since 1999 we have integrated diagnostic and treatment feature of severe sepsis into our berlin simulation training in order to optimize team functions. lectures and interactive simulation scenarios are combined and discussed. participants are postgraduates with differing professional experience in icm (pe). to evaluate the structure, content and impact of those courses participants of four simulation courses in 2004 were given anonymised questionnaires both in advance and immediately after the course. 28 pairs of items were defined to measure the acquirement of knowledge in sepsis and the impact of several teaching methods. participants were also asked whether the course content should have been given earlier or later during their postgraduate training. answers were given on a five point scale (likert-like) and results are given as median and interquartile range (iqr). participants' pe in icm varied from two months to 14 years. all participants expressed benefits from the course. both lectures and scenarios were evaluated helpful to identify sepsis patients earlier. most of the participants thought that the course was at a right point of time during their postgraduate training (pt) (n=15, median of icm/pe 1.5a, iqr 1-4a) and 12 participants thought that this course would have been even more helpful if had been given earlier during their pt [n=12, pe 2.5a (1.5-5.5a)]. "the course was being helpful concerning future identification of sepsis patients" -2 (2-3). "the lectures were being helpful concerning future identification of sepsis patients" -2 (1.5 -2) "the scenarios were being helpful concerning future identification of sepsis patients" -2 (1.5-3) "the scenarios were realistic" -2 (2-3) and "i enjoyed the course" -1 (1-2) conclusion. simulation courses to train early identification and timely treatment of septic patients are very helpful and appreciated at every stage of pe in icm. simulation courses should be integrated as early as possible. jermin s p 1 , kapila i 1 , dyson m 1 1 critical care, south manchester university hospital, manchester, united kingdom there is a recognised shortage of icu beds in the uk.critical care outreach services help reduce pressures on critical care by providing clinical support, increasing staff skills and by providing educational support(1). early identification of sick patients may lead to a reduction in number of admissions to icu, length of in hospital and icu stay (2) . this study aims to compare the level of care of all inpatients on a normal 'in hours'working day(tuesday) with those of all in patients on an average winter 'out of hours' day(sunday) data was collected from every inpatient in the hospital(excluding psychiatric,paediatric and long term rehabilitation patients) on an average tuesday in april 2004 between the hours of 0900-1600 and then on a sunday in january 2005 between the hours 0900-1600 . data consisted of levels of care(using uk intensive care society definitions)(3) during both periods but also included demographic details for the second period.presence of respiratory rate(used as an index of deterioration) recording was also noted for the second period. conclusion. the current complement of level 2 and 3 beds in the hospital is 18 and 20(dependant on staffing levels)respectively.despite some flexibilty in using level 3 beds for level 2 patients and assuming an 80% bed occupancy, there is a considerable need for more level 2 capacity particularly during the winter period.extension of the current theatre recovery area into a 4 bedded post-operative hdu could provide additional beds .outreach services would also need to be vastly extended. abizanda r 1 , nicolás-picó j 1 , mateu-campos l 1 , carregui-tusón r 1 , sánchez-morán f 1 , mas-font s 1 , ferrándiz-sellés a 1 1 intensive care department, hospital universitario asociado general de castelló, castelló, spain when no icu specific analytical accounting is available, the only indicators of direct costs are the number of icu stays per patient, and pharmacy costs. it is usually accepted that these pharmacy costs represent between 10 and 15 % of total costs, and that they are very much influenced by therapeutic attitudes of the attending teams and the introduction of new pharmacological options or the change in the already existing ones. our aim is to analyze the changes in pharmacy costs occurred during the interval between 1996 and 2003. methods. this is a retrospective analysis performed on a multidisciplinary 19 beds icu activity, in a teaching referral hospital. the analysis has been performed through data coming from the managerial departments and the pharmacy service, and costs have been classify as related to therapeutic group (pharmacy instructions from the spanish national health system) and to individual active drugs. the analysis collects information raised from the icu daily patients chart. pharmacy costs amount ranged between 16,8 % in 1996 and 12,3 % in 2002. since then a slight increment in pharmacy costs has been detected up to 14,6 %. the reasons for cost decrements are linked to the progressive control on albumin use and antibiotic policies. by the contrary, increasing percentages are associated to the introduction of new sepsis therapeutic approaches (drotecogin) and the routine introduction of antiplatelet agents in non elevated st coronary syndromes. the "top twenty" drugs cost evolution is presented, and in a constant fashion the two firs places represent the use of sedatives (propofol) and fibrinolytic agents in ami (tecneplase). factors that allow or avoid to keep the stability of what pharmacy costs represent are strictly linked to changes in physician attitudes (abandon of non demonstrable efficacy of certain agents -albumin -, the incorporation of new options -drotrecogin, antiplatelets -and the maintenance of consolidated practices -fibrinolytic agents, sedatives, nutritional strategies. physician teams are obliged to keep this information "alive" in order to avoid unnecessary raises in direct costs. cotogni p 1 , bini r 2 , forno g 3 , porta c 3 , aliffi s 3 , ranieri v m 1 , pittiruti m 4 1 anestesia e rianimazione, 2 chirurgia d'urgenza, 3 school of nursing, university of turin, turin, 4 chirurgia generale, catholic university, rome, italy enteral nutrition (en) is the preferred method for nutrient delivery in icu critically ill patients. nonetheless, there is always a significant gap between prescribed and delivered feed. this is partly due to 'patient-related' problems, e.g. gastrointestinal (gi) intolerance to en, but also by logistic 'management-related' events which imply transient nutrient delivery interruptions, which are often mandatory but sometimes avoidable. the aims of this study (prospective, descriptive study of en delivery in 4 teaching hospital icus) were (a) to analyze the causes for en transient interruptions; (b) to assess whether a specific nurse training might be associated with better nutrient delivery. in two icus (group a), all nurses had been previously trained in en through a 16 h education module, while nurses of other icus (group b) had not. over a period of 30 months, we studied all icu pts receiving en (either alone or combined to parenteral nutrition). pts receiving en for < 7 days were excluded. en was administered as a continuous (24/24 h) intragastric infusion of a standard polymeric diet. we recorded any transient interruption of nutrient delivery lasting more than 15 min, noting the duration and the cause. we examined 244 pts fed by en accounting for 2537 en days (10.4+2.6 days/pt). in the groups, patient populations were similar in saps, diagnosis on admission to icu, complications, days of mechanical ventilation and mortality. the main causes of transient en delivery discontinuation were mechanical (11%), or secondary to diagnostic and therapeutic procedures (59%), or related to true gi intolerance to en (30%). comparing groups, we found that group a was characterized by a lower incidence of discontinuations for mechanical causes (p<.01), as well as by a shorter duration of interruption due to mechanical causes (p<.001), to procedures (p<.001), or to intolerance (p<.01). also, the difference between prescribed and delivered feed was significantly lower in group a (p<.001). our study shows that (a) the majority of discontinuations of en delivery is secondary to 'management-related' causes and not to patient's intolerance; (b) a specific training in artificial nutrition of the icu nurses may be effective in increasing nutrient delivery by reducing incidence and duration of those en discontinuations which are not 'patient-related'. saura p 1 , ortiz d 2 , prat r 2 , fernández r 2 , artigas a 2 1 critical care center, hospital de sabadell, sabadell, spain, 2 critical care center, hospital de sabadell, sabadell, the role of icu staff on cost containment is a matter of debate being drugs consumption, diagnostic test and fungible the main able to be improved. we hypothesised that these items could have the major impact in cost variability per patient in the icu. our objective was to prospectively evaluate the relative role of these variables compared with other classical items as length of stay, quality of life, age, severity of illness. design: prospective cohort study setting: 26-bed intensive care unit patients: 200 consecutive patients with a length of stay longer than 24 hours. measurements: we prospectively recorded: demographic data, spas ii score and diagnostic related group on admission, length of icu stay, health-related quality of life (euroqol 5d), and consumption of fungible, pharmaceutical and diagnostic procedures. the costs of the fungible, pharmaceutical and diagnostic tests were recorded from the hospital administrative database as cost per unit. we elaborated a multivariate linear predictive model in order to analyse the variables causing the variability of the cost per patient. patient transfer between hospitals is associated with increased mortality (1), and patients transferred from intensive care unit (icu) to icu have also been shown to have increased mortality (2) . the aim of our analysis was to compare our mortality figures with those of published data. . a retrospective analysis of 2,481 patients admitted to a 13-bedded unit in a university teaching hospital over a 6-year period. we compared those patients admitted from our own hospital (internal) with patients transferred from other icus (external icu) and those transferred from other hospitals from an area outside icu (external other). we compared icu mortality with apache ii predicted mortality and calculated the standardized mortality ratio (smr). results. over the 6 year period, 2,481 patients were admitted into the icu. forty-nine(2%)were transferred from another icu, 124(5%)were transferred from areas outside the icu in other hospitals and 2308 (93%) were admitted from our own hospital. mortality figures are shown in the table. conclusion. lipid solutions enriched with w-3 fatty acids are safe, well tolerated in patients with ards, and without changes in the hemodynamic or gas exchange of these patients. cdc defintions for nosocomial infections nosocomial infections in pediatric intensive care units in united states nosocomial respiratory infections picu ventilator-associated pneumonia nosocomial pneumonia in the picu (abstract k-452) pediatric ventlator-associated pneumonia last's anatomy the impact of malnutrition on morbidity, mortality, length of hospital stay and costs evaluated through a multivariate model analysis incidence of nutritional risk and causes of inadequate nutritional care in hospitals nutritional risk screening (nrs 2002): a new method based on an analysis of controlled clinical trials r:an update on perioperative management of diabetes intensive insulin therapy in critically ill patients introduction. the objective of this study was to evaluate the use of human resources in icu comparing the planned level with the operative level. prospective study involving all the patients admitted in the icu between 2001 and 2004. simplified therapeutic intervention scoring system (tiss 28) was used to assess nurse workload in the icu. provision of resources was measured as the number of nurses per icu bed (patient nurse ratio -p/n). the operative level of care was calculated dividing the measured tiss points equivalent to the nursing activities of one nurse per shift. the efficiency in the use of nursing manpower was based on the number of available nurses, the amount of work that one nurse can perform per shift and the level of tiss during the study. the work utilization ratio was calculated. severity of illness was evaluated by the apache ii score. conclusion. the apache ii score remained elevated through the study period. the measured tiss was higher than planned and as a result the work utilization ration was above 100%. nevertheless the number of patients admitted increased every year and the mortality remained lower that the expected by the apache ii score. adverse drug reactions (adr) are common in hospitalised patients, but few empirical data are avalaible regarding patients with serious adr requiring intensive medical care. as morbidity linked to adr remain underappreciated, delay for diagnosis may contribute to organ failure requiring artificial life support. the aim of this study was to determined the proportion of admissions related to serious adr and potential avoidability. we have prospectively included all adults patients coming from university hospital admitted in a 14-bed medical intensive care unit (icu) in a french university hospital in bordeaux, france, during a 6-month period. for each patient, we have determined if serious adr have contributed to organ(s) failure(s) requiring admission by follow-up and with 2 independant clinical pharmacologists. clinical pharmacologists have estimated the strenght of relationship between drug(s) prescribed in hospital and potential avoidable adr as the cause of organ failure.results. of 344 patients admitted from medical icu between may and october 2003, 48 (13,4%) were hospitalised because of almost one organ failure related to adr, 31% of cases adr were potentially avoidable. coma, seizures with acute respiratory failure and metabolic life-threatening disorders were the most frequently avoidable adr and linked to drugs prescribed in a short delay before admission. artificial life support was required in 83% of cases.vasopressives drugs were prescribed in 12 cases , hemodialysis in 5, non invasive ventilation in 12, and mechanical ventilation was needed in 16 cases.the mean of simplified acute physiologic score ii of work load omega score, of length of stay and the rate of mortality were not significantly different between patients with or without adr. serious adr were a frequent reason of admission (13,4%) and were often potentially avoidable if cautions of prescriptions would have been taken into account. measures are needed to improve adr detection and reduce drug-induced morbidity. performing every interventional procedure strictly lege artis is of significant importance not only to the patient, but also to the medical personnel. ensuring the maximum alertness to stay strict to the rules and performing by book leads to minimal complications, more wise decisions and reduction of the cost as it minimizes the single use material waste. : in our polyvalent six bed icu we installed cameras providing full surveillance of each bed. all of the cameras were in use for two weeks registering every interventional procedure on 24hour basis. medical and nursing stuff were aware of the registration process. the whole medical team reviewed the collected videos every five days. we totally registered 62 central vein catheterizations, 16 oro-tracheal intubations, three tracheostomy operations, 55 radial artery catheterizations, 58 rhinogastric tube insertions, 43 urinary bladder catheterizations, 4 pulmonary artery catheterizations 5 bronchoscopies, and six chest drainage procedures. all procedures were graded on 1 to 10 scale, in respect with asepsia,, antisepsia ,speed of performance, material waste and complications. procedure days 1 -5 days 6 -10 days 11 -15 central veins access 8 9 9 radial artery access 7 8 8 pulmonary artery access 9 8 10 tracheal intubations 8 9 9 bladder catheteriasations 8 9 10 levin tube insertion 7 8 9 bronchoscopy 8 9 10 tracheostomy 9 9 10 bulau insertion 8 9 9conclusion. increased stress among the medical personnel was noted due to the presence of the cameras, although procedures were more exact as the time advanced in spite of the fact that the duration in time of each procedure seemed to be longer. complication rates were constant. [1] , thereby increasing providers' financial risk considerably [2] . in future, reimbursement in germany will be based on a special procedure (ops 301) which may be quantified through a specific, patientdependent cost predictor score. the aim of this study was to develop and validate such a score capable of predicting the total direct costs of intensive care services in large teaching hospitals. individualized clinical as well as economic information was collected for all consecutive patients across 15 mostly surgical icus in 10 university hospitals across germany during a 2 month period. resource consumption covered hotel and personnel costs, medication, laboratory tests, diagnostic and invasive procedures. resources were valued with local costs through bottom up costing. an "icu cost predictor score" (icu-cps) was devised by combining a routine measure of severity of illness (daily saps ii score without gcs) with a daily measure of selected medical interventions (10 highly rated parameters of tiss-28: mechanical ventilation, multiple catecholamines, >5 l daily fluid replacement, peripheral artery catheter, pulmonary artery catheter, haemofiltration, intracranial pressure measurement, alkalosis/acidosis treatment, special interventions, actions outside icu) during the entire icu stay. based on a preliminary analysis of 892 patients from 12 icus, the icu-cps demonstrated a strong positive correlation of 0.871 (p < 0.01, 2-tailed) with total icu costs. this coefficient varied from 0.778 to 0.921 between icus. the correlation of the icu-cps score with costs was better than that of saps ii (without gcs) (0.850; p<0.01, 2-tailed). the mean icu-cps per day was 37 ± 15 (mean ± sd). average costs per day were € 1,272 ± 5,611. on average, each score point of the icu-cps thus corresponded to a cost of 34. the preliminary results of this study indicate that intensive care services may be adequately reimbursed on the basis of the icu-cps predictor score, taking into account patients' acute severity of illness as well as required medical interventions. the aim of our study was to analyse variable cost determinants of severe sepsis treated in intensive care units in hungary. we selected a non-random sample of 6 intensive care units. each unit identified 10 patient retrospectively, who were treated with severe sepsis. the resource use of variable costs were collected on a daily basis (for day 1-3) from medical and nursing documents. these costs were divided into disposables, radiology, biochemistry, blood products and drugs&fluids. personnel costs were calculated from annual salary report and the indirect costs were calculated by the financial directors. the mortality of severe sepsis in our sample (n=60) was found to be 61.6 %, with average lenght of stay 15.9 (sd 9.2). mean icu cost per day of severe sepsis was 429 euro. there were no differences found between day 1-3 cost of radiology, biochemistry and blood products, however, disposables had much higher cost on day 1 (p=0.002). drugs&fluids costs were higher on day 1 only for those patients who did not survive. analysing drugs&fluids by grouping them into 7 categories, we found that colloid use was significantly higher on day 1 in those, who died later (1013 ml vs. 1244 ml, p=0.045). there was no correlation found between apache ii scores and any cost components. egdt has shown significant reduction in mortality and health care resource consumption 1 and is recommended by the surviving sepsis campaign 2 . this study assessed data from severe sepsis and septic shock patients prior to implementation of an egdt program and projected the potential impact on resource utilization at our hospital. we queried the clinical data repository and found 1081 emergency department(ed)patients admitted from jan 2002-dec 2004 meeting search criteria including: patients >18 years, hospital admission from the ed with documented infection, antibiotic treatment and requiring vasopressors (day 0,1), ventilator assistance (day 0,1), new dialysis (day 0-7) or a serum lactate >4 mmol/l. exclusion criteria were admission gi bleed or traumatic injury. based on resource utilization data from the henry ford health systems corporate data stores, percent differences between egdt and non-egdt groups were calculated and applied. assuming constant mortality, the projected impact on hospital resource consumption and costs was assessed. cost savings was assoicated with survivors. increased costs were noted in non-survivors. cost benefit favored egdt. . four consecutive tptd measurements were performed with ice-cold saline. the volume of the injectate varied between 3 and 5 ml depending on bodyweight. the mean of 4 consecutive measurements with a normal td curve and injectate temperature lower than 11°celsius was considered as the gold standard. a total of 117 quadruple measurements fulfilled the quality control criteria. mean cardiac index (ci) was 4,45 l/min/m2 (sd 1,5). the mean coefficient of variation (percentage of the sd of the mean) for quadruple tptd measurements of ci was 10,5% (sd 6,6). the table shows the differences between 1 measurement, the mean of 2 and the mean of 3 measurements in comparison with the mean of 4 measurements. measurements were performed at our catheterisation laboratory in seven children with a bodyweight of 5 -13,1 kg. evlw was measured with the cold system (cold, pulsion medical systems) incorporating both tptd and tpdd techniques. ice-cold indocyanine green was injected close to the right atrium. changes in temperature and dye concentration were measured using a special catheter located in the distal aorta. mean cardiac index (ci) was 4,1 l/min/m2 (sd 0,9) and mean evlw-tpdd was 11,9 ml/kg (7,5 -22,0). repeatability (1.96 x sd of the difference between repeated measurements) for ci, evlwi-tptd and evlwi-tpdd were 0,52 l/min/m2, 1,63 ml/kg and 2,1 ml/kg respectively. the bias between the two methods is -2,36 ml/kg with a precision of 1,89.conclusion. transpulmonary thermodilution appears to be an adequate method to measure evlw in children. children may have higher normal values of evlw compared to adults. loh t f 1 1 children intensive care unit, kk hospital, sin, singapore a common approach for insertion of central venous catheter is to access the subclavian vein via subclavian approach. this approach is associated with arterial puncture and air leak 1,2. local pressure is difficult to apply as the vein runs under the clavicle. we describe an axillary approach to access the subclavian vein in paediatric patients. patients were selected for this approach when conventional approaches for central venous access were exhausted or contraindicated. the patient's arm is kept abducted with slight external rotation perpendicular to the thorax with the dorsum of the palm flat to the bed. head is turned to the contralateral side. the axillary artery is palpated and followed as it inserts into the apex of the axilla lateral to the teres minor when it becomes the subclavian artery. the axilla vein runs medial to the artery becoming anterior to the artery as it enters the axilla apex to become the subclavian vein3. a puncture is made medial to the artery at the base of axilla and directed towards the axilla apex. the needle is punctured 10-15 degrees to the skin and limited to the apex of the axilla. confirmation of venous access is made by free flow of blood. the catheter is inserted using the seldinger technique and secured. chest xr done to confirm placement. . 5 paediatric patients were selected for this approach. arterial puncture was made in one patient and hemostasis secured with direct local pressure and subsequent insertion was successful. access required a mean of 2.2 attempts. one patient hand was swollen 6 days after the line inserted but doppler study did not reveal any venous thrombosis and the line left in situ. routine limb neuromuscular and vascular assessments were made. no malposition or air leak was seen on cxr.no local or line related infections were documented. catheters were removed after 8.3 days. follow up (mean of 2 weeks) after the catheter was removed showed normal hand power and movement in all patients. axillary approach maybe a novel alternative to central venous catheter insertion in paediatric patients when conventional approaches are not possible. change was recorded on capnography in all 281 tests performed. radiographs confirmed correct placement of ngt throughout study period. in the subgroup of children(n=13)who had an endotracheal placement the time to complete capnograph colour change was ≤ 15 seconds, (median 7 (5-15)) in all cases. in critically ill children sufficient gastric aspirate can be obtained for ph testing and capnography rapidly discriminates between ngt placed correctly from those passed in to the trachea. a one year prospective & observational study included all admissions (n=216) until 48h after discharge. cultures for bacteria and fungi and antibiotic sensitivity tests (19 antibiotic using bauer-kirby disc diffusion method) were obtained on admission [ blood, stool, urine & cerebrospinal fluid (if needed)] and repeated on suspicion of nis .all cannulae, endotracheal tube (et) aspirates & tips, nasogastric tubes & different catheters were cultured. all picu health care workers (hcws) were subjected to throat & under-finger nails culture as well as inanimate objects , both on bimonthly basis. the referral place (ward or emergency), prism iii score, length of stay (los) & fate, were recorded. reports of the dutch society of pediatrics concerning transport and stabilizing critically ill children, resulted in a reorganization of the transport of critically ill children in the netherlands. as of february 2004, all children in the northwestern region of the netherlands requiring mechanical ventilation were transported by pediatric intensive care teams of the vu and amc university medical centers. these teams consist of a pediatric intensivist or anesthesiologist (in training) and pediatric intensive care unit (picu) nurse and were on call 24 hours, 7 days per week. the objective is to report the first year results and to compare an experienced (amc) and a novice (vumc) center. demographic data, diagnosis at admission and severity of illness score (prism) and duration of transport (preparation, travel time, intervention in other hospital and complications during transport) were prospectively collected. all data were analyzed per center and in total in order to identify any difference between both picu-teams. transport frequency was divided according to picu-capacity (40% vumc and 60% amc). statistic analysis included student t-test for continuous variables and chi2 test for dichotomous variables. in total 100 patients were transported by either picu-team. half of the transports took place during the evening or night. demographics, prism-score and admission diagnosis were comparable. mean transport time was 2 hours and 17 minutes. there was a significant difference in preparation time . results concerning other transportation variables are similar for both clinics. neither picu-team reported complications during transport. a continuous picu transport system carried out by two specialized centers is feasible and efficient. apart from a difference in preparation time, which may be influenced by a multitude of factors, there were no differences concerning other transport variables between an experienced and inexperienced team. after cardiac surgery it is not uncommon that a solitary collapse of a lobe, e.g., the left lower lobe develops. it has been difficult to experimentally study therapeutic interventions for lobar atelectasis due to lack of suitable animal models. the aim of this study was therefore to develop a reproducible model in pigs.methods. 10 anesthetized pigs were tracheotomized and ventilated vcv, fio2 1.0, peep 10 cmh2o, vt 8 ml/kg. this ventilation was maintained under the experiment except during the lung recruitment maneuver (lrm). a bronchial blocker (cook c-aebs-7.0) was inserted in the right lower lobe (about 50 cm from the et-tube opening) by the use of a fiberoptic bronchoscope. to ensure a correct position, the balloon of the blocker was inflated shortly and thereafter deflated under inspection via the bronchoscope. thereafter, a lrm (pcv with peak pressure of 40 cmh2o, peep 10 cmh2o, i:e 1:1 and rr of 6/min during 2 min) was performed to optimize the lung volume history after which end-expiratory lung volume (eelv), quasistatic compliance of the respiratory system (crs) were measured and blood gases (mixed venous and arterial) were obtained. the balloon of the bronchial blocker was inflated, the air of the isolated lobe exsufflated and measured ("lobe volume"). thereafter the lobe was selectively lavaged (with a "lobe volume" of 37°c 0.9% nacl) using a syringe 15 times or until no frothing of the lavaged fluid was seen. eelv, crs and blood gases were obtained. in one pig ct thorax was done and another pig was thoracotomized and the lungs were inspected. statistics:wilcoxon. the "lobe volume" was 66±21 ml (mean±sd). after the selective lobe lavage, eelv decreased from 886±170 to 698±166 (p<0.002), pao2 from 76±9 to 40±14 (p< 0.004)and crs decreased from 32±6 to 22±7 (p<0.002).both ct and the inspection of the lung showed atelectasis of the right lower lobe. a reproducible experimental lobe atelectasis can be obtained by selective lobe lavage in pigs. this method may be used experimentally for studying methods treating atelectasis. garcia-hernandez r 1 , perez-vela j l 1 , corres m a 1 , hernandez-sanchez e 1 , renes e 1 , gutierrez j 1 , arribas p 1 , perales n 1 1 postoperativecardiac unit, hospital doce de octubre, madrid, spain in the literature donor´s norepinephrine (ne) usage was considered high vasoactive support and leads to refuse heart graft implantation.the sortage of available donor hearts limits cardiac transplantation and nowadays some authors point that vasoactive drugs (vad) could be useful to improve donors hemodynamics and so graft function in the recipient. objetive: to assess graft function and icu evolution in patients who underwent cardiac transplantation depending on donor´s ne dose. retrospective study from 1998 until 2004 of intrahospital donors and theirs recipients two groups were set: low ne dose: donors who received <0.2mcg/kg/min or no ne; high ne dose: donors who received >/= 0.2 mcg/kg/min. we assessed in the donors: number, type, length and dosage of vad; volume intake and clamp time; in the recipients: presurgical left ventricular eyection fraction (lvef); extracorporeal circulation (ecc) time; incidende of ventricular disfunction, cardiogenic shock, primary graft failure (pgf); mortality and others icu evolution parameters (incidence of acute renal failure-arf-, acute lung injury/respiratory distress -ali/ards-, sepsis, length of dva usage, mechanical ventilation and icu admission, etc...). statistical analysis was done with t-student´s test and chi(2) (using yates´ or fisher´s modification when indicated). conclusion. in our serie, the donor´s ne dose did not have an influence on the heart graft disfunction or in the others items assessed. the ne use for hemodynamic management on heart donors could not worsen the recipients evolution, but new studies with high number of patiemts should be developed in order to set a clear limit in the dosage used. jacquet l 1 , rubay j 2 , vancaenegem o 1 , laarbaui f 1 , lovat r 1 , noirhomme p 2 1 cardio-vascular intensive care, 2 cardio-vascular surgery, saint-luc university hospital, brussels, belgium many patients with complex congenital heart disease,the majority having been operated on during their first years of live, are now adults and pose unusual problems for cardiologists, surgeons and intensivists caring for adult patients. we have reviewed the charts of patients >15 years old who were admitted in our cardiovascular icu after operation for guch from january 2000 to december 2004 in order to describe their specific outcome. during this 5 years period, data from 38 pts (15 males,12 females and 1 xxyy karyotype) were collected. the mean age was 31 y (rang 15-59).among these, 35 had tetralogy of fallot and had been already operated before, 17 having had 2 previous surgical procedures. the main indication for surgery was pulmonary insufficiency and 34 pts received a pulmonary homograft. retrospective review of all echo examinations in this setting over a one-year period (jan2004 -jan2005) we performed 135 echo in 77 patients (15% of 489). echos were carried out during the first 24 hours in 16% and on the next day in 13%. sixty-eight percent were performed during the first 5 postop days. echo was performed as urgent in 20%, semi-urgent (>8 hours) in 29% and for control in 51% of cases (64% tte, 36% tee). 76% were carried out by cardiologists and 24% by anesthesiologists. however, 52% of the urgent echos were performed by anesthesiologists. indications were: hemodynamic instability (23%), cardiac transplantation follow-up (13%), cardiac ischemia (13%), cardiac function follow-up (11%), and suspected cardiac tamponade (9%). findings were left ventricular dysfunction (26%), hyperdynamic left ventricle (23%), right ventricle dysfunction (21%), new segmental wall motion abnormalities (12%) and hypovolemia/vasodilation (8%). 33% were normal or similar to previous echos. new myocardial infarction was diagnosed in 22%. echo induced changes in patient management in 60%: resternotomy (4%), medical therapy (48%) and others (8%)(iabp insertion/removal, anti-rejection therapy). main changes in therapy were: inotropic agents (59% increase, 34% decrease) and iv fluid administration (27%). in 23% echo findings were unexpected/unrelated to the symptoms. 58% of patients were also managed with a pulmonary artery catheter(pac). in 6% of these there was no agreement between two techniques. number of echo carried out by the anesthesiologists increased from 9% in the first four months of the study to 27% in the second four months and to 49% during the last four months. in this study, echo provided important diagnostic and therapeutic data on postoperative cardiac surgical patients. findings led to management changes in 60% of patients. echo should be included in training of physicians working in csicu. is 7.39%. the mean age is 59 ± 19 years. the mean bmi is 27 ± 3.9 kg/m2. patients with normal bmi (20-25 kg/m2) and patients with more than normal weight(above 25 bmi) had a similar outcome. (fig.1 ) we ruled out that younger age compensates for a possible higher mortality in heavier patients.however, the age turned out to be similar in the different bmi groups and cannot be held accountable for lack of increased mortality in patients with more than normal weight. (fig.2) .conclusion. bmi does not show to increase mortality in cardiac surgical patients, despite of possible increased comorbidities. samalavicius r 1 , misiuriene i 1 , norkiene i 1 , juozaitis m 1 , baublys a 1 1 department of cardiac anaesthesia, vilnius university hospital, vilnius, lithuania obesity is one of the risk factors for adverse outcomes of major surgery. we assesed the influence of obesity on outcomes of cabg in our institution. the data of 3178 consecutive patients, who underwent coronary artery bypass grafting at vilnius university heart surgery clinic between january 1, 2000 and december 31, 2004 were analysed. obesity was defined as body mass index > 30.0 kg/m2. obese patients (n=887) were compared to remaining group of patients. preoperative risk factors, postoperative outcomes, mortality rates were analysed. associations between obesity and postoperative outcomes were analysed. in a prospective observational study we assesed nutritional status of 211 consecutive cardiac surgery patients with a nutritional risk screening form, which contained bmi, food intake, weight lost and stress factor. we evaluated mortality, icu stay and frequency of impaired healing in the groups in nutritional risk and with normal nutrition. we identified 54 from 211 (25,6%) patients as in nutritional risk. both groups did not significantly differ in age, bmi, left ventricle function, preoperative serum albumin level, prevalence of chronic renal failure or perifery vascular disease. there were significantly more diabetics (51,9% vs. 34,2%, p<0,05) and patients with copd (30,8% vs. 10,3%, p<0,001) in risk group. we found out the rate of complicated wound healing 12,3% (all sites and grades). there was significantly higher rate of complicated wound healing (28,3% vs. 7% p<0,0001) and longer icu stay (51,6 vs. 24,2 hrs., p<0,01) in group in risk compared to group without risk. there was trend to higher mortality in risk group, statisticaly nonsignificant(9,4% vs. 3,2%). we identified diabetes, copd and nutritional risk as to be preoperative independent risk factors of impaired healing in elective cardiac surgery by multivariate analysis.conclusion. cardiac surgery patients have a similar prevalence of nutritional risk as general population of patients. simple screening form is able to identify group of patients in increased risk of impaired healing. maximum sofa during first three days (maxsofa3d) and deltasofa between first and third postoperative days (deltasofa31) revealed to have strongest correlation to mortality (p=0.005, roc area 0.793 and p=0.006, roc area 0.0784 respectively). the maxsofa3d of 15 points corresponded to mortality with sensitivity of 0.000 and specificity of 0.012. maxsofa3d correlated to the icu stay (p=0.001).conclusion. the sequential assessment of organ dysfunction during the first three days postoperatively is an independent predictor of mortality and morbidity in cardiac surgery patients. hájek r 1 , rùžièková j 1 , zezula r 1 , fluger i 1 , nìmec p 1 , jarkovský j 2 , nemethová d 2 1 cardiac surgery, university hospital olomouc, olomouc, 2 center of biostatistics, masaryk university, brno, czech republic introduction. thromboleastography (teg) is reliable and extensively used method of haemostasis monitoring. using teg as a bed-side method, we are able to detect a coagulation disorders, especially hypercoagulation and fibrinolysis methods. in prospective randomized study two groups of elective cardiac surgery patients were compared. patients of group a (n=144) were monitored both conventional lab tests and simultaneously with teg. the following teg measurements were performed: 1st -baseline after the anesthesia induction, 2th-at rewarming on cpb (with heparinase) and 3th-immediately after icu admission (both nativ and heparinase). patients of group b (n=146) were monitored only using lab tests. pre and postoperative coagulation status, incidence of thrombocytopenia, fibrinolysis,blood loss , transfusion therapy, surgical reexploration were evaluated. changes of hemostatic profile using teg diagnostic algorithm and also changes of pre-and postop.lab tests were evaluated results. both groups were comparable by age (66,1/67,4) , male gender (72%,7/ 68,5%) and surgery type. the lab coagulation tests including platelet count were within normal range in both groups before surgery. no diference between both groups were recorded in : average blood loss during and postoperative, incidence of surgical reexploration because of bleednig , red blood cell, fresh frozen plasma and platelet transfusion and using of aprotinin. in both groups lab values of quick test, platelet count and fibrinogen were lower and aptt and tt were higher after surgery. the changes of teg parameters characterised by coagulation index : ci1>ci2, ci1<ci3. frequency of coagulagulation patterns: teg 1 : enzymatic hypercoaguability 14,1%, platelet hypercoag. 13,3%, sec.fibrinolysis 9,4%. teg 2 : low platelet function 14,1%, primary fibrinolysis 14,1%, low clotting factors 7,8%. teg 3 (heparinase): enzymatic hypercoag.. 9,4%, platelet hypercoag. 9,4%, low platelet function 9,4%. only one parameter-quick test value(%) correlated with ecc duration (r-0,23, p<0,01)in both groups. this study shows no diference of therapy between groups monitored with teg compared with conventional lab tests. most of patients in teg group showed the signs of hypercoagulation on teg before and after elective surgery. . to achieve a better glucose control in diabetic cardiac surgery patients a three to five-fold increase of basal insuline dose has been suggested. (1) .our objective was to determine wether three or five-fold increase in basal insulin-dose during hypothermic cpb makes a better glycemic control without risks.methods. 74 type ii diabetes patients were included in this prospective study. two hours before surgery a continuous endovenous insulin infusion was started to achieve the target glucose level results. (median+/-sd)29 pts were excluded due to off-pump surgery. one due to continuous hypoglycemia .itg showed lower blood glucose levels(160 +/43 mgdl-1)than cg(194+/-69mgdl-1) during intraoperative period (p<0.05). 35% of itg and 12% of cg patients presented at least one blood glucose level.mean time with blood glucose levels conclusion. 1-in type ii diabetic patients sheduled to cardiac surgery a five-fold increase in insulin dose at the begining of cpb achived intraoperatively safe and better glycemic control than three-fold increase;.2-patients with better glycemic control had less infections and shorter hospital stay as it was also demonstrated by others (2) .this results give support to stablish insulin protocols that allow a tight glycemic control in the perioperative setting. giuliano k k 1 1 patient monitoring, philips medical systems, andover, united states introduction. the purpose of this research was to develop a predictive model for the early identification of sepsis using the currently recommended physiologic parameters that are continuously or frequently monitored in the critical care setting. data from the project impact ® international dataset were used to assess whether a combination of the physiologic parameters of heart rate, mean arterial blood pressure, body temperature and respiratory rate could distinguish between critically ill adult patients with a diagnosis of sepsis and those without a diagnosis of sepsis in the first 24 hours of intensive care unit admission. all four predictor variables used in the analyses (respiratory rate, heart rate, mean arterial blood pressure, and temperature) differed significantly between the septic and nonseptic patient groups. however, using logistic regression, only two out of the four predictor variables (mean arterial blood pressure and temperature) were independently associated with being septic. according to hosmer and lemeshow goodness-of-fit tests, the logistic regression model was a good fit and explained between 10.2% and 13.6% of the variance. using the significant predictors of temperature and mean arterial blood pressure, the odds ratio for having sepsis was 2.126 for patients with a temperature of 38oc or higher and 3.874 for patients with a mean arterial blood pressure of less than 70mmhg. in addition, the odds of having sepsis were 4.63 times greater if the patient had both of these physiologic conditions present. the results of this research provide support for the use of some of the currently recommended clinical monitoring guidelines for patients with sepsis. however, the results also underscore the limitations of using these variables for sepsis monitoring, since this group of variables alone was not sufficient for discriminating between critically ill patients with and without sepsis. more work needs to be done to see if these variables would perform better if different cut-off values were used. zubani f 1 , de peri e 1 , rasulo f 1 , candiani a 1 , latronico n 1 1 intensive care unit, spedali civili, university hospital of brescia, brescia, italy introduction. more than 70 percent of patients with sepsis syndrome or septic shock present altered mental status manifested by decreasing attention, disorientation, agitation, obtundation and in worst cases profound stupor and coma (septic encephalopathy). we don't know the exact physiopathological mechanism which cause se, but many clinical studies have shown that cerebral blood flow (cbf) is reduced in these patients. the current study was performed to evaluate the behaviour of cerebrovascular autoregulation during systemic inflammatory response syndrome (sirs), sepsis syndrome, septic shock and multiple organ failure (mof). we have studied all patients with se admitted into the intensive care unit of the spedali civili university hospital of brescia from january 2004 to september 2004. cerebral autoregulation has been evaluated bilaterally in the mca territory with thetransient hyperemic response test (thrt). twelve patients were included in this study and 50 thrt have been totally performed. cerebral autoregulation was impaired in 64% of thrt: the two conditions most associated with worst cerebrovascular response are sepsis (66,6%) and mof (80%); patients with septic shock have shown unexpectedly the best autoregulatory response. in patients with infections caused by gramnegative organisms cerebral autoregulation was impaired in 100% of measurements: there are many evidences in literature about endotoxin's ability to influence cerebral blood flow. another important factor able to influence the behaviour of cerebral blood-vessels is paco2: the effect of hypercapnia is more evident in patients with septic shock and with mof, particularly, the share of patients with septic shock with cerebral autoregulation completely impaired, was hypercapnic in 100% of measurements. cerebral autoregulation was impaired in the most part of measurements. gramnegative organisms and hypercapnia play a fundamentally roll in the loss of this mechanism. conclusion. platelet transfusion is associated with longer icu stay and 15% increased risk of death. rudiger a 1 , fischler m 2 , harpes p 3 , gasser s 4 , hornemann t 5 , von eckardstein a 5 , maggiorini m 2 1 wolfson institute of biomedical research, university college london, london, united kingdom, 2 medical icu, university hospital, 3 department of biostatistics, university, zurich, 4 medicine, regional hospital, zofingen, 5 clinical chemistry, university hospital, zurich, switzerland introduction. b-type natriuretic peptide (bnp) and its n-terminal portion (nt-probnp) may be useful in the assessment of cardiac preload in patients with chronic heart failure. however, it remains controversial whether or not this option is still valid in critically ill patients. to resolve this question we sought to establish the relationship between natriuretic peptide levels and surrogates of cardiac preload and inflammation in this kind of population.methods. this retrospective study included 12 critically ill patients with heart failure and/or refractory shock, all monitored with a pulmonary artery catheter (pac). linear correlations between hemodynamic and laboratory parameters were performed using relative differences and described by the pearson correlation coefficient (r). a mixed linear model for repeated measurement was used to control for repeated effects. the analysis included 75 measurements from patients with heart failure (n=6) and septic shock (n=6). mean (sd) age was 65 (15) years, and 67% were men. icu-and hospital mortalities of the population were 59% and 67%. bnp and nt-probnp correlated (p<0.001) well with c-reactive protein (r=0.65 and r=0.67), to a lesser extent with leukocyte count (r=0.40 and r=0.47) and central venous pressure (r=0.39 and r=0.27), but not (p>0.05) with the pulmonary artery occlusion pressure (r=0.12 and r=0.12). these relationships were confirmed in mixed linear model analyses for repeated measurements. supported by other clinical observations and evidence from laboratory studies, our results suggest that inflammation is a important stimulus for bnp and nt-probnp elevations in humans. natriuretic peptide levels may therefore not be used as surrogates of cardiac preload in critically ill patients with heart failure or shock. animal studies suggest that melatonin plays an adjunctive role in defence mechanisms to overcome severe illness and, accordingly, melatonin seem to affect morbidity and mortality.we report on correlations between nocturnal melatonin serum levels and measures of illness severity in 302 patients consecutively admitted to a medical intensive care unit. on the day of admittance at 02:00 h am blood for the determination of serum melatonin levels was obtained and illness severity was assessed according to the acute physiology and chronic health evaluation score (apache) and the therapeutic intervention scoring system (tiss). for the entire study group there was a weak negative correlation between tiss and nocturnal melatonin concentration (r = -1.22, p<0.04) while such correlation was not observed for melatonin and apache. subgroup analysis revealed that in patients with sepsis both apache and tiss scores correlated negatively with nocturnal melatonin concentrations (n = 14, apache: r = -0.656, p<0.02; tiss: r = -0.544, p<0.05). such correlation did not occur in other disease entities like coronary syndromes or intoxications. our study indicates that melatonin is specifically affected by serious infectious disease and low melatonin levels may contribute to the adverse outcome of sepsis. baykara n 1 , aydemir e 1 , solak m 1 , toker k 1 1 anesthesiology and reanimation, university of kocaeli, school of medicine, kocaeli, turkey the purpose of the present study to assess changes in antidiuretic hormone (adh), growth hormone(gh) levels and hemodynamic response during a standart weaning protocol in patients with copd. this study was carried out in 15 patients undergoing ventilatory treatment with synchronized intermittent mandatory ventilation (simv)+peep for respiratory failure due to copd. their durations of mechanical ventilation (mv) were between 3-7 days. exclusion criteria were:abnormal left or right ventricular function,abnormal liver or renal function,diabetes mellitus,cns disease or mv exceeding one week. weaning was carried out in 3 stages of 60 min each, from 1 / 2 of the initial rate of simv (simv1/2 ) +peep, to continuous positive airway pressure (cpap), to spontaneous breathing. systolic blood pressure, diastolic blood pressure,heart rate,central venous pressure,pulmonary capillary wedge pressure,cardiac output,hourly urine output,plasma osmolality and adh, gh were measured during at each ventilatory condition. hemodynamic parameters did not change significantly among the ventilatory conditions. adh concentrations during simv+ peep and simv i /2+peep were similar and were significantly higher than during spontaneous breathing. adh concentration during cpap was not significantly different from spontaneous breathing. even though statisticallyinsignificant,hourly urine output was higher during cpap and spontaneus breathing than during simv+peep and simv1/2+peep modes. gh level did not change significantly among ventilatory conditions. accordingly, weaning appears to be well tolerated from a hemodynamic standpoint in copd patients with normal cardiac function after short term mv. cpap is the ventilator mode causing the least adh secretion in patients with copd. jukes a l 1 , saayman a g 1 1 critical care directorate, university hospital of wales, cardiff, united kingdom enteral feeding is the preferred method of nutritional support in the critically ill patient (jolliett et.al., 1999) . the enteral feeding protocol within our unit advocates prompt replacement of wide-bore tubes with fine-bore feeding tubes once enteral tube feeding is established to maximise patient comfort and safety. the aim of this review was to compare the current fine-bore feeding tube used within the critical care directorate (ccd), medicina (entrafeed, 7fg or eng) with that manufactured by merck, (corflo, 8fg or cng). it was hypothesised that as a result of the specific features of the cng tube, it would be easier to aspirate; reduce the incidence of occlusions; and have increased radio opacity when compared with the eng.methods. an audit proforma was completed for 41 patients who had a fine-bore feeding tube placed within the ccd: 20(eng); 21(cng) placed. the patients were followed until feeding was stopped due to a complication, or no longer required. chest x-rays were reviewed by a consultant at the end of the study, unaware of ng type. all nasogastric feeding tubes were placed by medical staff. very few measured the tube length required to insert prior to placement. auscultation, was used in 24% of tubes placed. aspiration of gastric contents was attempted in 68% of tubes but only obtained in 10(24%)tubes (7 eng, 3cng). only 5 of these had a ph of 4 or less, confirming gastric placement. all patients received a chest x-ray, visibility comparable (17 eng and 16 cng clearly visible on x-ray). there were 5 occlusions (4 eng, 1 cng). many tubes were accidentally displaced or pulled out by patients (6 eng, 11 cng). the majority of tubes (76%) remained insitu for 14 days or less (14 eng, 17 cng). the results of the review did not warrant a change in the type of nasogastric feeding tube used within the ccd. it has highlighted that education and training of doctors is required within the ccd regarding the placement, and appropriate methods used to confirm correct ng position. radiological confirmation of ng tube position is advised on initial placement in critically ill patients. however, attempts should be made to aspiration and ph test to assist subsequent confirmation, avoiding unnecessary x-rays. administration of lipid solutions to critically ill patients may be associated with changes in laboratory and gas exchange parameters. lipid solution composure may impact in these changes. methods. investigate gas exchange and hemodynamic changes in patients with ards treated with a lipid solution enriched with w-3 fat acids. prospective, randomise, double blind study of parallel groups. sixteen patients with ards within 48 hours of diagnosis were randomised in two groups. group a (n=8) received lipid solution lipoplus® 20% b.braun medical (50% mct, 40% lct, 10% w-3) and group b (n=8) intralipid® 20% (100% lct). lipid solution was given over 12 hour at 0.12 mg/kg/h. hemodymanic and gas exchange parameters were analysed before treatment and at 12 and 24 h of lipid solution infusion. statistics: bmdp, wilcoxon and sign tests. the following table shows the percentage of change after lipid solution infusions compared with baseline levels. no side effects were observed with both lipid solutions in the patients studied. immunonutrition is a balanced nutritional support containing immune enhancing substances like arginine and omega-3-fatty acids. the aim of this study is to find out if immunonutrition can reduce the number of blood transfusions and blood loss in cardiac patients. in this prospective and double-blind study we randomised 130 patients who either received immunonutrition or an isocaloric placebo. comparison of the group was done with repeatedmeasures anova. we could not find a difference concerning postoperative blood loss and blood transfusions. 1086 ± 609 1018 ± 834 (mean ± sd) number of blood transfusions 1,3 ± 2 1,7 ± 2,7 per patient (mean ± sd) infection rate (%) 12 13 length of stay (hospital) 9 ± 7 9 ± 14 (median ± sd) conclusion. we could not prove a significant advantage of immunonutrition as reported in the literature. (1) . the objective was to apply rifle in the postoperative cardiac population and to analyze outcome, length of icu stay (los) and mortality for each subgroup. we stratified patients according to their preoperative plasmatic creatinine (ppc in mg/dl). theoretic plasmatic creatinine is obtained according to simplified formula mdrd (modification of diet in renal disease) (2) . the expected mortality was calculated using logistic euroscore (european system for cardiac operative risk evaluation) (3). we evaluated 1814 patients: 977 with ppc < 1; 629 with ppc < 1'5; and 208 patients were classified according to "r" (ppc 1'5-2), "i" (ppc 2-3), "f" (>3 without renal replacement therapy (rrt)), "fo" (oliguria treated by rrt). "fo" subgroup suffered major complications than non-oliguria subgroups (p< 0'05). conclusion. 1. mortality was equal in "i" and "f" patients, but higher than "r" group (p < 0'0001) and lower than "fo" (p < 0'0001). 2. los was similar in "rif". 3. "fo" los was longer than "rif" los. 4. "fo" group suffered more severe complications and developed acute renal failure as a part of multi-organ dysfunction syndrome (mods), which needed multiple organ support therapy (most). standard hemofiltration is reported to improve hemodynamics and survival in animal models of septic or endotoxic shock. in humans, despite the lack of convincing data, hemofiltration is thought to be the gold standard to treat acute renal failure (arf) in case of septic shock. we compared survival of septic arf treated with ihd or continuous veno-venous hemodiafiltration (cvvhdf) in the prospective randomised hemodiafe study. we performed post-hoc analysis of data from a prospective, multicenter (21 centers) randomised study. patients with arf (urea > 36 mmol/l or serum creatinine > 310 micromol/l or oliguria) associated with mods (lod > 5) and needing renal replacement therapy were enrolled. they were randomised to receive ihd or cvvhdf performed with the same membrane (polyacrylonitrile, an69) and a bicarbonate based buffer. guidelines to improve hemodynamic tolerance and efficiency were provided. primary endpoint was 60-day survival evaluated in an intention-to-treat analysis. septic arf was defined if any sepsis was diagnosed before the occurrence of arf. data are presented as mean±sem among the 360 patients enrolled in hemodiafe, the overall septic population consisted of 224 pts (66±13 y.o., m164/f60, saps ii 65±14, lod score 9.8±2.4) randomised in the ihd (n=126) or in the cvvhdf group (n=98). eighty-nine percent of patients had septic shock and 97 % were under mechanical ventilation. mean serum urea and mean serum creatinine were respectively 30.4±13 mmol/l and 388±151 micromol/l just before the first session. the 60-day survival in the whole population of the study was 32 % with no significant difference between the two groups (respectively 32,6 % and 31,5 % in cvvhdf and ihd). survival was significantly lower in septic patients compared to non septic (26,3% versus 41,8 % p = 0,007). in septic patients, we found no significant difference in survival between the two treatment groups (respectively 23,5 % versus 28,6 % in cvvhdf and ihd p = 0,23). standard cvvhdf does not offer any survival benefit compared to ihd to treat septic arf associated with mods. methods for evaluation of glomerular filtration rate -gfr-(24-hours creatinine clearance -24hcrcl-or cockcroft-gault formula -cg-) are not well suited for critically ill patients: 24hcrcl requires a steady state and cg has not been completely validated. shorter time crcl can be used but this method has not been evaluated in unstable patients. we intend to demonstrate that 2hours crcl (2hcrcl) is similar to 24hcrcl even in unstable patients prospective study on adult icu patients. we calculate 2hcrcl, 24hcrcl and cg estimate. 2hcrcl was measured at the beginning of the 24hcrcl interval. age, sex, weight and diagnosis were recorded and for the 24 hours period registered sofa, nutrition, diuretics, nephrotoxics, hypotension or hypoxemia, use of vasopressors and regularity of urine flow. we defined 2 groups: patients recently admitted (less than 24 hours) and in stable condition and expected stable renal function. statistical analysis: paired t-test, pearson correlation coefficient and partial correlation coefficients results. 93 patients, 41 (44.1%) on admission and 52 (55.9%) in stable condition. in 2 cases (2.2%) 2hcrcl was lost and in 11 (11.8%) 24hcrcl because methodological problems. 80 patients completed the protocol (43 stable and 37 on admission) and were included for analysis. no differences were detected in both groups. mean 2hcrcl was 124.08±88.2 and 24hcrcl 117.46±72.9 ml/min with a mean difference of 6.6±62.1 (p 0.34). 2hcrcl correlated well with 24hcrcl (coefficient 0.72, p<0.001) and less well with cg formula (coefficient 0.67, p<0.01). these coefficients were not affected by group of patient, antecedents, sex, age, sofa score, and use of diuretics, nutrition or nephrotoxic drugs, hypotensive episodes, hypoxemia, use of vasopressors and irregular urine flow. we observed less aggregation for values in the high range of clearance; analysing only patients with crcl below 80 (n=31) the correlation was even higher (0.73, p<0.05)conclusion. 2hclcr correlates well with 24hcrcl, is easier to obtain, is most reproducible and eliminates unnecessary delays and methodological problems complicating 24hcrcl. 2hcrcl can be a good estimate of gfr in icu, even in unstable patients key: cord-017309-pt27efu1 authors: gupta, g. s. title: selectins and associated adhesion proteins in inflammatory disorders date: 2012-03-20 journal: animal lectins: form, function and clinical applications doi: 10.1007/978-3-7091-1065-2_44 sha: doc_id: 17309 cord_uid: pt27efu1 inflammation is defined as the normal response of living tissue to injury or infection. it is important to emphasize two components of this definition. first, that inflammation is a normal response and, as such, is expected to occur when tissue is damaged. infact, if injured tissue does not exhibit signs of inflammation this would be considered abnormal and wounds and infections would never heal without inflammation. secondly, inflammation occurs in living tissue, hence there is need for an adequate blood supply to the tissues in order to exhibit an inflammatory response. the inflammatory response may be triggered by mechanical injury, chemical toxins, and invasion by microorganisms, and hypersensitivity reactions. three major events occur during the inflammatory response: the blood supply to the affected area is increased substantially, capillary permeability is increased, and leucocytes migrate from the capillary vessels into the surrounding interstitial spaces to the site of inflammation or injury. the inflammatory response represents a complex biological and biochemical process involving cells of the immune system and a plethora of biological mediators. cell-to-cell communication molecules such as cytokines play an extremely important role in mediating the process of inflammation. inflammation and platelet activation are critical phenomena in the setting of acute coronary syndromes. an extensive exposition of this complex phenomenon is beyond the scope of this article (rankin 2004). play fairly broad roles in the generation of immune responses. the three selectins act in concert with other cell adhesion molecules e.g., intracellular adhesion molecule (icam-1), vascular cell adhesion molecule-1 (vcam-1), and leukocyte integrins to effect adhesive interactions of leukocytes, platelets, and endothelial cells. the structure and functions of selectins, which belong to c-type lectins family, have been reviewed in chaps. 26, 27, and 28. the selectin family of lectins consists of three closely related cell-surface molecules with differential expression by leukocytes (l-selectin), platelets (p-selectin), and vascular endothelium (e-and p-selectin). structural identity of a selectins resides in its unique domain composition (chap. 26). e-, p-, and l-selectin are >60 % identical in their nh2 terminus of 120 amino acids, which represent the lectin domain (chaps. 26, 27, and 28) . the ligands (counter structures) of selectins are sialylated and fucosylated carbohydrate molecules which, in most cases, decorate mucin-like glycoprotein membrane receptors. their common structure consists of an n-terminal ca 2+ -dependent lectin-type domain, an epidermal growth factor (egf)-like domain, multiple short consensus repeat (scr) domains similar to those found in complement regulatory proteins, a transmembrane region, and a short cytoplasmic c-terminal domain. together this arrangement results in an elongated structure which projects from the cell surface, ideal for initiating interactions with circulating leucocytes. the lectin domain forms the main ligand binding site, interacting with a carbohydrate determinant typified by fucosylated, sialylated, and usually sulphated glycans such as sialyl lewis x (s-le x ). the egf domain may also play a role in ligand recognition. the short consensus repeat (scr) domains (two for l-selectin, six for e-selectin, and nine for p-selectin) probably act as spacer elements, ensuring optimum positioning of the lectin and egf domains for ligand interaction. the egf repeats have comparable sequence similarity. each complement regulatory-like module is 60 amino acids in length and contains six cysteinyl residues capable of disulfide bond formation. this feature distinguishes the selectin modules from those found in complement binding proteins, such as complement receptors 1 and 2, which contain four cysteines (chap. 26). the selectins cell-surface receptors play a key role in the initial adhesive interaction between leukocytes and endothelial cells at sites of inflammation. selectins (p, e and l) and their ligands (mainly p-selectin ligand) are involved in the rolling and tethering of leukocytes on the vascular wall. activation of endothelial cells (ec) with different stimuli induces the expression of e-and p-selectins, and other adhesion molecules (icam-1, vcam-1), involved in their interaction with circulating cells. lymphocytes home to peripheral lymph nodes (plns) via high endothelial venules (hevs) in the subcortex and incrementally larger collecting venules in the medulla. hevs express ligands for l-selectin, which mediates lymphocyte rolling (horstman et al. 2004) . for structure and functions of selectins, the readers are advised to consult chaps 26-28. in this chapter we will emphasize mainly on the role of selectins in inflammatory disorders including cancer. atherothrombosis, defined as atherosclerotic plaque disruption with superimposed thrombosis, is the leading cause of mortality in the western world. atherosclerosis is a diffuse process that starts early in childhood and progresses asymptomatically through adult life. later in life, it is clinically manifested as coronary artery disease (cad), stroke, transient ischaemic attack (tia), and peripheral arterial disease. from the clinical point of view, we should envision this disease as a single pathologic entity that affects different vascular territories. a suggestive analogy is that tia and intermittent claudication are the unstable angina of the brain and lower limbs, respectively; and stroke and gangrene are the myocardial infarction. circulating platelets display reversible interactions with atherosclerotic lesions. atherosclerotic arterial disease is associated with an increased share of platelets unable to express p-selectin and an increased fraction of platelets that microaggregate in citrate anticoagulant. these platelet alterations are not completely explained by either focal arterial injury or abnormal rheology associated with arterial stenosis but appear to be an effect of the atherosclerotic process (mcbane et al. 2004 ). the pathogenesis of arterial thrombotic disease involves multiple genetic and environmental factors related to atherosclerosis and thrombosis. venous thrombosis is a world wide health problem in the general population. injury to the endothelium leads to dysfunction. the causes of injury include lipids, immune complexes, microorganisms, smoking, hypertension, aging, diabetes mellitus and trauma. the selectins are thought to be largely responsible for the initial attachment and rolling of leukocytes on stimulated vascular endothelium. platelet activation is an important process in the pathogenesis of atherothrombosis. platelet adhesion, activation, and aggregation at the sites of vascular endothelial disruption caused by atherosclerosis are key events in arterial thrombus formation. platelet tethering and adhesion to the arterial wall, particularly under high shear forces, are achieved through multiple high-affinity interactions between platelet membrane receptors (integrins) and ligands within the exposed subendothelium, most notably collagen and von willebrand factor (vwf) . platelet adhesion to collagen occurs both indirectly, via binding of the platelet glycoprotein (gp) ib-v-ix receptor to circulating vwf, which binds to exposed collagen, and directly, via interaction with platelet receptors gp vi and gp ia/iib. platelet activation, initiated by exposed collagen and locally generated soluble platelet agonists (primarily thrombin, adp, and thromboxane a2), provides the stimulus for the release of platelet-derived growth factors, adhesion molecules and coagulation factors, activation of adjacent platelets, and conformational changes in the platelet a(iib)b3 integrin (gp iib/iiia receptor). platelet aggregation, mediated primarily by interaction between the activated platelet gp iib/iiia receptor and its ligands, fibrinogen and vwf, results in the formation of a plateletrich thrombus (steinhubl and moliterno 2005) . p-selectin expression in platelets is elevated in disorders associated with arterial thrombosis such as coronary artery disease, acute myocardial infarction, stroke, and peripheral artery disease. during thrombosis, p-selectin is expressed on the surface of activated endothelial cells and platelets. p-selectin mediates rolling of platelets and leukocytes on activated endothelial cells as well as interactions of platelets with leukocytes. platelet p-selectin interacts with psgl-1 on leukocytes to form platelet-leukocyte aggregates. furthermore, this interaction of p-selectin with psgl-1 induces the upregulation of tissue factor, several cytokines in leukocytes and the production of procoagulant microparticles, thereby contributing to a prothrombotic state. p-selectin is also involved in platelet-platelet interactions, i. e. platelet aggregation which is a major factor in arterial thrombosis. p-selectin interacts with platelet sulfatides, thereby stabilizing initial platelet aggregates formed by gpiib/iiia-fibrinogen bridges. inhibtion of the p-selectin-sulfatide interaction leads to a reversal of platelet aggregation. thus, p-selectin plays a significant role in platelet aggregation and platelet-leukocyte interactions, both important mechanisms in the development of arterial thrombosis. following activation, p-selectin is rapidly translocated to the cell surface (merten and thiagarajan 2004; wang et al. 2005 ). platelet activation occurs in peripheral blood of patients with rheumatic mitral stenosis (ms). the plasma levels of soluble p-selectin are elevated in permanent atrial fibrillation (af) patients; the plasma levels of soluble p-selectin in the left atrium do not significantly differ from those in the right atrium, femoral vein, or femoral artery. the venous plasma levels of sp-selectin in patients with moderate-to-severe ms are significantly higher than those in healthy volunteers or patients with lone af. in addition, in patients with ms, there was no difference in the plasma levels of sp-selectin between the left and right atrial blood and between peripheral and atrial blood. moreover, there was no change in spselectin levels as a result of percutaneous transluminal mitral valvuloplasty (ptmv) (chen et al. 2004) . lip et al. (2005) studied the relations of plasma vwf (an index of endothelial damage and dysfunction) and sp-selectin levels in relation to the presence and onset of clinical congestive heart failure (chf) and degree of left ventricular dysfunction in patients taking part in spaf (stroke prevention in af). while plasma vwf was higher among patients with af and chf, plasma p-selectin concentrations were not affected by presence, onset, or severity of heart failure. atherosclerosis is a complex chronic inflammatory disease of the arterial wall. though the inflammatory nature of atherosclerosis has been established, the initial events that trigger this response in the arterial intima remain obscure. studies reveal a significant rate of genomic alterations in human atheromas. the accumulation of genomic rearrangements in vascular endothelium and smooth muscle cells are important for disease development. it is well accepted that the induction of ec adhesion molecules is a critical component in acute inflammatory responses as well as allogeneic interactions in vascularized allografts and, possibly, atherogenesis. inflammation and genetics are both prominent mechanisms in the pathogenesis of atherosclerosis and arterial thrombosis. accordingly, population studies have explored the association of ischaemic heart disease with gene polymorphisms of the inflammatory molecules: tumor necrosis factors (tnf) a and b, transforming growth factors (tgf) b1 and 2, p and e selectins, and platelet endothelial cell adhesion molecule (pecam) 1. the partly conflicting data provide some evidence that alterations in the genetics of the inflammatory system may modify the risk of ischaemic heart disease. (ros) as an initial event in recent years, reactive oxygen species (ros) are considered as initial event in causing atherosclerosis. ros are a family of molecules including molecular oxygen and its derivatives produced in all aerobic cells. excessive production of ros, outstripping endogenous antioxidant defense mechanisms, has been implicated in processes in which they oxidize biological macromolecules, such as dna, protein, carbohydrates, and lipids. many ros possess unpaired electrons and thus are free radicals. these include molecules such as superoxide anion (o 2 à ), hydroxyl radical (ho • ), nitric oxide (no • ), and lipid radicals. other reactive oxygen species, such as hydrogen peroxide (h2o2), peroxynitrite (onoo à ), and hypochlorous acid (hocl), are not free radicals per se but have oxidizing effects that contribute to oxidant stress. the cellular production of one ros may lead to the production of several others via radical chain reactions. for example, reactions between radicals and polyunsaturated fatty acids within cell membrane may result in a fatty acid peroxyl radical (r-coo • ) that can attack adjacent fatty acid side chains and initiate production of other lipid radicals. lipid radicals produced in this chain reaction accumulate in the cell membrane and may have a myriad of effects on cellular function, including leakage of the plasmolemma and dysfunction of membrane-bound receptors. of note, end products of lipid peroxidation, including unsaturated aldehydes and other metabolites, have cytotoxic and mutagenic properties. a decline in no bioavailability may be caused by decreased expression of the endothelial cell no synthase (enos), a lack of substrate or cofactors for enos (fig. 44.1 and 44.2) . in mammalian cells, potential enzymatic sources of ros include the mitochondrial respiration, arachidonic acid pathway enzymes lipoxygenase and cyclooxygenase, cytochrome p450s, xanthine oxidase, nadh/nadph oxidases, no synthase, peroxidases, and other hemoproteins. although many of these sources could potentially produce ros that inactivate no • , 3 sources have been studied extensively in cardiovascular system. these include xanthine oxidase, nadh/nadph oxidase, and no synthase (cai and harrison 2000; hamilton et al. 2004; vijya lakshmi et al. 2009 ). during initial step in atherosclerosis, there is rapid targeting of monocytes to the sites of inflammation and endothelial injury; the adhesion of leukocytes to activated endothelial cells is mediated by icam-1. the induction of ec adhesion molecules is a critical component in acute inflammatory responses as well as allogeneic interactions in vascularized allografts and, possibly, atherogenesis. the "inflammatory triad" of il-1, tnf, and lps are potent stimulators of the ec activation and adhesion molecules e-selectin or elam-1 (or also known as cd62e), icam-1 and vcam-1. pecam-1 plays also a key role in the transendothelial migration of circulating leukocytes (diapedesis) during vascular inflammation. icam-1 and vcam-1 are inflammatory predicators of adverse prognosis in patients with acute coronary syndromes (acs) (postadzhiyan et al. 2008) (fig. 44.2) . levels of p-selectin are increased in the blood of patients with familial hypercholesterolemia (fh) in spite of long-term intensive extracorporeal ldl-elimination, documenting the activity of atherosclerosis. low levels of p-selectin and mcp-1 after hypolidemic procedure can be used as a marker showing the effectivity of the extracorporeal ldl-cholesterol elimination (blaha et al. 2004 ). in an extended study, the levels of expression of tissue factor, icam-1, p-and e-selectin, and pai-1 were found low, whereas those of endothelial protein c receptor and vcam-1 were high (merlini et al. 2004 ). polymorphisms in the e-selectin gene are associated with accelerated atherosclerosis in young (age <40 years) patients, further suggesting a role of inflammation in atherosclerosis. a further change in endothelial physiology is an increase in the surface expression of e-selectin, which regulate adhesive interactions between certain blood cells and endothelium. intravascular fibrinolysis induced by tissuetype plasminogen activator or urokinase may contribute to the initiation of atherosclerosis by inducing p-selectin and platelet activating factor as well as to plaque rupture, either directly or indirectly, by activating metalloproteinases. as e-selectin is only expressed on activated endothelium, it provides an opportunity to study pathophysiological aspects of this cell in cardiovascular and other disease. however, seselectin can be found in the plasma, which has potential role in the pathogenesis of cardiovascular disease as raised levels have been found in hypertension, diabetes and hyperlipidemia, although its association in established atherosclerosis disease and its value as a prognostic factor is more controversial (holvoet and collen 1997) . polymorphisms for three genes, p-selectin, l-selectin, and e-selectin (genes p-sel, l-sel, and e-sel, respectively) showed that the selectin cluster is linked to markers at chromosome 1q23 (vora et al. 1994 ). significant genomic alterations were found on 1q22-q25 in sel-l gene. the message indicated somatic dna rearrangements, on loci associated to leukocyte adhesion, vascular smooth muscle cells growth, differentiation and migration, to atherosclerosis development as an inflammatory condition (arvanitis et al. 2005) . wenzel et al. (1999) and yoshida et al. (2003) described an adenine to cytosine (a/c) substitution for cdna position 561 resulting in an amino acid exchange from serine to arginine at position 128 (s/r or ser 128 arg) was detected in the epidermal growth factor (egf) domain. a higher mutation frequency was observed in patients aged 50 years or less with proven severe atherosclerosis as well as in patients aged 40 years or less. if ser 128 arg substitution had an effect on the adhesion of blood cells to the endothelium, the polymorphism could be of interest with respect to association studies in a number of pathological conditions, such as cardiovascular diseases. the ser 128 arg polymorphism is associated with a higher risk for early severe atherosclerosis. yoshida et al. (2003) suggested that the eselectin ser 128 arg polymorphism could functionally alter leukocyte-endothelial interactions as well as biochemical and biological consequences, which may account for the pathogenesis of myocardial infarction . leu 554 phe e-selectin mutations in hypertension and cad wenzel et al. (1996 wenzel et al. ( , 1999 detected 17 mutations, five of which resulted in an amino acid substitution. in e-selectin, exchange at ser 128 arg in egf domain and leu 554 phe in membrane domain, and a dna mutation from guanine to thymine (position 98) presented different allele frequencies in young patients with severe atherosclerosis, compared with an unselected population. the bi-allelic a/c polymorphism in the e-selectin gene may be implicated in the clinical expression of erythema nodosum (en) secondary to sarcoidosis (amoli et al. 2004 ). however, the e-selectin polymorphism may be associated with severity of atherosclerotic disease, but it is unknown if it is actually a risk factor for atherosclerosis (ghilardi et al. 2004) . a strong relationship was confirmed between 561a > c and 98g > t polymorphisms of e-selectin gene and susceptibility to cad by zak et al. (2008) . a body mass index (bmi)specific effect of leu 554 phe polymorphism of e-selectin gene on blood pressure has been reported by marteau et al. (2004) who strengthened the view that e-selectin is implicated in hypertension (marteau et al. 2004) . serum levels of e-and p-selectin in patients with essential hypertension (eh) are significantly higher than in controls, where as differences in serum levels of soluble l-selectin, vcam-1, or icam-1 between the patients with eh and the controls were not different (sanada et al. 2005 ). gene p-selectin thr 715 pro (a/c) polymorphism genetic analyses of p-selectin in the progression of atherosclerosis have provided conflicting results regarding the role of variation within the p-selectin gene and risk for heart disease. miller et al. (2004b) suggested that the thr 715 pro c allele was rare in blacks (0.8 %) and intermediate in south asians (3.0 %) compared to whites (11.2 %). sp-selectin levels were significantly lower in the individuals with the ac or cc compared to the aa genotype in both whites and south asians. thus, in whites and south asians the c allele of the thr 715 pro p-selectin polymorphism is associated with (hamilton et al. 2004) . excessive production of ros has been implicated in processes in which they oxidize biological macromolecules, such as dna, protein, carbohydrates, and lipids. many ros possess unpaired electrons and thus are free radicals. these include molecules such as superoxide anion (o 2 à ), hydroxyl racial (ho • ), nitric oxide (no • ), and lipid radicals. the cellular production of one ros may lead to the production of several others via radical chain reactions. a decline in no bioavailability may be caused by decreased expression of the endothelial cell no synthase (enos), a lack of substrate or cofactors required for enos action. low-density lipoprotein (ldl) is oxidized to oxidized form of ldl (ox-ldl) and initiates the atherosclerotic process in the vessel wall (see fig. 44 .2). abbreviations: o2à, superoxide; no, nitric oxide; onooà, peroxynitrite; h2o2, hydrogen peroxide; ohà, hydroxyl radical; sod, superoxide dismutase; gsh, reduced glutathione; gssg, oxidised glutathione; vsm, vascular smooth muscle lower sp-selectin levels (miller et al. 2004b ). the p-selectin thr 715 pro polymorphism is not associated with incident chd or ischemic stroke in either whites or african-americans (volcik et al. 2006 ). in cad, inflammatory biomarkers have been extensively investigated; more evidence exists for c-reactive protein (crp; chap. 8). fatty acid (fa) composition in serum has been associated with crp and e-selectin but not with other inflammatory markers (petersson et al. 2009 ). studies suggest that, besides crp, other inflammatory biomarkers such as cytokines, s-cd40 ligand, serum amyloid a (saa), selectins (e-selectin, p-selectin), icam-1, vcam-1, and several others may have a potential role for the prediction of risk for developing cad and may correlate with severity of cad (eikemo et al. 2004; fang et al. 2004; potapov et al. 2005; zakynthinos and pappa 2009) . the combination of natriuretic peptide (bnp) and e-selectin offers increased predictive value. plasma levels of adhesive molecules are correlated in patients with stable ihd. cigarette smoke condensate (csc)-induced surface expression of icam-1, e-selectin, and vcam-1 in huvec. fang et al. (2005) reported a significant decrease in c allele frequency of pecam-1 gene and showed that leu 125 val polymorphism of pecam-1 gene and elevated soluble pecam-1 were related to severe coronary artery stenosis in cad patients. sp-selectin is associated with myocardial damage: platelets are known to be activated during myocardial infarction (mi). though, the levels of sp-selectin, se-selectin and specam-1 did not differ significantly in the pathogenesis of atherosclerosis, sp-selectin was substantially increased in patients with acute myocardial infarction (ami). yip et al. (2006) tested the hypothesis that platelet activity shown by cd62p is enhanced and predictive of both the extent of myocardial damage and 30-day clinical outcome in patients with st-se ami undergoing primary coronary stenting. xu et al. (2006) suggested that activated-platelets play an important role in the process of myocardial ischemiareperfusion injury, and platelet-derived p-selectin is a critical mediator. p-selectin expression, along with cd40 ligand and tissue factor is significantly increased in infarcted rabbits with respect to controls. clopidogrel administration reduced p-selectin expression and cd40 ligand (molero et al. 2005) . ox-ldl also augments expression of monocyte chemoattractant protein 1 (mcp-1) and macrophage colony stimulating factor (m-csf). mcp-1 mediates the attraction of monocytes and leukocytes and their diapedesis through the endothelium into the intima. m-csf plays an important role in the transformation of monocytes to macrophage foam cells. macrophages express scavenger receptors, which internalize oxldl in their transformation into foam cells. migration of smooth muscle cells (smcs) from the intima into the media is another early event initiating a sequence that leads to formation of a fibrous atheroma hyperhomocysteinemia and selecins in mi: hyperhomocysteinemia is regarded as an independent risk factor for vascular diseases, and homocysteine is supposed to contribute to oxidative stress and endothelial damage. hyperhomocysteinemia is significantly associated with mi in comparison with controls with an odd ratio of 6.26 (khare et al. 2005) . folic acid corrected and reduced hyperhomocysteinemia in a large majority of the cases. although the levels of sp-selectin, se-selectin and specam-1 decrease after folic acid therapy, it was only se-selectin which was significantly reduced. apart from their lipid-lowering capacity, statins also exert anti-inflammatory and antioxidant effects. dna polymorphism in mi: some polymorphisms may increase the risk of mi within specific ethnic groups or in certain populations. p-selectin expression is increased in atherosclerotic plaques, and high plasma levels of this molecule have been observed in patients with unstable angina. dna polymorphisms in p-selectin gene may be a possible candidate for mi. the p-selectin gene is situated on chromosome 1q21-q24, spans >50 kb and contains 17 exons. four polymorphisms (ser 290 asn, asn 562 asp, leu 599 val and thr 715 pro) predicted a change in the amino acid sequence of p-selectin. in patients with mi from four regions of france and northern ireland (the ectim study) the p-selectin polymorphisms provided a heterozygosity of 91 %. the polymorphisms were tightly associated with one another and displayed patterns of linkage disequilibrium suggesting the existence of highly conserved ancestral haplotypes. study illustrates the complexity of the relationship between gene variability and disease and the necessity to explore in detail the polymorphisms of candidate genes (herrmann et al. 1998; tregouet et al. 2002) . the e-selectin gene arg128, 98 t, and phe554 alleles and pecam1 leu1 25 val and ser 563 asn polymorphisms may increase the risk of atherosclerosis, but not necessarily the risk of mi. this association seems to be more pronounced in younger patients and may be especially important in patients with a low risk for developing atherosclerosis. reports indicated that screening for cd14-260 c/t genotypes is unlikely to be a useful tool for risk assessment and it remains unclear whether cd14 polymorphisms significantly increase the risk of mi. the a 252 g polymorphism of lymphotoxin-a (lta) gene, a member of the tnf family, is strongly related with the onset of ami (auer et al. 2003) . quantitative real-time rt-pcr confirmed that lta increased the expressions of e-selectin and vcam1 both in huvec and hcaec, suggesting the roles of lta in the development of atherosclerosis. aminian et al. (2007) determined the possible role of gly 241 arg and lys 469 glu polymorphisms in development of cad and acute or chronic mi. although the frequency of gly-arg and arg-arg genotypes were higher in the control group compared to the chd patients, no strong corelation was found between gly 241 arg and lys 469 glu polymorphisms and occurrence of chd and mi in population from iran. in ischemic event in patients with atherosclerotic ischemic stroke, though the platelet aggregability was decreased after day 3 compared to that at day 1 of stroke onset, platelet cd63 and p-selectin/cd62p expression remained high even 90 days after the events. this suggested that platelet hyperactivation in atherosclerotic ischemic stroke might be sustained for a considerable period (cha et al. 2004; nadar et al. 2004b; yip et al. 2006 ). blood levels of icam-1 and cd62p expression in different typing of patients with ischemic stroke are different. evidences suggest that mps (meridian-phlegm stagnancy) group of patients is the key pathogenic factor of ischemic stroke. mucosal tolerance to e-selectin after booster tolerization can relieve cerebral ischemia-reperfusion injury and induce ischemia tolerance in rats. the mechanisms may involve decreased frequencies of cd8 + t cells, heightened mrna expression of il-10 and lowered mrna expression of e-selectin in the ischemic hemisphere (yun et al. 2008) . selakovic et al. (2009) defined changes of soluble cams in cerebrospinal fluid and plasma in the patients with the acute brain infarction, in which significant increase in the level of soluble adhesion molecules occurs within the first seven days. studies show that hypoxia/reoxygenation stimulates icam-1 and apoptosis (antonova et al. 2009 ). cerebral arteriovenous malformations (avms) showed significant upregulation of e-selectin, vcam-1 and icam-1 (storer et al. 2008; chan and sukhatme 2008; tuttolomondo et al. 2009 ). li et al. (2008) showed that icam-1 lys 469 glu polymorphism was involved in the causation of ischemic stroke, especially in female but not in male (rodrigues et al. 2008) . two allelic variants were related to ischemic stroke. multivariable regression analysis after adjustment for vascular risk factors demonstrated that alleles arg of ser 128 arg and phe of leu 554 phe polymorphisms are independent risk factors for ischemic stroke. the combination of two minor alleles of e-selectin genes appeared to be the strongest susceptibility factor for ischemic stroke (haidari et al. 2009 ). sarecka-hujar et al. (2010) could not confirm the relationship between the 98 g > t polymorphism of the e-selectin gene and childhood ischemic stroke. the g allele of the e-selectin 98 g > t polymorphism was more frequently transmitted to the children after stroke compared to the t allele. there is a need for further studies in these areas. the association between blood pressure and different adhesion molecules appeared to be present in women younger than 50 years, who were likely to be pre-menopausal (miller et al. 2004a) . serum levels of e-and p-selectin in patients with essential hypertension (eh) are significantly higher than in the controls (sanada et al. 2005) . after adjustment for age, only se-selectin concentrations were significantly associated with blood pressure. higher levels of plasma sp-selectin were confirmed in hypertensive patients alone with vegf (nadar et al. 2004a) . it is stated that decrease in blood pressure may reduce the rate of progression of atherosclerosis by affecting the expression of e-and p-selectin in the endothelium, the platelets, or both. in vitro studies indicate that complement activation regulates the expression of p-selectin on endothelial cells. this suggests that in disorders such as ischemia/reperfusion injury, in which both complement and p-selectin have been shown to play a role, complement activation is a primary event and the effects of p-selectin are secondary. in mouse kidney model of i/r injury, results indicated that complement and p-selectin-mediated pathways of renal reperfusion injury are mutually independent (farrar et al. 2004 ). induction of circulating polymorphonuclear neutrophils (pmns) might contribute to the superior outcome following stenting and early intervention compared to conventional balloon angioplasty (ptca). a substantial increase in se-selectin levels early after ptca and stent implantation may predict development of restenosis (heider et al. 2006; kilickap et al. 2004) . after reperfusion of myocardial vessels, p-selectin expressed on majority of vessels (77 %) though the expression decreased during subsequent remaining duration of reperfusion (chukwuemeka et al. 2005) . in rats, the mrna expression for several genes was associated with inflammation after transient middle cerebral artery occlusion (mcao). gene expression increased in the injured hemisphere for il-1b, il-6 and icam-1. tnf-a mrna was upregulated in the injured versus uninjured hemisphere, while e-selectin mrna showed a significant increase from 6 to 24 h after mcao (berti et al. 2002) . both p-selectin and lfa-1 may be important targets to control pathologic inflammation in i/r-induced tissue injury in the colon (riaz et al. 2002) . the study in intestinal ischemia and reperfusion injury (ir/i) using murine models demonstrated the importance of p-selectin in warm and cold ir/i. the blockade of p-selectin using rpsgl1-lg or the absence of p-selectin ko mice confers a survival advantage and reduction in tissue injury. the mechanism appears to be independent of neutrophil infiltration (carmody et al. 2004 ). enterocyte apoptosis is increased following intestinal i/r injury. hyperoxia following intestinal i/r in rat increased e-selectin expression in the jejunum and ileum and a concomitant increase in neutrophil recruitment in the ileum, accompanied by increased cell apoptosis (braun et al. 2004; sukhotnik et al. 2008) . germ cell-specific apoptosis that occurs after i/r of murine testis is dependent on neutrophil recruitment to the testis and is dependent on e-selectin. blockage of e-selectin may be a strategy to treat postischaemic testis (celebi and paul 2008) . allergic inflammation is characterized by recruitment of specific leukocyte subpopulations from blood into tissue and requires a series of cell adhesion-molecule-mediated interactions between postcapillary vascular endothelium and the leukocyte cell surface. three major groups are involved: selectins, integrins, and the immunoglobulin gene superfamily. p-and e-selectin mediate initial leukocyte adhesion, whereas beta 2-integrin/icam-1 and vla-4/ vcam-1 pathways mediate leukocyte arrest and transendothelial migration. because vla-4 expression is restricted to eosinophils and lymphocytes, vcam-1 has been implicated in selective eosinophil recruitment characterizing allergic inflammation. however, additional factors such as profile of cytokine release are likely to operate since tissue eosinophilia has been observed in the absence of vcam-1 expression (smith et al. 1993a ). e-selectin is highly expressed on vascular endothelium in atopic dermatitis and psoriasis, and in patients with measles. the cutaneous lymphocyte-associated antigen (cla), which is expressed on peripheral skin-homing helper memory t cells in healthy persons, is at least partly the sialyl 6-sulfo le x determinant (ohmori et al. 2006 ) and a ligand for selectins. the differential polyadenylation of e-selectin transcripts may provide the molecular basis for the observed chronic expression of e-selectin in human dermal disorders. in atopic dermatitis, patients express icam-1 and icam-3, e-selectin and l-selectin (60 %) in the dermis, without expression of e-and l-selectins in the epidermis. a high expression of adhesion molecules in the skin lesions of atopic dermatitis patients may play an important role in the pathogenesis of atopic dermatitis (lugovic et al. 2006) . the blood markers for atopic dermatitis, including soluble forms of eselectin, vcam-1 and icam-1 were reduced after treatment with cetiridine (izu and tokura 2005) . the extracts from dust mites, dermatophagoides farinae, d. pteronyssinus and euroglyphus maynei with and without endotoxin (lps) stimulated endothelial cells to express icam-1, vcam-1, and e-selectin and to secrete il-6, il-8, mcp-1, and gm-csf. serum levels of se-selectin are higher in children with measles than in children with atopic dermatitis, atopic asthma and healthy controls. but it was not correlated with measles. there was no correlation between se-selectin and tnf-a level (park et al. 2008) . pollinosis from parietaria judaica is one of the main causes of allergy in the mediterranean area. the treatment of endothelial cells with pollen extract causes an increase of e-selectin and vcam-1 protein levels as well as an increase of il-8 production. the stimulation of cell adhesion molecules was paralleled by an increase of adhesion of polymorphonuclear cells (pmns) to hmvec-l monolayer (taverna et al. 2008 ). allergic rhinitis is an inflammatory disease of the nasal mucosa, caused by an ige-mediated reaction after exposure to the allergen. persistent inflammation is induced by the presence of an inflammatory cell infiltrate, together with icam-1 expression in the epithelial cells of the mucosa exposed to the allergen to which they are sensitized, in the absence of clinical symptoms (montoro et al. 2007 ). nasal polyposis is a chronic non-infectious inflammatory disease of the nasal and paranasal cavity mucosa. eosinophil migration from blood stream to nasal polyps involves different molecules such as icam-1, vcam-1, and l-, p-and eselectins. patients with nasal polyposis exhibit a higher expression of vcam-1, e-selectin, and l-selectin compared to healthy controls . staphylococcal enterotoxin a (sea) and staphylococcal enterotoxin b (seb) infection increased icam-1 expression and cytokine secretion (wang et al. 2007 ). excessive leukocyte accumulation is involved in the pathogenesis of the sepsis-induced acute lung injury. studies suggest that p-selectin has a substantial role in the pathogenesis of the lung injury induced by lps (ohnishi et al. 1999 ). in bleomycin-induced fibrosis in mice, the l-selectin and/or icam-1 deficiency inhibited skin and lung fibrosis with decreased th2 and th17 cytokines and increased th1 cytokines. in contrast, p-selectin deficiency, e-selectin deficiency with or without p-selectin blockade, or psgl-1 deficiency augmented the fibrosis in parallel with increased th2 and th17 cytokines and decreased th1 cytokines. yoshizaki et al. (2010) suggest that l-selectin and icam-1 regulate th2 and th17 cell accumulation in skin and the lung, leading to the development of fibrosis, and that p-selectin, e-selectin, and psgl-1 regulate th1 cell infiltration, resulting in the inhibition of fibrosis (yoshizaki et al. 2010) . adult respiratory distress syndrome (ards) appears to develop as the acute lung injury in the course of many severe diseases, as the result of damage of alveolar-capillary barrier. clinical observations suggest that analysis of e-, p-selectin and icam-1 concentrations in the serum of patients with ards may be helpful in monitoring the course and treatment of the disease (skiba-choińska and rogowski 1996). in the pathogenesis of paracoccidioidomycosis, gonzalez et al. (2005) suggest that during early stages, up-regulation of icam-1, vcam-1, cd18 and mac-1 expression may participate in the inflammatory process. the house dust mite (hdm) is the common indoor allergen associated with bronchial asthma. icam-1, vcam-1, and e-selectin are newly synthesized prior to spontaneous asthma attacks, and their expression may play a key role in eosinophil infiltration into the airway (ohkawara et al. 1995) . crude extract of d. farinae induces icam-1 expression in eol-1 cells through signaling pathways involving both nf-kb and jnk ). kirchberger et al. (2006) demonstrated that signaling via icam-1 induces adhesiveness of mononuclear phagocytes, which critically involves pecam-1 and is mediated via lfa-1/icam-3. the most common acute infection in humans, human rhinovirus (hrv) is a leading cause of exacerbations of asthma and chronic obstruction pulmonary disease. icam-1 is a critical target-docking molecule on epithelial cells for 90 % hrv serotypes. icam-1 regulates not only viral entry and replication but also signaling pathways that lead to inflammatory mediator production (lau et al. 2008; lee et al. 2008 ). the sicam-1 but not se-selectin from patients with asthma is significantly higher than healthy controls. although serum levels of sicam-1 are higher in asthmatics, it may be necessary to establish individual baseline values for serial estimation to evaluate their clinical relevance (bijanzadeh et al. 2009 ). the serum levels of sicam-1 were significantly higher in obese nonasthmatic and obese asthmatic children versus control and lean asthmatic children . p-selectin is an important controller of the inflammation by mediating selective eosinophil cell influx to the lung. it can be used as a sensitive marker in mild asthma (sjosward et al. 2004 ). adhesion molecule expression and interactions are involved in initiation and propagation of autoimmune diseases including rheumatoid arthritis (ra), systemic lupus erythematosus, sj€ ogren's syndrome, autoimmune thyroid disease, multiple sclerosis, systemic sclerosis (ssc) and diabetes mellitus. increased adhesion molecule expression and avidity changes occurring with cellular activation are the principal methods regulating leukocyte adhesion. although differences between specific autoimmune diseases exist, key interactions facilitating the development of autoimmune inflammation appear to include l-selectin/p-selectin/e-selectin, lfa-1/ icam-1, very late antigen-4 (vla-4)/vcam-1, and a4b7/ madcam or vcam-1 adhesion. a vast array of adhesive interactions occurs between immunocompetent cells, endothelium, extracellular matrix, and target tissues during the evolution of an autoimmune disease. dermatitis herpetiformis (dh) and bullous pemphigoid (bp), the autoimmune diseases, are characterized by destruction of the basement membrane zone (bmz) and anchoring fibres by autoantibodies and infiltration. skin biopsies from patients with dh, with bp, and from healthy subjects showed the expression of e and l selectins mainly in the skin leukocytes in all samples where as b1, b3 integrins was detected mainly in basal keratinocytes. integrins and selectins seem to play an important role in the destruction of bmz in dh and bp (erkiert-polguj et al. 2009 ). p-selectin levels were significantly higher than normal in ra and ssc, but not in sle. in contrast, mean l-selectin levels were significantly higher than normal in sle, but not in ra or ssc. where as soluble il-2 receptors in patients with active ra, ssc and sle were almost double the normal level, showing a strong positive correlation only between l-selectin and sil-2r, and only in patients with sle. these findings indicated a distinct pattern of immune cell activation in chronic diseases that share an over-activation of t-lymphocytes (sfikakis and mavrikakis 1999). adhesion molecules have been implicated in the development and progression of cardiovascular disease, particularly in people with diabetes. diabetes mellitus type 1 (type 1 diabetes or t1dm, also called insulin-dependent diabetes mellitus-iddm, or, formerly, juvenile diabetes) is a form of diabetes mellitus that results from autoimmune destruction of insulin-producing b cells of the pancreas. the chronic hyperglycemic state in t1dm patients produces an aggression to vascular endothelium leading to a premature development of atherosclerosis. in both boys and girls, seselectin is an early marker of endothelial dysfunction and a probable risk marker of atherosclerosis in children with t1dm (carrizo et al. 2008) . the levels of c-reactive protein, e-selectin, and cytokines in association with severity index were significantly increased in t1dm and type 1 diabetic patients with microvascular complications (t1dm-mv patients) compared with control subjects (devaraj et al. 2007 ). nerve microvasculitis and ischemic injury appear to be the primary and important pathogenic alterations in lumbosacral radiculoplexus neuropathy (lrpn) of patients with diabetes mellitus (dlrpn) and without diabetes mellitus (lrpn). the up-regulation of inflammatory mediators target different cells at different disease stages and that these mediators may be sequentially involved in an immune-mediated inflammatory process that is shared by both dlrpn and lrpn (kawamura et al. 2008) . adhesion molecules are upregulated in endothelial cells of the placental bed in pregnancies complicated by t1dm in association with increased adherence of peripheral blood monocytes. the increase in monocyte adhesion to decidual endothelial cells from diabetic pregnancies was associated with increased endothelial cell expression of icam-1, but not vcam-1. icam-1 expression in normal decidual endothelial cells was stimulated by pro-atherogenic and proinflammatory stimuli (xie et al. 2008; telejko et al. 2009 ). type 2 diabetes: in contrast to t1dm, type 2 diabetes mellitus (t2dm) results from insulin resistance, a condition in which cells fail to use insulin properly, sometimes combined with an absolute insulin deficiency (formerly referred to as non-insulin-dependent diabetes mellitus, niddm for short). endothelial dysfunction in type 2 diabetic patients is associated with inflammation, increased levels of circulating soluble adhesion molecules (vcam-1 and e-selectin), and inducing production of ros, and urinary albumin excretion (potenza et al. 2009 ). diabetic patients have increased susceptibility to infection, which may be related to impaired inflammatory response observed in experimental models of diabetes, and restored by insulin treatment (riad et al. 2008; west et al. 2008) . serum levels of cams in diabetic patients: abnormal levels some of serum icam-1, vicam-1, e-selectin, pselectin, l-selectin have been detected in t2dm. high-fat load and glucose alone produce an increase of nitrotyrosine, icam-1, vcam-1, and e-selectin plasma levels in normal and diabetic subjects. a decrease in neutrophil surface cd62l expression and significantly higher concentrations of sicam-1, svcam-1, se-selectin, vwf, hscrp, il-6 and fibrinogen in patients with diabetic microangiopathy in comparison with diabetic group without microangiopathic complications and healthy controls suggested that: (1) diabetic microangiopathy is accompanied by increase in cd11b expression and decrease in cd62l (l-selectin) expression on peripheral blood neutrophils; (2) neutrophil activation and intensified adhesion; (3) the development of diabetic microangiopathy is accompanied by an increase in soluble adhesion molecules and inflammatory markers concentrations in the blood (lim et al. 2004; mastej and adamiec; 2008) . levels of e-selectin positively correlated with high triglyceride levels in type 2 diabetic subjects with silent ischemia (adamikova et al. 2008; okapcova and gabor 2004; rubio-guerra et al. 2008) . though, baseline plasma levels of vascular markers (hscrp, sicam-1, svcam-1, e-selectin and p-selectin) were significantly elevated, they did not improve after aerobic exercise. the se-selectin and vwf are elevated in chronic heart failure patients with dm but not in those without dm. high seselectin levels may be associated with ischaemic events in patients with dm (kistorp et al. 2008) . diabetic nephropathy (dn) and diabetic heart: diabetic nephropathy (dn) is the leading cause of end stage renal disease (esrd) (malatino et al. 2007) . although the pathogenesis of dn is multifactorial, local inflammatory stress may result from both the metabolic and hemodynamic derangements observed in dn. the current evidence supporting the role of inflammation in the early phases of clinical and experimental dn has been reviewed (fornoni et al. 2008) . inflammatory markers such as il-18 and tnf-a are increased in the serum of patients with diabetes and dn. this occurs at an early stage of disease, and correlates with the degree of albuminuria. the pharmacologic interventions for dn by angiotensin converting enzyme inhibitors, angiotensin receptor blockers and aldosterone antagonists may have anti-inflammatory effects, which are independent of their hemodynamic effect. diabetic retinopathy: the association between soluble adhesion molecules levels and retinopathy in type 2 diabetic patients has been clarified. se-selectin levels are elevated in diabetic patients compared to control subjects, with no significant difference in sicam-1 and svcam-1 levels. the progression of retinopathy was not associated with an increase in soluble adhesion molecules. however, nowak et al. (2008) observed that serum levels of sicam-1 and selam-1 were significantly elevated and the concentration svcam-1 was elevated but not significantly in diabetic patients. increase in sicam-1 and svcam-1 levels, as well as their correlation with high vitreous il-6 and tnfa concentrations in patients with diabetic retinopathy seems to confirm the inflammatory-immune nature of this process. significantly increased tnf-a concentration in the vitreous body was related to the rise of vcam-1 (adamiec-mroczek and oficjalska-młyńczak 2008; leal et al. 2008; khalfaoui et al. 2008) . intravitreal injection of corticosteroid has been used to treat diabetic macular edema. plasma levels of vwf and sp-selectin (but not se-selectin) are significantly higher among rheumatoid disease (rd) patients compared to controls. levels of vwf progressively rise with increasing cardiovascular risk (bhatia et al. 2009 ). serum levels of icam-1, icam-3, vcam-1, l-selectin, and e-selectin have been detemined in children with a variety of pediatric rheumatic diseases. a trend toward higher levels of se-selectin was found in vasculitis vs other diagnoses. the sicam-1 was higher in patients with active vs inactive disease across all diagnoses. report suggests that (1) elevated e-selectin levels in vasculitis likely reflect the high degree of endothelial activation and possibly overt vascular damage in those conditions. (2) the correlation of sl-selectin with c4 in sle may indicate that downregulation of shedding of cell surface l-selectin is involved in continued adherence of leukocytes to endothelium, possibly causing further damage and immune complex deposition in this condition. (3) the trend toward inverse correlation between se-selectin and vwf:ag in diabetes mellitus is interesting. (4) levels of sicam-i may be a useful marker of active vs quiescent disease in general in the pediatric rheumatic diseases, although lack of correlation with disease activity indices indicates that it is too insensitive to smaller differences in disease activity to be recommended for routine clinical use (bloom et al. 2002) . considerable evidence indicates that patients with rheumatoid arthritis (ra) are at greater risk of developing atherosclerosis and cardiovascular disease. atherosclerotic cardiovascular mortality is increased in ra patients. the markers proposed for assessing ra activity include rheumatoid factor, anti-citrullinated protein/peptide antibodies, igm anti-igg advanced glycation end products, markers of bone/ cartilage metabolism, mannose-binding lectin, e-selectin, il-6, and leptin. various studies have investigated the correlation between some of these markers and other variables that might indicate disease activity, e.g., inflammatory activity tests and disease activity scores. however, there is as yet insufficient evidence that any of these markers, in isolation or in combination, are useful in the assessment of ra activity. many numerous endothelial cells become positive for eselectin and e-selectin mrna in ra synovial membranes and the e-selectin expression appeared to correlate with inflammatory activity. p-selectin deficiency in mice resulted in accelerated onset of joint inflammation in the murine collagen-immunized arthritis model. mice deficient either in e-selectin or in e-selectin and p-selectin (e/p-selectin mutant) also exhibit accelerated development of arthritis compared with wild type mice in cia model. the strong vascular expression of e-selectin indicates an activation of endothelial cells in the recruitment of cells associated with the chronic inflammation of ra (foster et al. 2009; da mota et al. 2009 ). e-selectin and icam-1 are upregulated on the synovial endothelium, while vcam-1 plays an important role in synovial lining layer cells and within the synovial stroma. the expression of cams may be blocked by mabs and modified by nonsteroidal anti-inflammatory drugs and disease-modifying antirheumatic drugs. (cobankara et al. 2004) . serum soluble adhesion molecules concentrations are down-regulated following anti-tnf-a antibody therapy combined with methotrexate (mtx) (klimiuk et al. 2004 (klimiuk et al. , 2007 lev€ alampi et al. 2007; bosello et al. 2008) . in comparison with osteoarthritis (oa), patients with early ra are characterized by high serum concentrations of sicam-1, svcam-1, and se-selectin (yildirim et al. 2005) , while ldl-cholesterol was decreased in all ra patients (pemberton et al. 2009 ). p-selectin deficiency in mice results in accelerated onset of joint inflammation in the murine collagen-immunized arthritis model. mice deficient either in e-selectin or in e-selectin and p-selectin (e/pselectin mutant) also exhibit accelerated development of arthritis compared with wild type mice, suggesting that these adhesion molecules perform overlapping functions in regulating joint disease. ruth et al. (2005) suggested that eselectin and p-selectin expression can significantly influence cytokine and chemokine production in joint tissue, and that these adhesion molecules play important regulatory roles in the development of ra in e/p-selectin mutant mice (singh et al. 2008) . in ra patients, p-selectin expression, pmc and scd40l levels were increased when compared with controls. the increase in markers of active platelets, p-selectin and scd40l, and platelet-monocyte levels might be associated with the increased cardiovascular mortality in ra. psoriatic arthritis (psa) is associated with the development of endothelial dysfunction and increased atherosclerotic complications. endothelial activation might have a role in the pathogenesis of both psoriasis and psa. among parameters of platelet activation, only pmc might play a role in the pathogenesis of psa (pamuk et al. 2008 (pamuk et al. , 2009 systemic lupus erythematosus (sle): elevated serum concentrations of et-1, s-thrombomodulin (tm), and seselectin reflect persisting endothelial cell activation in sle, and point to an important role of et-1 in the pathogenesis of internal organ involvement (kuryliszyn-moskal et al. 2008 ). the s-e-selectin, tm and s-vcam-1 are significantly elevated in lupus nephritis (ln) with renal vascular lesions (vls) than in ln without vls. a positive correlation was found between tm and serum creatinine in patients with vascular lesions. therefore, serum tm and s-vcam-1 can be biomarkers of vls in ln patients (yao et al. 2008 , rho et al. 2008 ). autoimmune thyroiditis: autoimmune thyroiditis is multifactorial in etiology with genetic and environmental factors contributions. patients with untreated graves' disease (gd) show high serum level of se-selectin, which correlated with the activity of the disease. the expression of icam-1 and vcam-1 was increased in ec from patients from graves' disease (gd) and hashimoto's thyroiditis (ht). results suggest that both the lfa-1/icam-1, icam-3 and vla-4/vcam-1 pathways could play a relevant role in autoimmune thyroid disorders (marazuela et al. 1994 ). in patients with gd, the 721 g-a polymorphism was associated with an earlier age of gd onset (before age 40) and that the 1405a-g polymorphism could predispose to graves ophthalmopathy. it was concluded that g241r and k469e amino acid substitutions in the icam1 molecule could influence the intensity/duration of the autoimmunity process and the infiltration of orbital tissues (kretowski et al. 2003) . chen et al. (2008) suggested that common sele variants may be associated with susceptibility to gd in chinese population, though the limitation of sample size and multiple test problems exists (chen et al. 2008 ). sjogren's syndrome: cams are involved in the lymphoid cell infiltration of the salivary and lacrimal glands in sjogren's syndrome (ss) patients. biopsies from ss patients showed a marked expression of vcam-1 and icam-1 in the venules surrounded by infiltrated cd4 + cd45ro + t cells. e-selectin was expressed on vascular endothelium with weak intensity (saito et al. 1993) . pisella et al. (2000) reported that a significant increase of hla-dr and icam1 expression by epithelial cells was consistently found in patients with keratoconjunctivitis sicca (sjogren syndrome). these markers were well correlated with each other and correlated inversely with tear break-up time and tear production. cytokine-mediated up-regulation of vcam-1 and icam-1 that facilitates the recruitment of vla-4 and lfa-1 expressing t cells might contribute to lymphoid cell infiltration in the salivary and lacrimal glands in ss cams mediate the extravasation of leukocytes and their accumulation in inflamed intestinal mucosa. eosinophilic inflammation is a common feature of numerous eosinophilassociated gastrointestinal (egid) diseases. increased intestinal expression of e-selectin has been associated with multiple organ failure and an adverse outcome. vcam-1 is not altered in in mucosa of patients with inflammatory bowel disease (ibd) regardless of the activity of the inflammatory process. in contrast, e-selectin was not detected in normal colonic mucosa or in colonic mucosa of patients with ibd. however, high levels of e-selectin were consistently found on endothelial surfaces in association with active inflammation in affected areas of colonic mucosa in patients with either ulcerative colitis or crohn's colitis. in addition, e-selectin appeared to be present within neutrophils which had migrated into crypt abscesses in affected mucosa. thus e-selectin may play an important role in facilitating leukocyte migration into sites of active ibd involvement (koizumi et al. 1992) icam-1 was expressed to a greater degree in ulcerative colitis (uc) specimens. serum icam-1 levels in uc patients showed lower levels than those in the control group and were found to vary according to degree of clinical severity (ogawa et al. 2008) . characterization of integrin expression on colonic eosinophils revealed that colonic cc chemokine receptor 3 + eosinophils express icam-1 counter-receptor integrins al, am, and b2. it appears that b2-integrin/icam-1-dependent pathways are integral to eosinophil recruitment in colon during gi inflammation associated with colonic injury (forbes et al. 2006) . mccafferty et al. (1999) examined the role of p-selectin in intestinal inflammation in p-selectin deficient mice alone or in combination with either icam-1 or e-selectin and suggested that anti-adhesion therapy might play only a limited, beneficial role and often a detrimental role in intestinal inflammation. the se-selectin levels of crohn's disease patients with active disease are higher than those with remission of the disease. l-selectin does not change in patients with active disease compared to those with remission. thus, determination of se-selectin in children with crohn's disease is of significance in estimation of inflammation activity (adamska et al. 2007 ). khazen et al. (2009) investigated mutations in cam genes in tunisian patients, implicated in determining susceptibility to ulcerative colitis (uc) and crohn's disease (cd). a significant increase in allele frequencies of 206 l of l-selectin and the associated genotype f/l was observed in patients with uc and cd compared with controls; the l206 allele and f/l206 genotype frequencies were significantly increased in uc patients with left-sided type; whereas, the f/l206 genotype was significant in cd patients with ileocolonic location. no significant differences in allele or genotype frequencies were observed for icam-1 k469e, e-selectin, and pecam-1 polymorphisms between uc patients, cd patients, and controls. khazen et al. (2009) suggest an association of inflammatory bowel disease with allele l206 of l-selectin gene, whereas genotype l/f was associated with a subgroup of uc (left-sided type) and cd patients with more extensive location of disease and stricturing behavior. however, vischer et al. (2008) did not reveal any difference in mrna and protein expression levels for any construct or a major impact of missense variants on icam-1 biological function. pulse-chase experiments showed that two variants, k469e and arg478 to trp (r478w), had a prolonged half-life compared with wildtype icam1, whereas two other variants, g241r and pro352 to leu (p352l), had a decreased half-life, implying differences in protein degradation. celiac disease: celiac disease is a chronic intestinal inflammatory disease that develops in genetically susceptible individuals after gluten ingestion. the icam-1 gene, located in the celiac disease linkage region 19p13, encodes icam-1 involved in inflammatory processes. increased levels of icam-1 were observed in intestinal biopsies and in sera of celiac disease patients. in addition, an association between the icam1 polymorphism g241r and celiac disease patients has been described in a french population.though, in spanish population results discard the importance of icam1 g241r in celiac disease (dema et al. 2008 ). behçet syndrome: behçet's disease/syndrome (bd/bs) is a multisystemic inflammatory disorder of which oral aphthous ulceration is a major feature. cd3 and gd t-cell expression and other adhesion molecules including vcam-1 and icam-1 were upregulated, whereas cd40 showed little change in bd. the changes in cell-cell and cellextracellular matrix interactions may affect cell homeostasis and participate in the formation of oral ulcers in bd (kose et al. 2008 ). however, demirkesen et al. (2008) found no significant differences between the bs and control groups in regard to e-selectin, p-selectin, vcam-1, pncam-1 except for icam-1. systemic sclerosis or systemic scleroderma: systemic sclerosis or systemic scleroderma is a systemic autoimmune disease or systemic connective tissue disease that is a subtype of scleroderma. severe fibrosis and increased expression of profibrotic cytokines are important hallmarks in the gastric wall of patients with systemic sclerosis (ssc; scleroderma). the cd4 + /cd8 + t cell ratio is significantly increased in ssc specimens. t cells strongly express the activation markers vla-4, lfa-1, and icam-1. endothelial cells showed corresponding surface activation with strong expression of vcam-1 and icam-1. these results provide the evidence that endothelial/lymphocyte activation leading to prominent cd4 + t cell infiltration may play a key pathogenetic role within the gastric wall of patients with ssc (manetti et al. 2008) . in patients with ssc with and without pulmonary arterial hypertension (pah), serum sicam-1, svcam-1, sp-selectin and specam-1 levels were higher than in healthy donors (hd) at baseline and fell to normal values after 12 months of bosentan therapy. endothelial activation occurs in ssc, and that changes in the t cell/endothelium interplay take place in ssc-associated pah. bosentan seems to be able to hamper these changes and restore t cell functions in these patients (iannone et al. 2008 ). soluble adhesion molecules play a significant role in hepatitis. biliary atresia (ba) is a congenital or acquired liver disease and one of the principle forms of chronic rejection of a transplanted liver allograft. in the congenital form, the common bile duct between the liver and the small intestine is blocked or absent. the acquired type most often occurs in the setting of autoimmune disease, and is one of the principle forms of chronic rejection of a transplanted liver allograft. the serum se-selectin of ba patients was higher than that of controls. subgroup analysis showed that there was an increase in se-selectin levels of ba patients with jaundice compared to those without jaundice. also, se-selectin was positively correlated with serum alanine transferase (alt), a marker for liver injury, but not with serum gamma glutamyl transpeptidase (ggt) (vejchapipat et al. 2008) . cholangitis without a modifier-from greek chol-, bile + ang-, vessel + itis-, inflammation) is an infection of the bile duct (cholangitis). in secondary cholangitis, icam-1 expression is increased along with de novo vcam-1 and e-selectin appearance on the endothelium of microvessels in chronic exacerbated cholangitis (gulubova et al. 2008) . primary biliary cirrhosis (pbc) is an autoimmune disease of the liver marked by the slow progressive destruction of the small bile ducts (bile canaliculi) within the liver. patients with pbc, primary sclerosing cholangitis and chronic active hepatitis (autoimmune) show significant increase in sicam-1 compared with normal healthy subjects. significant elevation in sicam-1 is also detected in patients with inactive alcoholic cirrhosis, suggesting that impaired liver may, in part, account for the increased serum level in patients with autoimmune liver disease. in contrast, se-selectin did not differ significantly from healthy controls. although, peripheral blood mononuclear cells (pbmc) may be a source of sicam-1, thomson et al. (1994) suggested that pbmc may not be a significant source of sicam-1 in this disease. the differential expression of cams in the liver is consistent with the suggestion of selectins involvement in neutrophil rolling in the vasculature and icam-1 in transendothelial migration and adherence to parenchymal cells (essani et al. 1995) . wu et al. (2009) investigated the relationships between the polymorphisms of e-selectin gene and plasma seselectin levels in relation to disease progression in a hepatitis b virus (hbv)-infected chinese han population. the frequency of c allele (ac or cc) of the a 561 c polymorphism was significantly increased in patients with liver cirrhosis (lc) compared to normal population. there was no difference in allele distribution of the g 98 t polymorphism. the a 561 c polymorphism of e-selectin gene may be associated with disease progression in patients with chronic hbv infection and control the expression of plasma soluble levels, while the g 98 t polymorphism may be related to fibrotic severity in chinese population (wu et al. 2009 ). mice with targeted deletion of the p-selectin gene developed unpolarized type 1/type 2 cytokine responses and severely aggravated liver pathology following infection pathogen schistosoma mansoni. liver fibrosis increased 6 fold, despite simultaneous induction of ifn-g and increase in inflammation in absence of p-selectin. this suggested a critical role of p-selectin in the progression of chronic liver disease caused by schistosome parasites (wynn et al. 2004 ). axonal degeneration was confirmed as the major pathological feature of critical illness polyneuropathy (cip). expression of e-selectin was significantly increased in endothelium of epineurial and endoneurial vessels, suggesting endothelial cell activation (fenzi et al. 2003) . increasing evidence indicates that inflammatory responses are implicated in the pathogenesis of cerebral vasospasm after aneurismal subarachnoid hemorrhage (sah). murine sah model provided the evidence of effective prevention of sah-induced vasospasm by a mab implied the possible role of e selectin in the pathogenesis of vasospasm after sah (lin et al. 2005) . neuroinflammation is present in the substantia nigra (sn) of patients of parkinson disease (pd). a large number of icam-1-positive reactive astrocytes have been observed in the sn of patients with neuropathologically confirmed pd, including three of familial origin. the icam-1-positive reactive astrocytes were mainly concentrated around residual neurons in areas of heavy neuronal loss and extracellular melanin accumulation (miklossy et al. 2006 ). the svcam-1 plasma levels were higher in late onset alzheimer's disease (load) and vascular dementia (vd) compared with controls. among patients (load, vd, and not-dementia (cdnd), se-selectin levels were higher in individuals with most severe cerebrovascular disease on ct scan. increased svcam-1 plasma levels in load and vd suggest the existence of endothelial dysfunction in both types of dementia. results support the possible role of e-selectin in the pathogenesis of cerebrovascular disease (zuliani et al. 2008 ). upregulation of icam-1, lfa-1, mac-1 and subsequent leukocyte infiltration appears to be significant events of pancreatic and pulmonary injuries in acute pancreatitis (ap) . proinflammatory cytokines and oxidative stress seem to be involved in the development of local and particularly systemic complications in ap patients. acute pancreatitis patients show vcam-1 and p-selectin concentrations significantly lower and l-selectin concentrations significantly higher than the healthy subjects. only e-selectin was significantly higher in severe than in mild disease (pezzilli et al. 2008) . kleinhans et al. (2009) showed that the endothelial cell expression of pecam-1, vcam, e-selectin, and p-selectin was upregulated in severe porcine pancreatitis. in acute pancreatitis, plasma levels of se-selectin and soluble thrombomodulin (stm) serve as endothelial markers; the former is an endothelial activation marker, while the latter is an endothelial injury marker (chooklin 2009; ida et al. 2009 ). in patients affected by microscopic polyangiitis (mpa) and associated with myeloperoxidase (mpo)-anti-neutrophil cytoplasmic antibodies (anca), higher sicam-1 and seselectin levels during active phase and their slower decline during the treatment period, could be a prognostic risk factor for chronic renal failure development (di lorenzo et al. 2004; musial et al. 2005 ). an increased level of se-selectin in patients susceptible to restenosis supports a role for white blood cell/endothelial interaction in restenosis after angioplasty (sainani and maru 2005) . the impairment of vascular endothelial function was obvious in uremic patients with maintaining hemodialysis (mhd). the changes of icam-1 and e-selectin could be accepted as biochemical criterions of vascular endothelial injury . diuresis, serum creatinine, urea, and enzyme elimination are pathological among patients with acute renal failure (arf). higher elimination rates of sicam-1 and higher values of se-selectin compared to patients without arf indicated additional parameters for early signs of kidney damage (dehne et al. 2008) . both circulating and urinary tnf-a levels are increased in inflammatory chronic renal diseases. tnf-a appeared to play a crucial role in the immunopathogenesis of nephritis by the induction of chemokine, icam-1 and vcam-1 expression via the activation of the intracellular mapk signaling pathway, which may contribute to macrophage and lymphocyte infiltration li et al. 2008) . snps in selectin genes and iga nephropathy: although intensive efforts have been made to elucidate the genetic basis of ig a nephropathy (igan), genetic factors associated with the pathogenesis of this disease are not well understood. a case-control study, based on linkage disequilibrium among snps in selectin gene cluster on chromosome 1q24-25 revealed two snps in the e-selectin gene (sele8 and sele13) and six snps in the l-selectin gene (sell1, sell4, sell5, sell6, sell10, and sell11), that were significantly associated with igan in japanese patients. sele8 and sell10 caused amino acid substitutions from his to tyr and from pro to ser for his-to-tyr substitutions; and sell1 could affect promoter activity of the l-selectin gene. the tgt haplotype at these three loci was associated significantly with igan. these snps in selectin genes may be useful for screening populations susceptible to the igan phenotype. (takei et al. 2002) transplant rejection: soluble adhesion molecules are not valuable markers for stable kidney graft (stx) rejection reaction. however, patients with chronic renal failure showed increased levels of adhesion molecules, which could reflect an impaired elimination (alcalde et al. 1995) . the expression levels of icam-1 and vcam-1 show positive correlation with the severity of graft rejection and can provide evidence for early diagnosis and prevention of cr. chronic allograft failure (caf) is the major cause for late graft loss in renal transplantation. icam-1 polymorphisms may represent a predetermined genetic risk factor for caf. this was substantiated by the polymorphism in exon 4 at the mac-1 binding site and in exon 6 at fifth ig-like domain (mclaren et al. 1999) . khazen et al. (2007) found no evidence for an association of any polymorphism with acute rejection in e-and l-selectin. during kidney reperfusion, eselectin, icam-1, and vcam-1 concentrations correlated positively with hypoxanthine concentrations during reperfusion, whereas concentrations of icam-1 correlated negatively with xanthine concentrations, indicating metabolic changes in renal tissue (domanski et al. 2009 ). serum cam levels have been analyzed in many organ diseases, including diseases of nervous system, endocrine disorders and others. immune dysfunction has been proposed as a mechanism for pathophysiology of autisticspectrum disorders. levels of sp-selectin and sl-selectin were significantly lower in patients than in controls. furthermore, sp-selectin levels were negatively correlated with impaired social development during early childhood (iwata et al. 2008) . in multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (eae), inflammatory cells migrate across the endothelial blood-brain barrier (bbb) and gain access to the cns. the role of e-and p-selectin in this process has been controversial. d€ oring et al. (2007) suggest that absence of e-and pselectin did neither influence the activation of myelinspecific t cells nor the composition of the cellular infiltrates in the cns during eae. thus, e-and p-selectin are not required for leukocyte recruitment across bbb and the development of eae in c57bl/6 and in sjl mice (d€ oring et al. 2007) . no significant differences in allelic or genotypic frequency in all the snps (rs6133, rs4987310 and rs5368 substitutions) tested were found in the italian population (fenoglio et al. 2009 ). the pathophysiology of cluster headache (ch) is supposed to involve the lower posterior part of the hypothalamus, the trigeminal nerve, autonomic nerves and vessels in the orbital/retro-orbital region. remahl et al. (2008) compared serum levels of sicam-1, svcam-1 and se-selectin in patients with episodic ch and in patients with biopsypositive giant cell arteritis (gca), a vasculitic disorder of large and medium-sized arteries. within the ch group, sicam-1, svcam-1 and se-selectin showed an increasing trend in remission compared with active cluster headache period, but se-selectin only was significant. remahl et al. (2008) suggest that cluster headache is not a vasculitic disorder of medium-sized arteries, but ch patients may have an immune response that reacts differently from that of healthy volunteers. adhesion molecules have a role in many vasculitic disorders. compared to controls, takayasu's arteritis (ta) patients had elevated levels of se-selectin, svcam-1, and sicam-1. compared to controls, patients with inactive ta also had elevated levels of se-selectin, svcam-1, and sicam-1. there was no difference between active ta and controls. the se-selectin had a trend towards increased levels in inactive versus active ta, but there was no difference in svcam-1 and sicam-1 levels between the groups. patients with inactive ta had elevated levels of se-selectin, svcam-1, and sicam-1 that might indicate persistent vasculopathy in clinically inactive disease (tripathy et al. 2008 ). serum levels of svcam-1, sicam-1, stm, p-selectin, eselectin and crp levels as inflammation markers are increased in patients of b-thalassemia intermedia and not influenced by treatment (kanavaki et al. 2009 ). pseudoxanthoma elasticum (pxe) is a hereditary disorder predominantly affecting the skin, retina and vascular system. p-selectin concentrations were increased in male and female pxe patients and levels correlated with the abcc6 gene status of the patients. patients harboring two mutant abcc6 alleles had 1.5-fold increased p-selectin concentrations in comparison to patients with at least one wild-type allele. eand l-selectin levels were within normal range and the allelic frequencies did not differ between from controls. elevated p-selectin levels in pxe patients are potentially due to oxidative stress and elevated protease activity in pxe (g€ otting et al. 2008) . fabry disease, an x-linked systemic vasculopathy, is caused by a deficiency of a-galactosidase a resulting in globotriaosylceramide (gb 3 ) storage in cells. accumulation of gb 3 in the vascular endothelium of fabry disease is associated with increased production of reactive oxygen species (ros) and increased expression of cams. increased gb 3 induces expression of icam-1, vcam-1, and e-selectin. reduction of endogenous gb 3 by treatment of the cells with an inhibitor of glycosphingolipid synthase or a-galactosidase a led to decreased expression of adhesion molecules. this study indicates that excess intracellular gb 3 induces oxidative stress and up-regulates the expression of cams in vascular endothelial cells (shen et al. 2008 ). recent reports have expanded the concept that inflammation is a critical component of tumor progression. many cancers arise from sites of infection, chronic irritation and inflammation. it is now becoming clear that the tumor microenvironment, which is largely orchestrated by inflammatory cells, is an indispensable participant in the neoplastic process, fostering proliferation, survival and migration. in addition, tumor cells have co-opted some of the signaling molecules of the innate immune system, such as selectins, chemokines and their receptors for invasion, migration and metastasis. these insights are fostering new anti-inflammatory therapeutic approaches to cancer development the complexity of the tumor microenvironment has been revealed in the past decade. the cams in the process of inflammation are responsible for recruiting leukocytes onto the vascular endothelium before extravasation to the injured tissues. some circulating cancer cells have been shown to extravasate to a secondary site using a process similar to inflammatory cells. the most studied ligands for cams expressed on cancer cells, s-lewis a and s-lewis x antigens, are shown to be involved in adhesion to endothelial cells by binding to e-selectin. this process, shared by inflammatory cells and cancer cells, may partially explain the link between inflammation and tumorigenesis. the adhesion of colon cancer cells to e-selectin can be directly affected by changes in the expression level of sialosyl le a antigen. the specific lack of expression of sialosyl le a carbohydrate structure on the surface of colon cancer cells completely abolished their adhesion to e-selectin. it is proposed that glycoproteins as well as gangliosides carrying sialosyl le a structures, when properly exposed and present in high density on surface of cancer cells, can effectively support the adhesion of cancer cells to e-selectin (klopocki et al. 1998; kobayashi et al. 2007 ). in addition to endogenous ligands for l-, p-, and e-selectins (chap. 26, 27, and 28), several proteins are found in cancer cell lines or solid tumors that act as ligands for e, l, and p selectins. selectin ligands present in cancers are: (1) glycodelin a (gda) is primarily produced in endometrial and decidual tissue and secreted to amniotic fluid. gda is expressed in ovarian cancer where it can act as an inhibitor of lymphocyte activation and/or adhesion (jeschke et al. 2009 ); (2) the cysteine-rich fibroblast growth factor receptor (fgf-r) represents the main e-selectin ligand (esl-1) on granulocytes. hepatic stellate cells (hsc) are pericytes of liver sinusoidal endothelial cells, which are involved in the repair of liver tissue injury and angiogenesis of liver metastases. hsc express fgf-r together with fuct7 and exhibit a functional e-selectin binding activity on their cell surface (antoine et al. 2009 ). (3) although bcell precursor acute lymphoblastic leukemia (bcp-all) cell lines do not express the ligand psgl-1, a major proportion of carbohydrate selectin ligand was carried by another sialomucin, cd43, in nall-1 cells. cd43 plays an important role in extravascular infiltration of nall-1 cells and the degree of tissue engraftment of bcp-all cells may be controlled by manipulating cd43 expression (nonomura et al. 2008 ). (4) thomas et al. (2009a) identified podocalyxin-like protein (pclp) as an alternative selectin ligand. pclp on ls174t colon carcinoma cells possesses e-/l-, but not p-, selectin binding activity. pclp functions as an alternative acceptor for selectin-binding glycans. the finding that pclp is an e-/l-selectin ligand on carcinoma cells offers a unifying perspective on the apparent enhanced metastatic potential associated with tumor cell pclp overexpression and the role of selectins in metastasis (thomas et al. 2009b ). (5) e-selectin has been shown to play a pivotal role in mediating cell-cell interactions between breast cancer cells and endothelial monolayers during tumor cell metastasis. the counterreceptor for e-selectin was found as cd44v4. however, cd44 variant (cd44v) isoforms was functional p-, but not e-/l-selectin ligands on colon carcinoma cells. furthermore, a~180-kda sialofucosylated glycoprotein(s) mediated selectin binding in cd44-knockdown cells. this glycoprotein was identified as carcinoembryonic antigen (cea). cea serves as an auxiliary l-selectin ligand, which stabilizes l-selectin-dependent cell rolling against fluid shear (thomas et al. 2009b ). zen et al. (2008) identified a~170 kda human cd44 variant 4 (cd44v4) as e-selectin ligand, which has a high affinity for e-selectin via sle x moieties. angiogenesis plays an important role in a variety of pathophysiologic processes, including tumor growth and rheumatoid arthritis. studies on capillary morphogenesis and angiogenesis in vitro have suggested a role for e-selectin in the process of differentiation into tube-like structures. soluble e-selectin is a potent mediator of human dermal microvascular endothelial cell (hmvec) chemotaxis, which is predominantly mediated through the src and the phosphatidylinositiol 3-kinase (pi3k) pathways (kumar et al. 2003) . gastrin-17 (g17) has marked proangiogenic effects in vivo on experimental gliomas and in vitro on huvecs and transiently decreased the expression of e-selectin, but not p-selectin, whereas il-8 increased the expression of e-selectin. specific antisense oligonucleotides against e-and p-selectin decreased huvec tubulogenesis processes in vitro. this showed that gastrin has marked proangiogenic effects in vivo on experimental gliomas and in vitro on huvecs. this effect depends in part on the level of e-selectin activation, but not on il-8 expression/release by huvecs (lefranc et al. 2004 ). adhesion molecules are thought to have a role in the host defense against carcinogenesis. significantly increased p-selectin, s-vcam-i and s-icam-i levels were observed in patients with bladder cancer, and s-vcam-i levels correlated with tumor stage (coskun et al. 2006) . selectins mediate attachment of leukocytes to activated endothelium as well as the adhesion reaction of tumor cells during malignancy (borsig 2007) . in a breast tumor xenograft model, the effect of combined tnf-a and ifn-g therapy involved the selective destruction of the tumor vasculature and death of tumor cells. concomitant with these changes rt-pcr analysis revealed the increase of stromal mrna levels for a series of stromal cytokines, cytokine receptors including tnf-a, sicam-1, vcam-1, p-selectin, which could be implicated in the observed events (de kossodo et al. 1995) . squamous cell carcinomas: in order to evaluate the risk of postoperative haematogenic recurrence of esophageal squamous cell carcinoma (scc) patients, shimada et al. (2003) examined the preoperative serum levels of seselectin and pathological status of the patients. the patients with a high serum soluble e-selectin concomitant with expression of s-lewis antigens had a significant risk of postoperative haematogenic recurrence. sccs of suninduced skin cancers are particularly numerous in patients on t cell immunosuppression. blood vessels in sccs did not express e-selectin, and tumors contained few cutaneous lymphocyte antigen (cla + ) t cells, the cell type thought to provide cutaneous immunosur-veillance. clark et al. (2008) found that sccs evade the immune response at least in part by down-regulating e-selectin and recruiting t reg cells. cutaneous t-cell lymphoma (ctcl): the ctcl is characterized by accumulation of malignant cd4 + t cells in the skin. in malignant t cells from sezary syndrome (ss), a leukemic variant of ctcl, in dermal microvessels in mouse skin, hoeller et al. (2009) found that ss cells rolled along dermal venules in a p-selectin-and e-selectin-dependent manner at ratios similar to cd4 + memory t cells from normal donors. chemokine ccl17/tarc was sufficient to induce the arrest of ss cells in the microvasculature. together, experiments suggested molecular adhesion cascade operant in ss cell homing to the skin in vivo. patients with ctcl showed increased levels of sicam-1 and sicam-3 when compared with healthy individuals and patients with inflammatory dermatosis. the se-selectin and svcam-1 levels were not affected (lópez-lerma and estrach 2009). hodgkin's disease: increased sicam-1 and se-selectin have been observed in hodgkin's disease/lymphoma (hd/ hl) patients at diagnosis and svcam-1 at diagnosis correlated with both sicam-1 and se-selectin levels. chemotherapy resulted in a significant decrease of sicam-1 and se-selectin (syrigos et al. 2004 ). serum sicam-1 level increases at advanced stages of untreated multiple myeloma (mm) patients, but did not differ significantly from controls. a positive correlation of il-6 appeared with sicam-1 and se-selectin (uchihara et al. 2006) . epstein-barr virus (ebv)-positive nk/t cells showed affinity to vascular components. ebv-positive nk lymphoma cells express icam-1 and vcam-1 at much higher levels than those in ebv-negative t cell lines. furthermore, nk lymphoma cell lines exhibited increased adhesion to cultured endothelial cells stimulated with tnf-a or il-1b. the up-regulated expression of vcam-1 on cytokine-stimulated endothelial cells can be important to initiate the vascular lesions (kanno et al. 2008) . non-small cell lung cancer: serum levels of icam-1 increased in advanced stage non-small cell lung cancer (nsclc) patients, whereas se-selectin levels were not significantly different from healthy controls reports suggest that higher serum icam-1 can be useful for diagnosis while e-selectin levels have prognostic significance and could be a potential prognostic factor in nsclc patients (dowlati et al. 2008; guney et al. 2008 ). the cyfra 21-1 and se-selectin showed good performance in detecting lung cancer from normal groups. however, cyfra 21-1 was superior to se-selectin in discriminating lung cancer from benign lung diseases (swellam et al. 2008 ). maspin, a serine protease inhibitor belonging to serpin family, is known as a tumor-suppressor protein and also exhibits an inhibitor effect on angiogenesis. positive correlations were found for maspin positivity and lymph node metastases; eselectin positivity and lymph node metastases, and p-selectin positivity and lymph node metastases and lymphovascular invasion. correlations do exist between maspin, e-and p-selectin expressions with each other and with tumor stage. inactive cytoplasmic maspin cannot act as a tumor suppressor. expression of e-and p-selectins in tumor cells facilitates the occurrence of metastases, lymphovascular invasion, and perithyroidal soft tissue invasion. further studies are needed to reveal detailed interactions between maspin, e-selectin, and p-selectin expression (bal et al. 2008) . primary hyperparathyroidism: patients with primary hyperparathyroidism (phpt) have impaired vasodilation. based on small number of patients, a study suggested that classic cardiovascular risk factors seem to be the main determinants for the high plasma levels of se-selectin and vwf in phpt. together with unaltered thrombomodulin and se-selectin levels, the vwf decrease in plasma after parathyroidectomy reflects a specific mechanism of its endothelial calcium-and/or pth-stimulated secretion in some phpt patients without risk factors (fallo et al. 2006 ). colorectal cancer: plasma level of sp-selectin, seselectin and icam-1 were significantly higher in colorectal cancer (crc) patients. the highest levels of se-selectin and icam-1 were observed in patients with liver metastasis. there was no correlation between sp-selectin and seselectin, but a significant correlation was seen between seselectin and icam-1 in all patients. plasma concentration of e-selectin and icam-1 may indicate tumor progression and liver metastasis (dymicka-piekarska and kemona 2009; sato et al. 2010) . the interaction between re-selectin and crc cells alters the gene expression profile of cancer cells. a dna microarry analysis indicated that e-selectin-mediated alterations were significantly more pronounced in metastatic crc variants sw620 and km12sm than in the corresponding non-metastatic local sw480 and km12c variants. the number of genes altered by e-selectin in metastatic variants was 10-fold higher than the number of genes altered in the corresponding local variants. analysis indicated that e-selectin down regulated (at least by 1.6folds) the expression of seven genes in a similar fashion, in both metastatic cells. confocal microscopy indicated that eselectin down-regulated the cellular expression of hmgb1 protein and enhanced the release of hmgb1 into the culture medium. the released hmgb1 in turn, activated endothelial cells to express e-selectin (aychek et al. 2008) . the entrapment of malignant cells within the hepatic sinusoids and their interactions with resident nonparenchymal cells are considered very important for the whole metastatic sequence. in the sinusoids, cell connection and signaling is mediated by multiple cell adhesion molecules, such as the selectins. the three members of the selectin family, e-, p-and l-selectin, in conjunction with sialylated lewis ligands and cd44 variants, regulate colorectal cell communication and adhesion with platelets, leucocytes, sinusoidal endothelial cells and stellate cells. therefore, trials have already commenced aiming to exploit selectins and their ligands in the treatment of benign and malignant diseases. multiple pharmacological agents have been developed that are being tested for potential therapeutic applications (schnaar et al. 2008; paschos et al. 2010; zigler et al. 2010 ). the degree of selectin ligand expression by cancer cells is well correlated with metastasis and poor prognosis for cancer patients. initial adhesion events of cancer cells facilitated by selectins result in activation of integrins, release of chemokines and are possibly associated with the formation of permissive metastatic microenvironment. while eselectin is one of the initiating adhesion events during metastasis, it is becoming apparent that p-selectin and lselectin-mediated interactions significantly contribute to this process as well (gout et al. 2008; l€ aubli and borsig 2010 ). extravasation of cancer cells is a pivotal step in the formation of hematogenous metastasis. extravasation is initiated by the loose adhesion of cancer cells to endothelial cells via an interaction between endothelial selectins and selectin ligands expressed by the tumor cells. metastatic spreading is a dreadful complication of neoplastic diseases that is responsible for most deaths due to cancer. it consists in the formation of secondary neoplasms from cancer cells that have detached from the primary site. leukocytes and tumor cells use e-selectin binding ligands to attach to activated endothelial cells expressing e-selectin during inflammation or metastasis. the formation of these secondary sites is not random and several clinical observations indicate that the metastatic colonization exhibits organ selectivity. this organ tropism relies mostly on the complementary adhesive interactions between cancer cells and their microenvironment. e-selectin and slewis antigens might play important role in breast tumor, lymph node and liver metastasis. high levels of se-selectin have been reported in melanoma and some epithelial tumors, especially in colorectal carcinoma. but se-selectin may not be used as a predictive marker of metastasis in colorectal carcinoma, though high levels of se-selectin may support diagnosis of liver metastasis (uner et al. 2004; eichbaum et al. 2004) . it appeared that serum levels of se-selectin are associated with the clinical course of liver metastases from breast cancer. eichbaum et al. (2004) observed a possible trend for certain unfavorable prognostic parameters (e.g., young women, low-graded tumors, human epidermal growth factor receptor 2 over-expression) that could be related to higher serum levels of se-selectin. role of e-selectin in diapedesis of cancer cells: diapedesis is a vital part of tumor metastasis, whereby tumor cells attach to and cross the endothelium to enter the circulation. e-selectin was found to regulate initial attachment and rolling of colon cancer cells and also the subsequent diapedesis through the endothelium. evidence indicates that eselectin-dependent paracellular extravasation is independent of icam and vcam and that it requires the activation of extracellular signal-regulated kinase (erk) mitogenactivated protein kinase downstream of e-selectin. studies establish the role of e-selectin in diapedesis of circulating cancer cells woodward 2008) . polymorphisms within e-selectin gene, especially the s 128 r polymorphism, may increase the risk of metastases by facilitating adhesion of tumor cells to endothelium. blood dna from patients treated for stage ii or iii colorectal cancer (crc) and from healthy controls was assessed for three polymorphisms within e-selectin gene (s 128 r, g 98 t and l 554 f) and one within the p-selectin gene (v 640 l). the s 128 r polymorphism was detected in 22.3 % patients and was correlated with g 98 t polymorphism. in multivariate analysis, the s 128 r polymorphism was associated with shorter event-free survival (efs) and overall survival (os) in whole population, in patients with stage ii crc, and in patients with stage iii crc. l 554 f and v 640 l polymorphisms had no prognostic value. the s 128 r polymorphism is a constitutional factor associated with a higher risk of relapse and death in patients treated for crc and its detection may permit better selection of patients suitable for adjuvant therapy, especially among those with stage ii disease (hebbar et al. 2009 ). metastasis is thought to involve the formation of tumor-platelet-leukocyte emboli and their interactions with the endothelium of distant organs. a link between these observations shows that p-selectin, which normally binds leukocyte ligands, can promote tumor growth and facilitate the metastatic seeding of a mucinproducing carcinoma. p-selectin-deficient (p-sele à/à ) mice showed three potential pathophysiological mechanisms: (1) intravenously injected tumor cells home to the lungs of psele à/à mice at a lower rate; (2) p-sele à/à mouse platelets fail to adhere to tumor cell-surface mucins; and (3) tumor cells lodged in lung vasculature after intravenous injection often are decorated with platelet clumps, and these are markedly diminished in p-selectin à/à animals (kim et al. 1998 ). however, the surgical procedure did not totally eliminate the factors responsible for platelet activation and did not normalize platelet activation (dymicka-piekarska et al. 2005; hanley et al. 2006 ). role of sialyl-lewis antigens: during inflammation, eand p-selectins appear on activated endothelial cells to interact with leukocytes through sialyl-lewis x (sle x ) and sialyl-lewis a (sle a ). these selectins can also interact with tumor cells in a sialyl-lewis-dependent manner and hence, they are thought to play a key role in metastasis. diverting the biosynthesis of sialyl-lewis antigens toward nonadhesive structures is an attractive gene therapy for preventing the hematogenous metastatic spread of cancers. the transduced a1,2-fucosyltransferase-1 (fut1) efficiently fucosylated the p-selectin ligand psgl-1 without altering p-selectin binding . the metastasis of cancer cells and leukocyte extravasation into inflamed tissues share common features. carbohydrate antigen sle a (ca19-9) is the most frequently applied serum tumor marker for diagnosis of cancers in the digestive organs. the normal counterpart of the determinant, namely disialyl-le a is predominantly expressed in non-malignant epithelial cells of the digestive organs. the disialyl-le a determinant carries one extra sialic residue attached through a 2 ! 6 linkage to glcnac moiety compared to cancerassociated sle a , which carries only one 2 ! 3 linked sialic acid residue (monosialyl lewis a) (fig. 44.3) . disialyl-le a in normal epithelial cells serves as a ligand for immunosuppressive receptors such as sialic acid binding ig-like lectins (siglec-7 and -9) expressed on resident monocytes/ macrophages and maintains immunological homeostasis of mucosal membranes in digestive organs. epigenetic silencing of a gene for a 2 ! 6 sialyl-transferase in the early stages of carcinogenesis results in impairment of 2 ! 6 sialylation, leading to incomplete synthesis and accumulation of sle a , which lacks the 2 ! 6 linked sialic acid residue, in cancer cells. simultaneous determination of serum levels of sle a and disialyl-le a , and calculation of the sle a / disialyl-le a ratio provides information useful for excluding a false-positive serum diagnosis. during cancer progression in locally advanced cancers, tumor hypoxia induces transcription of several glyco genes involved in sle a synthesis. expression of the determinant, consequently, is further accelerated in more malignant hypoxia-resistant cancer cell clones, which become predominant clones in advanced stage cancers and frequently develop hematogenous metastasis. sle a , as well as its positional isomer sle a , serves as a ligand for vascular e-selectin and facilitates hematogenous metastasis through mediating adhesion of circulating cancer cells to vascular endothelium. patients having both strong sle a expression on cancer cells and enhanced e-selectin expression on vascular beds are at a greater risk of developing distant hematogenous metastasis (kannagi 2007) . in a human-mouse model, the selectin ligand s-le a is involved in in vivo extravasation of colorectal carcinoma (crc) cells. highly metastatic crc cells expressing high levels of s-le a extravasate more efficiently than non-metastatic crc cells expressing low levels of s-le a . down-regulating the expression of s-le a in crc cells by genetic manipulations, significantly reduced crc extravasation. the arrest and adhesion of crc cells, and possibly of other types of cancer cells as well, to endothelium depend on the expression of the selectin ligand sle a by the tumor cells (ben-david et al. 2008) . 3'-sulfo-le a is known to be the potent ligand of e-selectin which is important in cell adhesion and migration. the serum 3'-sulfo-le a can provide important information in patients with primary gastric cancer, which might be useful as a predictive marker especially for the detection of tumor metastasis (zheng et al. 2009 ). specialized carbohydrates modified with sle x antigens on leukocyte membranes are ligands for selectin adhesion molecules on activated vascular endothelial cells at inflammatory sites. the sle x expression of invasive micropapillary carcinoma was higher than that of invasive ductal carcinoma, which was also associated with lymph node metastasis. e-selectin combined with sle x might play an important role in lymph node metastasis in invasive micropapillary carcinoma. the expression pattern of sle x in invasive micropapillary carcinoma suggested that the reversal of cell polarity of invasive micropapillary carcinoma might be as an important factor for the morphogenesis and possibly the pathogenesis, especially their higher rates of lymph node metastasis (wei et al. 2010) . the activity of core 2 b1,6 n-acetylglucosaminyltransferase (c2gnt1) in leukocytes greatly increases their ability to bind to endothelial selectins. c2gnt1 is essential for the synthesis of core 2-branched o-linked carbohydrates terminated with sle x (c2-o-sle x ). e-selectin and its ligand-sle x are closely correlated with the metastasis of hepatocellular carcinoma. c2-o-sle x is a potentially useful early predictor of metastasis (zhang et al. 2002) . the expression profiles of c2-o-sle x in the malignant progression and metastasis of colorectal adenocarcinomas is upregulated in colorectal adenocarcinomas and metastatic liver tumors (st hill et al. 2009 ). endothelial p-selectin as a target of heparin action: metastasis can be effectively inhibited by the anticoagulant heparin in different tumor models. at the cellular level, many of the antimetastatic effects of heparin in vivo are due to its action on p-selectin-mediated binding. ludwig et al. (2007) addressed the potential contribution of endothelial p-selectin expression to adhesive events between the microvasculature and melanoma cells in vivo. heparin not only inhibits p-selectin-mediated melanoma cell rolling but also attenuates melanoma metastasis formation in vivo, supporting the concept that endothelial p-selectin expression may represent an additional target of heparin in experimental melanoma lung metastasis (ludwig et al. 2007) . the low molecular weight heparin (lmwh) significantly improved colonic inflammation in rats with trinitrobenzene sulphonic acid (tnbs) induced colitis. the effect is possibly related to inhibition of proinflammatory cytokine il-8, but not involved platelet surface p-selectin expression (xia et al. 2004 ). the survival benefits in patients with cancer treated with lmwh may result from a lmwhmediated effect on the immune system or on the cross-talk between platelets and tumor cells. however, survival observed with lmwh in patients with cancer apparently cannot be explained by a lmwh effect on these circulating markers (di nisio et al. 2005) . nonetheless, in vivo antimetastatic effects of heparins reflect their action on pselectin-mediated binding. therefore, these commonly used anticoagulants widely differ in their potential to interfere with p-selectin mediated cell binding. importantly, the superior inhibitory capacity on p-selectin function of unfractionated heparin and lmwh nadroparin as opposed to lmwh enoxaparin and synthetic heparin pentasaccharide fondaparinux strongly correlated to the inhibitory potency of each in inhibiting experimental lung metastasis in vivo. hence, p-selectin inhibition constitutes a valuable feature to identify anticoagulants that are suitable for anticancer therapy (ludwig et al. 2006) . stevenson et al. (2005) studied metastasis inhibition by clinically relevant levels of various heparins and investigated the structural basis for selectin inhibition differences. five clinically approved heparins were evaluated for inhibition of p-selectin and l-selectin binding to carcinoma cells and showed differing abilities to inhibit selectins, likely explained by size distribution. it should be possible to size fractionate heparins and inhibit selectins at concentrations that do not have a large effect on coagulation. gao et al. (2005) prepared periodate-oxidized, borohydride-reduced heparin (ro-heparin) and tested its anticoagulant and anti-inflammatory activities. compared with heparin, ro-heparin had greatly reduced anticoagulant activity. intravenous administration of this compound led to reduction in the peritoneal infiltration of neutrophils in a fig. 44.3 structures of three carbohydrate determinants, disialyl lewis x (le x ), sialyl lewis a and lewis a (le a ). in panel, note that the only difference between the three determinants is the linkage of sialic acid residues. the a(2 ! 6) sialic acid residue in disialyl le a is synthesized by a sialyltransferase st6galnacvi, which shows a significant decrease in its mrna level upon malignant transformation mouse acute inflammation model. in vitro studies showed that the effect of ro-heparin on inflammatory responses was mainly due to inhibiting the interaction of p-selectin with its ligands. these results indicate that ro-heparin may be a safer treatment for inflammation than heparin, especially when selectin is targeted. to clarify the mechanism of heparin antimetastatic activity, several biological effects are being investigated. cancer progression and metastasis are associated with enhanced expression of heparanase, which is inhibited efficiently by heparin. heparin is also a potent inhibitor of selectinmediated interactions. p-and l-selectin were shown to contribute to the early stages of metastasis, which is associated with platelet-tumor cell thrombi formation. low anticoagulant heparin preparations still inhibited metastasis efficiently indicating that anticoagulation is not a necessary component for heparin attenuation of metastasis. modified heparins characterized for heparanase inhibitory activity are also potential inhibitors of selectins. selectin inhibition is a clear component of heparin inhibition of metastasis. the contribution of selectin or heparanase inhibition by heparin can provide evidence about its antimetastatic activity (borsig 2007) . one of the mechanisms by which heparin inhibits metastasis is by blocking the p-selectin-based interaction of platelets with tumor cell. the sulfate groups at c6/ n and especially c6, but not c2 and c3, of heparin play a critical role in p-selectin recognition and that 2-o,3-odesulfated heparin can block p-selectin-mediated a375 human melanoma cell adhesion. thus chemical modification of heparin, especially 2-o,3-o-desulfation, may result in a therapeutic agent that is anti-metastatic because it blocks unwanted p-selectin-dependent adhesion but that lacks dose-limiting anticoagulant effects . heparin-induced thrombocytopenia: the pathophysiology of heparin-induced thrombocytopenia (hit) is a complex process which involves platelets, vascular endothelium, and leukocytes. the activation products from these sites also contribute to the activation of coagulation and to the fibrinolytic deficit. many of the markers of hemostatic activation processes have been found to be at increased levels during acute phases of the hit syndromes. since the pathophysiology of hit involves the activation of platelets, endothelium, and leukocytes, it is expected that activation products related to these hemostatic systems, including soluble selectins, will also be increased in circulating blood. these alterations may provide an index of the pathophysiologic process. fareed et al. (1999) reviewed on the circulating levels of p-, e-, and l-selectins in hit patients and their modulation after therapeutic intervention. with the availability of recombinant hirudin, it is now possible to provide alternate anticoagulants to hit patients. however, fareed et al. (1999) suggest that the immunoactivation of platelets and other cells may require additional adjunct therapeutic approaches. activated protein c (apc) is the major physiological anticoagulant with concomitant anti-inflammatory properties. turunen et al. (2005) suggest that apc has an antiinflammatory role in i/r injury in clinical renal transplantation (turunen et al. 2005) . bimosiamose prolongs survival of kidney allografts. binding of the p-, l-, and e-selectins to sle x retards circulating leukocytes, thereby facilitating their attachment to the blood vessels of allografts. selectin inhibitor bimosiamose (bimo) inhibits the rejection process of kidney allografts in a rat model in association with reduced intragraft expression of p-selectin glycoprotein ligand-1, cx (3)cl1, ccl19, ccl20, and ccl2. thus, bimo blocks allograft rejection by reduction of intragraft expression of cytokines and chemokines (langer et al. 2004 ). brain death (bd), a significant antigen-independent process, the donor-related injury up-regulates variety of inflammatory mediators in peripheral organs. one of the immediate responses is the expression of selectins by endothelial cells of the transplanted tissues, which in turn trigger a cascade of nonspecific events that may enhance host alloresponses. using a rat model in which donor bd accentuates subsequent renal allograft injury, gasser et al. (2005) tested the effects of therapy with rpsgl-ig alone, or in combination with sirolimus (srl) and cyclosporin a. it was found that in contrast to the effects of standard doses of srl or cyclosporine, rpsgl-ig decreased inflammation in the early posttransplant period such that lower doses of maintenance immunosuppression were sufficient to maintain long-term graft function. intestinal transplantation (itx) is severely limited by ischemia-reperfusion (i/r) injury. t lymphocyte is an important regulatory cell in this inflammatory process (farmer et al. 2005a) . rpsgl-ig treatment leads to marked improvement in the outcome. the mechanism of action seems to involve the blockade of neutrophil and lymphocyte infiltration that leads to a decreased inflammatory response possibly driven by th2 cytokines (farmer et al. 2005b) . it was suggested that liver transplantation and liver resection, together with portal clamping time, might be a potential stimulus for platelet activation. becker et al. (2004) indicated that neither liver transplantation nor liver resection influences gpiib/iiia and p-selectin expression on circulating platelets (becker et al. 2004 ). endothelial activation contributes significantly to the systemic inflammatory response to bacteraemia. release of soluble endothelial markers into the circulation has been demonstrated together with elevated plasma levels of cams and has been reported in bacteraemic patients. it has been proposed that the infection of endothelial cells with staphylococcus aureus, streptococcus sanguis, or staphylococcus epidermidis induces surface expression of icam-1 and vcam-1 and monocyte adhesion. in general, leukocyte/endothelial cell interactions such as capture, rolling, and firm adhesion should be viewed as a series of overlapping synergistic interactions among adhesion molecules resulting in an adhesion cascade. these cascades thereby direct leukocyte migration, which is essential for the generation of effective inflammatory responses and the development of rapid immune responses (golias et al. 2007) . helicobacter pylori is a common bacterial pathogen that infects world's population up to 50 %. carbohydrate components on h. pylori (sequences related to le x or le a antigens) are responsible for the persistent inflammation through interactions with leukocyte-endothelial adhesion molecules of the host. h. pylori isolates from patients with chronic gastritis, duodenal ulcer and gastric cancer interact with e-and l-selectins (galustian et al. 2003) . expression of e-selectin was specifically upregulated in h. pylori-induced gastritis but not in gastritis induced by acetylsalicylic acid or pouchitis. the upregulated e-selectin expression was localized to the gastric mucosa rather than being a systemic response to the infection (svensson et al. 2009 ). although mice with mutations in individual selectins showed no spontaneous disease and had a mild or negligible deficiencies of inflammatory responses, bullard et al. (1996) , in contrast, found that mice with null mutations in both endothelial selectins (p and e) develop a phenotype of leukocyte adhesion deficiency characterized by mucocutaneous infections in response to intraperitoneal s. pneumoniae peritonitis. these mice provide strong evidence for the functional importance of selectins in vivo (bullard et al. 1996) . anthrax lethal toxin (lt), a key virulence factor of bacillus anthracis, enhanced vcam-1 expression on primary human endothelial cells suggesting a causative link between dysregulated adhesion molecule expression and the poor immune response and vasculitis associated with anthrax. results suggest that lt can differentially modulate nf-kb target genes and highlight the importance of vcam-1 enhancement (warfel and d'agnillo 2008) . vascular endothelium stimulation in vitro that lead to the upregulation of cams is known for the pathogenic spirochaetes, including rlic10365 of leptospira interrogans. the recombinant proteins of l. interrogans in e. coli as a host were capable to promote the upregulation of icam-1 and e-selectin on monolayers of huvecs. in addition, pathogenic and non-pathogenic leptospira are both capable to stimulate endothelium e-selectin and icam-1, but the pathogenic l. interrogans serovar copenhageni strain promoted a higher activation than the non-pathogenic l. biflexa serovar patoc (atzingen et al. 2009; gómez et al. 2008) . chlamydia pneumoniae has been associated with cardiovascular disease and atherosclerosis. to determine the ability of c. pneumoniae to elicit inflammation, h€ ogdahl et al. (2008) infected human coronary artery endothelial cells (hcaec) with c. pneumoniae. secretion of il-8, mcp-1, and icam-1 was significantly increased after c. pneumoniae infection of hcaec in comparison with uninfected controls, where as release of eselectin or mmp-1did not change. this suggested that c. pneumoniae initiates and propagates vascular inflammation in ways that contribute to coronary artery disease (h€ ogdahl et al. 2008) . the sicam-1, svcam-1, and se-selectin are present in gingival crevicular fluid (gcf) and changes in their levels may be a sensitive indicator to differentiate healthy sites from those with periodontitis (hannigan et al. 2004; tamai et al. 2007) . porphyromonas gingivalis is a gram-negative bacterium that is an important etiologic agent of human adult periodontitis. e. coli lps and isoforms of p. gingivalis lps were potent in stimulating the expression of inflammatory markers, with e. coli lps being more potent . dna samples from blood of periodontitis patients genotyped for e-selectin ser 128 arg and l-selectin phe 206 leu revealed a significant difference in the ser 128 arg polymorphism of e-selectin, but not in l-selectin, between periodontal patients and controls; the 128arg allele was present more frequently in patients. houshmand et al. (2009) suggested that ser 128 arg polymorphism of e-selectin might contribute to the susceptibility of iranian individuals to periodontitis. cams in subjects with hiv disease: swingler et al. (2003) suggested that while both soluble cd23 and icam1 promote resting cell hiv1 infection, productive infection of cycling cells requires soluble icam1. swingler et al. (2003) noted that these results may explain in part the existence of a resting t-cell reservoir infected with hiv-1. subjects with hiv disease have multiple risk factors for cardiovascular disease, including elevated levels of icam-1 and vcam-1. many of the variables associated with icam-1 and vcam-1 levels can be related to their impact on inflammation (melendez et al. 2008 ). the lfa-1, icam-1, and icam-3 are enriched at virological synapse (vs). the cognate adhesion molecule interactions at vs are important for hiv-1 spread between t cells (jolly et al. 2007 ). zuccarello et al. (2002) described a distinct form of familial chronic mucocutaneous candidiasis characterized by earlyonset infections by different species of candida, restricted to the nails of the hands and feet and associated with low serum concentration of icam-1. phan and filler (2009) measured the effects of c. albicans on the endothelial cell production of e-selectin and tnf-a in vitro. during invasive pulmonary aspergillosis, a. fumigatus hyphae invade the abluminal endothelial cell surface, whereas they invade the luminal endothelial cell surface during haematogenous dissemination. infection with hyphae stimulates endothelial cells to synthesize e-selectin, vcam-1, il-8, and tnf-a in vitro. in neutropenic mice infected with wild-type a. fumigatus, increased pulmonary expression of e-selectin and tnf-a occurred only when neutropenia had resolved. in nonneutropenic mice immunosuppressed with corticosteroids, a. fumigatus stimulated earlier pulmonary expression of eselectin and vcam-1, while expression of icam-1 and tnf-a was suppressed. in both mouse models, expression of e-selectin was associated with high pulmonary fungal burden, angioinvasion, and neutrophil adherence to endothelial cells (chiang et al. 2008; kamai et al. 2009 ). 44.9.3.1 falciparum malaria significant differences are observed between falciparum malaria patients and the healthy people in term of levels of both se-selectin and thrombomodulin (tm). the levels of both se-selectin and tm correlated positively with temperature, levels of ifn-g and levels of tnf-a; and negatively with hemoglobin levels. trends of positive correlations were observed between sp-selectin or vwf and temperature (matondo et al. 2008) . evidence from autopsy and in vitro binding studies suggests that adhesion of erythrocytes infected with plasmodium falciparum to the human host icam-1 receptor is important in the pathogenesis of severe malaria. fernandez-reyes et al. (1997) identified a mutation (k29m) in the icam1 gene, which they designated 'icam1 kilifi,' that was associated with susceptibility to cerebral malaria with relative risks of 2.23 and 1.39 for homozygotes and heterozygotes, respectively. the available epidemiological, population genetic and functional evidence link icam-1(kilifi) to severe malaria susceptibility (fry et al. 2008; cojean et al. 2008) . increased serum concentrations of soluble sicam-1, cd54 and of soluble e-, but not soluble p-and l-selectins were detected in malagasy patients living in hyperendemic focus of schistosoma mansoni. serum levels of icam-1 were significantly correlated with the disease severity (esterre et al. 1998) . studies in several models of inflammation have underscored the importance of p-and e-selectins in the migration of t cells to inflamed tissues. cd4 + t cells recruited to the cutaneous compartment during infection with leishmania major express p-and e-selectin ligands. results suggest that by blocking p-and e-selectins, the immune pathology associated with cutaneous leishmaniasis might be ameliorated without compromising immunity to infection (zaph and scott 2003) . invasive amebiasis offers a new model that poses an inadequate immune response leading to a continuous and prolonged activation of endothelial cells (ecs) by amebas, amebic molecules and cytokines, leading to necrosis. hyperactivated endothelial cells continuously express icam-1 and e-selectin, pro-coagulant molecules (tissue factor, vwf, and the plasminogen activator inhibitor), resulting in ever greater inflammation and thrombosis (campos-rodríguez et al. 2009) sepsis is a multifactorial, and often fatal, disorder typically characterized by widespread inflammation and immune activation with resultant endothelial activation. though bacterial sepsis is most common, sepsis occurs with fungal, parastic and mycobacterial organisms. during bacterial sepsis in vivo, in wild-type mice and mice with e-, p-, or e-/ p-selectin deficiencies, a phenotypic abnormality in e-selectin-deficient mice suggested that e-and p-selectin are important in the host defense against s. pneumoniae infection (munoz et al. 1997) . p-selectin is an important mediator of eosinophil recruitment to the cornea from limbal vessels to the corneal stroma, suggesting that p-selectin interactions may be potential targets for immunotherapy in eosinophil-mediated ocular inflammation (kaifi et al. 2000) . staphylococcus aureus is one of the most significant pathogens in human sepsis and endocarditis. peptidoglycan induced surface expression of ec inflammation markers icam-1 and vcam-1, which supported the adhesion of monocytes to these ecs (mattsson et al. 2008) . teoh et al. (2008) assigned adiponectin as a modulator of survival and endothelial inflammation in experimental sepsis and a potential mechanistic link between adiposity and increased sepsis. newborn infants with clinical diagnosis of sepsis demonstrated significantly higher plasma se-selectin levels in infected infants. infants with gram-negative sepsis had higher se-selectin levels than did those with gram-positive sepsis. c-reactive protein was the best test for diagnosis of neonatal sepsis (zaki and el-sayed 2009). hofer et al. (2008) compared two different models of sepsis lps-induced endotoxemia and cecal ligation perforation (clp) bacteremia in rats with respect to changes in endothelial expression of cams as a marker for capillary breakdown of the blood brain barrier. increased icam-1 expression might be an early factor involved in these pathogenic events. although the role of pecam-1 could not be determined, it was possible to show its expression on cerebral endothelium in all groups (hofer et al. 2008) . in mouse models of sepsis, shapiro et al. (2009) demonstrated increased circulating levels of se-selectin, sicam-1, svcam-1 and sp-selectin at 24 h, while clp was associated with increased levels of se-selectin alone. in real-time pcr, mrna levels for p-selectin, icam-1 and pai-1 were increased in skin from endotoxemic mice. in clp, mrna levels for p-selectin, icam-1, e-selectin and pai-1 were elevated, while vcam-1 expression was reduced in skin. most, but not all of these changes correlated with alterations in immunohistochemical staining (shapiro et al. 2009 ). the field of selectin inhibition has matured significantly in recent years in the ability to inhibit selectin/ligand interactions with drug-like molecules and to demonstrate disease modification in human trials. a comprehensive review of new developments in the field of selectin inhibition through discussion of patents/patent applications from 2003 to august 2009 has been reported by bedard and kaila (2010) treatment of human endothelial cells with cytokines such as il-1, tnf-a or ifn-g induces the expression of specific leukocyte adhesion molecules on the endothelial cell surface. interfering with either leukocyte adhesion or upregulation of adhesion protein is an important therapeutic target as evidenced by the potent anti-inflammatory actions of neutralizing antibodies to these ligands in various animal models and in patients. the induction of e-selectin, vcam-1, and icam-1 genes requires the transcription factor nf-kb. pharmaceutical agents, which prevent the induced expression of one or more of cell adhesion molecules on endothelium, might be expected to provide a novel mechanism to attenuate the inflammatory responses associated with chronic inflammatory diseases. e-selectin expression is induced on the endothelial cell surface of vessels in response to inflammatory stimuli but is absent in the normal vessels. thus, e-selectin is an attractive molecular target, and high affinity ligands for e-selectin could be powerful tools for the delivery of therapeutics and/or imaging agents to inflamed vessels. zimmerman and blanco (2008) reviewed the structure and regulation of lfa-1 and different classes of inhibitors that interfere lfa-1/icam-1 interactions. alicaforsen (isis 2302), an antisense to icam-1, designed to inhibit icam-1 expression did not reveal significant effect in crohn's disease. however, topical enemas for ulcerative colitis demonstrated some effect in secondary outcomes, and initial studies in pouchitis are promising (philpott and miner 2008) . icam-1 antibody (uv3) was highly effective at slowing the growth of tumors and/or prolonging survival in scid mice xenografted with human multiple myeloma, lymphoma, melanoma and other cell lines (brooks et al. 2008) . a structurally diverse collection of small molecule inhibitors has been characterized and developed either to bind the idas site of a l i-domain or to the midas of the b 2 i-like domain. cams play important roles in a critical step of tumor metastasis and arrest of tumor cells onto the venous or capillary bed of the target organ. in this process, il-1b induces nuclear translocation of nf-kb in huve cells, followed by induction of cell surface expression of e-selectin, icam-1, and vam-1, and subsequent adhesion of those cancer cells expressing sialyl le x antigen, which is a ligand to e-selectin. the adhesion of tumor cells to il-1b-treated huve cells can be inhibited by anti-nf-kb reagents such as n-acetyl l-cysteine, aspirin, or pentoxifylline. these observations indicate the involvement of nf-kb in cancer metastasis and the feasibility of using anti-nf-kb reagents in preventing metastasis (tozawa et al. 1995) . incubation of huvec with n,n,n-trimethylsphingosine (tms) resulted in a dose-dependent inhibition of il-1b-induced e-selectin expression. sphingosine or n,n-dimethylsphingosine had no effects on the expression. this inhibitory effect of tms on il-1b-dependent endothelial cell activation may partly explain the known anti-inflammatory or anti-metastatic effect of tms in vivo (masamune et al. 1995) . cimetidine inhibits the expression of e-selectin on vascular endothelial cells in gastric-and colorectal cancer patients, treated for chemotherapy (kawase et al. 2005) . since the expression of e-selectin and mac-1 is regulated either directly or indirectly by nf-kb, studies provide in vivo evidence that tepoxalin is a potent inhibitor of nf-kb mediated events in animal models and this novel molecular mechanism clearly defines it as a new class of anti-inflammatory compounds. e-selectin transcription requires binding of transcription factors, nf-kb, atf-2, and hmg-i(y). huve cells treated with tnf-a showed e-selectin surface expression, which peaked at 4 h and then declined. however, atf-2 binding was unchanged after stimulation with tnf-a. the termination of e-selectin expression is controlled at the level of transcription, with loss of protein-dna interactions at only one of three nf-kb-binding sites in the e-selectin promoter (boyle et al. 1999 ). e-selectin is synthesized following x-ray exposure to doses as low as 0.5 gy. x-ray-induced expression of eselectin and icam-1 has been proposed to contribute to radiation injury in normal tissues. e-selectin expression does not require cytokine synthesis, but involves nf-kb activation (hallahan et al. 1995) . nfkb inhibition using nfkb inhibitors abrogates x-ray induced inflammatory mediators (hallahan et al. 1998) . andrographolide, the principal component of medicinal plant andrographis paniculata, has been shown to inhibit nf-kb activity and may attenuate allergic asthma via inhibition of the nf-kb signaling pathway. andrographolide attenuated ovainduced lung tissue eosinophilia and airway mucus production, mrna expression of e-selectin, and inducible nos in lung tissues. findings implicate a potential therapeutic value of andrographolide in the treatment of asthma jiang et al. 2007 ). transforming growth factor (tgf-b) has been shown to decrease the adhesiveness of endothelial cells for neutrophils, lymphocytes, and tumor cells. tgf-b inhibits the basal e-selectin expression and tnf-stimulated expression. while tgf-b had no effect on the expression of vcam-1 and icam-1, the effect was additive with il-4 in inhibiting the expression of e-selectin. thus, perivascular tgf-b appears to act as an inhibitor of inflammatory responses involving neutrophils and a subset of lymphocytes (gamble et al. 1993) . ifn-g down-regulates the induction by a viral mimetic, polyinosinic-polycytidylic acid [poly-(i:c)], of e-selectin. the inhibitory effect of ifn-g on poly(i:c)induced e-selectin was specific for dsrna. results indicated the role for ifn-g in the regulation of e-selectin gene expression in response to dsrna by a transcriptional mechanism independent of nf-kb, as well as by a minor decrease in message stability (faruqi and dicorleto 1997) . retinoic acid inhibits the expression of vcam-1 but not e-selectin: several genes are regulated by tocopherols which can be categorized, based on their function. genes that are related to inflammation, cell adhesion and platelet aggregation include e-selectin, icam-1, and others (azzi et al. 2004) . retinoic acid and synthetic derivatives are known to exert anti-inflammatory effects in cutaneous diseases. pretreatment with all-trans-retinoic acid (t-ra) specifically prevented tnfa-induced vcam-1 expression, but not icam-1 and e-selectin induction (gille et al. 1997) . the tnfa-mediated activation of the human vcam-1 promoter was also inhibited after t-ra treatment, while the icam-1 promoter activation was unaffected, indicating that the selective inhibition of cam expression is regulated in part at the level of gene transcription. furthermore, the transcriptional inhibition by t-ra appears to be mediated by its effects upon the activation of nf-kb-dependent complex formation. the specific inhibition of cytokine-mediated vcam-1 gene expression in vitro provides a potential basis by which retinoids exert their biological effects at sites of inflammation in vivo (gille et al. 1997) . radiationinduced expression of e-selectin was also blocked by t-ra, whereas 9-cis retinoic acid was ineffective. application of statins and t-ra might have clinical impact in protecting against e-selectin-promoted metastasis, which might arise as an unwanted side effect from radiation treatment (holler et al. 2009; nubel et al. 2004 ). methylation of e-selectin promoter gene represses nf-kb transactivation: the e-selectin promoter in cultured endothelial cells is under-methylated in comparison with non-expressing hela cells. thus, methylation is likely to play a role in blocking e-selectin expression in nonendothelial cells (smith et al. 1993) . in intestine, muc2 is the main mucin carrying s-le x , which interacts with eselectin. this interaction may contribute to the extravasation of tumor cells and thus to the metastases. in several colorectal carcinoma cell lines the methylation of the 5'-flanking region of muc2 correlated with the suppression of the muc2 gene. the increase in muc2 expression after the inhibition of the methylation with 5-aza-2' deoxycytidine strongly supports the notion that the suppression of muc2 gene is related to the methylation of the promoter (riede et al. 1998 ). the advances in the development of adhesion molecule blocking agents, as well as an insight into the potential of these molecules in cardiovascular therapy have been reviewed from time to time (lutters et al. 2004 ). prophylactic dosing of a recombinant p-selectin ligand decreases venous thrombosis in a dose-dependent fashion in both feline and nonhuman primate animal models. additionally, treatment of 2-day iliac thrombi with a recombinant protein, p-selectin inhibitor, significantly improves vein reopening in nonhuman primates (register 2009 ). it is interesting to note that p-selectin inhibition decreases thrombosis without adverse anticoagulation. myers et al. (2005) evaluated an orally bioavailable inhibitor of p-selectin (psi-697), which decreased thrombosis. since, p-selectin is expressed on the surface of activated endothelial cells and platelets during thrombosis, targeting the plasminogen activator (pa) to pselectin would enhance local thrombolysis and reduce bleeding risk. a urokinase (upa)/anti-p-selectin antibody (husz51) fusion protein is known to increase fibrinolysis in a hamster pulmonary embolism (dong et al. 2004) aspirin reduces risks of myocardial infarction, stroke and cardiovascular death (serebruany et al. 2004 ). the impact of cyclooxygenase (cox)-2 antagonist treatment on acute coronary risk is controversial. prolonged cox-2 inhibition attenuates crp and il-6, does not modify p-selectin and mmp-9, and has no deleterious effect on endothelial function in stable patients with a history of recurrent acute coronary events and raised c-reactive protein (crp) (bogaty et al. 2004) . statins used in the control of hypercholesterolemia exert a protective effect on the endothelium reflected by a reduced level of circulating adhesion molecules. statins exert a beneficial effects on endothelial function and atherosclerotic plaque, modulating oxidative stress and inflammation, with subsequent, well documented, primary and secondary prevention of cad. following statin treatment, sp-selectin, and icam-1 and highly sensitive crp decreased compared to baseline levels. other proteins (svcam-1, se-selectin and platelet ecam-1) did not show significant changes. in contrast to crp, the reduction of spselectin concentrations correlated directly with the lowering of total cholesterol and inversely with the progression of cad (marschang et al. 2006) while diclofenac is capable of inhibiting the expression of e-selectin, icam-1 and vcam-1, the sjc13 is selective in inhibiting the expression of e-selectin and vcam-1, but not icam-1 in endothelial cells. nonsteroidal anti-inflammatory agents, such as sodium salicylate and aspirin, inhibit nf-kbdependent gene activation. salicylate blocked the tnf-ainduced increase in mrna levels of adhesion molecules and gave a dose-dependent inhibition of tnf-a-induced surface expression of vcam-1 and icam-1 with higher doses required to inhibit e-selectin expression. ibuprofen appeared a potent inhibitor of il-1a and tnf-a-induced surface expression of vcam-1 and a less potent inhibitor of icam-1. indomethacin, a nonsalicylate cyclooxygenase inhibitor, had no effect on surface expression of adhesion molecules, suggesting that the effects were not due to inhibition of cyclooxygenase (pierce et al. 1996) . methimazole, used in treating autoimmune diseases, may also diminish pathological inflammation by suppressing e-selectin expression. the phenyl methimazole can also reduce cytokineinduced e-selectin expression and consequent leukocyte adhesion. compound 10, which dramatically inhibits tnfa-induced vcam-1 mrna and protein expression in human aortic endothelial cells, has a modest inhibitory effect on tnf-a induced e-selectin expression and has no effect on icam-1 expression (dagia et al. 2004) . a thieno(2,3-d)pyrimidine, a-155918 inhibits the tnfainduced expression of e-selectin, icam-1, or vcam-1 on hevcs (stewart et al. 2001) . co-treatment of human endothelial cells with certain hydroxyflavones and flavanols blocks cytokine-induced icam-1, vcam-1, and e-selectin expression on human endothelial cells. one of the potent flavones, apigenin, exhibited a dose-and time-dependent, reversible effect on adhesion protein expression as well as inhibiting adhesion protein upregulation at the transcriptional level (gerritsen et al. 1996) . enalapril and losartan but not placebo induced a small but stable decrease of cardiovascular icam-1 and vcam-1, while e-selectin and leukocyte expression of icam-1 remained unchanged. the lowering of plasma adhesion molecules may indicate an antiatherogenic effect of angiotensin ii blockade in hypercholesterolemia (graninger et al. 2004 ). targeting interaction of selectins and appropriate carbohydrate ligand is a promising approach to treat chronic inflammation. b-1,3-glucan sulfate (ps3) has inhibitory activity toward l and p-selectins under static conditions (alban et al. 2009 ). access to synthetic carbohydrates is an urgent need for the development of carbohydrate-based drugs, vaccines, adjuvants as well as novel drug delivery systems. besides traditional synthesis in solution, synthetic carbohydrates have been generated by chemoenzymatic methods as well as automated solid-phase synthesis. synthetic oligosaccharides have proven to be useful for identifying ligands of carbohydrate-binding proteins such as c-type lectins and siglecs using glycan arrays. furthermore, glyconanoparticles and glycodendrimers have been used for specific targeting of lectins of the immune system such as selectins, dc-sign, and cd22 (lepenies et al. 2010) . compounds that target both heparanase and selectins offer a promising approach for cancer therapy. borsig et al. (2011) reported semisynthetic sulfated tri mannose c-clinked dimers (stmcs) which are endowed with heparanase and selectin inhibitory activity. stmc hexasaccharide is an effective inhibitor of p-selectin in vivo. p-selectin-specific stmc attenuated metastasis in animal models, indicating that inhibition of tumor cell interaction with the vascular endothelium is critical for cancer dissemination. the small size, the stability of the c-c bond, and the chemically defined structure of stmcs make them superior to heparin derivatives and signify stmcs as valuable candidates for further evaluation. steroids down-regulate the expression of cams in endothelial cells stimulated by lps in vitro. low-dose hydrocortisone is a new treatment of patients with septic shock, a state that is characterized by an endothelial injury. treatment with glucocorticoids differently affected the pattern of evolution of scams, with se-selectin being decreased and sicam-1 being increased. expression of sp-selectin and svcam-1 was not affected (leone et al. 2004) . methotrexate (mtx) markedly reduces the expression of vascular e-selectin. a positive correlation between disease severity and the frequency of cutaneous lymphocyte-associated antigen (cla)-positive t cells in the blood of untreated patients with psoriasis has been observed. it is suggested that mtx decreases the expression of cla and e-selectin and that this may be a major mechanism for the therapeutic effect of mtx on psoriatic skin lesions (sigmundsdottir et al. 2004) . assessment of selected adhesion molecule and proinflammatory cytokine levels in the vitreous body of patients with type 2 diabetes-role of the inflammatory-immune process in the pathogenesis of proliferative diabetic retinopathy levels of adhesion molecules bear a relationship to triglyceride levels in type 2 diabetic subjects with proven silent ischemia value of e-selectin and l-selectin determination in children and youth with inflammatory bowel disease ps3, a semisynthetic b-1,3-glucan sulfate, diminishes contact hypersensitivity responses through inhibition of l-and p-selectin functions circulating adhesion molecules during kidney allograft rejection icam-1 polymorphisms (g241r, k469e), in coronary artery disease and myocardial infarction e-selectin polymorphism in erythema nodosum secondary to sarcoidosis expression of e-selectin ligand-1 (cfr/esl-1) on hepatic stellate cells: implications for leukocyte extravasation and liver metastasis activation and damage of endothelial cells upon hypoxia/reoxygenation. effect of extracellular ph genomic rearrangements on vcam1, sele, apeg1and aif1 loci in atherosclerosis lp95, a novel leptospiral protein that binds extracellular matrix components and activates e-selectin on endothelial cells genetic polymorphisms in cytokine and adhesion molecule genes in coronary artery disease e-selectin regulates gene expression in metastatic colorectal carcinoma cells and enhances hmgb1 release regulation of gene expression by a-tocopherol e-selectin, and p-selectin expressions in papillary thyroid carcinomas and their correlation with prognostic parameters a novel antiinflammatory role for andrographolide in asthma via inhibition of the nuclear factor-kb pathway platelet p-selectin and gpiib/iiia expression after liver transplantation and resection selectin inhibitors: a patent review the involvement of the sle-a selectin ligand in the extravasation of human colorectal carcinoma cells quantitative real-time rt-pcr analysis of inflammatory gene expression associated with ischemia-reperfusion brain injury plasma indices of endothelial and platelet activation in rheumatoid disease: relationship to cardiovascular co-morbidity soluble intercellular adhesion molecule-1 and e-selectin in patients with asthma exacerbation selectins and monocyte chemotactic peptide as the markers of atherosclerosis activity soluble adhesion molecules in pediatric rheumatic diseases impact of prolonged cyclooxygenase-2 inhibition on inflammatory markers and endothelial function in patients with ischemic heart disease and raised creactive protein: a randomized placebo-controlled study antimetastatic activities of modified heparins: selectin inhibition by heparin attenuates metastasis sulfated hexasaccharides attenuate metastasis by inhibition of p-selectin and heparanase tnf-a blockade induces a reversible but transient effect on endothelial dysfunction in patients with long-standing severe rheumatoid arthritis transcriptional arrest of the human e-selectin gene expression of e-selectin and its transcripts during intestinal ischemia-reperfusion injury in pigs the antitumor activity of an anti-cd54 antibody in scid mice xenografted with human breast, prostate, non-small cell lung, and pancreatic tumor cell lines infectious susceptibility and severe deficiency of leukocyte rolling and recruitment in e-selectin and p-selectin double mutant mice endothelial dysfunction in cardiovascular diseases: the role of oxidant stress invasive amebiasis: a microcirculatory disorder? p-selectin knockout mice have improved outcomes with both warm ischemia and small bowel transplantation soluble e-selectin in children and adolescents with type 1 diabetes blocking e-selectin inhibits ischaemiareperfusion-induced neutrophil recruitment to the murine testis increased platelet cd63 and pselectin expression persist in atherosclerotic ischemic stroke receptor tyrosine kinase epha2 mediates thrombin-induced upregulation of icam-1 in endothelial cells in vitro increased plasma levels of soluble p-selectin in rheumatic mitral stenosis the common variants of e-selectin gene in graves' disease aspergillus fumigatus stimulates leukocyte adhesion molecules and cytokine production by endothelial cells in vitro and during invasive pulmonary disease pathogenic aspects of pulmonary complications in acute pancreatitis patients effect of rosiglitazone treatment on circulating vascular and inflammatory markers in insulin-resistant subjects changes in pselectin expression on cardiac microvessels in blood-perfused rat hearts subjected to ischemia-reperfusion human squamous cell carcinomas evade the immune response by down-regulation of vascular e-selectin and recruitment of regulatory t cells successful treatment of rheumatoid arthritis is associated with a reduction in serum seselectin and thrombomodulin level cytoadherence characteristics to endothelial receptors icam-1 and cd36 of plasmodium falciparum populations from severe and uncomplicated malaria cases p-selectin or intercellular adhesion molecule (icam)-1 deficiency substantially protects against atherosclerosis in apolipoprotein e-deficient mice circulating e-selectin and tumor necrosis factor-alpha in extraarticular involvement and joint disease activity in rheumatoid arthritis protein biochip array of adhesion molecule expression in peripheral blood of patients with nasal polyposis serum p-selectin, soluble vascular cell adhesion molecule-i (s-vcam-i) and soluble intercellular adhesion molecule-i (s-icam-i) levels in bladder carcinoma patients with different stages autoantibodies and other serological markers in rheumatoid arthritis: predictors of disease activity? phenyl methimazole inhibits tnf-a-induced vcam-1 expression in an ifn regulatory factor-1-dependent manner and reduces monocytic cell adhesion to endothelial cells changes in endogenous cytokines, adhesion molecules and platelets during cytokine-induced tumor necrosis evaluation of se-selectin and sicam-1 as parameters for renal function icam1 r241 is not associated with celiac disease in the spanish population comparative study of adhesion molecule expression in nodular lesions of behçet syndrome and other forms of panniculitis evidence of increased inflammation and microcirculatory abnormalities in patients with type 1 diabetes and their role in microvascular complications circulating levels of soluble adhesion molecules in patients with anca-associated vasculitis plasma cytokine and p-selectin levels in advanced malignancy: prognostic value and impact of low-molecular weight heparin administration circulating adhesion molecules and purine nucleotides during kidney allograft reperfusion p-selectin-targeting of the fibrin selective thrombolytic desmodus rotundus salivary plasminogen activator alpha1 e-and p-selectin are not required for the development of experimental autoimmune encephalomyelitis in c57bl/6 and sjl mice cell adhesion molecules, vascular endothelial growth factor, and basic fibroblast growth factor in patients with non-small cell lung cancer treated with chemotherapy with or without bevacizumab-an eastern cooperative oncology group study does colorectal cancer clinical advancement affect adhesion molecules (sp-selectin, se-selectin and icam-1) concentration? soluble p-selectin concentration in patients with colorectal cancer serum levels of soluble e-selectin are associated with the clinical course of metastatic disease in patients with liver metastases from breast cancer markers for endothelial activation during open heart surgery cell adhesion molecules-update expression of selected adhesion molecules in dermatitis herpetiformis and bullous pemphigoid differential induction of mrna for icam-1 and selectins in hepatocytes, kupffer cells and endothelial cells during endotoxemia serum concentrations of sicam-1, se-, sp-and sl-selectins in patients with schistosoma mansoni infection and association with disease severity biochemical markers of endothelial activation in primary hyperparathyroidism markers of low-grade inflammation and soluble cell adhesion molecules in chinese patients with coronary artery disease association of leu125val polymorphism of platelet endothelial cell adhesion molecule-1 (pecam-1) gene & soluble level of pecam-1 with coronary artery disease in asian indians selectins in the hit syndrome: pathophysiologic role and therapeutic modulation disruption of pselectin signaling modulates cell trafficking and results in improved outcomes after mouse warm intestinal ischemia and reperfusion injury cd62 blockade with p-selectin glycoprotein ligand-immunoglobulin fusion protein reduces ischemia-reperfusion injury after rat intestinal transplantation independent pathways of p-selectin and complement-mediated renal ischemia/reperfusion injury ifn-g inhibits double-stranded rnainduced e-selectin expression in human endothelial cells candidate gene analysis of selectin cluster in patients with multiple sclerosis enhanced expression of e-selectin on the vascular endothelium of peripheral nerve in critically ill patients with neuromuscular disorders a high frequency african coding polymorphism in the n-terminal domain of icam-1 predisposing to cerebral malaria in kenya icam-1-dependent pathways regulate colonic eosinophilic inflammation role of inflammation in diabetic nephropathy circulating endothelial cells and rheumatoid arthritis: relationship with plasma markers of endothelial damage/dysfunction variation in the icam1 gene is not associated with severe malaria phenotypes interactions of the gastrotropic bacterium helicobacter pylori with the leukocyteendothelium adhesion molecules, the selectins-a preliminary report transforming growth factor-beta inhibits e-selectin expression on human endothelial cells p-selectin-mediated acute inflammation can be blocked by chemically modified heparin, ro-heparin selectin blockade plus therapy with low-dose sirolimus and cyclosporin a prevent brain death-induced renal allograft dysfunction microvascular endothelial cells from e-selectin-deficient mice form tubes in vitro ser128arg gene polymorphism for e-selectin and severity of atherosclerotic arterial disease retinoic acid inhibits the regulated expression of vascular cell adhesion molecule-1 by cultured dermal microvascular endothelial cells leukocyte and endothelial cell adhesion molecules in inflammation focusing on inflammatory heart disease putative outer membrane proteins of leptospira interrogans stimulate human umbilical vein endothelial cells (huvecs) and express during infection expression of adhesion molecules in lungs of mice infected with paracoccidioides brasiliensis conidia circulating p-,l-and e-selectins in pseudoxanthoma elasticum patients selectins and selectin ligands in extravasation of cancer cells and organ selectivity of metastasis angiotensin receptor blockade decreases markers of vascular inflammation implication of adhesion molecules in inflammation of the common bile duct in patients with secondary cholangitis due to biliary obstruction serum levels of intercellular adhesion molecule icam-1 and e-selectin in advanced stage non-small cell lung cancer e-selectin genetic variation as a susceptibility factor for ischemic stroke e-selectin gene induction by ionizing radiation is independent of cytokine induction nuclear factor kappab dominant negative genetic constructs inhibit x-ray induction of cell adhesion molecules in the vascular endothelium strategies to reduce oxidative stress in cardiovascular disease variant isoforms of cd44 are p-and l-selectin ligands on colon carcinoma cells soluble cell adhesion molecules in gingival crevicular fluid in periodontal health and disease e-selectin gene s128r polymorphism is associated with poor prognosis in patients with stage ii or iii colorectal cancer role of adhesion molecules in the induction of restenosis after angioplasty in the lower limb the p-selectin gene is highly polymorphic: reduced frequency of the pro715 allele carriers in patients with myocardial infarction tumor necrosis factor-alpha upregulates the expression of ccl2 and adhesion molecules of human proximal tubular epithelial cells through mapk signaling pathways in vivo imaging of cutaneous t-cell lymphoma migration to the skin injury of the blood brain barrier and up-regulation of icam-1 in polymicrobial sepsis expression of chemokines and adhesion molecules in human coronary artery endothelial cells infected with chlamydia (chlamydophila) pneumoniae pravastatin limits radiation-induced vascular dysfunction in the skin thrombosis and atherosclerosis endothelial microparticles as markers of endothelial dysfunction e-selectin and lselectin polymorphisms in patients with periodontitis endothelial activation and systemic inflammation in obese asthmatic children bosentan regulates the expression of adhesion molecules on circulating t cells and serum soluble adhesion molecules in systemic sclerosisassociated pulmonary arterial hypertension significance of endothelial molecular markers in the evaluation of the severity of acute pancreatitis serum levels of p-selectin in men with high-functioning autism the various effects of four h1-antagonists on serum substance p levels in patients with atopic dermatitis immunohistochemistry, glycosylation and immunosuppression of glycodelin in human ovarian cancer andrographolide inhibits the adhesion of gastric cancer cells to endothelial cells by blocking eselectin expression adhesion molecule interactions facilitate human immunodeficiency virus type 1-induced virological synapse formation between t cells impaired eosinophil recruitment to the cornea in p-selectin-deficient mice in onchocerca volvulus keratitis (river blindness) polarized response of endothelial cells to invasion by aspergillus fumigatus soluble endothelial adhesion molecules and inflammation markers in patients with b-thalassemia intermedia carbohydrate antigen sialyl lewis a-its pathophysiological significance and induction mechanism in cancer progression adhesion of epstein-barr virus-positive natural killer cell lines to cultured endothelial cells stimulated with inflammatory cytokines inflammatory mediators in diabetic and non-diabetic lumbosacral radiculoplexus neuropathy increase in e-selectin expression in umbilical vein endothelial cells by anticancer drugs and inhibition by cimetidine adhesion molecules (icam-1 and vcam-1) and diabetic retinopathy in type 2 diabetes evaluation of markers of endothelial damage in cases of young myocardial infarction adhesion molecule polymorphisms in acute renal allograft rejection polymorphism in icam-1, pecam-1, e-selectin, and l-selectin genes in tunisian patients with inflammatory bowel disease increase in soluble eselectin level after ptca and stent implantation: a potential marker of restenosis p-selectin deficiency attenuates tumor growth and metastasis engagement of icam-1 by major group rhinoviruses activates the lfa-1/icam-3 cell adhesion pathway in mononuclear phagocytes biomarkers of endothelial dysfunction are elevated and related to prognosis in chronic heart failure patients with diabetes but not in those without diabetes the role of vascular adhesion molecules pecam-1 (cd 31), vcam-1 (cd 106), e-selectin (cd62e) and p-selectin (cd62p) in severe porcine pancreatitis reduction of soluble adhesion molecules (sicam-1, svcam-1, and se-selectin) and vascular endothelial growth factor levels in serum of rheumatoid arthritis patients following multiple intravenous infusions of infliximab soluble cell adhesion molecules (sicam-1, svcam-1, and se-selectin) in patients with early rheumatoid arthritis role of sialosyl lewis(a) in adhesion of colon cancer cells-the antisense rna approach endothelial cell adhesion molecules and cancer progression expression of vascular adhesion molecules in inflammatory bowel disease expression of cytokeratins, adhesion and activation molecules in oral ulcers of behçet's disease intercellular adhesion molecule 1 gene polymorphisms in graves' disease src and phosphatidylinositol 3-kinase mediate soluble e-selectin-induced angiogenesis clinical significance of selected endothelial activation markers in patients with systemic lupus erythematosus house dust mite induces expression of intercellular adhesion molecule-1 in eol-1 human eosinophilic leukemic cells selectin inhibitor bimosiamose prolongs survival of kidney allografts by reduction in intragraft production of cytokines and chemokines syk associates with clathrin and mediates phosphatidylinositol 3-kinase activation during human rhinovirus internalization selectins promote tumor metastasis m€ uller cells do not influence leukocyte adhesion to retinal endothelial cells contribution of soluble intercellular adhesion molecule-1 to the migration of vascular smooth muscle cells characterization of gastrin-induced proangiogenic effects in vivo in orthotopic u373 experimental human glioblastomas and in vitro in human umbilical vein endothelial cells cell adhesion molecules as a marker reflecting the reduction of endothelial activation induced by glucocorticoids applications of synthetic carbohydrates to chemical biology effects of infliximab on cytokines, myeloperoxidase, and soluble adhesion molecules in patients with juvenile idiopathic arthritis association between the ser128arg variant of the e-selectin and risk of coronary artery disease in the central china intercellular adhesion molecule-1 gene k469e polymorphism and ischemic stroke: a case-control study in a chinese population soluble cd40 ligand, soluble p-selectin, interleukin-6, and tissue factor in diabetes mellitus: relationships to cardiovascular disease and risk factor intervention monoclonal antibody against e selectin attenuates subarachnoid hemorrhage-induced cerebral vasospasm effects of congestive heart failure on plasma von willebrand factor and soluble p-selectin concentrations in patients with non-valvar atrial fibrillation p. gingivalis and e. coli lipopolysaccharides exhibit different systemic but similar local induction of inflammatory markers a distinct profile of serum levels of soluble intercellular adhesion molecule-1 and intercellular adhesion molecule-3 in mycosis fungoides and sézary syndrome the ability of different forms of heparins to suppress p-selectin function in vitro correlates to their inhibitory capacity on bloodborne metastasis in vivo p-selectin: a common therapeutic target for cardiovascular disorders, inflammation and tumour metastasis the role of adhesion molecules in atopic dermatitis blocking endothelial adhesion molecules: a potential therapeutic strategy to combat atherogenesis circulating e-selectin as a risk marker in patients with end-stage renal disease endothelial/lymphocyte activation leads to prominent cd4+ t cell infiltration in the gastric mucosa of patients with systemic sclerosis adhesion molecules from the lfa-1/icam-1,3 and vla-4/vcam-1 pathways on t lymphocytes and vascular endothelium in graves' and hashimoto's thyroid glands reduction of soluble p-selectin by statins is inversely correlated with the progression of coronary artery disease the leu554phe polymorphism in the e-selectin gene is associated with blood pressure in overweight people n,n,ntrimethylsphingosine inhibits interleukin-1 beta-induced nf-kb activation and consequent e-selectin expression in human umbilical vein endothelial cells neutrophil surface expression of cd11b and cd62l in diabetic microangiopathy transgene expression of a(1,2)-fucosyltransferase-i (fut1) in tumor cells selectively inhibits sialyl-lewis x expression and binding to e-selectin without affecting synthesis of sialyl-lewis a or binding to p-selectin markers of vascular endothelial cell damage and p. falciparum malaria: association between levels of both se-selectin and thrombomodulin, and cytokines, hemoglobin and clinical presentation staphylococcal peptidoglycan initiates an inflammatory response and procoagulant activity in human vascular endothelial cells: a comparison with highly purified lipoteichoic acid and tsst-1 the impact of peripheral arterial disease on circulating platelets intestinal inflammation in adhesion molecule-deficient mice: an assessment of p-selectin alone and in combination with icam-1 or e-selectin adhesion molecule polymorphisms in chronic renal allograft failure endothelial adhesion molecules are associated with inflammation in subjects with hiv disease evaluation of microbiologic and hematologic parameters and e-selectin as early predictors for outcome of neonatal sepsis expression of endothelial protein c receptor and thrombomodulin in human coronary atherosclerotic plaques p-selectin in arterial thrombosis role of icam-1 in persisting inflammation in parkinson disease and mptp monkeys cellular adhesion molecules and blood pressure: interaction with sex in a multi-ethnic population association between the thr715pro p-selectin gene polymorphism and soluble p-selectin levels in a multiethnic population in south london effect of clopidogrel on the expression of inflammatory markers in rabbit ischemic coronary artery allergic rhinitis: continuous or on demand antihistamine therapy? host defense against systemic infection with streptococcus pneumoniae is impaired in e-, p-, and e-/p-selectin-deficient mice serum vcam-1, icam-1, and l-selectin levels in children and young adults with chronic renal failure decreased venous thrombosis with an oral inhibitor of p selectin plasma and platelet-derived vascular endothelial growth factor and angiopoietin-1 in hypertension: effects of antihypertensive therapy platelet morphology, soluble p selectin and platelet p-selectin in acute ischaemic stroke. the west birmingham stroke project cd43, but not pselectin glycoprotein ligand-1, functions as an e-selectin counterreceptor in human pre-b-cell leukemia nall-1 blood serum levels of vascular cell adhesion molecule (svcam-1), intercellular adhesion molecule (sicam-1) and endothelial leucocyte adhesion molecule-1 (elam-1) in diabetic retinopathy ionizing radiationinduced e-selectin gene expression and tumor cell adhesion is inhibited by lovastatin and all-trans retinoic acid clinical significance of intercellular adhesion molecule-1 in ulcerative colitis in situ expression of the cell adhesion molecules in bronchial tissues from asthmatics with air flow limitation: in vivo evidence of vcam-1/vla-4 interaction in selective eosinophil infiltration identification of cutaneous lymphocyte-associated antigen as sialyl 6-sulfo lewis x, a selectin ligand expressed on a subset of skin-homing helper memory t cells protective effect of anti-pselectin monoclonal antibody in lipopolysaccharide-induced lung hemorrhage the levels of soluble adhesion molecules in diabetic and nondiabetic patients with combined hyperlipoproteinemia and the effect of ciprofibrate therapy increased platelet activation markers in rheumatoid arthritis: are they related with subclinical atherosclerosis? elevated plateletmonocyte complexes in patients with psoriatic arthritis elevated serum soluble eselectin levels in korean children with measles the engagement of selectins and their ligands in colorectal cancer liver metastases biomarkers of oxidant stress, insulin sensitivity and endothelial activation in rheumatoid arthritis: a cross-sectional study of their association with accelerated atherosclerosis relationships between serum fatty acid composition and multiple markers of inflammation and endothelial function in an elderly population serum adhesion molecules in acute pancreatitis: time course and early assessment of disease severity endothelial cell stimulation by candida albicans antisense inhibition of icam-1 expression as therapy provides insight into basic inflammatory pathways through early experiences in ibd salicylates inhibit i kb-a phosphorylation, endothelial-leukocyte adhesion molecule expression, and neutrophil transmigration flow cytometric analysis of conjunctival epithelium in ocular rosacea and keratoconjunctivitis sicca circulating soluble adhesion molecules icam-1 and vcam-1 and their association with clinical outcome, troponin t and creactive protein in patients with acute coronary syndromes natriuretic peptides and e-selectin as predictors of acute deterioration in patients with inotrope-dependent heart failure endothelial dysfunction in diabetes: from mechanisms to therapeutic targets biological mediators of acute inflammation primate models in women's health: inflammation and atherogenesis in female cynomolgus macaques (macaca fascicularis) comparison of soluble icam-1, vcam-1 and e-selectin levels in patients with episodic cluster headache and giant cell arteritis novel cardiovascular risk factors in premature coronary atherosclerosis associated with systemic lupus erythematosus enhancement of endothelial nitric oxide synthase production reverses vascular dysfunction and inflammation in the hindlimbs of a rat model of diabetes fundamental and distinct roles of p-selectin and lfa-1 in ischemia/reperfusion-induced leukocyte-endothelium interactions in the mouse colon increased methylation of promotor region suppresses expression of muc2 gene in colon carcinoma cells hydralazine reduces leukocyte migration through different mechanisms in spontaneously hypertensive and normotensive rats the effect of trandolapril and its fixed-dose combination with verapamil on circulating adhesion molecules levels in hypertensive patients with type 2 diabetes accelerated development of arthritis in mice lacking endothelial selectins the endothelial leukocyte adhesion molecule. role in coronary artery disease expression of cell adhesion molecules in the salivary and lacrimal glands of sjogren's syndrome elevation of serum soluble e-and p-selectin in patients with hypertension is reversed by benidipine, a long-acting calcium channel blocker association analysis of the e-selectin 98 g > t polymorphism and the risk of childhood ischemic stroke and e-selectin as markers of hematogenous metastases and as predictors of prognosis in colorectal cancer carcinoembryonic antigen and cd44 variant isoforms cooperate to mediate colon carcinoma cell adhesion to e-and l-selectin in shear flow temporal patterns of soluble adhesion molecules in cerebrospinal fluid and plasma in patients with the acute brain infraction lack of uniform platelet activation in patients after ischemic stroke and choice of antiplatelet therapy adhesion and lymphocyte costimulatory molecules in systemic rheumatic diseases skin biopsies demonstrate site-specific endothelial activation in mouse models of sepsis globotriaosylceramide induces oxidative stress and up-regulates cell adhesion molecule expression in fabry disease endothelial cells high serum soluble e-selectin levels are associated with postoperative haematogenic recurrence in esophageal squamous cell carcinoma patients methotrexate markedly reduces the expression of vascular e-selectin, cutaneous lymphocyte-associated antigen and the numbers of mononuclear leucocytes in psoriatic skin synoviocyte stimulation by the lfa-1-intercellular adhesion molecule-2-ezrin-akt pathway in rheumatoid arthritis decreased serum levels of p-selectin and eosinophil cationic protein in patients with mild asthma after inhaled salbutamol adhesion molecules and their role in pathogenesis of ards adhesion molecules in allergic inflammation dna-methylation of the e-selectin promoter represses nf-kb transactivation the high affinity selectin glycan ligand c2-o-slex and mrna transcripts of the core 2 b-1,6-n acetylglucosaminyl-transferase (c2gnt1) gene are highly expressed in human colorectal adenocarcinomas the role of the platelet in the pathogenesis of atherothrombosis differential metastasis inhibition by clinically relevant levels of heparins-correlation with selectin inhibition, not antithrombotic activity discovery of inhibitors of cell adhesion molecule expression in human endothelial cells. 1. selective inhibition of icam-1 and e-selectin expression inflammatory molecule expression in cerebral arteriovenous malformations effect of 100 % oxygen on e-selectin expression, recruitment of neutrophils and enterocyte apoptosis following intestinal ischemia-reperfusion in a rat expression and significance of icam-1 and its counter receptors lfa-1 and mac-1 in experimental acute pancreatitis of rats selective upregulation of endothelial e-selectin in response to helicobacter pylori-induced gastritis soluble cytokeratin-19 and e-selectin biomarkers: their relevance for lung cancer detection when tested independently or in combinations hiv-1 nef intersects the macrophage cd40l signaling pathway to promote resting-cell infection prognostic significance of soluble adhesion molecules in hodgkin's disease association between singlenucleotide polymorphisms in selectin genes and immunoglobulin a nephropathy possible requirement of intercellular adhesion molecule-1 for invasion of gingival epithelial cells by treponema medium effects of parietaria judaica pollen extract on human microvascular endothelial cells circulating asymmetric dimethylarginine, endothelin-1 and cell adhesion molecules in women with gestational diabetes adiponectin deficiency promotes endothelial activation and profoundly exacerbates sepsis-related mortality podocalyxin-like protein is an e-/l-selectin ligand on colon carcinoma cells: comparative biochemical properties of selectin ligands in host and tumor cells different roles of galectin-9 isoforms in modulating e-selectin expression and adhesion function in lovo colon carcinoma cells circulating intercellular adhesion molecule-1 (icam-1) in autoimmune liver disease and evidence for the production of icam-1 by cytokine-stimulated human hepatocytes effects of antinuclear factor kb reagents in blocking adhesion of human cancer cells to vascular endothelial cells specific haplotypes of the p-selectin gene are associated with myocardial infarction mechanisms by which eselectin regulates diapedesis of colon cancer cells under flow conditions soluble endothelial cell adhesion molecules and their relationship to disease activity in takayasu's arteritis activated protein c reduces graft neutrophil activation in clinical renal transplantation immuno-inflammatory and thrombotic/fibrinolytic variables associated with acute ischemic stroke diagnosis transactivation of the icam-1 gene by cd30 in hodgkin's lymphoma serum levels of soluble e-selectin in colorectal cancer elevated serum soluble e-selectin is associated with poor outcome and correlated with serum alt in biliary atresia oxidative stress in cardiovascular disease molecular investigation of the functional relevance of missense variants of icam-1 p-selectin thr715pro polymorphism predicts p-selectin levels but not risk of incident coronary heart disease or ischemic stroke in a cohort of 14595 participants: the atherosclerosis risk in communities study polymorphisms and linkage analysis for icam-1 and the selectin gene cluster platelet, not endothelial, p-selectin is required for neointimal formation after vascular injury staphylococcal enterotoxins a and b enhance rhinovirus replication in a549 cells anthrax lethal toxin enhances tnf-induced endothelial vcam-1 expression via an ifn regulatory factor-1-dependent mechanism selectively desulfated heparin inhibits p-selectin-mediated adhesion of human melanoma cells e-selectin and sialyl lewis x expression is associated with lymph node metastasis of invasive micropapillary carcinoma of the breast dna polymorphisms in adhesion molecule genes-a new risk factor for early atherosclerosis functional characterization of atherosclerosis-associated ser128arg and leu554phe e-selectin mutations l-arginine prevents metabolic effects of high glucose in diabetic mice crossing the endothelium: e-selectin regulates tumor cell migration under flow conditions polymorphisms and plasma soluble levels of e-selectin in patients with chronic hepatitis b virus infection p-selectin suppresses hepatic inflammation and fibrosis in mice by regulating interferon-g and the il-13 decoy receptor effects of low molecular weight heparin on platelet surface p-selectin expression and serum interleukin-8 production in rats with trinitrobenzene sulphonic acid-induced colitis intercellular adhesion molecule-1 (icam-1) expression is necessary for monocyte adhesion to the placental bed endothelium and is increased in type 1 diabetic human pregnancy activated platelets contribute importantly to myocardial reperfusion injury circulating thrombomodulin and vascular cell adhesion molecule-1 and renal vascular lesion in patients with lupus nephritis serum e-selectin and erythrocyte membrane na + k + atpase levels in patients with rheumatoid arthritis platelet activity is a biomarker of cardiac necrosis and predictive of untoward clinical outcomes in patients with acute myocardial infarction undergoing primary coronary stenting e-selectin polymorphism associated with myocardial infarction causes enhanced leukocyteendothelial interactions under flow conditions cell adhesion molecules regulate fibrotic process via th1/th2/th17 cell balance in a bleomycin-induced scleroderma model mucosal tolerance to e-selectin provides protection against cerebral ischemiareperfusion injury in rats synergistic effects between 561a > c and 98 g > t polymorphisms of e-selectin gene and hypercholesterolemia in determining the susceptibility to coronary artery disease inflammatory biomarkers in coronary artery disease th1 cell-mediated resistance to cutaneous infection with leishmania major is independent of p-and eselectins cd44v4 is a major e-selectin ligand that mediates breast cancer cell transendothelial migration e-selectin and its ligand-slex in the metastasis of hepatocellular carcinoma serum 3'-sulfo-le a indication of gastric cancer metastasis cell adhesion: implication in tumor progression inhibitors targeting the lfa-1/ icam-1 cell-adhesion interaction: design and mechanism of action familial chronic nail candidiasis with icam-1 deficiency: a new form of chronic mucocutaneous candidiasis markers of endothelial dysfunction in older subjects with late onset alzheimer's disease or vascular dementia key: cord-022353-q2k2krnm authors: w. quimby, fred; h. luong, richard title: clinical chemistry of the laboratory mouse date: 2007-09-02 journal: the mouse in biomedical research doi: 10.1016/b978-012369454-6/50060-1 sha: doc_id: 22353 cord_uid: q2k2krnm the frontier of clinical chemistry in the mouse has advanced and expanded because of two major events such as, the increasing reliance on mice in biomedical research, and increasing availability of practical yet sophisticated techniques and instrumentations that have allowed for the detection of a wider variety of biomarkers of disease. the progression of these two events is partially driven by the increasing regulatory demands related to safety/toxicity assessment of novel drug development. the availability of inbred strains has led to major breakthroughs in cancer, biology, and immunology. in addition, outbred stocks continue to be utilized in a wide variety of studies but particularly in the fields of toxicology and pharmacology. the power of these models to elucidate the genetic basis of disease cannot be overemphasized. this provided complete nucleotide sequences for each genome allowing investigators to quickly develop the equivalent murine model for many of the inherited human diseases. transgenic and knockout mice have helped clarify disease pathogenesis in virtually every area of medicine and often elucidated biochemical pathways, previously unknown, which are now subject to testing and quantification. it has been more than 20 years since publication of the first edition of the mouse in biomedical research, and since that time new emphasis has been placed on the mouse as a model for the pathophysiology and treatment of human diseases. during this time, the frontier of clinical chemistry in the mouse has advanced and expanded because of two major events: the increasing reliance on mice in biomedical research, and increasing availability of practical yet sophisticated techniques and instrumentations that have allowed for the detection of a wider variety of biomarkers of disease. the progression of these two events has been partially driven by the increasing regulatory demands related to safety/toxicity assessment of novel drug development. abbreviations used in this chapter are summarized in appendix 6-1. for the last quarter century, mice have been the most frequently used mammal in biomedical research, rising from 61% of all mammals used in 1983 to 71% in 1993 (national center for research resources 1997 . the nature of their use has changed dramatically during this time. during the first half of the 20th century, a great deal of emphasis was placed on the development of inbred strains. the availability of inbred strains has led to major breakthroughs in cancer, biology, and immunology (quimby 2002) . by 1970 there were approximately 250 inbred strains available. in addition, outbred stocks continue to be utilized in a wide variety of studies but particularly in the fields of toxicology and pharmacology. during the second half of the 20th century, investigators developed congenic lines and recombinant inbred strains, each having an impact on elucidating the role of individual genes and assigning new traits to linkage groups respectively (paigen 2003a ). however, beginning in the early 1980s scientists began to genetically engineer mice by either adding a new gene (transgenic) or deleting a normal mouse gene (knockout, ko) (paigen 2003b) . the power of these models to elucidate the genetic basis of disease cannot be overemphasized. over the past 15 years, publications citing transgenic and ko mice have increased exponentially and at the time of this writing the mutant mouse resource of the jackson laboratory listed more than 10,000 such unique lines. during this same period the results of both the human and mouse genome projects were published (international human genome sequencing consortium 2001; mouse genome sequencing consortium 2002) . this provided complete nucleotide sequences for each genome allowing investigators to quickly develop the equivalent murine model for many of the inherited human diseases. transgenic and ko mice have helped clarify disease pathogenesis in virtually every area of medicine and often elucidated biochemical pathways, previously unknown, which are now subject to testing and quantification. as a result, clinical chemistry in the mouse has grown from evaluation of 15-20 analytes found in plasma (or urine), to hundreds of biomarkers that can monitor disease status at the cellular level. due to the massive amount of new information characterizing each of the standard strains and mutant lines of mice, a mouse phenome database has been developed to manage data and provide researchers with the ability to explore raw phenotypic data (including clinical chemistry) and summary analyses. the resource allows investigators to select the appropriate murine model for physiology testing, drug discovery, toxicology studies, mutagenesis, modeling human disease, quantitative trait loci (qtl) analyses, identification of new genes, and unraveling the influence of environment on genotype (bogue and grubb 2004) . by far, the most important new technology in the rapidly growing and changing field of clinical chemistry is the discipline of proteomics. proteomics combines the disciplines of molecular biology, biochemistry, and genetics to the analysis of the structure, function, and interactions of proteins produced by the genes of a particular cell, tissue, or organism. the field of proteomics has grown mainly due to the development of new instruments that are based on sophisticated techniques, yet amenable to practical implementation. for example, the development of surface-enhanced laser desorption/ionization (seldi) platform time-of-flight (tof) mass spectroscopy (ms) allowed petricoin et al. (2002a) to identify five peptides in the sera of women with ovarian cancer that were not found in women without ovarian cancer, even though the structure and function of some of these peptides were not actually known. building on these findings, zhang et al. (2004) combined the seldi-tof ms with protein separation procedures to develop a multianalyte immunoassay for rapidly screening potential cancer patients. this process of identifying proteins (biomarkers) that are predictive for a disease is known as plasma protein profiling. similar technology has been used to develop plasma protein profiles for neoplastic conditions in humans, such as prostate cancer petricoin et al. 2002b; qu et al. 2002) and bladder cancer (adam et al. 2001; vlahou et al. 2001) , as well as a variety of non-neoplastic conditions, such as ischemic versus hemorrhagic stroke , severe acute respiratory syndrome (kang et al. 2005 ), alzheimer's disease (carrette et al. 2003) , and creutzfeldt-jakob disease (sanchez et al. 2004) . plasma protein profiling has also begun in mouse models. xiang et al. (2004) used a combination of two-dimensional gel electrophoresis (2d-ge) and matrix-assisted laser desorption/ionization (maldi) tof ms to quantify serum protein profiles of c57bl/6 mice harboring the lewis carcinoma with and without treatment using acetazolamide. they found upregulation of many peptides associated with tumor growth and metastasis, and many of these same peptides were modified during treatment. two specific targets of acetazolamide antitumor activity were subsequently identified as histone h2b and croci (a ubc-like peptide). park et al. (2004) used similar technology to determine plasma protein profiles for high (c57bl/6) and low (c3h) atherosclerosis prone strains of mice on normal or atherogenic diets. they identified 30 proteins in which the levels had changed after eating an atherogenic diet. of these, 14 were differentially changed in c57bl/6 mice and an additional 16 were changed in both strains. in addition, 28 proteins were differentially expressed between the two strains regardless of diets. four of these proteins were upregulated in c57bl/6 and 11 were upregulated in c3h. nine of those protein markers were definitively identified and their roles in the pathogenesis of atherosclerosis discussed in the "atherosclerosis" section. plasma protein profiling in mice has the potential to become an important discovery tool for translational research, particularly in identifying cancer-associated plasma biomarkers in humans. for example, juan et al. (2004) also used 2d-ge and maldi tof ms to identify plasma biomarkers in balb/c-nude mice harboring human xenotransplanted tumors. in mice bearing a human stomach cancer cell line, serum amyloid a (saa) was elevated. the authors then went back to screen the sera of human stomach cancer patients and were able to demonstrate that, when compared to controls, humans with stomach cancer also had significantly elevated levels of saa. all of these examples illustrate the power of proteomics. proteomic pattern diagnostics offers a means to look at molecular diagnostic information in human or mouse serum, without preconceived assumptions about the existence or identity of the biomarkers. this allows for the development of sensitive and specific peptide assays for identified biomarkers of disease. practical applications of proteomics have been facilitated by the creation of instrumentation that can analyze multiple analytes from small sample volumes with fast through-put (such as multiplex technology, which are discussed in the "multiplex technology" section). in an attempt to manage all the experimental data arising from the fields of proteomics, metabolomics and transcriptomics (gene array), the chemical effects in biological systems (cebs) knowledge base was developed (xirasagar et al. 2004 ). this is a useful, searchable database that integrates experimental findings from all three disciplines, by biosource identification (animal, test article, genotype, and investigator). by making the cross correlation from data arising from three different streams it is hoped that combination groups of predictive markers for disease and toxicity will emerge. the cost of bringing a new drug to market is estimated at $0.8-1.7 billion (food and drug administration 2004), of which a large proportion is spent on safety/toxicity assessments during the preclinical phase of the development of a drug. although expensive, safety/toxicity regulations are necessary, as illustrated by the fact that a new drug entering phase i clinical trials has only an 8% chance of reaching the marketplace due to toxicity issues. the ability to decrease the costs of drug development due to safety/toxicity issues is becoming increasingly reliant on the use of the laboratory mouse as a model for human disease and the identification of sensitive biomarkers for toxicity and disease. the underlying basis of using laboratory mice as models for human disease was demonstrated by everett and harrison (1983) , who showed that the predictive reliability of mice for quantitative toxicity of five chemotherapeutic drugs in humans was at least as good, if not better, that the predictive reliability demonstrative by dogs and monkeys. more recently, newell et al. (2004) presented data on predictive performance of rats and mice to demonstrate qualitative human toxicity when given 39 novel cancer chemotherapeutic agents. they claim that nonrodent species are unnecessary for identification of a safe phase 1 starting dose for human trials. furthermore, the two rodent species (rats and mice) used gave similar quantitative and qualitative results. the arrival of genetically engineered mice has further enhanced the relevance of using mice in research, especially in terms of basic mechanistic research and applied screening for genotoxicity and carcinogenicity. transgenic mice carrying a human gene and expressing the protein are said to be "humanized." these animals are invaluable in drug assessment especially when the drug interacts only with the human protein (bolon 2004) . zambrowicz and sands (2003a) showed that the ko phenotype of mice correlated well with the molecular targets of the 100 best selling drugs available to the u.s. market. zambrowicz et al. (2003b) compared the physiology of ko mice, where the deleted gene was known to produce a novel target for each of the 24 new drugs in the developmental pipeline of the 10 largest pharmaceutical companies, and found that 85% of these targets demonstrated a sound biologic rationale for the selected disease. transgenic and ko mice have been used successfully to screen new drug candidates for safety and to elucidate basic mechanisms of toxicity. a large number of drug metabolizing enzyme (dme) ko lines have been employed in safety screening. dmes may be involved in the safe metabolism of a drug or they may generate toxic intermediates. removal of the dme gene may result in the animal being more sensitive to the test article, and it may provide protection against toxic effects of the drug. for instance, ko mice lacking the nqo-1 gene have increased menadione toxicity, and mice lacking the cyp2e1 gene are resistant to acetaminophen hepatotoxicity (henderson and wolf 2003) . murine models are also available for evaluation of chemical mutagenicity (big blue mouse; stratagene, la jolla, ca and muta mouse; covance research products, denver, pa). these marker genes are present in mice of different genetic backgrounds (bolon 2004) . ko lines have been created with the increased sensitivity to chemically induced carcinogenesis. mice carrying a single p53 allele, or over expressing ha-ras, or having a complete deletion of the xpa (xeroderma pigmentosum) gene, have all been used for screening xenobiotics in vivo (bolon 2004) . these same transgenic and ko models are useful in screening environmental chemicals for toxicities (jacobson-kram et al. 2004) . it is clear that the mouse will continue to be essential for discovery and in evaluating the safety of new drugs. equally relevant is the development of clinical chemistry assays needed to study the associated metabolic events in mice (especially in transgenic and ko lines) and assess serum/plasma biomarkers. these biomarkers should either be quantitative measures of the biologic effects (which provide informative links between mechanism of action and clinical effectiveness) or surrogate markers (which are quantitative measures that predict effectiveness). in this arena, the field of proteomics is likely to make a dramatic impact on clinical chemistry. although we hope all readers of this chapter will benefit from this section on assays and instruments, the primary purpose of this chapter is to briefly introduce the reader to areas where methods in clinical chemistry are changing and provide sources of information for services, reagents (including test kits), and instrumentation, specifically for testing biomarkers (including traditional analytes) in mouse serum, plasma, or urine. those seeking a detailed description of clinical chemistry methods and instruments should refer to the fundamentals of clinical chemistry (burtis and ashwood 2001) . some dramatic changes have occurred over the past two decades that have revolutionized the discipline of clinical chemistry. historically, most analytes were measured by a colorimetric end point assay (based on the binding of an analyte to another molecule creating a new substance that absorbs light of a specific wavelength). some of these analytes and the newer biomarkers are now measured by enzymatic tests that have higher specificities and sensitivities. another change has been the substitution of electrochemical assays, such as ion-selective electrodes, for flame photometry used in the quantitation of sodium and potassium. perhaps the change that has had the largest impact on clinical chemistry is the development of monoclonal antibodies and their use as reagents in immunoassays (to be discussed later). many commercial companies now offer services that measure analytes and biomarkers in the blood of mice using a combination of colorimetric-, enzymatic-, electrochemical-, and immunologic-based methods, and many of the instruments used are capable of running multiple assay types simultaneously. the use of laboratories that offer validated assays specifically for mouse blood is important to ensure accurate and precise results. a summary of a few laboratories that provide validated murine assays is presented in table 6 -1. the increasing availability of mouse-specific reagents has resulted in many new assay techniques that provide a high degree of sensitivity and specificity. growth in the number of reagents capable of quantitating analytes in mouse serum is illustrated in table 6 -2. techniques employing nonisotopic labels for detection such as enzyme cascade, fluorescence, chemiluminescence, and electrochemiluminescence, are rapidly replacing older radioimmunoassay (ria) techniques. enzyme immunoassays, such as enzyme-linked immunosorbent assay (elisa), enzyme-multiplier immunoassay technique (emit), and cloned enzyme donor immunoassay (cedia), provide quantitative results based on photometric methods. enzyme immunoassay is popular because it generates compounds that can be quantitated photometrically. typical enzymatic labels include ~-galactosidase, horseradish peroxidase, alkaline phosphatase, and glucose-6-dehydrogenase. in addition these elisa test kits are compact, easy to use, and quantitated with inexpensive instruments. these kits (usually in a well format) are available for quantitation of a wide range of mouse serum and plasma biomarkers (table 6-3) . fluoroimmunoassay (fia), which utilizes a fluorescent molecule as an indicator label, was previously subject to problems associated with background fluorescence. today this problem has been largely resolved by using chelates of lanthanide as a label. modifications of fia have eliminated the need to separate free from bound label (homogenous assay). chemiluminescent and electrochemilumiscent immunoassays are similar to enzyme immunoassays, except that quantitation of results is based on the emission of light after a chemical label is exposed to an oxidation reaction (chemiluminescence) or to an electrochemical reaction (electrochemiluminescence). the number of biomarkers that can be quantified by commercially available test kits based on immunoassays is likely to grow at unparalleled rates as a result of proteomics research where mice are the favored model. mulitplex immunoassay is a unique technology that combines four distinct components in a manner that allows for simultaneous analysis of up to 90 different analytes from 50 ktl of serum/plasma. the core component is an inexpensive, consumable, 5.6 ~tm diameter polystyrene microsphere that are encoded into 100 different fluorescent color sets using two fluorophores (red and infrared) at multiple concentrations. the second component is a biologic assay (combined with an mmp-3 (matrix metalloproteinases) pro-mmp-9 mmp-9 tissue inhibitor of metalloprotease 1 (timp-1) orange fluorescent reporter molecule) that is built onto the surface of the microspheres. a diverse range of biologic assays can be built onto the microsphere surface, including immunoassays, nucleic acid assays, enzymatic reaction assays, or receptor-ligand analysis assays. the third component is a flow cytometer that focuses the microspheres into a single file in front of a two interrogating lasers, which allow for high throughout. one laser is a red diode emitting at 635 nm, which illuminates each microsphere. the resulting red and infrared fluorescence provides classification information for that particular microsphere set. the other laser is a green yag diode emitting at 532 nm that excites the orange fluorescent reporter molecules of the surface of the microsphere, providing a quantitative signal for that particular biologic assay. the last component is digital signal processing data acquisition hardware that provides the speed necessary to read the microspheres at up to 5000 per second. because multiplex technology can analyze a wide range of biomarkers simultaneously, dynamic reference results (plasma profiles) can be developed based on the changing concentrations of the biomarkers during the course of a disease. the known and potential applications of developing plasma profiles of diseases are powerful. plasma profiles can be used to screen and identify diseases (especially during the early development of a disease) and characterize the efficacy of drugs targeted against these diseases. multiplex technology can help elucidate new biomarkers of disease. furthermore, plasma profiles could also potentially be developed to monitor the blood concentrations of drugs as well. investigators seeking additional information on availability of reagents and test kits should refer to "clinical laboratory reference" (nelson 2006 ) and the linscott's directory of immunologic and biologic reagents (linscott 2005) . sections of these two references provide the names and addresses of sources. the catalogs of individual companies may be fruitful, but many reagents for use in human blood and have not been validated for use in other species (including the mouse). each of the various instruments mentioned in this section may be found in the 8th annual analyzers buyer's guide (advance for administrator of the laboratory 2003). this guide lists all manufacturers and gives detailed information on each instrument including technology platforms, methods used, through-put capability, purchase price, maintenance costs, reagent package cost, as well as a variety of options available. contact information, including web site, is available for each manufacturer. additional information regarding clinical chemistry on animal blood is available on the web site of the division of animal clinical chemistry at the american association of clinical chemistry (www.aacc.org/divisions/animal). with a mean body size of 35 g, adult mice have approximately 2.4 ml of blood volume, allowing 75 ~tl to be removed weekly without consequence to health and welfare (loeb 1997; loeb and quimby 1999) . four blood collection sites for acquiring 75 ~tl of blood repetitively from adult mice include the orbital plexus, the tail vein, the jugular vein, and cardiac puncture (quimby 1999b) . general anesthesia is recommended for orbital plexus and cardiac puncture, although terrilrobb et al. (1996) claim that topical application of proparacaine hydrochloride is an acceptable procedure for collecting orbital plexus blood. the jugular vein is punctured using a lancet directed at the rear of the jaw, exposing the jugular vein and its tributaries and the submandibular and facial veins (golde et al. 2005) . collection of blood from any of these veins at this site works well, and the animal does not require anesthesia or a restraint device. the blood should be collected with a small centrifuge tube or capillary tube. nerenberg and zedler (1975) describe a vacuum apparatus used to collect larger volumes of blood from the tail vein. lewis et al. (1976) found that heparinization of mice before tail bleeding increases the yield. prewarming the mouse under a lamp or through immersion in warm water facilitates tail bleeding. the use of heparinized microhematocrit tubes, during tail or orbital plexus bleeding can maximize the plasma volume with minimal hemolysis (quimby 1999b) . hem and smith (1998) recommend a saphenous vein prick method over the orbital plexus method for collecting repeated small volumes from conscious mice. macleod and shapiro (1988) used indwelling fight atrial catheters for repetitive bleeding of conscious, unstressed mice. disadvantages of specific procedures have been described. sakaki et al. (1961) reported sympathetic nervous system stimulation associated with tail bleeding and the mixing of venous and arterial blood with the orbital plexus method. patrick et al. (1983) found that, compared to jugular vein collection, cardiac puncture was associated with higher plasma glucose concentrations and lower creatine kinase (ck) activity. plasma glucose concentrations are higher in blood collected from the orbital sinus compared to the tail vein. differences associated with the method of anesthesia are also important. halothane, methoxyflurane, isoflurane, and pentobarbital sodium have been widely used and are considered safe. however, caution should be exercised in the interpretation of certain chemistry values. higher plasma glucose levels are reported with tail vein sampling in mice anesthetized with pentobarbital or methoxyflurane compared with conscious mice (chuang and luo 1997) . plasma glucose levels are higher after collection from the orbital plexus if pentobarbital or proparacaine hydrochloride is administered. methoxyflurane has no glucose elevating effect on samples collected from the orbital plexus. cunliffe-beamer (1983) claims that carbon dioxide narcosis provides sedation and analgesic for 1-2 minutes and is an appropriate anesthetic for orbital plexus bleeding. when greater volumes of blood are required, four collection sites can be used that require anesthesia and subsequent euthanasia of the mouse. these sites include the jugular vein (ambrus et al. 1951) , the abdominal aorta (lushbough and moline 1961) , the brachial artery (young and chambers 1973) , and the heart (cubitt and barrett 1978; mitruka and rawnsley 1977) . blood collection from a newborn mouse can be accomplished by decapitation. injection of two units of heparin subcutaneously several minutes before decapitation may allow collection of up to 40 ~tl of blood (20 ~tl of plasma). although collection of urine is possible in mice, it is impractical due to anatomic and physiologic restrictions. the total urinary bladder volume is less than 0.5 ml, and total urinary output per day is less than 2 ml (jung et al. 2003) , meaning that mice frequently micturate. this restricts the amount urine that can be collected at any one time point and, therefore, will also restrict the number of analytes that can be assessed from any one urine sample. to be able to collect enough urine for assessment from a single live mouse, mice can be placed in a plastic cage without absorbent bedding material and urine then collected from the bottom of the cage after 4-6 h. however, a disadvantage of this technique is that the urine is exposed to the contamination by feces and other environmental contaminants and organisms. pooled urine samples collected from a group of live mice using the same technique can increase the total amount of urine collected but will not allow specific results to be related back to specific individuals from that group. mice removed quickly from their cage and held over a piece of parafilm will frequently void and the parafilm helps prevent the urine from spreading. urine can also be collected directly from the urinary bladder from mice after euthanasia. this technique allows for a sterile sampling, but the volume that can be collected is restricted by total urinary bladder volume (0.5 ml). often, the volume is much less because mice typically void urine from the urinary bladder at the time of death. reference ranges refer to the range of an analyte or biomarker in a population that has not been selected for the presence of disease or abnormality. reference ranges are usually generated from a large number of individuals from a population, so reference ranges for the same analyte or biomarker can vary between two different populations. table 6-4 lists the reference ranges for selected analytes in two inbred strains and an outbred stock. in some situations, reference ranges that have been published or generated by laboratories cannot be relied on to accurately some variables known to adversely affect the host include environmental factors, pathogens, and shipment. in addition, nutrition, time of sample collection, and storage techniques may all contribute to variability. age has an effect on analytes in the mouse. in an early study, barrett et al. (1975) found that serum calcium levels in 4-monthold c3h/fg and a/fg inbred mice were significant higher than 7-month-old mice of the same strains. more recent and more comprehensive analyses completed by loeb et al. (1996) reinforces the effect that age has on clinical chemistry parameters in five inbred strains and two f1 hybrids, including serum protein. the effect sex has on chemistry parameters is most demonstrable with sex hormones, including follicle-stimulating hormone (fsh), luteinizing hormone (lh), and progesterone. table 6 -3 illustrates these differences between male and female mice, and between female mice in estrus and out of estrus. strain-associated changes appearing in healthy animals have been documented for complement components (goldman and goldman 1976) , cholesterol (dunnington et al. 1981; meade and gore 1982) , testosterone (ivanyi et al. 1972) , cortisol-binding protein (goldman et al. 1977) , and serum protein (borovkov and svirdov 1975; loeb 1997) . cyclic biorhythms, whether circadian or ultradian, influence blood levels of various analytes in mice. blood levels of adrenocorticotropic hormone (acth), corticosterone, growth hormone (gh), and lh may peak one or more times daily, and thus special attention must be given to both the time of collection and the order of sampling individuals between groups if between-group comparisons are to be made (loeb 1997) . the degree of hydration, exposure to noise, degree of confinement, and environmental temperatures has all been shown to affect serum chemistry analytes (quimby 1999b) . diet is known to influence the blood levels of many analytes. perhaps the best studied is the effect of atherogenic diets on serum cholesterol. similarly, significant differences in both serum cholesterol and urea nitrogen are seen in mice maindetermine the presence or absence of disease or abnormality, tained on a semipurified (ain-76) diet. the mouse is unique these situations include evaluating analytes and biomarkers in j among mammals because murine muscles do not contain transgenic and ko mouse populations (especially in experiments in which population sizes are small) and evaluating certain analytes and biomarkers (such as those assessing immune function). instead, baseline data compiled from controls (the population of wild-type mice from which the transgenic and ko mice were derived from) should be used. the usefulness of compiled baseline data depends on controlling a large number of variables known to influence chemistry determinations. several studies have demonstrated significant differences in selected serum analyte concentrations with age difference in the same strain, between sexes in the same strain, and among strains (everett and harrison 1983) . carnosine or anserine. carnosine serves as a source of histidine when histidine is restricted. unlike other mammals, mice on histidine-free diets show signs of histidine deficiency. fasting may also affect the levels of certain analytes (quimby 1999b) . the presence or absence (axenic mice) of intestinal microbial flora is associated with dramatic changes in immunoglobulin (ig) levels and may be associated with changes in other analytes as well. for example, axenic mice have significantly lower levels of iga compared to conventional mice (moreau et al. 1982) . the presence of pathogens may be associated with dramatic changes in various analytes, even with subclinical infection. for instance, infection with lactic dehydrogenase-elevating virus (ldv) is associated with major elevations in serum lactic dehydrogenase, isocitric dehydrogenase, malic dehydrogenase, aspartate aminotransferase, and glutathione reductase activities (quimby 1999b ). asummarized from loeb and quimby (1999) . alterations in clinicopathologic parameters can be attributed to stress in mice. landi et al. (1982) found that plasma corticosterone concentrations in mice tested within 48 h after arrival (by plane or truck transport) were significantly higher than tested after 48 h post arrival. mice sensitized on arrival with sheep red cells as an antigen had significantly lower antibody titers, fewer plaque-forming cells, and a decreased delayed type of hypersensitivity reaction when compared to normal mice allowed to acclimate to the facility for 48 h before being sensitized. elevated serum corticosterone levels can arise from excessive handling of mice prior to blood collection. the effect of sample storage at various temperatures and for varying periods has been described. falk et al. (1981) evaluated the effect of storage time (after freezing) on 20 serum analytes in six laboratory species. they found that in the mouse, only creatine kinase activity changed significantly with storage up to 28 days. hemolysis is a common problem during blood collection in mice. hemolyzed samples are associated with changes in various enzymes, such as increased ck activity and decreased lipase activity. to minimize this, everett and harrison (1983) recommend heparinized plasma and careful selection of collection sites for routine chemical determinations. the effect of lipemia, various anticoagulants, and pharmacologic agents on clinical chemistry values has been reviewed for domestic animals (meyer and harvey 1998) . using several methods. regardless of the distribution of data, it is generally useful to describe the limits that include 95% of the test results in a disease-free population. for values exhibiting a gaussian distribution, parametric methods (such as mean and standard deviation) are appropriate. for gaussian distributed data, this is the range that includes two standard deviations above and below the mean. certain murine analytes have non-gaussian distributions and must be evaluated using nonparametric methods. a variety of methods are available, including the percentile method and logarithm-transformed data analyzed with parametric methods. the method of percentile estimates is more vulnerable to bias due to extreme values (outliers) than is the log-transformed parametric method. boyd (1985) asserts that a sample size of at least 120 is required to give 90% confidence intervals using the percentile method, whereas a sample size of 50 may give reliable ranges if parametric analyses are used. neither statistical method just described will replace raw data in certain situations, such as when assessing analytes for immune function, nor when using data derived from wild-type mice as comparison baseline data for transgenic and ko mice. the issue of quality control and test validity has been thoroughly discussed for common domestic animals and is applicable to chemistry determinations in mice (meyer and harvey 1998) . quality assurance in clinical chemistry determinations is important to ensure that consistently accurate and precise results are achieved. everett and harrison (1983) stressed the importance of a clinical pathology quality assurance program that includes regular assays of pooled and commercially prepared pre-assayed sera. in addition, they encourage participation in a subscription quality assurance program, such as with a veterinary laboratory association. the within-day and day-to-day coefficient of variation should be known for each analyte measured. due to the tremendous number of variables known to influence clinical chemistry values in mice, it is often prudent to test adequate numbers of control specimens along with the experimental samples. this technique is often impractical when tests are being conducted strictly for diagnostic purposes, and in those situations, compiled values that are controlled for as many variables as possible may be sufficient. the values that define a reference range for a particular analyte or biomarker in normal or healthy mice may be described this section is intended to serve as a review of specific tests for routine analytes. additionally, a more comprehensive discussion is presented for analytes and biomarkers involved in two areas of translational research of intense interest (obesity/diabetes and atherosclerosis) and novel biomarkers of disease (such as immune function tests). only analytes and biomarkers for which there are currently available commercial tests for mice serum, plasma, and/or urine are discussed; certain analytes and biomarkers for which commercially available tests are not available for the mouse (such as calcitonin) are not covered. for more complete information for the routine analytes, please refer to loeb and quimby (1999) . glucose is the main source of energy in mice. blood glucose concentrations depend on the rates of entry and the removal rate from the blood. the rate of entry is dependent on intestinal absorption of dietary sources of glucose, the breakdown of body glycogen stores (glycogenolysis), and synthesis from gluconeogenic metabolites (gluconeogenesis). the removal rate is mainly dependent on insulin, which is released from [3-cells of the pancreatic islets. insulin promotes cellular uptake of blood glucose (mainly in muscle, liver, and fat) by stimulating the translocation of glucose transporters, glut-1 to glut-7 to the cell membrane. when removed from circulation, blood glucose may either be utilized (to maintain cell function) or converted to fat and glycogen in liver and muscle as an energy store. however, the effect of insulin is modulated by other hormones (such as glucagon, corticosterone, gh, epinephrine, somatostatin, and amylin) that ultimately result in the tight control the levels of blood glucose, depending on tissue demands for energy. glucagon is released from t~-cells of the pancreatic islets in response to low circulating glucose, stimulating the liver to increase circulating glucose through glycogenolysis and gluconeogenesis. corticosterone and gh antagonize the action of insulin. epinephrine suppresses insulin release and stimulates glucagon release and glycogenolysis. somatostatin suppresses both insulin and glucagon secretion, whereas amylin increases blood glucose, blood insulin, and insulin resistance (burtis and ashwood 2001; kaneko 1999) . glucose determinations are generally conducted on fresh serum. however, plasma glucose determination is acceptable if delay of greater than 30 minutes before separation of erythrocytes is anticipated. in this case, fluoride should be used as the anticoagulant because it inhibits glycolysis by erythrocytes. blood glucose in mice is measured using the hexokinase or glucose oxidase. these tests may be performed as an analytical method or by using glucose oxidase coated test strips (for urine glucose), or small portable analyzers (seidelmann et al. 2005) . assessment of long-term average blood glucose levels in mice is also available by rias measuring glycosylated hemoglobin and glycosylated serum proteins (collectively known as fructosamines) (gould et al. 1986 ). the mouse phenome database lists serum glucose levels of 41 strains with blood collected after a 4-h fast on 7-9 week old males and females on a standard laboratory diet. for all mice, the overall mean was 179 + 30.9 mg/dl (naggert et al. 2003) , but blood glucose levels varied with age, sex, and strain. females of a strain tended to have lower levels than males, with lp/j mice showing the lowest values (f = 125 + 22.0 and m = 146 _+ 18.8 mg/dl) and c57b1/10j mice showing the highest values (f = 230 _+ 25.5; m = 263 _+ 57.3 mg/dl). in mice serum glucose levels decrease between the 3rd and 12th month of age and in c57bl/6 and balb/c strains the glucose level rises again after 24 months (loeb 1997) . serum or plasma glucose levels may also vary depending on the site of collection and anesthetic used (quimby 1999b) . patrick et al. (1983) found that compared to jugular vein collection, cardiac puncture was associated with higher blood glucose. differences associated with the method of anesthesia are also important. higher plasma glucose levels are reported after tail vein sampling under either pentobarbital or methoxyflurane compared to sampling conscious mice (chuang and luo 1997) . plasma glucose levels were higher after orbital plexus sampling of mice if pentobarbital or proparacaine hydrochloride was provided. methoxyflurane had no glucose elevating effect on samples collected from the orbital plexus; however, in conscious animals, plasma glucose levels were higher in blood collected by retro-orbital sinus compared to tail bleeding. hyperglycemia (increased blood glucose levels) in the mouse can be due to increased peripheral resistance of tissues to insulin (such as with exogenous corticosteroid administration, increased endogenous corticosterone release, glucagons administration, and gh administration), and diabetes (see discussion later). hypoglycemia (decreased blood glucose levels) can be due to excessive circulating insulin (such as with excessive insulin administration and transgenic mice with [3-cell tumors of the pancreatic islets), reduced glycogen stores (such as with advanced liver disease), excessive glucose use (such as with pregnancy, septicemia, and neoplasia), and reduced glucose intake (such as with starvation and diseases of malabsorption). diabetes in mice is defined as persistent blood glucose levels of greater than 300 mg/dl in fasting mice. this level also corresponds to the renal threshold for glucose urine excretion in mice, so urine glucose assessment is useful in this regard for mice. nonobese diabetic mice (a model of type 1 diabetes) have nonfasting plasma glucose levels in young prediabetic mice between 130-180 mg/dl, which rises to >300 mg/dl between 10-30 weeks of age (leiter 1997) . genetically modified mice have been identified for virtually every ligand and receptor regulating glucose metabolism and, depending on the gene mutation, may exhibit altered levels of plasma glucose. for example, adenosine monophosphate-activated protein kinase (ampk) is a critical enzyme in energy metabolism (including cellular glucose uptake and fatty acid oxidation in muscle, and fatty acid synthesis and gluconeogenesis in hepatocytes). using ko mice, shaw et al. (2005) demonstrated that kinase lkb 1 is an important activator of ampk in the liver under energy-stress conditions, and that ko mice deficient in kinase lkb1 were persistently hyperglycemic. the authors also demonstrated that kinase lkb 1 is the target of the type 2 diabetic therapeutic drug, metformin. glucose tolerance tests (gtt) have been performed in mice. one-, 3-, and 4-h tests have been conducted. in the 1-h test, glucose concentrations are evaluated before and 1 h following the administration of 2 mg/g glucose administered intraperitoneally (ip) (oldstone et al. 1984) . the 3-h test compares pre-injection serum to serum collected at 15, 30, 60, and 120 minutes following administration of 3 mg/g glucose given ip (hotamisligil et al. 1996) . a sensitive 4-h gtt has also been described in which mice are given 10 ml/kg of 10% glucose orally (gates et al. 1972) . the hypoglycemic response to insulin in mice has also been described (hotamisligil et al. 1996) . kaku et al. (1988) measured glucose, glycosylated hemoglobin and insulin levels in six inbred strains undergoing gtt (either in fed or fasted mice). to estimate the number of genes involved in phenotypic differences in glucose tolerance, the least glucose tolerant strain (c57bl/6) was bred to the most tolerant strain (c3h/hej) and f1 hybrids and backcross animals tested. the authors concluded that glucose tolerance in six commonly used inbred strains is a polygenic trait. thrifty genes have been hypothesized to give early human hunter-gatherers a survival advantage by providing an economic mechanism to store energy during periods of famine (zimmet and thomas 2003) . because the most efficient mechanism for energy storage is through promotion of adipose tissue, it seems reasonable that at least some of these "thrifty" genes may function in this manner. another hypothesis holds that during periods of famine it is essential to conserve glucose for use by the brain and that the mechanism responsible involves insulin resistance in peripheral tissues (neel 1962) . should both hypotheses be true it is easy to envision an association between obesity and type 2 diabetes, especially where sedentary lifestyle and unrestricted access to food occur together (lazar 2005) . one candidate thrifty gene encodes the hormone leptin. leptin is produced by adipose tissue, and its absence leads to obesity and insulin resistance. during times of high adipose storage blood levels of leptin are high and it promotes energy metabolism and inhibits food intake. the opposite occurs during starvation. but leptin is only one of a number of adipokines, secreted by adipose tissue, which aid in the regulating appetite and metabolism. in fact the location of the adipose tissue, the size of average adipocytes, and adipocyte metabolism of glucose and corticosteroids each modify the endocrine function of adipose tissue (lazar 2005) . among the proteins secreted by adipose tissue are adiponectin, adipsin, resistin, visfatin, tumor necrosis factor-a (tnf-a), interleukin-6 (il-6), macrophage chemoattractant protein-1 (mcp-1), plasminogen activator inhibitor-1 (pai-1), angiotensinogen, saa, and a-acid glycoprotein. like adipocyte-derived free fatty acids, which have been shown to contribute to insulin resistance in liver and muscle, most of these proteins are capable of modulating glucose metabolism and insulin action. adiponectin and visfatin work synergistically with insulin to enhance glucose uptake by muscle and block glucose synthesis by the liver (hug and lodish 2005) . blood levels of visfatin increase in obesity and the cytokine can bind to and stimulate the insulin receptor (fukuhara et al. 2005) . blood levels of adiponectin negatively correlate with body mass and are lower in obese humans and mice, suggesting that reduced mrna expression of the adiponectin gene may be involved with obesity (masaki et al. 2004) . increases in adiponectin downregulate the hepatic expression of tnf-a. it mediates its antidiabetogenic effects via receptors on peripheral tissues, especially liver. tnf-a, resistin, and il-6 each induce resistance to insulin. however tnf-a also suppresses expression of adipocyte specific fred w. quimby and richard h. luong t genes; resistin maintains glucose during fasting; and il-6 production increases in the obese. the cytokines tnf-~ and il-6 are proinflammatory, are also produced by monocytes, and act on the liver to produce acute phase reactants. they also induce suppressor of cytokine signaling-3 (socs-3), an intracellular signaling molecule that impairs neuronal signaling by leptin and insulin, and thus causes resistance to the central actions of both hormones (schwartz and porte 2005) . resistin mediates its effects principally by decreasing the expression of gluconeogenic enzymes in the liver (banerjee et al. 2004 ). certain cytokines, increased endoplasmic reticulum stress, chronic hyperglycemia, chronic hyperlipidemia, and oxidative stress may all induce apoptosis of insulin producing ~l-cells in the islets of the pancreas (rhodes 2005) . insulin receptor substrate-2 (irs-2) is a key molecule promoting ~-cell growth and survival. these molecules act immediately downstream from surface receptors for insulin and insulin-like growth factor-1 and inhibition of irs-2 has been shown to lead to insulin resistance. inhibition may result from accumulation of reactive oxygen species in ~-cells chronically exposed to increased glucose metabolism or from chronic exposure to elevated fatty acid (which through production of long chain acyl-coa active protein kinase c-isoforms degrade irs-2). leptin has been shown to modulate interleukin-l~ (il-i~), a potent inducer of apoptosis. tnf-t~ and il-6 induce ~-cell apoptosis by activating the transcription factor nuclear factor k:i] (nf-~:[3). centrally the actions of both insulin and leptin take place in the mediobasal hypothalamus, where the neurons exert potent effects on food intake and energy expenditure. here neurons co-express neuropeptide y (npy) and agouti-related peptide (agrp), which stimulate food intake and reduce energy expenditure. leptin and insulin inhibit these neurons. under conditions of reduced leptin and insulin signaling, npy increases, inducing hyperphagia, weight gain, insulin resistance, and glucose intolerance. the anabolic effects of agrp arise from its antagonism of the melanocortin receptors mc3r and mc4r, which serve to limit food intake. blockage of mc3r and mc4r leads to weight gain and insulin resistance. precursor proopiomelanocortin (pomc) and pomc neurons in the arcuate nucleus are stimulated by leptin and insulin and the resultant production of melanocortin and its binding to mc3r and mc4r inhibits food intake and promotes weight loss (schwartz and porte 2005) . however, in the absence of leptin, as seen in lep ~176 mice, neurons of the arcuate nucleus of the hypothalamus are permanently disrupted and treatment in adulthood cannot reverse this defect (bouret et al. 2004) . orexin a (hypocretin-1) and orexin b (hypocretin-2) are neuropeptides produced in the lateral hypothalamus by neurons with axonal projections to many sites including those that control feeding behavior and sleep/wakefulness (taylor and samson 2003) . orexin ko mice develop hypophagia with obesity and insulin-resistant diabetes (hara et al. 2001 ). ghrelin, a peptide made predominantly by the stomach, is also known to act centrally and affect food intake and increase secretion of gh (ghigo et al. 2004; korbonits et al. 2004 ). in the periphery leptin has been shown to specifically repress rna levels and enzymatic activity of hepatic stearoyl-coa desaturase-1 (sld-1), which catalyzes the biosynthesis of monounsaturated fatty acids. this effect was found to be an important metabolic action of leptin (cohen et al. 2002) . leptin resistance, a common feature of obesity in mice and humans, has also been shown to result, in part, from the shedding of membrane-bound hepatic leptin receptors into the plasma, where soluble receptors modulate circulating leptin levels and possibly its biologic activity (cohen et al. 2005) . thus the connections between factors regulating obesity and insulin resistance (diabetes) are complex and occur both centrally and peripherally. further investigations in mice will involve quantifying glucose, adipokines, insulin, leptin, soluble leptin receptor, and sld-1. for further discussion of mouse models of obesity and diabetes please refer to chapter 19 in this text. metabolism and food intake insulin, leptin, amylin (also called islet amyloid polypeptide, iapp), glucagon, ghrelin, obestatin, orexin a, orexin b, gh, and corticosterone have been measured in mice, and ria and elisa kits are commercially available (see table 6 -3). in addition, these hormones may be measured as part of a multiplex panel (see "multiplex technology" section). leptin values in c57bl/6, 129, and fvb/n strains range from 1-3 ng/ml, insulin values range from 2.2-5.2 ~t iu/ml, corticosterone levels range from 5-40 ~tg/dl, and gh ranges from 1-90 ~tg/ml. epinephrine is measured by ria, and the range for mice is 0-200 pg/dl (depaolo and masoro 1989). adipokines secreted entirely by adipose tissue include adiponectin, adipsin, and resistin. adiponectin and resistin have been measured in mouse serum and commercial elisa test kits are available for this species. a polyclonal antibody that cross-reacts with a conserved sequence of mouse adipsin has been created (searfoss et al. 2003) . tnf-ct, il-6, and visfatin are adipokines that are synthesized also by macrophages and lymphocytes; as such they provide a common link between regulation of obesity, resistance to insulin and inflammation (lazar 2005) . both tnf-t~ and il-6 may be measured by commercially available elisa kits (see the "cytokines and chemokines" section). a method has also been published for measurement of visfatin (fukuhara et al. 2005 ). reagents for quantifying the soluble leptin receptor and sdl-1 are not commercially available at this time, although methods for measurement of these analytes in mouse serum have been published (cohen et al. 2002; cohen et al. 2005 ). the four main types of lipids in plasma are free cholesterol, esterified cholesterol, triglycerides, and phospholipids. lipids are derived from the diet (mainly from long-chain fatty acids), although endogenous recycling (mainly in the liver) occurs. plasma lipids have poor water solubility; thus, they require water-soluble protein molecules for their transport in plasma. the complex of plasma lipids and proteins are known as apoproteins (also known as apolipoproteins), which contain a core of nonpolar lipids surrounded by a surface layer of phospholipids, free cholesterol, and apoproteins. apoproteins are classified according to physical and chemical parameters, and include (in order of increasing density) chylomicrons, very low-density lipoproteins (vldl), intermediate density lipoproteins (idl), low-density lipoproteins (ldl), and high-density lipoprotein (hdl). particle density is proportionally related to the amounts of phospholipid, protein, and triglycerides they contain; increasing particle density corresponds with increasing proportions of phospholipid and protein, and decreasing density corresponds with decreasing proportions of triglyceride. chylomicrons and vldl are often referred to as triglyceride-rich lipoproteins. apoproteins on the surface of the particles serve as ligands for receptors, cofactors of enzyme interaction and structural components (wagner et al. 1999 ). table 6 -5 lists the mouse apoproteins and describes their known function. the largest lipoprotein particle is the chylomicron, which transports dietary lipids in the form of triglycerides from the intestines. chylomicrons contain apoproteins a and b48 and are absorbed into the lymphatics from the intestine, eventually entering the blood. they deliver fatty acids to the tissues with assistance from lipoprotein lipase (lpl) and release glycerol into the blood. as chylomicrons lose triglycerides they become smaller and are called remnants. these remnants acquire apoprotein e from plasma hdl and are rapidly cleared by the liver by the apoprotein e receptor or chylomicron remnant receptor. vldl is the second most prevalent lipoprotein particle in normal mouse blood (after hdl) and it transports triglyceride from liver to extrahepatic tissues. vldl is synthesized in the liver with apoprotein b 100, c, and e attached. lpl aids in the release of fatty acids (from triglycerides) to peripheral tissues. ldl, which is present in very low concentrations in normal mice, carries cholesterol to extrahepatic tissues. ldl contains apoprotein b 100 and is the primary source of cholesterol deposited in the intima of arteries in mice. apoprotein b 100 binds to the ldl receptor (ldlr). hdl is synthesized in the liver and contains large amounts of free cholesterol and apoproteins a, c, and e. during metabolism hdl free cholesterol is esterified by the action of lecithin:cholesterol acyltransferase (lcat). the exchange of cholesterol ester for triglycerides (from vldl) results in a less dense hdl subfraction that is completely removed from the circulation by the liver. exchange of esterified cholesterol for triglycerides is mediated by cholesteryl ester transfer protein (cetp) in humans; however, this activity is absent in mice. phospholipid transfer from vldl to hdl is mediated by phospholipids transfer protein (pltp). high levels of soluble hepatic lipase are found in mouse plasma (lusis, 2000; wagner et al. 1999) . hdl is thought to transport cholesterol from peripheral tissue to liver by a process known as reverse cholesterol transport (rct). this process is believed to be facilitated by the adenosine triphosphate (atp)binding cassette transporter a1 (abca1), which transports phospholipids and cholesterol to the acceptors apoprotein a-1 and apoprotein e (aiello et al. 2002) . hdl also serves as a reserve of apoprotein c and apoprotein e necessary for vldl metabolism. table 6 -6 lists the apolipoprotein receptors found in mice and describes their ligands and functions. atherosclerosis is a major factor in heart disease, stroke, and peripheral vascular disease in humans, and as such, is the principal cause of death in western countries. the etiology is complex, involving both genetic and environmental components. although there are some examples of single gene defects in humans that lead to atherosclerosis, these conditions are rare and do not explain disease prevalence. atherosclerosis is characterized by the formation of plaques in the intima of large and medium size arteries, usually in locations where blood flow is disturbed. the plaques contain a variety of cells including endothelial cells, monocytes or macrophages, smooth muscle cells, and lymphocytes, which secrete products that modulate progression of plaque formation. in addition plaques contain a complex mix of collagen, proteoglycans, occasional cartilaginous tissue with calcification and lipoprotein (primarily ldl). disease risk correlates directly with elevated circulating levels of ldl cholesterol and risk is indirectly correlated to levels of hdl. thus, although ldl promotes atherosclerosis, hdl protects against it (national cholesterol education program 1993). the combination of high ldl and low hdl is termed dyslipidemia and is found in human patients with insulin resistance syndrome. mice do not develop spontaneous atherosclerosis, because they have low circulating levels of ldl and lack cetp activity. however, genetically engineered mice that over-or under-express genes involved in lipid metabolism have contributed greatly to our understanding of lipoprotein metabolism (marschang and herz 2003) . in particular, transgenic mice have been developed with plasma lipid profiles that are similar to those of humans with atherosclerosis (breslow 1994 (breslow , 1996 . in humans and transgenic mice with elevated ldl (or low hdl), ldl passively diffuses across the endothelium, especially in areas where endothelial cell morphology has been altered by the sheer forces generated by blood turbulence. once ldl is within the intima there is an interaction between ldlapoprotein b and matrix proteoglycans resulting in ldl trapping. ldl undergoes modifications associated with oxidation, lipolysis, proteolysis, and aggregation that contribute to inflammation and uptake of ldl by tissue macrophages. lipoxygenases (los) insert molecular oxygen into polyenoic fatty acids producing hydroperoxyeicosatetraenoic acid (hete). endothelial cells release hete into the vessel wall, where it initiates oxidation of ldl. further oxidation of ldl is aided by myeloperoxidase and sphingomyelinase. mice lacking los have diminished atherosclerosis. macrophages and monocytes recognize oxidized ldl via their surface scavenger receptors and become loaded with cholesterol ester, forming a fatty streak appearing along the vessel wall. hdl, on the other hand, removes excess cholesterol from peripheral tissue and inhibits lipoprotein oxidation. hdl carries paraoxonase, which degrades oxidized phospholipids (lusis 2000) and protects against subsequent oxidative damage. oxidized ldl stimulates endothelial cells to express adhesion molecules (such as icam, vla-4, and vcam) and monocyte colony-stimulating factor (m-csf) on their luminal surface, which attracts additional monocytes and lymphocytes. oxidized ldl also inhibits nitric oxide production. infiltrating thymic-derived lymphocytes (t cells) and macrophages initiate migration of smooth muscle cells from the media to the intima, where they synthesize matrix components. mcp-1 is also released by macrophages, inducing monocyte differentiation, migration, and scavenger receptor expression. the accumulation of excess free cholesterol can be inhibited by activation of acyl coa:cholesterol acyltransferase (acat)mediated cholesterol esterification and cellular cholesterol effiux. one mechanism responsible for cholesterol effiux is secretion of apoprotein e by macrophages that promotes effiux via hdl. if this fails, as in the case of cholesterol-laden macrophages in plaques, apoptosis of the macrophages ensues. loading of the endoplasmic reticulum with free cholesterol activates er resident protein kinase and the unfolded protein response (upr) that initiates apoptosis through activation of caspase-12 (feng et al. 2003a) . as macrophages undergo apoptosis they create a necrotic core in the plaque, promoting extracellular cholesterol cleft formation. smooth muscle cells create a fibrous cap over the plaque near the luminal surface. the lesion advances as t cells, activated by cd40-cd40l ligation, release cytokines such as interferon-3, (ifn-y) and tnf-ct. these substances activate matrix degrading proteases and adhesion molecules, promoting additional inflammation. as the inflammation progresses the fibrous cap becomes compromised, leading to physical rupture and the generation of a thrombogenic surface. oxidized ldl increases the production of tissue factor that, on the thrombogenic surface, initiates the coagulation cascade and thrombus formation. each of these details have been elucidated using genetically modified mice (auerbach et al. 1992; choudhury et al. 2004; feng et al. 2003b; lusis 2000; reardon and getz 2001; tailleux et al. 2003; trigatti et al. 2004) . for a further discussion of these models readers are urged to read chapter 16 in this book. for measurements of plasma lipids, serum is the preferred sample. serum can be stored at 4~ for 5-7 days without adverse effect on measurements. a. serum cholesterol and triglycerides serum cholesterol in mice can be measured using the enzymatic oxidation method of roschlay (meade and gore 1982) , the lipid research clinic program protocol (morrisett et al. 1982) , or the abell technique (mitruka and rawnsley 1977) . the most common enzymatic method employs cholesterol ester hydrolase (to convert cholesterol ester to cholesterol), cholesterol oxidase (which oxidizes cholesterol to hydrogen peroxide and cholest-4-en-3-one) and peroxidase (which catalyzes a reaction involving hydrogen peroxide, phenol and 4-aminoantipyrine to form the dye quinonelmine). all of these components of this test are combined in a single reagent mix. the dye absorbance is measured at 500 nm (choudhury et al. 2004) . triglyceride (tg) levels, which are mainly reflective of the tg content of chylomicrons and vldl in the mouse, are quantified using a variety of enzymatic methods. the most popular method combines lipoprotein lipase, glycerol kinase, and glycerol-phosphate oxidase with peroxidase and 4-aminoantipyrine (usually in two reagents). in this method, serum tgs are converted to glycerol by lpl via hydrolysis. next the glycerol is phosphorylated in an atp-dependent reaction catalyzed by glycerol kinase. glycerol-phosphate is catalyzed to dihydroxyacetone and hydrogen peroxide by glycerophosphate oxidase and peroxidase catalyzes the final reaction in which peroxide and 4-aminoantipyrine are converted to a stable dye and absorbance can be read at 500 nm (tsimikas et al. 2000) . reagents for both total cholesterol and total tgs may be used in an automated analyzer or purchased directly from a chemical company with instructions for manual laboratory analysis. total serum cholesterol and tg levels vary by strain, gender, age, length of fast, and diet. jiao et al. (1990) studied total plasma cholesterol (tpc) and tg levels of various inbred strains fed a standard diet. after 18-20 h of fasting, tpc levels ranged from 55 mg/dl (akr/j) to 128 mg/dl (nzb/b 1nj), and tg levels ranged from 13 mg/dl (c57bl/6) to 67 mg/dl c3h/hej; each much lower than seen in normal humans. albers and piagen (1999) quantified total cholesterol levels in 15 strains give a standard laboratory diet and fasted for 4 h before blood collection. levels (in 6-to 8-week-old mice) varied greatly between strains with c57blks/j females having the lowest level (39 mg/dl) and nzb/b 1nj males having the highest levels (127 mg/dl). for a given strain, males always had higher levels than females. trends in total hdl levels in the same strains paralleled total cholesterol levels. triglyceride levels also varied greatly among strains but did not parallel cholesterol levels. among the strains tested, c57bl/6j mice had the lowest tg levels (71-80 mg/dl) and c3h/hesnj the highest (f, 162 mg/dl; m, 231 mg/dl). differences between genders of the same strain were less pronounced, compared to cholesterol and males generally had higher levels than females. a transient cause of plasma lipid elevation (hyperlipidemia) in the mouse is related to recent feeding (postprandial hyperlipidemia), especially on a high-fat diet. causes of persistent hyperlipidemia include diabetes, sustained feeding of a high-fat diet, and nephrotic syndrome. b. hdl, ldl, idl, and vldl levels of individual apolipoproteins can be measured by density gradient ultracentrifugation combined with either electrophoretic, immunologic, chemical, or morphologic analyses. although published reference ranges are not available, the distribution and characterization of murine apoproteins has been reported (camus et al. 1983 ). similar methods were used to evaluate plasma apoproteins in mice consuming atherogenic diets (moltisett et al. 1982) . more recently the profile of murine plasma lipoprotein cholesterol has been determined by fast protein liquid chromatography with on-line post-column analysis of superose 6 gel-filtration eluates (sehayek et al. 2003; strauss et al. 2001) . in contrast to humans, mice normally have low levels of ldl in their plasma (15 mg/dl), with the major plasma lipoprotein being hdl. the low ldl concentration is due to editing of 70% of apoprotein b mrna transcripts in mouse liver leading to apoprotein b48 containing particles that are cleared much faster than ldl in humans (lusis 2000) . in contrast, human apoprotein b mrna transcript editing only occurs in the intestine. a much simpler methodology has been developed to measure hdl and non-hdl lipoproteins in mice, based on precipitation and enzymatic analysis. when phosphotungstate and magnesium salt is added to serum (or plasma), all lipoprotein, except hdl, is precipitated and can be removed. the remaining solution is then tested for cholesterol as described previously. the non-hdl-cholesterol component is calculated by subtracting hdl-cholesterol from total cholesterol in the nonprecipitated sample (sehayek et al. 2003) . some strains develop elevated cholesterol levels associated with increases in non-hdl levels after consuming high fat diets (breslow 1994) . c. mouse apoproteins commercial antibodies against mouse apoprotein a1 and apoprotein a2 are available and elisas have been described for the measurement of these apoproteins in plasma and in hdl fractions of plasma (dansky et al. 1999) . plasma apoprotein a1 levels can also be measured by multiplex analysis. an assay for mouse apoprotein j (apoj or clusterin) has also been described (navab et al. 1997) . apoj, which is a ubiquitous glycoprotein postulated to have multiple functions, is associated with hdl and is the amyloid-associated protein associated with amyloid plaque formation in alzheimer's disease in humans. d. other analytes associated with lipid metabolism and atherosclerosis in mice elisa kits are commercially available for the quantitation of many mouse coagulation proteins including: fibrinogen, factor vii, d-dimer, tissue factor, and von willebrand's factor antigen. elisa kits are also available to quantify many murine products of arachidonic acid metabolism including hete. reagents, elisa kits, and multiplex assays are available for murine inflammatory cytokines and chemokines (see the "cytokines and chemokines" section), as well as monocyte colony stimulating factor. dansky et al. (1999) have described assays for the quantitation of murine aryl esterase and paraoxonase. table 6 -7 lists the mouse enzymes involved in lipid metabolism with references that describe their measurement. the innate and adaptive immune responses have been extensively studied (see volume 4 of this series) and therefore we will not attempt to describe all the features of mouse immunity in detail here. readers are directed to volume 4, molecular and cellular immunology of the mouse, for a general overview, and the 15 chapters that follow for a more detailed description. this section describes the growing number of quantifiable soluble serum proteins and lipids associated with immunity and inflammation in the mouse (see table 6 -3). furthermore, in addition to immunodeficiency, autoimmunity, and allergy, investigations of atherosclerosis, obesity, diabetes, cancer, as well as infectious diseases, each have immunologic and inflammatory components. 1. immunoglobulins (ig) as in humans, mouse immunoglobulins (ig) are molecules composed of four polypeptide chains; two of lower molecular weight called light (l) chains, and two with higher molecular weight called heavy (h) chains. disulfide bonds link one l chain to one h chain, and the two h chains to each other. immunoglobulin h chains are composed of four to five domains, including an n-terminal variable region domain and four constant-region domains. in addition, structural differences in the constant-region domains of the heavy chain are used to classify the five different classes of immunoglobulin, igm, igg, iga, ige, and igd. l chains are composed of only two domains and structural differences in these domains are used to classify l chains as either kappa or lambda type. in the mouse, 95% of serum ig has kappa l chains. any individual antibody secreting b cell (or plasma cell) will make a single ig molecule composed of two identical h chains and two identical l chains. mice make four subtypes of igg: igg1, igg2a, igg2b, and igg3. certain strains, c57bl/6, c57b1/10, sjl, and nod, do not make igg2a but rather make a novel igg2c. the igg subtypes in mice are not exact homologues of human subtypes (mestas and hughes 2004) . prenatal (transplacental) transfer of maternal ig as well as postnatal transfer across the intestinal epithelium is limited to the igg2a, 2b, and 3 subclasses of immunoglobulin and is mediated by neonatal fc receptors (fcrn) located in the placenta or on the intestinal brush border of the proximal small intestine (bankert and mazzaferro 1999) . mouse igg2b fixes complement by the classical pathway and igg 1 and igg2a fix complement by the alternative pathway. ige and igg1 are homocytotropic antibodies capable of binding to receptors on mast cells and basophils and mediate immediate hypersensitivity reactions. although igg and ige circulate in the mouse as monomers, igm circulates as a pentamer and iga circulates as a polymeric molecule. normally very little igd can be detected in serum. serum levels of igm, iga, igg, and ige are influenced by the rate of synthesis and rate of catabolism. like humans, the rate of igg catabolism in mice is directly proportional to the serum concentration of the subclass. the average half-life of murine igg is 4.5 days. the catabolic rate of iga is independent of serum concentration. mice synthesize from 50-130 mg/kg/day of total ig, although this is dependent on strain and level of antigenic stimulation. certain strains have a propensity to develop specific t-helper (th, cd4+) cell subclasses in response to antigenic stimulation, and these strains are referred to as having principally a th1 or th2-1ike phenotype. typically cd4+ lymphocytes modulate immune responses by the cytokines they secrete. those secreting il-1, ifn-y, and lymphotoxin are generally referred to as th1 (and are favored responses for immunity against viruses and intracellular pathogens) and those secreting il-4, il-5, il-10, and il-13 are referred to as th2 (and enhance humoral immunity while suppressing cell mediated immunity). when confronted with the same antigen, balb/c mice exhibit a th2-dominant response, and c57bl/6 mice exhibit a thl-dominant response. il-4 participates in immunoglobulin (antibody) class switching (see volume 4, chapter 5). consequently, th2 strains are the models of choice for investigations of allergic inflammation because they produce higher concentrations of il-4 induced immunoglobulin classes (ige and igg1). the cba/n strain is deficient in its ability to produce igm and igg3. immunoglobulin levels are also greatly reduced in germ-free mice, offspring of mice on zinc-deficient diets, and mice on protein-deficient diets (quimby 1999b) . quantifying the various classes and subclasses of murine ig can be done using various immunoassays including: radial immunodiffusion, ria, or enzyme immunoassay. both ria and enzyme immunoassay have the higher degrees of sensitivity that are needed to accurately quantitate levels of ige and igd in murine serum. enzyme immunoassay has become the favored assay to avoid isotope handling and disposal. the reported concentrations of normal balb/c mice are: igg1 (6.5 mg/ml), igg2a (4.2 mg/ml), igg2b (1.2 mg/ml), igg3 (0.1-0.2 mg/ml), iga (0.7 mg/ml), igm (1.0 mg/ml), and ige and igd are both less than 0.01 mg/ml (bankert and mazzaferro 1999) . in addition, there are many commercial sources of enzyme-linked antibodies that supply reagents (and instructions) for developing assays to measure antigen-specific antibody concentrations by subclass of antibody. the complement system is composed of 40 or more chemically and immunologically distinct proteins capable of interacting with antibodies, certain bacterial products, and cell membranes. a brief summary of this system is described later. please refer to two recent publications (quimby 1999a; turnberg and botto 2003) for more details about the structural and functional aspects of each protein of the complement system. the role of the complement system in mouse immunity is described in volume 4 (overview) of this series. the sequential activation of individual complement proteins from inactive to active substances is a dynamic event called the complement cascade. the ability of the first component of complement, c1, to bind specific sites on the heavy chain of mouse igg2b and activate a sequence of reactions leading to production of a molecular unit capable of lysing a target cell membrane has established the complement system as the primary mediator of antibody-antigen reactions. recent findings suggest that the pentraxins, c-reactive protein (crp), serum amyloid protein (sap), and pentraxin 3 (ptx3) can bind to complement component clq and activate the classical pathway. this may be an important mechanism for removal of apoptotic cells that might otherwise predispose to autoimmune disease (nauta et al. 2003) . each protein of the complement system is normally present in the circulation as an inactive molecule. although the complement cascade may be activated by any of four separate pathways, the central event for each is activation of c3 to c3b yielding a small c3 cz chain fragment. the two c3 convertases are c4b2a (for the classical, lectin, or pentraxin pathways) and c3bbb for the alternative pathway. each c3 convertase cleaves c3 and adds the c3b fragment to the convertase complex forming c5 convertase. cleavage of c5 leads to the membrane attack complex (mac) common to all activation pathways (goldsby et al. 2003) . assemblage of the mac on the surface of a target cell leads to the formation of a large channel enabling ions and other small molecules to diffuse out leading to cell death. the activated components of complement also participate in chemotaxis, phagocytosis, cell adhesion, and b-cell differentiation. there are many notable differences between mice and humans regarding expression of complement components and regulation of cascade activation. mice have both an active and inactive form of circulating c4 and the genes (both on chromosome 17) are designated ss and slp, respectively. many inbred strains do not synthesize active c5 (e.g., dba/2 and a/j strain) due to a post-translational defect and therefore they cannot generate a mac. some strains are deficient in the production of c8 (e.g., dba/2j strain). complement component 6 exists as two allelic forms in mice with 90 and 100 kda molecular weights. similarly there are several notable differences in the regulators of complement activation. most of these regulators are found in the regulator of complement activation (rca) locus on murine chromosome 1 and, as in humans, restrict assembly and stability of convertase enzymes. in mice, decay acceleration factor (daf) is encoded by two genes. however, only the product of daf-1 is widely dispersed on all tissues. this membrane-anchored protein inhibits c3 cleavage by accelerating decay of c3 convertases. in mice it is the major regulator of c3 in the skin (not kidney) and is a ligand for cd97 which, on cross-linking, leads to lymphocyte activation. cd59 is a membrane-anchored inhibitor of c5b-9 formation (mac) and prevents c9 from binding the c5b-8 complex. deficiencies of cd59 in humans lead to hemolytic anemia but not in mice. membrane cofactor protein (mcp) is a major cofactor for factor i in humans causing cleavage of c3b and c4b deposited on self-tissue. in mouse mcp is only expressed in the testis. complement receptors 1 and 2 are encoded by separategenes in humans but are produced by alternative splicing of a single gene in mouse. mice have a complement receptor 1 (cr-1) related gene/protein y (crry) not found in humans, which is a membrane-anchored c3 inhibitor. it is the major regulator of c3 in mouse kidney and has some of the functions as mcp and cr1 in humans. crry has daf activity and serves as a cofactor for factor i (which enzymatically cleaves c3b). the mouse has been widely used in studies on the biosynthesis and molecular biology of individual components of complement. the genes encoding 39 components, subcomponents, receptors, and inhibitors have been identified in mice. the complement components may be quantified by assays designed to measure the functional properties of these proteins or their antigenic properties. tests designed to measure antigenic properties of complement are generally simpler, less subject to error, and less expensive; however, they have the disadvantage of measuring both active and inactive forms of their proteins and therefore may not correlate well with functionally active protein. functional assays measure the ability of the entire classical or alternative pathway, or individual components of pathways to lyse (hemolyze) antibody-coated (sensitized) or noncoated (for the alternative pathway) red cells in suspension or in agarose gel. these assays are precise and sensitive (quimby 1999a) . antigenic assays include radial immunodiffusion, electroimmunodiffusion (rocket electrophoresis), automated immunoprecipitation, crossed immunoelectrophoresis, and more recently elisa (quimby 1999a) . elisa kits are commercially available for quantifying murine c lq, c3, c4, c3a (desarg), and c3a. in addition antibodies are commercially available for these as well as murine c4a, c4d, c5, and c6. complement components c2, c5, c6, c7, and factor b may be quantified using functional assays (quimby 1999a) . membrane-bound complement regulators, crry, cd59, and daf, can all be detected using previously published antibodies (lin et al. 2001 (lin et al. , 2002 . the principle mediator of the lectin activation pathway is mannose-binding lectin (mbl). after binding to mannose residues on the surface of microorganisms, two mbl-associated serine proteases (masps), masp-1 and masp-2, bind to mbl. this complex causes cleavage and activation of c4 and c2 (masp1 and 2 mimic the activities of c14 and cls). commercially available elisa kits are available for murine mbl-a and mbl-c quantitation. other activators of complement include members of the pentraxin family. in mice this may include crp or sap, although the concentration of crp in normal mice is very low. immunologic reagents are available to quantify both pentraxins in mice (gentry 1999; quimby 1999b ). circulating immune complexes (cic) are multimolecular substances composed of antigen, antibody, and activated complement components. in mice, igm, igg1, igg2a, and igg2b have complement activation regions. although cic may form following exposure to circulating foreign antigens, such as those associated with microorganisms, they are also common manifestations of spontaneous autoimmune disease such as that seen in (nzb x nzw)f1, mrl/lpr, bxsb, and krn strains. cic are cleared from the circulation by both cr-1, cr-2, and fcr. tissue deposition of immune complexes may lead to vasculitis. cic have been quantified in mice by capitalizing on their binding to various complement receptors, by precipitation of clq with polyethylene glycol, or by immunoassay. commercially available elisa kits are commonly employed today (quimby 1999b ). more than 100 inbred strains or mutant lines spontaneously develop autoimmune disease or are susceptible to autoimmune disease induction. the details of many of these lines may be found in volume 4, chapters l l and 12 in this series. autoimmune diseases in mice include: thyroiditis, rheumatoid arthritis, sj6gren's syndrome (ss), hemolytic anemia, lupus erythematosus, type 1 diabetes mellitus, experimental allergic encephalitis, oophoritis, orchitis, gastritis, ulcerative colitis, and polyendocrine disease (boyton and altmann 2002; ravirajan and isenberg 2002; sakaguchi 2000) . antibodies directed to self-antigens in the mouse have been quantified using a wide range of methods from immunofluorescence to elisa. table 6 -3 lists the commercially available elisa kits used to quantify murine autoantibodies. those antigenic targets associated with systemic lupus erythematosus include: cardiolipin, double-stranded deoxyribonucleic acid (dsdna), single-stranded deoxyribonucleic acid (ssdna), histone, [~-2 glycoprotein, proliferating cell nuclear antigen (pcna), neutrophil cytoplasmic antibody (canca), ribosomal p, and smith. antigenic targets for arthritis include: rheumatoid factor (rf), collagen type 1, collagen type 2, and ssdna. antibodies against insulin and glutamic acid decarboxylase are seen in type 1 (juvenile) diabetes. mixed connective tissue diseases are characterized by antibodies to ribonuclearprotein (rnp). antibodies to myeloperoxidase (mpo) may be observed in vasculitis. mice with ss develop autoantibodies to ss antigens a and b (ssa and ssb, respectively). panels containing 14 autoantigens are available as multiplex assays for quantifying murine autoantibodies. a. interleukins (il) (ils are cytokines that are secreted by leukocytes and act on other leukocytes. interleukins have been classified based on the secretory cell type (i.e., monokines vs. lymphokines), and they have been classified based on whether they are primarily involved in innate (il-1, il-6, il-12, tnf-t~, ifn-t~), or adaptive (il-2, il-4, il-5, il-10) immunity. commercially available elisa kits are available to quantify most murine interleukins, as demonstrated in table 6 -3. i. interleukin-1 (il-1) il-1 is a name for two proteins, il-lt~ and il-1 [3, that are encoded by separate genes. along with il-1 receptor antagonist, il-18, il-6, and tnf-t~, these proteins modulate acute inflammation. the effects of il-1 are pleotrophic and involve bone remodeling, insulin secretion, appetite regulation, fever induction, neuronal development, and many others. both il-1 o~ and il-1 [3 are secreted as 269-271 amino acid (aa) pro-cytokines that are enzymatically cleaved into bioactive 17-kda segments. unlike il-1 [3, the intact procytokine of il-1 ct is also bioactive, both within the cytoplasm and on the cell surface, where it is anchored to the cell membrane via a mannose glycosylation residue that attaches to the membrane-associated lectin. there is 78% sequence identify between mouse and human il-i~ genes and 58% identity between mouse and human il-1 t~ genes. a third gene encodes il-1 receptor antagonist, a soluble 25-kda molecule with 19% sequence homology to il-lt~ and 26% homology with il-113. mouse il-lra is 75% homologous with human il-lra. there are two il-1 receptors, types i and ii, but only il-1ri is capable of signal transduction. il-lra inhibits the action of il-lt~ and il-1b by binding il-1ri. a 60-kda form of il-1ri has also been described that is soluble and preferentially binds il-lra. il-1rii has no signal transducing associated protein and serves to modulate levels of il-1 t~, il-i[3, and il-lra by binding them on the cell surface. it can also occur as a soluble receptor. with the recent finding of six new members in the il-1 ligand family (in humans), a revised nomenclature for both il-1 ligands fred w. quimby and richard h. luong and il-1/il-18 receptor families has been developed (sims 2002) . further details may be found in volume 4 (overview and chapter 8) of this series. ii. interleukin-2 (il-2) il-2 is a lymphokine secreted by activated t-helper cells. it acts in an autocrine fashion to induce the expression of il-2 receptor on t cells, resulting in t-cell proliferation (cogoli-greuter et al. 2004) . il-2 also acts in a paracrine fashion modulating the activities of b cells, natural killer (nk) cells, and lymphocyte activated killer (lak) cells. il-2 is a glycoprotein of 133 amino acids (in humans) with 63% homology between mouse and human. the il-2 receptor (il-2r) is a multisubunit cellular receptor belonging to the class 1 cytokine receptor family (hematopoietin receptor family). the il-2r has a-, [~-, and y-chains. [3-and y-chains interact to transduce the il-2r signal and the y-chain is shared with receptors for il-4, il-7, il-9, and il-15. iii igg1 in mice and is responsible for the downstream events leading to differentiation and activation of th2 cells. il-4 also induces expression of adhesion molecules like vcam, th2 cytokines such as il-5, il-6, and il-9 and chemokines like eotaxin-1 and-2. il-4 primes mast cells and basophils leading to enhanced activation during allergic challenge (mueller et al. 2002) . homology between mouse and human molecules is low (25%) and each is species-specific in its biologic activity. it induces the growth of b-1 progenitors and igm production by b-1 cells. il-5 induces class switch, favoring production of iga, igg1, and ige. on eosinophils, il-5 induces iga and igg receptors and stimulates leukotriene (lt), c4, and paf secretion, in addition to inducing eosinophil growth and maturation. the receptors for il-5 consist of a ligand binding tx-subunit and a non-ligand binding (common) signal transducing ~-subunit that is shared by receptors for il-3 and granulocyte-monocyte colony-stimulating factor (gm-csf) (sato and miyajima 1994) . vi. interleukin-6 (il-6) il-6 is secreted by a wide variety of cells including t cells, b cells, monocytes, fibroblasts, hepatocytes, keratinocytes, astrocytes, and endothelial cells. it has broad pleiotropic effects on host defense, acute phase responses, immune responses, and hematopoiesis. il-6 is classified as an inflammatory cytokine and based on a helical cytokine structure and subunit makeup, il-6 is the prototypic member of a family of molecules that includes leukemia inhibitory factor (lif), oncostatin m (osm), ciliary neurotrophic factor (cntf), cardiotrophin (ct-1), and il-11. mouse il-6 is 25 kda and contains four cysteines and contains o-glycosylation sites and shares 40% homology with the human molecule (van snick 1990). the il-6 receptor has two subunits, a nonsignal transducing subunit binding with low affinity (~-subunit), and a signal transducing subunit (~-subunit) that does not bind il-6 by itself but participates in high-affinity binding. the soluble il-6r~ chain binds il-6 and the complex induces expression of mcp-1, which attracts monocytes into areas of inflammation (kaplanski et al. 2003) . vii. interleukin-7 (il-7) il-7, previously called lymphopoietin-1, is expressed by stromal cells, especially in the bone marrow and thymus, where it promotes thymopoiesis of t cells and the differentiation of pro-b cells into pre-b cells (aspinall et al. 2004; goldsby et al. 2003) . mouse il-7 has 65% amino acid sequence homology with human il-7 and both proteins exhibit cross-species activity. viii. interleukin-8 (il-8) il-8 is not expressed in the mouse; however, another protein, kc, is secreted and has many properties of the human chemokine, gro, which is known to bind the il-8 receptor (see the "cytokines and chemokines" section). ix. inrelclevlcln-io (il-io) il-10 is the prototypic member of the il-10 cytokine family comprising ill0, il-19, il-20, il-22, il-24 (fisp), and il-26. il-10 is a 178 amino acid protein with an 18 amino acid signaling sequence. both mouse and human il-10s have two intrachain disulfide bonds and form non-disulfide linked homodimers. mouse and human il-10 are 72% homologous. il-10 is a th2 cytokine, which inhibits ifn-y and gm-csf production by th1 cells. additionally it induces cd8 + t-cell chemotaxis, inhibits t-cell apoptosis, participates in iga class switch in b cells, induces histamine release from mast cells, and promotes tnf-tx and gm-csf production by nk cells. il-10 inhibits secretion of the neutrophil chemokines mip-lt~ and mip-i~ and blocks production of il-1~ and tnf-ct by neutrophils. it is immunosuppressive to dendritic cells and induces the differentiation of a subset of regulatory cd4 + t cells (trl) (grouz and cottrez 2003; morel et al. 1997 ). x. il-11, also known as adipogenesis inhibitory factor (agif), is a pleiotropic cytokine with effects that overlap that of il-6. il-11 is a member of the il-6 cytokine family and as such has a four-helix bundle fold motif. it binds to the multimeric il-11 receptor that shares the promiscuous gpl30 signaling ~-subunit with other receptors in this family. the il-11r ~ chain is unique and binds il-11 but does not have a cytoplasmic domain; instead binding leads to homodimerization of the ~-chain that activates the janus kinases. il-11 stimulates proliferation and differentiation of monocytes and megakaryocytes causing thrombopoiesis. it also activates osteoclasts and enhances bone resorption, decreases new bone formation, and stimulates chondrocyte and synoviocyte production. il-11 protects small intestinal epithelial cells from chemotherapy and radiation injury and ameliorates inflammatory bowel disease (schwertschlag et al. 1999) . il-11 inhibits adipogenesis, regulates neuronal differentiation, and regulates t-cell function (enhances th2 and inhibits th1 cytokine production). overexpression of il-11 in the lung causes airway remodeling, fibrosis, and mononuclear nodules analogous to the clinical picture in chronic asthma. il-11 is also required for the uterine decidualization response (robb et al. 2002; zheng et al. 2001) . xi. il-12, also known as natural killer cell stimulatory factor (nksf), is a heterodimeric cytokine composed of a 40-kda (p40) subunit and a 35 kda (p35) subunit. the p40 subunit is shared by il-23, a cytokine with similar activities. macrophage, monocytes, and dendritic cells produce il-12 after activation of toll-like receptors (tlr) on these cells by bacterial ligands. il-12 induces production of ifn-7by th1 and nk cells and intact il-12 skews the balance between th subsets in favor of th1 cells. il-12 binds to the il-12 receptor that is composed of two subunits, [31 and ~2, on the surface of nk and th1 cells. il-12p40 interacts with il-12r[31, and il-12p35 binds il-12r[32. negative feedback regulation of il-12 production involves down regulation of tlr signaling by phosphoinositide 3-kinases (piks). thus il-12 is centrally involved at the interface of innate and adaptive immunity (fukao and koyasu 2003" ottenhoff et al. 2002) . xit, il-13, along with il-4 and il-5, is a member of the type-2 cytokine family and as such is involved in inflammation, mucus production, tissue remodeling, and fibrosis. this single-chain protein shares 58% amino acid sequence homology with human il-13. il-13 is produced by activated t cells, mast cells, and nk cells and promotes th2 responses including synthesis of ige. signaling is mediated by the type-2 il-4 receptor which consists of il-4r~ and il-13rc~i chains. another il-13 binding protein, il-13r~2, strongly inhibits the activity of il-13 (goldsby et al. 2003; mentink-kane and wynn, 2004) . xiii. il-17, also known as cytotoxic t lymphocyte-associated antigen-8 (ctla-8), is produced by t cells and is pleiotropic in activity. il-17 is a 158 amino acid residue polypeptide with a 21 amino acid signal sequence and a mature polypeptide of 137 amino acids. it is a disulfide-linked homodimer. based on the presence of spatially conserved cysteine residues in the il-17 family of proteins, there are six family members in humans and mice, il-17, il-17b, il-17c, il-17d, il-17e, and il-17f (aggarwal and gurney 2002) . like il-17 itself, several of the family members appear to modulate immune function. produced by mouse cd4 ⧠t cells, il-17 induces il-6, mcp-1, prostaglandin-e2 (pge2) and granulocyte colony-stimulating factor (g-csf) by fibroblasts, keratinocytes, epithelial cells, and endothelial cells. it induces icam-1 surface expression, proliferation of t cells, and the differentiation of cd34 + marrow progenitors into neutrophils (fossiez et al. 1998 ). the ubiquitously distributed receptor is a type 1 transmembrane glycoprotein of 830 amino acids in length. xiv. il-18 is a 24-kda, nonglycosylated polypeptide that lacks a classical signaling sequence. its structure resembles il-1 and the propeptide undergoes proteolytic cleavage by interleukin-1 [3-converting enzyme (ice) or another caspase to produce an 18-kda bioactive molecule. there is 64% sequence homology between mouse and human il-18. il-18 induces the production of ifn-7 by t cells and nk cells and the expression of fas ligand (fasl) on a variety of all types. il-18 activates nf-~:[3 and the induction of various chemokines. il-18 plays an important role in the early antibacterial host response (weijer et al. 2003) . xv. and il-20 induces keratinocyte differentiation and proliferation. il-21 is a four-helix-bundle cytokine similar in structure to il-15 and sharing sequence homology with il-2 and il-4. murine il-21 is 57% homologous to human il-21. the il-21 receptor utilizes the common 7-chain. il-21 is produced by the th2 cells. the actions of il-21 are pleiotropic and seen on b cells, t cells, nk cells, and dendritic cells. il-21 induces apoptosis in resting and activated b cells, an effect counteracted by activation of cd40. it also upregulates production of igg1 and inhibits ige, in fact it inhibits many il-4 activities. il-21 also inhibits dendritic cell differentiation. il-21 enhances the activity of activated nk cells and mediates the proliferation and expansion of t-cell subsets. il-22 induces acute phase reactants by hepatocytes and reduces il-4 production by th2 (mehta et al. 2004) . recombinant antigens and antibodies are commercially available for murine il-20, il-21, and il-22. b. the transforming growth factor-~ superfamily (tgf-~sf) of cytoi~nes members of tgf-~ family share 25-40% sequence homology with tfg-~i and a monomeric structure that consists of two antiparallel pairs of [3-strands forming a flat curved surface, a separate long cz-helix, and a disulfide rich core with a characteristic cysteine knot. most tgf-~sf members are disulfide-linked homodimers; however, three members lack the seventh conserved cysteine residue and are not covalent homodimers. members of the tgf-[3sf include tgf-[31, tgf-132, tgf-133, activins, inhibins, bone morphogenic proteins (bmp), growth differentiation factors (gdf), glial-derived neutrophic factors (gdnf), and mtillerian inhibiting substance (mis). tgf-[31 is stimulatory for cells of mesenchymal origin and inhibitory for cells of epithelial or neuroectodermal origin. in the murine immune system tgf-[31 is the mediator of immune suppression via cd4+cd25+tr cells and, at least in the case of suppressing cd8 + effector t cells, involves tgf-[3 receptor ii on these cells (powrie 2004; von boehmer 2005) . in addition tgf-[3 is known to inhibit b-cell proliferation and it promotes isotype switch to iga. oral tolerance to th2 responses (against food allergins) is mediated by tgf-~i1 (mucida et al. 2005 ). tgf-[3 has a wide range of effects on cell growth differentiation and malignant transformation (letterio 2005) . murine tgf-~i 1 may be quantified in serum or plasma using commercially available elisa kits. antibodies are also available which specifically bind murine bmp, activin a, activin c, and gdf-1,-3,-5,-8, and-9, although they are not recommended for elisa development. c. the tumor necrosis factor superfamily (tnfsf) tnf-related ligands share many features but high amino acid sequence homology is not one of them. with the exception of nerve growth factor and tnf-~, all ligands are type ii tramsmembrane proteins (extracellular c-terminus) that contain a short cytoplasmic segment and a long extracellular region. tnf-~ is fully secreted and has a nonfunctional transmembrane segment. tnfsf members form trimeric structures and their monomers are composed of ~-strands oriented into a twosheet structure. receptors for the tnfsf ligands also belong to a superfamily, tnfrsf (gruss and dower 1995) , and are characterized as type i transmembrane proteins (with their amino termini outside of the cell), with extracellular cysteine-rich structural motifs. tnfrsf members exist both as membrane and soluble forms. commercially available elisa kits or matched antibody for development of assays are available to quantify the murine receptors and ligands of the tnfsf discussed in this section. i. tumor necrosis factor-ix (tnf-ix) tnf-tx is expressed as a 26-kda membrane glycoprotein and the soluble glycoprotein is generated by proteolytic cleavage via tnf-ct converting enzyme (tace). the 17-kda homotrimer cleavage product circulates. mouse tnf-tx has 79% sequence homology with human. tnf-tx is expressed widely on tissues throughout the body (goetz et al. 2004) . tnf-tx is a strong mediator of inflammation and immune function, or regulates on growth and differentiation, and is cytotoxic for many transformed cells. ii iii. cd40l cd40l (tnfsf5, cd154) is a 39-kda, type ii, transmembrane glycoprotein that can be proteolytically cleaved to 15-to 18-kda soluble forms with full biologic activity. it forms natural trimeric structures and the mouse cd40l shares 73% sequence identity with human cd40l. cells expressing cd40l include b cells, cd4 + and cd8 ⧠t cells, monocytes, nk cells, and y t cells (toubi and shoenfeld 2004) . on binding cd40, the complex initiates signals important for cell proliferation or apoptosis. cross-linkage between t and b cells allows cd40 to transduce the tyrosine kinases lyn and syk, and activate phospholipase c, ip3, and dag. when combined with other cytokines, ligation of b-cell cd40 provides the second signal allowing differentiation of b cells to plasma cells (goldsby et al. 2003) . iv. cd30l cd30l (tnf5f8, cd153) is a 40-kda glycoprotein with 72% sequence homology between murine and human molecules. cd30l is expressed on monocytes, macrophages, b cells, activated t cells, neutrophils, megakaryocytes, resting cd2 ⧠t cells, erythroid precursors, and eosinophils. ligation to cd30 can induce either proliferation or apoptosis. v. fas ligand (fasl) fasl (also known as tnfsf6), is a 40-kda glycoprotein that, after cleavage, forms a 70-kda homotrimer that is active only in membrane form in the mouse. polymorphisms in fasl also exist and a single amino acid substitution in position 273 (phe to leu) results in the generalized lymphoproliferative disease (gld) mutation. fasl is expressed on cells of the adaptive and innate immune systems, as well as cells of the lung and intestine. there is 77% sequence homology between murine fasl and human fasl (lynch et al. 1994) . ligation of fas by fasl on mature t cells leads to activation of the caspase cascade and apoptosis. this is a major homeostatic mechanism regulating the size of the t-cell pool and for eliminating t cells that repeatedly encounter self-antigens (goldsby et al. 2003) . vi. tnf-relateo activation-induced cytokines (trance) trance, also called rank ligand and osteoprotegerin ligand (opgl), is an osteoclast differentiation factor. mouse and human share 85% sequence homology, and trance is expressed on t cells and t-cell rich organs such as thymus and lymph nodes. vii. tnf-receeror superfamily (tnfrsf) tnfrsf members mediate the cellular effects of tnfsf members. tnfri and tnfrii bind tnf-~ and it appears tnf-~ complexes with lt-[3 and the complex binds to tnfri or lt-[~ receptor. cd40 is associated with b-cell proliferation but is expressed on many cells throughout the body. mouse cd40 shares 62% sequence homology with human cd40; however, the mouse molecule has a 28 amino acid extension of its cytoplasmic tail. cd30 has 480 amino acid residues and a 90 amino acid deletion in the extracellular region compared to human. it is expressed on cd4 + and cd5 + t cells and ligation results in production of il-5. murine fas lacks 8 amino acid residues found in human and shares only 50% sequence homology. soluble forms result from alternative gene splicing and circulate as dimers or trimers. fas is expressed by cd34 stem cells, fibroblasts, nk cells, keratinocytes, hepatocytes, b and t cells (and their precursors), and eosinophils. osteoprotegerin (opg) inhibits the action of osteoclasts and is a secreted member of the tnfrse although it has no transmembrane segment and circulates as a disulfide-linked homodimer. murine troy, also named toxicity and jnk inducer (taj) and tnfrsf19, shares 92% homology with human in its extracellular domains. d. interferons interferons are a group of related but distinct proteins that share more than 95% amino acid sequence homology. members of the type i interferon family share a common cell surface receptor composed of two subunits. commercially available elisa kits may be used to quantify murine io interferon-~ (ifn-~) ifn-~ is induced in a wide variety of cells, including monocytes and macrophages, in response to viral infection. one known inducer is double stranded ribonucleic acid (dsrna). induce resistance to viral replication by binding the ifn-~/~ receptor, which activates the jak-stat pathway, inducing several genes. one of those genes is ribonuclease, which degrades viral rna. binding of ifn-~ to nk cells enhances their lytic activity for virally infected cells. ifn-~ is secreted by leukocytes. iii inreturelcon-'t(ifn-y) ifn-y is secreted by th1 cells, nk cells, and cytotoxic t cells (tc), which activates macrophages to secrete tnf-ct, express class ii major histocompatibility complex (mhc) molecules, and produce antimicrobial activities. ifn-y secretion by th1 also induces antibody-class switch to igg2a, which supports phagocytosis and complement fixation. ifn-y promotes differentiation of tc from cd8 + precursors that will be involved in the effector response to viral infections and intracellular pathogens. ifn-y also inhibits the expansion of th2 cells. ifn-y secretion is induced by successful stimulation of t cells by antigen presenting cells. e. chemo~s chemokines, along with adhesion molecules, are the principle controllers of leukocyte migration and as such directly affect leukocyte retention and relocation during hematopoiesis and at sites of immune defense and inflammatory disease (moser et al. 2004) . chemokine-induced signaling is via g-protein coupled cell surface receptors. although most chemokines are secreted proteins, two chemokines, cxcl16 and cx3cl 1, are membrane bound. two primary subfamilies are recognized based on the arrangement of two nh2-terminal cysteine residues that are either located adjacent to each other (cc) or are separated by a single amino acid (cxc). two minor subfamilies include chemokines with a single cysteine resides (xcl1, xcl2) and a chemokine with three amino acids separating the cysteine residues (cx3cl1). functionally conserved regions of the n-terminus of each member mediate receptor binding and extracellular matrix fixation (or binding cell surface glycosaminoglycans). many chemokines are designated with a name given at the time of their identification; all have also been assigned a name based on their structural motif (cc, cxc, xc, cx3c) followed by l for ligand. receptors are heterotrimers and their activated g-protein subunits stimulate phospholipase c[3, piks, and c-src tyrosine kinases (moser et al. 2004) . receptors are designated by the type of chemokine they bind (i.e., cxc, cc, xc, or cx3c), followed by r. mice lack the cxcr1 family of chemokines and receptors found in humans. table 6 -8 lists the murine chemokine receptors and the known ligands. table 6 -3 lists the murine chemokines for which there are either commercial test kits or matched antibodies for kit development. two main functional groups define chemokines. inflammatory chemokines recruit effector leukocytes to sites of infection, inflammation, and repair. homeostatic chemokines control the navigation of leukocytes during hematopoiesis in the bone marrow and thymus, control homing of cells to spleen, lymph nodes, and peyer's patches during the adaptive immune response and control immune surveillance in peripheral tissues. some chemokines participate in both inflammation and homeostasis and are called dual-function chemokines. many dual function chemokines are highly selective for the recruitment of t cells. other chemokines have ill-defined functions regarding homeostasis and inflammation but participate in other vital activities such as the role of pf4 (cxcl4) in thrombosis and the role of cxcl 10 in gut epithelial cell turnover (cliffe et al. 2005) . most inflammatory chemokines are thought to be induced and the variety of stimuli that induce their expression is broad. by contrast most homeostatic chemokines are thought to be constitutively expressed. an exception is the inducing effect of lymphotoxin and tnf-~ on b-cell attracting chemokine-1 (bca-i, cxcl13), ccl19, and secondary lymphoid tissue chemokine (slc, ccl21), which also participate in inflammation. leukocytes also release several kinds of proteases that degrade chemokines at their n-terminus, resulting in the loss of receptor binding, antagonist generation, or enhancing their biologic function. leukocyte cd26, dipeptidyl peptidase iv is known to act on cxcl9, cxcl10, cxcl11, and cxcl12. murine sulphostin inhibits the action of cd26 and stimulates g-csf and granulopoiesis (abe et al. 2005) . matrix metalloproteases (mmps) are enzymes that degrade extracellular matrix proteins, including stromal cell derived factor-1 (sdf-1, cxcl12) and mcp-1. table 6 -3 lists the murine mmps that can be quantified by commercially available elisa kits. in addition to mmps, cathepsins have been shown to modify chemokines. table 6 -9 lists the murine chemokines and the proteases that degrade them and stimuli that induce them. certain chemokines may antagonize the activity of other chemokines; for instance, three agonists for the receptor cxcr3 are also antagonists for receptor ccr3 (which is agonized by eotaxin [ccl11]). because cxcr3 and ccr3 are differentially expressed on th1 and th2 cells, these chemokines [monokine induced by ifn-y (mig/cxcl9), ifninducible protein-10 (ip-10/cxcl10), and ifn-inducible t-cell chemoattractant (i-tac/cxcl11)] modulate the th subpopulation allowed to enter tissue sites favoring th1 immune polarization (moser et al. 2004) . table 6 -10 summarizes some known effects of murine chemokines. because chemokines and their receptors are known to modulate many inflammatory diseases including the autoimmune diseases, they have become target for new therapeutics. the chemokine antagonist, met-rantes, has been shown to be an effective inhibitor of allergic airway disease in mice. likewise, deficiencies of chemokines and their receptors in mice have modified disease progression in atherosclerosis, autoimmunity, and also prolong allograft survival (mackay 2001 ). in addition, many viral genomes are known to encode structural genes for chemokine antagonists, which appears to be a principal mechanism used by many viruses to evade the host immune system. these present another target for drug intervention for viral infections. finally, pepducins, derived from the intracellular loops of cxcr1, cxcr2, and cxcr4, specifically inhibit receptor g-protein signaling in mice and prevent fatal sepsis and disseminated intravascular coagulation (kaneider et al. 2005 ). certain growth factors (see table 6 -3) and cytokines activate phospholipases after binding their cell surface receptors. these phospholipases act on membrane phospholipids to release arachidonic acid, a precursor for several eicosanoids. arachidonic acid is metabolized by any one of three biochemical pathways: the cyclooxygenase (cox) pathway, which forms pgs and thromboxane, the lo pathway, which forms hetes and leukotrienes (lts), and the cytochrome p-450 monooxygenase pathway, which forms epoxides and hetes. the cox enzymes, cox-1 and cox-2, catalyze the first step in the synthesis of pgs by converting arachidonic acid to prostaglandin h 2 (pgh2). pgh 2 is chemically unstable and is the precursor for enzymatic and nonenzymatic production of pgd 2, pge 2, and pgfza. thromboxane synthetase converts pgh 2 to thromboxane a 2 (txa2) that is quickly converted to thromboxane b 2 (txb2). in vascular tissue, pgh 2 is converted to pgi 2 or prostacyclin by prostacyclin synthetase (natarajan and nadler 2004; reimers 1999) . although cox-1 is constitutively expressed in most tissues, cox-2 is induced by bacterial lipopolysaccharides, il-i~, il-ll~, and tnf-~ (see the "cytokines and chemokines" section). in the circulation, pge 2 and pgi 2 cause vasodilation, whereas pgfza and txb 2 are potent vasoconstrictors. in the kidney, pge1, pge2, pgd2, pgg2, pgi2, and pgh2 produce vasodilation, increased renal blood flow, and urinary excretion of sodium. renal production is increased by pgd2, pge2, and pgi2. pgg2, pgh2, and txa 2 modulate platelet aggregation and, following platelet adhesion, they release catecholamines, serotonin, and adenosine diphosphate, which enhance platelet aggregation. pgi 2 is a potent inhibitor of platelet aggregation. pge1 increases, whereas pge2 decreases, the ability of red cells to pass through capillaries. pge2 and pgfza inhibit the activities of t and b cells and the production of ils and chemokines, which attract monocytes. pgd 2 is a potent inducer of chemotaxis for th 2 cells and plays a major role in allergic airway disease. through the varied activities of vasodilation, vascular permeability, and leukocyte migration, the pgs are potent modulators of inflammation. in addition pge1 inhibits mackay 2001 rossi et al. 1999 niess et al. 2005 mackay 2001 huang and xiang 2004 yamaguchi et al. 2005 insulin secretion and release after glucose challenge (see the "glucose and carbohydrate metabolism" section), and it inhibits the lipolytic effects of glucagons, adrenocorticotropic hormone, and epinephrine (see the lipid metabolism section) and inhibits the secretion of corticosterone, prolactin (prl), gh, thyroid stimulating hormone (tsh), and lh. in fact, pgs have a multitude of effects associated with female reproduction (reimers 1999 table 6 under the action of 5-lo, arachidonic acid is converted to 5-hydroxyperoxyeicosatetraenoic acid (5-hpete) and then leukotriene a4 (lta4), which is unstable. lta4 is metabolized to ltb4 by lta4 hydrolase or to cysteinyl leukotrienes (ltc4, ltd4h, and ltc45) by ltc4 synthase. 5-lo expression is limited to neutrophils, eosinophils, monocytes, and mast cells; however, lta4 hydrolase is expressed in erythrocytes, t cells, platelets, airway and intestinal epithelial cells, fibroblasts, heart, kidney, and adrenal cortex. because the latter cells and tissues do not express 5-lo, lta4 must be delivered to them via myeloid cells by a process known as transcellular biosynthesis (maclouf 1993) . receptors for ltb4 (blt1 and blt2) have been identified and are either widely expressed (blt2) or are confined to myeloid cells, t cells, and lung cells (blt1). in addition to attracting myeloid cells to sites of inflammation, ltb4 is a potent inducer of th1 and th2 t effector cell chemotaxis (as potent as cxcl12 in the mouse) and cd8+t-effector cell chemotaxis (as potent as rantes). like pgd2, ltb4 plays a major role in allergic airway disease (luster and tager 2004) . additional information on murine prostanoids and their receptors is available in recent reviews (jala and haribabu 2004; kobayashi and narumiya 2002) . all the arachidonic acid metabolites discussed in this section may be quantified using commercially available elisa kits (see table 6 -3). e. enzymes ap is an inducible enzyme in which serum activity is increased due to increased synthesis. the exact physiologic function of ap is not known but is thought to transport metabolites across cell membranes. quantification of serum ap is based on a reaction between ap and a suitable phosphorylated substrate that is susceptible to ap activity. as in other species, there are two major forms of ap in mice: intestinal ap (lap) and tissue nonspecific ap (tnap). unlike other domestic animal species, iap activity contributes to serum ap activity, in addition to tnap. lap is located along the brush-border of the enterocytes. intestinal ap activity was shown to vary four-fold between two strains of swiss mice (nayudu and moog 1967) . this difference in activity was under polygenic control and influenced by a strain-specific factor in milk. tnap is found in various tissues, and in each location, post-translation modifications may result in a different isoenzyme. hoshi et al. (1997) used immunohistochemistry to localize these isoenzymes in mice to the following locations: bone tissue (specifically the entire cell surface of preosteoblasts and the basolateral cell membrane of osteoblasts), cartilage (mostly in chondrocytes of the proliferative and hypertrophic zones), the incisors (particularly the cells of the stratum intermedium, the subodontoblastic layer, the proximal portion of secretory ameloblasts, and the basolateral portion of odontoblasts), kidneys (on the brush borders of proximal renal tubules in kidney), liver (on cell membrane of the biliary canaliculi), and the placenta (on trophoblasts). serum ap activity can vary due to the type of assay used, age, sex, and strain. different ap assays vary in the type of substrate used, the ph of the reaction, and the incubation temperature of the reaction, all of which can affect quantitation of ap activity. for example, the hausamen technique can detect renal and intestinal ap activity, but not hepatic ap activity (hausamen et al. 1967 ). loeb et al. (1996 ) and frith et al. (1980 demonstrated that serum ap activity decrease significantly after 3 months of age in balb/c and c57bl/6 mice of both sexes but increase again in very old (36 month old) mice. the high levels of serum ap seen in juveniles is related to increases in the bone ap isoenzymes, which is associated with osteoblastic activity due to rapid growth. picketing and picketing (1984) showed that serum ap activity is lower in males than in females. quantification of serum ap measures the total ap activity from all sources, and therefore elevations in ap activity can be a nonspecific determinant of tissue dysfunction, depending on the tissue (and therefore ap isoenzyme) involved. for example, changes in the serum activity of ap due to liver disease only occur if cholestasis is concurrently involved. mice lacking the gene that modifies tnap into the bone isoenzyme suffer from skeletal hypomineralization (anderson et al. 2004 ). alt is a cytoplasmic enzyme in which serum activity is increased due to leakage across damaged cytoplasmic membranes. alt (also known as glutamic pyruvic transaminase [gpt]) reversibly catalyzes the conversion of alanine to pyruvate. quantification of serum alt is based on a reaction between alt and a suitable substrate (such as alanine). in mice, alt is found in the highest concentrations in the liver, although activity has also been demonstrated in intestine, kidney, heart, muscle, and brain (clampitt and hart 1978) . despite its widespread tissue distribution, alt is mostly used as an analyte to assess hepatocellular damage (masaki et al. 2005; taieb et al. 2005 ). an 11,000% increase in serum alt activity has been reported following infection with mouse hepatitis virus, and significant increases follow infection by helicobacter hepaticus (mccathey et al. 1997 ). ast is a cytoplasmic and mitochondrial enzyme in which serum activity is increased due to leakage across damaged cytoplasmic and mitochondrial membranes. ast (also known as glutamic oxaloacetate transaminase [got]) reversibly catalyzes the conversion of aspartate to oxaloacetate. quantification of serum ast is based on a reaction between ast and a suitable substrate (such as aspartate). in mice, ast is found in a variety of tissues, including liver, skeletal muscle, cardiac muscle, erythrocytes, blood vessels, brain, intestine, kidney, lung, testes (papadimitriou and van duijn 1970) . the highest specific activity of ast is found in mouse cardiac muscle and lowest in skeletal muscle (herzfeld and knox 1971) . in the liver, ast is found mainly in periportal hepatocytes based on histochemical studies (papadimitriou and van duijn 1970) . activity in lung, kidney, intestine, and skeletal muscle is very low when measured by the technique of bergmeyer and bernt (1974) . loeb et al. (1996) demonstrate significant age-associated increases in serum ast activity in two inbred and two f1 hybrid strains. although widely distributed, alt is mainly used to assess hepatocellular damage, cardiac muscle damage (naraoka et al. 2005; ray et al. 2005) , and testicular injury (santos et al. 2005) . alt activity has been used as an indicator of hepatic injury of mice infected with mouse hepatitis virus (fassati et al. 1969 ). ldh is a cytoplasmic enzyme in which serum activity is increased due to leakage across damaged cytoplasmic membranes. ldh reversibly catalyzes the conversion of pyruvate to lactate. quantification of serum ldh is based on a reaction between ldh and a suitable substrate (such as pyruvate or lactate). as in other species, ldh in the mouse is a tetrameric enzyme consisting of either a or b subunits. there are five isoenzymes of ldh (based on differences subunit a and b composition), and these are ldh-1 (b4), ldh-2 (a1b3), ldh-3 (a2b2), ldh-4 (a3b1), and ldh-5 (a4). specific isoenzyme distribution depends on differential expression of either the a and b subunits (quimby 1999b) . for instance, all embryonic murine tissues contain ldh-5 because the a subunit is only expressed during early fetal development. as the embryo matures, subunit expression can involve both a or b subunits in different tissues, meaning that by birth and sexual maturity, each tissue contain a characteristic ldh subunit profile. in adult mice, the heart and erythrocytes contain ldh-1 and ldh-2, whereas most other tissues have ldh-3. skeletal muscle and hepatocytes fail to express subunit b and therefore are composed predominantly of ldh-5. serum ldh activity can vary due to age and sex. ldh levels have been shown to be higher in males versus females of the balb/c strain (frith et al. 1980) . serum ldh levels increase with age in balb/c and c57bl/6 mice of both sexes (frith et al. 1980) . serum ldh can also be elevated falsely by hemolysis. quantification of serum ldh measures the total ldh activity from all sources. however, damage to a particular tissue will result in increased activity of that isoenzyme in serum. in general, the highest activity of ldh in the mouse is in skeletal muscle, with decreasing activity in the heart, liver, kidney, and intestine. serum ldh-5 activity rises within 72 h after inoculating mice with the mouse hepatitis virus (fassati et al. 1969) . mice infected with the ldh virus (ldv) exhibit increased serum concentrations of ldh, isocitric dehydrogenase, malic dehydrogenase, phosphohexase isomerase, and ast (notkins 1965) . along with ast and ck, ldh is considered an excellent marker for cardiac injury (naraoka et al. 2005; ray et al. 2005) . otc is a mitochondrial enzyme in which serum activity is increased due to leakage across damaged mitochondrial membranes. otc is found primarily in the liver of mice, and there increases in serum activity reflect severe injury to hepatocytes. abnormal otc activity has been described in mice having the sparse-fur (spf/y) mutation. they serve as a model for the most common inborn error of urea synthesis in humans. assays for mouse otc have been developed to monitor activity levels following gene transfer or liver transplantation (batshaw et al. 1999; ye et al. 2001 ). ck is a cytoplasmic and mitochondrial enzyme in which serum activity is increased due to leakage across damaged cytoplasmic and mitochondrial membranes. ck reversibly catalyzes the phosphorylation of adenosine diphosphate (adp) to ate using creatine phosphate as the donor for the phosphate group. quantification of serum ck is based on a reaction between ck and a suitable substrate (such as creatine phosphate). as in other species, cytoplasmic ck is a dimeric enzyme consisting of either m or b subunits. there are three isoenzymes of ck (based on differences subunit m and b composition), and these are ck-1 (bb), ck-2 (mb), and ck-3 (mm). brain contains ck-1, skeletal muscle contains ck-3 (mm), and cardiac muscle contains ck-1, ck-3, and ck-2 (mb) (quimby 1999b ). in the mouse, the greatest ck activity is found in skeletal muscle, with much less activity found in the heart and brain. mitochondrial ck (ck-mt) is found in mitochondria of many tissues. serum ck activity is affected by age, sex, and method of collection and anesthesia. patrick et al. (1983) found that compared to jugular vein collection, cardiac puncture was associated with lower ck activity. levels of serum ck activity have been reported for balc/cann mice and c57bl/10 mice of varying ages and sex (sub et al. 1994) . serum ck activity is a useful and specific marker enzyme of muscle injury, because ck in central nervous tissue does not cross the blood-brain barrier. sub et al (1994) compared normal c57bl/10 mice with heterozygous male and homozygous female mice carrying the mdx (mutant dystrophin) allele, and found that homozygous females have 12-to 15-fold increases and heterozygous males (mdx/y) have 30 fold increases in plasma ck compared to wild-type mice. plasma ck levels correlated with skeletal muscle necrosis in these dystrophic mice. mice have been engineered that lack cytoplasmic ck and ck-mt . mitochondria from heart or skeletal muscle from double kos had higher adp concentrations compared to wild-type animals, suggesting the higher concentrations contribute to the control of the reduced cytosolic atp free energy potentials seen in double kos. aldolase is a cytosolic enzyme that can alter its distribution between soluble and particulate forms, according to the metabolic status of the tissue. in adult mice, nine aldolase isoenzymes are known to occur in tissues with significant activities in the muscle, brain, liver, kidney, and spleen. in the mouse liver aldolase, together with fructokinase and triokinase, metabolize fructose (hagopian et al. 2005) . everett and harrison (1983) report no apparent advantages in mice in the measurement of aldolase over other enzymes known to have specific liver or muscle activity. sdh is a cytoplasmic and mitochondrial enzyme in which serum activity is increased due to leakage across damaged cytoplasmic and mitochondrial membranes. sdh (also known as iditol dehydrogenase [idh]) reversibly catalyzes the conversion of fructose to sorbitol. quantification of serum sdh is based on a reaction between sdh and a suitable substrate (such as fructose). sdh is located primarily liver, kidney, and seminal vesicles. the activity of sdh is usually low in the serum and rises during hepatic injury. however, the labile nature of sdh during handling makes is less suitable overall as a indicator of hepatic dysfunction compared to over liver-specific enzymes (such as ast). mice with the gene for sdh knocked out have been used to study the role of sorbitol accumulation in diabetic albuminuria (ii et al. 2004 ). amylase is a cytoplasmic enzyme in which serum activity is increased due to leakage across damaged cytoplasmic membranes. amylase hydrolyzes complex carbohydrates to form maltose and glucose in the presence of free calcium ions. quantification of serum amylase is based on a reaction between amylase and a suitable substrate (such as starch). similar to humans and pigs (but not dogs, cats, cattle, and horses), expression of amylase in mice is related to two distinct but closely linked loci (meisler et al. 1983) . salivary amylase is the gene product of amy-1 (salivary), and appears to be a single enzyme. pancreatic amylase is the gene product of amy-2, and based on electrophoretic studies in inbred mice, there appear to four isoenzyme classes: a1, a2, b 1, and b2. similar to other domestic animal species, pancreatic amylase is filtered through the glomerulus, but unlike other domestic species, pancreatic amylase is not resorbed by renal tubular epithelial cells and is excreted rapidly in the urine. therefore normal serum amylase activity in mice consists mainly of salivary amylase (mackenzie and messer 1976) . despite this, elevations in serum amylase activity is usually considered a reliable marker for pancreatitis in mice (nathan et al. 2005) . ross et al. (1974) reported two-to three-fold increases in serum amylase activity in mice infected with coxsackievirus of salivary and pancreatic trophism. alterations in the activity of specific pancreatic isoenzymes have been shown in streptozotocin-induced diabetes in mice (quimby 1999b) . there are two pancreatic lipase isoenzymes in mice. serum lipase activity has been used to monitor cerulean-induced acute pancreatitis in mice (cuzzocrea et al. 2004) . recently a new colipase-dependent lipase has been described in suckling mice (d'agostino and lowe 2004) . the enzyme 5'-nucleotidase was measured in the serum of normal mice using a simple one-step kinetic method (dooley and racich 1980) . a reference range of 10.9 + 4.5 (sd) u/1 has been recorded in 100 mice, and it is thought but not proven to be a good indicator of hepatic injury (clampitt and hart 1978) . glutamate dehydrogenase (gdh) has been measured in the tissues and serum of mice. gdh is known to play a key role in insulin secretion (carobbio et al 2004) . the activity of gdh is fivefold greater in the liver than in the kidney and brain, and the authors speculated that its measurement in serum would be a sensitive indicator of hepatic cell injury. serum gdh activity is also elevated in mice on caloric restriction (hagopian et al. 2003 ). corticosterone is the major glucocorticoid secreted by the adrenal cortex of mice. it functions as a regulator of carbohydrate, protein, and fat metabolism and modifies the host response to stress. the male mouse has a well-defined diurnal concentration pattern, with a maximum concentration of 9 ktg/dl at the start of the dark cycle and a minimum concentration of 5 btg/dl shortly before the end of the dark cycle (ottenweller et al. 1979) . in contrast, female mice have a minimum concentration of 13.5 ktg/dl at the beginning of the dark cycle and a maximum of 40 ~tg/dl well into the dark period (scheving et al. 1983) . the length of the dark cycle was different in each study. it may be measured using radioimmunoassay, elisa, or fluorometric assay in mouse serum or plasma. corticosterone circulates in both free and protein bound forms. in the mouse, greater than 99% circulates bound to cortisol-binding globulin (cbg) and albumin. the diurnal variation in corticosterone levels is paralleled by the diurnal pattern of cbg. urinary and salivary corticosterone is derived only from the free plasma fraction. corticosterone synthesis and secretion may be influenced by many drugs (woodman 1997) . measurements of corticosterone in unrestrained mice using indwelling catheters have elucidated the necessity of eliminating stress for accurate interpretation of data (macleod and shapiro 1988) . both handling and crowding laboratory mice can cause elevations in corticosterone (balcombe et al. 2004; fullwood et al. 1998) . c57bl/6 ob/ob mice have elevated corticosterone levels that increase markedly after 40 weeks of age, preceding elevations in serum glucose (garthwaite et al. 1980) . lean c57bl/6 controls had lower serum concentrations of corticosterone that declined between 5 and 20 weeks of age. food restriction (to 60% of ad libitum) profoundly affects the diurnal increase in plasma corticosterone in mice. at 20:00 hours (8 p.m.), the daily maximum in this study, food restricted balb/c mice had 300% more corticosterone compared to controls. these mice also had significantly reduced thymic weight and inflammation in response to the injection of carrageenan subcutaneously. the authors report that the magnitude of carrageenan-induced inflammation fluctuates with a diurnal trough, which coincides with peak corticosterone levels (klebanov et al. 1995) . corticosterone is synthesized in response to acth, which is made in the anterior pituitary. acth release is episodic (not at fixed intervals) and does not involve steroid feedback. acth concentration peaks in the early evening in mice and can be reversed by reversing the light cycle. acth is highly conserved with the mouse acth differing from human by only two amino acid residues. ria may be used to measure acth in the mouse (daily levels vary from 2.6-5.4 ng/ml) and to demonstrate rhythmic cycling. samples must be collected at 5-to 30-minute intervals. commercial elisa kits for acth are also available (quimby 1999b) ko mice incapable of synthesizing corticotrophin releasing hormone or acth may require corticosterone supplementation in drinking water and are particularly susceptible to hypoglycemia during fasting. fetuses of homozygous ko crh null mothers must be supplemented from gestational day 12 to weaning with 30 mg/ml corticosterone in drinking water (mugila et al. 1995) . oxytocin gene ko mice respond to a psychogenic stressor with more anxiety-related behavior and more corticosterone production (amico et al. 2004 ). lh promotes follicular development, increases estradiol secretion in the preovulatory follicle, causes follicular rupture, converts the preovulatory follicle into a corpus luteum, increases progesterone production by the corpus luteum, and, in the male, increases production of testosterone from leydig cells (woodman 1997) . lh is a glycoprotein composed of t~-and ~-chains. the amino acid sequence of the t~-chain is identical to that of follicle-stimulating hormone (fsh); however, the ~l-chain is specific and confers the receptor binding properties on the hormone. a homologous ria for rat lh using antisera to rat lh and purified rat lh has been employed to measure lh in mice (quimby 1999b) . lh and folliclestimulating hormone (fsh) are secreted by the same anterior pituitary cell in response to stimulation by gonadotrophinreleasing hormone (gnrh), which is synthesized in the hypothalamus. gnrh is identical in mouse and humans, and its differential stimulatory effect on gonadotropes, producing fsh or lh is controlled through gnrh pulse frequency. pulsatile secretion of lh leads to great variations in blood concentration. in female mice, there is a 10-to 25-fold increase in basal lh levels in the afternoon of proestrus. in a study comparing young cycling c57bl/6 female mice to old females (40% of which not cycling), flurkey et al. (1982) found lower lh levels in the older group and a more rapid rise in lh among younger cycling mice. these authors found that reproductive failure in male mice correlated with the loss of episodic release of lh. female homozygous ko mice lacking the gene for the immediate early transcription factor ngfi-a were infertile secondary to lh-[3 deficiency. although ovaries from these mice had a similar number of follicles, they lacked corpora lutea (lee et al. 1996) . basal levels of serum progesterone were lower in ngfi-a females (6.3 + 2.4 ng/ml) compared to wild type (11.3 +_ 3.6 ng/ml), whereas serum estradiol levels were similar in deficient (50.7 + 36.9 pg/ml) and wild-type (52.0 _+ 34.3 pg/ml) mice. decreased amounts of mrna encoding lh-[3, but not fsh-[3, were observed in ngfi-a-deficient mice; no changes were observed in mrna levels for prl or gnrh receptor between deficient and wild-type mice. an ovariectomy normally removes gonadal feedback inhibition, allowing for increased amounts of lh-~ and fsh-[3 in the pituitary; however, ovariectomy of ngfi-a deficient female mice lead to an increase in fsh-~ only. homozygous ngfi-a deficient males did make lower amounts of lh-~ compared to wild-type males, but they made significantly more lh-[3 than did ngfi-a-deficient females. the levels of lh in males appeared to be sufficient for fertility, and they had normal serum testosterone levels. these results suggest that ngfi-a acts synergistically with transcription factor sf-1 to regulate the promoter for lh-[3 gene expression. the simultaneous activation of both sf-1 and ngfi-a by gnrh appears to confer the specificity of lh-~ synthesis. female transgenic mice overexpressing lh are anovulatory, develop granulosa cell tumors, and undergo precocious puberty (mann et al. 2003) . excellent reviews detailing the use of transgenic and ko mice in elucidating the secretion and function of lh have been published (bums and matzuk 2002; huhtaniemi et al. 2002; wells and murphy 2003) . fsh stimulates the growth and maturation of ovarian follicles in the female and promotes the latter stages of spermatogenesis in males. it acts principally on the sertoli cells of the seminiferous tubules of the testis and induces the secretion of androgen-binding protein and inhibin. in females, fsh and lh have distinct secretory patterns that are synchronized to the estrous cycle; mouse estrous cycles have a 4-to 5-day periodicity. in addition to estrous cycle dependent rhythms, both lh and fsh have ultradian pulses and show circadian periodicity, with highest levels occurring during the dark period of the light cycle. testosterone, estrogen, and progesterone all provide feedback regulation of lh and fsh secretion (woodman 1997) . because rat and mice share considerable sequence homology for the fsh-~ chain, the rat ria kit from national institute of diabetes and digestive and kidney diseases (niddk) can be used to measure fsh in the mouse (dalterio et al. 1981) . a mouse specific ria is commercially available. in cycling female mice, the plasma levels of fsh increase from 80 to 120 ng/ml during proestrus and 250-300 ng/ml during estrus. although bronson and desjardins (1977) found age associated decreases in serum lh and testosterone in male cbf 1 mice experiencing decreased sperm production, no similar decreases were observed in fsh concentration. no significant differences in the twofold increased fsh levels were seen among young versus old males following castration. this appears to differ from results obtained from young versus old rats (finch et al. 1977) . the regulation and function of fsh has been studied in transgenic and ko mice (cooke and saunders 2002; wells and murphy 2003) . testosterone promotes spermatogenesis and provides feedback regulation of gonadotrophin synthesis. through its action on the epididymis, proteins necessary for spermatozoal maturation are synthesized. dihydrotestosterone (dht) promotes the growth and differentiation of accessory sex organs (depaolo and masoro 1989) . in addition to their effects on reproductive organs, testosterone and dht have physiologic functions on the central nervous system, cardiac tissue, and the liver. in the mouse, testosterone stimulates the growth of kidneys and synthesis of erythropoietin (woodman 1997) . lh stimulates synthesis of testosterone from cholesterol by the interstitial leydig cells of the testis. in target tissues, the enzyme 5r converts testosterone to dht. in the fetal testes, leydig cells first become identifiable during the rise of testosterone. in most mammalian species, leydig cells disappear when testosterone levels fall during gestation. however, the mouse is an exception and leydig cells do not undergo regression postnatally (aoki 1970) . testosterone and dht circulate in both free and protein bound forms. ninety-eight percent of total testosterone is bound to protein, mainly albumin. mice, unlike humans, dogs, monkeys, and cats, do not have circulating sex hormone-binding globulin. in mice, a pulse release pattern may be seen within their diurnal rhythm of testosterone. testosterone can be measured in mice using ria or elisa. plasma testosterone was not found to change during the average lifespan of c57bl/6 and dba/2j mice (finch et al. 1977) . this is in contrast to cbf1 mice, wistar and long-evans rats, and humans, which experience greater than a 30% decrease in testosterone at midlife (bronson and desjardins 1977) . in contrast to testosterone levels in older mice, castration-induced elevation of lh was impaired in 28-month-old c57bl/6j mice compared to 12-month-old mice. androgen receptor ko mice have been described (cooke and saunders 2002) . 5. estradiol (e2) the granulosa cells of the mature graafian follicle are the main source of 17[3-estradiol (e2) in mammals. fsh stimulates the activation of aromatase in follicles, which is responsible for the conversion of androgens to e2. e 2 is also synthesized by the testis, adrenal, liver, and skin, although in much lower amounts than by the ovary. e2 provides negative feedback control over lh secretion, and it stimulates prl secretion in mice. e 2 promotes the growth and development of the female reproductive tract, external genitalia, and the ductal system of the mammary glands. in addition, e 2 sensitizes the follicle to fsh. e 2 is measured in mice using ria and elisa. high circulating levels of e 2 precede the preovulatory surge in lh. ryan and schwartz (1980) reported basal levels of e 2 in mice of 1 to 5 pg/ml. holinka et al. (1978) studied circulating levels of progesterone and estradiol in young and old c57bl/6 female mice during gestation. they found that the old multiparous females have delayed and reduced preparturitional rise is plasma e 2 compared to young females. because preparturitional e 2 is thought to regulate uterine progesterone levels, the decline in e 2 seen in older females coupled with elevated progesterone may delay the onset of labor and lead to prolonged gestation. when sex steroid hormones such as e 2 bind their receptor, they induce a conformational change that allows the complex to bind dna response elements on nuclear target genes and associate with coactivators and transcription factors to form an active transcriptional complex. this complex is responsible for initiating the transcription of the target gene. one coactivator that associates with the active transcriptional complex involving sex hormones is steroid receptor coactivator-1 (src-1). to better understand its physiologic role during sex hormone-receptor binding, ko mice that were deficient in src-1 were created (xu et al. 1998 ). deficient male mice had a 34% reduction in testosterone-stimulated prostate growth and small testes compared to wild-type mice. deficient females had a half-normal uterine growth response to exogenous e 2 and only a partial ductal growth response (in the mammary gland) following exogenous e 2 and progesterone. serum concentrations of e 2 and testosterone were elevated 1.2 to 1.5 times wild-type levels, indicating an abnormality in the endocrine feedback control system. although src-1 was shown to clearly be necessary for optimal sex hormone-induced cellular activation, the lesion created in this ko mouse was not nearly as profound as that seen in mice with disrupted e 2 receptors (korach et al. 1996) . mice with the genes for the e 2 receptors and aromatase knocked out have been described (simpson et al. 2005 synthesized by the corpus luteum before implantation and by both corpus luteum and placenta following implantation, progesterone is necessary for preparing the uterus for implantation and maintaining pregnancy. in addition, a small amount of progesterone is synthesized by the adrenal gland. activation of the corpus luteum to secrete progesterone requires both lh and prl. progesterone provides negative feedback control over lh. in mice, progesterone is measured using ria or elisa. in cycling mice, the levels of progesterone increased from 5 to 35 ng/ml on proestrus and returned back to baseline on estrus. flurkey et al. (1982) compared the plasma levels of lh, progesterone, and prl in cycling young (10-week) and old (8month) female c57bl/6j mice. they showed age-related deficits in the preovulatory levels of all three hormones. the lh elevations during the ascending and descending portions of the preovulatory surge were smaller; the slower rises in lh during the ascending phase of the surge correlated with decreased progesterone in older females. there was no correlation between lh or progesterone level and length of estrus cycle. holinka et al. (1978) observed changes in plasma progesterone levels in young and old female mice during gestation. older mothers had a much slower decline in circulating progesterone than younger mothers between gestational days 17-23. because a major decrease in plasma progesterone is thought to be essential for the onset of uterine contractions and parturition in mice, the authors hypothesized that the attenuated decline in older mothers may account for their prolonged gestation. surgimoto et al. (1997) engineered mice lacking the pgfzareceptor (fp) and found that deficient female mice had normal estrous cycles, ovulation, fertilization, and implantation but did not undergo parturition. these pregnant females were characterized by a decline in preparturition progesterone levels due to delayed luteolysis and failure to develop labor. close examination revealed that near term there was no expression of the oxytocin receptor in the uterus of fp-deficient mice. when the ovaries of fp-deficient pregnant females were removed on day 19 of pregnancy, progesterone levels fell, uterine oxytocin receptors were expressed, and pups were born alive within 24 h. this study demonstrated that the role of pgf2~ is to initiate luteolysis, resulting in an immediate decline in progesterone levels. the subsequent induction of oxytocin receptors and their activation by bound oxytocin initiate labor. it is feasible that the age-associated prolongation of gestation in old female mice may involve a defect in pgfz,~-induced luteolysis. this mechanism for the induction of labor also explains the well-known observation that aspirin-like drugs (that inhibit cox metabolism) may delay parturition in women. prl, a 23-kda single-chain polypeptide, is principally made by the pituitary gland but may be also be found in brain, thymus, spleen, lymph nodes, mammary gland, myometrium, sweat gland, lacrimal gland, and bone marrow. more than 300 functions have been attributed to prl, involving reproduction and lactation, growth and development, endocrinology and metabolism, brain and behavior, immunomodulation, and electrolyte balance. it mediates effects by endocrine, paracrine, and autocrine mechanisms. in mice, prl is of major importance for maintenance of the structure, life span, and function of the corpora lutea and the development and maintenance of the mammary gland and lactation. however, prl-receptor ko males have no major defect in fertility. prl plays a role in anxiety-related behaviors, bone development, and abdominal fat deposition in both sexes (kelly et al. 2001) . its role in immune function is more controversial, although it appears to modulate immunity during times of stress (dorshkind and horseman 2000) . prl mediates its effects by binding the prl receptor (prlr). mice have two major forms of prlr that are created via alternative splicing of a single gene. the prlrs are members of the class i cytokine receptor superfamily. although most functions of prl are mediated by the unmodified 23-kda peptide, post-translational modification allows variants of prl to bind its receptor eliciting transcription of genes necessary for tissue specific changes (harris et al. 2004) . prl secretion is under inhibitory control by dopamine and among its secretagogues are thyrotropin-releasing hormone (trh), vasoactive intestinal peptide, gastrin, serotonin, ~-endorphin, oxytocin, angiotensin ii, gnrh, and arginine vasopressin. prl in mice is quantified using ria using the nih rp-1 reference standard. male mice measured during the light phase have <1 ng/ml whereas the range during the dark phase was 10-20 ng/ml (depaolo and masoro 989). oxytocin, a 21-kda nonapeptide, is synthesized as a prohormone in neurons whose bodies are located in the supraoptic and paraventricular nuclei at the base of the hypothalamus. once synthesized, the prohormone is packaged into neurosecretory granules and transported down the axons of these neurons transport synthesized oxytocin to the pars nervosa of the pituitary gland. during transport, the molecule is cleaved to yield the 10-kda carrier proteins, neurophysin i, and the l l-kda oxytocin. the primary stimulus for oxytocin release is mechanical stimulation of the mammary gland and distention of the reproductive tract (reimers 1999) . there is a single oxytocin receptor (otr). investigations using ko mice have shown that oxytocin is required for milk let down by the mammary gland as well as for postpartum alveolar proliferation. in males, oxytocin is required for normal spermiation and sperm transfer. in both genders, oxytocin helps control normal blood pressure and salt intake. kos display reduced aggression and a striking deficit in the ability to recognize a previously encountered mouse (young and gainer 2003) . oxytocin may be quantified in mice using commercially available elisa kits or by species-specific ria. values in one recent publication showed plasma oxytocin at 1.5 _+ 0.6 pg/ml in male c57bl/6 mice ). avp is synthesized and released by the same neurons previously described for oxytocin, although the two secretory granules rarely colocalize in the same neuron. the carrier protein for avp is neurophysin ii. avp is released from neuron secretory granules by electrical signals from osmoreceptors measuring the osmolarity of extracellular fluid. action potentials, generated in the receptors, cause calcium influx into axon terminals and exocytosis of ave avp is transported in the blood to the kidneys, where it binds receptors in the distal segment of the nephron and collecting ducts leading to increased resorption of water (reimers 1999) . there are three avp receptors, v l ar, vlbr, and v2r; and these are all g-protein coupled receptors. nonsense mutations of the avp gene (as seen in the brattleboro rat) or kd mice develop neurohypophysial (central) diabetes insipidus. mice with the v2r knocked out develop nephrogenic diabetes insipidus characterized by polyuria, polydipsia, and failure to concentrate urine. when the v1 ar gene is knocked out, mice develop an enhanced proliferation of splenic b cells and enhanced igg1 and igg2b production to thymicdependent antigens. when v lbr is knocked out the mice display a marked reduction in social aggression and a deficit in social recognition (young and gainer 2003) . avp can be measured by ria or elisa and the reported values in normal male c57bl/6 mice are 5.1 + 1.5 pg/ml in plasma ). tsh is a 28-kda glycoprotein made by the anterior pituitary gland on stimulation by thyrotropin-releasing hormone (trh). trh is made in the hypothalamus. tsh is secreted in a pulsatile manner, similar to acth. tsh secretion is regulated by triiodothyronine (t3), which acts on the hypothalamus to inhibit secretion of trh and acts on the pituitary gland to inhibit tsh synthesis (reimers 1999) . tsh plays a role in stimulating the thyroid gland to produce thyroxine and it influences the outcome of t-cell development in thymus and intestine. in addition to the thyroid gland, tsh receptors are found on many different populations of hematopoietic cells in bone marrow, subsets of dendritic cells, monocytes, and lymphocytes in the spleen and lymph nodes (klein 2003; scofield et al. 2005) . ko mice have been created in which genes for trh (wells and murphy 2003; yamada et al. 2003) and tsh receptors (biesiada et al. 1996) have been deleted. in addition, spontaneous mutations in the pitl gene lead to deficiencies of gh, prl, and tsh (yeap and leedman 1999) . tsh is measured by ria and reference ranges established around a 3.1 u/mg reference standard were 1-90 ng/ml (depaolo and masoro 1989). 11. thyroxine (t4) and triiodothyronine (t3) t 4 is synthesized entirely in the thyroid gland, whereas t 3 is produced primarily by conversion of t 4 to t 3 in extrathyroidal tissues. in the thyroid, thyroglobulin is synthesized and stored, and later hydrolyzed to form t 4. the tyrosine residues, held in peptide linkage within thyroglobulin, are first iodinated and later iodotyrosines are chemically coupled to form hormonally active t 4 and t 3. production of t 4 depends on adequate supplies of iodine. both t 3 and t 4 circulate in the blood primarily bound to albumin and m-globulin; however, mouse transthyretin does not bind t 4. approximately 85% of t3, the active thyroid hormone, in blood is made through the monodeiodination of the outer ring of t 4 in a variety of tissues. certain drugs, food deprivation, and illness can all effect monodeiodinase activity (reimers 1999) . t3 actions are mediated via two t3 receptors, tr~x and tr[3, which act as hormone-inducible transcription factors and belong to a large superfamily of nuclear hormone receptors including the steroid hormone, vitamin d, retinoic acid, and peroxisomal proliferator receptors (yen 2003) . in mouse and human tr~ and tr[3 encode nine mrna isoforms through alternative splicing; four isoforms tr~i, trc~2, tr[31, and tri]2, are expressed as proteins. trc~ ~ ko mice, which lack all products from the tr~ locus, are fertile and have normal basal thyroid status but have increased sensitivity to thyroid hormones in the pituitary and liver following provocative testing with increasing doses of t3. tr~x -/-have a disruption of the first coding exon in the tr~x locus, which prevents transcription of tr~i and try2 mrnas but not tra~i or tra~2. these pups die shortly after weaning unless supplemented with t3 for the first 2.5 months of life, after which they develop normal thyroxine levels. however, both genders are infertile. try2 -/-overexpresses tr~i and are hypothyroid but have inappropriately normal tsh levels. they exhibit some signs of hyperthyroidism, such as increased heart rate, weight loss, and elevated body temperature. tr~ -/-lack all tr[3 isoforms and display resistance to thyroid hormones demonstrating the key role of tr~ in set-point control of the pituitary-thyroid feedback axis. tr[32 -/-ko mice have resistance to thyroid hormones and elevated t3, t4, and tsh. they have defective tsh suppression by t3. several double kos (both tr~ and tri]) have been developed with profound resistance to t3. these targeted mutants have helped to elucidate the full function of thyroid hormones involving: bone formation and mineralization, abnormal development of skeletal muscle, disrupted development of the cones in the retina, abnormalities in the auditory system, cochlear and vestibular structures, delayed small intestine development, impaired thermogenesis, and altered development of the central nervous system and immune system (o'shea and williams 2002). both total t 4 and t3 can be measured by ria in mice, and reference ranges vary greatly by strain and age. although t3 is the active hormone, its serum levels are so low that it is a less reliable indicator of thyroid status. total t 4 ranges in swiss webster and icr mice are 5.5 _+ 0.7 and 4.7 + 0.3 jag/ml, respectively. total t3 levels range from 65-85 ng/100 ml in both sw and icr stocks (depaolo and masoro 1989). many factors, including autoantibodies, are known to interfere with thyroid hormone assays (despres and grant 1998; reimers 1999) . 12. parathyroid hormone (pth) pth is synthesized by the chief cells of the parathyroid gland and secreted as an 84 amino acid peptide. circulating levels of ionized calcium induce synthesis of pth. low calcium stimulates pth synthesis, and high calcium inhibits secretion. the primary function of pth is to control calcium concentrations in extracellular fluid and prevent hypocalcemia by increasing calcium resorption from bone, glomerular filtrate, and intestines (reimers 1999) . pth mediates its effect via its g-protein coupled receptor, pth1r. interestingly a second protein, pth related protein (pthrp), which is secreted by a variety of tissues and acts by autocrine and paracrine signaling, uses the same pth1r. pthrp modulates a wide range of physiologic and developmental responses (goltzman and white 2000) . mice with targeted mutations of the pth, pthrp, and pth1r genes have demonstrated the critical role of these proteins in regulating both the switch between proliferation and differentiation of chondrocytes and their replacement by bone cells (schipani and provost 2003) . serum levels of pth may be quantified in mice using a commercially available elisa kit, which can also be used to quantify the protein in rats. 13. other hormones elisa kits are commercially available to quantify many additional hormones in the serum of mice, including insulin, leptin, gh, epinephrine, orexin a, orexin b, adiponectin, adipsin, and resistin. each of these hormones have been briefly discussed in the "glucose and carbohydrate metabolism" section. the liver has many complex functions including protein metabolism, carbohydrate metabolism, and lipid metabolism. the liver is involved with the synthesis of many plasma proteins (including albumin), the conversion of ammonia to urea, and the production of glucose from glucogenic amino acids). the liver is implicated with the regulation of blood glucose levels (via glycogenolysis and gluconeogenesis, as discussed in the "glucose and carbohydrate metabolism" section), the removal of glucose from the blood via glut-1 and glut-2 membrane transporters, and storage in the form of glycogen. the liver is also involved in cholesterol and triglyceride synthesis, the formation of lipoproteins (discussed in the "lipid metabolism" section), and the synthesis of carbohydrates from fats. there are also other more specific functions that the liver facilitates, including the synthesis of heme, the synthesis of many coenzymes, detoxification/biotransformation of exogenous and endogenous substances via the cytochrome p-450 microsomal enzymes, and the synthesis of bile. the role of the liver in these functions for various animals has been reviewed (tennant 1999; woodman 1988 ). the elevation of plasma or serum enzymes usually confined to the cytosol or mitochondria of hepatocytes is helpful in elucidating liver injury. many factors influence the duration of elevated serum enzyme levels including molecular size, intracellular location, rate of plasma clearance, rate of enzyme inactivation, and hepatic production. hepatic necrosis is associated with elevations of alt, ast, sd, and otc in mice, and extrahepatic cholestasis is associated with elevated ap (tennant 1999) . please refer to the "enzymes" section for a description of each enzyme. bilirubin is a pigment that is produced by the degradation of the hemoglobin by cells of the mononuclear phagocyte system. there are two main types of bilirubin. unconjugated bilirubin is a non-water soluble molecule that is transported in blood bound to albumin. hepatocytes uptake unconjugated bilirubin, where it undergoes glucuronidation to produce the water-soluble form, conjugated bilirubin. conjugated bilirubin is then excreted via the hepatobiliary system and excreted in bile. quantification of serum bilirubin levels is based on the van den bergh or diazo reaction. diazo reacts directly with conjugated bilirubin, and with unconjugated bilirubin only after addition of alcohol to the reaction. therefore, measuring serum bilirubin involves the following steps. first, the level of conjugated bilirubin is measured. second, alcohol is added to the reactants, which allows quantification of total bilirubin levels. finally, the level of unconjugated bilirubin is determined by subtracting the conjugated measurement from the total measurement. the excretory capacity of the liver may be assessed by measuring serum bilirubin. elevated levels of unconjugated bilirubin are usually observed in situations of increased erythrocyte breakdown, such as in hemolytic diseases. unconjugated hyperbilirubinemia is also seen in disease in which hepatic uptake, conjugation, and secretion of bilirubin are diminished. increased levels of conjugated bilirubin are usually associated with intrahepatic cholestasis or extrahepatic bile duct obstruction. conjugated bilirubin in blood is normally filtered by the glomerulus in small amounts. conjugated hyperbilirubinemia may result in bilirubinuria (tennant 1999) . bilirubin clearance from blood and the role of the constitutive androstane receptor in this process has been studied in normal and transgenic mice (huang et al. 2003; . studies documenting the bilirubin-metabolism/detoxifying enzymes, their regulatory nuclear receptors, and lipid transporters in mice have been reported (wagner et al. 2005) . bile acids are cholesterol breakdown products that are secreted by hepatocytes into the hepatobiliary system, and ultimately into the intestinal tract. bile acids aid digestion by emulsifying dietary lipid aggregates, and solubilizing and transporting lipids in an aqueous environment (in particular fat-soluble vitamins). in the liver, bile acids also play a role regulating the secretion of apolipoprotein b (elzinga et al. 2003) . extensive enterohepatic recirculation results in almost 99% return of bile acids secreted by the liver from the intestinal tract. absorption occurs mainly in the ileum under the influence of the apical na ⧠bile acid transporter on epithelial cells (kida et al. 2003) . some absorption also takes place in the large intestine. absorbed conjugated bile acids (from ileum) pass unaltered to the portal circulation where they are removed by na+-taurocholate cotransporting polypeptide located on the basolateral hepatocyte membrane (jung et al 2004) . there are two types of bile acids, cholic acid and chenodeoxycholic acid. in mice, they are conjugated with taurine before secretion by the liver. the liver-specific enzymes, bile acid coa ligase and bile acid-coa:amino acid n-acyltransferase, are responsible for conjugation (inoue et al. 2004 ). stedman et al. (2004) described normal serum bile acid levels in normal and transgenic mice. elevated serum bile acid levels in mice is usually due to decreased bile acid recycling by the liver, and mainly includes diseases associated with decreased hepatic functional mass and cholestasis (tennant 1999) . hoda and green (2003) measured increased levels of serum bile acids after bile duct ligation. albumin is a nonglycosylated protein and is the most abundant protein in plasma. it serves as the most important determinant of plasma oncotic pressure, and it is a major transport protein for both endogenous metabolites and xenobiotics. it is made exclusively by the liver. serum albumin may be measured by radial immunodiffusion, dye binding reactions (using bromcresol green or bromcresol purple), electrophoresis, and elisa. serum albumin levels decrease with age in many inbred strains (quimby 1999b) . hypoalbuminemia is usually reflective of decreased hepatic synthesis (due to hepatic disease, inflammation, and malabsorptive/maldigestive diseases), increased loss due to hemorrhage or via the intestinal tract (protein-losing enteropathies) and kidneys (protein-losing nephropathies) (tennant 1999) . most of the proteins associated with coagulation are synthesized by the liver (except factor viii), and measurement of fibrinogen or prothrombin time has served as a marker for decreased synthesis due to hepatic injury (tennant 1999) . prothrombin time will also be increased due to consumption of clotting components and in vitamin k deficiency. fibrinogen is also an acute phase reactant, and plasma elevations are seen during inflammation (tennant 1999) . fibrinogen may be quantified in mouse plasma by elisa. ceruloplasmin is copper-containing acute phase reactant and iron transport protein made exclusively in hepatocytes of mice. its serum levels may be decreased during hepatic injury and increased in response to inflammatory stimuli (min et al. 1991; shim and harris 2003) . many of the complement components are also made in the mouse liver, especially c2, c3, c4, and factor b. decreased circulating levels may be seen due to hepatic injury or due to consumption during activation. please refer to the "complement" section for further details on complement function and measurement. two dyes, sulfobromophthalein and indocyanine green, have been used to assess hepatocellular or bile tract function. following an intravenous bolus, these dyes are rapidly cleared from the plasma by hepatocytes and excreted into the bile. both dyes have been used to assess liver function in mice (hurwitz et al. 1994; huang and vore 2001) . the kidney plays a complex role in maintaining homeostasis in the body and is involved in such functions as water and electrolyte balance, nutrient conservation, maintenance of blood ph, and removal of the end products of nitrogen metabolism (such as urea, creatinine, and allantoin). in addition, the kidney produces and responds to a variety of hormones. however, assessing renal function is complicated by the large functional reserve of the kidneys. for instance, increases in urea nitrogen do not occur until 70-75% of renal mass has become functionally compromised. additionally, assessment of analytes in urine is difficult due to the small size of mice (as discussed in the "sampling" section). urea is produced in the liver as a waste product of protein catabolism (specifically a breakdown product of ammonia). urea is a small molecule that freely diffuses across cell membranes, and therefore the urea concentration is the same in blood, serum, and plasma. traditionally, urea concentrations is measured in terms of urea nitrogen (the amount of nitrogen contained within urea). determination of urea nitrogen is usually made from serum, but whole blood can be used (hence the term blood urea nitrogen [bun] ). quantification of urea nitrogen is based on spectrophotometry assays, measuring the amount of urea that is hydrolyzed by urease. frith et al. (1980) investigated urea nitrogen levels in balb/c and c57bl/6 mice. urea nitrogen levels decreased after 3 months of age but increased again after 12 months in both sexes. in balb/c mice, levels were higher in males than females, whereas in c57bl/6 mice, females had significantly higher levels than males. elevated urea nitrogen (azotemia) can be caused by prerenal, renal, and postrenal conditions. prerenal causes include increased protein catabolism (such as with inflammation, starvation, and high-protein diets). renal causes usually are associated with conditions that compromise more than 70-75% of functional renal mass and include conditions such as renal amyloidosis, glomerular immune complex disease, and polycystic disease. postrenal include any cause that results in obstruction of the lower urinary system. decreased urea nitrogen is found with disease associated with hepatic insufficiency and low-protein diets. creatinine is a degradation product of creatine and creatine phosphate and represents an end-product of muscle metabolism. quantification of creatinine is based on spectrophotometry assays. baseline serum levels are directly related to muscular conditioning and total muscular mass, which varies between individuals. pathologically elevated serum creatinine levels are caused by the same prerenal, renal, and postrenal causes that elevate urea nitrogen in serum. therefore, quantification of serum creatinine offers no interpretative advantage over urea nitrogen (everett and harrison 1983) . proteinuria is a common finding in normal mice, and includes rodent-specific proteins such as uromucoid, small quantities of c~-and [3-globulins, and a family of prealbumins known as major urinary protein (mup). the mup has three electrophoretic variants, designated as mup-1, mup-2, and mup-3, that are under both genetic and hormonal control. a regulatory locus designated mup-1 with codominantly expressed alleles a and b (located on chromosome 4) controls the urinary levels of the three variants. the mup is synthesized in the liver, secreted into blood, and excreted into urine. males are have higher levels of proteinuria than are females, with levels of 5 mg/ml, and age-related increases are seen in mice of both sexes. increases in other urinary proteins have been associated with a variety of renal diseases in mice. the concentration of urine (amount of solutes dissolved in urine) can be measured by urine specific gravity (usg) or osmolality. urine concentration in healthy mice is very high. watts (1975) determined the usg of healthy cba mice, which ranged from 1.060 to 1.080. the author also found the usg increased from 20 to 80 days of age. the urine concentrating ability of transgenic mice expressing human sickle cell hemoglobin (kos for mouse hb) was assessed by ryan et al. (1997) . they found that sickled cells caused vascular, tubular, and glomerular changes, as well as corresponding hyposthenuria (4-h water deprived osmolality of affected mice was 807 + 285 mosm compared to 1541 + 360 mosm in controls). i. electrolytes 1. sodium, potassium, chloride, and phosphorus sodium and potassium levels are easily measured in murine serum using flame photometry (lithium reference) or ion-specific electrodes. serum sodium levels are slightly higher in mice than in most other mammalian species, with reported values of 174 + 23 (sd) meq/1 (finch and foster 1973) , 147 + 15 (sd) meq/l (everett and harrison 1983) , and 155-161 meq/1 (loeb et al. 1996) . no differences in serum sodium were seen during aging, between sexes, or among strains. serum chloride has been measured using mercuric thiocyanate and the chloridimetric or ion-specific electrode techniques, and inorganic phosphorus in mice has been measured using the phosphomolybdate technique. serum inorganic phosphorus levels decreased as mice aged between 1 and 12 months. this change was documented in balb/c and c57bl/6 strains, as well as in outbred mice (loeb et al. 1996) . total serum calcium reflects both ionized (active calcium) and protein-bound calcium (mainly bound to albumin). ionized calcium is biologically active in bone formation, neuromuscular activity, cellular biochemical processes, and blood coagulation. decreased serum albumin levels are also expected to decrease total calcium levels, by decreasing the amount of protein-bound calcium. however, hypoalbuminemia does not result in clinical signs of hypocalcemia. in mice, total serum calcium has been measured using the sodium alizarin sulfonate technique or atomic absorption spectrometry. two reports, using different techniques, list similar reference ranges of 9 + 1 (sd) mg/dl, whereas a third report lists reference ranges of 5.6 + 0.4 (sd) mg/dl for male and 7.4 + 0.50 (sd) mg/dl for female albino mice. the latter values reported for male mice were significantly lower than those for six inbred strains of mice in two different age-groups using the same techniques (quimby 1999b) . likewise, no differences associated with sex were reported by everett and harrison (1983) , who also examined random bred albino mice. however, bonella et al. (1968) demonstrated significantly higher levels in female swiss albino mice. serum calcium levels appear to decline between 3 and 12 months of age in the balb/c and c57bl/6 strains as well as in outbred mice . frith et al. (1980) found that female c57bl/6 mice had lower calcium levels than males. bonella et al. (1968) demonstrated significantly elevated calcium levels when blood was collected by orbital puncture versus cardiac puncture. hypercalcemia in mice can results as a response to increased levels of pth (such as with hyperparathyroidism) or pthrp (such as with certain neoplasms like multiple myeloma). in mice, the response of parathyroid chief cells to hypercalcemia appears different from other species. grone et al. (1992) studied this response in nude mice with pthrp secreting tumor cells or infusions of pthrp, and found prominent membranous whorls and increased cytoplasmic area of chief cells in mice with hypercalcemia (17.0 + 3.1 mg/dl), which marked these cells as distinctly different from parathyroid chief cells of other species. hypocalcemia can be due to hypoalbuminemia (as previously discussed), renal disease, pancreatitis, and hypomagnesemia. alcock and shils (1974) found that mice fed magnesium-deficient diets developed hypocalcemia, as did mice receiving intramuscular injections of heparin. total serum protein has been evaluated in mice using either the lowry or biuret methods, although the lowry method is preferred for analysis of small volume samples. hypoproteinemia is seen in hepatic injury (also see the "liver function tests" section) or during protein-losing enteropathies or nephropathies. hyperproteinemia is seen during dehydration. age-related changes have been described in a number of strains, such as increased total serum protein in older balb/c and b6 mice and lower levels in dba/2 mice (loeb et al. 1996) . each of the major classes of serum protein ((xl-, (x2-, ~-, and y-globulins) in mice can be distinguished by electrophoresis. changes in serum levels have been associated with a wide variety of diseases in mice. for instance, hypergammaglobulinemia was found in scid mice injected with human cag multiple myeloma cells . the acute phase reactants ceruloplasmin and fibrinogen have been discussed in the "serum proteins" section and crp and sap in the "complement" section. saa is an acute phase reactant synthesized by the liver and may be induced by the inflammatory cytokines il-1 or il-6 (see the "cytokines and chemokines" section). normally saa is quickly cleaved to a small molecular weight product, but during chronic infection or failure in the degradation pathway, tissue deposition of amyloid may occur. differences in serum concentrations have been described in various mouse strains and during disease. commercial ria and elisa kits are available for its quantitation in mice (quimby 1999b) . alpha-1-fetoprotein (afp) is encoded by the afp gene which is nearly identical in its organization to the mouse albumin gene, albl. it is likely afp arose due to gene duplication. afp is present in fetal serum but the synthesis declines shortly after birth. serum levels are regulated by two loci, afr-1 and afr-2, which are not linked to afp. the action of these genes may be modified by tgf-~ and p53 (wilkinson et al. 2005) . levels in mouse serum have been quantified by immunoelectrophoresis (zizkovsky 1975 ) and both polyclonal antibodies and recombinant dna-derived mouse afp are available for assay development (boismenu et al. 1997) and are elevated in mice with certain neoplasms and during hepatic regeneration (jin et al. 2005; quimby 1999b ). sulphostin, a novel inhibitor of dipeptidyl peptidases iv (dppiv) that stimulates hematopoiesis in mice proteomic approaches to biomarker discovery in prostate and bladder cancers 8th annual analyzers buyer's guide il-17: prototype member of an emerging cytokine family increased atherosclerosis in hypeflipidemic mice with inactivation of abca-1 in microphages diet-induced changes in plasma phospholipids transfer protein activity, lipids, and lipoproteins. mpd: 801, 802, 828. mouse phenome database web site comparison of magnesium deficiency in the rat and mouse apoc-1 and apoc-iii as potential plasmatic markers to distinguish between ischemic and hemorrhagic stroke comparison of methods for obtaining blood from mice anxiety and stress responses in female oxytocin deficient mice impaired calcification around matrix vesicles of growth plate and bone in alkaline phosphatase-deficient mice hormonal control of leydig cell differentiation interleukin-7 an interleukin for rejuvenating the immune system a spectrophotometric microtiter-based assay for the detection of hydroperoxy derivatives of linoleic acid mast cell lineage development and phenotypic regulation laboratory routines cause animal stress regulation of fasting blood glucose by resistin biochemistry of immunoglobulins variations in serum calcium between strains of inbred mice correction of ureagenesis after gene transfer in an animal model and after liver transplantation in humans with ornithine transcarbamylase deficiency biology of the congenitally hypothyroid hyt/hyt mouse the chemokine stromal cell-derived factor-1 regulates the migration of sensory neutron progenitors methods of enzymatic analysis the mouse phenome project purification and characterization of human and mouse recombinant alpha-fetoproteins expressed in escherichia coli genetically engineered animals in drug discovery and development: a maturing resource for toxicologic research effects of heparin on serum calcium in mice albumin content in blood of inbred mice strains lentiviral shrna silencing of murine bone marrow cell ccr2 leads to persistent knockdown of ccr2 function in vivo trophic action of leptin on hypothalamic neurons that regulate feeding the interpretation of serum biochemistry test results in domestic animals transgenic models of autoimmune disease lipoprotein metabolism and atherosclerosis susceptibility in transgenic mic mouse models of atherosclerosis reproductive failure in aged cbf male mice: interrelationships between pituitary gonadotropic hormones, testicular function, and mating success stromal cell derived factor 1/cxcl12 selectively counteracts inhibitory effects of myelosuppressive chemokines on hematopoieteic progenitor cell proliferation in vitro minireview: genetic models for the study of gonadotropin actions recruitment of cxcr3 and ccr5 t-cells and production of interferon-gamma inducible chemokines in rejecting human arteries tietz fundamentals of clinical chemistry distribution and characterization of the serum lipoproteins and apolipoproteins in the mouse, mus musculus disparate 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dehydrogenase, and aspartate aminotransferase and their isoenzymes as indicators of the development of experimental hepatitis in mic the endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages niemann-pick c heterozygosity confers resistance to lesional necrosis and macrophage apoptosis in murine atherosclerosis hematologic and serum electrolyte values of the c57bl/6j male mouse in maturity and senescence hormone production by the pituitary and testes of male c57bl/6j mice during aging age effects of luteinizing hormone, progesterone, and prolactin in proestrus and acyclic c57bl/6j mice innovation~stagnation: challenge and opportunity on the critical path to new medical products interleukin-17 hematologic and clinical chemistry findings in control balb/c and c57bl/6 mice pi3k and negative regulation of tlr signaling visfatin: a protein secreted by visceral fat that mimics the effects of insulin floor space needs for laboratory mice: c57bl/6males in solid-bottom cages with bedding a longitudinal hormonal profile of the genetically obese mouse return to normal of blood-glucose, plasma insulin, and weight gain in new zealand obese mice after implantation of islets of langerhans acute phase proteins ghrelin: more than a new frontier in neuroendocrinology tumor necrosis factors a rapid, simple, and humane method for submandibular bleeding of mice using a lancet palatal cytosol cortisol-binding protein associated with cleft palate susceptibility and h-2 genotype relationship in the functional levels of early components of complement to the h-2 complex of mice developmental and tissue-specific regulation of parathyroid hormone (pth)/pth-related peptide receptor gene expression ccl1-ccr8 interactions: an axis mediating the recruitment of t-cells and langerhans-type dendritic cells to sites of atopic skin inflammation measurement of glycosylated haemoglobins and glycosylated plasma proteins in animal models with diabetes or inappropriate 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luteinizing hormone reception function loperamide effects on hepatobiliary function, intestinal transit, and analgesia in mice redox state-dependent and sorbitol accumulationindependent diabetic albuminaria in mice with transgene-derived human aldose reductase and sorbitol dehydrogenase deficiency hepatocyte nuclear factor 4 alpha is a central regulator of bile acid conjugation genetic association between h-2 gene and testosterone metabolism in mice use of transgenic mice in carcinogenicity hazard assessment leukotrienes and atherosclerosis: new roles for old mediators endothelial lipase and hdl metabolism targeted mutation of plasma phospholipids transfer protein gene markedly reduces high density lipoprotein levels genetic heterogeneity of lipoproteins in inbred strains of mice: analysis by gel permeation chromatography afp gene expression after acute diethylnitrosamine intoxication is not afr2 regulated identification of tumor-associated plasma biomarkers using proteomic techniques: 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between the development of the intestinal iga immune system and the establishment of microbial flora in the digestive tract of young holoxenic mice split activity of interleukin-10 on antigen capture and antigen presentation by human dendritic cells: definition of a maturative step genetic susceptibility and resistance to diet-induced atherosclerosis and hyperlipoproteinemia chemokines; multiple levels of leukocyte migration control initial sequencing and comparative analysis of the mouse genome oral tolerance in the absence of naturally occurring tregs structure, binding, and antagonists in the il-4/il-13 receptor system corticotropinreleasing hormone deficiency reveals major fetal, but not adult, glucocorticoid need diet effects on bone mineral density and content, body composition, and plasma glucose, leptin and insulin levels. mpd: 143. mouse phenome database web site evaluation of h-fabp as a marker of ongoing myocardial damage using hgh transgenic mice lipid inflammatory mediators in diabetic vascular disease transgenic expression of pancreatic secretory trypsin inhibitor-1 ameliorates secretagogue-induced pancreatitis in mice summary of the second report of the national cholesterol education program (ncep) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults recognition and clearance of apoptotic cells: a role for complement and pentraxins mildly oxidized ldl induces an increase apolipoprotein j/paraoxonase ration the genetic control of alkaline phosphatase activity in the duodenum of the mouse diabetes mellitus: a "thrifty" genotype rendered detrimental by "progress"? cx3crl-mediated dendritic cell access to the intestinal lumen and bacterial clearance clinical laboratory reference sequential blood samples from the tail vein of rats and mice obtained with modified liebig condenser jackets and vacuum the cancer research uk experience of pre-clinical toxicology studies to support early clinical trials with novel cancer therapies lactic dehydrogenase virus insight into the physiological actions of thyroid hormone receptors from genetically modified mice ccr7 governs skin dendritic cell migration under inflammatory and steady state conditions viral pertubation of endocrine function: disordered cell function leads to disturbed homeostasis and disease targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity genetics, cytokines, and human infectious disease: lessons from weekly pathogenic mycobacteria and salmonellae circadian rhythms of plasma corticosterone binding activity in the rat and mouse one hundred years of mouse genetics: an intellectual history. i. the classical period one hundred years of mouse genetics: an intellectual history. ii. the molecular revolution the ultrastructural localization of the isoenzymes of aspartate aminotransferase in murine tissues proteomic analysis of diet-induced hypercholesterolemic mic clinical chemistry values of the n:nih(s) mice and parameter variations due to sampling techniques use of proteomic patterns in serum to identify ovarian cancer lessons from kitty hawk: from feasibility to routine clinical use for the field of proteomic pattern diagnostics alkaline phosphatase activity of the mouse immune regulation in the intestine boosted decision tree analysis of surface enhanced laser desorption/ionization mass spectral serum profiles discriminates prostate cancer from non-cancer patients animal models in biomedical research complement the mouse transgenic models of tolerance and autoimmunity: with special reference to systemic lupus erythematosus short-term and long-term in vivo exposure to an ephedra-and caffeine-containing metabolic nutrition system does not induce cardiotoxicity in b6c3f1 mice model models of atherosclerosis hormones type 2 diabetes: a matter of beta-cell life and death leukemia inhibitory factor and interleukin-ll: cytokines with key roles in implantation virus induced pancreatic disease; alterations in concentrations of glucose and amylase in blood lungkine, a novel cxc chemokine, specifically expressed by lung bronchoepithelial cells changes in serum hormone levels associated with male-induced ovulation in group housed adult female mice ko transgenic mouse model of sickle cell disease animal models of autoimmunity and their relevance to human diseases hematological comparison of the mouse blood taken from the eye and tail cystatin c as a potential cerebrospinal fluid marker for the diagnosis of creutzfeldt-jakob disease efficacy of 2, 3-dimercapto-l-propanesulfonic acid (dmps) and diphenyl diselenide on cadmium induced testicular damage in mice multimeric cytokine receptors: common versus specific functions bilateral lesions in the suprachiasmatic nuclei affect circadian rhythms and h-thymidine incorporation into deoxyribonucleic acid in mouse intestinal tract, mitotic index of corneal epithelium, and serum corticosterone pthrp, pth, and the pth/pthrp receptor in endochondral bone development diabetes, obesity, and the brain hematopoietic, immunomodulatory and epithelial effects of interleukin-11 intestinal tsh production is localized in crypt enterocytes and in villus 'hotblocks' and is coupled to il-7 production: evidence for involvement of tsh during acute enteric virus infection adipsin, a biomarker of gastrointestinal toxicity mediated by a functional gamma-secretase inhibitor loci controlling plasma non-hdl and hdl cholesterol levels in a c57bl/6j x casa/rk intercross quantitative trait loci mapping of genetic modifiers of metabolic syndrome and atherosclerosis in low-density lipoprotein receptor-deficient mice the kinase lkb 1 mediates glucose homeostasis in liver and therapeutic effects of metformin genetic defects in copper metabolism estrogen: the good, the bad, and the unexpected il-1 and il-18 receptors, and their extended family b-cell-and monocyte-activating chemokine (bmac), a novel non-elr ct-chemokine feed-forward regulation of bile acid detoxification oby cyp3a4: studies in humanized transgenic mice adenovirus-mediated rescue of lipoprotein lipase-deficient mice breeding of the gad-mdx mouse: influence of genetically induced denervation on dystrophic muscle fibers failure of parturition in mice lacking the prostaglandin f receptor inactivation of stress protein p8 increases murine carbon tetrachloride hepatotoxicity via preserved cyp2e1 activity murine models to investigate pharmacological compounds acting as ligands of ppars in dyslipidemia and atherosclerosis the other side of the orexins: endocrine and metabolic actions assessment of hepatic function mitochondrial affinity for adp is twofold lower in creatine kinase ko muscles. possible role in rescuing cellular energy homeostasis evaluation of proparacaine hydrochloride as a topical anesthetic for the collection of blood from the orbital sinus of mice mitochondrial affinity for adp is twofold lower in creatine kinase knockout muscles. possible role in rescuing cellular energy homeostasis the role of cd40-cd154 interactions in autoimmunity and the benefit of disrupting this pathway scavenger receptor class b type 1 in high-density lipoprotein metabolism, atherosclerosis, and heart disease: lessons from gene targeted mice cxc chemokine receptor cxcr2 is essential for protective innate host response in murine pseudomonas aeruginosa pneumonia in vivo uptake of radiolabeled mda2, an oxidation-specific monoclonal antibody, provides an accurate measure of atherosclerotic lesions rich in oxidized ldl and is highly sensitive to their regression the regulation of the complement system: insights from genetically-engineered mice interleukin-6: an overview development of a novel proteomic approach for the detection of transitional cell carcinoma of the bladder in urine mechanisms of suppression by suppressor t-cells lipids and lipoproteins car and pxr agonists stimulate hepatic bile acid and bilirubin detoxification and elimination in mice pneumocystis carinii activates the nf-kappa[~ signaling pathway in alveolar epithelial cells a simple capillary tube method for the determination of the specific gravity of 25 and 50 micro 1 quantities of urine interleukin-18 facilitates the early antimicrobial host response to escherichia coli peritonitis transgenic studies on the regulation of the anterior pituitary gland function by the hypothalamus cxcr3 and its ligand participate in the host response to bordetella bronchiseptica infection of the mouse respiratory tract but are not required for clearance of bacteria from the lung a direct intersection between p53 and transforming growth factor beta pathways targets chromatin modification and transcription repression of the alpha-fetoprotein gene assessment of hepatic function and damage in animal species laboratory animal endocrinology investigation of antitumor effects of synthetic epothilone analogs in human myeloma models in vitro and in vivo the protein profile of acetazolamidetreated sera in mice bearing lewis neoplasm cebs object modes for systems biology data, sysbio-om partal hormone resistance in mice with disruption of the steroid receptor coactivator-1 (src-1) gene platelet factor 4 gene transfection into tumor cells inhibits angrogenesis tumor growth and metastasis mice lacking the thyrotropin-releasing hormone gene: what do they tell us? differences in the human and mouse amino-terminal leader peptides of ornithine transcarbamylase affect mitochondrial import and efficacy of adenoviral vectors combined pituitary hormone deficiency: lessons from the murine models molecular basis of resistance to thyroid hormone a mouse bleeding technique yielding consistent volume with minimal hemolysis transgenesis and the study of expression, cellular targeting, and function of oxytocin, vasopressin and their receptors kos model the 100 best-selling drugs: will they model the next 100? predicting drug efficacy: kos model pipeline drugs of the pharmaceutical industry three biomarkers identified from serum proteomic analysis for the detection of early stage ovarian cancer cxc chemokine ligand 129 stromal cell-derived factor 1 alpha and cxcr4-dependent migration of ctls toward melanoma cells in organotypic culture il-ii: insights in asthma from overexpression transgenic modeling genotype, obesity, and cardiovascular disease: has technical and social advance outstripped evolution? specific fetal serum proteins of 13 mammalian species t3 t4 tace taj tc tg tgf-i3 tgf-~isf th tlr tnap tnf tnfrsf tnfsf tof tpc trance trh tsh tx upr usg vldl serum amyloid a serum amyloid protein standard deviation stromal cell-derived factor-1 sorbitol dehydrogenase surface-enhanced laser desorption/ionization secondary lymphoid tissue chemokine stearoyl-coa desaturase-1 suppressor of cytokine signaling-3 steroid receptor coactivator-1 sj6gren's syndrome single-strand deoxyribonucleic acid triiodothyronine thyroxine tumor necrosis factor-a converting enzyme toxicity and jnk inducer cytotoxic t-cells triglyceride transforming growth factor-j3 transforming growth factor-13 superfamily t-helper toll-like receptors tissue nonspecific alkaline phosphatase tumor necrosis factor tumor necrosis factor receptor superfamily tumor necrosis factor superfamily time-of-flight total plasma cholesterol tumor necrosis factor-related activation-induced cytokines thyrotropin-releasing hormone thyroid-stimulating hormone thromboxane unfolded protein response urine specific gravity very low-density lipoprotein key: cord-015082-l629n8is authors: nan title: poster sessions 323-461 date: 2002-08-29 journal: intensive care med doi: 10.1007/s00134-002-1455-7 sha: doc_id: 15082 cord_uid: l629n8is nan total protein concentration in bal increased significantly and led to peak at 1689±323 mg/ml 1hour after intubations . mucin concentration was highest at 1hour after ventilation (155.75±31.6mg/ml). bal sp-a concentration ratio increased about 20 times after 1hour ventilation. compare to 100mg/ml total protein, the ratio was 2.16±1.14 in 1hour later, and 4.31±2.8 in 3hours after ventilation.the change of bal wbc level led to peak in after 1hour ventilation, but blood wbc level led to peak in 3hours later. for elastase level both peak were 3hours later in bal and blood.in the caller components of bal, the neutrophyl cells were dominant in 1hour after intubation, but 3hours after ventilation, mast cells with phagocyted mucine and dusts were dominant. just introduction. coronary disease is prevalent in diabetic patients resulting in a frequency of invasive cardiac procedures four times that of non-diabetics. after cardiac surgery diabetics have twice the mortality and morbidity in early and late phases after operation. the reasons for this increased risk are poorly understood. diabetics exhibit complex abnormalities of lung structure and of the control of the cardiorespiratory system. these include pulmonary micro-vascular disease, autonomic neuropathy associated with an increased cardiovascular instability, an increased incidence of central and obstructive sleep apnoea and a reduced response to hypercapnia. this study was undertaken to determine whether at risk diabetic patients could be identified pre-operatively. methods. 14 patients awaiting urgent cardiac surgical re-vascularisation were studied with measurement of: spirometry; percentage increase in transfer factor from sitting to lying position (tf) as an indicator of micro-vascular lung disease; overnight oximetry on air; and 24hour holter monitoring at present arf is one of the most spread and serious complication of postoperation period. practically the experience of carring nimv on patients with arf on early stadies of mosf is absent. until now, the criteria of uneffectiveness of nimv and indications for cessation of mask ventilation and moving of patients to mechanical ventilation are not determined. methods. there were included 48 patients with ali in the examination. the cause of this condition was the mosf, developed in postoperative period. diagnosis of ali/ards was stated on the criteria adopted the american european consensus conference on ards (1). presence of organs failure was determined on multiple organ dysfunction score (table 1) . nimv was carried out by seances from 5 to 8 hours. average duration of nimv consisted 39.5±5.3 hours. results. improvement of gases change was determined on 28 patients (58%) out of 48. though 20 patients with mosf were reintubated, out of which 13 patients (27%) died lately as the result of mosf progressing. the condition of gases changing functions before intubation is one of the determining factors of prognosis. the patients reintubated under satisfactory indices of gase blood composition and early symptoms of mosf survived. patients, who were reintubated on decreased indices of arterial oxygenation under mosf progressing died in 100% cases ( nimv is effective method in complex therapy of arf, developing in postoperative period after cardiac surgery, that leads to significant improvement of lungs biomechanics and gases change function. progressing of mosf and storage disturbance of lung oxygenation is absolute indication for intubation and applications of special regimes of mechanical ventilation. references. 1. bernard gr, artigas a, brigham kl. am j respir crit care med -1994: 149:818 introduction. several bioimpedance cardiac output systems have been developed in the past in order to measure cardiac output in a wide variety of clinical situations. however, open thorax surgery negatively influences the accuracy of the measurement of thoracic electrical bioimpedance cardiac output (teb-co) (1). the purpose of the present study was to evaluate the performance of a new bioimpedance cardiograph hl-4 (vrije universiteit medical centre amsterdam and hemologic amersfoort, the netherlands), using a new algorithm and a new electrode configuration, during open and closed chest in cabg patients, comparing teb-co with transcardiopulmonary thermodilution (tcpco). methods. after hospital ethics committee approval and written informed consent, fourteen patients with preserved lv-function at cineangiography or echocardiography, scheduled for coronary artery bypass grafting were included. for the teb system two current injecting electrodes were placed on the forehead and the left thigh respectively and two voltage sensing electrodes were used: one above the left clavicle at the base of the neck and the other at the level of the xyphoid in the left midaxillary line. for tcpco, the picco-system (pulsion, munich, germany) was used. hemodynamic measurements were recorded at three time points: t1 before the operation, t2 after weaning from bypass before sternal closure and t3 after sternal closure. teb-co and tcpco data were compared with pearson's r correlation coeficient. p<0.05 was considered significant. bland-altman analysis (2) with bias and precision was carried out at each of the three time points. results. ten males and 4 females with age 64±9 yr, body weight 78 ± 13 kg and height 172±8 cm were included. a total of 34 matched data pairs were available for analysis. table 1 shows the results of correlation, bias and precision of the measurements at the three different time points. teb consistently underestimated tcpco. at all time points, there was a good correlation between both techniques. introduction. isoflurane sedation of icu patients has previously been shown to be useful but has not come into wide clinical use for a number of reasons.a new device(the anesthetic conserving device,"acd") enables easy and safe administration of isoflurane in the icu setting.we conducted a randomised, controlled study to evaluate efficacy of sedation and environmental safety during administration of isoflurane with the acd. the acd is a modified heat and moisture exchanger connected to the breathing circuit at the endotracheal tube.isoflurane is administered via a syringe pump to a vaporiser rod in the acd.due to the physical properties of the acd most of the exhaled isoflurane is returned to the patient.5 mechanically ventilated patients were randomised to receive isoflurane via the acd.4 control patients received midazolam intravenously. all patients received morphine analgesia. quality of sedation was assessed hourly in all patients."adequate sedation" was pre-defined as a set interval on the bloomsbury sedation scale. additionally, the patient's nurse determined if sedation over the previous hour in general had been adequate or not. time from discontinuation of the sedative drug until the patient followed verbal command and to extubation was compared between groups. in the isoflurane group a gas evacuation system was used during isoflurane administration. athmospheric concentration of isoflurane was measured at 0.5 m from the acd. results. in the isoflurane group patients were adequately sedated by the bloomsbury scale for 58 ± 9 % of the study period, compared to 50 ± 28 % in the control group.nurse satisfaction in the isoflurane group was 88 % of time and 57% of time in the control group.mean time to extubation after cessation of sedative administration was 11 min in the isoflurane group and 1864 min in the control group, mean time to patient cooperation was 55 min in the isoflurane group, and 1866 min in the control group. no significant hemodynamic changes were noted at initiation of the sedation in either of the groups. no serious complications related to sedation were noted in either group.opioid requirements in the isoflurane group were lower, with a mean rate of 1.6 0.9 mg/hr, compared with a mean rate of 4.2 2.4 mg/hr in the control group.mean isoflurane infusion rate was 2.5 ml/hr, with mean end-tidal isoflurane concentrations of 0.37% (0.19-0.82%).environmental levels of isoflurane were generally low,with a mean of 0.43 ± 0.27 ppm, well below the recommended long-term exposure limit of 2 ppm. brief peaks (<2min) between 2 and 15 ppm were noted during endotracheal suctioning, etc on an average of 0.1 times/hour of exposure. conclusion. isoflurane administered via the acd for sedation of icu patients is environmentally safe, requires small volumes of isoflurane and may provide better quality of sedation than midazolam. it appears to be more titratable with a shorter time from adequate sedation to extubation and ability to cooperate. references. millane ta, bennett ed,grounds rm,anaesthesia 1992;47:768-774.spencer em,willatts sm,intensive care brudney c. s. 1 , gosling p. 2 , manji m. 3 1 anaesthesia, 2 biochemistry, 3 anaesthesia, university of birmingham, birmingham, united kingdom increased capillary permeability has been implicated in the pathogenesis of ards and organ failures. surgery and ischaemia-reperfusion injury are both associated with stimulation of the acute inflammatory response, an early feature of which is an increase in systemic capillary permeability. the kidneys amplify small changes in systemic capillary permeability (1).the aim of this study was to explore any association between acr during and after cardiopulmonary bypass (cpb) and subsequent pulmonary and renal function. methods. forty patients (9 female) mean (range) age 67.8 (50-85) yrs undergoing coronary artery bypass grafting were enrolled. patients with severely impaired left ventricular function (<35% ef) were excluded. ten ml of urine was collected at intervals from the start of surgery until 48 hours post cpb. microalbuminuria was measured by automated immunoturbidimetry and expressed as the albumin creatinine ratio (acr: ref. range <2.3 mg/mmol). acr was compared with po2/fio2 ratio, hours on ippv, renal function and duration of inotropic support, using spearman's rank correlation procedure. results. two patients were excluded (death at 6 hours and acute renal failure post cpb). the median (range) duration of ippv was 15 (4-72) hours. 22 patients required inotropic support for median (range) 12 (2-96) hours. median (range) acr increased during surgery and was maximal 10 minutes post cpb. (table) two hour acr was inversely correlated with the mean po2/fio2 ratio up to 12 hours (rs = -0.46 p = 0.0044). two and 12 hour acrs were both positively associated with duration of ippv (rs = 0.46 p = 0.0071 and 0.62 p <0.0001 respectively). acr at 4 and 12 hours were associated with serum creatinine 24 hours post cpb, (rs = 0.35 p = 0.045, rs = 0.50 p = 0.003 respectively). acr at 2, 4 and 12 hours post cpb were associated with serum creatinine 48 hours post cpb (rs = 0.45 p = 0.033, rs = 0.59 p = 0.003 and rs = 0.49 p = 0.016 respectively). there was no significant association between duration of inotropic support and acr at any time point up to 48 hours. conclusion. cpb leads to a perioperative microvascular insult, causing increased capillary permeability which influences later pulmonary and renal function. these rapid changes in microvascular permeability can be monitored as the acr, and in the patient group studied, the magnitude of the acr as early as 2 hrs post cpb is associated with later organ function. acr may provide a tool allowing early identification of patients at risk of developing organ dysfunction, who may benefit from early intervention aimed at modifying the inflammatory response. acute lung injury (ali) is a major complication of gram-negative bacterial sepsis. to date, bacterial lipopolysaccharide has been held responsible for triggering ali (1). whether additional bacterial toxins play a role in the development of acute pulmonary inflammation during gram-negative sepsis remains an unresolved issue. flagellin, a principal component of bacterial flagella, has been recently shown to elicit immune responses via activation of the toll-like receptor 5 (2). we have newly found that flagellin induces an expression of icam-1 and a massive production of il-8 by human lung epithelial cells. in mice, flagellin produces a severe acute lung inflammation with local release of pro-inflammatory cytokines, accumulation of inflammatory cells and increased pulmonary permeability that was more pronounced than following endotoxin (3). the purpose of the present investigation was to evaluate the influence of flagellin on lung fluid filtration in rats. wistar rats (250-300 g) were exposed either to intravenous injection of flagellin 0.1-2 mg/kg or corresponding volume of normal saline (controls). after 8-20 h, the rats were anesthetized and the lungs were isolated. the isolated lungs were ventilated under a normoxic condition and perfused with homologous blood (37ºc) at a constant flow for 4 h or until development of irreversible edema. airway pressure, pulmonary arterial pressure, pulmonary vascular resistance, and changes in the lung weight were assessed. the increments in outflow pressure of 0.77 kpa for 6 min were used to determine the fluid filtration rate and filtration coefficient in the lungs every 30 min (4). flagellin induced a dose-and time-dependent increment in the lung fluid filtration rate. in parallel, flagellin markedly increased airway pressure, pulmonary arterial pressure, pulmonary vascular resistance, and filtration coefficient. in contrast to the control lungs, all the lung preparations from flagellin-treated animals developed irreversible edema within the first two hours of perfusion. in isolated blood-perfused rat lungs, flagellin enhances fluid filtration, most likely, through elevation both of pulmonary microvascular permeability and hydrostatic pressure. the present study provides further evidence that flagellin may contribute to the development of sepsis-associated ali. . whether protein c conversely affects eosinophil function has not yet been reported. we investigated the effects of protein c and activated protein c on chemotaxis of eosinophils. possible involvement of endothelial protein c receptor (epcr) in the regulation was studied by using specific epcr antibodies. for preparation of eosinophils we used macs cd+16 microbeads according to the manufactor's protocol. chemotaxis assays were performed using a 48-well boyden microchemotaxis chamber in which a 5-micrometer pore sized cellulose nitrate filter separates the upper and the lower chamber. eosinophils were pretreated by various protein c preparations with or without epcr antibodies, followed by washing and assessment of their migratory responses toward eotaxin. protein c and activated protein c exerted no significant chemotactic effect on eosinophils. however, eosinophils pretreated with protein c or activated protein c showed a sigificantly reduced response to the specific chemoattractant, eotaxin. moreover, this effects of protein c and activated protein c were inhibited using an antibody against epcr. conclusion. protein c as well as activated protein c inhibit the chemotactic effect of eotaxin on eosinophils via mechanisms involving epcr. this result indicates that protein c as well as activated protein c may decrease the number of eosinophils in tissue and thereby inhibiting inflammation and coagulation. deleterious effect of severe sepsis may be related to an oxidative stress, particularly related to peroxynitrite. selenium (se) toxicity is supposed to be related to oxidative stress through reaction with thiols. we perform a study to compare these toxicities. methods. 100 wistar rats were studied. after 8 day quarantine lipopolysaccharide (lps) or se was administered intraperitoneally in 3 ml saline water. lps and se were administered in groups of 10 rats with increasing doses from 28 to 34 mg/kg for lps, and from 0.35 to 4.5 mg/kg for se. mortality was observed at 48 hours. animals were sacrificed under halothane. blood samples were taken in 2 surviving rats of each group. nitric oxide (no) and nitrotyrosine (nit), a marker of oxidative stress especially related to peroxynitrite, were measured by elisa techniques, and plasma se concentration using atomic flame absorption. results. septic rats were rapidly sick. they rolled up into a ball. their fur was dull, and stood on end. they were asthenic and had diarrhea. at autopsy, intestinal abnormalities, and in some rats echymotics dots and hemolytic plasma were observed. rats were dehydrated. se rats developed an encephalopathy the first day and later recovered. se rats were lively, and seemed to required higher level of halothane for induction. (1)however the mechanisms responsible for this alteration remains under investigation. depressed micochondial respiration has also been found in different tissues during sepsis. (2) the objective of this work was to study diaphragmatic function in rats after peritoneal sepsis and to correlate these findings with diaphragmatic mitocondrial respiration. cecal ligation and perforation was done under general anesthesia in wistar rats (septic group, n=7) . after 48 hours the animals were monitored for arterial blood gases, systemic hemodynamia and body temperature. then, they were sacrified and the diaphragm force-frequency curves were obtained in vitro before and after fatigue. contraction time and relaxation time were also measured. mitochondrias were isolated from the diaphragm and oxygen consumption and other respiratory indexes were studied in septic animals. the results were compared to sham operated animals (control group, n=7). the septic group showed significantly lower values of aortic blood flow, arterial oxygen partial pressure, body temperature and arterial bicarbonate (p<0.05) when compared to the control group. the forces measured at the different frequencies of stimulation were lower in the septic diaphragms both before and after fatigue when compared to controls (p<0.05). mitochondrial respiration evaluated by oxygen consumption and rcr indexes was found decreased in the septic animals (p<0.05). diaphragmatic contractile failure along with hemodynamic, respiratory and metabolic dysfunctions was found in peritoneal sepsis in rats. diaphragmatic dysfunction could be explained by mitochondrial damage during sepsis. we speculate that mitochondrial injury and dysfunction could be related to oxidative stress in this animal model. introduction. protein c is activated by thrombin bound to thrombomodulin and this effect is enhanced in the presence of the endothelial protein c receptor (epcr). in vivo and in vitro studies have revealed that components of this pathway may also inhibit inflammatory responses. protein c was able to inhibit leukocyte adhesion to vascular endothelial cells and to reduce neutrophil accumulation in rat lungs [1] . protein c inhibits proinflammatory cytokine release in monocytes [2] that were shown to express epcr [3] . soluble epcr binds to proteinase-3 and cd11b/cd18 of activated neutrophils [4] , which were previously shown to synthesize thrombomodulin but not to promote thrombin-dependent protein c activation [5] . if protein c directly affects neutrophil functions has not jet been sufficiently demonstrated. we investigated the in vitro effects of protein c and activated protein c on chemotaxis of isolated human neutrophils and explored wether epcr may be involved. neutrophils were obtained from forearm venous blood by standard methods. leukocyte migration toward gradients of soluble attractants into cellulose nitrate micropore filters was measured using a 48-well microchemotaxis chamber. cells were either directly exposed to gradients of protein c or were pretreated with protein c followed by washing; then chemotaxis toward typical attractants was tested. neither protein c nor activated protein c induce chemotaxis of neutrophils. both inhibit neutrophil chemotaxis toward interleukin-8, fmlp and c5a and there is no significant difference in the effects of these two substances. a blocking antibody against the epcr is able to diminish the effects of protein c and activated protein c. conclusion. protein c as well as activated protein c is able to inhibit neutrophil chemotaxis. this indicates that an activation of protein c is not necessary for effects on neutrophils to occur or that neutrophils are able to activate protein c followed by migration. the reduction of the protein c effects by an antibody against the endothelial protein c receptor suggests that neutrophils express epcr capable to signal anti-migratory stimuli. during sepsis increased vascular permeability results in fluid extravasation and edema. lymphatics contribute in draining interstitial fluid from the abdomen to central circulation, but several factors (outflow venous pressure, pattern of mechanical ventilation) can act upon flow in the thoracic duct (1, 2). we have tested if lymph flow is affected by endotoxin infusion under different ventilatory conditions. methods. 9 anesthetized pigs (29.4±3 kg) were studied. septic damage was induced by continuous infusion of endotoxin (lipopolysaccharide e.coli, lps). abdominal lymph flow was continuously recorded by an ultrasound flow probe positioned on the thoracic duct at the diaphragm level; hemodynamics, respiratory system data, bga and intra-abdominal pressure (iap) were registered. during the first 2.5 hours of lps infusion animals were ventilated in volume controlled mode tv 10-11 ml/kg, rr 20 bpm, peep 5, fio2 0.5; during the next 2 hours animals were divided in group 1 (control, peep 5), 2 (peep 15) and 3 (spontaneous breathing, cpap peep 5). during lps infusion lymph flow significantly increased from 2.3 to 4.6 ml/min (p<0.05), cardiac output and compliance decreased from 3.4 to 2.8 l/min * and 32 to 21 ml/cmh2o * respectively, while mean pulmonary artery pressure and iap increased from 20 to 47 mmhg * and 13 to 20 cmh2o * (* p<0.05). in all the pigs a positive correlation was found between iap and lymph flow (mean pearson´s coefficient 0.59). no correlation was found between lymph flow and central venous pressure and airway pressure (mean pearson´s coefficient 0.20 and 0.13). in group 1 and 2 lymph flow changes averaged -9% and +19% (versus value before randomization). cpap increased lymph flow by 55%. lymph flow from the abdomen increases during lps infusion: role of lymphatics in draining abdominal fluid could thus be significant during sepsis (~ 280 ml/h are drained). these preliminary results suggest that spontaneous breathing could improve lymphatic flow from the abdomen. despite the following rise in intra-thoracic pressure, increase of peep is not associated with lymph flow reduction. animals in peep 15 group have however shown different patterns of response, and more data are needed to clarify this aspect. introduction. : ischemia/reperfusion or sepsis is initially responsible of an acute activation of pro-inflammatory cytokines (e.g. tumour necrosis factor (tnf-)). it is followed by a rise of anti-inflammatory cytokines (e.g. interleukin-10 (il-10)). in human umbilical vein endothelial cell (huvec) tnf-induces a mitochondrial release of reactive oxygen species (ros) in a dosedependent manner. the signalisation pathway which links tnf-at mitochondria involves ceramide pathway (1).the goal of our study is to evaluate the action of il-10 on the oxidative stress induced by tnf-in huvec and to define the mechanism of this interaction. huvec were grown on plastic cover slides. at confluence they were placed in a perfusion chamber under a microscope equipped with a digital camera connected to acquisition software. cells were perfused with krebs solution containing two fluorescent probes: dichlorodihydrofluorescein diacetate (dcfh) to study the release of reactive oxygen species (ros) and propidium iodide (pi) to study cell mortality. three cell groups were studied: a reference group, a tnf-group where, after one hour stabilisation, tnf-was added (1 ng/ml) in perfusion medium during one hour, · a group tnf-+ il-10 where il-10 was added to perfusion medium 30 minutes before tnf-. variations in fluorescence were recorded each 10 minutes for dcfh and each one hour for pi. for a non lethal concentration (pi remaining unchanged), il-10 reduces significantly the ros production induced by tnf-(anova for repeated measures). interleukin-10 has an inhibitory effect on the release of ros induced by tnfin huvec. this effect could be the result of an interaction with acid sphingomyelinase. (1) am. j. cell. molecular biol. 24: 762-8, 2001 . the immunosuppresive drug cyclosporine a (csa) is an inhibitor of mitochondrial permeability transition (mpt) which could afford protection against cell death [1] .to test whether csa protects against endotoxin-induced myocardial apoptosis [2], we produced 123i-annexin v [3], a marker of apoptotic cells, and measured its myocardial uptake during endotoxaemia in csa-treated rats. the specificity of the signal has been previously verified with caspase inhibitors and 123i-human serum albumin. methods. 1) 123i-annexin v was produced with a radiochemical purity higher than 97% as confirmed by hplc. 2) young male sprague-dawley rats were either given iv : saline (0.5ml) : control group, n=10, or lipopolysaccharide (lps) from e coli (30mg/kg) ± csa (10mg/kg): lps group, n=10 and lps+csa group, n=8. 6h later, all animals were given 123i-annexin v (18mbq, 10mg protein). after 12h, hearts were harvested and divided into apex, septum, right and left ventricle (rv, lv) for determination of 123i-annexin v myocardial uptake with a lkb gamma counter. results were expressed as a mean percentage ± sd of the injected dose per gram of tissue (%id/g). statistical analysis was performed by mann-withney test; a p value <0.05 was considered as significant (*). 123i-annexin v myocardial uptake is significantly increased in the lps group compared to control group; there is no significant difference between the septic groups . control lps lps+csa mean + -sd 0.05 + -0.01 0.18 + -0.06 * 0.19 + -0.05 ns mortality 0% 25% 0% 123i-annexin v myocardial uptake conclusion. our results confirm that endotoxaemia is associated with significant myocardial apoptosis but fail to demonstrate that csa can reduce the cell death signal detected by 123i-annexin v . in spite of its action on mpt and its myocardial dysfunction reducing effect in septic rats [4] , csa provides no myocardial protection in this model . a reducing effect of csa on endotoxin-induced mortality is not excluded but remains to be demonstrated. further investigations are needed to clarify the effect of csa on the inflammatory responses due to endotoxaemia. sepsis induced alterations in hemostasis with dysbalances in fibrinolysis may lead to capillary obstruction due to fibrin deposition. the aim was therefore to investigate regional net fluxes of the fibrinolytic enzyme tissue-type plasminogen activator, tpa, and its main inhibitor plasminogen activator inhibitor type-1, pai-1, in response to endotoxemia. methods. anesthetized pigs (n=8) were instrumented for registration of cardiac output (co, thermodilution) and portal (qpv), hepatic (qha) and renal (qra) blood flows (ultrasound flowmetry, transonic). blood samples were collected from the aorta and pulmonary artery as well as the portal, hepatic and renal veins. after baseline registrations, all animals were subjected to an e. coli endotoxin infusion for 90 min, followed by a volume/norepinephrine resuscitation for 210 min targeting baseline co levels. plasma concentrations of both total and active tpa and pai-1 were determined as described [1, 2] and net organ fluxes (ng/min) were calculated based on in-/outflowing plasma concentrations and local plasma flow [1]. results. endotoxemia induced a low co state and a decrease in qpv. total liver blood flow was preserved due to a concomitant increase in qha. during resuscitation co and qpv were restored to baseline values. systemic plasma levels of total tpa increased over time during endotoxemia, peaking at 90 min, whereupon a decline occurred. however, plasma levels of total tpa had not returned to baseline values at the end of the registration period (300 min). changes in systemic levels of active tpa mirrored changes in total tpa. a marked (8-fold) increase in mesenteric net release of total tpa was observed. this response was paralleled by a pronounced increase in hepatic uptake of tpa. pai-1 described a different response to endotoxemia. by the end of the experiment plasma levels of both active and total pai-1 increased. in contrast, no significant net fluxes of pai-1 were observed across any of the investigated vascular beds except for the hepatic vascular bed, where a net release of both total and active pai-1 occurred at approximately 150 min. hepatic pai-1 release rates then increased progressively. conclusion. endotoxemia induced a marked increase in mesenteric release of tpa which however was not entirely responsible for the increase in systemic plasma level of tpa. the results indicate that this profribrinolytic response at later stages are counteracted by increased plasma levels of pai-1 and this increase is mainly derived from the hepatic vascular bed. thus, patients with altered regional endothelial functions or liver capacity prior to a septic challenge can be expected to demonstrate varying susceptibility to thrombotic events. antithrombin has been shown to reduce mesenteric venular leukocyte interactions and intestine injury in a leukocyte-dependent model of endotoxemia (1). however, endothelial damage during early endotoxemia has been shown to be leukocyte-independent (2). the role of antithrombin in this setting is still unknown. therefore, it was the aim of the study to investigate the effects of antithrombin on leukocyte-independent endothelial damage. in male wistar rats, microvascular permeability (mp) and leukocyte-endothelialinteraction (leukocyte rolling, lr) were determined in mesenteric postcapillary venules using intravital microscopy at baseline, 60 and 120 min after start of a continuous infusion of endotoxin (etx; 2mg/kg/hr, e.coli o26:b6) (group a, n=8). therefore animals were laparotomized and the mesentery was exposed beneath an in-vivo videomicroscope. mp was measured using fluorescein isothiocyanate (fitc) labelled albumin. leukocyte-endothelial interaction was blocked in all groups by fucoidin (25 mg/kg b.w.), a l-selectin-binding carbohydrate, 10 min before laparotomy. animals in group b (n=8) received antithrombin (kybernin®, aventis-behring, germany; 500 ie/kg b.w.) prior to baseline measurement and additionally to the procedure described above. animals in group c (n=8) received equivalent volumes of nacl 0.9 % instead of antithrombin and endotoxin. statistical analysis was performed using two-way repeated measures anova followed by the scheffé test. a p-value <0.05 was considered significant. in groups a-c, fucoidin prevented lr during the entire experiment. however, in all groups mp increased significantly, starting at 60 min. animals in group a were characterized by a stronger increase in mp and showed significantly higher values in mp in comparison to groups b and c at 120 min. there were no significant differences in mp between groups b and c. leukocyte-independent endothelial damage during early endotoxemia is attenuated by antithrombin. endothelial damage during early endotoxemia has been shown to be leukocyte-independent (1). paf (platelet-activating factor)-and serotonin-receptor antagonism has been shown to reduce leukocyte-independent macromolecular leakage significantly (2, 3). nevertheless, the exact mechanisms involved in leukocyte-independent endothelial dysfunction are unknown. therefore, it was the aim of the study to investigate the effects of nitric oxide (no) on leukocyte-independent endothelial damage during endotoxemia methods. in male wistar rats, microvascular permeability (mp) and leukocyte rolling (lr) were determined in mesenteric postcapillary venules using intravital microscopy at baseline, 60 and 120 min after start of the experiment. in all groups, leukocyte-endothelial interaction was blocked by fucoidin. rats were randomized into 7 groups, 11 animals each. the experiments were divided into two parts. part i (no-inhibitor): in group a, the mesentery was superfused with a l-name superfusion (100 mmol/l) combined with a continuous infusion of endotoxin (etx; 2mg/kg/hr) after baseline measurement. group b received a l-name superfusion of the mesentery combined with a continuous infusion of saline 0.9 %. groups c and d were treated like groups a and b but without l-name. part ii (no-donator): group x received sin-1 (initial bolus of 1 mg/kg b.w. followed by 0.5 mg/kg b.w. after 60 min-measurement) followed by a continuous infusion of endotoxin (etx; 2mg/kg/hr). group y was treated similar to group c and group z was treated similar to group d. statistical analysis was performed using two-way repeated measures anova followed by the scheffé test. a p-value < 0.05 was considered significant. fucoidin prevented leukocyte-endothelial-interaction in all groups. part i: pe increased in all groups, being significant in group d at 120 min (p<0.05 vs. baseline) and being significant in groups a-c starting at 60 min. animals in group d were characterized by a slighter increase in mp and showed significantly lower values in mp in comparison to groups a and b at 60 min, and to groups a-c at 120 min. there were no significant differences in mp between groups a-c at 120 min. part ii: pe increased in all groups being significant in group z at 120 min (p<0.05 vs. baseline) and being significant in groups x and y starting at 60 min. animals in group y were characterized by a stronger increase in mp and showed significantly higher values in mp in comparison to groups x and z at 120 min. there were no significant differences in mp between groups x and z. leukocyte-independent endothelial damage during early endotoxemia is a nitricoxide mediated event. overproduction of nitric oxide (no) is thought to be a principal cause of the hypotension of septic shock. two nitric oxide synthase (nos) enzymes have been described in blood vessels: endothelial nos (enos) and inducible nos (inos). constitutive activity of enos in the endothelium is a major determinant of blood vessel tone in health; however, in experimental sepsis it appears endothelial enos expression is reduced while smooth muscle inos expression is increased (1). in contrast, another model of human sepsis found an increase in enos but not inos in the vessel wall (2). to resolve this discrepancy, we studied enos and inos protein concentrations in arterial smooth muscle (asm) from patients with clinical sepsis. asm was isolated from mesenteric vessels from patients undergoing bowel resection for perforated viscus (who in the perioperative period met the accp/sccm criteria for septic shock), and from controls with bowel cancer. after mechanical removal of endothelium and adventitia, the tissue was homogenised in protease inhibitor and frozen until sufficient samples had been accumulated. western blotting was performed under reducing conditions, with membranes incubated in 1:2000 (inos) or 1:500 (enos) primary antibody followed by 1:2000 peroxidase labelled secondary antibody. protein bands were quantified by computer analysis of the chemiluminescence detection film, then normalised to the protein concentration of the sample prior to dilution. . enos protein was increased in arterial smooth muscle from patients with septic shock (control 39.3 14.7 units/mg, septic 96.5 27.1 units/mg; n=13 controls and 11 septics; p = 0.04, student's t test). in contrast, there was no increase in concentration of inos; indeed inos protein was only detectable in asm from 2 control and 3 septic patients. we suggest that overexpression of enos, rather than inos, in the arterial smooth muscle of patients with septic shock may be responsible for the hypotension observed in these patients. introduction. data published in the literature concerning the effect of sepsis on intestinal motility found a reduction as well as a stimulation of intestinal motility . the settings used are mostly in vivo settings, and therefore not usable to investigate intestinal motility independent from circulatory changes. the aim of our study was to evaluate the direct effect of endotoxinemia on guinea-pig small bowel motility in vitro, independent from circulatory changes, and in a second step to evaluate the effect of vasoactive drugs on motility of these septic animals. two groups of guinea-pigs received 1 mg/kg e. coli lps intraperitoneally 4 or 20 hours before the experiments started. in the following hours the animals developed severe symptoms of sepsis. a control group did not receive lps before the experiments started. the small bowel of sacrificed guinea-pigs was excised, cleaned and kept in tyrode's solution. after a resting period segments of 8 cm length were set up in parallel organ bathes containing oxygenated tyrode's solution. peristaltic contractions were elicited by perfusion of the segments with tyrode's solution at a rate of 0.5 ml/min, against an aboral resistance of 400 pascal. the intraluminal pressure increased gradually until it reached a pressure threshold (pt) which triggered peristaltic contractions. these contractions were recorded via a pressure transducer at the aboral end of the segments. increasing concentrations of epinephrine, norepinephrine, dopamine, dobutamine, clonidine and dexmedetomidine were cumulatively added to the organ bath at 15 min intervals. each drug was tested on 8 different segments. statistics was performed using ncss for windows, one-way and two-way anova for repeated measures were used, p values <0.05 were considered statistically significant. in the control group all tested vasoactive drugs had a dose-and substance-dependent inhibitory effect on peristalsis. higher concentrations of all tested substances led to a complete block of peristalsis. 4 hours after lps application a pronounced reduction of the inhibitory effects of clonidine, epinephrine, norepinephrine and dopamine were found. the reduced inhibitory effect of dexmedetomidine was not significant. 20 hours after lps application the inhibitory effect was reduced again, but for most substances this reduction was not statistically significant. dobutamine was the only tested substance with a more pronounced effect after 4 hours than after 20 hours. endotoxinemia per se did not affect small bowel motility in vitro. a possible explanation for the controversy to in vivo data demonstrating an inhibitory effect on peristalsis might be that intestinal ischemia is a common event during sepsis, and ischemia in turn might cause paralysis. a described reduced sensitivity of alpha-adrenoceptors during sepsis, or a central effect of lps additionally inhibiting peristalsis (1), might also be responsible for our findings. high cytokine levels in patients admitted to the emergency department are associated with an increased incidence of sepsis/septic shock. patients with cardiogenic shock (cs) who often develop sepsis during icu-stay,have not been particularly studied. we studied whether plasma levels of cytokines are better predictors of sepsis/septic shock than routinely determined laboratory parameters. il-1,il-6 and il-10 plasma levels were determined in 51 pts with cs(cardiac index <2.2 l/min/m²,pcwp >15, mean arterial pressure <60mmhg or need for vasopressor therapy and signs of organ hypoperfusion) on admission to the icu (median 16hrs after shock onset). 36 patients who were not surgically treated during icu stay were eligible for the study and evaluated for development of sepsis or septic shock within 1 week after onset of cs. c-reactive protein (crp) levels and white blood cell (wbc)-counts were routinely evaluated once daily in all patients until discharge. data are given as median and interquartile range. all pts with cs were free of demonstrable infection at time of blood sampling. nevertheless 60% had a crp-level >5mg/dl at time of enrollment.11 pts (31%) developed septic shock within 1 week after onset of cs. pneumonia (54%, n=6) and catheter related infections (27%, n=3) were the leading causes of sepsis. sepsis after cs was not associated with a higher mortality rate (82% vs. 80%, p=ns) and sirs that was encountered in 69% of cs pts at the time of blood sampling did not predispose for development of sepsis (64 vs. 72%, p=ns).crp levels,and wbc-counts as well as il-1, il-6 and il-10 plasma levels on admission to the icu did not differ significantly between cs-pts who developed sepsis and cs-patients without sepsis ( in pts who survived for more than 24hrs (n=28) the absolute crp levels 24hrs after admission (crp24hrs) and the increase in crp levels over 24hrs following icu admission (dcrp) were significantly higher in pts who developed sepsis as compared to pts without sepsis. (crp24hrs: 23.2mg/dl [11-35.6] vs. 9.4 [5.9-13.1], p=0.002; dcrp: 9.7mg/dl [7.5-11.8] vs. 4.1[0.5-5.6], p=0.011). a dcrp >6.5mg/dl in 24hrs was more sensitive than an absolute crp level >20 mg/dl 24hrs after icu-admission for predicting sepsis (82 vs. 73%), but both parameters had equal specificity (88%). conclusion. although many pts with cs exhibit elevated crp levels the increase in crp over 24hrs (dcrp24hrs)is a valuable parameter to identify pts at risk for sepsis. single-point determination of cytokines on admission to the icu is not superior to follow-up determinations of crp for predicting sepsis. mitochondrial dysfunction may be implicated in sepsis-induced multi-organ failure. glycolytically-generated atp may thus be an important alternative energy source if aerobic respiration is compromised. little is known about glycolysis during sepsis, though both up-and down-regulation are reported 1,2 . we therefore examined changes in glycolytic activity in a longterm sepsis model. an instrumented, fluid-resuscitated, faecal peritonitis rat model was used. this has a 72-hour mortality rate of approx. 50%. septic (n=57) and sham (n=35) rats were sacrificed at various time points (0, 24, 48, 72h) and liver samples harvested and assayed for maximal activity of the rate-limiting glycolytic enzymes, hexokinase (hk), phosphofructokinase (pfk) pyruvate kinase (pk). we demonstrate an initial rise (albeit non-significant) then significant downregulation in two rate-limiting glycolytic enzymes during sepsis. the lack of difference at 72 h may reflect prior demise of the severely ill animals. whether the degree of glycolytic down-regulation is related to subsequent death requires further study. we presume the interesting finding of upregulation seen in the sham animals to be a response to surgery and/or fluid loading. recent studies have shown that low-dose vasopressin infusion or terlipressin bolus (tp, its long acting analogue; o'brien, 2002) restores blood pressure and reduces norepinephrine (ne) requirements in septic shock. however they have no effect upon blood pressure in non-septic patients. exact mechanisms underlying this hyperreactive effect in sepsis patients remain unknown. we chose to investigate this using our established in vivo and in vitro models of endotoxic shock in rats. in vivo -spontaneously breathing anaesthetised male wistar rats was given either saline (sham) or endotoxin (lps) (klebsiella 40mg kg -1 ) over 30 mins and then fluid resuscitated with colloid 25mls kg -1 hr -1 for 210 mins. at 120 mins either a bolus of tp (1.5mg kg -1 ) or a bolus and infusion of ne (0.5mg kg-1 and 1mg kg hr -1 ) was administered. measurement of flow and pressure (mean arterial pressure -map) were made from appropriately sited probes and transducers. in vitro -rings of rat mesenteric artery (rma) were harvested, cleaned and incubated for 20 h with or without 1mg ml -1 lps (s. typhosa). they were then mounted in organ baths for measurement of isometric tension. cumulative concentration-response curves to phenylephrine (pe: 10 -9 to 10 -5 m) or vasopressin (vp: 10 -12 to 10 -6 m) were then constructed. statistical analysis was by anova. results. in vivo -while ne had a significantly greater effect upon map in shams compared with lps rats (p=0.042), tp caused a greater increase in lps animals than shams. a bolus of tp lasted approximately 75 mins. in vitro -lps significantly depressed contractile responses to pe compared to control tissues (max contraction controls -1.35±0.14g, lps -0.48±0.11g, p<0.001, anova). however there was virtually no contractile response to vp even in control tissues after 20 h incubation. the cytokine cascade activated in response to injury consists of a complex biochemical network with diverse effects on the injured host. leukocyte activation after trauma is essential for inflammation. it is a multistep process in which chemokine -interleukin (il)-8 has pivotal role. in two-hit hypothesis, sepsis represent a second insult to a previously injured and primed host, converting a low-grade or regulated host response into an accelerated or dysregulated host response, triggering new or progressive organ dysfunction (1). aim of this study was to assess pro-inflammatory response to trauma with or without sepsis as a second insult. twenty five patients with severe trauma (explosive and sclopetarious) who developed sepsis and 10 patients with same kind of severe trauma without sepsis were enrolled in this study. in the trauma+sepsis group 21 patients developed multiple organ dysfunction syndrome (mods) and 14 died. in trauma group 4 developed mods and 2 died. blood was drown on the first, third and fifth day of trauma. concentrations of il-8, il-12, tumor necrosis factor (tnf)alpha and interferon (ifn)-gamma were determined in plasma using elisa assays. when compared trauma+sepsis group with trauma group we found statistically highly significant difference (p<0.01) in il-8 and ifn-gamma and statistically significant difference (p<0.05) in tnf-alpha concentrations; mean values of il-8 were 230-fold higher, ifn-gamma 360-fold higher and tnf-alpha 17-fold higher in patients with trauma with sepsis. il-12 was not statistically different (p>0.05) between two groups. when compared mods group with group without mods, we found statistically highly significant difference (p<0.01) in il-8 and tnfalpha concentrations; mean values of il-8 were 60-fold higher and tnf-alpha 43.5-fold higher in patients with mods; il-12 and ifn-gamma were not statistically different (p>0.05) between two groups. when compared non-survivors with survivors, we found statistically highly significant difference (p<0.01) in il-8 and tnf-alpha and statistically significant difference (p<0.05) in il-12 concentrations; mean values of il-8 were 2.3-fold higher in non-survivors, mean values of tnfalpha were 2.2-fold higher in survivors, il-12 was also higher in survivors. ifn-gamma was not statistically different (p>0.05) between two groups. there is augmented pro-inflammatory response after trauma with secondary sepsis. high concentrations of il-8 and tnf-alpha indicated higher severity (mods). but, fatal outcome was predicted with high concentrations of il-8 only; survivors had higher concentrations of tnf-alpha and il-12. therefore, pro-inflammatory response was partly beneficial and partly detrimental to the host. in patients with shock hypoxia is considered to be the most important cause of organ failure and death. the goal of treatment therefore is to restore tissue oxygen delivery (tdo2). due to impaired oxygen extraction in distributive (septic shock) the relation between tdo2 and tissue oxygenation is less conclusive. direct measurement of tissue oxygen pressure (pto2) could be of great importance in gaining a better insight in tissue oxygenation in these patients. previously published data concerning pto2 in patients with sepsis/septic shock are contradictory (1,2). furthermore the techniques used were not easily applicable at the bedside. in a prospective observational study we performed bedside pto2 measurements in 8 patients with sepsis/septic shock to gain insight in pto2 values and their dynamic changes related to the course of the illness, as well as investigating the practical applicability of tissue oxygen measurement in the icu setting. pto2 was measured continuously during the course of the illness using polarographic clark-type o2 electrodes (licox catheter measurement system, gms), which were placed subcutaneous in the upper arm. disease progression over time was expressed as the daily calculated sequential organ failure assessment (sofa) score. results. five men and 3 women with septic shock n=6 or sepsis n=2 were included. the median (range) age was 39 years (21-85), median apache-score on the day of admission was 15 (9-29), median duration of pto2 measurement per patient was 5,0 days (3-7). in none of the patients technical problems were encountered during the pto2 measurements. the first day of measurement the median pto2 of the eight patients was 49 (18-92) mmhg. in the six surviving patients the sofa score decreased over time and this was associated with a concomitant decrease in pto2 to a median of 30 (16-69) mm hg. in the 2 nonsurvivors an increasing sofa score was associated with an increase in the mean pto2 to 57 mmhg on the day of death. in seven patients linear regression analysis showed a positive correlation between the daily sofa scores and the daily mean pto2: r= 0.71, 0.75, 0.88, 0.93, 0.85, 0.87, 0.61. in one patient no correlation was found. conclusion. bedside pto2 measurements in the icu using the licox measurement system are easily performed. pto2 in septic patients is variable but changes with the clinical course reflected by the sofa score: clinical improvement was associated with a decrease in pto2 while deterioration was associated with an increase of pto2. these findings suggest that in patients with septic shock decreased oxygen utilisation may play a more important role than tissue hypoxia as such. to precise the diagnostic value of macrophage migration inhibitory factor (mif) as a marker of severity in patients with sepsis and to determine relations between mif and interleukin 6 (il-6), we conduced a prospective, observational, cohort study, in two general intensive care units. we analyzed 20 patients with septic shock, 17 patients with sepsis, and 10 healthy volunteers. the median mif serum level was significantly higher in septic shock patients (2.01 ng/ml, range 0.77-12.0) than in sepsis patients (1.10 ng/ml, range 0.31-5.11) or in healthy volunteers (0.45 ng/ml, range 0.28-1.54). there was a direct correlation between mif and il-6 concentrations (r=0.693, p<0.01). the area under the curve (auc) of the receiver-operating characteristic (roc) for prediction of septic shock was 0.81 (p<0.01) for mif and 0.85 (p<0.01) for il-6. the auc under the roc curve for prediction mortality was 0.714 (p<0.05) for mif and 0.707 (p<0.05) for il-6. in this trial we found significant elevated serum levels of mif in patients with septic shock and sepsis. moreover, mif levels were discriminative for septic shock and mortality, and had a direct correlation with levels of il-6 with a similar diagnostic accuracy. in conclusion, mif appear to be a promissory marker of severity in sepsis. high density lipoprotein (hdl) modulates the inflammatory response to injury and infection via several pathways. hdl also directly binds and neutralises lps. administration of reconstituted hdl reduces cytokine release and attenuates shock in experimental endotoxaemia (1). the hdl associated enzymes paraoxonase (pon) and lecithin cholesterol acyl transferase (lcat), destroy oxidised lipids that induce inflammatory changes in vascular endothelium (2). incorporation of serum amyloid a (saa), an acute phase protein, into the hdl particle during the inflammatory response, may displace these protective enzymes producing a particle with proinflammatory properties (3). alterations in hdl composition may, therefore, be implicated in dysregulation of the inflammatory response and could influence outcome from septic shock. methods. patients with septic shock, not given tpn or propofol, were recruited. apache ii scores and icu mortality were recorded. plasma and serum samples were taken within 48 hours of the onset of shock. hdl cholesterol was measured by microenzymatic colorimetric assay. apolipoprotein ai (apo ai) was quantified by liquid phase radioimmunoassay. pon activity was determined by measuring the rate of paraoxon hydrolysis and described as percent of a control serum pool. lcat activity was quantified by measuring the esterification rate of 14 c labelled cholesterol. saa was measured by elisa. results were compared with those of a pool of healthy volunteers and between survivors and nonsurvivors. (mann whitney u test). results. 20 patients were recruited. there were 13 survivors (s) and 7 nonsurvivors (ns). pon activity was significantly higher in s than ns: 47.6 (11.5-135.6) vs. 22.6 (5.7-39.43), p<0.02. saa concentration was significantly higher in s than ns: 1915 (90.9-14300) severe trauma and sepsis are the major sources of morbidity and mortality despite the rapid development of intensive therapy. studies have indicated that there are marked alterations in immune response in patients exposed to major trauma or prolonged surgical procedures, including altered pro-and anti-inflammatory mediator/cytokine release (1). traumatic injury results in profound immunosuppression which predisposes the patients to sepsis and/or multiple organ dysfunction syndrome (mods). aim of this study was to assess the prognostic value of anti-inflammatory cytokines: interleukin (il)-1 receptor antagonist (il-1ra) , il-4, il-10 and transforming growth factor (tgf)-beta 1 regarding severity and outcome in patients with trauma and sepsis, trauma only and sepsis only. twenty five patients with severe trauma (explosive and sclopetarious) who developed sepsis, 10 patients with same kind of severe trauma without sepsis and 5 patients with severe sepsis were enrolled in this study. twenty nine patients developed mods (of all 40 patients), 19 died. blood was drown on the first, third and fifth day of trauma or sepsis. concentrations of il-1ra, il-4, il-10 and tgf-beta 1 were determined in plasma using elisa assays. when compared mods group (regardless of initiating insult -trauma or sepsis) with group without mods, we found statistically highly significant difference (p<0.01) in il-1ra and il-10 concentrations; mean values of il-1ra were 6-fold higher and il-10 70-fold higher in patients with mods; il-4 and tgf-beta 1 were not statistically different (p>0.05) between two groups. when compared non-survivors with survivors, we found statistically highly significant difference (p<0.01) in il-1ra and il-10 concentrations; mean values of il-1ra were 2.7-fold higher and il-10 1.4-fold higher in non-survivors; il-4 and tgf-beta 1 were not statistically different (p>0.05) between two groups. when compared trauma+sepsis group with trauma group, we found statistically highly significant difference (p<0.01) in il-1ra and il-10 concentrations, they were higher in trauma+sepsis group (il-1ra 3.7-fold, il-10 42-fold). il-4 and tgf-beta 1 were not statistically different (p>0.05) between two groups. when compared trauma+sepsis group with sepsis group and trauma group with sepsis group, we found no statistically significant difference in either one of 4 anti-inflammatory cytokines. our study shows that il-1ra and il-10 are excellent predictors of severity and outcome of critical illness; higher concentrations were found in group with more severe clinical status (mods) and in non-survivors. il-4 and tgf-beta 1 had no significance as predictors of severity and outcome what so ever. fifty-eight patients admitted to two medical intensive care units for reasons other than acute coronary syndrome were consecutively included and analyzed according to their troponin status. thirty-day mortality, left ventricular ejection fraction, the presence or absence of underlying coronary artery disease, and a panel of inflammatory cytokines were compared between troponin-positive and troponin-negative patients. thirty-two of 58 critically ill patients (55%) without evidence for an acute coronary syndrome were troponin-positive. positive troponin levels were associated with higher mortality (22.4% vs. 5.2%, p < 0.018) and lower left ventricular ejection fraction (p = 0.0006). troponinpositive patients had significantly higher median levels of tumor necrosis factor a, its soluble receptor and interleukin-6. a subgroup of ten aplastic patients was troponin-negative at study entrance. three became troponin-positive during leukocyte recovery and subsequently died, whereas all the others stayed troponin-negative and survived. conclusion. elevated troponin is a mortality risk factor for medical intensive care patients admitted for reasons other than acute coronary syndromes. it is associated with decreased left ventricular function, and this may be mediated by tumor necrosis factor a and mediators produced by neutrophilic granulocytes. it is very interesting to notice the high correlation among protein c and atiii activity levels and sofa scores (p< .01) and the dramatic decrease of the protein c system is already firmly present 48 hours before negative outcome (not survivors). we also register the significant alterations of c1 inhibitor specially in the group (ns) patients with severe candidemia and this marker assumes a significant role with an interesting clinical future . tumour necrosis factor-a (tnf) is an important pro-inflammatory mediator and high levels of this cytokine have been associated with a poor outcome from sepsis. recently, genetic polymorphisms of the tnf locus and its promoter region have been associated with the incidence and outcome of severe sepsis 1;2 , although the results have been conflicting 3 . we chose to investigate the association between a known functional single nucleotide polymorphism (snp) in the tnf gene promoter (-308 g/a, guanine to adenine substitution) and outcome in severe sepsis and septic shock. caucasian adult patients with a diagnosis of severe sepsis or septic shock on 6 icus in the uk and from an icu in sydney, australia were recruited. whole blood was collected in edta, dna extracted and amplified by pcr using specific primers and digested with the restriction endonuclease nco1. this enzyme cuts the wild type (allele g) but this cutting site is abolished by the polymorphism (allele a). the restriction fragments were then size separated, visualised and scored on agarose gels. fisher's exact test was used for statistical analysis. shedding of membrane bound tumour necrosis factor receptors to produce soluble molecules (stnfrsf1a and 1b) is an important inflammatory control mechanism 1 . we and others have previously demonstrated that increased levels of stnfrsf1a and stnfrsf1b are associated with decreased survival from sepsis. furthermore, there appears to be an association between polymorphisms of the tnfrsf1b locus and plasma levels of stnfrsf1b 2 . we have therefore investigated whether polymorphisms of the tnfrsf1b gene and its promoter region might influence outcome from severe sepsis and septic shock. 170 caucasian adult patients with a diagnosis of severe sepsis or septic shock from 6 icus in the uk and from an icu in sydney, australia were recruited. we analysed 3 polymorphisms of the tnfrsf1b gene. a single nucleotide polymorphism in exon 6 (snp 196 t/g) was studied by pcr-rflp, a microsatellite in intron 4 (ms4) using an abi373a sequencer and a 15 base pair insertion/deletion in the promoter region (indel) by polyacrylamide gel electrophoresis. analyses of associations between genotype and allele frequencies and outcome were by fisher's exact test. . icu mortality was 28%. overall genotype and allele frequencies for each of the polymorphisms were similar to published population frequencies. there were no statistically significant differences in allele frequencies in any of the three polymorphisms between survivors and non-survivors (snp196 p=0.19, ms4 p=0.52, indel, p=0.42). the mortality was lower in patients homozygous for the 15 base pair repeat in the microsatellite polymorphism in intron 4 (the genotype associated with high levels of stnfrsf1b) (mortality 20% v 32.1%) and was higher in those with the snp196 (t/g or g/g) (mortality 34.8% v 23.2%). these differences, however, did not reach conventional levels of significance (p=0.14 and 0.12 respectively). larger studies will be required to confirm or refute associations between tnfrsf1b gene polymorphisms, particularly ms4 15 homozygosity, and outcome from sepsis. when associated with end organ dysfunction, sirs is a major cause of morbidity and mortality in the intensive care unit (icu) population (1). lps concentrations in the gastro-intestinal tracts of these patients are elevated as a consequence of bacterial overgrowth. lps processing in the mesenteric circulation may influence the systemic inflammatory response (2). tlr4 is an integral part of the lps receptor complex. a tlr4 polymorphism (asp299gly) is associated with hypo-responsiveness to lps in human bronchial epithelial cells. we examined the association of this polymorphism with clinical outcome in icu patients with severe sirs. methods. adult icu patients with evidence of severe sirs were studied. patient demographics, apache ii data, length of stay and outcome data were collected. genotype was determined using pcr amplification. statistical analysis was performed using spss 11.0. results. 67 patients have been genotyped of whom 2 are still in icu. of the remaining patients, 20/65 (31%) died in icu and 9 died in hospital after discharge from icu, giving an overall hospital mortality rate of 29/65 (45%). mean (sd) apache ii score was 21 (5).the tlr4 genotype frequencies were asp/asp 88.1% (59/67), asp/gly 10.4% (7/67) and gly/gly 1.5% (1/67). the allele frequencies were asp 93% and gly 7%, similar to previously reported frequencies in caucasians. preliminary analysis revealed no significant differences between apache ii scores in patients with the asp/asp genotype (mean 20.7, sd 5.6) and those with asp/gly or gly/gly genotypes (mean 19.0, sd 2.8) (p=0.40, student's t-test). 5/29 (17%) of patients who died during the hospital episode carried the gly polymorphism, compared to 3/36 (5%) of those who survived the hospital episode (p=0.45, fisher's exact test, or 2.3, 95% ci 0.5-10.5). conclusion. no associations with severity of illness on admission to icu, icu length of stay or hospital outcome were detected with the present sample size. recruitment is ongoing, to attain sufficient power. we aim to study genes coding for components of the lps receptor complex, which are biologically relevant to innate immunity and the development of sirs. detailed, prospective study of the role of polymorphisms in innate immunity has the potential to improve our understanding of the pathogenesis of sirs, and to influence risk stratification and management of this severe complication of life-threatening infection. the present study was designed to evaluate the effect of low dose albumin infusion vs. control on the local inflammatory response following abdominal surgery. albumin loss during surgery is a well described phenomenon. in previous experiments a loss of plasma proteins, resp. albumin was observed during abdominal surgery. intravital microscopy for five hours was used to evaluate the effect of low dose albumin on the mesenteric microcirculation. urethan-anesthetized sprague-dawley rats underwent median laparatomy and placement of a doppler flow probe around the abdominal aorta. an ileal loop was prepared for eventration onto a microscopic stage using a plastic foil technique and the mesentery was immersed with krebs-henseleit buffer(5%co2 in n2). low dose albumin (0.001 g/(kg bw*h)) was given vs. control (nacl 0.9%) during the experiment. heart rate, map, aortic blood flow were registered on a beat-to-beat basis. abg's were drawn hourly for analysis of metabolic(be), respiratory (po2, pco2) and hct values. rolling and adherent leukocytes significantly increased in the control group until the end of the experiment, whereas they constantly remained on a low level in the albumin group. velocity and shear rate in the mesenteric microcirculation were significantly higher in the albumin group which was supported by increased abdominal flow and stroke volume vs. control. low dose albumin infusion significantly reduces the inflammatory response on the mesenteric microcirculation following abdominal surgery. beneficial effects on systemic hemodynamics, mesenteric microcirculation and attenuation of leukocyte rolling and adhesion in mesenteric venules could only be observed in the albumin group, whereas the inflammation progressed in the control group. iasonidou c. 1 , pertsas e. 1 , koletsos k. 1 , kapravelos n. 1 , tsagalof s. 1 , riggos d. 1 1 icu, g.papanikolaou, thessaloniki, greece optimizing patient's hemodynamics in the icu can be challenging.the pa catheter has been used to determine preload, afterload and myocardial performance. however,insertion of a catheter is not a risk-free procedure and the values obtained can in some circumstances be misleading.the use of tee in icu has been increasing.previous studies have examined the correlation between the pulmonary vein (pv) velocities and mitral valve (mv) velocities and pcwp. the purpose of our study was to evaluate the relationship between these variables during different loading conditions as assessed by tee in icu patients. (11) patients,with a mean age of 65± 10 years, requiring mechanical ventilation were prospectively studied. in all patients a pa catheter was inserted and baseline measurements were obtained.the pv velocities and mv velocities were evaluated during three different loading conditions:1) in a control situation 2) in a state of decreased preload by intravenous administration of nitroglycerin 3)in a state of increased preload by administration of fluids.in all patients we used the following indices from the pv velocity : s (systolic),d (diastolic), decelaration time(dt) of d wave, apv (atrial reversal) and from mv velocity: e,a wave and deceleration time of e wave. the decrease in preload resulted in a trend toward a lower amplidute of d wave peak velocity as compared with the control state and a significant prolongation of the deceleration time (p<0,05).there was a decrease in height of the systolic (s) wave (p<0,001)and the apv (p<0,05). the mv curve demonstrated a significant decrease in e velocity (p<0,05) and prolongation of deceleration time (p<0,05).the increase in preload resulted in a significant increase in systolic and diastolic wave in pv (p<0.01) with a shortening of the dt of d wave.the apv became significantly higher (p<0,05).the mv curves demonstrated a significant increase in e wave (p<0,05) with a decrease in dt.there was a good correlation between d wave and pcwp (r:0,6),apv wave and pcwp (r:0,7) and e wave and pcwp (r:0,6).a direct correlation was present between changes in e and d waves (r:0,68) and changes in dt of e and dt of d velocities (r:0.78). this study provides evidence that tee gives information additive to the pa catheter in the assessment of preload in an icu population. examination of pv velocities and mv velocities and their changes during different loading conditions provide additional information regarding diastolic function. this may prove useful in minimizing the use of invasive methods for hemodynamic monitoring in icu patients. further investigation is required to correlate these doppler measures with the invasive hemodynamic measurements. methods. 26 patients with spe (19 women and 7 men), with a mean age of 59 years (sd of 17; range: 21 to 83 years), were studied prospectively. coloured doppler-echocardiography was performed in all cases at admission, confirming that diagnosis by perfusion gammagraphy and / or helycoidal ct scan. emboli were observed in six patients (23%): 3 in right atrial chamber, 2 in pulmonary artery and 1 in output tract of right ventricle. in 17 patients (65%) right ventricular dilatation with a mean value of 38.8 mm (sd 5), and tricuspid insufficiency in 20 (74%) with mean estimated systolic pulmonary arterial pressure of 60 mmhg (sd 16). pulmonary acceleration time was measured in 13 patients and found shortened in all of them: 52 milliseconds (sd 16), and septal abnormal movement was detected in 10 patients (39%). 25 out of 26 patients had more than one sign of severe pulmonary embolism (spe), 10 had two sign and the other 15 had three or more signs. echocardiography is a simple technique, which allows the diagnosis of spe by the detection of emboli in the right heart cavity and / or the objectivation of indirect signs of functional alteration of right ventricle. coagulopathy and systemic inflammatory response have been previously reported in patients after cpr (1). the coagulopathy includes activation of coagulation and inhibition of fibrinolysis, alterations similar to those reported in sepsis where profound depletion of anticoagulation proteins have been evidenced, and had significant therapeutic consequences (2). however, anticoagulation proteins: protein c and s (pc -ps), as well as antithrombin (at) levels were not reported after cpr. consequently, serial measurements of markers of coagulation (thrombin-at [tat], d-dimers), fibrinolysis (plasminogen-activator inhibitor 1: pai-1), inflammation (il-6) and endothelial injury (soluble thrombomodulin: stm) were performed in 50 patients (age: 61±13 years; saps: 64±13) after successful cpr.analyses on biomarker levels by anova were performed. the aim was to evaluate the effect of thrombolytic therapy in massive and submassive pulmonary embolism methods. 22 patients (16 women and 6 men), with a mean age of 59 years (sd 18), range: 21 to 83, studied prospectively. diagnosis at admission was confirmed with spiral ct scan and/or ventilation-perfusion (v/p) gammagraphy. a study protocol was performed in all patients consisting of: complete analysis, electrocardiography, thorax radiography and echocardiography.one hundred milligrams of rt-pa was infused in 2 hours due to severity of the clinical presentation: haemodynamic instability (4 cases) and/or severe hypoxemia or echocardiographic abnormalities (8 patients). clinical improvement was seen in the entire group. studied variables pre and postthrombolysis are shown in the table. mean arterial pressure (map); right ventricular diameter (rvd), systolic pulmonary arterial pressure (spap), acceleration pulmonary time (apt), oxygen saturation (os), fibrinogen, hematocrit and heart rate (hr). postthrombolytic changes in electrocardiogram were objectivated, showing that some abnormalities had disappeared, such as: right bundle heart block in 8 out of 19 patients (42%), s1q3t3 pattern in 5 out of 20 (25%), t wave alterations in 6 out of 21 (29%), and the pulmonary p in 2 out of 4 (50%). minor hemorrhagic complications were observed in 7 cases; only one needed transfusion. one patient had hematuria, one other hemarthrosis, and another one suffered pericardial blood effusion (after coronary by-pass graft). we have previously shown that the measurement of gut intraluminal redox potential (eh) during progressive bleeding and reperfusion is useful to monitor changes in oxygen transport 1 . eh could provide with a different type of information from other parameters of tissue oxygenation, such as lactate and intramucosal ph. our goal was to show the rate of decrease of eh after the occlusion of superior mesenteric artery blood flow (qintestinal). eight anesthetized and mechanically ventilated sheep were studied. eh was measured as a voltage difference using a milivoltmeter with a platinum electrode, against a reference electrode. qintestinal was measured with an electromagnetic flowmeter. after basal measurements, superior mesenteric artery was occluded and eh was continuously registered during 30 minutes. data (mean ± sd) were analyzed with repeated measures of anova followed by dunnett's test. response time was defined as a decline greater than three sd from basal values. assessment of heart rate variability (hrv) has been used in risk stratification after acute myocardial infarction, in congestive heart failure, and in the early diagnosis of diabetic neuropathy. patients with end-stage renal disease (esrd) constitute a population of increased cardiovascular morbidity and mortality. hemodialysis patients often show signs of autonomic neuropathy. data on hrv are usually derived from 24-hour holter recordings. however, short term rr interval variation as measured on standard 12-lead ecg holds important prognostic implications in subjects with dilated cardiomyopathy. the purpose of this study was to look at short term rr variation in esrd patients, and its modification after dialysis. methods. 47 (32 male, 15 female) patients were included in the study.all of them were in three times a week hospital hemodialysis. twenty control subjects, of similar age and gender distribution, with normal renal function and blood pressure, were recruited among ward stuff. the rr intervals were measured from a continuous 2-min recording of lead ii. rr variation was calculated as the standard deviation of the rr intervals (rrsd), and the coefficient of variance of the rr (rrcv), i.e. standard deviation divided by the mean rr and expressed as percentage. ecgs were also analysed for left ventricular hypertrophy (lvh). . rrsd and rrcv were significantly decreased in dialysis patients compared to controls. rrsd was 11.28±8.13 vs 39.832.12 p=0.000, and rrcv was 1.56±1.13 vs 4.63±2.75, p=0.000. rrsd and rrcv were not affected by dialysis, but were significantly decreased in those with ecg evidence of lvh, compared to those without. rrsd was 6.87±6.1 vs 10.9±8.8 (p=0.043), and rrcv was 0.95±0.76 vs 1.36±1.26, (p=0.033). rrcv was associated with mg and k postdialysis. rrsd and rrcv were inversely correlated with cornell voltage, an ecg index of lvh. hemodialysis patients present with low short term rr variation in comparison with control subjects. electrocardiographically detected lvh among esrd patients is also associated with depressed rr variability. increased intracranial pressure,as that was seen in patients with large cerebral tumors, reduces frequency of the pulse.prolapsus of the brain masses in tentorial incissura of foramen magnum and consequently bradycardia,respiratory arrest,coma and death might occur in these patients. the aim of this study is to examine the ecg changes in patients with brain tumors in regard to the kind of tumors and localisation . the study group was consisted of 31 patients (15 male and 16 female) of average age 57,9 years (range 30 to 83). there were 10 patients with temporal lobe tumor (4 left and 6 right), 9 patients with frontal lobe tumor (5 left and 4 right), 9 with parietal lobe tumor (3 left and 6 right) and 3 with occipital lobe tumor (2 left and 1 right). ecg changes were evaluated during the first 72 hours from receiving in the icu. large cerebral tumors confirmed with ct, and definitive diagnosis was made pathohystologicaly. the most common ecg abnormalities associated with central lesions that we found were: prolongation of the q-t interval in 64.7% patients with right and 50% with left cerebral hemisphere tumor; elevated, peaked, or notched t waves in 52.9% patients with right and 42.8% with left cerebral hemisphere tumor; and increased p-wave amplitude in 23.5% with right and 28.5% patients with left cerebral hemisphere tumor. the most frequent ecg changes that we registered among rhythm and conduction disturbances were: narrow-qrs tachycardia with regular rhythm; sinus tachycardia in 35% with right and 28% patients with left cerebral hemisphere tumor, sinus bradycardia in 11.7% with right and 14% with left cerebral hemisphere tumor, and incomplete/complete right bundle branch block (bbb) in 28% patients with left cerebral hemisphere tumor. we did not find any specific differences according to the pathohystologicaly type of the tumors. conclusion. ecg abnormalities associated with central lesions in the patients with brain tumors are not depend from the kind of tumors and side of the brain where tumor is located. in the patients with brain tumors on left side of the brain prevails incomplete and complete right bundle branch block (bbb). prolonged mechanical ventilation support (mv) is associated with increased morbidity and less cost -effective admissions in the postoperative period (po) of heart surgery (hs). study conducted to identify variables associated with prolonged mv in patients that underwent hs. methods. cohort study; 293 consecutive patients enrolled from 1/12 to 11/17 of 2001. inclusion criteria: patients submitted to hs and admitted to intensive care unit (icu) in use of mv. exclusion criteria: non-cardiac surgery, admission to icu in spontaneous ventilation or death during the first 12 hours of the po. variables that could be associated with prolonged mv were pre-selected for analysis and grouped according to the period it represented. preoperative period: in-hospital stay duration, age, body mass index, gender, severity of left ventricular dysfunction (lvd), pulmonary hypertension, chronic obstructive pulmonary disease, redo, urgency for the procedure. peroperative period: surgery, extracorporeal circulation (ecc) and aortic clamping duration, fluid and blood input/output differences, type of surgical procedure, combined procedures, need for post-ecc intraaortic balloon counterpulsation (iab). admission to the icu: oxygen alveolar/arterial difference, blood oxygen partial pressure/oxygen inspired fraction ratio (p/f). first 24 hours of po: dobutamine or norepinephrine (nor) use, blood drainage volume, lowest blood lactate measurement and prognostic scores sofa, tiss and mods. for dichotomyc variables, mann-whitney test was applied; for continuous variables we used kendall's tau non-parametric correlation test; cuzick tendency test was used to evaluate association with lvd. results. median mv duration: 8 hours; mean duration time 37.8 hours (1 to 742). increased mv duration was associated with emergency surgery (p=0.0117), coronary artery bypass graft surgery (p=0.0307), need of iab counterpulsation after ecc (p=0.0005), use of nor (p<0.0001). increases in mv duration were associated with increasing values of some variables (positive correlation): age (p<0.0001), surgery duration (p=0.0058), blood input/output difference (p<0.0001) and sofa, tiss and mods scores (p<0.0001). negative correlation was presented for fluids input/output difference (p=0.0142) and p/f (p<0.0001). increased linear tendency for mv duration correlates with worsening of lvd (p=0.012). conclusion. more sophisticated statistical analysis should be applied in order to determine the cause-effect correlation for these variables. interventional studies will be conducted in the following months. cerebral vasospasm is a, potentially, life threatening phenomenon after aneurysmal subarachnoid hemorrhage (sah). as part of "triple h" therapy, fenylephrine and norepinephrine in combination with dobutamine and dopamine, are used most frequently to elevate systemic arterial blood pressure (abp) in order to preserve optimal cerebral blood flow. in obtaining increased arterial blood pressure during episodes of vasospasm we altered medical therapy from fenylephrine to norepinephrine but experienced an increasing incidence of paralytic ileus. in a retrospective cohort study we evaluated clinical outcome and the incidence of paralytic ileus. methods. in 1991-1999, a consecutive series of 146 patients had surgery (aneurysmal clip ligation) within 72 hours after sah. 80 patients with clinical vasospasm, were subdivided into two groups with respect to medication used. group a (n=48) was treated with a combination of dobutamine, dopamine and fenylephrine (mean increase syst. abp 40.0±20.5 mm hg). in group b (n=32) norepinephrine was used instead of fenylephrine (mean increase in syst. abp 57.8 ± 27.3 mm hg). we compared basic variables of the two treatment groups, and investigated the clinical outcome using the glasgow outcome scale (gos), one year after initial sah. complications were registered and compared between these treatment groups. results. the two treatment groups were evenly matched concerning age (p=0.28), wfnsscore at admission (p=0.89), amount of subarachnoid blood on ct-scan (fisher) (p=0.75), and observed prognostic variables as hypertension (p=0.50) and smoking (p=0.65). the clinical outcome, was not influenced by the kind of medication used (p=0.55). the incidence of paralytic ileus differed between the two groups (group a: 2/48 vs group b: 12/32, p<0,001). paralytic ileus occurred mainly in patients treated with norepinephrine (12/32=37.5%, odds ratio=13.8). no relationship was found in height of systolic abp or dosage of norepinephrine administered to these patients. we observed a significant difference in duration of administration of norepinephrine in patients, who did develop a paralytic ileus (see -12.14 / -3.42 p=0.002 table: norepinephrine use in cerebral vasospasm; patients with or without paralyticileus. conclusion. -the use of norepinephrine in patients with cerebral vasospasm after sah did not influence clinical outcome, although higher blood pressure levels were reached. -norepinephrine administered during longer periods than 12 days, increases the risk of paralytic ileus.-fenylephrine is recommended in the treatment of cerebral vasospasm after sah. amigues l. 1 , klouche k. 1 , massanet p. 1 , canaud b. 1 , béraud j. j. 1 1 intensive care unit, lapeyronie university hospital, montpellier, france introduction. slow discontinuous ultrafiltration (sduf) is nown recognized as an effective complementary treatment for congestive refractory end stage cardiac failure(escf). however, an organic kidney disease is often associated with heart failure and may worsen the prognosis. this study was undertaken to compare the effects of sduf in escf patients with and without previous kidney disease. methods. 39 patients fullfilling escf criteria with fluid overload and oliguria (grade iv nyha) were treated by sduf. sduf was performed with a double head pump monitor (bsm 22, hospal), blood flow rate: 150-200 ml/mn, ultrafltration rate: 0.2 to 1 l/h. vascular access was provided by femoral silicone twin catheters. a renal replacement therapy was institued when indicated. age, sex, cardiopathy, and nephropathy were collected in each patient. patient follow up before and after sduf: systolic arterial pressure, heart rate, diuresis, total fluid volume removed, cardiothoracic index, creatinemia, blood urea nitrogen, natremia, natriuresis, mortality and average survival. datas were compared between two groups: group 1without and group 2 with nephropathy. mean age was 65±9 yo and sex ratio was 1/4,3. myocardiopathy origin was: ischemia (11), hypertension (6), valvulopathy (4), primary non-obstructive cardiopathy (7), multifactorial (11). oliguric renal failure: fonctional in 20 patients (group 1) and associated with mild chronic nephropathy in 19 patients prior to heart attack (group 2). no significant differences in clinical and biological datas were observed between the two groups except for blood urea nitrogen: 26,2±15 in group 1 vs 39,2±16,8 mmol/l in group 2. during scud, hemodynamic stability was observed in both groups; diuresis and natriuresis significantly increased and remained stable at the end of the treatment despite significant decreased diuretic doses. mean sessions of sduf was 3,8±2,4 in group 1and 5,6±5,6 in group 2 (ns). renal replacement therapy was institued in both groups but the number of sessions was significantly higher in group 2: 3,8±4,7 vs 0,95±1,3. mortality during hospitalisation was 25% in group 1 and 21%in group 2. from the surviving patients, 4/15 patients in group 1 and 6/15 patients in group 2 underwent a chronic hemodialysis treatment. average survival was higher but not significant in group 2 (21,3±36 vs 7,7±9 months). our study sugests that sduf remains a long term beneficent treatment for patients with both escf and renal failure. paradoxically, prognosis is slightly better than in patients with isolated refractory congestive heart failure. organic renal failure could artificially worsens cardiac function by increasing diuretic resistance which may be improved by sduf. (1). this study aims to evaluate the influence of factors affecting renal blood flow including map, cvp, pulmonary artery wedge pressure (pawp), cardiac index (ci), systemic vascular resistance (svr) and pulmonary vascular resistance (pvr) on immediate graft function. methods. 15 consecutive patients undergoing live-related kidney transplant were included. prior to anaesthesia, a 7.5f continuous cardiac output pulmonary artery catheter was placed via the right internal jugular vein. a continuous cardiac output monitor (baxter edwards px 1800) was used for haemodynamic monitoring. baseline values of the map, cvp, pawp, ci, svr and pvr were computed. data was collected at 30-minute intervals thereafter and immediately before and after release of the vascular clamps. the ischemia time, intravenous fluids, dopamine use and blood transfusion were noted. if warranted by low preoperative haemoglobin or increased surgical blood loss, blood was administered as 200 ml packed cell units using leukocyte filters. the outcomes chosen for graft function were the urine output on table (uo-ot), 24-hour urine output (uo-24), fall of serum creatinine from the preoperative value on day 1 (creat-1) and day 7 (creat-7). using spss statistical software, multiple linear regression analysis was done to find the variables significantly affecting the outcome. results. the only variable found to have a statistically significant influence on uo-ot was the map. no variable had any effect on the uo-24. blood transfusion had a negative influence on the fall of creatinine on day 1 and day 7 ( since clinically adopted during cardiac surgery, cardiopulmonary bypass (cpb) has been implicated in complement activation and postoperative acute phase reaction. corticosteroids are usually employed as an attempt to dampen these phenomena and related postoperative morbidity. methods. after informed consent previously approved by the local ethical committee, we included 62 adult patients submitted to cardiac surgery under cpb at a non-emergency setting. preoperative risk stratification employed euroscore (es) and cleveland clinic score (ccs). methylprednisolone (mp) -15mg/kg, was added to cpb priming solution for group 1 (n=31) but not for group 2 (n=31). blood samples were collected from all patients at anaesthesia induction (t0), 3(t3), 6(t6) and 24-hour postoperative (t24) for measurement of total c3 and c-reactive protein (crp) levels, by nephelometric technique. postoperative multiple organ score (mods) were daily registered. results. the groups were considered comparable concerning to preoperative risk stratification, length of cpb and postoperative organ dysfunction at 72h postoperative (mod72, as well. starting from similar levels of c3 and crp, we did not observe significant differences between groups 1 and 2 concerning to postoperative levels of c3. nevertheless, patients treated with mp (group 1) exhibited higher crp levels at 24h postoperative, as shown bellow: 11.24 ± 6.34 6.57 ± 6.20 0.001 perioperative administration of mp failed to show evidences of beneficial effect over postoperative organ failure and complement activation. the acute phase response, expressed as crp systemic levels, instead of softened, was significantly enhanced among patients to whose cpb priming solutions was added mp. these results support a larger randomised trial to reassess routine use of corticosteroids during cpb. objective: to evaluate the influence of enteral application of an immunoglobulin enriched bovine milk preparation on endotoxin plasma levels, endotoxin neutralizing capacity of plasma (enc) and the acute phase response (il-6, crp) during and after cardiac surgery in a pilot study. design, patients and methods. 60 patients who were treated by coronary bypass operation were enrolled in a controlled randomized study. the patients were treated by enteral application of 40g of a bovine colostrum milk preparation per day for 2 days preoperatively. endotoxin and enc were sequentially determined intra-and postoperatively by a chromogenic modification of the limulus amebocyte lysate test. interleukin-6, crp, transferrin, alpha-2macroglobulin, albumin, apo-a, apo-b, igg, iga, igm were determined by "elisa" and nephelometrically. the clinical course was followed up by daily evaluation of the apache-ii-score. main results: demographic data were comparable in both groups. no differences of the apache-ii-score (6.5 1.9 verum group, and 6.8 1,8 control group, on admission) were observed. endotoxin plasma levels and enc showed high levels at the end of the procedure and seemed to have a trigger function for the acute phase response but were not significantly reduced throughout the observation period in patients receiving the milk preparation as calculated by comparing the area under the curve. plasma levels of endotoxin binding proteins did not differ significantly. plasma levels of il-6 increased to maximal median values of 655 pg/ml in the verum and 786 pg/ml in the control group 2 and 6 h after surgery. a tendency to lowered il-6 levels was observed throughout the whole observation period for the verum group. crp-levels showed their maximum values 48h after the procedure and were significantly reduced in patients of the verum group (p = 0.034). conclusion. this study revealed that endotoxemia occurs early during an elective surgical intervention, which is followed by a subsequent increase in mediators of the acute phase reaction. the prophylactic enteral application of a bovine milk preparation for two days in cardiac patients did reduce postoperative crp-plasma levels but contrary to a former prospective double blind study in abdominal surgery did not reduce perioperative endotoxemia. one reason could be the too low application of the bovine colostrum milk preparation. to compare a possible effect of improved therapeutical approaches in head trauma, epidemiological data should be compared at certain time points. due to the legal obligation to document all in-patient treatments in germany and to forward these data in an anonymous form to the office for statistical affairs (statistisches bundesamt) it is possible to provide a nationwide epidemiological analysis of head trauma and to compare the yearly obtained data. the incidence, the mortality, and the duration of hospital stay for the treatment of all hospitalized patients suffering from head trauma were calculated and compared to the data from 1996 while considering the data obtained from the office for statistical affairs in bonn and wiesbaden. the data were investigated while separating them according to the international classification of diseases 9 (icd-9; no. 800-804 and 850-854). to further elucidate the causes of altered mortality and duration of hospital stay the number of cts and mris in german hospitals in 1996 and 1999 were compared. in addition, data indicating the number of patients admitted to neurological rehabilitation centers were analyzed. the incidence of head trauma did not change between 1996 and 1999 and was calculated to be at 340/100.000. the mortality, however, decreased from 11.6/100.000 in 1996 to 9.4/100.000 in 1999 (9412 vs. 7705 patients). in addition, the duration of hospital stay declined in all icd-9 encoded subgroups including mild brain trauma. this could be due to the increased number of ct devices and mris in german hospitals (ct: 732 vs 904/ mri: 227 vs 350) while comparing 1996 and 1999. the number of patients transferred from hospitals to neurological rehabilitation centers increased from 35800 in 1996 to 61088 in 1999 (+ 71%). it could be speculated that both improved knowledge on the field of brain trauma therapy and a higher number of technical devices allowing rapid diagnosis of brain injury or potential intracranial complications following head trauma accounted for the reduction in mortality due to brain trauma in germany from 1996 through 1999. the decline of the duration of hospital stay especially in patients with more severe head injury could also be due to a more rapid transfer of patients with head trauma from hospitals to rehabilitation centers. therapeutic hypothermia may improve outcome in patients with severe head injury, but clinical studies have produced conflicting results. we hypothesised that severe side effects of artificial cooling might have masked positive effects in earlier studies, and treated a large group of patients with severe head injury with hypothermia, using a strict protocol to prevent the occurrence of cooling-induced side effects. methods. 136 consecutive patients admitted to our hospital with severe head injury (glasgow coma scale (gcs) <8) in whom icp remained above 20 mmhg in spite of therapy according to a step-up protocol described previously [1] were included in our study. those who responded to the last step of our protocol (barbiturate coma) constituted the control group (n=72). those who did not respond to barbiturate coma (n=64) were treated with moderate hypothermia (32oc-34oc). average apache ii scores were higher, and average gcs at admission slightly lower in the hypothermia group, indicating greater severity of illness and more severe neurologic injury. predicted mortality was 86% for the hypothermia group vs. 80% in controls. actual mortality rates were significantly lower: 62% vs. 72%, p<0.02. the difference in overall mortality between hypothermic patients and controls was statistically significant (p<0.05). the number of patients with good neurologic outcome was also higher in the hypothermia group: 15.7% vs. 9.7% for hypothermic patients vs. controls, respectively (p<0.02). these differences were explained almost entirely by the subgroup of patients with gcs of 5 or 6 at admission (mortality 52% vs. 76%, p<0.01; good neurologic outcome 29% vs. 8%, p<0.01). artificial cooling can significantly improve survival and neurologic outcome in patients with severe head injury, when used in a protocol with great attention for the prevention of side effects. these effects are especially clear in patients with gcs of 5 or 6 at admission. because there is likely to have been bias against the hypothermia group in this study, the positive effects of hypothermia might even have been underestimated. introduction. s 100 b, a glial calcium-binding protein, is a serum marker of cerebral damage. posttraumatically, however, s 100 b in increased in all patients suffereing from hemorrhagic-traumatic shock, regardless of whether trauma is cerebral or extra-cerebral. the aim of this experimental study was to determine whether the posttraumatic s 100 b increase is caused by extra-cerebral trauma or by hemorrhagic shock and whether it is influenced by the severity of shock. hemorrhagic shock was achieved by bleeding anesthesized rats to a mean arterial pressure (map) of 30-35 mm hg through a femoral catheter and maintaining this map until incipient decompensation. subsequently, map was either increased immediately to 40-45 mm hg (moderate shock) or maintained at 30-35 mm hg until 40% of shed blood had been returned (severe shock), and then increased to 40-45 mm hg. resuscitation was provided after 40-45 mm hg map had been maintained for 40 min. trauma was achieved by midline laparotomy. hemorrhagic-traumatic shock caused an early s 100 b increase at the onset of decompensation. s 100 b in serum was highest at the end of the 40 min. period during which map was maintained at 40-45 mm hg and was significantly higher at all time points after severe shock than after moderate shock. in contrast, trauma (laparotomy)without hemorrhagic shock did not cause any increase of s 100 b in serum. the posttraumatic s 100 b increase in serum appears to be caused by hemorrhagic shock rather than by extra-cerebral trauma. regardless of whether the source of s 100 b is cerebral, indicating cerebral damage linked to shock, or extra-cerebral, the main determinant in the clinical setting remains the severity of shock. romera m. a. 1 , chamorro c. 1 , silva j. a. 1 , pardo c. 1 , marquez j. 1 , ortega a. 1 1 intensive care unit, clínica puerta de hierro, madrid, spain in patients with non-traumatic subarachnoid hemorrhage (sah), the development of myocardial abnormalities has been widely described. however, the true incidence of myocardial injury in this group of patients is unknown yet. we analyze the incidence of myocardial injury, in this population, using cardiac troponin i (tn i) assay and also we assess if the increase in tn i concentration has prognostic value. prospective study, including all patients with non-traumatic sah admitted to our intensive care unit (icu), from december 1999 to december 2001. serum tn i concentration was measured, at least once, within the first 72 hours after onset of symptoms. inmunoassay based on the "sandwich" principle was employed. the chi-squared test and fisher exact test were used for statistical analysis. of the 96 patients admitted, 14 were excluded ( admission later than 72 hours, absence of tni determination, previous cardiopathy or renal failure ). eighty-two patients were included in the study (50 women ). mean age 50±15 years. the tni concentration was increased in 24/82 patients (29% ). sixteen (19.5% ) patients died in icu. twelve of the 24 (50% ) with a high tni concentration and 4/58 (7% ) with a normal tni concentration died [ relative risk (rr) 7.25 (2.6 to 20.2; 95 % confidence interval (ci); p<0.001 ]. thirty-seven ( 45 % ) patients had a hunt-hess (hh ) grade greater or equal to iii. poor grades of sah ( hh>or = iii ) were associated with a higher incidence of raised tni concentration ; ci 95 %); p<0.001 ]. among this group of poor grade patients, elevated tn i levels were associated with a higher mortality [12/21 (57%) with a raised tni compared with 3/16 (19% ) with a normal tni concentration; rr 3 (1.03-9; ci 95% ); p<0.05]. however, mortality in every case was related to neurological problems. seven patients (8.5%) suffered from pulmonary edema and all had elevated tni levels. echocardiography was performed in all 7 patients, being abnormal in 5 of them. conclusion. in our series, the incidence of myocardial injury in sah was 29 %. this cardiac injury was more frequent among patients with severe grades of sah. elevations in tn i levels had prognostic value, being associated with a higher mortality. therefore, we should closely monitor those patients with sah who develop an increase in the tni levels. renaud e. 1 , matéo . j. 2 , benlolo . s. 2 , payen . d. 2 1 dept of anesthesiology and critical care, lariboisiere hospital, 2 department anesthesiology intensive care, lariboisiere, paris, france respiratory failure is one of the major complication of acute stroke (1). we have investigated the impact of the location stroke on respiratory failure incidence, cause of intubation and outcome. we reviewed 40 consecutive patients with acute stroke admitted to icu from 1998 to 2001. following data were collected, glasgow coma score (gcs), cause of icu admission, presence of acute respiratory failure (arf), reason for intubation, presence of aspiration, length of mechanical ventilation (lomv), severity of hypoxia, length of stay in icu (los) and mortality. continuous data were compared by paired t-test and nominal data by chi-test. explicative variables for arf were assessed by univariate analysis. . 24 patients had a middle cerebral artery (mca) stroke and 16 had brainstem stroke (bs). age (mca 50±13sd yrs vs bs 52±13sd for), gsg score (mca 8±3sd vs bs 10±3sd for), length of stay in icu (12±15sd days for mca vs 15±13sd) were not significantly different. 68% bs and 33% mca patients were admitted in icu for respiratory failure (p=0.01). admission to icu with loss of consciousness was significantly higher in mca (19/24, 80%) than in bs (0/16) (p=0.001). indication for intubation was always for aspiration pneumonia that was the leading cause of arf (0.0007) associated with swallowing paralysis in bs (p=0.001) and to unconsciousness in mca (p=0.03). there was no difference for the lomv, the severity of hypoxia between the 2 groups. arf, intubation or reason for intubation were not associated with mortality in the 2 groups (p=0.1). the major cause of death was the presence of cerebral herniation in the 2 groups (p=0.004). pulmonary complication due to aspiration more predominant in bs than mca stroke, represents the major cause of intubation and arf for bs patients. in the contrary, loss of consciousness in mca stroke group predominates for icu admission. outcome in all patients (mca and bs) was not influenced by presence of respiratory failure or reason for intubation. the major cause of death for stroke's patients is the neurologic state, and especially the presence of herniation. stroke code (sc) is a guidelines of united actuatio between out of hospital enmergency services from barcelone and the most four important hospitals of the city; which aim is to optimize the sequence time for stroke treatment; this allows to increase the number of candidates for reperfusion therapy. the present study aim is to evalute differents times sequences in the acute strokes in which trombolysis has been practised according to the acute stroke code first priority; and secundary to describe findings in the ct scan of these patients pro-inflammatory cytokines, such as tnf and il-1 are released in the brain within hours after closed head injury (chi). they were shown to have deleterious effects, mainly when active in the early post-injury period. a variety of anti-inflammatory and anti-apoptotic modalities have been shown to ameliorate the outcome of chi. erythropoietin (epo) is a kidneyderived cytokine regulating haematopoiesis both by acting as a growth factor and by inhibiting apoptotic cell death. recently it has been shown to be produced in cultured neurons, brain astrocytes and neurons under hypoxic/ischemic conditions and in response to oxidative stress. other studies have shown that the erythropoietin receptor (epor) is present under normal conditions on neuronal and brain capillary endothelial cells. epo has been found to have newly discovered neuroprotective properties in different models. these models include neuronal cultures against glutamate toxicity, global glutamate toxicity and rodent models of cerebral ischemia. in addition it induces brain endothelial cell proliferation and stimulates neovascularization in vivo. the present study was designed to test the protective effects of epo in rats udergoing controlled chi. methods. chi in rats was induced using a weight-drop device. clinical status was evaluated by the neurological severity score (nss), which tests 10 tasks including reflexes, behavior and motor activity. a point is awarded for failing to perform a task so a higher score corresponds to a more severe trauma. study animals were treated with 2 doses of i.p. 5000 units/dose (1 ml) of rhu-epo, 1 h and 24 h after chi (treatment group) or with 1 ml of vehicle injected i.p. at the same time points (control group). nss was evaluated by an observer blinded to the different groups at 1, 3 and 7 days post chi. nss scores were compared using a two tailed student t-test. control and study rats were subjected to chi of similar severity, (1h nss 10.4+0.78 and 10.22+0.46 respectively, p=0.86) and followed at 1d, 3d and 7d following chi. clinical recovery was facilitated in the treatment group starting at 24 h after chi and reached statistical significance at 7 days post chi. the treatment group's 7 d nss was 5.0 (n=8) vs. 6.625 in control animals (n=9) p=0.037. the present findings point to a neuroprotective role of epo in traumatic brain injury. brain tissue of treated and control animals is currently being analyzed for parenchymal cytokine levels. we have examined the role of post trauma treatment with epo of rats undergoing chi. as has been shown in other models of brain injury (stroke, ischemia, glutamate toxicity) epo seems to have a neuroprotective effect in head trauma. the exact mechanism of this protection has yet to be elucidated. this is the first time, to our knowledge, that epo has been studied in an animal model of traumatic head injury. (1998) (1999) (2000) is 4.7%. it is difficult to know how to apply these figures to individual patients. we have used the anaerobic threshold in a prospective observational study to try and identify patients with an increased risk of mortality. forty-five patients scheduled for elective aaa repair had their anaerobic thresholds measured pre-operatively. the anaerobic threshold is the patient's oxygen consumption in ml/kg/min when anaerobic metabolism occurs(reference 2). it is calculated by using a bicycle ergometer and a metabolic cart. clinical presentation and evolution of valvular heart disease (vhd) patients have great significance in determining the best moment for surgical correction but lacks correlation with surgical outcome in most cases. this study tries to determine the preoperative variables associated with mortality in the course of surgical treatment of vhd. cohort study conducted from january 2001 to february 2002. inclusion criteria: patients submitted to vhd surgery during the period of study. exclusion criteria: vhd surgery combined with non -vhd procedures. data were analyzed with chi -squared, fisher and mann -whitney tests. one hundred five patients met the inclusion criteria. the preoperative variables associated with surgical mortality were: systemic arterial hypertension (p=0.016), peripheral vascular disease (p=0.031), redo (p=0.031), age (p=0.051), blood creatinine level (p=0.042), left ventricular dysfunction (p=0.02). conclusion. based on these data, efforts will be held in order to develop a prognostic score index for mortality in vhd surgical patients. in our setting, the diffusion of institutional education in basic cardiopulmonary resuscitation (bcpr) is low. the number of patients admitted to our units after resuscitation following cardiac arrest is rising due to the population demand on the out-of-hospital emergency services, 061. the patients with neurological sequelae secondary to incorrect bcpr in the first 10 minutes are common. through the association of ex-patients of the intensive care medicine department, and with the psycho-social support of voluntary helpers on patient discharge, relatives are offered bcpr as part of the quality care programme. every three months, professionals from the department organise this course for 20 relatives in the form of a 5 hour module. the concepts of the prevention of ischaemic heart disease are presented together with the content of the national plan for bcpr. practical sessions are undertaken in small groups of 6 to 8 persons, using dummies. a total of 294 relatives in 13 courses have received this training over the past 5 years. the mean age of the students was 38.3 (sd 15) years (11-75), 72% women, 32% with middle and higher education, 20% housewives, and 15% manual labourers. the evaluation of the scores obtained in the 16 item test before and after the course is shown in the tables below. multiorgan system failure (mosf) is an infrequent but very serious complications after cardiac surgery, with high rates of mortality. this study was undertaken to determine the frequency, prognosis and risk factors for mosf this study was performed in a twelve-bed cardiac surgery intensive care unit over a 24-month period. all adult consecutive patients undergoing coronary, valvular and combined (valvular and coronary) surgery were prospectively studied (n = 2102). all patients were assessed by the "modified" parsonnet score results. mosf developed in 138 (6.57%) patients, of whom 51 (36.9%) died. this was the main cause of overall hospital mortality (51/92, 55.4%). in a logistic-regression anlysis, the development of sepsis, postoperative low cardiac output syndrome, mechanical ventilation more than 72 hours, a "modified" parsonnet score more than 25 and and preoperative ventilatory support were independantly associated with the development of mosf. an organ system failure index (osfi) of 3 or more was most significantly associated with icu mortality (p<0.001). conclusion. in our series mosf was a leading cause of mortality after open-heart surgery. the development of mosf with an osfi of 3 or more was the main predictor of postoperative mortality. we studied 211 patients who underwent cabg surgery. fifteen patients (13 male and 2 female) were younger than 45 years and 196 patients (147 male and 49 female) were older than 65 years. perioperative death occured in one patient from the <45 years group and in 10 patients from the >65 years group (p=ns). categorical data were compared using the chi-square test and numerical data were analysed using the student t-test. differences were considered significant at p<0.05. in a n investigation conducted in ours icu, 38% of patients hospitalised after elective cardiac surgery presented a pain score > 30 ( min score 0 ; max 100) . these results were considered inadequate. a quality improvement initiative was undertake. the aim of the present study was to test if pain evaluation and treatement improved following pain guidelines implementation in a surgical icu. the design consisted in observing de pain evaluation both before and 1 month after implementation of guidelines. these guideline are divided in two item : first introduction of a regular pain evaluation using a visual analogue scale (vas) and second in a proportional vasderived analgesic prescription protocol. recommendation were given during repetitive meetings, feedback sessions and regular poster information on the icu walls. pocket guideline and vas tool was distributed. pain intensity evaluation of the nursing team was checked by an independent observer and compared with the nurses-charted vas. improvement of pain control was tested based on the following criteria: utilisation of the algorithm at least twice per working shift; corresponding analgesic drug to observed vas; and follow up check of vas after analgesic administration. the independent observer measure vas at 8 a.m. and at 4 p.m. postoperative day 2 and 3. proportion of algorithm adherence before and after introduction of the recommendation were tested using fisher's exact test. variance of median vas was tested using mann-whitney test. these preliminary results indicate that the implementation of an algorithm on pain intensity evaluation and treatment increases the number of pain evaluation and re-evaluation after drugs administration. although the administration of analgesic drugs increased, the number of patients with insufficient pain treatment stays still high. the prognosis of liver transplant has improved the last few years due to advance in surgical techniques and immunosuppressive regimes, but early complications show a high prevalence affecting morbi-mortality in these patients a 42 beds icu in a teaching 3rd level hospital. prospective observational study on all patients with the mentioned condition treated in our centre from october 2000 to october 2001. follow up during icu stay. we have collected data from 46 patients (47 grafts) with a mean age of 52.4±9.6 years, 28.3% women, mean apache ii on admission 17±4.1, median child score 7,5 (7-8) and mean sofa score 5.8±1.8. surgical data were as follows: fluids balance 478±2570, hours of graft ischemia 7.6±2.1, reperfusion syndrome in 21% and fibrinolysis in 4.3%. at admission mean core temperature was 35.3±1 ºc. median icu stay 3.5 (3-4, max. 17) days and median hours under mechanical ventilation 10 (7.5-16, max 67). the prescribed immunosuppression was cyclosporine in 50% and tacrolimus in 50% of patients results. icu mortality was 6.5% (3 patients). complications were present in 78% (45.7% of them more than two episodes). 2 patients had to be reoperated, one because early graft dysfunction treated with mars and retransplantation (death because a new graft dysfunction), and the other because abdominal haemorrhage. one patient developed an early rejection. metabolic complic 60.9% (high insulin requirements 41.3%) -renal failure 45.7% (renal replacement 6,5%)-cardiac complic 30.4% (chf 8.7%, hbp 10.9%) -respiratory complic 19.6% (10.9% sdra) -bleeding 13% -neurological complic 10.9% (myelinolysis 1 patient) -infection 2.2%. patients who died had higher apache ii, child and sofa scores, lower serum albumin levels, longer graft ischemia, higher percentage of fibrinolysis and reperfusion syndrome during surgery and higher percentage of acute renal failure an need for renal replacement (not statistical analysis due to the low mortality rate we report the effects of substitution with a virus-inactivated protein c (pc) concentrate in disseminated intravascular coagulation (dic) in preterm infants and children with sepsis (meningococcal in the children and aldolecent; staphylococcal and enterobacter in the preterms) associated with purpura fulminans. this was a prospective open-label study. a total of 16 patients,9 paediatric and adolescent patients age 0.2 to 18.25 years with dic associated with severe acquired pc deficiency (range 0.02 to 0.48 iu/ml; median, 0.22 iu/ml) in meningococcal septic shock and purpura fulminans; and 7 preterm infants with severe acquired pc deficiency (range 0.01 to 0.1iu/ml; median, 0.02 iu/ml) due to staphylococcal and enterobacter sepsis were studied. replacement therapy was initiated with a virus-inactivated pc concentrate with an initial intravenous bolus of 80 to 120 iu/kg followed by 50 iu/kg up to six times per day as an adjunctive therapeutic regimen to otherwise optimal intensive care treatment. after initial pc administration, plasma pc levels rose to normal ranges and were maintained under pc replacement therapy. improving or even normalising global hemostatic parameters were assessed in all patients. markedly elevated plasminogen activator inhibitor type 1 (pai-1) levels prior to treatment, reflecting a reduced fibrinolytic potential, decreased rapidly under pc substitution. concomitantly improving signs of purpura fulminans reflected by decreasing size of skin lesions, demonstrated a restoring microcirculation. seven of the nine paediatric and all of the neonatal patients survived. one patient (paediatric) required limb amputation; two patients died because of multiorgan failure. both presented with a severely low plasma pc activity of 0.02 iu/ml on admission to the hospital. no adverse effects were observed with the pc concentrate administration. ait can be concluded that the administration of pc concentrate had a marked benefit on the deranged coagulation status of patients with purpura fulminans and septicaemia. normalisation or even partial correction of haemostasis as well as improvement of microcirculation accompanied by improving signs of purpura fulminans were demonstrated in all patients the main purpose of this study is to report medical and surgical complications of spine surgery in a third level universitary pediatric hospital with a reference spine surgical program. methods. study design is a retrospective clinical series of 73 pediatric spinal surgeries.all spine surgeries performed on children under 18 years of age between january 2000 and january 2002 were included. patient were grouped in four diagnostic categories (idiopathic, neuromuscular, congenital scoliosis and miscellaneous) and procedure performed (posterior (p) fusion, anterior/posterior (ap) fusion, anterior (a) fusion, (iw) instrumental withdrawal). next data were recorded from clinical chart:age, gender, needs of transfusion products, volume demands during first postoperative day,days on mechanical ventilation,medical and surgical complications. results. study sample included 73 patients, 47 female and 26 male. age ranged between 4 and 18 years with average of 12.7 years. characteristics were: idiopathic 29, neuromuscular 26,congenital scoliosis 7,miscellaneous 11. procedures performed were:p fusion 53,ap fusion 17,a fusion 1,iw 2. .average lenght of stay in pediatric intensive care unit were 1.3 days (range 0-25).average days on ventilatory support 0.46( range 0-2.5). no patient required intubation after weaning.major complications were: deep wound infection(2), respiratory distress(1), large intraoperative blood loss (1),and paraplegia (1).no deaths were observed.minor complications were: atelectasis(6), pleural effusion(4), pneumonia(2), pneumotorax(1), superficial wound infection(4), urinary tract infection(3) and electrolitical disturbances(17). postoperative transfusion needs were 8.4 ml/kg (95% confidence interval (ci) 4.1-12.8) for ap fusion, 6.8 ml/kg (95% ci 4.8-8.7)for p fusion; a fusion and iw doesn't need postoperative blood replacement. total blood transfusion was 40.7 ml/kg (95% ci 29.7-51.8)for ap fusion, 23.2 ml/kg (95% ci19.3-27.1)for p fusion; 6.9ml/kg for a fusion and 7.6 ml/kg for iw.volume demands(no blood products)during first postoperative day were 81.4 ml/kg (95% ci 50.5-112.2)for ap fusion, 59.1 ml/kg (95% ci 48-69.7) for p fusion;38ml/kg for a fusion and 44.8 ml/kg for iw. conclusion. spine surgery has few major complications rate in a reference spine surgery pediatric hospital. minor respiratory complications affect 15% of our patients without repercussion in outcome. total blood loss is greater in ap fusion than in other procedures, but postoperative blood replacement in picu didn't differ between procedures. background elevated intra-abdominal pressure (iap) adversely affects pulmonary, cardiovascular, renal, splanchnic and central nervous system physiology, and it determines the common clinical picture called "the abdominal compartment syndrome". nevertheless the direct monitoring of iap is not always practicable, because it requires an abdominal drainage. a lot of authors demonstrated in the adults that the bladder pressure is a reliable index of iap, but there are not studies on pediatric population. the aim of this study is to evaluate the level of significance of this index in a pediatric population. population: we enlisted a group of pediatric patients, sedated and paralysed (3 oltx, 3 abdominal surgery, 1 cardiac surgery), age 20.8 ± 16,3 (range 4-48) months. methods. the bladder pressure was measured with the patient in supine position, with a trasduction circuit connected to the bladder catheter and to the abdominal drainage (3 jpratt, 3 pig tail, 1 catheter for peritoneal dialysis). to obtain a good transduction of pressure, a volume of saline was pushed into the bladder. the volume of saline was variable according to the weigth and age: we obtained a scheme (table 1 ) from our empirical evaluation of the pediatric bladder compliance and urodinamic data. table 2 , there aren't significative differences between the level of pressure measured in the bladder and in the peritoneal cavity ( p= 0.50). mean:0,00 ds:1,15 from 1998 to 2001, 7 pediatric patients (age range 0.58 to 10 years, mean 3.91 years) were treated using nppv during distinct episodes of acute respiratory failure (arf) of neuromuscular origin. in all patients immediate intubation for an acute, life-threatening presentation was avoided and respiratory status improvement was achieved. few data are available up to now about nppv application and indications in the acute setting in infants affected by neuromuscular disorders (nmd). a prospective observational study was carried out on 7 non-consecutive neuromuscular patients admitted to picu because of arf and managed with nppv in the acute phase;remarkably, 4 out of 7 were <15 months aged. all the patients were treated by a flowtriggered intensive care mechanical ventilator (siemens servo 300 ventilator, siemens-elema, sweden) through a tight fitting face mask. nppv was administered for at least 24 hours postadmission. a pressure-control mode was adopted for better compensation of leaks around the mask. flow-sensitive trigger permitted a better synchronization of patient's spontaneous breathing, limiting the need for deeper patient sedation (low-dose midazolam drip). initially, a relatively low ventilator frequency delivery was set (8-10 b/min). peak inspiratory pressure was tritrated upward to obtain an exhaled tidal volume of 5-6 ml/kg maintaining a paco2 value <65 mmhg and a ph >7.25; peep value was adjusted to maintain an oxygen saturation >91-92% with a required fio2 <0.6. results. all patients were referred to picu on spontaneous breathing: those admitted with et tube already positioned were not considered eligible for this study. an oxygenation improvement was obtained in all patients within 3 hours from the onset of nppv . the pao2/fio2 increased from 86.29 ±16.13 to 235.6±32.32 (p<0.05) and 292.6 ± 25.75 (p<0.001) on selected time points (3 and 12 hours after nppv introduction, respectively); conformly, alveolar-to-arterial oxygenation difference (a-ado2) decreased from a 462.3 ± 77.77 to 195.7 ± 59.67 (p<0.05) and 101.8 ± 23.92 (p<0.001) respectively. conclusion. nppv resulted a safe and effective therapeutic approach in both hypoxemic and hypercapneic arf episodes in this children group affected by nmd. even in cases of emergency presentation or when resuscitation is needed, it is of importance to identify nmd children with residual ventilator-free breathing ability thus performing a nppv trial. life-threatening respiratory distress and young age should not preclude nppv application in a picu setting. pulse oximeters are widely used in paediatric intensive care but they have some severe limitations. the technique relies on the presence of adequate peripheral arterial pulsations, which are detected as photoplethysmographic signals (ppg). when peripheral perfusion is poor as in states of hypovolaemia, hypothermia and vasoconstriction oxygenation readings become extremely unreliable. hence, pulse oximetry becomes unreliable in a significant group of children just at the time when accurate readings are most needed. to overcome this limitation, the oesophagus has been investigated as a potential measurement site on the hypothesis that perfusion may well be better preserved at this central site. studies on adult patients have shown that measurable ppg signals at red and infrared wavelengths can be detected within the whole depth of the oesophagus. a new system to investigate the quality of oesophageal ppg signals is being constructed with the aim of developing a neonatal and paediatric oesophageal pulse oximeter. a reflectance optical sensor has been constructed comprising miniature infrared and red emitters and a photodetector. the sensor was design to fit into a conventional disposable transparent stomach tube, 12 french gauge. the oesophageal ppg sensor within the stomach tube was inserted through the nose into the oesophagus of a 2 kg, 17 day old neonate. the stomach tube was advanced into the oesophagus under direct vision until the probe was 25 cm from the nose. ppg traces from the oesophagus were recorded for approximately 5 minutes at this depth on a laptop computer. measurements were repeated at 20 and 15 cm from the nose. measurable ppg traces of good quality were obtained in the oesophagus at all three depths. the ppg signals in the mid to lower region of the oesophagus on average had larger amplitudes at both red and infrared wavelengths than the ppgs recorded in the upper oesophagus. artefacts on both wavelengths due to oscillations as a result of high frequency ventilation. filtering successfully eliminates the artefact. the new oesophageal reflectance optical sensor has allowed ppg measurements to be made within the whole length of the neonatal oesophagus. the red and infrared wavelengths used are suitable for pulse oximetry. these results are the first to demonstrate that pulse oximetry may be feasible in the neonatal or paediatric oesophagus. further studies are required to develop a neonatal/paediatric pulse oximeter. we used protein c (ceprotein; baxter -immuno) in two patients with moderate or severe, therapy-resistant vod. . patient 1 (e.g, swiss, 1,75 y) suffered from an acute myelogenous leukemia (m 7 with t(1;22) of early infancy after complete first remission by conventional chemotherapy an allogeneic stem cell transplantation with a matched unrelated donor was performed. conditioning comprised busulfan, vp16 and cyclophosphamide. patient 2 (m.k, iranian, 11 y) suffered from beta-thalassemia major with secondary moderate hemosiderosis, as well as chronic persisting hepatitis c infection with liver fibrosis. he received a matched related bone marrow transplantation, using i.v. busulfan , reduced cyclophosphamide dose and fludarabine. both patients received low dose heparin (100 iu/kg) and antithrombin iii substitution. in addition, pat. 2 got prophylactical defibrotide (20 mg/kg) and n-acetylcystein. two weeks after transplant both patients developed vod (severe (pat 1); moderate to severe (pat 2))with weight gain, hepatomegaly, massive ascites and severe thrombocytopenia. maximal bilirubin was 15 mg/dl (pat 1) and 2 mg/dl)(pat 2). therapy with defibrotide (60 mg/kg) was started immediately. in pat.1 the pulmonary situation deteriorated rapidly with massive aszites and oxygen need and a reversed portal venous flow. defibrotide was stopped after 5 days. thrombolytic therapy using rtpa and a continuous pc substitution (pc level 16%; bolus 100 iu/kg, followed by 100 iu/kg every 4h)) were started. lysis therapy had to be abandoned due to respiratory tract bleeding global coagulation (pt 34 %, aptt 250 sec) and pc level normalized within hours after pc substitution. a normal centripetal portal flow could be achieved by high dose defibrotide (120 mg/kg) and continued pc substitution after several weeks. pat. 2 showed only a temporary improvement under defibrotide treatment. due to clinical deterioration (hepatic pain, increased ascites) and low pc level (38%) a continuous pc substitution (50 iu/kg every 6 h) was initiated. there was a prompt recovery after adding pc with a dramatic reduction of ascites, weight and abdominal pain within 2-3 days after start of pc infusion. elevated bilirubin levels returned to normal in both patients. in our 2 patients neither prophylactic administration of at iii nor of defibrotide were able to prevent moderate to severe vod.our data indicate that pc substitution may be a useful adjunctive treatment in severe vod. until controlled studied will be initiated we recommend a stratified treatment in vod, starting with defibrotide, and adding pc in unresponsive cases. (1) showed recently that restrictive strategy of red-cell transfusion could be at least as effective as and possibly superior to a liberal transfusion strategy in critically ill patients. the aim of this study was to assess the impact of local transfusion guidelines emphasizing restrictive strategy on patients undergoing heart surgery and the prognostic value of transfusion following those restrictive criteria. methods. two groups of 100 heart surgery patients were compared before and after the introduction of local transfusion guidelines. these guidelines involved general information on blood transfusion risks and obligation for the physician to respect predetermined transfusion criteria (hb <7g/dl or >7 g/dl associated to systolic arterial pressure <90 mmhg or age over 70yrs or hr >100/min or ci <2.2 l/mim/m² or other associated disease 5.5 5.2 6 6.4* 5.6 5.6 8.5 9.9* <0.05 mortality (%) 4 6* 6 19* <0.05 conclusion. introduction of local restrictive transfusion guidelines was associated to a significant reduction in red cell transfusion during the postoperative period of heart surgery. the global morbidity and mortality rates in the whole group of patients were not affected. however patients who required blood transfusion following the restrictive strategy had a worse outcome. transfusion was probably more the consequence than the cause of this worse prognosis. if transfusion was the cause of the worse prognosis, then morbidity and mortality rates would have been higher among patients requiring transfusion during liberal period than in the whole group of patient. (1) hebert pc and col. a multicenter, randomized, controlled clinical trial of transfusion requirements in critical care. n engl j med 1999 ;340 :409-17. risk factors and outcome in european cardiac surgery higgins tl. quantifying risk and assessing outcome in cardiac surgery 3 1 intensive care medicine, hospital del mar, 2 servicio de microbiología to analyze if morbid obesity (mo) is associated with critical pathology in relation to patients undergoing vertical banded gastroplasty (vbg). all critical patients (cp) suffering mo, with a mean body mass index = 50,61, receiving programmed surgical treatment, and admitted in the icu during the next period: 1st oct. 96 to 25th feb. 02, were prospectively included. · surgery procedures. *restrictive: vbg according to masson's technic. *derivative: vbg + gip according to salmon's technic. vbg + gip according to capella's technic. ·type of study: descriptive. -vbg in association to other surgical procedures: 16 cp (16 cholecystectomies, 1 right inguinal herniorhaphy, and other 3 procedures). · mortality: 2 cp: -septic shock -multiorganic disfunction · readmission: 2 cp (subphrenic abcess and ards). complications 1. hypoxemia: 69 cp (60% of the total) 1.1. 1.1. not secondary to hypoventilation: 52 cp (72.5%). 1.2. 1.2. associated to hypoventilation: 52 cp (72.5%) 2. need for noninvasive mechanical ventilation: 25 cp (22%) 3. high blood pressure: 61 cp (52.0%). 4. disturbances of cardiac rythm and conduction: 8 cp (7%). 5. metabolic acidosis: 13 cp (11%). 6. other complications: 16 cp (14%). conclusion. 1-the mo patient undergoing vbg, with or without gip, rather than a patient bound to the reanimation or recovery room, is indeed a patient who requires admission in the icu for, at least, 24-36 hours. 2-hypertension of difficult management and hypoxemia not due to hypoventilation nor shunt are the most frequent complications. 3-an important percentage of cp requires also mechanical ventilation. 4-complications related to surgery are exceptional. karlicek a. 1 , haveman j. w. 1 , verhoeven e. 1 , van den dungen j. j. a. m. 1 , tielliu . i. f. n. 1 , hulsebos r. g. 1 , nijsten m. w. n. 1 1 surgery, groningen university hospital, groningen, netherlands introduction. the mortality in acute abdominal aortic aneurysms remains high. recent series still report a hospital mortality rate of more than 50% (1,2). despite the large number of published studies on hospital outcome, long-term outcome after icu admission has hardly been studied. here we present hospital survival and long-term outcome in 308 patients with an acute abdominal aortic aneurysm. the records of all patients operated for aneurysm surgery between 1990 and 2001 were retrospectively reviewed. in 308 patients surgery was performed for an acute abdominal aortic aneurysm. all operation reports were analysed. for complete follow-up the general practitioner was contacted if necessary. after arrival in the emergency department and confirmation of the diagnosis by physical examination and/or ultrasound all patients were immediately brought to the operation room. in our hospital even patients with cardiac arrest on arrival in the operation room are treated without delay, and were thus included in our study. all surviving patients were admitted at the intensive care. in case of postoperative haemodynamic instability, multiple organ failure, sepsis or diarrhea a sigmoidoscopy was performed to assess the presence of ischemia or infarction. three hundred and eight patients were operated for an acute abdominal aortic aneurysm, 266 men and 42 women. operative mortality was 13% (39/308). calculated from the moment of icu admission, 30 day survival was 78%. cumulative survival rates calculated with the kaplan meier method at 1, 3, 5 and 10 years were 65%, 55%, 46% and 28% respectively. in 23 patients in whom sigmoid resection was performed, 30 day survival was 35% compared to 83% in the other patients. mortality in ruptured abdominal aortic aneurysm remains high, 30 day survival was 78% in our group. sigmoid resection was associated with lower survival but sigmoidoscopy should be augmented to exclude sigmoid necrosis. outcome in these patients is not invariably poor. long term follow-up shows that also after discharge from the hospital these patients have a high mortality. carvalho a. g. r. 1 , gomes r. v. 1 , santos jr. b. 1 , barbosa o. n. 1 , weksler a. 1 , pontes a. p. 1 , camara a. c. 1 1 surgical intensive care unit, instituto nacional de cardiologia laranjeiras, rio de janeiro, brazil introduction. prognostic markers developed in europe and north america cannot be applied in latin america where life expectancy is 30 % lower according to world health organization. the objective of this study is to analyze patients profile submitted to heart surgery (hs), type of surgery distribution and the impact of variables, previously reported in medical literature, in the mortality and duration of intensive care unit (icu) stay in a public tertiary hospital. cohort study of patients submitted to hs from january 1998 to april 2001. patients profile, type of surgery distribution and many variables were analyzed. variables that were studied: age, gender, body mass index (bmi), body area (ba), preoperative in-hospital stay (preop), extracorporeal circulation (ecc) duration, ventricular function (vf), surgical indication, combined procedures (comb), urgency for the surgery, presence of diabetes mellitus (dm), systemic arterial hypertension (ah) and cigarette smoke (cs). the profile and patients variables were analyzed and compared in two different groups. group a (ga): patients discharged from icu or in-icu stay lower than or equal to 3 days (median in-icu stay in this study). group b (gb): death during icu admission or in-icu stay longer than 3 days. t-student, mann-whitney, chi-squared and fisher tests were used in the statistical analysis. conclusion. there is a rather singular distribution of surgeries in this group. many of the previously described variables showed correlation with mortality or longer admission in the icu. prospective studies will be held in order to adjust these variables and determine new ones more relevant to underdeveloped countries. pierce c. m. 1 , fortune p. 2 , petros a. j. 1 1 picu, great ormond street hospital, london, united kingdom, 2 picu, royal children's hospital, melbourne, australia there are anecdotal reports of sildenafil, a type 4 phospodiesterase inhibitor, being used to reduce pulmonary artery pressure in children with mainly cardiac induced pulmonary hypertension (pht). we have given oral sildenafil to 10 children on our paediatric intensive care units with pht from various causes. diaphragmatic hernia (3), avsd (2) vsd (1) pda (1), pphn (2) pulmonary hypoplasia (1). the median age of the group was 1m (iqr 1-6m). 7 were receiving inhaled nitric oxide during sildenafil. median dose was 0.5mg/kg (iqr 0.3-0.53mg/kg 8hrly) and duration was 7 days (iqr 3-29). pulmonary artery pressures were directly measured in 3 of the 4 cardiac children and deduced from doppler echocardiographic measurements of the tr jet in 5 children.results. pap decreased significantly (p<0.05) following oral doses of sildenafil (n=7). mean pulmonary/system (p/s) ratios decreased from 0.99 to 0.75 (n=8) within 3 hours of the oral dose. systemic pressure was unaltered in all children. in one child with pulmonary hypoplasia the p/s ratio was unaffected. oral sildenafil can significantly reduce raised pap in children when there is a reversible etiology. this may be particularly useful in children and neonate with pphn. central venous catheters (cvc) are an important means of securing intravascular access in pediatric intensive care unit patients. one of the major morbidity's in use of cvc is catheter-related infection (cri). the incidence of bacteremia with cvc use is approximately 5.3/1000 catheter days and mortality as high as 25%. one approach to reduce the incidence of cri has been to decrease catheter bacterial colonization (cbc). reduction in cbc is achieved by coating or impregnating antimicrobial substances into the catheter material. use of minocycline/rifampin treated catheters has been shown to reduce the rate of cri in critically ill patients. the concern in pediatric population is the use of minocycline. tetracycline and its derivatives (minocycline), when used in young children, carry the risk of dental and sceletal abnormalities. the problem of potential eluting of minocycline from minocycline-impregnated catheters may pose a risk for young children. our study examined whether detectable levels of minocycline and rifampin were present in the serum of the pediatric intensive care unit patients with indwelling minocycline/rifampin impregnated cvc. methods. patients admitted to pccu age 8-18 years and in need of cvc were eligible for study. six patients were enrolled. each patient had two samples of blood 2 and 2.5 ml withdrawn for rifampin and minocycline assays respectively. collection times were at the time of catheter insertion and 24h thereafter for seven days or until catheter removal, whichever came first. rifampin serum samples were processed prospectively soon after colection by standard hplc. minocycline serum samples were stored frozen in -80 centigrade and assayed in one batch using reverse phase hplc. results. demographic data are in table. ranges with mean values in ( ). none of the minocycline samples had detectable level of antibiotic. the limit of sensitivity for minocycline was 0.4 mg/l. therapeutic levels are 1.4-1.8 mcg/ml. one patient had 4 consecutive samples 3 to 6 with low therapeutic levels of rifampin (4-5 mcg/ml). therapeutic levels of rifampin are 4-32 mcg/ml. rifampin sensitivity was 1 mcg/ml. rifampin has distinct peak time and no interfering substances were identified.sex (3), the emergence of this consequences required fast and corrective treatment. an inotropic agents commonly used in vlbw infants such as dopamine and norepinephrine in some cases do not produce elevation in blood pressure despite using of very high dose. in this study i would like to exam the influence of hydrocortisone administration in vlbw infants with hypotension unresponsive to stndard catecholamine treatment. i have reviewed the cardiovascular response to hydrocortisone therapy in 16 preterm infants. mean gestation age was 27.1 (25-29) weeks, postnatal age 4.8 (1-9)days, mean birth weight 1070g (710-1420). eight of them suffered from respiratory distress syndrome and eight from sepsis. the first line of hypotension therapy was always volume administration (normal saline or albumine) and catecholamine infusion. hydrocortisone at the dose 2mg/kg was administered when dopamine at the dose 10mcg/kg/min (12 patients or norepinephrine 0.5mcg/kg/min (4 patients)failed to normalized arterial blood pressure. pneumococcal meningitis is an important cause of morbility and mortality in children. we describe the epidemiological characteristics and clinical features of pneumococcal meinigitis in children admitted to a children's hospital in barcelona. medical records of 32 children with a diagnosis of pneumococcal meningitis based on identification of s. pneumoniae in the blood or cerebrospinal fluid between january, 1993, to april, 2002, were retrospectively reviewed. results. 32 cases of pneumococcal meningitis were diagnosed in 29 patients. median age was 24 months (range 1.8 m-9.8 y). 24 children were younger than 5 years old (75%). male-female ratio was 1.2:1. none of the children had a previous immunological deficit. thirteen patients (41%) were pre-treated with antibiotics. the most frequent signs on admission were fever (100%), vomiting (72%), headache and irritability (34%), othalgia (22%) and shock (16%). neurological findings were lowered level of consciousness in 18 patients (52%), signs of meningismus in 24 patients (75%) and arreactive mydriasis in 3 patients (9%). the mean leukocyte counts in blood were 19164/mm3 and the mean c-reactive protein was 141mg/l. cerebral spine fluid indices on admission were: white blood cell=2301 (50-9800) /mm3; protein=148 (43-396) mg/dl; and glucose= 38 (0-262) mg/dl. main serogroups were: 18 (16%), 6 (13%), 4 (9%), 19 (6%), 23 (6%), 7 (3%), 14 (3%) and 9 (3%). overall, 50% of the pneumococcal isolates were penicilin-nonsusceptible, 28% cefotaxim-nonsusceptible and 3% were vancomycinnonsusceptible. an initial abnormal cranial computed tomography was found in 6 patients. the median duration of parenteral antibiotic therapy was 10 days. all patients were empirically treated initially with cefotaxime (associated to vancomycin in 22 of them). twenty-six patients (81%) received dexamethasone. the administration of mannitol was necessary in 6 patients (19%) and anticonvulsants were administrated in 8 patients (25%). only 8 patients (25%) needed inotropic support (no longer than 48 hours). mechanical ventilation was required in 15 patients (47%) during a mean of 1.5 days (range 0-10). acute complications were: metabolic acidosis (3/32), disseminated intravascular coagulopathy (6/32), seizures (7/32), siadh (3/32) and diabetes insipidus (2/32). twelve patients (37%) suffered deafness, three patients (3%) hemiparesia and four (12%) were exitus. the mean hospital stay was 18.5days and mean intensive care stay was 3.8days. there is an increased prevalence of pneumococco with decreased susceptibility to penicillin and to cefotaxime. deafness is one of the most common and serious sequelae of pneumococcal meningitis. corticotherapy has reduced the incidence of hearing loss. the new, antipneumococcal conjugated vaccine will confer effective prevention from the age of two months and will reduce the incidence of this meningitis. aims : to analize sedation/anesthesy methods used in our hospital for painfull or unconfortable procedures in children in relation to : 1)patient confort, 2)sedation complications, 3)and efficacy of the procedure a prospective study was conducted from january to march 2002 in 154 disconfortable procedures in 154 children. mean age was 40m ; their asa score was 4 in 7%, 3 in 16%, 2 in 57%, and 1 in 20%. more frequent procedures were : 54 lumbar punctures (lp), 23 thoracentesis or drainages, 21 central catheters insertion, endoscopys 18. we identified 3 different groups in relation to methods of sedation/analgesy : 1-procedures done in the emergency department with local anesthesia; 2-procedures done with administration of intravenous midazolan+ketamine; 3-procedures done with anesthesic support. we used the ramsay scale to classify the degree of anesthesia and the serna behavioral scale to classify the reaction to the procedure.results. group1(n=36):42%patients fighted against the procedure (serna scale 1) and in 25% of the patients, complications of the procedure were found to be related to inadequate sedation. group2 (n=67): in 10%, sedation was considered inadequate -serna level 1 (n=1) and 2 (n=5)-and in 1 case there were complications of the procedure related to unsufficient sedation; there were 7 (10%)cases of minor complications sedation-related; group3(n=51): patient confort and adequacy of the sedation were found in 100%, with 2 (4%) complications of the anesthesic method.conclusion. sedation/anesthesia were needed for the confort of the patients; only minor complications of sedation/anesthesy were found ; efficacy of the procedure was best achieved with the anesthesic method. introduction. the goals of emergency airway management are to antecipate and recognize respiratory problems and support therapy. the endotracheal intubation ( et) is not a routine procedure and it requests planning and personnel qualified to reduce the complications associated to this technique 1 . the purpose of this study is to evaluate early complications associated with endotracheal intubation methods. data were collected prospectively from february 1995 to january 2000 in tertiary teaching hospital. the variables were obtained in four age groups: group 1 (>1 month); group 2 (between 1 month to 11 months); group 3 (between 12 months to 144 months)and group 4 (>144 months). the data were collected as demographic data, reason for endotracheal (et) intubation, sedation administered, local of et, physician responsible for et, complications associated with airway management. the major complications were defined as technical problems that resulted increased morbidity. minor complications were incidents that should be avoided. the complications were compared between emergency or elective et intubation. statistical analysis by chi-square, fisher exact test . we evaluated 612 (45% female and 55% male) no consecutive patients. indication for intubation were: respiratory failure (31%), coma or depressed sensorium (7.5%), post-operative (45%) and shock (17%). sedation and/or analgesic were used in 54% of patients and 3.5% did not receive a sedative or analgesic for et intubation. a total 125 et emergency intubation ( we report an outbreak due to rsv in a 20 bedded picu with an annual admission rate of approximately 1000 patients, cardiac and medical patients accounting each for 40% of the population and 20% surgical.methods. an outbreak is defined as an event in which minimally 2 patients develop bronchiolitis due to rsv following transmission via hands of carers within a limited period of 1 week.nasopharyngeal aspirates were obtained from children with symptoms of lower airway infection, all samples were tested for rsv using the enzyme immuno assay, followed by tissue culture when the assay was negative. rsv positive children were isolated in cubicles and strict standards of hygiene were implemented. introduction.the objective of the study was to investigate the validity of outcome prediction after severe head injury using serum levels of protein s-100 b and of neuron specific enolase nse. methods.fifteen patients with severe head injury were included in this prospective study (9 men and 6 women) mean age 38 yrs (18-69). none of the above patients had spinal cord injury or any other neurological disease. venous blood samples were taken on admission and consecutively the 1, 2, 3, 4, and 5 day. immunoluminometric assay was used for the specimens. we tried to correlate the s-100 b and nse serum concentrations with the ct scan intacerebral pathology as well with the age, gender and outcome. results. all patients had elevated s-100 b and nse serum concentrations, with a gradual reduction towards the 5th day of icu stay. the mean values of day 1, for s-100 b were 2.4 ìg/l and for nse were 32.5 ìg/l. of day 5, they were for s-100 b 0.57ìg/l and for nse 14.9 ìg/l. patients who died had the first day mean values of s-100 b 4.2ìg/l and nse 47.2 ìg/l, whereas the survivors had mean values of s-100 b1.32 ìg/l and of nse 27.4 ìg/l (p <0.05). there was no strong correlation between the ct scan findings, the initial serum s-100 b and nse values and the gcs, on admission. conclusion. the protein s-100 b and nse are biochemical markers that seems to be elevated during the first days of injury, in patients with severe head trauma and could be used as markers of he severity of the injury. if protein s-100 b and nse could be used as a prognostic factor of the patient outcome, needs more investigation. our study is continued. estimates such as 28-day survival may be grossly misleading for assessment of intensive care utility. late mortality and morbidity may severely affect overall outcome. we studied 100-day survival rate in addition to survivors´ general health evaluation and prevalence of signs indicating post-traumatic stress disorder (ptsd). the setting is a university general intensive care unit. during the study period all adult patients who had been intubated and mechanically ventilated for at least 48 hours were included (patients who died before 48 hours are excluded). three to six months after their critical illness, survival data were retrieved from hospital and national registers. all patients surviving at this time were sent a health survey questionnaire (sf-36) and the post-traumatic stress syndrome 10-questions inventory (ptss-10). results. 153 patients fulfilled the inclusion criteria. the mean age was 69 years, 40% were women. health questionnaires were returned by 36 (58 %) of the 62 survivors at follow-up time. 28-day survival rate was 58 %, at 100 days survival rate had fallen to 46%. among the 36 responding survivors the frequency of a response pattern compatible with ptsd was 28%. survivors without signs of ptsd had sf-36 mean scores more than 1 standard deviation (sd) below the swedish norm in the domains of physical functioning, role-physical and social functioning. survivors with signs of ptsd scored below non-ptsd survivors in every domain, and were more than 3 sd below the swedish norm in the domains of social functioning, roleemotional and mental health. in total, there were only five persons (14 % of respondents) who scored at or above the swedish norm for both the physical and the mental health summary scales. assuming the same outcome in non-respondents this figure would correspond to about 6% of all the 153 included patients. conclusion. in this cohort of severely ill patients 28-day mortality was in the expected range but much mortality (another 12 %) occurs in the following 10 weeks, indicating a number of patients who have been subjected to long-lasting care with very meagre benefits. at 3-6 months following onset of their disease, survivors show considerably reduced subjective rating of their general health and life quality. as much as 28 % of the survivors show signs compatible with ptsd. it could be estimated that about 6 % of all patients included will both survive and within 3 -6 months reach a level of general health comparable to that of the general population. the aim of this study is to probe that critically ill patients gender is not associated with differences in severity of illness and related mortality. we had tested the premise in front of a controversial evidence offered by several years of our icu activity. observational study. retrospective analysis using data prospectively collected in a medical-surgical icu of 15 beds, in a teaching reference hospital, from november 1997 to july 2001. we analyzed 3152 consecutive admissions considering reason for admission, age, icu length of stay, severity of illness (mpm0, mpm24, saps ii and spanish version of apache iii) and related risk of death. cases were analyzed according to gender and age decades. therapeutic effort was analyzed according nems system. standarized mortality ratio (smr) and its 95% ci was determined. one thousand and twelve cases out of 3152 were women. mean age (sd) was 60 (17) years. significative differences were founded in mpm0 prognostic values (0.243±0.249 for men and 0.267±0.264 for women, p 0.003). the rest of epidemiological data do not offer significant differences. smr for men was 0.96, and for women 1.21, but 95% ci overlapped 0.86-1.07 vs. 1.04 -1.41, p ns. the same differences were found when different age intervals were analyzed. only 3 admission diagnostic (ischemic cardiopathy, post cardiopulmonar arrest and multiple trauma with no head trauma) showed greater mortality rates in women, but these differences disappeared when age intervals were considered. in spite of certain confusing data about greater mortality ratios in women admitted to our icu, accurate analysis does not show significant differences in severity of illness, associated prognosis and mortality, and therapeutic effort between male and female. bacterial infection is one of the most frequent and most feared complications in patients with a hematologic malignancy (phm). in a retrospective study, we found that bacteremia precipitating icu admission in phm was associated with a better outcome [1]. however, it remained unclear whether this finding could be extrapolated to all bacterial infections. the aim of this prospective study was to evaluate whether bacterial pneumonia (bp) and bacterial sepsis or other bacterial infections (bs) had a better outcome compared to non-bacterial or noninfectious complications (nbc) in critically ill phm.methods. 106 consecutive phm admitted to the icu over a 2 year period were categorized into bp (n=32), bs (n=24) or nbc (n=50) according to strict diagnostic criteria by an independent panel of physicians who were blinded for the outcome. the impact of bp and bs on the inhospital mortality was assessed by logistic regression after adjustment for severity of critical and underlying hematologic illness, duration of hospitalization before icu admission and other potentially important prognostic factors. two models were tested, the first using a classical severity of illness score (apache iii) and the second using a score system especially designed for cancer patients (groeger score) [2] . bacterial infection is one of the more favourable complications precipitating icu admission in phm and is associated with comparable mortality rates as in general icu patients. therefore, reluctance to admit phm to the icu for advance support is unjustified, especially when a bacterial infection is suspected to be the cause of deterioration. (1,2) the cleveland clinic score is the only one, to compile intraoperative data until the timepoint of icu admission.(3,4) we wanted to find out, whether the combination of pre and postoperative score, in alliance with additional parameters, improves the predictability of outcome. from 1995 from until 1999 adult cardiothoracic patients were examined. logistic regression was used for analyzing those variables, dealing with mortality. the selection of significant factors is based on a stepwise forward procedure(p<0,05). the accuracy of multivariate analysis is shown as roc(receiver-operator characteristic) curve. . 21 variables, pre as well as intraoperative parameters proved to be statistically significant in the analysis, 9 in the multivariate analysis: both scores, operation and aox time, preop at iii, assessment of intraop course, hb at icu admission, blood loss 24h<1155ml and rethoracotomy for bleeding. the pre and the postoperative cleveland clinic risk score were both statistically significant in the uni and multivariate analysis, but their combination improved roc. additional parameters had only little further impact. pre and postoperative cleveland clinic score are reliable in predicting the risk of cardiothoracic patients. adding further intra and postoperative data, risk stratification becomes more precise. the appearance of unexpected intraoperative difficulties was highly significant for adverse outcome. the collection of data should be continued on the icu and therapy should be reevaluated and modified any time. objective: to describe the frequency, etiologies, forms of presentation, and foci of bacteremia identified in patients admitted to the icu. prospective epidemiological surveillance study carried out from april 1998 to march 2001. bacteremia was defined as the isolation of a pathogenic microorganism in one or more blood samples. bacteremias were classified into contaminating or true according to clinical manifestations. a descriptive analysis of variables including mean values, ranges, and standard deviations is presented. a total of 332 episodes of bacteremia were identified, 228 of which were true bacteremias (27.8 episodes per 100 patients). the characteristics of patients with true bacteremia were as follows: mean (sd) age 64 (16.3) years; male sex 58.8%; mean apache ii score on admission 14.9 (5.8); and mean length of previous hospitalization 22 (27) days. in 153 (67.1%) cases, bacteremias were acquired in the icu and in 49 (21.5%) episodes were polymicrobial. a total of 289 pathogens were cultured. these included gram-positive cocci in 173 (59.9%) cases, gram-negative bacteria in 94 (32.5%), and fungi in 17 (5.9%). initial presentation included severe sepsis in 36 (15.8%) cases and septic shock in 51 (22.4%). the most frequent origin of intra-icu true bacteremias was unknown in 42.5% of cases (primary bacteremia) followed by catheterrelated bacteremia. crude mortality was 45.2% and bacteremia-related mortality 21.5%. primary bacteremia and catheter-related bacteremia were the most common. a total of 21.5% bacteremias were polymicrobial. gram-positive cocci were the predominant causative pathogens. gyurov e. g. 1 , milanov m. s. 1 , milanov s. g. 1 , neichev p. g. 1 1 general icu, emergency medicine hospital "pirogov", sofia, bulgaria intensive care units are unique because they house seriously ill patients in confined environments where antibiotic use is extremely common. since our last publication (1) there is a substantial rise in emergence of nosocomial infection namely gram-positive as well as changes of pattern of emergence. to study the frequency of emergence of nosocomial infection (nci) in intensive care unit (icu) we studied retrospectively data from case records and flow sheets of 1451 postoperative patients in our icu during 1999-2000 and compared data with last period. results. of 1451 patients in our icu during two years, we include those 613 (42.2%) who stayed for more than 72 hours. according to results from cultures we divided them to three groups. group one included 355 (57.9%) patients without bacterial growth. group 2 included patients with proved nosocomial infections /nci/. we obtained samples: 898 from urinary catheters (376 positive-41.9%), 552 from tracheal tube (457 positive-82.8%), 597 from blood (282 positive-47.2%), 64 intradermal segments from central venous lines (34 positive-53.1%), and 17 from sputum (15 positive-88.2%). the most common place for emergence of nci in our icu is respiratory tract. on 5-th icu day the tract became infected in almost 56% of the patients. the major role among pathogens played acinetobacter spp. (27.4%), citrobacter spp. (20.3%), p.aeruginosa (12% and serratia spp. (10%). the second place for emergence of nci is "reserved" for blood-stream infections. almost the half of the cultures (47.2%) showed bacterial growth. the isolated pathogens were the same: acinetobacter spp (19%), serratia spp. (16%), but there was substantial rise in frequency of emergence of s. epidermidis during the last years (see figure) . its frequency almost equalized that of acinetobacter spp. the other two main sources for nci were urine catheters and cv catheters. they remained on 3-rd and 4-th place. group 3 included patients with endogenous surgical wound infections. in this group we obtained samples from surgical wounds and drainages. in 1999 25.6% of cultures showed bacterial growth. during next 2000 this figure rose nearly twice (48.3%). the leading role played the same acinetobacter spp., citrobacter spp., p. aeruginosa, enterococcus spp. and e. coli. the role of s. epidermidis increased greatly during this period de waele j. j. h. c. 1 , hoste e. 1 , blot s. 1 , colardyn f. 1 1 icu, ghent university hospital, gent, belgium introduction. intra abdominal infections frequently complicate the postoperative course of patients with acute necrotizing pancreatitis. the objective of this study was to analyze the incidence of pancreatic surinfection after surgery for acute necrotizing pancreatitis, describe its characteristics and identify associated risk factors. we retrospectively (1995) (1996) (1997) (1998) (1999) (2000) (2001) analyzed 46 patients treated surgically for acute pancreatitis. surgical treatment consisted of debridement and postoperative continuous lavage. we recorded demographic characteristics, incidence of organ failure, data on surgical and infectious complications, data on surgical and medical treatment and disease severity by ranson and apache ii score. surinfection of the pancreatic necrosis was present in 30 out of 46 patients (65%). the surinfection was polymicrobial in 17 patients. most of the organisms were gram-negative (54%), the others were gram-positives (28%) or fungi (17%). patients with surinfected necrosis were younger (50 y vs. 64, p<0.01), had surgical complications more often (70% vs. 26.7%, p=0.01), needed retroperitoneal lavage for a longer time (28 days vs. 9, p<0.01), and had a longer hospital stay (84 days vs. 29, p<0.001) than patients without surinfection. multivariate analysis demonstrated that age (or 0.90; 95% ci: 0.83-0.97, p<0.01) and the occurrence of a surgical complication or 10; 95% ci 1.53-66.6, p<0.01) were independently associated with pancreatic surinfection. the mortality in patients with infected necrosis was higher (72% vs. 40%, p=0.04), although in multivariate analysis no association was found. pancreatic surinfection is high after debridement and retroperitoneal lavage, with mainly gram negative bacteria involved. surgical complications and younger age are significant risk factors for surinfection. the aim of this report is to describe the current status of sap in spanish icu's methods. sap cases are identified in accordance with generally accepted criteria in each icu, such as ranson, imrie, pcr and ct-dynamic criteria. sap was selected from the data base of the national study of spanish nosocomial infection monitoring (envin). this study covered the period from 1997 to 2000. envin is an observational, prospective and multicentre study. sap patients hospitalized during more than 24 hours in all the participating icu's have been included in the study. these patients were monitored until their discharge from the icu or up to a maximum of 30-60 days. secondary infections have also been monitored. severity is measured by means of apache ii. infections, mortality, epidemiological data and antibiotics used as a means of prevention are described. the statistical analysis used the chi x2 test for the association of qualitative variables, the student t for the comparison of averages and the 5% statistical significance level results. 199 patients (1.18%) of the 16,927 patients monitored by envin were found to have sap. the average apache ii was 14.9 and the average stay was 12.2 days. the base illness was medical (94.5%) and surgical (5%). 30.7% of the patients underwent emergency surgery. ni accumulated incidence was 48.7% and density incidence was 40/1000 hospitalization days. crude mortality was 31% and ni-related mortality was 45%. 97 infections were detected: 27 of abdominal origin (27.8%), 17 ventilator-associated pneumonias (18.5%), 12 secondary bacteremias related to abdominal infection (12.4%), 12 catheter-associated urinary tract infections 12.4%; primary bacteremias (10.3%); 3 central venous catheter-associated bacteremia (1.5%). a total of 66 pathogens were isolated. bgn 58.5%, cgp 24.5% (mrsa 4.2%), fungii 17% (principally candidas), enterococci 15% and anaerobes 3.7%. 85.4% of the sap patients received antibiotic treatment. the antibiotic most frequently used in prophylaxis was imipenem-cilastatine (70%) and piperaciline-tazobactam (10%). the antibiotics most frequently used in absolute indication were imipenem 70%, piperaciline/tazobactam 19.4%, metronidazol 14%, vancomicina 13.5%, ciprofloxacino13%, amikacina in 13% and fluconazol 7.6% conclusion. sap cases in spanish icu's account for little more than 1% of all hospital cases, but they result in high levels of severity, morbidity and mortality. crude mortality and sap septic complication-related mortality in spanish icu's are much higher than the average indicated in the literature (29.7% and 14.9%). imipenem is the antibiotic most frequently used in prophlaxis. the irruption of candidas has been detected. fluid /blood warmers help to prevent hypothermia by raising the temperature of intravenously administered fluids & blood. the hl-90 hotline fluid warmer is the model used in our hospital. it consists of disposable tubing set with a central channel through which the fluid is infused and outer tubing through which heated water circulates. the water is contained in a reservoir, which is heated by an electric element. the manufacturer's instructions recommend changing the water in the reservoir every 30 days. this water is a potential source of infection and we therefore sampled the water in the reservoir for microbiological contamination. this study was conducted at royal london hospital during the month of december 2001.there are 10 fluid warmers, all hotline in our operating theatres. samples of water were taken from each of the reservoirs at the end of the working day. using aseptic techniques 20 ml of water were added to a labeled blood culture bottle. each sample was cultured for 48 hours. after one week we repeated procedure results. after 48 hours of incubation, pseudomonas sp. grown in 7 out of 10 culture bottles. the results from the second sets also grew pseudomonas sp. in the same 7 out of 10 water reservoirs.conclusion. the water in the reservoir is heated to 41-42 degree celsius. this temperature does not inhibit the growth of pseudomonas. each time the disposable tube is disconnected from the reservoir approximately 5 ml of water is spilled potentially spreading microorganisms. in addition there are case reports of cracks/splits in the inner tubing of the disposable tubing potentially exposing infused fluid or blood to heated water from the reservoir (1). methods. 161 (17.26%) of 933 consecutive cardiac surgery patients operated at onassis cardiac surgery center, from january 1st to june 30th 2001, developed t>38.5 c and leucocytosis, without evidence of specific site of infection. those patients were examined for possible catheter related infection, by removing central and arterial catheters and sending them along with blood specimens for culture. infections within the first postoperative 48h were defined as early, whereas those developed after the first 48h were defined as late. we examined the relation between the incidence of catheter related infection and the type of microorganism isolated, the type of operation performed, the icu stay and the hospital mortality. . 610 coronary artery bypass grafting(cabg), 202 valve or ascending aorta replacement(vr), 86 combined(cabg+vr), 13 acute dissecting aneurysm(ada) and 22 other operations were carried out. positive blood or catheter cultures were found in 36 patients (3.85%). staphylococcus epidermidis was cultured from all patients with early(n=6) and 83% of those with late(n=30) infection, while candida was found in 30% of those with late infection. icu stay and hospital mortality was ten times higher in patients with positive blood or catheter cultures compared to the general icu population (21.4 vs 2.1 days and 27.7% vs 2.36%, respectively). finally, mortality was higher in patients with late compared to those with early infection(30% vs 17%). (pts) who had suffered traumatic brain injury (tbi) as well as the immune response of these pts. pts with moderate to severe tbi (gcs =<11) and age >17 were enrolled under the presupposition they remained on mechanical ventilation (mv) >4 days. a total of 35 tbi pts were followed-up; infected pts were identified and associated factors were studied. in addition, serum immunoglobulin (sig) levels and soluble interleukin-2 receptors (sil-2r) were measured in infected pts. c. albicans species. candidemia in icu patients is associated with a high mortality rate. c. albicans was the most common yeast isolated from blood. non-c. albicans species have a frequent occurence among candidemic icu patients. the moderate susceptibility of azoles against non-c. albicans species indicates the usefulness of susceptibility testing for antifungal treatment. prospective, cohort, observational, and multicenter study. urine cultures were performed once a week to all patients admitted to the icu. samples were processed at the different clinical microbiology laboratories of the participating hospital using specific culture medium (sabouraud) and the bactec technique and the a20c (biomerieux) system for the identification of species. candiduria was defined as < 104 cfu of candida spp. in the urine. frequencies are expressed as cumulative incidence (%) and incidence density (episodes per 1000 days of urinary catheter). . results. a total of 1765 patients admitted >7 days to the 70 participating icus between may 1998 to january 1999 were included in the study. of these patients, 1730 (98%) had a urinary catheter inserted, with 40,273 urinary catheter days. one or more candida spp. in the urine were detected in 389 patients. the rate of candiduria was 22 per 100 patients/icu and the incidence density 9.5 per 1000 days of urinary catheter. in 23 cases, candida spp. in association with different bacteria (5.9%) were found, mostly gram-negative pathogens (13 cases), in particular p. aeruginosa (n=5) and e. coli (n=3), and gram-positive pathogens (10 cases) especially enterococcos (n=7). in respect to candida spp., c. albicans predominated (68.4%) followed by c. glabrata (8.2%), c. tropicalis (3.6%), c. parapsilosis (2.3%), and c. krusei (1.3%), independently of the week in which isolation of pathogens was made. conclusions: candiduria was diagnosed in 22% of critically ill patients admitted for more than 7 days in the icu. candida albicans was the pathogen most frequently recovered (68.4%), although c. non-albicans was isolated in one out of each three cases. a retrospective study was done over the last year that included 19 neonatal patients admitted at the intensive care unit. all patients had congenital anomalies (6 patients with gastroschisis (31.5%), 3 patients with esophageal atresia (15.7%), and others with intestinal obstruction, duodenal atresia and malrotation). 90.9% of the patients were on total parenteral nutrition and mechanical ventilation. the average stay in the icu was 9.6 days. candida albicans was checked for in swabs of wound, in blood-culture, stool-culture, urine-culture, tracheal aspirate, gastric asp results candida albicans was identified in 11 patients (57.8%). it usually appeared 3-4 days after the introduction of the antibiotic therapy. it was most commonly found in gastric aspirate (63.6%), stool-culture (36.3%) etc. it would first appear in gastrointestinal tract (stool-culture and gastric aspirate after 8 days). in respiratory and urinary tract candida was identified after 12 days, and in blood-culture after 18 days. 54.5% of the patients received cephtriaxon or ampicillin, and 36.3% amikacin or gentamycin and metronidazol. morbidity in patients with yeast infection is very high. the most common causative agent is candida, and the predilection organ is digestive tract. risk factors are: prematurity, mechanical ventilation, total parenteral nutrition, longer hospital stay and widespectrum antibiotics. due to unspecific clinical picture early diagnosis is usually made according to the results of taken cultures. there are still many dilemmas regarding systemic antimycotic profilaxys. key: cord-015372-76xvzvdg authors: nan title: national scientific medical meeting 1996 abstracts date: 1996 journal: ir j med sci doi: 10.1007/bf02945204 sha: doc_id: 15372 cord_uid: 76xvzvdg nan zero months months months months group a 5.53 5.09* 5.66** 6.11"* 6.26** 4 zero months group b 6.06 6.4** values are means; *p<0.05 and **p<0.0l~ most other studies show neutral effects on insulin sensitivity, with minimal incidence of glucose intolerance. this may be partly explained by the diversity of age, diagnosis (whether insufficiency or deficiency state), other pituitary deficiencies and replacement therapy, and possibly by dosage ofgh utilised in these studies. clostridium difficile-associated disease (cdad) is primarily a nosocomial condition. community-acquired disease has been described but the incidence is low "). during a recent outbreak, we prospectively reviewed all new cases of cdad to determine what proportion of cytotoxin positive cases were community or hospital acquired. during a 4 month study period, 73 cases were identified. history of diarrhoeal episodes were recorded for each case. selected isolates were typed using pyrolysis mass spectrometry (pms). community-acquired cdad was defined as diarrhoea, on or within 72 hours of admission, in association with a positive stool cytotoxin test for c.difficile and in the absence of hospitalisation within 60 days. sixty-five cases (89%) were hospital acquired. fifteen patients (20.6%) had cdad on admission; 8 (11%) were community acquired, 7 (9.6%) had been recently hospitalised (4 at st. james's hospital, 3 at other hospitals). pms typing of faecal isolates revealed that 2 predominant strains were responsible for the hospital outbreak; one of these strains was also isolated in community-acquired cases. this study suggests that the incidence of communityacquired cdad may be higher than previously reported. we suggest that all newly admitted or transferred patients with diarrhoea should be screened for this organism. several studies have confirmed that activated protein c resistance (apc-r) occurs in 20-60% of thrombosis patients and is therefore more common than congenital deficiencies in the inhibitors of coagulation such as atii1, proteins c and s. homozygosity for the factor v (fv) gene mutation is associated with a 50-100 fold increased risk of venous thrombosis while heterozygosity is associated with a 5-10 fold increased risk. the mutation, however, is highly prevalent in the general population, a prevalence of 5% has been reported in several european countries. its frequency in the population of northern ireland (ni) has not yet been reported. we screened a group of 72 generally healthy elderly individuals (av. age 81.3; range 76-97 yr on several occasions for apc-r using an assay based on the prolongation of activated partial thromboplastin time by the addition of apc. a mean ratio of 2.64 (range 1.72-3.46) was measured. seven individuals (9.8%) had ratios <2.3 (av. 2.11; range 1.72-2.28). these subjects were then analysed for the fv mutation by pcr amplification and restriction analysis. the 4 individuals with the lowest ratios (av. 2.0; range 1.72-2.15) were found to be heterozygous for the mutation. none of these 4 individuals were deficient for protein c, protein s or atiii. the frequency of apc-r (5.8%) within this ni elderly group is similar to that reported by others in the uk whose studies would have included a generally younger population. the successful ageing of these individuals perhaps underlines the low risk associated with heterozygosity. alternatively a higher prevalence of the mutation may exist in the general population of ni, where the incidence of heart disease is one of the highest worldwide. the insulin-like growth factor ii gene (igf2) is imprinted. thus, in contrast to most genes where both maternal and paternal copies (alleles) are transcribed into rna and expressed, 1gf2 is normally only expressed from the paternal copy (monoallelic expression). alterations causing biallelic expression of igf2 may lead to excess growth factor production, and thus, to tumourigenesis. this study evaluated igf2 expression in a series of childhood cancers. to date 41 tumours have been evaluated using pcr based methodology (26 wilm's tumours, 12 rhabdomyosarcomas, 3 miscellaneous). dna was extracted and a polymorphic site apal within the igf2 gene was amplified and digested. this identified 10 samples as heterozygous for igf2, meaning that separate maternal and paternal alleles were distinguishable. rna from these informative samples was extracted, pcr amplified and restriction digested, to identify monoallelic versus biallelic profiles at the expression level. samples with normal imprinting (monoallelic) displayed allele a (236bp) or allele bl/b2 (176/63 doublet). biallelic samples displayed both alleles. using this approach 3/5 informative wilm's tumours and 2/ 4 rhabdomyosarcomas demonstrated biallelic expression. in conclusion, biallelic expression of igf2 was detected in a significant number of wilm's tumours and rhabdomyosarcomas, and should be considered; with other genetic alterations, as a candidate mechanism in tumourigenesis. the proinflammatory cytokines, tnf~, il8 and il-6, may mediate host metabolic and immune responses to cancer possibly leading to paraneoplastic phenomena such as cachexia. the cellular origin of these cytokines in the cancer patient, in many cases, remains unknown. we examined proinflammatory cytokine levels intracellularly, using flow cytometry, in pbmcs from oesophageal cancer patients (n=14) and age and sex matched controls (n=10). tnf~ and il-6 levels were significantly increased (p<0.05) in pma stimulated t cells and monocytes from the cancer patients when compared to the healthy controls. these results were confirmed using standard elisa assays. following cotlagenase digestion, increased levels of tnfa and il-6,were detected in oesophageal tumour infiltrating t cells when compared to cells from normal mucosa. there was also increased production of tnf~ and il-6, but not il-ib, in malignant epithelial cells when compared to normal and halothane and maintained by 50% nitrous oxide-oxygen, 0.75-1.5% halothane, with spontaneous ventilation. tc rose marginally in group 1 and fell in group 2 (not significant, ns). tp in groups 1 and 2 at induction and 20 and 30 minutes were, respectively, (mean + sem, celsius) 31.4+0.33 vs 31.6+0.3, ns; 33.9+0.3 vs 33.4+0.3, ns and 34.5+0.4 vs 33.2+0.3, (p<0.05). overall incidence of shivering-was 14.%, but not significantly different between the groups. the data suggests that preemptive application of the space blanket increases tp in paediatric patients during general anaesthesia and tends to conserve tc. chronic actinic dermatitis (cad) an uncommon, eczematous, photosensitive eruption is diagnosed on history, clinical examination, skin biopsy and abnormal light tests. drug induced photosensitivity may look identical clinically, have a similar history and patients with cad may be treated with potentially photosensitising drugs. we therefore reviewed all patients with cad and compared their monochrumator light tests with patients who had drug induced photosensitivity. phototesting was performed on unaffected skin of the back with an irradiation monochromator; the minimal erythema dose (med) determined for a series of wavelengths between 300 and 400 nm, in 14 patients with drug induced photosensitivity and 7 patients with cad. of ten females, four males with drug induced photosensitivity, age range 40-77 (mean 61 yrs), ten (71%), were photosensitive in the uva range (320-370nm), the implicated drugs including, quinine, sparfloxacin, amiodarone, doxycycline, mefenamic acid, nalidixic acid, fenbrufen, diclofenac, enalpril and prochlorperazine maleate. three patients with rosacea were photosensitive to doxycycline. the re/nainder (29%), were tested after discontinuation of the drug and their light tests were normal. in the cad group, (four males and three females), age range 38-86 (mean 71.5 yrs), three patients (43%), were sensitive to uva, uvb and visible light, four (57%) to uva and uvb. in conclusion, uva dissociated from uvb photosensitivity seems a relative but not absolute sign of drug induced photosensitivity. this pattern of light tests should prompt a detailed drug history to elucidate the causative agent. phototesting should be performed while on the offending drug as testing days or weeks after discontinuation will give normal results. patients at risk of bone fractures by measuring bone mineral density (bmd) and markers of bone turnover and to assess the correlation of these with the severity of cld. twenty three patients with cld had bmd measured by dual-energy x-ray absorptiometry scanning of hip and lumbar spine. bone formation was assessed using serum levels of procollagen type 1 peptide, osteocalcin and bone alkaline phosphatase, and bone resorption was assessed using 2 hour urinary excretion of hydroxyproline, pyridinoline and deoxypyridinoline. 48% and 39% of patients had evidence of osteoporosis at the lumbar spine and femoral neck respectively. biochemical results showed that 74% of patients had an increase in all 3 bone resorption markers and 42% had an increase in markers for bone formation. bmd at the lumbar spine was lower in patients with cholestatic liver disease compared to patients with other types of'liver disease (p=0.004). no correlation was found between bmd and patient age, bilirubin, albumin, inr or duration of liver disease. conclusions: osteopenia occurs in up to 62% of patients with cld due to a high bone turnover state where bone resorption exceeds formation. osteoporosis is most severe in those patients with cholestasis. a detailed profile is presented of all 632 leukaemia and multiple myeloma (icd-o code 169) patients registered by the southern tumour registry during the 8-year period 1983/1990 "). annual age-adjusted rates of 13.9 and 8.7 per 100,000 were seen for males and females respectively. these levels indicate a lifetime (up to 75 yr) risk of 1 in 68 for males and 1 in 116 for females. the main morphological sub-types registered were multiple myeloma (31%), cll (25%), aml (18%) cml (9%) and all (8%). one, two and five-year survival rates were examined; age at diagnosis and lesion type were extremely significant factors in relation to patient outcome. the overall incidence levels indicate that irish rates were relatively high by internatiomil standards r we assessed effects of reducing the volume of hyperbaric bupivacaine by giving half the volume as isobaric bupivacaine. when using 0.5% hyperbaric bupivacaine for spinal blockade, the segmental spread and cardiovascular effects of the block have been shown to be dependent on the volume of local anaesthetic injected. 40 patients undergoing elective surgery were studied in a prospective, randomised, double-blind trial: group 1 (20 patients) received their local anaesthetic as two equal aliquots of 0.5% hyperbaric bupivacaine and 0.5% isobaric bupivacaine respectively; group 2 (20 patients) received their local anaesthetic as two equal aliquots of 0.5% hyperbaric bupivacaine. there was no significant difference found between the two groups with regard to maximal height of block (group 1, mean (range), t7 (t4-ti 1); group 2, t8 (ts-t11)), rate of onset of blockade, or time to maximal blockade (group 1, mean (sem), 23.55 (2.41) rain; group 2:20.89 (2.95) min). there was no difference found between each group in either cardiovascular stability or vasopregsor usage. the administration of a mixture of 0.5% isobaric bupivacaine and 0.5% hyperbaric bupiv/~caine confers no advantages over administration of the same volume of 0.5% hyperbaric bupivacaine alone. propofolis used as a sedative during regional anaesthesia. providing titrateable sedation, it can compromise haemodynamic stability. a propofol ketamine combination provides stable haemodynamics during total intravenous anaesthesia, avoiding emergence phenomena m. we compared two sedative regimes in patients having spinal anaesthesia. following informed consent, 40 patients, asa i-ii, undergoing spinal anaesthesia were randomized to one of two groups (n=20). group i (propofol-ketamine) received loading doses of 0.4mg/kg propofol, 0.1mg/kg ketamine followed by an infusion of 1.2mg/kg/h and 0.3mg/kg/h respectively. group ii (propofol) received bolus 0.5mg/kg and infusion 1.5mg/kg/h. subsequent infusion rates were titrated to effect using a sedation score. heart rate, blood pressure, oxygen saturation, end tidal c02 and oxygen requirements were recorded. observation continued for the recovery period and patients visited the following day. data were analysed using t-test, chi 2 test and anova. groups were demographically comparable. sedative and respiratory indices were similar for both groups. there was no difference in total propofol requirements between the groups; group i -146_+194mg, group ii -137_+1:52mg (mean _+ sd). there was a large difference in mean arterial pressure, being much lower in the propofol only group. both groups had an uneventful recovery without emergence phenomena. our results do not confirm the described additive effect of andketammc . ketamine.with propofol for sedation propofol ' =~2) confers haemodynamic stability during spinal anaesthesia. we designed a controlled study to investigate whether there is a direct relationship between the degree of postoperative pain and the development of negative middle ear pressure in adults following tonsillectomy. middle ear pressure was measured by tympanometry. pressures were classified as type a (o to -99 mmh20), type b (flat) or type c (-100 to -350 mmh20) tympanograms. 30 patients with type a tympanograms, undergoing tonsillectomy were enrolled in the study. patients had daily tympanometry whilst in hospital and then weekly until amrmalisatign. a questionnaire incorporating visual analogue pain scores was filled in at the same time. a control group of 26 patients with type a tympanograms, undergoing appendicectomy and endotracheal intubation was used. follo~v up was available on 26 patients. 13 patients (50%) developed type c tympanograms, 5 patients 09%) type b and 8 (31%) patients remained unchanged. no member of the control group developed any change in middle ear pressure (chi squared = 27.5, p < vol. 165, 1996 irish journal of supplement no. 2 medical science 0.001). there was no relationship between pain scores for throat pain or otalgia and the development of negative middle ear pressure. 8 patients recorded higher pain scores for throat pain at day 7 then day 1, only 3 of this group had negative middle ear pressure. middle ear pressure reverted to normal at day 7 in 15/18 patients and in the remaining 3/18 it was normal at day 14. this study demonstrates the development of transient negative middle ear pressure following tonsillectomy in 69% of patients. this change is unrelated to the degree of postoperative pain nor is it associated with otalgia. postoperative ward analgesia remains suboptimal. this may be partially related to inadequate early use of opioids to attain minimum effective analgesic concentration (meac). we examined the incidence and predictors of severe postoperative pain on admission and discharge from our postoperative recovery room (rr). verbal pain scores were obtained in a pilot study of 202 patients on rr admission and discharge. procedures were classed as open cavitary, laparoscopic, orthopaedic, ent or body surface surgery. intraoperative use and dosage of narcotics and nonsteroidal (nsaid) analgesics, anaesthetists' experience (prefellowship or post-fellowship nchd, consultant) were noted, and rr opioid usage recorded. pain scores and analgesic use were examined using mann-whitney, ~2 analysis and logistic regression. moderate or severe pain was experienced by 25% of patients on either arrival or discharge. median intraoperative morphine dosage was 5 mg. opioid use was slightly (median morphine dosage 8 mg, p < 0.05) higher in patients undergoing cavitary surgery; these patients had the highest pain scores on rr arrival and departure. 21 patients (10%) received > 10 mg morphine intraoperatively. discharge scores of 6/10 or higher occurred in 24 patients (12%). opioid usage and pain scores were unrelated to level of training. nsaid use/nonuse was unassociated with differences in opioid use or rr pain scores. no morbidity attributable to analgesic use (desaturation, slow respiratory rate) occurred. nonattainment of meac is frequent after open cavitary surgery. conservative opioid dosages continue to be employed despite inadequate early postoperative pain relief; this does not change with increasing experience. reporting such findings in departmental audit may help to alter perioperative management; such data may serve as a baseline for future interventional studies. diclofenac is frequently used for analgesia after tonsillectomy. recently concern has been expressed about the effect of diclofenac on prolonging bleeding time. one recent retrospective study found its use in tonsillectomy was associated with an increase in reactionary haemorrhage. we designed a randomised controlled study to compare the effects of rectal diclofenac and im pethidine given at induction with pethidine alone, in children undergoing tonsillectomy. fifty nine patients were entered into the study. there were 26 males and 33 females, mean age 6 years, range (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) . patients were randomised according to chart number. thirty five patients received rectal diclofenac after induction. twenty four patients acted as controls. there were no significant differences in operating time or operative blood loss between the two groups. in the recovery room the diclofenac group was significantly less restless than tl~e control group (p < 0.05, chi squared test), with less crying, movement and agitation. there was no difference in postoperative recovery and no primary or reactionary haemorrhage. one patient in the diclofenac group developed a secondary haemorrhage. this study demonstrates a significant reduction in restlessness in the recovery room in children receiving rectal diclofenac. no increase in reactionary haemorrhage was demonstrated. diclofenac remains a safe and effective analgesic agent in children undergoing tonsillectomy. elderly patients have decreased dose requirements for many drugs compared to the young. few studies have examined dose requirements of opioids in the elderly when administered via patient controlled analgesia (pca) 1. we compared the pca morphine requirements between young and elderly patients. records were retrospectively analysed from 1,000 consecutive patients receiving pca for post-operative pain. inclusion criteria (i) age less than 10 years or greater than 60 years, (ii) upper abdominal surgery, and (iii) morphine pca usage. 238 patients fulfilled the inclusion criteria. 89 patients were young and 149 were elderly. the mean age in the young was 29 years and in the elderly was 69 years. 51% were female in the younger group, 40% were female in the older group. pain scores at rest and on movement were similar in both groups 3.8 and 6.7 respectively in the young, 3.2 and 6.2 in the elderly. (p > 0.05, students t-test). morphine usage over 48 hours was 58 + 32.1mg in the young, and 37 + 20.3 in the elderly. (mean + s.d.) (p.< 0.05, students t-test). elderly patients required significantly less morphine via pca to achieve the same pain scores as the young. these findings are consistent with studies showing decreased requirements of other drugs in elderly. the erythrocyte sodium-lithium countertransport (slc) is abnormal in essential hypertension and some other forms of cardiovascular disease (cvd) but the considerable overlap in its activity in patients with these conditions and in normotensive healthy subjects remains a strong point against its possible utility as a marker for cvd. we sought to address this issue in greater detail. twenty-nine hypertensive patients (19 with family history of cvd) aged 56.4+1.9 years (mean + se) and 32 normotensive subjects (12 with family history of cvd) aged 45.9 + 1.9 years, participated after informed consent. slc were determined in 35, 50, 70, 100 and 140mm sodium chloride; and the vmax and km of the transporter determined. hypertensive and normotensive individuals with family history of cvd (n = 31) had higher slc activity (0.331 + 0.011 vs 0.238 + 0.011, p < 0.0005), greater vmax (0.473 + 0.019 vs 0.397 + 0.018 mmol/lcell.h, p < 0.006) and lower km 56.0mm (median) vs 95.5 (p < 0.001) than hypertensive and normotensive subjects without such a history (n = 30). however, none of these parameters was sufficiently discriminatory as evidenced by the considerable overlap in the scattergrams for the two groups. on the other hand not only was the median quotient vmax/km significantly different 7.89 vs 4.20 (p < 0.001), but also the scattergram separated the two groups. this may reflect an effect of hereditary factors on the identified rate-limiting step in the transport system. capsaicinadministration results in depletion of substance-p sensitive nerves. this study was carried out to observe the impact on the morphology of mitral valve endothelium. the experimental group received capsaicin i.p. on day four of life; control animals received drug vehicle only. animals were anaesthetised by chloral hydrate and hearts were removed following perfusion of 4% glutaraldehyde, and were routinely processed for scanning electron microscopy. normal endothelial morphology showed an ordered and structured pattern, with large raised nuclei covered in discrete microappendages: no zoning was observed over the valve surface. following capsaicin administration, valves were seen to be torn and possessed a denuded endothelium. nuclear bulges changed in both apparent height and area, with the surface partially denuded of microappendages. one month following systemic administration of capsaicin to neonatal rats, a serious alteration of mitral valve endothelium morphology and integrity had occurred. depletion of substance-p may have resulted in mechanical insufficiency of the mitral valve. this study was funded by the health research board. with expanding applications and increasingly aggressive stress protocols, concerns about the safety of dobutamine stress echocardiography (dse) have arisen. the purpose of this study was to analyse prospectively the safety, adverse event profile, and complication rate of dse. prospective data was recorded in a consecutive series of 309 patients undergoing dse for diagnostic evaluation of chest pain, for risk assessment following myocardial infarction or for detection of hibernating myocardium. the maximum dose of dobutamine used was 50 mcg/kg/min in 24.6% of patients and 40 mcg/kg/min in 73.1%. atropine was used in 6. 8% long-term outcome following coronary artery bypass grafting may be related to the prevalence of major risk factors and their treatment following surgery. we aimed to establish the prevalence and current management of coronary risk factors in a group of consecutive patients attending our hospital. this is a report of the first 100 patients. data was collected by a structured patient interview, chart review, physical measurements and blood sampling. there were 74 male and 26 female patients, average age 61.5 years. the mean length of time since surgery was 2.2 years. thirty-nine patients had a recurrence of angina and this occurred on average ll months after surgery. as regards risk factors, 22 were active smokers, 56 were ex-smokers and only 22 had never smoked. two thirds of the patients were taking regular exercise; only 6 took no exercise at all. seventy-two percent of patients had a cholesterol greater than 5.5mmol/l, yet only 10 of the patients were on lipid lowering drug therapy and a further 16 were on a lipid towering diet. twenty-nine patients had a systolic bp > 160 or a diastolic bp > 90 and 15 of these were on antihypertensive therapy. seventy-seven patients were overweight but most of these had received specific advice regarding weight reduction in the preceding year. our results show a high prevalence of treatable risk factors in this high-risk group with inadequate treatment in many cases. new combined primary and hospital care strategies for cardiac rehabilitation and long-term secondary prevention of coronary heart disease are required. if the goal of modern therapy of acute myocardial infarction (ami) is preservation of myocardium, the occurrence of cardiac failure could be regarded as a treatment failure. in recent studies of iv thrombolysis and primary ptca the frequency of left ventricular failure (lvf) following ami has been as low as 3.5%. this very low rate might be explained by selection bias in patients recruited to randomized trials. the purpose of this study was to examine the frequency of lvf in a consecutive alterations in nitric oxide (no) synthesis have been implicated in cardiomyopathy, ischaemic heart disease and septic shock. recent work has suggested a possible role for nitric oxide in cardiac arrhythmogenesis. the effects of inhibiting no synthesis with l-name (n c -nitro -l -arginine methyl ester) on cardiac electrophysiology have not been fully determined. the dominant frequency of electrically induced ventricular fibrillation was determined in l0 anaesthetised pigs (30-42 kg) using a fourier transform. the dominant frequency (7.8 _+ 0.5 hz in lead ii) was not altered by treatment (8.5 _+ 0.5 hz) with l-name in a group of 16 pigs (45-58 kg) the effect of l-name (10 mg/kg) was assessed in relation to energy required to defibrillate. there was no significant difference in the energies required to achieve successful defibrillation on 80% of attempts between the l-name group (104.9 _+ 8.0 j) and the control group (111.1 _ 11.9 j), and on 100% of occasions, l-name (122.5 + 11.5 j) and control (137.5 _+ 13.1j). the results show that inhibition of no synthesis has no significant effect on the dominant frequency of ventricular fibrillation or on the efficacy of defibrillation in the pig heart. received either 100mg (7pts) or 200mg (19 pts) oral flecainide followed by a maintenance dose of 100mg bd. all pts were euthyroid. none had significant hypertensive, valvular or ischaemic heart disease. 23 pts had normal echocardiograms, 3 had mildly dilated atria. of the 26 pts 17 (65%) converted to sinus rhythm, 15 within 72 hrs and 2 at 12 and 21 days respectively. 2 subsequently, he was found to have paralysis of all the muscle groups of his right upper limb apart from some flexor movements in his fingers with areflexia and sensory loss over the c5/c6 dermatomes. the findings were thought to be in keeping with a brachial plexus.lesion. an mri scan showed a "haematoma" or "fibrosis" around the brachial plexus~ emg studies revealed a complete lesion from c5/c7 with evidence of partial function at c8 and a good function at c4. despite physiotherapy, at follow up 2 months later there was no improvement in the emg findings. though brachia~ plexus injury has always been considered a complication of central venous line insertion <l), there have been no cases described, in the literature in the last 10 years. central lines are mandatory irt major surgical cases but one must always be aware of the possible complications caused by them. body weight (bw) in icu patients fluctuates due to alterations in body mass (1) and water (2) and is usually estimated rather than measured. we hypothesised that (i) estimated weights are inaccurate and (ii) changes in true body mass (tbm) are masked by changes in water content. on admission, icu patients whose expected length of stay (los) was > 5 days were placed on a bed incorporating 4 load cells (accuracy of _ 0.1 kg). bw was estimated by icu staff. mbw was then recorded 12 hourly under standard conditions. changes in mbw due to pack insertion/dressing changes were excluded. we calculated tbm as mbw minus cumulative fluid excess, corrected for insensible losses c2~. patients received standardized nutritional support from day 3 and urinary nitrogen on day 6 was calculated we studied 20 patients whose mean (sd) age, mbw, los and apache ii score were 44.3 yr (18.54), 70.5kg (14.39), 10.0 days"(6.78) and 18 (3.8) respectively. mean error in estimated weight on admission was 10.3% (nurses) and 12.2% (doctors). mean protein and calorie intake was 64g and 1670 kilocals/day. mean decrease in mbw during icu stay 0.22 kg/day (range 0.21 kg (gain) to 0.49 kg). mean reduction in tbm was 0.56 kg/day (range 0.28-0.86 kg/ day) and this correlated with urinary nitrogen loss (r=0.7). in icu, (i) estimated weight is significantly inaccurate and should not be used in physiological calculations and (ii) rapid and significant decreases in body mass occur which may be underestimated due to fluid accumulation. dopamine appears to influence pituitary function and is associated with decreased circulating human growth hormone and insulin-like growth factor i m which could exacerbate catabolism, and therefore wasting, during critical illness. at 12 hrly intervals from admission, patients whose expected length of stay exceeded 5 days, were weighed (measured body weight, mbw) under standardized conditions,-using a bed incorporating 4 electronic load cells (accuracy + o.1 kg). standard demographics, apache ii score and use of dot~amine by infusion was recorded. changes in weight due to removal./ insertion of prostheses, packs/dressings were excluded mbw was converted to true body mass (tbm) using measurements of cumulative fluid balance (1000 ml = 1 kg) and insensible lossr patients received nutritional support, starting on day 3, based on their admission mbw. there were no significant differences in mean age, weight, length of icu stay, admission apache ii scores and mean daily protein/calorie intake between 14 patients who had received dopamine by infusion (group d) and 18 who had not (group nd). mean (sd) decreases in mbw during icu stay were 0.42 (0.08) kg/day (group d) and 0.20 (0.05) kg/day (group nd) (p<0.0i). mean decrease in tbm was 0.55 (0.10) kg/day and 0.35 (0.07) kg/day respectively (p<0.05). thus group d were losing an additional 1.4 kg body mass per week relative to group nd. the use of dopamine by infusion is associated with an accelerated loss of lean body mass during critical illness. adhesion of polymorphonuclear leucocytes (pmns) to pulmonary endothelial cells is an initial step in the inflammatory process characterising the adult respiratory distress syndrome. previous studies using human umbilical vein endothelial cells (huvecs) have suggested that lipopolysaccharide (lps) is a potent stimulus for pmn adhesion to endothelial cells. the aim of this study was to investigate the effect of lps on pmn adhesion to human pulmonary artery endothelial cells (hpaecs). human pmns were coincubated with hpaecs _+ lps (0.001-10mg/ml) with 2% serum for 1 hour. the effect of phorbol myristate acetate (pma) (125 ng/ml) was also examined. percentage adhesion stimulated by pma was 66+10sd. lps did not significantly increase adhesion at any of the concentrations used. to confirm the activity of lps pmns were incubated at 37~ with 0.01-10mg/ml lps + 2% serum for 1 hour and labelled with flourescent antibodies to the mac1 adhesion molecule complex (cd 18/cd 1 lb). facs analysis indicated upregulation of both cd18 and cdllb. thus in the system used, lps did stimulate pmn adhesion molecule expression, indicating that the lack of adhesion reflects a difference in hpaec response to lps compared to that reported for huvecs. this work was supported by the health research board ireland. although inhaled corticosteroid therapy is of undoubted benefit in the management of asthma, dysphonia is a recognised sequela. this study was designed to examine longitudinally the effect of inhaled steroids on the voice and vocal cords of newly diagnosed and previously untreated asthmatics. twenty subjects were recruited and underwent voice and vocal cord assessment prior to and 3 months after starting inhaled steroid treatment. the assessment consisted of 1) rating dysphonia using a visual analogue scale, 2) acoustical analysis of the voice and 3) videostroboscopic examination of vocal cord activity. prior to commencing inhaled steroid therapy for their asthma 14 subjects had normal voices, 5 subjects were mildly hoarse and one was moderately hoarse. vocal cord pathology was noted in 12 subjects, 2 patients had vocal cord nodules and the remainder were noted to have mildly oedematous cords together with a glottic chink. at 3 month follow up, improvement in voice was noted in 2 subjects, one patient felt more dysphonic but there was no change in vocal cord appearance. one subject was noted to have developed a mid glottic chink with no associated change in voice. one subject had clearing of mild vocal cord oedema and improvement in voice. this study demonstrates that 60% of subjects commencing inhaled steroid therapy for asthma have mild vocal cord pathology. voice is more likely to be improved following use of inhaled steroids for 3 months then made worse. although the relationship between elastin degradation and emphysema is well known, recent evidence suggests that a more complex process of pulmonary remodelling occurs within the emphysematous lung. the aim of this study was to assess the extent of extracellular matrix remodelling by ultrastructural examination of its two major components, elastin and collagen. emphysema was induced in rats by the intratracheal administration of porcine pancreatic elastase (1.2u/g body weigh0 and human lungs were obtained at surgical resection for lung carcinoma. emphysema was confirmed histologically in both animal and human samples by measurement of the mean. linear intercept. matching sections were immersed in 2.5m naoh and 88% formic acid to digest elastin and collagen respectively. scanning electron microscopy with stereo-pair imaging allowed 3-d visualisation of elastin and collagen frameworks. the distribution of emphysema was primarily panacinar in rat lungs a'nd centriacinar in human lungs. as expected in both types of emphysema, elastic lamellae were disrupted and perforated with multiple fenestrations. accompanying this disintegration was a marked increase in thickness of collagen fibrils which in some cases coalesced irish journal of medical science imparting a sheet-like appearance to the airspace walls. unique to human centriacinar emphysema, collagen formed helices which spiralled around alveolar septae to form bulky walls between adjacent airspaces. in conclusion, these findings lend support to the novel concept of aberrant collagen remodelling in the pathogenesis of emphysema. small cell lung carcinoma (sclc) is the most aggressive of the four common cell types of lung carcinoma. less than 10% of patients with sclc are alive two years after diagnosis. staging procedures, treatment regimens and survival results were reviewed in a small regional centre to make a comparison with larger treatment centres. thirty-one cases of sclc seen by one physician from 1985 to 1993 were reviewed. staging was clinical. treatment was undertaken in conjunction with the local oncology and radiotherapy services. 42% of patients had limited disease where as 58% had extensive disease at diagnosis. in patients with limited disease, 20% were alive at 18 months and there was a 10% long term survival rate i.e. greater than three years. average length of survival in limited disease was 456 days. survival results were comparable with those treated with chemotherapy alone and combination chemotherapy and radiotherapy. in patients with extensive disease the best results were from those treated with a combination of chemotherapy and radiotherapy with art average survival of 353 days. these figures compare favourably with those from larger multidisciplinary centres. the factors contributing to our relative success may relate to continuity of care achieved in a smaller centre. nasal cpap is a very effective therapy for osa, but is cumbersome, and compliance varies. we prospectively evaluated 91 consecutive osa patients treated with ncpap, who were asked to complete questionnaires before, and 2 to 12 months after starting therapy. this stttdy intended to examine both the patient's subjective response to ncpap and their bed-partner's impressions also. replies were received in 84 (92%) patients. patients were divided i~to 4 groups depending on whether they had a bed-partner, and according to their response to an initiat question assessing overall improvement in sleep quality and daytime al'ertness with ncpap, ranging from 0 (minimal/none) to +4 (excellent). patients were called responders if they scored > 2. group a (45 pts, 54%) were responders with bed-partners; group b (10 pts, 12%) non-responders with bed-partners; group c (15 pts, 18%), were responders (12 pts, 14%) and nonresponders (3 pts, 4%) without bed-partners; and group d (14 pts, 17%) had stopped ncpap. nine questions were directed at the bed-partner, and assessed their perception of changes in both the patient's and their own sleep quality, daytime alertness, mood and quality of life, and also to changes in the relationship between patient and bed-partner following institution of ncpap. these questions scored from -1 (worse) to +3 (marked improvement). significant improvement in all parameters for the patient (mean + sd = 2.4 + 0.7) were noted in group a. in addition, group a bed-partners reported ~ubjective improvement in the same parameters (1.7 + 1.1). group b improvements were less, (1.8 + 1.0 in patients, and 0.6 + 1.1 in partners). overall, the data indicate a subjective success of therapy in 68% of patients, but the bed-partner's replies indicate this figure underestimates the true response rate. furthermore, the results show significant improvements in the bed-partner's sleep quality, daytime alertness, mood and quality of life, indicating that successful treatment of osa patients with ncpap also gives significant benefits to their bed-partners. vincent's hospital, dublin. neutrophil collagenase (mmp 8) is a member of the matrixmetalioproteinases (mmps), a family of highly homologous zinc endopeptidases which play a crucial role in many physiological processes. the aim of this project was to develop a purification system for mmp 8 from purulent sputum and raise polyclonal antibodies. after initial extraction, contaminating proteins were removed with a zinc chelate affinity column. mmp 8 was then separated, from another closely related mmp, gelatinase b, on a q sepharose ion exchange column using a nacl gradient. the final purification step was carried out with an orange sepharose affinity column. sds-page analysis indicated the presence of purified protein with bands corresponding to latent neutrophil collagenase (85kd) and products of coll~igenase autodegradation at lower molecular weights (55kd & 57kd). a 10fold increase in specific activity was observed, with a 20% final yield, which provided mg quantities of pure enzyme. this work is funded by forbairt and the. irish american partnership. cd30 is a protein first described as a surface marker on hodgkin's lymphoma cells. recently cd30 has been demonstrated on th2-type t lymphocytes (produce il-4, 5, 6, l0 and 13), which have a pro-inflammatory cytokine profile. but is not found on thl-type t lymphocytes (produce il-2 and ifn-gamma). its ligand, cd30l, has also been described, cd30-cd30l interaction has been shown to aid the development oft lymphocyte clones into a th2 rather than a th 1 phenotype. th2-type cytokines are inextricably linked to the aetiology of inflammatory airway disease. firstly we investigated serum cd30 levels in various patient groups. we have demonstrated significant differences in serum cd30 levels in the following groups, atopic asthmatics (mean = 149 iu/l, n = 62), non-atopic asthmatics (mean = 107 iu/l n = 13) and atopic rhinitis/dermatitis (mean = 90 iu/l n = 36) and normal controls (mean = 14 iu/l, n = 9). secondly we cultured peripheral blood mononuclear cells from allergic individuals and normals. when these cultures were stimulated with house dust mite antigen (der p 1) and il-2 or der p 1 with both il-2 and i1-4, surface expression of cd30 on t lymphocytes could be demonstrated using fluorescent staining and flow cytometric analysis, after 9 days culture in the allergic individuals but not in normals. the presence of i1-4 in the culture increased the degree of surface cd30 expression. these results are important as they show that allergic individuals have an expandable population of memory t lymphocytes which respond to allergen by expressing cd30 and developing th-2 phenotype. most work on cd30 and th-2 cytokines has hitherto been carried out an t cell clones. we have developed a relatively simple in vitro system of looking at t lymphocyte response to allergen which will allow the testing of novel therapeutic interventions with a view to modulating the immune response in allergic disease. our work also suggests that even non-allergic patients with inflammatory airway disease may have increased th2 activity, which has not been shown previously. scimitar syndrome is a rare congenital disorder consisting of a spectrum of abnormalities including hypoplasia of the right pulmonary artery, dextroposition of the heart, anomalous pulmonary venous drainage of the right lung into the inferior vena cava and anomalities of the right diaphragm. bronchiectasis and respiratory tract infections on the right side are the usual clinical presenting features. a 70 year old male patient was referred to the outpatient clinic with a history of recurrent chest infections which were slow to resolve following antibiotic therapy. physical examination revealed decreased air entry, coarse crepitations and a prolonged expiratory wheeze in the right lower lobe. the only abnormalities on routine biochemical and haematological screening were an elevated esr of 69 and a slightly raised white cell count of 15. a chest x-ray revealed hypoplasia of the right lung when compared to the left. in addition to this there was a vascular shadow present in the right lower robe representing an anomalous pulmonary vein which appeared to drain to below the diaphragm on the night side. bronchoscopy showed a normal left bronchial tree. on the right, no apical segment was detected in the right lower lobe. otherwise no endobronchial lesion was seen. a dynamic computerised axial tomographic scan of the thorax was performed. this showed a dilated anomalous right lower pulmonary vein which was clearly seen to enter the inferior vena cava below the diaphragm. in addition the right lung was again noted to be hypoplastic when compared to the left. these findings were pathognomonic of the scimitar syndrome. "the patient was treated symptomatically and is presently stable. conclusion: scimitar syndrome, with its wide spectrum of abnormalities should be considered when reviewing plain chest x-ray in patients with recurrent right lower lobe respiratory tract infection. recent studies indicate that the ability of circulating neutrophils to regulate surface levels of adhesion molecules may be altered in disease situations. the aim of this study was to determine if changes in neutrophil responsiveness accompanies chronic inflammation in cf. neutrophils in blood samples from 17 cf patients and 13 age-matched control subjects were analysed by flow cytometry for expression of l-selectin and mac-1 (cd1 lb) following stimulation by interleukin-8 (il-8) and fmlp. as expected, both il-8 and fmlp provoked a decrease in surface levels of l-selectin and an increase in cdi lb levels. however, the magnitude of these changes was significantly lower in cf patients than in control subjects (table) . these results suggest that chronic exposure to inflammatory stimulii in vivo may alter neutrophil responsiveness in cf. given the emphasis on rational prescribing, we reviewed drug use in a 208 bedded long-stay unit. prescribing patterns were analysed on an appointed day thereby obtaining a "snapshot" of prescribing practices. one hundred and ninety four long-stay residents, with a mean age of 78, were on 1188 drugs, the maximum number of drugs per patient was 12, the minimum 1 and the average 6.1. sixty percent of prescriptions fell within one of the following therapeutic categories:-central nervous system (cns) preparations (321 prescriptions), analgesics (145), gastrointestinal preparations (139) and cardiovascular preparations (i 25). there were 19 ,prescriptions for respiratory drugs and only 36 prescriptions were for antibiotics. the most commonly prescribed cns preparations were anti-psychotics (127), benzodiazepincs (1 t 3), anti-depressants (30). 43% of all analgesics prescribed (63) were nsaids. the most commonly prescribed h2 blocker was cimetidine (33). nuseals aspirin (43), digoxin (35) and captopril (24) were the most commonly prescribed cardiovascular drugs. 25% of drugs were issued on an as required basis, i.e. "prn". the most commonly prescribed prn therapeutic classes were analgesics (100 prescriptions) followed by gastrointestinal (74) and cns preparations (68). these results contrast with prescribing patterns in hospitals and general practice and may provide an insight into the challenges and realities of management in long-stay units. supported by the health research board. evaluation of physician requests to hospital based clinical pharmacist for (1) drug information, (2) possible adverse drug reaction (adr) was undertaken over a two year period from jan '94 to dec '95 (admissions -1,667, opd attendances -5,908). overall 55 requests were made. (1) drug information: advice/information on new drugs, formulation, dosage, safety consideration prior to drug prescribing was given in 23 cases. (2) suspected adr: a total of 32 suspected adverse drug reactions were investigated. in 4 cases, no adr link was established, after extensive literature/data base search. adr's were confirmed in 28 cases of which 11 were reported to n.d.a.b. regular on-going interaction between physicians and clinical pharmacy allowed critical analysis of new drugs and heightened awareness of, potential adverse drug reactions in current clinical practice. we previously demonstrated that commonly prescribed medications are not easily identified by patients, doctors or nurses in the hospital setting o,2~. we then investigated the ability of hospital pharmacists, 33 in all, to identify the same 12 commonly prescribed branded and generic drugs. correct identification as follows:-bendrofluazide k (32/33), cimetidine (33/33), diazepam 5mg (31/33), diazepam 5mg generic (30/33), digoxin (24/33), ferrous sulphate (14/33), frusemide (16/33), mefenamic acid (33/33), paracetamol (27/33), prednisolone (1 l/ 33), temezepam (33/33), theoph3/lline (25/33). pharmacists had 78% correct answers compared with 62% for nurses and 42% correct for doctors. the pharmacists had no difficulty recognising drugs with brand names written on them e.g. cimetidine, but like nurses and doctors had difficulty identifying the plain white tablets e.g. prednisolone. generic drugs were tess well recognised. a number stated that they were unwilling to definitively identify medication with no clear marking. pharmacists were also asked to list the top 5 prescribed drugs, l0 got 1/5 correct and 23 got 0/5 correct. in contrast 36 out of 80 doctors got 1/5 correct, 25 got 2 right and only 4 got 3 right. we conclude that hospital pharmacists are generally better than doctors or nurses at identifying commonly prescribed drugs but their knowledge of the top 5 prescribed drugs is not as good at that of doctors. all professionals need assistant in this important task. suggesting reduced activity or more iranians with inherited variants of cholinesterase. one iranian subject with very low activity (dibucaine number below 20, atypical) had a history of apnea. these data indicate that the frequency of atypical and heterozygote genes for cholinesterase activity leading to prolonged apnea with succinylcholine (suxamethonium) is much higher in iranian than irish populations. this study emphasises the importance of ethnic pharmacology. it is has been advocated that funding for the prescibing of methadone in general practice should be provided separate from the indicative drugs budgeting scheme on the assumption that this may act as a disincentive to g.p.s to take on care of drug addicts. the objective was to analyse the current level and cost of methadone prescribing in 550 general practices in the eastern health board over a six month period. there was a review of methadone prescriptions for gms patients from jan. to jun. 1995. 1,087 persons received prescriptions. 3,945 scripts were issued. the age-specific prescribing rate for the total population was 6/10,000 (males 10/i 0,000, females 3/10,000). males aged 25-34 years had the highest age specific rates (44/100,000). the cost of methadone prescriptions amounted to s for the six months there was a trend towards an increase in the number of g.p.s who prescribed methadone over the period. only four of the 70 g.p.s (5.7%) who prescribed methadone issued in excess of 50 scripts for the period studied. for a small number of g.p.s methadone prescribing is a significant cost item on their budget. in the light of this, government policy should be reviewed with a view to excluding methadone from the indicative drug budgeting scheme. sciences, mashhed, lran. there is increasing evidence that some of the wide variation in the response to medicines has a genetic origin which may be expressed on a racial basis. to further study inter-ethnic differences in pharmacology we compared the activity of an enzyme responsible for the breakdown of endogenous substances and drugs -serum cholinesterase (pseudocholinesterase), dibucaine and fluoride numbers -in 200 irish and 200 iranian healthy subjects. irish subjects had significantly higher serum cholinesterase activity (7.28 + 0.14 vs 5.22 + 0.09 u/ml, mean + sem, p < 0.01 drug prescribing data may reflect changes in therapy and disease pattern. we reviewed current drug use among patients (n = 578) in a dublin teaching hospital in "snapshot" fashion on a designated day, and compared it with that obtained in 1985. in 1985, patients received an average of 7.8 different drugs each, with 41% on 5 or more and 3% on none. by 1995, the average was 6.8 (range i -19), with 62% on 5 or more. the percentage of patients receiving heparin fell from 42% to 13%, due mainly to a reduction in use on the medical side. the proportion of patients prescribed hypnotics fell from 55% to 37%, while ssri's are now the most used anti-depressants. antibiotic choice changed from amp/amoxicillin to coamoxiclav and the cephalosporins. diuretics remained the most frequently used cardiovascular agents, accounting for 4% of all drugs used and prescribed for around one quarter of patients. digoxin use remained constant, and by 1995, 20% of patients were on anti-platelet doses of aspirin. at least four different agents were in use in each of calcium antagonist, beta blocker and ace inhibitor classes. some of these changes in therapy reflect therapeutic advances, changes in disease management, greater choice of therapy and amendment of less than desirable therapeutic practices. on the other hand, some may reflect fashion or pharmaceutical promotion, rather than change as a consequence of evidence-based practice. acknowledgements: pharmacy staff, st. james's hospital and the health research board. studies of in vivo endothelial function in humans have usually involved intraarterial cannulation and the subsequent administration of substances that stimulate the endothelium to produce nitric oxide (no). such techniques are invasive and potentially hazardous. an alternative non-invasive method would be of benefit. animal studies have indicated that reactive dilation of vascular beds may be at least partially endothelium dependent. this study aimed to determine whether reactive hyperaemia in the human forearm was an endothelial dependent process with the potential to be used as a non-invasive method of stimulating the endothelium. ten volunteers underwent brachial artery cannulation and randomly received either placebo or n-monomethyl-l-arginine (l-nmma) (8~mol/min), an no synthase inhibitor, for 10 minutes. following this reactive hyperaemia was induced by the inflation of an arm cuff to 200 mmhg for 5 minutes and the response to this was measured by strain-gauge plethysmography. when flows had returned to baseline the process was repeated with the remaining substance. results were analysed by repeated me~isures anova. l-nmma resulted in significant reduction of basal forearm blood flow (p<0.01). there was no significant difference in reactive hyperaemia with either l-nmma or placebo. in conclusion, no does not contribute to reactive hyperaemia in the human forearm. dublin 8. home-based infusion therapy has been widely recognised as the optimum for treatment of disease states that require daily intravenous therapy from a patient-care aspect. conditions necessitating intravenous therapies in hiv disease include: cmv retinitis, intractable cryptosporidial diarrhoea, azole-resistent candidosis, nutritional support with total parenteral nutrition, chemotherapy for aids-related malignancies and palliative care in the terminal phase of the disease. the need for such therapies is increasing as patient survival improves. in 1993, the home-infusion service was set up in recognition of the need to treat patients, requiring intravenous therapy, in the home environment. this has been brought about by the development of small, light-weight pumps suitable for ambulatory use, the development of a service for aseptic compounding and the availability of permanent in-dwelling venous catheters. we describe the impact of this service on our patient cohort. to date, sixty-five hiv positive patients have received parenteral therapy at home. patients' age, sex, risk group, cdc stage, cd4 count, indication for therapy, complication rate and response to treatment are described. the provision of this service has reduced the number and length of patient admissions with associated improvement of quality of life. in addition, it recognises that patients prefer to be treated in the home environment aided by a co-ordinated multidisciplinary approach. since blood alcohol levels over 80 mg/100 ml are now illegal for vehicle drivers we have investigated if the commonly held "safe" limit of two drinks will bring the young adult over the legal limit and if this amount of alcohol will affect their psychomotor skills. following informed consent 33 healthy volunteers, nonhabitual drinkers on no medication (16 male, 17 female), with a median age of 24 (range 20-27) years participated and refrained from alcohol for at least 2 days. each drank within 15 minutes two standard drinks (35.5 ml each ) of 37.5% vodka (2.7 units of alcohol) plus 60 ml of orange juice at about 90 minutes after a standard mid-day meal. their psychomotor performance was estimated by the number connecting technique at 45 minutes after alcohol consumption and they were also asked to rate their feelings (which included alertness, clear-headedness, competence and attentiveness) using a visual analogue scale of 1 to 10. blood samples at one and two hours later were collected from the antecubital vein and analysed on the same day for alcohol content using enzymatic methods. mean (+ sem) blood concentrations of alcohol at 1 and 2 hours respectively were 25.39 + 6.19 and 21.62 + 4.58 mg/100 ml. in males and 36.43 + 11.89 and 38.66 + 3.43 mg/l-00 ml in females. values were significantly higher in females. blood concentrations in females were also higher (p < 0.01) than in males when expressed per kg body weight. while the blood alcohol in both the genders was considerably lower than the current legal limit in ireland their psychomotor skills as estimated from their task completion time and their answers to questionnaires were indicative of an impaired cns function. thus while 2 drinks may keep many subjects below the legal limit, there is considerable inter-individual variation with females showing higher concentrations and both genders have evidence of impaired performance at these lower levels. a non-linear approach was used to develop an hrv parameter, robust to both data non'-st~itionary and missing data points. unlike the standard chaos approach, using higher dimensional embeddings and time-delays, we employed a onedimensional correlation integral plot. the parameter thus obtained, allows an estimate of the spatial spreading of the attractor (ssa) or spatial variation of rr intervals along a straight line. heart rate data from 18 volunteers (22:00 to 06:00 hr), ~ifter oral placebo or propranolol 80 mg, investigated the ability to detect drug effect. vitamin e (~-tocopherol) is the most important dietary antioxidant in lipid and cell membranes and its intake reversely relates to the incidence of coronary heart disease and certain cancers. estrogen regenerates oxidized tocopherol radical in vitro m but such interaction has not been investigated in postmenopausal women receiving estrogen containing hormone replacement therapy (hrt) although estrogen containing oral contraceptive may reduce plasma vitamin e levep. we studied 21 healthy post-menopausal women (aged 46 -56) ammenorrheic for at least one year. fifteen subjects took a combination of harmogen provera therapy and 6 acted as a control group. blood samples were taken from all subject at baseline and after 4 weeks. in the hrt group, serum fsh levels were greatly reduced (86.46 + 25.27 vs 68.67 + 11.59 iu/1, p < 0.04, mean + sd, after hrt) with an increased serum oestradiol level (< 20 -28.68 vs 603.67 + 343.56 umol/1, p < 0.0001). no change occurred in the control group. vitamin e status, measured either as plasma or red cell ~-tocopherol respectively showed no change in both groups (hrt group 26.48 + 5.42 vs 27.17 + 3.39, 23.79 + 5.19 vs 24.93 + 4.37 gmol/1, p > 0.05). we conclude that in post-menopausal women, 4 weeks estrogen containing hrt did not alter vftamin e concentrations in vivo. we assessed the clinical benefit of the newer markers of bone formation: osteocalcin (oc), procollagen 1 carboxyterminal peptide (picp), bone alkaline phosphatase (balp), and bone resorption: carboxyterminal telopeptide of type 1 collagen (ictp) and urinary deoxypyridinoline crosslinks (dpd) over traditional assays such as total alkaline phosphatase (talp) and urinary hydroxyproline (oh/pr) in 23 patients with primary hyperparathyroidism (phpt). patients were sampled basally, then at 2, 6, 24, 48 and 72 hours post surgery and again at 1.5, 3, 6 and 12 months post op. the mean basal p1cp level was 89+26 ug/l (normal: 38-202) this increased to a peak at 48 h (168+_74 ug/l), then declined to normal at 6 weeks (116+56 ug/l). mean basal urinary dpd levels were raised at 8.6+1.2 nm/mm cr. (normal 2.0-6,0), they had normalised by 6 months to 5.9-+1.4 nm/mm cr. mean balp levels were always normal, although normal the yearly mean oc level was significantly lower than the basal value. mean 1ctp, oh/pr and talp levels were always normal. therefore bone turnover in phpt is best assessed by the newer markers picp and dpd. we have previously described seasonal variation in fibrinogen with higher levels in winter. as fibrinogen is an acute phase reactant, the winter rise may be a response to seasonal infections. the present study investigates this hypothesis by examining seasonality infibrinogen and markers associated with infection: white cell count (wcc), interleukin-6 (1l-6), human herpes virus 6 (hhv6) and herpes simplex virus (hsv) antibodies. monthly blood samples from healthy volunteers age 75 and over were measured for fibrinogen, wcc, il-6, hsv and hhv6 reactivation over a 1 year time period. a rhythmometric method was used to examine the data for seasonality. statistical significance was measured using the fstatistic. a highly significant seasonal variation (sv), peaking in mid-february, was found for fibrinogen (n=24; sv=0.629 g/ 1; f=76.148; p<0.01). no significant seasonal variation was present for measures of wcc (n=24; sv=0.209 e9/l; f= 1.940; p>0.05), hhv6 (n=24; sv=0.065 au; f=0.653; p>0.05), hsv (n=7; sv=6.055 au; f=2.048; p>0.05) or il-6 (n=7; sv=1.235 pg/ml; f=0.558; p>0.05). the present investigation does not support the hypothesis that seasonal variation in fibrinogen is a direct effect of the acute phase response, initiated by a seasonal variation in level of infection. the explanation for the seasonal changes in fibrinogen remains unknown. increased plasma homocysteine and reduced plasma antioxidants are risk factors in the development of vascular disease. design: 131 subjects drawn from elderly people living in the community (median age 85 yr, range 60-102 yr; 81 female). total plasma homocysteine, vitamin c, gamma tocopherol, retinol and beta carotene were measured by high pressure liquid chromatography. homocysteine levels in elderly males [median (range) = 12.35 um (4-18.1), n=241 were significantly higher than in vol. 165, 1996 supplement no. 2 irish journal of medical science elderly females [8.45 um (4.8-24.8), n=30]. these values were also higher than in a younger (30-49 years) male cohort [mean = 7.85 um, n= 610]. no correlations to vitamin concentrations were found, nor was there a correlation to age within the elderly cohort. within the elderly females, a significant negative correlation with age was found in vitamin c, gamma tocopherol and beta carotene (p<0.05). however a significant increase in retinol was noted. a very strong correlation between vitamin c and gamma tocopherol levels was noted in the elderly population sample (p<0.001 after multiple regression). conclusion. homocysteine levels in the elderly are higher than in samples of a younger population. a gender difference is maintained in the elderly. the provision of extended care forms one part of a spectrum of health care for older people. in the eastern health board area all patients over the age of 65 must be assessed by the multidisciplinary geriatric team prior to placement. we report on the experience of the total number of referrals for assessment for extended care to one department of geriatric medicine in a 268 bed teaching hospital. ninety-eight patients listed for extended care in 1994. the mean number of days between listing for long term care and placement was 49 _+ 45 days (range 3 to 206). almost one quarter of patients died while in hospital awaiting long term care: this underlines the frailty of patients who are admitted to hospital and request long term care. two patients were transferred to other institutions and 10 patients were able to get home. of the remaining patients 35 (63%) were placed in statutory or voluntary long term care accommodation and only 37% were eligible (usually financially but in some cases due to significant disability) for nursing home care using the terms of the 1993 nursing home act. patients who are listed for long term care through a general hospital are in general very frail, they tend to have a very extended length of stay and the provisions of the nursing home act only apply to a minority. these findings underline the need for provision of adequate statutory and voluntary extended-care places within the eastern health board area. there are over 40 screening assessments for cognitive function and choosing the most appropriate may be difficult. increasingly the importance of behavioural dysfunction is recognised. can any of the cognitive assessments help to predict behavioural dysfunction.'? we compared and contrasted the folstein mini mental state examination (mmse) and the cognitive assessment schedule (cas) of the clifton assessment procedures for the elderly (cape) and compared them with the behavioural rating scale (brs) of the cape. the study was carried out on a total of 21 referrals to the occupational therapy departments by geriatricians in the meath hospital and st. james's hospital. all subjects were over 65 and medically stable. the time scale involved was may-july 1995. the mmse and the cas were administered within the one sitting and each was timed. brs was rated the same day by either a staff or family, member. the average time to complete the mmse (416 + 124 s) was longer than the cas (342 + 171 s) but this was not statistically significant. the mmse and cas were significantly correlated (r = 8208, p < 0.0001). the cas was significantly correlated with the behaviour scale (r = 0,551, p < 0.01) whereas the mmse was not. these results suggest that equivalent assessments of cognitive function may be made with the mmse or cas, but a low cas score will be a better prediction of behavioural dysfunction. a spectrum of neurological and myopathological changes are associated with patients in intensive care units. we observed several patients post discharge from icu who presented with unexplained dysphagia which we suspected may be associated with the neurological complications of sepsis. the particular complication of dysphagia as a neurological manifestation of sepsis has not been documented. our descriptive study presents a series of three patients with persistent dysphagia which may represent a similar phenomenon. we selected patients for the study ranging from 75-87 years of age and on the basis of medical history including icu stay, sepsis, and intubation. all patients presented with dysphagia as observed on videofluoroscopy. we studied the video findings in-depth in order to ascertain if similar swailow patterns were present in these patients and if this could be correlated with their medical history. each of the three patients presented with similar dysphagia signs. the oral phase of the swallow was moderately atypical but the pharyngeal phase was significantly atypical. it was felt that intubation alone was not the sole causative factor of this dysphagia. the polyneuropathy associated with sepsis in icu may explain the atypical swallow patterns observed in these patients. the severity of the persistent dysphagia can cause serious respiratory and medical consequences. there is a need for further investigation of this phenomenon to identify patients who are at risk. little attention has been paid to the prevaience and phenomenology of behavioural disturbances among medical patients despite awareness of the high prevalence of cognitive vol. 165, 1996 supplement no. 2 impairment in this patient population. we screened 79 consecutive admissions to a department of acute geriatric medicine. patients were evaluated over a 2 week period using a modified version of the brief agitation rating scale. medication use, cognitive function and impact on nursing time were also measured. the prevalence of behavioural disturbance in this population was 19/79 (24%). the most frequent behavioural abnormalities were restlessness (12), complaining (12) and screaming (6). the most common underlying disorders were dementia, stroke disease, personality disorder and paranoid psychosis. the behavioural disturbance was only documented in the medical notes in 7 patients (37%) and in only 5 cases was a psychiatric consultation sought. these findings demonstrate that behavioural disturbances are not only common but also under-documented in elderly medical patients and there is a need for training in the detection and management of behavioural symptoms in this patient group. in lower limb trauma where there is severe compound fracture, the successful treatment of this depends on adequate bone and soft tissue debridement. as a result, subsequent bone defects can lead to instability and often require large amounts of bone grafts, and major soft tissue reconstruction is reaquired to obtain skin cover. large soft defects can by reduced bv primary bone resection and shortening of the limb. this will improve the chance of bone healing if performed in the presence of an external fixator, then lengthening at a site away from the traumatised area can gradually restore limb length. two cases are presented to demonstrate .the effect of compression / distraction techniques on soft tissue and bone injuries in these difficult situations. wegener's granulomatosis -wg (15), churg strauss syndrome -css (4), polyarteritis nodosa -pan (2) and unclassified (5). using the chc definitions, the diagnoses were wg (5), microscopic polyangiitis -mpa (10), pan (1) and undefined (10). there was concordance in only 5 patients (all wg). there is significant discordance between these two criteria sets. since the acr criteria does not recognise mpa, they tend to overdiagnose wg. in addition, the chc criteria cannot be applied without a biopsy and therefore surrogate features which predict the underlying histology are required to allow more practical application of the chc definitions. the objective was to determine the value of examination of dried freshly produced saliva, under light microscopy, in patients with xerostomia related to secondary sjogrens syndrome. ten patients with known connective tissue disease or rheumatoid arthritis attending rheumatology clinic were enrolled into the study, all with symptomatic xerostomia and dry eyes. all had an abnormal schirmer's test. five normal patients were enrolled, all of whom were without clinical evidence of rheumatological disease. control patients were enrolled who had no clinical evidence of rheumatological disease, a salivary sample was collected and examined by light microscopy. serum was also examined for the presence of anti-ro/la, rheumatoid factor, and anti-nuclear factor. all ten patients demonstrated 'reindeer horn' type ferning of saliva, a pattern of shorter thicker clubbed branches of crystallised mucus, in contrast to the normal ferning pattern of the healthy subjects. conclusion: we have shown in this preliminary report that light salivary microscopy is a simple test easily performed in an outpatient setting which could be a useful diagnostic procedure in sjogrens syndrome. recently, two sets of criteria have been proposed for the nomeclature of primary vasculitides, the 1990 american college of rheumatology (acr) classification criteria and the 1992 chapel hill consensus conference (chc) definitions. the aim of this study was to determine the concordance of these two systems in a cohort of patients with primary systemic vasculitis. patients with systemic vasculitis were recruited who had a biopsy proven diagnosis or, who had typical clinical features associated with a postive antineutrophil cytoplasmic antibody (anca). the case notes were reviewed and patients were classified according to both sets of criteria. twenty-six patients were recruited, 21 of whom had a positive biopsy. applying the 1990 acr criteria, the diagnoses were, primary pulmonary hypertension (pph) typically affects young individuals, and has a high morbidity and mortality. secondary pulmonary hypertension complicating connective tissue diseases likewise carries a poor prognosis. we evaluated the acute and chronic effects of ketanserin, a selective serotonin type-2 receptor antagonist in 2 patients with pulmonary hypertension in the acute study ketanserin was administered as a peripheral venous infusion during right heart catheterisation. following encouraging results during catheterisation oral administration of ketanserin 160mg daily in divided doses was instituted. in patient 1, a 30 year old female with probable pph, serial cardiac catheterisations over a 1 year period showed a significant, sustained reduction in both mean pulmonary artery pressure from 49 mmhg at baseline to 24 mmhg at 1 year (normal 10-20 mmhg) and pulmonary vascular resistance 1118 units at baseline to 288 units at 1 year (normal <200 units). in patient 2, a 61 year old female with limited scleroderma (crest) echocardiography after 1 month's oral ketanserin showed a reduction in estimated peak right ventricular systolic pressure from 60 mmhg at baseline to 13 mmhg (normal range 15-30 mmhg). the acute and long term response to ketanserin with improyement in pulmonary haemodynamics in these patients suggests that if a beneficial effect is detected during catheterisation long term oral therapy may be worthwhile. levels were low (< 7.9 iu/1); normal though above average (>-8 -<10 iu/l) and moderate high (>10 -<15 iu/l) respectively. gonadotrophins for ovarian stimulation were commencing initially at 225 iu for group a & b and at 450 iu for group c. ivf performance was poor in most aspects (total follicles, oocytes & embryos transferred) in group b comparing with group a or c; the cumulative ongoing pregnancy rate (pr) over 2 ivf cycles in group b; was 15% comparing with 27.6% in group a (p < 0.05) however there was no significant difference in pr in group c (20%) comparing with other two groups. cycle day 3 fsh screening is predictive of follicular development in ivf. high initial dose of gonadotropins help to improve the pregnancy rate in the presence of moderate high level of fsh. the purpose of this study was to evaluate patient satisfaction with antenatal care provided in the perinatal day centre (pndc). a self administered questionnaire was administered to 61 consecutive patients. the main indications for referral were suspected small-fordates (34%), non-proteinuric hypertension (23%), glucose tolerance testing (15%), reduced fetal movements (10%) and post-term evaluation (7.5%); 51% were nulliparae. thirty-two percent of patients were reviewed in the pndc on the day of referral; the rest within 7 days. twenty eight percent of patients lived more than 10 miles from the hospital and 48% spent more than 30 minutes in travelling there. eighty five percent of patients scored their level of satisfaction with the service provided in the pndc as > 7 out of 10; only 6.5% would have preferred admission; 42% said that they would prefer to visit the pndc 5 times per week to avoid admission. the main area of dissatisfaction related to the waiting time for review prior to discharge, with 69.5% of patients waiting over 3 hours. patients attending the pndc report a high level of satisfaction; changes to reduce the visit duration have been introduced. to examine the change of taking-up the essential preconceptual measurements; rubella immune status, cervical cytology and prophylactic folic acid intake; following specific advice and publicity through 3 general public meetings with new patients prior to in vitro fertilization (ivf) programme. in 1994 we studied 281 new couples for ivf for the presence of some specific pre-conceptual data (group a). in this study we follow-up the same intake in another 229 new women interviewed to commence ivf programme from january 1995 till september 1995 (group b). in group (b) the taking-up measurements were dramatically improved. however, 30% and 18% stilldid not have rubella immunity test and cervical cytology performed; compared to 54% and 36% in group (ai respectively (p<0.001). while folic acid intake was sustained at >90% in both groups. following specific advice the rate of taking-up of preconceptual measurements prior commencing ivf programme was improved. there is a future need for continuous enhancement of the publicity and advice regarding the importance of preconceptual measurements. the aim of this study was to introduce icsi to ireland for treatment of specific cases of male factor infertility. following an introductory proving period using the bovine model, thirtyeight couples with infertility attributed to the male were selected for an icsi attempt. ovulation induction, oocyte retrieval and luteal management were as described for conventional ivf tm. the average age of patients selected for icsi were 34.5 + 3.7 years and 36.4 + 4.6 years for the female and male respectively, with an average duration of infertility of 4.8 + 2.6 years. a 64 year old woman presented with a three day history of parasthesia in her lower limbs and difficulty walking. neurological examination revealed sensory loss in her limbs and truncal ataxia. rombergs sign was positive. pelvic examination revealed a large pelvic mass that was distinct from the uterus. routine blood investigations were normal. csf culture, ct brain and serum electrophoresis were negative. anti-purkinjie cell antibodies were not present. ca-125 levels were elevated at 159 micrograms/litre. laparotomy revealed a 20 cm left ovarian tumour. a total abdominal hysterectomy, bilateral salpingo-oophorectomy and omentectomy was performed. histology revealed a poorly differentiated clear cell adeno-carcinoma of the left ovary. the capsule was intact and peritoneal washings were negative. she made a good postoperative recovery. she received six courses of carboplatin without ill effect. her neurological symptoms resolved. subacute cerebellar degeneration can occur as a paraneoplastic disorder in ovarian carcinoma. the mechanism by which cancer can cause neurological disorders is not fully understood. paraneoplastic cerebellar degeneration occurs with or without the presence of purkinjie cell antibodies. the aim was to review all red cell transfusions in gynaecological surgery in 1994. a retrospective review of blood bank records and individual charts was carried out. 4611 patients underwent gynaecological surgery; 97 (21%) were cross matched and 60 (1.3%) were transfused. 184 units were transfused. there were no single unit transfusions. the mean number of units transfused per patient was 3. this accounted for 33% of all units transfused this year. 92% of patients were undergoing elective surgery. the overall cross match/transfusion ratio was 2. intraoperative difficulty was recorded in 56% of cases. 64% of patients were transfused perioperatively and 36% postoperatively. the percentage of patients requiring blood transfusions in the the main individual operation categories was as follows: radical surgery: 57%; total abdominal hysterectomy and salpingo-oopherectomy, 17%; vaginal hysterectomy and repair, 16%; subtotal abdominal hysterectomy, 30%; vaginal hysterectomy alone 12.5%; and total abdominal hysterectomy alone 5%. adverse reactions to transfusions were seen in 5% of patients. conclusion: the majority of patients transfused were undergoing elective surgery. vaginal hysterectomy was associated with greater blood loss than abdominal hysterectomy. only half of all units cross matched were transfused. dilatation and curettage (d+c) is the most common operation performed in the u.k. the liberal use of d+c has been criticised. the objective of this study was to evaluate the use of outpatient endometrial pipelle biopsy and determine its safety in terms of detecting abnormalities. complications and financial costs were also evaluated. data was reviewed from an active gynaecological unit from february 1993 to january 1995, using theatre and outpatient records. a total of 303 d+cs and 104 endometrial pipelle biopsies were performed in this period. 9 malignancies were detected by d+c and 1 by pipelle biopsy. a total of 24 and 3 benign abnormalities were detected by each method respectively. there was a higher complication rate in the d+c group but the failure rate was higher in the endometrial pipelle biopsy group. the monetary savings over this period is estimated at s there were no missed malignancies to our knowledge over the 8 year period since endometrial pipelle bioposy was introduced to this hospital. our study indicates that outpatient endometrial pipelle biopsy appears to be safe, efficacious and economical. while ultrasound findings may sometimes be in conflict with clinical examination, it is the case that there are instances when ultrasound findings have, following subsequent laparotomy, been found to be wholly incorrect. it is therefore not surprising that there remain some gynaecologists who view ultrasound with scepticism, preferring to rely solely of their clinical findings. there have been few studies that directly compare clinical, ultrasound and surgical findings in the detection of pelvic masses. the objective of the study was firstly to directly compare the reliability of clinical and ultrasound examination findings in the detection of pelvic masses proven by subsequent laparotomy and secondly to determine the accuracy of ultrasound in detecting malignancy. this was as a retrospective review of 86 women who underwent a laparotomy because of a pelvic mass between january 1993 to february 1995. information was obtained from theatre and patient records. real time abdominal ultrasound was used. findings at laparotomy were correlated with clinical and ultrasound findings. the sensitivity and specificity of ultrasound in detecting a uterine mass was 94% and 99% respectively. this contrasts sharply with clinical examination (sensitivity = 74% and specificity = 94%). similar findings were obtained when ultrasound was compared to clinical examination in detecting ovarian masses. ultrasouud is capable of predicting benign disease with reasonable confidence but the prediction of malignancy is less reliable. in conclusion, ultrasound is more sensitive and specific in detecting pelvic masses compared to clinical examination. vincent's hospital, dublin. osteoporosis occurring during pregnancy or lactation is a rare event despite the homeostatic demands of the foetus for calcium. we investigated the case of a 24 year old woman who, immediately following vaginal delivery of her first child, developed severe back pain due to a vertebral compression deformity of the second lumbar vertebrae. bone mineral density (bmd) was measured by dual-energy x-ray absorptiometry. calcium metabolism and bone turnover were studied. there was a severe reduction in bmd in the spine (z-score = -4.8) and f e m o r a l neck ( z -s c o r e = -3.6); but, serial measurements showed no further reduction in bmd. indices of calcium metabolism and bone turnover were normal. pregnancy-induced osteoporosis is a severe but self-limited disorder in calcium homeostasis of unknown aetiology. women with low bmd prior to pregnancy may be at increased risk. in view of increased demand, supplemental calcium and vitamin d should be considered during pregnancy and lactation. crumlin, dublin 12. the aim of this study was to assess the clinical status on admission and the critical care m a n a g e m e n t of children p r e s e n t i n g with m e n i n g o c o c c a l i n f e c t i o n . t h i s was a retrospective study of the charts of 46 consecutive admissions. mean age was 3.43 years (+3.46). the average duration of symptoms prior to admission was 20.4 hours (+11.09). on admission 17.4% were hypotensive, 45.6% had clinical signs of haemodynamic instability and 54.8% of cases that had a wood gas analysis on admission had a metabolic acidosis (bases excess < -5.0). the mortality rate was 10.9%. 80% of deaths were hypotensive on admission and all had a metabolic acidosis. of the 41 survivors 9.7% were hypotensive on admission, 39% had clinical signs of cardiovascular compromise, 78% were admitted to the high dependency unit, 25% required invasive pressure monitoring and 7.3% were ventilated and received inotropic support. in this study children presenting with m e n i n g o c o c c a l infection have a high incidence of cardiovascular instability. successful management is dependent on early presentation and initiation of therapy and on aggressive intensive care monitoring and support of the cardiovascular i11 and vital organ systems. the normal crying curve and incidence of colic for term infants are well known. we studied prospectively the crying pattern and the incidence of colic in preterm'infants to determine if prematurity influenced these behaviours. the subjects were consecutive preterm infants admitted to the cork neonatal units for two and a half months from july 1995. a continuous 24 hour diary was completed on each infant by the neonatal nurses, when the babies were on full oral feeding and no longer required intensive care. the parents completed the diaries 'after discharge. colic was defined according to wessel's rule of threes. two unwell babies were excluded. the duration of follow up was from 2 to 16 weeks. fifty infants were recruited and 45 completed the study (4 lost to follow up and one withdi'awn due to sepsis). their mean (range) gestational age was 31 (26-36) weeks and birthweight was 1.52 (0.64-2.4) kg. the mean (range) age of crying onset was x(y-z) weeks; crying, peak 'was x(y-z) weeks and crying offset was x(y-z) weeks. one baby developed colic in the period of follow up. conclusions: the incidence of wessel's colic was less than expected in these preterm babies. the crying pattern according to chronological age was different from that clescribed in term babies. in general preterm babies had a delayed onset of crying, but the pattern became similar to term babies when allowance was made for gestational age. the findings'suggest that the crying patterns of early infancy have a developmental basis. we re-evaluated 18 children who had had rs (1979-86), with their closest-age siblings using the wechsler scales, coopersmith self esteem inventory and achenback child behaviour checklist (acbc) (duffy j. et al 1991). the rs patients' means were consistently lower than that of their sibs. however, comparison of mean raw data, using "t-tests", yielded significant differences only in the acbc scores, in that rs children exhibited significantly more problem behaviours than their sibs (p=0.033). after categorisation of iq data, further comparisons between the groups (using x2), found rs patients were significantly more likely to score "below average" in tests of verbal iq compared to their sibs (p=0.04). age of onset and clinical stage were also found to be more important predictors of outcome. children less than 1 year of age at onset of rs had significantly lower iq scores on all measures of cognitive ability (p=0.003) and more problem behaviours (p=0.01) than children over 1 year of age. no significant differences were found in comparison with sibs. clinical stage to which rs progressed affected only verbal iq scores. children in whom consciousness had been impaired had significantly lower iq scores than both their sibs and rs children in whom consciousness was less impaired (p=0.02). in conclusion outcome remains cautiously positive, with 13/18 rs children attending mainstream schools or in employment without apparent difficulties. a national breastfeeding policy was introduced by the department of health in 1994. factors identified for promotion of breastfeeding were based on the who/unicef "ten steps for successful breastfeeding". we present clinical cases which suggest that one step may need to be modified. the charts of breastfed babies admitted to the special care baby unit were reviewed for one year following the introduction of the national breastfeeding policy to this hospital. thirteen term breastfed babies were admitted because of fever and dehydration. none of the babies had water or bottle feed supplements. ten of the thirteen mothers were primigravida. eleven babies were admitted in the six months following the introduction of an exclusive breastfeeding policy. the nursing staff were then alerted to the risk of dehydration, but two further babies of mums committed to exclusive breastfeeding were admitted in the subsequent six months. routine biochemistry, haematology and a limited septic screen was performed in all babies. three of the thirteen babies had lumbar punctures. the mean (range) weight loss on admission was 9.5 (4.1-16)%. the mean (range) plasma sodium level was 147.6 (138-156)meq/1 and the mean (range) urea was 6.5(2.1-13.4)meq/t. there was no growth from the cultures of the blood, urine, csf and swabs. all the babies were given intravenous fluids and parenteral antibiotics for 48 hours. the outcome was satisfactory in all babies and breastfeeding was reestablished in eleven of the thirteen babies. conclusions: the common factor to these babies was inadequate fluid intake prior to admission associated with stricl~ adherence to the policy, and avoidance of all supplements including water. we conclude that the who/unicef step 6 "not to give food or drink other than breastmilk unless medically indicated" is too restrictive in the immediate postpartum period. 42.5% of children reported headache in the previous 12 months 44.8% of girls and 40.5% of boys reported headache (p < 0.0001). 0.6% of children reported daily headache 5.9% of children reported weekly headache 9.4% of children reported monthly headache 22.5% of children reported headache less often than monthly the percentage of children with headache at each frequency, other than daily, increased with increasing age. in girls headache showed a marked rise at ages 14, 15 and 16 years. reported prevalence of headache in the past year in 5 to 15 year old aberdeen children was 66% and 48% was recorded for swedish children aged 8, 11, 13 and 17 years old. this study is the first community based prevalence study of headache in irish schoolchildren. our aim was to test the hypothesis that there is no correlation between the type of feeding & swallowing disorder the child has with the neurological diagnosis or the radiological findings. a further purpose was to develop a classification of the feeding & swallowing disorders which would guide us towards a management plan. a retrospective analysis of the data collected between the years 1988-94 from the feeding & swallowing clinic at booth hall children's hospital was done. 73 children were included in our study 9 ages ranged from 4 months to 17 yrs. all the children were assessed by the members of the feeding & swallowing team and had videofluroscopic assessment by the same radiologist. neurological signs, speech therapy assessments & videofluroscopy findings were compared between children with spastic quadriparesis & those without. significant differences were noted. a clinical classification was devised using cluster analysis. we conclude that there is no causal relationship between the neurological diagnosis & the type of dysphagia. there are three distinct groups of children who require different strategies of clinical management. surveillance commenced in january 1995 to continuously monitor the incidence of surgical site infections (ssi). employing modern optical scanning technology (formic for windows version 2.0, formic limited, london) a questionnaire was designed, which required minimal completion time. the questionnaire includes relevant data based on the american national nosocomial infections surveillance system for ssi including the ssi risk index. surveillance commences at the time of surgery and continues until the patient's discharge. optical scanning technology allows rapid reading of surveillance questionnaires thereby bypassing the bottleneck of manual data entry. by october 1995, details of 2,156 procedures had been recorded. the crude ssi rate for these patients was 2.7%. the patient risk index used demonstrated that there were increased chances of developing ssi in certain patient groups. seventy-nine per cent of ssi had presented by the 10th post-operative day. the length of stay increased by an average of 4 days in patients developing ssi. regular feedback to individual surgeons, theatre and ward staff maintains awareness and highlights possible problems. we recommend optical scanning technology to all those engaged in surveillance work. this system would be especially useful were data collected is transported from outlying hospitals to a central receiving centre for collation and analysis. in 1872 francis crumpe published a paper ~ in which he described the therapeutic effect of poisonous mussels (psp) on a case of tetanus. he obtained the mussels from tralee ship canal on the occasion of its infrequent emptying, and entertained the idea of using them in tetanus after treating a young girl with psp who recovered after 24 hours. prior to the use of psp he described it's paralytic effect in two cockrels who both recovered. in concluding his successful use of psp he speculated as to the clinical nature and role of the toxin tralee ship canal was opened in 1846. the water was relatively stagnant and would have contained plenty of nutrients. as such it would have been an ideal habitat for toxic algae which may have been brought across the atlantic as spores in bilge water 2. the emptying of the canal may well have been done at times of algal blooming. this is the first irish report of psp and a most remarkable use of saxitoxin (?) in the treatment of tetanus, antedating the current management by seventy years. this study was carried out to quantify the published research on smoking in irish medical journals; to ascertain the type of research carried out; and to identify the authors of that research. during the 35 years under study, 50 papers explicitly dealing with smoking were published. only 2 papers appeared in forum. there was a decline in published papers in the eighties with a resurgence in the early nineties. of the 50 papers, a majority were observational and ten were editorials. only one paper dealt with smoking cessation, and one with preventive work. general practitioners were poorly represented as authors. one doctor (prof. r. mulcahy) published at least one paper on smoking in each quinquennium since 1960. this study underlines the relative insignificance of smoking as a topic for research in ireland. a major sea change in attitude will be required if the government's targets for smoking cessation are to be realised, particularly if they are to be achieved by relying on the medical profession. radiology represents a major cost centre within a hospital. lack of awareness of cost amongst doctors may result in the inappropriate requesting of radiology services. this study assesses doctors knowledge of the cost to a tertiary referral hospital of commonly performed radioj'ogical procedures and investigations. doctors were asked to estimate the cost of 9 items namely -chest,x-ray, arch aortogram, ultrasound abdo., lumbosacral spine, barium enema, ct brain, ct abdo., ultrasound abdomen, i.v.p. and percutaneous gastrostomy tube insertion under radiological screening. 60 doctors in st. vincent's hospital were surveyed. doctors as a group overestimated the cost of all 9 individual tests, by margins ranging from 10% (i.v.p. & gastrostomy insertion) to 100% (ct brain/ct abdo.) the total cost to the hospital of all 9 items was s consultants'overestimated this total cost by 107%, followed by registrars, interns and s.h.o's who overestimated the total cost by margins of 51%, 43% and 12% respectively . conclusion: doctors tend to overestimate what radiological procedures and investigations cost a public hospital, often by quite wide margins. thus, any excessive requesting of radiology services by docto.rs is not due to a lack of awareness of their true cost to the hospital. (ded) in dublin. secondly, to identify the major cancers contributing to years of potential life lost (ypll) for the ehb, each cca and ded in males and females. in ireland little work has been done to date on disease specific premature mortality. crude death rates weight all deaths equally; in comparison, ypll emphasise deaths among younger persons and provide a measure of the burden of premature mortality. premature mortality for the 322 deds in dublin for the years 19988 and 1989 was estimated using ypll, which was calculated by subtracting the date of death form 65. ypll due to each of the major disease groups were ranked for each ded. seventy-one thousand four hundred and sixty-eight &471468) years of potential life were lost in dublin in the 1988 and 1989. 21.9% of ypll was due to injury and poisoning, 20.64% to cancer, 15.6% to circulatory disease. however, when the major cause of ypll was established for each ded, injury and poisoning was the number one cause of death in 25.5% of the deds; cancers 24%, congenital and perinatal conditions 21.5% and circulatory disease 14%. by emphasising deaths in younger individuals ypll is a valuable tool for planning and monitoring local health promotion initiatives. serum total homocysteine (thcy) levels are inversely associated with dietary intake of folic acid and b vitamins and raised levels have been linked with chd. we have examined the association between thcy concentration and the risk of chd in middle-aged men in 18 british towns. we used a nested case control study design, within an ongoing prospective study, thcy concentration was measured in serum samples, stored at entry to the study, from 110 incident cases of myocardial infarction and 118 controls. cases and controls were frequency matched by town and age group. levels of homocysteine [geometric mean (95%c1) were significantly higher in cases than'controls: homocysteine 13.5 (12.6 -14.3)gmol/l vs 11.9 (11.3 -12.6)lamol/l; p = 0,005. there was a graded increase in the relative risk (odds ratio; or) of chd in the 2nd, 3rd and 4th quartile of thcy (or 1.4, 1.9, 2.2; trend p = 0.006) relative to the first quartile. adjustment for age, town, social class, body mass index, smoking, physical activity, alcohol intake, hypertensive status, serum cholesterol and serum creatinine did not attenuate this association, (or 2.1,2.3, 2.7; trend p = 0.04). the findings suggest that thcy is an independent risk factor for chd with no threshold level. in summer 1995, a diagnosis of cryptosporidiosis was made 25 in a child who had visited a pet farm. this child had participated in a summer project involving 161 children and nine adults. reports of a similar illness among other project members, prompted an outbreak investigation. a cohort study consisting of two phases was initiated. 95% (162/170) of project participants responded to a self-administered questionnaire in the first phase. thirteen children met the case definition, of whom seven had cryptosporidium detected in their stools. illness was significantly associated with having visited a pet farm. (p<0.000005). 75% of those ill sought medical attention, of whom two were hospitalised the second phase of the cohort study was conducted among those who had visited the pet farm. 95% (52155) were interviewed. illness was significantly associated with play in sand, to which animals had access, at a stream's edge beside a picnic area (p<0.005). contact with various animals was not statistically significantly associated with illness. however the small numbers involved may have obscured such an association. this outbreak highlights a potential hazard for children visiting pet farms and also that cryptosporidiosis is a significant but often overlooked cause of morbidity in healthy children. managers of pet farms need to be aware of the potential for transmission of disease to visiting children. strict implementation of hygiene measures is essential to minimise risk. the mrc vitamin trial highlighted the importance of folic acid in the prevention of neural tube defects(l). since 1993, the department of health has recommended periconceptional folic acid supplements. the objective of this study was to document the knowledge and behaviour of women in child bearing years to periconceptional folic acid. a cross sectional community survey was conducted using an interviewer administered questionnaire in dublin. three hundred and thirty five women took part in the study. approximately two thirds 213/335 (63.6%) had heard of folic acid. knowledge was significantly associated with higher social class and higher education (p<0.o5). few 18/335 (5.4%) had been advised to take folic acid before pregnancy. only 9/335 (2.7%) of the women in the study were currently taking folic acid supplements. three quarters of the group (75.9%) would be willing to take periconceptional folic acid supplements if they knew it would reduce the risk of malformations. the majority (77.4%) would prefer to take folic acid in tablet form. this study clearly shows that few women in childbearing years have been advised on folic acid. however, if advised appropriately the majority would be willing to take periconceptional folic acid in tablet form. future publicity campaigns involving all health professionals should address these issues. unstable intra-articular fractures with or without dislocation of the phalangeal joints often lead to joint stiffness ana loss of function. nine patients with comminuted intra-articular phalangeal fractures were treated in our unit by dynamic external fixator using "pins and rubber bands traction system". the mean age was 32.3 years, and the follow-up average was 4.6 months. five patients had full and good range of motion in the involved joints. three patients had poor results, and one patient underwent open reduction one week following the original procedure. the technique and our results are discussed. this dynamic frame is compact, comfortable for the patient, easy to apply and allows early mobilisation. careful selection of patients and close follow-up in the first few weeks are needed. this study examines how gp's store and handle vaccines. all 144 gp's in a health board region were invited to take part. gp's were interviewed in their practice premises about how they dealt with vaccines fridges were examined and temperature recorded post interview. oral polio was taken from 20 randomly selected fridges for potency testing. cold chain monitors and freeze watch indicators were used to monitor batches of vaccine stored. of the 144 gp's, 142(98.6%) agreed to participate, 140 used 111 fridges to store vaccine, 2 store vaccine at room temperature. of the 111 fridges, 6 (5.4%) had the power supply safeguarded, 9 (8.1%) had thermometers, 36 (32.4%) had vaccine only stored therein. during defrosting, vaccine was inadequately protected in 22 (19.8%). of the 138 gp's who use multi-dose vaccine vials, 133 (97.2%) keep them for further use at the end of a day/session, 3 store them at room temperature. 42 (37.8%) fridges had temperatures outside the recommended range. 13 (28.2%) coldchain monitors indicated vaccine exposed to more than 10~ 18 (90%) of the oral polio samples showed a reduction in total titre of live virus, however, none were below the minimum acceptable. this study indicates that vaccine potency could be seriously compromised due to breaks in the cold-chain and suggests the need for guidelines to be drawn up, implemented and monitored to ensure the integrity of immunisation schemes. comparison was made with a study carried out in 1983. in addition the range of antimicrobial agents tested included new oral cephalsporins and quinolones that were not then available. three hundred microorganisms isolated from mid stream urine (msu) samples were examined by standard microbiological techniques. antimicrobial susceptibility testing to 12 antimicrobial agents was performed on significant pathogens (>105 organisms per ml) by disc diffusion test, and minimum inhibitory concentrations of antibiotics was carried out by e test on organisms found resistant by disc testing. by comparison with 1983 resistance amongst e.coli, the most commonly isolated pathogen, had increased for the following: ampicillin by 6% to 61%, co amoxyclav by 4.4% from 0%, trimethoprim by 18% to 28% and nitrofuradantin by 5% from 0%. no increase in resistance occurred to cephradine (4%), or nalidixic acid (5.4%). resistance to cefixime, ofloxacin and ciprofloxacin, was 3%, no resistance was encountered to cefotaxime. for proteus species resistance to ofloxacin, ciprofloxacin and cefotaxime was 4%, and for enterobacter sp 20%. enterococci were sensitive to ampicillin and augmentin but the numbers were small. pathogens isolated from patients domiciled in the inner city were significantly more resistant to nalidixic acid (20%), cefotaxime (6%), cefixime (14%), ofloxacin and ciprofloxacin (12%) than those isolated from patients in rural areas. the purpose of this study is to examine the relative importance of obstetric complications (0c%) in the aetiology of schizophrenia and mania. using the dublin psychiatric case register, birth records of 635 patients with an icd-9 diagnosis of schizophreniaor mania were obtained. these records were evaluated, for obstetric complications using two scales, the lewis, owen and murray scale (lom) it~ and the parnas scale 121. the mothers of those going on to develop schizophrenia did not differ from those going on to mania as regards maternal age, parity, social class, or period of pregnancy. however, males who developed schizophrenia when compared to males developing mania, experienced significantly more oc's when rated by the lom scale (p=0.007) and.more frequent oc's on the parnas scale (p<0.001) of greater severity (p=0.018). no significant differences were found between females with schizophrenia and those with mania. dublin. the aim was to evaluate the diagnosis, symptomatology and level of functioning of patients presenting with a first episode of psychosis to a catchment area service and a private psychiatric hospital. all patients presenting with a first episode of psychosis were assessed using the scid-p,,the positive and negative syndrome scale (panss) and the global assessment of functioning scale (gaf). fifty-eight patients (34 male, 24 female) ranging in age from 15 to 65 years (mean + sd = 28.4 + 10.8) were included in the study. the mean total panss score was 84.8 (sd + 21.6) and was strongly correlated with the gaf score (p < 0.001) but independent of age (p = 0.16). males had a significantly lower gaf score compared to females (p = 0.04) but there was no gender difference in the total panss score (p = 0.20), twenty-five patients (43%) had a lifetime prevalence of drug abuse or dependence but only 12 patients (21%) had signs of drug abuse or dependence in the month prior to presentation. level of functioning was strongly influenced by the severity of psychopathology. substance abuse is common in individuals presenting with a first episode of psychosis. the aim was to evaluate the presence of involuntary movements in patients presenting with first episode psychosis to a catchment area service and a private psychiatric hospital. patients presenting with first episode schizophrenia and schizophreniform psychosis were assessed for involuntary movements using the involuntary movements scale (a.i.m.s.). 28 patients (17m., llf.), age range 15-67 years (mean=30.5 years.) were included in the study. one patient (3.5%) satisfied the strict criteria of schooter and kane for spontaneous dyskinesia. 7 patients (6m., if.), had minimal involuntary movements in at least 2 body areas, predominantly orofacial. the total a.i.m.s. score was positively correlated with the number of days spent in hospital per year of follow up (p=0.01). the group with involuntary movements were found to have spent more days in hospital per year of follow up, 94 v. 55 days (p=0.047). for patients with first episode schizophrenia or schizophreniform disorder spontaneous dyskinesia is not common. however involuntary movements at presentation may be a predictor of poorer outcome. psychiatry has moved from custodialcare towards care in the community. adequate reprovision will have to be made in order to discharge the remaining continuously hospitalized patients. the objectives of this study were to describe a.n entire long-stay hospital population, to examine the differences between the old and new long stay groups within this populatian and to evaluate the needs for community residential and day care facilities in order for hospital closure to take place. the study group consisted of the total long-stay population of st. davnet's hospital, monaghan. the patients were assessed using the community placement questionnaire (cpq) one hundred and twenty four patients were included in the study. fifty-six were female and 89% were single. the mean age of the total group was 68.7 years. the majority suffered from schizophrenia. the assessment revealed a globally disabled group with multiple handicaps. the new long-stay group were disabled as the old long-stay group. the patients were characterised into four groups with regard to placement recommendations. these were a specialist unit for chronically disturbed geriatrics, a geriatric unit, a high support hostel and a medium support hostel. the remaining population of this hospital were highly dependent with multiple handicaps but would live in community with adequate support. there is little difference between the needs of the old long-stay and those of the new long-stay. failure of the immune system to identify self peptides is likely to lead to the development of an autoimmune reaction. susceptibility to autoimmunity is strongly influenced by genes clustered in the hla region (chromosome 6p) particularly class i (a, b and c) and class ii (d, q and p). it has been suggested that there is an autoimmune component in the aetiology of schizophrenia. of many conflicting reports from case/control studies using hla antigens the most consistent finding has been an increased frequency of hla-a9 (now split into a23/a24). additionally, a susceptibility locus for schizophrenia has been reported near the hla locus. to attempt to confirm the hla a9 hypothesis, we have genotyped a preliminary sample of 63 familial schizophrenic probands and 77 unrelated controls at the hla-a region, using a pcr-ssop technique. the frequency of hla-a24 (the major component of a9) in patients and controls respectively was 12.5% vs 15.5%. these findings do not support the hypothesis. some of the discrepancy may be due to unspecific cross-reactions produced by commercial antisera used in the microlymphocytotoxicity method of previous studies. however it is also possible that the hla associatton with schizophrenia may reflect linkage disequilibrium with unidentified gene(s) within the hla region which is less strong in the irish population. schizophrenia is a common mental disorder affecting about 1% of the general population with a devastating disturbance of mind and personality. family, twin and adoption studies have demonstrated that the disease is largely genetic with a polygenic mode of: transmission. dopamine receptors have been implicated in the aetiology of the disease. as yet 5 dopamine genes have been identified (d1-d5). in particular the d3 receptor is expressed in the limbic regions of the brain, implicated in the control of emotions. association studies of a d3 polymorphism (glycine to serine substitution at position 9,) with schizophrenia have produced conflicting findings, many of which, however, have demonstrated a significant excess of homozygosity, or excess of the 1 -l genotype at this polymorphism. in this study, 200 familial schizophrenics and 239 irish unrelated controls were genotyped. the result show a small increase in the frequency of the 1-1 genotype which did not attain statistical significance (patients, 41.5% vs. controls, 38.5%). homozygosity (alleles 1-1 and 2-2) was also slightly increased in the patients (patients, 55.5% vs. controls, 48.9%). the small increase in the frequency of the 1-1 genotype and of homozygosity in the patients is in keeping with earlier findings but suggests that the effect, if any, of d3 sequence variation in the development of the disease is small. lack of information about general practitioners' (gp's) ability to prescribe psychotropic medication may affect patients' compliance. in this study, 73 out of 75 patients attending a psychiatry out-patient clinic completed a questionnaire which documented how many had run out of medication, the steps taken if they had and the role each patient thought their gp played in their treatment. 82% indicated that their gp knew what their current medication was but only 46% thought that their gp could provide them with a prescription if they did not have one from the clinic. this figure was similar in those who had (21%) and had not (79%) run out of medication in the past. on running out of medication, 42% of patients waited until their next appointment, 29% attended their gp and the remainder either contacted the department or went to a chemist. in conclusion, many patients do not appreciate the entitlement of their gp's to prescribe psychotropics for them. literature regarding whether or not the social class distribution of patients with psychiatric illness may differ from the general population remains controversial. we sought to clarify this by examining social class at the time of birth, to see whether patients with serious psychiatric illnesses (schizophrenia and mania) differ from the general population. paternal occupation of 450 schizophrenic patients, and 77 manic patients, from the dublin psychiatric case register, were obtained from birth registration details and categorised according to central statistics office criteria, the same-sex previous live birth was used as a matched control. there was no difference between the social class of patients with schizophrenia or mania (p=0.32). neither patients with schizophrenia (p=b.31) nor mania (p=0.153) differed from controls in social class distribution. paternal social class was found to be related to amount of time spenr in hospital (p<0.001', mean=439.62), and educational age (p<0.001, mean=15.01) and "age at onset of the illness" (p=0.03, mean=31.59). these results suggest that social class of origin may not be related to the development of either schizophrenia or mania. however, social class of origin may be relevant in terms of presentation of schizophrenia for treatment. cognitive function is widely recognised to be impaired in schizophrenia but there is an ongoing debate as to whether this impairment is generalised or localised, progressive or static, similar in both sexes, or related to symptoms. using the positive and negative syndrome scale (panss)'we measured psychopathology in 48 chronic in-patients (27m, 21 f; mean age 68.0+12.7) who satisfied feighner criteria for schizophrenia. subsequent to this, we assessed their global cognitive function using the mini-mental state examination (mmse) and their frontal cognitive function using a new instrument, the executive interview (exit). poor performance on the exit was associated strongly with increasing severity of negative (r=-0.74, p<0.001) but not positive (r=--0.09, ns) symptoms,'in both males (r=--0.73, p<0.001) and females (r=--0.76, p<0.001). overall exit performance declined modestly with increasing age (r=--0.32, p<0.05) but this phenomenon was co'nfined to females (r=--0.64, p<0.01; males: r=--0.13, ns). mmse performance was also associated with negative symptoms (r=--0.67, p<0.001) but decreased mm:e prominently with age (r=--0.59, p<0.001) and showed no gender difference. frontal dysfunction in schizophrenia appears to be intimately related to negative symptoms over the course of severe chronic illness, and may reflect among males a more static' trait deficit than is accessed by the mmse. this study was supported by the stanley foundation. while determinants of the course of schizophrenia are unclear, emerging evidence suggests that the longer psychosis proceeds unchecked before initiation of anti-psychotic therapy, the poorer may be long-term outcome. we have reported that, among older in-patients, increasing duration of initially untreated psychosis in the pre-neuroleptic era was associated with a deterioration to a state of muteness (after controlling for intervening variables). the current survivors of this population have now been examined more extensively using the positive and negative syndrome scale (panss), the mini-mental state examination (mmse) and the executive interview (exit). among these 48 patients (mean age 68.0+12.7), after controlling for age and for the duration and continuity of subsequent antipsychotic treatment, increasing duration of initially untreated psychosis was associated with greater severity of negative symptoms (p<0.005) and with lower scores on the mmse (p<0.05) but not with executive dysfunction on the exit (p=0.3). increasing duration of initially untreated psychosis appears to be associated with the evolution of more prominent negative symptoms and cognitive impairment in a manner consistent with an active, morbid process in schizophrenia that can be ameliorated by anti-psychotic drugs. this study was supported by the stanley foundation and the health research board. patients who are selected and who agree to participate in the royal college examinations play an important role. as psychiatrists and exam organisers, we should be aware of the potentially stressful experience which this might present. the purpose of our study was to elicit attitudes to the exam, and also knowledge of the examination procedure. a questionnaire comprising 10 questions was circulated to 30 patients who had participated in the royal college examinations. responses were received from 28 (93%) of the 30 patients. there were 14 males and 10 females in the responding group. none of the patients had previously participated in the examinations. all of the respondents (n=28) felt that the candidate had been polite towards them during the interview. 38% (n=9) of the patients were nervous prior to the examination, and this group was predominantly female (n=6). 21% of the patients (n=5) did not know that they would receive payment for their participation. 67% (n=l 6) did not know that they might be physically examined as part of the examination procedure. 17% (n=4) of the patients described experiences which had been upsetting for them the results of our study suggest that on the whole patients tolerate the exam procedure quite well. one of the central issues concerning physiotherapists in stroke rehabilitation is the emergence of abnormal tone. rehabilitation involves re-establishing a normal postural control mechanism (ncpm)"~. abnormal tone may develop in the presence of severe sensory and proprioceptive loss. the patients' attempts to move and find a stable base can lead to compensatory movement patterns and asymmetrical postures. positioning is used by physiotherapists to influence the distribution of muscle tone and facilitate symmetrical postures. it is essential that the patient is made aware of his 'position in space' as failure to do so presents no feedback regarding movement resulting in inertia ~2~. standardised positioning charts have been used in hospitals. the physiotherapist liaises with nursing staff regarding correct use on a 24 hour basis. this study looked at the role of a more individualised approach to positioning in the form of a photograph. patients were randomised to two positioning groups group a standard vs group b photograph, and their positioning was scored by a 'blinded' research physiotherapist over an eight week period. the results of a pilot study on a small number of patients revealed that nursing staff preferred a positioning chart individually tailored to the patient's problems. from a physiotherapy perspective, improved postural awareness correlated with better positioning scores in group b. the prevention of compensatory movements, posture is critical in stroke rehabilitation, the use of an individualised positioning chart requires further evaluation. we discuss the case of a fourteen year old boy who presented with bilateral ptosis present since birth, microcephaly and pigmentory retinopathy"!. he was found to have mild facial and proximal limb weakness. creatinine kinase and ldh were raised. muscle biopsy showed ragged red fibres consistent with a mitochondrial myopathy ~2~. electron microscopy showed abnormal mitochondria. the term mitochondrial myopathy describes a diverse range of clinical disease ~3~ and this is discussed. she developed a vasculitic skin rash with pruritis and oedema associated adenopathy, low grade fever and mouth ulcers. lab tests showed leucocytosis, eosinophilia and abnormal liver function. skin biopsy indicated an inflammatory picture without vasculitis. ct thorax confirmed axillary and para-aortic adenopathy. lymph node biopsy confirmed a reactive lymphadenopathy. the aim of. this study was to assess the characteristics of patients referred for pudendal nerve studies over a one year period. 31 consecutive patients were asked a standard questionnaire and nerve studies were performed as described by kiff and swash m and swash and snook~2k of the 31 patients, only 3 were male. the age range was from 21 to 79 (mean 50). 10 presented with constipation and 19 with faecal incontinence. two had both symptoms. bladder incontinence was presented in 10 of 31 patients. of these, faecal incontinence was the cardinal symptom in 8 patients, constipation in 2. 12 patients were nulliparous. of the remaining 17, 14 had a history of complicated births involving forceps (7), caesarean section (4), post partum haemorrhage (1), breech without forceps (2) . 14 patients had pelvic surgery and one had major trauma. 22 of 31 patients had bilaterally delayed pudental nerve terminal motor latency (pntml). 7 of 31 people had unilaterally delayed pntml, 2 were right sided, 5 were left sided. 2 had normal studi~s. the range of measurements was 1.8 -9.2 ms with a mean of 3.65 ms. in conclusionl delayed pntml was seen in 11 of 12 patients with constipation and 19 of 20 with faecal incontinence. pelvic surgery and a complicated obstetric history were significant. urological symptoms were also a common association. the p50 component of the middle latency-auditory evoked potential is attenuated in response to the second of paired clicks in a normal population. in schizophrenia, this attenuation is minimal. in alzheimer's disease (ad), the results have varied between centres depending on the frequency of the stimuli and the interclick interval. we studied 10 ad patients, 10 elderly controls (ec) and 10 young controls (yc) using a paradigm of 32 sets of paired clicks. in contrast to previous studies, our study demonstrates significantly larger absolute p50 generation and recovery amplitudes in ad patients compared to elderly controls and young controls. the purpose of study was to establish a simple screening vol. 165, 1996 irish journal of supplement no. 2 medical science test to identify asymptomatic intracranial aneurysms (icas). an association between atherosclerosis and icas is recognised. elevated serum lipoprotein (a) [lp(a)] is an independent risk factor for atherogenesis. we aimed to assess the degree of correlation between serum lp(a) and the occurrence of sporadic ruptured aneurysms and familial asymptomatic aneurysms. lp(a) levels were measured in (a) 50 patients with icas and 42 normal controls, (b) 25 first degree relatives of patients with familial subarachnoid haemorrhage (sah). icas were detected by cerebral angiography. patients with sporadic icas had significantly elevated lp(a) levels when compared with matched controls. mean level was 20.1 mg/dl in patients and 10.8mg/dl in controls. in the familial studies, 10 out of i1 subjects with asymptomatic icas had elevated lp(a) levels. one young female with elevated lp(a) had a pre-aneurysmal dilatation at operation. six out of 14 subjects without icas had elevated lp(a) levels; four of these were in the second or third decade of life and may yet develop aneurysms. conclusions: lp(a) has potential as a biological marker for icas. follow-up studies are required on angiographically negative subjects. we have begun a genetic case-control study to establish if particular apoprotein (a) gene polymorphisms can be correlated with the occurrence of icas. post mastectomy breast reconstruction has undergone several changes in the recent years. attitudes have changed towards the problem from both the patient and the reconstructive surgeon, as aesthetic outcome receives a greater emphasis than previously. there is a shift towards using autologous tissue as a means of reconstruction; these new technically difficult procedures entail a longer learning. centralization of this type of reconstruction in highly specialized centres only will serve the patient better. we share our experience of post mastectomy breast reconstruction spread out over the past five years. seventy-eight consecutive cases of breast reconstruction are included in the study. different techniques of breast reconstruction were used with a recent switch to transverse rectus abdominis myocutaneous (tram) flap; we feet that tram flap is the gold standard of breast reconstruction as far as the ultimate cosmetic result is concerned. ours is only a moderate sized study compared to some published, yet it is representative of the experience of most of the plastic surgery units in the british isles. clinically significant paraneoplastic neurologicaldisorders are rare, most are associated with small cell lung, female genital tract and breast carcinoma. the malignancy is often silent and the neurological manifestations vary from encephalomyelitis, cerebellar degeneration, sensory neuropathy to neuromuscular block. prognosis is usually poor. pathogenesis is thought to be related to cross reaction with neurons of antibody produced to tumour antigens. detection of these antineuronal antibodies in serum has assisted diagnosis of paraneoplastic encephalomyelitis in which anti-hu antibodies are present and cerebellar degeneration, in which anti-yo are found. in our lab, we used avidin-biotin-complex immunocytochemistry to detect anti-hu (cortical neuron antibodies) and anti-yo (purkinje cell antibodies) in patients' sera. tests were performed on human frontal cortex and cerebellum, at 1 in 500 and 1 in 4,000 dilutions, with positive and negative controls. of 68 sera, 6 were positive. two patients had repeat positives; in one, antibody titre rose in the second sample. subsequent patient review showed 3 positives (2 patients) had identifiable carcinoma with paraneoplastic cns signs, 2 had no identifiable malignancy but had no other cause of their cns disorder and are being followed up; details of one patient were unavailable. these results are similar to other centres. the proliferation of tumour cells despite the presence of tumouricidal mediators could be due induction of a heat shock response, a universal cellular defence mechanism in host cells and possibly tumour cells. protection may be mediated either by increasing intracellular levels or surface expression of heat shock proteins (hsp). the aim was to assess the effect of heat shock induction on tumour cell protection against host effector cells. the heat shock response was induced in sw707 colorectal cells by either sodium arsenite (0-320~tm for 6hr) or by hyperthermia (42~ for 20 rain). monocyte (m0)-mediated cytotoxicity or flow cytometry to evaluate surface expression of hsp60 and hsp70 were assessed. cytotoxicity showed a significant decrease in all treated groups (p<0.002) when compared to the control value. there was also a significant decrease in all groups (p<0.001) when compared to the 42~ value. no significant alteration in surface expression of either hsp60 or hsp70 was seen. conclusion: heat shocking tumour cells significantly protects them from m0-mediated tumour cell lysis. since the flow cytometric data indicate that there is no concomitant increase in surface expression of hsp60 and hsp70 on the tumour cell following heat shock, it can be inferred that induction of intracellular hsp levels are responsible for the protective effect on the tumour cells. a 4 week qol study in 157 consecutive advanced cancer patients was undertaken to compare the subjective 28 question fact-g with simple subjective global tools (visual analog, categorical scales: vas, cas), objective tools (spitzer qli and ecog performance status) and verbatim patient description. we anticipated the high drop out rate enrolling 157 to achieve 87 complete study patients for statistical purposes. the study sample appeared representative of the advanced cancer population in the usa. generally qol was satisfactory despite the severity of illness. there were significant differences in all measures between those who described qol in verbatim responses as positive and negative, particularly cas, vas, and qli (p<0.0001). there were significant intercorrelations between qli and ps (observer rated), vas and cas (subject rated) respectively (p<0.0001). taking patient description as the gold standard, simple, global qol measures e.g. vas or cas are as effective as multidimensional ones (fact-g and qli). irish journal of medical science males had more dysphagia. survival from diagnosis was greater for females </=65 years (female median 18 months, male median 13 months, p<0.0016). anorexia was associated with reduced survival since diagnosis in the total population and in males. anorexia anddysphagia were associated with reduced survival from referral. advanced cancer patients were polysymptomatic. symptom prevalence differed with age, gender and cancer site. the pattern of g1 symptoms was related to gender and severe weight loss. specific symptoms were associated with reduced survival. there was a gender difference in survival favoring females. the administration of recombinant interleukin-2 (rll-2) is limited by the induction of increased microvascular permeability. the in vivo antineoplastic effects of taurine in combination with rll-2 were investigated and its impact on the associated vascular leak was examined. lung metastases were established in mice via tail vein injection. ten days after injection mice were randomized into treatment groups. on day 18 the mice were sacrificed; lungs were removed, weighed and metastases counted. treatment of tumour-bearing mice with rll-2 alone resulted in a significant reduction in tumour nodule incidence compared to a control group, while the~group recei'~ing rll-2 + taurine showed an even fuither reduction in the incidence of lung nodules. animals receiving rll-2 showed a significant increase in mean wet lung weight compared to control lung weight, while mean wet lung weight of the rll-2 + taurine group was significantly less than that of the rll-2 group.and comparable to control values. animals receiving ril-2 + taurine in vivo demonstrated significantly enhanced splenocyte-mediated antimelanoma activity ex vivo compared to animals receiving rll-2 alone. thus taurine may have an important role in modulating both metastatic growth and the associated toxicity of rll-2 immunotherapy. a prospective analysis of symptoms in 1,000 patients on initial referral to the palliative care service of the cleveland clinic was made using the paradox relational database. the median number of symptoms per patient was 11 (range 1-27). the 10 most frequent symptoms were pain 82%, easy fatigue 67%, weakness 64%. anorexia 64%, >10% weight loss 60%, lack of energy 59%, dry mouth 55%,'eonstipation 51%, dyspnea 51% and early satiety 50%. patients 65 years and under had more pain, sleep problems, depression, anxiety, vomiting and headache (all p<0.01 ).'the prevalence of early satiety, nausea, vomiting and anxiety were greater in females; dysphagia 3nd hoarseness in males. patients with >/= 10% weight loss had more gi symptoms; of these females had more nausea, early satiety; the progn.o'stic significance of abnormalities in the p53 tumour suppressor gene and in the expression of its protein in colorectal carcinoma may be influenced by the method of analysis used. we studied p53 abnormalities in 66 patients with colorectal cancer followed for more than 10 years. single-strand conformation polymorphism analysis (sscp) was used to detect alterations in exons 5-8 of the p53 gene. paraffin sections were examined immunohistochemically for p53 overe,xpression with the monoclonal antibody do-7 (dako) both with and without microwave antigen retrieval. abnormalities of the p53 gene were found in 41% of cases by sscp analysis but were unrelated to age, sex, tumour size or differentiation. outcome was unrelated to sscp abnormalities (p=0.19). overexpression of p53 protein was seen in 47% of cases by immunohistochemistry without microwave antigen retrieval and in 52% of cases with microwaving. poor long-term survival was related to immunohistochemical expression of p53 protein either with (p=0.03) or without (p=0.02) microwave antigen retrieval. these results suggest that immunohistochemical detection of the p53 protein product may be more useful than sscp analysis of the encoding p53 gene in identifying those at high risk of colorectal cancer recurrence and death. dublin 9. the anti-tumour activity of tumour infiltrating lymphocytes (tils) is known to be poor and therapeutic manipulation of these cells has met with little success. suppressor macrophages (smo) influence t cell cytotoxicity and proliferation. we hypothesized that smo are a component of the lymphoreticular infiltrate and that these cells may be related to lymphocyte numbers within the tumour. tgf-b may influence macrophage phenotype. colorectal and breast tumours were obtained within an hour of resection. tumours were dissaggregated with collagenase and dnase for three hours. antibodies was used to identify smo (rfd 1 and rfd7) and t cell subsets (cd4 and cd8) by flow cytometry on the resulting cell suspension. pre-op blood was collected from patients and tgf-b levelsdetermined by elisa. conclusions: we have shown for the first time that smo, defined by the antibodies rfd1 and rfd7, are present within breast tumours. we have also shown that the balance of t cell subsets is different in these tumours and may be related to smo content. circulating tgf-b levels are increased in breast cancer and associated with greater smo numbers. this was not found to be the case in colorectal cancers. these results imply the existence of a fundamental difference in the make-up of the lymphoreticular infiltrate between these cancers. swelling of the upper limb is an uncommon but well irish journal of medical science recognised complication of breast cancer treatment. in severe cases, patients have limited arm function and feel disfigured. in a pilot study, the incidence of arm swelling following complete axillary clearance in the immediate post-operative period and at long term follow-up was investigated. arm volume measurements were performed using an opto-electronic volometer (bosl medizintechnick, hamburg). both ipsilateral and contralateral arm volumes were assessed. the expected volume of the ipsilateral arm volume was calculated using the formula vr = v1 = 31 mls for right handed people and v1 = vr = 25 mls for left handed people (vr and v1 = volume of the right and left arms respectively). the difference between the expected and actual volume of the ipsilateral arm was expressed as a percentage of the expected volume. twelve patients undergoing axillary clearance for breast cancer were prospectively evaluated pre-operatively, 48 hours and 5 days post-operatively. a second group of 48 patients who had had axillary clearance at least 3 months previously (range 3 to 116 months) were also evaluated. there was no significant change in arm volume in the immediate post operative period. clinically detectable arm welling was found on 3 patients who had undergone axillary clearance at least 3 months previously but none had any impairment of arm function. we conclude that axillary clearance can be performed safely and that arm swelling is an uncommon complication. a larger study is planned to investigate factors such as the influence of pectoralis minor division, duration of the operation and the number of axillary nodes retrieved on upper limb volume. epidermal growth factor (egf) is a potent mitogen and has been shown to accelerate healing of epithelial damage both in the skin an.d the gut. in the skin egf is not produced locally as the requisite mrna is not present but egf receptors are present on the surface of basal keratinocytes. egf is produced in various sites in the gi tract including the submandibular salivary glands. we have hypothesised that as there is upregulation of salivary egf production in some enteropathies a similar situation may occur in disorders of the skin with an associated enteropathy. using a sensitive radio-immunoassay, egf activity was estimated in stimulated saliva from 87 patients with various skin disorders, 49 patients with gastrointestinal disease, 8 patients with mixed dermatological and gut disease and 41 normal healthy volunteer controls. elevated egf activity was found in the following groups of patients : skin cancers, psoriasis, acne, oesophagitis and ulcerative colitis. the hypothesis of up-regulation of salivary egf production in skin associated enteropathy was rejected but the discovery of elevated egf activity in skin cancers and psoriasis may have aetiological and therapeutic implications. the malignant fibrous histiocytoma (mfh) is considered an uncommon malignancy. its potential for invasion, metastasis and death of patient has been reported in literature. it can be confused with other tumours including fibrosarcoma. salient histologic features include cells of both the fibrocytic and histiocytic series. mfh with its high recurrence rate and lethal potential merits an aggressive evaluation and treatment. we present unusual case of recurrent mfh treated in our unit with an open question as to what qualifies to be adequate primary surgical excision. the recommended management of localised merkel cell carcinoma has been wide surgical excision, combined with adjuvant radiotherapy in selected cases. the risk of recurrent regional disease is reported to be between 30% and 45%. a 70 year old woman with merkel cell tumour on the cheek is presented; this patient was treated exclusively with radiotherapy to a total dose of 50 gy over 18 days. the tumour regressed rapidly during the treatment, and there were no signs of local or regional recurrence. the patient is still alive and free of disease for 45 months. immune in origin with a heightened cutaneous immune response to ultraviolet light. the coexistence of cad and pbc is a new association which has not previously been documented and may not be fortuitous given the similar pathogenesis of both diseases. chronic actinic dermatitis (cad) is a rare photosensitive disorder which primarily affects elderly men resulting in an eczematous reaction to ultraviolet-radiation and sometimes visible light. the pathogenesis has been attributed to an autoimmune process, possibly in response to a photoallergen which has yet not been identified. we report a 71 year old female patient who developed cad four years after being diagnosed with primary biliary cirrhosis (pbc). abnormal monochromator irradiation tests were detected with narrow band ubv, uva and in addition visible light wavelengths. phot0provocation tests induced florid vesicular eczema and multiple patch and photo-patch tests were positive, findings typical of cad. immunoglobulin g was elevated at 2290 mg/dl and liver histology was typical of pbc with an elevated anti-mitochondrial antibody. routine biochemical and immunological tests were ~ormal and porphyrin screen was negative. azathioprine 50 mg/day induced remission of cad. pbc is an auto-immune disorder where cell-mediated immunity is impaired, suggesting that sensitized t lymphocytes may cause damage to bile ducts. the pathogenesis of cad may be autowe present two cases of cutaneous polyarteritis nodosa (pan) associated with seronegative arthritis: the first patient, a 50 year old male presented in 1993 complaining of a7 year history of pain, stiffness and swelling affecting his right ankle. he also noted intermittent tender nodules on the dorsum of his foot and over his ankle over the preceding three years. the second patient, a 51 year old male, presented .in 1994 complaining of a 5 month history of tender nodules on his shins, and pain and swelling of his right ankle. skin biopsies of the nodules in both cases showed medium vessel vasculitis consistent with polyarteritis nodosa. neither patient had any symptoms or signs to suggest systemic involvement. the only abnormality.0n laboratory investigations for vasculitis was elevated esr. x-rays showed periosteal elevation and new bone formation in case 1, and were normal in case.2. bone scan demonstrated increased uptake at the talo-navicular joint in case 1 and at the fight ankle in case 2. synovial biopsy and mri confirmed the presence of an inflammatory arthropathy in patient 1. joint involvement has been a prominent feature throughout the course in both cases requiring aggressive treatment with vol. 165, 1996 irish journal of supplement no. 2 medical science cyclophosphamide and systemic corticosteroids in case 1. cutaneous pan is a localised cutaneous vascular disorder with a benign chronic relapsing course. in one reviewl, 12 of 23 patients had arthralgias but an association with arthritis has not been emphasized in the literature to date. we conclude that this condition may present as a seronegative arthropathy in which the joint symptoms may be the most prominent feature and aggressive immunosuppresive therapy may be required for control. cardiac transplantation patients have an increased risk of skin disease. in our centre, 119 heart transplants were performed with a 5 year survival of 75%. eighty three patients are now alive and 39 have required dermatological assessment. the mean age of patient was 48.3 years, (range 11-64 years); 99 males, 20 females. skin infections were diagnosed in 13 of 39 patients. drug side effects, including sebaceous hyperplasia and steroid acne, were common. in the patients who developed skin cancer, mean time from transplant to development of lesions was 3.1 years. eleven of 36 patients had 32 non melanoma skin cancers (nmsc), 28 squamous cell carcinomas (scc), 4 basal cell carcinomas (bcc), giving scc/bcc ratio of 7:1. three of 11 patients had multiple skin cancers, one had 12 tumours. nine of 39 patients had actinic keratoses, two thirds of whom had sccs. nineteen of 39 patients had viral warts, two of whom had sccs. viral warts, premalignant and malignant lesions were located on sun exposed sites. skin complications of cardiac transplantation though mild were very common. the observed incidence of nmsc in age matched cardiac transplant recipients, appears much higher than the expected incidence of 171.38 per 100,000 population (national tumour registry 1994). regular dermatological assessment of cardiac transplant patients is necessary to detect skin disease and early skin cancer. the increased incidence of warts and skin cancer in renal transplant recipients {rtr} is well known. the oncogenic potential of unusual human papilloma virus {hpv} types has been postulated from warts and in both premalignant and nonmelanoma skin cancer (nmsc). the possible etiological role of sun-exposure in facilitating the development of hpv associated skin disorders is also suggested. a clinical study to assess the risk factors for development of these lesions in 50 rtr attending the dermatology servic& age and sex matched haemodialysis patients were similarly examined as controls. 40 male and 10 female patients with a mean duration of transplant of 10.6 years, range 1 to 25 years. a total of 191 nmsc (range 1 to 57),175 scc and 16 bcc, ratio 10.9:1, were excised from 26 rtr of which over 95% had viral warts, mosle commonly occurring on sun exposed sites and always predated the development of neoplastic lesions. both were associated with mean duration from transplantation, 4 years for warts and 10.8 for skin cancer and not the type of immunosuppressive treatment. none of the control patients had similar findings. conclusions: the close clinical association of viral wart lesions and development of skin cancer in these patients suggests a close relationship to immunosuppression, in addition to exposure to ultraviolet radiation. this study highlights the high rate of nmsc in rtr. these patients justify early and regular skin assessments soon after transplantation with advice on sun protection. sensitivity to ultraviolet (uv) light may be established by exposure to broad band uva and uvb radiation. the minimal erythema dose (med) can be determined at individual wavelengths using a monochromator. uv action spectra of photosensitive disorders may thus be constructed. we examine the value of this process in distinguishing two clinically similar photosensitive disorders. the radiation from a xenon arc is separated into component wavelengths using the monochromator. each wavelength is focused on unaffected skin, on the patient's back. the patient is exposed to a range of doses of w radiation. the med is determined for a series of wavelengths from 300 to 400 nm. chronic actinic dermatitis (cad) and drug induced photosensitivity are photosensitive disorders which may have similar clinical history and presentation. ten cad and 14 drug induced photosensitivity patients were tested. uva photosensitivity was seen in 71% of the latter group. the remaining 29% had normal mlts as the implicated drug had been discontinued prior to testing. cad patients were sensitive to both wa and wb radiation. forty-three percent of these patients were also sensitive to visible light (400 to 800 nm). monochromator light test (mlt) results show that uva photosensitivity dissociated from wb photosensitivity is indicative of a drug induced light sensitive disorder. sensitivity to both wa and wb however indicates a diagnosis of cad. mlts can therefore distinguish between clinically similar photosensitive disorders. bartholomew's hospitals, london. patients with mpd have an increased incidence of both thrombosis and haemorrhage suggesting a pivotal role for; platelets in these conditions. this study aimed to examine platelet activation antigen expression in stable patients with mpd and to examine the predictive value of these antigens prospectively. 52 patients with mpd had p selectin and gp53 measured using a refined minimally manipulative flow cytometric technique. expression of p selectin -median 5.1% (inter quartile range 2.1-10.9), control 2.0% (1.3.-4.0) and gp53 -median 4.0% (1.8-5.7), control-2.1% (1.0-2.9), were significantly elevated p<0.01. patients were followed for a median of 26 months. 15% experienced thrombosis and 6% bleeding during follow up. at entry to the study 48% of patients had previously experienced thrombosis, median disease duration 8 deaths, 3 of which were caused by thrombotic events in which the mpd was a major risk factor. increased expression of p selectin or gp53 expression failed to predict thrombosis or bleeding in this study. nor was any significant retrospective relationship demonstrated. however, previous thrombotic events were strongly associated with future events (p<0.05). this association was independent of disease, duration, age and medication. not surprisingly disease duration was also correlated with thrombotic/bleeding events. taurine levels fall in gut mucosal cells during critical illness. however, taurine transport into human intestinal cells is poorly understood. the aim was to establish the efficiency of taurine uptake by enterocytes, and to examine uptake under stressful conditions. to investigate efficiency of taurine uptake, confluent caco-2 cells were incubated for time points up to 10h. in a second study, cells were incubated for 9h with medium containing dexamethasone and / or cytokines. media for both studies was supplemented with [3h]-taurine. radioactivity was related to mg/ml protein to calculate rate of taurine uptake for each time point. study 1: uptake exhibited a steady linear response which approached saturation at 7h. maximal uptake occurred at 9h after which the rate levelled off. study 2: dexamethasone alone reduced taurine uptake by 75.4% (p<0.001) and in combination with tnf-c~ and ifn ~/ it decreased transport by 66.3%. (p<0.001). lps alone impaired uptake by 56% (p<0.001). conclusion: we have established the time course over which taurine transport reaches its maximum rate in caco-2 cells, and that corticosteroids and cytokines significantly impair uptake of taurine in these cells. elderly individuals have an increased risk of infection suggesting that immune responsiveness is altered with age. changes in the level of proinflammatory cytokine production may be an important indication of any such age related change. using flow cytometry we examined intracellular tnfet, il-lf~ and il-6 in pbmcs from normal healthy volunteers of different ages ranging from 22 up to 85 yr (n=28). tnf and il6 levels from pma stimulated cd3 positive cells (t cells) were shown, using this technique, to increase in an age dependent manner (p<0.05). no il-113 was detected in any t cell sample. no significant differences were observed between the different age groups for tnfa, il-1 g or il-6 in cd 14+ cells (monocytes). the age related changes detected by flow cytometry have been confirmed using conventional elisas. this novel method of proinflammatory cytokine detection has detected increased tnf and il6 levels in t cells from elderly healthy volunteers which may help explain some of the exaggerated inflammatory responses seen in elderly patients. detection of proinflammatory cytokines by conventional elisa or bioassay is problematic due to the presence of naturally occurring biological inhibitors. flow cytometry allows the simultaneous detection of both intra and extracellular antigens thus intracellular cytokine levels can be quantified while cell surface markers allow cell type identification. a range of monoclonal antibodies were examined for tnfcx, il-lg and il-6 using saponin permeabilisation oft cells (cd3), monocytes (cd14) and epithelial cells (ber-ep4). t cells and monocytes were grown in ~culture, t~p :to 72 hr with or without pma activation, and intracellular cytokine levels were shown to increase with time, with the stimulated samples producing more cytol<ine than the spontaneous.samples (p<0.05). elisas performed on these same samples (n--:24) correlated well with the flow cytometric results (r=0.52). tnf~x, il-113 and il-6 are detectable !n monocytes while only tnfa and il-6 have been detected in tcells and epithelial cells using this methodology. this novel technique will allow us to identify the cytokine producing cell while also avoiding the influence of the extracellular millieu, thus being useful for a number of research and diagnostic applications. all the viral hepatitides are said to be common in the middle east. hepatitis a viru s (hav) seroprevalence is said to be 100% in the adult population of gulf countries. hepatitis b, c and d seroprevalence are taken from blood banks data which are biased by selection criteria and the background of blood donors. very little is known about hev in the uae. vol. 165, 1996 supplement no. 2 irish journal of medical science the aim was to investigate the seroprevalence of hav, hbv, hcv, hdv & hev in the uae. we prospectively recruited volunteers drawn from all the uae, socioeconomic classes and from different age groups in both sexes. each donated 10ml. of venous blood and using an indirect enzyme immunoassay were tested for total ig to hav; hbsag using monoclonal antibody t.o hbsag; for hcv antibodies using recombinant antigens hc-34, hc-43, c-100 and ns-5, (3rd generations); for total ig to hdv and for total ig to hev. positive samples were retested in duplicate for confirmation. this large sero-epidemiological study clearly shows that hav is decreasing in the uae. hbv is low but significant, hcv is similar to southern european countries, hev is lower than expected and hdv does not exist in hbsag carriers in the uae. reactive oxygen and nitrogen species produced by inflammatory cells plays a role in tissue injury and inflammation. taurine, a bamino acid present in high concentrations in leukocytes functions as an anti-oxidant by scavenging the mpo derived oxidant, hypochlorous acid, to form taurinechloramine. experimental colitis was induced in male sprague-dawley rats and randomised into two groups. one group received tap water whilst the second group was supplemented with taurine (4%). these groups were sub-divided into those receiving tnbs/ ethanol (n=8) or saline (n=8). an increase in wcc count, particularly neutrophils was found in colitis. tat~rine was found to reduce the severity of the disease (ns) usin.g a colon macroscopic score. in the first group, respiratory burst activity was reduced in animals that had developed colitis (p<0.05) although mpo activity was augmented (p<0.05). nitric oxide production was 2.2 and 1.8 fold respectively higher in inflamed and non-inflamed areas of the colon than that by normal colonic mucosa. animals supplemented with taurine demonstrated attenuated m po activity (p=0.001). administration of taurine did not lead to a reduction in nitric oxide production. thus, taurine appears to have a beneficial role in reducing the severity of disease. a reduction in mpo and respiratory burst may be beneficial in reducing tissue damage. to women subsequently identified by a national screening programme which revealed 438 to be positive for rna of hepatitis c, type lb. 94 were referred to this centre for evaluation. 10 had moderate activity by modified knodell index, mean 8 (_+1.4), bridging fibrosis and a mean alt of 77 (42-176). six months dual therapy was administered. two patients withdrew due to depression (2) and hyperthyroidism (1). one patient di d not respond. seven responded with normalisation of alt and negative pcr. eight patients showed improved histology (kai 4 +_ 1.5), fibrosis was unchanged. adverse reactions in the treated group included marrow suppression (1) and thyrotoxicosis (1) . at six months follow up four remain in remission with normal alt negative pcr, three have relapsed, (pcr positive, mean alt 87(50-150). we conclude that despite adverse prognostic indicators in this homogenous group with chronic hepatitis c, lb and long disease duration, dual therapy with interferon and ribavirin achieved results comparable to prolonged high dose interferon in patients with more favourable initial prognoses. the aim of the study is to evaluate a 5 day course of azithromycin in combination with omeprazole in eradication of h.pylori. twenty-four patients with proved h.pylori infection and peptic ulcer disease were treated with omeprazole 40 mg daily for 2 months plus azithromycin 500 mg daily for 5 days. h.pylori was diagnosed on the basis of clo test and histology. this was done at the beginning and 3 months after the treatment. eradication was defined as negative clo test as well as negative histology. out of 24 patients, 2 patients were lost to follow-up. only 21 patients were analysed. sixteen patients responded to treatment and had successful eradication as well as healing of their ulcers. these included 8 patients with gastric ulcers (79.95%), 4 patients with duodenal ulcers, 2 patients with gastric and duodenal ulcers and 3 patients with non-ulcer dyspepsia. five patients did not respond including 2 with non-ulcerative dyspepsia. 2 duodenal ulcers and 1 gastric ulcer. however their ulcers have healed and symptoms relieved and they were treated with other h.pylori eradication regimes. conclusion. in this small study we have shown that azithromycin 5 mg oncedaily for 5 days is an alternative safe effective and well tolerated monotherapy for eradication of h.pylori. it has been suggested that hepatitis c virus (hcv) infection is associated with a high prevalence of autoimmune disease, but that this risk may be diminished in patients who received a low viral load at initial infection. the aim was to determine the prevalence of symptoms, signs and markers of autoimmune disease in a group of women vol. 165, 1996 irish journal of supplement no, 2 medical science infected with hcv contaminated anti-d immunoglobulin. 50 patients, mean age 44.3 years (range 32 -65 yrs) were examined. 7 patients (14%) were rheumatoid factor positive. ana was positive in 6 patients (12%) but titres were 1/10 in 4/6. thyroid globulin antibodies were found in 3 patients (6%) with 1 also being tm positive. 2 further patients were positive for thyroid microsomal ab. prevalence of apa, ara, sm were 4%, 2% and 2% respectively. one patient had cryoglobulins detected while antibodies against mitochondria and lkm-i were not found only one patient of 50 was symptomatic. these levels of antibodies are no greater than levels in the general population and similar to other groups infected with hcv contaminated anti-d. these results further support the hypothesis that either low viral innoculum or long duration of infection may in some way result in a low level of autoimmunity. a previous study (ilan et al, arch int. med., 1993; 3: 1588-1592) suggested that lga deficiency disposes towards hepatitis c virus (hcv) infection in some patients. it has also been suggested.that iga deficiency may occur as a result of the viral infection as a secondary event. the aim was to determine the prevalence of selective and partial lga deficiency in patients with hcv infection using a radio-immunodiffusion technique. serum lgg and igm levels were also examined. immunoglobulin levels were measured in 67 patients, 59 women and 8 men, mean age 45.3 years (range 29 -72 years) infected with blood between 1977 and 1990. none of the patients were found to have selective or partial iga deficiency. mean iga = 2.59 g/l (range 0.86 g.1 -5.19 g/l). furthermore no patient exhibited deficiency of igg or igm. conclusion : no evidence of iga or other immunoglobulin deficiency exists among this group of patients with hcv infection. while it may be possible that lga levels decrease following infection, no evidence exists that this phenomenon persists in the long term. hereditary non-polyposis colorectal cancer (hnpcc) is the most common form of hereditary colon cancer and accounts for up to 10% of the overall cancer incidence. the disease is inherited in an autosomal dominant manner andis characterised by predominantly right-sided tumours. hnpcc is caused by mutations in one of four mismatch repair genes. these genes are homologues of the bacterial mismatch repair systerfi and their function is to detect and correct defects in dna. the hmsh2 gene is reported to account for up to 60% of cases, hmlhi, hpms1 and hpms2 account for 30%, 5% and 5% of cases respectively. we have collected eighteen irish hnpcc families which satisfy the amsterdam criteria for the disease. we have screened the entire hmsh2 gene in eight of these families. a combination of single strand conformational analysis (sscp) followed by dideoxy sequencing was used to detect defects in the dna. sequence analysis revealed the presence of a novel polymorphismin exon 5 in one family and a possible splice site mutation in the remaining ten families, using a novel mutation detection technique. departments of medicine and immunology, university college hospital, galway. serial iga gliadin antibody (igaga) studies are used as a surrogate for repeated small intestinal biopsy to monitor dietary compliance in treated coeliac patients. we have correlated igaga levels with indices of hyposplenism (platelets and erythrocyte "pits") to determine if either index can be used to monitor adherence to a gluten free diet. the study population of 129 biopsy proven coeliacs was stratified according to dietary status into 114 gluten free diet (groups a) and 15 normal diet (group b). the iga gliadin antibody titres were significantly lower in group a than group b (44.5 : 126 units p = 0.007). in group a gliadin antibody titres showed a statistically significant correlation with both platelet (p = 0.039) and pits count (p --0.04) ; in group b gliadin antibody titres correlated with platelet counts only (p -0.003). those ona gluten free diet were further subdivided according t ~ reported degree of adherence to the diet (strict diet -sgfd, lax'diet -lgfd). significantly higher gliadin antibody titres were found in lgfd than sgfd (103.6; 27.9 p = 0.003), but the ptatelet and.pit counts were similar in the two groups. igaga is superior to platelet or pit coums in monitoring dietary'compliance in coeliac disease. hospital, galway. iga endomysial antibodies (igaea) are said to be more specific and sensitive indicators of coeliac disease than iga gliadin antibodies (igaga). initially the igaea substrate was monkey oesophagus and more recently umbilical cord. a new commercial product has becomeavailable using primate smooth muscle (accu track -diagnostic slides). we have undertaken a preliminary study to compare the performance of this lgaea assay and igaga elisa in treated and untreated coeliacs, coeliac relatives and controls. the study population consisted of 127 biopsy-proven coeliacs who were stratified according to "reported" dietary status into 89 good gluten free diet (group a), 24 poor gluten free diet (group b) 14 normal diet (group c) and 10 unaffected coeliac relatives (group d) and 5 controls (group e). elevated igaga (>25 units) were found in the following groups : a 37%, b46%, c 50%, d 20%. igaea were positive in a 27%, b 33%, c 50%. with respect to igaga positivity, there were 11 false positive igaeaa 18%, b 12.5%, c 14% and 23 false negative -a 8%, b 8%, c 14%, d 20%. in this preliminary study in untreated coeliac patients the performance of the igaea test was on a par with the igaga assay. alt and fibrosis may be associated with a non-alcohol steatohepatitis and these processes may be synergistic. finally, number and type of riba bands is not a predictor of inflammatory activity. ~stepping hill hospital, stockport, uk sk2 7je. 2royal oldham hospital, rochdale rd., oldham ol1 2jh. we investigated upper gut bleeding in patients aged 75 years and over. a proforma addressing demography, drug therapy, clinical status, timing of endoscopy / surgery, and outcome was used. 109 consecutive patients (median age 80 years, range 75-92) were studied over 6 months. 106 (97%) underwent gastroscopy with a 97% diagnostic yield. 32 patients had severe oesophagitis, 2 had oesophageal malignancy, 29 had gastric ulcers -5 of which are malignant and 23 had duodenal ulcers. ulcerogenic drugs were implicated in 52 patients. 38 patients were referred for surgery, 8 operated upon with one postoperative death. 51 had haemoglobins of 10g/dl or less. all malignant lesions were inoperable. the overall mortality was 6% reducing to 2% if neoplasla were excluded. co-morbidity influenced mortality. 92 patients were discharged with a median hospital stay of 15 days. information on cause of bleeding greatly influenced management. the prognosis of gut haemorrhage in the very old need not be so poor. a few require surgery but the majority respond to active medical resuscitation which is a key factor in determining outcome. we advocate low threshold for endoscopy, judicious use of ulcerogens and adherence to guidelines on management of upper gut haemorrhage. haemochromatosis (hh), a common recessively inherited disorder of iron metabolism is closely linked to the hla-a locus on chromosome 6. linkage studies have demonstrated a close association between (hh) and the hla alleles, a3 and b7 ("ancestral haplotype"). heterogeneity at the molecular level may account for the variance in clinical phenotypic expression. the aim was to evaluate phenotypic expression of hh in the presence/absence of the a3-b7 ancestral haplotype. 32 probands (26m:6f) from unrelated irish families were investigated. phenotypic variability was assessed with regard to l) age; 2) % trans.sat.; 3) serum ferritin; 4) liver bx iron grade; 5) body iron stores and 6)symptomatology. three males were homozygous for a3b7, 15 were heterozygous for a3b7 and 2 were non-a3b7. symptomatology, trans, sat., serum ferritin and liver bx grade were not influenced by homozygousity or heterozygousity for a3b7. conclusion: there were no significant differences in phenotypic expression on comparison of the three haplotype groups. no predominant genotype appears to be responsible for phenotypic severity in irish families indicating the possibility of multiple mutagenicity of the hh gene. of 1013 riba positive anti-d associated chronic hepatitis c patients, 438 were pcr positive but had surprisingly mild disease. the disease status of the pcr negative patients was hitherto uncertain and is the subject of this study. 20/72 riba positive patients referred to this centre were biopsied because of elevated alt (4) or florid symptoms which dated from inoculation (16). 1 2 3 4 histological activity index * 5,1,0,1 3,2,5(f), 2(f), 0,4(f), 2,1,2 0,2,1,2 2,2,3 no bile duct damage, lymphoid follicles or aggregates was observed. 3/20 had mild periportal fibrosis (f), 2 of these had steatohepatitis with obesity (1) and impaired glucose tolerance (1) suggesting dual pathology. we conclude that riba positive, pcr negative patients have minimal disease activity. elevated the association between the hla locus and haemochromatosis (hh) has allowed early identification of affected siblings. it is unclear what proportion of subjects who are predicted to be homozygous or heterozygous for the disease by hla typing develop the disease. studies correlating clinical features with hla type in families from ireland -a putative source of this celtic trait have not been described. the aim was to correlate clinical, biochemical and pathologic features of hh with hla typing in 67 first degree relatives of 12 probands. initial analyses identified 12 homozygous (hh), 40 heterozygous (hn) and 15 normal (nn) individuals. however, 11/40 hn individuals had stainable iron on liver biopsy, confirming hh. further hla analysis revealed 7 homozygous x heterozygous matings and identification of all disease haplotypes within each pedigree allowed final classification of 30 hh, 25 hn and 12 nn individuals. vol. 165. 1996 supplement no. 2 conclusion: this study demonstrates the importance of hla typing in the clinical management of families with hh, furthermore, in multiply affected families the incidertce of homozygous x heterozygous matings is high indicating the high degree of "pseudodominance" in the irish population. the degree of acute hepatic failure after severe trauma and sepsis is related to the extent of hepatocyte (hc) damage and cell death resulting from either necrosis or apoptosis. we have previously demonstrated that tnf-ct and lps can directly lead to hc necrosis, but not apoptosis. recent, studies have shown that reactive oxygen intermediates (roi) and nitric oxide (no) are capable of inducing apoptosis in eukariotic cells. however, it is unclear whether roi or no are involved in hc cell death. the aims of this study were to evaluate the role of no and roi in hc cell death (apoptosis vs necrosis). hcs were isolated from sprague-dawley rats, and cultured with the no donor, sodium nitroprusside (snp) or the roi generation system, hypoxanthine-xanthine oxidase (hx-xod) and h202. the effect of lps, tnf-t~, and ifn-y alone or in combitmtion with different antioxidants and the no synthase inhibitor, n-methyl arginine (nma) on hcs was also assessed. snp caused a dose-dependent increase in hc apoptosis. roi generated by hx-xod and h202 did not induce hc apoptosis, but were responsible for hc necrosis. tnf-ct alone failed to induce hc apoptosis, but when ~combined with antioxidants resulted in increased hc no production and apoptosis. this effect was attenuated by nma. snp also induced hc damage and hc necrosis. moreover, tnf-ct-mediated hc damage and necrosis could be further reduced by the combination of antioxidants and nma. these results indicate that roi preferentially induce hc necrosis, but not apoptosis. induction of no resulted in both hc apoptosis as well as hc necrosis, which suggest that overproduction of no may be detrimental during the sirs. irish journal of medical science intervention was not uniform. mean albumin was 34.3g/1. mean weight was 57.8kg. poor cognitive status greatly increased the requirement for dietetic consultation time. lack of dietetic resources results in inadequate monitoring of these patients following discharge. this study highlights the need for a dedicated clinical nutrition service, for medical services for older people. periconceptual consumption of folic acid has been shown to decrease the incidence of neural tube defects. the preventative strategy of universal food fortification with folic acid presents the possible risk of masking the diagnosis of cobalamin deficiency in pernicious anaemia. in addition, the ultimate longterm effect of universal exposure of adult or foetal cells to a synthetic substance, ie. folic acid, is unknown. in this study, the threshold oral dose of folic acid in a number of foods above which metabolically-unaltered vitamin appeared in serum postprandially was determined in a young and elderly population by microbiological assay of serum pre-fractionated by hplc. subjects on a five-day regime of fortified cereal and bread along with their normal unfortified diet. were shown to have a threshold level of 400~g/d, abovewhich unaltered folic acid appeared in the serum. individuals given folic acid in either isotonic saline, milkor white bread exhibited a threshold level of 200~tg per serving. from patterns of food consumption in ireland, even moderate levels of fortification are likely to lead to some population groups being exposedto excessive amounts of un-altered folic acid in serum. many older people are nutritionally compromised. there is clear evidence that: nutrition intervention reduces morbidity m and mortality in older patients. to identify the spectrum of nutritional abnormalities referred for dietetic intervention and the problems associated with nutritional assessment, 50 elderly patients were alphanumerically selected from files of the department. of nutrition and dietetics. the most common dietetj.c interventions were: use of supplements 60%; high protein high calorie diet 46%; nasogastric feeding 26%; reduction fat 8%; iron/thiamine assessment 6%; high fibre diet 6%; diabetic diet 4%; nutrition swall6w programme 2%; lipid lowering diet 2%. some 60% referred required nutritional supplements, but the profile of an increase in oxidative stress in cystic fibrosis patients has been suggested. activated neutrophjls in the presence of chronic lung inflammation in addition to increased activity of the electron transport chain in cfmay. increase free radical generation. antioxidant protection against free radical attack is likely to be compromised as a i'esult of deficiencies in fat soluble antioxidants vitamins. in the present study stimulated thiobarbituric aoid reacting substances (tbars) were measured to determine the ability of plasma to withstand lipid peroxidation. copper was used to in!tiate the breakdown of lipids to lipid hydroperoxides and eventually to aldehydes, mainly malonyldialdehyde (mda). pooled cf plasm a and pooled control plasma were incubated for 0, 30, 60, 90, 140 and 200 min. mda complexes with thiobarbituric acid which absorbs at approximately 535 rim. there is a lag phase where antioxidartts vol. 165, 1996 irish journal of supplement no. 2 medical science in the plasma or tissue protect against lipid peroxidation, then a log phase where the protective effect is overcome and finally the reaction reaches a plateau when lipid peroxidation is complete. absorbance at 532 nm was measured in all samples and zero order and first derivative spectra were obtained. the lag phase appears to be longer in the pooled cf plasma compared with controls. plasma t~-tocopherol levels were within the normal range in both groups, indicatin~ an alternative protective effect in cf. mild hyperhomocysteinaemia is an established risk factor for heart disease. a source of homocysteine in humans is the essential amino acid methionine found in protein of animal origin. in an 8-week study weekly fasting plasma homocysteine levels were examined in a group of healthy male subjects (n=6) under normal dietary conditions (weeks 1 to 4) and in response to graded increased methionine intakes (weeks 5, 6, 7). nutrient intakes, including methionine, were calculated from 4x3-day food records. under normal dietary conditions weekly mean plasma homocysteine levels were not significantly different (anova) from each other ranging from 6.82+1.77 to 9.42+2.73~tm/1. doubling daily methionine intakes (supplementing with 25mg/ kg/d) did not result in a significant increase in plasma homocysteine (8.56+3.68~m/1), however, significant increases were achieved when diets were supplemented with methionine at levels of 50 and 75mg/kg/d resulting in mean plasma homocysteine levels of 13.37+5 9 and 18.051am/1+11.08, respectively. mean plasma homocysteine levels returned to baseline (8.76 + 3.42 ~tm/l) 10 days post supplementation. we conclude that supplementary methionine results in a significant increase in plasma homocysteine only when levels of five times the normal dietary intake are reached. this study is evaluating the use of a synthetic construct which encompasses primer binding sites for lpl and a variety of cytokine and other transcripts, to quantify lpl expression in cultured human monocyte-derived macrophages. following isolation of total rna at various times during cell culture, its reverse transcription (rt) using random hexamer primers generating first strand cdna and specific amplification of lpl cdna targets by polymerase chain reaction (pcr), generates products identifiable on gel electrophoresis. quantitation of message is obtained by incorporating the pawl08 construct in the rt assay in varying quantities as an internal standard with known amounts of monocyte-macrophage rna (l~tg). pcr amplification of this construct yields size distinguishable products from that produced by the monocyte-macrophage lpl cdna transcript. pcr conditions for the assay have been optimised at 29 cycles of denaturation (tmin @94~ annealing (1.5min@55~ and extension (lmin@72~ lpl mrna has been detected in cultured monocytes and macrophages throughout their differentiation. also increased expression of monocyte lpl mrna has been observed following 15 hr incubations with chylomicrons (30t~g/6x 106 mononuclear cells/ ml)-when compared with controls. interestingly, little or no lipase mrna was detectable in circulating monocytes using identical pcr conditions to preparations of mrna from the day 4 and day 8 cultured cells. this methodology will now permit investigation of the factors controlling lpl expression in cultured human monocytic cells. replacement growth hormone (gh) therapy in adult hypopituitarism is attracting increasing interest. in 1992 markussis (l) detected premature atherosclerosis by ultrasonography in the untreated patient 9 we have shown plaque regression with patients on replacement gh (norditropin) in a 4-month trial. 11 females and 9 males were recruited, mean age 49.6 years. at each timepoint plaque characteristics were measured by duplex ultrasound. 6 patients showed a large reduction in plaque size (mean 21%) after four months treatment (p value < 0~00l). similarly, highly significant values in cholesterol, hdl and ldl and apo a1 are achieved. chol hdl ldl apo a 1 pretreatment 6.54 1.23 5.04 119 posttreatment 5.64 w 1.24 w 3.79 w 109 ~ w achieve a high degree of statistical significance (p< 0.001). the significant reductions achieved in plaque characteristics in six patients studied who showed plaque formation correlates with other parameters traditionally accepted as reducing cardiovascular risk. hypertension is found in approximately 70% of patients with cushing's syndrome, but the mechanism is poorly understood. previous studies in our unit have examined levels of exchangeable sodium, plasma renin and angiotensin ii and cardiac sensitivity to phenylephrine. one previous study has demonstrated enhanced pressor responsiveness to noradrenaline in a group of patients with cushing's syndrome due to adrenal adenoma. we have investigated the blood pressure response to noradrenaline in 7 patients with pituitary dependent cushing's syndrome and in 7 controls matched for age, sex and bmi. noradrenaline was infused for 10 minute intervals at five different concentrations between 0.01 and 0.18 mcg.kg.min -~ multiple systolic and diastolic readings were recorded and the infusion was stopped if the systolic pressure became > 200 mmhg, diastolic _> 1 l0 mmhg or the systolic pressure rose > 35 mmhg. basehne blood pressure in the patients with cushing's disease (cd) was 140/88 + 6/3 compared with 122/83 + 6/6 mmhg in the normal controls (nc). in 6 of the 7 patients with cushing's disease, the test had to be stopped before completion of the protocol, whereas this was necessary in only one control subject the change in blood pressure from basehne to the blood pressure value recorded either at the time the test was stopped or at the peak blood pressure reading during equivalent noradrenaline infusions was compared between the matched pairs. the mean change in diastolic pressure was 22 + 5 mmhg in cd compared with 7 + 2 in nc (p<0.05). there was no statistically slgmficant difference in either systolic pressure (28 + 5 vs 26 + 5 mmhg) or mean arterial pressure (23 + 5 vs 14 + 3 mmhg). these results demonstrate an increased diastolic pressor response to noradrenahne tn cushing's disease. increased pressor sensitiwty to uoradrenaline may contribute to the elevated blood pressure seen in cushing's disease. increased plasma homocysteine (thcy) is an independent risk factor for premature vascular disease. patients with insulindependent diabetes have an increased prevalence of cardiovascular disease. accordingly, we measured plasma thcy concentration in 119 such patients (15-59y), randomly selected, and in 51 control subjects. in controls, thcy was higher in males than in females (supine: geometric mean (95% ci): ,8.3 (7.2,9 6) v 5.9 (5.1,7.0) i.tmol/l, p<0.001), as previously described, but there was no gender difference in patients. male patients, without microvascular complications, had lower thcy than controls (supine: 6.5 (5.4, 7.8) v 8.3 (7.2,9 6) ~mol/l, p<0.05), but values in female patients without complications were similar to those of female controls, thcy significantly correlated with age in diabetics but not in controls, thcy increased in patients with increased severity of microvascular complications, partly due to the effect of age. thcy was higher when standing.than when supine in both controls (8 0 (7.0,9.1) v 7 1 (6.4,8.0)lalmol/l, p<0.05) and patients (6.9 (6.5,7.4) v 6.4 (6.0,6.9)l.tmol/l, p<0.02). the absence of gender difference, the association between thcy and age, and higher levels with increasing microvascular complications suggest thcy could be of pathogemc significance in iddm patients, despite unexpectedly low levels in male patients without complications. differences.between supine and erect samples may be due to haemodilution of albumin-hound thcy in the latter a review of the treatment outcome of thyrotoxicosis with standard dose/doses of radio-active iodine (sdrai) in 67 consecutive patients presented to the endocrinology department, uchg, from december 1992-december 1993 was analysed. the mean pre-treatment levels of free thyoxine (ft4) was correlated with the treatment outcome. there was statistically significant difference in the pre-treatment ft4 between responders and nonresponders to the first dose rai (p = 0.001). response with hypothyroidism and/or euthyroidlsm was considered successful treatment 43/67 (64 2%) responded to a single dose rai; 26/ 67 (38.8%) and 17/67 (25 4%) responded with euthyroldism and hypothyroidism respectively 19/67 (28.4%) and 3/67 (4.5%) responded to the second and third doses of rai respectively giving a total response rate of 92.5% and 97% respectively. interestingly, 2/67 (2.99%) patients failed to respond even to the fourth dose rai 4/67 (5.97%) patients with t3 toxicosis (3 females and 1 male). two responded to the first dose (one with hypothyroidism and the other with euthyroidism), the remaining 2 required a second dose, which produced the same results. no statistically significant difference in the response rate between t3 and t4 toxicosis (p = 0.9) was observed. inherent in the st. vincent declaration targets is the need for continuous data collection and audit. we present preliminary information from the mater database, the first prospective audit of patients from a homogenous irish population. 1,051 iddms (527m:524f) were identified with the following characteristics (mean + sd), age 37.3 + 14.4 years, duration of dm 185 + 11.2 years, bmi 24.4 + 3.0 (males) and 24.2 + 4.0 kg/m2 (females), hbalc 8.6 + 1.97% (n<6.2%). no male:females differences existed in the above nor in macrovascular complication rate 10.1% (predominantly peripheral vascular &sease and lschaemic heart disease). however, males were more likely to be current smokers (34% vs 27%, p = 0 01). hypertension rates (17.2m vs 15.4%f), cholesterol > 6.5 mmol/1 (6.2m vs 9.8%f) were similar but more males had cholesterol < 5.2 mmol/l (64.8m vs 53.8%f, p < 0.01). clinical nephropathy was present in 11.4% of males vs 8.7% in females (p<0 05) 11.9% had clinical peripheral neuropathy. retinopathy will be described elsewhere. 10.4% of females and 2.3% of males had a history of hyperthyroidism and 2.5% of females vs 1.4% of males of hypothyroidism. 7.8% had history of psychiatric disease. conclusion although not a population based study, care of iddm in ireland is almost totally hospital clinic based cigarette smoking is identified as the major problem to be addressed patients with diabetes meltitus (dm) are at a higher risk of developing vascular complications, including coronary artery disease (cad). we performed a detailed analysis of predictors of cad and its seventy in patients with dm and chest pain 19 patients in total single vessel cad (svd) in 4, double vessel (dvd) in 5, and triple vessel (tyd) in 5 on cine contrast angiography clinical, biochemical and dobutamme stress echocardiographic findings are tabulated below for patients with angiographically proven coronary artery disease. patients with tvd had a longer duration of dm (19 4 years) and were more likely to have retinopathy (100%) the sensitivity of dse was excellent for severe disease. conclusion' duration of dm, retinoapthy, and a positive dse were the best predictors of severe cad in a diabetic population with chest pain the haemodynamic hypothesis for the pathogenesis of diabetic microangiopathy argues that an initial increase in microvascular flow leads to sclerosis and disturbed microvascular autoregulation. we have recently demonstrated impairment of vasoconstrictor responses to endothelin-1, a potent endothelium-derived constrictor substance, in niddm and have suggested that this could contribute to the initiation of microangiopathy the purpose of this study was to determine whether responsiveness to endothelin-i is also impaired in iddm. non-specific vascular smooth muscle contraction was assessed using high dose serotonin eleven patients with iddm and 11 control subjects underwent forearm blood flow (fbf) measurement by venous occlusion plethysmography in response to local infusions of endothelin-1 (5 pmol/min for 60 minutes) and serotonln (30 la g/min for 2 minutes) control subjects showed slow onset vasoconstriction in response to endothehn-1 reaching maximum at 35 minutes (p<0.01) the diabetic group did not respond to endothelin-i group differences were significant (p=0.02). the two groups showed similar vasoconstriction in response to serotonln. in conclusion, vasoconstriction in response to endothelin-t is impaired m iddm non-specific vascular smooth muscle contraction is preserved. impaired vascular responsiveness to endothehn-i is a possible common mechanism for the pathogenesls of microanglopathy in 1ddm and niddm. we measured total corrected (tca) and standardised ionised calcium (lca) in a population of 76 intensive care (1cu) patients (with a mean age of 57+22 years, 59% male) to determine the prevalence of abnormalities in circulating calcium and its possible determinants severity of illness was measured by the apache ii score (acute physiological and chronic health evaluation). for comparison of ica we examined 20 subjects undergoing arterial gases which proved to be normal and 40 non-critically 111 hypoxlc subjects ica was measured on arterial gas samples and corrected for ph 84% of icu patients had a total ca (unadjusted) of < 2 2mmol/l. after adjustment for serum albumin, 55% of icu patients had an tca <2 2 mmol/1 30% of icu patients had a serum phosphate of <0.8 mmol/1 ica in controls was 1.44 + 0.017 mmol/1 and 1.38 + 0.015 mmol/1 in hypoxlc non icu patments (ns) ica in icu was lower: 1.34 +0.016 (<0.003). tca and lca were not slgmficantly related. tca and ica did not sigmficantly differ between patients who died and who survived in the icu, and they were not related to apache ii score. belfield, dublin 4. from july 1987 to june 1995 there was a 16-fold increase m the annual number of specimens submitted to the virus reference laboratory because of a perceived risk of contracting hiv through a needlestick injury, blood splash, human bite, or through occupational exposure. needlestick-associated specimens also comprise an increasing proportion of 'at-risk' specimens, rising from 0.9% m the year july 1987-june 1988 to 9.4% in the year july 1994-june 1995. between july 1992 and june 1995 a total of 1787 patients had specimens submitted for hiv antibody testing after a perceived'exposure to hiv of these only 209 patients had more than 1 specimen taken. although the time of putative exposure is rarely avadable, the median interval between 1st and 2nd postexposure specimens for these 209 patients is 3 months with 136/ 209 (65%) lying between 1 to 6 months. if the risk of hiv refection from a needlestlck injury is assessed as sufficient to warrant serological investigation, the timing and number of blood samples are important. a negative report from a single early specimen may not indicate an absence of infection a basehne specimen and follow-up specimens at 6 weeks, 3 months and at a minimum of 6 months post-exposure are recommended appropriate serology for other viral mfections (hepatitis b and hepatitis c) should also be considered. since the beginning of 1995 a significant sustained increase in the numbers of hepatitis a cases has occurred. the number of cases m 1995 was 229 against 121 in 1994. this reverses the continuous fall observed over previous years. the reason for this increase remains unidentified at present this increase has occurred following a dramatic increase in the total number of hepatitis a igm tests carried out by the vrl since february/ march 1994 the increase in the number of tests carried out since this time is primarily attributable to increased hepatitis testing following receipt of hepatitis c-contaminated rhesus anti-d immunoglobuhn. analysis of the age profiles of the positive patients and of all the referrals indicates that 95.5% of positive results were found m patients aged 50 yr or less whdst 31% of all hav igm tests were performed on individuals aged 51 yr or greater. this raises the question whether greater selectivity should be employed when requesting hepatitis a tests studies report compliance rates ranging from 30 to 50% in hiv negative patients there has been no comprehensive study of compliance in hiv positive patients. accurate measurements of compliance are not easy; easy measurements of comphance are not accurate "). to determine the compliance rate in hiv posmve patients attending st. james hospital, dublin, one hundred consecutive patients attending the service were interviewed (homosexual 46, ivdu 45, heterosexual 9). the questionnaire was divided into three sections. firstly, a medical review was completed by the clinicmn which included demographic data, cd4 count, cdc staging, karnofsky index and prescribed medication. secondly, the pharmacy detailed the medication dispensed to each patient. the third section comprised a patient interwew to determine adherence to, and understanding of prescribed drug therapy. we report an overall comphance rate of 61%. this was unevenly distrtbuted between the two main patient groups (89% in homosexuals, 24% in ivdu) the following factors were found to mfluence compliance: number of medications, cdc stage, karnofsky index, dysphagia, educational and socio economic factors. we also found that poor patient understanding of the prescribed therapy significantly affected compliance. the aim was to determine the incidence of stds in patients presenting for hiv testing at the department of genito-urinary medicine, saint james's hospital. a retrospective analysis of all patient notes who presented for hiv testing between july '94 and december '94 was undertaken. according to clinic policy all patients had been screened for the following stds; neisseria gonorrhoea, chlamydia trachomatls trlchomonas vagmalis, candida, human papilloma virus, herpes simplex virus syphilis and hepatitis b in addition intravenous drug users (ivdus) were also screened for hepatttls c all patients underwent pre test counselhng. sex, age, risk groups and diagnoses were noted. 504 patients presented for hiv testing, of whom 66% were male, 34% female, wtth an average age of 28.5 years. of the total, 8% were, or had previously been ivdus. of the total 46 ivdus, 37 were heterosexual males 2 were bisexual and 4 were females 10.2% of the male patients were homosexual and 4 4% btsexual there were 15 posmve hiv tests (3% of total); 14 males and 1 female. in this group there were 5 patients with hepatitis c, all of whom were ivdus. no other stds were detected in the hiv negative group hepatitis c was diagnosed in 20, hepatttis b in 4, anogenital warts in 58, herpes gemtalis in 10, syphilis in 3, n. gonorrhoea in 9, t. vagmalis in i, c. trachomatls m 12, g vaginalis in 5 and candldlasis in 47. this study confirms the importance of std screening in all patients requesting a hiv test. of the total testing for hiv, 30% had a concurrent std diagnosis although no stds were identified in the hiv positive group this may be more a reflection on the makeup of the irish hiv positive population, the majority being ivdus, rather than a difference m the mode of sexual toxoplasmosis is the most common opportumstlc infection of the central nervous system in aids patients. in clinical practice the diagnosis depends on clinical, radiographic and serological findings coupled to chnical response to therapy brain biopsy is not routinely performed. in this retrospective review, we describe our experience with diagnosing toxoplasmosis we examined (a) the clinical demographics and presentation, radiographic findings, response to therapy and patient outcome (b) role of polymerase chain reaction (pcr) in detecting toxoplasma gondii from blood and csf samples (c) the usefulness of serology in diagnosing acute infection. all cases diagnosed as toxoplasmosls based on the above criteria were reviewed. pcr to detect toxoplasma gondn dna used primers to the b 1 gene giving a 223bp amphficatton product serological tests used were sabln-feldmen dye test and latex agglutination. there were 25 cases diagnosed (17 m, 8f, cd4 0-300; cdc iv 19). 3 panents unknown to be hiv posmve presented with cerebral toxoplasmosis 22 patients were not receiving continuous systemic prophylaxis against pce diagnostic value oft gondii pcr in blood and csf showed a sensmvlty of 20%, specificity of 100%, ppv was 100% and npv was 40% determination of lg subtype was of limited value 25% (6) of patients were seronegatlve of whom 50% (3) had histologically proven disease 2 of these latter 3 cases were pcr negative the dye test was of poor predictive value this review confirms the need to combine all parameters in making a diagnosis of toxoplasmosis in lmmunocompromlsed hosts. the performance of the newly developed, rapid and fully automated m~croparticle enzyme immunoassay abbott imx hiv-1/hiv-2 3rd generation plus assay for the detection of antibody to hiv-1 and hiv-2 including subtype o m human serum or plasma was assessed the assay was evaluated by testmg specimens from blood donors, diagnostic populations and hospitalized patients, hiv seroconverslon panels confirmed hiv-positive specimens, and potentially interfering specimens. the abbott imx hiv-1/hiv-2 3rd generation plus assay showed an overall apparent specificity of 99.97% (lower limit of 95% ci 99.84%) in the tested blood donor populations (n=347t). this comparable to the specificity found for the abbott imx hiv-i/hiv-2 3rd generation plus eia (99 93%) and the axsym h1v-1/hiv-2 assay (99 90%). the apparent sensitivity of the abbott imx hiv-i/hiv-2 3rd. generation plus assay is at least equivalent to that of the abbott imx hiv-i/hiv-2 3rd generation plus eia and the axsym hiv-i/hiv-2 assay. of 21 hiv-i seroconversion panels tested, the abbott imx hiv-1/hiv-2 3rd generation plus assay detected seroconverslon earlier on up to 6 panels, depending on the comparison assay. among 785 specimens from asymptomatic and symptomatic hiv patients, the abbott imx hiv-1/hiv-2 3rd generation plus assay detected 100 (785) including 7 specimens characterized as hiv-1 subtype o. the abbott imx hiv-1/hiv-2 3rd generation plus assay is an extremely sensltlve and highly specific assay for the early detection of antibody to hiv-1/hiv-2 and shows at least an equivalent performance to the abbott imx hiv-i/hiv-2 3rd generation plus eia and the axsym hiv-1/hiv-2 assay. the fully automated imx instrument system offers ease of use and rapid results on a widely accepted and reliable platform. streptokinase (sk), a 47kd protein produced by group c b hemolytic streptococci, is a widely used thrombolytic agent. anti-sk antibodies arise either as a result of therapeutic administration of sk or following natural mfection with streptococci although the clinical significance of antl-sk antibodies is not clear, there is evidence that some anti-sk antibodies arising from natural infections can interfere with sk activity tn vtvo, resulting in thrombolytlc failure. to facilitate further investigations of these antibodies, we have developed and validated a highly sensitive functional assay, which measures sk neutralisatlon activity of serum independently of other circulating inhibitory factors in the sample, and a rapid and convenient enzymeimmunoassay for the detection of anti-sk antibodies. analysis of over 200 random serum samples from the local blood bank with the enzymeimmunoassay showed the prevalence of antl-sk antibodies to be approximately 13%. all the positive samples and an equal number of the negative samples randomly selected were analysed by the functional assay the agreement between the results of the two assays was excellent indicating that our enzymelmmunoassay was a convement method for detection of anti-sk antibodies which could neutrahse sk activity m vitro irish journal of medical science tuberculosis drug therapy, isolation precautions and prophylaxis. conventional methods of detection such as microscopy and culture either lack sensitivity and specificity or are timeconsuming. in this study we investigated the use of a pcr based diagnostic assay for the detection of m. tuberculosis in sputum samples this assay has been developed by bioresearch ireland (bri) and raggio italgene. sputum samples were lysed and pcr amplified using an m. tuberculosis complex-specific primers. the results obtained using the bri/c-trak tm technology were initially compared to the amplicor system (hoffman la roche). both probe detection methods represent fast and reliable methods for the detection of m. tuberculosts in clinical samples. this test is designed to eliminate the possibility of obtaining false negatives. strongyloldes stercoralis infection in humans ts endemic in the tropics. as travel is becoming more common, it will be seen more frequently. two cases of this infection in irish people are described. case i. a 21 year old women had travelled and worked in poor rural areas of mexico for one month, three years before presentation. two and a half years later she developed abdominal discomfort, anorexia and sore throat. myalgia, arthralgta and a transient skin rash began to appear in the next month. eosinophilia, mild anaemia and raised liver blood tests were noted. elisa test for strongyloides was positive but parasites were not seeri in the faeces. ivermectin was given and the patient feels better. case ii. a 31 year old nurse had arthralgia, fatigue and some weight loss for 18 months'. on two occasions in the last four years, she had been travelling extensively in s.e. asia for a total of four months. she was admitted to hospital because of acute fever and loin pain. a urinary tract infection was diagnosed. absolute eosinophil count was i'aised 0.63x10^9/1. esr was 17mm/hr. strongyloides elisa was positive and treatment administered as above. strongylotdes is the most important nematode in the returned tropical traveller. it can multiply and persist within the body for long periods of time and it can cause hypertnfection syndrome, a protean fulminating infection of bowel, lungs, blood stream and brain, in those who are lmmunocompromised. diagnosis can be difficult by stool microscopy. thlabendazole has side effects but ivermectin is safe and effective. june 1995, there was an outbreak of c.difficde-associated diarrhoea (cdad) at st. james's hospital. the aims of this study were to determine the incidence and outcome of cdad in hiv positive and negative patients we prospectively reviewed all patients with diarrhoea, a positive c.difftcile cytotoxin assay, and in whom no other infectious cause for diarrhoea was identified demographic data, history of diarrhoeal episodes, risk factors and outcome were recorded. the incidence of cdad in hiv negative patients was 1.2 per 100 hospital admissions, compared to 1 per 100 admissions m hiv positive patients. the average number of courses of antibtotlcs received, in hiv negative patients prior to the onset of symptoms was 2.3, and 76% of this group were exposed to third generation cephalosporins. hiv positive patients received an average of 3.4 courses of antibiotics and no patients received third generation cephalosporins. there were no deaths due to cdad in hiv positive patients however 4 hiv negative patients died from severe pseudomembranous colitis in conclusion we documented a unexpectedly low incidence and complication rate of cdad in hiv positive patients this is surprising considering their multiple hospital admissions and exposure to ant~microbial and chemotherapeutic agents. the number of new positive hiv specimens detected at the virus reference laboratory has risen from a cumulative total of 638 m july of 1987 to 1597 in september of 1995. we examined our data to determine the proportional make up of these positives by major risk group. in august 1987 64.7% of positive specimens were from intra venous drug abusers (ivda) by september 1995 ivda made up 49% of the total positive hiv specimens. positive specimens from homosexual individuals rose fro.m l0 9% of total positives in august 1987 to 20.6% in september 1995 there were no recorded positive specimens from heterosexual exposure in august 1987 but in september 1995 8.9% of positive specimens recorded heterosexual exposure. a further category which includes blood donors, haemophiliacs, transplant patients and organ donors made up 25% of total positives in august 1987 and in september 1995 made up 22.4% of total positives. we further examined our data in order to show when these changes occurred by ascertaining how many new positive patients have been discovered per year in each of the main risk groups. see in the united kingdom echovlrus type 22 (echo-22) is regularly isolated, with nearly 200 reports annually to the central public health laboratory. reports increased during 1986-1988 and overall it is the second most commonly reported echovlrus in the u.k. echo-22 epldemiology is different to that of other enteroviruses; over 90% of patients with echo-22 tsolated are less than 2 years of age echo-22 shows distinctive and unique cytopathogenic features in tissue culture, and based on sequence analyses, it seems to belong to a separate subgroup of picornaviruses. echo-22 has been associated with respiratory symptoms in premature infants, myocarditis and severe encephalitis. in 1989 an outbreak of acute flaccid paralysis associated with echo-22 was described in jamaica in six patients, four of whom died. we describe three cases of sudden death in infants associated with echovirus type 22 infection case i: s d. born 20/7/94; birth asphyxia and death at two days of age; echo-22 isolated on 2217194 from spleen. this study assessed the antibiotic sensitivity of organisms causing urinary tract infections (uti) among genito-urinary medicine (gum) clinic attenders in order to determine whether it is worthwhile giving tetracycline for dipstick (nitrite) positivity, even in the absence of clinical features of uti. we looked retrospectively at 100 laboratory confirmed uti's diagnosed among gum clinic attenders over a period of eight months. we assessed antibiotic sensitivities of the organisms involved, and determined how many dipstick positive urines which were left untreated turned out to be real uti's. 86% of uti's were due to coliforms and 66% of these were sensitive to tetracycline. 4% of uti's were due to staphylococcus saprophyticus, 3% due to beta haemolytic streptococcus group b, 2% due to enterococcus, 2% due to proteus species and 2% due to coagulase negative staphylococci. 25% of nephur positive urines were left untreated. 32% of these were nitrite positive. failure to treat a positive urine dipstick which turned out to be a uti necessitated a further clinic visit for adequate treatment. nitrite positive urines should be treated as a uti, even in the absence of clinical features of uti, either with trimethoprim or tetracycline. the number of untreated uti's and unnecessary extra visits to gum clinics would have been reduced with the use of judicious antibiotic therapy for nitrite positive urines. strains of enterococci resistant vancomycin have been reported with increasing frequency. in 1995, we investigated an increase in the frequency of vancomycin-resistant enterococcus faecium (vref) among patients in the haematology/oncology unit using pulse-field gel electrophoresis (pfge) to genotype these isolates and to assist in establishing the source of these vref. eighteen clinical isolates of vref from blood, urine sputum and-faeces and two environmental isolates were collected from separate patients between march and july 1995. minimum inhibitory concentrations (mics) to several antibiotics including teicoplanin and vancomycin were determined by agar dilution. pfge were performed following smal restriction endonuclease digestion. antimicrobial susceptibility testing revealed high level resistance to vancomycin and teicoplanin; mics >128 mg/ l and >32 mg/l respectively. this antibiogram is consistent with the van a phenotype. pfge of all 20 isolates revealed identical patterns indicating clonal spread of vree subsequent implementation or infection control measures reduced the frequency of vref isolation. pfge proved useful in demonstrating clonal spread of vref and.in emphasising the need for infection control measures. a prospective audit of baeteraemia in our 600 bed teaching hospital was carried out from february 1991 to march 1993. clinical and microbiological data were collected on 520 episodes of bacteraemia in 78 patients. of these 230 (62%) were hospital acquired and 200 (38%) community acquired. urinary tract and respiratory tract sources were implicated in 30% and 14% of community acquired episodes, making e. coli and s. pneumoniae the commonest community acquired isolates (41% and 16% respectively). other gram negative bacilli accounted for 13% and s. aureus for 11%. coagulase negative staphylococci were the commonest hospital acquired isolate (24%) followed by s. aureus (22%), e. coli (i5%) and enterococcus spp. (10%). enterobacter spp. were the second commonest gram negative isolate (5%). central venous cannulae were implicated in 43% of hospital acquired cases. urinary tract infections accounted for 20%. 63% of which were catheter related. invasive diagnostic procedures (angiography, prostate and liver biopsies, sinography) were implicated in t0 episodes. gentamicin resistance was found in 9% of hospital acquired aerobic gram negative bacilli and mrsa accounted for 13% of hospital acquired s. aureus. these figures are higher than expected but may be explained by outbreak of mrsa and gentamicin resistant entercobacter spp. which occurred during the study period. the past severalyears have seen a significant increase in the recognition of moraxella (branhamella) catarrhalis as a respiratory pathogen (~). the pathogenic mechanisms employed by the organism are largely unknown, but adherence may play a role ~2~. in our investigation the haemagglutinating ' activity of 40 isolates of m. catarrhalis was determined by a microtitre method. no isolate agglutinated horse, chick or sheep red blood cells (rbc). seventeen isolates agglutinated human rbc, x~hile 7 of these 17 isolates also agglutinated rabbit red. blood cells. haemagglutination of human and rabbit red blood cells was inhibited by porcine mucin. galactose inhibited the haemagglutiriating activity of the 7 isolates which agglutinate both human and rabbit rbc and yet bad no effect on the haemagglutinating activity of the isolates which haemagglutinate human rbc alone. .electron microscopy studies of the bacteria demonstrated a diffuse outer fibrillar layer on the surface of haemagglutinating positive isolates, thislayer was subsequently removed following trypsin treatment, as was the haemagglutinating activity. a 200 kda trypsin sensitive protein appears to be associated wfth haemagglutinating properties. mrsa is an increasingly important cause of morbidity, and is spreading from large hospitals to smaller community-based facilities and nursing homes. the objective of this survey was to obtain an indication of the size of the mrsa problem in ireland prior to introducing national mrsa control guidelines. a survey of all microbiology laboratories in ireland was carried out over two weeks in spring 1995. for patients from whom mrsa was isolated during the study period standard demographic and clinical data were requested and period prevalence/1000 discharges was calculated. all 45 microbiology laboratories surveyed responded. mrsa was isolated from 448 patients during the 2 week period. the period prevalence of mrsa/1,000 discharges was 16.5. males aged 65+ had the highest rate of infection (50/1000 discharges). half of all isolates were from patients in surgical or medical wards, but 4% were from community-based sources e.g. gps, nursing homes, hospices. thirty-two percent of mrsa patients were infected rather than colonised. mrsa is clearly a substantial problem in ireland. while it is largely a hospital problem at present, the increasing trend for day procedures and shorter stays means that infection will increase in the community. a survey in a university hospital in the usa revealed 41% of mrsa cases to be communityacquired. tonsil core specimens were cultured for bacteria including mycoplasma, chlamydia and ureaplasma urealyticum in 70 children undergoing tonsillectomy for recurrent acute tonsillitis. serology for chlamydia and mycoplasma pneumoniae was obtained in 55 of the children. the polymerase chain reaction (pcr) was used to investigate the presence of chlamydia pneumoniae in core tonsil tissue. ureaplasma urealyticum was cultured in three children (4.3%) and mycoplasma salivarium in two children (2.8%). culture was negative for chlamydia pneumoniae and mycoplasma pneumoniae. the complement fixation test for chlamydia species was positive in 16/55 children (29%) indicating previous infection. specific immunofluorescence testing for c. pneumoniae was positive for lgg (titre> 16) in 10/55 (18%). igm antibody to c. pneumoniae and antibodies to c. trachomatis and c. psittaci were not detected. ninechildren (16%) had titres > 32 to m. pneumoniae. pcr failed to demonstrate c. pneumoniae. aerobic and anaerobic bacteria were cultured from all specimens. the culture of ureaplasma urealyticum in 4.3% of our patients indicates a higher rate of colonisation then previously thought. this study 49 irish journal of medical science demonstrates past infection with c. pneumoniae in 18% and with m. pneumoniae in 16% of children with recurrent tonsillitis. however c. pneumoniae and m. hominis do not play a significant role in childhood recurrent tonsillitis. multiply resistant enterococci are increasingly common causes of serious infection in hospitalized patients. high level gentamicin resistance (mic > 1000 mga) in enterococci further compromises the therapy of such infections. we have identified seven clinical isolates of enterococcus hirae demonstrating high-level gentamicin resistance (hlgr: mic > i000 mg/1). to our knowledge this is the first report of hlgr for this enterococcus species. plasmid analysis has demonstrated the presence of a single, large plasmid in all seven isolates, as well as several smaller plasmids in some of the isolates. filter mating experiments have revealed that in all seven cases, hlgr was transferred to a laboratory recipient e. faecalis jh-22 by conjugation. plasmid analysis of transconjugant strains confirmed transfer of the large plasmid in all cases. based on restriction enzyme profiles, two distinct conjugative plasmids were identified for the e. hirae isolates investigated. at present we are using southern blot techniques with oligonucleotide probes designed to hybridise to the hlgr determinant found in other species of enterococcus. the results will confirm whether or not the same resistance determinant is responsible for the dissemination of hlgr in the genus enterococcus. dublin 8. aminoglycosides remain commonly used in the treatment of severe gram negative infection and have conventionally been given on a twice or thrice daily basis. single daily dosing offers advantages with respect to less nephrotoxicity, better bactericidal activity, convenience, nursing time, cost and should avoid subtherapeutic dosing which has a significant impact on outcome. we reviewed serum gentamicin assays from january to december 1995 to assess potential toxicity and subtherapeutic dosing in patients who received once daily gentamicin and those who received multiple daily dosing. 4346 assays were performed in the study period. 520 of those were random assays and not included. there was a trend towards significantly less potentially toxic levels in the once daily group compared to the multiple daily group (p<0.1). once daily dosing produced significantly less subtherapeutic dosing (p<0.001). over 98% of peak assays in the once daily group were in the recommended range. we conclude that current practice of multiple daily dosing of gentamicin leads to significant underdosing and more potentially toxic trough levels. measurement of trough assays only in patients who are treated with once daily aminoglycosides is sufficient and will have considerable cost savings. respiratory, syncytial virus (rs virus) is a major respiratory pathogen of infants less than 1 year old. it occurs in annual epidemics during the winter and early spring in temperate climates. during rs virus epidemics a significant number of infants less than 6 months old are hospitalised with symptoms of bronchiolitis and pneumonia. rs virus exists in two antigenically distinct subgroups, a and b which are known to cocirculate in the same community during the same rs virus season. there is much debate regarding the virulence of one strain over the other. using a panel of monoclonal antibodies specifically directed against the two rs virus strains, 167 rs virus isolates from specimens sent to the virus reference laboratory, university college dublin, over seven consecutive rs virus seasons (1987) (1988) (1989) (1990) (1991) (1992) (1993) (1994) were typed and the rs virus subgroup predominance monitored. subgroup a was the most predominant of the rs virus isolates accounting for 67.7% of the total and was found to be the predominant rs virus strain in six out of the seven rs virus seasons studied. subgroup b predominated in a season in which the number of rs virus detections peaked much later than normal. treatment of fungal infections in patients in intensive care unit (icu.) is usually empiric. the aim of this study was to identify candida species isolated from i.c.u. patients and to test their susceptibility to antifungal agents to enable more directed therapy. forty candida sp. from 23 patients in i.c.u. were isolated from the following sites, blood culture (5), central venous catheter (7), chest drain fluid (5), wounds (8), catheter urine (8), bronchial lavage (3), sputum (4). strains were identified by standard procedures. minimum inhibitory concentrations of amphotoeracin b, 5-flucytosine and fluconazole were obtained by agar dilution test and e test. the isolates were identified as c.albicans (22), c.glabrata (8), c.krusei (5), c.tropicalis (2), c.parapsilosis (2), c.kefyr (i). all the candida species were sensitive to amphoteracin b (mic = 1-<025mg/l), and 5 flucytosine (mi c= <025mg/l). c.albicans and c.parapsilosis were fluconazole sensitive (mic = 16-15mg/l). four of eight c.glabrata were fluconazole resistant (mic = >256mg/l). c.krusei, c.tropicalis and c.kefyr were also resistant (mic = >256 mg/l). wbilst there were no major discrepancies between the agar dilution test and the e test, the agar dilution test was laborious and required a high degree of skill. the e test was easier to read and more reliable results were obtained provided the inoculum was carefully standardised. this study shows that azole antifungals should not be ganglion is probably the commonest tumour encountered in the hand and the wrist. it often arises from tendon sheath or lining of a joint capsul. the treatment can be surgical or nonsurgical, the latter includes aspiration with or without injection of steroids. surgical treatment of ganglion can pose a difficult situation to deal with. it requires hand surgeon to deal with one such problem, we present a 46 year old man with tender mass in the hypothenar eminence. during surgical exploration it was obvious that the ganglion was infiltrating the wall of the ulnar artery, and the histology proved this later. the clinical features, management arid the outcome of this unusual case are discussed. ischaemic preconditioning (ipc) of the myocardium with repeated brief periods of ischaemia and reperfusion (i-r) prior to prolonged ischaemia significantly reduces subsequent infarction. following ipc two "windows of opportunity" (early and late) exist during which prolonged ischaemia can occur with reduced myocardial infarction, we investigated if ipc of skeletal muscle prior to flap creation improved subsequent flap survival and perfusion in either early or late windows. the latissimus dorsi muscles (ldm) of sprague-dawley rats were used. group 1: (control, n=12). the ldm was elevated as a thoracodorsatly based island flap group 2: (early ipc, n=8). the ldm was preconditioned with two 30 minute episodes of normothermic global ischaemia with intervening 10 minute episodes of reperfusion prior to elevation. group 3: (late ipc, n=8). the ldm was elevated 24 hours after ipc ischaemia was created by occlusion of the thoracodorsal artery and vein and the intercostal perforators having previously isolated the muscle on these vessels. muscle perfusion was assessed by a laser doppler perfusion imager. one week after flap elevation the percentage of muscle necrosis was measured by computer-assisted planimetry ipc significantly reduced muscle flap necrosis (table) in both early and late windows. muscle flap perfusion was similar in all groups. this study compares the biochemical, serological and histopathological findings in 20 women with chronic hepatitis c virus (hcv) infection with 20 age-matched women with established autoimmune hepatitis (aih). there is increasing evidence of autoimmunity in hcv liver pathology. because of different treatment regimens for hcv (d-interferon) and aih (steroids, immunosuppression), clear distinction between the two diseases is desirable. liver enzymes (alt, ast, alkaline phosphatase), antinuclear factor (anf), anti-smooth muscle (asm) and antimitochondrial (ama) antibodies are compared for both groups of patients. liver biopsies from all women were compared using the grading and staging system of ishak et al (1995) . the results show that while some women in both groups show elevated liver enzymes, positive anf and asm autoantibodies, the values are much higher in aih. similarly aih patients show overall more severe disease on liver histology. both groups demonstrate poor correlation between histological features and autoantibody titre. we conclude that distinction between hcv and a!h is usually possible with liver histology and serology. some women with chronic hcv and positive autoantibodies however," demonstrate a histological and serological picture suggesting that chronic hcv may be mediated by an immunopathogenic mechanism. irish journal of medical science terminal changes only, 5 showed lymphocytic meningitis and 4 of these also had perivascular lymphocytic inflammation (cd3 positive) in the subependymal regions, brainstem and choroid plexus. two brains showed purulent meningitis and one case had central pontine myelinolysis probably related to profound metabolic disturbance. basal ganglia mineralization, hiv encephalitis (hive) or hiv leucoencephalopathy (hivl) were not present. these findings differ considerably from those described in us cases, in whom the majority have evidence of hiv within the cns. relatively early death from systemic infection may account for the lack of hive/hivl in these cases. the lymphocytic meningitis and perivascular inflammation may represent an immuno-allergic reaction, previously reported as "early" changes, regarded as important in inducing vascular damage which allows subsequent entry of h1v into the brain. the aim of this study is to assess apoptosis in areas of interface hepatitis and spotty necrosis in hepatitis c virus (hcv) infected liver biopsies, and to correlate the degree of apoptosis with severity of histological activity. 25 liver biopsies were randomly selected from a group of type lb hcv positive women. these patients were diagnosed by recombinant immunoblot assay (riba) test and the presence of hcv rna was confirmed using the polymerase chain reaction (pcr). apoptosis was demonstrated in the biopsies by the oncor apoptag in-situ hybridisation technique. the average number of apoptotic hepatocytes per portal tract, and within the parenchyma per 10x objective, was determined. the modified histological activity index (h.a.i.) was used to score each biopsy. comparison of the results shows that increasing numbers of apoptotic hepatocytes are consistently associated with increasing scores for interface hepatitis and spotty necrosis. it is concluded that apoptosis occurs in hcv infected livers and that it correlates with increasing histological activity .indicating a significant role for apoptosis in the pathogenesis of hcv liver disease. and university of edinburgh, scotland. paediatric aids represents only 5% of cases worldwide, in most published series parental iv drug use was the main risk factor, cns pathology was a late feature of the disease and antiretroviral treatment had been given. we studied eleven brains from romanian children with probable postnatal hiv infection using standard neuropathological stains and immunostains for lymphocyte markers and hiv p24 antigen. death was due to systemic infection, mostly pneumonia or gastroenteritis; antiretroviral treatment had not been given. three cases showed we studied the role of the p53 and bcl-2 genes in the pathogenesis of post-transplant lymphoproliferative disease (ptl). ten cases were examined by immunohistochemical and molecular methods. immunohistochemistry was performed using standard and antigen retrieval methods, with the p53 do-7 and the bcl-2 oncoprotein clone 124 antibodies. dna was extracted from paraffin blocks and subjected to pcr, and single-strand conformation polymorphism (sscp) analysis searching for mutated p53 genes. samples showing any evidence of aberrant migrations were further analysed by direct sequencing. pcr was also used to detect bcl-2 gene rearrangements. we employed a technique called representational difference analysis to search for previously undescribed translocations or deletions which may be involved in breast carcinogenesis. dna was isolated from both invasive ductal carcinoma of the breast and normal tissue from the same patient. following restriction enzyme digestion and tigation of oligonucleotide linkers, pcr was carried out on both tumour and normal dna using oligonucleotide specific primers to obtain a representation of each genome (amplicons). digestion with the same restriction enzyme removed the original linkers, and a second set of oligonucleotides were ligated to normal amplicons only. the tumour derived amplicons were subtractively hybridised to the normal and subsequent pcr was used to isolate fragments unique to the normal dna (difference products). this was possible since oligonucleotides were ligated to normal dna only. following a series of further subtractive hybridisations and subsequent amplifications, purified difference product was obtained. difference products in the size range 500-1500 bp were obtained. following further rounds of subtractive hybridisation and amplification, purified difference product will be sequenced and characterised by comparison with known gene sequences. the chromosomal location of the affected gene will be established by in-situ hybridisation and somatic ceil hybridisation, using the difference product as probe. protein s is secreted by osteoblasts and case reports of reduced bone mineral density in patients with total protein s deficiency have lead to the hypothesis that this inherited disorder is associated with generalised osteoblast dysfunction predisposing to osteoporosis ~. we have assessed bone formation in 8 patients (2 male and 6 female) with total protein s deficiency and 4 controls (2 male and 2 female) using two recently available sensitive markers; serum osteocalcin (oc) and serum procollagen 1 carboxyterminal peptide (p1cp), both secreted by the osteoblast. the mean total protein s level amongst the patients was 40 _+ 12% (ref range 62-135%), mean oc was 14.2 ng/ml (ref ~'ange 5.2-59 ng/ml) and the mean picp was 81.5 + 15 uga (ref range 38-02 ug/1). in the control group, mean oc was 12.6 _+ 5 ng/ml and the picp was 124 + 37 ug/1. there was no statistical difference between both groups using either marker. in conclusion bone formation as assessed by serum osteocalcin and p1cp appears to be normal in patients with total protein s deficiency. hereditary spastic paraparesis (hsp) is a variably expressed neurodegenerative disorder which exhibits clinical and genetic heterogeneity. hsp can be inherited in an autosomal dominant (ad), autosomal recessive (ar) or x-linked manner. ad-hsp has been linked to a number of loci. we have ruled out linkage to these loci in a large irish family affected with ad-hsp. the aim of this study was to determine whether ad-hsp is linked to spinocerebellar ataxia loci (sca), sca-i & sca-ii. ad-hsp can be clinically similar to sca. dna was extracted from blood taken from 45 co-operating family members. microsatellite markers spanning the sca-i and sca-ii loci were amplified by pcr. individuals were genotyped and linkage analysis was carried out using the linkage set of programs. significantly negative lod scores were obtained for both sca-i and sca-ii loci. d6s274 gave maximum exclusion of 18cm on either side of the sca-i locus with a lod of -2.11 at a recombination fraction of 0.18. d12s105 gave maximum exclusion of 13cm on either side of the sca-ii locus with a lod of -2.08 at a recombination fraction of0.13. other markers examined also outruled linkage to these loci. we conclude that the gene for ad-hsp is unlinked to the major sca loci. serum vitamin b12 is frequently measured in the investigation of anaemia, and in screening neurological and other disorders. frequently, patients are found with low serum vitamin b12 level with a normal hb and without clinical abnormalities relevant to vitamin b 12 deficiency. this study was carried out to determine the significance of a low serum vitamin b 12 level. 959 vitamin b12 measurements were carried out over an 8 month period using a chemiluminescence method (abbott imx). clinical data was obtained retrospectively. of the 959 samples 81 (8.4%) representing 65 patients had a low serum vitamin b12 level (>180 pg/ml) with a mean of 152 pg/ml (39-179). data was available on 44 patients. 20 (45%) had a hb below the normal range with median serum vitamin b12 level of 158 pg/ ml (75-184). 19 (45%) had a normal hb, mcv and mchc with a median serum vitamin b12 of 152 pg/ml (52-210), and 5 had a normal hb with an abnormal mcv or mch. lft's, autoantibodies, schillings test and bone marrow examination data will be presented. in conclusion in patients with a low serum vitamin b 12 level, there was no significant difference in the b12 levels in those patients with a normal or a low hb concentration. it would appear that serum vitamin b12 is a poor discriminatory test but that changing the normal range may not help in screening. low serum vitamin b12 on its own may not appear to provide adequate grounds for lifelong replacement therapy. of 1 in 133 for males and 1 in 172 for females. as expected the overall incidence of hodgkin's was lower with one third of male and one quarter of female lymphoma cases affected by the disease. a distinct age specific pattern is evident depending on lesion type. marked variation in incidence levels were noted throughout the study region. an extremely varied pattern is evident in the survival rates for lymphoma patients. the cork and kerry rates for malignant lymphoma are relatively low when compared with international levelstzl obstetric complications and schizophrenia: methodology and mechanisms perinatal complications and clinical outcome within the schizophrenia spectrum 96) negative symptoms, cognitive impairment and duration of initially untreated psychosis in schizophrenia davnet's hospital, monaghan, and royal college of surgeons in ireland retinal pathology in kearns sayre syndrome mitochondrial dna and disease mitochondrial myopathies: clinical features, investigation, treatment and genetic counselling phenytoin induced pseudolymphoma. a report of a case and review of the literature cutaneous reactions in head injured patients receiving phenytoin for seizure prophylazis hydantoin induced pseudopseudolymphoma branhamella catarrhalis: an organism gaining respect as a pathogen correlation between branhamella catarrhalis adherence to oropharyngeal cells and seasonal incidence of lower respiratory tract infections 206) epidem1ology and survival rates for all 324 lymphoma patients registered in cork and kerry over the eight year period an indepth review of all 324 lymphoma (icd -o code 196) patients registered by the sourthern tumour registry during the eight year period 1983/1990~l annual age adjusted rates of 7.6. and 5.4 per 100,000 were seen for males and females respectively. these levels indicate a lifetime (up to 75 yr) risk references 1. cancer, the irish experience cancer incidence in five continents volume v immunohistochemistry for bcl-2 oncoprotein without antigen retrieval gave negative results, but with antigen retrieval, showed positive staining in 6 out of 8 cases. no bcl-2 rearrangements were detected by pcr. the combination of sscp and sequencing confirmed only wild type dna in all cases, p53 immunohistochemistry by standard methods revealed positive staining in only one out of nine samples analysed. when the antigen retrieval method was employed for this antibody, positive staining was seen in > 10% of tumour cells in four further cases.our results suggest that p53 does not play major role in ptld. bcl-2 overexpression but not rearrangement may contribute to the development of ptld. transplant arteriopathy (ta) is the major cause of death in cardiac allograft recipients. the pathogenesis is unclear. we have previously shown a plasma cell predominance in the infiltrate of ta, leading us to hypothesise a role for epstein-barr virus (ebv) infection in its pathogenesis. an association between cytomegalovirus (cmv) and ta has previously been suggested. the aim of the study was to investigate the role of epstein-barr virus (ebv) and cytomegalovirus (cmv) in the pathogenesis of ta.we performed pcr for cmv and ebv dna and protein (lmp) in seven cases of ta, involving cardiac allografts. restriction mapping was used to confirm that pcr products were either cmv or ebv dna respectively.cmv dna was found in four cases. ebv dna was found in six of the seven cases and ebv lmp staining was present in six cases. ebv was detected in all cases by either pcr or ihc.our results suggest that ebv infection may play a pathogenic role in transplant arteriopathy. the evidence for a similar role for cmv ~s less strong. st. vincent's hospital. hereditary spastic paraplegia (hsp) is a neurodegenerative disorder characterised by progressive spasticity, primarily of the lower limbs. it can be inherited in an autosomal dominant (ad), autosomal recessive or x-linked manner~ we have identified a large irish family (family a) affected with ad hsp that cosegregates with dementia. three. loci have previously been identified that are linked to ad hsp in families of different ethnic origin. the locus on chromosome 2 is reported to be the major hsp locus. the aim of the present study was to examine family a for linkage to the chromosome 2 hsp locus.dna has been extracted from blood taken from all 45 co-operating family members for genotyping. polymorphic microsatellite markers from chromosomal region 2p24-21 have been amplified by pcr, electrophoresed on a denaturing polyacrylamide gel and detected by silver staining. linkage analysis was carried out using the linkage series of programs. linkage analysis excluded the hsp gene from the chromosome 2p locusl the most significant marker was d2s367, with a lod score of-2.12 for recombination fraction 0.19, thereby excluding approximately 19cm either side of this marker. negative lod scores were also obtained for the other markers chosen (d2s391, d2s392, d2s352, d2s 174) excluding 20cm, 8cm, 4cm and 8cm respectively.the current study has therefore successfully excluded linkage of ad hsp in family a to the major locus on chromosome 2p. further studies are underway to exclude linkage of hsp in this family from other candidate loci, prior to carrying out a genome wide search. the presence of dementia in this family in association with hsp suggests that a new and as yet unidentified gene is responsible. vincent's hospital. eye and ear hospital, dublin. ched is a corneal endothelial dystrophy characterised by diffuse bilateral corneal opacities resulting in impaired vision. both autosomal dominant and autosomal recessive modes of inheritance have been described. another endothelial dystrophy, posterior polymorphous dystrophy (ppmd) has been linked to 20qll.we have used homozygosity mapping to analyse a pedigree with autosomal recessive ched for linkage to 20ql.1. all affected individuals are offspring of consanguinous matings. homozygosity mapping is based on the principle that these offspring would be homozygous for genetic markers near the disease gene. homozygous regions would be random between different offspring of these matings, except at the di-sease locus shared by affected offspring.dna was extracted from blood taken from 22 family members, 14 of which have ched. allele frequencies were determined in pooled dna from affected individuals. pooled dna from unaffected individuals was used as a control. at the disease locus, a shift in allele frequencies towards a single homozygous allele would be observed in the affected dna pool. pooled dna was genotyped by pcr for 3 polymorphic microsatellite markers in the region of20qll. pcr products were separated on a polyacrylamide gel and visualised by silver staining. similar allele frequencies were observed at these loci in both dna pools demonstrating independent assortment of alleles. in addition, affected and unaffected family members were individually genotyped at these loci and no significant loss of heterogeneity in the affected individuals at these loci was observed. these data indicate exclusion of linkage of the ched gene to 20qll. key: cord-006391-esnsa4u5 authors: nan title: abstracts 5(th) tripartite meeting salzburg/austria, september 9–11,1982 date: 1982 journal: langenbecks arch chir doi: 10.1007/bf01279099 sha: doc_id: 6391 cord_uid: esnsa4u5 nan the gastric secretion capacity increases during the first year after all types of vagotomy which is assumed to be important for the development of recurrent ulceration. however, the cause of this reestablishment of the preoperative gastric function is not adequately explained. the purpose of this study was to investigate the vagal innervation of the parietal cell mass in dogs 11/2 year after truncal vagotomy (tv), selective gastric vagotomy (sgv) and parietal cell vagotomy (pcv). material and methods: mongrel dogs supplied with a gastric fistula were used. the groups comprised 5 dogs with tv, 6 dogs with sgv and 6 dogs with pcv. the acid secretion was measured before and one month and one year after vagotomy following insulin and pentagastrin stimulations. operative technique: a gastroduodenostomy was performed in addition to the gastric fistula operation in the dogs randomised for either tv or sgv. the vagotomy operations were performed after the prevagotomy secretion experiments. tv by a 4 cm resection of the main truncs through a left side thoracotomy and sgv as described by amdrup [1] . the pcv dogs had no gastroduodenostomy added but the technique at the lower esophagus was identical with the sgv and the dissection of the lesser curvature was extended 1 cm distal of the sharp physiological and histologically determined borderline between the fundus and the antrum. about 11/2 year after the vagotomy the dogs were sacrificed by an acute experiment. the persistent or the regenerated vagal innervation of the parietal cell area was mapped out by neutral red excretion elicited by electrical stimulation of either the thoracic or the cervical vagal nerves [2] . results: one year after vagotomy no increase in insulin response could be demonstrated in the tv and sgv groups, but a gradual increase to about 50 % of prevagotomy output occured in all pcv dogs. reliable neutral red experiments were performed in 2 tv, 6 sgv and 4 pcv dogs. the stimmulated neutral red excretion was scanty to the same extent at the cardia region in all three groups, but a distinct antralfundic border was outlined in all 4 pcv dogs. the border was, however, only suggested in one of the sgv dogs. conclusion: pcv including denervation of 1 cm distal of the antral-fundic border do not prevent a functional important reinnervation of the distal part of the parietal cell area. no important reinnervation seems to occur at the cardia region after any of the three vagotomy procedures. 1 and insulin (0.2 ugkg -1) i.v. bolus; after uni-and then bilateral truncal vagotomy. each study was 120 minutes and blood was taken at 1, 3, 5, 7 and then 10 min intervals. gastric acid was measured by autobiuret titration. plasma was stored at -20°c until assayed for pp by ria using an antibody sensitive to 3 fmol ml-1. results are expressed as mean total increment _+ se. significance: p < 0.01". bombesin-stimulated gastric acid secretion was not significantly altered by vagotomy (p < 0.05), whereas that stimulated by insulin was significantly inhibited by bilateral truncal vagotomy (p < 0.01). bilateral and right hemi-vagotomy significantly inhibited pp release by bombesin (17 < 0.01), however; only bilateral truncal vagotomy significantly inhibited pp release by insulin (p < 0.01). these results suggest that the measurement of pp release by insulin or bombesin is a sensitive index of vagal integrity and that bombesin-released pp may specifically delineate the integrity of the right vagus. since the measurement of gastric acid secretion after operation is both uncomfortable and often difficult to interpret, the value of a simple blood test to determine vagal integrity may be of considerable clinical relevance. glucose (g) or oleate (o) empty more slowly from the stomach than 0.15m nac1 (s). this chemoregulation may be achieved by resistance beyond the proximal stomach [1] . we sougth to define the site of this resistance. expt. 1: gastric emptying of s and 600 mm g was measured in 5 dogs with intestinal cannulas 45 cm distal to the pylorus while gastric pressure was maintained at 10, 18 or 26 cm h20 with a barostat. the dogs were studied either with an uninterrupted intestinal stream or with duodenal effluent diverted through a second barostat prior to its return downstream. since the latter ensured a constant pressure at the ligament of treitz, any observed effects on emptying could not be due to resistance further distally. expt. 2: emptying of s, g, and 40 mm oleate emulsion was measurd in 5 dogs without cannulas after control antroduodenal transection and after antrectomy while gastric pressure was controlled at 11, 17 and 23 cm h20. emptying rates expressed as the average of the volumes emptied at the 3 pressures. under both conditions in expt. 1 emptying rose with pressure yet s emptied faster than g. thus a major site of chemoselective resistance to emptying of liquids lies proximal to the ligament of treitz. after antrectomy s and o emptied faster than before, however, the differences between saline and nutrients were maintained. resistance, therefore, does not reside in the antrum or pylorus and clearly the duodenum is implicated. gastric emptying of liquids may be abnormally rapid in du. this abnormality my be due to a selective loss of inhibition in response to acid since previous studies [1] have demonstrated rapid emptying of acid solutions but normal emptying of glucose and fat. previous investigations, however, used meals of only one concentration, so differences between dus and normals (n) may have been minimized by selection of a supra-maximal inhibitory dose. furthermore intragastric ph was not maintained constant so emptying could have been slowed by higher rates of acidification in du. we therefore studied emptying of graded concentrations of citric acid, glucose and oleate in 500 ml meals. gastric volumes were measured by the george technique in dus and n at 5 rain intervals for 30 min after ingestion. gastric ph was maintained constant by intragastric titration (3.0 for acid; 7.0 for nutrients). subjects received one type of meal on separate days and the doses were randomly administered. dose dependent inhibition of emptying in response to acid and nutrients occured in n and du, but emptying was faster in du (p<0.05). with glucose and acid the difference occured within 0-5 min whereas with fat the difference occurred between 5-30 min. thus (1) dus do not have a selective loss of inhibition of emptying in response to acid, and (2) the mechanisms which control emptying of fat and which are disturbed in du differ from those which control emptying of glucose and acid. 1 gross ra, isenberg ji, hogan d, samloff im (1978) the effect of fat on meal-stimulated duodenal acid load, duodenal pepsin load and serum gastrin in duodenal ulcer and normal subjects. a means of reducing the requirement for postoperative nasogastric intubation would be advantageous. we report the effect of cimetidine on postoperative nasogastric aspirates. thirty patients undergoing elective abdominal aortic surgery or colonic resection were entered into a double blind randomised study, receiving cimetidine 200 mg 4 hourly or placebo (saline) by continuous intravenous infusion from the morning of the first postoperative day. volumes of 4 hourly aspirates were recorded and ph and (h ÷) of samples measured. nasogastric tube removal accorded to standard clinical practice. it has been shown, that the duodenum has a better ability to resist acid than jejunum or ileum. this resistance was attributed to alkaline secretion (a.s.) of the mucosa, because pancreatic and bile secretion were excluded. recent in vitro studies demonstrated an energy dependent hco3-transport (flemstroem 1980 am j physiol g 239: 198). we were interested in studying the role of blood flow (bf) in the production of alkali in an in vivo preparation. min. three groups of animals were studied: i) controis with normovolemia for 120 min. ii) normovolemia for 60 min, thereafter 60 min of shock induced by bleeding to 40 mmhg mean arterial blood pressure. iii) 60 min normovolemia and 60 min vasopressin (0.2 i. u./kg/min) under normovolemia. in controls a small decrease in bfand a.s. as well was observed. both shock and vasopressin induced a significant reduction in a.s. and bf. the relation ofbf to a.s. was found to be exponential (y = 2.71 + 0.21x-0.003x 2 r= 0.71; p<0.05). the reduction in a.s. during shock was much less, when shock induced acidosis was compensated by i.v.-infusion of hco 3 (0.3 ,uequ/kg/min). conclusion: the alkaline secretion of the proximal duodenum is dependent on blood flow and the arterial [hco~. there has been considerable controversy as to the nature of the offending agent in the etiology in reflux oesophagitis in man. in rat studies, surgically induced reflux oesophagitis was shown to be correlated with the presence of active trypsin in the reflucting juice. reflux of bile and gastric juice with a ph of 4 did not induce oesophagitis. even short periods of oesophagitis (1-6 weeks) showed an increasing amount ofpanmural fibrosis which even further increased after a reflux abolishing roux-y operation. the pattern of the collagen content of the full thikkness oesophageal wall was similar to that found in human reflux oesophagitis. the hypothesis that active trypsin is also in man responsible for reflux oesophagitis prompted to the following investigation. 24 patients with typical gastrooesophageal reflux symptoms and 17 control patients were endoscopically examined. on entering the stomach with the endoscope samples of gastric juice were taken for ph and active trypsin determinations. trypsin activity was determined with a kinetic method using s-2160 (kabi vitrum b.v. amsterdam) as substrate. results: in all patients some trypsin could be detected in the gastric juice. in those patients with endoscopically erosive and/or ulcerative changes (n = 14) the trypsin content was significantly elevated compared to the controls (/7<0.05 wilcoxon). conclusions: the composition of gastric juice is determined by gastric secretion and duodenogastric reflux. duodenogastric reflux is increased in patients with symptoms of reflux oesophagitis [1] . trypsin in gastric juice (ph 3.5-7) will stable and partly active over prolonged periods [2] . so from our study in man one may conclude that the high concentration of trypsin in the gastric juice in patients with reflux oesophagitis, may well be the etiological factor of the oesophagitis. disinfection, using artificial contamination of hands with escherichia coil and 'surgical' disinfection directed against the resident microflora of the hands. the authors who developed the procedure have reported unexpectedly high potency for n-propanol compared to iso-propanol, and, in turn, for iso-propanol compared to povidone-iodine and chlorhexidine preparations [1] . however, using the same procedure we have been unable to find any significant differences in activity between these agents. we have identified several potential sources of error including the method of applying e. coliand its spontaneous loss of viability, the use of neutralizers before disinfection, the differing surfactant effects of the agents, and the absence both of a control untreated area and of a cross-over of disinfectants studied sequentially. in our parallel tests using an excision-sample technique [2] which is considerably more sensitive than the dghm procedure, we have observed the following mean reductions in the counts of accessible bacteria: iodine in ethanol, 96%; povidone-iodine, 89%; chlorhexidine in ethanol, 88%; iso-propanol, the purpose of this study was to compare radiation injury in guinea pig small bowel (1) devoid of contents (2) containing bile (3) containing pancreatic juice. one group was intact control animals not radiated. another was intact animals radiated. in group 3 a roux y cholecystojejunostomy was constructed and the bile duct ligated. thus one limb contained bile, the other pancreatic juice. in group 4 a blind roux y was constructed such that one limb was empty of contents and the other contained both bile and pancreatic juice. each animal was subjected to a single dose of 1600 rads via an abdominal port and sacrificed four days later. the severity of injury was judged by counting the number of surviving crypts per circumference. damage was further evaluated by histologic criteria -mucosal loss, edema, inflammation, hypervascularity and loss of mucus. the maximum histologlc grading score on this scale was 20. the microscopist was ,,blinded" as to the origin of each tissue section. for histologic grading all radiated groups were significantly different from control (p<0.02) and from one another (/r<0.05) except pancreatic juice vs. empty. intestine devoid of contents sustains a mild, but significant radiation injury. the presence of pancreatic juice enhances the damage. pure bile makes for yet more severe injury but still significantly less than normal whole intestinal contents which contain both bile and pancreatic juice. the main problem facing patients with ulcerative colitis after mucosal proctectomy and ileo-anal anastomosis is severe frequency of bowel action. our hypothesis was that either an artificial valve [1] or reversed ileal loop might improve intestinal absorption and slow transit. we tested it in dogs after colectomy and low ileo-rectal anastomosis (ira). body weight, xylose absorption, serum albumin, folate, vitamin b12, calcium, urea and electrolytes, full blood count, faecal weight and mouth to anus transit time [2] were measured before operation. the dogs then randomly underwent either ira alone (control, c, n = 7), ira with a 10 cm reversed loop (ira + rl, n = 6) or ira with an artifical valve (ira + v, n = 6). body weight, haematological estimations and faecal chemistry were measured weekly for 3 months post operatively. xylose absorption and transit time were measured a minimum of 2 months after operation. body weight decreased significantly after each type of operation. there was no difference however, between (ira + rl) and c or (ira + v) and c. likewise, haematological and faecal measurements after both test operations did not differ significantly from c or from pre-op, measurements. the grosfeld valve prevented the reduction in transit time that occurred after the control operation, whereas the reversed loop did not. use of such a valve in combination with mucosal proctectomy might slow transit, but is unlikely to improve absorption. it seems worthy of further study. histologic grading___ sem crypt count-+-sem we have examined the effects of changes in intestinal blood flow induced by hypovolaemia, the mesenteric vasoconstrictors somatostatin and vasopressin and the mesenteric vasodilator prostaglandin e1 (pge1), on intestinal absorption, electrical activity and volume. in each of 7 dogs a 75 cm jejunal segment was isolated and serosal electrodes attached. in absorption experiments the segments were perfused at 2.9 ml/ rain with a solution of 90 retool/1 sodium and 100 retool/1 glucose. to measure segment volume, a solution of 50.7 g/1 mannitol was perfused. this solution shows zero net absorption but does not alter electrical activity. intestinal motility and absolute volume were monitored by gamma camera. experiments were performed after a steady state was reached. absorption and transit time were measured using non-absorbable markers. electrical fast wave activity was reduced by (a) intravenous somatostatin 2.5 mcg/kg/h (28+1.4 to 11_+3.4; mean_+sem/5 rain) and abolished by (b) intravenous vasopressin 1.2 u/kg/h (p<0.001). motility was inhibited and mean transit time was prolonged (a) 7.4 to 15.3 rain and (b) 4.4 to 19.9 rain (/r<0.001). intestinal volume rose by (a) 12.3___ 3.7 ml and (b) 15.4-+4.7 ml (p<0,001). absorption was unchanged by somatostatin and fell with vasopressin (5.58---0.7 to 4.35-+0.68 ml/5 min). acute blood loss sufficient to lower the central venous pressure by 5 cm of water produced identical changes to somatostatin. pge1 produced no alteration in electrical activity or absorption. intestinal absorption is resistant to changes in intestinal blood flow whilst electrical activity is depressed and intestinal volume increased by mesentric vasoconstriction. the accepted views on the pathogenesis of amoebic lesions in complicated amoebic colitis are that amoebae produce a toxin resulting in cytolysis and necrosis. this concept may adversely affect the management of patients with complicated amoebic colitis by implying that once the amoebae are killed, the disease process is arrested and colonic perforation is unlikely. we present an alternative hypothesis that 'complicated amoebic colitis is the result of vascular compromise'. transmural disease is caused by amoebic invasion of vessels supplying a segment of the colon with subsequent thrombosis and ischaemic necrosis of the affected area. the ischaemic nature of the necrosis is suggested by its shape, and the demonstration of vascular thrombosis on dissection ofresected colons which have perforated. amoebic invasion of blood vessels can be demonstrated histologically. the ischaemic nature of the lesions can be confirmed by angiographic examination of the resected colon. vascular occlusion can be demonstrated pre-operatively with the use of selective mesenteric angiography, which clearly delineates the ischaemic segment of the colon. selective rnesenteric angiography in 27 patients with fulminating amoebic colitis aided the preoperative diagnosis of colonic infarction before signs of visceral perforation had developed and permitted life saving surgical intervention. there is accurate correlation between angiographic and operative findings. in postdysenteric colitits associated with amoebic strictures of the colon, mesenteric angiography will demonstrate the ischaemic nature of the stricture and accurately define the extent of resection. after ca. the mean age at operation was 33 and 61 years respectively. anal canal length was 3.5___0.1 cm (mean + s.e.m.) after ia and 3.7___0.1 cm after ca. a resting tone of 66___ 3 cm water after ia and 53 -----10 cm water after cawas recorded. squeeze pressure was 123___9 cm water after ia and 79-----3 cm water after ca. the recto-anal reflex was present in 54% of the ia patients and in 75 % of the ca patients. a mean daily bowel frequency of 3.5___0.2 followed ia and 4+_1 after ca. these findings show that after ia anal pressures are within the normal reference range for our laboratory [2] . lower pressures were found after ca, probably due to the older age of the patients in this group [3] . normal anal canal length, the integritiy of the striated sphincter and in most cases preservation of the recto-anal reflex contribute to satisfactory continence following peranal anastomoses. perineal descent is found in patients with idiopathic faecal incontinence (ifi) and patients presenting with symptoms of the descending perineum syndrome (dps), who have no incontinence. manometric, radiological and neurophysiological studies were performed in 17 patients with ifi, all of whom leaked during rectal infusion of 1500 ml saline, 18 patients with dps, who were continent of rectally infused saline, and 14 matched controls. both patient groups exhibited similar degrees of perineal descent below the pubococcygeal line of straining (ifi, -5.1___2.1 cm; dps, -5.9 + 1.7 cm) compared with controis (-2.4__+ 1.5 cm;p<0.005 in both cases), and similar prolongation of both the latency of the cutaneoanal reflex (ifi, 13___5 ms; dps, 16+6 ms; controls, 9+4 ms;/,<0.005 in both cases), and motor unit potential duration (ifi, 9.1___2.6 ms; dps, 8.4___2.0 ms, control, 6.7+1.5 ms; p<0.01 in both cases). moreover, both groups had an abnormal ano-rectal angle (/,<0.005), though this was more obtuse in ifi than dps (120___21 ° vs 107___ 15°;p<0.05). however, while patients with ifi had lower sphincter pressures than normal (basal; 39___17 vs 81___29 cm water; /7<0.005; squeeze, 96___18 vs 208___84 cm water; /7<0.001), and required a lower rectal volume to inhibit sphincter tone for more than one minute (32 + 18 vs 76___29 ml; p<0.00l), these values were normal in patients with dps (basal; 87___ 30 cm water; squeeze, 198_+90 cm water; rectal volume, 52___46 ml). these findings suggest that perineal descent and neuropathy are not necessarily associated with incontinence if sphincter pressures remain normal. aminoacids administered either enterally or intravenously stimulate gastric secretion in both dog and man. it has been suggested that absorption of amino acids from the gut into the circulation might contribute to the intestinal phase of gastric secretion. the mechanism of this stimulation by amino acids remains uncertain but in an earlier communication we reported the direct action on the parietal cell of phenylalanine, glutamine and alanine [1] . in the present study using (14c) aminopyrine uptake as an index of dispersed parietal cell secretory function, the interaction between histamine and individual amino acids and the effect of the histamine h2-receptor blocker ranitidine (10-4 molar) on these interactions have been examined. results (percentage of maximal stimulation produced by 10-4 molar histamine) results (percentage of maximal stimulation produced by 10-4molar histamine) methionine glutamine alanine amino acids only (10-2molar) 26 32 29 amino acids and histamine (10-4molar) 175 -137 amino acids and ranitidine (10-4molar) 10 17 7 histamine and ranitidine -complete inhibition -25 125 1. the response to the combination of amino acids and histamine was greater than the sum of the maximal responses to each of these agents alone. 2. the histamine h2-receptor antagonist ranitidine completely inhibited the histamine stimulated response but only partially inhibited secretion stimulated by amino acids. we conclude that amino acids act directly on the parietal cell by a mechanism which is not solely dependent on histamine. 1 sex related differences in gastric acid secretion have been documented (lilja et al, 1967) . it has been also reported that male rats have a significantly higher serum gastrin concentration than females and that oophorectomy increases serum gastrin to male levels (lichtenberger et al., 1976) . estimation of serum gastrin levels after administration of androgens has not been reported. aim. the aim of this study was to investigate the serum gastrin levels before and after oophorectomy and administration of estrogens and testosterone in female guinea pigs. method. 4 groups of guinea pigs were used for this study, 8 animals as controls, 6 were given estrogens for 2 weeks, 8 were given testosterone for 2 weeks and 5 underwent oophorectomy. results. the mean serum (-+sem) gastrin value in controls was found 35+ 1.63 pg/ml, after estrogens 13.33-+1.22 pg/ml, after testosterone 55_+1.89 pg/ ml, after oophorectomy 46-+2.45 pg/ml. conclusion. it is concluded that estrogens decrease the serum gastrin, and that the increase of serum gastrin after administration of androgens and oophorectomy is statistically significant (/r<0.01). the mechanism by which the ovarian hormones influence gastrin levels may be a sex-dependent change in the synthesis or secretion of gastrin. the reported inhibitory influence of estrogen on food consumption may be also the predominant factor in serum gastrin alterations. mean caloric intake was 2336--+427 kcal/day. moderate to severe dumping occurred in one patient. faecal fat was normal in 11 while 7 patients had greater than 10 grams fat loss per day. weight loss was 14.6_+8.6% of recall weight. muscle mass measured by arm muscle circumference (25.7_+3.9 cm) and creatinine heigth index (99___26%) was normal for our patients but triceps skin fold was 30% less than normal (8.6_+2.2 mm) indicating that in these patients fat stores were reduced. in six patients haemoglobin was less than 13 grams per cent but serum iron, iron binding capacity and serum folate were normal. red cell folate was depressed in six patients whose haemoglobin values were normal. it is concluded that with this reconstruction total gastrectomy produces satisfactory digestive and "nutritional results. examination of factors affecting faecal fat loss and folate metabolism may help improve the nutritional status of these patients. in order to assess the optimal conditions for segmental pancreatic graft viability the following experiment was conducted. the duct obliterated splenic lobe of the canine pancreas was autotransplanted by end to side fashion to the femoral vessels (n = 10). the graft was lodged in a subcutaneous pocket. a month later total pancreatectomy was completed. the dogs were supplemented with pancreatic enzymes (viokase). mean survival was 42___ 12 days. deaths were due to hyperglycemia when grafts lysed from local infection and thrombosis. in the second experimental group (n= 10) the autografts were anastomosed to the iliac vessels and placed intraperitoneally followed by total pancreatectomy one month later. construction of a distal splenic arterio-venous fistula increased blood flow from 31.4 cc/min to 108 cc/min. the pancreatic duct was obliterated with neoprene (n=4) silastic (n=3) or left open (n=3). three dogs died one month to four months due to graft fibrosis and failure. seven dogs had been followed from four months up to seven months when sacrificed. all were normoglycemic. mean k values for ivgtt were 1.5. the normoglycemic dogs had mean plasma concentrations of amino acids similar to healthy controls. a two to threefold elevation was observed in pancreatectomized diabetic dogs for plasma leucine, isoleucine and valine. immunoreactivity of pancreatic polypeptide was measured by radioimmunoassay with an antibody raised against the human hormone by re. chance, lilly research laboratories. mean plasma levels rose form 256--+28 s.e. pg/ml to over 1000 pg/ml following protein meal (20g ground lean beef/kg) in all normal control dogs (n=4), and to levels in the range of 390 to over 1000 pg/ml in the same dogs following infusion ofbombesin (1 pg/kg/ h) for 15 min. mean basal levels were lower (42___ 2.2 s.e. pg/ml) in dogs with autotransplants and did not increase significantly during any stimulatory test. the levels of pancreatic polypeptide did not distinguish between viable and failing grafts. in successful grafts histology showed fibrosis of the exocrine component. transmission electron microscopy revealed the presence of clusters of functional islet cells in the six-month implants. a and b cells were common. d cells were much less frequent. release of secretion granules into the perivascular connective tissue space was observed. the results indicate that vascularized segmental pancreatic graft comprising 25 % to 30 % of the pancreatic mass maintains normal carbohydrate and amino acid metabolism. graft survival was extended by intraperitoneal location and creation od distal splenic a-v fistula, but chronic graft fibrosis occurred regardless of the method of pancreatic duct treatment. the results further suggest the necessity of an intact vagal innervation for the physiological or pharmacological stimulation of pancreatic polypeptide release from thendocrine pancreas which is otherwise apparently functional. besides, these considerations rule out pancreatic polypeptide levels as a useful marker for evaluation of pancreas graft. polyisoprene ductal occlusion allows segmental pancreatic transplantation in man with satisfactory early islet cell function. however acute rejection remains a problem as the diagnosis, based on hyperglycaemia and glycosuria, represents a late manifestation of rejection. although llqndiumlabelled platelets have been used in the diagnosis of renal rejection [1] , their use in pancreas transplants has not been studied. ~ in 6 patients, autologous llqn-labelled platelets were injected on the second, sixth and tenth days following combined renal and pancreatic transplantation. graft radioactivity was expressed as the ratio of counts from the pancreas over a reference area on daily gamma images for 14 days. one patient developed hyperglycaemia coinciding with renal rejection on day 5. over the previous 24 h pancreas radioactivity had increased by 94 %. in a further patient whose pancreas continued to function, a perigraft haematoma was recognised on the gamma image. the remaining 4 patients had good pancreatic function and no 11xin-platelet accumulation: mean (_s.c. mean) pancreatic radioactivity was 1.76+0.190 on the third postoperative day and 1.75_+0.135 at the end of study. these results demonstrate that ductal occlusion with polyisoprene does not cause significant platelet accumulation. hence min-platelets may potentially be used for the earlier diagnosis of pancreatic rejection. isograft models of pancreatic transplantation, methods involving closure of the duct system result in severe inflammatory changes and eventual fibrosis [1] . these changes can be avoided by formal drainage of the duct into the bowel of urinary tract. inflammatory changes initiated by duct closure may contribute to and enhance allograft rejection. pancreas transplants were performed in rats using lewis (rt11) donors and streptozotocin-induced diabetic da(rt1 a) recipients, using duct-ligation, open duct and ureteric duct drainage. cyclophosphamide produced significant prolongation ofnormoglycaemia in all groups and although there was a tendency towards longer function in the unligated groups there was not statistical difference (wilcoxon's unpaired test) between the methods of duct management. management of the pancreatic duct did not seem to have any immunological consequences for graft survival but septic complications, associated with the normoglycaemic deaths, were more common in immunosuppressed animals with draining ducts. between july 1, 1980 and march 30, 1982 combined pancreatic and kidney transplantation was performed in 9 patients with type i diabetes who had all been on insulin therapy for at least 16 years. age at the time of transplantation was 28 to 45 years. the pancreatic segment used for transplantation consisted of body and tail of the organ based on a vascular pedicle of the celiac axis and the splenic vein. imediately prior to intraperitoneal transplantation to the iliac vessels the ductal system was filled with 4 to 6 ml of prolamine, a rapidly solidifying alcoholic protein solution. simultaneous kidney transplantation was performed through a separate incision. five pancreas transplants were lost for non-immunological reasons. four of them never had any useful endocrine function, one graft was lost of venous thrombosis after 48 hours of excellent function. in none of these patients did failure of the graft lead to any substantial complication. in the 4 remaining patients initial graft function was excellent with plasma glucose normalizing within 12 hours and subsequent normoglycemia on a regular diet without exogenous insulin administration. plasma glucose did not rise spontaneously during rejection episodes of the kidney and eleva-tions due antirejection treatment were promptly; reversed as high dose prednisolone was discontinued. in oral glucose tolerance test (ogtt) performed at 4 to 6 weeks in 3 patients median glucose rose from a basal 5.1 mmol/l to 8.6 mmol/1 and was back in normal range (4.7 mmol/1) at 3 h. basal insulin was 43-237 pmol/1 and also returned to the normal range after 3 h after a peak of 201-531 pmol/1 at 50-210 rain. c-peptide showed a significant 2 peaked rise over basal values (230, 233 pmol/1) in 2 patients while all values were above 4500 pmol/l in the third. two of these patients have since rejected both organs. two tranplants still function very well after 12 and 21 months, respectively. in one of these patients the ogtt after 13 months is even slightly better than the above -mentioned first test, and he shows a norreal insulin and c-peptide response. in the fourth patient an ivgtt performed at 7 months and an ogtt at sixteen months were normal with insulin peaking at 251 pmol/1 and a c-peptide peak of 1267 pmol/1 at 60 min in the latter. -results at 26 and 17 months, respectively, will be presented. three problems persist in clinical organ preservation. these are failure of current systems to replenish the ischemically injured organ, to reliably extend the period of organ preservation and to definitely determine organ viability. previous studies have documented the value of electrochemical redox control in rejuvenation of the ischemically injured kidney during perfusion preservation. this study was undertaken to develop the cost effective technique applicable to current organ preservation systems, to test the reliability of redox measurement in prediction of organ viability and to determine the ability ofredox maintenance to safely extend the period of organ preservation. a disposable cell has been developed using reticulated vitreous carbon as an electrode which is driven by a potentiostat powered by a nine volt transistor battery. short term preservation studies using ischemically injured dog kidneys (60 rain in-situ warm ischemia) were auto transplanted after 24 h of pulsatile preservation to determine the optimum redox level of the hypothermic kidney. this was determined to be a -20 mv vs the standard calomel electrode. twenty-one adult mongrel dogs were equally divided into three groups. pulsatile perfusion preservation was extended to four days in group a with redox level monitored only. group b was treated with electrochemical reduction for four days and group c for six days. three of seven dogs survived in group a following auto transplantation and immediate contralateral nephrectomy. the kidneys of all survivors were able to bring perfusate redox potential under control and maintain this level throughout the preservation period. six of seven dogs in group b survived with a mean posttransplant peak serum creatinine of 4.5 rag/100 ml. in group c four of seven kidneys supported life immediately. all redox controlled kidneys made copious amounts of urine. our data indicate that perfusate potential may be either monitored as a reliable index of organ viability or controlled to allow extended safe preservation. antithymocyte globulin (atg) has been shown to be an effective agent in combination with increased doses of steroids in reversing renal allograft rejection. since it is frequently undesirable to employ raised doses of steroids, the following study evaluated the effect of 15-21 i.v. daily doses of horse atg alone without additional steroids, on 2nd-5th rejection episodes in 10 recipients of cadaveric renal allografts (28 rejections: 25 biopsy-proven) detected within 3-18 mos. of transplantation. of 28 rejection episodes, 25 were successfully reversed, with return of serum creatinine to pre-rejection levels in 18 episodes (7 patients). three patients had primarily humoral rejections and returned to dialysis 2-4 mos. after treatment of the last rejection. levels of circulating horse immunoglobulins were obtained in all 10 patients during and follwing administration of atg. recurrent rejections (3rd-5th) following last treatment with atg (3-11 mos.) were seen in 5/10 patients. all 5 patients had rapid immune eliminiation of atg (1/2 life 2-4 days) as compared to the 5 patients who had no more than 2 rejection episodes (1/2 life 6-14 days). atg without additional steroids is an effective agent for reversal of multiple renal allograft rejections which by biopsy are primarily cell-mediated. to be effective, heterologous atg must be given in adequate total doses and/or from appropriate heterologous source, to prevent rapid immune elimination by the recipient. the use of atg alone for treatment of recurrent allograft rejections is particularly recommended for its steroidsparing effect in treatment of multiple rejections and for those patients at high risk from steroid side effects. the significance of the monocyte crossmatch in lrd recipients of hla identical kidney grafts j. cerilli, l. brasile and s. rogers ohio state university hospitals, columbus, ohio, usa in a preliminary study from our transplant center, the presence of pre-formed antibody in recipient sera directed against monocytes from their respective living-related donors correlated with a poor clinical course. a poor prognosis for graft survival was found regardless of the hla match grade. to minimize the role of the hla system, only those living-related recipient/donor pairs who were hla identical at the a, b, c, d and dr antigen loci, and who exhibited severe immunological types of rejection were evaluated. due to the small numbers found in this category at any one center, this abstract represents an international study from 12 different transplant centers. 26 patients who met the criteria were studied for the presence of antibody directed against their respective donor's monocytes both pre-and posttransplant. in eighteen of these patients, cytotoxic antibody against their donor's monocytes was found in their pre-transplant sera. there was no detectable cytotoxic activity against their donor's t or b lymphocytes. two additional transplant recipients exhibited this antibody in post-transplant sera. again, no t or b lymphocyte cytotoxicity was detected. a control group of hla identically matched siblings who incurred no or minimal rejection demonstrated no anti-donor monocyte antibody. the results of this international study points towards a correlation between a high incidence of graft rejection and the presence of antibody directed against their respective donor's monocytes. therefore, in our view, the presence of anti-monocyte antibody to the prospective donor pre-transplant is a contraindication to transplantation. in patients with different liver diseases the reduced concentration of the peripheral blood t-cells and altered immune response were observed. the purpose of our studies was to investigate the mechanism of the immunological modification of the lymphoid system in the hepatic injury. studies were carried out in 3 groups: 1) lew rates were treated with dimethylnitrosamine (dmna), 35 mg/kg b.w., i.v. for acute liver necrosis induction, 2) cc14 (0.1 ml/100g b.w., i.v.) twice weekly, over 4 weeks for chronic liver damage, 3) ccl 4 over 10 weeks for liver cirrhosis (histologically examined). serum and thymus, spleen, mesenteric lymph nodes (ln) were removed 2 and 32 days after dmna treatment and 5 and 35 days after last cc14 injection. the total number of thymocytes and spleen cells was counted and the reactivity to pha and cona was measured. normal liver perfusate (lp) was prepared 6 h after removing (20°c) by 5 times perfusion in 30 rain intervals (flow rate 1 ml/g/min with 2 ml/g of ringer). the effect oflp, sera, dmna and cc14 on the viability and pha response of normal thymocytes was tested in vitro. liver enzymes were evaluated, we found the decreased total number of thymocytes immediately after the stopping of medication (group 1: 9,2___ 1,8 %, group 2: 4,6___ 1%, group 3: less than 1% of control). proliferative response of the remaining cells in thymus to pha was much higher than normal thymocytes (group 1:325 ± 30 % group 2: 608___164%, group 3: nd). the total number of spleen cells was not changed but their response to cona was altered in the all groups and to pha only in group 3. pha response of ln-cells was decreased in the all groups. liver perfusate was cytotoxic (53___3 % of viable cells) and suppressive for pha response of thymocytes (6_+5% of control). the sera of rats with the hepatic damage showed an enhanced suppressive activity. cc14 and dmna did not have any effect on thymocyte in vitro. one month after last medication we observed the recovery of thymus involution (total i in the acute and partial in the chronic hepatic damage and cirrhosis). our results suggest that the liver origin cytotoxic and immunosuppressiv e factor(s) can be released from the damaged liver into the circulation (like to lp) and can destroy the thymus leading to the secondary changes in the other lymphoid organs. the grade of thymus alteration is dependent on the degree and the duration of hepatic damage and is reversable. the hepatic factor (s) showed the similar effect on the cortical population of thymocytes like the steroid immunosuppressants. liver grafts in the rat are in certain strain combinations not rejected and in this situation there is evidence for spontaneous donor specific tolerance [1] . we have developed a model of auxiliary liver transplantation which would allow us to study the immunosuppressive properties apparently produced by a liver allograft. the portal vein is anastomosed to the left renal artery, the i.v.c. to the renal vein and the bile duct to the ureter. simultaneous kidney or heart allografts were performed. conclusions:auxiliary liver grafts are rejected. survival of heart or kidney grafts is not influenced by a simultaneous kidney or heart allograft. heart and kidney grafts are prolonged by simultaneous auxiliary liver grafts. [1] . the role of suppressor cells in the initiation and propagation of malignant tumours in man, is less clearly defined. the present study, using in vitro mitogen assays (pha, cona, pwm) and various rosetting assays [2] with specific monoclonal antibodies to lymphocytic helper (okt4) and suppressor (okt8) cells, has revealed the presence of such suppressor lymphocytes in women with clinically localised (breast and axilla) mammary carcinoma. lymplaocyte hyporeactivity to mitogens was found in 30% of lymphocyte preparations from blood and axillary lymph nodes of patients with breast cancer. in 10% of patients nodal lymphocytes were totally anergic. the most profound hyporeactivity, however, was detected in the lymphocyte subsets (<75 %) of specimens isolated from the breast carcinomas by collagenase digestion and sephadex g-10 column passage [3] . the lymphocyte preparative techniques were not responsible for the low levels of responses detected. also, in situ prostaglandin synthesis and release did not appear to be involved in depressing lymphocyte reactivity [4] . comparable percentages of suppressor cells (okt8+) were detected within these different lymphocyte preparations. suppressor cells were not found in the lymphocyte preparations from the blood and lymph nodes of appropriate controls. from clinical and experimental studies it is known that blood transfusions may have immunosupressive as well as immunostimulating consequences. the effect of transfusions on graft survival has been extensively studied by our group in the bn to wag rat model. in this donor-host combination it was found that a donor specific pre-transplant blood transfusion could lead to a marked prolongation of heart and kidney graft survival, whereas the similar pretreatment resulted in accelerated rejection of bn skin allografts. this specific model was used to investigate the influence of a single bn transfusion on the growth of 2 different syngeneic transplantable tumors in wag rats. the first tumor was a radiation induced basal cell carcinoma of the skin (t 1), the second tumor was a chemically induced adenocarcinoma of the duodenum (t 2). the antigenicity of both tumors was assessed in vivo, using classical challenge-protection experiments. it was observed that t i exhibited strong immunogenetic properties, whereas t 2 was only weakly immunogenic. the doubling time oft i was 2.5 days, the doubling-time of the adenocarcinoma was 14 days. intravenous inoculation of isolated t 1 cells led to development of lung nodules which could be counted after 14 days. wag rats were injected i.e. with 1 ml of bn blood or syngeneic blood (controls) at 7-14 days before tumor challenge. t l was given in two different ways: a) sc. implantation of+2x2 mm pieces, b) i.e. injection of 106 isolated tumor cells. t 2 was implanted sc. only. each experimental group consisted of 8 animals. for t 1 it was found that allogeneic blood transfusions caused a slight (but not significant) inhibition of subcutaneous tumor growth. however, in the t 1 lung-metastasis model it was observed that a single bn blood transfusion led to a 50 % reduction of nodules, counted at 3 weeks after inoculation. this reduction in number and size of nodules was highly significant (p<0.01). for tumor t 2, the bn blood transfusions evoked a strong inhibitory effect on the growth of the sc. implanted tumor. at 8 weeks after implantation all tumors in the control group had grown to a diameter of 10-16 rnm (average diameter 12.2 ram). in the group pretreated with bn blood, only 3 of 8 tumors were palpable at that time (average diamter 4 mm). for t 1 it was further investigated whether a single bn transfusion, given one week after i.e. tumor cell inoculation, would have any influence on tumor growth. no significant effect on number or size of the lung nodules could be noticed, if anything, the transfusion appeared to have a stimulatory effect. the results indicate that allogeneic transfusions can lead to a substantial modification of tumor growth, depending on tumor type and site of implantation. this observation may have important clinical implications. we report here the serial study of circulating immune complexes (cic) in two human tumor systems, colorectal cancer and gestational trophoblastic neoplasia (gtn). cic were assayed by antigen nonspecific insolubilization induced by 3.75 % polyethylene glycol (peg) and monitored as a od45o changes by spectrophotometry. all 11 of the serially studied colorectal cancer patients presented with elevated cic levels (mean = 610 + 396 zt od450) as compared to our standard cic level for pooled normal human sera (202--+_4 aod450, p<0.05). initial values in these patients range from 304 to 1407 a od450 with no correlation to tumor load, site of presentation, or subsequent clinical course. in 6/7 patients who underwent ,,curative" resection of primary or metastatic colorectal cancers, serial cic elevations occured only when antigen excess (measured by simultaneous carci-noembryonic antigen [cea] assay) decreased. immunoglobulin components of fractionated cic showed predominantly iga subclass. in 30 gtn patients followed with serial cic and simultaneous human chorionic gonadotropin (hcg) assay, only those patients documented to enter hcg remission after molar evacuation showed significant elevation of cic. chromatographic fractionation of peak cic in one such patient defined three irnmunoglobulin containing fractions showing immunoreactivity to one of four paternal hla haplotypes (aw32). one ,,antigen" fraction (<25.000 mw) from this complex completely inhibited reference anti-aw32 binding. as in the colorectal cancer patients, these data show that cic rise only when antigen excess decreases (reflected in the gtn patients by hcg normalization). in addition, some gtn patients may react to immunogenic paternal hla haplotypes as part of their response to molar pregnancy. dfmo, an enzyme-activated irreversible inhibitor of ornithine decarboxylase (odc), reduces tumor polyamine levels, inhibits growth of emt6 sarcomas and hepatomas in experimental animals, and induces remission in human leukemia. a renal adenocarcinoma (ra) cell suspension, or a 1 mm segment ofwilms' (wm) tumor was transplanted intrarenally into balb/c mice (n--105) or subcutaneously into wistar-furth rats (n=80) respectively. dfmo (2 %) in drinking water was administered to half the animals in each group throughout the experiment. at 28 days ra tumors in dfmo-fed mice weighed 72 % less than tumors in control animals (p<0.001); wm tumor weight at 28 days was not affected by dfmo feeding. the mean number of lung metastases in dfmo-fed r.a-bearing mice was 0.1 and in ra-bearing control mice was 1.4 (p<0.001). dfmo caused 72-75 % inactivation of tumor odc, reduced ra putrescine levels by 710/o (p<0.001), reduced wm putrescine levels by 65% (d7<0.01), reduced wm spermidine levels by 58% (p<0.001) and increased wm spermine levels by 37% (p<0.01). dfmo feeding did not alter dna content of ra or wm tumors. although final carcass weight was similar in all animals, dfmo feeding progressively reduced total body weight (tbw) of mice, but not rats, until at day 20 the tbw of dfmo-fed mice was 10.5% less than tumor-bearing control mice (p<0.01). dfmo-fed mice bearing ra tumors survived 3.0-t-0.8 days longer than control mice (p<0.05). reduction of polyamine levels in wilms' tumors does not affect tumor growth. lowering of renal ade-nocarcinoma putrescine levels by continuous feeding of dfmo to tumor-bearing animals decreases tumor growth, reduces lung metastases, and increases host survival. we have previously demonstrated that vagal nerve stimulation releases 5-ht into the lumen of the feline gut. this study was initiated to: 1. determine if substance p (sp) and motilin (mt), other enterochromaffin cell products, are released simultaneously, 2. to evaluate if this release is under cholinergic or adrenergic control. in 6 cats, 15cm isolated in situ segments of proximal jejunum were perfused with saline at 1 ml/min (37 ~c). perfusate samples were evaluated at 5 rain intervals and concentrations of 5-ht, sp, motilin were measured by ria's developed in our laboratory. after two 5-min basal periods, the supradiaphragmatic sectioned vagus nerves were stimulated electrically (10v, 5m s, 10hz). the output from the loop in ng/5 min (5-ht) and pg/5 rain (sp and mt) was: introduced into the jejunum of conscious dogs through an external small bowel fistula. the gut was perfused at 5 ml min-1 with a physiological electrolyte solution containing the non-absorbable marker polyethylene glycol (mol. wt. 4000; 5 g 1-1); water and electrolyte absorption and transit time (tt) were measured during intravenous (i.v.) administration of each peptide, and during preceding and succeeding i.v. control infusions of 0.15m naci. separate studies showed jejunal absorption and tt to be constant over prolonged periods during i.v. nac1 administration. bombesin (10 pmol kg-lmin-1) and neurotensin (1 pmol kg-~min 1) significantly reduced jejunal water absorption; bombesin and enkephalin (0.5 nmol kag-1rain-1) significantly prolonged tt; and enkephalin encreased water absorption (/7<0.05 in all cases). measurement of plasma neurotensin during i.v. infusion indicated that physiological blood levels were not exceeded during these studies. conclusion: a number of peptides may be involved in the regulation of small bowel function. the effect of neurotensin on jejunal water transport provides a possible mechanism linking raised blood neurotensin levels with intestinal intraluminal fluid accumulation in the dumping syndrome. in order to establish whether this release was under adrenergic control, 4 cats had cervical ganglionectomies. using the same electrical paramenters, stimulation of the cut cervical vagus nerves resulted in identical 5-ht responses as above. in 4 additional cats atropine administration (lmg/kg iv) totally abolished the 5-ht responses to vagal nerve stimulation. the paralled release of 5-ht, sp, and mt following vagal nerve stimulation, strongly suggest that the ec cell is the source of these luminal hormones. this release appears to be under cholinergic control. since diabetes mellitus is markedly improved immediately after jejunoileal bypass before significant weight loss, but only gradually and often incompletely changed after gastric bypass, it seemed appropriate to investigate the effects of intestinal exclusion on experimental diabetes. studies were performed on alloxan diabetic sprague-dawley rats. two days after jejunal exclusion (je) in 8 rats (resection of proximal 1/3 of small intestine), fasting blood sugars (fbs) decreased 580, from 344 to 146mg/dl, and averaged 164 mg/dl at 3 weeks. after ileal exclusion (ie) in 7 rats (resection of distal 1/3 of small intestine), fbs fell 39% in 2 days, from 250 to 153 mg/dl, but increased 40% above preoperative levels to 350 mg/ dl at 3 weeks. sham operated rats responded similarly to ie rats. after alloxan and operations, all groups lost weight, but only je rats began to increase weight at a normal rate. increased water intake, polyuria (140 ml/24 h), and glycosuria (2756 rag/all), were present in ie rats; je rats were normal (urinary output <20 ml/24 h, and urinary glucose 36 mg/dl). oral glucose tolerance tests (gtt) were extremely abnormal in ie rats, similar to those in non-operated alloxan rats, while gtt curves in je rats were similar to normal animals, with some elevation at 30, 60 and 120 min. serum insulin levels remained low in all alloxan treated rats after jejunal exclusion. possible mechanisms, currently under study, relate to carbohydrate malabsorption and changes in enteric chemical mediators. the purpose of the present study was to investigate the changes in somatostatin release and somatostatin-containing cells of the pancreas and stomach of the streptozotocin (stz) -induced diabetic rat after the amelioration of diabetes by whole pancreatic transplantation. highly inbred lewis rats were divided into three groups: (1) normal rats, (2) stzinduced diabetic rats and (3) transplanted rats. diabetes was induced by the administration of stz (60 mg/kg). on the seventh day after stz treatment, pancreatic transplantation was performed. four weeks after the transplantation, in vivo and in vitro, studies were performed. pancreatic d cells and gastric somatostatin-containing cells were stained with antibody enzyme method. studies in vivo showed marked improvement of the impaired arginine-induced insulin release by the transplantation. studies in vitro employing isolated perfused rat pancreas and stomach revealed following results: mean basal pancreatic somatostatin release in normal, diabetic and transplanted rats were 12___3, 24-t-7, and 17__+4 pg/ml, respectively. total amount of pancreatic somatostatin release in each group during arginine stimulation (19.2. mm) were 946--+200, 2321___266, and 1013-+126 pg/15 min, respectively. significantly higher somatostatin release was obtained from the diabetic pancreas, which was, however, reduced to normal after the whole pancreatic transplantation. on the other hand, insulin release from the diabetic pancreas was severly impaired and pancreatic transplantation had not effect on insulin release from the host pancreas in the transplanted rats. as to the glucagon release, there was not significant difference among them. mean basal gastric somatostatin release in normal, diabetic and transplanted rats were 179-t-5, 173___5, and 135 + 5 pg/ml, respectively. there was no significant difference between normal and diabetic rats, though the significant decreased value was obtained in the transplanted rats. (vs. normal;/7<0.01, and diabetes; /7<0.01). on the other hand, glucagon-stimulated peak values in these groups were 498_ 36, 662___47, and 412+25pg/ml, respectively. glucagon-stimulated gastric somatostatin release in diabetic rats was significantly increased, but reduced to normal value by pancreatic transplantation. also, a number of pancreatic d cells and gastric somatostatin-containing cells were markedly increased on the diabetic rats. on the other hand, a number of these cells in the transplanted rats were descreased to normal levels. in summary, enhanced pancreatic and gastric somatostatin release and cells in the diabetic rats were both normalized after the amelioration of diabetes by the whole pancreatic transplantation. from these results, it is suggested that pancreatic and gastric somatostation are regulated by circulation and/or metablic of nutrients. doppler velocity recordings are widely used for the non-invasive diagnosis of carotid arterial disease. although detailed analysis of carotid doppler spectral information has been suggested as a method for improving diagnostic sensitivity, the accuracy of the relationship between the doppler recording and the true instantaneous velocity profile has not been established. the purpose of this study is to determine if a cw doppler velocitymeter can accurately transduce the true instantaneous blood flow velocity information. methods: a pulsatile flow model has been constructed in which it is possible to record the instantaneous doppler spectrum and simultaneously photograph and measure the true velocity profile. a computer controlled pump generates a carotid waveform in tubes without stenoses and with, symmetrical stenoses. flow is visualized using the photochromic dye tracer technique. a short burst of uv light from a laser is passed across the tube. a narrow band of the fluid turns blue, its movement is photographed, and the instantaneous velocity profile determined every 10 msec throughout the pulse cycle. at the same time, the instantaneous doppler spectral information is recorded by a frequency analyzer. the results follow. for pulsatile laminar flow, the doppler spectrum correctly recorded the true velocity spectrum, including the instant/~neous maximum velocity and mean velocity. for disturbed flow, it was not possible to show the same direct relationship between the doppler spectral recordings and the blood flow velocity. in conclusion doppler velocitymeters accurately transduce velocity information when flow is laminar but when flow is disturbed there is not a direct relationship between doppler recordings and the true velocity profile. consequently, one should be cautious in attempting to relate doppler measurements of disturbed flow directly to the true changes in the velocity pattern. early failure of arterial reconstruction may originate in poor patient selection. in aorto-iliac stenosis (ais) selection for operation relies upon clinical examination of the femoral pulse and radiology. since single plane arteriography is inadequate for accurate definition ofiliac stenosis [1, 2] , this paper compares clinical examination, doppler ankle systolic pressure (aspi) and femoral signal analysis (laplace transform damping, ltd, and pulsatility index, pi) [3] with biplanar contrast studies. i at biplanar angiography 66 of 102 ischaemic lower limbs had ais with diameter reduction from 25-84 %, of the remainder, 25 were normal (<25 % stenosis) and 11 were ,,occluded" ('_--850/0). nearly two thirds (61%) of limbs with a clinically normal femoral pulse had identifiable arteriographic stenosis (~-__.250/0), upstream abnormality was predicted incorrectly in 15 % and the overall accuracy of clinical examination was 67 %, both for detecting stenosis and predicting its severtiy. aspi (median 0.61; 95% confidence ,limits 0.3-1.0) and pi (5.4; 2.3-0.05) although correlated with stenosis (aspir = 0.65; p = 0.05, pir = 0.62; p=0.05 variance analysis for linear regression), did not aid the clinician further (accuracy 62 %). however ltd (0.72; 0.37-1.0) was well correlated (r=0.76, p=0.001) and did improve assessment ofiliac stenosis (accuracy 84 %). the need for biplanar arteriography is reiterated and its use with doppler signal analysis should improve the evaluation of aorto-iliac disease. in the absence ofa non-invasive method for estimating volume flow in an individual artery, local blood pressure measurement has proved, with certain limitations, useful in assessing the cardiovascular system. now ultrasound technology has progressed to enable blood flow in an artery to be measured noninvasively. we report the results o four evaluation ofa 5 mhz, computerized, 30 channel, pulsed doppler vessel imaging and flow measuring instrument in in-vivo experiments. computed blood flow was compared to actual blood flow (calculated by timed collection) in 17 anaesthetised dogs. correlation between computed and actual blood flow was stronger in the larger abdominal aorta than in the smaller common carotid arteries. from the regression plot, the coefficients of determination, p, were: 0.927 (exposed aorta scans); 0.857 (transcutaneous carotid scans); and 0.784 (exposed carotid scans). stepwise regression analysis showed the computed flow values to be independent of probevessel angle, depth and lumen diameter for vessels greater than 2.5 mm in diameter. these results suggest that this pulsed doppler instrument has the versatility and accuracy essential for diagnostic flow measurements in the main conducting arteries of the neck and limbs and in vascular bypass grafts. in the assessment of patients undergoing carotid artery surgery, many laboratory methods are available in addition to angiography. in a series of 210 patients experience has been gained with the use of eeg, tc 99m isotope scanning, opg, ct scanning and doppler. in a 10 year follow up over 85% of patients had a satisfactory outcome. an early mortality of 2% in the beginning of the series has been eliminated due to improved selection. in this report the application of multi-gated pulsed doppler techniques is reported. this allows a non invasive measurement of mean volume flow in the common carotid artery with a method reproducibility of + 5 %. mean volume flow in 50 undiseased arteries (25 subjects mean age 46 years) was found to be 536± 104 (s.d.) ml/min. from this a lower range for normal flow of 300 ml per minute (2 x s.d.) was selected. 30 patients were investigated before surgery and a follow up examination was performed at a mean interval of 4v2 months post operatively. 3 groups were defined. group a >300 ml/min; group b 200-300 ml/min; group c <200 ml/min, of 14 arteries in group a before surgery, 11 remained in the group and 3 dropped to group b. in group b, of 8 arteries, 5 go to group a, 2 remain and 1 dropped to group c. in group c, 7 out of 8 arteries moved to group a and 1 to group b. thus of 16 arteries with below normal volume flow before surgery, 12 were returned to normal range and further 1 improved. 2 remain unchanged and 1 disimproved. of the 30 arteries examined 4;/2 months after surgery, 23 are in the normal range and a further 1 improved. 2 remain unchanged and 1 disimproved. of the 30 arteries examined 41/2 months after surgery, 23 are in the normal range in flow values and a further 2 remain unchanged. the non-invasive and isotope techniques have a valuable and practical application in assessment of carotid artery surgery. timing is influenced by the finding fo infarction on ct or isotope scanning. doppler techniques are useful not only in defining severity of diseas and sub-radiological plaques, but valuable flow information can be obtained by pulsed doppler pre and post operatively. this may help in identifying patients who need further medical or surgical treatment. stepwise logistic regression-amodel for predicing success of femorai-popliteal bypass grafts the objectives of this study were to identify the preoperative factors that influenced postoperative patency of femoral-popliteal grafts and to develop a model that could be used prospectively to determine the probability of successful outcome. data base material consisting of history, physical examination, laboratory data, angiography, and operative findings in 199 patients undergoing femoral-popliteal bypass grafting was entered into a computer programmed for stepwise logistic regression analysis. the computer identified and ranked 24 factors that influenced outcome. the top five factors (other than technical problems) included quantity of runoff, previous ipsilateral femoral-popliteal bypass, preoperative prediction of potential amputation level, concurrent proximal vascular reconstruction, and the location for distal graft anastomosis. having established the computer data base, it is now possible to enter information from new patients into the computer which will weigh all factors and indicate the likelihood of surgical success. in addition, tables can be generated which will look at simple combinations of variables to predict patency. for example, in a patient about to undergo a primary femoral-popliteal bypass with no anticipated technical problems, the likelihood of success as a function of runoff and preoperative amputation level is as follows: irreversibility of shock and ischemic injury is generally considered a consequence of extensive cellular injury. to study the role of intravascular coagulation in shock, 56 rats were bled to a mean arterial pressure of 50 mm hg for 3 hrs or 25% uptake of shed blood, whichever occured first. return of shed blood with these data provide the patient and the surgeon with a quantitative prediction for success and permit an informed decision when considering therapeutic alternatives. potential cytoprotection by heparin was studied by similarly bleeding 52 additional rats; controls were only cannulated. twenty pairs were heparinized (h); 32 were not (nh). paired-bled and control rats were sacrificed following hemorrhage, liver (l) and kidney (k) mitochondria were isolated, and the isolates were studied by the polarographic technic with glutamate and succinate to determine the respiratory control index (rci) as a measure of cellular injury. results were: an equal volume of isotonic saline resulted in a 43 % survival; % uptake of blood during shock allowed prediction of survival. an additional 55 rats were then randomized (coin toss) to heparinization versus no heparin prior to shock, and were similarly bled and resuscitated. significantly improved survival (p<0.025) was seen in heparinized (17/23; 740/0) versus nonheparinized rats (13/32; 41%). uncoupling and inhibition of mitochondrail function were noted in both h and nh rats with rci being significantly reduced from control. however, there was no difference between h and nh compared to each other. heparin does not provide cytoprotection during shock; improved survival with heparin may rather be a consequence of improved reperfusion of tissues following the shock episode. fl-endorphin (b-end) has been postulated to play a role in the pathogenesis of shock because the opiate "antagonist naloxone improves the macrohemodynamics in various shock models [1] . however, plasma levels ofopioid peptides have not been determined as yet. the aime of our study was to measure the plasma concentrations of various peptides and to evaluate the influence of naloxone particularly on the plasma concentration of r-end. in 16 anesthetized foxhounds, the adrenolumbar vein was cannulated and hemorrhagic shock (map = 4 mm hg for 3 h) was induced according to wiggerstechnique. the plasma levels offl-end, methioninenkephalin (m-enk), and leucine-enkephaline (l-enk) were simultaneously determined in c.v. and/ or adrenal venous blood by a specific ria. crossreactivity of r-end withfl-lipotropin was about 4 %. the enk-antibodies crossreacted with less than 5 %. five dogs received an i.v. bolus of naloxone (2 mg/ kg) and a subsequent naloxone infusion of 2 mg/kg/ h after 2 h of hypovolemia. eleven dogs served as control and received equivalent volumes (1 mg/kg per h) of ringer solution. hemorrhage resulted in a sharp rise of central venous plasma levels particularly of m-enk and l-enic this effect was even more pronounced in the adrenal's effluent system.fl-endorphin levels remain elevated whereas the enk secretion began to decrease 1 h after hemorrhage. naloxone treatment inhibited any spontaneous fall of adrenal enkephalin release during the shock phase and the values remained elevated 10-15 fold. volume substitution with autologous blood resulted in a normalization of all peptide levels. these data demonstrate that hemorrhagic shock will cause stimulation of endogenous opioid peptides. the high levels of enkephalins in the adrenolumbar vein indicate that the adrenal gland is the main source of these peptides in the circulation. in addition toil-end, the enk seem to play a role in the pathogenesis of shock as well. at our present state of knowledge, however, it is difficult to design a coherent concept of mechanisms involved. this shows that cp treated cells bound nearly as much [125t]-acth analog as control cells but there was very little specific binding to sp treated cells. low concentrations of acth effectively displaced the acth analog whereas exposure of adrenocortical cells to sp resulted in a significant decrease in acth receptors. this suggests that sp has a factor(s) that binds to acth receptors of adrenocortical ceils which may adversely affect the stress response of shock. f-43 has been proposed as superior to other asanguinous fluids due to increased oxygen carrying capacity. evaluation to date has been largely uncontrolled and at extremes of hemodilution (hct. <2 %) rarely seen clinically. near infrared spectrophotometric monitoring of brain cytochrome a, a3 redox state, a sensitive indicator ofintramitochondrial oxygen availability, offers a unique opportunity to contrast f-43 with balanced salt-albumin (bsa) and whole blood saline (wbs) as a resuscitative regimen in a clinically relevant model. fifteen rats were subjected to 30 minutes of hypoxia (fio2 = 7,5%) and hemorrhagic hypotension (map = 30 mmhc), then randomly allocated to one of three groups and resuscitated by fio2 = 100% and infusion of either f-43, bsa, or wbs. cytochrome a, a3 redox state was monitored continuously at 813 run. thirty additional rats were sacrificed at baseline, end shock and 20 and 120 minutes post resuscitation for cerebral cortical atp and lactate assay. despite hematocrits as low as 15 % in the bsa and f-43 groups, there were no significant differences /7<0.05 between groups in the parameters of oxygen sufficiency; atp, lactate, and cytochrome a, a3 redox state. we assume differences in cardiac output compensated for differences in arterial oxygen content. on this basis we suggest perfluorochemical utilization should be limited to situations in which hematocrits are <15 % and when cardiac reserve is limited. metabolites of the prostaglandin endoperoxide h2 (pgh2) affect both vascular tone and platelet aggregation and thereby may influence blood flow. we, therefore, determined the metabolites formed from pgh2 by microsomes isolated from human saphenous vein used for aortocoronary bypass surgery. in the absence of reduced glutathione (gsh), the enzymatic metabolism of 14c-pgh2 produced only prostacyclin (pgi2) as measured by the formation of its stable breakdown product 6-keto-pgfla. the amount of pgi2 formed varied from 10-50% of the substrate depending upon the microsomal protein and pgh2 concentrations. in addition, the nonenzymatic breakdown of pgh2 resulted in the formation of pgf2a, pge2, pgd2 and heptadecatrienoic acid (hht). there was no formation of thromboxane a2 (txa2) as measured by the absence of its stable breakdown product txb2. in the presence ofgsh, a required cofactor for microsomal pge2 isomerase activity, the formation of pge2 was augmented 2 fold (up to 35 % of the substrate) indicating enzymatic for-mation of pge2. the gsh either did not alter or augmented (less than 1 fold) the formation of pgi2. the increased formation of pge2 in the presence of gsh was at the expense of decreased nonenzymatic breakdown of pgh2 to pgf2a, pgd2 and hht. these data suggest that prostacyclin synthetase activity may serve to protect the vessel graft from platelet aggregation and/or vessel spasms and may possibly serve as an indicator of graft viability. thrombosis is a frequent cause of early arterial bypass graft failure and platelets are known to be major determinants ofthrombus formation on arterial surfaces. pgi2 and flbriolytic activators from the vascular wall counteract intravascular thrombosis. the aim of this work was to study the effect of arterial grafting on the aforementioned mechanisms. 6 cm lengths of tanned human umbilical vein grafts (huvg) with an internal diameter of 5 mm were interposed end-to-end in the carotid arteries (c.a.) and jugular veins (j.v.) of sheep. placed in the c.a.'s, 12 grafts with restricted flow (50 cc/min) were removed on the 10th postoperative day (group i); 18 grafts with unrestricted flow (140___20 cc/min after placement) were taken out 90 days later (group ii) and 4 grafts placed in the j.v.'s were removed 10 days after surgery (group iii). upon removal, the grafts were checked for patency and sections from the proximal and distal anastomoses and midgraft were obtained for determination of pgi2 production (ria) fibrinolysis activators activity (histochemical method) and for light and scanning electron microscopy. sections from the femoral arteries were also obtained. the results of pgi2 generation are expressed in ng/ml/cm% all grafts showed fibrinolytic activity in the adventitia but 4 grafts in group ii also showed fibrinolytic activity in the intima. early neointimal fibrous hyperplasia (nfh) characterized by proliferation of smooth muscle cells was present in group ii. the, occluded grafts showed organizing thrombus material and inflammatory cells and the patent ones showed fibrin and scattered inflammatory cells. in groups i and iii, sem revealed numerous platelets and rbc's incorporated into a proteinaceous material overlying the anastomoses and in some specimens obvious thrombus material was present. in group ii, the anastomotic areas were covered with large endothelial cells, nonetheless, some areas were denuded and small thrombi were occasionally noticed. in conclusion: 1. anastomotic sites create a strong stimulus for thrombus formation despite a high production of pgi2. this suggests that antithrombotic therapy may be necessary to prevent early failures. 2. huvg develop the capacity to produce pgi2 and fibrinolytic activators and 3. although the etiology of nfh remains obscure, the decreased levels of pgi2 in group ii suggest that exhaustion of pgi2 generation from the endothelium might occur leading to proliferation of smooth muscle cells (nfh). these cells will in turn supply pgi2 if a persistent stimulus exists. permeability of intestinal capillaries to fibrinolytic products d. manwaring and p. william curreri department of surgery, university of south alabama college of medicine, usa fibrin/fibrinogen degradation product d (fdp-d) is significantly elevated in the serum of patients after trauma or sepsis. purified fdp-d infused into nontraumatized rabbits precipitates thrombocytopenia, complement depletion, pulmonary dysfunction and increased permeability of lung capillaries to i12s-albu -composite fibrin plate assay size oflytic zones (mmx) after 17 h incubation at 37 °c rain. in order to determin of products of fibrinolysis alter fluid filtration or permeability, either purified fdp-d or fdp-e were tested in an isolated, autoperfused cat ileum preparation. steady-state lymphatic: plasma protein concentration ratio (cl/cp) and lymph flows (ql) were measured at a venous outflow pressure of 5 mmhg. data was analyzed for each animal group by the paired student t test for ql, cl/cp and protein clearance (ql x cl/cp). in cats which received fdp-d (n=7), ql and clearance increased five-fold (p<0.002), but cl/cpwas not altered, which suggests a permeability change. ileal mucosal biopsies prepared for histology had villi that were de-epithelialized and platelet clots in blood vessels. fdp-e (n=7) provoked a slight increase in ql (p<0.01), but not in cl/cp or clearance. histology was normal. (fdp-e causes no pathological lung change in awake rabbits). fdp-d may contribute to various organ pathologies after trauma. the effect of aspirin on the fibrinolytic activity of viable granulocytes r.c. franz, w.j.c. goetzee, b. rotunno and r. anderson department of surgery, university of pretoria, rsa although several influential authors have suggested that low dose (150 rag) acetyl salicylic acid (asa) represents a balanced daily antithrombotic regimen probably by both inactivating thromboxane a2 production and enhancing prostacyclin synthesis little is known about the effect of aspirin on the fibrinolytic activity of live granulocytes [1] . the present study was designed to evaluate this effect in vivo. methods: granulocytes from fasting samples of heparinized venous blood taken from male volunteers were separated from monomuclear cells and platelets by density gradient centrifugation (ficoll: sodium metrizoate). viable granulocyte suspensions and plasma samples were placed as drops on a coinbefore aspirin after aspirin p = [2] . the experiment was repeated 3 h after each subject had ingested 1.8g of aspirin. the results are summarized in table 1 . 1. there appears to be a significant increase in granulocyte fibrinolytic activity 3 h after the ingestion of 1.sg of aspirin. 2. this increment is insufficient to overcome the resting inhibitor potential of plasma on granulocyte fibrinolysis. 3. aspirin does not evoke a significant increase in plasminogen activator-(urokinase) induced flbrinolysis in platelet free plasma or in the combined system of granulocyte-plasma mictures. 4. the optimal dosage of aspirin as a fibrinolytic agent requires further study. the terminal vascular bed of malignant tumors is characterized by a lack of organization, differentiation and sufficient developement of nutritional capillaries. as a result, malignant tumors reveal consistently small regions of low or even no perfusion. pre-vious data in a melanoma indicate that due to the rarefication of capillaries, the full impact of tumor treatment ±st diminished by an elevated microvascu lar resistance, which could significantly affect the impact of tumor therapy. since the improvement of the blood's fluidity has been shown as one therapeutic modality to increase significantly the capillary blood flow, it was assumed that this measure might enhance the accessibility of tumor tissue for bloodborne drugs. this study was aimed to investigate the effects of the improvement,of microcirculatory flow on tumor growth and tissue oxygenation. moreover, the response of the melanoma to chemotherapy was evaluated when isovolemic hemodilution was employed in conjunction with chemotherapy. a transparent chamber technique, intravital microscopy, a platinum multiwire electrode (local po2 measurement) and quantitative television image analysis (capillary blood cell velocity and diameter) were employed to study the microvasculature in the amelanotic melanoma a-mel-3 of 18 hamsters in the event of hemodilution without and in conjunction with chemotherapy (200 mg/m 2 dtic, dimethyl-triazeno-imidazol-carboxamid). permanent indwelling catheters in carotid artery and jugular vein served for measuring systemicpressures, heart rate, for withdrawing blood and the infusion of dtic and/or dextran 60. after inoculation of 4 x 104 cells of the amelanotic hamster melanoma a-mel-3 into s.c. tissue in the preparation, this tumor re~ched a diameter of approx 3 mm within five days. the reduction of systemic hematocrit from 0.45 to 0.31 (1.3 ±0.2 ml blood vs dextran 60, 7 animals) at a tumor diameter of 3 mm increased the growth rate of the melanoma by about 30 % while enhancing significantly the volume flow through capillaries and the mean local po2. table 1 control hemodilution capillary velocity (ram/s) 0.39 _ 0.12 0.50 ± 0.12 capillary blood flow (ml/min x 10 -5) 5.4 ± 1.7 8.9 ± 1.2 mean local po2 (mmhg) 9.4 (0 -28) 15.7 (0 -60) the frequency distribution of local po2 on the tumor's surface showed a distinct shift toward higher po2 values with still some hypoxic regions remaining. intravital microscopy, however, revealed petechial bleeding and localized, interstitial edema which compressed a small number of capillaries. by contrast, the tumor's diameter remained at app. 3 mm for a period of ten days with chemotherapy alone (3 animals). in one of the animals, a complete stop in the melanoma microcirculation was seen within four hours after infusion of dtic followed by a significant decrease of tumor diameter. when chemotherapy was initiated in hemodiluted animals, neither retardation of tumor developement nor vascular obstruction was observed (5 animals). conclusion: capillary blood flow of the melanoma can be enhanced by hemodilution thus diminishing tissue hypoxia. this measure, however, was associat-ed with an increase in melanoma diameter of 30%. at the present, we investigate whether, in hemodiluted animals, a reduction of tumor size can be obtained with a higher dose of dtic. ). however, the etiology of stress hyperglucagonemia in the immobilized rat is only poorly defined. since during restraint stress, catecholamines (ca) are elevated and stimulation ofglucagon by ca is accepted (woods sc d jr [1974] physiol rev. 54: 596), we decided to study by surgical means the the relative contribution to glucagonemia of different sources of ca, i.e. peripheral sympathetic nervous system and adrenal medulla. methods: male sprague-dawley gastric fistula rats (n=74), weight approx. 250 g, were subjected to either sham-op or various sympathectomies [microsurgical splanchnicotomy = s-sx; chemical sympathectomy = c-sx (150 mg/kg 6-oh-dopamine ip two days prior to the experiment); adrenal demedullation = amx; combinations: s-sx + amx; c-sx + amx]. gastric acid secretory trials (duration 8 h), preceded by a 24 h fasting period were carried out 6-7 days following the operation. at the start of the experiment an intraperitoneal polyethylene tube, was inserted into the abdominal cavity of the rats, allowing a constant infusion of physiological saline (4 ml/ kg/h). in addition, stress was performed by pairwise restraint of the extremities and small electric shock waves applied by a tail electrode. at the end of the experiment, blood was drawn from the vena portae and the abdominal aorta for plasma and serum. hormones (glucagon, insulin) were measured by radioimmunoassay, glucose enzymatically, volume was read to the next 0.1 ml, acidity by microtitration. results (see table) : volume and acidity are not changed by the various sympathectomies, when 179 compared to the sham group. the same is true for acid secretion, except in s-sx + amx, where it is elevated. glucagon in peripheral plasma is elevated in c-sx, amx and c-sx + amx. in the portal vein, glucagon is dccreased in s-sx + amx (408___99 pg/ ml) and elevated in c-sx + amx (2625___405 pg/ml) when compared with sham rats (930--.195 pg/ml). the portal/aortal glucagon ratio is significantly decreased only in c-sx and amx (1.7--+0.9, 1.7--.1.0, resp.) when compared with sham (3.8--_ 2.1). insulin is increased only in amx, glucose decreased in amx, s-sx + amx and c-sx + amx, insulin and glucose are unchanged in the other groups. conclusion: 1. stress hyperglucagonemia in the rat is confirmed (levels during zero stress 30-180 pg/ml) and also the rise in insulin following removal ofadrenomedullary ca (but not other sympathectomy); 2. the blood glucose fall (amx; s-sx + amx; c-sx + amx) is not uniformly paralleled by hyperglucagonemia, but in the case of amx it may be secondary to relative stress hypoglycemia owing to removal of adrenal medullary ca or reactive insulin release; 3. mechanism underlying the increased systemic glucagon despite partial (c-sx; amx) or total (c-sx + amx) sympathectomy are yet unknown. 4. during stress the enterogastrone component of hyperglucagonemia may be of minor importance. evlw showed good agreement with gravimetric lung water determinations. significant lung water accumulation was produced by pressure elevations over 20 mmhg. reductions in plasma oncotic pressure significantly increased transvascular fluxes at each level of pressure elevation. however, fluid accumulation was not significantly greater during hypoproteinemia. we conclude that a 40-50% reduction in plasma oncotic pressure does not contribute to increased high pressure edema because the lymphatic safety factor is augmented. this phenomenon may explain the well tolerated state of hypoproteinemia in patients after hemorrhagic shock. computerized gamma scintigraphy is a new technique for the analysis of albumin flux in the acute respiratory distress syndrome (ards). the objectives of this study were to obtain normal control values and to determine the method's validity in patients with cardiogenic vs. permeability pulmonary edema. methods: following 10 mci99mtechnetium-human serum albumin, lung :heart radioactivity ratios were determined. this ratio remains constant unless there is a leak o falbumin, when a rising ratio is seen, called the ,,slope index" (si). si's were determined in 5 control individuals who had :> 50 % left ventricular ej ection fraction and < 7s pulmonary circulation. thirtythree studies were obtained in 27 patients using a portable gamma camera. fourteen patients had clinical evidence of ards. results: studies were considered positive if the si was 2 s.d. > control mean ( -0.4___0.5 x 10-3 units/ min). among 15 positive studies, all had diffuse air space disease on chest radiographs. their average pulmonary capillary wedge pressure (pcwp) was 14.6_+4.6 mmhg. the average artertial: alveolar oxygen tension ratio (a/a)o2 was 0.31+--0.15 on 10.8 cm h20 peep, which were both significantly (p<0.01) different from patients with normal si's. positive si's were present from 24 hours to 6 days following the apparent onset of ards in 6 patients. recovery of gas exchange was associated with normal si's on repeat studies in 4 patients. of 6 patients with cardiogenic pulmonary edema, 5 had negative studies (18-29 mmhg pcwp) and i a positive study (40 mmhg pcwp). conclusion: gamma scintigraphy was a sensitive, non-invasive tool for the detection of a pathological increase in pulmonary protein flux, which was usually normal in cardiogenic pulmonary edema. positive scintigraphy was associated with significantly impaired gas exchange. the method documented that the leak of albumin in ards may last for days but resolves with recovery. cancellous bone thermocoagulation ph. dumontier, r. benichoux and a. vidrequin institut de recheres chirurgicales -c.h.u. de brabois 54511 vandoeuvre les nancy cedex, france electrocoagulation can not stop bleeding from the cancellous part of a sectioned bone. therefore we tested the efficiency ofthermocoagulation by hot air. the hot air generator delivers a flow, of non illtercd air, at 25-30 1/min at a fixed temperature of 300°c measured at the exit of a 3 mm diameter pipe and 60 °c at the site of bleeding. the generator sustains usual steam sterilization. 14 dogs were operated on both patellae, femoral short segments and iliac crests giving 53 different site of cancellous bone. in vitro sterility studies: the conduit of hot air was applied at various distances, from 10 cm to 2 meters, above a petri plate containing culture material. thus the turbulence in the atmosphere around the zone of thermocoagulation has been bacteriologically controlled and a particle counter used. in vivo thermocoagulation: three sites of cancellous bone were used in 14 anesthetized dogs, using a sterile procedure: the iliac crest, a small segment of the femoral bone and the divided patella. 1. five iliac crests were divided and bleeding measured after thermocoagulation. 2. the 2 segmental femoral resections were thermocoagulated. 3. the patellae were vertically divided and each section submitted separately either to thermo or electrocoagulation. the pipe of thermo-181 coagulation was directed to the bleeding surface at a 2 cm distane, sweeping it during 5 to 6 seconds. the bleeding was compared by photography and the two fragments were approximated by a wire synthesis to provide a bone fusion. in few cases both sides were thermocoagulated. results: in vitro: no contamination was found in the atmosphere, up to a distance of 2 meters. there was a significant decrease of particles around the operating site (60 % less, at 80 cm and 30 % less, at 2 meters). in vivo: the bleeding was weighted around 50% less than with conventional coagulation. thermocoagulation did not delay or disturb the healing of the patella after wire synthesis. the in vitro nucleation time of cholesterol crystals from gallbladder bile of patients with gallstones is more rapid than that from normal persons, (holan rt [1979] gastroenterology 77: 611). this study determined whether this was due to a gallbladder or liver defect and wether the defect was the addition of a nucleating factor or the deletion of an antinucleating factor. hepatic and gallbladder bile were gathered at surgery in stone patients and gallbladder bile in patients with a normal biliary tract. after ultracentrifugation, the isotropic phase was observed daily by polarizing microscopy until cholesterol crystals appeared. in gallstone patients, the nucleation time of gallbladder bile was significantly more rapid, 2.5 days+0.9 sem, than that of hepatic bile 7.9+2.1 days, although hepatic bile was significantly more saturated with cholesterol [cholesterol saturation index (csi), 2.12_+0.36], than gallbladder bile, (csi, 1.31_+ 0.12). thus the characteristic short nucleation time of stone formers is due to an alteration in bile after it enters the gallbladder. to determine whether the gallbladder defect was due to addition of a nucleating factor or the deletion of an antinucleating factor, isotropic phases of normal gallbladder bile and that from stone formers were mixed and nucleation time determined. mixtures of up to 95 % normal bile had pathological nucleation times demonstrating that the defect is the addition of a nucleating factor by the gallbladder, and that this factor is potent. the rate of formation ofgaustone precursor crystals in bile, although faster in gallstone patients than in controls, is unrelated to the degree of cholesterol supersaturation [1] , implying that other factors are involved. two competing factors seem likely; (a) secondary seed crystals in bile may trigger and accelerate gallstone crystal formation from supersaturated solution; (b) "poisons" in bile may retard or inhibit crystal growth. because of the complexity of bile itself, experiments were performed in highly purified mixtures of bile salt, lecithin and cholesterol, in concentrations closely resembling those of gallbladder bile. (a) lipid solutions were seeded with calcium carbonate, hydroxy-apatite, calcium bilirubinate and biliary mucus, all of which are found in gallstones [2] . cholesterol crystal formation was significantly faster in_ the presence of all of the seed compounds tested (x= 221.7/~g ml-lh-1) than in unseeded controls (128.0/ag ml-~h-1) (/7<0.05). (b) substances with "crystal-poisoning" properties included heparin, chondroitin sulphate and bile salt. these and changes in ph altered the quantitiy (20-80 % decrease) and rate of calcium carbonate and calcium phosphate crystal formation. we suggest that gallstone precursor crystal formation may be affected by a subtle balance between crystal seeding and crystal growth-inhibition, both due to the presence of other compounds in bile. at the last tripartite meeting we reported experimental data on a new method to destroy concrements of the kidney in situ by shockwaves allowing for spontaneous excretion via the urinary tract [1] . the shockwaves are generated externally by underwater discharge of a condensor with sparking electrodes which are localized in a focus o fan elliptic cavity. for treatment the renal concrement must be positioned exactly in the second, virtual focus opposite to the elliptic cavity. since then, altogether 126 patients were subjected to this form of treatment in our institute by the colleagues of the department of urology at the university of munich. 90% of the patients got rid of their concrements within a few days, in 8.5 % small remnants remained in the renal pelvis, and in 1.5% (2 cases) additional surgery became necessary. meanwhile this technique is employed on a routine basis in the department of urology of the university of munich [2] . the experimental as well as clinical results were considered encouraging enough to extend the technique for the treatment of biliary concrements. for this purpose, human gallbladder concrements of different composition (bilirubin, cholesterol) were implanted into the gallbladder of dogs for exposure to shockwave treatment after wound healing. under in vitro-conditions the biliary concrements could be crushed into any size desired, irrespective of their composition, while only in 8 out of 10 experiments this was accomplished under in vivo-conditions. blockage of the biliaiy duct after shockwave exposure was never observed. concrements which were experimentally implanted into the bile duct could be visualized without difficulties by contrast medium. here, destruction by shockwaves was accomplished as well. currently experiments are conducted to dissolve remnants of biliary concrements after treatment by administration of desoxycholic acid. precise positioning of the gallbladder concrements in the second virtual focus is a problem which has not been satisfactorily solved so far, because the concrements cannot be visualized by conventional x-ray techniques. alternatively it is attempted to employ ultrasound, or visualization by retrograde injection of xray contrast medium through a catheter. in experimental animals, we leave a t-formed drain in the gallbladder for injection of contrast medium. in animal experiments conducted so far, histological or clinical evidence for tissue damage has not been obtained, as is the case with shockwave treatment of kidney stones. we are convinced that treatment of biliary concrements by shockwave exposure can be employed under clinical conditions in the near future. exploration of the common bile duct (cbd) for calculi, particularly in the presence of obstructive jaundice, is a procedure with considerable mortality and 183 morbidity. to avoid the problem of retained stones, choledochoduodenostomy and transduodenal sphincteroplasty have been recommended, but have their own complications. this morbidity might be reduced by removal of cbd calculi prior to surgery. endoscopic sphincterotomy (es) allows this. a review of 123 cases of es performed for calculi indicated that this was a safe (complications 8% no deaths) and reliable procedure (85 % success rate). a study was conducted of patients with known cbd stones who had either preliminary es followed by operation at a later date (group i) or operation alone (group ii). this study showed a lower morbidity in group i. a prospective randomised controlled study has begun on the basis of these findings and the data from both studies are shown in the table. these results suggest that pre-operative endoscopic sphincterotomy my reduce the morbidity of cbd stones. group ii n = 28 two controversies regarding the physiology of the biliary sphincter (bs) concern its functional independence from the duodenum [1] and those aspects of its acitivity which control bile flow [2] . the rabbit was chosen as the experimental animal as it has an easily identifiable sphincter. during anaesthesia induced by intravenous pentobarbital sodium, recordings of the electrical and mechanical activity of the bs and duodenum were made from (a) starved, (b) fed and (c) starved animals during administration of cholecystokinin, pentagastrin, secretin and glucagon. spike complexes (sc) were ordinarily associated with mechanical acitivity of the sphincter and duode-num. of 2.172 sphincter sc, recorded in 18 animals, 775 (36%) were not associated with duodenal acitivity, whereas 1.397 of 1400 (99.8 %) duodenal sc were accompanied by synchronous bs activity. this supports the hypothesis that the rabbit's bs can contract independently of the duodenum but that duodenal contraction is usually accompanied by simultaneous contraction of the bs. sphincter scs correspond to its phasic acitivity. food and cholecystokinin increased the number of sc without altering the baseline pressure of the perfused common bile duct. pentagastrin produced a transitory increase in sphincter activity whereas :secretin and glucagon were without effect. phasic activity of the spincter may influence bile flow through the choledochoduodenal junction. natural blood coagulation finally results in the formation of fibrin, which is one of the most important components of hemostasis in the human organism and thus provides the basis of all reparative procedures that are part of wound healing. it stands to reason to utilize the properties of fibrin for hemostasis during surgery and for joining severed tissue. first attempts of this kind were made at the beginning of this century. but only after greater insight had been gained into the coagulation proc-185 ess and the manufacturing techniques of blood derivatives had become more sophisticated, the essential breakthrough was made. a biological adhesive system has been developed, which consists of highly concentrated fibrinogen, thrombin and clotting factor xiii. this tissue sealant is completely resorbable and of high adhesive property. further advantages are elasticity of consistence and excellent tissue compatibility. after extensive animal experimentation, first clinical experience was made in 1973. in the meantime the fibrin-adhesive-system (fas) has been introduced into numerous surgical disciplines with excellent results. the outstanding properties are: atraumatic tissue synthesis; enhancement of fibroblast proliferation and promotion of rapid wound healing; obtaining of local hemostasis by sealing bleeding surfaces, which is of special importance in the treatment of patients suffering from hemophilia or during operations under heparinization. the authors experience in using the fas within the last 10 years is reported and a review over indications, techniques and advantages of this method is given. bile salts have been shown to enhance the stability and prolong the activity of intraluminal pancreatic enzymes and may therefore influence the effects of impaired exocrine secretion in patients with pancreatitis [1] . individual bile salts in the peak 15 min collection of duodenal fluid following cck/secretin administration have been quantitated by high performance liquid chromatography in 7 patients without pancreatic or hepatic impairment (group c), 6 patients with acute pancreatitis (group ap) and 8 patients with chronic pancreatitis (group cp) all with functioning gallbladders, and 4 patients with gallstone related acute pancreatitis (group gs). the peak total bile salt output in moles and the trihydroxy: dihydroxy (tri:di), primary:secondary (p:s) and glycine:taurine (g:t) bile salt ratios are shown below (mean___ sem). the duodenal aspirates contained detectable amounts of taurine and glycine conjugates of cholate, chenodeoxycholate, deoxycholate and ursodeoxycholate but not lithocholate or free bile salts. the low total bile salt output in groups cp and gs were due to decreased levels of all the individual bile salts. although the bile salt pattern in groups c and ap were similar, the relative proportions of trihydfoxy and secondary bile salts were higher in groups cp and gs respectively. these results indicate that patients with chronic pancreatitis without obvious large bile duct obstruction have an impaired bile salt output into the duodenum and this may exacerbate the effects of pancreatic exocrine insufficiency. an elevated amylase creatinine clearance ratio (accr) was considered a specific test for the diagnosis of acute pancreatitis (ap). however, it has been found elevated in other diseases as well as after surgery. the aim of this study was to evaluate prospectively the accr levels in patients with ap and in several groups of surgical patients. we studied 197 subjects divided into 6 groups: group a: acute pancreatitis (n=67). group b: patients undergoing biliary tract surgery (n=38). group c: peptid ulcer patients undergoing gastric surgery (n=25). group e: patients undergoing cardiac surgery under extracorporeal circulation (n = 16). group f: control group of healthy subjects (n = 40). the accr was determinated using the levitt method. in the surgical groups the accr was measured before and after the operation. amylase was determinated by the phadebas amylase test. ap was diagnosed on the basis of both clinical and radiological findings and the presence of high serum amylase levels. this diagnosis was confirmed through laparotomy in 18 cases (26%). the accr was 1.96 + 0.8 % (mean___ sd) in group f, control group, and 7.5+5.7% in group a, acute pancreatitits p<0.01. accr values below 4 % were found in 21 cases of acute pancreatitis (31%). among those patients whose ap diagnosis was confirmed through surgery the accr was 10.6+7.9% (mean_+sd), higher than in the rest of the ap patients, 6.4±4.2% (p<0,05). in groups b,c,d and e (surgical groups) the accr before the operation was 1.83±0.8%, 2.34±0.9%, 2.28+ 1.6% and 1.54+ 1.06% (mean___ sd), respectively. after the operation it was: 3.85+2.2%, 3.18+2%, 2.83± 1.5% and 2.05+ 1.6%, respectively. on the average, we found an increase in the accr levels after the operation in the biliary tract group (p<0.01), but not in the other surgical groups. in 22 patients (24 %), the accr after operation was above the upper limit of normal. none of these patients had symptoms compatible with clinical pancreatitis. in conclusion: 1. the accr increase in acute pancreatitis (sensitivity: 69o/0) 2. the average accr increase after biliary tract surgery, but not after either gastric or thyroid surgery or after cardiac surgery under extracorporeal circulation. however, it is possible to find isolated cases with high accr after any type of surgery without any symptoms of pancreatitis, suggesting that an increase of accr may be an unspecific finding in postoperative patients which require further investigations. out of 217 patients with acute pancreatitis 25 developed a fulminant type. 18 were males and 7 females, mean age 45 years (range 23-83 years). all 25 patients were primarily treated by peritoneal lavage applied at laparotomy. indication for laparotomy was sudden deterioration with (20) or without (5) organ failure. a necrotic or hemorrhagic pancreas was found in every patient. the pancreas was exposed and 2 soft and large catheters were placed close to the pancreas. mean duration of lavage (ll/h) was 9 days. due to secondary deterioration a pancreas resection was performed 2-10 days later in nine patients. 7 patients with acute fulminant pancreatitis died, a mortality of 27.7%. all 192 patients with the mild type of acute pancreatitis survived, thus the overall mortality was 3.2%. none treated by peritoneal lavage only developed diabetes mellitus, whereas 4 out of 5 surviving patients with an additional pancreas resection had this complication. 19 patients with acute fulminant pancreatitis displayed 3 or more ranson criteria [1] and six died. however, no less than 32 of those with a mild type of acute pancreatitis fulfilled 3 or more of these criteria and they all survived. a laparotomy -not a laboratory test -is necessary to confirm the diagnosis of acute fulminant pancreatitis. the indication for laparotomy is mainly clinical and therefore such a patient should preferably be handled by surgeons or physicians experienced in this disease. after confirmation of a correct diagnosis peritoneal lavage is one of the methods by which the mortality of acute fulminant pancreatits -which by conservative means is 80-100% -can be reduced. coincidences ofhyperparathyroidism and pancreatitis have been given up by different author varying between 1% and 19%. frequently a causal relationship has been defended. recently, however, any causality has been queried [1] . we analysed our own series of patients with surgically and histopathologically confirmed primary hyperparathyroidism (phpt) (n=686) and found a coincidence with a coexisting or prior pancreatitis of 1.5 % (n = 10). from this a causal relationship cannot be concluded. however, 3 out of these 10 patients (i.e. 0.4%) had an acute onset or exacerbation of a pancreatitis immediately following the parathyroidectomy, which is strikingly more than one would expect after an operation without any anatomical relation to the pancreas (<0.01%) [2] . none of these 3 patients had another cause of the pancreatitis such as cholelithiasis or alcohol abuse. hence a causal relationship cannot be excluded. it is imaginable that excessive amounts of parathyroid hormone are released during the surgical manipulation of the pathologically altered parathyroids. the postoperative pancreatitis may be caused by the acute elevation of parathyroid hormone levels in the presence of hypercalcemia. in our series the parathyroidectomy was combined with a partial or total thyroidectomy in 27 patients. in another 7 patients a thyroid operation was performed prior to the parathyroidectomy. although calcitonin has been employed as a possible therapy in patients with acute pancreatitis (ap), the normal levels of this substance in ap are not well documented and have not been correlated with pth. furthermore no definitive role in human calcium homeostasis is accepted for calcitonin, whereas high pth levels have previously been correlated with hypocalcaemia [1] . in 21 patients with ap the mean serum calcitonin on admission was 121 ng/1, and the peak level mean 173 lag/1 (upper limit of normal 75 ng/1). calcitonin levels were higher in severe than mild ap, and 5 of 6 patients with levels >200 ng/1, were objectively graded as severe ap [1] . in the 3 hypocalcaemic patients, with corrected serum calcium <2.0 mmol/1 all recorded calcitonin levels >100 ng/1 and had elevated pth. nine of the 21 patients had significantly elevated pth ('>700 rig/l) and of these only 1 did not have an associated elevation in calcitonin. the high calcitonin levels in patients with ap suggest that supplementary calcitonin intended to inhibit pancreatic secretion is unnecessary. at present it is merely speculative to suggest a role for calcitonin in ap but intriguing to report a tendency to parallel the elevations of pth. nuclide labelled microspheres were used to measure pancreatic and other visceral blood flow in two groups of conscious dogs before and after intravenous alcohol infusion. blood alcohol concentrations at the time of blood flow measurements were similar to those encountered in intoxicated humans. thus 8 dogs (groups a) were given a somewhat low dose of alcohol to produce a mean blood alcohol level of 0.16 gm d1-1, while 6 dogs (group b), received substantially greater doses of alcohol, and reached a mean blood alcohol of 0.32 gm dl-1. wilcoxon matched pairs signed rank test confirmed a biphasic, concentration-related, response of pancreatic blood flow after alcohol infusion; no such response was found in blood flow to other viscera. moderate alcohol levels (group a) were associated with a decrease in pancreatic blood flow (p<0.03), while high blood alcohol concentrations resulted in increased pancreatic blood flow (p'<0.02). colonic blood flow increased in both groups a and b, but blood flow to the gallbladder, small intestine and the parotid gland increased in group b only. gastric, duodenal, renal, hepatic (arterial) and cerebral perfusion did not change. in addition, direct observations of the surface of the pancreas showed occasional haemorrhagic areas and mottling. these findlings however could not be confirmed by objective attempts to measure blood flow in such discrete areas in conclusion pancreatic blood flow shows a biphasic, concentration-related, response shortly after intoxication. this response appears to be peculiar to the pancreas and does not occur in other viscera. we recently showed that acute ethanol (e) and/or aspirin (a) ingestion increased the permeability of the pancreatic duct to large molecules, this suggested that pancreatic enzymes might leak from the duct into the parenchyma, causing pancreatic disease. this is a new concept in the study ot he pathophysiology of this organ (to be presented at aga, may, 1982, plenary session). in the present experiments wie studied the effects of chronic e/a ingestion on pancreatic function in dogs. methods: 6 dogs were fitted with duodenal and gastric cannulas. after recovery, baseline secretory sutdies were performed by cannulating the pancreatic duct and collecting pancreatic juice during secretin infusion (0.1 (submax) or 2.0 u/kg-hr. (maximal) iv). at least 2 studies were performed in each dog at each dose. after baseline studies, 3 dogs (group e) were given daily intragastric ethanol (2 gm/kg-day). 3 dogs (group a/e) were given e and a (100 mg/kgday). after 36-48 weeks, secretory studies were repeated. results: all dogs gained weight (x = 1.28 kg). the pancreases appeared normal by light microscopy. drug treatment increased volume and hco3 output by 47 and 67%, respectively, in group e, submax secretin, but decreased them by 29 and 34 % in group a/e animals. (p=0.0001 vs. predrug values and group e vs. a/e values). drug treatment decreased volume and hco3 output in both groups by 5-10°/c (p=0.0001) after maximal secretin stimulus. (manova test for all statistical analyses). conclusions: consumption of e alone increased volume and hco 3 output after submaximal and decreased them after a maximal secretin stimulus. this confirms the work of sarles. consumption of e and a reduced volume an i-ico3 output after secretin at both doses. thus chronic a ingestion further impaired pancreatic function in these animals. since only a small proportion of chronic alcoholics develop clinically significant pancreatic disease, an aggravating "cofactor" may be operating in this group. chronic asa ingestion, not uncommon in alcoholics, may represent such a cofactor. the development of diabetes mellitus in pancreatic cancer is well known but the standard oral glucose tolerance test is not recognised as a useful diagnostic indicator. glucose homeostasis, insulin and c-peptide secretion in response to intravenous glucagon were studied prospectively in patients with suspected pancreatic cancer and assessed as to their diagnostic value. 34 fasting patients were given glucagon, 1 m.g., i.v., and serial measurements of blood glucose, plasma insulin and c-peptide concentrations made for 60 min. subsequently it was shown that 12 patients had pancreatic cancer and the remainder constituted a control group. there was not significant difference in the rise in blood glucose between the groups after glucagon. the mean plasma insulin concentrations rose rapidly in both groups peaking between 2 and 5 rain but the values were sginificantly lower in the pancreatic cancer group (p<0.01 at 5 min: p<0.001 at 15 min: p<0.01 at 60 min). a similar pattern was observed with c-peptide. in patients with obstructive jaundice the plasma insulin response was a better discriminator of pancreatic cancer. we conclude that abnormal pancreatic beta cell funktion exists in patients with pancreatic carcinoma, detectable before any change in glucose homeostasis, particularly in patients with obstructive jaundice. the glucagon stimulation test may have a useful role in the diagnosis of pancreatic cancer. t-suppressor cells (t~) have previously been implicated as mediators of graft surival in baboons tolerant to their renal grafts [1] . in addition, it seems that t-helper cells (th) are also affected by totallymphoid irradiation in that they are unable to provide help in mitogen-induced t-cell responses or pokeweed mitogen induced immunoglobulin synthesis in bcells. in this study the evolution of t~ and th is followed in baboons undergoing graft rejection at different rates. peripheral blood was collected from the animals at weekly intervals after renal transplantation, defibrinated and the lymphoid cells isolated by flotation on ficol hypaque. the t-lymphocyte fraction was purified by filtration through a column packed with nylon wool. th, ts and total t-cells were enumerated using monoclonal antibodies okt4, 8 and 11 respectively (ortho). the th/ts rations reported were taken immediately before a rapid rise in serum creatinine occurred. in longlived baboons who maintained normal creatinine values, a mean of the ratios over the last month was used. b rats are produced by sublethal (750 rad) x-irradiation of thymectomized animals, reconstituted with bone marrow from syngeneic, thymectomized, thoracic duct drained donors. in these lew rats, (lew x bn)f1 cardiac allografts survice indefinitely (>100 days); unmodified lew rats acutely reject such allografts (8-+ 1 days). in this study, we have tried to restore the processes of acute rejection in b recipients. graft survival appeared independent of blocking factors or suppressor cells, as transfer of serum or lymphocytes from b recipients into syngeneic normal animals failed to increase survival of test allografts, placed subsequently. similarly, immunogenicity of long surviving grafts was unchanged; such grafts functioning >100 days and retransplanted into normal animals were rejected acutely. adoptive transfer of 108 unseparated spleen cells (sl) from nonimmune syngeneic animals produced slow rejection (28--+5 days) in b rats; 108 sensitized slwas somewhat more effective (22_+ 1 day). transfer of 2 x 107 syngeneic peritoneal exudate (pe) cells plus 108 sensitized sl caused acute rejection in 50 % orb recipients (11 _ 1 days), the remainder experiencing rejection at c 3 weeks. pe cells harvested from rats injected ip with thioglycollate 3 days previously, were primarily macrophages/adherent cells, as the cells used were that fraction sticking to plastic dishes and removed with lidocaine (purity >90 %). in vitro, b rat macrophages were abnormal, having only 50 % of capacity of normal macrophages to promote production of interleukin 2 (il2) when co-cultured with purified t lymphocytes. however, b recipients experienced acute graft rejection (10-+ 1 days) after transfer of 108 sensitized sl plus semipurified il2, thus bypassing the above defect. addition ofll2 to the t cell (purity >95°/0) equivalent of 108 sl (purified over degalan bead columns coated with rabbit antirat lgg, nonadherent fraction) failed to reestablish acute rejection (17-+7 days), while further addition of b lymphocytes (degalan bead adherent fraction, purity >90%) or macrophages was uninfluential (16___2 days and 16___3 days, respectively). transfer of il-2 alone never produced rejection. acute rejection can be re-established, however, by increasing the number oft lymphocytes (108, transferred concomitantly with il2). thus, the state of unresponsiveness in b rats can be reversed in vivo by adoptive transfer of particular cellular elements in the presence of growth factors; increased graft survival seems dependent ultimately upon il-2 production by sensitized t cells, presumably t helper cells. the relative inability of b rat macrophages to promote production of il-2 by t cells may be primarily responsible for the immunological deficit of the b rat. one of the most intriguing findings ofcyclosporin a (cya) immunosuppression is that in some species a short course of treatment will produce very prolonged allograft survival. we have tested the ability of cya to prolong the survival ofvascularized heart, kidney and pancreas allografts by direct comparison in a da (rt1 a) to lew (rt1 ~) rat allograft model. accessory abdominal heart and orthotopic left kidney transplantation were performed using standard microsurgical techniques. in renal transplantation the left kidney was removed at the time of transplantation, the remaining right kidney 5 days thereafter. streptozotocin-diabetic animals received ductligated pancreas whole organ grafts isolated on the portal vein and a segment of the aorta giving offthe coeliac axis and the superior mesenteric artery. rejection was taken as complete stop of palpable pulsations in heart transplantation, the day of death in renal transplantation and recurrance of hyperglycemia above 14 mmol/1 in pancreas transplantation, respectively. cyclosporin a 15 mg/kg body weight, dissolved in olive oil, was administered intramuscularly for 14 days starting with the day of transplantation. in all instances functional demonstration of rejection was confirmed by histological examination. cyclosporin a is effective to prolong the survival of vascularized heart, kidney and pancreas allografts. while cya is administered none of the grafts has been rejected. however, following withdrawal of the drug pancreas grafts are rejected within 14 days and heart grafts within 33 days. none of the kidney grafts has been rejected so far. the differential susceptibility of vascularized heart, kidney and pancreas allografts to cya immunosuppression may be caused by differences in immunogenicity due to organ specific alloantigens or a differential representation of spezialized antigen presenting cells. it may also reflect different patterns of rejection of the various organs. during cya administration all rejection processes are effectively suppressed. in the maintaince phase after withdrawal of cya such immune responses may prevail and ultimately lead to rejection of pancreas and to a lesser degree of heart allografts. the venous allograft still remains an attractive alternative for the reconstruction of small caliber vessels. however, when the venous graft is introduced to a non-histocompatible host, rejection and early occlusion is the rule. this study evaluates the use of cyclosporin a (cya) as a graft pretreatment, or systemic immunosuppressant for venous allografts. in addi-tion, cryopreservation techniques for pretreated venous allografts was investigated. 45 adult mongrel dogs, weighing between 12 and 24 kg, were used as recipients for donor jugular vein segments (5-6 cm) which had been excised and flushed with 100 cc of plasma protein fraction (ppf) at 4°c. these venous allografts were anastamosed end-to-end into a carotid artery of the recipients. the animals were divided into five groups as follows: group i (n=6) received untreated venous allografts without subsequent immunosuppression, group ii (n = 5) was the same as group i with minimal immunosuppression (azathioprine 2.5 mg/kg/day). in group iii (n= 10) the animals were transplanted with venous grafts stored in 25 cc of plasma protein fraction (ppf) containing cy a (50 mg/1) at 4 ° c for 24 hours, immunosuppression was as in group ii. in group 1v (n=9) the animals received allografts that had been cryopreserved in a 15% dmso solution at -196 ° c for 25-35 days and then the animals had azathioprine as in group ii. in group v (n= 15) venous allografts recipients were treated with systemic cya (20 mg/kg/day x 2 weeks, followed by 15 mg/kg/day x 2 weeks) as the only immunosuppression. the patency of the allografts was evaluated at 1, 2, 3, 4 and 8 weeks post transplantation. patency results at one month showed that azathioprine alone failed to improve the patency rate (gr. i and ii = 0% patency). cya graft pretreatment, however, significantly improved the one month patency (gr. iii = 57%). in addition, cryopreservation appeared to enhance the graft pretreatment effect of cya (gr. iv; one month patency = 85.7%) of the allografts. finally, systemic cya proved very effective in preventing rejection and occlusion (gr. v; one month patency = 100 %). grafts that remained patent for a initial critical period of 4-6 weeks, all showed long term patency. the effect of cya in preventing graft rejection was further documented by histiological studies of the allografts which showed a marked cellular infiltration and degenerative changes in all the grafts of the control group as compared to minimal or no cell infiltration in the patent grafts of the treatment groups. in summary, it appears that cya used as a graft pretreatment with minimal immunosuppression of the recipient, in conjunction with cryopreservation or given systemically as the sole immunosuppressant can significantly improve the survival of venous allografts. in our previous reports, it was shown that isolated hepatocytes transplanted into the splenic parenchyma of syngeneic rats, proliferated markedly and recomposed the hepatic tissue. this experimental system provided a new model to elucidate the mechanism of hepatic regeneration which could not be obtained in in vitro cell culture experiments. in the present paper, fetal hepatic tissue instead of isolated adult rat hepatocytes were transplanted into the rat spleen. we document briefly long-term morphological observations on the transplanted fetal hepatic tissue with special reference to proliferation of the hepatocytes and bile ducts. materials andmethods:wistar rats were mated in our laboratory for a fetal liver source. gestation day 0 was when a plug or sperm were observed in the vaginal smear. fetuses used were of 18 to 20 days gestation. about ten fetal livers which were obtained from one maternal rat, were minced with scissors. the liver fragments were washed three times with saline solution. transplantation was carried out by direct injection into the spleens of syngeneic adult rats using a 15 gauge needle. half of the liver fragments obtained from one maternal rat were innoculated into the spleen of one animal. a total of approximately 30 rats with transplanted liver fragments were killed 1, 3, 7, 14 and 30 days and then every two to three months until one year after transplantation. the spleens removed were stained by h.e., pas and silver nitrate for histological examination. results: fetal livers exhibited no lobular architecture or hepatic cord structure. the very sparse cytoplasm of the hepatocytes and many hemopoetic cells among the hepatocytes were characteristically found only in the fetuses. one week after transplantation, the survived hepatocytes revealed almost the same morphological features as in fetal liver except for the presence of several proliferated bile ducts around the hepatocytes. two weeks later, the hepatocytes formed apparent hepatic cord structures and the extoplasm of each hepatocyte increased abunduntly and became acidophilic as seen in normal neonatal hepatocytes. hemopoetic cells disappeared. four weeks later, hepatocytes began to proliferate sporadically among the markedly proliferated bile ducts, groups of survived hepatocytes with cord structure were very similar to a neonatal liver except for the lack of the glisson's area. two or three months later, proliferation of the hepatocytes became prominent. there seemed to be no interrelationship between proliferated hepatocytes and bile ducts. one year after transplantation, a white nodule was observed on the spleen macroscopically and it consisted of numerous bile ducts and hepatocytes with or without cord structure on histology. summary: 1. fetal hepatocytes transplanted into the spleen, differentiated to almost normal neonatal hepatocytes two weeks after transplantation. 2. hepatocytes began to proliferate about 4 weeks after transplantation. 3. three days after transplantation, proliferation of bile ducts was already observed independent of the transplanted hepatic tissue. 4. when comparing the difference in proliferation between fetal hepatic tissue and isolated hepatocyte transplantation, marked proliferation of the bile ducts in fetal hepatic tissue was observed and fetal hepatocytes proliferated more rapidly, while there were no proliferated bile ducts in isolated hepatocyte transplantation. pretransplant splenectomy (sx) has been of disputed benefit since its introduction two decades ago. 220 of 315 patients with first cadaver transplants treated at our institution between dec. 1968 and dec. 1981 have had pretransplant sx. at six monts, sx patients had 10% better kidney survival, but this benefit was lost shortly after 1 year and by 5 and 10 years was 10% and 15% worse in sx patients. patient survival for sx and no sx was identical for the first year but was 10% and 20% worse by 5 and 10 years respectively in sx patients. thus, the early improvement in kidney survival was more than offset by a late high mortality. a rational basis for selecting patients who might benefit most from pretansplant splenectomy is urgently needed. since july, 1978, 34 patients ages 16-45 have received first cadaver transplants after having been tested for reactivity to dncb. nine of 20 dncb negative patients had splenectomy as did 6 of 14 dncb positive patients. kidney survival at 1 year for dncb negative patients without sx was 79%; for dncb negative with sx, 29%; for dncb positive without sx, 21%; for dncb positive with sx, 62%. rejection was the sole cause for kidneyloss in dncb positive patients without sx. however, 5 of 9 dncb negative patients with splenectomy died, primarily of septic complications. since 1978 survival of sx patients has been 60% compared to 95% in non sx patients (p<0.04). sx appears to be beneficial in dncb positive patients but has an adverse effect in dncb negative patients because of an increased susceptibility to fatal infections. prior blood transfusion improves renal graft survival [1] . plasma from uraemic patients suppresses the in vitro responses of normal lymphocytes to antigen (plasma suppressive activity, psa) and this effect is mainly attributable to the plasma protein macroglobulin (a2m) [2] . the aims of the present study were: a) to identify changes in psa and a2m concentration in uraemic subjects following primary blood transfusion. b) to correlate the psa of transfused renal transplant recipients with subsequent graft survival. a) ten potential transplant recipients were studied before and after their first blood transfusion. following blood transfusions the psa increased significantly (p<0.01) reaching a maximum at two months. there was no significant change in the plasma a2m concentration over the same period. b) the plasma of 137 consecutive chronic renal failure patients was tested for psa prior to renal transplantation and before institution of immunosuppressive therapy. all but two patients had received previous blood transfusions. after transplantation patients were followed for a minimum of 3 months and a maximum of 42 months. 16 grafts failed for non-immunological reasons and were excluded from the study group. patients were divided into two groups according to the degree of suppressive activity of their plasma. a volume of5/t 1, producing a 50% inhibition of normal lymphocytes was used as a treshold to differentiate those with a high or low suppressive activity. graft survival in the first three months was significantly better, 92% (/7<0.05) for those recipients with a high psa as compared to 74% for those with a low psa. we conclude that blood transfusion causes a significant increase in psa although not a2m concentration and that patients with high psa have a better graft survival. the effect of in vitro steroid on antibody dependent cellular cytotoxicity (adcc) was studied in patients awaiting renal allotransplantation and the results were correlated with transplant outcome. 85 recipients of primary cadaveric allografts were classified as steroidsensitive or steroid-resistant from the degree of adcc suppression induced in vitro by methylprednisolone, 36 patients being steroid-sensitive and 49 steroid-resistant. following transplantation patients received azathioprine and prednisone, and rejection crises were treated with bolus doses of methyl-prednisolone. graft failure occured in 5 of the 36 steroid-sensitive patients, and in 34 of the 49 steroid-resistant patients. the observed one year graft survival rate was 57.6% for the whole group, 83.1% for the patients with steroid-sensitive adcc and 36.9 % for those with steroid-resistant adcc, the difference between the two groups being highly significant (xz=23.6). a high incidence of early graft failure was seen in steroid-resistant adcc patients, 49.7% of grafts being lost in the three months after transplantation, as compared with only 2 of 36 graft failures in the steroid-sensitive adcc group in the same period. analysis of hla-a, hla-b and hla-dr incompatibilities showed no significant difference between the groups, and since all patients had received deliberate pregraft blood transfusion, the difference in survival rates between the two groups appears to be independent of these two variables. these findings confirm our preliminary observation that pregraft assay of adcc response to in vitro steroids identifies those patients who are unlikely to respond to steroid therapy in the treatment of rejection, and in whom alternative forms of therapy may be appropriate. post-operative dxt, whilst not influencing survival, protected patients from loco-regional recurrence 07<0.001, hazard ratio (hr) = 3.1). interestingly it was found to be most effective against axiallary node recurrence (p<0.001, hr = 4.0), reasonably effective against chest wall recurrence (/7<0.001, hr=2.1) but conferred no protection against supraclavicular node recurrence (hr = 1.2) in spite of a supraclavicular field being routinely employed in the radiotherapy technique. with such large numbers involved, this trial has facilitated the study of the prognostic significance of sub-groups of patients with different patterns oflocoregional recurrence as first evidence of treatment failure (see table) . of those patients developing loco-regional recurrence who have since died (222 out of 356 in wp group; 73 out of 128 in dxt group) 63% in the wp group and 69% in the dxt group did so with evidence of persistent loco-reglonal disease. however, the incidence of uncontrolled local disease at death was higher in the wp group overall. stress as well as dietary fatty acids have been shown to prolong allograft survival in rats [1] . poly unsaturated fatty acids (linoleic acid, arachnoidic acid) have been reported to depress immune response [2] . depressed immune response was suggested to correlate with a higher incidence of spontaneous tumor [3] as well as with an increased growth rate of inoculated tumors [4] . the objective of this study was to elucidate the effect of two environmental factors i.e. chronic stress (change in light/dark pattern) and diets low and high in linoleic acid on immune response and growth of transplantable tumors in bn rats. immune response: four experimental groups (n >10) were used in immune response studies. group i: high linoleic acid dietl; group i1: low linoleic diet 2, group iii: l/d shift weekly, normal diet and group iv: controls on normal diet, normal lighting. seven weeks after the start of the experiment the immune response was measured. the results showed that corticosterone levels were slightly increased in all experimental groups, although only the high linoleic group showed statistic significant difference with the control group. cellular immune response (con a stimulation and popliteal node assay) was decreased in all experimental groups compaired to controls. transplantable tumors: 1 x 106 leukemia cells were injected i.v. and pieces of 1 mm 3 of an spontaneous adrenal cortical carcinoma, a urethral squamous cell carcinoma and a round cell cervix sarcoma were implanted subcutaneously. all tumors were inoculated in groups of 10 animals each. spleen weight as a measure of leukemia growth was high in the control group and low in the experimental group. the same pattern was seen in the growth of the subcutaneously implanted adrenal cortical carcinomas. both the urethral squamous cell carcinoma and the round cell cervix sarcoma, being non-immunogenic, did not show any difference in growth. so far, it can be concluded, that the immunosuppression as induced by mild chronic stress or dietary fatty acids does not lead to enhanced tumor growth. in contrary, the results of both leukemia and adrenal cortical carcinoma show a possible reserve effect. little is known of the derivation or content of human breast cysts. recent reports have shown wide variations in the content of steroid hormones, particularly dehydroepiandrosterone sulphate (dhas) [1, 2] . no explanation for this is apparent. to confirm the large variation in dhas concentrations and to further define the contents of cyst fluids, 100 cysts from 85 patients have been analysed for dhas, sodium and potassium. dhas concentrations ranged from 1.5-1155 pmol/1. both sodium and potassium content also varied widely (sodium 30-200 pmol/1 and potassium 3-158 ~umol/1). there was a significant direct correlation between the content of potassium and dhas in cyst fluid (p<0.001) and a significant negative correlation with sodium content (/7<0.001). three separate subpopulations of cysts could be identified according to the sodium and potassium content and these were, predominantly potassium cysts (47), predominantly sodium cysts (44) and mixed cationic cysts (9). the median dhas concentration of the potassium cysts was 215 pmol/1 similar to the levels found in human breast secretions [3]. in contrast the median concentration of dhas in the predominantly sodium cysts was 14 pmol/1 and significantly different (p<0.0005), with many of these cysts having dhas concentrations in the same range as those found in plasma. the remaining mixed cysts had a median dhas concentration intermediate between the two main groups. it may be that the variation in cationic content and dhas concentration in these two major subpopula-tions of human breast cysts represents either, derivation from two different sources, namely breast secretions and plasma or marked differences in the secretory activity of the epithelium lining these two groups of cysts. there is no uniform agreement on the correct management of patients with invasive lobular carcinoma (ilc). it is widely considered that in ilc there is an increased risk of developing a contra-lateral carcinoma and the major controversy surrounds the management of the second breast. the survival of patients with ilc was significantly better than that of idc fp<:0.025). six patients had bilateral carcinomata at diagnosis and a further 14 developed a contra-lateral carcinoma during the period of follow-up (10 to 21 years). survival data showed poor survival for patients with simultaneous bilateral disease, but no difference in survival for patients with metachronous bilateral or unilateral disease. this suggests that the later development of a second carcinoma does not necessarily reduce the probability of survival for patients with ilc. the major factor predicting patients at risk of developing a contralateral carcinoma was histologi-195 cal type. of 22 patients with a particular histological pattern of ilc [4] with a classical pattern of spread but showing nuclear pleomorphism and cellular cohesion, 10 developed a contralateral carcinome, compared with a further 10 in the remaining 81 patients (p<0.001). if bilateral mastectomy is justified it ought to be restricted to patients with this histological type of ilc. both the anti-oestrogen tamoxifen and cyclical combined chemotherapy will provide significant palliation in advanced breast cancer. the optimal use of these agents requires further evaluation and thus this trial was designed to compare a combination ofcytotoxic therapy and tamoxifen, against cytotoxics alone in patients with advanced breast cancer. post-menopausal patients presenting with metastatic breast cancer, locally advanced cancer extending beyond the breast and regional nodes, or with tumor recurrence following primary local treatment were allocated to the 3 treatment arms via sequential manner. doxorubicin, cyclophosphamide, 5-fluouracil, and vincristine were given intravenously once every 3 weeks. tamoxifen was prescribed in a dose of 20 mg. b.d. on failure or relapse from one of the single modality arms, a crossover of those arms occurred. the combination consisted of both the above therapies. assessment of therapies was made in terms of objective response (uicc criteria), duration of response, and survival. we have previously reported that the combination results in a significantly greater response rate [1] . as a result of stenosis reducing flow or by platelet embolisation [1] . as neither aniography nor ultrasound can identify thrombotic activity we have evaluated gamma camera neck imaging using n 1indium platelets. labelled platelets on endarterectomy specimens were also measured and the activities found were then examined in a theoretical model. twentyfive patients with tia received rain platelets and sequential gamma images were interpreted by two observers, carotid endarterectomy in 11 patients allowed measurement of specimen radioactivities. angiography and doppler spectral analysis [2] were also performed. all endarterectomy specimens contained labelled platelet deposits with the most active equivalent to platelets from 1.8 ml of blood. this activity level was at the threshold of resolution in the theoretical model. both observers agreed that 22 of the 50 carotid bifurcations showed platelet accumulation on imaging. of the 12 atheromatous ulcers demonstrated by angiography 11 were visualised, but only 5 of 10 stenoses greater than 80 per cent were detectable~ since ultrasound identified all stenoses only one angiographically diseased carotid was not detected by combining doppler and platelet imaging. diseased carotids accumulate rain platelets with the more thrombogenic ulcerated plaques identified more frequently than stenoses. long term follow-up is required to establish the clinical relevance of platelet deposition. major problem in vascular endoscopy is the existence of blood which prevents clear visualization. we devised a new technique using a combination of balloon catheter and slender fiberoptic endoscope, by which clear visualization was obtained experimentally and clinically. three to four pairs of orifices of intercostal arteries were also visualized in one visual field. in some dogs, acute aortic dissection was experimentally created by means of blanton's method. the entry, which was located at the descending aorta just distal to the left subclavian artery, was clearly identified. complete occlusion of blood flow and clear visualization could be obtained when balloon pressure exceeded systemic blood pressure. clinical study: in six patients requiring major vascular reconstruction of the aorta (abdominal aneurysm 4, leriche's syndrome 1, dissecting aneurysm 1), vascular endoscopy was performed intraoperatively. in five patients, balloon catheter was introduced through the one of the limbs of y graft after proximal anastomosis. in each case, orifices of the major abdominal aortic branches were clearly observed. irregular orifices and atheromatous plaque of the aortic intima which were not expected from aortogram, were also identified in all patients. intimal tears by vascular claps were more extensive than expected and anastomotic suture lines were able to be checked from inside. in a case of dissecting aneurysm, balloon catheter was advanced through the 11 mm graft which was sutured to the common femoral artery with finding the entry just above the left renal artery. using fiberoptic endoscope and balloon catheter was useful to observe orifices of the major aortic branches, unexpected intimal tears by vascular clamps and atheromatus plaques. it was particularly usbful to check the anastomotic suture line from inside of the aorta and to identify the exact location of the entry in dissecting aneurysm. vascular endoscopy could be one of the invaluable methods to examine, diagnose and treat the patients requiring aortic, caval and other major vascular surgery. (3) produced endothelial injury and a local increase in shear stress in 14 cynomolgus monkeys by suture plicating and constricting the aorta and then feeding an atherogenic diet for 6 months. our findings reveal that carotid plaques localize on the outer wall of the internal carotid (plaque thickness 0.7--+0.1 mm) which is an area of low flow velocity ( -4___ 1 cm/s at re 800) and shear stress (0-+2 dynes/cm 2) and not at the flow divider (thickness 0.1___0.1 mm, p<0.01) which is an area of high flow velocity (81---3 cm/s) and shear (161-+48 dynes/cm2). distal to the carotid bulb, velocity and shear increased on the outer wall and little or no plaque was observed. in experimental coarctations, no endothelial damage was observed (sem and tem) within the high-shear coarct channel and the channel was noted to be free ofatherosclerotic plaque despite the development of extensive diet-induced lesions proximally and distally. thus, high flow velocity and shear stress do not appear to produce endothelial damage in vivo. in addition, plaques were minimal in high shear areas in the human carotid bifurcation and high shear appears to have an inhibitory effect on experimental plaque formation. these data contradict previous investigations implicating high shear stress in plaque pathogenesis. in contrast, host aortic endothelium (ae) fails to cover large vp by pannus ingrowth even over much longer times. to see if iaes succeeds because of inherent differences in growth potential between ae and ve, we used ae to seed 8 cm x 6 mm diameter dacron velour infrarenal vp in dogs. an average of 4 x 105 cells obtained by trypsin/collagenase digestion of the bypassed aortic segment was used to seed each vp by a 4 step preclotting method. the identity of ae was confirmed by stains for factor viii antigen. viability of seeded ae was verified by growth of subaliquots in tissue culture. six weeks after surgery central segments of aeseeded (n = 9) and control unseeded (n = 9) vp were compared by light and scanning electron microscopy using an endothelial coverage score range of -1 (for fibrin/platelet thrombi) to +1 (for confluent endothelial coverage). ae-seeded vp had a score of+0.89 ___ 0.33 (mean___ sd) versus. -0.67___ 0.70 for controls (p<0.001). in addition to endothelial coverage, the subluminal smooth muscle and intramural vasa vasorum previously reported in ve-seeded vp were also seen in ae-seeded vp. since ae and ve seeding give identical results, the success of iaes with ve cannot be due to inherent biological differences in mitotic potential between ae and ve. iaes must instead achieve additional endothelial growth either through a) the action of the proteolytic enzymes used for cell harvest or b) mitogenic stimuli to nonconfluent cells at the edges of seeded cell clusters on the vp. further improvement of the efficiency of iaes to allow use of less harvested vein per cm 2 of vp should come from enhancing one or both of these effects. pyrolytic carbon is a crystalline form of carbon that has been extensively used in the construction of cardiac and bone prostheses. since it has also been suggested that pyrolytic carbon will prevent thrombosis from occuring in vascular prostheses, the aim of the present study performed in dogs was to test the immediate blood compatibility of this material and to evaluate its biocompatibility when inserted as vascular substitute. after pryolysis of a gazeous hydrocarbon, the carbone crystalite was deposited on a knitted textile surface or tube. its surface examined by scanning electron microscopy (sem) was rough and porous to a depth of 40p. this material was tested 1 °) for immediate hemocompatibility as inserts within the vascular lumen (aorta and inferior vena cava). the specimens were examined sequentially by sem and histology at 10, 20, 30, 180 s and 10 min after reestablishment of the blood flow, 2 ° ) for long term biocompatibility as vascular cylinders (7 mm id) inserted either in the aorta or inferior vena cava or as intraatrial (left or right) implants. patency of vascular cylinders was tested during 2 postoperative month by doppler ultrasound investigations, specimens were examined by histology, electron microscopy (scanning transmission) at 15, 30 and 60 days following implantation. satellite lymph nodes were examined by histology. already 10 s after establishement of the blood flow, platelet adhesion and limited fibrin mesh with few erythrocytes developed on the material. platelet aggregates of limited extent were only observed on intravenous implants. plasmatic protein deposition, an early event on polymeric vascular material was not observed. after 30 s a fibrino-erythrocytic membrane recovers completely the material. except in the case of intravenous insert, no thrombosis developed at the contact of intraarterial or intracardiac implant. after 15 days it was completely recovered by a 5-7 fibrocellular layer consisting of large myofibroblasts with microfilaments, newly synthetized collagen and elastin. the blood interface was of fibrous nature. at one month by sem, endotheliallike cells developed in a mosaic-like pattern, characterized by transmission e.m., by microvillous projections, numerous pinocytic vesicles and intercellular tight junctions. this endothelial-like cell lining was complete 2 months after implantation. their immunocytochemical properties are now under investigation using specific anti-dog factor viii-rag sera. although preliminary, the present results suggest that among the numerous vascular biomaterials tested, pyrolytic carbon may represent a unique feature of rapid cell development and differentiation of endothelial lining at the blood material interface. department of connective tissue biology, institute of anatomy, university of aarhus, aarhus, denmark in the surgical clinic a significant number of patients report that their incision wound has burst, even though the scar appears to be intact. by mechanical testing of strips from skin wounds we have noticed a breaking pattern, in which the deepest layer of the wound ruptures earlier than the superficial part. therefore, we have investigated the strength and extensibility of rat skin wounds at different levels (superficial-deep) of the epidermis (0.03 mm), dermis (1.8-2.2 mm) and m. panniculus carneous (0.6-1.5 mm), average thickness is indicated. 10, 20 and 60 day old standardized skin wounds from the dorsal region of rats have been used. strips were punched out at right angle to the wound line and mounted in a materials testing machine. the strips were stretched until rupture and load-strain curves registered continuously. simultaneously, the strips were transluminated and the breaking pattern was studied by taking photographs of the wound specimens during the mechanical testing (30-35 photographs of each specimen). the photographs were marked on the load-strain curve by means of a connection between camera and x-y-recorder. from the load-strain curves the maximum load and the failure energy were calculated. the breaking patterns of 10, 20 and 60 day old wounds were found to be similar. the deep part of the wounds ruptured first. the force required to break the deep part was less than that required to break the superficial part of the wounds. the musculus panniculus carneous was very extensible and did not break. however, it possessed only minimal strength. quantitative measurements of the strength of the combined superficial-deep layers were performed on 2 mm wound strips. specimens contained the superficial 0.5, 1.0 and 1.5 mm of the wound area and were produced by cutting off the deep layer parallel to the skin surface. specimens containing the total wound area down to the musculus panniculus carneous were produced by cutting off the muscular tissue. these specimens were mechanically tested as described above. the present studies demonstrate the mechanical inhomogeneity of incision wounds. a new method for testing the mechanical properties of the tissue of incision wounds at various levels (superficial-deep) is presented. the superficial layer of an incision wound contributes a major part of the strength of the wound and is more extensible than the deep layers. these results may explain the clinical observations. the effect on wound healing of different kinds of vitamins is worth investigating, since the efficiency of vitamin c has been dearly demonstrated. the possible action of vitamin bs-whose trophic effect on skin is well known -has been experimentally studied on skin and aponeurosis healing after a standard laparotomy. materials and methods: experiments were carried out on 42 five months old rabbits which were randomly divided into three groups: in group i, 18 animals served as controls (3 animals in sequence of 5 days from the 5th to the 30th post-operative day), group ii, 12 animals injected with vit b5 (25 mg/kg of body weight/24 h) and group iii, 12 animals injected with a placebo (2 animals in sequence of 5 days for each group). in each case four samples were tested of skin and aponeurosis for determinating tensile strength, directly recorded with an original technic [1] : this new apparatus allowed us to obtain simultaneously two dynamic parameters, the healing tensile strength and stretching of the scar. results: 1. no significant difference was found between controls (group i) and the placebo group (group iii) both for resistance of skin and the aponeurosis. 2. as far as vitamin b5 treated animals were concerned (group ii) there was no significant difference regarding skin resistance when compared with the other two groups. 3. inversely aponeurosis resistance become significantly greater when measured on the 20th (p<0.050), 25th (/r<0.025) and 30th (p<0.050) post-operative day. in 17 mongrel dogs (7x 15 cm cranial based rectus abdominis) mc and corresponding rp flaps were raised. in group i (9 dogs) skin bf was determined from the clearance curve for ~33xenon injected intradermally and measured with a computer-linked gamma camera. in group ii (8 dogs) subcutaneous pro2 was determined by a recently developed method using a silastic tonometer, subcutaneously implanted. the pro 2 inside the tonometer was measured in infused saline, by a platinum oxygen needle electrode and a silver/silver chloride reference electrode. b f and pto2 were measured before and after the flaps were raised and on postoperative days (pod) 1, 3, 6 and 15. pto2 were taken at 6 various inspiratoric oxygen levels (f~o2) ranging from 21% (air) to 100 % oxygen. intact areas lateral to the flaps and in flap regions prior to surgery served as controls. immediately after surgery bf in the mc increased while in the rp flaps was 24 %, 23 % and 31% of the flow in the mc flaps, in lateral intact area and in the preoperative areas (p<0.001). during pod 1-2 bf in the rp flaps increased to the preoperative level, but not to the increased levels found in the mc flaps and the lateral intact areas. by pod 6 there were no differences in bf between the two types of flaps and the lateral areas, but all were higher than corresponding preoperative values (/'<0.005). tissue oxygen tension showed a dramatic fall pod 1, 3 and 6 in the rp flaps for all fio2, and for all days the values were lower than the preoperative level (p<0.001). one rp developed pod 3 distal necrosis and the pro 2 was then 0 even with a fio2 of 100%. the mc flap showed an increased pro2 on the operative day but at pod 3 the values were slightly lower than the preoperative level, but pod 1, 3 and 6 the values for all fio2 were higher than for rp flaps (p<0,005). at pod 15 the pro2 reached preoperative level for rp as well as mc flaps. lateral intact areas showed comparable changes to that observed in the mc flaps. it is concluded that the mc flap demonstrates superior bf as well as pro2 when compared to the rp flap. early postoperative pro 2 in the distal part of the rp flaps is critically low despite of increasing f~o2 to 100 % and increasing bf. differences in bf and pto2 may be the biologic factors responsible for the superior healing characteristics of the mc flap. (1) atp~adp + pi (inorganic phosphate) (2) pcr + adp~atp the net result ofreaotjoxas 1 and 2 is a fall in pcr and a rise in pi while atp ~'emains relatively constant. all of the phosphorus metabolites are easily measured in gastrocnemiaas muscle using 31pnmr spectroscopy. normal volunteers and patients with angiographically documented arterial occlusions were studied in a 101/4" bore oxford research systems tmr-32 spectrometer at rest and after exercising each limb separately. normal resting values ofpcr/p iwere >10 and the nmr index = p/(p~+pcr) was 0.07_+0.03 (s.d.). limbs with femoral arterial occlusions whose ankle systolic pressure index was <0.5 had nmr index which was significantly elevated above norreals (0.18 + 0.09p<0.01) indicating a failure of metabolic compensation for reduced bloodflow and oxygen delivery, although atp concentration was norreal. exercise produced a five-fold rise in nmr index in both normal and diseased legs. spectra were taken over one minute intervals during the recovery period and in normal limbs returned to resting values within 2 rain. the recovery period was considerably slower in the diseased limbs indicating abnormal mitochondrial oxygen delivery and impaired mitochondrial formation of atp. these data demonstrate the feasibility of using 31pnmr to non-invasively probe the biochemical abnormalities of energy metabolism in patients with peripheral vascular disease. the incidence of urinary calculous disease (ucd) in the south african black population is very low in comparison with the white population group. no biochemical differences in serum nor urine account for this discrepancy and no other measurable parameters have demonstrated any difference between the two groups. urinary particulate activity measurements have demonstrated differences between normal persons and those with ucd who are otherwise biochemically similar, and it would therefore seem rational to expect such measurements to demonstrate differences between the two population groups. urinary particulate activity was measured in the urin of 100 normal whites and 100 normal blacks, the two groups being matched for age, height and weight, and monitored under normal dietary hydrational and environmental conditions. the three parameters of particulate nucleation, growth and aggregation were measured and the two groups compared. particulate nucleation demonstrated the most significant contrast between the two groups with the production of new particles through nucleation being far greater in the white group than that which occurred in the black group (p<0.001). particulate growth occurred at similar rates in the two groups although at slightly higher rates in the white group. particulate aggregation occurred at a greater rate in the white group but the difference between the two groups was not statistically significant. the differences between the two groups are shown to occur as a consequence of differing rates of particulate nucleation although the rates of particulate growth and aggregation are parallel. whilst the factors responsible for the low nucleation rate in black person remain unknown their effect can now be measured quantitatively through the parameters of urinary particulate activity. blood levels of ketone bodies appear to determine skeletal muscle amino acid release; high levels conserve protein and attenuate gluconeogenesis. starvation indt/ced ketosis is suppressed by infection [1] . to determine if the relative hypoketonaemia following sepsi s in turn contributes to increased glucogenesis, arterial substrates and glucose production (constant infusion 6-3h(n)-glucose) were measured before and after infusion of na-dl-./3-hydroxybutyrate (/3oh) to raise levels three-to fourfold in fed (n= 11), in fasted (n = 8) and in fasted-infected (n= 9) animals. in fasted-infected animals before infusion ketosis did not occur (/3oh 0.81±0.1 mm/1 fasted; 0.45 ±0.55 fasted-infected) and basal glucose turnover was increased (5.05±0.18 /tm/kg/min fasted; 9.5±0.83 fastedinfected). with infusion of glucose and alanine concentrations decreased as expected in fed and fasted animals but not in fasted-infected (glucose 2.33±0.17 ram/1 befor; 2.44+0.11 mm/1 after). glucose production also fell significantly in the fed (10.11±1.33 /~m/kg/min before; 8.44±1.05 after) and fasted (5.05±0.28 v. 4.11±0.33) groups but was unaffected by infusion in the fasted-infected group (9.5+-1.83 v. 9.11+1.44). the accelerated rate of gluconeogenesis in infection is thus not a consequence of hypoketonaemia. the usual reciprocal relationship between glucose and ketone utilisation during feeding and fasting has not been demonstrated~in sepsis. preliminary experiments in a hindlimb model support the hypothesis 201 that during infection amino acid release from muscle is not affected by ketone levels. we have developed a technique for measuring the total body carbon of the living subject which is suitable for measuring the critically-ill as well as the ambulatory patient. by combining this measurement with that of total body nitrogen and calcium [1] an estimate of total body fat is derived. measurement at the beginning and end of a given period enables the changes in total body protein and fat to be obtained, as well as the patient's energy expenditure if energy intake is also known. the method is a radiation technique. the supine patient is irradiated laterally with a horizontal beam of fast neutrons and the resulting gamma rays from the body are detected by a radiation detector placed unterneath the subject. the nuclear reaction employed is the inelastic scattering of fast neutrons by the carbon nuclei of the body with the emitted gamma rays having an energy of 4.43mevi in the initial application, measures of total body fat obtained using the technique were compared with those derived from skinfold thicknesses in six volunteers: there was no significant differences between the two measurements, (see table) . the method is being employed in studying the changes in total body protein and fat, and the energy requirements of surgical patients receiving nutrition. in order to investigate the mechanism of this effect, normal monocytes were incubated at 37°c for 30 min (with intralipid 40/a/ml) and their function assessed by three different techniques (chemotaxis, phagocytosis and chemiluminescence). all three methods showed impairment of function following exposure to intralipid. in order to try and prevent this potentially damaging effect, heparin was added to the various in vitro tests and found to cause marked impairment of phagocytosis. (p<0.01) to assess its effect in vivo, 11 volunteers were given 5,000 units of subcutaneous heparin 2 h prior to intravenous intralipid (as above). although the use of heparin did not affect either immunological function, it completely prevented the fall of monocyte chemotaxis following intralipid alone. these findings suggest that monocyte function may be impaired by the presence ofintracellular lipid particles. the use of s.c. heparin may help to alleviate this problem and could, therefore, be beneficial to ill and often septic patients requiring intravenous nutrition. to investigate the effect of elevated glucocorticoids of stress and trauma on peripheral glutamine metabolism, 0.44 mg/kg bw dexamethasone was injected daily intramuscularely in adult mongroel dogs over a period of 2 weeks. at least 3 weeks prior to the experiments catheters were placed into the animal's abdominal aorta (2) and caval vein (1) in order to measure a-v differences and hindquarter blood flow. during dexamethasone treatment nitrogen balances were negative, -7.1-4-1 g n per day, whereas slightly positive n-balances were observed during the control period (3.1 +__ 1.7 g n/day). muscle glutarnine concentrations declined constantly from 22.1 +2.3 mmol/1 intracellular water to 10.0-4-1.2 by 56% within two weeks. whole blood arterial and venous plasma concentrations remained constant. to test the hypothesis of increased peripheral glutamine utilisation or decreased glutamine formation, the activities of glutaminase and glutamine synthetase were measured in a muscle homogenate obtained before and 8 days after dexamethasone treatment. both enzyme activities were found to be unchanged. hindquarter glutamine efflux increased from 20.3+22.6 pmol/min in the control state to 80.0+24.1 during dexamethasone treatment indicating a 4 fold muscle glutamine output. this increased glutamine output was enirely due to increased a-v differences and despite decreased hindquaarter blood flow during dexamethasone. it is concluded that dexamethasone reproduces the metabolic response of trauma and sepsis in terms of negative nitrogen balance and muscle glutamine depletion. muscle glutamine is shifted from peripheral tissues to visceral organs with muscle compensating for visceral demands rather than skeletal muscle being the primary target of corticoid action. it has been suggested that there is abnormal glucose utilisation in malnourished patients and that this may explain the adverse clinical sequelae of high rates of glucose infusion during intravenous feeding. we have investigated the hypothesis that there is a depression of the key enzymes of glucose oxidation in the muscle of malnourished patients which is due to an alteration of muscle fibre type proportions. 14 malnourished patients (p) (9m,5f 56+12 yrs) our results demonstrate that there is a positive correlation between preoperative cp and stage of cancer 0' =34.4+-3.88x; r=0.62;/9<0.001). nevertheless before surgery there is no difference between cp values in the two groups considered (g.c.= 46.2___9.7 mg %;p.u. =44.4----9.0 mg%), but after surgical trauma cp presents a positive response in patients with p.u. (mean increase +14.9 %), whereas it acts as a negative ap protein in g.c. patients (mean decrease 5-60/0) (p<0.02). moreover malnourished g.c. patients present a reduction of cp values ( -10 %) which is greater than g.c. patients with albumin >3.5 g% ( -1.5%); this difference is not statistically significant. cancer patients undergoing palliative (n=10) or radical surgical procedure (n=31) show parallel decrease of preoperative cp ( -6%), the first group presenting higher preoperative values in relation to the tumor diffusion. in conclusion our results demonstrate that cp is not only a positive ap protein, but in some circumstances it may act as a negative pa protein depending on the underlying disease and the preoperative nutritional status. in this study the free aa concentration in liver tissue of 4 non septic patients (cholecystectomy) were compared with those of 7 septic patients (abdominal sepsis). the liver specimens were taken intraoperatively..the nature and possible risk involved in this study were explained to the patients and their consent obtained. the data presented in this abstract are part of a metabolic screening program of septic patients including the determination of aa (plasma, muscle), hormones (insulin, glucagon, cortisol), nutritional parameters (prealbumin, retinol-binding protein, transferrin), and of energy metabolism (atp, adp, glucose, free fatty acids). for the determination of the free aa the intra-and extracellular water content (chlorid method) and of fat content of the liver specimen were analysed. a membrane potential o f -45mv was assumed. the aa analysis were performed with an automatic aa analyser (kontron, svitzerland) by means of an ionexchange resin (durrum dc-4) and a lithium buffer system (durrum-pico buffers). conclusions: 1. this study reveals decreased concentrations of nearly all aa in liver tissue of septic patients (exception: phenylalanine, tyrosine, cystathionine). 2. the significantly decreased concentrations of the gluconeogenetic aa (thr, ser, ala) indicate that the gluconeogenetic capacity of the liver is not exhausted through an increased uptake of those aa as shown earlier by wilmore et al. [4] 3. an increased administration of gluconeogenetic and basic aa (lys, his) may normalise the aa pattern in the liver of septic patients. the liver is being increasingly recognized as a critical organ in postoperative multiple organ failure. the principle factors precipitating postoperative multiple organ failure were sepsis, hypotension and injury to the liver. previous studies from our laboratory have shown that hepatic failure, which has a high mortality rate, is linked to the marked decrease in energy charge. in order to evaluate the possible presence of metabolic blocks, the changes in the ratio of acetoacetate to fl-hydroxybutyrate (ketone body ratio), which reflects the hepatic mitochondrial redox potential, were analyzed in relation to energy charge in hepatectomized, jaundiced, hemorrhagic-shokked and septic animals, as well as patients with postoperative multiple organ failure. experimental: 1. in hepatectomized rabbits, mitochondrial phosphorylative activity increased to 170 % of the control and the energy charge level decreased from the normal level of 0.87 to 0.76 at 24 h after hepatectomy (/7<0.001). afterward, these values returned to preoperative levels within a week. the ketone body ratio in arterial blood was positively correlated with hepatic energy charge (r=0.696, p~0.01). 2. in jaundiced rabbits, the hepatic energy charge decreased rapidly after the bile duct ligation along with the decrease of mitochondrial pbospborylative activity. the hepatic energy charge fell from 0.87 to 0.73 at 48 h postoperatively with a maximum incidence of mortality. moreover, changes in the blood ketone body ratio were positively correlated with the hepatic energy charge (r= 0.844,/~0.01). the decrease in the blood ketone body ratio was attributed to the restricted mitochondrial reoxidation of nadh due to an inhibition of oxidative phosphorylation. 3. in hemorrhagic-shocked rabbit with a mean arterial blood pressure of 40mmhg, the changes in the blood ketone body ratio were correlated with hepatic energy charge (r=0.772, p<0.01). 4. in septic pigs subjected to the ligation and perforation of the cecum, the hepatic energy charge level decreased gradually from 0.86 to 0.80 and the mitochondrial phosphorylative activity was enhanced to 150% of controls in the hyperdynamic state. in the hypodynamic state, the hepatic energy charge level fell drastically 0.69 concomitant with the decrease in mitochondrial phosphorylative activity and blood ketone body ratio. from these results, the blood ketone body ratio may be regarded as a reliable indicator for assessing the degree of decreased energy charge. clinical: changes in the blood ketone body ratio were measured in 55 patients who underwent major surgery such as hepatectomy. these patients were classified into 4 groups according to the postoperative changes in blood ketone body ratio: group a without decrease to below 0.7, group b with transient decrease to 0.4, group c with progressive decrease to below 0.4 and group d with terminal decrease to below 0.25. all 35 group a and b patients tolerated the operation well. by contrast, the 20 group c patients showed multiple organ failure with 85 % mortality rate, which involved pulmonary failure (70%), hepatic failure (70%), gastrointestinal bleeding (30%), renal failure (600/0), cerebral failure (70%) and coagulopathy (65%). all patients who transitioned to the terminal stage of group d died of cardiogenic decompensation. in 5 patients of group c, the decreased blood ketone body ratio was restored with the amelioration of clinical symptoms after ex vivo pig or baboon liver crosshemodialysis and 3 patients of them were later discharged. evidence presented indicates that the decreased blood ketone body ratio has a direct bearing on multiple organ failure. conclusion: sepsis, hypotension or injury to the liver are a metabolic burden to the liver mitochondria which can result in mitochondrial impairment leading to a marked decrease in hepatic energy charge. such impairment ultimately leads to multiple organ failure as a result of the critically decreased energy and substrate store and the reduced protein synthesis relative to demand in the various organs. 110 in interferon-treated cells, the 2'-5'a synthetase, activated by double stranded rna, polymerizes atp into a series of oligonucleotides characterized by 2'-5' phosphodiester bonds and collectively designated 2'-5'a. these activate an endoribonuclease which cleaves rna. other regulatory functions of this enzyme may be expected because of its wide occurence in mammalian cells (untreated with interferon), where its activity appears, in vitro, to be dependent on the growth conditions, hormone responses regenerating liver after partial hepatectomy is often used as a model for the study of control growth and cell proliferation in vivo. in order to evaluate the role of the 2'-5'a synthetase in the processes leading to initiation of cell division, we measured this enzymatic activity in the rat liver during the first 40 h after partial hepatectomy. partial hepatectomy (50 rats) was performed under neuroleptanalgesia by removing the median and the left lateral lobes of the liver according to the method of higgins and anderson. control animals (3 rats) were subjected to a sham operation. after selected time intervals, the animals were sacrificed and the enzymatic activity in the regenerated liver was measured. the 2'-5'a synthetase activity present in the two first removed lobes was defined as 100%. we observe a very rapid decrease of enzymatic activity which reaches 50 % already 10 h after partial hepatectomy. the lower level of enzymatic activity (25 %) is measured between 20 and 24 h after partial hepatectomy. this minimum is followed by a slow restoration of the activity (at 40 h: 60 %). during this early phase of liver regeneration, a maximal incorporation of tritiated thymidine in dna takes place 24 h after surgery. so well differentiated liver cells have elevated levels of2'-5'a synthetase. but, after partial hepatec-tomy, the 2'-5'a synthetase activity decreases dramatically before the first wave of cell mitosis. these observations clearly illustrate the relationship between 2'-5'a synthetase activity and the growth status. moreover, this drop of enzymatic activity may be a trigger for the initiation of cell division. the primary event or events setting in motion the process of liver regeneration after partial hepatectomy (ph) remain unsettled. regarding the so called hepatotrophic factors, i.e. insulin, glucagon and recently egf, present evidence suggests that they would play mainly a promoting rather than a initiating role. early changes such as glycogen breakdown, fat infiltration and changes in adenine nucleotides and mitochondrial phosphorylative activity are usually, at least the first two, ascribed to metabolic overload of the remaining liver (bucher et al (1969) johns hopkins med, j, 125: 250). so far, however, little attention has been paid to a possible involvement of this phenomenon in the initiation of liver regeneration. attempts were therefore made to modify metabolic overload through early changes in energy metabolism in order to study their influence on the pattern of dna synthesis. fed or 16 h fffsting male wistar rats weighing 200+20 g were examined after ph at 4, 12, 18, 24, 30 and 36 h for adenine nucleotides, oxidative phosphorylation and dna synthesis, based on the rate of~h thymidine incorporation. fasting animals received continuous infusion of 20 % dextrose for 24 h at the rate of 0.45 ml/100 g/h. in addition to the above mentionned parameters hepatic glycogen and fatty acids were measured. within 12 h partial hepatectomy caused a decrease in hepatic atp which was maximal at 3 h (from 2.99___0.13 to 2.36+0.07 /~ moles/g p<0.001); in energy charge (atp + 0.5 adp/atp + adp + amp) from 0.851-+0.01 to 0.816___0.03 (p<0.01) and increase in phosphorylation potential which was maximal at 3 h (from 76+3 to 106_2p<0.001). dna synthesis began at 18 h reaching a peak by 24 h. glucose infusion to ph rats suppressed the decrease of hepatic atp, 2.97___ 0.07/1 mole/g at 3 h vs 2.99_ 0.13 in the control group, prevented glycogen depletion (histochemical estimation) and the increase in fatty acids (two folds increase in ffa and triglycerides (tg) at 24 h vs 10 folds in ffa and 8 folds in tg in the control group),with little effect on mitochondrial activity. the initiation of dna synthesis was delayed and the whole pattern was considerably modified. cessation of glucose infusion restored the usual rate of 3h thymidine incorporation after a late fall of hepatic atp. in conclusion, glucose infusion was shown by one of us to modify the hormonal response to ph, but insulin and glucagon administration to glucose treated animals failed to normalize the pattern of dna synthesis. it is suggested that metabolic overload as estimated on the basis of early changes in energy matabolism may account for one of the events involved in the initiation of dna synthesis after ph. infusion on liver cell regeneration after partial hepatectomy in the rat b. de hemptinne, j.f. ngala and l. lambotte university of louvain, laboratory of experimental surgery ucl 5570, 1200 brussels, belgium after partial hepatectomy (ph) the portal and the peripheral blood serum concentration of immunoreactive insulin suffers a drastic fall and levels ofglucagon show a rapid increase which is maximal 4 h after the liver resection. as these changes appear closely correlated to the blood glucose levels which show a 30 % decrease at 4 h and progressive restoration towards normal values up to 24 h, attempts have been made to alter the insulin/glucagon ratio by glucose infusion after ph and study its relation to liver regeneration. the purpose of this work is thus to determine after ph and hypertonic (20%) glucose infusion: 1. the effect of glucose on insulin and glucagon blood levels over 24 h; 2. the repercussion of the insulin/glucagon modified ratio on dna synthesis; 3. the possible improvement of dna synthesis by extensive glucagon infusion. male fisher rats underwent a standard 70 % ph. a 20 % glucose solution or isotonic salin was infused at a constant rate of 0.9 ml/h through a cannula placed in the iliac vein. the rats were sacrified at 0, 4, 8, 12, 22 and 24 h. (3h) thymidyne (27..3/~ci/mmol) was injected 2 h prior to sacrifice and the liver caudate lobes removed for analysis of (3h) thymidine uptake into nuclear dna. blood samples were withdrawn from the portal vein, the inferior vena cava and the aorta for glucose, insulin and glucagon assays. compared to the salin treated group, the infusion of glucose while keeping a normal steady blood glycemia was responsible of a marked increase of insulin (0.38--+0.01 ng vs 2.24_+0.4 ng,/7<0.01) and decrease of glucagon (0.615-+0.085 ng vs 0.335+0.037 ng, p<0.01), with a major switch of the insulin/glucagon ratio at 4 h after ph (from 6.68 to 0.61). dna synthesis started at 22 h in both series, but was very significantly impaired at 24 h in the glucose infused group (12800-+2280 cpm/mg dna vs 27450+2050 cpm/mg dna, p<0.01). infusion of increasing doses of glucagon (from 0.01 to 2 mg/kg/day could not restore the impaired dna synthesis. only a slight improvement was recorded at 0.5 mg/kg/day as the insulin/glucagon ratio tended to approach that of the control group. fractionation of various doses of glucagon over the 24 h perfusion time, in such a way that the changes in concentration of glucagon after partial hepatectomy was imitated, remained unsuccessful to improve thymidine incorporation. in conclusion: 1. the infusion of hypertonic glucose which impairs dna synthesis after ph, modifies markedly the insulin and glucagon secretion. 2. if a specific insulin/glucagon ratio after ph is important to sustain normal regeneration, its modification does not seem to be the major factor contributing to the blunted dna synthesis response in the hypertonic glucose infusion model. operative mortality of emergency shunt operations or esophageal transection during acute hemorrhage from ruptured esophageal varices in cirrhotic patients (child's category c) has been intolerably high. hence, the emergency operations in these patients should be avoided when the bleeding could be stopped by non-operative measures. when the emergency operation eventually becomes inevitable, the operative procedures should be simple and of short duration. in 1976, we have introduced the endoscopic balloon tamponade method for the management of esophageal variceal bleeding. the new balloon tube used in this method has essentially the similar structure as the sengstaken-blakemore tube, but is made of translucent plastic materials, and has a larger internal diameter so that a small caliber optic fiberscope (ex. bronchofiberscope) can be passed through the tube. by this method, the endoscopic observation of the esophagus is possible through the translucent balloon tube during tamponade of the ruptured varices. it is possible to know directly whether the bleeding from varices has been successfully stopped or not, which seems to be an advantage over the blind tamponade method using the sengstaken-blakemore tube. this endoscopic tamponade method has been used for emergency hemostasis of 166 acute bleeding from ruptured esophageal varices in 66 patients. the mean initial tamponade pressure by the esophageal balloon necessary to stop bleeding was 30_+12 mrnhg and the average duration of tamponade was 50 h. the location of the ruptured v~ices observed by this method was in the lower esophagus within 10 cm of the esophagocardiac junction in 86% of the cases. the bleeding has been stopped in 156 occasions (93.9 %) by this method. no significant complications other than atelectasis in 2 patients have been observed. in 4 patients, the bleeding was initially stopped by the new translucent balloon tube, but recurred within 24 h after decompression of the esophageal balloon. tamponade was repeated for several times since these patients were in the category c of the child's classification, however, the emergency operation eventually became necessary. transthoracic esophageal transection was performed using autosu-ture apparatus, eea, in these patients. one patient died of liver failure in the 6th postoperative day, but others survived the operation and recovered. the use of eea in transthoracic esophageal transection simplifies the esophageal anastomosis, and shortens the duration of operation by 40 rain. we have so far used the autosuture apparatus, eea (28 mm cartridge) in elective esophageal transection of 33 patients. neither anastomotic bleeding or insufficiency has been encountered. acute variceal bleeding in category c patients should be treated initially by endoscopic balloon tamponade, when emergency operation is inevitable, transthoracic esophageal transection using eea is the operation of choice. arterialisation of the portal vein in conjunction with an end-to-side portacaval shunt has been proposed as a method of improving survival following shunting [1] . however, there is little experimental evidence to support this suggestion and so we have examined the effects of arterialisation of the portal stump in cirrhotic rats. 24 rats with dimenthylnitrosamine-induced cirrhosis were used in this study. 8 of the rats received an end-to-side portacaval shunt, 8 had a portacaval shunt and the portal stump arterialised with the left gastric artery, eight were sham-operated. liver blood flow (lbf) and wedged hepatic venous pressure (whvp) were measured before and after shunting. three weeks after surgery lbf and whvp measurements were repeated, the animals bled and the liver removed and weighed. an end-to-side portacaval shunt led to an immediate fall in lbf (31.3_+5.6 to 15.5_+4.3 mls/min/ 100g-~) and whvp (7.6_+0.8 to 3.1-+0.5 mmhg). however, if the portal stump was arterialised lbf (34.6-+2.9 to 45.3+5.7) and whvp (7.9_+1.0 to 7.3_+1.3) did not change significantly. no further changes in lbf and whvp were observed three weeks after operation in any group of animals. arterialisation of the portal stump prevented the loss in body weight, loss in liver weight deterioration of liver function tests and hyperammonemia observed in animals with a portacaval shunt alone. these findings suggest that arterialisation of the portal stump may prevent some of the deleterious effects after shunting. departments of surgery and pathology, university of virginia hospital, charlottesville, virginia 22908, usa we have hadexperience with operative restoration ofhepatopedal portal blood flow in five patients intolerant of total splanchnic shunting. hepatopedal flow was reestablished by takedown of the total shunt and construction of a selective, distal splenorenal shunt, or by isolation and arterialization of the hepatic limb of the shunted portal vein. in two patients, shunt revision was undertaken electively for chronic encephalopathy, unresponsive to low protein diet, intestinal antibiosis and oral lactulose. each individual had been hospitalized more than eight times for encephalopathy or coma. nine and 34 months postoperatively, both patients have had no encephalopathy on unrestricted protein intake, and work. actively as homemakers. serial liver needle biopsies have shown bilobed nuclei and enhanced mitotic activity suggesting hepatocyte regeneration. in three patients, shunt conversion or arterialization was undertaken in desperate circumstances, characterized by liver failure (bilirubin >10 mg/dl, albumin <2.5 girl, prothrombin time >1"6 s)~ coma and respirator dependency. although two patients showed immediate, marked improvement in mentation, all three died of intraabdominal hemorrhage in the first few postoperative days in spite of prolonged attempts to achieve hemostasis and maximum blood product support. three conclusions can be drawn from this limited experience: 1. total shunt procedures which are anatomically suitable for subsequent conversion to a selective configuration or for hepatic limb arterialization should be favored over those not offering such potential; 2. at a time of election, restoration of hepatopedal portal flow can be accomplished in patients with side-to-side portacaval or hemodynamically equivalent shunts with considerable benefit; and 3. similar procedures in patients with fulminant liver failure are unlikely to succeed. peritoneal-venous (leveen) shunts are associated with a significant incidence of disseminated intravascular coagulation (d.i.c.). this study identifies a site of origin, a pathogenic mechanism, and the hemostatic pathway which accounts for the thrombogenicity of human ascites. 500 ml of peritoneal fluid were removed percutaneously from individuals with malignant and cirrhotic ascites. ficoll-hypaque column chromatography and ultracentrifugation were utilized to prepare four fractions: cellular; a low speed cell-free fluid; a high speed supernatant; and the precipitate from the high speed centrifugation. the cellular fraction from both types demonstrated an ability to shorten a one stage clotting time by 60 % relative to saline and endotoxin controls. similarly, low speed cell-free fluid shortened the clotting time of pooled normal plasma by 61%; was also effective in factor viii (required for intrinsic pathway of coagulation) deficient plasma; but had no effect on factor vii (required for extrinsic pathway of coagulation) deficient plasma or platelet aggregation and release. the high speed supernatant was demonstrably less thrombogenic. the resuspended precipitate shortened the clotting time of pooled normal plasman by 67 % and of factor viii deficient plasma from infinity to 99 s. in contrast, this material was ineffective on factor vii deficient plasma. we conclude that the thrombotic potential of human ascites derives from peritoneal cells, either leucocytes or malignant cells. consistently, the thrombotic factor exists in suspension and is thromboplastin-like in its behavior, operating through the extrinsic pathway of coagulation. thus, a site of origin and a pathway of activity for the thrombotic agent in human ascites is identified. the effect of somatostatin in hepatic haemodynamics in the cirrhotic rat s. jenkins, p. devitt and r. shields department of surgery, university of liverpool, liverpool, u.k. although somatostatin has been suggested as an alternative treatment to vasopressin in the emergency control ofbleeding oesophageal varices [1] , recent studies on its effect on wedged hepatic venous pressure in cirrhotic patients have provided conflicting results [2, 3] . therefore we have examined the effect of somatostatin on hepatic heamodynamics in cirrhotic rats. rats with dimethylnitrosamine-induced cirrhosis received a bolus injection of 2, 4 or 8 pg/kg body weight of somatostatin followed by a 30 min infusion of either 2, 4 or 8 pg/h/kg body weight. control rats received saline only. portal venous flow (pvp) portal pressure (pp), liver blood flow (lbf) and wedged hepatic venous pressure (whvp) were measured before, during and after somatostatin infusion. at the lowest rate of somatostatin administration (bolus injection of 2 pg/kg body weight followed by an infusion of 2 pg/h/kg body weight)there was a rapid decrease in pp (9.8+0.5 to 8.0--+0.4 mmhg), whvp (7.5___0.4 to 5.8---0.3 mmhg) and pvf (31.2 ___ 4.9 to 21.0___ 3.8 ml/min). 30 rain after the start of the infusion pp, whvp and pvf were still signaflcantly lower than preinfusion levels. at higher rates of somatostatin infusion there was no furhter decrease in pp, whvp and pvf. lbf was significantly reduced at all the doses of somatostatin. the highest rate of infusion of somatostatin (8 pg) produced a significantly greater reduction in lbf than either 2 or 4 pg. these data suggest that somatostatin may have a role in the management of portal hypertension, but that higher doses may have a detrimental effect on lbf. er positive breast cancers preferentially metastasize to bone (1) prostaglandin e2 (pgez) is synthesized by breast cancers and potentiates bone resorption in vitro (2) . this study investigates the relationship between er status and pge2 synthesis in breast cancer cells. pge2 was measured by radioimmunoassay in 75 primary breast cancers: a) after ethanol inhibition of further prostaglandin (pg) production -,,basal pg" b) after stimulating pg production with excess arachidonic acid -,,total pg". ,,total" minus ,,basal" = ,,synthesized pg". in order to relate pg production to breast cancer cells, measured pge2 values have been corrected for the epithelial cellularity of each tumour. pge2 x 100 corrected pge2 = actual (%) cellularity cellularity was evaluated by a proportional count of cancer cells expressed as a percentage against non cellular material in all fields of 3-5 histological sections at 63x magnification. we conclude that er positive tumour cells synthesize greater amonts pge2 than er negative cells. this may account for the greater tendency of er positive cancers to recur in bone. oestrogen receptor activity (er) is of prognostic value in breast cancer [1] . however, the chosen cutoff between er-negative and -positive is arbitrary and likely to affect its prognostic value. in 201 patients with invasive breast cancer treated by mastectomy, tumour er was determined [2] and the patients were followed up until first recurrence. using a computer program to perform repeated logrank analysis, the effect of varying the cut-off on prognostic value of erwas studied between 1 and 25 fmol/mg protein. er levels were higher in postmenopausal patients (0-1301, median 72, n=121) than in pre-menopausal patients (0-201, median 25, n=80). for the whole group, optimal prognostic discrimination was achieved with a cut-off 6 fmol/mg protein. in premenopausal patients, the effect of varying cut-off was complex, leading to an optimum of 17 fmol/mg protein, whilst in post-menopausal patients the optimum was 3 fmol/mg protein. these findings were unexpected and may relate to the relationship of er to tumour cellularity [3] . it is concluded that: 1. the failure of some centres to relate er to prognosis may be due to the inappropriate cut-off point chosen; 2. in our patients, the optimal cut-off was 6 fmol, which agrees with the level suggested by the british breast group [4], though it differed between pre-and post-menopausal patients; 3. optimal cut-off for prognosis may differ from that for predicting response to endocrine therapy. previous studies have reported that the use of adjuvant chemotherapy improves relapse free survival following mastectomy. the effect on overall survival is less certain. 290 patients with histologically confirmed axillary node metastases were randomised to receive postoperative radiotherapy (rt), chemoterapy (cmf) or radiotherapy plus chemotherapy (rt + cmf). 244 patients have now been followed for between 6 months and 5 years. patients initially treated with rt received chemotherapy on failure where possible. of 79 patients receiving rt alone, 28 have developed distant recurrence and 20 have died. of 78 receiving cmf alone 22 have developed distant recurrence and 15 have died (see table) . although the use of adjuvant chemotherapy significantly prolongs the relaps free interval there is no corresponding improvement in overall survival. a sample of 89 patients with histologically proven prostatic carcinoma underwent transrectal find needle aspiration at six month intervals, for a mean follow-up of 18 months, as an out-patient procedure, without significant side effects. 49 patients were followed from time of initial diagnosis. the others had been on treatment for a mean period of 36 months before the study commenced. in 25 of the new patients, cytology was positive at the time of histological diagnosis, and correlated with the histological grade. there were no false positives. repeat cytology on 15 patients after 12 months showed 6 positive and in 5 of these the disease was progressing, and in one it was stable. of the 9 who were negative, 8 were stable and one progressing. 16 of the 48 patients already on therapy had positive cytology at their initial aspiration -9 showing progressive disease and 7 being stable. 5 patients whose cytology was initially negative became positive 12 months later and all had disease progression at this time_ 19 patients had negative cytology on 3 or more occasions and in only 2 cases was there evidence of disease progression. cytology showed evidence of squamous metaplasia on follow-up in 13 of the patients whose condition was stable. the prognosis for all patients was assessed on the basis of gleeson's score and aspiration biopsy has been shown to be a safe and useful procedure for monitoring disease activity and response to treatment. the optimum method of restoring the ability to swallow in patients with unresectable carcinoma of the oesophagus remains controversial. this study evaluates the palliative potential of pulsion intubation versus retrostemal gastric bypass of the excluded oesophagus in unresectable carcinoma of the upper thoracic segment (20-32 cm from the incisor teeth). patients and methods: 71 patients were prospectively randomised for treatment by pulsion intubation (37 patients) or gastric bypass (34 patients). non-resectability was indicated by (1) tumour lengths greater than 6 cm, (2) tracheal or bronchial invasion, (3) disrant dissemination, (4) mediastinal invasion. the operative mortality, morbidity, palliation of dysphagia and postoperative nutritional status was compared in the two groups. results: mortality: intubation resulted in 2 deaths, a mortality rate of 5,4%. gastric bypass resulted in 3 deaths, a mortality rate of 8,8 %. complications: intubation was complicated by respiratory infection (8), tube migration (1), respiratory obstruction (1), oesophageal perforation (1), bleeding (1). gastric bypass was complicated by chest infection (9), pneumothorax (3), wound infection (5), subphrenic abscess (1), anastomotic leak (4), pulmonary embolism (1), purulent neck discharge (3). ability to swallow: palliation of dysphagia was achieved in 92 % of patients following intubation and 91% of patients following bypass. postoperative nutritional status: improvement in nutritional status was more rapid following intubation. conclusion: pulsion intubation is the preferred palliative procedure because of fewer complications and lesser degree of postoperative catabolism. the current technique for investigating the response of vascular prosthetic materials to infection is by challenge with a sub-lethal dose of bacteria, usually an intravenous infusion of 108 organisms in an animal model. this large bacterial innoculum, however, obscures any difference in the infectibility of prostheses that may be inherent in the material, its incorporation into host tissues, or its resistance to infection. we have developed a sensitive method for determining the susceptibility to infection of vascular prostheses based on calculation of the number of bacteria required to infect a specific prosthesis in 50% of trials (ids0). following implantation of the prosthesis to be tested in the canine infra-renal aorta, a dose-response curve was generated by the intravenous injection of known innocula of s. aureus (at log intervals from 102 to 108 bacteria). at six weeks the prosthesis was harvested and cultured to document infection with s. aureus. a characteristic sigmoid curve resulted from which the ids0 was determined. to test this method, a comparison was made between commercial human umbilical vein grafts (huvg) and huvg impregnated with silver sulfadiazine in 35 dogs. although both prostheses were infected by the standard 108 innoculum, a greater than tenfold increase in the resistance to infection by the treated huvg (<102 to 101) was demonstrated in the dose-response curves. since the number of bacteria in a postimplant bacteremia rarely exceeds 102 organisms/ml, such differences in infectibility are clinically significant. ids0 determination provides a sensitive, reproducible method for quantitating resistance to infection in vascular protheses. prosthetic infection following reconstructive vascular operations is an infrequent but often fatal complication which generally persists until the graft is removed. it is accepted that infection arises from operative contamination, bacteraemic seeding and abscesses or viscus eroding into the graft. this study investigates the role played by distal sepsis on groin grafts. ten dogs had a specific strain of staphyloccocus aureus inoculated onto a surgical wound in the right foot pad. five days later interposition 6 mm dacron grafts were implanted into the ipsilateral and contralateral groin in continuity with the superficial femoral artery. ten days following this the grafts were removed for bacteriological and histological examination. blood cultures and lymphnode cultures were also taken at this time. in seven dogs the specific staph, aureus, was grown from both grafts. two dogs failed to grow the specific staph, aureus from either graft. these results are significant at the 1% level using fischer's exact test. blood cultures grew staph, aureus from only one dog. ipsilateral lymphnode cultures yielded the specific staph, aureus in seven dogs. we believe that distal sepsis plays an important role in the estabishment of graft sepsis. the mechanism of spread would appear to be via the lymphatics. the influence of tumor growth on wound healing p.w. de graaf and a. zwaveling laboratory experimental surgery, state university, leyden, the netherlands leakage of a colonic anastomosis is a complication each surgeon fears, because it usually leads to a prolonged hospital stay and is accompanied by a high mortality. carcinoma as an indication for operation and preoperative malnutrition are considered to be high risk factors. the subject of this study was to measure the influence of malignant tumor growth at a distant site on woundhealing in colon and skin, and to find a correlation between the influence of the nutritional state of the experimental animal, and the influence of malignant tumorgrowth on woundhealing. we also tried to find ways to compensate the possible unfavourable influence a malignant tumor might have on woundhealing. the study was performed in rats, the tumor used was a rhabdomyosarcoma, transplanted subcutaneously in the rats right flank. parameters for woundhealing were tensile strength of the skin-incision, and bursting pressure of a colonic anastomosis. six groups of rats were studied, each rat undergoing a standardized colonic operation after two or four weeks. the groups consisted of: control animals (20); tumorbearing animals (20), without tumor removal; malnutrltioned animals (20); tumorbearing animals receiving intravenous hyperalimentation (20), without tumor removal; animals undergoing tumor removal and colonic operation in one session (10); animals undergoing the colonic operation two weeks after tumor removal (10). woundhealing was negatively influenced in an early stage of tumorgrowth (f<0.05). four weeks of tumor growth did not impair woundhealing to a greater degree than two weeks. laboratory determinations showed no deviations. malnutrition, leading to less weight gain than in control and tumor bearing animals took much longer to influence woundhealing than tumor pearing. this is in accordance with publications by irvin and hunt (1974), devereux et al (1979) and garattini and guaitani (1981) , which led us to the conclusion that the negative effect of tumor bearing on woundhealing was not solely the result of anorexia. hyperalimentation in tumorbearing rats resulted in undisturbed woundhealing, despite the fact that hyperalimentation as practised by us (with amino-acids and carbohydrates) stimulated tumorgrowth. we concluded from these experiments that tumorgrowth caused a metabolic chaos in the animal, which in turn led to a disturbance in woundhealing. intravenous hyperalimentation could apparently compensate this. woundhealing in rats operated in one session was significantly more disturbed than woundhealing in rats operated in one session was significantly more disturbed than woundhealing in rats operated in two sessions (p<0.05). irvin an hunt (1974) have demonstrated however that extraabdominal trauma had no influence on colonic healing. this led to the conclusion that the diminished woundhealing found in rats operated in one session was an effect of the tumor. obviously this influence is not instantly removed with tumor excision. this study offers a theoretical foundation for the clinical observation that in operations for colonic carcinoma with a substantial operative trauma, the anastomosis needs extra protection and/or needs to be performed in a second session. the aorta of the blotchy mouse undergoes dilatation during adult life, resulting in the formation of fusiform aortic aneurysms. the mutation is x-linked, so only the males are affected. we have found a shift in the fluorescent spectrum of insoluble, hydrolyzed, dermal collagen in these mice; suggesting the presence of an abnormal crosslinkage compound. the purpose of this report is to describe additional studies carried out on the soluble collagen fraction in these animals, which lend support to the hypothesis of a crosslinkage abnormality. dermal collagen was prepared from one-monthold mutant mice and their normal male littermates by sequential extractions in salt solution, acetic acid, and urea. amino acid analyis and sds gel electrophoreses of the salt-soluble fractions revealed similar profiles of amino acids and collagen-chain types, suggesting that there is no abnormality of the basic building blocks of collagen in the mutants. fluorescent spectroscopy of the acid hydrolysates of these fractions also demonstrated similar absorption and emission peaks. however, reduction with sodium porohydride abolished fluorescence in the collagen fraction from the mutants. results (2emission m a x ± sem): normal blotchy before reduction 424.3 ± 3.0 420.7 -----2.9 after reduction 420.0 + 2.1 none sodium borohydride is used experimentally to stabilize labile collagen cross-linkages. the present studies indicate that the salt-soluble collagen from the blotchy mice is borohydride-reactive and is thus chemically ,,unstable". these studies suggest a rational for developing probes based on these fluorescent croperties of investigate collagen from human subjects affected with aneurysms. one third of testes are removed after torsion (1, 2) because of delay in presentation and diagnosis. in a retrospective study of 89 patients with torsion our salvage rate was 64%. nineteen percent were misdiagnosed as epididymitis by a junior doctor and 7 % by a surgical registrar. doctors could not distinguish between torsion of the testis and its appendages. in the literature, eight factors have been reported to be of discriminant value (table 1) . a computer program, using these parameters and bayes' theorem, was written to calculate the most likely diagnosis. data from the 89 patients with torsion and 96 patients with acute epididymitis were analysed by this program ( table 2) . the program correctly diagnosed all 96 cases of epididymitis. of 75 patients with torsion of the testis . in this study we determined the critical ischemic time for the development of an arrhythmogenic potential. egg and bipolar electrograms were recorded from the his bundle, ventricular endocardium and epicardium in twelve anesthetized dogs. short bursts ofventricular pacing (3 beats; 240-420 beats/min) were used to induce ventricular arrhythmias before and after lidocaine administration (2, 4, 6 mg/kg). previously, lidocaine has been shown to exacerbate conduction delay in ischemic myocardium leading to reentrant ventricular arrhythmias. in the control state, ventricular pacing with or without lidocaine induced either no response or single or repetitive ventricular responses. sustained ventricular tachycardia was never evoked in the control studies. in six dogs, after 20 min of left anterior descending coronary artery ligation and reperfusion, ventricular pacing with and without lidocaine produced sustained ventricular tachycardia in one animal. in another six dogs, after 30 rain ofligation and reperfusion, one dog showed sustained vertricular tachycardia in response to ventricular pacing alone. the other 5 showed sustaine~l vertricular tachycardia after ventricular pacing with lidocaine. sustained ventricular tachycardia averaged 400/min and degenerated into ventricular fibrillation within 1-2 rain. in conclusion 20-30 min of ischemia appears to be the critical period for development of malignant arrhythmogenic potential in the canine heart. administration of lidocaine during this critical period enhances the inducibility of cardiac arrhythmias, which may have clinical relevance in the management of acute myocardial infarction. untreated coronary artery air embolism in the experiments without ecc (group i) resulted in a transmural myocardial ischemia with a mortality rate of 28 % in contrast there were no death in the experiment when extracorporeal circulation was used (group ii to v). the best therapeutic effect with regard to the reversibility of temporary myocard ischemia following coronary artery air embolism was achieved by increasc of the postembolic perfusion pressure (group v), a finding being significantly different (p<0.05) to groups i through iv. also volume depletion of the left ventricle on ecc (group ii) resulted in a faster regression of the left ventricular hypothermic area compared to group i (p<:0.05). the application of dipyridamole (group iii) and st.thomas cardioplegic solution (group iv) was not able to produce a significant therapeutic effect. the increase of perfusion pressure following coronary artery air embolism has demonstrated to be the most effective procedure to achieve reversibility of myocardial ischemia. to transfer these experimental findings into clinical application is suggested because of its practicability and convenience. transmural biopsies were obtained before and 5, 15, 30, 60, 90, 120 min after acc for biochemical and structural analysis. myocardial temperature (t) ph (mph), and pco2) were measured continously with a thermistor, a new ph electrode, and a mass spectrometer respectively. in group i (n=8) acc was under normothermia. in group ii mean t was reduced to 19°c by the administration of cold potassium cardioplegia immediately after acc and consecutively every 30 min. mph at the end of acc reached 5.39-t-0.08 group i) and 6.49___0.13 (group ii) (/7<0.001); pmco2 rose from 41___4 to 234_+13 mmhg in group i and did not change in group ii. tissue content of atp decreases by 51% group i) and by 14°/0 (group ii) (f<0.001); tissue creatine phosphate fell by 98 % (group i) and by 48 % (group ii) (p<0.001). changes in atp and creatine phosphate as well as in tissue glucose and glycogen related ton concomitant changes in ph. the degree of ischemic damage assessed histologically by a mean ischemic score was 2.5+--0.06 in group i and 0.75_+ 0.19 in grup ii (p<0.01). irreversible structural demage assessed by electron microscopy occurred in group i 60-90 min after acc and was associated with a mph below 6.2. no such damage was observed in group ii. we conclude: 1. irreversible damage during normothermic arrest occurs considerably later than has been reported for regional ischemia in the beating heart; 2. during 2 h of acc cold potassium cardioplegia does not completely protect against id when mean t is 19°c; and 3. on-line oaeasurement of mph reflects the inadequacy of' mp more accurately than do either pmco2 or t and thus, provides a useful potential method for intraoperative monitoring of mp. in rabbits, infusion cardioplegia was installed at 37, 27, and 17°c respectively. the infusate contained na 27, k 5, 30, 120, 160 or 240, ca 0, 1 or 5 mmol/1, and varying amounts of glucose. measured osmolality varied from 305 to 500 mosmol/kg. the hearts were excised at the end of a 5 rain infusion period; one half was immediately frozen, the other half was incubated for varying periods of time. specimens of left ventricular myocardium were analyzed for metabolite and electrolyte contents. at termination of the cardiplegic perfusion, total adenine nucleotides (tan) and total creatine (tcr) were within control ranges under all conditions. however, atp and ecp = (atp + 0.5 adp)/tan as well as creatine phosphate (crp) and the ratio crp/tcr decreased significantly with increasing dosages of k and ca and higher temperatures. there were corresponding increases in adp, amp, and free creatine. at 17°c, there were no such changes except in hearts perfused with 240 retool/1 k and 5 mmol/1 ca. on the contrary, crp and the ratio crp/ tcr were elevated above the control values in hearts perfused with ca-free solutions containing between 30 and 120 mmol/1 k. the decrease in atp served as the parameter of the ischemia-induced metabolic alterations and the energy deficit developing during cardiac arrest. at 37°c, it averaged 1.67 and 2.55/zmol/g (p<0.02) after 10 min of arrest induced by 30 and 240 mmol/l k respectively. ca, 1 or 5 retool/1 respectively, significantly increased the atp-decay to 2.41 and more than 3.63 pmol/g/lo min respectively (/7<0.02 and p<0.05). hypothermia of 27°c reduced the rate of metabolic deterioration and suspended the influence of the k-dosage. however, the ca-effect was still visible: induction of cardiac arrest by 30 and 240 mmol/l k without/with ca decreased atp by 2.07/ 2.58 pmol/g (p<0.025) and 2.07/2.41 pmol/g (n.s.) respectively within 20 rain. at 17°c, the rate of metabolic alterations was further reduced. the effects of k-dosage and of ca were completely abolished. the atp-decreased 2.2 pmol/g during 40 min of arrest. although higher concentrated k-solutions for infusion cardioplegia do not enhance the ischemiainduced myocardial metabolic alterations in deep hypothermia, they are not recommended for practi-despite the advantages of cbk carrdioplegia, the severely impaired myocardium and/or long ischemia time continue to be a challenge. because of the association of ca ~ with cell injury and death the use of ca ~ channel blockers is logical. investigation of cbd revealied no advantages over cbk. the combination of k and d is appropriate because of their different mechanisms of action. ten dogs had one hour of myocardial ischemia (mi) with topical ice (temp 7___2°c) after coronary perfusion with 200 ml cb (5___ l___c) containing k, 30 meq/l and d, 400 pg/kg. eight dogs had two hours of mi after perfusion with 200 ml cb containing k, 30 meq/l and d, 200/zg/kg. six dogs received the same treatment as the previous group except that d was increased to 1600/zg/kg for all four perfusions. baseline studies were repeated after 60 min ofreperfusion without the use of ca ~ or inotropic agents. heart rate, peak systolic pressure, vc¢, v~,~x, peak + dp/dt, peak -dp/dt, dp/dt over common peak isovolumic pressure, left ventricular compliance, stiffness and elasticity and great water were unchanged from control. coronary vascular resistance was unchanged in groups one and two but declined in group 3. creatine phosphate returned to control but atp, adp and the adenosine pool were depressed. ultrastructure was well preserved. in 16/24 dogs defibrillation was not required whereas 48/48 dogs with cbk and 13/13 with cbd required defibrillation. these data suggest that d is a worthwhile addition to cbk. early one-stage repair of some congenital vascular anomalies might be accompleshed readily if the material used for grafts grew along with the reconstructed tissues. we have evaluated the capacity of tubular grafts constructed from anterior rectus sheath to serve as growing segmental replacements of the descending thoracic aorta of puppies (age 9 weeks). grafts measuring 3 cm in length were placed in 17 animals; 10 were implanted with attention to tissue preservation (live grafts) while 7 received grafts subjected to prior freezing and thawing in order to kill the donor tissue cells (devitalized grafts). grafts were measured initially and at sacrifice 1, 2 or 3 months later. each months 3 animals with live grafts and 2 with devitalized grafts were sacrificed. no aneurysmal dilatations or grafts ruptures were observed in any of the specimens. by 3 months, the live grafts had increased 26.9-1-1% in length and 27.3___ 3 % in diameter, while length and diameter of the devitalized grafts showed minimal growth (2.1___20/o and 5.3___5o/o respectively). during the same period the length of the thoracic aorta increased 4.5___5% in the live graft group and 41.1___ 8 % in the devitalized graft group. grafts were lined by endothelium and organized into 3 transmural zones. the thickness of each zone in the live grafts remained static from 1 to 3 months, whereas thickness of the middle and outer zones in the devitalized grafts increased 4 to 6 fold. in living grafts the layers consisted principally of newly formed fibrocellular tissue; the rectus sheath was reduced to a atrophic fibrous band. by contrast, the rectus sheath remained prominent in the devitalized graft specimens. cellularity (cells/field) of the live grafts was much greater than that of the devitalized grafts (ratio 15.9___3 : 6.0-----2). a newly formed, fibrocellular layered structure replaces the rectus sheath in the live grafts. these findings indicate that rectus sheath grafts are capable of growth in the young animal, providing that at implantation the tissue is well preserved. it is well known that durability of valvular bioprostheses (vbp) depends mainly on the structure of constituent biomaterial and long-term preservation of the collagen network. to our knowledge, no studies on ultrastructure of tissues currently used for vbp has been reported so far after a long time of chemical preservation. the presentation of a new anysotropic tissue, xenogenic cervical duramater (xcd), with a collagen tridimensional network, and a comparative study of scanning (sem) and transmission (tem) electron microscopy of bovine pericardium (bp), human duramater (hd) and procine aortic-cusp (pao) represent the purpose of the present communication. samples of the tissues were obtained in semisterile fashion, rinsed and fixed in karnovsky medium at 4°c for 24 h. materials were then minced on paraphine plates and washed with 0.2m cachodylate buffer for 1 h and dehydrated in increasing concentrations of ethanol (30 %-100 %) and intermediate exposure to 1% uranyl acetate for the in-bloc contrasts. were conducted, and results are exposed in table 1 . as a general rule, histologic evaluation was by far, more satisfactory for tissues preserved with 0.5 % glutaraldehyde (xcd, pap) than those with 98 % glycerol (hd) and 4 % formaldehyde (bp) in combination. it is concluded that the new anysotropic tissue herein presented, xcd, can be appropriately preserved with 0.5 % glutaraldehyde as collagen preservation is concerned, and it's anysotropic and ultrastructural features, maintained for as long as 12 months as assessed by sem and tem. albeit, clinical use of this new biomaterial is subjected to further experimental and animal trials. tetralogy of fallot and absent pulmonary valve (tapv) is associated with massively dilated pulmonary arteries which cause tracheobronchial compression in the newborn and heart failure and cyanosis in older patients. corrective operations have been attended by mortality rates exceeding 40% due to pulmonary insufficiency causing right heart failure (rhf) and pulmonary complications. pulmonic valve insertion (pvi) with complete repair has resulted in improved survival. the purpose of this paper is to assess the results of total correction and pvi in ten patients. during the last five years, 152 patients with tetralogy were corrected. of these, ten patients (ages 51 days to 34 years) had absent pulmonary valve. one patient (51 days) present with severe rhf and pul-monary insufficiency and nine patients presented with mild rhf and cyanosis. chest roentgenographs showed increased cardiothoracic ratio and pulmonary prominence in all. arteriography revealed massively enlarged pulmonary arteries with a mean right pulmonary artery to aorta size ratio of 2.7 to 1. associated pulmonic stenosis and insufficiency was present in all. seven patients underwent closure of ventricular septal defect (vsd) and pvi. of these, three had pvi (2 tissue and 1 prosthetic) with outflow patch and four had right ventricle to pulmonary artery (rv-pa) tissue valved conduits. two patients had repair without pvi, and one had repair with a monocusp pericardial valve patch. nine patients have done well with no episodes of thromboembolism or infection. death occurred in a 51 day old infant who had vsd closure and relief of pulmonic stenosis. pulmonary valve insertion seems indicated in these patients at it lowers peak pulmonary artery pressure and thus reduces compression effects on the trachea and bronchi. as well, when pvi was used right heart failure was not noted postoperatively. although in experimental acute myocardial ischemia intraaortic balloon pumping (iabp) appears to increase regional contractility of ischemic segments of the left ventricle by increasing the collateral flow, evidence for this effect of iabp in patients with refreactory myocardial ischemia is not available. this study was done to analyze the effect of labp on global and segmental left ventricular performance in patients with refractory, acute myocardial ischemia using gated blood pool scanning with tc-99 albium tracer. eight patients were studied on-and-off iabp prior to and following coronary bypass surgery. left ventricular (lv) wall motion analysis was undertaken and both cardiac output (co) and left ventricular filling pressure (lvfp) were determined from thermistor-tipped pulmonary artery catheters. when placed on iabp, mean preoperative global ejection fraction (gef) increased (0.264-0.05 (off) to 0.354-0.05 (on); p<0.01) abut no changes in co or lvfp were observed. when comparisons were made praend postoperatively, co increased after operation (p<0.05), due chiefly to an increase in heart rate (824-6 to 1074-8 beats/rain; p<0.01), but gef was unchanged whether or not iabp was on. only segmental wall motion analysis pre-and postoperatively revealed that iabp increased regional ejection fraction (ref) and contraction velocity by 50% (mean) in the angiographically determined regions of myocardial ischemia and these regional changes were maximal during the last one-third of the lv contraction cycle. it appears that ref is a more sensitive indicator of changes in lv performance than co and gef in these patients. further, this study indicates that iabp does increase the regional wall contracility in the ischemic areas of the myocardium in man. the use of aspirin (asa) to inhibit platelet thromboxane synthesis (tbx2) in patients undergoing coronary artery bypass grafting (cabg) has been advocated to reduce the potential for graft occlusion. however, asa in conventional doses also inhibits the venous synthesis of prostacyclin (pgi2) a potent anti-thrombotic factor, and may contribute to excessive surgical bleeding. to investigate the relationship between asa dose, blood loss and inhibition of venous synthesis of pgi2, 27 patients undergoing cabg were studied. control patients (no asa) were compared to those receiving 80 or 325 mg. asa as a single dose 8-16 hours prior to surgery. production of pgi2 by saphenous vein specimens removed at the time of surgery was measured by radioimmunoassay (ria) of 6-keto pgf~a following incubation with 25 um na arachidonate. blood loss was assessed by chest tube drainage and transfusion requirement in the perioperative period. serum tbx2 was measured by ria following asa ingestion. transfusion (units), drainage (cc), vein pgi2 (pg/mg tissue) and serum tbx2 (ng/ml) were (mean 4-sem): therefore, asa 325 mg or 80 mg did not increase blood loss during cabg. however, asa 325 mg, significantly reduced saphenous vein pgi2 synthesis (/7<0.01) while the lower dose asa 80 mg spared the production of pgi2. asa is both doses inhibited tbx2 (p<0.001) and blocked platelet aggregation. low dose asa deserves further investigation as an anti-thrombotic agent since it does not appear to increase operative bleeding or significantly inhibit venous pgi2 production. transfusion postoperatively, igg increased progressively in patients in group a, reaching preoperative levels on the 6th or 7th day, whereas in patients in group b, igg remained at lower than preoperative levels during the first week. the number of patients with abnormally low lavels of igg during the whole po period was significantly higher in group b (p"~.01). no significant differences were found in the others studied variables. one patient in group a had infection. three patients in group b had infections. conclusion: high doses of fab2igg administered during ohs and the first po days are effective in lessening the po decrease oflgg and thus may be beneficial in preventing po infection. a water insoluble but very hygroscopic polyvinyl alcohol powder, zy-15044", is capable of significantly stimulating the cicatrization of open, contaminated skin wounds in rats. when it is compared to other substances in clinical use such as powders containing antibiotics, antiseptics, amino-acids, enzymes or a dextranomer, no other agent tested was capable of producing a similar beneficial effect. these excellent experimental result justified pilot clinical trials on 30 patients: 18 varicose vein ulcers, 4 atherosclerotic leg ulcers, 4 infected traumatic wounds and 4 infected postoperative wounds. whether in rats or in patients, zy-15044 powder removed secretion, bacteria and tissue debris; it produced an early and increased proliferation of fibroblasts with the appearance of dense granulation tissue; it reduced wound size after less than three applications permitting very early grafting of the wound of eventually, cicatrization advanced to complete epithellzation. regardless of whether the treatments were applied daily in the hospital bed or every other day in outpatients, there were not complaints of unpleasant sensations such as itching, burning or pain. few studies have dealt with long term healing in stomach and none is available for duodenum. this study evaluates morphology and development of mechanical strength and collagen concentration in and adjacent to wounds after 20 to ]g0 days of healing. incisions were made in the non-glandular (rumen) and in the glandular oxyntic (corpus) part of the stomach and in duodenum of 64 male wistar rats, 23 intact rat served as controls. the wounds were dosed with 6-0 polypropylene sutures using a single sutur technique. after 20, 40 and 80 days of healing complete load deformation curves were determined on strips perpendicular to the incision line after the sutures were cut. collagen distribution was measured as hydroxyproline in strips parallel to the incision line. wound tissue continued to gain strength up to 80 days of healing (breaking energy significantly increased in all tissues from 20 to 80 days of healing). no such increase was found for wounds tested togetherwith adjacent tissue, because the "point of maximum weakness" had moved laterally from the incision line with healing time. wound collagen concentration increased in corpus and duodenum between 20 and 40 days of healing, but was unchanged between 40 and 80 days. the biochemical active zone around the incision line was unchanged 4-5 mm on each side from day 20 to 80. conclusion: while the wound tissue itself continues to gain strength during the 80 days ofheaiing studied, the increase seen after 20 days does hot,enhance the functional properties of the organ as a'whole. the "point of maximum weakness" has at that time moved to the borderline of the biochemically active zone, which lies outside the tissue area enclosed by sutures. myocutaneous flaps based on either the inferior or superior epigastric artery may be used to cover extensive soft tissue defects from the mid thigh to the clavicle. we report our experience with 33 rectus abdominis flaps, 29 based on the superior epigastric artery and 4 based on the ingerior epigastric artery. the flap is easily elevated, no skin loss has occurred and primary closure of the donor defect has been achieved in every case. in a follow-up period of between 1 month and 15 months no patient has developed an incisional hernia. the flaps have been used to cover extensive chest wall resection for recurrent carcinoma of the breast (6 patients), extensive radionecrosis of the chest wall (1 case) as part of a primary breast reconstructive procedure (22 patients) and to replace soft tissue overlying the femoral vessels after radical dissection of the groin for metastatic tumour (4 patients). of the 22 patients undergoing primary reconstruction 20 required additional subpectoral prosthesis but in 2 adequate bulk was provided by the flap itself. primary healing occurred in all cases. this easily raised and reliable flap will shorten hospital stay after major ablative surgery for recurrent disease and provides a useful addition to methods of breast reconstruction. a. watson department of surgery, royal lancester in girmary, lancaster, u.k. occult gastro-intenstinal bleeding which defies readiological and endoscopic diagnosis provides one of the greatest diagnostic challenges in surgery. lesions producing this clinical situation are often very small by definition and not infrequently mucosal angiodysplastic lesions, localisation of which may be extremely difficult pre-operatively and indeed at laparotomy. various methods have been described in an attempt to improve pre-operative localisation of such lesions. string tests an dthe detection of sicr labelled red cells in the faeces are notoriously inaccurate. arterioghraphy is invasive and usually necessitates a bleeding rate in excess of 500 ml per day. scintiscanning after red cell tagging with 99mtc gastro-intestinal blood loss, but localisation is difficult. a method oflocalisation of such lesions using slcr labelled red cells and intestinal intubation is described. after labelling of the red cells, a miller-abbot intestinal tube with the balloon inflated and rendered radio-opaque is passed and samples of gastrointestinal secretions aspirated at each 5 cm during passage down the gastro-intestinal tract. samples are counted on a well scintillator counter and when a sample of high radioactivity is obtained, localisation is assessed both by the length of tube passed and by instilling dilute barium down the miller-abbot tube underfluoroscopic control. this method has been used successfully in five patients which were subsequently treated surgically with localised resection resulting in cessation of bleeding. case histories together with photographs of the method and respected specimens will be presented in poster form. postoperative sepsis is tlae most frequent complication of surgery and is the commponest cause ofprolungation of hospital stay. purpose of the study is to prospectively evaluate incidence predisposing factors, bacteriology and costs of postoperative infections. 758 consecutive surgical patients admitted to our institute from may 79 to july 81 were studied. patients undergoing minor surgical procedures (wound less than 2 cm) were excluded from the study. patients were evaluated daily during hospital stay for onset of infection and results recorded in a data sheet. hemocultures in septic patients and free plasma, activated partial thromboplastin time, prothrombin time, and thrombin time, ristocetin test for soluble monomer complexes and fibrin degradation products (fdp, welco test) euglobulin lysis time (elt) and platelet counts. conclusion: 1. thrombocytopenia is not a typical feature of boomslang coagulopathy. 2. the demonstration of soluble monomer complexes is blood samples (positive ristocetin test) appears to be an early and consistent indication of intravascular coagulation. 3. despite unequivocal evidence of advanced consumption, platelet function seems to be maintained, thus preventing complete heamostatic failure. 4. support for this view is found in the clinical observation that bleeding is essentially mild and late in onset. 5. thrombelastographic hyperretractility the "spinning top" appearance is a constant feature and is probably mediated via the platelets. 6. although d. (vpus venom is extremely potent and often fatal, the antivenom is rapidly effective despite advanced consumption. this mechanism which may involve the platelet release reaction has not been fully elucidated and deserves detailed study. in most cases artificial ventricles are driven pneumatically with the advantage of easy handling and the disadvantages of regulation problems, high weight and volume of the driving units and the danger of air embolism in the case of membrane rupture. the driving unit developed at innsbruck universiy consists of a microprocessor-controlled electromagnet driving a pump that functions as a safety chamber (sk). force transmission to the artificial ventricle (ellipsoid heart-eh) is effected hydraulically. thus there is a fixed connection between armature stroke and membrane movement in the eh. the armature position is measured by the microprocessor by means of a position sensor with programmable switch-over points determining the change from systole to diastole so that idling and beat volumes, respectively, of the eh can be programmed. the sk is a newly designed double rolling membrane pump for pressure and suction with very low compliance. pressure and suction are determined by the armature force which is proportional to the microprocessor-controlled current in the solenoids. thus a very positive control of the pumping action is possible. in mock circulation experiments with the hydraulic safety driving unit the cardiac output (co) was between 21/min and 121/min at beat frequencies of 40 to 130 bpm. with constant piston-stroke a direct relation between frequency and co was observed. the influence of preload (starling's law) and afterload decreases with increasing armature force (decreasing periods of systole and diastole, respectively) which is due to friction in the hydraulic transmission system. improvements in the geometry of the next system should reduce these losses. in vivo experiments confirmed the hemodynamic efficiency of the system. applied as lvad the system proved to relieve the beating heart considerably. in the case of heart fibrilation the circulation could be maintained by the safety driving unit. the variable height differences between driving unit and eh as they occur in long-term experiments (animal lying or standing) affect only the ratio systole/diastole periods with the co remaining constant. mechanically assisted circulation is indicated in patients with cardiac failure after open heart operations. the clinical experiments show that left ventricular assist devices are capable only in a few cases to maintain full circulation. the limiting factor is the additional right heart failure syndrome. in patients with postoperative cardiac failure, a distinction must be made between isolated left heart failure and total heart failure. in patients with total heart failurebased on diffuse coronary sclerosis or on an increased pulmonary resistance -left ventricular assistance alone is not effective and right ventricular support is desired. therefore, a simple, quick and safe biventricular assist device is necessary. with the biventricular bypass it is possible to maintain complete circulation in cases of cardiac insufficiency. for a successful outcome in patients with low cardiac output after cardiopulmonary bypass, the e-bvad was evaluated in animal experiments (with 9 calves in 4 acute and 5 survival experiments) in cardiac failure situations. the cannulas for the inflow are put into the left and right ventricle, the outflow tracts into the descending aorta and the pulmonary artery. both parts are connected with the ellipsoid hearts. with the e-bvad it is possible to have a replacement of the heart -a total heart assistance similar to the total artificial heart. in cases of clinical emergency this device can be recommended because of the satisfactory hemodynamic effects achieved and the small degree of traumatic hemolysis (1,8 mg% free hemoglobin). it represents an easy and quick implantable system for total functional heart replacement. the realtionship between acute pancreatitis (ap) and the enzyme and hormone level in blood and gastric and duodenal juices is the factor that had driven us to do this experiment that would complete the study of the physiopathology of this illness. material and methods. we used eight 5-year-old dogs. a) production of two antiperistaltic fistula in the mann and bullman way; one gastric and the other duodenal (first part). 1. blockage of the gastric and duodenal compartments: by the gastric fstula we introduced one foley's bucket with closed point and inflatable globe to block the pylorus. by means of the duodenal fistula we introduced another foley's bucket into the duodenum. once inflated, the ball blocks the first duodenal portion. 2. juice extraction: by adjacent openings in the gastric bucket blocking the pylorum, we extracted the gastric juice separately. with another thin foley's bucket, that was introduced by the duodenal fistula near the already blocked one, to block the third part of duodenum when the globes inflates. b) production of ap: 15 days after the first surgical operation, by the morandeira method with intraparenchymal injection of a mixture of autologous bile and olive oil. results. one of the animals died on the fifth day after the first operation. + no animal died spontaneously after the second operation. they were killed on the 4th day. in each one acute necrotizing pancreatitis could be verified. + the radiograph showed that the gastric and duodenal compartments were sealed. -it was easy to separately extract blood and gastric and duodenal juices. conclusion. we believe that this method is complete, simple, with good results and very useful for the study of the physiopathology of ap. liver samples were taken form 18 patients operated for cholelithiasis and common bile duct obstruction by gallstones or pancreatic tumour. early evident histoenzymatic deficiency was found even without jaundice or liver structural lesions. in the bilio-obstructive jaundice, the histochemical methods revealed more marked liver impairment, as compared to the histological picture. the degree of the enzymatic depression could explain the occurence of the postoperative liver failure in some patients. experimentally, the effect of common bile duct ligation was studied in 80 rats. group i = 10 healthy controls. group ii = aspartate (1 ml 5% sol.) was injected i.p. in 10 rats daily, for a week. group iii = the common bile duct was ligated under other anes-thesia, in 20 rats 48 h before killing, and group iv=in another 20 rats 7 days before killing. group iv =in 20 rats aspartate was administered 3 h before an twice after choledocus ligation, and group vi = in another 20 rats aspartate was injected daily for a week after surgery. the liver slices frozen in liquid nitrogen were cut in a slee-type cryostat and the histochemical reactions were performed according to arnold, chayen-bitensky and lojda-gossrau-schiebler's methods. the biochemical reactions were performed using merckotests and merz-dade test-kits. after 48 h, and still more after 7 days, very severe structural, histochemical and enzymatic lesions developed. the liver cell glycogen and rna, as well as the "marker" enzymes of infrastructures markedly diminished: nach2-tetrazolium reductase -21.33%, glucose-6-phosphatase -48.33%, alphanaphthylacetat esterase -31.33%, atp-ase -71.0%, 5'-nucleotidase -38.67%, pas-reaction -56.0%, mgp-staining -30.67%. aspartate treatment seems to exert a protecting effect against the noxious action of retained bilirubin and conjugated bile salts upon the liver cells: nadh2-tetrazolium reductase +17.37%, gsp-ase +40.64%, esterase + 24.27%, atp-ase +67.81%, 5'n-ase +41.85%, pas + 25.05%, mgp +29.32% and lipids -33.12%. but it does not influence the biochemical signs ofcholestasis: bilirubinemia, alkaline phosphatase, leucinearylamidase, gamma-glutamyltransferase, lactate dehydrogenase. aspartate prevented partially but significantly only the increase of cytolysis enzymes: asat -40.48% and alat -52.97%. the histochemical and enzymatic results are in agreement with the severity ofmorphologic changes. therefore, aspartate treatment might be adequate for the preoperative improvement of the liver function in cases of obstructive-jaundice, in order to reduce the incidence of liver failure, without influencing cholestasis. oral glucose tolerance tests (ogtt) y. yamoaka, a. sugitani, ic kimura, ic ozawa and y. tobe department of surgery, kyoto university medical school, sakyo-ku, kyoto, 606 japan two patients with cholecystographic evidence of septate gallbladder underwent dynamic hepatobiliary radionuclide scanning with 99mtc-ehida. when a steady state of emission had been reached from both gallbladder lobes, cholecystokinin was infused incrementally in four doses (0.005, 0.01, 0.03 and 0.06 ivy-dog-units kg-lmin -1) and counts from the gallbladder analysed by computer. in each an obvious functional difference was revealed between the lobes: both distal lobes ceased emptying abruptly during the third hormone infusion whereas the proximal lobes continued to empty predictably [2] . in one case, histology revealed the septum to be a well-developed smooth muscle ring with cholecystitis glandularis proliferans confined to the distal lobe; the other case awaits surgery. the cause of the difference in response was probably contraction of the muscle in the septum acting as a sphincter. this study provides indirect confirmation that pain in septate gallbladder is due to septum contraction and raised distal intralobe pressure. it is known that the gallbladder stone passes into the common bile duct. furthermore, common bile duct stones pass to the duodenum. these gallstone movements were analysed in 334 cases during the past six years. the phenomenon in which gallbladder stones pass through the cystic duct down to the common bile duct are seen in cases where stones are small and numerous, the cystic duct is wide and connected with the common bile duct angularly or in parallel. in 13.75% of our cases common bile duct stones passed into the duodenum. there were neither inflammatory stenosis of the terminal choledouchous, high grade pappilitis nor severe obstruction of the common bile duct in these cases. it appears that the movement of gall stone is related to size and number of stone and morphological variations in the biliary system. the study was conducted in order to investigate whether intraoperative mano~etry of the bile ducts could be of additional valtie in diagnosis of afflictions of biliary tract or of the papilla. in contrast to current methods of water manometry it seemed important to the authors to use a simple and safe method which could deliver precise results. method: electromanometrics studies were carried out with a cannula placed in the common duct. three manometric parameters were obtained for evaluation. 1. resting pressure (rp); 2. pressure increase after injection of 10 ml saline (lml/s) (pi); 3. time in seconds needed for return of initial pressure (bile flow) (tr). for pressure measurements a pressure transducer with a recorder was used. results: (table) manometry results in patients with calculi in the common duct or with organic stenosis of the papillawere significantly changed as compared with normal findings in the bile ducts. false positive results (proven by cholangiography and biopsies of the papilla) were found in 3.5% of cases, false negatives in 6 % 97 % of all patients with patho-histological changes in the papilla had pathological bile pres-. sures. although false positive and negative results were mainly caused by methodical errors, the described method delivers a high percentage of correct results in agreement with the final diagnosis. it is avery sensitive method in indicating impaired and reduced bile flow and discovers early pathological changes in the papilla as proven by histological examinations. a functional anhepatic state in experimental animals for biochemical studies and as a model for treatment of acute liver failure can be achieved by different surgical procedures like portocaval shunt and complete arterial devascularization or by replacement of the liver with vascular prosthesis and protocaval shunt. we developed a simple hepatectomy procedure using t/4 inch silastic tubing, carbon-y-connector and specially designed "vascular spirales" to restore normal portal and caval blood flow. this model was used for auxiliary liver perfusion studies in heparinized animals; the operation takes 15 min, requires no surgical skill, vascular occlusion is less than 1 min, no signs ofsplanchic hypertension are present and blood loss in fully heparinized animals over 24 h is insignificant. one of the main problems in atypical and anatomical liver resections consists of achieving appropriate hemostasis. also because bile capillaries are opened when liver tissue is separated there is the danger of infection and subphrenic or parahepatic abscesses. the authors have used the human fibrinogen clue to seal the surface of the resected liver in 7 cases (3 hemihepatectomies and 4 resections). even in anatomical resections exists, in the majority of cases, a diffuse bleeding or oozing of the resected area. this can be completely controlled by sealing the surface with the fihrinogen adhesive. also one of the main advantages of the fibrinogen consists of having the possibility to avoid the insertion of numerous tubes for drainage. all our cases were drained by a single silicon tube. the postoperative courses were uneventful in all cases no major complications were observed. liver regeneration is thought to be stimulated by changes in portal blood constituents, or flow or by a substance in the hepatocytes. rice et al. have documented increases in total hepatic perfusion in rats during liver regeneration. this study has measured the portal and arterial components sequentially in large animals. young pigs (6-8 weeks) were subjected to shamoperation or 50% partial hepatectomy (ph). blood flow in the portal vein or hepatic artery were measured pre-operatively and at intervals of 24 h postoperatively using cuff probes of an s.e.m. electromagnetic flowmeter; these were positioned daily under light anaesthesia. systemic arterial and portal venous pressures were measured via indwelling catheters. previous studies have shown that the peak of regenerative response occurs in pigs 3 to 4 days after ph. the mean pre-operative total liver blood flow increased from 1.02+0.36 (s.e.m.) ml.g-lmin-1 to a peak of 226+ 33 % on day 2. thereafter flow declined towards normal by day 6. the pre-operative portal component was 71 ___3 %; this increased to 85+2.5 % on day 2 and returned to normal level within 4 to 6 days. it seems that both total hepatic flow and the portal component increase markedly just preceding the peak of regeneration. this response may stimulate regeneration, or merely accompany it, or may be proyoked by similar stimulators. the analysis of individual bile acids is relevant to several clinical investigations although established techniques have a number of disadvantages. to overcome these we have developed a simple hplc method using a waters associates rcm100 radial compression module with a radial-pak c18, 10,u, 8mm id, reverse-phase cartridge (column). bile acids were eluted from the column at a flow of 2.0 ml min-1 with a mobile phase of methanol:water (75:25 v/v) containing 2.5 % (v/v) acetic acid and adjusted to ph 5.25 with 10 m naoh. a mixture of standards of cholic, chenodeoxycholic, deoxycholic, lithocholic and ursodeoxycholic acids and their glycine and taurine conjugates were resolved, while the 10 conjugates alone were completely separated in under 20 rain. the technique has been applied to the study of human bile from the common duct, gallbladder, tube and duodenal fluid, and serum from patients with hepatobiliary disorders. bile acids in these samples were rapidly extracted using a sep-pak c18 cartridge before being applied (10/a to 200pl) to the hplc system, although bile could be analysed directly without extraction. quantitation (~mol ml-1 sample) of separated bile acids was achieved by comparison with standards using an on-line integrating computer and less than 5 nmol could be detected using a refractive index detector. acute hepatic failure induced by total liver devascularization in pigs -amino acid uptake by combined charcoal -resin hemoperfusion support system where losses averaged 38 %. jib patients had progressively greater losses with increasing preoperative weight (/250 lbs-30 %, 250-299 lbs-35%, 300-349 lbs-38 %, 350-399 lbs-43 %, 1400 lbs-50 %). weight loss in patients dbs was statistically and clinically greater with jib. 2. diabetics, especially insulin-dependent, were rapidly cured after jib. normal plasma glucoses, serum insulins, and oral glucose tolerance curves were usually seen within l postoperative mouth, unrelated to weight loss. all 13 patients requiring insulin or oral medication preoperatively could discontinue mediation usuallywithin a weekpostoperatively. improvement after gb was gradual, appeared related to weight loss, and was often incomplete. 3. hypercholesterolemic patients had 51% decreases, from 275 to 134 mg/dl, after jib, but only 19 % decreases after gb. all serum cholesterols were normal after jib, but 23 % remained elevated after gb. because of complications after jib, some needed reoperation and conversion to gb. fortunately, most benefits were retained after conversion to gb. we suggest considering jib for "superobese", diabetic and hypercholesterolemic patients. in the past 9 years 140jenunoileal bypass procedures were performed. the most common complications and side effects were frequent abdominal pain, or discomfort and flatulence in almost half of the cases. in addition kidney stones, blind loop invagination, electrolyte and liver dysfunction problems could be observed. the over all complication rate was 20%, the postoperative mortality rate 2 %. to eliminate the blind loop of the small intestine, to maintain enterohepatic circulation of bile, and to diminish undesirable side effects in the conventional jejunoileal bypass, biliointestinal bypass was introduced in 1980. twenty patients with a mean weight of 128 kg were subjected to primary biliointestinal bypass within the last two and a half years. three additional patients had secondary biliointestinal bypass due to side effects, especially diarrhea and flatulence, of jejunoileal bypass, performed two to four years previously. the surgical procedure entailed establishment of an end-to-side jejunoileostomy. 25 cm of the jejunum and 25 cm of the ileum were left in the continuity. the blind loop of the jejunum was anastomosed to a functioning gallbladder. so far the above mentioned complications of conventional jejunoileal bypass could be remarkebly diminished. there have been no deaths in this material and no metabolic side effects. frequent diarrhea is avoided by reduced bile spill-over to the colon. the weight reduction has been satisfactory. in summary: due to a lesser complication rate biliointestinal bypass seems to be superior to the simple jejunoileostomy in the treatment of morbid obesity. hyperplasia or neoplasia g.w. geelhoed, c.j. schaeffer and p. daudu department of surgery, george washington university medical center, washington, usa patients with various thyroid disorders who were undergoing thyroid operation were studied for the presence of absence of estradiol and progesteronebinding proteins in thyroid tissue. steroid receptor assays were carried out using similar techniques and standards as those routinely employed for study of breast cancer specimens. quantitative data were collected by coded specimen number by an observer unaware of the patients' clinical diagnoses, and diagnostic correltions were drawn following de-coding. there was no correlation with age or sex and the presence or quantitative value of steroid-binding site. specimens with the histologic diagnosis of follicular adenoma had estradiol binding sites, and had them at high levels (14.19 to 143.83) femtomoles/mg cystosol protein). patients with the histologic diagnosis of thyroid hyperplasia had marginally positive estradiol-binding and lacked progesterone binding. patients with adenocarcinoma of the thyroid exhibited neither estradiol nor progesterone-binding in significant quantity. presence or absence of steroid binding sites in thyroid tissue appears independent of sex or age, but correlates with benign neoplasia of the thyroid, suggesting a possible etiologic association for thyroid adenomas. 77 patients with esophageal cancer underwent resection and primary reconstruction of the esophagus by posterior invagination esophagogastrostomy which we devised. the anastomosis was made in the cervical level in 58 cases and in the left thoracic cavity in 19 cases. functions of stomach placed in the posterior mediastinum were examined in 7 patients surviving more than 5 years and in 13 patients surviving less than 3 years. within one year postoperatively, the absorption ofvitamine b12 decreased markedly. one and a half year after operation, however, the secretion of castle's instrinsic factor recovered and it showed normal values in patients surviving over 5 years. in secretion of gastric acid by tetragastrin, both total acidity and free hydrochloric acid increased in the progress of the postoperative period and showed normal values in all 4 patients surviving over 9 years. this fact was also confirmed by endoscopic observation of color development by congo-red in the gastric mucosa; its coloring area was scattered in 2 patients surviving 5 years, but extended to the whole surface in patients surviving over 9 years. roentgenographycally in head-down position, the surgically created fornix with a sharp angle of his was effective in preventing gastroesophageal reflux completely. the strain combination of donor and recipients. the nature of the mechanisms determing the different fate of allografts remains obscure and all interpretations of the above findings must be speculative. however, the long-term acceptance of hepatic allografts in the rat appears to be similar to that in other species and emphasizes the role of the liver as an immunologically favoured organ. footnote: part of the work (intrahepatic bile duct proliferation!) contained in this abstract will be presented at the xvii essr meeting 1982. peoples' friendship university, tamojenii pr. 4, department of operative surgery, moscow, ussr a transplanted kidney, besides tissue incompatibility reaction, is influenced by non-specific injuring factors, including decentralisation. for normal functioning of a kidney transplant over a long period of time we carried out an experimental study. the purpose of the study was the investigation of operative reinnervation possibilities and its influence upon the functional state of a transplanted kidney. for the kidney reinnervation kirpatovski's method for suturing perivascular fascial tissue flaps of anastomosed vessels with branches of vegetative plexus nervosus on them was used. experiments were carried out on 175 mongrel dogs, both male and female. in the first group of experiments a kidney was transplanted into the pelvis while the opposite kidney was preserved or ablated. in the second group the kidney autotransplanted into pelvis was reirmerved by the described method with preserving the contralateral kidney in part of the animals and ablating the kidney in the rest of them. the third, fourth and fifth were control groups. in the third group the kidney was denerved, in the fourth it was denerved and reinnerved by the mentioned method, in the fifth group unilateral nephrectomy was performed. after the operation the animals, in different periods of time (from 3 days to 2 years), were studied by isotopic renography and by scanning kidney's by jbl-hippuran and neohydrin-hg 2°3 intravenous injections. results of the isotopic renography were estimated qualitatively and quantitatively by universal renographic index (hirakawa-corcoran, 1963). results of the experiments indicate that autotransplantation and denervation of a kidney without its reinnervation were followed by lowering of a kidney vascularisation and its secretory and excretory functions. during the first 2 or 3 months after the operation a tendency to improvement of the kidney function took place; later those functions gradually oppressed and the renographic index lowered down to 13.9+__2.1, 16.6+--.5.5 (p<0.001) respectively according to the groups. after operative reirmervation of a kidney autotransplant and denervation of a kidney without its transplantation gradual improvement of separate functions of the kidney took place: in 1 or 2 months the kidney circulation was restored; in 2 or 4, and in 4 or 6 months respectively, secretory and excretory functions of the kidney restored; renographic index reached its norm after 4 or 6 months and remained stable during 2 years (according to the groups 54.9+___5.5, 52.3-1-4.3, p>0.05 respectively). thus, the operative reirmervation of a kidney prevented depression of its functions and provided its normal functioning during a long period of time. introduction of fine needle aspiration cytology in renal transplantation gives the possibility not only to distinguish acute tabular necrosis, arterial and venous obstruction and viral infection from acute rejection, fnc also seems to allow judgement of the effectiveness of various types ofimmunosuppressive therapy during rejection episodes. method: after sterile fine needle biopsy the cytological evaluation of the aspirate was performed on cytocentrifuged smears after staining the cells by the method of may-grtinwald-giemsa. thus normal and "activated" mononuclear cell populations as well as parenchymal cells such as tabular and endothelial cells and their pathological changes could easily be recognized. the white blood cell types in fnc were compared with those of the peripheral blood. the difference in number due to the graft invading cells was expressed as "increment". the findings of fnc were correlated with clinical signs and serum creatinine values. results: fnc allows to judge the in situ inflammation in the rejecting kidneys within 2 h. non-rejection grafts show cell counts comparable to peripheral blood. with onset of rejection (grade 1) t-and bblasts and monocytes appear in the graft. with sever-ity of the rejection (grade 2) monocytes and lymphocytes as well as blood cells increase in numbers. acute rejection (grade 3) is heralded by high numbers of monocytes and macrophages. acute tubular necrosis (atn) and virus infections can be distinguished from rejection episodes to be treated. in all cortisone and azathioprine resistant cases where alg was used in order to suppress inflammation during rejection episodes it reduced not only the peripheral white blood cells, but also within 12 h after start of alg therapy the number of in situ cells. beside reduction of inflammatory cells typical changes in shape of the nucleus and density of chromatine in up to 10 % of the lymphocytes and granulocytes could be detected. these changes look closest to what has been described as apoptoses. conclusion: cortisone and azathioprine resistant rejection episodes could be monitored within 12-24 h using fine needle aspiration cytology. this method is safe, cheep, and it provides good information about the in situ situation of the graft, it allows to distinguish acute rejection from atn and other situations where higher immuno-suppressive therapy, especially with cortisone, could only be harmful for the patient. criteria of the border of normothermic ischemic tolerance in dog kidneys are first normal pah-and exogenic creatinine-clearances compared with unilateral nephrectomized dogs in neuroleptic analgesia under excessive water and sodium load and second perfectly normal kidneys after two weeks in pathological examination. the protective solution htk ® by bretschneider has a superior buffering capacity compared to euro-collins-solution ® , which, by itself, enhances anaerobic glycolysis by substrate stimulation (glucose) especially between 15 ° and 35°c. without preservation in normothermia oligo-anuric arf is observed at the border of ischemic tolerance (15-20 min; 36°c). the obligo-anuric arf is dependend on ischemia which will result in necrosis of the kidney after 45 rain and 36°c ischemic temperature. the border ofischemic tolerance corresponds to an intrarenal ph of less than 6.2 (outer stripe of medulla), and medullary lactate levels of 60 to 80 pmol/gdw. substrate stimulation of anaerobic glycolysis limits the effectiveness of euro-collins-solution ® above 15°c earlier than anaerobic glycolysis in pure ischemia. after warm ischemia (>--__..27°c; 60 min) all kidneys protected with euro-collins-solution ® show anuric arf, whereas all htk®-protected kidneys (60 to 240 min of warm ischemia; 32°c) are having polyuric arf. under these conditions, renal ph will not increase above 250 nmol/1 h+-concentra -tion. the border of ischemic tolerance for htk ®protection ofkidneys is 120rain-31°c and seems to be limited by a transitory secondary postischemic oliguria (3-48 h). in summary: intrarenal anaerobic glycolysis limits tolerance of pure warm ischemia by inducing anuric arf. in contrast polyuric renal failure is observed at the border ofischemic tolerance by using the protective procedure with the htk®-solution mentioned above. at least 60 % of unrelated pigs respond to liver allografting with a rejection episode which they overcome without immunosuppression; kidney transplants are rejected uniformly within 9 to 14 days. in this study, mlc responses were measured at weekly intervals in such animals. non-litter mate pigs were subjected to autograft, exchange liver allograft, or exchange renal allograft. blood from mlc responses were taken from unaesthetised pigs before and at weekly intervals after operemained the same for each pair of transplants. in 5 of 8 pairs of liver allografts, mlc responses were depressed up to 10 fold for 3 weeks post-operatively. in all these pigs, the mlc responses were initially high. in 2 pairs where the mlc response was initially low, there was no change after the transplant, and in one pair with a low pre-operative response, there was a marked increase post-operatively. in 3 pairs of kidney allografted pigs, there was a similar depression in the single post-operative sample available. in 3 liver autografted pigs, there was a slight post-operative rise in mlc response. it appears that liver and kidney transplantation but not hepatic autografting, markedly depress sequential post-operative mlc responses. the process of ischaemic kidney degeneration was estimated by measuring the adenine nucleotide (atp)levels and the effect of inosine and naftidrofuryl (praxilene) was assessed. 50 min ischaemia was induced on both dissected kidneys of wistar male rats. then, either both clamps were removed and the right kidney was excised after i0 rain reperfusion the role of mononuclear phagocytes in various disease states has been extensively studied by phagocytosis, fc and complement receptor sites, and enzyme content. chemotaxis of peripheral blood mononuclear phagocytes, however, has not been studied to any great extent. we have therefore investigated a method for quantifying chemotaxis by mononuclear phagocytes. mononuclear phagocytes were purified from peripheral blood by centrifugation over ficoll-hypaque and a discontinuous percol gradient. chemotaxis was quantified by the 'under-agarose method'. mononuclear phagocytes were allowed to migrate towards the chemotactic agent zymosan activated serum (zas), and the control non-chemotactic agent eagles medium. the distance of cell migration was measured after 20 hours incubation at 37°c in 5 % co2 with the aid of a microscope eyepiece graftcule. using non-sepcific esterase staining the purity of mononuclear phagocytes obtained was 85%___ 10% and the viability by trypan blue dye exclusion was at least 99 % preliminary results have shown selective migration by purified human peripheral mononuclear phagocytes towards zas. this method may therefore represent a means of investigating an important role of these cells in disease states. the intrathoracic pressure rises when a person exhales into a manometer to such an extent that finally the venous blood-flow to the heart stops. it is supposed that this venous stop flow pressure (vsfp), measured by an ultrasound device, is equal to the central venous pressure. in a prospective clinical trial by two independant examiners in 97 % of the cases (n= 100) the correlation was within 5 cm h20. therefore, the central venous pressure (cvp) can be measured non-invasively with an ultrasound device. we have previously reported that opiate receptor blockade with naloxone (nal) significantly improves cardiovascular function and survival in canine hemorrhagic shock [1, 2] . since species differences do exist, we investigated the effects of nal in cynomulgus monkeys anesthetized with n20/o2. blood was withdrawn to achieve a mean arterial pressure (map) of 45 mmhg (t---0) which was maintained until t= 1 h when the reservoir was clamped and the animals treated with either nal at 2 mg/kg bolus plus 2 mg/kg.h infusion i.v. or 0.9% nac1 in equivalent volumes. there were no significant differences between nal (n=5) and con (n=5) in map, left ventricular contractility (lv dp/dt max, mmhg.10vs), and survival; the nal animals, however, were acidotic (pha 7.24-----0.09) and colder (37.95-0.3°c) than con (7.42+__0.02, 38.5-+-0.1). map responses to nal were proportional to ph a complications of vascular bypass grafts, especiallyin the femoro-popliteal region are common. the most frequent of these by far is occlusion. other late complications iclude leaking and false aneurysm formation at the anastomotic site, dilatation of the graft material and stenosis ofanastomotic sites. the most commonly employed non-invasive studies render little information of value in the diagnosis of these complications. we have recently undertaken a study to evaluate arterial grafts at various intervals after implantation with the use of a duplex ultrasonic scanner. imaging of graft material with this system ist excellent and patency of the lumen can easily be established. graft diameter can be measured and accumulation of material within the lumen can be measured and quantitated. although anastomoses are not always clearly visualised they are often seen satisfactorily for diagnostic purposes. graft dilatation, false aneurysm formation and occlusions can be accurately diagnosed with this method. we have also studied the behaviour of femoro-popliteal grafts across the knee joint using the duplex scanner. as a non-invasive technique scanning with a duplex system has many advantages enabling frequent investigations and therefore early recognition of complications where synthetic material vascular grafts have been used for arterial bypass. an experimental model for dissecting aortic aneurysm has been designed to obtain much better understanding of the disease, leading to more effective methods of surgical treatment. dissection of the descending thoracic aorta was successfully performed by modified blanton's prodedure in 85 % of more than 100 mongrel dogs. fals lumens ruptured distally into true lumens to form a doublebarreled aorta in 70% of the dogs. the method of surgical treatment performed were (1) closure of entry by direct suture, (2) closure of entry by inserting dacron vascular prosthesis with a stainless steel ring, and (3) bypass grafting with vascular prosthesis and ligation of the thoracic aorta. in 10 dogs treated by methods (1) or (2) at the time of performing the dissection, cine angiography revealed that false lumens hat thrombosed within 1 month and dissection was found to have completely healed on autopsy. in 10 chronic cases treated by methods (1) or (3), false lumens were all patent with various degrees of formation of mural thrombi at observation of 3 to 6 months after surgical treatment. these results indicate that closure of entry for acute dissecting aneurysm should be a curative procedure. studies are now continuing on chronic dissecting aneurysm. the lack of controlled trials not rarely resulted in the incorrect acceptance o fan ineffective operation as an effective one 0igation of internal mammary arteries for relief of angina pectoris, for example). this failure also explains the non-stop controversies over the appropriate operation (and whether the question operate at all) for various myocardial revascularization procedures, over the thymectomy for myasthenia gravis, the limited vs radical mastectomy for breast cancer etc. several clinicians and surgeons assert that controlled clinical trials for new operations are unrealistic and naive because of some important differences between operations and drugs. compared with medical trials surgical ones are often really more difficult to be carded out. they are subject to more restrictions, ethical, statistical and practical, and may require longer or even remarkably long times. in any case, patients must nevertheless be sheltered from harmful and ineffective surgical operations since luckily not all the undoubted difficulties are unsurmountable, and prospective controlled trials of surgery, including random allocation of patients, are quite feasible. comparison between surgical and non-surgical treatment is a really particularly suitable field for these studies. comparison of effectiveness of two operations of the same type or of different types can also be carried out, but only on certain conditions and using suitable and appropriate expedients. also the doubleblind approach may be feasible, although remarkable restrictions and adequate cooperation are necessary. acceptance in trials of surgery as placebo is objectively very difficult, and obviously it has to be considered only when an effective operation for the considered disease does not exist. the ethical sound of this delicate aspect of clinical research can be minimized enough only if (1) there are substantial doubts about the effectiveness of the intended operation and (2) requested placebo-surgery is of very slight importance. posters for scientific meetings mary evans and a.v. pollock scarborough hospital, north yorkshire y012 6ql1, u.k. the display of information in posters antecedes even the,art of writing. they have been used to advertise, to educate and to inform and their function is to disseminate information to a wide audience quickly, simply and effectively. in medicine they have been used since the thirteenth century for the promotion of health education. in recent years poster sessions have been introduced into scientific meetings as an alternative to the spoken paper for the presentation of original work. following this experimental procedure hepatic coma supervenes in 6-8 h and animals die after ca. 24 h. five male pigs, aged 3-4 months, weighing 30-35 kg were studied ca. 20 h after operation. the detoxifying system consisted of activated charcoal (norit ¢ (lmm x 2ram) and ionexchange resin (dowex lx2), subsequently inserted in an extracorporeal circulation. plasma amino acids were determined serially (3, 20, 40, 60 rain) in and out the detoxifying system. plasma extraction of individual amino acids (means_ se in/zmol/min) is reported in the table a remarkable extraction was present in the first 40 rain and chiefly involved aromatic amino acids and tryptophan which did not further increase. during hemoperfusion, the molar ratios between neutral amino acids improved. the ratio of branched-chain to aromatic amino acids increased from 1 late hepatic effects of small intestinal bypass (sibp) for obesity plasmafl-endorphin (/3-end, pg/ml, by ria) was related to t:fl-end = 334 (t-37) + 597, r=0.67, p<0.05. when acid-base balance and temperature were maintained, nal (n=6) significantly (*p<0.05) improved map and lv dp pg/ml in nal and 969 + 94 pg/ml in con). r-end is released in and contributes to the cardiovascular depression of primate hemorrhagic shock. nal reverses this depression and improves survival but only when arterial ph and core temperature samples of exudates at site of infection were taken wherever possible for aerobic and anaerobic cultures. in following infections were recorded: wound, respiratory tract, thrombophlebitis, indwelling intravenous catheter, unidentified origin fever (>38 °c lasting more than 3 days) (fuo) and miscellaneous. incidences: 310 patients (41%) had postoperative septic complications. wound infection was recorded in 145 patients (19.1%), respiratory tract infection in 105 (13 %), urinary tract infection in 100 (13 %), fuo in 55 (7.2%) thrombophlebitis in 18 (2.3 %) and miscellaneous infections in 49 (6.4o/0). predisposing factors: wound infections were 53/413 (12.8 %) in clean operations, 41/215 (22.7 %) in potentially contaminated, 19/65 (29%) in contaminated and 24/65 (36.9o/0) in dirty.wound infections were diagnosed later (mean 9th day) in clean than in dirty operations (mean 5th day) (p<0.05). a statistically significant correlation was found between wound infection and leght of preoperative hospital stay: from 5.4% in patients operated on within 0-6 days to 42.8% for patients operated after 28 days or more (p<0.001). no correlation was found between wound infection and age. urinary infections were more frequent when the patients were catheterized at least once in the postoperative period (34 % vs 11%). a statistically significant correlation was found between the incidence of respiratory infections and duration of anaesthesia (3.2 %----<60 min, 120/0>60<120 min, 270/0>120 min;/~0.05). bacterology: 181 out of 295 cultures gave positive results (61%): aerobes were isolated in 129 samples (490/0); mixed aerobes and anaerobe in 29 (9.8o/0); anaerobes in 23 (7.7 %). bacteroides fr. was the commonest isolated anaerobe in all types of sample except hemocultures were propionibacterium aches was the most frequent. a statistically significant correlation was found between the incidence of recvery of anaerobes and intraoperative contamination (7.4 % in clean operations, 13.5 % in potentially contaminated, 12.5% in contaminated and 20.8% in dirty; p<0.05).in wound infections the most frequent aerobes were staphylococcus in clean and e. coli in contaiminated operations. costs: mean postoperative hospital stay of patients with septic complications was 15 days, whereas patients with no postoperative sepsis were discharged after an average of 10 days (p<0.05). mean daily cost in our hospital was extimated $100; accordingly, the mean postoperative hospital stay of patients with sepsis costs $1500 vs $1000 of that o~ the patients without postoperative infections. overwhelming postsplenectomy sepsis/infection has been accepted the highest risk with more than 50 percent mortality due to pneumococcus species. however, e. coli, pseudomonas and staph. aureus have been reported a deadful threat to the splenectomized individual also. pneumococcal challenge after splenectomy in animal experiment and after partial salvage has been well examined. staph. aureus-challenge has not been tried after splenic repair, so far. material and method: a total of 150 nmri-mice (18-20 g) have been subjected to: 1 sham-operation, 2 splenectomy, 3 peritoneal splenosis, 4 controls as non-operated group.the technique was similar to the one performed in 90 rabbits, prior reported. 6 weeks postoperatively, the mice were exposed to intraperitoneal injection of staph.aureus concentrations of 101°c/ml down to 104c/ml. the peritoneal cavity of mice has a high resistance to staph.aureus, as only no. 36835 proved lethal. results: whereas the macroscopic view post mortem seemed promissing, showing almost total salvage of the splenic particles 6 weeks postoperatively bacterial challenge with staph.aureus was contradictory: 3 x 10 s c/ml was survived by all splenectomized mice, by only 25percent of the splenosis micecand by none of the sham and nonoperated mice. due to the small number of only 48 mice within this mice-pathologenic staph. aureus species, we may not draw definite conclusions. as for one, the intraperitoneal application may not be the natural way of septicemia. on the other hand, splenic remnants may not be as effective in the protection of staph.aureus-sepsis as in a pneumococcal challenge. therefore, further studies with staph.aureus species in other animals and by other application route will be needed! studies of the coagulant effects of boomslang (dispholidus typus) venom have indicated that the coagulant effect was mainly due to its ability to activate prothrombin. it also activates prethrombin i, factor x and possible factor ix as well [1] . although boomslang envenomation is said to represent a classic model of disseminated intravascular coagulation, certain features are worthy of critical thought. the coagulation profile of a nine year old lad, who was admitted to the h.f. verwoerd-hospital, 48 h after a reputed boomslang bite, provided the impetus to explore the in vivo effect ofcurde. d. typusvenom on the thrombelastographic and other haemostatic changes in the chacma beboon. methods: 0.0005 mg, 0.0015rag and 0.05mg respectively of crude. d. typus venom were injected subcutaneously in 3 adult baboons. serial determinations of the flow parameters were done over 3-6 days. thrombelastography on whole blood an platelet this report presents the ten year survey of liver mitochondria analized in 385 patients.1. two mechanisms were of major importance in the augmentation of mitochondrial ability to synthesize atp: a) the enhancement of atp-generating capacity per unit of respiratory assemblies and b) the increase in respiratory enzyme contents. those compensatory mechanisms were functional within a certain range of contents in cytochrome a (0.5-1.5 nmol/mg protein, normal; 0.8). hepatic insufficiency was observed in patients with cytochrome a contents less than 0.5 or more than 1.5.2. in all patients with cytochrome a of 0.7-1.0 nmol/mg, the blood glucose level after an oral glucose load (50g) returned toward normal within 2 h (parabolic pattern). in 25 patients with cytochrome a of more than 1.5, the blood glucose level did not return toward normal within 2 h (linear pattern). parabolic and linear patterns were intermingled in the patients with cytochrome a form 1.0 to 1.5.in this report, we will emphasize the importance of preoperative ogtt and measurement of cytochrome a contents during operation for the prediction of operative prognosis, better than any other currently used index of liver function, in the patients receiving major surgery such as hepatectomy or operation for esophageal varices in severe cirrhosis. the reticulo-endothelial system (res) has largely been ignored in studies of liver regeneration. in this study, the res was "blocked" with colloidal carbon, or was stimulated with olive oil or with glucan. indices of liver regeneration were the thymidine kinase acitivtiy (tk) and the number of mitotic figures (mi) in liver biopsies. fibronectin was measured as a putative index of kupffer cell function.four animals in each group were sacrified at 6, 12, 24, 36, 48, 72 and 96 h after sham-operation or 68% partial hepatectomy (ph). group 1: carbon suspension (pelikan ink, wagner) 16 mg/100 g rat single injection pre-operative; group 2: olive oil (10 % in 5 % dextrose water + 0.1% tween 20) 0.25 ml/20 g rat single injection pre-operative; group 3: as group 2 given at intervals of 24 h; group 4: glucan 1.0 rag/ 100 g rat pre-op, and at intervals of 24 h.results: glucan or a single dose of oil increased tk in ph and sham-operated rats but did not influence m.i. carbon inhibited mi but did not influence tk. fibronectin levels were increased in sham-operated and ph rats after daily oil or glucan.in conclusion, no administration enhanced both tk and mi but stimulation of the res with glucan or oil did increase fibronectin levels in both sham and ph rats. it appears that the res does not have a role in liver regeneration as assessed above, but fibronectin appears to have been an indicator of res activity. anatomical abnormalities of the gallbladder include multiseptate and bilobed organs, and the more common transverse septate variant. patients with the latter type often have typical biliary symptoms which are thought to be caused initially by raised intracystic pressure and subsequently by inflammation and calculi formation in the distal lobe [1] .this study evaluate~ the long term effects of sibp on hepatic lipid and fibrous tissue accumulation. liver was obtained at the time of abdominal operation from 6 normal weight patients, 18 morbidly obese patients (mean body weight 163+21 kg) and 27 patients 4 or more years (mean 69-+ 19 months) postoperative from sibp. from wedge liver biopsies hepatic total lipid, triglyceride, cholesterol, phospholipid and protein contents were determined as well as the activities of acetyl coa carboxylase and fatty acid synthetase. histologic evaluation of needle biopsies of the liver was used to quantitatively estimate the amount of hepatic steatosis and fibrosis present.there was also a significant histopathologic increase in hepatic fibrosis present in the liver tissue from sibp patients when compared to hepatic fibrosis in liver from obese patients as well as when compared to liver specimens obtained from the sibp patients at the time of their sibp. the effect of reversal of the sibp on hepatic fat content and fibrosis was determined by transcutaneous liver biopsy 6-22 mos. following take down of sibp. post-reversal histologic quantitation of hepatic fibrosis and inflammation was significantly decreased from pre-reversal values and the improvement was greater in patients who lost weight. patients year after sibp have persistent hepatic steatosis with hepatic fibrosis and inflammation. improvement in these parameters may be anticipated following reconstruction of the intestinal tract particularly if weight loss is maintained. the aim of this paper is to study the metabolic effects of gastric bypass in dogs. we used dogs weighing between 21 and 25 kg. the animals were divided into three groups: a) five dogs without surgical operation (control); b) six dogs with a fundoj ejunal bypass, and c) six dogs with a gastroplasty. in group b and c the exclusion was 9/10 of the stomach. in all of the animals and groups were determined: cholesterol , live r function tests, calcium, magnesium-and electrolyte levels, the body weight evolution and the apparent-digestibility coefficient (adc) of fat, protein, minerals and glucosides for a type of diet. the composition of this diet in dry substance was: fat 35.4%, protein 35.2%, glucosides 26.5% and minerals 2.9%. these analyses were determined five times during the twelve months of control. the animals were kept in individual metabolism cages which allow feces and urine to be gathered separately as well as the food consumed to be controlled. the cages were housed in a room thermoregulated at 18 °c±2. the cholesterol, calcium, magnesium and electrolyte levels were normal during the twelve months of control in the three groups. the liver function tests were normal in all groups with the exception of serum aspartate transaminase initially deteriored in group b. the body weight evolution was significantly diminished in groups b and c compared with the control group (p<0.002 and p<0.002). the results of adc expressed in % ± sd were.by 15roviding a functional gastric capacity of less than 100 ml and consequently a forced reduction in food intake the gastric bypass produces an effective loss of weigth. our results suggest that the gastric bypass can be considered as an effective and safe alternative to intestinal bypass for treatment of morbidly obese subjects who have failed nonsurgical treatment; however, a great and deeper research is necessary to discover the possible side effects of 227 gastric bypass surgery; then its ultimate benefits will be fully understood. there was no operative mortality. the percent of the excess weight lost following the two operations is shown and compared with weight lost following gastric bypass. it is concluded that: 1. complications occured more often following gastroplasty, 25.5 % compared with 10.4 % of the patients treated by gastrogastrostomy. 2. the amount of excess weith lost was greater after gastroplasty, a mean of 61% for 30 patients followed for two years, than after gastrogastrostomy, a mean of 41% for 13 patients followed for two years. 3. because of the incidence of stomal obstruction requiring reoperation following gastroplasty, 14.8 % compared with 1.4% after gastrogastrostomy, is our preferred operation. 4. to improve weight loss after gastrogastrostomy the ta-55 is now used instead of the ta-90 stapling instrument. the proximal gastric pouch is reduced in size to 20-25 ml and the stoma made smaller to 9 ram. an artifical esophagus was made of silicone rubber tube covered with a dacron mesh. a segment of thoracic esophagus of 16 dogs was replaced with this graft using three different types ofamastomosis, i.e., overlayer end-to-end anastomosis, two layer end-toend anastomosis both using a flanged tube, and monolayer end-to-end anastomosis with noflange tube. seven of 16 dogs (44%) survived more than 12 months without complications, and 4 of them more than 6 years. in 6 of 7 of the prolonged survivors, extrusion of the graft was recognized in the 3rd to 6th month after operation. esophageal stenosis increased slightly up to the 6th month after extrusion of the graft, but it did not further advance until sacrifice. in these dogs, mucosal regeneration of the neoesophagus was complete with muscle layers and mucous glands in the submucosa recognized microscopically. proximal esophagus from the replaced portion was apparently dilatated more than that of the distal portion. there was no definite difference between the anastomotic techniques with regard to complication or prognosis. these results suggest a possibility for clinical trials. paper withdrawn 171 attempts to reduce nephrovascular hypertension by surgical techniques deviating renal venous blood and renin directly to the hepatic filter are at present discouraging. experimental data with portocaval transposition are contradictory, due mostly to the use of heterogeneous biological models (mongrel dogs) and collateral circulation developement. renal to portal vein end-to-side (ets) and end-to-end (ete) anastomoses in the rat were therefore tested as possible experimental models. we observed that even after right kidney decapsulation, collateral vein circulation develops through the periureteral plexus, particularly after ets shunts, one month after surgery. nevertheless, collateral cirulation in the male rat can be prevented by ligature and cutting offthis plexus near the kidney. in the female rat instead, collateral circulation is still possible. in fact, newly formed pericapsular veins join the right kidney to the right ovaric vein and therefore preventive ligature and eradication of these vessels is also necessary. in conclusion, both models (ete-ets shunt) appear feasible in the rat, and reliable for studies dealing with nephrovascular hypertension and hepatic metabolism of renin. the handling of the exocrine system is one of the main problems inpancreas transplantation. one trial to overcome this problem consists of the occlusion of the pancreatic duct system. a the theoretical disadvantage is the induction of a secretion oedema which may lead to blood flow disturbance followed by venous thrombosis. the aim of the present experiments was to study the effect of an anti-oedematous drug (cumarin and rutin sulphate (venalot ®) on the blood flow of autologous segmental pancreatic transplantation was tested by chosing the cervical region of the dog as the site of grafting. material and methods: 10 dogs weighting 20-30 kg were used. these dogs were divided into two groups (control group, n=5; treated group, n=5, venalot ® dose: 1 rag, cumarin/kg/b.w.). the body and the left limb of the pancreas was removed, perfusion with eurocollins was started wia the splenic artery and the duct system was injected with prolamine. a distal a. v. fistula of the splenic vessels was performed. this conditioned graft was transplanted at the neck of the same dog, performing the anastomoses between the carotid and the splenic vessels. the blood flow of the pancreatic graft was measured with radioactively labelled microsperes of 15/~m immediately after, as well as 4 days after grafting. the developing oedema of the graft was estimated by weight gain, histological and electron microscopic investigation. residual exocrine function of the graft was measured by determining the serum lipase and amylase levels.results: the autotransplantation of an segmental pancreatic graft to the neck of a dog is a feasable and technically easy surgical procedure, without major venalot -treated dogs local complications. in the ® there was a higher weight increase of the graft in comparison to the controls. as tested so far there was no difference in the behaviour of the i~lood flow of the grafts in both experimental groups. there was a significant increase of serum amylase and lipase values in the venalot®-trea'ted group. conclusion: the technique of cervical segmental pancreatic transplantation in dogs is recommended in cases where immunological monitoring (aspiration cytology, biopsies) is the aim of the study (good access to needles). the technique of microspheres injection is a useful method for the examination of the blood flow in segmental pancreatic grafts in dogs. the prolamine-induced secretion-oedema of ductoccluded pancreatic grafts is resistant to venalot ®treatment. graft pretreatment has been used in various organs to prolong auograft survival. we have recently demonstrated that graft pretreatment of canine renal allografts with cyclosporin a (ca a) led to improved animal and graft survival. our present study assesses the effect ofcy a as a graft pretreatment on pancreatic islet cell allografts. pancreases were removed from unrelated donor mongrel dogs and placed in iced saline (4°c) after the collagenase digestion. the tissue fragments were washed once more and then another 5 mg cy a was added to the preparation prior to intrasplenic injection of the islet cell allografts. three groups were studied: group i (n= 15) served as pancreatectomized non-transplanted controls, group ii (n = 15) received a non-pretreated islet cell allograft after total pancreatectomy and group iii (n= 10) received a cy a graft pretreated islet cell transplant after total pancreatectomy. all animals were given minimal immunosuppression with azathioprine (5 rng/kg/day x 3, followed by 2,5 mg/kg/ day). blood glucose values were monitored to determine engraftment and subsequent rejection. hyperglycemia was considered when plasma glucose values rose above 150 mg/dl. all pancreatectomized controls (group i) became hyperglycemic by the first post-operative day. non-pretreated islet cell allografts in group ii had variable function and became hyperglycemic between one and nine days after transplantation. only five of ten animals in group iii, receiving cy a pretreated islet cells, became hyperglycemic levels greater than 90 days and one died of unknown causes immediately after transplantation. the following table presents the animal survival data for the 90 day follow-up period.although the exact mechanism of action is not known, this study indicates that cy a graft pretreatmerit can be beneficial in prolonging pancreatic islet cell allograft survival, further studies will optimize the use ofcy a in this application and hopefully contribute to the improvement of pancreatic islet cell transplantation. grafting of vascularised organs has become a standard procedure in surgical research. however, only few data of the fate oforthotopicliver transplantation in the rat are available, probably because of the difficult ~iargery involved. this communication gives an account of the outcome of 40 liver allografts and 16 liver isografts in four inbred strains of rat. the following groups of allo and isografts are formed; the survival time is given in days postoperatively.the technical details of the operative procedure of orthotopic liver transplantation in the rat are described elsewhere (eur surg res 13:236 (1981). the distribution of death among allallograft groups show 3 blocks of survivors. the first block includes animals surviving up to 30 days. acute rejection and infection are the diagnosed causes of death in this compartment. the second block includes animals surviving up to 110 days. in the grafts, which belong to this compartment a intrahepatic bile duct proliferation is a frequent and dominant histological feature. because of the development of identical lesions in isograft surviving the same periode and the induction of this lesions by common bile duct ligation experiments, chronic rejection is excludet as the cause of death. the third block includes all so called long-term survivors, in which the structure of the grafts are remarkably well preserved and very few abnormalities are present. the occurence of fatal acute rejection and long-term survival in each allograft group demonstrates, that the fate of the orthotopically transplanted livers in not dependent upon key: cord-022653-qa1uph35 authors: nan title: poster discussion session pds date: 2017-08-30 journal: allergy doi: 10.1111/all.13251 sha: doc_id: 22653 cord_uid: qa1uph35 nan objectives: since bradykinin is a short-lived, low-abundance mediator in the systemic circulation, the discovery of additional biochemical biomarkers correlating hae disease activity with contact system dysregulation may be useful for further elucidation of hae pathophysiology and pharmacodynamic therapeutic modulation of the contact system. results: activated pkal cleaves single chain high molecular weight kininogen to generate bradykinin and cleaved 2chain hmwk. cleaved 2-chain hmwk was measured in human plasma using both a semi-quantitative western blot assay with fluorescent detection (licor) and a novel elisa with a capture antibody that specifically binds 2-chain hmwk. the western blot assay was previously used to monitor pharmacodynamic activity in hae patients treated with lanadelumab, a fully human antibody inhibitor of pkal that is in clinical development for hae attack prophylaxis. lanadelumab inhibited 2-chain hmwk generation following contact system activation in vitro, confirming that 2-chain hmwk is a product of pkal activity. plasma 2-chain hmwk levels from hae patients during or between attacks were compared to that from healthy volunteers using both the western blot assay and elisa. roc curve analyses with both methods suggest that 2-chain hmwk is a trait-specific biomarker capable of differentiating hae patients from healthy volunteers. the elisa was able to differentiate samples from hae patients collected during an attack from those collected between attacks with moderate sensitivity and specificity (c-statis-tic=0.8176). a comparison of 2-chain hmwk levels in citrated plasma versus plasma that contains protease inhibitors (scat169 plasma) provides estimates of endogenous versus ex vivo activation. the measurement of 2-chain hmwk using the specific assays described may find use in further dissecting the role of the contact system in disease pathology, to identify additional indications for modulators of this pathway, and to investigate therapeutics targeting the contact system. 0206 | g protein coupled receptor kinase 2 (grk2) regulates endothelial permeability induced by bradykinin 0208 | pharmacokinetics (pk) and pharmacodynamics (pd) of c1 esterase inhibitor of chronic urticaria challenges most commonly identified were the following: time of onset of disease; frequency/duration of and provoking factors for wheals; diurnal variation; occurrence in relation to weekends, holidays, and foreign travel; shape, size, and distribution of wheals; associated angioedema; associated subjective symptoms of lesions; family and personal history regarding urticaria, atopy; previous or current allergies, infections, internal diseases, or other possible causes; psychosomatic and psychiatric diseases; surgical implantations and events during surgery; gastric/ intestinal problems; induction by physical agents or exercise; use of drugs; food allergies; relationship to the menstrual cycle; smoking habits; type of work, hobbies; stress; quality of life and emotional impact; previous therapy and response to therapy, and previous diagnostic procedures/results. we included all of these aspects in our guide and as a result we developed a chronic urticaria check list. conclusions: our guide of clinical history for chronic urticaria (gur) contributes to have an easy tool in order to achieve a better diagnosis and evaluation of chronic urticaria. 0213 | clinical and diagnostic features in acquired cold urticaria patients in a coruña sanitary area, spain physicians and dermatologists/allergists; c/sa patients were more likely to visit dermatologists/allergists (51% vs. 47%) and less likely to visit general physicians (32% vs. 57%) than european patients. emergency room visits due to cu were more common in c/sa (40%, mean [sd] number=23.2 [124.3] ) than europe (29%, mean [sd] number=3.7 [11.4] ). conversely, hospital admissions due to cu were more likely to occur in europe (22%) than c/sa (8%), but the average (sd) number of admissions among those hospitalised was greater in c/sa (3.3 [4.7] vs. 2.0 [3.1]). variations were seen in subregion comparisons. mean (sd) overall wpai scores were 7.0 (18.9), 25. 1 (26.8), 27.3 (28.5), and 33.3 (30.8) for absenteeism, presenteeism, work productivity loss, and activity impairment, respectively; patients in c/sa reported a higher rate of impairment (range, 13%-36%) on all domains compared with patients in europe. conclusions: cu is associated with substantial hru and work and activity impairment in both europe and c/sa. general physicians should be considered key members of the treatment team in the care of patients with cu in these regions. objectives: cu patients (n=15) received monthly subcutaneous injections of omalizumab for up to six months. urticaria-related symptoms were assessed by both the urticaria control test (uct) and the chronic urticaria quality of life score (cu-q 2 ol). peripheral blood was drawn prior to each injection for determining the concentration-dependent reactivity of patients' basophils to specific anti-fceri and unspecific fmlp stimulation by basophil activation test. furthermore, the impact of anti-ige treatment on ige-bearing cell populations was characterized by flow cytometry analyzing the surface expression of both fceri (e.g. on monocytes, dendritic cells, basophils) and the low-affinity receptor for ige, fcerii (e.g. on b cells, eosinophils). results: anti-ige treatment of cu patients significantly improved clinical symptoms of cu already after the first injection as evaluated by cu-q 2 ol and uct, the latter of which correlated with an increase of basophil numbers and a decrease of basophil surface bound ige. of note, cell-bound ige on fcerii-expressing cells was not altered. furthermore, while clinical amelioration also was accompanied by reduced fceri expression on basophils, the basophil responsiveness to anti-fceri stimulation increased in 73% (11/15) of treated patients. in contrast, ige-independent activation of basophils by fmlp was unchanged. conclusions: clinical improvement of cu patients treated with omalizumab is associated with characteristic immune alterations in basophils, like rapid, cell-specific reduction of surface bound ige and fceri-expression as well as normalization of basophil responsiveness to fceri-stimulation. while our findings might help to better understand the mode of action of anti-ige therapy in cu, they also can shed new light on the pathomechanisms underlying cu. 0216 | omalizumab in patients with severe active chronic spontaneous urticaria (csu) heavily treated with corticosteroids and cyclosporine introduction: increased levels of blood d-dimers (d-d), the byproducts of fibrin degradation, is linked to the severity of chronic spontaneous urticaria (csu) and to poor response to antihistamines h1 (ah1). omalizumab (oma) is a human monoclonal anti-immunoglobulin-e antibody registered as an add-on treatment of csu in adults and adolescent (≥ 12 years old) and with insufficient response to ah1. the sunrise study assessed the efficacy of omalizumab on csu symptoms and the correlation between d-d levels and response over time to treatment with oma to explore its potential predictive value. objectives: sunrise was a french prospective non comparative phase 4 study. included patients had to have been diagnosed with csu for at least 6 months, be resistant to ah1 treatment, and have a uct score (assessed by patient over the 4 last weeks, values from 0 (maximal disease)to 16 (full control)) <8, indicative of a poorly controlled disease. the widely used uas score (assessed by patients over 1 week which captures intensity of pruritus and number of hives, values ranging from 0 (no disease) to 42 (severe disease)) was further used to evaluate the proportion of patients achieving a well controlled disease(uas≤6). all patients received 300 mg oma by sub-cutaneous injections at day 0, weeks (w) 4 and 8. blood levels of d-d were assessed (turbidimetric immunoassay) at d0, w4 and w8. response to treatment was evaluated at w12 by means of the uas7. results: median level of d-d assessed at d0 in 112 patients was increased at baseline (618 ng/ml, extremes 108-5170) and normalized as early as w4 reaching 286 ng/ml (108-481) at w8. correlation between d-d concentration and uas7 score at w12 was weakly positive (spearman coefficient 0.108). among the 10 patients with a very high baseline de d-d level (>3000 ng/ml) 8 were responder (uas7≤6) at w12. conclusions: baseline d-d levels were increased in more than half of patients of this study in line with relevant literature. a fast normalization was observed with oma as early as w4 of treatment. d-d levels at w8 were weakly correlated with uas7. subgroup analyses may help to better understand the link between d-d and clinical response, as these preliminary results do not yet allow the predictive use as a biomarker. the sunrise study explored for the first time in a prospective way the impact of oma on dd and found weak correlations were measured between dd level and response to oma. further studies will be needed to evaluate its predictive value for response. (crp, esr, il-6, il-10, il-33, ccl2/mcp-1), and the disease severity in patients with chronic spontaneous urticaria introduction: pru p 3 is the primary sensitizer of some fruits and responsible for severe reactions in the mediterranean area. sublingual immunotherapy (slit) using peach extract enriched in pru p 3 (prup3-enriched-slit) brings a new perspective to treat patients with reactions to peach considering that currently the treatment of the allergy to peach is based on avoidable ingestion of fruit. we performed a pilot study to examine the immune modulation by slit in patients with peach allergy over a 1-year treatment period. objectives: we aimed to evaluate the effect of the slit during one year in patients with peach allergy. we analysed the capacity pru p 3enriched-slit to modulate immune response, from a th2 to th1 response with increases of treg cells. we studied three groups of subjects: peach allergic patients who received prup3-enriched-slit for 1 year, peach allergic patients non treated, and healthy controls who tolerated peach. monocyte-derived dendritic cells (dcs) maturation, peripheral blood mononuclear cell phenotype and lymphocyte proliferation after prup3 stimulation were assessed by flow cytometry from samples obtained before, and 1, 6 and 12 months during slit. results: we found statistically significant differences in dcs activation and maturation between allergic patients and controls at the basal state. when we analyzed the effect of prup3-enriched-slit over time, we found a significant reduction of activation and maturation markers (ccr7, cd40, cd80, cd83 and cd86) at the first month of treatment that was maintained after 1 year. concerning lymphocytes, we observed a significant decrease of effector cells immune cells. recent studies showed that fructo-oligosaccharides (fos) increase the efficacy of oral immunotherapy (oit) in a mouse model for cow's milk allergy, however, the mechanism is unknown. objectives: investigating the effect of oit+fos on the effector response and the process of tolerance induction. methods: female c3h/heouj mice (5-6 weeks old, n=8/group) were sensitized to the cow's milk protein whey (20 mg in pbs, intragastrically (i.g.)) with cholera toxin (15 lg) once a week for 5 weeks (d0-d28). the mice received a diet with 1% fos or a control diet from d35-d70. oit (10 mg in pbs or pbs) was provided 5 days a week for 3 weeks (d41-d59). intradermal (i.d.) and i.g. challenges were performed to measure the acute allergic response. serum, bone marrow, caecum content, small intestines and mesenteric lymph nodes (mln) were collected at d50, d63 and d70. spleen-derived t cell fractions (whole spleen, cd4+cd25-and cd4+cd25+, using macs) were transferred to na€ ıve recipient mice at d70. the recipients were sensitized and challenged as described for the donor mice. conclusions: this study shows that oit+fos results in an early induction of functional tregs and a reduction of mast cell degranulation upon challenge. the latter may be caused by inhibition of mast cell activation by galectin-9 and/or butyric acid. moreover, the effect of fos on bone marrow suggests possible epigenetic changes reducing development of mast cells. further research is needed to investigate if this approach may be of potential value to treat food allergies. 0348 | safety and feasibility of slow low-dose oral immunotherapy sugiura s; kitamura k; tajima n; takasato y; kato t; tajima i; ono m; tagami k; sakai k; nakagawa t; ito k aichi children's health and medical center, obu, japan introduction: slow low-dose oral immunotherapy (sloit) is an ongoing clinical trial conducted in our institute (umin registry number 000017416). this is a type of oral immunotherapy with a low dose antigen increased very slowly. objectives: to evaluate the safety and feasibility of the protocol. results: sloit enrolled the patients who were diagnosed as severe egg, milk or wheat allergies, with a threshold dose of 5 g (or ml) or less in the oral food challenge (ofc, 20-minutes boiled egg white, whole milk, udon noodle). subjects were divided into two groups, intervention (sloit) group or elimination (control) group, based on their preference. sloit group began to ingest 1/2 to 1/10 amount of the final dose of the ofc based on the severity of provoked symptoms. intake was continued everyday with an increasing dose less than 1.5 times/ month, expecting a 10 times increase from the starting dose after 12 months. feasibility was evaluated based on the proportion of patients who complied the protocol. safety was evaluated based on the frequency of immediate allergic reactions observed in the programmed intakes. fifty-nine patients were enrolled from april to december 2015, and 36 of them (egg: 23, milk: 4, wheat: 9) preferred the sloit group. among them, 11 patients (30.6%) dropped out from the study protocol because of provoked allergic symptoms (n=2) or the other personal reasons (n=9). among a total of 7090 ingestions by 25 patients who continued the protocol over 10 months, mild symptoms were observed 25 times (0.35%), to which rescue medicine such as oral antihistamines was used in 9 cases (0.13%). no one needed an emergency visit or an adrenalin auto-injector. low threshold dose at the initial ofc (1.0 g or less, n=16) was associated with higher proportion of provoked allergic symptoms in the protocol (low threshold: 0.50%, non-low threshold: 0.12%, p=.019). the level of specific ige titer was not associated with the safety. conclusions: sloit protocol has sufficient safety and feasibility, but low threshold patients should be monitored closely. the efficacy of this protocol is being evaluated by an ofc after 12-15 months of the treatment, and the overall data will be presented after march 2018. objectives: the aim of this phase i, randomized, non-controlled, multicenter, opened, with parallel groups clinical trial, is to evaluate the safety and tolerability of subcutaneous immunotherapy (scit), in a polymerized mixture (100/100) depot presentation. patients with rhino-conjunctivitis polysensitized to olea europaea/ phleum pratense received a schedule consisting of two weeks of initiation with three weekly injections; or a program comprising two administrations in the same day separated by 30 minutes. both treatments continued with a maintenance period of three months with a monthly administration. the primary outcomes are the number, percentage, and severity of adverse reactions. secondary endpoint included subrogate efficacy parameters evaluation: changes in immunoglobulin titers (specific ige, igg and igg4) and changes in cutaneous reactivity at different concentrations. 2 systemic reactions were registered, representing 4.3% of the included patients: one grade 0, described as general discomfort plus dizziness and one grade i, such as rhinoconjunctivitis. there were no local reactions. all were classified as of mild intensity and took place with the cluster schedule. symptomatic treatment was not required. conclusions: both schedules with polymerized mixture of phleum pratense/olea europaea, (100/100), presented a good safety and tolerability. a statistically significant decrease in cutaneous reactivity to olive and grass allergens was observed after immunotherapy. 0351a | design of the pivotal phase iii study to assess the efficacy and safety of subcutaneous hdm allergoid immunotherapy in patients with hdm induced allergic rhinitis/ rhinoconjunctivitis introduction: in order to comply with ema guidelines on development of allergen immunotherapy products, a clinical development program was started to obtain full marketing authorization for a allergoid subcutaneous immunotherapy (scit) product for the treatment of house dust mite (hdm) allergy. previously, the safety and tolerability of increasing doses of this allergoid scit product was evaluated in patients with hdm-induced allergic rhinitis/rhinoconjunctivitis (arc) [eudract 2008-006261-81] . no safety or tolerability issues were identified for doses up to 40 000 aueq. subsequently, a dose-finding study to identify the optimal, i.e. effective and safe dose in hdm arc with or without concomitant asthma was performed [eudract 2011-000393-61; pfaar, allergy 2016] . this study demonstrated a dose response relationship with doses of 10 000 aueq (0.5 ml of 20 000 aueq/ml) up to 50 000 aueq (0.5 ml of 100 000 aueq/ml) showing significant improvements compared to placebo. the current pivotal phase iii study [eudract 2016-000051-27] aims to confirm safety and efficacy of this hdm allergoid scit product at a dose level of 50 000 aueq/ml (0.5 ml) compared to placebo after one year of treatment in patients with hdm-induced arc. objectives: the current study is a multi-center (80 clinical study centers in 7 european countries) randomized, double-blind, placebocontrolled, parallel-group study in 730 adult patients, with moderate to severe hdm induced arc with or without mild to moderate persistent asthma. the primary outcome of the study is the difference in mean combined symptom and medication score (nasal symptoms only) (csms (n)) between 50 000 aueq/ml allergoid scit and placebo treatment, assessed during the last 8 weeks of the approximately 1 year treatment period. results: 211 patients from 14 centers, (mean age 32.9 years) have been included and analyzed. the 49.8% are men, 65.9% presented associated asthma. a large majority of patients have received subcutaneous sit (98.6%), and 58.8% of them containing a single allergenic source. 55.8% in polymerized formulation and 40.9% in depot. accelerated schedule has been the most prescribed (56.9%), followed by clustered one (32.2%). from the 81 patients who have completed the study, 45.7% of them improved from persistent to intermittent rhinoconjunctivitis (p<.01) and 49.4% from moderate/severe to mild intensity (aria) (p<.01). moreover, 21% of asthmatic patients at baseline, did not have any bronchial symptoms after 1-year treatment. the improvement in quality of life was possible to be analyzed in 53 patients. mean values in rqlq questionnaire (total score) decreased from 3.1 to 1.5 points (51.6% of score reduction) in final visit, reflecting a statistically significant improvement (p<.01). mean value of treatment satisfaction was 7.3 (sd=1.7) and 7.2 (sd=1.9) for physician and patients respectively. for safety assessment, out of 199 analyzed patients, only 8 systemic reactions were reported in 8 patients (4.02%). seven of them were classified as grade i, and one as grade ii according to eaaci grading system. strasbourg is a 147 m 3 chamber, located into the university hospital of strasbourg at less than to 5 minutes to the intensive care unit. one of the characteristics of this unit is that the maximum parameters are controlled: temperature, relative humidity, ventilation rate, particles number and particles size, concentration of airborne of der p 1 and airborne voc. mite allergens extract were nebulized through a nebulizer. airborne der p1 concentrations were sampled using 5 glass fiber filters and measured with an elisa assay. particles number and particles size were monitored continuously during nebulization, using 10 particles counters distributed inside the exposure room. the cleaning process was also controlled and validated. objectives: to validate the technical parameters of the environmental exposure chamber (eec) of strasbourg with mite allergens. results: the reproducibility was excellent for the indoor temperature, relative humidity and airflow (5 cv interassay <20%). three concentrations of der p1 were measured: 63, 76, 105 ng/m 3 (n=45). for all 3 concentrations, the cv intra-assay of airborne der p1 was 22ae1.3%, the interassay was less than 30%. for the particle size 0.5-5 and 5-10 lm, the cv interassay was 8 and 13%, respectively (n=19). the cv of the mmad was 2.2% (n=10). no measurable airborne der p1 was detected in the toilets and the medical supervision room (n=6) neither in the exposure room 10 minutes after the end of the allergen exposure (n=9). no airborne voc was measured in the chamber and the other rooms of the clinical unit (n=10). there was no significant change in particles size and number when 2 to 6 persons entered the room (n=4). introduction: pathogenesis of systemic sclerosis (ssc) includes vasculopathy with endothelial dysfunction which is considered to be one of the earliest changes in the pathogenesis of ssc. several biological molecules, including e-selectin (e-sel), inter-cellular adhesion molecule 1 (icam-1), endothelin 1 (et-1), von willebrand factor (vwf) and interleukin 6 (il-6) have been associated with endothelial activation. objectives: we aimed to determine if these vascular biomarkers are associated with distinct capillaroscopic ssc patterns and/or more severe disease in ssc patients. results: correlations between serum levels of all 5 vascular biomarkers were good to moderate and statistically significant, with r indices varying between 0.660 and 0.332, the only exception being et-1 which did not correlate with e-sel. good correlations (r 0.465 to 0.727) were also found between all 5 biomarkers and crp. patients with severe vasculopathy, as reflected by the nfc "late" pattern, had higher levels of il-6 (median 12.06 vs 3.08 pg/ml, p=.001), et-1 (median 2.06 vs 1.59 pg/ml, p=.029), vwf (median 3284 vs 2730 iu/ml, p=.013) and e-sel (median 52.6 vs 42.3 ng/ml, p>.05), compared to patients with nfc "early" or "active" patterns. there was a significant, negative correlation between lung transfer for carbon monoxide (dlco) and e-sel, icam-1 (both p<.001) and vwf (p=.013). et-1 was higher in patients with more severe disease (dcssc, patients positive for anti-topoisomerase antibodies and patients with a history of digital ulcers-all p<.05). conclusions: serum endothelial activation biomarkers are elevated in patients with more severe ssc-associated vasculopathy and correlate with serum crp. together with nfc data they may be used for assessing vasculopathy severity in ssc. objectives: here we present the imagination findings of eye involvement in a family whose 11 members have mws. method: clinical data was collected during the course of ongoing patient care. results: we evaluated the clinical features of 11 patients who were referred to our center. the median age of the patients was 25 years (range: 9-65 years). the ratio of females /males was 1.2 (6/5). all patients had arthritis with exacerbation on exposure to cold and ocular involvement, mostly in the form of conjunctivitis and far less other forms. the median age of onset of ocular involvement was 8 years (2-45 y). chronic eye damage were detected in three patients. corneal involvement and clouding was detected in four patient. two conclusions: in this study, it has been shown that eye findings related to mws can vary from conjunctivitis to severe uveitis. we want to emphasize that ocular involvement in mws should be carefully assessed, since it can lead to visual impairment. 0485 | antiretroviral activity of the conjugates 3'-azido-3'deoxythymidine and derivatives of 1,3-diacylglycerides case report: aids epidemics remain the critical problem due to both their emergent and long development. treatment of aids with azt reduces p24 antigenemia, increases cd4 + lymphocyte counts, reduces the frequency and severity of opportunistic infections and prolongs life. however, zidovudine and other dideoxynucleotides do not decrease the ability to isolate hiv from pbmc, and in addition, these drugs are very toxic. this phenomenon is caused by insufficient inhibition of hiv due to low levels of nucleoside kinases in ccr5-positive cells, including in macrophages, which are a major reservoir of hiv. potential advantages of these liponucleotide prodrugs include: greater in vivo efficacy and lower toxicity due to a greater delivery to monocytes/macrophages, ability to bypass the initial anabolic phosphorylation due to the presence of the phosphorous center in the structure, and the prospect of improved pharmacokinetics and prolonged intracellular persistence. the aim of this work is the study of cytotoxicity and anti-hiv activity of glycerolipids derivatives of azt. evaluation of the cytotoxicity of azt and test compounds was per0488 | bone mineralisation defect in patients with hax-1 deficiency 4 and osteopenia (z score <à1) in 3 patients. bone mineralisation defect was found in all female patients while only one male had osteopenia (table 1) . conclusions: in this study, a significant decrease (87.5%) of bone mineralization was observed in patients with hax-1 deficiency. female patients were found more prone to bone mineralisation defect. to conclude on this subject, more studies are needed with large number of patients having not only hax-1 deficiency but also ela-2 mutations. gene mutation age ( introduction: bronchopulmonary diseases are kept as one of the actual problems of the pediatricians. nevertheless fulfilment of several scientific works on study of these diseases, presently we meet the complication of the respiratory diseases, recurrency, changing to the chronical type. objectives: the main purpose of our work to study the cytokine status and substance p, mutual connection they in frequently ill chilresults: in fic with respiratory diseases in the acute period of the disease increase of levels proinflammatory cytokines il-1beta, il-6, il-8, tnf-alpha and substance p,decrease of levels il-2 and ifngamma was marked. clinical remission in these children is not accompanied by normalization of cytokine status and substance p. the high level of proinflammatory cytokines and substance p testifies to proceeding of inflammatory process that is possible connected with persistence of the infections agent. acute decrease in level of cytokines il-2 and ifn-gamma in these children,most likely, is caused by the presence of a immunodeficiency of cellular type. conclusions: in this connection it is necessary to carry out an adequate therapy of fic with arvi. introduction: atopic diseases are known to be characterized by a t helper (th) 2-skewed immunity. th2-skewed immunity at birth, th2-associated cc chemokine ligand (ccl)-22, appears to be associated with high total ige levels but not of allergic outcomes later in life. the prevalence of asthma increases with a rapid upward trend after age 2; however, there are few studies addressing the changes of ccl22 chemokine levels during infancy related to the development of atopic diseases in early childhood. objectives: we investigated 182 children followed up regularly at the clinic for 5 years in a birth cohort study. the levels of th2 related chemokine ccl22 were quantified in cord blood and age 1.5 by multiplex luminex kits. specific immunoglobulin e antibodies against food (egg white, milk, and wheat) and inhalant allergens (d. pteronyssinus, d. farina, and c. herbarum) were measured at 6 months as well as 1.5, 3, 4 and 5 years of age. results: a total of 125 pairs of ccl22 chemokine levels from birth to age 1.5 were recruited in this study. k-means clustering was performed using r software and package mfuzz and this resulted in 3 groups of ccl22 chemokine levels that declined from around 2000 to 500 pg/ml (cluster a, n=51), from around 1200 to 400 pg/ml (cluster b, n=46) , and raised from around 400 to 600 pg/ml (cluster c, n=28) . in children with raised ccl22 chemokine levels appeared to be associated with a higher prevalence of house dust mite sensitization at age 1.5. furthermore, raised ccl22 levels during infancy were significantly associated with higher risk of asthma at age 3 (p=.026). conclusions: raised ccl22 chemokine levels during infancy appear not only to be associated with an increase in the prevalence of house dust mite sensitization but also the risk of asthma in early childhood. 0520 | serum periostin is "not" a biomarker for pediatric asthma suzuki n 1 ; hirayama j 1 ; nagao m 1 ; kameda k 1 ; kuwabara y 1 ; kainuma k 1 ; ono j 2 ; ohta s 3 ; izuhara k 3 ; fujisawa t 1 1 mie national hospital, tsu, japan; 2 shino-test corporation, kanagawa, japan; 3 saga medical school, saga, japan introduction: periostin is a matricellular protein induced by type 2 helper t-cell cytokines, expressed by airway structural cells and is thought to contribute to airway remodeling and progressive lung function decline in severe asthma in adults. we sought a possible clinical utility of serum periostin in children with asthma. objectives: we recruited volunteer children and adolescents (age range, 3 to 17 years) who were otherwise healthy except for allergic diseases including bronchial asthma. allergic diseases were classified with isaac questionnaire and serum levels of periostin were measured with elisa. abstracts | 167 results: a total of 661 volunteers were enrolled. among them 161 had no allergic diseases (na), 215 had only allergic rhinitis (ar) and the rest of 285 had bronchial asthma (ba) and/or atopic dermatitis (ad) . serum levels of periostin in na younger than 15 were significantly higher than the older counterpart and the levels in children <15 years old were similar across each age. data distribution of serum periostin in ar were very similar with that in na and we defined na and ar groups as reference population. reference values, geometric mean (+2 geometric sd range), for serum periostin were 101 (179) and 82 (151) ng/ml in children/adolescents <15 and ≥15, respectively. there were no gender differences in serum periostin in the reference population. we then compared the serum levels of periostin in ad and/or ba with the reference group and found that serum periostin in ad and ad+ba, not ba, in <15 years old were significantly higher than the reference group. in addition, severity of asthma had no association with serum periostin levels in age group of <15. on the other hand, the levels in ba aged ≥15 were slightly higher than non-ba (statistically significant). conclusions: serum periostin is physiologically high in children and adolescents, possibly in growing age, and the levels are elevated in those with ad, not ba. serum periostin is "not" a useful biomarker for pediatric asthma. 0521 | clinical, biochemical and radiological factors for response to aspirin desensitization in patients with aspirin exacerbated respiratory disease-pilot study introduction: aspirin desensitization is regarded as an effective and well-tolerated therapy for patients with aspirin exacerbated respiratory disease (aerd). despite many studies investigating the pathophysiology of aerd, the underlying mechanism responsible for the beneficial effects of aspirin therapy has not yet been clarified. the aim of the study was to evaluate the influence of aspirin desensitization on clinical, biochemical and radiological changes in aerd individuals. objectives: this is a prospective study of twenty-one aerd patients subjected to one-year aspirin desensitization. all participants were hospitalized three times over the period of one year. at baseline and during each follow-up visit (2 nd and 12 th month) blood, urine, induced sputum (is) and nasal lavage (nl) were collected from all participants. the acquired material was processed in order to evaluate 1) is and nl cell count 2) concentrations of is and nl eicosanoids 3) leukotriene e4 (lte4) in urine and 4) periostin in blood. additionally, participants underwent a ct scan of the paranasal sinuses at baseline and after 12 months of aspirin therapy. the lund-mackay score values were compared. for statistical analysis, summary statistics and repeated measures anova with post-hoc test were applied. results: twenty participants completed a one-year aspirin therapy. there was a statistically significant decrease in the number (p=.007) and percentage (p=.02) of eosinophils in is between baseline and after aspirin desensitisation. significant increase of urine lte4 in the course of aspirin therapy was observed (p=.02). the levels of prostaglandins and leukotrienes in is and nl as well as blood periostin level and the differential cell count in nl did not change during aspirin desensitization. in ct scan images the regression of the lesions in paranasal sinuses was observed in 32%, the worsening in 42% and in 26% of patients no changes were noted. in is count of eosinophils in aspirin-sensitive individuals, which may potentially be used in disease monitoring and tailoring asthma therapy. aspirin desensitization did not lead to significant changes in local eicosanoid levels in is and nl. blood periostin level is not a good marker for patient's response to aspirin desensitization. introduction: one of the main severe asthma phenotype is the "severe eosinophilic" or "eosinophilic refractory" asthma, for which novel biologic agents are emerging as therapeutical options. in this context, blood eosinophils count are one of the most reliable biomarker. objectives: the aim of our study is to evaluate the performance of a point-of-care peripheral blood counter in a clinical setting of severe asthmatics. seventy-six patients with severe asthma were evaluated, for blood cells count, by both a point-of-care and a standard analyser. results: the inter-and intra-assay variation was acceptable for leukocytes, neutrophils, lymphocytes and eosinophils. this was not the case of monocytes and basophils. a significant correlation between blood eosinophils assessed by the two devices was found (r 2 = 0.854, p<.0001); similar correlations were found also for white blood cells, neutrophils and lymphocytes. the point-of-care device showed ability to predict blood eosinophils cutoffs used to select patients for biologic treatments for severe eosinophilic asthma, and the elen index, a composite score useful to predict sputum eosinophilia. introduction: over 10 years ago there was revealed periostin should play an important role in pathogenesis of allergic inflammation including asthma and processes of tissue remodeling and fibrosis. its expression has been observed in the thickened basement membrane as well as in serum of asthmatic patients. thus, measuring of periostin serum levels may shed some light on these elusive asthma features. periostin has already demonstrated a convenient value in clinical studies as a companion diagnostics for lebrikizumab, tralokinumab or omalizumab treatment. however, to date, the changes of periostin serum levels following asthma therapy except for inhaled corticosteroids remain unclear. objectives: to emphasize this issue we have collected clinical and laboratory data including serum periostin of 48 asthma patients (19 males/29 females) in a cross-sectional study. all patients were treated either by conventional therapy comprising inhaled corticosteroids (n=38) or by inhaled corticosteroids with additional biological therapy (omalizumab) (n=10). asthma phenotype, control, complications, comorbidities and other available biomarkers have been assessed and statistically analysed. results: we have observed a weak but statistically significant correlation between serum periostin and total ige levels (p=.039) and absolute eosinophil count (p=.045). association between periostin and total ige had nonlinear character (p=.007), and was expressed particularly in non-severe asthma patients without omalizumab treatment (p=.018). despite mutual correlation between serum periostin a total ige levels, both biomarkers showed different reaction on asthma treatment. multivariate analysis demonstrated, that only periostin levels, in contrast to all other assessed biomarkers, were significantly decreased in severe asthma patients treated by omalizumab (p=.013). this relationship was independent of asthma control (assessed by asthma control test -act), exacerbation rate, hrct or spirometry measurement results or comorbidities, except chronic rhinosinusitis with nasal polyps (crswnp) (p<.001). conclusions: we demonstrate, serum periostin levels are dependent on therapy, thus it may contribute not only to asthma phenotype stratification, prediction of treatment responsiveness, complications such as remodeling but probably more precise monitoring of therapy effect too. 0527 | usefulness of serum pteridines as a biomarker for childhood asthma kasuga s 1 ; hamazaki t 1 ; fujitani h 1 ; fujikawa s 1 ; niihira s 2 ; shintaku h 1 1 department of pediatrics, osaka city university graduate school of medicine, osaka, japan; 2 the national institute of special education, tokyo, japan introduction: reliable and stable biomarker of airway inflammation is essential to determine intensity of asthma treatment. exhaled nitric oxide (feno) has been introduced to assess useful marker of airway inflammation but it fluctuates depending on steroid inhalation. nitric oxide is produced by nitric oxide synthase which requires tetrahydrobiopterin (one of pteridines) as a cofactor. objectives: to assess pteridines as a biomarker of childhood asthma control. results: asthmatic children were recruited for periodical asthma checkup program in japan to assess asthma control status by using objective questionnaire, respiratory function tests, airway resistance, feno, and serum pteridine levels. serum pteridine levels were measured by high performance liquid chromatography. total 131 japanese children (4-17 years) were participated in this program. 36 children who have no asthma attack over three years were divided as remission group to evaluate the long-term asthma control. the other 95 children were divided as asthma group. furthermore, we divided asthma group for three groups by childhood asthma control test (c-act) scores to evaluate the short-term asthma control. and we had 61 age matched children as control group. pteridine levels tended to decrease in patients who showed higher feno in asthma group. asthmatic children showed lower pteridine levels than other groups. there are significant differences between control group and remission groups and asthma groups (p<.001, tukey's honestly significant difference test). but there are no significant differences between the three groups in asthma group (well-controlled group, partly-controlled groups and uncontrolled groups) which were divided by c-act scores. the low concentration of serum pteridines in children with stable asthma may indicate poor long-term control of asthma. but that not indicate poor short-term control of asthma. since pteridine biosynthesis pathways were suppressed by th2 mediated immune response, these results suggest that th2 mediated immune response was dominating the th1 response in asthmatic children. therefore, serum pteridines could be a novel biomarker of stable asthmatic children. introduction: asthma is a syndrome with chronic airway inflammation. the goal in the treatment of asthma is to control the inflammation. however, there has been a scarcity of useful noninvasive tests for monitoring the airway inflammation in clinical setting. metabolites of the eicosanoids pathways in induced sputum of the patients with asthma could be valuable biomarkers that can reflect the airway inflammation. objectives: to investigate eicosanoid metabolites and to find out their phenotypic differences, induced sputum supernatants from 37 patients with refractory asthma, 40 patients with controlled asthma, and 6 normal control subjects were analyzed by using liquid chromatography tandem mass spectrometry. in addition, we evaluated the relationship between asthma exacerbation and eicosanoid metabolites. results: among 43 metabolites which were measured, 26 metabolites were detected in the induced sputum. we found that normal control subjects had higher concentrations of prostaglandin (pg) d2 (normal subjects vs controlled asthma vs refractory asthma, median conclusions: eicosanoid profiles in induced sputum supernatant were different between patients with refractory asthma, those with controlled asthma, and normal control subjects. our findings suggest that they could be biomarkers to differentiate refractory asthma from controllable asthma or non-asthma. this work was supported by the research program funded by the korea centers for disease control and prevention (2016-er7402-00) . 0529 | serum periostin in asthma is related to disease severity, eosinophilia and il-33 introduction: introduction: asthma is a chronic inflammatory disease where more than 100 powerful inflammatory mediators are associated with airway hyperresponsiveness, mucus hypersecretion, activation of fibroblasts and hyperplasia and hypertrophy of smooth muscles of the airways and if they do not use preventive antiasthma treatment can cause irreversible airway remodeling. objectives: the aim of this study was to determine the effect of adding montelukast to combined therapy icss/labas in patients with uncontrolled asthma by analyzing of serum level of il-13, il-5, eosinophils and symptom score. methods: in study we included 29 patients, they were treated with icss/labas (500/50 mcg-twice daily) plus montelukast (10 mgdaily). in each of them were measured serum levels of il-13 and il-5 by the elisa method, value of eosinophils were obtained with visual examination of peripheral blood smear, and assessing symptom scores with 5-point likert scale of breathlessness at the beginning and after 6 months of therapy. objectives: this study has been focussed on a detailed comparison of two samplers-cyclone and chemvol-and on the parameters that could influence their efficiency. results: airborne concentrations of two key olive and grass allergens, ole e 1 and phl p 5, respectively, were monitored over two years with different weather patterns, 2012 and 2014, in c ordoba, located in south-western spain. allergenic particles were quantified by elisa assay and results were compared with pollen concentrations monitored using a hirst-type volumetric spore trap over the same study periods. the influence of weather-related parameters on local airborne pollen and allergen concentrations was also analysed. inter-year differences were observed with regard to pollen season timing and intensity. for both olive and grass, the pollen season was longer and the pollen index (pi) higher in 2014; that year the peak value was higher and it was recorded earlier. although a positive correlation was detected between results obtained using the two samplers during the pollen season, results for the cumulative annual allergen index varied considerably. the two samplers revealed a positive correlation between pollen concentrations and both minimum temperature during the warmer year (2012) and maximum temperature during the cooler year (2014); a negative significant correlation was observed in both cases with rainfall and relative humidity. conclusions: in summary, although differences were observed between the two samplers studied, both samplers may be suitable for allergen detection. objectives: the scientific council of rnsa was asked to update the allergy potency (ap) of plant species that can be established in urban areas. to update the allergy potency of plant species, the rnsa used scientific work on the subject, and also the opinions of allergists and botanists. the pollen grains of anemophilous species are transported by wind; they produce very large quantities of pollen grains so that the fertilization of female flowers has a greater chance of being effective. the majority of allergenic species are anemophilous. results: the pollen allergy potency of a plant species is the ability of its pollen to cause an allergy to a significant part of the population. the allergenic potential can be: low or negligible: no problem to plant them in urban garden moderate: only a few species can be planted in the same garden high: this species cannot be planted in urban places. the table presented on the poster will permit to know the level of allergy potency of more than 30 species. conclusions: species or genus with a strong ap should be labeled as "not to be planted in habitation or residence area ", those with moderate ap should be labeled as "not to be planted in big quantities in habitation or residence area". other species with low or negligible ap may not be affected by public information. gadermaier g 1 ; metz-favre c 2 ; stemeseder t 1 ; de blay f 2 ; pauli g 2 1 universit e de salzbourg, salzbourg, austria; 2 chru strasbourg-allergologie, strasbourg, france introduction: the purpose is to study the profile of cutaneous and molecular sensitization of patients sensitized to plantain pollen living in the north-east of france. objectives: the sera of 28 patients with seasonal pollinosis and cutaneous polysensitization including a positive test to plantain, are investigated through immunoblot (ib), elisa and immunocap. the plantain immunoblot is inhibited with plantain, grass, ash and birch pollen extracts as well as with pla l 1. the specific iges against pla l 1, phl p 1-5 and profilin are measured. results: pla l 1, the major plantain allergen is detected by ib in 10 cases out of 28, either in glycosylated or non-glycosylated form. an allergen of 35 kda is detected in 21 out of 28 cases, always and exclusively inhibited by the grass extract. the intensity of the spot is proportional to the anti-phl p 1 / phl p 5 ige levels, and corresponds to an allergen which is cross-reacting with grass. other cross-reacting allergens with grasses are detected at 30 kda (14/28), and at 50-54 kda (5/28). at the molecular weights of 15 to 17 kda, we detected in addition to pla l 1, cross-reacting allergens with grass, ash and birch pollen which predominantly corresponded to profilin which was confirmed by specific ige measurements in 20 cases. conclusions: among the patients included in this study, only 28% had genuine sensitization to plantago, which never corresponded to a monosensitization in our cohort. most often cutaneous sensitizations to plantain pollen were based on ige cross-reactivity with grass pollen allergens mainly through a 35 kda allergen and/or profilin. the strong environmental grass pollen pressure, compared to the low plantain pollen exposure, seems to be at the origin of this profile of molecular sensitization. these results reinforce the superiority of molecular diagnosis in patients with pollinosis, who are polysensitized. 0536 | sensitisation to peach tree pollen in a highly exposed population results: the 14% of subjects were sensitized to pp, which was the most prevalent after olive tree, grass and cypress pollens, respectively. sensitization to peach fruit was 8%. pru p 3 spt was tested in 30/78 pp positive cases, being 46% positive (14/30). specific ige to pru p 3 was detected in 4/10 pp sensitized individuals. immunoblot showed specific ige to different components in the pp extract, being the most frequent band recognised a 20-25 kda protein. conclusions: pp is a prevalent inhalant allergen in highly exposure areas. in our population pru p 3 was not identified as the major allergen. other pp molecular components need to be identified and their clinical relevance should be further investigated. introduction: allergic respiratory diseases increase after an exposure to airborne pollen, as asthma and allergic rhinitis, are deeply increasing and nowadays, they represent one of the major public health problems. olive pollen is one of the main causes of allergic disease in the mediterranean area, especially in northwest and west of the turkey. olive pollen has been characterized 20 proteins with allergic activity and ole e 1 is the major allergen of olea pollen. objectives: the aim of this study was to estimate the correlation between daily airborne olive pollen and ole e 1 in the atmosphere. aeroallergen load of ole e 1 detected by cascade impactor (chemvol) using prewashed polyurethane foam and pollen counts recorded by hirst trap. chemvol sampler collects particles at 800 l/minutes and it contains 2 impaction stages pm>10 micron and 10>pm>2.5 lm. this sampler is being tested in the frame of the project hialine. results: generally, similar behaviour of pollen count and total allergenic load of ole e1 was observed during the main pollen season. nevertheless, in some occasions, before and later of main period, airborne ole e 1 activity was recorded and some differences between pollen grain/m3 and allergen concentration/m3 were detected. pollen from different days released 11-fold different amounts of ole e1 per pollen. average allergen release from pollen was much higher in 2011 (1.11 pg ole e 1/pollen, r 2 =.44) than in 2012 (0.65 pg ole e 1/pollen, r 2 =.3148). indeed, yearly olive pollen counts in 2011 were 3.8 times higher than in 2012, but ole e1 concentrations were 2.2 times higher. these results have shown that ole e 1 is mostly associated with olive pollen grains but aeroallergen load was not always directly proportional to airborne pollen counts. this suggests that ole e 1 quantification is a better marker for olive allergen exposure. in conclusion, aeroallergen monitoring may contribute to a better understanding of the ole e 1 exposure from airborne pollen. objectives: to describe the clinical profile of the patients sensitized to alt at 6 and to assess the sensitivity of the prick test with the alternaria extract in comparison to the patients sensitized to both allergens alt a 6 and alt a 1. out of the 726 patients, 94 (12.9%) were sensitized to alternaria with specific ige of ≥0.3 isu to alt at 1 or alt a 6. twenty patients (11 females and 9 males, age 6-57 years old) who were sensitized to alt a 6 were selected. we analyzed the clinical profile, total ige values, other co sensitizations as well as the clinical relevance of alternaria sensitization in these patients. : of the 94 patients sensitized to alternaria, 20 (21.3%) were sensitized to alt a 6, of which 9 (9.6%) were monosensitized to alt at 6. the mean age was lower in monosensitized (ẋ 16.2 vs 25.2). among the 9 patients monosensitized to alt a 6, 4 (44%) had prick test negative with the alternaria extract. eight out of 9 patients were polysensitized to more than three different aeroallergens and were asthmatics (6 persistent). the median total ige was higher in monosensitized (859 ku/l vs 479 ku/l). of these 8 patients, alternaria sensitization has clinical relevance in 5 (62%), of which 3 have positive prick test to alternaria. all the patients (12/ 12) sensitized to both allergens have positive prick test to alternaria. ten of them were polysensitized, 8 were asthmatic (3 persistent) and 5 sensitized patients showed clinical relevance. introduction: the role of vitamin d as a potential immune-modulator has been recently elucidated. dendritic cell, a key regulator driving towards th2 immune response in allergic diseases is known to be affected by vitamin d. however, the role of vitamin d in the pathogenesis of allergic rhinitis is unclear and its anti-allergic effect has not been established yet, especially in the mouse model. objectives: the aims of this study are to evaluate 1) the anti-allergic effect of topically applied vitamin d in the allergic rhinitis mouse model, and 2) the effect of vitamin d on dendritic cell activation. results: balb/c mice were intraperitoneally sensitized with ovalbumin (ova) and alum, and they were intranasally challenged with ova. intranasal 1, 25-dihydroxyvitamin d3 was given to treatment group and solvent was given intranasally to sham treatment group. allergic symptom scores, eosinophil infiltration, cytokine mrna levels (il-4, il-5, il-10, il-13, ifn-c) in the nasal mucosa, serum total and ova-specific ige, igg1, and igg2a were analyzed and compared with negative and positive controls. cervical lymph nodes were harvested for flow cytometry analysis. in the treatment group, allergic symptom scores, eosinophil infiltration, and the mrna levels of il-4 and il-13 were significantly reduced compared to positive control. il-5 mrna level, serum total ige, and ova-specific ige and igg1 levels showed a tendency to decrease in the treatment group, but did not reach to a significant level. il-10 did not show a significant difference between groups. cd11c + , mhcii hi , cd86 + activated dendritic cells were significantly reduced in the treatment group. cd4 + , cd25 + , foxp3 + treg cells tended to increase in the treatment group, however it was not significant. the intranasal instillation of 1, 25-dihydroxyvitamin d3 has an anti-allergic effect in the allergic rhinitis mouse model. we believe that the anti-allergic effect of vitamin d is mediated by the inhibition of dendritic cell activation, and therefore decreased objectives: we wanted to assess whether treatment with cyclosporine and tacrolimus allows children with vkc to improve the level of vitamin d due to the higher summer sun exposure for good control of the ocular symptoms. objectives: our objective was to assess the expression of smad2 and smad5 proteins in patients with asthma in correlation with clinical parameters and the expression's changes in response to allergen and methacholine challenge test. the study included 151 patients with asthma and 77 healthy volunteers. spirometry, skin prick tests (spt), allergen and methacholine provocation tests were performed in compliance with standards. personalized clinic surveys including act tm were collected. venous blood was collected before and after provocation. the expression levels of il-5 and il-15 and smads were evaluated by qrt-pcr using isoform-specific primers. results: we showed correlation between the mrna expression of smad2 and the asthma control according to act tm (p<.001). the expression of smad2 is higher in the group of uncontrolled (2 -δct =0.52, p<.001) and in the group of partially controlled patients (2 -δct =0.50, p<.05) in comparison to the group of patients with controlled asthma (2 -δct =0.40). we proved that expression of mrna of smad5 correlates significantly with expression of il-5 and il-15 in patients with asthma (il-5 r=0.34; il-15 r=0.36) and in the control group (il-5 r=0.36; il-15 r=0.27) (p<.05). expression of smad5 elevates more after methacholine provocation test (median 2 -δct =0.69) rather than after allergen provocation test (median 2 -δct =0.51; p=.04). we showed also that skin prick test results correlate positively with smad5 level after the provocation (p<.001). conclusions: the loss of asthma control is connected with expression of smad2, which is part of tgf-ßrii related pathway. il-5 and il-15 correlates with smad5 expression, which can indicate the participation of these cytokines in the regulation of this pathway. the expression of smad5 elevates after methacholine and allergen provocation as well as it correlates positively with skin prick test results, which can be useful clinically. to sum up, tgf-ß-tgfßrii-smad proteins are an important mediators of inflammation in asthma. 0545 | deletion of nfatc1 in t lymphocytes affects th2 and th17 cell differentiation as well as il-9-mediated mast cell activation in allergic asthma objectives: analysing the role of nfatc1 in the allergic trait of human asthma. additionally, we investigated the influence of nfatc1 on t cell differentiation and immunoglobulin class switch and explored its impact on mast cell differentiation in experimental asthma. with the children 0 s hospital in erlangen, we studied nfatc1 mrna expression in freshly isolated pbmcs from pre-school children with and without asthma. in asthmatic children, we found increased nfatc1 mrna expression, especially in those with a positive skin test. these results were confirmed in the asthma bio-repository for integrative genomic exploration (asthma bridge) study, where the isoform a and d of nfatc1 were found significantly increased in asthmatic adults with a positive skin test. moreover, il-9 was also found increased in the supernatants of pbmcs of asthmatic children with a positive skin test. furthermore, mice with a deficiency of nfatc1 in cd4 + t cells display significantly lower levels of il-9. additionally, these mice show reduced numbers of lung th2 and th17 cells. moreover, basic leucine zipper atf like (batf), which is known as an important transcription factor for t cell differentiation as well as immunoglobulin class switch, was found decreased in these mice. consequently, ova-specific ige and total igg1 levels were found significantly decreased after allergen exposure and in the absence of nfatc1. furthermore, nfatc1 deletion also resulted in decreased mast cell numbers. we then analyzed the effect of il-9 on mast cell differentiation and histamine release. we observed that bone marrow differentiated mast cells incubated with ova and il-9 had an induced histamine release. conclusions: thus, nfatc1 deficiency in t cells results in defective ige production affecting ige-orchestrated mast cell activation mediated through il-9. therefore, nfatc1 emerges as a novel target for anti-allergy intervention. 0546 | efficiency of the use of nitric oxide donors for the treatment of bronchial asthma bazarova s; djambekova g center of therapy, tashkent, uzbekistan introduction: to study the influence of nitric oxide donor-l-arginine-on indicators of endothelial system in patients with bronchial asthma. objectives: 58 patients aged 27-55 years (41 ae 4.12 years) with moderate persistent bronchial asthma were examined. ratio of men to women was 30/28. two age-and sex-matched groups were randomly selected. main group (32 patients) received nitric oxide donor -l-arginine in addition to standard background therapy (gina, 2012) . the medication (100 ml of 4.2% solution, tivortin, "yuria-farm", ukraine) was administered intravenously once daily for 10 days. the control group (26 patients) only received the background therapy. the efficiency was assessed with the use of conventional methods of study (clinical laboratory methods, instrumental methods: spirography, peak flow monitoring, bronchomotor tests). concentration of nitric oxide stable metabolites in exhaled breath condensate (ebc) and in blood was studied. their ratio was also assessed. results: baseline data in patients of both groups demonstrated the increase of level of nitric oxide stable metabolites in blood (0.78 ae 0.02 mmol/l) and in ebc (0.43 ae 0.01 mmol/l). after the treatment the positive clinical efficiency of inclusion of l-arginine was much higher than that in the control group (p<.05). in the main group, reduction of requirement for beta2-agonists, decrease of frequency of night-time symptoms, decrease of frequency of lowest pef rate in the morning and improvement of respiratory function were observed on average on the 3rd/4th day compared to the control group were such improvements were evident on the 7th/8th day. in the main group the concentration of nitric oxide stable metabolites significantly increased in blood (0.59 ae 0.02 mmol /l, p<.05) and in ebc (0.21 ae 0.03 mmol/l, p<.05). in the control group the concentration of nitric oxide stable metabolites changed in blood (0.69 ae 0.06 mmol/l) and in ebc (0.32 ae 0.05 mmol/l), but not significantly. introduction: abpa is currently believed to be an exaggerated form of aspergillus sensitization, and is probably the first step in its development. objectives: the aim of this study was to investigate the clinical and immunologic characteristics of fungal-sensitive asthma (fa), nonfungal-sensitive asthma(nfa) and abpa. conclusions: there were different clinical and immunological features among nfa, fa and abpa. the abpa had worse function as well as higher percentage of bronchiectasis, and higher dose of oral corticosteroid. besides, the sensitivity to aspergillus was more severe in abpa. the level of sige-a.f was associated with the damage of lung function. 0548 | self-reported allergic rhinitis and/or allergic conjunctivitis associate with il13 rs20541 genotypes in finnish adult asthma patients introduction: the increased prevalence of asthma and allergic diseases is a major public health problem worldwide. atopy, family history, inhaled irritants, and upper airway inflammation are known risk factors of asthma. a population-based sample of finnish adult asthma patients (n=1156) and matched controls (n=1792) filled a questionnaire. asthma was diagnosed based on a typical history of asthma symptoms and lung function tests. skin prick tests (spt) with 17 aeroallergens and blood tests including analysis of interleukin 13 (il13) rs20541 (g/a) genotypes were performed for a subsample (n=193). objectives: our aim was to observe in adult asthmatics with and without allergic co-morbidities e.g. subject-reported allergic rhinitis and/or allergic conjunctivitis (ar/ac) association with il13 rs20541 genotypes and other factors. results: the proportion of asthmatics reporting ar was 61.9% and ac was 40.7%. after adjustments, the presence of il13 rs20541aallele (or=3.06 ci=1.42-6.58, p=.004) or multi-sensitization (adjusted or=4.59, ci=1.48-14.26, p=.008) associated with ar/ac-asthma. nasal polyps and aspirin-exacerbated respiratory disease associated also with ar/ac-asthma. conclusions: adult ar/ac-asthma could putatively be a phenotype, characterized by the presence of atopic and/or eosinophilic factors and a high prevalence of the il13 rs20541a-allele. studies on the mechanisms behind this and in other populations are needed. 0549 | immunoregulatory role of nfatinteracting protein (nip) 45 in adaptive and innate immune responses in allergic asthma introduction: nfat-interacting protein (nip) 45 is a th2 associated transcription factor. after t cell receptor stimulation, the arginine methylation domain of nip45 supports the interaction with nfat, thereby enhancing the production of the th2 cytokine il-4. moreover, nip45 deficient mice have been shown to be deficient in il-4 and ifn-gamma production indicating that nip45 controls both th1 and th2 cytokine production and might therefore play a protective role in allergic asthma . objectives: we wanted to analyze the importance of nip45 in allergic asthma in pre-school children as well as adults. furthermore, we investigated the role of nip45 in a murine model of allergic asthma to find out more about its contribution to adaptive as well as innate immune responses in the disease. results: in the european study predicta, in collaboration with the children 0 s hospital in erlangen, we analyzed nip45 mrna expression by using quantitative real time pcr in rna extracted from pbmcs isolated from pre-school children with and without asthma. in the pbmcs of the asthmatic children, nip45 mrna expression was found significantly increased compared to healthy control children. furthermore, nip45 mrna expression was also found induced in cd4 + t cells in adult asthmatics from the asthma bio-repository for integrative genomic exploration (asthma bridge) study. we further analyzed the importance of nip45 in a murine model of allergic asthma. after allergen sensitization and challenge nip45 (-/-) mice showed decreased airway hyperresponsiveness, inflammation and mucus production, three of the main patho-physiological features of asthma. additionally, we discovered that nip45 (-/-) mice released decreased th2 type cytokines and also expressed less st2, which is the receptor for il-33, after allergen challenge. furthermore, a defect in innate lymphoid cell type 2 (ilc2) differentiation was observed in the absence of nip45 in allergic asthma and in bone marrow differentiated ilc2s indicating a crucial role for nip45 in mediating asthma via ilc2s. conclusions: in summary, we found that the lack of nip45 influences not only immune responses of the adaptive immune system but also influences components of innate immunity resulting in a abstracts | 181 protective phenotype to allergic diseases such as asthma. objectives: in the study were included 40 ap and 10 healthy controls (hc). fraction of exhaled no (feno), standard lung function parameters, complete blood count and absolute count of cells, serum ige, crp, il-6, il-17a and periostin were measured. results: four clusters were identified by cluster analysis: cluster 1 (c1)(n=14)-late-onset, non-atopic, eosinophilic asthma with impaired lung function, cluster 2 (c2)(n=13)-late-onset, atopic asthma, cluster 3 (c3)(n=6)-late-onset, aspirin sensitivity, eosinophilic asthma and cluster 4 (c4)(n=7)-early-onset, atopic asthma. we have found higher levels of il-6 in all clusters ap as compared to hc (c1: p=.001, c2: p=.017, c3: p=.012 and c4: p=.003). tendency for higher levels of serum il-6 in c1 compared with c2 or c4 (p=.089 or p=.062, respectively) was observed. periostin levels were significantly higher in c1 (p<.001), c2 (p<.001) and c4 (p<.01) as compared to hc. there were no differences in periostin levels between all clusters (anova, p=.389). il-17a levels were significantly higher only in c4 as compared to hc or to c1 and c2 (p<.05, for each comparison). we have found correlation between il-6 and crp (r=.640; p=.014) in c1, il-17a and periostin in c1(r=.631; p=.016) and in c2 (r=.641; p=.018). interestingly, we have observed negative correlation between the duration of asthma and il-17a (r=à.886; p=.019), but positive between the duration of asthma and crp (r=.886, p=.019) in c3. our results have shown higher levels of il-6 in all clusters as compared to hc that are associated with marker for systemic inflammation. periostin levels were significantly elevated in c1, c2 and c4 as compared to hc with no differences between the clusters. a positive correlation between periostin and il-17a in c1 and c2 was observed, that rising the question about their interrelationship in the pathogenesis of late-onset asthma. serum il-17a was significantly higher in c4 in comparison with hc or c1 and c2 suggesting that th17 mediated immunity may be involved in early-onset, atopic asthma. these data support the concept of heterogeneity of the bronchial asthma. 0551 | eosinophil activation with autophagy and extracellular dna traps is involved in severe asthma results: il-5+lps treatment significantly increased autophagy and eet levels from pbes of the study subjects (p<.05 for all), which were in a positive correlation (r=.802, p<.001). compared to nsa patients, both untreated and il-5+lps-treated pbes from sa patients had significantly higher autophagy levels (p=.007 and .002, respectively), while only il-5+lps-treated pbes from sa patients had higher eet level (p=.036). eet level from untreated pbes was correlated with serum eosinophil cationic protein level (r=0.608, p=0.036) and fev1% predicted value (r=à.620, p=.056). co-culture of aec with pbes slightly increased il-8 production, which was significantly enhanced by il-5+lps treatment (p<.001). the il-8 production in co-culture system was reduced by pretreatments with dexamethasone (1 mm, p=.001), antibodies against major basic protein (200 ng/ml), il-5 receptor (1 mm) and il-4 receptor (1 mm, p=.05 for all), but increased by cotreatment with micrococcal nuclease (10 iu/ml, p=.001). conclusions: pbes from sa patients are highly susceptible to be activated to produce high levels of autophagy and eet, which could enhance and maintain airway inflammation. we suggest that steroid and anti-il-5/il-4 receptor antibodies may be beneficial to control airway inflammation in severe eosinophilic asthma via inhibition of eet production, while dna digesting reagents may increase airway inflammation. introduction: it has been demonstrated that exposure to stress induce hyporeactivity of the hpa system by modifying the secretion of cortisol and also that psychosocial stressors such as poor social status are associated with an increased risk of childhood asthma, decreased serum cortisol and high ige response. thus, from this concept it could be postulated that a blunted hpa axis response may increase the risk for allergic inflammatory reaction. objectives: aim of this study was to evaluate the association of serum cortisol in pediatric allergic bronchial asthma and its influence on the ige immune response in a poor children community with psychosocial chronic stress. results: this was a pilot analytical case control study(50 ipa positive subjects and 50 healthy control paired by age and gender, both from poor areas of barranquilla objectives: in order to characterize bp14 an immunoproteomics analysis was conducted, i.e. electrophoretic separation of cypress pollen extracted proteins, ige western blotting using cypress pollen allergic patient's sera and mass spectrometry (lc/ms/ms) for identification of ige-binding proteins. results: ms analysis using chymotrypsin identified in bp14 a peptide also found in the family of protein snakin/gibberellin-regulated protein (grp). the snakin-1, an anti-microbial peptide of potato, produced as a 63 amino-acid recombinant protein (homologous to the c-terminal part of grp), is recognized by ige from cypress pollen allergic patients with ige to bp14. this ige reactivity is abolished after reduction of disulfide bridges and is inhibited by a cypress pollen extract. ige reactivity to bp14 is however barely inhibited by the recombinant potato snakin-1. conclusions: bp14 exhibits a molecular mass closer to grp than to snakin and the absence or very low inhibition of the ige reactivity to bp14 with snakin may be explained by peptidic ige epitopes on the n-terminal part of bp14. the potato snakin-1 share 83% sequence identity with peamaclein, the peach allergen pru p 7, also shown in citrus. these results might explain the peach/cypress and citrus/cypress syndromes described and point out bp14 as the cross reactive allergen. the proteins of snakin/grp family present in many fruits and vegetables might include allergens involved in pollen/food associated syndromes. introduction: exposure to high levels of grass pollen may lead to a high degree of allergic inflammation, sensitization to minor allergens, such as profilin, and development of severe profilin mediated food reactions, similar to those described due to sublingual immunotherapy (slit). objectives: our objective here was to identify genetic biomarkers in order to generate a model that can improve the classification and treatment of patients with a higher probability of developing severe adverse reactions. results: 6 healthy subjects (group 1, control) and 17 patients with mild (group 2) or severe (group 3) profilin mediated food reactions were studied. rna extraction was performed on ficoll-isolated pbmcs using the rneasy ® mini kit (qiagen) and its integrity was analyzed with experion rna stdsens analysis kit (bio-rad). the gene expression profile of all the samples was analyzed using the gene-chip ® wt plus reagent kit (affymetrix) and two specific software: affymetrix ® expression console ™ and affymetrix ® transcriptome analysis console (tac). finally, the microarray data was validated by quantitative real-time pcr (qpcr). the hierarchical clustering of the samples shows the separation of the patients into three clusters coincident with the three established clinical groups (control, mild and severe). genetic profile of patients with mild reactions is similar to healthy patients while severe subjects were significantly different from the other two groups. genes regulated in the severe group were related to histone modification pathways, human complement system, cell adhesion and tgf-b and its receptor. these changes may be associated with the different degree of inflammation between patients in each experimental group. in the course of our study we found out that severe patients had different rna expression patterns compared to mild and non-allergic patients. this lead to the identification of genetic biomarkers useful for the generation of a model able to predict severe reactions and/or adverse reaction during immunotherapy, thus improving the diagnosis and treatment of this type of allergic results: his-tagged recombinant allergens, namely parvalbumin, aldolase, enolase and tropomyosin from c. idella and l. crocea, as well as cod parvalbumin, were synthesized in e. coli and purified using immobilized metal-chelate chromatography. 14 children with history of immediate-type fish allergy were included in this study and their serological ige reactivity to the fish allergen components were measured by elisa. 11 children were positive to at least one allergen component, with nine of them being positive to parvalbumin. despite the high similarity between l. crocea, c. idella and cod parvalbumins (70.6-81.6%), three children only reacted to l. crocea and c. idella parvalbumin but not to cod parvalbumin, while the other six children were positive to all three parvalbumins. competitive inhibition elisa revealed that c. idella parvalbumin inhibited >85% of the binding of specific ige to both l. crocea and cod parvalbumin, while reciprocally only inhibition of 60% and 50% could be achieved respectively. two children were reactive to aldolase and enolase but not parvalbumin. one of them had positive sige to both enolase and aldolase from l. crocea, while the other reacted to aldolase from both species. these two children exhibited relatively mild subjective allergy symptoms (itchy skin and throat). notably, three children were non-reactive to all components tested, and two of them were outgrowing fish allergy clinically. introduction: anti-a-gal antibodies are naturally produced in response to the gastrointestinal flora. in red meat allergy, patients develop ige antibodies towards the a-gal epitope, which itself are structurally closely related to the blood group b antigen. objectives: this study aimed to explore the relationship between a-galand b-antigen-specific antibodies in red meat allergic patients compared to healthy individuals with blood group b or a/o. sera from 39 red meat allergic patients and 84 healthy blood donors of whom 52 belonged to blood group b and 32 to blood group a or o were included. ige reactivity against a-gal and the b-antigen were determined using immunocap. allergen-specific igg, igg1, igg2, igg3, igg4 and ige were assessed by indirect elisa assay. statistical analysis was performed using spearman rank correlation and unpaired t-tests. results: all red meat allergic patients, 12 of the healthy a/o and 3 of the healthy b donors were ige positive to a-gal. however, the ige levels to a-gal were significantly higher in the allergic group compared to the healthy a/o and b individuals. the majority of the meat allergic patients, but none of the healthy individuals had ige antibodies against the b-antigen. a moderate correlation between a-gal and b-antigen specific ige was noted (r 2 =.48). the red meat allergic patients had significantly higher igg levels against a-gal with igg1 and igg4 antibodies as the predominant difference compared to the healthy individuals. the 12 healthy a/o ige positive individuals had significantly higher igg1, igg3 and igg4 compared to the ige negative individuals. the igg response to the b-antigen followed the same pattern as to a-gal. there was a low correlation between the igg levels against a-gal and the b-antigen in both meat allergic patients and healthy a/o individuals (r 2 =.30 and .33). introduction: fx5, a food mixture of milk, egg white, fish, peanut, wheat and soybean, is largely used for food allergy detection. besides the fact that it is questionable if this is the better approach to identify a food allergy, the information that the test provides may represent a pitfall because a positive or negative result does not mean food allergy presence or absence, respectively. objectives: the goal of our study was to access the reason for fx5 request in a pediatric hospital and to analyze its suitability. methods: the fx5 requests, performed in a pediatric population of d. estefânia hospital over a five months' period, were analyzed, concerning demographic data, reason for request and attitude taken due to the result. this test was requested due to respiratory symptoms in 37 patients, gastrointestinal symptoms in 23 patients, cutaneous symptoms in 20 patients and nonspecific complaints in 48 patients. all of these symptoms were not directly related with food intake. a positive result was obtained in 25 patients; of those, only 15 were referenced to our immunoallergology department. in all of them a detailed clinical history was obtained and diagnostic tests (skin prick tests and specific ige) were performed in the ones considered suitable. food allergy was diagnosed in only one patient. conclusions: in the vast majority of patients, fx5 was asked for nonspecific complaints and often without a clinical history suggestive of food allergy. moreover, a positive fx5 test does not mean clinical reactivity or food allergy. on the other hand, the clinical history allows us to identify a suspect trigger in most of the children with food allergy. in such cases, it is preferable to request the specific ige towards the allergen, which gives a more precise and accurate result, instead of the fx5. as our results showed, in the majority of the cases, the fx5 request was often made without complying with a reasonable criterion, implying unnecessary costs. the high number of requests verified may be explained because it is an easily accessible analysis but this attitude should be discouraged. tuppo l 1 ; alessandri c 2 ; pasquariello s 3 ; petriccione m 3 ; giangrieco i 1 ; tamburrini m 1 ; rafaiani c 2 ; ciancamerla m 2 ; mari a 2 ; ciardiello ma 1 1 istituto di bioscienze e biorisorse -ibbr-cnr, naples, italy; 2 caam -centri associati di allergologia molecolare, rome, italy; 3 cra, research unit on fruit trees, caserta, italy introduction: pomegranate, punica granatum l., is one of the oldest cultivated fruit trees. the fruit contains the arils, which are seeds covered by a red pulp, that is a juice sac. the arils are surrounded by the white and fleshy mesocarp. pomegranate can trigger allergic reactions, but the allergenic pattern of this fruit is still poorly characterized and only one allergen, the ltp pun g 1, was reported. objectives: the aim of this study was the investigation of the allergen pattern in pomegranate tissues and cultivars. reported symptoms were food impaction (68%), dysphagia (65%), heartburn (33%) and vomiting (30%). the first 2 symptoms were more frequent in adolescents and adults (96%). children's main complaint was vomiting (73%) and 4 cases presented failure to thrive. most patients had associated allergic diseases (88%), 23% had previous food allergy and 74% were sensitized to aeroallergens. endoscopic evaluation revealed esophageal stricture in 5 patients. at least half of diagnostic esophageal biopsies had >20 eosinophils per highpower field and 33% showed microabscesses. food sensitization was found in 61% of the patients, mainly to cow's milk (46%), nuts and peanut (33%), cereals (30%) and egg (21%). considering therapeutic approach, 88% were treated with swallowed fluticasone and dietary elimination was recommended in 54%. oral corticosteroids were prescribed in 8 patients. at present time 42 patients had done endoscopic reevaluation, 30 (71%) showed histologic resolution. five patients had clinical and histologic relapse during follow-up. conclusions: eoe has a balanced distribution but a distinctly clinical presentation accordingly to age group: children may present unspecific symptoms like vomiting, whereas adolescents and adults complain of food impaction and dysphagia. other atopic diseases and food sensitization is very common. introduction: air pollution, particularly ambient air particulate matter (pm), is considered as one of the most important environmental risks for human health. pm could potentially disrupt immune regulatory mechanisms and predispose exposed individuals to asthma. in contrast, children exposed to traditional farm environment seem to have a natural resistance to asthma. this phenomenon links with exposure to stable dust and subsequent immune regulatory mechanisms initiated in the airways. the underlying exposure agents and definitive pathways determining the risk of asthma are still to be identified. objectives: our aim was to investigate the effect of urban air pm (high risk environment) and farm dust (protective environment) on immune responses in finnish children. briefly, peripheral blood mononuclear cells (pbmcs) of 4-year-old children (n=18) were stimulated with farm dust extract (40 lg/ml, stable in northern savonia, finland) and pm samples (75 lg/ml, pm 2.5-1 , pm 1-0.2 or pm <0.2 , nanjing, china) for 18 hours. expression of immunogenic cd80 and tolerogenic ilt4 in circulating myeloid dendritic cells (mdcs) and plasmacytoid dcs (pdcs) and monocytes were analyzed by flow cytometry. pm samples were analyzed for polycyclic aromatic hydrocarbons (pahs), inorganic ions and elements. farm dust sample will be analyzed for microbial content. results: pm stimulation decreased the percentage of cd80+ monocytes and dcs among children's pbmcs, whereas farm dust stimulation increased the percentage of cells positive for this marker. the percentage of tolerogenic ilt4+ was decreased in all cell types after stimulation with pm. farm dust also decreased the percentage of ilt4+ monocytes, but not dcs. specific metals and pahs in pm associated with the studied immunological parameters. conclusions: samples from high risk and protective environments have differing capacities to influence immunogenic and tolerogenic properties in children's immune cells. the importance of these findings in relation to the risk of asthma in exposed populations will be studied further. introduction: alterations in cell surface glycosylation pattern is a common feature of tumor cells that might be related to immune evasion and malignancy. objectives: to study the capacity of the carbohydrate a10 (ca10) located in the surface of murine ehrlich tumor (et) cells and also in certain human adenocarcinomas to condition the phenotype and function of human dendritic cells (dcs) and the capacity to polarize t cell responses. results: nf-jb/ap-1 are not activated in thp1 cells by ca10, however, this carbohydrate partially impairs the activation of these transcription factors induced by the tlr2 ligand pam3csk. ca10 induces the expression of the tolerogenic marker pdl1 in human monocyte-derived dcs (hmodcs) as well as the production of il-6 and il-10, analyzed by flow cytometry and elisa assays respectively. ca10-activated hmodcs generate functional il-10-producing cd4 + cd25 high cd127 -foxp3 + regulatory t (treg) cells that were able to inhibit the proliferation of cd4 + t cells from pbmcs in a dose-dependent manner. supporting the role of ca10 in the induction of treg cells, our in vivo data showed that the ca10 is present in the sera of tumor bearing mice and the percentage of foxp3 + treg cells is increased in the regional (inguinal) lymph nodes from tumor bearers. our results showed that ca10-activated hmodcs induce the generation of foxp3 + treg cells both in vitro and in vivo, which might well condition the immune response against the tumor and promote the tumor escape. 0567 | assessment of changes in expression of immune system biomarkers to assist the differential diagnosis of acute bacterial infections introduction: biomarkers for acute infections include c-reactive protein, mmp-9, sicam-1, procalcitonin, and neutrophil band counts for bacterial infection. a rapid means of assessment of acute bacterial infections via biomarker assessment was sought. objectives: the expression of toll-like receptors (cd282 and cd284), complement receptors (cd35 and cd88), integrins (cd11b and cd11c), fc-receptors (cd32 and cd64) and l-selectin (cd62l) on the surface of blood neutrophils and monocytes stimulated with inactivated e. coli, l. acidophilus, e. coli derived bacterial ghosts, e. coli lps and l. acidophilus cell walls was assayed using flow cytometry. both the percent of expression and mean fluorescence intensity (mfi) were analyzed for each molecule. results: all the bacterial components used exerted similar activation capabilities even in low concentrations. while the expression of cd11b, cd11c, cd32, cd35 and cd88 was enhanced by both neutrophils and monocytes under activation, the expression of cd64 significantly increased only in neutrophils. the expression of tlr2 and tlr4 was slightly increased by neutrophils and monocytes. the expression of cd62l by monocytes and neutrophils (the percent of activated cells as well as the mfi) was decreased during activation. there was a negative correlation between cd62l expression and integrins (cd11b and cd11b). the activation index was calculated for each molecule as a ratio of expression of molecule by activated cells vs cells used as a negative control (resting). the highest values for the activation index was seen with cd11b, cd11c, cd32, cd35, cd62l and cd88 mfi by neutrophils and monocytes, and the percent of cd64 expression by neutrophils. conclusions: e. coli and bacterial ghosts significantly increased the expression of cd11b, cd11c, cd32, cd35, cd62l and cd88 by neutrophils and monocytes even in very low concentrations, suggesting use as potential biomarkers in the differential diagnosis of the etiology of acute infections. objectives: the aim of this study was to evaluate changes in peripheral blood monocyte expression of cd16, cd163, cd206, cd209, hla-dr, and cd47 in kidney allograft recipients. in total, 88 patients who underwent renal transplantation from a deceased donor were enrolled in the study. the phenotype was evaluated by a multicolor flow cytometry in defined time points and in the case of complications requiring fine needle aspiration biopsy procedure. the results confirmed our pilot data, proportions of peripheral cd14+cd16+ monocytes were downregulated during the first week after the kidney transplantation while the percentage of cd14+cd163+ monocytes dramatically increased early after the kidney transplantation and remained high for at least four months in most patients. the expression of cd206 (marker of m2 macrophages) was limited only to a small population of monocytes (less than 5% in most patients) but the receiver operating characteristic (roc) curve analysis showed its potential importance by significant correlation with acute rejection with a sensitivity of 70% and specificity of 80.33% (area under the roc curve 0.7787, p-value: 0.004973). no correlation between two different m2 markers cd163 and cd206 has been found. the expression of cd209 (dc-sign) was low and did not show any changes in time or association with acute rejection. hla-dr (mhc ii) and cd47 (integrin associated protein) were constitutively expressed without any significant changes in patients with acute rejection of the allograft. we assume from our data that kidney allograft transplantation is associated with early reciprocal modulation of monocyte subpopulations (cd14+cd16+ and cd14+cd163+). a decreased proportion of cd206 positive blood monocytes seems to be associated with an increased risk of acute rejection of kidney allograft. introduction: thioredoxin (trx), a 12-kda oxidoreductase enzyme, is well known to be a redox-active protein that regulates reactive oxidative metabolism. in addition to its anti-oxidative activ0570 | progranulin-dependent regulation of th2 airway inflammation by house dust mite allergen introduction: progranulin is a growth factor that consists of 593 amino acids including 71/2 repeats of cysteine-rich motifs, and produced by variety kinds of cell. progranulin is involved in the regulation and maintenance of inflammatory response, and its role is wellstudied in neuronal and metabolic diseases such as neurodegeneration and type 2 diabetes. however, the role of progranulin during the development of airway inflammation induced by inhaled allergen is still obscure. objectives: in this study, we evaluated the role of progranulin in the development of th2 airway inflammation induced by house dust mite allergen. results: to find the main source of progranulin, we stimulated each cell line with various doses of house dust mite allergens. the production of each cytokine, including progranulin, was estimated in culture supernatant by elisa. to investigate the role of progranulin in airway inflammation, we intranasally administrated house dust mite allergens to 6-week-old female progranulin knock-out mice (macrophage-specific) or littermate mice. lung inflammation and immunological parameters were evaluated at 15 h after first sensitization with allergen or 24 h after final allergen challenge. the production of progranulin was significantly elevated by house dust mite allergen stimulation in innate immune cells, especially in alveolar macrophages over other cells. in the house dust mite allergeninduced airway inflammation model, we found that the level of progranulin increased earlier than other pro-inflammatory cytokines. in addition, in macrophage-specific progranulin knock-out mice, airway inflammation was down-regulated in the earlier phase after exposure to house dust mite allergen. moreover, we stimulated mice with house dust mite allergen for a longer period to observe the changes in the adaptive immune response of th2 airway inflammation, which was found to be decreased in conditional knock-out mice. conclusions: these findings indicate that th2 airway inflammation induced by house dust mite allergen is dependent on progranulin. objectives: the aim of the present study was therefore to investigate the immunomodulatory effect of epinephrine on m2a macrophages and its consequence on cross talk to mast cells in a human model of allergic inflammation. results: primary monocytes from healthy pbmcs were first differentiated into m2a macrophages using m-csf in the presence of il-4 and il-13 cytokines. after overnight incubation with epinephrine, supernatants were collected and analyzed by elisa for il-10, tnf, il-6, ccl1, il-12 and ifn-c, whereas cell surface markers including cd206, cd163 and cd86 were evaluated using flow cytometry. subsequently, both m2a and epinephrine-treated m2a supernatants were transferred onto cord blood-derived mast cells (cbmcs) for further overnight incubation, after which ige-mediated degranulation was assessed by the ß-hexosaminidase release assay. after overnight epinephrine treatment, m2a macrophages showed an increase in il-10, ccl1, tnf and il-6 production, but no ifn-c and il-12 expression was observed. epinephrine treatment also downregulated surface markers cd206 and cd163 and upregulated cd86. when supernatants from epinephrine-treated m2a macrophages were added to cbmc cultures, ige-mediated degranulation was impaired compared to cbmcs treated with supernatants of unstimulated m2a macrophages. conclusions: taken together, epinephrine promoted a phenotypic shift of m2a polarized human macrophages toward an m2b-like regulatory phenotype that was able to reduce the ige-mediated degranulation of cbmcs. we conclude that prolonged acute stress exposure in allergic patients may attenuate symptoms of acute allergy by directing macrophages towards an immunosuppressive phenotype, which can further dampen mast cell degranulation. objectives: a murine local lymph node assay was used to investigate the effect of oa on the immune response to the known skin sensitizer hexyl cinnamic aldehyde (hca, 25% w/v). the ear lobes tape stripped prior to immunization. test solutions (25 ll) were applied on the dorsal side of each ear on three consecutive days. female balb/c mice (8 groups a 6 mice), were exposed to the vehicle acetone:olive oil (4:1) alone, or in combination with hca, with or without oa in the concentrations 5, 10 and 20%, or oa alone (5, 10 and 20%). on day 5, the animals were weighed and exsanguinated by cardiac puncture. the auricular lymph nodes were harvested for single cell preparation, stimulation with cona and cytokine release of il-2 and il-17. the earlobes were excised and fixed for immunohistochemistry. results: no group differences were found for bodyweights or bodyweight change, number of lymph node cells or il-17 secretion. il-2 showed a tendency of dose-related increase, but a significant difference were only found between hca and hca+10% oa (p=.034) in one out of the two experiments. in he stained sections, the epidermal thickness was significantly increased in groups given hca + 10 and 20% oa (p≤0.001). sections immunostained with anti-ly6g showed significant increase in neutrophil influx for the same groups as above (p≤0.001). oa alone showed no effects or effects significantly lower than hca + oa. objectives: we hypothesized that plasma s1p levels in cf patients might be associated with cftr mutations and could influence disease presentation. results: plasma was collected with a defined standard operation procedure to impede unspecific s1p release from blood cells from 20 double lung transplanted adult cf patients as well as 20 sex-and age-matched, non-allergic healthy controls all being fasted overnight. total plasma s1p was measured by mass spectrometry and unbound plasma s1p by elisa. levels were correlated with cftr mutation status, routine laboratory parameters and clinical symptoms. we observed higher total and unbound plasma s1p levels in healthy controls compared to cf patients with the latter value reaching statistical significance (p=.044) after exclusion of two statistical outliers. unbound plasma s1p levels were significantly higher in df508 homozygous cf patients compared to patients with df508 heterozygosity (p=.029). 2 patients with other mutations were excluded. levels of unbound s1p were positively correlated with hemoglobin and negatively correlated with triglyceride levels. additionally, we observed a positive correlation of total plasma s1p levels in cf patients with hba1c. gastrointestinal symptoms were more common in df508 heterozygous (6/8) compared to df508 homozygous cf patients (4/10). fecal calprotectin levels were found to be significantly higher in df508 homozygous compared to heterozygous cf patients (p=.047). differences in unbound s1p levels were not correlated with immunosuppressive treatment after transplantation. conclusions: to the best of our knowledge this is the first clinical study directly correlating plasma s1p levels with cf genotypes and clinical presentation in cf patients. we emphasize to evaluate s1p as a potential novel disease biomarker as well as a therapeutic target for cf in future studies. supported by the austria science fund grant kli 284 (to eu). ochoa-grull on j 1 ; tejera-alhambra m 2 ; guevara k 1 ; guzm an-fulgencio m 2 ; benavente cm 3 ; mart ınez r 3 ; p erez c 3 ; peña a 3 ; rodr ıguez de la peña a 1 ; llano hern andez k 1 ; rodr ıguez-fr ıas e 1 ; s anchez-ram on s 1 objectives: we show preliminary data of one study aimed to evaluate the use of this strategy in the prevention of rrti in infants and preschool children. results: patients: 121 children (70 male and 51 female, age range 6-35 months) were included in a randomized double blind and placebo-controlled study (eudract 2012-002450-24) . all of them showed negative in vivo and in vitro allergy tests. active treatment consisted in a suspension of a mixture of selected strains, grown and inactivated in optimal conditions, of s. aureus (15%), s. epidermidis (15%), s. pneumoniae (60%), k. pneumoniae (4%), m. catarrhalis (3%) and h. influenzae (3%) in physiologic saline solution with 50% glycerol at a concentration of 300 formazin turbidity units (ftu)/ml (equivalent to 10 9 bacteria/ml). placebo did not contain any bacteria. patient were treated for a period of 6 months, receiving 2 daily sublingual puffs of active or placebo and with a follow-up of other 6 months (total period of evaluation was 1 year). symptom (cough, dyspnea, wheeze, mucus, fever, discomfort) and medication (inhaled corticosteroids, beta adrenergics, montelukast, antibiotics) scores were evaluated since the first day of treatment to the end of the study (1 year) . any adverse event was recorded to assess safety. for the comparison between both groups, t test was used. patients who received active treatment experienced an improvement of 39% over placebo in overall symptoms and 38% in medications scores (p<.01). in the combination of symptoms and medication scores the improvement was 38% (p<.0001). no adverse events related to treatment were recorded. conclusions: immunostimulation with these selected strains of bacteria is safe and can be successfully used in infants and preschool children in order to prevent rrti. introduction: to reduce the duration and the risk of the allergen specific immunotherapy using commonly used allergen extracts, new highly immunogenic and non reactogenic vaccines are needed. objectives: the goal of the present study was to employ the ap205 spytag/catcher system to develop a virus-like particle (vlp) vaccine based on the major house dust mite (hdm) allergen der p 2 and to evaluate its reactogenicity in vitro. spycatcher-ap205 vlps and recombinant der p 2, fused at the c-terminus to the 13 amino acid spytag binding-partner, were expressed in e. coli. purified spytagged der p 2 was mixed with spycatcher-vlps, which resulted in covalent conjugation of der p 2 to the surface of spycatcher-vlps. excess unbound der p 2 was removed by dialysis. dynamic lightscattering (dls) was used to analyse the size and aggregation state of vlp-der p 2. the ige reactivity of vlp-der p 2 was assayed by direct elisa and by rat basophil degranulation assays. conclusions: our results demonstrate that coupling of spy-tagged der p 2 to ap205 spycatcher-vlps dramatically reduces the reactogenicity of the allergen, suggesting that vaccination with ap205 vlp-der p 2 may be a safe and effective treatment for hdm allergy. objectives: the qm1s hybrid protein is a qm1 variant where cysteine amino acids have been replaced by serine. the expression of qm1s hybrid protein was performed in e. coli bl21(de3) after iptg induction. the purification of qm1s protein was performed from inclusion bodies by a three-step chromatography process. the stability of qm1s was analyzed by sds-page and total protein assay. qm1s ige-binding capacity was compared with natural der p 1 and der p 2 by elisa-inhibition and allergenicity was studied by mediator release from rbl cells. immunogenicity was evaluated in mice by analysis of the specific igg response to der p 1 and der p 2. results: qm1s was expressed in complex media as inclusion bodies that were solubilized in urea. soluble protein was purified by anionic ion exchange, hydrophobic interaction, and size exclusion chromatography in the presence of a detergent. qm1s purification process was shorter and more efficient than that of qm1. the purity obtained was >95%. elisa inhibition assay showed that qm1s hybrid protein was almost unable to inhibit ige-binding to the hdm extract, less than 10% in all the range of concentrations tested (0.1-10 000 ng/ml) and representing a 40-fold reduction as compared to qm1. qm1s showed a great reduction of the b-hexosaminidase release in rbl cells, compared to der p 1 and der p 2. qm1s was able to induce der p 1-and der p 2-specific lgg antibodies responses comparable with those induced by the mixture of wildtype allergens. mouse igg antibodies induced by the hybrid proteins qm1s and qm1 showed similar ige-blocking antibodies properties to mixture of der p 1 and der p 2. the stability of qm1s was studied in solution at 25°c, 4°c, and -20°c and lyophilized at 4°c, being the frozen and lyophilized forms the best conservation conditions. the qm1s hybrid exhibited less ige-binding activity than qm1 and the natural der p 1 and der p 2 while retained the immunogenicity. these properties together with the improved manufacturability made qm1s a good candidate for sit to hdm allergy. objectives: slit tablets of cockroach, slit tablets of parthenium, slit tablets containing both allergens together (mix) and slit bilayer tablets containing one layer with parthenium allergen and other layer with cockroach allergen, compress to single tablet were prepared. punches and dies of 11 mm were used for compression. slit drops containing cockroach, slit drops of parthenium and slit drops containing both allergens together, were prepared and filled in 10 ml amber colored dropper vials. results: tablet formulations were evaluated for thickness (3.5-3.6 mm), weight variation (357-402 mg), hardness (2.0-2.5 kg/cm 2 ), disintegration time (not more than 2 min.), and biologically active content (90%-110% of the stated label claim). in-vitro dissolution test was performed as per usp using distilled water as the medium and the release was shown between 70 to 80% in 5 minutes. the liquid formulations were analyzed for ph (7.0 -7.5), the biological content (90% -110% of the label claim), specific gravity ( introduction: virtually all patients suffering from the common birch pollen allergy exhibit ige against the bet v 1 relevant allergen. as such, an elisa method for the quantification of bet v 1 was selected and validated as part of the bsp090 project, aiming to establish reference methods for the european pharmacopoeia. herein, we report the mapping of the epitopes recognized on recombinant bet v 1 allergen by the two specific murine monoclonal antibodies (mabs) used for the accurate and precise quantification of bet v 1 within birch pollen extracts. objectives: in order to investigate the ability of mabs 5b4 and 6h4 to recognize various bet v 1 isoallergens, we first carried out immunochromatography combined with electrophoresis. epitope mapping was performed by hydrogen/deuterium exchange (hdx) coupled with mass spectrometry (ms) analysis, using a gmp-grade purified rbet v 1 molecule. results: immunochromatography unveiled that both mab 5b4 and mab 6h4 capture most of bet v 1 isoallergens present within birch pollen natural extracts. those two mabs cross-react with the aln g 1 allergen from alder pollen but do not react with the cas s 1 chestnut pollen allergen. hdx-ms experiments combined with site-directed mutagenesis evidenced that mabs 5b4 and 6h4 target two distinct epitopes. the hdx-defined 5b4 epitope is discontinuous and contains a dominating sequential element (i.e., loop ile56-lys68). the hdx-defined 6h4 epitope is also discontinuous and mainly composed of regions ile44-lys55 and arg70-phe79. conclusions: overall, this study provides a precise molecular characterization of epitopes within bet v 1 recognized by mabs 5b4 and 6h4, confirming that these two antibodies target distinct non-overlapping epitopes and recognize the vast majority of currently introduction: short or common ragweed (ambrosia artemisiifolia), belonging to the aster plant family, sheds enormous amounts of highly allergenic pollen late in the summer. due to its high allergenic potential ambrosia artemisiifolia is becoming a health threat in north america and europe. hal allergy is developing a subcutaneous immunotherapy for patients suffering from ragweed pollen allergy. a standardized ragweed pollen extract, chemically modified and adsorbed to aluminium hydroxide (al(oh) 3 ), is being investigated for its potential use in immunotherapy. objectives: ragweed extract (re) was modified by glutaraldehyde followed by adsorption to al(oh 3 ). in vitro, a mediator release assay (mra) using hurbl (humanized rat basophil leukemia) cells was performed. hurbl cells were pre-sensitized using individual sera of ragweed-allergic patients and challenged with serial dilutions of re and modified re starting at 10 lg/ml followed by eight 1/10 dilutions (0-10.000 ng/ml). antigen-specific release of ß-hexosaminidase was measured and calculated in relation to 100% release values. in vivo, the immunogenic potency of modified re was evaluated by measuring the induction of re-specific igg in mice. female balb/c mice (n=10 per group) were subcutaneously (s.c.) injected with 20.000 aueq/ml re or modified re adsorbed to al(oh) 3 (0.8 mg/ml) per mouse on days 0, 7, 14 and 21. control mice (n=6) were injected with matrix only. specific igg titres were determined in serum obtained at days -1, 13 and 28. results: the potency of modified re in mra was drastically reduced in all patients, with a mean reduction of 1000 fold or more. chemical modification resulted in a later onset of activation as well as a lowering of the maximum release of ß-hexosaminidase. in vivo, both re and modified re show comparable levels of re-specific igg antibodies in mice at day 28 of the immunogenicity model. shown that chemical modification impairs the capacity of re to activate basophils while retaining its capability to be immunogenic. therefore, chemical modification of re may be a promising approach for the development of a safe and effective immunotherapy for ragweed allergy. objectives: the children who had taken subcutaneous conventional venom immunotherapy in our pediatric allergy outpatient clinic between 2002 and 2015 were evaluated with respect to the side effects. in addition, each child was called to ask if the patient was exposed to bee sting and the result of a sting during immunotherapy. introduction: the major unmet needs for allergen immunotherapy (ait) are improved efficacy with good tolerability, and high adherence. to achieve these, allergoids, peptides and recombinant proteins are potentially the answer but their low rate of systemic aes make selection of the optimal dose difficult. to select the dose for an ultra-short course subcutaneous birch ait, the company has adopted the use of a conjunctival provocation test (cpt), a wide range of doses and the multiple comparison procedure -modelling (mcp-mod) statistical analysis to test for a dose response and to determine the shape of the dose response curve. objectives: a range of dose regimens of 5100 su, 15300, 20100 and 27300 su were compared with placebo with respect to reduction of total symptom score elicited by cpt after treatment. patients were administered 6 weekly injections outside the pollen season. cpt was performed at screening, at baseline and 4 weeks after completion of treatment. the study was undertaken in 28 sites in germany and austria with 370 patients. the primary efficacy analysis was performed on a modified full analysis set (fas). the mcp-mod methodology was used to test for a dose-response using the placebo and above doses to describe the shape of the dose response curve. three candidate models were pre-specified: a maximum possible effect for the agonist (emax) model, a logistic model, and a linear in log-dose model. a statistically significant dose-response (p<.01) was shown for the range of cumulative doses, which approached a plateau with the 27300 su dose. the median effective dose (ed-50) was 2600 su. only minor differences were observed between the six active treatment groups in prevalence of treatment-emergent adverse events (between 70.9% and 83.9% of patients with overlapping 95% two-sided confidence intervals), majority of which were local reactions, short-lived and mild. teaes classified as systemic reactions were seen in 2.0% (20100 su group) and in up to 15.1% (5000 su group) of patients in the active treatment groups, and in 11.3% of patients in the placebo group. no treatment related saes were observed. adherence was >90% in all treatment arms. the ed-50 was 2600 su, demonstrating that the currently marketed dose (5100 su) is effective. the highest 27300 su dose will be further investigated in a pivotal phase iii trial having achieved an increase in efficacy by 50% without differences in the onset of aes between the treatment arms. introduction: in order for allergen immunotherapy (ait) to induce long-term immunological and clinical effects prolonged administration is required. therefore adherence to treatment is crucial for its efficacy. there is currently limited data available on ait adherence beyond clinical trials i.e. in real-life clinical practice. objectives: this eaaci immunotherapy interest group endorsed survey aimed to prospectively evaluate adherence to sublingual and subcutaneous immunotherapy in adults with allergic respiratory diseases and hymenoptera venom allergy in real life practice across different european countries. in addition, the reasons for lack of adherence and discontinuation of treatment were explored. this was a prospective, multi-centre, observational survey which took place in eight countries: czech republic, georgia, germany, greece, italy, poland, portugal and spain. data collection involved an online survey that followed participants four-monthly for a period of 36 months from the start date of ait. results: a total of 1336 participants were included in the analysis. introduction: allergic rhinitis is a multiple gene-regulated disease involved in many immune cells such as mast cells and eosinophils, and various inflammatory mediators, and mirna probably plays a critical regulatory role in its pathogenesis. therefore, studies on the functions of critical mirna and its regulatory mechanisms in activated mast cells will lay an important theoretical foundation for our understanding of ar pathogenesis and the development of therapeutic strategies. objectives: to investigate the effect of mir-125a-3p on mast cell activation in an ar mouse model. the number of sneezes and the frequency of nasal rubbing in ar+mir-125a-3p group were significantly reduced compared to those for ar+mir-nc group (p<.05). histological examination showed that inflammation in the nasal mucosa from ar+mir-125a-3p group was slighter than that in ar+mir-nc group. the number of mast cells in ar+mir-125a-3p group was increased compared to ar+mir-nc group (p<.05). the levels of histamine and il-13 in nasal lavage fluid supernatants, histamine in plasmas and il-13 in sera were significantly decreased in ar+mir-125a-3p group compared to ar+mir-nc group (p<.05). conclusions: upregulation of mir-125a-3p can reduce allergic inflammation in the nasal mucosa of ar and alleviate ar symptoms through inhibiting mast cell activation in vivo. mir-125a-3p probably becomes a new target for gene therapy of ar. 0222 | correlation between chronic cough and chronic rhinosinusitis in adults: nationwide, population-based, and cross-sectional study the second hospital of shandong university, ji nan, china; 2 national university of singapore, singapore, singapore introduction: nasal polyp implies a refractory clinical course in case of chronic rhinosinusitis (crs). although hypoxia is believed to be associated with nasal polyposis, little is known about the mechanism underlying polypogenesis. objectives: the aim of this study was to assess mrna and protein introduction: nasal polyp is a multi-factorial disease commonly associated with inactive ciliary beating frequency (cbf), a condition partly attributed to the mis-localization of dnah5, a component of the outer dynein arm in ciliary axoneme. so far however, there have yet to be a systematic histopathological investigation directly linking dnah5 localization pattern in nasal polyps. therefore, we sought to examine the localization of dnah5 in cilia structure of both nasal polyps and inferior turbinates from healthy individuals, and assess whether there are any localization changes that can account for the extensive inactive cbf observed in nasal polyps. objectives: the focus of this work is to compare the localization of dnah5 from the nasal polyps biopsies (n=80) and normal inferior turbinates (n=19) by immunofluorescence. the characterization of each sample was obtained from an average of 10 fields at 4009 magnification. results: from the samples, we observed three distinct localization patterns of dnah5 in the nasal cilia. the three patterns were as follows: 1) the localization of dnah5 in normal cilia is present throughout the entire axoneme (pattern a); 2) the localization of dnah5 is within the axoneme except at their proximal regions (pattern b); 3) the localization of dnah5 is restricted exclusively at the ciliary base and not present in the entire axoneme (pattern c). approximately 96% of the samples exhibited more than one distinct localization patterns for dnah5 within the observed fields. the percentage of pattern a, pattern b and pattern c were observed in 36.5%, 24.7%, and 38.8% fields for samples from nasal polyps. correspondingly, 75.1%, 19.3%, and 5.6% were observed for samples from healthy controls. the results indicated that the predominance of "pattern c" in nasal polyps countered by "pattern a" in inferior turbinates from healthy individuals. conclusions: our study indicated that there is a significant increase in the mis-localization of dnah5 among the cilia in nasal polyps as compared to controls. this mis-localization may account for the inactive cbf, a hallmark characteristic, observed in nasal polyps. zhao l 1 ; zhi l 2 ; jin p 1 ; zi x 1 ; tu y 1 ; li a 1 ; li t 1 ; shi l 1 (3.64, 2.28-5.01, p<.05) and +gc np patients (10.39, 6.00-14.77, p<.05 the results showed that the number of th17 + cells were correlated with eosinophil cells and macrophage (r=.3210, p<.05, r=.3269, p <.05), but no correlation was found between th17 + cells and neutrophil cells. the significantly correlation were found between ilobjectives: this study aimed to reveal if some features of the sinus wall and content (as homogeneity and density of the sinus content, or the continuity, thickness and density of the sinus wall), differ between the afrs and other forms of crs. we tried also to establish early diagnostic parameters for recognition of fungal rhinosinusitis on ct. results: the study included 36 adult patients (mean age: 45.58ae14.29 years, m:f ratio=2.1:1) with clinically diagnosed crs. out of all maxillary sinuses (n=72) from study patients, 62 (86.11%) were opacified, and only these sinuses were included in further analyses. we found out that: (1) positive fungal finding had 33% (12/36) crs patients and 86% (31/36) of these patients had severe forms of crs, (2) patients with positive fungal finding had more often positive specific ige ab than those without fungi in sinuses (43.9% vs. 21.3%, p=.027), (3) foci of non-homogeneity, mean and maximum densities and wall density were more common found in maxillary sinuses with present fungi than those without fungi (p=.037, p=.05, p=.05; respectively) and (4) patients with crs lasting more than 10 years had more often foci of non-homogeneity and presence of hyperattenuation centres, than patients with shorter length of crs. results: neutrophil-related gene mpo and eosinophil recruitment genes ccl13 and ccl18 showed higher expression level in acp than in controls, which were in line with the significantly elevated neutrophils and eosinophils infiltration in acp compared to control. increased cd8 + t cells, macrophages and cd4 + t cells infiltration in acp were also observed. the expression level of t-reg transcription factor foxp3 was significantly higher in patients with acp than in controls, but the expression of th1/th2/th17 transcription factor tbet, gata3 and rorc were significantly decreased in acp vs controls. we further investigated the relationships between these t-cellassociated genes in acp. the expression of foxp3 was positively correlated with t-bet, gata3, il17r and il12a, while no significant correlation with rorc was evident. il6 was observed positively correlated with t-bet, gata3, foxp3, and il17r. il10 had significant correlation with t-bet and il12a. objectives: the aim of this study was to find the olfactory change pattern of crs after ess in short-term and the differences between crswnps and crssnps, secondary aim were to identify the relationship among olfactory dysfunction, ct scores and quality of life(qol). in this study, 48 crs patients who underwent ess were evaluated preoperative by t&t recognition threshold tests, snot-20 score and lund-mackay ct score. patient outcomes were re-evaluated at clinical follow-up 1 month, 3 months and 6 months postoperative. analysis of variance was performed and correlation was calculated, with results analysed separately for crswnp and crssnp subgroups. 1. subgroups of crs differed in the degree of olfactory dysfunction reported before and after the ess. a significant difference in the changes of olfactory dysfunction between the two groups was found at 6 month postoperative. 2. the mean t&t and snot-20 scores showed significant improvement within 6 months after ess in both crswnp and crssnp subgroups, however, no significantly recovery of olfactory dysfunction was observed at 3 months compared to 1 month postoperative. there is a plateau of olfactory recovery at 3 months postoperative. 3. in crswnps, the mean t&t scores preoperative were correlated with lund-mackay ct score significantly(r=.649, p<.001; r=.625, p<.001). however, no relations were found in crssnps and the changes of olfactory dysfunction at the 6 months postoperative with lund-mackay ct score. 4. olfactory scores, before and after the ess, and their changes did not correlate with sont-20 scores. conclusions: olfactory dysfunction was more severe in crswnps. olfaction and qol of crs patients were significantly improved after ess in both groups, but there was a plateau of olfactory recovery at 3 months postoperative. ct scan may predict olfactory disorder, but the olfactory scores were not related with the qol. objectives: in this study, we investigated the effect of hgf, tgf-b1, and pge 2 as effective components for allergic rhinitis treatment using in vitro and in vivo mouse model studies. results: pge 2 decreased infiltration of eosinophil in nasal mucosa. tgf-b1 decreased the infiltration of eosinophil in nasal tissue and increased the number of treg in spleen. however, there was no antiallergic effect of hgf in this experiment condition. in case of the combination treatment group (tgf-b1+pge 2 +hgf), eosinophil infiltrations and the expression of eotaxin-2 were reduced in the nasal tissue, and treg was increased in the spleen. in all treatment group, serum ige and systemic cytokine levels were not decreased due to intranasal administration rather than systemic administration. in vitro study showed that phosphorylation of map kinases such as erk, jnk, and p38 and translocation of p65 were inhibited after treatment of hgf, tgf-b1 and pge 2 , suggesting their anti-allergic mechanism. conclusions: we found that tgf-b1, and pge 2 decreased allergic inflammation and these effects might be derived from changes in the frequency of treg and the activation of map kinase and p65 in the t cell receptor signaling pathway. furthermore, we hypothesized that tgf-b1, and pge 2 would be effective components for allergic rhinitis therapy. introduction: interleukin (il)-10 is implicated in suppression of allergic inflammation. the role of il-10 in the early-phase reaction in type 1 hypersensitivity has been unclear, however. we investigated the contribution of il-10 in a mouse model of the ige-mediated early-phase reaction in allergic conjunctivitis. objectives: ige-mediated allergic conjunctivitis was induced in c57bl/6-kit(+/+) wild-type mice, kit(+/+) il-10-deficient mice, and kit(w-sh/w-sh) mast cell-deficient mice by means of passive conjunctival anaphylaxis. the mice were thus subjected to subconjunctival injection with anti-dinitrophenol ige (dnp-ige) followed after 24 h by intravenous injection with dnp antigen. kit(w-sh/w-sh) mice that had received a subconjunctival graft of cultured bone marrowderived mast cells from kit(+/+) wild-type mice or kit(+/+) il-10deficient mice were similarly treated. vascular permeability of the conjunctiva was examined 30 min after antigen injection by colorimetric evaluation of the extravasation of evans blue dye. results: passive transfer of dnp-ige followed by intravenous antigen injection increased vascular permeability in the conjunctiva of kit(+/+) wild-type mice but not in that of kit(w-sh/w-sh) mice, suggesting that this effect was dependent on mast cells. vascular permeability was increased to a significantly greater extent in kit(+/+) il-10-deficient mice than in kit(+/+) wild-type mice. reconstitution of kit(w-sh/w-sh) mice with kit(+/+) wild-type or kit(+/+) il-10deficient mast cells restored the dnp-ige-and dnp-induced increase in vascular permeability to similar extents. our results suggest that il-10 produced by cells other that mast cells suppresses the mast cell-mediated early-phase reaction in ocular allergy. objectives: our aim was to evaluate the effectiveness and the safety of ccl treatment for keratoconus in children with vkc. forty-two boys (mean age 13.5ae3.3 years) and 17 girls (mean age 11.8ae4 years) with vkc were included in the study. tarsal, limbal and mixed vkc were detected in 55.9%, 8.5% and 35.6% of the subjects, respectively. evaporative dry eye was detected in 23 children out of 43 (53.5%), schirmer test results were <10 mm/ 5 minutes in 20.5% and <5 mm (severe dry eye) in 4 out of 44 children (9.1%) and 39% of the subjects (n=23) had confirmed keratoconus/forme fruste keratoconus with corneal topography (sirius, cso, italy). allergic symptoms were controlled with topical steroids, cyclosporine, dual action antihistaminic/mast cell stabilizers and lubricant agents before the procedure. ccl surgery was performed under topical anesthesia. the children were followed-up at least 1 year and preoperative and postoperative corneal topographic parameters were compared using paired sample t test. results: the visual acuity was between 0.4 and 0.6 (moderate visual loss) in 16.9% of the subjects and less than 0.3 (severe visual loss) in 6.8% of the children. ccl procedure was performed to 19 eyes of 15 children. at the end of one year, the disease was stable in all children with no differences in k1 and k2 corneal parameters before and after cxl (p>.05). there was a statistically significant improvement in maximum keratometry value after the procedure (before 56.57ae4d, after 55.37ae3.5d, p=.005). in one subject, a corneal infiltrate was detected 3 days after ccl, which was treated successfully with topical moxifloxacin. otherwise, no complications were observed in the postoperative period. conclusions: as keratoconus is common in vkc, these children should be referred to ophthalmologists for an eye examination and corneal topography. ccl seems to be a safe and effective option to halt the progression of keratoconus, which might be very aggressive in children with vkc. results: surprisingly, we found among them 11 cases of celiac disease, 7 cases of thyroid dysfunction (thyroiditis), 3 cases of crohn's disease and 1 case of ulcerative colitis, 1 case of anterior uveitis and 1 case of pemphigus respectively. we realized that an average percentage of 7% of the total of vkc cases are affected by an "autoimmune" systemic disease. limbal form of vkc was prevalent and more than 70% of children showed it. we can suppose that a racial and genetic predisposition to systemic diseases can coexist with vkc or that there is a group of vkc affected subjects in which the immune disorder is predominant on the allergic disease. introduction: nasal polyposis (np) is a heterogeneous inflammatory disease of nasal mucosa affecting 1-4% of the population with a high rate of recidivism. polyps arise from nasal sinuses to nasal cavity and are often associated with a strong local eosinophilia. the pathophysiology of np remains controversial, as it seems to be a phenotypic manifestation of multiple possible immunologic processes, such as respiratory allergy, despite a lack of correlated systemic response. here we propose a multiparametric assessment of np patients, in order to shed light on the underlying mechanisms of the disease in allergic and non-allergic patients and aiming to find new predictive biomarkers. objectives: our main aim was to unravel the link between systemic and local allergic inflammation and polyp development, as well as the nasal epithelium condition in polyps and surrounding healthy tissue. methods: four groups of patients were included in the study: healthy donors with or without allergy and np patients with or without allergy. in this regard, several different approaches were followed: a metabolomics serum study, a polyp and nonpolypous nasal epithelium histology and transcriptomic study. results: as for the histological study, luna staining revealed differences in eosinophilia between allergic and non-allergic patients, especially when patients were polysensitized, including perennial allergens; and between nonpolypus tissue and polyps being higher in polyps. pas staining showed differences in epithelium integrity and submucous and goblet cell (pas positive) distribution. immunohistochemistry for cd4+ and cd11c+ reveal a significant inflammatory infiltration in polyps. this inflammatory response was also asses by abstracts | 209 gene expression quantitation. no differences were seen in the metabolic profile in patient sera between groups. for the first time, nasal epithelium from polyps and neighboring tissue were studied. histology techniques and image analysis revealed differences in eosinophil concentration in both mucosa and submucosa areas, as well as different features in epithelium and submucous tissue structure. some of these findings were confirmed by gene expression quantitation. conclusions: our data show an increased eosinophilia and inflammatory infiltration in polyp tissue suggesting a role for allergic inflammation in the progression of np. additionally, we provide clues for the role of inflammation in the damage on nasal mucosa and the following progression of the disease. 0235 | is specific immunotherapy effective in subjects suffering from vkc? a tertiary referral center ten years experience. objectives: our work shows the results of sit additional to usual treatments, in children suffering from vkc and followed in our tertiary referral center (lavagna hospital, genova, italy). we retrospectively analyzed the clinical data of 37 subjects (25 males and 12 females); their mean age at the beginning of treatment was 8 ae 1.2 years. the patients were treated both with scit (sub-cutaneous immune-therapy-56.7%) and slit (sub-lingual immune-therapy -43.3%) depending on patient's wishes. they had to be mono-sensitized to one of the usually more frequent allergens (dust-mites, grass pollen, and pellitory) which was detected by means of recombinant rast, prick test and conjunctival provocation test (cpt); these tests were performed after a complete ophthalmological and allergological history and examination. children selected for sit needed to be positive to all performed allergy tests. systemic involvement included 25 cases of asthma, 5 cases of atopic dermatitis and 1 case of rhinitis; the remaining 6 cases were asymptomatic. local involvement included only vkc cases, the 62% of which were of the limbal type and only 14 subjects were suffering from the tarsal papillary type. mean follow-up was 7.2 years. all the patients included into the study completed their treatment and followed the therapeutic protocol. after one year of sit, no variation in clinical course and treatment was recorded. after the third year of sit, an average improvement in symptoms and signs score (43%) and an average decreased need for allergy systemic medications (63%.) (i.e. antihistamines and corticosteroids) was registered. also topical therapy (including steroid and cyclosporine eye-drops) was discontinued in 43% of children, in this group, short courses of steroid drops were necessary in less than 30% of children (as rescue treatment in the acute phases of the disease). these positive results after sit treatment were stable for the following 5 years. few (only local sub-cutaneous) side effects were recorded and the treatment was generally well-tolerated. conclusions: our experience shows positive results with sit in vkc which can have sensitive-to sit-treatment subtypes. results: 39 deaths occurred in children (21 boys, 54%). median age at death was 11 years (iqr 2-15). pamr of any cause was 0.08 (95%ci, 0.05-0.10) per 10 6 children per year, with a decreasing rate over time (annual change: -2.5%; 95%ci, -5.6 to 0.9). triggers were iatrogenic causes (n=18, 46%), insect venom (n=3, 8%) and food (n=2, 5%). unspecified causes were frequently reported (n=16, 41%). there was no difference in overall pamr between boys and girls (p=.74). there was no age group related differences in fatalities: preschool children (<7 years) (n=14, 36%), school children (7-12 years) (n=12, 31%), adolescent and toddlers (>12 years) (n=13, 39%). the number of fatal cases was similar comparing the southern (n=16, 41%) and the northern regions of france (n=23, 59%) (p=.15). the first episode of anaphylaxis for each patient was captured to calculate incidence. we estimated incidence rate ratios using poisson regression models. results: between 2001 and 2015 there were 523 anaphylaxis episodes in 481 patients younger than 18 years in hong kong. the incidence of admission for anaphylaxis increased markedly from 2.46 to 6.63 per 100 000 person-years during the study period (p<.001). the incidence of food-related anaphylaxis increased significantly from 0.21 to 1.88 (p<.001). increases in anaphylaxis and food-related anaphylaxis were seen in all age groups, with the largest increase in those aged 0 to 4 years. at the beginning of the study period (2001), medication was a more common trigger for anaphylaxis than food (1.61 vs 0.21 per 100 000 person-years). by 2015, food had become the predominant trigger (1.88 vs 0.54 per 100 000 personyears for medication). the incidence of medication-related anaphylaxis decreased significantly (p<.05). the incidence rate of anaphylaxis was significantly higher in boys than girls in the 5-14 and 15-18 year age groups, while there was no significant gender difference in the 0-4 year age group. the most common food triggers of anaphylaxis were peanuts, seafood, eggs, milk products, tree nuts & seeds (in descending order). conclusions: even though the incidence of anaphylaxis among children in hong kong is lower compared with other western countries, it has recently increased significantly, with food-related anaphylaxis predominant. 0238 | prevalence of anaphylaxis and prescription rates of epinephrine self-injector in korea based on national health insurance data results: the prevalence of anaphylaxis over the 5 years were 0.02%. the annual prevalence of anaphylaxis increased over the 5 years. anaphylaxis was more prevalent in male than female (56% vs. 44%) and in population aged 50-59 years old. for the regional prevalence of anaphylaxis in korea, gangwon province showed the highest prevalence of anaphylaxis (41.5 per 100 000 individuals) and relatively low prescription rates (5.8%) of epinephrine self-injector for the patients with anaphylaxis. on the contrary, seoul showed relatively low prevalence of anaphylaxis (14.3 per 100 000 individuals) and the highest prescription rates (26.6%) of epinephrine self-injector for patients with anaphylaxis conclusions: the prevalence of anaphylaxis has increased annually in korea. the prevalence of anaphylaxis and prescription rates of epinephrine self-injector showed regional difference in korea. objectives: the aim of the study was to analyze the prevalence of allergic symptoms and anaphylaxis in mastocytosis patients analyzed in the registry of the ecnm. results: methods: a total of 1513 patient with mastocytosis were enrolled. in these patients, the prevalence of allergy, anaphylaxis, triggers of allergic reaction, and disease subtypes were analyzed. results: symptoms of allergy were observed in 28% of all patients. the most affected group were patients with bone marrow mastocytosis (bmm: 64.52%) and indolent systemic mastocytosis (ism: 32.78%). insect venom allergy (iva) was reported in 14.14% of all subjects. in ism/bmm patients iva affected 22.30% of the cases, while in other patient groups, only 4.3% of the cases were affected (p<.00001). most patients (64%) had wasp allergy, 18% had bee allergy, 2% polistes allergy, and 6.8% allergy to more than one venom. in 9.2% the culprit insect was not identified. food allergy was reported in 3.56%, drug intolerance/allergy in 4.7%, inhalant allergy in 4.43%, and physical triggers in 0.8% of patients. in mastocytosis patients iva is the most prevalent cause of anaphylactic reactions exceeding the prevalence of iva in the general population by far. iva affects mainly bmm and ism patients (22.3% of cases). but 267 (66.1%) subjects didn't know whether adrenaline was administered. only 6 within 44 patients who had adrenaline autoinjectors used their autoinjectors during an anaphylaxis attack. most common symptoms were skin (n=363, 89.9%) and respiratory symptoms (n=327, 80.9%). syncope, hypotension or hypoxemia were present in 273 cases(63.9%), at least three organ dysfunctions in 258 (63.9%) cases; 56 patients (13.9%) had to be hospitalized (f:36, m:20).nearly a third (26.2%) of the patients had stage 1-2 anaphylaxis and 298 patients(73.8%) had stage 3-4 reactions. in 184 cases (45.6%), basal tryptase levels were examined and the average value was correlated with the severity. concomitant drugs being used by the patients were antihypertensives (20.5%),oral antidiabetics (6.2%); angiotensin converting enzyme inhibitors or angiotensin receptor blockers(11.1%), beta blockers(8.4%), diuretics(4.7%) and nsaid's (7.2%). conclusions: male sex was noted as a risk factor for severe reactions and recurrent anaphylaxis. anaphylaxis requiring hospitalization was more frequent in the patients using oral antidiabetics or diuretics. baseline tryptase levels were higher in patients with neurological and gastrointestinal symptoms. cardiovascular symptoms were found to be higher if a cofactor was present. skin symptoms were seen more frequently and higher rate of hospitalization occurred in anaphylaxis in the presence of infections or nsaid use. this study is important to elucidate the factors affecting anaphylaxis severity. 0241 | serum levels of 9a,11ß-pgf2 in combination with apolipoprotein a1 or cysleukotrienes are reliable biomarkers of anaphylaxis objectives: we analyzed mast cell mediators in sera derived from patients with acute anaphylactic symptoms (n=18) versus patients with acute cardiovascular or febrile reactions (n=12) and patients with a history of anaphylaxis but without displaying any symptoms when sera were taken (n=27). in addition, we identified proteins with substantial changes during anaphylaxis. matched serum samples were used to compare basal mediator levels with corresponding levels during acute anaphylaxis in the same patient (n=9). roc curve analysis was performed to determine the sensitivity/specificity of each mediator. results: serum levels of histamine and tryptase were not increased upon anaphylaxis and showed no relation to anaphylaxis severity. however, serum 9a,11ß-pgf 2 , a metabolite of pgd 2 , was significantly increased in acute anaphylactic patients (~8-fold) and abstracts | 213 correlated well with anaphylaxis severity. 9a,11ß-pgf 2 distinguished anaphylaxis from cardiovascular or febrile reactions and showed the highest diagnostic power observed by roc curve analysis. cys-leukotrienes (cys-lts=ltc 4 , ltd 4 , lte 4 ) were increased upon anaphylaxis while apolipoprotein (apo) a1 was significantly decreased. the highest diagnostic power was observed with the combination of 9a,11ß-pgf 2 and apoa1. conclusions: in conclusion, histamine might only be used to detect anaphylaxis when assessed shortly after onset of an anaphylactic response because of its short half-life, whereas tryptase is a useful biomarker if the baseline level of the same patient is known. 9a,11ß-pgf 2 seems to be the most reliable marker as demonstrated by the distinct increase upon anaphylaxis and could be supported by apoa1 or cys-lts. further investigations are needed to prove the suitability of these markers. objectives: objective of this study was to estimate the long-term bv of tryptase using certain chronic disease models and to compare it with those in food and drug allergy. results: serial determinations of tryptase concentrations (n≥5 data points per patient) obtained from patients diagnosed with mastocytosis (n=10) or chronic urticaria (n=1) during a period of sometimes several years were measured by the immunocap assay and evaluated using sigmaplot software. polynomial curve fitting was performed and data points outside the 95% confidence interval of the curve were appointed as outliers and excluded. because the data points were not normally distributed due to long-term fluctuations in homeostatic set-point, a non-linear fitting was applied and used to compute the standard error of the estimate. these standard errors of the fit divided by the estimates themselves were used to calculate the total coefficient of variation within a subject (cv t ). the analytical cv (cv a ) was calculated based on quality control samples (3 levels, n=149 data points) in a conventional way, while the within-subject bv (cv i ) was defined as cv l =(cv t 2 à cv a 2 ) ½ . eleven patients with a chronic disease were selected, of which 1 patient was treated as a potential outlier and 2 patients had to be excluded because of cv t <cv a . the between-subject bv (cv g ) was without the outlier 3.68ae1.92% (n=85 data points during a period 5.6ae2.3 years) and with the outlier 5.75ae6.46% (n=92 data points during a period of 5.3ae2.4 years). as a food and drug allergy control group 2 additional patients were selected with an uncomprehended food (n=1) or drug allergy (n=1). this cv g was calculated under the same conditions and proved to be 9.61ae1.64% (n=16 data points during a period of 4.1ae1.2 years). the long-term between-subject biological variation of tryptase concentrations of patients in chronic disease models was smaller than that of patients with an uncomprehended food or drug allergy. chronic disease models may be of interest to determine the long-term "physiological" biological variation of tryptase. vilà-nadal g; rodr ıguez-sanz a; s anchez-villanueva r; fiandor-roman a; dom ınguez-ortega j; bell on-heredia t introduction: hypersensitivity reactions during hemodialysis sessions have been reported. our aim was to study the mechanisms of the allergic or "pseudoallergic" reactions to synthetic polysulfone (ps) dialysis membranes in "ps-allergic" patients who tolerated cellulose triacetate (cta) membranes. objectives: ten patients with adverse reactions to ps and 8 control non-allergic patients in hemodialysis were studied. 50 ml of blood were collected and an ex-vivo hd were performed on experimental external circuits with low or high priming volumes and pediatric membranes (fx-paed helixone 0.2 m 2 and sureflux). serum tryptase levels, basophil degranulation (hla-dr à cd123 + cd63 + leukocytes), and t cell activation (cd69 expression on cd4 + and cd8 + subpopulations) were analysed in pre-and post-dialysis samples. objectives: the aim was to investigate the influence of asa and alcohol on egg allergy. donor sera from 4 egg allergic patients (i-iv) with s-ige to ovalbumin at (i) 0.1, (ii) 8.87, (iii) 19.5, and (iv) 170 ku/l were injected intracutaneously to the forearm of 11 healthy/nonallergic volunteers. they were then orally challenged separately with: 1) egg white 2) egg white + asa and 3) egg white + alcohol. 'time to wheal' and 'wheal size' were compared between the three experiments. results: two sera (i-ii) with the lowest s-ige to ovalbumin did not react sufficiently and where excluded from further analysis. the 2 remaining sera (iii-iv) significantly decreased 'time to wheal' with both asa (p=.001) and alcohol (p=.019) added as co-factor compared to baseline. increase in 'wheal size' was only found with co-factors added for the less reactive serum (iii) and not for the serum with the highest s-ige (iv). introduction: eaaci have published numerous guidance documents providing evidence-based recommendations for the recognition, risk factor assessment and management of anaphylaxis. these guidelines clearly advocate intra-muscular adrenaline firstline for the treatment of anaphylaxis, and list absolute indications when an adrenaline auto-injector (aai) must always be prescribed; eg for those who have previously experienced an anaphylactic event, those who have a mast cell disorder and for those with co-existing unstable or moderate/severe asthma and a food allergy. furthermore, eaaci recommends that an aai should be prescribed to those at risk of anaphylaxis (termed relative indications), and that 2 aais should be prescribed and carried by patients at all times. despite widespread dissemination of these guidelines, anaphylaxis hospitalizations continue to rise in many european countries. objectives: in this environment eaaci developed the anaphylaxis survey (axis) as part of its allergy awareness initiative to gain insight into guideline awareness. this survey was sent by email to 8720 eaaci members. 873 members from 89 countries responded. data were anonymous and analysed descriptively. results: 81.4% (n=711) reported familiarly with the eaaci anaphylaxis management guidelines, and of these 96.8% (n=688) reported following them. 97.0% (n=847) responded that they would use adrenaline firstline for the treatment of anaphylaxis (although 9.6% (n=84) would still administer it subcutaneously). however, when confronted with a list of indications, only 41.8% (n=365) of eaaci members would prescribe an aai for all absolute indications. furthermore, although 90.1% (n=787) of respondents considered they would prescribe an aai for those at risk, when provided with a list of relative indications, 0% would prescribe an aai for all of them. there was also a lack of consensus about when an aai should actually be used, with 23.8% of respondents (n=208) considering aai use when individuals experienced breathing difficulties and felt that they were going to collapse. conclusions: these results reflect the latest findings that aais are under-prescribed in daily practice. there appears to be a mismatch between perception of guideline compliance and aai prescription behaviour. continued training, further simplification and wider dissemination of guideline messages are required. objectives: the aim of this study was to analyse the clinical practice of emergency physicians concerning the management of anaphylaxis in alsace and lorraine, a region of 3.5 million inhabitants, in france via an electronic survey. results: the response rate was 18.3% (44). fifty percent of physicians were female. 84.1% (37) (1). in case of grade 2 reactions, intramuscular adrenaline injection was performed by 11.2% (5) of physicians as first-line treatment, intravenous adrenaline injection by 8.4% (4), antihistamines (oral route or iv) by 16% (7) and corticosteroids (oral route or iv) by 17.8% (8). in case of grade 3 reactions, intramuscular adrenaline injection was performed by 13.6% (6) of physicians as first-line treatment, intravenous adrenaline injection by 64.3% (28), iv antihistamines by 6.8% (3) and corticosteroids (oral route or iv) by 16% (7). following an acute anaphylaxis episode, only 2.3% (1) of physicians systematically recommended an allergy assessment, 11.2% (5) sometimes, and 86.4% (38) never. conclusions: this study highlights the discrepancies between current guidelines for the management of anaphylaxis in emergency departments and common clinical practice. our results emphasised the need for specific programs to improve clinical management of anaphylaxis in ed. conclusions: a significant number of individuals among those surveyed reported a severe allergic reaction requiring an eai, yet less than a quarter used an eai. the study also identifies most common challenges faced by at-risk individuals including eai availability, cost, and concerns over short expiry dates. objectives: to evaluate the implementation of a stock epinephrine (stock epi) program in a mall setting. stock epinephrine devices are not prescribed for a particular person and can be used in anaphylactic emergencies. methods: a mixed methods approach was used to evaluate the implementation of the stock epinephrine program regarding its acceptability and adoption by diners, food service, mall administration and security personnel; and the appropriateness, costs (included costs for personnel, epinephrine auto-injector devices, and training materials), fidelity, reach, and sustainability (measured by the national health service (nhs) sustainability model, united kingdom) of the program in this setting (hamilton, ontario, canada). the study revealed that individuals with food allergy and food service establishments were in favour of a stock epi program. a total of 1580 individuals with food allergy completed our national online survey. approximately 42% dine out once or twice per month, and one-third had experienced an allergic reaction. we also completed in-person surveys with 120 diners at the mall food court. of these, 24% had a food allergy and 35% reported carrying their epinephrine auto-injector when dining out. the mean nhs sustainability score was 87 among survey participants, well above the mean considered a sustainable program (>55). total cost of the stock epi program over a one-year period varied by ontarian stakeholders: $2155 for the mall, $987 for fast-food restaurants, and $715 for sitdown restaurants. conclusions: this is the first study to evaluate the implementation of a stock epi program. the stock epi program was well received abstracts | 217 and sustainable. implementing a stock epi program provides enhanced access to emergency medication, however it does not replace the responsibility of individuals with food allergy to self-manage. objectives: to identify the optimal needle length for epinephrine prefilled syringe. results: three hundred seventy-two children aged 1 month to 18 years were enrolled. skin to muscle depth (stmd) and skin to bone depth (stbd), which can represent the minimum and maximum needle length respectively, were measured using an ultrasonography at the mid-anterolateral thigh. number of children who had stbd less than needle length (too long needle) and stmd greater than needle length (too short needle) were calculated. one hundred thirty-seven children weight <15 kg, 80 children weight >15-30 kg, and 155 children weight >30 kg were enrolled: 196 (52.7%) children were male. one inch needle was too long in 38 (27.7%) children weight <15 kg, 1 (1.3%) children weight >15-30 kg. it was too short in 8 (5.2%) children weight >30 kg. age≥3 months, weight≥6 kg, height≥59 cm, bmi≥13 kg/m 2 and thigh circumfer-ence≥23 cm, provided the sensitivity of 97-99% in predicting the appropriateness for using 1 inch needle for children weight <15 kg. in children weight >30 kg, thigh circumference≥48.75 cm provided the sensitivity of 86% and specificity of 75% for predicting the inappropriateness for using 1 inch needle. objectives: we present a patient with a probable mdh syndrome to unusual drugs, including ah and cct. results: we report a case of a 23-years-old female, with history of moderate-persistent asthma and chronic urticaria, who experienced angioedema and exacerbation of urticaria hours after the administration of multiple ah (desloratadine, loratadine, cetirizine), systemic cct (hydrocortisone, methylprednisolone, deflazacort) and nonsteroidal anti-inflammatories (nsaids) (paracetamol, ibuprofen, flurbiprofen). patch tests (pt) with excipients (bial arestegui ® ) and drug provocation test (dpt) with placebo were negative. skin prick tests (spt) and intradermal tests (id) with hydroxyzine, hydrocortisone, methylprednisolone and prednisolone were positive to hydroxyzine (5mg/ml) and pt with corticosteroids (bial arestegui ® ) and hydroxyzine were negative. dpts with desloratadine and an alternative ah, dimetindene, were positive with facial angioedema and generalized urticaria within 5 hours. lymphocytic transformation test (ltt) was positive to desloratadine, ebastine and clemastine. dpt with dexamethasone was negative, however, when administered as treatment, a reproducible reaction occurred. since dpt with montelukast was also positive, omalizumab 300mg was initiated to control angioedema and chronic urticaria. after 1 year of treatment, dpt with nimesulide was negative. omalizumab dose was reduced to half after the patient found out she was pregnant. there were no further episodes after anti-ige therapy introduction and pregnancy went uneventfully. conclusions: mdh syndrome is rare, more so when the drugs reported are ah and cct. hr to ah was confirmed, but diagnostic workup remains incomplete, postponed due to the patient's pregnancy. this case is as challenging in terms of diagnosis as it is in terms of therapeutic, so much so that omalizumab was initiated as an off label therapy, maintained during pregnancy based on the premise that risk was lower than benefit. objectives: in this study, we aimed to present our patients who were admitted with oral iron hypersensitivity. conclusions: according to our clinical experience, we think that oral iron salts with different conjugates are safe and acceptable option in patients with suspected oral iron hypersensitivity. introduction: antineoplastic agents are consider nowadays an essential treatment for many kinds of cancer. the increased use of these drugs in recent years is in parallel with a high rise of hypersensitivity reactions to them. these reactions range from mild to severe and as other allergic reactions, are not predictable. a nursing protocol in the desensitization schedules with these drugs is essential in allergy daily hospitals. objectives: the aim of this study are to describe a nursing protocol during desensitization schedules with antineoplastic agents carried out in our allergy unit in order to detect symptoms suggestive an allergic reaction during drug administration and to assess a correct intervention in case of reaction. conclusions: an appropriate nursing protocol in desensitization schedules with antineoplastic agents is essential in order to achieve the correct administration of the treatment in safety conditions. 0255 | long term clinical effects of aspirin desensitization in patients with nerd: comparison of maintenance doses of 300 mg vs 600 mg aspirin çelik ge 1 ; karakaya g 2 ; erkekol f € o 3 ; dursun ba 4 ; gelincik a 5 ; celebioglu e 6 ; y€ ucel t 7 ; yorulmaz i 8 ; dursun e 9 ; tezcaner ç 8 ; s€ ozener zç 10 ; b€ uy€ uk€ ozt€ urk s 11 ; kalyoncu f 12 ; aydin ö 1 introduction: aspirin desensitization (ad) treatment has been shown to be effective in relieving the respiratory symptoms as well in reducing recurrency of nasal polyps in patients with nsaids exacerbated respiratory diseases (nerd). however, a conflict occurs about effective maintenance doses of aspirin on clinical parameters. objectives: in this study, our aim was to compare the effects of two different maintenance doses of aspirin on clinical outcomes for 3 years of ad. this was a multicenter study which involved 4 tertiary centers. patients who completed at least one year of ad treatment were included to analysis. study outcomes were number of nasal surgery, sinus infections, asthma morbidity (number of severe asthma attacks, hospitalization) as well as medication uses for both clinical conditions. the study included 114 subjects, 33 of whom were under 300 mg aspirin daily as maintenance treatment whereas remaining 81 on 600 mg aspirin for a mean of 48.2ae32 months of ad duration. regardless of maintenance doses, number of nasal polyp surgery gradually decreased at 1 (0.04ae0.03/year) and 3 years (0.02ae0.01/year) compared to that of before ad (0.41ae0.06/year) (p<.0001) in all subjects and were comparable in 300 and 600 mg. considering asthma outcomes, decrease in asthma attacks were observed only in 600 mg aspirin group (p<.01) at 1 and 3 years whereas hospitalization due to asthma and systemic corticosteroid use decreased in both groups at 1 and 3 years. conclusions: ad has a reducing effect on nasal polyp recurrence for at least 3 years in patients with nerd. this effect was similar for both 300 and 600 mg maintenance doses of aspirin. considering both treatment arms provided decreased hospitalization due to asthma and systemic corticosteroid use, we think that at 3 years evaluation both 300 and 600 mg/day aspirin has comparable effects on asthma as well. however, reducing effect of ad on asthma attacks was only existed in patients taking 600 mg. aspirin. objectives: pregnant women with syphilis and history of immediate hypersensitivity reaction (hsr) to penicillin were enrolled. according to the risk stratification, which was based on the initial hsr, serum specific ige and skin tests, patients were re-exposed to penicillin either through desensitization or provocation. patients with a clinical history suggestive of penicillin-anaphylaxis and/or positive serum specific ige to penicillin and/or positive immediate skin test were considered at high risk and were desensitized. the remaining patients underwent penicillin provocation test. results: we evaluated 21 pregnant women with latent syphilis and history of penicillin allergy. clinical history was suggestive of immediate hsr in 13 out of these 21 (62%) patients, who were desensitized. all of them had negative serum specific ige to penicillin. intradermal tests were positive in 4/13 (30%). three out of those four were desensitized with an oral protocol and reacted during the procedure. one patient had a severe breakthrough reaction with uterine contraction and did not finish the procedure. the only patient with positive intradermal test that didn't react during the rdd underwent an intravenous protocol. the remaining 9/13 (70%) patients had negative skin tests and an uneventfully rdd. there was a statistically significant association between positive intradermal tests and breakthrough reactions during the rdd (p=.02). the other 8/21 (38%) patients with inconclusive history and negative skin test were submitted to penicillin provocation, which were negative in all of them. conclusions: risk stratification based on the initial clinical reaction and skin testing to guide penicillin re-introduction was safe and effective, as well as rdd. skin testing identified allergic patients to penicillin with increased risk of reactions during rdd. 0257 | utility of basophil activation test for monitoring the acquisition of clinical tolerance after subcutaneous desensitization to brentuximab-vedotin in two patients many hypersensitivity reactions (hsrs) produced by biologic agents have been seen and their true incidence is unknown. desensitization is a method to counter hsrs from monoclonal antibodies in patients with no other adequate alternative options. objectives: we describe a successful rapid desensitization to bv in two patients with scleronodular type hl, refractory to several lines of chemotherapy and asct. this clinical tolerance to bv is easily observed through the basophil activation test (bat) as a decrease in activated basophils after desensitization was done as a treatment of hsrs. because there was no therapeutic alternative in the two patients, we planned to pursue bv administration using a rapid desensitization 12-step protocol. a total dose of 125 mg of bv was given through increasing rate and concentration. the patients completed their infusion without difficulty. after desensitization to bv, bat was done in both patients. the percentage of activated stimulated basophils with bv descended in both patients. both values are similar to their corresponding negative controls. conclusions: the bat continues to be a useful in vitro tool for the study of drug allergic disease. also, the bat in flow cytometry is able to monitor an acquired tolerance induced by a desensitization treatment in hsrs to bv. however, studies involving a larger number of patients will be required to assess the safety and efficacy of this approach to bat as a method to validate rapid desensitization in patients with hsr to bv. objectives: we retrospectively reviewed 166 desensitizations in patients with a history of ihsrs to chemotherapy agents performed in our center from january 2014 to december 2016. the protocol consists of increases in infusion rate every 10 to 15 minutes, in a 10 to 12 steps depending on the drug. in all cases the protocol was performed without premedication (only using regular medication according to instructions for every drug). results: a total of 61 patients with a history of (ihsrs) received 166 desensitization protocol without premedication to chemotherapy agents. the most common involved drug was carboplatin in 24 (39.3%) patients (of these 66% presented positive skin test (st)), followed by paclitaxel in 15 (24.9%) patients (of these 46.6% presented a positive st) and oxaliplatin in 9 (14.75%) patients (of these 11.11% o the st were positive). other chemotherapy agents involved were cetuximab, rituximab, irinotecan, epirrubicin, etoposide, cisplatin and cyclophosphamide. all patients were able to successfully complete the desensitization protocol without premedication and none of them need to withdraw the drug. conclusions: this protocol for rapid desensitization to chemotherapy without premedication is safe and effective. in addition, minimizes secondary effects of the premedication in these patients that are polymedicated. 0259 | rapid desensitization for the management of hypersensitivity reaction to biologicals-infliximab and adalimumab in inflammatory bowel disease patients objectives: to identify barriers to best practice with regards to drug allergy history taking and documentation, and to elaborate the potential strategies to overcome them. results: a total 164 prescribers responded to the survey: doctors in training 47.6% consultants 44.5% non-medical prescribers 7.9% most respondents 56.1% (95%ci 47.9-64.3%) were not aware of the availability of penicillin allergy testing in our trust. among those that were aware of it, 63.9% (95%ci 51.9-76%) had not referred any penicillin-allergic patient to immunology during the past year. barriers to accurate allergy history collection: 55.2% (95%ci 47-63.4%) concurred that often it is not possible to draw a firm conclusion based on history alone. 59.4% (95%ci 51.4-67.5%) agreed with the statement saying that, regardless of the details of the allergy history, it is always better to "play it safe" and not to use alternative beta-lactams in patients labelled as being penicillin-allergic (figure will be attached in the poster). among the interventions proposed; practical educational sessions, an interactive questionnaire to guide allergy history taking and classification and a modified antibiotic policy to guide prescribing based on the allergy classification, were all rated as useful (average score >7 on a scale from 1-not useful at all-to 10-very useful). 0262 | the regulatory role of germinal center maturation during the early b cell response to inhalant allergens investigated in the piama cohort using the medall allergen microarray introduction: in contrast to common belief, igg to airborne allergens is higher in allergic subjects, even before immunotherapy. one of the confusing aspects of the allergic immune response is that not only the igg response, but also the ige response can follow more than a single trajectory (with or without gc maturation). for ige we assume that the direct isotype-switching pathway (igm to ige) is the most relevant for the initial, mature-gc independent phase of sensitization to low-dose airborne allergens (such a pollen, mite). in later phases and for higher exposure situations as well as for other immunization routes, indirect switching is assumed to be the more relevant pathway. methods: ige, igg1 and igg4 antibodies were measured using the medall allergen microarray in 105 children from the piama cohort at age 1 and 4. these results were analyzed in relation to the ige levels at age 4 and 12. objectives: to find support for the hypothesis that the ige/igg ratio reflects not only exposure, but also details on immunological processes during sensitization, such as germinal center maturation. results: sigg1 and sigg4 levels to the major inhalant allergens were low at age 1 and remained in general low at age 4. however, children who at that time were positive for ige an allergen had a significant increased sigg1 to the allergen in question. sigg4 also appeared, but this response was low. the sigg level at age 4 in sigenegative children was not consistently predictive for sige at age 12. conclusions: the initial igg1 response to inhalant allergens is synchronized with the ige response. this result fits with the hypothesis abstracts | 225 that in the initial phase of sensitization to inhalant allergens the allergen initiates a weak and incomplete germinal center response that allows parallel ige-and igg1 production. one of the consequences of the multiplicity of b cell developmental pathways is that the igg/ige ratio is potentially diagnostic: if a subject has sigg levels in the microgram range, and thus a high sigg/sige ratio, this indicates involvement of mature gcs and the indirect class-switching pathway for some or all of the sige in this subject. 0263 | synthetic allergoid consisted of plga nanoparticles covered with synthetic peptides from bet v1 objectives: the aim of this study was to test a hypothesis that the ahr signaling is critical in controlling sl homeostasis through the regulation of key sphingolipid enzymes involved in the s1p synthesis. results: we found that an ahr ligand and a tryptophan photoproduct, 6-formylindolo (3, 2-b) carbazole (ficz; 1 nm), induced a increase in s1p level in a ros and ca 2+ -dependent manner, leading to the degranulation as well as il-13 secretion in mast cells, when compared to those seen in vehicle-treated cells. this was concomitant with an increased level of sphk1 phosphorylation and with a reduction in the enzymatic activity of s1p lyase, which could be reversed by the addition of an anti-oxidant, nac. moreover, s1pl was found to be directly oxidized by ros in vitro and in vivo. conclusions: our findings suggested that ahr-mediated ros and ca 2+ signals are critical for controlling sl homeostasis through regulating sphk1 and s1pl metabolic pathways, providing a new regulatory pathway in mast cells. methods: peptide cytokine mimetics were selected by phage display technology. flow cytometry, elisa, elispot, t cell proliferation, reporter gene, mediator release, intravital microscopy and peritonitis assays were conducted to evaluate the capacity of the mimetic peptides to modulate the immune response. results: the synthetic tgf-b1-like peptide was able to down-regulate the production of tnf-a, il-4, ifn-c and il-8, up-regulate il-10, decrease basophil degranulation and induce t reg cell differentiation. furthermore, this peptide was able to decrease leukocyte rolling and neutrophil migration during an inflammatory condition in vivo. the synthetic il-10-like peptide was able to decrease basophil degranulation and to inhibit the proliferation of allergen-specific t cell lines established from the peripheral blood of birch pollen-allergic patients. conclusions: the peptide cytokine mimetics tested herein, were able to modulate the immune response in the tested conditions. they, thus, represent promising novel candidates for therapeutic approaches. nonetheless, most studies focus on changes occurring early in life and there are rare data on differences in responses between allergic and non allergic subjects. objectives: we aimed to evaluate i) the maturation trajectory of the tlr3 antiviral pathway ii) if this trajectory varies between atopics and non atopics. peripheral blood mononuclear cells (pbmcs) were isolated from otherwise healthy atopic and non atopic subjects. atopy was assessed by medical history and skin prick testing to 8 common aeroallergens and egg white. selected cytokines involved in the antiviral response were measured by luminex multiplexing technology in 24 hour culture supernatants of poly:ic-stimulated pbmcs. data were analyzed by estimating the non-parametric correlation between age and cytokine expression in atopics and non atopics and comparison of regression curves for each cytokine between the 2 groups was performed. results: the analysis comprised data from 39 atopic and 39 non atopic patients (mean age 10.8 years, age range 0-45 and mean 10.3 years, range 0-43.3, respectively). significant age-related increases in the production of ifn-a2, ifn-c, il-1b, il-17a, tnf-a, and mip-1b were found only in non atopics and of il-9 and il-10 in both groups. significant differences in the trajectories (slopes) of cytokine responses over time between atopics and non atopics were observed for ifn-a2, ifn-c, il-10, il-1b, tnf-a and mip-1b, with suboptimal production in atopics. conclusions: age-related increases in cytokines implicated in innate antiviral responses were observed mostly for non atopics. atopy was associated with suboptimal trl-3 induced cytokine responses. differences in the developmental pattern of those cytokines between atopics and non atopics may account for the reported increased susceptibility of atopics to infections. 0271 | a systems-immunology approach identifies a set of micrornas in shaping the th2 phenotype in allergic airway inflammation introduction: mouse allergy is a common disease in inner city households, affecting up to 18% of children who are exposed as determined by house dust analysis. it is associated with allergic rhinitis, atopic dermatitis and asthma and it has been reported that exposure and sensitization to mouse allergens is a strong predictor for asthmatic disease. despite a strong link between mouse exposure and asthmatic disease, the allergic immune response to mouse has been significantly understudied. to date, only one major allergen in mouse, mus m 1, has been identified and very little is known about the targets of the allergic immune response against mouse. objectives: using a proteomic/transcriptomic approach, we sought to identify t cell targets in 24 mouse allergic and asthmatic patients. results: mouse urine and epithelial extracts were analyzed by 2d-ige/igg immunoblots using pooled sera from mouse-sensitized donors. mass spectrometry of selected protein spots identified 30 novel antibody reactive proteins. predicted mhc binding peptides from these novel proteins and mouse homologs to mammalian allergens were screened for t cell reactivity in pbmcs from mouse allergic patients. overlapping peptides from the major mouse allergen mus m 1 and its major urinary protein isoforms were screened in parallel. our screen for t cell responses in pbmc from mouse allergic donors demonstrated that major urinary proteins account for >75% of the total t cell response but they are not the only target of mouse-specific t cell responses. reactivity to mouse peptides homologous to other mammalian allergens, specifically guinea pig, was also detected. conclusions: in summary, our data demonstrates that the cellular and serological targets of the allergic response overlap, with mus m 1 being the major target for both t cells and antibodies. to the best of our knowledge, this is one of the first comprehensive studies of t cell epitope targets in mouse allergy, which provides important insights into cellular and serological targets. this data may form the basis for the development of a mouse-specific immunotherapeutic approach. introduction: food allergy has a complex etiology with many potential underlying factors proposed to contribute to and modify its development and progression. the use of a databases to collect and analyse all relevant data related to incidents of food allergy is essential to fully understand causal factors and improve treatment. objectives: we developed a database using sql, hibernate and java server pages (jsp) that was designed to allow allergy professionals to easily add and modify patient data, including medical history, reaction details and in vitro/in vivo test results. we then filled this database with clinical data relating to reactions to plant-based foods for patients who visited the allergy service of the regional university hospital of malaga between 2012-2016. these data were then analysed in various ways. cluster analysis of skin test results was used to search for relationships between different allergens based on similarities between patient sensitisation profiles; descriptive statistics and graphical analysis were performed to search for relationships between food type, age of first reaction, number of reactions and reaction severity. results: cluster analysis placed the different skin test allergens in distinct groups, which generally correlated with the type of allergen. for example, nuts, rosacea plant-food, mites, trees and weeds formed distinct clusters. analysis of patient history data showed that the first reaction occurred most frequently between ages 10-20, with a right skewed distribution. a relationship was also found between age at first reaction and reaction severity, being urticaria and angioedema more common when the initial reaction occurs at a younger age, and anaphylaxis when the initial reaction occurs later in life. oas remained relatively prevalent at all ranges (around a quarter of all reactions in all age groups). we found fruits to be the most frequent triggers, followed by nuts; within fruits peach and banana were the most frequent. conclusions: this preliminary study show the importance and utility of recording patient allergy information in a well-structured and easily managed database. future work is currently underway to collect a new set of patients from the same geographical area and to analyse similar data from a different area in order to identify what results are replicable within our population and which results are generalizable to other areas. introduction: cow's milk allergy is very common in children and its correct diagnosis is important to prevent possible dangerous allergic reactions. the aim of the present work was to evaluate the prevalence of allergic sensitization to both cow's milk and to its main proteins. with medcalc 9 ® . normality distribution of data was evaluated through the kolmogorov-smirnov test. patients were divided into three age groups (0-2 years, 3-6 years-7-16 years). chi-squared test was performed to verify a statistical difference between sensitization to whole milk and to its main proteins and patients' age. introduction: the order fagales represents an important cause of tree pollen allergy, which is ubiquitous in the northern hemisphere. a high degree of allergenic cross-reactivity has been observed among allergens from these plants, mainly represented by pathogenesis-related protein class 10 (pr-10) pr-10s, including inhalant and food allergens. conclusions: testing ige reactivity to a panel of pr-10s unveils important associations between sensitization profiles and clinical presentation, and allows the identification of novel cluster patterns potentially useful to predict disease severity in patients with pr-10 allergy. results: 11 patients were included, seven boys and four girls, with a median age at diagnosis of six months. the most common offending foods were cow's milk protein (cmp, n=5) and rice (n=2). other foods were fish, egg, chicken, wheat, carrot and potato. average time of symptom onset was 2.5 hours. the most frequent symptoms were vomiting (n=10) and diarrhea (n=4). six patients had a history of hospital admission related to this problem. seven patients had concomitant atopic diseases, being the most frequent allergic comobility atopic eczema (n=5). skin prick tests and/or specific ige to culprit foods were negative at diagnosis, except for one patient with low specific ige to cmp. another patient become sensitized to cmp during follow-up. open food challenges were performed in 10 patients starting from six months of age. resolution was achieved in 6 patients, at a medium age of 36 months. results: a total of 2901 cases of immediate-type fa among 2056 children were reported, with 92.5% involving patients younger than 7 years of age. the major 7 causative foods were hen's egg (27.4%), cow's milk (26.6%), walnut (7.2%), wheat (6.2%), peanut (5.5%), soybean (2.4%), and shrimp (2.2%). the most common causative food in each age group was cow's milk (0-2 years), walnut (3-6 years), walnut and hen's egg (7-12 years), and buckwheat (13-18 years), respectively. the symptom onset time was less than 30 minutes in 65%. food-induced anaphylaxis was reported in 732 (25.2%) out of 2901 cases, and the major 7 causes were cow's milk (26.9%), hen's egg (20.1%), walnut (12.0%), wheat (9.6%), peanut (7.0%), buckwheat (4.0%), and shrimp (2.5%). the proportion of anaphylaxis was highest in buckwheat (60.4%), followed by walnut and pine nut (42.1% each). korean children were hen's egg, cow's milk, walnut, wheat, and peanut, with distinctive distributions according to different age groups. anaphylaxis was reported in 25.2% among immediate-type fa. results: a total of 263 children with a median (inter-quartile) age of 6.13 years (4.27-8.43) were enrolled to the study; (male 66.5%). their ages at diagnosis were 0.50 years (0.40-0.67); follow-up times were 5.63 years (3.57-7.69) and milk specific ige levels at diagnosis were 7.6 ku/l (1.9-27.2). in 30.8% of the children there was only cma; the other children were polyallergic to different foods having most frequently egg white allergy. concomitant diseases were 56.7% atopic dermatitis, 35.4% were asthma, 12.2% were allergic rhinitis. during the follow-up milk tolerance was developed in 43.1%, 65.2% and 68.1% at the ages of 3, 5 and 6 years respectively. the specific ige level at the beginning of the disease was found to be a risk factor for the persistence of the disease (p<.001). conclusions: cma is frequently present with other food allergies. nearly half of the patients develop tolerance to cm up to the age of 3 years; whereas 2/3 becomes tolerant when they are at the age of 5 years. most frequent concomitant diseases are atopic dermatitis and asthma. objectives: two survey tools were used; a questionnaire based on similar surveys done overseas, and the validated food allergy quality of life questionnaire (faqlq). this was distributed throughout paediatric allergy clinics at two metropolitan centres. children and adolescents aged 10-19 completed the questionnaire independently, whilst parents assisted with children aged 5-9 years. results: 102 surveys have been collected at the time of writing of which 64 were answered by parents for young children. overall, 44/ 97 (45%) reported bullying, with a higher portion in older children and adolescents (22/37; 59%). of this group, 10/20 (50%) reported being bullied or teased because of their food allergies. from parental reports, 11/19 (57%) stated that their child had experienced bullying or teasing because of food allergies. for those not bullied, parents mentioned that this may be due to their child having friends at school, being too young for bullying or because other children at school had a good understanding of the severity of allergies and were educated by teachers. the most common location for bullying was "in the playground or sportsground" (36/39). the most common form of bullying involved being "teased, called names or someone has said mean things to me" (31/39). whilst food allergens were involved in bullying in many cases (24/39), there were no reports of children being forced to eat food to which they are allergic. of concern however, two adolescents reported experiencing an allergic reaction as a result of the bullying. the majority reported experiences of sadness from bullying (30/39) while seven stated that it had no effect. conclusions: our current research shows that 45% of children and adolescents with food allergies experience bullying, and that 22% (21/97) experience bullying specifically because of their food allergies. this indicates a significant social problem that requires addressing to positively assist those children living with food allergies. introduction: recently we demonstrated that intake of specific foods, types of fat and micronutrients was associated with inflammation and mucosal integrity in adults with eosinophilic oesophagitis (eoe). the current study aimed to compare dietary intake of these patients with dietary guidelines and intakes of the general dutch population to further investigate our hypotheses on the protective or allergy-provoking role of specific nutrients in eoe. results: the total faqlq score was low when assessed by teenagers and children (4.0 and 3.9, respectively) and moderately low when assessed by parents (2.7). experience of anaphylaxis and having multiple food allergies impaired hrql according to faqlq parent form (p<.05). gender, having prescribed an adrenaline-auto injector, experience of food provocation test, peanut allergy and faim did not contribute to different hrql. hrql in kindergarten and schools were moderately diminished (sum score 2.6 in schools and 2.2 in kindergartens) (p>.05). perceived disease severity was moderately present with total faim scores being 3.4, 3.6 and 3.9, when assessed by children, teenagers and parents, respectively (p>.05). 68% of participants' reported at least some possibility of dying if child/teenagers would accidently eat a food allergen. after fulfilling faqlq and faim, all participants expressed content, ten children/teenagers decided to approach food provocation tests de novo, employees of children's schools/kindergartens were encouraged in written invitations to assess anaphylaxis training programs, and four families accepted additional psychological support. conclusions: food allergies impair hrql in children and teenagers. allergies to multiple foods and experience of anaphylaxis were associated with more severe impairment of hrql. hrqlq and faim are useful, additional tools to assess and discuss child's/teenager's/parent's fears and obstacles because of food allergy and identify further needs of support. introduction: fruit allergy is the most common cause of food allergy in children older than 5 years and adults. regional variations have been observed in europe but there are few studies in pediatric population. objectives: in the context of a prospective and multicentric study on pollen and vegetable food allergy in spain, we enrolled 45 patients (median age 12, range 2-18, female 64%), who had suffered at least two episodes of immediate symptoms after ingestion of fruits and had positive skin test to the implicated fruits. immunocap isac was analyzed in all patients. our aim was to describe the clinical characteristics with the fruits involved and the usefulness of the allergens included in the immunocap isac to improve its characterization. symptoms were categorized into oral allergy syndrome (oas), systemic symptoms (ss) and anaphylaxis. results: a total of 45 patients were included. all of them had symptoms with more than one fruit. 42 patients had pollen sensitization. the main offending food associated with allergic reactions were peach (49%), kiwi (27%), melon (24%), apple (22%) and banana (15%). 5 allergic patients to peach had oas, 13 ss and 4 anaphylaxis, were recognized prup3 in all patients with anaphylaxis, in 4 patients witch oas and in 10 with ss, also prup3 associated with abstracts | 235 prup1 in 2 patients with ss. for allergy to kiwi, 9 patients had oas and 3 ss, were recognized actd2 in 1 patient with sao and 3 patients with ss. actd1 in 1 patient with ss. conclusions: in our population, the most prevalent fruit allergy was the peach, as in spanish adults. patients allergic to peach were the ones that presented the most ss and anaphylaxis, followed by allergic to apple. as previously have been reported most of them had sige to its components in isac; being prup3 the most prevalent (9% had prup1). only patients with ss with kiwi were sensitized to kiwi allergens .the majority of patients allergic to apple, melon and banana were not diagnosed by immunocap isac. introduction: studies have shown that asthma and allergy are prevalent among production workers processing seafood, particularly in workers processing crustaceans. a major ige-reactive proteins is the muscle protein tropomyosin. specific ige to tropomyosin is suggested as a central marker for crustacean allergy, however it is not the only protein characterised as an allergen in crab processing. objectives: the aim of our study was to characterise tropomyosin exposure and prevalence of sensitisation to allergens in workers processing king crab (paralithodes camtschaticus) and edible crab (cancer pagurus) in land based processing plants in norway. results: personal air samplers collected air from the workers' breathing zone during crab processing. workers' blood was collected for ige testing. extracts of both king crab and edible crab raw meat, cooked meat, intestines and shell were made in our lab and used for skin prick testing and immunoblotting. while processing cooked crab yielded highest tropomyosin levels in the edible crab plant, processing raw crab yielded highest levels in king crab plants. ten (12.8%) edible crab and 11 (9.7%) king crab workers had positive ige test (>0.35 ku/l blood, immunocap systems) to crab. four (10%) of skin prick tested king crab workers and 15 (19%) of skin prick tested edible crab workers had one or more positive reactions to edible crab extracts. more workers reacted to cooked crabmeat extracts than to raw crabmeat extracts. immunoblotting showed ige binding to a large number of proteins in all four extracts of both king and edible crab. binding was found to high molecular weight proteins in all four extracts of the crab tested, and the ige-reactive proteins differed between king crab and edible crab. conclusions: workers are exposed to tropomyosin in their breathing zone during crab processing. both king crab and edible crab workers are sensitised to crab, shown with immunocap specific ige test to crab, as well as positive skin prick tests and immunoblots to four different crab extracts made in our lab. workers processing crab in norwegian processing plants have an increased risk for developing sensitisation to crab. objectives: the aim of the study was to assess frequency of skin symptoms in surgery clinic employees, to evaluate burnout as a predictor of the frequency of skin symptoms, and to determine latexspecific ige in surgery nurses with skin symptoms. results: skin symptoms were significantly more frequent in surgery nurses (25%) than in surgeons (2.5%), other physicians (0), and other nurses (6.7%) (v 2 =18.16; p=.001). skin symptoms were also significantly more frequent in workers with high/medium than in workers with low emotional exhaustion (14.3% vs 5.2%; v 2 =4.32; p=.038), as well as in participants with burnout than in subjects without burnout introduction: baker's asthma sensitization pattern is changing due to the introduction of different types of grains and seeds. objectives: a 43 year-old ecuadorian man showing ocular, nasal and pulmonary symptoms when handling grain flours (with or without seeds), while baking for the last 8 years. he tolerated grain flours oral intake, but had oropharyngeal symptoms, rhinoconjunctivitis and dyspnea when eating sunflower and sesame seeds, mustard, and beer with alcohol. he tolerated alcohol-free beer. we performed an allergy study: prick-test with commercial extracts of pollens, dust and storage mite, fungus, animal danders, cereals, yeasts and mustard. prick by prick with patient's products: wheat, multicereals and seeded flour, sunflower seeds, regular and alcoholfree beer spirometry and niox. serial peak flow measurement at and away from work, handling tests with wheat flour and sunflower seeds. laboratory studies: specific ig e to cereals and seeds (cap-isac-microarrays). immunoblot with regular beer (at room temperature and boiled), wheat flour and sunflower seeds, and sequential chromatography. results: skin tests were positive with commercial mustard and all provided products except for the alcohol-free beer. spirometry was normal. niox: 116 ppb. peak-flow monitoring showed a 27% variability during working period, remaining stable during holidays. handling tests with wheat flour and sunflower seeds were positive. specific ig e was positive for grains, malt, gluten, mustard and sunflower seed. the specific determinants were positive to 7s-viciline, 11-s globulin, several prolamins (2s-albumine, alfa-amylase inhibitors and gliadin) and ltp. immunoblot detected a band lower than 14 kda in both regular beer extracts (not detected in alcohol-free beer) identified as barley's ltp, a band of 18 kda in the sunflower seed extract (2salbumine), and two bands lower than 14 kda in wheat flour extract (two kinds of alfa-amylase inhibitors). we present a non-atopic baker with occupational rhinoconjunctivitis and asthma due to prolamins (alfa-amylase inhibitor and gliadin of wheat flour together with 2s-albumin sunflower seed), and anaphylaxis when eating seeds (2s-albumins, 7s-vicilin and 11s-globulin) or drinking beer (sensitization to barley's ltp). it is interesting that the manufacturing process of non-alcoholic beer (high temperature and high pressure) seems to degrade barley's ltp, as suggested by both tolerance to its ingestion and loss of immunoblot band. objectives: the main objective of this study is to evaluate longitudinal change of lung function in workers employed in food preparation and distribution potentially exposed to food allergens. spirometries performed between 2012 and 2015 as part of medical surveillance of 58 food-handlers workers were evaluated. data about occupational task, work years, smoking habits and diagnosis of atopy, asthma and copd were collected from a clinical database. differences in prevalence were calculated by chi-square test, differences in means were calculated using spirola software referred to european predicted values. results: the majority of workers were females (n=49; 84.5%) and caucasians (n=57; 98.3%). 25 (43.1%) subjects were current smokers, 6 (10.3%) were ex smokers, 9 (15.5%) were atopic and no one reported a diagnosis of asthma or chronic obstructive pulmonary disease. 69% workers were canteen service employees and 31% were cookery employees. mean yearly values of the pair-wise within person variation of fev1 and fvc were respectively à248 ml and à266 ml. 6.9% of last observations had a fev1 below lln and 8.6% of last observations had a fvc below lln. conclusions: this study may help in planning preventive programs and in facilitating early recognition and diagnosis of work-related respiratory diseases. wheat, foods and latex allergens may determine decline in fev1 and fvc; furthermore, in our study, a significant proportion of workers reported exposure to tobacco smoke. excessive loss in fev1 over time should be evaluated using a percentage decline (15% plus loss expected due to aging) that we will make afterwards adding more years of follow up spirometries. intervention of smoke avoidance are needed. 0288 | prevalence of wood dust sensitization in occupationally exposed workers in germany-what can be tested? objectives: in 373 serum samples from patients with suspiciously allergic symptoms to wood dust overall 2797 specific (s)ige-tests with standardized wood-dust extracts coupled to streptavidin-immu-nocaps were conducted. additionally, ccd as known source of non-protein ige-target was evaluated. sensitization rates were calculated for 21 different wood species. most frequently requested wood dust allergens were obeche (n=198), beech (n=186), oak (n=173), spruce (n=175) and pine wood (n=147) followed by 80-100 requested sige-tests to mahogany, ash, larch and maple wood. results: overall wood dust sensitization rate was about 11% (range: 0-29%) with obeche, box wood and kambala as most prominent sensitization sources obtaining each more than 20% sensitization. no sensitizations were detectable to red cedar and meranti wood in more than 50 requested tests, respectively. in 81 patients at least one sige-sensitization to any wood was measured. there from 77 were additionally tested with ccd resulting in 47% positive and 53% negative ige responses to ccd. some wood species were exclusively recognized by ccd-positive subjects: ash, maple, alder, mahogany, teak, mansonia and palisander. whereas other woods were recognized by sige of subjects with / or without sige to ccd: obeche, box wood, spruce, oak, beech, limba, pine and kambala. relevance of wood sensitization next to ccd was investigated in eight double positive subjects (wood + / ccd +). specific ige-binding to wood allergens was completely inhibited by ccd in three samples. these subjects were supposed to have no clinically relevant wood sensitization. whereas in five samples sige-binding to selected wood species was not significantly reduced by ccd. in four of these patients skin prick tests (spt) and challenge tests (bronchial and/or nasal) with corresponding wood allergens were performed. three of four challenge tests were positive with the respective wood extract and all spts with wood extracts whose sige-binding was not affected by ccd inhibition showed positive reactions. here clinical relevance of sige-mediated wood sensitization could be demonstrated. conclusions: in summary, our data demonstrate that standardized wood extracts and ccd tools are necessary for valid in-vitro diagnosis of wood dust sensitization. introduction: respiratory symptoms have been reported frequently among seafood processing workers. since seafood processing workers handle the raw material and participate in processing activities during work, they are exposed to inhalable bioaerosols. this put them at risk of developing respiratory symptoms, asthma and allergy. there is little knowledge about the respiratory health status among fish production workers on board fishing trawlers. objectives: the aim of this study was to assess the respiratory health status among norwegian fish production workers, processing fish on board fishing trawlers. the study population consisted of 92 fish production workers, 21 machinists, 22 support crew members and 38 non exposed controls, all were males. written informed consent was the fish production workers had a significantly decreased fev 1% predicted compared to the non-exposed control group, b=à5.9, 95% ci [à11.2, à0.6], when controlling for age, pack-years and family history of asthma/allergy/eczema. the effect did not change when controlling for doctor diagnosed asthma or after dividing fish production workers by doctor diagnosed asthma. machinists and support crew members showed a similar decrease in fev 1% predicted, but the difference from the non-exposed control group was not statistically significant. furthermore fish production workers, reported a non-significantly increased prevalence of wheezing and daily morning cough compared to the non-exposed control group. conclusions: fish production workers, processing fish on board fishing trawlers, showed reduced lung function values compared to a non-exposed control group, and this finding is in accordance with previous findings in seafood industry worker populations from our research group. results: study group comprised 264 patients with work-related respiratory symptoms suggesting wra. the research completion with sic allowed to recognise wra in 164 persons (108 oa and 56 wea) and to exclude asthma in 100 cases qualified to reference group (gr). workers with wea occupationally exposed do lmw-a manifested the highest level of baseline non-specific bronchial hyperresponsiveness (nsbhr) in comparison to the other groups (table 1) . patients with oa exposed to lmw-a more frequently than exposed to hmw-a revealed nsbhr before sic (p=.036) with lower level of median (me) provocative methacholine concentration value causing 20% fall in fev 1 (pc 20 induced sputum (is) was obtained before and 24h after sic from 159 patients (38 gr and 121 wra). in all gr samples and samples possessed before sic from wea subjects exposed to hmw-a, intermediate profile of is (neutrophils (ne)<61% and eosinophils (eo)<2%) dominated ( results: eg: in "granulation" exposure is relatively high and the number of different enzymes handled is low; here the risk of sensitization is highest. in contrast in the pilot plants the exposure compared to granulation in general is lower but the number of enzymes handled concurrently is higher. still the sensitization risk is lower than the range for granulation. conclusions: even though this approach may seem crude and not free from bias and potential misclassification, data does not support the hypothesis that the number of enzymes increases risk of sensitization, whereas increasing exposure level seems to be a risk factor. this suggests that each enzyme exposure acts as a risk in its own right and that the "cocktail effect" seems to be of minor relevance. phospholipids. bioinformatic studies of sequence homology conducted prior to this study showed no similarity between the mpla1s and known allergens including ves v 1. however, the common enzymatic activity in ves v 1 and the mpla1s might still lead to crossreactivity. the goal of this project was therefore to test for possible cross-reactions between sige towards ves v 1 and three different mpla1s. methods: serum from 10 known wasp allergic persons with sige towards ves v 1 spanning from 1.57 ku/l to 1734 ku/l were used for inhibition studies. from each, 125 ll serum was incubated with 125ll of either saline solution (negative control), 50 lg/ml alk802 soluprick solution (positive control) or one of three mpla1s, each in three concentrations (either 0.5 lg/ml, 5 lg/ml and 50 lg/ml (n=3) or 5 lg/ml, 50 lg/ml and 500 lg/ml (n=7)). the level of sige towards ves v 1 was measured using the i211 immunocap, and a decrease in this level was calculated as %inhibition compared to the sige measured from serum incubated with the negative control. inhibition by mpla1s would indicate cross-reactivity. results: the positive control caused 62.5ae28.6% (n=10) inhibition of sige towards ves v 1. this was lower than expected but was found to be caused by a few sera where the fraction of sige towards ves v 1 was <10% of all sige towards wasp venom. in the remaining sera, %inhibition was 78.1ae14.9% (n=7). for all three mpla1s, no inhibition was found for any serum tested (n=10) at the highest concentration tested with %inhibition being 3.5ae2.5%, 3.3ae2.9% and 1.8ae6.0% respectively. conclusions: no inhibition of sige to ves v 1 was found to any of the three microbial phospholipases tested. this indicates that no cross-reaction is found between the phospholipase a1 in wasp, ves v 1, and phospholipase a1 from microorganisms despite the common enzymatic activity. objectives: the aim of study was to evaluate the prevalence and the impact of polyvalent ige-mediated allergy on the course of ad and the occurrence of allergic symptoms from other organs and systems in infants and young children. conclusions: polyvalent ige-mediated allergy is common in young children with ad and seems to be a risk factor for the severe course of the disease. introduction: non-steroid anti-inflammatory drugs (nsaid) are the second most common cause of drug allergies in childhood. objectives: the aim of the study is to determine the frequency of nsaid hypersensitivity in asthmatic children. results: 976 patients who were being followed up for asthma were included in this study. the mean age of the patients was 10.61ae4.21 years, while 59.5% (575) were male. 1% (n=10) of the patients had a reaction history to nsaid (ibuprofen in 4, flurbiprofen in 2, diclofenac potassium in 1, metamizole+acetylsalicylic acid in 1, paracetamol+acetylsalicylic acid in 1 and ibuprofen+acetylsalicylic acid in 1). nsaid sensitivity was confirmed in 9 (0.9%; 9/976) patients who were tested with suspected drugs, while the provocation test was found negative in one patient who described reaction with ibuprofen. of the 2000 children who were assessed as a control group, only 1 had a reaction history to acetylsalicylic acid and no reaction developed in the provocation test. conclusions: nsaid hypersensitivity is more common in patients with asthma. thus, these patients should be evaluated for nsaid hypersensitivity. results: from jan 2012 to dec 2013, 8840 pediatric cases were received by kaers. of 8840 pediatric patients, 56.4% were male, 42.0% were female and 1.6% were unknown. these 8840 pediatric cases included a total of 17 319 adr events with an approximate average of 1.9 adr events per report. of those 8840 cases, 21.2% were in infants (age 0-1 years), 27.8% were in young children (age 2-7 years), 24.3% were in old children (age 8-13 years), 22.7% were in adolescent.(age 14-18 years) and unknown were 4.0%. male to female ratio was 1:0.8 and the mean age was 8.3ae6.3 years. regarding categorical ranking of reported adr agent groups, the most common group were antibiotics (24.4%) followed by antineoplastic agents (14.8%), vaccine (11.6%), antipyretics (7.1%), opioids (5.4%), sedatives (3.1%), antiepileptic drugs (2.9%), contrast media (2.3%), steroids (2.0%). the most common adr symptoms were gastrointestinal system disorder in 33.2%, skin-appendage disorder in 19.5%, abstracts | 249 body as a whole-general disorder in 9.5%, central-peripheral nervous system disorder in 7.5%. regarding seriousness of adrs, 610 events (6.9%) had episodes requiring hospitalization and were considered life threatening. of these, 126 cases had anaphylaxis or anaphylactoid reactions. introduction: multiple drug allergy is an adverse reaction to two or more structurally unrelated drugs that appears to occur by immune mediated mechanism. patients with a history of reaction to two or more drugs often apply to allergy clinics. objectives: the aim of this study is to evaluate the test results of the patients who have a history of multiple drug allergies and underwent drug provocation tests. results: during the study period, drug provocation tests were performed in 889 patients (1014 drug provocation tests). of these patients, 106 (11.9%) had a history of drug reactions to 2 or more drugs. the mean age of the patients who had a history of reactions to two or more drugs was 8.48ae3.94 years, and 58.5% (n=62) toms, and autoimmune manifestation in comparison to igm/iga responders (respectively, pneumonia: 64%, 31% and 0%; chronic diarrhea: 25%, 14% and 0%; autoimmunity 41%, 29% and 0%; autoimmune cytopenias: 17%, 8% and 0%). malignancies were found more frequently in the non-responders and igm-only responders groups in comparison to igm/iga responders (respectively, 23%, 14% and 0%). eleven (15%) patients died during the study time. survival analysis according to the igm/iga responder status showed that the 6-years estimated survival for non-responders vs igm-only vs igm/iga responders was respectively after one year 98%, 87% and 100%; after two year: 93%, 87% and 100%; after three years: 91%, 80% and 100%%; after 4 years: 87%, 80% and 100%; after 5 years: 87%, 80% and 100%; after 6 years: 83%, 80% and 100%. interesting, in our series only two deaths were due to infective complications: five were consequent to malignancies, one to autoimmune cytopenias and three to not-cvid related conditions. between-infusions intervals (10-14 days) than pid patients (6-7 days). finally, a small number of patients with anti-cd20-related sid was able to discontinue scig replacement therapy after recovery of spontaneous igg production. conclusions: this is, to our knowledge, the biggest single center cohort of scig-treated patients ever described. results suggest that safety and effectiveness of scig is similar in pid and sid, irrespective of the mechanisms underlying igg depletion. moreover, in sid a lower igg dosage is required and ig replacement does not always need to be lifelong, with obvious pharmacoeconomic implications. 0685a | should we screen children with bronchial asthma for primary immune deficiencies? miteva d 1 ; perenovska p 1 ; papochieva v 1 ; georgieva b 1 ; lazova s 1 ; naumova e 2 ; petrova g 1 the patient became febrile and cultures were repeated, being positive to campylobacter jejuni, resistant only to ciprofloxacin (blood) and to campylobacter coli, susceptible only to gentamicin and amoxicillin/ clavulanic acid (stools). treatment was thus switched to amoxicillin/ clavulanic acid (1875/125mg 8/8h). the patient became apiretic at day 2 and improvement of local inflammatory signs was noticed, treatment was prolonged for six weeks. one week after cessation, skin lesion worsened again in the same location, in association with fever. blood and stool cultures were repeated and gentamicin (240mg/day; iv) and cefixime (200mg 12/12h) were started, in agreement with previous cultures results. curiously, there was no history of diarrhea, but the patient referred a period of recurrent abdominal colicly pain, before cutaneous lesions appear. conclusion: bacteremia with campylobacter species requires specific laboratory workup. 7diagnosis of campylobacter jejuni bacteremia should be considered in hypogammaglobulinemic patients with recurrent fever, particularly when typical copper color erysipela-like skin lesions occur. campylobacter eradication can only be achieved with prolonged antibiotic therapy guided by antibiogram in cultures. conclusions: in this study, genetic defects of five higm patients have been identified, for other patients further genetic investigation such as next generation sequence (ngs) is required. the study results can help diagnose of the disease more definitively and also can provide valuable information for genetic counseling especially for those who have a history of immunodeficiency in their families and also for prenatal testing. conclusions: mutation analysis of unc13d gene can help the families with hlh patients give genetics counseling for carrier detection and prenatal diagnosis. woelke s 1 ; valesky e 2 ; bakhtiar s 3 ; bader p 3 ; schubert r 1 ; zielen s 1 1 department for children and adolescents, division of allergology, pulmonology and cystic fibrosis, goethe university hospital, frankfurt, germany; 2 department of dermatology, venereology and allergology, goethe university hospital, frankfurt, germany; 3 department for children and adolescents, division for stem cell transplantation and immunology, goethe university hospital, frankfurt, germany introduction: ataxia telangiectasia (a-t) is a devastating multi-system disorder characterized by progressive cerebellar ataxia, growth retardation, immunodeficiency, chronic pulmonary disease and genetic instability with an increased risk for malignoma. as described in other primary immunodeficiencies cutaneous granulomas are a known phenomenon also in a-t. still treatment indication and strategies remain controversial. objectives: from our cohort of 60 classical a-t patients, eight patients in the aged 2 to 11 years presented with granulomas. histopathology of the lesions confirmed the presence of granulomatous inflammation without detection of any microbiological agent in all patients. five patients suffered from cutaneous manifestation, in two patients we detected a bone and in one a joint involvement. both patients with bone involvement (patients 1 and 2) as well as one patient with massive skin manifestation (patient 3) were treated with tnf inhibitors (infliximab). the patient with granulomas in his finger joint (patient 4) was bone marrow transplant (bmt) for other reasons. year led to a total remission for three years now. in patients 2 and 3 treatment with tnf inhibitors led to a partial regression of granulomas. treatment interruptions caused deterioration again. in the course of treatment the effects of infliximab diminished most likely caused by drug antibodies. after changing treatment to subcutaneous adalimumab a further regression could be detected. in patient 4 granulomas totally disappeared with immune reconstitution after successful bmt. partially successful in treatment of granulomas. due to the known immunodeficiency in a-t patients, indication for immunosuppressive therapies as tnfa inhibitors should be held strictly. woelke s 1 ; hess u 1 ; knop v 2 ; krausskopf d 3 ; kieslich m 3 ; schubert r 1 ; zielen s 1 of 1 to 38 years regarding c-reactive protein (crp), liver enzymes, abdominal ultrasound and neurological status (ataxia score). we divided the patients into two age groups of 27 a-t patients aged 1 to 11 years and 26 a-t patients aged 12 years to 38 years. ataxia score (r=0.34), although the underlying pathomechanism is unclear. ultrasound revealed nonalcoholic fatty liver disease in only one young patient (3.7%) compared to 11 older patients (42.3%). one female patient aged 37 years died due to a hcc. conclusions: liver disease is present in almost all older a-t patients. structural changes, nonalcoholic fatty liver disease and fibrosis are frequent findings. there is a considerable risk for hcc. prospective studies are necessary using noninvasive techniques for the assessment of liver fibrosis (eg transient elastography) and to establish the risk of hcc in a-t patients. objectives: in this study, it was aimed to determine the frequency of pollen-food syndrome in children who have sensitization to pollens. results: 672 pollen-sensitized patients were included in this study. the mean age of the patients was 11.82ae3.90 years, while 63.1% (n=424) were male. in 45.2% (n=304) of the patients, allergic rhinitis was concomitant with asthma. 4.3% (n=29) of the patients described symptoms related to pfs. 31% (9/29) of them had a history of anaphylaxis with suspected food. the mean age of the patients describing pfs was 12.22ae4.26 years and 55% (n=16) of them were female. in 17 (58.6%) of these 29 patients, skin tests performed with suspected food was positive, but in one patient the skin test was negative while specific ige was positive. suspected food was fruit/ vegetables in 21 patients. in patients with pollen allergy, oropharyngeal symptoms related to fresh fruit, vegetables, dried fruits and nuts should be enquired. it should be noted that these patients might experience serious systemic reactions including anaphylaxis. patel nb 1 ; vazquez-ortiz m 1 ; lindsley s 1 ; abrantes g 1 ; bartra j 1 ; dunn galvin a 2 ; turner pj 1 conclusions: there is no evidence that the occurrence of anaphylaxis at fc, and self-treatment with an adrenaline auto-injector device, result in adverse impact on hrql measures. the impact of a reaction at food challenge appears to confer greater benefit on the parent than the child. the relationship between confidence in management and hrql needs further assessment, since it is likely that these outcomes will be affected in different ways following therapeutic interventions. results: fifty-one patients (23 females, 28 males) (median age: 6.5 years, range 0-18) with cma were studied. spt to cm was 7.3ae3.3 mm as mean diameter. forty-eight out of fifty-one (94.1%) patients underwent dm-opt (3 patients refused to underwent opt due to positive spt or s-ige to dm introduction: bovine milk is the most common food allergen in children under 3 years of age. milk allergy is treated by eliminating milk from the diet. the milk elimination diet endangers the child's energy intake and also exposes the child to shortage of multiple nutrients. this study was needed because there are no previous systematic reviews about this subject. objectives: the aim of the present study was to examine if the milk allergy or the other factors associated with milk elimination diet have an influence on child's growth. the present study was conducted according to international guidelines for systematic reviews. results: a total of 646 studies were initially identified, of which three fulfilled our criteria. these three studies included 186 children with cow's milk allergy and 122 control children. in all these three studies, children with cow's milk allergy were lighter than controls. in addition, in two studies, the growth of the children with cow's milk allergy was stunted. in two studies the milk elimination was substituted with special infant formula. also in one study where both the growth and the weight were stunted, no major differences in energy or nutrient intake between cow's milk allergic cases and controls were reported. conclusions: current evidence suggests that milk allergy is associated with stunted growth in childhood, but reasons for this are unclear. in order to clarify the effect of cow's milk elimination diet on growth in childhood and the underpinning mechanisms, more studies need to be conducted. in addition, special attention to the diet and growth of children with cow's milk allergy is needed. results: a total of 158 817 children were surveyed in this 6-year period (annual average: 26 470). in 2011, 704 children (2.6%) were diagnosed with food allergy, including 159 cases of pfas, 376 cases of egg allergy, and 235 cases of dairy-product allergy. in 2016, 939 children (3.6%) were diagnosed with food allergy, including 344 cases of pfas, 385 cases of egg allergy, and 249 cases of dairy-product allergy. over this 6-year period, the prevalence of pfas increased 2.2fold (from 0.59% to 1.31%). among the pfas cases, the prevalence of rosacea and apple allergy increased 2.9-and 3.3-fold, respectively. the most prevalent was apple allergy (0.76%), followed by peach (0.59%), loquat (0.53%), plum (0.44%), pear (0.40%), and strawberry (0.18%). the prevalence increased significantly for pfas but not for other types of food allergy (egg and dairy-product allergy). in the future, nationwide studies are needed to further elucidate the relationship between pfas and allergic rhinitis. 0327 | immune profile after oit in children with cow 0 s milk allergy patients with elimination diet, group 3: patients with natural tolerance, group 4: healthy control). in all groups specified laboratory tests were performed at onset and also at 6 month to treatment groups. in desensitization group at 6 month of treatment we evaluated increase in total ige level, sp iga and igg4 antibody responses, decrease in cow's milk spige levels and an increase in il-2, il-10, tgf-b cytokine responses without a difference in cd4+cd25+fox-p3% levels. il-13 levels were similar with pre-treatment levels whereas foxp3 mrna expression was similar with tolerance group. in elimination group at 6 month treatment, there was a decrease in cow's milk spige whereas there was no change in sp iga levels and a minimal increase in igg4 levels. there was no change in il-2 and il-10 cytokine levels. and an increase in tgf-b cytokine response less than group 1, decreased il13 response, a foxp3 mrna expression differed from tolerance group was identified. objectives: although hyperuricemia has a significant prevalence in the general population, and has been related to exercise-induced asthma, could be related to bronchial asthma and nasal polyposis. hitherto, its possible association with hypersensitivity to nsaids and its convenience as biomarker have not been acquainted. conclusions: in our population, hyperuricemia has not demonstrated to be a reliable biomarker related to nsaids allergy and could not be used as a risk factor for assessing the triad nsaids allergy, asthma and nasal polyps. to study the vas of the total score was seen significant variance in: nsaid-cu (3.38pt) and nsaid-pa (6.57pt) (p=.046), nsaid-cu conclusions: data support that cutaneous manifestations have a common response with aerd to cox inhibitors. conclusions: this study showed that in a positive drug oral provocation test, respiratory symptoms were accompanied with nitric oxide changes in both nasal and exhaled way. barrionuevo e 1 ; doña i 1 ; salas m 1 ; bogas g 1 ; guerrero ma 1 ; sanchez mi 1 ; cornejo-garcia ja 2 ; torres mj 1 1 allergy unit, regional university hospital of malaga-ibima, malaga, spain; 2 research laboratory, ibima-regional university hospital of malaga-uma, malaga, spain introduction: non-steroidal anti-inflammatory drugs (nsaids) are the most frequent triggers of drug hypersensitivity reactions, being cross-hypersensitivity reactions (chr) the most frequent. the categories included in chr are nsaids-exacerbated respiratory disease (nerd); nsaids-exacerbated cutaneous disease (necd) and nsaids induced urticaria/angioedema (niua). however, it has been reported patients with chr to nsaids who developed a combination of skin and respiratory symptomatology (blended reactions). objectives: our aim was to analyse the characteristics of patients with blended reactions and compare with those developing symptoms exclusively respiratory (nerd) or cutaneous (niua). episodes of cutaneous and/or respiratory symptoms after the intake abstracts | 265 of ≥3 different nsaids included strong cox-1 inhibitor (acetylsalicylic acid (asa) and/or indomethacin); ii) if they had <3 episodes of cutaneous and/or respiratory symptoms induced by <3 different nsaids, a positive drug provocation test (dpt) with asa was required; iii) if patients had respiratory symptoms accompanied or not by cutaneous involvement, a positive nasal provocation test with lysine aspirin (npt-lasa) was required. atopy was assessed by skin prick test using a panel of inhalant and food allergens. objectives: subjects with nsaids hypersensitivity were divided into two groups: a) those from 2 to 14 years and b) those from 15-20 years. diagnosis was established by a clinical history and controlled challenge with asa. atopic status was verified with a detailed allergological study including skin testing to inhalant allergens. clinical entities were classified in three categories: urticaria/angioedema, anaphylaxis and respiratory (asthma and/or rhinitis). cases. no differences were observed in the atopic status between both groups. there were significant differences between males/ females (p<.05). when we compared the clinical entities there were more cutaneous manifestations, mainly angioedema, in the group a) and more anaphylaxis in group b) although there were no significant differences due to sample size. conclusions: significant sex differences between hypersensitivity reactions to nsaid occurs with predominance of males in the first group (a). although there are also a predominance of clinical entities, an increase number of cases is needed to establish significance. studies on this direction are in progress. show an increase in all age groups. etiologic analysis was limited as the study was carried out using the icd-10 code (nhic records) and database of self reporting systems (kaers). so, further study was needed. introduction: genetic variants from the 17q21 locus are the strongest known genetic determinant for early-onset childhood asthma, and have also been associated with uncontrolled asthma despite asthma treatment. objectives: the aim of the study was to assess whether there is an association between a single nucleotide polymorphism (snp) in the 17q21 locus (rs7216389) and asthma exacerbations despite the objectives: our hypothesis is that the arg16 allele is associated with increased use of prescribed asthma medication, over a 9-year period. to explore this hypothesis, we have undertaken a secondary analysis of breathe, a study of gene-environment associations with asthma severity. breathe data were collected on participants with asthma, aged 3-22 years, between 2003 and 2005, in tayside conclusions: in children and adults, the homozygous arg/arg status is associated with long-term increased prescribing for asthma medication compared to those carrying at least one gly allele. defining subgroups of individuals requiring more medicines could help predict treatment costs and develop targeted management strategies. objectives: considering the role of ptgdr in allergy, the goal of this study was to analyze the effect of ptgdr expression on cytokine levels in the a549 cell line. analyze ptgdr expression in a549 cell cultures. cytokine production assays in the culture supernatant were measured by cytometric bead assay using the bioplexpro tm human cytokine standard 27-plex, group i. the assays were conducted with bioplex high-throughput fluidics system, powered by the luminex technology. every sample was run at least in triplicate. the ptgdr expression in the transfected cells with the haplotypic variants differed by 5 orders of magnitude relative to control cells (p<.001). we found significant differences in ptgdr expression between ctct and cccc haplotypic variants. the cccc (à613 c, à549 c, à441 c, and à197 c) introduction: c159t polymorphism in the cd14 gene has been suggested in susceptibility to asthma. cd14 is a multifunctional receptor endotoxin, which is expressed on the surface of macrophages, monocytes and neutrophils. it is likely to play a role in the inflammation pathway. though data is available regarding association of cd14 gene with asthma but independent studies are in conflict. objectives: the present study was conducted to examine the association of promoter c159t single nucleotide polymorphism (snp) in the cd14 gene for indian children with atopic asthma. we characterize the c159t polymorphism in children with asthma (50), cohort group (20) and healthy control group (50) by pcr rflp. association analysis was performed using v2 tests. we also analyzed the association of cd14 (c159t) with total ige levels by elisa and foxp3 expression using flow cytometry. in this snp a 497 base pair (bp) pcr product was generated using the standard primers. after restriction products showed that homozygous c allele was appeared as a single 497bp band, the homozygous t allele as bands of 144 bp and 353 bp, and heterozygous exhibits all three bands (144, 353 and 497 bp). the or of cc genotype frequency abstracts | 271 was 0.84 in study group and 0.78 in cohort group and the or of c allele frequency was 2.38 in study group and 2.52 in cohort group. total ige level were found to be significantly higher in cc genotype compared to ct and tt genotype. foxp3 level is significantly higher in control group in all genotypes compared to cohort and study group. conclusions: the present study concludes that in cd14 gene polymorphism cc genotype was not significantly associated with asthma but other factors ie total ige and foxp3 showed significant association of cc genotype with asthma. on the other hand, there was significant association of c allele with asthma. 0362 | the disbalance of tlr2, tlr4 gene expression and cytokines production in children with bronchial asthma svitich oa 1 ; gankovskaya lv 2 ; namazova-baranova ls 3 ; bragvadze bg 2 ; alekseeva aa 3 ; gankovskii va 3 objectives: examined were 38 patients with bronchial asthma aged from 3 to 12 years and 10 healthy children of the same age. cytokines were determined by elisa. determination of mrna expression in scrapings from the mucous membranes of the respiratory tract and in peripheral leukocytes was carried out polymerase chain reaction in real time. results: in scrapings from the mucous in patients with moderate to severe asthma found a significant increase in the gene expression of tlr2, 3 times, the gene tlr4 in 10 times in comparison with the control group. in children with severe asthma also found a significant increase in the gene expression of tlr2 4.8 times compared to healthy children, p≤.05). indicators of tlr4 gene expression in this group of patients have a tendency to increase, but not statistically significant. when comparing the indicators of innate immunity in samples with a leukocyte mass in the group of children with severe asthma showed a decreasing expression of tlr2 compared with the index in healthy children. also decreased the expression of tlr4. in children with moderate ba similarly, a significant decrease of tlr4 gene expression in 18 times. the trend towards reduced expression of tlr2 remains in this group, but is not reliable. in washings from the nasopharynx shows that patients with bronchial asthma il-1 is increased 4.5 times compared to the norm, to 45 pcg/ml, tnf increased in 6 times and 18 pcg/mg in severe asthma, and 7.7 pkg/ mg-for mild, il-17 increased in 3 times (16 pcg/mg), pkg of 7.5/ mild blood, il-10 also increased 4.5-fold and equal to 22.5 pkg/mgwith heavier with easy-7.5 pkg/mg, normal à5 pkg/mg. ie is an increase in proinflammatory and anti-inflammatory cytokines. conclusions: the overexpression of tlr2, tlr4 accompanied by increasing, the production of cytokines. this is a violation of mechanisms of innate immunity at the level of the mucous membrane of the nasal cavity on the role of inflammation in the pathogenesis of tlr. 0364 | sustained reduction in risk of experiencing asthma symptoms and using asthma medication in years following grass slittablet treatment-results from the paediatric gap trial were: wheezing, cough (for more than 10 consecutive days), shortness of breath, chest tightness. the odds ratio for having asthma symptoms, using asthma medication, or having asthma symptoms and using asthma medication was significantly lower in the grass slit-tablet group during the 2 followup years. for asthma symptoms, the results were: or=0.52, p=.016 days with csms<2 were defined as "no or minimal symptoms" and days with csms >6 were defined as severe symptoms. csms score was significant lower in the ilit group than in the placebo group for 2014, 2015 and 2016. in 2014 mean csms was 4.05 roth-walter f 1 ; schmutz r 2 ; mothes-luksch n 2 ; zieglmayreer p 2 ; zieglmayer r 2 ; jensen-jarolim e 3 objectives: here, we investigated whether the immune molecule lipocalin 2 (lcn2) may discriminate between allergic and sensitized individuals, and between responding and non-responding patients. results: lcn2-concentrations were assessed in sera of healthy and allergic subjects (n=160) as well as of house dust mite (hdm) allergies that underwent hdm-sublingual immunotherapy (slit) in a randomized, double-blind, placebo controlled trial for 24 weeks. sera pre -, post-slit and at least 3 months after slit were assessed for lcn2 and correlated with total nasal symptom score (tnss) obtained during chamber challenge at week 24 in patients receiving hdm-(n=30) or placebo-slit (n=10). allergic individuals had significant lower lcn2-levels than healthy controls, with women having lower lcn2-levels than men in the patient cohort. hdm-allergic patients who received hdm-slit had a significant increase in hlcn2 6 months after termination of hdm-slit, whereas in subjects receiving placebo no increase in hlcn2 was observed. within the hdm-slit treated group, lcn2-levels were significantly higher in patients whose symptoms improved during slit in contrast to those in which symptoms became more severe. hence, time-course of lcn2 in an allergic individual was predictive to assess clinical reactivity to hdm. objectives: here, we present the benefits of treatment in terms of nnt to prevent one additional child from having asthma symptoms and asthma medication use. children treated with grass slit-tablet had a reduced risk of asthma symptoms and asthma medication use during the 2-year follow-up period compared with placebo (or=0.28 [0.14, 0.57] for sq grass slit-tablet (n=377) vs placebo (n=398), p<.001, relative risk reduc-tion=71%). the risk reduction was independent of age at treatmentstart. younger children had a higher predicted probability of developing asthma symptoms and asthma medication use than older children. thus, the younger the children were at treatment-start, the greater the percentage was prevented from having asthma symptoms and asthma medication use during follow-up off treatment. for children aged 5 at treatment-start, the risk was reduced from 40% to 24% and for those aged 12 it was reduced from 10% to 5%. consequently, the nnt to prevent one additional child from having asthma symptoms and asthma medication use during the 2-year follow-up increased with age, with nnt=6 for children aged 5 and nnt=20 for children aged 12. conclusions: the grass slit-tablet reduced the risk of asthma symptoms and asthma medication use during the 2-year follow-up period; the risk reduction was independent of age. however, the nnt increased with age as younger children had a higher risk of developing asthma symptoms and asthma medication use, emphasising the importance of treatment-start early in life. results: 226 participants were screened for birch and grass allergy, of whom 93 ultimately met randomization criteria and were treated with either slit-t or placebo for 4 months. treatment was preceded by a successful baseline birch pollen challenge in the eeu where a minimum tnss of 6 was achieved in the first 2 of 5 hours of pollen exposure. 87 participants attended the post treatment challenge in the eeu, also 5 hours in duration. no significant differences were noted in the reduction of birch-induced tnss compared to baseline between the slit-t and the placebo treated participants (the primary outcome measure). adverse events with a minimum 5% frequency occurred in 50% of participants in the placebo arm and 70% of participants in the active arm, with upper respiratory tract infection (32% in active arm and 24% in placebo arm) being the most common. oropharyngeal itch was the most common adverse event with causality at least possibly related to study medication (23% in active arm and 2% in placebo). no serious adverse events occurred including no anaphylaxis. objectives: here, we present a pooled subgroup analysis in adolescents from 2 phase ii/iii-iii trials with the hdm slit-tablet (12 sq-hdm dose), p001 in north america and to-203-3-2 in japan. results: to-203-3-2 was a randomised, dbpc phase ii/iii trial investigating the efficacy and safety of the hdm slit-tablet in japanese adolescents and adults (12 to 64 years) with moderate-tosevere hdm ar (n=946, of which 302 were adolescents). subjects were randomised to treatment with the hdm slit-tablet in doses of 6 sq-hdm, 12 sq-hdm or placebo for 1 year. p001 was a randomised dbpc phase iii trial investigating the efficacy and safety of the hdm slit-tablet in north american adolescents and adults (≥12 years) with moderate-severe hdm ar with or without asthma (n=1482, of which 189 were adolescents). subjects were randomised to treatment with 12 sq-hdm or placebo for up to 1 year. in both trials, the primary endpoint was the average total combined rhinitis score (tcrs) during the last 8 weeks of treatment. the pooled analysis was performed on mean values using a linear mixedeffects model. treatment with 12 sq-hdm of the hdm slit-tablet resulted in a statistically significant reduction in the average tcrs of 1.04 (22%, p=.002) compared to placebo in the last 8 weeks of treatment. statistically significant differences were seen for both components of the tcrs, the rhinitis medication score (difference=0.06, p=.038) and rhinitis symptom score (difference=0.87, p=.004). furthermore, treatment with 12 sq-hdm resulted in a statistically significant reduction of the conjunctivitis symptom score compared to placebo (differ-ence=1.10, p=.026). treatment was well tolerated. the most frequent adverse events were mild-to-moderate local allergic reactions. the pooled subgroup analysis showed that the hdm slit-tablet (12 sq-hdm) was effective in treating hdm allergic rhinitis in adolescents (12-17 years old). the reduction in the primary endpoint tcrs was statistically significant and comparable to what has been observed in adults. results: at the time of the data-cut for this analysis, a total of 179 european patients were screened (58% male). 56% of the subjects were aged 4-11, 28% were aged 12-17, and 16% were adults. the mean age of the sample was 11.4 [sd 6.9] years, with a minimum of 4 and a maximum of 49 years. in europe were children. many were allergic to foods other than peanut, and most had at least one other atopic condition. even if allergic to multiple foods, patients and their families were nonetheless keen to participate in the trial. the majority of the screened patients were highly sensitive to peanut, reacting to 144mg (cumulative) or less of peanut protein but a significant proportion of screening failures were due to lack of reactivity to this dose. 0372 | efficacy of 300ir 5-grass pollen sublingual tablet: improvement in subjects with grass pollen-induced allergic rhinoconjunctivitis based on well days and severe days evaluation objectives: four phase ii/iii dbpc studies, 3 in adults and one in children/adolescents, were conducted worldwide. participants were abstracts | 277 randomised to receive the 300ir tablet or placebo starting 4 months (4m) prior to the pollen season and continuing for its duration. data from the first season of the 3 trials in adults were pooled. the well days were defined as those with not more than one moderate symptom or 2 mild symptoms of the 6 evaluated symptoms (sneezing, rhinorrhoea, nasal pruritus, nasal congestion, ocular pruritus, watery eyes) and no use of rescue medication. the severe days were defined as either at least one moderate symptom in subjects using rescue medication, or at least 2 moderate symptoms or one severe symptom whether rescue medication was taken or not. for each subject, the proportions of well days and those of severe days were evaluated during the pollen period while on treatment. treatment groups were compared using a wilcoxon 2-sample test. introduction: the safety of subcutaneous immunotherapy (scit) has been proven in several studies, but the occurrence of side effects (se) remains a concern in daily clinical practice and understanding of their risk factors and avoidance strategies remains limited. objectives: the aim of presented study was to assess the incidence and risk factors of early and late side effects in patients undergoing scit. we conducted a 2 year-long observation of over 1300 patients undergoing scit in our outpatient clinic. we recorded detailed information for each administration and screened subjects for both immediate and late reactions. we compiled the records with medical histories to build a database, which was analyzed using multiple logistic regression. casein is a major cow 0 s milk allergen and very resistant to high temperatures. higher levels of ige towards caseins have been reported to associate with persistent cow 0 s milk allergy and higher risk of adverse reactions before and during the milk oit. a follow-up study on oit to milk started in 2005 with a six-month built-up phase. seventy-six children (mean age 9.7 years) with challenge-verified cma participated the milk oit and their immunological changes were analyzed employing crd. during the year 2016, long-term follow-up report was collected by questionnaires (73%, n=49) and blood samples (46%, n=35). specific antibody responses were characterized before oit and on long term follow up and compared to long-term questionnaire results: milk consumption (yes/no) and side-effects from milk from past 12 months (yes/no). objectives: to define the utility of crd in long-term follow-up after milk oit. results: mean follow-up time was 8 (5-11) years. ten patients (20%) avoided milk completely and 39 patients (80%) reported routine consumption of dairy products. side effects after milk consumption from last 12 months were reported by 39% of the patients. increased specific ige levels towards caseins were seen among patients who did not consume milk at the long-term (p=.03*). there was no significant association between the long-term milk consumption and ige levels to whole milk (p=.08), caseins (p=.06), alpha-lactalbumin (p=.25) and beta-lactoglobulin (p=.08) measured before the oit in this small sample. patients who consumed milk at the longterm follow-up and reported side effects had higher specific ige levels towards caseins before oit (p=.02*). conclusions: there was a high incidence of side effects even after eight years of milk consumption, which may indicate desensitization instead of true tolerance. component-resolved diagnostics may corroborate in predicting prognosis, as suggested by higher incidence of side effects among patients with high levels of ige towards caseins also in the long-term follow-up. objectives: retrospective study of 346 patients who underwent subcutaneous ait for allergic rhinitis (20% had concomitant asthma). patients with less than 4 months of treatment were excluded. large local reactions (wheal≥25 mm) and systemic reactions were defined according to who grading system. patient's satisfaction was defined by vas score. results: the mean age of patients (54% males) was 28ae13 (range 5-72) years (11%<16 years).the most prevalent immunizing allergens were house dust mite (92%), olive (26%) mix of grasses (23%) and pets (17%). 47% of patients were immunized against a single allergen whereas 12% were immunized with≥4 allergens. patients were followed for 24ae18 months. following ait the vas score decreased from 7.2ae2.3 to 3.6ae2.5 (p<.0005). 92% of the patients declared that they will recommend immunotherapy to their relatives. systemic adverse reactions (37% class i, 62% class ii) were observed in 120 introduction: metabolomics, one of the core disciplines of system biology, is a high-dimensional biology method that may allow hypothesis-free profiling of biomarkers, rather than a traditional hypothesis-driven approach. objectives: this study aimed to apply the metabolomic approach to serum to longitudinal alterations during allergy immunotherapy (ait) in dermatophagoides pteronyssinus (der p) sensitized asthmatic children. results: a robust hydroxyeicosatetraenoic acids (hetes) of inflammatory responses was found for discriminating during ait, including 5-hete, 12-hete and 15-hete. 12-hete and 15-hete were significantly decreased continuously from 6 through 12 months of ait. moreover, compared with baseline of ait 5-hete was detected in very low level during 6 and 12 months. the metabolomic profiling could clearly longitudinal alterations biochemical-metabolic profiles in der p-sensitized children during ait. these markers might be involved in asthma pathophysiology but also represent the therapeutic target for ait. background: metabolomics, one of the core disciplines of system biology, is a high-dimensional biology method that may allow hypothesis-free profiling of biomarkers, rather than a traditional hypothesis-driven approach. this study aimed to apply the metabolomic approach to serum to longitudinal alterations during allergy immunotherapy (ait) in dermatophagoides pteronyssinus (der p) sensitized asthmatic children. methods: in this longitudinal study, we recruited 58 der p-sensitized asthmatic children with ait for 12 months. serum samples were analyzed using a metabolomic approach based on mass spectrometry. results: a robust hydroxyeicosatetraenoic acids (hetes) of inflammatory responses was found for discriminating during ait, including 5-hete, 12-hete and 15-hete. 12-hete and 15-hete were significantly decreased continuously from 6 through 12 months of ait. moreover, compared with baseline of ait 5-hete was detected in very low level during 6 and 12 months. the metabolomic profiling could clearly longitudinal alterations biochemical-metabolic profiles in der p-sensitized children during ait. these markers might be involved in asthma pathophysiology but also represent the therapeutic target for ait. we performed desensitization with asa according to the wong protocol (doses of 0. 1, 0.3, 1, 3, 10, 30, 40, 81, 162, 325 mg of asa at intervals of 10-20 min). we pre-medicate with cetirizine 10 mg. our patient successfully reached the necessary dose: 164 mg of asa (0. 1, 0.3, 1, 3, 10, 30, 40, and 80 mg) conclusions: approximately 10% of patients with asthma undergo respiratory symptoms due to exposure to aspirin, meanwhile 0.07% and 0.2% of the population shall undergo hives when exposed to it. desensitization with aspirin is an efficient and safe treatment in patients with hypersensitivity type i, iii and iv. due to the benefits and low toxicity of the wong protocol, we recommend this protocol in order to desensitize patients with allergy to aspirin who undergo rheumatologic or cardiovascular disorders and need antiplatelet treatment. we finally recommend our patients to take 150 mg of asa premedicated with 10 mg of cetirizine every day. we warn that if you interrupt the administration of asa for more than 48 hours, it shall have to be administered again under medical supervision. semedo fm 1 ; cruz c 2 ; reis r 2 ; tomaz e 2 ; in acio f 2 horiuchi t 1 ; takazawa t 2 ; saito s 1 1 department of anesthesiology, gunma university hospital, maebashi, japan; 2 intensive care unit, gunma university hospital, maebashi, japan introduction: sugammadex is a synthetic c-dextrin derivative that is designed to selectively bind to steroidal neuromuscular blocking agent molecules. although sugammadex has been used in many cases of general anesthesia, there are several reports of anaphylaxis following its use. skin testing is the gold standard for detecting the causative agent of anaphylaxis. however, the test itself sometimes precipitates serious complications, including recurrence of anaphylaxis. hence, development of a novel test that can be performed in vitro without causing such complications is desired. recently, the basophil activation test (bat) has been established as a tool to detect the causative agent of anaphylaxis with high sensitivity and specificity. yet, there are few studies examining the utility of the bat in diagnosing sugammadex-induced anaphylaxis, besides our previous report. although both cd63 and cd203c are currently used as the major markers for activated basophils, which one of them is more suitable for the bat depends on the targeted drugs. objectives: the aim of this study was to investigate whether bat could be utilized to diagnose sugammadex-induced anaphylaxis. in addition, we compared the capability of cd203c and cd63 as markers for activated basophils. seven patients with perioperative anaphylaxis demonstrating a positive skin test for sugammadex were included. furthermore, 21 individuals who tolerated sugammadex and had a negative skin test for allergy to this drug were enrolled as controls. results: the ratios of activated basophils in the patients were much higher than those in controls (mann-whitney u test, p<.005). cd203c up-regulation. this was also true for cd63. in the case of cd203c, the sensitivity of bat for sugammadex was 83% and specificity was 100%, while sensitivity and specificity for cd63 were 71% and 100%, respectively. there were no significant differences between cd203c and cd63 in the areas under the roc curve. conclusions: this study showed that bat is a reliable instrument to diagnose sugammadex-induced anaphylaxis. we did not find any difference between cd203c and cd63 as markers for activated basophils in the bat for sugammadex. objectives: we report a retrospective study of 10 patients who were referred to our allergology departments between 2012 and 2016 with a suggestive history of immediate hypersensitivity to ppis. our purpose was the analysis of the clinical presentations and the allergological investigation performed in order to confirm the diagnosis of drug hypersensitivity to ppis and to study the cross-reactivity among ppis. results: the culprit drugs were pantoprazole (n=5), omeprazole (n=3) and lansoprazole (n=2). the allergological investigation confirmed the diagnosis of hypersensitivity to ppis in 5 patients (4 by skin tests and 1 oral challenge). we observed cross-reactions between omeprazole, pantoprazole and esomeprazole (n=1), omeprazole and pantoprazole (n=2), respectively omeprazole and esomeprazole (n=1). in 5 patients the severity and the recurrence of the reaction did not allowed the provocation test with the culprit drug. two oral challenges allowed the use of an alternative ppi (based on the pattern of less cross-reactivity depending on the chemical structure: pantoprazole for a patient who had a grade iii anaphylaxis to lansoprazole and lansoprazole for a patient who had a grade ii anaphylaxis to pantoprazole). conclusions: a complete allergological investigation (skin tests and cautiously, oral challenge) is needed in order to confirm the diagnosis of drug hypersensitivity to ppis and to take a therapeutic decision. the diagnostic approach is limited by the low sensitivity of skin tests and the patient's background (comorbidities and severity of the reaction) which do not always allow the oral provocation test. an ige dependent mechanism may be involved in hypersensitivity reactions to ppis or their metabolites. herrero-lifona l 1 ; muñoz-rom an c 2 1 hospital quironsalud campo de gibraltar, los barrios, spain; 2 hospital regional universitario de m alaga, m alaga, spain introduction: hypersensitivity reactions to beta-lactams are an important problem to study, especially in children, given that these antibiotics are the gold standard for the treatment of many infectious diseases in the infancy. most hypersensitivity reactions to betalactams in children are due to non-immediate response and to diagnose them is essential to perform a drug challenge test. although hypersensitivity is usually ruled out in children by drug challenge test, it is positive test up to 5%-10%. objectives: a 6 year-old boy is suspected to have experienced two drug reactions after the intake of amoxicillin with and without clavulanic acid for pharyngitis treatment. in the first one, he presented a maculopapular eruption in the back after one day treatment with amoxicillin-clavulanic acid. in the second reaction, two hours after the intake of amoxicillin, it appeared an itching maculopapular eruption in the back that was resolved with symptomatic treatment. in both cases, the patient tolerated treatment with cefuroxime afterwards. results: intradermal test with amoxicillin was negative in immediate and delayed reading. oral drug challenge test with amoxicillin (50 mg/kg/day, total cumuoral drug challenge test with penicillin v (total cumulative dose 250 mg): it was well tolerated and the patient continued taking it for epicutaneous patch testing with amoxicillin in the lesion area and in an unaffected area: in the lesion area, it appeared mild erythema, but it was considered a negative result in both areas. conclusions: we report a case of an amoxicillin-induced multiple fixed exanthema: an unusual non-immediate reaction in childhood. most cases of non-immediate hypersensibility need to be confirmed by drug challenge test, given that skin testing lacks sensitivity both intradermal and patch tests. the patient presents a selective hypersensitivity to amoxicillin, being tolerant to penicillin and cephalosporins. jimenez-rodriguez t 1 ; soriano-gomis v 1 ; gonzalez-delgado p 1 ; cueva-oliver b 1 ; venegas-diaz ij 1 ; fernandez j 2 results: data from 62 patients were analyzed, of which 64.5% (40) were women. the overall mean age was 46.1ae16.9 years, with no statistical difference between sexes. the 69.4% (43) of the patients presented symptoms with a single group of nsaids, and 30.7% (19) with 2 or more different groups. the 90% (56) of patients presented a history of atopy. in patients with rhinitis 58% had symptoms with only one nsaid and 42% with two or more groups of nsaid, while patients with asthma, 83% reacted to one nsaid and 17% to two or more groups. the most frequent clinical manifestations were: urticaria/angioedema in 71% (44), pruritus in 35.5% (22), bronchospasm in 19.4% (12), other respiratory symptoms in 16% (10) and anaphylaxis in 13% (7) patients. the most frequently involved nsaids were: metamizole (46.8%) and ibuprofen (37.1%). skin tests were performed in only 24 patients, of whom 13 (54%) were positive to metamizol. dpt was performed in 66.1% (41) objectives: fifty patients diagnosed with "dress" (2012-2016), in a tertiary center were retrospectively analyzed, with 31 cases meeting the regiscar criteria. we collected demographic, clinical, laboratory and therapeutic data from the electronic medical records. results: all cases occurred during hospitalization and in several hospital areas. the mean age was 49.16 years (7-94) with 58.06% women and 95% of caucasian origin. reported clinical manifestations were skin rash (97%), fever (48.39%), digestive symptoms (22.58%), respiratory (12.9%), neurological (9.68%), head and neck (6.45%), urological (6.45%), ophthalmologic (3.23%) and musculoskeletal (3.23%). the most relevant laboratory findings were eosinophilia (77%), elevated transaminase levels (77%), lymphocytosis (53%), altered coagulation (34%) and altered renal function (32%). virus serology was positive in 4 cases (12.9%) involving hhv-6, ebv, cmv and hiv. antinuclear antibodies were positive in 2/13 cases (15.38%). skin biopsy, performed in 12 cases (38.7%) revealed findings suggestive of drug induced skin reaction. suspected culprit drugs were antibiotics (45%), nsaids (26%), antiepileptic drugs (13%), and antiparasitic agents (10%). one case was related to the administration of heparins and other to a monoclonal antibody. in 10% of cases, the episode was not related to a particular drug. treatment was accomplished by the administration of corticosteroids (97%), antihistamines (96%), and immunosuppressant drugs in 2 cases. fluids and other medications were administered attending to symptoms. death occurred in 3 patients, although only in 1 case it was related to dress syndrome. the average time from reaction to death was 4.33 months. allergy evaluation, performed in 11 cases addressed the potential role of drugs. skin tests were positive in 3/11 patients (28.6%) and basophil activation test was negative (2/2 cases). results: from 17 patients admitted with possible diagnosis of scar, only 8 met the inclusion criteria (6f/2m, age 15-81, average 49.1 years-old). they were all admitted to a medicine ward. five patients had diagnosis criteria of dress, 2 of sjs and 1 of ten. as for the drugs related with the reaction, antiepileptic drugs were the most frequent (4 patients); allopurinol (1), betahistine (1), ciprofloxacin+nimesulide (1) were the causative drugs in the remaining patients. average time to the beginning of symptoms was 30.7 days (2 patients unknown). in 1 patient the cause was unknown (he had criteria of ten and also diagnosis of malaria and was transferred to another hospital). there were no deaths. objectives: the objectives of the present study were to determine the efficacy of nfeno to: 1. establish the diagnosis of rhinitis; and 2. discriminate allergic rhinitis (ar) from non-allergic rhinitis (nar). material and methods: prospective and controlled study with healthy subjects, which included patients with rhinitis. ar and nar were phenotypically defined according to positive or negative prick test results, respectively; the control group was collected from people without upper or lower respiratory tract disease and the prick test was negative. in all sample included the following measurements were performed: feno and nfeno (novario analyzer) were performed; spirometry; eosinophil counts in blood and nose (by nasal brushing); bilateral nasal endoscopy; and acoustic rhinometry. results: we included 147 cases (ar=61, nar=53 and controls=33), with a mean age of 37 (sd 11.2) years, 66% men. the table below shows the results of the variables analyzed by group. patients with rhinitis compared to controls had significantly greater feno production, as well as a higher proportion of eosinophils in blood and nose, but not in nfeno production. however, when the two subgroups of rhinitis were compared, ar patients had significantly higher feno and nfeno than nar, in addition to blood and nose eosinophils. there were no differences in acoustic rhinometry values between groups. conclusions: although nfeno does not appear to identify patients with rhinitis, it may be useful in discriminating ar from nar. in routine clinical practice, it could help guide to identify an ar in cases with inconclusive results of the complementary tests commonly used in its diagnosis. introduction: fractionated exhaled nitric oxide (feno) is used as a marker of eosinophilic airway inflammation in asthma, whereas clinical presentation of nasal no (nno) is unknown. objectives: the objective of this study was to evaluate the factors influencing nno levels. in patients with chronic nasal symptoms, total-nasal-symptom-scores (tnss) were calculated; skin-prick-test conclusions: in conclusion nasal no is useful in allergic inflammation in the absence of sinus obstruction in nasal cavity. however, its value is limited with paranasal sinus ostium occlusion. were recruited across australia and the uk via a patient panel. the aim was to assess which ar treatment attribute(s) drove patient preference. results: table 1 shows the willingness to pay (wtp) for each attribute. all attributes were significant predictors of treatment choice, except administration method. however, patients in both countries showed a considerable preference for treatments that were more efficacious, fast acting and affordable. conclusions: although treatment relief remains of primary concern, time to treatment benefit and cost could dictate treatment preference. these data may be of value in optimizing the acceptability of future ar treatments and informing trial endpoint selection. australia ( izquierdo l; chiriac am; molinari n; demoly p introduction: allergic rhinitis is a frequent disease with an important impact on quality of life. self-administrated control assessment tests can help achieve a better management of this disease. however, none of these tools has been validated in teenagers. test in its original version, following the same protocol as the original study in adults. we designed a multicentre, observational, crossconclusions: while the number of included patients is low, the first results are promising. indeed, a significant improvement of the rhinitis control score was found between d1 and d15. this led us to the hypothesis that this questionnaire could be used as is to estimate the control of the allergic rhinitis in teenagers. analyses concerning the validation of the questionnaire itself do not reach at the moment the threshold of significance, the recruitment is on-going, to increase its power. objectives: the objective of this study was to evaluate the role of serum vitamin d in patients with symptomatic allergic rhinitis and active asthma during the allergy season and observe the effect of montelukast 10 mg daily as treatment. results: this study included 130 asthmatic and seasonal allergic rhinitis patients following a single-blind, placebo run-in period of 3-5 days, patients were randomized to oral montelukast 10 mg (n=70) or placebo (n=60) daily during the 2-week, double-blind, active-treatment period. the serum vitamin d was also evaluated in both the groups. the serum vitamin d levels were found to be higher in patients taking montelukast compared to placebo after 2 weeks (p<.001). montelukast reduced the daily rhinitis symptoms score: difference between montelukast and placebo (p<.001). similar improvements were seen in daytime nasal symptoms (p<.001) and nighttime symptoms (p<.001). conclusions: montelukast provides significant relief from symptoms of seasonal allergic rhinitis, while also conferring a benefit for asthma, in patients with both allergic rhinitis and asthma. further, it has a beneficial role in improving vitamin d levels. venegas-diaz ij 1 ; jimenez-rodriguez t 1 ; canto-reig vj 1 ; lindo-gutarra m 1 ; soriano-gomis v 1 ; gonzalez-delgado p 1 ; cueva-oliver b 1 ; fernández j 2 1 allergy section. general hospital alicante. isabial, alicante, spain; 2 allergy section. general hospital alicante. isabial-umh, alicante, spain introduction: local allergic rhinitis is an accepted entity, which only can be recognized by nasal provocation test (npt) in patients with clinical rhinitis with negative skin tests or specific ige (sige). our aim was study this entity in our patients in alicante, spain. objectives: 100 patients with symptoms of rhinitis with skin tests, and sige negative for common aeroallergens were selected, since 2015. half of them (47) most were young adults, 50% belonging to the range of age between 18-30 years; more than 43% had family history of atopy; the mean age of onset of symptoms was 25 years and up to 18.7% had had symptoms before the age of 14 years; 56.2% were nonsmokers. regarding comorbidity, 62.5% of the patients presented with concomitant symptoms of conjunctivitis and 6.2% of asthma. symptoms in 93.7% of the patients were persistent and most of moderate (50%) to severe (43.7%) intensity. the majority of patients (87.5%) presented perennial symptoms and a tpn positive to mites in 37.5%, to salsola pollen in 25% and to cypress pollen in 12.5%. but 3 patients (18.75%) were simultaneously positive to 2 allergens. conclusions: local allergic rhinitis represents an important proportion of patients with clinical rhinitis with negative skin tests and sige. we have to pay attention to identify clinical and demographic factors of ral and to use npt to demonstrate it. objectives: we report the extent of sinus involvement at baseline (bl) in pts with bilateral np refractory to intranasal corticosteroids from a dupilumab phase 2a study (nct01920893). ct scans from enrolled pts, 59 adults <65 years with nasal polyp score of ≥5/8, were pooled and analyzed at bl. for opacification, standard lund-mackay (lmk) scoring (0=normal, 1=partial opacification, 2=total opacification) in maxillary, anterior/posterior ethmoid, sphenoid, frontal sinuses was used. ten sinus scores plus bilateral ostiomeatal complex (omc) score (0=not occluded, 2=occluded) were summed to a bilateral total lmk (0-24). zinreich modified lmk score (zlmk), providing more granularity on opacification degree, was evaluated post-hoc; each sinus was given a 0-5 score based on opacification % from mucosal thickening (0=0%, 1=1%-25%, 2=26%-50%, 3=51%-75%, 4=76%-99%, 5=100%); omc results: the prevalence of asthma at the age of 12 years was 6.4%. 15% of them had an act tm value below 20, ie uncontrolled asthma, median 22.0 (range 12-25). independent risk factors for uncontrolled asthma at the age of 12 were current doctor diagnosed rhinitis (adjusted or, aor 2.8; 95% ci 1.1-7.0), wheeze triggered by exercise (aor 5.6; 1.9-16.6) and cat at home (aor 3.5; 1.2-10.0). if at least one of the parents had higher education the risk of uncontrolled asthma was decreased (aor 0.3; 95% ci 0.1-0.8). six children reported hospitalisation due to asthma during the last 12 months (ie 2.6%) at 12 years of age. hospitalisation was more common in individuals with uncontrolled asthma (or 12.3; 2.2-70.5) or when mites (or 9.8; 1.9-51.5) or pollen (or 5.8; 1.0-32.5) was reported as trigger factors. also, oral corticosteroids (betamethasone), in the last 12 months increased the risk for hospitalisation (or 8.2; 1.4-48.7). to have a parent with asthma (17% compared to 37%, or 0.3; 0.04-2.9) or higher education (40% compared to 62%, or 0.3; 0.07-2.5) was numerically less common for children who were hospitalised for asthma but did not reach statistical significance. conclusions: uncontrolled asthma was associated with current allergic rhinitis, having a cat and exercise as trigger factor. at least one parent with higher education reduced the risk. hospitalisation due to asthma was more common in individuals with uncontrolled asthma. abstracts 0404 | adolescent asthma in relation to severity and gender-data from a prospective population based cohort study introduction: asthma often debuts early in life, but recent studies show that the development of asthma is a dynamic process with disease turnover throughout child-and young adulthood. objectives: to describe adolescents' asthma with focus on severity and gender. the study population consisted of 3115 adolescents participating in the cohort, followed since birth up to 16 years of age with questionnaires and clinical investigations. blood samples from 2452 adolescents (78.7%), sera analyzed for specific ige against common inhalant allergens. asthma at 16 years was defined as fulfilling at least 2 of the following 3 criteria: symptoms of wheeze and/or breathing difficulties in the last 12 months, ever doctor's diagnosis of asthma and/or asthma medicine occasionally or regularly last 12 months. uncontrolled asthma was based on parental information on symptoms of asthma in the last 12 months prior the 16-year follow-up, including 3 or 4 features: nocturnal asthma, activity limitation, wheeze 4 times and hospitalization due to acute asthma. we looked into 2 phenotypes; late-onset, defined as asthma at 8, 12 or 16, but not at 1, 2 and 4 years of age and persistent, defined as asthma at 1, 2 or 4 and 8, 12 or 16 years of age. severe asthma, defined as asthma as above combined with prescribed and dispensed corticosteroids used within the last 18 months plus uncontrolled asthma and/or lung function (fev 1 <80% of predicted). results: at 16 years of age, 14.2% (n=437) fulfilled the study definition of asthma, 49.3% late onset and 50.7% persistent asthma. persistent asthma was more frequent among boys than girls 59.1% vs 43.3% (p=.001). overall ige sensitization was common among adolescents with asthma, 70.1% were sensitised to common inhalant allergens, and equally common in late-onset and persistent asthma (71.7% vs 68.8%, p=.54). however, more boys than girls were sensitised (79.9% vs 62.6%, p<.001). in total 8.1% (n=25) adolescents had severe asthma, 11.2% (n=15) boys and 5.8% (n=10) girls (p=.08). the overall prevalence of severe asthma in the cohort was estimated to 1.0%. severe asthma did not significantly differ among adolescents with late-onset compared to persistent asthma (17/25, 8/25, p=.09). conclusions: among adolescents with asthma, late-onset (age ≥8 years) and persistent asthma were equally common. persistent asthma were more common among boys. there were no difference in asthma severity, or proportion of ige sensitization among adolescents with late-onset or persistent asthma. however, data investigating associations with wheeze and asthma in later childhood are scarce. objectives: our aim was to explore the association of maternal milk fatty acid composition with childhood wheezing phenotypes and asthma up to age 13 years. breast milk was collected 6 weeks and 6 months post-delivery in the ulm birth cohort study (n=720 and n=454, respectively). concentrations of 10-24 carbon atom chain length fatty acids were measured by high-resolution capillary gas-liquid chromatography. to control for constant sum constraint, concentration data were transformed using the centered log ratio method. compositional biplots and correlation matrices were used to group fatty acids based on within sample correlation. adjusted risk ratios with parent-reported wheezing phenotypes and doctor-diagnosed asthma were computed using a modified poisson regression. results: we observed no straightforward evidence of associations between overall breast milk fatty acid composition and specific wheeze phenotypes or doctor-diagnosed asthma. conclusions: despite our use of sophisticated statistical methodology, our results may have been biased toward the null by several cohort-specific factors associated with breast milk collection and fatty acid composition. to overcome potential selection bias by maternal lifestyle, further research should investigate fatty acid intake during the first year of life including sources other than breast milk among children who were never or not exclusively breastfed. introduction: fall is the most common season for asthma exacerbation. previous studies found that exposure and sensitization to house dust mites (hdms) can exacerbate asthma. the seasonal variation in indoor hdm concentration is highest in the fall, correlating with the incidence of asthma exacerbation. most of the studies conducted to date have focused on the effect of hdm exposure on the incidence of acute asthma exacerbation, but reports about the effect of hdm sensitization are few. thus, we aimed to determine whether sensitization to hdms acts as a risk factor of asthma exacerbation in the fall. in addition, we investigated whether asthma exacerbation in the fall had any distinctive features by comparing levels of various cytokines and chemokines with those in other seasons. objectives: we enrolled 55 children aged 3-14 who visited the emergency department because of acute asthma exacerbation from january 2008 to december 2013 (63.6%, males; mean age, 6.0ae2.8 years). they were treated in accordance with the standardized treatment protocol. blood samples were collected from the children during the course of treatment. by using residual sera from the blood samples, we measured the levels of total immunoglobulin e (ige), hdm-specific ige (sige), eosinophil cationic protein (ecp), and various cytokines and chemokines, and classified them according to season. we compared the date divided into fall group (from september to november, n=31) and other season group (from december to august, n=24). ci, confidence interval; or, odds ratio; der f-sige, dermatophagoides farinae -specific immunoglobulin e; der p-sige, dermatophagoides pteronyssinus-specific immunoglobulin e; ecp, eosinophil cationic protein; ige, immunoglobulin e. data are presented as n (%) or as meansaesems and median (range). p value refers to the difference between the fall and "other season" groups and was calculate by the chi-square statistics, fisher's exact test, student's t-test, or wilcoxon rank-sum test. a adjusted for total ige. results: this retrospective study obtained archived nasopharyngeal aspirate (npa) samples from patients aged below 18 years who were hospitalized for acute respiratory illnesses in a university-affiliated hospital during the periods september-november 2014 and january-april 2015. their clinical information was retrieved from computerised record. hrv was detected by rt-pcr, and isolates were sequenced to determine the genogroups and serotypes. ninety patients whose npa was positive for hrv and 160 patients being negative for an extended panel of respiratory viruses by multiplex pcr method were identified. mean age of these groups was 3.6 years and 3.5 years respectively. hrv infection was significantly associated with asthma exacerbation (or 16.54, results: a total of 203 children were included: 73.4% were boys; 26.6% aged 6 months to 2 years, 51.0% aged 3-6 years, 20.1% aged 7-13 years and 2.4% aged 14-18 years; 44.0% were hospitalized, results: the long-term remission (≥5 years without treatment) rate 16 years after initiating early anti-inflammatory therapy was 88.1% (intermittent asthma, 100%; mild persistent asthma, 72.2%; moderate persistent asthma, 90.0%; severe persistent asthma, 66.7%). longterm remission rates improved compared with past asthmatic convaobjectives: the aim of this study is to explore the potential value of feno level for diagnosing chronic cough in children. objectives: in this study, we aimed to compare the efficacy of classical spirometry and impulse oscillometry (ios) in evaluation of late reversibility in children who received treatment with the diagnosis of atopic asthma. we enrolled 83 patients aged 7-17 years who were diagnosed with atopic asthma. exclusion criteria were having received asthma treatment during the previous two months, having acute asthma exacerbation findings, having any other respiratory disease or cardiac disease that may affects the lung function test results. allergic sensitization was determined by skin prick test performed according to eeaci guidelines. lung function test measurements were performed at enrollment and after two months of inhaled steroid treatment. conclusions: classical spirometry is more valuable compared to ios in evaluation of late airway hyperreactivity in children with atopic asthma in children older than seven years age. 0413 | computer bronchophonographyfrequency analysis of the respiratory cycle. objectives: we performed mct in 144 children with symptoms suggestive of asthma and 30 without respiratory symptoms. after each inhalation step oscillometry and spirometry was performed. parameters analysed for ios were z5, r5, r20, x5, x15 and ax (the integrated impedance reactance at r5 and above) and fev1 for spirometry. a fall of 20% in fev1 from baseline after mtc was considered as a positive challenge. pc20-fev1 and pc40 r5, x5 and ax were calculated. results: a total of 174 patients, 79 female, with a mean age of 9.5 (ae3.03) years were enrolled. 132 had a ≥ 20% fall in fev1 after mtc. the mean variation in fev1 was 7% (-12.2%/ +45.8%), the mean variation in z5, r5, r20, x5, x15, ax and fres were 21.88% objectives: the objective of our study was to synthesize cationic peptides and study their antiviral activity (aa) in vitro. results: 16 peptides were synthesized by solid phase method with different structures (linear, helical and dendrimeric). cytotoxicity of the peptides was studied by mtt assay using hela cells. objectives: the aim of the study is to evaluate the changes of ilresults: 48 subjects aged 18-65 years were enrolled in this study, which were divided into 4 groups: 23 patients with diagnosed moderate to severe ba (1st group), 6 ba patients with rvi (2nd group), 8 subjects with rvi only (3rd group) and 11 healthy volunteers (4th group). all patients with ba from group 1 and 2 received inhaled corticosteroids. clinical blood test revealed increase of eosinophils in patients with ba and ba accompanied with rvi up to 7% and 4%, respectively. fev1 was decreased in 1st and 2nd groups to 77% and 79% compared 98% and 97% for 3rd and 4th groups, respectively. conclusions: this study is alarming for asthmatic nosocomial hazards in this govt. hospital. identification of ige specific reactive components of predominant fungal allergens and cross-reactivity among each other, delined in this study could minimize the hazard of therapeutic and diagnostic use of these cross-reactive components, in fungal allergen-specific immunotherapy. objectives: we conducted a longitudinal prospective study to examine the development, composition and diversity of the gut microbiota in healthy and allergic children. we followed 93 children from 4 months to 8 years of age with clinical evaluation; specific ige levels and skin prick testing. fecal samples were collected at 4, 6, 13 months and 8 years. 16s rrna sequencing was used to profile the gut microbiome using illumina miseq. the composition and diversity of the gut microbiome were assessed using quantitative insights into microbial ecology (qiime). comparisons between groups were made using the lefse pipeline; non-parametric factorial kruskal-wallis test, unpaired wilcoxon rank test and linear discriminant analyses (lda) with score >2.0. to assess the interaction effect, the likelihood ratio test (lrt) was applied using r statistical program. p<.05 was considered significant after correction for multiple testing. results: to achieve our aim we divided balb/c mice into 4 groups: mice with viba (1st group), mice with viba treated with nonspecific sirna against gfp (sigfp) (2nd group) and against il-33 (siil-33) (3d group). 4th group was intact mice. groups 1-3 were i.p. sensitized on days 1, 14, 28 with ovalbumin (ova) mixed with aluminum hydroxide and i.n. challenged with ova on days 40-42. the same mice were i.n. infected with 5910 6 tcid 50 /mouse rsv strain a2 on day 39. mice from group 2 and 3 were i.n. treated by sirnas on days 38-42 in dose 120 lg/mouse. on day 43 hyperresponsiveness (ahr) to methacholine was measured. on day 44 and lungs were removed for histological analysis. viral rna (vrna) load and il-33 gene expression were evaluated by qpcr in lungs. bronchoalveolar lavage (bal) was collected for differential cell count by light microscopy. so i.n. administration of siil-33 suppressed il-33 gene expression in lungs by 50% compared to sigfp treated mice. there were no significant changes in body weight and lung vrna amounts between mice received sigfp and siil-33. mice treated with siil-33 demonstrated the tendency to improve lung function compared to mice of group 1-2, that expressed in 13% reduction of specific resistance of airways and 15% increase of peak expiratory flow. bal cell count revealed decrease of total cell number, eosinophils and lymphocytes in mice received siil-33 by 55%, 90% and 63% compared to sigfp treated mice, that indicate reduction of inflammation, that was confirmed by histopathological studies showed 50% leukocyte reduction. downregulation of il-33 resulted in 2-and 1.7-fold decrease of bronchial epithelium metaplasia and hyperplasia. and femur length measurements were collected from routine antenatal screenings. these and derived head to abdominal circumference ratio and estimated fetal weight were converted into z-scores adjusted for gestational age and gender and categorized as "low" (≤1 sd below mean), "normal," or "high" (≥1 sd above mean). ad cases were children with parent-or pediatrician-report of physician ad diagnosis assessed yearly up to age 3 years and supplemented by clinical diagnoses during dermatological exams at 0.5, 1, and 2 years. modified poisson regression models were used to compute risk ratios (rr) adjusted for potential confounders. conclusions: these results provide further evidence for a role of fetal growth as an influence on atopic disease outcomes. unlike previous studies, our data suggests several patterns of fetal growth beginning as early as the 1st trimester may influence ad outcomes. objectives: we aimed to examine the role of eosinophil cationic protein (ecp), eosinophil derived neurotoxin (edn) and total immunoglobulin (ig) e as a bio-marker of disease severity. we examined the difference in level of total ige, ecp and edn between the two groups and whether any correlation existed between disease severity and ecp or edn. objectives: we aimed to identify the subgroup of ad patients with a good clinical response to probiotic treatment. we recruited children who suffered from moderate to severe ad with the scoring ad (scorad) index of 20 or higher. after 2 weeks of washout period, all patients were given lactobacillus plantarum cjlp133 at a dosage of 1910 10 colony-forming units once a day for 12 weeks. we measured eosinophil counts in the peripheral blood, the proportion of cd4 + cd25 + foxp3 + regulatory t (treg) cells in cd4 + t cells, serum total ige levels, and specific ige to common allergens before the start of the treatment (t1) and at discontinuation (t2). logistic regression models were used for the statistical analysis. seventy-six patients (48 boys and 28 girls) with a mean age of 8.7ae4.7 years completed the study. there were 36 responders and 40 non-responders after probiotic treatment. the median scorad was reduced from 29.5 (range 20.6-46.3) at t1 to 16.4 (range 6.3-30.8) at t2 in the responder group (p<.001). in multivariable logistic regression analysis, a good clinical response was significantly associated with high total ige levels (aor 5.1, 95% ci 1.1-23.6), increased expression of , and high proportion of cd4 + cd25 + foxp3 + cells in cd4 + t cells (aor 3.7, 95% ci 1.1-12.7). in responder group, the proportion of cd4 + cd25 + foxp3 + cells of cd4 + t cells were significantly increased after 12 weeks of treatment (p=.004), while the levels of tgf-b mrna expression were decreased (p=.017). there were no differences in total ige levels between t1 and t2 (p=.414) conclusions: the therapeutic effect of l. plantarum cjlp133 on ad is more pronounced in children with high total ige levels, objectives: the objective of the study was to examine the effect of a specific synbiotic mixture of short-chain galacto-, long-chain fructo-oligosaccharides (scgos/lcfos, ratio 9:1) and bifidobacterium breve m-16v on the severity of ad and correlation to serum chemokines in infants with moderate to severe ad and elevated ige. in an exploratory randomized, double-blind, placebo-controlled trial the effect of extensively-hydrolyzed whey-based formula without intervention. serum obtained prior to start and at the end of intervention were analysed using luminex. six chemokines and nine ratio's thereof were correlated to ad severity (sample size=24). introduction: dyshidrotic eczema is one of the most common skin conditions. contact allergy is often associated with dyshidrotic eczema although the exact impact and the influence of contact allergens in different forms of dyshidrotic eczema remain unknown. hypersensitization to nickel is one of the most common contact allergies associated with pompholyx. the standard of care protocol is to use a medical treatment with topical corticosteroids and calcineurin inhibitors to treat the symptoms, together with occlusive barrier creams to avoid skin exposure to the allergens. after the symptoms have been cleared with the topical treatment, the recommendation is to use occlusive barrier creams to prevent recurrence of the symptoms. objectives: a new emollient with specific metal-scavenging agents and no occlusive ingredients has recently been developed and made commercially available. the aim of this study was to evaluate the effect of such cream to provide relief for patients with dyshidrotic eczema associated with nickel allergy. results: thirty-two subjects with dyshidrotic eczema and a positive patch test ppt (contact sensitized) reaction to nickel were selected. these were divided into two randomized groups, group-a was given nickel-scavenging cream (skintifique creamtm, paris) after medical treatment, (n=9) and group-b followed the standard protocol for pompholyx, (n=23). hand eczema was scored according to the dyshidrotic eczema area and severity index (dasi). dasi scores were evaluated at the beginning of the study (day-0), after the medical treatment (day-15) and two months after the end of medical treatment (day-75). results show a significant difference in the efficacy of treatment between the two groups at day-75. a higher percentage of at least 75% reduction of initial dasi score (77.8%) and a higher percentage of total clearance (56%) in patients using nickel-scavenging nonocclusive moisturizing cream was observed as compared to standard-of-care occlusive creams (26.1% and 22%, respectively). objectives: the goal of this study was to investigate whether long-term emollient therapy is associated with alterations of skin barrier function and shifts of the skin microbiome in infants at high risk for developing ad. we prospectively enrolled newborns with a family history of ad to be randomized to either emollient treatment group or control group. at 6 months of age, we tested the skin barrier (transepidermal water loss/tewl, water capacitance/cap, ph) and skin microbiome (16s rdna sequencing of skin swabs from cheek, dorsal and volar forearm). results: the emollient group (n=10) had significantly lower skin ph compared to controls (n=9) (p=.02), but without a statistically significant difference in tewl or cap. the emollient group had higher numbers of different bacterial taxa (chao richness) at cheeks (p=.003), dorsal forearms (p=.008), and volar forearms (p=.003) as compared to controls. both streptococcus pneumoniae and s. salivarius statistically significantly contributed to the observed skin microbiome differences between patient groups. s. salivarius was significantly more abundant in emollient subjects at all sampling sites (p=.02). we then analyzed our previous larger cohort of older children with ad and also observed higher s. salivarius proportions in ad patients with treated and less severe disease (p=.01). objectives: to evaluate the effect of overnight treatment with a temperature-controlled laminar airflow (tla) device in children/adolescents with severe eczema over a 12-month period. in an open-label study, 15 subjects aged 2-16 years (median 10 years) with longstanding severe eczema attended 3 visits during the run-in period lasting 6-10 weeks (median 7.14 weeks) to optimize eczema management. the run-in was followed by a 12month treatment period using overnight tla device (airsonett ® , sweden), which included 8 study visits. we used scorad-index results: the median duration of eczema was 116.5 months (interquartile range 82-145.5). all subjects were sensitised to ≥1 perennial allergen, and had multiple comorbidities (15/15 rhino-conjunctivitis, 14/15 food allergy, 11/15 asthma). there were no significant changes during the run-in period in any of the outcome measures. we observed a significant improvement in scorad after the 12-month tla-treatment period, from 34.9 [28.75-45.15 ] to 17.2 [12.95-32.3] , p=.015. iga improved significantly from a median of 4 [3-4] to 2 [1] [2] [3] , p=.001. improvement in symptoms was paralleled by a significant reduction in medication usage. by 12 months, there was a significant improvement in .0] to 12 [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] , p=.032), and an improvement in cdqli (marginal, p=.084). however, we observed no changes in poem (p=.196) . post-hoc cluster analysis of the patterns of changes in scorad over the 12month treatment period identified two clusters, with 9 participants classified as responders and 6 as non-responders. introduction: intestinal degradation has been shown to determine allergenicity of food allergens. however, it is unclear how allergens are degraded inside pivotal immune cells such as dendritic cells (dc), and how this affects the subsequent immune response to these allergens. in our studies, we determined whether we are able to measure uptake and degradation of allergens inside dc and whether differences in above mentioned factors exist between allergens. objectives: using mouse bone marrow-derived dc and fluorescent-labeled proteins, we studied the cellular uptake of the peanut proteins ara h 1, 2, 3, and 6. results: first, we observed that dc uptake of ara h1 was much higher than ara h2, 3 and 6. using blocking reagents and receptor binding assays for various uptake routes in dc, we observed that one of the principal routes of uptake for ara h 1 was the mannose receptor. other uptake routes, and the routes of uptake for ara h2, 3 and 6 are currently under investigation. second, using proteins coupled to beads we observed that intracellular protein degradation was higher for ara h1 and ara h3 than for ara h2 and 6. finally, cd4+t cells from ara h 1, 2, 3 or 6 sensitized mice were added to matching allergen-pulsed dc. we observed that while ara h1, 3 and to lesser extend ara h6 elicited strong th2 type responses, ara h2 did not elicit any t cell responses. conclusions: together, we show that allergenicity of (peanut) proteins may be, at least partly, determined at the level of allergen uptake and breakdown inside the antigen presenting cell. these findings may be relevant to risk evaluation of existing allergens but also of novel or modified proteins. this also illustrates the usefulness of in vitro, cell based assays to examine initial processes of allergenic sensitization to proteins. 0445 | sensitising capacity of unmodified and acid hydrolysed gluten through the skin-a comparative study in na€ ıve vs tolerant brown norway rats ballegaard ar; madsen cb; bøgh kl national food institute, technical university of denmark, søborg, denmark introduction: allergic sensitisation to foods may occur in infancy without prior oral exposure to the offending food. this has led to the assumption that food allergy sensitisation may occur through alternative routes, such as the skin, supported by the observed correlation between skin barrier disruption and food allergy. recently, concerns have been raised regarding the safety of use of cosmetic and personal care products containing hydrolysed wheat proteins, since these products have been shown to induce allergy towards acid hydrolysed wheat through the skin, and even to cause an abrogation of the already established oral tolerance against unmodified wheat. objectives: the aim of the study was to compare the sensitising capacity of an unmodified and an acid hydrolysed wheat product via slightly damaged skin, in order to evaluate differences in conditions necessary for skin sensitisation in na€ ıve vs tolerant individuals. brown norway rats were raised and bred on either (1) a diet free from wheat, resembling individuals with a na€ ıve immune system, or (2) a conventional wheat containing rat chow, resembling individuals tolerant to wheat. results: in the na€ ıve rats both products were able to induce a statistically significant specific antibody response after application of the products on the slightly damaged skin, whereas in the wheat tolerant rats, only the acid hydrolysed wheat product was able to induce a statistically significant antibody response. for both the na€ ıve and the wheat tolerant rats the response was dose-dependent. in the na€ ıve rats both products were able to sensitise through the skin, inducing a specific ige response, whereas in the tolerant rats only the acid hydrolysed product were able to induce a specific ige response, though this ige response was much lower than in the na€ ıve rats. results from competitive elisas demonstrated that new epitopes had developed as a result of acid hydrolysis, though original epitopes were maintained at the same time. this may explain why only the acid hydrolysed wheat could induce specific antibody responses in the tolerant animals. conclusions: this study showed that the sensitising capacity through the skin of two different wheat products is heavily influenced by the tolerance status of the immune system and the degree of modification of the wheat products. results: using a germ-free c3h/hen mouse model of food allergy, we examined the presence of a major peanut allergen, ara h 2, in the blood after intraperitoneal injection in previously sensitized and control (non-sensitized) mice using an untargeted, quantitative proteomic approach. previously sensitized mice underwent physiological (core temperature decrease, clinical symptoms) and biochemical (mast cell protease increase, ara h 2 specific ige positivity) changes associated with severe allergic reactions. we were able to confidently detect multiple peptides derived from the ara h 2 protein after intraperitoneal injection in both control and ara h 2 sensitized mice. however, the ara h 2 protein was present at 15-40 fold higher levels if mice were previously sensitized, suggesting increased transit across the peritoneal mesothelium with sensitization. an untargeted proteomic approach also allowed changes in the blood proteome of mice undergoing a severe allergic response to be examined. we identified proteins with significantly altered quantity in serum between control and sensitized mice and were therefore apparently associated with the allergic response. we demonstrate the applicability of untargeted proteomics to the study of the allergen transport and proteomic changes which co-occur with a resultant allergic reaction. the transit of allergens into the bloodstream is heavily dependent upon previous sensitization. we are continuing this work to examine the specificity of observed increases in trans-mesothelial transport and to address transport of allergens after intragastric challenge. 0447 | iga to cow's milk differs between breast milk and serum for its epitope specificity objectives: we sought to assess whether the profile of epitopespecific iga differs between mother's serum and bm. we also examined how infants' food epitope-specific iga develops in early infancy and the relationship of iga epitope recognition with development of cow's milk allergy (cma). results: . we measured iga specific to an array of overlapping peptides in major cow's milk allergens (alpha s1-, alpha s2 -, betaand kappa-caseins and beta-lactoglobulin). diversity of peptide-specific iga (ie epitope diversity) was determined in paired maternal and infant sera as well as breast milk samples in 31 mother-infant dyads within the first 15 postpartum months utilizing peptide microarray. microarray data was converted to z-scores and filtered for noise. peptide epitopes were determined based on jaccard distance between neighboring peptides, and intra individual correlation between sample types was estimated using phi coefficient. comparisons between groups and individuals was done using non-parametric tests. we noted marked discordance in epitope recognition in paired breast milk and maternal serum samples. at least one shared epitope was recognized by both milk and serum samples ranging from 13% of mothers for alpha-s1 casein to 73% for kappa-casein. epitopespecific iga was detectable in infants' sera starting at less than 3 months of age. sera of mothers with a cma infant had increased binding of epitope-specific iga to cow's milk proteins compared to those with a non-cma infant (p<.05 for all five proteins). conclusions: these findings support the concept that mothers' milk represents a product of the mucosal immune system that has an antibody repertoire distinct from that of peripheral blood. results: multiple ige-binding proteins were detected in the different wn preparations and many of which were dissemblance in 2dblotting. profiles of detectable proteins (peptides) of raw, roasted and boiled wn extracts were profoundly different in emi analysis. introduction: consumption of tree nuts is on the rise due to their beneficial health effects. however, tree nuts can led to severe allergic reactions in sensitized patients. thermal processing can modify the structure and function of food proteins and may alter (increase or decrease) their allergenic properties. knowledge about the effects of thermal processing on tree nuts such as cashew or pistachio is rare and based on traditional in vitro immunoassays. objectives: to elucidate the influence of thermal treatments (boiling and heat / pressure) on the ige reactivity of cashew and pistachio proteins, by means of traditional in vitro immunoassays and mediator release assays (mra). results: the allergenicity of untreated and treated cashew and pistachio nuts was evaluated by ige-elisa, ige-immunoblot and elisa inhibition assays using sera from spanish patients with clinical allergy to cashew and pistachio. rat basophilic leukaemia cell line transfected with the a-chain from the human high-affinity ige receptor fceri, was sensitized with a pooled sera and used for mra. sensitized cells were stimulated with untreated and treated protein extracts, in order to investigate the capability of untreated and treated cashew and pistachio proteins to cross-link ige on effector cells. the results showed that heat and pressure treatment at the harshest conditions considered in this study produced a higher decrease of the ige-binding capacity of cashew and pistachio proteins than boiling without pressure or soft conditions of heat and pressure. interestingly, although the treatments of heat and pressure seemed to affect cashew allergens to a greater extent than pistachio allergens (evaluated by ige-elisa and elisa inhibition), the results of mra using the cell line rbl-48 indicated that cashew proteins treated with heat and pressure, still retained some capacity to cross-link ige. introduction: previous research has indicated an important role for dietary non-digestible oligosaccharides in decreasing the incidence of atopic dermatitis in children at risk of allergy. it is assumed that these prebiotics promote colonization of beneficial bacteria in the gut. children with atopic dermatitis receiving a diet of galacto-and long chain fructo-oligosaccharides (scgos/lcfos) with bifidobacterium breve m-16v had enhanced serum galectin-9 levels. galectin-9 has ige-binding capacities and can hereby suppress degranulation of mast cells and basophils. next to their function in the gut, oligosaccharides may also affect other immune cells directly, since they were found in plasma and urine. objectives: we investigated whether non-digestible oligosaccharides or galectin-9 can have a direct effect on immune cells, by determining the effect on basophil degranulation in peanut-allergic patients. whole heparinized blood samples were collected from 12 peanut-allergic adult patients and incubated for 24 hours with either a mixture of 0.05% 9:1 scgos/lcfos or scfos/lcfos, or galectin-9 (1 or 5 lg/ml) at 37°c in the presence of il-3 (0.75 ng/ml). after 24 hours, a basophil activation test (bat) was performed. basophils were stimulated for 30 minutes at 37°c with increasing concentrations of whole roasted peanut extract or human anti-ige. degranulating basophils were determined as cd63+ cells and calculated as percentage positive cells. results: in each patient, the concentration of anti-ige or peanut extract that induced maximal degranulation in the untreated control sample was used as reference value to compare degranulation of the samples pretreated with scgos/lcfos, scfos/lcfos, or galectin-9. pre-treatment of whole blood with scgos/lcfos resulted in an average decrease in degranulation of approximately 8%, while a significant reduction of 19% was observed after pre-treatment with scfos/lcfos (p<.05). pre-treatment with 1 lg/ml galectin-9 decreased basophil degranulation with 12%, whereas 5 lg/ml galectin-9 caused a significant decrease of 25% (p<.05). no differences were observed in the ec50, indicating that the basophils are not becoming less sensitive to the peanut extract or anti-ige. the prebiotic mixture scfos/lcfos and galectin-9 can contribute to decreased degranulation of basophils in a igemediated bat assay using whole blood. further analysis is warranted to define the exact working mechanism of these oligosaccharides. introduction: the intestinal mucosa plays a key role in the development of food allergies. we studied the interaction between intestinal epithelial cells (iecs) and pbmcs of peanut-allergic patients in a transwell co-culture model. exposure of iecs to a mixture of galacto-and/or long chain fructo-oligosaccharides (scgos/lcfos) in combination with synthetic cpg dna (tlr9 ligand),modulated the cytokine response of activated pbmcs, driving away from the allergic phenotype. objectives: our aim was to compare the efficacy of scgos/lcfos with a scfos/lcfos mixture and to evaluate these effects in an allergen-specific co-culture model. iecs (ht-29, human colon adenocarcinoma) were grown on transwell filters until confluence. iecs were apically exposed to 0.5% 9:1 scgos/lcfos or scfos/lcfos either or not in combination with 2.5 lmol l à1 cpg dna to mimic presence of dna of beneficial bacterial in the gut. these iecs were co-cultured with basolateral pbmcs from peanut-allergic patients, either activated with anti-cd3/cd28 (24 hours) or peanut-extract (pe, 6 days).cytokines ifn-c, il-10, il-13 and tnf-a were measured in the basolateral supernatant and t-cell polarization of the pbmcs was determined (th2, th1, treg and tfh). results: apical exposure of iecs to cpg dna increased basolateral ifn-c and il-10 production by anti-cd3/cd28 activated pbmcs (p<.01), and was enhanced by both oligosaccharide mixtures (p<.01). cpg exposure in transwells with pe stimulated pbmcs also increased ifn-c and il-10 production, which was further enhanced by scfos/lcfos (p<.05). in this pe-specific model, percentages of th1 and treg cells increased upon cpg exposure of iecs, and th1 frequency was increased by scfos/lcfos (p<.05). cpg dna exposed iecs suppressed il-13 and tnf-a production by anti-cd3/ cd28 activated pbmcs. both scgos/lcfos and scfos/lcfos reduced tnf-a production in combination with cpg dna (p<.01). tfh cells can produce il-21, inhibiting class-switching to ige2. upon cpg exposure of iecs, we observed an increase in tfh cells (p<.01) and similar tendency for cd4 + il-21 + cell frequency in anti-cd3/ cd28 activated pbmcs. conclusions: epithelial exposure to both scgos/lcfos and scfos/lcfos enhances the cpg dna induced th1 and regulatory il-10 response in an anti-cd3/cd28 co-culture model, whereas only scfos/lcfos was effective in an peanut-specific co-culture model. introduction: in areas of spain with high level of grass pollen exposure, 60% of grass allergic patients were sensitized to profilin, and most of them developed severe profilin mediated food reactions. this specific population of patients constitute an ideal model to study the relation among respiratory and food allergy objectives: our aim here was to analyze the links between epithelial barrier integrity and inflammation in oral mucosa. methods: 30 allergic patients and 6 controls were included in the study. allergic patients were stratified into mild or severe according to their clinical history and response to profilin oral challenge test. immunohistochemistry (ihc) for cd11c, cd3, cd4, claudin-1; and dapi nuclear staining were performed in formalin-fixed, paraffin embedded sections of oral mucosa biopsies. rt-pcr was performed to analyze il1b and il33 gene expression and the number of circulating cd4+ cells in pbmc was measured by flow cytometry. additionally, basophil activation test was carried out in whole blood samples upon profilin stimulation and the ec50 was calculated. results: regarding epithelial barrier integrity, claudin-1 expression resulted inversely proportional to pollen-associated food allergy severity. furthermore, by dapi staining, we noticed a lower number of epithelial cells in allergic patients than in non-allergic, and the gene expression of il1b and il33 resulted significantly increased in oral mucosa from severe group. as for immune response in oral mucosa, the number of cd11c and cd3+ cells resulted significantly higher in severe group. in allergic patients, double ihc for cd11c-cd4 showed an increase colocalization of t cells and apcs in the interface between epithelium and connective tissue. a decrease in blood circulating cd4 + cells was detected in allergic patients compared to non-allergic. as for the basophil sensitivity, ec50 in severe patients was 10-times lower than in mild. our results show that damage in oral mucosa epithelium, probably induced by high grass pollen exposure, might allow profilin to penetrate inside oral mucosa and induce inflammation with local recruitment of immune cells. furthermore, analyzing the immune response developed by effector cells, basophils from severe group result more sensitive to profilin as they react to a lower allergen concentration. these data explain the differences in food allergy severity between mild and severe group and propose oral mucosa as a new sensitization route. introduction: th2 cells producing the hallmark cytokines il-4, il-5 and il-13 have been found to constitute the majority of the allergen-specific th cell responses in allergic diseases. subpopulations of the th2 responses have been described with an early primed th2 subtype characterized by production of il-4 and il-13, and a highly differentiated th2 subtype, which in addition produce at least il-5. objectives: we aimed to investigate the polarity of tree nut and peanut allergen-specific th cells in subjects with confirmed tolerance or allergy to multiple nuts, and hereby detect differences in allergenspecific th cell responses within the same study population. we also wanted to stress the question whether a th2 phenotype dominates in asymptomatic sensitization. methods: pbmcs from 35 donors all assessed for clinical reactivity to hazelnut, walnut, cashew nut, pistachio nut and peanut was stained with cfse and stimulated (2910 6 cells/ml) with and without whole nut extracts (50-200 lg/ml) for 7 days. allergen-specific cd4+ t cell phenotypes and cytokine release was analyzed with flow cytometry and luminex, respectively. the allergen-specific th cells of the allergic donors showed a trend of more highly differentiated il-4+il-5+ th2 cells and higher abstracts | 319 release of il-5 than in the tolerant donors. unexpected, except in the cashew nut stimulated cells, no difference was found in the relative percentage of the less differentiated th2 cells (single il-4+ allergen-specific cd4+ t cells) when comparing allergic with tolerant donors. when subdividing the tolerant donors into asymptomatically sensitized or ige-negative (<0.35 kua/l), increased frequency of highly differentiated il-4+il-5+ allergen-specific th2 cells were found in asymptomatically sensitized compared to tolerant-ige-negative donors. interestingly, a positive association of the allergen-specific ige level and the frequencies of allergen-specific il-4+il-5+ and il-4+ th2 cells were found in hazelnut, pistachio nut and cashew nut but not in walnut and peanut allergic subjects. conclusions: an overrepresentation of allergen-specific highly differentiated il-4+il-5+ th2 cells and an elevated il-5 production were observed in allergic subjects compared to subjects that tolerated the nuts. we furthermore found a trend that subjects asymptomatically sensitized to nuts differed from tolerant-ige-negative subjects by having relatively more highly differentiated il-4+il-5+ allergen-specific th2 cells. introduction: it remains largely unknown which features of food proteins that render them allergenic vs tolerogenic. however, it has been suggested that the protein-chemical features affects protein uptake in the intestine, and that protein uptake route may impact on the risk of sensitisation. objectives: the aim of this study was to investigate the interplay between protein-chemical characteristics, the allergenic vs tolerogenic properties and the intestinal uptake of two protein products. the allergenic vs tolerogenic capacity of a heat-treated whey product, consisting of partly denatured and aggregated proteins, was compared to an unmodified whey product in: (1) results: though this study showed that both unmodified and heattreated whey had immunogenic, sensitising and eliciting capacities as well as tolerance inducing capacity, significant differences between the two products were observed. the heat-treated product was found to have a lower allergenicity combined with high tolerogenicity compared to the unmodified product. competitive igg1 elisas indicated that heat-treatment of whey induced de novo epitopes while the original epitopes were maintained. newly established methods to study in vivo intestinal uptake were successfully applied to compare the uptake kinetics of the two products in different small intestinal tissues and serum. collectively the in vivo and in vitro uptake experiments suggested that uptake kinetics and the major intestinal uptake route differed between the heattreated and unmodified product. conclusions: this study showed that heat-treatment, which induces partly protein denaturation and aggregation, changes the immunological properties and intestinal uptake of a whey protein product. the heat-treated product was found to have a lower allergenicity combined with high tolerogenicity compared to the unmodified product, which highlights this products promising potential for induction of cow's milk tolerance. objectives: in this study, we enumerated tregs in esophageal tissue of patient with eoe, gerd and normal controls. ten patients with eoe, 10 patients with gastroesophageal reflux disease (gerd), and 8 patients with normal endoscopy and normal esophageal tissue were included. tregs were enumerated in paraffin embedded esophageal biopsy blocks, using immunohistochemistry assay. tregs were identified as foxp3+, cd3+ cells. results: tregs were counted in 3 high power fields (hpf, 9400) for 28 patients and the average of 3 hpf was recorded. the number of tregs in esophageal tissue of patient with eoe (mean 10.9 cells/ hpf) was significantly more than gerd(mean 2.77 cells/hpf) and control groups(mean 0.37 cells/hpf) (p value <.001). conclusions: there is an increase in number of regulatory t cells in esophagus of patients with eoe in comparison to gerd and control groups. the presence of these cells might be due to eosinophilic inflammation and help controlling the inflammation. objectives: to evaluate whether anti-cd38 (daratumumab) treatment results in a reduction of total and specific ige levels. results: samples from patients with relapsed/refractory multiple myeloma, treated with daratumumab monotherapy or daratumumab plus lenalidomide-dexamethasone in the umc utrecht between april and august 2014, were tested for total ige levels as well as presence of specific ige against common inhalant allergens. in patients with detectable ige at baseline, the total and specific ige levels were evaluated during treatment up to 20 weeks. of eight patients receiving treatment, four had detectable ige levels at baseline. one patient demonstrated sensitization to common inhalant allergens. for this patient, levels of total ige gradually decreased during 20 weeks of treatment (from 356 to 44 ku/l; 88%), as well as specific ige against timothy grass (9.8-1.7 ku/l; 83%) and house dust mite (3.2-0.4 ku/l; 88%). a second patient, not sensitized to common inhalant allergens but with ige levels of 41 ku/l at baseline, also demonstrated a decrease in ige levels, to 5 ku/l (88%). the last two patients had total ige levels <10 ku/l at baseline, which dropped below detection limit after eight weeks of treatment. conclusions: this proof of concept demonstrates that (specific) ige depletion occurred during treatment with anti-cd38 (daratumumab). anti-cd38 could potentially play a role in the management of severe ige-mediated diseases. objectives: following individual and assessment, patients established and stable on omalizumab have been commenced on home therapy. training and education in self-administration has been provided. a service assessment has been carried out to review the ongoing safety, quality and patient experience of the service. results: to date, over 90 doses of omalizumab have been self administered in the community with no adverse events or incidents related to home therapy. conclusions: self-administration of omalizumab at home by patients offers the potential to improve quality of life while also providing efficiency savings. introduction: chronic idiopathic/spontaneous urticaria (csu) is a chronic urticarial subtype, defined as itchy hives that last for at least 6 weeks, with or without angioedema, and that have no apparent external trigger. although csu is more frequent in adult populationup to 0.5%-1%-, it can affect children and generally has a prolonged duration and has a detrimental effect on patients' quality of life. before the fda and ema approval of the anti-ige monoclonal therapy omalizumab for adults and children 12 years and above, nonsedating h1-antihistamines were the only agents licensed for use in patients with csu. however, a majority of patients did not respond to these drugs, even when they were administered at three to four times their licensed dose. objectives: we present 3 pediatric patients (≥12 years old) with csu non-responding to antihistamines, treated with omalizumab. results: at the diagnosis of csu, the patients were 11, 13, 14 years old, respectively. a trial with non-sedating h1-antihistamines, administered up to three to four times their licensed dose, were performed for all patients, without clinical efficacy. omalizumab was started at 12, 14, 14 years, respectively. before anti-ige therapy, the urticaria activity score (uas) was 5, 4, 5 respectively, indicating poor symptom control. omalizumab therapy was administered every 4 weeks for 6 months at the dosage of 300 mg s.c. after 1 month of therapy, all 3 patients were symptom free with uas 0; the patients remained asymptomatic for all the 6 months of duration of monoclonal therapy and antihistamines were discontinued. by now, after 2, 5, 6 months respectively from discontinuation of omalizumab, all 3 patients are asymptomatic with uas 0. introduction: mepolizumab is a humanized monoclonal antibody directed against il-5, and is licensed for the treatment of severe asthma in patients aged >12 years (eu only adults) with an eosinophilic phenotype as an add-on treatment. we were interested to know whether the substance has an antiallergic effect, too. objectives: here, we report the case of a 60 year old man (nonresults: in march 2016 he was switched from omalizumab to mepolizumab (100 mg/month) 4 weeks after the last omalizumab because of two asthma exacerbations under omalizumab in the last 6 months and 380 eosinophils/ll blood under 10 mg prednisolone/day. after two injections of mepolizumab his fev1 increased from 55% to 64% pred., the act from 14 to 21 points, feno decreased from 48 to 32 ppb and the amount of prednisolone from 10 mg to 5 mg/day. but -the patient reported new nasal symptoms after the 2nd injection of mepolizumab, mainly blocked nose which has not been observed under omalizumab. he agreed to be challenged in the mobile gae²len exposure chamber* with house dust mite allergen for 40 min. the total symptom score increased from 2 to 7 points after 20 min exposure time remaining stable till the end, the positive nasal inspiratory flow decreased from 90 to 50 l after 30 min and 40 l after 40 min, the fev1 decreased from 62% to 51% pred. following the inhalation of salbutamol the fev1 reversed. conclusions: mepolizumab has an anti-asthmatic effect but the anti-allergic efficacy seems to be small or absent. methods: all patients treated with omalizumab for severe allergic asthma or chronic spontaneous urticaria at the department of respiratory medicine and allergy at aarhus university hospital (n=64) were grouped after their home municipality. the number of omalizumab treated patients/inhabitant in these municipalities was correlated to the distance to the treatment centre at aarhus university hospital. results: mean distance to the treatment centre was 53.5 km for all inhabitants in the central region, while patients treated with omalizumab lived in average 38.7 km away. we found a negative linear correlation between the number of patients treated with omalizumab/inhabitant and the distance from the municipality to the treatment centre (slope: à0.038 95%ci: à0.072 to à0.003), p=.034, n=64). conclusions: patients living at long distance from the treatment centre are less likely to be offered omalizumab treatment for severe allergic asthma or chronic spontaneous urticaria. objectives: demographics, clinical characteristics, and response to mepolizumab, were evaluated for atopic and non-atopic subgroups. sensitization to any one of the following; house dust mite, dog dander, cat dander, alternaria, or cockroach was considered as atopic, as assessed by specific serum ige of ≥0.35 ku/l. mensa (nct01691521) was a gsk sponsored study. results: of the 576 severe asthma patients, 275 (48%) were considered atopic, 272 (47%) were considered non-atopic and atopic status was missing for 29 (5%) patients. the majority of atopic patients (n=147) were sensitized to ≥3 antigens. compared to the non-atopic sub-group the atopic sub-group was younger with a mean age of 46 vs 54 years of age, had a longer mean duration of asthma (21.6 vs 18.4 years), and a higher total baseline ige level (293 u/ml vs 85 u/ml). mean number of exacerbations in the 12 months prior to the study was similar in the atopic and non-atopic subgroups (3.6 vs 3.8) as was the mean baseline peripheral blood eosinophil level (300 cells/ll vs 300 cells/ll). with mepolizumab an 80% reduction in eosinophils was achieved by week 4 irrespective of atopic status and maintained throughout the treatment period. mepolizumab reduced the rate of exacerbations relative to placebo by 48% in the atopic subgroup and by 54% in the non-atopic subgroup. conclusions: the mensa study recruited an equivalent portion of atopic and non-atopic severe asthma patients. the atopic population was younger and had a longer duration of asthma than the non-atopic subgroup. while the baseline total ige level was >3 times greater in the atopic subgroup, the pharmacodynamic and efficacy response to mepolizumab treatment was similar. introduction: a treatment goal in the management of oral corticosteroid (ocs) dependent severe asthma is to reduce daily ocs use due to the side effects associated with long term use. anti ige treatment is used in atopic patients to reduce daily ocs. this analysis characterizes ocs reduction and asthma control in the sub-set of atopic and non-atopic ocs-dependent severe eosinophilic asthma (sea) patients from the 24-week sirius ocs reduction study. objectives: sirius study participants (n=135) were sub-grouped by atopic status in a post-hoc analysis; demographics, clinical characteristics, and response to mepolizumab were evaluated. sensitization to any one of the following; house dust mite, dog dander, cat dander, alternaria, or cockroach was considered as atopic, as assessed by specific serum ige of ≥0.35 ku/l. sirius (nct01691508) was a gsk sponsored study. results: of the 135 ocs-dependent sea patients, 49 (36%) were considered atopic (61% female), 77 (57%) were considered non-atopic (51% female), atopic status was missing for 9 patients (7%). compared to the non-atopic sub-group, the atopic sub-group was younger; mean age of 46 vs 52 years of age, while the mean duration of asthma was the same irrespective of atopic status (19 years). the mean ocs daily dose and acq-5 score at baseline were similar between subgroups (12.6 mg vs 13.2 mg and 2.16 vs 2.04, in the atopic and non-atopic subgroups respectively). the mean baseline peripheral blood eosinophil level was comparable in both subgroups (250 cells/ll vs 220 cells/ll) while the total ige level was higher in the atopic subgroup (182 u/ml vs 86 u/ml). mepolizumab lead to a ≥ 80% reduction in eosinophils by week 4 irrespective of atopic status and eosinophil reduction was maintained throughout the study. mepolizumab reduced the ocs dose in the atopic group and non-atopic subgroups (odds ratio of a greater ocs reduction category of 5.6 (95% ci 1.71, 18.49) and 1.6 (95% ci 0.66, 3.93), respectively) and led to improvement in asthma control with a change in acq-5 of à0.60 (95% ci à1.20, 0.00) in the atopic subgroup and à0.45 (95% ci à0.94, 0.04) in the non-atopic subgroup. in an ocs dependent sea population mepolizumab reduced eosinophils independent of ige level and atopic status. in this limited post-hoc analysis, mepolizumab was an effective ocs sparing agent while improving asthma control in both atopic and non-atopic patients. patients who responded to retreatment had a similar mean time to response between the 1st dosing period (3.5 weeks) and 2nd dosing period (3.1 weeks). of all patients who were retreated (n=56), symptom control (uas7 ≤ 6) after two doses was achieved in 80% (1st period) and 85% (2nd period) of patients; complete response (uas7=0) occurred in 63% (1st period) and 56% (2nd period) of these patients. omalizumab was well-tolerated during both dosing periods. conclusions: omalizumab retreatment is safe and effective in patients with ciu/csu who respond to initial treatment and relapse after withdrawal, with most patients regaining symptom control after a 2nd course of omalizumab. : total ige reactivity of the grass allergoid formulation was diminished compared with native unmodified extracts. a difference in igg profiles was observed and indicated enhancement of accessible reactive igg epitopes across size distribution profiles of the grass allergoid formulation. detailed analysis of the epitope specificity showed retention of six lol p 1 igg binding epitopes in the grass modified extract. all epitopes were mapped on the solvent exposed area of lol p 1 homology model accessible for igg binding. one of the epitopes was identified as an "immunodominant" lol p 1 igg binding epitope (62-ifkdgrgcgscfeik-76) and classified as a novel epitope. lastly, lol p 1 igg antibodies showed functional capacity to block up to 50% of ige binding sites which provides evidence in protective function of immunotherapy induced antibodies against native allergens. the structural and immunological changes which take place following the grass allergen modification process were further unravelled revealing distinct igg immunological profiles. the results from this study support the concept that modification not only enhances the safety profile of scit but allows shorter-course objectives: design targeted sirna delivery system into liver cells. results: liposome surface was modified by lactose derivatives with different structures: lac(c 16 ) 2 and (lac) 7 lac(c 16 ) 2 . the basic physicochemical and biological properties of the carriers have been examined. it is shown that glycoconjugate introduction has no effect on the physico-chemical characteristics. however the carbohydrate modification of the liposomal surface leads to increasing in transfection efficiency on a specific cell line hepg2. moreover, the introduction of mono-carbohydrate derivative increases the transfection efficiency better than using a branched derivative of lactose. in the pharmacokinetic study target effect also was shown. unmodified liposomes start to be detected in the liver at 10 minutes after administration, whereas modified liposomes were detected at 2 minutes after administration. similarly, the excretion of modified liposomes from the liver was more slowly. also worth noting the high concentration of modified liposomes in the liver. furthermore liposomes modified by carbohydrates reduces the intensity of its accumulation in the lung. conclusions: it was shown that increasing attraction of drugs to the liver cells can be achieved by using liposome modification with lactose derivative. 0472 | sialylated fetuin-a is a candidate predictive biomarker for successful grass pollen allergen immunotherapy objectives: to identify new biomarkers of ait efficacy, pre-treatment sera obtained from 82 grass pollen allergic patients responding or not to sublingual ait were differentially assessed by 2d-dige or label-free mass spectrometry. the role of fetuin-a in allergy physiopathology was studied by using gene silencing in a mouse asthma model, human dendritic cell stimulation assays and surface plasmon resonance. results: using comparative proteomics, we provide evidence for an increased o-linked sialylation of fetuin-a in sera collected before treatment from patients exhibiting clinical responses, when compared with low responders. whereas feta may either inhibit or promote chronic inflammation, no specific role in allergy had been ascribed to it until now. in ovalbumin-sensitized mice, silencing of the fetuin-a gene increased airway hyperresponsiveness and th2 responses. fetuin-a, but not its non sialytated counterpart, synergizes with lps and grass pollen or mite allergens in a tlr4-dependent pathway to enhance the proallergic profile of human monocyte-derived dendritic cells. conclusions: quantification of sialylated fetuin-a glycoforms in the blood before treatment allows to identify patients more likely to benefit from grass pollen immunotherapy. validation of the hypothesis that this marker associated with "an inflammation status" can be used to predict ait efficacy is ongoing in larger cohorts of patients. introduction: kawasaki disease (kd) is a vasculitis that mainly affects small to medium-sized vessels, particularly the coronary arteries, and is a leading cause of acquired heart disease in children. previously, we found that the development of kd is associated with an elevation of both th1 and th2 immunity. gene hypomethylation is abstracts | 329 an important form of epigenetic regulation, which results in increased gene expression. because m1 macrophages are associated with inflammation and th1 immunity while m2 macrophages are associated with immune regulation and th2 immunity, we hypothesize that kd is associated with hypomethylation of both m1 and m2 macrophage related genes. objectives: our objective was to investigate the methylation profile of m1 and m2 related genes in patients with kawasaki disease. twenty-four patients with kd and 12 age-matched healthy controls (hc) were included in this study. in patients with kd, blood was sampled 24 hours before ivig therapy (kd1) and 21 days after ivig therapy (kd2). after dna extraction, samples were analyzed using human methylation bead chip 450 (illumina) to examine the methylation ratios of genes related to m1 macrophages (64 genes, 1536 cpg sites) and m2 macrophages (46 genes, 1329 cpg sites). cpg sites with more than 10% difference in methylation levels and a pvalue less than 0.05 between groups were considered significant. objectives: primary aim of the study is the identification of differentially expressed genes (deg) associated with allergic rhinitis (ar) by using genome-wide transcriptomics data from blood immune cells. this set of candidate genes will then be used to define gene networks relevant for onset, severity and potential treatment outcome of house dust mite associated ar. with different ethnicity or populations exposed to a different environment is currently in preparation. introduction: allergic patients display abnormal immune responses to harmless antigens, leading to various symptoms from hay fever to life-threatening conditions. these responses include abnormal type 2 immunity polarization and induction of allergen-specific memory t and b cells, resulting in development of allergy instead of immune tolerance. allergen-specific immunotherapy (ait) is currently the only causative treatment of allergic disorders. yet, in depth understanding of the underlying differences in allergen-specific cd4+ t cell and treg responses between allergy and tolerance and their changes during immunotherapy is lacking. objectives: we investigated whole-genome transcriptomics of circulating birch (bet v 1) and grass (phl p 5a)-specific cd4+ t cells in allergic patients before and at 3, 6, and 12 months of ait, as well in non-allergic healthy controls in corresponding seasonal time points. detailed immunophenotyping with flow cytometry and cd4+ mhc class ii tetramer staining with low rna/single cell next generation sequencing were performed. results: at baseline, out of the pollen season, there were more allergen-specific cd4+ t cells in allergic patients than in controls. at this time, over 1500 genes were significantly changed in allergenspecific as compared to total cd4+t cells in patients, yet we found substantial differences in signal transduction and inflammatory response gene expression when compared to healthy controls. during ait we noted significant increase of allergen-specific cd4+ cells in patients with subsequent gene expression changes in the immune tolerance pathways. finally, we found increase in allergen-specific treg cells in patients upon ait, but not in tolerant controls in corresponding seasons. of interest yet, at early ait time points, allergenspecific treg cells displayed gene profiles suggesting their insufficient suppressive functions. conclusions: in summary, in vivo allergen exposure causes profound changes in the transcriptomic profiles of allergen-specific t cells. these gene profiles seem to be deficient at baseline in allergic patients, but ait is skewing them into the tolerant controls levels. introduction: the variability of the pharmacological response to beta 2 (b2)-agonists may be due to the polymorphism of the gene of results: singled out statistically significant differences of the genotype distribution. the gly/gly16 homozygous allele was discovered twice as often in the group with an insufficient response to b2-agonists than in the group with a good response (66 vs 38%, p<.001), while the distribution of heterozygous allele was detected the opposite pattern (55 vs 28%, p<.001). in the arg/arg16 genotype distribution, there were no considerable differences in both groups (6% in each group). in the subgroups of children, receiving the high doses of the inhaled glucocorticoids, there was a trend for the prevalence of gly/gly16 homozygous allele prevalence. we have discovered the association of the gly/ gly16 genotype of the adrb2 gene with an insufficient effect of broncholytic therapy by means of short-term b2-agonists; we also revealed the participation of the gly16 allele in the phenotype formation with the severe run of bronchial asthma and tolerance towards the therapy both by b2-agonists and inhaled glucocorticoids. mckenna oe 1 ; posselt g 1 ; lackner p 1 ; schmitt a 2 ; h€ ollbacher b 1 ; briza p 1 ; wessler s 1 ; gadermaier g 1 ; ferreira f 1 1 universit€ at salzburg, salzburg, austria; 2 georg august-universit€ at g€ ottingen, g€ ottingen, germany introduction: an excess of 100 million people worldwide have a reported allergy to birch pollen. proteases in such allergen sources have been suggested to contribute to primary sensitisation and exacerbation of allergic disorders. until now the protease content of betula pendula, a species endemic to the northern hemisphere, has not been studied in detail. hence, we aim to identify and characterise pollen and bacterial derived proteases found within betula pendula pollen. objectives: birch pollen transcriptome was constructed via de novo transcriptome sequencing. reads were assembled using the trinity software suite.. analysis of the birch pollen proteome was achieved via mass spectrometry and use of zymographic gels with the embedded substrates gelatinase and casein, which enabled visualisation of proteolytic activity. further to this, protease activity was quantified using a fluorescently labelled casein substrate protease assay. results: by using mass spectrometry, we were able to identify up to 26 proteases within birch pollen. we could cluster the proteases into specific families based on their distinct proteolytic activities. further comparative analysis of the proteome and transcriptome revealed the relationship between transcript levels and the proteins they encode. zymographic methods enabled distinct visualisation of proteolytic activity for both casein and gelatin substrates. using fluorescently labelled casein, the birch pollen protease activity was quantified as 0.68 lg/ml when compared to a trypsin standard curve. additionally, 26 bacterial isolates of the birch pollen were identified, and the proteolytic activity analysed. we report successful discovery of pollen and bacterial derived proteases endogenous to betula pendula. whilst none of the known birch pollen allergens have been recognised as a protease, we aim to investigate the role of tight junction degradation within epithelial cells and further enhance understanding of proteolytic activity on immune-polarization. objectives: to investigate the frequency and reasons of shortand long-term reintroduction failure in adults after a negative fc. method:: these are preliminary results of an ongoing prospective study. after a negative fc, patients receive standardized aftercare consisting of an introduction scheme to introduce the food at home and consultation by phone for advice and support 24 hours, 1-3 weeks and 6 months after the fc. short-term data about the frequency that patients failed introduction, defined as not completing the introduction scheme or patient-reported allergic complaints repeatedly during introduction, was obtained using a telephone interview. long-term data (5-12 months after negative fc) about frequency and reasons of reintroduction failure in daily diet, defined as not eating the food or only eating products with traces of the food, was obtained using a patient-reported questionnaire. results: from 2014 until now 38 patients were included (mean age: 34 years, male: 26%), who underwent a total of 110 fcs with: hazelnut (23%), other nuts (17%), fruit (7%), composite meals (7%), fish and crustaceans (7%), cow's milk (6%), hen's egg (5%) and other (28%). in 96 (87%) of the negative fcs, introduction using an introduction scheme was advised. in 13%, patients did not receive standardized aftercare for different reasons, eg negative fc with a composite meal. in 19%, introduction with an introduction scheme failed. long-term information was available for 40 fcs. introduction failed in 12 fcs (30%), including 1 patient that even avoided traces of the food. patient-reported reasons for introduction failure were (n=11): symptoms after ingestion of the food (n=8), fear for allergic reactions (n=2) and not like the taste of the food (n=1). conclusions: short term introduction with an introduction scheme failed in 19%. however on the long-term in almost one third of the negative fcs, patients failed to reintroduce the food in daily diet, despite careful aftercare. it is recommended to give these patient even more intensive and tailored support. introduction: in order to help allergic patients manage often severe symptoms, food manufacturers are required to list allergens on labels and take precautions to avoid inadvertent contamination of foods with allergens. existing tools using generic immunoassays do not provide precise identification or quantification of specific food allergens. furthermore, existing elisa immunoassays are not able to be run simultaneously and are often unreliable. objectives: our goal was to develop and validate an accurate, sensitive and high throughput immunoassay that would enable simultaneous quantification of multiple allergens in foods. fluorescent beads coupled to allergen specific monoclonal antibodies were used to develop a multiplex array for simultaneous quantification of major allergens from peanut (ara h 6), milk (bos d 5), egg (gal d 2), and shrimp tropomyosin (pen a 1). target allergens were detected using biotinylated antibodies and streptavidin conjugated fluorochrome. the array was quantified using highly purified natural allergens as standards. allergen content was measured in various samples including samples spiked with purified allergen and allergen incurred food matrices. the results were compared to elisa. a multi laboratory validation was performed to validate the performance characteristics of the assay. results: there was a high correlation between the multiplex array and allergen specific elisa. the limit of detection of the array was as low as 20 pg/ml. no significant cross reactivity was observed between the various food allergen assays. the recovery of allergen from spiked samples was generally between 70 and 130%. inter and intra assay variability was observed to be less than 15%. in conclusion, an accurate, sensitive and reliable multiplex array for major food allergens was developed and validated. the flexibility of the microsphere technology allows for further expansion to produce a comprehensive array for the most important food allergens. this quantitative multiplex array may help to reduce the risk of inadvertent contamination of food. objectives: our aim was to create a food allergy web-based educational program for both schools and restaurants professionals. results: an interactive platform that hosts a free learning program, conclusions: the program and the toolkit are currently available online and are being implemented in schools at the north of portugal. we expect that fac program will give us an important insight on the professionals' knowledge about food allergy. additionally, acting as free integrated service and awareness effort, this program could be an educational tool easily adapted and disseminated, which may improve professionals 'commitment and skills to deal with food allergy in the community. 0495 | development of parallel reaction monitoring (prm) methods for soy and milk detection: consideration of allergen-derived ingredients chen s 1 ; krawitzky m 1 ; yang ct 2 ; downs m 1 1 university of nebraska-lincoln, lincoln, united states; 2 thermo fisher scientific, san jose, united states introduction: the presence of undeclared food allergens poses both regulatory and health risks. in order to assess the magnitude of these risks, accurate quantitative detection methods are required. for some allergenic foods, the food industry uses not only the allergenic source but also a number of source-derived ingredients. some detection methods (both immunoassays and mass spectrometry methods) may fail to accurately detect and quantify these allergen-derived ingredients if they are not taken into consideration during development. objectives: the objective of this work was to select peptide targets for parallel reaction monitoring (prm) mass spectrometry methods that are suitable for detecting a variety of soy-and milk-derived ingredients. six soy-derived and six milk-derived ingredients were obtained for this study. the ingredients were extracted (50 mmol l à1 tris-hcl, ph 8.6, with 6 mol l à1 urea, 20 mmol l à1 dtt, and 1% pvpp) and subjected to in solution trypsin digestion. discovery proteomics analysis was conducted by lc-ms/ms using a q exactive tm plus orbitrap tm running in top 10 data-dependent acquisition mode. peptides were identified using proteome discoverer 2.1 and relative, labelfree quantification was conducted on high-confidence (fdr<0.01) peptides. selected peptides were subsequently analyzed in a targeted prm mode. results: the relative abundance of identified peptides varied among both the soy-derived and milk-derived ingredients. in the case of soy ingredients, for example, there were particularly marked decreases in the abundances of numerous peptides, across different proteins, in a hydrolyzed soy protein isolate. in the milk ingredients, variation in peptide abundance could be more directly attributed to differences in product protein fractionation (eg a decrease in abundance of whey proteins in a sodium caseinate product), although some peptides and proteins maintained consistent abundance across ingredients. after applying specificity and performance criteria, 57 peptides from 14 soy proteins and 64 peptides from 10 milk proteins were selected for further analysis in a targeted prm method. conclusions: peptide abundances vary widely among ingredients derived from allergenic foods. consideration of these peptide differences during ms method development by incorporating discovery proteomics may lead to more versatile and widely-applicable quantitative detection methods for food allergens. introduction: milk and its derivates are usual ingredients in many food products, which must be excluded by cow's milk allergic (cma) patients. oit protocols in patients with cma appear to be safe and effective in inducing desensitization and milk introduction in the diet. objectives: we conducted a survey in cma patients after successful completion of an oit programme, to assess their dietetic profile after introduction of fresh milk and dairy products (unrestricted diet) we considered 42 cma patients (pts), 32 males and 10 females, age 5-29 (median 12.5), who successfully completed the oit protocol. these patients were on an unrestricted diet for milk and derivates, and they had been recommended to assume at least 100 ml of fresh milk or yogurt every day. patients were given an ad hoc questionnaire to assess the milk/dairy products intake at least 24 months after oit completion. results: from our investigation all of the pts have been assuming milk protein everyday without significant reactions after oit. 71% of the interviewee (30 pts) referred to be drinking fresh milk at least three times a week, in a quantity of 100 ml or higher. of note, 85% have to add cocoa powder (14 pts) or coffee (11 pts) to mask milk taste. dislike of milk taste was the cause of the refuse to take fresh milk in 10 pts (over 12 patients that didn't take milk at all); 5 pts who didn't drink milk had at least 125 ml yogurt instead, almost every day. fresh cheese was eaten at least once a week by 50% of all the pts; hard cheese was eaten as main course almost once a week by 62%. 86% of patients consumed biscuits and sweet baked products containing milk/derivates at least once a week, 62% every day. pizza was also present in the diet of the majority of pts (86%), once a week/month. ice cream was appreciated by all the patients and regularly assumed especially during the summer time. conclusions: taste seems to be the main factor that directs the daily choice of milk derivates or milk containing products. all pts easily consume baked products containing milk/derivates, and the majority of them accept fresh milk as advised in order to maintain unresponsiveness. our survey confirms that oit is effective in expanding food choice, but for some patients the change of dietetic habits is hampered due to taste reasons. a more detailed, qualitative and quantitative analysis of the milkunrestricted diet will derive from the 7-days food diary given to these patients. 0497 | frozen-defrosted dried skimmed milk is a suitable product for sublingual immunotherapy for cow's milk allergy introduction: sublingual immunotherapy (slit) is a promising treatment for cow's milk allergy (cma) due to its favourable safety profile. however, its efficacy is limited-probably due to the small volumes and doses that can be delivered sublingually, especially in children. milk products with preserved allergenicity that allow higher protein concentrations in smaller volumes might potentially improve the efficacy of slit for cma. dried skimmed milk (dsm) could fulfill these characteristics. in addition, once dsm is reconstituted, freezing individual vials from the same batch for further administrations could increase dose-consistency throughout the treatment, which would help ensure safety. however, little is known about whether processing to produce dsm, and further freezing-defrosting, may alter its allergenicity. objectives: to evaluate the allergenic protein composition of dsm, including once frozen-defrosted, in comparison to fully-allergenic usually consumed fresh milk. methods: sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) followed by silver staining was performed following the laemmli method to determine the soluble protein composition of the following products: fresh pasteurized milk (friesland campina, amersfoort, the netherlands), dsm (marvel, the premier foods group ltd, london, uk) and frozen-defrosted dsm to identify all protein bands present. western blot with anti-whey and anti-casein antibodies was performed to identify the specific allergenic proteins. results: no significant differences were found amongst the milk products tested, with sds-page displaying all the bands corresponding to the major allergenic proteins in milk (alpha-lactalbumin, beta-lactoglobulin and alpha-, beta-and kappa-caseins) and the western blot showing recognition to all proteins with similar intensity. the major allergenic proteins present in unprocessed milk are well-preserved in dsm, even after freezing-defrosting, making it a convenient product for sublingual immunotherapy. results: nineteen children were randomized into the low dose group (n=11) and the elimination group (n=8). there were no significant differences in background between these groups. after 24 weeks of therapy, the rate of passing the oral food challenge test with 1/8 th of a whole egg was significantly higher in the low dose group (11/11 (100%) vs 4/8 (50%), p=.018). ovomucoid-specific ige levels in the low dose group after 24 weeks were significantly lower than those at baseline. adverse events associated with both therapies did not occur during the study period. conclusions: these results show that low dose ingestion of egg is safe and effective for tolerance or desensitization induction in children with egg allergy, without the need for dose elevation. 0499 | another brick in the wall: toward a national food allergy strategy for canada introduction: the world is experiencing an apparent epidemic of food allergies. with rising prevalence and increasing global spread, basic scientists continue to search for causes while clinical and social scientists continue to study the consequences. these include life-long chronic illness, fear and anxiety, social exclusion and stigma, all of which are underscored by inequalities across vulnerable groups (eg, low income families, immigrants, indigenous peoples). concomitantly, consumer organizations and patient support groups attempt to influence policy in order to maximize choice and minimize risk for individuals and families affected by food allergy, both directly and indirectly. this includes food labeling legislation, food safety regulations, and policies and practices in public places such as schools, restaurants and transportation modes (eg, airplanes). objectives: in canada, with the support of 10+ years of science undertaken by the allergy genes and environment (allergen) networks of centre of excellence in allergic disease, we are leading a national team that aims to establish the building blocks for a national food allergy strategy. conclusions: the greatest challenges to building a national food allergy strategy are not those related to the basic or clinical science, but rather the activities and commitment of individual stakeholders. while gaps remain in the science, the challenges of transdisciplinary integrated knowledge translation hinder progress. we present lesions learned regarding culture shifts that will facilitate the progress we need to maximize choice and minimize risk for canadians affected by food allergy. 0500 | component resolved diagnostics reduces diet restrictions by half among finnish school children savolainen j 1 ; mascialino b 2 ; pensamo e 3 ; hermansson l 4 ; silvan m 3 ; borres mp 4 ; korhonen k 5 1 university of turku, turku, finland; 2 department of allergology, uppsala, sweden; 3 thermo fisher scientific-immunodiagnostics, turku, finland; 4 university of turku, uppsala, sweden; 5 thermo fisher scientific-immunodiagnostics, lieto, finland introduction: there are 50 000-100 000 special diets because of food allergy in finnish schools. the finnish allergy program 2008-2018 was launched to reduce problems and costs induced by allergies. one of the special aims was to reduce diets caused by food allergy will by 50%. the h€ ark€ atie social and health care service region schools have required doctor's certificates for diets for over abstracts | 337 10 years. all diets are listed in a register and checked on a regular basis. objectives: the purposes of this study were to evaluate whether it is possible reduce the special diets when the special diet system appears to be working well and to assess the added value of immu-nocap ® isac allergen component diagnostics in the intervention. results: there were 2885 children attending the schools in the h€ ark€ atie region. there were 205 children with diet due to food allergy. children were recruited to study from the diet register and contacted by a nurse's phone interview. there were 48 children who refused the study leaving 157 children for the assessment of the diet. 23 children were not following any diet and in 44 children the diet was not regarded necessary by the study nurse. 90 children agreed to take part in the study and were referred to laboratory tests for food specific ige and isac. 7 children dropped out leaving 83 children in the study. after the food specific ige results were available, children were contacted by physician. based on the results and/or interview a free diet was allowed for 3 children. 15 children were eating small amounts of the food and were encouraged to increase the use of the foods. 48 children were advised to avoid the foods only if they caused symptoms. only 17 children were advised to continue diet. after the results of isac were available, children conclusions: the study confirmed that it is possible to have diets decreased over 50% in accordance to the finnish allergy program. additionally, the study showed that food allergic children can benefit from the use of component resolved diagnostics. 0501 | omalizumab treatment for severe food allergy caused by lipid transfer protein: a preliminary case series mari a; zennaro d; ferrara r; bernardi ml; alessandri c caam-centri associati di allergologia molecolare, rome, italy introduction: lipid transfer proteins (ltp) are allergens from plantderived foods causing symptoms ranging from oas to anaphylaxis. ltp are present in almost all kind of plant species used for human feeding. ltp allergic patients suffer of reactions to a broad variety of foods, with a very personal reactivity profile which can involve few or many foods. in addition, patients who start to record severe reaction to several foods begin to be afraid of eating any other related or non-related food, leading to a poor diet. omalizumab (ozm) has been documented to be effective in treating food allergies with sensitization to other allergens. objectives: to document the approach for recruiting, studying, monitoring ltp allergic patients, and do a preliminary evaluation of the clinical impact of ozm therapy. results: six adult patients with documented reactions to plantderived foods in the last 12 months and diagnosed to be ige positive to multiple ltp by using multiplex testing, namely isac112 at the beginning and faber244 during the follow up, were recruited. ozm was offered as an off label treatment adopting an open label study design. a questionnaire was submitted to each patient before, during and after the treatment. foods listed as "no more eaten" where considered for reintroduction, starting from those excluded because of fear, going for those with an increasing risk of reactions. re-introduction was done at home after receiving detailed instructions, having all rescue medication at hand. the allergist was connected real time with the patients using one or more ict tools. one patient dropped out because was not complying with the scheduled reintroduction. three patients completed the treatment with a successful re-introduction of all or almost all excluded foods, in two cases peach was reintroduced. the last two patients reintroduced 50%-60% of the excluded foods, including some of those previously causing reactions. the attempt of re-introducing the most risky foods failed causing local allergic reactions promptly controlled by the therapy. all foods re-introduced were tolerated once the treatment was stopped. conclusions: ozm seems to be a promising treatment for severe ltp allergics, improving their qol. starting from the recruitment phase to the re-introduction, the overall procedure looks quite complicated and deserve much resources. ige sensitization to ltp, carefully defined before treatment, can be monitored during the ozm course as the drug doesn't interfere with faber test ige detection. 0502 | oral immunotherapy with peach-juice in lipid transfer protein (ltp) allergy: is it possible to reach tolerance? introduction: food allergy to rosaceae fruits and nuts, due to sensitization to lipid-transfer protein (ltp), is frequent in the mediterranean area countries. based on milk allergy oral immunotherapy (oit) protocols, a study is proposed to obtain an oit regimen in patients allergic to ltp. objectives: we included patients with anaphylactic reactions due to ltp-food allergy, from january/2015 to october/2016. skin prick test (spt) was performed with a food and inhalant battery, ltp bial-aristegui ® (0.1 mg/ml), prick by prick (pbp) with a specific commercial peach-juice (dilutions to endpoint), and determination of pru p3 ige levels (immunocap). the presence of pru p 3 protein in the peach-juice, was confirmed by sds-page and elisa method, and quantified with anti-pru p 3. we elaborated an oit guideline with progressive administration of peach-juice, 1-2-4-5 drops (5drops=0.13 ml), sublingually, starting concentration chosen according to the endpoint spt results, up to 5 ml daily dose. the dose reached at the allergy service, was continued at home, daily, for 1-2 weeks. all the increasing doses were performed at the allergy service, after pbp with peach-juice and ltp control spt. after a time, an oral challenge up to 250 ml peach-juice was performed. patients were instructed on the importance of maintaining regular intakes and avoiding cofactors. results: 16 patients started this protocol (6 male/10 female), aged 7-38 (media 19.67). peach-juice analysis in agarose gel electrophoresis, showed a 10 kda band quantified as 21.16 lg/ml of ltp. there was no difference between wheal diameter on spt for ltp bial-aristegui ® and pbp with commercial peach-juice along all the study. average concentration on pbp to endpoint: 1/1000 (4), 1/100 (8), 1/10 (4). pru p 3 ige level average: 30.22 ku/l. none anaphylactic or serious reactions during the oit protocol appeared. only five patients (31%) reported mild occasional symptoms (oral pruritus and mild located urticaria). 11 patients reached 5 ml daily dose (68.75%), and 4 of them (25%) completed oral food challenge with peach-juice up to 250 ml, with good tolerance. this pattern also allowed us to reintroduce withdrawn foods from the diet due to previous reactions, with good tolerance. results: 60 children (age 10-18 years) sensitized to dog dander extract (median 11.5 ku a /l), with or without known dog induced allergic airway disease, were included. nasal provocation testing with dog dander extract was performed. measurement of ige to dog dander extract and to can f 1-can f 6 was performed with immunocap. an ige level ≥0.1 ku a /l was considered positive. among all 60 children sensitization to can f 1 (65%) and can f5 (62%) was most frequent. corresponding numbers for can f 4, can f 2, can f 6 and can f 3 were 48%, 47%, 47% and 27% respectively. based on the results from the nasal challenges three groups were identified; no (n=21), mild (n=14) and strong reaction (n=25). the median ige levels to dog dander and to all 6 allergen molecules were higher in the strong reaction group than in the mild and the no reaction group. among children with a strong reaction, ige to can f 4 was found in 68% (17/25) compared to 24% (5/21) among children with no reaction (p=.003). ige to can f 6 showed a similar pattern (p=.006). 40% (10/25) of the children with a strong reaction were sensitized to can f 3 compared to 9.5% (2/21) of the patients with no reaction (p=.01). no associations were found between the ige levels to can f 1, can f 2 and can f 5 and the no reaction or strong reaction groups. using multiple regression analysis, with all 6 allergens in the model, sensitization to can f 4 showed a statistically significant difference between the groups. conclusions: molecular allergology may improve diagnostics of dog allergy in children. sensitization to the lipocalin can f 4 was significantly associated to a strong positive nasal reaction implying its usefulness as a marker of clinically relevant dog allergy. tsolakis n 1 ; malinovschi a 2 ; nordvall l 1 ; janson c 2 ; mattson l 3 ; lidholm j 3 ; borres m 3 ; alving k 1 1 uppsala university, women's and children's health, uppsala, sweden; 2 uppsala university, medical sciences, uppsala, sweden; 3 thermo fisher scientific, uppsala, sweden introduction: cat allergy is common worldwide and a major trigger of asthma in many countries. molecular patterns of cat sensitisation vary between individuals but their relationship with allergic inflammation has not been extensively studied. objectives: the aim was to investigate the prevalence of ige to different cat allergen components and their relationship to type-2 inflammation and bronchial responsiveness in young asthmatics. conclusions: ige sensitisation to cat serum albumin (fel d 2) and cat lipocalins (fel d 4, fel d 7) , but not to secretoglobin (fel d 1) or cat dander extract, were independently associated with feno and b-eos count. we suggest that cat serum albumin and cat lipocalins can be used as markers for increased risk of type-2 inflammation in young asthmatics, as shown in multiple regression analyses. 0505 | complete sequence and recombinant production of horse dander allergen equ c 2 lidholm j; lundgren t; larsson h; mattsson l thermo fisher scientific, uppsala, sweden introduction: horse dander is an increasingly important cause of respiratory allergy. equ c 2 was one of the first horse allergens to be recognised but only a small part of its amino acid sequence has been reported. objectives: the aim of this study was to determine the complete sequence of equ c 2 and express it as an immunoreactive recombinant protein. methods: equ c 2 was purified by size exclusion, hydrophobic interaction, anion exchange and reversed phase chromatography. recombinant equ c 2 was expressed as a hexahistidine tagged protein in e. coli and purified by immobilized metal ion affinity and ion exchange chromatography. ige antibody reactivity to natural and recombinant equ c 2 and other horse dander allergens was determined in sera of 25 subjects allergic to horse. results: a putative equ c 2 sequence predicted from genomic data revealed a lipocalin protein of 159 amino acids with a highest sequence identity among known lipocalin allergens of 33% to can f 4. n-terminal sequencing and mass spectrometric analysis of purified natural equ c 2 confirmed 97.5% (155/159 residues) of the predicted sequence. recombinant equ c 2 displayed ige antibody binding activity comparable to that of purified natural equ c 2 (r=.98). of the 25 horse allergic subjects studied, 19 (76%) showed ige antibody binding to equ c 1, 13 (52%) to equ c 2, 5 (20%) to equ c 3 and 7 (28%) to equ c 4. the complete sequence of equ c 2 was established. as a fully immunoreactive recombinant protein, it represents an important addition to the panel of allergens useful in the diagnosis of allergy to horse. 0506 | component resolved diagnosis using guinea-pig allergens elucidates allergen sensitization profiles in allergy to furry animals objectives: to identify major guinea-pig allergens and to evaluate their potential as reliable markers for a specific ige-diagnosis in comparison to dander extracts. results: forty-three patients with a clinical history of allergy to guinea-pig and 45 patients allergic to cat and/or dog were recruited for the study. major guinea-pig allergens were identified by ige-immunoblot and n-terminal sequencing of ige-reactive proteins. corresponding cdnas were cloned and allergens were expressed as recombinant proteins in e. coli. specific ige to animal dander, fel d 2 and can f 3 were determined, specific ige to fel d 4, can f 6 and to guinea-pig allergens were quantified by elisa. two new guinea-pig lipocalin allergens, cav p 1 and cav p 6, were identified in guineapig dander. the combination of 4 guinea-pig allergens, the 2 new allergens and the previously isolated lipocalins cav p 2 and cav p 3, enabled the identification of 39 out of 43 patients sensitized to guinea-pig. the vast majority of the patients had specific ige to cav p 1 (84%). cav p 6 shares 53% sequence identity with fel d 4 and can f 6 and was found to be cross-reactive with these cat and dog allergens. in the group of cat and/or dog allergic patients, 87% had also specific ige to guinea-pig dander. nearly half of those (47%) had ige to cat serum albumin fel d 2 or to fel d 4 (58%) and to can f 6 (56%), explaining the high degree of cross-reactivity to guinea-pig dander. only 25% of the cat/dog allergic patients with a positive isle test to guinea-pig dander had specific ige to any of the non crossreactive guinea-pig allergens cav p 1, cav p 2 or cav p 3. however, none allergen has been characterized from mongolian oak. objectives: in this study, we tried to characterize a major allergen from mongolian oak. results: a molecule homologous to pathogenesis-related protein 10, a putative que m 1, was cloned by rt-pcr and its recombinant protein along with que a 1, an allergen from white oak (q. alba) was produced. cloned que m 1 sequence shares 57.5%-96.2% amino acid sequence identity (96.2%) with pr-10-like allergens from various plants. allergenicity and diagnostic value of recombinant que m abstracts | 341 1, que a 1 and bet v 1 proteins were compared by elisa using sera from oak sensitized subjects. specific ige to recombinant que m 1, que a 1, and bet v 1 were detected in 90.0%, 78.0%, and 94.0% of 50 serum samples from korean tree pollinosis patients. recombinant que m 1 was able to inhibit ige reactivity to que a 1 and bet v 1, indicating its strong cross-reactivity. activation pattern of basophils from five patients was similar in terms of cd203c expression and protein concentration of challenged bet v 1 and que m 1. objectives: over the course of one year, all patients who were seen at our immunoallergology clinic for the first time underwent spt with our standard series, to which four olive tree cultivar extracts were added (arbequina, blanqueta, hojiblanca e picual). we then recorded wheal size diameters, considering a wheal >3 mm to correspond to a positive test. we recorded 832 individual sessions of spts, in which 613 presented at least one positive result. two hundred and thirtysix of these patients (38.5%) had a positive spt for olive tree pollen, only one of them being monosensitized. when looking only at pollen-sensitized patients, twenty-two patients (5.9%) tested positive solely for olive tree pollen. in all allergic patients, the most frequent cultivar sensitization was to the cultivars hojiblanca (31.5%) and arbequina (31.3%), followed by the cultivars picual (29.2%) and blanqueta (27%). twenty-six patients (11% of all olive pollen sensitizations) had a positive spt for the conventional extract and negative spt for the cultivars; 192 patients (81.4%) had a positive spt for both. we noted that 7.6% (n=18) of patients sensitized to olive tree pollen had a negative spt with the conventional extract and positive with one of the cultivar extracts. the cultivar hojiblanca was the most frequent in this group (66.7%, n=12), followed by the arbequina cultivar (55.6%, n=10). in the group as a whole, there was a positive correlation between the results of the spts with the conventional extract and each of the cultivar extracts. this correlation was weaker with the cultivar hojiblanca (r=.785). conclusions: some patients, sensitized only to the pollen of certain olive tree cultivars, are not identified with the conventional extract. spt with the hojiblanca cultivar could identify most of these patients in our country, and therefore should be considered in patients with a history of pollinosis and a negative spt for the common extract of olive tree pollen. 0509 | structural and immunological comparison of heat treated pru p 3 and art v 3, the non-specific lipid transfer proteins of peach and mugwort pollen wildner s 1 ; stock l 1 ; regl c 2 ; alessandri c 3 ; mari a 3 ; huber c 2 ; stutz h 2 ; gadermaier g 4 objectives: recombinant art v 3.0201 and pru p 3.0102 were expressed in e. coli rosetta-gamib plyss and purified using cation exchange chromatography. proteins were analyzed in gel electrophoresis and mass spectrometry. proteins were incubated at 95°c in time intervals up to 120 min using buffers at ph 3.4 and 7.3. physico-chemical properties of native and heated allergens were analyzed by circular dichroism spectroscopy, dynamic light scattering and size exclusion chromatography (sec). using sera from italian patients sensitized to pru p 3 and art v 3 (n=26), ige binding to native and heat-denatured allergens was investigated in elisa. results: highly pure recombinant pru p 3 and art v 3 were obtained as non-tagged proteins from e. coli. identity and formation of disulfide bonds was verified by mass spectrometry. circular dichroism showed high thermal stability of both proteins at acidic ph. the alpha-helical fold of art v 3 was lost upon heating for 15 min at ph 7.3 while pru p 3 was already altered after 5 min. purified pru p 3 and art v 3 are monomeric molecules with a hydrodynamic radius of 1.6 and 1.8 nm, respectively. structural relaxation is observed upon heat treatment which is not attributed to protein aggregation as determined by sec. the ige reactivity to both allergens was largely unaffected upon heating at ph 3.4. notably, ige reactivity to art v 3 was already significantly decreased upon 15 min heating and was completely abrogated to both proteins after 120 min denaturation at neutral conditions. conclusions: even though the fold of pru p 3 is more compact compared to art v 3, susceptibility to structural changes upon thermal treatment at neutral conditions are more pronounced which do however not directly translate to lower ige binding capacities. particularly the buffer environment needs to be considered when formulating ltp-containing products which undergo heat treatment. objectives: we sought to determine the ige binding capacity and potential diagnostic value of a recombinant hybrid molecule. results: the codon-optimized nucleotide sequences of a hybrid molecule comprising the full sequences of blo t 5 and der p 2 at the amino and carboxyl ends respectively, here named bp-2, was cloned into a plasmid vector and expressed in escherichia coli as a 6xhis tag protein. two hundred and thirty three sera from colombian (n=118) and cuban (n=115) allergic patients were tested by elisa for ige reactivity. thirty seven sera from non-allergic subjects and negative skin test (spt) to mites were used as controls. all subjects provided written informed consent to their participation in this study. hdm allergy was diagnosed on the basis of clinical symptoms in combination with mite extract spt. potential diagnostic value of bp-2 specific ige was determined by receiver operating characteristic (roc) analysis and area under roc curve (auc) calculated. positive serum ige values to hybrid molecule were defined as optical density (od) higher than 0.13 (mean od plus 3 standard deviations in 15 nonallergic subjects). in respiratory allergic patients, the overall frequency of positive ige reactivity to bp-2 was 70.9%, in non-allergic subjects the frequency was 21%. serum ige levels to bp-2 were positively correlated to spt to b. tropicalis (spearman r=.54, p=.001), and to d. pteronyssinus (spearman r=.4, p=.001). using spt to mite extracts as gold standard, the sensitivity and specificity of serum ige levels to bp-2 were 71.4% and 94.1% respectively, with an auc of 0.82 (95% confidence interval 0.74-0.89). conclusions: these data suggest that bp-2 could be a useful reagent for identifying allergic patients sensitized to b. tropicalis and/or d. pteronyssinus. 0511 | igg, ige and igg4 specific antibodies to molecular allergens of aspergillus fumigatus introduction: in clinical allergy, alongside with skin prick tests, in vitro determination of specific ige for a particular patient contributes to the diagnosis and helps to estimate the risk associated with different food allergens. however, with commercial methods of specific ige antibodies detection (component-resolved diagnosis, crd), the clinician is typically limited by the list of the available allergens. objectives: to overcome this limitation, we developed two component-resolved diagnostic tests for food allergy in which natural extracts can be used. in the first developed method, the crd is performed using immunoaffinity capillary electrophoresis (iace) coupled with matrixassisted laser desorption/ionization mass spectrometry. meanwhile, the second method is based on in-tube immunomagnetic separation (ims) with mass spectrometry identification (mass spectrometry or peptide mass fingerprinting). in both techniques, magnetic beads coated with antihuman ige antibodies are used to extract the ige antibodies from the blood serum of the allergic patient. then, the immunocomplex, obtained on the magnetic beads, is used to quantify the total ige level or to probe the ige binding with standard allergens or natural allergenic extracts. afterwards, the identification of the extracted proteins, ie potential allergens, is performed by mass spectrometry with or without ce separation. after optimisation, the proposed methods have been successfully applied to a commercial blood sample of a patient with a known allergy to cow's milk, with results confirmed by standard tests. as a proof-of-concept, the sensitization profile of a patient suffering from protein contact dermatitis to the cow's whey fraction has been determined. we confirmed the presence of circulating ige antibodies binding lactoferrin and bovine serum albumin. cross-reactivity tests were also performed using goat and sheep milk and revealed the patient sensitivity to serum albumins from these two milks. such approaches open the possibility for direct identification of ige-bound allergens molecular mass and structure. these methods allow the discovery of yet unknown allergens and could be useful for precise personalized allergy diagnosis, allergens epitope mapping, and cross-reactivity studies. objectives: the objective of this study was to investigate the validity of cord blood ige for predicting atopy at 6 years of age. methods: a total of 385 children born in 2010 participated in the longitudinal investigation of global health in taiwanese schoolchildren (lights) cohort. total ige was measured in umbilical cord blood at birth. perinatal history was collected from medical records in the chang gung memorial hospital, taiwan. total and specific serum ige and questionnaires were carried out at 6 years of age. receiver-operating characteristic (roc) curves were used to determine the validity of cord blood ige for predicting atopy at 6 years of age. logistic regression models were applied to assess the association between cord blood ige and atopy at 6 years of age. results: cord blood ige levels was significantly associated with total serum ige level at 6 years of age (r=.314, p<.001). the cord blood ige levels in atopic children aged 6 years (meanaesd, 1.61ae6.2 ku/l) were significantly higher than those in nonatopic children (0.36ae0.94 ku/l) (p<.001). the area under the receiveroperating characteristic (roc) curve of cord blood ige for predicting atopy at 6 years of age was 0.702. the sensitivity, specificity, and positive and negative predictive values of cord blood at the optimal cutoff of 0.34 ku/l on the roc curve for predicting atopy were 50.9%, 88.1%, 92.1%, and 39.8%, respectively. higher cord blood ige levels (≥0.34 ku/l) was associated with a higher likelihood of atopy at 6 years of age (or=7.66; 95% ci: 2.81-20.90; p<.001). our results indicate that cord blood ige is a potential predictor of atopy at school age, with an optimal cutoff of 0.34 ku/l. bogomolov a vinnitsa national pirogov memorial medical university, vinnitsa, ukraine introduction: allergen sensitization is being diagnosed by commonly available methods in clinical practice-skin prick tests (spts) and specific immunoglobulin e test (sige). recently, a new thermographic (th) method for the assessment of spt was developed, and it was demonstrated that the th measurements of forearm temperature distribution during spt, supported by a mathematical model, offer a new quantification method of allergen-induced skin reactions. objectives: the aim of this study is a comprehensive comparison of the th method with spt and sige techniques. the group of patients who were participated in this study consist of 45 patients. among them were 24 patients (53.3%) with allergic rhinitis and 21 patient (46.7%) with asthma, 35.5% of them were men and 64.5% were women aged 18-53 years (mean age 38.3ae6.5 years). spt and sige testings were performed by the standard techniques. for th analyses, set of thermograms of both forearms were acquired after prick and analyzed with the use of developed software. all results were converted into categorized scale for comparison. after counting patients who were true positive (tp), true negative (tn), false positive (fp), and false negative (fn), the sensitivity, specificity, and accuracy were calculated according to the following formula using the results of spt as the standard; sensitivity=tp/(tp+fn), specificity=tn/(tn+fp), and efficacy=(tp+tn)/(tp+tn+fp+fn). the results showed high correlation coefficients between the methods equal to 0.73-0.97. the sensitivity and accuracy of the th assessment in respect of both the classical methods is at a good level (0.70-0.94). the acceptable level of specificity 0.60-0.88 was also achieved for the majority of allergic reactions. the best accordance was observed between th and sige results (r=.92), while th-spt was the most divergent pair r=.85. in case of particular allergens, the biggest correlation was 0.99, while the smallest value amounted to 0.76. the results of diagnostic indicators of thermographic measurement of skin reactivity in comparison with the classical methods of determining the sensitivity to allergens show the prospect of using the method in routine practice. the main advantages of this method are its higher measuring ability and objectivity, by which the possibility of making error in diagnosis is significantly reduced. introduction: chronic urticaria symptoms may be worsened by factors such as temperature, exercise, hormones and stress. a salicylate rich diet has been reported to worsen symptoms in these patients. the mechanism by which natural salicylates do this is unclear but, like aspirin, is thought to be due to their ability to interfere with the arachidonic pathway via cyclooxygenase inhibition. studies have shown that low dose aspirin increases serum il-3 levels in patients with antiphospholipid syndrome. il-3 is important in basophil and mast cell function, inducing mediator release and cd203c upregulation in the absence of ige stimuli. objectives: the gold standard for diagnosing salicylate exacerbated chronic urticaria is by challenge testing. there is no in vitro laboratory test approved for routine diagnosis. this study investigated whether il-3 levels are raised in patients with chronic urticaria and if these levels were affected by salicylate intake. we aimed to find an optimum method of measuring il-3 levels by comparing levels in serum and salivary samples. the quantikine human il-3 enzyme linked immunosorbent assay (elisa) was validated and used on both saliva and serum of patients with chronic urticaria and normal controls. this was a case control study of 19 medicated patients with chronic urticaria and 26 controls at university hospital southampton, to see whether there were any correlations with il-3, and degree of salicylate intake (based on a questionnaire). introduction: since the introduction of molecular components in allergy, a big challenge of allergy diagnostics is to connect clinical symptoms with optimal test use and correct interpretation of results. objectives: the aim of the study was to (1) develop an algorithm that would meet that need, and (2) to evaluate the effect of introduction of algorithm to clinical practice. the algorithm was developed which groups clinical symptoms into six categories: rhinoconjunctivitis/ asthma, oral allergy syndrome (oas), urticaria/angioedema, eczema, anaphylaxis, and a combination of symptoms and combines them with knowledge of possible allergen specificity. this information is combined with two basic allergen mixtures (panels), reflex testing of selected food molecular components and accompanied by interpretative comments. the introduction of our algorithm led to less inhalation screenings, more food screenings and an increase in the requested molecular components. the oas was seldom recognized or used as a symptom by specialists. the reduction in costs, by using the possibility that the disease presentation may be a consequence of an relatively not dangerous oas, was therefore not achieved. all pr-10 positive proteins in various allergen sources showed also positivity for birch antigen. the screening based on this algorithm has potential to enable clinicians/general practitioners with a tool to increase the pre-test probability of allergy for the most frequently occurring allergens. allergy diagnostics may be more efficient if pr-10 component of birch (r bet v 1) is included in early screening and can help in early recognition of oas. helbling a 2 department of otorhinolaryngology-head and neck surgery 0492 | clinical and immunological evolution of patients who failed milk-oral immunotherapy there is lack of evidence on evolution among failures. objectives: to analyze clinical and immunological evolution of patients who failed milk-oral immunotherapy. data were obtained from medical records of a cohort of 20 patients who failed moit in the past 10 years in hospital infantil universitario niño jesus 14(70%) patients failed during build-up phase [13(92%) due to adverse events (ae) and 1(8%) to family decision. 6 failed during maintenance phase: 3(50%) due to eosinophilic esophagitis, 1(16%) to family decision, 1 to psychiatric disorder and (range 5-1023). the most frequent aes were cutaneous and gastrointestinal symptoms. 10/20 (50%) patients underwent a second moit and was successful in 5. the second moit was performed between 2 and 9 years after the first one. there was no statistical differences between specific ige(ku/l) levels at baseline and 12 months after the moit end portugal introduction: fish allergy is common in countries where consumption is high. parvalbumins present in fish muscle are the major allergens. allergy to multiple fish species is caused by parvalbuminspecific cross-reactive ige. cross-reactivity with parvalbumin from baltic cod retrospective study of patients with fish allergy followed in our immunoallergology department. fish tolerance acquisition was evaluated by oral food challenge. statistical analysis was performed using spss version 23 (descriptive statistics, student test results: 81 pts were included (55 male, 26 female), 62 children and 19 adults (age 14ae9 years). 63(78%) had previous history of rhinitis, 35(43%) of asthma and 54(67%) of eczema age of first contact with fish averaged 8.9ae4.0 months (min4, max36) and the possible types of contact were: oral in 78(96%), cutaneous in 22(27%) and inhalation of cooking fish vapours age of first clinical manifestation (excluding the 4 pts who developed allergy in adulthood) was at 22ae25 months (min4, max132) the clinical manifestation of the reaction was: angioedema/urticaria 58(72%), gastrointestinal symptoms 28(35%), eczema 27(33%), respiratory symptoms 19(23%), oral allergy syndrome 10 (12%), cardiovascular symptoms 2 (2%) age at the first immunoallergology out-patient clinic consult averaged 7ae9 years (min 0.4, max 48) and time from first symptom to first thermo-fisher) was evaluated before and after acquisition of tolerance to at least 1 fish. before tolerance, sige (ku/l) averaged 21 roc curve (area under curve 0.854) showed that, for gad c 1 105 ku/l, pts had a sensitivity of 90.2% and specificity of 62.5% that they would have a negative oral food challenge to a fish an sige<0.635 ku/l had sensitivity of 98% of a negative challenge 34 ku/l had specificity of 92.9% of a positive challenge. conclusions: fish allergy is a common allergy in early childhood however, acquisition of tolerance is possible. rgad c 1 appears to be a good marker for fish tolerance and could help allergologists as to when start testing for fish tolerance key: cord-015306-us58wwmp authors: nan title: abstracts for the ipna congress, 30 august 3 september 2013, shanghai, china date: 2013-06-21 journal: pediatr nephrol doi: 10.1007/s00467-013-2518-4 sha: doc_id: 15306 cord_uid: us58wwmp nan first 5 days after the diagnosis. dwi-mri was performed without application of contrast medium and without general anaesthesia. results: dwi-mri examination confirmed the inflammatory infiltration in kidney parenchyma in all our patients (100 %). on the other hand, static renal scintigraphy confirmed inflammation only in 15 children (60%). six months later, none of the two follow-up examinations showed any signs of inflammation or scarring in 17 children examined so far. conclusion: in conclusion, nuclear magnetic resonance (dwi-mri) imaging seems more beneficial and accurate in the diagnostics of acute pyelonephritis when compared with static renal scintigraphy. moreover, dwi-mri provides more accurate information on the extent of kidney damage. objective: to evaluate the most proper radiologic investigation algorithm in detecting high grade vesicoureteral reflux (vur) and renal cortical scarring after first febrile urinary tract infections in infants aged less than one year . methods: a total of 408 infants aged less than one year with a first febrile urinary tract infection who completed the diagnostic follow up of renal bladder ultrasound (rbus), voiding cystourethrography (vcug) and late 6 months technetium 99 dimercaptosuccinic acid renal scan (dmsa) were enrolled in the study.the most proper radiologic investigation algorithm that could highly detected both high grade vesicoureteral reflux and renal scar in infants after the first febrile urinary tract infection considering high benefit, low cost, low radiation exposure were assessed. results: abnormal renal bladder ultrasound (rbus) was identified in 101 (24.7 %) infants. vesicoureteral reflux (vcug) was identified in 92 (22.5%) with high grade reflux (grade 4 and 5) in 18 (4.4%) infants.abnormal renal parenchyma including renal scar was identified in 32 (7.8%) infants.the top down approach with late 6 months dmsa scan showed high benefit in detecting all abnormal renal parenchyma including renal scars, with high sensitivity ( 72%) in detecting high grade vesicoureteral reflux (vur) whereas reducing the unnecessary investigation for low grade vur (94.6%) and radiation exposure per patient (0.89 msv). conclusion: currently, there is no ideal diagnostic radiologic investigation after a first febrile urinary tract infection in infants and children.the study suggest performing the top down approach with late 6 months dmsa scan that could detect all renal scars and highly detection of high grade vur. abstract# p-sat010 association between postvoid urine bladder volume and urinary tract infection in infants and children: a retrospective cohort study orpheus monakil, ivy avilla department of pediatrics, da la salle university medical, philippines objective: to determine the association between post void urine bladder volume residual and urinary tract infection(uti) in children. methods: medical records of pediatric patients aged 0 to 18 years old with diagnosis of urinary tract infection were reviewed. demographic data, urinalysis and urine culture results and kidney and urinary bladder(kub) ultrasound findings were tabulated and analyzed using the 95% confidence interval. relative risks were computed with a level of significance of p value < 0.05. results: a total of 803 patients were included in the study. five hundred forty-four(67.8%) belongs to age group < 5 years old. majority were females with a 0.97:1 male to female ratio. escherichia coli is the most common organism isolated. patients with post void urine bladder residual are at risk for having growth in the urine culture. in sub group analysis for age and sex, statistically significant results were noted among patients belonging to the 3-5 years with the rr of 1.337, 95% ci (1.082, 1.651) p-value of 0.003 and males with rr 1.186 (95% ci 1.024, 1.374) p value 0.0168. conclusion: there is an association between the occurrence of urinary tract infection and the presence of post void bladder volume residual. males and those patients aged 3 to 5 years of age are more at risk for urinary infection. objective: to evaluate if a relationship exists between renal ultrasound and voiding cystourethrography (vcug) findings among children with uti and determine whether the renal ultrasound findings/results can serve as a guide if a procedure such as vcug is needed to be done in the patient. methods: medical records of infants and children diagnosed as having uti who underwent renal ultrasound and vcug were reviewed. demographic data, urine culture and results of imaging studies were tabulated and analyzed. kappa statistics was used to determine the agreement between renal ultrasound and vcug results. mcnemars test was utilized to determine the statistical significance of the agreement. level of significance was place at p value of < 0.05. results: a total of 146 patients were included in the study. thirtyeight(26%) had vesicoureteral reflux. there were more females with a male to female ratio of 1:1.3 while cases with reflux had a 1:1.5 male to female ratio. most common chief complaint was fever. e. coli is the most commonly isolated etiologic organism from the urine. twenty(52.7%) patients had primary vur while 18(47.3%) were secondary. fifty two of 146 children had abnormal sonogram; of duplex collecting system (dcs) diagnosed during postnatal usc on objective: the aim was to evaluate the relationship of laboratory investigations, therapeutic delay time (tdt), and therapeutic response time (trt) with acute renal damage and to verify these parameters in the presence of non refluxing and refluxing urinary tract infection (uti). methods: a prospective study was conducted in 67 children. all patients received voiding cystourethrography (vcug) and dimercaptosuccinic acid (dmsa) renal scintigraphy. statistical analyses were applied to assess all parameters with dmsa renal scintigraphy. results: abnormal dmsa renal scintigraphy was detected in 20/67 (29.9%) patients. there were no significant differences in peak temperature, tdt and treatment duration. however, white blood cells (wbc) count, percentage of serum polymorphonuclear cells (%pmn) and trt had significant differences at p-values 0.042, <0.001 and 0.001, respectively. the area under roc curve for wbc count, %pmn and trt was 0.653 (95%ci 0.509-0.798) at p-values 0.055, 0.799 (95%ci 0.681-0.888) at p-values <0.001 and 0.760 (95%ci 0.637-0.858) at p-value 0.001, respectively. overall, the optimal cutoff value for %pmn was 56.0 with sensitivity 84.2% (60. 4-96.4 ) and specificity 60. 9% (45.4-74.9 ). the optimal cut-off value for trt was 22 hours with sensitivity 80.0% (56.3-94.1) and specificity 63. 6% (47.8-77.6) . in 50 patients with no vesicoureteral reflux (vur), there was significant difference in trt at p-values 0.002. the area under roc curve for trt was 0.824 (95%ci 0.693-0.955) at p-value 0.004. the optimal cut-off value for trt was 25 hours with sensitivity 88.9 % (95%ci 51.7-98.2) and specificity 68.4% . in vur patients, there were no significant differences in tdt, %pmn and trt between normal and abnormal dmsa renal scintigraphy at p-value 0.750, 0.191 and 0.313, respectively. conclusion: %pmn ≥56% and trt ≥22 hours predict renal damage in the first episode of uti. however, in patients with no vur, trt ≥25 hours predict renal damage. dmsa renal scintigraphy in the first episode of uti should be considered in those patients. susceptibility data. knowledge of local antimicrobial susceptibility all children with the first episode of febrile urinary tract infection traditionally. nowadays it has been revealed that the presence of renal scar is more important than the presence of vur regarding renal and patient outcome. our aim was to assess the relationship between the severity of vur and the severity of dmsa scan changes which is performed in the first week of the first febrile uti. methods: children with the first febrile uti who were admitted in ali asghar children hospital were evaluated prospectively. all dmsa scans have been observed and graded by our nuclear medicine specialist without any knowledge of presence or absence of vur. renal damages in dmsa scan were classified to 8 grades as follows: grade 0: normal kidney, grade 1: decreased uptake in one pole with intact border, grade 2: decreased uptake in two poles with intact borders, grade 3: diffuse decreased uptake with intact border, grade 4: decreased uptake in one pole with scar, grade 5: decreased uptake in two poles with scar, grade 6: multiple scars, grade 7: diffuse decreased uptake with one pole scar, grade 8: diffuse decreased uptake with multiple scars. then dmsa findings in patients with no vur, with low grade vur and high grade or dilating vur were compared with each other. results and conclusion: one hundred and six patients with the first febrile uti were included in this study, thus 212 kidneys were evaluated for the presence or absence of vur and dmsa grading. the mean age of our patients was 4±2.9 years old. 12.3% of our patients were male. 44.8% of kidneys did not have any vur, low grade vur (grades 1,2,3) was seen in 34.4% of kidneys and high grade vur 9grades 4, 5) was found in 20.3% of kidneys. 76.2% of patients with low grade vur and/or without vur had dmsa scoring. abstract# p-sat040 fibronection in reflux nephropathy, is it a marker of grade of reflux? nahid rahimzadeh tehran university of medical science, associated professor, tehran, iran objective: vesicoureteral reflux (vur) is one of the most common urinary tract abnormalities in patients with urinary tract infection. nowadays noninvasive diagnostic methods are suggested to recognize vur and its severity. methods: we measured urinary and serum fibronectin in 51 children with vur. results: the mean serum fibronectin was 318.3±112.1 in children with low grade vur versus 356.1±189.9 in children with high grade vur (pv>0.05). the mean urinary fibronectin was also 31.5±12.9 in low grade vur and 25.9±14.2 in high grade vur (pv>0.05). thus we didn't find any association between the severity of vur and the amount of fibronectin in serum and urine of patients. we also didn't find any relationship between dmsa changes at the acute phase of uti and serum and urine fibronectin. conclusion: in contrast to some previous studies, we showed the serum and urinary fibronectin cannot preclude the severity and grade of vur and hence it is not suitable surrogate marker for imaging techniques for vur diagnosis. abstract# p-sat041 the use of serum procalcitonin level in the prediction of high grade vesicoureteral reflux in urinary tract infection nahid rahimzadeh tehran university of medical science, associated professor, tehran, iran objective: procalcitonin is a reliable and specific marker of bacterial infection such as urinary tract infection. some authors suggest measurement of serum procalcitonin as a predictor of vesicoureteral reflux. we investigated this association in children who admitted because of acute pyelonephritis. methods: forty eight children with the first febrile uti were included. twelve patients had low grade vur, nine patients had high grade vur ((≥ 3) and twenty seven patients didn't have any vur in their imaging assessment. results: there was a significant association between high grade vur and higher levels of procalcitonin (pv=0.04). the sensitivity of procalcitonin level ≥ 0.31 ng/ml was 90% and specificity was 32% for diagnosis of high grade vur. conclusion: we concluded that serum procalcitonin concentration is a sensitive and promising predictor of high grade vesicoureteral reflux. hydronephrosis, parenchymal scarring and to study the rate of resolution of vur on follow up. methods: this was a retrospective study conducted by reviewing case records of all infants and children with primary vur who had minimum follow up of 3 years, at our nephro-urology clinic over the last 10years.the imaging evaluation (renal ultrasound, voiding cystourethrogram, dmsa scan) was done based on the indian society of pediatric nephrology guidelines. severity of vur was classified as mild (grade i, ii), moderate (grade ii, iii) and severe (grade v) results: of the 218 children screened for primary vur, 107 with complete data were included for analysis. the mean age was 25.68 ±22.45 months with male to female ratio of 1.4:1. the age at presentation was significantly lower for moderate to severe vur as compared to mild vur (p=0.033). thirty children (28%) had abnormal antenatal scans. majorities (80%) of children were diagnosed to have vur during evaluation for urinary tract infection.among107 patients, six had solitary kidneys and thus the total number of systems evaluated was 208. of these, 164 systems had vur (24% had mild, 66% had moderate and 10% had severe vur). hydronephrosis was seen in 56% of patients with vur. of these, 45% were unilateral and 55% were bilateral.there was no significant difference in the presence of hydronephrosis and grade of vur. there was no significant difference in presence of scars between mild and mod-severe vur. at the end of 3 years of follow up, complete resolution of reflux was seen in 53%, 43% and 55% of children with mild, moderate and severe vur respectively. conclusion: severity of vur did not necessarily correlate with hydronephrosis and parenchymal scarring. over a 3 year follow up, complete resolution of reflux was seen in nearly 50% of children irrespective of the grade of reflux. abstract# p-sat044 growth in children with dilating vur -a follow up of the swedish reflux trial per brandstrom, sverker hansson pediatricuronephrologic center, university of gothenburg, gothenburg, sweden objective: the swedish reflux trial included 203 children, 1-2 years of age, with vur grade 3-4, diagnosed after uti in 194 and prenatal urinary tract dilatation in 9. dmsa was abnormal at start in 124 children (61%). the children were randomized to antibiotic prophylaxis, endoscopic injection with deflux™ or surveillance. there have been reports on growth retardation in children with vur and catch up after vur resolution. the mechanism behind these findings is unclear. the children of the swedish reflux trial constitute a high risk group with dilating vur, recurrent febrile uti and high prevalence of renal parenchymal defects. we have searched the growth pattern of these children for differences related to gender, treatment group, uti recurrence, vur grade at follow up or renal defects on dmsa. methods: height and weight z-scores, compared to standardized swedish growth charts, were registered during the 2 year follow up in 199 of the children in the trial and at outpatient visits thereafter in 129 patients to the age of 5.3 years (median, range 2.0-11.8). change in height z-score between first and last visit was used to measure growth over time. results: the first and last recorded height and weight z-scores were all within normal range. there was a larger gain of height in children with renal defects compared to those without (z-score difference 0.42 vs. 0.13, p=0.009). there were no differences in growth related to gender, treatment group, vur-grade at follow up or recurrent uti. conclusion: the children of the swedish reflux trial constitute a high risk group with dilating vur, recurrent febrile uti and high prevalence of renal parenchymal defects. they have normal weight, height and height gain at follow up for up to 10 years. there was no sign of growth inhibition in these children related to dilating vur. the larger height gain seen in those with renal defects seem to be due to their slightly shorter stature at study start, although within normal range, which could be related to more severe urinary tract and renal problems during their first 1-2 years of life. engin kose, caner alparslan, serdar saritas, cengizhan elmas, fatma mutlubas ozsan, onder yavascan, nejat aksu tepecik training and research hospital, pediatric nephrology, izmir, turkey objective: the management of vesicoureteral reflux (vur) is varied and remains controversial. conservative therapy is based on the understanding that vur can resolve spontaneously, mostly in young patients with low-grade reflux. in this study, we wanted to evaluate the spontaneousresolution rate of low-grade vur (grades i and ii) in children. methods: children with low-grade (i-ii) vur treated in our hospital from may 2010 to may 2012 were prospectively studied. patients with low-grade (i-ii) vur and those who showed normal dmsa findings were included into the study. initially, a dmsa scintigraphy was performed in all patients and those who experienced acute febrile urinary tract infection (uti) during the follow-up period. treatment success was defined as complete vur resolution. no patients were prescribed antibiotic prophylaxis. all parents were informed by being given an explanation of the clinical significance of personal hygiene methods used after urinating or defecating. our institutional review board approved to collect the data, retrospectively. statistical analysis was made by using ibm spss 20.0 software. results: the study sample comprised 21 infants (10 boys and 11 girls) all of whom showed low-grade reflux (grades i-ii). bilateral reflux was seen in 7 (33.3 %) of cases. median age at diagnosis was 8 months (range: 1-37 months). median follow-up time was 14 months (range: 9-28 months). the spontaneousresolution rate of reflux was 89.3 % (25 out of 28 renal units). the frequency of febrile uti was 0.74±0.7 episode/year (median: 0.8 episode/year). during the follow-up no patients with febrile uti experienced scar on dmsa scintigraphy. conclusion: infants with low-grade reflux show a low risk of febrile uti and a high spontaneous resolution rate without antibiotic prophylaxis. therefore, these children should be managed primarily by conservative therapy. ji-nan sheu 1,2 , hai-lun sun 1, 2 , shan-ming chen 1, 2 , yu-hua chao 1, 2 , min-sho ku 1, 2 , pen-fen liao 1 , ko-huang lue 1, 2 1 pediatrics, chung shan medical unversity hospital, taichung, taiwan 2 school of medicine, chung shan medical university taichung, taiwan objective: to assess the usefulness of procalcitonin (pct) as a marker for predicting dilating (grades iii-v) vesicoureteral reflux (vur) in young children with a first febrile urinary tract infection (uti). methods: children aged≤ 2 years old with a first febrile uti were prospectively evaluated. serum samples were tested for pct measurements upon admission to a tertiary hospital. all children underwent renal ultrasonography (us), 99m tc-dimercaptosuccinic acid renal scan, and voiding cystourethrography. the diagnostic characteristics of pct test for acute pyelonephritis (apn) and dilating vur were calculated. results: of 272 children analyzed (168 boys and 104 girls; median age, 5 months), 169 (62.1%) had apn. there was vur in 97 (35.7%), including 70 (25.7%) with dilating vur. the median pct value was significantly higher in children with vur than in those without (p< 0.001). using a pct cutoff value of ≥1.0ng/ml, the sensitivity and negative predictive value for predicting dilating vur were 94.3% and 95.4%, respectively, for pct, and 97.1% and 97.8%, respectively, for the combined pct and us studies, whereas the positive and negative likelihood ratios were 2.03 and 0.107, respectively, for pct, and 1.72 and 0.067, respectively, for the combined studies. by multivariate analysis, high pct values and abnormalities on us were independent predictors of dilating vur. conclusion: pct is useful for diagnosing apn and predicting dilating vur in young children with a first febrile uti. a voiding cystourethrography is indicated only in children with high pct values (≥1.0 ng/ml) and/or abnormalities found on a us. objective: to evaluate the accuracy of acute 99m tc-dimercaptosuccinic acid (dmsa) scan in predicting dilating vesicoureteral reflux (vur) among young children with febrile urinary tract infection (uti). methods: the medical records of children (age≤2 years), presenting with febrile uti between january 2000 and december 2011, were retrospectively reviewed. the sensitivity, specificity, positive and negative predictive value, positive and negative likelihood ratio of acute dmsa scan in predicting dilating vur in young children with febrile uti were calculated. results: a total of 523 children were included, of which 397(75.9%) had abnormal dmsa results and 178(34.0%) were identified as vur on micturatingcystourethrography (mcu). among all the patients, the number of dilating vur was 151(28.9%). the rate of abnormal results on dmsa of dilating vur group was significantly higher than the rates of non-vur and low-grade vur groups (p<0.01). in age<6 months group and age≥6 months group, the sensitivities of dmsa in predicting dilating vur were 96.15%, 100.0% respectively, while the negative predictive value were 97.26%, 100.0% and negative likelihood ratio were 0.0911,0.0000, respectively. conclusion: for children of age≤2 years with febrile uti, acute dmsa scan possesses certain values in excluding dilating vur. the possibility to detect dilating vur by mcu is rather low when the result of dmsa is negative. cakut: voiding disorders abstract# p-sat048 mono-symptomatic and non-mono symptomatic nocturnal enuresis: a clinical evaluation mitra naseri 1 , mehran hiradfar 2 1 dr sheikh children hospital/pediatric nephrology department, mashhad university of medical sciences, mashhad, iran 2 dr sheikh children hospital/pediatric surgery department, mashhad university of medical sciences, mashhad, iran objective: nocturnal enuresis is divided into mono-symptomatic nocturnal enuresis(mne) and non mono-symptomatic nocturnal enuresis(nmne).this study was conducted to review clinical and ultrasonography findings in enuretic children, and compare organic and functional pathologies of lower urinary tract (lut) in children with mono-mne with those who have nmne. methods: 111 neurologically normal children with chief complaint of enuresis enrolled in the study including 60 boys and 51 girls, aged 5-17 years old, 43 (38.8%) with mne and 68 (61.2%) with nmne. urine analysis, urine culture and kidney bladder ultra sonography was done for all .some patients underwent voiding cystoureterography (vcug), urodynamic study (uds), or both. results: patients were divided in to 3 groups: mne, nmne daytime incontinence and nmne +daytime incontinence. constipation, encopresis and urge incontinence were significantly more frequent in patients with nmne +daytime incontinence (p= 0.011, 0.003, 0.001 respectively) . bladder wall thickness was the most common us findings..one patient with mne and 9 with nmne+ daytime incontinence had vesico -ureteral reflux(vur) )(p=0.016).posterior urethral valve was reported in one patient with nmne. evidences of bladder dysfunction were noted in about half of the patients who underwent uds, with higher prevalence in cases with nmne + daytime urinary incontinence(p=0.297). bowel symptoms and vur were significantly more prevalent in cases with nmne +daytime incontinence. conclusion: we recommend doing vcug in enuretic children who have daytime incontinence.in addition our study revealed that symptoms suggestive of over active bladder are not good indicators for bladder dysfunction. abstract# p-sat049 correspondence between urinary calcium, ca 2+ concentration and osmolality in enuretic children agata korzeniecka-kozerska, tadeusz porowski department of paediatrics and nephrology, medical university of bialystok, bialystok, poland objective: among many factors predisposing to enuresis hypercalciuria may play an important role. hypercalciuria is be observed in patients both in patients with nocturnal polyuria and without. hence, we decided to assess urine concentration of calcium (mmol/l) and ca 2+ (mmol/l) in patients with monosymptomatic enuresis and answer the question if patients with enuresis present calcium balance disturbances methods: the study was conducted on 204 children (83 enuretic aged median 9.66 (4.16-16.98 ) yrs diagnosed with monosymptomatic enuresis after 6 months of unsuccessful non farmacological treatment and 121 healthy children aged median 9.99 (4.15-16.86 ) yrs. we collected 24-h urine samples from all children enrolled to the study. calcium concentration, ca 2+ , ph, osmolality of urine and additionally daily sodium excretion were estimated and compared between two groups. statistical analysis were performed using statistica ver. 10.0 (statsoft,tulsa, ok). the mann-whitney u test was used for comparisons between two independent parameters. correlations were made with spearman test. a p value of <0.05 was considered to be statistically significant. results: there were no differences in age, gender and parameters of physical development between both studied groups and between girls and boys among groups. urinary calcium concentration in enuretic children did not differ compared to reference group ( p=0.993). we found statistically significant differences in urinary ca 2+ concentration (p= 0.001) and osmolality (p=0.02) between both studied group. ca 2+ in urine correlated negatively with age and parameters of physical physical development only in enuretic patients. additionally, positive correlation was found between ca 2+ and calcium concentration (r = 0.7923; p<0.005) and between ca 2+ and osmolality (r=0.2795; p<0.05) in urine of enuretic children. positive correlation was also observed between ca 2+ and daily natrium excretion (r=0.318; p<0.05) in enuretic children. conclusion: disturbed calcium balance may play an important role in pathogenesis of monosymtomatic enuresis. it's a need to assess calcium and natrium excretion in enuretic children. abstract# p-sat050 usefulness of the application of questionnaires to detect attention deficit hyperactivity disorder (adhd) and other psychiatric disorders in children with functional voiding disorders sandra gautreaux pediatric nephrology, complejo asistencial universitario de leon, spain objective: although the relationship between functional voiding disorders (fvd) and the presence of a psychological problem remains controversial, the greater frequency of adhd among children with this condition is well known. the purpose of this study was to determine the diagnostic performance of the application of questionnaires to detect adhd and other psychiatric disorders in children with functional voiding disorders in general pediatrics consultations methods: the study was conducted on 32 children between 6 and 13 years of age (20 males) diagnosed with fvd (patient group) and 32 children of the same age (21 males) who had no urinary symptoms (control group). the parents of these children responded to the questions in two standardized questionnaires: the strengths and difficulties questionnaire (sdq) to screen for mental health disorders and the questionnaire for the detection of adhd of the dsm-iv psychiatric disorders of american academy of psychiatry manual. the variables obtained from the questionnaire responses were compared between the two groups of children using the student's t-test for unpaired samples when variables were quantitative and the chi-square test if the variables were qualitative. it was considered significant when p<0.05 results: no significant differences were found between the two groups in the sdq questionnaire in any of its sections (emotional symptoms, conduct problems, hyperactivity, peer problems and prosocial behavior) or global assessment test, in which only 2 patients of each group had an abnormal result. the questionnaire for the detection of adhd, presented an altered overall result of 17.18% and 14.06% patients in the control group (p=ns). there were also no differences between the two groups on the results of this test concerning the inattention or hyperactivity-impulsivity sections. conclusion: the results of the application of questionnaires to detect adhd and other psychiatric disorders in children with fvd are similar to those of the general population. the routine application of this type of questionnaires to all the patients in pediatrics consultations does not seem necessary. abstract# p-sat051 characterization of voiding dysfunction in chidren with attention deficit-hyperactivity disorder jun yonug kim, kun hee lee, jung won lee department of pediatrics, hallym university kangnam sacred heart hospital, seoul, korea objective: attention deficit hyperacitivity disorder (adhd) has been associated with impairment of frontal inhibitory function and catecholaminergic system. adhd is diagnosed in 3~5 % of children. children with adhd seem to suffer from various forms of urinary problems such as nocturnal enuresis, dysfunctional voiding and diurnal incontinence. however, no data exist to confirm in korean adhd children. we investigate the clinical findings of voiding dysfunction in adhd children. methods: between october 2009 and march 2011, a total of 63 children (33 children with adhd and the other 30 children with upper respiratory infection as control group) were enrolled in gangnam sacred heart hospital, hallymuniversity. adhd children were diagnosed under diagnostic and statistical manual of mental disorders (dsm)-iv criteria. a comprehensive survey of voiding and defecating were administered. results: the patient group included 28 boys and 5 girls, and the control group 15 boys and 15 girls. mean age were 9.09+/−2.8 year in adhd group and 8.58+/−3.1 in control group. children with adhd had statistically significant higher incidence of enuresis (p=0.017), urgency (p=0.017), urge incontinence (p=0.033) and constipation (p=0.007). there was no significant differences in straining, intermitteny, holding maneuvers (p>0.05). conclusion: children with adhd in korea have significantly higher rates of enuresis, urgency, urge incontinence and constipation than those without adhd. the psychological correlates of primary nocturnal enuresis weiran zhou, xiaomei liu, ying shen nephrology, beijing children's hospital, beijing, china objective: previous studies based on clinical samples have reported that enuresis in children is associated with behavioural problems and reduced self-esteem, anxiety, but the relationship remains controversial. this study investigated psychological correlates of enuresis in a group of children suffering primary nocturnal enuresis(pne). methods: this survey involved 98 parents and their children with pne aged 7-14 years. clinical datas of enuresis were collected through parents' reports and individual administrations to all children. parents completed the child behaviour checklist. children completed piers harris children'sself concept scale, the screen for child anxiety-related emotional disorders (scared)and depression self rating scale for children (dsrsc) . results: of the 98 children, 60 are boys and 38 are girls. 65 are monosymptomatic primary nocturnal enuresis and, 33 are nonmonosymptomatic. 72 children accord with the severest form of bedwetting(>=7 times/week). 64.3%(n=63)children have behaviour problems and, girls get higher scores in withdraw and internalizing problems than boys. boys are more aggressive. 42.9%(n=42) get positive results through scared. 9.2%(n=9) get positive results in dsrsc. children with the severest form of bedwetting are likely to have more complex form of psychological problems. they have greater social problems, get higher scores in behaviour problem (p=0.026) and lower scores in piers harris children's self concept scale (p=0.034). but children with or without the severest form of bedwetting have no significant difference in scared . mne have no significant difference with nmne in scores of all the three children's self-evaluated scales. conclusion: most children with pne had different psychological problems. the severer the symptoms are, the more complex the psychological correlates are. these preliminary findings support the view of enuresis closely related with psychological problems. psychological problems are affected by many factors except for enuresis, so further researches need to be conducted to determine whether there is a causal relationship between psychopathology and enuresis. the current situation of treatment of primary nocturnal enuresis in children xiaomei liu, lu chen, weiran zhou nephrology, beijing children's hospital, beijing, china objective: we would like to discuss the treatment of primary nocturnal enuresis in children. methods: children, diagnosed with primary nocturnal enuresis, paid outpatient visits to beijing children's hospital from january 2011 to may 2012, are enrolled in the study. the information of previous clinical experience, compliance and expected treatment goals are collected and analyzed. results: 623 children, with the mean age of 8.69±2.33 are enrolled. 449 children have visited doctors before, 295 have unsatisfied clinical experience, such as late intervention, less standardized treatment and over-treatment. 64.36% have poor compliance with doctor's suggestion of behaviour therapy and alarm therapy. 262 children have been suggested to take medicines such as chinese traditional medicine ddavp, but 17.8% of them refused the suggestion anxious of the side effects or stop the usage on their own. the primary expected treatment goal of parents is to improve the symptoms, accounting for 86.04%. excluding the underlying diseases, alleviating parents' burden, treating the symptoms of hyperactivity or inattention and avoiding adverse effects on intellectual and fertility are also included. for children, they'd like to improve the symptoms so they won't be published by parents or laughed by their fellows. most parents (79.94%) preferred medicines as the first choice. 12.40% parents and children are inconsistent with the behavior therapy (the rate of newly diagnosed and non newly diagnosed children were 8.04%, 14.03% respectly. χ 2 test p>0.05). 38 (6.10%) don't accept alarm therapy (the rate of newly diagnosed and non newly diagnosed children were 2.30%, 8.02% respectly. χ 2 test p>0.05). conclusion: children with primary enuresis need early intervention and standardized treatment. the effect of treatment largely depends on cooperation and joint participation of children and parents. this study found that the compliance of behaviour training and alarm therapy is lower than medicine. the goals of children and parents, living conditions and other factors should be fully considered in the treatment of enuresis to improve short-term remission rates and long-term cure rate of the disease. beware of the sleeping bladder in monosymptomatic nocturnal enuresis (mne) britt borg, konstantinos kamperis, birgitte mahler, soren rittig pediatrics, aarhus university hospital, skejbv, denmark objective: bladder reservoir function in children with nocturnal enuresis is assessed by maximal voided volumes (mvv) registered on frequency-volume charts. although a degree of association is evident, mvv does not always reflect the nocturnal bladder reservoir function in mne. we aimed to evaluate the nocturnal bladder reservoir function during the night in children with apparently normal mvv. methods: data from 557 children aged 5-15 treated for mne in a tertiary referral centre was analyzed. data from 135 children was excluded due to reduced mvv according to iccs standardization and 156 due to lack of home recordings. the remaining were divided into two groups, based on whether mvv was above (n=91) or below (n=175) the average nocturnal urine production during wet nights (nupw) . first morning voids were not included in mvv values. results: 34 % of the children with mne and a normal bladder capacity had an average nocturnal urine production during wet nights below their mvv, one third of these had nupw below 65 % of mvv expected for age. these children did not differ significantly in terms of demographic characteristics, frequency of wet nights or treatment time needed to achieve dryness. urine output during dry nights was not statistical significant between the groups (p=0.077, mean diff.=28.47 ml). the group with mvv>nupwet shared significantly higher mvv to mvv expected for age ratios (mean diff=0.16, p<0.05). urine output during wet nights was lower in the group of children with mvv>nupw (diff: 96.45 ml, p<0.05) . conclusion: children with mne and apparently normal bladder reservoir function during daytime may experience wet nights with urine volumes well below their mvv and mvv expected for age. the fact indicates bladder reservoir function abnormalities during sleep that is not assessed by day recordings. physicians treating children with mne should consider anticholinergic treatment. one year experience of a multidisciplinary investigation on nocturnal enuresis in a brazilian tertiary care facility objective: to characterize a cohort of children, 6-16 yrs old, with nocturnal enuresis, defined by 2010 iccs criteria on the basis of a multidisciplinary evaluation including renal, neurological, psychological and physical therapy approaches. chronic clinical conditions and genetic disorders constituted exclusion criteria. methods: after irb approval, families were invited to participate in the project through press releases, 130 children completed quality of life evaluation through clinically validated questionnaires, followed by a one -day multidisciplinary clinical evaluation. sleep, urinary and intestinal diaries were evaluated. urinary sonography, nocturnal polysomnography, urinary and blood analysis were scheduled. results: 96/130 participants (pts)were male (73,8%), mean age 8 ±1 yrs. twenty two children were excluded due to noncompliance /chronic clinical conditions. 118/130 pts were evaluated. 93/130 parents of children/adolescents, answered the cbcl questionnaire, 25/93 (27%) of which resulted in scores compatible with clinical psychological conditions. monosymptomatic enuresis (moe) was diagnosed in 89/118 children (75.4%). in the moe group, 67/89 pts were diagnosed with intestinal constipation; 7/89 pts with obstructive sleep apnea, 2/89 pts with hipercalciuria and 2/89 were characterized with adhd. area and velocity of center or pressure displacement (vm) were used for postural control evaluation and resulted in 3,67 ±2,95 cm 2 and 20,35±13,04 cm.s-1, respectively; 22/89 pts were also assessed for postural alignment exhibiting pelvic anteversion (10,27 ±3,71 grades) and head protusion (0,40±1,86 grades). these results corroborate with a smaller range of motion found for hip flexion (94,86±17,14 grades) and hip extension (2±3,89 grades) assessed using a goniometer. conclusion: multidisciplinary evaluation of enuretic children may be the key to optimize therapy on the basis of the underlying etiology of the process abstract# p-sat056 weight depending dosing of desmopressin (ddavp) in nocturnal enuresis pauline de bruyne 1 , ann de guchtenaere 1 , charlotte van herzeele 1 , ann raes 1 , jo dehoorne 1 piet hoebeke 2 , erik van laecke 2 , johan vandewalle 1 1 paediatric nephrology, ghent university hospital, ghent, belgium 2 department of urology, ghent university hospital, ghent, belgium objective: in children as well as in adults, a uniform starting dose of desmopressin is prescribed. this uniformity is based on the inability to detect a weight-dependent dose-concentration correlation as this correlation is probably blurred by the wide intra-and interindividual differences in plasma concentration for a fixed desmopressin dose. recently, a smaller variation in plasma concentration was shown for the oral lyophilisate formulation of desmopressin (compared to tablet formulation). therefore, this study assessed a possible correlation between weight-corrected dose and plasma concentration for both formulations. methods: 23 children with monosymptomatic nocturnal enuresis were recruited in a tertiary centre. two tests were performed on two separate days (at two weeks interval) in identical, standardized conditions: on day 1 desmopressin tablet 200μg and on day 2 desmopressin oral lyophilisate 120μg was administered. plasma concentrations were measured at one, two and six hours post dosing. the nonparametric spearman's rank correlation coefficient was used for assessing the correlation between weight corrected dose and plasma concentration. results: mean (sd) age and body weight of the patients were respectively 12.7 (2.9) years and 50.1 (15. 2) kg. a positive correlation between plasma concentration of ddavp was found for the oral lyophilisate formulation at 2 hours (p-value 0,021) and 6 hours (pvalue 0,005) post dosing. this is not the case for the tablet formulation. results are shown graphically in figure 1 and 2. figure 1 and 2: correlation of dose corrected by weight to plasma concentrations at 2 and 6 hours post dosing conclusion: to the best of our knowledge, this is the first pharmacokinetic study showing a significant dose (normalized for size) -concentration correlation for desmopressin. nevertheless, this correlation was only significant for the oral lyophilisate group. this result is clinically important as it is a strong indication for more predictable plasma concentrations for the oral lyophilisate formulation, and thus preventing elevated concentrations of desmopressin. objective: increased nocturnal urine production and/or bladder hyperactivity in primary nocturnal enuretic (ne) patients could possibly be associated with autonomic nervous system dysfunction. reports of studies on autonomic nervous system dysfunction in ne are limited. to investigate autonomic nervous system function in enuretic children by performing ambulatory blood pressure monitorisation (abpm) for 24 hours. methods: children with primary ne were enrolled in this study and they get 24 hour abpm. their results were compared with healthy children. urinalysis,urine electrolyte levels, urinary culture and urinary system ultrasound were carried out in all the children. they also requested to have a diary about daily fluid intake and volume of daily urine. results: the enuretic group consisted of 28 children (m/f:19/9) and the control group of 27 healthy children (m/f:14/13). the mean ages were 7.9±2.2 years and 8.77±2.65 years, respectively. the mean 24-hour bp and daytime dbp did not differ between the groups however, the mean systolic bp was significantly higher in enuretic children (p<0.05). the mean nighttime systolic bp, dbp and map were significantly higher as well in the patient group. daytime bp load did not found to be elevated in both groups.elevated night time systolic bp load was observed in 4 patients and was not observed in control group (14.2 % vs 0%) (p<0.01). nighttime diastolic bp load was elevated in 7 of 28 patients, compared with 1 of 27 control subjects (p<0.01). the nocturnal bp dip was significantly reduced in the patient group compared with control group for diastolic blood pressure ( 67.8 % vs 14.7 %, respectively; p=0.037). patients with elevated night time bp load was found to have higher frequency of urinary incontinence per week as well as per night while compared with enuretic children with normal night time bp load (r=0.72, p<0.01; r=0.69, p<0.01 respectively). conclusion: nocturnal bp loads were significantly higher in children with enuresis. these subtle abnormalities of circadian blood pressure regulation, loss of nocturnal dip and observation of decreased nocturnal pulse rate may reflect autonomic nervous system dysfunction and pathogenesis of en. objective: bladder dysfunction and especially oab plays a major role in nocturnal enuresis, not only in the non monosymptomatic (nmne) but as well in the monosymptomatic patients (mne) . if the enuresis is related to a mismatch in nocturnal diuresis and maximal functional bladder capacity, then the bladder volume should be a major parameter, but is sofar not taken as a parameter for subtyping into nmne in the iccs standardization. maximal voided volume in a diary is the golden standard, although relation with outcome is poorly studied. methods: aim of the study was to evaluate the optimal parameter for estimation of bladder volume as maximal voided volume in a diary, during forced diuresis, bladder volume during 3 uroflow + uroflow), correlating with cystomanometry (if indicated). studypopulation 398 patients age 5 to 18 years, >6/7days wet, only 48 cystomanometries. results: if we compare the data from the bladder volume against the reference frame from rittig (aarhus), then 60,4% of patients had a mvv in diary < 2,5% percentile demonstrating that a majority of patients had a small for age voided volume in their diary. correlation between mvvdiary, mvv forced diuresis, bladder capacity (uroflow+ residu) and cystomanometry show that there is a strong correlation between the 4, but especially for the last 3 parameters (r 2 0.48-0.62, p <0.01),but results with mvv diary are worse (r 2 0.34-0.48, p 0.04-0.018) . there is no sex or gender difference in this observation. correlation with response to therapy at 1 year shows a superior correlation with mvv forced diuresis and during uroflow than mvv diary (p0.04). since cystomanometry is only performed in refractory cases, this voided value had no correlation with clinical outcome. conclusion: bladder volume can be estimated in several ways, each with their advantages and pitfalls. our data demonstrate that the alternative non invasive methods during force diuresis and in center during 3 uroflow correlate best to each other and to the cystomanometricvalues. but as well to the one year outcome (p 0.04). abstract# p-sat060 evaluating nocturnal diuresis (polyuria) claire debusschere, delphine guenter, sophie wouters, johan vandewalle, ann raes, joke dehoorne pediatric, nephrology, gent, belgium objective: nocturnal enuresis is more than bedwetting, but a symptom of a disorder involving multiple pathogenetic factors in circadian rhythm of diuresis/solute excretion and bladder dysfunction. where the sum of diaper weight and morning voided volume, is the standard to evaluate nocturnal diuresis (polyuria), it does not give indices about pathophysiology. the aarhus concept : in center studies during standardised intake, offers the advantage of standardized conditions, and reliability of the values, but is restricted to specific research centres in limited patients for budgetary reasons). the ghent concept : a home based 24 hours concentration prophyle with 4 time daytime and 4 nighttime collections offers an alternative. this one day test was not validated against 14 days diuresis nighttime registration, and was criticized since waking up the patient overnight might increase diuresis and solute excretion, as is demonstrated in sleep deprivation aim of the study: to validate nocturnal diuresis and solution excretion to evaluate nocturnal polyuria against 14 days nighttime diary and 2 days daytime diary. methods: to the study the incidence of nocturnal polyuria, and to evaluate the value of the morning osmolality in the study of nocturnal polyuria. results and conclusion: 401 children (262m), mean age 8 y (5-18y) , 24 h diuresis 1023+/−445 ml, daytime 663+/−367 ml nighttime 365+/−203ml, no sex difference. correlation between volumes in diary and 24 h concentration prophyle during 24 h (p 0.036), night (r 2 0.336 p 0.013) and day (p 0.012). only 13% of patients have a nocturnal polyuria (&gt;130%ebv= absolute nocturnal polyuria), but up to 28% have a nocturnal diuresis higher than 100% ebv and &gt; than their mvv (relative polyuria). there is no correlation between early morning osmolality and nocturnal diuresis-rate (nocturnal polyuria). objectives, methods and results: we present a 16 months old albanian female with fever, urinary retention and constipation. this problems has occurred one week before hospitalization. no history of trauma. life history has no remarkable data. no history of uti or constipation before. physical examination: weight and stature on 50 centile. no respiratory problems. the big overfilled bladder was palpated. on the left sacral region, was a tumorous mass very painful and worm. perianal reflex, bulbocavernosus were weak. other reflexes were normal. there were a hard stools in the rectum. other systems examination was unremarkable. high es rate and pathological urine sed with infection indicators. other laboratory examination was unremarkable. ultrasound examination resulted with dilatation of the left kidney pyelon (9mm), distended and overfilled bladder lumen (>400ml) with thin bladder wall. rectum distended and full with fecal masses.lateroposterior there is a sonolucent cystic formation with (volume 7ml). that cystic formation was supected for ureterocellae first. the urinary catheter and rectal clismas were done. that formation was abscess and it was drained. and two weeks after was better. voiding cystourethrogram and computer tomography was done and resulted that are no anatomical defects in the urinary tract. because of the collapsed bowels there's not good visualization of the region around the sigma and rectum. irigografy resulted: dolihosigma and suspected for hirschprung. two months later the abscess relapsed and needed drainage again. conclusion: since the abscess has repeated the next step will be biopsy. hirschprung, tailgut cysts and other presacral masses should be included in the differential for patients with recurrent abscess in the perisacral space with clinical manifestation of urinary retention and constipation. abstract# p-sat062 treatment failure to enuresis alarms: challenges and factors influencing adherence to treatment indra ganesan 1 , jessica xj hooi 2 , jasmine jy goh 2 , yh ng 1 , sm chao 1 1 nephrology service, kk women's and children's hospital, singapore, singapore 2 department of pediatrics, kk women's and children's hospital, singapore, singapore objective: the study aims to identify factors influencing treatment failure to enuresis alarm and the factors influencing adherence to treatment. methods: all patients aged 6 to 16 years referred to the voiding clinic in kk women's and children's hospital, singapore with primary nocturnal enuresis who opted for enuresis alarm treatment, over 6 year period from 2007-2013, were prospectively studied. data, via direct interview and parental questionnaire was collected on demographics, age at presentation, presence of family history, frequency of nocturnal enuresis per week and per night, on whether child awakens after wetting, or associated constipation in the child and parental perception of why primary enuresis happens. results: seventy-nine of 111 (71%) children with primary nocturnal enuresis with complete data were included. mean age at presentation was 9.6±2.1 years and 45 (57%) were male. twenty-five children had a family history of nocturnal enuresis in a 1 st degree relative. eighteen children had constipation. fifty-one (64 %) children achieved the targeted 21 consecutive dry nights. in the remaining 28 children, the main reasons for treatment failure were non adherence to usage of enuresis alarms mainly due to an inability to wake up to the alarm in deep sleep (71%) and underlying neurobehavioural conditions (21%) (adhd, autism, depression and a chromosomal disorder). the only significant clinical factor predicting treatment failure was the presence of an underlying neurobehavioural condition (or 2.8, and developmental delay . factors such as male gender, family history in first degree relatives, frequency of enuresis, arousal when wet, and the presence of constipation, were not significant. conclusion: in our population, 64% of children achieved nocturnal urinary continence with enuresis alarms. treatment failure of enuresis alarms is higher in the presence of an underlying neurobehavioural condition and developmental delay. non adherence to treatment is mainly due to inability to arouse from deep sleep. long-term follow-up of children with nocturnal enuresis -do enuretics become nocturics? an-sofie goessaert 1 , bente schoenaers 2 , olivier opdenakker 2 , karel everaert 1 , johan vandewalle 3 1 urology, ghent university hospital, ghent, belgium 2 ghent university, ghent, belgium 3 pediatric nephrology, ghent university hospital, ghent, belgium objective: although an overlap between both nocturnal enuresis (ne) and nocturia is known, research on the occurrence of both conditions in one patient is lacking. this study aims to investigate the prevalence of nocturia and other urinary symptoms in patients who have suffered from ne. methods: a questionnaire was sent to 1265 patients treated more than 3 years ago in the university hospital of ghent for ne evaluating the history and current status of enuresis and validated questionnaires on urinary incontinence (iciq-ui) and overactive bladder symptoms (iciq-oab). medical files of all subjects were analysed on the history of enuresis. all subjects were asked to sign an informed consent. results: of the 516 (41%) subjects who completed the questionnaire, 183 reported nocturia (36%), with a sex ratio (m/f) of 101/81 in the nocturic group versus 230/103 in the non-nocturic group, mean age is 18 versus 17 years old, respectively. comparing the nocturic and nonnocturic group, retrospective analysis of the history of enuresis shows an older age at which the subjects were cured (10 vs 9.5, respectively) and a higher percentage of non-monosymptomatic ne (74% vs 63%, respectively) in the nocturic group. no differences in past treatment for ne were found between both groups. prospective analysis shows a significantly higher prevalence (p<0.001) of voiding frequency during daytime, urge and urinary incontinence in nocturics compared to nonnocturics. with an increase in number of nocturic episodes per night, the percentage of female subjects increases (p<0.001) and the percentage of subjects with non-monosymptomatic ne increases as well (p<0.011). conclusion: over one out of 3 former enuretic patients develops nocturia, often accompanied by other urinary symptoms and with significant bother. cure of nocturnal enuresis does not necessarily equals cure of the urological pathology and some of the nocturic patients, who remain to suffer from bothersome symptoms, might benefit from continuous treatment for the underlying urological condition, such as overactive bladder syndrome or nocturnal polyuria. objectives, methods and results: a 3 year old girl was admitted to our hospital due to unusual pattern of voiding. when she wanted to urinate, she was writhing in pain; she cried, became pale and sweated. since the newborn period parents have observed sudden episodes when the girl started to cry and curled up her legs. she stopped when she began to urinate. the girl's father occasionally had pains in the jaw between yawning. the girl's voiding was like parents described: when she was trying to begin urinating, she was squatting on the toilet seat, crying and became pale and sweated. when voiding occurred the girl seemed to relax. the voiding volume was small and the urinary stream was weak. all investigations results were normal. according to the clinical course we assumed that the girl had an unusual expression of paroxysmal extreme pain disorder (pepd). the diagnosis was confirmed by the detection of the heterozygous pathogenic mutation in the scn9a gene, a missense mutation in exon 5. the same mutation was also confirmed in her father. the girl was started on carbamazepine and the pain attacks almost disappeared. pepd is a rare autosomal dominant neuropathy linked to mutation in the scn9a gene which encodes voltage-gated sodium channels. abnormal pain sensitivity occurs due to changes in the properties of the channels. pepd's onset is in the neonatal period with the most characteristic clinical features being attacks of excruciating pain in the rectum, genitalia, face and limbs. in literature we did not find any citation of patients with attacks triggered by the voiding stimulus. carbamazepine has been effective in relieving symptoms but the response is often incomplete (1) (2) (3) , as in our patient. conclusion: the case described is interesting because of the rare clinical expression of pepd with pain triggered by voiding. the same mutation was recently found in patients with sfn (4) . the study of functional importance of this mutation showed that the result of the mutation is increased frequency of firing pain-signaling neurons (5) . rita pavione rodrigues pereira 1 , vera herminakalika koch 2 , simone nascimento fagundes 2 , aline rossi 1 , juliane de oliveira marques vieira 1 , clarice tanaka 1 1 physical therapy, university of são paulo, são paulo, brazil 2 communication disorders and occupational therapy, university of são paulo, são paulo, brazil objective: to assess the postural control in children and teenagers with enuresis compared to a control group methods: 22 enuretic (eg) patients (11 boys and 11 girls) with mean± sd of 10.52±2.77 years, ranging from 7 to 16 years old, and 19.39±3.50 of bmi were paired with assyntomatic kids (cg) of equal age, gender and bmi. three trials of 20 seconds were collected while standing on a forceplate at 60 hz of frequency with standard position of feet in four different sensorial conditions: (1) open eyes and stable surface; (2) closed eyes and stable surface; (3) open eyes and unstable surface, and; (4) closed eyes and unstable surface. the analized variables were area (a) and velocity (vm) of center of pressure displacement (cop). student t-tests were used to compare variables between groups for each sensorial condition. the level of significance was 95% for all the analysis. results: when compared to cg, the eg revealed larger area of cop displacement in the condition (1) (mean± sd of 2,46±2,11 cm 2 for cg and 4,16±2,76 cm 2 for eg; p=0,027), and in condition (4) (mean± sd of 4,29±2,45 cm 2 for cg and 7,33±6,46 cm 2 for eg; p=0,05). no difference in vm were identified. conclusion: when compared to assymptomatic control group, the enuretics children and teenagers presented larger area of cop displacement under normal sensorial input (condition 1) and when vestibular input alone was offered (condition 4). the hip and spine mobility is diminished in enuretic children and teenagers rita pavione rodrigues pereira 1 , vera herminakalika koch 2 , monica maria ribeiro goncalves 2 , thais de souza milhoratti 1 , daniela castro pacheco 1 , clarice tanaka 1 1 physical therapy, university of são paulo, são paulo, brazil 2 communication disorders and occupational therapy, university of são paulo, são paulo, brazil objective: to assess the hip and spine mobility in children and enuretic teenagers compared to a control group. methods: 22 enuretic (eg) patients (11 boys and 11 girls) with mean ± sd of 10.52±2.77 years, ranging from 7 to 16 years old, and 19.39±3.50 of bmi were paired with assyntomatic kids (cg) of equal age, gender and bmi. the range of motion of hip flexion and extension was measured using a goniometer. spine flexibility was measured using schober e stibor test and bank of wells. student t-tests were used to compare variables between groups. the level of significance was 95% for all the analysis. results: when compared to cg, the eg revealed diminished range of motion of hip extension bilaterally (2±3.89 for eg and 10±7.99 for cg at right side; p<0.01; ge 2.05±3.88 for eg and 10.4±7.22 for cg at left side; p<0.01). no difference was found for hip flexion range of motion, however compared to cg, eg showed lower values bilaterally (94.9±17.1 for eg and 100.32±8.52 for cg at right side; 98.3±11.7 for eg and 101±11.1 for cg at left side). the spine flexibility did not presente any difference for schober (14.88±3.18 for eg and 16.18±1.16 for cg; p =0.07), and stibor (50.21±6.2 for eg and 51.93±7.37 for cg; p=0.40) and bank of wells (25.58±7.67 for and 23.66±7.42 for cg; p=0.63). conclusion: when compared to assymptomatic control group, the enuretics children and teenagers presented diminished range of motion of hip extension suggesting transversal misalignment of the pelvis. more concern about enuresis children: an epidemiological study of primary nocturnal enuresis in elementary schools in shanghai yibing zheng 1 , yinv gong 1 , hong xu 1 , keli wang 1, 2 , zhonghui ni 1,2 , dandan he 1,3 1 nephrology and rheumatism, shanghai, china 2 xuhui health bureau, shanghai, china 3 minhang center for disease control and prevention, shanghai, china objective: (1) to assess the prevalence of primary nocturnal enuresis (pne) and its risk factors in children of elementary school age in shanghai. (2) to evaluate the impact of enuresis on these children and their parents, and to identify the methods and effectiveness of managing enuresis. methods: a randomly selected cross-sectional study was conducted in four elementary schools in two districts inshanghai. the parents of these children were asked to complete questionnaires anonymously which included items about the presence and frequency of enuresis, its risk factors and its perceived impact and management. the distress caused to the family by enuresis and the outcome of any management was evaluated using a 5-point visual analogue scale (vas). pns was defined as an involuntary voiding of urine during sleep, with a frequency of more than two times a week for three consecutive months, in the absence of congenital or acquired defects of the central nervous system. results: a total of 7600 questionnaires were distributed. the overall response rate to the questionnaire was 97.1%, girls 45.2% and boys 51.8%. the prevalence of pne declined with age from 6.3% at 7 years old to 0.5% at 11 years old. of all enuresis children, 10.1% had daytime urinary symptoms and 11.7% had a positive family history. 58.8% of parents were concerned and 17.6% were very concerned about enuresis. 41.2% of children were worried about and 5.9% were very worried about enuresis. only 35.3% of pne children sought for treatment and the common strategies (23.5%) were adjusting lifestyle such as restriction of water intake at night, 11.9% used alarm clock and 5.8% were take medication. 48.6% of parents felt that the current treatment was effective. conclusion: we conducted a relatively scientific epidemiological survey on the prevalence of pne in two districts in shanghai. we find enuresis still has great impact on children and their parents, but only a small part of them will seek for treatment. and only half of the parents feel that the current treatment is effective. therefore, it would be desirable to create a series of standardized management and follow-up processes for enuresis children. abstract# p-sat068 renal manifestations of tuberous sclerosis: a descriptive analysis sophy korula 1 , alka ekbote 2 , naresh kumar 1 , sumita danda 2 , indira agarwal 1 , swasti chaturvedi 1 1 paediatrics, christian medical college, vellore, india 2 clinical genetics, christian medical college, vellore, india objective: to describe the renal manifestations in children 0-18 years of age diagnosed with tuberous sclerosis complex (tsc) at a tertiary hospital in south india. methods: data of children with tsc who presented to christian medical college vellore hospital from january 2008 onwards, were analysed by a retrospective chart review. the cases were identified from outpatient records and underwent ultrasonography, urine analysis and serum creatinine to recognize renal involvement. results: twenty-five children with tsc were identified. two children did not imaging studies available and were excluded from the analysis. the age of included children ranged from 5 days to 15 years with a median of 8 years. seventy four percent (17/23) were males. ten of the 23 children had evidence of renal involvement (43.5%). of the ten children with renal involvement, six had angiomyolipoma (60%), five had renal cysts (50%) and one had suspected renal cell carcinoma. in two children both angiomyolipoma (aml) and cysts were noted. none were symptomatic. one child was found to have proteinuria and another had reduced creatinine clearance of 52.5 ml/min/m 2 with normal bp and urinalysis. the rest of children had no evidence of proteinuria and had normal creatinine clearance. conclusion: we conclude that all children with tsc should be screened for renal involvement and regular follow up should be arranged. abstract# p-sat069 sonographic growth charts for kidney length in normal korean children: a prospective observational study objective and methods: kidney length was measured by sonography in a prospective observational study of 343 normal children from 0 to 12 years of age. results and conclusion: there was a good correlation between kidney length and somatic values including age, weight, and height. the rapid growth of height during the first 2 years of life was intimately associated with a similar increase in kidney length. the values of weight and height showed good correlation with kidney length in children from 2 to 12 years of age. height should be considered the important factor to correlate with kidney length. for the children aged 2 years or older, the regression equation was obtained: kidney length (cm) = 2.677 + 0.046 x height (cm). for the younger than 2 years, the equation was: kidney length (cm) = 1.120 + 0.066 x height (cm). objective: the outcome of children with unilateral nonfunctioning mcdk. methods: 24 children (13 pts with right mcdk), afunction confirmed by renal scan, 12 boys, mean age of 6 years (range 1.5 month -21.5 years), mean observation period of 6.7 years with a range of 1.5 month -21.5 years. prenatally diagnosis was made in 83 %. abnormalities of the contralateral kidney were found in 4 /24 pts (13 %) -dystopia (n=2), hydronephrosis due to ureterovesical obstruction (n=1), vacter syndrome (n=1). in 2 pts mcdk had been removed (in period 1991-1997) . collected data include egfr (calculated by cystatin c), urine protein/creatinine ratio, microalbumin/creatinine ratio, a1microglobulin (a1m)/creatinine ratio, urine beta-2microglobulin (b2m), urine ngal, urine nag, blood pressure (bp), renal length. results: hypetrophy of the contralateral kidney (length >2 sds) was detected in 19/24 pts (79%) to 2 years of age. complete involution (in 33 %) or decrease of size (in 30%) of mcdk occured in the first 5 years. hyperfiltration (defined as egfr of 149 ml/min/1.73m2) was seen in 4 pts (17 %). none of 24 pts demonstrated decreased egfr, proteinuria (mean value of 24.8 mg/mmolcreatinine), microalbuminuria (mean value of 2.0 mg/mmolcreatinine).urine a1m (mean value of 5.8 mg/mmolcreatinine), urine b2m (mean value of 0.2 mg/l), urine nag (mean value of 7.3 ukat/mmolcreatinine), urine ngal (mean value of 10.4 ng/l) were in a normal range. all pts were normotensive -bp defined as <95th percentile for age and gender. the combination of hepatoblastoma with renal failure due to congenital dysplastic kidneys in children is extremely rare. we report two children in renal failure since birth due to congenital cystic dysplastic kidneys. hepatoblastoma was subsequently diagnosed in both of them. methods and results: case1 a male infant was born at 35 weeks gestation with an antenatal diagnosis of bilateral dysplastic kidneys and bladder outflow obstruction secondary to a prolapsing ureterocele, anhydramnios, and pulmonary hypoplasia. he underwent surgical repair of other congeniatal abnormality after birth. peritoneal dialysis was initiated at 3 months. treatment with erythropoietin (epo) was commenced at 6 weeks of age. case 2 a male infant with congenital cystic renal dysplasia, bilateral vesicoureteric reflux pulmonary hypoplasia and oligohydramnios was born at 36 weeks gestation. peritoneal dialysis was commenced at day 20 due to anuria from birth. epo was commenced at 6 months of age. both of them required high dose epo to maintain adequate haemoglobin. hepatoblastoma was diagnosed in both of them at 2-3 years, following an incidental finding in abdominal imaging done for different reasons. conclusion: hepatoblastoma has been reported in association with cystic renal dysplasia in 5 children in the literature to date. none of these children had evidence of impaired renal function prior to the diagnosis of hepatoblastoma. in our report both the child were treated with high dose erythropoietin prior to diagnosis of hepatoblastoma. erythropoietin (epo) is the primary regulator of erythropoiesis through specific interaction with its receptor (epo r). epor expression has been demonstrated in common paediatrictumour cells and such expression is reported to promote tumour cell survival through release of angiogenic growth factors. angiogenesis is the primary requirement for tumour growth. epo production has been observed in patients with hepatomas and in the hepg-2 cell line in hepatoblastoma. we report two cases of hepatoblastoma detected incidentally in children with established renal failure and propose a putative role of epo in the development and / or progression of hepatoblastoma in this population. objective: hypercalciuria is the most common metabolic cause of renal stone. long term immobilization is associated with hypercalciuria and bone loss. the effect of short term immobilization on hypercalciuria was the main objective of this study. methods: in a prospective study all orthopedic patients less than 40 years with pelvic fracture who were assigned for immobilization with traction were enrolled in this study. serum (calcium, phosphorous, alkaline phosphatase, sodium, potassium, uric acid, bun, creatinine)and fasting urine calcium, creatinine, sodium, potassium and uric acid were checked within 48hours of hospitalization and also in 1st,2nd and 3rd weeks of immobilization and then after 2to 3months of mobilization. student's t-tests used for statistical analysis. results: fifty five patients 45 male and 10 female with the mean age of 19.4 ± 12.7 years were studied. urine calcium /creatinine (u ca/cr)* ratio before immobilization u ca/cr0 was 0.13±0.06. one, two and 3 weeks following immobilization u ca/cr ratio was 0.17±0.11, 0.22±0.12 and 0.29±0.17respectively.multivariate tests revealed a significant rise in u ca/cr ratio during hospital stay when this value checked before immobilization and throughout immobilization as soon as end of 1st week and following in 2nd and 3rd week ( objective: importance of renal biopsy(rb) in diagnosis and treatment of kidney disease is the same for children as for adults. however there are some differences between institutions in practice of this invasive method. the aim of this study is to identify the current status and complications associated with the renal biopsy of childhood kidney disease. methods: a retrospective study was conducted based on medical records from all patients who had undergone native renal biopsies with 16-gauge needles from april 1997 to november 2012 in our center. we analyzed the number of experiments, sex, age, indication, the types of harvest (percutaneous or open) and anesthesia (general or local), histopathological findings, and complications. results: a total of 158 renal biopsies were performed in 141 patients (82 boys, 59 girls). the mean age at first procedure was 9.4 years. about 70 % of the indications for a biopsy showed moderate proteinuria and hematuria in the annual urinary screening program in japan, and approximately 20 percent of those were refractory childhood nephrotic syndrome. open renal biopsies were practiced in only 3 cases, and the rest of children underwent percutaneous ultrasound-guided kidney biopsies. the most procedures of patients aged 10 or younger were executed under general anesthesia due to the non-cooperation of them. about 30 % of the biopsy-proven kidney diseases showed immunoglobulin a nephropathy, and approximately 20 percent of those were henoch-schonlein purpura nephritis. for the post biopsy complications, 5 out of all performances administered hemostatic agents for moderate hematoma, but none of them required blood transfusion or surgical intervention. conclusion: we suggested that renal biopsy could be supportively performed under local anesthesia in the patients more than the upper grades of elementary school. in addition, 17 patients aged 3 or younger, including 12 months, could undergo the percutaneous ultrasound-guided renal biopsies without procedural and general anesthesia complications. therefore, our results revealed that this procedure was considered safe even in infants. abstract# p-sat084 fifteen years review of indications and results of renal biopsy in children from a single center in egypt there were 20 insufficient biopsies. in pathologically diagnosed 1226 specimens primary glomerulonephritis was the most common finding (n=826, 67.4%) followed by secondary glomerulonephritis (n=238, 19.4%) and end stage renal disease (n=50, 4.1%). the most common causes of primary glomerulonephritis were minimal change disease (n=267, 21.8%), diffuse proliferative glomerulonephritis (n=188, 15.3%), focal proliferative glomerulonephritis (n=164, 13.3%) and focal segmental glomerulosclerosis (n=129, 10.5%). lupus nephritis (n=209, 17%) was the most common cause of secondary glomerulonephritis followed by hemolytic uremic syndrome (n=16, 1.3%) and amyloidosis (n=10, 0.8%). only one mortality has been reported as a complication of renal biopsy (uncontrolled bleeding). conclusion: this study introduces the first biopsy based epidemiological information of pattern of renal diseases in egyptian children from a single tertiary pediatric center in which minimal change disease was the most common histopathological finding and steroid resistant nephrotic syndrome was the most frequent indication for biopsy. results: a total of 112 children were enrolled in the study. most of them (71%) had isolated microhematuria, and were proved mild lesion of glomeruli (60%) by renal biopsy. about 30% of them, however, might have progressive glomerulonephritis, such as iga nephropathy, focal segmental glomerulosclerosis, alport syndrome and so on. these asymptomatic children were found urine abnormalities due to either school urine screening study, health examination, or during diagnosis of non-renal diseases. in center a, iga nephropathy was the most case diagnosed, while in center b and c, minor glomerular abnormalities was the most case diagnosed. the difference among three centers was significant and this variation might be due to different indications of renal biopsy. conclusion: our findings confirm that urinalysis may help early detecting progressive glomerulonephritis in asymptomatic children. children with isolated microhematuria has relatively low risk of severe pathologic lesion of glomeruli, thus isolated microhematuria per se might not be suggested as indication of early renal biopsy. long-term follow-up with appropriate further examination is of great importance for these asymptomatic children. abstract# p-sat089 twenty-three-year review of disease patterns from renal biopsies: an experience from a pediatric renal center there were 59 episode of peritonitis with an incidence of 1 episode per 16.8 patient/months. the commonest organisms isolated were gram negative rods 12(20.3%), followed by staph aureus 11(18.6%). sixteen (27.1%) were culture negative and 3(5.1%) were fungal peritonitis. catheter removal related to peritonitis was performed in 20 patients in whom only one patient returned back to pd. exit site infection (21 episodes) and tunnel infection were reported in 20 and 10 patients respectively. catheter malfunction was the most common non infectious complication seen in 27 (61.3%) children. other noninfectious complications include haemorrahgic effluent 5 (11.3%) children while catheter leak, umbilical hernia and inflow pain where seen in 2 (4.5%) children each. at the end of the study 17 (18.7%) children remained pd active, 26 (28.6%) transferred to hd, 13(14.3%) transplanted, 9(9.9%) suspended from pd, 5(5.5%) lost follow up and 21(23.1%) children died. conclusion: in the setting of our country with limited resources and investment, capd is efficient and successful with complications comparable to most parts of the world. attempts to produce pd fluids locally and to train and educate health care workers will greatly improve the use of pd in developing countries. profile and response to therapy in patients with idiopathic membranous nephropathy from march 2010 to december 2012. renal biopsies were studied for light microscopy, immunofluorescence and electron microscopy. antibodies to the m-type phospholipase a2 receptor (pla2r), bovine serum albumin (bsa), and cationic bsa were measured by western blotting with patient sera (when available) and then detecting for both totaligg as well as specifically for the igg4 subclass. results: 80 renal biopsies were performed during the study period, out of which 11 patients had membranous nephropathy. five were secondary to sle and 6 patients (7.5 %) had idiopathic membranous nephropathy. mean age at presentation was 13.16+/-1.94 years with 50 % having hematuria and 66.6% hypertension at presentation. estimated gfr was 160.57+/-49.33. renal biopsy showed findings consistent with membranous nephropathy on light microscopy with with<5% of the interstitium showing tubular atrophy and interstitial fibrosis. immunofluorescence studies showed granular igg and c3 along the peripheral capillary walls. electron microscopy showed subepithelial deposits, with formation of basement membrane spikes between the deposits. few intramembranous deposits were also present. subendothelial and mesangial deposits were not present. there were no antibodies to bsa or cationic bsa detected in any of the five sera available. one of the five patients had circulating antibodies to pla2r. all patients received prednisolone, angiotensin converting enzyme inhibitors and oral calcium supplements. two patients continued to have nephrotic range proteinuria inspite of prednisolone and were started on cyclosporine. none of the patients developed any complications. conclusion: immunosuppressive therapy is beneficial in children with idiopathic membranous nephropathy. anti-pla2r antibodies can be seen as early as 13 years of age. abstract# p-sat108 hypercalcemic crisis and nephrogenic diabetes insipidus due to vitamin d intoxication methods: twenty-three children (46 kidneys) with first uti with p-fimbriatede.coli were enrolled in the study. children were aged between few months and 10 years. all children were treated with adequate antibiotic therapy. dmsa scintigraphy was performed in a few days after infection and control scintigraphy was performed 5 months to 36 months later. dmsa findings were categorised as normal, generally diminished uptake of activity, focally diminished uptake of activity and clearly pathologic with renal scars. results: the first dmsa scintigraphy immediately after infection was normal in 12 kidneys (26.1%), generally diminished activity was found in 21 kidneys (45.7%) and focally diminished activity in 6 kidneys (13% ). renal scars were present in 7 kidneys (15.2%) . on the control scintigraphy 20 kidneys (43.5%) had normal findings. generally diminished activity were found in 6 kidneys (13%) and focal diminished uptake in 8 kidneys (17.4%). significantly higher number of scars, in 12 kidneys (26.1%), despite of antibiotic treatment were found on the control scintigraphy (p< 0.01). conclusion: dmsa scintigraphy demonstrated that the infection with p-fimbriatede.coli can result in permanent renal damage, which clearly points to uti with p-fimbriatede.coli as a risk factor in renal damage. our results confirm obligatory scintigraphic follow-up of children with positive p-fimbriatede.coli infection. objective: efficacy of mannitol with furosemide was compared with that of albumin with furosemide in the treatment of diuretic resistant oedema in childhood nephrotic syndrome. methods: forty patients fulfilling the criteria for "resistant oedema" in nephrotic syndrome cases were enrolled in this descriptive cross sectional study. resistant oedema was diagnosed based on failure to achieve therapeutic response to diuretics or a weight loss of <1% body weight daily. all nephrotic syndrome patients with severe oedema, age 1 year to 15 years of both sexes were hospitalized and were managed with fluid restriction, no added salt and bed rest. beside these 2 mg/kg/day oral furosemide or combination of furosemide and spironolactone, were given for 3 days to achieve desired diuresis. those patients who did not get response were divided into two groups (group-a, group-b) in consecutive fashion. the group-a study population, was with intravenous mannitol 0.5-1 gm/kg/day in single daily dose over 1-2 hrs followed by intravenous furosemide 1 mg/kg/day for 5 days. the group-b study population was with intravenous albumin, 0.5 -1 gm/ kg /day in single daily dose over 1-2 hrs followed by intravenous furosemide 1 mg/kg/day in every alternate day, total 3 doses. conclusion: this boy presented with rhabdomyolysis, myohemoglobinuria and arf. (similar pr similar presentation can be rarely seen in human being, but often seen in dogs). blood exchange transfusion in time must be necessary for preventing "water fall effect" established, for it can eliminate inflammatory factor and toxic products such as bun, ccr, and creatinasein blood effectively. blood tranfusion in combination with azithromycin and clindamycin must be a good choice replace the "babesia-infected" rbc and eliminate pathogens in blood. beta-2-microglobulinuria in human immunodeficiency virus infected children objective: to determine whether hiv infection in children is associated with increased urinary excretion of β2-microglobulin, which is a marker of tubular defect. methods: a prospective observational study was done of hiv infected children attending outpatient immunology clinics to investigate urinary β2-microglobulin excretion by measuring urine β2-microglobulin to creatinine ratio. in addition serum sodium, potassium, urea, bicarbonate and creatinine levels were documented and urine dipstick test was done to quantify proteinuria. results: thirteen (13) children aged 2 months to 49 months (mean 20+/-4 months) were enrolled. all children had normal estimated glomerular filtration rate. eleven children were on different haart regimes (see table 1), one was receiving nevirapine prophylaxis and one was not on any treatment. the duration of haart use was 1-16 months (mean of 8+/-2 months). seven (7) children were on cotrimoxazole for pneumocystis jiroveci prophylaxis and 4 on anti-tuberculosis drugs, namely rifampicin, ethambutol, isoniazid and pyrazinamide. none received any nephrotoxic drugs. the hiv viral load was >1000 copies/ml in 8 children and <1000 copies/ml in 5 children. of 13 hiv-infected children, 4(31%) had no abnormal urine protein excretion and nine (69%) had elevated urinary levels of β2-microglobulin.of note only 2 (22%) of the 9 children with β2-microglobulinuria had associated proteinuria. ten children ( 76%) had metabolic acidosis with the mean serum bicarbonate (co 2 ) level of 16.9 mmol/l ( range 10-20 mmol/l). conclusion: proteinuria is believed to be the earliest finding for the diagnosis of hiv associated renal diseases. in our study β2microglobulinuria and metabolic acidosis were the prominent findings suggesting that hiv induces tubular dysfunction in children without clinical evidence of renal disease. objective: acute kidney injury (aki) is an important cause of mortality in sub saharan africa because access to renal replacement therapy is limited. there are few reports on haemodialysis in childhood aki in the sub region. we therefore performed a preliminary review of data on children who received intermittent haemodialysis for aki in our centre. method: a retrospective review of case records and haemodialysis registers of children in aki who received haemodialysis in our centre from january 2006 to december 2012. results: 62 children, including a child aged 3years, received haemodialysis for aki over the period but full details were available for 23 children and were further reviewed for this study. there were 13 males (56.5%). the children were aged 5-13 (8.4+/-2.4) years. the primary aetiology of aki was related to intravascular haemolysis (ivh) with massive haemoglobinuria (n=12), septicaemia (n=4), acute glomerulonephritis (n=2), malignancies (n=2), malaria (n=1), hiv (n=1) and haemolyticuraemic syndrome (n=1). the aetiology of ivh was secondary to glucose-6-phosphate dehydrogenase (g6pd) deficiency in 4 patients, autoimmune haemolyticanaemia in 1 and was unknown in the others. one of the patients with ivh secondary to g6pd deficiency also had malaria. the number of sessions of dialysis ranged from 1 to 5 sessions per patient with a modal value of 2 objectives, methods and results: a twelve-day old, formula-fed, full term neonate with unremarkable perinatal history was admitted for poor oral feeding and significant (12%) weight loss. physical examination was otherwise unremarkable. investigation showed normal anion gap metabolic acidosis, hyperkalemia (6.5mmol/l), hyperurecemia (14.5mmol/l) but normal creatinine (28umol/l). metabolic screen and other investigation was unremarkable. on further questioning parents has been feeding baby with goat milkbased formula since day 5. baby was switched back to normal cow milk-based formula with prompt resolution of metabolic disturbance and without recurrence of acidosis on follow-up. conclusions: goat milk is becoming increasing popular to parents because of the myths associated (organic, easier digested, hypoallergenic, better nutrient content etc). however there is good evidence suggesting against these misbelief and there are even reported morbidities associated with ingestion of goat milkbased formula. with increasing appealing but not necessarily accurate information available on the internet, what the public need is education. objective: there have been great inroads made in controlling the scourge of hiv in south africa. the national pmtct program has reduced the mtct rate to 3.5% and children infected prior to these interventions are now on effective treatment programs. these children are now surviving longer and so we are seeing increased numbers of hiv related renal pathology presenting to our clinics. before the widespread availability of arv's in south africa these children would have been denied access to our program but now we are able to offer them crrtand a transplant. the biggest challenge facing our program is one of the social disintergration of much of our indigent population. the vast majority of our patients come from social backgrounds that are incompatible with adhering to the rigours of a chronic renal program. this is compounded when the care givers are themselves ill or when the child has a second serious illness such as hiv to deal with. methods: we would like to present our experience with admitting 8 hiv positive children with ckd5 to our chronic program and will highlight the challenges that face these families and their health care providers. we will describe the social issues affecting each family and how they impact on the child's care. results: all 8 families had serious social issues on top of having to deal with their hiv disease. all 8 families had extreme difficulties in adhering to our program. 2/7 died as a result of non adherence and of the remaining 5 children only 1 is really coping well with the program. 3/7 children have no living parents and 1 is foster care. the rest are either with a single parent or with a family relative. conclusion: the huge difficulties facing these patients is forcing us to rethink the criteria that should be applied to admitting these patients to our program. restricting the program to children with these problem seems cruel and we feel that all options should be considered before denying these children access to life saving care. abstract# most of the patients presented during autumn and winter. the incidence of apsgn has decreased in the past 11 years in shenyang, but the proportion of the patients with acute kidney injury (aki) or nephroticrange proteinuria was increased. the proportion of children with macroscopic hematuria remained almost the same during these years. macroscopic hematuria resolved in 2-3 weeks and non-nephroticproteinuria resolved in 1-3 weeks. the treatment of apsgn patients with nephrotic-proteinuria was the same with the children with primary nephrotic syndrome and prednisone was withdrawn in 3-4 months. some aki patients have been followed up for 5 years and no patients with chronic renal failure. all patients with microscopic hematuria at onset and 30% patients with microscopic hematuria after 2 years. conclusion: the incidence of apsgn was decreased during recent years. the proportion of apsgn patients with aki or nephrotic-range proteinuria was increased. the short-term prognosis of apsgn was good and minority of the patients persisted with microscopic hematuria. abstract# p-sat122 analysis of clinical manifestations and prognosis in 43 children of acute poststreptococcal glomerulonephritis objective: crescentic glomerulonephritis (gn) is characterized by severe infiltration of massive inflammatory cells into glomeruli and crescent formation. although renin angiotensin system (ras) is the key player in renal injury, the impact of direct renin inhibitor on glomerular crescent formation is not elucidated yet. methods: to examine whether direct renin inhibitor ameliorate renal injury in crescentic gn, we investigated renal injury induced by antiglomerular basement membrane (gbm) antibodies in wistar kyoto rats treated with direct renin inhibitor, aliskiren. in addition, using cultured glomerular mesangial cell (mcs) and parietal epithelial cell (pecs), we examined whether recombinant renin could induce monocyte chemoattractant protein-1 (mcp-1) expression and cell proliferation, respectively. results: an anti-gbm nephritis model developed progressive proteinuria and glomerular crescent formation, accompanied by increased expression of mcp-1 and (pro)renin receptor. interestingly, (pro)renin receptor expressed strongly in the crescent formation area in diseased glomeruli. proteinuria was significantly reduced by the treatment of aliskiren. then, aliskiren markedly ameliorated renal injury (% glomerular crescent: 26.0 +/− 1.7 %) compared to vehicle treatment (59.6 +/− 3.6 %). excretion of urinary protein in a group of rats treated with aliskiren were significantly reduced compared to vehicle-treated rats. aliskiren treatment markedly decreased mcp-1 and (pro)renin receptor mrna levels in the diseased kidney. next, primary cultured mcs stimulated by recombinant renin showed significant increases of mcp-1 mrna expression. furthermore, primary cultured pecs showed an increase in recombinant renininduced cell proliferation. conclusion: these data suggest that therapeutic strategy of direct renin inhibitor may prove beneficial for crescentic gn by the suppression of the ras activation and the decrease of inflammation and cell proliferation in glomerular crescent via (pro)renin receptor. an update on management of acute glomerulonephritis in children objective: to analyze the clinical effects, safety, and significance of leflunomide combined with hormone in the treatment of children with refractory nephrotic syndrome. methods: collected 60 cases patients in our hospital in children with refractory nephroticsyndrome,and randomly divided into 30 cases of the control group and treatment group. control group was treated with mycophenolatemofetil (mmf)andhormone,the treatment group was treated with leflunomide (let) and hormone.measured and compared the changes before and after treatment of 24h urinary protein excretion,tc,alt,cr,bun clinical efficacy and complications. results: the two groups after 6 months of treatment, 24 h urinary protein, plasma albumin, total serum cholesterol, serum creatinine and blood urea nitrogen and other indicators were significantly better than before treatment and has a statistically significant (p <0.05); the overall response rate in the control group and treatment group were 83.33% and 86.67%. conclusion: the application of leflunomide combined with hormone treatment of children with refractory nephrotic syndrome can make the disease to be effectively alleviated, the clinical results were satisfactory, safe, and fewer complications, it is worthy of promotion. objective: post-streptococcal acute glomerulonephritis (psagn) is a familiar disease in children. misdiagnosis might occur when it is presented with atypical symptoms. here we report a 12-year-old girl presented with acute heart failure which was finally diagnosed comorbidity of psagn and graves' disease. methods: retrospectively review the history of the patient. the girl was admitted to our hospital with cough, shortness of breath and chest tightness, without any other obvious symptoms and history of illness. the examination of cardiac color ultrasound showed mitral insufficiency with severe mitral regurgitation and heart enlargement. other examinations also found she had hypertention, mild anomalies of urinalysis, hypocomplementemia, hypoalbuminemia, hyperthyroidism and elevated antistreptolysin o. first we diagnosed her illness as rheumatic disease. after two weeks treatment with rest, fluid and sodium restriction, controlling blood pression, diuresis and methimazole, the girl recovered very well. she had no any symptom with normal urinalysis and blood pressure, the result of reexamination of cardiac color ultrasound was normal, her hypocomplementemia and hyperthyroidism were improved significantly. finally she was diagnosed as co-morbidity of psagn and graves' disease. conclusion: psagn with atypical symptoms at presentation should be paid more attention, especially when it is also has co-morbidity. abstract# p-sat128 report of a child with idiopathic cryoglobulinemia idiopathic cryoglobulinemia is rare to see in child. here we report a child with typical manifestations of cryoglobulinemia. the subjected child was male, 6 years and 4 months, he had repeated rashes, itching, desquamation on his fingertips for more than two years, accompanied with recurrent miliary reddish rashes on the trunk and limbs for more than one year, transient arthralgia for 2 weeks, and edema and oliguria for 10 days before he was admitted to our hospital. he was treated as atopic dermatitis all the time. but the rashes were recurrent. erythema could be seen after desquamation. 2 weeks before admission, he had arthralgia on the right knee, 2 days later on left knee without swelling, both were retrieved spontaneously. x-ray of the knees revealed no abnormal. ten days before admission, edema and decreased urine output was notice without gross hematuria. physical examination in local hospital revealed blood pressure 140/108 mmhg, needlelike erythematous maculopapules in his hands and feet. urine test revealed protein 5g/l, rbc 164/ul, no casts. serum bun was 32.41mmol/l, cr 116umol/l, tco2 11mmol/l, c3 0.47g/l. he was sent to another hospital 2 days later. there, prednisone 25mg per day was given. as a result, the urine output increased gradually, but serum bun and cr remained high, so he was referred to our hospital. renal biopsy indicated membrane proliferation glomerular nephritis with abundant microthrombus. the diagnosis of cryoglobulinemia was suggested. after that serum cryoglobulin and serum protein electrophoresis were tested 18 days after prednisone was given, both were normal. etiological examinations, included hepatitis virus a, b, c, d, e, g were all negative; antibodies of hiv, syphilis, toxo, cmv, rvb, rsv, hsv, eb, adv, and rickett's organism were all negative, mp-igm 1:40. antinuclear antibodies of sle, anca and antiphospholipid antibodies were all negative. so idiopathic cyoglobulinemia was more favored. the treatment of prednisone 25mg per day was continued after admission. he restored gradually. prednisone was taped down gradually. now prednisone has been stopped for more than one month, he seems well during that period of time. methods: children with hsp over 3 years of age were enrolled in the study. they were evaluated for demographic, anthropometric, clinical and laboratory data including urinalysis, complete blood count, serum albumin, creatinine, iga levels. in addition, anti-tissue transglutaminase iga (elisa), anti-endomysium iga (ifat), antigliadin (gaf3x, deaminated) iga (ifat) and anti-gliadin (gaf3x, deaminated) igg (elisa) antibody levels were determined. seropositive patients were evaluated by endoscopic small bowel biopsy. the rate of cd seropositivity in hsp patients was compared to the rate in healthy turksih children by the test for the statistical significance of two percentages. results: celiac serology was evaluated in 42 children (25 male, mean age 11.2 +/-3.6 years) with hsp. there was no patient with growth failure or having symptoms associated with cd like abdominal pain, abdominal distention or diarrhea. in addition, none of the patients had iga deficiency, anemia or hypoalbuminemia. celiac serology was positive in 5 (12%) children. endoscopic evaluation was performed in 3 patients and one of them was diagnosed as cd. prevalence of cd in children with hsp was significantly higher compared to healthy turkish children (p<0.001). conclusions: celiac seropositivity was 12% in children with hsp and this rate is significantly higher than the rate in healthy children. although the number of children with hsp is small in this preliminary study, this result suggests that celiac screening may be considered in children with hsp. (14) groups. the glomerular and tubulointerstitial lesions were scored by katafuchi criteria and oxford classification of igan,respectively. results:1.the baseline data of different clincal or pathological groups were no significant differences (p more than 0.05),but 24h protienuria were significant increased in grade 3 and 4 groups than grade 2 group(p less than 0.05).2.according katafuchi critiria,the severity scores of glomerullar and tubulointerstitial lesions were positively correlated with either different clincal or pathological groups(p less than 0.01).3.according to oxford classification, only the severity scores of mesangial hypercellularity and segmental glomerulosclerosis/adhension were positively correlated with different clinical groups(p less than 0.01),but the severity of mesangial hypercellularity, endocapilarity hypercellularity, segmental glomerulosclerosis/adhension,tubular atrophy/interstitial fibrosis and cellular/fibrocellular crecents were all positively correlated with different pathological groups (p less than 0.01). [1990] [1991] [1992] [1993] [1994] [1995] [1996] [1997] [1998] [1999] [2000] [2001] [2002] [2003] [2004] [2005] [2006] [2007] [2008] [2009] were evaluated retrospectively. patients were analyzed comparing those hospitalized during the first and the second ten-year period.the long-term prognosis was evaluated in 60/64 patients (94%) with hsp nephritis followed-up for 1 to 11 years (mean 5.7±3.6 years) based on urine analyses, serum creatinine and blood pressure measurements. results: of 156 patients (mean age 6.9±3.5 years, range 1-17 years, m: f ratio 1.0) with palpable purpura (100%), other symptoms were more common during the first ten-year period including arthralgias (62% vs. 52%, p=0.282), gastrointestinal symptoms (63% vs. 44 %, p=0,018) and renal involvement (55% vs. 21%, p=0.001). among 64 patients (41%) with hsp nephritis, 42 had isolated hematuria and/or non-nephrotic range proteinuria (66%), 15 nephritic syndrome (23%) and 6 nephritic/nephrotic syndrome (10%). hematuria and/or nonnephritic proteinuria was less common (p=0.0005) in the second tenyear period while the incidence of nephritic/nephrotic syndrome was insignificant (p=0.807). recurrences of purpura had 11 patients within 2 years. 6 patients had renal biopsy including one with 30% of crescents and one another with iga nephropathy diagnosed seven years after initial presentation of hsp. long-term prognosis was favourable in most patients with abnormal urine analyses in 12 (20%), in one with hypertension but normal serum creatinine and in all with nephritic/nephrotic syndrome at disease onset. conclusion: clinical evaluation of patients with hsp nephritis treated at our institution showed a good long-term prognosis. urine and blood pressure abnormalities in followed-up patients were associated with nephritis at disease onset. -up (f-up) . egfr was estimated using schwartz formula; decline in renal function was defined as the slope of egfr over the f-up. results: 72% were males, with mean age at renal biopsy of 12.7 ± 3.7 y and a median f-up of 4.7 y (iq range 2.4-7.8 y); more than 80% presented with egfr > 90 ml/min/1.73m 2 . end-stage renal disesase (esrd) was reached by 4% of children, 50% loss of initial egfr by 5%; 7% reached the combined end point (esrd or 50% loss of initial egfr). at renal biopsy 57% presented with mesangial proliferation (m1), 24% with endocapillary proliferation (e1), 35% with segmental glomerulosclerosis (s1) and 9% with tubular atrophy/interstitial fibrosis (ta/if; t1/2). patients with segmental sclerosis and ta/if showed a significantly worse egfr slope over the f-up (s0 vs s1 p=0.04; t0 vs t1/2 p=0.003). at univariate linear regression, clinical data at renal biopsy (egfr, proteinuria and map) were not associated with renal function decline, while data at 6-12 and 12-24 months and ta-proteinuria and map significantly predicted egfr slope.a multivariate linear regression model (including proteinuria and map at 12-24 months together with the difference of egfr at renal biopsy and at 12-24 months as independent variables) performed well in predicting egfr slope (r 2 =0.39). this model was used to derive a formula able to estimate egfr slope with good performance (mean bias between estimated and really observed egfr slope of 0.05 ± 6.6 ml/min/1.73m 2 ). conclusion: the oxford classification of igan was well applicable to this pediatric population. a formula was developed that estimates renal function decline over the f-up based on proteinuria, map and egfr loss after 1-2 years of observation, which will need a validation on other cohorts. sixteen-year experience with pediatric iga nephropathy and validation of the oxford classification as a risk predictor (m1), segmental sclerosis (s1), endocapillary proliferation (e1), and tubulointerstitial fibrosis (t1 or t2) was 32 (35.2%), 15 (16.5%), 9 (9.9%), and 6 (6.6%), respectively. there was no significant decrease in gfr within the entire cohort. however, five patients showed decreased renal function, and of them, two reached stage iii or iv ckd. the five patients' changes in the k/doqi ckd stage were 2 to 4, 1 to 3, 1 to 2, 1 to 2, and 1 to 2. their pathological classifications were haas 4/m1 s1 e0 t0, haas 4/m1 s0 e1 t1 with global sclerosis, haas 1/m0 s1 e0 t0, haas 1/m0 s0 e0 t0, and haas 1/m0 s0 e0 t0 with global sclerosis. in the oxford classification, global sclerosis was the only factor correlated with decreased renal function, whereas the haas classification showed no significant correlation with renal function. conclusions: global sclerosis was correlated with decreased renal function. however, in our cohort, there was no significant decrease in renal function during the follow-up period (p = 0.216). thus, we could not clearly correlate the haas and oxford classifications with renal outcome. the value of the oxford classification as a predictor of renal outcome remains unclear in korean children with iga nephropathy. meng-jie jiang, xiao-yun jiang, ying mo, li-zhi chen, li-ping rong pediatric, the first affiliated hospital of sun yat-sen university, guangzhou, china objective: to analyze the clinicopathological characteristics of primary immunoglobulin a nephropathy (igan) manifested as macroscopic hematuria in children. the clinicopathological characteristics of 48 cases with primary igan manifested as macroscopic hematuria were analyzed retrospectively. according to the lasting time of macroscopic hematuria, 15 cases were assigned to group a (no more than seven days) and 33 cases were assigned to group b (more than seven days). results: the manifestations comprised: acute glomerulonephritis (40% in group a, 33.3% in group b), nephrotic syndrome (20% in group a, 15.1% in group b), hematuria and proteinuria (6.7% in group a, 12.1% in group b) and isolated hematuria (33.3% in group a, 39.4% in group b). 20.0% cases in group a and 21.2% cases in group b were accompanied with abnormal renal function. there was no significant difference between group a and b (p>0.05). the urinary lysozyme or β2-mg increased (6.7% in group a, 30.3% in group b). in group a, subclass ii was the most common histopathological type (53.3%), followed by iii (33.3%) and i (13.3%) while the predominant histopathological types in group b were subclass iii (45.5%), iv (27.3%), ii (21.2%) and i (6.1%). none in group a and 15.2% cases in group b showed crescent. besides, only one glomerulosclerosis was in group a while 8 (24.2%) cases of glomerulosclerosis were in group b. the difference between group a and group b was statistically significant (p<0.05).no differences were found in balloon stenosis between group a (6.7%) and group b (12.1%), as well as the renal tubular and interstitial damage (33.3% in group a, 39.4% in group b). 46.7% cases in group a and 60.1% cases in group b were found with simple iga deposition in mesangial area. there was no significant difference between group a and group b (p>0.05). the children with primary igan manifested as macroscopic hematuria lasting for more than seven days are easily appearing renal tubular damage, crescent formation and glomerulosclerosis. subclass iii and iv are the most common histopathological types. a case report of iga nephropathy accompanying crohn's disease akihiko shirasu 1 , akira ashida 2 , hideki matsumura 2 , hyogo nakakura 2 , tomoki aomatsu 2 , atsushi yoden 2 , motoshi hattori 3 , hiroshi tamai 2 , 1 pediatrics, hirakata city hospital, hirakata, japan 2 pediatrics, osaka medical college, takatsuki, japan 3 pediatric nephrology, tokyo wemen's medical university, tokyo, japan there have been several reports of iga nephropathy accompanying crohn's disease in which the clinical course of the two diseases was linked. we recently experienced a case of iga nephropathy with deterioration of renal function complicated by crohn's disease. a 15year-old boy diagnosed as having crohn's disease underwent total colectomy at the age of 7 years. the patient was referred to pediatric gastroenterology unit at the age of 13, and thereafter maintained a state of remission form crohn's disease while receiving 5-aminosalicylic acid (5-asa). two months prior to referral to our pediatric nephrology unit, when the patient was 15 years old, a school health examination had revealed microscopic hematuria and mild proteinuria. at that time, laboratory tests suggested deterioration of renal function. renal biopsy was performed and histological examination by light microscopy demonstrated focal segmental mesangial proliferation and moderate tubule atrophy with a diffuse interstitial inflammatory infiltrate consisting predominantly of lymphocytes. immunofluorescence assays revealed 2+ diffuse granular staining for iga and 1+ staining for c3 in the mesangium. therefore, the patient was diagnosed as having iga nephropathy and chronic interstitial nephritis. the 5-asa therapy was stopped considering the possibility that it had induced interstitial nephritis. the renal function of the patient has since remained quiescent for 7 months. patients with crohn's disease who present with abnormal urinalysis findings commonly have urological complications, such as urolithiasis and urinary tract infections. however, the possibility of renal parenchymal disease including iga nephropathy should also be considered, even if there is no apparent aggregative linkage of urinalysis findings with gastrointestinal symptoms of crohn's disease. the duration of heavy proteinuria determined long-term outcome of henoch-schönlein purpura nephritis in children results: among the 9 patients, 7 (77.78%) cases presented with hematuria and nephrotic syndrome (ns); 1 (11.11%) with hematuria and nephrotic range proteinuria (>50mg/kg/24h); 1 (11.11%) with hematuria and moderate proteinuria (25~50mg/kg/24h). the typical pathological features, such as the diffuse glomerular mesangial and endocapillary proliferation, mesangial interposition, and double contour formation, were shown in all specimens. moreover, the podocyte hypertrophy, shedding, and cytoplasmic absorption dropletswere also observed in most specimens under light microscopy. the percentage of small cellular crescents varied from 4.25-25%. 9 patients were followed up for 2 to 4 years, and all had recovered. conclusion: the predominant clinical manifestation of iskdc grade vi hspn in children was ns accompanying with hematuria. the well outcome might be associated with the prompt use of steroids and/or immunosuppressive drugs at the onset of iskdc grade vi hspn with very mild glomerulosclerosis and tubulointerstitial lesion. in our future study, all patients should be followed for five or ten years, and more specimens should be observed under electron microscope to investigate whether the podocyte lesions of iskdc grade vihspn should be taken into account in the clinical pathological report and a future new histologic classification of hspn. objective: the aim of the study was to assess efficacy of immunosuppressive treatment in children with iga nephropathy (igan) and henoch-schoenlein nephritis (hsn) based on clinical manifestation and histopathological oxford (o-c) and who (who-c) classifications. the study group consisted of 18 children with igan-9 and hsn -9, (mean age 10.44 ±3.94 years) , treated with azathioprine (aza) 2 mg/kg/day -12 months with prednison (pred) 2mg/kg/day (nephrotic syndrome-ns), 1mg/kg/day (nephritic syndrome -nps) 8 weeks, gradually diminished within 15 months. it recognize clinical remission at normal renal function and absence of proteinuria at the end of treatment. in all patients we estimated: proteinuria, gfrs (calculated using schwartz's formula) at the onset of study and after 15 months. they had performed renal biopsy on average 0.69 ± 0.95 years after the onset of illness and at the end of treatment. we were analyzed the histopathologic evaluation of renal biopsy specimens using the who-c (grade i-v) and oxford classification : (mmesangial hypercellularity, e-endocapillary hypercellularity, ssegmental sclerosis, t-tubular atrophy/interstitial fibrosis; absent =0, present=1). results: in patients with igan we observed: ns-1, nps-8; with hsn : ns-7, nps-2. after the treatment aza/pred we noted clinical remission in all children with igan and 89% with hsn. gfr was normal before and after treatment. at the onset of illness igan patients had: grade iii-6, grade iv-3 according to who-c; hsn had: grade ii -2, grade iii-6, grade iv -1; at the end of therapy we observed positive effect in who-c only in 2 (22%) igan patients and in 5 (56%) hsn , without progression properly 6 (67%) and 4 (44%). in o-c of igan patients we observed nobody with total regression of changes, only 3 children had m0 or e0 or s0 after treatment. in patients with hsn we noted total regression of changes in 4 (44%), in other 3-m0 or e0 or t0. conclusion: aza/pred therapy used in children with igan and hsn can make clinical remission in both groups, but histopathological regression-more frequent in hsn. oxford and who classifications are very useful for estimation of efficiency of treatment. 10-year follow-up of pediatric hennoch schönlein purpura with renal involvement elena tudorache 1 , christine azema 1 , stéphane decramer 2 , georges deschênes 3 , tim ulinski 1 1 pediatric nephrology, armand trousseau hospital, paris, france 2 pediatric nephrology, chu toulouse, toulouse, france 3 pediatric nephrology, robert debré hospital, paris, france objective: the aim of this study was to determine the long-term outcome in children with hsp nephritis who underwent a renal biopsy and to identify possible correlation between disease parameters and treatment. we included retrospectively all patients with renal biopsy proven iga nephropathy related to hsp of three pediatric nephrology centres over a 10-year period. results: 142 patients were included. nephrotic range proteinuria was present in 28% with grade ii, 60% with grade iii and 90% with grade iv lesions. renal function was impaired in 14%. significant proteinuria >500 mg/l was found in 9/48 patients 3 years post renal biopsy, in 8/25 patients at 5 years and in 3/14 patients at 10 years. there was no correlation between risk for proteinuria at 3, 5, or 10 years with initial histological lesions. there was a tendency to higher residual proteinuria in patients with nephrotic proteinuria at disease onset and also for patients who were not treated with steroid pulses at disease onset with 310 vs. 175 mg/l (p=0.06) at three months, 100 vs. 50 mg/l (p=0.93) at one year, 720 vs. 150 mg/l (p=0.1) at five years, and 520 vs. 355 mg/l (p=0.35) at ten years. there was a tendency to less proteinuria in the long term for those with early steroid pulses (<15 days vs. >16 days after proteinuria onset (p=0.16).in patients with acei/arb treatment within 15 days after renal disease onset, compared to those who were treated later (>one month), proteinuria was significantly lower at 6 (p=0.02) and 12 months (p=0.03). among 18 control kidney biopsies, 50% show fibrosis of different degree unrelated to initial histology. conclusion: there is a risk to underestimate long term disease severity in patients with low iskdc classes. there is need for prospective long term studies to explore the benefit of early acei/arb and/or steroid pulses. abstract# p-sat147 good outcome of biopsy-proven henoch-schonlein purpura nephritis in children in shanghai single center objective: severe hsp nephritis considered a risk factor for chronic kidneys disease development especially in patients with nephrotic syndrome (ns). methods: we present our experience with tacrolimus (tac) as a steroid sparing agent for a treatment of severe hsp nephritis in 17 years boy. our patient has been observing at the republic center of pediatric nephrology minsk for 5 years. he presented with a typical purpuric rash with recurrences after infections. one year later during acute pneumonia macrohematuria, proteinuria (1-1.5 g/24h) occured and ace inhibitors (iace) were prescribed. despite this, boy developed nephrotic range proteinuria (3.8-7.7 g/24h), biochemical protein 52g/l, albumin 27g/l, cholesterin 6,5mmol/l. renal biopsie showed mesangial proliferation with iga deposits. prednisolon (pred) 80mg/daily with heparin failed to decrease proteinuria and activity. cyclosporin a 200 mg/daily plus pred on alternate day were started and followed during 8 months with positive dynamic (proteinuria 1,7g/24h, decreased clinical and laboratory activity) after which pathogenic treatment was stopped. arterial hypertension (ag) was treated by iace. 6 months later proteinuria (3.5g/24h) and activity increased again. with the presence of ns resistance to steroids combined with hematuria and ag, medrol (64mg/daily) plus tac 1 mg twice daily were prescribed. duration of tac therapy was 5 months. tac was succesfully withdrawn with no rebound disease activity observed (during 9 months). at present the patient receiving iace only. our results indicate that tac may be a promissing steroid sparing agent for treatment of severe hsp nephritis. anti-proteinuric effect of cyclosporine a treatment for iga deposit diseases objective: to evaluate the therapeutic effect of cyclosporine a (csa) on nephrotic-range proteinuria in children with immunoglobulin a (iga) deposit diseases in mesangium such as iga nephropathy (igan) and henoch-schonlein purpura nephritis (hspn). methods: fifty four children (36 children with igan, 18 children with hspn) who were diagnosed with iga deposit diseases in mesangium at renal biopsy were analyzed retrospectively. all the patients developed nephrotic-range proteinuria (> 40 mg/m2/day) for more than 3 months. the starting dose of csa was 5 mg/kg per day that was given in two divided doses, and the drug level was maintained between 100 and 200 ng/ml. the degree of proteinuria was measured before and after csa treatment. steroids were tapered off and stopped gradually after initiation of csa. results: mean duration of csa treatment was 10.7 +/-5.6 months (range 1.4 -32.7 months). mean follow-up duration was 3.7 +/-3.1 yr (range 0.7 -15.2 yr) from the beginning of csa treatment. the mean ratios of protein to creatinine decreased from 3.7 +/-1.5 to 0.6 +/-0.4 after csa treatment for 12 months. thirty-two (59.2%) patients achieved complete remission. renal function was preserved in all patients 12 months after csa treatment. there were no severe complications in patients with csa treatment. conclusion: our findings indicate that csa is an effective agent to treat nephrotic-range proteinuria of iga deposition glomerular diseases such as igan and hspn and the duration of csa treatment should be at least more than 3 months for efficacy. objective: to observe the therapeutic effects of hemoperfusion combined with corticosteroid in treating children with henoch-schonlein purpura. methods: a total of 180 patients (101 inthe hemoperfusion group (hp) and79 inthe control group) were included and followed up for 12 months. both groups were treated with corticosteroids and other supportive therapy. patients in the hp group received 2 hours of hemoperfusion each day for 3 consecutive days. clinical features at the acute phase, relapses in a year and renal involvement at 1, 3, 6, 12 months were compared. the blood levels of iga and such cytokines as, tnf-alpha-il-1, il-6, ltb4 were measured by elisa technique before and 1, 2, 3 days after hemoperfusion. results: hemoperfusion significantly reduced the severity and duration of abdominal and joint pains. for patients with renal involvement at acute phase the hp group had quicker relief than the control (log rank p=0.000). for patients without renal involvement at acute phase the hp group had less occurrences of renal involvement compared with the control during follow-ups(log rank p=0.001). the rash was also alleviated after hemoperfusion though the difference was not significant. patients' levels of iga were elevated compared with the healthy control before treatment ( day0) patients'levels of cytokines also significantly increased compared with the healthy control on day0, they began to decrease 1 day after hemoperfusion and there were significant differences among those groups. less relapses were seen in the hp group compared with the control (p=0.011). conclusion: hemoperfusion combined with corticosteroid was more effective than corticosteroid alone in improving extrarenal symptoms, alleviating and preventing renal involvement. the effects may be achieved by iga and cytokine reduction after hemoperfusion. the treatment of henoch-schonlein purpura nephritis in children combined with mycophenolate mofetil and corticosteroid nephritic syndrome type, 13 cases(39.4%). about histological degrees, 32 cases were in grade 3 (mesangial proliferation, all accompanied by <50% crescent formation), 1 case was in grade 5 (moderate to severe mesangial proliferation, accompanied by 88% crescent formation). treated with new therapy, 30 cases were complete remission (91%), 2 cases were improved, and one died. the complete remission rate of this therapy was significantly higher than that of the past treatment. objective: steroid pulse therapy (spt) has been reported to be effective for improving urinary abnormalities and preventing renal deterioration in patients with iga nephropathy. however, some patients are refractory to spt or have relapse after spt, resulting in renal impairment. the efficacy of tonsillectomy combined with spt has been discussed. the aim of our study is to evaluate the efficacy of tonsillectomy combined with spt in childhood iga nephropathy. methods: 30 children, aged 3 to 14 years, who had been followed up for more than 3 years after the first biopsy between 2001 and 2009, were enrolled in this study. all patients received spt (methylprednisolone, 20~30 mg/kg intravenously 3 times per week for consecutive 3 weeks). 8 patients received tonsillectomy combined with spt (group a), and 22 patients received spt alone (group b). tonsillectomy was performed one month before or after spt. the therapy was followed by oral prednisolone and tapered off for 12 to 24 months, with warfarin, dipyridamole and ras inhibitor. all of 30 patients underwent repeat biopsy. clinical features and pathological findings were retrospectively analyzed. results: the disappearance rate of proteinuria was 87.5% for group a vs 100% for group b at 1 year, and that of hematuria was 100% vs 72.7% at 2 years. for group a, all the cases which of urinary abnormalities had improved by the treatment could maintain remission, but for group b, 27.3% of the cases had recurrence of proteinuria and 18.2% had recurrence or did not achieve disappearance of hematuria at the last observation. histlogically, the percentage of glomeruli that showed crescents was significantly reduced in both groups. but the cases which still showed crescent formation at the second biopsy were 0% for group a vs 22.7% for group b. conclusion: the differences of recurrence rate for urinary abnormalities after the treatment and disappearance rate of crescents at second biopsy may suggest that tonsillectomy can improve the long-term prognosis. objective: to observe the efficacy and security of tripterygium wilfordii polyglycosidium (twp) in treating children with severe henoch-schonlein purpura nephritis (hspn). methods: 25 children failed to intravenous pulse methylprednisolone for 6 days for their severe hspn entered our study and were given oral twp for 3-6 month. we evaluated the efficacy and security of twp through examinations of urinalysis, blood routine, function of liver and renal, myocardial enzyme series, level of sex hormone and electrocardiogram (ecg) before treatment, 1-2 month after giving twf and 4 weeks after stopping medication respectively. results: proteinuria disappeared in all patients. the remission time was 4 weeks in 22 children, 12 weeks in 2, 15 weeks in 1 respectively. during treatment, elevated glutamic-pyruvic transaminase and glutamic-oxaloacetic occurred in 2 patients and returned to normal after stopping medication. abnormal findings in examination of ecg were observed in 2 patients, one with left ventricular high voltage, another with knot parallel rhythm of the heart. after stopping medication, one patient still had t wave change in ecg. we didn't find any abnormalities of blood routine, renal function and level of sex hormone. conclusion: the twp has good efficacy and security on the treating children with severe hspn. the efficacy and side effects of glucocorticoids in treating children with henoch-schonlein purpura kai-yun liu, ling lu pediatrics, irst affiliated hospital of anhui medical university, anhui, china objective: to observe the efficacy and safety of glucocorticoid in the treatment of children with henoch-schonlein purpura. methods: 50 patients with hsp were divided into mild and severe group according to clinical manifestations. 30 children in mild group were given hydrocortisone sodium succinate (hcss) therapy. 20 children in severe group were given methylprednisolone ( mp ) therapy. a follow-up record system was set up for every patient. we observed the recovery time of clinical symptoms and side effects of glucocorticoid. results: symptoms of skin rashes, joint and gastrointestinal in all children were disappeared within 3 months of treatment. the urinalysis in 13 of 14 patients with renal involvement returned to normal in 4 weeks. the other patient failed to mp therapy was given oral tripterygium and got normal urinalysis in 8 weeks after disease onset. the body mass index (bmi) values in all children after glucocorticoid treatment were significantly higher than before treatment. after stopping medication, there was no significant difference in bmi value compared with before treatment and between two groups. during the treatment, 1 patient in each group had ecg abnormal, and their ecg returned to normal after stopping drug. we didn't find any abnormalities of blood routine, renal function and level of sex hormone. objective: to investigate the role of th17/treg imbalance in the pathogenesis of childhood henoch-schonlein purpura(hsp). and further explore the immunomodulatory affects of compound/lycyrrhizin(gl) on the th17/treg deviation in hsp. methods: 31 hsp patients were chose as gl-treated group, another 5 patients were used as conventional therapy group. besides (0.5-2)ml/kg/l was enjected in l-treated group, other treatments were same, any steroid or immunosuppressants treatment was prohibited. 15 age and sex-matched healthy children were used as healthy controls. blood samples of patients were obtained at the acute stage and after 5 days treatment. using intracellular staining, the frequency of peripheral cd3+cd8-il17+(th17) and cd4+cd25+foxp3+ treg cells was detected by flow cytometry all patients received methyl-prednisone pulses iv (1 g/1.73 m2 for 3 consecutives days) and subsequently oral prednisone with gradual withdrawal in six months, and mmf at dose of 10-20 mg/kg/12 h for 24 months. results: after six months of therapy all patients but one had persistent microscopic hematuria (me), only 3/11 patients had recurrent ma. patients with grade ii hspn showed significant reduction or normal proteinuria. only 3/11 patients with grade iii hspn had proteinuria still >1gr/24 h, 4/11 patients had proteinuria <1g/24 h, in 4/11 proteinuria was normal. mean period of follow-up was 3 years. after one year, only 1 patient had recurrent ma, 10 patients showed persistent me. two patients with persistent proteinuria >1 g/24 h showed a significant reduction (< 1g/24 h in 2 pts, normal in 1 pt). proteinuria did not reappear in all patients.the effect persisted after 2 years and after therapy withdrawal. no side effects was recorded. conclusion: our experience shows the efficacy and safety of mmf in hspn and has to be confirmed by larger controlled studies. the analysis of blood perfusion therapeutic effect on 45 cases of allergic purpura cheng guo-qiang xi 'an children's hospital urology department, xi'an, shanxi, china objective: to observe the auxiliary therapeutic effect on severe allergic purpura. methods: 45 children with severe allergic purpura were randomly divided into the control group (19 cases) and the treatment group (26 cases). the children of control group were treated with conventional therapy (anti-inflammatory, anti-allergic, anticoagulation and symptomatic treatment). the children of treatment group were treated with conventional therapy and blood perfusion. two groups of patients were both treated for 14 days, and were followed up for 9 months. the amelioration time and the regression time of clinical symptoms such as abdominal pain, bloody stool, rash, joint symptoms, visible hematuria and proteinuria, was compared in two groups before and after the treatment. the amelioration time of the clinical symptoms of children in the treatment group, such as acute abdominal pain, bloody stool, rash, joint symptoms, abdominal pain and bloody stool, visible hematuria, proteinuria, and other clinical symptoms, was shorter than the time in the control group (p < 0.05), and the late symptoms such as joint symptoms, abdominal pain and bloody stool iterations was also less than those of the control group (p < 0.05). methods: iga nephropathy model was established in male sprague-dawley rats. at week 10, rats in the model group were randomly assigned to either remain in the model group (n=5), or to receive treatment with rednisone (n=6), 1,25(oh) 2 d 3 group (n=6), or prednisone plus 1,25(oh)2d3 (n=6). at week 12, serum interleukin-17 level was detected by elisa, and the level of treg cells in the blood was assayed using a flow cytometry method. results: (1) the proteinurine and the number of blood cells in urine from rats in the model group was significantly higher than in rats in the control, prednisone, 1,25(oh)2d3, and prednisone plus 1,25(oh)2d3 treatment groups (p < 0.01). (2) serum interleukin-17 in the model group was significantly increased compared with control and treatment groups (p < 0.01), and the levels were decreased in turn in the prednisone treatment group, 1,25(oh)2d3 treatment group, and prednisone plus 1,25(oh)2d3 treatment group. in addition, the 1,25(oh)2d3 and prednisone plus 1,25(oh)2d3 treatment groups showed lower levels than the prednisone treatment group (p < 0.05). (3) the level of treg cells in the model group was significantly decreased compared with the control and treatment groups (p < 0.01), with the levels showing a slight increase in the prednisone treatment group, a larger increase in the 1,25(oh)2d3 treatment group, and the greatest increase in the prednisone plus 1,25(oh)2d3 treatment group when compared with the model group. furthermore, the 1,25(oh)2d3 group and prednisone plus 1,25(oh)2d3 treatment groups showed higher levels than the prednisone treatment group (p < 0.01). conclusion: a th17/treg disorder exists in rats with iga nephropathy, with the levels of interleukin-17 increased and the levels of treg cells decreased. vitamin d3 can regulate the th17/treg balance and reduce the level of protein and blood in the urine in rats with iga nephropathy. objective: to explore the therapeutic effect of prednisone (pred) combined with mycophenolate mofetil (mmf) and cyclosiporin a(csa) in children with severe purpura nephritis (hspn). methods: we collected 6 patients with severe hspn (from iskdc iiia to i) whose proteinuria symptom of nephrotic syndrome did not significantly relieve after 4 weeks which treated with oral pred and mmf (20-30 mg/(kg.d)); and (or) the gross hematuria did not disappear after 2 courses which treated with large doses of methylprednisolone. all patients were treated with oral csa 2-4mg/(kg.d) to induce remission therapy for 3-6 months, then gradually reduced csa to 1-3 mg/(kg.d). the follow-up was 8-30 months. results: combined therapy with immunosuppressant treated a month later, gross hematuria were disappeared in all 5 patients, 1 patient (17%) was partial remission (urinary protein and urine erythrocyte continued to decrease 25%-49% than before),5 patients (83%) were significantly remission (urinary protein and urine erythrocyte continued to decrease over 50% than before), 24-hour urinary protein was 30.50±19.35mg/kg, urine erythrocyte was 15.23±11.39×10 4 /ml.3 months later, 5 patients (83%) were significantly remission, 1 patient was complete remission (urinary protein was completely negative, urinary sediment red blood cells <8000/ml), 24-hour urinary protein was 18.67±10.10mg/kg, urine erythrocyte was 10.71±8.55×10 4 /ml. 6 months later, 5 patients (83%) were significantly remission, 1 patient was complete remission, 24-hour urinary protein was 7.66±6.31mg/kg, urine erythrocyte was 5.18±3.81×10 4 /ml. 4 patients were complete remission at last and the median duration of complete remission was 8.5±4.93 months. 2 patients had not been reached complete remission, which follow-up was 8-9 months. 3 patients were relapse during withdraw or reduction, other side effects included hairy (4 patients objective: increasing evidence suggest that the cosmc(1-beta-3galactosyltransferase-specific molecular chaperone ) might play a vital role in the pathogenesis of iga nephropathy (igan). however, the mechanism is not clear. methods: we investigated whether cosmc gene( c1galt1c1 )methylation is an important mechanism in igan in children. methods 33(m:f=25:8) primaryigan children and 39(m:f=28:11) healthy controls were enrolled randomly. the methylation status of the c1galt1c1 was detected using bisulfite-specific polymerase chain reaction (bsp)-based sequencing analysis. results: the male patients had obviousely high methylation percentage than the male controls(p=0.047). three significant hypermethylation sites were identified in the male patients: cpg site 20 (p = 0.034), site 21 (p = 0.039), andsite 26 (p = 0.047). in the female groups,the patients and the normal controls all had high level methylation without significance (p=0.947). conclusion: methylation of c1galt1c1 might have the vital significance in the susceptibility to the male igan patients. objective: the aim of this research is to investigate the value of serum and urinary tgf-β1and mmp-9 level, the noninvasive and easy repeatable biomarker, for assessing the severity and disease progression in igan children. methods: 54 children with biopsy-proven igan and 55 normal children(control group) were enrolled between july 2009 and january 2013.(fingure 1)the igan group was divided into 3 clinical groups according to their clinical features: isolated hematuria group (ih group, 20 patients), hematuria and proteinuria group (hp group, 16), and nephritic syndrome group (ns group, 18). patients were divided into two groups according to their lee's pathologic classification: grade i+ii (lee's i+ii, 39 patients), grade iii+ iv (lee's iii+ iv ,15) groups. igan group was classified according to the renal function into two groups(renal failure group vs normal group). igan group was also divided into four groups according to their immunophenotype: iga(7patients),iga+igg(9),iga+igm(10),iga+igg+igm(28) group. results: the level of tgf-β1 and mmp-9 inserum and urinary of the igan group was significantly higher than that in the control group (p<0.05). both the level of tgf-β1 and mmp-9 inserum and urinary of the three clinical groups in the igan children were not statistically different. the serum tgf-β1 and mmp-9 levels were significantly higher in lee' s iii+ iv group compared to that in lee's i+ii group(p<0.05). the difference was not statistically significant between urinary tgf-β1 and mmp-9 levels in the two lee's group.the serum tgf-β1 and mmp-9 levels of renal failure group were significantly higher than that of normal renal function group in igan children(p<0.05),and the urinary tgf-β1 and mmp-9 levels of the two group were not statistically significant different. the serum and urinary tgf-β1 and mmp-9 levels were not statistically different in all immune classification. conclusion: the serum tgf-β1 and mmp-9 plays an important role in the severity and progression of children with igan. tgf-β1 and mmp-9 may be two more noninvasive and easy repeatable biomarkers to be used in evaluating the progression of igan in children. abstract# p-sat163 glomerular ace2 expression is enhanced in pediatric iga nephropathy yusuke seki, maki urushihara, takahiro tayama, takashi nagai, ariunbold jamba, shuji kondo, shoji kagami department of pediatrics, the university of tokushima graduate school, tokushima, japan objective: angiotensin converting enzyme (ace)2 is a homolog of ace and is thought to be a potent counter-regulator against ace activity. while renin-angiotensin system (ras) plays a critical role in the progression of iga nephropathy (igan), the role of ace2 has not been investigated in pediatric patients with igan. this study was performed to examine the relationship between ace2 expression and the development of pediatric igan. methods: we performed immunohistochemical analysis of ace2 and ace in kidney tissues from 39 patients with pediatric igan and 14 patients with minor glomerular abnormalities (mga) using specific antibodies and examined the correlation with clinical parameters and pathological findings. in addition, we elucidated the effects of various cytokines on ace2 expression in cultured human mesangial cells (mcs) by quantitative real time pcr and western blot analyses. results: ace2 expression levels in glomeruli (r = 0.5732, p < 0.0001) and tubules (r = 0.4917, p = 0.0002) were positively correlated with the mesangial hypercellularity score, while ace expression levels in glomeruli (r = 0.1331, p = 0.3420) and tubules (r = 0.0743, p < 0.5959) are not. multiple regression analysis showed that the mesangial hypercellularity score correlated with ace2 expression level in glomeruli and urinary protein-creatinine ratio (r = 0.6295, p < 0.0001). in igan patients not treated with a ras blocker (ace inhibitors or angiotensin ii type 1 receptor blockers), ace2 expression levels in glomeruli were significantly increased compared to patients with mga (p < 0.0001). iga patients treated with a ras blocker did not show this increase in ace2 exrepssion. furthermore, ace2 mrna and protein expression was enhanced by interleukin-1 beta, a pro-inlammatory cytokine, and suppressed by transforming growth factor-beta 1 , a pro-fibotic factor in the progression of igan. conclusion: these data indicate that ace2 expression in glomeruli is associated with mesangial hypercellularity in the active phase of pediatric igan. abstract# p-sat164 increased serum interleukin-17 and peripheral th17 cells in children with henoch-schonlein purpura nephrology (hspn) xiaoshan shao nephrology, guiyang children's hospital, guizhou, china objective: interleukin (il)-17 and th17 cells have been involved in many autoimmune diseases. the aim of this study is to investigate the involvement ofil-17 and th17 cells in the pathogenesis of childhood henoch-schonlein purpura (hspn). methods: serum and supernatant levels ofcytokines and chemokines were analyzed by enzyme-linked immunosorbent assay (elisa). using intracellular staining, the frequency ofperipheral th17 and th1 cells was studied by flow cytometry. results: children with hspn had significantly higher serum levels of il-17, il-6and transforming growth factor-beta than healthy controls. the il-17 levels in culture supernatants of peripheral blood mononuclear cells with anti-cd3 and cd28 antibody stimulation were much higher in patients with hspn (212.2 +/− 71.4 vs. 34.7 +/− 12.6 pg/ml, p = 0.021). thepatients also had more th17 cells (1.47 +/− 0.23% vs. 0.61 +/− 0.10%, p = 0.012) but not th1 cells in peripheral blood. moreover, il-17 could promote human endothelial cells to produce chemoattractants il-8 and monocyte chemotactic protein-1. the increased frequency of peripheral th17 cells and serum il-17 levels are shown in childhood hspn that may in part contribute to vascular inflammation, suggesting cellular immunity is likely to be involved in the process of hspn. expression and significance of tgf-beta, smad7, fn, ub and smurf2 in the renal tissues of children with iga nephropathy chen li-zhi, jiang xiao-yun, ling yi-hong, mo ying, sun liang-zhong pediatrics, the first affiliated hospital of sun yat-sen university, guangzhou, china objective: in this study, we investigated the expression of tgf-beta 1, smad7, snon, fn, ub and smurf2 inthe renal tissues in children with igan, in order to explore the potential cross-talk between ubiquitinproteasome pathway and tgf-beta 1 signaling and even their role in the progressive renal fibrogenesis of igan. methods: sixty children with igan were divided into three groups according to their clinical features: isolated haematuria group (ihgroup, 20 patients), hematuria and proteinuria group (hp group, 21), and nephrotic syndrome group (ns group, 19) . patients were also divided into three groups according to pathologic grades: grade 2 (26 patients), grade 3 (20) and grade 4 (14) groups. six normal renal specimens (four was non-tumor kidney tissues from patients who had renal tumor and underwent nehprectomy, another two was healthmismatched donor renal tissues before renal transplantation) were used as the control group. the expression of ub, smurf2, tgf-1, smad7, snon, and fn in renal tissues were determined by immunohistochemistry (two-step powervisiontm) and semi-quantitatively analyzed by the software of image-pro plus 6.0. the degrees of glomerular and tubulointerstitial lesions were scored according to the katafuchi semi-quantitative criteria. results: the expression of tgf-beta 1, fn, ub and smurf2 inigan kidneys was significantly higher than that in the control group(pless than 0.01), while the increased expression of smad7 was only found in glomeruli but not in renal tubular interstitium(p less than 0.01). the highest expression of tgf-beta 1, fn, and ub were found in the ns and grade 4 groups(p less than 0.01), smurf2 in the ih and grade 2 groups(p less than 0.01), while the lowest expression of smad7 was found in the grade 4 group(p less than 0.01), closely associated with different clinical and pathological groups. (p less than 0.01) conclusion: these results demonstrate that upp and tgf-beta 1/smads signaling pathway were both activated in children with igan.both upp and tgf-beta 1/smads signaling pathway may play an important role in the progress of glomerular sclerosis, renal tubular injury and renal interstitial fibrosis in children with igan. abstract# p-sat166 objective: the proteasome (ps) plays a key role in the activation of transcriptional factors, cytokines and presentation of hla-i restricted peptides. in immune mature cells, particularly dendritic cells, the ps, under the action of interferon gamma and alpha, becomes an immunoproteasome (ips), by substituting 3 catalytic units beta1, beta2, beta5 with other low molecular weight proteins (lmp2 and lmp7) and an endopeptidase-like complex (mecl-1). this modification confers an optimal catalytic property for professional presentation of specific peptides to mhc class i. our group previously demonstrated that adult patients with iga-nephropathy (igan) have an increased expression of ips catalytic subunit, which correlated with the severity of renal disease.aim of this study was to investigate the switch from ps to ips in children with primary igan and with henoch-schoenlein purpura (hsp), a vasculitis sharing with igan several immune system abnormalities. methods: peripheral lymphomonocytes (pbmc) from 18 children with igan, 57 with hsp and 33 healthy control subjects (hc), isolated by centrifugation gradient, were tested with real time prc (taqman) to assess quantitatively the mrna levels of ps alpha subunit (constitutive), of the active subunit of ps (beta1, beta2, beta5) and of ips (lmp2, lmp7 and mecl-1). results: objective: recent studies suggest that dysregulated innate immunity plays an important role in the pathogenesis of iganephropathy (igan). interleukin-20 subfamily and its receptor, interleukin-22 receptor alpha-1 (il-22r1), were recently identified as immunomodulators in human diseases, acting as mediators of mucosal host defense. however, the potential role of il-22r1 in the pathogenesis of igan has not been explored. methods: in the current study, one hundred ninety four patients with igan and 287 normal controls were genotyped for coding polymorphisms of the il-22r1 gene and the association between the polymorphisms and igan was investigated. local expression of il-22r1 was examined in patients with igan and healthy controls using immunohistochemistry. results: our case-control analysis showed that genotypes of rs3795299 were associated with childhood igan. individuals with the cc genotype of rs3795299 had about three fold reduced risk of igan compared with those with the gg genotype in the codominant model (p=0.0028) and those with the genotypes containing g allele (gg or gc) in the recessive model (p=0.002).after bonferroni correction, the association between rs3795299 cc genotype and reduced risk of developing igan remained significant. furthermore, the renal expression of il-22r1 was significantly higher in healthy controls compared with subjects with igan. conclusion: our data suggest that the cc genotype of rs3795299 polymorphism in il-22r1 gene is associated with the reduced risk of igan, and this genetic association was supported by the higher renal expression of il-22r1 in healthy controls compared with patients with igan. abstract# p-sat168 toll-like receptor 9 gene polymorphisms contribute to development of proteinuria in childhood iga nephropathy henoch-schoenlein purpura (hsp) is the most common form of immune-mediated systemic vasculitis in children, which mainly affects skin, joints, gastrointestinal tract and kidney. the overall prognosis of hsp is favorable, but the long-term outcome is dependent on the degree of renal involvement. the incidence of renal involvement varies from 20 to 60% and there have been some reports showing that nephritis might be related to an older age at onset, persistent purpura (> 1 month), severe abdominal pain, and relapsing disease.recently, several studies have shown that galactose-deficient iga1 (gd-iga1) is recognized by anti-glycan antibodies, resulting in the formation of the circulating immune complexes and their mesangial deposition causing renal injury in hsp nephritis and serum galactose-deficient iga1 levels were highly inherited in children with hsp nephritis.regarding the treatment of hsp, one randomized double-blinded controlled study recently showed that patients with abdiminal pain or arthralgia may benefit from early treatment with prednisone, but the drug has not been proven to be capable of preventing the development of renal symptoms. however, it was effective in altering the course of renal involvementmild henoch-schoenlein purpura nephritis (hspn) generally does not require aggressive treatment due to a favorable course of the disease, but severe nephritis has a high risk of progression to end stage renal failure. although several intensive therapies, such as intravenous high-dose methylprednisolone pulse, immunosuppressive/cytotoxic drugs, fibrinolytic therapy, anticoagulants, antiplatelet agent and plasma exchange, have been used in children with severe hspn, treatment of hspn still remains controversial due to the rarity of randomized controlled studies in this field. expressions and significance of mtor, p70s6k1 and e-cadherin in glomeruli of children with iga nephropathy objective: to investigated the expressions of mtor, p70s6k1, and e-cadherin in glomeruli of children with igan to explore the role of mtor/p70s6k1 signaling pathway in the progressive renal glomeruli fibrosis of igan. methods: seventy-two children with igan were divided into three groups according to their clinical features: isolated hematuria group (ih group, 24 patients), hematuria and proteinuria(hp group, 26 patients), and nephritic syndrome group (ns group, 22 patients). patients were also also divided into three groups according to their pathologic grades: grade ii(33 patients), grade iii(25 patients) and grade iv(14 patients). six normal renal specimens were used as control group. the expressions of mtor, p70s6k1, and e-cadherin in glomeruli were determined by immunohistochemistry method and the relationship between these indexes and the clinical pathology indexes were analyzed. results: the highest expressions of mtor and p70s6k1 inglomeruli were found in the ns group, both of which in tubulointerstitium were higher in grade iii and iv group than that in grade ii group and control group while the lowest expressions of e-cadherin was found in the grade iv group. the glomerular lesion level degree was positively correlated with expressions of mtor and p70s6k1 inglomeruli. the quantitative urinary protein of 24 h was positively correlated with expressionsss of mtor in glomeruli. the concentration of serum iga was negatively correlated with the expressionss level of mtor in glomeruli. there was no significant correlation between the expressions of ang ii, mtor, p70s6k1, e-cadherin and α-sma in igan glomeruli and their courses from duration of illness as well as iga depositional strength. conclusion: the mtor/p70s6k1 signaling pathway was actived in the children with igan and it may play an important role in the progress of renal glomeruli fibrosis in children with igan. abstract# p-sat171 iga1 from hsp patients trigger apoptosis and inhibit cytoskeletal proteins in huvec objective: henoch-schonlein purpura (hsp) is the most common form of small-vessel vasculitis in children, and its etiology is believed to be associated with immune injury. a polymorphism in the gene encoding heat shock protein 70-2 (hsp70-2) is known to be associated with immune diseases. the purpose of this study was to investigate the correlation between the hsp70-2gene polymorphism (+ 1267 a/g) and hsp in children. the polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) method was used to detect the hsp70-2 polymorphism in 205 cases of children with hsp and 53 controls, and the association of this polymorphism with hsp and hsp nephritis (hspn) was analyzed. results: in patients with hsp, the a/a, a/g, and g/g genotypic frequencies at the +1267a/g position of hsp70-2 were 25.9%, 51.2%, and 22.9%, respectively. in the control group, the a/a, a/g, and g/g genotypic frequencies at the +1267a/g position of hsp70-2 were 30.2%, 60.4%, and 9.4%, respectively. thus, the g/g genotypic frequency in the hsp group was significantly higher than that in the healthy control group (χ2=4.764, p<0.05). the frequencies of the a and the g allele were 51.5% and 48.5%, respectively, in the hsp group and were 60.4% and 39.6%, respectively, in the control group. the frequencies of the a/a, a/g, and g/g genotypes in patients with hsp were 24.8%, 52.9%, and 22.3%, respectively. in hspn, the frequencies of the a/a, a/g, and g/g genotype were 27.4%, 48.8%, and 23.8%, respectively. conclusion: the +1267a/g polymorphism in the hsp70-2 gene is associated with hsp in children. the g/g homozygous genotype may be a genetic factor that predisposes individuals to hsp, but it is not significantly associated with the development of renal impairment. abstract# p-sat174 investigation of the correlation between tumor necrosis factoralpha gene polymorphism and henoch-schonlein purpura in children fei zhao 1,2 , songming huang 1, 2 , huaying bao 1 , guixia ding 1 , ying chen 1 , hongmei wu 1 , aihua zhang 1 1 nephrology, nanjing children's hospital, nanjing, china 2 institute of pediatrics, nanjing medical university, nanjing, china objective: to investigate the association between serum tumor necrosis factor-α (tnf-α) gene polymorphism and the occurrence and development of henoch-schoenlein purpura (hsp) in children. the polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) method was utilized to detect and compare the genotype and allele frequencies at the tnf-α -308 locus in 205 hsp child patients and 53 non-hsp child patients. furthermore, the relationship between the genotypic and allelic frequencies at the tnf-α -308 locus and the susceptibility to hsp and henoch-schonlein purpura nephritis (hspn) was analyzed. results: 1. the g/g, g/a, and a/a genotype frequencies at the tnf-α gene (−308g/a) locus in the hsp group and the control group were 59.5%, 32.2%, and 8.3% and 77.4%, 20.8%, and 1.9%, respectively; among them, the a/a genotype frequency in the hsp group was higher than that in the healthy control group, with a statistically significant difference (x 2 = 6.447, p < 0.05). the g and a allele frequencies in the hsp group and the control group were 75.6% and 24.4% and 87.7% and 12.3%, respectively. the a allele frequency in the hsp group was higher than that in the healthy control group, with a statistically significant difference (x 2 = 7.241, p < 0.05). 2. the g/g, g/a, and a/a genotype frequencies in the hsp non-renal impairment group and the nephritis group were 64.5%, 26.4%, and 9.1% and 52.4%, 40.5%, and 7.1%, respectively, but these differences were not statistically significant (x 2 = 4.474, p > 0.05). the g and a allele frequencies in the hsp non-renal impairment group and the nephritis group were 77.7% and 22.3% and 72.6% and 27.4%, respectively; the difference in the frequency of the a allele in the hsp non-renal-damage group and the nephritis group was not statistically significant (x 2 = 0.240, p > 0.05). conclusion: the tnf-α (−308g/a) gene polymorphism was associated with hsp in children. a/a homozygosity may be a genetic predisposing factor for hsp; however, this factor was not significantly correlated with the development of kidney damage. abstract# p-sat175 objective: the nephrotic syndrome (ns) is characterized by proteinuria, hypoalbuminemia and generalized edema. although the pathophysiological mechanisms of ns remain unknown, studies with animal models and patients have associated the ns with changes in immune response. the present study investigated the expression of molecules related to cell activation, such as the beta-2 integrin (cd18) and cd80 on peripheral blood leukocytes and renal production of reactive oxygen species in rats with ns induced by doxorubicin. meathods: male wistar rats, 250-300g, were divided into two groups: animals receiving intravenous injection of doxorubicin (7.5 mg/kg) (dox, n=32) and control animals that received saline (con, n=32). the animals were sacrificed at days 7, 14, 21 and 28 after injection, and 24 hour urine and blood samples were collected for biochemical and immunological analyzes. the phenotypic analysis of leukocytes was performed by flow cytometry. the expression of cd18 in monocytes, cd4+, cd8+ and nk cells and the expression of cd80 in monocytes were measured. in renal tissue samples, the oxidative activity was evaluated by tbars production and antioxidant activity of sod and catalase. results: the dox group animals showed significant increase in cellular cd18 expression and in the percentage of cytotoxic t lymphocytes, nk cells and monocytes that expressed cd18 as well as raised cd80 expression on peripheral blood monocytes when compared to the con group. the increased production of reactive oxygen species in renal tissue of dox group animals was positively correlated with the cd80 expression on monocytes and serum levels of creatinine. conclusion: these findings indicate a potential link between increased activation of peripheral monocytes and kidney damage in animals with ns. additional studies analyzing the effects of the blockade of integrins and co-stimulatory molecules may offer new therapeutic opportunities to treat human ns. abstract# p-sat182 cyclosporine a protects podocyte via upregulating the expression of cofilin-1 li xiaoyan, zhang xiaoyan, li xuejuan, wang xuejing, wang suxia, ding jie pediatric, peking university first hospital, beijing, china objective: podocyte foot process is dysregulated in nephrotic syndrome. the effacement of podocyte foot processes typically arises owing to perturbations in the actin cytoskeleton. calcineurin inhibitor cyclosporien a (csa) is currently used in the treatment of nephrotic syndrome. recent data suggest that the effects of csa on nephrotic syndrome are independent of its effects on the immune system. they identified that csa can stabilize the actin cytoskeleton through stabilizing synaptopodin in podocytes and thereby reduce proteinuria directly. other studies also showed that csa induced cofilin phosphorylation and promoted stress fiber generation in proximal tubular cells. however, whether the antiproteinuric role of csa is played by regulating cofilin-1 in podocyte has not been studied. methods: acute podocyte injury and nephrotic syndrome were induced by puromycin aminonucleoside (pan) injection in rats with or without csa. cultured podocytes were exposed to pan with or without csa treatment. cofilin-1, nephrin, synaptopodin expression were determined by western blot or immunofluorescence. the cofilin-1 specific effect was determined using cofilin-1 sirna. results: csa reduced proteinuria, restored expression of cofilin-1, nephrin, syanptopodin and repaired foot process effacement of pan induced nephropathy in vivo. in vitro studies showed that exposure of csa restored the expression of cofilin-1 and nephrin which decreased by pan. csa also repaired actin cytoskeleton impaired by pan. the protective effect of csa was disappeared partially when cultured podocytes were exposed to cofilin-1 sirna. objective: to find a probably mechanism of immune disorder in rsv nephropathy in rats which resembled the change of minimal change nephrotic syndrome. methods: 1. rsv mrnas (g, f, m2, ns1, ns2) and proteins (g and f) were detected with fluorogenic quantitative rt-pcr and indirect immunofluorescence assay (ifa) to find the evidence of viral persistence. 2. dc-sign mrna and protein were detected with rt-pcr and ifa to find the trend of its change. 3. the levels of il-12/il-10 and cd4/cd8 were measured by elisa and flowcytometry. results: the course could be divided into two stages: 1. first stage (4d-14d) the mrna of rsv f, g, m2, ns-1 and ns-2 in kidneys, lungs and spleens expressed and arrived at the highest on 14d, which accorded with the expression of rsv f and g proteins in ifa. the positively fluorescent luminance and mrna of dc-sign expressed stronger than that in the control, especially from 8d (ct kidney =18.50±3.12) to 14d (ct kidney =11.40±2.07). compared with the control, the secretion of il-12 in renal tissues was rising from 4d (1205.46±262.07pg/ml) to 14d (1054.68±154.18pg/ml). the expression of cd4 and cd8 in peripheral blood was lower than that in normal control. 2. second stage (30d-120d) rsv mrna and viral proteins in the tissues were persistently expressed; it showed that partial rsv survived through immune escape. after 30d, the fluorescent luminance of dc-sign was gradually reduced in the three tissues. the expression of dc-sign mrna in kidneys, lungs and spleens on 60d (ct kidney =14.10±8.32) and 120d (ct kidney =14.92±1.80) was higher, except that in spleens on 30d (ct= 21.90±1.43) was significantly lower than the control. the secretion of il-12 in tissues decreased after 30d (583.40±199.26pg/ml), but that of il-10 gradully increased after 30d (3832.38±88.20pg/ml). although the expression of cd4 and cd8 in peripheral blood was increased gradually after inoculation, the level was still lower than that in normal control. conclusion: there was rsv persistence in rats which likely attributed to the rsv-induced immune escape. rsv interacts with dc-sign to make dcs inhibit the immune ability of cytotoxic t cells, caused t cell immune dysfunction and finally induced the immune tolerance. abstract# p-sat185 evaluation of the migratory and regulatory profile of leukocytes from peripheral blood in pediatric patients with idiopathic nephrotic syndrome objective: dynamic regulation of the podocyte cytoskeleton plays an important role in maintaining the glomerular filtration barrier. severe glomerular disorders are associated with perturbed organization of the podocyte actin cytoskeleton. the therapy is based on immunosuppressive agents, in particular cyclosporine a, whose beneficial effect seems also related to the stabilization of the actin cytoskeleton in podocytes. previous studies of our group demonstrated, that the mtor inhibitor everolimus (ev) might similarly involve the recovery of the actin cytoskeleton in a puromycin (pan) experimental model of proteinuric disease. methods: in the present study, inhibitory effects of ev on mtor complex 1 (mtorc1) and −2 (mtorc2) were analyzed by western blotting in human differentiated podocytes. downstream of mtorc2 the activity of rhoa (gtpase pull-down assay) and myosin light chain (mlc) was studied. to identify further signaling pathways affected by ev, we performed affymetrix microarray expression analysis followed by verification using real-time rt-pcr. results: biochemical studies revealed a substantially decreased phosphorylation level of both mtor effector proteins mtorc1 (reduced p-akt) and mtorc2 (reduced p-p70s6k) by ev. objective: notch constitutes an evolutionarily conserved intercellular signaling pathway that determines cell fate in various organs. activation of notch1 and notch2 has been implicated in human glomerular diseases. notch1 was reported to play a critical role in development of glomerular diseases; however, a function of notch2 remains unclear. our aim of the study is to clarify notch2 pathway's contribution to developing proteinuria and glomerulosclerosis. methods: we injected either jagged1 antagonistic antibody (mab) or notch1 agonistic mab or notch2 agonistic mab intraperitoneally to mice with adriamycin (adr) nephropathy, a model of nephrotic syndrome and focal segmental glomerulosclerosis, and evaluated the levels of proteinuria and the ratios of sclerotic glomeruli. next, we treated cultured podocytes with adr in the presence or absence of notch2 agonistic mab, and we assessed the effect on cell survival and examined the pathways involved. then, we evaluated a correlation between notch2 activation and podocyte loss in human kidney specimen of nephrosis, minimal change disease (mcns) and focal segmental glomerulosclerosis (fsgs). results: administration of notch2 agonistic mab ameliorated nephrosis and glomerulosclerosis in mice with adr nephropathy, even when notch2 agonistic mab was administered therapeutically after the onset of nephrosis. in vitro, notch2 agonistic mab protected adr-damaged podocytes from apoptotic cell death. the specific knockdown of notch2 led to increased apoptosis in damaged podocytes. notch2 rescued damaged podocytes from apoptosis through akt pathway. human kidney specimens of nephrosis showed a positive linear correlation between the number of podocytes expressing activated notch2 and the number of residual podocytes. the glomeruli with mcns showed more activated notch2 and more podocytes, conclusions: whereas those with fsgs showed less activated notch2 and less podocytes. notch2 pathway has a pivotal role in preventing apoptosis of damaged podocytes. specific activation of notch2 may represent a novel clinical strategy for the amelioration of nephrosis and glomerulosclerosis. abstract# p-sat191 proliferation and apoptosis of tubular cells in initial and advanced stages of focal segmental glomerulosclerosis (fsgs) objective: to analyze processes of proliferation, apoptosis and ciliogenesis in proximal tubular cells during initial and advanced stages of fsgs associated with cystogenesis. methods: normal kidney tissues and tissues of fsgs kidneys were immunohistochemically analyzed using ki-67 proliferation marker and caspase-3 apoptotic marker. diameters of dilated and cystic proximal tubules were measured and correlated with proliferation index of tubular cells. data were analysed by the kruskal-wallis and dunn's post hoc test and expressed as mean+/−sd. significance was accepted at p<0.05. results: normal kidney tissue showed absence of proliferation in proximal tubules. initial stages of fsgs were characterized by intermingling areas of healthy non-proliferating proximal tubules, and pathologically changes, mildly dilated (15um diameter) proximal tubules showing proliferation index of 15.92%. advances stages of fsgs displayed different stages of cystogenesis: while mildly dilated tubules (diameter 12+/−1.4um) contained 36,18% of proliferating cells, their number increased to 45,23% in cysts with diameters from 21+/−1.8um to 54+/−1.1um, and then decreased to 13,76% in cysts with diameter 73+/−2.1um. while in the largest cysts proliferation index was lowest and the primary cilia short and distorted, in distal tubules and collecting ducts, primary cilia were extremely long and branching. apoptotic caspase-3 positive cells were observed within the tubular and interstitial cells. conclusion: deterioration of proliferation and apoptosis, and primary cilia formation characterizes cystogenesis in fsgs kidneys. we suggest that changes in primary cilia length might cause alterations in transfer of signaling pathways which control proliferation, differentiation and apoptosis of proximal tubules cells. advanced stages of fsgs are associated with cyst formation and increased proteinuria leading to nephrotic syndorme objective: to investigate effect of prednisone on the expression of ezrin and neph1 in rats with adriamycin-induced nephrosis. methods: adr model was induced by a tail intravenous injection of adr.the rats were divided into three groups, which were control group, model group and prednisone group. serum index and 24h urinary protein were measured at 4 and 8 weeks. observe pathologic changes of renal tissues at 8 weeks. the expression of ezrin and neph1 in glomerulus was evaluated by immunohistochemistry respectively. results: compared with control group, at 4 weeks, 24h urinary protein and total cholesterol in model group and prednisone group were significantly increased(p<0.05),albumin and total protein were significantly decreased(p<0.01),which indicated that the model was successfully established;compared with control group, at 8 weeks, the expression of ezrin and neph1 in the model group were significantly decreased(p<0.01).compared with the model group, the expression of ezrin and neph1 in prednisone group were significantly increased(p<0.01),24h urinary protein and total cholesterol were significantly decreased(p<0.01),albumin and total protein were significantly increased(p<0.05), renal pathology and ecm/ga were significantly increased(p<0.01). conclusion: the expression of ezrin and neph1 were reduced in rats with adriamycin-induced nephrosis which were negatively related to proteinuria and renal pathological damage. prednisone can reduce the proteinuria and relieve the renal pathological damage by improving the expression of ezrin and neph1. objective: the activation of the complement system plays an important role in various kidney diseases, such as antibody-mediated rejection or membranous glomerulopathy. the majority of the circulating complement components are produced in the liver. but in the last decade the local production of complement components by other cells is highly debated. the aim of our study was to proof the ability of human podocytes to produce and secrete complement components. methods: immortalized human podocytes were analyzed with western blot (wb), immunofluorescence (if) or/and pcr for their ability to produce components (c1/ c1q, c2, c3, c4, c5) inhibitors (factor h, mcp, cd 55, cd59) and activators (factor b, properdin) of the complement system. secretion of components was measured in the medium, and functionality of c3 was tested in a specific c3convertase assay. stimulation of the cells was done with interferon-γ, interleukin 6 and human albumin. results: pcr-studies revealed that human podocytes express on mrna level the components c2, c3, c4, c5, the inhibitors factor h, mcp, cd 55 and cd59, and the activators properdin and factor b. on protein levels c2, c3, factor b, properdin, factor h, cd 55 and cd59 were detected. in immunofluorescence all the components showed either intra-plasmatic, perinuclear or peri-membranous distribution. in addition, we could show that podocytes secrete the factors c2, c3, c4, c5, factor b and factor h into the medium. the secreted c3 was clearly functionally active and could further enhanced by stimulation with interferon-γ. objective: to construct the eukaryotic expression plasmid and its shrna plasmids of il-17 of mice, and to investigate their ability of expression or inhibitory effects on il-17. methods: the rna from spleen cells of bal b/c mouse was reversed to cdna, and the full-length cds fragment of il-17 was amplified. the target segment was cloned into the expression vector plvx-ires-zsgreen1, then, the recombinant plasmid plvx-il-17-ires-zsgreen1 was obtained. three pairs of shrna chains targeting il-17 gene and one pair control shrna chain were designed and synthesized, then annealed to form double strand ,and inserted into the expression vector plvx-shrna2. three shrna plasmids and one control plasmid were constructed ,which were called shrna1/shrna2/shrna3/shrnac, respectively. these plasmids were identified by restriction analysis and sequencing, and then they were transfected or co-transfected into 293t cells in mediation of liposome. the transcription levels of il-17 mrna were detected by fluorescent quantitative pcr, while expression levels of il-17 protein were detected by elisa. results: restriction analysis and sequencing proved that the recombinant plasmid were constructed correctly. the plasmid of plvx-il-17-ires-zsg reen1 could expression il-17; when cotransfected with expression plasmid, il-17 expression levels were significantly reduced in the group of shrna1/shrna2/shrna3 as compared with the shrnac group, and the group of shrna1 had the least expression. conclusion: il-17 eukaryotic expression plasmid and its specific shrna plasmids were constructed successfully, and plasmid shrna1 had the best inhibitory effects. abstract# p-sat195 reno-protective effect of all-trans retinoic acid on adriamycininduced nephropathy mice xiaoli wang, jingjing cui, qiu li nephrology and immunology department, children's hospital of chongqing medical university, chongqing, china objective: to investigate the reno-protective effect of all-trans retinoic acid on adriamycin-induced nephropathy mice. methods: balb/c mice were randomly divided into nephrosis(a single adriamycin 10.5mg/kg,n=20) and control(tail intravenous injection the same amount of saline,n=10). after two weeks nephrosis divided into atra-treated(intraperitoneal injection all-trans retinoic acid 20mg/kg, three times a week,n=10) and adr nephrosis. then, we chose five mice each group randomly after treatment 6 weeks and 10 weeks to collect 24-hours-urine, blood, kidney for detection. results: all-trans retinoic acid significantly decreased 24-h urinary protein excretion, serum tc and tg(p<0.05). fas/fasl shows by immunohistochemistry and fluorescence quantitative pcr is decreased sifnificantly(p<0.01). conclusion: all-trans retinoic acid has anti-apoptosis on adriamycin induced nephropathy mice to protect kidney damage and delay fibrosis process. abstract# p-sat196 molecular mechanism of ers induced by lipid accumulation in human mesangial cells (hmcs) objective: dehydroxymethylepoxyquinomicin (dhmeq) is a novel nf-κb inhibitor that potently inhibits the dna-binding activity of nf-κb, resulting in therapeutic effects for various pathological conditions. to elucidate the pathogenetic role of nf-κb in minimalchange nephrotic syndrome (mcns), we examined whether dhmeq could ameliorate the nephrosis in mice induced by puromycin aminonucleoside (pan), which is considered to be an animal model for mcns. methods: mice were injected with dhmeq or only vehicle 2 hours before pan injection on day 0. mice without pan injection served as controls. the dhmeq or vehicle was injected on 5 consecutive days. the daily urine was collected from day 0 to day 30, and the urinary concentrations of albumin and creatinine were measured. some mice were sacrificed 7 days after pan-injection, and their serum concentrations of cholesterol, total protein, albumin and il-6 were measured and the pathological changes of the kidneys were observed. results: pan injection without dhmeq in the mice induced albuminuria which gradually increased up to day 10 and gradually decreased thereafter. in contrast, pre-treatment with dhmeq was associated with no significant increase in the amount of albuminuria during the first 2 weeks. in the serological tests, pan-injection induced an increase in the cholesterol level, a decrease in the total protein, a decrease in the albumin and an increase in the il-6 in the serum compared to controls 7 days after injection, but dhmeq ameliorated these changes. an electron microscopic analysis indicated an effacement of the foot processes of the podocytes in the pan-injected mice, but this was rarely observed in the pan-injected mice pre-treated with dhmeq. further immunohistochemical analysis showed that dhmeq can inhibit the pan-induced translocation of nf-κb from cytoplasm into nucleus. conclusion: these results suggest that dhmeq can be a candidate therapeutic agent for mcns, because the activation of nf-κb of podocytes may be associated with pan-induced nephrosis. knocking-down stim1 gene expression inhibits some podocyte molecules jingjing yan, meigui wang, siguang lu the first people's hospital, lianyungang, jiangsu, china objective: to study the effect of knocking-down stim1 on nephrin,podocin,cd-2ap and α-actinin-4 in murine podocytes in vitro. methods: conditionally immortalized murine podocyte cells were cultured in rpmi1640 medium at 33°c permissive condition.then the cells were shifted to non-permissive condition at37°c and cultured for ten-fourteen days, and were transfected with stim1 small interfering rna(sirna) using transfection reagent lipofectamine 2000.transfection efficiency was measured by flow cytometer and inhibitory effect of stim1 sirna was determined by rt-pcr and western blot at fourty-eight, senventy-two hours after transfection respectively. results: (1)the transfection efficiency of fam-sirna was about 75.5%. (2)after transfection with specific sirna,the expression levels of stim1 mrna and protein were down-regulated by 80.7% and 49.8% respectively. (3) the expression levels of nephrin,podocin and α-actinin-4 mrna were decreased by 62%, 35%, 60% respectively, whereas cd-2ap showed no-change.both podocin and α-actinin-4 protein were decreased by 45%, 20% respectively. objective: one of the most common causes of nephrotic syndromes in children is the minimal-change-glomerulonephritis (mcgn). typically, mcgn results in foot processes effacement of podocytes. their plate-shaped flattening very likely results in dysfunction of the filtration barrier and may underlie the increase in urine albumin concentrations. notably, mcgn often disappears at the onset of puberty, thus in parallel with increasing peripheral hormone levels. this is particularly true in girls. methods: podocytes were studied by western blot analysis, immunofluorescence and by radioimmunoassay for their ability to secrete and bind estrogens. results: podocytes express estrogen receptors both, estrogen receptor alpha and beta, suggesting that increasing levels of estradiol during puberty potentially maintains and restores morphological integrity of podocytes. this protective function could be mediated by stabilization of the podocyte cytoskeleton by estradiol, as evidenced in vitro by the increase in phosphorylation of cofilin, very similar to the effects of estradiol in neurons. furthermore, we show that dissociated podocytes express aromatase, the final enzyme of estrogen synthesis, and that podocytes in fact synthesize estradiol, as evidenced by measuring estradiol content in the supernatant. conclusion: as it was shown that estrogen synthesis in cells other than those in gonads becomes stimulated during puberty, an autocrine mechanism of estrogen action could underlie decreasing frequency of mcgn at the onset of puberty. overexpression of pgc-1α inhibits aldosterone-induced podocyte phenotypic changes and detachment by blocking mitochondrial dysfunction objective: to explore the role of heparanase in the pathogenesis of rat respiratory syncytial virus (rsv) nephropathy. methods: 150~200g sprague-dawley(sd) rats (n=5 per group) were inoculated with 6×10 6 (plaque-forming units, pfu) rsv and sacrificed on days 4,8,14 and 28 postinoculation (rsv 4 ,rsv 8 ,rsv l4 and rsv 28 ). five normal sd rats inoculated with dulbecco's minimum essential medium were served as normal control. the expression level of heparanase protein and mrna in kidney of each group was determined by immunohistochemical staining and real-time quantitative rt-pcr respectively. the proteinurina was measured and the relationship between the expression level of heparanase and the 24hour urinary protein was studied. results: rats with rsv nephropathy exhibited higher proteinuria by comparison with normal rats. there was a significant difference between each group(rsv 14 >rsv 8 >rsv 28 >rsv 4 ). compared with normal control, rats with rsv nephropathy showed up-regulated expression of heparanase protein in glomeruli. the expression level of heparanase protein in rsv 8 and rsv 14 group was higher than that in rsv 4 and rsv 28 group. there was a linear positive correlation between the expression level of glomerular heparanase protein and the quantity of urinary protein(r =0.783,p<0.05).compared with normal control group, the expression level of heparanase mrna in kidney f r o m r s v 4 , rsv 8 , rsv 1 4 a n d r s v 2 8 g r o u p w a s elevated(rsv 14 >rsv 8 >rsv 4 >rsv 28 ).there was a linear positive correlation between the expression level of renal heparanase mrna and the 24-hour urinary protein(r=0.725,p<0.05). the increased heparanase in kidney may be important to the loss of glomerular negative charge in glomerular basement membrane and is involved in the pathogenesis of rsv nephropathy. podocyte morphology andautophagosomes were viewed using electron microscopy. podocyte numerical density was estimated by weibel-gomez method. expressions of autophagy markers and ersassociated proteins were analyzed by western blot. results: the expression level of ers-associated protein grp78 was up-regulated from day 4 to day 21 post-injection. the results also showed that autophagosomes were massively accumulated and the autophagy marker lc3 was up-regulated in the models on day 7 and 14. furthermore, rapamycin was given to phn rats to further explore the role of autophagy in the process of complement-dependent podocyte injury. results revealed that rapamycin, which enhanced autophagy in podocyte, could reduce proteinuria, lighten podocyte lesionsand prevent podocyte loss on day 21. conclusion: taken together, our results demonstrated that ers plays an important role in completment-dependent podocyte damage, and in another aspect, autophagy induced byers can alleviate injury as a protective mechanism. this provides an important basis for a thorough understanding of the role of autophagy in the process of podocyte damage and the pathogenesis of mn. abstract# p-sat209 the cytoprotective role of autophagy under oxidative stress in human podocyte objective: autophagy is a ubiquitous catabolic process involving selective degradation of cellular components. it shows cytoprotective effects in different cell types and helps to maintain cell homeostasis. some studies demonstrated that cell stress, generated by starvation or some chemical reagents, can initiate autophagy which responsively resists harmful stimuli at the early stage. however, little is known about autophagy in human podocytes under oxidative stress. since the apoptosis and autophagy pathways share some common molecules, we investigated the role of autophagy induced by puromycin aminonucleoside (pan) in human podocytes. methods: human conditional immortalized podocytes were treated with pan, the generation of reactive oxygen species (ros) was measured by immunofluorescence. then, autophagy was assayed by immunofluorecence staining for lc3 puncta and western blotting for lc3. in addition, the mammalian target of rapamycin (mtor) and its substrates which play critical roles in autophagy inhibition were investigated for elucidating the mechanism of pan induced autophagy. podocyte apoptosis was assessed by flow cytometry (yo-pro-1/pi and active caspase 3 assays). to study the effects of autophagy on podocyte apoptosis, 3-methyladenine (3-ma) and chloroquine were used to inhibit autophagy. results: autophagy was detected in pan-treated podocytes in a dose and time dependent manner and prior to apoptosis which was accompanied with increased ros generation. lc3 aggregates were observed in the cytoplasm and the expression of lc3 ii was significantly elevated. intriguingly, phospho-mtor and its substrates (phospho-p70 s6 kinase and phospho-4e-bp1) increased when the autophagy was activated. when autophagy was inhibited by 3ma and chloroquine, podocyte apoptosis increased significantly. conclusion: pan induced both apoptosis and autophagy in human podocytes. our results suggested that this in vitro model will be useful for the study of crosstalk between apoptosis and autophagy in podocytes. autophagy may be the adaptive cytoprotective mechanism for podocytes under oxidative stress. further studies directed at identifying the role of mtor are essential. abstract# p-sat210 impact of angptl3 knock-out on adriamycin-induced nephropathy mice objective: idiopathic focal segmental glomerulosclerosis (fsgs) is associated with recurrence after transplantation due to a circulating permeability factor or factors (n engl j med, 334:878-883, 1996) . we have shown the effects of fsgs plasma and its fractions on glomerular permeability in vitro and in vivo and have used state-of-the-art proteomics to identify cardiotrophin-like cytokine-1 (clcf1), a member of the il-6 family, as a candidate for the active substance. we hypothesize that a new model of fsgs can be based on the effects of clcf1 in mice. methods: rclcf1 (r&d systems) was injected intraperitoneally (ip), one dose, 10 μg/kg, or infused by minipump for 28 days, 40 μg /kg/day. a construct containing clcf-1 was administered by electroporation. all studies were done in c57bl6 mice. urinary albumin/creatinine, pjak1, pstat3, patk, and perk in peripheral blood cells (pbc) and in kidney homogenate were measured and glomerular histology was assessed. results: albuminuria was induced promptly by rclcf1 after either injection or electroporation. peak albuminuria occurred by 7 days of expression and was 3-5 fold increased vs. baseline. ip administration of rclcf-1 increased pjak2 and pstat3 of pbc within 15 min. and renal pjak1 and pstat3 remained upregulated for at least 72 hours after injection. kidney pjak2 and pstat3 as well as perk/12 and pakt were markedly increased after 28 days of infusion. mesangial matrix was increased at that time. conclusion: we conclude that clcf1 mimics many of the renal effects of the active fraction of plasma from patients with idiopathic fsgs and may play an important role in its etiology. its relationship to other candidates such as circulating urokinase receptor (supar) is not known. a murine model based on administration or overexpression of clcf1 may permit us to define mechanisms of injury and to test potential therapeutic agents prior to use in clinical trials. over-expression of myo1e in mouse podocytes enhance cellular endocytosis, migration, and adhesion objective: to investigate the effects of tacrolimus (fk506) on hepatocyte growth factor and transforming growth factor β1 in kidneys of fsgs rats. methods: establish unilateral nephrectomy combined adriamycin double tail vein injection of fsgs model in rats divided to model group and treatment group ,then treatment group rats were treated withtacrolimus 8 weeks. the protein expression and site of hgf and tgf-β1 in renal of each group were assayed by western blot and immunohistochemistry after animals were sacrificed. urine protein, serum albumin, blood lipoids and kidney function were tested automatic biochemical analysis system. results compared with the normal control group , model group and treatment group rats 24h urinary protein excretion , serum creatinine , blood urea nitrogen , cholesterol significantly increased serum albumin significantly reduced severe renal pathological changes and tgf-β1 protein expression was significantly increased , and the differences were statistically significant ( p <0.05 ) ; compared with the model group , in experiments 9 weekend , the treated rats 24h urinary protein excretion , relevant serum biochemical indicators of renal pathological changes varying degree of improvement in renal tissue hgf protein expression was increased and tgf-β1 protein expression was decreased , the above differences were statistically significant ( p < 0.05 ). objective: the cellular mechanisms of kidney injury caused by obstructive nephropathy are interstitial inflammation, fibrosis, and apoptosis. rodent models can be used to simulate obstructive nephropathy in the human kidney. we developed shear-thinning hydrogels for the local delivery of il-10 to abate the progression of inflammation and fibrosis that leads to ckd. methods: injectable dock-n-lock gels were developed as previously reported 1 (fig. 1) . il-10 was added to the gel or phosphate buffered saline solution at a concentration of 0.33 ug/ul. study design: eight cohorts were studied (7, 21, 35 days, n=4): healthy, sham operation, healthy injected with msa, healthy + gel, unilateral ureteral obstruction (uuo), uuo + il-10, uuo + gel, uuo + gel/il-10. 15 ul of il-10 solution, gel, or gel/il-10 was injected into the left kidney via retroperitoneal approach 3 days after the initial uuo or sham operation. histology: immunohistochemistry (ihc) was performed on paraffin sections to identify macrophages and apoptotic cells, and trichrome stain was used to evaluate fibrosis. cells and total area were quantified for ihc and total fibrotic area was quantified for trichrome. results: comparing the treatment groups to the untreated uuo, macrophage infiltration and apoptosis were significantly reduced at day 21 and 35. by day 35, adding the il-10 via gel injection reduced macrophage infiltration more than il-10 alone and il-10 alone did not reduce apoptosis. fibrosis was decreased by day 35 in all three treatment groups (fig. 2) . conclusion: injectable hydrogels were synthesized that permit facile local delivery of immunotherapy to both healthy and obstructed kidneys. renal inflammation and scarring was reduced in an animal model of ckd by using an injectable hydrogel for drug delivery. logistic regression reveled that apd was the main parameter significantly associated with surgery treated upjo cases (roc plot was 0.79). a possible threshold of 14mm apd may be used as a cut-off value of surgery treated upjo group with a sensitivity of 77% and a specificity of 69%. conclusion: apd dilatation was the strongest predictor of surgery treated upjo. pt and renal length also significantly discriminate the two groups and correlate with apd, only with lower predictive power. our findings expand the clinical knowledge in the field of prenatal consult by highlighting a threshold of apd, which predicts the need for surgery in prenatally detected hn cases. abstract# p-sat221 survival and renal outcome in fetuses with lower urinary obstruction (luto) with and without intra-uterine vesicoamniotic shunting.a ten years experience of a cohort. objective: ureteropelvic junction obstruction (upjo) is the most common cause of hydronephrosis in children. the significant role of the surgical relieving of high grade obstruction is indisputable. nevertheless, the effect of pyeloplasty on the function of the involved kidney remains controversial, especially in upjo with significantly reduced relative renal function (rrf) before surgery. to evaluate the effect of pyeloplasty on the relative function of kidneys in children with upjo and decreased rrf. methods: the records of children who underwent pyeloplasty for upjo during a 10 year period in schneider children's medical center of israel were reviewed. the study group included 40 children who underwent pyeloplasty for upjo and had an initial rrf <40%. children with bilateral upjo, solitary kidney or other genito urinary abnormalities were excluded. the control group included 38 children with non obstructive hydronephrosis and an initial rrf<40%. results: the average initial rrf in the study group was 26% (range 8-39%), and the final renal function was 32% (1-55%). in the control group, the initial rrf was 23% (2-40%), and the final function was 22% (0-44%). the final rrf of the study group was significantly higher comparing to the control group (p value<0.05). in the subgroup of patients with upjo and initial rrf<30% (24 patients) the average initial function was 20% (8-30%) and the final function was 27% (3-55%). in the control subgroup with initial rrf<30% (26 patients) the initial average function was 17% (2-30%) and the final function was 17% (0-44%). conclusion: pyeloplasty is associated with an improvement of renal function in children with upjo and an initial rrf <40%. this is also true for patients with rrf<30% before surgery. our results support the need for pyeloplasty in children with upjo and reduced rrf. objective: common cause of chronic kidney disease (ckd) in children. kidney damage occurs in utero, and kidney disease progresses postnatally. the objective of this project was to identify clinical biomarkers, in particular antenatal variables, which predicted long-term renal outcome in boys with puv. methods: this was a retrospective cohort analysis. primary outcome was the development of end stage renal disease (esrd) as defined as starting dialysis or preemptive transplantation. clinical variables studied included antenatal factors, postnatal renal function, and modifiable variables. continuous data for the two outcome groups were compared, receiver-operating characteristic (roc), kaplan-meier, and logistic regression analyses were conducted to assess the robustness of each candidate biomarker as a predictor of outcome. results: in this cohort 15 cases reached the primary outcome of end stage renal disease at a mean age of 7.0 ± 6.7 yrs. compared to those who didn't, those who progressed to esrd had younger age at diagnosis (0.6 ± 1.5 vs 3.3 ± 7.3 yrs, p<0.05), valve ablation ( . fsgs rate was found as 60% in biopsied subjects. nephrotic syndrome was defined by edema, massive proteinuria (>40 mg/m 2 per hour or a protein/creatinine ratio >2.0 mg/mg), hypoalbuminemia (<2.5 g/dl), and hyperlipidemia. remission was defined as a urinary protein excretion below 4 mg/m 2 per hour or a protein/creatinine ratio below 0.2 mg/mg for three consecutive days. steroid resistance was accepted as no achievement of remission in spite of treatment with prednisolone, 2 mg/kg per day for 4 weeks. if steroid resistance was seen, patients were also treated with cyclosporine a (csa) (3-5mg/kg per day for least 6 months) and, thereafter, if required, with cyclophosphamide(cp) (2.5-3.0 mg/kg per day for 10-12 weeks). renal failure was defined as a glomerular filtration rate (gfr) below 80 ml/min per 1.73 m 2 body surface area, and esrd was defined as a gfr below 10 ml/min per 1.73 m 2 or the necessity for any renal replacement therapy. bidirectional dna cycle sequencing analysis of entire coding exons and adjacent intronic segments of nphs2 gene was performed.nphs2 gene mutation analysis has been also performed in 100 healthy children. results: pathogenic nphs2 mutations were found in 127 patients (18%) from the totally 700 srns children group. mutation rate was 29% in familial group, and 16.5% in sporadic group. a total of 53 mutations were determined in the nphs2 gene, and 37 of which were characterized as a novel mutation presented at hgmd databank. in the mutation positive nphs2 group, most of the mutations were found out to harbor in exons 1, 4, and 5 while no mutation were found in exon 6 of the respective gene. patients with p118l, r138q, r138x, r168h, s211a, a212t, v218g, h228d, ivs7+5g>a, c.460-467inst, c.503delg mutations were progressed to end-stage renal disease (esrd). also, age at onset of proteinuria (years) was 4.2+/-0.5 in the mutation (-) (n=573) group; 3.6+/-3.08 in the mutation (+) (n=127) group. for the mutation negative patients, effects of other disease causing genes involving nphs1, wt1, trpc6, cd2ap, and actn4 for different molecular subtypes of srns may be considered. nphs1 mutation screening was performed for all the patients who had proteinuria up to two years of age. as a causative srns gene, we should consider nphs2 gene mutation screening in early diagnosis and the follow-up of the clinical course. conclusion: in relation to homozygous or compound heterozygous nphs2 mutated patients who have the lack of response to standard steroid therapy we suggest to perform nphs2 gene mutation analysis for every child (if consent can be obtained) soon after the first episode of ns. for the newly diagnosed patients, the crucial certain determination of the causative disease gene mutation will enable clinicians to avoid redundant immunosuppressive therapeutic trials. we then performed next generation sequencing in the 5 affected patients and in one of their unaffected relatives. exome enrichment was conducted using the agilent sureselect human all exon v4+ utrs capture kit. the multiplex libraries were then sequenced on an illumina hiseq2000 instrument with a 2x76 bp read length. results: we unexpectedly identified a novel lmx1b mutation segregating with the disease in the family. subsequently, we screened 74 additional unrelated families from our international cohort of autosomal dominant fsgs and found mutations of the same residue in 2 families. none of the probands had any sign of dysplasia of nails, patellae or elbows, iliac horns or glaucoma, or any ultrastructural changes suggestive of nail-patella-like renal lesions. lmx1b encodes a homeodomain-containing transcription factor that is essential during development. a lmx1b in silicohomology model suggests the mutated residue plays an important role in strengthening the interaction between the lmx1b homeodomain and dna. both mutations are expected to diminish such interactions. conclusions: our data demonstrate that isolated fsgs could be due to mutations in genes also involved in syndromic forms and highlights the need to include these genes in all next-generation sequencing diagnosis approaches in fsgs. .in 24/37 patients mutations were identified by ngs (detection rate 65%). in 8/24 patients sequence variants were found in two different genes (33%) suggesting oligogenic inheritance. in 6 of these 8 patients the causative mutation was located in a gene following an autosomal dominant inheritance pattern. these patients showed a more severe form of the disease compared to affected family members who showed only the causative mutation without additional modifier variants. one additional patient of this cohort with pierson syndrome had two single heterozygous mutations in nphs1 and lamb2, respectively (both genes following an autosomal recessive pattern of inheritance). a further patient showed two mutations in nphs1 and the non-neutral polymorphism p.arg229gln in nphs2. structural and/or functional analysis of all mutations identified in patients with mutations in two genes suggested impaired protein function of the particular gene products. conclusion: the influence of modifier genes or digenic inheritance seems to play an important role in the pathogenicity of srns. the application of ngs is therefore of special interest and highly efficient in the diagnostics of patients with srns. abstract# p-sat232 mutation analysis in japanese patients with congenital and infantile nephrotic syndrome objective: mutations in podocyte genes (nphs1, nphs2, wt1, and lamb2) are associated with congenital (<3 months) and infantile (3-12 months) nephrotic syndrome (ns). the purpose of this study is to investigate the frequency of causative mutations in these genes in ns manifesting in the first year of life in japan. methods: all exons and exon-intron boundaries were investigated in consecutive, unrelated 37 patients from regional pediatric kidney disease centers, by pcr-direct sequencing. results: we detected disease-causing mutations in 64.9% (24 of 37) patients (75% in congenital and 25% in infantile) (table) . objective: hypercoagulability along with thrombosis are prevalent complications of nephrotic syndrome. in cases of refractory nephrotic syndrome, intracardiac thrombus, although rare, is a serious complication because of its association with morbidity and high mortality herein, we report a case of a patient with a right atrial thrombus associated with nephrotic syndrome who responded well to corticosteroid therapy and attained quick remission. case presentation: a 12-year-old japanese boy was referred to our hospital for a right atrial thrombus associated with idiopathic nephrotic syndrome diagnosed 1 month ago. the patient had attained the partial remission within the 10 days of the corticosteroid therapy initiation. he was asymptomatic otherwise at the time of admission. the patient's vitals were stable and laboratory values were almost within the normal range. an echocardiogram showed a large isolated hyperechoic mass in the right atrium, which originated from the superior right atrial wall. the mass measured 31 × 26 mm in diameter and traversed through the tricuspid valve in a to-and-fro motion. the patient underwent emergency surgical thrombectomy on day 1 and anticoagulation therapy was started 7 days after the operation. the subsequent hospital stay was uneventful, and the patient was discharged on day 87. objective: nephrotic syndrome features proteinuria and severe sodium retention which is implicated in ascites and edema formation. previous works realized in nephrotic rats (pan) clearly highlighted that, the increase of sodium absorption in the ccd is associated with an increase activity of the na/k atpase; sodium absorption is independent of aldosterone, activation of epithelial sodium channel enac is not necessary and sodium absorption is inhibited by amiloride. our hypothesis is that, in nephrotic syndrome, another apical sodium channel sensitive to amiloride and independent of saar pathway is implicated in sodium absorption in the ccd. methods: transcriptome of ccds from control rats (ct) and pan treated clamped rats (adx pan) was analyzed using a high-resolution quantitative and comparative analysis of gene expression. subsequently, transcriptional (rt-qpcr) and proteic expression (western blot and immunohistochemistry) of channel were analyzed. sodium handling in vivo and in vitro microperfused collecting ducts in different conditions (ph 6 and 7.4, apical zinc 0.3mm and amiloride 0.1mm) was analyzed. results: metabolic study confirmed previous results: sodium absorption and ascites were similar in pan and adxpan rats suggesting that sodium absorption in nephrotic syndrome was independent of saar. transcriptome analyses highlighted an increased expression of accn1 mrna only in adx pan at day 6. rt-qpcr confirmed an increased expression of accn1 only in the ccd of adx pan (about eight time compared to adx ct). subsequently, protein shows an increase expression of accn1 (adx pan 0.72+/-0.12 vs adx ct 0.31+/-0.03, p 0.03) and immunohistochemistry on microdissecated ccd highlighted a strong apical expression of accn1 only in adx pan rats. schimke immuno-osseous dysplasia (siod) is a rare autosomalrecessive multisystem disorder, characterised by: disproportionate growth deficiency, defective cellular immunity, nephrotic syndrome and progressive renal disease.siod is caused by bi-allelic mutations of smarcal1 gene, which encodes the hepa-related protein (harp), a member of the snf2 family of atpases, acting as chromatin remodelers within multi-protein complexes. a 5-year-old patient was hospitalized in november 2011, after detection of proteinuria (1.5 g/day). at our first clinical examination: height-weightgrowth retardation, thinning hair, thin and hypopigmented skin, arched palate and normal neuro-psychological development.serological examination showed proteinuria (1.7 g/day), hyperchol. (col tot 242 mg/dl, ldl-col 165 mg/dl) and lymphocyte deficiency (17.6%).renal function and complement levels were normal. lymphocyte subpopulations showed a decrease of t lymphocytes and an increase of nk cells(cd16 + cd56 +).the patient had also a detour back-lumbar scoliosis and normal kidneys in size, but with reduced corticomedullarydifferentiation. we started therapy with ramipril 2.5 mg/day. the 24 hrs urine exams performed monthly showed persistent proteinuria (500-800 mg/day). at the end of april 2012, for the persistence of proteinuria, we performed renal biopsy, which showed focal and segmental glomerulosclerosis. at discharge, she started therapy with irbesartan 75 mg/day with significant reduction of proteinuria (140 mg/day). after 6 months of therapy, thepatient did not present proteinuria and her renal function was normal. the molecular genetic study of smarcal1 gene revealed that patient was compound heterozygous for two mutations: a novel missense mutation in exon 3, inherited by mother, and a nonsense paternally-derived mutation in exon 17, leading to a truncated smarcal1 protein. our data allow us to diagnose a schimke immuno-osseous dysplasia (siod). in most patients, life expectancy is limited to childhood or early adolescence, due to the onset of stroke, infections,hematopoietic bone marrow failure and renal failure. only patients with milder and late onset forms can survive until adulthood. gain of glycosylation in integrin-alpha-3 causes nephrotic syndrome and lung disease objective: congenital nephrotic syndrome and interstitial lung disease is a rare multiorgan disorder, characterized by disrupted basement membrane structures. itga3 gene mutations were recently identified as the genetic cause of this disorder, but the disease mechanism remains poorly understood. methods: we describe a patient who presented with neonatal respiratory distress, glomerulosclerosis, proteinuria, pulmonary hypoplasia and alveolar glycogenosis, who died 7 months after birth due to respiratory insufficiency. a genome-wide screening for deletions and duplications revealed a large homozygous region that included the itga3 gene. we sequenced the gene and then conductedin vitro characterization studies to investigate the effect of the variant on the protein function. results: a novel homozygous missense mutation was identified in the coding region of the itga3 gene, which introduces an n-glycosylation motif to the protein sequence. thereby, the mutant integrin alpha-3 protein becomes hyperglycosylated. functional studies demonstrated that the conformation of integrin alpha-3 is affected and the mutant alpha-3 precursor is targeted for degradation. consistent with these findings, alpha-3 integrin was not detected in the patient glomeruli. furthermore, integrin alpha-3 protein expression was absent in murine podocytes that lack endogenous integrin alpha-3 and transfected with mutant itga3. conclusion: our findings underscore the role of the integrin alpha-3beta-1 complex as the main regulator of podocyte basement membrane integrity. here, we show that hyperglycosylation of the integrin alpha-3 subunit, causing the complete lack of alpha-3-beta-1 expression on the basement membrane, is a new pathogenic mechanism underlying congenital nephrotic syndrome and interstitial lung disease. itga3 mutation screening was directly implemented in dna diagnostics, facilitating early diagnosis, recurrence risk estimation, and genetic counselling. the clinical study on pkhd1 gene-based testing for the diagnosis of arpkd objective: in order to study the implications of pkhd1 gene-based testing for arpkd diagnosis in clinical practice, we performed a prospective study to apply pkhd1-based genetic testing to the suspected arpkd patients. methods: 12 suspected arpkd patients from 10 unrelated families were detected on mutations of pkhd1 gene by pcr direct sequencing. these patients were evaluated by combining the testing results and their clinical materials. results: in the 6 detected children, 4 mutations (p137s, v836a, q1574h, l2658x) were novel and 7 mutations (t36m, r559w, r760c, n830s, a1262v, q3899r, q4048r) were previously described. 4 of the 11 variants were definite pathogenic mutations. two pathogenic mutations in both chromosomes were found in 2 out of 6 families (30%), which greatly aid to making definite diagnoses. the 4 families with perinatal presentation consisted of 2 fetuses and 2 couples. one definite pathogenic mutation (s3457c) was found in the wife of family no.9, however, it was still difficult in conforming the diagnosis owing to lack of the pathogenic mutation from the husband. for the other families, only some potential mutations were found.two in four children with congenital hepatic fibrosis were detected two pathogenic mutations in both chromosomes, and their liver functions were obviously lower than the other two children without pathogenic mutations. conclusion: pkhd1 gene-based testing is an effective means for the diagnosis of arpkd, at the same time it could improve the understanding of this disease by analysing the testing results and their clinical materials. objective: to present a case of schimke immuno-osseous dysplasia (siod) which is the first to be reported from egypt. methods and results: this article presents a case from egypt with mild form of siod presented at the age of 14.5 years with disproportionate short stature, srns (focal segmental glomerulosclerosis), laboratory evidence of cellular immune deficiency and radiologic characteristics of spondyloepiphyseal dysplasia and died at the age of 16.5 years with bone marrow failure and severe pneumonia. conclusion: we emphasize that siod is to be considered in children with growth retardation, srns and bone abnormalities and that inherited nephrotic syndrome may be presented in late childhood or even adolescence and siod suspected patients even before full picture development should be closely monitored for proteinuria, hypertension, cellular immunity and opportunistic infections especially with the need to start immune suppressive therapy for nephrotic syndrome. abstract# p-sat244 denys-drash syndrome presenting with polycystic kidneys objective: denys-drash syndrome (dds) is characterized by a congenital or infantile nephrotic syndrome due to diffuse mesangial sclerosis, male pseudohermaphroditism and a strong predisposition to develop wilm's tumors and gonadoblastoma's. the syndrome is caused by a dominant mutation in the wt1 gene. we report a case of dds associated with renal cysts, which has never been described so far. we report a case of dds in a 3 months old girl presenting with proteinuria and bilateral cortical cysts on renal ultrasound. autosomal recessive polycystic kidney disease was excluded by a normal liver biopsy. a kidney biopsy showed diffuse mesangial sclerosis. at the age of 7 months, she developed a unilateral wilm's tumor, which was treated by heminephrectomy and chemotherapy. the renal cysts were still present on ultrasound. histological examination of a cyst (located in the resected specimen, but separate from the wilm's tumor) showed one layer of flattened epithelial cells, without any evidence of malignancy. the combination of diffuse mesangial sclerosis and a wilm's tumor pointed to the diagnosis of dds. genetic analysis showed a de novo heterozygous missense mutation c.1186g>a (p.asp396asn) in the wt1 gene, previously described in patients with dds, confirming the diagnosis. because polycystic kidneys have never been reported in dds, we explored several genes responsible for these renal manifestations, such as hnf-1β, pax2, pkd1 and pkd2. remarkably, we identified a heterozygous missense variant c.12439a>g (p.lys4147glu) in the pkd1 gene. mutations in pkd1 lead to autosomal dominant polycystic kidney disease (adpkd). the pathogenicity of the newly identified missense variant in our case was evaluated by different mutation prediction software programs and was classified as being 'likely pathogenic'. the same variant was found in the patient's mother, having no renal cysts on ultrasound and in the grandfather, having bilaterally renal cysts. conclusion: this is the first case of dds in combination with polycystic kidneys. we hypothesize that pkd1 c.12439a>g variant might be an incompletely penetrant allele that can cause a severe phenotype of adpkd in association with a wt1 gene mutation. the phosphatase and tensine homolog (pten) gene is a tumor suppressor gene located on the long arm of chromosome 10 (10q22-23). the pten protein is broadly expressed in cells throughout the body, acting as both lipid and protein phosphatases, and regulates intracellular signaling via various pathways. reduced pten protein resulting from a pten gene mutation may enhance cell proliferation, resist from apoptosis. in addition, it has became clear that, in vascular endothelial cells, pten gene abnormalities lead to increased expressions of vascular growth factors, such as angiotensin ii (ang ii), that are essential to angiogenesis. recent reports revealed a germline mutation of the pten gene to cause several syndromes with generalized, multiple hamartomatous lesions of tridermic origin. thus, the new concept of pten hamartoma tumor syndrome (phts) has been proposed. to the best of our knowledge, we describe here for the first time a case of focal segmental glomerulosclerosis (fsgs) with phts. a 3-year-old girl was found to have proteinuria and hematuria on health examination and referred to us for persistent urinary abnormalities. igm nephropathy were diagnosed by renal biopsy, and she started taking an angiotensin-converting enzyme inhibitor. however, proteinuria was not reduced, and an oral angiotensin ii receptor blocker (arb) was administered. proteinuria persisted and she underwent renal biopsy again, yielding a diagnosis of fsgs. despite continued oral arb treatment, proteinuria is ongoing with a urine protein-to-creatinine ratio of approximately 0.5, and mild renal impairment. at age 13 years, 2 years ago, a neck mass prompted a detailed examination which revealed multiple thyroid nodules, ovarian cysts (mucinous cystadenoma), hemangiomas in the plantar and femoral regions, spinal cord lipoma, and renal nodular lesions, necessitating follow-up. subsequently, additional workup detected a pten gene heterozygous mutation (exon 8 codon 335 cga(arg) tga(stop)) and she was diagnosed with phts. methods: a 7-day-old girl, the first child of non-consanguineous chinese parents, was hospitalized due to edema, gross proteinuria, and progressive renal failure. she was delivered spontaneously at 36 weeks of gestation with a birth weight of 2700gand a body length of48cm. during the delivery, she was experienced mild asphyxia. placental weight was large, and the amniotic fluid was meconium-stained. pregnancy was unremarkable and had included routine prenatal ultrasound evaluation. the family history of proteinuria or renal failure was negative. on admission, the findings from physical examination showed the patient had bilateral microcoria and limb hypotonia. results of serological testing for torch infections were negative. since pierson syndrome was suspected, a kidney biopsy was performed when she was aged 10 days. meanwhile, all coding exons of lamb2 were analyzed by using pcr and direct sequencing. results: electron microscopy revealed some glomerular basement membranes were thick and thinning, with splitting of the lamina densa. in addition, the foot processes of podocytes were diffusely effaced, and the number of podocytes increased. two different novel nonsense mutations (trp16x and gln748x) of lamb2 were detected in the patient. these mutations were inherited from her parents respectively. conclusions: newborn with unexplained renal insufficiency or nephrotic syndrome should consider pierson syndrome. our report extends the genotypic spectrum of pierson syndrome. a familial wt1 mutation associated with incomplete denys-drash syndrome we report a familial wt1 missense mutation in exon 9 (1180c>t, r394w) in three members of one family. patient 1, a 2 years old boy, was born at ambiguous genitalia (46, xy karyotype), penoscrotal hypospadias and bilateral inguinal hernias. he was found proteinuria on the preoperative examination and renal biopsy showed diffuse mesangial sclerosis. patient 2, a 3 years old, is the older sister of patient 1, who has normal genitalia. she was found proteinuria and renal biopsy showed focal mesangial sclerosis. wilms's tumor was not found in both of them. a wt1 mutation was detected in both the two patients and their farther. the patients are considered as incomplete dds and they can inherit mutations from their father. so, this finding will provide new evidence for a better understanding of dds and further familial studies in patients of dds are need. ying chen 1 , songming huang 1, 2 , guixia ding 1, 2 , hongmei wu 1 , aihua zhang 1, 2 1 nephrology, nanjing children's hospital, nanjing, china 2 institute of pediatrics, nanjing medical university, nanjing, china objective: to investigate the correlations between peroxisome proliferator-activated receptor (ppar)-α, ppar-γ, and ppar-γ coactivator-1α (pgc-1α) gene polymorphisms and susceptibility to primary nephrotic syndrome (pns). methods: patient genotypes were determined using the polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) technique for the pro12ala and val290met polymorphisms of the ppar-γ gene, the gly482ser polymorphism of the pgc-1α gene, and the leu62val polymorphism of the ppar-α gene. the gene polymorphisms in 111 cases of pns in children and 111 normal controls (nc) were analyzed and compared to examine the differences in the clinical metabolism index, proteinuria, renal pathological types, and hormone treatment responses among pns children with different genotypes. results: the ppar-γ pro12ala and pgc-1α gly482ser mutations were not associated with the occurrence, blood pressure, total cholesterol, a decrease in glomerular filtration rate (gfr), urine protein excretion at the onset of disease, renal pathological type, or hormone treatment response in children with pns. although the homa-ir values for children with the a allele did not differ significantly from those of children with the pp genotype of the ppar-γ gene, their insulin levels were decreased and their isi values were significantly increased (p=0.012 and 0.006, respectively). the triglycerides (tg) levels of children with the aa genotype of the pgc-1α gene were significantly increased (p=0.026). in addition, the ppar-γ (val290met) and ppar-α (leu162val) gene polymorphisms did not show any mutations in the 111 cases of pns in children or the 111 nc children. conclusion: the pro12ala mutation of the ppar-γ gene may be correlated with a decrease in insulin secretion and an increase in insulin sensitivity and the pgc-1α (gly482ser) gene polymorphism may be a causative genetic factor for the triglyceride abnormalities in children with pns. the relationship between endothelin-1 gene polymorphisms and primary nephrotic syndrome in children objective: nphs1 mutations have been reported in cns. however, no hot mutation has been described in chinese family. in this study, nphs1 mutations were analyzed in a chinese family with two siblings died of cns. methods: genomic dna samples were extracted from peripheral blood of the proband, her parents, and 150 unrelated normal individuals. all 29 exons of nphs1 were detected by polymerase chain reaction(pcr) and direct dna sequencing. results: the proband, a 15-day-old girl, weighed 3500g at birth. the placenta was one time heavier than usual. she was hospitalized with edema of legs, heavy proteinuria(+++), and hematuresis(+++). two compound heterozygous mutations were identified in exon 15 (c.2020c>t, p.p674s), which was detected in her father, and in exon 16(c.2207t>c, p.v736a), which was also detected in her mother. these two new mutations were not found in the 150 chinese controls. conclusion: new compound heterozygous nphs1 mutations (c.2020c>t and c.2207t>c) were identified in a chinese family with two siblings of congenital nephrotic syndrome, which were suggested to be the causative mutations in this family. the compound heterozygous mutations (c.2020c>t and c.2207t>c) lie between ig6 and ig7 domain which has a free cysteine residues. these mutations might cause misfolding and defective intracellular transport, with consequent absence of the mutant nephrin on the plasma membrane. abstract# p-sat255 sporadic myh9-related disease: a case report and mutational analysis of myh9 gene .genetic analysis confirmed that these three cases had intron 9 (+kts) mutation.after confirmation of fs, cytogenetic analysis showed xy in girls though phenotypically there were females.abdominal ultrasound in them showed normal uterus with streak gonads and gonadectomy was done for both and histologically reported as dysgerminoma stage i. familial girl aged 12 entered esrd and received live related renal transplant from her mother. the sporadic girl aged 9 years also entered end stage renal disease rapidly and died. the third was a nephrotic boy aged 9 years with fs. he had bilateral hypoplastic testes. his chromosome was xy. renal biopsy showed mcd with igm deposits and partially responding to therapy. a six year old girl presented at the emergency department with a history of 3 episodes of vomiting in 24 hours. she had a history of polyuria and polydipsia with a current daily intake of 3 liters. physical examination revealed signs of mild dehydration. plasma electrolytes revealed hyponatremia (128 mmol/l), hypokaliemia (1.6 mmol/l), metabolic acidosis with normal anion gap (14 mmol/l), hypophosphatemia (0.86 mmol/l), hypouricemia (90 umol/l) and normal serum creatinine (29 umol/l). urinalysis confirmed the na, k, ph and acid uric losses: na (67 mmol/l, fractional excretion 2%), k (83 mmol/l, ttkg 30), tubular phosphate reabsorption of 18%, and fractional excretion of uric acid of 30%. cystine level in leucocytes was normal. she was discharged six days later with oral supplementation of potassium and phosphorus. laboratory studies 3 weeks later revealed surprising findings with persistent hypokaliemia (3 mmol/l), metabolic alcalosis (hco3 30 mmol/l), hypomagnesemia (0.65 mmol/l), and hypocalciuria (calcium/creatinine 0.06 mmol/mmol). we therefore sequenced the slc12a3 gene and we found that she is compound heterozygous for 2 known missense mutations (p.thr304met and p.gly439ser). conclusion: gs can have atypical presentations including severe polyuria; it should be suspected in case of severe hypokaliemia even in the absence of the other classical features. this case report emphasizes the need for repeated laboratory tests in unclear tubular disorders, especially outside of confusing intercurrent illness. a single basepair mutation causes cystinosis in the majority of western cape patients. conclusion: renaltube is being used by pediatric nephrologists over the world for the study of their patients with primary tubular disorders. renaltube will likely contribute to a better care of these children and to a better scientific understanding of primary tubular diseases. the creation and development of this sort of international collaborative efforts must be encouraged within the pediatric nephrology community. objective: to study molecular mechanism of hypercalcemia-induced nephrogenic diabetes insipidus (ndi) by identification of proteins in inner medullary collecting duct (imcd) responsible for this syndrome. methods: the effect on rat kidney medullary collecting duct of early onset ndi resulting from parathyroid hormone-induced hypercalcemia was studied using proteomics and phosphoproteomics in native inner medullary collecting duct (imcd) cells. imcd tryptic peptides and phosphopeptides were identified and quantified by mass spectrometry using a label-free methodology. the major findings were confirmed by selected reaction monitoring study, immunoblot, and immunohistochemistry. results: a total of 5,866 peptides corresponding to 1,107 proteins and 1,388 phosphopeptides of 580 proteins were identified, with significant changes in abundance of 69 proteins and 49 phosphopeptides in early onset ndi versus vehicle controls. gene ontology terms and pathway analysis revealed that hypercalcemiaaffected proteins and phosphoproteins are associated with integrin signaling, and actin cytoskeleton organization. immunoblot and selected reaction monitoring lc-ms/ms studies confirmed the hypercalcemia-regulated proteins (agrin, arcp1b, capg, erm, itgb1, lamb2, and lamc1) and phosphoproteins (myh9, add1, dync1li1, cgnl1, aqp2). hypercalcemia induced changes in abundance of vasopressin-regulated phosphorylation of aquaporin-2 (aqp2) at ser256 and ser261, but not total aqp2, and decreased abundances of slc14a2 urea transporters ut-a1, ut-a3, and phospho-ser486-ut-a1. the major findings were also identified in imcd of rats with vitamin d-induced hypercalcemia. filamentous actin aggregation was firstly demonstrated in imcd of rats with hypercalcemia-induced ndi from both pth and vitamin d. conclusions: early increasing in water excretion in response to hypercalcemia are dependent of changes in abundances of aqp2 phosphorylations, ut-a and its phosphorylations are consistent with prior literature pointing to key roles of the integrin signaling and the actin cytoskeleton in maintenance of collecting duct function. objective: to study molecular mechanism of hypokalemia-induced nephrogenic diabetes insipidus (ndi) by identification of proteins in inner medullary collecting duct (imcd) responsible for this syndrome. methods: the effect on rat kidney medullary collecting duct of early onset ndi resulting from hypokalemia was studied using proteomics in native inner medullary collecting duct (imcd) cells. imcd tryptic peptides were identified and quantified by mass spectrometry using a label-free methodology. the major findings were confirmed by selected reaction monitoring study, immunoblot and immunohistochemistry. results: a total of 2,477 peptides corresponding to 821 proteins were identified with significant changes in abundance of 189 proteins in early onset ndi versus vehicle controls.gene ontology terms and pathway analysis revealed that hypokalemia-affected proteins are associated with generation of precursor metabolite and energy and regulation of actin cytoskeleton. immunoblot and selected reaction objective: inherited distal rta (drta) is a rare condition and is almost always observed in children as a primary entity. mutations in genes encoding transporter or channel proteins operating along the renal tubule may result in a variety of functional defects. the identification of the molecular defects in drta may provide a basis for future design of targeted therapeutic interventions and, possibly, strategies for gene therapy of these complex disorders. for this reason, we evaluated mutations in slc4a1 and atp6v1b1 genes. methods genomic dna from 20 pediatric patients diagnosed with drta was extracted from peripheral blood samples. sequencing was performed for five exons of slc4a1 (14, 15, 17, 19, 20) and two exons of atp6v1b1 (1, 7). all the selected exons are considered hotspots for mutations associated to this tubular disorder. results silent mutations were identified in both studied genes. the mutation c.2688t>c was identified in exon 20 of slc4a1 in one patient and the second variation (c.27t>c) was found in exon 1 of atp6v1b1 in two patients. although previously described, these silent variations are not highly frequent in the overall population. also in slc4a1, exon 19, two nonsynonymous variations were identified in the same patient (p.pro854leu and p.val862ile). the p.val862ile variation was also found in three other patients. it has been shown that these variations are rare polymorphisms in the population, but they have already been associated to diego group erythrocyte antigen. in exon 1 of atp6v1b1 the variation p.met2? was presented in twelve patients and the mutation p.thr30ile was found in six patients. although they occur in the coding region of atp6v1b1, these variations have been described as highly frequent in the population. dent's disease is an x -linked renal proximal tubulopathy associated with mutations in clcn5 (dent's type 1) and ocrl1 (dent's type 2).ocrl1 mutations also cause the oculocerebrorenal syndrome of lowe. we report the case of a 5 year old boy with dent's disease associated with a novel hemizygous change, c.2t>c p.(met1) in exon 1 of ocrl. our patient presented at 3 years of age with incidental finding of non-nephrotic range proteinuria. he had elevated urinary beta -2 microglobulin, retinol binding protein and hypercalciuria. he was developmentally normal and is otherwise of good general health. slit lamp examination and vision were normal. he had normal renal function but had very poor renal accumulation of 99mtc-dmsa on renoscintigraphy. genetic testing identified a hemizygous change, c.2t>c p.(met1), in exon 1 of ocrl, which affects the translation initiation codon. his healthy 8 year old brother who was also found to have low -molecular weight proteinuria and hypercalciuria, is currently being investigated. to the best of our knowledge, the hemizygous change, c.2t>c p.(met1?) in exon 1 of the ocrl gene which was identified in our patient with dent's disease represents a novel variant which has not been previously reported in a dent's disease patient. abstract# p-sat274 enamel-renal syndrome associated to splenic and ovarian calcifications: a case report sameh mabrouk, noura zouari, houda ajmi, jalel chemli, menair tfifha, saida hassayoun, saoussen abroug , abdelaziz harbi pediatrics, university hospital of sahloul, sousse, tunisia enamel-renal syndrome (omim204690) is a rare condition characterized by amelogenosis imperfecta and nephrocalcinosis.we report a new pediatric case of this rare association with the particularity of associated splenic and ovarian calcifications. this report concerns a 13-year-old girl, born to consanguineous parents (first degree cousins). she has no family history of nephrocalcinosis or kidneydisease. her unique brother, aged 8 years, is in good health and has no dental or renal problems. she was first seen at the age of 12 years in dentistrydepartment because of dental abnormalities, the diagnosis of amelogenosis imperfecta was made and the patient was further investigated. a renal ultrasound showed a bilateral medullary nephrocalcinosis. ct confirmed bilateral nephrocalcinosis which was associated to ovarian and splenic calcifications. besides this young girl has a normal growth, her puberty began at the age of 12 years. laboratory findings including serum electrolytes, urea, creatinine, calcium, phosphate, parathormone and alkaline phosphatase were within normal ranges. on urine examination we found a hypocalciuria, with normal sodium and phosphate excretion. the patient is regularly seen in both dentistry department and pediatrics. the aim of this report is to highlight the important role of pediatricians as well as dentists in recognizing this rare and uncommon syndrome. objective: ipex syndrome, a hereditary (x-linked) immune dysregulation with autoimmune polyendocrinopathy and enteropathy, as the basic manifestations, presents a rare and severe disease. the objective of this case report is to highlight the pleomorphism of the syndrome. methods: the authors report the case of a male infant, with a family history of three male siblings affected by ipex syndrome. the patients' medical records were reviewed in order to describe the case of the youngest one. results: during the follow-up of the youngest of three siblings, who presented eczema and intestinal manifestation, without compromised pancreatic and thyroid function, different from other two siblings, it was noticed the pattern of dent's disease. we registered hypophosphatemia, hypercalciuria, glycosuria, low molecular weight proteinuria and ultrasound revealed second stage bilateral nephrocalcinosis. in this child there was no apparent glomerular involvement, as it was seen in the eldest sibling. conclusion: dent's disease is an x-linked renal proximal tubulopathy associated with mutations in the chloride channel gene clcn5 (xp11.22), which is next to foxp3 gene on the x chromosome (xp11.23-q13.3). it seems that in this sibling mutations occurred in exons of both of these genes. this case is to remind on pleomorphic potential of mutations that occur near the coding regions of the foxp3 gene. objective: multiple target organ defects (mtod) is a subgroup of pseudohypoaldosteronism (pha) type i and salt wasting is more severe in this form of pha i. it has a poorer outcome than the renal form, and therapy must be maintained throughout life. here, we reported an infant with mtod who had severe hyperkalemia resistant to classical treatment of pha. hyperkalemia was normalized only by peritoneal dialysis (pd). case: a 4 month-old boy was admitted to our hospital with persistent vomiting, metabolic acidosis, and severe hyperkalemia. there was consanguinity between parents and he had a healthy sister and brother. there was no history of medication. physical examination revealed the signs of volume depletion and acidosis. results: in laboratory examination, blood urea nitrogen was 71 mg/dl, creatinine 0.43 mg/dl, sodium 114meq/l, potassium 9.5meq/l, and chloride 97meq/l. blood ph was 7.33, base excess -12meq/l, and hco3 11meq/l. urinary analysis revealed low specific gravity (1003) with ph 8.0, and normal urinary sediment. urinary sodium was 68meq/l, potassium 1meq/l, and plasma renin activity 80.2ng/ml (2.7-37.0), aldosterone 2800pg/ml (50-900), cortisol 11.1 mg/dl (7-17.5), and acth 5.5pg/ml (10-469). renal ultrasonography did not show any abnormalities. sweet-test showed high sodium waste from sweet glands (144meq/l). the patient diagnosed as mtod. severe hyperkalemia persisted and was resistant to all treatment options for pha, including high-sodium and low-potassium diet, fluid resuscitation, potassiumbinding resins, indomethacin, fludrocortisone, and hydrochlorothiazide. nutritional status of the patient was also not good, and parents did not accept gastrostomy. hyperkalemia was corrected only after pd treatment, and his nutritional status improved dramatically. conclusion: in the case of type iv renal tubular acidosis resistant to therapy, mtod could be considered as a cause of acidosis. if the classical treatment options fail to correct hyperkalemia in these patients, pd may be a reasonable choice to normalize severe hyperkalemia. gülsün gülay yılmaz 1 erta yilmaz 1, 2 1 pediatric nephrology, md, antalya, turkey 2 akdeniz univercity, prof. dr, antalya, turkey tuberous sclerosis, which has an autosomal dominant hereditary disease with a high rate of spotaneous mutation, especially occupies the nervous system and many other organs like kidney, heart and lung. the case presented here was initially diagnosed as congenital heart and polycycystic kidney diasease, because of its characteristics which were quite different than expected for tuberous sclerosis. finally, it was evaluated at our pediatric department together with its dermatological and radiological aspects and defined as a tuberous sclerosis of delateddiagnosis. the case was discussed with its former and actual findings and presented as a warning for early diagnosis. abstract# p-sat285 the effect of bone marrow stem cells mobilization on expression of hif-1α and egf in ischemia/reperfusion-induced renal injury objective: to investigate the therapeutic effects of bone marrow stem cells which has been mobilized by g-csf and stem scf on expression of acute tubular necrosis hypoxia inducible factor-1a (hif-1α) and epidermal growth factor (egf), and to investigate the mechanism of scf and g-csf on the treatment of ischemia/reperfusion-induced renal injury. methods: 160 male sprague-dawley rats that are 8-10 weeks old were randomly allocated into 4 groups (n =40 ineach group): control group (group a), ischemic-reperfusion group (group b), scf + g-csf + ischemic-reperfusion group (group c) and scf + g-csf + control group (group d). detection index: hif-1α was measured by immunohistochemistry technique, the expression of egf and cd34 + cells in kidney was measured by strept avidin-biotin-peroxidase(sabc) and egf mrna by rt-pcr . results: (1) at 5 days postoperative, the cd34 + cells of group b and c was conspicuous higher than group a and d(p<0.05), group c was higher than group b(p<0.05). they gradually descended from 5 days with the time prolonged. (2)at 5 days postoperative, there was significant difference of the expression of hif-1α between group a and d (p<0.05). the expression of hif-1α of group b and c showed higher positive reaction at 5 days postoperative then decent to normal. at each time the expression of hif-1α of group c was significant higher than other groups (p<0.05). (3)the egf expression of group b and c showed higher positive reaction at 5 days postoperative and decent to normal with the time prolonged, group b peaks at 17 days and higher than group a, group c peaks at 10 days, and it is the highest (p<0.05 results: in contrast to model group, atn treated with adscs displayed: cells expressing green fluorescent protein were dectected in injured tubule in kidney . treatment group were significantly higher than model group, p<0.05; renal damage is lighter; kidney damages were relative gently and histopathologic lesion scores was relative lower; while ki-67 positivece cells in treatment group were more than those in model group (p<0.05); the expression of bax, bcl-2 and cell apoptotic index(ai) in treatment group was lower than those in model group , p<0.05, but bax/bcl-2 ratio was higher than those in model group. conclusion: allogenic adscs transplantation can accelerate proliferation of renal tubular epithelial cell and suppress apeptosis injury through up-regulating the ratio of bcl-2/ bax and down-regulating the expression of bax protein in acute kidney injury. objective: previously we showed that pretreatment with the antidepressant fluvoxamine (flu) improves postischemic survival and ameliorates functional and structural kidney damage after renal ischemia/reperfusion (ir). in heart ir injury flu is protective through activating the sigma-1 receptor (s1r) -nitric-oxide synthase (nos) system. here we studied the intrarenal vasoregulatory effect of flu and analyzed the renal s1r-nos cascade. methods: male wistar rats were were treated i.p. either with (1) vehiculum (veh); (2) flu (20 mg/bwkg; flu); (3) flu+ s1r antagonist ne-100 (1mg/bwkg; fn); (4) flu+ non-selective nos blocker l-name (10mg/bwkg); (5) flu + selective endothelial (e) nos blocker l-nio (20mg/bwkg); (6) flu and selective neuronal (n) nos blocker 7-ni (25mg/bwkg). 30 minutes later rats were either harvested or subjected to 50 minutes of ischemia followed by 24 hours of reperfusion. sham-operated animals served as controls (n=10/group). renal s1r, akt, enos and nnos protein levels were measured, the alteration of renal capillary diameters was determined in vivo using muliphoton microscopy. results: ir induced renal vasoconstriction, which was ameliorated by flu. this increase was neutralized by all nos blockers, mostly by 7-ni. after ir all measured protein levels increased. s1r expression was similar in all treatment groups. akt and enos levels were lower, while nnos levels were higher in the flu treated group compared to veh and fn. the acute vasodilatative effect of flu 30 minutes after treatment was suspended by l-name and 7-ni and even reversed by l-nio. s1r, akt and enos protein levels were elevated 30min after flu treatment, while nnos levels remained unchanged. discussion: pretreatment with the s1r agonist flu -used chronically without notable side-effects -improves postischemic renal perfusion through the activation of s1r -nos system in a time and nos isoform specific manner. based on this data one can hope to find a new therapeutic target in the treatment of renal ir damage through the modulation of the s1r. methods: 36 sd rats were randomly divided into model group, intervention group and normal group, 12 rats in each group. intraperitoneal injection with gentamicin for 7 days to establish renal tubular injury rat model. then the intervention group rats were transplanted with 4×10 6 /ml bm-mscs via tail vein. 7 days after transplanted with bm-mscs, took blood and kidney specimen of rats for testing. serum creatinine (scr), urea (bun), malondialdehyde (mda), superoxide dismutase (sod), glutathione peroxidase (gpx), heme oxygenase -1 (ho-1) were detected by spectrophotometry. he staining was used to evaluate the change of renal tissue pathology, then score the renal tubular injury according to the nangaku semiquantitative scoring method. tunel method was used to detect the apoptosis of epithelial cell rate in renal tubular, while immunohistochemistry was used to detect the proliferation of renal tubular epithelial cells (pcna labeling index). results: 1. scr and bun of intervention group rats were lower than those in the model group, the difference was statistically significant (p <0.01); 2. renal tubule pathological score of intervention group rats were lower than those in the model group, the difference was statistically significant (p< 0.01); 3. apoptosis ratio of renal tubular epithelial cell in intervention group rats were lower than the model group, the difference was statistically significant (p<0.01); 4. the pcna labeling index of intervention group rats were higher than those in the model group, the difference was significant (p<0.05); 5. the mda level was significantly reduced in intervention group rats than in the model group (p < 0.01); 6. the sod, gpx and ho-1 inintervention group rats were significantly increased than those in the model group (p < 0.01). conclusion: antioxidant factors such as sod, gpx and ho-1 were significant increased after bm-mscs transplanted into rats. it means that bm-mscs may have antioxidant effect to heal the renal tubular damage. the effect on regeneration and repair in renal tubular epithelial cell after injury deal with at2r antagonist pd123319 objective: to study the effect of proliferation of renal tubular epithelial cell after injury deal with at2r antagonist pd123319,and to explore the function and mechanism of at2r in the regeneration and repair after acute kidney injury. methods: to establish the human renal proximal tubule cells (hk-2 cells) hypoxia /reoxygenation model. hk-2 cells were divided into two groups. (1) (1) these results suggest that the renal proximal tubule epithelial cells after hypoxia/reoxygenation can be simulated tubular epithelial cell process damage; (2) the proliferation of renal tubular epithelial cell after injury was inhibited by pd123319.at2r may play an important role in the regeneration and repair in the kidney by means of promoting the proliferation of renal tubular epithelial cells. (3) arb have no significantly inhibit the proliferation of renal tubular epithelial cell after injury, suggesting that at1r may not have major role in regeneration and repair after acute kidney injury. the effect and mechanism on regeneration and repair in renal tubular epithelial cell after injury by the inhibitory proliferation effect of acei and arb objective: to study the effect of regeneration and repair of renal tubular epithelial cell after injury deal with fosinopril and valsartan, and to explore the function and mechanism of acei and arb induced acute kidney injury by suppressing proliferation. objective: ischemia-reperfusion injury (iri) is a major cause of acute kidney injury (aki) and both innate and adaptive immunity contribute to the pathogenesis. t cell immunoglobulin-3 (tim-3) has been reported as an important regulatory molecule and plays a pivotal role in several inflammatory diseases. however, it keeps unknown whether tim-3 is involved in renal iri. to investigate the expression of tim-3 on kidney mononuclear cells (kmncs) from mice with renal iri and explored its role in the pathogenesis of renal iri. methods: the left renal pedicle was clamped in c57bl6 mice for 45 min, followed by reperfusion. animals were sacrificed at baseline, day 1,3. flow cytometry was used to quantify tim-3 expression on kmncs ,cd4 + t cell,cd8 + t cell,foxp3 + tregs and cd19 + b cell. the levels of tnf-α, ifn-γ, il-4 and il-10 in kidney tissue were measured using elisa. results: at day 1, the increased expression of tim-3 on kmncs ,cd4 + t cell,cd8 + t cell,foxp3 + tregs in the injured kidney from mice with renal iri compared to those from uninjured kidney tissues and baseline (p<0. 05). percentage of tim-3 + cells in kmncs showed an inverse correlation with kidney injury score and kidney tnf-α level. similar negative correlations were found between kidney injury score and tim-3 levels on cd4 + t, cd8 + t cells. consistently, tim-3 expression on cd3 + t cells was further increased in mice at day 3. tim-3 expression on foxp3 + tregs negatively correlates with kindey tnf-α. conclusion: tim-3 might participate in the proceeding of renal iri by regulation on various cd4 + t cell subsets. tim-3 might be a potential new marker for assessing severity of renal iri. expression of galectin-9 and tim-3 in kidney of mice with renal ischemia reperfusion injury objective: t cell immunoglobulin-3 (tim-3) is a surface molecule expressed on various immune cells which plays a central role in immune regulation. identification of galectin-9 (gal-9) as a ligand for tim-3 has established that the tim-3/gal-9 pathway has been linked to several inflammatory diseases by regulating adaptive and innate immunity. to study the expression of gal-9 and tim-3 in kidney of mice with renal ischemia reperfusion injury (iri). methods: thirty c57bl6 male mice were randomized into renal iri groups with and without recombinant gal-9. the left renal pedicle was clamped in c57bl6 mice for 45 min, followed by reperfusion. animals were sacrificed at baseline, day 1,3,10, 21 after iri. gal-9 and tim-3 mrna levels in kidney tissues were determined using real-time rt-pcr. expression of gal-9 in kidney were detected by immunohistochemistry staining .the levels of tnf-α, ifn-γ,il-4 and il-10 in kidney tissue were measured using elisa. results: the expression of gal-9 and tim-3 mrna in the injured kidney tissues increased significantly compared with uninjured kidney tissues and baseline (p<0. 05). compared with the uninjured control and baseline control, the expression of tnf-α and ifn-γ increased significantly in the injured kidney. the expression of gal-9 and tim-3 mrna was positively correlated with renal il-4 and il-10 level (r=0.792, r=0.79 respectively; p<0. 05), but negatively correlated with kidney tnf-α and ifn-γ level (r=-0.69,r=-0.75 respectively,p<0.05). after recombinant gal-9 treatment for three days, the kidney injury ameliorated and inflammatory cytokines (tnf-α and ifn-γ) decreased. the expression of gal-9 and tim-3 in kidney tissues increase in mice with kidney iri. tim-3/gal-9 pathway are closely related to inflammatory process in renal iri. objective: we previously found that rgc-32(response gene to complement-32), a key factor in regulating cell cycle, plays an important role in dealing with epithelial-mesenchymal transition (emt). this study aimed to evaluate the effects of rgc-32 regulating cell cycle in renal tubular epithelial cells injury and repair. methods: (1) objective: autophagy is a lysosomal degradation pathway that is essential for cellular stress adaptation and normal homeostasis. increased level of autophagy has been reported in the post-ischemic kidneys by static analysis. this study aimed to understand the dynamics of epithelial autophagy in kidneys following acute ischemic injury and during renal repair. methods: taking the advantage of differential ph sensitivity of rfp (pka 4.5) and egfp (pka 5.9) fluorescence, we generated a new strain of cag-rfp-egfp-lc3 mice to distinguish early autophagic vacuoles from autolysosomes and to monitor autophagic process in the kidneys with ischemia-reperfusion injury (iri). results: renal epithelial cells responded to nutrient deprivation with easily detectable fluorescent puncta that represented autophagic vacuoles and corresponded to lc3-ii and atg5 protein levels. the majority of the egfp lost its fluorescence in the acidic environment of the autolysosomes where bright rfp signals remained. in normal kidneys, few egfp and rfp puncta were present in the nephron. iri led to dynamic changes in autophagic process in the proximal tubules with the number of egfp puncta reaching the peak at 1 day and returning to the control level at 3 days whereas rfp puncta persisted at a high level through 3 days post injury, indicating autophagy initiation at 1 day but autophagosome clearance at 3 days as kidneys were recovering. since rfp puncta persisted in cells with recent autophagy, we examined ki67 expression and found significantly lower proliferation in cells that contained rfp puncta, suggesting that autophagic cells were less likely to divide for tubular repair. furthermore, 87% proximal tubular cells with mtor activation indicated by p-s6 kinase expression contained no rfp puncta. inhibition of mtorc1 activity with rapamycin caused a 2-fold decrease in cell proliferation. conclusion: our results highlight the dynamic regulation of autophagy in post-ischemic kidneys and suggest a role of mtor in autophagy resolution during renal repair. abstract# p-sat297 novel mechanisms by which heparin can regulate the vascular activity of angiotensin ii (ang-ii) and fibroblast growth factor-2 (fgf-2) and affect the outcome of acute kidney injury (aki) objective: critically ill children treated with extracorporeal membrane oxygenation (ecmo) and/or cardiopulmonary bypass (cpb) frequently develop hypertension, endothelial dysfunction, and acute kidney injury (aki). these patients show high serum levels of ang-ii and fgf-2, and are treated with heparin to prevent clotting disorders. however, the mechanisms by which heparin may affect the vascular activity of ang-ii and fgf-2 are not clearly understood. we carried out this study to determine whether heparin can modulate the vascular activity of ang-ii and fgf-2 by affecting the rho-a, rac-1, src, and pkc signaling pathways. methods: normal fvb/n mice were injected with adenoviral vectors carrying a secreted form of human fgf-2 or lac z vectors, and treated with heparin (5,000 u/kg) or control buffer (n=5 per group). vascular contractility was studied in pressurized isolated resistance-sized mouse mesenteric arteries, in the presence and absence of ang-ii and fgf-2. the activation of the src, rho-a, rac-1, and pkc signaling pathways were assessed by pull-down assays and western blots, both in vivo and in vitro. permeability changes were explored in cultured human renal glomerular endothelial cells (hrgec) using fitc-dextran. results: heparin significantly enhanced the fgf-2-induced activation of rho-a and rac-1 in the kidney and isolated mouse mesenteric vessels. in addition, heparin antagonized the ang ii-induced contractility of isolated mouse mesenteric vessels through rho-a and pkc-dependent pathways. these changes were reverted by fgf-2, and abolished by the rho kinase inhibitor y27632. both heparin and fgf-2, acting in a synergistic manner, increased the permeability of hrec by activating the rho-a and src signaling pathways. conclusion: we conclude that heparin and fgf-2, acting in a synergistic manner, can modulate the activity of ang-ii in the kidney, isolated resistance vessels, and cultured hrgec. these findings clearly identify novel mechanisms by which heparin, acting alone or in combination with fgf-2, may affect the control of blood pressure, renal perfussion, and capillary permeability in critically ill children treated with ecmo and cpb. abstract# p-sat298 cisplatinum (cis) toxicity in immortalized human kidney (hk-2) proximal tubular epithelial cells is independent of dna strand breaks and has implications for biology and therapy of renal cell carcinoma (rcc) results: viability of huh-7 and hk-2 cells decreased similarly after ic50 doses of cis with morphology and mtt assays. many dna damage genes, including atm-related genes, were expressed less at mrna and protein levels in both cell types. however, p53 expression decreased in hk-2 cells. comet assays showed extensive dna strand breaks in huh-7 but not in hk-2 cells. atm promoter activity increased in only huh-7 cells. also, s and g2/m populations were depleted by facs in huh-7 but not hk-2 cells. conclusion: hk-2 cells transformed by hpv e6/7 oncogenes displayed independence from cis-induced dna breaks and atmmediated cell cycle arrest. such independence from genotoxicity will help explain mechanisms imparting resistance to chemo-or radiotherapy and invasiveness in rcc. abstract# p-sat299 renal neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 expression in children with acute kidney injury on henoch-schonlein purpura nephritis yue du, jinjie guo, ling hou, yubin wu, tingting sun paediatrics, post graduate trainee, china objective: to evaluate serum, urinary and renal neutrophil gelatinaseassociated lipocalin(ngal) and kidney injury molecule-1(kim-1) in children with aki on hspn by prifle or hspn with nephroticrange proteinuria. methods: we performed a prospective single-center evaluation of serum, urinary and renal ngal and kim-1 in a cohort of children. we recorded any relevant data including age, gender, weight, hemoglobin(hb), serum creatinine(scr), cystatine c(cysc), serum beta2-mg, albumine, urine beta2-mg, urine protein in all patients. each patient's estimated creatinine clearance was calculated using the original schwartz formula. blood sample and five ml of urine sample from each participating patient were collected for ngal and kim-1 using an enzyme-linked immunosorbent assay. analysis of proteins ngal and kim-1 using sabc immunohistochemical assay. results: twenty five patients were enrolled in the study. nine patients with aki-on-hspn(a-on-c) and six were done renal biopsy, sixteen patients with hspn with nephrotic-range proteinuria and ten were done renal biopsy. blood cystatinc, beta2-mg , scr, ngal and kim-1 were increased significantly in patients with a-on-c than those with hspn, and so do urine ngal, kim-1 and beta2-mg. there was no significant difference of proteinuria between the patient with a-on-c and the patients with hspn. immunohistochemical results showed that ngal and kim-1 were expressed in proximal tubule and their expression were significantly higher in a-on-c group than in hspn group. the correlation analysis showed that urine ngal and kim-1 were negative correlated with gfr and they were uncorrelated with proteinuria. conclusion: blood, urine and renal ngal and kim-1 were significantly increased in patients with a-on-c than those with hspn, and they were negative correlated with gfr. we may conclude that ngal and kim-1 may diagnose a-on-c patients more sensitive than scr. abstract# p-sat300 objective: neonatal period is an important stage in the development of renal function at the children. the frequency of renal damage in infants undergoing resuscitation is high, but non-specific clinical symptoms and low information existing survey methods impede their timely diagnosis. purpose -evaluation of diagnostic importance of determining the level of carbonic anhydrase ix (ca9) in the urine of newborns in critical states. methods: we evaluated human carbonic anhydrase ix in the urine of 40 newborns who had asphyxia at birth and receiving treatment in the intensive care unit -the main group. the control group consisted from healthy newborns (13 people). for the study used a sample of newborn urine collected at 1-2 days of age. carbonic anhydrase was determined using enzyme immunoassay kit. results: human carbonic anhydrase ix (ca9) -trans membrane protein, the main physiological function of which is to regulate the ph by the reversible hydration of carbon dioxide. the main pathogenic factor causing kidney damage of fetus is chronic fetal hypoxia. there is a single study hif-i alpha (hypoxia inducible factor-i alpha) in the blood, increased expression of which occurs during hypoxia. this is connected with damage of tubules and interstitial cells of the kidneys, their proliferation, synthesis of cytokines and extracellular matrix. in this case, activity of ca9 is reduced in the blood. normally, ca9 is missing in the urine, so its definition can be a marker of early renal dysfunction. the authentic increase of ca9 level in the main group was revealed in comparison with the control group ( and β2-mg, 59,723/ 32,616/ 1511/ 996/ 1084/ 600 μg/g cr. levels of each marker were very high in nb and 1m children, and subsequently decreased gradually. urinary α1-mg levels reduced the quickest, and became the same as at >=3 years old by 6 months after birth. conclusion: renal tubular function can be evaluated in children <3 years old using these normal values. the most stable and useful marker from early infancy seems to be urinary α1-mg. urinary biomarkers for gentamicin-induced acute kidney injury in the neonatal intensive care unit objective: gentamicin (gm) is an aminoglycoside frequently used in the neonatal intensive care unit (nicu). gm is nephrotoxic and may cause acute kidney injury (aki). serum creatinine (scr) appears to be an insensitive and unreliable marker for detecting aki. to determine whether urine biomarkers are useful for early detection of gm-induced aki in neonates in the nicu. methods: prospective, clinical, observational study. forty-six neonates (32m/14f) without pre-existent kidney disease were divided in a gm group (n=26) and a reference group (n=20). demographics, vital signs and clinical conditions were recorded. only neonates with a bladder catheter in place were included. urine samples were collected every two hours. biomarkers (gsta1-1, gstp1-1, kim-1, nag, ngal) were determined. residual blood samples were used to measure scr. results: the gm and reference group were comparable for gestational age, weight and mortality. neonates treated with gm are admitted longer than neonates in the reference group. treatment with gm resulted in higher scr compared to the reference group (58. 5 [44.8-58.5] vs. 34.0 [28.3-58.8]; p<0.05). higher levels of scr correspond with higher urinary excretion of all biomarkers, especially in neonates treated with gm. the average time until the highest peak was shorter for all biomarkers compared with scr (p<0.05). there was no difference in produced urine volume between the gm and the reference group. conclusion: higher scr levels correspond with higher urinary excretion of all biomarkers, especially in neonates treated with gm. gstp1-1 seems the most useful marker for early detection of aki in neonates. seong heon kim, sang wook mun, su young kim 1 1. pediatrics, pusan national university children's hospital, yangsan, korea objective: spontaneous tumor lysis syndrome (stls) before cancer treatment is rare and develop mostly in burkitt lymphoma and non-hodgkin lymphoma. here we report a case of stls secondary to tcell acute lymphoblastic leukemia(all) which presented with renal stone and subsequent aki. methods and results: a 6-year-old boy was admitted to our hospital for generalized tonic clonic seizure. one month ago, he visited other hospital for episodic right-sided flank pain and evaluations revealed microscopic hematuria, hyperuricemia and stone on right uretero-pelvic junction. his blood pressure was 160/90 mmhg and he did not have any dysmorphic features and he had developed normally. physical examination showed tender hepatomegaly (2 cm below costal margin) and no splenomegaly. his initial laboratory findings were as follows : bun 119.3 mg/dl, creatinine 4.77 mg/dl, uric acid 47.6 mg/dl, ldh 460 iu/l, wbc 11,900 /ul, hb 9.9 g/d l, platelet 242k /u l. there was no blast on his peripheral blood smear test. emergent hemodialysis was started because of aki, hypertension and seizure. his renal function, blood pressure, uric acid and electrolyte abnormalities gradually improved with appropriate therapy after 2 times of hemodialysis. but serum uric acid level increased again (from 6.1 mg/dl to 12.9 mg/dl), so allopurinol was added on. the cause of aki was unclear and we thought that aki presumed to be secondary to acute uric acid nephropathy caused by stls. after few days, bone marrow biopsy was done and demonstrated normocellular marrow without evidence of malignancy. after day 15 of hospitalization, abdominal pain with hepatomegaly was getting worse than before and uncontrolled high fever occurred. 20% of blasts were seen on his peripheral blood smear and subsequent second bone marrow biopsy demonstrated acute t cell lymphoblastic leukemia. then, he was referred to division of pediatric hemato-oncology on our hospital and induction chemotherapy was started. conclusion: stls with aki is very uncommon initial presentation of leukemia and stls presenting with renal stone is extremely rare. in a clinical situation of marked hyperuricemia with acute kidney injury, we need to consider occult malignancy and stls. the application of serum beta-2-microglobulin (beta-2-mg) and cystatin c(cysc)concentration to evaluation of renal function impairment in patients withneonatal jaundice yanan xin, cairong jiang, junfeng yang, hui xu, yuan zhang the fourth hospital of baotou, baotou, china objective: to explore the application of serum beta-2-microglobulin (beta-2-mg) and cystatin c(cysc)concentration to evaluation of renal function impairment in patients with neonatal jaundice. methods: 60 neonates with hyperbilirubinemia from jan. of 2012 to dec.of 2012 in pediatric department and 34 full term infants were chosen as research object, hyperbilirubinemia divided into mild group (bilirubin<256.5umol/l) and moderate severe (bilirubin> 256.5umol/l) group according to the level of bilirubin. the different of sex and age were not significant between neonates with hyperbilirubinemia and control group. serum beta-2-mg, cysc, cr and bun were measured in the two groups. results: serum beta-2-mg and cysc in 60 neonates of hyperbilirubinemia were significantly higher (p<0.01) than healthy neonates, meanwhile, serum cr and bun were significantly lower (p<0.01) than healthy neonates. after treatment, serum beta-2-mg and cysc of neonates with hyperbilirubinemia were significantly lower (p<0.01) than pretreatment. however, serum beta-2-mg were higher (p>0.05) abeta-d cysc were significantly higher (p<0.01) than normal group. but there was no significant difference between mild and moderate severe group in the level of serum beta-2-mg, cysc, cr and bun(p>0.05). the result implied that neonates with hyperbilirubinemia suffered for renal function to varying degrees. howerer, the damage can partially recovered after energetic treatment. clinical and pathological analysis of iga nephropathy with acute kidney injury minguang chen, xiaohua ye, qing yang nephrology, yuying children's hospital, wenzhou, china objective: to investigate the incidence, etiology, clinical pathological characteristics and prognosis in primary iga nephropathy(igan) children with acute kidney injury(aki). methods: retrospectively analysis the clinical and pathological manifestations and follow-up results of chlidren with primary igan and aki in our department from january,1996 to jun, 2012. results: there were 19 cases with aki in 196 chlidren with igan(9.7%), the peak serum creatinine were from 94.5umol/ to 282 umol/l. histological changes: with the formation of crescent in 10 cases, diffuse endocapillary proliferation in 5 example, 15 cases of renal tubular injury, 10 cases of red blood cell and protein cast, 1 cases with acute interstitial nephritis. multivariate logistic regression analysis showed: with massive proteinuria were independent risk factors of igan in children with aki (or = 27.370, 95% confidence interval was 3.151-237.740, p<0.01). the etiology of aki except with massive proteinuria, include: 1. iga nephropathy with severe glomerular damage, including crescentic glomerulonephritis and diffuse endocapillary proliferation; 2 complicated acute interstitial nephritis; 3 drugs causing decreased glomerular filtration rate; 4. renal tubular injury induced by gross hematuria. all of the patients were not on dialysis, hormone therapy in 13 cases (including 7 cases of methylprednisolone pulse therapy), 6 cases combined with cyclophosphamide treatment. except 1 cases no significant improvement, the renal functiones of all patients recovered or improved within 1-2 months after treatment. follow-up from 1 month to 7 years, 3 cases had renal function improved, but 2 were lost to follow-up and after 3 years enter to the chronic renal failure, 1 case with renal function loss after 32 months and repeated renal biopsy showed glomerular sclerosis of 31.6% during the follow-up period. conclusion: aki is not uncommon in children with igan, the causes are varied and massive proteinuria is independent risk factor among them. we should adopt different treatment strategy according to different causes and the short term prognosis is good. abstract# p-sat307 biopsy proven acute interstitial nephritis in children objective: biopsy proven acute interstitial nephritis (ain) is an uncommon cause of acute renal failure in children. the cause of ain most is commonly due to medications such as antibiotics or infections. we reviewed our experience of biopsy proven ain in children. the biopsy database of all native renal biopsies over a 15 year period was reviewed. all biopsies which listed acute interstitial nephritis were selected and clinical and laboratory data were extracted from clinical records. results: 16 cases of ain from 540 biopsies,(2.9%) was identified. median age 11.5 years, range 1-14 12/16 cases presented with acute renal failure without an obvious cause. 13/16 cases had non specific constitutional symptoms and oliguria was reported by 6 patients. antibiotics were the most common medications implicated (5/16 patients) three patients were on multiple medications with one having chronic epilepsy and the other crohn's disease. five patients required short term dialysis ranging from 2 to 7 days.13 patients were treated with corticosteroids and at last follow up, 4 had reduced gfr 45-74ml/min/1.73m 2 . conclusion: acute interstitial nephritis is uncommon in childhood but when it occurs, antibiotics are the single commonest cause. there is significant morbidity associated with the illness with not all patients making a full recovery. abstract# p-sat308 acute kidney injury as presentation of burkitt's lymphoma eva greta ter haar 1 , anne uyttebroeck 2 , marleen renard 2 , veerle labarque 2 , djalila mekahli 1 1 pediatric nephorlogy, university hospital leuven, leuven, belgium 2 pediatric hemato-oncology, university hospitals leuven, belgium objective: acute kidney injury (aki) has become increasingly prevalent. approximately 2-3% of children admitted to pediatric tertiary care centers present with this life-threatening condition. the most common causes are post-operative septic shock, organ or bone marrow transplantations and intrinsic renal disease. the latter comprises multiple disorders, but tumor invasion due to a lymphoproliferative malignancy is very exceptional. methods and results: we report two cases of aki caused by infiltration of burkitt's lymphoma. both four-year-old male patients presented with abdominal pain, nausea, vomiting, weight loss and overall weakness since a few weeks. clinical examination showed hepatosplenomegaly and impressive bilateral nephromegaly. both had malignant hypertension. blood analysis showed severe renal impairment in both patients. the first had creatinine of 2.9 mg/dl (egfr 21 ml/min/1.73m 2 ), urea of 97 mg/dl and uric acid of 8.9mg/dl. the second had creatinine of 5.97mg/dl (egfr 10 ml/min/1.73m 2 ), urea of 215mg/dl and uric acid of 12mg/dl. lactate dehydrogenase was extremely elevated in both cases (5860 u/l; 758 u/l respectively). urinary sediment was normal. ultrasound showed bilateral nephromegaly in both patients (patient 1:+12sd and +11sd; patient 2: +10sd and +9sd, for right and left kidney respectively). mri demonstrated a homogenous renal enlargement with features of an infiltrative lesion. bone marrow was inconclusive and diagnosis of burkitt's lymphoma was confirmed by a renal biopsy. after starting chemotherapy according to the inter-b-nhl ritux 2010 protocol, both children developed a tumor lysis syndrome and required hemodialysis. at last follow-up both children still had hypertension. nevertheless, renal size and function were normalized. conclusion: lymphomatous infiltration due to burkitt's lymphoma is a rare cause of aki. however, it should be considered in a patient presenting with unexplained renal failure and bilateral nephromegaly with normal urinary sediment. renal biopsy may be needed to confirm diagnosis. abstract# p-sat309 does malnutrition interact with acute kidney injury in children? objective: to investigate the interaction between malnutrition and acute kidney injury (aki) assessed by prifle criteria and to assess the effects of these factors on the outcomes of pediatric intensive care unit (picu) patients. methods: prospective cohort study conducted on children. outcome variables: mortality, need for dialysis, picu free days (the number of days alive from icu discharge to day 28) and ventilator-free days (the number of days alive and breathing without assistance from admission to day 28). exposure variables: malnutrition on admission (who growth standards) and any change in prifle criteria during the first icu 14 days. results: of 98 patients (median age =19 iqr =50 months, 35 girls) enrolled, 39 (40%) were malnourished and 34 (35%) developed aki. among the malnourished, 19/39 cases (49%) had aki, while this complication occurred in 15/59 (25%) of patients without malnutrition resulting in a risk ratio of 1.9 (95%ci 1.1 -3.3). the 28 days mortality rate was 10/98 cases (10%) and aki was associated with higher risk (8/34 versus 2/64 cases -rr=7.5 95%ci 1.7-33.5). concurrent malnutrition and aki was present in 19/98 cases (19%) producing a tendency (p=0.2) to a further increased risk of death (rr=9.4 95%ci 1.5-58.0). 9/98 (9%) patients needed dialysis and malnutrition increased the risk for this outcome (rr=5.3 95%ci 1.2-24.2). aki was associated with significantly higher icu free days (19±7 vs. 15±10) and ventilator-free days (22±9 vs. 13±12) but concomitant malnutrition did not reduce either of these outcomes. conclusion: malnutrition and aki are common in children admitted to the icu and the presence of malnutrition is associated with increased risk of developing aki. the need for dialysis was increased in malnourished patients and more powered studies are demanded to test whether the tendency to higher mortality observed in patients with concomitant aki and malnutrition is confirmed. urine erythropoietin level is associated with kidney and brain injury in critically ill neonates yanhong li, jie yan, xiaozhong li department of nephrology, children's hospital affiliated to soochow university, suzhou, china objective: erythropoietin (epo) is a glycoprotein hormone produced predominantly in the kidneys. the protective effect of exogenous epo in hypoxic-ischemic brain injury has been thoroughly examined in neonates. however, the metabolism of endogenous epo in neonates remains unclear. we aimed to evaluate the concentration of urinary epo (uepo) in critically ill neonates and to identify possible clinical and laboratory variables that may be associated with uepo levels. methods: the concentrations of epo, cystatin-c, microalbumin, and α 1 -microglobulin in the first available urine sample during the initial 72 hours of life were measured in 103 critically ill neonates. clinical and laboratory data were collected for each neonate. results: there was a positive correlation between uepo levels and urinary levels of cystatin-c (r =0.265, p =0.008), microalbumin (r =0.422, p <0.001), andα 1 -microglobulin (r =0.421, p <0.001). the concentration of uepo was elevated in neonates who developed acute kidney injury (aki) during the first week of life compared with those without aki (p =0.002) and was also elevated in neonates with brain injury, as demonstrated by ultrasound or magnetic resonance imaging, compared to neonates without brain injury (p =0.008). an increased log 10 uepo level was associated with the occurrence of aki (odds ratio of 2.70, p =0.007) and brain injury (odds ratio of 2.33, p =0.016). conclusion: an increased urinary epo level in the early postnatal period is significantly associated with kidney and brain injury in critically ill neonates. abstract# p-sat311 acute kidney injury following extracorporeal membrane oxygenation support and concomitant hemofiltration -the role of diuretics objective: fluid overload (fo) is common during extracorporeal membrane oxygenation (ecmo) and can be managed by continuous hemofiltration (hf) and/or diuretic therapy. although combination therapy of hf and diuretics in particular can be most effectively for fo removal during ecmo, it may also increase the risk for prerenal acute kidney injury (aki) post-ecmo. our objective was to describe the incidence of aki post-ecmo in patients treated with ecmo and hf who received concurrent diuretics. methods: in this cohort study all neonates (≤28 days after birth) treated with ecmo and concomitant hf between 2007 and 2011 were included. patients were divided into two groups based on diuretic regimen: group-1, patients who received no or one single diuretic dose during ecmo and group-2, patients who repeatedly received diuretics. both groups were compared using mann-whitney u test for nonparametric data and chisquare test for categorical data. aki was defined as the highest serum creatinine (scr)-based rifle class reached on post-ecmo day 1 up to post-ecmo day 4 (risk, injury, or failure being 150%, 200% or 300% of median scr reference values for age). results: 56 neonates received ecmo support with hf, 6 died immediately following decannulation and 6 others were lost to followup since they were transferred back to their referring hospitals within 3 days post-ecmo. of the 18 patients in group-1, 3 (17%) qualified as risk and 1 (6%) as injury. of the 26 patients in group-2, 3 (12%) qualified as risk, 4 (15%) as injury and 9 (35%) as failure (group-1 vs. group-2, p=0.039). patients in group-2 had an increased ecmo duration (p=0.001) and number of ventilator days (p=0.002). no differences were observed in baseline characteristics (e.g. age at the start of ecmo, underlying diagnosis, severity of illness scoring using the pim and prism risk adjustment systems), hf flow rate, and fluid balance between both study groups. conclusion: the incidence and severity of aki immediately post-ecmo was significantly higher in patients who repeatedly received diuretics in addition to hf during ecmo. valentina sitnikova, yuliya pashkova, tatiana zvyagina, elena kulakova, alexandra nastausheva voronezh state medical academy, voronezh, russia objective: it is actually to find the new markers of kidney damage and spread them in practice. cystatine c (cys) is one of these, but there are only few data about it in children with urinary tract infection (uti) according to the age. the aim of our study was to evaluate serum concentration of cys in children with uti of different age. we investigated 83 patients (47 girls and 36 boys) with uti from 1 month to 17 years among them 32 children were under 1 years old. methods: concentration of cys were measured by immunoenzime method (elisa) with test-system biovendor (check republic). results: the mean cys concentration in children with uti under 1 year was 1.43± 0.37 mg/l, in older children it was lower: 1.23 ± 0.36 mg/l, p= 0.025 (mann-whitney). in 16 girls before 1 year cys concentration was 1.47± 0.32 mg/l and in 16 boys of the same age it was 1.40±0.43 mg/l, p>0.05. in 31 girls over 1 year cys concentration in serum was 1.31± 0.41 mg/l, in 20 boys -1.3± 0.33 mg/l, p>0.05. the results of our study showed that among children with uti serum concentration of cys was higher in children of the first year of life and did not depend on sex. probably, higher level of cys in infants connects with low glomerular filtration rate in children of this age. microalbuminuria can prognosticate outcome in the critically ill children biplab maji, surupa basu, rajiv sinha paediatrics, post graduate trainee, kolkata, india objective: microalbuminuria [albumin creatinine ratio (acr) >30 mg/g] increases in acute inflammatory conditions as a result of glomerular endothelial dysfunction. the study evaluated its prognostic potential in the critically ill child. methods: an ongoing prospective observational study (oct 2012-mar 2012) of random urine acr (mg/g) estimated on day1 and day 3 of pediatric intensive care unit (picu) admission, of a tertiary care pediatric hospital. demographic, laboratory data, vasopressor use, mechanical ventilation, length of stay, outcome and pelod scores were recorded. results: of 113 children, 72 children (median age 4.5years, 68% male) recruited. the commonest cause of admission was pneumonia, infection associated hemophagocytic lymphohistiocytosis followed by meningitis. day1 acr (median 70.8 mg/g) significantly correlated with day 3 total leukocyte count (p=0.016). both day 1 and day 3 acr correlated with pelod scores [median 8] (p=0.002 & p= 0.003 respectively) and duration of mechanical ventilation (p=0.003 and p=0.001 respectively). both median day 1 acr, and day 3 acr were significantly different between survivors (n= 29) and non-survivors (n=11) (p=0.0001 and p<0.0001, respectively). roc curve analysis for mortality prediction revealed the highest area under curve (auc) of 0.963 for acr3, followed by 0.928 for acr1 and 0.867 for pelod scoring (p>0.05, non-significant for auc comparisions) (fig. 1.) . a cut-off value of day 3 acr of 102.4mg/g had a positive predictive value of 95.8% for negative outcome conclusion: microalbuminuria is an early inflammatory marker that correlates with organ dysfunction. significant levels reliably predict negative outcome early into intensive care admission, as accurately as pelod scores, which can help counsel patients, plan treatment and triage, and allocate resources judiciously. abstract# p-sat314 an unusual presentation of congenital nephrotic syndrome caused by wt1 mutation ann raes, sofie maebe, joke dehoorne, bert callewaert, johan vande walle pediatric nephrology, university hospital, gent, belgium objective, methods and results: a caucasian girl was admitted at the age of 28 days because of progressive lethargy, poor feeding, oliguria en peri-orbital edema. extreme hyponatremia (89 mmol/l), severe renal insufficiency (creat 1.9 mg/dl, p 10.7 mg/dl), low plasma albumin (1.5 g/dl), respiratory compensated metabolic acidosis (arterial ph 7.31 pco 2 27 mmhg, bicarbonate 13.5 mmol/l, be -10.9 mmol/l) and massive proteinuria (12 g/l). clinical examination was normal without anomalies of the external genitals or other dysmorphic features. despite supportive therapy (sodium, bicarbonate, albumin and furosemide) she progressed to respiratory failure and renal failure with need for mechanical ventilation and peritoneal dialysis. congenital infections and thrombosis was ruled out. histological examination showed diffuse mesangial sclerosis (dms). the combination of congenital nephritic syndrome and dms on biopsy was suggestive for mutations in the wilms' tumor 1 (wt1) gene. genetic analysis showed normal female karyotype and conformed a heterozygosity for the c.1223c>g or p.his441gln mutation within the zinc finger 2 domain. after a long and complicated icu episode, a stable situation with home choice peritoneal dialysis was achieved. due to the important risk for comparision of auc of pelod with acr 3, p= 0.766 for comparison with acr 1) for the prediction of mortality in the pediatric intensive care unit. development of wilms' tumor, bilateral nefrectomy was performed and kidney transplantation will be planned. we present an unusual case of congenital nephrotic syndrome with extreme hyponatremia at presentation and fast progression to esrd, due to mutation in wt1 gene. several genes have been implicated in congenital nephrotic syndrome. genetic testing is mandatory and can add important information such as risk of malignancy. renal histology can play an important role in the approach for appropriate mutational screening. mutations in the wt1 gene are associated with denish drash syndrome and frasier syndrome, but also with isolated and sporadic steroid resistant nephrotic syndrome. in the last category the majority of patients is female, the age of onset and time to esrd is variable, although the vast majority of cases are older at presentation and time to esrd is longer than in our case. objective, methods and results: a 8-year-old girl, affected by b thalassemia major, was admitted for proteinuria detected at the emergency unit. fever, hyporexia and diarrhea had been present in the last three days. on physical examination she presented with decreased skin turgor and hypertension. laboratory examinations showed: hyponatremia (124 meq/l), hypokalaemia (2.9 meq/l), hypophosphatemia (1.1 mg/dl), hyperazotemia (60 mg/dl), reduced creatinine clearance (crcl = 68 ml/min/1.73 m 2 ) and metabolic acidosis (hco 3 -12.8 mmol/l). urine analysis revealed: proteinuria, glycosuria, hypercalciuria, hyperuricuria, hyperphosphaturia and aminoaciduria. she had been under treatment for five years with deferasirox (dfx) 26 mg/kg/die. we stopped dfx and gave supplementation of potassium, phosphate, sodium bicarbonate and sodium chloride with electrolyte and renal function (crcl 130 ml/min/1.73 m 2 ) normalization in 6 days. after one month only minimal proteinuria persisted. conclusion: in so far a fanconi syndrome (fs) has been reported in 11 patients (6 children), while mild renal insufficiency (ri) has been documented only in 2 children. in our case dehydration seems to be responsible for the ri, while an interstitial nephritis cannot be excluded. close monitoring of renal function should be done in children on dfx. objective: to test the hypothesis that neutrophil gelatinase-associated lipocalin (ngal) and interleukin-18 (il-18) are early biomarkers for aki in critically ill patients and evaluate the predictive value of them in patients with established aki at inception of crrt. methods: children from picu and health examination center in guangzhou women's and children's medical center were divided into four groups: critically ill patients with aki recevied crrt group (group1 ), critically ill patients with non-aki recevied crrt group(group2) , critically ill patients with aki don't recevie crrt group (group 3),healthy control group (group4). 1.5 ml venous blood and urine specimens were collected and was kept under -70°c.from each patient in picu untill they were discharged, transferred or die . serum creatinine (scr) and urine ngal and urine il-18 were analyzed . results: compared with group2 and group4, the urine ngal and urine il-18 increases obviously in group1 and group3 (p<0.05). there is no significance of urine ngal and urine il-18 between group2 and group 4.(p> 0.05).the concentration of urine ngal increased more than10 times obviously 2 days before diagnosed of aki under the akin standard with auc 0.841(p <0.05) , and the concentration of urine il-18 increased more than 5 times 2 day before aki with auc 0.808(p <0.05). the auc was 0.943(p <0.01) when they were combined. the level of urine ngaland il-18 at initiation of rrt were higher in in non-survivors compared to survivors. conclusion: urine ngal and urine il-18 are useful indicators to predict and early diagnose aki. the level of urine ngal and il-18 at initiation of rrt.were negative correlation with the outcome of renal prognosis and survival rate . objective: the aim of this study was to evaluate the rate of early kidney injury in infants with congenital heart disease (chd). neutrophil gelatinase associated with lipocalin (ngal) which can be measured both in serum (sngal) and in urine (ungal), is at the moment the most promising marker directed to discover early kidney injury within the glomeruli and distal and proximal tubules. less known is cathepsine l as a marker of tubule necrosis. methods: the study group consisted of patients (22 boys and 12 girls) with congenital heart disease. all patients were under 3 years of age (mean 8.2 months). blood and urine samples were obtained during routine checkups. all patients had normal serum creatinine level at the day of collecting samples. the control group (n = 20) was age-and gender-matched. levels of sngal, ungal and cathepsine were compared in whole group. the study group was divided depending on the nature of the defect (cyanotic/acyanotic) and the treatment (surgical/conservative treatment). results: chlidren with congenital heart disease had significantly higher concentration of sngal, ungal and urine cathepsine than the control group. no significant difference was observed in the urine cathepsine and urine and serum ngal level between patients with acyanotic and cyanotic chd. there were also no significant differences in patients who underwent surgery and on conservative treatment in ngal level (both in urine and serum). cathepsine level was significantly higher in group after surgical treatment. conclusion: patients with chd are at higher risk of early kidney damage. this process is independent on the etiology and nature of the defect. ngal determined in the serum and urine of these patients may be used to detect kidney injury or monitoring disease progression. urine cathepsine is more efficient marker of early kidney injury in patients after cardiac surgery. abstract# p-sat318 tim-3 expression in kidney mononuclear cells and its relationship with foxp3+ tregs from mice with renal ischemia reperfusion injury yamei wang, yuhong tao, li ye department of pediatrics, west china second university hospital, chengdu, china objective: foxp3 + tregs participate in the repair of renal ischemia reperfusion injury. t cell immunoglobulin-3 (tim-3) plays a pivotal role in several inflammatory diseases by modulation of foxp3 + regulatory t (treg) cells. to detect the expression of tim-3 in kidney mononuclear cells (kmncs) from mice with renal iri and analyze the relationship with foxp3 + tregs, to explore the role of tim-3 in repair of renal iri. methods: the left renal pedicle was clamped in c57bl6 mice for 45 min, followed by reperfusion. animals were sacrificed at baseline, day 1,3,10, 21 after iri. tim-3 expression in kmncs were determined using real-time rt-pcr and flow cytometry. the percentage of foxp3 + treg in cd4 + t cells was quantified by flow cytometry. the levels of tnf-α、ifn-γ、il-4 and il-10 in kidney tissue were measured using elisa. the correlation among tim-3 expression, foxp3 + treg and cytokine level was analyzed. results: the expression of tim-3 expression in kmncs from injured kidney at acute stage(day 1) was significantly higher than those from uninjured kidney at acute stage and lower than those from injured kidney at repair stage (day 3,10, 21). the percentage of foxp3 + treg in cd4 + t cells was up-regulated with time. compared with the uninjured kidney, the expression of tnf-α and ifn-γ increased significantly in the injured kidney at acute stage and the expression of il-4 and il-10 increased significantly in the injured kidney at repair stage. the tim-3 expression in kmncs at iri repair stage was positively related with the level of foxp3 + treg, il-10 (r=0.81, r=0.79 respectively; p<0. 05), but negatively related with the level of tnf-α and ifn-γ. conclusion: the increased expression of tim-3 in kmncs may take part in the repair of renal iri and contribute to the development of foxp3 + treg. objective: (d+)hus is a critical health problem in argentina since it is the main cause of acute renal failure in children and the second cause of chronic renal failure. fecal contamination of food and drinking water by asymptomatic cattle is often the source although secondary infection through personto-person contact may also occur. occasionally in (d+) hus, the onset among affected siblings occurred within a short time of each other. to evaluate the risk and clinical severity of illness for childhood (d+)hus in siblings. methods: we retrospectively analyzed the clinical records of 133 children with d+hus that were admitted in our pediatric department between march 1997 and december 2012. results: (d+) hus occurred in 2 siblings in 4 of the 129 families studied (3%). 16 patients had an affected sibling with diarrhea and 4 progressed to hus (25%). family cases: mean age: 28 months, 5 were girls. the mean duration of interval between hus episodes was 4 days. second family members had prolonged oligoanuria (3 vs 12 days) and a most of them developed neurological complications. long-term renal complications were more frequent in this group, but differences were not statistically significant. conclusion: these findings emphasize the potential for extensive intra-familiar transmission of stec, especially between siblings. a second family member might also develop an even more severe hus episode, so siblings should be kept under close surveillance. objective: kawasaki disease (kd) is a common cause of systemic vasculitis in children. other than well known complications like coronary artery aneurysm, there have been few reports of this disease involvement in renal system. according to the study from wang jn et.al, 52% (26 out of 50) of the patients who suffered from kd showed renal inflammatory foci in dmsa renal spect which suggested a possibility of renal scar formation subsequent to kd. therefore, this study was performed to verify the renal inflammation following kd. methods: from march 2011 to october 2011, 15 patients who were diagnosed as kd at national health insurance service ilsan hospital were enrolled to the study. all of the patients underwent dmsa renal spect to evaluate renal involvement during their acute phase of kd. echocardiography was performed to assess cardiac involvement such as coronary artery aneurysm. complete blood cell counts, aspartate amino-transferase, alanin amino-transferase, albumin, c-reactive protein, and bun/creatinine were measured. also urine β2microglobulin was measured to assess renal tubular function. addition to fever of more than five days of duration, 4 of the following symptoms, rash, conjunctival injection, changes of lips or oral mucosa, erythema and swelling of hands and feet and cervical lymphadenopathy, was the diagnostic criteria for kd. results: among the 15 patients, 80% showed increased white blood cell s (wbc) and 47% showed elevated ast/alt level. serum albumin was below 4.0 g/dl in 93% of the patients. all of the patients presented normal renal function test. in urinalysis, hematuria and pyuria were observed in 13% and 33% respectively. echocardiography revealed coronary artery aneurysm in 33% of the patients. urine β2-microglobulin was elevated in 46%. regardless abnormal findings in urinalysis and elevated β2microglobulin, no significant findings were observed in dmsa renal spect. conclusion: according to the study, mild abnormality in the urinalysis and elevated β2-microglobulin were the only findings of renal involvement in kd. however there was no aggressive renal manifestation which could be detected in dmsa renal spect. objective: in this study, the question remains if ngal is a culprit or only bystander in aki due to sepsis in children. methods: twenty seven children, (m-17, f-10) admitted to intensive care diagnosed with sepsis, severe sepsis or septic shock were enrolled in this study. the concentration of ngal, protein c and s, antithrombin iii and basic parameters were measured in the plasma at diagnosis and after 10 days of treatment. results: mean creatinine concentration in septic children at admission was higher than in reference group (p <0.05). there was statistically significant difference in aptt, prothrombin time, prothrombin ratio, thrombocytes count, heart rate, and systolic blood pressure between study groups in first and after 10 days (p<0.005, p<0.001, p<0.001, p<0.01, p<0.05, p<0.05 respectively). the pc concentration in the septic patients was significantly lower than those of the references (p<0.0001), and during intensive 10 days treatment raised (p<0.01). also protein s concentration rose during the treatment (p<0.005), and was lower in septic children then reference group (p<0.0001). ngal concentration was significantly higher in study group on the admission day comparing with reference group (p<0.05). plasma ngal was correlated at admission and after 10 days of treatment with protein c (respectively: r=-0.48, p<0.00005; r=-0.53, p<0.05). multivariate regression revealed that ngal was significantly predicted by creatinine (β=0.39, p<0.01) and protein c (β=-0.38; p<0.01), yielding a model r2 =0.34 (p<0.001). conclusions: it is possible that kidney binding of pathogenic antibodies stimulates local expression of ngal, which plays a crucial role in the pathogenesis of aki. objective: the aim of the study was to assess the impact of perinatal risk factors (prfs) on serum ngal level in term neonates. methods: the study group consist of 62 term neonates with prfs, and 14 healthy neonatescontrol group (cg). serum ngal (elisa) was measured in samples of cord blood (cngal) and peripheral blood (pngal) taken within first 5 days of life, and stored in -80°c until elisa procedure was performed. prfs were divided into 3 groups: 1. pathology of pregnancy or mother: diabetes (d) n=23, infection (i) -elevated crp or wbc n=20, positive vaginal culture (vc) n=21, hypertension (ht) n=7, others n=5; 2. labor pathology: instrumental delivery (id) n=41, premature rupture of membranes, >6 hours (prom) n=26, fetal distress (fd) n=26; 3. neonatal pathology: intrauterine infection (ii) n=10, cakut n=5, perinatal asphyxia (pa) n=4. the results were shown as median; values of cngal, pngal were logtransformed before analysis. statistical analysis was performed with t-student, manova tests with help of statistica 10. statistical significance: p<0.05. results: in 62 term neonates, median cngal was 117.69ng/ml vs 64.37ng/ml in cg. mean log cngal was significantly higher (p<0.01) vs cg, pngal was ns. mean log cngal correlated positively with mean log pngal (r=0.36, p<0.01). univariate analyzes of log ngal in neonates with chosen prfs vs neonates without particular factor showed significantly higher (p<0.05) mean log cngal in id and fd group (median cngal: 126.47 vs 67.50 and 122.30 vs 77.01ng/ml respectively), whereas in ii group significantly higher were mean log cngal and mean log pngal (median cngal 209.69 vs 96.52ng/ml, pngal 457.35 vs 156.37ng/ml). multivariate analysis showed that the impact of id was significant in groups: d (f=9.84; p<0.002), i (f=4.56; p<0.04), prom (f=6.01; p<0.02) and fd (f=5.36; p<0.02). conclusion: 1. intrauterine infection may have a significant influence on ngal in cord and peripheral blood whereas fetal distress and instrumental delivery only in cord blood. 2. the instrumental termination of pregnancy has an import impact on ngal level in cord blood of neonates whose mothers had diabetes or infection, with prom or fetal distress. objective: acute kidney injury (aki) is a common problem and associated with significant morbidity and mortality in neonates. pediatric-modified rifle (prifle) classification system was developed to standardize the definition of aki in children. we aimed to evaluate the performance of prifle score diagnosis, severity and prognosis of aki in term and preterm neonates. methods: in this retrospective study, charts of 820 patients who were admitted to neonatal intensive care unit (nicu) over a 4 year period were reviewed for development of aki. a diagnosis of aki was determined for 254 patients (30.9 %) according to the prifle criteria results: of the 254patients included in this study, 101 of them were girls and 153-were boys. mean age was 4.12days (min-max: 0-49), mean birth week was 33.6 ±5.1 weeks (min-max: 24-42), mean birth weight was 2166 ± 1045 gr (min-max: 580-4600 gr), length of nicu stay was 37.3 ± 33.5(min-max: 2-165days). two hundred fifty four patients with aki were classified according to prifle criteria. ninetyeight patients (38.5%) achieved an 'r' level of aki severity, 86 patients (33.5%) an "i" level, 66 patients (26%) an "f" level, 4 patients (1,5%) an "l" level. the most common etiologies of aki -were prematurity, congenital heart disease, hypoxic ischemic injury, sepsis and usage of nephrotoxic agents. patients classified with a higher prifle score had a longer nicu stay and required a greater number of mechanical ventilation (p<0.05). renal replacement therapy (rrt) was needed for 30 patients (11.8%). mortality rate was 26% and all deceased patients were classified as prifle 'i','f' or 'l',p < 0.05) conclusion: using prifle criteria in neonates with aki is an efficient way to assess the severity and prognosis of the disease. objective, methods and results: i present a case of a 11 years albanian old boy, 25th centile of development. presented with abdominal pain, vomiting. four days after, was addmited in the surgery clinic for uregent apendicitectomy. wbc: 23.0, se: 64/, bun: 6.1, cre: 60, urine: normal. after the intervention medications that were used were: gentamycin, metronidazol, cephtriaxon, h2 blocators. two days after he was better. thereafter he had polyuria and frequent urinating. the fifth day he had oliguria to seventh day, when he couldn't urinate. nativ rtg of abdomen resulted normal. he was transferede to pediatric clinic-nephrology department as an akute kidney injory. he looked very sick, consious, stratified tongue, ecg: 50b/min, bp: 115/60 mmhg, breathing sounds were normal, periferal edemas and cold extremities. lab resultrs: cre: 624, bun: 16.6, k: 5.1, na: 131, egfr: 8.8 ml/min oer 1.73m2. ultrasound resulterd with urinary stasis: hydronephrosis, huge bladder. first was suspected for acute obstruction and aki couse of a medication. we changed a bladder cathether which we find out that was blocked and took out 2700 ml urine/24h. we dialysed him for three days and cre was stabilized to 266, when we stoped dialyse. bp was stabile, his weight droped from 32kg to 27.5 kg. he had proteinuria 2 g/24 h. schintigraphy was done (biopsy we couldn't do) and resulted: egfr was 12% less than adecuat for age, suspected for nephritis tubulointestialis. he had some toxoalergic exanthema spread to the body. repeted us resulted with ascaridosis in the gallbladder, which was treated with mebendazole and in the feces were found the parasites. imune ab were negativ, ige: positive, periferal blood smear had eosinophils, wastage of na and mg with urine. cre after four weeks was 58, bun:5, negative proteinuria. no changes of uveitis in the eyes. importance of this case is cause this is the first case in kosova that was dialysed and has recovered and now is free of dialyse (all other cases have traveled abroad until then). conclusion: we have to think for parasites as a cause of a tubulointesticial nephritis but also of a abdominal pain or fatigue. surgery team should have a close cooperation with pediatricians, in order to identifye in time complications of the surgery. urine output in first 48 hours after birth in relation to akin criteria saroj kumar patnaik, gaurav aggarwal, uday kumar, vempati venkateshwar, shamsher singh dalal pediatrics, command hospital air force, bangalore, india objective: applicability of akin urinary output (uo) criteria in neonates during first 48 hours of life remains contentious since nonpassage of urine during this time has traditionally been considered 'normal'. we hypothesize that 'physiological' oliguria in first 48 hours of life is mostly prerenal in origin related to poor fluid intake during transition and uo criteria should be applicable in the 1st 48 hours of life too. we aimed a) to investigate pattern of uo in babies admitted to nicu with underlying morbidity but regulated fluid intake versus roomed-in babies in postnatal ward on breastfeeds in first 48 hours of life; b) to relate uo till 48 hrs of life with development of aki methods: prospectively timing of first void and 6 hourly uo from birth till 48 hours age was compared between consecutive postnatal ward and nicu admissions. neonates with structural malformations and requiring ventilatory/vasopressor support were excluded. outcomes-primary : time of first void after birth secondary: uo in 1st 48 hrs in relation to subsequent aki results: amongst eligible 87 postnatal breastfed babies (mean bwt 2.86 kg ; sga 18; 27 lscs born) and 110 nicu admissions (median birthweight 2.01 kg,mean ga 34 wks (26-43 wks); 67 prematures (10 < 30 wks); 82 sga (6 elbw) recruited after informed consent, first voiding was significantly earlier for nicu babies (mean(95%ci) (hrs) postnatal 4.12 (3.29, 4 .93) vs nicu 2.31 (1.50,3.12) hrs). nonvaginal delivery(lscs 3.00 (1.89,4.11) vs vaginal 4.68 (3.60,5.75 ) and normal birthweight (aga 4.08(3.10, 5.06) vs sga 4.22(2.81, 5.63) ) had a trend for earlier voiding. 80% babies voided within 6 hrs of birth -more in nicu babies (90.4% nicu vs 76.7% postnatal (p<0.004) (fig) .asphyxia with mas in nicu and iugr and poor feeding in postnatal ward had delayed first void beyond 12 hours. 6 postnatal and 7 nicu neonates anuric in 1st 6hrs met stage i akin creatinine criteria. 2 asphyxiated babies anuric beyond 12 hours in nicu developed stage iii aki. conclusion: 'physiological' anuria in 1st 48 hrs of life is misnomer and is related to fluid intake. most babies are nonoliguric by 6 h; anuria >12 h needs investigation. akin urinary criteria should be applicable in neonates. odillha morales maglalang-reed nephrology, philippine children's medical center, quezon city, philippines objective: the precise mechanism of renal injury among dengue patients is not known. patients who have atn will usually require early dialysis. however, on admission to the hospital, it is difficult to distinguish dss patients with atn from patients with reversible prerenal causes that will respond to simple hydration. our understanding of the complex pathogenesis of tubular injury in dengue aki is very limited that until it is sufficiently increased, therapeutic strategies will continue to fail.therefore we sought to explore the limitations of serum creatinine in this setting. general objective is to determine the clinical and diagnostic factors which are predictive for the need for dialysis among dss patients at pcmc. specific objective is to determine if the following factors are predictive of the need for dialysis: decrease in estimated creatinine clearance by 75% or <35 ml/min/bsa with urine output of <0.3ml/kg/hr. x 24 hours or anuria of 12 hours, assess the usefulness of urinary sediment scoring (uss) in predicting the need for dialysis, methods: this retrospective study covered 60 newly admitted cases of dengue shock syndrome lll and lv at the philippine children's medical center between january 2010 to december 2011. results: data from 60 patients were available for analysis. comparison of the demographic characteristics between patients who required dialysis and those who did not showed no significant difference as proven by all p values >0.05. of the differentclinical and laboratory parameters, there was a significant difference in the hr, rr, o 2 saturation, bicarbonate and base excess as proven by all p values <0.05. the hr an rr were significantly higher among those who needed dialysis than those who did not o 2 saturation, bicarbonate and base excess were significantly lower among those who needed dialysis than those who did not. the estimated creatinine clearance was significantly lower among those who needed dialysis than those who did not. urine sediment score (uss) ≥3 was significantly higher among those who needed dialysis than those who did not. conclusion: our data indicate that a decrease in estimated creatinine clearance by 75% or <35 ml/min/bsa with urine output of <0.3ml/kg/hr. x 24 hours or anuria of 12 hours is not predictive of dialysis among dss-induced aki as well as other clinical and laboratory indices. but rather, relatively lower serum creatinine level among dssinduced aki who needed dialysis is associated with greater urine sediment scorecompared with those who did not. a uss ≥ 3 correlated with atn and was predictive of early dialysis.however, estimated crea < 50 is the only factor predictive of dialysis, since this is the only one that was positive after logistic regression. abstract# p-sat327 pre-operative fgf23 predicts acute kidney injury in pediatric cardiac surgery patients: a prospective study mark r hanudel, myke federman, barbara gales, georgina ramos, vicky campbell, kristen ethridge, mary scotti, brian reemtsen, isidro b salusky, katherine wesseling-perry pediatrics, ucla, los angeles, usa objective: fibroblast growth factor 23 (fgf23) is a phosphaturic hormone that predicts renal disease progression in ckd. however, in the setting of aki, there is a paucity of prospective data on fgf23, especially in the pediatric population. thus, we prospectively measured fgf23 levels in pediatric patients undergoing cardiopulmonary bypass (cpb) and assessed the ability of fgf23 to predict aki. methods: pediatric patients, age newborn to 21 years, without underlying ckd, undergoing cardiac surgery requiring cpb were eligible for the study. plasma fgf23 levels (2nd generation cterminal, immunotopics) were measured pre-operatively and at 2, 6, 12, 24, 48, and 96 hours post-reperfusion. serum creatinine was obtained at baseline and daily post-reperfusion. aki was defined by the akin criteria, estimated gfr was calculated using the schwartz formula, and cardiac surgery complexity was classified via rachs-1 score. results: of the 20 enrolled patients, 13 developed at least stage 1 aki. gender, weight sds, height sds, baseline egfr, cardiac surgery complexity, and cpb duration did not differ between the aki group and the non-aki group. patients who developed postoperative aki were younger than those who did not. preoperative fgf23 levels were inversely related to age (r = -0.67, p = 0.001). pre-operative fgf23 levels were significantly higher in patients who developed aki than in those who did not; this remained significant upon correcting for age. post-operatively, fgf23 levels increased in all patients. conclusion: pre-operative fgf23 levels predict the development of post-operative cpb-associated aki. fgf23 levels increase in non-ckd patients undergoing cpb, even in patients without significant changes in serum creatinine, suggesting that fgf23 may be a more sensitive marker of aki than serum creatinine. non-aki pateints p value objective: improved perinatal care increased not only the survival rate but also the frequency of acute kidney injury (aki) in newborns. we aimed to determine the frequency, etiology, clinical course and mortality of aki in a third level neonatal intensive care unit (nicu). methods: medical records of all patients admitted to a nicu in western turkey during 2007-2011 were evaluated and those having aki within 0-30 days of life were determined. birth weight, gestational age, mode of delivery, gender, maternal morbidity, hospitalization period, accompanying morbidities and mortality of all patients; and primary disease causing aki, highest serum creatinine and prognosis of patients with aki were recorded. results: there were 677 patients (m/f:392/285) and 94 (13,9%) had aki of which 80% developed during 0-7 days of life mostly due to birth asphyxia, hypovolemia, cardiac disease, sepsis and urinary system anomalies. aki incidence and total mortality rate were higher in patients with birth weight <1000 g and with gestational age <28 week. however, mortality in patients with aki was increased independent on birth weight (42,1% in <1000 g vs 32,1% in >1000 g; p=0,324) and gestational age (44,1% in <28 week vs 31,7% in >28 week; p=0,227). mortality tended to increase in the presence of aki independent on the underlying cause, but this was significant only for sepsis (33, 3% vs 5, 9%, p<0, 001; or 7, 9) and cardiac diseases (50,0 %vs 15,0%, p=0,001; or 5, 6) . hospitalization period was increased if aki was present (39,1 vs 20,5 days, p<0,001). serum creatinine in deceased patients with aki was higher than those who survived (2,0 vs 1,6 mg/dl, p<0,05). chronic kidney disease was developed in 6 (10%) patients of whom 3 had urinary tract anomalies and 3 had birth asphyxia. conclusion: aki incidence in nicu was 14%, and 80% of aki developed during the first week of life. aki is associated with low birth weight, prematurity, birth asphyxia, sepsis, hypovolemia, cardiac diseases and urinary tract anomalies. mortality in patients with aki is increased independent on the birth weight, gestational age and underlying etiology. objective: cisplatin (cddp) is one of the antineoplastic agents widely used for solid tumors in adults and children. an excessive dose due to medical error causes severe nephrotoxicity, ototoxicity and myelosuppression. previous reports described the effects of plasma exchange, sodium thiosulfate, n-acetylcysteine and other interventions. however, the strategy for over-dose patients, as regards the selection or intensity of therapies and the target of cisplatin removal, remains uncertain in children. a 12 year-old girl was admitted for headache and vomiting. she was found to have a right cerebellar tumor and underwent total extirpation of this medulloblastoma. next, we administered chemotherapy mainly using cddp. however, she developed renal insufficiency and hearing loss on the fourth day. we then realized that we had been administering cddp 90 mg/m 2 for 4 days, instead of the intended 1 day. we promptly discontinued the cddp and began plasma exchange and sodium thiosulfate administration. nonoligiric renal failure was confirmed by a urea nitrogen value of 63.6 mg/dl and creatinine level of 4.4 mg/dl. with 4 cycles of plasma exchange and 2 weeks of sodium thiosulfate, renal function improved and the cddp concentration decreased from 6.04 to 0.99 mg/ml. after 1 month, the concentration was 0.34 mg/ml. we switched from cddp to carboplatin, and chemotherapy was continued for 4 courses and then radiation therapy was added. ultimately, she had mild renal insufficiency and moderate hearing loss. we evaluated the relationship between renal function and the cddp concentration. severe renal failure and hyponatremia were observed at a cddp concentration of 1 mg/ml or more. creatinine clearance of 60 ml/min/1.73 m 2 was maintained at a cddp concentration below 0.5 mg/ml. conclusion: in children with a cddp over-dose, plasma exchange and sodium thiosulfate administration are effective. also, monitoring of cddp concentrations is recommended in patients with acute renal failure. in over-dose patients, early severe renal failure can be prevented when the cddp concentration is reduced to less than 0.5 mg/ml with these therapies. abstract# p-sat331 peritoneal dialysis in children with acute kidney injury: a developing country experience om p mishra 1 , aditya k gupta 1 , vishal pooniya 1 , rajniti prasad 1 , narendra k tiwary 1 , franz schaefer 2 1 pediatrics and medicine, institute of medical sciences, varanasi, india 2 division of pediatric nephrology, heidelberg university medical centre, heidelberg, germany objective: peritoneal dialysis (pd) is the preferred and convenient treatment modality for acute kidney injury (aki) in children and hemodynamically unstable patients. the present study analyzed the efficacy of pd in patients with aki and factors contributing to mortality. methods: the outcome of acute pd was studied in 57 children (39 males) with aki, aged 1 month to 12 years, at a tertiary care centre of a teaching hospital in india. results: there were 14 patients less than 1 year of age, 23 patients 1-5 years of age, and 20 patients more than 5 years of age. per the rifle criteria, 5 patients were classified at the risk stage; 13, at the injury stage; and 39, at the failure stage at the time of the decision to start pd. hemolytic uremic syndrome was the most common cause of aki (36.8%), followed by septicemia (24.6%) and acute tubular necrosis (19.3%). treatment with pd was highly effective in lowering retention markers (blood urea decreased by 40% and serum creatinine by 34% during the course of dialysis therapy, both trends significant at p < 0.001). overall mortality was 36.8%. deaths occurred 2-7 days after hospitalization. significantly higher proportion of non-survivors had fluid overload (66.7% vs 25%, p= 0.002) and septicemia (47.6% vs 11.1% , p< 0.001) than survivors at presentation. the risk of mortality by multivariate analysis was higher when patients were anuric [odds ratio (or) 8.2, 95% confidence interval (ci) 1.3-49, p<0.05), had septicemia (or 3.79, 95% ci 1.55-25.8, p<0.05), or severe infectious complications (or 8.2, 95% ci 1.5-42.9, p<001). conclusion: because of its simplicity and feasibility, acute pd is still an appropriate treatment choice for children with aki in resource-poor settings. septicemia and severity of aki are contributory factors to high mortality in pediatric acute kidney injury. outcome of acute kidney injury managed in a regional paediatric nephrology centre shivaram hegde, sabina pahari paediatric nephrology, university hospital of wales, cardiff, uk objective: this study reviewed the aetiology, treatment modalities and outcome of children with aki managed in our tertiary paediatric nephrology unit. method: retrospective analysis of referral practices, aetiology, and management of 38 children treated for aki over the last 5 years. children primarily treated in intensive care units were excluded. outcomes noted as complete recovery, residual renal injury, renal replacement therapy (rrt) dependency or death. they were followed up until their renal function normalised and any proteinuria or hypertension resolved. result: out of the total 38 children aged 5 months to 16 years, 34% were under 5 years. haemolytic uremic syndrome (hus) was the commonest aetiology in 18 cases (47%), 15 of them secondary to ecoli 0157 and 3 with atypical hus. obstruction was second most common (5) and renal function improved following relief of obstruction. supportive management sufficed in 23 (60%) cases and 15 (37.5%) received renal replacement therapy (rrt); peritoneal dialysis being the commonest mode. most children needing dialysis were oliguric (14). at discharge there were no deaths, 5 patients showed complete recovery of renal function, one was dialysis dependant and renal function was improving in the rest. at 3 months we found normal renal function in 26 (68%) children and chronic kidney disease (ckd) stage ii in 7 (18%) and ckd stage iii in 4. the dialysis dependent child underwent renal transplantation. based on the data from 12 patients currently under follow up (for 12-62 months, mean 41 months), 32 (84%) children have recovered completely and 5 have developed ckd; stage i in 2 and stage ii in 3. conclusion: prognosis following aki was excellent in our patients, probably because of lack of multiorgan dysfunction. hus was the commonest cause and urgent renal imaging needed when obstruction is suspected. oliguric patients are more likely to require dialysis and need early referral to the regional unit. all cases should have long-term follow up to ensure renal recovery and detect delayed complications. clinical course and outcome of acute kidney injury (aki) due to childhood haemolytic uremic syndrome (hus): a single centre experience shivaram hegde, sabina pahari paediatric nephrology, university hospital of wales, cardiff, uk objective: clinical data, along with the management and outcome of 15 children with aki due to hus, caused by shigatoxin-producing ecoli (stx-hus) are described. method: we analysed the data of children with stx-hus induced aki, managed in our unit over the last 5 years. outcomes noted as complete recovery, residual renal injury, dialysis dependency or death. all patients were followed up at least for a year and further monitoring continued until their renal function normalised and any proteinuria or hypertension resolved. methods and results: stx-hus was the commonest cause of aki in our unit, accounting for 15 of the total 38 cases (39.5 %) treated for aki during this period. 11 patients were less than 10 years of age. all children presented with blood in stool and 11 with oligoanuria. all showed microangiopathic haemolytic anaemia and thrombocytopenia. positive stool cultures (for ecoli 0157) were obtained in 11 and 4 had ecoli 0157 lipopolysaccharide serum antibodies. 10 patients required dialysis (peritoneal dialysis in 7, haemodialysis in 1 and both modes in 2) and one child needed plasma exchange. the remaining responded to supportive management. morbidities encountered included bowel perforation (1), hypertension (2), seizures (2) and diabetes mellitus (1) and three of these children needed intensive care management. at discharge there were no deaths, none with dialysis dependency or complete renal recovery but all showing improving renal function. at 3 months we found normal renal function in 12 (80%) and chronic kidney disease (ckd) stage ii in 2 and ckd stage iii in 1. a 13 year old girl with an unknown myopathy presented for the third time with clinical and laboratory features of rhabdomyolysis. on day two of admission, her renal function deteriorated with decreased urine output, increasing creatinine of 380 μmol/l and ck of 240,220 iu/l. she was commenced on haemodialysis and started on calcium supplements, as her calcium levels were 1.6 mmol/l. three week into her illness, she was symptomatic with high blood pressure of 170 mm of hg leading to a generalised seizure. her serum calcium was 4.01 mmol/l. the ct brain did not reveal any evidence of haemorrhage or infarction. there were no other trigger factors identified for the seizure and the hypertension, apart from the hypercalcaemia. the hypercalcaemia was managed with low calcium dialysate, calcitonin and sevalamer. despite the above treatments, she continued to be hypercalcemic and developed erythematous palms and soles and an injected conjunctiva. in view of the refractory hypercalcaemia, she received two doses of intravenous pamidronate (0.5 -1 mg/kg) when the creatinine was 317 mmol/l. following this, her calcium levels normalised to 2.59 mmol/l. her renal function improved and was discharged a week later. there was no nephrocalcinosis seen on follow up. bisphosphonates are used in children with caution, as there is little evidence on its safety and efficacy. they act by binding to the surface of calcium phosphate crystals and inhibiting osteoclast formation, aggregation and dissolution. it is nephrotoxic causing acute tubular necrosis and collapsing focal segmental glomerulosclerosis. pamidronate is usually used in children with chronic kidney disease but this was the first time it was used in a child recovering from acute kidney injury secondary to rhabdomyolysis. there were no complications with the use of pamidronate in our patient. chronic kidney disease during long-term follow-up in children treated with neonatal extracorporeal membrane oxygenation: do we need to worry? objective: acute kidney injury (aki) is a common complication in children receiving extracorporeal membrane oxygenation (ecmo) support. as aki may cause loss of a significant number of functioning nephrons, these children are at risk of developing chronic kidney disease (ckd) post-ecmo. therapeutic interventions might be needed to prevent further renal function deterioration or comorbidity of ckd in these patients. the objective of our study was to determine the prevalence of ckd during long-term follow-up (fup) of children treated with ecmo. methods: this was a cross-sectional study performed between 2010 and 2013. all children previously treated with neonatal ecmo who visited our fup clinic at the age of 1, 2, 5, 8, 12 and 18 years were screened for ckd. if more check-ups were available per patient, only the latter one was used for the study. ckd screening included height (ht) and blood pressure measurements (bp), and laboratory parameters including serum creatinine (scr) (schwartz formula [0.413*ht (cm)/scr (mg/dl)] or mdrd formula were used to estimate gfr) and urinary protein/creatinine (up/c) ratio. ckd was suspected in patients with hypertension (>95th percentile of reference values according to height and age), abnormal egfr (<90 ml/min/1.73m 2 ) or proteinuria (up/c ratio >30 mg/mmol creatinine). patients were excluded if scr was lacking. results: to date, 132 children visited the fup clinic. of these, 4 (3%) were excluded because of pre-existent kidney disease and 6 (5%) because of missing scr data. hence, 122 children (44% female) were screened for ckd. the number of patients per fup age category was 17 <5 years, 56 between 5-12 years, and 49 >12 years. bp was within normal ranges in all children. in 13 (11%) children either an abnormal up/c ratio or egfr was observed. up/c ratio was increased (median 36 [iqr 32-38 mg/mmol creatinine]) in 8 (7%) children, of which 1 was explained by low muscle mass. only 5 (4%) children had an abnormal egfr but all >60 ml/min/1.73m 2 . conclusion: the prevalence of ckd and its clinical implication in children previously treated with neonatal ecmo seems to be limited. future research will focus on identifying risk factors for ckd following ecmo support. abstract# p-sat336 10 years clinical retrospective analysis in children of acute poisoning inpatient man jiang, qiu li nephrology and immunology department, children's hospital of chongqing medical university, chongqing, china objective: acute poisoning is the common critical and emergency disease in children. since childhood is the special life stage with continuous growth, clinical features are different from the adult and changes were happened in resent years. the change rules, clinical features, treatments and the outcomes of acute pediatric poisonings inpatients were investigated in this article. methods: retrospective evaluated the 1005 cases of pediatric acute poisonings admitted to the children's hospital of chongqing medical university. cases were divided into 5 groups by ages, or divided into 2 groups based on different origins (urban or suburban), different causes and routes of poisonings in different groups were calculated. the clinical manifestations, treatments and prognosis in acute intoxication were also studied. results and conclusion: pediatric acute poisoning mainly happened in 1-4 years old children (50.02%), there was no statistical difference between genders. the common causes of poisonings were pharmacological poisoning (26.07%), food poisoning (19.50%), animal bites and stings (16.92%) and pesticide poisoning (14.73%). in the resent five years, pharmacological poisoning rose to be the top reason of poisonings, and was mainly composed by children≤3 years old (p < 0.001),and urban children were more than children from suburban(p < 0.001). neurological and psychiatric drugs were the most common (32.06%) in drug poisoning. rural children with animal bites and stings, pesticide poisonings and rodenticide poisonings were more than the urban children (p < 0.001, p < 0.001, p < 0.05). the main poisoning causes in 6-12 years old group and >12 years old group children were food poisoning (29.20%) and pesticide poisoning ( objective: little is known about cardiac surgery-associated acute kidney injury (cs-aki) in children in developing regions of the world. the study aimed to determine the prevalence of cs-aki, associated factors and its impact on mortality and utilization of hospital services. methods: hospital records of children aged 0-17 years that underwent cardiac surgery (other than device closure procedures and pacemaker insertion only) at an indian hospital between 2011 and 2012 were reviewed. cs-aki was defined as a rise in serum creatinine of ≥0.3 mg/dl in any 48 hours and or by urine output less than 0.5ml/kg/hr for an 8-hour period in the first 5 days after cardiac surgery. results: the study included 323 children with a median age of 1 year (0.04-17), of which 22 (6.8%) were neonates and 18.3% had single ventricle. about 60% of the children had rachs-1 1 or 2 interventions. cs-aki occurred in 39 children (12.1%), most often in the first 48 hours after cardiac surgery. on univariate analysis cs-aki was associated with sepsis and intra and post-operative hypotension. in-hospital mortality was 6-fold higher in children who developed cs-aki. cs-aki was associated with 2-3 days longer duration of mechanical ventilation, inotropic support and icu stay. conclusion: cs-aki occurs in children in developing countries but at a lower frequency mainly due to older children with less complex congenital heart disease undergoing cardiac surgery. cs-aki was associated with higher in-hospital mortality and increased utilization of hospital services. factors associated with cs-aki included intra and post-operative hypotension and sepsis. objective: data on long term effects of newborns after acute kidney injury is limited. the aim of the study was to evaluate long term effects of acute kidney injury (aki) in neonatal period. methods: inclusion criteria were as follows: oligo-anuria or plasma creatinine >1.5 mg/dl during first three postnatal days or >0.7 mg/dl after third postnatal day or ≥25% increase in plasma creatinine within 48 hours during hospitalization in neonatal period. three hundred sixty children who had aki during neonatal period (between january 2000 and december 2009) and survived to hospital discharge were invited; 106 of them accepted to participate. patients' characteristics during aki and during follow-up were recorded; a physical examination and laboratory studies, including acr (urine microalbumin/creatinine ratio) were performed. schwartz formula was used to estimate gfr; hyperfiltration was defined as a gfr >160 ml/min/1.73 m 2 . hypertension was defined as blood pressure ≥95 th percentile for age, gender and length. results: the mean age on evaluation was 6.78±2.9 years. 15.8-15.8-12 .2% of patients. all these children were dialyzed for more than 7 days on acute phase of the disease. at time of discharge from hospital proteinuria remained in 53 (85.5%), hypertension -in 47 (75.8%) patients. after 1 year, 5years and >10 years period proteinuria was detected accordingly to 26.3-7.9-33.3%, hypertension to 11.3-6.5-12.9% of patients. after >10 years from the onset of disease hypertension was detected more often in the group of children who were < 1 years old on acute phase of the disease (40% vs 11.1%, p=0.05). in the group of patients who hadn't proteinuria on the acute phase, after >10 years period proteinuria was observed to 25% of them. after 1 year period renal impairment of various degree was observed to 26.3%, after 5 years -to 39.5 %, after >10 years-to 33. . on admission his mean arterial pressure was 35 mmhg, his creatinine was 5.8, potassium 6.5 and sodium 121. usual medical management for aki was initiated but even by late d5 he continued to be anuric with a rising creatinine. his uric acid was grossly elevated at 14 mg/dl. dialysis was considered but as the parents were not in favour of it they were offered the option of rasburicase. rasburicase was given on late d5 and within 12 hrs he started to produce urine which peaked at 4 ml/kg/hr by d9. the creatinine also showed a concomitant fall and by discharge (d10) it was 2.1 mg/dl and normalized at 0.5 mg/dl by d30. uric acid was 0.5 mg/dl on d7 and 2.8 mg/dl on d9. case 2: 6 years old boy with multiple special needs was transferred from another hospital intubated, anuric and in multi organ failure (mof). creatinine was elevated at 3.7 mg/dl and uric acid was also found to be raised at 9.1 mg/dl. in view of mof along with disseminated intravascular coagulopathy (dic) dialysis was considered a risky process and parents were offered the option of rasburicase. rasburicase was given on d2 of admission and although the repeat uric acid was 0.3 mg/dl and he did produce 5 ml of urine within 6 hours no further improvement in renal parameters was noticed. dialysis (sled) was initiated on d3. although the creatinine improved with sled he continued to be oligo-anuric and died of mof by d5. conclusion: although rasburicase seemed to work in case1, it was not beneficial in case 2. the exact mechanism for rasburicase is still hypothetical and the few case reports have all documented positive results. although it seemed to have worked in case 1, to the best of our knowledge case 2 is the first documented case wherein it did not work. proper randomized control trial is needed before advocating rasburicase as a novel therapy for aki. results: age, sex, diagnosis, baseline and post-surgery hemoglobin, total leukocyte count, platelet count and biochemistry were recorded. baseline and post-operative, urea (mg/dl), creatinine (mg/dl), urine output (ml/kg/hr) and inotrope dose were also recorded daily. the duration of cpb was noted. post-operatively cardiac, renal, hepatic, neurological and respiratory dysfunctions were recorded. conclusion: fifteen (7.2%) children developed aki stage i, one child (0.5%) developed aki stage ii and four children developed aki stage iii (2%). all patients with aki had a longer stay in hospital. eight children required dialysis for aki; two required dialysis to maintain the fluid balance post operatively. none developed chronic renal impairment. using stepwise regression, younger age (<1 year), weight < 10kg, pump failure, sepsis and duration of cpb more than 60 minutes were significant risk factor for developing aki. results: most of the patients were male, with less than 28 days old and weighing less than 5kg.in the majority of cases (77%), arf was diagnosed in the first 6 days after surgery, demonstrating influence of the procedure itself. all of them were treated by peritoneal dialysis(pd) for a mean time of 255 hours; 58.3% of them died. there was no statistical significant difference between age, time on extracorporeal circulation and duration of dialysis, comparing the patients who survived and the deceased ones. conclusion: acute renal failure is a common complication in children with congenital heart disease and pd is a safe dialytic method. the mortality rate is high and influenced by aspects related to the child, underlying disease, type of surgery and many other associated aspects. abstract# p-sat344 acute kidney injury epidemiology and associated factors in a pediatric intensive care unit. conclusion: acute kidney injury (aki) is associated with significant morbidity and mortality in pediatric patients with critical illness. the main causes associated to aki were sepsis and shock. we found no association with cardiac surgery, probably explained by the low level of complexity that is performed in our center. almost half of our patients with aki were in failure and the mortality was high as previously reported. objective: fibroblast growth factor-23 (fgf-23), a phosphaturic hormone involved in calcium phosphate homeostasis, appears to predict renal disease progression in adults with non-diabetic ckd (fliser, jasn 2007) . this study aimed to determine renal survival according to fgf-23 serum levels in children with ckd stage ii-iv. methods: 232 children participating in the escape trial (age 11.5±4 yrs, gfr 45±18 ml/min/1.73m 2 ; underlying renal disease: hypo/dysplasia (71%), glomerulopathies (12%), hereditary or other (17%)) were analyzed. all patients received fixed dose ace inhibition and were followed prospectively by 2-monthly examinations for up to 5 years. the study endpoint was defined by egfr loss >50% from baseline, gfr <10ml or start of renal replacement therapy. fgf-23 levels were determined at baseline (c-terminal human fgf-23 elisa (immutopics, san clemente,ca,usa objective: the long-term outcome of patients born with unilateral renal agenesis (ura) and of those who underwent therapeutic unilateral nephrectomy (un) remains a topic of concern and debate. children with a solitary functioning kidney (sfk) have an increased risk of developing hypertension, albuminuria and chronic kidney disease in later life. the purpose of this study was to identify whether plasma symmetric dimethylarginine (psdma) is an useful biomarker reflecting the level of renal injury in children with solitary functioning kidney (sfk). methods: we measured circulating psdma in 51 patients with sfk and no other urinary defects. patients were subdivided for two groups: primary sfk (psfk)-unilateral renal agenesis (ura) and secondary sfk (ssfk) after unilateral nephrectomy. the control group (c) consisted of 21 healthy children, aged mean 9.92 ± 4.85 yrs. immunoenzymatic elisa commercial kits was used to measure psdma concentration. data analysis was performed using computer program statistica 9.0. results: the age and sex of studied children did not differ from healthy controls (p> 0.05). plasma sdma levels in sfk children were higher than in healthy participants (p< 0.05). there was no difference in psdma concentrations between psfk and ssfk patients (p> 0.05). sdma plasma levels correlated significantly with c cr (r= -0.32, p< 0.01) in all participants. roc analyses performed in order to define the diagnostic efficiency of serum creatinine and psdma in identifying children with c cr < 90ml/ min/ 1.73m 2 among sfk and healthy participants revealed no difference between all two aucs (p> 0.05). conclusion: in children with a solitary functioning kidney increased psdma levels were observed, however the sensitivity and specificity of this marker in detecting the decrease in c cr was not better than creatinine. objective: renal osteodystrophy encompasses a variety of skeletal disorder ranging from high turnover lesions of secondary hyperparathyroidism (shpt), to low turnover lesionsof diverse etiology that are usually associated with normal or reduced pth levels. so our aim was to assess by dexa which is a precise , rapid and noninvasive procedure the degree of osteopenia in patients with crf. methods: 53 children aged (11-3.84) years, 17 on conservative treatment and 36 on hemodialysis were included in the study bmd of lumbar spine and wrist were measured by (dexa) and compared with age and sex matched controls. results: shows that out of 53 patients, 25 (47%) are osteopenic 22 (88%) on hemodialysis and 3 (12%) on conservative treatment, of these 13 (24.4%) had severe osteopenia as regard bmd of the spine, while dexawrist shows 11 (21%) are osteopenic, 7 (64%) on regular hemodialysis and 4 (36%) on conservative iii, of these 3 (5.66%) had severe osteopenia ,correlation between z-score spine in the osteopenic group and different biochemical parameters shows non-significant correlation except -ve correlation with duration and age of the patients, while z-score wrist of the same group shows +ve correlation with bicarbonate. our result shows that group with ipth >= 200 pg/ml are more osteopenic than those with lower ipht levels, although difference did not reach level of statistical significance p > 0.05. conclusion: we can conclude that osteopenia is frequent in patients with crf more in the dialyzed group, with longer duration of the disease, older age and severe acidosis, irrespective of the severity of the disease. although, degree of osteopenia is not correlated with biochemical findings of (shpt) but still patients with (shpt) are more osteopenic and have lower cortical bone density. mineral and bone disorders were defined according to k/doqi guidelines. results: during the study period 95% of stage i-iv ckd patients and all esrd patients and renal allograft recipients had at least one type of mbd. high turnover bone disease with hyperphosphatemia, hyperparathyroidism and hypocalcemia was the most frequent type of mbd in stage i-iv ckd and esrd patient. adynamic bone disease was seen in only one esrd patient. hypophasphatemia was the most common disorder in renal allograft recipients. vitamin d deficiency/insufficiency was seen in 55% of stage i-iv ckd, 65% of esrd patients and all renal allograft recipients. all patients with stage i-iv ckd and esrd were treated with calsitriol and renal allograft recipients were treated with vitamin d3 supplementation. conclusion: mineral and bone disorders especially high turnerover type is common in children with ckd and esrd. although successful kidney transplantation corrects many of the metabolic abnormalities associated with the development of mbds, renal allograft recipients had increased risk of vitamin d deficiency/insufficiency. objective: calciphylaxis is a complication of chronic kidney disease that characterized by necrotic lesion in the skin and in histological examination reveals microcalcification of medium sized blood vessels. materials and methods: we report on a 21-month old girl with end stage renal disease due to diffuse mesangial sclerosis referred for tenckhoff catheter implantation. laboratory tests on admission were: bun = 150 mg/dl, cr= 5 mg/dl, uric acid = 10 mg/dl, calcium= 4 mg/dl, phosphate = 40 mg/dl, alkaline phosphatase= 1017, pth= 700 pm/l,vitd= 21 mg/ml. calciphylaxis process started when she received calcium gluconate iv the day before surgery and continued progressively when oral calcium carbonate and oral calcium gluconate were given. hourly peritoneal dialysis was started and all calcium containing medications were stopped. but the process continued progressively. surgical detriment of the necrotic tissue of right wrist was done. comfeel dressing had no effect and the circulation of tissue was poor. therefore the left wrist was kept as control. pamidronate 0.5 mg/kg/day prescribed for 6 days and then once a week for 5 weeks. after one week of starting pamidronate skin lesion began to heal, circulation improved and after six weeks all skin lesions completely recovered. as shown in figure one debridement cause skin scar on right wrist. conclusion: pamidronate is effective to stop calciphylaxis in children with advance renal insufficiency and severe calcium-phosphate imbalance. medical or surgical debridement are not suggested and lesions will recover without scar by pamidronate objective: fibroblast growth factor 23 (fgf23) is a key player in kidney-bone axis and regulation of calcium and phosphate homeostasis. most of the recent research has focused on fgf23 in chronic kidney disease and there is paucity of data in healthy children. hence we initiated a study on the various biomarkers of calcium-phosphate metabolism including fgf23 in healthy children and present here the interim report of our findings as of now. methods: a total 100 healthy school children and adolescents aged 6-16 years, were enrolled in this cross-sectional study, after a written consent from their parents. height, weight and pubertal staging were assessed. blood was collected and calcium, phosphate, pth and vitamin d were estimated by standard methods. plasma c-terminal fragment of fgf23 was quantitated using a commercial elisa kit (immutopics, usa). three day dietary recall was done to calculate mean daily calcium, phosphate and protein intake. results: of the 100 children enrolled 60% were males. the mean age was 12.3 yrs, mean height was144 cms and mean weight was 34kgs. the mean blood biochemistry values of the children tested so far for ca, p, pth and vitamin d shown in table 1. the parameters of the children were normal for their respective ages however fgf23 levels were found to be lower compared to reports in literature. conclusions: from the results available as of now all of the children had normal biochemistry for their ages. the fgf23 levels were found to be lower than reports in the literature. whether it can be attributed to the dietary differences from the children of the west needs further investigation. testing of the entire cohort will reveal if this lower fgf23 is a universal phenomenon among all children in our study. on the contrary, all dxa parameters showed a decreased in their mean zscore bmd values that reached statistically significance. finally, multivariate stepwise regression analyses showed that estimated glomerular filtration rate at the beginning of the study was the best predictor of the difference in bmd z-scores measured at lumbar spine. additionally, values of ipth at the beginning of the study and the change of ipth throughout the study predicted the 72.3% of the difference in z-score of sos measured at radius with an inverse relationship. conclusion: the use of two different techniques have shown bipolar changes of bone properties after renal transplantation. reversal of renal osteodystrophy and consequently correction of many of the underlying metabolic abnormalities, mainly normalization of ipth, strengths cortical bone as this is best illustrated by qus assessment. however, reduction in bmd, is predominantly considered as an adverse effect of steroid immunosuppression. age 3.3 yr) (group 1), 33 patients with ckd stage 5 on pd or hemo (mean age 9 yr, mean time on dialysis 2 yr) (group 2) and 29 patients with ckd stages 2-4 (mean age 7 yr, mean gfr 37 ml/min/1.73 m 2 ) (group 3). 25ohd levels were deficient in 20% and insufficient in 35% of the group 1 patients, 48% were deficient and 15% were insufficient in group 2, and 7 % were deficient and 17% were insufficient in group 3. results: intact pth (ipth) was above the target level by different ckd stages in 52 % of the group 1 patients, in 58% of the group 2 and in 52% of the group 3 and showed an inverse correlation with 25ohd levels (p<0.05, r=−0.25). there was inverse correlation with age in the complete cohort of patients (p=0.0005, r =−0.33) height standard deviation score (sds) was associated with 25ohd only in group 2 (p=0.025, r=0.45). there was direct correlation between 25ohd levels and gfr in group 1 and 2. conclusion: our data suggest that 25ohd deficiency is common in pediatric renal transplant and ckd children of any stage even in an area with year-long sunshine, especially in adolescents. hyperparathyroidism was also frequent in all groups. it would be advisable to monitor vitamin d status in these patients. 3) years, 28 with ckd 5 (2 on hd, 9 on pd and 17 tx) mean age 13 (5.4-17.5) and 113 healthy children, mean age 11.5 (5-17.9 objective: the aim of our study was to analyze twelve single nucleotide polymorphisms (snp) of gene lrp5 potentially associated with osteoporosis risk in children with routine steroidotherapy in the course of the idiopathic nephrotic syndrome. methods and results: glucocorticosteroids are important risk factors for drug induced osteoporosis. decrease in bone mineral density (bmd) and increased risk for pathological fractures are caused by direct division of steroids at the cellular level by inhibiting the replication of osteoblasts and stimulation of its apoptosis. also proven its effects on the inhibition of type i collagen synthesis. lrp5 is one of the wnt signaling pathway proteins coreceptors involved through the rank-rankl system in regulation of the osteoblasts function. lrp5 is included to the osteoporosis phenotype genes group and its selected single nucleotide polymorphisms can be responsible for bmd decrease in patients with glucocorticoid therapy. the study group was composed of 38 children with idiopathic nephrotic syndrome, 21 boys and 17 girls beetwen the age of five to twelve years old , 14 with osteoporosis and 24 with normal bone mass density. study also included the control group consists of 102 healthy individuals at the same age not treated with glucocorticoids. the analysis was carried out with polimerase chain reaction (pcr) and taqman molecular probe designed for 12 single nucleotide polymorphisms (snps) nearby investigated mutations in lrp5 gene that can be connected with osteoporosis fenotype. odds ratio value (or) was based on frequencies of single nucleotide polymorphisms in lrp5 gene and its haplotype analysis. the results showed significant differences in or value among the three groups. it was also found the differences in the lrp5 gene structure. based on gabriel algorytm single nucleotide polymorphisms pairs analysis in children with osteoporosis and nephrotic syndrome we proved the correlation between selected snps pairs presence and decretion of bone mineral density in studied group. objective: growth hormone (gh) and insulin-like growth factors are essential for normal growth and development. chronic renal failure (crf) results in major changes in the circulating growth hormone /insulin-like growth factor (igf) system. our aim isto study: to assess clinical and laboratory parameters of growth and osteodystrophy including igf1 and igfbp2 as part of the somatotropic hormone axis in egyptian children suffering from crf on conservative therapy. methods: 62 egyptian children (47 boys and 15 girls) with a mean age of 9.7y (0.47 to 21.12y) suffering from crf on conservative therapy and 21 controls were included in the study. ht, wt and tsf were measured ,pubertal staging was assessed and followed up for a period of 6 months. at the end of the follow up period serum for igf1 and igfbp2, renal function, electrolytes, ca, p ,and alkaline phosphatase and acid base balance were measured and an x-ray of the left hand and wrist was done to determine their bone age by tanner and whitehouse. results: our study shows that children suffering from crf in egyptian conservative therapy have growth retardation with a mean ht of -3.7 sds, a mean wt of -2.24 sds. tsf mean was -1.3 sds. on the average the patients had a delay of 2.95y (+/-2.0) in their bone age. their height was retarded more than their bone age with a height age/bone age of 0.8 (+/-0.18). alkaline phosphatase as a markers of renal osteodystrophy is significantly correlated to the height, height age , bone age and to the ph. the mean igf1sds (-0.6 +/-1.8) did not differ from that of controls while the mean igfbp2sds (2.4 +/-4.6) was significantly higher in patients with crf than in controls. height and weight were significantly correlated to igf1 but not igfbp2. there is a significant correlation between igfbp2 level and the glomerular filtration rate. conclusion: the imbalance between normal insulin-like growth factor-i (igf-i) and markedly increased igfbp2 plasma levels plays a pathogenic role for growth retardation in children with chronic renal failure. the lower the gfr the higher the igfbp2 level. the latters inhibitory action may provide hope for improving growth in cases of crf by reducing the level of igfbp2 or displacing igf1 from it. objective: anemia is one of the most common complications of chronic kidney disease (ckd) and renal transplantation. this study evaluated the prevelance and the etiology of anemia in children with stage i-iv ckd, end-stage renal disease (esrd) and renal allograft recipients. methods: between 2010 and 2012, we prospectively followed 21 pediatric stage i-iv ckd, 20 esrd patients and 27 renal allograft recipients. anemia was defined according to k/doqi guidelines as hb is less than the fifth percentile of the normal, adjusted for age and sex. results: during the study period52.3% of stage i-iv ckd, 100% of esrd patients and 48.1% of renal allograft recipients developed anemia. the mean hb levels were 11.30+/-1.72 g/dl, 9.62+/-1.94 g/dl and 11.95+/-1.88 g/dl in stage i-iv ckd, esrd patients and renal allograft recipients, respectively. the mean hb levels were significantly lower in esrd patients as compared to stage i-iv ckd patients and renal allograft recipients (p<0.0081, p<0.002). erythropoetine (epo) deficiency was the most frequent cause of anemia in stage i-iv ckd and esrd patients, followed by iron deficiency. in addition to epo and iron deficiency, bone marrow suppression and parvovirus infection were the causes of anemia in renal allograft recipients. erythropoietic stimulating agents were used in 27% of stage i-iv ckd, 100% of esrd patients and 23% of the renal allograft recipients. conclusion: although anemia is one the most common problems seen in children with ckd and esrd, the prevelance is also high in renal allograft recipients. these patients should be evaluated and treated accordingly to the underlying causes. abstract# p-sat375 determinants and prevalence of anaemia among preschool children in highly focussed states of india results: about more than 60 % child is anaemic among all eag states. the prevalence of severe anaemia is 6.7 % in rajasthan followed by uttar pradesh (3.6 %) and madhya pradesh (3.4 %). those children aged 12 to 17 months are 7 times significantly more likely to be severe anaemic as compare to the 36 to 59 months aged children. those mothers have severe anaemia, their children are also found to be severe anaemic (16 times more than not anaemic mothers). mothers who are highly educated and belong to richest quintile their children are less likely to be anaemic. the finding of the study shows that demographic and socio-economic indicators play significant role in determining the prevalence of anaemia in eag states which needs focused programme to reduce the prevalence of anaemia in preschool children. abstract# p-sat376 impaired renal growth hormone mediated jak/stat5 signaling in juvenile rats with chronic kidney disease objective: linear growth retardation is a major problem in children with chronic kidney disease (ckd) and is ascribed to gh insensitivity. treatment with exogenous gh has been accepted as standard therapy in children with ckd and short stature. however, concerns have been raised in the past on the potential fibrogenic effects of gh. there is no information regarding renal ghr signaling pathway in ckd. methods and results: to investigate this pathway, surgically 5/6 nephrectomized (ckd) and pair-fed control (c) juvenile (3 wk old) rats were sacrificed after 2 weeks of ckd. serum creatinine and albumin excretion were significantly elevated (associated with glomerulomegaly and early fibrosis) while body weight and length gain were reduced in ckd rats. serum igf-i levels were decreased in ckd, even though serum gh levels were unchanged. kidney ghr mrna and protein levels were reduced and phosphorylation of jak2 and stat5 was significantly impaired. supressor of cytokine signaling (socs3) mrna was increased in association of increase in renal il6 mrna. renal igf-1 mrna was unaltered in ckd. thus, in the remnant kidney of ckd growth retarded juvenile rats there is impaired gh mediated jak2/stat5 signaling. this defect may be due to a reduction in gh receptor expression and signaling together with an increase in socs3 expression. conclusion: we suggest that the insensitivity of the remnant kidney to gh may serve to protect against the potential adverse renal effects of exogenous gh in ckd patients. objective: concerns have been raised about possible adverse effects of growth hormone(gh) in short children with chronic renal diseases. six cases of short children with chronic renal diseases ckd were studied in our hospital, the microalbuminuria as a sensitive parameter of early glomerular damage was detected during the treatment of recombinant human growth hormone. the individual growth response to gh was also observed. methods: of the 6 cases, male were 4 , female 2, the onset age ranged from 4 to 14 years old, all were diagnosed as growth retarded children with various chronic renal diseases. the dosage of gh was 0.1 iu/kg/d. the follow-up period was from 3 months to 1years. microalbuminuria was measured by a commercially available elisa kit, as a sensitive parameter of early glomerular damage in children being treated with recombinant human growth hormone. serum insulin-like growth factor igf-i concentration and igfbp3 were also measured before and during the treatment with gh. results: there was no statistically difference on the level of microalbuminuria compared with that of pretreatment. linear growth were 1.0 cm/month with an increment in height sds by 1.5 at the end of the first year of gh treatment. growth retardation in children with ckd is associated with normal to slightly low concentrations of (igf)-i and igfbp3. objective: iron deficiency anaemia is common in chronic haemodialysis patients and is the most common factor of resistance to erythropoeitin treatment. kdoqi clinical practice guidelines and recommendations for anaemia in ckd suggest maintaining hb levels between 11-12 g/dl, tsats >20% and serum ferritin above 100 ng/ml. our unit's previous practice provided 1-2 mg/kg/week of elemental iron intravenously to our haemodialysis population and monitored blood parameters monthly. from july 2012, we changed our practice to withhold routine administration of iv iron, unless blood parameters (monitored fortnightly) suggested iron deficiency. we assessed the change in our practice in safely reducing the frequency of iv iron administration in our haemodialysis population. methods: we included all 9 children attending our haemodialysis unit (july 2012). we used %hypochromia of >4%, correlating with hb level of <11 g/dl as a marker for initiating iv iron sucrose (3mg/kg/week) until improvement of the above parameters. serum hb and %hypochromia were monitored at the start of change-over and fortnightly thereafter. all patients received routine erythropoietin at recommended doses of between 50-300 units/kg/week. results: at initiation of change-over, there were 9 patients with mean hb 11.3 g/dl, mean %hypochromia 1.2% and mean tsats 27.7%. from july 2012 -mar 2013, four patients required iv iron therapy (mean hb 9.2 g/dl, mean %hypochromia 6.2%, mean tsats 16.3%). three patients had 4 weeks and one patient had 7 weeks of iv iron sucrose 3 mg/kg/week to achieve normalisation of serum hb (mean 11.5 g/dl), %hypochromia (mean 2.73%) and tsats (mean 26%). at time of reporting (mar 2013), there were 7 patients with mean hb 11.22 g/dl, mean %hypochromia 2.07% and mean tsats 28.2%. two patients received transplants. conclusion: a novel change in our practice has resulted in a safe reduction of iv iron therapy in our population of children undergoing haemodialysis. from our findings, we suggest avoiding routine iron infusions in paediatrichaemodialysis, which is unlikely to have an adverse effect on the incidence of anaemia and will reduce the risk of inadvertent iron overload. abstract# p-sat380 reversible portal ascitis after bilateral nephrectomy in infant with polycystic kidney disease we report here on a newborn with moderate renal failure and severe hypertension due to autosomal recessive polycystic kidney disease (arpkd). antihypertensive therapy associating minoxidil, labetalol, amlodipine and lasilix were not able to control adequately blood pressure. due to massive kidney enlargement, inadequate respiratory function and inability to tolerate enteral nutrition, parenteral nutrition was initiated at 2 months to maintain adequate calories intake. at 4 months of life, the infant presented severe viral pneumonia complicated by cardio respiratory arrest . he required cardio respiratory resuscitation and hospitalization in icu for several days. unilateral right nephrectomy was decided after this severe complication in order to avoid similar accidents due to massive kidney volume and to improve respiratory and nutrition status. one week after unilateral nephrectomy, we noted rapid increase volume of the left kidney and important ascitis having the biological characteristics of portal hypertension. repeated aspiration of more than 100 ml of portal ascitis was performed every day. in parallel daily perfusion of albumin for more than 3 weeks and complete parenteral nutrition did not succeed in treating ascitis. faced to resistant ascitis and persisting difficulties in maintaining enteral nutrition and adequate respiratory ventilation , left nepherctomy was decided and performed. automated peritoneal dialysis was rapidly initiated in our anuric infant (weight after binephrectomy4kgs). portal ascitis disappeared in 48 hours with rapid normalization of respiratory ventilation and return to normal and well tolerated enteral nutrition. conclusion: to our knowledge, this is the first case reported of portal ascitis noted after palliative unilateral nephrectomy to facilitate pulmonary expansion and gastrointestinal function in arpkd. ascitis was resistant to daily albumin perfusion and repeated aspirations but was completely reversible after bilateral nephrectomy and initiation of peritoneal dialysis. early bilateral nephrectomy at 2 months of life was probably the ideal choice to avoid many complications in our infant due to abdominal and thoracic compression by massive kidney compression. ethical considerations, therapeutic possibilities, and parent consent should be discussed in such challenging and very difficult cases. abstract# p-sat381 serum hepcidin 25 levels and anemia in pediatric chronic kidney disease objective: hepcidin restricts the availability of iron from its stores for erythropoiesis resulting in a functional deficiency of iron and hyporesponsiveness to erythropoiesis stimulating agents. the objectives were to estimate the serum hepcidin 25 in children with chronic kidney disease stage ii to iv and correlate it with hemoglobin, iron status (serum ferritin and transferrin saturation), inflammation (c reactive protein) and estimated glomerular filtration rate (egfr). methods: it was a cross sectional study. children aged 2 to 18 years with ckd stage 2-4 on stable dose of iron and or erythropoetin for the last 4 weeks were included. children with anemia due to other causes, severe acute infection, chronic cardiac or respiratory conditions and those who received parenteral iron and or blood transfusion in last 4 weeks were excluded. hemoglobin, serum ferritin, transferrin saturation, c reactive protein were assessed. serum hepcidin 25 levels were estimated using a elisa based kit in the cohort as well as in 15 age and sex matched normal children to estimate the levels in normal population. multivariate regression analysis was used to assess the relationship between hepcidin and hemoglobin, iron status, inflammation and egfr. results: forty children were recruited into the study. the mean age of children was 9.1+ 4.5 years. around 30%, 28% and 42% of children had ckd stage ii, iii and iv respectively. the mean serum hepcidinlevels in children with ckd (55.87+14.27ng/ml) was significantly higher than controls (11.17+4.38ng/ml). conclusion: serum hepcidin levels showed an increasing trend with decrease in egfr, but this was not statistically significant. the mean haemoglobin was 10.76+1.59g/dl, the mean ferritin was 43.27+38.3ng/ml , the mean transferrin saturation was 21.68+11.5%. anemia and absolute iron deficiency was seen in 100% of our cohort and worsened with increasing stages of ckd. serumhepcidin levels did not correlate significantly with haemoglobin, ferritin or with c-reactive protein. objectives, methods and results: presenting a 16-year old armenian patient who developed a secondary amyloidosis on top of a familial mediterranean fever (fmf) and moved to germany 4 years ago. at the time of the move the patient had already a terminal renal insufficiency and received intermittent hemodialysis. despite the therapy with colchicine moderate attacks of the fmf occurred with an interval of 4 weeks and inflammatory parameters were permanently elevated. the dosage of colchicine was titrated up to 3 mg per day. as a consequence diarrhea occurred and the dosage had to be decreased. over the course the amyloidosis progressed and affected all internal organs including heart and bowel. the combination of inflammation, uremia and amyloidosis led to malnutrition (bmi 14 kg/m2). at the age of 16 years the most severe colitis with peritonitis and paralytic ileus occurred. the treatment with colchicine was paused in assumption of a toxic effect. after improvement of the diarrhea but still persisting abdominal pain the colchicine treatment was slowly started again. shortly afterwards a very severe attack of the fmf developed with an accompanied cardiovascular insufficiency. the treatment with a single high-dose of methylprednisolone and the inhibition of interleukin 1 with anakinra (1 mg/kg, given after dialysis) led only to a short remission. the high-dose methylprednisolone treatment was therefore repeated three times and anakinra was given on a daily basis. since then no new attack of the fmf occurred and the combination of lowdose colchicine and anakinra improved the general status of the patient the elevated inflammatory parameters including serum amyloid a have almost normalized. objectives: invasive aspergillosis is almost exclusively occurs in immunocompromised hosts. the central nervous system (cns) is one of the most frequent sites of invaziveaspergillozis after the lungs. we report a case of invasive aspergillosis in a boy with end stage renal failure. methods and results: a 15-year-old boy was admitted to the hospital for abdominal pain, fewer and cough. he suffered from chronic kidney failure (crf) known for a year and he was on peritoneal dialysis for seven months. he was initially treated with iv. ceftriaxone for lobar pneumonia, intraperitonealceftazidime and cefazolin for peritonitis. the peritoneal fluid was also cultured for fungus and mycobacteria and all cultures were negative. on the seventh day, the patient complained for lower extremity weakness, and bilateral hyperactive deep tendon reflexes and positive babinski reflex were found. cranial magnetic resonance imaging (mri) was normal, but the spinal cord was compressed between c7-t5 by a solid mass which was originated from right lung on spinal mri. computed tomography-guided tru-cut lung biopsy was performed for the solid mass. repeated peritoneal fluid culteres were also negative. two days later, when unconsiousness and convulsions were seen, intraparenchymal multiple hemorrhagic abscesses were found on his second mri. typical hyphae of aspergillusfumigatus was present in pathologic specimens and galactomannan test was positive. cerebral abscesses were evaluated as cns involvement of invasive aspergillosis. anti fungal therapy was began immediately as amphotericin b and caspofungin. the peritoneal catheter was removed and continuous veno-venous hemodiafiltration was performed. however his condition kept deteriorating and death ensued on the fourth day of anti fungal therapy. conclusion: invasive aspergillosis may rarely occur in immunocompetent patients and diagnosis may be missed or delayed due to lack of the particular clinical signs. we should be careful about diagnosis of fungal infections in patients with crf since early treatment before the invasion and dissemination of aspergillosis to adjacent tissues and/or organs offers a higher survival chance for the patient. questionnaire and to find a way to increase reliability for healthy children aged 8-12 years especially, according to iranian culture. objective: we lunch this study to compare the parent and children's behavior problem in pediatric patients suffering from ckd referring arak amir kabir hospital. methods: to perform this case control study, we recruit 116 children with ckd and compared them with other 116 non affected children age between 5 to 16 years old. the child behavior checklist cbcl4/18 for child behavior assessment and general assessment function gaf for the evaluation of their parent's behavior were completed by the parent's data was analyzed using qualitative variables and chi-square formula. results: among 116 patients with ckd, 10 case 8.6% showed behavioral problem while this figure was 3 case 2.6% in the control group, denoting a significant difference p-0.04. moreover 20 children 17/2% in the case group and 9 children 7.8% in the control group had internalizing problem p-0.02. 22 children 19% with ckd and 8 children 6.9% in the healthy group had externalizing problem which was also a significant difference p-0.0003. as a significant p-0.0001 the parent's average stress and behavior scores in case and control were 3.65 & 3.76, respectively. conclusion: the higher prevalence of behavioral problem in the children suffering from ckd and their parent's functional impairment highlights the important of early treatment and subsequently prevention of future behavioral problem in their sibling. abstract# p-sat391 development in 6-year-old children with and without ckd objective: ckd is a pathophysiologic process with multiple etiologies, resulting in the inexorable attrition of nephron number and function and frequently leading to esrd. a number of studies have suggested that children with ckd have problem in development than other children our goal was to compare development of children with ckd with normal children within the common age range for the disease. methods: in this descriptive-analytical study, we selected 30 children with 6-year-old children whom were diagnosed with ckd as our case group and studied their developmental status by ages and stages questionnaire (asq). the control group was selected from 6-year-old children who attended the clinic for reasons other than ckd. conclusion & application to practice: the eleven key areas of responsibility used to measure sc in a periodic evaluation demonstrated a strong correlation to the increasing extent of qpdo. additionally, as the nurses progressed to becoming expert a direct correlation to the qpdo was notable. the study became the foundation for staff training and developing a competency appraisal framework in renal nursing practice thereby promoting quality assurance procedures while attaining qpdo. objective: urinary incontinence is a child health problem that affect both the child and the family in psychological and social aspect and that leads to a decrease in quality of life of the child and his caregiver. patients with higher ckd stage had significantly lower qol score in all domains in the child-self reports, but not parent-proxy reports. whereas there was no difference between patents with stage i and stage ii. according to gender, boys had a tendency to present better qol than girls, but there was no significant difference between these two groups. age discrepancy was not a significant factor to decide qol in children with ckd. in addition, there was significant difference between parent-proxy reports and child-self reports and qol scores in the child-self reports was significantly higher than in the parent-proxy reports, especially in the domains of emotional, school functioning and psychosocial health score. conclusion: residual renal function in children with ckd is a important factor to decide qol. in addition, emotional and psychosocial support for their parents can be necessary to improve qol in children with ckd. abstract# p-sat398 results: the mean period of post-transplant follow-up was 4.7+/-2.1years(range: 0.8-9 years). in the first year of transplantation, 14 (15.2%) recipients had obesity obese patients had higher serum ggt levels than non-obese patients (21.2+/-6.2 u/l vs 14.9+/-3.8u/l, p=0.01). there was a positive correlation between bmi and ggt and tg levels in obese patients (r=0.61, and r=0.5, p<0.01 respectively). patients with dyslipidemia were found to have significantly higher mean ggt level (14.2+/-3.6 u/l vs. 19.2+/-6.2 u/l, p=0.016) and bmi (18.6+/-3.4 vs. 29.4+/-6.3 p=0.04). we could not find any correlation between bmi, and gender, post-transplant follow-up, duration of crf, primary disease, donor status and graft loss. in the first year of transplantation, gfr was significantly lower in obese patients than non-obese recipients (p<0.05) conclusion: our data has suggested that, even within its normal range, elevated serum ggt concentrations and hyperlipidemia are closely associated with obesity in transplanted patients and obesity has negative effects on graft functions. the relationship between the graft function and the serum and urine ngal in children with renal transplantation tehran university of medical science, associated professor, tehran, iran objective: the evaluation of graft function long term after transplantation is important. serum creatinine is the most common used marker for graft function in renal transplantation. the level of serum and urine ngal may predict graft function in renal transplant recipients. methods: we evaluated 21 pediatric renal transplant recipients by measurement of serum and urine ngal, serum creatinine concurrently. the gfr of the patients was also estimated by schwartz formula. results and conclusion: the time after transplantation was 3-12 years (mean: 6.8+/-2.47 years). the mean serum ngal was 140+/-94.2 ng/ml (15.4-324) . the mean urine ngal was 17.8+/-20 ng/ml (32-68). we didn't find any association between serum creatinine, gfr estimated by schwarz and serum ngal. we also didn'y find any association between urine creatinine, gfr estimated by schwarz and serum ngal. glomerular filtration rate measured by dtpa scan of fourteen patients were available. we also didn't any association between gfr measured by dtpa scan and serum and urine ngal. abstract# p-sat409 racial disparities in paediatric kidney transplantation in australia glomerulonephritis and late referral were more common in non-caucasian patients. among patients who did receive a transplant, caucasians had overall better hla matches to their graft and were less sensitised. conclusion: caucasian australian paediatric patients have greater access to kidney transplantation, particularly from living donors, and are likely to have the better graft survival. some racial disparities may be inevitable due to differences in kidney diseases, however further work is required to understand barriers to live donation and address disparities. abstract# p-sat410 cost-effectiveness of renal transplantation in paediatric and adult transplant recipients. [1.4-4.8] ), were associated with a lower inscription probability 6 months after reaching esrd. the national inscription rate was 69% with a significant center variability (median 69% [iqr 51-80]) that remained after adjustment on patient characteristics (p<0.0001). preemptive transplantation rate explained 19% of the inter-center variability (p=0.06). probability of transplantation 12 months after inscription decreased with time of waiting list inactivity (p<0.0001) and with the probability of finding a compatible transplant considering patient's hla and abo groups. underweight (bmi <-2sd) was associated with lower transplant probability (or 0.3 [0.1-0.9]). at 12 months, 81% of patients were transplanted with a significant center variability (median 80% [iqr 72-88] that remained after adjustment (p=0.02). conclusion: as expected patients' characteristics contribute to intercenter variability as well as centers' attitude towards the inscription of younger children, management of specific primary renal diseases and preemptive transplantation. despite national allocation rules we found a significant inter-center variability in the probability of being transplanted after inscription on the list, which was not explained by either the patient or center characteristics of our study. abstract# p-sat414 ten year outcomes of paediatric renal transplantation: the irish experience objective: focal segmental glomerulosclerosis is the most common cause of steroid-resistant nephrotic syndrome in children and adolescents. within 10 years of initial presentation, 50-70% of patients will progress to end stage kidney disease requiring kidney transplant. recurrence rate after transplantation is high and may be associated with early graft loss. methods: we analysed demographic and transplant data collected by the australian and new zealand dialysis and transplant registry (anzdata) on children and adolescents with a diagnosis of primary fsgs who were 20 years of age or younger at the time of transplant. kaplan-meier analysis was performed to compare graft survival according to recurrence of fsgs and donor characteristics. results: during the 20-year period from 1st january 1992 to 31st december 2011, 80 transplants were performed in 74 patients (age range 3-20 years, median 15 years). twenty seven patients developed recurrent fsgs, with one child developing recurrent disease in 2 grafts (overall recurrence rate 36%). the median time to recurrence was 15 days, with loss of graft function in 12/28 transplants (43%). median graft survival in patients with recurrence was significantly shorter than in patients with no recurrence (2.3 years vs >10 years, respectively; p<0.01). there was no significant difference in recurrence rate in recipients receiving deceased donor (dd) versus live donor (ld) transplants (p=0.24) and median graft survival was significantly better in recipients with live donors (11.7 yrs vs 1.6 yrs for dd, p<0.01) conclusion: the rate of recurrent fsgs and graft loss in this retrospective cohort study of children and adolescents transplanted within the australasian region, was similar to previously reported studies. however, in contrast to previous studies, there was no significant difference in rate of recurrence for live versus deceased donors. additionally, live donor recipients had longer median graft survival compared to deceased donor recipients. outcomes of calcineurin inhibitors conversion to mammalian target of rapamycin inhibitors in children with renal transplantation nuntawan piyaphanee, suroj supavekin, anirut pattaragarn pediatrics, siriaj hospital, bangkok, thailand objective: to study outcomes of pediatric renal transplant recipients who underwent cni elimination and conversion to mtor inhibitors. methods: we performed retrospective analysis of all pediatric renal transplant recipients who underwent cni elimination and conversion to mtor inhibitor at siriraj hospital, a tertiary care center in thailand. indications for conversion were defined. graft function prior and post conversion, history of acute rejection, and cni elimination failure were reviewed. results: nine of 32 pediatric renal transplant recipients underwent complete cni conversion to mtor inhibitor. triple-drug regimen including prednisolone, cni and mmf/mpa was basically prescribed to the recipients. eight with tacrolimus and 1 with cyclosporine were conversed to everolimus. mean ages at transplantation, conversion and last follow up were 12.1±2.4, 13.6±2.1 and 14.5±1.8 years, respectively. everolimus was initiated within 3 months in 4 patients. each patient due to cni induced hemolytic uremic syndrome, posttransplant diabetes mellitus, graft impairment with early onset nephrocalcinosis and graft impairment with renal artery stenosis. everolimus was initiated after 3 months in 5 patients due to 1 chronic cni nephrotoxicity, 2 chronic allograft nephropathy (can) and 2 unfavorable graft functions with history of delay graft function. median duration from transplantation to cni conversion was 4.5 months (range 0.8-88) and median time from conversion to the last follow-up was 8 months (range 2-26). mean creatinine clearance (crcl) prior to the cni elimination was 33.0±13.2, as compared to mean crcl of 49.0±16.3 ml/min/1.73m 2 at last follow up (p =0.069). three patients had acute rejection (ar) before the conversion, but only 1 patient had ar post cni elimination. there were no patients with graft loss or cni-elimination failure. conclusion: conversion from cni to everolimus was safe without increasing risk of acute rejection in pediatric kidney transplant patients who experienced early and late cni associated complications. objective: due to a severe shortage of suitable deceased-donor kidneys for children awaiting kidney transplants (kt), we have performed a series of abo-incompatible (abo-i) living kt since 1989. historically, abo-i kt was performed using several session of plasmapheresis (pp) to remove existing anti-a or anti-b antibodies, followed by splenectomy to prevent rebound of antibodies. because splenectomy had risks of surgical complications including bleeding and pancreatic pseudocyst, we introduced a new protocol, for abo-i kt, in january 2006. the new protocol without splenectomy utilizes the anti-cd20 monoclonal antibody (rituximab) and pp. this study retrospectivery examined the efficacy and safety of this protocol. methods: eight de novo abo-i kt (5 males and 3 females) were performed between january 2006 and december 2012. the mean age at transplantation was 15.3±4.0 years (range 9.9-20.5 years). the immunosuppressive protocol consisted of cyclospolin or tacrolimus, mycophenolate mofetil, and methylprednisolone. all patients received induction therapy with basiliximab. the preconditioning protocol included pp or double filtration plasmapheresis (dfpp) and a single dose of rituximab (average dosage 179 mg/m 2 ). all patients who underwent kt achieved a isoagglutinin titer less than 1:16 with 1-4 sessions of pp/dfpp treatment before transplant. results: the patients were followed for 5 to 82 months with a mean follow-up of 36 months. patient and graft survival rates were 100%. one non-adherent patient experienced antibody mediated rejection. figure1 ). in the rtr who had graft loss, there was no significant difference in graft survival time (6.0, 3.1 years, dsa negative vs dsa positive), grade 1 rejection (0.5, 0.7 episodes per patient), grade 2 rejection (0.5, 0.7 episodes per patient) or c4d staining (100%, 57% patients). one patient in each group had bk virus associated nephropathy. two patients had plasma cell infiltrates on biopsy and lost their grafts within 6 months of dsa detection. results: fifty-one kidney recipients (male 32 cases; female 19cases) were enrolled in this study. the median age was 15 years, the youngest kidney recipient was 3years old with a body weight of 10.0kg). there were 11 recipients less than equal 10 years, including one children with congenital renal dysplasia and one with congenital renal artery malformation. about 73.7% kidney recipients came from guangdongprovince. about 1 to 7 pediatric patients accepted renal transplatation each year from 2003 to 2011, but there were13 in2012, which showed a significant increase than in each previous sigle year.the policy that children had a kidney recipients priority may contribute to this increase. the kidney donors were cadaveric kidneys(72.5%) and living kidneys(27.4%), only one patient accepted auto-renal-transplatation. all kidney recipients were survival to present with the treatment of glucocorticoid and or immunosuppressants,such as csa, fk506, mmf,ect. only two patients accepted retransplatation due to losing function of the renal grafts. conclusion: renal transplantation in children increased significantly in 2012 in our hospital, and it seemed an effective treatment to children with esrd. recent adult studies have shown that low levels of immunosuppression (is) are associated with dndsa. however, limited data is available on significance of dndsa and its management in paediatric population. to assess relationship between dndsa with renal function, histological findings, immunosuppression levels and graft outcome in paediatric renal transplant recipients (rtr). in our centre, at the time of the study, dsa were tested if deterioration in graft function was found necessitating renal biopsy. methods: retrospective review of all rtrs in a single tertiary nephrology centre tested for dndsa. data collected included dndsa, histological findings, egfr as marker of renal function, baseline immunosuppression, subsequent treatment and outcome. results: 27/88 patients had dsa tested; 12/27 had dndsa detected. median time for measured of dndsa was 7.6 years post-transplant (range 1.07-13.02).histology: 8/12 biopsies in dndsa+ve subgroup had antibody mediated rejection (abmr) of which 6 were active abmr (3 were c4d positive) compared to 1/16 in dndsa-ve subgroup. the following treatments were used in the dndsa+ve group: increase in is+ pulsed methylprednisolone (mp) (5 patients), increase in existing is (2), rituximab (2), pulsed mp (1), sirolimus added+pulsed mp (1) . in one patient no change was made. at last follow up, in 9/12 patients with dndsa an improvement in egfr was observed. on re-testing for dndsas, 3/12 patients tested negative, 2/12 had lower and 3/12 increased levels. 3/12 had not yet had repeat dndsa testing. conclusion: despite previous reports of dndsa conferring poor prognosis, in our cohort with allograft dysfunction and positive dndsa an improvement in renal function was observed in 75%. however, larger prospective studies are required to further evaluate these findings. abstract# p-sat425 anuria since birth: does it impact outcome of kidney transplant in infants? shefali vyas, maria isabel roberti pediatric nephrology, saint barnabas medical center, livingston, usa objective: scarcity of data exists for allograft outcomes and urological problems in children with long term de functionalized bladders. however, even less is known about the outcome of infants anuric since birth and whose bladders underwent "forced rehabilitation" after a successful renal transplant. methods: in this retrospective study we compared urological events and allograft outcome in infants with esrd mainly due to urological problems. they were grouped according to the history of pre-txp urine out put: group a with urine output prior to txp and group b anuric since birth (all had visible small bladders by ultra sonogram). results: there were no significant differences regarding birth history or ethnicity . group a had 13 boys (87%) and group b had all girls; all but one child in group b received lrd txp. all received induction followed by triple therapy (tacrolimus, steroids and mmf). group a: 8/15 had v-u reflux and 13/15 were on pd prior to txp (mean time= 7 mos). all patients in group b were started on pd in the neonatal period (mean =9.8 days). pre txp native nephrectomies were done in 3 patients (2 in group a and 1 in group b). group a had 2 acute rejections and none in group b. conclusion: anuric young infants had higher rates of post txp utis and v-u reflux, a direct consequence of their inherent small bladders. however, the 3 year graft survival, patient survival and gfr remained excellent in anuric infants (group b )compared to young infants transplanted with higher rates of v-u reflux and dysfunctional bladders pre txp. abstract# p-sat426 kidney transplantation in a child with bladder dysfunction who underwent prior bladder augmentation: a case report 1 tepecik training and research hospital, pediatric nephrology, they were divided into 2 groups; 12pts with esrd on dialysis (d), 4/12 with history of kidney transplant, and 9 kidney transplant recipients with good graft function (t). all subjects were tested for anti-hla antibody 1 month prior to and 1 month and 6 months after administration of the combination vaccine of influenza a/ h1n1. results: among the t group, no pt tested positive for either anti-hla class i or class ii antibodies before or after influenza a/ h1n1 vaccine. in the d group, of the 4 pts with a history of graft failure who were sensitized before immunization; 2 showed no change in class i & ii, one patient had mild increase in class i after vaccination, and one patient had an increase in class i by 24%. [figure] conclusion: none of the t pts had clinical evidence of either cell mediated or humoral rejection after the influenza a/h1n1 vaccine. in the d group, no pt had any statistically significant increase in anti-hla antibody following vaccination. our study suggests that influenza a/h1n1 vaccination may be safe and tolerable in pediatric dialysis pts with or without a failed kidney allograft. the effect of dipping blood pressure status on structural and diastolic heart function in renal transplant recipients mitra basiratnia 1 , gholamhossein ajami 2 1 shiraz nephrology urology research center, shiraz university of medical sciences, shiraz, iran 2 department of pediatric cardiology, shiraz university of medical sciences, shiraz, iran objectives: non dipping has been linked to cardiovascular disease in adults, however the impact of non-dipping on cardiovascular status of the adolescents with renal transplantation has not been well established. the aim of this study was to evaluate the influence of non dipping status on left ventricular mass index and diastolic function in subjects with renal transplantation. methods: sixty six stable renal transplant patients (34 females, 32 males), aged 7 to 25 years (mean 17.4±4.3 years) were enrolled in this study. cardiac function assessed by tissue doppler echocardiography and blood pressure measurement performed using ambulatory method. dipping was defined as at least 10% bp decline during the night and was calculated as (mean daytime -mean night time/mean daytime)*% 100. left ventricular mass (lvm) was calculated by standard 2dimensional directed m-mode echocardiography according to the formula of devereux and was indexed to height in meters to the 2.7 power to allow the comparison between recipients of different sizes. left ventricular hypertrophy was defined as lvmi>38.6g/m 2.7 in patients younger than 18 years and 51g/m 2.7 in patients > 18 years. data analysis was performed by spss-15. a p<0.05 was considered statistically significant. results: non-dipping was identified in 48 (73%) patients. five recipients were systolic non-dippers, 1 diastolic non-dipper, and 42 both systolic and diastolic non-dippers. left ventricular hypertrophy (lvh) was found in 37.1% of the renal allograft recipients. lvh was present in 41% of the systolic nondippers and 27.8% of the systolic dippers (p=0.33) . forty five percent of the diastolic non dippers and 22.7% of the diastolic dippers had lvh (p=0.08) . there was no correlation between systolic and diastolic dipping status and lvh, respectively (p=0.33, p= 0.08). there were no significant differences in terms of diastolic function [measured by early diastolic inflow velocity (e), e/a ratio, and early diastolic mitral inflow velocity to earlydiastolic annular velocity (e/ea)] between dipper and non dippergroups(p= >0.05). conclusion: non dipping is common among renal transplant recipients, but is not always related to diastolic dysfunction and lvh. prospective longitudinal studies are required to determine the impact of dipping status on diastolic and structural heart function in renal transplant recipients. parsa yousefi chaijan, parvin soltani, farshid haghverdi, masood fazelimoslehabadee nephrology, arak university of medical sciences, arak, iran objective: nephrolithiasis in renal grafts is a relatively common phenomenon which can induce organ damage; hence early diagnosis and management of predisposing factors can preclude subsequent complications. the aim of this analytic cross-sectional study is to determine the contributory factors to nephrolithiasis after renal transplantation. method: 56 renal-transplanted patients (10-40 years old) were enrolled in the study, being divided into two groups of 28 transplanted patients suffering from nephrolithiasis (within first 5 years after surgery) and 28 transplanted patients free from renal stone with the same age and gender and similar gfr. data were collected and arranged in excellmicrosoft program and the statistic analysis was carried by spss v17. pvalue < 0.05 were considered statistically significant. result: the studied showed that male gender (p = 0.048), age group of 15-25 years (p=0.021), hyper cholesterolemia (p=0.007), hyper triglyceridemia (p=0.031), 3 rd year after surgery (p=0.023), hyperuricosuria (p=0.012) , hypocitraturia (p=0.001) and anemia (p=0.006) were significantly more common in patients with nephrolithiasis. conclusion: it is better to evaluate hyperuricosuria and hypocitraturia in kidney donors, moreover all patients had better undergo serial sonography for early screen and management of renal stone as well as treatment for hyperlipidemia and anemia. it also seems prudent to further assess the donors of transplanted patients suffering from renal stone and possbile relation between cni & nephrolithiasis ( regardinghyperuricusuria) objective: epstein-barr virus-associated smooth muscle tumor (ebv-smt) in immuno-compromised patients has recently been reported. but there were no evidence about the treatment of ebv-smt. we report a 6-year-old girl treated for multiple ebv-smt by using rituximab. methods: case report results: she was suffering from end stage renal disease due to congenital nephrotic syndrome induced by wt1 mutation. renal transplantation (tx) was performed at the age of 2 years, and immunosuppressive therapy was performed her. soon after performing tx, she was infected cytomegalovirus(cmv), bkv and ebv. she was reduced in amount of immunosuppresive therapy, and she recovered from viremia of cmv and bkv. her ebv titer did not become negative, but we did not perform any medication because of no symptom. four years after tx, she was pointed out 1cm of cholecystic polyp in a protocol abdomen ultrasonic examination. we soon performed cholecystectomy and extirpation of swelling mesenteric lymph nodes. the pathological finding of cholecystic polyp was ebv encoded small rna (eber) positive smooth muscle tumor but there was no finding about post-transplant lymphoproliferative disorder in mesenteric lymph nodes. positron-emission tomography (pet) showed accumulation of 1cm mass around 12th of thoracic vertebra and ct scan showed three small masses in the lung and one in the liver. we diagnosed her suffering multiple infection of ebv-smt. immunosuppression therapy was reduced, but there was no change about size of her tumor. rituximab was administered for keep away from repetition of tumor after proving infection of ebv only in b cells by using flow cytometry. after medication of rituximab, the ebv dna counts were normalized. ultrasonic examination detected three small masses in liver and one lymph node around main artery. but her mass around 12th of thoracic vertebra was disappeared and whole body ct scan detected only one small residual mass in the lung. we report a case of an ebv-smt presenting multiple infections, which was treated with rituximab. rituximab may be an effective treatment for multiple ebv-smt but careful observation is also needed for the reappearence of ebv-smt. the usefulness of monitoring of epstein-barr viral load after renal transplantation in pediatric recipients with ebv seronegative objective: the purpose of this study are to establish a protocol for monitoring epstein-barr virus (ebv) infection for identification of pediatric renal transplant recipients with a high risk of developing posttransplantlymphoproliferative disorder (ptld) and to predict the development of ptld. methods: peripheral blood mononuclear cells (pbmcs) and plasma ebv loads were measured by nested pcr (n-pcr) and real-time pcr (r-pcr) every 1-3 months after grafting in 17 pediatric recipients who were seronegative for ebv before grafting (4 with ebv-associated symptoms, including 2 with ptld (group a); 6 with asymptomatic persistent high ebv loads in pbmcs of >1,000 copies/ug dna for over 6 months (group b); and 7 with neither ebv-associated symptoms nor persistent high ebv loads in pbmcs (group c). ebv-ctls were also measured in 13 patients without ebv-associated symptoms. results: the ebv genome detected by n-pcr was present in plasma in 3 (75%), 1 (17%), and 0 (0%) in groups a, b and c (p<0.01 for a vs. b and a vs. c). ebv loads detected by r-pcr in pbmcs were significantly higher in groups a (p<0.05) and b (p<0.01) compared to group c. ebv genomes in plasma were detected by n-and r-pcr in only the 2 cases with ptld. one patient with lymphadenitis in group a and 1 patient in group b had ebv-dna in plasma based on n-pcr, but the viral loads using r-pcr were <250 copies/ml. the ctls' percentage was significantly lower in group b when ebv loads first rose above 100 copies/ugdna. conclusion: plasma ebv loads (over 250 copies/ml) estimated by r-pcr and ctls' monitoring may be useful to distinguish ptld from other ebv-associated diseases or asymptomatic viremia, and to avoid ptld as patients with asymptomatic persistent high ebv loads had higher ebv loads and lower percentages of ctls. abstract# p-sat437 risk factors for post-transplant lymphoproliferative disorder (ptld) in children with kidney transplantation (ktx) -a single center survey since the introduction of tacrolimus (tac) since pre-transplant evaluation showed complete obstruction of ivc/ iliac vein below diaphragm, she had been managed with peritoneal dialysis for 7 years until january 2012 when she received a living donor kidney allograft from her mother. the graft was transplanted in a left orthotopic position. venous drainage was to the left ascending lumber vein. in addition, a venous bypass was made using donor ovarian vein between graft vein and splenc vein after splenectomy. such double venous drainage was working very well after transplantation. there was no surgical complication and the serum creatinine (s-cr) during the hospital stay was 0.54mg/dl. at second month after the transplantation, she developed an increase of s-cr with edema and hypertention. however, biopsies of kidney allograft performed at 2 nd and 4 th month after the transplantation revealed no evidence of acute rejection. hypertention was difficult to control even with a maximum dose of calcium blocker, and a temporary increase of the s-cr up to 1.30 mg/dl was noted when angiotensin ii receptor blocker was administrated. by the ultrasonography, the maximum arterial blood flow of the kidney allograft was high enough (459cm/s) to suggest renal arterial stenosis. moreover, severe renal arterial stenosis was clearly shown for about 10mm length from the anastomosed site of aorta by the contrasting ct.for the treatment of artery stenosis of the kidney allograft, percutaneous transluminal renal angioplasty (ptra) was performed at 8 th month after the transplantation. the stenotic lesion was expanded using the special dilatation balloon from 1.7mm through 2.0mm in diameter. because intravascular ultrasonography showed no intimal thickening of the blood vessel, a vascular stent was not placed. after the ptra, s-cr was decreased to 0.5mg/dl, and the blood pressure became controllable by antihypertensive agents. moreover, the angiography performed 4 months after the ptra revealed no progression of arterial stenosis. the s-cr of this patient is now stable in 0.7 mg/dl. post-transplant encapsulating peritoneal sclerosis in children: a single center experience kei nishiyama tokyo women's mdical university, pediatric nephrology, tokyo, japan objective: a substantial proportion of encapsulating peritoneal sclerosis (eps) cases develop after renal transplantation (rt), an entity known as post-transplant eps. although risk factors for eps include prolonged pd, recurrent peritonitis, decreased ultrafiltration and prolonged administration of hypertonic glucose or icodextrin, the pathophysiology of post-transplant eps is largely unknown. however it has been postulated that the use of calcineurin inhibitors (cnis) after transplantation may promote eps, as these drugs are considered profibrotic. a recent scottish study showed that the contribution of post-transplant eps might be even as much as 50% of the total eps patients. by contrast pediatric cases are very rare. therefore we examined the incidence of post-transplant eps in pediatric renal transplant recipients. methods: in this study, we retrospectively investigated clinical records from 52 consecutive pediatric renal transplant recipients in our center between 2007 to 2012, who performed pd before transplantation. clinical parameters and pd-related risk factors of eps were collected at the time of rt. transplant-related variables were also collected. eps cases who met ispd diagnostic criteria including clinical feature and either radiologic and/or histopathological confirmation were examined. results: the median duration of pd was 2.0 yr (range 0.5 to 7.9). twelve patients (23.1%) had at least one episode of peritonitis. ten patients (19.2%) were administered hypertonic glucose and/or icodextrin. the median follow-up period after transplantation was 2.3 yr (range 0.3 to 5.8). all patients were administered cni, and 2 patients discontinued corticosteroid during the follow-up period. although 5 patients had ultrafiltration failure at the time of rt, there was no case who developed post-transplant eps. conclusion: the case of eps after rt was not seen in this study. possible explanation of this result might be associated with shorter durations of pd and small sample size. since post-transplant eps is rare but carries a high mortality, caution should be paid to developing post-transplant eps even in pediatric patients with a relatively long pre-transplant duration of pd. asli kantar, kaan gulleroglu, esra baskin, umut bayrakci, zafer ecevit, hande arslan, aydincan akdur, gokhan moray, mehmet haberal pediatric nephrology, baskent university, ankara, turkey objective: viral infections remain a significant cause of morbidity and mortality following renal transplantation. although cytomegalovirus is the most common opportunisticpathogenesis in transplant recipients, numerous other viruses may affect clinical outcome. viral infections are potentially severe complications of transplantation, as they not only induce specific diseases, but they also favor the development of allograft damage, opportunistic infections and acute rejection. we evaluated the major viral infections seen following kidney transplantation and allograft outcomes in our pediatric patients. methods: we evaluated retrospectively 94 pediatric renal transplant recipients for the occurrence of viral infections and compared outcomes among these patients. patients were divided in to two groups those who developed an infection and those who did not. inthese groups, we recorded induction therapy used at transplantation, immunosuppressive therapy given at discharge, acute rejection rate, patient and graft survival rates. results: the mean age of the patients was15.5±5.3 years. viral infection was found in 32 patients. cytomegalovirus infections were the leading causes; ebv and bk virus were following causes in our study. any significant correlation could not be shown between viral infections and immunosuppressive therapy. we did not observe any correlation between acute rejection and viral infections. lowest gfr at 6 th month was shown in patients with bk virus infection. a significant correlation was shown between viral infection and graftloss (r: 0.22 p: 0.028). conclusion: viral infections are common after kidney transplantation. patients should be monitored more carefully for provide against damage in transplanted kidney. objective: anemia is a frequent condition in kidney transplant recipients and it has a negative long term impact on graft and patient outcomes. recently it has been shown that treatment with angiotensinconverting enzyme inhibitors (acei), angiotensin ii receptor blockers (arb) and mtor inhibitors could be the leading causes of anemia in renal transplant recipients.to study the association of hemoglobin (hb) and ferric parameters with gfr, immunosuppressive drugs, acei, arb and clinical features of kidney recipients. method: hospital records of 94 (f/m:48/46) kidney recipients were reviewed retrospectively. the mean age of the study group was 15.5±5.3 years. the mean follow-up was 52.6±36.31 months. results: thirty six (38%) patients were found to be anemic. mean hb levels of anemic and non anemic patients were 10.2±1.15 mg/dl vs 13.87±3.41 mg/dl respectively. ferritin and iron levels as well as transferrin saturation index, rdw and mcv did not differ among the groups. anemia was not found to be correlated with immunosuppressive or antihypertensive drugs including acei and arb. donor status did not also have any influence on anemia. mean gfr of patients at posttransplant 6 months and 1 year follow-up was found to be significantly lover than patients without anemia (74.4±34.1 vs 99.1±28.9 ml/min/1.73m2 respectively). graft loss was also found to be significantly higher in anemic patients (16% vs 6%, p<0.018). conclusion: in this study we examined the prevalence of anemia and its risk factors in kidney transplanted patients. its incidence is quite high in our patients. we found that anemia in kidney recipients is neither related to iron status nor medications like acei, arb or immunosuppressive drugs. the major determinant of hb level, especially during the first year of transplantation is the graft function. the most important conclusion of this study is the considerable controversial impact of anemia on graft survival. there was no significant effect on blood pressure ; 4 patients 7±2 modigraf® administration days in the converted group. in the whole series, mean creatinine level was 0.7±0.5 mg/dl throughout the observational period, and no rejection episodes were detected. blood pressure was well controlled and no proteinuria was seen. the equivalent dose ratio between modigraf® and liquid tacrolimus was 1.28. conclusion: modigraf® appears to be a safe and sustainable way to administer tacrolimus in kidney transplanted infants. in our experience required modigraf® dose was 1.28 times the oral liquid tacrolimus one, and therapeutic levels were attained in one week. objective: prescribing of medications in paediatric practice is problematic as many drugs remain unlicensed. this is especially true of immunosuppressive medications. however, there is now a licensed product for tacrolimus called modigraf® with data available on bioavailability. we undertook a single centre prospective study of conversion to modigraf®, an oral liquid available in granule formulation (1mg and 0.2mg) that allows for dosing according to body weight. methods: all paediatric renal transplant recipients (rtr) under the care of a single centre were considered for conversion to modigraf® from their current tacrolimus regimen. inclusion criteria included all rtr under 18 years of age who were on tacrolimus suspension. exclusion criteria included recipients of multi-organ grafts, patients with lactose intolerance and patient choice to continue with their current suspension. patients were then seen by the multi-disciplinary team. after equivalent dose conversion, patients were monitored with blood tests one week after conversion with subsequent doses adjusted accordingly. patients continue to be monitored with blood levels over a 3 month period after conversion with renal allograft function, renal allograft loss and side-effect profiles recorded. results: forty-three (27% of 158) rtr were considered for conversion to modigraf®. the families of four patients requested to stay on their current tacrolimus suspension, three patients were deemed unsuitable due to lactose intolerance and three patients were excluded due to low doses (incompatibility with the granule dosing). thirty-three patients were then converted to modigraf® and closely monitored. after blood level monitoring one week after conversion, 12% (4) patients had their doses increased due to lower than anticipated 12-hour trough tacrolimus levels. there was stable renal allograft function without renal allograft loss after conversion. conclusion: conversion to modigraf® immunosuppression can be safely undertaken in paediatric rtr, although regular monitoring in the immediate period of conversion is required to provide accurate immunosuppression dosing. escort trial -effects of strict control of blood pressure in pediatric renal transplant recipients -baseline characteristics of patients from a randomized controlled trial tomas seeman, jiri dusek, nadezda simankova, karel vondrak, jakub zieg dpt. of pediatrics, university hospital motol, prague, czech republic objective: arterial hypertension is a known risk factor for impaired graft survival in patients after renal transplantation (rtx). strict control of blood pressure (bp <50 th percentile) delays progression of chronic kidney diseases in children (escape trial). it is not known whether strict bp control has renoprotective effect also in children after rtx. the aim of this randomized controlled trial was to investigate whether strict bp control can protect kidney graft in children after rtx. methods: all children from our pediatric renal transplantation center were screened for eligibility for the study (children 3-16 years at least 1 year after rtx, no acute rejection in the last 3 months, egfr>15 ml/min/1.73m2, 24hr mean bp >50 th percentile using ambulatory blood pressure monitoring abpm). altogether 23 children fulfilled the inclusion criteria. they were randomized to intensified bp control group (intens, target 24hr map <50 th percentile, n=12) or standard bp control group (stand, target 24hr map 50-95 th percentile, n=11). all antihypertensive drugs are allowed to reach the target bp. the study period is 3 years. the primary endpoint is the yearly change in egfr (schwartz formula, ml/min/1.73m2/year), the secondary endpoints are graft failure, change in proteinuria, left ventricular mass and safety of strict control of bp. results: the baseline characteristics of the patients are given in the allelic variants. tac dose (mg/kg/day) and tac exposure normalized for dose (tac co/d) (ng/ml/mg/kg/day) were analyzed with respect to cyp3a5 genotype and for interaction with azoles and corticosteroids, for a period of one year post-transplant. over time, tac co/d was significantly lower in recipients with a cyp3a5*1/*3 genotype compared to those being homozygous for the cyp3a5*3 allele (55.33 +/-21.30 versus 83.07 +/-9.9 ng/ml/mg/kg/day, p= 0.0068). the dose requirement was significantly higher in children with a cyp3a5*1/*3 genotype compared to those being cyp3*3/*3 (0.21 +/-0.02 versus 0.17 +/-0.01 mg/kg day, p=0.0079). the tac co/d was significantly higher for patients receiving azoles (n=17) than those not receiving azoles (n=40) (117.21 +/-1.06 and 62.01 +/-0.52 ng/ml/mg/kg/day, p= 0.002) and consequently the required tac dose was lower in patients receiving azoles overtime (0.08 +/-0.00 versus 0.22 +/-0.01 mg/kg/day, p < 0.001). in children receiving steroids without azoles the tac co/d was significantly different between the cyp3a5*1/*3and cyp3a5*3/*3 genotypes, respectively (52.86 +/-1.71 vs. 58.68 +/-0.85 ng/ml/mg/kg/day, p=0.0044) but the tac co/d was not different for those receiving steroid versus those not receiving steroid with cyp3a5*3/*3 genotype ( 84.91 +/-12.7 and 84.21 +/-1.41 ng/ml/mg/kg/day, p=0.16). conclusion: in conclusion, the tac dose is influenced by the cyp3a5 genotype and drugs such as azoles, while steroids may not impact tac dose. while therapeutic drug monitoring of tac remains necessary, integrated knowledge of patient genotype and comedication use provides the opportunity to refine tac dosing in children receiving kidney transplant. abstract# p-sat458 basiliximab induction therapy in pediatric renal transplantation, a double blind clinical trial hasan otukesh tehran university of medical science, associated professor, tehran, iran this is an open, single center, randomized study to compare induction therapy with basilixamb with no induction therapy in children with renal living transplantation. in this trial 20 pediatric renal transplant recipients enrolled randomly to one group with basiliximab as induction therapy and another group without basiliximab induction therapy. both group received prednisolone, cyclosporine and cellcept. we assessed graft function at 3 months after transplant and compared this item between these two groups. in the congress the data and results of this randomized trial will be presented. abstract# p-sat459 legalization of the organ donation and optimal utilization of young pediatric donor kidneys into pediatric recipients in china objective: china has started to establish a new national system for organ donation and transplantation since march 2010 by the ministry of health and the red cross society of china. the aim of this study was to describe our initial experience of pediatric renal transplantation using organ donations from pediatric patients no more than 6 years old. methods: the procedure of organ donation includes: 1. judgment of brain death, or circulatory death, or brain death followed by circulatory death by doctors; 2. organ donation informed consent form signed by family (children's parents); 3. approval by the hospital ethics committee; 4.organ donation to the red cross society and allocation by the china organ transplant response system (cotrs). the red cross society has been commissioned by the ministry of health to run this system. clinical data of 8 children who underwent renal transplantation using organ donations from pediatric patients no more than 6 years between september 2011 and march 2013 were retrospectively analyzed. results: the age at transplantation for these 8 patients was 4.5 years to 14 years and the weight was 14 kg to 35 kg. among these 8 cases, 7 donors were used aged from 33 days to 6 years and all diagnosed with circulatory death. 5 recipients received en bloc kidney transplantation and the other 3 recipients received one single kidney according to the size of both recipient and donor. the duration of follow-up after the transplantation was 1 month to 18 months. patient survival rate was 100% and graft survival rate was 7/9 (77.8%, one graft loss due to the hemorrhagic complication and the other one due to the thrombosis). at latest follow-up, the median serum creatinine level was 92umol/l and the median egfr was 80ml/(min·1.73m 2 ). conclusion: organ transplantation legislation is necessary to ensure the rights and obligations of donors, recipients and medical institutions. we believe young pediatric donors can be expanded further to increase the number of pediatric renal transplants. this pediatric to pediatric combination on the one hand efficiently lower the discarding ratio of the kidneys from small donor, and give more chances to the younger recipient on the other. to assess the normalization of serum 25 (oh)d level (>30 ng/ml) after standard treatment dosing among primary hypertension (ph) and chronic kidney disease (ckd) methods: we enrolled 144 patients aged 2-19 yrs we collected retrospective data on age, sex,race, cause of kidney disease, egfr, ht, wt, bmi, bp z-scores, lipid panel, 25(oh) d level, pth, calcium, phosphorus, magnesium, medications, type and dosing of vitamin d supplements and follow up serum vitamin d level three months post treatment. results: mean age (yrs) was (12.99±5.05). white(2) prevalence of vitamin d deficiency was (88%); 60% had level <20 ng/ml. mean pretreatment 25(oh) d (ng/ml) was (19.25±10.69) after completion of standard treatment almost 64 % patients had 25 (oh) d level <30 ng/ml; ph(28.02±9.22) and ckd(25.38±9.8). none were in toxic range. conclusion: the standard treatment dose of vitamin d doesn abstract# p-sat360 mineral metabolism in european children with end-stage renal disease marjolein bonthuis 1 the netherlands 2 pediatrics, nephrology and dialysis unit results: hypocalcaemia was found in 22% of hd, 18% of pd, and 48% of transplanted patients, with a mean time on transplantation of 4.3 years. hyperphosphataemia was found in 47% of hd, 37% of pd, and 5% of transplanted patients. pth was outside target in 56% of hd, 58% of pd and 14% of transplanted patients. in dialysis patients, calcium and pth were inversely associated with age; 53% of adolescents were hyperphosphataemic resulting in a significantly higher risk compared to 3-5 year olds (or:1.77, 95%ci:1.43-2.19). patients transplanted pre-emptively had a lower risk of hypocalcaemia compared fgf23 is modulated by calcium in children under chronic peritoneal dialysis azocar 1 , maria l. ceballos 1 , angelica m. rojo 1 luis calvo mackenna children's hospital human intact fgf-23 levels (pg/ml, immutopics) were determined through a 2-site elisa kit. klotho levels were determined by a solid phase sandwich elisa kit (pg/ml). descriptive statistics, univariate and multivariate analysis were performed methods: we measured serum calcium, phosphorus, intact parathyroid hormone, alkaline phosphatase (alp), 25-hydroxy vitamin d3 (25d3), 1, 25-hydroxy vitamin d3 (1,25d3), and fgf-23 from 233 children (male:female = 157:76, mean age 10.2 years) with ckd stage i-v predialysis in korea with 0.5-1 year intervals since 2011. results: hypocalcemia was observed in 23.8%, 32.4%, 21.8%, 22.4% and 31.8% of patients with cdk i to v (the rest is the same as above) 3%, 8.3%, 47.1%, 68.1% and 40.9%. 25d3 level was below fgf-23 (ru/ml) was 38.0, 44.9, 54.4, 67.3 and 119.6. serum p had positive correlation with alp (p < 0.0001), ipth (p = 0.0489) and fgf-23 levels (p < 0.0001) and was inversely correlated with urine phosphorus conclusion: the prevalence of hypocalcemia, hyperphosphatemia and hyperparathyroidism increased as ckd progressed. serum ipth and fgf-23 increased and 1,25d3 level decreased in proportion to the progression of ckd malaysia 6 national transplant resource centre, national transplant resource centre, kuala lumpur, malaysia objective: to analyse the cost-effectiveness of paediatric and adult living related renal transplantation (lrt) and deceased donor renal transplantation (drt) the time horizon was the lifetime of transplant recipient from transplant to death. data for survival analysis was obtained from national renal registry. statistical analysis was performed using stata se version 11.2. the costs was discounted at 3% p.a. results: we reviewed 206 medical records. there were 88 children (39 lrt, 49 drt) and 118 adults (63 lrt, 55 drt). the paediatric recipients mean age was 12.3 +/-3.4 (lrt) and 14.0 +/-2.4 years (drt), whereas the adult recipients mean age was 33.4 +/-10.3 (lrt) and 41.8 ±8.9 years (drt). the 5 and 10-year patient survival rates for both paediatric lrt and drt were 95% and 85% respectively. the 5 and 10-year patient survival rate for adult lrt was 92.5% and 90% respectively, whereas adult drt was 77.5% and 70% respectively. mean cost per paediatric transplant at first year was rm 81,000 (lrt) and 90,000 (crt), from second year onwards was at 20,000 per year (lrt) and 37,000 per year (drt) . the total lifetime cost was rm 650,000 (lrt) and 630,000 (drt) their serum creatinine, egfr, cholesterol, ldl, proteinuria, full blood counts and liver function were profiled to compare the pre-/post-conversion changes. common adverse effects, acute rejection, opportunistic infection and need of treatment for hypercholesterolaemia and proteinuria were investigated. results: we have 15 eligible patients with m: f = 7 : 8. the mean age at renal transplantation was 12 sirolimus was started at a mean age of 15.4 +/-5.0, at a mean time period of +/-2.5 years after renal transplantation, and used for a mean duration of we observed new onset hyperlipidaemia in 9 patients (60%) and needed statin treatment. we found significant proteinuria (spot pr/cr > 1.0 mg/mg) in 4 (27%) patients and needed acei treatment. there was no adverse effect on blood counts and liver function. there was no opportunistic infection observed after conversion to sirolimus in the study period. there was one acute cellular rejection (ia) one month after conversion to sirolimus and responded to pulse methylprednisolone. conclusion: converting from a cni based immunosuppressant protocol for paediatric renal transplantation patients to a sirolimus based one was effective and the benefit was shown up to 54 months post-transplantation one patient received a desensitisation regime with rituximab and plasmaexhange pre-transplant and iv immunoglobulin post-operatively. surgical complications occurred in 7 patients; 2 lymphocoeles, 3 ureteric leaks and 2 renal vein thromboses. early post-operative medical complications included hypertension (47), pulmonary oedema (13), seizures (9), reaction to basiliximab (3), sepsis (5), acute tubular necrosis (8), delayed graft function (5), acute rejection (2) and diabetic ketoacidosis (1). primary ebv infection occurred in 38 patients -1 developed ptld. there were 9 (12%) graft failures; renal vein thrombosis (2), acute humoral rejection (1), acute tubulointerstitial nephritis and tacrolimus toxicity (1), de-novo acute glomerulonephritis (1), chronic rejection (2) and non-compliance (2). graft survival rates were 95% at one year, 93% at 3 years, 89% at 5 years and 88% at 10 years. patient survival was 100% at 10 years. conclusion: our outcomes compare well with international figures a desensitisation program for paediatric renal transplant patients in nsw one child received atg in addition. results: complications/ patient outcomes:1: 2 yo boy, donation from mother. dsa antibodies mfi 658. no complications. no infections. good renal function, creatinine 45 at 2yrs. 2: 14 yo boy, previous transplant from biological father. donation from foster father. dsa antibodies mfi 2602. post operative complication of pseudomonas pneumonia/bronchiectasis, cmv, acute cellular rejection x2. good renal function creatinine 120 at 1 yr. 3: 17 yo boy, donation from father. dsa antibodies mfi 724. early e-coli urine infection. no other complications, creatinine 117 at 1 yr. 4: 14 yo girl, recipient of 3 liver transplants. maternal donor with positive b cell cxm and dsa mfi 6488. no complications. good renal function four of them had subsequently increasing needs in bicarbonate and/or sodium supplementation, and f was reintroduced in 3 of them. conclusion: f is effective in most cases of severe tubulopathy after rtx. however, side-effects can occur. further prospective studies are needed to validate this indication methods: eighty-one (f/m: 41/40)pediatrictransplant patients were included to the study.demographic characteristics and laboratory parameters were recorded.risk factors for hyperuricemia and the effects of plasma uric acid levels at 3 rd and 6t h months, 1 st and 3 th years on allograft outcomes were evaluated. results: mean age was 16.9±5.6 years.mean follow-up time after transplant was 3.5±0.47 years. hyperuricemia was detected in 17.6% of patients. a significant negative correlation was found between 6 th month uric acid leveland 3 th year of gfr value (r = -0.33, p = 0.04 and r = -0.33, p = 0.017). a significant positive correlation between 3 th and 6 th months uric acid levels and3 th year plasma creatinine level was demonstrated conclusion: uric acid levels may have predictive value in the long term assessment of renal function.posttransplanthyperuricemia can be used as a long term prognostic marker of poor renal outcome.patients with hyperuricemia should be monitored closely for renal functions abstract# p-sat447 tuberculosis in paediatric renal transplant patients -single centre experience methods: retrospective descriptive folder review of tb in paediatric renal transplantation at red cross children's hospital, university of cape town from 1994 -2012. results: 14 paediatric renal transplant with tb identified. male 11: female 3. ages 3.4 -18.8yrs(mean 12.0; median 13.2). all patients screened for tb prior to transplant polycystic kidney disease, 2 dysplasia, 2 chronic glomerular nephritis, 2 unknown cause, 1 systemic lupus erythematosis and 1 all patients were on steroids and 8 had recent intensification of immunosuppression. tb treatment included conventional drugs in all but 1 case. levels of calcineurin inhibitors were affected in 12/14 patients and required increased dosing(up to 3 times baseline) according to levels in all. rejection was seen in 7 patients(all biopsy proven). successful treatment of tb in all patients with retention of graft. graft and patient survival 100% post treatment. conclusion: tb is a significant problem in paediatric renal transplants in developing countries. our series shows that with careful investigation and diagnosis of tb as well as careful monitoring of immunosuppressant levels objective: to evaluate the reliability, validity and feasibility of the persian version of the pediatric quality of life inventory (pedsql tm 4.0tm 4.0) generic core scales in iranian healthy students ages 7-15 and chronically ill children ages 2-18. methods: we followed the translation methodology proposed by developer to validate persian version of pedsql tm 4.0tm 4.0 generic core scales for children. six hundred and sixty children and adolescents and their parents were enrolled. sample of 160 healthy students were chosen by random cluster method between 4 regions of isfahan education offices and 60 chronically ill children were recruited from st. alzahra hospital private clinics. the questionnaires were fulfilled by the participants. results: the persian version of pedsql tm 4.0tm 4.0 generic core scales discriminated between healthy and chronically ill children (healthy students mean score was 12.3 better than chronically ill children, p< 0.001). cronbachs alpha internal consistency values exceeded 0.7 for children self reports and proxy reports of children 5-7 years old and 13-18 years old. reliability of proxy reports for 2-4 years old was much lower than 0.7. although, proxy reports for chronically ill children 8-12 years old was more than 0.7, these reports for healthy children with same age group was slightly lower than 0.7. constructive, criterion face and content validity were acceptable. in addition, the persian version of pedsql tm 4.0tm 4.0 generic core scales was feasible and easy to complete. conclusion: results showed that persian version of pedsql tm 4.0tm 4.0 generic core scales is valid and acceptable for pediatric health researches. it is necessary to alternate scoring for 2-4 years old objective: bisphosphonates are widely used in the management of children with steroid induced osteoporosis (sio). with the increasing use of bisphosphonates, there have been reports of abnormal radiological findings in the growing skeleton. therefore, their use in pediatric patients remains controversial. the present study was conducted to evaluate the long term follow-up results of radiographic features especially metaphyseal sclerotic lines, associated with pamidronate therapy in pediatric patients with nephropathy. methods: twenty two children with nephropathy receiving oral calcium and pamidronate (mean duration: 7.9 months, dose: 125mg daily) were evaluated restrospectively. all patients had soi because of chronic glucocorticoid therapy for the treatment of nephropathy. biochemical tests, long bone radiography and bone mineral density (bmd) were performed before the treatment of pamidronate and followed up several years later. the physeal growth rates were estimated by measuring the distance that the sclerotic lines moved on the radiographs during the corresponding time intervals. results: the mean follow-up period was 9.5 years. in all patients, the well-defined sclerotic lines at the metaphyseal ends were observed and progressively moved from physeal plate to diaphysis on the radiographs of long bones. the mean moving rates of the sclerotic lines was 7.95 mm per year and in twelve patients, the lines disappeared. and the mean growth rate of height was 4.50 cm per year. conclusion: our long-term follow-up results suggest that the metaphyseal sclerotic lines associated with pamidronate treatment tend to disappear without affecting the skeletal growth. bisphosphonate treatment for soi in pediatric patients with nephropathy seems to be safe although further studies for larger number of patients are needed abstract# p-sat366 clinical effectiveness and safety of the high dose active vitamin d therapy on severe hyperparathyroidism in children with esrd eun gu kang, su-yon kim, joo hoon lee, young seo park department of pediatrics, asan medical center children's hospital, seoul, korea objective: a potent inhibiting effect of active vitamin d on parathyroid hormone is well known, but there is uncertainty on the dose to be used in hyperparathyroidism. the aim of this study is to assess the effectiveness and safety of high dose active vitamin d treatment on severe hyperparathyroidism in children with end stage renal disease (esrd). methods: fifty-four patients underwent dialysis for more than 1 year between may 2002 and feb 2013 in asan medical center. among them, patients who were administered high dose of active vitamin d (dose of alfacalcidol 1mcg/day to 3mcg/day) with severe hyperparathyroidism(intact parathyroid hormone (ipth) >800pg/ml) were selected. changes of ipth, plasma albumin-corrected calcium, phosphorus and 1,25 (oh)2 vitamin d were analysed. results: fourteen patients (10 boys and 4 girls) with median age of 12.5 years (6-19 years) were included. the mean duration of dialysis was 49±32 months and the median duration of the high dose alfacalcidol therapy was 5 months(3-22months). the ipth level significantly decreased in 10 patients (71%) from 1206.3±387.6 to 215.2±117 during the high dose alfacalcidol therapy (p<0.001). serum phosphorus (5.9±1.3mg/dl vs. 6.0±1.8mg/dl, p=0.88) and calciumphosphorus product (53.4±11.9mg2/dl2 vs. 59.8±18.4mg2/dl2, p=0.20) were not significantly changed and 1,25 (oh)2 vitamin d was low or normal after therapy. plasma albumin-corrected calcium significantly increased (9.4±0.64mg/dl vs. 10.2±0.75mg/dl, p=0.016). four patients (29%) had persistent severe hyperparathyroidism despite treatment with high dose of alfacalcidol, which was controlled after kidney transplantation (2 patients) or after parathyroidectomy (2 patients).conclusion: high dose active vitamin d therapy controlled severe hyperparathyroidism in the most children with esrd without significant adverse events. clinical features and treatment of ckd-mbd in children with ckd i-v predialysis objective: vitamin d is known to have multiple effects on the cardiovascular system, renal function, hyperparathyroidism and growth, its deficiency is common in adults and children with chronic renal disease (ckd), but data in kidney transplant children are scarce. the aim of the present study was to investigate the vitamin d status in 3 groups of children and adolescents with ckd and to establish the association between 25(oh) vitamin d (25ohd) levels, age, hyperparathyroidism, short stature, and renal function. methods: we recruited 105 children: 44 renal transplant patients with a functioning graft for at least 6 months (mean age 12 yr, mean graft abstract# p-sat386 the expression and significance of il-6 、ip-10 and il-17 in serum and synovial fluid with juvenile idiopathic arthritis. objective: to detect the disparity of three cytokines about interleukin-6(il-6),interferon-inducible protein 10(ip-10) and in peripheral blood(pb) and synovial fluid(sf) of patients with juvenile idiopathic arthritis (jia). method: serum concentrations of the three cytokines were measured in 27 patients with 13 systemic-onset jia(sjia), 14 polyarticular jia(pjia) and 28 healthy controls using enzyme-linked immunoabsorbent assays (elisa). 19 patients being no marked arthritis symptom or only temporary arthralgia enrolled in probable sjia group. sf from 18 patients with 7 sjia,11pjia were examined for cytokine levels. objective: the aim of the present study is to evaluate serum concentrations of ghrelin and leptin, and their associations with fat mass and insulin homeostasis in children undergoing chronic dialysis. methods: the study population consisted of 40 patients on maintenance dialysis (22 pd and 18 hd) aged between 5-19 years and 20 age-and sex-matched healthy children. serum levels of total ghrelin and leptin were measured in all patients and controls. fasting serum glucose and insulin levels were also measured in the patients; insulin resistance was estimated by the homeostasis model assessment of insulin resistance (homa-ir). nutritional status was assessed by measuring body mass index (bmi), triceps skinfold thickness (tsf) and multi-frequency bioimpedance analysis (bia). body fat mass was estimated by the bia method. results: the mean total ghrelin level was significantly higher in the patients than the controls (1671±1317 vs. 543±365 pg/ml, p<0.001). higher total ghrelin levels in dialysis patients were significantly associated with younger age (p=0.015), lower bmi-sds (p=0.050) and lower bia-based fat mass-z score (p=0.007). the mean leptin level was also higher in dialysis patients compared to the controls but the difference was not statistically significant (15.5±29.2 vs. 6.48±5.51 ng/ml). however, the ratio of leptin levels to fat mass was significantly higher in dialysis patients than the controls (1.16±1.31 vs. 0.50±0.12, p=0.041). serum levels of leptin in dialysis patients positively correlated with bmi-sds, tsf-z score and bia-based fat mass-z score (p<0.001 for all). serum leptin levels also had a positive correlation with serum insulin (p=0.001) and homa-ir (p<0.001), and an inverse correlation with total ghrelin level (p=0.005). conclusion: children on maintenance dialysis have high levels of total ghrelin that are closely related to decreased fat mass and poor nutritional status. in contrast to ghrelin, leptin is associated with increased fat mass and insulin resistance; however, these patients have inappropriately elevated leptin levels in relation to body fat mass that may be related to wasting. objective: this study investigated the influence of social support and other psychosocial factors upon mortality, adherence to medical care recommendations, and physical qol amongst hemodialysis patients. method: 272 hd patients were examined using the qol questionnaire to determine self-reported inclinations. logistics regression through weighted k was used to analyze data. results: 53.5% of patients reported health had interfered with their social activities demonstrating a strong associated with risk towards all-cause (sp=1.33) and cause-specific mortality including cardiac diseases (sp=1.28). these patients had a greater risk of withdrawing (sp=1.67) from treatment, non-adherence to phosphorus (sp=1.06) greater than 7.5 mg/dl and increased risk towards an albumin of less than 3.5 g/dl (sp=1.23). patients reporting dissatisfied with family support (12.0%) were at highest risk to non-adherence to intra-dialytic weight gain (sp=1.27), shortening the dialysis session (sp=1.21) and increased risk of potassium level greater than 6 meq/l (sp=1.14). however, patients reporting dissatisfied with staff support (14.1%) revealed a higher risk of decreased physical qol (sp=0.76). conclusion: this study demonstrated that physical qol was not only affected by medications and other laboratory work-ups but also with additional psychosocial support. the study led to the development of programs empowering patients and families to participate in their treatment plans. the program includes various counselling approaches directed to patient, families, and health team. objective: the aim of the study was to analyze health-related quality of live (hrqol) in children with chronic kidney disease (ckd) dependent on the ckd stage, treatment modality and selected social life elements in families of the patients. furthermore, potential differences between self-and parent/proxy reports and the factors influencing them were assessed. methods: 203 ckd children (on hemodialysis-hd, peritoneal dialysis-pd and conservative treatment-ct) and their 388 parentproxies were enrolled into a cross-sectional national study. a semistructured interview form was used to determine the demographic and social characteristics of the participants. we used the pediatric quality of life inventory (pedsql) 4.0 generic core scales to assess the hrqol in children. results: hrqol scores for all ckd groups were significantly lower in all domains compared with population norms, the lowest one being in the hd group. in ct children, hrqol did not depend on the ckd stage. children with ckd reported problems with education and emotional functioning. both parents assessed the hrqol of their children differently depending on their involvement in the care. there are differences between the hrqol scores of the children and their parents. conclusion: the hrqol in children with ckd is lower than in healthy children. this is already observed in the early stages of the disease. the disease itself influences the child's mental state. children on hd require special support on account of the lowest demonstrated overall hrqol. children's lower rating of the quality of life observed by their parents may render the patients unmotivated and adversely affect their adjustment to life in later years. it may also create conflicts between the parents and the children. objective: chronic medical illness is a significant risk factor for the development of psychiatric disorders. the aims of the study were: to investigate the level of anxiety in children with chronic kidney disease (ckd) and to identify factors associated with the presence of that emotional problem. methods: ckd children on hemodialysis (hd, n=22), peritoneal dialysis (pd, n=20,) and on conservative treatment (ct, n=95), and healthy subjects (n=100) were enrolled in the study. we used state-trait anxiety inventory (stai) for adolescents and stai-c for children. socio-demographic and physical factors were assessed. results: there was a significantly higher level of anxiety-state among hd children (8-12 years) compared with other groups of participants of the same age. the level of anxiety among adolescents (13-18 years), both anxiety-state and anxiety-trait, was significantly higher in the hd group compared with other groups, which did not differ among themselves. in the hd adolescents, there was a correlation between the anxiety-state and the duration of the disease as well as with the number of hospitalizations. pd adolescents in the mainstream education had higher levels of anxietystate and anxiety-trait compared with home schooled patients. conclusion: even though children and adolescents with ckd are at risk of developing a variety of emotional disorders, the level of anxiety among the researched group, with the exception of hd patients, was not significantly different than the level of anxiety among healthy subjects. adolescents on hd who present a high level of anxiety should undergo long-term psychological treatment. ipek akil objective: compare medication adherence and kidney graft loss rate before and after transition in a single-center cohort of pediatric patients. methods: records of the patients transplanted in our center between 1990 and 2011 were screened and patients who remained in the program by their 18th birthday were included in the analysis. adherence and graft function were assessed for the period of 2 years prior and 2 years after transition. undetectable and/or sub-therapeutic levels of calcineurin inhibitors and their level variability were used as measures of adherence. graft survival analysis was performed for the period of 4 years before and 4 years after transition. results: out of 197 screened patients, 71 were eligible and 25 of them were transitioned (tg -transitioned group). the remaining 46 patients were used as a comparison group (cg), 27 of them did not reach the age of transition by the time of the analysis. the median age at transplantation was 17.7 [15.1-19.1] years in the tg and 15.8 [13.5-17.6] years in the cg, median age at transition was 22. 5 [21.7-23 .3] years. overall, there was no significant difference in adherence within tg before and after transition (p=0.5 for low drug levels and p=0.6 for drug levels variability); however, patients with lower pre-transition adherence (tertile i) showed improvement of their adherence following transition when compared with those with high pre-transition adherence (tertile iii), p=0.02. there were 4 graft losses per 67.9 patient-years in tg vs 19 losses per 102.4 person-years in cg (p=0.02). the peak graft loss in all pediatric transplant recipients in our center (n=105 losses) occurred 3 years (sd 5.7 years) prior to the transition. conclusion: at our institution, transition was not associated with worsening adherence or graft loss. this may be in part do to the fact that the transition occurred at a later age than in other institutions. a report on 7-years experience in the use of mtor-inhibitor (sirolimus) in paediatric renal transplantation patients with calcineurin inhibitor (cni) toxicity the management of children with end-stage renal disease (esrd) deu to congenital urological abnormalities is more problematic and difficult than in patients with esrd due to other causes. kidney transplantation in neurogenic bladder patients with small capacity and defunctionionalized urinary bladders is a challenging issue in the field of pediatric transplantation. in these patients with severe bladder dysfunction, augmentation cystoplasty can protect the transplanted kidney by reducing intravesical pressure and creating an appropriate capacity. 8-year-old boy who presented urinary tract infection with fever at 2 year-old and while his investigation, stage 3 chronic kidney disease secondary to bilateral grade 5 reflux disease was found (blood urea: 81 mg/dl, creatinin: 1.5 mg/dl, creatinin clerance: 32 ml/min/1.73m 2 ). his mixiocystoureterographic and urodynamic study were showed bladder wall irregularity and trabeculation; decreased bladder capacity and compliance, respectively. at 8-month of followup he was putted into dialysis programme about 6 years. thereafter, normal bladder capacity was achieved after bladder augmentation implementation. he was achieved renal transplantation from his mother, after 6-month of augmentation operation. now, he still got uneventfull follow-up period about 24 months. consequently, bladder augmentation application with timely and rationally could be a chance to kidney transplantation in children with bladder dysfunction. the effect of anti-hla antibodies on renal graft functions esra baskin 1 , asli kantar 1 umut bayrakci 1 , kaan gulleroglu 1 , mahir kirnap 1, 2 , feza karakayali 1, 2 , aysegul haberal 1, 3 , gokhan moray 1, 2 , mehmet haberal 1, 2 1 pediatric nephrology, baskent university, ankara, turkey 2 general surgery, baskent university, ankara, turkey 3 immunology, baskent university, ankara, turkeyobjective: the identification of suitable donor kidneys for transplant candidates with high levels of circulating antibodies against human leukocyte antigen (hla) is a major challenge and results in adverse graft outcome. methods: seventy four kidney transplanted children without any shown hla antibody in the pre-transplant period were enrolled in the study. their anti hla antibody status was checked by luminex during post transplant period and its relation with the graft function and prognosis of the patients is studied. results: mean age of the patients was 13.5±5.2 years. mean follow-up time was 3.8±1.1 years. pre-transplant cytotoxicity tests and pra was negative in all patients. nine (12.1%) patients were found to have anti hla antibodies after kidney transplantation. mean time for the detection of antibodies was found as 11±4.8 months. patients with anti hla antibodies were similar with patients without antibodies in the terms of age, sex, hla mismatch, transfusions and immunosuppressive drugs as well as the presence of viral infections. mean serum creatinine level was found to be higher in patients with anti hla antibodies. the antibody mediated rejection rate was found to be 7.2% (5/65) in patients without anti hla antibodies while it was 55.1% (5/9) and remained dialysis dependent. all 6 were dialysis dependent by 6 months post diagnosis. time from anca gn diagnosis to kidney transplant (mean±sd) was 31±12 months (range 17 -48 months). all patients received induction therapy and maintenance immunosuppression with prednisone, mycophenolate mofetil, and tacrolimus. median duration of follow up post transplantation was 3.5 years (range 1.25 -6.9). egfr at last follow up was 71.9 ± 34.7 ml/min/1.73m 2 (range 5.7 -100.5). 1 patient lost her transplant to biopsy-proven, severe acute cellular rejection due to complete non-adherence to medications after 21 months of stable transplant function. no patient had recurrence of vasculitis. conclusion: short-term patient and allograft survival in paediatric patients with eskd secondary to anca gn is excellent despite aggressive disease, with no recurrence of vasculitis post transplant.abstract# p-sat429 en-bloc kidneys from infant donors less than 5 kg transplantation into pediatric recipients at school age objective: given the shortage of donor kidneys in china, the use of grafts from deceased infant donors (weight < 5kg) is a potential approach to expand the donor pool. in this study, we reviewed the results of the first cohort of en bloc kidney transplantation of infant donors to pediatric recipients at school age in our center.methods: from february 2012 to march 2013, 4 infant en bloc kidney transplants in pediatric recipients were performed in our center. en bloc kidneys from 4 infant donors (maastricht category iii) who died of severe congenital disease were recovered and donated to the red cross society of tianjin and allocated to our center by china organ transplant response system (corts). donor age ranged from 33 to 56 days with weight ranging from 2.5 to 5.0 kg. recipients included 2 females and 2 males with age ranging from 5 to 11 yr. the en bloc graft was implanted extraperitoneally in the right iliac fossa. the distal end of the donor aorta was anastomosed end-to-end to the internal iliac artery, while the donor vena cava was anastomosed to the external iliac vein. the donor ureters were implanted separately onto the bladder with double-j stents placement. after the operation, the recipients received basiliximab as an induction therapy. maintenance immunosuppression consisted of tacrolimus and myfortic. prophylactic anticoagulation with heparin was used for the first week after transplantation. results: patient survival was 100%. complications included delayed graft function in 1 patient (managed by pd for one week), urine leak in 1, and anticoagulation-related hemorrhage in 1. due to discontinued anticoagulation, one graft was lost early from vascular thrombosis. of the remaining 3 recipients, all had immediate and excellent long-term function with average creatinine of 1.13±0.31 mg/dl at 5 months follow-up (range 1-12 months). conclusion: this is the first report of en bloc kidney transplantation from infant donors into pediatric recipients in china. many improvements by our transplant teams had improved the survival of patients and grafts. based on our experience, albeit very limited, we concluded that favorable outcomes can be obtained from en bloc transplantation from infant donors. objective: to describe the rates and outcomes of renal transplantation in children with intellectual disability (id). methods: we performed a retrospective analysis of all children receiving a first kidney alone transplant in the united network for organ sharing (unos) dataset from january 1, 2008 to october 31, 2011. recipients with definite, probable, and without id were compared using chi-square and fisher's exact tests. kaplan meier curves were constructed for patient and graft survival. results: over the study period, 218 children with definite (90) or probable (128) intellectual disability underwent first renal transplant accounting for 17% of all first pediatric renal transplants (total n=1280). children with definite or probable id did not significantly differ from other recipients on the basis of gender or ethnicity but tended to be younger. children with definite id had higher rates of structural kidney disease and lower rates of glomerulonephritis. children with id were not significantly different than children without id with respect to rate of preemptive transplant, donor source, or number of episodes of acute rejection. graft and patient survival were similar between children with definite or probable id and without id. in cox regression, intellectual disability was not significantly associated with patient or graft survival. conclusion: in this first large-scale study, 17% of all first pediatric renal transplants are performed in children identified as having intellectual disability. early outcomes after transplant appear to be equivalent between children with and without id. further research is needed on long-term outcomes and quality of life effects of transplant in this population. mariana guerra duarte rosa de lima, ana cristina simoes e silva, nadine marcia de faria, eleonora moreira lima pediatrics, universidade federal de minas gerais, belo horizonte, brazilobjective: the aim of this study was to evaluate the clinical course of children and adolescents undergoing renal transplantation at federal university of minas gerais between 2000 and 2011 and to identify possible factors that could interfere with graft survival. methods: a retrospective observational cohort study through analysis of medical records of patients below 18 years submitted to renal transplant were performed. data were analyzed in spss, version 19.0. the results were expressed by descriptive variables and survival analysis was performed using the kaplan-meier method. comparisons between subgroups were made using the log-rank test. p level was set at below 5%. results: we analyzed 64 patients who underwent 66 transplants. the mean age was 10.5 ± 3.9 years. the main causes of chronic kidney disease were cakut (32.8%) and glomerular diseases (26.6%). most patients were submitted to dialysis before transplantation (81.9%) with a mean duration of 36.5 ± 25.5 months. deceased donor was used in 57.6% and the average cold ischemia time was 22.2 ± 6.3 hours. dialysis after renal transplantation was performed in 22.7% of cases. the median survival of patients 1, 5 and 10 years was 98.3%, 95.8% and 92.8%. there were four deaths in this population, two after graft loss. twelve patients lost their grafts (19.7%). the median survival of the graft was 9.2 years and 1, 5 and 10 years survival was 91.7%, 81.2% and 75.6%, respectively. no statistical difference was detected in graft survival between the deceased versus. living donor recipients, the different age groups, the occurrence of hypertension or not, the preemptive transplantation vs. preceded by dialysis (p=0.97) and those with more than three versus less than three hla mismatches. a significant reduction in graft survival was associated with cold ischemia time associated with 24h, need for dialysis after transplantation, early acute or late rejection and creatinine greater than 1mg per dl after the first year os transplantation. conclusion: in our center, graft survival was similar to that described in the literature. however, further studies are needed to address the variables that negatively impacted the results of transplantation. objective: the aim of this study is to assess whether there is an increase in anti-hla antibodies after the influenza a/h1n1 vaccine given to pediatric pts with esrd on dialysis (d) and with kidney transplant (t). methods: 21 pediatric pts were enrolled. mean age was 15.5 years old, 29% female, 71% male, 57% african american, 29% hispanic.objective: ptld is the most common malignancy and important complication of pediatric solid organ transplantation. the rates of pltd have been increasing through 1990s in ktx recipients. we conducted a survey of ptld in children who received ktx since the introduction of tac. methods: from 1975, ktx was performed in 463 recipients at our center. among them, we analyzed data from children who had undergone ktx since the introduction of tac and retrospectively studied incidence of ptld and risk factors, including immunosuppression protocol and pre-ktx ebv serology. results: we retrieved the data of 188 pediatric recipients since 1997 (110 boys, median age at ktx 8.2 years, living ktx 170, pre-emptive ktx 26, ebv-seronegative 82). among them, 11 cases (10 ebvrelated, 1 non-ebv) of ptld were diagnosed in 10 recipients (5.3%), and all cases were ebv-seronegative at ktx. the median duration from ktx to the onset of ptld was 7 months (range: 4-101 months). the most common presenting symptom of ptld was abdominal complaints in 9 cases, followed by fever in 3, superficial lymphadenopathy in 3 and nasal congestion in 1. immunosuppressants were reduced in all cases; the subsequent treatment consisted of antiviral agents + ivig (3/11), anti-cd20 monoclonal antibody (5/11), and anti-cd20 monoclonal antibody + chemotherapy (2/11). only one recipient died due to ptld. assessing the correlation between ptld and immunosuppressants in 82 ebv-seronegative recipients (median age at ktx 5.7 years, cya 39, tac 43, mzr 26, mmf 56, il-2 receptor antibody 56), a significantly higher incidence of ptld was associated with tac (9/43) than cya (1/39) [or 18.3, p=0 .003] in multiple logistic regression analysis. we compared the incidence of ptld, median age at ktx, and rate of ebv-seronegativity in 3 eras (a: 1997-2001, b: 2002-2006, c: 2007-2011) of the study period and found 4%/8.2 yrs/32% in a, 3%/7.1 yrs/43% in b, and 10%/9.0 yrs/55% in c. thus the incidence of ptld increased with the increase in ebv-seronegative recipients. conclusion: in our study, maintenance treatment with tac (compared with cya) is associated with a higher risk for developing ptld. attention should be paid to the increase in the ebv-seronegative recipients.abstract# p-sat438 acute graft dysfunction and encephalitis post renal transplant: the role of epstein-barr virus objective: epstein-barr virus (ebv) has been described as a rare cause of acute kidney injury and encephalitis in children. methods: we report a case of a 5 year old renal transplant recipient who developed acute graft dysfunction and a significant neurological insult with evidence of primary ebv infection. results: the patient had been diagnosed with posterior urethral valves prenatally and received a live related renal transplant aged 4 years. seven months post-transplant he presented fluid overloaded, oliguric, with significant uraemia and hyponatraemia. there was a history of lower urine output and sore throat 4 days before admission with evidence of anaemia with red blood cell fragments, thrombocytopenia, mild monocytosis, raised ldh, alt and splenomegaly on presentation. haemodialysis was commenced. primary ebv infection was subsequently detected by serology and pcr.renal biopsy showed a picture of acute glomerular thrombotic microangiopathy(figure above) and acute tubular injury secondary to haemoglobinuria.scanty eber+ lymphocytes containing ebvencoded small nuclear rna were also present. within 3 days of presentation patient deteriorated neurologically developing a reduced conscious level, dysarthria, decreased motor power peripherally and an inability to upward gaze. an mri brain demonstrated increased t2 signal and mild diffusion restriction in in the caudate nuclei, putamina and ventro-lateral thalami. this picture has been previously described in ebv encephalitis. there was a gradual neurological improvement and by day 7 of admission, and 6 doses of i.v. methylprednisolone, the patient had a full neurological recovery. the number of ebv copies has subsequently decreased on lower dose of immunosuppression. by week 4 after presentation he was no longer dialysis dependent with creatinine 3-fold higher than the previous baseline. conclusion: this is the first report of acute graft dysfunction and encephalitis due to primary ebv infection in renal transplant recipient.abstract# p-sat439 indoleamine 2,3-dioxygenase (ido) as a new immunological marker in kidney elisa loiacono 1 , barbara votta 2 , alessandro amore 1 , licia peruzzi 1 , maria paola puccinelli 2 , roberta camilla 1 , luca vergano 1 , giuliana guido 3 , maria elena donadi 1 , rosanna coppo 1 1 nephrology, cittàdella salute e dellascienza. regina margherita children's hospital, turin, italy 2 nephrology, cittàdella salute e dellascienza, turin, italy 3 nephrology, sapienza university of rome, rome, italyobjective: the enzyme indoleamine 2,3dioxygenase (ido), induced by interferon-gamma (ifn-gamma) and toll-like receptors (tlrs) ligands in dendritic cells, degrades the essential aminoacid tryptophan (trp) to kinurenine (kyn). its activity is estimated by the ratio of kyn to trp concentration (kyn/trp).t-cell activation and proliferation are affected by trp deprivation and accumulation of kyn. therefore, activating ido during immune responses counter balances mechanism of negative feedback loop of ifn-gamma and acts as a tool to downregulate overwhelming immune activation. ido activation has been reported to be increased in acute rejection and downregulated in vitro by the immunesuppressants adopted for organ transplantation. objective: to determine the prevalence of mycophenolic acid (myfortic) use as part of the immunosuppressant regime in our current paediatric renal transplant recipients in a single tertiary paediatric nephrology centre and examine the demography of these cases. method: case note and pharmacy record review of all paediatric renal transplant recipients' history, immunosuppressant medication and current renal allograft function in january 2013. indication for medication switch from mycophenolatemofetil to enteric-coated mycophenolic acid tablets (myfortic) was noted. data presented as median (range). results: 14 (10 male) out of a total of 88 patients with renal transplants are currently receiving myfortic in our centre. age and time from transplant was 12.3 (8. 1-17.4 ) years and 4.3 (0.68-10.6) years. the most common cause of end stage renal disease requiring transplantation in this subgroup was posterior urethral valves in 5/14 cases. two of these recipients had abo incompatible living related transplants. current egfr was 46.7 (20.9-82.3) ml/min/1.73m2. 13/14 patients receiving myfortic were also receiving prednisolone and tacrolimus as part of their immunosuppression regime. one patient was on tacrolimus and myforctic only (history of idiopathic intracranial hypertension). dosage of myfortic varied based on patient size from 180mg twice daily to 720mg twice daily. the indication for carrying out a medication switch from mycophenolatemofetil to myfortic was related to gastrointestinal symptoms in all cases; specifically diarrhoea in 9 cases, abdominal pain in 2 cases and both symptoms in 3 cases. these symptoms improved on myfortic with renal allograft function remaining stable, however in view of neutropenia in one patient her myfortic is currently suspended and under review. we have found that enteric-coated mycophenolic acid (myfortic) was a well-tolerated alternative in paediatric renal allograft recipients who developed gastrointestinal symptoms whilst receiving mycophenolatemofetil. its usage should be considered in such patients who are able to take tablet preparations. ferretti alfonso, ilaria luongo, bruno minale, gabriele malgieri, carmine pecoraro nephrology and urology, santobono children' hospital, naples, italyobjective: uti represent one of the main complications after kt and have an important role in graft funcion impairment. this retrospective report is aimed to evaluate the incidence of uti in a group of patients attending our hospital for post transplant usual medical controls. methods and results: we incuded in our study 75 children who underwent kt between 2000 and 2011: 42 m and 33 f, mean age 11.6 +/-5.3 . four children received a graft from a living related donor and 71 from a deceased donor. first morning sterile urine sample to the microbiology laboratory was obtained when attending the hospital for its usual medical control or immunesuppressive drug detection levels. results: in 41 patients (54.7%) esrd was secondary to cakut. thirty-six patients (48%) presented at least one episode of uti occurred in the first 6 months after kt, mainly in females (1.2:1) . uti developed in the first period were caused mainly by gram negative bacteria (91%). e. coli was the main agent (66.1%), the other uropathogens involved were: proteus (9%), enterobacter cloacae (9%), pseudomonas aeruginosa (6%), candida albicans (3.3%) and klebsiella (3.3%). later infections were caused mainly by candida, klebsiella, proteusansdenterobacterfaecalis. one patient at the 15th month post-transplantation manifested uti caused by corinebacterium urealyticum associated with concretions of baldder mucosae. eight patients (5m and 3 f) experienced the graft loss: 4 patients were affected by cakut and had febrile uti posttx. one patient left the second graft by a an acute deterioration of renal function during an uti by bkv. conclusion: uti in kidney transplanted children represent a main complaint as demonstrated by the high incidence (48%) in total population mainly in females. a significant role is played by primary uropathy and immunosuppression as demonstrated by the high frequency also in non uropathic patients. the role of uti in longterm outcome of kt remains controversial. factors associated with elevated pulse wave velocity in children after renal transplantation objective: even after successful transplantation children with underlying chronic kidney disease (ckd) still carry a high cardiovascular risk. this is also documented by cardiovascular death being the second leading cause of death after transplantation in this patient cohort.methods: in a cross-sectional approach 109 renal transplant recipients at three german transplant centres were enrolled. we measured pulse wave velocity (pwv), a strong predictor for cardiovascular events, as our primary end point. in all participants we assessed classical (e.g. blood pressure, cholesterol) and non-classical (e.g. crp, pth) cardiovascular risk parameters. results: patients were 13 ± 3 years of age and had received their transplant 5 ± 4 years ago. pwv-sds adjusted to height was 0.4 ± 1.5. in order to identify predictors of elevated pwv, we performed a univariate screen and introduced factors with a p-value below 0.2 into a stepwise forward linear regression analysis. we found that systolic blood pressure in the ambulatory blood pressure measurement and gfr independently predicted pwv-sds (table) . conclusion: our results show a large variability in aortic stiffness in children after renal transplantation. we found elevated pwv to be associated with classical as well as non-classical risk factors. the wide range of cardiovascular comorbidity seen in these patients highlights the need for individualized risk factor monitoring and management. objective: renal transplantation is the optimal treatment for end-stage renal disease in children,. however, it remains technically challenging in small recipients, especially with adult-sized graft. we report on two 4-year-old children, undergoing living donor kidney transplantation, and describe our management after failure to close the abdominal wall. method: both children weighed < 15kg and received their father's kidney. the recipient/donor weight ratios were 1/5 and 1/6. surgical procedure was an extra peritoneal approach to the iliac fossa, with vascular anastomosis on the aorta and vena cava. they recieved intensive fluid replacement during surgery to optimize hemodynamic status and graft perfusion. after initial successful reperfusion, all attempts to close the abdominal wall (even simple skin closure or superficial muscle layer closure) led to graft hypoperfusion, diagnosed on graft colour change and doppler ultrasound. closure with resorbablevicryl?mesh gave the same results and failure. finally, we had to close with a synthetic non resorbable plate (goretex?), without skin closure.results: the first patient presented acute tubular necrosis and required continuous hemofiltration from day 2 to 6. the second one had immediate diuresis and with a decrease of serum creatinine to 65μmol/l at day 2. in both cases, kidney was successfully replaced intra abdominally at day 6, with superficial closure of facia and skin in one child and with vicryl? mesh and skin in the second one. no effect on clinical graft perfusion or doppler parameters were observed after final closure. in patient 2, a limited necrotic area 3x3cm, on direct contact with the goretex?plate, was noticed during the second surgery procedure but it remained very superficial. finally, with a follow-up of respectively 1 year and 1 month, recipients and grafts are doing well without any other events and a serum creatinine of 40-50 μmol/l. conclusion: in conclusion, in small patients kidney size discrepancy and tissue oedema can lead to renal allograft compartment syndrome. in this situation, goretex? plate is an option for initial wound closure and graft salvaging. objective: amr in the transplant is one of the most complicated form of rejection, which do not respond to the standard therapy. survival of the transplant exposed to amr is still low. this is a clinical report of an amr-treatment in children in our centre. methods: 4 girls with amr after the deceased donor renal transplantation were observed of age10, 13, 15, 16 from 2009 to 2012 in our centre. all had a negative "cross-match" at the time of transplantation, the quantity of discordant antigens varied from 3 to 6. for 1 girl it was the 2 transplantation. 3 children had an immediate function of the graft,1-delayed. induction of the immunosuppressive therapy: polyclonal antibodies and methylprednisolone, maintenance: tacrolimus, prednisolone, mmf. 2 children had high level of preexisting antibodies (more than 30%) at the time of transplantation. there were morphological confirmation and decrease of the graft function for the moment of amr. the treatment of amr included: pulse therapy of methylprednisolone, polyclonal antibodies, rituximab, immunoglobulin iv. function of the graft was evaluated by the cr levels, gfr, the level of dsa.results: the cr of children was 483±256 umol/l for the moment of developing of amr. function of the graft has been restored to satisfied: cr fall to 121±28 umol/l, there were no proteinuria. it was noted a significant reduction of dsa i and ii class in 3 from 4 patients. conclusion: thus, treatment of an amr of the graft with methylprednisolone, rituximab and immunoglobulin intravenously let us achieve 100% 1-year graft and recipient survival after deceased donor kidney transplantation in children. since the risk of amrdevelopment is high in children with high levels of preexisting antibodies, addition of one-time rituximab and immunoglobulin intraveneously in therapy seems reasonable.abstract# p-sat454 tacrolimus granules use in kidney transplanted infants. dose and safety.ramon vilalta, enrique lara, alvaro madrid, marina munoz, sara chocron, gema ariceta paediatric nephrology, hospvalld'hebron, barcelona, spainobjective: tacrolimus in infants has been administered as an extemporaneously compounded oral liquid made by hospital pharmacy. modigraf® is a new tacrolimus formulation available as sachets containing granules (0.2 mg and 1 mg), that are made up with water, prepared by parents or caregivers. modigraf® is considered at least as safe and practical than the compounded oral tacrolimus liquid. furthermore, it is easier to be stored and ready to use, and promotes patient's autonomy, preventing potential medication errors. 9 kidney transplanted infants, 3 de-novo and 6 converted from oral liquid to modigraf® were evaluated to assess its safety and adequate dose. methods: after induction with basiliximab or atg, de novo's3 patients were treated with modigraf® associated with mmf and with tapered steroids. 6 infants with similar immunosuppression regimen were converted from oral tacrolimus to modigraf®. target tacrolimus through level was 8-10 ng/ml. mean patients' age at transplantation was 2.2 years old, and mean follow-up period with modigraf® was 10±8 months.results: in the de-novogroup the stable modigraf® dose needed to reach target levels was 0.28±0.12 mg/kg/day. in the converted group, the initial oral tacrolimus liquid dose was 0.20±0.12 mg/kg/day, whereas modigraf® stable required dose was higher: 0.30±0.10 mg/kg/day (p =0.0003). target levels of tacrolimus were reached after key: cord-005646-xhx9pzhj authors: nan title: 2nd world congress on pediatric intensive care 1996 rotterdam, the netherlands, 23–26 june 1996 abstracts of oral presentations, posters and nursing programme date: 1996 journal: intensive care med doi: 10.1007/bf02316512 sha: doc_id: 5646 cord_uid: xhx9pzhj nan we present the results of a prospective population-based audit of paediatric intensive care activity in two comparable communities with markedly different delivery systems. in the trent region of the uk (4.2 million people), children receive intensive care largely without the supervision of a paediatric intensivist in a variety of hospitals, few of which have designated paediatric intensive care units (picus). critically ill children otherwise receive intensive care in children's wards, special care baby units (scbus) or adult intensive care units. in the australian state of victoria (4.5 million people), children receive intensive care almost exclusively in one centre -a picu staffed by full time paediatric intensivists. the two regions are otherwise demographically comparable. in both groups, data were collected on all children admitted to an intensive care unit between 1/4/94 and 31/3/95 and children who received intensive care (defined by levels of intervention and nurse dependency) in other sites during the same period. values of each variable at first contact with the icu, and the highest and lowest values over the first 24 hours were recorded. the principal outcome was survival to discharge from the intensive care unit. severity of illness was assessed using pim (paediatric index of mortality) and prism. risk-adjusted mortality was compared using flora's z test and logistic regression. the rate of utilisation of intensive care (>1000 admissions in each region) were similar. there was some variation in case mix between the two groups, but crude mortality rates were similar (7.4% in trent and 6.6% in victoria). however severity corrected data and other measures of picu performance were dramatically better in' the centralised delivery system. the substantial excess mortality in the trent region provides strong evidence for the benefits of centralisation of paediatric intensive care services. there are considerable difficulties in evaluating the efficiency and effectiveness oflcare in children presenting with respiratory failure during acute medical illness. optimal outcomes for such episodes include survival and the shortest length of stay (los) in intensive care with negligible risk of readmission. we have tried to determine whether or not the time course of acute severe medical illness with respiratory failure is predictable. study i (n=1000): a retrospective study of intubated and mechanically ventilated children (>28 days, <17 years) with acute severe medical illness. measures: diagnosis, intensive care los in calender days, and survival. results: the underlying diagnosis fell within one of three broad categories: respiratory disease (n=521, mortality 19.2%), central nervous system (cns) disease (n=342, mortality 38.7%), and systemic inflammation or multisystem (sims) disease (n=137, mortality 47.5%. the los in survivors was: respiratory -median (interquartile range) 8(4-16) days, cns 4(3-8) days, £p,4£ 5(7-g) days. 5:i'~'-+cen diag~,~is-rc!ated-grnnp~ (drgs) were identified (8 respiratory, 5 cns, 3 sims disease) and each have been characterised by mortality and los. study ii (n=300): a prospective study of patients supported by the hypothesis that los for the above drgs was predictable (compared with study i data). in certain instances attributable causes for variances in los were identified: e.g. disease severity, timing ofdrug therapy, and associated disease. with daily paediatric risk of morality scoring within each drg, four profiles of instability were identified. discussion: the time course of acute severe medical illness with respiratory failure is predictable and variance may be attributable to specific care or diagnostic factors. we are now developing a means of linking drg-specific clinical care pathways with an integrated computerised decision support and education facility at the bedside. the objective of this open, prospective study was to assess the relation between basic patient characteristics as well as effectiveness of treatment on the one hand and resource utilization in pediatric intensive care on the other. as universal, non-monetary indicators of resource utilization we used the therapeutic intervention score system (tiss) and length-ofstay (los), from which indicators for total resource utilization per admission (tisstot) and average daily resource utilization (tiss-mean = tisstot/los) were obtained. overall 593 admissions, totalling 3130 days, were included. mortality was 8.4%; non-survivors accounted for 14.1% of overall resource utilization. in non-survivors, both total resource utilization per admission and average daily resource utilization were higher, whereas los was not different from survivors'. severity of illness, surgical status, the presence of substantial chronic comorbidity, emergency admission and transfer from another hospital constituted the major predictive determinants of tisstot (r:=0.19) and tissmean (ra=0.45) in multiple regression analysis (p<0.0001). hence these indicators are appropriate non-monetary measures of resource utilization, a considerable proportion of which are determined by a concise set of basic clinical characteristics. subsequently we analysed the relation between effectiveness of care and resource utilization by assessing severity of illness corrected mortality in low, medium and high resource users, respectively. these 3 categories were delineated by percer/tiles of resource utilization (< p20, p20-ps0, > ps0). despite on average long los and high resource utilization in the high risk group, a relatively low standardized mortality was found, probably warranting prolonged intensive treatment in this patient category. summary: objective:the primary purposes of intensive care are to provide treatments to patients with life-threatening physiological dysfunction or to monitor and observe patients perceived to be at significant risk of dying. this collaborative study was performed to describe our patients and their outcome. in order to improve our results we tried to identit~ high risk groups, patients and methods: 13 picus entered the study, the data included all the admissions with >12 hs. during a 60 days period between the l°june and the 30th september 1993. the records included: age, sex, weight, mechanical ventilation (mv), post-operative condition (p.op), malnutrition, diagnosis, length of stay, prism score and outcome. student test, mann-whitney or wileoxon were performed for univariate analysis. fisher exact test or chi square for dicotomic variables. risk group analysis was performed by logistic regression, odds ratio and 95% confidence interval. results: 650 patients entered the study. mean age was 47.6 months (ds hh¢# 60) and median 18 months. we found significant statistical differences in calculated ,is observed mortality rate comparing malnourished with euthrofic patients; mechanical ventilated (mv) with non mv patients. no differences in ter ~,h of stay or di~ noses were found. effect of the un sanctions on the morbidity rate araong the iraqi small children ( below 3 years old of age ) in bagdad. abdulsamad a.abood / institute of medical technology, bagdad. meningitis is essentially a childhood disease (i). the risk of infection are increased by powerty and overcrowding (7). the impah'ed immunity may be an important pathogenic factor underlying the susceptibility to infections in undernourished subjects (5). in general, malnutrition is a man made disease and it begins quite in the womb and ends in the grave (i). 1918 small children, below 3 years of age were admitted to the pediatric hospital in washash with meningitis over 4 cold months in i994, in contrast to only 176 child admitted with meningitis over the same period in1989. all of the children who admitted in 1994 were frankly undernourished, 45% of them were infected with enterobacteriae, because they were exposed to faulty hygiene and lack of asepsis. these facts showed precisely that our small children had suffered at most from the un_ sanctions against iraq, because of food, milk and drug shortage, since 4 years which had resulted a severe undernutrition among them, which impaired their immune status. m wells, of riera-fanego, j lipman. baragwanath intensive care unit, university of the witwatersrand, south africa. background the use of prism or other scoring systems in the icu is of great importance for evaluating the efficacy and efficiency of a particular icu, the prism score was developed and validated in the usa and europe but has recently been shown to be inaccurate in a south american population, a south african population as well as several european studies. part of the poor performance of the prism score is as a result of differences in the case mix between the reference population and other paediatric icus. since scoring systems should generally be used only in populations similar to the reference population from which the prediction model was developed, a modification of the prism score is necessary to improve its discriminatory ability in a wide range of patient groups, aim to improve the predictive power of the prism score in a south african paediatdc icu population. patients & methods we analysed prism, demographic and clinical data collected prospectively from 1528 consecutive paediatrie icu admissions. the prediction of actual mortality by prism was evaluated by standard statistical methodology (goodness-of-fit test and receiver operating characteristic (roc) analysis), the components of the prism logistic regression equation (prism score, operative status and age) and the 14 physiological variables making up the prism score in addition 10 new variables analysed (nutritional index, the need for inotropes and institution of mechanical ventilation) were subjected to discriminant analysis to determine their association with outcome. results the goodness-of-fit test showed a significant failure of prism to accurately predict mortality over a wide range of expected mortality (chi2[8] = 195, p = 0). prism underpredicted mortality at lower prism scores, but overpredicted mortality in patients with high prisms. similarly roc annysis indicated apoor predic~jve power (az = 0.73 ± 0.01), with an area under the curve significantly less than that for the prism reference population (p = 0), prism showed equally poor discriminatory function at all age groups and diagnosfic categories. '~mth the addition of an index of nutrifional status (proportional weight-far-age), and indicators of early respiratory and cardiovascular failure to the logistic regression formula, and a recalibration of the acute physiological score component, the roc can be improved to 0.83 ± 0.02, with a good fit described by the goodness-of-fit test (cn218] = 3, p = 0.89). discussion the prism score is not accurate in our patient population has been recalibrated in view of the poor discriminatory function that we have shown. part of the inaccuracy derives from the different demographic characteristics of our icu population and a different pattern of diseases. in addition to assessments of acute physiological aberrations, an assessment of nutritional status and early respiratory and cardiovascular failure significantly improve the discriminatory ability of the prism score, these parameters have been devised with a view to improving the accuracy of prism in our population, while not decreasing its accuracy in icus similar to the reference population. in interviewing parents regarding how physicians have communicated bad news, the response i have received is that it has not infrequently been done without appropriate care, understanding and compassion. personal experience and the lessons learned from parents, chaplains and others who deal extensively with these situations have provided me with an approach that has been supportive, compassionate, and caring. an especially difficult communication situation for the intensivist occurs when the parents have to be informed of the death of their child. for the parent, death is the hardest loss of all -the ultimate unalterable loss. circumstances surrounding the death are an important consideration (e.g., a fatal crash caused by a drunken driver, a prolonged illness, a suicide, aids). each produces a different grief reaction. the physician needs to inform parents of their child's death sympathetically coming right out with the news and leaving details until later. allow pauses and time for the paren~ to express sorrow and grief, the best communication may be thoughtful silence and a tender touch. there is disbelief that this happened. it is necessary to repeat oneself. acknowledgment of the parent's "feeling terrible" and the physician's acknowledgment of how terrible he/she feels that the life of the child could not be saved is an important first step in the parent's dealing with this tragic loss. with prolonged resuscitation, it is helpful to have a member of the icu team talk to the parents while the resuscitative efforts are ongoing so that the parents are not left unsupported at this time. a progress report should be delivered in a caring, lucid, and sensitive.manner, indicating that every effort is being made to save the life of their desperately injured child. after a child has died, it is helpful to the family if the physician maintains some contact with them. this should take the form of follow-up telephone calls at approximately 6, 12, and 24 months. this can help to screen for depression in the parents. in giving bad news to the family and making every effort to support them through this tragic time, it is necessary to remind oneself that the intensivist has personal needs for dealing with grief and will also require support to pass through this stage. direct evidence that child mortality is lower in specialist pediatric icus comes from 3 studies. a study in oregon (ccm 1981; 19:150-9) found that mortality adjusted for severity of illness was 102% of expected in 3 pediatric units and 139% of expected in 71 general units (p<0.05). a study in holland (ccm 1995; 23:238-45) found that mortality in high risk patients was 85% of expected in 6 tertiary pediatric units, and 143% of expected in 4 nontertiary units (p<0.05). a third unpublished study, has found that children in victoria (who almost all receive intensive care in a pediatric icu) have a much lower standardised mortality rate than children in the trent region of the uk (where many children receive intensive care in adult icus). there is indirect evidence that icus looking after many children are likely, on average, to perform better than icus looking after few children: numerous studies in many specialities have found that units looking after many cases of a particular disease have better results than units with few cases. see luft hs, "hospital volume, physician volume, and patient outcomes", happ, 1990; and farley d, medical care 1992; 30:77-94. compared to general icus, medical and nursing staff in pediatric icus are likely to be better at looking ~fter children, and plcu rmos have greater skills in pediatric intubation, ventilation, iv drip insertion and drug doses. picus are more likely to have appropriate equipment to manage children -especially for uncommon but life-threatening situations. icus in pediatric hospitals are more likely to have physicians and surgeons with pediatric expertise available for consultation at all times. the american academy of pediatrics, the society of critical care medicine, the british paediatric association and the australian nh&mrc have all said that children should receive intensive care in'specialist pediatric units. the weight of authoritative opinion, and direct and indirect evidence is strongly in favour of looking after children in dedicated pediatric icus. neurological deficit showed higher cbf values (125.7/115.2 ml/100g/ rain.) than the 11 patients with good outcome (mean cbf 1 17.5 sd +8.1; cbf 2 19.9 sd _+9.1 ml/100g/rain}. discussion: in asphyxia decrease of ph is due to reduced tissue oxygenation and indicates the severity of metabolic derangements. co2reactivity in newborns with perinatal asphyxia correlates with the lowest ph and therefore may reflect severity of asphyxia. continuous monitoring of cerebral activity is carried out in our unit on all admissions at risk of cerebral dysfunction, a number of monitors are commercially available and we report our experience with the cfam2 which provides in addition to amplitude integrated eeg analysis, continuous raw eeg display and frequency distribution. bilateral recordings are commenced as soon as possible and continued while clinically indicated. forty one children ranging in ages from 4 weeks to 16 years were monitored for periods from 3 hours to i0 days, diagnoses included traumatic brain injury (11), sepsis/meningitis/encephalitis (1 t), status epilepticus (8) and miscellanous others (11). results are tabulated below. patients 13 12 16 status epilepticus 10 4 1 * beta activity 1 8 15 * background voltage 10 3 1 * < i o/zv 2 or more of above 12 2 1 * (*z2 p < 0,001) asymmetry developed in 4 children, all of whom died. positive predictors of good outcome included a mean background activity of >10zzv, the presence of faster frequencies (usually 13) in response to sedative drugs and the absence of seizures. all monitoring is performed by the picu staff and increasing expertise in interpretation has resulted in earlier therapeutic and diagnostic interventions. regional it was previously found that histamine, a vasoactive mediator, accumulated in brain compartments (kov~ics et al 1995 neurosci lett 195:25) , and antihistamines prevented brain edema formation (dux et al. 1987 neuroscience 22:317) in asphyxiated newborn pigs. in the present study we investigated the effect of intracarotid histamine injection on the blood-brain barrier (bbb) permeability, left internal carotid artery of 30 newborn pigs (4-8 h; 1,180-1,530g; ketamine anesthesia, 10 mg x kg 4) was catheterized through the external branch and different doses of histamine (0, 10 -6, 5xi0 -6, 10 -5, 5x104, 104 m, respectively, in 6 groups of animals; n=5 in each) diluted in 1.0 ml isotonic saline was injected into the vessel through 1 rain. bbb permeability was determined for a small (sodium fluorescein, sf, 376 da) and a large (evans blue/albumin, eba, 67 kda) tracer (2%, 5 mlxkg 4, 30 rain circulation time for both dyes) concomitantly in frontal, parietal and occipital cortex, hippocampus, and periventrieular white matter both on left and right sides 1 h after the challenge. then, intravascular dyes were removed by perfusion and bbb permeability for both tracers was quantified by fluorescence spectrophotometry (wavelengths for excitation and emission were 440 nm and 525 nm for sf; and 620 nm and 680 nm for eba, respectively). histamine injection, in doses higher than 10 .6 m, significantly (p<0.05; kruskal-wallis one way anova on ranks followed by dunn's test) increased bbb permeability for both tracers in each brain region. changes in left hemisphere were more intense (p<0.05) than those in right one after the doses of 5xi0 -6 and 10 -5 m in each region, i0 4 m histamine administration induced similar edema in both sides. increased intracarotid histamine levels resulted in a dose-dependent vasogenic brain edema formation. histamine might have a pathogenetic role in neonatal hypoxicischemic cerebral injuries. supported by otka f-12722 and h-u.s,-jfno.392, $162 in coma caused by traumatic brain jnjury, an indication of the likely outcome is provided by the best motor response to pain in the first .$ hours after the insult. in a study in our picu, the proportion of children who died or had a severe disability was 100% in 35 who had no response to pain, 40% in 47 with an extensor response, 14% in 64 with a flexor response, and 1% in 61 who localized in response to pain. the long term outcome of traumatic brain injury appears to be worse in children <4 years old. other risk factors in traumatic brain injury are absent basal cisterns, midline shift or subdural haemorrhage on ct scan (or loss of grey-white differentiation in nontraumatic injury); or an intracranial pressure >30 mmhg despite hyperventilation, mannitol and barbiturate infusion. apart from brain death, there are two findings implying such a poor prognosis that consideration should be given to stopping treatment: first, after traumatic injury, the absence of any motor response to painful stimulus in the cranial nerve distribution (providing drug effects and a post-ictal state have been excluded); and second, in acute brain injury from trauma, infection, hypoxia, or ischaemia, the b{lateral absence of short-latency somatosensory evoked potentials (providing brain stem haemorrhage, subdural and extradural effusions, and decompressive craniectomy have been excluded). in children over 2 months of age, recovery from prolonged coma or a vegetative state is exceedingly rare when more than 12 months have elapsed after traumatic brain injury, and when more than 3 months have elapsed after nontraumatic injury. overproduction of nitric oxide (no) via an inducible isoform of" no synthasc (inos) produces profound vasodilatation in adult septic shock. high nitrate levels have been reported in hypotensive children with sepsis syndrome ]. cardiovascular collapse is a prominent feature of severe meningocoecai disease (mcd). however, systemic vascular resistance (svr) was slightly higher in a group of non-survivors ~ and the rote of no in ivicd remains unclear. children with a presumptive diagnosis of mcd were enrolled. parental consent was obtained. blood was drawn on admission and 12hrly thereafter. plasma was separated immediately and stored at -80°c. the final concentrations reported represent the product of nitrite and nitrate (nox). nox was measured spectrophotometrically using the greiss reaction. 21 children were studied (median age (range); 27m (5-203)). the diagnosis of mcd was confirmed in 18 children, 12 of whom had a glasgow meningococcal score (gms) of" ~8. in this group with severe mcd there were 3 deaths. peak nox was significantly higher (,.54(27-78) vs 96(50-363)nmol/ml, median) and systolic btood pressure was significantly lower in children with severe mcd than mild mcd (p<0.05. wilcoxon rank test). there was a significant correlation between peak nox and gms (spearman's rank correlation r=0.6 (p=0.01)) and prism (r=0.6 (p:0.01)). nox production from adm.ission onwards was also higher in the severe mcd group (p:0.002, kmskal ~wallis). we have demonstrated that plasma nox levels are elevated in children with mcd, correlate directly with the severit 3' of disease and are inversly related to systolic blood presssure. similar to hypotensive septic syndrome, mcd appears to be associated with an up-regulation of the l-arginine-no pathway.. non-survivors with mcd have higher svrs and may be relatively hypovolaemic. in our group of severe mcd there was a significantly lower systolic pressure and increased no formation. excess inos expression at different stages in mcd may contribute to the pathology of the disease. the identification of agents which can boost and/or inhibit no reiease may therefore represent different treatment strategies for mcd. u. merz, th. peschgens, g. kusenbach, m. b6hle, h. h6rnchen in this controlled, prospective study 30 ventilated premature infants with a birth weight < 1250 g were randomized to receive treatment with dexamethasone (dex) either on day 7 of life or on day 14 of life. dex was given over 16 days tapering from 0.5 mg/kg/day to 0.1 mg/kg/day. the infants treated with dex on day 7 of life could be weaned earlier from the ventilator -in median after 14 days (range 10 -34) versus 24 days (range 8 -44) in the [ate treatment group (p = 0.01). the need for supplemental oxygen was shorter in the early treatment group -in median 24 days (range 10 -50) versus 40 days (range 10 -70) (p = 0.2, ns). the incidence of chronic lung disease was lower in the early treatment group -6 of 14 infants (42.9%) versus 10 of 16 patients (62.5%) (ns). to evaluate the long-term efficacy of early dex treatment we performed a respiratory function test in the age of 3 -6 months using an infant whole body-plethysmograph. the intrathoracic gas volume (itgv), the airway resistance (r.w) and the airway conductance (gaw) were measured and no significant differences could be detected between the groups. the frequency of adverse effects due to dex therapy was found to be without significant differences between the early and the late treatment group. we conclude that early dex treatment had short-term improvements in pulmonary outcome in our study population, long-term efficacy however, remained unproven. several factors contribute to the development of chronic lung disease (cld) in premature infants including structural immaturity of the lung, mechanical ventilation, and oxidative stress. reactive oxygen species are formed during normal cellular metabolism but they are generated in higher concentrations during inflammation or inhalation of high oxygen concentrations. to study the relationship between increased oxidative stress, antioxidants and the development of cld we examined 102 ventilated premature infants with birth weights below t500g. 32 infants developed severe chronic lung disease of prematurity (cld), defined by radiological signs of cld and an increased oxygen requirement at a postconceptional age of 36 weeks, and 29 infants had moderate cld with an increased oxygen requirement on day 28 but not at an age of 36 weeks. ventilator settings (fio2, peak inspiratory and mean airway pressure) and the incidence of early-onset-sepsis were significantly higher in the severe cld group than in infants with moderate cld or without cld (n=41) during the first week of life. plasma concentrations of the two antioxidative substances bilirubin and uric acid (ua) were comparable in all groups during the first days of life. however, on day seven bilirubin and ua were significantly decreased in the plasma of infants with severe and moderate cld compared to the non cld group (p<o.001). there was no difference between both cld groups. in serially obtained tracheal aspirate fluids (taf) malondialdehyde (mda) was measured by hplc as an indicator of lipid peroxidation reflecting oxygen injury of the lung. ua was found in higher concentrations in taf than bilirubin. ua has been proven to be a potent antioxidant, capable to react especially with hydroxyl radicals and hydrochlerous acid, which play an important role in the induction of lipid peroxidation. infants with severe cld had significantly higher mdnua ratios in taf from day 5 to 14 compared to the non-cld group (p<o.01). mdnua ratios in taf of infants with moderate cld were significantly higher from day 7 on but still lower than in the severe cld group. in addition, the ratio oxidized / reduced glutathione was significantly increased in taf of infants with cld compared to infants without cld between day 5 and 14. the data support the hypothesis that an imbalance of oxidative stress and antioxidant defense contributs to the onset of cld at the end of the first week of life. introduction. rocuronium bromide (roe), a neuromuscular blocking agent with an onset time dose to that of succinyleholine l'z, has been studied in american society of anesthesiologists' physical status (asa) i or ii pedlatrie patients under non-critical elective conditions. there is a paucity of studies looking at the pharmacodynamics of roc under emergent conditions, involving pediatric patients who are asa iii or iv, without the use of adjuvant inhaled anesthetics ~. methods. with approval of our committee on clinical investigations (irb), roe 1.2 mg/kg was administered to 15 asa iii/iv pediatric intensive care patients (age 40 days to 23 months; m:f=8:7) on intravenous sedation. the time to reach 25% a~d 10% of b~elinc tetanie stimulus response was recorded using a r.elaxogra'ph/qmt-100 nerve stimulator/recorder (datex, helsiaki). depth of clinical relaxation at the 25% level was recorded. results. the time from rapid bolus to 25% was 25.4+12.6 see with a range of 10-50 see; and to 10% was 30.1+17.6 see, with a range of 10-69 see. at the 25% level, excellent clinical relaxation was seen. conduslon. in critically ill children, use of roe at 1.2 mg/kg produces rapid onset of neuromuscular blockade and excellent clinical relaxation, such that roe should be considered for rapid sequence intubation. int anesth clin. 1995;33(1):39-60. anesthesiology. 1994;81 (5) computed tomography of the chest (ct) in mechanically ventilated adults can detect intrathoracic pathology not appreciated on chest radiographs (cxr) and influence intensive care management. no such data exists for ventilated children. aims: 1) to assess if ct provides additional information over cxr regarding extent and nature of intrathoracic disease in ventilated children. 2) to determine whether such information alters clinical management, inclusion criteria :10 ventilated children with cxr changes inconsistent with their respiratory status underwent ct if either a) pao2:fio2 ratio <30 and/or mean paw>15 cm h20 or b) there was an unexplained increase in ventilatory requirement. methods : high resolution ct was performed in 3 patients and spiral ct in 7 patierits, to ensure minimal transport related morbidity, patients were transferred to the ct scanner by a specialised mobile intensive care team. results: in 2/10 patients ct demonstrated greater extent of disease than appreciated on cxr but did not significantly alter clinical management. in 7/10 patients ct provided additional information regarding the nature of disease present, in 2/7 children this involved a further diagnosis and in 5/7 children the exclusion of a suspected pathology. new information led to a positive therapeutic intervention in 2 children, prevented inappropriate manoeuvres in 3, and had no significant effect on acute management in 2 children. conclusions: initial data suggests that in a selected group of mechanically ventilated children chest ct can add to the sensitivity and specificity of intrathoracic diagnosis provided by the chest radiograph and directly influence acute management. case selection criteria and choice of the most appropriate protocol requires further study. pressure control ventilation (pcv) utilizes a decelerating flow pattern which may improve gas distribution and lead to alveolar recruitment. in contrast, volume control ventilation (vcv) employs a constant flow. in children, the effects of pcv as compared to vcv are unclear. the purpose of this study was to determine how these two modes compare in terms of dynamic compliance (cdyn). peak iaspiratory pressure (pip), and mean airway pressure (paw) at equivalent minute ventilation. methods: sixteen infants and pediatric patients ranging in age from 1 day to 13 years were studied. diagnoses included ards (6), postoperative cardiac surgery (7), head trauma (1), and resfrictive lung disease (2). patients were randomized to pcv (9) or vcv (7). initial measurements of gas exchange (abg's) and respiratory mechanics (ventrak, novametrix medical systems) were obtained after a 20 minute stabilizadon period. respiratory mechanics included pip, peep, paw, delivered tidal volume, and cdyn (avolume/apressure). the patients were then crossed over to the alternate mode of ventilation holding delivered tidal volume, peep, inspiratory time, minute ventilation, and fio2 constant. data were collected after 20 minutes, in each mode the absence of intrinsic peep was confirmed. to assure that the measurements were not affected by changes in clinical status, the patients were returned to the initial mode of ventilation and measurements repeated (final) . patients were ventilated with a siemens 900c or sv300. reselts: data were analyzed using 2-way analysis of variance with repeated measures. ~ <0.05 vs. vcv) vcv pcv ~ initial ] final ! cdljn 3.5_+0.7 4.3_+0.8 * 3.7_+0.6 3.9_+0.7 i , pip 32+1.0 30l-_t.0 * 31_+1,0 31+-1,0 paw 9.2_+0.6 10.9i-_0.7 * 9.7+0.7 10.0-!-_0.8 pao2 97_+14 92+-10 87_+9 97_+14 discussion: at the same minute ventilation, the decelerating flow pattern of pcv resulted in a 23% increase in cdyn and an 18% increase in paw while decreasing pip by 6%. the lack of a significant change in oxygenation may be a result of the limited time in each ventilator mode as well as the inclusion of patients with both normal and abnormal lungs. there was no significant difference in initial and final measurements indicating patient stability. the beneficial effects of iecre~l~iug cdyn and paw while decreasing pip indicate that pcv may be a preferable mode of ventilation in patients with lung injury. further randomized studies examining the effect of pcv on respiratory outcome measures in pediatrics are indicated. prolonged positive pressure ventilation following repair of cdh is associated with a high prevalence of iatrogenic lung injury, in our unit dudng 1981-1990 314 late deaths after repair of cdh were due to chronic lung disease. since 1990 babies requiring assisted ventilation for more than 7days following surgery were transferred to a cnep chamber to limit lung injury. cnep of -6cm of h20 was combined with positive pressure ventilation via an endotracheal tube dudng the transition phase. immediate reduction of peak inspiratory and positive end pressures were possible and following extubation respiratory support was maintained by cnep v~th appropriate inspired oxygen. overall outcome: [1981] [1982] [1983] [1984] [1985] [1986] [1987] [1988] [1989] [1990] n=68 deaths before surgery (%) 11 ( ecmo during 1990 -1995 /16 who were ventilated for more than 7 days received cnep and there were no deaths and no chronic lung disease in that group. cnep assisted ventilation may be an important management option for babies who require prolonged respiratory support to avoid the adverse effects of chronic positive pressure ventilation, introduction so far 2 modes of liquid ventilation (lv) have been used in experimental animals and, exceptionally, in humans: 1. total liquid ventilation (tlv)-functional residual capacity (frc) is filled by perfluorocarbons (pfc), and slow tidal volume (tv) breathing is performed by pfc. 2. partial liquid ve,0ti,la~ion (page) -only frc is filled by pfc. gas tv is delivered by conventional mechanical ventilation (cmv), high frequency jet ventilation (hfjv) or high frequency oscillation (hfo). the aim of our study is to present our limited experience with page in newborns and infants. page was used in two groups of infants: 1, in 2 infants with brain death before disconnection from cmv, because recipients for organ transplantation were not available. these infants have relatively normal lungs (fio~ less than 0.4). infants stayed on page for 1 hour, during that period no ventdator manipulations were made. after page, infant were switched to cmv for next 6 hours. 2. very critically diseased infants with ards (rds) -2 on ecmo more than 5 days, 1 before cannulation for ecmo, 4 on hfo because of intractable respiratory failure, preoxygenated rm 101 (miteni, italy) was used in the doses up to 40 ml/kg intratrachealy. blood gases and parameters of pulmonary mechanics were followed (dynamic compliance -c dyn, airway resistance -raw, bicore monitor). page was combined with no inhalation (5-80 p.p.m, in 2 infants). in both groups ad hoc an approvement from e local ethical commission and informed parental consent were obtained. in the first qroud with relatively normal lung parameters of oxygenation drops after pfc instilation intratracheally and stayed depressed for 4-6 hours. slight pco2 retention occured in both cases during page. c dyn increased almost double during page period, raw drops transitorily after pfc instilation but in 10 minutes they were identical like in prepage period, parameters of oxygenation (peo2/fio2) after 4-6 hours after page improved and were better than in prepage period. after that time infants were disconnected and died. in the second group no improvement of oxygenation was seen in one ecmo baby, in spite ()f transient improvement of c dyn. in the second ecmo baby, oxygenation improved and flow of pump could be decreased by more than 20%. none of these babies, however, survived, improvement was only transient in spite of repeated dosis of pfc. in these babies serious problems were to maintain the adequate frc by liquid, because of severe air leak, in 5 babies on hfo/hfjv with severe ards/rds the improvement of oxygenation were seen in all the cases immediately after pfc instiletion for the period of 4-5 hours. after that period, pfc dose had to be repeated. two babies of this group survived. conclusion. page is going steadily from tabs to clinical practice. it is simple, could be performed anywhere, cheaper than tlv. however, because liquivent -perflubren (aliance pharmaceutical) is not available in europe, rm 101 of 82 (mitenti, italy) is the only solution, which could be currently used here. before the widespread use of page in clinics, liquid network among most nicus and picus must be built up, the criteria for page must be defined and ethinal-legal problems resolved as well. after resolution of these particular problems page can be life saving procedure for very special part of critically ill newborns end infants. catherine caronia, peter silver, laura nimkoff, cad quinn, jack gorvoy, and mayer san. division of pediartic critical care, medici,, schneider children's hospital, new hyde park, ny 11040, imroduetiun: cystic fibrosis (cf) patients awaiting lung transplantation present a therapeutic dilenuna when severe respir, aory decompemalion occurs, endotracheal intubation and mechanical ventilation is known to have no long term benefits and is associated with high morbidity and mortality. noninvasive respiratory support appears to be a beneficial alternative. methods: we instituted bipap (respironics, inc,, murrayville, pa) in 9 end-stage cf patients who were admitted to the pediatric icu with severe respiratory decompeusation. all patients were awaiting tung transplantation. after a control period, bipap was applied via a tight fitting nasal or facial mask, using the spo~aneous breathing mode, expiratory pressures were set at 4-8 cm hhzo. inspiratory pressures were started at 8 cm ~i o and increased in 2 cm i-i20 increments until the patient's respiratory comfort was achieved and substantiated by non-invasive monitoring. patients were instructed to use bipap during night sleep and whenever subjectively required, data are reported as mean _+ s.d. results: all 9 patiems utilized nocturnal bipap for 6-10 hours/day during a follow-up period of 2-19 months. compared to their pre-bipap status, the patiems' oxygen requirement and respiratory rate both oz~ cundusion: bipap tl~rapy improves the respiratory status of decompeusatir!g end-stage cf paacnts. it is well tolerated for long term use at home, and provides an extended period of respiratory comfort and stability for cf patients awaiting lung transplantation. l. bindl*, g. kiihl**, p. lasch***, appel**, j.m611er**** and the "arbeitsgemeinschaft ards im kindesalter" background acute respiratory distress syndrome (ards) is a therapeutic challenge in pediatric intensive care in view of the high mortality, in 1992 about 50 german paediatlic hospitals founded a working group aiming on collaborative clinical research in this field. aims and methods the aim of both a prospective and retrospective survey conducted in german pediatric intensive care units in 1993 was to accumulate data on the epidemiology, risk factors, natural history and treatment strategies in a large group of pediatric ards patients who were treated in the tt~ee year period from 1991 to 1993.all patients had acute bilateral alveolar infiltration of noncardiogenic origin and a po2~io2 ratio < 150mmhg. the influence of sex, underlying disease and single organ failure was analyzed using the fischer's exact test, the influence of additional organ failure on mortality was tested with the cochran-mantel-haenszet statistics. results 112 patients were reported giving an incidence of 7 cases per 1000 admissions to pediatric icus. median age was 24 month. in 43% of the cases, ards was associated with a pulmonary, in 39% with a systemic underlying disease. in 20% immunocompetence was impaired. mortality was 46% and not dependent on age, sex and triggering event. the number of associated organ failures, however, strongly influenced mortalib,. mortafity in immuno-compromised patients was 8 t %. the analysis of treatment modalifies employed in the patients revealed a lack of uniform therapeutic strategies. on the other hand, the patients were exposed to interventions not yet supported by controlled trials. conclusions the observation of the lack of uniform treatment strategies led to the elaboration of recommendations on ventilator therapy and patient monitoring within the working group. the data gathered in this survey provide the basis for the design of prospective multicenter studies urgently needed to evaluate innovative treatment modafities in pediatric ards. recurrent apnea and respiratory failnre due to severe lower respiratory tract disorders such as bronchiolitis or pneumonia are the most common reasons for mechanical ventilation during respiratory syncytial virus (rsv) infection. acute respiratory distress syndrome (ards) has been described as a complication of severe rsv infectionj in contrast to the low mortality rates associated with rsv infection (< 5 %), mortality rates in the range of 40-70 % have been reported in pediatric patients with ards. however, studies on ards are usually lumped in respect to causation and the disease course of rsv induced ards has not been previously studied. we examined the lung function abnormalities of 37 infants with rsv induced respiratory failure requiring assisted ventilation, measurements included respiratory mechanics, maximal expiratory flow-volume curves and lung volumes, ards was defined clinically using the criteria which were recently proposed by the american-european consensus conference on ards~: acute disease onset, pao2/fio~ ratio _< 200 mrn hg, bilateral infiltrates on chest radiograph and absence of clinical evidence of left atrial hypertension. we calculated the murray lung injury scores modified for use in pediatric patients 3 from total respiratory system compliance, radiographic findings, ventilator settings and blood gas results. we identified 10 infants with severe restrictive lung disease that fialfilled the clinical criteria fbr classification as ards. all had lung injury scores above 2.5 which is the recommended cut-off for a diagnosis of ards, twenty-seven infants had obstructive disease consistent with a clinical diagnosis of bronchiolitis. the ards patients were significantly younger, had a longer time of assisted ventilation (p <0.05) and a greater proportion of infants with preexisting illnesses (p=0.023, odds ratio =6.67) when compared to the patients with obstructive disease. with the exception of one immunodeficient patient, none of these infants died. given the low mortality despite a clinical picture of severe lung injury, there is evidence that rsv induced respiratory failure may represent a relatively benign cause of ards in pediatric patients, bachmann an audit of patients with severe acute bypoxic respiratory failure (ahrf) receiving highfrequency oscillatory ventilation (hfov) in our unit ( n=32, mortality 75%) revealed that sub-groups with severe underlying disease (n=14, mortality 100%)and those with mu~pie organ failure ( > 2 systems failing, n=7 mortality 100%) accounted for all the deaths beyond the neonatal period. v~ therefore hypothesized that in a modem paedistric intensive care unit (picu): a) children greater than one month of age with ahrf do not die in the absence of severe, pre-existing disease or multi-organ dysfunction syndrome, b) respiratory parameters alone will predict outcome poorly in ahrf. method prospect~/e sty/of all adm~ns to our tertiary picu. data it, citing the respiratory parameters (oxygena~n index [ol] , aiveolar-artedal oxygen tension gradient , pao2/fio2 ratio) were collected hourly from the bedside charts throughout admission. patients were included in the study if ahrf was present at admission either none or in combination with other organ dysfun~on. ahrf was defined as the acute (<48hour) onset of respiratory dysfunctk:~l with a pao2/fio2 ratio.< 200 for six consecutive hours dunng the first 24 hours of admission (with no evidence of left anal hypertension), x-ray review defined a sub-group of patients with acute respiratory distress syndrome (ards) by the presence of bilateral interstitial infiltrates. results to date 59 children (ages 1-168 months, weight 1.2-70 kg) have been admitted in ahrf. 18 of these also had ards. the overall mortality was 23.7% (14/59), and greater in the ards group than the non-ards group (10t18, 55.5% vs, 4141, 9.7%, p< o.01) . it was not possible to predict survivors from non-survivors on the basis of the seventy of the respiratory failure alone, the a-ado2 on the day of admission (best in 24 hours) was not significantly different between survivors and non-survivors: (mean, + sd)(174 mmhg +_108, vs 304 mmhg _+_156). kdl non-survivors were immunodeficient (n=8), previously extmrnsly premature infants (<28140),(n=3) or suffedng fcom chronic metabolic or gastrointestinal disease (n=3). no previously normal child died. conclusion the severity of respiratory failure does not allow predioljon of outcome in our patients. we believe that this reflects that modem picu is so effective at providing respiratory support that pre-existing pathology alone de~ prognosis. this suggests that an abnormally regulated host response or abnormal persistence of a pathogen may be required to induce lung injury of sufficient severity that the resulting respiratory failure cannot be supported in a modem picu. introduction: postural changes (supine to prone) is a therapeutic intervention that could be useful in children with adult respiratory distress syndrome. objective: to determine the effects of postural changes in the oxygenation of young children with ards. method,s: a prospective stud3," was performed in eleven subjects aged 6 to 120 months (mean=33) with the diagnosis of ardsreceiving vendlatory support. (mean peep and fio2 of 9 and 0.75 respectively). postural changes was performed every 8-12 hours, during a period of time ranging from 5 to 16 days. arterial blood gases were determined before and 30-60 n~n after the postural change, no modification in the mechattical ventilation other that changes in the fio2 were performed. the oxygenation was determined by the index pao2/fi02 (p/f). to study the differences between the oxygenation mean, before and after the postural changes the wilcoxon test for paired samples was used, results: 184 changes were performed (104 from supine to prone and 80 from prone to supine). a9% increased p/f ratio was obtained after the change from supine to prune. although, not all the patients receiving postural changes improved their p/f. six of them (group i) showed an improve in the p/f when changed from supine to prone, returning to their base line when positioned from prone to supine. no improvement on the p/f was observed in the remaining 5 subjects (group ii)after postural changes (table 1) . during the maneuver no complications were observed. two patients had a pneumothorax, not related with the postural change. conclusions: postural changes (supine to prone) is an easy way to improve oxygenation in some children with ards. change to prone change to supine introduction: the common noninvasive diagnostic efforts to identify possible obstruction of the intrathorucic airway, are of limited value. invasive procedures such as bronchoscopy and bronchography may also be noncontributory and entail risks. we evaluated the usefulness of 3d-ct in the diagnosis and management of pediatric patients with suspected intrathoracic airway obstruction (itao). methods: we used a diagnostic algorithm (see diagram) in patients with suspected itao resulting in respiratory distress. three-dimensioual imaging of the tracheobronchial tree was reconstructed, following high speed spiral ct scan, by specific computer software (advantage window computer work station, general electric, milwaukee, wisconsin). non-ionic contrast medium was injected, in some patients, to delineate the intrathoracie large vessels.. results: eight patients were studied. in 5 patients the 3d-ct revealed intrathoracic airway abnormalities. these patients underwent further invesive studies which confirmed the following diagnoses: 2 patients had bronchomalacia, 1 had bronchial stennsis due to a dilated pulmonary artery mad 2 patients had subglottie stenosis extending to the thoracic cavity. three patients had no significant disruption in the configuration of the tracheobronchial tree and thus did not require invasive diagnostic procedures. conclusion: computer reconstruction of three dimensional images of the tracheobronehial tree is a safe and reliable diagnostic tool for itao. ards and ecmo; preliminary data from a randomized clinical trial. j fackler, c steinhart, d nichols, d bohn, m heulitt, t green, l martin, k newth, m klein, j ware. many suggest ecmo be considered experimental for ards and undertaken only with careful data collection and reporting. a mtflticenter pediatric rct is in progress to determine whether 1) ecmo and/or 2) permissive hypercapnia, offer significant advantage for the treatment of ards. methods: all patients aged 2 wk to 18 yr (without congenital heart disease) are eligible for study. data collection begins when a patient receives at least 50% oxygen and a peep of 6 cm h20 for 12 hours (stage t). if the predicted mortality reaches 60% within 7 days (stage 2), eligible patients are asked for written consent for randomization. patients are excluded from randomization with significant chronic lung disease, immune compromise, cardiac disease; or profound acute central nervous system damage. the prime outcome variable is survival. at the studies onset, 400 pts were estimated to be required so that 65 pts were randomized per arm. results: 131 patients are enrolled from 9 centers. data are complete on 85. 66 patients never reached stage 2 (i.e. 60% mortality). 47 patients improved and 19 died. of the latter, 13 had randomization exclusion criteria even if stage 2 was reached. 19 patients reached stage 2. 11 had exclusions from randomization and all died. eight patients (4 survivors were eligible for randomization; consent was obtained in no case. two patients received ecmo. overall survival is 60% (51/85). in patients without randomization exclusions, survival is 77% (34/44). morbidity m survivors (discharge -admission popc or pcpc score >_2) was seen in none of the 4 stage 2 surviviors and 15% (7/41) of those who reached only stage !. conclusion: the rct requires completion. the records of hospital in-patients at king faisal specialist hospital and research center who received external cardiac massage as part of their cardiopulmonary resuscitation were reviewed. success of resuscitation was analyzed as (1) short term (restoration of spontaneous circulation), and (2) long term (discharge from hospital). of 234 such patients, 171 (73.1%) survived the initial resuscitation, and 66 (28.2%) were discharged. success of outcome was not related to age, location of patient, time of day, or rhythm at arrest, including asystole. longer resuscitation time was associated with less chance of restoration of spontaneous circulation (p<0.001), but not associated with hospital discharge rate. results for patients with congenital heart disease were similar to those with other medical or surgical conditions. in this series, 36.7% of ward in-patients survived to discharge, compared to two 5"*;'~r ~r;~'9 ,.,.'her,, the r-e~ult~ were 0c/ "'~d ~, ~,°(. overall, 39 7% of patients who survived the initial resuscitation were discharged from hospital. where resuscitation continued for more than 30 minutes, 18.9% of patients had tong term survival. outcome from asystole was no worse than for other cardiac rhythms, we believe that previous reports of poor outcome from asystole in pediatric cardiac arrest should noi influence decisions to stop resuscitation for pediatric in-patients prematurely. successful restoration of spontaneous circulation with long term survival can be achieved after prolonged resuscitation. abdelmoniem~ lindsey jahusou~,mariano fiallos, university of florida, 820 prudential drive, suite 203 jacksonville, florida 32207 usa central acidosis is well recognized as a marker of inadequate tissue perfusiou, and ventilation. however, obtaining central venous blcod is difficult and fraught with complications in the child undergoing cardiopuimonary resuscitation. intraosseous blood may be used instead of central venous blood to judge ph and pcoz during short durations of cardiopulmonary resuscitation and during hemorrhagic shock. the purpose of this study is to compare the ph and pcoz status of intraosseous and central venous during prolonged cardiopulmonary resuscitation after fluid and drug infusion. we hypotbesized that there would be no difference in ph and pco2 values of simultanecusly obtained intraosseous and central venous blood samples. eighteen (18) introduction: cardiopulmonary arrest (cpa) in children is usually preceded by a deterioration of cardiac or respiratory function due to sepsis, dehydration and hypovolemia. early recognition of clinical and laboratory signs followed by immediate intervention are essential for prevention of cpa. the purpose of the present study was to identify factors which contributed to high rates of mortality from cpa in patients admitted to a paediatric intensive care unit (p1cu). methods: a prospective study was done of all non-surgical patients with cpa who were admitted to the picu, hospital baca ortiz, quito ecuador from january to october 1995. clinical and laboratory variables before and after admission to the picu, time from hospital admission to picu admission and the pediatric risk of mortality score (prism) were recorded on a questionnaire designed specifically for this study. results: of the 70 non-surgical patients admitted to the picu, 14 (20%) were admitted after developing cpa on the general pediatric wards. mean age was 16 + 19.1 months, with 13 of 14 patients under 20 months of age. initial diagnoses upon picu admission included meningitis (n=3), respiratory failure (n=2), congenital heart disease (n=2), severe neurological impairment (n=2), end stage neoplastic disease (n=2), hypovolaemic shock (n=l), peritonitis (n=l) and sepsis (n=l). mean time from hospital admission to p1cu admission was 16 _+ 19.2 hours. the mean prism score upon hospital admission was 30+ 13.7 (score > 20 = > 50% mortality). 79% (11/14) of the patients died. one of the three survivors had severe neurologie injury. prior to picu admission, patients experienced tac~,cardia (n=9), hypotension (n=8), neurological deterioration (n=8), respiratory, distress (n=7), oliguria (n=5), bradycardia (n=3), metabolic acidosis (n=7), hyponatremia (n=4), hypokalemia (n=2), hypocalcemia (n=2) and severe hypoglycemia (n=2). there were serious delays from the time of development of clinical and laboratory abnormalities to the time of admission to picu. conclusion: in the critically ill pediatric patient, rapid recognition of clinical and laboratory signs of deterioration, followed by immediate intervention, are required to prevent end stage shock and cpa. we found serious delays in intervention following development of important premonitory clinical and laboratory abnormalities in patients less than 20 months of age on the general pediatric wards, which iikely contributed to the dismal 79% mortality rate. hospitals throughout ecuador should institute immediate improvements in ctinical supervision, and provide training in paediatric advanced life support (pals) to decrease excessively high rates of and mortality from cpa. intraosscous access is recommended by the american heart association and american academy of pediatries as a means of rapid access to the vascular system for childhood emergencies. bone marrow and fat embolism is a concern and has been reported post intraosseous infusion in stable animals but has never been studied in animals subjected to cardiopuimonary resuscitation. we undertook this study to investigate the incidence and magnitude of lat and bone marrow embolism with the use of intraosseous infusion during prolonged cardiopuhaonary resuscitation and after fluid and drug infusion. we hypothesized that there will be no difference in the magnitude of fat embolism between cardiopulmonary resuscitation only and other cxperirnental conditions. thirty-one (31) piglets were anesthetized, mechanically ventilated, and instrumented (carotid artery, pulmonary artery and intraosseous earmulas ). the animals then underwent bypoxic cardiac arrest followed by chest compressions with the mechanical thumper (michigan insmunents) and mechanical ventilation for a minimum of 45 minutes. the animals were divided in groups: a (n=5) which had no intraosseous, ~'oup b (n=6) had intraosscous with no infi~ion, and groups c (n=6), d (n=6), e (n=8) had intraosseous with infusion of adrenaline, normal saline and sodium bicarbonate, at cessation ofcardiopulmonary resuscitation, representative lung samples were collected fi'om upper and lower lobes of each lung, embedded in ocp and firozen immediately. ltmg specimens were stained using oil red-o dye and observed for fat globules and bone marrow elements. the amount of emboli present was rated as a percentage in relationship to iung tissue, by a pathologist blinded to the experimental groups. buffy coat specimens were collected before and at cessation of cardiopuimonary resuscitation, stained with oil red-o dye and observed for fat globules. percentage of fat present were compared using analysis of variance. fat globules were seen in the prebronchial blood vessels and in intravascular areas throughout all lung fields. there was no difference in appearance or distribution of fat globules between groups. quantity varied in the different groups[(a) 45%, (b) 44%, (c) 30% (d) 23%, (e) 25%], but were not statistically significant (p = .097). fat globules in the buffy coat were few and inconsistent with lung findings. fat and bone marrow emboli were present in all experimental conditions, the use of the intraosseous cannula does not increase the magnitude of embolization during cardiopuimonary resuscitation. the decision to use the intraosscous route should not be influenced by the risk of embolization. tzareva iv/,, md*, nedialkova r, md**, *dept. of pathophysiol, *~dept. of child surg. and icu, emergency medical institute pirogov, sofia, among 566 children with blunt abdominal trauma, treated in emi pirogov during the last five years, 79 children had serious disturbances of the basic vital functions, connected with the trauma, and most often with massive haemorrhage, for this reason being an object of reanimation and intensive care. in the group of children who survived -37, predominated the trauma of only one abdominal organ (mainly the spleen, rarely the kidneys, the intestine) and only 15 children had injuries of more than one abdominal organ. in the same group, in 15 children the abdominal trauma was combined with chest or head trauma or bone fractures. in the group of children who died -12, a profound combined trauma was present. the haemodynamic parameters in all children showed a characteristically significant tachycardia along with normal or even high blood pressure, while hypotonia was present in only 64% of the children on the first trauma day. despite the fact that only 13.4% of the children had direct chest injury as well, the gas exchange was considerably disturbed -899'0 of the children were hypoxemic during the first, and 100% during the third trauma day -in 25% significant -below 8.0 kpa (60 mmhg). together with the markable decrease in haemoglobin levels, this determines the pronounced disturbance in oxygen transport. during the first trauma day all the children were acldo~c, and a metabolic alkalosis was present during the following days. twelve of the children with severe combined trauma died within several hours, with the symptoms of irreversible haemorrhagic shock, or in the next 2-3 days, developing multiple organ failure. in conclusion, the intensive therapy of children with severe abdominal and combined trauma, should take in consideration the special haemodynamical trauma answer in children, and requires dynamic monitoring of the most influenced homeostatic parameters -blood gases, acid-base metabolism, haemostasis. introduction: endocrine emergencies, other than diabetic ketoacidosis, are uncommon causes of pediatric intensive care unit (picu) admissions. we report our experience of children diagnosed of adrenal insuficiency (ai) admitted in the picu, during the last four years. subjects: five eases of ai requiring 7 intensive care unit admissions are presented. four females anna 1 male, with ages ranging from 11 days to 7 years, none of them had a previous systemic or endocrine diseases that could suggest al the initial clinical manifestations were: dehydration (5), vomits (3), abdominal pain (2), seizures (2), lethargy (2) and hyperpigmentation in the muco-genitat area in a newborn male and ambigna genitalia in a newborn female. the reason for their admission in the p1cu were: shock in two subjects; three because of hyperkalemia and hyponatremia (k/na: 5.6/123; 9/126; 7,1/134 meq/l); and two with severe hyponatremia (na: 117; 113 meq/l). laboratory findings: severe hyponatremia (5), increased concentration of urinary sodium and chloride (4); metabolic acidosis (4); hyperkalemia (3); increased levels of urea (3) and hypoglycemia (2). in all of them, the electrolytes abnormalities did not normalize with replacement and only normalized after the administration of hydrocortisone. tile ai was due to: autoimmtme disease in two subjects, congenital adrenal hypoplasia, congenital adrenal hyperplasia secondary to 21 alia hydroxylase deficiency and in one no etiology was found, at the present time, comments: aiis an uncommon disease in the pediatric age. anearly diagnosis is crucial, as if the treatment is delayed could lead to patients death. in subjects with arterial hypotension and electrolytes abnormalities refractory to the usual treatment, they should be treated with corticosteroids, if no etiology is found. although, previously samples must be obtained to make the diagnosis, 0: denotes the number of cases. gerbaka b; hakme c; akatcherian c. toxics are frequently involved in domestic accidents during childhood; among non medical products ingestion, carbohydrate poisoning is a serious injury often made possible by inadequate stocking. over 10 years, 43 children aged 10 years and less were examined in the emergency department of hotel-dieu de france hospital for carbohydrate ingestion. 62,8% are boys; age goes from 13 months to 6 years (moan = 2,5years). kerosene is found in 35,8% of cases; all were admitted (mean = 2,8 days). 79,1% were symptomatic on first examination but 93% of all children presented signs of gastric (58%) or respiratory (69,8%) irritation sometime during their history; 37,2% had neurological signs and 41,9% presented some fever. leucocytosis is found in 65% of cases; 25,6% of the children received antibiotics. chest x ray was abnormal in 48,8% of cases: mainly parahilar infiltrates were found, all children survived; 76,7% with a normal course (1,9 days of hospital stay) whereas those who presented complications (severe pneumonia, coma) stayed in the hospital for 6 days (mean) with short course of assisted ventilation for two of them; long term follow up was not possible. we fonnd nick's criteria for hospital admission to be of value: -symptomatic children with normal x ray } 6 to 8 hours monitoring -asymptomatie children with x ray abnormality } -symptomatic children with x ray abnormality: hospital admission -asymptomatic children with normal x ray : no admission. these criteria would have helped to avoid admission in 8 children and would have allowed a short t2 hours stay for 6 more. we found chest x ray to be mandatory in carbohydrate ingestion; other tests were not helpful, aside arterial blood gases measurement in case of respiratory involvement; we now also advocate more restriction in antibiotic use. prevention remains efficient and should be stressed on. severe liver failure [slf] is a rare but severe condition in infants. we report our experience. patients: slf was defined as liver insufficiency with hepatic encephalopathy and a decrease in the level of factor v to below 25 %. between 1984 and 1996, 29 infants (mean : 4 mo) were admitted for slf (neonates excluded). main causes were metabolic disorders (41.3%) (tyrosinemian=5, hemochromatosis n=2, reye's syndrome n=2, other n=3), virus-induced flf (20.6%) and hematologic diseases (13.7%). in 4 cases, the causes remained undetermined. results: olt was contraindicated in 12 cases because of multiple organ failure (n=10), or underlying disease. all of them died within 6 days after admission. 7 patients had no indications for olt, all but one are alive. (1 of them was transplanted later for tyrosinemia and 1 died lately (virus induced-slf). among the t0 infants who underwent emergency olt, 6 are alive and 4 died because of primary non function of the graft. conclusion: slf in infants admitted before their first birthday is a severe condition with an overall mortality rate reaching 60%. inherited metabolic disorders are the first cause of slf at this age. contraindications for olt are frequent because of underlying disease or multiple organ failure. a number of children undergo primary graft failure after liver transplantation. it is unknown if there is any increased morbidity or mortality following retransplantation. this study seeks to explore these issues. methods: a pediatric intensive care/iiver transplant database is in formation. records of all liver transplant patients are reviewed and abstracted. this data is then computerized to allow analysis. this data provides the source for this study. statistical analysis was performed via student's t-test where appropriate. results: of the 350 patients who have thus far received at our center orthotopic liver ransplants, the records of 112 who underwent 140 transplants form the basis for this review. twenty-three patients underwent multiple transplants, 19 required one additional, three required 3 organs, and one patient survived after a fourth organ transplant, there was no significant difference in age at first transplant between those who received multiple organs and those who did not (40 vs, 44 months, p=ns). the anesthesia time for the procedure did not significantly increase tbr subsequent transplants (8.3 vs, 7,3 hours), nor did time in the intensive care unit (t6.6 vs. 22.2 days), nor did time on the ventilator (8.4 vs. 15.3 days) subsequent transplants did not predispose to having more bleeding in the intensive care unit for usage of packed red blood cells or platalets was not significantly altered (299 vs 306 ml and 127 vs 207 ml respectively). patients who required retransplantatior~ did receive mere fresh frozen plasma (ffp)daring their first transplant than in the subsequent ones (275 vs 81 ec, p < 0.05). however ffp use was not significantly different than patients who did not require retransplant. patients who underwent retransplant had a markedly increased mortality (47%) than the overall mortality for liver transplants at our center (20%), conclusion: children who require another liver transplant have a markedly increased mortality. bleeding and prolonged icu stay is not significantly different between the first and subsequent transplants, fulminant hepatic failure and ortothopic liver transplantation.dr.sasb6n,j;centeno,m;entin,e;acarenza,m;ciocca, m:gofii,j;bianco,g;weller, g;imventarza,o. unidad de cuidados intensivos.hospital de pediatria "dr.j.p. garrahan"1245.buenos aires.argentina. introduction:fulminant hepatic failure (fhf) is a clinical syndrome, defined by the development of hepatic encefalopathy within 8 weeks from onset of illness in a previously healthy person.by far,the most comun cause of pediatric fhf in all series, is acute viral hepatitis.we report our experiences with the pediatric fhf and ortothopic liver transplantation (olt) as attemative of treatment. patients:30 childrens with fhf diagnosis were admitted at the picu from 1/1/1993 to 1/12/1995.symptomatic treatment was given to all children and all were put on list for olt,) following the king's college criterion (protrombina time,age,atiologies,bilirrubin,and encefalopathy state). results:etiologic causes corresponded to the 30 childrens were:23, hav (76%); 6, noa nob (20%);1 ,autoinmune (4%).the age was mean:4 years (range:16 month-10 years).seventeen patients were transplanted,13 chidmn were discarded because:no donors:5;withdrow of the list:3,because sepsis in 2 and bleeding of cns 1;and no admission at list:5 because genetic syndrome 1 ,massive intestinal necrosis, 1 ,mitral valvulopathy 1 and sepsis,2. 25 patients (86%) had at least one complication dudng the post operative period.the most frequent was the acute renal insufficiency(ari) and 4 patients requiered continuos hemofiltration.the gtobal mortality rate was 75%.the mortality of patients without olt was 100% and the mortality of patients with olt was 41%,4 patients dayed because sepsis, (2 candidiasis) and the others 3 because mof.the actuarial survival at 1 year is 54% and the follow up of 8 months. conclusions:the fhf is a very severe and frequent disease at picu. supportive treatment only is associated with a very poor prognosis and high mortality rate.the most frequent etiology in our country is the hav. the olt is applicable in this cases and is a valid alternative of treatment (mortality in our series 41%).the ari is the most frequent complication during the post opeative period.in argentina,due the high prevalence of hav,prevention must be considered the main and only way to avoid this catastrophic illness.to assess the efficacy of gastric intramucosal ph (phi) for evaluation of tissular perfusion and prediction of hemodynamic complications m critically ill children. patients and methods: thirty critically ill children (16 boys and 14 girls) whose age ranged from 3 month and 12 years old were studied. a tonometry catheter was placed in the stomach of all patients at their °admission in pediatric icu. intramucosal ph measures were made at the admission and each 6-12 hours during the study: a total of 202 determinations were made. the catheter was removed after extubation and/or checking of hemodyrmmic stability of the patient. the intramucosal ph was derived from application of the henderson-hasselbaeh formula using the pco2 value from the tonometer and the arterial bicarbonate. values of phi between 7.30 and 7.45 were considered normal. the relationship between phi and severity of patient measured through prism, presence of major (cardiorespiratory arrest, shock) and minor (hypotension, hypovolemia or arrhytlmtias) hemodynamic complications, mortality and stay in the picu, was analysed. results: the admission value of phi was 7.48 -t-0.15 (range 7.04-7.68). five patients (16%) had an admission phi < 7.30. no relationship was found between an admission phi < 7.30 and a higher incidence of hemodynamic complications. sixteen patients (53%) showed some values of phi < 730 during their evolution. patients with phi < 7.30 had a higher number of hemodynanuc complications than the rest (p< 0.0001). every cardiorespiratory arrest (cra) and shock cases were related to a phi < 7.30. patients with major complications (cra and shock) had a phi lower (p= 0.03), as well as a higher number of measurements of low phi (p= 0.003) than patients with minor hemodynamie complications. the value of phi lower than 730 presented a 90% of sensibility and 98% of specificity with regard to hemodymanic complications. there was no relationship between phi < 7.30 and prims score and stay in picu. patients with phi < 7.20 presented a prims higher than the rest of patients (p< 0.05). conclusions: the phi value may be an early sign of presence of hem0dyaaimc complications in the critically ill child. we tested the hypothesis that gastric intramural ph (phi) can be used as an early sign of failure m weaning pediatric patients because the blood flow from nonvital areas is diverted to meet the increased demands of respiratory muscles. methods: 24 children (mean age (4.2_+0.3) years + sd) who were thought by their physicians to be weanable from mechanical ventilation (mv.). these patients were ventilated on serve 900c ventilators, receiving ranitidine, and had intestinal tonometer (tonometrics, inc.) 60 minutes before obtaining a sample.. all children were placed on pressure support (ps) at levels judged to overcome the resistance of the endotracheal tube and ventilatory circuit (2 em h.,o). a sample of arterial blood and a sample oftonometer were obtained during vm and weaning (ps). phi, hemodynamic and respiratory data were recorded during vm and weaning we did not interfere with the primary caretaker's decisions regarding extubation. patients were considered to be successfully weaned if they were able to sustain spontaneous ventilation for more than 24 hours after extubation. paired t-test were used to compare the values obtained during mechanical ventilation with those obtained during weaning trials. unpaired ttest were used to compare values from the group that was successfully weaned (a=i5) with those from the group that were not (b=9). results: we did not find statistical differences in any of those variables mesured during mv for patients who were successfully weaned(group a) and those who were not (group b). gastric phi was in group a: 7.35 + 0.03 (vm) and 739 + 0.02 (weaning); in group b: 7.40 _+ 0.04 (vm) and 7.4t _+ 0.02 (weaning). discussion: although we did not find differences in gastric phi during vm, the group a had a lower value than group b because of the number of cardiac patients (70%) and transfusion therapy, in fins group. in group b 75% of patients showed a problem in upper airway (subglottic edema, and enlarged tonsils). we found it after extubation. conclusion: 1) gastric phi is a good predictor of risk in critically ill patients but maybe because of the small size of the sample, in our study is not of practical value as a predictor of failure in weaning pediatric patients from vm. 2) this test is not a predictor of problems in upper airway~ important etiology of failure weaning in children. objectives: i-to determine the prognostic value of the gastric intramueesal phi in mortality and multiple organ dysfunction (sdmo) in critically ill children. 2-to compare this value, with the pediatrics risk index mortality score (prims). methods: aprospective study was performed with 51 critically illcbildren, aged from 1 mouth to 16 years. the athnittiug diagnosis was: 26 post-surgery (13 neurosurgery, 9 spinal fusion and 4 thoracic or abdominal surgery), 7 sepsis, 6 polytraumatism, 5 adult respiratory distress syndrome and 8 with miscellaneous. all the subjects were monitorized on picu admission and treated for their underlying condition. gastric intramucnsal pt{ was measured following the tonometric method, ou admission and every 4-8 hours depending on the patients state. the severity of the clinical condition was evaluated using the the prims, on admission (prims-i) and during the first 24 hours, when the clinical condition deteriorate, the worse score was utilized for the statistical analysis (prims-2). to perform the statistical analysis the subjects were divided in two groups, one with the phi<7.30and the other with phi>7.30.aunivariate analysis (student's tand wilcoxon two tailed test, chi-square) and multivariate analysis were used. results: 12 out of the 51 subjects dyed. of 14 children developing multiorgan failure (mof) 9 expired. 50% of the patients admitted to the picu with sepsis, ards and miscellaneous had a phi < 7.30. in contrast, with 27 % of post-surgical and none of the postqraan~atism. the mortaliry rate, in children with a phi<7.30was 47% (ci 95%:26.16; 69,04) and 11.76% (ci 95%:4,67; 26.62) in children with phi>7.30 (p=0.011). mofwas observed in41,18% of children withphi<7.30v.s, 20.6% with phi >7.30.no relatiouship was observed between the phi and the score of prims-i and 2. perforating an unconditional logistic regression analysis, two independent variables have mortality predictive value: the phi and the prism-2. (table i) following induction of anaesthesia, a laser doppler probe (moorsoft instruments ltd) was inserted 7cm into the patient's rectum, the probe's special design ensuring that the optical prism lay against the mucosa. continuous monitoring of rectal mucosal perfusion ("flux") was continued throughout the operation. after 10 rain cpb at 35°c, "steady state" readings of nasopharyngeal temperature, mean femoral arterial pressure (map) and flux were recorded over a further 5 min before cpbinduced core cooling to 14-24°c. steady state was defined as a 5 rain period with no change in core temperatures or map. other 5 rain steady state recordings were taken immediately prior to low flow, immediately prior to rewarming and after rewarming to 35°c, before initiation of any vasoactive drugs. the cpb flow rate was kept at 100 m l k g -1 min q, the pcv at 25_+3%, the p~co 2 at 5.3+0.5 kpa and the pro2 at 20+5 kpa. results: initial warm and rewarm map (both 46 mmhg) were significantly lower (19=0.008) than during the 2 cold cpb periods (63 & 64 mmhg). the mean cold flux before (152) and after (159) low flow were both significantly lower (p=0.001) than the mean initial warm cpb flux (211). the mean rewarm cpb flux (127) was significantly lower than all other flux values (p=0.001). there were no siglaificant correlations between map and flux except at the first warm cpb period (r=0,33, p=0.04). conclusions: although hypothermia significantly reduces rectal mucosal perfusion, rewarming produces an even greater reduction in gut perfusion which, considering that mucosal oxygen constmaption is highest during this time, may prove crucial in the postoperative development of mof. therapy aimed at improving gut perfusion during cpb should be directed at the rewanning period in particular. abstract this work is aimed at establishing a clinical procedure for the diagnosis of enteritis necroticans (en), even at the communal level, and to define criteria for diagnosis able to distinguish between acute forms. subjects and method : 100 cases admitted at the institute for protection of children's health dpch), having characteristic symptoms, were examined clinically, by roentgenography of the abdominal cavity, with the analysis of the blood (total protein, electrolytes, hematocrite) and cultures of intestinal fluid and faeces. through surgical operations, the pathological lesions were observed and recorded. results: common epidemiological features: the average age is 6-8 years old (3-15) ; male/female : 1.85; in 70% of the cases, the disease occurred after a meal rich in protides. the acute toxic form accounted for 15% : severe shock appearing early, with very severe dehydration associated with profoundly decreased blood protein concentration and lowered natriemia as well. the lesions of the small intestine were expanded, all of them were necrotic. in the surgical form (20%), the predominant feature was an obstruction -peritonitis syndrome, the peritoneal fluid showed a characteristic inflammatory reaction. for the rest of cases 65% were the internal form, the shock syndrome was less severe, the abdominal distention was light and disappears gradually, the inflammatory reaction of the peritoneal fluid was not so characteristic. conclusion (ino) is a selective pulmonary vesodilator that is rapidly inactivated compared to intravenous vasodilators. these qualities make ino an attractive agent for the treatment of pulmonary hypertension (pittn). the efficacy of ino has been studied in persistent fetal circulation, acute respiratory distress syndrome (ards), and congenital heart disease (chd). potential adverse effects oflno include: nitrogen dioxide (no0 toxicity, methemoglobinemia, and platelet dysfimction. our objective was to evaluate the safety of ino in pediatric patients (pts). methods: pediatric pts. with phtn from ards or chd were studied under an established, approved protocol conforming to fda guidelines tbr an investigational new drug. informed consent was obtained for each child prior to treatment. 1no was sequentially titratad from 10 parts per million (ppm) to 20, 40, 60, and 80 ppm at ten minute intervals. parameters monitored before and during therapy included nitric oxide (no) and no~ concentrations (cone.), mean arterial blood pressure (map), and percent methemoglobin (mhg). no and noz levels were continuously monitored using an inline dr~ger electrochemical detection device. ~,litp was continuously measured with an indwelling arterial catheter. mhg was measured by co-oximetry. a mhg level e 5% or no2 cone. ~ 5 ppm were considered adverse effects by study criteria. pretreatment map was compared to map at 40 and 80 ppm ino using paired t-tests. ap value < 0.05 was considered statistically significant. results: thirty-two mechanically ventilated children with phtn (16 with ards, 16 with chd) were studied. five pts. were treated following cardiopulmonary bypass. methemoglobin (met-hb) levels were routinely measured in two prospective clinical studies on no inhalation in 25 pediatric patients with pulmonary hypertension following heart surgery with extracorporeal circulation and in 19 pediatric and neonatal ards patients, the observed differences between the groups prompted in an in vitro study, red blood cells (rbc) of 20 patients sampled before and after surgery with and without extracorporeal circulation (ecc), respectively, were incubated with 32 ppm no for 100 rain, met-hb, atp, and nadht nadph concentrations were compared, during therapeutic exposure no increased met-hb from 0.2 -2-_ 0.1 to 1.2 _+ 0.7 % in cardiac surgery patients and from 0.2 ± 0,1 to 0.5 ± 0.4 % in ards patients (p < 0.01 ). rbc's having undergone ecc were more susceptible to met-hb formation (p< 0,001 ) whereas intracellular coenzymes did not differ neither between the groups (table) nor before and after no exposure. ecc predisposes to increased methemoglobinemia upon exposure to no both in vivo and in vitro. our data suggest a reduced activity of met-hb reducing enzymes rather than diminished availability of energetic substrates, variation of the inhaled nitric oxide concentration with the use of a continuous flow ventilator. anne pmc de jaegere ~, frans im jacobs 2, nico gc laheij 2, john n van den anker t . dept. of paediatrics ~, central instrumentation 2, sophia children's hospital, erasmus university rotterdam, rotterdam, the netherlands. objective: to investigate the homogeneity of nitric oxide (no) concentration in a delivery system with a continuous flow ventilator. design: bench study, setting: biomedical laboratory. interventions: a nitrogen/nitric oxide (njno) gas mixture was injected at three different sites in the patient circuit: just before and just behind the humidifier, and 20 centimetres before the y-connector. ventilator flow (12, 15, 20 l/rain), ventilator rate (40 to 110, increments of 10) and compliance of the testlung (0.36; 0.5; 1.0 ml/cm h20) were changed. carbon dioxide (co2) instead of n2/no was injected at the same points in the circuit. measurements and main results: a) though the flow ratio of the njno and the ventilator gas were kept constant, the no concentration ([no]) raised with increasing ventilator rates. the increase in [no] was up to 40% when the n2/no injection site was close to the y-connector of the ventilator circuit. minimal changes in [no] were noticed when the n~/no was mixed to the ventilator gas before the humidifier. b) analysis of the ventilator flow pattern showed variations at different places in the ventilator circuit. the magnitude cf the p, ow change depended on the meas~:rement site. the closer to the expiratory valve the highest the flow change was. the duration of the flow change was inversely proportional to the adjusted ventilator flow. c) real time measurements of the co 2 concentration ([coz]) showed variations during tile respiratory cycle. these [co2] variations were higher when the co2 gas was blended closer to the yconnector. conclusions: the ventilator flow variations in relation to the fixed side flow of the n2/no gasmixture result in changes of the inhaled [no] during the respiratory cycle. the no concentration during inspiration is always higher then during expiration. this could not be detected with the available monitoring system. to ensure a constant [no] by blending a njno gas balance in a continuous flow ventilator, the site of injection should be as close as possible to the inspiratory outlet. nitric oxide, a potent and selective pulmonary vasodilator, has recently been successfully used to treat pulmonary hypertension of variable etiology in infants and children. side-effects and complications in infants are so far not well known. we describe here two cases in which prolonged (5 and-7 days respectively) high-dose (50 -80 ppm) nitric oxide was used to treat refractor~¢ pulmonary hypertension. one patient was a newborn infant with pulmonary hypertension secondary to a large leftsided diaphragmatic hernia. nitric oxide was begun under conventional ventilation (babylog 8000) at 7 hours of life with a slight initial improvement in oxygenation. he was then placed on oscillation with the same nitric oxide concentration due to worsening respiratory failure. he died on 5th day of life. monitored nitric dioxide concentration never exceeded 4 ppm. the other patient was a 3 months old infant with severe pulmonary hypertension due to a complete atrioventricular septal defect. he required high-dose nitric oxide to come off cardiopulmonary bypass after surgical repair of his heart defect. he slowly improved over the week following surgery but developped suddenly respiratory failure due to massive pulmonary hemorrhage and died. surprisingly, a particular autopsy finding in both infants was a massive acute necrotizing tracheobronchitis. we conclude that nitric oxide is an excellent and sometimes lifesaving treatment of pulmonary hypertension in infants. tracheobronchitis has not yet been reported as a possible complication of nitric oxide administration. we suggest that caution needs to be taken with prolonged high-dose administration and this possible complication to be looked for at autopsy. introduction: permissive hypereapnia (ph) is a beneficial strategy for patients with acute respiratory distress syndrome (ards) to minimize barotrauma by decreasing the peak inspiratory pressure (pip). hypercapnia and hypoxia cause pulmonary vasoconstriction, pulmonary artery (pa) hypertension, and, thus, an increased afterload to the right ventricle. this increased afterload may result in increased right ventricular (rv) work load and subsequent rv dysfunction. one therapeutic approach is the use of inhaled nitric oxide (inn), a selective pa vasodilator. the objectives of this study were to test the hypothesis that in a swine model of ards with ph, inn would improve rv work load and not change intrinsic rv contractility. methods: in 11 swine (25-35 kg), ards was induced by surfactant depletion. hypercapnia was achieved by decreasing the pip while increasing the peep to maintain a constant mean airway pressure, inn was administered in concentrations of 2, 5, and 10 ppm in a random order. pulmonary blood flow (qpa) was determined by an ultrasonic flow probe. rv total power (tp) and stroke work (sw) were calculated by fourier transformation of the pa pressure (ppa) and qpa data. preload recruitable stroke work (prsw), a preload and afterload independent measure of ventriculur contractility, was determined by a shen-subtraction method and vena caval occlusion. respiratory failure with pulmonary hypertension in piglets gerfried zobel*, bernd urlesberger*, drago dacar**, siegfried rtdl*, fritz reiterer* and ingeborg friehs** depamnents of pediatrics* and cardiac surgery**, university of graz,austria objective: to evaluate gas exchange, pulmonary mechanics and bemodynamic data during partial liquid ventilation (plv) combined with inhaled nitric oxide (no) in acute respiratory failure with pulmonary hypertension. design: prospecfive~ randomized, controlled study. setting: university research laboratory. subjects: twelve piglets weighing 9 to 13 kg. interventions: acute respiratory failure with pulmonary hypertension was induced by repented lung lavages and a continuous infusion of the stable endoperoxane analogue of thromboxane. thereafter the animals were randomly assigned either for plv or conventional mechanical ventilation. initially perfhiorocarbon liquid (30ml/kg) was instilled into the endotracheal tube over 5 min followed by 5-10ml/kg~. all animals were treated with different concentrations of no ( 1-10-20 ppm) inhaled in random order. measurements and results: continuous monitoring included ecg, cvp, mpap, map, san2 and svo2 measurements. during plv pao2/fio2 increased significantly from 62_+3.2 mmhg to 193±44 mmhg (p<0.01) within 10 rain, while pao2]fio2 remained constant at 61 -+3.3mmhg. qs/qt decreased significantly from 48-+4% to 25-+5% (p<0.01) during plv and did not change during conventional mechanical ventilation. static pulmonary compliance (cstat) increased significantly ff~m 0.4r±0.07 to 0.75_+0.03 ml/cmh20/kg (p<0.01) during plv and decreased slightly from 0.58_+0.08 to 0.46e0.04 ml/cmh20/kg during conventional mechanical ventilation. the infusion of the endoperoxane analogue resulted in a sudden decrease of pao2/fio2 from 262_+44 to 106_+8.0 mmhg in the plv group and from 71±7 to 52+_2.0 mmhg in the control group. inhaled no significandy improved oxygenation in both groups (pao2/fio2:344_+38 mmhg during plv and 196+_.56 mmhg during conventional mechanical ventilation). during inhalation of no mpap decreased significantly from 57-+2 m 35±2 mmhg (p<0.01) in both groups. there was no significant change in oxygenation and mpap during inhalation of 1 and 20 ppm no. conclusions : plv significantly improves oxygenation and pulmonary compliance in acute respiratory failure. the additional application of inhaled no further improves oxygenation and pulmonary hemodynamics when acute respiratory failure is associated with severe pulmonary hypertension. inhaled no is very effective in improving oxygenation and pulmonary blood flow even at low doses. the work was supported in part by grants of the austrian nationalbank nr 5545. as in neonates, severe respiratory failure in infants and children can be aggravated by pulmonary hypertension, resulting in further deterioration of oxygenation due to increasing intrapulmonary shunting. we analysed the influence of inhalational nitric oxide (ino) in treatment, course and outcome of severe ards in a pediatric population. since 1993 20 infants and children (age: 1-107 months) with ards and oi > 15 (mean value: 32.5± 11) underwent a trial with ino (concentration: 3, 10, 30, 60 and 100 ppm) to prevent further respiratory failure. 11 patients had a significant improvement of their oxygenation (rise of pa09 > 15 mm hg) for at least 24 hours (responders); mean best ~fficient no dose: 24.6 ppm. the non-responders had only a short-term improvement or ino had no effect. in responders and nonresponders there was no significant difference with regard to age, underlying disease, ards severity, time on mechanical ventilation, blood gases and ventilator settings before notrial, nor was there a different grade of pulmonary hypertension (estimated by echocardiography). the only difference was an higher ol in the group of the non-responders: 40.9 ± 9.i vs. 25.6 ~ 6.7, p < 0.002. in the group of the 11 respenders there was a secondary deterioration of lung function after i -6 days on ino in 5 children (transient responders): in these patients, as well as in the group of the non-responders, alternative modalities of treatment (hfov and/or ecmo) became necessary. 6 children (30 %) died: 2 transient respenders and 4 non-responders. in infants and children with ards due to different underlying diseases ino can acutely lead to a significant improvement of oxygenation in about 50 % of the cases. the right selection of patients for no therapy and the influence of ino on the survival rate of ards in childhood has to be evaluated in further studies. and pediatric cardiology, university of graz, a-8036 graz purpose: after fontan procedure cardiac output is critically dependent on the pulmonary vascular resistance. even minor elevations of the pulmonary vascular resistance may significantly decrease cardiac output. inhaled no is an effective, selective pulmonary vasodilator in experimental and clinical situations of pulmonary hypertension. the aim of this study is to evaluate the effects of inhaled no on oxygenation and pulmonm 3, circulation in children after a bidirectional glenn-anastomosis (n-~) or a fontan-like operation (n=9). material and methods: from june t993 to january 1996 13 children with a mean age of 7.1+~2.1 (sem) yrs and a mean body weight of 24.3-+5.8 (sem) kg were treated with inhaled no after glenn-or fontan-like operations. all but one had complex cardiac malformations with single ventricle. all children were mechanically ventilated with an fin2 >0.75. inhaled (no) was applied using a rrdcrdproeessor based system which additionally allowed measurement of no/nox using the chemihimniscence method. methemogtobin concentrations were determined 3 times a day. the major indication for postoperative inhalation of no was a high (>10mmhg) transpulmonary pressure gradient (tpg--cvp-lap). severe myocardial dysfunction of the single ventricle was excluded by echocardiography. results: the mean duration of mechanical ventilation was 8.1_+2.2 (sem) days the. mean dose of inhaled no was 4.4-+0.8 (sem) ppm, the mean duration of no-inhalation was 106_+19 (sem) hours. the mean methemoglobin concentration was 1.2-+0.2 (sem)%. hemodynamic data and arterial oxygen saturation before inhaling no and 15 minutes later are given in table 1 acute hypoxaemic respiratory failure (ahrf) in children occurs in a heterogenous group of diseases with pulmonary pathophysiological processes ranging from reversible physiological intrapulmonary shunting to fixed structural lung damage. we hypothesized that inhaled nitric oxide (ino), a selective pulmonary vasodilator, might identify those patients with potentially reversible disease, i,e, large response may indicate a greater likelihood ef reversibility and thus survival. a retrospective review of the early response to ino in 30 infants and children (aged 1 month to 13 years, median 7 months) with severe ahrf(18 with ards). the mean p(a-a)o2, pao2 / fio2, oxygenation index (oi) and acute lung injury (all) score prior to the commencement of ino were 568 +_9.3, 56 +_2.3, 41 _+3,8 and 2.8 +_0.1 respectively, the magnitude of response to ino was quantified as the % change in oi occurring within 60 minutes of 20 ppm ino therapy. this response was compared to patient outcome data. results. there was a significant correlation between response to ino and patient outcome, kendall tau b r=0,43, p<0.02 (table) conclusion. in ahrf response to ino appears te define a subgroup of patients with improved outcome compared to nonresponders. we speculate that response to ino may be useful in selecting patients with potentially reversible lung disease for special support therapies such as ecmo. randomised controlled trials are needed to define the role of ino in paediatric ahrf. between may 1994 and december 1995, 22 patients (pts) were treated for mas. treatment groups were: group i only 02:6 pts; group i1 conventional mechanioal ventilation (cmv): 11 pts; group ii1 hfo: 1 pt; group iv hfo+no: 4 pts. therapy was stepwise intensified until oxygenation improved ( i -) ii -) iii --) iv). "high volume strategy" was used with hfo (mawp 18-24 cm h20). the initial no-concentration was 20-30 ppm, with rapid reduction down to 5-10 ppm once oxygenation improved. results: one pt (group it) died of hypoxic-ischemic encephaiopathy (termination of therapy); all other newborn babies survived. in group iv pt 1 and 2 showed barotrauma prior to hfo. pt 1,2 and 4 were treated with additional mgci2 (max. mg serum concentration 2.8 -6.5 mmol/i). following the identification of inhaled nitric oxide 0"no) as a selective pulmonary vasodilator (frostell et al 1992) [ 617 .+6,3 626+6.3 data are compared to baseline values within each group. *=p<0.05, **=p<0.03, ***=p<0.0l among 12 patients who fulfilled ecmo criteria, 6 improved with no and did not required extracorporeal life support. tltree out of 6 ecmo patients eventually survived. conclusions: m our study low-dose of irthaled no showed a variable effect on oxygenation in newborns with acute respiratory failure. an acute response to no appeared to be correlated with a better short-term outcome and the avoidance of extracorporeal support in ecmo candidates. differently, lack of acute and/or sustained response was associated with death or need for ecmo. although the nature and severity of the underlying disease or the degree of prematurity may play an important role in these patients, we believe lack of acute response to no may be an early predictor of bad outcome, prompting toward alternative treatments such as ecmo or liquid ventilation. *picea s., °bartuli a.,°dionisi-vici c., *dello strologo l., §villani a., §bianchi r., ^salvatori g.,*rizzoni g, °sabetta g. *div. of nephrology, °div. of metabolism, §intensive care unit, ^div. of neonatology. "bambino gesfl" children research hospital. rome, italy. successful prevention of handicaps or death in newborns with ~ depends on rapidity and efficiency of treatment. poor response to nutritional and/or pharmacological treatment requires extracorporeal removal of nh4. efficiency and cardiovascular tolerance are often difficult to obtain with peritoneal or hemodialysis in neonates. we report the results of cavhd in 3 newborns with hc. methods: vascular access: femoral vessels. blood flow: 10-35 ml/min, dialysate flow: 200-500 ml/h. filter: amicon minifilter plusrm(polysulfone membrane; 0.08 sq.m.). no ultrafiltrate(uf) production, patients: case 1 with carbamoytphosphate synthetase deficiency (body weight -bw-: 3.2 kg) showed hc at day 4, a relapse of hc occurred at day 14 due to an infectious event. case 2 and 3 (bw: 3.0 and 2.8 kg), both affected by propionic aeidemia, showed hc at day 5 and day 7, respectively. plasma nh4 (~tg/dl) decrease is shown in the complications: transitory ischemia of arterial cannulation limb and transitory thrombocytopenia occurred in case 1; surgical repairing of artery after cavt-id was necessary in case 3; no cardiovascular instability was observed during cavhd . outcome,'all patients recovered from hc in less than 1 day: case 1: alive, mild b)iootonia at 34 mos; case 2: dead after 10 days from cavhd withdrawal for pulmonary hemorrhage; case 3: alive, normal development at 7 mos. conclusions: 1) in newborns with hc, ca~q-id provides good cardiovascular tolerance,high efficiency and quick removal of nh4, even without uf production (i.e. only by diffusion). this allows easier management (no need of fluid and electrolyte balance). 2) arterial complications seem frequent in neonates treated by cavhd. venovenous circulation could overcome this problem. vb nguyen, m jokie, c leeaeheux paediatric intensive case service, hospital university centre, avenue c6te de nacre, 14033 caen cedex, france background, the implication of polymorphonuclear neutrophils (pmns) in the physiopathology of children's haemolytic.uraemie syndrome (hus) becomes more and more evident. the purpose of the present study is to role out their impact among other pronostie elements during the course of the disease. patients and methods. diarrheal prodrome and its duration, patient's age, maximal blood nitrogen level, anuria and dialysis time, extra.renal involvements, white enll and pmn counts and thrombopenia duration have been retrospectively analysed in 18 infants with good outcome and in 8 another children with unfavorable outcome. results. neither diarrhoea or its duration, nor children's age, nor blood nitrogen level, nor anuria or dialysis time had any predictive value for the disease evolution in the acute phase of our patients. adversely, extra-nenal involvements was accompanied by severe and complicated courses of the disease (p<0,02). the elevation of white cells and pmns (heyon 20 x 109/i) and pmns (more than 15 x 109/1) as well as its persistence beyon a week were most frequently observed in complicated forms (p<0,001, p<0,001 and p<0,01, respectively). a transient thrombopenia (less than 5 day@ in patients with elevated counts of white cells may be a filrther obvious sign of an unfavorable course of the disease (13<0,02). conclusion. the elevated count of white cells and pmns, either alone or associated to one rapid regeneration of platelets, seems enabled to predict an unfavorable evolution of the hus in children. msud results from an inherited impairement of catabolic pathway of branch chain amino-acids. high leucine blood levels may induce acute brain dysfunction. this dramatic complication led us to propose leucine removal procedures as continuous hemofiltration. patients and methods three newborns in acute msud onset were treated by hf, hdf and hd. extracorporeal circulation was performed through a 6.5 fr catheter, a circuit with a blood pump (priming volume = 40 ml). patients and procedures characteristics are summarized below in the sucralfate (an aluminium salt of sucrose octa sulfate) is used to prevent and treat upper gastrointestinal bleeding in critically ill patients. with minimal absorption, the potential for side effects is thought to be limited, though aluminium toxicity has been reported in patients with chronic renal failure. these patients may already have had high body stores of aluminium. we report 5 critically ill children with high serum concentrations of aluminium following sucralfate therapy. all 5 had renal impairment. the normal aluminium level is < 0.4 gmol/l and in patients with chronic renal failure < 2.2 ].tmol/l. none of these patients had known preexisting chronic renal disease. cpb was conducted under deep hypothermia (t,°16°c) and cardiocirculatory arrest (cca) or under hypothermia (t,°24°c) and low-flow perfusion. continuous holter-electrocardiograms (h-ecg) were recorded from the ilranediate postoperative (po) period on for 72 hours. h-ecg were also recorded prior to the operation and before discharge. following dr were observed: snpraventricutar (sv) and ventricular (v) extrasystoles (es) (>50/24h), sv and v tachycardia (svt and vt), accelerated junctional rhythm (ajr) and junctional ectopic tachycardja (jet), and 2nd and 3rd degree atrioventricular block (avb2 and avb3). the incidence of po dr was 20% in the pre-op h-ecg, 74% on the 1st, 33% on the 2rid, 34% on the 3rd po day and 21% befbre discharge. compared to the pre-op findings, an increased incidence of sves, ves, svt and avb3 on the 1st po day was observed, whereas vt and a jr or jet were exclusively observed po. all types of dr were observed up to the 3rd po day. ty23e of dr before discharge was similar to pre-op findings and there was no definitive avb3. considering patient groups according to the most frequent isolated op-procedure, the incidence of dr on the first po day was 56% after asd ii-closure (n=23), 74% after stthaortal vsd-closure (n=lg), 75% after correction of a complete avsd (n=8), 80% after correction of a tetralogy of fallot (n=20) and 100% after fontan-operation (n=10). incidence and type of dr were not significantly different between groups. longer cpb-dttration and use of cca were risk factors for po ves and vt (p<0,005 and p<0,05, respectively) whereas use of cca and degree of hypothermia were risk factors for the development of a jr and jet (p<0,02 and p<0,0001, respectively). -our results indicate that po dr after cpb in children m'e frequent but mainly transient. in our series, specific cpb-related parameters are of greater influence than surgical procedure itseif for the development of dr and are discriminant risk factors for particular types of dr. the course of anp, cgmp/anp (as indicator for atrial natriurefic peptide biological activity), and no2 and no3 (as indicator for endogenous nitric oxide (no) synthesis) was investigated in i9 infants (median age 4 months) undergoing cardiopulmonary bypass (cpb). patients were divided into 2 groups according to whether they had (group 1, n=13) or not (group 2, n=6) preoperative heart failure (hf) and pulmonary hypertension (pht). group 1 patients had preoperatively significantly higher levels of anp (p<0.005), cgmp (p<0.02) and no2 and no3 (,p<0.02) but had significantly lower cgmp/anp (i0<0.05) than group 2 patients. during cpb, anp was significantly higher in group 1 patients ~<0.02). as compared with prebypass values, cgmp/anp was reduced in both groups during cpb (p<0.0001). cgmp/anp inversely correlated with duration of cpb and aortic clamping time (p<0.001, respectively). no2 and no3 were significantly higher in group 1 than in group 2 patients (p<0.05) without any intraindividual change during cpb. from the early postoperative period on anp, cgmp/anp and no2 and no3 were similar in both groups. after cpb, anp correlated in both groups with blood pressure (p<0,001) and diuresis (p<0.05). no2 and no3 inversely correlated with pulmonary arterial pressure immediately after cpb (19<0.05 patients after a fontan-type of procedure have elevated central venous pressures (cvp) leading to congestion in the gastrointestinal system and often ascites. purpose of this study was to evaluate whether this causes a different postoperative gastric mucosal ph (phi). methods: we evaluated a series of 35 patients, who underwent cardiac surgery with cardiopulmonary bypass (age: 5 days to 16 years (mean 2,2 yrs), weight: 3.2 to 37kg (mean 10.2 kg). a commercially available tonometer (tonometics®) for sigmoidal use in adults was inserted into the stomach after induction of anesthesia. the phi measurements were done according to manufacturer recommendations we compared three groups of patients: 1) aeyanotic (n=20), among them 9 p with vsd and 5 p with avsd; 2) cyanotic (n=10): tof: 6p, tga: 4p; 3) cyanotic after a fontan-type procedure (n=5). phi were measured at picu arrival and after 6h. fudhermore we compared lactat levels at these time points. differences between the groups were evaluated with one way anova on ranks with pairwaise multiple comparisons (dunn's method). the relationship between cvp and phi was investigated by regression analysis. results: the median phi for groups i, 2 and 3 were 7.28, 7.27 and 7.13 at ardval and 7.30, 7.25 and 7.21 after 6h respectively. at picu arrival group 3 was significantly (p<0.05) different from groups 1 and 2. there was no significant difference between the latter two groups, after 6h group 1 was different from group 3, there were no other significant differences. the median lactate levels for groups t, 2 and 3 were 2.2, 3,2 and 4.1 at ardval and 1.6, 3.1 and 3.3 after 6h respectively. at ptcu arrival group 3 was significantly (p<0.05) different from group 1, after 6h there were no significant differences. there was a weak negative correlation between cvp and phi: r= -0.21; p<0.05. conclusion: patients after a fontan-type of procedure have lower phi than patients after other cardiac surgical procedures, however, this is only in part due to the elevated cvp and venous congestion. eleven children were investigated 32 months (median) after postoperative mof. iviof was defined as the failure of at least two vital organ systems (kidney, liver, lung, central nervous system) in addition to cardiac insufficiency and high fever. underlying surgical procedure was repair of tetralogy of fallot (n=3), fontan-(n=7) or seuning procedure (n=l). all patients fulfilled criteria for mof in the 3 first postoperative (po) days. six patients needed peritoneal or hemodialysis for 31 days (median) during the po period. one patient showed cerebral infarction due to thromboembolism in the territory of the right internal carotid artery immediately after the operation. the follow-up protocol consisted of extensive investigations of heart-, renalliver-, and lung functions as well as complete neurological and psychological examinations. all patients had adequate cardiac examination. lung function was normal in all but 2 patients who had an obstructive syndrome. only 1 patient showed an isolated decreased creatinine clearance. abnormalities of the liver ftmction tests were only noticed in patients after fontan procedure. severe neurological sequels such as paraplegia (n = 1) and diplegia (n-i) were observed in 2 of the 11 patients. the remaining 9 children presented with a delayed graphomotorical and speech development associated with normal intelligence. -in our series the most frequent and severe sequels after postoperative mof were neurological. -abnormal liver fimction tests are more likely to be a consequence of the fontan hemodynamics than a sequel of mof. the optimal dosing schedule of surfactant therapy for the treatment of neonatal respiratory distress syndrome (rds) remains unclear. goal: surfaetant function and the concentration of phospholipids (pl) in tracheal aspirates are compared in a prospective randomized trial involving neonates with rds who received either two or more (3 or 4) doses of survanta. methods; ventilated neonates <35w with rds were treated with survanta 1oo mg/kg if fio 2 >_40% or mean airway pressure _>7,5 cm hzo, after 6h a 2nd dose was given (same criteria), if the support still exceeded the criteria 12h after the 2nd dose, the patient was randomized to no extra dose (two}, or to an extra dose of survanta (morel (and a 4th dose 12h later; same criteria), pl was measured in tracheal aspirates and corrected for dilution with the urea method. "active" large aggregates and "non-active" small aggregates of surfactant were separated by centrifugation and quantified. surface tension of the large aggregate fraction was measured by pulsating bubble surfactometer, results: 13 neonates were randomized, 6x two and 7x more (5x3 and 2x4 doses), gestational age was 31,7±2,4w and birth weight 1582±568g. most patients had severe rds with initial ventilation: rate 63.1_+11,1, peak inspiratory pressure (pip) 24,3-+6.4 cm hzo, fio 2 75.3±21.0%. at randomization: rate 63.5±6.9, pip 20.3-+2.5 cm hzo, fio 2 29.5±15.7%, and 24 h after randomization: rate 45.9±17.1, pip 18.7_+2.2 cm hzo, fio 2 26.8±6.6%, without signif, differences between the groups. there was 1 relapse (again fio2_>60% within 72h) in group two and t bpd in group more. in total, 112 tracheal aspirates were analyzed. pl was not signif, different before randomization (two 27.5 ± 15.7 vs more 24.5 ± 11.4 /jmol/ml), but neither after randomization (two 21.2-+ 11.0 vs more 19.3±7,o /~mol/ml). there was no difference in the % small aggregates (two 4.2±1.9 vs more 6.9±5.5%), the surface tensions (ran/m) were not signif, different (each time two vs more): before randomization 10.0±2,3 vs 14.2-+7.2, in the 24h after randomization 12.6±5.0 vs 11.2-+3,8, or 24-48h after randomization 17.0-+5.5 vs 12.8±9.8, or 48-72h after randomization 15.7_+0.4 vs 13.7-+5.6. conclusion: neonates who received more than two doses of survanta did not have higher pl, nor a better surfactant function than neonates who received only two doses of survanta. continuation of the trial is necessary to evaluate clinical outcome. may not indicate need for treatment p.c. clemens s.j. neumann university of hamburg, department of pediatrics, klinikum schwerin, wismarsche str.. 397, d-19049 schwerin. aim of the study: the finding of elevated tsh and decreased t4 in the newborn usually is classified as "transient hypothyroidism", thus the elevation of tsh is classified as consequence of the lowered t4. but on the other hand several data sets show that tsh elevation as well as low t4, one independently of the other one, are associated with different kinds of perinatal stress. each of these laboratory deviations, if not associated with the other value being abnormal too, is generally accepted not to be an indication for treatment. from this we conclude, that more pefinatal stress, as in intensive care neonates, may produce tsh elevation as well as low t4, but only coincidentially, not the tsh elevation being the consequence of low t4, thus not to be classified as "hypothyroidism", thus not indicating treatment. if this hypothesis is right, we should find an association of increasing pefinatal stress with an increasing number of neonates from tsh and t4 normal via tsh or t4 abnormal to high tsh and low t4. method: in the newborn screening program in germa w we determine primarily tsh, and only in the neonates with elevated tsh, in addition we determine t4. thus in our study we asked whether we find an association of increasing perinatal stress with an increasing number of neonates from tsh normal via tsh abnormal while t4 normal to high tsh and low t4. definitions for this study were: tsh elevation = >20 mu/1 (as usual in the german screening programs), t4 lowered = < 6 p_g/dl perinatal stress score was 0 or 1 or 2 or 3 in dependency of the neonate having stress in none to all of the following three categories: (a) forceps or vacuum extraction or sectio co) birth weight below 2500 g (c) at the 5th day existence of a relevant neonatal disorder (rds, ictems gravis, infection/sepsis, vitium cordis with hemodynamic relevance, severe malformation). results: our data of 1131 neonates show a high significant association (chi2 = 84, p <0.001) of, on one hand, perinatal stress score 0 with normal tsh, versus, on the other hand, perinatal stress score 2 or 3 with high tsh and low t4. discussion: facing the background given above, in the intensive care newborn, the constellation of high tsh and low t4 may be only a coincidential addition of two independent abnormalities. in tbese cases -the high tsh not being the consequence of low t4 -the classification as "hypothyroidism" is not justified, thus a therapy not indicated. on the other hand of course there exist rare cases with high tsh as consequence of low t4 thus with hypothyroidism tlms with indication for therapy. unfortunately we have no criteria, that enable a certain discrimination of these two categories thus in respect to the question of therapy or not. conclusion: further research has to be done to learn how to discriminate the coincidential high tsh and low t4 from the causal constellation of high tsh and low t4. until we have certain discrimination criteria we have to treat both groups of neonates. few studies have focused on fa composition of surfactant pc in preterm infants before and after surfactant therapy. methods: tracheal aspirates were collected in 7 venttlated mfants from birth until extubatlon (27/7_1 /twk ga, 859.+ 155g bw). after lipid extraction, t.l.c,, and methylation, fas of pc were quantified by gaschromatography. intralipid a (53.2 % linoleic acid,18:2•6) was started 48h after birth. results: six infants developed respiratory distress syndrome (rds) and received survanta r i00mg/kg (sr), all doses within 18h after birth (ix s r n=l, 2x s r~ n=3, 3x s r n=2). one child did not develop rds. in alt patients, the patmitate % in pc was ~ 65% (before sr<=natural composition), increased to ~ 85% after s r, and remained >80% for i5h after lx s a, 22.3.+i1.8h after 2x, and 38.5.+3.3h after 3 doses. in 4 patients, intubated long enough, the palmitate % decreased with a half-life of 78.7_+42.8h to a new plateau which was still higher than baseline after 1 week. linoleic acid % was 5.85_+2.3 (with rds), decreased after s r~ and returned to baseline due to the decrease in patmitate %. thereafter the linoleic acid % increased linearly with 0.021% per h, in 1 patient even up to 15.1%. other fas did not increase after return to baseline. in neonatal medicine the current parameters, arterial oxygen saturation and arterial oxygen pressure, are poor indicators for oxygen delivery and oxygen demand. the purpose of this study was to obtain venous blood samples from the inferior vena cava in stable neonates with respiratory failure and to determine a parameter that reflects more adequately the balance between oxygen delivery and oxygen demand. "l~e study included 22 neonates requiring mechanical ventilation tbr severe respiratory insufficiency. an umbilical venous and arterial catheter were inserted in the inferior vena cava and in the aorta respectively. paired blood samples were obtained at the time that the patients were hemodynamically stable. fifty paired arterial and mixed venous blood samples were analyzed. 1jnear regression analysis showed the following correlations: in a neonatal intensive care unit adjacent to a delivery room caring for 4000 mothers per year, (with a referral of 400 mostly for preterm delivery), virtually every neonate <ls00g was seen by the same ophtalmologist, j.o. the incidence of retinopathy of prematurity (rop) was in 1976-80 of ii cases (464 preterms <ls00g with 58% surviving to discharge, and 4% rop in survivors; 8% if less than 30 weeks). in 1987-1993, 57 cases of rop were diagnosed by the same ophtalmologist. 679 were born at less than 30 weeks, of which 452 (66%) survived to discharge, and among those survivors, rop was present in 53 (12%), with rates varying from 58% at 24 weeks to 5% at 29. objective: to compare a method of teicoplanine (teico) infusion using an electrical syringe-pump with a manual injection. methods: infusion of teico was simulated through a standart neonatal i.v. system. the infusion system consisted of an life care 4 infusion pump (abbo'it lab.) with its i.v. set for maintenance intravenous fluid (flow < 6 mi/h) connected to a 3-way stopcock. an 1 meter extension tubing (vygon lab.) was placed between the stopcock and a neonatal catheter, an another 1 meter tubing (injection tubing) was connected to the stopcock. the volume of the injection circuit was 2.6 ml. simulations were realised for 2 weights (1 or 3 kg), with a doses of 8 mg/kg and a injected volume of 0,8 ml to 3 ml. our goal was to perform a complete infusion in 10 minutes. we compare one method of infusion with an electrical syringe-pump with 2 manual methods: a: teico was infused with a syringe-pump pilot c (becton & dickinson lab.) according to a protocol using 1) a priming before connecting the syringe to the tubing (for immediate starting of infusion), 2) a programmed volume injected in 5 minutes (the drug volume was greater than the dose prescribed to avoid loss of teico into the syringe), 3) a 5 ml wash out was performed over 5 minutes with the syringe pump. b: teico was manually injected in a few seconds in the tubing followed by a 5 ml wash out over 10 minutes. c: idem as b but a nu site valve (medex) was connected to the proximal end of injection tubing to avoid leakage between removal of the teico syringe and connection of the wash out syringe. during each run, serial samples were collected every ten minutes over a one hour period. the samples were assessed using hplc method. results: the amount of drug delivred at 10 minutes was calculated and expressed as a perventage of the total amount of teico prescribed. results are a mean of 5 to 8 rons. a b c 1kg 94.2+17,9% 86,8+9.1% 94,4-+6.4% 3kg 95-+4,9% 72-+13,4% 90.9-+6% conclusion: with a drug volume injected < circuit volume and a valve, a direct i.v. administration of the drug in the circuit followed by a wash out seems an accurate alternative to drug infusion conbroled by a syringe pump and is easier to perform. doxapram hydrochloride is a central and respiratory stimulant which is used in neonatal intensive care units to treat caffeine-resistant apnoes of the newborn. routinely, doxapram is administered intravenously. oral administration would be much easier but data on bioavailability after oral administration are limited (two studies) and show a large variation. we therefore studied the oral bioavailability of doxapram in 8 preterm infants. doxapram treatment was started with an intravenous loading dose of 2,5 mg/kg during 15 minutes, followed by a continuous infusion of 1 mg/kg/h. after 24 h the intravenous administration was changed to continuous oral administration with the same dosage, blood samples were collected 24 h after both the intravenous and oral administration and analyzed by hplc-assay. apnoea rates per 6 hours were listed before and after starting doxapram treatment. the median apnoea rate per 6 hours declined from 9 to 2 after both the intravenous and oral administration. the oral bioavailability of doxapram is between 43 and 65%, indicating that the oral route can be used effectively in these infants 2. there were no differences in the decline of the apnoea rate after intravenous versus oral administration of doxapram, infasurf r has been comnared with exosurf r in two studies: one as drodhvlaxis and the other a rescue trial. similar. althouah non-sianificant benefits were found for the natural surfactant. in 6 trials there was a sianificant reduction in dulmonarv air leaks for 0.53: 95% ci 00.41-0.641 for babies treated with natural comnared to svnthetic surfactants. for 7 trials (3554 babies~ comdarina natural and synthetic surfactants for rds {6 comdarina survanta r and exosurf r. and one infasurf r and exesurfr% neonatal mortalitv was sianificantlv reduced (or 0.80: 95% ci 0.66-0.971 with natural compared to synthetic surfactant treatment. in two further trials different natural surfactant drenarations were compared. reduced oxvaen needs for 24 hours after treatment were found for r r infasurf and curosurf respectively when each was compared to survanta r. andarent lonaer-term benefits from these surfactants were not statisticallv sianificant. further trials will be needed to ascertain differences between various surfactant preparations. introduction bacterial meningitis is a common cause of admission in the paediatric age group, and one which still carries a high morbidity and mortality. these complications are strongly associated with a delay in antibacterial therapy. a significant proportion of these patients require mechanical ventilation; it is in this particular group of patients that survival is greatly diminished. the identification of risk factors for mechanical ventilation associated with bacterial meninges may help improving survival by shortening the delay to icu referral. the paediatdc risk of mortality score (prism) is the most widely used scoring system in the paediatric icu literature; the accuracy of prism on predicting outcome in meningitis has been shown to be poor. aim we aimed to describe our population of patients with bacterial meningitis requiring ventilation, and then to identify predictors of survival. methods this study was conducted at the baragwanath hospital in south africa which is an universityaffiliated institution with end average annual paediathc admission of 4500 patients. baragwanath icu admits 250 non-neonatal paediatdc patients per year. a retrospective chart review from january 1991 to december 1995 of 42 consecutive paedistdc icu admissions with bacterial meningitis was performed. results approximately 150 cases of bacterial meningitis are admitted to the general paediatric wards every year; this constitutes ± 3% of all admissions. the mortality of these patients is + 14%. during this time 42 (23m, 19f) patients (7%) were accepted for icu admission, with a mortality of 42.2%. the median age was 9 months (range 15 days to 17 years). the median delay to hospital admission was 48 hours, and the median delay to icu admission was a further 4 hours. the median duration of tcu stay was 48 hours (40 for non-survivors, 120 for survivors). the main presenting features were convulsions (34%), altered mental state (40%), fever (50%), respiratory symptoms (24%), headache (18%), and diarrhoea and vomiting (32%). the most common indications for icu admission were seizures (55%), coma (36%), shock (26%), respiratory failure (29%) and acidosis (21%). in 29% of patients there was a cardiorespirstory arrest prior to admission. the most common organisms in the csf was pneumococcus (57%), haemophilus influenza (15%) and e coli (12%). the presence of leucopenia (wcc < 5), a low platelat count (<100), acidaemia (ph<7.2), and the need for inotropic support were strongly associated with nonsurvival. tachycardia and hypotension were not significantly essorjsted with poor outcome, as has been previously reported. discussion early diagnosis of meningitis and prompt institution of antibiotic therapy requires a high level of clinical suspicion. paediatric patients with bacterial meningitis that require mechanical ventilation have a poor prognosis. there is little evidence to suggest that nomsurviva[ can be predicted prior to icu admission, but a rapid deterioration requiring ventilation and inotrepic support with evidence of severe sepsis (low platelet count, leucopenia) is nmost invariably associated with a fatal outcome. the long term functional outcome of these patients make this disease even more devastating; the rote of icu in the management of these patients has yet to be fully estanished. objective and study design: a retrospective study was pertbrmed for all patients diagnosed with hemorrhagic shock and encephalopathy syndrome (hse) during 1984 to 1994. soroka medical center is the only medical facility in the southern negev region of israel serving a population of -400,000 residents, consisting primarily of jews and bedouins. results: 20 patients (17 bedouins and 3 jews) were diagnosed with hse. main features on arrival included profound shock and coma with con~alsions. active bleeding and/or disturbing coagulation tests with falling heuroglobin levels and thrombocytopenia was noted in every case. all infants developed diarrhea shortly after arrival. elevated urea, creatinine and liver enzymes was noted in all cases. annual incidence tbr inlhnts <1 year of age was 5:10,000 for bedouins and 0.6:10,000 for jews. patients ranged in age from 6-32 wks and arrived at the hospital late night/early morning (2:00am-ll:00am), during winter/early spring (november-april). all were healthy prior to admission, with short prodromal symptoms of upper respiratory tract or gastrointestinal infection noted in 10 cases. most infants had markedly elevated body temperature on arrival. a history of overwrapping and/or excessive hearing was obtained in 4 of 20 infants. bacteriological and virological cultures were negative in all infants. one infant died and neurological sequelae were observed in all survivors. the high prevalence of hyperpyrexia during sleep in the presence of negative microbiological results with no evidence of excessive hearing, and the high incidence of rise among a closed, culturally isolated society known to have a high incidence of congenital malformations, may support previous assumptions that hse results from hyqperpyrexia, originating in most cases from a "physiologic" heat induced trigger which starts and peaks during the night in previously healthy infants with susceptible, predisposing genetic underlie. approximately 10% of hospitalised infants with respiratory syaacytial virus (rsv) infection require mechanical ventilation. our general practice is not to use specific antiviral therapy. we have undertaken studies of rsv-infected mechanically ventilated patients without underlying congenital heart disease or immunodeficiency. study i (n=45): a retrospective review of 45 infants (mean post-conceptual age 48 weeks; median duration of intubation was 8 days (interquartile range 5-11 ). blinded review of chest x-rays during the first three days of mechanical ventilation revealed a spectrmn of lower respiratory tract findings from marked diffiase consolidation in all zones without hyper-inflation (n=10) to gross hyperinfiation without consolidation (n=5), with the remaining patients have an intermediate picture (n=30). death occurred only in patients who were at the poles of this continuum (4/10 consolidation and 1/5 hyperinflation). patients at these poles could be differentiated by gender predominance, birth gestation, alveolar-artarial (a-a) oxygen gradient (p<0.0 l), oxygenation index (p<0.0 l), peak inspiratory pressure (p<0.05), and intubation days (p<0.01). study ii (n=28): prospective audit of infants with the aim of verifying the above differentiation. separating patients based on their early x-rays and a-a gradient we confirmed the above predictable difference in duration of supportive therapy (consolidation v intermediate: 6(4-7) v 14( 12-33 ) p<0.01 ). severe lower respiratory tract rsv-infecrion in young infants results in different distinctive partems of disease with characteristic radiological and clinical features, and each has a predictable timecot~se. conflicting reports on special therapy should be interpreted with respect to these observations before making conclusions about the efficacy of any specific treatment. the clinical data of 186 children wich suffered from a severe mycosis between jan.1990-dac.1994 and wich were treated with amphotericin b in 10 spanish hospitals were collected. mean age was 6+3.9 years; 157 were mate (84%) and 29 female (16%). the most common underlying diseases were leukemimymphoma (31%) and congenital heart defects (20%). the major pathogens isolated were candida albieans (61%), other candida (18%) and aspergillus (17%). and liposomal amphotericin (la) in 68 (37%). in the ca group, the starting dose was 0,3~0,1 mg/kg, reaching a maximal dose of 0.9+0,3 mg/kg after 4.7+3 days. maintenance time was 14+10 days and the cumulative dose: 12~17 rag. in the la group, the starting dose was 1.1±1 mg/kg, reaching a maximal dose of 2.9_+2.4 mg/kg (p<0.01) after 5+5 days. maintenance time was 19~16 days (p<0.05) and the cumulative dose 42_+37 mg (p<0.05). the reasons for la use were: elective in 66% of cases, previous renal failure in 29%, and ca inefficacy in 4%. hepatic or renal function impairment was two times more frequent in la group than in ca group, the antifungal efficacy was 62% for ca and 67% for la. therapeutic failure or toxicity induced withdraw of ca in 7% of cases, vs, 1,5% for la (p<0,01). mortality was not different in both groups (21 vs. 22%), adverse effects were related to ca in 28 events vs, only one in la group (p<0,01). the commonest side effects in ca patients were fever (18%), chilis (10%) and nausea (9%). in ca group, 44 cases (37%) developped analitical anormalities related with amphotericin, vs. 10 cases (15%) in la patients (p<0.001). in the first group, the commonest serum alterations were hypokaliemia (25%), raised creatinine (14%) and bilirrubin (6%). in the second one, hypokaliemia presented in 7% of cases (p<0.01), raised bilirrubin in 6% and creatinine in 3% (p<0,05), the antifungal efficacy of la is at least the same of ca. from the clinical and analitical points of view, la is much less toxic than ca. la can be used at a greater dose than ca and is safe in children with renal failure. laurence desplanques -serge gottot -christian dageville d~artement de sant~ publique -facult~ xavier bichat -16 rue henri huchard 75018 paris -france -the french << reaped ~> network was created to implement a nosecomial infections (ni) quality care program in nicu and picu, the first objective was to describe the annual ni incidence rate in each icu population : all patients stayed more than 48 hours in icu. methods : n] criteria were defined by the reaped group according to cdc criteria. all data were collected by a medical and nursing team. all infection data were validated by an external investigator. results : 4525 patients were admitted over a 14 months period. 68% were newborns. 371 ni were identified among 311 patients. the overall ni incidence rate (ir) was 8.2% and 5.9°/00 person day (from 5.0 to 8.2°/00 according to age, lowest rate for newborns). septicemia (50% of ni) and pneumonia (41% of ni) were the two main ni. according to age, the septicemia ir varied from 6.8 to 10.9°/oo catheter day (lowest rate for newborns) and the pneumonia ir from 3.9 to 7.4°/00 ventilator day (lowest rate for newborns). there were very few other infections (uti : 4%, ir : 7.4°/00 catheter day). gram positive cocci were isolated in 73% of septicemia ( 70% of them were coagulase negative staphylococcal). gram negative bacilli were isolated in 53% of pneumonia (40% of them were pseudomonas). 5% of ni were caused by candida, mostly septicemia. the septicemia and pneumonia ir varied according to unit even after adjustment for age. discussion the aminoglycoside antibiotics are frequently used in newborns for the treatment of severe infection and sepsis due to gram-negative microorganisms. the currently recommended dosage schedule for tobra (2.5 mg/kg q18h) does not take into account differences in gestational or postnatal age during the first 4 weeks of life. we questioned the validity of these recommendations and studied the population kinetics of tobramycin to establish predictive equations that enables the clinician to select the appropriate initial dosing schedule. methods tobra trough (t=0) and peak values (t= 1) were taken on day 2-4 after birth in 460 newborns. tobra was administered as a 30-minute intravenous infusion already in an adapted dosage schedule: 3.5 mg/kg q24h in infants with gas < 28 weeks; 2.5 mg/kg q18h in infants with gas between 28-36 weeks and 2.5 mg/kg q12h in infants with gas > 36 wks, tobra concentrations were analyzed by tdx-assay, a one-compartment model was assumed and non-linear mixed effect modelling (using nonmem) was applied to the data, a trough level < 2 mg/l and a peak level between 6 and 10 mg/l was required, with the present dosage scheme 40% of the trough levels were too high and almost 60% of the peak levels too low. calculations showed that the following dosage schedule should result in optimal levels of tobra. preterm infants gas < 28 wks: 6 mg q48h preterm infants gas 28-36 wks: 4.5 mg q36h preterm infants gas > 36 wks: the currently recommended dosage schedules for toeira result in high trough and low peak levels. prolongation of the dosing interval and increasing the amount of drug per dose according to the above scheme will improve tobra level control. since january 1993 british clinicians have been conducting a randomized controlled trial of neonatal ecmo. mature infants (>-35 weeks gestation and birthweight 2 2 kg) with severe cardiopulmonary failure have been randomized to receive continued care in their referring institution or referral to a designated ecmo centre for further management. we now present the preliminary results which have prompted closure of recruitment to this trial. the final outcome will be assessed as intact survival against death or severe disability at one year of age for all the recruited patients. patients were categorised by diagnosis such as isolated persistent fetal circulation, secondary persistent pulmonary hypertension of the newborn or congenital diaphragmatic hernia and by severity of illness at the point of first contact with the clinical coordinators of the trial -judged primarily by the oxygenation index (240 before randomization). 180 patients were randomized (90 in each arm). hospital outcome data are reported for all patients and 1 year outcomes on t18 (65 survivors). at this stage 26 of the babies allocated to ecmo are known to have died compared to 52 of those allocated to conventional management (rr 0.5; 95% ci 0.35-0.72; p=0.0002). fewer deaths have been obsea-ved amongst ecmo allocated babies in all the diagnostic categories used. a 28% incidence of disability and impah~nent has been observed amongst survivors. this rate is similar in both groups and the survival advantage is not offset by an increased rate of disability or impairment following allocation to ecmo. we consider that these data combined with those available from other studies provide conclusive evidence that the survival to discharge from hospital is substantially higher in patients allocated to ecmo than in comparable infants not so allocated. therefore recruitment to this trial has been closed whist awaiting complete one year outcome data. sigston pe, goldman ap. #keating j. crook r. ~e dj~. great ormond street hospital for children nhs trust, and ~biochemistry department, kings college hospital, london, united kingdom. isoflurane is a safe and effective means of long term sedation in both children and adults in the intensive care setting. the use of isoflurane, by adding it to the sweep gas allows the use of this volatile anaesthetic agent in patients on ecmo, enabling rapid control and weaning of sedation. a potential problem with the long term use of isoflurane is fluoride ion accumulation with the possibility of renal toxicity, the purpose of this study was to assess plasma fluoride levels in patients receiving prolonged isoflurane on ecmo. method: fifteen infants and children (aged 1 day -10 years, median 2 weeks) receiving ecmo support for either cardiac or respiratory failure were recruited to this study. the patients were sedated with isoflurane as well as intravenous agents (morphine and midazolam). isoflurane was administered (0% -3%) via a calibrated vaporiser to the sweep gas, adjusting the level to maintain adequate sedation. blood samples were obtained on a daily basis for plasma inorganic fluoride assay. the relationship between plasma fluoride and amount of isoflurane administered, as %-hours (vaporiser setting in % x hours) was calculated by linear regression. results: the duration of ecmo ranged from 42 to 532 (mean 207) hours, during which the amount of isoflurane administered varied from 7 to 418 (mean 168) %-hours. 75 blood samples were anaiysed, demonstrating individual peak plasma fluoride levels of 2.7 to 16.5#mol/1, mean 7,1p.molli (toxic threshold = 50gruel/f). the plasma fluoride positively co;related with the %-hours of isoflurane (r = 0.65, p = < 0.001). conclusion: this study shows that although there is a dose related accumulation of inorganic fluoride ions in patients sedated with isoflurane on ecmq, the peak fluoride levels are well below the suggested toxic threshold. merzel y, lev a, bar yosef g, halbertal m, lorber a ecmo center, picu, emek medical center, israel. the mortality rate of pediatric patients with acute myocarditis is 20-60% according to the severity of myocardial damage. a 15 month old gzrl presented with high fever, respiratory and cardiac failure. diagnosis of acute myocarditis was made and the patient was ventilated with high pressures and fio2 of 1.0. she required high doses of inotropes. echocardiography revealed a dilated la and lv with severe mr. lvedd was 41 mm and lvsf 9%. calculated oxygenation index was 55. she was resuscitated after a cardiac arrest. she was commenced on ecmo (using biomedicus centrifugal pump and avecor 800 oxygenator) at a flow of 100 ml/kg/mm with immediate improvement of hemodynamlcs, oxygenation and pc02. resptratory assistance and vasoactive drugs were reduced. the patient was transported by air, on ecmo, to the ecmo cevter. she developed arf and cvvh-d was performed. cardiac fimction started to improve after 12 days. ecmo was discontinued on day 18. echo revealed lvedd 34 mm and lvsf 24%. ippv was discontinued on day 20. on discharge, a month later, her lvedd was 29 mm and lvsf 28%. she behaves normally for age without neurologic or other medical sequellae. literature search revealed no case of acute myocarditis, as severe, that was treated successfully. survavors of disease this severe usually suffer dilated cardiomyopathy and permanent disability. the use of ecmo allows myocardial rest which prevents long term myocardial damage. introduction ecmo is increasingly used in the care of critically ill newborns. despite the frequent use of betalactam antibiotics in the treatment of these infants there are no data available on the dispbsition of cefotaxime (ctx) and amoxicilfin (am) d0ring ecmo. the purposes of this study were to determine the pharmacokinetics of these two drugs in infants on ecmo and consequently formulate appropriate dosing regimens. we therefore studied the pharmacokinetics of ctx (100 mg/kg ql 2h) and am (50 mg/kg q6h) in 8 term infants on day 3 after birth, blood samples were taken before (t-o) and 0.5,1,2,4,6 (am) and t2 h (ctx) after the intravenous bolus injection and analyzed by hplc-assays. 2. ctx 100 mg/kg q12h results in adequate serum levels of ctx in fullterm infants on ecmo, am 50 mg/kg q6h results in very high serum trough levels. recalculation based on the known volume of distribution and elimination serum half-life of these infants resulted in the following dosage recommendation: 50 mg/kg q12h. persistent pulmonary hypertension of the new-born (pphn) is characterised by rapid fluctuations in pulmonary artery pressure (pap) and a clinical impression of stifflungs. lung mechanics were measured in 35 term infants, mean age 1.5 +_ 0.7 days who were paralysed and ventilated within the first three days of life. fourteen infants had pphn with systemic or suprasystemic pap measured by echocardiography. in these patients, the respiratory system resistance was 29.4% higher (p < 0.001) and compliance 22.4 % lower (p = 0.03) during systemic or suprasystemic pap compared to when the pulmonary hypertension had resolved. in contrast, there were no changes in resistance in the 14 infants with respiratory distress syndrome (rds) and no pulmonary hypertension or in the seven infants with normal lungs, where two readings were taken 24 hours apart. the changes in lung mechanics interfered with mechanical ventilation, resulting in a 12.5 mmhg rise in paco2 (p=0.007) during pulmonary hypertension. inhalation of nitric oxide 10 ppm resulted in a 16% decrease in respiratory system resistance and an improvement in oxygenation. the bronchial and vascular smooth muscle was increased by 120% in postmortem lung samples from eight infants with pphn compared to six age matched post-mortem controls with normal lungs (p<0.001). these findings suggest a co-constriction and co-hypertrophy of bronchial and vascular smooth muscle during pphn. anatomically the pulmonary vasculature and bronchi lie in close proximity to each other. thus mediators such as endothelin-1 released locally may act on both vascular and bronchial smooth muscle to produce the observed vasoconstriction, bronchoconstriction and smooth muscle hypertrophy. prince of wales children's hospital university of new south wales, randwick, n.s.w. australia. introduction an increasing mortality in asthmatic children has been reported. the increased severity of asthmatic illness leads to an increased demand for icu admission, and a corresponding increased need for mechanical ventilation. geographic end environmental factors are thought to be partly responsible for differences in disease sevedty throughout the wodd. for this reason, epidemiological studies from diverse areas are important, risk factors for icu admission, and for the institution of mechanical ventilation should be identified, to optimise icu admission criteria and to avoid unnecessary delays in admitting at-risk patients. aim to document the clinical characteristics of ventilated and non-ventilated asthmatic patients admitted to icu. methods this is a retrospective study of all paediatric asthma icu admissions from january 1990 to december 1995. results there were 65 patients admitted to the icu for acute severe asthma in the study period. the male:female ratio was 33:32, the mean age 76.1 • 57.3 months, the mean prism 8.5 4-11.1%, and the mean duration of admission 135 4. 129 hours. there was no seasonal variation in admissions. only 40% (26/65) patients required mechanical ventilation. in 22% of all patients this was the first presentation with asthma. there were some significant differences between ventilated and non-ventilated patients (see table) . there was a significantly higher incidence of concomitant and nosocomial pneumonias in the ventilated patients (84.0% vs 21.1%) as well as segmental lung collapse (68.0% vs 26.3%). there were no deaths. discussion the need of mechanical ventilation significantly increases the morbidity of and duration of icu stay of asthmatic patients. younger asthmatic paediatdc patients have a significantly higher risk of ventilation. the need for ventilation is predicted principally from a worsening pco2 and respiratory acidaemia, which is often independently interpreted by the clinician as respira4ory exhaustion. this study has shown that icu admission is important in the management of young paediatdc patients with acute severe asthma and respiratgry fa!!ure. intravenous salbutamoi in the emergency, department management of severe asthma in children. g.j.browne,a. perma,x. phung,m.soo westmead hospital, sydney, australia. it is postulate that if an initial intravenous loading dose of salbutamol is given in severe asthma, a more rapid clinical response will occur, reducing requirements for continued high doses of nebulised salbutamoi with fewer side effects. this double blinded study was conducted in the emergency department of westmead hospital a university hospital in sydney, australia. all children with severe asthma had initial nebuliser therapy (5rag of salbutamol with 4ml of saline). if asthma remained severe 20 minutes later, they were given a dose of intravenous hydrocortisone (5mg/kg) and either normal saline or salbutamol 15microgm/kg intravenously. frequent nebulised salbutamoi therapy continued during the initial first hour if clinically indicated. continuous respiratory and haemodynamic monitoring occurred in the first 2 hours. serum potassium and glucose determinations were made at study commencement and 1 hour after intravenous therapy. salbutamol determination was made at study commencement. children remained clinically monitored for the next 22 hours, with their ongoing treatment determined by clinical response. 29 children with severe asthma 12 months to 12 years of age were studied, with 14 given intravenous salbutamol and 15 given intravenous saline. the intravenous satbutamol group (ivsg) showed rapid reduction in asthma severity scale in the first 2 hours, with reduced need for high frequency nebuliser therapy ( _<2 hourly), occurring 8.78 hours.earlier. no clinically significant side-effects were found in either group, although, tremor more frequent in the [vsg. biochemistry and salbutamol concentrations were similar in both groups. the use of intravenous salbutamol (i5 microgm/kg) in the management of severe childhood asthma is a safe and effective therapy with no significant side-effects and the potential to abort severe asthma attacks in the emergency department. intravenous terbutaline in picu piva j., amantra s, rosso a., zambonato s, giugno k, maia t. introduction: the admission to a picu of children with respiratory failure secondary to an acute obstructive lower airway disease is a common event, especially during winter seasons. these diseases have several causes, but most of them (especially asthma and chronic airway disease) have a good response to the administration of b2-adrenergic drugs. objective: to find the dosis of intravenous terbutaline that is safe, efficient and with minimal adverse effects when used in children admitted to a picu with acute obstructive lower airway disease and respiratory failure. material and methods: we study the records of all children that were admitted to our picu during the winter of 1995. only the patients that had respiratory failure and acute lower airway disease and who needed the use of iv terbutaline were selected. the records were divided in two groups: less than 12 months and more than a year old these two groups were compared in the following aspects: the minimal and maximal dosis, and the length of time of use of iv terbutaline, frequency of tachycardia, hypokalemia, and mechanical ventilation. to establish any difference in the two groups we use the t exact test of fisher and x2, with p< 0.05, results: during the period of study were admitted 367 patients to the picu, and 38 (10,3%) of them used of iv terbutaline. the mean age was 14.2 +12.2 month, used iv terbutaline during 7.24 +3.75 days (0.5 to 17 days), the initial rate was 0.55 +0.26p~g/kg/min, and the means of therapeutic dosis was 2.48 +l.181~g/kg/min (ranged from 0.5 to 4.4). twelve (31.5%) patients had tachycardia art obstacle to the increases in the rate of use of iv terbutaline during any time. mechanical ventilation was necessary in 22 patients (57.8%) and 11 (28.9%) patients died. the children under 1 year of age used initial dosis of iv terbutaline lower than the children up of 1 year old (0.45 p.g/ kghnin x 0.57 ~tg &g/rain, p<0.001), but without difference in the length of use, the maximal dosis, the rate of mechanical ventilation and tachycardia. the frequency of hypokalemia was most common in the group of children under year of age. acute respiratory failure during status asthmaticus may require mechanical ventilation. current therapy includes paralysis, pressure control ventilation (pcv) and permissive hypercapnia to limit pulmonary barotranma and its hemodynamic consequences. asthmatic children exert a significant amount of respiratory effort during exhalation. with paralysis, this expiratory effort is lost. unloading the inspiratory work of breathing while maintaining the patient's expiratory eftbrt using pressure support ventilation (psv), may be beneficial. methods: children receiving pcv (peak inspiratory pressure (pip) = 4 kpa. rate 10 breaths/min) and pco2 > 8 kpa were switched to psv. children were initially ventilated with psv 3.7 kpa and peep = 0.3 kpa (servo 900c). all children received beta agonist therapy, ipratropium and anesthesia with ketamine or inhalational anesthesia, and were breathing spontaneously. respiratory parameters and blood gases are shown be~bre psv, within 30 minutes (start) and when the ph had normalized (during). data are presented as median and range, * p < 0.03 compared to before psv. results: children with hypercarbia during pcv responded to psv, normalizing pcos and ph within 6 hours. the mean respiratory rate decreased from a median of 45 (31-46) to 35 (22-35) while the pip was decreased to 3.2 (2.5-4.0) kpa within 6 hours. the i:e ratio also significantly decreased. conclusion: psv permitted patients to active/y exhale while unloading the inspiratory work of breathing. perhaps this strategy shifts the patient's respiratory effort from inspiration to exhalation, thus permitting the child to meet the excess work of breathing caused by bronchoconstriction. maged z. youssef, peter silver, laura nimkoff, and mayer sagv. division of pediatric critical care medicine, schneider children's hospital, new hyde park, ny 11040. introduction: mechanical vemiladon of patients with severe bronchospasm can be difficult, due to poor chest compliance and increased airway resistance. ketarmne is a cormnonly used anesthetic agent that has been shown to have bronchodilator properties. the purpose of this study was to determine ifa continuous infusion of ketamine had an effect on the oxygenation and chest compliance of children with severe lironchospasm who were mechanically ventilated. methods: a retrospective chart review was conducted of pediatric patients in severe bronchospasm who were mechanically ventilated in our picu and treated with a continuous ketamine infusion. all patients were receiving aggressive bronchodilator therapy and adequate sedation prior to keramine. patients were excluded if any new bronchodilator or sedative agents were started within 24 hours of initiation of ketamine treatment. all patients were simultaneously treated with benzodiazepines. for each patient, the pao2/fio ~ ratio and dynamic compliance [tidal volume/(peak imp. pressure -peep)] was determined immediately prior to ketamine, and at 1, 8, and 24 hours post-ketsmine initiation. data are presented as mean ± s.d., and were a~yzed using one way anova and the multiple comparison method of bonferroni. patients (age 6.0 ± 5.7 yrs.) received * p<0.05 ketamine for severe bronchospastu during mechanical ventilation in our picu. both . .xto-* * the pao2/fio2 ratio and dynamic . . -.... . compliance increased significantly following initiation of the ketamine 200infusion (see figure) . the mean ketamine dose was 32 ± 10 mcg/kg/min, and the -, mean infusion duration was 40 ± 31 too-[/ hours. one patient required glycopyrrotate 6 ~' to control excessive airway secretions, and " one patient required an additional dose of o--j i ~-~4 ~/me diazepam to control hallucinations after i 8 cessation of ketamine. all patients were t~n~,mr~ *~am~ successfully weaned off mechanical ~l~s ~,~s~on ventilation and discharged from the picu. conclusion: continuous ketamine infusion to mechanically ventilated pediatric patients with refractory broncliospasm results in a significant improvement in oxygenation and dynamic compliance of the chest. reports of adults with status nsthraaticus document significant morbidity and mortality, whereas studies in children have had more varied results. different centers report mechanical ventilation (mv) in 10 to 33% of admissions, occurrence of pneumothoraces or paeutuomediastinums in 2 to 11%, and mortality in up to 7% of patients ~'t3. we retrospectively reviewed 113 status asthmaticus admissions to the pediatric intensive care unit (picu) between january 1993 and december 1995. seventy-five of these patients were admitted fr~an the emergency department of chla (er admit). the mean length of stay in the picu was 2.1 days and the mean length of stay in the hospital was 4.6 days. based on 95 patients who had arterial blood analyses, 36 patients had hyperoapnia (pco2 > 45). all patients received oxygen, inhaled albuterol (alb), and cortieosteroid therapy. ninety-five percent of patients also received methylxanthine (mx) therapy. of the 113 admissions, 12 patients (11%) required mv. only 4 of these patients were admitted through our emergency department, whereas the remaining 8 patients were intuhated at outside facilities. twenty-three cases required intr:wenous beta-agonist therapy, either isoproterenol osop) or terbutaline (terb). h~ff of the ea.~es re~%wed were complicated with hypokalemia (k+< 3.5). c,', ,~lications ofpoeumothoraces or pneumomediastinums were seen in 10% of ,'r:u~ported patients, but in only 4% of er admit patients. only 2% of these were in mechanic.all, )atients. there were no deaths in the review. respiratory mechanics measurements 'are useful in mechanically ventilated children to optimize ventilator settings. nevertheless, the transducers used to measure flow (f) and pressure (p) remain expensive. objective. to evaluate the performances of piezoelectric p transducers (350 us dollar) in measuring f and p. methods. we used a previously described monitoring system measuring respiratory parameters [ 1] . in this study f was obtained by a differential piezoelectric p transducer (_+ 12.7 cmi-i20, honeywell) whose sensitivity has been reduced to +_ 2 cmh20 by an electronic amplification equipment and p by a piezoelectric p transducer (_+ 7().3 cmhzo, honeywell) connected to a grid pneumotachymeter &nt) ffleisch 0 or 1 ). volume (v) (5 to 400 ml) obtained by numeric integration off (0.125 to 10 l/rnin ) and p (2 to 70 cmh20) were respectively delivered through a calibrated seringe and an electronical manometer (pic 400 premier) and calculated by the computer. bland and altman analysis was used for assessment of results bias. coefficient of repeatability (cr) was estimated by the standard deviation of repeated measurements of the parameters as calculated in a oneway analysis of variance. results. mean difference (mdi 0 between injected v (5 to 50 ml) and measured v using pnt 0 was 0.15 ml, sd = 0.13 ml. difference and mean v were not correlated. sd of repeated v measurements were not correlated to v. cr was 0.4 ml. mdif between injected v (25 to 400 ml) and measured v using pnt 1 was 3 lrd, sd = 6 ml sd of repeated v measurements were not correlated to mean v. cr was 6 ml. mdif between injected p and measured p was 0.3 cmi-i20, sd 0.4 cm h20 sd of repeated p measurements were not correlated to mean p. cr was 0.3 cmh20. conclusion. inexpensive piezoelectrical transducers can be used to measure f and p and evaluate respiratory mechanics in ventilated children. previous studies have already shown the problem of the reproducibility of pft in preterm ventilated babies. were studied 10 preterm ventilated babies {mean weight 1128 gr) in the first week of life in clinically stable condition, measuring flow, airway pressure and esophageal pressure simultaneously. each baby was studied twice with an interval of one hour and each study was done increasing the rate till 60 to inhibit spontaneous breaths. none sedative has been used. only mechanical breaths were analyzed. compliance and resistence were calculated with a computer system using the linear regression method. we expressed quantitatively the intrapatient variability as the percentage of variation of tidal volume, compliance and resistence between the two studies in each baby. then intraclass correlation coefficient test (icc) was applied to confirm qualitatively our results (total agreement =1, good reproducibjtity > 0.75). we h~£ed, an a6eept~ble ~efiabirl¢, ~-~r;= '~ . during mechanical ventilation, an air leak (al) and plateau phase duration (pl) may influence dynamic and static compliance (cdy and cst, respectively). this study evaluated the effect of al and pl on two methods of measuring c.dy and est. methods. 13 intubated, ventilated patients in a pediatric intensive care unit were evaluated after obtaining informed consent. patients were intuhated with a cuffed endotracheal tube and ventilated with a serve 900(2 ventilator. cdy and cst were determined using the serve ands~rmedics 2600. objective: evaluate the repercussion in respiratory mechanics and arterial blood gases and the impact of the ventilator adjustments on the auto-peep magnitude. material and methods: the measurement of the auto-peep was performed using an eletronic-pneumatic controlled device with a oclasion valve installed between endotracheal canutla and the ventilator circuit. the d~'ice was connected to a solenoid to detecte the end of inspiratuo phase and thus, the activation of the oclusion valve. the signs of pressure and flow were monitorized using a diferential transducer and it was processed using a pc computer and tmeumoview® software. the stud3 were divided in 2 phases: phase a. where the ventilator adjustments was performed using the routine of the unit and phase b, where the targets of mechanical ventilation were to minimize the auto-peep. static compliance (crs) was ineasured by the single-breath occlusion technique, using a mean of ten occlusions for analysis. passive respiratory resistance measurements and the tidal breathing flow-volume loops were also obtained., while the ventilatory settings were siguificantly reduced soon atier ecmo was started. before ecmo crs measured in all patienls was 0.23_+t).03 ml/cmh20/kg (mean_+sem). for each patient the ecmo course was divided into four periods, proportional to the duration of the treatment, and the best ~alue of crs in each period was chosen for analysis. as shown on the figure. crs significantly improved (*p<0,05) from the second half of the ecmo course in the group of patient that finally were successfidly weaned from ecmo. no change ill compliance was measured in the group of patients who failed to respond to the extracorporeal hmg support our data suggest that compliance measurements during ecmo can be useful togelher with overall clinical evaluation to predict both outcome and duration of cxtracorporeai support in the neonatal and pediatric population. objectives: brain temperature determines the amount of neuronal damage caused by hypoxic insults. thus measuring brain temperature at standardised conditions is in request. we investigated whether brain temperature of neonates varies with head insulation environmental temperature, body activity and time course. patients and methods: we investigated non-invasive brain temperature analogues in 19 healthy prematures tess than two weeks of age in an incubator (gestational age 31.5 + 2.1 wks; x + sd, weight 1653 + 370 g). we measured nasopharyngeal temperature (tnasoph) by a thermistor placed in the nasopharynx via a feeding tube, zero-heatflux temperature (zht) at the temple by a thermistor and healflux transducer, insulated by two pads, as well as rectal and incubator temperatures. patient activity was documented by video taping. measurements were performed during periods of increased insulation 1) by turning the head with its measuring site on to the mattress ( (5) 3 (5) -2 (6) 4 (5) 0 (6) 4 (3) 2.5 2 4(5) 25(10) 20{12) 8(5) 5(10) -2(7) 5 6 38 (22) 112(34)i70 (48)51(27) 20 (18) 5(15) 7.5 3 38 (19) 125(21) t85(29) 120(30) 70(30) 30(20) 10 4 53 (30) 133(28) 182(33) 157(24) 154(34) 110(45) web 2170 (lmg/kg) 5 at 30 rain 3 3 (5) -4 (6) 5 (6) 4 (3) 5 (4) -3 (6) the vehicle had no effect. paf caused dose dependent rise in ao and pa pressure and reduction in flow to lpa (up to 80% like the vascular endothelium, the endocardial endothelium (ee) has a significant impact on adjacent myocytes, and may critically alter myocardial function.~ we have previously shown that ee cells are capable of sensing and responding to hypoxia by the release of prostacyclin (pgl). 2 potassium channels in other cell types have been reported to be oxygen sensitive. to determine whether potassium channels modulate the ee hypoxic response, we investigated the effects of three potassium channel inhibitors on hypoxia-induced pg] 2 release from ee cells. methods: ovine endothelial cells were harvested and passaged onto 30 ,~ microcarriers. cells were constantly perfused with normoxic and hypoxic kreb's solution, and with three potassium channel blockers: glibenclamide (gb, 3 #g/ml), tetraethyl-antmonium (tea, 10 ram) and 4 aminopyridine (4ap, i0 mm), perfusate was assayed for prostacyclin (ria). data were compared by analysis of variance. * p<.05 compared to 3normoxic control; # p< .05 compared to hypoxic control. adrenaline is extensively used for resuscitation in neonates with rds. however, effects of adrenaline on systemic, pulmonary and cerebral hemodynamics have not been defined in newborns with rds. thirteen anesthetized, and ventilated newborn piglets were subjected to repeated saline lung-lavage series while mean systemic arterial pressure (abp), mean pulmonary arteriat pressure (pap), mean left atrial pressure (lap) and mean central venous pressure (cvp), cardiac output and blood flow in the internal carotid artery (ica) were measured. systemic vascular resistance (s~), pulmonary vascular resistance (pvr) and cardiac index (ci) were calculated. sixty minutes after luug-lavage, the adrenaline group (a) (n=6) received adrenaline as a continuous infusion of 1.2 lag/kg/mi, while the control group (c) (n=7) received saline. none of the varlables were changed by saline. however, significant increases in abp (p<0.0001), pap (p<0.0001), ci (p<0.001) and svr (p<0.01) were observed after administration of adrenaline, whiie pvr and ica were not modified. mean±sd for abp/pap (p/a), fvr/svr (p/s) and ci (ml/mirdkg) were: ratios of pap/abp and pvpjsvr significantly increased following infusion of adrenaline. these data suggest: 1) the cerebral perfusion is preserved during the infusion of adrenaline; 2) effect of the adrenaline infusion on the systemic circulation is more pronounced than its effect on the pulmonary circulation in newborn piglets with surfactant deficiency. s demirak~a, ch knothe, kj hagel, j bauer department of pediatrics, justus-liebig-university giessen, frg inhaled no is a short acting selective pulmonary vasodilator. we studied the effects of 80 ppm no and 100% oxygen during heart catheterization in 16 children (age 1 -6 years, median 9 years) with heart defects and elevated pulmonary vascular resistance index (pvri) in order to asses the value of no as a tool of decision making for corrective cardiac surgery. patients were eligible for testing when they were more than one year old and had a pathologically elevated pvri in a previous heart catheterization. intubation, 'anesthesia and muscle paralysis were performed in all patients during testing of pulmonary reagibility. calculations of pulmonary vascular resistance and flow were based on the fick method. response to no was assumed when pvri declined more than 30%, 9 of the 16 patients were responders to no. effects of no and oxygen on pvri, mean pulmonary arterial pressure (mpap) and pulmonary vascular flow (qp) in all responders are described in the table below. cardiac surgery was offered to all responders, and 5 of them were successfully operated. surgery is planned in another 3 patients and parental consent for surgery was not given in one patient. in ebstein disease, during the first days of life, the ability of right ventricle to propel blood to the pulmonary artery is impaired due to high pulmonary vascular resistances. the flow is mainly directed to left atrium through tricuspid insufficiency, right atrium and foramen ovale. to decrease pulmonary resistances and increase pulmonary blood flow, high frequency oscillations, mechanical ventilation, nitric oxide and prostaglandin are required. after few days, a forward circulation is normally established. we cared two newborns with ebstein disease where this approach was hindered by a large pulmonary valve insufficiency. both of them were diagnosed in utero, showing a large tricuspid insufficiency with a non opened pulmonary valve and a ductal left to right shunt. one fetus was hydropic. at birth, blood stream from the ductus arteriosus was directed to the right ventricle through the pulmonary valve insufficiency then to right atrium, left atrium and ventricle, aorta and ductus arteriosus. a low pulmonary blood flow was demonstrated by low mean velocities (10cm/sec). a high reverse flow was seen in descending aorta with a negative flow in the renal artery. both of these newborns were oliguric because of ductus arteriosus steal. pulmonary blood flow doppler evaluation allowed different strategies of ventilation, switching between hfo and conventional ventilation, modulation of pge1 doses, inhaled pulmonary vasodilators (nitric oxide) and surfactant. the hydropic baby died, the other survived after 3 weeks of intensive care complicated by supraventricular arythmia (wpw). in conclusion, during neonatal period, in ebstein disease, a large pulmonary insufficiency leads to a vicious circle where lungs are excluded, inducing severe asphyxia and high pulmonary resistances. the blood is backward propeled from the aorta through the ductus arteriosus to the right ventricle and atria, then left cavities to aorta. arec must be considered when pulmonary blood flow does not increase despite optimal therapy. guti~rrez-larraya f*, mandoza a*, velasco jm*, zavaneua (3**, gatindo a ~, s&nchez-andrede r, s&nchez jl***, mellon a***, mar f***. pediatric cardiology*, pediatric cardiac surgery**, pediatric intensive care unit***. hospital 12 de octubre. madrid. background: transesophageal pacing (tp) is effective and sate both for diagnosis and treatment of pediatric arrhythmias. material and methods. eleven consecutive patients are included. a tri or quaddpolar 6 or 7f temporal transvenous catheter with an interpolar distance of 13 to 22 mm was advanced through the nares and positioned to the point with the largest amplitude of atrial deflection, surface ecg and a bi or monopolar electregram were recorded simultaneously, selecting filters when needed (5 to 100 mhz). pacing was performed with a programmable stimulator (medtronic 5328) beginning with 2 ms and increasing ma to 10 and then increasing up to 9.9 ms. narula method was selected to diagnose sinusal node disfunction (snd) and overdrive pacing to treat tachyarrhythmias. results. tp was useful in all the 11 patients and no complications were observed: in 3 patients a snd was diagnosed (one needing a definitive pacemaker), in two patients with atrial ratter (ripe 1) sinus rhythm was recovered, in one patient with a postoperative junctional ectopic tachycadia we were able to get atrial synchrony with marked bemodinamic improvement, and 5 patients with paroxysmal supraventricular tachycardia sinus rhythm was easily and quickly restored (2 of them recquirad repited episodes of tp until pharmacelogycal levels of antiarrhythmic drugs were raised). mean age and weight were 31 months and 12.7 kg (one patient had 2.1 kg). there was a close relation between height and depht insertion (r= 0.98). mean stimulation parameters were 9,1 ms and 13.5 ma. discussion. in experiencied hands tp is an effective and safe way to treat and diagnose cardiac arrhythmias even in newborns. it should be tried before endovenous pacing is stablished and it is faster than pharmacologycal treatment. bailing g., eicken a., sebening w., vogt m., schumacher g., bl~hlmeyer k.; kinderkardiologie, deutsches herzzentrum m0nchen, germany to assess the outcome of balloon valvuloplasty in infants with cardiac failure caused by critical aortic stenosis a retrospective study was performed. between 1986 and 1995 33 neonates, aged 1 -28 days (median 9 d), weight 2.t -4,1 kg (median 3,3 kg) with critical valvar aortic stenosis were dilated by balloon (aovp) as the first line treatment. 21 patients received prostaglandin el, 18 needed inotropic drugs and 16 mechanical ventilation. associated cardiac lesions : persistent ductus arteriosus (pda) in 27 patients (restrictive pda in 8 cases), a mitral regurgitation (mivr) in 27 cases (15 severe and 12 moderate or mild mivr), angiographic findings of endocardial fibroelastosis (efe) in 12 patients, mitral stenosis (mivs) in 8, coarctation of the aorta (coa) in 2, and finally a small musculary ventricular septum defect (vsd) in i patient. vascular approach for ballooning : a. axitfaris in 20 cases (61%) a. femoralis in t 0 (30%) and v. femoralis in 3 cases (9%). the median ratio between inflated balloon and aortic valve diameter was 0,99. dilatation was achieved in all 33 cases. the peak systolic gradient across the aortic valve (pre aovp) ranged from 0 to 137 mmhg (median 50 mmhg) and was reduced to 0 to 55 mmhg (median 15; gradient reduction is significant (p < 0,01)). aortic regurgitation (aovr) was absent or mild in 30, moderate in 2 and severe in 1 patient after aovp. 23 children survived (actual suwival rate: 70%; early mortalffy: n = 3; late mortality: n = 7). mid term follow up (0-8,8 years; mean 2,7 years) showed an increase of the systolic peak doppler gradient across the aortic valve (median 41 mmhg) but no increase of aovr. 10 re-interventions (re-aovp: n = 3, commissurotomy: n = 2, mitral valve replacement n = 2, resection of subaortic stenosis: n = 1, resection of coarctation: n = 2,vsd-closura: n = 1 ) were performed in 6 patients. rv contractility and pulmonary vascular mechanics(pvm) in immature animal models are poorly underslood. we developed an acute rv injury model to measure rv contractility and pvm in response to commonly used cateehalamines. ten anesthetized piglets (9-12kg) were instrumented with micromanometers in the lv, rv, pa, and la. a pulmonary artery flow probe was placed to measure cardiac output(qpa). ultrasonic dimension crystals were sutured to the myocardium and dynamic chamber volumes estimated using shell subtraction methodology. rv injury was induced with 3-7 cryoprobe injuries at -50 to -70°c for 3-4 minmes each. da at 10mg/kg/min, db at 10mg/kg/min, and ep at 0.1 mg/kg/min were infused in random order. rv contractility was evaluated by calculating a load independent measure of contractility, the preload recmitable stroke work(prsw), during vena caval occlusions. to describe pvm, input resistances), characteristic impedance(z0), total pewer(tp), and efficieacy 03f=qimo"p) were measured. measurements were made pre-and post-injury, during infusions, and between infusions. clyoablation decreased prsw (22.8_+7.8 to 13.8+4.1, p<0.001). at the end of the experiment, prsw remained depressed to this level indicating stability of the model. one factor contributing to organ dysfunction for infants undergoing repair of congenital heart defects (chd) is their "inflammatory response" to cardiopulmonary bypass (cpb). this response is characterized by an increase in cytokine release, complement activation and endothelial injury. modified ultrafiltration (muf) is a method for removing tissue water and inflammatory mediators by rapid ultrafiltration followin~ cpb, muf may acutely improve post-operative end organ function. in this study, we evaluated the effects of muf on the pulmonary and cerebral function of infants undergoing cpb for repair of chd. we prosnecrivety randomized 30 infants (.~ 5 mos) to either muf (n=16) or no muf (n=14)(control) following correction for chd. the study intervals were 1) before cpb, 2) immediately after cpb, and 3) 20minutes after cpb. pulmonary function was evaluated by measuring dynamic compliance (cdyn) and airway resistance (raw). for 13 pts (mue=6 pts; control=7 pts) exposed to a period of deep hypothermie circulatory arrest (dhca), cerebral metabolism (cmro2) was calculated at each interval using the xe 133 clearance technique for cerebral blood flow measurements and arterial and jugular bulb saturation measurements to calculate cmro2. a reduction in cmro2 has been consistently demonstrated after dhca. the effects of muf on cdyn and on cmro2 are shown below: p<0.05 vs pre-cpb; # p<0.05 vs post-cpb • p--o.06 vs. post-cpb this study demonstrates that immediately following exposure to cpb, muf will improve pulmonary compliance. raw was not different between groups. there was no significant difference in hours of post-op ventilation for either group. in those pts exposed to dhca a trend towards better cerebral metabolic recovery compared to control was demonstrated. this is the first technique applied to infants undergoing dhca where cmro2 after cpb was greater than precpb measm~s. although this may be beneficial to postoperative hemodynamics, ventilatory management and long-term neurologic recovery, more patients and longer follow up will be necessary to verify such an effect. the effects of conventional mechanical ventilation (cmv) on left ventricular (lv). diastolic filling in neonates are not well established. one approach to improve lv filling is the use of cmv to provide a phasic increase in airway pressure {thoracic augmentation). this phasic increase in airway pressure may result in an increase in lv filling similar to that which occurs with cpr. thoracic augmentation has not been evaluated in neonates with ventricular dysfunction who frequently demonstrate increased heart rates. attempts to maintain low peak airway pressures during cmv may result in a prolonged inspiratory time that occurs over multiple cardiac cycles. this may alter lv filling in the later cardiac cycles. to determine the effects of inspiratory time on lv diastolic filling, 10 infants were examined with doppler echocardiography less than 24 hrs after surgery for the arterial switch procedtme. pulsed doppler recordings of the millal valve (mv) were obtained with the inspiratory time adjusted to occur over 3 cardiac cycles (21 sec.). a pressure transducer was placed in line with the ventilator, and the respiratory cycle was recorded superimposed on the doppler tracing to provide accurate determination of inspiration and expiration. doppler recordings were obtained from the apical 4-chamber view and the following measurements were made: peak e and peak a velocities, eia ratio, and deceleration time. compared to the expiratory phase of cmv, the initial beat during the iuspiratory phase of cmv resulted in an increase in mv peak e (.53 +-.06 vs .65 -+ .08 m/s, p<0.05) and peak a (.47 + .07 vs .63 -+ .09 m/s, p<0.05) velocities with no change in mv deceleration times (p<.01). compared to the initial beat during tile inspiratory phase, the third beat during the inspiratory phase resulted in decreased peak e (.65 + .08 vs .40 + .05 m/s, p<0.05) and peak a (.63 + .09 vs .40 + .05 m/s, p<0.05) velocities with no difference in deceleration times. thus, cmv augments lv filling during the initial phase of inspiration. however, as the increase in airway pressure is distributed over multiple cardiac cycles, lv filling falls below baseline levels. these observations indicate that while thoracic augmentation may be beneficial, to optimize lv filling the inspiratory time of cmv must be < 3 cardiac cycles. energy expenditure in pediatric orthotopic liver tranaplantat~on, to determine the actual calorie requirements of critically ill children and evniuate the correlations between measured, stress-p~lictod and repleted energy exponditttm and the severity of illness. des/gn: a prospective, dinlcal study. se~ng: tertiary care pediatric icu in a university hospital. patients: ten patients aged 6 to 210 months with disorders prompting picu admission, including sepsis, respiratory failure, solid organ transplantation, and cardiovascular surgery. inta~entions: all patients were studied within 24 hrs of major surgery or transplantation, or following acute illness. all patienls were severely stressed clinically and all but two were intubated by cuffed tubes, in three of them, still in a stress state, the study repeated on the third day of the disease, energy expenditure mensurements (mee), as well as illness seventy scoring systems, mtfltisystern organ failure scores and various anthropemetric and clinical indices of nutritional status, the stress-predicted energy expenditure (s-pee), the basal metabufie rote (pbmr), the repleted energy (re) and the recommended dietary allowances (rda) were measured or calculated in each patient. multiple regression analysis was used to analyze the data. measurements and main results: although the mean mee was significantly lower than the mean s-pee (37.6+11 kcal/kg/day vs. 50.35:16 kcal/kg/day, p<.002), it did not differ significantly from the pbmr (mean difference -2.62 kcal/kg/day, range -10.07 to +9.06 kcal/kg/day). the s-pee/mee ratio ranged from 1.04 to 2.07, while the re/rda ratio (21.25:4 kcal/kg/day)/(75.85:7 kcal/kg/dny) ranged from only .1 to .5. the prism/tiss ratio was not correlated better with mee than the diagnostic category (r~=.36 vs..38, respectively). the re was positively correlated withthe mee (rz=.65, i)=.07) while negative oarrelatian has been found between mee and age, mid-arm circumference, triceps skinfotd and the use of vaseactive agents (r~.81, -88, -.67, p<.005 and -.71 resp~lively). concl.m~: if s-pee is used for caloric repletion in the stressed oritic~ly fll el~d, these patients will be substantially overfed by as much as 100%. although pbmr appears to approximate the mee by ±10%, other clinical and nutritional indices should also be ennsidered. objective: to deter .mine..t.he metabpli.c and.nutritional state of mechanically ventilated intants and children m relatmn wlm severity or msease. patients and methods: 37 mechanically ventilated infants and children, median age 7 months (range 3 days to 13years), were studied. severity of illness was assessed using prism, prism-ii~ and fiss-scores. oxygen consumption (vo2), energy expenditure (mee) and respiratory quotient (rq) were determmed by mdirect calorimetry. total urinary nitroger(tun) and creatinine excretion, levels of albumin and crp were aetermmed in 16 patients. in these patients daily caloric intake and substrate utilization were assessed. they were categorized in subgroups: a partial feeding (recent admission to p1cu); b complete feeding. results: mee of the total group (n=37) 0a) i=intake g/kg/day (% total intake); u=utilization g/kg/day (% total production). nitrogenba]ance was negative in all patients in group a (mean -227.7 --176:4 mffkg/day) and positive in all but one patient in group b (.mean 84.9±109.d n~g/..kg/day;p=0.001). no significant correlations were round between creatinine height index, crp, albumine, jun vs v u2/kg conclusions: the mean measured energy expenditure does not exceed predicted resting energy expenditure, but ~ere is a wide range. in a majority ot patients with complete feeding h.igh carbohydrate intake resulted, in high kq and lipogenesis. in patients witla partial teeding the highly negatwe nitrogen'balance suggests that in the early phase of diseasean higher protein intake should be provided. severity of illness scores ann oiocnemicm markers of physiologic stress correlatedpoorly with oxygen consumption. leite,hp; iglesias, s; faria, c; ikeda, a; albuquerque, mp; carvalho, wb pediatric icu -s~o paulo federal university -s~o paulo, brazil objectives: 1 ) to evaluate patterns of use and monitoring of nutritional support in critically ill children; 2) to evaluate an education program in nutrition support given throughout the resident physician training in the pediatric icu. patients and methods: records of 37 patients receiving nutritional support during 1993 were reviewed. aider this first phase, knowledge and understanding of the role of nutrition support was conveyed to the residents through didactic lectures. in a second phase thedata were reevaluated in 35 children who were given nutrition support in 1995. results: from a total of 425 days ofthempy, the single parenteral route was utilized in 80,5%, the digestive route (tube feeding or oral route) in 19,5%. of this time. a previous nutr~ional assessment was performed in 3 children; no patient had the nutr~on goals set. the nitrogen to nonprotein calories ratio ranged among 1:80 and 1:250. only 29,7% of the patients had their estimated caloric needs supplied and this goal was achieved only in those patients who were on enteral tube feeding. patients did not achieved their goals for vitamins. the supply ofoligonleme~s was adequate except the zinc. nutritional monitoring parameters including weight, serum albumin and serum triglycerides were performed in almost all the patients but without uniformity. the reevaluation ofthase parameters showed adequacy of protein and micronutrients supply; however deficiency in nutritional monitoring and infrequent enteral feeding were still detected. conclusion: there were lacks in the implementation of nutritional support, which were partially corrected in the 2rid phase of the study, although the training of residents may have contributed to give them cognitive skills, it didn't changed policies and procedures as desired. we recommend reinforcement of the education program concerning basic nutritional aspects, and the organization ofa multidisciplinary team in charge of coordinating the providing of nutritional support. plasme free fatty acids (ffa) are the meier energy source for mast tissues. during fasting ffa are released from the breakdown af triglycefides in edipose lissue (at). lipalysis, le. the rote of release o/ ffa, has been megsured in humans by means of stable isotope techniques using labeled pa or glyeerd as traces. no information is avoilob!e io dale on the ro of la. we infused albumin hound u13c-pa and u13c-la in 7 critically ill infants, receiving 20 kcel/kg/doy of iv glucose end na oral feeding (weight 3.6,i.,3 kg;, range 1.9-5.8; ego 57:64 days, range 1 149) and measured simultaneously the ra of pa and la from (he isotopic enrichment of plasma fea by gas chromatography-mass speclrome|ry ai 1:50, 2:00 and 2:10 hours from tile shod of the infusion. a subcutaneous gluted at biopsy was obtained far fatty acid (fa) composition. we intended to (1) in fie infants sbjdied atipa ~'os hi9her than attla (~p<o.o01)' and ihe ra of pa was higher then thai of la (~p=0.00 §). however the ratio el the re's to the respeclive fa in at was higher for la than far pa (*p=0.02). in conclusion the ra af la was higher then expected from ~he fa composition of at. we speculate [hat la is preferentially released during tipolysis and its contributuion to the energy metnbolism of the sick infonl could io !orger than what: is normai!y assumed from ihe fa composilion of at and of plasma fea. preoperative starvation can be prevented in infants undergoing non gi surgery by continuing feeds till as long as it is safe to do so. some gi disorders require withholding of feeds for a long period. for these and for infants already suffering from pem~ parenteral feeds are given preoperatively. peroperatively nutrition is maintained by ensuring adequate glucose supply in the infusion fluid and its smooth metabolism. proper placement of feeding tubes are also carried out peroperatively. postoperative early resumption of oral feeds is ensured by new techniques of anaesthesia and pain relief and methods to ensure early return of peristalsis. enteral feeds of pre digested substances and feeds through transanastomotic tubes and through gastrostomies and jejunostomies can prevent starvation in many situations. parenteral feeding is carried out only when enteral feeding is not possible, for example in necrotising enterocolitis~ gastroschisis, short gut syndrome, high enteric fistula and acute pancreatltls. the artedal ketone body ratio (akbr) is established as a valuable tool in the management of adult patients undergoing surgery for severe liver disease.the redox theory emphasises the central role of the liver in preserving homeostasis and the importance of the hepatic mitochonddal redox potential (nad+/nadh) in monitoring liver function and integrity.the akbr (plasma acetoacetate/3hydroxybutyrate) is a measure of hepatic mitochonddal redox status, reduced ratios associated with poorer outcome. the venous ketone body ratio (vkbr) is influenced by peripheral ketone utilisation and therefore thought unreliable in assessing hepatic redox its use has been dismissed as no correlation with the ratio in hepatic venous blood was found. inspite of this. vkbr is used in the diagnosis of lactic acidoses and respiratory chain defects. the objective of this study was to determine the relationship between the akbr and vkbr in a paediatnc intensive care population. 31 children admitted to the paediatdc intensive care unit, with indwelling arterial and central venous lines as part of their routine care, were recruited for the study. the median (range) age was 2.0 years (0-16.6 years) with prism score 10 (0-36), simultaneous akbr and vkbr were measured. suppression of ketogenesis was confirmed using a rapid 3-hydroxybutyrate strip test (ketofilm, genzyme diagnostics) pdor to sampling. the median vkbr 0.50 (range 0.13-1.46) was lower than median akbr 0.56 (range 0.11-1,33), there was good agreement between artedal and venous ratios, bland-altman analysis giving a 95% confidence interval for relative bias of -0.13 to -0.03. there was a significant correlation between vkbr and akbr r 2 0,8271, p,0.001 (intercept 0.02, slope 0,85) conclusions; values for akbr tend to be higher than vkbr, however there is a consistent relationship in critically ill children. in paediatdc patients in whom ketogenesis is suppressed, vkbr may be considered equivalent to akbr. this simple test may prove to be a useful adjunct in predicting mortality in cdt{cally ill children. to understand the present status of pediatric intensive care in china, we conducted a survey between october and december, 1993, involving 20 hospitals in 14 provinces and municipalities. the results showed that there were totally 41 icus for pediatric patients, including 19 pediatric icus (picu), 18 neonatal icus (nicu) and 4 pediatric surgical icus (sicu), with total of 403 beds. the physicians to bed and nurses to bed ratios were 1:1.5 and 1:0.91 respectively. the average number of equipment per bed was 0.47 ventilator, 0.34 multi-fnnction monitor and 0.47 infusion pump. very few of the icus had portable x-ray machine, biochemical and blood gas analyzers, and hemodialysis machines. the most frequently treated diseases/conditions were pneumonia, intracraniat infections, post-operation and sepsis in picus, and neonatal pneumonia, hypoxic ischemic encephalopathy and sclerema in nicus. pneumonia and respiratory failure accounted for 33.29% and 26.50% of all the diseases/conditions treated in the icus and the mean case fatality rate of respiratory failure was 24.50%. the results of the survey suggest that there is shortage of tcu beds and modern equipment, and treatment is often delayed due to excessively strict criteria for mechanical ventilation. a set of simple, and nationally acceptable criteria for evaluation of severity and cure of the diseases/conditions is urgently required. the medical reports of 286 children were reviewed and each admission was analysed in terms of age, sex, diagnosis, management within the hosp~al, length of hospital stay and type of poisoning. the youngest age was 23 days and the oldest was 16 years. hundred and twelve (39.5%) of the children were girls and 174 (60.5%) were boys. the most common (26.5%) reason of admission was intoxication among all of the cases and the greatest group (73.7%) of the poisoned children had ingested medication which was followed by another group of patients (9.2%) who had eaten mushrooms. the peak incidence of drug ingestion was salicylate intoxication (20%). forty drug poisoninigs were accidental while 16 was intentional. the majority (82.4%) of the cases that committed suicide was between 12 and t7 years old, and the main cause of suicide was being unsuccessful at school. we conclude that poisoning -especially salicylate intoxication -is still a major problem in turkey and we believe that emphasis on the need to stere all kinds of drugs in a secure place and re-examination of a chin resistant packaging should help to reduce childhood poisoning significantly which is a preventable condition. included were all patients who died in the hospital, except those treated in the neonatal icu (predominantly premature and sga newborns) and those who died in the emergency room. among a total of 7179 hospital admissions (excluding the neonatal icu and emergency room), 99 patients died (1.4%). of these 99 patients 73 (74%) died in the pediatric icu, 18 (18%) in the ward and 8 (8%) in the operating room. a chronic underlying disease was present in 66 (67%). withholding or withdrawal of therapy was implemented in 48 (48%) children, 27 (27%) died due to failed cpr, 20 (20%) were brain dead and 4 (4%) died following a dnr order. justification for therapeutic restrictions in the 52 patients of the dnr and w/w groups was imminent death in 32 (62%), lack of future relational potential in 12 (23%) and excessive health burden in 8 (15%). withdrawal or withholding of therapy was carried out by extubation in 46%, vasoactive drugs were stopped in 25 %, and mechanical ventilation was withdrawn in 21%, analgetics and sedatives were frequently used (in 73% and 79%, respectively). hence decisions concerning restrictions of treatment are common in pediatric practice, mostly due to imminent death. patients in which treatment was restricted were characterised by a longer hospital stay, a worse prognosis, a higher frequency of chronic underlying diseases and a lower acute mortality risk. despite restrictions of intensive therapy, most patients were allowed to die in the pediatric icu. background microalbuminuria (mca), a subclinical increase in urinary albumin, reflects glomerular and overall vascular permeability 1"2. an increase in urinary excretion of albumin occurs after burns and trauma. transcanillary albumin escape rate is also increased in response to elective surgery 3 and in critically ill adult patients 4. we investigated a possible relationship between urinary albumin levels and clinical instability, as measured by pediatric risk of mortality (prism) scores. method we studied 26 consecutive patients (median age of 13 months, range 2-100). patients whith nephropathies or any abnormality of urine analysis were excluded from the analysis. prism scores, mca (mg.1-1) (immunonepbelometric) and urinary creatinine (cu) (mmol.l -!) (jaffb) (48 hour collection sample) were determined within 48 hours from admission to the picu. the mca/cu ratio (mg.mmol.1 "l) was used to correct for urine output variability. diagnoses included respiratory failure (6), postoperative (5), neurologic (5), sepsis (4), trauma (3) and miscellaneous cases (3). pearson's correlation was performed to correlate data. results and conclusions. mean prism score and mca/cu were 16.9 ± 5.9 sd and 1004-39 sd, respectively. a significant correlation was found between mca/cu and prism scores (r=0.80, p<0.001). our observations show that, independently of the initial insult, the paediatric unstable patient may have increased capillary permeability, which is correlated with the degree of physiological derangement, as measured by prism scores. microalbuminuria can be rapidly determined since it is routinely used in the management of diabetic patients, it is inexpensive, simple to measure and blood-spearing. therefore, it might have a role in the clinical assessment of capillary permeability and transcapillary albumin escape worldwide the demand for paediatric intensive care services exceeds the supply. in developing countries sporadic access to such services alters expectation of care and can lead to children being either mechanically or handventilated in general paediatric wards. the outcome of children requiring ventilation in a major teaching hospital in india was reviewed.children were ventilated on an adult intensive care unit (aicu) if a bed was available, otherwise in the general paediatric wards. over a 4 year period 109 children were ventilated on aicu with 54 deaths. yearly mortality rates varied between 43-58%. over a 3 month period 37 patients were ventilated on the paediatric wards. of the 15 p~tients over 4 weeks of age 11 died (chi squared 0.t >p>0.05) reasons for the higher mortality rate on the paediatric ward likely include the higher patient:nurse ratio, and more limited resources. a predictor of mortality based on simple physiological observations without the need for expensive blood tests and including chronic health status would be a useful tool. the establishment of a paediatric intensive care unit is proposed to redress the balance of care. to assess the performance of the pediatric intensive care unit of hospital dona estef~nia by an international standard score, the authors did a prospective study of 1149 consecutive admissions to the unit during a period of 29 months. mean age was 50.63 _+ 54.07 months; mean lengh of stay was 3.16 + 5.59 days. the effectiveness and efficiency were determined by the admission prism. admission efficiency was defined by two criteria: a) mortality risk > 1% or b) the administration of at least one intensive care unit-dependent therapy. the cumulative observed mortality was 5.57% and the expected mortality was 5.97%, with a standardized mortality ratio (smr) = 0.933. the overall performance of the prism score-based predictive model was found to be good (goodness-of-fit test x2 [5] = 6.387;p=0.271). of 1149 patients admitted, combining the two criteria (icudependent therapy and mortality risk) an admission efficiency of 825 (71.8%) was found, equating to 3263 (89.94%) of 3628 1cu days. conclusion: in our study the assessment of the admission efficiency and of the effectiveness of the unit was possible by using the prism score of admission. there was no significant difference between mean values for otiss and ntiss)in level l patients (p=0.12 paired t-test).for level 2 and 3 patients mean value of ntiss was greater than otiss (p<0.0001). there was a significant correlation between levels using either ntiss or otiss (mean difference level 1 and 2, level 2 and 3, ( p < o.oool). conclusions: a new tiss has been developed and used in a picu. nurses were able to accurately score the interventions on their shift. the assignment of patients to intensive care levels correlates with tiss values allowing a quantitative measure of severity. objective : to compare the rate of cerebral palsy (cp) between monochorionic-twins, dichorionic-twins and singletons born at 25 to 32 weeks' gestation. design : two-year prospective cohort study. setting : geographically defined study (region of franche-comt~., france). main outcome measures : type of plasentation was obtained by anatomopathological, or macroscopic examination of placenta and comparison of 6 twins' blood-groups. neurological assessment was performed at two years of age (uncorrected for gestational age) by family doctor (pediatrician or physician), or neonatologist of the icu at tertiary center. sample : 167 of 17i survivors aged of two years (98% follow-up rate), born between 09/30/90 and 10/01192. triplets and chromosomic malformation were non included. results : thirteen (11%) of the 119 singletons had cp.vs 3/29 (10%) of dichorionic twins and 6/19 (32%) of monochorionic twins (p=0.04). four of the 19 monochorionic twins (21%), 2/29 dichorionic twins (10%) and 4/119 (3%) nngletons suffer from quadriplegia (p<0.01).in a multivariate approach, monochorionic twin placentation was the strongest risk-factor of cerebral palsy (or=9.7, ic 95% = 2a-39, p<6.01). others risk-factors of cp were : lack of father's profession (or 11, p<0 .03), maternal antecedent of abortion (or 3.2, 1-10, p<0.04), vaginal delivery (or 3.4, 1-11, p<0.03), hyaline membrane disease (or 3.4, 1.2-t0, ~0.02). discussion : this is the first population-based study to uplight the role of monochorial twin-placentation as a strong risk factor of cp for premature infants. cp is more severe in monochodonic twins than in other infants. mecanism of cerebrat deficiency is not clear since none of our infants with cp was survivor of an in utero cotwin's death, and none of these infants was exposed to twin to twin transfusion syndrome. were these monochorionic-twins affected by an undiagnosed neurological structural defect that could lead both to prematurity and handicap remains an open question, a vital role of the intensivist is to ensure that knowledge and practice are imparted to trainees in the icu so that patients receive optimal care. teaching effectiveness varies widely leaving gaps in knowledge and practice in the trainee. being an effective teacher should not be a "gift" of a privileged few. the icu provides a fertile ground for using a variety of methods for teaching, e.g. didactic, at the bedside, emergencies, and in the performance ofproeeaures. in this environment, much can be learned. we have embarked upon a program to facilitate this learning process. i) teaching needs to be recognized as the foundation of good clinical care, i.e., patient related, and in its ability to generate discussion and research investigation. 2) teaching structurally has many components including the speaker, audience, varying situations, and the message delivered. 3) establishment of a program using these components to enhance teaching abilities at all levels, a) evaluate base-line teaching skills initially, b) individualize interventions to improve teaching skills, e) demonstration of learned skills with re-evaluation. this process is analogous to the analysis of a clinical disorder in a patient which, once recognized, interventions are then instituted and then re-evaluated. 4) instill the desire to use these attained skills to teach and interest others to teach. teaching excellence should be recognized through awards, honors, and academic advancement. a major emphasis of this program is to provide participants with skills necessary to teach thought processes, decision-making skills (what to do, what to avoid) and implementing appropriate management during stressful emergency situations common to the picu. introduction: many" e-mail based discussion groups exist on the internet to provide medical professionals with a rapidly responsive medium for the international exchange of ideas relating to patient care. several such lists each serve more than a thousand professionals in more than 30 countries, each distributing a dozen or more messages each day to every subscriber. there is very little known about the time being spent by professionals interacting with these lists, and very little known about the impact of the discussions on patient care. we wished to test the hypothesis that these discussion groups provide infortuation which is being used to change the care of individual patients and the general approach to patient problems. methods: in early january 1996 a pilot electronic survey was sent to a small fraction (n=63) of the memberships of 2 e-mail discussion groups, picu@its.mew.edu, and nicu-net@u.washington.edu (the full memberships of both. groups (n=t439 for nicu-net, n=1045 for picu) will be surveyed in early february of 1996). participants were asked for demographic information, experience and skill level relating to e-mail, time spent with the discussion groups, perceived usefulness of different types of discussions, and the ways in which the discussions were used clinically. the pilot study was analyzed for construct validity by correlating an overall assessment question with a summary of the specific questions. scale reliability was measured by cronbach's alpha statistic. results: the pilot survey response rate was 30163 (48%). the majority of respondents were male physicians, with an average age of 39+_5 years, who had completed subspecialty training in intensive care, and were working at a university-affiliated hospital. most had been using e-malt for more than 6 months, and considered themselves moderately adept in that use. 63% felt that the list helped weekly to keep them informed about current issues and practices in their field(s), and 57% felt that, at least monthly, they used information from the list(s) that was not readily available in medical journals. overall, 75% agreed that the list improved their professional competency. when asked to compare the value of 6 months of membership on an e-mail discussion group with more traditional educational media, 34% compared it with attending a national conference, and 26% compared it to a journal subscription. cronbach's alpha was .76, construct validity testing yielded coeff=.50, p <.05. conclusior~: internet-based e-mail discussion groups for health care professionals can be an important part of a strategy for maintaining professional competency. despite the very low cost of this medium for most, the value is felt to be comparable to that of t~r more expensive forums for education. further study will include distribution of the full survey in early february of 1996. fronk shann, tony slater, gale pearson and the pim study group we have developed a new score for predicting the risk of mortality in children admitted to intensive care. the score is calculated from only seven variables collected at the time of admission to icu: mechanical ventilation (yes/no), booked admission after elective surgery (yes/no), the presence of any one of 14 specified underlying conditions, both pupils fixed to light (yes/no), the base excess, the pao 2 divided by the fio2, and the systolic blood pressure. most scores used to predict outcome in intensive care require the collection of a large number of variables (so many icus do not calculate them routinely), and they use the worst value of each variable in the first 24 hours in intensive care. this means they appear to be more accurate than they really are (about 40% of child deaths in icu occur in the first 24 hours -so they are diagnosing these deaths rather than predicting them), and they blurr the differences between traits (a child admitted to a good unit who recovers will have a low score; but the same child who is mismanaged in a bad unit will have a high score -the bad unit's high mortality rate will be incorrectly attributed to its having sicker patients). pim was developed in the picu at the royal children's hospital in melbourne, and has been tested in six other picus in australia and one in the uk. objectives: to study the characteristics of the muhiorgan dysfunction syndrome (mds) in children. methods: a retrospective study with all the children with mds diagnosed from january 1990 to june 1995 is presented. 173 children fulfilled the wilkinson criteria (i). in all of them the number of organs affected and the prims score were determined during the first 24 hours. several groups were performed according to the clinical diagnosis, the hospital of origin and the order of organs affected. results: the 173 subjects studied were an 8% of the pediatric intensive care unit admissions. 100 of them expired (58%). no differences in age, sex and weight were observed between the children dying and the survivals. the most common causes of mds were sepsis, both nosocomial (25%) and medingococcal (i4%) and acute respiratory failure. sixty-fivepercent of the patients were from the hospital wards and the remaining were directly admitted to the pigu from the emergency room. the systems affected were: respiratory (93%), cardiovascular (92%), hematologic (61%), central nervous system (52%), renal (43%) and (hepatic) liver (28%). the organs initially failing were: heart (39%), tung (28%) and central nervous system (18%). the children dying had a larger number of organs with failure than the survivors (3.89 v,s. 3.34, p<0.001).the prmis score was higher in the children expiring than in the survivors (22.4 v.s. 17, p <0.001). s.mmary: the mds is a common pathology in picu, with a high mortality, the mortality is higher in children with a larger number of organs affected and a higher prism score. sepsis is the most common etiulogy. methods : from june ist to july 15th 1995, all patients admitted to the pediatric icu were included. the score was measured at day 1 (d1) and day 3 (d3) and we used 10 variables. for each organ system, we defined 2 categories : dysfunction or failure, which we respectively confered 1 or 4 points. results : 56 patients were admitted : 22 newborns, 34 children. 23 were medical and 33 were surgical patients. 36 (64 %) patients had two or more organ failure at the admission, 12 (21,4 %) patients died, which 6 (50 %) in the first 48 hours. the mortality rate was the same for children with two or more organ faiiure at d1 and d3 : 6/36 (16,6 %) at d1, 4/22 (18,2 %) at d3. the mean score is different for children who survived or who died : 8,6 versus 17,9 at d1 ; 10,6 versus 18,2 at 133. when the score is > 15, the mortality rate is significant. conclusion : in this study, there is a good correlation between the score of severity and the mortality rate but we have few included patients. we need a prospective multicentric study to assess these results and we must compare this score to other scores of severity used in picu. back.qround: injury to the central nervous system is the cause of death in the majority of pediatric trauma victims, studies have identified a wide range of factors associated with poor outcome from brain injury. however, when single features are analyzed, they are not sufficiently accurate predictors. few studies have used a multivariate analysis of these factors and pediatric outcome, methods: clinical and radiographic features of 164 comatose children after traumatic brain injury were analyzed, clinical parameters, the initial cranial ct scan, and demographic characteristics were analyzed for an association with death or vegetative survival at 6 months. a tree diagram in which risk factors may differ within the study subpopulations was constructed using recursive partitioning. results: chitdren with a motor score _<2 had an 11-fold increased risk of poor outcome compared to those with motor scores >2. among patients with scores of _<2, those with abnormal pupillary reflexes experienced a 13-fold increased risk of death compared to those with normal pupillary reflexes. among patients with a motor score >2, an intracranial diagnosis code (no pathology, mild shift _<5 mm, swelling, shift >5 mm, surgical mass lesions, or non-operative mass lesions) was highly predicative of poor outcome at 6 months. children with ct findings other than normal or mild swelling had a 4-fold increased risk of poor outcome. of children with swelling, shift or mass lesions, the pupillary light reflex was associated with outcome. children with abnormal pupils had a 6-fold increased risk of poor outcome. discussion: a few clinical and radiographic features stratified comatose children into fairly distinct risk groups. information available early after traumatic brain injury in comatose children provides useful prognostic information on the likelihood of death or devastating injury. a retrospective study of 70 children with the diagnosis of epidural hematoma was made during 1990-1995 period. ages ranged between 7 days and 17 years (18% less than 1 year, 40% between 1 and 10 years, and 42% older than 10 years), 82% of them were admitted at the picu. 51% of the cases were due to falls, 35% to road traffic accident and 14% to other causes. on admission gcs was less than 8 in 19% of the cases and more than 14 in 53%. diagnosis was made during first 4 hours in 63% of patients and delayed more than 12 hours in 28% of them. neurologic impairment was present at admission in 33% of patients, and delayed in 30%. even so,27% remained without impairment. radiological findings at first ct were skull fracture (68%); epidural hematoma localization was: in the right side (63%), frontal area (24%), temporoparietal (66%) and occipital (t0%). associated lesions were: several (13%) or unilateral (51%) cerebral contusions, diffuse brain oedema (10%), unilateral hemispheric oedema (14%) and 38% showed shifted middle line. four patients died, half of them during the first 24 hours. 41 fully recovered (58.6%) and 25 have sequelae of different nature :7 were left with severe motor disability (10%); at the follow-up t3 have some degree of neurodisability. next datas keep correlation with death or neurosurgical impairment: only were significative multiple cerebral contusion (p=0.002) and brain oedema (p=0.05), gcs less than 8 at the admission (p--0.002), shock (p=0.003) and remaining cerebral contusion in control ct correlated with death or diasability at discharge. on the other hand, neither surgical drainage volume nor first or highest levels of icp (12 cases),nor pupillary abnormalities (10 cases) correlated with worse prognosis. conclusion: gcs equal or less than 8 an shock are main factors related to worse prognosis, also multiple cerebral contusions in ct and diffuse brain oedema. the results of a modified gcs were compared to outcome and intensive therapy in 78 children (mean age 8,5t4,7 years) with head and associated injuries (53,6% of all cases) of different causes (traffic accidents, falls). the gcs was regularly used inn the course of intensive therapy. according to our own and other experiences the gcs was divided in 3 stages: stage 1 (4-8 points), stage 2 (9-12 points) und stage 3 (13-19 points) palhuiugy wile sp, tdhlg c~'lcb1al blood ~0w. sabgcqucntl}. rhc slat,: rerltncd to t1011tl,91 iiltlils. the p0st,~pem~v~ b}i~g wij!!,:q1! ,:_a!~p!ica!j0n~:. 4 ri~;¢ ill the level of sensibflizatjou lo tile cerebn~ anhgrns up to 1t.4-o7 was flofcd iu 9 i,alicnts. there wa.~ al~ iuclt~a~e ill cerebral vdociij,. ~m d~;'ati0a il~ p¢fiphc~ai re~ista/isc of the large ce~'bral ve~ds. neur0h;~c ~:yn'.pt,m~at0!a~, (s::mno!en~', _r_uscu!~r l~:pot0ni& !ryper*'flema) was nbserwed tu lt~ese pal~enls o. cbruc~l ~0nnds. rile ple~c.ut abse~vafion~ suggesl ihal die ~tttdy at" ihe stale ~f hematocr~chcplm/itic bm~ic~ in ckil&en with 31110on emergensy is of abviou.~ !?ece~sib; in co~.te ctin g severe pa~0lo2~-i~mnediately f0u0wing ne ,:~per,'~fion. background: reconstruction of the heart by three-dimensional (3d) echocardiography provided new information on anatomy of complex congenital heart defects, we assessed the utility of 3d ultrasound in detecting morphological changes in cerebral anatomy in newborns before and after cardiac surgery. methods: transfontanel cross-sectional ultrasound, scans were obtained in standardized coronal and median sagittal planes. subsequently, rotational scanning was used to acquire the multiple sequential crosssections of the brain. for rotational scanning, a conventional 5 mhz transducer was rotated 180 degrees.scanning took less than one minute and required no sedation, data was stored in the image processing computer which allowed for off-line three dimensional reconstruction of different brain regions.twelve infants aged 3 -21 (median 7) days were assessed before and after cardiac surgery, results: cavity of lateral ventricle, choroid plexus and the periventricular brain parenchyma could be reconstructed in all. accurate estimation of size and volume of lateral ventricle, aqueduct, and other ultrasonographic visible pathological brain lesions could be performed. reconstruction of various brain areas was accomplished in 3-10 minutes. the localisation and extension of severe periventricular hemorrhage which was detected preoperatively in one infants was better visualized than in conventional ultrasonography. epicortical and subarachnoidal space could be reconstructed in all and allowed detection of hemorrhage in one case which was not detected by conventional ultrasound. conclusion: 3d reconstruction of different areas of the brain may provide additional quantitative information on size and volume of the internal ventricle and choroid plexus, and better understanding of the topographical aspects and the extension of intra-and periventricular hemorrhage than conventional cross-sectional ultrasound. introduction: intracranial cerebral blood has been estimated to be 70% venous, the invasive measurment of venous blood saturation in the jugular bulb provides quantitative information on cerebral oxygen supply and consumption. however, routine oxymetric measurement of blood saturation in the jugular bulb by insertion of a catheter line into the internal jugtdar vein is an invasive procedure which has limited use especially in infants and young children. thus the aim of this study was to investigate the correlation between the non-invasive spectroscopic measurement of rso2 and the oxymetric determination of the blood saturation in the jugular bulb in infants and children undergoing routine cardiac catheterization.. methods: during routine cardiac catheterization 30 infants and children (age 5 day-16 year, median 4,5 year) the rso2 was measured continuously using a two chanel cerebral oxymeter (invos 3100a). the sensor was placed in standardized location at the left temporal head side. after the routine oxymetric blood sampling in the superior vena cava the oxymetric catheter was manupilated into the left jugular bulb. after control of the catheter position simultenuous values of the rso2 were documented. results: over a range of (33-87%) sjo2, a significant linear correlation was found between the spectroscopic measurement of rso2 and the oxymetric determination of venous blood saturation in the jugular bulb (r=0,83, p<0,001) and the superior vena cava (r=0,65, p<0,05). no significant correlation was found between rso2 and the arterial blood saturation in the descending aorta and as well as to the standared hemodynamic parameters. conclusion: meusurement of rso2 by mrs may provide continuous non-invasive information on cerebral venous blood saturation and thereby possibly on cerebral oxygen supply and consumption in infants and children. these may be of clinical value particulary during and immediately after heart surgery by means of non-pulsatile cardiopulmonary bypass. information on refractory status epilepticus (rse) from developing countries is scarce. we analysed 43 cases of rse admitted over last 2 yrs. the objective was to study etiology end evaluate efficacy of diezepam infusion. median age of the patients was 1.25 years irange 1.5 months to t 1.5 yrs); 70% were boys. onset of seizures was 1-t44 hours (median 24 hours) prior to hespitalisation. the glasgow coma scale score ranged from 3.11 (mean+sd 5 + 2). the commonest underlying causes were acute cns infections (26/43, 60%; bacterial meningitis, 16, encephalitis, 10) and epilepsy (8/43, 10%). oiazepam infusion in incremental dose (range 0.01-0.025 mg/kg/min) was used in 38 patients over 3.4_+2.1 days. seizures were controlled n 31 (82%), mechanical ventilation was required in 10 (26%)only, while none had hypotension; 84% patients survived. thiopental infusion (holus 5 mg/kg followed by 0.2 mglkg/min, and increments of 0.1 mg/kg/min till seizure control) was used in 8 patients over 1.7_+0.7 days; seizure were controlled in all, but five patients needed mechanical ventilation, six developed hypotension needing infusion of vasopressoi drugs, 3 out of 8 (38%) died, overall mortality was 26%, mainly due to acute cns infections (n-6) and prolonged se. the patient was a 2-year-old gift di~aosed of dov,~'s s~drom¢, tetralogy of fallot. (t.f.) before admission a vasovagal crisis after coughing and vomiting was seen, and she was taken to the emergency room. mother said she had eyanosis in the mucous membranes of the mouth with exercise.on physical examination, she ~as afebrile, normal fundi and neurologic examination was normal. a harsh systolic murmur was hear~ with decrased intensity during bradycardia. chest rx disclosed a decreased pulmonary vascular markings. ecg: synus rhythm, with bradycardia and nodal escape rhyflmas. she was transferred to our picu because of severe h3,pertomc seizure, lost conciousness, and deeembrate poslamng~ ~t cyancx~is. the episode lasted for ~weral seconds, and ceased v~th diazepam. on admission she was lethargy, and neurologlc exammation showed weakness of left leg without babinski, and normal funduscopic. the patient had two episodes of bradycardia and isoproterenol was begun. during those episodes the patient was cyanotic, and the murmur was heard with the same intensity. act scan disclosed a tight parieto-temporai abscess with midline shift, lnmediately after the diagnostic ct, we administered antibiotics, antiedema treatment and it was drained. the abscess culture was negative. a ct control disclosed air and midlme shift. ~ the next two days she had three episodes of h39oxia and c'yauosis ceased with o@gen, morphine and propanolol the patient died during a fourth episode. discussion: arrhytmias are uncommon in patients with tetralogy of fallot before surgery. in our case the first diagnosis was sick sinus syndrome vs bradycardia secondary to cyanotic episodes. the incidence of cerebral abscess in children with congenital heart disease (chd) is approximately 5%. tetralogy of fallot is the most common associated lesion, and is unusual in children under 2 years of age. conclusion: 1) brain abscess is a rare complication of patients with cyanotic chd, but should be suggested in patients with °'apparent" sick sinus syndrome. in patients with down's syndrome, t.f.,with cyanotic episodes, and difficult neurologic exploration, a brain ct scan is recommended. guillain-ba~re syndrome (gbs) is an acute autoimmune reaction, directed primarily toward the myelin encasing the peripheral motor nerves= this reaction causes a delay or block in nerve conduction. the presentation often can be very subtle but is followed by rapid loss of neuromuscular power, leading to acute respiratory distress, resulting from weakness of muscles and aspiration pneumonia. there were 3 boys -4, 8, and i i years old with gbs, treated in our icu. two of them due to the respiratory distress were intubated nasotracheally and ventilated mechanically with servo-9ooc (siemens-elema, sweden) ventilator. duration of ventilation was i i and 34 days, respectively. plasma exchange was performed in all cases. the numbers of plasma exchange sessions were 2-4 in each case. mean amount of plasma exchanged per session was 28,24 ml/kg. plasma was substituted with albumin, plasma or saline. the most important aspect of the management of patients with gbs in the icu involves the airway care, prevention and treatment of aspiration pneumonia and the mechanical ventilation if respiratory distress presents. endotracheal intubation should be performed whenever there is evidence of retention of pulmonary secretions, refractory to chest physical therapy, weakness of protective reflexes of the airway, leading to aspiration pneumonia and (or) atelecr~sis. cardiac arrhithmias too, is a main threat to the circulatory stability in gbs. therapeutic plasmapharesis has been shown to be beneficial, reducing the time for weaning from the ventilator and for achieving independent ambulation. however, plasma exchange is expensive and not without significant risks for the patient. some authors find that plasmapheresis is not effective for patients with fulminant course of gbs and blocking of nerve conduction. recent studies have demonstrated that intravenous high-dose immunoglobulin can be equally effective. there were no significant complications associated with plasma exchange. all presented patients survived without residual disability. tetraparesis associated with long-term paneuronium use in an infant. paneuronium is a muscle relaxant used in ventilatory management of patients with respiratory distress in intensive care unit. after the end of sedation some patients were found to have severe tetraparesis. paresis was accompanied by complete areflexia and diffuse atrophy of alt extremity muscles. this neuromuscular complication is caused by prolonged high-dosage pancuronium treatment. in the last 5 years, numerous reports have linked the use of pancuronium bromide with prolonged paralysis, disuse atrophy and areflexia. this side-effect is well known in adults patients but rare in a pediatric intensive care unit. we describe one pediatric observation of tetraparesis after prolonged pancuronium treatment in a 9-month-old girl, this female infant developed respiratory distress syndrome and was intubated and mechanically ventilated. to decrease chest wall rigidity pancuronium bromide was administered during 11 days. (she received approximately 120 mg of pancuronium bromide). on day 12 the drug was discontinued and the patient had severe tetraplegia and areflexia with normal head movements. electromyograpliy showed absence of any disorder of neuromuscular transmission. this infant showed a recovely of muscles after 3 months. the other causes of peripheral neuropathies were eliminated. electroencephalograms and head scans were normal. the recovery pattern observed in our patient correspond to the process of regeneration after axonal degeneration. it is suggested that these neuromuscular complications were caused by prolonged high-dosage pancuronium treatment (associated with cortieoid and aminoglucosides). polyneuropathy syndrome in adult lc.u. appeared in literature in 1984 and is extremely common in long stay cases. the etiology of these disorders remains elusive. it is tempting to ascribe them to administration of drugs (muscle relaxants, steroids, aminoglycosidea), plolonged immobility, malutrition, sepsis and ischemia associated with reperfusion injury. to our knowledge there is only one case report of similar condition in a children i.c.u. (pascucci 1990) we present a serie of 16 previously healthy children, aged 9 months to 13 years, who admitted in i.c.u with respiratory failure and who following weaning from m.v, remained in profound diffuse hypotonia with proximal and distal muscle weakness for various length of time, recovery of muscle strength occured in a week or months {the longest i0 months), all children, except one, 3-4 days before admission developed symptoms of either respiratory or upper airway infection with fever. on admission viral and bacterial cultures were positive in 2 cases (haemophilus influenze, herpes virus). during treatment 9 patients became septic. muscle histological and neurophusiological investigations have not been done. considering the multifactorial nature of the aquired nmd in adult critically ill pts, is impossible to attribute the muscle weakness of our pts to any specific cause, in conclusion, our findings suggest the need for further investigation of nmd in critically ill children treated in i.c.u. a van esch, ha van steen~l-m011, ir ramtal, g derksen-lubsen, idf habbema. febrile status epilepticus (fse) is a prolonged and serious febrile seizure. little is known about the outcome of fse in neurologically normal children. this survey involved patients between 6 months and 6 years of age who had visited due to their first fse, the sophia children's hospital during the period of january 1981 till december 1991. patients with a history of neurologic disorders were excluded. 57 patients were identified, 65% were male. the cause of the fever remained unknown in 51% of the cases. in all case the fse was generalized and it most frequently occurred at night (47%). the mean age at fse was t.6 years (0.5-4.7), the mean temperature 39.6°c (38.5-40°c). the mean follow up time was 1.7 year. twelve children (21%) had neurologic sequelea. the neurologic sequelae varied from speech deficit (4 case mild, v2 -1 year delayed; 4 case moderate > 1 year delayed) to severe retardation and epilepsy (4 cases). speech deficit was detected after a mean period of 6 months (range 0-18), age, gender, temperature, family history and time of onset were no significant risk factors for neurologic sequelae. duration of seizure [rr 3.0 (0.8-11.3)] and more than two drugs to treat fse (rr 5.2 (t.5-18.1) were related to neurologic sequelae. we recommend that fse children should be followed for at least a year to detect possible speech disorders properly and start early intervention. unusual presentation of myasthenlg gra%qs ibtza e. modesto ,v~ abe~gochea a, sanch]s 1l all, go l varas k folgado s, garcia e. p.1.c.u. la fe, valencia. spain case report: the patient was a 2-year-o!d gift transferred to our pic because of severe respiratory failure. the patient, convaleseem of ehiekenpox, came into contact with horse manure previous afternoon. in the morning, she was lethargy, and irritability, with poor finding, and ~ an episode of coughing, cyanosis and acute respiratory failure after mucous vomiting when she was drinking milk. on admission she had severe respiratory distress, respiratory acidosis, and the sat 02 was 86%. she was mtubated without difficulty, and was transferred to our p.i.c.u. physical examination reveals stable hemodynamies, pupils equal, round, reactive to light, normal fandi, and muscle relaxation. crusted vesicles diseminats~d. rhonehi over both lungs. hepatomegaly (+) and splenomegaly (+). ~lhe urine, hematologic, and c.s.f. laboratory findings were normal. c.t. scan of the brain, e.e.g., and ekg. revealed no'abnormalities. rx chest disclosed a retrocardiac atelectasis. speci~ts of stool and blood were obtained for cultures and study of c. botul#num toxins. pending receipt of these results, a broad-speotmm antibiotic and acyctovir was begun. the initial differennal diagnosis consisted of laryngospasm associated with aspiraqlon, botulism, and postmfecfious varicella encephalitis. after 15 hours, weatm~ was begun. the neurologic examination showed a low modified glasgow coma ~ale (mgcs), generalized hypotouia and muscle weakness. these data suggested three diagnoses, posfnfecfious encephalitis, residual neuroumsoaar blockade, and excessive doses of sedative and analgesic drugs. after 20 hours she regained skeletal muscle poxver and ufltlcient respiratory effort, the mcgs was acceptable, and blood gases were normal. she was given n~-tigmine and atropine, and her tr~ma was extubated. an acute respiratory failure ocurrs 120 ram. after. chest radioga'aph disclosed a left inferior lobe atelectasis. after 20 hours weaning begun~and the same episode w~as seen. at this point her mother stated that the girl showed weakness of the eyelids or extraneular muscles. it suggested myasthenic syndrome vs ~-barr6 syndrome. c. botul#num toxins were negative, chotinesterase level ~as normal. edrofoinum test ~as positive. anti-acetyleholine receptor antibodies were negatives. e.m.g. confirmed myasthenia gravis (congenital vs juvenile serenegative). pyridostigmine was begun and the trachea was extubated without complications. conclusion: din the differential diagnosis of weamng failure we must consider ~c gravis~ 2)myasthenia gravis could resemble encephalitis, because of low ocs, overall if is triggered by viral infection. 3)in some diseases (this case) gcs could not he an aemuate index of mental state. a burguet*, a menget*, e monnet**, a gasca-avanzi*, c fromentin*, h allemand**, jy pauchard*, ml dalphin*. * r4animation infantile potyvaiente chu st jacques 25030 besancon cedex. ** d~padement de sant6 publique 25030 besancon cedex, france, objective : to point out that strabism is) of one-year-old premature is a good predictor of a poor neurological outcome at two years of age. design and setting : two-year prospective cohort study and geographically defined study (region of franche-comte, france). main outcome measures : neurological assessment was performed at one and two years of age (uncorrected for gestationnal age). a mailing questionnaire was sent to the famity and fuu-filled by thefamily doctor (pediatrician or physician), or neonatologist of the icu at tertiary center, s was diagnosed at one year of age by the examinator but s was not used to diagnose cerebral palsy (cp). sample : 161 of 171 survivors (94%) evaluated at one and two years of age. results : correlation of one and two years neurological evaluation is weak (kappa=0.5). correlation of s at one year and cp at two year is fair (kappa=0,72). the goal of this paper is to review evidence related to hypothesis that the "waiting" axons and cells of the transient subplate zone may participate in the structural plasticity of the human cerebral cortex after perinatai brain damage (kostovic et al, metabot brain res4:17, t989) and to correlate this phenomenon with different forms and mechanisms of structural plasticity. it is our basic assumption that all lesions occuring during cortical histogenesis will lead to more or less pronounced structural reorganization. here we show that various components of the subplate zone participate in several forms of the structural "plastic" responses in the human cortex: modification of convolutional pattern, changes in size of cytoarchitecturat areas~ columnar reorganization, dendritic and synaptic plasticity. the etiological factors which induce lesions and subsequent plastic changes act via the following pathogenetic mechanisms: * disturbances of radial unit formation (rakic); * changes in ingrowth of afferent fibres; * changes in the rate of normally occuring reorganisational events, depending on the critical period for a given histogenetic event. in the present study developmental lesions (localized perlventricular leukomalacia and haemorrhages) were demonstrated by ultrasound in live-born infants ranging between 26 to 40 weeks of gestation. in younger infants (24-34 w) who died shortly after birth, examination revealed lesions of the white matter with the preservation of the subplate zone. in infants who died one week of more after the lesion, we have observed localized micropolygyria, cavities, condensed layer vi -subplate zone, and columnations of the cortical plate. these changes are less prominent if the lesion occurs after diminishment of the subplate zone (after 34 w). since in the fetal cortex the subplate zone serves as predominant source of growing fibers, transient neurons, trophic factors and contains cellular substrata for migration, this zone is the most likely candidate for major types of structural plasticity. in conclusion, cerebral cortex of the low -birthweight infants is more susceptible to the various lesions but shows vigorous structural plasticity and conspicuous functional recovery due to the growing, transiently located neuron at elements. the mortality due to meningoccocal sepsis is high in spite of important progress in emergency and intensive care medicine. during the last decade multiple scoring-systems have been developed in order to establish a therapeutic approach and to evaluate the final outcome of a meningococcal infection. different clinical and biological data (shock, ecchymosis, peripheral wbc and platelet count, coagulopathy, acidosis, meningism, etc) are taken into consideration and the importance given to these data depends on the scoring-system used. a review of the different scoring-systems is given and a clinical case is presented. we report the case of a 4 year old male, who was transfered to our icu 12 hours after onset of temperature and skin rash. the parents described a fast deterioration of his condition. the boy presented wide spread ecchymosis, high temperature, no signs of meningism, circulatory insufficiency and shock, coagulopathy and low peripheral wbc and platetet count. disseminated intravascular coagulopathy developed promptly. the glasgow meningococcal septicemia prognostic score (gmss) was used and the obtained score reached the highest level (15/15). this corresponds to a 100% mortality. the patient required mechanical ventilation for 5 days. at admission he received human albumine, fresh frozen plasma, dexamethason, dopamine, dobutamine and a continuous infusion of adrenaline. antibiotical treatment consisted of ceftdaxone. the evolution was favorable and the infant fully recovered. retrospectively the gmss was compared to other meningococcal scoring scales which gave the same mortality (100%). we conclude that the scoring-systems are important to evaluate the seriousness and to assess the therapeutic approach, but they should be used cautiously even when 100% mortality is predicted by several risk evaluations scoring-systems. the aim of this study was to assess the haemodynamic status on admission and the critical care management of children presenting with meningococcat infection. this was a retrospective study of the charts of 46 consecutive admissions. mean age was 3.43 years (+/-3.46). the average duration of symptoms prior to admission was 20.4 hours (+/-14.09). on admission 17.4% were hypotensive, 45.6% had clinical signs of haemodynamic instability and 54.8% of cases that had a blood gas analysis on admission had a metabolic acidosis (bases excess < -5.q): the mortality rate was 10.9%. 80% of patients that died were hypotensive on admission and all had a metabolic acidosis. of the 41 survivors 9.7% were hypotensive on admission, 39% had clinical signs of haemodynamic instability, 25% required invasive pressure monitoring and 7.3% were ventilated and received inotropic support. this study demonstrates that at the time of presentation with meningococcal infection children had a high incidence of established haemodynamic instability. successful management of this infection is dependent on early presentation and initiation of therapy and on aggressive support of the cardiovascular and vital organ systems. dept. of intensive care medicine and dept of infectious diseases, our lady's hospital for sick children, crumlin, dublinl2, ireland. jude. pediatric intensive care unit, ch&u, 59037 lille-france. more than 10% of children surviving sip (defined as purpura with shock) have snli. objective. to search for a specific hemostatic profile in children with snli. patients and methods. between may 1989 and march 1995, 34 children with sip were admitted to our picu : 6 (17.6%) died and 28 (82.4%) ranged in age from 1 to 185 months (mean : 29) survived, 5 of them (17.8%) with snli (defined as the need of a surgical procedure). in survivors, two hemostasis studies (between h0 and h12, and 24 h later) included the determination of coagulation factors (routine tests), protein c (pc : amidolytic activity, biogenic), total protein s (ps : elisa, stago), c4b binding protein (c4bbp : laurell's technique, stago), antithrombin3 (at3 : chomogenic test, stago), and plasminogen activator inhibitorl (pail : chromogenic test, biopool). three severity scores were determined at admission : french group of pediatric intensive care, gedde-dahl, and crp. statistical analysis used the wilcoxon's test. results. at admission (lst sample) severity scores and at3, pc, ps, c4bbp levels were not different between the group with snli and the group without snli ; quick time (22 4-5% vs 35 ± 14% ; p = .025), vti+x (20 4. 3% vs 30 4-10% ; p = .04i) and pall (105 4-157 ui/m! vs 580 4. 570 ui/ml ; p = .028) were lower in the group with snli. on the 2nd sample there was no difference between the two groups. kinetics of hemostatic abnormalities was not different between the two groups. conclusion. in the literature, intravascular coagulation (dic), low fibronectin and at3 were identified as predictors of snli, and a negative correlation was found between the mean size of the skin lesions and pc activity, at3, and total ps. in this series, apart from dic, there were no specific hemostatic abnormalities that support the use of treatments such as pc, at3, and pail antibodies administration to prevent snli. further studies including more children are needed. the aim of study was to investigate the efficacy of intravenous immunglobulin with enriched igm content pentaglob/n /biotest/. in our pediatric intensive care unit ten septic children /group i/-their average age 2,6 years /sd:o,6/, 7 of them with gramm negative and one with gramm positive blood cultures, and two with unindentified bacteria-were treated with basis sepsis therapy and pentaglobin. the application of pentaglobin was as follows: 1,5 ml/kg loading dose for one hour, followed by a continuous intravenous infusion 0,1-0,4 ml/kg/hour depending on body temperatura /lanser scheme/ for 72-96 hours. another ten septic patients /control-group ii/the mean age 2,5 years/sd:o,65/, their blood cultures were gramm negative bacteria 6, positive 2, and the bacteria was not indentified in two cases -were treated with only the basis therapy. results: the duration of intensive treatment decreased from an average 22,7 days /sd:8, min 12-max 38 days/ to 19,5 days /sd:5,2 min 9-max 25 days/ in the group treated wit pentaglobin. the difference was significant /x 2 p<0,01/. in the group i nobody died, but three in the group ii. conclusion: the pentaglobin therapy can improve the efficacy of the basis therapy of sepsis. sinus bradycardia after an episode of sepsis is a rare symptom complex decribed in children with hematologic malignancies. we present a case of postsepsis bradycardia following severe typhlitis and septic shock in a 12 year old boy with relapse common all. blood and ascitic fluid specimen grew clostridium species and pseudomonas aeruginosa. at surgery there was a necrotic gangrenous terminal ileum and cecum, requiring ileocecal bowel resection with ileostoma. while clinically recovering from sepsis he developed bradycardia for 120 hours. extensive diagnositic procedures was given and the heart rate slowly increased to normal range of age. postsepsis bradycardia in children with hematologic malignancies after an episode of sepsis is self-limiting and after careful differential diagnostics warrants an expectative attitude. nitrate level is known to be enhanced during sepsis. serum nitrate is the stable metabolic end-product of endogenous nitric oxide generation. nitric oxide has demonstrated to be a powerful anti microbial final mediator and also a key molecule driving to the lethality of one of the most common complication of sepsis; the endotoxic shock. such facts prompted us to investigate the possible diagnostic and/or prognostic value of monitoring serum level in high risk, presumptive and confirmed sepsis patients. additionally we have explored the usefulness of this mediator as index of therapeutic response. in our study it is demonstrated that there is an important relationship between nitrate level and the occurrence of neonatal sepsis. septic newborn group showed 6 fold higher nitrate level than that of healthy control group. in addition, the group of patients with high risk of sepsis which finally became septics, exhibited 3 fold higher nitrate level at 24-72 hours before the first symptoms appeared, when compare with those who did not develop sepsis. however in the presumptive sepsis group, there was no difference between the patients which finaliy ,&'ere considered septics and those which not. in all septic cases, after 7 days of a successful therapy with antibiotics, the level of nitrate diminish 3 fold. our results suggest the utility of monitoring nitrate as index for the diagnosis of neonatal sepsis. the potential benefits of exchange transfusion, plasma exchange, and haemofiltration have all been described in children with overwhelming sepsis. however, little hard evidence exists to prove the benefits of any of these techniques. i have treated five patients with plasma exchange (pe), having been asked to see all these patients at a point when it was felt death was inevitable. two of the patients had staphylococcal, two meningococcal and one enterococcal septicaemia. all patients showed a dramatic haemodynamic improvement following pe with improvement in blood pressure, reduction in inotrope requirement and improvement in tissue perfusion. three patients survived. one of the patients with staphylococcal sepsis and both of the patients with meningococeal sepsis had developing gangrene of the limbs which showed remarkable reperfusion with pe. in two of the patients measurements of cardiac output (co) and systemic vascular resistance (svr) showed ~a reduction in co and a rise in svr over the course of a pe despite the reduction or cessation of vasoconstricting inotropes. many believe haemofiltration is of value in septic shock. a trial with a no treatment limb is difficult to achieve. i believe we now have enough evidence to justify a controlled trial of haemofiltration versus plasma exchange in patients with septic shock and unstable haemodynamic status whilst on inotropic support. during the next several days, cough and chest pain suggested pulmonary embolism confirmed by radiologic evaluation. echocardiographic examination showed multiple thrombosis of the superior vena cava, right atrium and ventricle and pulmonary artery. estimated protein c level was 50.7 % (normal range 70-140%); identical deficiency was found in patient's mother and elder sister. cvc was removed, and alter 2-month heparin therapy and supstitution of protein c with fresh frozen plasma, there was almost complete thrombolysis of the great vessels and cardiac chambers. we conclude that invasive diagnostic and therapeutic procedures in such patients may result in higher risk for severe thrombosis at unusual sites, and numeuos further complications bronchopulmonary dysptasia (bdp) is a chronic pulmonary disease of preterm and term babies treated with mechanical ventilation for respiratory problems of different origin and requiring oxygen therapy 28 days after birth. bpd is a disease affecting the growth and development of pulmonary tissue. such pulmonary }esions heal by squamous metaplasia leading to scar formation and fibrous tkssue r~growth, the pediatric intensive care unit makes the survival of babies w~h very low birth weight (500 -999 g) possible. with the increase in their aulyival, the number of complications in low birth weight babies increases as well. bdp is a very serious complication. therefore the importance of early diagnosis and treatment of bdp must be stressed in order to reduce the consequences. babies with bdp must be under medical suveillance for at least 3 years as the disease needs at least that long for complete resolution. tn the icu of pediatric department at madbor teaching hospital: during the past two years (1994-95) 154 newborns were treated with mechanical ventilation. the neonatal and postnatal death rate of all newborns admitted to our icu was 7,1%o.ln the two years from 1994 to 1995, 16 newborns were admitted to our icu (2 %~ of all newborn babies at maribor teaching hospital), with birth weight 500-999 g. in the icu, the survival of these babies and parallel to it the number of complications is increasing. during the mentioned 2-year period, 8 babies with very low birth weight (500-999 g) survived: 5 in 1994 and 3 in t995. in 45-50 %, first or second stage bdp was treated,there was no case of third of fourth stage bdp. the treatment consisted of eary removal from mechanical ventilation, oxygen therapy~ intensive treatment of infection, volume and caloric intake contro}, corticosteroid treatment throught 6 weeks with decreasing doses, diuretic end antioxydant therapy. the children are to be reevaluated at the age of 3 and 6 months and again at i and 3 years. oeure j van der, markhorst do, haasnoot k department of pediatrics, pediatric intensive care unit, free university hospital, amsterdam, the netherlands. case summary a 4%-month 6.5 kg girl of african origin was admitted to the pedfatric irtensive care unit with pneumonia and progressive respiratory irlsuffjderey. she was intubated and ventilated by pressure regulated volume controijed ventilation (servo 300c, siemens, soma, sweden). maximum conditions were inspiratory minute volume 3.2 l, peep 10 cm h~o ahd 100% 0~. chest x-ray showed bilateral interstitial consolidation. material obtained by broncho-alveolar lavage showed preumocystis car}nil htv-serology (elisa and westerll blott) and p24-antigerl were positive, confirming the diagnosis of pediatric aids. she was then treated with high dose co-tllmoxazoie, penthamldine, z{(~ovudire and steroids iv. because of thee x-ray features, high need for o 2 (100%, pad 2 56 mm hg), not responding to elevatiofi of peep (max 10 cm h=o) and pao2/fio = <200 (s6). m acute respiratory distress syhdrome (ards) was diagnosed. because conventional ventilation (cv) failure, hfo-v (31ooa, serisor medics,yorba linda, ca) was initiated. starting mean airway pressure (map) of 19 cm h~o was based or map of the cv, oscillatory pressure amplitude (dp) of 47 was, at ii~itial frequency of 7.5 hz, adjusted ur~til chest wall vibrations were visible, it was required to raise map to 26 cm h20 and dp to 66 before optimal lung volume and ventilation were achieved and need for o 2 reduced within hours, this was monitored by frequent blood-gas analysis and chest x-rays. map and dp could slowly be reduced, after a good response the first day, gradually 02demand reduced and the patient could be weaned from the ventilation. map, dp, fi02 and oxygenation index (map x pa0~jfio 2) are shown in table i. chest x-ray follow-up showed gradually improving lung features, with marked improvement of aereation. after 10 days hf0-v she could be succesfully detubated when a map of 10 cm h20 was acmeved. results : sianificant increase in ventilato~ rate and mean airway pressure was noticed after the change to savi. no differences in oxygenation, co 2 partial pressure and systolic, diastolic or mean blood pressure between imv and savi periods were noted. in 6 infants however an improvement in pao2/p43.ol/ and decrease in paco 2 was observed after the switch to savi. these babies had a lower initial a/a oxygen tension ratio and required higher initial ventilator rate /p<o.05/ in comparison to the rest of the studied patients. conclusion:infants with low a/a po2ratio who need high initial ventilator rate are likely to obtain benefit from savi.. fumimare hatori, haruo uchida, masao katayama, and rika muto department of anesthesia and critical care medicine chiba children's hospital, chiba city, japan. purpose: this study was made to find out whether the heat and moisture exchangers (hme) was effective enough to humidify in the respiratory management for children. patients: the study population consisted of 21 patients who were provided with artificial airway. they were divided into the two groups: ii with heated humidifier (a) and i0 with hme (b). methods: hme could be used for those without pulmonary complication and with less leakage around the endotracheal tube. in both a and b groups, (i) we have measured relative humidity, temperature, and absolute humidity at the endotracheal tube connector. for these measurements, humidity sensor system was utilized. (2) any troubles and complications caused by the artificial airway were investigated. results: (i) in group a, we confirmed the mean values as the relative humidity of 96.5%, the temperature of 32.8~c, and the absolute humidity of 34.0mg/l. in group b, they were 92.7%, 29.5°c, and 28.7mg/l respectively. no difference was noted between the two groups as to the relative humidity, but the temperature and absolute humidity were lower in group b with a significant difference (p<0.0001). (2) in neither of the groups, any respiratory complications such as obstruction or stenosis of the artificial airway were noted. in pediatric icu, hme can be used without any problems under given conditions. titei: objective: evaluate the precision, reproductibility and aplicability of a new device to measure auto-peep in pediatric patients during mechanical ventilation. material and methods: it's an electromc-pneumatic eontolled device with an oclusion valve and a pressure monitor installed between endotracheal canulla and the ventilator circuit. the measurements were performed with a sollenoid conected with to the ventilator to detect the end of inspiration phase and the actwation of the oclusion valve. the signs of pressure and flow were moniturized using a difere~rtial transducer and it was processed using a pc computer and a pneumoviaw® software. the auto-peep also displayed in the ventilator pressure monitor. results: we submitted 17 children with neuromuscular disease or respiratory distress in this study. the incidence of auto-peep was 76%, with variability between 1,5 to 13 cmh20. all the measurements were performed 3 times to verify the precision and the reproduetibility and evaluation of the pressure and flow curves. conclusion: the device is low cost, easy to use and can be used without the pneumotacograph. the auto-peep measurement can be done during mechanical ventilation with time cycled, pressure limited and continuous flow, the most commom ventilator used in pediatric patient. resistance was modeled by 3 endotracheal tube (ett) sizes (2.5, 3, 3,5), and compliance by 2 test lungs (1 and 5 ml/crnh20), for each resistance -compliance arrangement, we measured tidal volume (vt) at various frequencies, peak to peak (p-p) and mean airway pressures. results vt increased when ett size increased with all 3 ventilators in accordance with fredberg results (jap, 1987; 62: 2485-90) . the maximum vt delivered was smaller with the hfv-babyiog 8000 than with the ohf 1 or the sm 3100a at a given frequency (figure on the left). increasing p-p resulted in a linear increase in vt delivery in the 0-30% range of maximum p-p (figure on the right). hfv-babylog 8000 didn't provide significant vt increase when p-p was set above 50% and vt deliven/decreased when mean airway pressure decreased. --i 5~ sm 3100a a number of ventilatory parameters had been analyzed with russia-produced monitor humitemp htm 902 (servisinstrument j,-s. co) in 24 newborn infants with rdsn of t-iv stages on cmv, cppv, imv, and cpap steps using respirators of various kinds (stephan-staxel, bear-2001, newport 8rezee, sechrist). parameters investigated were: temperature of inspired mixture (t), relative and absolute humidity (rh and ah, respectively), static compliance (c), expired tidital volume (vte), expired minute volume of ventilation (mve). the monitor read these parameters in following ranges; rh-from 10 to 100%; ah-up to 55mg/i; mve-up to 3.0 i/mini vte-from 3 to 130 ml; t-accurate to 3%; c-from 0.1 to t0 ml/cm h20. continuous monitoring time ranged from 2 to 96 hours, it was noted, that during this time the monitor and transducers had no malfunctions, and it's using didn't require additional calibration, it was found, that humidifiers mr 498 and mr 410 (fisher, paykel) couldn't maintain parameters t and ah continuously (these values varied from 34,3 to 38.2 c and from 37,5 to 47.1 mg/i, respectively, when heating regulator position was unchanged). changes of parameters c depended clearly on cppv conditions and rdsn severity (with rdsn of stages i-ii c value ranged from 2.4 to 1.2 ml/cm h20; and with stages iit-tv-from 0.3 to 1.1). tn 6 newborn suffering from rdsn of stages ii-ltl and ill-lv with pip>25 mbar, fi02>0,7, peep=4-7 mber, c-from 0.3 to 1.2 ml/cm h20, effectivity of exosurf therapy was studied. in 4 newborns in 4-12 hours of therapy pip decreased to 0.3-0.4, and c increased to 1,7-2.4 ml/cm h20. in 2 newborn infants with aad02>500 mmhg and c from 0,3 to 0.8 mltcm h20 positive effects of exosurf on lung compliance were not observed. in 3 newborns the monitor had revealed decreased of c (from 3.4-2.9 to 1,8-1.3 ml/cm h20), manifested clinically by pneumothorax. in general, monitor htm 902 made possible; 1), to estimate the adequacy of cmv-parameters and regimes in newborn infants; 2). to select optimal t and ah values in the respiratory outline in dependence on lung damage severity and infused volume; 3). to reveal rdsn severity; 4), to optimize indications and adequacy of surfactaot therapy; 5). to diagnostieate the air leakage syndrome; 6). to effects to some agents (broncholytics, spasmolytics); 7). to obtain objective indications for imv/simv and cpap regimes. albano communication is an important aspect of human development and existence, and an inability to vocalise can be a problem in ventilatordependent patients. we present our experience with speaking aids as a means of enhancing verbal communication in four ventilatordependent children in our paediatric intensive care unit. the age of the children ranged from 7 months to 5 years, and the period of ventilation ranged from 3 months to 21 months via a tracheostnmy. they require continuous flow generated pressure limited or control ventilation at rates of 13-20 bpm. the reasons for ventilation include tetraptegia following a shrapnel injury; tetraplegia following congenital cervical spine damage; tetraplegia following atlanto-axial subluxation; and critical illness polyneuropathy following adult respiratory distress syndrome from prolonged ventilation for a severe head injury. the first three patients have passy-mnir one-way speaking valves and the final patient has a bivona foam cuffed tmcheostomy tube with a talk attachment in view of recurrent aspiration. an improvement in quaiity of speech has been shown by independent assessment. we will review the present literature on this subject and discuss the advantages and disadvantages of these two types of speaking aids in the light of our experience. the prognosis of antenatally diagnosed cdh is closely related to the degree of ph. there have been attempts to correlate antenatal or postnatal criteria to mortality: none have been demonstrated to be predictive of lethal ph. the aim of this retrospective study was to determine whether antenatal or early postnatal data could correlate with the findings of post-mortem examinations. patients and methods: between july 1990 and july 1994, 32 cdh patients have been antenatally and postnatally managed at our institution. twentythree infants underwent a post-mortem examination. ph was assessed by using the lung weight to body weight ratio (lw/bw) and the radial alveolar count (rac). antenatal results: cdh diagnosis was made at 24 weeks of gestation (wg) (15-37). twenty-eight patients had a left sided cdh, 3 had a right sided cdh, and one had a bilateral cdh. herniated organs were stomach none (n=21), or liver alone (n=4), or both stomach and liver (n=5 the patient was a 3-yenr-old girl with chronic renal insufficiency see~ to renal dysptasm, two months before admission a kidney trar~ptant was performed. one morah later she showed acute graft rejection with serum ereafinine (cr) level of 0.7 mg%. the rejection was unreslxmsive to an increased steroid dosage, and okt3 was begun with resolution of the rejection. one week arer, new rejection episode was seen marestxmsive to an increased steroid dosage, and transp~ ~s performed five days before admission to our ptc. hemedialysis and peritoneal dialysis (p.d.) each other day, was indicated (g.r.f.< 10 ml/rnin). four days before admission t ~ rose to 38°c. "lhe diagnosis of opporttmistic pneumoma was made on the basis of tach3,pr',e~ hypoxi~ and diffuse interstitial infiltrates. senma ~ was positive for cytomegaloviras (cmv), and stool culture for c albicans. pentamidine, ganciclovir (dhpg), arai-cmv gamma globulin, eritromicine and amphotericin b was administered. on admission in our picu, trachea was mmbated, (a-a) o2 gradient was 600, paofffio~: 65, lung injury score > 3 with peep level of 8 cm hzo. she had normal fiver function. during te next days she had fever and developed ards. bal was negative. p.d. was of little efficiency. we adjusted pentanfdine, and dhpg doses for severe renal failure, with supplements after hero, sis, and at~rp.d.. during ~ next days she was afebrile, and the chest became radiologlcally normal. after ten days on menhani~al ventilation (mv.), the patient was extubated. cr. level was 3.2 rag%, (a-a) oz gradient was 20, and paoyfioz was 375, the patiem was discharged with chronic ambulatory p.d. discussion: opportunistic pneumonia is a major complicalaou in imm~romised children, specially after kidney tvansplaraafion. c m.v. infection can result at~r okt3 administration. in the treatment dhik} dose must be adapted to the degree of renal insu~cieney, with supplements after hemedialysis, and after pd. pneu~y~tis cann# tmeumov~ is ehemeterized by ventilafion-perfusion mistmaeh, decreased pulmonary compliance, hypoxia arld elevated (a-a) oz gradient, with diffuse interstitial infiltrates. in our ease bal was negative. although we did not find the etiology the prevoclons eombh~ation of arairmcrobiat therapy, along with m.v., and supportive measures were the most effective trealme~. conclusion: 1) in patients with severe renal failure and life-threatening infections, we must co~ider drug adjuslments. 2) in our patient we gave dhpg supplements at~r pd. with excett~at results, although p.d. was of little effiele~. introduction: endotracheal intubation and mechanical ventilation have become an important treatmem for many diseases accompanied by respiratory failure. with the frequent use of this treatment modality, an increasing number of complications associated with endotracheal intubation have gained clinical significance. material and methods: a transversal study was realized to find the prevalence of pulmonary aspiration with endotracheat tubes in 36 infants and children. aspiration was assessed by applying two dyes (evans blue, er)¢rosine sodic) on the tongue and searching for the dye during suctioning in the endotracheal aspirate. the factors, that potentially have influenced the aspiration, including weight, age, sex, cause of respiratory failure, main pressure airway (map), level of consciousness, presence of swallowing and body position were evaluated. all the variables studied had their association with aspiration tested by chi-square method with relative risk considering a confidence interval of 95%. the results were adjusted by multivariate analysis. results: the overall prevalence of aspiration was 36.1%. among all children who aspirated, compared to those who did not, there was a statistically significant difference in the presence of swallowing (p=0.005). the odds ratio to aspiration in the presence of swallowing was 38.4 (t.75 -100 c.i.95%) and the relative risk 55.5. aspiration was not significantly affected by sex, weight, age, cause of respiratory failure, map, level of consciousness and position of the body during the ventilation. conclusion: the endotracheal intubated children frequently aspirate as intubated adults and that preventive measures are ineffective. the presence of swallowing movements is the main risk factor to aspiration of oropharingeal content in intubated patients. clinical features and shortterm outcome skling, rp gie pneumonia is the second most important cause of death in young south african children. the clinical features, intensive care course and outcome of children being ventilated for pneumonia in the developing world is unreported. aim: to describe the clinical findings, aetiology and shortterm outcome of children younger than 6 months with pneumonia requiring ventilation. the data of all babies under the age of six months with a lower respiratory tract infection admitted to the paediatric icu for ventilation were prospectively collected over a period of 14 months. tracheal aspirates and blood specimens were submitted for viral and bacterial cultures. results: forty-seven babies aged 14 to 174 days were ventilated for pneumonia. twenty-six infants had been born prematurely; t2 had been ventilated during the neonatal period and 4 had bpd. the median duration of symptoms was 1 day, the most common being cough, tachypnoea, apnoea and cyanosis. five babies (10%) died. the mean duration of ventilation was 8 days (range 1-85 days) and of ward stay after icu discharge 19 days (range 1-161 days), blood euttures were positive in 7 children (15%). viruses were cultured in 14 children (30%). conclusion: 1) fifty-five percent of children below 6 months requiring ventilation for pneumonia were premature infants, of whom 46% had been ventilated during the neonatal period. 2) the median duration of symptoms prior to admission was 1 day. 3) ninety percent of the children survived and were discharged from hospital. 4) viral pneumonia was responsible for 30% of the admissions. mechanical ventilation and atrial natriuretic factor release ulloa santamarfa, e, p6rez navero jl, ibarra de la rosa i, espino hernladez m, velasco jabalquinto mj, frfas p6rez m. picu. reina sofia children's llospital. c6rdoba. spain. mechanical ventilation effects on renal function decreased diuresis and natriuresis due several factors including anf. several studies have demostrated anf released due increaasing pressure in right atrium. on the other hand, mechanical ventilation, overall peep modality, inhibits peptide release althougt cvp increased is found. this study was designed to demostrate anf stimulation is due rigth atrium stretch which be higher during mechanical ventilation instead of atrium pressure. we desing a prospective study including 14 patients, age range 16 months-13 years with congenital heart disease. all of them were admitted at pediatric intensive care unit after extracorporeal surgery and were assisted by mechanical ventilation. hemodinamic state was stabilized in all patients and nor renal neither neurological diseases were found. after 24 hours with mechanical ventilation, plasmatic levels of anf were measurement, pvc, pericardical pressure were assessment; all patient were sedated with midazolan and paralized with neuromuscular blocking agent; mechanical ventilation technique was as follow: imv between 20 and 30, tidal volume and fi o2 enough to mantain respiratory parameters in normal range. afterwards, at least twentyfour hours in spontaneous breathing, the study was made again in each patient. atrial stretch was assesssment according to following equation: transmural pressure= cvp -pericardial pressure. cvp were significantly higher with mechanical ventilation than when the patient was breathing by himself. (5.4+__ 2.2 vs 3.8 + 1.8 mm hg; p<0.01). however, transmural pressure during mechanical ventilation were lower than during spontaneous breathing (8.92 +__ 3.86 vs 11.76 +__ 3.32 mm hg; p < 0.01) equal, plasmatic anf levels were lower during mechanical ventilation ( 87.77 + 46.55 vs 108.92 + 49.06 pg/rnl; p<0.01). in conclusion, anf secretion decreases during mechanical ventilation, even with cvp higher. anf release would depend on atrial stretch meassured by transmural pressure, lower in patients with mechanical ventilation and it would not depend on atrial pressure. the paediatric intensive care unit shaikh zayed hospital, lahore is an acute care area devoted to the care of critically sick children upto the age of 13 years. in a 6 bedded unit with limited equipment, constant care is ensured by the presence of at least one nurse aed one doctor round the clock. in this setup we have the facility to ventilate 2-3 children at one time, between sep. 93 and dec. 95, out of 885 patients admitted to icu, 171 (19.32%) were below 1 yr of age, while 48 (28%) were below 1 month of age. life support was discontinued in 17 (9.9%). total mortality was 56 (32.7%), major mortality was in 0-1 month age group 22 (12.8%), and 1 month to 6 month 15 (8.7%). majority of the patients were of sepsis (36.2%), cns disorder (22,2%) followed by respiratory problems (14.6%). it seems therefore that the major indicatiou for ventilation was overwhelming septicemia leading to multiple organ failure, rather than purely respiratory problems. high frequency oscillation (hfo) in the therapy for ards in pediatric patients requiring aggressive conventional mechanical ventilation (cmv) -routine or experimental mode ef pre ecmo therapy. fedora m., nekvasi~ r, vobruba v., srnsky p,, zapadlo m. dpt. critical care medicine, nicu and ecmo center, university children's hospita! brne, nicu of university hospital prague, czech republic. introduction: 9 pediatric patients (8 males, 1 female, average age 4.7 months, average body weight 5,8 kg) with severe ards ventilated with aggressive regimen of pcv or prvc were connected to hfo (sensormedics 3100) as the last "rescue" therapy due to uncontrollable respiratory failure before intended ecmo. in the course of hfo 2 of them were given no in the concentrations of 5-80 p.p.m., 3 were subjected repeatedly to surfactant replacement therapy (alveofact). results: ecmo was needed in no patient, 8 patients survived, 1 patient was disconnected from the ventilator because of brain death in spite of conspicuous improvement of oxygenation and other parameters, some relevant parameters 48 hours before and 48 hours after starting hfo are given in table 1~ in all the cases, the disconnection from hfo was carried out through the simv regimen, never directly to cpap. table 1 : the levels of blood gases, oxygenation index (oi), aado2,map,fio2 and pao2/fio2 ratio 48 hours before and 48 hours after starting hfo. conclusion: although none of the patient had to be subjected to pediatric ecmo, hfo should be carried out only in workplaces having the immediate possibility of using this method in the case of hfo failure. speculation: should not hfo be used ir pediatric patients with ards earlier than aggressive cmv? can hfo ce considered standard, not experimental method of therapy? refractory hypoxemia in premature patients is characterized in a persistent elevation of pulmonary vascular resistance, with right to left shunt through the ductus arteriosus and or foramen oval. we report the case of a vlbw patient (ga 27w, bw 1010g) who present a severe hypoxemia related to hyaline membrane disease and a pulmonary and systemic infection to group b streptococcus, refractory to conventional ventilatory support and surfactant therapy, associated to hemodynamic failure falling in ecmo criteria used for term infants. a rescue therapy with hfov (sensor medics 3100a) is decided at 5 h of live, the table resume the patient's evolution before and after hfov. at 36w of postgestational age the patient present a fio2 of 0.23 with a chest x ray compatible with a cld type l at discharge no oxygen requirements was needed and actually he's doing well. conclusion: hfov, using an adequate alveolar recruitment strategy, was effective in the rescue of a severe hypoxemic respiratory failure with a rapid off of ecmo criteria entry in our vlbw premature patient, during the united nmioffs embargo ~nst yugoslavia the prevalence of the ast}nnafic ~acks in c~dldren aratsed. the mo~t common causes have beem dramm~e worsening of life standard, ecom~c disaster in global community, gr~ number of refugees from the other parts of former yugodavia. it wm obviom that mcio-ecoumnical conditions took a part in the exacerbations of previously known cldldhood asthra~, ~av~ of micro-and m~mclimaflc changes, psychosocis] and emotional cryses, lack of medics-m~nts for p~ve~on and tl~rspy of acute asflanatic attacks. about 10% of d-dldv~ tmslod in our picu for these year~ exp~dvncod ~vcr~ attack for the flint time iu ~jzeir lifts. it has been cu~ 1~%~ children in mspir~ry picu of our hos~mt. the scut~ revere attack (more ~asn ~/o of hight clinical score) was detected in 62% of all children admitted with respirak~ problems. from tl~ mmlysss we exclu&d: bmncldolifis, ~i anomalies, ~eve~ i~ccqions. concerning our drug supplies (which wc~e reduced), we started our therapy by administration of oxygen, ~ta2-ago~dst inhalations (but sometimes we had the solution for jet nebulizcm only for o~e inhalation per p~cnt), mwinophyllin and mefl~ylpr~ini~done in/ravenously. 48% of ih~ asthmatics needed repea~ doses of muinophyl~n pinch.ally, tnch.,ding the fluids. the bronchodilak)r msponm was poor ~r~cl slow, hospital stay in picu was for 4 days and for 14 days in other units sl~rwsvds. tim ~ of their stable condifio~ was hard at borne (or refugees camps), without p~ventkm, so they came bsvk to hospital for morn than 3 times in 27% of cases, dtrdng ~e4je last motlfl~s file dtustion improved, concerning tim drugs supply for prevention, and we hope that these lifc~restening conditions wouldd~ introduction: the incidence of ards is increasing as survival of critically ill patients is higher. the application of new therapeutic modalities have increased the survival rates in (ards) adult patients. objective: to study the therapeutic efficacy of new tleamlents in children with ards material and methods: a retros~ctive study was conducted from 1990 to 1995. 17 children with severe ards, (lung severity score > 2,5) (r), aged 15 days to 16 years, were included. the diagnosis were as follows: 9 interstitial pneumonitis, 5 non interstitial lung infection, 2 with lung aspiration and 1 with clinical sepsis. 5 patients had different tipes of cancer and 4 to suffer inmunodeficiency disease, the first 8 subjects (group t) were treated with conventional measures. from october of 1994new therapeutic modalities were introduced, including: less agressive ventilatory support, postural changes (prone to supine) in 9 subjects, administration of corticosteroids in 8 patients, rfitric oxide in 3, pe~ssive hypercapnia and administration of exogeans sarfactant in one, pao2/fio2, d(a-a)o2, oxigenation index (oi) and the score of respirator), severity disease were similar in both groups. the two groups evolntiou was compared. results: -ten patients died, 6 from group i and 4 from group ii (75% v.s.44:4%,ns). -the evolution time, either to exitus or weaning from ventilatory support was higher in group ii (22.9 v.s. 13.6days in group i, ns), -the incidence of barotrauma was observed in 12 subjects (70.6%), 6 from group i and 6 from ii. of these patients 75% expired. -during the course of the disease, 15 (88%) patients had more than one damaged organ. only in one subjet mof was considered to be the main cause of death. the majority of the patients expired because of their respiratory disease, although, 80% of them met criteria of mof. -fifty percent of the subjects were infected at the time of death. stmmry: a trend toward a higher survival rate is observed in the subjects receiving the new modalifies therapeutic intervention (corticosteroides, postural changes and permissive hypercapnia). our results are not significative,probably because of the small number of subjects studied. a new doubleaurae~t two-stage et-tube (dl-ett) was desig~aed and tested in the rabbits with acute king injury under conventional mechanical ~entilation_ ventilation efficiency of dl-ett was emrrpared with that of canveniionally t~sed single lumen et-tube (sl-ett). meth~s: dl-ett was specially made out of two sl-ett. vertical crosssections at the distal end of two et-tube (td 3_0 rmn portax) were adhered with each other to form a tracheal stage lumen wifu id 3.0mm the two remained uncut parts of the tubes corlntithted the oval s~ge with two separate imnens. dl-ett and sl-ett were randomly applied to five adult paralyzed rabbits with acute lung injury (by 0.1 nffkg oleic acid. iv). a bird inter 3 vetffttator (bird products corporation) was used for time-cycled pressure-limited ventilation at 40/min of respiratory rate, 10 ern h20 of peak i_~piratory pressure, l: 1 of ire ratio, 6 ljmin. of flow rate and 0.21 of fich. peak inspirntory pressure, mean mrway pressure, posi6ve end-expiratory pressure at tip of et-mbe and bemodynamics were measured and recorded continuously. arterial blood and expired gas were measured ~by avl 993 blood gas analyzer) after each stabilization t.~iod of 30 minntes. _analysis w~as by prated t test. result: dl-ett acaltety improve cos removal at all amman. pa(?oz was decreased by t0.6+_t.5 (p<0.0l) and physiologic dead space fraction (v~zvt) reduced by 22% +-1.8% (p<0.0t), compared with dl-ett. there were no significant change in arterial oxygenation. conelus|on: the double-lumen two-stage et-tabe significantly increases ventilation effmiency with simple operation in rabbits v, ith acute hmg injury, lts availability may influence future clinical management of ~ennated patient~. this ~muly was fimded by the science and technology. commiuee of beijing municipality. analis of hemostasis alterations on different coagulation cascades in 46 children with septic shock has shown that coagulation disorder character is dependent on lung affection rate. the initial manifestation of the respiratory distress-syndrome (rds) are characterized by the obvious activation of blood thrombin potential, moderate coagulopathy and not sharply marked endoteliosis, the witlebrand's factor (wf) increase tot 140-220%. progress in the clinical picture of "shock lung" leads to chronometric and structural hypocoagulation with potential hypercoagulation in "mix-test", high level of firbin derivative, thrombocytopenia with thrombocytopaty and the wf increase to 210~315%, terminal stages of the rds, as a rule, are characterized by potential hypercoaguletion absense, depletion of at-lit and plasminogen, prevalence of antithrombin and antiaggregating activity, obvious endoteliosis (the wf to increase250-540%). the arteriowenous difference according to index of the thromboelastography (teg) in the rds ill-iv rates was 69,8% less than in the 1-11 rates, disorder of lung filtering ability in severe rds is confimed also by minimal arterio-venous difference of activated euglobulin lyses (ael) in children with the rds ill-iv rates is only 11,4%, while the patients whit rds i-i1 rates have the ael-activity in arterial blood 2,1 times as much than in venous blood. the use of then allows to determine the potential hypercoagulation rate, the at-ill level and fibrinogen quantity during the anticoagulant therapy and also the character of the x-factor activation and thrombocytic hemostasis. the effective therapy component of septic genesis rds in children is the controled coagulation method with the use of the individual selected heparin doses in according to desagregants, kryoplasma, proteolisis inhibitors and trombolytics. it is necessary to avoid the heparintherapy for children with the rds complicated with producting coagulopaties and termal phases of blood disseminated intravascular coagulation (dic). bronchoseopy has been used for evaluation of the potential problems of the airways and for investigation the bronchial specimens for diagnostic purposes. regent technical advances result in performing this procedure at the bedside manner and in critically ill patients. we have performed 150 hronehoaeopy during last three years on 1362 pediatric patients with respiratory problems, in 90% of cases the opentube hroneh0seopy was performed (for diagnostic as well as for therapeutic reasons) and collected secretions or bioptic material were examined. the indieatiuns were: acute upper respiratory problems, chronic wheezing, inspiratory strider, tracheal or bronchial bleeding, chronic eongh, retractable atelectssis, severe pulmonary infections, lymph node perforation in lung tuberculosis and soquells like bronehiectssis and fibrosis. our results were: anatomical malformations in 10%, mueosal oedema with chronic inflammation and thick secretions in 56%, easuos masses in 11%, granulation tissue and purulent secretions in foreign bodies and bronehieetasis in 16%, and only 7% of eases were normal finding. our exlxdenees pointed that this invasive procedure in carefully selected patients has important role in establishing the diagnosis and in theintroduction: tbg has been a useful investigation in the management of ventilator-dependent infants in our experience. one ml of contrast was hand ventilated into the respiratory tree via their nasotracheal tubes and their anatomy and dynamics demonstrated on radiological screening. case descriptions: three infants who were difficult to ventilate requiring high airway pressures, high peep and a significant oxygen requirement had tbgs. the ages ranged from 3 to 9 months. two cases were complicated by complex cardiac lesions. in all cases there were frequent episodes of desaturation, where hand ventilation proved difficult and various intermittent lobar collapses occurred. microlaryngobronchoscopies (mlb) performed on the infants by experienced paediatric ent surgeons failed to identify the airway problems. more than one mlb was frequently done. concern about introducing contrast into the airways of infants with limited cardiorespiratory reserve combined with an uncertainty about how much extra intbrmafion would be gained often led to a delay in investigation. when performed these fears proved groundless, the anatomy and pathology of the airways were demonstrated in full and the correct therapeutic plan started. in two cases tracheostomy and peep producing patency of bronchomalacic segments allowed weaning to low levels of ventitatory support. in one case tracheal reconstruction was undertaken and in the cardiac cases the respiratory component of the ventilatory dependence was fully assessed. at the age of 4 months, a baby boy with a history of minor respiratory problems, was admitted to hospital with an upper airway infection and severe dyspnoea. shortly after arrival at the icu he had a total airway obstruction. after intubation there were still difficulties to establish a normal gas exchange, and he was tranferred to the regional picu. ct scan and bronchoscopy verified a congenital tracheal stenosis affecting the whole trachea except the upper 15 mm below the vocal cords. the diameter was estimated to less than 2 ram. an unsuccessful attempt was made to dilate the extremely rigid stenosis with a balloon. after the procedure he had a respiratory and circulatory arrest, and he was put on ecmo as a bridge to surgical correction. after 4 stable days on ecmo, surgery was performed during ecmo with a tracheal homograft transplantation. immediately after surgery, ecmo was discontinued. a silastic dumont type stcnt was inserted inside the homogra~, and a nasotracheal tube was placed inside the stent for assisted intermittent mechanical ventilation. repeated bronchoscopies were performed to remove granulation tissue and secretions. at 9 months of age, the stem was removed with an endoscopic procedure. however, the trachea was still soft and collapsable, and another silicon stent was placed inside the trachea for another 4 months period, after removal he had some respiratory problems and he was treated with nebulized salbutamol, mcemic epinephrine and steroids. he was discharged from the hospital at 14 months of age and his condition is now stable. this is the first procedure of its kind in sweden. it was accomplished by international and multidisciplinary collaboration. ecmo may be a bridge to corrective surgery and long time stenting may be necessary in the postoperative period. post mtubation laryngitis ( pil ) is still a frequent complication, occurmg in l -6 % of intubated patients. inhaled racemic epinephrine has for long been used as an accepted therapy, but this drug is not always available. the authors undertook a randomized, double-blind, placebo-controlled trial to determine the efficacy of inhaled l-epinephrine(le) in the treatment of plu in the period between july/93 and may/95, 289 patients were submitted to endotracheal intubation for ventilatory support. atter the extubation procedure patients were considered for enrollement if they met the following criteria: clinical signs of laryngeal estridor and a downes and rafaelly score for upper respiratory obstruction equal to or higher than 4 patients with primary upper respiratory disease were excluded all patients enrolled reeieved either inhaled l-epinephrine 1% or normal saline. dexametasene ( 0,6 mg/kg/day) was given to all patients in both groups. after 2 inhalations, au patients were monitored for a period of 1-20 minutes and monitoring included cardiac and respiratory rate, mean arterial blood pressure, arterial blood gases and the dowries and rafaelly score. statistical analysis included, qui-square with the fisher correction test and the z-test for paired variables. thirty eight patients ( 13,1% ) met the criteria for enrollment, 18 to the le group and 20 to the placebo group.there were no significant differences in both groups in regard to age, sex, initial score ( 5,05 x 5,1 ) and endotracheal tube diameter. the period of ventilatory support and tracheal intubation was significantly higher in the le group (8,06 x 4,54, p = 0,01). the follow-up score showed a significant drop only at 30 minutes after the inhalations (p = 0,03). re-intubation due to laryngitis, occured in 1 patient of the le group and in 4 of the placebo group with no statistical sxgnificance (p = 0,2). no difference was observed on the monitored hemodynamic variables during the 120 minutes, except for the mean arterial pressure at 60 minutes, being heighar on the placebo group (p = 0,05). we concluded that, although the l-epinephrine group showed a trend in better scores post-inhalation and fewer re-intubations due to laryngitis, the results were not statistically significant. we especulate that the period of intubation may have affected our results. similarlly there were no differences in the incidence of adverse effects between both groups. objectives:to evaluate the complications of endotracheal intubation in children with upper airway obstruction due to epiglottitis or croup. methodes: during a 5 year period (1991 -1995) all patients with epiglottifis or croup were reviewed to determine the complications of endotracheal intubation, especially upper airway obstruction due to granulomas. results: 33 patients were reviewed. in 17 children (mean age 2.5 years) with epiglottitis the mean duration of intubation was 4.0 days (3 -5). no complications were seen. in 16 patients (mean age 2.3 years) with croup the mean duration of intubation until the first extubation was 8.1 days (1 -15 days). elective extubation was performed if an airleak was present or after 7 days without airleak but in the absence of fever and obvious secretion. reintubation was not necessary in 10 children (62.5%). in this group the mean duration of intubation was 6.4 days (1 -12). in 6 patients (37.5%) reintubation was necessary because of severe upper airway obstruction due to granulomas. mean duration of intubation until the first extubation was 10.8 days (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) . there seems to be a difference in duration of intubation between these two groups with croup, however it is not significant (p > 0.1). all the patients with granulomas could be successfully extubated after microlaryngeal surgery, with a mean intubation period of 35.3 days (21 -47). revealed no complications, where as endotracheal intubation in children suffering from croup showed a high incidence (37.5%) of granulomas. however1 laryngeal steepsis and other serious complications were not sesn~ 3 patients (42 days averagely] was obviously seen in ~he peak =one of fl, f2 resonance and in the zone of high freq,-~ncy :r, ~;~e composition while 12 cases(3 day~ average;y] :~bowed no abnormality both clinically and isryngoscopica!~y. 7/10 patients with catheter placement for more than 6 week~ end 1126 p~tie,~ts for less than 5 weeks had t;~ryngeal abnormal change in their larynges,abnormal changes of sound spectrogram were all seen in 3 patients with placement for mope than 5 weeks. our data suggest= ca] the complication of endotracheal intubation was increases with increasing length of time of catheter placsm. entjbut aeriuoa complication is rare i (b] the time limit of pernasal endotraoheal catheter placement is 5 weeks within which the procedure is • comparatively safe and effective means for maintaining e tong term artificial airway. in a 6-year period (1986) (1987) (1988) (1989) (1990) (1991) (1992) we diagnosed tbm as an apparent dilatation of the trachea and main bronchi ih four premature infants on continued mv for respiratory distress syndrome (rds). the infants were three boys and one girl with gestational age (ga) 26-33 weeks and body weight (bw) 1100-1965 g. mv was provided by bourns 2001 cub time-cycled and pressure-limited ventilator to attain normal gas tensions. no jet ventilation was used. chest radiographs were reviewed for a complete evaluation, and for the evaluation of the airway. after the intial subjective diagnosis of tbm, the width of the tracheal and main bronchial air column was measured at the lower level of the first and the third thoracic vertebal body it1, t3) and near the carina; the width of the main bronchi below the carina was also measured. in all infants, tbm became apparent close to the 20lh day, that is, after 2-3 weeks of mv. therefore, for the time period from birth to the 20th day the following ventilatory parameters were reviewed and analyzed: (1) the percentage of total ventilation time when more than 40% o2 concentration was required, (2) the peak inspiratory pressure, (3) the positive end-expiratory pressure, and (4) the duration of high frequency ventilation (80-160 breaths per minute). also noted were the apgar scores (1 and 5 min after birth), the duration of hypotension (systolic bp below 40 mmhg) and circulatory instability, the presence of systemic or tracheal conatal or later infection, the duration of mv, and the final clinical outcome. the records were also reviewed for other possible pertinent data. rigid respiratory endoscopy in children fraga j, amant6a s, piva j, nogueira a, palombini b. introduction: the respiratory endoscopy is an important procedure to diagnose and treat many airway's diseases in children. although have had advances in radiologic investigation exams and pulmonary function tests, the direct anatomic visualization of airway is important to the management of many respiratory problems. objective: evaluation the respiratory endoscopies performed with a rigid bronchoscope in a pediatric reference hospital. material and methods: we study the records of all children that were submitted to respiratory endoscopy under general anesthesia from march 1989 to march 1992. age, sex, clinical to indicate the procedure, diagnosis and complications of endoscopy were registered. results: three hundred and fifty six respiratory endoscopies were performed. the most common indications for endoscopy were strider (52%), suspected foreign body (16%), atelectasis (16%) and difficult tracheal extubation (8%). the most frequent diagnosis were laryngomalacia (36%) and subglottic stenosis (6%) in the glottic and subglottic areas, and foreign body (9%) and tracheomalacia (7%) in the tracheobronchial area. normal endoscopy was performed in 54 (21%) of the children. only three slight complications of the endoscopy were observed. two patients presented bradycardia during the exam, and the third need tracheal intubation due to post-endoscopic subglottic edema. conclusion: the rigid endoscopy in children is efficient and has no serious complications. near drowning; indicators of acute and long term prognosis bernardien t.mj. thunnissen t, reinoud j.b.j. gemke 1, loes veenhuizer?, krijn haasnoot 3, a.johannes van vugh0 department of pediatrics, ~wilhelmina children's hospital, utrecht, 2sophia hospital, zwolle, and ~free university hospital, amsterdam, the netherlands. in this retrospective study factors that affect short and long term prognosis after submersion were analysed. all patients that were admitted to a tertiary pediatric icu between january i, 1986 and january i, 1992 were included. of 34 patients, aged 0-13 years, 8 died in the icu, one after hospital discharge. survivors and non-survivors showed significant differences with respect to central temperature, pupillary reactions, arterial ph, pediatric risk of mortality (prism) score and therapeutic intervention scoring system (tiss) upon admission (p < 0.05). non-survivors more frequently required mechanical ventilation, bicarbonate administration and active reheating. ards was seen in 22 patients (65 %), invariably within 6 hours after admission. no patients with cardiac arrest on" admission snrvived without sequelae. hypothermia appeared to have no protective effect on hypoxic damage. survivors with persistent sequelae _> 6 months after discharge had significantly higher prism and t1ss scores (mean 27 and 34, respectively) than those with complete recovery (mean 14 and 23, respectively). long term cognitive problems were present in 7/25 survivors (28%) and emotional disturbances in 5/25 (20%). in conclusion, a concise number of clinical and laboratory parameters, representing acute severity of illness, are important prognostic indicators for survival and health status of children after submersion. there were 59 (91%) bronchoscopies, and 6 (9%) were oesophagoscopies.the average age was 2,8 years for bronchoscopies, and 4 years for oesophagoscopies. the outcome of the patients was good. no complications were observed. extraction is recomended in every symptomatic patient. orphenadrine is an anticholinergic drug mainly used to decrease symptoms of parkinson disease. orphenadrine has a peripheral and central effect and overdose can result in athetoid movements, convulsions, cyanosis, coma, arrhythmias, shock and cardiac arrest. physostigmine is a specific antagonist of the peripheral and central effects and can be a useful antidote. we report the case of a two and a half year old female who was transfered to our icu for general convulsions. the little girl had, three hours before admission, accidently ingested 400rag of orphenadrinehydrochlodde (disipal®), which was her grandmothers anti-parkinson medication. three hours after ingestion she presented neurological signs: confusion, unstable walking, and periods of aggression. generalized tonic-clonic seizures appeared who were rebel to administration of multiple anti epileptica but ceased after iv administration of diazepam and endotracheal intubation and ventilation. an episode of ventdcular tachycardia responded well to the iv administration of tidocaine. the levels of orphenaddne in the serum were high at admission (3550pg/l) and were present in the blood up to 96 hours after ingestion. high serum levels are, in the literature, associated to a high mortality rate. physostigmine was administered three times at a 0.02mg/kg dose in the first 24 hours. we decribe the noted effects of physostigmine on the different symptoms. the patient survived and could leave the icu after one week. in conclusion: orphenadrine poisoning is a very complicated medical problem associated with high mortality. in severe intoxication, the benefit of physostigmine more than counterbalances its side effects. objective: to define the optimal volume of dilution for endotracheal (et) administration of epinephrine (epi) design: prospective, randomized, laboratory comparison of four different volumes of dilution of endotracheal epinephrine (1.2, 5, and 10 ml of saline) setting large animal research facility ofa universi~ medical center subjects and interventions: epinephrine (0.02 mg/kg) diluted with four different volumes ( 1, 2.5. and i 0 rot) of normal saline was injected into the et tube of five anesthehzed dogs. each dog served as its own control and received all four volumes in different sequences at ieast one week apart. arterial blood samples for plasma epinephrine concentration and blood gases.were collected before and 0.25, 0.5. 0.75_ 1.2.3, 4. 5. 10, 15.20, 25.30 , and 60 minutes after drug administration. heart rate and arterial blood pressure were continuously monitored. measurements and main results: higher volumes of diluent (5 and i0 ml) caused a significant decrease of pao2, from 147:!:8 tort to 106±i0 torr, compared to the tower volumes of diluent (1 and 2 ml), from 136±10 torr tu135+_7 torr (p<0.05). these effects persisted for over 30 minutes. mean plasma epinephrine concentrations significantly increased within 15 seconds following administration for all the volumes of diluent. mean plasma epinephrine concentrations, maximal epinephrine concentration (cmax), and the coefficient of absorption (ka) were higher in the 5 ml and 10 ml groups. the time interval to reach maximal concentration (tmax) was shorter in the 5 ml and 10 ml groups. yet these results were not significantly different. heart rate. systolic and diastolic blood pressures did not differ significantly between the groups throughout the study. conclusions: dilution of endotracheal epinephrine into a 5 ml volume with saline optimizes drug uptake and delivery, without adversely affecting oxygenation and ventilation. the aetiology and outcome of paediatric out-of-hospital cardiac arrest was studied during a 10-year period in southern finland served by physician staffed emergency care units. the files of 100 prehospital patients less than 16 years old without palpable pulse and spontaneous respiration were analysed retrospectively. fifty patients were declared dead on the scene (dos) and resuscitation (cpr) was initiated in 50 patients. the sudden infant death syndrome was the most common cause of arrest (68%) in the dos patients as well as in patients receiving cpr (36%). asystole was the initial cardiac rhythm in 70% of the patients in whom cpr was attempted. eight of the 13 hospitalised patients were discharged, 6 of them with mild or no disability, 1 with moderate disability and one in vegetative state. in multivariate analysis the short duration of cpr (<16 minutes) was the only factor significantly associated with better survival. due to various aetiologies the survival rate from prehospital paediatric cardiac arrest is quite low. on the other hand, hypothermic near-drowning victims seem to have a relatively good prognosis. duration of cpr less than 16 minutes was the best predictor of intact survival, our study supports the previous findings of the importance of early and effective resuscitation efforts for establishing ventilation and perfusion on the scene. in our system well trained physician staffed emergency care units are able to provide immediate and effective als on the scene. on the other hand, these units also appear to be able to refrain from resuscitation when the prognosis is pessimistic. objective: to assess the normal ,gastric intramucosal ph ~hi) by tonometry in healthy children patients and methods: twelve healthy children (6 males and 6 females) with age rmaged from 6 months to 12 years scheduled for minor plastic or urologic surgery. children were previously medicated with midazolam (0.25 mg/kg) and atropine (0.02 mg~) both i.m.. anaesthetic induction was standardized with 02 -n20 (75%) administered via facial mask and increased halotane concentrations (up to 2%). all patients got an endotraeheal tube after iv. administration of femanile (2 mcg:jkg) and vecuronium (0.1 mg/kg) or suxametonio (1 mg/kg), pmaesthesia was maintained with o 2 -n20 (60-75%) and isofluorane (0.5-1%). during surgery, 8 children needed mechanical ventilation and the others maintained spontaneous breathing. ekg, heart rate, blood pressure, and pulse oximetry were moniterized. after anaesthesia, a sigmoid tenometry catheter (tonometrics, inc.) was inserted in the stomach of the patients by direct visualization with laryngoscope and magyll clamps. children were all maintained normoventilated and with normal cardiorespiratery variables. cadet's balloon was £~led with 2.5 ml of saline. thirty minutes after the insertion 1 rrd was extracted and rejected, just afterwards the remanent 1.5 ml was extracted and immediately analyzed. simultaneously an arterial gasometry by puncture was performed. gastric phi was calculated by the henderson-hasselbalch's equation using the pco 2 obtained from the tenometry catheter and the bicarbonate value obtained from the arterial gasometry. results: average gastric phi was 7.34 -i-0.027, range (7.29-7.46). objective: demons~ating intramucesai ph (phi) alterations during transport of patients from operative room to pediatric intensive care unit (picu), material and methods: phi measurements were performed with gastric tonometer catheter in t4 patients undergoing cardiac surgery with cardiopulmona d" bypass (cpb), there was 9 mate and 5 female, the average age = 3yl0ra, average weight = 12,5 kg, average time of cpb = 70 rain. the measurements were made at the end of the surged' and when the patients had arrived in the picu statistical aualysis: average and ~andart deviation and test "t" student. objetive: to asses the efficacy of gastric iatramucosad ptt (phi) and arterial lactate levels to evaluate splacalc tissular perfusion in an experimental model of intestinal ischemia. suneets ~nd methods: twelve piglets weights t3-20 kgs. undergoing orthot~ie liver trasplantation. the intestinal ischemia was induced by aortic damping. tonometry catheter (tonometrics inc.) w~s placed in the stomach after artaesthesia and ot intubation. phi ~s determined 7 times and lactate levels was determined fi times in 3 stages: i) pre-ae~hepatic stage (twice: before surgery and before aortic clamping ); ii) end anhepatic stage (only phi): iii) reperfusion stage (a 30, 90, 120 and 180 minutes). the phi was derived from application of the henderson-hassdbach formula using the pco2 value from the tonometer and the arterial bic~rbonate. all pipets received raaitidiila before sttrgery. values of phi above 7,35 and lactate levels between 6 and 15 mg/dl were considered nortrm. the results were statistically anaj.izated with anova and bonferroni tests. results: the phi was normal on pre anhepatic stage (> 7,35) and lactate levels were slightly increased (21, 5 +_ 8, 9 and 19, 5 ±5, 9mg/dl ns) . in relalion to we-anhepatics values, phi decreased signncatly at the mid of anhevatic stage (7,39_+0,14 vs 6,94_+0,1 p<0,001), phi remain low in stage iii, at 30 rain (6,86+0,12 p< 0,001) and 90 min(g94-+o, 12 p< 0,001). arterial lactate levels increased significatly in relation to levels in stage i, at 30 rain (63,6_+9,7 p<0,o01) arid 90 rain (65,8±9,9 p<0,001) of reperfusion stage. there is a slight improvement on phi and lactate ievels at 120 and t80 rain althought the differences did not reach significance. cnmments: phi and arterial lactate levels propperly reflect hypoperfusion on the experimental model of acute intestinal isdlemia. b~kground : the paediatrie gallbladder diseases generally described are calculous ¢hol~tstitis, cystic duct obstruction, congenital anomaly of the biliary tract, and inflammation. in the neonatal period, noulithogenie gallbladder disease could be also due to erythroblastosis or hyperalimentation. obieetive : we describe an other type of disease affecting the gallbladder in neonates thought to be related to their vascular vulnerability. methods : four patients with abnormal gallbladder ultrasound not related to classical observations were included. we have studied and reviewed the biological and clinical data, the ultrasound findings and their evolutions. results : four patients, 30 to 32 ~.k-old neonates ~ffth a birthweight be~,een 1,3 and 1,9 kg, were intubated and under total parenteral nutrition for 10 to 35 days. none of them were symptomatic on repeated clinical evaluations. one newborn developped hypotensien on umbilical bleeding at 3 hours of life. in two cases, signs of cholestasis were discovered : the total bilirubin level has risen to 5 mg/dl; the direct bilirubin level was 1,5 mg/dl while the urina were dark and the ~o~,ls :mcolour~. the c~mplct~ ~crology as a!! the culvare~ remained negative. the ultrasound explorations were atypical : in the four eases, an initial increasing broad and thickness of the wall of the gallbladder with an hyperecbogenie inside content, which was not sludge, was discovered. in three eases the images resolved in ten to fifteen days. in one ease, an asymptomatie thrombosis of the vena portu which remained patent was discovered. in this case, at one month, the ultrasound showed images encountered in chronic ebolecystitis and, at one year, the gallbladder appeared atrophic. none of them underwent surgery. conelusiou : the gallbladder diseases are multifactorial. besides the prematurity, the infections, the total parenteral nutrition, the premature neonate is exposed to vascular vulnerability affecting also the gallbladder and this may explain our findings. progress in prognosis of pts with b-nhl had followed the use of multimodality chemotherapy (ct). with the prolonged survival, there are comlications due to myetosupression & desease process. the syndrome of neutropenic enterocolitis (ne) is one of the ominous problems because ofpts increased susceptibility to infection & overwhelming sepsis. this material included 25 neutropenic pts (4-14 years) with the stages iil& iv of b-nhl who were treated with the modifired bfm-90 (mtx 1 g/m 2 in 24-h inf.); 22 males, 3 females. seventeen episodes of ne were observed & only after the first 2 courses of ct (13 of 25 after tst, 53%; 4 of 24 after 2nd, 17%). the symptoms existed 3 to 14 days. wbc ranged from 50 to 600 in l~tl (median, 100). the first signs of ne were directly correlated to the beginning of the neutropenia & the recovery of neutrophils led to the disappearance of abdominal recovery of neutrophils led to the disappearance of abdominal pain. the conservative treatment included gastrointestinal tract decompression, broad spectrum antibiotics initially, volume & electrolyte substitution, nutritional support, correction of acid-base balance, symptomatic treatment. sixteen pts were treated nonoperatively, 1 died. on autopsy the transmural bowel necrosis due to thrombosis of branches of a.mes.sup, was found. the bowel perforation occurred in one patient, he was undergone laparotomy & hemicolonectomy & survived. we conclude that ne is a frequent complication in neutropenic pts with the st. lii& iv of b-nhl. it occurs after the induction courses of ct. close observation by surgeons, oncologists & pediatric intensivists is mandatory. conservative treatment is effective & more preferable until leucopenia resolves. operation is necessary only for those.with perforation. near infrared spectroscopy as a tool for evaluation of intestinal perfusionpresentation of an animal model. c. scheibenpflug, p. buxbaum and a.m. rokitansky the recent development of and investigations in the so called near infrared spectroscopy ( nirs --transcutanous emission and simultaneous registration of intensity of spectralcolours depending upon modulations of tissue perfusion ) enable physicians to measure and qualify organ perfusion and nowadays is mainly used to control cerebral as well as skeleton muscular blood flow in trauma patients at intensive care units ( icu ). today intestinal perfusion, hypoperfusion , cell damage caused by reperfusion injury, bacterial and toxin translocation are serious problems in critically ill patients at an icu. paediatric intensive care physicians put major concern on intestinal perfusion, which for. instance gains more and more importance, especially in the neonatal period for example as an etiologic factor for necrotizing enterocolitis. we established an animal model, in which we measured intestinal perfusion by nirs under various invasive and noninvasive conditions. methods and results will be referred. for preliminary conclusion we propose near infrared spectroscopy ( nirs ) also as a potent diagnostic tool to determine early intestinal malperfusion in order to prevent lethal outcome. fm'ther investigations in animals as well in paediatric iritensive care patients should be done to estimate our efforts. introduction: following the acute phase of necrotising enterocolitis (nec) starvation of the gut for a period up to 3 weeks is a generally accepted treatment modality in many centres. objective criteria to refeed these patients are hardly available. recently the double sugar test has become available as a parameter for (ab)normal gut permeability ~'2. aim of the study: to evaluate the changes in permeability of the small bowel in patients with nec and controls before introduction of enteral feeding. methods: a lactulose! rbarrmose (i/r) test was performed in two groups. group 1 was studied 2-3 times within a 5-week period of starvation (n=5, mean gest. age 35, range 31-40 weeks). in group 2 seven different control patients were studied (mean gest.age 33, range 28-38 weeks). the test was performed by giving a patient after at least a 4 hour fast 1 ml/kg bodyweight l/r solution and determination of the 1/r ratio in a 4-hour urine sample by chromatography. results: objective: to evaluate the prognostic factors in the response to nitric oxide (no) in children with acute respirator/ distress syndrome (ards) and/or pulmonary hypertension (pht). patients and methods: 23 critically ill children received no inhaled for ands and/or pht treatment. 14 patient before and after cardiac surgery (2 cardiac transplants), 5 with bronchopneu~onia, 2 multiple trauma, 1 sepsis and 1 cardiorespiratory arrest. 15 patients showed /j~ds and 8 pht, in 4 with associated ards. we analyzed age, sex, diagnosis, pao2, pa02/fi02, oxygenation index, pht, shock, and sepsis as prognostic factors and response factors to n0. results : after no administration oxygenation did not improve in 2 patients (8.6 %) and pht did not diminishe in one children (12 %). 12 patients survived (52 %), 8/15 (53.3 % with /d%ds) and 4 /8 (50 %) with pht. the four patients with isolated pht survived , and the 4 patients with pht and ards dead. patients after cardiac surgery presented less mortality (35.7 %) than the rest of patients (66.6 %). patients with shock presented higher mortality (64.2 %) than the rest of patients (22.2 %). there are no differences in response to no in respect of sex, age, diagnosis, shock, and sepsis. survivors showed higher increase of pao2/fi02 64.3 ± 58.4 to no than non-survivors 48.4 ± 51.1 (n.s). patients with pht showed higher increase in pa02/fi02 to no administration ( 88 ± 47.1) than patients with ards (43.4 ± 50.8), (n.s), but patients with ards showed a higher increase in 0!, 15 ± 6.7, than patients with pht 4.8 ± 4 (p < 0.05). patients with pa02/fi02 < i00 showed less increase in pa02/fi02, 47.8 ± 46.3, than the rest of patients 82.8 ± 65.5 (n.s) conclusions: i. mortality of isolated pht treated with no is less than patients with ap~s. patients with shock and those with pht and ards showed higher mortality. 2. we have not found any clinical or analytical factor to predict clinical response to no administration. 14 patients showed ards, and 9 severe pht after cardiovascular surgery, in 5 with associated ards. we registered respiratory assistance, blood gases, pao2/fi02, the oxygenation index (oil, and mean pulmonary pressure/ mean systemic pressure (pap/sap) before and after no inhalation. we measured continuous concentration of no and no2 by electrochemical method (noxbox, bedfont, airliquide). results: no administration improved oxygenation mean pao2 from 74 ± 17 tm~g to i19 ± 54 ~g (p < 0.01), mean pa02/fi02 fr<xa 83 ~ 30 to 13£± 72 (p < 0.01)and 0i from 28 ± 14 to 20 ± 12 (p < 0.01). 2 patients did not improved with no administration. the oxygenation improved in the first five minutes and the best oxygenation was achieved with 5 -15 ppm. there is no significant change in pac02. pap/sap diminished from 62 ± 17 % to 42 ± 9 % ( p< 0.05), in one patient there is no response. the no administration was maintained between ~5 minutes to 47 days. the effect of no on pulmonary pressure and oxygenation was maintained without change during all the time it was administrated. n02 concentration were always less than 2 ppm y metahemoglobinaemia less than 3.5 %. there are no side effects secondary to no administration. 12 patients survived (52 %). concluaions:l.administration of no improves oxygenation in children with ~/eds and diminishes pht after cardiac surgery. 2.no effect is fast and maintained during the evolution. high frequency oscillatory ventilation in combination with inhaled nitric oxide g~thberg sylvia, md, edberg k e md, phi), dept of paediatric intensive care, children's hospital, s-416 85 g6teborg, sweden background: for man), years severely sick infants with congenital heart malformations or acute lung disorders have died due to pulmonary hypertension, hypoxia and multiple organ failure. today there are several therapeutic facilities coming up for these infants. ecmo progranunes have been introduced in severul centres as well as improved venlllatory techniques, as high frequency oscillatory ventilatior~ (hfov), which provides adequate ventilation with less risk for hmg injury.. in 1987, the endothelium derived relaxing factor was identified as nitric oxide (no), and extensive studies have sho~-a that inhaled no reduces pnimonm3 o vascular resistance and improves oxygenation in hypoxic infants with pulmona~ hypertension. material.. from july 1994 to january 1996, we have treated 13 severely hypoxic children with combined ttfov-no. seven were newborn, 3 with cdh, one with mas, one with irds, one with paediatric ards due to rsv infection and one with poor oxygenation atler open heart surgery. 6 were between 1 month and 9 years and all had ards of differentorigin but one who was hypoxic after open heart surgery. method: hfov was given by means of sensormedics 3100a oscillatory ventilator to 12 patients, and dr~ger babylog 8000 was used in one case. mean airway pwssures varied from 10-34 cm h20. oscillatory pressures varied from 25-85 cm h20 and ventilatory rates from 6-15 hz. fi o2 varied from 0,21 -1,0. no was administered by nomius classic. no and no2 was measured in the inspiratory limb with an electrcchemieal device. noconcentration varied from 2 to 20 ppm and the no2 -concentration between 0-0,2 ppm. methemoglobin was never more than 3,7 % (average 1,7). duration of treatment varied from 1 to 20 days. combined trealment with hfov and inhaled no improved oxygenation and carbon dioxide elimination in 12 out of 13 treated childretl 5 patients died, 3 due to their underlying congenital heart disease, one of asphyxia due to rsv-infection and one of cdh with progressive hypoxia and multiple organ failure. combined treatment with hfov and inhaled no improves oxygenation in severely hypoxic children. treatment should be slarted early to reduce the risk for chronic lung injury following barotranma and high oxygen concentrations. we speculate that the combined hfov-no may reduce the use of ecmo, and that it may improve outcome in ceotres where ecmo programmes are not introduced. apart from its vasodilative properties nitric oxide appears to act also in physiologic immune defense. intracetlular concentrations of no synthesized by macrophages are in the range of 103 above those produced by vascular endothelium. we investigated the bacteriostatic effect of nitric oxide at concentrations used during inhaled therapy for pulmonary vasodilation in neonates. ten different strains of five species were used (staph. anrens, staph. epidermidis, strop. group b [gbs], e. coil and pseudomenas aeruginosa), which are ~e most often tracheally isolated bacteria in mechanically ventilated premature infants and neonates. we compared bacterial growth of cultures applying three different concentrations of nitric oxide (40 ppm. 80 ppm, 120 ppm) m the growth of the same strains in room air for a duration of 24 hours. no bacteriostatic effect was demonstrable at no concentratioes of 40 ppm. e. coil showed decreased bacterial growth at 80 ppm and 120 ppm, however without reaching statistical significance (p=0.058). at nitric oxide concentrations of i20 ppm staph. epidermidis and gbs grew significantly less as compared to colonies of the same strains in room air. no effects were found regarding the growth of staph. aureus and pseudomonas aneruginosa. we conclude that nitric oxide has a selective bacteriostatic effect on some of the most often trachealb, isolated bacteria in premature infants and neonates. this effect appears to be dose-dependant and occurs in the upper range of dosages used with inhaled no therapy. further research is required in order to examine the mechanisms of action as well as specific interactions between different strains of bacteria and nitric oxide. the no,box monitor (bedfont, kent, uk) is used to monitor nitric oxide (no) and nitdc dioxide (no 2) during no administration in ventilated neonates. according to the manufacturers specifications the monitor can be applied in cases where airway pressures range from 5 to 50 mbar. we investigated in-vitro the accuracy of the no monitor in a range of ventilatory conditions. using a dr~ger babylog 8000 we ventilated an artificial lung. no was administered {800 ppm no in 100% nitrogen) with a mass flow controller to the inspiratory tube, at a distance of 20 cm from the y-piece. the no target value was set to 10 ppm. the ventilator was operated in both cpap and ippv modes at different settings. the no sampling tube was placed on two different locations; in the inspiratory tube dose to the y-piece; in the expiratory tube haft way between the y-piece and the ventilator. the no-monitor was ca)ibrated with 84 ppm no at 30 mbar. fig. 1 and 2 show the sensitivity of the no-monitor for respectively static pressure (cpap) and pulsatile pressure (ippv) with the sampling tube located in the inspiratory tube. fig. 3 shows the resur for pulsatile pressure with sampling in the expiratory tube. we conclude that the accuracy of the bedfont no-monitor is dependent upon airway pressure and sampling site, the latter possibly caused by incomplete mixing, pressure and no are linearly related when sampling occured further away from the administration site. based on this study we suggest to place the sampling tube in the expiratory tube. during neonatal care the clinician should be aware of varying inaccuracies depending on the respiratory pressures. hypothesis: availability of therapy with inhaled nitric oxide (ino) decreases ecmo use in patients referred .to a tertiary care hospital for treatment of respiratory, failure unresponsive to conventional therapies. background: at children's hospital of wisconsin (chw) treatment for patients who have failed conventional treatment for respiratory failure, sometimes called "rescue" therapy, has included ecmo since 1986 and high frequency oscillatory ventilation since 1992. ino has been in experimental use at chw since may of i994 and has resulted in the perception of a decreased need for ecmo therapy in those patients referred for rescue therapy. this study was designed to tbst the validity of that perception. study type: retrospective cohort. methods: the medical records of all 105 patients referred to chw from 1/lj93 to 4/1/95 for rescue therapy for severe respiratory failure were included in the chart review. data were collected regarding diagnoses, illness severity, hospital course including interventions and complications, and outcomes. exclusion criteria w~ere the finding of structural heart disease or congenital diaphragmatic hernia as the basis for hypoxemia. qualification for ino treatment included an a-ado2 -600 or oi ~-40 for two hours or -> 25 for twelve hours and echocardiographic demonstration of persistent pulmonary hypertension of the newborn. patients were classified into two groups based on the availability of ino at the time of their hospitalization. results: in the time period of the study, 105 patients were referred for possible ecmo therapy. twelve patients greater than 4 weeks old, 31 with congenital diaphragmatic hernia and 12 with congenital heart disease were excluded from this analysis, leaving 50 patients for study, ino availability reduced ecmo use from 16 of 34 (47%) patients in the ~ino unavailable" group to 2 out of 16 (12.5%) patients in the "ino available" group, p=&026 by fisher's exact test. the fact that the two groups were composed of patients of similar severity of illness is reflected by comparable rates of ecmo and ino rescue therapy (47% vs. 56%). conclusion: by providing an alternative rescue therapy, ino has reduced the need for ecmo in this group of neonates referred for respiratory failure. introduction: true hepatnrenal syndrome (his) is defined an acute renal failure {arf) in the presence of severe liver disease without other known causes of renal failure. hrs is frequently seen in the course of hepatic cirrhosis• in children, cirrhosis is rare; however, arf can be seen in combination with aseites and liver dysfunction• we describe 3 patients with hepatic dysfunction and aseites in combination with ar~ and abnormal sodium-water handling, leading to the diagnosis of hrs. pathophysiology: three factors are considered in the pathogenesis of hr~: i) hepatic dysfunction, 2) deranged hemodynamics, including abnormal blood pressure, reduced effective arterial blood volume and abnormal blood flew distribution, and 3) neuro-humoral dysrsgulatiom, including elevated levels of aldosteron, renin, angiotensin-ll, ade, vasodilatim 9 nitric oxide and vasoconstrictor peptide endothelin-l. the main pathogenetic feature is decreased cortical renal blood flow, decrease of glomerulur filtration rate (gfr), vastly increased sodium retention, uliguria, and azotemia. treatment: therapy is based on counteracting sodium and fluid retention by highdose aldosteron antagonists and loop diuretics, improving renal perfusion by lowdose dopamin, and strict restriction of fluid and sodium. interventions as paracenteals of aacites or n peritoneo-systemic shunt are associated with high morbidity and poor outcome in children. reversal of hem by conservative measures can only be attained at early stages of hrl liver transplantation is the only definitive treatment that can reverse ere at advanced stages. patients: the described patients developed severe ascites with insidious renal dysfunction and abnormal sodium-water handling during admission at picu and fullfilled clinical criteria fur hrs. treated according to the cited principles, all patients showed improvement of gfr, with increased natriuresis and gradual decrease of ascites. eventually, renal function normalised completly. conclusion: ere deserves greater recogmitimn in the picu population; diagnosis can be suspected on clinical criteria. with this increased awareness, therapy tun be instituted at an early phase, with better prospects for recovery. positive outcome of hem depends on early recognition of the clinical picture, understanding of the pathophysiology, and early institution of consistent treatment. mtx is an antimetatxflite widely used as chemotherapeutic agents. high dose ivitx (i to 30~m2) administered as a prolonged intravenous infusion (over 4-42 hours), is often used to treat malignant paediatric diseases. major complications of this treatment are myelosuppression, orointestinal mucositis, dermatitis and impairment of anal function. we report two cases of mtx overd~age occurred in two children (5-year-old. 14 month-old) t~ted for acute lymphoblastic leukaemia. they were treated by cavh and the mtx bhk~d levels rapidly decreasedavoiding multisystemic involvement. establishment of alkaline diuresis and monitoring of plasma mtx levels during treatment is essential to prevent nephrotoxicity. however. leuco',cnn rescue may not prevent the development of potentially lethal toxicities in patients with mtx concentrations persistantl} exceeding t0mm. in theses cases, em'ly treatment of mtx intoxication may pm~cnt myelosuppression and reducerenal damage. the goal is to lower the concentration to below 10 mmoll, at which time rescue agents aleme would be expected to be cllcctive. respective indications of these remo',at mctny.:is are still discussed : hacmt~ialysis t~ eharc(~l haemoperfusion should be prolx',sed for massive and acute intoxication. however, rebound has been reported after combined hcmodialysis and hemoperfusion. exchange transfusion may be proposed as a treatment for prolonged and moderate intoxication. peritoneal dialysis is an incflbedve method for remo~ al of mtx. cavh was used in our icu. cavh is a simple method for blood purification and n':dy iluid control. use of cavh was never be reported in this indication to our knowledge. simplicity, rap~d application and gco.l clinical tolerance are the main advantages of this technique. the technique presents ~peclal advantages in terms of low priming volume of extracorporeal circuit, low blood flow, low rate heparinisation. our results show a decreaseof plasma mtx concentration and a rapid reduction of halfqite of elimination (t5 hours over the period of cavh). moreover, we didn't delec~d rebound after stopping prc,xedure. small size of the i:ratients may present sometime special problems, but these technical problems can be overcome, no severe complication (needing, inlection) were observed during filtration, in summary, aggressive intravenous fluid hydration and alkaliniaation of the urine coupled with careful monitoring of renal function and plasma mtx concentrations during and al'tcr infusion along with lem~overin rescue has reduced the inndcace of life-threatening toxicity after highdose mtx. however, some mtx inu>xication still occurred, leading to se~em toxicity, particularly nephrotoxicity. in these cases, we think that cavh (or cavhd) is a reliable, rapid method without rcix~und increase in plasma mtx concentration or important adverses effects compared to other procedure removal. gouyon jb, germain jf, semama d, pr6vot a, desgres j preliminary limited data suggested that hemofiltration and hemodiafiltration may be valuable in some neonates with decompensation of maple syrup urine disease (msud). venovenous hemofiltration (vvhf) and hemodiafiltration (vvhdf) were performed with a new neonatal hemo(dia)filter (miniflow 10, hospal) on 8 anesthetized rabbits infused with branched-chain amino acids (leucine, isoleucine and valine) and c~-keto-isocaproate. the bcaa and aketo-isocaproate blood levels were close to those previously observed in neonates with msud when extracorporeal blood purification was required. vvhf and vvhdf performances were assessed with two different blood flows (qb = 8.3 and 16.6 ml/min). vvhdf was performed with 4 dialysate flow rates (qd = 0,5, 1.0, 2.0 and 3.0 l/h). thus, each animal was submitted to 10 successive procedures. within each studied period, clearances of the 3 bcaa were strictly similar. bcaa clearances obtained by vvhf were similar to ultrafiltrate rates (respectively, 0.78 4-0.14 and 1.79 4-0.28 ml/min at high and low qb ; p < 0.05). the ~x-keto-isocaproate clearances obtained by vvhf were 0.39 4-0.17 and 0.92 4-0.43 ml/min at low and high qb (not significantly different). whatever qd value, the vvhdf procedures always allowed higher bcaa and c~-keto-isocaproate clearances as compared with the corresponding v'~hf period with similar qb. bcaa clearances obtained by vvhdf with a 0.5 l/h dialysate flow, were 4.1 4-0.5 mljmin and 5.4 4-0.5 ml/min at iow and high qb, respectively. the concurrent a-keto-isocaproate clearances were 2.5 4-,. 0,8 ml/min and 2.9 _+ 1,0 ml/min. at both qb regimens, bcaa clearances provided by vvhdf were markedly higher than values previously obtained with peritoneal dialysis in human neonates with msud. the management of renal failure in the newborn is difficult. when dialysis is instituted peritoneal dialysis (pd) is usually the technique of choice. this is can be problematic and impossible in some patients with pre-existing intra-abdominal pathology. continuous arterio-venous haemofiltration (cavh) has been described in infants but sick preterm infants are not able to support the circuit. i have devised a means of having pumped haemofiltration in small/preterm infants (phis/pi) and describe its use in nine patients ranging in size from 750 to 3000gms for periods of 1 to 7 days. vascular access was achieved through 24 or 22 guage cannulae in either a peripheral artery and a central vein or through two central veins. blood was pumped out using an ivac 572 infusion pump and through a gambro fh22 haemofilter. a second ivac pump was used to remove haemofiltrate from the filter and a third to infuse replacement solution. removal rate was set to give a clearance of 15mls/min/1.73sq.m and blood flow rate set to between 5 and 10 times the removal rate. heparin was infused into the circuit to prevent clotting of the filter. biochemical and fluid balance control was achieved in all infants. guaranteed fluid removal allowed the administration of full nutritional support. four patients died when treatment was withdrawn because of an untreatable underlying problem. one recovered renal function but died some weeks later from unrelated problems, three survived and recovered renal function and one patient is still on treatment. this system allows a secure means of achieving fluid and electrolyte control in the preterm infant. the use of this technique may allow haemofiltration to become as applicable to preterm infants as it is to older children and adults. unibrtunately, children often receive no treatment, or inadequate treatment for pain and painful procedures. this prospective, multicentric study focuses on the efficacy, safety and side effects of novalgin (metamizol sodium) for this indication. patients and method: novalgin was administered to 56 children, aged between 6-16 years, with acute, postoperative or procedural pain. novalgin (10-15 mg/kg) was given 6-8 hourly iv or im respectively, in some cases (15) in combination with opioids (tramadol 10, piritramid 3, butorphanol 2). the pain relief was assessed by six-step verbal rating scale (vrs) from 0 to 5, vital signs were monitored, the side effects, that occured were recorded. results: pain relief was good (vrs less 2) in 53 children -94.6 % of study patients. novalgin was very well tolerated, only one patient had adverse reaction -hyperpyrexia following intravenous application of the drug. discussion: novalgin (metamizol sodium) is safe and effective drug in the management of acute pain in children with low incidence of side effects. obie~qve: a prostx~tive study comparing simultaneous, indepeadent ratings conducted by intensi~4sts using an american (comfort) and an european chartwig) sedation scale for mechanically ventilated pediatric patients. measurements and results: the study comprised 30 observations in 18 mechanically ventilated pediatric patients (aged 16 days to 5 years) in a pediatric intensive care unit (from march 1995 to january 1996 . each patient was sedated by his/her managing physician with opiates, benzodiazepines, barbiturates, used isolated or in combination. each observation consisted of a 3-mid period of oly~ervatien of the patient in his or her pediatric icu bed, after each observation, the comfort (analyses 8 dimensional physiologic and behavioral subscores -range 8 to 40 paints) and hartwig (analyses 4 dimensional behavioral subsenres -range 8 to 25 points) were performed by the intensivist. we established the comfort scores ~ correspanding to adequate (range 17 to 26), excessive (range 8 to 16), and inadequate (range 27 to 40) sedation; and, hartwlg scores z correslxmding to adequate (range 15 to t8), excessive (range 8 to 141, and inadequate (range 19 to 25). statistical mmlysisj: agreement rate (kappa) and p <.01 was considere d s!l~nificant. comfort 18 (60.9%) 2 (6,6%/ 10 (33.4%) hap, twig , 17 (56.6%) 7 (23.4%) 6 (20.0%) to the comfort score, the average for adequately sedated, inadequately sedated, and too sedated was 20.28+-2.78, 2z5_+0.70, and 15a.+_l10, respectively. and to the ha~twig scorn, the average for adequately sedated, inadequately sedated, and too sedated was 16.35:k-'0.77, 20.85-&l57, and 13.0l-0.89, respectively. conclnsion: in our study there were no significantly statistical difference when you apply a more complex scale (conff'ort) or a less complex scale (hartwig) to assess the sedation of mechanically vemilated pediatric patients. the application of local and intravenous morphine infusion after surgery of urinary tract eva nemeth , m.d. semmelweis medical university , first oepartment of paediatrics , budapest , hungary in±roduction:continuous analgesia with morphine may be ~egaroed as a safe and effective method of pain relief during postoperative period. subjects and methods: 24 children /mean age 2.3 years/ underwent elective ureteroneoimplanta±ion were randomly selected to receive either morphin intravenously of lo ug/kg/h /group one/ or bladder morphineinfusion 50 ug/kg/h /group two/ after surgery. all patients were prospectively evaluated during their s±ay in the postanaesthetic care unit. cardiac and respirafory rates,blood pressure,sa 02 ~,degree of alertness,pain perception and complaints of the paticnto ~cr~ recorded hourly. pruritus,nausea and vomiting,voiding difficul-±ies,sedation,dysphoria were systematically sough and quoted. statistical analysis was performed by chi square test. results:postoperative analgesia was the same in the two groups,but side effects were less in the bladder morphine group,because of the lower se morphine concentration.the differentes weren't significant in two groups. conclusions:the administration of bladder morphine infusion is a safe and effective method in children. objetive: compare the evaluations of sedation level made by physicians and nurses with the visual analog scale (vas) and the comfort scale (cs) in pediatrics patients receiving difforents modes of intravenous sedation. material ~ method." file evaluations were made by an attending physician and nurse with the vas and by another physician (always the same) using the cs. the observations were divided following the sedation mode: one drug (fentanyl or midazolan), two continuous drugs, one continuous and one intermi~ent drug and two intermittent drug (fentanyl and midazolan). the groups were compared using the t-student test. the groups also were compared between the percentual of agreement of the evaluations of sedation level made by physicians and nurses with the cs and vas using the x 2 . results: we didnk find any statistical difference between the observations made by physicians and nurses with the vas in the differmts modes of intravenous sedation, the average of the observations using the cs betwom one drug and two drugs modes didnk exhibit also statistical difference. the observations made by physicians mad nurses using the the vas when compared with the cs didn't show statistical difference between the sedation level. we found statistical difference only in percentual of concordance of sedation level between physicians and nurses when compared the one and two drugs modes of sedation. conclusion: we didn't find differences in the observations made by physicians and nurses in the sedation level, only in concordance pereentua/ of observations when compared two modes of sedation. the observations using the cs (more complex) didnk show differences when compared with the vas. effects of age, concurrent administration of other pharmacologic agents, and disease [cardiac(n=31) & pulmonary(n=22)] on the pk & pd of b were evaluated in volume overloaded infants aged 4 days-6 mo (n=53). single doses of 0.005,0.01,0.015,0.02,0.025, 0,03, 0.035,0.05 & 0.10 mg/kg iv were given over 1-2 min after baseline evaluation. age was used as a continuous vadable to determine its effects on the variability in the pk & pd of b. values for pk parameters were compared between patients in cardiac and pulmonary disease groups. hierarchical multiple regression analyses were used to determine the effects of age, disease and other pharmacologic agents on the variability of bumetanide excretion rate (ber) and pd responses, e.g. urine flow rate (ufr) & electrolyte excretion. cit, cir & cinr increased with age (p<0.05) while t,2decreased markedly in the first monthe of life (p<0.05). ber normalized for dose increased with increasing age. patients with pulmonary disease exhibited significantly greater clearance and shorter t~= (p<0.05) than those with cardiac disease whereas vd~ was similar in both groups. the administered dose of b was the primary determinant of ber but increasing age also contributed. penicillin antibiotics decreased ber. dose response curves for ufr and electrolyte excretion were similar between disease groups. more of the variability in ber and pd responses could be accounted for in the pulmonary group than the cardiac group but this was not statistically significant. conclusion: the pk of bumetanide were influenced significantly by age and disease. differences in pk between patients with pulmonary and cardiac disease were primarily due to differences in total clearance. age and the administered dose of b were positive determinants of ber and pd responses while penicillin antibiotics had a negative impact on both, once b reached its site of action, no differences in pd responses were detected between disease groups. the pharmacodynamic effects of bumetanide were evaluated in volume overloaded infants (n=56) aged 4 days-6 months. single doses of 0.005, 0.01,0.015, 0.02, 0,025, 0.03, 0.035, 0.05 & 0.10 mg/kg iv were given over 1-2 rain. bumetanide concentration in blood (n=l 0) & urine (n=6) samples were quantified by hplc. baseline urine samples were collected over 2-4 hours prior to drug administration. determinations of urine volume, electrolytes (na ", k +, ci, ca ++ and mg++), creatinine and osmolality were performed before and at 0-1, 1-2, 2-3, 3-4, 4-6 and 6-12 hours after bumetanide dosing. changes in urine flow rate and electrolyte excretion were plotted as a function of bumetanide excretion rate which was considered the effective dose of the drug. peak bumetanide excretion rate increased linearly with increasing doses of drug and showed no evidence of approaching a maximum. time course patterns for urine flow rate and electrolyte excretion were similar for all dosage groups. urine flow rate and electrolyte excretion increased lineady up to a bumetanide excretion rate of approximately 7 #g/kg/hr and either plateaued (urine flow rate) or declined at bumetanide excretion rates > 10 #g/kg/hr. bumetanide had no detectable effect on serum electrolyte concentrations, conclusion: maximal diuretic responses occurred at a bumetanide excretion rate of about 7 ;~g/kg/hr. higher bumetanide excretion rates produced no increased diuretic effect. peak bumetanide excretion rate of about 7 #g/kg/hr corresponded to bumetanide doses of 0.035-0.050 mg/kg. neonates using an electrical syringe-pump. authors: tr~luyer j.m., sertin a., bastard v., settegrana, c., bourget p., hubert p. background and objective: many problems can be observed with drug administration by i.v. route, especially in neonates. so we evaluate different protocols of teico delivery using an electrical syringe-pump. methods: we simulate infusion of teico with a syrlnge-pump (pilot c, becton & dickinson lab.) trough d standart neonatal i.v. system. for 2 weights (1 or 3 kg) we used 2 doses of teico (8 mg and 16 mg/kg) and a dose volume _<4.2 ml. our goal was to perform a complete infusion in 10 minutes. the infusion system consisted of an life care 4 infusion pump (abbott lab.) with its lv. set for maintenance intravenous fluid (flow _< 6 ml/h) connected to a 3-way stopcock. an 1 meter extension tubing was placed between the stopcock and a neonatal catheter. an another 1 meter tubing (injection tubing) connected the teicoplanine syringe to the stopc, ock. the volume of the injection circuit (from the syringe to the distal part of the catheter was 2.6 ml 4 methods of injections were assessed: a: injection of the predetermined volume of teico in 10 minutes with no wash out. b: idem as a but the teico was injected in 5 minutes, followed by a wash out (5 ml / 5 minutes). c: twice the required volume was introduced in the syringe and the volume to infuse was programed in 5 minutes, followed by a wash out (5 ml/5 minutes). d: ]dem as c but a priming was performed before connecting theteico syringe to the tubing. during each run, serial samples were collected every ten minutes over a one hour period. the samples were assessed using hplc method. results: the amount of drug delivred at 10 minutes were calculated. the results are a mean of 2 to 6 runs and expressed as the percentage of the total amount of teico prescribed. a 2,8 % 6,4 % b 47 % 62,3 % c 82a % 86,8 % d 94,2% 95 % conclusiom for accurate and reliable intermittent drug infusion with a syringe pump it is mandatory to use a precise protocol of administration and to take in account 1) a priming (for immediate starting of infusion), 2) a drug volume greater than the dose prescribed and a programmed volume injected, 3) a wash out of the tubing (with a volume ~ 1,5 x volume of tubing injection) caz is an antibiotic with activity against the major pathogens responsible for neonatal bacterial infections. we previously reported the pharmacokinetics of caz in 136 preterm infants on day 3 of life which showed that the clearance of caz increased with increasing gestationat age (ga). mean serum half-life of infants with gas < 32 wks was 8.7 h. we wanted to investigate the effect of postnatal age on caz pharmacokinetics, we therefore studied caz pharmacokinetics on day 19-21 of life in 10 preterm infants with gas < 32 wks. caz (25 mg/kg) was administered as an intravenous bolus injection. blood samples were coilected before (t =0), and 0.5,1,2,4,8 and 12 h after the caz dose and analyzed by hplcassay, the pharmacokinetics of caz followed a one-compartment open model. during 1995 11 newborns with complex congenital heart defects requiering either htx or palliative staged single ventricle repair were admitted to our hospital: hlh n=8, unbalanced cavsd, tga with hypopl. rv and hypoplastic aoa. tga with hypopl. rv, sas and dextrocardia. 8/i 1 children had been admitted with cardiogenic shuck and mukiorgan failure due to intermittend closure ofductus arteriosus; in 3/8 stabilization failed. parents were informed about the known and unknown risks of the always palliative surgery; in 2 cases parents denied further therapy. one pafiem with hlh underwent orthotopic htx at the age of 5 month after the ducms art. had been stunted in the newborn period. 9 month later he is still in favourable condition and without any sign of acute organ rejection. 5/11 underwent first stage of palliative single ventricle repair: norwood -op. ( 3 ) ( n=3 ), damus-kaye-stansel -procedure ( 2 ). the clue to adequate postoperative management was to archieve a balanced distribution of flow to systemic and pulm circulation, that is to protect the single ventricle from volume overload and to guarantee sufficient oxygenation and pulmonary development as well. with the centralvenous sato2 at about 50% provided maintaining the arterial sato2 at about 75_+5% is corresponding with a qp/qs of 1:1. using modified bt-shunts of3.5mm resp. a central anrtopulm, shunt of 4mm in one case l severe puim. hypertension, surgery at 6 weeks of age ) there was no excessive pulm. blood flow and no need to increase pvr with inspired co2. one child ( norwood at 5 weeks, preexisting pnim_ edema ) developed severe pulur hypertension and parenchymal pulm. dysfunction after prolonged bypass and multiple transfusions due to intraoperative bleeding: hypoxemia could be managed successfully by implanting a second shunt of4mm 18hh later and temporarily using prostacyclin and no; at sternum closure 6 dd later the second shtmt was banded to 3ram. follow-up ranges 5-5 month: all 5 children are at home being assigned for second stage operation at about 6 month of age. establishing clinical practice guidelines has become increasingly important in the current health care environment. significant effort has been focused upon development of post-operetive critical care pathways. however, benchmark data upon which such pathways should be based has not been well reported. length of mechanical ventilation (lmv) and length of stay (los) for children following cardiac surgery, for example, is poorly described. we prospectively recorded the lmv and los in 168 patients who underwent cardiothoracic surgery between 9/1/93 to 6/30/95. only patients who belonged in any one of five categories of congenital heart disease (ventricular septal defect _+ other septal defects (vsd), atrioventricular (av) canal, tetralogy of fallot (tof), transposition of great arteries (tga), and single ventricle physiology (fontan)) were included. eight non-survivors were excluded from the analysis. all patients were admitted to an intensive care unit 0cu) post-operatively where mechanical ventilation was managed by 4 pediatric intensivists. lmv was defined as the period from post-operative admission to planned extubation. length of stay (los) was defined to be from le from the icu. cytokine patterns during and after cardiac surgery in young children. especially in children, cardiac surgery with cardiopulmonary bypass (cpb) can cause a systemic inflammatory response. this process is thought to be mainly a result of inflammation induced by surgery and exposure of blood to an artificial surface, and of reperfusion injury during weaning of bypass. complement activation, degranulation of granulocytes, induction of free oxygen radicals, endotoxemia and release of cytokines, are important contributing factors. we studied cytokine patterns before, during and after cpb in young children admitted for complex surgery or for septal defect correction. in the first group, significant amounts of il-6 and il-lra could be detected preoperatively. these findings could reflect the already existing hemodynamic dysregutation. in both groups, cpb procedure upregulated the circulating pro-inflammatory cytokines il-6/8, but not il-1b. at the same time, il-lra became detectable. therefore, we suggest that in these patients the production of the anti-inflammatory cytokine il-ira was not induced by the preceding acnvity ot pro-inflammatory cytoidnes. during cpb, we noticed a sharp decline in the capacity of the leucocytes to secrete il-6/8. the ex-vivo production of il-lra however, was only slightly attenuated. we conclude that there is a differential regulatory pathway for the induction of il-6/8 and il-lra. in addition, we studied the influence of dexamethasone administration on the cytokine pattern. administration delayed the appearance of il-6/8 and il-ira in the plasma, interestingly, it did only interfere with the ex-vivo production of pro-inflammatory cytokines. the latter supports our hypothesis that production of il-6/8 and of il-lra is regulated by two independent pathways, (60%) of 43 pts. 82% ofpts < 12 months of age developed metabolic alkalosis as compared with 38% ofpts > 12 months of age.the infants with metabolic alkalosis received more citrated blood products and furosemide. following cardiac pulmonary bypass the highest ph-values and be-values were observed 24-48 hours and 48-72 hours, respectively. ii. prospective study: metabolic alkalosis was registerd in 2t children (70%), 8 of those <12 month (75%) developed metabolic alkalosis and 67% of those elder than 12 monms.durmg the postoperative course patients younger than 12 months developed the highest ph-and base excess values after 102 and t05 hours, in the subset of the older patients maximum ph and base excess was found after 48 and 81 hours, respectively. in one case the top level ofph-value exceeded 7.6, the base excess +20 mvalb. conclusion: children undergoing cardiac surgery with cardiopulmonary bypass often develop metabolic alkalosis.in contrast to previous reports, we did not observe an association between metabolic alkalosis and mortality, nor greater frequency of cardiac arrythmias or prolonged mechanical ventilation. in context with decreasing serum lactate levels, our data show positive correlation of metabolic alkalosis with postoperative improvement of liver function. respirator, mechanics and weaning outcome in children undergoing cardipvascular surgery. vassallo j., cernadas c., saporiti a., landry l., rivello g., buamsha d., rufach d., magliola r. mechanical ventilation (mv) and acute respiratory failure are common events in children unergolg cardiovascular surgery (cvs), the development of new techniques helped to measure some of the main respiratory mechanics (rm) in a non invasive fashion. our goal was to evaluate the predictive value of these measurements in weaning (w) outcome in these patients, patients and methods: we prospectively evaluated children considered clinically to be ready for w with < 20 kg and > 24 hs mv. patients with diaphragm paralysis and those who failed w because of upper airway obstruction were excluded. before patient extubation the following measurements were recorded during spontaneous ventilation (cpap/t piece) using the cp 100 neonatal pulmonary monitor bicore (lrvine, ca): total respiratory system static compliance (cssr) and resistance (rts), rapid shallow breathing index (rsbi). maximal inspiratory negative pressure (pi max) was measured using an unidirectional expiratory valve. threshold values predicting w success (ws) were: cssr > 0.5 ml/cm h20, rts < 75 cm h20 /l/sec, rsbi 160 and pi max > -30 cm t120. w failures (wf) -patient reintubation within the following 48 hs, these values were compared between w success and failures using fisher exact test. an apriori level of statistical significance was chosen at p < 0.05. 4 considered, an increase in tnf-a levels is observed after cardiac surgery (p<0.001) with a return to previous values after 24 hours (p<0.005). 72 hours after cpb, similar values are observed in groups ii and ill, but there is a further increase in serum tnf-a levels in group i when compared with both other groups (p<0.03). we found no statistically differences in any other moment. there was a significant correlation between serum tnf-o levels determined 72 hours after surgery and cpb duration (p<0,003). conclusions: cpb in childhood provokes a significant increase in serum tnfa levels, in newborns the inflammatory response is maintained 72 hours after surgery. this enhancement of serum tnf-e levels indicates the existence of a relevant inflammatory response in these patients. introduction: cardiac surgery appears to induce a systemic inflammatory response. we have investigated the behaviour of il-1 i~ and il-6 before and after cardiac surgery. patients and methods: we studied serum il-1 6 and il-6 levels from 20 children with congenital heart disease (10 boys and 10 girls), aged from 7 days to 14 years, undergoing open heart surgery, before cpb (d we found no statistically differences in the il-i levels in the different groups and moments. there is a significant increase in il-6 immediately after surgery (p<0,01) with similar levels 24 hours after cpb and a significant decrease (p<0.01) 72 hours after cpb. preoperatory il-6 levels were higher in the groups i and tl than in group i11 (p<0.05). 24 hours after cpb serum il-6 levels in group 1 were significantly higher when compared with group 111 (p<0.05). conclusions: cpb in childhood induces a significant transient increase in serum il-6 levels, strongly relevant in newborns. cpb was not associated to a significant modification in serum il-1 6 levels. thus, cpb in childhood induces a dissociated behaviour in the proinflammatory il-6 and il-1 & pathways. obiective, to evaluate the effects of amg receipt on the clinical condition during the first 12 hours after birth (t2), the morbidity and mortality in immature outborn neonates. methods. we studied 44 outborn neonates with ga 26 to 29 wks, admitted during the years 1993 to 1995. eighteen neonates exposed to amg (ga:27,6+lwks, bw: 1066_+195g) and 26 neonates did not (ga: 27,7_+1wks, bw: 1042_+187g). results. amg-exposed neonates compared to those not exposed had lower incidence of apgar score at 5 min _< 3 (6% vs 35%, p<.05), lower incidence of ph t2 <7.20 (11% vs 48%, p<.05), decrease need of bicarbonate 12 (22% vs 54%, p<.05), lower fio212 (fio212min>40: 17% vs 48%, p<.05 and fio212max >80: 17% vs 52%, p<.05), lower incidence of intubation (67% vs 92%, p<.05), lower requirements of surfactant (50% vs 79%, p<.05) and lower mortality (11% vs 50% p<.01). there were no differences between the two groups for the following parameters: type of delivery, hypothermia hypoglycemia and anemia during admission, hypernatremia, hypotension 12 (map<30mmhg), need of dopamine and or plasma 12, incidences of ptx pda sepsis nec severe rop major ivh (plus pvl) and bpd and duration of intubation. conclusions. the main beneficial effects of amg receipt on the immature outborn neonates were the decrease of mortality and the decrease of surfactant need. there was no effect of amg receipt upon other severe morbidity in this high risk group of neonates. premature babies are very sensitive on homeostatic disturbances, and often develope intracranial haemorrhage (ich). ultrasound scan of the bram shows four grades of ich: -grade i -only periventricular hyperechogenic areas -grade ii -haemorrhage ham the lateral ventricles -grade ili-dilated lateral ventricles -gtrade iv -intracerebral haemorrhage. the purposes of this study were: 1 to show the incidence of ich in premature babies and its correlation with the gestational age, 2. to determine the severity of ich 3. to present the outcome &those babies. in the study were included 393 premature babies successively-born at the department of gynecology and obstetrics before 37 gestational week (g.w.) and grouped in three groups: less than 28 g.w., 28-32 g.w., 33-36 g.w. to all of them was performed ultrasound scan of the brain. results : 1. the incidence of ich hi premature babies is 49 % and there is ingh level of correlation with the gestational age: -babies born before 28 t~ g.w. have 100% incidence of ich and graduated : i grade -5%, ii grade -65%, iii grade -25%, iv grade -5% -babies old between 28-32 g.w. have incidence of 61% : i grade -24%, i[ grade -62%, iii grade -14%. -babies older than 32 g.w. have incidence of 33%: i grade -46%, ii grade 48%, iii grade -6% 2. sixty of 393 premature babies have died and it is 15.2% lethality. in all died ilffant was confirmed the grade of ich diagnosed by ultrasotmd scan of the brain. d. maksimo~5c. z.braiko~ic, n.vunjak. p. ivanovski (5~iversi~, children's hospital. belgrade, yugosla~, ia infantile intracranial hemorrhage is the most frequent and serious manifestation of late hemorrhagic disease of the newborn caused by ,,~tamm k deficiency in earl?,, ti~fancy. in the last two years, we recorded five cases of infantile intracranial hemorrhage due to "dtamin k deficiency, despite routine prophylax~s (intramuscular vitamin k, 1 mg) , with bpieal clinical presentation: age was 18 -65 days (average 40 days): vomiting, poor feeding, lethar~'irritabiljty, palor, bulging t0ntanelle and convatsiones were present in most cases.two patients developed signs of hemorrhagic shock, with hemoglobin level less than 70 g.1. in 3~5 f \qi level was less than 30 % of predicted value. there was no evidence of head trauma or liver disease in none of patients. four inlants were breast fed, while one, who had diarrheal disea.se, was on adapted milk formula. routine therapy wa.s given (including vitamin k and fresh frozen plasma). two patients were discharged with no sequellae, one developed posthemon'hagic hydrocephalus as a complication and two patients died. late hemorrhagic diseo.se of the newborn is sill/ a significant cause of morbidib' and mortality in earl3' infancy, despite different approaches to prophylaxis developed in recent years. background: neonatal hearing screening in at risk newborns can detect 50% of the children with a congenital hearing loss. automated abr hearing screening (algo-1) has been introduced for healthy newborns. the aim of this study is to test the validity of this algo-1 screener in at risk newborns in a neonatal intensive care unit. subjects: 250 at risk newborns (median gest.age: 30.0 wks, median birthweight 1350 g) selected according to the criteria of the american joint committee on infant hearing. interventions: algo-i automated abr-hearing screening at a level of 35 db was performed in the neonatal intensive care unit. when bilaterally referred, further audiologic screening and/or therapeutic intervention took place. when passed uni-or bilaterally, children enrolled in a) a nation wide screening programme (ewlng) at the age of 9 months and b) in a half yearly follow-up programme in which hearing and speech-and language development were observed according to egan an illingworth. results: screening without disturbance from ambient noise or from routine technical equipment was possible in the incubator, even during nasal cpap therapy. 245 (98%) newborns passed algo-1 screening. 5 (2%) did not pass bilaterally. 1 of 5 with a congenital rubella died shortly after screening.in 4 of 5 bilateral congenital hearing loss of ->35 db was confirmed. 235 of the newborns passed were still alive at the age of 1 year. ewing screening was performed in 183 of 235 (77,9%). 161/183 passed, 15 of 183 had passagere conductive hearing loss, in 7/183 no further investigation was performed. all 235 children enrolled in the i/2 yearly follow-up programme had normal speech-and language development. in this study all 4 at risk newborns with bilateral congeni "tai hearing loss were detected with algo-1 screening. screening results showed no false negatives at follow-up. the algo-1 infant hearing screener can be used as an valid automated abr-screener to detect hearing loss in at risk newborns in a neonatal intensive care unit. gancia gp, bruschi l pnlito e, ferrari g, rondini g -divisione di patologia nc~matate e turapia intensiva -irccs policlinico s. mattco -pavia, italy latrogenic esophageal perforations (iep) in preterm and term infants are seldom reported in litteraturc, in association with difficult endotracheal (et) intubation (with or without stylets), insertion of gastric tube, and pharyngeal suctioning with stiff catheters. crieopharyngeal muscle spasm caused by instrumentation may also lead m a narrowing of lumen, with increased risk of local injury. we report 4 iep observed in intubatcd, mechanically ventilated newborn infants (2 male, 2 female, all outborn). a common feature of iep was inability to pass a nasogastric (ng) tube into the stomach, mimicking e~)phageal atresia.~se 1: birth weight (bw) 185(i g, gestational age (ga) 37 wk, sepsis. before admission to n1cu, the baby underwent multiple et inmbations, because of inappropriate securing of et robe. bloody secretions in pharynx were observed. the endoscopy showed a large lesion at the end of proximal third of the esophagus, case 2: bw 1080 g, ga 32 wk, rds. chest x-ray (cxr) showed a retrostcrnal air leak: the ng tube was stopped }~etwcen d8 and d9 and soluble contrast was seen in upper mediastinum.case 3: bw 76(/g, ga 26 wk, rds. the endo~opy showed an esophageal lesion. cxr showed a paravertebral route of ng tube and a right pneumothorax.case 4: bw 102(i g, cz 22 ,.v!:. rd c. ~!,'.::;;: ::':'_'rvt!~'2s l" ~k':.rvrx. cwr, d,,,,vs ~,,mr~e, ~n rhe upper mediastinum and abnormal route of ng tube through a false passage. surgical intervention is needed in case of mediastinitis or mediastinal abscess: conservative management included broad spectrum antibiotics, total parenteral nutrition, antireflux therapy and, if necessary, drainage of air leaks. enteral feeding has been stopped lor 15 days and cautiously resumed after radiographic study. [x~cal sequelae and death are uncommon, but iep occur in newborns with high risk of death due to prematurity and other diseases. in our patients, et intubation has been performed by experienced personnel: therefore the lack of skills in resu~itative procedures is not always the main factor of iep. prevention of iep requires appropriate materials (et tubes, laryngoscope blades, suction catheters), and procedures (positioning of the infant with correct neck estension, firm et placement). sedation and pain control may help to prevent the muscle spasm. aggressive treatment has improved the tong-term outcome of extremely low birth weight neonates (elbw) but it has also increased the chances of iatrogenic lesions. reviewing the charts of our neonates we observed a high number of vascular injuries. from 1987 to 1994, 2898 neonates were admitted to the neonatal intensive care unit (nicu); 335 of them were elbw (11.5%). studying the charts of these elbw we observed 9 cases (4 m -5 f) with vascular lesions (2.6%). mean gestational age of these patients was 28.7 weeks (rain 24-max33). mean weight at birth was 880g . mean weight at diagnosis was 1825g (1230-2700). in the same period 10 patients with vascular injuries were reported in the 2563 neonates over 1500g (0.3%). the injuries observed in elbw group were: 6 arteriovenous fistula (2 bilateral) at femoral,level, 1 carotid lesion and 2 limb ischemic lesions. aetiology was in 7 cases by venipuncture, in one case umbilical catheter and in the case of carotid lesion a wrong surgical maneuver. no general simptoms were observed. the vessels were repaired with microsurgical technique in six cases: the carotid lesion and five arteriovenous fistula; one case was solved with thrombolitic drugs; an amputation at knee level was required in one case after a long period of medical treatment. the last neonate with an arteriovenous fistula was only observed for parent's will. at follow-up (clinical and by ecodoppler) 7 out of 9 neonates presented normal vascular function without sequelae. from our experience elbw neonates have more chances than older neonates to develop iatrogenic vascular lesions. we advocate an aggressive microsurgery and/or medical treatment to obtain good results and prevent late sequelae. a retrospective comparison between 2 natural surfactants l.j.i.zimmermang m.c.m,van oosten. dept. pediatrics, div. neonatofogy, sophia children's hospital/erasmus university, rotterdam, the netherlands. aim: retrospective comparison of alvofact (in 1993) versus survanta (in 1994) as rescue treatment for neonatal respiratory distress syndrome (rds). methods: both surfactants were given at an initial dose of 100 mg/kg (except for alvofact 50 mg/kg for mild rds grade mi). repeat doses were attowed (survanta 100 mg/kg, alvofact 50 mg/kg) up to a maximum of 200 mg/kg, all parameters and outcome criteria were strictly defined beforehand. the initial response (good,mild,no response,relapse) to surfactam therapy was defined on the basis of the decrease in fio 2. results: there were no signif. differences in patient population and initial parameters: ga (29.9+_2.2 vs 29a _+2,6 wks), birth weight (t332_+431 vs 1227-+444 g), severity of rds (grade ill-iv: 78.6% vs 80.3%), apgar scores, cord blood gases, initial ventilatory settings. in '93 however, the initial surfactant dose was administered earlier than in '94 (14.4-+ 17.4 vs 6.5_+7.8 hrs postpartum, p= 0.025). although the average total cumulative dose was equal in '93 and '94 (169.3-+65,8 vs 167.4_+69 .4 mg/kg), more doses of alvofact were given compared to survanta {2.3_+1.1 vs 1.7_+0.6, p=o.o01) and more patients in '93 received more than two doses than in '94 (46% vs 18 % of patients). there was no difference in the incidence of non-putmonarycomplications. aivofact ( there was a better initial response to survanta and a better respiratory outcome in 1994: in the group < 1250g the duration of ventilation was half in 1994, and in the group >~125og the duration of extra o 2 need was half in 1994 as compared to 1993. we speculate that the main reason for this difference is the earlier and initially higher dosing used with survanta compared to that used with alvofact which was given in the same total cumulative dose but over a larger time span. background: e×ogerlous sur&ct~t raplacem~t treatmem has become rou~ne k~ the t~eatme~t of respira~"¢ dim'~ syndrome (i~ds) of pr~e~tur~, wh~eas its effica w th odi~ respiratory diseoses is sdi1 being wader mvesugatio~. objective: "eac~ mt ereat isto report ottr results of prospect/re, non-randomized "re~-o.e" study oe suffact~t replacement in outhom premamae infa~t~ with rds reruirmg me~aical ventilatioa (nfv). p~tien~ and metho0.s: from j-aly 1993 to june 1995, 18/58; (31%) out~ ~¢ infaats, at a mesa age of 22 z 2,7 horn's ( 13 boys, 5 ~rls; ~ gestafioan age 32-+2.8 weeks, mera~ birth weight 1846 _+ 544 g, ~ 7.2 i" 17 at 5 minutes) with rds, requiring mv, received bov~e-suff~amt (survanta, ros~/aboti, laboratotie~ columbus, ohio) eadotracheally, as was recomm~aded by maaufacturer. as the c,~:ttrol group 19 o~bom premature infants (ot~ of 49; 39%, admitted with rds from euiy 1991 to eune 1991) were saelected ~d who did not receive surfaaam, compared with ~hctant ~'oup they were admitted for treatmeat e~'li~" aft~" daliv~:y (at the age 6.4::2.2 hours vs. 11.7+-13 hours), but they did not diff~ in othe~ baseline dam'a~eri~cs at ~ti~ion. entry crkeda for ~¢fa~aut ~hcadou were fractional i~firat o~ oxtgem r~emeats -fio2 > 0.50 -0.60, ratio au-lerlal to alveolar oxygea pre~are~ao2~ao2 < 0,20 ~ad oxyge~at,~ i~.dex -ol > 10. primary o~comes were deter~caned by ~hanges m exs'ge~ab, c~ ~r~d vmtilatic~ ~ the following variable~; (1) fi'aaic~ of i~spired oxtge~ (fio2); (2) mesa nnvay presmzre (map) (3) pag2 ~ao2 ratio, (4) oxyge~ion index (oi). commo~ comphcadces of prem,musty ~d con~ol mechamcal v~ati]al~on (pater dumas merios.s, intracr~nlal haemcrn:hage, air leak, br onchop ulmrmm'y dy~pl~a ~d death) were reg~ded as sec~d,~y outcomes. r~suas: in warfactaat group we observed slg~5.c~t improve~aeat (p<0.05) in oxygea~thia md veaatilation at 24 hours all~ e~try k~to the m~dy in compari~ion to nons~fa~m" group. compa~on of secondao' outcomes in ~ts with p,.ds showes table l we did not observe ~y major acute hfe fl:u-eattming complicatlola,s m sxlrlhct~mt grou~ tr/lmediately after stu'~actsmt rcplacemev_t therapy. the duramm of mechmucal ven~ation ~ad oxygen lreau~ent m survivals of both groups did not dafter 51gmficautl y a-ore ead~ other. condusion: l!a premature mthats with rds treated with surfaaaat replacemeaat therapy we observed decrease m mc~de~ce of tme'~m~o~oraces add de~th (p<0.01 and p<0.05), whe~e~s m othe~ observed variables thee was uo ,igmfi~t d~=ecce infectious complications during the therapy of respiratory insufficiency in neonates with birth weight less than 1500 g in the course of 3 yearsretrospective study. zitek infants on cmv, cppv, and imv were administered exosurf in dose of 50-60 mg/kg twice endotracheally (see table) . in 32 newborns (86.4%) 2 hours after surfactant admin fi02 value decreased by 20.8%, and after 6 hours -by 28.1% compared with initial value; pip and peep values decreased by 3-5 cm h20 and 1-2 cm h20 after 6 hours, and by 4-7 cm h20 and 2-3 cm h20 after 1 day, respectively accompanied by mean decrease of aado2 from 486,2 to 240.2 mmhg, qs/qt decrease from 24.9 to 13.2% (see table) . mean time of cmv, cppv was 7.8 days, imv-14-36 hours, cpap -10-24 hours. respiratory therapy in 5 newborns (13.5%) was complicated by pneumothorax (bilateral -in 2 infants chorioangioma is a rela~ively rare placentai malformation associated with considerable mortality and morbidity. a chorioangioma can be regarded as an arterio-venous shunt in the circulatory system of the fetus. this causes volume loading eventually resulting in cardiomegaly and high output cardiac failure. a female neonate (gest age 40 wk, birth weight 2290 g, -2.6 sd) was born with an apgar score of 4 and 7 after 1 and 5 rain respectively. the placenta showed multiple chorioangioma. ultrasound of the heart showed a hypertrophic cardiomyopathy. she developed severe hypertension (100/70 mm hg), treated with nitroglycerine and nitropruside. finally blood pressure decreased when enalaprillic acid was given (0.15 mg.kg4). we measuered the activity of the renin-angiotensinsystem. an elevation in renin-angiotensin system is shown probably to compensate for the low resistance circulation before birth, hypothesis: the instantaneous cut off of a large arteriovenous shunt did not result in a fast downregulation of the renin-angiotensin system resulting in hypertension. hypertension should be added to the list of complications of chorioangioma of the placenta. the authors studied 75 cases of children's septicemia with blood culture yielding staphylocucetts aurens. the age of patients varied from 2 months to 15 years (51,3% from 3 years downward), 74% of the children caught their disease in the hot season (may to october). the deaths also occured in this season: 87,5% (21/24). following were the anatomo-dinical lesions. -skin 42%, muscle 60,0%, bone 21,3%, joint 9.3%. -viscera : lung 50%, heart 33.3%, cerebrum 22.6%, kidney 60.6%, fiver 17,3%. -simple lesion skin-muscle-bone joint: 12%, no death in this group. the concomitant lesions of the soft tissue,bone-joint and viscera : 34% with one viscera, 26% with two viscera, 18% with three viscera and 9% with four viscera. -bone lesion : mainly on the long bones (50% on the tibia, 25% on the femur, the remainder being the mandible (3) and the humerus), inflammation of' the hip joint was the main one. -i,ung lesion had forms pneumatocele (4 cases), bronchopneumonia (6 cases), pleural effusion (7 cases), multimicroabcess bursting into the pleura (8 cases), most multimicroabcesses were lethal : 20/22 (90,9%), -heart: all thethreelay~rs got le@~r~, 20% had 2 or 3 layers alrected and death ensued. -cerebrum : the meninges had three forms of lesions purulent meningitis (13 cases), obturafing embolns of brain vessels (2 cases) and cerebral abcess (one case). the characteristic clinical sign was paralysis and meningismus, phlebothrombosis of eavcrnous finus (13 cases)was mually ther~sultofalxil vdfi:h burst there were 6 cases of death with lesion of the meninges and 2 cases of obturating embolns of brain vessels. -the main sign of lesion of the kidney was a change in the components of urine: 60% got proteinuria, 75% had leucocytes in their urine, 42% had erythrocytes in their urine, the urea in their blood increased (over 60rag%) in 21.4% of cases.the lesion of the kidney seemingly had little relation to death. seven cases of ictertts due to an increase of direct bilirubinemia and a decrease of blood-albumin. -the biological characteristics of the pathogen staphylococci showed that all the 75 isolated specimens had positive coagulaza ; the specimens from the dead patients were less semiti~e to, mad ~t to mali~ overag death rate was 34.7 % (24/75). the fungal infection to fusariun species in immunocompromissed child have been reported in the literature with a rare, severe and high, mortality rate in spite.of the use of antifungal drugs. we report a case of successful treatment of a severe disseminated fusariun infection in a ll-year-old boy with acute lymphocytic leukemia (lla-l3), after use a chemotherapy followed by absolute granuloeytopenia. the patient developed fever, skin lesions, pneumonia and fungaemia. fusariun species was cultured from the blood, necrotic skin lesions and lung secretion. the child developed multiple organ system disfunctiou in spite of use broad spectrum antibiotcs and antimycotic therapy needing. uci during 18 days. the patient receive suport treatment (mechanical ventilation, inotropie d~.ugs, diuretics, imunestimulants, blood components, a broad spectrum antibiotes and antifungal agents). we absorved a gradual recovery in the white blood cell count and regression on the sites of infection. the association of preeoce diagnostic and the terapentic with increase in the white blood cell count was the most important in a successful treatment. a 5 year old african-american child suffered a severe pulmonary injury in a house fire. initial survey revealed 1% total body surface burns, soot on the face, and bloody endotracheal secretions. initial chest radiograph revealed diffuse, bilateral infiltrates. severe respiratory failure with an oxygenation ratio of 73 rapidly developed. he developed a pneumomediastinum and subcutaneous emphysema. although transient improvement occurred with inverse i:e ventilation and surfactant, he became more hypoxic (sac2 as low as 47%) and acidotic. on day 2 post injury, he was placed on venc~venous extracorporeal life support (ecls). on ecls day 30 he was decannulated. chest radiograph on ecls day 15 showed an opacity in the left chest. ultrasound of the left chest was consistent with atelectasis rather than pleural fluid. flexible bronchoseopy failed to reveal any obstruction in the left lung. a computed tomography (ct) seen of the chest, which was performed after decannulation, revealed a large loculated collection of fluid in the left, anterior chest. under ct guidance, a 14 f cope loop catheter was inserted and 40 cc of thick blood was removed, follow-up ct performed immediately after this procedure revealed minimal change in the size of the fluid cavity. over the next 48 hr, we instilled urokinase 20,000 units over 20 minutes every two hours. a 30 minute dwell time was allowed before draining the fluid. repeat ct scan done at the end of the urokinase infusion showed a marked decrease in the size of the fluid cavity. act scan was not performed prior to decarmulation because the ecls circuit tubing was too short to allow appropriate positioning of the child in the ct scanner. after a ct scan revealed loculated pleural fluid, a simple drainage procedure was diagnostic but inadequate treatment. we were able to successfully dissolve the thrombus after 48 hr of urokinase therapy even though the thrombus was > 14 days old. we suggest that large loculated plenral thrombi which develop as a complication of ecls therapy may be successfully managed with urokinase infusion. introduction: haemorrhages, particularly intracranial, are major complications experienced in 10-35% of neonates treated with extracorporeal circulation. an induced thrombocytopenia and impaired platelet function play a key role in the increased bleeding tendency observed in these patients. the aim of the present study was to establish a dose-respons curve for the effect of a synthetic protease inhibiting agent, nafamostat mesilate (fut-175), on platelet membrane glycoprotein density and platelet activation during experimental perfusion. methods: two identical extracorporeal life support (ecls) circuits were primed with fresh, heparinized human blood and circulated for 24 h. four different concentrations of fut-175 (7.12 mg/l blood/h; 14.25 mg/l/h; 14.25 mg/l/h+25% bolus at the start of the perfusion and 2&5mg/l/h+25% bolus) were used in different perfusion experiments. a total of eight paired experiments were performed. platelet count, plasma betathromboglobulin levels and platelet membrane density of glycoprotein ib and lib/ilia were followed as well as plasma concentration of haemoglobin. results: a protective effect of the agent on platelet count, plasma concentration of btg and platelet membrane gpib could be observed during the first 3 hours of the perfusion when a bolus dose was added. no positive effect could be recorded with the two lower doses used. plasma concentration of haemoglobin was higher in all the fut-circuits compared to the control circuits. conclusion: the addition of a bolus dose of fut-175 at the start of the perfusion seem to induce a protective effect on platelets during the first hours of perfusion. extracorporeal membrane oxygenation (emco) is a form of invasive cardiopulmonary support that can provide imporary physiologic stabilisation in reversible circulatory failure and or respiratory failure. we reviewed our expierence with extra corporeal membrane oxygenetion in 4 children aged 1 day to 4 year between 1991 and 1995. two neonates was succesfully decanulated, but died 1-2 well after decanulation due to septic complictions. one child 4 years old, one neonates died on day 5 and day" 7 respectively while still on emco. complication which were and encountered were heavy bleeding in case 1 (child), 4 (neonate) and raceway rupture in case 2 (neonate). problems which are specific developing countries like indonisia are: high cost (20.000 us for 7 days) difficulty in transportation (transporting intubated baby) from the orgin hospital, lack of knowledge and understanding of the primary physician and nm-ses and difficulty organizing in 24 hours emco team. resnratory mon1tor/ng in picu z,zjvkovic, s. mihailovic, o, tosev respiratory monitoring in pediatric intensive care unit 0picu) provide the importartt informations for understanding of the pathophysiology of the clinical signs, aid with the diagnosis, and assist in therapeutic management and predicting prognosis. pien in children's hospital for ~flmonary diseases and tuberentosis remained for the t~s't two end a half years relatively limited for diagnomic tools and therapeutic regimens, mostly because of the poor fmnaeial suptx~rt. the number of children admitted for aurae asthmatic at.lzek~ severe pneumonias, bronehiolitis, complicated pulmonary tuberculosis, foreign bodies and exacerbations of ehronit'. pulatonary diseases was t362. for all patients the respirator' monitoring system means: physie~d examination, ehe~ x rays, capillary bltxxl gas mmlyses (vevv few ehiktren experienced itwasive arterial blt~.~'i gases), noninvasive oxyntctry, measuring of the vital capacity in coopo-able patients, as~d capnography. later on, after the imtial critical illness, a complete hmg fimction tests was performed, as well ,~s bronehoscopy in selected eases, (~lr experience revealed that abotrt 60% of ehil&en heos suecessthl outcome, without s~lllens , instead they had been tremted in limited conditions. ']'he rest of our patients were previously diagnosed ~s ettronie pulmonary patients, with high risk score system ibr having seqnells 'llae mortality rate were 0,5%. the continuous blood gas monitor, pasatrend 7 (biomedical sensors, ltd., high wycombe, bucks, england) has the capability of measuring ph, pco2, and po2 via an indwelling optical absorption optodelclark electrode sensor that is placed through an intra-arterial catheter. we evaluated the accuracy of the sensor in radial and femoral locations in critically ill pediatric patients. methods: the simultaneous values of ph, pcoz, and po2 recorded from the paratrend 7 monitor were compared to values measured by standard arterial blood gas analyzer (coming 278, ciba-corning diagnostics, medfield, ma). criteria for the elimination of data points included a core vs. sensor temp. gradient, and sensor pulled back beyond accepted insertion distance. mean time of monitoring per sensor was 108 hours (range 0.75-403.7 hrs). mean time of radial monitoring was 35 hrs (range 0.75-160.5hrs) and of femoral monitoring was 137.2 hrs (range 12.8-403.7 hrs.). linear regression and bland-altman analysis for bias and precision for each parameter were calculated. results: a total of 49 patients (age range 2 weeks to 18 years) had paired samples of ph, pens, and poz made by the sensor and blood g&s analyzer. the range of measurements were ph 6.99-7.66, pco, 16.0-i14.2 t(n r, and po2 34-480 torr. the paratrend 7 monitor demonstrated accuracy that is comparable to the accepted standard of blood gas analysis in a group of critically ill pediatric patients manifesting wide variation in ph, pen2, and poz..this technique appears m be very useful especially in the extreme values of the parameters measured. funding provided by biomedical sensors. understanding of pulse oximetry d.semple, l.e.wilson. royal hospital for sick children, edinburgh, eh9 1lf, scotland, uk. pulse oximetry is a useful, non-invasive monitor, routinely used on the itu and increasingly often on the general wards. we used a questionnaire incorporating questions on the theory and clinical uses of the pulse oximeter to assess understanding of pulse oximetry in medical and paramedical staff doctors indicated grade, speciality, pulse oximetry tuition and neonatology experience. 45 doctors, 15 itu nurses, t9 medical students and 4 physiotherapists completed the questionnaire. some confusion existed between the principles of pulse oximetry and transcutaneous oxygen measurement. wide variations in the lowest acceptable saturation in fit children were seen (80-95%), with around 20% of respondents in all groups accepting values of 90% or less. some potentially serious mistakes were made in the evaluation of oxygen saturations in the clinical scenarios. there were widespread variations in correct responses at all grades of medical staffing. nurses scored well on more clinically-orientated questions but relatively poorly on theory. only 15% of doctors (mostly senior grades) had received tuition in putse oximetry. neonatology rotations appeared to confer little additional knowledge on pulse oximetry. few doctors and nurses receive tuition in the use of pulse oximetry a significant proportion of nurses and doctors, of all grades, exhibited a lack o{" understanding of the principles of pulse oximetry. this may result in unsafe use of the equipment and put patients at risk. one can see from the table that blood composition in uv and ua differens in some characteristics, and similar in sgp magnitude. venous-asterlal gradients "gas functiomals" between uv and ua represent the measure of difference in this characteristics. the gradient cari be positive, zero -order or negative and change both in value and in sign but not reach apo2 (positive) and apco2 (negative) in absolute significance.minimization of "gas functionals" deviations atom the zero is achieved due to"mutual replacement acts" between po2 and pco2 in uv and ua blood. we suggest that presented tests can be useful in full evaluation of gas exchange in newborns. (pap) in the context of pulmonary hypertension is oft desired but rarely achieved. inhaled nitric oxide (no) has been shown to produce this desirable effect, but is relatively difficult to administer or monitor. we wondered whether np, chemicaily related to no but more stable in solution, would produce similar physiologic effects when administered in the convenient modality of nebulization. methods: 9 piglets were anesthetized, mechanically ventilated, and surgically instrumented. systemic blood pressure (bp), pap, and cardiac output (co) were monitored continuously. after postoperative stabilization, 0.9% nac} nebulization was begun, and pulmonary hypertensiorr was induced by reducing fio2 from 0.30 to 0.07. the piglets were monitored for 15 minutes during this hypoxic phase, next, without altering fio2 or ventilator settings, np (10 mg/ml, dissolved in 0.9% nacl, flow 4 ipm) was substitued for 0.9% nacl in the nebulizer circuit. np was nebulized for 15 mins. results: during hypoxia, pao2 fell from 159 to 29 mm hg. pap rose during hypoxia from 14 to 31 torr (p< 0.01). ,^fhile bp and co did not change significantly. pap fell during nebulized np in each piglet, (mean apap = 31 to 21 torr; p< 0.01; mean reduction of hypoxia-induced rise in pap = 61%; range: 36 to 78%; p < 0.01). pvr/svr fell by 28% during np nebulization (p< 0.01), while bp and co did not fall significantly (90 to 86 tort; 653 to 636 mllkg-min), the reduction in pap began within 2 minutes of the onset of nebulized np, and appeared to reach a plateau by 15 minutes. no tachyphylaxis to nebulized np was noted. nebulized np did not significantly affect pap, bp, or co under normoxic conditions. conclusions: 1) like no, np selectively reduced hypoxia-induced pulmonary hypertension without altering systemic bp, 2 ) unlike no, np can be administered by nebulizer, a technique familiar to virtually all health-care providers, and potentially adaptable to both intubated and non-intubated patients. 3 } nebulized np may be beneficial in clinical contexts where inhaled no is impractical. dang phuong kiet and nguyen xuan thu examining 6 cases of purulent pericarditis with various clinical forms treated by surgery, the authors drew the following experiences for their diagnosis. t. clinical factors. purulent pericarditis appeared like a cardiac tamponade in a septicemia due to staphylococci with dassieal symptoms: severe dyspnea, tachycardia, faint heartsound, big liver, prominent cervical vein ; rentgenography of the chest showing enlargement of the cardiac silhouette, a diminution of ventricular pulsations, ~i clear lung field. by an emergency operation, 500ml of diluted blood were drained. purulent pericarditis and pleural effusion appeared at the same time but at first tile symptoms of purulent pericarditis were masked by the predominant symptoms of plearal efihsion. after the pleura was drained, its pus was no more, the general state was relatively stabilized but there still were big liver, dyspnea, enlargement of the cardiac silhouette while central venous pressure increased. purulent pericarditis appeared late. in the first stage (about 2 weeks) there was no suspected sign. later on gradually appeared such symptoms as dyspnea (during serum transfusion for instance). central veinous pressure also raised. the heart chest diametre increased at first (up to 60-65%) then decreased (down to below 50% ) but the liver kept on swelling together with the particular changes of electroeaediegramme. now the pericardium had no more pus but get fibrous (up to 3ram) thus constricting the heart and its main arteries 0ike pick syndrome). 2. diagnostic values of electrocardiograms : common signs of ecg related of these purulent pericarditis were: a diminution of voltage, a widespread elevation of the st segment, the tf wave flattened and inverted. however, what should be stressed was : the diagnostic values of an electrocardiogram for purulent pericarditis was mainly in the dynamics of their signs: in the first week, the voltage diminished corresponding to a pericardium containing pus, while the st segment went up then seemed parallel to the fibrosis of the epicardium, the liver swelled, the central velnous pressure increased, the heart/chest dimension ratio decreased, the st segment went down, the t wave became more flat and inverted. between 1986 and 1995 23 neonates, aged 2 -23 days (median 5), weight 2,38 -4kg (median 3,28) with critical valvar pulmonary stenosis were scheduled for balloon dilation (psvp), 19 children (83%) were on pge1 and 13 (57%) needed mechanical ventilation. after stepwise dilation a final balloon : pulmonary valve (pav) ratio of 114% (25-150) was achieved, there was a significant correlation (p<0,01) between an adequately sized balloon and freedom of reintervention. two valves could not be passed, four neonates underwent surgical procedures (brock n = 3, commissurotomy n = 1), two children (10%) died of sepsis. 17/23 patients (73%) were successfully palliated by psvp in the first month of life. the rv : systemic pressure value fell from 132% (75-231) to 58% (40-87), complications included 2 transient dysrhythmias, 1 transient hypoxia, 3 vessel occlusions;-1 right ventricular outflow tract perforation. in 16/17 patients follow up data is available. the residual systolic peak doppler gradient over the pav on the last out patient visit (5-103 months after psvp) was 10-41 mmhg (median 20). four children needed repea.ted psvp 26 to 72 months after the initial intervention. conclusion: psvp of critically ill newborns is possible. the risk of mortality is relatively low. psvp in neonates with an adequately sized balloon is a challenging alternative to surgical treatment. post hypoxic-ischemic (hi) reperfusion induces the formation of non protein bound iron (npbi), leading to production of the reactive hydroxyl radical. it was investigated if the ironchelator deferoxamine (dfo) could reduce free radical production and improve neonatal myocardial performance after hi. severe hi was produced in 13 newborn lambs and changes from pre-hl values were measured at 15, 60 and 120 min post-hi for (mean) aortic pressure (mean pao), cardiac output (co) and stroke work (sw). left ventricular (lv) contractility and co were assessed by measuring lv pressure (tip-manometer) and volume (conductance catheter), using inferior caval vein occlusion to obtain slope (ees) and intercept of the end systolic pv relationship (v10). npbi, reduced and oxidized vitamine c ratio (vcred/ox) and lipid peroxidation (mda) were measured from sinus coronarius blood. 7 lambs received dfo (10 mg/kg i.v.) immediately post-hi, control lambs (cont) received a placebo. results: mean pao was stable, co and sw decreased up to 60 and 40% respectively in cont as compared to pre-hi. in both dfo-groups co and sw remained within the normal range. ees and v10 decreased in all groups post hi, but did not differ between groups. npbi and mda were higher at 15 min post hi (p<o.05), vcred/ox was lower at 15 min post-hi (p<o.o5) in cont as compared to dfo-group, indicating more oxidative stress in cont. conclusions: dfo reduced the oxidative stress of the heart and prevented co and sw to drop, suggesting a positive effect of iron chelation on the myocardium after h{. from 1987 we published reports with analysis of performance of the heart as a whole organ.while analysing the heart performance as a whole, we recognize three block in it located intrapericardially: l."atrial"block -left (la) and rigth (ra) atriums; 2."aorta-pulmonary" block-aorta 'bulb (a) and pulmonary artery(pa); 3.ventricular "three-chambered" block (vb) consisting of: -left (lv) and right (rv) myocardial chambers, both frith blood outflow into "aorta-pulmonary" block vessels, and -spongy (venous) myocardial chamber with the blood outflow through coronary sinus (cs) and thebesiau vein (tv) into "atrial" block. the following conceps are introduced for vb functions assessment "common" systole and "common" diastole of "three-chambered" vb. the process of normal "common" vb systole: -begins with blood ejection from vb spongy chamber into the "atrial" block; -continues with blood outflow from rv and lv into "aorta-pulmonary" block wi~ venous minimums -x-coiiapses -~btmation iu "a~riai" block; -completes with "three-chambered" vb general emptying. at this period the following blood volumes are transferring: -two-from rv and lv(their stroke volumes)into"aor~o-pulmonary"block; -two-from "spongy" chamber .into "atrial" block; -two-from systemic and pulmonary veins into "atrial" block during the process of so called "systolic" membrane suction (at pulling atrio-ventricular valves into rv and lv chambers as blood ejects out of them). it appears to be the regulation of blood inflow to "atrial" block by blond outflow from "three-chambered" vb into "aorto-pulmonary" block. objective: to evaluate the efficacy and complications of transpyloric enteral nutrition(ten) in critically ill children patients and methods~ from june 1994 to january 1996 23 children (i0 boys and 13 girls), aged from 5 days to 11 years (mean l.8 ± 3.4years) were treated with ten. 18 patients were included after cardiac surgery, 3 with acute respiratory failure, and 2 after cerebral surgery. the indication of ten was mechanical ventilation in 22 patients and failure of nasogastric nutrition in 1 patient.19 children (82 %) had previously received parenteral nutrition. 8 and i0 fg enteral tubes was inserted into duodenus through nasogastric intubation en 21 patients and by endoscopy in 2 patients. rx and ph determination were used as methods to control tube situation. 18 children received adapted formula, 7 caseine hidrolisate, and 3 enteral formulas. (in 5 patients the nutrition formula was changed during ten). 22 patients were supported with mechanical ventilation during ten, 17 received midazolamperfussion(range 2 to 15 mcg/kg/min), 15 fentanyl (i -12.5 mcg/kg/h), and 6 vecuronium (0.1 -0.3 mg/kg/h). resulte: ten was used during 3 to 73 days (mean 18.3 ± 19.3 days). mean maximmnvolume ad~iniateredwas 132 ± 47 ml/kg/day (range 34 208) .patients with midazolam, fentanyl and vecuronitml tolerated ten similar than children without sedatives. complications were diarrhoea 1 patient, abdominal distension and/or important gastric fed residuals 2 patients. pulmonary aspiration or respiratory complications were no registered. ten was ended in one patient due to diarrhoea, in 18 due to change to oral or nasogastric nutrition, 3 children continue yet with ten and 1 was discharged of picuwith ten. conolusion: ten is an useful method of nutritional support in critically ill children with mechanical ventilation and/or nasogastric intolerance. introduction: the enteral nutritional support of the critically ill child may reduce the morbidity and mortality by attenuating, the hypermetabolic and catabolic response, decreasing the gut translocation and the septic complications, improving the bowel mucosa integrity and the wound healing. the aim of this study was to show the safety and tolerance of en. methods: we enrolled 36 consecutive patients admitted to our unit from may to december 1995, mean age 3.4 years (range imonth to i7 years). the prism, mechanical ventilation, inotropic use, opiates and other drugs were recorded. the en was delivered by continuous infusion across a polyuretane tube.we kept the patients head elevated 30 °, and we used cisapride routinely. the gap betweeen admission and the en beginning, average time to meet the nutritional objective, en duration, selected formula, patients outcome and complications were recorded. results: prism groups were 0-1=0 patients; 1-5=1; 5-10=13; 10-15=7; 15-30=12. two patients with mof (multiorgan failure) died. 72% needed mechanical ventilation.the average time elapsed from picu admission to initiation of en was 2.1 days (range 1 to 7 ) and the average duration of the en was 7.3 days (range 3 to t4). the average time to meet the nutritional objective was 2 days (range 1 to 3). the selected formula were: lactose free infant formula in 10 patients, elemental diet in 8, pediasure in 16 and jevity in 2. we recorded vomiting in 4 patients, diarrhea in 5 and constipation in 4. none of them forced the en suspension. we didn't recorded any case of ~spiration or pneumonia. we found no relation between drugs, selected formula and complications. we think that en may be a safe and well tolerated procedure at the picu setting. generation of free radicals in iipid emulsions used in parenterai nutrition (pn) has been described recently. this seems to be due to high polyunsaturated fatty acid content. we studied lipoperoxidation status and antioxidant parameters presurgically and through the postoperative period (5 days) in a group of twelve children (ranging from 4 months to 12 years of age), who underwent heart surgery and received nutritional support with pn. malondiaidehyde (mda), an end product of lipid peroxidation was used as a marker of oxidative stress. it was determined by the yagi spectrofluorometric method. antioxidant activity was measured with an assay based on the inhibition of spontaneous autooxidation of a brain homogenate, and vitamin e in serum by the technique of hansen and warick. erythrocyte mda release fonowing incubation in hzo 2 in vitro was used as a functional measure of tissue vitamin e levels. data were compared using one-way variance analysis. there was not significant differences in the plasma mda production among the values obtained before surgery (3 :t: 0.5 nmol/ml), postoperative pre-pn (3.5 +_ 0.6 nmol/ml) and through the course of pn (3.5 5:0.6 nmol/ml). levels of vitamin e previous to surgery were 5,1 5:1.1 #g/ml and decreased to 3.2 _+ 1.4 #g/ml at 24 hours post-surgery before starting pn. while receiving pn, concentrations of vitamin e increased progressively up to 7 + 4. l #g/ml (p = 0.08). pre-surgicai plasma antioxidaut activity values (72 :i: 11%) dimiuished in the first postoperative day (56 _+ 9.5%) and then showed a clear increasing tendency, directly correlated with that observed in vitamin e levels. the measurements of the maximal percentage of mda release of erythrocytes in vitro, showed an inverse relationship with plasma vitamin e levels and with the plasma antioxidant capacity: preoperative 5.8 + 2.4 % and postoperative descending from 20.5 + 14% to 12 _+ 9.5%. our results indicate that during surgery and within 24 hours postsurgery, a decline in antioxidant defenses occurs, in the postoperative period, while patients are on pn and taldng into account our obtained data: the unaltered mda values, the rise of plasma vitamin e levels, the recovery of both functional erythrocyte membrane antioxidant protection and the plasma antioxidant activity, they do reflect that during the intravenous administration of lipid infusions, oxidative damage does not occur, probably due to the addition of vitamin e to pn solutions in order to avoid autooxidation of lipid emulsions. garnitine is one of the essential nutrient in the diet of newborn infants. mother's milk represents the natural eamitine source for the newborn infants. we studied enteral earnitine intake in 71 premature infants 35 eutrophic (eu) and 36 hypotrophic (hy), fed with mothers milk trough 5 consecutive days-from fifth to tenth day of life. mean gestational age was 33 (range 32-36) weeks for eu and 34 (range 32-36) weeks for hy. birth weight was ranged 1430-2450 g for eu and 1250-2300 g for hy (p < 0,05). breast milk carnitine concentration was 78,88 i.tmol/i in mothers milk delivered eutrophic babies, and singnificant higher mean coenetration 113,88 l.tmol/i, of hypotrophic premature infants (p<0,05). breast milk earnitine concentration was ranged between 39,15-163,71 ~moi,'l, and daily carnitine excretion was between 9,7-165,48 p.mol/i, mean 60,6 +-38,7i p.molll. the daily mean carnitine intake was from 1-1,93 mgkg/d, in eutrophic and 1,23-1,93 mg/kg/d, in hypotrophic premature infants, in milk volumene intake from 150-200 mllkgld., the results of this study suggest that premature infants should be fed with own mothers mitk in eady neonatal pedod in attempt to prevent camitine deficiency. keto-acids and parenteral lipid admlnistration. castorina m., antuzzi d., ricci r., rendeli c., polidori g. pediatric intensive care unit -catholic university -roma (italy). some metabolic studies have suggested that the administration of mediumchain tltgticerides (mct) might induce a marked increase in ketone body concentrations; the aim of this study is to evaluate the effective ketogenetic risk of infusing mct emulsions in total parenteral nutrition (tpn). two groups of seriously infected children were examined: a-12 septic infants were given to tpn with a 100 % of long-chain tryglicerides (100 lct). b-10 septic infants, in wich the lipid source in tpn consisted in a 50-50 mixture of lct and mct. the children in the different groups showed similar severity and therapeutic scores (prism, tiss, ~, f~2, f~3); the lipid intake was the same, corresponding to 1-1.5 g, ckg bodyweight/day. in all patients a sample of urine was taken six times a day for the whole time of tpn the urine samples were screened by the diphenylhydrazone test, and the ketoacids excretion was confirmed by a chromatography. the urinary excretion of 13-keto-acids equivalent to p-ohphenyl-pymvate (mcg/ml urine) is summarized in the no significant increases in frequence and/or in severity of acid-base disturbances were found in all studied patients in consequence of the lipid infusion. the patients of b-group showed a lower excretion of 13-ketoacids. the c~-keto-acids excretion was ever unsignificant and seemed to be correlated with the illness severity more than with the lenght of the tryglicerides chain. this observations let us suppose that the mct, at employed doses, can be safely administred also in childen with metabolic acidosis and/or serious illness. reye syndrome cayetano heredia hospital, lima, peru five patients with reye syndrome were studied; included were those diagnosed from january 1991 to march 1994 and treated at cayetano heredia hospital. the age of presentation varied from z.5 to 7 months. the syndrome occurred more frequently in males (4/5); the time of illness at the presentation varied from 2 to lo days, and the following features were found: fever (3/5), akeration in mental status (5/5), seizures (4/5), gastrointestinal illnesses (diarrhea) (4/5), and respiratory insufficiency (1/5) --in this case ventilator)-support was needed, in all cases hepatomegaly, intracranial hypertension, hypokalemia hyponatremia, metabolic acidosis, hyperammonemia and elevation of hepatic enzymes were found. coagulation blood tests were abnormal in three patients. cerebrospinal fluid (csf) showed hypocellularity in all cases (less than 8 cells/ram3). the cultives were negative, and the final diagnosis was confirmed by hepatic biopsy. no deaths were due to reye syndrome. a contribuhon to the study of reye's syndrome the aulhors analysed 38 records of patients meeting all the clinical, biochemical and anatomic criteria put forward by hutrealoch~ and samaha (1975) who were ireated at the emergency and resusdlation depammlt tithe ipch in 10 years. the average age of the children was 3 years (the 3~umgest 11 monks and the driest 7 year~ most of lhon (34/38) came from lhe countryside to lhe lmfia~ willfin 24 hours of file oulbreak. a few days previously the patients w¢~ld have fever (some of ahem wilh diarrhea or cough) ,,rod vomit then rapidly fidl into coma and c~nvuksien. they ~me to the imtia~ with the signs of typical increased inlracranial pressure ( stages 2-4 according to lovejiy for most cases) but the liver was unllkely big. the main biocheafical dmnge of the blood was acute hepatic thilure -the bllod glucose decreased : (ha the aver~ 23,46n~% (26) ~ not measured in five cases (glucose a"aces). -the blood ammonia ino~ased riven 158 to 275 microg/dl fin lhe average 220, n = 3) -the percentage of p~in was low, in the average 44,5% (n--9), 11% at the lowest (a case ofmekaa lasling five days was ctwed with vitamin k). -got and gpt enzymes increased 3 -4 times (n=14) besides, tht~re was decompcmated addt~is with lhe average valnes ph = 7.19; pcoz = 2~6mmhg and eli = -1~6 ~, (n~). the charadaislic dumge in the ¢~'ebrospinal fluid was a low glucose tx~acenwalien h the average of 1k65mg/dl (3 cases of glucose it'aces) ; meanwhile protein decreased in the average of 14mg/dl (4 cases below 10mg/dl). besides ~here was no inflammatory lesion ~tion) : very serious brain oedona and fatty degeneration of live~ scatteredly or parllally.microso~ically (biopsy and necropsy) in file brain appeared bright space around blood arteries and glioma extended v'lrdmw -robin space, with no inflammatory reaction. liver.-the sa~dure was intact there was no " "mtlammal~s3' cell, the kupffer cells did not show hyperplasia but show a heterogenous deg~nerafion~eart: also showed a fatty degencralion in some areas of the myocard eellkidney: the ihtty degenerati~l scattetedly in the tubular cell. pano~as:fatty degeneration scattetedly in file pancreatic cell. two cases of liver biopsy for lhe 2rid lime (check before leaving file inslitute) found that fatty degeneration was r~tta~ nearty ff~pletely. the exact muse remained unknown. objectives: to analyse whether there exists serum and urine electrolyte disorder in children with respiratory failure, methods: we have made prospective study of 48 children in our icu during a 12 month period, electrolyte concentrations were measured in serum and urine collected during 24 hours (sodium-na, potassium k, chloride-ci, calcium-ca, magnesium-mg and phosphorus-p). results: average values in serum were: na 139.28 +/-3,20 (rv:133-146) mmol/i, k 4.22 +/-0.55 (3.3-5.2)mmol/i), cl 95.20 +/-3.98 (93-106) mmol/l, ca 2.t2 +/-0.12 (2.20 2.65)mm01/i), mg 0.82 +/-0.22 (0.77-1.12)mmol/i. average electrolyte levels in 24 hours urine were: na 64.23 ~/~ 44.11 (42-202)mmol/day. k 22.14 +/-10.33 (22-112)mmol/day, ci 56.14 +/-27.22 (100-244)mmol/day, ca 1.17 +/-0.13 (1.68-6.8)mmol/day and p 11.89 +/-7.12 (11.7-32)mmol/day. conclusions: average serum ci and ca levels were decreased in children with respiratory failure. in urine, average k, ci, ca and mg levels were decreased and urine p concentration was increased. we concluded that serum and urine electrolite disbalance may be expected in children with respiratory failure. the nurse is going to kill me! (icu syndrome) anita duvndam ~, sonia v.d,sluis 2 ~dpt. of pediatrics, div. pediatric intensive care, sophia children's hospital, rotterdam 2dpt. of pediatrics, div. pediatric tntensive care, juliana children's hospital, the hague. content description: this presentation will provide the critical nurse with background information concerning the identification, the prevention and management of the critical ill child who has developed the icu syndrome. the results of a questionnaire concerning the occurrence of icu syndrome on a picu will be presented. behavioral objectives: at the end of this session the participant will be able to: 1. describe four clinical symptoms of the tcu syndrome, 2. identify major sources of picu environmental stress, 3. discuss nursing's unique role in helping the child to cope with the icu syndrome, 4. discuss nursing's unique role in preventing icu syndrome. content outline: the icu syndrome is a situation in which the individual is not able to deal with changes in observation and interpretation of both quantity and of patterns of sensory perceptions. in other words the borders between the inner and outer world becomes unclear for the person. the exact incidence of the icu syndrome in the critical care setting is unknown, we have limited knowledge of the occurrence of the icu syndrome in critical ill children. yet critical ill children in our hospitals sometimes show the symptoms of icu syndromes. to be able to identify the occurrence of icu syndrome we developed a questionnaire. the thesis of the questionnaire was: does the icu syndrome occur in critical ill children in the age between three and ten, who have been intubated and/or ventilated in a picu? eighteen questionnaires have been send out to parents of critical ill children in two hospitals. the response was 90 percent. conclusions: 1. most of the children were anxious (n=11), showed regression (n=8) or had sleeping problems (n-lo) during the stay on the picu. 2. children showed the same problems after discharge. 3. there is no relationship between environmental factors and symptoms of the tcu syndrome during the stay. 4. there is no relationship between preexistential behavior and symptoms of the icu syndrome during the stay. because the lack of a scientific definition for the icu syndrome in children and good criteria for diagnosis, we were not able to get an answer to our thesis. more research is needed. continuous veno venous haemofiltration (cvvh) was adopted as a first line renal replacement therapy in our paediatric intensive care unit (picu) some three years ago. ~/he process of introduction and development of cvvh proved to be an exciting challenge for nurses as indeed it was for the whole multidisciplinary team involved. we have successfully used cvvh in the treatment of over 20 infants and children, weighing between 3-85 kg. the range of conditions treated is ever increasing. to date we have not only used cvvh for patients in renal failure and fluid overload, but also to gain biochemical control in tumour lysis syndrome-and metabolic disorders. other distinct patient benefits in comparison with more conventional means of renal replacement therapies are that, it is a continuous controlled treatment being extremely effective in creating 'space' for nutrition, which is especially important in the fluid restricted, catabolic patient. of paramount importance in providing this level of service is the education and training necessary to impart the skills and knowledge for nurses to become expert practitioners in this field. we now have a teem of highly advanced practitioners who have developed their nursing skills to provide even more holistic care for their patients. this group are embracing new challenges and responding positively to the developing service whilst expanding their knowledge base. this paper will discuss the advantages and disadvantages of cvvh as we have experienced, relating i t to the wide variety of patients we have treated. we wish to share how cvvh has become an accepted role of the picu nurse and how the service has been successfully implemented. integrated care pathways (icp's) define the optimum course of events in the care of a patient with a specific condition, within a set timescale. aims and methods: icp's and case management (cm) were introduced to evaluate and improve clinical practice. icp's were developed for patients undergoing surgery for atrial septal defect (asd) ventricular septal defect (vsd) and fallots tetralogy. they were based on the median experiences of the last 31 patients. 108 patients have been managed using icp's. results: analysis of variation from the icp's shows a reduction in the following potentially avoidable causes of variation: delay in extubation has been reduced from 29% to 10%, and 31 patients (29%) were extubated earlier than the median. prolonged stay on the intensive care unit (icu) has decreased from 13% to 0%, and 24 patients (22%) were discharged to the ward a day earlier than the median. the number of patients receiving inadequate post operative analgesia has decreased from 35% to 15%. delayed feeding after operation has been reduced from 52% to 28%. unavoidable delays in extubation and discharge from icu occurred in 22 patients (20%) because of haemodynamic instability" or lung problems. cm utilising individualised pathways for these patients has reduced variation in care which can improve patient outcomes. pathways have been developed for other conditions and it is anticipated that approximately, 80% of patients will be treated using an icp revision of icp's results in continuous evaluation and improvement of the care provided, conclusions: the use of icp's and cm has improved patient care and decreased avoidable delays and variations. the future development of other icp's, combined with a case managed model for selected patients, is seen as a viable method of continuously improving patient care. this paper is a retrospective audit of 24 children who received continuous veno venous haemofiltration (cwh) whilst on the paediatrie intensive care unit (picu), data was collected over a 21 month period from may 1994 to january 1996. the 9 girls and 15 boys were aged from 1 day to 15 ½ years (median 4½ years) and weighed between 2,5 and 85 kg (median 17.75 kg). length of admission to picu was 2 to 47 days (median 9 days). sepsis syndrome was diagnosed in 11 cases: 4 of these had bowel necrosis and 3 meningoceccal disease. eight patients had an underlying haematological, oncolngical or immune disorder. the remaining children had inborn errors of metabolism (2), nephrotic syndrome (2), or cardiac failure (1). cvvh was instituted for a variety of reasons. muttiorgan dysfunction was present in 46% of patients, 29% had acute renal failure, 12.5% tumour lysis syndrome, 8.5% uncontrollable metabolic acidosis with hyperammoneemia and 4% required fluid management. treatment was continued for between 4 hours and 26 days (median 4½ days). haemofiltration was successful in achieving its desired effect in all except one patient. 87.5% of children with haematological, ontological or immune disorders died despite successful haemofiltration. survival was higher in cases of septicaemia (45.5%), nephrotic syndrome (50%) and errors of metabolism (50%). overall mortality was 62.5%. this was attributed to the severity of the underlying disease process rather than the effectiveness of cvvh as a renal replacement therapy. asynchronous defibrillation and synchronized cardioversion deliver direct currents of high amplitude through the chest, to stop ventricular fibrillation or to convert life-threatening arrhythmias. since the human body is partially conductant, it should be possible that after a brief delay of time, a derivation of this monophasic defibrillation pulse is measurable in regions of the defibrillated body, other than the traject between anodal ("sternum") and cathodai ("ape~;") paddles. one could also estimate that health care personnel in the immediate environment of defibrillatoty shocks, are always at risk to possible electric injury. accidents might occur when health care personnel do not stand clear from the patient during firing and create an additional electric path from this patient through their own body. most likely, the nonisolated parts creating a transversal eiectric path, will be the hands of the caretaker. since defibrillation generates touch voltages up to 5000 v and 60-70 a in ved, short delay's (5 to 10 millisec), the total body resistance during skin to skin or skin to paddle contact, is calculated to be as [ow as 750 ohm (percentile tank 50 for the entire population). an experimental protocol was developed to evaluate the conducted currents during defibrillation. mature pigs with a weight of 100 up to 150 kg. were used as animal models. the:,, had barbiturate anaesthesia, inotropic support with dobutamine at l 0 micmgrams/kg bodyweight and all had a sinasat rhythm at the begining of the test-rounds. synchronized defibrillation at 100 j (approx. 0.75 j/kg) and asynchronized -at 360 j (whenever ventricular fibrillation or ventricular tachycardia occurred), were attempted, through latero-lateral placed and firmly pressed defibrillation paddles and/or adhesive multi-function electrodes. gel pads were choosen as contactmedium to cross the skinpaddle barrier. subdermal electro-myography needle electrodes were put at different parts and regions of the pig's body, but not between the paddles. these electrodes were coupled to a resistance device of 800 ohm and a measuring computer. we measured monophasic and low current pulses up to 0,10 a during the defibrillation burst. thus conducted currents are high enough to produce accidental electric shocks when additional electric paths are created by the caretaker. "llaese are usually harmless, but in some "worst case" (wet or transpiring hands, rings,...) or in association with defectuous equipment, peak currents might be harmfull, and can produce pain, bums, apnae or cardiac (transient or persistant) arrhythmias. maria skogbv. karin mellgren, lars-g6ran friberg, g6sta mellgren, hans wadenvik. g6teborg, sweden. bleeding complications in extracorporeal circulation is partly due to perfusion-induced platelet dysfunction. the aim of this study was to evaluate an in vitro model for investigation of platelet function parameters in an extracorporeat system. study: two complete extracorporeal life support systems were perfused with fresh heparinized human blood for 24 hours. piatetet membrane glycoprotein changes, platelet granule release and platelet count were followed. eight paired experiments were performed. one of the circuits was perfused by a roller pump (polystan) and the other by a centrifugal pump (biomecicus), other components were identical. results: a continuous increase in glycoprotein (gp) ib-negative platelets was seen in both circuits. a marked increase of plasma beta-thromboglobulin concentration and a decrease of the intracellular pool of serotonin was observed, indicating a marked release of alpha as well as of dense granules. the plasma concentration of glycocalioin increased in parallel with a reduced platelet surface expression of gpib, suggesting that the loss of gpib is caused by proteolysis rather than by a downregulation of this receptor protein. conclusion: 24 hours of ecls perfusion induces pronounced platelet degranutation and causes changes of important membrane receptors. this is in accordance with clinical studies, suggesting that our in vitro model does mirror the platelet exhaustion observed in a clinical reality. no significant difference was observed between the two pumps. the purpose of this study was to examine signs of distress exhibited by several patients following the discontinuation of opioids and benzodiazepines (o & b) in a 7-bed pediatric intensive care unit. the authors compiled a list of all cases of possible withdrawal reactions reported by nurses in the study setting over a one-year period. five cases were selected for study in terms of their wide diversity of relevant circumstances. the 5 cases were examined through a retrospective chart review. data pertaining to analgesic and sedative administration, along with nurses' reports of behavioral distress, were transcribed and coded. a remarkable pattern of behavioral distress was clearly associated with discontinuation of o & b. cessation of benzodiazepines was associated with severe distress. these reactions were characterized by various combinations of crying, irritability, tremors, grimacing, gagging, vomiting, or feeding problems. some of these persisted in spite of large amounts of bolus drug administration. these signs were manifested for 2 to 9 days following cessation of o & b. these findings demonstrate that the rapid weaning of o & b infusions, sometimes following short-term therapy, can cause severe withdrawal reactions. the particular course that a specific patient will follow will likely be modulated by underlying manifestations of "icu psychosis" and unresolved pain and emotional distress. hermana tezano8 mt, pilaf orive j, latorre garcia j, lizarraga azparren ma, lopez bayon j ucip hospital de cruces. bilbao. spain a female child, one year old, was referred to our unit from another hospital because she had ischemic syntomsjn her right forearm wich started three days before. she had ~i downs syndrome and fallot's tetralogy with a systemic-pulmonary by-pass. nine days before the admission in our hospital she was undergone to a cardiac catheterization by arterial and venous femoral punctures with no incidences. on the admission to the picu her forearm was cold and pale with absence of cubital and radial pulses and slow capilary filling. treatment with heparine was started unsucessfully in the firsts 24 hours, so uroquinase was added that later was changed to rtpa plus brachial plexus blockade. by the time me arterial tlux became more and more evident (doppi~) il bccuute aiat) evident a regional aedema witch made necessary a fasciectomy &the wrist. she also developped necrotic delimitted areas that included her first and fifth fingers. she was discharged from the picu after 13 days and a week later she went to the operating room where an amputation of the distal phalange of the fifth finger was made. the etiolgy of this ischemic picture can't be attributed to the catheterization itself, but probably it should be due to some attempts in arterial puncture in its course, attempts that could be guess by the little marks noticed at the elbow flexure when she was admitted. long maintenance of cardiovascular funtion by assisted ventricular device with membrane oxigenator hermana tezanos mt, pilar orive j. latorre garcia j, lizarraga azparren ma lopez bay6n j. ucip. hospital de cruces. bilbao. spain a little girl of three and a half years came to the intensive care unit from the operating room. affected of great vessels transposition with a pulmona~' banding corrected vith rastelli technique, it became impossible to discharge her from the by-pass circulation. it was assumed that this factt was due to a transient myocardial disfunction dfter the surgeon x~ent through the technique and found no wrong step in it. at the picu admission she had an ejection fraction belox~ 20% (echncardiografy), and was manteined on veutricular assistance with a circuit consisting ofa plate's membrane oxigenator, a bio-medicus pump and pvc rubber tubes, with monitoring of pulmonary', right and left atrial pressures. initially she needed a ventncular support of 1.2 l/min with dopamine. dobutamine and adrenaline; gradually the ejection fraction rose to 60% aliowing a decrease in the mechanical and inotropic support. the organic function ,.'.-as preserved but x-ray of the thorax showed images of pulmonary aedema with an increase in the 02 needs until a fi02 of 0.4. even though she was placed in a transplant program, she was remouved when the ejection fraction rose to 50% on the 1 lth da 3 of the evolution. there were no signs of infection (profilactic coverage). on the fifhteen postsurgery day. with a good cardiac sound and an ejection fraction of at least 60%. it was considered the withdrawal of the ventficular support, but unsucessfully despite the increase ofinotropic drugs, so she died. we think that our patient is a good example of the posibiti~ to mantain x~ith ecmo during a prolonged period of time a potential transplant receiver without major complicatios. joan wierema; ma0)an mourik. sophia children's hospital, department of pediatric surger,j, rotterdam, the netherlands. the success of an ecmo program is heavily determined by the organizational structure and the daily availability of both well trained nurses and physicians. until now there is no general guideline to determine the optimal training schedule to set up an ecmo program. objective: to evaluate the set-up of an ecmo program in an area without direct availability of perfusionists of cardiothoracic surgeons. description of the progress: during the preparation of the ecmo program which started november 1991, different phases are identified. first phase, acquisition of experience by training abroad of one physician, staff member of the icu, physician acting as a fellow and one icu nurse with theprimary task to serve as an ecmo coordinator and train the nursing start. in the second phase 12 animal experiments were performed by 2 paediatric surgeons, 2 staff physicians, 2 fellows and 12 nurses. third phase, start of the actual ecmo program, priming by one trained nurse and ecmo fellow, daily care of the patient by 2 nurses, 1 icu nurse taking care of the patient related activities, 1 ecmo trained nurse taking care of the circuit. fourth phase, transition of the tasks for priming the system from the ecmo fellow to the ecmo nurse and changing daily care for the ecmo patient now including the circuit by 1 trained icu nurse with special legislation for ecmo. ongoing training of 6 to 8 nurses working for at least half a year in the icu. during an ecmo run direct contact between the ecmo nurse and one of the staff members; patient data: in a 4 year period 52 neonatal ecmo cases were performed with an overall mortality of 74%, ranging from over 95% in meconium aspiration to 45% in congenital diaphragmatic hernia. in 7 other patients pediatric ecmo (age range 1 month -14 months) mortality was 50%. conclusions: ecmo can be established without perfusionists or cardio surgical back up once a predetermined training schedule is available, resulting in comparable results with the international ecmo registry both for neonatal as well as for pediatric cases. mechanical ventilation at home makes normal development of the child with chronic respiratory failure possible. the parents must be encouraged to accept the treatment at home; therefore, they should be enabled to take care of their children all by themselves and the continuous professional support has to be offered to them. it must be stressed that the nurse is the link between the diseased child and his family introducing them into the process of the medical care at home. all the equipment needed for the mechanical ventilation of the child at home (ventilator, suction device, pulse oxymeter, oxygen tanks) has to be provided before the child is discharged. it is paid by the national insurance company. the maintainance service workers will make the necessary installations available and they will renew oxygen and compressed air weekly. the ventilation assembly must not interfere with the childs activities. the related dispensary and nursing service should be notified of the child's condition and the list of the required material (suction tubes, cannulas, desinfectants etc.) has to be made. our experience is so far -from the nursing and medical point of view -satisfactory, however the social status of the parents needs further regulations, omphalocele. mourik m, sophia children's hospital, department of pediatric surgery, rotterdam, the netherlands. in patients with (giant) omphalocde operative closure is impossible due to the lack of intra-abdominal space and a variable degree of pulmonary hypoplasia. consequently conservative treatment allowing epithelialization is the treatment of choice with a high risk of infection and/or sepsis due to a sometimes very long stay in the icu. this conservative treatment consists of daily application of an antibiotic containing powder on the cele which is covered by sterile gauzes. furthermore the enteral intake is started as soon possible. objective: to evaluate whether this treatment renders a high risk for serious infections. patient characteristics: in a 10 year period 23 patients with a giant omphalocele were admitted; mean birth weight 2800g (1500-3990g) mean gestational age 38.5 (33-42 weeks). overall mortality 6 (19%). 11 patients had to be ventilated for a median duration of t6 days (2,5-164). results: patients were discharged at the mean of 68 days following birth (17-260) following complete epithelialization of the omphalocele. following the initial stabilisation period parents were involved in daily care of all patients even during artificial ventialation. enteral intake was started at a median of 2 days (1-18). colonization of the omphalocele was observed within 2 -4 days in all patients. although infectious periods were observed at regular intervals, sepsis was the primarily cause of death in only 3 of the 6 patients who died, in conclusion: nursing care to prevent sepsis in patients with (giant) omphalocele is feasible for a long period of time and can be performed with simple measures, since june 1994, we have been using nasal cpap in the treatment of respiratory disorders in newborn infants (severe apnea syndrom, tachypnea, hyaline membrane disease). in newborns presenting hyaline membrane disease, we have used cpap and administered surfactant therapy to 25 premature babies out of 70. clinical data were : mean ga : 30.8 + 23 weeks. mean bw : 1686 + 551 g. mean administration time was 11 hours. before administration, mean fio2 was 0.4 + 0.07, mean pao2 6.2 + 1.8 kpa and mean a/ao2 ratio 0.21. all babies were intubated for administration of surfactant (curosurf) and were extubated after half an hour to 3 hours after administration. this treatment failed for seven babies and they were ventilated by oscillation ventilation ; all babies survived. complications : we have observed pneumothorax in 2 cases and 1 cerebral hemorrage. mean duration time of nasal cpap was 71 hours for the 18 babies without assisted ventilation. nurses in charge of babies with nasal cpap should be aware of neonatal care, of the possibility of surfactant administration and of complications during this type of treatment. therefore nurses should know very well the use of nasal cpap, the monitoring during this treatment, the fixation of the system on the baby, nursing of the babies during this treatment and finally should take care of the baby physical and psychological well being. as a conclusion, nurses in intensive care neonatal units should know the possibility of treatment of rds by nasal cpap and should be aware of baby-nursing. emco in the postcardiotomy infan_t : a simplified circuit and reduced management grillet lsabelle, bosson christa, goelz andrea, helmer pascale, icu nurses, tschaut rudy perfusianist aldo castaneda institute, clinique de genolier, suisse to improve postoperative survival of infants with repaired cardiac tessions but severe residual cardiopnlmonary dysfunction, emco was used in 4 infants, ranging in age from l0 days to 9 months and in weight from 2,8 kg to 7,8 kg. diagnosis included tga / intact ventricular septum (2) (both subiected to rapid arterial switch operation because of unsuitable lv function) and tga +vsd (2). "['he emco circuit included a sarns roller 15ump, a medtronic minimax plus hollow fiber oxygenator and tubing (1/4 -1/16 inch) with bioactive surfaces, an arterial canula 10 fr., 2 venous canulas : right atrial 20 fr, left atrial 12 fr., an air oxygen mixer and a sarns heater. the act was kept between 180-200 sec. after the infant was connected to emco by the surgical team and the perfusion technologist, the patients were being cared for and controlled exclusively by the 1cu nurse assigned to the patient and the physician on call for the icu, pump flows of 100-150ml / kg / mln were targeted to insure an adequate urine output, a brisk capillary fill and physiologic left and right atrial pressures. sodium nitroprusside was used to control systemic hypertension while all other inotropic drugs were discontinued. inspired oxygen fraction on the ventilator was reduced to 0,25 at a respiratory rate of 10-15 breaths per minute. body temperature was maintened close to normal. when the heart started to eject again, inspired oxygen was increased to about 0,40. the preparation for weaning from emco was done by the icu nurse: including oxygen fraction and ventilator rate. weaning from emcowas coordinated by the medical / surgical team and the perfusion technologist. the duration of emco in the 4 patients ranged from 2 to 9 days. three patients (75%) were saccessflly weaned from emco. one of the three died within 24 hours from neurologic complications, unrelated to the emco. the other 2 patients ( both with tga + vsd ) are long term survivors and are doing well. neither the survivors nor the patients who died bad hemorrhagic complications, despite the fact that 3 of the 4 were placed on emco because of failure to wean from cardiopulmonary bypass. the simplified emco circuit worked reliably throughout this experience and no complications occurred nor could any shortcoming be ascribed to the use of this reduced, cost effective management team. 10 years of praciical experience wiih exiracorporeal membrane oxygenation (echo) -the viewpoint of the nursing siaef monika schindler in 1985 a training program was started for the introduction of echo in the kinderklinik of mannheim/germsny. 2 years later (1987) the first european echo patient, a 3 days old newborn baby, ~as treated successfully in our institution. with echo, more treatment modalities ~ere available after failure of conventional therapy in eases of severe lung diseases like meeonium aspiration syndrome, congenital diaphragmatic hernia, sepsis/pneumonia, primary persistent pulmonary hypertension of the newborn and -in infants and children -ards (acquired respiratory distress syndrome) due to multiple underlying illnesses. in 1989 the first ards patient, a 5 years old girl, was treated successfully ~ith ecmo in mannheim. until now 137 patients (age: i day -10 years) were treated with echo in our institution; 111 additional patients were transferred to us from other institutions for therapy with echo, but could be treated ~ieh alternative moda]ities of therapy like improved conventional ventilatory support, high frequency oscillatory ventilation (hfov) and inhaled nieric oxide (no), unexpectedly the more ~ide-spread use of hfov and no -also in other ~ntensive care units -did not lead to a reduction of the echo frequency in our hospital. due to this long ecmo practice, which is helpful in many critical situations, a certain routine came up in the nursing staff of our intensive care units; but ecmo continues to be a maximum strain for all co-workers and implies an high personnel and material/financial-expense. but in our opinion the improved survival rates in severe respiratory failure-(70 % in neonates and 50 % in infants and children, estimated survival rates under continued conventional management: < 20 %) by the use of echo justifies this expense, even in times of less financial support for public health. • analyse the differences between each urdt. method : ni criteria were defined by the rt~a ped group according to cdc criteria. all data were collected by a nursing and medical team. data 3vere dealed with epi info software. all u-dections data were validated by an external mvestigatol : 4 525 patients who stay more than 48 hours in an icu were included over a 14 months period. 68 % were newborns, 19 % infants and ,13% children. 371 ni were idenlified among sll patients, the overall ni incidence rates was 8,2 % and 5,9 person day, septicemia was the first cause of ni (50 %). staphylococcus epidermidis were isolated in 60 % of septicemia cases. pneumonia was the other main ni (41%) with gram negatwe bacille isolated m 53 % (40 % of them beei,ng fseudomonas) accordmg to age, the septicemia mcidence rate varied from 6,8 ~., to 10.9 %,, catheter/day and pneumonia incidence rate from 3,9 %, to 7,3 % ventilation/daywith the lowest rate for newborns. : this survey was possible thanks to the collaboration between laboratories, bacteriologist, physicians and nurses, and allowed each concerned unit to work together in~tead of every one in his own field. 'lhe results of this survey bring changes in attlhades and empower the different team work the next step is to analyse the ditferent nurses procedures of indwelling central venous catheter in each unit ar)d implement a ni quatity care. program in all nicu anti picu. v~,~jl~j~'v~y : new born in~ra-v~6nous drugs ~md lheir pr~taration mcounter a lot of risk ~)f utters, due to inadapted conditionn~ng to podia)eric and minimal amount injec~.~d, th~ study t~ed to evaluate degrees of do.~age er~o~ dye tt) dru~ preparaek, n i~ nc,:,~atol~gy and to come out w~th ~ method of dihttion able to reduce risk to minima (errors _< 5 %) at the ~eme t~me, ¢o~,~x-tueaces on cost were studioj methnd~ : amikacine was choosen because this antibiotic is regularly ~,~l in neonatology unit and its dosage is easily used by standardiaud method, 30 amikacine doses (15, 23 mg doses and 15, 12 mg doses) were prepared" by 15 nurse~ form 50 mg for 1 m) vial. each dose w'a~ diluted to obtain 2 mi volume, tallowing usual ward method, for each dilution, amikacine con<entrati('m and it,s volume was measured and used equipment for preparation wa~ regi~tred. 10 "first s01utj(m" were prepared diluting ~t'~ mg amikacine pouder inn 51) ml glucnse solution bag (viaflex baxter bag with transfer cap), from tho~e "first solution" 15 doses at 23 mg and 15 doses of ~2 rng were prepared, amik~cine coc, cenir,)tmn for each preparabon and their volume were measured, ai,~n ntttl~,ber of seringe were tounted. first soiuhon staeiiity was conrroieo aher one week conservation at + 4'>c. amjkacine concentration were measured by fluorescence process (tdx abbott) after sample dilurion. on a 10 mg/l sample, tovhnical reliahility show~ > 9~ % of result mpmductlon and < 5 % of variation due to dilutions. results : when amikacine injection werv pro.pared from araikacme 5/) mg for 1 mt vial > 10 % do~ge, ermr~ were found in 19/40 cases ; ~ 30 % in ,t1,to cases. if preparation is done from amikacine "~it'st soltltion", les.--concenvr~tcd, it i~ more preci,,,e and only one dosage error ~ 5 % (6,3 %} is found in eli 30 studied doses. in add)inn to )hal if 10 doses were wep,m-'d from one "first soiatiol~' bag, the cost economy sl~ouid b~" of 32 fr~, and ii 20 dos~$ were prepared tram the same bag the saving mtmey should be o{ i72 its .cencluslon : .ur survey shows th~t h' ntu)nato|ogy the u~ of a "first sohation which can be kept fi~r one week is enable to reduce dosage erroes and i~ co,~tsavmg, regarding [,v. admimst'rahon method the survey is still on, introduction: so-called vein of galen m~iformations ale rare in~racranial embryologycal anomalies, repl~senti~g tess than 1 of symptomatic intracranied artefiovenoas l~alform~tions. the spontlneous prognosis is ~s~u~lly fatal, because of cardiac frilure due to left-to-right shunt thrq~ugh the fistula. recent developments of new techniques of treatment of the malformation and its cardiac consequence have led to a revolution in the practical approach of children w~th galen malformation. our fukfose is to contribute, with our persoaal series of 7s newborns and infal~ts admitted in our unit after endov~,scular embolization, to a better management of these children. such a management requ!res a rnultidisciplinary approach. intensive care are required prior to embollzation for patients with cardiac failure or cardiogenic shock and after cmbolization in order to insure cardiac and cerebral hemodyna.mic stabilities. this overlooking suppose for the nursing team to understand: prior to embolization : heart failure and cardiogenic shock. after cmbolization : evaluation of neurological and hemodynamic consequences of this proccdure, without forgetting the nursing and psychologic aspects, in concl'iision, this last ten yerrs, these new approaches give to the patients and their famitiy a good reason to hope a total recovew, in our exl)erience, the global mortality is 9 % aad 66 % of children #j-e neurologically normal after embolizafion, ii ii~ i ~ii i ii i i l i iiii~ i ~i iii i background: venous oxygen saturation (svo z) reflects the residuai oxygen after tissue oxygen extraction and represents the relation between tissue oxygen supply and demand. we studied svo 2 and arterial lactate during progressive isovolemic anemia to assess the relation between svo2 and tissue hypoxia. subjects: ten 8-10 day old anesthetized ventilated piglets sao 2 and svq were measured continuously by a fiberoptic catheter (oximetrix, abbott lab.) in the carotid and pulmonary a~epy tissue hypoxia was confirmed by a reduced vo, and an increase in lactate. conclusion: svo 2 reflects better a reduced dp obtained by progressive anemia surfactant replacement improves gas exchange in early-stage adult respiratory syndrome (ards) [1,2], but not in late-stage ards [3] . we report the first case of successfull treatment of ards after repeated instillation of surfactant.a ten year old boy, weighing 32 kg, presented with hemorragic shock. biphasic-positive-airways-pressure ventilation was performed (evita ii, dr~ger, germany). he had recieved nine units of packed red blood cells and underwent surgical exeresis of two bleeding gastric ulcus. post-operatively, a cardiac arrest required cardiopulmonary resuscitation for three minutes. hemodynamic status was subsequently stabilised. the chest-radiograph showed infiltrates of both lungs without signs of cardiac failure. on the third day, the patient became severely hypoxic with a pao2/fio 2 ratio of 30. gas exchange was not improved by high ventilator settings. peak inspiratory pressure (pip) and ventilatory rates were 40 cmh~o and 18 breaths/min respectively. inspiratory:expiratory time was 1:1 and the positive end expiratory pressure (peep) 8 cmh20. after increasing the peep level to 11 cmh20, we instilled over 2 minutes, 80 mg/kg of porcine surfactant (curosurf, serene france), in two equal volumes in both main bronchus,the spo~ rose to 97% within 15 rain, the fie 2 could be reduced to 0.6. twenty four hours later, gas exchange worsened again (pao2/fio2 ratio 90). we increased the peep from 8 to 11 cmh20, and instilled a second dose of surfactant (60 mg/kg). again, fie 2 could be reduced within 15 minutes (spo 2 95; fie 2 0.6.). the patient was weaned from the ventilator and extubated on the tenth day. follow-up at four month showed normal lung function.we demonstrate improvement in oxygenation after repeated exogenous surfactant administrations. we assume that in early-stage ards, surfactant may potentiate shunt-reducing effect of peep as it has been demonstrated in experimental model of ards [4] , and allow decrease in fie2. in case of secondary deterioration, we think that a second dose of surfactant should be administered. 1. weg jg, balk ra, tharratt rs, et al. ,lama 1994 : 272: 1433 -8. 2. spragg rg, gillard n, pdchman p, et al. chest t994: 105: 195-202. 3. haslam pl, hughes da, mcnaughton pd. et al. lancet 1994 343:1009 -11. 4. huang yc, caimulti sp, fawcett ta, et al. jappl physiol 1994 76:991-1001 43% (ref) . the aim of this study was to verify these data: patients/~lethods: all pts admitted to our multidisciplinary nicu/picu in 1995 were included if they were in respiratory failure recruiting conventional mechanical ventilation (cmv) with peep >_ 6 and 'fig2 -:2 50% or high-frequency oscillation ventilation (hfo) with mean airway pressure _> t8cm h20 for 12 or more houm. diagnosis, maximal ventilatory parameters, barotrauma, organ/ system failures, mechanism of death and glasgow oulcome scale (gos) 1 and 6 months after study entry were prospectively collected. results: 685 patients were admitted to the unit, o1 whom 337 required mechanical ventilation for a mean duration of 4.0 days. overall mortality was 5%, 22 patients fulfilled study criteria. 17 survivors had gos 5, 2 pts with preexisting neurological impairment survived with gos 3. neonatal diseases included hyaline membrane disease (7), meconium aspiration syndrome (4) and cardiovascular surgery (1), pediatric diseases included bacterial (1) and viral (5) pneumonia, aspiration (1) and cardiovascular surgery beyond the neonatal period (3). 1990 -1994) . patients and methods: cefotaxim was used as a prophylactic agent in 43 patients in life threatening situations (e.g. multitrauma, neurosurgery atc.). more than 85 % children required cefotaxim for the treatment of severe infections (epiglotitis, meningitis, sepsis, pneumonia mainly in immunodeficient and neutropenic patients) in monotherapy or in the combination with the other antimicrobial agents. results: cefotaxim as a prophylactic drug was successful in all 43 cases (100 %). the effectivity of treatment of infections was 82.8 % (313 patients). the change of antibiotic therapy required 9 patients (2.4 %). 40 patients (10.6 %) died, but only in 12 of them (3.2 %) the obduction confirmed infection. conclusion: we conclude that cefotaxim is very effective and safe antibiotic and represents "golden standard" in the treatment of severe infections in childhood. in order to improve nursing quality, we recently adapted nursing care to the "five nursing functions" (activities of daily living, accompagnment in crisis, treatment, prevention and research) as described by the swiss red cross in accordance to the new educational guidelines of the european community, the aim of this study was to document complications of "treatment nursing function".methods: all treatment complications were prospectively collected by the nursing and medical staff. the nursing staff included patient (pt) name, time of occurence and exact description of complication, proposal for prevention and information of parents. the medical staff reported type of complication together with pt information, diagnosis, medication, treatment and interventions, outcome and referral, all complications were discussed in monthly meetings including nursing and medical staff.results: from january until december 1995, 685 pts were admitted to the picu/nicu for 3233 nursing days (81% of total bed occupancy). 337 pts needed endotracheal intubation for an average of 4.0 days and 47 pts required nasal cpap. 26 complications in 21 pts were noted (1 per 26 pi): inadequate check-up of equipment 11; accidental extubation 4 (1 in 85 intubated pts); bedsores 3; false drug dosing 2; wrong drug 2; umbilical bleeding 2; wrong transfusion setup 1; nasal septal necrosis 1). there was no mortality due to these complications. exact documention of treatment complications and their meticulous discussion within the medical and nursing staff may improve "treatment nursing function". however, documentation and evaluation of nursing within all "five nursing functions" will be nessecary in order to achieve optimal nursing care. cardiac output determination by thermodilution, using iced injectate has been shown to be valid and reliable in pediatric patients. it has been demonstrated in adult patients that there is no difference in cardiac output values when using room temperature injectate as compared to iced temperature injectate. the purpose of this study is to examine the effect of injectate temperature on cardiac output values in pediatric patients. our study consisted of sixteen pediatric patients who had oximetric thermodilution catheters in place after cardiac surgery and who had cardiac output determined using both iced and room temperature injectate. with each patient, cardiac output was measured once on the day of surgery and again the following day. in each case cardiac output was measured using both iced and room temperature injectate. statistical analysis included a two-way, repeated measures analysis of variance for each individual injectate administered and no significant differences were found in cardiac output. no statistically significant differences were found between groups with regard to the order of injectate administration or volume of injectate used (i,e., 3 or 5 cc's). the correlation coefficients between groups for cardiac output measurements at each injectate administration time, and for the average measurements across times, ranged between 0.81 to 0.94 (p < .0005). preliminary data analysis suggests that cardiac output measurements for children are not effected by the temperature of injectate. a lenghty stay at a paediatric intensive care unit will always have sideeffects on a child's well-being and will put a high strain on the parents. in order to minimize the side-effects longterm intensive care unit opened in 1990 at the childrens' hospital. admitted children are all ~ongterm-ill and technically-dependent and the ventilatory support can alter from a tracheostoma to cpap or portable volume ventilator. nutritional support is applied by gastrostomies. a homelike atmosphere surrounds the children, they share a dormitory, a living-room and a dining-room the main purpose is to send the child home with or without technical equipment. this can only be implemented by giving structured education (theory and practice) to all categories involved. the multi-disciplinary team consists of one anaesthesiologist, head nurse, clinical specialist, rn nurses, nurses, one habilitation doctor, one social worker and therapists. twenty-four patients have been admitted to licu during these six years. length of stay was from one day to four years. four are presently staying at the trait. the assessment of pain in children (0-3 yrs) is still difficult, because children of this age have limited language and cognitive skills. to standardize the assessment of postoperative pain and distress in the intensive care unit an observational mstrument was needed that met several criteria. it should be easy to use in daily routine care. be suitable for the i.c. situation, and in children of 0-3 hrs of age. the comfort scale, an observational instrument designed to assess distress in infants in i.c. units, met these criteria. to accommodate the use of the comfort scale in the i.c. units and in research, nurses should be trained to use the scale. an additional requirement was that the inter-rater reliability should be sufficiently high, (cohen's kappa > .60). objectives: 1) to introduce the comfort scale in the i.c.u.; 2) to examine whether this instrument can easily, be incorporated into routine care; 3) to investigate the inter-rater retiabtlity. methods: the comfort scale is an 8-item instrument specifically designed for use in pediatric i,c, units and contains both physiological items (heart rate, blood pressure) and behavioral items (e.g., alertness behavior, calmness/agitation, body movement, facial expression respiratory response, muscle tension). the observation period is 2 minutes. the scale is supplemented with an item on crying tbr children who are not mechanically ventilated. groups of 8 t.c. nurses were trained by means of video's and observations at the wards. after the training, each nurse completed 10 scores with other nurses, after which the cohen's kappa was computed. when the kappa's for the items met or exceeded our .60 criterium, a new group of nurses was trained. results: to date, 30 nurses have been trained. nurses find the comfort scale easy" to administer and a valuable addition to routine care in the i.c. unit. the cohen's kappa's were higher than .60 for all items that the inter-rater reliability was high. the comfort scale is feasible in postoperative care in the i.v. and is considered a valuable instrument to improve and maintain high postoperative quality of care in the i.c. unit. introduction:children with neuro-muscular disease are believed to have a higher resting energy expenditure (ree), because of their increasedwork of breathing.the influence of nocturnal nasal mask ventilation on energy metabolism and nutritional state of these children has not been studied so far.objective:l,ls the ree inereased?2.1s there an influence of nasal mask ventilation on the ree?3.what is the nutritional state?4.what is the estimated total energy expenditure(ete) in relation to the caloric intake? methods:a pilot study of 4 patients(12-16 years) .the following measurements were performed:l.anthropometry.2.bioelectric impedance-3.ree was measured by indirect calorimetry during the day (in bed) with and without nasal mask ventuation,ree was compared with predicted ree according to schofield(pee),4.caloric-intake and activities were recorded during 48 hour before measurement.5.total energy expenditure was calculated as follows:measured ree x estimated activity factor. results:tin all children weight for height was too low, <p3.2.aii children had a low % of fat.3.according to the healthy group, ree is not increased.4,nasal mask ventilation lowered ree in 2 patients, in 1 patient we measured a clear decrease.conclueion:l.anthropometry and indirect calorimetry were helpful in evaluating the nutritional state.2.there is no increased ree,in t patient there is clearly effect of nasal mask ventilation.3.when caloric intake was related to ree and level of activity 2 patients had too low caloric intake.4.the influence of nocturnal nasal mask ventilation and nutritional assessment should be evaluated longitudinally. increasing awareness that the neonate can percept pain and suffer following surgical procedures, has major impact on pain management. in our unit continuous morphine infusion (t0/~g/kg/hr) was introduced from january 1990 as a standard medicatton following surgical procedures. before 1990 morphine was given as a bolus (i.m., i.v., rectal) of 100/~g/kg up till four times a day. repeated gifts of morphine were only gwen following observation by the nurse that the child experienced pain. aim of the study: to evaluate pain management before and tbllowing introduction of a continuous morphine infusion for postoperative pain in our icu. patients and methods: term born babies with isolated oesophageal atresia and tracheo-oesophageal fistula were included in the study. the total amount of morphine (/~£/kg/24 hours); number of days morphine was given; duration of artificial ventilation was evaluated in a case control study. res ults: 1985 -1990 1991 -1994 central venous catheters (cvc) have been increasingly used also at our picu, university medical centre ljubljana. besides advantages, the cvc has brought numerous risks of complications; the catheter sepsis occurs most often and can be fatal for the patient. for example, in 1995 we dealt with 400 children aged 0 -12 years or in other words 1.14 cvp per a child treated at our picu. a specialized unit for preparing parenteral natrition started to function in the university medical centre pharmacy in 1977. after that we equipped a special room for preparing other necessary infusion mixtures and organized a team of nurses -"catheter nurses".their tasks comprise: managing cvcs, replacing and installing infusion systems and mixtures made at pharmacy, preparing drugs and some necessary infusion mixtures in special room, permanent training and co-operation with related disciplines, pharmacy, epidemiology, microbiology, etc. all registered nurses at picu have been trained for this work. the routine is as follows: in the morningafter the doctor's visit, we first check the cvc and change the dressings; according to the nurse's observations, the doctor decides whether there is a need to remove or replace the cvc. if the catheter site is inflamed, swollen, or purulent, a swab is taken for microbiological culture and the area cleaned. in case of systemic infection signs, cvc has to be replaced, blood culture taken and the infection treated with antibiotics. the dressings are always changed by two tmcatheter nurses together. afterwards the infusion mixtures for the next 24 hours are prepared for each child. the infusion mixtures which will not be used immediately are kept on +4°c; before application they are, of course, warmed to at least room temperature. finally the "catheter nurse" supplies the cvc with new infusion mixtures made at our pharmacy. our observations show that such work requires responsibility and is demanding; but our pemanent care lessens the number of complications. thus the described scheme fulfilled our expectations. perl-0perative management of neonates with hypoplastic left heart syndrome muir warren, blondel maryse, foltz rhonda, farine martine, icu nurses, the aldo castaneda institute, clinique de genolier, switzerland both the pre-operative and post-operative physiology of patient with hypoplastic left heart syndrome is critically determined by the puhnonary-tosystemic resistance ratio. a high ratio results in minimum volume work for the right ventricle but is at the expense of important hypoxemia. a low ratio is reflected in minimum hypoxemia but an unfavorable increase in excess ventricular volume work. a resistance ratio of approximately 1 results in an arterial oxygen saturation of 75% -80%, a qp/qs of 1, and ventricular volume work only twice normal. this latter physiology appears best for oxygen delivery and systemic perfusion with tolerable volume work. carbon dioxide (co2) in the inspired gas is known to increase pulmonary vascular resistance without significantly influencing systemic resistance in the range of 1 to 20 tort partial pressure. hyperventilation with no co2 in inspired gas decreases pulmonary vascular resistance. thus, managing co2 may be useful to modulate the pulmonary-to-systemic resistance ratio, in the tast 16 months, 20 patients with hypoplastic left heart syndrome were managed by a norwood procedur e. 10 patients with endotracheal entubation pre-operatively had co2 added to inspired gas to modulate the pulmonary-to-systemic flow ratio. 17 patients had co2 added to inspired gas post-operatively. all but 1 patient had characteristically large pulmonaary-to-systemic flow ratio with oxygen saturation mean 90 % preoperatively, even in those patients receiving co2, co2 was accompanied by elimination of metabolic acidosis pre-operatively. the most comlnon partial pressure of co2 added post-operatively was 14 torr. po2 ranged from 25 mmhg to 40 mmhg. all surviving patients developed a metabolic alkalosis (10 mean) when treated with co2. there were 4 deaths, one from drug toxicity, one from excessive post-operative hemorrhage, two from sepsis or mocardial insufficiency. only one patient benefited from catecholamine support which can increase systemic resistance unfaborably. in this experience co2 was felt to be an important adjunct in some patients pre-operatively and most patients postoperatively to faborably modulate the critically important systemic-topulmonary flow ratio, to maximize oxygen delivery and systemic perfusion, while minimizing excessive right vantricular volume work. key: cord-004948-ad3i9wgj authors: nan title: 7th international congress on amino acids and proteins : vienna, austria, august 6–10, 2001 date: 2001 journal: amino acids doi: 10.1007/s007260170030 sha: doc_id: 4948 cord_uid: ad3i9wgj nan the raised concentration of protein bound homocysteine in homocystinuric (hcu) patients displaces protein bound cysteine and increases the free/bound cysteine ratio in plasma. this ratio is independent of albumin concentration. results from 31 hcu patients were compared to 40 controls. free cystine concentrations in hcu were poorly discriminated from the control range but the total cysteine results were almost invariably lower than control data. this appears to result from an increased free/bound cysteine ratio in hcu [mean (range) for control 0.50 (0.22-0.71) and for hcu 0.76 (0.32-1.75); p ϭ 0.0005]. ex vivo protein binding experiments in albumin solution revealed the free/bound cysteine ratio to be linearly related to the amount of homocysteine bound (r ϭ 0.907, p ͻ 0.001). we conclude that measurement of total cysteine is essential for assessment of the true cysteine status in hcu. however, any cysteine deficit, or alteration to free/bound cysteine ratios, does not obviously effect glutathione synthesis as assessed by measurement of plasma total glutathione. (339 nmol/g; 21.6%); sprague-dawley rat (rattus norvegicus, rodentia) (n ϭ 6; 98-381 nmol/g; 9.8-13.7%); rabbit (152 nmol/ g; 11.3%); pig (sus scrofa f. domestica, artiodactyla) (221 nmol/ g; 13.4%); bovine (bos primigenius f. taurus, artiodactyla) (284 nmol/g; 23.6%); seal (phoca vitulina, carnivora) (136 nmol/g; 3.8%), and rob (halichoerus grypus, carnivora) (218 nmol/g; 5.4%). from the date it is concluded that d-aas are common in body fluids and certain tissues of vertebrates. in order to determine the quantity of cyst(e)ine and methionine, the oxidation of cyst(e)ine and methionine (ϫ) refers to not detected or not determinable; asx ϭ asp ϩ asn; glx ϭ glu ϩ gln; his, arg, trp, cys not determined; a) feed fortified with dl-met; b) nmol/g lyophilized serum. mentation and subsequent purification. the separation of the enantiomers of cysteic acid, methionine sulphone, aspartic acid and glutamic acid is displayed on the chromatogram. into cysteic acid and methionine sulphone with performic acid is often applied before hydrolysis of protein. the authors examined the applicability of this process in case of quantification of cyst(e)ine and methionine enantiomers. the rp-hplc analytical method was developed for the determination of the amount of cysteic acid and methionine sulphone enantiomers. the rate of conversion during oxidation from cyst(e)ine into cystic acid and from methionine into methionine sulphone was determined. the racemisation of l-cyst(e)ine and l-methionine was negligible during oxidation with performic acid, therefore this process can be applied before hydrolysis during quantification of cyst(e)ine and methionine enantiomers. after the performic acid oxidation and the 6 m hcl hydrolysis of the protein, opa/tatg (o-phthaldialdehyde/tetra-o-acetyl-1-thio--d-glucopiranoside) precolumn derivatisation method was used, and the enantiomers of sulphur containing amino acids were separated by rp-hplc (lichrosphere 100 rp-18e, 125 ϫ 4 mm, 5 µm column, merck-hitachi lachrom hplc). the resolution of the peak of cysteic acid and methionine sulphone enantiomers was better than 1,5. the method was used to determine the amount of l-and d-cyst(e)ine and land d-methionine containing preparations prepared by ferd/l rate of aspartic acid and the individual age of specimens. a method for age determination based on d-aspartic acid content and on the racemisation of l-aspartic acid of teeth was developed. d-glutamic acid, beside d-aspartic acid, was found to be eminently suitable for the estimation of individual age, as it showed a sufficiently high sensitivity. calibration curves based on these investigations were used for the age estimation of 65 adults (39 males and 26 females) of unknown individual age from the avar period series of kereki-homokbánya (hungary). the age distribution of the sample was the following: 39 individuals (60%) belonged to the adult age group, 22 persons (34%) to the mature and 4 (6%) to the senile one. the correlation between our results and those obtained using standard paleoanthropological methods was over 0.9. quantitative determination of free and bound 3-nitrotyrosine in rat plasma and tissues using isotope dilution liquid chromatography-electrospray tandem mass spectrometry t. delatour, p. a. guy, j. richoz, j. vuichoud, and nestlé research centre, nestec ltd, vers-chez-les-blanc, lausanne, switzerland since 3-nitrotyrosine was reported to be readily formed in proteins by reactions with nitrite or nitrogen dioxide, it has been postulated to be a possible marker for investigating peroxynitrite-mediated nitration of proteins. thus, several methods were developed to assess nitration of tyrosine in proteins and determine 3-nitrotyrosine in physiological fluids. methods based on hplc or gc/ms techniques were described to quantify 3-nitrotyrosine within tissues or biological fluids. unfortunately, it has been demonstrated that an artifactual nitration of tyrosine occurs with gc/ms assays leading to an overestimation of the response. in the present work, lc-esi-ms/ms methods for quantification of free 3-nitrotyrosine in rat plasma as well as bound 3-nitrotyrosine in tissue samples are reported. plasma samples were spiked with 2,5,6-d 3 -3-nitrotyrosine and the following steps were applied prior to injection into the lc-esi-ms/ms system used in selected reaction monitoring (srm) mode (m/z 283 ae 181 for the analyte and m/z 286 ae 184 for the internal standard): protein precipitation, solid phase extraction on aminopropyl cartridge and derivatization in nbutanol in hcl 3 n. 3-nitrotyrosine butyl ester has lead to a dramatic increase of the sensitivity (ca. 5-fold) by comparison with 3-nitrotyrosine. under such conditions, calibration curves exhibited excellent linearity (r 2 ͼ 0.99) within concentration range 0.3 to 28.5 nm (equivalent to 47.3-4,730 fmol on column) and recoveries above 85%. inter-and intra-assay precision was determined below 15% over the concentration range 1.4 to 28.5 nm. no artifactual nitration of tyrosine occurring during sample clean-up was observed. this was unambiguously established by plotting experimental ratio of analyte response/ internal standard response versus expected within the range 0.3-28.5 nm. this curve strongly correlated with a linear model (r 2 ͼ 0.99) and slope was 1.07 ϯ 0.06 (mean ϯ sd). basal level of 3-nitrotyrosine in rat plasma was measured to be within concentration range ͻ lod to 1.5 nm. 3-nitrotyrosine basal level in rat plasma, kidney and liver proteins was established by performing enzymatic hydrolysis in order to avoid artifactual nitration of tyrosine which may occur under strong acidic conditions (hcl 6 n at 120°c). resulting hydrolysates were analysed by lc-esi-ms/ms and 3nitrotyrosine was monitored in srm mode (m/z 227 ae 181 for the analyte and m/z 230 ae 184 for the internal standard). t. guszczynski 1 , r. b. kapust 2 , d. s. waugh 2 , and t. d. copeland 1 1 basic research laboratory, and 2 macromolecular crystallography laboratory, national cancer institute at frederick, maryland, u.s.a. the set-can fusion gene was first detected as associated with acute undifferentiated leukemia. set (also called phap ii) is a nuclear phosphoprotein with a long acidic tail. set has been shown to inhibit phosphatase pp2a and is a substrate of human granzyme a. in order to determine any zn(ii) binding properties of set, we utilized affinity capillary electrophoresis (ace) to detect shifts in mobility as zn(ii) ions bind to the protein. we have earlier employed ace to measure the binding constants of zn(ii) to the nucleocapsid protein of hiv-1. with a constant concentration of recombinant set as a receptor and varying concentrations of zn(ii) as ligand in the sample buffer, we observed changes in electrophoretic mobilities of set when complexes were formed with zn(ii). scatchard analysis of the mobility provided the stoichiometry and binding constant of zn(ii) to set. interdisciplinary research center, institute of nutritional science, department of food sciences, university of giessen, germany peptaibols are defined as fungal polypeptides containing a high proportion of aib (α-aminoisobutyric acid) and a cterminal bound amino acohol. the mold trichoderma aureoviride (strain imi 91968; commonwealth mycological institute, kew, uk) was cultured in complex medium consisting of casein peptone, 17 g; soy peptone, 3 g; yeast extract, 5 g; dglucose, 2.5 g; nacl, 5 g; dipotassium hydrogen phosphate, 2.5 g in 1 l demineralized water adjusted to ph 6.8. fermentation was conducted in nineteen 2-l shake flasks, each containing 400 ml medium, for 7 d at 27°c. mycelia were obtained by filtration and extracted with meoh and meoh/chloroform. extracts were evaporated to dryness and subjected to sephadex and silica gel chromatography (eluent chloroform/meoh/acoh/water 65 : 25 : 3 : 4) yielding 2.83 g and 0.9 g, respectively, crude peptaibol mixture named trichoaureocins. the peptide mixture was uniform on tlc but could be separated by analytical (fig. 1 ) and semipreparative hplc (nucleosil 100 c-18; 250 ϫ 8 mm id; 3 µm). six peptides could be isolated each of which was subjected to sequencing using on-line hplc (fluorocarbon stationary phase) esi-ms/ ms (lcq, thermoquest, finnigan mat) as described for peptaibols trichovirins and antiamoebins. sequences are presented in fig. 2 . the 20-residue peptaibols represent a natural peptide library and cause hemolysis of sheep erythrocytes and exert antibiotic activity against bacillus subtilis and staphylococcus aureus. national institute of chemistry, ljubljana, slovenia wine consists of several hundred components present at different concentrations. the dominant ones are water, ethanol, glycerol, sugars, organic acids, and various ions, while amino acids are present at much lower concentration. the composition of amino acids is of great importance in wine production. they act as a source of nitrogen for yeast during fermentation, they influence the aromatic composition of wine and their composition can be used to differentiate wines according to vine variety, geographical origin, and year of production. among already established analytical methods high-field nmr has been shown to be a promising method for the nondestructive analysis of low-molecular mass compounds in complex mixtures like wine due to its selectivity and capability of simultaneously detecting a great number of compounds. 1 h and 13 c one-dimensional nmr spectra of wine are very crowded and many signals are overlapped. due to a great difference in concentration levels the signal intensities of particular compounds may vary for the factor of 25. the tails of the dominant frequencies of water, ethanol and glycerol obscure weak signals of minor compounds like amino acids in the near surroundings. the use of 2d homo-and heteronuclear experiments and the suppression of strong signals are a prerequisite for a successful 1 h and 13 c signal assignment. a complete assignment of 1 h and 13 c nmr resonances of seventeen amino acids commonly present in wine and of γ-aminobutyric acid at ph 3 was accomplished using gradient-selected cosy, tocsy, gradientselected hsqc and hmqc experiments with incorporated wet pulse sequence for the supression of large signals. unambiguous assignment of 1 h and 13 c nmr resonances of amino acids is necessary for the selection of appropriate signals in fast and simple one-dimensional nmr that can serve as parameters in the chemometric classification of wines according to the provenance, vine variety, and year of production. institute of medical biochemistry, jagiellonian university collegium medicum, kraków, poland highly sensitive colorimetric method for determination of aldehydes in the reaction with n-methyl benzothiazolone hydrazone (mbth) turned out to be not very specific for such carbonyl compounds. namely, it has been found that tryptophan and to higher degree its n-derivatives (n-acetyl-trp, ala-trp, gly-trp) and also tripeptides (gly-trp-gly and leu-trp-leu) in the reaction with mbth and fe 3ϩ are converted to coloured products, with maximum wavelength at 595 nm. the properties of the products and the kinetics of the reaction under defined conditions are described in the spectrophotometric procedure. proteins containing tryptophan are also substrates in the reaction with mbth. comparison of molar extinction coefficients of mbth-fe 3ϩ -treated various proteins with those of simple n-derivatives of tryptophan shows, that not all molecules of tryptophan in proteins are accessible to the reagents, and in order to determine all tryptophan moieties partial unfolding of protein has to be performed. it should be emphasized that aldehydes cannot be detected and accurately determined in the presence of tryptophan derivatives and protein, and also aldehydes interfere with determination of tryptophan derivatives. natural product laboratory, department of chemistry, the university of burdwan, w. bengal, india detection of protein amino is of utmost importance for the evaluation of protein structure and also their presence in numerous natural products. several specific and non-specific reagents have been used for their detection using thin-layer chromatography, an important tool for such purpose. of the reagents in general use, ninhydrin is the most popular for its high sensibility, however, nihydrin produces same purple color with most of the amino acids (only proline and hydroxproline produce yellow color). an endeavour has been made to resolve this color problem with a reagent which is capable of developing various distinguishable colors with many of the protein amino acids and also shows its high sensitivity comparable to ninhydrin. a probable mechanism for such color formation has also been proposed. measuring enrichments below the sensitivity range of conventional gc-ms. the gc-c-irms technique combines the resolution capabilities of gc with the accuracy and precision of irms. at low abundance gc-c-irms analysis it is superior in terms of time, labor, and sample requirement as compared to the conventional off-line analysis. we discuss some latest advancements and applications of gc-c-irms amino acid analysis related to nutrition research. plasma amino acids in omnivorous human subjects show a characteristic 15 n-isotopic pattern with phenylalanine and threonine showing the lowest abundance, whereas e.g. alanine and leucine are higher by 25‰ δ 15 n. in rats fed diets containing intrinsically labeled 13 c casein or the corresponding amino acid mixture labeled with 13 c leucine and 15 n lysine whole-body protein homeostasis is better supported by casein-bound than free amino acids. there is no adaptation to a low lysine diet by an enhanced bioavailability of intestinal microbial lysine to extra-splanchnic tissues in minipigs. highly selective hplc determination of tyrosine, tryptophan and their related compounds based on precolumn derivatization followed by intramolecular fluorescence resonance energy transfer detection h. nohta 1 , m. yoshitake 1 , h. yoshida 1 , t. yoshitake 2 , and m. yamaguchi 1 1 faculty of pharmaceutical sciences, fukuoka university, nanakuma, johnan-ku, fukuoka, and 2 chemical evaluation and research institute, ishii machi, hita, oita, japan we have developed highly selective hplc method for the determination of tyrosine, tryptophan and their related compounds (l-dopa, catecholamines, 5-hydroxytryptamine, etc.). the compounds were precolumn-derivatized with a commercially available fluorogenic reagent for amines by usual manner. each derivative afforded intramolecular fluorescence resonance energy transfer (fret) from the tyrosyl or tryptophoryl moiety (donor) to the labeled fluorophore (acceptor); the acceptor fluorescence was observed with the excitation of the donor at 280 nm. the derivatives were separated on a reversedphase column and then effectively detected by monitoring their fret. through the screening study of 11 fluorogenic reagents, o-phthalaldehyde (with 2-mercaptoethanol) and dansyl chloride gave the best results for the purpose. the fret detection method was highly selective and sensitive by comparison with the previous methods detecting native fluorescence of the compounds or typical fluorescence of the acceptor. the presented study was devoted to determination of the energetic effect of interactions in aqueous solutions between urea and neutral amino acid derivatives. the principal reason for studying of interactions of peptides with urea is the hope that such investigations will give insight into the factors affecting protein denaturation in aqueous solutions. the enthalpies of solution of n-acetylglycinamide, n-acetyl-l-alaninamide and n-acetyl-l-leucinamide were measured in water and in aqueous solutions of urea of molality 0.25 to 3.0 mol·kg ϫ1 using the "isoperibol" type calorimeter at 298.15 k. from the obtained standard dissolution enthalpies ∆ sol h ϱ m the enthalpic pair interaction coefficients h xy for urea-nacetylamino acid amide pairs in water were calculated. these parameters derived from mcmillan-mayer theory are regarded as a measure of effect of interactions between solute molecules in solution. the h xy values for the systems investigated suggest that the interactions between urea and amide molecules dominate the effects of dehydration of nonelectrolyte and of peptides. the replacement of the hydrogen atom in the hydrocarbon chain with a methyl group causes a positive change in the value of the enthalpic pair interaction coefficient. the obtained results were compared with those of earlier studies of interactions between electrolytes, namely sodium chloride, potassium chloride and sodium iodide and the same n-acetylamino acid amides. the effect of the solute type on the magnitude of the interaction parameter was also analysed. the side chains of amino acids in solution react in various ways with the water molecules which surround them as well as with other components of solution depending on the fact whether they possess non-polar, polar or ionic groups. many research laboratories carry out studies intended to describe precisely the intermolecular interactions with the participation of amino acid side chains. such a description may allow one to describe better the spatial structures of protein and the mechanisms of folding its surface area. the present work reports the results of calorimetric measurements of the dilution enthalpies of l-α-amino acids in water. using modified mcmillan-mayer's theory, these results served to calculate the enthalpic homogeneous interaction coefficients which characterise interactions between the amino acid zwitterions with the competitive participation of water molecules. thus, these coefficients illustrate the differences in amino acid molecules interactions both with the homogeneous amino acid molecules and water molecules around them, and consequently they may play the part of a parameter which differentiates the hydrophobic/hydrophilic properties of amino acid side chains. the enthalpic interaction coefficients of the homogeneous pairs of l-α-amino acids were compared also with the hydrophobicity parameters obtained by fauchere et al., which describe the side substituents of natural amino acids as well as aminobutiric acid (aba). based on the above statement, one may conclude that the obtained enthalpic homogeneous pair interaction coefficients of l-α-amino acids in water make it possible to systematise amino acid side chains according to their affinity to water or their hydrophobic-hydrophilic properties. thus the enthalpic homogeneous pair interaction coefficients may play the role of parameter describing the lipophilicity (hydrophobicity) of amino acid side chains. compounds (iii) with amino acid ligands. in this work we present results of x-ray investigation of fourth amino acid complexes of rhenium (iii), which have different coordination of amino acids around binuclear complexforming center -re 6ϩ . substances (glyh) 4 . 2h 2 o -in inner, but gaba has cisposition according to re -re bond. influences of fatty radical length in the amino acid ligand on week interaction between binuclear anion [re 2 cl 8 ] 2ϫ and protonized amino acid are discussed. role of hydrogen bonds in formation of crystal unit cell of investigated substances is shown. these two factors are the reason of formation of staggered conformation of an anion [re 2 cl 8 ] 2ϫ in the substance (glyh) 4 [re 2 cl 8 ]cl 2 together with existence of quadruple re -re bond that is described first. in the substance [re 2 (gaba) 2 cl 5 (h 2 o)]cl . 2h 2 o axial position of re 2 6ϩ fragment are substituted by ligands of different kind: h 2 o and cl ϫ -that says about possibilities to coordinate a substrate of biological nature exactly to these position. a precise, sensitive and reliable rp-hplc/uv method was developed to enable determination of α, and k caseins in cow's milk. the optimised method using a chrompack p-300-rp column allowed separation of caseins in 30 min. this column differs from conventional alkyl-bonded silica rp matrices in that it is an underivatised polystyrene-divinylbenzene matrix, a material which proved excellent chemical and ph stability. gradient elution was carried out at a flow rate of 1 ml/min and a temperature of 46°c, using a mixture of two solvents. solvent a 0.1% trifluoroacetic acid in water and solvent b was 95% acetonitrile-5% water-0.1% trifluoroacetic acid. the effluent was monitored by a uv detector at 280 nm. the determinations were performed in the linear range of 0.038-0.38 mg/ml for k-casein, 0.19-1.9 mg/ml for α-casein and 0.15-1.5 mg/ml for -casein. the detection limits were 0.037, 0.03 and 0.0075 mg/ml, respectively. the validity of the method was verified. the recoveries ranged from 91 to 100% for cow's milk. the precision of the method was also evaluated, the % cv being less then 3.67%. the developed methodology was also applied with success to the separation of caseins in ewe and goat milks. different chromatographic profiles were obtained for the three kinds of milk. department of aquatic biosciences, the university of tokyo, bunkyo-ku, tokyo, japan several aquatic crustaceans and bivalve molluscs accumulate a large amount of free d-alanine (3-50 µmol/g wet wt.) in their muscle tissues. during seawater acclimation from freshwater to 75% seawater, red swamp crayfish procambarus clarkii largely accumulated d-and l-alanine by 6.8-and 4.5fold, respectively, together with l-glutamine, l-proline, and glycine. the percentage of d-alanine to total alanine increased from 38% in freshwater to 48% in 75% seawater. these data indicate that d-and l-alanine are the major compatible osmolytes responsible for the intracellular isosmotic regulation of this species as well as other crustaceans. under anoxia stress for 12 h in freshwater, 50 and 75% seawater, crayfish increased d-and l-alanine in muscle and hepatopancreas in addition to the increase of lactate. the increase was much higher in seawater than in freshwater. thus, d-and l-alanine may be anaerobic end products during prolonged anoxia of this species. alanine racemase [ec 5.1.1.1] has been proved to catalyze the interconversion of d-and l-alanine in crustaceans and bivalve molluscs. this enzyme was isolated to homogeneity from the muscle of black tiger prawn penaeus monodon. the purification was 127,600-fold with 16% yield. the molecular weight of the enzyme was estimated to be 45 kda on sds-page and 90 kda on gel filtration, suggesting the dimeric nature of this enzyme. the amino acid sequences of the peptide fragments obtained from the isolated enzyme showed low homology below 50% with those of microbial enzymes. syntheses and immunological effect of thymic humoral factor-γ2 analogues research laboratory, global shinwa pharmaceutical co. ltd., yoriki, matsuomura, iwate-gun, iwate-ken, japan nine analogues of thymic humoral factor (thf)-γ2, were prepared by the solid-phase method and their in vitro restoring effect on the impaired blastogenic response of phytohemagglutinin (pha)-stimulated t-lymphocytes of uremic patients with infectious diseases were examined. the results were as follows: [arg6]-thf-γ2 exhibited higher restoring activity than that of our synthetic thf-γ2. phylos has developed a powerful combinatorial biology platform for peptide and protein selections. phylos' proprietary profusion tm technology enables the selection of peptides and proteins with desired properties. the fundamental advance represented in this unique platform is the in vitro covalent linkage of a peptide or protein (phenotype) to the encoding messenger rna (genotype). this linkage permits the selection of a protein based on its characteristics and allows the recovery and amplification of that protein through pcr, an efficient means of bring the desired proteins to easily detectable levels. profusion tm technology has routinely selected peptide and protein binders with affinity constants in the nanomolar to picomolar range. the starting library size of randomized peptide or protein profusion tm constructs is typically 10 13 . linear and constrained loop peptide libraries, for ligand generation, enzyme: substrate interaction, peptidomimetic design, and epitope mapping have been successfully used. randomized constrained loops have also been incorporated in a betasandwich scaffold, resulting in the successful selection of binders against targets of therapeutic interest. antigenic properties of three biological active de novo proteins were investigated by peptide scanning approach, using noncleavable multipin technology. a de novo protein albebetin (pid caa47376) was engineered to attain a pre-designed 3d structure and later modified by grafting short peptide fragments from human α 2 interferon (aag59605), and insulin molecules (aag59606). such protein constructs carrying important biological activities may be used in future as potential protein pharmaceuticals. despite artificial proteins are investigated for more than 10 years, immunological properties of these substances are not known. in our experiments we applied an innovative approach of raising antibodies in yolks of egg-laying hens. three continuous antigenic determinants with different immunogenic potentials have been revealed in two proteins with partially overlapping sequences. it was shown that the octapeptide interferon fragment is the immunodominant site in albeferon and albeferon-insulin molecules. on the contrary, the hexapeptides, corresponding to the insulin fragment displayed low immunogenic activity. thus we recognise that the fragments attached to the de novo frame could essentially govern immunological properties of resulting construct. no preference of any type of secondary structure was observed in antigenic determinants. nevertheless, all of them are located at the boundaries of the secondary structure elements and on the predicted surface-located sites of albebetin molecule. peptide fragments from human α 2 -interferon and insulin corresponding to the functionally important sites of their molecules were grafted into de novo protein albebetin (pid caa47376) engineered to attain a pre-designed tertiary structure with a unique topology that has not been observed in natural proteins. by means of genetic engineering the dna fragments corresponding to these peptides were inserted into the albebetin gene to obtain two variants of albebetin with antiviral fragment of human α 2 -interferon and two variants of albebetin with insulin-like peptide. the chimerical genes were expressed in escherichia coli in a fusion expression system with thioredoxin based on the plasmid pet-32 (novagen). the fusion proteins were digested by highly specific protease factor xa and the target chimerical proteins were purified and tested for their structure and biological activity. according to the cd spectroscopy study the chimerical proteins maintained the pre-designed structural properties of albebetin. toxicological testing of the proteins in the mtt-test did not reveal their cytotoxicity. antiviral activity of de novo proteins with human α 2 -interferon fragments was studied in vitro using human fibroblasts cell line l-41 and simian cell line vero. treatment of these cell lines with the proteins revealed the dose-dependent stimulated antiviral activity on fibroblasts and direct dose-dependent antiviral activity on the vero cells. one of two de novo proteins including insulin-like fragment (pid aag59607) acquired ability to stimulate glucose uptake by l-929 cells although the efficacy of stimulation was lower than that for the synthetic peptide and insulin. these results demonstrated that albebetin can be used as a scaffold for constructing of the functionally active de novo proteins possessing the pre-designed tertiary fold of albebetin and various biological activities. the identification of genes encoding unique tumor associated antigens (taas) has facilitated the development of novel immunotherapeutic strategies in cancer patients. clinical investigations have focused on targeting these cancer antigens for the generation of anti-tumor t-cell responses. taa epitopes come from differentiation antigens, from embryonal reexpressed or overexpressed proteins, from mutated proteins and from viral proteins in viraly associated tumors. we have recently developed a novel screening system for identification of immunogenic and antigenic ctl peptide epitopes using d b-/-x 2m -/double knockout mice, transgenic for a single-chain hla-a2-2m molecule (hhd mice). specific ctl were derived by immunization of hhd mice with tumor peptide extracts loaded on antigen presenting cells and with hhd transfected human tumor cell lines ctl induced against peptides from various tumors recognized tumor peptides more effectively than peptides extracted from normal tissues and also reacted with a serie of peptides derived from overexpressed candidate proteins, identified by differential display methods (sage, microarrays) comparison of ctl derived from hhd mice to ctl induced from patient's pbmc showed overlapping recognition of many candidate peptides. using these hhd mouse derived ctl we identified novel peptide sequences from prostate, bladder, breast and colon carcinomas, antigens pap and steap, from breast carcinoma antigens muc1 and ba46-1. analysis of tumor differentially expressed genes by the sage method in colon, followed by screening for hla-a2 binding peptides resulted in 500 candidate peptides for immunogenicity screening. we have identified 22 antigenic peptides of which 7 peptides were found to be immunogenic in hhd mice. interestingly 3 of these peptides are derived from the same protein. differential expression studies, using "dna chips" were performed on prostate and bladder tumors versus normal tissues. ten new candidate genes from tcc were analysed for expression and potential immunogenic peptides. novel peptides from uroplakins and from mage-8 were identified. surface plasmon resonance biosensing in the study of viral antigenic sites mimicked by synthetic peptides p. gomes 1 , e. giralt 2 , and d. andreu 2 1 centro de investigação em química da universidade do porto, portugal 2 department de química orgànica, universitat de barcelona, spain antigen-antibody binding has been regarded as one of the most representative examples of specific molecular recognition in nature. the simplistic view of antigenic recognition in terms of a lock-and-key mechanism is superseded, since it is now evident that both antigens and antibodies are flexible and can undergo substantial mutual adaptation. this flexibility is the source of complexities such as degeneracy and non-additivity in antigenic recognition. we have used surface plasmon resonance to study the effects of combining multiple amino acid replacements within the sequence of the antigenic gh loop of foot and mouth disease virus. our aim was two-fold: to explore to what extent can antigenic degeneracy be extended in this particular case, and to search for potential non-additive effects in introducing multiple amino acid replacements. combined analysis of one such multiply substituted peptide by spr, solution nmr and x-ray diffraction shows that antigenic degeneracy can be expected as long as residues directly interacting with the paratope are conserved and the peptide bioactive folding is unaltered. structural properties of creatine kinase from amphioxus, branchiostoma belcheri gray f. inoue, s. obase, t. suzuki, and t. imai department of physiological chemistry, faculty of sciences, toho university, funabashi, japan to further our knowledge of creatine kinase (ck) in the fields of molecular evolution and comparative enzymology, we analyzed the ck gene of the protochordate amphioxus. amphioxus is thought to be the phylogenetic predecessor of vertebrates and thus possesses characteristics, such as enzymological properties, that are associated with ancestral vertebrates. the results clarified the sequence of 789 bases including the active site. the homology of the active site and the surrounding 48 bases for the amphioxus ck gene to that of the human and electric ray ck-m gene was 89.6% and 87.5%, respectively. the amino acid sequence of this region of amphioxus ck was also identical to that of human and electric ray ck-m. in addition, the estimated secondary structure of amphioxus ck was compared to that of human and electric ray. there were no marked differences in the relative ratio of the α helix, sheet and turn structures for the peptide structure of ck consisting of 263 amino acid residues. there was a high degree of homology in the sequence of 25 amino acid residues (met271ϳhis295) near the active site of ck between amphioxus and other organisms, suggesting that this region of ck is functionally essential for transphosphorylation. gelsolin is a ca 2ϩ -activated and phosphoinsitide-regulated cytoskeletal actin-binding-and-severing protein, its fragments 135-142: ksglkykk (g135-142) and 150-169 khvvpnev vvqrlfqvkgrr (g150-169), are responsible for the binding of this protein to actin and the cellular messenger phosphatidylinositol 4,5-bisphosphate (pip2). the binding of peptides g135-142 and g150-169 to a cluster of four pip2 molecules in a dimyristoyl-phosphatidylcholine lipid was in vestigated by means of molecular-dynamics (md) simulations of 1,600 ps. the binding of the pip2 molecules to the peptides g135-142, g150-169 showed both electrostatic and hydrophobic nature: lysine residues of the peptides formed salt bridges with the phosphate groups of the pip2 molecules, while hydrophobic interactions occurred between the nonpolar residues of the peptides and the fatty-acid tails of pip2. during the binding some of the pip2 molecules were dragged out of the lipid, thus disrupting the bilayer. after the binding dissociated a draggen-out pip2 molecule tend to incooporate back to the lipid. division of applied physiology, institute of veterinary physiology, university of zürich, switzerland chemical modification of the proteins: bovine serum albumin, α-lactalbumin, -lactoglobulin and chicken egg white lysozyme by 3-hydroxyphthalic anhydride (3hp) yielded compounds which exerted antiviral activity in vitro as compared with the native unmodified proteins. of the three enveloped viruses tested: human herpes simplex virus 1 (hsv-1), bovine parainfluenza virus 3 (pi-3) and porcine respiratory corona virus (prcv), the 3hp proteins were shown to be active against human herpes simplex virus 1 only indicating that a perturbation of the viral envelope is unlikely. pre-incubation of vero cells with 3hp-albumin, 3hp--lactoglobulin and 3hplysozyme resulted in protection against hsv-1 infection whereas pre-incubation with 3hp-α-lactalbumin had no antiviral effect. however, all 3hp modified proteins showed a more significant inhibition when present during or after the viral infection step. thus multiple mechanisms appear to be involved in the inhibition of hsv-1 infection. the blocking of cell receptors may contribute to the antiviral activity as shown by the preincubation data. however, a direct interaction between the modified proteins and the hsv-1 glycoproteins responsible for viral entry and spread, seems to play a more important role, as indicated by the smaller ec 50 values obtained during and after the infection. a dummy or a protagonist on the stage of inflammation? r&d department, zambon group, bresso, milan, italy amino acids are usually present in large excess in healthy and the excess is used as source of calories. however, metabolic alterations are observed in ill patients and preferential retention of sulphur amino acids (saa) occurs during the inflammatory response. the metabolism of cysteine is modified during the acute phase of sepsis in rats. sulphate production is lower, whereas the higher liver production of taurine seems to play a protective role; glutathione concentration is greater in liver, kidney and other organs and cysteine incorporation into proteins was higher in spleen, lung and plasma (acute phase proteins) while albumin level decreases. another important phenomenon is the impairment of methionine conversion to cysteine during stressed condition. premature infants or hiv patients synthesise cysteine from methionine at a much lower rate. thus, the metabolic flow through the trans-sulphuration pathway may be insufficient to meet the glutathione and cysteine requirement in critical conditions. the pro-inflammatory cytokines, interleukin-1, interleukin-6 and tnf-α are the main initiates that alter protein and amino acid metabolism. in this complex picture, saa supply may contribute to the immune system regulation. our previous investigations showed some biological activity of newly synthesized cluster rhenium compoundtetrachlorodi-µ-(γ-aminobutirato)dirhenium(iii) chloride -i such as antitumour activity, cell-stabilizing activity against osmotic hemolysis, changing of morphology of cells, and other. there exists some information about stabilizing effects of some metal-organic substances with antitumour properties on the isolated ishaemic-reperfused rat heart (leperre a. 1995) throughout decrease of malonaldehyde (mda) production. some new investigations showed the influence of metal-organic substances on apoptotic processes (winter b. 1998 , syrkin a. 1998 , that are considered now as the main mechanism of such tissue damages as ishaemia, myocardial infarct, etc. thus we tried to analyze such activity of i. two models of hemolytic anemia was used: a -on rabbits by introducing of pbac 2 -solutions; this model permits to investigate dynamics of anemia in one experimental animal; bon rats by introducing of phenylhydrazine chloride. i was administrated as in solution as in lyposomic (lyp) forms. all measurements and models were accomplished according to described procedures. administration of i led to: increase of hemoglobin and resistance of erythrocytes and to prolonging of life for hemolytic animals; significant decrease in quantities of mda and increase in quantities of reduced glutathion (gsh), glutathionreductase (gsr) and glutathionperoxidase (gsp) in myocardium, blood, brain, liver, splenic and entherocites of anemic animals. the most effective was i in lyposomic form. mechanism of antioxidant action of rhenium cluster compound is speculated and experiments with some well-known antioxidants to compare with i are working out. at present problem of finding remedies against the mostly dangerous human disease -aids is one of higher interest. the aim of this work was the investigation of inhibiting effect of high-pure l-lysine-α-oxidase (lo) e.c.1.4.3.2, extracted from trichoderma sp., on hiv-virus reproduction, comparatively to azidotymidin (azt), being now in use for treatment of aids-patients. for studying of inhibiting effect of lo, the mt-4 cells, sensitive to citopathical action of virus, were used. the experimental studying has shown, that the enzyme at concentration 7-70 ng/ml suppresses hiv reproduction and synthesis of virus' proteins, not exerting toxical effect on mt-4 cells. toxical dose of lo has been determined preliminary. a comparison with standard preparation -azidotymidin, which causes suppression of virus reproduction at concentration 3 mkm (1,2 mg/ml) not exerting toxical effect on mt-4 cells. the same effect is attained having used lo in doses 7-70 ng/ml. using lower concentrations of enzyme leads to partial increasing of virus' titre comparatively to control cultures. obtained data allow to conclude that lo from trichoderma sp. is more high specific agent than azidotymidin, because it needs 1000 times lower concentration for the same action. comparison of azt and lo action on synthesis of virus' antigens presenting in cultural media of mt-4 cells infected with virus, leads to conclusion, that lo has inhibitory action both on virus' reproduction and virus' protein synthesis. department of microbiology, dokkyo university school of medicine, mibu, tochigi, japan our previous studies showed that the cellular amino acid composition obtained by amino acid analysis of whole cells, differs in various organisms. these results suggest that the difference in the cellular amino acid composition reflects biological evolution. however, the basic pattern of cellular amino acid composition is relatively constant in all organisms, and the cellular amino acid compositions of the archaeobacteria are quite similar to those determined from codon usage data, based on the complete genomes. in the present study, the free amino acid compositions in archaeobacteria, eubacteria, protozoa, blue-green alga, green alga, slime mold, plants and mammalian cells were analyzed, to investigate whether changes in their free amino acid compositions reflected biological evolution. cell homogenates were treated with 80-90% ethanol to separate cellular proteins and free amino acids contained in the cells. rat hepatoma cells (r-y121b) were cultured in eagle's minimum essential medium (mem) containing 5% serum or in a modified mem lacking arginine, tyrosine and glutamine. no significant difference in the free amino acid composition was observed between the two cell groups cultured under two different conditions. the patterns of the free amino acid compositions differed completely from those of the cellular amino acid compositions, and from each other in various organisms. characteristic differences were observed between plant and mammalian cells, and between archaeobacteria and eubacteria. the patterns of the free amino acid composition in blue-green alga, green alga, protozoa and slime mold differed from each other and from those of eubacteria and archaea cells. it has been suggested that the free amino acid composition reflects apparent biological changes as the result of evolution. 2) catalyzes the hydrolysis of gamma-glutamyl compounds such as glutathione, and the transfer of their gamma-glutamyl moieties to amino acids and peptides. we previously developed enzymatic methods for the synthesis of various gammaglutamylamino acids using the transfer reaction of ggt from e. coli k-12 as a catalyst. it has been reported that gamma-lglutamyltaurine has a potent and long-lasting antiepileptic action, and its chemical synthesis has also been reported, but it required protecting and deblocking of reactive groups. thus, the purpose of this study was to develop an enzymatic method for the synthesis of gamma-l-glutamyltaurine using ggt. the optimum reaction condition was 200 mm l-glutamine, 200 mm taurine and 0.2 unit/ml ggt, ph 10, and 1-hr incubation of 37°c. forty-three mm gamma-glutamyltaurine was obtained and the yield was 21.%. gamma-glutamyltaurine was purified by dowex 1 ϫ 8 column and c18 column, and then identified with gamma-l-glutamyltaurine by nmr and polarimeter. in this study the yield of gamma-l-glutamyltaurine was comparatively low because synthesized gamma-lglutamyltaurine was promptly converted into the by-product, gamma-l-glutamyl-gamma-l-glutamyltaurine. the production of antimicrobial peptides is an important aspect of host defense in animals ranging from insects to mammals. they do not target specific molecular receptors on the microbial surface, but rather assume amphipathic structures that allow them to interact directly with microbial membranes, which they can rapidly permeabilize. they are thus perceived to be one promising solution to the growing problem of microbial resistance to conventional antibiotics. insects express a battery of potent antimicrobial proteins in response to injury and infec-tion. until now, approximately 170 immune peptides have been characterized from insects and other invertebrates. an antimicrobial gene (md-cecropin) belonging to cecropin family was cloned from the bacteria-charged adult house fly, musca domestica. expressed in the vector pgex-4t1. mrna was isolated and degenerated primers were designed according to the conserved sequences of cecropins. the full-length cdna encoding md-cecropin was cloned by rt-pcr and 5ј, 3ј-race and sequenced. the deduced amino acid sequence indicated that a prepeptide with 63 amino acid residues is first translated and then processed to a mature peptide with 40 amino acids. the dna encoding the mature peptide was subcloned into expression vector pgex-4t1, and expressed efficiently in e. coli bl21 as a fusion protein. the fusion protein was purified and specifically digested and the md-cecropin was further purified to homogeneity and the activity spectrum was investigated. escherichia coli with metabolic engineering methods l. yun, x. zhang, s. wang, q. xu, and l. ma biotechnology laboratory, institute of beijing radiation medicine, beijing, p.r. china a bioengineering escherichia coli strain was obtained by metabolic engineering method. three genes related to the biosynthesis of phenylalanine, arog, phea, and tyrb encoded key enzymes: 3-deoxy-d-arabino-heptulonate-7-phosphate synthetase (ds), a bifunctional protein-chorismate mutase (cm)/prephenate dehydratase (pd) and aminotransferase (at), respectively. in this work, the feedback inhibition of ds and cm/pd were relieved by site-directed mutagenesis on bases of homology comparison of related sequences of the key enzyme. the feedback inhibition resistant genes encoding ratelimiting enzymes in the main and terminal pathways were amplified by co-expressed in order of arog-phea-tyrb on the plasmid by their own operator plpr, pl, and pr. in the recombinant strain showed great resistant to the l-phenylalanine analogues, the specific activities of ds, cm, pd and at were increased by 3.10, 3.29, 4.91 and 8.16 folds, respectively. as the result, the amount of phenyalalnine biosynthesis of the bioengineered strain was increased greatly compared with that of the host strain. an enzymatic approach for the mapping of phosphoproteins resolved on two-dimensional polyacrylamide gels hiroshima proteome laboratory, regional science promoter program, kagamiyama higashihiroshima, japan an enzymatic approach for high-throughput mapping of phosphorylated proteins resolved on two-dimensional (2-d) polyacrylamide gels is presented. proteins of cultured rat skin fibroblasts were divided into two aliquots, one of which was dephosphorylated using recombinant 1 protein phosphatase. the two aliquots were then subjected to 2-d electrophoresis. the phosphoproteins could be mapped on the 2-d gel by com-paring the gels of the phosphatase-and non-treated samples, because the dephosphorylated proteins shifted to more basic positions on the gel. this technique revealed that approximately 5% of the detectable proteins were phosphorylated. fifteen phosphoproteins were identified by mass spectrometry, including proteasome component c8 and small glutamine-rich tetratricopeptide repeat-containing protein. furthermore, the extent of phosphorylation of two actin-modulating proteins, destrin and cofilin, was found to be significantly reduced when the cells were chemically or enzymatically detached from the culture dishes. the presented technique can be applied to all biological materials because it requires no protein-labeling step, and is therefore useful for high-throughput mapping of phosphoproteins in proteome research. with the completion of the human genome sequence maldi-tof-ms is increasingly becoming an established method for identification of proteins separated by 2d gel electrophoresis. mono-isotopic peptide mass fingerprinting (pmf) has been previously shown to be amenable to full automation encompassing the process of acquisition, data processing and databank searching under full software control. until now the throughput of maldi-tof-ms for proteomics has been limited to several hundred samples in a working day and this represents approximately 5-10% of the total proteins resolved by a large format 2d gel. to reduce the number of proteins to be identified the 2d gels are imaged and analysed to determine differences in expression levels within a set of gels. although much of the image processing is semiautomated the comparison is labour intensive as manual pattern matching has a role in the gel alignments (land marking). increased ms sample throughput allows the possibility of identifying every protein spot in a 2d gel within a day. this could eliminate the potentially erroneous step of human gel image alignment, whereby land marking could be achieved using the ms data. increased sample throughput requires greater capacity and robust unattended instrument operation. in this poster we describe an integrated robotic multiple plate loader that allows overnight unattended ms operation. other improvements include an increased laser repetition rate that allows the data capture rate to increase four fold. sample tracking, data archiving and data reporting are essential attributes of this new technology and these aspects are outlined in the presentation. the proteinchip tm biology system for ciphergen biosystems: a novel proteomics platform for rapid biomarker discovery, validation and identification ciphergen biosystems ltd., surrey technology centre, the surrey research park, guildford, surrey, u.k. the proteinchip system uses seldi (surface enhanced laser desorption/ionization) proteinchip technology to perform the separation, mass detection and analysis of proteins at the femtomole level directly from biological samples. surfaces are based on either chromatographic based chemistry (ion exchange, reverse phase, imac etc.) that bind large classes of proteins or biologically defined surfaces (antibodies, dna, receptors, etc.) that are used to investigate specific proteininteraction events. as with conventional elution chromatography each type of surface is designed to bind a different subset of proteins from a crude mixture. sample complexity is reduced on the surface by washing with standard biological buffers compatible with the chosen proteinchip array. unlike elution chromatography, proteins are detected directly from the stationary phase using laser based mass spectrometry greatly increasing throughput whilst reducing sample loss and improving reproducibility. multiple proteinchip surface and wash conditions are explored with a small sample set to resolve hundreds of proteins and establish assay conditions that reveal candidate biomarkers or diagnostic protein profiles. the resulting custom built assay is then used to monitor disease processes or drug toxicity profiles by screening large banks of samples such as tissue extracts or physiological fluids (serum, urine, csf, etc.). pharmaceutical research, genomics technologies, f. hoffmann-la roche ltd., basel, switzerland to the present, samples representing the total protein mixture have been usually analyzed by proteomics technologies mainly only the abundant, hydrophilic components have been visualized. these proteins could be solubilized with reagents compatible with isoelectric focusing, for example urea and chaps. such an analysis provides us with a limited image of the proteome, which is insufficient for the detection of the majority of the proteins. in a 2-d gel, where about 1 mg of protein amount has been resolved, 1,000-3,000 protein spots can be detected, using coomassie blue staining. the spots represent the products of only 200-300 different genes. other gene products, not visualized, are most likely expressed at too low levels for detection or they can not be identified because of limitations of the current technology, they are too small, too large, basic or hydrophobic. here we will discuss protein enrichment approaches prior to the analysis, which we have applied for the enrichment of bacterial and eukaryotic proteins. proteomic analysis of the rat liver mitochondrial proteins m. fountoulakis 1 , j.-f. juranville 1 , and l. suter 2 1 genomics technologies, and 2 drug safety, f. hoffmann-la roche ltd., pharmaceutical research, basel, switzerland subcellular fractionation increases the probability of detection of low-abundance proteins. we prepared mitochondrial, microsomal and cytosolic protein fractions from total liver of male rats. the proteins of the three fractions were analyzed by two-dimensional electrophoresis using broad and narrow ph range immobilized ph gradient strips. the proteins were identified by martix-assisted laser desorption ionization mass spectrometry. in the mitochondrial fraction, 190 different gene products were detected. approximately 70% of the identified mitochondrial proteins are enzymes with a broad spectrum of catalytic activities. most of the identified proteins had been detected before in other samples as well, analyzed in our laboratory. eight gene products were detected for the first time. these were represented by one spot each, whereas most of the frequently detected proteins were represented by multiple spots. in average, approximately 15 spots corresponded to one gene product. centre for molecular medicine, university college london, u.k. three kinds of experiments have been carried out successfully in our labs. (1) identification of post-translational modifications of the endothelin a and b receptors (etar and etbr) including both phosphorylation and acylation. we have developed new, very efficient methods for single step isolation of highly pure etar and etbr from cells. this has allowed us to obtain evidence that the post-translational modifications are very complex and result in multiple phenotypes showing different forms of modification for receptor. as with other systems, e.g. insulin-like growth factors, it is probable that these multiple phenotypes of the et receptors correspond to different forms of signalling dependent on cellular state, e.g. the cell cycle. it is, for example, already clear from the phosphorylation of the receptor that a series of different kinases must be involved. (2) following stimulation of fibroblasts with endothelin, phosphorylation/dephosphorylation signalling cascades involving several hundred proteins have been observed by use of high resolution 2d electrophoresis and detection of phosphorylated proteins labeled with 32 p by autoradiography or immunological methods. the large number of proteins involved are being identified by mass spectrometric methods such as mass fingerprinting or sequencing by mass spectropmetry. (3) differential gene expression has been followed by using 35 s met pulse chase labelling concurrently with endothelin stimulation. at least 50 proteins showed significant changes in expression of 2d gels and these proteins are also being identified. these experiments demonstrate that it is now possible to use proteomics methods to investigate the integration of response to an extracellular signal at the levels of the receptor itself, the subsequent signalling cascades and the ensuing gene expression. the proteomics technology permits concurrent monitoring of large numbers of protein phenotypes (the forms and amounts of individual proteins and is therefore able to provide a global overview of signalling processes which greatly augments more traditional investigations of individual proteins or pathways. furthermore, these new methods will allow quantitative determination of the changes in protein phenotypes, which is very important in view of the highly non-linear amplification properties of such signalling processes. an integrated approach to automated high throughput protein identification by 2d gel electrophoresis and mass spectrometry d. gostick 1 , s. cohen 2 , p. young 1 , b. karol 2 , j. langridge 1 , j. randell 3 , t. slyker 3 , and a. jacobson 3 1 micromass, manchester, u.k. 2 waters corporation, milford, massachusetts, and 3 bio-rad laboratories, hercules, california, u.s.a. establishing the function of gene products is the major challenge of the post genomic era. the rate-limiting step in this endeavour is the speed with which proteins can be isolated and identified. separation of proteins from cell lysates or sub-cellular domains by 2d gel electrophoresis is an established method of visualising these complex systems. recently mass spectrometry has proved to be a powerful method of further characterising these proteins. from the mass spectrum of the enzyme digest of a 2d gel spot, the resulting digest map is compared with the theoretical maps from the databases and the protein identified when these correlate. maldi-tof is of great benefit in these studies since it requires a minimal amount of sample, is relatively tolerant to salts and other contaminants arising from the gel and may be configured for automated sample analysis. high sample throughput with automated analyses including data processing and client-server database searching are already available. our system automatically acquires the data and processes the maldi mass spectrum into a monoisotopic peak list. this peak list is then automatically sent to a networked database for protein identification. when proteins are not identified from the maldi analysis or an ambiguous result is obtained, then further analysis of the sample by electrospray caplc-ms-ms is required. the development of a hybrid quadrupole orthogonal acceleration timeof-flight mass spectrometer (micromass, q-tof) has facilitated the generation of unambiguous amino acid sequences from the ms-ms analyses of tryptic peptides. these ms-ms spectra can be automatically searched against protein, nucleotide or est databases. thus enabling protein identification from gel spots, despite non-specific enzymatic cleavage, protein co-migration and post transitional modifications. for organisms who's genome sequences are poorly represented in the data bases de novo amino acid sequencing may be required. inferring de novo peptide sequences from ms-ms data is complex and is often the rate-determining step in this method. however, it is now possible to interpret the ms-ms spectrum automatically. in our approach the raw ms-ms spectrum is reduced to the plausible single-charge, monoisotopic mass spectrum. sequence interpretation is achieved by generating "trial sequences" consistent with the experimentally determined molecular weight. a probabilistic fragmentation model is used to transform the trial sequences to predicted spectra for comparison to the single-charge, monoisotopic spectrum and to calculate the likelihood that the trial sequence would account for the observed data. the possible number of trial sequences for any peptide is large, for example there are 20 10 possible sequences for a peptide containing any of the 20 naturally occurring amino acids and having 10 residues. to reduce the scale of the problem a terminated markov chain monte carlo algorithm is used to produce sequences. this bayesian method simulates an exhaustive search of all sequences having the correct mass. the huge increase in genomic sequence information available, combined with the increased sensitivity and selectivity provided by mass spectrometry, has allowed large-scale protein identification. however the analysis of the post translational modifications present on the identified proteins is a more challenging problem. currently the approach that offers the most expedient and specific solution, to determine modified peptides, is precursor ion scanning. this approach has primarily been performed on a triple quadrupole mass spectrometer where the rear quadrupole, (ms2) is set to transmit only the fragment ion of interest. the ms1 quadrupole is then scanned across the appropriate mass to charge range. in this paper we describe a method that allows specific post translationally modified peptides to be identified and sequenced during the course of an hplc experiment on the q-tof mass spectrometer. during the hplc run the instrument is switched alternately at one-second intervals between low and high collision energy with argon in the collision cell. the quadrupole, ms1 is not mass selective, operating in the rf only mode. the first data set at low energy (4ev) shows only the normal pseudo molecular ions. the second at higher energy shows their fragments. wherever a product ion of interest occurs in the high-energy data all its possible precursors are revealed by the corresponding 4ev data. since the two data sets contain the entire set of precursor and product ions that can be formed it is clearly possible to generate the equivalent of a constant neutral loss scan. this is invaluable in the case of phosphorylated peptides where the neutral loss of 98da (h 3 po 4 ) occurs via -elimination from the phosphoserine and phosphothreonine residues. this allows the q-tof mass spectrometer to switch from the ms mode to the ms/ms mode of operation when a potential pseudo molecular ion exhibits a neutral loss of 98 da between the high energy and low energy data sets. the product ion ms/ ms spectrum can then be acquired on the phosphorylated precursor ion. in the case of phosphotyrosine, neutral loss of the h 3 po 4 moiety is not observed, however a low mass immonium ion at m/z 216 can be detected. this characteristic ion (from the high energy data) is used to direct the mass spectrometer to fragment potential phosphopeptide precursor ions, which are selected from the low energy data. in this case several precursor ions may require ms/ms interrogation at one decision making time-point. with the first draft of the human genome completed largescale protein identification by mass spectrometry, even for samples originating from higher organisms has become relatively straightforward. this requires a high throughput facility to identify proteins that have usually been separated by 2d page. the approach providing the highest level of automated sample throughput, in terms of samples per hour, is currently maldi-tof-ms. this technique provides a peptide mass fingerprint of the protein digests and allows the rapid and accurate identification of the parent protein by comparison to a databank. however, under some circumstances, for example if the number of peptides detected is small or if the sequence coverage is poor, it is advantageous to be able to include even a short piece of sequence information to provide added specificity. in a conventional maldi-tof-ms instrument post source decay (psd) can be used to try and generate sequence information, however this approach is notoriously unreliable in producing good quality ms/ms data. one reason for this is that the peptide ions do not undergo fragmentation in a controlled environment such as a gas cell with selected collision gas and collision energy. an alternative approach is to use the predictable fragmentation obtained from a hybrid quadrupole ortho maldi source has been fitted to a hybrid quadrupole orthogonal acceleration time-of-flight (q-tof) mass spectrometer. in contrast to a conventional maldi-tof-ms instrument the resolution and mass measurement accuracy of the data is comparable between the ms and ms/ms modes. this allows superior data acquisition in the ms-ms mode compared to conventional maldi-tof-ms. a number of modifications have been made to optimise the system for high throughput proteomics. the maldi source has been configured with a high-density target plate, compatible with a 96 well microtiter plate. the acquisition software has been modified for automated data acquisition in both the ms mode and the ms to ms/ms switching mode. dedicated processing software has been developed to fully automate the post acquisition and databank searching. this software has been optimised to consider the unique nature of the data acquired from this configuration of instrument. in this paper we demonstrate the how an maldi-q-tof instrument can be used for high throughput proteomics. we also compare and contrast is functionality in comparison with alternative strategies for high throughput proteomics, namely conventional maldi-tof-ms and electrospray lc-ms/ms. pseudomonas putida is an ubiquitous, metabolically and physiologically extremely variable soil bacterium. it is kown to be a good colonizer of plant roots and a plant growth promoter. now, after the sequencing of the total genomic dna has been finished we have focused on the functional analysis of this strain. plant growth promotion is achieved in different ways. one is the inhibition of fungal and bacterial phytopathogens, which is known to be a multifactorial mechanism. an important factor of this mechanism is the production of siderophores (iron-transport-agents), small linear or cyclic peptides, which are synthesized in a ribosomal-independent manner by special synthetases. the siderophore production is induced by iron limitation. the regulation of this process was investigated by pulselabelling with [ 33 p] inorganic phosphate. 2d-protein patterns generated from cells grown with and without fesupplementation were compared. proteins which were phosphorylated under iron limitating conditions were analysed by maldi-tof peptide mass fingerprint. for the identification of the proteins we used an in-house peptide mass database which has been built based on the genomic sequence data. bio-rad laboratories, inc., hercules, california, u.s.a. worksbase software for proteomics is a platform independent information management system encompassing laboratory experimental workflow and bioinformatics for protein and biochemical research. the worksbase system is designed to allow direct internal integration between laboratory experimental data and background biological knowledge found in reference and in-house data, such as gene, protein and functional annotation databases. worksbase provides a crossdisciplinary research infrastructure for drawing together multiple lines of evidence for characterization of proteins, and integration of this data with domains such as gene expression, pharmacological screening, structure and related areas. while the focus is on the biology underpinning the experimental work, the system is also designed with the capability of providing a sample and workflow tracking system for use in the wet lab, effectively a proteome lims (laboratory information management system). as experimentation proceeds in the laboratory, worksbase software can be used for development of hypotheses on protein, biochemical pathway, and post-translational processing involvement in biological systems and disease processes. as such, identifications that are derived from lab work and user observation can be used to augment the reference data repository. however, unlike databases ands systems where the methods and reasoning for assignment of annotations are obscure, by maintaining the link between the source data and the biological roles derived from them, the accuracy and integrity of any information stored in the worksbase system can be directly ascertained. changes in the brain protein levels following administration of kainic acid k. krapfenbauer 1,3 , m. berger 2 , g. lubec 3 , and m. fountoulakis 1 1 f. hoffmann-la roche ltd., pharmaceutical research, genomics technologies, basel, switzerland 2 institute of cancer research, and 3 department of pediatrics, university of vienna, austria kainic acid (ka), a potent neurotoxin and excitatory amino acid, leads to derangements and modulation of brain proteins. no global brain protein expression pattern induced by ka-treatment has been reported yet. we studied the effect of systemic ka administration on the levels of brain proteins. rats were injected placebo or ka intraperitoneally and brain was taken after one week. the mitochondrial and cytosolic fractions of the brain proteins were analyzed by proteomics technologies. heat shock protein hsp 27 was exclusively detected in brains of animals treated with ka. the levels of neurofilaments and alpha-internexin were significantly decreased and a fragment of tubulin alpha-1 chain was manifold increased in ka-brains. the mitochondrial enzymes dihydrolipoamide dehydrogenase, atp synthase beta chain and isocitrate dehydrogenase were reduced and pyruvate kinase m1 was increased following ka treatment. the results indicate altered regulation of heat shock proteins, neuronal death, cytoskeletal disruption and mitochondrial derangement by systemic ka administration. this report confirms and extends previous studies on the effect of ka on the expression of brain proteins and suggests that our analytical system can serve as a model for neurotoxicological, neurobiological and neuropathological proteome studies. the rat brain mitochondrial proteins genomics technologies, f. hoffmann-la roche ltd., pharmaceutical research, basel, switzerland we constructed a two-dimensional database for rat brain mitochondrial proteins. rat is a useful model of human diseases of the central nervous system. in order to detect alterations in the levels of the low abundance brain proteins, the mitochondrial, microsomal and cytosolic fractions were prepared. the proteins of each fraction were analyzed by two-dimensional electrophoresis, followed by martix-assisted laser desorption ionization mass spectrometry. approximately 500 proteins were identified in the mitochondrial fraction, which were the products of 165 different genes. about 75% of the identified proteins were detected in the mitochondrial fraction only and the rest were detected in the cytosolic and about 2% were found in the microsomal fraction as well. 98 of the 165 proteins had not been detected before in our laboratory. the identified proteins were in the majority enzymes or enzyme subunits with a broad spectrum of catalytic activities and heat shock proteins. whilst lc-ms/ms has been utilised for the identification of proteins from complexes and cell lysates (qualitative proteomics), the quantitative study of gene expression using differential display has until recently been the preserve of a 2d gel based proteomic experiment. however, recently a great deal of interest has been generated on the use of isotope coded affinity tags (icat) for the quantitative study of gene expression at the proteome level. the technique is based upon chemically modifying the cysteine residues of proteins isolated from cells in two different states with light and heavy isotopically labeled reagents. the two cell states are then combined, digested with trypsin and the cysteine containing peptides preferentially selected by binding to an avidin column, prior to analysis by mass spectrometry. the eluent from this column is then analysed by capillary lc esi-ms/ms. interrogation of the eluting peptides by tandem mass spectrometry and databank searching results in the identification of the associated protein. we describe how icat data analysis has been automated within a software environment. the ms and msms data acquired using the qtof instrument are processed and analysed using a new algorithm which recognises related isotope clusters and quantifies their relative intensities. based on a user defined ratio threshold the software will automatically carry out an lc-ms/ms experiment and databank search in a client-server mode and provide a report of the identified proteins and their expression ratio in the two cell states. deterioration of the transcriptional, splicing and elongation machinery in brain of fetal down syndrome b. lubec 1 and m. fountoulakis 2 1 department of neonatology, university of vienna, austria 2 gene technologies, cns research, f. hoffmann la roche, basle, switzerland perturbation of brain development i.e. regulation of gene expression, differentiation, growth and migration in down syndrom (ds) has been reported to occur early in life pointing to impairment of the complex system of transcription and or translation and indeed, altered expression of transcription factors has been reported in adult ds brain. we therefore decided to compare the transcriptional and translational machinery in cortex of brains of controls and fetuses with down syndrome in the second trimenon of gestation. we determined a series of transcription/translation factors by 2 d-electrophoresis followed by maldi -identification and quantification with specific software. the protooncogene c-crk, crk-like protein, elongation factor 1-alpha 1, elongation factor 2, elongation factor tu and two out of four spots representing ptb-associated splicing factor psf were significantly downregulated in brain of fetal ds fetuses as compared to controls. the finding of reduced transcription and translation factors may indicate deranged protein synthesis. the underlying cause for individual reduced transcription, splicing and translation factors may be explained by chromosomal imbalance or by posttranslational modifications as e.g. phosphorylation, known to be aberrant in ds. reduced expression of transcription factors in fetal ds during early life may be responsible or reflecting impaired brain development and deficient wiring of the brain in ds. r. mazzoli 1 , m. g. giuffrida 2 , e. pessione 1 , g. dellavalle 2 , c. barello 2 , e. griva 1 , and c. giunta 1 1 dipartimento di biologia animale e dell'uomo, università di torino, and 2 csaapz-cnr. c/o bioindustry park canavese colleretto giacosa (to), italy a fast phenol degrading acinetobacter radioresistens strain was isolated in our laboratories and selected for bioremediation applications. this bacterium is also able to grow on benzoate and catechol as sole carbon-energy sources, metabolizing them via the ortho route. in previous researches we detected, by means of proteome analysis, some marker enzymes of the phenol and benzoate degradative pathways. in the present work we extend the identification of the proteins involved in the aromatic-ring opening (the different components of the phenol hydroxylase and benzoate dioxygenase, the catechol dioxygenase isozymes) together with other satellite proteins specifically induced by the aromatic growth substrate. of these last proteins some are probably related to the cellular uptake of benzoate and phenol while others are ascribed to the groel family of heat-shock chaperonines, involved in proteins processing and folding. aromatic substrates may thus act as stress-agents like heat or cold. proteomic studies on rat body fluids i. miller 1 , r. wait 2 , l. sironi 3 , i. eberini 3 , m. gemeiner 1 , e. tremoli 3 , and e. gianazza 3 1 veterinärmedizinische universität, wien, austria 2 imperial college school of medicine, hammersmith, london, u.k. 3 universita' degli studi, milano, italy previously, we have characterized rat serum proteins, both under "normal conditions" and during experimental inflammation, using two-dimensional electrophoretic separation, densitometric quantitation and identification by mass spectrometry and immunological procedures (http://linux.farma.unimi.it/ homeframed.html). we have now extended these studies to the protein composition of cerebrospinal fluid (csf) and urine, and have identified several proteins specific to these fluids, including major urinary protein, uromodulin, and prostaglandin d synthase. these baseline data provide a useful comparison to the biological fluids of stroke-prone spontaneously hypertensive rats, an inbred strain, which develops cerebrovascular abnormalities following high blood pressure. our studies have detected signs of an inflammatory condition several weeks prior to stroke. we have confirmed the sharp rise in proteinuria preceding stoke onset, and have identified the excreted proteins. following stroke we observe a massive increase in csf protein concentration as serum proteins, even those of large molecular size, cross an impaired blood-brain barrier. as a first step to discover useful disease markers from the urinary proteome, we have developed a unique and systematic approach for detection of low molecular weight urinary proteins by using high resolution two-dimensional (2d) electrophoresis and mass spectrometric methods. unlike previous studies on urinary proteins, and most importantly as observed in present study, our results show that a large number of low molecular weight protein spots can be visualized in the 2d electrophoresis pattern. it was observed that protein concentration and fractionation methods were critical for our ability to detect many proteins in the gel pattern. therefore, several approaches were carefully considered to concentrate and fractionate proteins in urine samples. initially, urine specimens from normal individuals were concentrated by using centrifugation and ultrafiltration methods. the concentrated samples of urine proteins were then fractionated by size exclusion and immunoaffinity chromatography. the size exclusion method was used to generate two fractions of proteins based on their native molecular weights. further, this method allowed us to enrich concentrations of less abundant proteins for each fraction. the immunoaffinity method was used to specifically remove well-known abundant urinary proteins (such as albumin) from the above mentioned two fractions. that the 2d pattern includes many native low molecular weight proteins was confirmed by analyzing both protein fractions from size exclusion chromatography. a detailed mass spectrometric analysis of the protein spots is carried out to identify the proteins observed in 2d pattern. since urine is an ultrafiltrate of plasma, many factors in urine are present in proportion to their rate of synthesis in the body. these factors include many low molecular weight proteins that remain undiscovered due to their low abundance. therefore, the present analysis of urinary proteins would serve as the most useful guide for the discovery of novel diagnostic markers in urinary proteins. i. pucci minafra 1,2 , s. fontana 2 , p. cancemi 1 , g. alaimo 1 , and s. minafra 1,3 1 centre of experimental oncobiology, 2 department of cell biology and development, and 3 institute of histology and embryology university of palermo, italy breast cancer is one of the leading causes of death for cancer among women. there are different types of breast cancers, grouped as invasive and non-invasive types. among the invasive types "infiltrating ductal carcinoma" (idc) accounts for about 80% of all breast cancers. in order to study some biological properties related to this type of cancer, we have developed and well characterized an "in vitro" system, consisting of an idc-derived cell line, 8701-bc (minafra et al., br. j. cancer, 60, 185-192, 1989 ) and some of its cloned cell lines, selected for their high and low invasive activity in matrigel. using this model we are producing proteomic maps to compare with that of non-tumoral breast epithelial cells and with breast tissue fragments, existing in our collection or available at the expasy proteomics server. protein identification is currently done by means of gel matching, edman-microsequencing and immuno-detection. to rationalize data we grouped proteins into functional categories: a) cytoskeletal proteins, b) metabolic enzymes, c) chaperonins and other functionally related proteins, d) peptides and enzymes with regulatory functions. a fifth group consists of peptides with unknown identity. among these sets of proteins we found that glycolitic enzymes and some chaperonins are overexpressed in cancer cells. in addition, new isoforms of potential interest as biomarkers for breast cancer, were identified by means of microsequencing. a. santucci 1 , l. trabalzini 1 , d. soldateschi 2 , e. ferro 1 , a. paffetti 1 , and p. martelli 1 1 dipartimento di biologia molecolare, sezione di chimica biologica, universita' degli studi di siena, and 2 diesse diagnostica senese srl, siena, italy human cytomegalovirus (hcmv) is an ubiquitous virus, belonging to the herpesviridae family, betaherpesvirinae subfamily, able to induce morbidity in immunocompromised patients and congenitally infected new-borns. hcmv has the largest genome among the herpes-viruses (240 kbp): ad169 strain genome was completely sequenced, containing about 200 open reading frames encoding polypeptides, most of which are not characterized. the viral genes are activated in a cascade fashion: 1) alpha, immediate-early genes, coding for regulatory proteins necessary for the activation of 2) beta, early genes, needed for dna replication, and, finally 3) gamma, late genes, coding for structural proteins of the mature virions. this latter category includes the virus surface antigenic proteins responsible for the main immune response during hcmv infections. although the sequencing of hcmv genome has been completed, very little is known about the actual nature of the viral proteins. the most appropriate approach to characterize hcmv phenotype is to study its protein expression as it is carried out within the host cell. for this purpose, we analyzed by two-dimensional electrophoresis (2d-page) the protein phenotipic repertoire of human fibroblasts and compared it with that of the same cell type following infection with hcmv strain ad169. the phenotypic 2d map of human fibroblasts dramatically changes following infection with hcmv. a relevant amount of newly appeared spots is attributable to hcmv proteins, mainly of the structural category, since we analyzed host cells at the 7-9th day of infection, when the late, gamma genes are supposed to be the only to be activated. on the other hand, a marked decrease of protein synthesis can be easily evidentiated in the infected fibroblasts respecting to uninfected cells. a temptative mapping of the main structural viral proteins (those against which patients sera are directed) was carried out by immunoblotting, microsequencing and mass spectrometry. comparative proteomics of cultured cells: identification of genetic defects and molecular mechanism of apoptosis regulation v. seyrantepe 1 , k. landry 1 , s. taurin 2 , s. n. orlov 2 , and a. v. pshezhetsky 1 1 sainte-justine hospital research centre, and 2 research centre, chum, university of montreal, montreal, pq, canada we employed a comparative proteomics of cultured cells to study mechanism of genetic disorders and for identification of key proteins involved in cell proliferation, differentiation, and death. in particular, this technology proved to be very useful to understand molecular basis of severe inherited diseases resulting from deficiency of lysosomal membrane transporters, and a role of programmed cell death (pcd) of vascular smooth muscle cells (vsmc) in cardiovascular disorders. to increase sensitivity of the identification of cellular proteins we have either have isolated cellular organelles such as lysosomal membranes or performed the differential extraction of soluble, membrane and cytoskeletal proteins. by comparison of pro-teomic cell maps from normal controls and individuals affected with lysosomal transport disorders we have selected and identified several candidate disease-causing proteins, which have to be further studied by mutation analysis and functional expression. for the second group of disorders we identified proteins, which de-novo synthesis could result in survival of vsmc including a two members of hsp70 family, a molecular chaperone grp78, and so-called mortalin (grp75) highly expressed in non proliferative tissues and associated with mortal cell phenotype. two-dimensional polyacrylamide gel electrophoresis (2d-page) is the established technology employed for the separation of proteins from a cell lysate, sub-cellular organelle or tissue sample prior to identification of the excised protein spots by mass spectrometry. in the order of several hundred to several thousand proteins, can be separated and visualised on a 2d gel by conventional staining or utilising fluorescent labelling techniques. the advantage of performing a two dimensional gel based separation is the ability to obtain quantitative information by comparing and contrasting two samples in a differential display experiment, for example, between a healthy and diseased state. the last stage however stipulates that the gels are reproducible which can be both difficult and time consuming to achieve. the relativity poor dynamic range that the gels exhibit also limits quantification. other restrictions include the under representation of certain classes of proteins, such as membrane proteins, large or small proteins and very acidic/basic proteins. for these reasons, amongst others, alternatives to 2d-page are being investigated. advances in both lc and mass spectrometry instrumentation have allowed the analysis of protein complexes, which have not been separated on a 2d gel. in this case protein identification is achieved via database searching of esi-ms/ms data. this provides qualitative information on the proteins that are present and has recently been coupled with isotope dilution experiments to provide relative quantiative information. these experiments normally involve separation of the complex digest mixture by microcapillary liquid chromatography connected to an instrument capable of data dependant switching between the ms and ms/ms modes. using this approach it has been demonstrated that hundreds of ms/ms spectra can be acquired in a fully automated fashion, resulting in the identification of significant numbers of proteins, including low copy number proteins, from a single lc-ms/ms experiment. if, however, a complex protein mixture is to be investigated then a fractionation step prior to separation of the peptides on the basis of their hydrophobicity would be advantageous. we have, therefore, adopted a 2d lc-ms/ms approach using a capillary lc system (caplc) operating at nanoliter per min flow rates coupled to a q-tof 2 mass spectrometer. by replacing the standard sample loop within this system with a strong cation exchange (scx) cartridge followed by a c18 trap cartridge it is possible to pre-fractionate the peptides before separation on a c18 column. after loading the sample, discreet fractions are sequentially eluted from the cation exchange cartridge using a salt step gradient; the eluted peptides are then retained on the trapping c18 cartridge whilst they are desalted. finally the peptides are eluted from the c18 pre-column, at 200 nl/min, onto a 75 um id ϫ 10 cm waters symmetry analytical column for separation and elution into the mass spectrometer. this analytical approach will be discussed with examples where this methodology has been used for the analysis of standard protein mixtures and also for the analysis of cell lysates and sub-cellular fractions. monoclonal igg are commonly observed in various b cell disorders, the most clinically relevant being multiple myeloma. in a series of 73 serum samples, immunofixation identified igg 1 , igg 2 , igg 3 , and igg 4 in 63, 4, 5, and 1 cases, respectively. their light-chains were k in 45 cases and λ in 28 cases. these monoclonal igg were further characterized by high resolution two-dimensional polyacrylamide gel electrophoresis (2-de) with various isoelectric focusing conditions as well as by 3-de (2-de of the proteins extracted from agarose after serum protein agarose electrophoresis). after 2-de or 3-de, the monoclonal γ-chains were not visualized in 29 out 73 cases, whatever the isoelectric focusing conditions that were tested. in 6 cases, γ-chains were only detected using alkaline ph 6-11 gradients. monoclonal γ-chains and light chains were highly heterogeneous in terms of pi and mr. however, a good correlation (p ͻ 0.05) was observed between the index of migration of the monoclonal igg in agarose gels and the pi of their γand of their light-chains (r ϭ 0.735, multiple linear regression). because of the extreme diversity of the different γ-chains as well as of the k-and γ-chains, it appears that a classification of monoclonal igg based only on their electrophoretic properties is not possible. alzheimer's disease (ad) is one of disorders caused by protein conformational changes and recent studies have shown that several chaperone proteins are involved in this process. as information of chaperone expression in ad brain is limited, we aimed to study the expressional pattern of chaperones in several brain regions as this may be essential to understand how folding defects can lead to disease. we studied the concomitant expressional patterns of molecular chaperones in seven brain regions of adults with ad using two-dimensional polyacrylamide gel electrophoresis (2-de) and matrixassociated laser desorption ionization mass spectroscopy (maldi-ms). we unambiguously identified and quantified nine different chaperone proteins. six chaperone proteins, heat shock protein 60 (hsp 60), hsp 70 ry, heat shock cognate (hsc) 71, alpha crystallin b chain, glucose regulated protein (grp) 75 and grp 94 showed aberrant expressional patterns depending on brain region. hsp 70.1, grp 78 and t-complex 1 (tcp-1) epsilon subunit did not show any significant expressional change. these findings are compatible with neuropathological and biochemical abnormalities in ad brain and this report presents the first approach to quantify nine different chaperones simultaneously at the protein level in individual ad brain regions providing evidence for the relevance of aberrant chaperone expression to ad neuropathology. the mainstream approach to protein separation, visualisation and identification has been to use two-dimensional gel electrophoresis coupled to mass spectrometry for the identification of the separated proteins. however this approach is limited with the level of protein that may be loaded onto the 2d gel and the nature of the proteins that may be incorporated onto the first dimension (ipg strip). an alternative approach for the qualitative analysis of complex protein mixtures is the use of tryptic digestion followed by electrospray lc-ms/ms. this approach is dependent on a high degree of chromatographic separation prior to the mass spectrometer, such that ideally individual peptides are eluted into the source. if this is the case then the dynamic range of protein identification can be increased and low copy number proteins can be identified. often, however there is a large degree of redundant sequence information acquired, as in theory one peptide ms/ms spectrum is sufficient to identify a protein from a sequence databank. if a protein identification is obtained from a databank search of an ms/ms spectrum, it is potentially valuable to exclude the rest of the theoretical tryptic peptides to "mine" deeper into the protein complex being studied. we have introduced a new protein databank search engine capable of matching a tryptic peptide from the swissprot/ trembl databank to an ms/ms spectrum in one second. using this search engine we are able to generate dynamic tryptic peptide exclude and include lists, based upon the theoretical tryptic peptides from the identified protein, which can be passed to the acquisition software of our q-tof mass spectrometer in real time. thus, we are able to automatically steer the q-tof, during acquisition, to select and switch to the ms/ms mode only on those peaks that meet the modified selection criteria. experiments can be designed in which peaks that belong to a protein already identified during acquisition can be avoided. this exclusion list is based upon m/z, charge state and a user definable mass tolerance. the mass measurement of the data from the q-tof mass spectrometer is typically better than 10 ppm and as a consequence of this a tight mass tolerance can be selected, thus making the exclude list extremely specific. alternatively, in the case of samples derived from 2d gel spots, the mass spectrometer may abandon the current sample, re-equilibrate the lc column and move on to the next sample. to illustrate this methodology we show examples, both on standard samples and complex protein mixtures where q-tof data acquisition has been directed based upon the results from a databank search. this data will be compared and contrasted to data acquired in the normal automated lc-ms/ms mode. the specific anti-cancer activity of green tea (؊)-epigallocatechin-3-gallate (egcg) department of molecular biology, hebrew university-hadassah medical school, jerusalem, israel the effect of the green tea polyphenol-(ϫ)epigallocatechin-3-gallate (egcg) was tested in cultures of normal and transformed nih-patmras fibroblasts. in this system transformation can be induced at will by the addition of dexamethasone, which induces the expression of h-ras by activating the mammary tumor virus long terminal repeat (mmtv-ltr) promoter. this facilitates a reliable comparison of the susceptibility of normal and transformed cells to egcg. it has been shown that egcg inhibited the growth of transformed but not of the normal fibroblasts. in an attempt to elucidate the mode of the preferential inhibitory activity of egcg, its effect on growth promoting factors has been examined. the level of ornithine decarboxylase (odc, ec 4.1.1.17), which is a signal for cellular proliferation, was reduced by egcg in the transformed but not in the normal cells. egcg also showed strong inhibition of tyrosine kinase and mitogen-activated protein kinase (mapk) activities, without affecting the kinases in the normal cells. similarly, egcg also preferentially decreased the levels of the oncogenes ras and jun in transformed cell. egcg preferentially induced apoptosis in the transformed fibroblasts. in vitro chemosensitivity tests demonstrated that egcg inhibited the proliferation of leukemic cells. these findings suggest that egcg has a therapeutic potential in the combat against cancer. objectives: to develop a safo, affordable immune supportive therapy for hivϩ patients. design: a randomised, double blind, placebo-controlled study, testing an internationally patented l-methionine combination (lmc), in approximately 400 hivϩ patients; not yet on anti-viral treatment (cd4 count 200 to 500). methods: parameters measured included: cd4 count, total lymphocyte court, viral load, several clinical, as well as mechanistic parameters. the difference in the change from the baseline (active -placebo) was determined for each parameter. the study is ongoing. results: within 3 months, significant trends are noted. the cd4 count of the patients on the active therapy, presented with a slower rate of decrease, compared to the placebo group, mean difference (md) in this change from baseline; 15.1/cmm and 95% confidence interval (c1), this was confirmed by the total lymphocyte court values. after 12 months the placebo group was placed on active, causing the difference to disappear. conclusions: although further trials are needed, these results already indicate t-methionine as an important role player in the immune system of patients with impaired immune function. c. chiarla 1 , i. giovannini 1 , j. h. siegel 2 , g. boldrini 1 , and m. castagneto 1 1 centro di studio per la fisiopatologia dello shock cnr, catholic university, rome, italy 2 department of surgery, umdnj, newark, new jersey, u.s.a. in critical illness and sepsis, changes in amino acid plasma levels (aapl) have been assessed extensively, while little is known about the relationship with changes in other plasma components, such as those involved in fluid-electrolyte and osmotic balance; their investigation is also limited, in large clinical samples, by inter-patient variability. we analyzed the relationships between plasma sodium (na ϩ pl, meq/l) and aapl (µm/ l) in eighty consecutive measurements performed in one single patient with post-traumatic sepsis and severe, prolonged illness. unique feature of plasma taurine (tau) was maintenance of a highly significant inverse correlation with na ϩ pl (r 2 ϭ 0.48, p ͻ 0.001). all other aapl were correlated directly, or unrelated, to na ϩ pl, the only exception being a weak inverse correlation between tryptophan and na ϩ pl. tau was correlated, strongly and directly, also to phosphoethanolamine (pea), glutamate (glu) and aspartate (asp): tau ϭ 707.1 ϩ 7.3(pea) ϫ 4.6(na ϩ pl) r 2 ϭ 0.86, p ͻ 0.001 tau ϭ 710.2 ϩ 11.4(asp) ϫ 4.7(na ϩ pl) r 2 ϭ 0.61, p ͻ 0.001 tau ϭ 677.4 ϩ 30.7(logglu) ϫ 4.7(na ϩ pl) r 2 ϭ 0.59, p ͻ 0.001 and unrelated, or weakly and inversely related, to other aapl (measurements of beta-alanine were not included). co-variation of na ϩ pl and these aapl (particularly tau and pea) was influenced by severity of illness, and more complex regressions were needed to quantify this effect. these results provide useful information on interdependency of tau, na ϩ pl and other aapl in critical illness. the central nervous system (cns) shows an exceptionally high degree of vulnerability to reactive oxygen species. considerable evidence suggests that free radical formation and oxidative stress might play an important role in the pathogenesis of parkinson's disease (pd). moreover, it has been reported that the levels of glutathione and vitamin e increase in the brain of patients with pd as a compensatory mechanism to deal with oxidative stress. since vitamin e is an effective free radical scavenger in the brain, its neuroprotective function is the issue of new therapeutic approaches in neurodegenerative diseases. to elucidate the possible role of vitamin e in the pathogenesis of pd, we assessed the plasma levels of vitamin e, measured by high-performance liquid chromatography, in 54 patients with pd. vitamin e concentrations were also assessed in 93 age and sex matched normal individuals. the mean plasma levels of vitamin e did not differ significantly between these two groups (22.5 ϯ 8.15 mmoli/l for pd patients and 21.0 ϯ 7.9 mmoli/l for controls). the results of our study suggest that plasma vitamin e concentrations do not play a major role in the pathogenesis of pd. vitamin e and cardiovascular disease: nutritional and intervention approaches f. galli 1,2 1 institute of biological chemistry, university of urbino, italy 2 department of cardiovascular research, st thomas' hospital, london, u.k. vitamin e is represented by a family of eight natural vitamers (4 tocopherols and 4 tocotrienols) of which αtocopherol (α-t) form has the highest biological activity. this vitamin accounts for most of the lipid-soluble, chain-breaking antioxidant activity in mammalian tissues and plasma. in addition, it shows nonantioxidant properties through which it modulates cell signaling and the expression of specific enzyme in cell models playing a role in atherogenesis (e.g. endothelial and inflammatory cells). the preventive effect of vitamin e on acdv is still a matter of debate. the largest epidemiological investigations and 4 out of 5 main intervention studies at yet available have suggested a correlation between levels of vitamin e and incidence of atherosclerotic cardiovascular disease (acvd) and related mortality. an overall conclusion rising from these studies is that the major effect (if any) of vitamin e is to be found with intakes higher than 100 iu (100 mg all-rac α-tocopheryl acetate) per day. however, other investigations have failed to demonstrate a beneficial effect of vitamin e against acvd, suggesting the need for more studies on its metabolism and function. recently a family of tocopherol binding and transport proteins has been identified. they play a key role in the selective uptake and delivery of tocopherols to lipoproteins and tissues. genetic abnormalities of these proteins have been demonstrated to be responsible for conditions of vitamin e deficiency in humans. their tissue distribution and regulation are now under investigation. the information available on vitamin e metabolism and its response to supplements or diet changes are at yet poorly characterized. the synthesis of stable isotopes and the characterization of major metabolites of main vitamers provide important advances in this research. in the last years, both plasma levels and urinary excretion of relevant metabolites of α-t have been characterized. little information is available on metabolites formed by other vitamers. the emerging role of γ-t and its main catabolite 2,7,8-trimethyl-2-(b-carboxyethyl)-6hydroxychroman (γ-cehc) in the defense against nitrogen oxide species formed during the activation of inflammatory cells is now well established and suggests the need for further studies on the bioavailability and transformation of this homologue of vitamin e in humans. at the same time, an oxidation byproduct of α-t found in human plasma, namely α-tocopherylquinone, has been proposed to be also de novo synthesized from phenylalanine with a role in the genesis of a defective polyunsaturated fatty acid metabolism observed in phenylketonuric patients. this suggests a possible, and at yet unexplored relationship between vitamin e and phenylalanine/fatty acid metabolism which might have also a role in atherosclerotic process. r. gaspari 1 , s. mensi 1 , g. mercurio 1 , c. callà 2 , l. colacicco 2 , e. sacco 2 , and s. lippa 2 1 department of anaesthesiology and intensive care medicine, and 2 department of biochemistry and clinical biochemistry, catholic university of rome, italy four patients (3 females, 1 male; aged from 21 to 45 years) affected by severe liver failure, were treated by a new blood purification method, namely molecular adsorbent recycling system (mars). mars removes albumin-bound toxins using a specific membrane with a dialysate solution containing albumin. in the patients the plasma levels of methionine (meth), branched chain and aromatic amino-acids and liposoluble antioxidants were measured. the fischer's index did not show any significant variation, whereas the plasma levels of meth were well correlated with the levels of liposoluble antioxidants (vitamin e and coq10). in fact, in the patients receiving just branched chain amino-acids, the plasma levels of both meth and antioxidants progressively decreased. on the contrary, if meth and branched chain amino-acids were administered, the plasma levels of coq10 and vitamin e showed a positive correlation with the plasma meth levels (p ͻ 0.02; r ϭ 0.63 and p ͻ 0.005; r ϭ 0.77, respectively). since vitamin e and coq10 are mutually dependent-molecules, the administration of meth, essential substance for coq10 synthesis, may be effective to maintain a good antioxidant status in patients with severe liver failure undergoing mars treatment. we obtained new synthetic peptide preparation epitalon to be widely applied as a pharmaceutical due to its properties important in medical care. epitalon was found to stimulate repair processes in retinal diseases via restoring the retinal functions, in particular its photoreceptors. this promising peptide drug is a linear tetrapeptide of formula h-ala-glu-asp-gly-oh (alanyl-glutamyl-aspartyl-glycine). the substance was obtained by classic peptide synthesis in a solution (scheme: (1 ϩ 2) ϩ 1) with n-oxysuccinimide activated esters. coohgroups of lateral radicals of glutamic and aspartic acids were defended as benzyl esters, benzyloxycarbonyl (ala) and tert.butyloxycarbonyl (glu) n-defending groups were employed, deblockade conducted by trifluoroacetic acid and catalytic hydrogenolysis. preparative hplc on a reverse phase was applied for purification. the product was fully characterised by the data of analytical hplc (substance content -99%), amino acid analysis, ir-and hmr-spectra. the ready drug form is ampoules containing 10 µg of the substance in 1 ml of isotonic solution. epitalon application in patients with pigmented retinal degeneration stopped the pathology development in 100% and increased visual functions in 80% of the cases. in 70% of the patients visual acuity raised by 0.1-0.3. electroretinography confirmed the retinal functional activity increase. an increasing number of proteins are implicated in apoptosis and several of them have been shown to be altered in alzheimer's disease (ad) brain. because of this apoptosis is thought to be the underlying mechanism of neuronal cell loss in ad. to further substantiate this hypothesis we investigated the expression of a recently identified apoptosis related proteins and other apoptosis regulators in frontal cortex and cerebellum of ad by western blot and elisa techniques. quantitative analysis revealed unaltered levels of bax and raidd (receptor interacting protein associated ich-1 (caspase-2)/ ced-3 (caenorhabditis elegans death protease-3)-homologous protein with death domain) in both regions. zip (zipper interacting protein) kinase, bim/bod (bcl-2 interacting mediator of cell death/bcl-2 related ovarian death gene) and p21 were significantly increased only in ad frontal cortex (p ͻ 0.05, in all cases). cerebellar bcl-2 levels were significantly increased in ad (p ͻ 0.01) while in ad frontal cortex, although the levels tended to increase did not reach significance level. the results indicate that apoptosis indeed account for the neuronal loss in ad. however, it does not seem to involve bax and raidd. a. magyar 1 , m. brózik 3 , r. tóbi 1 , t. szabó 1 , j. szakonyi 2 , b. rojkovich 3 , p. gergely 2 , and f. hudecz 1 1 research group of peptide chemistry hungarian academy of science, budapest, 2 central laboratory of immunology, semmelweis university, budapest, and 3 national institute of rheumatology, budapest, hungary rheumatoid arthritis (ra) is a systemic autoimmune disease of unknown etiology. it is the most common of the inflammatory joint diseases, affecting 1-2% of the world population. anti-filaggrin antibodies (afa) directed against the epidermal protein, filaggrin, belongs to the most specific markers of ra. epitopes, containing citrulline within the sequence of filaggrin, have been recently identified as major antigenic sites recognised by afa. the aim of our study was to identify these epitopes of filaggrin derived-peptides targeted by ra specific antibodies to provide further information about the nature of the initial autoantigenic substance. the most immunogenic six sequences of filaggrin and further, on the n-and c-terminal, shortened version of the original peptide ( 306 shqestrgrsrgrsgrsgs 324 ) were synthesized. we used conventional solid-phase peptide synthesis (fmoc strategy) carried out on "multipin ncp" noncleavable kit. in elisa experiments the presence of afa was deter-mined using serum samples of ra patients and healthy blood donors. in conclusion our results provide further evidence that not simply the presence of citrulline but also the nature of its surrounding amino acids have important role in the creation of autoantigenic epitope reactive with anti-filaggrin antibodies. the autoimmune nature of multiple sclerosis (ms) has introduced cytokine genes as logical candidates for the loci determining susceptibility to the disease and/or influencing disease progression. interleukin (il)-1alpha and 1beta are major proinflammatory cytokines that have been related with several chronic inflammatory diseases such as ms. the il 1-receptor antagonist (il-1ra) is a protein structurally related to il-1beta that effectively inhibits the proinflammatory effects of il-1. a polymorphism in the 5ј-flanking regulatory region at ϫ889 of the il-1alpha gene, which may cause an overexpression of il-1alpha and a variable number tandem repeats (vntr) polymorphism in the il-1ra gene have been also associated with several inflammatory diseases. two biallelic base change polymorphisms in the il-1beta gene have been reported to influence the protein production: one is located in the promoter region at position ϫ511 and the other is in exon 5 at position ϩ3953. to analyze the contribution of il-1alpha, il-1beta and il-1ra genes in the genetics predisposition to ms, we have examined four polymorphic genetic markers in 132 italian patients with clinically definite ms and 130 healthy controls. in summary, no significant differences in genotypes and allele frequencies were found between ms patients and healthy controls. fibronectin -the extracellular matrix protein is oxidatively modified with oxygen reactive species (ros) in inflammation site. activated neutrophiles release the hypochlorite acid (hocl) and chloramines as products of myeloperoxidase/ h 2 o 2 /cl ϫ system. these reactive chlorine species chlorinate in turn matrix proteins. the resulting changes of tertiary protein structure could be evaluated by monitoring the antigen/antibody complex formation. the formation of the complexes between native/chlorinated fibronectin and igg class antibodies were examined by means of elisa with luminol chemiluminescence detection. the degree of fibronectin modification was monitored with spectroscopic methods. since the oxidation leads to the fibronectin aggregation -the tryptophane contents in resulting aggregates were evaluated with stern-volmer approach (acrylamide quenching). moreover, the aldehydes influence on the ag/ab complex formation was examined -since aldehydes are known products of amino acids n-chloramines deamination. also the native and modified fibronectin adherence to the matrix proteins was monitored with use of hrp labeled antifibronectin antibodies. the preliminary results suggest that chlorination impairs the ab/ag complex recognition but also prove that igg bounded chlorinated fibronectin promotes igg clusters formation. it was found also that mm concentration of the serine derived glycoaldehyde decreases the fibronectin/igg recognition and the effect could be attributed to the igg aggregates formation. we demonstrate also that hrp-labeled iggs detect the collagen and fibrynogen adherent fibronectin in a dose dependent manner-details of the elisa method are discussed. in subjects with rheumatoid arthritis (ra) oxidized low density lipoproteins (ldl) are supposed to serve as mediators for joint damage, further exacerbating the inflammatory process. to better understand mechanisms of ldl oxidation in ra a specific marker of oxidative modification of apolipoprotein (apo) b-100 proline and arginine residues, 5hydroxy-2-aminovaleric acid (hava), had been measured in plasma and synovial fluid ldl subfractions (ldl 1 , svedberg units (s f ) 7-12 and ldl 2 , s f 0-7) by gc-ms. paired knee synovial fluid and plasma samples were collected from 10 subjects with ra. additionally, plasma samples were collected from 10 healthy controls. the ldl 1 hava content in plasma was not different between the groups (ra, 0.004 ϯ 0.001 vs controls, 0.004 ϯ 0.001 mol/mol apob-100, p ϭ 0.748). the ldl 2 hava content in plasma was significantly higher in ra (0.145 ϯ 0.051 vs 0.013 ϯ 0.002 mol/mol apob-100, p ϭ 0.000). furthermore, synovial fluid ldl 1 and ldl 2 in ra contained elevated hava levels when compared with plasma concentrations (ldl 1syn , 0.023 ϯ 0.012 mol/mol apob-100 (p ͻ 0.001) and ldl 2syn , 0.434 ϯ 0.129 mol/mol apob-100 (p ͻ 0.001)). results suggest that proline and arginine residues of apob-100 are highly reactive toward oxygen radicals in both plasma and synovial fluid in ra. furthermore, susceptibility of apob-100 to oxidative modification increases along the lipoprotein metabolic cascade. particularly small dense ldl 2 were prone to direct oxidation of apob-100. correlation between hava content in plasma and synovial fluid ldl 1 and ldl 2 in ra may allow the use of hava as a clinical marker of antioxidant barrier impairment in ra. vascular collagen accumulation contributes to development of hypoxic pulmonary hypertension (ph). we have shown that injections of a polymer of the proline analogue cis-4-hydroxy-l-proline (chyp) in liposomes attenuated acute ph in rats (ajrccm 1997; 155 : 1384) . we now treated rats with established ph with a new polymer containing an increased "payload" of chyp. chyp was conjugated to a low mw poly(ethylene glycol)-lysine carrier {poly (peg1000)-lys-chyp} to increase the % by wt of the analogue. rats were exposed to 10% o 2 for 7 da to induce ph. on da 0, 7 and 14 after 7 da of hypoxia, animals were injected iv with chyp polymer in liposomes (hc) or bioinactive trans-hyp polymer in liposomes (ht). air controls received thyp polymer in liposomes (at). at 0 and 21 da, we measured mean right ventricular pressure (rvp) and hydroxyproline (hyp) content in main pulmonary arteries. on da 0, rvp (mmhg) was 9 ϯ 1 and hyp (µg/vessel) was 88 ϯ 6 in at. rvp and hyp increased to 17 ϯ 1* and 139 ϯ 4*, respectively, in hypoxic animals (n ϭ 4; *p ͻ 0.05 vs. at). on da 21, rvps were at 10 ϯ 1, ht 24 ϯ 1*, hc 15 ϯ 1* †; hyps were at 91 ϯ 9, ht 176 ϯ 15*, hc 122 ϯ 1* † (n ϭ 4; *p ͻ 0.05 vs. at; †p ͻ 0.05 vs. ht). from da 0 to 21, rvp did not increase and hyp decreased in the hc group vs. ht. we conclude that weekly injections of polymeric chyp prevented progression of established hypoxic ph and reversed hyp accumulation. targeted delivery of antifibrotic polymers may prevent and reverse the progression of ph. (support: phs, barbara cornwall foundation). glucosinolates are amino acid-derived natural plant products found throughout the capparales order, which includes agriculturally important crops such as oilseed rape, brassica vegetables and the model plant arabidopsis. glucosinolates and their degradation products have a wide range of biological activities, e.g. in plant defense as deterrents against insect and fungi and as attractants to insects that are specialized feeders in brassicaceae. the conversion of amino acids to oximes is a key step in glucosinolate biosynthesis. we have recently shown that cytochromes p450 belonging to the cyp79 family catalyze the conversion of aliphatic, aromatic as well as indole amino acids to the corresponding oximes. cyp71e1 catalyzes the oxime-metabolizing step in the biosynthesis of the cyanogenic glucoside dhurrin. we have recently shown that the oxime-metabolizing enzyme in the glucosinolate biosynthetic pathway is a cytochrome p450 homologous to cyp71e1. the post-oxime enzymes in the glucosinolate pathway have high substrate-specificity for the functional groups, and low substrate-specificity for the side chain. therefore, we have been able to metabolically engineer new glucosinolate profiles into arabidopsis by altering the level of endogenous cyp79s and by introducing new cyp79s. the approach has great potential for design of "biotech crops" with improved pest resistance and increased nutritional value. hypercalcemia as a potential threat in the dietary treatment of maternal phenylketonuria f. eyskens 1 and s. beernaert 2 1 pediatrician, metabolic diseases and 2 dietitian, azm-koningin paola childrens hospital, metabolic lab pcma, antwerp, belgium over 90% of infants born to mothers with blood phenylalanine (phe) concentrations above 1200 µmol/l exhibit evi-dence of foetal dammage, low birth weight, microcephaly, dysmorphic facies, slow postnatal growth and development and long-term intellectual impairment. keeping maternal phe concentrations below 250 µmol/l before conception and throughout pregnancy reduces significantly the risk of abnormalities in the offspring of women with phenylketonuria (pku). we describe a woman, 31 years old, who showed phe blood levels of 150-200 µmol/l under a strict diet (total protein content of 1.13 g/kg body weight/day with 0.54 g/kg natural proteins and 0.59 g/kg proteins provided by the aminoacid mixture pku3 (milupa, germany); 1,655 cal/day) at the beginning of her first pregnancy. the first weeks she developed vomiting which gradually increased in severity. at 8 weeks of pregnancy, she had diarrhea, severe bouts of vomiting and manifested a deficient nutritional status with intake of 0.2 g/kg bw proteins and 1,178 cal/day. she was hospitalized to start refeeding using continue drip feeding administered by nasogastric tube. after 2 days on this regimen she developed vomiting, heart palpitations and mental confusion. her serum calcium level, that was normal at admission in the hospital, showed an elevation to 6.5-7 meq/l (ref. value 4.2-5.1 meq/l). the feeding was stopped immediately and under an intravenous infusion and gradually introducing a feeding composed of pku3, carbohydrates and mct fats the serum calcium and the blood phe levels dropped to normal values. and volatile components of caramel obtained by heating commercial maltose solution for different time intervals. one sample containing maltose only was used as control, the caramelization was conducted at 130c° for total time period 90 minutes and subjected to sensory analysis and isolation of volatile components. the odour and colour sensory tests were evaluated according to the international standard methods (iso). the results showed that addition of lysine as a catalyst gave rise to a significant (p ͻ 0.05) increase in intensity of the whole flavour in comparison with the control sample. the sweet and caramel notes, the most characteristic attributes of caramel, showed remarkable increase. on the other hand the increase in heating time in presence of lysine as a catalyst resulted in high significant increase in browning rate of caramel solution. the volatile components of each sample were isolated by using the new technique, solid phase microextraction (spme) and subjected to gc and gc-ms analysis. over 250 volatile components were separated, however only the most important component for caramel flavour were reported. maltol and 5hydroxymethyl-2-furfural (hmf) and 4. h-pyran-4-one,2,3dihydro-3,5-dihydroxy-6-methyl (dihydro dihydroxy maltol), the main characteristic caramelization products were present in high concentration in samples contaning lysine heated for 60 minutes. in addition one pyrazine was only identified in the samples contaning lysine. a comparative study between the present results and those of our previous study concerning addition of alanine as a catalyst was carried out. short-term exposure of human umbilical vein endothelial cells (huvecs) to hyperglycemia increases l-arginine transport (system y ϩ /cats) and nitric oxide (no) production (via enos). it has been reported that enos could also be activated by a ca 2ϩ -independent mechanism involving phosphorylation of ser 1177 by a phosphatidylinositol 3-kinase (pi3-kinase) dependent pathway. we investigated the involvement of pi3kinase on the stimulatory effect of acute hyperglycemia on enos and l-arginine transport in huvecs. l-arginine transport, no synthesis and phosphorylation of ser 1177 in enos were increased by d-glucose (25 mm, 2 min). similar results were obtained in huvecs exposed to insulin. incubation of cells with wortmannin (pi3-kinase inhibitor) prevented the effects of d-glucose and insulin. no changes in the intracellular ca 2ϩ and enos protein levels were detected. thus, acute hyperglycemia increases l-arginine transport and enos activity through a pi3-kinase dependent, ca 2ϩ independent mechanism in huvecs. [ the hypercalcemia in this patient was due to a very high content in calcium of the feeding administered (2-3 times the adh value) associated with a high vitamine d concentration (see table) and a clinical state of dehydratation. the further pregnancy was uncomplicated and a healthy girl was born who developed normal. • the aminoacid mixtures used in the treatment of pku contain a high level of calcium, phosphate, magnesium and iron. they also contain a high concentration of vitamine d. • nutritional monitoring of pregnant pku patients should include the calcium, phosphate, iron, zinc and vitamins status. • vitamins a and d suppletion is contraindicated in these patients based on the high concentrations of these vitamins in the aminoacid mixtures used in the dietary treatment. flavour and aroma chemistry department, national research centre, dokki, cairo, egypt caramelization of various carbohydrates leads to product with a high tinctorial strength provided by different additives catalyzing the process. the present study was conducted to evaluate the catalytic effect of lysine on the sensory attributes administered pku3 (80 g ϭ adh 1, 2 g/kg/bw) lipoic acid is a prosthetic group of h-protein of the glycine cleavage system and e2 components of the pyruvate, 2oxoglutarate and branched-chain 2-oxoacid dehydrogenase complexes. in mammals, attachment of lipoic acid to these proteins requires two enzymes. lipoate-activating enzyme (lae) catalyzes the activation of lipoate to lipoyl-nucleoside monophosphate. then, lipoyltransferase transfers the lipoyl moiety to the specific lysine residue of the proteins. we purified lae from bovine liver mitochondria. lae activated lipoate with gtp at a 1000-fold higher rate than with atp. the reaction absolutely required lipoate and mggtp, and the reaction product was lipoyl-gmp. lae activated both r-and senantiomers of lipoate to the respective lipoyl-gmp although preference for r-lipoate was observed. lipoyltransferase equally transferred both r-and s-lipoyl moiety from respective activated lipoate to apoh-protein. however, only h-protein carrying r-lipoate was active in the glycine cleavage reaction. cdna clones encoding a precursor lae with a mitochondrial presequence were isolated. amino acid sequence of lae was identical with that of xenobiotic-metabolizing/medium-chain fatty acid : coa ligase-iii, but an amino acid substitution due to snp was found. these results indicate that the medium-chain acyl-coa synthetase in mitochondria plays a novel function with gtp, the activation of lipoate. instituto di chimica biologica "g. fornaini", università di urbino, italy nitric oxide (no) can modulate red blood cells (rbc) glycolysis by translocation of the enzyme glyceraldehyde-3phosphate dehydrogenase (gapd) [e.c. 1.2.1.12] from the cytosolic domain of the membrane protein band 3 (cdb3) in the cytosol. in this study we have investigated which no-reactive thiols might be involved influencing gapd translocation, and which is the role of glutathione (gsh) in this context. two highly reactive cys residues (k 2 ϭ 73.7 m ϫ1 s ϫ1 and 101.5 m ϫ1 s ϫ1 , respectively) were identified by transnitrosylation with nitrosoglutathione (gsno) of cdb3 and gapd. the cys 149 in the catalytic site of gapd is exclusively involved in this gsno-induced nitrosylation. reassociation experiments carried out at equilibrium with preparations of rbc membranes and gapd revealed that different no-donors may form ϫsno on, and decrease the affinity between, gapd and cdb3. in intact rbc, both the no-donors 3-morpholino-sydnonimine (sin-1) and peroxynitrite (onoo ϫ ) significantly increased gapd activity in the cytosol and glycolysis measured as lactate production and energy charge levels. however, we obtained data suggesting that onoo ϫ is the main no-derivative able to cross the rbc membrane leading to gapd translocation and ϫsno formation. both in cell-free experiments and intact rbc, diamide (a thiol oxidant able to inhibit gapd activity) was observed to reverse the effect of sin-1 on gapd translocation. the results demonstrate that cdb3 and gapd contain reactive thiols that can be transnitrosylation mainly by means of gsno, these can ultimately influence gapd translocation/ activity and the glycolytic flux. abteilung für allgemein-viszeral-und gefässchirurgie, kliniken dr. erler gmbh, nürnberg, germany new surgical procedures like minimal-invasive-surgery brought many advantages for the surgical patient: less pain and shorter hospitalization. regarding nutrition, patients gets normal food on the ward still on the operation-day and need only saline-infusions overnight for fluid and electrolyte substitution but no hypocaloric parenteral nutrition. hypocaloric parenteral nutrition had been developped as a peripheral intravenous nutritional concept for patients with a normal body mass index over a period not longer than 4-5 days. multiple clinical studies showed that bowel movements increase earlier after an early postoperative enteral feeding which allows an earlier discharge of the patient. the result is a remarkable decrease of costs and an increase in patient benefit. still some years before surgeons preferred in visceral surgery parenteral nutrition over a period of 4-5 days under the opinion not to stress an anastomosis. this opinion changed in the last years under the aspect that about 1,000-1,500 ml of bile fluid, 1,000-1,500 ml pancreatic juice and 1,000-1,500 ml gastric juice per day are passing a small intestine anastomosis without any complications. concerning colon-anastomoses, the colon is preoperatively washed out, so it lasts until 5 days until defecation. multiple studies also showed a benefit for the patient regarding immunostimulation by early postoperative enteral feeding. conclusion: in our hospital with 244 surgical patients we recommend postoperatively either early normal enteral feeding or a high caloric parenteral nutrition if parenteral nutrition is needed for longer than 5 days. if artificial nutrition is necessary for more than 14 days we recommend enteral nutrition given by a tube or peg (percutaneous endoscopic gastrotomy). department of food technology, national research centre, dokki, cairo, egypt in the near east, "frekeh" has been known for many centuries as a stable food made from wheat. it is generally claimed that "frekeh" is better than wheat regarding its storage stability. the protein quality of parched immature durum wheat (frekeh) produced from 2 variety was evaluated. frekeh from four maturing levels during the dough stage of the seed development, were analyzed for approximate analysis. results showed that "frekeh" produced at the beginning of the dough stage was of better nutritional value than that produced at the following maturity levels, since the former was higher in protein, fat, minerals and crude fiber as well as in reducing sugar content. in addition, it was shown that these results confirm well with the sensory quality evaluation of the cooked product. further more, it was found that the cooking time was suitable to produce a "freqkeh" meal with high levels of acceptability. the observed decrease in protein content with increasing maturity level raised the question of how the protein quality of "frekeh" versus that of nature wheat grains varied. in this investigation, the amino acid of "frekeh" was determined. dietary treatment and carnitine supplementation has greatly improved long-term outcome of patients with ppa and (vitamin b12 unresponsive) mma. however, metabolic decompensation may be frequent and final outcome in most patients show various handicaps. to investigate the usefulness of measuring free carnitine and acylcarnitines in dried blood by tandem mass spectrometry, we investigated 9 patients with ppa and 5 with mma in a period of 8 months by weekly capillary blood punctures performed by the parents. age of the patients were from 0.5 until 18 years. clinical status at the time of blood drawing was evaluated by regular phone calls. free carnitine in all patients substituted by oral carnitine treatment (50-100 mg/kg/day bw) was normal. the parameter best reflecting clinical status was the c 3 /c 16 -acylcarnitine quotient. mean value in mma and ppa patients showed a range of 11.5-29.4 (normal 1.5 ϩ/ϫ 0.36, n ϭ 18), there was no difference between ppa and mma patients. individual mean values of the patients significantly increased when the patient was ranked higher in the clinical score system or during decompensation. since measurement of acylcarnitines in dried blood by tandem mass spectrometry is easy to perform, this method may be used for home monitoring of patients with mma and ppa. influence of acute treatment with 1,2,3,4tetrahydroisoquinoline on the levels of glutathione and reactive oxygen species, and on the enzymatic activity of γ-glutamyl transpeptidase in dopaminergic structures of rat brain e. lorenc-koci 1 , m. sokoĺowska 2 , m. zapaĺa 2 , and l. wĺodek 2 1 institute of pharmacology, polish academy of sciences, kraków, and 2 institute of medical biochemistry, collegium medicum, jagiellonian university, kraków, poland 1,2,3,4-tetrahydroisoquinoline (tiq) and its derivatives generated considerable interest as molecular species that may be implicated in the pathogenesis of parkinson's disease (pd). in pd, apart from the lack of dopamine in the striatum, a decreased concentration of glutathione (gsh) is found in the substantia nigra (sn). it is also known that gsh depletion potentiates the toxicity of mptp and 6-hydroxydopamine. however, there are no data available on the tiq influence on gsh metabolism. the aim of the present study was to exemine the effect of acute tiq administration on the levels of gsh and reactive oxygen species (ros), and on the enzymatic activity of γ-glutamyl transpeptidase (γ-gt) in dopaminergic structures of rat brain. the investigation was carried out 4 h after a single dose of tiq (100 mg/kg i.p.). at that time, a marked increase in the tissue gsh level and simultaneous significant inhibition of γ-gt were found in the structures studied. in tiq-treated rats, the production of ros was reduced in the sn, but it was markedly enhanced in the striatum. our results suggest that the increase in gsh level in dopaminergic structures stems from inhibition of γ-gt and refers to the extracellular pool of this peptide. apparently, the tiq-mediated alterations in the levels of gsh and ros may have some implications for the etiology of pd. tetrahydrobiopterin-responsive phenylalanine-hydroxylase deficiency with mutations distant from the tetrahydrobiopterin binding site z. lukacs 1 , r. steinfeld 1 , a. kohlschütter 1 , j. zschocke 2 , and k. ullrich 1 1 department of pediatrics, university of hamburg, and 2 university-children's hospital, heidelberg, germany phenylalanine hydroxylase (e.c. 1.14.16.1) catalyzes the hydroxylation of phenylalanine to tyrosine in the presence of oxygen and the cofactor (6r)-l-erythro-5,6,7,8tetrahydrobiopterin (bh 4 ). mutations in the phenylalanine hydroxylase gene may cause phenylketonuria or hyperphenylalaninemia. alternatively, disorders in bh 4metabolism also result in an increase in phenylalanine concentrations but simultaneously affect other bh 4 -dependent enzymes, consequently, causing a severe neurological disorder. recently, several patients with a phenylalanine-hydroxylase deficiency but with normal bh 4 -metabolism were reported who showed a significant decrease in blood phenylalanine concentrations upon treatment with bh 4 . indeed, two such patients in our hospital were also sensitive to daily oral doses of 5-10 mg bh 4 /kg. the subsequent molecular genetic analysis revealed that patient 1 was homozygous for the widespread mutation y414c and patient 2 was compound heterozygous for the mutations a104d and k320n. it is striking, that all mutations are located distant from the known bh 4 -binding site and thus, should not be associated with bh 4 -sensitivity. additionally, further patients who share the same genotype are not sensitive to bh 4 . therefore, it must be concluded that factors independent of the phenylalanine hydroxylase gene, like e.g. individual chaperone proteins, influence the three-dimensional structure of the enzyme and thereby, enhance enzymatic activity in the presence of elevated concentrations of bh 4 . retrotransposons are structurally similar to retroviral gag and pol which are required for their replication via reverse transcription, and seem to be an ancestral form of specialized retroviruses. reverse transcription of retrotransposons was assumed to occur in virus-like particles as well as in retroviruses. rna-packaging in this particles suggests a possibility of infection. presumably, the formation of functional virus-like particles requires the interaction of gypsy rna with a protein encoded by gypsy first open reading frame (orf1) or a product of its processing. the objective of this work was to study whether the protein by this frame can bind with nucleic acids similarly to retroviral gag-protein and how phosphorilation of that protein may influence to this interaction. then gypsy orf1 was cloned and expressed in escherichia coli, and its protein product was purified by ion-exchange chromatography on deae-cellulose and affinity chromatography on heparin-sepharose and tested electrophoretically. it was shown that recombinant protein bound with its own mrna and with dna. the affinity for ssdna bing higher than for dsdna. the binding constant was estimated with rna. the method utilizes the ability of nitrocellulose to bind proteins but not nucleic acids. binding of 50% gypsy rna was achieved with about 100 ng of the protein in 50 ml of the reaction mixture. the binding constant was 5&times; 108 m-1, which is consistent. the structure of the putative nucleic acid-binding domain suggests that the protein is more similar to the core proteins of spumaviruses of the family retroviridae that to those of other retroviruses. phosphorilation of gag-like protein encoded by first open reading frame of retrotrasposon gypsy (mdg4) affects to interaction with nucleic acid. tryptophan (trp) in humans is catabolized by several pathways leading to various metabolites of kynurenine and indolic compounds formation. a number of diseases are connected with abnormalities in its excretion, but relationship of cause and effect is usually unclear. we introduced a two-step procedure for the detection of defects in metabolism of trp: 1) tlc is employed when starting the investigation, 2) two hplc methods were proposed and used at the next step, when pathological findings are to be proved and the individual metabolites quantified. the first hplc procedure enables the assessment of tryptophan, indolylacry-loylglycine (iag) and other five indolic compounds. the second method is intended to the monitoring of kynurenine and seven of its catabolites. the same sep-pack pre-treated sample of plasma and urine is used for all methods. the reference values and the excretion pattern in some groups of patients (350 in total) were assessed. hepathopathy, gastrointestinal defects, myopathy and seizures with other neurological symptoms were the conditions connected with changes in the excretion of some metabolites of trp. significant decrease of iag excretion was found in burn patients early after the injury. urine analyses were performed at patient with hartnup disease and benign xanthurenic aciduria, inherited metabolic defects of trp. in other experiments, trp effect on the decarboxylation of other aromatic amino acids in the liver was investigated; only weak inhibition under physiological conditions was recognised. ( two hypothetical proteins of escherichia coli, ybbq and yhae, show high sequence similarity to d-threonine dehydrogenase from pseudomonas cruciviae ifo 12047. we cloned each gene encoding ybbq and yhae into e. coli jm109. both ybbq and yhae showed no d-threonine dehydrogenase activity and showed significant activities for d-serine in the presence of nad. ybbq and yhae were purified to homogeneity from the e. coli clones. ybbq consisted of two identical subunits with a molecular mass of 31 kda, whereas yhae was a tetramer (native molecular mass, 124 kda). ybbq showed the maximum activity at ph 11.0 for the oxidation of d-serine. whereas optimum ph of yhae was ph 10.5. they catalyzed oxidation of glycerate and 3-hydroxyisobutyrate. d-glycerate was the best substrate for both enzymes. both enzymes also catalyzed reduction of tartronate semialdehyde in the presence of nadh. at physiological ph, the rate of tartronate semialdehyde reduction was much higher than that of d-glycerate oxidation. the ybbq gene is in the operon of glyoxylate utilization and the yhae gene is in the operon for d-glucarate/galactarate utilization. these results suggest that both ybbq and yhae are dglycerate 3-dehydrogenases and function physiologically in conversion of tartronate semialdehyde into d-glycerate. a serine protease inhibitor model: synthesis and biology z. mucsi 1 , á. bódi 2 , l. gráf 2 , a. perczel 1 , a. patthy 3 , and g. orosz 4 1 department of organic chemistry, 2 biochemistry, eötvös university, budapest, 3 agricultural biotechnology centre, gödölló´, and 4 research group of peptide chemistry, hungarian academy of sciences, budapest, hungary sgci is structurally related to the pmpd-2 family of canonical serine protease inhibitors. in these peptides, there is a p1-p1ј position which is responsible for reversible binding to chymotrypsin. their structure is characterized by structural compactness: the molecule contains three -sheets and three disulfide bonds. in the sgci molecule the p1-p1ј corresponds to lys-leu bond, which is cleaved by chymotrypsin extremely slowly. the question arises why an excellent substrate behaves at the same time as inhibitor. it was assumed that the threedimensional structure of the molecule is responsible for the inhibitory activity. a model was designed to include all the known features of the inhibitor: the structurally necessary -sheet structure and the fragment containing the p1-p1ј environment. three model peptide were synthesized. two model peptides had no inhibiting effect and were cleaved by chymotrypsin. one of the cleavage points is the expected p1-p1ј position, while the other positions found to be chymotrypsin preferred positions after the first cleavage. the three-dimensional structures of the model peptides were mapped by nmr. on the basis of nmr structures obtained it has been shown that the cyclopeptide part is more flexible in the models than in sgci. the initial process in the reaction mechanism of a bisubstrate enzyme, rat mercaptopyruvate sulfurttransferase: inactivation study by using chloropyruvate n. nagahara 1 , t. nakagawa 2 , and m. minami 1 1 department of hygiene and public health, nippon medical school, sendagi bunkyo-ku, tokyo, japan 2 institute for organic chemistry, darmstadt university of technology, darmstadt, germany to investigate the reaction mechanism of a bisubstrate enzyme, rat mercaptopyruvate sulfurtransferase (ec 2.8.1.2, mst), inactivation kinetics with 3-chloropyruvate (chloropyruvate) was studied; each inactivation reaction was completed in a preincubation procedure. chloropyruvate is an analog of 3-mercaptopyruvate (mercaptopyruvate) and irreversibly inhibits mst. the inactivation depended on incubation time and the concentration of chloropyruvate and showed saturation kinetics. the plot for the logarithm of % activity remaining versus preincubation time showed pseudo-first-order. the kinact is 8.0 ϫ 10 ϫ2 min ϫ1 and k1 is 3.1 mm. these suggest that chloropyruvate serves as a mechanism-based inactivator. mercaptoethanol , so that chloropyruvate can approach cys247 via the donor substrate route and acceptor substrate one, and a ternary complex may be formed prior to the inactivation. these findings suggest that a donor substrate enters the catalytic cavity prior to an acceptor one in the initial process of the mst reaction: mst follows an ordered sequential mechanism. polyketides are natural products of bacteria, fungi, marine organisms and higher plants, many of which have clinical usage. actinorhodin (1) is an antibiotic produced by streptomyces coelicolor via an iterative type ii polyketide synthase (pks) system. this consists of a multi-enzyme complex with a single catalytic function for each enzyme. departments of 1 chemistry and 2 pediatric surgery, school of medicine, fujita health university, toyoake, japan it has been reported that, in rats with a single intoxication of α-naphthylisothiocyanate (anit), acute liver injury develops with enhanced lipid peroxidation and neutrophil infiltration in the liver tissue. melatonin functions as an antioxidant. melatonin is known to inhibit neutrophil infiltration into damaged liver tissues. therefore, we examined whether melatonin exerts a protective or preventive effect on anit-induced acute liver injury. male wistar rats received a single i.p. injection of anit (75 mg/kg) and oral administraton of melatonin (100 mg/kg) at 12 or 24 h after anit injection. animals administered with melatonin at 12 and 24 h after anit injection were sacrificed 24 and 48 h, respectively, after the injection. liver injury appeared 24 h after anit injection and developed at 48 h. melatonin administered at 12 h after anit injection prevented liver injury formation with attenuation of increases in hepatic lipid peroxide level and myeloperoxidase activity, an index of neutrophil infiltration. melatonin administered at 24 h after anit injection prevented liver injury development with attenuation of further increase in hepatic lipid peroxide level. thus, melatonin protects against and prevents anit-induced acute liver injury in rats possibly through its antioxidant action and/or its inhibitory action against neutrophil infiltration in the liver tissue. k. okamura-ikeda, s. katayama, k. fujiwara, and y. motokawa t-protein is a component of the glycine cleavage system and catalyzes the tetrahydrofolate-dependent reaction. mutation in human t-protein (ht) gene results in clinical nonketotic hyperglycinemia (nkh). eight point mutations have been identified so far in nkh patients with t-protein deficiency. to understand the structure and function of ht, the wild-type (wtht) and three mutant t-proteins (g47r, g269d and r320h) were expressed in escherichia coli with chaperons groel and groes which facilitated the recovery of the expressed proteins as a soluble form. levels of expression of these proteins were similar but the recovered soluble forms of mutants were about one-third of wtht. g47r showed comparable specific activity to wtht, whereas g269d and r320h mutants exhibited remarkable reduction in specific activity. since homoallelism for g269d mutation and heteroallelism for g47r and r320h mutation were identified in typical and atypical nkh, respectively, these results suggest that g269 and r320h mutations are highly deleterious in the aspects of not only protein folding and/or stability but also catalytsis. on the other hand, g47r mutation might affect mainly on the protein stability. detailed characterization of these mutants is now in progress. laboratory of animal nutrition and biochemistry, miyazaki university, miyazaki-shi, japan the minimal actinorhodin pks, shown in black below, consists of the ketosynthase (ks), chain length factor (clf) and acyl carrier protein (acp) and is the minimum set of enzymes required for polyketide production. we have investigated the stoichiometry of the ks-clf complex and the ks-clf:acp minimal system using three methods: 1. native gel electrophoresis. 2. cross-linking of proteins using dibromoacetone. 3. radical cross-linking of proteins. this new method has also been used with wild type s. coelicolor cell free extract with 10% ks-clf in order to elucidate which proteins are in close proximity to ks-clf during in vitro actinorhodin production. in ruminant animals, essential amino acids have never been completely established, because of the difficulty of its estimation due to the presence of microorganisms such as bacteria and protozoa in the first stomach called rumen. in our previous paper, histidine was shown to be the first limiting amino acid in the rumen contents when evaluated by chemical score. recently we have also reported that rumen microorganisms cannot synthesize histidine from histidinol. on the other hand, there have been some reports which showed that nitrogen balance of ruminants was not improved by supplementation of histidine to rumen microbial protein together with methionine, lysine and threonine which had been known to improve. based on these facts, we have a hypothesis that histidine may not be an essential amino acid for ruminants. in the present paper, we will report about the abilities of cattle liver and kidney to synthesize histidine from histidinol comparing with those of swine liver and kidney. the ability was demonstrated by examining the activities of histidinol dehydrogenase (crude enzyme) by means of direct measurement of an increase in histidine and decrease in histidinol. the amount of histidine produced from histidinol by the enzyme seemed sufficient for meeting the histidine requirement of cattle. the browning reaction is the sequence of events which begins with the reaction of amino group in amino acids, peptides or proteins with glycosidic hydroxyl group of sugars; the sequence terminates with the formation of brown nitrogenous compounds or melanoidines. this reaction gives rise to tremendous number of components such as volatile alcohols, ketones, aldehydes, esters, ethers and sulfur and nitrogen containing heterocycles in addition to nonvolatile amadori compounds and complex brown pigments of medium to high molecular weights. the present study was designed to choose a currently occurring system (aspartic acid -fructose) as a model system, since aspartic acid was found to be one of the most important amino acids in many kinds of food varieties. the reaction was done under controlled conditions of reactants ratios, temperature and time. the reaction mixtures were subjected to successive extractions with suitable solvents where the obtained corresponding flavour concentrates were thoroughly investigated. the results indicated different classes of compounds such as aldehydes, furans, alcohols and alkylated pyrazines varying in quantities depending on the reaction conditions. these products were also investigated concerning their toxicological effects. so, such products of nonenzymatic reactions showed different chemical and biological properties. purification and characterization p. piyarat 1 , s. nagata 2 , h. misono 2 , and k. packdibamrung 1 1 department of biochemistry, faculty of science, chulalongkorn university, bankok, thailand 2 department of bioresources science, faculty of agriculture, kochi university, nankoku, kochi, japan nad ϩ dependent alanine dehydrogenase was purified 100 fold to homogeneity from aeromonas hydrophila. molecular mass of 230,000 daltons was estimated for alanine dehydrogenase by sephadex g-200 chromatography. sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified en-zyme showed 1 polypeptide band with molecular mass of 40,000 daltons, indicating that the enzyme is hexamer. the enzyme is highly specific for alanine and nad ϩ . sulfhydryl group of the enzyme plays an important role in the catalysis. the enzyme retained its activity on heating at 55°c for 16 h. optimum ph for reductive amination and oxidative deamination were 8.0 and 10.5, respectively. the steady state kinetic studies including product inhibition on the enzyme reaction indicated that the oxidative deamination proceeds through a sequential ordered binary-ternary mechanism in which nad ϩ binds first to the enzyme followed by l-alanine and products are released in the order of pyruvate, ammonia and nadh, respectively. the k m values for nad ϩ , l-alanine, pyruvate, ammonia and nadh were 0.17, 20, 1.33, 77 and 0.25 mm, respectively. an elevation of apolipoprotein (apo) b-100 concentrations is a particular feature of several metabolic disorders, such as type 2 diabetes (t2d), impaired glucose tolerance (igt), and familial combined hyperlipidemia (fchl). to further understand the in vivo turnover of apolipoprotein b-100 of very low density lipoprotein subfractions (vldl 1 , svedberg units (s f ) 60-400 and vldl 2 , s f 20-60) kinetic studies were performed in subjects with t2d, igt, fchl, and healthy controls using a tracer of either l-[ring-13 c 6 ]-phenylalanine or l-[5,5,5-2 h 3 ]leucine. these studies showed direct hepatic vldl 1 apob-100 secretion to be increased in patients with t2d and igt when compared with controls. in contrast, patients with fchl showed a discrete increase in hepatic vldl 2 apob-100 secretion. in all patients vldl catabolism is not essentially impaired. vldl 1 apob-100 secretion is associated with plasma insulin and free fatty acid (ffa) concentrations, resp., whereas vldl 2 apob-100 secretion is correlated with plasma mevalonate and lathosterol levels. in conclusion, vldl overproduction is supposed to be completely responsible for higher triglyceride (tg) levels found in patients with t2d, igt, and fchl. vldl 1 overproduction seems to be regulated by tg and ffa substrate and appears to be an indicator of decreased insulin sensitivity. in contrast, vldl 2 overproduction is more likely to be regulated by the availability of cholesterol substrate. these data give further in vivo evidence that vldl 1 and vldl 2 secretion is regulated independently. arabidopsis resulted in enhanced production of cysteine and glutathione graduate school of pharmaceutical sciences, chiba university, chiba, japan serine acetyltransferase (satase) catalyzes the formation of o-acetyl-l-serine (oas) which is the key intermediate of cysteine biosynthesis. oas is not only a dominant limiting factor but recently suggested as a possible signal molecule for gene expression in cysteine biosynthesis. in has been shown that the activity of cytosolic satase from watermelon was feedback inhibited by l-cysteine. to enhance the ability of cysteine biosynthesis in plants and to reveal the role of oas in the regulation of sulfur assimilation, we made the point-mutated watermelon satase gene (satg277c) whose product was not inhibited by cysteine, and introduced satg227c into arabidopsis. the contents of oas, cysteine, and glutathione in transgenic arabidopsis were increased significantly as compared to the wild-type arabidopsis. we are currently dealing with the expression analysis of sulfur-related genes in transgenic arabidopsis accumulating oas due to the overexpression of satase. certain amino acids as source of specific branched chain fatty acids in fish sauce manufacture n. g. sanceda 1 , e. suzuki 2 , and t. kurata 1 1 institute of environmental science for human life, and 2 department of human biological studies, ochanomizu university, tokyo, japan the source of some branched volatile fatty acids (vfa) during the fermentation process in the manufacture of fish sauce was investigated. we previously reported that straight chain volatile acids seemed to have been derived from fish fats but unlikely for branched fatty acids which was believed to be derived from other sources. to clarify the source of branched volatile acids, specific amino acids, alanine, leucine, iso-leucine and valine were used in this study. these amino acids were first mixed with salt and added to fish. the fish mixtures were then aerobically and anaerobically incubated for one and a half months. results showed that addition of valine significantly increased the production of iso-butyric and iso-hexanoic acids and leucine increased that of iso-valeric in the aerobically fermented fish mixtures. a similar tendency was observed in the anaerobically fermented fish mixture except that an increase in the amount of iso-hexanoic acid was observed in the leucine added mixture, which was not observed in the aerobically fermented one. it seemed that specific branched volatile fatty acids were derived from certain amino acids. glutathione (gsh) is an important component of the cellular defense mechanisms that protect cells from oxidative injury. in the retina, the glial (müller) cells have been shown to synthesize and transport gsh, and thus are likely to be involved in regulating gsh levels. in the present study, we have characterized gsh transport system in a müller cell line using 35 s-gsh uptake. the results showed that gsh was taken up in a na ϩ -and concentration-dependent manner with a k m of 0.31 mm. moreover, cellular gsh had no effect on the rate of gsh uptake. in related studies, we found that oxidative stress induced the expression of γ-glutamylcysteine synthetase (gcs) subunits, and that gcs mrna levels were correlated with the degree of gsh depletion. because organic anion transporters (oatps) have been implicated in glutathione cotransport, we examined expression of oatp members using rt-pcr. we found that the müller cell line expressed transcripts for oatp1, oatp2 and oatp3. these studies indicate that the müller cell plays important role in gsh homeostasis in the retina. in the active site of human porphobilinogen synthase (ec 4.2.1.24, pbgs), two zinc ions are coordinated by cys 122 , cys 123 and cys 132 , and his 131 and cys 223 , respectively. the fomer zinc ion, closer to catalytic site lys 252 , plays an important role in catalysis. on the other hand, a role of the latter (distal) one has not been clarified. interestingly, in human hep3b cell, his 131 was replaced with arg (h131r). to elucidate the role of his 131 in catalysis, the kinetic properties of wild type and h131r mutant enzymes were studied. these cdnas were cloned by rt-pcr with total rna from human peripheral lymphocyte and hep3b cell, respectively. each cdna encoding pbgs with 3ј non-coding region was inserted into pet-22b(ϩ) vector and then the constract was transformed into e. coli strain bl21(de3). the cells were cultured in lb medium containing 50 mg/ml ampicillin and 10 µm zn ion for 3 h at 37°c. after addition of 1 mm isopropyl--d(ϫ)-thiogalactopyranoside, cells were further cultured for 24 h at 20°c. the highly purified pbgss were obtained by ultora centrifugion, fractionation with ammonium sulfate and column chromatographies with deaecellulose, hydroxylapatite and superdex 200, serially. we are now investigating molecular properties of these pbgss. agriculture and agri-food canada, lacombe research centre, lacombe, alberta, canada handling and management procedures such as capture and restraint can be significant stressors for recently domesticated animals such as elk (cervidae elaphus). the objective of the current study was to investigate the use of pre capture nutritional therapy in attenuating hpa response and improving animal welfare. fourty eight adult male elk stags ranging in age from 2-4 years and raised on pasture were used in the study with 23 as control and 25 as nutritionally treated. twenty four hours prior to capture the elk were offered either 1 kg of a cereal grain based dietary supplement or 1 kg of a cereal grain based nutritional therapy product containing specified amino acids (usa patent # 5505968). the amino acid content of the nutritional therapy product was minimally 0.5 g per 500 kg animal weight of ala, lys, phen, meth, thre, isoleu, val and tryp plus 15 g per 500 kg weight of leu and 40 g/500 kg weight of glut. the animals were subsequently captured and held in appropriate facilities designed to handle elk. saliva samples were collected on all animals immediately following capture and salivary cortisol was monitored by ria. animals offered the nutritional therapy product containing the amino acid mixture displayed lower cortisol levels (11.8 nmol/l) compared to the untreated controls (14.9 nmol/l; p ͻ 0.05). the data suggest that amino acid therapy can be used to attenuate hpa response to a stressor in captured elk. department of bioengeneering and technology, delhi, new delhi, india resistance to analogues of methionine by corynebacterium lilium results in the partial de-repression of methionine biosynthetic enzymes. the levels of enzymes involved in methionine biosynthesis also increased step-wise by successive endowing the resistant markers, resulting in the overproduction of methionine. moreover, the repressibilities of the enzymes were also reduced by the addition of methionine analogue resistance. analogue resistant mutants were developed by uv induced mutagenesis of corynebacterium lilium (wild type) strain. the single analogue (norleucine) resistant mutant c. lilium nl-87 produced 372 µg/ml methionine in shake flasks with methionine yield at 0.068 g methionine/g glucose and specific methionine production at 0.237 mg/g dcw, while double analogue (norleucine and triazole) resistant mutant c. lilium nt-33 produced 521 µg/ml methionine. a triple analogue (norleucine, triazole and ethionine) resistant mutant c. lilium nte-99 produced 1.848 g/l methionine. the methionine yield was 0.248 g methionine/g glucose and its specific productivity was 1.04 g methionine/g dcw. clinical biochemistry, laboratory 22, luxemburg, grand duchy of luxemburg blood plasma glucose level was compared on fast and 60 minutes after oral administration of 1200 mg of acetylcysteine. in the group of 49 healthy persons the plasma glucose level feel by 7.6% over the 60 minute period. in the 30 diabetics on the contrary, the plasma glucose level observed 60 minutes after administration of acetylcysteine was 11.8% higher than in blood plasma taken on fast. similar tests were carried out "in vitro" to interpret these different results. the control group consisted of 1 ml of distilled water ϩ0.2 ml 10% glucose ϩ0.2 ml god pad (boringer mannheim gmbh). in the acetylcysteine group the distilled water was replaced by 1 ml 0.01% solution of acetylcysteine. in the glucagon group the distilled water was replaced by 0.01% solution of glucagen hypokit novo nordisk. spectrometric determination was carried out after 60 minutes of incubation. a 27% diminution of glucose was observed in the acetylcysteine group in comparison with the control group. a 32.2% increase in glucose was observed in the glucagon group in relation to the controls. the results with healthy persons and the tests "in vitro" indicate that acetylcysteine lowers the level of glucose. but it elevates the level of glucose in the blood plasma of diabetics. it may be presumed that acetylcysteine modifies the insulinglucagon balance in favour of glucagon. the objective of this study was to fortify yogurt with three oilseed protein hydrolysates prepared from soybean (glycine max), sesame (sesanum indicum) and rice bran (oryza sativa) flours. hydrolysis was carried with two enzymes one of plant origin (papain) and the other of microbial origin (alcalase). a yogurt fortification experiment was then carried using the previous hydrolysates. the hydrolysates were added to yoghurt at 5, 10 and 15% levels of fortification and the fortified yoghurt was analyzed fresh, and after 7 and 15 days of consuming period. fortified yogurt was chemically examined for fermentation activity (ph values, acidity and proteolysis) as well as its organoleptic properties. results of this experiment indicate that the addition of soybean hydrolysates with papain (8.9 units/g) for 5 minutes (tb) and rice bran hydrolysates with alcalase (27.6 units/g) for 5 minutes (te) to yoghurt can ex-ceed 5-10%, while fortification with sesame hydrolysed with papain (8.9 units/g) for 30 minutes (td) and soybean hydrolysed with papain (8.9 units/g) for 30 minutes (tc) can not reach up to 15%. it is well known that dna is fragile to reactive oxygen intermediates (rois) damage. evidences that dna fragmentation and apoptosis occur in cardiomyopathies, in the failing heart and in cultured cells under hyperbaric oxygen (hbo) stress, demonstrated that oxygen free radicals also play a critical role in heart failure. as a consequence, myocardial cell survival depends on response to oxidative stress. experimental data obtained in vitro suggested that polyamines, by acting as rois scavengers, play a role in prevention of endonucleasemediated dna fragmentation and inhibition of alkylating agents-mediated damage, potentially exerting a protective role against rois damage. thus we studied polyamine metabolism and superoxide dismutase (sod) expression in an in vivo model of heart oxidative stress, such as rats subjected to hbo. four experimental groups were used: 1) controls; 2) rats subjected to hbo for 15 min once and immediately sacrificed; 3) rats treated as group 2 but for 3 consecutive days and immediately sacrificed; 4) rats treated as group 3 but sacrificed 24 h later (recovery). northern blot analyses showed that odc mrna accumulation increased immediately (paralleled by activity) in groups 2-4, while ssat mrna decreased remarkably, thus leading to higher polyamine concentration in rois-stressed hearts. contrariwise, sod mrna level decreased rapidly in groups 2-4. this suggests that hbo-induced compensatory mechanism in rat heart is based on specific and rapid boosting of polyamine concentration, caused by coordinate induction of biosynthesis and inhibition of catabolism, and not of enzymes known to metabolise rois such as sod. amino acids oxidation was greater in tumor-bearing rats muscle. leucine is an important ketogenic amino acid that proves energy to the skeletal muscle. leucine supplemented diet was used to analyze the effects produced by walker 256 growing in pregnant rats which were distributed into six groups. three groups received normal diet (18% protein): control (c), tumor-bearing (w), pair-fed rats (cp). three groups were fed with diet supplemented with 3% leucine (15% protein plus 3% leucine): pregnant fed with leucine (l), tumor-bearing with leucine (wl) and pair-fed with leucine (lp). after 21 days, the animals were submitted to intestinal perfusion to measure leucine, methionine and glucose absorption. leucine absorption increased in w and wl groups. glucose absorption reduced in tumor-bearing. in pregnancy with cancer, metabolic changes provided both reduced fetal and tumor development. tumor-bearing rats showed increase in methionine and leucine absorption, probably diverting this nutrients to tumor cells. glucose absorption reduced in w and wl. leucine supplemented diet group promoted high leucine absorption which could be used by neoplasic cells, and mainly by fetus and host. probably, the transamination of the branch long chain amino acid provided energy substract for the skeletal muscle, keeping the nitrogen offered to host carcass. ( undernutrition cause several changes as body weight loss, in biochemical parameters, even microscopic alteration in absorptive epithelium. this means the nutrients absorption process has been harmfully and consequently increase the damages caused by malnourished. knowing leucine is used as a ketonic and oxidative amino acid our main propose was to recovery the malnourished young rats with normal (rc) and leucine supplemented diet (rl, 3% of leucine) for 60 days. it was measured body, liver, and muscle weight, intestinal absorption of glucose, methionine and leucine, and body chemical composition. the body weight gain in rc and rl was higher than control group, suggesting that nutritional replacement for these groups could provided nutrients to support the body weight recovery, reaching as the same weight as the control. methionine and glucose absorption was reduced in malnourished group, but it was recovered (glucose, methionine and leucine) after nutritional replacement. leucine supplemented diet promoted a good recovery of carcass collagen nitrogen, keeping the carcass structural nitrogen. further studies are necessary to investigate this mechanism. [financial support: fapesp ( we diagnosed the very rare autosomal recessive disorder hyperprolinaemia type ii (deficiency of ∆ 1 -pyrroline-5carboxylate dehydrogenase, ec 1.5.1.12) in a girl aged 20 months presenting with seizures and encephalopathy. l-∆ 1pyrroline-5-carboxylic acid accumulates in this disorder and there is a 10-15-fold increase in plasma proline. surprisingly, she also had vitamin b 6 deficiency. this was an unrecognised association, which was not explained by her diet or medications. we hypothesised that pyridoxal phosphate (vitamin b 6 coenzyme) was de-activated by l-∆ 1 -pyrroline-5-carboxylic acid. with high resolution 1 h nuclear magnetic resonance spectroscopy and mass spectrometry, we have shown that these two compounds react at ph 7.4 and 37°c in vitro to form three novel adducts, which we characterised. they are products of a claisen condensation (or knoevenagel type of reaction) of the activated c4 carbon of the pyrroline ring with the aldehyde carbon of pyridoxal phosphate. if this previously unreported interaction occurs in vivo, pyrroline-5-carboxylic acid is a unique endogenous vitamin antagonist. preliminary observations show that pyrroline-5-carboxylic acid also condenses with other biologically important aldehydes and ketones. some of these reactions may contribute to the brain disturbances in hyperprolinaemia type ii. we have already identified adducts with acetoacetic acid in urine from our child, which is evidence that condensation can occur in vivo. the kidneys are characterized by a high activity of γglutamyl transpeptidase (γ-gt), as well as by a high cysteine level. the present paper was aimed to obtain information on how the activity of γ-gt and the levels of non-protein sulfhydryl compounds (npsh) changed with age in rat kidneys. simultaneously, protein-bound cysteine (pb-cys) and sulfane sulfur compounds were estimated. the kidneys were from following rats groups: young (3-month-old), middle-aged (19month-old) and old (31-month-old). the obtained results showed that the activity of γgt and npsh levels in the kidneys fell with age. at the same time, a significant increase in the level of protein-bound cysteine was observed. on the other hand, the content of sulfane sulfur compounds was elevated in the group of the oldest animals. these findings indicate that -due to disturbances in the γ-glutamyl cycle -the capacity for extracellular glutathione degradation and, in consequence, the availability of cysteine for intracellular gsh biosynthesis may be impaired. the increased pb-cys level indicates potentiation of the thiolation reaction, i.e. development of protein-mixed disulphides, cysteine, sulfane sulfur compounds, oxygen reactive species. national research centre, dokki, cairo, egypt in the past few years, many attempts have been made to prepare a synthetic insulin. the biological activity of insulin is known to be closely related to the c-terminal octapeptide fragment of its b-chain. this does not necessarily mean, however, that each of the amino acid residues of the octapeptide fragment is essential for its activity. it was found that b 23 gly and b 24 phe were present in all insulins so far obtained from various animal species indicating the significance of these two residues. it would therefore seem desirable to study the effect of each of these two amino acid residues or both on biological activity of the octapeptiede fragment of the b-chain. weitzel et al. found that the substitution of arginine b 22 with another amino acid resulted in a very large decrease in biological activity, which indicates that it participates in the action of insulin. also it was found that the aromatic amino acid residues (b 24 -b 25 ) participate in the action of insulin. a heptapeptide arg-phe-tyr-thr-pro-lys-ala-och 3 , corresponding to (b 22 -b 30 ) insulin des gly 23 -phe 24 , and an octapeptide arg-phe-phe-tyr-thr-pro-lys-ala-och 3 , des gly 23 were synthesized using the solid phase method. the c-termenal ends of both peptide were converted to methyl ester by transesterification cleavage from the resin. the side chain protecting groups were removed by hf. manual counter current distribution method was used for purification of the free peptides. the way to solve the evaluation of tyrosine containing peptide was studied. the free methyl ester peptides were administrated for insulin-like activity test by glucose metabolism in the rat fat cells technique in vitro. nitric oxide synthase inhibitors influence dynorphin immunoreactivity in the rat brain following hyperthermia p. alm 1 and h. s. sharma 2 1 department of pathology, university hospital, lund university, lund, and 2 laboratory of neuroanatomy, department of medical cell biology, biomedical centre, uppsala university, uppsala, sweden nitric oxide (no) is a free radical gas that influences neuronal communication in the central nervous system (cns). recent reports suggest that no can influence dynorphin neurotransmission in the normal brain as well as in several pathological states. previous reports from our laboratory show that the enzyme nitric oxide synthase (nos) responsible for no formation is upregulated in several brain regions following hyperthermia. the present investigation was carried out to find, whether hyperthermia can influence dynorphin immunoreactivity in the brain, and if so, whether inhibition of nitric oxide synthesis will alter its distribution in heat stressed rats. rats subjected to hyperthermia at 38°c for 4 h in a biological oxygen demand incubator (bod) resulted in marked redistribution of dynorphin immunoreactivity in several brain regions e.g., cerebral cortex, hippocampus, cerebellum and brain stem. pretreatment with two potent nos inhibitors, l-name (50 mg/kg, i.p.) and l-nmma (30 mg/kg, i.p.) 30 min before heat stress significantly altered the dynorphin immunoreactivity in the brain. these drugs alone however, did not influence the peptide expression in normal rats. the results suggest that (i) hyperthermia has the capacity to influence dynorphin immunoreactivity in the brain, and (ii) inhibition of nitric oxide synthase considerably influences the dynorphin immunoreaction in hyperthermia, not reported earlier. the functional changes induced by uncompetitive and competitive nmda antagonists, memantine, amantadine and mk-801, and cgp 40116, respectively, were studied in both saline-pretreated and mptp-pretreated c57 bl/6 mice. the nmda antagonists were administered acutely by themselves or in combinations of either: nmda antagonist plus subthreshold l-dopa dose or nmda antagonist plus suprathreshold l-dopa dose, to either the mptp-pretreated or the salinetreated mice. activity-enhancing or functional restorative effects of the nmda antagonists were variable with memantine and mk-801 distinguished from amantadine and cgp 40116. in the study of long-term effect of nmda antagonists mk-801 was administered postnatally and spontaneous motor behaviour and motor activity in response to several pharmacological interventions was assessed. marked alterations associated possible with apoptotic penchance are discussed. t. archer 1 and a. fredriksson 2 1 department of psychology, university of göteborg, and 2 department of psychiatry, university of uppsala, ulleråkers hospital, uppsala, sweden synergistic antiparkinsonian actions of different classes of putative therapeutic agents co-administered with a subthreshold dose of l-dopa (5 mg/kg) in drug-naive mptp-treated mice as well as the restorative actions of those compounds in suprathreshold l-dopa-tolerant mptp-treated mice subjected to "wearing-off" of l-dopa efficacy were assessed in a series of experiments. the classes of compounds studied included the noncompetitive nmda antagonists, memantine, amantadine and mk-801, the anticonvulsive and putative anticonvulsive agents, lamotrigine, fce 26743, phenytoin, the monoamine oxidase inhibitors, l-deprenyl, amiflamine, α-ethyltryptamine, clorgyline and phenelzine, and the α 2 -adrenoceptor agonists, clonidine and guanfacine. in this final case, the restorative effects of clonidine and guanfacine were antagonised by the α 2 -adrenoceptor antagonist, yohimbine, but not the α 1adrenoceptor antagonist, prazosin. within each class of potentially therapeutic agents a differential restorative efficacy was obtained, but the combination of different doses of apomorphine with clondine failed to restore motor activity. in vivo proton mr-spectroscopy of the human brain: assessment of n-acetylaspartate (naa) reduction as a marker for neurodegeneration w. block 1 , f. träber 1 , s. flacke 1 , f. jessen 2 , ch. pohl 3 , and h. h. schild 1 1 department of radiology, 2 department of psychiatry, and 3 department of neurology, university of bonn, germany proton magnetic resonance spectroscopy ( 1 h-mrs) is a well accepted non-invasive method to investigate changes in brain metabolite composition in different types of cerebral disease. we performed proton spectroscopy in patients with dementia of the alzheimer's type (ad) and in patients with motor neuron disease (mnd) with the aim to detect a specific metabolic pattern for each of these two neurodegenerative disorders. overall, more than 150 spectroscopic data sets of patients with mnd and more than 100 data sets of ad patients were acquired within the last 5 years. in the mnd group we found a significant reduction of naa/tcr metabolite ratios in the central region, which correlates with the disease severity and the clinical lateralisation of neurological symptoms and increases in the time course of the disease. in ad patients a similar reduction in relative naa contents was observed in the medial temporal lobe. the observed regional metabolic alterations correlate well with the characteristic neurological symptoms in ad (dementia) and mnd (muscular palsy) and seem to follow the disease process over time. since naa is exclusively expressed in neurons as shown by immunohistochemical studies, reduced naa levels suggest neuronal loss or dysfunction in the observed regions. center for molecular imaging research, massachusetts general hospital, boston, massachusetts, u.s.a. non-invasive measurement of hemodynamic parameters and imaging neovasculature architecture during angiogenesis is highly important in determining tumor prognosis and in assessing treatment efficacy. we suggested a technique to map the tumor vascular (vvf) and interstitial volume fraction (ivf) noninvasively in vivo. a poly-l-lysine based macromolecular probe (mpeg-pl-gddtpa) with extended circulation in the bloodstream designed to shield chelated paramagnetic lantanide with poly(ethylene glycol) chains. we hypothesized that a magnetic resonance signal after intravenous administration of a vascular paramagnetic probe can be maximized so the signal change after administration of a second comound (gddtpa) reflects the ivf but not the vvf. the method and its assumptions were verified in animal models of cancer. tumoral vvf and ivf values were consistent with histology data and literature values. imaging showed heterogeneity of both parameters at submillimeter pixel resolution. this technique was used for characterizing differential angiogenesis in human mammary adenocarcinoma lines as well as for imaging anti-angiogenic drug effects. anti-angiogenesis was induced using synthetic d-reverse peptides derived from thrombospondin-1. this study showed that peptide treatment results in slower brain tumor growth due to inhibition of de novo blood vessel formation and synergistic anti-proliferative effect on tumor cells. in conclusion, in vivo mr imaging can be used for non-invasive treatment assessment of novel antiangiogenic drugs. wallenberg neuroscience centre, lund university, lund, sweden we have recently found that 6-hydroxydopamine lesioned rats gradually develop dyskinetic-and dystonic-like movements upon repeated administration of a therapeutic dose of l-dopa. such movements simulate the time course of peak-dose dyskinesia in parkinson's disease. in this rat model, the severity of l-dopa-induced dyskinesia is strongly correlated with an upregulated expression of the prodynorphin gene in striatal neurons. using antisense technology and gel-shift assay analyses, we have addressed the role of transcription factors which may mediate this response. we have found that the camp response-element binding protein (creb) is essential in maintaining a basal expression of prodynorphin mrna in the intact striatum, but it is not required for l-dopa to induce the prodynorphin gene in dopamine-denervated striatal neurons. we have thus addressed the role of fos-and jun family tran-scription factors, and found very high levels of fosb-and jundlike proteins in the striata of dyskinetic animals. these proteins could bind to both ap1 and cre sites in the prodynorphin promoter. moreover, intrastriatal fosb knockdown could inhibit both the upregulation of prodynorphin gene expression and the development of dyskinesias under chronic l-dopa treatment. we propose that dimers of fosb-and jund-like proteins mediate abnormal changes in striatal gene expression which are linked to the development of l-dopa-induced dyskinesia. department of pharmacology, grünenthal gmbh r&d, aachen, germany glutamate plays important roles in both normal and pathophysiological nocieception. upon physiological conditions, glutamate release from primary afferents in the spinal cord activates largely ampa receptors. as those are ubiquitously involved in fast transmission in the cns, ampa antagonists have a broad side-effect profile. prolonged activation of nociceptors by tissue damage, inflammation or nerve injury evokes a long-lasting release of glutamate and neuropeptides, activating nmda receptors in the spinal cord. this mechanism appears to play a key role in pain chronification. the nmda receptor is, therefore, an important target for chronic pain treatment. both animal and human studies confirm the efficacy of nmda antagonists in chronic pain, however, clinically available compounds are weak or have unacceptable side-effects. glycine b antagonists and compounds selectively blocking nr2b-containing receptors appear to be safer, the reasons for this remain unclear. central side-effects could potentially be avoided by using nmda antagonists with restricted central access. peripheral nmda receptors (as well as some other subtypes of glurs) could be activated by glutamate released from the site of injury, thus contributing to peripheral hyperexcitability. some other subtypes of glurs can also contribute to peripheral sensitisation. of ionotropic glurs, kainate receptors appear important in inflammatory and neuropathic pain. they can be activated by high intensity stimulation of nociceptive afferents, and may act as autoreceptors controlling release of glutamate. group i metabotropic glurs are also present on primary afferents and on second order neurones in the spinal cord, and may play a similar role. antagonists of these subtypes of glurs are active in some models of chronic pain. specific upregulation of group ii metabotropic glurs in some pain-relevant structures could reflect a possible adaptive role of these inhibitory receptors under chronic pain conditions; their selective agonists also have a potential for the treatment of chronic pain. we have performed a series of studies of the distribution and function of mglur subtypes in the basal ganglia that suggest that members of this receptor family could serve as targets for novel therapeutic agents that would be effective in treatment of pd. for instance, two group ii mglurs (mglur4 and mglur7) are localized on presynaptic terminals of striatal neurons in the globus pallidus where they could reduce gaba release. furthermore, activation of group i mglurs results in a depolarization and increased cell firing of neurons in the subthalamic nucleus (stn) and projection neurons of the substantia nigra pars reticulata (snpr). interestingly, this effect is mediated by mglur1 in snpr projection neurons and mglur5 in stn neurons. finally, activation of group ii mglurs results in inhibition of glutamate release from stn terminals in the snpr. furthermore, selective agonists of group ii mglurs inhibit haloperidol-induced catalepsy in rats, suggesting an antiparkinsonian effect of these compounds. the rich distribution and diverse physiological roles of mglurs in basal ganglia raises the possibility that these receptors may provide targets for novel therapeutic agents that could be used for treatment of pd and related disorders. a. cupello 1 , m. parodi 2 , and m. balestrino 2 1 centro di neurofisiologia cerebrale, cnr, genova and 2 dipartimento di scienze neurologiche, university of genova, italy in vitro rat hippocampal slices are commonly used to study the effects of hypoxia in the central nervous system, because they allow to differentiate the effects of hypoxia in the brain from that of systemic (e.g., respiratory and cardiac) failure that may accompany hypoxia. we used electrophysiology to monitor and evaluate the damage caused by transient hypoxia to the nervous tissue. a few minutes after oxygen deprivation brain tissue suddenly depolarizes. this event, which is termed ìanoxic depolarizationî is accompanied by dramatic metabolic changes: transmembrane ionic gradients disappear (na ϩ enters, k ϩ exits the neurons), neurons swell, there is intra-and extra-cellular acidosis. this event is caused by functional inactivation of (na ϩ / k ϩ )atpase caused by decreased atp content, as it is suggested by the fact that it is mimicked by ouabain treatment. one of us has contributed to show that if this event is not promptly reversed by reoxygenation it causes irreversible damage, mainly by determining a massive entry of ca 2ϩ into neurons. pretreatment of tissue with creatine (1 mm or more) both increases neuronal energy store by increasing neuronal phosphocreatine and protects brain tissue from irreversible damage. in vivo increase in phosphocreatine has been shown using lower (0.5 mm) creatine concentration, injected directly into the lateral ventricle. a different type of hypoxia-induced damage is observed when hypoxia is of shorter duration. in this case upon reoxygenation one does not observe disappearance of evoked potentials but their increase. this phenomenon, originally described as ìpost-hypoxic hyperexcitabilityî has been later called ìanoxic long-term potentiation (ltp)î. we showed that this event can be prevented by inactivating the nuclear protein s-100. while this damage is milder than that induced by anoxic depolarization, it may explain stroke-induced epileptic fits. we are currently investigating what role, if any, pretreatment with creatine may have in preventing also this type of damage. cytoskeleton is subject to continuous modification to yield changes in cell shape and function of plasmamembrane proteins linked to the cytoskeleton. gelsolin (gsn) depolymerizes filamentous actin and thus causes dynamic uncoupling of membrane ion channels. we have studied alteration of neuronal ca 2ϩ influx by the absence of gsn and its pathophysiological consequences during cerebral ischemia. cytosolic ca 2ϩ concentrations were determined ratiometrically in synaptosomes preloaded with fura-2am. glutamate release from synaptosomes superfused with krebs' buffer was measured by hplc. transient focal cerebral ischemia was induced by 2 h occlusion of the middle cerebral artery (mca). in gsn deficient mouse brain cortical synaptosomes [ca 2ϩ ] i increase in response to k ϩ (30 mm) depolarization was 42% higher than in wild-type. ω-agatoxin iva 0.2 µm decreased ca 2ϩ -influx in neocortical wild-type synaptosomes by 53%, and abolished differences between gsnϩ/ϩ and ϫ/ϫ genotype. k ϩinduced release of glutamate in neocortical synaptosomes was 78% higher and lesion size after mca occlusion was 45% higher than in wild-type. it is concluded that presynaptic ca 2ϩ influx is increased in gsn deficient nerve terminals which, together with subsequently increased glutamate release, increases neuronal vulnerability. in vivo assessment of tissue alteration in cerebrovascular and neurodegenerative diseases s. flacke, w. block, f. träber, p. mürtz, h. urbach, and h. schild the combined used of perfusion imaging (pi) and molecular diffusion imaging (dwi) are opening a new window into the processes that occur during the first hours of ischemia. dwi detects early changes of proton diffusion associated with cytotoxic edema. pi has the potential to characterize the degree of regional hypoperfusion. mismatches between dwi and pi, i.e. hypoperfused areas with normal adc are considered potentially salvageable. we present results of 11 patients with an angiographically defined thrombembolus in the middle cerebral artery and a spontaneous stroke evolution. whereas the infarct core was clearly visible on both dwi and pi, tissue at risk of infarction could only be detected by an increased blood volume and transit time. however only in a subgroup of patients (n ϭ 3) these areas were incorporated into the final infarct. in these patients perfusion parameter of tissue at risk of infarction were more pronouncedly altered than in those where the tissue at risk was spared from infarction (ratios of tissue at risk vs normal (rcbv 2.17 ϯ 0.59, mtt 1.67 ϯ 0.22, ttp 1.32 ϯ 0.11, p ͻ .05). these human data show that a detailed analysis of diffusion/ perfusion mismatches allow the identification of tissue at risk of damage. glucose deprivation enhances the sensitivity of cerebellar granule cells to die by excitotoxicity. we have previously reported that neither 70 min of glucose deprivation, a treatment that depletes cell energy resources, nor exposure to 20 µm glutamate (glu) for 30 min, induce significant cell death in cerebellar granule cell cultures, 24 h after treatment. in contrast, the combined treatment with 20 µm glu and glucose deprivation induces both cell death and choline (cho) release. we investigated whether the neurotoxic effect of this treatment is related with inhibition of phosphatidylcholine (pc) synthesis. we found that exposure to 20 µm glu alone for 30 min, glucose deprivation for 70 min, and the combination of both treatments inhibited pc synthesis when measured at the end of treatment by 71%, 92% and 91%, respectively. furthermore, we found that exposure to either 20 µm glu, glucose deprivation or 20 µm glu ϩ glucose deprivation decreased incorporation of [ 3 h]cho into phosphocholine and increased the intracellular content of free [ 3 h]cho, indicating that all these treatments inhibit the synthesis of pc by inhibiting choline kinase activity. since only the combined treatment with 20 µm glu plus glucose deprivation evoked cho release and excitotoxic cell death, the present results indicate that other factors in addition to inhibition of pc synthesis are required to induce cho release and excitotoxic cell death in cerebellar granule cells. (supported by cicyt, saf98-0063.) the role of striatal metabotropic glutamate receptors in degeneration of dopamine neurons k. goĺembiowska, j. konieczny, k. ossowska, and institute of pharmacology, polish academy of sciences, kraków, poland the present study was undertaken to characterize the effect of blockade of the mglu 5 receptor subtype by 2-methyl-6-phenylethynylpyridine (mpep), as well as the effect of stimulation of the mglu 2/3 receptor by (ϫ)-2-oxa-4aminobicyclo[3.1.0]hexane-4,6-dicarboxylic acid (ly379268) on spontaneous and stimulated dopamine (da) release in rat striatum using an in vivo microdialysis. mpep (100-500 µm), perfused through a microdialysis probe affected neither the basal nor the veratridine (100 µm)-stimulated striatal da release. however, mpep given intraperitoneally (5 mg/kg) diminished either the basal or the veratridine-evoked da release. ly379268 (100-250 µm) administered locally also inhibited the veratridine-evoked da release in rat striatum. antagonists of mglur-i and agonists of mglur-ii have been shown to have neuroprotective properties in several models of neurotoxicity in animals. we have approached this issue using a selective mglu 5 antagonist in an animal model of neurotoxicity induced by methamphetamine. in our preliminary experiments, methamphetamine (5 ϫ 10 mg/kg sc every two hours) decreased the tissue content of striatal da and its metabolites dopac and hva.mpep (5 ϫ 5 mg/kg ip) given before every methamphetamine injection reversed its action. the effect exerted by the mglu 5 antagonist mpep seem to be mediated by sites located outside the striatum due to relieving da neurons of the facilitatory influence of glutamate. in turn, the attenuation of da release from nigrostriatal terminals by ly379269 may be a consequence of activation of striatal mglu 2/3 receptors. reversal of the methampetamine-induced da depletion suggests a potential for neuroprotective activity of mpep. o. golubnitschaja 1 , h. h. schild 1 , and j. flammer 2 1 department of radiology, university of bonn, germany 2 university eye clinic, basel, switzerland glaucoma remains a major eye illness with unknown etiology. although elevated intraocular pressure has been shown to be the major risk factor, there is a cohort of relatively young patients developing normal-tension glaucoma (ntg). assymptomatic ischemic events in brain have been shown to be often attributable to galucoma. perfusion of the retina and optic nerve head suffering from observed vasospastic dysfunction may be further reduced by changes in the intraocular pressure. ocular ischemia developed due to these blood flow deficits may play a major role in initiation of glaucoma. possibly secondary to ischemia the autoimmunogenic capacity is activated by ntg patients having an increased prevalence of systemic autoimmune diseases. therefore, the determination of potential molecular markers in blood lymphocytes could be useful for early diagnostics of ntg. our recent study using "gent hunting"-techniques showed indeed altered gene expression in lymphocytes of ntg patients. the demonstrated downregulation of xpgc gene expression which subsequently leads to the accumulation of damaged dna and an elevated p53 expression, together with the upregulation of a new abc-transporter seem to be specific for the pathogenesis of ntg. molecular imaging of ntg provides insights in mechanisms of disease initiation and allows the early diagnostics and preventive treatment. (supported by "bio-rad" and "amersham pharmacia aggregate cell cultures prepared from fetal rat telencephalon were used to study neuronal amino acid consumption during glucose restriction. to that end, both mixed (neuronglia) and neuron-enriched cultures were grown in chemically defined medium and tested at an advanced maturational stage. it was found that 6 h of exposure to reduced glucose (0.25 mm instead of 25 mm) caused significant increases in the consumption of several amino acids and the accumulation of ammonia. it also greatly changed the intracellular level of several amino acids in neurons, particularly of aspartate and glutamate. irreversible neuron-specific damage was observed one week after the insult. elevated glutamine media concentrations (4 mm instead of 0.25 mm) during glucose restriction further increased ammonia production and neuronal damage, although the overall rate of glutamine metabolism remained practically unchanged. taken together, our findings suggest that glucose deficiency caused (i) the dysfunction of crucial transamination pathways; (ii) a shift towards the oxidative deamination of glutamine and several other amino acids used by neurons as alternative energy substrates; and (iii) the accumulation of neurotoxic ammonia levels. institute for brain research, university of vienna, austria the racemic (d,l) mixture of the naturally occurring neutral aromatic amino acid 3,4-dihydroxy-l-phenylalanine (l-dopa) was first synthetized in 1911. in 1913, the natural levorotatory isomer was isolated from vicia faba beans and declared to be biologically inactive. however, in 1930 l-dopa was observed to lower the blood pressure in the rabbit, an effect opposite to the vasopressor effect of adrenaline. following the discovery, in 1938, of the enzyme l-dopa decarboxylase, ldopa's conversion in tissues (by decarboxylation) to dopamine (da), the first biologically active substance in the biosynthetic pathway of catecholamines, was demonstrated. subsequent pharmacological studies, done between 1942 and 1957, showed that the biological actions of l-dopa were, in principle, due to da formed from it in the body. in 1957, the central antireserpine effect of d,l-dopa was described in mice and confirmed in 1960 with l-dopa in humans. following the demonstration of da's occurrence in the brain in the years 1957/58, d,l-dopa was found (in rabbits) to restore brain da levels, reduced by reserpine. in 1960, the severe brain da deficit, confined to patients with parkinson's disease (pd) was reported and a year later l-dopa's superior anti-akinesia effect in patients with pd demonstrated. finally, in 1967 the high-dose oral l-dopa regimen was successfully introduced into clinical practise. in contrast to these supreme achievements, two related early studies remained, for different reasons, without consequence. despite some initial doubts about its mechanism of action, there is now convincing evidence for l-dopa therapy being a classic example of a central neurotransmitter replacement therapy, with the severe brain da deficit furnishing a rational basis for the amino acid's clinical use and high efficacy in patients suffering from pd. b. jakobsen 1 , a. tasker 2 , and j. zimmer 1 1 anatomy and neurobiology, sdu-odense university, odense, denmark 2 department of anatomy and physiology, university of prince edward island, canada the toxicity of domoic acid (dom) was studied in rat hippocampal slice cultures, prepared from 7-days old rats and grown on semipermeable membranes for 2-3 weeks before exposure. dom (0.1-100 µm) was added to the medium, alone or together with the glutamate receptor antagonists ns-102, nbqx or mk-801, for 48 hrs followed by 48 hrs in normal medium. neuronal degeneration was monitored and ec 50 values estimated by densitometric measurements of the cellular uptake of the propidium iodide (pi) at 24, 48, 72 and 96 hrs. the lowest ec 50 values, obtained at 72 hrs, were: ca1 (6 µm), dentate granule cells (dg) and ca3ab (10 µm),ca3c (12 µm). protective effects of 10 µm nbqx at 72 hrs were seen against 3 µm dom in dg, ca1 and ca3c and against 10 µm dom in ca1 and ca3c. 10 µm ns102 and mk801 only displayed protective effects together with nbqx. mk801 thus significantly increased the protective effect of 10 µm nbqx in ca1 against 10 µm dom in combination with 10 µm nbqx and 10 µm ns102. we can confirm that dom neurotoxicity primarily involves ampa/kainate receptors, but also nmda receptors at high concentrations (glutamate release). department of physiology/neuroscience, medical university of south carolina, charleston, south carolina, u.s.a. although dopamine has been most clearly tied to the development of addiction to drugs of abuse, recent studies indicate that once addiction has been established the expression of addictive behaviors, such as drug craving, is mediated more by long-term neuroadaptaions in glutamate transmission. data will be presented which supports and extends this hypothesis. repeated cocaine injections were given for one week and three weeks after the last drug injection a number of molecular, neurochemical and behavioral neuroadaptations were measured. it was found that in the nucleus accumbens there is a increase in the expression of genes encoding mglur2/3 and glur1, and a decrease in the expression of mglur5 and its accompanying scaffolding proteins homer1bc. this was accompanied by an increase in the capacity of mglur2/3 receptors to regulate presynaptic glutamate release and a blunting in the effects of stimulating mglur1/5 receptors. in addition, there is reduced activity in the cystine/glutamate exchanger 3 wks after repeated cocaine. as a result of these changes there is a decrease in the basal release of glutamate, and a relative increase in the releasibility of glutamate upon stimulation. by using the reinstatement model of drug seeking behavior, it was shown that glutamate transmission in the projection from the prelimbic cortex to the core of the nucleus accumbens was particularly affected by the cocaine-induced changes in gene expression. taken together, these findings support the use of glutamate autoreceptor agonists as possible therapeutic adjuvants in treating the cravings associated with addiction. dopamine (da), a catechol that autoxidizes to an oquinone, is implicated as an endogenous pro-toxin, however, the following studies suggest that da has dual neurodegenerative and neuroprotective roles. in rats treated as neonates with 6-hydroxydopamine (6-ohda; 134 :g icv), there was a 99% reduction in striatal tissue da content in adulthood, and a 3 to 5fold increase in spontaneous hydroxyl radical (ho*) formation (indirect salicylate trapping method: dihydroxybenzoic acid analysis). additionally, systemic l-dopa (60 mg/kg i.p.) suppressed ho* formation. however, when glutamate (50 mm) was added to an in vivo microdialysate, ho* formation was increased substantially more in the microdialysate from dainnervated striatum. these findings indicate that da innervation is inherently neuroprotective, but in the presence of a high level of an excitatory amino acid, da innervation predisposes to formation of reactive oxygen species. ongoing neuronal activity is likely to interact with and to determine the role of da as a neurotoxic or neuroprotective substance. (supported by ns 39272.) the glutamate hypothesis of schizophrenia along with the dopamine hypothesis was intensively discussed in the past. the last years however suggest more and more that neither a hypofunction of the glutamatergic system alone nor a hypofunction of the dopaminergic system alone is responsible for symptoms found in schizophrenia. the basal ganglia (bg) as the critical structures mediating symptoms of schizophrenia are innervated by dopaminergic fibers from the mesencephalon as well as by glutamatergic fibers from limbic structures; like prefrontal cortex, hippocampus, entorhinal cortex and amygdala. thus, limbic input is able to modulate information processing in each structure of the bg and by this way control dopaminergic functions through feedback mechanisms. dysfunction in limbic structrues may result in an imbalance of information processing via the bg and terminates in behavioral symptoms of schizophrenia. we showed in recent neurochemical studies in combination with behavioral analysis that a simple, generalized hypofunction of limbic glutamatergic input on bg nuclei is not the key mechanism inducing schizophrenic behavior. a dysfunction of a particular limbic structure or pathway seems to be responsible for an imbalanced information processing via the bg and imbalanced behavioral adaptation terminating in schizophrenic symptoms. [ there is a need to identify subtype-specific ligands for mglu receptors to elucidate the potential of these receptors for the treatment of nervous system disorders. to date, most mglur antagonists are amino acid-like compounds acting as competitive antagonists at the glutamate binding site located in the large extracellular n-terminal domain. we have investigated novel subtype-selective mglur5 antagonists which are structurally unrelated to competitive mglur ligands. using a series of chimeric receptors and point mutations we demonstrate that these antagonists interact with novel allosteric binding sites in the tm domain via a noncompetitive mechanism of action. recent studies in animal models implicate mglu 5 receptors as a potentially important therapeutic target particularly for the treatment of pain and anxiety. vascular endothelial growth factor (vegf) is a major mediator in angiogenesis and vascular permeability. in central nervous system (cns) vegf plays pivotal roles such e.g., inductor of endothelial cell proliferation, migration and inhibition of apoptosis, as well as mediator of blood brain barrier (bbb) breakdown and subsequently of brain edema formation. these ubiquitous epiphenomena are major complications in several cns pathologies, including head trauma and stroke. reduced tissue oxygen tension (hypoxia) and hypoglycaemia triggers vegf expression that occurs in ischemic regions around postraumatic or postinfarct necrosis. after brain injury, the expression of vegf is increased contributing to disruption of the bbb. vegf increases the permeability of bbb via the synthesis/release of nitric oxide and subsequent activation of soluble guanylate cyclase. the immunohistochemistry shows an increase of stained astrocytes around a cortical micronecrosis. vegf participates in the response of the cns to injury in a dose dependent way. immunostaining correlates with infarct volume and clinical disability. vegf-antagonists reduce ischemic brain edema and injury, involving vegf in pathogenesis and eventually in treatment of stroke and related disorders. this cytokine also exerts a neuroprotective effect mediated by its receptor flk-1. functions related to the inflammatory response, co-expression with proteins of the ecm and interaction with the two main receptors, flk-1 and flt-1, will be discussed. n-methyl-d-aspartate (nmda) receptors can mediate excitotoxic or neuroprotective responses. one of the molecular mechanisms responsible for nmda neuroprotection involves the release of brain-derived neurotrophic factor (bdnf) which in turn binds to and activates its cognate receptor trkb. bdnf levels in the neuronal culture medium increased 2-fold when cells were preincubated for three hours with nmda. at three hours, the increase in bdnf protein levels in the medium was accompanied by a concomitant increase in bdnf mrna. thus, nmda elicited two temporally distinct responses: an early release of bdnf protein followed by a later transcriptional activation of dbnf mrna and protein release. these results suggest that nmda activates the trkb receptor via a bdnf autocrine loop resulting in neuronal survival. in addition, extracellular regulated kinases (erk 1/2) were rapidly activated, which peaked within six hours of nmda treatment. erk 1/2 activation is completely blocked by mk-801 and partially blocked by k252a, suggesting the nmda and trkb receptors act in a coordinated fashion to activate erk 1/2. as an extension of this work, we discovered a single nucleotide polymorphism in the human nr1 gene that, when transfected into hek cells, alters the electrophysiological properties of the nmda receptor complex. possible consequences of this nmda receptor variant in signaling will be discussed. this overview summarizes our recent knowledge of the role that tyrosyl radical (tyro • ) can play in neurochemical systems of brain and thereby lead to neural disorders (pd, ad, als). these could involve the interactions of tyrosine and tyro • with reactive oxygen species (ros) and reactive nitrogen species (rns), via radical mechanisms and chain processes in the presence of o 2 and endogenous brain antioxidants. concentrations of tyro • , ros and rns can increase dramatically under conditions of generalized stress: oxidative, nitrative or reductive. this in turn can directly damage (by lipid peroxidation) or indirectly damage (by protein oxidation and/or nitration) cellular substructures which ultimately can lead to apoptotic neuronal cell death or autoschizis. enzymatically (classical peroxidase mechanisms) or non-enzymatically formed tyro • can react with no • and this reversible and intrinsic "combination" acts to "buffer' tyro • concentrations. the reaction of tyro • with superoxide (o 2 •ϫ ) is a scavenging reaction which proceeds rather by addition, not by electron transfer; and major resultant products are tyrosine hydroperoxides (tyrooh). however, the decay of tyro • can be also terminated by self-termination (dimerization) resulting in dityrosine (dt) formation. tyro • can catalyze ldl oxidation, although the precise mechanisms of this reaction in vivo remain unknown. nitration of tyrosine to 3-nitrotyrosine (3-nt) requires a one-electron oxidation as a primary step, with formation of tyro • , followed by addition of the nitrogen dioxide radical (no • 2 ). the promoting effect of carbon dioxide on peroxynitrite-mediated tyrosine nitration (via radical mechanisms) (tyro • /no • /o 2 •ϫ /no • 2 system) is due to the selective reactivity of the putative carbonate radical anion, as compared to that of the oxidizing hydroxyl radicals ( • oh). moreover, once formed, 3-nt may act to promote repetitive redox cycling; it may be reduced to the corresponding nitroanion radical, which is then oxidized by molecular o 2 to o 2 •ϫ and parent 3-nt. one-electron oxidation of 3-nt can result in catalytically active imminoxyl radical. dt formation can outcompete tyrosine nitration at lowsteady state concentrations of peroxynitrite. it is unquestionable that very high fluxes of no • and o 2 •ϫ are requisite intermediates of peroxynitrite, a tyrosine nitration agent formed via tyro • . evidence for the existence of generalized stress within neurons includes the presence of protein peroxides (tyrooh), dt, and 3-nt. the nitration/denitration processes can be pathologic, but these also may play: 1) a signal transduction role; 2) a role of "blocker" for radical-radical reactions (scavenging of no • , no • 2 and co 3 •ϫ by tyro • ); or 3) a role of delimiting factors for peroxynitrite formation. it is still unknown whether oxidation/nitration of tyrosine (as dopamine precursor or protein residue) via tyro • formation, is a footprint of generalized stress and neuronal disorders, an important part of o 2 •ϫ and no • metabolism, or just a part of integral processes for maintaining neuronal homeostasis. the complete answer of these questions should be the first priority task of our recent search, wherein the problem of increased free radical formation in the brain and/or the imbalance of ratios: ros/rns/tyro • may be all important in determining neural cell and tissue injuries under pathological conditions resulted from generalized stress. [acknowledgements. this work was supported in part by kbn ( more than 50% of patients with type 2 diabetes have coronary heart disease, related to silent ischemia, caused by an autonomic denervation of the heart in diabetic patients. oxidative damage to dna has been well documented in cardiac cells isolated from diabetic patients and rats with streptozotocin-induced diabetes mellitus (dm) . this dmmodel shows already seven days after onset of disease structural changes in vascular tissue typical for the development of atherosclerosis. this study evaluates possible molecular mechanisms for early events in the development of dm-induced cardiomyopathy. methods: using "expression array" we examined the activation of cardiac cell death in heart of dm-rats. ms-pcr was used to examine a differential dna methylation. results: an increased expression of genes encoding renin, angiotensinogen and p53 was detected in heart of dm-rats. substantial changes in the methylation status of the p53dependent p21 waf1/cip1 -gene and the cyclin d1-gene were detected in dm-rats. conclusions: the renin-angiotensin system is upregulated with diabetes, and this may contribute to the development of cardiomyopathy via oxidative damage and p53-dependent activation of cardiac cell death. this pathway includes de novo methylation of the p53-inducible p21 waf1/cip1 -gene encoding a protein which binds to and inhibits a broad range of cyclincyclin-dependent kinase complexes. (supported by "bio-rad" and "amersham pharmacia biotech") department of pharmaceutical biosciences, uppsala university, uppsala, sweden during the past decade studies have indicated that growth hormone (gh) may exert effects on the central nervous system (cns). for instance, gh replacement therapy was found to improve the psychological capabilities in adult gh deficient (ghd) patients. furthermore, beneficial effects of the hormone on certain functions, including memory, mental alertness, motivation and working capacity have been reported. likewise gh treatment of ghd children has been observed to produce significant improvement in many behavioural problems seen in these individuals. studies also indicated that gh therapy affects the cerebrospinal fluid (csf) levels of various hormones and neurotransmitters. further support that the cns is a target for gh emerges from observations indicating that the hormone may cross the blood-brain-barrier (bbb) and from studies confirming the presence of gh receptors in the brain. it was previously shown that specific binding sites for gh are present in discrete areas in the cns of both humans and rats. in peripheral tissues gh is shown to elicit its effects through a second mediator insulin-like growth factor 1 (igf-1). igf-1 is well recognized as a protective agent against neural injury in the cns. the neuroprotective effect of this peptide has a broad spectrum affecting many brain regions and acts through its antiapoptopic effect. the production of igf-1 is upregulated in areas of brain damage and the igf-1 system may be an important part of an endogenous neuroprotective system. in spinal cord injuries, however, the content of igf-1 is reduced. we recently observed a neuroprotective effect of topical application of igf-1 in animals subjected to spinal cord trauma. the observed effect may be mediated via a mechanism involving nitric oxide. in the same animal model we have very recently observed a neuroprotective effect of gh. recent reports suggest that the level of gh is drastically reduced in patients with spinal cord injury. in victims of spinal cord injury the secretion of gh and igf-1, as well, is known to be decreased. therefore, exogenous substitution of gh and igf-1 might be a promising approach in the future therapy of spinal cord injury victims. in fact, there is one report indicating that prolonged treatment with synthetic gh of spinal cord injured rats attenuates some of the neurological motor dysfunction seen in these animals 3 weeks following trauma. in our animal model we observed that topical application of rgh significantly reduced traumainduced disturbances in the fluid micro-environment. we also noted that gh was capable of attenuating the trauma-induced depression of spinal cord evoked potentials. the mechanism by which gh exerts it neuroprotective effects will be discussed. chronically administered levodopa in parkinson's disease (pd) treatment is ultimately associated with alterations in motor response. in 6-hydroxydopamine lesioned hemiparkinsonian rats, chronic twice-daily administration of levodopa progressively shortens duration of contralateral turning and augments the period of turning at or below 20% of peak turning rate. the pathogenesis of the response alterations involves in part sensitization of the corticostriatal glutamatergic synaptic activity. characteristic changes involving interactions between striatal kinase and phosphatase signaling now appear to contribute to sensitization of spiny-neuron glutamatergic receptors. glutamate-mediated striatal dysregulation, subsequently, modifies basal ganglia output system in ways that favor the appearance of parkinsonian motor response complications. at a molecular level, transcriptional activation of striatal creb contributes to the persistent expression of the levodopa-induced motor response alterations. conceivably, a safer and more effective therapy for all stages of pd can be provided by drugs that target intracellularly on striatal kinases or phosphotases, or by agents that interact extracellularly on non-dopaminergic striatal receptors such as ampa and nmda, adenosine a2, adrenergic a2, opiod, and serotonergic 2b. the primary cause of parkinson's disease is a loss of dopamine in the corpus striatum. it has been postulated that this effect leads to disinhibition of the striopallidal pathway and, secondarily, to a functional shift towards glutamatergic stimulation. the aim of the present study was to find out whether inhibition of glutamatergic transmission at a level of metabotropic glutamate receptors (mglurs) in the striatum may alleviate parkinsonian-like symptoms in rats. the non-competitive antagonist of receptor subtype 5 (mglur5), mpep (1.0-10 mg/kg ip), or the agonist of group ii mglurs, ly354740 (5-10 mg/kg ip), reduced the haloperidolinduced muscle rigidity and catalepsy. intrastriatal injections of the antagonist of mglur1, (rs) aida (7.5-15 µg/0.5 µl), but not of the agonist of group ii mglurs, 2r,4r-apdc (7.5-15 µg/0.5 µl), inhibited the muscle rigidity induced by haloperidol. in order to search for an influence of mglurs on the striopallidal pathway, the effect of mpep or of the agonist of group ii mglurs, dcg-iv, on the preproenkephalin mrna expression in the striatum was examined. the obtained results suggest that blockade of group i mglurs, or stimulation of group ii mglurs may be important to the amelioration of parkinsonian symptoms. striatal mglurs may contribute to at least some of these effects. several lines of evidence suggest an important role of glutamate in depression. the involvement of group i mglurs in depression has also been proposed. thus, we decided to evaluate whether group i mglurs antagonists have antidepressantlike effects. we also investigated if antidepressant treatment influences group i mglu receptors in the brain. the experiments were performed on male wistar rats (200-250 g) and male c57bl/6 mice (22-26 g). aida (group i mglurs antagonist) given i.v. in the dose of 50 µg, decreased the immobility time in the despair test in rats. mpep (noncompetitive, systemically active mglur5 antagonist) given i.p., was not effective in the despair test in rats. however, in doses of 1.0, 10 and 20 mg/kg, it significantly decreased the immobility time of mice in the tail suspension test. moreover, the deficit in passive-avoidance learning, which was observed in bulbectomized rats, was reversed by chronic, but not acute mpep (10 mg/kg) treatment. prolonged imipramine treatment resulted in significant increase of the level of expression of mglu5 receptors in the ca1 field of the hippocampus, while prolonged electroconvulsive shock treatment (ect) enhanced significantly the chemiluminescence of mglu5 receptors in the ca3 field. the results indicate that group i mglu receptors are modified by chronic antidepressant treatment and that group i metabotropic glutamate receptors antagonists may play a role in the therapy of depression. (this study was supported by kbn grant no. 4.po5a.091.17) institute of pharmacology, polish academy of sciences, krakow, poland chronic exposure to nicotine, alcohol, opioids, sedatives, and cannabis results in development of drug dependence that becomes evident upon a cessation of drug administration and expresses itself as a withdrawal syndrome (with its physiological and motivational manifestations). adaptations at the nmethyl-d-aspartate receptor (nmda-r) complex have been observed in different brain areas during chronic exposure to, and upon withdrawal from, opioids, ethanol, benzodiazepines and barbiturates. behavioral studies employ the assessment of the effects of nmda-r antagonists on: a) the development of dependence (nmda-r antagonists are co-administered with the drug), b) the maintenance of dependence (nmda-r antagonists are administered to animals with pre-established dependence, and -most relevant to the clinical situation -c) on the expression of drug dependence (assessment of the withdrawal severity in subjects with nmda-r antagonists administered just before the expected emergence of withdrawal). the development of dependence to opioids and benzodiazepines is significantly retarded by nmda-r antagonists. studies from this laboratory demonstrate similar inhibition by nmda-r antagonists of the maintenance of opioid dependence. both in rodents and humans, the expression of opioid antagonist-precipitated as well as spontaneous (natural) withdrawal is inhibited by nmda-r antagonists, and animal data demonstrate similar inhibition of the expression of dependence produced by ethanol, barbiturates and benzodiazepines. the involvement of the excitatory amino acid, glutamate and the inhibitiory amino acid, gamma-amino butyric acid (gaba) in the pathophysiology of spinal cord trauma is not known in details. this investigation is focused on the involve-ment of glutamate and gaba in a rat model of spinal cord injury using immunohistochemistry. spinal cord injury induced by an incision into the right dorsal horn of the t10-11 segments resulted in profound edema formation and cell damage in the adjacent t9 and t12 segments at 5 h. pretreatment with h-290/51 (50 mg/kg, p.o.), a potent antioxidant compound, effectively reduced the edema formation and cell injury following trauma. at this time period, untreated traumatised rats exhibited a marked increase in glutamate immunoreactivity and a distinct decrease in gaba immunostaining in the t9 and t12 segments compared to the control group. the changes in glutamate and gaba immunoreactivity in traumatised rats were considerably attenuated by pretreatment with h-290/51. these results suggest that (i) oxidative stress contributes to alterations in glutamate and gaba in spinal cord injury, (ii) glutamate and gaba are contributing to edema formation and cell damage and (iii) the antioxidant compound h-290/51 has a potential therapeutic value in the treatment of spinal cord injuries. dov pharmaceutical, inc., hackensack, new jersey, u.s.a. both preclinical (i.e., behavioral despair models) and clinical studies indicate that compounds reducing transmission at nmda receptors are antidepressant. conventional antidepressants may be viewed as "monoamine-based", increasing the synaptic availability of serotonin, norepinephrine, and/or dopamine. however, chronic administration of of conventional antidepressants alters both mrna levels encoding nmda receptor subunits and radioligand binding to this family of ligand-gated ion channels in circumscribed areas of the cns indicating that nmda receptors may be a downstream target of these monoamine-based agents. we have recently reported (li, et al., neuropharmacology, in press ) that a class of ampa receptor potentiators also exhibits antidepressant-like actions in preclinical models. in this presentation, i will describe how these two distinct, and (at a cellular level) seemingly diametric approaches employing glutamatergic mechanisms converge on intracellular targets that are also impacted by chronic treatment with biogenic amine-based agents. kainic acid is an essential pharmacological tool for many forms of neurobiological research. until several years ago, all commercially available kainic acid was derived from a single biological source (digenia simplex). commercial isolation of kainic acid in japan ceased in 1999, creating a void in the marketplace. recently several different companies have become providers of kainic acid, but each uses a different source of the compound (2 biological and 1 synthetic) and different isolation procedures. our objective was to use three common assay systems to evaluate the comparative pharmacological and neurotoxicological properties of these three sources of kainic acid. dose response curves, both alone and in the presence of receptor selective antagonists, were constructed for each kainate formulation using (a) cerebellar granule neurons in culture, (b) isolated hippocampal slice preparations, and (c) whole animal behavioural toxicity studies. preliminary results reveal many similarities, but also distinct differences between the three formulations, especially when challenged with antagonists for different eaa receptors. full results will be presented and discussed with respect to their implications for both extending the known kainite literature and for future studies employing kainic acid as a ligand in both mechanistic investigations and in animal models of neurodegenerative disease. our results from in vitro studies further elucidate the role of cell-cycle related proteins in neuronal apoptosis induced by excitotoxins. exposure of primary cerebellar neurons to toxic concentrations of glutamate was found to produce a significant, short lasting increase in the expression of p53 and cdc2. transcriptional activity of p53 was shown by increased p53 dna binding activity and by the concomitant induction of the cdk inhibitor p21, the cell cycle regulator gadd 45 and the apoptotic induced bax. cell-cycle proteins are also expressed concomitantly to dna damage in neurons undergoing excitotoxic degeneration. we found that excessive activation of glutamate receptor by nmda results in the formation of 8-oh-deoxyguanosine, which is a marker of oxidative dna damage. in addition, the expression of the dna repair factor msh2 increases in cultured cerebellar neurons or in ca3 pyramidal cells that have been challenged with excitotoxins. excitotoxicity may thus provide a further example of how re-expression of cell-cycle proteins might be tightly connected to dna damage and repair in neurons. rush-presbyterian-st. luke's medical center, chicago, u.s.a. patients with parkinson's disease by definition benefit from levodopa therapy. however, after 5 years of therapy 50% of patients experience motor response complications (mrc's): the benefit from each dose becomes shorter (wearing-off), more unpredictable (on-off) and associated with involuntary movements (dyskinesias). when dyskinesias first arise, they are associated with high levodopa levels and may be prevented or minimized by lowering levodopa intake. later on, the therapeutic window of levodopa narrows progressively and dyskinesias occur at doses equal to those needed to induce an antiparkinson effect. while the pathogenesis of motor complications remains incompletely understood, recent clinical studies implicate mechanisms downstream from the degenerating nigrostriatal dopamine system, possibly involving glutamatergic projections to the basal ganglia. in a rat model of pd, blockade of striatal glutamate receptors of the n-methyl-d-aspartate (nmda) subtype reverses levodopa-induced motor fluctuations. similarly, in 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (mptp) lesioned primates, several nmda-antagonists reduce levodopa-associated dyskinesias. in parkinsonian patients the nmda-antagonists dextromethorphan, dextrorphan and amantadine improve dyskinesias as well. these findings have lead to the suggestion that hyperfunction of nmda receptors on striatal efferent neurons, as a consequence of chronic non-physiologic dopaminergic stimulation, contributes to the pathogenesis of motor response complications. protein misfolding and aberrant polymerization are salient features of virtually all central neurodegenerative disorders, including alzheimer's disease (ad), parkinson's disease, polyglutamine diseases, tauopathies, and prion diseases. in many instances, a single amino acid change can predispose to disease by increasing the production and/or changing the biophysical properties of a specific protein. possible pathogenic similarities among the cerebral proteopathies suggest that therapeutic agents interfering with the proteopathic cascade might be effective against a wide spectrum of diseases. however, testing compounds preclinically will require diseaserelevant animal models. numerous transgenic mouse models of ad-like pathology have now been produced. our studies have found that tg2576 mice overexpressing human -amyloid precursor protein (huapp695k670n/m671l) produce copious deposits of diffuse and compact -amyloid as they age, and that females are more susceptible than are males (callahan et al., am. j. pathol. 158, 1173 -1177 , 2001 . recently, we also found that the overexpression of p25 protein, an activator of the kinase cdk5, results in tau hyperphosphorylation, axonopathy and severe motor deficits in transgenic mice, in the absence of neurofibrillary tangles. none of the existing transgenic models of -amyloidosis or tauopathy fully recapitulates the pathology of ad. in an attempt to more authentically model the human disease, we infused dilute ad-brain extracts into tg2576 mice at 3-months of age (i.e. 5-6 months prior to the usual onset ofamyloid deposition). we found that infusion of ad brain extracts results in: 1) earlier and more abundant deposition of -amyloid in app-transgenic mice (kane et al., j. neurosci. 20, 3606-3611); 2) evidence for the spread of pathology to other brain areas, possibly by neuronal transport mechanisms; and 3) tau hyperphosphorylation (but not neurofibrillary pathology) in axons passing through the injection site. the seeding ofamyloid by ad brain extracts suggests pathogenic similarities between -amyloidoses such as ad and other cerebral proteopathies, and could provide a new model for studying the proteopathic cascade and its neuronal consequences in neurodegenerative diseases. supported by warner-lambert/pfizer. purpose: the effects of essential amino acid deficiencies on function of cornea and lens were investigated. methods: dietary deficiencies of tryptophane and methionine were studied in young rats over 3 months. transparency of cornea and lens were evaluated using slitlamp microscope and scheimpflug camera. after sacrifice, lens fresh weight and crystallin patterns were determined to evaluate effects on lens growth and protein synthesis. results: methionine deficiency had no effect on the parameters investigated. tryptophane deficiency caused severe loss of body weight in both rat strains (brown-norway, bn; sprague-dawley, sd), sd rats also lost their hair. they developed corneal neovascularisations and cortical cataracts. bn rats developed faint neovascularisations and a discontinuity zone in the lens. diet intermission arrested pathological processes restarting when feeding diet again. this observation is supported by lens fresh weight data. dna staining evidenced that tryptophane deficiency arrested lens fiber maturation. conclusion: a difference has been found for 2 essential amino acids in their effects on transparency of cornea and lens. tryptophane deficiency stimulated corneal neovasculariseration, but arrested lens fiber cell maturation. the difference in reaction of cornea and lens to tryptophane deficiency between bn and sd rat eyes remains to be elucidated. dynorphin is a neuropeptide that is present in the dorsal horn of the spinal cord. the peptide is actively involved in pain processing pathways. however, its involvement in spinal cord injury is not well known. alteration in dynorphin immunoreactivity occurs following a focal trauma to the rat spinal cord. infusion of dynorphin into the intrathecal space of the cord results in ischemia, cell damage and abnormal motor function. antibodies to dynorphin when injected into the intrathecal space of the spinal cord following trauma improves motor recovery and reduces edema and cell changes. however, influence of dynorphin on trauma induced alteration in spinal cord bioelectrical activity is still not known. spinal cord evoked potentials (scep) are good indicator of spinal cord conduction that are altered following trauma. therefore, in present investigation, influence of dynorphin antibodies on trauma induced changes in scep was examined in our rat model. in addition, spinal cord edema formation and microvascular permeability disturbances were also investigated. our results show that intrathecal administration of dynorphin antiserum prior to injury has a beneficial effect on trauma induced electrical activity, microvascular permeability disturbances, and edema formation. these observations indicate that dynorphin is somehow involved in the altered bioelectrical activity of the spinal cord and participates in the pathophysiological processes leading to cell injury. fatty-acid binding proteins (fabps) are involved in the intracellular binding, targeting and transport of long-chain fatty acids (fas) to modulate cell growth and/or differentiation. fabp form a family of proteins displaying tissue-specific expression. the expression of brain type fabp (b-fabp) is spatially and temporally correlated with neuronal differentiation during brain development. heart type fabp (h-fabp) is widely distributed and present in skeletal muscles, kidney, lung, brain and endothelial cells. it is neuron-specific in postnatal brain and participates in neurite formation and synapse maturation. epidermal type fabp (e-fabp) is expressed at high levels during neurogenesis, neuronal migration, and terminal differentiation. although all three fabps could be involved in normal brain function in prenatal and postnatal life, a neurobiological role of fabps in neurodegenerative diseases has not been reported yet. these made us evaluate the protein levels of fabps in brains from patients with down syndrome (ds) and alzheimer's disease (ad) and fetal cerebral cortex with ds using two-dimensional (2-d) gel electrophoresis with subsequent matrix-assisted laser desorption ionization mass spectroscopy (maldi-ms) identification and specific software for quantification of proteins. in fetal brain, b-fabp and e-fabp levels were comparable between control and ds. in adult brain, b-fabp was significantly increased in occipital cortex of ds, and h-fabp was significantly decreased in ds (frontal, occipital, parietal cortices) and ad (frontal, temporal, occipital and parietal cortices). we conclude that aberrant expression of fabps, especially h-fabp in neurodegenerative diseases could be involved in impaired neurite outgrowth and synapse maturation. in our previous paper, it was shown that gaba-a receptor antagonist picrotoxin suppressed etoh (ethanol) selfadministration. recently, several authors indicated that systemic injection of dopamine or serotonine agonists reduced ethanol drinking in rats. therefore, in the present study we investigated the effects of thip (4,5,6,7-tetrahydroizokasazolo, 5,4-c pyridin-3-ol) gaba-a receptor agonist in naive and long-term ethanol-experienced rats on etoh selfadministration and on cardiovascular system. adult 13-17week-old male, normotensive wistar-kyoto (wky) and spontaneously hypertensive rats (shr) were used. naive rats were examined according to smith method. long-term ethanolexperienced rats were studied according to boyle method. thip was injected in naive rats at a dose of 8 and 16 mg/kg i.p. metabotropic glutamate receptors are coupled to phospholipase c stimulation and adenylyl cyclase inhibition through g-proteins. c6 glioma cells, that endogenously express the phospholipase c coupled metabotropic glutamate receptor type, were treated with different specific agonists of these receptors and the effect of these treatments on different components of metabotropic glutamate receptor pathway was studied by radioligand binding, phospholipase c activity and rt-pcr assays. agonists treatment caused a decrease in l-[ 3 h]glutamate binding to intact cells and membranes in a time dependent manner being maximum at 3-6 hours and recovered at 24-36 hours. this decrease was associated with a significant increase in the mrna level coding mglurs. no changes on g q/11 mrna level were detected in any case. however, a significant decrease in l-glutamate stimulated phospholipase c activity was detected after agonist treatments in both membranes and intact cells. this decrease was not associated to significant variations in mrna level coding phospholipase c 1 isoform. all these results suggest that agonist exposure causes a desensitisation of glial metabotropic glutamate receptor decreasing not only receptors number but its functionality. in this study the interaction between these two nuclei were investigated by means of microinjection and microdialysis techniques in sprague-dawley rats. steroetaxic surgery was performed by placing intracerebral parenchymal microinjection cannula into the right dmh and microdialysis probe into the left pvn. iliac artery was also cannulated to monitor the pulsatile blood pressure and heart rate by means of pressure transducer connected to a polygraph microinjection of 50 pmol nmda into the dmh was performed and microdialysis perfusates were collected simultaneously from the pvn in conscious rat model. γ-aminobutyric acid (gaba) and l-glutamic acid levels were analyzed by an isocratic hplc (high pressure liquid chromatography) method with the aid of a fluorescent detector. microinjection of 50 pmol nmda into dmh produced significant increases in mean arterial pressure and heart rate. nmda microinjection into the dmh produced significant increase in l-glutamic acid release in the pvn, but no significant change in gaba release was observed. these results suggest that stimulation of dmh by nmda results in subsequent stimulation of the pvn. [this study was sponsored by marmara university research foundation (project no: 1998/sag/38).] and in long-term ethanol-experienced rats only at a dose 16 mg/ kg i.p. control group (cg) received saline 3 ml/kg i.p. as can be seen in fig. 1 and table 1 the lower consumption of ethanol in shr in comparison to wky rats was observed. systemic injection of thip decreased dosedependently etoh intake in naive rats of both strains. this effect was more pronounced in shr (fig. 1) . similar phenomenon was observed after thip injection in long-term ethanolexperienced rats. there were no effect on systolic blood pressure and heart rate after thip treatment. born-bunge foundation, university of antwerp, and university of ghent, belgium increased neuronal excitability may underlie some of the neurological complications in uremic patients. in an effort to identify candidate neuroexcitatory compounds, 17 different uremic retention solutes, including several amino acids and amino acid derivatives, were applied to mouse spinal cord neurons in primary dissociated cell cultures. using the tight-seal whole-cell technique, a few of the candidate toxins were shown to evoke whole-cell currents in cells clamped at ϫ60 mv. in a first survey, each of the solutes was briefly applied in a concentration of 5 mm. significant inward whole-cell currents were evoked by guanidinosuccinate, spermine, and 3-indoxyl sulfate, whereas phenol evoked an outward current. further experiments indicated that guanidinosuccinate-evoked whole-cell currents were due to activation of nmda-type glutamate receptors in concentrations similar to those found in uremic patients. high (mm) concentrations of spermine activated voltage-gated calcium channels, whereas low (µm) concentrations were found to potentiate guanidinosuccinate-evoked currents through its action on the nmda receptor-associated polyamine binding site. whole-cell currents evoked by 3indoxyl sulfate or phenol seemed to be due to complex interaction with several different ion channels. we conclude that guanidinosuccinate-evoked nmda receptor activation, possi-bly potentiated by the neuroexcitatory effects of polyamines and other putative uremic neurotoxins, could be an important mechanism underlying the increased neuroexcitability in uremic brain. glutamine (gln) is one of the key metabolites in the cns (energy metabolite, precursor of neurotransmitter amino acids, end product of ammonia detoxication, osmolyte), and as such is a routine supplement of cns cell culture media. c6 glioma cells relatively easily adapt to culturing in a gln-deprived medium. the present study investigated the effects of gln deprivation on the characteristics of the different systems that mediate gln cell membrane transport in the cells. in contrast to a variety of cns and non-cns cells, the absence of gln did not derepress the methyl-amino-isobutyric acid (meaib)-sensitive ("system adependent") uptake. system asc became relatively more-, and system n less active than in cells grown in the presence of gln, but the ion -and substrate specificity of the uptake remained unaltered. system asc in c6 cells grown in a glnsupplemented medium shows two features distinct from most other cell types: a) strong ph sensitivity and b) partial tolerance of lithium substitution, pointing to domination of system asct2 -an asc variant strongly expressed in cultured astrocytes. cells grown in gln-deprived medium lost lithium tolerance, but not ph-dependence of the uptake, their properties thus resembling system glnt (sat1), a neuron-specific variant of system a. by contrast, transport of threonine, a standard asc system substrate, was not affected by gln deprivation and showed neither ph dependence nor lithium tolerance, which is typical of an asc in all the non-cns tissues. (supported by scsr grant no. 4 p05a 060 18.) the classical the hypothalamic-neurohypophysial system (hns) is comprised of neurons originating within the supraoptic nucleus (son) which project to the neurohypophysis to release the nonapeptides oxytocin (oxt) and vasopressin into the blood after appropriate stimulation. previous experiments have shown that a single social defeat experience triggers the release of oxt from somata and dendrites into the extracellular fluid of the son, but not from axon terminals in the neurohypophysis. to further investigate the regulatory mechanisms underlying this dissociated release, we exposed male wistar rats to a 30-min social defeat experience and monitored the release of the inhibitory amino acids gamma amino butyric acid (gaba) and taurine into the son using microdialysis. social defeat caused a significant increase of the intra-son pre-stress basal release). to reveal the physiological significance of the intrahypothalamically released gababicuculline, a specific gaba a -receptor antagonist -was administered into the son by retrodialysis. this treatment increased significantly the release of oxt both within the son (200%; p ͻ 0.05 vs. pre-stress basal release) and -as measured via chronically implanted jugular venous catheters -into blood under basal and stress conditions (up to 200%; p ͻ 0.01 vs. prestress basal release). however, bicuculline did not affect plasma vasopressin. these data demonstrate that gaba is released within the son during social defeat to act as an inhibitor of both, central and peripheral oxt secretion during emotional stress. the mechanism described here contributes to the regulatory capacity of the hns to ensure the appropriate involvement of oxt in the stress response of the animal (supported by dfg, en 366-21). down syndrome (ds) is the most common human chromosomal abnormality caused by an extra copy of chromosome 21 and characterized clinically by somatic anomalies, mental retardation and precocious dementia. the phenotype of ds is thought to result from overexpression of a gene or genes located on the triplicated chromosome or chromosome region. reports that challenge this notion, however, have been published. to add to this body of evidence, the expression ofamyloid precursor protein (app), ets-2 and collagen α1 (vi) chain precursor, encoded on chromosome 21, was investigated in fetal brain by western blot and two-dimensional electrophoresis (2-de). western blot detected app and ets-2 that migrated at ϳ75 and 50 kda, respectively. subsequent densitometric analysis of app and ets-2 immunoreactivity did not produce any significant change between controls and ds. since the metabolic fate of app determines the propensity of amyloid production, the expression of the secreted forms of app (sapp) had been examined. neither the expression of sappα nor sapp showed any detectable changes among the two groups. collagen α1 (vi) chain precursor, a protein resolved as a single spot on 2d gel was identified by matrix associated laser desorption ionization mass spectroscopy. quantitative analysis of this spot using the 2d image master software revealed a significant decrease in fetal ds (p ͻ 0.01) compared to controls. linear regression analysis did not show any correlation between protein levels and age. the current data suggest that overexpression per se can not fully explain the ds phenotype. apoptosis is the mechanism by which cells are programmed to die under a wide range of physiological and developmental stimuli. accumulating evidence indicates that enhanced apoptosis (programmed cell death) in down syndrome (ds) may play a role in mental retardation and precocious neurodegeneration of the alzheimer-type. in this regard, alteration of several apoptosis related proteins have been reported in adult ds brain. fetal ds neurons exhibited increased reactive oxygen species leading to early apoptosis, however, expression of apoptosis related proteins in fetal ds, has never been considered. to address this issue, we investigated the expression of proteins involved in apoptosis including fas (cd95, apo-1), caspase-3, bcl-2 and annexins in the cerebral cortex of control and ds fetal brain by western blot and two dimensional electrophoresis. here, we report that no detectable changes were obtained in fetal ds brain in the expression of fas, caspase-3, bcl-2 and annexins (i, ii, v, and vi) compared to controls. in parallel experiment, we also examined the expression of neuron specific enolase (nse), a neuronal marker found to be decreased in adult ds brain, to see if there is any neuronal loss and no difference was observed between the two groups. protein expression did not correlate with age. the unchanged levels of fas, bcl-2 and annexins together with unaltered caspase-3 expression, a predominant caspase that executes apoptosis in the developing nervous system, suggest that enhanced apoptosis may not be apparent in fetal ds brain as demonstrated for adult ds brain. introduction. among the various metabolites indicating neuronal damage, amino acids are regarded particularly important. detection of amino acids by microdialysis is currently introduced as a neuromonitoring tool in patient care. here, we present changes in the extracellular concentrations of various amino acids in stroke patients and in experimental stroke in cats. method. cat focal ischemia was produced by occlusion of the middle cerebral artery (mca) for 1 h followed by 2 h reperfusion. glutamate, aspartate, gaba, taurine, glycine, serine, glutamine, methionine, threonine, tyrosine, asparagine, valine, phenylanaine, isoleucine and leucine were sampled by microdialysis in the ischemic core and subsequently analyzed by hplc. human microdialysis was performed in patients with large mca infarction. the microdialysis probes were inserted into primarily non-infarcted tissue in the border zone of the ischemic territory. results. transmitter amino acids rose immediately after occlusion in the cat model. correspondingly, these substances increased sharply in the human brain, when the tissue around the probes became infarcted, as shown by positron emission tomography (pet) and ct scan. in contrast, structural amino acids did not show marked increases or even decreased during severe ischemia in both, experimental ischemia and stroke patients. these substances did increase, however, when the brain tissue was only slightly ischemic, i.e. after reperfusion of the cat brain, when brain swelling occurred, or in human brain, when tissue did not show any infarction in the ct scan but hypoperfusion in the pet image. conclusion. extracellular amino acids detected by microdialysis can serve as markers for secondary ischemia. severe ischemia is reflected by rapid increases of transmitter amino acids, due to various mechanisms including synaptic release and reversal of reuptake systems. oligemia seems to be reflected by slow increases of structural amino acids, possibly due to a reduction in cerebral protein synthesis. apoptosis has been implicated in the selective neuronal loss of down syndrome (ds). apoptosis activates a family of cysteine proteases with specificity for aspartic acid residues referred to as, caspases that play a key role in dismantling a cell committed to die. caspases activity is regulated by a variety of proteins that possess a domain resembling the prodomains of caspases. little is known, however, about the changes of caspases and their regulatory proteins in ds. here, we investigated levels of nine such different proteins by western blot technique in frontal cortex and cerebellum of control and ds subjects. the protein levels of dff45 (dna fragmentation factor 45), and flip (fadd like interleukin-1 -converting enzyme inhibitory proteins) were significantly decreased whereas that of rick (rip-like interacting clarp kinase) increased in both regions of ds. in contrast, cytochrome c, apaf-1 (apoptosis protease activating factor-1), procaspase-9 and arc (apoptosis repressor with caspase recruitment domain) were unchanged. procaspase-3 and -8 were significantly decreased in frontal cortex but no significant change was observed in cerebellum. regression analysis revealed no correlation between postmortem interval and levels of the investigated proteins. however, inconsistent correlation was found between age and levels of proteins as well as amongst the density of individual proteins. these findings demonstrate that dysregulation of apoptotic proteins does exist in ds brain and may underlie the neuropathology of ds. the study further suggests that apoptosis in ds may occur via the death receptor pathway independent of cytochrome c. hence, therapeutic strategies that target caspase activation may prove useful in combating neuronal loss in this disorder. in order to examine the differential roles of nitric oxide (no) induced by either endothelial no synthase (enos) or neuronal no synthase (nnos) after transient cerebral ischemia, we investigated the effects of the relatively selective cnos inhibitor, l-n 5 -(1-iminoethyl)ornithine (l-nio), the relatively selective nnos inhibitor, 7-nitroindazole (7-ni) and the no scavenger, 2-(4-carboxyphenyl)-4,4,5,5tetramethylimidazole-1-oxyl 3-oxide (ptio) on hippocampal dysfunction caused by cerebral ischemia. we measured no concentration, mean arterial blood pressure (mabp), hippocampal blood flow, direct current potential, ca1 population spike (ps) and release of amino acids from rat hippocampus after transient forebrain ischemia, which was induced by 4vessel occlusion for 10 min. l-nio (20 mg/kg), 7-ni (25 mg/kg) and ptio (1 mg/kg) were administered intraperitoncally 20 min before ischemia. ptio, 7-ni and l-nio reduced ischemiainduced no production in the hippocampus during the early period of reperfusion. the rank order of inhibitory potency was ptio ͼ 7-ni ͼ l-nio. l-nio, but not 7-ni, reduced hippocampal blood flow during ischemia and increased mabp before, during and after ischemia, compared with the vehicle group. ptio increased mabp during and after ischemia. ptio and 7-ni, but not l-nio, reduced amplitude of anoxic depolarization induced by ischemia. 7-ni recovered in part ps amplitude 60 min after ischemia. 7-ni, but not l-nio, reduced ischemiainduced release of aspartate and glutamate, but not taurine. the present study provides further evidence for the idea that in the early stages of transient forebrain ischemia, enos-derived no has a neuroprotective effect in the hippocampus, while nnos-derived no has a neurotoxic effect. the estrogen affects brain protein synthesis in ovariectomized female rats k. hayase 1 , m. tanaka 1 , k. tujioka 1 , e. hirano 1 , and h. yokogoshi 2 1 department of home economics, aichi university of education, kariya, aichi, and 2 laboratory of nutritional biochemistry, school of food and nutritional sciences, the university of shizuoka, japan the purpose of this study was to determine whether 17-estradiol affected the rate of brain protein synthesis in ovariectomized female rats. experiments were conducted on three groups of 12 wk old female rats: group 1. ovariectomized to reduce the level of plasma estradiol; group 2. ovariectomized and treated with estradiol; and group 3. sham-operated control. the fractional rates of protein synthesis in brain of ovariectomized rats treated with estradiol were significantly greater than in ovariectomized rats without estradiol treatment. in brain, the rna activity [g protein synthesized/(g rnaᮀd)] significantly correlated with the fractional rate of protein synthesis. the rna concentration (mg rna/g protein) was not related to the fractional rate of protein synthesis in any organ. the results suggest that estrogen treatment of ovariectomized female rats is likely to increase the rate of protein synthesis in the brain, and that rna activity is at least partly related to the fractional rate of brain protein synthesis. we have synthesized a series of new peptides that have demonstrated potent antidepressant activity in animal models for depression and in phase iia and iib clinical trials. mif-1 (prolyl-leucyl-glycinamide) an endogenous brain peptide has been reported to have some clinical activity in patients with unipolar depression with few apparent side effects. we have undertaken a study to determine the effect of molecular structural changes on the antidepressant activity of this peptide. we evaluated our new derivatives in a stress-induced animal model for depression, i.e. porsolt test, we have found that 4-f-phe-4-oh-pro-arg-gly-trp-nh 2 (inn 00835) is superior in all the statistical parameters used. in comparative testing inn 00835 was more active than prozac (fluoxetine) and zoloft (sertraline) in our antidepressant model. a u.s. patent has been granted on these compounds. the clinical results of inn 00835 show that it is effective in over 80% of depressed subjects when blood levels exceeded therapeutic threshold with no significant side effects. inn 00835 has a rapid onset of action, 3-5 days (vs. 2-6 weeks for currently marketed products) with sustained effects for months following 5 to 10 doses over 1-2 weeks. h. iwama 1 , 2 , a. umino 2 , a. hashimoto 2 , k. takahashi 2 , n. yamamoto 2 , and t. nishikawa 1,2 1 section of psychiatry and behavioral science, tokyo medical and dental university graduate school, and 2 department of mental disorder research, national institute of neuroscience, ncnp, tokyo, japan using an in vivo dialysis technique, we have studied the extracellular contents of endogenous free d-serine in comparison with that of l-serine, glycine and l-glutamate in the brain of the freely moving rat. a high amount of d-serine was detected in the dialysate obtained from the medial prefrontal cortex and striatum, whereas the cerebellar dialysate contained only a trace concentration of the d-amino acid. intra-medial prefrontal cortex perfusion of a sodium channel activator, veratrine, augmented the extracellular release of glycine and l-glutamate but a slight decrease in that of d-serine in a tetrodotoxin-sensitive manner in the prefrontal area. moreover, selective destruction of neuronal cell bodies in the medial frontal region by means of local infusion of an excitotoxin quinolinate resulted in a marked reduction of extracellular and tissue levels of d-serine in the infused prefrontal region. these findings suggest that endogenous d-serine might be liberated into the extracellular space from non-neuronal cells or certain exceptional neuronal cells probably by a carrier-mediated process in the mammalian prefrontal cortex. also, the endogenous d-amino acid has been indicated to be accumulated or synthesized, at least in part, in the neuronal cells. nucleoside diphosphate kinase (ndpk) catalyzes a transfer of the terminal phosphate from nucleoside triphosphates ((d)ntps) to nucleoside diphosphates ((d)ndps) and has been suggested to be involved in the regulation of wide variety of cellular functions. in addition, ndpk isoforms (a and b) are encoded by nm23 genes (h1 and h2) , which are related with the metastatic potential of some tumors. although ndpk/ nm23 has been also implicated to modulate neuronal cell proliferation, differentiation and neurite outgrowth, a neurobiological role of ndpk/nm23 in neurodegenerative diseases has not been reported yet. here we evaluated the protein levels of ndpk-a/nm23-h1 in brains from patients with down syndrome (ds) and alzheimer's disease (ad) using twodimensional (2-d) gel electrophoresis with subsequent matrixassisted laser desorption ionization mass spectroscopy (maldi-ms) identification and specific software for quantification of proteins. ndpk-a/nm23-h1 was significantly decreased in brain regions (frontal, occipital, parietal cortices) of both ds and ad compared to controls. we conclude that the down-regulated ndpk-a/nm23-h1 upon neurodegeneration could play a pivotal role in a wide range of neurobiological functions such as neurite outgrowth and consequently these could result in functional disturbance of the nervous system in ds and ad. brain α-endosulfine is manifold decreased in brains from patients with alzheimer's disease: a tentative marker? and drug target? α-endosulfine has the sulfonylurea-like ability to block atp-sensitive potassium (k atp ) channels and is expressed in a wide range of tissues. although the blockade of k atp channels has been reported to be involved in the release of neurotransmitters, the neurobiological role of α-endosulfine has not been studied yet. we examined the levels of αendosulfine protein in frontal cortex and cerebellum from patients with alzheimer's disease (ad). α-endosulfine was extremely decreased in both regions of ad compared to controls. this could result in the continuous opening of k atp channels with subsequent decrease of neurotransmitters release and change of potassium fluxes. this study is of great significance for providing a neurobiological function of α-endosulfine in brain and furthermore, α-endosulfine could serve as a useful marker for the diagnosis of ad and a target for drug treatment. children's hospital heidelberg, and department of pharmacology and toxicology, university of marburg, germany d-2-hydroxyglutaric aciduria is an inherited neurometabolic disorder of unknown etiology characterized by progressive neurodegeneration of vulnerable brain regions during infancy and early childhood, resulting in psychomotor retardation, hypotonia, seizures, macrocephaly, enlarged lateral ventricles, delayed cerebral maturation as frequent neurological presentation in affected children. the disease is biochemically characterized by the accumulation of d-2hydroxyglutarate (d-2), which is structurally similar to lglutamate (ϭ 2-amino-glutarate). we therefore investigated in primary neuronal cultures from chick and rat, whether d-2 induces excitotoxic neuronal damage. here we report that d-2 decreased cell viability in a concentration-and time-dependent fashion by disturbance of intracellular calcium homeostasis as determined by fura-2 measurement. furthermore, fluorescence microscopy using dihydrorhodamine-123 revealed an increased generation of reactive oxygen species (ros) elicited by expo-sure to d-2. n-methyl-d-aspartate (nmda) receptor blockade specifically prevented excitotoxic neuronal damage as well as increased calcium influx and ros production, suggesting that d-2 is an agonist at nmda receptors. patch-clamp investigation confirmed that d-2 activated recombinant nmda receptors in hek293 cells. furthermore, activity measurement of single respiratory chain complexes revealed a specific inhibition of complex v activity by d-2. we conclude that excitoxic mechanisms contribute to the neuropathology of d-2hydroxyglutaric aciduria, highlighting new neuroprotective strategies for this neurometabolic diseases. these studies were designed to determine the effects of aging and an aging intervention on nmda subunit expression. in situ hybridization and receptor autoradiography were performed on naïve or behaviorally-tested c57bl/6 mice of different ages (3, 10-15, and 26-30 months old) and diet groups (ad lib-fed and diet restricted). there were age-related decreases in both e2 and z1 mrna density in naïve, ad lib-fed mice. correlations were found between changes in e2 subunit mrna and agonist binding and z1 mrna expression and antagonist binding. diet restriction significantly improved learning ability, slightly spared glutamate binding to nmda sites and improved z1 mrna expression in older mice. significant correlations were found between agonist binding and both learning ability and e2 and e1 mrna density. learning ability in the old mice also correlated with the ratios of mrna expression for e1 and e2 and/or z1 subunits. these results suggest that changes in nmda receptor binding and the relationship between subunit expression levels are important for maintaining memory functions in older animals. extracts of st john's wort (hypericum perforatum l.) are widely prescribed for the treatment of mild to moderate depression and the putative antidepressant constituent is probably hyperforin. in this study the effect of hyperforin was investigated on the release of neurotransmitter amino acids. coronal cortical slices (400 mm) were cut and perfused with gassed (95% o2, 5% co2) acsf at 37°c. two-minute samples of perfusate were collected and aspartate and glutamate were assayed by hplc. potassium-and veratridine-stimulated release was elicited by administering 2 pulses of kϩ (60 mm) or veratridine (20 mm) 30 minutes apart. in control experiments the second kϩ pulse elicited glutamate release which was 80% of the first pulse. hyperforin (5 mm) perfused for 30 minutes prior to, and during, the second kϩ pulse significantly increased glutamate release to 170% (p ͻ 0.001, n ϭ 6-8). release elicited by the second veratridine pulse was 70% of the first pulse for both glutamate and aspartate. hyperforin (5 mm) increased this release to the second pulse to 160% and 130% respectively (p ͻ 0.001, n ϭ 6-8). when perfused on its own for 30 minutes, hyperforin (5 mm) increased the basal release of glutamate (p ͻ 0.001, n ϭ 4-5). in conclusion, the increase in the release of neurotransmitter amino acids observed following hyperforin is possibly mediated through a facilitatory action on voltage-operated ca2ϩ or naϩ channels. glaxosmithkline group, glaxo wellcome s.p.a., medicines research centre, verona, italy n-methyl-d-aspartate (nmda) receptors are ligand gated ion channels widely distributed in mammalian brain, which play a crucial role in important physiological mechanisms, such as excitatory transmission, neuronal migration and memory formation. a peculiar feature of nmda receptors is the absolute requirement of l-glutamic acid and glycine for the opening of the channel. noteworthy, these two aminoacids reciprocally modulate binding at their respective recognition sites. aim of this work was to study nmda receptor glycine/glutamate interactions in rat and human brain. binding of the nmda antagonist [ 3 h]cgp39653 to rat cerebral cortical membranes was inhibited by glycine. the overall effect of glycine consisted in a decrease of [ 3 h]cgp39653 affinity, with a parallel increase of the receptor affinity for glutamate. the glycine antagonist gv150526a competitively reversed glycine inhibition, proving that the modulation was via the glycine binding site. [ 3 h]cgp39653 binding to rat brain sections revealed the existence of regionally distinct nmda receptor subtypes with difference glycine/glutamate interactions. in most regions of the human brain nmda receptors presented the same allosteric modulation of [ 3 h]cgp39653 binding, as revealed by large section autoradiography technique. nevertheless, detection of any regional variation was not possible, probably due to the high intersubject variability. the effect of long-term high k ϩ -treatment on neuronal survival, cellular maturation, nmda receptor (nr) splice variant expression, and receptor function was investigated in primary cultures of rat cortical neurones. long-term incubation (up to 15 days) with 25 mm k ϩ significantly increased neuronal survival and induced multiple morphological changes associated with promoted cellular maturation. cultures grown in medium containing 25 mm k ϩ also exhibited multiple changes in nr1 splice variant expression according to rt-pcr studies performed with primer pairs flanking the alternatively spliced regions, in order to estimate the ratios of the corresponding 3ј and 5ј splice variants. nr1-1 and nr1-3 (each containing exon 21) were decreased, whereas nr1-2 and nr1-4 (each lacking exon 21) were increased, accordingly. the predominant expression of nr1-b was further increased. after administration of ttx, each of the k ϩ -induced changes on mrna expression was virtually abolished. in voltage-clamp recordings (holding potential: ϫ70 mv), nmda induced inward currents in a concentration-dependent manner with a maximum effect of ϫ745 pa under control conditions. neurones treated with 25 mm k ϩ showed a significantly diminished response to nmda (max. response: ϫ368 pa). in conclusion, the present data indicate that a sustained increase in neuronal activity induces adaptive changes in nr1 splice variant expression and a decrease in receptor function. thus, alternative splicing associated with a diminished receptorcytoskeletal linkage may be important compensatory mechanism in preventing cellular damage due to long-term activation of excitatory nr. it seems conceivable that this mechanism contributes to the promoting effects of 25 mm k ϩ on neuronal survival and maturation. (supported by bmbf grant 01gg9818/0) there is accumulating reports that kainate-induced seizures elicit expression of various heat-shock proteins (hsps) in the brain, such as hsp27, hsp32, and hsp70. however, no investigation has been carried out on changes in level of apg-2, a member of hsp 110 family, after excitatory amino acid-induced seizures. by means of an immunoblot assay, we determined the levels of hsp70 and apg-2 in discrete brain structures of mice after a single intraperitoneal injection of kainate or nmda. apg-2 level was significantly decreased in the frontal cortex, hippocampus, and striatum 3 days after kainate administration, while hsp70 level was increased in these regions following the administration. decreased level of apg-2 returned to the control levels in the three regions 10 days after kainate administration. no significant changes were observed in levels of both hsp70 and apg-2 in hypothalamus, midbrain, medulla-pons, and cerebellum of kainate-treated mice. by contrast, nmda administration did not significantly affect both levels in any of the regions examined. these results suggest that, unlike the case of hsp70, apg-2 expression could be temporarily down regulated by signals peculiar to kainate, but not by those peculiar to nmda, in murine telencephalon. high concentrations of glucagon-like peptide-1 (7-36) amide (glp-1) and its specific receptor (glp-1r) have been found in the rat hypothalamus. in this study the actions of glp-1 and its related peptides, exendin-4 (glp-1r agonist), exendin (9-39) (glp-1r antagonist) and glp-1(9-36)amide (major glp-1 metabolite) on levels of amino acids (glu, asp, gln, gly, tyr, trp, gaba) in the hypothalamus were investigated. 125 i-glp-1 binding in rat hypothalamic membranes was competed by the peptides in the following order of potency; glp-1 ͼ exending-4 ͼ exendin (9-39) ͼ glp-1(9-36)amide. intracerebroventricular (icv) glp-1 (4 nmoles) produced a statistically significant reduction in levels of all measured amino acids compared with saline injected controls, whereas 4 nmoles of exendin (9-39) was ineffective. exendin-4 produced a statistically significant reduction in the levels of trp, glu, and tyr. glp-1(9-36)amide showed a statistically significant increase in the level all the amino acids tested in this study. prior administration of exendin (9-39) or glp-1(9-36)amide blocked the effects of glp-1 on the levels of the amino acids. these data are consistent with exendin-4 being a glp-1r agonist and exendin (9-39) being a specific glp-1r antagonist. glp-1(9-36)amide, a primary metabolite of glp-1, appears to act as an endogenous antagonist at the glp-1r. department of biophysics, instituto de fisiología celular, universidad nacional autónoma de méxico, méxico city, méxico cholecystokinin (cck), a family of neuropeptides, seems to be involved in anxiety. evidence from several laboratories indicates that the ansiogenic effects of cck are mediated by cck b receptors. however it has been reported that cck a receptors have been found in brain and cck a receptor antagonists have ansiolytic properties. the aim of this work was to study whether or not cck a receptors are also involved in the modulation of anxiety. male rats were cannulated in the lateral ventricle and cck (9 fmol) and/or cck antagonists (900 fmol) were injected 7 days after surgery. anxiety was evaluated in the elevated plus-maze test and locomotion in an open-field test. ansiogenic effects were observed when cck b receptor agonists (cck8ns; cck4) or a mixed cck b and cck a receptor agonist (cck8s) were injected. in contrast, cck33, a cck a receptor agonist or cck 1-21 and cck 26-29 were uneffective. furthermore, the ansiogenic effects of cck8s were prevented by the previous (15 min) administration of l365,260 (cck b receptor antagonist) but not by devazepide (cck a receptor antagonist). no effects on locomotion were observed in any condition. these results indicate that cck a receptors are not involved in anxiety, as measured by the elevated plus-maze test. congenital conditions (i.e. neural tube defects: ntg) have a multifactorial aetiology. deficiencies in the folate and transsulfuration pathways have, in recent years, been positively linked to ntd and other dysmorhogenic syndromes. efficient one-carbon metabolism is crucial for the synthesis of dna precursors, the remethylation of homocysteine and biomethylation of dna. more than 80% of the one-carbon units that flow through the metabolic system in mammals and birds are derived from l-serine and glycine, the natural substrates for shmt. the mitochondrial glycine cleavage enzyme system (gces) can potentially compete with shmt for tetrahydrofolate (thf) in the generation of the methylenetetrahydrofolate pool. valproate (depakene, epilim), an anti-epileptic agent, appears to be strongly associated with hyperhomocysteinemia, several other induced metabolic conditions, the inhibition of the gces and an increased incidence of ntd in epileptic women of child-bearing age. the exact mechanisms of valproate-induced ntd are not yet clear. we investigated the association of the teratogenic properties of valproate with the inhibition of shmt and/or the gces in developing embryos. chicken embryos were treated with sodium valproate (vpa) and pregnant female mice (c57bl) received intraperitoneal injections of vpa, during the critical period of embryonic neural tube development. control embryos were treated with sterilised saline solution. harvested embryos were subsequently investigated for congenital abnormalities and hepatic shmt and gces activities quantified with radiometric assays. the effect of vpa on hepatic dna synthesis was monitored ( 3 h-thymidine incorporation into embryonic dna) and the dna-methylation status determined (dna n 5 -methylcytosine levels). dose-responsive incidences of ntd were observed in vpa treated embryos. very few defects occurred in control embryos. shmt and gces appeared to be inhibited in liver extracts of vpa-treated embryos. hepatic dna synthesis was significantly compromised and 5-mc levels were altered in vpa-treated embryos. the inhibition of either shmt and/or gces activities appeared to be associated with valproateinduced ntd in the chicken and mouse embryo models. the primary mechanism of this effect can probably be ascribed to a restriction in the flow of one-carbon units through the metabolic system, decreased synthesis of dna precursors and alterations in the methylation status of dna. department of neuroscience, university of cagliari, italy ethanol is long known to cause dose-related biphasic effects and we recently found that ethanol bidirectionally affects also working memory. the euphoriant and excitatory effects produced at low doses are associated with the rewarding action of ethanol and are thought to be mediated by the activation of the mesolimbic dopamine (da) system. however, ethanol monophasically stimulates mesolimbic da release in the nucleus accumbens, even at doses that cause hypnosis and coma. in contrast, ethanol biphasically modulates mesocortical da release in the prefrontal cortex (pfc). the changes in da release induced by ethanol are time locked with corresponding changes in extracellular glutamate levels. these biphasic effects of ethanol on pfc da and glutamate are matched by biphasic changes in the performance in a spatial delayed alternation task -a working memory test that is sensitive to proper function of the pfc -suggesting a link between da and glutamate transmission in the cognitive effects of ethanol. focal application in the pfc of the competitive ampa/kainate receptor antagonist cnqx suppresses both da release and the improvement of working memory induced by low doses of ethanol. these results suggest that ethanol may increase da transmission in the pfc and enhance working memory functions by increasing the release of glutamate, thereby stimulating non-nmda glutamate receptors. the enhancing effect on working memory by low, excitatory doses of ethanol may be perceived as rewarding and could constitute an important neurobiological mechanism for excessive ethanol drinking. physiology department, faculty of medicine, al-quds university, jerusalem, palestine glutamate and asparate are considered as the main excitatory neurotransmitters in brain and spinal cord, in addition to their role in energy metabolism, synthesis of proteins and detoxification of ammonia. glutamate and aspartate are centrally involved in basic mechanisms generating epileptic seizures and in epileptogenesis. stimulated release of glutamate and aspartate was detected in vivo and in vitro following neuronal depolarization. photic stimuli has increased glutamate release from visual cortex, and afferent brachial stimulation has increased the endogeneous release of glutamate from contra-lateral sensorimotor cortex compared to ipsi-lateral side. similar results were achieved after local application of tityustoxin or veratridine to the sensorimotor cortex. implantation of cobalt powder over the right sensorimotor cortex of rats produced an epileptogenic lesions characterized by contra-lateral fore and hind limb jerks and an increase in the frequency of eeg spikes. the jerks started after 6 days with maximum myoclonic activity (15 jerks/min). the concentration of glutamate in the epileptogenic focus was decreased significantly by 29% (p ͻ 0.01) compared to the non-epileptogenic area on the left sensorimotor cortex, which was dissected but not treated with cobalt. part of the decrease in glutamate could be related to the enhancement of in-vivo release from the epileptogenic lesion to the extra-cellular fluid. kindling is the best model for studying the development of the epileptic focus (epileptogenesis), it could be achieved by repeated intra-cerebral micro-injection of glutamate (1.5 µ mol), aspartate (0.75 µ mol) or nmda (2-4 n mol), or repeated electrical stimulations of specific brain regions. in addition, glutamate antagonists particularly those specifically acting on the nmda receptor type e.g. 2-amino-5-phosphonovaleric acid (ap5) and 2-amino-7-phosphonopheptanoic acid (ap7) have been found to inhibit seizures in epileptic animals and inhibit the development of electrically kindled epilepsy. pre-synaptic glutamate receptor agonists like (1s, 3s)-acpd the agonist of group ii, and l-ap 4 the agonist of group iii receptors has reduced ca ϩϩ uptake and glutamate release, thus it has inhibited epileptogenesis by preventing the increase in both seizure score and after-discharge duration. injection into fully kindled animals has produced an anti-epileptic effect by reducing the mean seizure score and by increasing the mean generalized seizure thresholds. this results suggest the mechanism by which pre-synaptically active glutamate receptor agonists block the development of the chronically epileptic state induced by electrical kinding, and indicate that their anticonvulsive activity is due to inhibition of pre-synaptic glutamate and/or asparate release following blockade of pre-synaptic ca ϩϩ entry. testing the changes in glutamate release from hyperactive brain tissues, and the effect of different glutamate agonists and antagonists, supports the role of glutamate in initiating the process of epileptogenesis, and contributes in developing new anti-epileptic agents. (this project was supported by a grant from alexo) the functional roles of cl(ϫ) and divalent cations in the na(ϩ)/cl(ϫ)/gaba cotransport were examined in xenopus oocytes expressing the human gat-1 (hgat-1) gaba transporter cdna. our results showed that cl(ϫ) was not absolutely required for na(ϩ)/gaba transport via the hgat-1 (loo et al., j biol. chem. 275:37414-37422, 2000) . the cl(ϫ) interacted with the transporter to modulate the binding of external na(ϩ). although hgat-1 transported cl(ϫ) across the membrane with a stoichiometry of 2na(ϩ) : 1cl(ϫ) : 1gaba, the transported cl(ϫ) did not contribute to the net charge translocated across the membrane, suggesting a cl(ϫ)/cl(ϫ) exchange mechanism during the gaba transport cycle. the gaba transport via the hgat-1 is also modulated by divalent cations. the uptake of [3h]-gaba was inhibited significantly when both ca(2ϩ) and mg(2ϩ) were removed from the uptake buffer. several divalent cations tested were individually able to sustain the gaba uptake. in contrast to uptake, the gaba efflux was enhanced significantly upon removal of both ca(2ϩ) and mg(2ϩ) from the efflux buffer. the gaba transporter inhibitor skf89976a blocked the enhanced efflux, suggesting that the hgat-1 operated faster in the reverse mode in the absence of external divalent cations. these results suggest a regulatory role for the divalent cations in gaba transport. merck sharp & dohme, neuroscience research centre, terlings park, harlow, essex, u.k. the role of alpha5 containing gaba a receptors in hippocampal synaptic function has been investigated using pharmacological and electrophysiological techniques, as well as following disruption of the alpha5 subunit gene in knockout mice (ko). in the ca1 region of the hippocampus the induction of long-term potentiation (ltp) is powerfully regulated by gaba mediated synaptic currents (ipscs). agents that inhibit gaba-mediated transmission potentiate ltp induction, whereas allosteric agonists such as benzodiazepine-site agonists which slow the decay kinetics of ipscs suppress ltp induction. in alpha5 ko mice paired pulse facilitation of the amplitude of excitatory synaptic potentials is selectively enhanced in the ca1 region but not dentate gyrus. likewise, the frequency and rise time of spontaneous ipscs were similar in wt and ko slices. however their amplitude was significantly smaller in ko mice. furthermore, a significantly greater proportion of ipscs were best fitted to a mono exponential function in ko mice compared to wt animals. thus alpha5 containing gaba a receptors contribute to functional postsynaptic receptors on ca1 pyramidal cells in the hippocampus and modulate a postsynaptic component of synaptic facilitation. pharmacological research institute, volgograd medical academy, volgograd, russia the purpose of the study is to investigate effect of phenil (karphedon, mephebut, gammoxin) and circle (pyracetam) gaba derivatives on reproductive function of stressed male rats. the adult male rats were stressed by immobilization exposure (2 hours) twice in week during 6 weeks. four from five groups of stressed males were given substances (daily) at doses: karphedon -50 mg/kg, mephebut -10 mg/kg, gammoxin -40 mg/kg, pyracetam -200 mg/kg. the treated males were mated with intact females during 12 days. after the mate the treated males and more in 10 days all the mated females were sacrificed and investigated. analysis of our data indicates that the time of spermatozoa motion and epididymal sperm counts were decreased 73.2% (p յ 0.05) and 49.1% (p յ 0.05) respectively when compared with their intact controls. gaba derivatives have a softening effect on functional parameters of spermatozoa stressed males. karphedon and pyracetam increased the time of motion spermatozoa 86.3% (p յ 0.05), karphedon and mephebut drew near sperm counts to intact control level. the result of mate show that pregnancy rate was increased (p յ 0.05) by stress exposure and pregnancy rate of females mated with gaba stressed males was some more (p ն 0.05) than that of intact controls. the general embryonic morality was increased twice by stress and so the number of embryos was reduced 48.8%. the gaba derivatives exposure to stressed male rats reduced the embryonic mortality of their posterity and increased the number of embryos to intact control level. our findings demonstrate that gaba derivatives administration has a protective effect on reproductive function of stressed male. transmission on the brain. the realization that glutamatergic pathways are involved in such diverse processes in epilepsy, ischemic brain damage and parkinsons' disease, is of a great practical interest. there are at least three functional classes of ionotropic glutamate receptors: n-methyl-d-aspartate (nmda), α-amino-3-hydroxy-5 methyl-4-isoxazolepropionic acid (ampa) and kainate (ka). other central neurotransmitter systems are under nmda influence. some data point on neuroprotective action of nmda antagonist on nigrostriatal pathway. in the present study female wistar rats were exposed during pregnancy with daily injected mk-801 (dizocilpine) 0.5 mg/kg sc. control rats received tap water only. behaviour of 3 month old male offsprings was investigated by several psychopharmacological methods. oral activity, yawning, locomotor activity, stereotypy and catalepsy were recorded following respective central dopamine receptors agonists and antagonists administration (skf 38393, quinpirole, apomorphine, haloperidol). our results indicate that mk-801 applied during pregnancy modulate reactivity of the central dopamine receptors in adult offspring rats. [ the development of mammalian ingestive behavior is characterized by a transition from suckling to chewing, two distinct motor behaviors. we hypothesize that this transition is accompanied by changes in brainstem circuitry underlying these movements. since glutamatergic neurotransmission is critical for the proper functioning of brainstem circuitry responsible for mastication, we investigated the development of glutamate receptors in trigeminal motoneurons (mo5) and mesencephalic trigeminal neurons (me5); neurons comprising the circuitry responsible for jaw movements. we conducted a series of receptor immunohistochemistry experiments that characterized the expression of iontotropic and metabotropic glutamate receptors (mglurs) during early postnatal development. the functional roles of nmda, ampa and mglurs in neonatal mo5 were investigated using in vitro electrophysiological experiments. results demonstrated that the spatial and temporal expression of ampa, nmda and group i and ii mglurs are developmentally regulated within and between mo5 and me5 during early development. electrophysiological data demonstrate that mglurs function pre-and postsynaptically to modulate synaptic transmission between trigeminal premotoneurons and mo5. furthermore, nmda induced bursting is developmentally regulated and coincident with the transition from suckling to chewing behaviors. our studies suggest that the transition from suckling to chewing is accompanied by changes in the composition and function of glutamate receptors. fetal life in down syndrome starts with normal neuronal density but impaired dendritic spines and synaptosomal structure r. weitzdoerfer 1 , m. dierssen 2 , m. fountoulakis 3 , and g. lubec 1 1 department of pediatrics, university of vienna, austria information on fetal brain in down syndrome (ds) is limited and there are only few histological, mainly anecdotal reports and no systematic study on the wiring of the brain in early prenatal life exist. histological methods are also hampered by inherent problems of morphometry of neuronal structures. it was therefore the aim of the study to evaluate neuronal loss, synaptic structures and dendritic spines in the fetus with down syndrome as compared to controls by biochemical measurements. 2 dimensional electrophoresis with subsequent mass spectroscopical identification of spots and their quantification with specific software was selected. this technique identifies proteins unambiguously and concomitantly on the same gel. fetal cortex samples were taken at autopsy with low postmortem time, homogenized and neuron specific enolase (nse) determined as a marker for neuronal density, the synaptosomal associated proteins alpha snap [soluble n-ethylmaleimidesensitive fusion (nsf) attachment protein], beta snap, snap 25 and the channel associated protein of synapse 110 (chapsyn 110) as markers for synaptosomal structures and drebrin (drb) as marker for dendritic spines. nse, chapsyn 110 and beta snap were comparable in the control fetus panel and in down syndrome fetuses. drebrin was significantly and remarkably reduced and not even detectable in several down syndrome brain samples. quantification of snap 25 revealed significantly reduced values in ds cortex and alpha snap was only present in half of the ds individuals. we conclude that at the time point of about 19 weeks of gestation (early second trimester) no neuronal loss can be detected but drebrin, a marker for dendritic spines and synaptosomal associated proteins alpha snap and snap 25 were significantly reduced indicating impaired synaptogenesis. early dendritic deterioration maybe leading to the degeneration of the dendritic tree and arborization, which is a hallmark of down syndrome from infancy. pathfinding of growing axons to reach their target during brain development is a subtle process needed to build up contacts between neurons. abnormalities in brain development in down syndrome (ds) are described in a couple of morphological reports but the molecular mechanisms underlying abnormal wiring in fetal ds brain are not yet elucidated. we therefore performed a study using the proteomic approach to show differences in protein levels involved in the guidance of axons between control and ds brain in early prenatal life. proteins obtained from autopsy of human fetal abortus were applied on 2-dimensional gel, identified and quantified. we quantified 5 members of the semaphorin/collapsin family, the dihydropyrimidinase related proteins 1-4 and the collapsin response mediator protein-5 (crmp-5) in 8 ds and 7 control cortex samples. drp-1 and crmp-5 levels were comparable in the control and ds samples. evaluation of drp-2, drp-3 and drp-4 revealed significantly decreased levels of 2 of the 15 spots assigned to drp-2 and increased levels of one spot assigned to drp-3 and increased drp-4 in ds brain. we conclude that as early as from the 19 th week of gestation pathfinding cues of the outgrowing axons are impaired in ds. these findings may help to elucidate mechanisms leading to abnormalities in neural migration of ds brain. inflammatory processes play an important role in the degeneration of basal forebrain cholinergic cells alzheimer's disease. the proinflammagen lipopolysaccharide (lps) was infused chronically into the basal forebrain of young rats. we then determined whether the administration of two novel nonsteroidal anti-inflammatory drugs or a pancaspase synthesis inhibitor, zvad, could provide neuroprotection from the cytotoxic effects of the neuroinflammation. we also determined whether the administration of the non-competitive n-methyl-d-aspartate (nmda) receptor antagonist, memantine, could provide neuroprotection from the cytotoxic effects of the neuroinflammation. chronic lps infusions decreased choline acetyltransferase activity and increased the number of activated microglia within the basal forebrain. caspases 3, 8 and 9 activity was increased in ventral caudate/putamen. non-steroidal antiinflammatory drug therapy attenuated the toxicity of the inflammation upon cholinergic cells and reduced caspases 3, 8, and 9 activity in the caudate/putamen. zvad significantly decreased the levels of caspases 3, 8 and 9 but did not provide neuroprotection for cholinergic neurons. memantine significantly attenuated the cytotoxic effects of chronic inflammation upon cholinergic cells. these results suggest that prostaglandins contribute to the degeneration of forebrain cholinergic neurons in alzheimer's disease and that the cytotoxic effects of prostaglandins occur upstream to nmda receptor activation. intracranial administration of n-methyl-d-aspartate (nmda) receptor antagonists block learning of classical and avoidance conditioning in goldfish. studies with goldfish have shown that nmda receptors are mostly dense in the telencephalon and telencephalon ablation impairs avoidance learning. the present study investigated amnestic effects of microinjection of nmda receptor antagonist ap5 to the goldfish telencephalon in avoidance conditioning. in experiment 1, fish received no injection or microinjections of saline or various doses of ap5 to their telencephalon 20 minutes before three semiweekly training sessions. fish were tested without injec-tions in session 4. a one-way anova with multiple comparisons on the test scores showed that ap5 produced anterograde amnesia in a dose-dependent manner. in experiment 2, fish received several training sessions and a microinjection of various doses of ap5 20 minutes before testing. the test scores showed that ap5 did not decrease avoidance responses, suggesting that microinjection of ap5 did not impair performance processes. in experiment 3, fish received microinjections of ap5 or saline to their telencephalon immediately following three semiweekly training sessions and were tested without injections in session 4. a one-way anova on the test scores showed that ap5 did not produce retrograde amnesia. (supported by gvsu grant-in-aid.) tryptophan modulates striatal serotonergic activity relative to fatigue t. yamamoto 1 and e. a. newsholme 2 1 health science laboratory, tezukayama university, nara, japan 2 department of biochemistry, university of oxford, u.k. we have been reported that mechanism of fatigue in the brain relates to enhanced extracellular tryptophan and serotonergic function. brain concentration of tryptophan is not only dependent on the change of tryptophan which originates from the centarl nervous system, but also enhance tryptophan entering the brain from the blood-brain barrier and peripheral circulating tryptophan which is a trigger. supplementation of ltryptophan (2 um) into the incubation medium with the synaptosomal striatum causes tryptophan to the extrasynaptosomal release by high kϩ stimulation. injecting l-tryptophan (1 mm/ 30 min) into the left striatum by microdialysis method can induce early fatigue for running time of rats. on the other hand, tryptophan deficiency rats (body weight average 200 g) were made by tryptophan free feeding for 2 weeks, and the rat's running time increased (ͼ100 min difference). these results suggests that tryptophan is a potent active substance for fatigue in the brain. the active zone may be presynaptic terminal and the tryptophan itself may be releasing neuromodulators. (we appreciate that tryptophan free diet was provided by ajinomoto co., inc., japan.) our recent studies on the distribution of free d-serine, together with the d-serine action on the glycine site of the nmda type glutamate receptor, suggest that the d-serine can be an endogenous modulator of the nmda receptor. to explore the possible removal systems for brain d-serine signaling, we have evaluated the uptake of [3h]d-serine into the synaptosomal p2 fraction from the rat cerebral cortex. the cortical p2 fraction was able to accumulate [3h]d-serine in a temperatureand ph-dependent and saturable manner. the kinetic analysis indicates that cortical d-serine transport occurs by an apparent single-component system with km value of 283 µm and a vmax value of 207 pmol/mg protein/min. depletion of na ϩ and cl ϫ ions remarkably decreased d-serine uptake into the cortical p2 fraction. the pharmacological profile of the inhibition of dserine uptake by various amino acids was different from those of glycine uptake system and other amino acid transporters reported. d-serine uptake activity was preferentially observed in the brain tissues such as cerebral cortex and cerebellum to the peripheral tissues. the present data support the view that the endogenous d-serine is taken up mainly through a carriermediated transport system to regulate the extracellular concentration in the mammalian brain. a. bocheva et al. the mammalian brain contains all the urea cycle intermediates, whereas enzymes participating in the conversion of lornithine (l-orn) into l-citrulline (l-cit) are absent, resulting in an incomplete urea cycle. the discovery of nitric oxide (no) synthase that catalyses the formation of no and l-citrulline as a co-product from l-arginine (l-arg) in the brain has indicated an additional pathway for l-arg metabolism. l-canavanine (l-cav), is a potent antimetabolite and structural analog of larginine, produced by legumes such as the jack bean, canavalia ensiformis. l-canaline (l-can) is a potent inhibitor of ornithine aminotransferase. our previous results indicated that l-cav, l-cit, l-arg, and l-orn exerted an antinociceptive effect, whereas l-canaline induced hyperalgesia in rat. l-canavanine exert stronger antinociceptive effect than l-arginine, l-ornithine and l-citrulline. the aim of the present study was to investigate are d-arg, l-cav and naloxone reversed the analdesic effects of l-ornithine, l-citrulline and l-arginine. the experiments were carried out on male wistar rats. the changes in the mechanical nociceptive threshold of the rats were measured by the radall-selitto paw pressure test using and analgesimeter (ugo basile). the amino acids were applied intracerebroventricularly (i.c.v.) at a dose 20 µg/rat. the present results shown that d-arg, l-cav and naloxone reversed antinociception. the regulation of lysine metabolism in cereal crops r. a. azevedo 1 , p. j. lea 2 , s. a. gaziola 1 , a. p. pellegrino 1 , and s. m. g. molina 1 1 departamento de genética, escola superior de agricultura luiz de queiroz, universidade de são paulo, brazil 2 department of biological sciences, university of lancaster, u.k. a major nutritional drawback of cereal seeds is a deficiency in some amino acids, in particular lysine. biochemical, molecular and genetic studies have considerably increased our knowledge concerning the regulation of the aspartate pathway, by which lysine is synthesized. among the enzymes involved in lysine metabolism, aspartate kinase (ak) and dihydrodipicolinate synthase (dhdps) control the regulation of lysine biosynthesis, whereas lysine: 2-oxoglutarate reductase (lor) and saccharopine dehydrogenase (sdh), have been shown to play a key role in the breakdown of lysine. in general, lysine overproduction can be obtained by altering the sensitivity of dhdps to lysine, but accumulation of this amino acid in cereal seeds requires further manipulation of lor and/or sdh. this suggestion is strongly supported by five main points: (1) cereal mutant or transgenic plants do not exhibit any significant accumulation of lysine in seeds, but only in other tissues. (2) the enzymes of lysine degradation, lor and sdh, are endosperm specific in cereals only. (3) the opaque-2 mutant, which exhibits higher concentration of soluble lysine and protein lysine in the seed, contains several-fold lower lor and 2-fold lower sdh activity when compared to the wild-type maize. this reduction in activity in the opaque-2 mutant is due to a reduced protein lor-sdh concentration by reduction of the zlkrsdh gene transcript. furthermore, the opaque-2 maize gene has been shown to regulate ak and lor activity. (4) intermediates of lysine catabolism accumulated in the seeds of soybean and canola lysine overproducing plants, suggesting the presence of reduced lor and/or sdh activities. (5) among cereals and although still below the recommend values by fao, rice exhibits the higher concentration of lysine, but lor and sdh are present in much lower activities. also, in phaseolus vulgaris, lor and sdh activities were shown to be around 10fold lower then in maize endosperm. the regulation of the lor activity is complex and involves a calcium dependent phosphorylation/dephosphorylation mechanism. it remains to be seen whether this latter mechanism can be controlled, so as to allow the production of more crop plants that contain elevated concentrations of lysine in the seed. the genetic progress for nue can be accelerated with the use of secondary traits that possess high inheritance and correlation with productivity. several traits have been studied such as chlorophyll concentration, plant height, leaf senescence, anthesis-silking interval, kernel number, activities of enzymes of n assimilation and loci of quantitative traits for assisted selection. (we are grateful for financial support from fapesp, brazil and the british council.) (termed hyperaccumulators) that grow on metalliferous soils, are able to translocate cd from the roots and accumulate it in high concentrations in the shoots. cd may be detoxified in plants by combination with a family of sulphur rich peptides termed phytochelatins. cd has the capacity to inhibit a range of enzyme activities in plants, in particular those of the calvin cycle and chlorophyll biosynthesis. evidence that cd causes the production of reactive oxygen species (ros) has also been obtained. we have investigated the antioxidant responses of radish, soybean and sugarcane to cd treatment. seedlings were grown in increasing concentrations of cdcl 2 , ranging from 0.01-1 mm, for up to 96 h in a hydroponic system. analysis of cd uptake indicated that most of the cd accumulated in the roots, but some was also translocated and accumulated in the leaves. roots and leaves were analysed for catalase (cat), glutathione reductase (gr) and superoxide dismutase (sod) activities. gr activity increased considerably in the roots of all plant species tested after exposure to the metal, indicating a direct correlation with cd accumulation. cat activity also increased in roots but to a much lesser extent when compared to gr and also varied depending upon the plant species. the analysis of native page enzyme activity staining, revealed several sod isoenzymes in leaves of all plant species, however, only in radish was a clear increase in activity observed. the results suggest that in these plants, the activity of antioxidant enzymes responds to cd treatment. the main response may be via the activation of the ascorbate-glutathione cycle for the removal of hydrogen peroxide, or to ensure the availability of glutathione for the synthesis of cd-binding proteins. (we are grateful for financial support from fapesp, brazil and the british council.) all plant cells, tissues and organs provide the biosynthetic machinery and capacity to synthesise aliphatic polyamines. however, in physiological conditions only some organs and tissues synthesise polyamines, such as apical buds and sprouts, root apex, lateral buds of branches and secondary roots, as well as superficial layers of young stems and leaves, like epidermis, subepidermis and parenchyma cells. apical roots can also synthesise polyamines, but these activities in physiological conditions are lower than that of the shoots. this patterns recalls the one of auxins. polyamines are accumulated in high concentrations in storage organs, such as seeds, but not in tubers like helianthus tuberosus, potato or tuberised roots such as the carrot. also some fruit, e.g. oranges, contain high level of free polyamines, putrescine in particular. all other organs obtain polyamines through translocation via phloem tubes and xylem vessels. in plants, in addition to free polyamines, many polyamines are conjugated to hydroxycinnamic acids, the hydroxycinnamic amines, that only rarely represented outside the plant kingdom. this compounds are paticularly abundant in solanaceae family, where they can represent as much as 90% of the total polyamine pool, but they can be detected in different concentrations in many other families. the role of free and conjugated polyamines and their importance in food is discussed. drought, salinity or other environmental stressors promote the accumulation of free amino acids, amines and other organic n-metabolites with low molecular weight. in this contribution the influence of drought on the accumulation of amino acids, polyamines and trigonelline in growing barley plants and barley grains was examined. in comparison to non-stressed plants we obtained in stressed plants, exposed to drought before flowering, a higher concentration of proline (increase: 4-fold), n-trimethylglycine (2-fold), histidine (3-fold), tryptophane (1,7-fold), putrescine (1,4-fold), spermine (1,7-fold) and trigonelline (2-fold) in the dry matter of barley sprouts. in addition to this, drought caused an increase of the n-content in the plant biomass (35%) as a result of growth inhibition (34%). six weeks later the content of soluble n-metabolites and protein was analyzed in non-stressed and pre-stressed barley plants again. during this reproductive period of plant development all the test groups were cultivated under the same moisture conditions. the analysis of n-metabolites in the ripening grains showed, surprisingly an after-effect of the drought stress. for example, in grains of pre-stressed barley the concentrations of free proline, histidine, tryptophane and asxϩglx were threefold to fivefold higher than in grains of non-stressed barley. depending on the resistance of barley cultivars to drought the biochemical response was different: in plants with low resistance the increase of amino acids and amines was higher than in resistant cultivars. however, resistant cultivars have already high genuine concentrations of n-metabolities in non-stressed plants. by treatments with choline or 2-aminoethanol the stresspromoted accumulation of amino acids and trigonelline was diminished. consequently, different biochemical responses of cereals to drought result in changes of product quality and nitrogen use. our goal is to increase the lysine content in corn. we have used genetic engineering to increase lysine synthesis and to prevent metabolic breakdown of lysine. to increase synthesis we circumvented the normal feedback control of a key enzyme in the lysine biosynthetic pathway, dihydrodipicolinic acid synthase (dhdps). lysine-feedback-insensitive dhdps, encoded by the corynebacterium dapa gene, was expressed from seed-specific promoters in transformed corn seeds. expression of dhdps in the corn embryo, but not in the corn endosperm, resulted in a 20 to 30-fold increase in the accumulation of free lysine in the seeds and the total seed lysine content nearly doubled. lysine breakdown products have been observed in transgenic seeds that accumulate high levels of free lysine. we isolated a corn gene for the bifunctional enzyme lysine ketoglutarate reductase (lkr)/saccharopine dehydrogenase (sdh), which catalyzes the first two steps in lysine breakdown. knockout of lkr/sdh in corn by either mutation or genetic engineering results in a 10-fold increase in seed free lysine. combination of feedback-insensitive dhdps with knockout of lkr/sdh results in 2 to 3-fold higher levels of free lysine than dhdps alone. no adverse effects on seed or plant agronomic performance are associated with the high lysine trait. biotechnology center for agricultural and the environment and the plant science department, rutgers university, new brunswick, new jersey, u.s.a. 5ј-adenylylsulfate (aps) reductase catalyzes a key reaction in the plant sulfate assimilation pathway leasing to the synthesis of cysteine and the antioxidant glutathione. in arabidopsis thaliana aps reductase is encoded by a family of 3 genes. in vitro studies revealed that the enzyme product derived from one of the aps reductase genes (apr1) is activated by oxidation, probably through the formation of a disulfide bond. redox titrations show that the regulation site has a midpoint potential of ϫ327 mv at ph 8.5 and involves a 2-electron redox reaction. exposure of a variety of plants to ozone induces a rapid increase in aps reductase activity that correlates with the oxidation of the glutathione pool and is followed by an increase in free cysteine and total glutathione. during the response to ozone the level of immuno-detectable aps reductase enzyme does not increase. treatment of a. thaliana seedlings with oxidized glutathione or paraquat induces aps reductase activity even when transcription or translation is blocked with inhibitors. the results suggest that a post-translational mechanism controls aps reductase. a model is proposed whereby redox regulation of aps reductase provides a rapidly responding, self-regulating mechanism to control the glutathione synthesis necessary to combat oxidative stress. in aspergillus nidulans the structural genes coding for nitrate reductase (niad) and nitrite reductase (niia), share a common promotor region of 1,200 bp. we have previously characterized in vitro and in vivo the physiologically relevant cisacting elements for the two synergistically acting transcriptional activators, nira and area. we have further shown that area is constitutively bound to a central cluster of four gata sites and is directly involved in opening the chromatin structure over the promoter region and thus making additional cis-acting binding sites accesible. here we show that the asymmetric mode of nira-dna interaction determined in vitro is also found in vivo. binding of the nira transactivator is not constitutive as in other binuclear c 6 -zn ϩϩ -cluster proteins but depends on nitrate induction and additionally, on the presence of a wild type area allele. dissecting the role of area further, we found that it is required for intracellular nitrate accumulation and therefore could indirectly excert its effect on nira via inducer exclusion. but in a strain accumulating nitrate independently of area nira binding and chromatin rearrangement is not triggered by nitrate in the absence of area. v. nikiforova 1 , m. zeh 1 , o. kreft 1 , s. maimann 1 , h. hesse 2 , and r. höfgen 1 1 max-planck-institut für molekulare pflanzenphysiologie, potsdam, and 2 institut für biologie, angewandte genetik, freie universität berlin, germany higher plants, being a source of reduced sulfur for animal nutrition, assimilate inorganic sulfate into cysteine which is subsequently converted to methionine, another sulfur-containing amino acid. in order to investigate the possible regulatory points of the cysteine and methionine biosynthesis pathway a series of transgenic potato plants was engineered using clones encoding enzymes of the branched pathway from serine to cysteine as a pathway intermediate and from aspartate further on to methionine. increased cysteine levels were obtained in the leaves of serine acetyltransferase (sat) sense and cystathionine -lyase (cbl) antisense transformants. furthermore, glutathione levels were elevated in sat plants while downregulation of cbl was desastrous for plant growth, eventually. increased methionine levels were successfully obtained in potato by antisense inhibition of threonine synthase (ts). accumulation of free methionine was not only observed in source leaf tissues but as well in tubers. this enzyme competes with cystathionine gamma-synthase for the common substrate o-phosphohomoserine at the branchpoint between threonine and methionine synthesis, respectively. important control points of the biosynthesis of cysteine and methionine in potato, thus, turned out to be sat and ts, while further studies on overexpression of cystathionine gamma-synthase, cbl and ms did not reveal any substantial effect on potato methionine biosynthesis. dniepropetrovsk national university, board of biophysics and biochemistry, ukraine amino acids in root exudates of plants may be chelate agents as an alpha-amino acid can act like a bidentate ligand, forming a five-membered heterocyclic ring with suitable metal cations thus increasing mobility of metals. recently we have showed that application of growth regulators led to sharp increase of root exudative activity of some cultural (zea maize l.) and wild cereals (festuca rubra l., lollium perenne l.) during first days of germination. in this work we present results obtained in experiments with lollium perenne l., grown on sterile sand and on soils contaminated with great quantities of zn. detailed analysis of amino acid content of root exudates of several types of maize (hybrid, several lines, an opaque-2 mutant line) showed that the specie had more certain amino acids (cysteine, aspartic and glutamic acids and their amides, serine) in root exudates than cultural ones. these amino acids has more possibility for chelation due to existance of one more polar or ionogenic functional groop. seeds of lollium perenne l. were treated with growth regulater and planted on soils contaminated with salts of zink. it was shown that during 15 days of germination quantity of zn in primary leaves increased from 121,46 to 243,75% and decreased in soil: in upper layer from 95,74 to 85,07, midde layer from 95,83 to 82,04, lower layer from 85,72 to 72,74 mkg/kg correspondingly. thus, it was shown that stimulation of root exudative activity by pretreatment with a growyh regulator may be succesful in cleaning of soils and basicly this is a good method for phytoremediation. erenol exerted the strongest effect. exercise completely abolished the levels of cysteine in the atrial heart muscle. propranolol, isoproterenol, caffeine and pentylenetetrazol increased the ratio of cysteine to the total free amino acids in the atrial muscle, while physical stress and all cardioactive drugs tested increased this ratio in the ventricle muscle. disappearance of cysteine from the heart's atrial muscle after intensive exercise may be attributed to its utilization for atrial natriuretic factor and/or for endothelin synthesis, during stress. on the other hand it seems that hypoxia and isoproterenol are strong stimulants of no production, and consequently decrease the tissue levels of l-arginine, which is the major endogenous donor of no acting as the endothelin antagonist. measurement of serum levels of vitamin b12 is a screening test for detection of deficiency of this vitamin but low levels do not always indicate a deficiency of the vitamin. measurements of serum homocysteine and methylmalonic acid (mma) are used to confirm this deficiency because two enzymes involved in their metabolism have been shown to require vitamin b12, but these results can also be inaccurate. vitamin b12 deficient white cells exhibit ultrascopic nuclear appenages which have been shown to contain dna; this finding could possibly be used as another confirmatory test of vitamin b12 deficiency. twenty-seven patients (mean age -76.6 years) with low serum b12 were studied by electron microscopic determination of the percent of neutrophils exhibiting these appendages and routine clinical parameters. only one patient did not have nuclear appendages; the others had a range of 0.8%-17.6% of neutrophils examined. there was a significant correlation of homocysteine (r ϭ .46, p ͻ .05) and mma (r ϭ .53, p ͻ .01) with serum b12 levels but no correlation of appendage number (r ϭ .18) with serum b12. there was no correlation of appendage number with homocysteine (r ϭ .25) or mma (r ϭ .04). these results suggest that b12-deficient white cell nuclear appendages do not measure the same metabolic pathways as homocysteine and methylmalonic acid and may be useful in confirmation of vitamin b12 deficiency. further extensive clinical evalution would be necessary to explore this possibility. the hypothesis: "l-theanine has relaxing effects of central nervous system of human beings", was verified by electroencephalographical methods. methods: 14 male, healthy sport-students, free of drugs or stimulants, participated weekly in a cross-over study. after exhaustive bicycle-ergometer test as an individual, reliable, stress model, the subjects recovered by lying in a segregated shaded room. three testdrinks with different l-theanine content (d1 ϭ placebo, d2 ϭ 50 mg, d3 ϭ 200 mg) were given in a randomised, double-blind order. all test-conditions were standardized strictly. eeg-recordings (closed eyes) were carried out (m1 ϭ 3 min. after stress/before testdrink, m2 ϭ 30 min.-, m3 ϭ 45 min.-, m4 ϭ 60 min.-, m5 ϭ 120 min. after testdrink) with the cateem ® system. absolute and relative eeg-spectralpower were examined. results: significant reductions in all frequencies (exception theta-power) were found in early recovery, being not significant influenced by testdrinks. qualitative different behavior trends were found in frontal-, central-, occipital-regions with increased alpha1, theta (frontal) and decreasing beta1 relative-power earlier in recovery with d3. these findings were related to relaxing effects. after ingestion of l-theanine alpha2-, beta1-power at occipital regions decreased faster (m2) to placebo recovery levels (m3/m4). thus it may be concluded that l-theanine has no pharmaceutical effect on the down regulation system but supports the physiological mechanisms during recovery after physical stress in human brain. arginine and cysteine in muscle cytosol of rats' heart after exercise, hypoxia or challenge with six selected cardioactive drugs r. brus 1 , j. gabryś 2 , j. konecki 2 , and j. shani 3 1 department of pharmacology and 2 department of histology and embriology, medical university of silesia, zabrze, poland 3 department of pharmacology, the hebrew university school of pharmacy, jerusalem, israel levels of the amino acid l-arginine (a major endogenous donor of nitric oxide-no), cysteine (sulfur-containing amino acid, important for atriopeptins and endothelins synthesis), and of total free amino acids, were assayed by gas-liquid chromatography in cytosols of rats' atrial and ventricular muscle cardiomyocytes. the tissues were assayed after the rats had been exposed to either exercise (swimming), hypoxia or one of six cardioactive drugs such as propranolol, digoxin, pentylenetetrazol, reserpine, isoproterenol and caffeine. physical stress and the examined drugs significantly reduced the total amount of cytosolic free amino acids in both cardiac muscles. in the cytosol of the heart atrial muscle, reserpine, propranolol and pentylenetetrazol increased the relative content of l-arginine, while hypoxia and digoxin decreased it. in the cytosol of the ventricular heart muscle, hypoxia and all six drugs used, decreased the relative levels of l-arginine. hypoxia and isoprot-addition of somatostatin-14 or of some of its analogs was found to cause a selective inhibition, up to 50%, of the uptake of large neutral amino acids by isolated brain microvessels. although the luminal and abluminal sides of brain endothelial cells are both capable of taking up large neutral amino acids, only the uptake from the abluminal side was apparently inhibited by somatostatin. the involvement of a type-2 somatostatin receptor was suggested by assays with a series of receptorspecific somatostatin agonists, and was confirmed by the inhibition release caused by a specific type-2 receptor antagonist. a type-2 specific mrna was indeed shown to be present both in bovine brain microvessels ex vivo and in primary cultures of endothelial cells from rat brain microvessels. hemorphins represent a bioactive peptide class which contents between 4 and 10 amino acids and generated from the proteolysis of an hemoglobin "strategic zone". many activities have been related to hemorphins such as in vitro anti tumour effect, analgesia effects in vivo, and a potential role in the renin angiotensin system (ras). as far as their activity towards the ras is concerned, it was demonstrated that they could inhibit angiotensin converting enzyme (ace) and aminopeptidase n activity. so they could reduce angiotensin ii formation and angiotensin iv degradation. moreover some hemorphins, lvv-hemorphin-7 and vvhemorphin-7, could behave like angiotensin iv receptor binding competitor. further it could be interesting to study the angiotensin iv potentiality to interact with ace. inhibition studies showed that it was possible that angiotensin iv could behave like a competitive inhibitor of ace. so some hemorphins could interact at different ras steps to inhibit ace. additionally to their inhibition of angiotensin i conversion, they could inhibit angiotensin iv degradation and consequently cause ace feedback inhibition. inhibition studies have been checked with ras natural substrate (angiotensin i) and confirmed that angiotensin iv, vv-hemorphin-7 and mainly lvv-hemorphin-7 could be natural ace inhibitors. so the hemorphins regulatory role in the ras appears to be more and more probable. the role of administration of each of methionine and finastride on the testicular function of both normal and prostate precancerous old male rats was investigated. for normal animals, neither methionine nor finastride has exerted any significant change in the hormonal profile after teatment for 20 days. however methionine alone could exert a significant change in both testosterone and prostatic specific antigen {psa} levels after treatment for 40 days. on the other hand, both methionine and finasteride significantly increased the levels of testosterone and androstenedione, whileas markedly reduced the levels of dihydrotestosterone and prostatic specific antigen {psa} after treatment of prostate precancerous old male rats for a period of 20 days. noteworthy, continuation of treatment for aperiod of 20 days realized marked improvement of hormonal profile of the prostate precancerous old male rats. several observations in our department point to some role of glycine in fatigue and exercise. 1) in the framework of a study on the involvement of one-carbon matabolism in patients suffering from a polymorphic episodic psychosis, amino acid loading tests with serine, glycine and alanine were performed. a few hours after the administration of glycine, approximately 40% of the patients reported overwhelming feelings of fatigue and/or showed vegetative symptoms. 2) in patients suffering from chronic fatigue syndrome, we found increased plasma levels of glycine in 20% of the female patients. moreover, 60-70% of these patients omplained about a distorted sensory perception of objects. 3) young soccer players were observed during a period of 10 months, while in the course of this period eight blood samples were taken for amino acid analysis. based on the number and severity of injuries this population was divided into injury-prone and not injury-prone soccer players. it was found that in injury-prone soccer players plasma glycine levels during the whole observation period were significantly lower than in subjects who were not injury-prone. the consequences of the above mentioned observations will be discussed. institute of sportsmedicine, university of paderborn, germany 50 percent of amino acids in green tea leaves are represented by l-theanine (5-n-ethylglutamine). previous rat experiments demonstrated effects of l-theanine to act on metabolism of neurotransmitters. it was therefore suggested that is causes the relaxing effects of green tea. to examine its influence as a component of a drink on the sympathetic nervous system after maximal physical exercise skin resistance measurements through electrosympathicography (esg) were used. after individual maximal exercise on a bicycle-ergometer testdrinks with different amounts of l-theanine (0, 50 and 200 mg) were administered to 14 healthy volunteers in a randomised cross-over double-blind distribution on a weekly base. esg was monitored before and immediately after exercise as well as 13, 30, 45, 60, 75 and 135 minutes after end of exercise. all testconditions were standardized strictly. a characteristic esgcourse with subsequent qualities could be shown: 1. decreasing skin resistances after exercise could be established in each volunteer. 2. esg-activation levels before exercise could not even be reached again after a period of regeneration of 2 1 / 4 hours. 3. maximal electrodermal activity did not appear immediately after exercise, but after 13 minutes. however, l-theanine could not significantly influence peripheral sympathetic electrodermal activity during the regeneration after maximal physical exercise. a. mero 1 and h. pitkänen 1,2 1 neuromuscular research center, department of biology of physical activity, university of jyväskylä, and 2 rehabilitation center of kankaanpää, finland essential amino acid leucine has many important roles in the body. therefore the purpose of the present study was to investigate if leucine supplementation has effects on serum amino acid profile and performance following training period or following single training sessions. all experiments were carried out in a randomized double blind cross-over procedure during a training season. thirty six adult male track and field power athletes served as subjects. in experiment 1 ten of them were given leucine (50 mg/kg body weight per day) as tablets. the concentration of leucine decreased significantly (20%) in the placebo group (p; n ϭ 10) during 5 weeks but not when leucine was taken. also total amino acids (taas) decreased strongly (19%) during 5 weeks when dally protein intake was 1.26 g/kg body weight. in experiment 2 the subjects (n ϭ 16) carried out a single strength training seasion (sts) and consumed a drink containing leucine 100 mg/kg body weight. following sts leucine in serum increased by 11% (ns) when leucine was taken but decreased strongly (30%) in p, in experiment 3 the subjects (n ϭ 12) underwent at 7 days interval two maximal anaerobic running exercise (mare) tests on treadmill (n ϫ 20s with a recovery of 100s between the runs) until exhaustion. the subjects consumed drinks containing leucine (200 mg/kg body weight) or placebo 50 min before the test runs. following mare the concentration of leucine strongly increased by 28% whereas isoleucine (25%) and valine (15%) strongly decreased with the supplementation but no changes occurred in p. there were no improvements in physical performance either in mare or in explosive strength (experiment 2) with leucine supplementation. the date suggest that leucine supplementation during a training period and before single training sessions prevents decreases in serum concentration of leucine and may have also effects on some other single amino acids. this may be beneficial during intensive training although improvements in performance were not observed in this study. since there are only limited data regarding effects of training period or training sessions on serum amino acid profile, the purpose of this study was to investigate serum amino acid changes following training period and following three different training sessions. the subjects consisted of 31 track and field adult male power athletes. in experiment 1 eleven of them performed a 5-week training period including a training sessions per week, which included sprint work, speed endurance work, endurance work, weight training, and jumps. significant decreases in the fasting concentrations of total amino acids (taas) (19%), branched chain amino acids (bcaas) (21%), essential amino acids (eaas) (19%) and leucine (20%) were observed following training with the daily protein intake of 1.26 g/kg body weight. in experiment 2 eleven subjects performed a short run session (srs) of 3 ϫ 4 ϫ 60 m with recoveries of 120 s and 360 s, and a long run session (lrs) of 20 s runs with recoveries of 100 s until exhaustion. there were no significant changes in taas following the sessions but bcaas decreased by 8% in srs and by 7% in lrs. leucine decreased by 10% following srs but only by 5% (ns) following lrs. the peak blood lactate concentrations after srs and lrs were 13.8 ϯ 1.9 mmol/l and 16.4 ϯ 1.3 mmol/l, respectively. in experiment 3 sixteen subjects carried out a strength training session (sts), which consisted of jumps and heavy resistance exercises (speed and maximum strength) during 90 minutes. the taas decreased significantly by 14%, bcaas by 24% and leucine by 30% following sts, while the peak blood lactate concentration was 2.2 ϯ 0.4 mmol/l. these data indicate that remarkable decreases occur in the concentration of amino acids during a training period with the daily protein intake of 1.26 g/kg body weight. the decreases in serum amino acids are more pronounced following a strength training session than following lactic anaerobic running sessions. glutamine acts as a multipurpose regulator of amino acid and peptide transport across the blood-brain barrier departments of cellular biotechnology and haematology and of biochemical sciences, university "la sapienza", rome, italy isolated brain microvessels, the in vitro equivalent of the blood-brain barrier, have distinct na ϩ -independent uptake systems for the uptake of large hydrophobic amino acids, of enkephalins and of deltorphins, as shown by the absence of reciprocal inhibition. both d-and l-glutamine were capable, if added to the extracellular buffer, of exerting a competitive inhibition on the uptake of all these substrates. a trans-stimulatory effect was instead induced, in all cases, by l-glutamine preloading of the microvessels -the d-stereoisomer being instead ineffective, probably because of only l-glutamine could be taken up, in a concentrative manner, by some na ϩ -dependent concentrative system(s). all the na ϩ -independent systems present in brain microvessels seem therefore to share some structural feature responsible for their common susceptibility to interference by l-glutamine. this amino acid, whose synthesis can take place in the astrocytes, in the pericytes and also in the endothelial cells of the microvessels, plays a critical role in regulating the movements of several different substrates across the blood-brain barrier. department of applied bioorganic chemistry, division of life science, graduate school of agricultural science, tohoku university, aoba-ku, sendai, japan isolation and structure analysis of two amino acids from bovine ligamentum nuchae elastin hydrolysates revealed the presence of pyridine cross-links in elastin. the structures of these amino acids were determined to have 3,4,5-and 2,3,5-trisubstituted pyridine skeletons both with three carboxylic acids and a mass of 396 (c 18 h 28 n 4 o 6 ), identified as 4-(4-amino-4-carboxybutyl)-3,5-di-(3-amino-3-carboxypropyl)pyridine and 2-(4-amino-4-carboxybutyl)-3,5-di-(3-amino-3carboxypropyl)-pyridine. we have named these pyridine cross-links, desmopyridine (desp) and isodesmopyridine (idp), respectively. structure analysis of these pyridine crosslinks implied that the formation of these cross-links involved the condensation reaction between ammonia and allysine. the elastin incubated with ammonium chloride showed desp and idp levels increased as the allysine content decreased. desp and idp were measured by hplc with uv detection and were found in a variety of bovine tissues. the desp/desmosine and idp/isodesmosine ratios in aorta elastin were higher than in other tissues. desp and idp contents in human aorta elastin were found to be gradually increased with age. the concentration of idp was significantly elevated in aorta elastin of rat with chronic liver cirrhosis induced by carbon tetrachloride when compared with normal rats. the provision of glutamine to marathon runners has resulted in a decreased, self-reported incidence of illness. increasing evidence -in vitro; and in vivo suggests that neutrophils in humans may benefit from exogenous glutamine. the provision of glutamine in vivo should replete the marked decrease in the blood concentration observed after stress such as clinical trauma or prolonged, strenuous exercise. beneficial effects of glutamine supplementation include increased phagocytic activity and reactive oxygen intermediate production in vitro; decreased neutrophilia and il-8 production (a chemoattractant for neutrophils) in vivo and ex vivo. the aim of the present study was to establish whether glutamine supplementation in vitro and in vivo affects neutrophil function at rest and after exhaustive exercise. in addition, it was planned to establish the presence of glutaminase in human neutrophils, which has not yet been achieved, although glutaminase is present in rat neutrophils. methods: blood samples were taken from marathon runners receiving either glutamine or placebo, immediately after and one hour after a race. measurements included the plasma concentration of glutamine (enzymatic assay), il-8 production (elisa), and neutrophil activity. the latter was measured with two different techniques for measuring oxidative burst in whole blood, one of which was a novel chemiluminescence assay (knight scientific ltd, u.k.) with the fluorescent label, pholasin, and two different stimuli, f-met-leu-phe (fmlp) and phorbol-myristate-acetate (pma). in addition, isolated whole cells and subcellular neutrophil fractions were assayed for the presence of glutaminase. results: the plasma glutamine concentration was reduced overall by 26% 1 hr after the race (p ͻ 0.02). there was an apparent decrease (only close to significance, p ͻ 0.13) in il-8 production in the glutamine group compared with the placebo group. neutrophil function did not change between groups at any stage. the incidence of illness was 46% higher in the placebo group than the glutamine group in the week after the race. neutrophils from four out of six subjects gave an increased response (39.2%) to fmlp when incubated with glutamine compared with no glutamine, and four out of four gave an increased response to pma (31.1%). in the fmlp experiments there were two individuals who did not respond to the addition of glutamine. however, the response was not diminished whether or not glutamine was present. in separate studies, the effect of glutamine on lipopolysaccharide-induced il-8 production was also monitored. conclusions: the provision of glutamine after prolonged, exhaustive exercise appears to modify exercise-induced neutrophilia via a reduction in il-8 production and to reduce the incidence of illness in the following week. in vitro data suggest a role for glutamine in neutrophil metabolism. disappointingly, little or no evidence of the presence of glutaminase was found in human neutrophils. the three different methods used, freeze-thaw, homogenisation, nebulisation were apparently not sufficient to break open the granules. current studies are addressing this problem. r. j. ward 1 and l. m. castell 2 departments of biochemistry, 1 university catholique de louvain, belgium 2 oxford university, oxford, u.k. it is essential that the developing muscle has adequate amino acids for the synthesis of actin and myosin as well as those required for a multitude of enzymes involved in muscle metabolism. with carbohydrates and lipids, the body is able to store a reserve as glycogen and triglycerides respectively; however this is not the case with amino acids creatine supplementation in increasingly being used as a dietary supplement by athletes during high intensity, short term exercise to improve physical performance since it is converted in the muscle to phosphocreatine. transporters which permit creatine to cross the muscle membrane namely crt1 and crt2 (a na ϩ and clј dependent mechanism) have now been identified. creatine uptake is enhanced by the ingestion of carbohydrate at the same time as supplementary creatine. this may be due to increased circulating levels of insulin or insulin-like growth factor 1. more recently attention has been focussed upon the various transporters for amino acids across the muscle membrane. certain criteria are needed for the amino acids to enter the blood which include the presence of specific carriers for its transport across cells of the gastrointestinal tract, such as enterocytes, as well as minimal metabolism within these cells. a wide number of different transporters has been identified, which include neutral amino acids and cationic amino acids. despite the evidence which suggests that supplementation with some amino acids can influence metabolism, and therefore athletic performance, much more experimental work is still required in this area. m. weiss 1 , t. barthel 1 , r. schnittker 1 , k. e. geiss 3 , w. falke 3 , and l. r. juneja 2 1 university of paderborn, germany 2 taiyo kagaku co., yokkaichi, japan, 3 isme gmbh mörfelden, germany in animal studies l-theanine was shown to influence neurotransmitter systems. thus it may be helpful in managing stress regulation. so we observed the down regulation after physical stress in the brain (measured by eeg-mapping) and in peripheral hormonal systems (plasma levels of catecholamines, cortisole, prolactine, serotonine, measured by hplc). n ϭ 14 healthy students consumed drinks containing either 0, 50 or 200 mg l-theanine in randomized double-blind trials in the min 6-14 after a near maximal bicycle step test. measurements were done directly after exercise (m1) and 30 (m2), 45 (m3), 60 (m4), 120 (m5) min after the drink. l-theanine seemed to accelerate the normalization of eeg spectral power in high frequency waves (barthel in this congress). the physiological return of increased hormon levels to basal levels / the circadianic rhythm up to m2 (catecholamines) or m5 (cortisole, serotonine, prolactine) was not influenced by the drinks. but in the l-theanine trials correlations between eeg spectral power and some hormones were altered (slow wave power/some catecholamines except norepinephrine/delta disappeared and new correlations with prolactine appeared). thus we conclude that l-theanine acts at the switch from the brain to the peripheral stress regulation and thereby supports physiological relaxing after severe exercise. polyamines the development from callus to plantlets, both activities increased, reaching the maximum at this latter stage. also sadenosylmethionine decarboxylase activity displayed a similar trend. all the activities were present in supernatant and in particulate fraction. higher activity of enzymes assayed in the small embryos rather than in the embryo with higher shape, was consistent with following polyamine accumulation. department of biology, laboratory of cell biochemistry, university of rome "tor vergata", rome, italy intracellular transglutaminase (tg, ec 2.3.2.13), which catalyzes the formation of ε-(γ-glutamyl)lysine isopeptide cross-links between polypeptides, has been related to a variety of important biological processes and in the development of senile cataract. the majority of the dry weight of the eye lens is composed of protein called crystallins. in the mammalian lens, these proteins are divided into three major classes: α-, -and γcrystallins. native -crystallins are oligomers, which elute in two or more size classes during gel filtration, ranging from 200-50 kda. they contain 7 different types of subunits, named b1, b2, b3, a1, a2, a3, ba4, ranging from 31-32 kda. in the rabbit eye lens two -crystallin subunits ( b2 and b3), among the water soluble proteins, have been shown to act selectively as acyl donors substrates for lens tg. calpains are cytoplamic ca ϩϩ -dependent cystine proteinases. the cleavage of αand -crystallins, the main substrates of lens calpain ii, has been associated to the increase of lens turbidity, due to insolubilization of peptides. we observed that tg-induced post-translational modification of b2-and b3-crystallins with polyamines, enhances their cleavage by calpain ii. this finding suggests that the enhancement of calpain ii activity, after conjugation of polyamines into -crystallins, could represent an important regulatory mechanism which may contribute to the opacification process of the eye lens, conducting to cataract formation. transglutaminases represent a family of enzymes, widely diffused in nature, from bacteria to plants and higher animals. the present discussion will focus on 4 isoenzymes in mammals, which have been well characterized from the structural and functional point of view. they act on tissular proteins catalyzing crosslinkage through isopeptide bonds at peptidyl glutamine and lysine residues or incorporation of small molecular weight primary amines, usually polyamines, in an irreversible, calcium dependent reaction. in several instances the expression of transglutaminases is regulated at the transcriptional level. these enzymes help in maintaining structural integrity of tissues intervening in wound repair and in cellular homeostasis at the levels of cell activation, receptor signaling, cell proliferation, differentiation and death. these general roles involve bis(guanylhydrazones) are a class of compounds known to interfere with the metabolism of polyamines by virtue of their ability to inhibit s-adenosylmethionine decarboxylase (samdc), a key enzyme of polyamine biosynthesis. this property has made them useful tools to study the biological functions of these compounds. a curious feature of bis(guanylhydrazones) is their structural relationship with two molecules involved in polyamine biosynthesis, namely spermidine and s-adenosylmethionine. the methylglyoxal derivative of bis(guanylhydrazones), mgbg, has been actively studied both in animal and plant systems. in the present work the male pollen from actinidia deliciosa has been utilized to investigate the role of polyamines on the pollen tube growth. the effect of several bis(guanylhydrazones) was tested on pollen germination, length of pollen tube, levels of free and conjugated polyamines and samdc activity. all bis(guanylhydrazones) tested (glyoxal-bis-guanylhydrazone, gbg, methylglyoxal-mgbg, methylpropylglyoxal-mpgbg, ethylmethylglyoxal-emgbg) inhibit pollen germination and their effect is dose-dependent. a clear reduction of spermidine, both in free and conjugated form, was observed, as well as a pronounced decrease in samdc activity. these results suggest that the mechanism by which bis(guanylhydrazones) reduce the germination of kiwi pollen is related to their effect on spermidine biosynthesis. molecules structurally related to polyamines (1,8diaminooctane, 1,9-diaminononane, 1,10-diaminodecane) and other inhibitors of their metabolism (cyclohexylamine, cha) are also tested on kiwi pollen germination. n. bagni 2 , d. bertoldi 1,2 , e. candioli 1 , l. martinelli 1 , and a. tassoni 2 1 istituto agrario, san michele a/adige, and 2 dipartimento di biologia, università di bologna, italy in the frame of the study aiming to enlighten developmental programs during regeneration in grapes, polyamine content (free and conjugated to hydroxycinnamic acids) and biosynthetic enzyme activities were assayed during somatic embryogenesis. aliphatic polyamines are growth regulators affecting plant growth and development both in vivo and during in vitro cultures, being involved in several morphogenic processes related ti their action in cell division. the study was conducted on samples of callus, embryogenic callus, embryo at different stages and plantlets of vitis vinifera brachetto and chardonnay cultivars induced from anthers and ovaries. polyamine content (putrescine, spermidine and spermine) free plus conjugated to percloric acid soluble fraction, referred to unit, was higher in the cv. brachetto than in the cv. chardonnay, and reached the higher levels in the fullydeveloped embryo stage. besides, ornithine decarboxylase activity resulted higher than arginine decarboxylase and during multiple catalytic processes: the receptor signaling activity (demonstrated only for isoenzyme 2) is related to an intrinsic gtp-ase activity of type 2 transglutaminase; the processes leading to control of cell proliferation, differentiation and death are mainly related to the protein crosslinking activity, while the cell activation is tentatively considered dependent on the polyamidation of endogenous proteins at glutamine residues. the knowledge on this last aspect lies far back in comparison to the other roles of transglutaminases and requires further accurate investigation, which must further extend to the role of the enzyme in human pathology. the examination of polyamine metabolism at the present time suggests that vitamin b12 is implicated in polyamines metabolism. literature data speak that spermine and spermidine stimulate activity of cobalamin-dependant methionine synthase, the enzyme that catalyses the recycling of homocysteine to methionine; polyamines inhibit methionine adenosyltransferase. beside the wellknow significance of vitamin b12, in transmethylation reaction, the significance of 5,-deoxyadenozyl cobalamin, except the conversion of methylmalonyl-coa to succinyl coa, is not well elucidates. methionine as s-adenosylmethionine (sam) is essential amino acid for polyamine biosynthesis. sam has frequently usage in treatment of liver diseases. according the mentioned facts the aim of our experiments is to exanimate the significance of application of vitamin b12 alone and altogether with methionine to rats without and with experimentally induced cholestasis. our preliminary results speak about the disturbance of polyamine metabolism in hepatic tissue of rats with cholestasis. application of methionine alone increases the amount of polyamine in rat liver tissue, in-group without cholestasis and with bile duct obstruction. the animal treatment with cobalamin has higher amount of polyamines and lower activity of polyamine oxidase in liver tissues in both groups. the effects of vitamin b12 may be in direct relation with the formation of 5,-methylthio deoxyadenosine (mta), the by-product of spermidine and spermine biosynthesis. the explanation the exact roles of vitamin b12 in polyamine metabolism of liver tissue need the futher investigations. department of molecular genetics, the weizmann institute of science, rehovot, israel exposure of mouse myeloma cells that massively overproduces ornithine decarboxylase (odc) but not of parental cells to ornithine results in a massive increase in the intracellular concentration of putrescine, followed by rapid cell death. the treated odc overproducing cells display fragmented nuclei, chromatin condensation and an oligonucleosome-size dna "ladder"; consequently, their death can be described as apoptosis. the apoptotic process induced by the accumulated putrescine involves the release of cytochrome c from the mito-chondria, activation of caspases cascades demonstrated by the cleavage of caspase-2 and parp, a substrate of caspase-3. the general inhibitor of caspases, bd-fmk, effectively inhibited parp cleavage but failed to inhibit cell death. the intracellular ca 2ϩ chelator bapta/am and the antioxidant bha inhibit parp cleavage. however, only bapta/am inhibit the induction of cell death. it seems that bha subverted the death into caspase independent pathway. treatment with bapta/am did not interfere with the accumulation of putresine following ornithine treatment, suggesting that the accumulated putrescine induces the elevation in the concentration of intracellular ca 2ϩ which then activates the apoptotic process. the dominant anti-apoptotic effect of bapta/am over egta suggests that internal stores are the main source of the elevated ca 2ϩ , but that putrescine is also capable of inducing influx of extracellular ca 2ϩ . extensive small intestine resection results in the loss of absorptive surfaces, acceleration of intestinal transit and, as a consequence, in malnutrition, weight loss, diarrhoea and other complications of short bowel syndrome. the availability of human recombinant growth hormone rgh and its stimulatory effects on gut growth suggested its use in the treatment of short bowel syndrome. the trophic response of gi tract epithelium to hormones such as growth hormone is mediated by polyamines, which are vital in cell proliferation. this study was undertaken in rats to: 1/ evaluate the effects of rgh by monitoring polyamine and amine metabolism parameters in the adapting short bowel and 2/ determine whether erythrocyte (rbc) polyamine concentrations reliably reflect the proliferative activity of the remaining bowel. seventy per cent resection of the small intestine of wistar rats was performed under ether anesthesia leaving equidistant lengths of bowel from pylorus and ileocecal valve. recombinant human gh (0.2 iu, s.c., saizen, serono, switzerland) was administered once daily for 5 or 10 days, to randomly selected rats on the second postoperative day. animals were sacrificed 8, 13 and 21 days after the operation. enzyme activities were measured with radioassays or fluorimetry. polymines were determined as dansyl derivatives by hplc/fluorimetry. gh treated animals had significantly higher intestinal odc and sat and lowel dao activities; higher (non-significant) mucosal growth index and polyamine concentrations than in untreated counterparts on 8 th postoperative day. thereafter the two groups did not differ in the investigated parameters. rbc polyamine concentrations were higher in operated verses control rats; rgh treatment had no significant effect. however, rgh treatment significantly reduced hepatic mao a and b activities. our results suggest gh accelerated the adaptive growth of the bowel remnant. they justify use of erythrocyte polyamine concentration measurement as the marker of small bowel proliferative activity. however, side-effects of this treatment must be considered. tissue transglutaminase (ttg) activity has been evaluated in different neural tissues, such as brain, spinal cord and peripheral ganglia, and appears to be expressed in cerebellar granule cells (cgn) as well as in astrocytes. the role of ttg in neuronal functioning is likely to be quite complex. other than the role during development, significant changes of enzyme activity have been evaluated in different neurodegenerative conditions. it is well known that nmda receptor activation may be able to trigger excitotoxicity. the nmda-induced injury is mainly associated to ion influx and subsequent calcium overload. the effects of nmda application to both, cerebellar granule cells and glial cell cultures, have been assessed. in cgn, ttg activity increased rapidly after a brief stimulation with 100 µm nmda, whereas in glial cell cultures, high levels of enzyme activity was obtained after incubation of 24 h in presence of the same concentration of nmda. such results rule out the possibility that excitotoxicity can modify numerous proteins making them better substrates of ttg, and this could contribute to enhanced ttg-modifications of proteins in response to excitotoxicity. the pote protein can catalyze both uptake and excretion of putrescine. the km values of putrescine for uptake and excretion (putrescine-ornithine antiport) are 1.8 µm and 73 µm, respectively. amino acid residues, cys62, trp201, glu207, trp292 and tyr425 are strongly involved in both activities, and that glu77, tyr92, cys210, cys285, cys286 and glu433 are moderately involved in the activities. mutations of tyr78, trp90 and trp422 mainly affected uptake activity, indicating that these amino acids are involved in the high affinity uptake of putrescine by pote. mutations of lys301 and tyr308 mainly affected excretion activity, indicating that these amino acids are involved in the recognition of ornithine. the putrescine and ornithine recognition site on pote was found to be located at the cytoplasmic surface and the vestibule of the pore consisting of twelve transmembrane segments. the cadb protein has 30% sequence homology with pote protein. cadb can catalyze both uptake and excretion of cadaverine. the km values of cadaverine for uptake and excretion (cadaverine-lysine antiport) are 20 µm and 300 µm, respectively. it was found that two glutamate residues (glu76 and glu204) and four tyrosine residues (tyr73, tyr89, tyr90 and tyr310) are involved in the both activities. the difference of the substrate recognition site on pote and cadb is discussed. a. lentini, b. provenzano, and s. beninati department of biology, laboratory of cell biochemistry, university of rome "tor vergata", rome, italy tissue transglutaminase (ttg, e.c. 2.3.2.13) is a protein cross-linking enzyme which catalyzes an acyl transfer reaction where the carboxamide group of a peptide-bound glutamine is the acyl donor, and a lysine residue the acyl acceptor. polyamines may act as acyl acceptors, leading to the formation of mono-and bis-(γ-glutamyl)derivatives. we provided evidence that ttg activity is directly associated to differentiation markers, and inversely related to cell proliferation and invasion. we have shown the in vivo reduction of experimental melanoma metastasis by i.v. injection of a plasmid (psg5) carrying the ttg gene sequence to c57bl6/n mice. tumor cell metastatization requires specific interactions with subendothelial basement membrane (bm) and migration through the endothelial wall, allowing the colonization of the target tissue. therefore, the investigation on the possible mechanisms responsible for ttg effects is focused on the posttranslational modification of bm proteins. we detected that "matrigel", a tumor-derived complex of bm proteins, modified with polyamines after ttg catalysis, reduces both melanoma cell adhesion and invasion in an in vitro metastatic assay. similar results were obtained using polyamines conjugated to laminin, one of the major bm components, as unique substrate. our findings suggest that the increase of bm proteins conjugated to polyamines may be responsible for impairments of the invasive properties of melanoma cells. we demonstrated that interferon-α (ifnα) induces apoptosis in human epidermoid cancer cells. tissue transglutaminase (ttgase) is an enzyme involved in the regulation of apoptosis through the inactivation of some cell components. among these eukaryotic initiation factor-5a (eif5a) is peculiar because its activity is modulated by the formation of the amino acid hypusine. recently, we found that growth inhibition induced by ttgase is paralleled by reduced hypusine levels. here we report the effects of ifnα on the apoptosis, ttgase modulation and eif5a activity in human epidermoid lung h1355 cancer cells. we have found that 48 h exposure to 2,500 iu/ml ifnα induces 50% growth inhibition and 15% apoptosis in h1355 cells. moreover, ifnα induced a 4-fold increase of ttgase activity and expression that already occurred after 24 h of exposure to the cytokine. this effect was paralleled by a 1.6-fold enhance of ttgase mrnas. ifnα induced also a 30% increased eif5a expression while an about 50% decrease of hypusine levels was observed. increased ttgase activity was paralleled by a decrease of hypusine content and of eif5a activity. therefore, ifnαinduced apoptosis could occur through an increase of ttgase activity and the mechanism by which ttgase regulates biological functions can be the reduction of eif5a activity. adometdc deficient mice k. nishimura 1 , f. nakatsu 2,3 , k. kashiwagi 1 , h. ohno 3 , t. saito 2 , and k. igarashi 1 1 graduate school of pharmaceutical sciences, chiba university, chiba, 2 graduate school of medicine, chiba university, chiba, and 3 cancer research institute, kanazawa university, kanazawa, japan the amd1 gene encodes s-adenosylmethionine decarboxylase (adometdc) that is one of the key enzymes of polyamine biosynthesis. to examine the physiological role of polyamines, we performed the targeted disruption of the gene in mice to generate spermidine-and spermine-free mice. although the level of adometdc mrna decreased by 50% in amd1 ϩ/ϫ mice, adometdc activity reduced only by 30% and spermidine and spermine contents did not change significantly. they showed normal phenotype and life span. to obtain amd1 ϫ/ϫ mice, we intercrossed amd1 ϩ/ϫ mice and determined the genotype of the resulting offspring. however, we could not obtain any amd1 ϫ/ϫ mice from 20 heterozygous intercrosses over. amd1 ϫ/ϫ embryos died early in development, between e3.5 and e6.5 days post coitum. in culture of blastocysts at e3.5, the shapes of all cell lines were normal, but amd1 ϫ/ϫ cells appeared to arrest the cell proliferation at day 3 after the onset of cell culture. the arrest of amd1 ϫ/ϫ cell proliferation was rescued by addition of spermidine. these data indicated that the lethal phenotype of amd1 ϫ/ϫ mice was caused by growth retardation by polyamine depletion at early developmental stage. the formation of active species such as h 2 o 2 and aldehydes during the oxidative deamination of biogenic amines by amine oxidases (ao) suggests for these enzymes a key role in cellular processes. the ability of bovine serum amine oxidase (bsao) to oxidase free amino groups of lysozyme and ribonuclease a has been observed indicating a possible ao involvement in the post-translational protein modification. furthermore, bsao inhibition by h 2 o 2 formed during substrate oxidation under limited turnover conditions was demonstrated, which may be relevant to cellular physiopathology. we have also observed that some inhibitors of mitochondrial amine oxidases (mao) protected human melanoma cell line (m14) against apoptosis. the protection by catalase of mao-substrates induced membrane permeability transition was also obtained in isolated rat liver mitochondria, thus confirming a role of mao-derived h 2 o 2 in apoptosis. enrichment in ao activity by treatment with vegetal ao has been obtained in a erythroleukemia cell line (k562), substaining the possibility to modulate the intracellular ao activity. an antiarrhythmic and cardioprotective effect of bsao has been also observed on isolated rat heart in reperfusion; a protective effect during anaphylaptic crisis has been shown "in vivo", thus suggesting aos as a possible therapeutic agents. tetrakis(3-aminopropyl)ammonium, a unique polyamine produced by an extreme thermophile, stabilizes nucleic acids at high temperature t. oshima and y. terui department of molecular biology, tokyo university of pharmacy and life science, hachioji, tokyo, japan an extreme thermophile, thermus thermophilus, produces tetrakis(3-aminopropyl)ammonium; a novel polyamine containing a quaternary ammonium nitrogen. to clarify the roles of the unique polyamine in thermophily, the effects of tetrakis(3aminopropyl)ammonium on biochemical reactions related to nucleic acids have been investigated. the unique polyamine stabilized both double and single stranded dnas and rnas. tm of a double stranded dna was raised by 8°c by the addition of 0.25 mm of tetrakis(3-aminopropyl)ammonium. at around the boiling temperature of water, depurination of dna takes place. other long polyamines produced by the thermophile such as caldopentaamine also stabilized dnas and rnas. we found that tetrakis(3-aminopropyl)ammonium prevents depurination most effectively. tetrakis(3aminopropyl)ammonium activated the protein biosynthesis catalyzed by a cell-free extract of the thermophile at high temperature. the effects of this unique polyamine on dna and rna polymerases are also being investigated and the results will be presented. tissue transglutaminase (ttg) catalyses the cross-link formation between glutamine (q) residues and nh 2 -donor molecules present in the cells (polyamines, lysine-donor proteins). recently, it has been correlated to neurodegenerative disorders characterised by polyglutamine (q n ) expansion, like huntington's disease. studies carried out on cell extracts revealed that glyceraldehyde 3-phosphate dehydrogenase (gapdh) was found covalently linked to q n domains. however, to date no structural data are available to solve the issue of which residues of gapdh are substrates for ttg. by coupling classical protein chemistry procedures and mass spectrometric techniques we achieved this goal by using as ttg substrates the substance p, an 11-aa peptide bearing the simplest q n domain (q 2 ), and polyamines of different size and shape as q-and nh 2 -donor, respectively. in the present study we report that out of the 26 lysines present in gapdh only three are sites of ttgasedependent cross-link formation in vitro. moreover, to characterize the ttg catalysed cross-link between gapdh and polyq protein we used a synthetic q 17 -peptide as ttg substrate in the catalysed reaction with polyamines. we found that any q residue is a potential ttg substrate, no matter the specific position in the sequence or the steric hindrance of the specific amine under investigation. cjf inserm 95-09, institut contre les cancers de l'apareil digestif (ircad), strasbourg, france as soon as the key role of odc in polyamine metabolism was recognised, it became the major target for selective inhibi-tion. s. harik presented in 1973 the first potent odc inhibitor, α-hydrazino ornithine. although efforts continued until today, with the aim to improve odc inactivation, 2-(difluoromethyl)ornithine (dfmo) remained the most important compound among all polyamine-directed drugs. a known anti-leukaemic drug, methylglyoxal-bis(guanylhdrazone), was recognised early on by g. williams-ashman and his collaborators as an inhibitor of adometdc, the other highly regulated biosynthetic decarboxylase, and served as matrix for more recent developments. in the course of the years selective inhibitors for all enzymes involved in polyamine biosynthesis and degradation were synthesised. moreover, a series of polyamineuptake inhibitors were reported. however, only some of these numerous compounds reached a stage above evaluation as growth inhibitors of cancer cells. owing to the sophisticated homeostatic regulation of the polyamines in cells and organs by de novo synthesis, degradation, uptake and release, and due to the fact that exogenous polyamines (i.e. gut polyamines) can be utilised by the vertebrate organism, the efficacy of selective enzyme and uptake inhibitors remained modest in cancer therapy. the fact the dfmo became the most important drug for the therapy of west and central african sleeping sickness relies on differences of vertebrate and parasite biochemistry. a novel approach, initiated by carl porter, involved the design and synthesis of structural analogues of spermidine and spermine, which do not share the growth-promoting effects of the natural polyamines. a very large variety of homologues, mostly of spermine, with different alkyl-substituents on the primary amino groups, have been studied systematically with regard to their ability to alter enzyme and polyamine patterns, and to inhibit cell growth. in addition polymine-like chains with interposed heteroatoms (0, s, si etc.), and analogues with rigid aliphatic chains (due to inbuilt double and triple bonds, or of small rings) have been explored. the structural analogues either mimic regulatory functions of the natural polyamines, and thus lead to the depletion of endogenous pools of putrescine, spermidine and of spermine, or they prevent growth effects of the natural polyamines by displacing them from functionally important binding sites. the later type may be considered as polyamine antagonists. the actual drugs usually exhibit to some extent polyamine mimetic and antagonist properties. at present several polyamine analogues are in clinical trial. however, after more than 25 years of active research, a polyaminerelated anticancer drug is still not available. one may conclude from this fact that the polyamines are an inappropriate target for cancer treatment. however, it is more likely that polyamine metabolism is a difficult target, because the differences between normal and cancer cells are mainly of quantitative nature. moreover, numerous mechanisms have developed in the course of evolution, which enable the vertebrate organism to prevent lethal polyamine losses. nine novel chemically modified polyamine (pa) analogs were evaluated for their ability to inhibit the pa biosynthesis in rat hepatoma g-27 cell-free system as well as the growth of caov tumor cells. the final concentration of oxy-and aminoadenosine pa analogs or two uracils modified pa analogs were 0.1 mm in the reaction mixture. bis(uracilyl)-analogs and 8-(2-oxyethyl)ami-no-9--d-xylofuranosyladenine supressed pa and putrescine synthesis and in the same conditions were more effective than dl-α-difluoromethylornithine (dfmo) -strong specific inhibitor of ornithine decarboxylase (odc). the other adenosine modified compounds could act both as activators of odc and inhibitors both diamine and polyamine oxidase activities in regenerating liver test system. in contrast to those mentioned above two uracils modified agents as well as dfmo were able to inhibit odc and to increase the rate of oxidative deamination of pa in the same system. thus bis(uracilyl) pa analogs were the most active and may be useful for further investigation as substances having potential antitumor and antiproliferative properties. several studies concerning the periodontal status in adult and adolescent patients treated with fixed ni-ti archwires have been performed, but until now it is not yet available any information about the influence of patient age on gingival tissue responses to ni-ti alloy. recently, researches by us demonstrated that the prolonged use for over 12 months of ni-ti appliances may contribute to local pathological proliferative processes early detectable only through salivary polyamine concentration increase. although other data from our laboratory showed that salivary polyamine amounts are age and sex-independent, nothing is known about the influence of the age on salivary polyamine content m subjects wearing ni-ti appliances. eighty patients, under orthodontic treatment for 12 months, were divided into four groups: the pre-, the mid-, the late-and the post-pubertal. salivary polyamine concentrations were determined by hplc. only the late pubertal group revealed a significant increase in both the spermine and spermidine content, while the other groups showed no modification. the results suggest that gingival pathological responses to a long-term appliance's use may be related to the endocrine modifications that occur in the late-pubertal age. sexual hormones appear to be in synergy with ni-ti alloy in promoting proliferative activity of gingival cells. the effects of polyamines on the synthesis of various σ subunits of rna polymerase were studied to determine how polyamines influence the functional specificity of transcription using western blot analysis. synthesis of σ 28 was stimulated 4.0-fold and that of σ 38 was stimulated 2.3-fold by polyamines, whereas synthesis of other σ subunits was not influenced by polyamines. stimulation of σ 28 synthesis by polyamines occurred at the level of transcription. since our hypothesis is that polyamines regulate macromolecular synthesis mainly at the translational level, we searched for a target protein, related to the polyamine stimulation of σ 28 synthesis, whose translation is altered by polyamines. stimulation of σ 28 synthesis was due to an increase in the level of camp, which occurred through polyamine stimulation of the synthesis of adenylate cyclase at the level of translation. polyamines were found to increase the translation of adenylate cyclase mrna by facilitating the uug codon-dependent initiation. analysis of rna secondary structure suggests that exposure of the shine-dalgarno (sd) sequence of mrna is a prerequisite for polyamine stimulation of the uug codon-dependent initiation. antitumor quinones are approved for clinical use and others antitumor quinones are in different stages of clinical and preclinical development. the efficiency of the quinonic compounds in inhibiting cancer cells growth is believed to stem from their participation in key cellular redox mechanisms with consequent generation of highly reactive oxygen species (ros). the ros is turn modify and degrade nucleic acids and proteins within the cells. recently, quinonic drugs were attached to the neurodecapeptide lh-rh and evaluated as potential drugs in the treatment of different tumours. we have synthesized several series of n-quinonyl amino acids in which five ω-amino acids are attached to p-quinones with different values of redox potentials. the attachment was made via michael-like reductive addition of the amino acids to the quinonic ring or via substitution of a chlorinated atom. the n-ω-quinonyl amino acids were characterized as to their ability to form semiquinone anion radicals by epr and cyclic voltammetry technique. the preparative methods, the redox potentials as well as the physical and spectral data ( 1 h-nmr, ir, uv-vis and hrms) of these n-ω-quinonyl amino acids will be presented. the de novo design of biologically active peptides and proteins, mostly has involved consideration and design of backbone conformations (secondary structures) such as α-helix, -sheets, -turns, etc. (η/ψ space). however, for many bioactive peptides and proteins, especially those critical for information transduction such as neurotransmitters, hormones, antigens, neurocrines, etc. molecular recognition via side chain moieties is of paramount importance. thus far, the specific three dimensional orientations of side chain groups ( angles; chi space) in terms of biological activity has received only modest attention. in part this may be due to the energetics of chi space compared to ramachandran space. in order to overcome the current limitations of evaluating the importance of chi space in critical biological functions related to disease and behavior, we have designed amino acids with novel structures and unique constraints in chi space ( 1 , 2 , etc.), with special attention to their ability to mimic the chi space of native proteins and peptides. we have developed novel and simple asymmetric synthetic methods for such amino acids, often with ees greater than 98%. incorporation of these novel amino acids into bioactive polypeptide neurotransmitters has provided ligands with unique biological activities that effect unique behaviors including feeding, sexual, pain, and addictive behaviors. (supported by grants from the usphs and nida.) protein technology, wallenberg laboratory ii, lund university, lund, sweden we describe a method for comparative quantitation and de novo peptide sequencing of proteins separated either by standard chromatographic methods or by one and two-dimensional polyacrylamide gel electrophoresis. the approach is based on the use of an isotopically labelled reagent to quantitate (by mass spectrometry) the ratio of peptides from digests of a protein being expressed under different conditions. the method allows quantitation of the changes occurring in spots or bands that contain more than one protein, and has a greater dynamic range than most staining methods. since the reagent carries a fixed positive charge under acidic conditions and labels only the n-terminal of peptides, the interpretation of tandem mass spectra to obtain sequence information is greatly simplified. the sequences can easily be extracted for homology searches instead of using indirect mass spectral based searches and are independent of post-translational modifications. dehydroamino acids and their derivatives play important roles as constituents of various natural products and as synthetic intermediates for the preparation of optically pure amino acids. a large number of amino acid derivatives containing a pyrazol-4-yl, isoxazol-4-yl and other heterocyclic moieties has been prepared as potential agonists or antagonists for central glutamate receptors in connection with (r,s)-2-amino-3-(3hydroxy-5-methylisoxazol-4-yl)propanoic acid (ampa), a bioisostere of (s)-glutamic acid. -hetaryl-α, -didehydroalanines might be considered as conformationally constrained ampa analogs and might be potential candidates for the synthesis of novel types of ampa analogs, for example, via their hydrogenation. compounds containing 2h-pyran-2-one ring are also very useful synthons in selective synthesis. recently we have shown their use for the preparation of (e)-α, -didehydroα-amino acid derivatives containing a pyrazolyl moiety (vraničar l, polanc s, kočevar m (1999) tetrahedron 55: 271). as a continuation of our investigation in this field we report here a detailed study of the transformation of 2h-pyran-2-one derivatives 1 with hydroxylamine (2, x ϭ o) and various hydrazines (2, x ϭ nr 2 ) towards novel types of (e)-and (z)α, -didehydroamino acid derivatives 3. in most cases, the reactions were performed under basic conditions in a mixture of ethanol and pyridine. depending on the substrate and the reagent used the reaction could be controlled to give either (e)-or (z)-isomers; in some cases decarboxylation to the corresponding enamines 4 also occured during the reaction course. some attempts to hydrogenate compounds 3 towards α-amino acid derivaties 5 by homogeneous or heterogeneous catalysis were also performed. analogs of endomorphin 1 and 2 were prepared to investigate the effect of the positional and c-terminal amide replacements and modifications on the biological activity. modifications in position 2 and 4 were studied. in position 2 several hydroxy-and serine related amino acids were incorporated, whereas in position 4 the amide bond was replaced by hydroxymethyl and allyl group. protected peptide derivatives were synthesized on 2chlorotrityl resin and further transformed to the corresponding derivatives in solution phase. among the analogs tested, in in vitro tests the most effective compound found was d-ser 2 -endomorphin ϫ2. quite surprisingly, the partial agonist/antagonist properties of the derivatives in receptor binding and g-protein stimulation tests have been shown behave differently. the differences in efficacy and receptor binding properties of the compounds may explain the discrepancies between the in vitro and receptor binding tests. we have been assessing the possible applications of substituted 2h-pyran-2-ones 1 containing α, -didehydroamino acid unit in their structure as dienes in [4ϩ2]-cycloaddition reactions. as dienophiles we have been using different acetylene derivatives as well as n-phenylmaleimide and maleic anhydride. as it is evident from the structure of 2h-pyran-2-ones upon the cycloaddition of acetylene derivatives the first intermediate formed (2) still contains the carbon dioxide bridge. in many cases 2 easily expels co 2 and substituted benzene derivative 3 is produced. when the alkenes are used, the first part of cycloaddition is the same as when acteylene derivatives are used, but after the extrusion of co 2 from the adduct 4 there are two possible paths: so formed cyclohexa-1,3-diene (5) is either aromatized into benzene derivative (3) or it acts as another diene with favourably positioned double bonds and unusual double cycloadducts (6) are formed. since co 2 -containing adducts are thermally unstable it is advantageous to use high pressure techniques. with the acetylene derivatives we have not been able to isolate co 2 -containing adducts (2), while with alkenes we have isolated, depending on the structure pattern of the compound 1, all three types of products: aromatized 3, co 2containing 4 and double adducts 6. especially the type 4 is suitable for further transformations into other heterocycles containing amino acid moiety. research group of peptide chemistry, hungarian academy of sciences, budapest, hungary among the opioid receptors family, the cloning of µ, k and δ receptors was followed by another member, named lc 132 or orl 1 . searching for an endogenous ligand for this receptor resulted in successful identification of a peptide (fggft garksarklanq) called noc or ofq. in vitro and in vivo studies have demonstrated that noc mediates a variety of biological actions. results from structure-activity experiments suggest that the whole sequence of noc is not required for binding to the lc 132 receptor and for full biological activities. noc(1-13)-oh seem to be the minimum and essential sequence for good interaction with the receptor. this neuropeptide, similarly other peptides, are unresisting for enzymatic degradation and the releasing metabolites are very weakly active or inactive. some previous experiments refer to that the c-terminal amidation may protect the peptide from degradation. we purposed to synthesize carbamoyl analogues of noc(1-13)-nh 2 , hoping that these derivatives retain the ability to bind lc 132 receptor and are resistant against biological degradation: phe-nh-co--ala-noc(4-13)-nh 2 phe--ala-nh-co-phe-noc(5-13)-nh 2 phe-gly-nh-co--hphe-noc(5-13)-nh 2 the first step in the synthesis of the carbamoyl analogues was the preparation of the building block [r-co-nh-co-nh-hc(rј)-cooh] by the classical method and then it was incorporated into the peptide by solid phase peptide synthesis. [ nonproteinogenic amino acids and their derivatives are valuable compounds from their pharmacological and biochemical effects. they can be used also in synthesis of peptides, as biomarkers, as the ligands in catalitically active transition metal complexes and so on. it is possible to prepare such amino acids by asymmetric hydrogenation of their prochiral precursors. however high enantioselektivities was reached only in the case of chiral phosphine-rhodium catalysts. recently we showed that high diastereoselectivity in the hydrogenation of linear dehydrodipeptides may be achieved over achiral catalyst in the catalytic system substrate -salts of ca ore mg -pd/c due to formation of dehydrodipeptides complexes with ions ñà 2ϩ or mg 2ϩ and hence increasing of the conformational rigidity of substrates. this phenomenon may as well happen in other dehydrodipeptides, containing nonproteinogenic amino acids. among unnatural amino acids those bearing heterocyclic rings have attracted considerable attention due to the possibility of the heteroatoms participation in coordination with ions of metals. we have received some n-acyldehydrodipeptides, containing in the prochiral unit of dipeptides nonproteinogenic dehydroamino acids. all this n-acyldehydrodipeptides form in alcohol solution complexes with cax 2 and mgx 2 where one metal ion binds together several (up to 5) substrate molecules. this kind of complexation leads to the increase of conformational rigidity and to the diastereoface shielding of cϭc bond. moreover the combination of cations (ca 2ϩ or mg 2ϩ ) and anions (x) and the sequence of their mixing with a substrate determine the assembly inside complex particles and hence the sign and degree of asymmetric induction. indeed hydrogenation of these complexes formed in situ over achiral heterogeneous catalyst (pd/c) gives two diastereomers of corresponding n-acyldipeptides with the substantial increase of the reaction diastereoselectivity (up to 80%). in living cells, glutamine represents one of the main storage forms of nitrogen and is a major physiological source of ammonia for the biosynthesis of aminoacids, aminosugars, purine and pyrimidine nucleotides and coenzymes. glutamine-dependent amidotransferases perform nitrogen transfer from the amide group of glutamine to various electrophiles. when the latter is fructose-6p, the product of the reaction catalysed by glucosamine-6p synthase is d-glucosamine 6-phosphate, a structural building block of peptidoglycane (bacteria) and of chitin and mannoproteins (fungi). fluorinated analogues of glutamine are expected to interfere with this biological process due to the strong electron withdrawing effect of fluorine atom (without significant steric consequence), inducing modulation of binding and/or electronic properties. these compounds might therefore behave as reversible or irreversible active site-directed enzyme inhibitors. synthesis of optically active 1 from d-serine will be described and first results in the biological evaluation on glucosamine 6-phosphate synthase will be included. o. melnyk 1 , d. bonnet 1 , e. loing 2 , l. bourel 1 , and h. gras-masse 1 1-umr 8525, 2-sedac-therapeutics, biological institute of lille, france lipopeptides, owing to their ability to cross passively the cell membrane or biological barriers, are unique tools for the intracellular delivery of bioactive peptides. the structure of the lipophilic moiety is known to have a profound effect upon the interaction with the membrane and its alteration. the stepwise solid phase synthesis of lipopeptides is limited by the necessity to perform a complex rp-hplc purification following the cleavage and deprotection step. in addition, the harsh conditions used during the final acidolysis procedure does not allow the introduction of unsaturated or sensitive fatty acids. to speed up the access to large lipopeptides modified by various fatty acid moieties or cholesterol derivatives, we have designed novel synthetic methods which involve the chemoselective reaction of fully deprotected and purified hydrazinopeptides with fatty acid succinimidyl esters or glyoxylyl derivatives. application of these methodologies to the c-terminal 95-135 portion of interferon (ifn)-γ allowed the selection of the optimal lipopeptide ifn-γ agonist, as determined by its ability to induce the expression of surface mhc-ii molecules through interaction with the intracellular components of ifn-γ receptor. graduate school of science, osaka city university, osaka, japan glutamate receptors in mammalian cns are implicated in the construction of memory and early learning as well as in the pathogenesis of neuron damage to cause various neuronal diseases. in recent years, we have studied the conformational role of l-glutamate when it binds to the receptors through the synthesis of l-2-(carboxycyclopropyl)glycines (ccgs) and their related analogs. the works have demonstrated that not only the receptors require a specific conformation of l-glutamate, but also these analogs can be used as important tools for the neuropharmacological research. among them, dcg-iv, a 3јsubstituted analog of ccg-i, is used as a potent and selective agonist of mglur2. as an extension of these works, next program was focused on the synthesis of α-substituted glutamate analogs which would enable to develop potent and subtype-selective ligands for mglurs and transporters. α-alkoxymethylglutamate and ly354740 and its c5 epimer were chosen for the synthetic targets, since the former slightly restricts the glutamate conformation to an extended form and the latter rigidly fix to an extended or a folded form on its bicyclo[3,1,0]hexane skeleton. the key to the synthesis was a stereoselective construction of the quarternary carbon center, which was efficiently performed based on an asymmetric version of the strecker synthesis. details of the synthesis and their neuropharmacological activities will be described. using a genetically modified organism a broad variety of linear unsaturated amino acids are now accessible in enantiomerically pure form via this methodology, which can be used as starting materials for the synthesis of highly functionalized pipecolic acid derivatives. these compounds can be used to restrict conformations in polypeptides or can serve as scaffolds in synthesizing libraries for drug discovery. the synthetic approach involved both a pd-catalyzed amidopalladation reaction of alkoxy-allenes, in which the nh is added across one allene double bond and a ruthenium catalyzed ring closing metathesis step, to form a benzyloxypipecolic acid. further reaction of this n-sulfonyl-iminium-ion precursor with a nucleophile results in the formation of cis-substituted pipecolic acids. due to the unique electronic properties of fluorine, incorporation of α-fluoroalkylated amino acids is a new approach to design biologically active peptides with increased metabolic stability and defined secondary structure and provides a powerful nmr label for spectroscopic investigations. the application of proteases especially for cn-ligations is an attractive alternative to chemical methods, because the enzymatic formation of peptide bonds is highly regio-and stereospecific and, therefore, does not require large efforts to protect side chains of trifunctional amino acids. recently, the enzyme-catalyzed incorporation of α-fluoromethyl amino acids into the p 2 , p 3 and p 2 ј-position (nomenclature according to schechter and berger) of peptide fragments has been successfully performed. carboxypeptidase y was now shown to be suitable to catalyze the incorporation of α-trifluoromethyl alanine into the p 1 position of peptides. furthermore, the general applicability of the substrate mimetic concept in enzymatic peptide synthesis was expanded to the transfer of c-terminal α-fluoroalkyl substituted amino acids. generally, each trifluoromethyl-and difluoromethyl amino acid 4guanidinophenyl esters can be applied as acyl donor in trypsin and chymotrypsin catalyzed peptide bond formation independently of the acyl moiety and the natural enzyme specificity, respectively. via these two approaches, incorporation of αfluoroalkylated amino acids into the p 1 position of peptides using enzymatic methods was successfully applied for the first time. this investigation was performed in search of new 2јdeoxynucleoside analogues modified at 3ј-and 5ј-positions with amino acids and possessing antiviral activity. substrate mimetics strategy: an efficient approach to protease-catalyzed peptide ligation n. wehofsky 1 and f. bordusa 1,2 1 max-planck-society, research unit "enzymology of protein folding", halle, and 2 institute of biochemistry, university of leipzig, germany two main drawbacks seriously restrict the synthetic value of proteases as reagents in peptide fragment coupling: (1) native proteolytic activity and, thus, risk of undesired peptide cleavage; (ii) limited enzyme specificities restricting the amino acid residues between which a peptide bond can be formed. the latter can be overcome by the use of substrate mimetics. contrary to common acyl donors, substrate mimetics bear a binding site specific ester leaving group instead of having a specific amino acid moiety at the c-terminus of the acyl residue. this replacement mediates the acylation of the protease by nonspecific acyl residues. deacylation of the artificial acyl enzyme intermediate by the amino component added results in peptide bond formation regardless of the primary specificity of proteases enabling nonspecific coded and noncoded amino acid derivatives and even non-amino acid-derived acyl moieties to be coupled. the successful application of these artificial substrates for model peptide ligations catalyzed by the argspecific trypsin, the glu-specific staphylococcus aureus strain v8 protease (v8 protease), and α-chymotrypsin, which is specific for aromatic amino acid moieties, will be demonstrated. new development in the tritium labelling of peptides and proteins using solid state catalytic isotopic exchange with spillover-tritium yu. a. zolotarev 1 , a. k. dadayan 1 , b. v. vaskovsky 2 , and n. f. myasoedov 1 1 institute of molecular genetics, russian academy of sciences, and 2 shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia the reaction of high temperature solid state catalytic isotope exchange (hscie) of hydrogen in peptides and proteins with spillover-tritium was studied. the reaction ability of amino fragments in hscie was shown to depend both of their structure and on the availability and the mobility of the polypeptide chain. [ 3 h] peptide analysis using 3 h nmr spectroscopy was carried out, and the modified fragment [ 3 h]actc 4-10 (met-glu-his-phe-gly-pro), with molar activity of 80 ci/mmol and [ 3 h] zervamicin iib (ac-trp-ile-gln-iva-ile-trh-aib-leu-aib-leu-hyp-gln-aib-hip-aib-pro-phl, where aib ϭ 2amino-isobutyric acid) with molar activity of 70 ci/mmol was produced. the obtained preparations completely retained their biological activity. with the -galactosidase protein from termoanaerobacter ethanolicus as example, the interrelation between the protein's tertiary structure and the isotopic label distribution incorporated due to the hscie reaction was used. the labeled protein with the molecular mass of 83 kda was brought to fragmentation by glu-proteinase. peptide fragments were separated by hplc and were identified by maldi mass spectrometry. a correlation between the position of the amino acid fragment in the protein tertiary structure and its reaction ability in the hscie reaction was obtained. data on the retention of thegalactosidase enzymatic activity in condition of tritium label introduction are supplied. taurine chloramine modulates cytokine production by peripheral blood mononuclear cells m. chorą z . y 1 , e. kontny 1 , j. marcinkiewicz 2 , and w. maśliń ski 1 1 institute of rheumatology, warsaw, and 2 jagiellonian university, cracow, poland objective. proinflammatory cytokines are produced in a cascade fashion, where monocyte-derived tnfα and il-1 trigger production of il-6 and il-8 also in the other cell types. we reported recently that taurine chloramine (tau-cl) inhibits production of the latter cytokines in fibroblast-like synoviocytes. in present study the effect of taurine (tau) and tau-cl on tnfα, il-1 and il-6 production was examined. methods. peripheral blood mononuclear cells from healthy volunteers were stimulated with lps (24 h) in the presence of tau or tau-cl (100-500 µm). cytokine production was measured in culture supernatants (secreted) and cell lysates (intracellular) using elisa. results. in lps-stimulated cells both secreted and intracellular il-1 and il-6 were inhibited by tau-cl with ic 50 ϸ 300 µm and 425 µm, respectively. however, tau-cl exerted dual effect on tnfα production, raising it slightly (1.5 times) at low (100-200 µm) while reducing it (ic 50 ϸ 450 µm) at higher concentration. tau did not significantly affect cytokine production. tau-cl modulates proinflammatory cytokine cascade and eventually might down-regulate it when present at high (ͼ300 µm) concentration. department of biology, division of general physiology, university of oslo, blindern, norway every living cell must deal with osmotic and hydrostatic pressure changes between its environment and its interior and counteract volume changes. swelling activated channels is one group of effectors in the cell membrane that is important in preventing excessive volume increases by releasing inorganic ions and organic solutes that include taurine. such channels are associated with several physiological processes, but little is known about their activation mechanisms. we have used a rat thyroid cell line (frtl-5) to investigate the activation of a swelling sensitive [3h]taurine efflux pathway. hypo-osmolality and thyrotropin (tsh, 500 µm) increased transiently the rate coefficient for [3h]taurine efflux with a similar pattern of activation. the phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine (100 µm) increased the swelling activated efflux rate coefficient 6.4 times above the control level and the camp analogue dibutyryl-camp (500 µm) activated the pathway. these results indicate that both swelling and tsh activation of the taurine efflux pathway are mediated by camp. other aspects of the signal transduction pathway will be discussed. based on the inclination of n-chloroamines to disproportionate, the endogenous bactericidal agent n-chlorotaurine (nct), mainly at ph ͻ 7, is accompanied by n, ndichlorotaurine (ndct). since pure ndct could be synthesized as crystalline sodium salt, a first evaluation of its chemical and bactericidal properties was possible. ndct-na (melting point: 162-4°c, decomp.) is very well soluble in water and poorly in ethanol where it can be recrystallized from. on storage the initial ph 5 of the aqueous solution decreases which correlates with a decrease of oxidation capacity of 1.8% per day, probably originated by the elimination reaction r-ch 2 -ncl 2 ae r-chϭncl ϩ h ϩ ϩ cl ϫ as a first step. contrary to nct-na an immediate decomposition occurs when ndct-na comes into contact with undiluted dmso. in aqueous solution, however, ndct does not react with dmso. the bactericidal activity of 55 mm ndct at ph 7 and 5 against the gram-positive bacteria s. epidermidiand two strains of s. aureus was the same as with equimolar nct though ndct bears twice the oxidation capacity. against the gramnegative bacteria e. coli, p. aeruginosa, and p. mirabilis, however, a significantly higher activity of ndct was observed at both ph. the mechanism of taurine chloramine inhibition of fibroblastlike synoviocytes growth e. kontny 1 , m. kurowska 1 , j. kowalczewski 1 , i. janicka 1 , j. marcinkiewicz 2 , and w. maśliń ski 1 1 institute of rheumatology, warsaw, and 2 jagiellonian university, cracow, poland objective. in rheumatoid arthritis (ra) enhanced proliferation of fibroblast-like synoviocytes (fls) leads to hyperplasia of synovial membrane (sm). therapeutic approaches to inhibit an excessive growth of these cells are not satisfactory. thus, we investigated the effect of taurine (tau) or taurine chloramine (tau-cl) on ra fls growth. methods. fls isolated from sm of ra patients were stimulated for 72 hours with rhpdgf or rhtnf-α. tau or tau-cl were added at 100-250 µm concentrations. cell proliferation was determined by incorporation of 3 h-thymidine into dna. expression of proteins regulating cell-cycle progression or apoptosis, was estimated by western blotting. results. at 250 µm concentration tau-cl inhibited (by ϸ 70%) both pdgf-and tnf-α-triggered cell proliferation and similarly reduced expression of pcna (a cofactor for dna polimerase δ). however, tau-cl affected neither the expression of cell-cycle inhibitors (p21, p27) nor anti-apoptotic bcl-2 protein. tau has no effect on tested responses. conclusion. we report that tau-cl inhibits proliferation of ra fls by affecting expression of pcna, that is critical for cell cycle progression. e. kontny 1 , k. szczepań ska 1 , j. kowalczewski 1 , m. kurowska 1 , i. janicka 1 , j. marcinkiewicz 2 , and w. maśliń ski 1 1 institute of rheumatology, warsaw, and 2 jagiellonian university, cracow, poland objective. proinflammatory cytokines play critical role in the pathogenesis of rheumatoid arthritis (ra). we reported recently that taurine chloramine (tau-cl), but not taurine (tau), inhibits production of il-6 and il-8 by fibroblast-like synoviocytes (fls). in present study the mechanism of tau-cl inhibitory action was investigated. methods. fls isolated from synovial membrane of ra patients were stimulated with rhil-1 . tau or tau-cl were added at 250-500 µm concentration. after 0.5-2 h or 4 h the dna binding activity of nfkb and ap-1 (emsa) and the expression of il-6 and il-8 mrnas (rt-pcr) was examined, respectively. results. il-1 raised nfkb and ap-1 activity, followed by the elevation of cytokine mrnas expression. tau-cl, but not tau, reduced both the expression of cytokine mrnas (il-6 ͼ il-8) and the activity of transcription factors (nfkb ͼ ap-1). conclusion. tau-cl inhibits transcription of il-6 and il-8 genes due to its ability to diminish the activity of key transcriptional factors, that regulate these proinflammatory cytokine expression. institut für organische chemie, universität bremen, germany the synthesis of taurine and hypotaurine from cysteine can be followed up in astroglia-rich primary cultures obtained from brain of neonatal wistar rats. using 1 h and 13 c nuclear magnetic resonance spectroscopy cell extracts of glia cells incubated with 13 c labelled cystein show the label subsequently in hypotaurine, taurine, lactate and gluthathione. within 72 h, 35% of the total intracellular hypotaurine and 22.5% of taurine were newly synthesized from cysteine. both metabolites were also released to the medium. neurones are capable to take up both metabolites from glia media to recruit their organic osmolite. part of newly synthesized glutathione and lactate are also exported to the medium. by this means lactate may serve as an energy substrate for neurons. in-vivo mrs of lactate is obscured by line splitting and signal overlay. using various two dimensional pulse sequences as spin preparation sequences prior to localized single voxel, in-vivo mrs or spectroscopic imaging sequences will provide homonuclear non-coupled resonance signal of taurine. these singlet signals are detectable and quantified. diffusion weighted spectroscopy is used to characterize the mobility of taurine in living tissue. department of pharmacology and ophthalmology and visual sciences, texas tech university health sciences center, lubbock, texas, u.s.a. taurine depletion, whether by removing taurine from the diet or by using a taurine transport inhibitor, has demonstrated various pathologies in various animal models including man. the first reported pathology associated with dietary taurine depletion was in the retina of the cat. in this animal model, taurine deficiency resulted in disorganization of the tapetum (the light reflecting membrane), disruption of the outer segments, photoreceptor dysfunction, and cell loss. when allowed to proceed for a number of months the result was blindness. subsequent studies demonstrated that taurine deficiency also had a profound effect on cardiac physiology. echocardiograms of the left ventricle of the cat heart depleted in taurine showed a dilated cardiomyopathy reflected in an extended end-diastolic diameter and an extended end-systolic diameter. dietary taurine supplementation resulted in the above parameters returning to normal. the cat is a difficult animal model to use for a variety of reasons and thus the rat was chosen to further probe the consequences of taurine depletion. unfortunately, the tissue taurine levels in the rat do not respond to dietary taurine depletion, and thus other experimental means had to be designed. guanidinoethanesulfonic acid (ges), an analogue of taurine and a taurine transport inhibitor, has been utilized for the last 20 years to deplete rat tissues of their taurine content (j. e. shaffer and j. j. kocsis, methods of reducing tissue taurine levels, and r. j. huxtable, h. e. laird, and s. lippincott, rapid depletion of tissue taurine content by guanidinoethyl sulfonate. in: the effects of taurine on excitable tissues, spectrum publications, new york, 1981) . ges, when administered to rats in their diet in the drinking water as a 1-1.5% solution, usually produces a significant decrease in the taurine content of all tissues within one week of treatment. within 3-5 weeks the levels of taurine reach their nadir (20-50% of control) and continued feeding of ges does not further reduce the levels of taurine. unfortunately, ges replaces taurine and thus one must always consider the effects of ges on physiological events that occur within the tissues in question. again, as in the cat, taurine depletion manifested itself in retinal pathology: disruption of the photoreceptor structure, dissociation of the disc membranes, and abnormal electroretinograms (erg). other animals models such as the monkey have also demonstrated structural disorganization of the photoreceptors and abnormal ergs. finally, the ultimate test is whether taurine deficiency has an effect in man. in 1985, koppel and associates (geggel et al., n. eng. j. med. 312: 142-146, 1985) demonstrated that children on long term parenteral nutrition devoid of taurine had abnormal erg. supplementation of the parenteral nutrition with taurine restored the ergs to normal in the majority of the children. because of these definitive studies, all infant formulae in the united states, europe and japan now contain taurine. (supported in part by a grant from the taisho pharmaceutical co., ltd., tokyo, japan.) department of pharmacology and ophthalmology and visual sciences, texas tech university health sciences center, lubbock, texas, u.s.a. it has been demonstrated previously in our laboratory that taurine inhibits the phosphorylation of an ϳ44 kdalton protein present in the mitochondrial fraction of the rat heart (j. mol. cell. cardiol. 26: 1675 -1689 , 1994 . upon administering 1.5% guanidinoethanesulfonic acid (ges) in the drinking water of rats for 6 weeks, the taurine levels in cardiac tissue decline by 75%. however, the phosphorylation of a ϳ44 kdalton protein in the mitochondrial fraction of the heart tissue increased by 85% (j. mol. cell. cardiol. 26: 1675-1689, 1994) . reversal of these effects could be accomplished by feeding the rat 1.5% taurine in the drinking water for 6 weeks. the ϳ44 kdalton protein was isolated by 1-dimensional polyacrylamide gel electrophoresis (page) using traditional glycine buffers followed by re-electrophoresing the cut out portion of the gel, which corresponds to the ϳ44 kdalton protein, on a tricine-buffered gel resulting in sufficient pure protein for digestion and sequence analysis. it was determined that the ϳ44 kdalton was pyruvate dehydrogenase (amino acids 12: 139-144, 1997) which indicates a significant regulatory role for taurine in energy metabolism in cardiac tissue. these data are of significant interest in that taurine may be an additional effector of this enzyme or of the enzyme complex. studies are in progress to determine if taurine has a direct effect on either the kinase (inhibition) or the phosphatase (stimulation) associated with the pyruvate dehydrogenase complex. it has also been demonstrated and now reported that taurine depletion utilizing ges in vivo in rats affects the phosphorylation of myelin basic protein (mbp). in these experiments the animals were given ges (1%) for 6 weeks in their water and then killed; the hearts were removed and homogenized. the homogenate was then incubated with buffer containing mbp (50 ì) and radioactive atp for 6 minutes. animals were also treated with taurine (1%) in their drinking water for 4-5 weeks or treated with taurine following the ges treatment. page of the incubation mixture, autoradiography on the dried gel, and densitometry of the mbp band gave the following results: relative % activity ϯ sem (normalized to mg protein) control 100 ϯ 20 (n ϭ 6) ges-treated 147 ϯ 28 (n ϭ 6) p ͻ 0.05 (paired 5-test) control 100 ϯ 16 (n ϭ 5) taurine-treated 95 ϯ 17 (n ϭ 5) p ͼ 0.05 control 100 ϯ 32 (n ϭ 4) ges followed by taurine 95 ϯ 37 (n ϭ 4) p ͼ 0.05 these data confirm previous reports that it is easier to deplete animals of their cardiac taurine content than it is to raise the levels of taurine. these data on the effects of taurine depletion (increase in mapkinase activity) and taurine supplementation (no change in mapkinase activity) on mapkinase activity reflect these past observations. (supported in part by a grant from the taisho pharmaceutical co., ltd., tokyo, japan.) act additively, or in the case of mpo negated each other's effects. regarding our results there is significance to pharmacological regimens which enhance the supply of propofol or taurine in whole blood. these regimens influence considerably pmn intracellular amino acid concentrations and it is this pmn "labile free amino acid pool" which may be one of the determinants in cell nutrition positively or adversely affecting pmn immune functions. taurine supplementation to pmn seems to interfere independently from the effects of propofol on pmn free amino acids and on immune functions tested. institut für hygiene, universität innsbruck, austria n-chlorotaurine (nct) is a long-lived oxidant produced by activated human leukocytes during the oxidative burst. it has activity against a broad spectrum of pathogens including bacteria, fungi, viruses and helminths. as a special feature, the killing of microbes by nct can be increased significantly in the presence of ammonium and also of some amino acids (alanine, glycine). this is explained by transfer of the active chlorine ("transhalogenation") from nct to ammonium and amino acids to form the corresponding, stronger microbicidal n-chloro derivatives monochloramine and n-chloro amino acids, respectively. especially addition of ammonium to nct provokes rapid inactivation of fungi and even mycobacteria. because of its good tolerability, nct solution can be applied to human tissue to treat infections. in ammoniumcontaining body fluids like nasal mucus and urine, fungi and bacteria are killed within minutes. therefore, amino compounds of human secretions can be transformed to the above quoted endogenous and highly microbicidal chloramines by nct via transhalogenation -a unique property of an antimicrobial agent. successful treatment in cases of urinary tract and otorhinolaryngological infections and conjunctivitis in phase iia clinical trials provides strong support for this concept. the endogenous sulfonated amino acid taurine has numerous functions in the central nervous system, including positive modulation of gaba a receptor function. recently we found that mice lacking protein kinase c -epsilon (pkcε) are behaviorally and biochemically supersensitive to ethanol and other positive allosteric modulators of the gaba a receptor. in addition, these mice consume 50-75% less ethanol and wildtype controls in two separate self-administration paradigms. microdialysis studies in pkcε-deficient mice revealed elevated extracellular levels of taurine, which may account for the supersensitivity of gaba a receptors in these mice and resulting decreases in ethanol intake. in light of the fact that the taurine derivative acamprosate (calcium acetylhornotaurinate) is moderately effective in reducing craving and relapse in detoxified alcoholics, we examined the effect of taurine-related compounds on acute ethanol consumption in a two-bottle choice paradigm in rats. taurine (10-200 mg/kg ip) was only slightly effective in reducing ethanol intake but not preference, while the highest dose of taurine (200 mg/kg) also suppressed water intake. the taurine precursor hypotaurine (10-100 mg/kg ip) was also weakly effective in reducing ethanol intake but not preference or water intake. the most effective compound tested was homotaurine (10-100 mg/kg ip), which suppressed ethanol intake and preference by approximately 50% without altering water intake. these data indicate that endogenous taurine may regulate sensitivity to ethanol and subsequent ethanol self-administration, and that taurine-related compounds may be effective in reducing alcohol intake in humans. we are currently exploring whether taurine and related compounds are able to suppress ethanol-stimulated mesolimbic dopamine release, a primary neural substrate of ethanol reinforcement. (this work was supported by funds provided by the state of california for medical research on alcohol and substance abuse through the university of california at san francisco.) organic osmolytes, such as taurine, regulate a cell's osmotic balance without directly altering either the cell's ionic composition or the membrane potential. this property of the organic osmolyte often renders the cell resistant to damage during a pathological insult. indeed, ischemia is associated with a massive efflux of taurine from the cell, an event that minimizes the severity of the osmotic imbalance that develops from the accumulation of lactate, inorganic phosphate and sodium. however, taurine depletion also activates specific signaling pathways that provide further protection to the cell. among the signaling pathways activated by taurine depletion is a pi 3-kinase (phosphatidylinositol 3-kinase) linked pathway that catalyzes the phosphorylation and inactivation of the pro-apoptotic factor, bad. taurine depletion also activates protein kinase c, which in turn elevates the intracellular content of the antiapoptotic factor, bcl-2. increases in the extracellular osmolality by either addition of 20 mm taurine or 25 mm mannitol to the incubation medium activates similar pathways. however, pi 3kinase assumes a more important role in the mannitol treated cell than the taurine depleted cell. moreover, p38 map kinase is activated by mannitol treatment but not by taurine depletion. despite these differences, both taurine depletion and mannitol treatment protect the cell against hypoxia-induced apoptosis. the data suggest that osmotic stress protects the cell against apoptosis by increasing cellular levels of bcl-2 and promoting the inactivation of bad. this work was supported by a grant from the american heart association. a dummy or a protagonist on the stage of inflammation? r&d department, zambon group, bresso, milan, italy amino acids are usually present in large excess in healthy and the excess is used as source of calories. however, metabolic alterations are observed in ill patients and preferential retention of sulphur amino acids (saa) occurs during the inflammatory response. the metabolism of cysteine is modified during the acute phase of sepsis in rats. sulphate production is lower, whereas the higher liver production of taurine seems to play a protective role; glutathione concentration is greater in liver, kidney and other organs and cysteine incorporation into proteins was higher in spleen, lung and plasma (acute phase proteins) while albumin level decreases. another important phenomenon is the impairment of methionine conversion to cysteine during stressed condition. premature infants or hiv patients synthesise cysteine from methionine at a much lower rate. thus, the metabolic flow through the trans-sulphuration pathway may be insufficient to meet the glutathione and cysteine requirement in critical conditions. the pro-inflammatory cytokines, interleukin-1, interleukin-6 and tnf-α are the main initiates that alter protein and amino acid metabolism. in this complex picture, saa supply may contribute to the immune system regulation. department of applied biological chemistry, the university of tokyo, japan the intracellular level of taurine is maintained not only by the taurine transporter that transports extracellular taurine inside cells but also by endogenous synthesis from methionine and cysteine. we therefore investigated the regulation of both the taurine transporter and the cysteine dioxygenase, one of the main taurine biosynthetic enzymes, in hepg2 human liver cells. the intracellular taurine content of hepg2 cells was extremely increased by culturing in a hypertonic medium. the activity of taurine transport was increased by hypertonic conditions, which was due to the increased expression of the taurine transporter gene. the expression level of the cysteine dioxygenase gene was also increased, suggesting that the expression levels of both the taurine transporter gene and the cysteine dioxygenase gene were regulated in harmony by hypertonic conditions to accumulate taurine inside cells. on the other hand, the activity of taurine transport in hepg2 cells was down-regulated on culturing the cells in taurine-rich medium, the expression level of the taurine transporter gene being also markedly decreased. however, the expression level of the cysteine dioxygenase gene was not significantly altered under taurine-rich conditions, indicating that the gene expression of the taurine transporter and that of the cysteine dioxygenase was independently regulated by extracellular concentration of taurine. the amino acid, taurine, is found in very high concentration in the heart. although its most important putative function is osmoregulation, it also serves as a regulator of cell growth. isolated cardiomyocytes exposed to medium containing 1 nm angiotensin ii undergo hypertrophy, a process blocked by 20 mm taurine. the amino acid also inhibits angiotensin iiinduced activation of c-fos, upregulation of atrial natriuretic factor and induction of tgf-betal. central to virtually all of these actions of angiotensin ii is the translocation and activation of key protein kinase c (pkc) isoforms. therefore, we proposed that taurine inhibited the hypertrophic actions of angiotensin ii by interfering with the translocation of one or more of the pkc isoforms. indeed, taurine and angiotensin ii exhibited different effects on the translocation of several pkc isoforms. while taurine promoted the translocation of pkcalpha, pkcdelta and pkcepsilon from the particulate fraction to the cytosol, the levels of the three isoforms in the particulate fraction were elevated following treatment with angiotensin ii. by contrast, both taurine and angiotensin ii increased the pkczeta content of the particulate fraction and the pkcbeta2 content of the cytosol. when the isolated cardiomyocytes were incubated with medium containing both angiotensin ii and taurine, the effects on pkc distribution were largely additive. these data support the notion that taurine prevents the hypertrophic effects of angiotensin ii by interfering with the translocation of either pkcalpha, pkcdelta, pkcepsilon or a combination of more than one of the isoforms. (the study was supported by a grant from taisho pharmaceutical co.) main final metabolites of l-cysteine in mammals are sulfate and taurine, and they are excreted in the urine. our previous studies in rats have shown that the ratio of urinary sulfate and taurine in rats fed diet containing sufficient methionine and cysteine is 10: 2-3. in the present study, we determined urinary sulfate and taurine in urine samples of 58 healthy japanese women after 12h starvation following usual meal. free (inorganic) and total (free ϩ ester) sulfate were determined with ion chromatography, and taurine by reversed-phase hplc after dabsylation. average excretions (micromols per mg of creatinine) were: total sulfate, 12.53 ϯ 3.85; free sulfate, 11.57 ϯ 3.69; ester sulfate, 0.96 ϯ 0.94; taurine, 0.78 ϯ 0.53; urea, 187.71 ϯ 66.13. the ratio of total sulfate and taurine was 10 : 0.62. this suggests that sulfate formation in humans is more dominant than taurine formation as in rats and this tendency is more evident in humans than in rats, which is in accordance with low cysteinesulfinate decarboxylase activity in humans. sum of sulfate and taurine excretions was significantly correlated with that of urea: correlation coefficient, 0.675. this indicates that sulfur metabolism in humans is in the state of sulfur equilibrium similar to that of nitrogen and reflects protein metabolism. h. yokogoshi 1 and h. oda 2 1 laboratory of nutritional biochemistry, school of food and nutritional sciences, the university of shizuoka, and 2 department of applied biological sciences, nagoya university, nagoya, japan the effect of taurine on hypercholesterolemia induced by feeding a high-cholesterol (hc) diet to rats was examined. when various amounts of taurine (0.25-50 g/kg) were supplemented to hc for 2 wk, serum total cholesterol gradually and significantly decreased in a dose-dependent manner, compared with the control (cholesterol free) diet group. by contrast, serum hdl-cholesterol was elevated by taurine supplementation. in the hypercholesterolemic rats fed the hc diet, the excretion of fecal bile acids and hepatic cholesterol 7α-hydroxylase (cyp7a1) activity and its mrna level increased significantly, and the supplementation of taurine further enhances these indexes, indicating an increase in cholesterol degradation. agarose gel electrophoresis revealed that, in hypercholesterolemic rats fed the hc diet, the serum level of the heavier vldl increased significantly, but taurine repressed this increase and normalized this pattern. significant correlations were observed between the time-and dose-dependent increases of cyp7a1 gene expression and the decrease of blood cholesterol concentration in rats fed the hc diet supplemented with taurine. these results suggest that the hypocholesterolemic effects of taurine observed in the hypercholesterolemic rats fed the hc diet were mainly due to the enhancement of cholesterol degradation and the excretion of bile acid. in vitro studies have shown that ammonia, which is responsible for neurological symptoms associated with hyperamonemia, causes a massive release of taurine from cultured cns cells and brain slices. in this study, taurine (tau) release was measured in vivo in rat striatum following direct application to the microdialysis tube of 60 mm ammonium chloride which renders the final ammonia concentration in the extracellular space of ϳ5 mm. various in vivo stimuli evoke taurine efflux either by opening osmosensitive anion channels and/or by a mechanism secondary to glu accumulation and its interaction with nmda or ampa receptors. the following compounds were coadministered with ammonia to distinguish between these mechanisms: anion/cation transport inhibitors -dids and furosemide, a glu transport inhibitor-pdc, and nmda and ampa/ka receptor antagonists dizocilpine and dnqx. ammonia stimulated tau accumulation in the microdialysates to ϳ250% of basal value. dids and furosemide moderately inhibited the effect of ammonia (furosemide by ϳ30%), albeit dids added alone induced massive accumulation of tau with a delayed onset as compared to ammonia. ammonia-dependent tau accumulation was increased by ϳ50% in the presence of pdc and reduced in an equal degree (ϳ35%) by dizocilpine and dnqx. none of the agents affected tau accumulation in the absence of ammonia. the results show that ammonia in vivo evokes tau accumulation both via anion channels, possibly secondary to cell volume changes, and in consequence of stimulation of both nmda and ampa/ka receptors. (supp. by a scsr grant no 4p05a05519 and cimo, the acad. of finland) biosciences department, university of hertfordshire, hatfield herts, u.k. the discovery in 1987 that endothelium-derived relaxing factor is nitric oxide (no) was followed a year later with reports that the cationic amino acid l-arginine is the physiological precursor for nitric oxide. it has since been established that the terminal guanidinium nitrogen of l-arginine is metabolised via a series of oxidation reactions resulting in no production, with citrulline being formed as a co-product. of interest was the parallel observation that uptake of l-arginine was enhanced in inos expressing cells and that this was due to de novo synthesis of carrier proteins. the precise signaling pathway that regulates the enhanced expression of these carriers has been the subject of intense studies in recent years. current literature suggests that activation of upstream signaling molecules such as protein kinase c may be critical. in addition, downstream kinases thought to be points of convergence for various signals originating from cell surface receptors have also been implicated. two of these downstream targets include the 42 and 44 kda forms of mitogen-activated protein kinase (mapk) and the stressactivated 38 kda mapk. it is worth noting however that the involvement of these different transduction pathways in the regulation of the induction of l-arginine transporters is not universal, and likely to be different from system to system. as a result there has been conflicting data on the relevance of these signaling proteins in inducing l-arginine transport in different cell. these issues will be discussed and the individual signaling pathways assessed on a cell type and species basis. moreover, the role of downstream signaling molecules will be examined in more detail, looking in particular at the critical dependency on the p38 mapk. this kinase currently exists in four different isoforms which are p38α, , γ and δ. the involvement of individual isoforms of p38 in enhancing the expression of carrier proteins for l-arginine will be discussed. gw274150 is an acetamidine derivative of heterosubstituted lysine which has been shown to have a marked selectivity for the human inducible nitric oxide synthase isoform (young et al. 2000. bioorg. med. chem. lett., 10: 6, 597-600) . the systems associated with transport of this compound have been investigated using the macrophage cell line j774. prior to each study, j774 cells were seeded in 96-well culture plates and allowed to adhere for 24 h in dulbecco's modified eagle's medium (dmem). transport studies were carried out using hepes buffered krebs solution (50 µl; 37°c) containing l-[ 14 c]gw274150 (1 µciml ϫ1 ) in the presence of either 0.1 mm or 0.025-1 mm unlabelled substrate. in parallel studies transport (1 µciml ϫ1 , 0.1 mm) was monitored in the presence of 1 mm excess of various other amino acids known to be substrates for distinct transport systems. time course experiments revealed that transport of 0.1 mm of l-[ 14 c]gw274150 occurred in a time-dependent manner and was linear for up to 5 min. in addition, uptake was only marginally dependent on extracellular na ϩ . kinetic studies revealed that transport was saturable, and michaelis-menten analysis revealed single affinity entry with an apparent k t of 0.31 mm and v max of 5.15 pmol·µg protein ϫ1 min ϫ1 . at 1 mm, 2-methylaminoisobutyric acid (meaib), lalanine, l-valine and -2-amino-bicyclo-(2,2,1)-heptane-2carboxylic acid (bch) caused little or no inhibition of l-[ 14 c]gw474150 (0.1 mm) uptake. in contrast, transport of l-[ 14 c]gw274150 was inhibited markedly by l-arginine, llysine, l-leucine, l-methionine, 6-diazo-5-oxo-l-norleucine (don) and l-glutamine. with the exception of l-arginine and l-lysine, the inhibition caused by the other substrates was critically dependent on extracellular na ϩ and was completely reversed when extracellular na ϩ was replaced with choline. in parallel kinetic inhibition experiments, transport of 0.1 mm l-[ 14 c]gw274150 was inhibited in a concentration dependent manner by l-arginine (ki ϭ 0.04 mm), l-leucine (ki ϭ 0.06), don (ki ϭ 0.18 mm) and l-glutamine (ki ϭ 0.13 mm). taken together, these data suggest that gw274150 may be transported, at least in part, by system y ϩ . however, the marked inhibition caused by l-leucine, l-glutamine and l-methionine, substrates for the relatively high affinity cationic amino acid transporter system y ϩ l, would suggest that this system may also contribute to the uptake of gw274150; if so, the monophasic substrate kinetics imply that the two systems handle gw274150 with similar affinity. other systems such as b 0,ϩ could be ruled out on the grounds that this transporter is critically na ϩ -dependent while uptake of gw274150 is largely (ϳ80%) na ϩ -independent. similarly, b 0,ϩ , another broadspectrum aminio acid transporter that may be capable of transporting gw274150 does not interact with l-glutamine and thus unlikely to be involved in transport of gw274150, at least in j774 cells. although a large number of different amino acid transporters have been identified on a molecular basis, some of themfunctionally described in mammalian cells -are still missing. in search of mammalian est sequences, which contain the signature of the aaap (amino acid/auxin permease) family, we identified a murine full length cdna, which encodes a membrane protein with 10-11 putative transmembrane domains. the transporter mrna is expressed in various murine tissues, including lung, heart and kidney. for functional characterization we used the xenopus laevis oocyte expression system and employed flux studies and electrophysiological analysis. oocytes injected with the crna showed an increased uptake of 3 h-l-alanine and 3 h-l-proline. detailed electrophysiological analysis revealed an electrogenic transport mode, independent of sodium and chloride ions. lowering the extracellular ph increased significantly substrate induced currents in crna injected oocytes. out of the 20 proteinogenic amino acids the transporter recognizes only small amino acids, such as gly, ala, pro and ser. distinct structural analogues of these amino acids also interact with the transporters substrate binding site. in conclusion, we describe the molecular and functional characteristics of the first electrogenic proton driven amino acid transporter of mammals. pharmacology department, dr. willmar schwabe gmbh, karlsruhe, germany it is now well established that transport of amino acid neurotransmitters (like glutamate, aspartate, gaba and glycine etc.) from and to the neurones is essential for their proper functioning. like in the case of other neurotransmitters, specific pre-and post-synaptic as well as vesicular transporters are involved in such processes. extensive efforts to clarify the mechanisms and processes involved in the control and/or proper functioning of the amino acid transporters are now, therefore, being made in numerous laboratories. such efforts have not only led to the identification of a few specific ligands and/or modulators of neuronal amino acid transporters, but also have started unravelling the complex and diverse processes regulating their functions. aim of this communication is to point out potential usefulness of some neuroactive constituents isolated from therapeutically used medicinal herbs for clarifying the mechanisms involved in neuronal amino acid transport. our interest in such studies was initially triggered by the observations made with hyperforin, i.e. quantitatively the major neuroactive component of hypericum perforatum extracts widely used for the treatment of mild to moderate depressive disorders. this acyl phloglucinol derivative not only modulate synaptic transports of biogenic amines but also of glutamate, aspartate and gaba. since it does not interact with any of the till now described transporters for these neurotransmitters, efforts were made to clarify the mechanisms involved in their observed effects (both in vitro and as well as in vivo). the results of the in vitro studies available to date strongly suggest that its effects on neuronal amino acid transport processes is mediated via some novel extracellular mechanism controlling the h ϩ (and/or other ionic) concentrations of neurones. these observations not only demonstrate that hyperforin represent a structurally and mechanistically novel class of therapeutically useful agent but also suggest that it could be useful tool for clarifying the complex mechanisms involved in the control of neuronal amino acid transport. these observations stimulated us to screen other putative psychoactive herbal extracts and their active constituents on neuronal amino acid transport and on the consequences of disturbances caused by malfunction of specific transporters. observations made with several such agents indicate that either modulation of mechanisms and/or processes involved in neuronal amino acid transport or reversal of pathologies caused by anomaly of transporter functions could be involved in their modes of actions. these observations reinforce our conviction that studies directed towards clarifying the effects of herbal constituents on neuronal amino acid transport might not only be a feasible way for identifying novel types of therapeutically interesting molecules but also could expedite our knowledge on these complex processes. glutamate-regulated sodium dynamics in cortical astrocytes: implications for cellular bioenergetics j.-y. chatton, p. marquet, and p. j. magistretti the mode of na ϩ entry and the dynamics of intracellular na ϩ concentration (na ϩ i ) changes consecutive to the application of the neurotransmitter glutamate were investigated in mouse cortical astrocytes in primary culture by video fluorescence microscopy. an elevation of na ϩ i was evoked by glutamate, whose amplitude and initial rate were concentration-dependent. the glutamate-evoked na ϩ increase was primarily due to na ϩ -glutamate cotransport. the rate of na ϩ influx decreased during glutamate application, with kinetics that correlate well with the increase in na ϩ i and which depend on the extracellular concentration of glutamate. a tight coupling between na ϩ entry and na ϩ /k ϩ atpase activity was revealed by the massive na ϩ i increase evoked by glutamate when pump activity was inhibited by ouabain. during prolonged glutamate application, na ϩ i remains elevated at a new steady-state where na ϩ influx through the transporter matches na ϩ extrusion through the na ϩ /k ϩ atpase. a mathematical model of the dynamics of na ϩ i homeostasis will be presented which precisely defines the critical role of na ϩ influx kinetics on the establishment of the elevated steady-state and its consequences on the cellular bioenergetics. indeed, extracellular glutamate concentrations as low as 10 µm approximately doubled the energetic demands of the astrocytes. department of biochemistry and molecular biology, faculty of biology, university of barcelona, spain in the last 5 years a new family of amino acid transporters composed by two different subunits has been defined. two heavy subunits (rbat and 4f2hc) and seven light subunits are known. rbat and the light subunits b0,ϩ at and y ϩ lat1 are responsible for the inherited aminoacidurias type i cystinuria, non-type i cystinuria and lysinuric protein intolerance, respectively. the heavy subunits are highly glycosylated type ii proteins, while light subunits are very hydrophobic unglycosylated membrane proteins, displaying a polytopic (generally 12 transmembrane domains) predicted structure. the specificity of the amino acid transport activity depends on the light chain expressed. this, together with its topology, indicates that the transport function mainly relies on the light subunits. i will summarize some of our current studies directed to the understanding of structure-function relationships of these heteromeric carriers, specially concerning their oligomeric structure and initial attempts to reconstitute them. ongoing work on the isolation of new rbat-associated light subunits and new b0,ϩ at-associated heavy subunits, which could also play a role in cystinuria, will also be discussed. department of pharmacology, joh. gutenberg university, mainz, germany mammalian cationic amino acid transporters (cats) catalyze the transport of basic amino acids through the plasma membrane. the cat family comprises at least five related carrier proteins (cat-1, -2a, -2b, -3 and -4) with cat-2a and -2b being splice variants. in humans, only the "old" members of the family have been characterized (hcat-1, -2a and -2b). hcat-1 and -2b exhibit high affinity for cationic amino acids and are sensitive to trans-stimulation, consistent with the classical system y ϩ . in contrast, hcat-2a is a low affinity carrier relatively insensitive to trans-stimulation. interestingly, hcat-2a and hcat-2b differ only in a region of 42 amino acids. cat-3, so far only identified in rat and mouse, exhibits also system y ϩ activity. however, the substrate recognition and maximal transport activity seems to differ from other y ϩ transporters. cat-3 expression has been reported to be restricted to the brain in adult animals. a cdna encoding for human hcat-4 has recently been isolated, however, the transport activity of hcat-4 has not been characterized. when optimally aligned, the amino acid sequence of hcat-4 shows only about 40% identity with the other hcat isoforms. in contrast, the amino acid sequences of hcat-1, -2(a or b) and -3 are about 60% identical. to elucidate which amino acids are responsible for the difference in the transport properties of the hcat proteins, we constructed chimeric proteins between hcat-1 and hcat-2a and performed site directed mutagenesis. using this approach, we identified two amino acid residues that are responsible for the different transport properties of hcat-2a compared to the high affinity cat-isoforms. to characterize the human cat-3, we cloned a cdna encoding hcat-3. when expressed in xenopus laevis oocytes, hcat-3 had a similar transport activity and affinity for l-arginine as hcat-1 or -2b. hcat-3mediated l-arginine transport was trans-stimulated and independent of extracellular na ϩ ions. expression studies demonstrated that hcat-3 is not only expressed in different regions of the human brain, but also in peripheral tissues. to investigate if hcat-4 also functions as an amino acid transporter, we measured the transport of cationic, neutral and acidic amino acids in xenopus laevis oocytes expressing hcat-4, but could not detect an transport activity for any substrate tested. a bright fluorescence could be detected in the plasma membrane of oocytes expressing hcat-4 with the green fluorescent protein attached to the c-terminus. therefore, hcat-4 might either need a complementary protein to function as an amino acid transporter or serve as a transporter for a yet unidentified substrate. renal amino acid reabsorption in immature and adult rats as a sensitive marker of heavy metal-induced nephrotoxicity (pt, cr, tl) institut für pharmakologie und toxikologie, klinikum der friedrich-schiller-universität jena, germany the effects of cis-platinum (cp; 0.6 mg/100 g b. wt. i. p.), sodium dichromate (cr; 1 mg/100 g b. wt. s. c.) and tl 2 so 4 (tl, 2 mg/100 g b. wt. i. p.) on renal amino acid excretion and plasma amino acid composition were investigated in 10-(both sexes) and 55-day-old (female) anaesthetised wistar rats (han : wist). on the basis of diuresis experiments on conscious rats (determination of urinary volume and protein excretion) the mentioned doses and times (1 st day after cr in both age groups and in 10-day-old rats after cp and 3 rd day after cp in adult rats; 2 nd [55-day-old rats] and 5 th -6 th day [10-day-old rats] after tl) were found out to be optimal for the characterisation of amino acid transport after heavy metal poisoning. interestingly, in conscious 10-day-old rats cr nephrotoxicity is not detectable after 1 mg/100 g b. wt. whereas all of the other experimental groups showed nephrotoxic effects of cr, tl and cp in conscious rats. urine volumes were lower, but not significantly, in anaesthetised immature rats, independently of the administered nephrotoxin. glomerular filtration rate (gfr) is significantly lower in 10-day-old rats compared to adults. after cp, cr and tl gfr is significantly reduced only in adult rats and age differences disappeared nearly completely. in principle the renal fractional excretion (fe aa ) of amino acids was distinctly higher in immature rats as a sign of lower amino acid reabsorption capacity. nevertheless, the amino acid plasma concentrations were relatively high in immature control rats. however, both cr and cp did not distinctly influence molecular cloning and functional characterization of ata3, a novel subtype of the amino acid transport system a medical college of georgia, augusta, georgia, u.s.a. recent molecular cloning studies have revealed that the amino acid transport system a consists of more than one subtype. two different system a subtypes, called ata1 and ata2, have been cloned and functionally characterized. ata1 is expressed primarily in the brain and placenta whereas ata2 is expressed ubiquitously. heterologous expression studies have shown that these two subtypes cannot be distinguished functionally based on substrate affinity nor substrate specificity. we have now cloned a third subtype of system a, designated ata3. it is expressed primarily in the liver. apart from the liver, detectable level of expression is noted only in the skeletal muscle. interestingly, ata3 can be easily differentiated from the other two subtypes of system a based on functional characteristics. we first isolated rat ata3 cdna from a skeletal muscle cdna library using rat ata2 cdna as the probe. rat ata3 consists of 547 amino acids and exhibits a high degree of homology in amino acid sequence to rat ata1 (47% identity) and rat ata2 (57% identity). interestingly, this new transporter also has a comparable degree of homology to sn1 and sn2, the two known subtypes of the amino acid transport system n. however, when expressed heterologously in xenopus laevis oocytes, rat ata3 transports α-(methylamino)isobutyric acid (meaib), a specific model substrate for system a, confirming that this transporter is definitely a subtype of system a. system n does not transport this system a model substrate. with two-microelectrode voltage-clamp technique, we have shown that exposure of rat ata3-expressing oocytes to neutral, short-chain aliphatic amino acids induces inward currents. the amino acid-induced current is na ϩ -dependent and phdependent. analysis of the currents with alanine as the substrate has shown that k 0.5 for alanine (i.e., concentration of the amino acid yielding half-maximal current) is 4.2 ϯ 0.1 mm and that the na ϩ : alanine stoichiometry is 1 : 1. subsequently, we have cloned the human homolog of rat ata3 from a liver cell line (hepg2) cdna library. human ata3 also contains 547 amino acids and shows 88% identity in amino acid sequence with rat ata3. the sequence identity of human ata3 with human ata1 and human ata2 is 47% and 57%, respectively. the homology of human ata3 with human sn1 and sn2 is also similar (56% and 51% identity, respectively). the gene coding for human ata3 contains 16 exons and is located on chromosome 12p13. in the human, ata3 is expressed almost exclusively in the liver. when expressed in mammalian cells heterologously, human ata3 mediates the transport of neutral amino acids, including meaib, in a na ϩ -dependent manner. interestingly, while characterizing the function of this clone, we have uncovered a unique feature of this system a subtype. human ata3 is capable of mediating the transport of cationic amino acids. in fact, the affinity of human ata3 for cationic amino acids is higher than for neutral amino acids. however, the human ata3-mediated cationic amino acid transport is na ϩ -independent. in this respect, ata3 is similar to transport system y ϩ l that also transports neutral amino acids in a na ϩ -coupled manner and cationic amino acids in a na ϩindependent manner. in contrast, ata1 and ata2 have not been shown to interact with cationic amino acids. in addition to this difference in substrate specificity, ata3 also differs from ata1 and ata2 in substrate affinity. ata1 and ata2 interact with meaib with a k t of ϳ0.3 mm whereas the affinity of ata3 for this model substrate is comparatively at least 20-fold lower (k t , ϳ8 mm). but, ata3 interacts with arginine with a k t value of 0.3 mm. since liver does not express any of the previously known high affinity cationic amino acid transporters, amino acid plasma concentrations. but in both age groups the administration of cr and cp significantly decreased amino acid reabsorption capacity (increase in fe aa ) as a sign of nephrotoxicity, most pronounced in adult rats after cp. on the other hand, after tl, the fe of amino acids was distinctly higher only in adult rats as a sign of lower amino acid reabsorption capacity and, thus, as a sign of higher nephrotoxicity. in immature animals fe aa was increased only for few amino acids. however, in both age groups tl administration significantly decreased plasma amino acid concentrations, more pronounced in immature rats. the investigation of renal amino acid handling confirmed: (1) cr, cp and tl were more nephrotoxic in 55-day-old animals compared to immature rats as could be demonstrated previously using other parameters for nephrotoxicity testing. (2) the extent of toxic effects of heavy metals on the kidney is related to the maturity of renal functions involved in the enrichment of the respective metal in renal tissue and in its toxicity mechanism. (3) changes in the fractional excretion of amino acids (reduction in renal amino acid reabsorption capacity, e.g. increase in fe aa ) and in amino acid plasma concentrations (especially decreases as a consequence of enhanced renal loss of amino acids) are early indicators of nephrotoxicity. (4) therefore, the determination of renal amino acid handling is a highly sensitive marker for nephrotoxicity testing, both in immature and in adult rats. the mammalian h ϩ /peptide cotransporter pept2 was initially identified in the brush border membrane of renal proximal tubular cells as a high affinity type ptr2-family member. here we describe the synthesis and functional analysis of novel high affinity inhibitors for pept2 that will be useful in further studies on structure and functions. starting from lys[z(no 2 )]-pro a series of different lysine-containing dipeptide derivates were synthesized and studied for interaction with pept2 based on transport competition assays in pichia pastoris yeast cells and in epithelial skpt cells, both expressing pept2. the twoelectrode-voltage-clamp technique in x. iaevis oocytes expressing pept2 was used to determine whether the compounds are transported electrogenically or block the uptake of dipeptides. synthesis and functional analysis of lys-lys derivates containing z(no 2 ) side chain protections provided a set of inhibitors that reversibly inhibited the uptake of dipeptides by pept2 with k i values as low as 10 nm. this is the highest affinity of a ligand of pept2 ever reported. moreover, based on the structure-function relationship we can conclude that the spatial location of the ε-amino protecting group in a lys containing dipeptide and its intramolecular distance from the alpha catom are key factors for the transformation of a substrate into an inhibitor of pept2. ata3 is likely to provide the major route for the uptake of arginine in this tissue. institute of pharmacology and therapeutics, faculty of medicine, porto, portugal the present study examined the nature and regulation of the l-dopa transporter in two functionally different clonal subpopulations of opossum kidney (ok lc and ok hc ) cells. the inward transfer of l-dopa was largely promoted through an energy-dependent and sodium-insensitive transporter, though a minor component (ϳ15%) was found to require extraceilular sodium. l-dopa uptake was insensitive to meaib, but competitively inhibited by bhc (ok lc , ic 50 ϭ 336 µm; ok hc , ic 50 ϭ 439 µm). l-and d-neutral amino acids and basic amino acids markedly inhibited l-dopa accumulation. l-dopa, lleucine, l-arginine, bhc or l-arginine plus bhc stimulated [ 14 c]-l-dopa efflux. the accumulation of l-dopa was significantly higher at an acidic ph, and incubation of cells with l-dopa (100 µm) resulted in marked intracellular acidification. modulators of pka, pkg, pkc and ptk failed to affect the accumulation of l-dopa. only the ca 2ϩ / calmodulin inhibitors inhibited l-dopa uptake. it is likely that system b 0,ϩ might be responsible for the sodium-dependent uptake of l-dopa in ok cells, whereas sodium-independent uptake of l-dopa may include systems b 0,ϩ and lat2, the activation of which results in trans-stimulation of l-dopa outward transfer. the trans-stimulation of l-dopa inward transfer by an imposed h ϩ gradient suggest that ok cells are provided with an l-dopa-h ϩ cotransport system. amino acids are essential nutrients for cell growth and maintenance. the essential amino acids arginine and lysine, are mainly transported via the cationic amino acid transporter 1 protein (cat1). the regulation of translation of the cat1 mrna during amino acid starvation was studied. an adaptive response to amino acid starvation and stress is a global decrease of protein synthesis, by phosphorylation of the translation initiation factor eif2a. translation of the transporter mrna increases when eif2a is phosphorylated, allowing synthesis of the essential for survival arginine/lysine transporter protein. the mechanism of increased translation of this mrna involves the induction of activity of a uorf-containing internal ribosomal entry sequence (ires). translation of the uorf and phosphorylation of eif2a are required for increased activity. we propose that eif2a phosphorylation triggers translational attenuation within the uorf, converting a relatively inactive, to a high activity ires. this study demonstrates that like yeast, mammalian cells have developed a sophisticated response to stress conditions: when expression of most genes decreases, synthesis of stress response proteins increases to support cell survival. amino acid transport, cell volume and the regulation of cell death f. lang, s. fillon, i. setiawan, p. lang, v. tanneur, d. häussinger, and s. bröer department for physiology, university of tübingen, germany cell volume regulatory mechanisms participate in a wide variety of cellular functions including regulation of epithelial transport, excitability, hormone and transmitter release, metabolism, migration, cell proliferation and apoptotic cell death. besides ion transport, polyols, betaine and glycerophosphorylcholine, cells utilize amino acids including taurine to balance extracellular osmolarity and regulate their volume. cells counteract shrinkage by uptake and swelling by release of amino acids including taurine. moreover, cell swelling stimulates synthesis and cell shrinkage favours breakdown of proteins which are osmotically less active than the sum of the amino acids thus generated. conversely, amino acid transport does influence cell volume. concentrative uptake of amino acids leads to cell swelling, amino acid release to cell shrinkage. through alterations of cell volume the amino acids participate in the regulation of protein metabolism. thus, concentrative amino acid transport inhibits and release of amino acids favours proteolysis. these mechanisms participate in the regulation of cell death. cd95 induced apoptotic death of jurkat t lymphocytes is paralleled by the release of taurine. the taurine release occurs with a delay of some 60 min following cd95 receptor triggering but immediately preceedes apoptic cell shrinkage and dna fragmentation. the signaling leading to taurine release is in large part elusive but requires at some stage activation of caspases. moreover, taurine release and apoptotic dna fragmentation are strongly inhibited by lowering of temperature. preloading of the cells with taurine retards cd95 induced dna fragmentation pointing to an active role of taurine in the regulation of apoptosis. peptide transporters of the ptr-family are integral plasma membrane proteins, that mediate the electrogenic protoncoupled transport of di-and tripeptides and peptide-like drugs across cell membranes. the physiological role of pept1, one member of this family in mammals, is mainly the uptake of small peptides into intestinal and renal tubular epithelial cells. in caenorhabditis elegans a homologue to mammalian pept1 is encoded by the pep-2 gene, which is expressed in the intestinal cells and a subset of sensory neurons in the head of the animal. to study the physiological role of the pep-2 transporter in vivo, a c. elegans pep-2 mutant was constructed. the animals deficient in pep-2 show a remarkable phenotype with pronounced signs of malnutrition, characterised by a delayed development, less eggs in the uterus, a smaller brood size and a prolonged mean life-span compared to wild-type animals. we rescued the phenotype by the expression of the wt pep-2 gene in the mutant. the observed starved phenotype in pep-2 mutants might be best explained by the reduced intestinal absorption of peptide bound amino acids that are required for protein synthesis and energy metabolism and provides the first direct evidence for the predominant role of the intestinal peptide transporter in amino acid absorption. adenosine is a potent vasodilator in many vascular beds and modulated tone via elevation of intracellular camp and/or release of nitric oxide (no). we have previously reported that adenosine (ado) stimulates l-arginine transport and no production in human cultured umbilical vein endothelial cells (sobrevia et al., j. physiol. 499, 135-140, 1997) , and here further characterise the signalling cascades. rt-pcr demonstrated that fetal endothelial cell possess mrna levels for a 2a , a 2b and a 3 -adenosine receptor subtype, whereas negligible levels were detected for the a 1 -receptor. adenosine (10 µm, 2 min) induced increases in l-arginine transport and no production were ca 2ϩ and camp independent and stimulated transport was abolished in cells depolarised with 80 mm k ϩ . whole-cell patch clamp experiments revealed that adenosine activated inward k ϩ currents, resulting in a membrane hyperpolarization and enhanced influx of the cation substrate l-arginine. adenosine induced l-arginine transport and no production were also abolished by inhibitors of tyrosine kinases (genistein), mek1/2 (pd98059, u0126) but unaffected by inhibitors of pkc (calphosin c) and pi-3 kinase (ly29002). these data suggest that adenosine induces membrane hyperpolarization by activating inward k ϩ currents, increasing the driving force for cationic amino acid influx via system y ϩ . the discovery of nocardicine a by aoki et al. and aztreonam showed that monocyclic -lactams, collectively known as monobactams, can have antibiotic activity. this activity is poor but compensated by the unique effect they can induce on certain microbial cell membranes. our quest for new non-conventional surfactants for various biomedical applications led us to synthesize bioactive compounds with structural similarities to nocardicins. we present here the preparation and the study of original trimodular biosurfactants of type i: spermine and amine oxidase induce a cytotoxic effect on multidrug resistant chinese hamster ovary cells e. agostinelli 1 , s. lord-fontaine 2 , e. przybytkowski 2 , and d. a. averill-bates 2 1 department of biochemical sciences "a. rossi fanelli", university of rome "la sapienza" and cnr, centre of molecular biology, rome, italy 2 department de chimie/biochimie and toxen (centre de recherche en toxicologie de l'environnement), université du québec à montréal, canada the occurrence of resistance to cytotoxic agents in tumor cells is a major obstacle to successful anticancer chemotherapy. multidrug resistance (mdr) is associated with several phenotypic alterations. cells with the mdr phenotype display decreased drug accumulation due to overexpression of pglycoprotein (p-gp), encoded by the mdr-1 gene, which acts as an energy-dependent pump involved in extrusion of drugs. we studied a new strategy to eliminate mdr cells using an enzyme, bovine serum amine oxidase, capable of forming cytotoxic products, h 2 o 2 and aldehyde(s), from polyamines (spermine). the involvement of both toxic products, formed by the bsao/spermine enzymatic system, in causing cytotoxicity was investigated in multidrug resistant chinese hamster ovary cells, ch r c5, at 37 and 42°c. we observed that hyperthermia, depletion of intracellular glutathione (by l-buthionine sulfoximine) and inhibition of glutathione s-transferase (by ethacrynic acid), sensitized ch r c5 cells to the cytotoxic effect of spermine enzymatic oxidation products. mdr cells showed no resistance to h 2 o 2 and aldehyde(s) relative to their drug-sensitive counterparts, auxb1 cells, in experimental conditions of: higher temperature, higher spermine concentration and longer incubation time. the inhibition of cellular detoxification systems led to increased cytotoxic effects of spermine enzymatic oxidation products on both mdr and sensitive cell lines. these results might be of great interest and suggest that toxic oxidation products formed from spermine and amine oxidase could be used in anticancer therapy, mainly against multidrug resistant tumor cells. [acknowledgements: this work was supported by cnr "target project on biotechnology", ministero della sanità tar these compounds present a hydrophobic part introduced by an ester or amide linkage with an aminoacid, a junction modulus which corresponds to -lactam, and a hydrophilic part which contains a triazole, well-known in pharmaceutical industry for its inhibitor effect against -lactamase. the compounds are synthesised from 2-hydroxymethyl-2methyl propionic acid in five steps. selective activation of one of the primary hydroxyl groups was accomplished by the formation of alkoxy tris(dimethylamino)phosphonium (atdp) salts 3 from the corresponding diol. treatment of 3 with excess potassium carbonate in refluxing anhydrous acetone yields the monobactams 4. activation by atdp salts followed by treat-ment with sodium azide and reflux in toluene gives the azido compound. the reaction with acetylenic derivatives allows to obtain the surfactants. the compounds show classical surfactant behavior and the evaluation of their biological properties give evidence for their antibacterial and antiviral activity, which corresponds apparently to antiprotease activity. a prodrug approach to glutathione derivatives with in vitro antiparasitic activity department of chemistry and materials manchester, faculty of science and engineering, metropolitan university, manchester, u.k. the potential chemotherapeutic activity of peptides are lost in many cases in vitro, due to their inability to cross cell plasma membranes. the recent identification of a series of glutathione diesters with high antiparastic activities in vitro against t.b.brucei (african sleeping sickness) lead us to investigate the determinants associated with their activity. a qsar study on some twenty-five diester derivatives against t.b.brucei and t.b. rhodesiense lead us to conclude that the mechanism of action of these compounds is related to membrane penetration and hydrolysis, controlled by hydrophobicity and steric factors. a hplc and sensor study have confirmed the de-esterified diacid as the active agent of these prodrugs. dietary taurine prevents oxidative stress and morphological alterations in the retina of diabetic rat f. franconi 1 , m. a. s. di leo 2 , s. caputo 2 , n. gentiloni silveri 2 , and g. ghirlanda 2 1 department of pharmacology, university of sassari, and 2 department of internal and geriatric medicine, catholic university, rome, italy diabetes mellitus can cause various complications including retinopathy, which is the earliest and most common complications of diabetes mellitus, affecting 90% of diabetics and progressing to blindness in about 5%. considerable evidence implicates oxidative stress in the pathogenesis of diabetic retinopathy. in fact, hyperglycemia generates reactive oxygen species and free radical defense is reduced in diabetic patients. thus, the prevention of oxidative stress may have important implications for pharmacological attempts to prevent diabetic retinopathy. at this regard, it has been found that taurine, a semi essential amino acid with antioxidant activity, is decreased both in type 1 and type 2 diabetes mellitus. moreover, taurine seems to have a peculiar role of taurine in terms of cellular physiology and pathophysiology of the retina. among others, taurine is thought to produce important physiological effects through osmoregulation, calcium modulation and antioxidant effects. therefore, we examined the effect of dietary chronic (4 months) taurine (2% and 5%) supplementation in diabetic rats in comparison with vitamin e (200 and 500 ui). dietary taurine supplementation, for 4 months, does not influence conjugated dienes (cd), lipid peroxides (lp) and na/k atpase activity in the retina of non diabetic rats. using rats streptotozocin (stz) induced diabetes of 4-month duration, we found that cd, lp are significantly increased and they remained elevated for 4 months. while, the na/k atpase is significantly decreased during the whole experimental time (4 months). moreover, an inverse correlation has been found among the cd and lp and atpase activity. in the retina of stz rats, these biochemical alterations are accomplished with marked profound morphological changes. in stz rats, taurine enriched diets decrease the lipid peroxidation and preserve the atpase activity, being 5% taurine more effective than 2% diets. the morphological examination reveals that in rats feed with 5% taurine no proliferative changes are present. moreover, the beneficial effects of taurine are more marked than of those of vitamin e. these results and previous findings encourage new investigations to evaluate the efficacy of taurine as an adjunctive agent ch ch 3 (ch 2 ) n xco iran applicated be (500 mg/kg -10 days) and the third -control. enzyme activities were determined spectrophotometrically in brain homogenate. results: polyamine oxidase activity decreased significantly lower dose of be didn't induce any significant change in diamine oxidase activity gaba-transaminase activity increased significantly (p ͻ 0.005; p ͻ 0.001) and dose dependently upon be treatment we have been examined the effects of propofol, taurine and propofol combined with taurine on free intracellular amino acid (aa) profiles, superoxide anion formation (o 2 ϫ ), hydrogen peroxide production (h 2 o 2 ) and released myeloperoxidase activity (mpo) in polymorphonuclear leucocytes (pmn). propofol led to significant changes in pmn free taurine, glutamine, glutamate, aspartate, methionine, basic, neutral (naa) and branched chain amino acid concentrations. exogenous taurine reduced pmn naa while increasing intracellular taurine. taurine supplemented to propofol significantly reversed the changes in taurine, naa and alanine only. regarding pmn immune functions propofol significantly decreased o 2 ϫ , h 2 o 2 formation and mpo. taurine decreased o 2 ϫ and h 2 o 2 production, while increasing released mpo. when propofol and taurine were combined they appeared to by reacting tyrosine with 1-nitroso-2-naphthol in the presence of nitric acid 1-2-benzyo-8-(alanyl)-3-phenoxazone (blp) an analog of actinomycin d is produced. the structural similarity of blp to actinomycin d prompted the national cancer institute (nci) to investigate its antitumor activities. the nci investigations revealed that blp exhibits growth inhibitory effects on various cancer cells and as a result blp has received the u.s. patent from the u.s. patent office. the purposed of this investigation was to synthesize similar benzo phenoxazone derivative by reacting 1-nitroso-2-naphthol with 4-(α-hydroxy -methylaminopropyl)phenol in the presence of nitric acid. during the study, it was found out that 1,2-benzo-3phenoxazone derivative is not produced but a hydrogenated form of 1,2-benzo-3-phenoxazone which is probably 1,2-benzo-8-(α-hydroxy -methylaminopropyl)-3-hydroxyphenoxazine (bhmhp) which has been suhhested from mass spectra obtained by electron ionization, ei, chemical ionization, ci and electro-spray ionization, esi, methods. 1 bhmhp was screened against various cancer cell lines by nci and has shown promising effect against three (3) breast cancer cell lines: mda-mb-435, mda-n and hs-578 t. the 50% growth inhibitory (gi 50 ) concentrations for these three cell lines were 4.60 ϫ 10 ϫ6 , 4.62 ϫ 10 ϫ6 and 5.74 ϫ 10 ϫ6 molar respectively. a. bocheva 1 and t. pajpanova 2 1 institute of molecular biology, bulgarian academy of sciences, and 2 institute of physiology, bulgarian academy of sciences, sofia, bulgariathe histamine is an endogenous substance with neurotransmitter and neuromodulator functions in the organism. its antagonists are used in the therapy of allergic diseases and inflammatory reactions and as antiulcer drugs.the limited potentialities of the antihistamine therapy together with the increasing number of the people suffering from allergic diseases give rise to the design and synthesis of new histamine analogues as a perspective area in the chemistry of therapeutic drugs.additionally, compounds containing the guanidine, oxyamino and sufonamide moieties are known to elicit a variety of pharmacological responses and are present in several marketing drugs or drug candidates.on the other hand, similar compounds, being a part of bigger structures (for instance peptides), can imitate the molecules of already known at ii-receptor antagonists.having in mind these data we aimed to synthesize new analogs of histamine containing sulfo-and oxy-guanidino groups with common formula: a. bocheva 1 , s. pancheva 2 , and t. pajpanova 2 1 institute of physiology, bulgarian academy of sciences, and 2 institute of molecular biology, bulgarian academy of sciences, sofia, bulgariathe problem of the efficient therapy of pain is important not only from clinical but from social and economic point of view. the great achievements in medicine are connected with the research on the development of antinociceptive drugs.melanocyte-inhibiting factor (mif) is a tripeptide (pro-leu-gly-nh 2 ) that was discovered in hypotalamus.the mif-1 exerted a weak analgesic effect. the synthesis of non-protein amino acids and their incorporation into biologically active peptides might become a powerful method for the design and development of modified analogues of natural peptides. having in mind these data we synthezied a number of new mif-analogues, containing unnatural amino acids such as cav, slys, sleu, slle and snie and in vivo experiments were performed to study their action on the nociception. the changes in nociceptive effects were examined in male wistar rats by the tail-flick (tf) and hot-plate (hp), as well as, the randall-seitto paw-pressure tests. the peptides were applied intaperitoneal (i.p) injection at a does 1 mg/kg. the results show that the newly sinthesized analogues exert an antinociceptive effects in all tests used. naloxone at a dose 1 mg/kg (i.p) antagonized the antinociceptive effects of mif-analogues. the interaction between platelets and fibrinogen is known to be mediated by the intergrin gp iib/iiia. the arg-gly-asp (rgd) sequence located on fibrinogen and other proteins of blood and extracellular matrix is the minimum requirement for cell attachment and adhesion. it has been found that peptides containing the rgd sequence can effectively inhibit the binding of fibrinogen to gp iib/iiia. in addition aspirin has been shown to be beneficial in the treatment of stable and unstable angina, acute myocardial infraction. aspirin acetylates and inhibits the enzyme cyclooxygenase, the first enzyme involved in thromboxane a 2 (txa 2 ) synthesis, an activator of platelet aggregation and adhesion.we have already reported that the combination in the same molecule of dipeptide amides, containing amino acid(s) of rgd sequence, with salicylic-residue 2-ro-c 6 h 4 -coϳ, {where rϭh or ch 3 co} at their n-terminal amino group have shown inhibitory activity on human platelet aggregation. continuing this research project on salicyl-peptides we have synthesized a series of rgd analogs, incorporating salicylic acid derivatives, by conventional solution techniques and/or by solid phase. the synthesized rgd analogs were identified by ir, nmr and es-ms spectra and tested for inhibitory activity on human platelet aggregation in vitro, by adding common aggregation reagents (collagen, adp, thrombin) to citrated platelet rich plasma (prp). platelets were obtained from venous blood of healthy donors and the prp was isolated by centrifugation at 200 g for 5 min at 37°c. the aggregation was determined using a dual channel electronic aggregometer. malonyl dialdehyde (mda) production was measured using thiobarbituric acid reagent. in order to confirm these results, flow cytometry with monoclonal antibodies against gpib, gpiib/iiia, gpiiia and gmp140 was used. the ic 50 values of the synthesized and tested compounds, as well as their mda production and flow cytometry results will be discussed. amino acids have a long tradition as building blocks, chiral auxiliaries and/or ligands in advanced organic synthesis and catalysis. at dsm an enzymatic kinetic resolution process has been developed, based on an aminopeptidase catalyzed stereoselective hydrolysis of racemic amino acid amides to form a mixture of l-amino acid and unchanged d-amino acid amide.several small peptides currently are under investigation as possible anti-tumor agents. neuropeptides such as substance p (sp) and neuropeptide y (npy), have been studied for their ability to prevent tumor growth or the proliferation of several cancer cell lines. these neuropeptides have been investigated for their effect to prostate cancer, small cell lung cancer (sclc) and breast cancer. the synthetic sp analog [d-arg 1 , d-phe 5 , d-trp 7,9 , leu 11 ]sp (antagonist d) and the c-terminal analog [arg 6 , d-trp 7,9 , mephe 8 ]sp 6-11 (antagonist g) inhibit sclc cell proliferation in vitro and in vivo, while the analogs [glp 6 , glu(bu t ) 11 ]sp 6-11 and [glp 5 , glu(bu t ) 11 ]sp 5-11 showed significant inhibition in the proliferation of the cancer cell lines hela and t47d.in the present study the c-terminal analogs of sp [glp 6 , d-trp 7 , glu(bu t ) 11 ]sp [6] [7] [8] [9] [10] [11] (1), [glp 6 , d-trp 7,9 , glu(bu t ) 11 ]sp [6] [7] [8] [9] [10] [11] (2), [glp 6 , d-trp 7,9 , mephe 8 , glu(bu t ) 11 ]sp 6-11 (3), [glp 6 , d-trp 7 , mephe 8 , glu(bu t ) 11 ]sp 6-11 (4), [glp 6 , trp 7 , mephe 8 , glu(bu t ) 11 ]sp 6-11 (5), [glp 6 , mephe 7 , d-trp 8 , glu(bu t ) 11 ]sp 6-11 (6), [glp 6 , d-trp 7 , mephe 8 , glu(bu t ) 11 -oh]sp 6-11 (7), [glp 6 , d-trp 7 , cys(acm) 11 -oh]sp 6-11 (8), [glp 6 , d-trp 7 , mephe 8 , cys(acm) 11 -oh]sp [6] [7] [8] [9] [10] [11] (9), [glp 6 , d-trp 7,9 , mephe 8 , cys(acm) 11 -oh]sp 6-11 (10) have been synthesized and tested for their antineoplastic properties in several cancer cell lines. they were also examined for their cytotoxicity to normal cells.the analogs 1-6 are peptide amides whereas the analogs 7-10 are peptide acids. they were performed using the stepwise synthesis either in solution, using the method of mixed anhydrides with carbonic acids or in spps using the fmoc/bu t methodology. the fragment condensation method in solution, using phosphonium reagents, such as pybop, was also applied. the analogs were purified (hplc) and identified (ft-ir, es-ms, 1 h-nmr).the antineoplastic properties of the analogs were studied using sister chromatide exchange (sce) and proliferation rate index (pri). as it is known the sce method is an indicator of dna damages or its repair mechanism, while the method of pri is a sensitive marker of cytotoxicity. the experiments were carried out using cultured human lymphocytes from healthy donors and these results will be discussed.semiempirical quantum chemical investigation of some thymidine derivatives modified with amino acids and peptides at 3ј, 5ј-positions j. velkov 1 , i. stankova 1 , a. ivanova 2 , and a. tadjer 2 1 department of chemistry, south-west university "neophit rilski", blagoevgrad, and 2 department of chemistry, sofia university "st. kl. ohridsky", sofia, bulgaria optimized geometry and electron charge distribution for some thymidine derivatives (3ј,5ј-bis-o-n-α-benzyloxycarbonyl-alanyl-, 3ј,5ј-bis-o-n-α-benzyloxycarbonyl-valyl ,3ј,5ј-bis-o-n-α-benzyloxy-carbonyl-glycyl-glycyl-glycyl,3ј,5јbis-o-n-α-benzyloxycarbonyl-phenylalanyl,3ј,5ј-bis-o-n-αbenzyloxycarbonyl-glycyl) were calculated at the semiempirical (am1) level. the choice of method is limited by the molecular size. in addition, the differences between the ground state energy of the compounds and that of the hydrolysis reaction intermediates were compared to the experimentally found stability towards hydrolysis.with a few notable exceptions, attempts to crystallise integral membrane proteins have failed due to the difficulties in finding appropriate conditions for proteins that have both hydrophobic and hydrophilic domains. thus structural information is largely limited to predictions of secondary structure from the amino acid sequence and computer modelling, neither of which can as yet give high resolution detail. thus alternative approaches are required, and one that we have employed is to look at the substrate binding/transport characteristics of compounds and predict what features the binding site might have. the membrane transport protein that we are interested in is the proton-coupled di/tri-peptide transporter, which has a wide range of natural substrates and is known to transport therapeutically important non-peptides such as ᮀ-lactam antibiotics and angiotensin converting enzyme inhibitors.the initial question that interested us was what makes a di/ tri-peptide a substrate, but not an amino acid? while the obvious answer is the peptide bond, studies with 'space mimic' compounds (which have the space filling properties of a dipeptide but no peptide bond) gave the surprising result that the peptide bond was not essential for binding and translocation. although these space mimics had n and c termini, studies from our laboratory and others have shown that the presence of free amino or carboxyl groups are not a prerequisite for binding or translocation either. this leaves the question of what does distinguish a pept1 substrate from a non-substrate?computer modelling of a large number of pept1 substrates has allowed the development of a substrate template, whereby potential substrates can be scored according to their predicted binding affinity. from this it is clear that it is a sum of energies derived from a number of substrate-transporter interactions that determine binding affinity, including the n-and c-termini, the peptide bond components and the substrate side-chain groups. further studies aim to refine this model through the complimentary approaches of novel substrate design and sitedirected mutagenesis of the transporter protein.why are we interested in this? a large number of promising therapeutic compounds are found to have little or no bioavailability. compared with most membrane transporters pept1 has a wide range of potential substrates, and amongst its non-peptide substrates are a range of peptidomimetic therapeutic compounds. the recent finding that a peptide bond is not a prerequisite for transport opens up the possibility of designing prodrugs to be substrates for pept1, and this has found to be an effective strategy for example with the antiviral drug valacyclovir.(we thank the wellcome trust for their generous support.) nuklearmedizinische klinik und poliklinik der technischen universität münchen, germanyaim: the high amino acid metabolism of tumor cells allows tumor imaging with radiolabeled amino acids as 11 c-methionine (met) by positron-emission-tomography (pet). however in recent experimental and clinical studies met uptake was also found in inflammatory tissue thus leading to false positive results. the aim of the study was to compare [ 18 f]fluoroethyltyrosine (fet), a new amino acid analogue, with met to assess their suitability for differentiating between tumor cells and inflammatory cells in vivo and in vitro.methods: popliteal lymph nodes of balb/c and dba/2 mice were stimulated either by streptocotocin (stz), causing chronic lymphadenitis, or by concanavalin a (con a), causing in acute lymphadenitis. tumor infiltrated lymph nodes were induced by inoculating cells from a lacz transfected t-cell mouse lymphoma line into the footpads of syngenic dba/2 mice. the uptake of met and fet was determined quantitatively in tumor infiltrated and inflammatory lymph nodes as well as in the lymph nodes of untreated mice. in vivo imaging of tracer uptake in mouse lymph nodes was performed using a high resolution (2.4 mm) small animal pet (madpet). in vitro the uptake of the amino acids met and fet was investigated in different cells, such as sw707 human colon carcinoma cells and c6 rat glioma cells, stimulated human lymphocytes and macrophages. about 5 ϫ 10 5 cells of each cell line were incubated in a buffered medium containing either different concentrations of unlabeled amino acids or con a (stimulation of lymphocytes) or the transport inhibitors 2amino-norbornane-carboxylic acid (bch, l-system), α-(methylamino)-isobutyric acid (meaib, a-system) or l-serin (asc-system). 0.37 mbq of each amino acid tracer were added and incubated. uptake was stopped by using ice-cold pbs, cells were washed three times and uptake was analyzed.results: in tumor infiltrated lymph nodes uptake of both tracers was higher than in control lymph nodes. met showed an increased uptake in both lymphadenitis models, whereas fet did not accumulate significantly. met and fet uptake in tumor infiltrated lymph nodes was also seen in madpet images, however inflammatory lymph nodes could only be detected in met images.the amount of tumor uptake was different in the various cell types investigated. c6 cells showed the highest uptake of all cells investigated and a slightly lower uptake was found in sw707 cells. in con a stimulated lymphocytes, the uptake of fet was negligible, while met uptake was significantly higher than in both tumor cell lines. since bch reduced the uptake of fet and met to approximately 10%, fet seems to be also predominantly transported into tumor cells by the l-system. the results indicate, that fet appears to differentiate between tumor and inflammatory tissue, as a result of the low uptake of fet in inflammatory cells. nuklearmedizinische klinik und poliklinik der technischen universität münchen, germanyover the past few years numerous studies have documented the high diagnostic accuracy of positron emission tomography (pet) using the glucose analogue f-18-fluordeoxyglucose (fdg) for detection and staging of malignant tumors. a significant limitation of fdg-pet, however, is that increased uptake is not only observed in malignant tumors but also in activated inflammatory cells. due to the high glucose utilization of the normal brain and the lower protein synthesis in the normal gray matter the radiolabelled amino acid c-11-methionine (met) gives higher contrast between brain tumors and normal tissue than fdg-pet. rapid uptake of met has been documented for several malignant tumors like gliomas, lung cancer, bladder cancer and malignant lymphomas since amino acid transport and protein synthesis are generally increased in malignancies. the application of met-pet however has been limited by the short half life of the radioactive label c-11 (20 min) in contrast to f-18 (110 min). amino acid analogous labeled with f-18 like f-18-fluoro-α-methyltyrosine (fmt), f-18-fluoro-ethyltyrosine (fet), f-18-fluoro-phenylanaline, f-18-fluore-proline will allow a more widespread application of amino acid pet in oncology. an other amino acid analogue i-123-iodo-α-methyltyrosine (imt) is of clinical interest because the radionuclid i-123 allows it applicability for single-photoemission-computer-tomography (spect). the uptake of the amino acid analogues can only be regarded as a measure for the increased amino acid transport in the tumor cells because they are not incorporated into proteins. clinical data show that radiolabelled amino acids that are only transported into the cells are not inferior to those that enter protein synthesis. this tracers may also help to differentiate tumor lesions from inflammatory lesions when the expression of the transport systems for amino acids in tumor cells and inflammatory cells is different.lysinuric protein intolerance: understanding the pathophysiology of a multi-system disorder of dibasic amino acid transport m. p. sperandeo 1,2 , v. fiorito 2 , a. pietrosanto 2 , a. pepe 2 , g. andria 2 , and g. sebastio 2 1 telethon foundation, rome, and 2 department of pediatrics, federico ii university, naples, italy lysinuric protein intolerance (lpi; mim 222700) is an autosomal recessive disease, mainly found in finland and italy. clinical findings of lpi include: vomiting, diarrhea, failure to thrive, hepatosplenomegaly, osteoporosis, episodes of coma, and mental retardation. a life-threatening lung involvement (alveolar proteinosis) and renal insufficiency were also reported. metabolic derangement of lpi includes: reduced intestinal absorption of cationic amino acids (lysine, ornithine, arginine, caa), increased renal excretion of caa and dysfunction of the urea cycle leading to hyperammonemia and orotic aciduria. most of the clinical findings cannot be explained by a selective deficiency of amino acid transport, as indeed observed for cystinuria (mim 220100), a cognate disease of lpi. the molecular basis of lpi resides in an abnormal caa carrier functioning at the level of basolateral membrane of epithelial cells in the intestine and the kidney. caa transport is mediated by y ϩ l system, that is exerted by heterodimers consisting of the 4f2 heavy chain (4f2hc) and a light chain represented by either the solute carrier family 7a, member 6 (slc7a6) or 7 (slc7a7). after excluding the 4f2hc as the causative gene of lpi, we identified slc7a7 as the lpi gene and characterized mutations in twenty-five patients from 21 families (16 italian, 2 japanese, 1 moroccan, 1 greek, and 1 pakistani; 34 independent alleles) affected by lpi. thirty-two of the 34 independent alleles (94.1%) were characterized and fourteen mutations were identified. only five mutations (namely 1625insatca, w242x, 1425delctct, ivs3 ϩ 1gaea, s386r) were identified in more than one independent family. most mutations are located in the slc7a7 coding region, except for two splicing mutations. the pathogenesis of some clinical findings of lpi, namely alveolar proteinosis and renal involvement, remains mostly unknown. we are currently investigating the role of slc7a6 gene in lpi, which, in addition to slc7a7, is responsible of the y ϩ l activity. in fact, the regulation of the y ϩ l system, exerted by either 4f2hc/ slc7a76 or 4f2hc/slc7a7, is still unknown. hypothetically, the activation of 4f2hc/slc7a76 in all tissues might be the "simple" way to a lpi gene-therapy.[acknowledgements: m. p. s. is supported by telethon-italy (grant n.29cp) and is an assistant telethon scientist.] pre-eclampsia (pe) is a potentially life threatening complication of pregnancy and is one of the leading causes of maternal and fetal morbidity and mortality. pe is associated with endothelial cell dysfunction and inadequate placental perfusion. fetal plasma l-arginine levels are decreased in pe and there is controversy as to whether nitric oxide (no) production is altered. we have investigated whether the kinetics of l-arginine transport via system y ϩ and no production are altered in fetal umbilical vein endothelial cells (huvec) from pe pregnancies. kinetics of l-arginine transport were similar in huvec isolated from normal, preterm and pe pregnancies, however nethylmaleimide inhibited transport in normal but not pe huvec. basal and histamine-stimulated no production was similar in normal and preterm huvec, whereas pe increased basal (25 ϯ 5 vs 5.3 ϫ 3 pmol/10 8 cell/5 min) and histaminestimulated (70 ϯ 12 vs 20 ϯ 5 pmol/10 8 /5 min) no production. whole-cell patch clamp measurements revealed similar inward rectifying k ϩ currents in normal and pe huvec, with resting membrane potentials of ϫ65 ϯ 4 and ϫ80 ϯ 18 mv in normal and pe huvec, respectively. increased enos activity in pe endothelial cells may serve as a compensatory mechanism to counteract the hypertension observed in pe, however, elevated no production is apparently not associated with enhanced larginine transport. department of pharmacology, university of cambridge, u.k.over the past years, concerns have heightened over the escalating numbers of pathogenic microorganisms that are resistant to multiple antibiotics. this phenomenon poses major problems in the treatment of patients with hospital or community-acquired infections caused by bacteria, yeast, fungi and parasitic organisms. particularly intriguing are the so-called multidrug transporters, which have specificity of compounds with very different chemical structures and cellular targets. this lecture will focus on the molecular properties of the atpbinding cassette multidrug transporter lmra in the lactic acid bacterium lactococcus lactis. lmra is a close homolog of the human multidrug resistance p-glycoprotein, overexpression of which is one of the major causes of resistance of human cancers to chemotherapy. surprisingly, lmra can even substitute for pglycoprotein in human lung fibroblast cells. recent biochemical and pharmacological studies on lmra suggest that the protein may operate by a two-cylinder engine mechanism to transport amphiphilic drugs from the inner leaflet of the plasma membrane. this mechanism will be discussed in more detail. bone and bone marrow are important sites of metastasis formation in breast cancer; so, we studied the level of bone sialoprotein (bsn) and fibronectin (fn), two key connective tissue antigens, in patients with metastatic breast carcinoma. our data reveled that bsn have a statistically significant association with bone metastases in that disease. fn level was also significantly changed in metastatic breast carcinoma when compared to the non metastatic cases. kharkov national university, radiophysical department, chair of molecular and applied biophysics, kharkov, ukraine * present address: institute of cell and molecular biology, university of edinburgh, edinburgh, scotland, u.k.in the work the temperature dependencies of dielectric parameters of human serum albumin (hsa) and fibrinogen solutions (0.15 m nacl, ph 7.2) were obtained in the temperature interval 5-50 c degrees. the measurements of the dielectric parameters were carried out at the frequency of 9.2 hhz, i.e. in the range of free water molecules dispersion. in contrast to dependencies for poor solvent, temperature dependencies of dielectric parameters for protein solutions are of nonmonotonous character; they have a number of peculiarities in the temperature ranges of 8-10, 22-24 and 34-36 c degrees. this fact means that at these temperatures redistribution of free and bound water in protein-water system occurs due to structural changes in protein molecules. the dependencies of hydration of hsa and fibrinogen on temperature were obtained as well.in the work the mechanism of temperature changes of spatial organisation of protein molecules was proposed. perhaps, this mechanism is responsible for maintenance of thermal stability of the functionally active conformation of native proteins. as peculiarities on temperature dependencies of dielectric parameters of solutions of globular (hsa) and fibrillar (fibrinogen) proteins were in the same temperature regions, one may to assume that the mechanism of proteins thermal stabilisation in physiological temperatures interval has a general character. laboratory of cell pharmacology, university of leuven, medical school, campus gasthuisberg (o&n), leuven, belgium n-pomc was purified from conditioned medium of att20 cells using a sequence of concentration, fractionation by ion exchange, rp-hplc and gel-filtration. twenty isoforms of n-pomc, for both 11 and 13 kda, were identified by means of mass spectrometry and n-terminal sequencing. these isoforms are assumed to be pomc1-74 or pomc1-95 with heterogeneous glycosylation.the n-pomc isoforms were tested on prolactin (prl) gene expression and lactotroph mitosis in pituitary cell aggregate cultures. prl mrna content was quantified by means of real time rt-pcr. three 11 kda n-pomc fractions enhanced prl mrna levels by 33-36%, while all other isoforms were inactive. this effect was abolished by immunoneutralization with n-pomc monoclonal antibody. only one fraction stimulated lactotroph proliferation (38.2 ϯ 7.5%) as assessed by brdu incorporation in prl-immunoreactive cells. several (but not all) 13 kda n-pomc fractions stimulated prl mrna level and lactotroph mitosis. on the other hand, all 11 and 13 kda isoforms activated the mc-3 and mc-5 receptor in cell lines in which these receptors were transfected. thus, att20 cells produce various n-pomc isoforms, only a part of which display an effect on prl mrna expression. even fewer isoforms affect lactotroph proliferation. since all isoforms activate the mc-3 and mc-5 receptor, it is suggested that the effect of the few isoforms on lactotrophs is mediated by (a) different receptor(s). are widely prescribed for the treatment of mild to moderate depression and the putative antidepressant constituent is probably hyperforin. in this study the effect of hyperforin was investigated on the release of neurotransmitter amino acids.coronal cortical slices (400 µm) were cut and perfused with gassed (95% o 2 , 5% co 2 ) acsf at 37°c. two-minute samples of perfusate were collected and aspartate and glutamate were assayed by hplc. potassium-and veratridine-stimulated release was elicited by administering 2 pulses of k ϩ (60 mm) or veratridine (20 µm) 30 minutes apart.in control experiments the second k ϩ pulse elicited glutamate release which was 80% of the first pulse. hyperforin (5 µm) perfused for 30 minutes prior to, and during, the second k ϩ pulse significantly increased glutamate release to 170% (p ͻ 0.001, n ϭ 6-8). release elicited by the second veratridine pulse was 70% of the first pulse for both glutamate and aspartate. hyperforin (5 µm) increased this release to the second pulse to 160% and 130% respectively (p ͻ 0.001, n ϭ 6-8). when perfused on its own for 30 minutes, hyperforin (5 µm) increased the basal release of glutamate (p ͻ 0.001, n ϭ 4-5).in conclusion, the increase in the release of neurotransmitter amino acids observed following hyperforin is possibly mediated through a facilitatory action on voltage-operated ca 2ϩ or na ϩ channels.glucagon-like peptide-1 (7-36) amide (glp1) is the main product of the glucagons gene expression in intestinal l cells into the cirulation in response to the ingestion of food and is the most potent stimulator of glucose-induced insulin secretion. glp1 receptors have also been detected in discrete areas of rat brain and intracerebroventricular glp1 has been shown to inhibit feeding in fasted rats. in this study hplc techniques were employed to evaluate the effects of glp1 on serotonin (5-ht) and γ aminobutyric acid (gaba) metabolism in rat brain. glp1 (0.5 µm) produced a significant decrease in levels of 5-ht by 20% after 15 minutes of incubation with combined hypothalamus and brain sterr. synaptosomes. levels of 5hydroxyindolacetic acid (5-hiaa), the principal metabolite of 5-ht, and tryptophan the amino acid precursor of 5-ht, were also decreased significantly by 21% and 37% respectively. gaba and its amino acid precursor glutamic acid were both measured at the same conditions as above, but a precolumn derivatization hplc technique was used. the increase in levels of gaba (14%) and glu (6%) by glp1 was not significant.the results suggest that decreased synaptosomal levels of 5-ht and 5-hiaa caused by glp1 are due to diminished availability of typtophan by the peptide. in experimental model of iron overload we obtained the following results: the concentration of carbonyl groups tended to increase, while mda level significantly increased after feso 4 treatment (1.66 ϯ 0.25 vs control 1.51 ϯ 0.50 µmol/mg prot.) and (2.13 ϯ 0.5 vs control 1.3 ϯ 0.3 nmol/mg protein p ͻ 0.01) respectively. it was associated with significantly increased iron content (0.89 ϯ 0.23 µg/mg prot. vs control 0.49 ϯ 0.17 p ͻ 0.001). it is clear that oxidative stress occurs in experimental iron overload, if sufficiently high levels of iron within hepatocytes are achieved. in group treated with feso 4 and spermine, iron content was significantly decreased (0.36 ϯ 0.07 p ͻ 0.01 compared with fe treated only) and carbonyl group content tended to be lower in comparison to feso 4 treated only (1.58 ϯ 0.24), but mda level didn't change (2.31 ϯ 0.72). in addition, treatment with spermine alone resulted in increase of mda level (2.74 ϯ 0.7 vs control p ͻ 0.01), iron content didn't change (0.59 ϯ 0.29), but carbonyl groups were decreased (0.99 ϯ 0.28 vs control p ͻ 0.05). feso 4 treatment increased gsh level (126.38 ϯ 34.11 nmol/mg prot. vs control 88 ϯ 22.77; p ͻ 0.05) while in combination with spermine this increase was more profound (235.48 ϯ 42.7; p ͻ 0.001 vs control, p ͻ 0.001 vs feso 4 ). spermine alone produced similar increase of gsh level (127.4 ϯ 34.11, p ͻ 0.05 vs control; p ͼ 0.05 vs feso 4 ). the results emanating from the human genome programme have required a reappraisal of protein science and have led to the rapid upsurge in interest in the area of proteomics. this sudden re-emergence of protein science, in fact, was predictable and should not have been surprising.recent experience of protecting group design with respect to lysine and aspartic acid will be discussed together with aspects of chemical synthesis of small proteins of biological significance and in the context of chemical synthesis methodology making contributions to the general field of proteomics. using a cell line permanently expressing the mouse taurine transporter (mtaut) as a fusion protein, we investigated the underlying mechanism by which the immunosuppressive drug cyclosporin a (csa) inhibits taurine transport. csa inhibited the recombinantly expressed mtaut function both in dose and time dependent manner. the inhibitory effect of csa was reversible. thus, washing out the csa resulted in almost complete recovery of taurine uptake. to obtain further insight, we examined the surface abundance of the mtaut as a function of csa treatment using a surface-labeling assay. our results demonstrated that csa treatment altered the surface expression of the mtaut without significantly altering its total expression level, and the reduction in the cell surface expression paralleled the decrease in taurine uptake. upon removal of csa, the virtual recovery in taurine uptake was due to the concomitant increase in the number of taurine transporters on the cell surface. taken together, our results suggest that csa induced inhibition of taurine uptake was either due to the impaired targeting of the taurine transporters to the cell surface or due to the removal of the transporters from the cell surface. polyamines are neuromodulators in a number of physiological and pathological conditions in cns. since application of ethylene glycols causes hypoactivity and lethargy of experimental animals, depression of cns and various neurological symptoms, the aim of this study was to examine the effects of 2butoxyetanol on polyamine and gaba catabolism, taking in account an alternative pathway of gaba synthesis from putrescine. methods: male wister rats were allocated into three groups: first treated by be (100 mg/kg -10 days), second key: cord-015147-h0o0yqv8 authors: nan title: oral communications and posters date: 2014-09-12 journal: inflamm res doi: 10.1007/bf03353884 sha: doc_id: 15147 cord_uid: h0o0yqv8 nan the drosophila host defense is a multifaceted process which involves reprogramming of gene expression, activation of proteolytic cascades in the blood and uptake of microrganismsby professionalphagocytes. most of the recent studies have focused on challenge-induced expression of antimicrobial peptides and have addressed the following questions : (1) which genes are upregulated during various types of bacterial, fungal or viral infections and what are their functions? (2),what is the nature of the intracellular signalling cascades which lead togene transcription during these infections; (3) how does drosophila recognize infections and does it discriminate between various types of aggressors (e.g. fungal versus bacterial or viral) to mount an appropriate response. over the last ten years we have gained significant insights into these various aspects and the presentation will review our current understanding of the drosophila immune response and put it into a phylogenetic perspective, namely by drawing some stringent parallels with the mammalian innate immune response. there is strong evidence that autoimmunity to myelin antigens plays a major role in the development of multiple sclerosis. several myelin-derived autoantigenic targets have been described and include myelin basic protein (mbp), proteolipid protein and myelin oligodendrocyte glycoprotein. there has been a particular focus on mbp for at least two reasons: mbp-specific cd4+ tcell receptors (tcrs) have been found in multiple sclerosis brains, and cells presenting an immunodominant mbp(85-99) peptide in complex with hla-dr2b have been shown to be present in multiple sclerosis lesions. also, humanized mice expressing the hla-dr2b gene and a human t-cell receptor (tcr) that recognises the mbp85-99 peptide in the context of hla-dr2b either spontaneously or after immunization with mbp85-99 develop experimental autoimmune encephalomyelitis, which has several features in common with multiple sclerosis. this talk will focus on, how humanized mice recently has been used to study the interplay between genetic and environmental risk factors in multiple sclerosis. to resolve the pathogenic mechanisms of rheumatoid arthritis (ra) we need to identify the causative genetic and environmental factors. this has however proven to be complex, with many factors and genes interacting. inbred animals are useful for studies of the identification of genes associated with complex traits and diseases such as ra. animal models offer a possibility to better define the traits, and to segregate the genes in a controlled way enabling linkage analysis. there are several arthritis models, which each may reflect various variants of the heterogeneity of ra in humans. examples are collagen induced arthritis (cia) and pristane induced arthritis (pia), which both fulfills the clinical diagnostic criteria for ra. both diseases are genetically complex and the susceptibility is, as ra, dependent on many polymorphic genes operating in concert. so far 2 genes in this concert have been identified; the mhc class ii ab gene in the mouse (1) and the ncf1 gene in the rat (2) and in the mouse (3) . the ncf1 protein is a part of the nadph oxidase complex mediating oxidative burst. the discovery of the ncf1 polymorphism led to a new proposed pathway in which oxygen radicals modify antigen presentation and the resulting activation of autoreactive t cells. mice with the deficient ncf1 allele are more susceptible to cia, and also developed a chronic form of arthritis. interestingly, the immune response to cii was enhanced by the ncf1 deficiency linking the ncf1 pathway to the adaptive immune response. oxidation of t cell membranes seem to be a key event in the pathogenic mechanism as reduction of t cell membranes induces arthritis in rats (4). on the basis of these findings a new type of therapy myasthenia gravis is a prototypic autoimmune disease, caused in most cases by autoantibodies to the muscle acetylcholine receptor (achr) at the neuromuscular junction. the antibodies reduce the number of achr leading to failure of neuromuscular transmission and muscle weakness. the achr antibodies as measured in conventional immunoprecipitation assays are igg, high affinity, polyclonal and specific for human achr. they reduce the numbers of achr by antigenic modulation and by complement-mediated damage to the neuromuscular junction. myasthenia gravis has a very intriguing relationship with the thymus gland. in many younger patients, the thymus is hyperplastic with immune cell infiltrates and germinal centre formation. around the germinal centres, within the medulla, there are rare muscle-like cells called myoid cells that express achrs. there are many b cells and plasma cells and thymic lymphocyte preparations synthesise achr antibodies. the possibility that the thymic achr induces the germinal centre formation and achr antibody synthesis is supported by much evidence. some patients, however, have thymic tumours and in these the role of the thymus is less clear. moreover, older patients with typical myasthenia usually have thymic tissue which is normal for their age. there are up to 20% of myasthenia patients that do not have the typical achr antibodies. some of these have instead antibodies to muscle specific kinase, a receptor tyrosine kinase that is restricted to the neuromuscular junction. the pathogenic mechanisms by which the antibodies cause disease are not yet clearly identified and the evidence will be discussed. finally, among the patients who have neither achr nor musk antibodies by conventional testing, we have evidence for lower affinity antibodies to achr which can now be detected using molecular approaches which will be described. arry-886 (azd6244) is an inhibitor of mek1/2 currently in development for cancer. phase 1 determined the msd (100 mg) and the safety of the compound given continuously. in decreasing frequency, common treatment-related side effects were rash, diarrhea, nausea, peripheral edema, and vomiting. paired pre-and postdose tumor biopsies showed a reduction in perk (-83%) and proliferative index (-46%). the trough plasma concentration (400 ng/ml) corresponded to~40% inhibition of perk. about 45% of pts had stable disease after 2 months. these results demonstrate that arry-886 (azd6244) is well-tolerated, hits its target, and produces a high incidence of long-lasting stable disease. there are several on-going phase 2 studies, in melanoma, colorectal, lung and pancreatic cancer. arry-162 is another potent, selective mek 1/2 inhibitor, currently in development for inflammatory diseases. when human whole blood was stimulated with tpa, arry-162 inhibited tnfa, il-1b and il-6 (ic50s of 23, 21 and 21 nm, respectively). 50% inhibition of perk required 280 nm. arry-162 was highly efficacious in cia and aia rat models, with ed50s of 3 and~10 mg/ kg, respectively. in normal volunteers arry-162 was well-tolerated and there was a dose-proportional increase in drug exposure. in ex vivo blood samples, there was a dramatic time-and concentration-dependent inhibition of tpa-induced tnfa and il1b. an on-going multiple ascending dose clinical study is further exploring the pharmacokinetics, pharmacodynamics and tolerability of arry-162 monotherapy. in addition, we have initiated a clinical trial designed to evaluate arry-162 in combination with methotrexate in patients with rheumatoid arthritis. rho kinases (rock) are involved in many physiological and pathological processes including smooth muscle contraction, cytoskeletal arrangement, cell adhesion, migration and proliferation.rocks prominent role in cytoskeletal architecture suggests that rock inhibitors should have therapeutic impact in oncology and fibrotic diseases which require cytoskeletal rearrangement to progress.we have synthesized small molecule inhibitors of rock which are specific for the rock-2 isoform.these rock-2 inhibitors, typified by slx-2119 and slx-3060, are potent (ic50 < 100 nm), selective for rock-2 (>100 fold selectivity for rock-2 over rock-1) and exhibit good oral bioavailability.this talk will focus on several areas in oncology and fibrotic disease where the ability to demonstrate an in vitro effect on the cytoskeleton translates into activity in the disease model in vivo.slx-2119 inhibits cell proliferation and migration in several tumor cell lines including ht-1080, panc-1 and mda-mb-231. moreover in xenograft studies using nude mice, slx-2119 significantly inhibited tumor growth with these same cell lines. in liver fibrosis, slx-2119 prevents the differentiation of rat primary hepatic stellate cells into myofibroblasts and inhibits the proliferation of myofibroblasts as well as inhibiting hepatic steatosis in an atherosclerosis model.slx-3060 is an effective antifibrotic agent in the kidney unilateral urethral obstruction model and inhibits renal fibroblast differentiation and proliferation.these data suggest that rock-2 selective inhibition of cytoskeletal modification in key cell types (e.g. tumor cells, stellate cells and fibroblasts) by compounds such as slx-2119 and slx-3060 will provide effective treatment for oncology and fibrotic disease. cyclooxygenases (cox) catalyze the first step in the synthesis of prostaglandins (pg) from arachidonic acid.cox-1 is constitutively expressed.the cox-2 gene is an immediate early-response gene that is induced by variety of mitogenic and inflammatory stimuli.levels of cox-2 are increased in both inflamed and malignant tissues.in inflamed tissues, there is both pharmacological and genetic evidence that targeting cox-2 can either improve (e.g., osteoarthritis) or exacerbate symptoms (e.g., inflammatory bowel disease).multiple lines of evidence suggest that cox-2 plays a significant role in carcinogenesis.the most specific data that support a cause-and effect relationship between cox-2 and tumorigenesis come from genetic studies.overexpression of cox-2 has been observed to drive tumor formation whereas cox-2 deficiency protects against several tumor types.selective cox-2 inhibitors protect against the formation and growth of experimental tumors.moreover, selective cox-2 inhibitors are active in preventing colorectal adenomas in humans.increased amounts of cox-2-derived pge2 are found in both inflamed and neoplastic tissues.the fact that pge2 can stimulate cell proliferation, inhibit apoptosis and induce angiogenesis fits with evidence that induction of cox-2 contributes to both wound healing and tumor growth.taken together, it seems likely that cox-2 induction contributes to wound healing in response to injury but reduces the threshold for carcinogenesis. (1), k hagihara(2), t nishikawa(1), j song(1), a matsumura (2) (1) health care center, osaka university, japan (2) osaka university graduate school of medicine, japan it is still less known about the actual pathogenic role of il-6 in the inflammatory status. to know the pathogenic role of il-6 and the efficacy of il-6 blockade in inflammation, a humanized anti-il-6 receptor antibody, tocilizumab, was used for the treatment in chronic inflammatory diseases, such as castlemans disease, rheumatoid arthritis and crohns disease. since il-6 blocking therapy improved the clinical symptoms and the laboratory findings, the il-6 function in inflammation could be analyzed for the induction of inflammatory molecules, such as serum amyloid a (saa). saamrna induction, saa promoter activity and assembling of transcriptional factors on saa promoter were analyzed by the real time rt-pcr, gel shift assay and dna affinity chromatography in hepatocyte stimulated with the proinflammatory cytokines, il-6, il-1 and tnf-alpha. in result, il-6 was an essential cytokine in induction of saamrna through the activation of stat3 which constructed the complex with nf-kappab p65 and a cofactor p300. although there was no stat3 consensus region on saa promoter, stat3 bound at the 3 site of nf-kappab re. the above research proved that il-6 signal is essential on the synergistic induction of saa via newly discovered stat3 transcriptional mechanism, suggesting the presence of this stat3 mechanism in inflammation, and confirming the normalization of serum saa level by il-6 blocking therapy in inflammatory diseases. this research method develops a subsequent therapy for serious aa amyloidosis by inhibition of saa production, and elucidates the cytokine mechanism on immunopathogenesis of chronic inflammatory diseases. takashi wada(1), k matsushima(2), s kaneko (1) (1) kanazawa university, japan (2) university of tokyo, japan accumulating evidence indicates that chemokine/chemokine receptor system plays a key role in the pathogenesis of various renal diseases via leukocyte migration. pathophysiological impacts of chemokines have shed light on molecular mechanisms of leukocyte trafficking and their activation in the inflammatory aspects of progressive renal injury.locally expressed chemokines are proven to be capable of inciting leukocyte migration to the kidney, resulting in initiating and promoting chronic kidney diseases.the possible positive amplification loop from cxc chemokines to cc chemokines may contribute to progressive renal injury, resulting in sclerosis/fibrosis.it is of note that monocyte chemoattractant protein (mcp)-1/ monocyte chemotactic and activating factor (mcaf)/ ccl2, a prototype of cc chemokines, promotes and escalates chronic kidney diseases with any etiologies via the infiltration and activation of monocytes/macrophages, proteinuria and collagen synthesis.interactions between infiltrated inflammatory cells and resident renal cells eventually lead to the progression of fibrosis. new insights into renal fibrosis have been uncovered by the regulation of fibrocytes dependent on chemokine system. in addition, recent studies demonstrate that chemokines have been expanding their universe beyond leukocyte migration to the kidney, including homeostasis, development and repair of the kidney.the selective intervention of chemokines might have the therapeutic potential to alter inflammatory responses, thereby halting the progression of renal injury. we focus on recent progresses on the role of chemokines and their cognate receptors in renal injury in this symposium. (1), p lacamera (1), b shea (1), g campanella (1), b karimi-shah (1) , n kim (1), z zhao(2), v polosukhin (3), y xu(2), t blackwell (3) aberrant wound-healing responses to injury have been implicated in the development of pulmonary fibrosis, but the mediators directing these pathologic responses remain to be fully identified.here we demonstrate that lysophosphatidic acid (lpa) is induced by lung injury in the bleomycin model of pulmonary fibrosis, and that mice deficient for one of its receptors, lpa1, are dramatically protected from pulmonary fibrosis and mortality following bleomycin challenge. the absence of lpa1 markedly reduced fibroblast responses to the chemotactic activity present in the airspaces following bleomycin, and attenuated the subsequent accumulation of fibroblasts in the lung.the increase in vascular permeability caused by lung injury was also markedly reduced in lpa1-deficient mice, whereas bleomycin-induced leukocyte recruitment was preserved.these results demonstrate that lpa1 links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak, two of the wound-healing responses that are thought to be inappropriately excessive when injury leads to fibrosis rather than repair.lpa1 therefore represents a new target for lung diseases in which aberrant responses to injury contribute to the development of fibrosis, such as idiopathic pulmonary fibrosis and the acute respiratory distress syndrome. we have reported that inflammation is detrimental for survival of new hippocampal neurons early after they have been born. our data now show that microglia activation, as an indicator of inflammation, is not pro-or antineurogenic per se but the net outcome is probably dependent on the balance between secreted molecules with pro-and antiinflammatory action. we have found that a substantial fraction of the new hippocampal neurons formed after status epilepticus survive despite chronic inflammation. we have started to explore the role of tnf-alpha for adult neurogenesis. infusion of an antibody to tnf-alpha was shown to reduce survival of new striatal and hippocampal neurons generated after stroke, probably by interfering with action of the ligand on the tnf-r2 receptor. we have shown that tnf-r1 is a negative regulator of progenitor proliferation in basal and insult-induced hippocampal neurogenesis. we have also used patch-clamp technique to explore whether a pathological environment influences the synaptic properties of new granule cells. rats were exposed to either a physiological stimulus, i.e., running, or a brain insult, i.e., status epilepticus which is associated with inflammation. we found that new granule cells in runners and status epilepticus animals had similar intrinsic membrane properties. in contrast, the new neurons which had developed in the physiological and pathological tissue environments differed with respect to tonic drive and short-term plasticity of both excitatory and inhibitory afferent synapses. the role of inflammation for these differences is currently being explored. proteinase-activated receptor-2 (par-2) is cleaved within its aminoterminal extracellular domain by serine protei-nases such as trypsin, unmasking a new aminoterminus starting with sligkv that binds intramolecularly and activates the receptor. par-2 is implicated in innate defense responses associated with lung inflammation. we showed that par-2 is expressed by human alveolar (a549) and bronchial (16hbe) epithelial cell lines, and is activated by trypsin and by the activating synthetic peptide sligkv-nh2. in cystic fibrosis patients, airspaces are invaded by polymorphonuclear neutrophils that release elastase and cathepsin g, two serine proteinases, and by pseudomonas aeruginosa that secretes an elastolytic metalloproteinase.we demonstrated that these three proteinases do not activate par-2, but rather disarm this receptor, preventing its further activation by trypsin but not by sligkv-nh2. preincubation of a par-2 transfected cell line with either proteinases leads to the disappearance of the cleavage/activation epitope. proteolysis by these three proteinases of synthetic peptides representing the aminoterminal extracellular domain encompassing the cleavage/activation sequence of par-2, generate fragments that would not by themselves act as receptor-activating ligands and that would not yield receptor-activating peptides upon further proteolysis by trypsin. our data indicate that neutrophiland pathogen-derived proteinases can potentially silence the function of par-2 in the respiratory tract, thereby altering the host innate defense mechanisms. caspase-3 -dependent killing of host cells and to disrupt intestinal barrier function, which, at least in the case of giardiasis, ultimately causes lymphocyte-dependent intestinal malfunction, and the production of diarrheal symptoms. ongoing research is investigating whether par agonists and microbial pathogens may cause epithelial apoptosis, increased permeability, and overall epithelial malfunction in the gastrointestinal tract, via common or intersecting pathways. the intestinal epithelium is exposed to a variety of proteases in both health and disease, including digestive proteases such as trypsin.given that protease-activated receptor 2 (par2) responds to trypsin and is expressed on intestinal epithelial cells, we investigated the effect of trypsin on intestinal epithelial barrier function.scbn, caco-ii and t84 epithelial cells were grown to confluence on filter supports and mounted in ussing chambers to study short circuit current (isc) and transepithelial resistance (rte).cell monolayers were incubated with inhibitors of transcellular ion transport in order to isolate the contribution of the paracellular pathway to rte.apical exposure to serine proteases including trypsin, elastase and chymotrypsin caused a rapid and sustained increase in rte and decreased the transepithelial flux of a 3000mw dextran.interestingly, the effect of trypsin could not be replicated by activators of pars 1, 2 and 4, suggesting that the effect on rte was not due to activation of pars.subsequent experiments showed that trypsin activated phosphatidylinositol-dependent phospholipase c.a trypsin-induced increase in intracellular calcium was not involved.inhibition of pkc-zeta, but not of typical pkcs, also blocked the response.our data point to a role for postprandial trypsin that extends beyond that of a digestive enzyme; it is also a participant in cellular pathways that control tight junction permeability. physiologically, the trypsin-induced increase in resistance could augment transcellular transport by reducing passive paracellular back-diffusion of ions. further studies will assess how these pathways might be disrupted in the barrier dysfunction characteristic of intestinal inflammation. clustering of inflammatory bowel disease in large families and the observation of an increased concordance between monozygotic twins suggests heritable components in these disorders. the high concordance in monozygotic twins (>55%), which is not seen in dizygotic twins (<5%) points to strong contribution of genetic susceptibility to the overall risk for disease. ibd represents a "complex disease" and may involve a large number of interacting disease genes. crohns disease has become an example for the successful molecular exploration of a polygenic etiology. crohns disease was not known before 1920. incidence has increased since now leading to a lifetime prevalence of up to 0.5% in western industrialized countries. the current hypotheses propose unknown trigger factors in the life style of western industrialized nations that interact with a polygenic susceptibility. it appears that increased expression and production of tnf and an enhanced state of activation of the nfkb system are main drivers of the mucosal inflammatory reaction. the exploration of inflammatory pathophysiology of crohns disease using full genome, cdna and oligonucleotide based arrays, respectively, has generated large sets of genes that are differentially expressed between inflamed mucosa and normal controls. while this may lead to new targets for a pathophysiology oriented therapy, it appears, however, that the dissection of the inflammatory pathophysiology does not allow to identify the multifactorial etiology of the disease. genome-wide linkage analysis has demonstrated eight confirmed susceptibility regions with the one on chromosome 16 being most consistent between different populations. in 2001 three coding variations in the card 15 gene were identified that are highly associated with development of the disease. all variants affect a part of the gene that codes for the leucin rich part of the protein, that appears to be involved in bacteria induced activation of nfkb in macrophages and epithelial cells. interestingly, the three disease associated snps are never found on the same haplotype. in compound heterozygotes or homozygotes they result in a rr of > 35 to develop crohns disease as an adult. a particular subphenotype with localization of the disease in the ileocecal region is highly associated with the variants in the card 15 gene. variations in the card 15 gene do not fully explain the linkage finding in the pericentromeric region of chromosome 16. after stratification for card 15 variants, the broad linkage peak is reduced to two more defined peaks on 16p and 16q, respectively. while the exploration of these regions has led to several association signals that are subject to further fine mapping a further disease gene progress has been greater in the other linkage regions (i.e. on chromosomes 10 and 5, respectively). dlg-5 is the example of a low-risk susceptibility gene with a modest associated odds ratio (1.2-1.5) . interestingly, the associa-tion signal appears to be confined to young males. slc22a4/5 which encode the kation-transporters octn1 and 2 have been suggested to represent the disease gene in the 200+ kb haplotype block on chromosome 5q31. mdr1 has also been implicated as a disease gene in ibd. although the human association studies have resulted in highly controversial findings a knockout mouse with a colitis phenotype makes mdr1 likely as a low risk susceptibility gene. with the advent of high-density, genome wide association studies enormous progress has been made to discover the remaining disease genes. recently a 330k illumina scan has been published identifying il-23r as a further disease gene. we used a genome wide candidate gene approach (with appr. 20.000 csnps) to identify atg16l1 as a further disease genes. both genes were confirmed and a further regulatory snp involving ptger4 was annotated by a belgian genome wide scan. by the time of presentation three further genome wide snp scans in crohn disease will most likely have entered the public domain. the further exploration of crohns disease (and other inflammatory conditions of barrier organs) will have to annotate the function and pathophysiologies based on genetic risk maps that are completed with amazing speed. the creation of a medical systems biology of disease will lead to new models and eventually new therapies. the chemokine receptor ccr9 plays a pivotal role in mediating the migration of t cells to the gastrointestinal mucosa. the ligand for ccr9, teck, s highly expressed in the gi tract. the pathogenicity of intestinal ccr9+/ cd4+ t cells has been demonstrated in animal models and this cell population is substantially increased in the peripheral circulation of crohns and celiac disease patients. ccx282-b is a highly selective and potent, orally bioavailable, small molecule antagonist of ccr9.the compound proved to be highly efficacious in the tnf-aare and mdr1a-/-murine models of inflammatory bowel disease (ibd). in phase i trials, ccx282-b was well-tolerated, and no drug-related saes were reported.a 28-day placebo-controlled phase ii study was recently completed in patients with moderate to severe crohns disease. ccx282-b was shown to be both safe and to have encouraging clinical results: 56% of patients on ccx282-b (cdai !250, crp >7.5mg/l) exhibited a 70-point drop in cdai compared to 29% on placebo. furthermore, a crp decrease of 11 mg/l was seen in the ccx282-b group compared to placebo. colonic biopsy samples were analyzed for expression of several pro-inflammatory cytokines. a mean decrease from baseline in the concentrations of tnf-alpha, il-12 p70, ifn-gamma, and the chemokine rantes was shown in the ccx282-b group while levels remained stable in the placebo group. ccx282-b is the first chemokine-based inhibitor of leukocyte trafficking to be tested in ibd. the compound shows anti-inflammatory activity and encouraging evidence for clinical benefit in the treatment of crohns disease. the activating receptor nkg2d seems to be implicated in the pathogenesis of several autoimmune diseases in humans and in animal models for type 1 diabetes and multiple sclerosis. the aim of this study was to asses the role of nkg2d in a model of inflammatory bowel disease, where cd4+cd25-t cells from balb/c mice are adoptively transferred to scid mice, and to evaluate the therapeutic effect of an anti-nkg2d antibody therapy. the expression of nkg2d was evaluated by flow cytometry, immunohistochemistry and by pcr. we found a marked up-regulation of nkg2d on the cell surface as well as increased levels of nkg2d mrna in cd4+ t cells from colitic scid mice as compared to normal balb/c mice. we next studied the effect of anti-nkg2d antibody (cx5) treatment initiated either before onset of colitis, when the colitis was mild, or when severe colitis was established. cx5 treatment decreased the cellsurface expression of nkg2d and prophylactic administration of cx5 attenuated the development of colitis significantly. a moderate reduction in the severity of disease was observed after cx5 administration to mildly colitic animals, whereas cx5 did not attenuate severe colitis. thus, nkg2d may be involved in the pathogenesis of colitis in this model, particularly in the early phases, since the expression of nkg2d in cd4+ t cells increased markedly during the development of disease and since administration of cx5 early but not late in the course attenuated the disease severity. proteins are used already for more than a century in the treatment of disease. the first generation were proteins derived from animals such as antisera used to treat infectious diseases as diphtheria and tetanus and later bovine and porcine insulin for the treatment ofdiabetes. the second generation were natural proteins from human source like the plasma derived clotting factors and human growth hormone. the development of the recombinant dna and cell fusion technology in the seventies of the 20th century opened up the possibilities to produce human proteins and monoclonal antibodies in unlimited amount in microbial and mammalian host cells. in 1982 human insulin was introduced as the first recombinant dna derived biopharmaceutical and since than more than 160 have gained approval. the pipeline contains many more potential biopharmaceuticals and at present 1 in 4 new drug applications concerns a biotechnology derived product. a major problem of therapeutic proteins is the induction of antibodies. for foreign proteins such as the murine derived monoclonal antibodies thisimmunogenicity was to be expected. however the humanization of monoclonal antibodies has reduced but not solved the problem of immunogenicity. and also the proteins which are homologues of endogenousfactors such as gm-csf, interferons etc. induce antibodies, sometimes even in the majority of patients. by definition we are immune tolerant to products which are copies of endogenous proteins. the products not necessarily need to be exact copies of the natural proteins to share this immune tolerance. when human therapeutic proteins induce antibodies, they are breaking b cell tolerance, which starts with the activation of autoreactive b-cells. presenting the self-epitopes in an array form is very potent activator of these b-cells. this explains why aggregates of human proteins are the most important factor in induction of antibodies. these aggregates may not be immediately present in the product, but may appear during storage making stability and formulation an important issue in predicting the immunogenicity. there are only a few studies in experimental model systems on the properties of the aggregates which break b-cell tolerance, indicating that only multiple order aggregates (>trimers) are involved. we study the capacity of a protein product to break b-cell tolerance in mice made transgenic for the specific protein. these mice are immune tolerant and there is a good correlation of an immune response in these mice and in patients. although these models have helped to identify the factors important for breaking b-cell tolerance and also have been useful in improving the formulation of products, there is not yet enough experience to use them as absolute predictors of immunogenicity of human proteins. they also allow to study the involvement of tcells in breaking b cell tolerance. all data obtained untilnow indicate this process to be t-cell independent. contact information: dr huub schellekens, central laboratory animal institute, utrecht university, utrecht, the netherlands e-mail: h.schellekens@gdl.uu.nl biomonitor aps, and institute for inflammation research (iir), rigshospitalet, copenhagen, denmark using recombinant technology, one can now produce protein drugs which are almost identical with naturally occurring human proteins, including antibodies (abs). many have assumed that these drugs may be administered with little or no risk of triggering specific t-and/or b-lymphocyte reactivities, because patients according to immunological dogma are tolerant towards their own proteins. unfortunately, this is not the case, and even socalled 100% human biologicals are potentially immunogenic (1) (2) . i shall discuss two examples: 1) recombinant human cytokines (ifn-beta-1a and -1b), and 2) anticytokine ab constructs (anti-tumor necrosis factor (tnf)-alpha). ifn-beta has been used for treatment of patients with multiple sclerosis since the early nineties. though initially neglected as a clinical problem, ifn-beta like many other human proteins is indeed immunogenic, especially those produced by recombinant gene technologies. the reported frequencies and titers of anti-ifn-beta ab vary considerably depending upon ifn-beta preparations and administration, and the types of assays being used (2-4). it took more than 10 years of clinical use before abmediated decrease in bioactivity of ifn-beta, a condition in which the clinical effect of continued injection of rec. ifn-beta is minimized or abrogated, was universally recognized (5, 6). 2) anti-tnf-alpha human ab constructs tnf-alpha is an inflammatory cytokine of central pathogenic importance in many immunoinflammatory conditions, and measures to diminish production and/or effects of tnf-alpha have long been a goal in the treatment of these conditions. currently, there are three approved and two other anti-tnf-alpha biopharmaceuticals in clinical use. unfortunately, response failure is frequently encountered. thus, 30-40% of patients are primary non-or lowresponders to the anti-tnf constructs, and secondary response failure is commonplace, mostly due to induction of anti-abs. several different methods have been used to assess circulating levels of anti-tnf drugs as well as anti-abs. most of these have been based on elisa technology, with their inherent problems of false positivity, susceptibility to nonspecific interference, etc. interferon beta (ifnbeta) has been an important step forward in the treatment of multiple sclerosis(ms), an inflammatory disease of the human central nervous system. however, one of the problems of ifnbeta is its immunogenicity; a substantial percentage of ms patients treated this recombinant protein develop anti-ifnã� antibodies, primarily of the igg class. the level of these antibodies tends to be low in the first month or two and peaks by six to eighteen months after initiation of therapy. most studies of these antibodies have measured their ability to neutralize ifnbetas effect in vitro, using assays in which sera from ms patients inhibit the protective effect of ifnbeta on viral killing of target cells. this antibody population is called neutralizing antibodies (nabs). tests measuring binding of antibodies to ifnã� in vitro are called binding antibody (bab) assay. anti-ifnbeta antibodies detected by bab assays are present in a high percentage of ms patients, and can occur at low levels without any apparent adverse effect on ifnbeta bioactivity. the distinction between babs and nabs is artificial, and all binding antibodies are likely neutralizing, if the neutralizing assay system is adequately sensitive; i.e., the development of babs and nabs is a continuum with the assay systems simply measuring the strength of the antibody response. in many treated patients, the anti-ifnbeta antibody response is strong, despite the resemblance of the injected protein to the human homologue, and high levels of neutralizing antibodies develop. high levels of anti-ifnbeta antibodies with high affinity results in loss of ifnbeta bioactivity, a phenomenon which has been called antibody-mediated decreased bioactivity or adb. adb can be considered the in vivo correlate of the neutralizing effect of the anti-ifnã� antibody population, while the nab assay measures the in vitro neutralization of this population of immunoglobulins in the serum. the three ifnbeta preparations have different incidences of nabs and different patterns of appearance and disappearance over time of nabs. because there is no direct correlation between nab levels and bioactivity at moderate levels of nab, in vivo bioactivity assays for ifnbeta have become increasingly utilized. in a large multicenter study in the us, called the insight study, bioactivity as measured by ifnbeta induced upregulation of the ifn-response genes mxa, viperin, and ifit1, was shown to be highly correlated with nab levels, confirming a single center study (pachner, a.r., pak,e., narayan, k., multiplex analysis of expression of three ifnbeta-inducible genes in antibody-positive ms patients, neurology, 66:444-446, 2006) . multiple studies, including a large multicenter study in denmark and a recent study from our center using high resolution mri of the brain once a month, have demonstrated that nabs abolish the salutary effects of ifnbeta on clinical aspects of ms, especially inflammation. recent guidelines for european neurologists recommend stopping ifnbeta in nab-positive patients. in order to maintain bioactivity of this important medication for ms, some neurologists have attempted to use immunosuppressives either to prevent the development of nabs, or to treat them once they have developed. however, at this point in time, there is no clearly optimal way to treat nabs. major efforts have been underway to decrease the immunogenicity of ifnbeta and a new formulation of one of the higher immunogenicity products has been recently developed and tested. proteinase-activated receptors (pars). endogenous serine proteinases such as thrombin, mast cell tryptase, trypsins, kallikreins, cathepsin g, for example, as well as exogenous proteases released by mites or bacteria are involved in cutaneous inflammation, host defense or tumor cell regulation. thus, the expression of pars on keratinocytes, endothelial cells, nerves, and immune cells suggest important role of pars as a part of the communication system in the skin during inflammation and the immune response. for example, par2 activates nf-kb in keratinocytes and endothelial cells, stimulates the release of chemokines and cytokines, and is involved in proliferation and differentiation. on sensory nerves, this receptor controls neurogenic inflammation by modulating edema and extravasation via release of neuropeptides into the inflammatory site. par1 and par2 also modulate leukocyte-endothelial interactions in the skin, thereby regulating inflammatory responses such as leukocyte trafficking through the vessel wall. they also stimulate signal transduction pathways involved in cutaneous inflammation. in sum, this novel receptor family requires a paradigm shift in thinking about the role of proteases in cutaneous biology and disease. novel compounds regulating protease and par function may be beneficial for the treatment of several skin diseases such as atopic dermatitis, psoriasis or pruritus. serine proteinases are upregulated in arthritic joints where their enzymatic activity participates in the destruction of articular soft tissues. in addition to their degradative functions, serine proteinases can also act as signalling molecules by activating members of the gprotein coupled receptor family called the proteinase activated receptors (pars). these receptors are known to regulate tissue inflammation and pain, although their function in joints is unclear. our study examined the effect of par4 activation in joint inflammation and pain. male c57bl/6 mice received an intra-articular injection of either the par4 activating peptide aypgkf-nh2 or the inactive peptide yapgkf-nh2 (100 mg) into the right knee. knee joint blood flow was then measured in these mice by laser doppler perfusion imaging while joint diameter measurements gave an indication of tissue oedema. mechanical allodynia was also assessed in these animals by application of von frey filaments onto the plantar surface of the ipsilateral hindpaw and a pain score was calculated. intra-articular injection of the par4 activating peptide caused knee joint blood flow to gradually increase by up to 25% over the succeeding 2hrs. knee joint swelling was also observed as well as the development of a mechanical allodynia. all responses could be blocked by pre-treatment with the selective par4 antagonist pepducin p4pal10 (100 mg i.p.). the control peptide yapgkf-nh2 had no discernible effect on joint inflammation or pain. these experiments show that peripheral activation of par4 receptors in mice knees causes joint inflammation and pain. vincent lagente (1), e boichot (2) (1) air liquide, centre de recherche claude-delorme, jouy en josas, france (2) inserm u620, universitã� de rennes 1 matrix metalloproteinases (mmps) are a major group of proteases known to regulate the turn-over of extracellular matrix and so they are suggested to be important in the process of lung disease associated with tissue remodelling. these led to the concept that modulation of airway remodeling including excessive proteolysis damage of the tissue may be of interest for future treatment. among metalloproteinases (mmps) family, macrophage elastase (mmp-12) is able to degrade extracellular matrix components such as elastin and is involved in tissue remodeling processes in chronic obstructive pulmonary disease (copd). pulmonary fibrosis has an aggressive course and is usually fatal for an average of three to six years after the onset of symptoms. pulmonary fibrosis is associated with deposition of extracellular matrix (ecm) components in the lung interstitium. the excessive airway remodeling as a result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of extracellular matrix components could be in favor of anti-protease treatments. indeed, the correlation of the differences in hydroxyproline levels in the lungs of bleomycin-treated mice strongly suggests that a reduced molar pro-mmp-9/timp-1 ratio in broncholaveolar lavage fluid is associated with collagen deposition, beginning as early as the inflammatory events at day 1 after bleomycin administration. finally, these observations emphasize those effective therapies for these disorders must be given early in the natural history of the disease, prior to the development of extensive lung destruction and fibrosis. in addition to their degradative properties, proteases can act as signalling molecules that send specific messages to cells.recent work has demonstrated that proteases are able to signal to peripheral sensory neurons, thereby participating to neurogenic inflammation processes and to the transmission or inhibition of pain messages. serine proteases cleaving specifically at an arginin site are able to activate protease-activated receptors (pars), which then send specific messages to cells. we have demonstrated that 3 members of the par family (par1, par2 and par4) are present on peripheral sensory neurons, where they can be activated by different proteases.the activation of par1 and par2 in isolated sensory neurons provokes calcium mobilization and the release of substance p and cgrp, while the activation of par4 inhibited bradykinin-and capsaicin-induced calcium signal, and neuropeptide release.thrombin and pancreatic trypsin caused inflammation respectively through a par1 and par2-dependent mechanism involving the release of neuropeptide.the extrapancreatic form of trypsin (mesotrypsin or trypsin iv) also caused neurogenic inflammation through a par2 and par1dependent mechanism, and causes inflammatory hyperalgesia and allodynia, through a par2-dependent mechanism.in contrast, activation of par4 on peripheral sensory neurons inhibited inflammatory hyperalgesia and allodynia. taken together, these results provide evidences that proteases can interfere with inflammatory and pain mechanisms through the activation of pars on peripheral sensory neurons.determining the role of each individual proteases and their receptors in sensory neuron signalling and above all inflammatory and pain mechanisms constitutes an important challenge to raise new anti-inflammatory and analgesic drugs. introduction: a scoring system for disseminated intravascular coagulation (dic) in humans has been proposed by the international society on thrombosis and haemostasis (isth). it was the objective of this study to develop and validate a similar scoring system for dic in dogs in order to establish the dog as a spontaneous animal model. methods: for the developmental study, 100 consecutive dogs admitted to the intensive care unit (icu) were enrolled prospectively (group a). blood samples were collected daily and a broad panel of coagulation assays performed. diagnosis of dic was based on the expert opinion of one human physician and two veterinarians. a multiple logistic regression model was developed with the coagulation parameters as explaining variables for the diagnosis of dic. integrity and diagnostic accuracy was subsequently evaluated in a separate prospective study according to the stard criteria. the validation study prospectively enrolled 50 consecutive dogs (group b). results: 37 dogs were excluded from group a where 23/63 dogs (37%) had dic. final multiple logistic regression model was based on aptt, pt, d-dimer and fibrinogen and had a very high diagnostic sensitivity and specificity. diagnostic accuracy of the model was sustained by prospective evaluation in group b. conclusion: based on generally available assays, it was possible to design an objective diagnostic model for canine dic, which has both a high sensitivity and specificity. such a model will provide basis for treatment optimization and make it possible to conduct multicentered therapy studies with a minimum risk of systematic misclassification of patients. in 1997, a coagulation independent change in light transmittance (biphasic waveform [bpw] ) was reported in automated activated partial thromboplastin time assays (aptt) in patients with disseminated intravascular coagulation (dic). a calcium-dependant precipitate of creactive protein and very-low-density-lipoprotein was causing the bpw. our group recently identified this phenomenon in dogs also. initially, bpw was introduced as a complementary tool to assist diagnosing dic. however, recent studies reported that bpw may have a stronger potential as a prognostic marker for survival. the aims of the study were to prospectively investigate (a) the diagnostic significance of bpw regarding dic and (b) the significance of bpw to outcome, in dogs with diseases known to predispose for dic. the study was performed as a prospective, observational study including 50 consecutive dogs with a final diagnosis known to predispose for dic (20% were finally diagnosed with dic). outcome was 28-day survival. bpw was assessed by means of a hirudin-modified aptt assay (kjelgaard-hansen et al., jvim 2006:20; 765-766) . relative risk according to bpw (rr [95% confidence interval]) for (a) a dic diagnosis and (b) 28-day mortality, were assessed. 28-day mortality in the study population was 44%. 28% were bpw positive. bpw was not a significant diagnostic factor for dic (rr=0.64 [0.16; 2.67] ), but strongly so for outcome (rr=2.57 [1.46;4.52] ) with a 79% (11/14) mortality amongst bpw positive dogs. in conclusion, bpw was observed in dogs predisposed to dic, with a strong potential as a risk factor for outcome, a finding in line with recent findings in humans. (1), b hideo (2) (1) department of molecular pathology, kumamoto university, japan (2) department of gastroenterological surgery, kumamoto university, japan aeromonas species are facultative anaerobic gramnegative rods that are ubiquitous, waterborne bacilli, most commonly implicated as causative agents of gastroenteritis. aeromonas infections often develop sepsis and disseminated intravascular coagulation syndrome (dic) is a life-threatening complication of sepsis patients, causing multiple organ failure.however, a mechanism leading to coagulation induction in the bacterial infection has not been known. to study the dic induction by aeromonas species infection, we investigated coagulation activity of a serine protease (asp) from aeromonas sobria, predominantly isolated in patients blood. proteolytically active asp shortened both activated partial thromboplastin time and prothrombin time of human plasma in a dose-dependent manner starting at an enzyme concentration of 30 nm. asp activated human prothrombin, releasing hydrolytic activity for thrombinspecific substrate boc-val-pro-arg-mca, but no enzymatic activity was produced from coagulation factors ix and x. analysis by sds-page revealed that asp released a prothrombin fragment with a molecular weight identical with that of fâ¿-thrombin in an incubation timedependent manner. western blotting using biotinylated phe-pro-arg-chloromethylketone, a thrombin inhibitor, showed that asp produced an enzymatically active fragment whose molecular weight was same as that of fâ¿-thrombin. prothrombin incubated with asp but not the protease itself caused platelet aggregation. these results indicate that asp activates prothrombin, producing fâ¿-thrombin that converts fibrinogen to fibrin clot, and suggest that asp coagulation-inducing activity contributes to dic development in sepsis caused by aeromonas sobria infection. the present study shows a link between inflammation and coagulation mediated by a bacterial protease. hemolytic episodes are often associated to high amounts of free heme in circulation (up to 20 um) and the development of an inflammatory response that may develop to a chronic inflammation. our group has shown that free heme is a prototypical proinflammatory molecule, able to induce neutrophil migration, actin cytoskeleton reorganization and nadph oxidasederived reactive oxygen species (ros) generation, as well as pkc activation and interlukin-8 expression (graã�a-souza et al., 2002) . moreover, free heme inhibits human neutrophil spontaneous apoptosis, a feature that is closely related to the impairment of resolution of inflammation and consequent promotion of chronic inflammatory status. heme protective effect requires nadph oxidase-derived ros and involves the activation of mapk, pi3k and nf-kb signaling cascades as well as heme oxygenase (ho) activity (arruda et al., 2004) . more recently, we have shown that heme antiapoptotic effect is closely related to the maintainance of mitochondrial stability, inhibition of bax insertion into mitochondria and a dramatic increase on bcl-xl/bad protein ratio in a ros-dependent manner, requiring the same signaling pathways that regulate heme anti-apoptotic effect. these findings attest to a prominent role of free heme in the onset of inflammation associated to hemolytic episodes as well as the statement of chronic inflammation related to these disorders. the recent advance on the study of free heme as a proinflammatory molecule brings up hope for the development of new strategies to ameliorate acute and chronic inflammation found during hemolytic episodes.financial support: faperj, cnpq, capes. (1) (1) university of melbourne, victoria, australia (2) monash university, victoria, australia we have previously demonstrated that mice lacking the anti-oxidative enzyme, glutathione peroxidase (gpx1), show significantly larger infarcts after stroke. recent studies have demonstrated that adhesion molecule-mediated leukocyte recruitment is associated with increased tissue damage in stroke, while mice lacking key adhesion molecules conferred neuro-protection. nevertheless, the involvement of oxidative stress in leukocyte recruitment and subsequent regulated cell injury is yet to be elucidated. to explore this, gpx1-/-mice were subjected to transient mid-cerebral artery occlusion (mcao) followed by cerebral intravital microscopy, for assessment of leukocyte-endothelium interactions in intact cerebral microvasculature. after 1hr mcao, leukocyte-endothelium interaction was significantly reduced in gpx1-/-mice compared to wt counterparts during the second hour of reperfusion. laser doppler and direct measurement of blood flow in pial postcapillary venules revealed a reduction of reperfusion in gpx1-/-mice following transient mcao. this suggests that the reduction in nutritive blood flow following stroke in gpx1-/-mice may explain the enhanced injury in these mice as well as the reduced leukocyte-endothelium interaction. furthermore, matrix metalloproteinase-9 (mmp9) which has previously been shown to be implicated in endothelial dysfunction and the pathogenesis of stroke was found to be up-regulated in gpx1-/-mice to a greater extent than in wt mice after mcao, suggesting a role for oxidative stress in cerebral microvascular injury. the data present here suggests oxidative stress may be one of the factors that contribute to reduced post-ischemic perfusion, via the disruption of the endothelial function as indicated by the increased level of mmp9. chris bolton(1), c paul(2), s barker (3), r mongru (3) (1) william harvey research institute, london, uk (2) university west of england, bristol (3) queen mary university of london adrenomedullin (am) acts as a vasodilator in many vascular beds including the cerebral circulation where the peptide is produced in larger amounts than in the periphery.in vitro work has shown that am beneficially regulates blood-brain barrier (bbb) characteristics including transendothelial electrical resistance, permeability and p-glycoprotein pump activation.our preliminary studies in acute experimental autoimmune encephalomyelitis (eae), a model of the human disease multiple sclerosis (ms), have demonstrated significant elevations in am peptide levels corresponding with am mrna changes during late, neurological disease where am production may be linked to the restoration of bbb function. however, am is not exclusively produced as result of am gene upregulation. furthermore, am peptide levels do not always match am mrna changes during other disease phases of eae.the current study has investigated, more closely, the relationship between am gene expression and subsequent levels of associated peptides. am mrna levels were determined, by rt-pcr, in the cerebellum, medulla-pons and spinal cord of normal and eae-inoculated lewis rats at the height of disease. am and proadrenomedullin peptide (pamp) levels were measured in the tissues by radioimmunoassay.all tissues examined showed an increase in am gene mrna compared to control levels.am and pamp changes were observed in the samples and differences between the peptide profiles were recorded.an understanding of alterations in the generation of am and related peptides during neuroinflammation may provide insight into mechanisms affecting bbb permeability and be of relevance to the changes in neurovascular function seen during ms. platelet-activating factor (paf) contributes to the robust inflammatory responses in acute phase and spread of secondary injury. although, paf is believed to be a potent edematous but non-painful mediator in peripheral tissues, we recently demonstrated that paf may be a mediator of noxious signaling in spinal cord in case of neuronal injury. paf-induced tactile allodynia may be mediated by atp, glutamate and the generation of nitric oxide (no). the present study elucidated down-stream signaling pathway for paf-induced tactile allodynia. paf-and glutamate-induced tactile allodynia was blocked by the pretreatment with no scavengers and inhibitors of no synthase, soluble guanylate cyclase or cgmp-dependent protein kinase (pkg). recent evidence attributes the generation of pain to specific disfunctions of inhibitory glycinergic neurotransmission. to explore the target molecule for induction of tactile allodynia, the effect of knockdown of glycine receptors containing the a3 subunit (glyr a3) by sirna spinally transfected with hvj-e vector was examined. in mice spinally transferred with sirna for glyr a3, the reduction of glyr a3 was demonstrated in superficial layer of dorsal horn by immunohistochemical analysis. pcpt-cgmp, paf, glutamate failed to induce tactile allodynia in mice spinally transferred with sirna of glyr a3, while these compounds produced tactile allodynia in mice transferred with mutant sirna of glyr a3 as a control. glycine tranporter inhibitors ameriolated paf-and pcpt-cgmp-induced allodynia. these results suggest that glutamate-no-cgmp-pkg pathway plays a key role for paf-induced tactile allodynia in spinal cord and glyr a3 may be a target molecule for pkg to induce allodynia. (1), r leite(2), ys bakhle (3) (1) federal university of minas gerais, belo horizonte, brazil (2) medical college of georgia, augusta, usa (3) imperial college, london, uk selective cyclooxygenase 2 inhibitors (coxibs) induce a characteristic increase in mechanical nociceptive threshold, referred to as "hypoalgesia", in inflammatory pain induced by carrageenan in rat paws.we have here assessed the role of the cytoskeleton in this hypoalgesia induced by celecoxib (cx). male holtzman rats (150-200g; 3-5 animals/group) were injected in the right hind paw (ipl) with a range of cytoskeletal inhibitors (selective inhibitors of microtubules (taxol, nocodazole, colchicine), of actin microfilaments (latrunculin b, cytochalasin b) or of intermediary filaments (acrylamide) (pico to nanomoles per paw) and 30 min later given cx (12mg/kg, s.c.). after a further 30 min, rats were injected (ipl) with the inflammatory stimulus, carrageenan (250 mg/paw). mechanical pain threshold was hourly measured over the next 4 h, using the randall-sellitto method. the cxinduced hypoalgesia was reversed by low doses of latrunculin b or cytochalasin (latrunculin 100% reversal = 1.26 nanomoles), higher doses of microtubule inhibitors (taxol 100% reversal = 23.4 nanomoles) with no effect of acrylamide (7 up to 141 nanomoles).we conclude that 1) local changes in (paw) cytoskeleton occurred during cxinduced hypoalgesia and 2) actin microfilaments were the cytoskeletal components most critically involved in this hypoalgesia.financial support: cnpq, fapemig and capes there are reports regarding the up-regulation of cyclooxygenase isoenzyme particularly inducible isoform i.e. cox-2 in brain during neurodegenerative or neuropsychiatric disorders.in the present study, we examined the effect of nimesulide (a preferential cox-2 inhibitor) in subchronic immobilization stress. mice were subjected to immobilization stress for 6 hrs daily for a period of seven days. nimesulide (2.5 mg/kg, i.p.) was administered daily for 7 days before challenging them to immobilization stress. behavioral analysis revealed the hyperlocomotor activity and increased anxious response. subchronic stress decreased % retention of memory and also caused hyperalgesic response in mice. biochemical analysis revealed that chronic immobilization stress significantly increased lipid peroxidation and nitrite levels and decreased the reduced glutathione and adrenal ascorbic acid levels. chronic treatment with nimesulide significantly attenuated the immobilization stress-induced behavioral and biochemical alterations. these results suggested that the use of nimesulide could be a useful neuroprotective strategy in the treatment of stress. there is accumulated evidence for ngf role as a peripheral pain mediator. ngf is upregulated in diverse inflammatory conditions and evokes hyperalgesia when injected in humans and rats. ngf increase was also observed in temporomandibular join (tmj) after cfa injection, indicating its possible involvement in local hyperalgesic states. therefore, the objective here was to evaluate if ngf participate in the tmj nociception. to test this hypothesis, the ngf was injected into the tmj alone or after carrageenan (cg) and the spontaneous nociceptive behavior of head flinches was counted for up 30min. further evidence for the ngf nociceptive activity was obtained quantifying the local production of ngf after cg injection, by elisa, and the fos-like immunolabeling in the trigeminal sensory nucleus (including the caudalis, interpolaris and oralis) after ngf injection. injections were performed in 0.25ul. ngf (0.2, 1 and 5ug) injected in the tmj challenged 1h prior by cg (100ug) induces a dose-dependent increase in the number of head flinches. this increase was reduced by k252a (1 and 2ug), indicating a trka receptor-mediated effect. we detected a significant increase in the ngf production 1 and 3h after the tmj cg (300ug) injection. the tmj injection of ngf (5ug) alone did not induce detectable spontaneous nociceptive behavior. however, the ngf (5ug) injection induces a significant increase in the fos like immunolabeling (fli) in the sensory trigeminal nucleus compared to the saline injection. these results indicate that the ngf participates in the nociceptive activity in the tmj, specially in inflammatory conditions. mif was reported as a key cytokine in the pathogenesis of rheumatoid arthritis (ra) several years ago, but it now clear that mif is also involved in the pathogenesis of systemic lupus erythematosus (sle) and atherosclerosis. mif-deficient lupus-prone mrl/lpr mice exhibit prolonged survival and reduced renal and skin disease compared to mif-expressing mice. similarly, mif-deficient atheroma-pone ldlr-deficient or apoe-deficient mice are significantly protected from disease and antimif mab therapy is beneficial. ra and sle are each characterised both by an increased prevalence of atherosclerotic vascular disease and by overexpression of mif. given the effects of mif on atherosclerosis it can be hypothesised that mif overexpression participates in the risk of atherosclerotic vascular disease in ra and sle. recent data have provided insights into mechanisms of action for mif relevant to all these concepts. firstly, the newly described role of mif in the selective recruitment of monocyte-macrophage lineage cells is of particular relevance to ra, sle, and atherosclerosis, with evidence that mif mediates macrophage recruitment in sle and atherosclerosis. secondly, glucocorticoid (gc) therapy is possible risk factor for atherosclerosis in patients with ra and sle, and it is now clear that gc increase the expression and release of mif, potentially implicating mif in gc-related increases in atherosclerosis in ra and sle. specific therapeutic targeting of mif in ra and sle may address not only primary disease pathways but also the increased risk of atherosclerosis in these diseases. to enter inflamed tissues, leukocytes must undergo adhesion molecule-mediated interactions with the endothelial surface of vessels at the site of inflammation.cytokines such as tumour necrosis factor (tnf) are established as important mediators capable of promoting leukocyte-endothelial cell interactions.however, in inflammatory diseases such as atherosclerosis and rheumatoid arthritis, elevated expression of another cytokine, macrophage migration inhibitory factor (mif) occurs, yet the role of this cytokine in leukocyte recruitment is unknown.therefore we explored the ability of mif to regulate leukocyte recruitment.this was achieved using intravital microscopy to examine the intact microvasculature in mice following local mif treatment. these experiments showed that mif induced leukocyte adhesion and transmigration in vivo, resulting in accumulation of predominantly cd68+/f4/80-ve/cd11c-ve monocyte/ macrophage lineage cells.mif did not induce upregulation of adhesion molecules p-selectin and vcam-1, although their constitutive expression contributed to recruitment.in contrast, mif-induced recruitment was blocked by antibodies to the monocyte-specific chemokine, ccl2/mcp-1, and its receptor ccr2, and in response to anti-cxcr2.this was supported by in vitro experiments showing that mif induced ccl2/mcp-1 release from cultured murine endothelial cells.finally, mice lacking cd74, the putative mif binding molecule, did not respond to mif.these data demonstrate a previously unrecognized function of this pleiotropic cytokine: induction of monocyte migration into tissues, and indicate the involvement of a pathway involving a complicated chemokine/chemokine receptor pathway with contribution from cd74.this function may be critical to the ability of mif to promote diseases in which macrophages are key participants. gm-csf and m-csf (csf-1) can enhance macrophage lineage numbers as well as modulate their differentiation and function. of recent potential significance for the therapy of inflammatory/autoimmune diseases, their blockade in relevant animal models leads to a reduction in disease activity. what the critical actions are of these csfs on macrophages during inflammatory reactions are unknown. to address this issue, adherent macrophages (gm-bmm and bmm) were first derived from bone marrow precursors by gm-csf and m-csf, respectively, and stimulated in vitro with lps to measure secreted cytokine production, as well as nf-kb and ap-1 activities. gm-bmm preferentially produced tnfa, il-6, il-12 and il-23 while, conversely, bmm generated more il-10 and ccl2; strikingly the latter population could not produce detectable il-12 and il-23. following lps stimulation, gm-bmm displayed rapid ikba degradation, rela nuclear translocation and nf-kb dna binding relative to bmm, as well as a faster and enhanced ap-1 activation. each macrophage population was also pre-treated with the other csf prior to lps stimulation and found to adopt the phenotype of the other population to some extent as judged by cytokine production and nf-kb activity. thus gm-csf and m-csf demonstrate at the level of macrophage cytokine production different and even competing responses with implications for their respective roles in inflammation including a possible dampening role for m-csf. granulocyte macrophage-colony stimulating factor (gm-csf), initially discovered for its role in the differentiation of haematopoietic cells into granulocytes and macrophages, can also affect mature cell function and may be considered proinflammatory. gm-csf is able to prime macrophages for increased pro-inflammatory responses, including the increased release of tnfa and il-12 following stimulation with, for example, lps. in addition, gm-csf has been shown in vivo, using murine disease models, to play a key role in a number of inflammatory diseases. gm-csf-/-mice have been shown to be resistant to several diseases, including arthritis, and, most notably, blockade of gm-csf with a neutralizing monoclonal antibody was effective at ameliorating arthritis when given either prophylactically or therapeutically. t cells appear to be the major cell type responsible for gm-csf production required for arthritis, and gm-csf appears important in the effector phase of disease, subsequent to t cell activation. blockade of gm-csf results in fewer inflammatory cells, particularly macrophages, and cytokines such as tnfa, at the site of inflammation. these findings suggest that blockade of gm-csf may be an effective treatment in a range of inflammatory diseases. the autoimmune disease type 1 diabetes mellitus (t1dm) is thought to be mediated by autoreactive t cells recognizing islet autoantigens, including gad65, ia-2 and proinsulin. this disease arises on a distinctive genetic background, mapping most notably to the mhc, and is also open to strong environmental influence. to investigate the pathogenesis of the disease, and in particular the prevailing paradigm that islet autoreactive t cells are important, we have developed an approach to epitope identification that is mhc allele and autoantigen specific, and operates for both cd4 and cd8 t cells. utilizing this, we have uncovered populations of islet antigen-specific t cells that have the immunological credentials to be both pathogenic (eg th1, tc1) and protective (treg) in the disease. we have cloned some of these cell types, enabling us to analyse their function and provide an insight that will be important for an understanding of disease mechanisms, as well as guiding novel therapeutic interventions. tcr transgenic targeting b:9-23 cause diabetes 2. knockouts of the insulin 2 gene (expressed in thymus as well as islets) accelerates diabetes while knockout of insulin 1 gene (islet expression) prevents 90% of diabetes 3. dual insulin knockout with transgenic insulin with altered peptide (b16:a) prevents all diabetes 4. islets with native b:9-23 sequence, but not altered sequence when transplanted into knockouts restore anti-insulin autoimmunity and diabetes transfer by t cells 5.anti-b9-23 t cells have conserved valpha and jalpha chain usage but no conservation n region or beta chain 6. alpha chain as transgene sufficient to engender anti-insulin autoantibodies 7. kay and coworkers demonstrate insulin reactivity "upstream" of igrp and igrp reactivity nonessential.future studies in nod directed at deleting specific conserved alpha chains to test diabetes prevention and develop therapeutic.in man we can now identify at birth genetic risk as high as 80% of activating anti-islet autoimmunity with mhc analysis and restricted heterogeneity suggesting dominant target.insulin autoantibodies in prospective studies such as daisy usually appear initially and levels are related to progression to diabetes.analysis of cadaveric donors is underway to elucidate primary targets. (t1d) is an autoimmune disease in which genes and environment contribute to cell-mediated immune destruction of insulin-producing beta cells in the islets of the pancreas. the holy grail of autoimmune disease prevention is negative vaccination against autoantigens to induce disease-specific immune tolerance. this has been achieved in rodents by administering autoantigen via a tolerogenic route (mucosal), cell type (stem cell or resting dendritic cell), mode (with blockade of t-cell co-stimulation molecules) or form (as an altered peptide ligand). compelling evidence demonstrates that proinsulin is the key autoantigen that drives beta-cell destruction in the non-obese diabetic (nod) mouse model of t1d, and possibly in humans. proinsulin/ insulin dna, protein or t-cell epitope peptides administered in a tolerogenic manner to the nod mouse can delay or prevent the development of diabetes, via one or more mechanisms (deletion or anergy of effector t cells, induction of regulatory t cells). administration of autoantigen via the mucosal route, which induces anti-diabetic regulatory t cells in the rodent, is the most immediately translatable approach to humans. initial human trials of vaccination with oral autoantigens lacked evidence of bioeffect, probably due to inadequate dosage in end-stage disease. recently, however, the first evidence for a therapeutic effect of mucosal autoantigen has been seen in trials of oral and nasal insulin in islet autoantibodypositive individuals at risk for t1d. combination autoantigen-specific vaccination also shows promise in combination with non-specific immunotherapy in established t1d. leukocyte extravazation is an integral process both physiologically (immunosurveillance) and pathophysiologically (inflammation). the initial paradigm of a 4-step process comprising tethering/rolling, activation, firm adhesion, and diapedesis, each involving specific adhesion molecules, has repeatedly been modified in the light of more recent findings. additionally, organ-specific differences regarding the role of distinct molecules were established. finally, the skin became a good "model" to study due to its accessability and availability of powerful animal models. in-vitro adhesion assays, flow-chamber systems, intravital microscopy, animal models for delayed-type hypersensitivity, and transplantation approaches have successfully been employed to investigate leukocyte extravazation. numerous molecular interactions such as the cutaneous lymphocyte-associated antigen and sialyl-lewisx, or icam-1 and lfa-1, have been proven sufficiently relevant to make them candidates for potential therapies. with the anti lfa-1 antibody efalizumab, approved for the treatment of psoriasis, the first therapeutic agent specifically targeting leukocyte extravazation is already on the market; other compounds are under development. moreover, novel data suggest that well-established anti-inflamamtory therapies such as fumarates also influence this process, thus contributing to their clinical efficacy. ongoing research aks for adopting a more "dynamic" view on leukocyte extravazation as several molecules obviously perform multiple tasks throughout this process rather than being limited to just one step of this multi-step cascade; this is particularly true for the so-called junctional adhesuã­on molecules which obviously mediate more than just diapedesis. finally, similarities between leukocyte extravazation and hematogenic metastases are emerging. consequently, certain anti-inflammatory compounds may turn out to also exhibit striking anti-metastatic efficacy, and vice versa. department of dermatology, heinrich-heine-university, dã¼sseldorf, germany atopic dermatitis, psoriasis vulagaris and cutaneous lupus erythematosus represent chronic inflammatory skin diseases showing distinct clinical phenotypes but sharing one aspect. the recruitment of pathogenic leukocyte subsets into the skin represents a prerequisite for their initiation and maintenance. during recent years, our knowledge of the immunopathogenesis of chronic inflammatory skin diseases increased significantly. with regard to the recruitment pathways of leukocytes, a superfamily of small cytokine-like proteins so called chemokines has attracted significant attention. here the complex interactions within the chemokine ligand-receptor network are introduced, the involvement of chemokines in memory t and dendritic cell trafficking is outlined and current concepts of their role in the immunopathogenesis of atopic dermatitis, psorasis vulgaris and cutaneous lupus erythematosus are summarized. the skin serves as a unique organ for studying general principles of inflammation because of its easy accessibility for clinical evaluation and tissue sampling. a network of pro-inflammatory cytokines including il-1 and tnf-a is known to play a key role in the pathogenesis of cutaneous inflammatory diseases through activation of specific signalling pathways. recently, progress in understanding the underlying mechanisms regulating inflammatory signalling pathways in the immunopathogenesis in skin carcinomas, psoriasis vulgaris and atopic dermatitis has been made. kinases have been identified to play a crucial role in regulating the expression and activation of inflammatory mediators in these inflammatory skin diseases. mitogen-activated protein kinases (mapks) are a family of serine/threonine protein kinases that mediate a wide variety of cellular behaviours in response to external stress signals. increased activity of mapks, in particular p38 mapk, and their involvement in the regulation of synthesis of inflammatory mediators at the transcriptional and translational level has recently been demonstrated. progress in our understanding of inflammatory signalling pathways has identified new targets for treating inflammatory diseases, but the challenge is to place a value on one target relative to another and to evolve strategies to target them. a careful examination of different signalling pathways in various inflammatory conditions is therefore needed. this presentation gathers recent advances in signal transduction in skin inflammation focusing interleukin-1, tnf-âµ, p38 mapk, msk1/2, mk2, nf-kappab and ap-1. histamine is an important inflammatory mediator in humans, and despite their relatively modest efficacy antihistamines are frequently used to treat allergic conditions, as well as other histamine-mediated reactions such as pruritus. in contrast, antihistamines are of very limited use for controlling other conditions where histamine production is abundant, including asthma. the discovery of the histamine h4 receptor (h4r) prompted us to reinvestigate the role of histamine in pulmonary allergic responses, as well as in pruritus. h4r deficient mice and mice treated with h4r antagonists exhibited decreased allergic lung inflammation in several models, with decreases in infiltrating lung eosinophils and lymphocytes and decreases in th2 responses. ex vivo restimulation of primed t cells showed decreases in th2 cytokine production, and in vitro experiments suggest that decreased cytokine and chemokine production by dendritic cells after blockade of the h4r was responsible for the the t cell effects. the influence of h4r on allergic or histamine-induced pruritus was explored in mice using selective histamine receptor antagonists and h4r deficient mice. the h4r was found to mediate the majority of histamine-mediated and allergic itching, while the contibution by the h1r was minor. surprisingly the h4r effect was independent of mast cells or other hematopoetic cells. this work suggests that the h4r can modulate both allergic responses via its influence on t cell activation, and pruritus through mechanisms that are independent of hematopoetic cells. the studies show that the h4r mediates previously uncharacterized effects of histamine and highlight the therapeutic potential of h4r antagonists. (1), bsp reddy (2) (1) nizams institute of medical sciences, hyderabad; india (2) genix pharma, india rupatidine, carries the majority of the histamine h1receptor -blocking activity and has been introducedfor the treatment of allergic rhinitis and urticaria. objectives: the aim of this study was to compare the effect of two by measure of inhibition of histamine induced wheal and flare response. methodology: 12 male volunteers were enrolled after written informed consent before to ethic committee approved protocol. in this randomised, double-blind, single oral dose, cross overstudy, they were randomized to receive either 10 mg rupatidineformulation after overnight fast. washout was 10 days. wheal and flare were induced on the forearm of the trial subjects, by histamine intradermally injection while the subject was lying comfortably with arm resting on the bed. ten minutes later, wheal and flare were visualized under a bright lamp. histamine induced wheal and flare skin test was performed before and regularly to 24hours after drug administration. results: administration of reference and test formulations of rupatidine, significantly inhibited the histamine induced cutaneous response in all the subjects. the least square mean ratio (%) t vs r for peak activity imax-% (maximum inhibition of histamine induced wheal and flare response); area under the activity time curve (auc0-24 mmsq/hr and auc 0-24 %/hr) both for untransformed and log transformed data were found to be within 80-125% of90% ci limits both formulations well tolerated. conclusions: it can thus be concluded that be concluded that test formulation of rupatidine tablet is bioequivalent to reference rupatidine tablet (1), h yoshimura(1), k ohara (1), y mastui (1), h hara (1), h inoue (1), h kitasato (1), c yokoyama(2), s narumiya (3), m majima (1) (1) kitasato university school of medicine, kanagawa, japan (2) tokyo dental medical colledge, tokyo, japan (3) kyoto university school of medicine, kyoto, japan thromboxane (tx) a2 is a potent stimulator of platelet activation and aggregation and vascular constriction. we have reported the magnitude of cytokine-mediated release of sdf-1 from platelets and the recruitment of nonendothelial cxcr4+ vegfr1+ hematopoietic progenitorsconstitute revascularization. we hypothesized that txa2 induces angiogenic response by stimulating sdf-1 and vegf which derived from platelet aggregainflamm. res., supplement 3 (2007) tion.to evaluate this hypothesis, we dissected the role of the txa2 in angiogenesis response using mouse hind limb model. recovery from acute hind limb ischemia, as assessed by the ratio between the treated ischemic limb and the untreated control right limb was assessed in wild type mice (c57bl/6 wt) , prostaglandin i2 receptor (ip) knock out(ipko) and thromboxane (tx) a2 receptor (tp) knock out(tpko). blood recovery in tp-/-significantly delayed compared to wt and ipko. immunohistochemical studiesrevealed that tp-/-mice were less stained against pecam positive cells compared to wt and ipkoplasma sdf-1 and vegf concentration were significantly reduced in tp-/-mice. we observed during in vivo fluorescence microscopic study that compared to tpko, ipko and wt significantly increased platelet attachment to the microvessels around ligated area. tpko translpanted wt bone marrow cells increased blood recovery compared to tpko transplanted tpko bone marrow cells. in addition, mice injected with txa2 synthase c-dna expressing fibroblast increased blood flow recovery compared to control mice. these results suggested that tp signaling rescues ischemic condition by inducing angiogenesis by secreting sdf-1 and vegf from platelet aggregation. purpose: the s100 calcium-binding proteins, a8, a9 and a12 are constitutively expressed in neutrophils and induced in activated macrophages. high levels are found in sera from patients with infection and several chronic inflammatory diseases. the calgranulin complex, a8/a9 is anti-microbial; a8 has oxidant-scavenging functions. a12 is chemotactic for monocytes, and recruits leukocytes in vivo by activating mast cells (mc). effects of these mediators on mc and monocyte function were compared. methods: human pbmc or murine mc were activated in vitro with s100 and mediator release and cytokine induction (assessed by quantitative rt-pcr/elisa), determined. a cys41 to ala41 a8 mutant was used to determine whether effects on mc are mediated by redox. immunohistochemistry was used to demonstrate s100s in asthmatic lung. the s100s were expressed in asthmatic lung, particularly in eosinophils and alveolar macrophages. strong reactivity occurred with an antibody recognising predominantly the hypochlorite-oxidised from of a8. a8, a9 or the a8/a9 complex had relatively low ability to induce il1, tnf, il6, and chemokines mrna from pbmc compared to a12. only a12 induced significant levels of il13; none induced il4 or gm-csf mrna compared to lps. in contrast to a12 which is activating, a8 significantly inhibited mc degranulation provoked by ige cross-linking; suppression was dependent on cys41. conclusions: the cytokine profile generated by a12 in mc and monocytes strongly supports a role the pathogenesis of asthma. in contrast, results strongly support a role from a8 in oxidant defence, particularly to hypobromite generated by activated eosinophils. (1), d mankuta(2), g gleich (3), f levi-schaffer (1) (1) hebrew university of jerusalem, israel (2) hadassah university hospital, israel (3) university of utah, usa the onset, amplitude and termination of allergic responses is regulated at the mast cell/eosinophil interface. eosinophil major basic protein (mbp), which activates mast cells in the late-chronic phase of allergic inflammation, is a central determinant in this interface. characterized more than two decades ago, the exact nature of this activation has not been clarified as yet. here we demonstrate that mbp exerts its activating effect on human mast cells and basophils through cd29 and hematopoietic cell kinase (hck). a genome-wide analysis showed that hck displays shifts in mrna levels specifically upon mbp-induced mast cell activation. hck also shows a unique priming pattern prior to this activation. cd29 is phosphorylated specifically upon activation with mbp and deploys a signaling complex that critically depends on hck. extracellular neutralization of cd29 interferes with mbp entry into the cell, and this as well as rna silencing of hck results in defective mbp-induced activation. finally, cd29 neutralization abrogates mbp-induced anaphylaxis in-vivo. these findings picture for the first time a chronic-phase specific pathway mediating eosinophil-induced mast cell activation with critical consequences for the therapy of chronic allergic inflammation. alexander robinson (1), d kashanin (2), f odowd(2), v williams(2), g walsh (1) (1) cellix ltd, institute of molecular medicine, dublin, ireland (2) university of aberdeen, scotland, uk leukocyte adhesion to endothelial cell bound proteins, such as icam-1 and vcam-1, is an initial step of the inflammatory response. we have developed an in vitro microfluidic system which mimics conditions found in blood vessels in vivo during an immune response. using this system, we can record leukocyte adhesion levels under physiologically relevant flow conditions (e.g. 0-5 dynes/cm2). the adhesion profiles of resting and pmastimulated peripheral blood lymphocytes (pbls) were recorded, with respect to vcam-1, icam-1, and bsa. images at each shear stress level were captured using a digital camera, and analysed using our in-house ducocell software package. distinct morphological changes in pma-stimulated pbls, compared to non-stimulated cells, can be observed. these include a less rounded appearance of the pma-stimulated pbls, and evidence of "uropod" formation, which anchor the t cell to the endothelium as part of the migration process. levels of adhesion to vcam-1 are high (80-90%, compared to control), but there appears to be little difference between the adhesion profiles of non-stimulated and pma-stimulated pbls.however, there is a distinct difference between the adhesion levels of non-stimulated and pma-stimulated pbls to icam-1, with pma-stimulated cells showing a higher affinity for icam-1 than nonstimulated cells (approx. 70% and 40%, respectively).pbl adhesion to bsa is negligible. we present a novel in vitro microfluidic pump system that can simulate leukocyte adhesion to the endothelium under flow conditions. this platform is a more efficient and economical system compared to those currently available, due to reduced material costs and style of construction. introduction: pulmonary aspiration of gastric contents is a common complication observed in icu patients and a potential trigger of ards. in this study we evaluated the course of lung inflammation induced by intranasal instillation of gastric juice (gj). methods: gj was obtained from donor rats (ph 1.6). male c57bl/6 were instilled with 2 ml/kg of gj. after 2 or 24 h, the animals were sacrificed and lung and balf were collected. control group consisted of non-manipulated mice. 287.3ae18.6, sg24h: 277.8ae63.8; pg/ml). discussion: gj aspiration induced an initial adherence of pmn to lung tissue that is correlated with increased tnf-a/il-10 ratio in balf at the 2nd h. the reduction of mpo activity is correlated with the decrease in tnf-a/il-10 ratio. the late increase of pmn in balf might be a consequence of the early production of tnf-a. the results are suggestive that the treatment of patients exposed to acid aspiration should be focused in the initial period of the insult and in the blockage of tnf-a. objectives: intestinal i/r is implicated as a prime initiating event in the development of acute respiratory distress syndrome (ards) after trauma and hemorrhagic shock. we investigated the effects of lps challenge to mice previously submitted to i-i/r, a two-hit model of acute lung injury. methods: male c57bl/6 mice were subjected to 45 min of intestinal ischemia and challenged with 0.1 mg/kg of intranasal lps at the 4th hour of reperfusion (two-hit). balf and culture of lung explants were performed 20 h after lps challenge. mice subjected to i-i/r or lps alone were used as controls. results: two-hit mice showed marked increase in lung evans blue dye leakage compared to i-i/r (375.5ae53.9 vs 48.8ae3.5, mg/mg). lung mpo was increased (0.30ae0.03 vs 0.15ae0.03; od 450nm) whereas the neutrophil recruitment to balf was inhibited in the two-hit group compared to lps group (11.5ae2.6 vs 28.5ae3.4; x 10e3cells/mouse). the levels of nox-in the two-hit group were significantly increased when compared to i-i/ r controls in balf (10.6ae0.5 vs 6.5ae0.8; mm) and in lung explants (3.1ae0.3 vs 1.3ae0.1; mm/mg of tissue). conclusions: intestinal i/r predisposes the animal to an exacerbated response to a low dose lps insult. the exacerbated production of nitric oxide observed in the two-hit group may cause endothelial damage, thereby explaining the major increase in vascular permeability in the two-hit group. the results are suggestive that patients exposed to systemic inflammatory response might develop ards when in contact with secondary inflammatory stimuli. nitric oxide may play an important role in this process. (1) (1) novartis institutes for biomedical research, horsham, uk (2) university of michigan, usa obligatory for using oxygen in energy transfer pathways was the simultaneous co-evolution of enzymes that detoxify the reactive species formed as by-products. thus, we hypothesized that individuals with low aerobic function will have reduced anti-oxidant capacity and, therefore, be more susceptible to smoking-related lung diseases like copd. to test this hypothesis, we exposed high capacity runner (hcr) and low capacity runner (lcr) rats to 3 months of whole-body smoke exposures.the animals, bred over successive generations on the same background strain for high or low running capacity, differ by over 500% (p<1x10-37) for exercise capacity, measured by running on a treadmill.after 3 months of exposures, inflammatory cells in bronchoalveolar lavage fluid were increased in both the hcr-and lcr-smokeexposed(se) animals compared to air-exposed controls (p<0.001); however there was a 2-3-fold increase in the number of neutrophils and lymphocytes in the lcr-over the hcr-se group (p<0.001).histopathology revealed there was greater inflammation and lung damage present in the lcr-versus hcr-se group (p<0.05). metabonomic (metabolite profiling) analysis revealed that while peroxidation of lung lipids occurred for both se groups, oxidative damage to the lung surfactant layer was significantly more extensive for the lcr-se. systemic oxidative damage was also more apparent in the lcr-se group, with metabolic profiling suggesting a reduced capacity to regenerate muscle glutathione. the metabolic data suggest that repair processes maybe more effective in the hcrs. in summary, these data support the concept that aerobic capacity may be central to ones susceptibility to developing smoking-related lung disease. (1), ap ligeiro-oliveira(1), jm ferreira-jr(4), sr almeida(1), w tavares de lima(1), shp farsky (1) (1) university of sâ¼o paulo, brazil (2) regional integrated university of alto uruguai and missã°es, brazil methods: male wistar rats were exposed to vehicle or hq (50 mg/kg; ip.;daily, 22 days, two-day interval every five days). on day 10, animals were ip sensitized with ovalbumin (oa). assays were performed on day 23. results: hq-exposed rats presented reduced number of leukocytes in the bronchoalveolar fluid and by impaired in vitro oa-induced tracheal contraction. the latter effect suggests reduction on mast cell degranulation, and it was corroborated by in vivo decreased mesenteric mast cell degranulation after topical application of oa. the oa-specificity response was confirmed by normal ability of mast cells to degranulate in both groups of animals after topical application of compound 48/80. in fact, lower levels of circulating oa-anaphylactic ige antibodies were found in hq-exposed rats. this latter effect was not dependent on number or proliferation of lymphocytes, nevertheless reduced expressions of costimulatory molecules cd45 and cd6 on oa-activated lymphocytes indicated the interference of hq exposure on signaling of the humoral response during an allergic inflammation. contact information: ms sandra manoela dias macedo, regional integrated university of alto uruguai and missã°es / university of sâ¼o p, department of clinical and toxicological analyses, sâ¼o paulo, brazil e-mail: smdmacedo@yahoo.com.br (1) (1) radboud university nijmegen, medical centre, nijmegen, the netherlands (2) university hospital, zã¼rich, switzerland toll-like receptors (tlr) are essential in the recognition of invading microorganisms. however, increasing evidence shows involvement of tlr in autoimmunity, such as rheumatoid arthritis (ra), as well. here we investigated whether synovial expression of tlr3 and tlr7 was associated with the expression of ifna, tnfa, il-1b, il-12, il-17, and il-18 and studied in what way these receptors and cytokines were associated in vitro. using immunohistochemistry, we found that tlr3 / tlr7 expression in synovial tissue was associated with the presence of ifna, il-1b and il-18, but not tnfa, il-12 and il-17. to investigate whether ifna, il-1b and il-18 could induce tlr3 / tlr7 upregulation in vitro, we incubated separate lymphocyte populations with these cytokines and subsequently determined tlr3/7 mrna expression. ifna incubation resulted in significant tlr3 /tlr7 upregulation, whereas il-1b and il-18 did not. pre-incubation with ifna and subsequent stimulation of tlr3 /tlr7 significantly enhanced il-6, tnfa and ifna/b production, indicating that the ifn-induced tlr upregulation was functional. low amounts of biologically active il-1b were produced upon stimulation with atp, but not upon tlr3 / tlr7 stimulation, although mrna levels were high. interestingly, ifna-priming significantly increased the atp-induced il-1b production. here, we demonstrated a dual role for ifna in vitro, which could explain the association between tlr and il-1b / il-18 in synovial tissue. first, involvement in tlr3 /tlr7 regulation and second, involvement in atp-induced production of biologically active il-1b. these results suggest involvement of anti-viral immune responses in ra and ifna as a key player in chronic inflammation. the pathogenesis of chronic joint inflammation remains unclear although the involvement of pathogen recognition receptors (ppr) has been suggested recently. here, we described the role of two members of the nacht-lrr (nlr) family, nod1 (nucleotide/ binding oligomerization domain) and nod2 in model of acute joint inflammation induced by intraarticular injection of tlr2 (toll-like receptor) agonist streptococcus pyogenes cell wall fragments. we found that nod2 deficiency resulted in reduced joint inflammation and protection against early cartilage damage. in contrast, nod1 gene deficient mice developed enhanced joint inflammation with concomitant elevated levels of proinflammatory cytokines and cartilage damage. to explore whether the different function of nod1 and nod2 occurs also in humans, we exposed pbmcs carrying either nod1frameshift or nod2frameshift mutation with scw fragments in vitro. both tnfa and il-1b production was clearly impaired in pbmcs carrying the nod2fs compared to pbmcs isolated from healthy controls. in line with the nod1 gene deficient mice, human pbmcs bearing the nod1 mutation produced enhanced levels of proinflammatory cytokines after 24h stimulation with scw fragments. these data indicated that the nlr family members nod1 and nod2 have a different function in controlling tlr2-mediated pathways. we hypothesize that intracellular nod1-nod2 interactions determine the cellular response to tlr2 triggers. whether lack of controlling tlr2-driven pathways by nod1 signalling is involved in the pathogenesis of autoinflammatory or autoimmune disease, such as rheumatoid arthritis (ra), remains to be elucidated. leukocyte immunoglobulin-like receptors (lilrs) are a family of receptors with potential immune-regulatory function. activating and inhibitory receptors play a role in maintaining immunological equilibrium and an imbalance may lead to the onset of autoimmune diseases such as rheumatoid arthritis (ra). ra is a chronic inflammatory disease of joints caused by mediators (i.e. tnf-a) produced by activated leukocytes. we recently demonstrated expression of activating lilra2 in synovial tissue macrophages from ra patients. the aim of this study was to determine expression and function of lilra2 in monocytes and macrophages. peripheral blood mononuclear cells (pbmc) were prepared by standard density gradient separation and in vitro-derived macrophages were generated by differentiating thp-1 cells with vitamin-d3. lilra2 expression was measured by flow cytometry before and after modulation with cytokines. differentiation to macrophages significantly up-regulated lilra2 expression (p=0.0239). treatment of macrophages with lps, tnf-a, il-1b and ifn-g but not il-10 caused significant down-regulation of lilra2 (p<0.01). function of lilra2 was assessed by cross-linking with plate-immobilised lilra2-specific mab. soluble tnf-a was measured by elisa. activation of cells elicited tnf-a production in a dose-dependent manner while time-course analysis shows maximal production at 18h. correlation between lilra2 expression and response to cross-linking indicates that level of expression may relate directly to the degree of activation. decrease expression in response to acute-phase cytokines suggests controlled regulation during inflammation. in ra, abnormal regulation of lilra2 could potentially exacerbate inflammation by inducing uncontrolled production of proinflammatory cytokines. pharmacological blocking of lilra2 could potentially provide therapeutic benefit. (1) (1) university of valencia, spain (2) northwick park institute for medical research, uk co-releasing molecules (co-rms) mimic the biological actions of co derived from heme oxygenase activity. in the present work we studied the effects of a water-soluble co-releasing molecule (corm-3) on an animal model of human rheumatoid arthritis. dba-1/j mice were treated with corm-3 (5, 10 or 20 mg/kg/day, i.p.) from day 22 to 31 after collagen-induced arthritis (cia) and sacrificed on day 32. administration of corm-3 resulted in a significant improvement of the clinical profile of this disease since it markedly reduced joint swelling and redness. histological analysis of the joints in control arthritic mice indicated the presence of granulocytes and mononuclear cells, cartilage erosion, chondrocyte death and proteoglycan depletion. all these parameters were significantly reduced by corm-3 treatment with the most pronounced protective effect observed at 10 mg/kg. the levels of pro-inflammatory mediators (pge2, il-1beta, tnfalpha, il-2 and il-6) in the hind paw homogenates were significantly inhibited by corm-3. in addition, comp levels in serum, a marker of cartilage degradation, was reduced by the co-releasing agent. our studies show that therapeutic administration of corm-3 alleviates the clinical features of murine cia at the late phase of this response. the beneficial action of co liberated from corm-3 appears to be associated with a decrease in inflammatory cytokines and reduction of cell infiltration into the synovial tissues ultimately leading to a protective effect on the cartilage. aim: to setup a bovine model for cytokine-induced articular cartilage collagen degradation, and characterize the model using a variety of compounds targeting different disease mechanisms relevant to arthritis. methods: full thickness bovine articular cartilage punches were cultured with or without 10 ng/ml il-1a, tnf-a and oncostatin m. after three weeks the cartilage and culture medium were analyzed for weight changes, water content, dna content, glycosaminoglycans (gag), hydroxyproline (hyp), damaged collagen molecules, mmp activity, ctx-ii and comp. diclofenac, dexamethasone, pioglitazone, remicade, risedronate, galardin and a77-1726 were tested for their effect on cartilage degradation. results: exposure of articular cartilage to cytokines resulted in a decreased cartilage weight, increased proteoglycans degradation, increased collagen degradation, increased percentage of denatured collagen, increased water content and increased levels of active mmps (all p < 0.01). comp release during the first week of culture showed a trend towards up regulation during the first week of culture for all three donors, this was however not significant due to the small number of donors. most of the described processes were modulated by one or more of the drugs tested, indicating that this model for articular cartilage destruction is sensitive to treatment. discussion: stimulation of bovine articular cartilage explants with a cocktail of il-1a, tnf-a and osm results in clear and consistent changes in the cartilage, highly reminiscent of cartilage destruction during arthritis. further research needs to establish whether the model is also sensitive to anabolic factors that potentially could repair the damage. toll-like receptors (tlrs) may contribute to the progression of rheumatoid arthritis through recognition of hostderived damage-associated ligands that have repeatedly been found in arthritic joints. involvement of tlr2 and tlr4 activation in the expression of arthritis was studied using interleukin-1 receptor antagonist deficient (il-1ra-/-) mice, which spontaneously develop an autoimmune t-cell mediated arthritis. spontaneous onset of arthritis was dependent on tlr activation by microbial flora, as germ-free mice did not develop arthritis. after crossing with tlr knockouts, il-1ra-/-tlr2-/-mice developed more severe arthritis compared to il-ra-/-tlr2+/+ littermates; whereas, tlr4-/-il-1ra-/-mice were protected against arthritis. to clarify the mechanism by which tlr2 and tlr4 differentially regulated the disease expression, we studied the role of these tlrs in il-23 production and th17 development, both important in il-1ra-/-arthritis. wild type bone-marrow-derived dendritic cells (bmdcs) produced similar levels of il-23 upon stimulation with tlr2 and tlr4 ligands; however, il-1ra-/-bmdcs produced less il-23 than wild type dcs upon tlr2 stimulation and more il-23 than wild type dcs upon tlr4 stimulation. furthermore, il-1ra-/-t cells produced lower amounts of il-17 when cultured with tlr2-activated apcs and higher amounts of il-17 when cultured with tlr4-stimulated apcs, both in combination with cd3 stimulation. facs analysis of th17 (cd4+/il-17+) cells from both spleen and draining lymph nodes revealed 70% reduction in il-1ra-/-tlr4-/-mice compared to il-1ra-/-tlr4+/+ littermates. specific cd3/cd28 stimulation of non-adherent splenocytes confirmed lower il-17 production in il-1ra-/-tlr4-/-. these findings suggest important roles for tlr2 and tlr4 in regulation of th17 development and expression of arthritis. prostaglandine2 (pge2) stimulates the transactivational activity of p53 through p38map kinase-dependent ser15 phosphorylation (jbc 2006) .p53 controls cell-cycle progression, in part, by differential regulation of ap-1 proto-oncogenes (jun/fos).currently we studied pge2 control of cyclin d1 promoter activity with particular attention to the role of ap-1 oncogenes.pge2 induced a 7.6 fold increase in junb mrna expression (northern blot), a 2.1-fold increase in junb promoter activity (luciferase assay), and increased ser259junb phosphorylation in human synovial fibroblasts (hsf) (western blot).c-jun was strongly inhibited while jund, c-fos, fra1/ 2, and fosb expression were upregulated by pge2.in cell-cycle experiments, transformation with a constitutively active ha-ras construct (ras g12v) resulted in a 3.9 fold increase in cyclin-d1 promoter activity, cyclin-d1 synthesis, thr202/tyr204 phosphorylation of erk1/2 (6.2 fold) and ap-1 (c-jun)-dependent transactivity (3.1 fold); cyclin d1/cdk4-6 inhibitor p16ink4a synthesis was suppressed. addition of excess rass17n dominant negative mutant construct to the plasmid mix abrogated the aforementioned processes.ectopic expression of c-jun, c-fos and especially jund expression constructs stimulated cyclin d1 promoter activity/protein synthesis, blocked p16ink4a synthesis; the latter effects were reversed by the addition of excess junb.pge2 exerted temporal and bi-phasic dose-dependent control of the cyclin d1 promoter activity, largely through differential ap-1 activation and promoted cell cycle arrest and apoptosis in hsf at high physiological concentrations.the results provide further insight into the biology of the cpla2/ cox/pges biosynthetic axis and highlight the complexity of pge2 action in terms of cell-cycle progression. di-glucopyranosylamine (diga) is an antikeratitic (roberts et al., 1990 , acvo conference, scottsdale) immunomodulatory pyranosyl disaccharide with parenteral anti-rheumatic activity (bolton et al., 2005 , inflammation res. 54(s2) s121) and unknown mechanism of action.interestingly, anti-tnf therapy is anti-keratitic.-diga hydrolyses to monoglucosylamine (mga) and glucose, which is prevented by n-acetylation (nacdi-ga).lider (1995, pnas. 92: 5037-41) showed that sulphated disaccharides are orally active, inhibit tnf synthesis and the dth reaction.we have investigated the anti-tnf and anti-rheumatic activity of the sulphated and free digas.human whole blood (hwb) was stimulated with pha (5mg/ml) to synthesise tnf.antigen induced arthritis (aia) was induced in methylated bovine serum albumin (mbsa) sensitised c57bl/6 mice challenged i.a. into the stifle joint.collagen arthritis (cia) induced in dba mice by sensitisation to bovine collagen, were boosted i.p. with collagen at day 21. hwb tnf synthesis was inhibited by diga, mga and nacdiga(ic50 <0.1mm). diga (10ml, 1mm) i.a. prevented 24 hour aia (-0.32+/-0.06mm).diga at 100mg/kg reduced aia when administered i.v. (-0.40+/-0.13mm, p<0.05) and i.p. (-0.34+/-0.13mm, p<0.05), but is hydrolysed p.o. (0.24+/-0.08mm ns). polysulphated diga (diga8s) was unstable, but stabilised by n-acetylation (nacdi-ga8s).tnf synthesis was potently inhibited by both nacdiga and nacdiga8s (ic50 <0.1mm).nacdiga8s (100mg/kg p.o.) inhibited aia (-0.43+/-0.13mm), and nacdiga8s with lower degrees of sulphation (mw 800 and 1200 kda) inhibited the development of mouse collagen induced arthritis as assessed by clinical score. sulphated diglucosylamines represent a new class of heparinoid which are potent inhibitors of tnf synthesis and possess oral anti-rheumatic activity. excessive no appears to play a key role in the pathogenesis of chronic inflammatory diseases. in this study we aimed to evaluate no synthesis in rheumatoid arthritis (ra) before and after therapy. it was performed on 92 persons, divided into 6 groups: a negative control group of healthy volunteers, a positive control group with ra, a group with ra and physiotherapy (phys), a group with ra and low doses of cimetidine (cim) + doxycycline (dox), a group with ra and combined treatment phys + cim + dox, and a group treated with usual doses of ibuprofen (ibu). serum nitrite/nitrate (griess) was measured in order to evaluate no synthesis. results: compared to the positive control group, in all the treated groups no synthesis decreased significantly. there was no significant difference between phys and cim+dox effect alone. the combined treatment, phys + cim + dox had a much better inhibitory effect on no synthesis. between the phys, cim + dox and phys + cim + dox groups and that treated with ibuprofen, there was no significant difference in reducing no synthesis. conclusions: 1) in ra phys + cim + dox treatment was as efficient as ibuprofen in reducing no synthesis. 2) the low doses of cim and dox may allow a longer treatment due to the lower side effects risk enhanced socs1 expression following exposure of murine macrophages to lps implicated socs1 in the control of lps-mediated signaling. socs1 regulates nfkb signaling in murine macrophages, blocking at the level of mal or ikba phosphorylation. we investigated the role of socs1 in regulating the production tnf by lps and pam3csk4-activated primary human monocytes. blood monocytes were isolated by centrifugal elutriation and either infected with an adenoviral vector expressing socs1 (adv-socs1), control vector (adv-gfp) or left untreated. adv-socs1 monocytes were exposed to tlr4 and tlr2 ligands, lps (500 ng/ml) or pam3csk4 (300 ng/ml). facs analysis demonstrated infection efficiencies of 50ae4% and 67ae4% (n=16, mean ae sem) of monocytes expressing adv-gfp or adv-socs1 at moi 50. adv-socs1 blocked lps and pam3csk4 induced tnf mrna and protein production in a dose-dependent manner. in contrast, il-6 and il-10 production by adv-socs1-infected monocytes was not blocked. adv-socs1 also blocked lps and pam3csk4induced tnf production by macrophages isolated from synovial fluid. infection efficiencies of 67ae1% or 72ae1% were obtained. quantitative western blot analysis revealed that the classically defined nfkb pathway was not altered at the level of ikba or p65 activation. furthermore, the kinetics of lps and pam3csk4induced ikba phosphorylation and degradation in adv-socs1 monocytes remained unaffected (n=5 and 2 donors, respectively). further, analysis of parallel mapk pathways demonstrated no block in p38 or erk mapk pathways. these data suggest that socs1 regulation of lps and pam3csk4-induced tnf production by human monocytes occurs downstream of tlrs, possibly at the level of transcription. recently, beta-nad+ has emerged as a novel extracellular player in the human urinary bladder. beta-nad+ is the natural substrate of cd38 which catalyzes the conversion of beta-nad+ to cadpr. under normal conditions in vivo, there is no or only very small quantities (submicromolar range) of extracellular beta-nad+ compared to intracellular levels (200-1000 mm). during inflammation cell lysis may cause bursts of high local beta-nad+ levels. however, the effect of beta-nad+ on the human detrusor smooth muscle cells (hdsmc) was unknown. the effect of beta-nad+ on cultured (explant technique) hdsmc was determined by: 1) measuring cytosolic free calcium ([ca2+] i) in fura-2 loaded hdsmc using spectrofluorometry and 2) force measurements in 10-20 mg detrusor strips. hdsmc responded to beta-nad+ (40-100 mm) with an immediate and transient increase in [ca2+] i. the ca2+ transient was followed by one or two much slower and transient increases in [ca2+] i, indicative of beta-nad+ enzymatic conversion into cadpr. the ca2+ responses persisted in the absence of extracellular calcium. the ca2+ responses to beta-nad+ were not affected by exposure of hdsmc to atp supporting the notion that the effects of beta-nad+ were not mediated via p2x7 purinoceptors. furthermore, beta-nad+ caused a concentration-dependent detrusor muscle relaxation. this is the first study to report that extracellular beta-nad+ affect intracellular calcium homeostasis and force in hdsmc. these powerful actions of beta-nad+ suggest a role for beta-nad+/cadpr system as a novel extracellular player in the human detrusor during inflammation. aids remains a worldwide threat more than two decades after identification of hiv as the etiological agent. its wide dissemination can be partly attributed to its successful suppression of immunity resulting in disease progression and concomitant opportunistic infections including mycobacterial and cytomegalovirus infections. hiv trans-activator (tat) is one of the regulatory proteins that mediates hiv replication and dysregulates cellular functions such as apoptosis and cytokine expression. for example, tat induces tumor necrosis factor (tnf) and enhances gp120-induced neurotoxicity. we recently showed that tat induces the overexpression of il-10 via cellular kinase pkr and activation of transcription factor ets-1. in this study, we examined whether tat plays a role in perturbing interferon-& (ifn&) signal transduction. we showed that tat impaired ifn&-induced stat1 tyrosine phosphorylation, but had no effects on the serine residue of stat1 and jak kinases in primary human blood monocytes. furthermore, we found that the nuclear translocation of phospho-stat1 was abrogated by tat. the inhibition of phospho-stat1 led to the deformation of stat1 homodimers and subsequent stat-dna complex. to investigate the cellular consequences, we measured the expression of ifn&-stimulated genes including human leukocyte antigen (hla) and 2,5oligoadenylate synthetase (2,5oas), a key enzyme in the activation of latent ribonuclease l. the results showed that tat inhibited transcriptional activation of 2,5oas and hla. taken together, we identified a new role for tat in which it impairs ifn& signal transduction and suppresses inflammation, thus crippling the immune system and contributing to hiv persistence, opportunistic infections and disease progression. caspase-1 belongs to the group of inflammatory caspases and is the activating enzyme for the pro-inflammatory cytokine interleukin-18 (il-18), a cytokine known to play an important role in the pathogenesis of psoriasis. the purpose of this study was to determine the expression of caspase-1 in psoriatic skin and the signaling mechanisms involved in stress induced activation of caspase-1 and il-18. interestingly, increased caspase-1 activity in lesional compared with nonlesional psoriatic skin was seen as determined by western blotting. in vitro experiments in cultured human keratinocytes demonstrated anisomycin induced, p38 mapk dependent increased secretion of procaspase-1 and active caspase-1. furthermore, anisomycin increased the mrna expression of il-18 through a p38 mapk dependent but caspase-1 independent mechanism, reaching a maximum level after 12 hours of stimulation. finally, anisomycin caused a rapid (4 hours) increase in the secretion of proil-18 and active il-18. secretion of active il-18 was mediated through a p38 mapk/caspase-1 dependent mechanism, whereas secretion of proil-18 was mediated by a p38 mapk dependent but capsase-1 independent mechanism. these data demonstrate that the activity of caspase-1 is increased in psoriatic skin and that il-18 secretion is regulated by a p38 mapk/caspase-1 dependent mechanism, making caspase-1 a potential target in the treatment of psoriasis. prostaglandin e2 (pge2) regulates the stability of cyclooxygenase-2 (cox-2) mrna through adenylate/uridylate-rich elements (ares) in the 3untranslated region (3utr) by a positive autocrine/paracrine feed-forward loop. the principal objective of this study was to elucidate the molecular mechanisms involved in the pge2dependent stabilization of cox-2 in human synovial fibroblasts (hsfs). transfection of well-known are binding proteins (aubps) demonstrated that tristetraprolin (ttp) potently destabilized a [luciferase-cox-2 3utr] reporter fusion mrna (71ae5.7% decrease in luciferase activity vs. control). ttp protein levels in hsfs remained constant despite il-1b-induced changes in ttp mrna levels, thus suggesting translational regulation of its expression. pge2 did not affect the transcription or translation of this gene in hsfs. western blot analysis of hsf ttp demonstrated the existence of a specific, covalent~55kda heterocomplex containing ttp (ttphcx). although ttphcxs exact composition and stoichiometry is yet to be defined, pge2 selectively regulated the amount of this heterocomplex in a time-dependent manner. furthermore, protein shuttling studies performed using real-time confocal microscopy revealed that pge2 can induce export of a small nuclear pool of ttp-gfp. finally, transfection of ttp into hsfs also influenced cox-2 gene transcription, thus enabling ttp to regulate cox-2 gene expression at both the transcriptional and post-transcriptional level. in conclusion, we have demonstrated that ttp is an rna binding protein capable of influencing cox-2 mrna stability and transcription and whose localization and interaction with other factors is regulated by pge2. these data can provide important insight into deciphering the role of pge2 in fine-tuning physiological and pathophysiological gene regulation. (1) (1) chinese academy of sciences, shanghai, pr china (2) ohio state university, usa mitogen-activated protein (map) kinases play a critical role in innate immune responses to microbial infection through eliciting the biosynthesis of proinflammatory cytokines. map phosphatases (mkp)-1 is an archetypical member of the dual-specificity phosphatase family that deactivates map kinases. induction of mkp-1 has been implicated in attenuating the lipopolysaccharide (lps) and peptidoglycan (pgn) responses, but how the expression of the mkp-1 is regulated is still not fully understood. here, we show that inhibition of p38 map kinase by specific inhibitor sb 203580 or rna interference (rnai) markedly reduced the expression of mkp-1 in lps or pgn-treated macrophages, which is correlated with prolonged activation of p38 and jnk. depletion of mapkap kinase 2 (mk2), a downstream substrate of p38, by rnai also inhibited the expression of mkp-1. the mrna level of mkp-1 is not affected by inhibition of p38, but the expression of mkp-1 is inhibited by treatment of cycloheximide. thus, p38 mapk plays a critical role in mediating expression of mkp-1 at a posttranscriptional level. furthermore, inhibition of p38 by sb 203580 prevented the expression of mkp-1 in lpstolerized macrophages, restored the activation of map kinases after lps restimulation. these results indicate a critical role of p38-mk2-dependent induction of mkp-1 in innate immune responses. the i-kb kinase (ikk) complex regulates the activation of nf-kb a key transcription factor in inflammation and immunity. whilst ikka activity is necessary for proinflammatory and anti-apoptotic gene expression, ikka has distinct roles in lymphorganogenesis and b cell maturation. here we describe a role for ikka in cell mediated immunity (cmi). paw inflammation in methylated bsa-induced cmi was significantly reduced in transgenic mice expressing a mutant ikka protein that cannot be activated (ikka aa/aa ) compared to wild-type (wt). antigen-induced il-2 and ifng production by ikka aa/aa splenocytes and ikka aa/aa t cell:dc cocultures were also significantly reduced ex vivo. this could be normalised by using wt t cell: ikka aa/aa dendritic cell (dc) but not ikka aa/aa t cell:wt dc combinations. this suggests that reduced cmi in ikka aa/ aa mice is due to a defect in ikka aa/aa t cells not dcs. this is not due to a requirement of ikka in tcrmediated activation of t cells, since anti-cd3/cd28 mediated activation of ikka aa/aa t cells was unaltered. however, lps-induced production of the important th1 cytokine il-12 is impaired in ikka aa/aa dcs. we are currently addressing the hypothesis that ikka activity may be required for the generation and maintenance of antigen-specific t cells in vivo. recently we described a role for ikkâµ in the negative regulation of innate immunity and acute inflammation, which is in contrast to its role shown here in promoting adaptive immunity and antigen-driven inflammation. ikka may represent an alternative target for the treatment of autoimmune disease which would not compromise host defence. as a latent transcription factor, nf-kb translocates from cytoplasm into nucleus upon stimulations and mediates expression of genes important in immunity, inflammation and development. although extensive studies have been done regarding how nf-kb is triggered into nucleus, little is known about how it is regulated inside nucleus. by twohybrid approach, we identify a prefolding-like protein snip that is expressed predominantly and interacts specifically with nf-kb inside nucleus. we show that rnai knockdown of snip leads to impaired nucleus activity of nf-kb and dramatically attenuates expression of nf-kb dependent genes. this interference also sensitizes cells to apoptosis by tnf-a. furthermore, snip forms a dynamic complex with nf-kb and is recruited to the nf-kb enhancesome upon stimulation. interestingly, snip protein level correlates with constitutive nf-kb activity in human prostate cancer cell lines. the presence of nf-kb within nucleus of stimulated or constitutive active cells is significantly diminished without endogenous snip. our results reveal that snip is an integral component of nf-kb enhancesome and essential for its stability in nucleus, which uncovers a new mechanism of nf-kb regulation. bone remodeling is a tightly regulated process that couples resorption of old bone by osteoclasts and the deposition of new bone by osteoblasts. an imbalance between bone formation and bone resorption can result in various metabolic bone diseases, such as rheumatoid arthritis and osteoporosis. osteoclasts are terminally differentiated cells that arise from a haematopoietic stem cell lineage, which also gives rise to monocytes and macrophages. osteoclast differentiation and regulation of this process to maintain bone homeostasis are central to the understanding of the pathogenesis and treatment of bone diseases, such as osteoporosis. in vitro, osteoclast formation from bone marrow macrophages is induced by rankl (receptor activator of nf-kappa b ligand) in the presence of m-csf (macrophage colony stimulating factor). osteoclastogenesis is markedly enhanced in bone marrow macrophages from ifnar1-/-and ifnar2-/-mice and results in increased number of multinucleated cells positive for osteoclast marker, trap (tartrate-resistant acid phosphatase). consequently, the mutant mice develop osteoporotic phenotype, characterised by reduced bone density. these findings suggest that the ifn alpha/beta system is critical for the negative feedback regulation of osteoclastogenesis and that rankl signaling is essential for the induction of osteoclast differentiation. atp acting on p2x7 receptors in macrophage is one of the main physiological signals that lead to the processing and release of the pro-inflammatory cytokine, interleukin-1beta (il-1b), their activation also leads to rapid opening of a membrane pore permeable to dyes such as ethidium. here we identify pannexin-1, a recently described mammalian protein that functions as an hemichannel when ectopically expressed, as this dye-uptake pathway and show that signalling through pannexin-1 is required for processing of caspase-1 and release of mature il-1b induced by p2x7 receptor activation. furthermore, maitotoxin and nigericin, two agents considered to evoke il-1b release via the same mechanism were studied. maitotoxin evoked dye uptake whose kinetics were similar to a slow pannexin-1-independent phase induced by p2x7 receptor activation, and this was unaltered by pannexin-1 inhibition.nigericin did not induce dye uptake.inhibition of pannexin-1 blocked caspase-1 and il-1b processing and release in response to this two stimuli.thus, while pannexin-1 is required for il-1b release in response to maitotoxin, nigericin and atp, a mechanism distinct from pannexin-1 hemichannel activation must underlie the former two processes. introduction: saa is a classic acute-phase protein upregulated during inflammatory response. saa is active on leukocytes and modulates inflammation and immunity through the induction of cytokines, including the chemokine il-8. here we verify the effect of saa on the mrna expression and release of mip-3alpha, a chemokyne involved in the recruitment of dendritic cells. methods: peripheral blood mononuclear cells (pbmc) isolated from peripheral blood by density gradient were cultured in rpmi medium in the presence of saa. mip-3alphaconcentration was determinated in the supernatant of cell cultures by elisa. mrnawas analyzed bythe ribonuclease protection assay (rpa). results: pbmc stimulated with saa (20 ug/ml) induced the expression of mip-3alpha mrna at 2, 4 and 12 hours. mip-3alpha protein was found in the suppernatant of 24 and 42 hours cultures (p<0,05) and the addition of sb 203580 (p38 inhibitor) and pd 98059 (erk 1/2 inhibitor) completely abolished the release of mip-3alpha. conclusions: saa is an inducer of mip-3alpha expression in pbmc and p38 and erk1/2 are important pathway signaling to this effect. saa is one of the factors responsible by the recruitment of dendritic cells. the p38 pathway is activated in numerous inflammatory conditions, including ra, ibd asthma, acute coronary syndrome, and copd, and its activation helps drive the production of inflammatory mediators. inhibitors of p38 decrease mediator production and therefore can produce profound anti-inflammatory effects. arry-797 is a potent inhibitor of p38 enzyme (ic50 < 5nm) with a novel pharmacophore and physiochemical properties distinct from those of other p38 inhibitors, being very water soluble. it is extremely potent in human whole blood, blocking lps-stimulated tnf production with an ic50 < 2nm.in animal models of rheumatoid arthritis (cia and aia) the compound significantly normalized histologic endpoints, such as inflammation, bone resorption and cartilage damage (ed50~3 mg/kg). a phase i single ascending dose clinical study was run in healthy volunteers. after an oral dose of 50, 100, 200, 300 or 400 mg), blood was drawn at various times, stimulated ex vivo with lps, and analyzed for cytokines and inflammatory mediatorsfj arry-797 was well-tolerated and drug exposure was proportional to dose. in ex vivo samples, there was both a time-and concentrationdependent inhibition of il1, pge2 and tnffz with >80% inhibition observed at the 50 mg dose level. the plasma concentrations of drug peaked at~20 ng/ml at the 50 mg dose and cytokine inhibition was sustained for >4 hours, showing that low doses of arry-797 produced profound effects on clinical biomarkers. further evaluation of arry-797 in patients with inflammatory diseases is planned. introduction: we demonstrated that in vivo chronicle blockage of nos (l-name, 20mg/kg; oral route; 14 days) impairs leukocyte-endothelial interactions and neutrophils migration into inflammatory focus. these effects may be depending, at least in part, on decreased expression of l-selectin on leukocytes and pecam-1 on endothelium. aimed to clarify the mechanisms involved on these inhibitory effects, we now investigated the role of l-name treatment on secretion of tnf and il-1b; by circulating leukocytes and migrated peritoneal neutrophils. methods: male wistar rats were treated with l-name (20mg/kg; oral route; 14 days) or sterile saline (control). circulating leukocytes were isolated from blood collected from abdominal aorta and migrated neutrophils were obtained 4 hours after i.p. injection of oyster glycogen (1%;10ml). no (griess reaction) and cytokines (elisa) were quantified in supernatants of 1 x 106 cultured cells before and 18 hours after lps stimulation (5m;g/ml). results: levels of no, tnf and il-1b; were reduced in circulating leukocytes from l-name-treated rats in both basal and lps stimulated conditions. on the other hand, only secretion of il-1b; was impaired by migrated neutrophils. conclusions: results show that in vivo l-name treatment, which partially reduces no production, decreases the secretion of pro-inflammatory cytokines by circulating leukocytes. however, the same pattern of inhibition is not detected if neutrophils are in vivo primed. objectives: to investigate the ability and mechanism of ifn-g to suppress interleukin-1 (il-1)-induced mmp-13 expression in articular chondrocytes. methods: human chondrocytes were treated with ifn-g or il-1beta alone or in combination. mmp-13 mrna was analyzed by rt-pcr. mmp-13 protein, phospho-stat1 and p44/42 mapk levels were measured by western blotting. mmp-13 promoter-luciferase, cmv-cbp/p300 plasmids and stat1 sirna were transfected by calcium phosphate method. ap-1 activity was monitored by elisa. stat1-cbp/p300 interaction was studied by immunoprecipitation. results: ifn-gpotently suppressed il-1-induced expression of mmp-13 and promoter activity. blockade with neutralizing ifn-gr1 antibody revealed that mmp-13 inhibition by ifn-â¼ was mediated by the ifn-â¼ receptor. ifn-beta-stimulated activation of stat1 was directly correlated with mmp-13 suppression. knockdown of stat1 gene by specific sirna or its inhibition with fludarabine partially restored the il-1 induction of mmp-13 expression and promoter activity. ifn-g did not alter activator protein (ap-1) binding ability but promoted physical interaction of stat1 and cbp/p300 co-activator. p300 overexpression reversed ifn-g inhibition of endogenous mmp-13 mrna expression and exogenous mmp-13 promoter activity. conclusions: ifn-g through its receptor activates stat1, which binds with cbp/p300 co-activator, sequesters it from the cell system and thus inhibits transcriptional induction of mmp-13 gene in chondrocytes. ifn-g and its signaling pathways could be targeted therapeutically for (1), p asmawidjaja (1), r hendriks(2), erik lubberts (1) (1) erasmus medical center, department of rheumatology, rotterdam, the netherlands (2) erasmus medical center, department of immunology, rotterdam, the netherlands the objective of this study was to identify the role of il-23 in th17 polarization in the prone autoimmune dba-1 mice with and without collagen-induced arthritis and to evaluate th17 specific cytokine and transcription factor expression. il-23 induced th17 cells in vitro from spleen cells of naã¯ve and collagen-type ii (cii) immunized dba-1 mice. the percentage of th17 cells is markedly higher in cii-immunized versus naã¯ve dba-1 mice. adding il-23 to tgf-beta/il-6 stimulated cd4 + t cells did not significantly increase the percentage of th17 cells. tgfbeta/il-6 in contrast to il-23 induced a relatively high percentage of il-17+/ifn-gamma-cells and low il-17-ifn-gamma+ cells. tgf-beta/il-6 did not increase il-23 receptor expression, which may explain why adding il-23 directly or two days after tgf-beta/il-6 did not result in an increase in the percentage of th17 cells. elevated expression of il-17a and il-17f as well as the th17 specific transcription factor rorgammat was found under il-23 as well as tgfbeta/il-6 conditions. interestingly, il-23 but not tgf-beta/il-6 is critical in the th17 cytokine il-22 expression in t cells from ciiimmunized dba-1 mice. these data show that il-23 was more pronounced in inducing il-17+/ifn-gamma-(th17) cells under cii-immunized conditions. furthermore, il-23 did not markedly increase the percentage of th17 cells induced by tgf-beta/il-6. however, il-23 is critical for the induction of il-22 expression, suggesting a unique role for il-23 in the induction of specific th17 cytokines ebi3 was initially discovered as a transcriptionally activated gene in epstein-barr virus-infected human b lymphocytes, and similar to p40 of il-12. ebi3 protein has been shown to form heterodimers with p28. p28/ebi3 termed il-27, can influence the function of multiple t cell subsets, including naive, effector, regulatory and memory t cells. however, previous studies showed that the overlapped expression of ebi3 and p28 is very limited. these data lead to the hypothesis that ebi3 may play a role independently from its association with p28. thus, to define the function of ebi3, we generated ebi3 transgenic (tg) mice expressing in multiple tissues. ebi3 tg mice exhibited no histologic abnormalities in various organs and normal numbers of naive and memory cd4+, cd8+ t cells, b cells, nk cells and nkt cells. cd4+t cells isolated from spleens of ebi3 tg mice, however, produced less ifn-g than cells from wt (wild type) control mice after in vitro stimulation with anti-cd3 and anti-cd28 antibodies. in vivo studies, delayed-type hypersensitivity (dth) and contact hypersensitivity (chs) responses were significantly reduced in ebi tg compared with wt mice. moreover, the chs responses in ebi3 tg mice were recovered with anti-ebi3 polyclonal antibody. notably, chs reaction in wt mice was increased by anti-ebi3 antibody. in contrast, anti-p28 antibody suppressed chs responses in wt mice. these data suggest that ebi3 acts in different from il-27, and reduces th1 responses. (1), o thaunat(2), x houard (1), o meilhac (1), g caligiuri(2), a nicoletti (2) (1) inserm u698 and university denis diderot-paris 7, chu xavier bichat, paris, france (2) inserm umr s 681, universitã� pierre et marie curie-paris 6, centre de recherche des cordeliers, paris, france arteries are composed of three concentric tissue layers which exhibit different structures and properties. because arterial injury is generally initiated at the interface with circulating blood, most studies performed to unravel the mechanisms involved in injury-induced arterial responses have been focused on the innermost layer (intima). in contrast, the role of the outermost tunica, the adventitia, has attracted relatively little attention and remains elusive. in the present review, we focus on involvement of the adventitia in the response to various types of arterial injury leading to vascular remodeling. several lines of evidence show that the initial insult and the early intimal response lead to the genesis of (neo-) mediators that are centrifugally conveyed by mass transport towards the adventitia. these mediators trigger local adventitial responses including angiogenesis, immuno-inflammation, and fibrosis. we propose that these three processes sequentially interact and that their net balance participates in producing each specific pathological condition. hence, an adventitial adaptive immune response predominates in chronic rejection. inflammatory phagocytic cell recruitment and initiation of a shift from innate to adaptive immunity characterize the adventitial response to proteolysis products in abdominal aortic aneurysm. centripetal adventitial sprouting of neovessels, leading to intraplaque hemorrhages, predominates in atherothrombosis. adventitial fibrosis mediated by low inflammation characterizes the response to mechanical stress and is responsible for constrictive remodeling of arterial segments and initiating interstitial fibrosis in perivascular tissues. these adventitial events thus impact not only on the vessel wall biology but also on the surrounding tissue. atherosclerosis has many of the characteristics of an inflammatory disease, and thus would classically involve endothelial cox-derived prostaglandins such as pge2 and prostacyclin acting on ep and ip receptors, respectively.activation of vascular ip receptors is especially important in limiting the atherogenic properties of thromboxane a2 acting on tp receptors.more recently, expression of ghrelin receptors has been shown to be increased in atherosclerotic plaques, and ghrelin itself has anti-inflammatory properties in addition to its classical role as a hunger hormone.as well as the complex crosstalk between g-protein-coupled receptors (gpcrs), recent evidence indicates that many gpcrs exist constitutively as homodimeric complexes, and that the formation of heterodimers not only influences the classical cell signalling pathways used by these receptors, but also affects their subcellular distribution.we have found that ep3-i, tp and ghrelin receptors readily form homodimers, but that co-transfection of hek293 cells with these receptors results in the formation of heterodimers with unpredictable effects on receptor distribution and cell signalling properties.since inflammatory conditions are thought to change the relative expression levels of gpcrs in the vasculature, and since varying the expression levels of gpcrs will affect their ability to form heterodimers, then one might predict that gpcr heterodimerization would indeed influence the reactivity of vascular tissue during inflammation. [this work was fully supported by grants from the research grants council of the hong kong special administrative region (cuhk4267/02m and 2041151 vascular inflammation leads to formation of leukotrienes through the 5-lipoxygenase pathway of arachidonic acid metabolism. leukotriene forming enzymes are expressed within atherosclerotic lesions and locally produced leukotrienes exert pro-inflammatory actions within the vascular wall by means of cell surface receptors of the blt and cyslt receptor subtypes. recent mechanistic studies have supported the notion of a major role of leukotriene signaling in atherosclerosis. leukotriene b4 (ltb4) is for example one of the most potent chemotactic mediators formed within the atherosclerotic lesion, inducing migration of a number different cell-types of both hematopoietic and non-hematopoietic origin. initially identified on neutrophils, blt receptor activation is involved in monocyte chemotaxis and adhesion as well as in vascular smooth muscle cell migration and proliferation, providing examples of potential mechanisms in ltb4-induced atherogenesis. targeting ltb4-induced activation of vascular smooth muscle cells has beneficial effects in models of intimal hyperplasia and restenosis after vascular injury. furthermore, blt receptor expression has been demonstrated on t-cells, suggesting ltb4 as a potential link between innate and adaptive immunological reactions. taken together, the local formation of leukotrienes within the atherosclerotic lesion and the potent pro-inflammatory effects of leukotriene receptor activation in target cells of atherosclerosis provide a rationale for a role leukotrienes in this disease. further experimental and clinical studies are however needed to develop therapeutic strategies of treatments targeting leukotriene signaling in atherosclerosis. in normal physiological conditions, the prostanoid (prostaglandin (pg) and thromboxane (tx)) synthesis is dependent on the constitutive isoform of cyclooxygenase (cox-1). this synthesis and release happen few minutes after cell or tissue stimulation. in vascular preparations submitted to pro-inflammatory conditions for some hours, the inducible isoform of cyclooxygenase (cox-2) and other prostanoid synthases can be observed. as an illustration of the previous experimental results, there is an increased presence of cox-2 and the inducible enzyme responsible for pge2 synthesis (mpges-1) detected by immunocytochemistry in the carotid atherosclerotic plaque with strong inflammation. in vascular cells in culture, pgi2 is the major biological active prostanoid produced in the normal physiological conditions. however, when cox-2 is induced, pgi2 and pge2 are equally produced. the role of cox-1, cox-2 and mpges-1 activities is also dependent on the expression of the various prostanoid receptors in the considered vessel. there is increasing evidence for the presence and a role of the ep receptor subtypes (ep1, ep2, ep3 or ep4) preferentially stimulated by pge2 in the vascular wall. for these reasons, we have characterized the receptors activated by pge2 in human mammary arteries. in these vessels incubated with a pro-inflammatory cytokine (interleukin-1ã¢) and lipopolysaccharides a reduced contractility to norepinephrine has been observed. this effect is abolished by treatment of the vascular preparations with a selective cox-2 inhibitor, suggesting that prostanoid synthesis and/or prostanoid receptors could be involved. rheumatoid arthritis is a syndrome which probably consists of a number of diseases for which the risk factors differ. two major processes were identified: the generation of the anti-citrullinated antigens immune response (highly sepcific for ra).we show that the different hla class ii alleles contribute to the development of anti-ccp-positive and anti-ccp negative ra.the se alleles do not independently contribute to the progression to ra, but rather contributed to the development of anti-ccp antibodies. next we determined the effect of smoking and observed that smoking only conferred risk to contract ra in the ccp-positive group and not in the anti-ccp negative group. for the risk factor ptpn22 (a gene that regulates treshold of lumphocyte activation) the allele c1858t only contributed to ccp-positive ra. in contrast to hla two other risk factors were found to be associated with both ccp-positive and ccp-negative ra. the risk factor in the fcrl-gene as has been identified in the japanese population was also tested in 931 dutch ra cases and 570 unrelated dutch controls. carrier analysis of the snp (rs7528684) revealed association of cc genotype with higher risk of developing ra as compared to tt & tc carriers (p =0.039 and or =1.31). in a meta-analysis of all studies comparing 9467 individuals, the or for the cc genotype to develop ra was 1.2 and the p-value < 0.001. in conclusion, different steps in pathogenesis of the syndrome ra can be delineated this talk will focus on recent advances in understanding primary genetic factors predisposing to inflammatory bowel disease (crohns disease and ulcerative colitis). proven genes containing genetic variants predisposing to crohns disease include ibd5/5q31, card15/nod2 and il23r. data is suggestive but not yet as convincing for many other genes. a common theme is of genetic variants influencing early innate immune responses to intestinal bacterial components, and subsequent adaptive immune responses, leading to intestinal inflammation. only for card15/nod2 is there (partial) understanding of how genetic variation influences biological function to cause chronic disease. some mouse models (gene targeted) of card15 appear to show opposite effects to other models and human systems. in humans, card15 mutations impair responses to bacterial components (muramyl dipeptide) mainly at low dose sensing. it is likely this receptor system normally maintains intestinal crypt sterility and protection from invasive infection. pathogen-recognition receptors (prrs) are key components of immune systems and are involved in innate effector mechanisms and activation of adaptive immunity. since their discovery in vertebrates, toll-like receptors (tlrs) have become the focus of extensive research that has revealed their significance in the regulation of many facets of our immune system. recently a new family of intracellular prrs, the nod-like receptors (nlrs), which include both nods and nalps have been described. mutations within the nalp3/cryopyrin/ cias1 gene are responsible for three autoinflammatory disorders: muckle-wells syndrome, familial cold autoinflammatory syndrome, and cinca/nomid. the nalp3 protein associates with asc and caspase-1 (thereby forming a molecular machine termed inflammasome that displays high proil-1beta-processing activity. macrophages from muckle-wells patients spontaneously secrete active il-1beta. increased inflammasome activity is therefore likely to be the molecular basis of the symptoms associated with nalp3-dependent autoinflammatory disorders. here we will emphasis on the ability of this protein complex to promote the development of autoinflammatory syndromes. allergic inflammation (ai) is a complex phenomenon initiated by allergen binding to ige sensitized mast cells and consequent mast cell activation. this causes the symptoms of the early phase of ai and the onset of the late phase characterized by the penetration in the inflamed tissue of inflammatory cells, notably the eosinophils. their subsequent activation is believed to cause tissue damage and to be the main responsible for the tissue remodeling, especially when the ai becomes a chronic process. we defined a novel functional unit that we termed the allergic synapse formed by mast celleosinophil couples. in the synapse these two old cellular players of ai have a cross talk via soluble mediators and receptor-ligand interactions. this results in mast celleosinophil functional synergism that consequently amplifies and prolongs the inflammatory response. in addition, mast cells and eosinophils are influenced and influence as in a sort of allergic niche the surrounding structural cells, i.e. fibroblasts and endothelial cells. we propose to view the allergic synapse/niche as a specialized effector unit worthy to be blocked for the treatment/prevention of allergic inflammation. (1) (1) erasmus mc, rotterdam, the netherlands (2) department of immunology weizmann institute of science, rehovot, israel allergic asthma is one of the most common chronic diseases in western society, characterized by reversible airway obstruction, mucus hypersecretion and infiltration of the airway wall with th2 cells, eosinophils, and mast cells. if we are to devise new therapies for this disease, it is important to elucidate how th2 cells are activated and respond to intrinsically harmless allergens. dendritic cells (dcs) are the most important antigen presenting cells in the lung and are mainly recognized for their exceptional potential to generate a primary immune response and sensitization to aeroallergens. we have shown that intratracheal injection of ovalbumin (ova) pulsed dcs induces sensitization leading to eosinophilic airway inflammation upon ova aerosol challenge. we investigated the role of dcs in the secondary immune response in a murine asthma model. ova aerosol challenge in ova-dc sensitized mice, induced an almost 100 fold increase in the number of airway dcs as well as an increase in eosinophils and t cells. to investigate the functional importance of dcs for the induction and maintenance of airway inflammation in response to allergen challenge, we conditionally knocked-out endogenous dcs in sensitised cd11c-diphtheria toxin (dt) receptor (cd11cdtr) transgenic mice by airway administration of dt 24 h before ova aerosol (4x) challenge or during an ongoing inflammation (depletion after 3x ova aerosols continued with 3 additional ova aerosols). numbers of balf eosinophils, th2 cytokine production by mediastinal lymph nodes and peribronchial and perivascular inflammatory infiltrates were dramatically decreased, illustrating an essential role for airway dcs during secondary challenge. karolinska institute, stockholm, sweden nk cells are innate lymphocytes with potent immunoregulatory functions. they are potent producers of several cytokines and chemokines, and also respond to similar molecules in the body and at inflammatory sites. even though traditionally best characterized for their role in anti-viral and anti-tumor immunity, they influence several other types of immune responses. for example, they are involved in, and affect, acute as well as chronic inflammatory responses. in the present talk, a general overview on our current knowledge of nk cell biology will be provided, with a special emphasis on the role of these cells in allergic inflammation. basophils are major effector cells in allergic reactions due to their ability to release substantial quantities of histamine and eicosanoids following activation of high affinity ige receptors (fc<epsilon>ri) with allergens. although these attributes are shared with their tissuefixed mast cell compatriots, basophils are unique in their ability to also rapidly elaborate th2-type cytokines (e.g. il-4 and il-13), subsequently supporting ige synthesis and underlying atopy. importantly, these mediators are additionally secreted following primary exposure to certain parasites (e.g. s. mansoni) and immunoglobulin superantigens, suggesting a role for basophils in innate immunity and in assisting developing th2-type adaptive immune responses. while we are beginning to understand the potential physiological functions of these cells regarding host defence, blocking their activity with respect to treating symptoms of allergic disease has remained an enigma. recent advances, however, have shed light upon the major intracellular signal transduction processes involved in fc<epsilon>ri activation and may lead to novel therapeutic strategies for inhibiting mediator secretions. an important discovery in this regard is the phosphatase ship, which downregulates pi 3-kinase signalling in both basophils and mast cells. recent data shows that ship expressions in basophils are reduced from donors with active allergic disease but that these levels may be increased, and the activity of basophils subsequently inhibited, by targeting receptors associated with ship recruitment (cd200r, cd300r). identifying the natural ligands for these inhibitory receptors may therefore pave the way for new therapies for the treatment of allergic inflammation. mitogenesis and proliferation of vsmc play an important role in atherogenesis. pro-inflammatory secretory phospholipases a2 (spla 2 ) hydrolyse glycerophospholipids of hdl and ldl and release pro-inflammatory agents, lyso-lipids, oxidized and non-oxidized fatty acids and isoprostanes.spla 2 s lipolysis products localize in vascular wall in vicinity of vsmc.we have tested the impact of spla 2 , hdl and ldl and of their hydrolysis products on mitogenesis and pge2 and ltb4 release from vsmc.mitogenesis was significantly enhanced by native hdl, and ldl, and by group v spla 2 . spla 2 hydrolysis of hdl and ldl enhanced mitogenic activity in order v>x>iia.the release of pge2 from vsmc was enhanced by group x spla 2 s but not iia or v.the greatest effect was seen for hdl hydrolysed by group v and x spla 2 .native ldl and its spla2 hydrolysis products enhanced the release of pge2 in order x>v>iia.the release of ltb4 from vsmc was markedly increased by native ldl and hdl, and hydrolysis products of group v and x, but not iia spla2.migration of vsmc was significantly enhanced by spla 2 iia and inhibited by hdl.this study demonstrates a complex interaction of hdl and ldl with pro-inflammatory spla2s, which affects mitogenesis, eicosanoid release and migration of vsmc.study of biocompatible spla2 blockers in the therapy of atherosclerosis is indicated. contact information: professor waldemar pruzanski, university of toronto, department of medicine, toronto, ontario, canada e-mail: drwpruzanski@bellnet.ca (1) (1) ipmc-cnrs umr6097, valbonne, france (2) university of washington, seattle, usa (3) inserm umrs 525, paris, france (4) university of naples, italy the superfamily of phospholipase a2 comprises at least 9 intracellular enzymes and up to 12 secreted pla2s (spla 2 s). elucidating the biological roles of each pla2 member is currently the most challenging issue in the pla2 field. the different spla 2 s are not isoforms and are likely to function either as enzymes producing key lipid mediators (eicosanoids and lysophospholipids) or as ligands that bind to specific soluble or membrane-bound proteins (like cytokines). increasing evidence suggests that spla 2 s iia, iii, v, and x are involved in inflammatory diseases including atherosclerosis. among spla2s, the human group x (hgx) enzyme has the highest enzymatic activity towards phosphatidylcholine, the major phospholipid of cellular membranes and low density lipoproteins (ldl). on human alveolar macrophages, hgx spla 2 can trigger secretion of tnf alpha, il1 and il6 in a non-enzymatic manner. on colorectal cancer cells, hgx spla 2 stimulates cell proliferation, produces potent eicosanoids including pge2, and activates the transcription of key genes involved in inflammation and cancer. the enzyme can also hydrolyze pc and platelet-activating factor (paf) of ldl particles very efficiently. finally, hgx spla 2 is present in human atherosclerotic lesions and converts ldl into a proinflammatory particle that induces macrophage foam cell formation, as well as map kinase activation, arachidonic acid release, and expression of adhesion molecules in huvec cells. some other key molecular features of spla 2 s including hgx will be presented. we have reported preferential release of polyunsaturated fatty acids during hydrolysis of lipoprotein phosphatidylcholine (ptdcho) by spla 2 s, but the mechanism of this selectivity is not known. since both sphingomyelin (sm) and lysoptdcho inhibit the activity while increasing fatty acid specificity of other pla2s, we have examined fatty acid release by spla2siia, v and x in relation to relative increases in proportion of endogenous sm and lysoptdcho during lipoprotein digestion. the analyses were performed by normal phase liquid chromatography with on-line electrospray mass spectrometry (lc/esi-ms) and lc/collision induced dissociation (cid)/esi-ms using conventional preparations ofldl and hdl. the highest preference for arachidonate release from ldl by group x spla 2 was observed when the residual sm/ pdcho molar ratio had reached 1.2 compared to a starting ratio of 0.4.group v spla 2 showed preferential release of linoleate at residual sm/ptdcho molar ratio 1.5, while at intermediate ratios, both arachidonate and linoleate were released at more comparable ratios. the relative increases in lysoptdcho and sm during the digestion with spla 2 iia were much more limited, and a preferential hydrolysis of polyunsaturated fatty acids was not observed. these results suggest a lipid phase separation as a likely basis for a differential hydrolysis of molecular species of ptdcho. the residual sm/ptdcho ratios reached during group v and x spla 2 digestion are similar to those observed for lesional ldl, which promote release of ceramides by smase leading to ldl aggregation. the above findings support a potential role of sphingomyelins in atherogenesis. although sphingomyelin (sm) is one of the most abundant phospholipids in lipoproteins and cell membranes, its physiological significance is unclear. because of its localization in the outer surface of the cells, and its structural similarity to phosphatidylcholine (pc),we proposed that it competitively inhibits phospholipolysis of cell membranes by external phospholipases (pla). we showed that sm inhibits several lipolytic enzymes including secretory pla2 iia, v, and x, and hepatic and endothelial lipases, all of which hydrolyze pc. treatment of sm in the substrate with smase c not only relieved the inhibition but also activated the pla2 reaction further, suggesting that ceramide, the product of smase c, independently stimulates pla2, possibly by disrupting the bilayer structure. smase d treatment, which produces ceramide phosphate, did not stimulate the spla2. the fatty acid specificity of pla2 is significantly affected by sm. thus spla2x exhibited enhanced specificity for the release of arachidonic acid (20:4) in presence of sm, due to a preferential inhibition of hydrolysis of other pc species. in contrast, sm inhibited the release of 20:4 by spla2v. ceramide selectively stimulated the release of 20:4 by both enzymes. only the long chain ceramides (> 10 carbons) were effective, while ceramide phosphate did not stimulate spla2 activity. sm-deficient cells released more 20:4 in response to spla2-treatment than normal cells, and pretreatment of normal cells with smase c increased their susceptibility to spla2 attack. these studies show that sm and ceramide regulate the activity and specificity of pla, and consequently the inflammatory response. secretory phospholipase a2 (spla2) types iia, v, or x, have been associated with inflammatory diseases and tissue injury including atherosclerosis in humans and mice.given the link between spla2 and atherogenesis, a mouse model of atherosclerosis (apoe-/-) was used to study the effects of a-002, an inhibitor of spla2 enzymes, on atherosclerosis and cholesterol levels over 16 weeks of treatment. mice were fed with a high-fat, high cholesterol diet alone during the study (21% fat; 0.15% cholesterol, 19.5% casein) and were treated with vehicle or a-002 bid at 30 mg/kg or 90 mg/kg by oral gavage. total cholesterol was significantly decreased after one month of treatment and remained lower throughout the study.treatment with a-002 significantly reduced aortic atherosclerotic plaque formation in apoe-/-mice fed a high fat diet when compared to the untreated control by approximately 40 %. in a different model that used angiotensin ii in conjunction with a high fat diet in a background of apoe-/-deficient mice for 4 weeks, oral dosing of a-002 (30 mg/kg bid) significantly reduced aortic atherosclerosis and aneurysm rate when compared to vehicle. these data suggest that a-002 is a potential novel therapeutic agent for the treatment of atherosclerosis. (1), s doty(2), c antonescu (3), c staniloae (1) (1) saint vincents hospital manhattan, new york, usa (2) hospital for special surgery, new york, usa (3) sloan-kettering institute for cancer research, new york, usa tnf-stimulated gene 6 (tsg-6) is induced by tnf-a during inflammation and its secreted product tsg-6 glycoprotein is involved in immune-mediated inflammatory diseases and fertility. it regulates cox-2 and prostaglandin synthesis, and participates in extracellular matrix remodeling. considering the chronic inflammatory nature of atherosclerosis we hypothesized that tsg-6 is expressed in atherosclerotic plaques and investigated tsg-6 protein expression and cellular distribution on 12 superficial femoral artery endarterectomy specimens from 6 diabetic and 6 non-diabetic patients with peripheral vascular disease. six histologically normal radial artery specimens were analyzed as control. paraffin embedded samples were studied by immunohistochemistry using a goat polyclonal anti-human-tsg-6 antibody. tsg-6 expression was consistently present in all atherectomy specimens but not in control specimens. a distinct, strong cytoplasmic staining pattern was uniformly detected in the endothelial lining of the intima, as well as in the neo-vessel proliferation of the plaque. cytoplasmic staining was also identified in the smooth muscle cell proliferation of the neo-intima. patchy tsg-6 expression was noted in the extracellular matrix. within the inflammatory plaques from diabetic patients, tsg-6 stained the foamy macrophages. tsg-6 expression was also confirmed and quantified by qrt-pcr that showed a significant up-regulation of tsg-6 gene (more that 100 fold induction compared to housekeeping genes). our study identifies for the first time the preferential expression of tsg-6 in atherosclerotic lesions and characterizes its distribution within the cellular and matrix components of the plaque. tsg-6 is a novel inflammatory mediator of atherosclerosis and a potentially new marker of endothelial / smooth muscle cell activation. (1), r krohn (1), h lue (1), jl gregory(2), a zernecke (1), rr koenen (1), t kooistra (3), p ghezzi(4), r kleemann (3), r bucala(5), mj hickey (2), c weber (1) (1) university hospital of the rwth aachen, germany (2) centre for inflammatory diseases, monash university, melbourne, australia mediators, which cannot be classified into chemokine subfamilies but share functional patterns, e.g. signaling through chemokine receptors, constitute a group termed chemokine-like function (clf)-chemokines. the pleiotropic cytokine macrophage migration inhibitory factor (mif) plays a critical role in inflammatory diseases and atherogenesis. the underlying molecular mechanisms are poorly understood, but, interestingly, mif displays structural features resembling chemokines. we have identified the chemokine receptor cxcr2 as a functional receptor for mif. mif triggered galphai/integrin-dependent arrest and chemotaxis of monocytes specifically through cxcr2, inducing rapid integrin activation. mif directly bound to cxcr2 with high affinity (kd of 1.4 nm). monocyte arrest mediated by mif in inflamed or atherosclerotic arteries involved cxcr2 as well as cd74, a recently identified membrane receptor moiety for mif. accordingly, cxcr2 and cd74 were found to occur in a receptor complex. in vivo, mif deficiency impaired monocyte adhesion to the aortic/arterial wall in atherosclerosis-prone mice, as evidenced by intravital microscopy. thus, mif displays chemokine-like functions by acting as a non-cognate ligand of cxcr2, serving as a regulator of inflammatory and atherogenic recruitment. these data harbor an intriguing novel therapeutic prospect by targeting mif in atherosclerosis and add a new dimension to mif and chemokine receptor biology. (1), r toes (3), h van bockel(2), paul quax (1,2) (1) tno bioscienses, leiden, the netherlands (2) department of vascular surgery, leiden university medical center, the netherlands (3) department of rheumatoly, leiden university medical center, the netherlands the immune system is thought to play a crucial role in regulating collateral circulation (arteriogenesis), a vital compensatory mechanism in patients with arterial obstructive disease. here, we studied the role of lymphocytes in a murine model for artiogenenesis after acutehind limb ischemia. arteriogenesis was impaired in c57bl/6 mice depleted for natural killer (nk)-cells by anti-nk1.1 antibodies and in nk-cell-deficient transgenic mice. arteriogenesis was, however, unaffected in jâµ281knockout mice that lack nk1.1+ natural killer t (nkt)cells, indicating that nk-cells, rather than nkt-cells are involved in arteriogenesis. furthermore, arteriogenesis was impaired in c57bl/6 mice depleted for cd4+ tlymphocytes by anti-cd4 antibodies, and in major histocompatibility complex (mhc)-class-ii-deficient mice that lack mature peripheral cd4+ t-lymphocytes. this impairment was even more profound in anti-nk1.1treated mhc-class-ii-deficient mice that lack both nkand cd4+ t-lymphocytes. finally, collateral growth was severely reduced in balb/c as compared with c57bl/6 mice, two strains with different bias in immune responsiveness. correspondingly, fewer cd3-positive lymphocytes accumulated around collaterals in balb/c mice. these data show that both nk-cells and cd4+ t-cells play an important role in arteriogenesis. moreover, our data hold promise for the development of novel clinical interventions as promoting lymphocyte activation might represent a powerful method to treat ischemic disease. post-interventional vascular remodeling in venous bypass grafts, seen as intimal hyperplasia (ih) and accelerated atherosclerosis, often causing graft failure. inflammation is an important trigger for these processe. complement is an important part of the immune system and participates in regulating inflammation. although involved in several other inflammatory diseases, the role of the complement cascade in vein graft remodeling is unknown. the involvement of the complement system in vein graft disease was studied here using a model in which caval veins are grafted in carotids arteries of hypercholesterolemic apoe3leiden mice. in these veins ih and accelerated atherosclerotic lesions develop within 28 days, consisting mainly of foamcells and smc. to study the functional role of complement in vein graft remodeling, cobra venom factor (cvf:20u daily) was used to deplete complement starting one day prior to vein graft surgery. cvf-treatment reduced vein graft thickening by 64% (p=0.02), when compared to saline treated controls (n=6).to confirm that the reduction by cvf was due to hampered complement function and not a direct effect of cvf, complement activation was blocked using crry-ig (inhibiting c3 convertases). crry-ig (3mg every other day) led to 50% decrease in vein graft thickening (p=0.03) compared to controls receiving non-relevant control igg. these data prove that complement activation plays a major in intimal hyperplasia and accelerated atherosclerosis in vein grafts. (1), m-c koutsing tine(1), p borgeat(2), h ong (1), sylvie marleau (1) (1) universite de montreal, quebec, canada (2) centre de recherche en rhumatologie et immunologie, canada we have previously shown that ep 80317, a growth hormone-releasing peptide (ghrp) analogue binding selectively to the scavenger receptor cd36, elicits a striking reduction in atherosclerosis development in apolipoprotein-deficient (apoe-/-), a condition associated with increased circulating numbers of primed/activated leukocytes. we investigated the effect of ghrp analogues on i/r-elicited remote lung injury in 18week-old apoe-/-mice fed a high fat high cholesterol (hfhc) diet from 4 weeks of age. at 18 weeks old, mice were treated daily with a s.c. injection of ep 80317 (300 mg/kg) for 14 days and were then subjected to unilateral hindlimb ischemia (by rubber band application) for 30 minutes, followed by 180 minutes reperfusion. our results show that ep 80317 significantly reduced leukocyte accumulation by 56% in the lungs, from 3.6 (ae 0.7) in vehicle-treated mice to 1.6 (ae 0.6) x 107 leukocytes/g lung in ep 80317-treated mice (n = 6-7 per group), as assessed by myeloperoxidase assay. this was associated with a 56% reduction of opsonized zymosan-elicited blood chemiluminescence. in contrast, neither blood chemiluminescence, nor leukocyte accumulation in the lungs were signicantly modulated in apoe-/-/cd36-/-deficient mice, from 1.9 (ae 0.5) in vehicle-treated mice to 1.9 (ae 0.8) x 107 leukocytes/g lung in ep 80317-treated mice. we conclude that ep 80317 protects i/r-elicited circulating leukocyte priming/activation and remote lung injury, possibly through a cd36-mediated pathway. glycogen synthase kinase 3beta (gsk-3beta) is a serine/ threonine protein kinase that has recently emerged as a key regulatory switch in the modulation of the inflammatory response. dysregulation of gsk-3beta has been implicated in the pathogenesis of several diseases including sepsis. here we investigate the effects of two chemically distinct inhibitors of gsk-3beta, tdzd-8 and sb216763, on the circulatory failure and the organ injury and dysfunction associated with hemorrhagic shock. male wistar rats were subjected to hemorrhage (sufficient to lower mean arterial blood pressure to 35 mmhg for 90 min) and subsequently resuscitated with shed blood for 4 h. hemorrhage and resuscitation resulted in an increase in serum levels of (a) creatinine and, hence, renal dysfunction, and (b) alanine aminotransferase and aspartate aminotransferase and, hence, hepatic injury. treatment of rats with either tdzd-8 (1 mg/kg, i.v.) or sb216763 (0.6 mg/kg, i.v.) 5 min before resuscitation abolished the renal dysfunction and liver injury caused by hemorrhagic shock. the protection afforded by these compounds was confirmed by histological observations of lung, kidney and liver samples. in addition, tdzd-8, but not sb216763, attenuated the increase caused by hemorrhage and resuscitation in plasma levels of the proinflammatory cytokine interleukin 6. neither of the gsk-3beta inhibitors however affected the delayed fall in blood pressure caused by hemorrhagic shock. thus, we propose that inhibition of gsk-3beta may represent a novel therapeutic approach in the therapy of hemorrhagic shock. (1), y ito (1), h yoshimura (1), h inoue (1), n kurouzu (1), h hara (1), y mastui (1), h kitasato (1), s narumiya(2), c yokoyama (3), m majima (1) (1) kitasato university school of medicine, japan (2) kyoto university school of medicine, japan (3) tokyo medical and dental university, japan thromboxane (tx) a2 is a potent stimulator of platelet activation and aggregation and vascular constriction. we have reported cytokine-mediated release of sdf-1 from platelets and the recruitment of nonendothelial cxcr4+ vegfr1+ hematopoietic progenitors constitute the major determinant of revascularization. we hypothesized txa2 induces angiogenic response by stimulating sdf-1 and vegf which derived from platelet aggregation. to evaluate this hypothesis, we dissected the role of the txa2 in angiogenesis response using mouse hind limb ischemia. recovery from acute hind limb ischemia, as assessed in wild type mice (c57bl/6 wt) , prostaglandin i2 receptor (ip) knock out mice (ipko) and thromboxane (tx) a2 receptor (tp) knock out mice (tpko) by using lase doppler. blood recovery in tpko significantly delayed compared to wt and ipko. immunohistochemical studies revealed that the number of cd31positive cells in the ischemic quadriceps were less stained in tpko compared to wt and ipko.plasma sdf-1 and vegf concentration were significantly reduced in tpko mice. we observed during in vivo fluorescence microscopic study that compared to tpko, ipko and wt significantly increased platelet attachment to the microvessels around ligated area. tpko translpanted wt bone marrow cells increased blood recovery compared to tpko transplanted tpko bone marrow cells. in addition, mice injected with txa2 synthase c-dna expressing fibroblast increased blood flow recovery compared to control mice. these results suggested that tp signaling rescues ischemic condition by inducing angiogenesis by secreting sdf-1 and vegf from platelet aggregation. administration of selective tp agonist may open new therapeutic strategy in regenerative cardiovascular medicine. during renal ischemia/reperfusion (i/r) injury, apoptosis has been reported as a very important contributor to the final kidney damage. the determinant role of cytoskeleton derangement in the development of apoptosis has been previously reported, but a clear description of the different mechanisms involved in this process has not been yet provided. the aim of the study is to know the role of peroxynitrite as inductor of cytoskeleton derangement and apoptosis during the inflammatory process associated to renal ischemia-reperfusion. based in a rat kidney i/r model, by experiments in which both the actin cytoskeleton and peroxynitrite generation were pharmacologically manipulated, results indicate that the peroxynitrite produced during the i/r derived oxidative stress state, is able to provoke cytoskeleton derangement and apoptosis development. thus, the control in the peroxynitrite generation during the i/r could be an effective tool for the improvement of cytoskeleton damage and reduction apoptosis incidence in the renal i/r injury. metabolomics, the global profiling of metabolites, may inform about the multiple interacting processes involved in inflammatory disease. using nmr spectroscopy we analysed metabolite fingerprints in serum from early arthritis, and at a site of inflammation, in the posterior segment of the eye. serum from 101 patients with synovitis of "t 3 months duration whose outcome was determined at clinical follow-up was used. vitreous samples were from 31 patients undergoing vitrectomy for vitreoretinal disorders. one dimensional 1h nmr spectra were acquired. principal components analysis (pca) of the processed data was conducted along with a supervised classification. with the arthritis serum there was a clear relationship between each samples score in the pca analysis and the level of crp. supervised classification of the initial samples was able to predict outcome, whether rheumatoid arthritis, other chronic arthritis or self-limiting arthritis, with high specificity and sensitivity. a similar approach using the eye fluids was able to give a clear discrimination between two pathologically similar conditions lens-induced and chronic uveitis. in this case differences were not due to a straightforward relationship with inflammatory markers (il-6, ccl2), which did not correlate with pca in these samples. similarly, certain molecules, such as lactate, were associated with ocular disease, but not rheumatoid arthritis. these results suggest that underlying inflammatory processes may differ in these conditions or may reflect predisposing metabolic patterns in individual patients. 1h-nmr-based metabolomics may provide a useful measure of outcome in inflammatory diseases and give novel insights into the pathological processes involved. (1), am artoli(2), a sequeira(2), c saldanha (1) (1) instituto de medicina molecular,faculdade de medicina de lisboa, portugal (2) cemat, instituto superior tã�cnico, universidade de tã�cnica de lisboa, portugal the recruitment of leukocytes from the blood stream and their subsequent adhesion to endothelial walls are essential stages to the immune response system during inflammation. the precise dynamic mechanisms by which molecular mediators facilitate leukocyte arrest are still unknown. in this study combined experimental results and computer simulations are used to investigate localized hydrodynamics of individual and collective behaviour of clusters of leukocytes. leukocyte-endothelial cell interactions in post-capillary venules of wistar rats cremaster muscle were monitorized by intravital microscopy. from these experiments the haemorheologic and haemodynamical measured parameters were used in time dependent three-dimensional computer simulations, using a mesoscopic lattice boltzmann solver for shear thinning fluids. the dynamics of leukocyte clusters under non-newtonian blood flow with shear thinning viscosity was computed and discussed. in this paper we present quantified distributions of velocity and shear stress on the surface of leukocytes and near vessel wall attachment points. we have also observed one region of maximum shear stress and two regions of minimum shear stress on the surface of leukocytes close to the endothelial wall. we verified that the collective hydrodynamic behaviour of the cluster of recruited leukocytes establishes a strong motive for additional leukocyte recruitment. it was found that the lattice boltzmann solver used here is fully adaptive to the measured experimental parameters. this study suggests that the influence of the leukocytes rolling on the increase of the endothelial wall shear stress may support the activation of more signalling mediators during inflammation. macrophages are essential for host defence, but when excessively and persistently activated, these cells contribute to the initiation and progression of inflammatory diseases such as rheumatoid arthritis. investigating the function of inflammatory genes in macrophages may identify novel therapeutic targets for inflammatory diseases. one family of transcripts that are highly expressed in activated macrophages are members of the schlafen (slfn) gene family; a recently identified family whose function is still unknown. this study examined the mrna expression of slfn in activated bone marrowderived macrophages in vitro, and in collagen-induced arthritis (cia) in vivo. real-time pcr expression analyses of bone marrow-derived macrophages stimulated with lipopolysaccharide (lps) over a time course, revealed differential expression of individual slfn family genes. in particular, slfn-1, slfn-2, and slfn-4 were maximally induced after 24 hours. the maximal induction of slfn-8 and slfn-9 was observed after 4 hours of lps treatment. individual members of the slfn family were also differentially expressed in cia, a model of rheumatoid arthritis. mrna levels of slfn-1, slfn-2, slfn-4 and slfn-5 were elevated in joints affected by cia. to investigate the role of slfn-4, we have generated a transgenic mouse line, which over expresses slfn-4 specifically in cells of the mononuclear phagocyte system, by using a novel binary expression based on the c-fms promoter and gal4. further characterisation of the slfn-4 over expressing mouse line will be used to assess the function of slfn-4 in macrophage biology and inflammation, and its potential as a therapeutic target. macrophages play an important role in resolving inflammation. it is known that the resolution of inflammation requires alternative activation of macrophages. but the precise events of phenotype switching in macrophages remain poorly understood. we show that lipocalin 2, lcn-2, is able to provoke a switch in macrophage activation. in an in vitro co-culture model for renal epithelial cells and macrophages, we detected by sirna technique that the presence or absence of lcn-2 determines proliferation processes in damaged renal epithelial cells. the proliferative response was dependent on proinflammatory or anti-inflammatory environment. as lcn-2 is an acute phase protein synthesized during inflammation and unregulated in a number of pathological conditions, it may play an important role in survival and regeneration. we anticipate here that our results could be relevant for further research on the mechanisms of the phenotype switch induced by lcn-2. (1), y cao (1), s adhikari (1), m wallig (2) (1) national university of singapore, department of pharmacology, singapore (2) university of illinois at urbana champaign, usa it has earlier been shown that the extent of apoptotic acinar cell death is inversely related to the severity of acute pancreatitis. our previous works have demonstrated that induction of pancreatic acinar cell apoptosis by crambene protects mice against acute pancreatitis. the current study aims to investigate the role of phagocytic receptors and the anti-inflammatory effect of phagocytosis in protecting mice against acute pancreatitis by crambene. acute pancreatitis was induced in the mouse by administering hourly injections of caerulein (50 mg/kg) for 3, 6 and 10 hours respectively. neutralizing monoclonal anti-il-10 antibody (2.5 mg/kg) was administered either with or without crambene (70 mg/kg) 12 hours before the first caerulein injection. rt-pcr, western blotting and immunostaining were performed to detect cd36 expression. apoptosis in pancreatic sections was visualized by tunel. severity of acute pancreatitis was evaluated by estimation of serum amylase, pancreatic myeloperoxidase (mpo), water content, and morphological examination. pancreatic levels of inflammatory mediators were examined by elisa. the protective effect of crambene is mediated by reducing production of pro-inflammatory cytokines such as mcp-1, tnf-a and il-1㢠and up-regulating anti-inflammatory mediators like il-10. phagocytotic clearance in mouse acute pancreatitis may be essentially through macrophage surface receptor cd36.the anti-inflammatory mediator il-10 plays an important role in crambene-induced protective action against acute pancreatitis. the release of anti-inflammatory mediator il-10 is downstream of phagocytosis. these results show that induction of pancreatic acinar cell apoptosis by crambene treatment protects mice against acute pancreatitis via induction of anti-inflammatory pathways. (1,2) (1) northern ontario school of medicine, thunder bay, ontario, canada (2) lakehead university, canada integrin receptors and their ligands are involved in adhesion and internalization of several human pathogens, including pseudomonas aeruginosa. we have recently established that beta1 integrins in lung epithelial cells (lec) provide co-stimulatory signals regulating inflammatory responses (ulanova et al, am j physiol, 2005, 288: l497-l507). we hypothesized that lec integrins serve as receptors to recognize pathogen-associated molecules and mediate the innate immune response to p. aeruginosa. to determine molecular mechanisms of integrin involvement in innate immunity, we used an in vitro model of p. aeruginosa infection of a549 cells. to investigate interactions of bacteria with lec, p. aeruginosa strain pak was chromosomally labeled with a green fluorescent protein gene using a mini-tn7 delivery system.using several fluorescence-based detection systems, we established that the natural beta1 integrin ligand, fibronectin, mediates bacterial adhesion to lec.p. aeruginosa infection caused rapid transcriptional upregulation of alphav and beta4 integrin expression followed by the increased cell surface protein expression. the surface expression of integrin beta1 increased shortly following bacterial exposure without alterations of mrna expression, suggesting rapid protein redistribution within the cells. the data indicate that p. aeruginosa are capable to modulate integrin gene/protein expression in lec, potentially using fibronectin to alleviate bacterial binding to beta1 integrins. upon their engagement, integrin receptors can initiate intracellular signaling involved in innate immune and inflammatory responses to the pathogen. integrin receptors in lec may represent significant therapeutic targets in pulmonary infection caused by p. aeruginosa. the purine nucleoside adenosine has a major modulatory impact on the inflammatory and immune systems. neutrophils, which are generally the first cells to migrate toward lesions and initiate host defense functions, are particularly responsive to the action of adenosine. through activation of the a2a receptor (a2ar) present on neutrophils, adenosine inhibits phagocytosis, generation of cytotoxic oxygen species, and adhesion. also, recent work showed that adenosine can transform the profile of lipid mediators generated by neutrophils, inhibiting leukotriene b4 formation while potentiating that of prostaglandin e2 through the up-regulation of the cyclooxygenase (cox)-2 pathway. moreover, our laboratory determined that a2ar engagement can dramatically modulate the generation and secretion of neutrophil-derived cytokines/chemokines, including tnf-and mips. in mice lacking the a2ar, migrated neutrophils expressed less cox-2 than their wild type counterpart while displaying higher mrna levels of tnf-and mip-1. mononuclear cells from a2ar knock out mice, which eventually replace neutrophils into the air pouch, also displayed a more pro-inflammatory phenotype than those from wild-type animals. signal transduction experiments, aiming to delineate the intracellular events leading to the modulation of neutrophil functions following a2ar engagement, implicate pivotal metabolic pathways such as intracellular cyclic amp, p38 and pi-3k. together, these results indicate that adenosine may have a profound and multi-pronged influence on the phenotype of neutrophils and present this cell as being pivotal in mediating adenosines anti-inflammatory effects. the newest developments regarding adenosines effects on neutrophil functions will be presented.this work is supported by the canadian institutes of health research (cihr). human skin serves not only as a physical barrier against infection, but also as a "chemical barrier" by constitutively and inducibly producing antimicrobial proteins (amps). to identify human skin amps, we analysed extracts of healthy persons stratum corneum by reversed phase-hplc and purified a novel 9 kda amp that showed sequence similarity to mouse hornerin. suggesting that it originates from the human ortholog, we cloned it. human hornerin encodes a 2850 amino acid protein that contains a s100 domain, an ef-hand calciumbinding domain, a spacer sequence and two types of tandem repeats, suggesting that it represents a novel member of the fused s100 protein family. strongest constitutive hornerin mrna expression was seen in differentiated keratinocyte cultures. to follow the hypothesis, that hornerin fragments represent amps, we recombinantly expressed three hornerin peptides, rhrnr2 (tandem repeat unit b), rhrnr3 (tandem repeat unit a) and rhrnr4 (c-terminus) and subsequently analysed their antimicrobial activity using the microdilution assay system. the rhrnr3 peptide, containing the sequence motif found in the purified natural hornerin fragment isolated from stratum corneum, exhibited antimicrobial activity at low micromolar concentrations against escherichia coli, pseudomonas aeruginosa and candida albicans. the other peptides were found to be not or nearly not antimicrobially active. our results suggest that hornerin may have a yet unknown protective function in healthy human skin as part of the "chemical barrier" with preformed amps, which are generated from parts of the tandem repeats of a hornerin precursor molecule by a yet unknown cleavage mechanism. (1), n lu(1), r jonsson(2), d gullberg (1) (1) department of biomedicine, university of bergen, norway (2) the gade institute, university of bergen, norway a11ã�1 is the latest addition to the integrin family of heterodimeric receptors for the extracellular matrix. previously, it has been shown that this collagen receptor takes part in processes such as cell migration and matrix contraction. in this study we investigated the factors that regulate mouse integrin a11ã�1 expression. specifically, we have analyzed the influence of cell passage, growth factors and the 3-d microenvironment. using sv40 immortalized as well as primary fibroblasts, we show that a11ã�1 integrin is up-regulated when these cells are cultured within stressed collagen type i lattices. however, a11ã�1is downregulated when the collagen gels are made under relaxed conditions, allowing cells contract the lattice diameter. we also show here that a11 is upregulated by tgf-a on planar substrates. these findings suggest that mechanical tension and tgf-a are important factors in the regulation of a11ã�1 that need to be to taken into consideration when evaluating the role of a11ã�1 in wound healing and fibrotic disorders. (1), n vergnolle (1), p andrade-gordon (2) (1) inflammation research network, university of calgary, canada (2) rw johnson pharmaceutical research institute, canada the objective of this study was to investigate the effects of par2 deficiency in various models of colonic inflammation in order to elucidate the role of endogenous par2 in the process of inflammation in the gut.colonic inflammation in c57bl6 wildtype and par2 -/-mice was induced by treatment with 2.5% dss (in drinking water) or tnbs (1mg or 2mg in 100ul of 40% ethanol, single intracolonic injection) or pre-sensitizing mice with 3% oxazolone (in olive oil) applied to the skin of the abdomen, and 7 days later, a single intracolonic injection of 1% oxazolone (dissolved in 50% ethanol).intravital microscopy was performed, 7 days (tnbs/dss) or 4 days (oxazolone) after induction of colitis on the colonic venules to assess changes in leukocyte rolling, adhesion and vessel diameter.lastly, various parameters of inflammation were assessed following the intravital microscopy.par2 -/-mice showed significantly lower leukocyte adherence and vessel dilation compared to the wildtype mice in dss, and tnbs challenge. in all three challenges, mpo activity, macroscopic damage score and bowel thickness were significantly higher in wild-type mice, compared to par2 -/-.our evidences indicate that deficiency in par2 attenuates inflammatory responses in the experimental models of colitis associated with either th1 (tnbs/dss) or th2 (oxazolone) cytokine profile.therefore, par2 deficiency in the gut exerts antiinflammatory properties that are independent of th1 or th2 cytokine profile.the present study further highlights par2 as a potential target for inflammatory bowel diseases. (1), n vergnolle (1), p andrade-gordon (2) (1) inflammation research network, university of calgary, canada (2) rw johnson pharmaceutical research institute, canada in a previous study, inflammatory responses induced by three different models of colitis (tnbs/dss/oxazolone) were significantly attenuated in mice deficient for par2 (par2-/-). among the inflammatory parameters observed, infiltration of granulocytes to the colon was consistently reduced by par2 deficiency. aim of this study was to assess the effects of par2 deficiency (via par2-/-mice) on the recruitment of leukocyte in colonic venules. in anaesthetized animals, leukocyte rolling/ adherence and vasodilation were induced, by topical administration of fmlp (100 mm) or paf (100 nm) or by intraperitoneal injection of tnf-a; (0.5 mg -given 3 hours before the intravital microscopy). using intravital microscopy, we evaluated the ability of various leukocyte stimuli to induce leukocyte trafficking and vasodilation in colonic venules of par2 -/-versus par2+/+ mice. fmlp and paf as well as tnf-a; induced significant vasodilation and an increase in rolling/adhesion of leukocytes in mouse colonic venules. par2 -/-mice showed significantly lower leukocyte rolling compared to the wildtype mice in response to fmlp topical administration. leukocyte adherence induced by fmlp and tnf-a; was significantly lower in par2 -/-mice compared to wild types as well. no difference was observed between par2 -/-and wildtype for leukocyte rolling/adherence-induced by paf. the lack of functional par2 attenuated leukocyte trafficking in response to fmlp and tnf-a; but not to paf. the involvement of par2 activation in mouse colon leukocyte trafficking highlights par2 as an important mediator of inflammatory cell recruitment and thereby a potential target for the treatment of inflammatory bowel diseases. (1), kk hansen(2), k chapman(2), n vergnolle (2), ep diamandis (1), md hollenberg (2) (1) advanced center for detection of cancer, mount sinai hospital, university of toronto, toronto, on, canada (2) proteinases and inflammation network, university of calgary, calgary, ab, canada kallikreins (klks) are secreted serine proteinases identified in many cancers and multiple sclerosis lesions. we have recently shown that klks can activate proteinaseactivated receptors (pars), a family of g-protein coupled receptors associated with inflammation. we hypothesized that like trypsin, kallikreins can trigger inflammation via the pars. we studied the ability of klks 6 and 14 to activate pars 1, 2 and 4 in vitro and to cause oedema in a mouse model of paw inflammation in vivo. we found that klk14 is able to activate both of pars 2 and 4 and to prevent thrombin from activating par1. on the other hand, klk6 was a specific activator of par2. kallikrein administration in vivo resulted in a paw oedema response comparable in magnitude and time to that generated by trypsin. the oedema was accompanied by a decreased threshold of mechanical and thermal nociception. our data demonstrate that by activating pars 2 and 4 and by inactivating par1, kallikreins, like klks 6 and 14, may play a role in regulating the inflammatory response and perception of pain. (1), d park (1), b short(2), n brouard(2), p simmons(2), s graves (1), j hamilton (1) (1) melbourne university, melbourne, victoria, (2) peter maccallum cancer institute. melbourne, australia mouse mesenchymal stem cell enriched populations can be isolated from bone tissue by employing lineage immuno-depletion followed by fluorescence-activated cell sorting based on the cell surface expression of the sca-1 antigen. such isolated cells can subsequently be cultured and differentiate towards the osteogenic, adipogenic or chondrogenic linage in vitro. using this model we investigated the influence of the proinflammatory cyto-kines, tnfa or il-1b, on early osteogenesis in vitro. under osteogenic conditions, il-1b was found to inhibit cell proliferation in a dose dependent manner, whereas tnfa exhibited no effect. histochemical examination revealed the presence of either tnfa or il-1b to dramatically decreased mineralization in a dose dependent manner. q-pcr analysis indicated that in the presence of il-1b, despite increased expression of bone-specific alkaline phosphatase (akp2) mrna, levels of other osteogenesis markers (runx2, col1a and sp7) were decreased. in the presence of tnfa, levels of akp2, runx2 and sp7 were all decreased. our findings indicate that the influence of early mesenchymal progenitor cells on bone remodelling may be substantially altered in the presence of proinflammatory cytokines. using are-driven and nf-kb-targeted reporter genes, transfection of the nf-kb p65 subunit and nrf2 into hepg2 or other cells, as well as sirna technique to knockdown endogenous p65 in cells, we found that nf-kb p65 subunit repressed the anti-inflammatory and anticarcinogenetic nrf2-are pathway at transcriptional level. in p65-overexpressed cells, the are-dependent expression of heme oxygenase-1 was strongly repressed. in the cells where nf-kb and nrf2 were simultaneously activated, p65 unidirectionally antagonized nrf2 transcriptional activity. the p65-mediated are inhibition was independent of the transcriptional and dna-binding activities of p65. co-transfection and rna interference experiments revealed two mechanisms which coordinate the p65-mediated repression of are: (1) p65 selectively deprives creb binding protein (cbp) from nrf2, but not mafk, by competitive interaction with the ch1-kix domain of cbp, resulting in inactivation of nrf2 transactivation domain and concomitant abrogation of the nrf2-stimulated coactivator activity of cbp; (2) p65 promotes recruitment of histone deacetylases 3 (hdac3) to are by enhancing the interaction of hdac3 with either cbp or mafk, leading to inactivation of cbp and deacetylation of mafk. this study may establish a novel pro-inflammatory and pro-carcinogenic model for the transrepression of the are-dependent gene expression by p65 subunit. since various inflammatory and tumor tissues constitutively overexpresses p65 in their nuclei, the finding in this study implies a strong repression of are-dependent gene expression must take place in those tissues. in this regard, the findings in this study may help to explain why oxidative stresses and toxic insults usually occur in those pathological loci. dendritic cells (dc) play a pivotal role in the induction of immune response and tolerance. it is less known that dc accumulate in atherosclerotic arteries, where they might activate t-cells and contribute to the progression of disease. the serine protease thrombin is the main effector protease of the coagulation cascade. thrombin is also generated at sites of vascular injury and during inflammation. hence, thrombin generation is observed within atherosclerotic and other inflammatory lesions including rheumatoid arthitis. thrombin activates various cells via protease-activated receptors (pars). immature dc do not express pars. upon maturation with lps, tnfalpha, or cd40l, only lps-matured dc expressed par1 and par3 on their surface. stimulation of dc with thrombin, par1-or par3-activating peptides elicited actin polymerization and concentration-dependent chemotactic responses in lps-, but not in tnf-alphamatured dc. the thrombin-induced migration was a true chemotaxis as assessed by checkerboard analysis. stimulation of pars with thrombin or respective receptoractivating peptides led to activation of erk1/2 and rho kinase i (rock-i) as well as subsequent phosphorylation of the regulatory myosin light chain 2 (mlc2). the erk1/2-and rock-i-mediated phosphorylation of mlc2 was indispensable for the par-mediated chemotaxis as shown by use of pharmacological inhibitors of rock, erk and mlc kinases. in addition, thrombin significantly increased the ability of mature dc to activate proliferation of naive t-lymphocytes in mixed leukocyte reactions. in conclusion, our work demonstrates expression of functionally active thrombin receptors on lps-matured dc. we identified thrombin as a potent chemoattractant for mature dc, acting via rho/ erk-signaling pathways. data concerning the role of circulating modified low density lipoproteins (modldl) in atherogenesis and other pathologies are scarce. one reason for this is the lack of suitable radiolabeling methods for direct assessment of metabolic pathways of modldl in vivo. we report a novel approach for specific labeling of human native ldl (nldl) and modldl (iron-, hypochloriteand myeloperoxidase-oxidized, nitrated, glycated, and homocysteinylated ldl) with the positron emitter fluorine-18 (18f) by either nh2-reactive n-succinimidyl-4-[18f]fluorobenzoate or sh-reactive n-[6-(4-[18f]fluorobenzylidene)-aminooxyhexyl]maleimide (radiochemical yields, 15-40%; specific radioactivity, 150-400 gbq/ mmol). radiolabeling itself caused neither additional oxidative structural modifications of ldl lipids and proteins nor adverse alterations of their biological activity and functionality in vitro. the approach was evaluated with respect to binding and uptake of 18f-nldl and 18f-modldl in cells overexpressing various lipoproteinrecognizing receptors. the metabolic fate of 18f-nldl and 18f-modldl in vivo was delineated by dynamic small animal pet studies in rats and mice. the in vivo distribution and kinetics of nldl and modldl correlated well with the anatomical localization and functional expression of ldl receptors, scavenger receptors, and receptors for advanced glycation end products. the study shows that ldl modification, depending on type and extent of modification, in part or fully blocks binding to the ldl receptor, and reroutes the modldl to tissuespecific disease associated pathways. in this line, 18flabeling of modldl and the use of small animal pet provide a valuable tool for imaging and functional characterization of these pathways and specific sites of pathologic processes, including inflammatory processes, in animal models in vivo. the p38 mapk signaling pathway, which regulates the activity of different transcriptions factors including nuclear factor-ã�b (nf-ã�b), is activated in lesional psoriatic skin. the purpose of the present study was to investigate the effect of fumaric acid esters on the p38 mapk and the down stream kinases msk1 and 2 in cultured human keratinocytes. cell cultures were incubated with dimethylfumarate (dmf), methylhydrogenfumarate (mhf) or fumaric acid (fa) and then stimulated with il-1b before kinase activation was determined by western blotting. a significant inhibition of both msk1 and 2 activations was seen after pre-incubation with dmf and stimulation with il-1b whereas mhf and fa had no effect. also, dmf decreased phosphorylation of nf-kb / p65 (ser276), which is known to be transactivated by msk1. furthermore, incubation with dmf before stimulation with il-1b resulted in a significant decrease in nf-kb binding to the il-8 kb and the il-20 kb binding sites as well as a subsequent decrease in il-8 and il-20 mrna expression. our results suggest that dmf specifically inhibits msk1 and 2 activations and subsequently inhibits nf-kb induced gene-transcriptions which are believed to be important in the pathogenesis of psoriasis. these effects of dmf explain the anti-psoriatic effect of fumaric acid esters. a humanized model of psoriasis was successfully established by transplanting non-lesional skin biopsies from psoriasis patients onto bg-nu-xid mice lacking b, t and nk cells. in this system, a psoriatic process is triggered by intradermal injection of activated autologous peripheral blood lymphocytes. inflammation is associated with the expression of activation markers and inflammatory medi-ators such as tnf-alpha, hla-dr and cd1a and this results in increased proliferation and differentiation of keratinocytes, demonstrated by increased expression of ki-67 and ck-16. epidermal hyperplasia is a typical readout in this model. in a series of studies, this model was found to be sensitive too a wide range of compounds, including inhibitors of tnf-alpha, antibodies directed against growth factors, mmp-inhibitors, calcipotriol, metothrexate, betamethasone and cyclosporine a.in addition, we showed that inhibition of fatty acid oxidation had an anti-psoriatic effect in this model (caspary et al. brit j dermatol 2005; 153, 937-944) . employing lesional skin it was demonstrated that inhibition can also be performed in a therapeutic setting.due to its humanized nature this model represents a powerful tool for the identification or validation of compounds with potential for the treatment of psoriasis. kristian otkjaer (1), e hasselager(2), j clausen(2), l iversen (1), k kragballe (1) (1) aarhus university hospital, denmark (2) novo nordisk a/s, denmark interleukin-20 (il-20) is assumed to be a key cytokine in the pathogenesis of psoriasis. increased levels of il-20 are present in lesional psoriatic skin compared with nonlesional skin where it is barely detectable. whether il-20 is derived from antigen-presenting cells or keratinocytes remains unsolved. the aim of the present study was, therefore, to characterize il-20 expression in non-lesional psoriatic skin ex vivo. 3 mm punch biopsies from nonlesional psoriatic skin were collected. biopsies were transferred to cacl enriched keratinocyte basal media and cultured with vehicle or il-1beta (10ng/ml) for 0, 1, 2, 4, 6, 12 and 24 hours, respectively. the samples were analyzed by in situ hybridisation, qrt-pcr, immunofluorescent staining and elisa. incubation with il-1beta rapidly induced il-20 mrna expression in the biopsies. the highest level of il-20 mrna was detected after 4 hours and in situ hybridisation revealed that basal as well as suprabasal keratinocytes throughout the epidermis were the only cellular source of il-20 mrna. increased levels of il-20 protein were detected in the supernatant of the il-1beta stimulated biopsies. immunofluorescent staining of the biopsies showed no il-20 protein in the keratinocytes, whereas the il-20 protein was present in epidermal cd1a positive dendritic cells. our data emphasize the keratinocyte as the cellular source of il-20 expression in human skin. interestingly, immunofluorescent staining of our cultured biopsies showed il-20 protein in epidermal dendritic cells whereas no il-20 was detected in the keratinocytes. this indicates that epidermal dendritic cells are the target for keratinocytederived il-20. one response of epidermal keratinocytes to inflammatory stress is the induction of matrix metalloproteinases (mmps) that participate in tissue remodeling. excessive proteolytic activity is associated with chronic wounds and tissue damage during persistent inflammation. calcitriol, the hormonally active form of vitamin d, is known to have beneficial effects during cutaneous inflammation. we hypothesized that one way in which calcitriol exerts its effect on inflamed skin is by attenuation of damages caused by excessive mmp proteolytic activity. our experimental model consists of hacat keratinocytes cultured with tnf to simulate an inflammatory state. pro-mmp-9 was quantified by gelatin zymography and mrna by real-time pcr. the levels and activation of signaling proteins were determined by immunoblotting. the increase in pro-mmp-9 activity and mrna levels induced by tnf was inhibited by~50% following 48h treatment with calcitriol. using specific inhibitors we established that the induction of mmp-9 was dependent upon the erk pathway, while p38-mapk and pkc inhibited, and jun-kinase, pi-3-kinase and src did not affect it. levels of c-fos, a component of ap-1 transcription complex known to mediate mmp-9 induction, were elevated by tnf and further increased by calcitriol. the induction of mmp-9 by tnf was abolished by inhibition of the egfr tyrosine kinase attesting to the requirement for egfr trans-activation. calcitriol also inhibited the induction of mmp-9 by egf. we conclude that calcitriol inhibits the induction of mmp-9 gene expression by tnf in keratinocytes by affecting an event downstream to the convergence of the egfr and the tnf signaling pathways. (1), p verzaal (1), t lagerweij (1), c persoon-deen (1), l havekes (1), a oranje (2) (1) tno pharma, department of inflammatory and degenerative diseases, leiden, the netherlands (2) erasmus medical center, department dermatology and venereology, rotterdam, the netherlands mice with transgenic overexpression of human apolipoprotein c1 in liver and skin display a strongly disturbed lipid metabolism. moreover, these mice show a loss of skin-barrier function evident from increased trans epidermal water loss. these mice develop symptoms of atopic dermatitis, i.e. scaling, lichenification, papules, excoriation and pruritus. both hyperplasia of epidermis and dermis are observed. histological analysis shows increased numbers of cd4+ t cells, eosinophils, mast cells and ige-positive cells in the dermis. serum levels of ige are increased as well. cytokine profiling of draining lymph nodes is in favor of a th2-mediated disease. development of atopic dermatis in this model was found to be sensitive to topical treatment with triamcinoloneacetonide, fluticasone-proprionate and tacrolimus. moreover, oral treatment with dexamethasone successfully inhibits the development of disease in this model. impairment of the skin barrier is most likely the underlying cause of the development of atopic dermatitis in this model.this model is useful for identifying new therapeutic strategies and obtaining new insight into the pathogenesis of atopic dermatitis. topical immunosupppressants such as elidel and protopic are highly efficacious therapeutics for the treatment of atopic dermatitis and other dermatological conditions.-when delivered topically, these calcinuerin inhibitors offer several advantages over topical steroids; however, these marketed drugs have received a controversial "black box warning" because of a potential cancer risk. we speculated that systemic exposure of these drugs over long term use may contribute to the cancer risk.accordingly, we have designed and discovered a series of "soft" cyclosporin a (csa) derivatives as potentially safer alternatives.in general, soft drugs are engineered, via medicinal chemistry, to be effective upon local delivery but upon systemic exposure they are rapidly inactivated by metabolic pathways.in this way, exposure of active drug to distal organs is greatly minimized resulting in a significant enhancement in therapeutic index.the results or our drug discovery efforts around soft csa derivatives will be presented. (1), y sawanobori (2), u bang-olsen (3), c vestergaard(4), c grã¸nhã¸j-larsen (1) background: a strain of japanese fancy-mice, nc/nga, serves as a model for atopic dermatitis. under specific pathogen-free conditions, the mice remain healthy, but when kept under non-sterile conditions, they exhibit pruritic lesions like atopic dermatitis. scratching behaviour of the mice precedes the development of dermatitis, and a correlation between registered scratching counts and expression of il-31 mrna has been shown. also, transgenic mice over-expressing il-31 exhibit increased scratching behaviour and develop severe dermatitis. consequently we decided to explore the therapeutic effect of an anti il-31 antibody on scratching behaviour and dermatitis in nc/nga mice. methods: prior to clinical manifestation of dermatitis, we commenced treatment of nc/nga mice with il-31 ratanti-mouse 10 mg/kg intraperitoneally every fifth day for seven weeks. clinical dermatitis, scratching behaviour and weight gain, was assessed throughout the intervention period. serum analysis for ige and il-13 as well as histopathological and immunohistochemistry analysis on skin biopsies were also performed at end-point. results: taken over the entire intervention period, treatment with anti il-31 antibody in nc/nga mice from age seven weeks did not meet the primary end points, which were scratch, dermatitis and body weight. however, post hoc analysis revealed a significant reduc-tion of scratch by the anti il-31 antibody treatment in the time interval day 22-43. our results suggest an anti pruritic role for il-31 antibody in an atopic dermatitis-like animal model. anti il-31 antibody is therefore a new therapeutic opportunity for the treatment of pruritus in atopic dermatitis and perhaps other pruritic diseases. (1), p ferro (1), hm asnagli (1), v ardissone (1), t ruckle (2), f altruda (3), ch ladel (1) (1) rbm merck serono/university of torino, italy (2) merck serono pharmaceutical research institute, geneva, switzerland (3) university of torino, dipartimento di genetica, biologia, biochimica, italy class-i phosphoinositide 3-kinases (pi3ks) play a critical role in modulating innate and adaptive immune responses, as they are important transducers of external stimuli to cells, such as granulocytes and lymphocytes. since pi3k-g plays a pivotal role in mediating leukocyte chemotaxis and activation, as well as mast cell degranulation, the pharmacological blockade of pi3k-g might offer an innovative rationale-based therapeutic strategy for inflammatory skin disorders. in our study the inhibitory properties of a selective pi3k-g inhibitor as605858 on inflammation was applied to murine models modeling skin diseases like psoriasis and dermatitis. two mouse models were used: the first, irritant contact dermatitis (icd), is an innate inflammatory skin condition arising from the release of pro-inflammatory cytokines in response to haptens, usually chemicals. the second, contact hypersensitivity (chs) is a t-cell dependent model, modeling in part t-cell-mediated skin diseases such as psoriasis. we demonstrated the therapeutic effect of pi3kg inhibition and subsequent inhibition of chemotaxis in models of skin diseases and showed that a selective pi3k-g inhibitor can excert an important therapeutic efficacy (dose-dependent) in models of innate immunity (icd) -effective dose 0,3mg/kg p.o. once -as well as in t-cell mediated skin pathology (chs)effective dose 1mg/kg p.o. bid. we conclude that the mechanism of action related to inhibition of pi3k-g are demonstrable after oral administration of selective inhibitors like as605858 in models of acute and chronic skin inflammation and are mediated by modulation of innate and acquired immunity. introduction: high mobility group box 1(hmgb1) has recently been identified as a late mediator of endotoxin lethality. we newly developed an extra corporeal hmgb1 absorber. the purpose of this study was to test the hypothesis that hmgb1 removal could prevent or reduce endotoxin induced lethality or tissue injury of rats. methods: all experiments were conducted in accordance with the institutional care and use committee.male wistar rats were randomly allocated into three groups; hmgb1 absorber group (group i),hmgb1 nonabsorber group (group ii), and vacant column group (group iii).we applied these columns for each groups at 6 hours after lps injection. the rats were sacrificed 48 hours after lps injection for pulmonary histology. we statistically analyzed survival rate with kaplan-meier and the levels of hmgb1 with anova. results: survival rate was 67% in the group i at 48 hours after lps injection, as compared with 0% in the group ii and 11% in the group iii. the pulmonary histology in both group ii and group iii showed acute inflammatory injuries, whereas group i showed less inflammatory changes.the level of hmgb1 in the group i was significantly lower than those of group ii and iii. discussions:these results demonstratethat specific absorption of endogenous hmgb1 therapeutically reverses lethality of established sepsis indicating that hmgb1 inhibitors and absorber can be treated in a clinically relevant therapeutic window that is significantly wider than for other known cytokines. contact information: dr hideo iwasaka, oita university, anesthesiology and intensive care unit, yufu city, japan e-mail: hiwasaka@med.oita-u.ac.jp (1) (1) department of pharmacology, national university of singapore, singapore (2) dso national laboratories, singapore hydrogen sulfide (h2s) is increasingly recognized as a proinflammatory mediator in various inflammatory conditions. in this study, we have investigated the role of h2s in regulating expression of some endothelial adhesion molecules and migration of leukocytes to inflamed sites in sepsis. male swiss mice were subjected to cecal ligation and puncture (clp) induced sepsis and treated with saline, dl-propargylglycine (pag, 50 mg/kg i.p.), an inhibitor of h2s formation or sodium hydrogen sulfide (nahs) (10mg/kg, i.p.), a h2s donor. pag was administered either 1 hour before or 1 hour after induction of sepsis while nahs was given at the time of clp. using intravital microcopy, we found that in sepsis, prophylactic and therapeutic administration of pag significantly reduced the leukocyte rolling and adherence in mesenteric venules coupled with decreased mrna and protein levels of adhesion molecules (icam-1, p-selectin and e-selectin) in lung and liver. in contrast, injection of nahs significantly upregulated leukocyte rolling and attachment as well as tissue levels of adhesion molecules in sepsis. on the other hand, normal mice were given nahs (10mg/kg i.p.) to induce lung inflammation with or without pretreatment of nf-xã�b inhibitor, bay 11-7082. h2s treatment enhanced the pulmonary level of adhesion molecules and neutrophil infiltration in lung. these alterations were reversed by pretreatment with bay11-7082. moreover, expression of cxcr2 in neutrophils obtained from h2s treated mice was significantly upregulated leading to an obvious elevation in mip-2 directed migration of neutrophils. therefore, h2s act as an important endogenous regulator of leukocyte trafficking during inflammatory response. transient receptor potential vanilloid 1 (trpv1) is primarily found on sensory nerves. we have demonstrated its pro-inflammatory potential in arthritis and now present evidence that it is protective in an endotoxininduced model of sepsis. selective trpv1 antagonists are not available for use in the mouse in vivo, thus established trpv1 knockout (-/-) mice were used. c57bl6 wt and trpv1-/-mice were matched for age and sex and injected intraperitoneally (i.p.) with lipopolysaccharide (lps). the response was monitored for 4h. blood pressure, measured before and at intervals after lps in conscious mice via a tail cuff was reduced in both wt and trpv1-/-mice, with trpv1-/-mice showing an enhanced drop at 4h. in a separate group temperature, a proposed pre-mortality marker was also reduced by 4h, again with a significantly increased drop in trpv1ko. furthermore higher levels of two inflammatory markers tnfa and nitrite (as an indicator of no) were measured in peritoneal lavage and higher levels measured intrpv1-/-as compared with wt samples. finally aspartate aminotransferase (ast) levels also enhanced in trpv1-/-versus wt mice, although markers for kidney and pancreatic damage were similar in both genotypes. we conclude that trpv1 plays a protective role in sepsis. trpv1 is known to be present on nonneuronal (e.g. vascular components) and their relative involvement in sepsis is unknown. (1), da souza-junior(2), l de paula (1), mc jamur (2), c oliver (2), sg ramos (2), cl silva (2), lucia helena faccioli (1) ( h37rv) . infected balb/c mice developed an acute pulmonary inflammation and higher levels of tnf-a, il-1, kc, mcp-1 and mip-2 were detected in the lungs by day 15. in vivo degranulation of mast cells by c48/80 led to a reduction of the inflammatory reaction associated to a marked decline proinflammatory cytokine and chemokine levels in the lungs. the magnitude of cellular immune response was also partially impaired in infected mice and treated with c48/80. histologically, the exacerbated granulomatous inflammation shown in the lung parenchyma of infected mice was attenuated in infected mice and treated with c48/80. of interest, the number of mycobacterial bacilli recovered from the lungs was 1 log higher after treatment of infected mice with c48/80. these findings suggest that mcs participate in host defense against m. tuberculosis infection through of the modulation of cytokines and chemokines, which are important for the recruitment and activation of inflammatory cells. (1), ms chadfield (2), db sã¸rensen (1), h offenberg (1), m bisgaard (1), he jensen (1) (1) department of veterinary pathobiology, faculty of life sciences, university of copenhagen, denmark (2) novo nordisk a/s, cell biology, gentofte, denmark introduction: pasteurella multocida is an important cause of pneumonia in several animal species and may spread systemically. the aim of this study was to evaluate initial inflammatory reactions and the inocula effect due to strains of p. multocida of different origin in an aerogenous murine model. materials and methods: 30 female balb/ c-j mice (app. 20 g, taconic, denmark) were infected intranasally with two clinical isolates of p. multocida of avian (vp161) and porcine (p934) origin, at three different levels of inocula concentration. after euthanasia, specimens of lung and liver tissue were collected for bacteriological and histopathological evaluation. furthermore, lung tissue samples were taken for measurement of expression of metalloproteinase mmp-9 and metalloproteinase inhibitor timp-1. results: all mice infected with the avian strain were euthanized after 24 hours. viable counts recovered from lung and liver tissue were high, and histopathology revealed pronounced acute bronchopneumonia. in the liver, disseminated necrosis with formation of microabscess was also seen. on the contrary, a dose response was observed with the porcine strain with regard to recovery of viable counts and development of lesions was apparent after 24, 48 and 72 h. furthermore, differences were seen in the nature of the lesions caused by the two strains. there was a difference in expression of mmp-9 and timp-1 between infected and noninfected mice. the model proved suitable for the evaluation of pulmonary inflammatory reactions between the two different host-derived strains as demonstrated through viable counts, histopathology and expression of mmp-9 and timp-1. (1), r molinaro (1), a franã�a (1), m bozza (1), f cunha(2), s kunkel (3) (1) universidade do rio de janeiro, brazil (2) universidade de sâ¼o paulo, brazil (3) university of michigan, usa introduction and objectives: studies reveal that regulatory t (treg) cells control immune responses; therefore these responses must be controlled to enable the effective protection against infections and cancer. ccr4 knockout mice (ccr4 -/-) are more resistant to lps shock. so, our aim is to study the mechanisms involved in the resistance of ccr4-/-subjected to severe sepsis by cecal ligation and puncture (clp) and how tregs modulate this effect. results: c57/bl6 mice were subjected to clp model, whereby the cecum was partially ligated and puncture nine times with a 21g needle. sham-operated mice were used as control. mice subjected to clp and sham surgery were treated with antibiotic since 6h after and until 3 days. ccr4 -/-mice subjected to clp presented an increase in survival rate (78%) compared with wildtype mice (17%), and a marked improvement in the innate response concern to neutrophil migration to peritoneum and lung, bacteria load and cytokine levels compared to wild-type mice. besides, tregs from ccr4-/-clp mice did not inhibit proliferation of t effector cells as observed for treg from wild clp mice, at a proportional ratio of teffector:treg. interesting, treg from ccr4-/-clp mice did not inhibit neutrophil migration to bal when co-injected with fungal challenge as secondary infection, while the treg from wild clp mice did, as expected. conclusions: these results suggest that treg cells from ccr4-/-mice did not present suppressive response and it could be an important factor in their survival. inflammation and oxidative stress are known to be one of the important causes that are responsible for many diseases. inflammation has been associated with diseases like cancer, diabetes and many other. proinflammatory cytokine (tnf -âµ and il-1ã¢) and no are considered as pivotal mediators in inflammatory conditions like rheumatoid arthritis, sepsis and cancer etc. thus inhibition of pro-inflammatory cytokines and no production are important targets for treatment of inflammatory disorders. nowadays due to the emerging side effects of cox inhibitors, these targets have been paid more attention for the treatment of these conditions. some medicinal plants such as curcuma longa (1), commiphora mukul (2) in humoral memory, antibodies secreted into serum and other body fluids protect an individual against repeated challenges of previously encountered pathogens. antibody-secreting plasmacells are mostly considered to be shortlived, terminally differentiated b lymphocytes, eliminated after a few days or weeks by apoptosis. however, in secondary lymphoid organs and in the bone marrow, plasma cells can survive for months and years, without dna-synthesis and refractory to signals from antigen or antigen-antibody complexes. the lifetime of these longlived plasmacells depends on an intrinsic competence to survive in the distinct environment of those organs, which defines a specific survival niche. the niche provides survival signals like il-6, cxcl12 and tnf. within a functional niche, the lifetime of a plasma cell is apparently not limited intrinsically. the number of niches in the body has to be limited, in order to maintain physiological concentrations of serum immunoglobulins. thus recruitment of new plasmacells to the pool of old memory plasma cells has to be competetive. this competition is probably controlled by a simple molecular mechanism, namely the dual functionality of chemokines like cxcl12, which attract newly generated plasmablasts to a survival niche and at the same time are a survival signal for the plasma cell. plasmablasts and plasma cells express cxcr4, the receptor for cxcl12. while plasmablasts migrate in response to cxcl12, plasma cells depend on it for survival in the niche, but are no longer migratory. thus once disloged from their niche, they will die. plasmablasts newly generated upon systemic secondary immunization, upon concommittant stimulation with interferon-gamma, can also express cxcr3, the receptor for the interferon-gamma-induced chemokines cxcl9, 10 and 11, which may lure the plasmablasts into inflamed tissue. the switch in the potential to migrate provides also an efficient means to eliminate plasma cells of the peak of an immune response, which as plasmablasts had migrated to the tissue inflamed in that pathogenic challenge. inflamed tissue contains survival niches for plasma cells. in the inflamed tissue, plasma cells provide high local antibody concentrations while the tissue is inflamed. upon resolution of the inflammation the plasma cells will be dislodged and die. longlived plasma cells provide longlasting antibody titers (protective memory) and leave memory b cells a role in reactive memory, generating memoryplasmacells in secondary challenges, and if serum titers are not sufficient to protect. in chronic inflammation, this mechanism can contribute to pathogenesis. thus in the nzb/w model of lupus, longlived autoreactive plasma cells are generated early in pathogenesis, which survive in bone marrow and spleen. later, in established disease, autoreactive plasma cells are shortlived and continuously generated. they do not compete with the longlived plasma cells, and both populations coexist as prominent populations. interestingly, longlived plasmacells are resistant to therapeutic immunosuppression, while the generation of shortlived plasma cells is blocked. this may be the reason for the failure to cure antibodymediated immunopathology, e.g. in autoimmunity and allergy, by conventional immunosuppression, (1), h lee (2) c5a is a potent inflammatory mediator produced during complement activation. unregulated c5a signalling through its receptor (c5ar) on neutrophils and other leukocytes is implicated in the pathogenesis of autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosus. considerable effort has gone into development of c5ar antagonists for human therapy. we took neutrophils from genetically modified human c5ar knock-in mice in which the mouse c5ar coding region was replaced with human c5ar sequences and immunized wild-type mice to generate high affinity antagonist monoclonal antibodies (mabs) to human c5ar. these mabs inhibit c5a-induced neutrophil migration and calcium-flux, and bind to a region of the 2nd extracellular loop of c5ar loop that seems to be critical for receptor activity. this study investigated the effectiveness of these mabs in the k/bxn serum-transfer model of inflammatory arthritis. human c5ar knock-in mice were given 1-10 mg/kg mab intraperitoneally, before or after inflammatory arthritis developed. mice treated with anti-c5ar mab one day before serum transfer did not develop swelling or clinical signs of arthritis in contrast to controls. histopathology of the joints in anti-c5ar mab-treated mice revealed a complete block of the massive influx of leukocytes and cartilage erosion seen in controls. furthermore, and most significantly, a single 1 mg/kg dose of anti-c5ar mab given 5 days after initiation of disease completely reversed inflammation. in the collagen-induced arthritis (cia) model, injection of anti-c5ar mab after development of inflammation also reversed inflammation to baseline. these potent new antibodies to human c5ar are in preclinical development. the cytokine macrophage migration inhibitory factor (mif) participates in fundamental events in innate and adaptive immunity. the profile of activities of mif in vivo and in vitro is strongly suggestive of a role for mif in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis (ra), asthma, and sepsis. mif also has a unique relationship with glucocorticoids, in that despite antagonizing their effects, the expression of mif is in fact induced by glucocorticoids. thus, mif functions as a physiological counter-regulator of the anti-inflammatory effects of glucocorticoids. therapeutic mif antagonist may therefore provide a specific means of steroid sparing. since mif are highly conserved among different species, it is hard to develop high affinity antibodies due to immune tolerance.we developed a proprietary technique to break the immune tolerance and selected high affinity mouse monoclonal antibodies against mif.the antibody can neutralize mif activity in cell based assays, and is very effective in a lps induced mouse sepsis model. using this antibody as a tool, we are studying the function of mif in comparison with the function of lps.we found that lps induced inos expression and no secretion are dependent on the secretion of mif.we also found that although both lps and mif induce g1 arrest in macrophage cell line raw264.7, their functions are independent to each other. structure-based small molecule drug design. an effective agent would be the first orally active cytokine antagonist. methods: collagen-induced arthritis (cia) was induced in dba-1 mice by immunisation with bovine type ii collagen/adjuvant on day 0 and 21. cor100140, synthesized on the basis of computer modeling of mif protein x-ray crystallographic data, was administered by daily oral gavage from day 21. etanercept (8mg/kg ip q2d) was used as a positive control. the mek-erk pathway is activated in numerous inflammatory conditions, including ra, ibd and copd. arry-162 is a potent (ic50 = 12 nm), selective, atp-uncompetitive mek1/2 inhibitor.arry-162 is highly efficacious in cia and aia rat models, with ed50s of 3 and 10 mg/kg, respectively, equal to or better than standard agents. addition of arry-162 to methotrexate, etanercept, ibuprofen or dexamethasone regimens in these models results in improved efficacy that is at least additive, if not synergistic. in tpa-stimulated human whole blood, this compound inhibited tnf, il-1 and il-6 production (ic50s of 23, 21 and 21 nm, respectively). in contrast, inhibition of perk required 280 nm to achieve a 50% reduction, demonstrating that inhibition of pro-inflammatory processes is very sensitive to perk inhibition.in clinical studies, healthy volunteers were administered a single oral dose of 10, 20, 30, 40 or 80 mg); blood was drawn at various times after dosing and stimulated ex vivo with tpa. arry-162 was well-tolerated and drug exposure was dose-proportional. in ex vivo blood samples, there was a dramatic time-and concentration-dependent inhibition of tpainduced il1 and tnffz with >80% inhibition observed at plasma concentrations of 50 and 150 ng/ml, respectively. similar inhibition of perk required 300 ng/ml. a multiple ascending dose clinical study has confirmed the pharmacokinetics and pharmacodynamics of arry-162 and helped define tolerability. clinical evaluation of arry-162 in combination with methotrexate in patients with rheumatoid arthritis is on-going. p38 (a mitogen-activated protein kinase) has been shown to play a key role in the release of cytokines such as tnfand il-1a from monocytes in signaling cascades that are initiated due to extra cellular stress stimuli. inhibition of p38 activity is expected to regulate the levels of tnf-a and il-1b thereby alleviating the effects of inflammation in ra. a new class of p38 inhibitors based on the naphthyridininone scaffold have been discovered. x-ray crystallography and site directed mutagenesis studies were critical tools that aided the evolution of the naphthyridinone lead class starting from a pyrido-pyrimidinone template. this presentation will discuss the derivation of key benchmark pre-clinical candidates in these novel scaffold classes (shown below) as influenced by structural biology studies, mutagenesis data and molecular modelling. efficacy studies in animal models for benchmark compounds will also be presented. (1), h aaes (1), w-h boehncke(2), j pfeffer(2), t skak-nielsen(1), i teige (1), k abell (1), ph kvist (1), e ottosen (1), tk petersen (1), lars svensson (1) (1) discovery, leo pharma, 55 industriparken, ballerup, denmark (2) department of dermatology, johann wolfgang goethe-university, frankfurt am main, germany p38 map kinase plays an important role in mediating an inflammatory response in mammalian cells. as a consequence of activation, several inflammatory mediators are released including il-1b] and tnfa. both cytokines have a central role in the pathogenesis of inflammatory conditions such as psoriasis. approximately 30% of psoriasis patients develop psoriatic arthritis. leo15520 is a member of newly developed class of selective p38 map kinase inhibitors. the compound was tested orally in in vivo models relevant for psoriasis and psoriatic arthritis. the in vivo models selected include the cia arthritis model, the human psoriasis xenograft scid mouse model, the uvb-induced dermatitis model, the lps induced tnfa model and a local gvh model. treatment with leo15520 led to an amelioration of the ongoing inflammation in all investigated in vivo models. in the cia model, a clear dose response effect was observed on the developing arthritis in both rats and mice (65 % reduction in mice and 77 % in rats at 10 mg/kg p.o.). in the humanised psoriasis model, leo15520 at a dose of 20 mg/kg, had an effect on both the hyperplastic epidermis (epidermal thickness reduced by 41%) and on the infiltrating inflammatory cells. the anti-inflammatory effect of leo15520 was even close to the effect of systemically delivered steroids in both models. we believe that the new highly selective class of p38map kinase inhibitors has a strong potential as an orally delivered therapy for systemic inflammation diseases such as psoriasis and arthritis. slx-2119 is a potent, selective, orally bioavailable inhibitor of the rho-kinase rock-2. its ic50 for rock-2 and rock-1 inhibition is 100 nm and >10 mm respectively. the ability of slx-2119 to inhibit septic liver injury was investigated in c57bl/6j mice challenged with lipopolysaccharide (lps) and d-galactosamine (d-gal). mice were given lps (10 mg/mouse) and d-gal (18 mg/ mouse) i.p and 5.5 hours later were sacrificed for analysis of liver injury. mice challenged with lps/d-gal had a >10 fold increase in serum alt and ast levels. this increase was reduced by >90% in mice pretreated with slx-2119 either orally (100 mg/kg, 2 and 20 hrs prechallenge) or i.p. (10-100 mg/kg, 15 min pre-challenge). slx-2119 inhibited the increase in hepatic levels of tnffalpha produced by lps/d-gal by >50%. to assess the kinetics of slx-2119s benefit, slx-2119 (100 mg/kg i.p.) was given 15 min prior to, or 15 or 60 min after the lps/ d-gal. slx-2119 was effective at inhibiting the rise in alt and ast levels at all 3 time points suggesting that inhibiting rock-2 even after the initiation of the lps/d-gal driven cascade protects against septic liver injury. in a survival study, 9 out of 10 mice given lps/d-gal were dead by 8 hrs whereas in mice given slx-2119 (100 mg/kg orally) 1 animal died at 10 hours and the remaining 9 mice were alive 48 hrs later. these results show that specific inhibitors of rock-2 may have therapeutic utility in the treatment of sepsis and subsequent liver injury. theta through three key hydrogen bond mediated interactions.they potently inhibit protein kinase c activity in vitro as demonstrated by inhibition of il-2 secretion in human purified t-cells stimulated with anti-cd3 and anti-cd28 or whole blood seb challenge.the pkc-theta inhibitors are orally bioavailable and demonstrate immunosuppressive activity in a mouse model of human delayed-type hypersensitivity responses. (1), j zhang (1), k henley (1), m white (1), d hilton (1), b kile (1) ( to investigate pathogenesis of rheumatoid arthritis, we used mice transgenic for the uniquely human fcgam-mariia in inducible and passively transferred models of arthritis. transgenic mice developed severe ra-like disease in both model systems, indicating that the transgene played a major role in arthritis pathogenesis. disease could be reduced by the administration of either specific monoclonal antibodies to fcgammariia or small chemical entities (sce) designed to bind to the fcgam-mariia dimer. to investigate the cause of this enhanced sensitivity to auto-immune stimuli, the phagocytic capacity of transgenic mice compared to c57bl/6 control mice was examined using phagocytosis of fluorescent beads coated with ova or ova/anti-ova immune complex or opsonised sheep red blood cells. in both assays macrophages from fcgammariia transgenic mice showed significantly increased phagocytosis comapred with cells from control mice at 4 hours.this difference diminished over time andwas only seen where particles were opsonised. treatment of macrophages with specific fcgammariia blocking monoclonal antibody fragments 8.7 f(ab)2 or iv.3 f(ab) reduced phagocytosis to background levels . macrophages from transgenic mice also showed significantly greater production of inflammatory cytokines tnf-alpha and il-1beta when stimulated in vitro with heat aggregated immunoglobulin (hagg). moreover, this response was also blocked by specific fcgammariia monoclonal antibody fragments or sce. thus, expression of the fcgammariia transgene in these mice leads to increased uptake of and reactivity to immune complexes, resulting in enhancement of inflammatory sequelae in the form of increased th1 cytokine secretion andamplification of the pro-inflammatory response leading to arthritic disease. harald burkhardt (1), u hã¼ffmeier (2), i kã§nig (3), j lacorz (2), a reis (2), k reich (4) (1) johann wolfgang goethe university, frankfurt, germany (2) friedrich-alexander-unisversity of erlangen-nuremberg, germany results: whereas the earlier described strong association of allele tnf*-238a with psoriasis could be confirmed, our study revealed that this association was completely dependent on carrying the psors1 risk allele. for psa, but not psoriasis vulgaris without joint involvement strong association with the allele tnf*-857t was detected (or=1.956, 95% ci 1.33-2.88; pcorr=0.0025) also in patients negative for the psors1 risk allele. our results indicate genetic differences between psoriasis vulgaris patients with and without joint manifestation. while the previously reported association between tnf*-238a and psoriasis seems to primarily reflect ld with psors1, tnf*-857t may represent a risk factor for psa independent of psors1. (1), n modi (1), m stanford (1), e kondeatis (1), r vaughan (1), f fortune (2), w madanat (3), c kanawati(4), p murray (5) methods: dna was obtained from 167 patients with bd, 61 from the uk and 106 from the middle east (me), and 159 controls individuals, 129 from the uk and 30 from me. dna was prepared by proteinase k digestion, and salt extraction and -318 and 49 snp were detected by a pcr-ssp. results: there was no significant difference in expression of -318c/t or 49a/g when all bd patients were compared to all control individuals, (p=0.3 and 0.9, respectively). as we have previously shown differences in snp expression in different patient groups we tested the uk and me patients separately. however, there was no significant difference in either snp in uk bd patients, (p=0.15, p=0.39) or me bd patients (p=0.7, p=0.6) when compared to the appropriate controls. (1), da brown (1), h johnen (1), mr qiu (1), t kuffner (1), pgm curmi (1), l brown (1), m mazzanti (2), sn breit (1) ( introduction: cerebral palsy (cp) is a nonprogressive motor disorder caused by white matter damage in the developing brain. it is often accompanied with neurocognitive and sensory disabilities. the cause and pathogenesis of cp is multifactorial and continues to be poorly understood. chorioamnionitis, clinically silent or manifest, has been reported to be a risk factor for cp both in term and preterm infants. il-16 gene is single copy gene located on chromosome 15q26.1-3 in humans. interleukin-16 is synthesized by a variety of immune (t cells, eosinophils and dendritic cells) and non-immune (fibroblasts, epithelial and neuronal) cells. it is also detected in organ-specific secretions in a number of inflammatory processes. amniotic fluid interleukin-16 concentrations decreased with advancing gestational age. but, women with preterm labor and women with chorioamnionitis have higher interleukin 16 amniotic fluid concentrations than those who delivered at term or those with sterile amniotic fluid. the aim of our study was to estimate allelic frequency for regulatory il16 -295 snp in the children with the cp. methods: dnas obtained from peripheral blood of 46 cp patients and 182 unrelated healthy volunteers were genotyped for the il16 -295 snp pcr-rflp method. results and conclusions: il16 -259 genotype fncc was more common in the population with cerebral palsy in the comparison to healthy volunteers. the significance of the association between il16 fngene polymorphisms and cerebral palsy has to be investigated in the future studies. (1), d delbro (1), e hansson (2) (1) kalmar university, sweden (2) gã§teborg university, sweden background: acetylcholine (ach) is a major signalling molecule, binding partly at nicotinic receptors (nachrs; a family of ions channels with nicotine as a selective ligand). one subtype, the alpha7nachrs, has antiinflammatory effects by way of down-regulation of tnfalpha release from macrophages. the alpha7nachrs have been demonstrated in neuronal as well nonneuronal tissues, e.g. astrocytes and microglia. aim. in rat astrocytes in primary culture, and in astrocytes cocultivated with primary microvessel cultures study: 1. the expression of alpha3nachrs and alpha7nachrs by immunofluorescence and western blot. 2. intracellular ca2+-transients spread within the astroglial networks after stimulation with nicotine. 3. pro-inflammatory cytokines, il-1b, il-6, and tnfalpha, released from microglial cells after stimulation with nicotine. results: alpha7nachrs expression was more evident in astrocytes co-cultivated with endothelial cells, suggesting that endothelial cells release factors, which increase the maturity of astrocytes. the ca2+ transient evoked by nicotine was also more pronounced in the co-cultured astrocytes. these ca2+ responses were blocked by the alpha7nachr antagonist alpha-bungarotoxin. the release of pro-inflammatory cytokines was down-regulated after stimulation by nicotine. conclusion: alpha7nachrs appear to be involved in some of the effects of nicotine administration to rat astrocytes. an anti-inflammatory action of cholinergic nerves on the astrocytes via nachrs seems probable, which may have therapeutic implications. university, department of natural sciences, kalmar, sweden e-mail: ann.pettersson@hik.se gedeon richter plc., budapest, hungary tramadol, an atypical opioid analgesic, is increasingly used for the treatment of osteoarthritis because it does not produce typical side effects of nsaids. review of clinical data show that it produces symptom relief and improves function, but these benefits are small and adverse events often cause participants to stop taking the medication (cohran database 2006). efficacy of tramadol in animal models of inflammatory pain is well established; however complex characterization of the drug regarding analgesic and side effects is missing in rats. our aim was to assess oral efficacy of tramadol at side effect free doses in rats. anti-hyperalgesic effect was determined in complete freunds adjuvant (cfa) induced thermal hyperalgesia test (th) and in model of cfa-induced knee joint arthritis. effect on inflammation was characterized in carrageenan induced paw edema test (et). for the characterization of side effect accelerating rotarod assay (arr) was used. significant impairment in arr was noted at 55 mg/kg dose, and above. in the th test weak 35% effect was seen at side effect free dose (40 mg/kg). in the arthritis model b.i.d. 40 mg/kg dose of tramadol given on days 3-7 after cfa caused a maximum 86% effect on weight bearing incapacitance (day 4). however, its effect seemed to diminish (34% on day 7) upon repeated treatment. in et tramadol had significant anti-inflammatory effect at 100 but not at 30 mg/kg dose. these results show that efficacy of tramadol in rat inflammatory pain models is limited by its side effects, in accordance with clinical data. chronic relapsing experimental allergic encephalomyelitis (cr eae) is a disease that bears striking similarities to the human condition multiple sclerosis (ms). in particular, cr eae and ms have major inflammatory events in the central nervous system (cns) that culminate in demyelination and disruption of axonal function.one group of mediators involved in the progressive cns inflammation are the prostaglandins (pgs).pg generation is regulated in experimental non-immune conditions of the cns by n-methyl-d-aspartate (nmda) receptor activation.nmda receptor-mediated events are also evident in eae and may therefore have the potential to influence pg production.the study was designed to profile pge2 and pgd2 in cns tissues during the development of cr eae and to examine the role of the nmda receptor in pg production through the use of the specific antagonist mk-801.biozzi mice were inoculated for cr eae and cns tissues were sampled during the course of disease.enzyme immunoassay of processed samples revealed pge2 and pgd2 levels within normal limits during the acute and subsequent remission phases of cr eae.in contrast, dramatic changes in pg concentrations were observed with a relapse of symptoms and a remission of disease.mk-801 was therapeutically administered to cr eae-diseased mice at the onset of relapse and changes in cns pg levels were recorded.the relapse phase of cr eae, but not the acute stage of disease, is characterised by an increase in cns pg production that may be influenced by nmda receptor activation. livia l camargo(1), lm yshii (1) (1), sk costa (1) (1) university of sâ¼o paulo, brazil (2) king's college, london, uk (3) butantan institute, sâ¼o paulo, brazil objectives: the neuropeptide substance p (sp) released by capsaicin-sensitive nerves (csn) plays a pivotal role in neurogenic inflammation. despite the prevalence of arthritis, the contribution of sp to the progression of arthritis has not been established. this study investigated the effect of csn ablation and sr140333, a sp antagonist, on knee joint inflammation and pain induced by intraarticular (i.a.) injection of kaolin (10 %, 5 h time course) in female wistar rats. the kaolin-injected knee (ipsilateral -ipsi) of vehicle-treated rats exhibited a significant, timedependent oedema as compared to the contralateral knee. in addition, increased pain score and high levels of myeloperoxidase (mpo, marker of neutrophil accumulation) and both pro-inflammatory (il-1b and il-6, but not tnfa) and anti-inflammatory (il-10) cytokines was detected in the ipsi synovial fluid of these animals. both destruction of knee joint csn fibres by neonatal capsaicin treatment and i.a. injection of rats with sr140333 (1 nmol/cavity) significantly attenuated the kaolin-induced pain score and knee oedema, suggesting that kaolin is acting, at least partially, via a neuronal mechanism. in contrast, the same treatment caused increased mpo activity and cytokine concentrations measured 5 h post kaolin injection. conclusions: peripheral release of sp after kaolin injection acts to increase pain generation, oedema formation and inflammatory cell influx. however, chronic tachykininergic depletion by capsaicin treatment up-regulates the production of pro-inflammatory cytokines that are important in triggering cell influx in the synovial cavity. (1) (1) university of sâ¼o paulo, sâ¼o paulo, brazil (2) butantan institute, sâ¼o paulo, brazil objectives: previously, intra-tracheal (i.tr.) injection of dep or 1,2-naphthoquinone (1,2-nq) evoked plasma extravasation and cell influx in rat airways. we now investigated whether simultaneous injection of these pollutants had a synergistic inflammatory action. we also determined the ability of dep and 1,2-nq-induced airway inflammation to evoke changes in the rat isolated thoracic aorta (rta) and corpus cavernosum (rcc) reactivity using organ bath assays. results: capsaicin-or vehicle-treated male wistar rats received i.tr. injection of dep (1 mg/kg) and 1,2-nq (35 nmol/kg). after 15 min, dep and 1,2-nq produced a potent (additive) plasma extravasation in the trachea and bronchi, but not lung, compared with each compound alone. in capsaicin-treated rats or treated with tachykinin antagonists, the response was inhibited, suggesting an important role for c-fibres, primarily tachykinins. increased mpo and cytokine levels were detected in bronchi of capsaicin-treated rats following 3 h treatment with pollutants. this treatment contributes to augment the ach (10-9-10-4 m)-induced relaxation in the rta but not in the rcc. in capsaicin-treated rats, the rta response to the pollutants was not affected but it was capable of evoking a marked relaxation in rcc in animals challenged with pollutants. conclusions: 1,2-nq exacerbates dep-induced plasma extravasation and mpo activity in the airways, indicating a neurogenic mechanism through tachykinins. the exposure to dep and 1,2-nq affects the endotheliumdependent response in rta without interfering with rcc. neuropeptides are unlikely to affect the pollutantsinduced changes in the rta. acknowledgements: capes, cnpq, fapesp. we thank ma alves for technical assistance. trans-resveratrol (rv) is a naturally occurring polyphenolic compound present in certain foods that has anticancer and anti-inflammatory properties. the purpose of this study was to determine the effect of rv on the production of proinflammatory cytokines and reactive oxygen species stimulated by lipopolysaccharides (lps) in glial cells. rt-pcr showed that rv (5, 25, 50 mm) dose-dependently inhibited 500 ng/ml lps induced tnfa, il-1b, il-6, mcp-1 and inducible nitric oxide synthase mrna expression. rv also inhibited lps-induced production of these cytokines (elisa), nitric oxide and reactive oxygen species in a dose-dependent manner. western blot analyses showed that resveratrol could inhibit lps-stimulated phosphorylation of erk1/2 and jnk but not p38. nf-kb reporter assay showed rv could inhibit nf-kb activation by lps in microglia and astrocytes. these results suggest that rv may inhibit lps-induced microglial and astrocyte activation through erk1/2, jnk and nf-kb signaling pathways. therefore, rv is a natural product with therapeutical potential against disease conditions in cns that involve an overproduction of proinflammatory cytokines and reactive oxygen species. (1), cs patil(2), sv padi (1), vp singh (1) (1) university institute of pharmaceutical sciences, panjab university, chandigarh, india (2) pharmacology r & d, panacea biotec ltd. lalru, punjab, india persistent stimulation of nociceptors and c-fibers by tissue injury causes hyperalgesia and allodynia by sensitization of nociceptors and facilitation of synaptic transmission in the spinal cord. the important participant in the inflammatory response of injured peripheral nerve may be nitric oxide (no). the aim of the present study was to test the sensitivity of pde5 inhibitor sildenafil in chronic constriction injury (cci) model, a rat model of neuropathic pain. sciatic nerve injury is associated with development of hyperalgesia 14 days after the nerve ligation. sildenafil (100 and 200fã� g/rat, i.t.) produced a significant decrease in pain threshold, which in lower dose did not alter the nociceptive threshold. the hyperalgesic effect of sildenafil was blocked by l-name and methylene blue (mb), which on per se treatment showed antinociceptive effect in nerve ligated rats. the results from the present study indicated that the major activation of no cgmp pathway in the chronic constriction injury model of neuropathic pain. the aggravation of hyperalgesic response might be due to the increased cgmp levels resulting in pkg-i activation and its upregulation. glycine transporter (glyt) 1 and glyt2 are expressed in glia and neurons, respectively. glyt1 make clearance of glycine released from glycinergic neuron in synapse and thus terminates the neurotransmission and also regulates over-stimulation by glycine spilled over to nmda receptors, while glyt2 supplies glycine into synaptic vesicles in glycinergic neurons. therefore, glyt inhibitors could modulate inhibitory glycinergic or excitatory glutamatergic neurotransmission. the present study examined the effects of glyt inhibitors on pain in animal models. inhibitors of glyt1 (sarcosine and org25935) and glyt2 (alx1393 and org25543) by intrathecal (i.t.) injection reduced formalin-induced nociceptive behaviors. glyt1 inhibitors reduced allodynia score and reversed the reduction of paw withdrawal threshold in complete freund adjuvant (cfa)-induced inflammation mice but the antiallodynia effects appeared after latent period. on the other hand, glyt2 inhibitors produced antiallodynia effects immediately after the i.t. injection in cfa-treated mice. these inhibitors produced the similar antiallodynia effects in the partial sciatic nerve ligationinjury or streptozotocin-induced diabetic neuropathic pain models, either by i.t. injection or i.v. injection.pretreatment of specific antagonists of glycine site of the nmda receptors disappeared the latent periods of glyt1 inhibitors and potentiated the antiallodynia effect. glycine receptor antagonist, strychnine by i.t. injected reversed the antiallodynia effect of i.v. injected glyt inhibitors. these results suggest that both glyt1 and glyt2 inhibitors by enforcing glycinergic inhibitory neurotransmission in spinal cord produce potent antinociceptive effect and may be novel candidate for medicament of pain control. the tumour microenvironment in particular tumour associated macrophages (tams) play a role in determining tumour outcome. despite strong causative links between inflammation and human gastric cancer progression, little is known of the role of tams in this disease. we have utilized our mouse model of gastric tumourigenesis, the gp130757ff mouse to assess the effect of the gp130757ff mutation on macrophage function and ascertain the role of macrophages in tumor formation. this mouse has a knockin mutation in the il-6 family cytokine receptor gp130 preventing shp2/ras/erk signaling and leading to constitutive stat3 transcriptional activation. tumour development is inflammation dependent, there is a requirement for il-11, and development is inhibited by nsaid treatment or microbial eradication, however an adaptive immune response is s410 inflamm. res., supplement 3 (2007) posters dispensable for tumorigenesis. in gastric antrum the gp130757ff mutation results, decreased erk1/2 activation and constitutive phosphorylation of stat3, abnormal signaling is replicated in macrophages. antral stat3 activation is unaffected by depletion of il-6. however in macrophages an absence of il-6 results in higher stat3 activation demonstrating the anti-stimulatory role of il-6 on macrophages. the gp130757ff mutation results in macrophages with decreased il-6 and increased inos mrna expression reflecting a more basally activated phenotype. manipulations of the gp130757ff mouse that reduce tumor size (eg. antibiotic or nsaid treatment or stat3 hemizygosity) coincidently result in reduced macrophage infiltration of antral mucosa. macrophages of gp130757ff mice display aberrant gp130 signaling potentially resulting in exaggerated response to stimuli. the key difference between mutant gastric mucosa and macrophages are changes in transcription of target genes. (1), y riffo-vasquez(2), s brain(2), s costa (1) (1) university of sâ¼o paulo, brazil (2) king's college london, uk objectives: we have previously shown that simultaneous intra-tracheal injection of diesel exhaust particles (dep) and 1,2-naphthoquinone (1,2-nq) caused a potent inflammation in rat airways partially dependent on neurogenic-mediated mechanism. this study investigates the mechanism of action of thee pollutants using inflammatory assays and histopathological approach in the lung and trachea of wild type (wt) and trpv1 knockout (ko) mice exposed to dep and 1,2-nq. dep (1 mg/kg) and 1,2-nq (35 nmol/kg)-induced airway inflammation was assessed via 125i-labelled albumin 1 h post pollutants injection. mpo assay and histopathology were performed 3 h after treatment. staining of lung and trachea specimens with h&e provided a profile of cell infiltration in both wt and ko animals. results: injection of dep and 1,2-nq evoked a potent plasma extravasation into the trachea and lung of wt, but not trpv1 ko, suggesting these pollutants act via a trpv1-mediated mechanism. in contrast, mpo activity in the airways of trpv1 ko mice was exacerbated compared to wt mice data. likewise, the histopathology revealed high numbers of leukocytes and macrophages infiltrated into the lungs and trachea of trpv1 ko mice compared to wt. conclusions: inhibition of increased microvascular permeability in the airways of trpv1 ko mice treated with pollutants suggests that these receptors are the predominant mechanism involved in the inflammation. however, neutrophils/macrophages accumulate more in trpv1 ko, indicating that the lack of trpv1 receptors up-regulates production of inflammatory cells in response to pollutants, thus supporting a protective role for trpv1 receptors. acknowledgements: capes, cnpq, fapesp. (1), n sato(1,2), y endo (1), s sugawara (1) (1) division of oral immunology, department of oral biology, tohoku university graduate school of dentistry, sendai, japan (2) division of fixed prosthodontics, department of restorative dentistry, tohoku university graduate school of dentistry, sendai, japan biotin is a water-soluble vitamin of the b complex and functions as a cofactor of carboxylases.biotin deficiency causes alopecia and scaly erythematous dermatitis.moreover, serum biotin levels are significantly lower in atopic dermatitis patients than in healthy subjects, indicating that biotin deficiency is involved in inflammatory diseases.however, immunological effects of biotin on allergic inflammation remain unclear.in this study, we investigated the effects of biotin-deficiency on metal allergy using nickel (ni)-allergy model mouse and a murine macrophage cell line j774.1. female balb/c mice (4 weeks old) received a basal diet or a biotin-deficient diet for 8 weeks.ten days after sensitization with intraperitoneal injection of lps and nicl2, the mice were challenged with intradermal injection of nicl2 into the pinnas.allergic inflammation was measured by ear swelling.the ethical board for nonhuman species of the tohoku university graduate school of medicine approved the experimental procedure followed in this study.j774.1 cells were cultured in biotin-sufficient or deficient medium for 4 weeks. ear swelling was significantly higher in biotin-deficient mice than biotin-sufficient mice.il-1 beta productions by splenocytes were significantly higher in biotin-deficient mice than in biotinsufficient mice.moreover, biotin-deficient j774.1 cells produced il-1 beta significantly higher than biotinsufficient j774.1 cells.to investigate the therapeutic effects of biotin-supplementation, biotin-deficient mice received biotin contained-water for 2 weeks.ear swelling was significantly lower in biotin-supplement mice than biotin-deficient mice.these results indicated that biotindeficiency deteriorates allergic inflammation.the augmentation of il-1 beta production is probably involved in those deteriorations. one of the most important targets of cytokine action is the blood vessels, which undergo some structural and functional changes that result in activation of endothelium. applying the elisa technique, levels of il-18, icam-1 and e-selectin were studied in 77 patients with acute pancreatitis. mediators levels were studied in arterial, venous and pancreatic ascites samples. according the atlanta criterion the mild pancreatitis was established in 33 patients and severe -in 44 patients. the highest levels of il-18 were noted in ascites and lowest in arterial samples. the highest concentration of adhesion molecules was in venous samples and lowest in ascites. it was a clear correlation between levels of il-18 and adhesion molecules and severity of pancreatitis. during first week the levels of il-18 gradually increased in patients with severe pancreatitis, while in patients with the edematous pancreatitis its levels decreased starting from the third day. icam-1 levels gradually increased during first three day with the following decrease after this term. the highest levels of e-selectin were noted at the time of admission. the clear correlation between il-18 and adhesion molecules was noted in both groups of patients. besides that, the clear strong correlation was observed between il-18 and quantity of circulating granulocytes and between e-selectin and hematocrite in patients with necrotizing pancreatitis. our study confirms the importance of activation of endothelium as a part of the systemic inflammatory response in patients with acute pancreatitis. subsequently eos infiltrate the tissues, are activated, and release mediators inducing the late phase response. this is characterized by tissue damage and repair, and in chronic reactions, tissue remodeling and fibrosis. mc/eos cross-talk by physical and non-physical contact is an essential feature of late-phase and chronic allergic reactions. we have previously described an mc/eos interaction facilitated by soluble mediators and shown to enhance allergic inflammation. still, pathways that mediate mc/eos cross-talk in allergy are not fully characterized. methods: human cord-blood derived mc were cocultured with peripheral blood eos, and activated with anti-ige. mc/eos couples in co-culture and in human nasal polyp tissue sections were specifically stained and counted using microscopy. expression of surface molecules was analyzed by facs. mc activation was measured by chromogenic assays for ã¢-hexosaminidase. relevant surface molecules were neutralized using antibodies, to assess interference with couple formation and activation. results: mc and eos physically interact, forming welldefined couples in-vitro and in-vivo. in the presence of eos, mc are more releasable under baseline or igeactivating conditions. this effect is partially mediated by cd48 and dnam-1 on mc, and 2b4 and nectin-2 on eos. we describe a novel physical interaction between mc and eos that we name "the allergic synapse". this synapse may upregulate allergic reactions, thus serving as a target for therapeutic intervention in allergic and inflammatory diseases. methods: mc were obtained from cord blood mononuclear cells (6-8 weeks with scf, interleukin-6 and prostaglandin e2, cbmc). cbmc were activated, after their culture for 5 days with myeloma ige (2mg/ml), by rabbit anti-human ige antibodies (5mg/ml), for 1, 3 and 6h at 378c. activation was measured by b-hexosaminidase release, determined by enzymatic-colorimetric assay. the expression of flip, mcl-1, bcl-2, bcl-xl, bak and bax was assessed by immunoblot analysis. results: two anti-apoptotic proteins were found to be upregulated: flip, which is involved in the extrinsic apoptotic pathway and mcl-1 that is mainly implicated in the intrinsic apoptotic pathway. in contrast, the expression of two other anti-apoptotic proteins that we have examined (i.e. bcl-2 and bcl-xl) was not altered. likewise, the expression of pro-apoptotic proteins from the bcl-2 family (i.e. bak and bax) was either undetectable or unchanged. conclusions: our findings reveal that ige-dependent activation of human mc mainly induces the selective increase in the expression of two pro-survival molecules. this may be one of the mechanisms that underlay mc hyperplasia in the chronic allergic inflammation. (1), c armishaw(2), z yang (1), h cai (1), p alewood(2), c geczy (1) (1) university of new south wales, faculty of medicne, sydney, australia (2) university of queensland, institute for molecular biosciences, st lucia, australia purpose: human s100a8, s100a9 and s100a12 are closely related proteins associated with inflammation. s100a12 is expressed in the human genome, but not in the mouse. s100a12 is a potent monocyte chemoattractant and mast cell (mc) activator. mouse (m) s100a8 and human (h) s100a8 share 58% structural identity, but the hinge domains are more divergent. the ms100a8 hinge (ms100a842-55) is a potent chemoattractant for leukocytes whereas this sequence in hs100a8 (hs100a843-56) is inactive. methods: s100a12 hinge domain and its alamine scan mutants were synthesized and their activities were tested by using thp1 cells or murine mc in vitro and mouse footpad injection. results: s100a12 hinge domain (s100a1238-53) was chemotactic for monocytes and mc and provoked mc degranulation in vitro and oedema, and leukocyte recruitment in vivo. in contrast to s100a12, the hinge domain only weakly induced cytokine production (il6, il8) by macrophages. residues essential for oedema were hydrophobic in nature (leu40, isoleu44, isoleu47 and isoleu53). n42 and i44 were essential for responses provoked by 10-12m s100a1238-53 whereas mutation of k38, l40, n46, i47, d49, k50 and i53 significantly reduced migration with 10-9m s100a1238-53. conclusions: s100a12 and ms100a8 may be functional chemattractant equivalents; s100a12 may have arisen by duplication of human s100a8. isoleucine residues in s100a12 hinge domain are essential for its proinflammatory properties. (1), g kiriakopoulou (1), e tsimara(2), a voultsou(2), k zarkadis (1) (1) general hospital of zakynthos, greece (2) medical centre of katastari, zakynthos, greece schistosome is a disease which pests in 74 tropical countries. the endemic areas are south america, far and middle east, and africa. our aim is to present an incident which concerning a parasitic infection, not endemic in greece. patient: man 30 years old who immigrated from pakistan (at the side of the aparkenar river) in greece, a year ago. he turned out with anlage, headache and weight loss. he is a swimmer. he mentioned also a fever before migrating to greece. clinically: largely-scaled paleness, stomach -mild and diffused sensitivity, with also lightly increased enteric sounds and a small degree of hepatomegaly, when palpated deeply. laboratory examination: leucocytes 9.030 eo 24.8%, hb 8.2g/dl, ht 27.2%, mcv 65,6fl, mch 19.1pg, thrombocytes 419.000, blood sedimentation 10mm, fe 10mg/ml, ferritin 2.98ng/ml. normal biochemical examinations. u/s: livers size lightly increased 16.5mm, hepatoportal vein with normal amplitude. parasitology of feces: in the immediate confection with lugol of the second sample, there were observed: big scattering oval ovules (130 -60mm) with a thin wall and quills on the side, as well as three imago worms (>10 mm): schistosoma mansoni. treatment: praziquantel was given. recheck in a months time: blood examinationleucosites 11.230, eo 9.8%, hb 10,2g/dl, ht 33.6%. parasitology of feces is negative. in the diagnosis of anemia combined with fever, to patients who are immigrants, schistosomiasis posters inflamm. res., supplement 3 (2007) should be taken into account, especially when sideropenic anemia is accompanied with intense eosinophile, because the disease is mostly a reaction of retardate supersensitivity. bronchial asthma is a chronic airway inflammatory disease caused by immune cells such as t lymphocytes and eosinophils. recently, high-sensitivity crp (hs-crp) has become available for detecting small changes in crp levels within the normal range, allowing for assessment of subclinical inflammation. this study was undertaken to investigate the relationship between hs-crp and bronchial asthma. we collected blood samples from 109 patients with bronchial asthma with or without attacks and measured serum eosinophil cationic protein (ecp) and pulmonary function as well as serum hs-crp. serum crp levels in patients without attacks (average 0.473 mg/ l; p < 0.001) and with attacks (average 0.908 mg/l; p < 0.001) were significantly higher than those of normal controls (average 0.262 mg/l). serum hs-crp levels were inversely correlated with fev1.0% in asthmatic patients (r = -0.4915, p < 0.01). in conclusion, these results indicate that serum hs-crp as well as ecp may be related to the state of asthma exacerbation and allergic inflammation. objectives: the pshr assay can be used to test the biologic activity of allergens since it mimics the effector phase of a type i hypersensitivity reaction. in this study we tested methods for removing ige-antibodies (stripping) from donor basophils. moreover a method was developed for improving the antigen specificity profile in pshr. proof-of-concept was provided using absorption with complete allergen extracts. methods: buffy coats were screened to exclude reactivity against relevant allergens. subsequently pbmcs were purified and basophils were stripped with either lactate or a phosphate buffer. cells were passively sensitized with patient sera and challenged with allergen extracts in various concentrations. released histamine was measured spectrofluorometrically (hr-test, reflab). cutoff was 10% hr. for absorption experiments patient sera (from a peanut allergic and a codfish allergic patient) were incubated with streptavidin-coated sepharose beads coupled with biotinylated allergens (peanut, and bsa as control). after centrifugation the supernatant was used to passively sensitize stripped basophils. hr was measured as described above. results: stripping experiments using the two buffers only partially removed surface ige but passive sensitization of stripped basophils was equally effective enabling determinations of sub-nanogram quantities of peanut allergen. absorption experiments showed that it was possible to specifically remove peanut specific ige from patient serum. removal of specific ige reactivity to peanut extract was verified by western blotting. conclusions: using peanut extract as a model it was demonstrated that in pshr antigen specificity can be modified. (1), sk bk(1), v sharma(2), rp bhandari (3) (1) nepal medical college teaching hospital, nepal (2) all india institute of medical sciences, new delhi, background: nepal has one of the highest maternalmortality rates in the world. this study was to evaluate the incidence, disease pattern, and risk-factors for thromboembolism in pregnant nepalese women. methods: women with thromboembolic diseases were identified and their case records retrieved and reviewed s414 inflamm. res., supplement 3 (2007) posters from january 1998 to december 2006. demographiccharacteristics were compared between women with and without thromboembolism. the total number of deliveries over the study period was 16,993, giving an incidence of 1.88 per 1000 deliveries. there were two cases of pulmonary-embolism and one resulted in a maternal-death. the others had deep-vein-thrombosis of which over 80% were limited to calf veins only. the ultrasound-examinations requested for suspected deep-venous-thrombosis before and after the event of maternal-death were 1.62 and 10.7 per 1000 deliveries (p <.001);the corresponding cases of deepvenous-thrombosis diagnosed were 0.29 and 2.94 per 1000 deliveries, respectively (p <.001). the majority (75%) of cases were diagnosed in the postpartum period, mainly after cesarean-delivery. women with venous-thromboembolism were older, had higher bmi, and a higherincidence of preeclampsia. there were approximately twice as many postpartum as antepartum events. bloodgroup a, multiple-pregnancy, caesarean-section, cardiacdisease, delivery at gestational-age of <36 weeks, a bmi of25, or more and maternal-age of 35 or over were all found to increase incidence of venous-thromboembolism. the long-standing belief that thromboembolism is rare among nepalese is at least partly because of undiagnosis most of these events are deep-veinthromboses occurring in the postpartum period and it is very essential for primary prevention developing country like nepal. (1), ch ladel (1), t ruckle(2), c rommel(2), r cirillo (1) (1) rbm-merck serono, colleretto giacosa, italy (2) serono pharmacological research institute, geneva, switzerland rheumatoid arthritis (ra) is a severe articular disease. massive leukocyte activation and infiltration into joints result in cartilage and bone destruction. blockade of pi3k signalling pathway has been demonstrated to be curative in a murine model of ra, collagen-induced arthritis (cia). in this study we explored the molecular mechanisms by which pi3k signalling inhibition resulted in clinical amelioration of disease symptoms. as605858, a novel isoform non-selective, yet specific class-i-pi3k inhibitor, administered at 15 mg/kg twice-a-day for 8 days, to mice showing signs of arthritis, paw swelling and inflamed digits, induced a significant amelioration of disease course. at the end of treatment, post-arthritic paws were removed and phosphrylation levels of akt (p-akt), downstream target in pi3k-mediated signal, were determined by semi-quantitative immunohistochemistry, also immunophenotyping of circulating cells by flowcytometry was conducted. akt phosporylation resulted to be significantly enhanced by disease induction and as605858 was able to decrease its levels down to values comparable with naã¯ve animals. controls and as605858treated mice were bled before treatment, after two days of treatment and at treatments end. no changes in cellular composition (morphology and hematology parameters) between experimental groups were observed. t cell number was not affected, however a significant decrease of natural-killer, memory and regulatory t cells was observed after as605858 administration. finally, a non-significant, moderate reduction of bcell number was also observed. these data demonstrate that efficacy of as605858 in arthritis models is mediated by direct modulation of the target resulting in a mixed anti-inflammatory (via pi3kg) and immunosuppressive (via pi3kd/a/b) activity. (1) (1) institute of biomedical science, university of sao paulo, brazil (2) ibilce -sao paulo state university, brazil epidemiologic data suggest that female sex hormones are involved in the pathophysiology of allergic asthma. we investigated in rats the immunomodulatory potential of estradiol and progesterone on the expression of allergic asthma. seven days after being ovariectomized (ovx), groups of rats were sensitized with ovalbumin (oa). fourteen days after sensitization, the animals were oachallenged and used 1 day thereafter. allergic,shamoperated animals were used as controls. some ovx animals were treated with estradiol (280 ã¬g/kg) or progesterone (200 ã¬g/kg) 24 h before being challenged. mast cell degranulation was quantified in samples of isolated, oa-challenged bronchi. the airway reactivity of inner bronchi to methacholine and the functional activity of cells we analysed. ovx caused reduction of the allergic lung inflammation and bronchial hyperresponsiveness with regard to intact female rats. estradiol reverted the reduced cellular recruitment into lungs, whereas progesterone reduced the pulmonary inflammatory response and reverted the bronchial hyperresponsiveness. cultured bal and bone marrow cells from allergic rats increased posters inflamm. res., supplement 3 (2007) s415 the release of il-10 and reduced that of il-1 and tnf. the release of il-4 by bone marrow cells was significantly reduced. these effects were reverted by estradiol, and progesterone reduced il-4 and increased il-10 production in bal and increased that of il-1 and tnf in bone marrow cells. bronchial mast cell degranulation upon direct contact with oa in ovx rats was less than in controls. it is suggested that female sex hormones can modulate the allergic lung inflammation in rats by acting on cellular migration/activity and airway responsiveness. objectives: to study the participation of fsh on modulation of e-and l-selectin, icam-1 and mac-1 expression in ovariectomized (ovx) rats made allergic. methods: female rats were sensitized (oa/alumen) after 1 (ovx-1) or 7 days (ovx-7) of ovx or sham-operated (sh). fourteen days thereafter animals were challenged (oa, 1%; aerosol) and sacrificed 24 h after. bronchoalveolar lavage (bal) was collected and flow citometry analyses oficam-1, mac-1 and l-selectin expression was studied. in parallel, lungs were frozen and sections were analysedfor e-selectin expression by immunohistochemistry. results: at day 1, e-selectin expression increased(ovx-1=1,8ae0,08) and at day 7, decreased (ovx-7=1,3ae0,1) as compared to respective controls (sh-1=1,45ae0,08; sh-7=1,9ae0,08). estrogen treatment reverted this profile in both groups (ovx-1+e=1,37ae0,09; ovx-7+e=1,8ae0,1). mean fluorescence intensity of bal cells showed increase of mac-1 expression (ovx-1=19ae0,5 vs sh-1=15,5ae1,0), icam-1 (ovx-1=18,1ae1,1 vs sh-1=13,3ae0,2) and l-selectin (ovx-1=11,2ae0,8 vs sh-1=9,5ae0,12) at day 1 i.e., ovx-1 group. on the other hand, we observed a decrease in icam-1 (ovx-7=14,2ae0,75 vs sh-7=19ae1,4) and mac-1 expression (ovx-7=20,7ae1,4 vs sh-7=27,5ae1,5) was seen in ovx-7 group. conclusions: oscillation of hormone levels during immunization with oa increased (ovx-1) and decreased (ovx-7) expression of adhesion molecules. estradiol treatment reverted this effect. this results suggest that fsh modulates the ali in rats by acting on cell (1), s lim (1), y lin (1), bp leung (1), c thiemermann (2), wsf wong (1) (1) national university of singapore (2) the william harvey research institute, london, uk glycogen synthase kinase 3b (gsk-3b) is known to regulate various cellular functions including inflammatory responses. we hypothesized that inhibition of gsk-3b may have anti-inflammatory effects in a mouse asthma model. balb/c mice were sensitized with ovalbumin (ova) and challenged with aerosolized ova. tdzd-8, a non-atp competitive gsk-3b inhibitor, was administrated by i.v. injection one hour before ova challenge. tdzd-8 significantly reduced the ova-induced eosinophilia in a dose-dependent manner and inhibited the levels of il-5, il-13 and eotaxin in bronchoalveolar lavage (bal) fluid. tdzd-8 also suppressed the mrna levels of icam-1, vcam-1 and chitinase proteins in the lung. histological studies revealed that tdzd-8 substantially reduced the inflammatory cell infiltration and mucus secretion in the lung tissue. tdzd-8 also decreased ova-specific ige level in the serum. in addition, ova-induced increase in airway resistance and reduction in dynamic compliance were attenuated by tdzd-8. our findings suggest that inhibition of gsk-3b may have therapeutic potential for the treatment of allergic airway inflammation. (1), a yildirim (1), f ercan (2), n gedik (3), m yuksel(4), inci alican (1) ( materials and methods: sprague-dawley rats (200-250 g) were exposed to 90 oc (burn group) or 25 oc water bath (control group) for 10 s under ether anesthesia. adm (100 ng/kg; s.c) was administered 10 min before burn and all rats were decapitated 24 h after the burn insult. trunk blood was collected for the measurement of tnf-alpha level and the lung, ileum and kidney samples were stored for microscopic scoring and for determination of lipid peroxidation (lp), myeloperoxidase (mpo) activity and formation of reactive oxygen metabolites (roms) using chemiluminescence assay. results: burn resulted in severe morphologic damage in tested tissues. lp increased in lung and kidney (p<0.01-0.001) and mpo activity showed a marked increase in all tested tissues (p<0.001) of the burn group. adm reversed these parameters effectively (p<0.05-0.001). luminol chemiluminescence levels showed increases in both ileum and lung (p<0.01-0.001) whereas lucigenin chemiluminescence levels increased in ileum and kidney (p<0.01) of the burn group. adm treatment was also beneficial in reducing chemiluminescence levels near to controls (p<0.01). adm reduced plasma tnf-alpha level (p<0.01) which showed a significant increase in burned animals compared to controls (p<0.001). conclusions: adm is beneficial in remote organ damage following burn insult via decreasing neutrophil infiltration, rom generation, lp, and the release of proinflammatory cytokine tnf-alpha. kalpana panday (1), sd joshi (2), kr reddy (3) ( we determined the crystal structure of human hematopoietic prostaglandin (pg) d synthase (h-pgds) as the quaternary complex with glutathione (gsh), mg2+, and an inhibitor, hql-79, with anti-inflammatory activities in vivo, at 1.45 resolution. hql-79 was found to reside within the catalytic cleft between trp104 and gsh in the quaternary complex. hql-79 inhibited h-pgds competitively against the substrate pgh2 as well as noncompetitively with respect to gsh. surface plasmon resonance analysis revealed that hql-79 bound to h-pgds with an affinity that was 10-fold higher in the presence of gsh and mg2+ than in their absence. hql-79 inhibited selectively pgd2 production by human h-pgds-expressing megakaryocytes but only marginally affected the production of other prostanoids, suggesting the tight functional engagement between h-pgds and cyclooxygenase. orally administered hql-79 inhibited antigen-induced production of pgd2, and airway inflammation in mice without affecting the production of pge2 and pgf2fâ¿. knowledge about this quaternary structure should accelerate the structure-based development of novel anti-inflammatory drugs that inhibit pgd2 production specifically. introduction: it has been proved that high levels of mechanical ventilation produce lung injury as well as local inflammation. this study was designed to evaluate how the generation of inflammatory mediators by an over-stretched lung affects the non-hyperventilated lung. methods: male wistar rats (250-275g) were anesthetized and paralyzed, and the two lungs were independently intubated. differential ventilation was applied for 3h (70 breath/min). one lung was subjected to hyper-ventilation (20ml/kg/lung) and the other was ventilated with a normal volume (5ml/kg/lung). in a control group, both lungs were ventilated with a normal volume. after sacrifice, samples of lung, plasma and liver were collected. the expression of the pro-inflammatory chemokine mip-2 was evaluated by rt-pcr and the edema was assessed by the ratio between the wet and dry weight of the lung. systemic inflammation was estimated in liver by measuring the expression of tnfa by rt-pcr as well as its levels in plasma. the hiper-ventilated lung showed an increase in the ratio between the wet and dry weight and in mip-2 expression compared to the normal ventilated lung. no differences were found in edema, neither expression of mip-2 between the normally ventilated lung and control lungs. no significant changes were observed in liver expression and plasma levels of tnfa as a consequence of unilateral lung hyper-ventilation. the over-straining caused to the hyperventilated lung leaded to a local inflammatory response without systemic effects. the normal ventilated lung is not affected by the inflammatory process triggered on the over-strained lung. (1), j-y gillon(2), v lagente (1), e boichot (1) (1) university of rennes 1, rennes, france (2) serono international s.a., geneva, switzerland macrophage elastase (mmp-12) is a metalloproteinase involved not only in emphysema but also in the inflammatory process associated with copd (chronic obstructive pulmonary disease). the mechanism of action of mmp-12 in the development of pulmonary inflammatory process is still unknown. in the present study, we investigated the effect of recombinant human mmp-12 (rhmmp12) on il-8/cxcl8 release from alveolar epithelial cell line a549 and we explored the underlying mechanisms. a549 cells were stimulated with rhmmp-12 (1.10-3-2.10-1 u/ml) during 6 hours and il-8/cxcl8 level in supernatant was determined by elisa. involvement of map (mitogen activated protein)-kinases was studied by western-blotting and also using chemical inhibitors. nfkb activation was examined with the transam nfkb p65 kit. we observed that mmp-12 elicited il-8/cxcl8 release in a dose-dependent manner. this production could be prevented by a pretreatment for 1hour with a selective mmp-12 inhibitor (as111793 1-30 mm) or with the nonselective inhibitor batimastat (1-30mm) . the il-8/cxcl8 production was also inhibited by actinomycin d (5mg/ml), erk1/2 inhibitors (u0126 5mm and pd98059 10mm) and nfkb inhibitors including bay11-7082 (10mm) and nfkb activation inhibitor (10nm), whereas p38 kinase inhibitor (sb203580) had no effect. stimulation with mmp-12 was rapidly followed by a phosphorylation of erk1/2 (5min) and an nfkb nuclear translocation and activation (1h). the nfkb activation was not inhibited by a treatment with u0126. these data suggest that alveolar epithelium is a target of mmp-12 since it upregulates gene expres-s418 inflamm. res., supplement 3 (2007) posters sion and release of il-8/cxcl8, via erk1/2 and nfkb activation, but these two pathways appears to be distinct. agents which are associated with lung inflammation, such as cigarette smoke and lipopolysaccharide (lps), induce the production of pro-inflammatory chemokines in lung epithelial cells in vitro, and the induction of interleukin (il)-8, in particular, is often used a measure of relative toxicity.in this study we have compared mrna expression and mediator release in nci-h292 human lung epithelial cells exposed to lung toxicants, namely: cigarette smoke total particulate matter (tpm), lps, bleomycin, diesel exhaust particles, residual oil fly ash (rofa), carbon black and vanadyl sulphate.polystyrene, poly(methyl-methacrylate) and the tpm vehicle, dimethyl-sulphoxide were used as negative controls.confluent monolayers of h292 cells were exposed to serial dilutions of test agents in serum-free medium for 24 hours.the conditioned medium was then removed and assayed for a range of pro-inflammatory cytokines and other selected mediators by luminex technology.the levels of gene expression of il-8, matrix-metalloprotease-1 (mmp-1), the gel-forming mucin muc5ac, heparinbinding epidermal growth factor-like growth factor (hb-egf) and the cytochrome p450s cyp1a1 and cyp1b1 were determined by quantitative-polymerase chain reaction.all of the toxicants induced similar responses whereas the negative controls were largely ineffective.-such a panel of biomarkers may enable an in vitro assessment of the potential to cause lung inflammation.-moreover the use of several biomarkers could give a more accurate picture of toxicity than the determination of il-8 alone, particularly in the case of agents such as tpm, where the conventional vehicle is found to have some biological activity. respiratory tract infections are a major public health issue. prevention in high risk populations relies mainly on vaccination. vaccination is highly recommended in decrease absenteeism. immunomodulating drugs are important tools in the treatment of infectious diseases. immunomodulatory agents are probably contributive in decreasing exacerbation rates. the authors present different classes of immunomodulators that are currently in use. the vaccine, created from a bacterial protein, reeducates the immune system to stop inflammation. by preventing infections, vaccines prevent the development of a strong t helper (th1) response. the challenge is now to inform about new possibilities of an optimal prevention respiratory tract infections. deoxypodophyllotoxin (dpt) is a medicinal herbal product that is isolated from anthriscus sylvestri. that inhibits cyclooxygenase-2 (cox-2) and cox-1dependent phases of prostaglandin d2 (pgd2) generation in bone marrow-derived mast cells (bmmc) in a concentration-dependent manner with ic50 values of 1.89mm and 65.3mm, respectively. this compound inhibited cox-1 and 2-dependent conversion of the exogenous arachidonic acid to pgd2 in a dose-dependent manner with an ic50 values of 0.01mm and 12.1mm, respectively. however, dpt did not inhibit cox-2 protein expression up to a concentration of 30mm in the bmmc, indicating that dpt directly inhibits cox-2 activity. furthermore, this compound consistently inhibited the production of leukotriene c4 (ltc4) in a dose dependent manner, with an ic50 value of 0.37mm. these posters inflamm. res., supplement 3 (2007) s419 results clearly demonstrate that dpt has a dual cox-2/5-lox inhibitory activity in vitro. therefore, this compound might provide the basis for novel anti-inflammatory drugs. in order to determine anti-allergic and antiasthmatic activity of dpt in vivo, we used rat pca model which was activated by anti-dinirophenyl ige and ovalbumin/alum induced mouse asthmatic animal model, respectively. as a result, dpt strongly inhibited pca reaction as well as it reduced infiltrated eosinophil numbers in bronchoalveolar lavage fluid. furthermore, dpt decreased the mrna levels of the th2 cytokines in a murine asthmatic model. in addition, northern blot analysis showed that dpt also reduced both the eotaxin and arginase i mrna levels in a dose dependent manner. these results suggest that dpt may be beneficial in regulating various inflammatory diseases. an imbalance of proteases and anti-proteases in the lung has been implicated in the pathogenesis of chronic obstructive pulmonary disease (copd), a smokingrelated disorder associated with accelerated lung function decline.in particular, the activity of matrix metalloproteases (mmps) have been implicated in driving both the inflammation and parenchymal destruction observed in copd patients.here, we tested whether a broad spectrum mmp inhibitor, pkf-242484, could block the inflammation induced by an acute exposure to cigarette smoke in 2 strains of mice, balb/c and c57/bl6.animals were administered the compound (1-10 mg/kg) either per os (p.o.) or intranasally (i.n.) 1 hour before and 5 hours after exposure to smoke on three consecutive days.bronchoalveolar lavage (bal) was performed and lungs were flash frozen for inflammatory marker analysis.pkf-242484 dose-dependently reduced lung neutrophilia in balb/c mice when dosed either p.o. (~45% at 10 mg/kg; p < 0.01)or i.n. (~60% at 10 mg/kg; p < 0.01).however, the compound had no clear effect on bal neutrophil infiltration in c57/bl6 mice by either route of administration.interestingly, in both strains bal macrophages dose-dependently trended towards an increase when the compound was dosed p.o. and decreased when dosed locally (p < 0.05). examination of lung tissue cytokine levels revealed that while smoke-exposure increases il-1beta, kc, and mip-2, pkf-242484 had little effect on these cytokines.these data suggests the ability of broad spectrum mmp inhibitors to inhibit smoke-induced acute neutrophil inflammation is strain-dependent, while its ability to limit macrophage infiltration may be route dependent. to investigate the role of seh in the regulation of the pulmonary inflammatory response, we have used seh deficient mice in a locally administered lps model. male seh deficient mice (ko) and their wildtype (wt) littermates were exposed to inhaled lps.four hours later they were sacrificed and bal was performed. differential counts and cytokine levels in bal were evaluated. results: lps induced a significant increase in total cell number and neutrophil number in bal in the seh deficient mice and in wt mice;no significant differences between the groups were seen (table 1 ) cytokine analysis showed significant increases in tnfalpha, il-6, kc, gm-csf, mcp-1, il-1beta and rantes in the lps-exposed wt mice. no significant differences were seen between lps exposed wt and ko mice except for a significant increase in tnfa in ko mice. our results show that seh has no pivotal role for the regulation of the acute inflammatory response to lung administered lps. fam.liliaceae. based on literature data which signaled the presence of steroidic saponins in the rhyzomes, isolation, identification and quantitative determination of these compounds were done. the antiinflammatory activity was tested in non-immune chronic inflammation model:the cotton-pellets granuloma test in rats, and in an immune chronic inflammation model:arthritis test induced by freunds adjuvant in rats. in the first test, the antiinflammatory effect of steroidic saponins 600 mg/kg is weak, statistically insignificant. in the arthritis test, steroidic saponins 600 mg/kg proved an antiinfalammatory activity, influencing especially the primary response, but also the secondary one, in the last part of the experiment (after 16 days). in both tests, the suprarenal glands weight was modified. objectives: dendritic cells (dcs) are professional antigen presenting cells. many types of dc with subtle difference in phenotype have been reported in several organs. existence of dc was reported in synovial tissue of rheumatoid arthritis (ra), however, the details in ra dc still remains unclear. in this study, we generated a new lineage of dc with gmcsf (+tnfa) and investigated their functions. furthermore the ability of osteoclastgenesis was examined. methods: monocyte-derived dcs or macrophage were generated in (1) tnfa + gm-csf (2) gm-csf (3) il-4 + gm-csf (4) mcsf. the phenotypes of these cells were analyzed by morphological examination and flow cytometry (facs calibur). cell proliferation was examined by wst-8 assay. dc functions were assessed in antigen presenting ability (mlr assay), cytokine production (elisa), and endocytosis (fitc-dextran uptake). concerning osteoclastgenesis, monocyte-derived dcs were incubated with rankl and mcsf. trap stain was performed and the resorption ability was assessed on osteologic cell culture system and dentine slices. results: these cells were dendritic-like and their surface markers were cd1a low cd11b + cd11c + cd14 + cd86 + cd83 low hla-dr + dc-sign low and different from conventional dc or macrophage. they had an antigen presenting ability to induce na*ve cd4+ t cell proliferation, il-12 production and endocytosis. in the presence of rankl and mcsf, they differentiated multinuclear trap-positive cells with bone resorption ability, which was strengthened by tnfa. we generated a new lineage of dc with gm-csf (+ tnfa). the dc seemed to play a pivotal role in inflammatory arthritis under tnf immunity. the clinical effectiveness of rituximab and other b cellattenuating rheumatoid arthritis (ra) therapies has increased interest in understanding the role of b cells in ra pathogenesis.the possible mechanisms underlying the effectiveness of rituximab were investigated by performing biosimulation research in the entelos ra physiolab platform, a mathematical model of the joint of an ra patient.the platform dynamically integrates the contributions of immune cells (t cells, b cells, and macrophages), resident cells (fibroblast-like synoviocytes and chondrocytes) and mediators to the joint inflammation and structural damage observed in ra.the b cell lifecycle is represented in the platform, as well as effector functions such as antigen presentation, mediator and autoantibody production, and immune complex formation.the dynamics of these b cell properties were calibrated to reproduce reported experimental behaviors and clinical outputs from ra patients (e.g., acr score and radiographic progression rates).an assessment of the contribution of individual b cell functions to clinical outcome suggests that plasma cell-derived immune complexes are key modulators of inflammation in ra patients. in contrast, proinflammatory cytokine production by b cells contributes minimally to synovial hyperplasia, but plays a role in the progression of structural damage.immune complex formation leads to monocyte activation, increased mediator production by macrophages and an increase in antigen availability to t cells.biosimulation research in the ra physiolab platform is advancing our understanding of the mechanisms underlying effective b cell-targeting ra therapies, and may guide the development of improved second-generation therapeutic approaches. methods: the hmscs were obtained at the operation.the mononuclear cells were extracted and the colony forming assay were performed after 2weeks. the hmscs were cultured and in passage1,the cell surface antigens of both groups were analyzed by flow cytometory.in passage2, in control group, the cells were cultured with beta glycerophosphate(bgp) and in osteogenic group, the cells were cultured with bgp and ascorbic acid(aa) and dexamethasone(dex).after 2weeks, alp staining and activity were measured in each group.after 3weeks, alizarin red s assays were performed.rna was extracted from the cells cultured with bgp and aa and dex for 2weeks and 3weeks and the gene expressions of bone formation markers were examined by real time pcr. the mann-whitney test was used for the statistical analyses. the colony forming assays showed no significant differences in oa and ra. in flow cytometory, the cell surface antigens in oa and ra were almost same.in alp activity,there were no significant differences in oa and ra.in alizarin red s, there were significant differences.in real time pcr,the gene expressions showed no significant differences in oa and ra. conclusions: the hmscs of ra will be able to use for regenerative therapy. silje vermedal hã¸gh (1), hm lindegaard (2), gl sorensen (1), a hã¸j (3), c bendixen (3), p junker (2), u holmskov (1) ( cytosolic phospholipase a2 (cpla2) plays a crucial role in eicosanoid production, by releasing an arachidonic acid from membrane phospholipids. in addition, cpla2 regulates the phagocyte nadph oxidase-releasing superoxides and that is the only isozyme responsible for the production of eicosanoid in phagocytic cells. collageninduced arthritis (cia) in mice is an experimental model of auto-immune diesease with several clinical and histological features resembling rheumatoid arthritis in humans. previous studies show that cpla2-deficient mice are resistant to cia. thus we aimed to study whether cpla2 is up-regulated during the development of cia and to detect its exact location in the inflamed joints. immunoblot analysis revealed an increase in the level of joint cpla2 protein during the development of the disease which correlates with the severity of the inflammation, as examined by paw thickness. immunohistochemistry with specific anti-cpla2 antibodies revealed low positive cpla2 protein levels in skeletal muscles, sebaceous glands and skin (epidermis, dermis) tissues of healthy paws. in the joints of the cia mice, large amounts of inflammatory infiltrate containing cpla2 were detected. in addition, robust cpla2 protein expression in the skeletal muscles surrounding the joints and strong cpla2 positive staining in sebaceous glands were detected. the high correlation between the severity of inflammation and the elevated cpla2 protein, due to an inflammatory infiltrate and increased cpla2 expression, suggests an important role of cpla2 in the development of arthritis. rheumatoid arthritis (ra) is the complex disease depending on environmental as well as genetic factors. in spite of the large research efforts we know in fact only small number of genes involved in this disease. animal models are useful tools for better understanding of the pathogenic mechanisms and genes leading to the disease process. the aim of the current project is to identify the genes and their functional role importance for arthritis. two loci associated with arthritis were identified using a cross between the b10.q (intermediate susceptible) and nod.q strains (resistant to arthritis). one on chromosome 2 a disease protective locus (cia2) and another on chromosome 1 a disease-promoting locus (cia9). nod.q allele at cia9 promotes arthritis whereas cia2, has a protective effect in contrast to the b10.q allele on chromosome 2. a promising candidate gene in cia2 locus is complement factor 5 (c5) as the nod.q allele produced defective c5 protein. cia9 locus contains several genes of potential importance for disease such as fc riib, fc riii, fc riv ncf2, fh. the results of cia (collagen induced arthritis) and caia (collagen antibody induced arthritis) experiments using the subcongenic mice generated that contains fc r region showed a significant difference in incidence and severity of arthritis. the disease is controlled in a recessive pattern. the fragment devoid of fc r region seems to be protective. the subcongenic for the cia2 locus has been generated recently which will be tested for cia and caia susceptibility. the results from these experiments will be discussed in detail. angela pizzolla, ka gelderman, r holmdahl ncf1 is a component of the nadph oxidase complex, which produces reactive oxygen species (ros) upon activation into phagosomes and extracellularly. polymorphisms in the ncf1 gene that impair the capacity to produce ros, enhance susceptibility of both mice and rats to arthritis. activation of autoreactive t cells drives arthritis development but neither ncf1 expression nor oxidative burst have been detected in t cells. we hypothesize that antigen presenting cells influence t cell activity by producing ros during antigen presentation. we aimed to clarify the role of ros produced by dendritic cells (dc) on t cell activation. dc were grown from bone marrow from ncf1 mutated and wildtype mice. we could show that ncf1 mutated dc proliferated and differentiated better, had higher expression levels of costimulatory molecules and mhc ii upon stimulation as compared to ncf1 wildtype dc. in addition, ncf1 mutated dc induced higher levels of il-2 production by hybridoma t cells. to analyze the role of ncf1 in dc on arthritis, mice were developed expressing functional ncf1 restricted to dc (b10.qdcn). these mice are characterized for burst, ncf1 expression and ability to present antigen. we published that immunization with myelin oligodendrocyte glycoprotein (mog) protein resulted in higher disease severity than immunization with mog peptide in ncf1 deficient mice. this suggests that ncf1 plays a role in the uptake and processing of posters inflamm. res., supplement 3 (2007) s423 antigens, probably by dc. this will be further investigated with the b10.qdcn mice using in vitro assays as well as in vivo models for arthritis and multiple sclerosis. purpose: macrophage migration inhibitory factor (mif) is a pro-inflammatory cytokine involved in both innate and adaptive immune responses. it is expressed in human ra synovial tissue and its suppression inhibits t or b cell dependent animal models of ra. we investigated the role of mif in k/bxn serum transfer arthritis. methods: arthritis was induced by injection of k/bxn serum on days 0 and 2 in littermate wt and mif-/-mice. arthritis was scored clinically, ankle thickness was measured using microcallipers, and joints collected for histology. sections were scored for synovitis, synovial fluid exudate, cartilage degradation and bone damage. results: wt mice exhibited arthritis as early as day 1 and 100% incidence was observed on day 7. mif -/-mice exhibited delayed arthritis, with onset on day 3 and 100% incidence on day 9. mif -/-mice exhibited significantly reduced disease severity as measured by clinical disease score ( methods: osteoarthritis was induced by bilateral transection of the medial meniscus in dunkin-hartley guinea pigs using minimal invasive surgery to avoid cartilage damage due to inflammation and/or intra-articular bleeding. results: the first signs of osteoarthritis development were macroscopically observed four weeks after meniscal transection. twelve weeks after surgery the lesions were still restricted to the medial side of the joint and did not reach into the subchondral bone. cartilage destruction due to meniscal transection was also histologically detected. however, biomarkers for cartilage destruction (ctx-ii, hp/lp ratio, comp) were not increased. treatments aiming at different processes in osteoarthritis, such as bone destruction (risedronate), inflammation (pioglitazone and anakinra), and cartilage destruction (galardin) were not effective in this model. the early degenerative changes in this transection model are probably too mild to be measured in the systemic circulation using classic biomarkers. further research into new biomarkers is needed to detect and monitor the early stages of osteoarthritis. the ineffectiveness of the compounds tested in this model underscores the urgent need for new strategies to treat the disease. the meniscal transection model might prove to be useful tool for identifying new biomarkers and treatments. livia l camargo (1), a denadai-souza (1), lm yshii (1), a schenka (2), ma barreto (1), d boletini-santos (3), c lima (3), v rioli (3), mn muscar (1), e ferro (1), sk costa (1) ( methods: aia was induced via intraarticular (i.a.) injection of methylated bovine serum albumin in immunized male rats. knee oedema and pain score were assessed daily in controls and animals treated with hemopressin (10 or 20 ã¬g/day; i.a.). histopathological changes, cell number and cytokines in the synovial fluid of aia rats were determined at day 4. results: aia rats developed a severe mono-arthritis characterized by a joint oedema and pain. at day 4, there was marked cellular infiltration, hyperplasia, pannus formation and destruction of bone and cartilage, but pro-inflammatory cytokines were undetectable by elisa. both doses of hemopressin significantly reduced the knee oedema, but only 20 mg of hemopressin attenuated the pain score. acute joint inflammation was significantly reduced by hemopressin, but failed to significantly affect chronic histopathological signs (hyperplasia, pannus etc.). conclusions: hemopressin has potential for treating acute signs of aia by reducing synovial plasma protein extravasation, alleviating pain and reducing acute joint histopathological changes, thus providing an alternative strategy for treatment of oedema and pain in arthritis. calcitriol, the hormonal metabolite of vitamin d and it synthetic analogs exert an anti-inflammatory action on psoriatic skin lesions while eliciting mild inflammation on healthy skin. the map kinase erk plays an important role in the induction of chemokines, cytokines and adhesion molecules in keratinocytes that maintain the epidermal inflammatory response.we hypothesized that the dual effect of calcitriol may be partially attributed to differential effects on erk activation in the presence or absence of inflammatory mediators. our experimental model was immortalized non-tumorigenic human hacat keratinocytes cultured in the absence of exogenous growth factors or active mediators. inflammation was mimicked by exposure to tnf. level and activation of signaling molecules were determined by immunoblotting. by using the specific egf receptor (egfr) tyrosine kinase inhibitor, ag1487, we established that tnf activates erk in an egfr-dependent and egfrindependent modes. the egfr-dependent activation resulted in the induction of the transcription factor, c-fos, while the egfr-independent activation was of a shorter duration and did not affect c-fos expression. treatment with calcitriol alone increased erk activation and c-fos induction. pretreatment with calcitriol enhanced egfr-dependent erk activation and tyrosine phosphorylation of the egfr, but completely abolished egfr-independent tnf-induced erk activation. pretreatment with calcitriol increased the rate of de-phosphorylation of activated erk, accounting for the inhibition of egfr-independent erk activation by tnf. it is possible that effects on the erk cascade underlie the dual action of calcitriol and its synthetic analogs on cutaneous inflammation. christina barja-fidalgo (1), r saldanha-gama (1), ja moraes (1), r zingali (2), c marcienkewicz (3) (1) universidade do estado do rio de janeiro, rio de janeiro, brazil (2) universidade federal do rio de janeiro, rio de janeiro, brazil (3) temple university, philadelphia, usa neutrophils adhere on vascular endothelium and directly migrate toward inflamed tissue to exert their primary defense function. integrin are receptors that drive cell adhesion and motility and interfere with cell activation, functions and survival.acting as both anchoring molecules and signaling receptor, transducing signals outsidein and inside-out, integrins are potential targets for therapeutic and diagnostic opportunities. disintegrins are a family of cystein-rich low-molecular weight peptides that usually contain an rgd sequence, a cell attachment site of ecm and cell surface proteins recognized by integrins. they are considered selective and competitive antagonists of integrins, being potent inhibitors of platelet aggregation and cell-cell/cell-ecm interactions. we reported that rgd-disintegrins, selectively interact with integrin amb2, a5b1 and/or avb3) on human neutrophils, interfering with cell functions through the activation of integrin-coupled intracellular signaling pathways. recently showed that, a selective ligand of a9/a4b1 integrins, vlo5, induces neutrophil chemotaxis, cytoskeleton mobilization and potently inhibiting neutrophil spontaneous apoptosis. these effects are mediated vlo5 interaction with a9b1 integrin, activating the focal adhesion cascade. vlo5 effects on the delay of neutrophil is modulated by pi3k, erk-2 mapk and nf-kb pathways that seems to interfere with the balance between anti-and pro-apoptotic bcl-2 family members and with mitochondrial membrane potential. data emphasize mechanistic details of the role of a9b1 integrins interactions on human neutrophils and support the use of disintegrins as prototypes to develop logical combinations of drugs to optimize or minimize the susceptibility of a selected target cell population to apoptosis during therapeutic interventions. (faperj, cnpq, capes, ifs-sweeden) (1), h serezani (2), m peters-golden(2), sonia jancar (1) '(1) university of sâ¼o paulo, brazil (2) university of michigan, usa it is been shown that leukotriene (lt) b4 and cysteinyl lt (ltc4, ltd4 and lte4) enhances fcr-mediated phagocytosis in alveolar macrophages (am), dependent on protein kinase c (pkc). in contrast, ltb4 but not cyslt effects are exclusive on syk activation. in the present study we investigated the role of specific pkc isoforms and its upstream and downstream targets involved in lt-enhanced fcr-mediated phagocytosis. to this purpose, ams were pretreated or not with inhibitors of pkc-d (rottlerin -6 um), pkc-a (ro-32-0432-9 nm), pi3k (ly290042-10 um and wortmannin-10nm), erk 1/ 2 (pd98059 -2 um), cpla2 (aacocf3-10 um), p38mapk (sb20190-10 um) and ca++ (bapta/am-10 um), before stimulation with ltb4 or ltd4 and addition of igg-opsonized red blood cells for 90 min. activation (phosphorylation) of signaling molecules by lts were analyzed by western blot. our results demonstrate that ltb4 -enhanced phagocytosis is dependent on pkc-a, while ltd4 effects are mediated by pkc-d. cell proliferation and differentiation, adhesion, cell activation and apoptosis. while galectin-1 mainly acts as an anti-inflammatory and pro-apoptotic molecule, galectin-3 is known as a strong pro-inflammatory and anti-apoptotic signal. we have recently recognized galectin-3 as a new molecular target of immunomodulatory drugs in monocyte/macrophage-like cells. in this study we investigated the effects of immunomodulatory drugs (aspirin, indomethacin, hydrocortisone and dexamethasone), applied in therapeutic ranges on the expression of galectin-1 at gene and protein level in monocytic thp-1 cells. we have also tested the effects of these drugs on both galectins in the cells activated by lipopolysaccharide from e. coli (lps). the targeted mrna level was evaluated using quantitative rt-pcr technique and the expression of both galectins in cell homogenates was determined by western-immunoblot analysis. the results showed that immunomodulatory drugs affected the expression of galectin-1 on both, gene and protein level, and that the effects were dependent on drug type and applied concentration as well as time of the exposure. the modulatory effects of the applied drugs on galectin-1 and -3 expressions were also observed in the cells activated by lps. these findings represent important step in the understanding of the effects of immunomodulatory drugs on galectin-1 and-3 expressions, as well as the role of these lectins in the physiology of monocytes. introduction: pancreatitis-associated protein (pap) has been recently described as an endogenous mechanism involved in the regulation of inflammation. in the present study, we show some of the molecular mechanisms implicated in the intracellular signaling pathways modulated by pap. the pancreatic human cell line panc1 was incubated with pap (500ng/ml) and/or tnfa (100ng/ml). total rna was obtained and the expression of tnfa was examined by rt-pcr. in addition, the effect of pap on nfkb activation was measured by inmunofluorescence in cells. western blot analysis was used to determine the expression of nfkb mediators: phosphorylated ikk, ikba and p65. results: we observed that pap administration to cells prevented nfkb translocation to the nucleus as well as the tnfa-induced tnfa gene expression. when tnfastimulated cells were treated with cycloheximide in order to block protein synthesis, the induction of tnfa gene expression was completely restored. on the other hand, pap had no effect on ikk phosphorylation or ikba degradation. conclusions:in this study we have provided evidence that pap modulates the inflammatory response by blocking nfkb translocation to the nucleus. this pap-induced nfkb inhibition requires a jak/stat-dependent de novo protein synthesis. objectives: this study investigated the inhibitory mechanism of hyaluronan (ha) on lipopolysaccharide (lps)stimulated production of proinflammatory cytokines in u937 macrophages. methods: ha was added to u937 macrophage cultures in the presence of lps, with or without pretreatment with anti-intercellular adhesion molecule-1 (icam-1) antibody. secreted levels of tumor necrosis factor a (tnfa), interleukin (il)-1b, and il-6 were determined by enzyme-linked immunosorbent assay. the phosphorylation of nuclear factor (nf)-kb, ikba, and mitogenactivated protein kinases (mapks) was analyzed by immunoblotting. results: lps stimulated production of tnfa, il-1b, and il-6. in contrast to 800 kda ha, 2700 kda ha at 1 mg/ ml inhibited lps-induced cytokine production. anti-icam-1 antibody blocked the effects of ha on the lps actions on u937 cells. lps activated nf-kb and mapk pathways, whereas ha down-regulated p65 nf-kb and ikba phosphorylation by lps without affecting mapks. inhibition studies revealed the requirement of nf-kb for lps-stimulated cytokine production. anti-icam-1 antibody reversed the inhibitory effects of ha on phosphorylation of p65 nf-kb and ikba. conclusions: ha of intrinsic molecular weight suppresses lps-stimulated production of proinflammatory cytokines via icam-1 through down-regulation of nf-kb and ikb. exogenous ha into arthritic joints could act as an anti-nf-kb agent by the mechanism demonstrated in the present study. the principal eicosanoid product of endothelial cox-2 is prostacyclin (pgi2), which is a potent vascular patency factor. induction of endothelial cox-2 under hypoxic conditions is well documented. this response, along with associated pgi2 release, is likely an important protective homeostatic response. in order to explore the role of candidate signalling agents in cox-2 expression in response to hypoxia, studies were undertaken using luciferase reporter constructs of the cox-2 promoter region in huvec. cox-2 induction under hypoxic conditions was confirmed with the wild type construct. strategic mutations of transcription factor binding sites showed that sites for hypoxia inducible factors (hifs) were more important for cox-2 expression than those for nfkb. furthermore, expression of cox-2 was increased under normoxic conditions by transfection of huvec with normoxia-stable hif mutants. emsa showed hif binding in nuclear extracts from untransfected hypoxic huvec. under these hypoxic conditions, increased release of pgi2 but not vaso-occlusive thromboxane a2 was seen. thus the putative protective induction of cox-2 in endothelial cells in response to hypoxia involves signalling by hifs. crescentic glomerulonephritis is characterized by crescent formation and rapid progress to renal failure, where the predominance of th1 immune response plays a crucial role. however, the therapeutic efficacy of the regulation of th1-predominant immune responses remains to be investigated. therefore, the effects of a th1 selective inhibitor tak-603 were investigated in a model of crescentic glomerulonephritis in wky rats. methods: tak-603 was administered orally, starting at the time of induction of glomerulonephritis. in group 1, the drug was administered daily for the initial 6 days. tak-603 was administered on day 0 only in group 2, and from day 3 to 5 in group 3. in each group, nephritic rats were killed on days 6 and 56. results: in group 1, glomerular damage, including crescent formation, was improved on day 6, with reductions in the numbers of cd4, cd8 and ed-1 positive cells, as well as in urinary protein excretion. protein and transcript levels of th1 cytokines in the diseased kidneys were markedly decreased by tak-603 treatment. renal pathology, including glomerulosclerosis and interstitial fibrosis, was ameliorated and proteinuria was markedly decreased. elevated levels of serum creatinine showed concomitant improvement. in group 3, in which treatment was initiated shortly after the appearance of glomerular abnormalities, glomerular damage was also diminished, resulting in a decrease in urinary protein excretion. treatment only on the first day in group 2, partially rescued renal dysfunction. conclusions: these results suggest that the initial inhibition of th1 immune response has an appealing therapeutic potential for crescentic glomerulonephritis. parasitic nematodes and their hosts are now known to produce a wide range of galectins. whilst host galectins have been shown to modulate the recruitment and effector function of inflammatory cells including mast cells, neutrophils and eosinophils, the role of secreted parasitic galectins is less well defined. studies at moredun have demonstrated that both the endoparasitic helminth, haemonchus contortus, and the ectoparasitic mite, psoroptes ovis, produce galectin-like factors which, in vitro, directly influence the migration and survival of eosinophils from their natural sheep host. excretory-secretory extracts from both parasites contained potent chemokinetic activity and were also able to promote eosinophil survival in the presence of dexamethasone. separation by affinity chromatography, as well as specific sugar inhibition and mass spectrometric profiling, revealed the active components to be galectins. in the case of h. contortus, there was homolgy with known est sequences, which allowed subsequent cloning and expression studies to be undertaken. a functional in vivo role for these parasitederived galectins awaits confirmation, but the possibility is raised that they could directly influence the host immune response following infection. this may have particular significance in mite infections in which exudates from the associated eosinophilc lesion appear s428 inflamm. res., supplement 3 (2007) posters to provide the primary nutrient source for their survival. moreover, the observation that two very different parasites may have evolved similar mechanisms for manipulating the host inflammatory response to their benefit, raises the further possibility that parasite galectins may provide potentially novel therapeutic targets. the aim of the study was to investigate the time course of cytokine gene expression in liver and lungs of mice with lipopolysaccharide (lps)-induced septic shock and to assess the effect of three different immunomodulatory agents on cytokine mrna levels in these tissues. male cd-1 mice were injected intraperitoneally with 50 mg/kg lps alone or concomitantly with an intravenous dose of pentoxifylline (100 mg/kg), lisofylline (100 mg/kg) or prednisolone (10 mg/kg). the tissues were harvested 0, 1, 4, 6, and 24 h following lps administration and stored at -808c. relative quantification of tumor necrosis factoralpha (tnf-alpha), interleukin-1beta (il-1beta), interleukin-6 (il-6), and interferon-gamma (ifn-gamma) mrna levels was performed by real-time rt-pcr. the highest levels of cytokine mrna were observed at 4 or 6 h after lps administration, whereas the expression of tnf-alpha and il-1beta in lungs and il-6 in liver reached the peak values at 1h and then decreased gradually. in addition, the lps effects on cytokine mrna were more pronounced in liver when compared to lungs. all administered compounds inhibited the lpsinduced tnf-alpha mrna expression (by up to approximately 50%), whereas lisofylline significantly increased ifn-gamma mrna levels in both tissues at most investigated time points. for other cytokines, the observed differences did not reached statistical significance. in conclusion, with the exception of ifn-gamma, the time course of cytokine mrnas differed considerably depending on the type of tissue. in the murine model of lps-induced septic shock, only tnf-alpha gene expression was suppressed by all compounds under investigation. maria sanz(1), m losada (1), c company (1), c lope-gines(1), l piqueras(2), j cortijo (3) the migration of leukocytes into inflamed tissues involves a cascade of molecular events finely regulated by cell adhesion molecules and chemokines. fractalkine/ cx3cl1 (fr) is a membrane-bound chemokine that functions as a mononuclear leukocyte chemoattractant and as an adhesion molecule. clinical studies and animal disease models have shown that fr is also involved in the pathogenesis atherosclerosis. we have demonstrated that angiotensin-ii (aii) has proinflammatory actions inducing the initial attachment of mononuclear cells to the arteriolar endothelium. in the present study we have investigated whether aii can cause the synthesis and expression of fr on human umbilical arterial endothelial cells (huaecs). huaecs were stimulated with ang-ii 1 microm or with tnfalpha (20 ng/ml) for 1, 4 and 24 h. fr was determined in the culture supernatants by conventional sandwich elisa. fr was only detected after 4 h and 24 h stimulation with tnfalphafnwhereas aii was unable to provoke the cleavage of the chemokine. semiquantitative rt-pcr analysis of huaecs showed increased fr mrna expression in aii-stimulated cells for 1 and 4 h. these effects were caused by the interaction of aii with its at1 receptor since they were abolished by losartan (at1 receptor antagonist). tnfalpha also increased fr mrna. immunohistochemical analysis of the cultured endothelial cells showed a clear expression of fr in huaecs stimulated with aii or tnfalpha for 4 and 24 h. these results suggest that fr could be a key chemokine in the selective adhesion of mononuclear leukocytes to the arterial endothelium elicited by aii. the lipophilic yeast malassezia is an exacerbating factor in atopic dermatitis (ad).among organisms of the malassezia species, m. globosa and m. restricta are particularly dominant on the skin of ad patients. our previous study has demonstrated that human keratinocytes responded to the two malassezia species with different th2-type cytokine profiles, i.e. m. globosa induced il-5, il-10, and il-13 secretion from the keratinocytes, whereas m. restricta induced il-4 secretion.these findings suggest that m. globosa and m. restricta play a synergistic role in triggering or exacerbating ad by stimulating the th2 immune response. pattern recognition receptors (prrs) of human keratinocytes play an important role in the induction of inflammatory and innate immune responses. in this study, we assessed the role of prrs for cytokine production by human keratinocytes in response to malassezia species. human keratinocytes were pretreated with various anti-prr monoclonal antibodies (mabs) and stimulated with m. globosa or m. restricta. cytokine secretion from keratinocytes was measured by using fast quant elisa kit. exposure of human keratinocytes to m. globosa and m. restricta resulted in enhanced secretion of il-5 and il-4, respectively. the m. globosainduced increase in il-5 secretion was inhibited by mabs against cd14 and cd91. in case of m. restricta, mabs against toll-like receptor 2 (tlr2) and cd14 suppressed significantly il-4 secretion from keratinocytes. these findings suggest that the distinct prrs interactions with fungal pathogen-associated molecular patterns (pamps) are key factors in differential cytokine secretion from keratinocytes stimulated with malassezia species. atopic dermatitis (ad) is a chronic, relapsing inflammatory skin disease associated with allergy. mdc (macrophage-derived chemokine/ccl22) and tarc (thymus and activation-regulated chemokine/ccl17) are th2type cytokines, and it has been reported that serum mdc and tarc levels are associated with ad disease. in present study, we investigated the effect of prunus yedoensis matsum barks on the inflammatory chemokines (mdc and tarc) and jak-stat pathway in hacat keratinocytes. as a result, etoac fraction and e5 sub-fraction inhibited the mrna expression and protein level of mdc and tarc in a dose-dependent manner. also, e5 sub-fraction showed inhibitory activity on the stat1 protein level. these results suggest that p. yedoensis may have an anti-atopic activity by suppressing the inflammatory chemokines (mdc & tarc). the il-1 family now consists of 11 members, most of which have assigned functions.there are 10 members of the il-1 receptor family (including the decoy receptor type ii il-1r).many of the il-1 family members possess neither a signal peptide nor an apparent prodomain, but nevertheless manage to exit the cell.the il-1 family members il-1f6, f8 and f9 signal through a complex of the il-1r family member rp2 in association with il-1r acp to activate common inflammatory pathways.the specific activity is is low, on the order of 1-10ug/ml ec50.we have found that removal of a few n-terminal amino acids from il-1f5, f6, f8 and f9 can increase their bioactivity approximately 1000-fold. the location of the n-terminus leading to increased specific activity is quite specific; removal of one more or one fewer amino acid eliminates the effect.in addition, n-terminally truncated il-1f5 is capable of antagonizing signaling via il-1rrp2, but full-length f5 is inactive. (1) university of ulsan, japan kyoto university, japan inflammation plays a pathogenic role in the development of obesity-related complications such as type ii diabetes and atherosclerosis. tumor necrosis factor alpha (tnfa) is closely associated with the enhanced inflammatory responses in obesity and the obesity-related pathologies. tr2 (hvem/tnfrsf14), which is a member of the tnf receptor superfamily and the receptor for lymphotoxins-related inducible ligand that competes for glycoprotein d binding to herpesvirus entry mediator on t cells (light/tnfsf14), is a potent mediator of inflammatory responses. the purpose of this study is to examine the hypothesis that obesity-induced inflammatory responses can be attenuated by inhibiting tr2 pathway. c57bl/6 tr2 knockout mice and their wild-type control were fed a high-fat diet for 12 weeks and the obesity phenotypes were determined in the obese tr2 knockout mice and the control. the obese tr2 mice fed a high-fat diet elicited the attenuation of body weight gain and insulin resistance relative to wild-type control mice. expression levels of inflammatory genes significantly decreased in the adipose tissue of the obese tr2 knockout mice compared with those of the control. our results demonstrate that obesity-induced inflammatory responses and insulin resistance can be attenuated in obese tr2 knockout mice fed a high-fat diet. objectives: the present study was undertaken to investigate the role of insulin on allergic airway inflammation. methods: diabetic male wistar rats (alloxan, 42 mg/kg, i.v.) and controls were sensitized with ova (100 ã¬g) and al(oh)3 (8 mg, s.c.) 10 days after alloxan or saline injection. the animals were challenged 14 days later by the intratracheal instillation of ova (1 mg/0.4 ml). the following analyses were performed 6 h thereafter: (a) total and differential cell counts in bronchoalveolar lavage (bal) fluid; (b) quantification of tnf-alpha and il-1 beta in the bal by elisa; and (c) immunohistochemistry for p-and e-selectins on lung vessels. results: compared to the control animals, diabetic rats exhibited reduced number of neutrophils (90%) and mononuclear cells (50%); reduced levels of tnf-alpha (45%) and il-1 beta (30%), and reduced p-selectin expression (30%) in response to ova challenge. these abnormalities were corrected after treatment of diabetic rats with a single dose of nph insulin (4 iu, s.c.), 2 h before ova challenge. although we did not find differences in e-selectin expression between diabetic rats and controls, expression of this molecule was amplified by insulin. conclusions: data presented show that insulin controls neutrophils migration during allergic airway inflammatory possibly by modulation of tnf-alpha and il-1 beta production and selectin expression. supported by fapesp and pronex. hormonally active vitamin d derivatives are beneficial in the treatment of cutaneous inflammatory disorders, particularly in psoriasis. their anti-inflammatory effect is usually attributed to inhibition of the activity of infiltrating immune cells.we examined whether vitamin d also interferes with the pro-inflammatory action of the keratinocytes themselves. human hacat keratinocytes cultured in the absence of exogenous growth factors or active mediators were exposed to tnf to simulate an inflammatory challenge and their response was monitored by assessing mrna levels of the cytokine tnf, the chemokine il-8 and the adhesion molecule icam-1 by real-time pcr. icam1 and il-8 were induced rapidly peaking after 2h, their mrna levels increased again from 8h to reach a plateau between 16h to 24h after exposure to tnf, whereas tnf mrna levels increased steadily between 2h and 24h. 24h pretreatment with calcitriol, the hormonal form of vitamin d, inhibited induction of il-8 but did not affect that of icam-1 or tnf 2h following exposure while calcitriol markedly inhibited the induction of all 3 pro-inflammatory genes 16h after the tnf challenge.calcitriol inhibits the activation of jun kinase (jnk) and p38 by tnf. this action was mimicked by the posters inflamm. res., supplement 3 (2007) s431 jnk inhibitor sp600125 and the p38 inhibitor sb203580.the combination of the two inhibitors fully reproduced the time and gene dependent modulatory effect of calcitriol. we conclude that vitamin d attenuates the active contribution of keratinocytes to cutaneous inflammation and that this modulatory effect is explained by inhibition of the jnk and p38 cascades. (1), cm lotufo (2), p borelli (1), zs ferreira (2), rp markus (2), shp farsky (1) (1) department of clinical and toxicological analyses, school of pharmaceutical sciences, university of sâ¼o paulo, brazil (2) department of physiology, bioscience institute,university of sâ¼o paulo, braziil introduction: we showed that endogenous glucocorticoids (eg) control neutrophil mobilizations from the bone marrow and peripheral compartment by modulating the expression of l-selectin on segmented cells. aims: we evaluated the role of eg on endothelial cells (ec) and the molecular mechanisms responsible for hormonal actions in neutrophils and ec. methods: neutrophils were collected from blood, segmented leukocytes from femoral bone marrow and ec were cultured from testis vessels. cells were obtained from adrenalectomized (adx), ru 38486-treated, shamoperated, vehicle-treated and non-manipulated (nm) wistar rats. results: circulating neutrophils and segmented cells from ru 38486-treated rats presented elevated and decreased expressions of l-selectin vs cells from control animals, respectively. the effects were not dependent on alterations of l-selectin mrna levels. ec from adx animals presented more ability to adhere neutrophils from nm rats and enhanced mrna levels and membrane expressions of icam-1, vcam-1 and pecam-1. participation of the glucocorticoid cytosolic receptor(gcr) on these effects was shown by similar results in cells from ru 38486 treated rats. nfkappab translocation in neutrophils was equivalent in all groups of animals, but it was enhanced in ec from adx or ru 38486-treated rats. conclusions: data show the participation of the gcr on events involved in neutrophil mobilizations, but nfkappab transcription is only involved on ec. (1), y naito(2), t okuda (3), k mizushima (3), t okayama (3), i hirata (3), h tsuboi (3), t suzuki (3), o handa (1) background: despite the inhalation of co at high concentrations had been considered as a toxic gas, the inhalation of co at low concentration has recently been shown the cytoprotective and anti-inflammatory effect against various animal models. however, it is unclear whether the direct exposure of co to the intestinal inflamed mucosa is effective or not. in this study, we investigated the therapeutic efficacy of the rectal co administration for rat colitis model. acute colitis was induced with trinitrobenzene sulfonic acid (tnbs) in male wistar rats. co(200ppm-10ml) was intrarectally administrated twice a day after the induction of colitis. rats were sacrificed at 3 days after the administration of tnbs. the distal colon was removed, and the ulcer lesions were measured. thiobarbituric acid (tba)-reactive substances and tissueassociated myeloperoxidase (mpo) activity were measured in the colonic mucosa as indices of lipid peroxidation and neutrophil infiltration, respectively. moreover, we evaluated the expressions of cinc-1 mrna/protein and p-p38 mapk protein. the intracolonic administration of co ameliorated tnbs-induced colonic ulceration. the increases in tba-reactive substances and mpo activity after tnbs administration were both significantly inhibited by treatment with co. moreover, the rectal administration of co significantly inhibited the increased expression of cinc-1 mrna/protein and p-p38 mapk protein after the induction of tnbs-induced colitis. the rectal administration of co protected from the intestinal inflammation in rats. based on these data, the beneficial effects of co on the intestinal mucosal injury may be attributed to its anti-inflammatory properties. alessandra gambero (1), m marã³stica (1), m saad(2), j pedrazzoli jr (1) (1) sâ¼o francisco university medical school, brazil (2) state university of campinas, brazil recent studies have shown that adipocytes produce and secrete several bioactive molecules like adipocytokines. the adipose tissue can also present short and long-term changes during inflammation and infectious pathologies. in this study, the alterations of mesenteric and perinodal mesenteric adipose tissue during experimental colitis induced by repeated intracolonic tnbs instillations were evaluated. the adipocyte size was measured after collagenase digestion. the basal lipolysis (glycerol release) and adipocytokine production was monitored after short time culture of adipose tissue. the colitis animals showed higher mesenteric fat mass (1.02+-0.06 and 0.78+-0.09 % of body weight for colitis and control, respectively; p<0.05) in despite of the lower body weight. the mesenteric adipocytes from colitis animals presented reduced diameter (31.67+-0.28 and 35.69+-0.37 um for colitis and control, respectively; p<0.01), higher basal lipolysis (41.67+-6.89 and 18.77+-3.66 ug.mg-1 for colitis and control, respectively; p<0.05) and tnf-alpha production (8.64+-0.77 and 5.51+-0.86 ng.mg-1 for colitis and control, respectively; p<0.05). perinodal mesenteric adipocytes presented normal diameters, higher basal lipolysis (72.48+-15.38 and 25.96.77+-3.05 ug.mg-1 for colitis and control, respectively; p<0.05), increased tnfalpha (46.34+-5.82 and 15.71+-2.15 ng.ml-1 for colitis and control, respectively; p<0.01), leptin (400.41+-77.10 and 201.01+-52.01 pg.ml-1 for colitis and control, respectively; p<0.05) and adiponectin production (15029+-2340 and 5918+-498 ng.ml-1 for colitis and control, respectively; p<0.01). the mesenteric adipose tissue was modified during the experimental inflammation, but some alterations were site specific. perinodal adipose tissue retained the ability to produce anti-inflammatory and pro-inflammatory cytokines, while mesenteric adipose tissue only the pro-inflammatory one. this work was financially supported by fapesp. inflammatory bowel disease (ibd) is a group of chronic inflammatory disorders of the intestine. the role of the pro-inflammatory p38 mapk signalling cascade in the pathogenesis of ibd is highly controversial. we therefore aimed to investigate the role of p38 mapk in chronic dextran sodium sulfate (dss) induced colitis, an experimental model of ibd. chronic intestinal inflammation was induced by oral cyclic administration of 3 % dss in sjl mice. clinically, the dss treatment produced episodes of colitis manifested by diarrhoea, gross intestinal bleeding, marked loss of body weight, and shortening of the colon. at the molecular level, this was accompanied by an up-regulation of tnfa, il-1ã¢, il-6, il-17, kc, cox-2, igg heavy chain, and phospho-stat3 in the dss treated mice.the clinical and molecular features described above recapitulate findings reported in human ibd. in order to assess the role of p38 mapk, the activation of the p38 mapk signalling cascade was analysed by western blot analysis. the expression and phosphorylation levels of both p38 mapk and of mk2 and hsp27, two down-stream targets, were not increased in dss treated animals compared to controls. leo 15520, a potent inhibitor of p38 activity in vivo, was dosed as pretreatment and after completion of dss treatment. pretreatment had a deteriorating effect on all measured cytokines, whereas treatment after disease induction had no effect on any measured parameters. collectively, these results strongly suggest that the p38 kinase pathway only plays a minor role, if any, in the dss model. (sp) were gmcsf differentiated, dcs purified through gr1+ cell depletion, and spleen tcells isolated by pan tcell negative selection. spdcs or bmdcs were stimulated +/-100ng/ml lps. for mlr, balb/c tcells were added for 5 days. cells were incubated with sb203580 (4-(4-fluorophenyl)-2-(4-ethylsulfinylphenyl)-5-(4-pyridyl)1h-imidazole, sb) or ml3403 ((rs)-{4-[5-(4-fluorophenyl)-2-methylsulfanyl-3h-imidazol-4-yl]pyridine-2-yl}-(1 -phenylethyl)amine, ml) and washed prior to lps stimulation (bmdcs) or cell mixing (t cells). 3hthymidine incorporation was measured, cell viability by mtt assay, tnfa and il-12 production by elisa. mlr tcell proliferation inspdcs or bmdcs was inhibited by sb (ic50 spdc 0.06mm, bmdc 1.0mm) and ml (ic50 spdc 0.03mm, bmdc 0.03mm). preincubation with dcs had no effect, despite reduced lps stimulated il-12 and tnfa synthesis by sb (ic50 il-12 1.2mm, tnf 0.16mm) and ml (ic50 il-12 0.9mm, tnf 0.11mm). preliminary data shows that preincubation of t cells with sb and ml modifies the mlr response. p38 plays a role in the interaction of dcs and t cells in antigen recognition. however, pre-incubation of drugs with dcs was ineffective. the role of t cell p38 mapk in the mlr is under investigation. p38 inhibitors may possess disease modifying properties because of reduced tcell antigen reactivity to dc antigen presentation. (1), s luik(2), s laufer(2), m seed (3), v holan(4), s fiorucci (5) (1) synovo gmbh, tã¼bingen, germany (2) university of tã¼bingen, germany in vivo anti-inflammatory activity of certain p38 kinase inhibitors is limited by bioavailability. however, it is possible that they may be useful in the therapy of ibd should it be possible to mediate there uptake in and around bowel lesions. we reasoned that activity could be especially increased if drug physical properties were altered to allow preferential partition into macrophages and neutrophils (wbc) associated with lesions. we prepared prodrugs of p38 inhibitors and screened them using whole human blood, murine spleenocytes and peritoneal macrophage. pharmacologically inert macrocycle (azilide) conjugates were assessed for enhanced efficacy in murine collagen induced arthritis either therapeutically (after onset of signs) or prophylactically (2 d post boost) or in a dss or tnbs model of ibd in the mouse. in both types of models, the prodrugs achieved improved suppression of arthritis and inflammatory score in colon sections at tolerated doses with optimal activity at 15mmolkg-1d-1. we propose that the prodrugs increase efficacy via improved pharmacokinetics partly related to biased disposition of the prodrug toward immune cells. despite the potent anti-inflammatory and immunosuppressive properties of glucocorticoids its applying in the management of severe necrotizing pancreatitis is still controversial. the plasma levels of interleukins (il-8, il-18) and adhesion molecules (e-selectin and icam-1) were measured in 36 patients with necrotizing pancreatitis. the measurement was performed immediately after admission, at the 3, 7, and 14 day. all patients were divided on two groups: first group compiled 20 patients, in which dexamethasone (16 mg/day during 7-8 days) was applied in the complex management of acute pancreatitis, and control group -16 patients that did not receive corticosteroids. all patients received the initial therapy. the increased levels of il-6, il-8, il-18, icam-1, and eselectin were noted in both groups of patients at the time of admission. the gradually increase of all proinflammatory mediators plasma levels up to seventh day was noted in patients of the control group. its levels clear correlated with the severity of mods and spreading of necrotic processes confirmed by ct. starting from the third day the gradually decrease of mediators levels were noted in the patients of the first group. the incidence of contamination of necrotic foci had no difference in both groups of patients. the ability of glucocorticoids to inhibit expression of proinflammatory mediators due to the glucocorticoids-mediated repression of nf-kappa b pathway provide the pathogenetic substantiation for the applying of glucocorticoids in the complex management of necrotizing pancreatitis. the objective of this study was to examine whether t cell specific overexpression of the th2 transcription factor gata-3 can inhibit th1/th17 cell mediated experimental mbsa arthritis. mbsa-immunized wild type mice developed joint inflammation which gradually increased in time with a maximum at day 7. at day 1, t cell specific gata-3 tg mice did not show any difference in arthritis score compared to wild type mice. however, at day 7, wild type mice had developed severe joint inflammation having the maximum arthritis score. in contrast, gata-3 tg mice showed only mild joint inflammation, suggesting that t cell specific overexpression of gata-3 protects against development of severe joint inflammation. facs analysis revealed low levels of il-17 +/ifn-gammacells in wild type as well as in gata-3 tg mice at day 1. as expected, il-4 positive cells were higher in gata-3 tg mice compared with wild type mice. interestingly, at day 7, the percentage of il-17 +/ ifn-gamma-cells were markedly increased in wild type mice but not in gata-3 tg mice, suggesting prevention of th17 expansion under gata-3 overexpression in vivo. these data revealed that t cell specific overexpression of the th2 transcription factor gata-3 protects against progression of severe joint inflammation during mbsainduced arthritis. furthermore, il-17 +/ifn-gammacells play a critical role in the progression of joint inflammation in this model and gata-3 overexpression in t cells prevents expansion of the il-17 +/ifn-gamma-t cell subset. pingping jiang (1), pt sangild(2), t thymann (2), hh-y ngai (1), w-h sit (1), k-l chan (3), jm-f wan (1) ( necrotizing enterocolitis (nec) is a severe intestinal inflammatory disease for which the disease etiology and progression remain unclear. preterm delivery and enteral milk formula feeding are factors predisposing to nec. to understand the pathophysiology of nec, two-dimensional gel electrophoresis (2d page) proteomic approach was applied in studying changes in intestinal protein pattern in preterm piglets with spontaneous nec occurring in response to 2 days of parenteral feeding followed by 1 day of enteral formula feeding. the intestinal proteomes of pigs with clinical symptoms of nec (n = 3) were compared with corresponding pigs remained healthy (n = 4). syproruby staining was used and differently expressed proteins were identified by maldi-tof ms or maldi-tof/tof ms. the proteins with significantly different expression between nec and healthy pigs involve in energy metabolism (sorbitol dehydrogenase, mitochondrial aldehyde dehydrogenase 2 and chain a, medium-chain acyl-coa dehydrogenase with 3-thiaoctanoyl-coa etc.), inflammation (peptide-binding protein 74 (pbp74) and snail homolog 3), signal transduction proteins (thyroid hormone binding protein precursor, park7 protein and chain b, structure of the rho family gtp-binding protein cdc42 in complex with the multifunctional regulator rhogdi etc.) and anti-oxidation (manganese-containing superoxide dismutase(sod)). these data underscore the significant impact of intestinal proteomics in unraveling nec pathophysiology and biomarker discovery. blood are used to monitor the progression of inflammation. the aim of our study were to investigate systemic markers of disease in a rat model of lps induced pulmonary inflammation to provide a link between preclinical in vivo research and early clinical research. animals were exposed to bacterial lipopolysacharide (e.coli 026:b6) by inhalation. the lungs were lavaged 6 hours post provocation and the level of cell influx was determined. relevant mediators of acute pulmonary inflammation were analysed with standard elisa technology in bronchoalveolar lavage fluid and in blood. in addition, measurement of changes in body temperature were performed at different time-points post provocation in order to monitor the systemic inflammatory responses to the local pulmonary inflammation manifested as alteration in body-temperature. results showing the effects of lps challenge on local and systemic parameters will be presented and the possible link to lps responses in man discussed. pulmonary inflammation models are widely used in pharmacological research. however, provocations and treatments aimed directly at the lung are often invasive which limits the possibility to perform repeated administrations of test agents and compounds. also, results derived from bronchioalvelar (bal) fluid are subject to variability if the retrieval techniques are non standardized. here we describe a non-invasive standardized method for retrieval of bal fluid to be used in mice. we present the characterisation of these techniques using the inflammatory response to lps and propose that this non invasive method can be used to refine lps and other challenge models. the objectives were to evaluate the dynamic response after a single intra-tracheal administration of of 5mg (50ml/animal) of lps (p.aeruginosa) to c57bl/6j mice. control animals received a single dose of sterile 50ml 0.9% saline/animal. the mice were terminated 4, 24, 48, 72 and 96h after instillation using a non invasive and operator independent lavage technique. results showing the effects of single lps challenge on bal parameters, excised lung gas volume and lung weight will be presented showing reliable dynamic responses. these techniques open the possibility to run repeated treatments and chronic provocations and are not subject to variability from bal fluid retrieval. the human psoriasis xenograft scid mouse model is one of the most accepted and well characterized models for screening of novel anti-psoriatic compounds. the model has primarily been applied for testing novel treatment principles via systemic or intradermal administration routes. in order to evaluate the model for topical treatment, psoriatic keratome biopsies were grafted to immune-deficient scid mice. transplanted mice were treated with daivonex /dovobet (calcipotriol) and bms (betamethasone). the results show a strong antipsoriatic efficacy after treatment with bms (epidermal thickness reduced by 68%). treatment with daivonex / dovobet also showed an anti psoriatic effect with a 31% reduction in epidermal thickness. serum did not contain test compounds, indicating that the observed effect were not due to systemic exposure. the observed effects are in concordance with clinical results of treatment of psoriasis. it is concluded that the model is useful for testing topical treatments. we have demonstrated that adult rats offspring of dams submitted to protein restriction during early lactation, presented impaired acute immune responses probably related to an imbalance in glucocorticoids and insulin secretion (barja-fidalgo; inflamm res 52 (11): 2003) . here, we evaluated the innate immunity mediated by neutrophils and host defense against infection in adult rats offspring of dams fed with either a protein free diet (un-group) or 22% protein diet (c-group) during the first 10 days of lactation. un rats showed lower number of blood pmn, though no difference in bone-marrow neutrophils number was observed. blood neutrophils from un-group presented a significantly reduced phagocytic activity against opsonized zymosan, constitutively expressed inos and spontaneously produced o2-, no and tnf-alpha. in vivo treatment with lps, at non-lethal doses, significantly increases tnf-alpha and superoxide production by neutrophils, compared with controls. lps increased no production by neutrophils from both groups, inducing inos expression in control cells, but no further increase in inos expression in un rats. nucleare nf-kb is constitutively augmented in un rats. un animals presented a higher survival rate in a model of clp-induced severe sepsis. these results indicate that a metabolic programming induced during early lactation affects the innate immune responses in adult rats, which are unable to properly mount an inflammatory response, may predispose to chronic diseases in adult life. transgenic mice over-expressing vascular endothelial growth factor (vegf) in the epidermal basal layer under the human keratin 14 (k14) promoter have previously been reported to develop a psoriasis-like inflammatory condition in the skin. important hallmarks of psoriasis are epidermal hyperplasia in association with infiltration of t-cells in the dermis and epidermis and also increased dermal angiogenesis. the aim of this study was to describe the epidermal hyperplasia and the infiltration of the skin with t-cells in transgenic k14/vegf mice. we induced a cutaneous inflammation in the ear skin by repeated topical treatments with 12-o-tetradecanoylphorbol-13-acetate (tpa), in order to investigate the inflammatory response. the in vivo pharmacological effect of topical treatment with a number of reference compounds, including betamethasone-17-valerate, was also investigated. the ear thickness was significantly increased in transgenic animals compared to wild type animals following tpa-induction. the epidermal thickness measured in histological sections of biopsies from the ear skin was also significantly increased in transgenic animals. furthermore, increased dermal vascularisation was observed in the histological sections of the ear skin. a marked infiltration with cd3-positive cells was observed in both dermis and epidermis, and this was highly correlated with the increase in epidermal thickness. finally, topical treatment with betamethasone-17-valerate significantly reduced the ear swelling and epidermal thickness. we conclude that over-expression of vegf in the epidermal basal layer plays an important role in skin inflammation and for the development of important psoriatic hallmarks. the model may furthermore be used as an in vivo screening tool for novel anti-psoriatic compounds. background and aims: the diabetes-prone bb (bbdp) rat spontaneously develops insulin-dependent diabetes resembling type 1 diabetes (t1d) in man. the bbdp rat is t-cell lymphopenic with a profound lack of regulatory t cells. the recent thymic emigrants in bbdp rapidly undergo apoptosis unless rescued from apoptosis by tcr stimulation. the increase in apoptosis is due to a frameshift mutation in gimap5 which causes a severe truncation of the protein. the mutation is the strongest genetic factor for rat t1d. we aim to detect how gimap5 affects the lifespan of t cells. results: overexpression in c58 cells of both wt gimap5 and gimap5 with the bbdp mutation causes an increase in apoptosis -the latter with a very rapid onset. reduction of human gimap5 by rna-interference in jurkat cells did not affect the number of apoptotic cells. overexpression of human gimap5 causes apoptosis in jurkat cells and primary naã¯ve t cells but not in activated t cells. finally, gimap5-mrna is upregulated in in vitro activated human primary t-cells (detected by rt-pcr). conclusions: based on the phenotype of the bbdp, rat gimap5 was expected to be anti-apoptotic. however, we report here that overexpression of both mutated and wt gimap5 causes rapid death of the cells. this suggests that gimap5 is pro-apoptotic. the results with human wt gimap5 support this conclusion: recently, much focus has been on the cellular cd38/ cadpr signaling system during inflammatory processes. the cd38/cadpr system has been shown to be regulated by interferon, estrogen and the proinflammatory cytokine il-8. to our knowledge, the expression and function of the cd38/cadpr signaling system in the human detrusor muscle have not been described. cd38 protein expression in cultured (explant technique) human detrusor smooth muscle cells (hdsmc) was demonstrated by western blot (wb) and confocal microscopy (cm). cytosolic free ca2+ concentration ([ca2+] i) in hdsmc and isometric force in human detrusor strips were measured by spectrofluorometry and myograph technique, respectively. wb and cm showed that hdsmc expressed cell surface cd38 which could be upregulated by il-8 (20 ng/ml). in hdsmc briefly activated with il-8 (20 ng/ml) cadpr induced a rapid, transient dose-dependent increase in [ca2+]i. cyclic adpr-mediated ca2+ increase was greatly reduced in ca2+ free medium suggesting ca2+ entry as well as ca2+ release. cyclic adpr -elicited ca2+ increase was mimicked by 3-deaza-cadpr, and blocked by 8-bromo-cadpr, a cadpr antagonist, but not by nifedipine or verapamil. in the presence of il-8, cadpr caused concentration-dependent relaxations of detrusor muscle. we report for the first time that 1) hdsmc express cell surface cd38, 2) the expression and function of cd38 are augmented by il-8, 3) externally added cadpr elicited a rapid, il-8 -dependent, and 8-bromo-cadpr-inhibitable ca2+ mobilization, 4) cadpr induces relaxation of human detrusor muscle. the study indicates a role of cd38/cadpr in human urinary bladder inflammation. miao lin is a formulation of sen miao san and lingzhi that consists of cortex phellodendri, atractylodisa rhizoma, radix achyranthis bidentatae, and ganoderma lucidum. these ingredients are reported to have anti-inflammatory and analgesic effects. in this study, we have investigated the anti-arthritic property of miao lin in an animal model of arthritis induced by unilateral injection of freunds complete adjuvant (fca) into rat knees. contents of the miao lin capsules were dissolved in saline and administered to the rats daily by intraperitoneal or oral route for 7 days before induction of arthritis and 7 days after. extension angle, size and blood flow of the rat knee joints were measured to give indexes of algesia, oedema, and hyperaemia, respectively. assessments of the extent of cell infiltration, tissue proliferation, and erosions of cartilage and bone provided additional indexes of the arthritis condition. single unilateral injection of fca into rat knees produced significant oedema, algesia, hyperaemia, immune cell infiltration, synovial tissue proliferation, and erosions of cartilage and bone in the ipsilateral knees compared with the contralateral saline-injected knees. intra-peritoneal injection of miao lin (50 mg/kg/day) suppressed oedema, pain and hyperaemia in the inflamed knees, and oral administration (500 mg/kg/day) suppressed oedema and hyperaemia. histological examination showed that both routes of administrations of miao lin reduced immune cell infiltration and erosions of cartilage and bone, and intraperitoneally administered miao lin also attenuated synovial tissue proliferation. these findings suggest treatment with intra-peritoneal or oral miao lin could provide significant anti-arthritic effects. an extract of the anti-arthritic thermalife cream contains 13 trace elements. diffusion studies were undertaken to assess the permeability of human epidermis to the trace elements. non-penetrating trace elements were discarded from the test formula (t2), and compared with the original formula (t1) for in vitro anti-inflammatory efficacy (tnf-a secretion in lps-challenged human monocytes). methods: human epidermis was mounted in vertical franz type diffusion cells (stratum corneum facing up). t1 cream (n=4) or no cream (n=4) was applied to the donor compartment of diffusion cells, with pbs in the receptor compartment (3.0ml ; stirred continuously at 37 c). 240 min after administration the receptor fluid was analysed for presence of metal ions by icp-ms. a replication study used a different skin donor. subsequently, human monocyte cultures (10% fcs, 5% co2) were either stimulated with 500ng/ml lps (e.coli 0111:b4,) or not in the presence of 10% t1, 10% t2, or no treatment.24 hours after incubation, culture media were collected, centrifuged, and assayed (cytokine elisa). statistical analyses used a treat by lps anova (p < 0.05). results: zinc was the only trace element to penetrate the human epidermis significantly. both formulations strongly suppressed lps-induced tnf-a secretion. t2 with zinc only was more effective than t1 (treat:f2,12 = 57.13, p < 0.0001; lps:f1,12 = 245.47, p < 0.0001; treat by lps:f2,12 = 70.01, p < 0.0001). conclusions: anti-tnf efficacy from thermalife extracts was retained with zinc chloride as the only trace element. (1) (1) osprey pharmaceuticals limited, canada (2) probetex, inc., texas, usa the ccl2/ccr2 chemokine/receptor axis, infiltrating monocytes/macrophages (m/m), th1 cells and mast cells play a pathological role in tissue damage and fibrosis in kidney diseases. the eradication of the supernumerary activated leukocytes should curb the production of inflammatory mediators and modulate chemokine communications, thus ameliorating disease. a recombinant fusion protein comprised of the human ccl2 chemokine fused to a truncated form of the enzymatically active a1 domain of shiga toxin has been developed. the ccl2 portion binds specifically to ccr2-bearing leukocytes and enters the cells, where the sa1 portion inhibits protein synthesis. the compound was tested in a model of anti-thymocyte serum (ats)-induced mesangioproliferative glomerulonephritis. male rats were injected with ats on day 0 and treated intravenously with vehicle, 50 or 100mg/kg of the recombinant protein q2d from day 2 until day 8. urine and blood collections were made prior to ats injection and on days 5 and 9. animals were sacrificed on day 9. no treatment related effects on body weight or signs of clinical toxicity were observed. urine protein levels were decreased in treated animals. histopathological analyses of kidney sections revealed maximum reductions of 40, 36, 38, and 28% for glomerular lesions, m/m count, fibronectin and âµ-smooth muscle actin, respectively. the latter two proteins are markers for extracellular matrix synthesis and mesangial cell activation, respectively. these results indicate a significant renal-protective effect in this model of nephritis. further observations suggest that different chemokine-ligand toxins may be used in the treatment of diseases modulated by other chemokine/receptor axes. inflamm. res., supplement 3 (2007) posters immuno-depletion followed by fluorescence-activated cell sorting based on the cell surface expression of the sca-1 antigen. such isolated cells can subsequently be cultured and differentiate towards the osteogenic, adipogenic or chondrogenic linage in vitro. using this model we investigated the influence of the proinflammatory cytokines, tnfa or il-1b, on early osteogenesis in vitro. under osteogenic conditions, il-1b was found to inhibit cell proliferation in a dose dependent manner, whereas tnfa exhibited no effect. histochemical examination revealed the presence of either tnfa or il-1b to dramatically decreased mineralization in a dose dependent manner. q-pcr analysis indicated that in the presence of il-1b, despite increased expression of bone-specific alkaline phosphatase (akp2) mrna, levels of other osteogenesis markers (runx2, col1a and sp7) were decreased. in the presence of tnfa, levels of akp2, runx2 and sp7 were all decreased. our findings indicate that the influence of early mesenchymal progenitor cells on bone remodelling may be substantially altered in the presence of proinflammatory cytokines. coronary artery disease (cad) is characterized by enrichment of inflammatory cells in the vessel wall. we hypothesized that an altered transmigration and activation of monocytes may contribute to plaque build up. in vivo transmigration was studied by use of a skin blister model. blisters are raised by suction and cells are analysed the following morning (0h blister) and after additional ten hours of incubation with pbs or autologous serum, corresponding to intermediate and intense blister. monocytes were analysed by flow cytometry for the expression of cd11b, before and after in vitro fmlp stimulation. chemokines in serum and blister fluid was analysed in parallel. cd11b expression on resting monocytes harvested from 0h blister was lower in patients as compared to controls (p=0.001). lower expression of cd11b in patients was also observed in the intermediate and intense blisters after stimulation with fmlp (p=0.04 and p= 0.005, respectively). the number of transmigrated cells was similar in both groups and increased with the intensity of inflammation. serum concentration of mip-1âµ was higher among patients (p=0.01) and similar levels were seen in blister fluids. concentration of mcp-1 was similar in both serum and blister fluid. we demonstrate that monocytes from patients with cad have a reduced expression and ability to up-regulate the adhesion molecule cd11b at sites of inflammation. these differences may modulate events related to the transmigration process and indicate a changed activation pattern. to which extent this feature might contribute to monocyte entrapment at the atherosclerotic site needs further studies to be delineated. in inflammation, nitric oxide (no) is produced by inducible nitric oxide synthase (inos) induced by bacterial products and cytokines, and no acts as a regulatory and proinflammatory mediator. one of the anti-inflammatory mechanisms of glucocorticoids is the inhibition of no production. the aim of the present study was to investigate the mechanisms how glucocorticoids inhibit inos expression and no production. dexamethasone and a dissociated glucocorticoid ru24858 inhibited no production, and inos protein and mrna expression in murine j774 macrophages exposed to bacterial lipopolysaccharide (lps). in the presence of a glucocorticoid receptor (gr) antagonist mifepristone, the effects of dexamethasone and ru24858 on no production were reduced. the role of histone deacetylation in the glucocorticoid effect was studied by using three inhibitors of histone deacetylases (hdacs); non-selective trichostatin a and apicidin, and hdac1 selective mc1293. hdac inhibitors reversed the effects of dexamethasone and ru24858 on inos expression or no production. stably transfected a549/8 cells containing human inos promoter were used in promoter-activity studies. cytokine-induced inos promoter activity was inhibited by dexamethasone and the inhibitory effect was reversed by trichostatin a. these results suggest that glucocorticoids inhibit inos expression and no production by a gr-mediated and gre-independent manner possibly through histone deacetylation and transcriptional silencing. we are investigating mechanisms involved in tnfainduced hyperalgesia in the mouse paw. previously, we have seen that tnfa causes a trpv1-dependent bilateral hyperalgesia. here we investigate the role of cox in this process. female cd1 mice (25-30g) were given intraplantar injections (i.pl.) of tnfa (10pmol/50microl) and tyrode (as vehicle, contralateral paw; 50microl). thermal hyperalgesic thresholds were measured using the hargreaves technique before and 1h after injection. indomethacin (20mg/kg) was co-injected with tnfa whilst contralateral paw was injected with tyrode and corresponding amounts of indomethacin vehicle (5% nahco3). another group of mice were injected with tnfa i.pl. plus 5% nahco3 with the contralateral paw injected with tyrode plus indomethacin (20mg/kg). results are expressed as mean ae s.e.m and statistical analysis performed using students t-test. tnfa (10pmol) leads to significantly reduced (p<0.05 compared to baseline values) paw withdrawal latency in both paws 1h after injection i.e bilateral hyperalgesia. however, local injection of indomethacin (20mg/kg) with tnfa prevented this reduction in paw withdrawal latency in both paws suggesting that prostaglandins are important in the development of hyperalgesia. interestingly, indomethacin co-injected with tyrode in the contralateral paw did not prevent the reduction in paw withdrawal latency in both paws. the same results were seen using the selective cox-2 inhibitor, nimesulide. in conclusion, cox-2 derived prostaglandins are important in the development of hyperalgesia. local cox-2 inhibition at the site of tnfa-induced inflammation prevents the bilateral hyperalgesia suggesting that local prostaglandin production is sufficient to cause hyperalgesia in the contralateral paw. hydrogen sulfide (h2s) is synthesized naturally in the body from cysteine by cystathionine g lyase (cse). h2s has been variously reported to exhibit both pro-and antiinflammatory activity. in an attempt to obtain further information about the role of h2s in inflammation we examined the effect of dexamethasone on lipopolysaccharide (lps)-mediated endotoxic shock. male sprague dawley rats (240-280g) were administered dexamathasone (1 mg/kg, i.p.) either 1 h before or 1 h after lps (4 mg/kg, i.p.) injection. animals were killed 3 h after lps administration and plasma and tissues harvested. as expected, lps injection significantly increased plasma tnfa and il-1b as well as liver and lung myeloperoxidase (mpo) activity. lps also increased plasma nitrate/ nitrite (nox), h2s concentration and liver and kidney h2s synthesis from exogenous cysteine indicative of upregulation of cse in these tissues. either pre-or post treatment of animals with dexamethasone reduced signs of inflammation and also reduced the increase in plasma h2s and tissue h2s synthesizing activity. in separate in vitro experiments, exposure of rat peripheral leucocytes to lps (100 ng/ml, 3 h, 37oc) resulted in upregulation of both cse and inos (measured by western blot). dexamethasone (100 nm) significantly (p<0.05) reduced expression of both cse and inos. these data provide further evidence that h2s is synthesised during endotoxic shock and suggest, for the first time, that at least part of the anti-inflammatory effect of dexamethsone may be related to inhibition of h2s production. (1), u jalonen (1), h kankaanranta (1), r tuominen(2), e moilanen (1) (1) the immunopharmacology research group, medical school, university of tampere and tampere university hospital, tampere, finland (2) the division of pharmacology and toxicology, faculty of pharmacy, university of helsinki, helsinki, finland tristetraprolin (ttp), also known as nup475, tis11, g0s24 and zfp36, is a factor that binds to 3utr of mrna of some transiently expressed inflammatory genes and regulates mrna stability. ttp has been implicated in the posttranscriptional regulation of e.g. tumor necrosis factor a and inducible nitric oxide synthase. however, the regulation of the expression of ttp itself is largely unknown. in the present study, we investigated the role of classical protein kinase c (cpkc) isoenzymes in the regulation of ttp expression. in j774 macrophages ttp expression is induced by lipopolysaccharide (lps) and this can be further enhanced by addition of 100 nm phorbol myristate acetate (pma). this additive effect of pma on ttp was abolished by a prolonged preincubation with a higher s442 inflamm. res., supplement 3 (2007) posters concentration of pma for 24 h, which also downregulated the expression of pkca, pkcbi and pkcbii isoenzymes. pkc inhibitors ro318220 (inhibits pkcb, & and e), gã�6976 (inhibits pkca, b and &) and cgp53353 (inhibits pkcbii) reduced lps + pma -induced ttp protein and ttp mrna expression. pkcbii inhibitor cgp53353 did not affect ttp mrna half-life and therefore we measured the effects of cgp53353 on the activation of transcription factors involved in ttp expression. cgp53353 had no effect on the activation of nf-kb, egr1 or sp1. in contrast, cgp53353 reduced the activation of transcription factor ap-2, which may explain its inhibitory action on ttp expression. the results suggest that pkcbii is involved in the regulation of ttp expression in activated macrophages, possibly through the activation of transcription factor ap-2. the most widespread gracilaria verrucosa in the sea of korea is the attached form of red algae growing on a rockly substrate. in this study, we isolated fourteen compounds from g. verrucosa and investigated their inhibitory effect on the production of inflammatory markers (tnf-a il-6, il-1 and no) in raw264.7 cells. among them, 10-oxooctadec-8-enoic acid and 11-oxooctadec-9-enoic acid inhibited the production of tnf-a, il-6, il-1 and no at the concentration of 20 mg. also, these two compounds showed inhibitory activity on the mrna expression and protein level of inflammatory markers (tnf-a il-6, il-1 and inos) in a dose-dependent manner. these results suggest that g. verrucosa may have anti-inflammatory activity through the inhibition of inflammatory cytokines and inos. lene jensen(1), p hjarnaa (1), j fensholdt (1), p-p elena(2), k abell (1), tk petersen (1) (1) discovery, leo pharma, ballerup, denmark (2) iris pharma, la gaude, france angiogenesis is known to play an important role in many inflammatory diseases including arthritis. additionally, inflammation is known to play a role in the angiogenesisdriven disease age-related macular degeneration (amd). we have synthesized a potent angiogenesis inhibitor, leo-a, targeting kinases related to angiogenesis, e.g. vegfr-2. additionally, leo-a has potent effects on a broad panel of other kinases, whose normal functions are related to inflammation and immunity. the compound was tested systemically in inflammatory in vivo models in mice and rats. the in vivo models selected include the cia arthritis model (mice and rats), the local gvh rat model, the lps induced tnfa model (mice and rats), the anti-cd3 induced il-2 response mouse model and the rat argon laser-induced choroidal neovascularisation (chnv) model, a model for amd. the following results were obtained after systemic treatment with doses of up to 30 mg/kg i.p. or 50 mg/kg p.o. once daily: in the local gvh model, leo-a significantly inhibited the growth of the local lymph node by 50 %. in the cia model, leo-a had a significant inhibitory effect on the progress of arthritis both in mice and in rats when dosed early (pretreatment). in the lps induced tnfa model in mice, high doses of leo-a were found to inhibit the tnfa release. in the chnv model, a significant effect was obtained following systemic treatment. in conclusion, leo-a has an interesting profile for the treatment of diseases in which inflammation and angiogenesis are involved. mice lacking pi3kg and d isoforms display severe impairment of thymocyte development, but the outcome of this developmental defect has not been investigated. we show here that mice harbor pi3kg gene depletion and pi3kd kinase-inactive mutation, pik3cgd koi, exhibited thymus atrophy, similar to previously reported pi3kg and d double knockout (p110g/d-/-) mice, and profound peripheral lymphoid depletion, markedly reduced lamda chain production and seemingly lymphopenia-provoked effector/memory t cell activity. in particular, serum igg1/ igg2a ratio and ige level were elevated in pik3cgd koi mice corresponding to a skewed th2 profile in vitro. histological analysis revealed eosionophil-and t celldominated inflammation in stomach and salivary gland as well as occasionally other organs of pik3cgd koi mice, but organ-specific auto-antibody was not detected in circulation. on the contrary, when mature wt t cells were treated with pi3k d or together with pi3k g selective inhibitors, while th1 cytokines were suppressed th2 cytokines were not augmented in vitro. thus, t cell development, but not peripheral t cell proliferation or cytokine production, requires cooperativity of pi3kg and d. genetic inactivation of these two isoforms leads to the development of severe lymphopenia, skewed type 2 ig and t cell response, and increased susceptibility to eosinophilic multiple organ inflammation; whereas pharmacological inhibition at the adult stage would probably not promote th2 reaction but attenuate th1 medicated disorders. platelet activating factor (paf) is an important mediator in several pathophysiological processes. paf receptor activation can causes a series of cellular and tissue modifications and can lead to the production and/or release of diverse molecules, including cytokines, chemokines and receptors, amongst others, which are capable of amplifying the inflammation. paf can up-regulate kinin b1 receptor expression by various mechanisms. our aim was to investigate the role for kinases in paf-induced kinin b1 receptor up-regulation. wistar rats were treated with paf, or left untreated as controls, 6h before i.d. injection of 0.1ml pbs containing des-arg9-bradykinin (dapk, 100nmol right hind paw) and 0.1ml pbs (for control, left paw). various kinase inhibitors were administered to the rats after paf treatment and oedema was measured by the use of a plethysmometer (ugo basile) 10-120 minutes after dapk-injection. oedema was expressed in ml as difference between right and left paws.additionally paw samples were taken for western blot analysis for total and phosphorylated forms of jnk and erk1/2. dabk-induced paw oedema after pafinjection is significantly inhibited by the selective jnk sp600125 and erk1/2 pd98059 inhibitors. western blot analysis shows that phosphorylation of jnk and erk1/2 is important in the up-regulation of b1 receptors. our results clearly show that the phosphorylation of both erk1/2 and jnk mapkinases is an important step for the in vivo up-regulation of b1 receptors by paf. however, the exact mechanisms (transcriptional and post-transcriptional) by which paf can trigger kinase phosphorylation and then up-regulate the b1 receptor require further investigation. continued interest in development of small molecule inhibitors of p38 mitogen-activated protein (map) kinase is based on the central role this enzyme plays in inflammatory cell signaling. activation of p38 leads to increase production of pro-inflammatory cytokines such as tnf-a and il-1b making it an prominent target for antiinflammatory drug discovery. a virtuell screening approach identified 3-(2-chlorophenyl)-6-((4-methoxyphenoxy)methyl)[1, 2, 4] triazolo [3,4-b] [1, 3, 4] thi adiazole as a potential hit. this was confirmed by synthesis and testing. to explore further sar, a first set of derivates was prepared by cyclization of the 5-substituted-4-amino-3-mercapto-4h-1,2,4-triazoles with carboxylic acids in presence of phosphorus oxychloride. the synthetic strategy used allows both variation at position 3 and 6. synthesis and sar will be presented. cytokines like il-1b and tnfa play central roles in inflammatory diseases like rheumatoid arthritis. production of cytokines in monocytes, macrophages and other cells is triggered by factors such as lps, uv-light, osmotic and cellular stress or physical and chemical attraction. in particular il-1b and tnfa are key regulators as they amplify inflammatory stimuli in cells by induction and upregulation of further cytokines. involved in this signal pathway, p38mapk as a pivotal enzyme is considered to be a validated drug target and therefore, p38mapkinhibitors are of therapeutical interest. in this study, we developed and validated an economic in vivo whole blood assay for optimization and characterization of small molecule p38mapk-inhibitors with promising in vitro activity. the assay procedure involves defined blood cell stimulation by lps and isolation of tnfa or il-1b, which are subsequently quantified by tmb-elisa technique via photometric measurement. the validation of the assay conditions involved well characterized p38mapk inhibitor sb203580 and a highly active compound developed in our lab. a data set was generated by determining 18 whole blood samples consisting of in each case three male and female individuals on three different days. statistical methods were used to analyze specificity, baseline-peak correlations, repeatability, robustness as well as gender specific intra-and interindividual differences. p38 mitogen-activated protein (map) kinase is required for the biosynthesis and release of pro-inflammatory cytokines il-1 and tnf a. inhibition of p38 map kinase could reduce the expression of these cytokines and is therefore a promising target for the treatment of many inflammatory disorders, like rheumatoid arthritis and inflammatory bowel disease. trisubstituted pyridinylimidazoles are potent inhibitors of the p38 map kinase. scope of this work was to investigate 2-thio-ether moiety as a position to link the inhibitors to macrocyclic drug carriers. we synthesised 2-alkylsulfanyl, 4-(4-fluorophenyl), 5-(2-aminopyridin-4-yl) substituted imidazoles as p38 map kinase inhibitors. as substitution at this pyridinyl moiety allows both increase the anti-inflammatory activity as well as selectivity. the synthesis and biological testing of effective the 2-aminopyridin-4-yl imidazoles with low inhibitory concentrations are described. biological data demonstrate both the imidazole derviates and the linked imidazoles lead to highly efficient inhibitors.variation at the 2-thio-ether moietywhich interacts in the phosphate binding region of the enzyme -with polar groups shows no loss of activity. studies underscored the importance of hydrogen bonding with the backbone nh group of met109, for inhibitory activity. less clear is the importance of the hydrogen bond between n3 of the imidazole ring and lys53 of p38 map kinase.to investigate the role of lys53 in interacting with the scaffold we prepared two sets of 4,5diaryl-substituted isoxazoles. these data suggest a dynamic interaction of the core heterocycle with lys53, contrary to the observation on the compound vk-19911 and p38 map kinase, that a nitrogen atom bearing a lone pair in position 3 of the imidazole ring could be necessary to avoid a repulsive interaction with the positively charged side chain of lys53 rather than to form an attractive interaction with p38 map kinase. to complete our study, we focused on the interdependency of biological effects exerted by substitution at the pyridine ring for a series of 3-substituted and unsubstituted 4,5-diarylisoxazoles investigating the interaction with the hydrophobic pocket ii of p38. these data indicate that the isoxazole has better scaffold properties compared with imidazoles, suggesting that heterocycles that are stable as regioisomers, such as isoxazole (in contrast to tautomeric imidazoles), are worthy of further investigations. despite of the intensive research effort, sepsis is still the leading cause of death in critically ill patients. it is a consequence of acute inflammatory response to lipopolysaccharide (lps), a major component of the outer membrane of gram-negative bacteria. natural products are known sources of bioactive components exerting antioxidative and anti-inflammatory effects. in this study, we investigated the effect of ferulaldehyde (fa), a natural compound of red wine, on lps-induced endotoxic shock in mice and on lps-stimulated murine macrophage-like raw 264.7 cells. treatment of c57bl/6 mice with fa significantly attenuated the lps-induced inflammatory response in the gastrointestinal tract, and decreased the level of the two major pro-inflammatory cytokines tnf-a and il-1b in the serum. the serum level of the anti-inflammatory cytokine il-10 was higher in mice treated with fa and lps compared to lps treatment alone. lps-induced phosphorylation and thereby activation of akt, and jnk was also strongly inhibited by fa treatment whereas the phosphorylation level of erk1/ 2 and p38 mapks remained unaltered. activation of nuclear factor-kappab (nf-kb) in liver of fa-treated mice were significantly suppressed. although fa had no effect on the production of inflammatory cytokines, or on inhibition of signal transduction pathways in raw 264.7 cells either, it decreased the lps-induced ros and nitrite production in a dose-dependent manner. our results suggest that fa has antioxidative and anti-inflammatory activities by enhancing antioxidative defense systems, which in turn decrease inflammatory cytokine response and suppress nf-kb activity via the down-regulation of akt and jnk. myeloperoxidase (mpo), stored in the azurophilic granules of the neutrophil granulocyte, is a heme enzyme with the unique property of oxidising chloride ion to the powerful reactant hypochlorous acid in the presence of hydrogen peroxide. therefore, it plays an important role at inflammatory loci in killing invading micro-organisms. on the other hand, hypochlorous acid reacts with a variety of biomolecules as amino acids or membrane lipids and causes therefore host tissue damage resulting in widespread diseases like atherosclerosis or rheumatoid arthritis, e.g. the formation of chloramines from taurine or ammonium ions is one possibility to reduce tissue toxicity while maintaining bactericidal properties. membrane charge alterations during apoptosis provide docking sites for the kationic enzyme myeloperoxidase and this close contact to the membrane lipids opens the possibility for lipid alteration pathways even though these reactions will normally not take place because of their slowness. we investigated alterations in phospholipids after reaction with hypochlorous acid or the myeloperoxidase-hydrogen peroxide-chloride system by matrixassisted laser desorption and ionisation time-of-flight mass spectrometry (maldi tof ms). specific reaction products play an important role in the modulation of the immune response. comparative pathobiology of the disease is also discussed within the context of current human and animal reoviral disease models. objectives: to study the safety and efficacy of infliximab plus leflunomide combination therapy in adult rheumatoid arthritis (ra). methods: twenty patients with active ra received leflunomide 100 mg for 3 days followed by 20 mg daily for 32 weeks. at week 2 all patients started infliximab 3 mg/kg, and received a further four infusions at weeks 4, 8, 16 and 24. results: the commonest adverse event was pruritis associated with an eczematous rash. there was no relationship between the serum concentration of a77 1726, the active metabolite of leflunomide, and adverse events. the mean disease activity score (das28) fell from 7.18 at week 0 to 5.18 (p<0.0001) at week 4 and remained between 3.85 and 4.85 up to week 32. in those patients remaining on treatment, more than 80% achieved an acr20 response from week 8 to week 28, and up to 46% achieved an acr70 response. conclusions: infliximab plus leflunomide combination therapy appears to be highly efficacious in the treatment of adult ra. however, widespread use may be limited by adverse events, which were common and in some cases severe. objective: the transcription factor nuclear factor-kb (nfkb) regulates the expression of proinflammatory cytokines such as tnfa and il-1 those play pivotal roles in pathogenesis in rheumatoid arthritis. parthenolide, a sesquiterpene lactone, was reported to inhibit the dna-binding of nfkb. the objective of this study is to investigate the potential of parathenolid to inhibit the pathogenesis of collagen-induced arthritis. methods: mice were injected i.p. with a cocktail of 4 anticollagentype ii mabs on day 0, followed by i.p. injection of lps on day 3 to induce anti-collagen mab-induced arthritis. the mice were orally administrated with parathenolide (50 mg/kg/day) starting on the day of first immunization (day 0) in prophylactic treatment group and after the onset of arthritis (day 4) in the therapeutic treatment group. clinical disease score, radiographic and histological scores were evaluated. mrna expression of il-1b and tnfa in the affected joints were measured by real-time pcr. results: clinical disease scores were significantly reduced both in prophylactic treatment group (7.4ae3.7) and therapeutic treatment group (7.25ae3.1) compared to untreated group (13.6ae2.7, p = 0.0163 and p = 0.0084 respectively). histological scores of joint destruction were significantly reduced in prophylactic treatment group compared to untreated mice (p<0.05). steady state mrna levels of il-1b and tnfa in isolated joints were significantly decreased in prophylactic treatment group compared to untreated mice (p<0.05). the results in this study suggest that nfkb is an important therapeutic molecular target for treatment of inflammatory arthritis. fibrinogen is a soluble plasma glycoprotein, multifunctional, that participates in haemostasis and has adhesive and inflammatory functions through specific interactions with other cells. the concentration of this glycoprotein increase in inflammatory conditions. a fundamental paradigm involved in the acute inflammatory response is neutrophil migration to the affected tissues to mount an initial innate response to the aggression. the objective of this study is to characterize how fibrinogen modulates the pattern of neutrophil activation. neutrophils from healthy donors were isolated from peripheral venous blood and loaded with the fluorescent probe dihydrorhodamine 123 (1ã¬m) to detect oxygen free radical production. the cells (1,0x 106 cell/ml) were then incubated with a range of concentrations of fibrinogen (0-400mg/dl) for 15 minutes. our results show that posters inflamm. res., supplement 3 (2007) s449 fibrinogen leads to an increase in neutrophil activation as measured by free radical production. this effect becomes evident at borderline-high concentrations (300-400mg/ dl), and in some of the individuals it was possible to differentiate two subpopulations of low-responsive and high responsive neutrophils to activation by fibrinogen. we hypothesize that, in this regard, the concentrations of fibrinogen identified as a risk factor might promote the setting of an inflammatory microenvironment in the circulation and facilitate cardiovascular disease progression. cyclooxygenases (cox-1 and cox-2) are isoenzymes involved in the first steps of the biosynthesis of prostanoids. the constitutively expressed isoform cox-1 is mainly involved in homeostatic processes, while the inducible isoform (cox-2) is associated with inflammatory reactions. various in vitro assays have been developed in order to define the selectivity against cox-1 and cox-2 of nonsteroidal anti-inflammatory drugs (nsaids). however, these in vitro assays can give discordant results related to several parameters. the aim of this study was to optimize and standardize two distinct in vitro methodologies to evaluate new nsaid candidates. first, in an enzymatic cox assay, the arachidonic acid concentration (aa; cox substrate), and the species of cox enzymes tested (ovine vs. human), two factors able to conceal the anti-cox activity of nsaids, have been evaluated and optimized. next, we developed an in vitro cell-based assay using human whole blood depleted from plasma and reconstituted in saline solution. this cell-based assay allows concomitant measurement of anti-cox-1 and anti-cox-2 effects by prostaglandin e2 (pge2) measurement after a23187 (calcimycin) and bacterial lipopolysaccharide (lps) stimulations, respectively. both assays have been calibrated and compared by testing 7 reference nsaids, selective or not for cox-1 or cox-2. fifty % inhibitory concentration (ic50) values against cox-1 and cox-2 and cox-2:cox-1 ratios obtained were in accordance with the previously described nsaid specificity and coherent between both assays (r= 0.93). in conclusion, both in vitro assays are optimized to determine the efficiency and the selectivity of new nsaid candidates against human cox-1 and cox-2. for increasing the success of preclinical drug candidate molecules, there is a need for translatable animal models. the human serum skin chamber technique and the rodent carrageenan induced air pouch model are two wellestablished methods for measuring interstitial inflammation in respective species. we aimed to study the translational aspects of these models. material and methods: in humans, epidermal skin chambers were stimulated with autologous serum for 10 hours. in rats, a dorsal subcutaneous air pouch was stimulated with autologous serum on day 6. the inflammatory response was measured after 4, 8 and 24 hours. the cellular distribution of in vivo transmigrated cells, the expression of cytokine receptors, adhesion molecules and inflammatory mediators was investigated. results: at 8/10 hours the cellular distribution was similar in air pouch and skin chambers. the major population constituted of granulocytes, followed by monocytes/macrophages and lymphocytes. both in human and rats the concentrations of mpo and mcp-1 were increased. furthermore, transmigrated cells displayed a different chemokine receptor pattern. in rats transmigrated cells expressed cd11b, were cd45lo, ssclo and rp-1+ (granulocyte marker). in humans, transmigrated granulocytes expressed cd16 and cd11b. these cells had a significantly higher cd11b expression compared to corresponding cells in peripheral circulation. our results indicate that the serum induced human skin chamber technique and rodent air pouch model translate well to each other. these models may be useful for bridgingpreclinical and clinical drug discovery. furthermore, they may work as translatable proof of mechanism (pom) models for drug candidates targeting different inflammatory components. objectives: to analyse if neopterin (a by-product of activated macrophage metabolism) is elevated in patients with systemic inflammatory insult at the time of ischemic stroke. material and methods: we investigated 86 consecutive patients with mean age 67ae7.8 years who were admitted within 24 h after ischemic stroke. a control group of 37 patients with mean age 58ae4.9 years without ischemic stroke was also tested. measurement of serum neopterin levels were performed using enzyme linked immunosorbent assay. results: patients with acute ischemic stroke had significantly higher serum levels (mean value+sd) of neopterin than those without acute ischemic stroke: 9.6ae1.2 and 7ae0.8 nmol/l. correlation analysis revealed p<0.01. discussion: immune mechanisms contribute to cerebral ischemic injury. the finding of higher serum levels of neopterin, which is regarded as a humoral component of the immune-mediated inflammatory response sustains the hypothesis that patients with ischemic stroke may show higher levels of inflammatory markers like neopterin. our results indicate increased macrophage activation after ischemic stroke. in patients with stroke it has been shown that neopterin was determinant of endothelium-dependent vascular dysfunction. however, these preliminary results need be confirmed by controlled studies. produced a marked (p<0.01) reduction in the number and duration of ventricular tachycardia (vt) during both ischemic and reperfusion phases. the total number of ischemic ventricular ectopic beats (vebs) reduced from 667ffae116 in the control to 66ffae22 at the concentration of 50 ffmg/ml (p<0.001). in the ischemic phase, cynodon dactylon (50 ffmg/ml) also decreased the incidence of vt from 100% (control) to 33%. in addition, incidences of reperfusion-induced vt, total vf and reversible vf duration were significantly lowered by the same concentration (p<0.01 for all). the results show that cynodon dactylon has a protective effect against i/r-induced cardiac arrhythmias in isolated rat hearts. regarding the presence of flavonoid glycosides confirmed during phytochemical screening of the extract and their potential role in the scavenging of oxygen free radicals, it seems that the cardioprotective effects of cynodon dactylon probably is due to its anti-inflammatory properties.key words: cynodon dactylon; arrhythmias; anti-inflammatory; isolated heart; rat objectives: intestinal ischemia-reperfusion (iri) is well known to be associated with distant organ dysfunction; but no evidences to date have focused either the brain or skeletal muscle. we thus decided to investigate the effects of iri on nos and cox isoforms, neutrophil infiltration (mpo), lipoperoxidation (tbars) and protein tyrosine nitration (nt) in different brain areas and diaphragm muscle of wistar rats. methods: iri comprised the occlusion of superior mesenteric artery during 45 min followed by 2 h of reperfusion. sham animals were submitted to the surgical procedure with no interference on the blood flow. results: iri resulted in increased expression of mrna for nnos (cortex) and cox-2 (hypothalamus) associated to a marked reduction of ca2+-dependent nos activity in cortex, hypothalamus and hippocampus (but not in cerebellum). tbars contents were also reduced in cortex and hypothalamus. neither mpo activity nor nt was altered by iri in the brain. diaphragms from animals with iri exhibited increased mpo and ca2+-dependent nos activities, as well as tbars content and nt. in contrast, enos protein expression and both gene and protein nnos expression were reduced. no effects were observed on cox isoforms or enos gene expression. conclusions: these findings suggest that, within the first 2 h of reperfusion following intestinal ischemia, an oxidative response is observed in diaphragm, involving both lipid and protein modifications. in the cns, distinctive susceptibility to the iri seems to occur in the different areas, probably as a defensive strategy aimed to counteract the iri-mediated systemic injury. anne-sofie johansson (1), h qui (2), m wang (2), i vedin (1), jz haeggstrã§m (2), j palmblad (1) ( (1), r carnuccio(2), p romagnoli (3), f rossi (1) (1) second university of naples, italy (2) university of naples, italy (3) university of florence, italy we previously found that several inflammatory markers, e.g., nuclear factor-kb (nf-kb), were increased and a neointima was formed in a model of carotid surgical injury (1) . the purpose of the present study was to determine if chronic treatment with rosiglitazone protects rat carotid artery from surgical injury induced by an incision of the vascular wall. to this aim we measured cox-2, nf-kb, platelet aggregation and neointima formation in rats administered rosiglitazone (10 mg/kg/ die, by gavage) for 7 days before carotid injury and 21 days after injury. control rats received physiological solution. 14 days after injury cox-2 expression, evaluated by western blot, was significantly lower in the treated carotid versus controls (p<0.0001). rosiglitazone also caused a significant decrease of nf-kb/dna binding activity, evaluated by electrophoretic mobility shift assay, in nuclear extracts of treated carotids at all time points considered. platelet aggregation was reduced by 30% in treated versus control carotids (p<0.0005). the influx of inflammatory cells in response to injury, monitored by electron microscopy and immunohistochemistry, was lower in treated than in control carotids starting 7 days after rosiglitazone treatment. the results indicate that rosiglitazone inhibits molecular and cellular inflammatory events induced by vascular injury. the aim of the present study was to investigate the relevance of peripheral macrophage activity for the susceptibility to the induction of experimental allergic encephalomyelitis (eae). rats of eae-susceptible dark agouti and eae-resistant albino oxford strain were immunized with guinea pig spinal cord homogenate (dagpsc and aogpsc), while non-immunized rats served as controls (danim, aonim). on day 15 after immunization rat peritoneal macrophages were tested for adherence capacity, zymosan phagocytosis and respiratory burst. macrophages from aonim rats exhibited lower adherence capacity and higher phagocytosis and h2o2 production then macrophages from danim rats. immunization decreased adherence and phagocytosis and increased h2o2 production in macrophages from ao rats, but did not influence these activities in macrophages from da rats. our results suggest that inflammatory activities of macrophages from ao rats could be considered as regulatory mechanisms connected with the resistance to eae induction ( (1), b sehnert (2), h lanig(2), s pã¤ã�ler(2), r holmdahl (3), h burkhardt (1) (1) johann wolfgang goethe university, frankfurt, germany (2) friedrich-alexander university of erlangen-nuremberg, germany (3) lund university, sweden objectives: the aim of the present study was to characterize the interaction sites between the prototypic arthritogenic murine igg mab ciic1that is highly somatically mutated and its epitope on type ii collagen (cii, aa359-369). methods: the establishment of a dynamic simulation modelling of a ciic1 single-chain fragment (scfv) in complex with the triple helical ciic1 epitope permitted structural insights into immune complex formation. the computer-based data were experimentally tested by mutations of predicted critical residues into alanine in the c1scfvs and the respective ciic1 epitope that were produced as recombinant constructs. the binding affinities of the mutated scfvs were determined by elisa and surface plasmon resonance measurements. the mutation experiments confirmed the predicted interaction sites of cii in the cdr2 and cdr3 regions of both heavy andlight chain. surprinsingly also the model prediction, that the conversion of the c1scfv sequence into the respective germline does not affect cii binding affinity (kd 3x 10-8) could be confirmed experimentally by the mutagenesis of 13(!) positions. our data indicate that potentially harmful cartilage specific humoral autoimmunity is germline encoded. the molecular modeling further demonstrate that the rigid collagen triple helix restricts the likelihood of molecular interactions with the corresponding cdrregions of the antibody considerably compared to globular antigens. these sterical constraints might provide an explanation why somatic mutations have no obvious impact on cii recognition by the arthritogenic autoantibody. moreover, the structural insights into cii-autoantibody interaction might be useful in future developments of collagenomimetic ligands for therapeutic and diagnosistic purposes. we observed a significant association between the mbp-elicited cd4+ t-cell proliferation and active brain lesions, on the one hand, and il-4, il-5 and ifn-gamma, on the other. when grown in the presence of standard serum from a healthy donor, pbmc from healthy individuals responded to mbp with a higher il-10 production than pbmc from ms patients. thus, normal pbmc respond to mbp with production of tnf-alpha, ifn-gamma and il-10, but ms is associated with enhanced tnf-alpha-, ifn-gammaand decreased il-10 responses, and disease activity is associated with mbp-induced proliferation of cd4+ t cells. (1), k goula (1), p georgakopoulos (2) (1) renal unit, st. anrdew hospital, patras, greece (2) intensive care unit, st. anrdew hospital, patras, greece background: urethritis is an infection of the urethra. most cases are sexually transmitted. haemodialysedpeople seem more prone to all kinds of urinary tract infectionsthan others. patients with underlying diabetes are also a specific population at risk. urethritis may be caused by some sexually transmitted diseases (chlamydia, gonorrhea, and ureaplasma urealyticum infections) and by the same organisms that cause urinary tract infections (e. coli or klebsiella). viral causes of urethritis include herpes simplex virus and cytomegalovirus. neisseria gonorrhoeae and c trachomatis account for most cases of urethritis in men (70%). the aim of our study was to determine all cases of urethritis of haemodialysed patients at our unit during the last five years. we also determined diabetes as a coexisting factor in the infected patients. we retrospectively reviewed all cases of urethritis of 72 maintenance haemodialysis patients at our center over the past 5 years. the diagnosis was made according to patients symptoms and signs but also using urine specimens for culture.12 patients (16.6%) from the study group were diabetic. results: 4 cases of urethritis were determined. all infected patients were diabetic. isolated microorganisms were e. coli (2 cases), enterobacter aerogenes (1 case objectives: to explore the ability to use paquinimod as a steroid sparing drug in an animal model for sle. methods: mice were initially treated with a high dose of prednisolone (2 mg/kg/day). thereafter the amount of steroid was reduced to 0.5 mg/kg/day and a low dose of paquinimod (0.2 mg/kg/day) was added. the development of glomerulonephritis was measured as hematuria during the experimental period. serum was collected for analysis of anti-dsdna antibodies. kidneys were collected and histopathological observations were performed. organ weight and lymphocyte sub-populations were assessed in the spleen. results: when treatment with high dose prednisolone was replaced by low dose prednisolone andpaquinimod a steroid sparing effect was seen in a number of variables. a significant reduction in the level of hematuria, in spleen enlargement and in the total number of cd4+, cd8+ and on cd4-cd8-t cells was observed in mice treated with paquinimod and low dose of prednisolone compared to mice treated with high dose prednisolone alone. the development of glomerulonephritis was also significantly reduced in these mice. an almost complete inhibition of anti-dsdna in serum was seen in all treated groups. conclusions: when high dose prednisolone was replaced by low dose prednisolone and paquinimod a steroid sparing effect was seen when a number of variables e.g., hematuria, t-cell sub-populations and development of glomerulonephritis were examined. this setting could be of great importance in future treatment of human sle in order to reduce the steroid dose needed in the treatment of this disease. and la(ssb). the purpose of this study was to screen for novel antibodies against cell surface antigens in primary sjs. proteins (mp) were isolated from cell membranes (hela cells), and were tested with sera from sjs patients or healthy blood donors individually in western blot (wb) at 1:100. mp were separated on 2-d gels and tested in wb (1:100) to locate the appropriate spots for mass spectrometry (ms) analysis. paraformaldehyde fixed hela cells were incubated with sera from patients or blood donors and examined by fluorescence microscopy. antigens were isolated at around 35, 50, 75, 100 kda (64 total positive/79 tested patients). the dominant antigen was at 50 kda. large quantities of endogenous proteins were obtained and the membrane fraction was enriched. one of the main obstacles to further study possible surface antigens as m3 muscarinic receptor was overcome. proteins were separated on 2d-gels and tested in wb to locate the relative spots for ms. the correct localization of the patients antibodies on the cell surface was confirmed by fluorescence microscopy. in conclusion, membrane or membrane-associated antigens were recognised by sera from sjs patients. one of them might correspond to m3 muscarinic receptor. this identification might help in developing a diagnostic assay for sjs. osamu handa, s kokura, k mizuahima, s akagiri, t takagi, y naito, n yoshida, t yoshikawa aim: various additives and preservatives are used in cosmetics, foods and medicines in order to prevent deterioration. however the precise mechanism of cytotoxicity of these additives are not known. in this study, we investigated the effects of ultraviolet-b (uvb) exposure on additives-treated human normal skin keratinocytes (hacat). most popularly used additives in cosmetics such as methylparaben (mp), octandiol (od) and phenoxyethanol (pe) were used. hacat keratinocyte was cultured in mp-containing medium for 24 h, exposed to uvb and further cultured for another 24 h. subsequent cellular viability was evaluated by fluorescent microscopy and flow cytometry using double staining method with hoechst 33342 and propidium iodide or annexin-v. same experiments were done using od and pe respectively instead of mp under same condition. in addition, gene chip analysis was performed in each group. results: uvb exposure enhances cytotoxicity of these additives even at low concentration. gene chip analysis showed that the expression of apoptosis-related genes, oxidative stress-related genes and transcription related genes were significantly upregulated in each group. these results indicate that some additives, which have been considered safe preservatives in cosmetics, may have harmful effects on human skin when exposed to sunlight. these kinases in the pathogenesis of psoriasis. recently, increased focal activation of the downstream target mitogen-and stress-activated protein kinase 1 (msk1) was demonstrated in psoriatic epidermis. the purpose of this study was to investigate msk2 and the transcription factor camp-response-element-binding protein (creb) in psoriatic skin and in cultured normal human keratinocytes. keratome and punch biopsies were taken from patients with plaque-type psoriasis. normal human keratinocytes were cultured and stimulated by interleukin-1㢠(il-1ã�) or anisomycin. some of anti-inflammatory plant flavonoids as a form of whole plant extracts have been used topically for skin inflammatory disorders. on human skin inflammation, matrix metalloproteinase-1 (mmp-1) plays a pivotal role on unbalanced turn-over or rapid breakdown of collagen molecules. in the present study, for establishing a therapeutic potential against skin inflammatory disorders, the effects of natural flavonoids on mmp-1 activity and mmp-1 expression were examined. from the results, the flavonols including quercetin and kaempferol were revealed to be strong inhibitors of human recombinant mmp-1 with the ic50s of 39.6 -43.7 ã¬m, while the flavones such as apigenin and wogonin showed weak inhibition. when the effects of flavonoids on mmp-1 induction were studied, it was found that quercetin, kaempferol, apigenin and wogonin (12.5 -25.0 ã¬m) strongly inhibited mmp-1 induction from tpa-treated human dermal fibroblasts, but naringenin (flavanone) did not. by gel shift assay, these flavonoids were also found to inhibit the activation of the transcription factor, ap-1, whereas naringenin did not. among mapks, quercetin inhibited the extracellular signal-regulated protein kinase (erk) and p38 mapk activation, and kaempferol inhibited the p38 mapk and c-jun n-terminal kinase (jnk) activation. on the contrary, the flavones and naringenin did not inhibit the activation of these three mapks. all these results indicate that the capacity of mmp-1 inhibition and mmp-1 down-regulation of flavonoids may block collagen breakdown in certain pathological conditions and certain flavonoids are useful to treat skin inflammation, especially by topical application. (1) (1) university of valencia, spain (2) istituto di chimica biomolecolare cnr, napoli, italy avarol is a marine sesquiterpenoid hydroquinone with several pharmacological properties including antioxidant, anti-inflammatory, and antipsoriatic effects. recently, its derivative avarol-3-thiosalicylate (ta) also demonstrated interesting perspectives as anti-inflammatory drug in vitro and in vivo.it is interesting to note that avarol and ta inhibited nf-ã�b activation in hacat keratinocytes. now, the effect of avarol and ta was investigated in the tpa-induced hyperplasia murine skin model, which presents some similarities with psoriatic lesions. topical treatment with ta (20 mg/ml) produced a 97 % inhibition of oedema and a strong reduction of pge2 (100 %), ltb4 (100 %) and mpo activity (65 %) in skin homogenates. the inhibitory effect of avarol at the same dose was 79 % for oedema, 90 % for pge2, and 50 % for ltb4 and mpo activity. histological study for both compounds showed a decrease in epidermal hyperplasia as well as leukocyte infiltration respect to tpa treatment. besides, the reduction of cutaneous tnf-a by avarol and ta was also detected by immunohistochemical analysis. these compounds were also capable of suppressing nf-ã�b nuclear translocation in mouse skin. in summary, our results suggest that inhibition of proinflammatory metabolites by ta and avarol might be beneficial for the treatment of the inflammatory component of psoriasis. its mechanism of action is related to the inhibition of nf-ã�b activation and can be mediated by the downregulation of intracellular signal-transduction (1), ams silva(2), cmm santos(2), dcga pinto(2), jas cavaleiro(2), jlfc lima (1) (1) requimte, departamento de quã­mica-fã­sica, faculdade de farmâµcia da universidade do porto, porto, portugal (2) departamento de quã­mica, universidade de aveiro, aveiro, portugal 2-styrylchromones are a novel class of chromones, vinylogues of flavones (2-phenylchromones), which have recently been found in nature. several natural and synthetic chromones have demonstrated to possess biological effects of potential therapeutic applications. however, the anti-inflammatory potential of 2-styrylchromones has not been explored so far. thus, the aim of this work was to evaluate the putative anti-inflammatory properties of several synthetic 2-styrylchromones by studding their influence on different systems that are related to the inflammatory process. the putative inhibitory effects of several 2-styrylchromones on the proinflammatory enzymes cyclooxygenase 1 (cox-1), cyclooxygenase 2 (cox-2) and 5-lipoxygenase (5-lox) was evaluated in vitro and compared with structurally related flavonoids. the capacity of the studied 2-styrylchromones to scavenge reactive oxygen (ros) and nitrogen species (rns) was also assessed by different in vitro assays, which allowed to identify the influence of those compounds in each reactive species, separately. from the tested 2-styrylchromones, those having a catecholic bring where shown to be the most effective scavengers of ros and rns, being, in some cases, more active than flavonoids. no considerable correlation was found between the scavenging profile of these compounds and their interactions with pro-inflammatory enzymes. the results obtained from the present study indicate that some of the tested compounds are promising molecules with potential therapeutic value. the usefulness of 2-styrylchromones in the prevention or control of inflammation can only be clarified with additional studies concerning their influence on other relevant mechanisms of this pathology. the importance of tumor-associated inflammatory cells, able to affect different aspects of neoplastic tissue, is a current matter of debate. primarily monocytes are recruited from the circulation into solid tumors and metastases where they differentiate into macrophages with several phenotypes and, e.g., may significantly contribute to uptake of certain radiotracers. we therefore sought to characterize the uptake of various radiotracers used for positron emission tomography (pet) in a well characterized in vitro model of human monocytes/macrophages in comparison with that in various human tumor cells. uptake of radiotracers 18f-fluorodeoxyglucose (fdg), 3-o-methyl-6-18f-fluoro-l-dopa (omfd), and 18f-labeled native/oxidized low density lipoproteins (nldl, oxldl) in single-or cocultivated human myeloid (monocytic) leukemia cell line thp-1 was compared with that by squamous cell carcinoma (fadu), mamma (mcf-7) and colorectal adenocarcinoma (ht29) cell lines (without or in the presence of specific inhibitiors). several thp-1 phenotypes along the monocytic pathway (monocytes, differentiated macrophages, retrodifferentiated cells) were studied before, during and after incubation with phorbol myristate acetate. differentiated thp-1 cells show, when compared with tumor cells, a comparable fdg accumulation, a considerably lower omfd uptake, and a significantly higher oxldl uptake. on the other hand, during differentiation and retrodifferentiation thp-1 cells obviously establish a distinct sequence of biological processes also reflected by considerable alterations in radiotracer uptake. the observed differences in uptake of several radiotracers in vitro in-between thp-1 phenotypes and between thp-1 phenotypes and tumor cells, respectively, stimulate studies on the contribution of macrophage radiotracer uptake to the overall uptake in neoplastic or inflammatory lesions in vivo. genomic and full-length cdna sequences provide opportunities for understanding human gene expression. determination of the mrna start sites would be the first step in identifying the promoter region, which pivotally regulates transcription of the gene. although the mrna start sites of most genes show heterogeneity, this may reflect physiological, developmental, and pathological states of the particular cells or tissues. recently, we have developed a 5-end sage (5sage) that can be used to globally identify the transcriptional start sites and frequency of individual mrnas. a strong association exists between states of chronic inflammation and cancer, and it is believed that mediators of inflammation may be responsible for this phenomenon. another important factor in tumor development seems to be the epigenetic effects on tumor suppressor genes. because of its ability to suppress tumor cell proliferation, angiogenesis, and inflammation, the epigenetic drug such as histone deacetylase (hdac) inhibitor is currently in clinical trials. however, how epigenetic drugs mediate its effects is poorly understood. to assess the effects of epigenetic drugs, the gene expression by 5sage in colon cancer cell lines treated with epigenetically affecting agents, 5-aza-2deoxycytidine, a potent inhibitor of genomic and promoter-specific dna methylation and trichostatin a, a hdac inhibitor was investigated. epigenetic modification induced not only the change of expression of several inflammation-associated genes and the cell cycle progression-associated genes in human colon cancer cells but also the gene expression with aberrant start sites. colon cancer is one of the most frequently diagnosed cancers in western societies. interleukin-6 (il-6) is a potent, pleiotropic, inflammatory cytokine that contributes to a multitude of physiological and pathophysiological processes. il-6 is produced by many different cell types. the main sources in vivo are stimulated monocytes, fibroblasts, and endothelial cells. a variety of studies have demonstrated that over expression of il-6 contributes to the pathogenesis of various inflammatory diseases as well as cancer. it has been reported that human colorectal cancer cells display a wide heterogeneity in their potential to express and produce il-6. serum levels of il-6 are elevated in patients with colorectal cancer, however serum levels of il-6 were found to be independent of il-6 mrna expression in tumor tissue. in this study we analyzed il-6 mrna expression by real-time pcr in 50 sporadic colon cancer tissue as well as corresponding normal mucous tissue. il-6 mrna expression in tumor tissue was lower than in the corresponding normal mucous tissue (p=0,0074). there was no correlation betweenil-6 mrna expression and tumor grade or stage. thus we can conclude that il-6 produced at the tumor site is not involved in sporadic colon cancer progression. (1), t aiamsa-ard (1), v chinswangwatanakul (2), ki techatraisak (3), s chotewuttakorn (1), a thaworn (1) ( material and methods: huvec were cultured as standard techniques and grown to confluence until use. serum was obtained from cholangiocarcinoma patients and normal healthy subjects. huvec were treated with 10% of serum and incubated for 24 hours. cells were analyzed by using [3h] thymidine and immunoblotting assay for cell proliferation and cox-2/nos-2 protein expression, respectively. results: serum of cca patients trend to have more effect on proliferation of endothelial cell than healthy control subjects. on the protein expression, cca serum significantly increased the expression of cox-2 but not nos-2 in hevec. however, the proliferate effect on endothelial cells by cca sera did not correlate with the expression of cox-2. conclusions: this result suggested that some factors in serum of cancer patients could induce cox-2 protein expression in huvec. the increasing of cox-2 might be one of various factors involve in the proliferation process. aim: superoxide is responsible for the neutrophil-mediated tumoricidal activity. the aim of our work was to monitor the changes of superoxide production from neutrophil attributed to tumor development from the early phase to the advanced stage, and to investigate the effects of ok-432@on neutrophil-derived superoxide production and tumor growth. methods: ah109a rat hepatocellular carcinoma cells were implanted into the hind leg of male donryu rats. pmns were harvested from rat peritoneal cavity 6 h after intraperitoneal injection of oyster glycogen. superoxide production were measured by the method of cladependent chemiluminescence, which has high sensitivity and specificity to superoxide. the counts of peripheral leukocytes were significantly increased during tumor progression, and there are significant difference between that of controls and tumor-bearing rats after 18 days of tumor inoculation. both pma and oz-induced superoxide generation derived from neutrophils became significantly reduced in the advanced stage of cancer. the suppression of neutrophil-derived superoxide generation was accompanied with tumor progression and an increased number of neutrophils in the peripheral blood. the subcutaneous administration of ok-432, a biological response modifier, prevented the suppression of neutrophil-derived superoxide generation during tumor progression, which might induce the tendency of tumor growth suppression. our results suggested that the decreased superoxide generation as well as the high leukocytes concentration in the peripheral blood could be considered as indicators of an advanced stage of cancer. furthermore, the effect of ok-432 on neutrophil-derived superoxide production in cancer-bearing rats may provide pharmacological evidence to the therapeutic effects of ok-432. (1), m jokic (2), v zjacic-rotkvic(1), s kapitanovic (2) (1) university hospital sestre milosrdnice, bucharest, romania (2) division of molecular medicine rudjer boskovic institute, bucharest, romania introduction: il-6 is a pleiotropic cytokine mapped to chromosome 7p21-24. its promoter snp -174 g/c is associated with high serum cytokine production, and according to current investigation can play a role in the development and progression of different gastrointestinal malignancies. we tested its genotypes in the gastrointestinal and pancreatic neuroendocrine tumors (gep-nets). patients and methods: dnas from 80 patients diagnosed with gep-net and 162 age and sex-matched volunteers were analyzed for -174 g/c snp of the il-6 gene. to analyze il6 -174 c/g polymorphism we used pcr -nlaiii rflp method. for statistical analysis ã�2 test and fishers exact test were used. the level of significance was 0.05. results: there were no differences observed in the frequencies of the -174 high expression (gc and gg) genotypes between the patients and healthy volunteers (p=0.5518), as well as between patients with gastrointestinal or pancreatic endocrine tumors (p=0.3762). -174 g/c genotype was statistically more frequent among patients with non-functional pancreatic endocrine tumors (pets) than in those with functional pets (p=0.0246). conclusions: high expression genotypes of il-6 -174 snp are more frequent in non-functional pets and may be a marker for the mentioned malignancies. are important in inflammation, are found around and within a variety of human tumors. their number correlates with tumor vascularity and aggressiveness and is a negative indicator for patient survival. how mast cells influence tumor growth is not well understood. the neuroendocrine peptide, neurotensin (nt) is a potent secretagogue of mc that has tumor-promoting effects in animals and promotes the growth of a variety of human cancer cells, acting via its gpcr nt-type 1 receptor (nts1). here we show that hmc-1 human mc express nt-precursor (pront) mrna and protein, and secrete immunoreactive nt when stimulated. rt-pcr on hmc-1 cell rna yielded a band with 99% sequence identity to pront and a band corresponding to the pront processing enzyme, pc5a.immunocytochemistry on hmc-1 cells showed specific staining for pront. stimulation of hmc-1 cells with a23187 + pma, pge2, c48/80 or mastoparan released immunoreactive nt.rt-pcr on hmc-1 cell rna yielded a band with 99% sequence identity to human nts1. western blotting gave bands corresponding to unglycosylated (49 kda) and glycosylated (55 kda) nts1.immunocytotochemistry on hmc-1 cells showed specific staining for nts1. these finding have significance for the role of mast cells in tumor growth. (1), j buddenkotte (1), mp schã§n(2), m steinhoff (1) (1) university hospital mã¼nster, germany (2) university hospital wã¼rzburg, germany the proteinase-activated receptor par-2 has been demonstrated to modulate tumor growth, invasion and metastasis in various tissues. however, the role of par-2 in cutaneous cancerogenesis is still unknown. here we could show a protective role of par-2 in the development of epidermal skin tumors: we established a mouse skin tumor model using chemically induced carcinogenesis. to this end, par-2-deficient and wild-type mice were painted once with dmba (7,12-dimethylbenz[a]anthracene) for sensibilization, followed by topical application of the phorbol ester pma (phorbol myristate acetate (12-o-tetradecanoylphorbol-13-acetate)) twice per week at the same sites. tumors started to appear after eight weeks. after 13 weeks, par-2-deficient mice showed a significantly increased number of skin tumors (14 per animal on the average) in contrast to the wild-type (eight tumors per mouse). analysis of possible signal transduction pathways activated upon par-2 stimulation in hacat keratinocytes showed an involvement of extracellular signal regulated kinase 1/2 (erk1/2) and profound epidermal growth factor receptor (egfr) transactivation, leading to secretion of the tumor-suppressing factor transforming growth factor-beta 1 (tgf-ã¢1). thus, our results provide the first experimental evidence for a tumor-protective role of par-2. (1), ma arbã³s (1), a fraga (1), i de torres(2), j reventã³s (1), j morote (3) ( pathogenesis of benign prostatic hyperplasia (bph) and prostate cancer (pca) is still unresolved, although chronic inflammation may play a significant role in disease progression. prostate stromal fibroblasts may be contributing to the inflammatory process through the expression and secretion of pro-inflammatory mediators, in particular proteoglycan-bound chemokines and other chemoatractants, and the interaction with inflammatory cells such as monocytes. to better understand molecular mechanisms underlying functional differences among prostate fibroblast populations, our primary objective was to characterize proteoglycan and chemokine gene expression in human fibroblasts of different histological/ pathological origin cultured in normal and monocyteconditioned media. we analysed 20 primary human fibroblast cultures from normal transition zone (tz), normal peripheral zone (pz), benign prostatic hyperplasia (bph), and pathologically confirmed prostate cancer (ca). cells of different origin displayed distinct mrna expression profiles for the core proteins of proteoglycans and both sdf1/cxcr4 and mcp1/ccr2 chemokine axis. when incubated with monocyte-conditioned medium all four cell types significantly changed sdf1/cxcr4 and mcp1/ccr2 expression in a fibroblast population dependent manner. monocyte-fibroblast cell adhesion and the chemotactic response of fibroblasts to human peripheral blood monocytes were investigated in a coculture system. monocytes adhered rapidly to fibroblasts and preferentially to bph and pz cells. in addition, chemotaxis was significantly induced in both fibroblast cultures after incubation with monocytes. our results suggest that prostatic fibroblasts have a key inflammatory role associated to a distinctive proteoglycan gene expression and chemokine induction, which is dependent on their histological and pathological source. supported by the spanish urology society (madrid, spain). we have recently shown that paf-receptor is involved in phagocytosis of apoptotic and necrotic but not viable cells, possibly through its interaction with paf-like molecules present on the surface of these cells. removal of altered cells by macrophages could modify the microenvironment at an inflammatory site, and thus influence tumor growth. in the present study we investigated the impact of apoptotic cells or treatment with paf-r antagonist on ehrlich ascitic tumor (eat, ip) and melanoma b16f10 (sc). paf-r antagonist, web2170 (5mg/kg, ip) was given daily for 6 days. we found that eat growth was significantly reduced by pretreatment with web2170, and that inoculation of apoptotic cells (thymocytes) before tumor implantation stimulated tumor growth, an effect reversed by web2170 pretreatment. eat growth was accompanied by increased production of prostaglandin e2, vegf and no which was reduced significantly by web2170 treatment. in b16f10 melanoma, web2170, alone or in association with an apoptosis inducer chemotherapeutic agent, dacarbazin (dcb, 40 ug/kg,ip) significantly reduced tumor mass volume and the number of intratumoral small vessels. in association with dcb, web-2170 reducedactive caspase-3 expression in the tumor andmarkedly increased the survival of tumor-bearing mice. the data obtained here show that during tumor growth, activation of paf-r by molecules present in the surface of apoptotic/necrotic cells, or by paf produced in the milieu, favors tumor growth and suggests that pafantagonists could be useful in tumor treatment, particularly when in association with chemotherapy. financial suport by fapesp and cnpq. (1), mt quiles (1), a figueras(2), r mangues(2), f vinals(2), jr germa(2), g capella (2) (1) institut de recerca vall de hebron, barcelona, spain (2) translational research laboratory, idibell -institut cataladoncologia, spain the malignant potential of tumor cells may be influenced by the molecular nature of k-ras mutations. we have previously shown that codon 12 mutations associate with an increased resistance to apoptosis. we hypothesized that their different malignant potential in vivo could be also related to the generation of a distinct angiogenic and inflammatory profile including vascular structure, macrophage infiltration and expression of angiogenic modulators, proteolytic mediators and the cxcl12(sdf-1)/ cxcr4 chemokine axis. to do so we have combined in vitro and in vivo studies using stable cys12 and asp13 nih3t3 transfectants. cys12 tumors showed a higher microvessel density associated with shorter latency period. prominent vessels with âµ-smooth muscle actin positives cells surrounded by f4/80 macrophages were only observed in asp13 tumors associated with a shorter growth period. asp13 tumors displayed increased vegf expression both at the rna and protein levels, mainly produced by tumor cells. tsp-1 protein levels were similarly diminished in both transfectants. higher mmp9 and mmp7 activities and expression were observed in asp13 tumors probably produced by macrophages or stromal cells. total and active mmp2 levels were higher in cys12 tumors. the expression of sdf-1 and cxcr4 remained unchanged while sdf-1g isoform was selectively induced in cys12 tumors, suggesting sdf-1a or b are induced in asp13 tumors. these results show distinct k-ras mutations induce specific angiogenic phenotypes. the differential stimulation of vegf expression, metalloprotease activities and sdf-1 expression observed is the result of the joint action of tumor cells and the local microenvironment. contact information: dr maria a arbos via, institut de recerca vall de hebron, unitat de recerca biomedica, barcelona, spain e-mail: maarbos@ir.vhebron.net incisional hernias (ihs) represent a common complication of laparotomies, involving remarkable healthcare costs. representative ih animal models are lacking and characterization of human tissue resources is scant. this limited understanding of fundamental mechanisms regulating the destruction of the abdominal wall currently limits the prevention and treatment of ihs. here, we compared tissue specimens (carefully obtained > 5cm of the defect) and primary fibroblasts cultures from fascia and skeletal muscle of subjects with/without ih hernia. the most prominent morphologic characteristics of ih tissue were: alterations of the microstructure of the connective tissue and loss of extracellular matrix (ecm), and a paucity of fibroblasts. in ih muscles, inflammatory infiltrates were observed. other significant changes were: decreased collagen type i/iii ratio; differential proteoglycans mrna expression; enhanced metalloproteinases/ endogenous inhibitors ratio (mmps/timps); and upregulation of apoptosis effectors (caspase-3 and substrates; tnf-alpha; il-6). in vitro, hernia fibroblasts (ihfs) exhibited significantly higher (2-fold) cellular proliferation and migration rates and decreased strength of adhesion as compared to control fibroblasts, even after several passages. moreover, ihfs ultrastructure analysis revealed accumulation of autophagic vacuoles, autophagolysomes-like structures and multilayered lamellar and fingerprint profiles, as well as mitochondrial swelling. based on these descriptive results in human tissues, a novel hypothesis emerges regarding ih formation. specifically we propose that inflammation-related mechanisms triggering proteolytic and apoptotic effectors regulate cell turnover and eventually contribute to atrophy and progressive tissue insuffiency. overall, this may be causally involved in the mechanisms of ecm destruction yielding ih (supported by fis pi_ 030290 and gencat_agaur_ xt_0417). (1), m spinola(2), c pignatiello(2), w cabrera (1), og ribeiro (1), n starobinas(1), t dragani (2) (1) butantan institute, sao paulo, brazil (2) istituto nazionale tumori, milan, italy airmax and airmin mice are phenotypically selected for maximal or minimal subcutaneous acute inflammatory response, respectively, and display high inter-line differences in protein exudates and neutrophil infiltration, as well as in bone marrow granulopoiesis, inflammatory cytokines, and neutrophil apoptosis. in a combined experiment of urethane-induced lung inflammatory response and lung tumorigenesis, airmin mice developed a persistent subacute lung inflammation and a 40fold higher lung tumor multiplicity than airmax mice, which showed a transient lung inflammatory response. we have analyzed gene expression profiles of these outbred lines in comparison to the lung cancer resistant c57bl/6 and lung cancer susceptible a/j mouse strains. gene expression profile analysis of urethane-treated and untreated animals was performed using the applied biosystems mouse genome survey microarray containing 32,000 mouse transcripts. mrna expression of candidate differentially expressed genes was validated by quantitative real-time pcr and the over-represented biological themes were analyzed with the ease software. urethane treatment modulated the gene expression profile in all four lines. among the confirmed genes, vanin3 (vnn3) and major histocompatibility antigen e alpha (h2-ea) resulted common to both mouse models. the most represented gene categories in air model were acute phase response, immunoresponse, electron and lipid transport, complement activation and tissue repair. mhc/antigen process and presentation and immunoresponse were the major themes in the inbred model. moreover, a gene cluster in chromosome 3 (45.0 cm) was observed. the study suggests that the expression of a subset of genes may show a strain-and line-specific modulation pattern during inflammatory response and lung tumorigenesis. inhibition of tumour induced angiogenesis constitutes very attractive anti-cancer therapeutic approach.it is well established that the vegf signal transduction pathway is one of the key drivers of deregulated angiogenesis and selective inhibition can lead to inhibition of tumour growth. however, multiple angiogenic growth factors and pathways are involved, leading to a phenomenon of redundancy and overcoming of an inhibition of vegf signalling only. we have developed a nanomolar inhibitor (compound a) of the receptor tyrosine kinase vegfr-r2 (kdr), which was subsequently shown to be a potent inhibitor of closely related kinases (vegfr-1 and -3, pdgfr, kit, csf-1r) but also unrelated soluble tyrosine kinases (src-familily of kinases and raf). compound a potently inhibits vegf stimulated endothelial cell proliferation but has no effect on non-ec proliferation, which is suggestive of a selective antiangiogenic potential. the unique kinase inhibitory profile of compound a combined with excellent oral bioavailability (87%) has translated into superior in vivo antiinflamm. res., supplement 3 (2007) posters tumour efficacy when compared to the relatively selective kdr inhibitor ptk787. thus, treatment of nude mice implanted with either commercial atcc derived tumour cells (a431 and du-145) or low passage patient derived tumors (cxf 1103; colon cancer, rxf 631; renal cancer) with compound a resulted in inhibition of tumour growth which was significantly better than for ptk787 treated mice. compound a is fairly well tolerated by rodents and extended toxicological studies have been initiated to determine the therapeutic index, which also may allow for exploration of other non-cancer indications. (1), p bobrowski(2), m shukla (3), t haqqi (3) (1) albany medical college, usa (2) rainforest nutritionals, inc, usa (3) case western reserve university school of medicine, usa background: the amazonian medicinal plant sangre de grado (croton palanostigma) has traditional applications for wound healing and inflammation. we sought to characterize an extract (progrado) in terms of safety, proanthocyanidin profile, antioxidant activity and anabolic/catabolic actions in human cartilage explants. methods: acute oral safety and toxicity was tested in rats according under oecd protocol #420. proanthocyanidin oligomers were quantified by hplc and progrados antioxidant activity assessed by the orac, norac and horac assays. human cartilage explants, obtained from surgical specimens, were treated with il-1b (10 ng/ ml) to induce matrix degradation and glycosaminoglycans (gag) release. progrado (2-10 mg/ml) was tested for its ability to maintain optimal igf-1 transcription and translation in cartilage explants and cultured chondrocytes. results: progrado displayed no evidence of toxicity (2000 mg/kg po) leading to gsh safety rating of 5/unclassifiable. oligomeric proanthocyanidin content was high (158 mg/kg) with the majority of oligomers > 10mers.progrado was a remarkably potent antioxidant and in an ex vivo model of inflammation-induced cartilage breakdown, progrado was exceptionally effective in reducing both basal and il-1b induced glycosaminoglycan release from human cartilage explants. progrado prevented il-1b induced suppression of igf-1 production from human cartilage explants as well as stimulating basal igf-1 production (p<0.05). comparable changes in igf-1 gene expression were noted in cultured human chondrocytes. conclusions: progrado has a promising safety profile, significant chondroprotective and antioxidant actions, and promotes the production of the cartilage repair factor, igf-1. this suggests that progrado may offer therapeutic benefits in joint health, wound healing and inflammation. the solvent extracts from korean fermented soybean (chungkukjang) were evaluated for their protective effects against the generation of free radicals and lipid peroxidation. the activities of chungkukjang were compared with several antioxidants and soybean isoflavones including genistein and daidzein. in addition, the protective effects against h 2 o 2 -induced cytotoxicity and oxidative dna damage in the nih/3t3 fibroblasts line were examined. the extracts from chungkukjang and soy isoflavones inhibited the generation of 1,1-diphenyl-2picryl hydrazine (dpph) radicals, and had an inhibitory effect on ldl oxidation. the extracts from chungkukjang and soy isoflavones strongly inhibited h 2 o 2 -induced dna damage in the presence or absence of endonuclease iii and fpg. furthermore, they showed cytoprotective effects against h 2 o 2 , without cytotoxicity except for the hexane extract at high concentrations (> 450 mg/ml). the ethanol and n-butanol extracts appeared to have most potent antioxidant activities. these in vitro results show that the extracts of chungkukjang may be a useful antigenotoxic antioxidant by scavenging free radicals, inhibiting lipid peroxidation and protecting against oxidative dna damage without having cytotoxic effect. moreover, the extracts of chungkukjang inhibited mda formation in the liver, dna damage assessed by comet assay and the microucleated reticulocyte formation of peripheral blood in kbro 3 -treated mice. these in vivo results were similar to those of in vitro experiments. therefore, chungkukjang containing soy isoflavones is a promising functional food that can prevent oxidative stress. (supported by bk21 project from korea research foundation). sirt1 is a histone deacetylase, involved in oxidative stress and aging. because the role of aging and exercise on sirtuins activity in rats is unknown, we investigated the effects of exercise on age-related changes in the sirt1 activity, comparing heart (h) and adipose (a) tissue of sedentary young (n10), sedentary old (n10) and trained old (n10) rats. the trained old rats performed a 8-weeks moderate training on treadmill. on h and a tissue of all rats sirt1 activity was evaluated by assay kit, peroxidative damage measuring malondialdehyde (mda) and protein-aldehyde adducts 4-hydroxynonenal (4-hne), mnsod, catalase and foxo3a by western blot, and gadd45a, cyclin d2 and foxo3a mrna by rt-pcr. aging reduced sirt1 activity in h (p<0.0001) without effects in a, producing an increase of mda (h, p<0.0005; a, p<0.0001) and 4-hne (h, p<0.005; a, p<0.0005), and a decrease of mn-sod (p<0.02) and catalase (p<0.0001) expression in both h and a. aging did not affect foxo3a protein expression in h, and foxo3a mrna in a. exercise produced an increase in h foxo3a protein expression (p<0.02) and in a foxo3a mrna, associated to higher mn-sod (h, p<0.01; a, p<0.005) and catalase (h, p<0.0001; a, p=0.01) levels in both h and a of aged rats. in heart exercise-induced higher sirt1 activity bring on decrease in cyclin d2 and increase in gadd45a mrna expression. in a we found a similar decrease in cyclin d2, without changes in gadd45a mrna expression. these findings suggest that exercise is able to increase sirt1 activity in aged rats. (1), t horiguchi(1), k abe(2), h inoue(2), t noma (1) (1) institute of health biosciences, the university of tokushima graduate school, tokushima, japan (2) minophagen pharmaceutical co. ltd, japan objectives: glycyrrhizin (gl) is a major component of glycyrrhizae radix (licorice) that is generally used for treatment of hepatitis. gl has a regulatory activity on arachidonic metabolism, immunological function, and anti-viral effects. however, the molecular mechanisms of the effects remain unclear. to analyze the molecular basis of gl signaling, we performed the microarray analysis using ccl4-induced mouse hepatitis models. methods: eight-week-old icr male mice were treated intraperitoneally with 100 fã�l/ kg bw of ccl4 w/wo 250 mg/ kg bw of gl. after 24 hours and 72 hours, livers and serum were collected and analyzed. for microarray analysis, the expression patterns of genes between 72 hour-treated-livers (ccl4 or ccl4 and gl) and no treated-livers were compared. results: gl-treatment dramatically decreased the gpt activity in plasma at 24 hours compared to that in ccl4treated plasma. however, the levels of mrna expression of inflammatory genes such as phospholipase a2, hsp47, and procollagen were still very high in gl-treated liver. after 72 hours, the mrna levels of them were significantly reduced in gl-treated mice compared to those of ccl4-treated liver. then, we screened 41,000 genes by microarray and found that 71 genes were up-regulated and 63 genes were down regulated in ccl4+gl compared to ccl4 treatment. interestingly, ros scavenger-related genes were significantly up-regulated in ccl4 + gl. detail analysis is currently ongoing. we found the unique relationship between gl activity and ros regulation. this finding suggests a novel way to treat inflammatory diseases including hepatitis. objectives: experimental autoimmune encephalomyelitis (eae) is a demyelinating autoimmune disease that results from an immunological reaction against different myelin components at the cns. it is widely employed as an animal model of human multiple sclerosis. interestingly, the number of studies relating these diseases with peripheral organs is limited. we thus investigated the consequences of eae on the degree of lipoperoxidation (tbars) and mpo activity in different rat peripheral organs (eg. lung, spleen, liver, stomach, duodenum, colon, ileum, kidney and bladder). university of waikato, hamilton, new zealand mitochondria play a fundamental role in the life and death of all eukaryotic cells. cells with dysfunctional mitochondria are known to have higher levels of a molecular stress protein (cpn60). this protein is increasingly being implicated to play a role in modulating cellular inflammation. we have developed an in vitro model cell system using thp-1 monocyte cells with compromised mitochondrial bioenergetic functions to investigate the relationships between mitochondrial dysfunction, cpn60 expression and modulation of proinflammatory cytokine responses. we have found that the ability of cpn60 to modulate tnf-a expression was strongly correlated with the loss of mitochondrial bioenergetic functions in our thp-1 cells. we also demonstrate that such modulation involves both erk1/2 and p38mapk pathways. the significance of these results in relation to the role of mitochondria as modulators of inflammation will be discussed. (1), b arnold(2), g opdenakker (3) (1) jagiellonian university, department of evolutionary immunobiology, krakow, poland (2) german cancer research center, department of molecular immunology, heidelberg, germany (3) rega institute for medical research, university of leuven, laboratory of immunobiology, leuven, belgium we showed that in mice genetically deprived of metalloproteinase 9 (mmp-9-/-) at least one compensatory mechanism operates as there are elevated levels of pge2 of cox-1 origin expressed by peritoneal macrophages during zymosan peritonitis; and this leads to increased early vascular permeability observed in those animals. also infiltration of peritoneal cavity by inflammatory neutrophils is changed in mmp-9-/-mice as at 6 hrs of inflammation, when otherwise highest numbers of neutrophils are detected in peritoneum, the cell numbers are significantly lower in the mice in comparison to their controls. in contrary, at 24 hrs of peritonitis, when normally resolution of peritonitis takes place, no decrease in neutrophil counts is observed. thus the aim of the present study was to evaluate if impairment of neutrophil apoptosis could account for this latter phenomenon in mmp-9-/-mice. for this numbers of apoptotic (annexin v) and necrotic (7-aad) peritoneal leukocytes were evaluated and levels of active caspases were tested by application of either caspase detecting antibodies or fluorochrome-labelled inhibitors; all analyses were performed by flow cytometry. the results revealed that both, numbers of apoptotic cells and levels of active caspase 3 were significantly lowered in mmp-9-/-mice while levels of caspase 7, 8 and 9 were significantly elevated in comparison to control animals. we conclude that an impairment of apoptosis is observed in mmp-9-/mice during zymosan peritonitis and it is due to the decreased levels of active caspase 3. the increased activity of other examined caspases is most probably independent of apoptosis. (1), h james (1) the selective inhibition of nitric oxide generation by inhibiting the activity of nitric oxide synthase(nos) isoforms represents a novel therapeutic target for the development of anti-inflammatory agents. the aim of this study was to evaluate the activity of nos inhibitors in experimentally induced inflammation, pain and hyperalgesia. the effect on acute inflammation was studied in carrageenan-induced paw edema in rats. the effects on carrageenan-induced hyperalgesia, tail flick response to radiant heat and acetic acid-induced writhing were also studied. nos inhibitor ng-nitro-l-arginine methylester (l-name), 10 and 20 mg/kg produced a dose-dependent inhibition of paw edema (42% and 57% at 2h; 45% and 57% at 4h). a marked reduction in paw edema was observed with ng-monomethyl-l-arginine acetate (l-nmma), 10 mg/kg(79% at 2h; 91% at 4h). selective inducible nos(inos) inhibitor aminoguanidine hemisulfate inhibited the paw edema at a dose of 20 mg/ kg(59% at 2h; 64% at 4h) but not with a dose of 10 mg/kg . the effects were comparable to nonselective cox inhibitor indomethacin 5 mg and 10 mg/kg (40% and 77% at 2h; 29% and 72% at 4h respectively) and selective cox-2 inhibitor rofecoxib, 5 mg/kg (49% and 78% respectively). nos and inos inhibitors significantly increased the pain threshold latency in the tail-flick test. these inhibited the acetic acid-induced writhes, the effect being comparable to indomethacin. however, carrageenan-induced paw hyperalgesia was not inhibited. the results suggest that nitric oxide plays a role in carrageenan-induced acute inflammation and both nosand inos inhibitors have a potential anti-inflammatoryand anti-nociceptive activity. (1), p hart(2), j edwards (1), c quirk (1) (1) molecular pharmacology limited (usa), australian division, perth, western australia (2) telethon institute for child health research, perth, western australia thermalife cream, an anti-arthritic biological product, has been successfully used off-label for sun burn recovery. a novel product, derived from thermalife, was assessed on its therapeutic potential in oxsoralen-uvb burns. as a possible mechanism for the sunburn efficacy, suppression of tnf-a and il-1㢠production by human monocytes was assessed in vitro. methods: sunburn: four sites were marked on the arm of the subject. three sites were exposed to oxsoralen (1%) plus uva/uvb light, one site was exposed to oxsoralen only. cream was applied at 5min, or at 4hrs after injury. a third injury site was not treated. photographs were taken before, 24hrs, and 7weeks after injury. cytokines: human monocyte cultures (10% fcs, 5% co2) were either stimulated with 500ng/ml lps (e.coli 0111:b4) or not in the presence of 0% or 10% active ingredient.24hrs after incubation, culture media was collected, centrifuged, and assayed (cytokine elisa). results: at 24hrs after oxsoralen-uv, the 5min treatment site showed slight erythema, the 4hr treatment site had pronounced erythema and slight blister formation, whereas the untreated site had pronounced erythema and strong blister formation. 7 weeks after injury, the 5min site was normal, the 4hr site was a dark colour, whereas the untreated site had a significant scar. oxsoralen alone had no effect on the skin. the novel product suppressed lps-induced tnf-a and il1㢠secretion by 48.7% and 71.1%, respectively. conclusions: a novel thermalife-derived product reduced total injury after oxsoralen enhanced uva/ uvb burns, which is possibly related to cytokine suppression. (1), p hart(2), j snowden (1), maud eijkenboom (1) (1) molecular pharmacology limited (usa), australian division,perth, western australia (2) telethon institute for child health research, perth, western australia a mixture of bovine plasma protein fractions and zinc chloride (bov-zn) was assessed for its ability to regulate cytokine production by lps-stimulated monocytes. dosereponse curves for tnf-a suppression were generated. further, competition with fcs in the culture medium and the metabolism of monocytes under influence of bov-zn were assessed. in all experiments the culture medium environment was similar. human monocyte cultures (10% fcs, 5% co2) were either stimulated with 500ng/ml lps (e.coli 0111:b4) or not in the presence of 0%, 2.5%, 5%, 7.5%, 10%, 20% or 40% bov-zn (two pooled experiments). 24 hours after incubation, culture media were collected, centrifuged, and assayed (cytokine elisa). a competitive inhibition design for the standard tnf-a assay was set up for 0%, 1%, 5%, 10% fcs against 0%, 2.5%, 5%, 10% bov-zn. the culture media were treated as above. metabolism of non-proliferating monocytes was measured via accumulation of bioreduced formazan (promega celltiter 96) in treated and untreated cell cultures over 0-45 hrs at intervals. the ic50 for tnf-a suppression was reached at 2.5% bov-zn in each of two experiments. fcs did not compete with bov-zn in suppressing tnf-a in lpsstimulated monocytes. at low fcs concentrations bov-zn stimulated tnf-a production in the absence of lps. this tnf-a increase was countered with increasing concentrations of fcs. metabolism of cells was not affected by 10% bov-zn. conclusions: bov-zn could reliably and effectively reduce tnf-a secretion in vitro, without competing with the fcs in the culture medium, and without disturbing the metabolism of monocytes. inflammatory diseases such as rheumatoid arthritis (ra) result from overproduction of cytokines including tnf-â£\ and il-1fã�. these cytokines are known to be regulated by the stress-activated p38fnfnmap kinase pathway. because of this, inhibition of p38 map kinase has been one of the most compelling targets for the treatment of inflammatory disease. over the last 10 years, numerous groups have reported on the development of p38 map kinase inhibitors. x-ray co-crystallization with the enzyme suggests a propensity to accommodate structurally diverse molecules. regions of the binding site are known to be unique to p38 vs other kinases, enabling the development of p38 selective molecules. inflamm. res., supplement 3 (2007) posters anti-inflammatory activities. a series of 11 labdane-type diterpenoids (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) with various patterns of substitution were tested for potential anti-inflammatory activity.of these compounds, 4 and 11 were selected to evaluate their influence in targets relevant to the regulation of the inflammatory response. these derivatives displayed good in vivo anti-inflammatory activity, and maximum inhibitions of 50 and 90 % were noted in the 12-o-tetradecanoyl-phorbol-13-acetate (tpa)-induced ear oedema in mice. in addition, inhibition of myeloperoxidase activity, an index of cellular infiltration, was also observed. the diterpenoids also reduced the production of nitric oxide, prostaglandin e2, and tumour necrosis factor-alpha in bacterial endotoxin-activated raw 264.7 macrophage cells with ic50 in the range 1-10 mm. inhibition of these inflammatory mediators was related to reductions in the expression of inducible nitric oxide synthase, and cyclooxygenase-2, as determined by western-blot analysis and rt-pcr. since nuclear factor-kappab (nf-kb) plays a central role in the transcriptional regulation of these proteins, we investigated the effects of these diterpenoids on this signalling pathway. our results indicate that both compounds interfere with the phosphorilation of ikbalpha and ikbã�, resulting in inhibition of their degradation. in summary, the anti-inflammatory effects of these labdane diterpenoids are related to the inhibition of inflammatory mediators by blocking nf-kb activation and provide potentially therapeutic perspectives in inflammatory conditions. the aim of the study was a research of mechanisms of inflammatory action of a new drug fepolen at a dosage of 100 mg/kg prepared from bee products (bee pollen and phenolic hydrophobic extract of propolis) for prostatitis treatment. to fulfill above mentioned task a model of zimozan induced oedema whose dynamics gives a possibility to estimate influence of a drug on both routs of arachidonic acid metabolism -via cyclooxiginase and lipooxigenase was used. a comparison with diclofenacum at a dosage of 8 mg/kg and substance ffw-755ã� (inhibitor of cyclooxygenase and lipooxygenase and has a high antiinflammatory effect (41%) at a dosage of 16 mg/kg) was made. the results of influence of drugs dynamics of zimozan inflammation show that anti-inflammatory action of fepolen is based on decrease of release of biogenic amines and activity of lipooxigenase and is higher then effect of diclofenacum. fepolen revealed the highest effect during first 30 min and 1 hour of inflammation that was higher then action of diclofenacum. these data proves that fepolen decreases lipooxygenase activity that is in charge for the inflammation during this period. during next hours therapeutic effects of fepolen and diclofenacum were at the same level. fepolen showed the same dynamics of anti-inflammatory action as ffw-755ã� that demonstrates a property to influence both routs of arachidonic acid metabolism. in summary with previous results in conditions of carageninic inflammation we can conclude that anti-inflammatory action of fepolen is based mostly on influence on lipooxygenase then cyclooxygenase. the aim of this study was to establish a method by which probiotic bacteria can be selected for their immuno modulatory properties, especifically the ability of certain strains to suppress an inflammatory response. the gastrointestinal inflammatory condition crohns disease involves a th1-response with increased levels of proinflammatory cytokines like tnfa and il12, and mouse models of crohns disease show that the balance of il10/12 is crucial for disease progression. we have used mouse bone marrow derived dendritic cells (bmdc) to model a proinflammatory crohns disease like condition in vitro with cocktail-induced bmdc secretion of il12, il6 and tnfaf jthe model was validated using anti-inflammatory molecules like dexamethasone and prostaglandin d2, which were able to suppress the cocktail induced il12 secretion. further validation of the model is confirmed by the fact, that probiotic strains which are able to suppress tnbs induced colitis in mice in preventive studies, also show potent anti-inflammatory activity in our model. among clinically relevant as well as novel probiotic strains, we have selected strains with potent anti-inflammatory properties, and are currently investigating the possible mechanism of action of these strains. in summary, our established model is suitable for identification of anti-inflammatory activity of probiotic strains and potentially other immune suppressing components, for rational selection of candidates for further preclinical and clinical evaluation and development. p11-12 inflammation and human incisional hernia pathophysiology maria antonia arbos via(1) eae was induced by immunization of female lewis rats with guinea-pig myelin basic protein (mbp) in complete freunds adjuvant (cfa) and the animals were studied at the stage iii of the disease (characterized by complete paralysis of the hind-limbs) compared to cfa rats, eae resulted in increased mpo activity (u/mg tissue) in kidney (280 ae 75 vs. 121 ae 14 179 ae 35; p<0.001), and higher tbars contents (nmol mda/mg tissue) in liver acknowledgements: capes, cnpq, fapesp. contact information: ms simone teixeira, university of sâ¼o paulo, department of pharmacology, campinas, brazil e-mail: mone@usp.br tolerability, investigator and subject global assessments and rescue medication consumption supported by bk21 project from korea research foundation) contact information: mr young hoon kim here we report on the development of the pge2 mimetic combination therapy (dp-837953) that inhibits basal and lps/tlr4 induced tnf-?, il-1ã�, and mmp-1, 8, 9, 13 in human and murine synovial membranes and peripheral macrophages.in a murine model of chronic synovitis (dorsal skin air pouch), dp837953 dramatically inhibited il-1ã�, tnf-?, mip2, mcp-2 and il-8 expression, delayed the profile of leukocyte/neutrophil extravasation and reduced exudate volume.in a model of inflammatory arthritis (collagen induced arthritis, cia), dp837953 markedly reversed the inflammatory pathology by reducing synovial hyperplasia, cartilage erosion and articular inflammation.in addition, tnf-?, il-1ã�, mmp-8 and to a lesser extent mmp-9 expression/synthesis levels were strongly suppressed as judged by rt-pcr and elisa measurements we have developed two rias, one for functional blood levels of the above mentioned anti-tnf-alpha constructs, and one for anti-abs (all isotypes), and we have used these methods to monitor patients treated with infliximab/remicade and etanercept/enbrel ; i shall present some of these data (7, 8 anatomy-physiology, faculty of medicine, laval university, quebec, canada neutrophils, which are often the first leukocytes to migrate at inflamed sites, can generate ltb4 from the 5-lipoxygenase pathway, pge2 through the inducible cyclooxygenase (cox-2) pathway and cytokines/chemokines as tnf-alpha, il-1beta, il-8, mip-1alpha, mip-1beta, mip-2alpha and mip-3alpha. engagement of the adenosine a2a receptor (a2ar) blocks the in vitro synthesis of ltb4 while it potentiates the cox-2 pathway in fmlp-treated neutrophils. in addition, it selectively prevents the expression and release of tnfalpha, mip-1alpha, mip-1beta, mip-2alpha and mip-3alpha in toll-like receptor-4-stimulated human neutrophils. little effect was observed on il-1beta and il-8. using the murine air pouch model of inflammation with a2ar-knockout mice, we observed that the activation of a2ar positively impacts the expression of cox-2 in vivo, with particular magnitude in inflammatory leukocytes. in mice lacking the a2a receptor, neutrophils that migrated into the air pouch 4h following lps injection expressed higher mrna levels of tnf-alpha, mip-1alpha and mip-1beta than neutrophils from wild type mice. together, these results indicate that neutrophils are important mediators of adenosines protective effects. given the uncontrolled inflammatory phenotype observed in a2ar-knockout mice and in view of the potent inhibitory actions of pge2 on inflammatory cells, an increased cox-2 expression and a prevented release of tnf-alpha, mip-1alpha and mip-1beta caused by a2ar activation, observed particularly in neutrophils, may take part in an early modulatory mechanism promoting anti-inflammatory activities of adenosine. sepsis induced by endotoxins including lipopolysaccharide (lps) is a big problem in clinical medicine. for a better insight into the molecular pathways and to assess markers of endotoxin-induced sepsis, we applied thetwo dimensional gel electrophoresis (2d-page) and maldi-tof to follow the changes of significant proteins in a murine macrophage cell line -raw 264.7 after challenged with lps (escherichia coli 026:b6) for 12, 18 and 24 hours. we identified 21 proteins from approximately 500 detected protein spots with either increased or decreased in relative abundance as a result of lps treatment. the proteins identified with increased expression are the retinoblastoma binding protein 7, capg protein, poly(rc) binding protein 1, isocitrate dehydrogenase 3 (nad+) alpha, lactate dehydrogenase 1, a chain, guanine nucleotide binding protein (g protein), beta polypeptide 2 like 1, triosephosphate isomerase 1 and proteasome alpha 5 subunit); and ones with decreased expression are the acidic ribosomal phosphoprotein p0, malate dehydrogenase, soluble, proliferating cell nuclear antigen, proteasome (prosome, macropain) subunit, alpha type 1 and rho, gdp dissociation inhibitor (gdi) beta). many of these altered proteins have interesting functions in inflammation. with the information obtained with the proteomic approach, it is possible to improve current methods of monitoring endotoxemia and to identify new therapeutic targets. the ubiquitous mitogen-activated protein (map) kinases are important enzymes in signal-tranduction cascades which regulates diverse cellular events such as cell transformation, proliferation, differentation, and apoptosis. they are therefore potential drug targets for therapeutic intervention in the treatment of inflammation, cancer, and other immune diseases. based on a virtual screening approach we identified 6-amino-1-benzyl-4-(4-bromophenyl)3-methyl-1,4-dihydropyrano[2,3c] pyrazole-5-c arbonitrile as a potential novel lead structure as p38 map kinase inhibitors. a set of compounds were prepared starting from different substituted pyrazol-5(4h)-ones via a base-catalyzed condensation with aldehydes and ch acids, such as malononitrile, to provide them for biological tests in a p38 enzyme assay. first structure-activity relationship confirm the value of this novel lead. this study was conducted to determine the physiological c-reactive protein (crp) and alpha 1-acid glycoprotein (aag) levels for two groups of beagle dogs: healthy dogs of various ages and pregnant dogs. serum crp levels were measured by elisa and aag levels were measured in healthy beagles of various ages by tia, and then separately -in pregnant beagles -by srid. serum crp levels ranged from 1.5 to 16.0 ã¬g/ml in male, and from 1.8 to 18.9 ã¬g/ml in female dogs. no significant sex-related differences were observed in the values. further, there were no significant age-related differences either. serum crp levels increased during pregnancy and peaked at 70.2-90.4 ã¬g/ml 30 or 45 days after ovulation, demonstrating two characteristic features of crp levels change in pregnant dogs. serum aag levels ranged from 40 to 960 ã¬g/ml in male, and from 47 to 833 ã¬g/ml in female dogs, without any significant sex-or age-related variation. serum aag levels increased in all pregnant beagles and peaked in the middle of gestation at 250-1,000 ã¬g/ml. despite a high value of 1,210 -1,360 ã¬g/ml being observed for serum aag levels in 3 pregnant beagles inoculated with staphylococcus aureus, its levels in umbilical cord blood were below the detection limit of srid (40 ã¬g/ml). no significant sex-/-age related differences were observed in serum both crp and aag levels and these levels increased during pregnancy. the results of aag levels in umbilical cords were below the detection levels suggest aag is not transported to the placenta. polymorphonuclear neutrophils (pmns) play a key role in the inflammatory response against infectious agents.however, they can elicit significant tissue damage and in this respect, anti-inflammatory drugs are of interest.in this study, we examined the effect of pbi-1393, a low molecular weight immunomodulatory molecule, on pmn activation by lps both in vitro and in vivo. we measured by elisa the production of tnf-a by human lps-activated pmn in the presence or absence of pbi-1393.the ability of pbi 1393 to modulate pmn activation and recruitment in vivo was assessed using a rat air pouch model of inflammation.exudates from different groups of animals (controls and pbi-1393 treated animals, n=6) were used to assess leukocyte infiltration and to measure by elisa tnf-âµ, mcp-1 and pge2 production.in vitro, pbi-1393 is able to significantly decrease by 28.8% ae 0.08% (p<0.05), tnf-a production by human lpsactivated pmn.in vivo, pbi-1393 significantly decreased the production of tnf-a (41.4% ae 7.2%; p<0.005), mcp-1 (16.3% ae 8.4%; p<0.05) and pge2 (29.2% ae 13.5%; p<0.05) induced by lps injection.however, pbi-1393 did not significantly inhibit leukocyte infiltration.these results show that pbi-1393 is able to modulate pmn activation and inflammatory response and suggest potential use as anti-inflammatory agent. (1), lj lowenstine,(2), aj norris(2), t spangler(2), lm woods (2) (1) zoological society of san diego, usa (2) department of pathology, microbiology, and immunology, university of california, davis, usa this study investigated the role of a novel reovirus in a 2004 outbreak of necrotizing typhlocolitis in american crows in california. included is a detailed characterization of the necrotizing and inflammatory characteristics of the disease, as well as a discussion of the implications of these findings upon proposed mechanisms of pathogenesis. complete histopathology including stains for lesion characterization and potential concurrent etiologic agents was performed on all outbreak crows. feces and ceca were submitted for culture, parasitology, and negative contrast electron microscopy. two control groups (n=10 each) were selected for parasitology and em (group 1), and gross and histopathology (group 2). all outbreak cases and group 2 controls tested negative for west nile virus by pcr.all outbreak crows had marked, necrotizing heterophilic typhlocolitis, fibrinonecrotizing splenitis, and variable intestinal lamina proprial necrosis and hemorrhage.two cases had multifocal hepatic necrosis. negative contrast em revealed reovirus particles in 100 % (4/4) of outbreak cases and in 0% (0/ 10) of controls. supplemental tests failed to suggest other concurrent or confounding etiologic agents.overall, the findings suggest association between the reovirus and the outbreak of typhlocolitis, and the absence of reovirus in controls suggests that it is not ubiquitous in the crow population.there was a noteable absence of similar typhlocolitis in 65 archived crows submitted to the vmth from 1994-2004, suggesting an emerging corvid disease in california, which bears further investigation. mitogen-activated protein kinase (mapks) pathways play an important role in the signalling system activated by proinflammatory cytokines. among the most important cascades the activation of erk1/2 by mek1/2 is reported to be responsible for inflammatory responses and degradation of osteoarthritic cartilage. as701820, a selective mek1 inhibitor, demonstrated anti-inflammatory properties in reducing tnf-alpha production induced by lps injection (ic50 2 mg/kg). therefore, primary aim of the present study was to assess the therapeutic strength of the as701820 in a mouse model of collagen-induced rheumatoid arthritis (cia) assessing the effect of the compound on structural changes related to the cartilage. cia is characterized by severe polyarthritis affecting peripheral joints, synovial hyperplasia with persistent inflammation and cartilage erosion. as701820 treatment was initiated when signs of arthritis were clinically visible (in terms of paw swelling and redness) and was continued for 7 days (twice daily), by oral route at the doses of 10, 30 and 100 mg/kg. as701820 at 30 and 100 mg/kg significantly reduced clinical arthritic read-outs such as clinical score and paw swelling. at histology, vehicle-treated animals showed severe inflammation and joint surface erosion. administration of as701820 significantly decreased inflammatory infiltrates and treated cartilage surfaces that presented normal levels of proteoglycan content. in conclusion, the results obtained in this study clearly demonstrate that the selective blockade of mek1 could be considered as an innovative therapeutic approach to treat rheumatoid arthritis. experimental evidences have shown that the toxicity of ni salts may involve inflammatory processes, with a subsequent overproduction of reactive oxygen species (ros) and carcinogenicity. neutrophils are the most abundant leukocytes of blood, and participate actively in the inflammatory innate host defense response. however, relatively little is known about the potential of nickel salts in activating human neutrophils.thus, the aim of the present study was to evaluate the putative stimulation of oxidative burst in isolated human neutrophils by nickel nitrate. the measurement of neutrophil burst was undertaken in vitro, by chemiluminescence, by monitoring the oxidation of luminol by neutrophil-generated ros and reactive nitrogen species (rns). enzymatic inhibitors and specific reactive species scavengers were used to evaluate which species were involved in neutrophils activation by nickel nitrate. the obtained results showed that nickel nitrate stimulates human neutrophils burst in a concentration-dependent manner, within levels that may be attained in vivo. in the present experimental conditions, the reactive species involved in neutrophils activation by nickel nitrate were superoxide radical (o2-.), hydrogen peroxide (h2o2), hydroxyl radical (ho.) and perchloric acic (hocl). the observed activation of isolated human neutrophils burst by nickel nitrate and subsequent tissue damage due to a sustained formation of reactive species may contribute for the deleterious effects attributed to this transition metal, though this assumption needs to be confirmed in vivo. (1), h spalteholz (1), u reibetanz (1), p salavei (1), m fischlechner (1), h-j glander(2), j arnhold (1) (1) university of leipzig, medical faculty, institute for medical physics and biophysics, germany (2) university of leipzig, derpartment of dermatology, andrology training centre of the european academy of andrology, germany unintentional childlessness often caused by common reasons as inflammation affects 15 -20 % of german couples. inflammations of the male genital tract lead to an infiltration of polymorphonuclear granulocytes (pmn), respectively induce a restricted spermatozoa quality associated with early triggered acrosome reaction (ar) and apoptosis as well as changes in the lipid structure and reduced mobility. stimulated pmn release the strongly cationic heme protein myeloperoxidase (mpo), which is able to bind to negatively charged membrane surfaces, e.g. apoptotic cell membranes with externalized phosphatidylserine (ps). a population of freshly prepared spermatozoa shows only a very small amount of cells with mpo binding ability as well as externalization of ps. the number of spermatozoa able to bind mpo raises considerably in samples containing predamaged cells or introducing the ar as could be observed with rhodamine b isothiocyanate (ritc)labelled mpo and antibody techniques by fluorescence microscopy as well as flowcytometry. the activation ofmpo with its substrate hydrogen peroxide (h2o2) in the presence of chloride ions generates the powerful oxidizing and chlorinating species hypochlorous acid (hocl) and enhanced markedly the number of annexin v positive and non-vital cells. components of seminal plasma as well as serum albumin can protect spermatozoa for the deleterious effects of mpo. the coincidence of ps externalization and mpo binding to spermatozoa surfaces indicates an up to now unknown role of this enzyme in recognition and removal of apoptotic cells during inflammation. recent findings suggest a crucial role of proteinaseactivated receptor-2 (par2) in inflammation and innate immunity. par2 is the second member of a novel g protein-coupled receptor subfamily with seven putative trans-membrane domains. this subfamily is characterized by a unique mechanism of receptor activation. accessible serine proteases cleave the receptor to expose a new, previously cryptic, n-terminal sequence ("tethered ligand") which further interacts with the same receptor and activates it. tryptase, trypsin, and bacterial serine proteases are capable of directly activating par2. par2 is expressed by human neutrophils, however its functions on these cells remained unclear. the data of our present study indicate that par2 agonists enhance interferon gamma (ifna)-induced up-regulation of cell surface fca;ri, one of the key receptors involved in neutrophil phagocytic activity. moreover, par2 agonists (serine proteases as well as synthetic activating peptide) and their receptor represent an additional system which controls neutrophil transendothelial migration and apoptosis in vitro. additionally, there is a significant increase of par2 expression on the neutrophil cell surface in the case of septic patients as compared to cells from healthy volunteers. together, our results indicate that par2 may be involved in the pathophysiology of acute bacteria-induced human diseases (sepsis or septic shock, for example) potentially by regulating neutrophil apoptosis, transendothelial migration and fca-receptor expression. aim: to ascertain the role of macrophages as direct inducers of regeneration after renal ischemia/reperfusion, and to establish whether inflammatory conditions contribute to the process. we determined whether adoptive transfer of macrophages at different stages of kidney inflammation after mouse renal i/r could restore reparation and assessed the influence of inflammation in the process.results: i/r provoked the increases in renal regeneration, as evaluated by inmunohistochemistry and pcr mrna of stathmin and pcna. the cytokine profile revealed the influence of the inflammatory environment on kidney repair. regeneration was macrophage-dependent, decreasing when depletion was provoked, and increasing with adoptive transfer of macrophages; however, administration of resting macrophages did not induce repair at the time points in which tissue was inflamed, and was only able to promote regeneration in the absence of inflammation (72 hours). pro-inflammatory cytokines increased at the early stages of reperfusion, coinciding with low regeneration, and anti-inflammatory cytokines increased during the longer periods of reperfusion when regeneration was more evident.conclusions: macrophages directly induce renal regeneration after ischemia/reperfusion in an inflammationdependent manner. (1), k bendtzen (1), f sellebjerg (2), ch nielsen (3) ( antibodies against myelin basic protein (mbp) are present in sera from patients with multiple sclerosis (ms), but the role of these antibodies is controversial. we collected sera from 22 ms patients and 17 healthy individuals and found that both groups contained igm anti-mbp antibodies, while ms sera contained small amounts of igg anti-mbp. however, the two groups of sera did not differ significantly with respect to the content of either antibody subclass. addition of mbp to the various sera and subsequent addition of the mixtures to normal peripheral blood mononuclear cells (pbmc) resulted in a significant deposition of igm on cd14+ monocytes, indicating that formation of mbp/igm complexes had occurred. this deposition was strongly inhibited by addition of 10 mm edta to the sera, indicating that it was complement dependent. the pbmc produced significant amounts of il-10, tnf-alpha and ifn-gamma upon stimulation with mbp, and the extent of the cytokine production did not depend upon whether sera from ms patients or from healthy controls were present. however, disruption of the tertiary structure of mbp by boiling significantly reduced the production of all three cytokines, supporting a role for antibodies in the induction of cytokine responses to mbp. we propose that natural igm autoantibodies may form complexes with mbp, facilitating the uptake of mbp by antigenpresenting cells (apc). since sera from ms patients did not enhance this uptake and the subsequent cytokine production, the mechanism may be part of an appropriate peripheral regulation of self-reactivity. we currently investigate this possibility. loredana postiglione(1), g tarantino(2), a spanã²(2), p ladogana (1), fl perrone(1), s padula(2), a riccio (2) (1) federico ii university medical school of naples, department of molecular and cellular biology and pathology l.califano, naples, italy (2) federico ii university medical school of naples, departmentof clinical and experimental medicine, naples, italybackground: hepatitis c virus (hcv) infection can induce immunological disorders with different clinical expression such asarthritis, sjogren sindrome and various form of vasculitis.aim: to study the prevalence of anti-cyclic citrullinated peptides antibodies (anti-ccp) in a group of patients affected by hcv-related arthritis and the eventual correlations with rheumatoid factor (rf) and/orantinuclear antibodies (ana), and articular involvement. study design: 30 patients with arthritis were selected in a population of 380 subjects affected by hcv infection. each patients was evaluated by clinical examination (23 denoted poliarticular and 7 mono-oligoarticualr involvement), by x-graphic aspects of joint involvement (8 patients presented join erosions), by ana, rf and anti-ccp positiveness.results: 33,3% of patients presented positivenessfor anti-ccp, without significant correlation between suchparameter and ana, rf and articular involvement. anti ccp resulted positive in 4 out of the 8 patients with joint erosions, and only in 6 out of the 22 patients without joint erosions. such frequency analyzed by chi square ended up in no significant differences. our patients presented an interesting prevalence of the positiveness for anti-ccp. these data suggest a consideration about the specificity, commonly attributed to this parameter in the diagnosis of rheumatoid arthritis. expression of nkg2d on cd4+ t cells is generally rare in both mice and humans, but has been reported in a number of inflammatory diseases, including rheumatoid arthritis, crohns disease and an animal model of type 1 diabetes. the monoclonal antibody cx5 recognizes murine nkg2d and has been shown to block ligandbinding and mediate internalization of nkg2d. furthermore, cx5 can inhibit and/or ameliorate disease in animal models of type 1 diabetes and inflammatory bowel disease. thus, it is very likely that nkg2d plays an important role in the development of inflammatory and autoimmune diseases. since little is known about the pharmacokinetics and pharmacodynamics of the cx5 antibody, we decided to study this in both regular balb/ c mice and immunodeficient cb17.scid mice. different doses of cx5 antibody was injected intraperitoneally and pk and pd was measured by elisa (anti-cx5 elisa in serum) and flow cytometry (down-regulation of nkg2d on cd49b+ nk cells) for up to two weeks after administration. we found that cx5 very efficiently down-regulate nkg2d on cd49b+ nk cells and that the effects of the antibody can be seen for more than two weeks after one single injection. finally, we propose a model which may be helpful in predicting the effects of different doses of cx5 antibody in vivo. (1), k mehta(2), n deo(2), j chaudhary(2), p bobrowski (3) (1) albany medical college, usa (2) vedic lifesciences, usa (3) rainforest nutritionals, inc, us background: the efficacy and safety of reparagen, in treating osteoarthritis was compared to glucosamine sulfate in a mumbai-based multi-center, randomized, double-blind study.methods: subjects (n=95) were screened and randomized to receive glucosamine sulfate (n= 41, 1500 mg/day) or reparagen (n=38, 1800 mg/day), a polyherbal consisting of vincaria (uncaria guianensis) and rni 249 (lepidium meyenii) administered orally, twice daily. primary efficacy variables were womac scores, visual analog score (vas) for pain, and response to treatment defined as a 20% improvement in womac pain, with assessments at 1, 2, 4, 6 and 8 weeks. secondary variables were results: subject randomization was effective and both treatments showed significant benefits in primary outcomes within one week (p<0.05), with a similar, progressive improvement over the course of the 8 week treatment protocol (42-49% reduction in total womac or vas scores). the response rate was substantial for both glucosamine (88%) and reparagen (92%), which exceeded placebo responses (55%, p <0.01) and supported by investigator and subject assessments. tolerability was excellent and safety parameters were unchanged. rescue medication use was significantly lower in the reparagen group (p <0.01), and serum igf-1 levels were unaltered.conclusions: both reparagen and glucosamine sulfate produced substantial improvements osteoarthritis symptoms. response rates were high and the safety profile was excellent, with significantly less rescue medication use with reparagen. we speculate that the high response rate to glucosamine sulfate may reflect higher baseline pain levels or synergy with dietary curcumin. inflammation accompanies and aggravates progression of all modern human chronic pathological conditions. growing evidence indicates the beneficial role of proper nutrition in controlling inflammation. we investigated the effects of selected essential nutrients in experimental inflammation and the molecular mechanisms involved. tested nutrient mixture (nm) consisted of green tea catechins, citrus flavonoids hesperidin, naringenin and quercetin, ascorbate, lysine, proline, arginine and cysteine. systemic inflammation in mice challenged with bacterial lipopolysaccharide (lps) was monitored by blood plasma levels of fourteen key inflammatory cytokines. two week supplementation with 250 mg nm/kg body weight prior to lps challenge provided significantly greater protection than did supplementation with ibuprofen. induction of interleukin-6 (il-6) and monocyte chemoattracting protein-1, two cytokines especially responsive to lps challenge, was reduced in nmsupplemented animals by 58% and 86%, respectively. corresponding reduction in ibuprofen group was 34% and 43%. protective mechanisms involved were assessed in human cultured u937 macrophages stimulated with lps.the cytokines most responsive were tumor necrosis factor alpha (71% and 17% reduction by supplementation with nm and ibuprofen, respectively) and il-12 (66% and 15% in corresponding reduction). nm supplementation dramatically reduced prostaglandin e2 secretion by stimulated macrophages along with cyclooxigenase-2 (cox2) cellular protein expression. mrna levels forcox2 and inflammatory cytokines were also dramatically reduced. quercetin was the most effective nutrient when tested individually. however, nm appeared to surplus the combined effect of individual components. we conclude that the tested combination of essential nutrients demonstrates strong beneficial effects in experimental inflammation by targeting responsible gene expression. (1), hp kim (1), kh son (2) (1) college of pharmacy, kangwon national university, south korea (2) department of food and nutrition, andong university, south korea chalcones belong to flavonoid family from plant origin and some of them possess anti-inflammatory activity. recently, several natural and synthetic chalcones were reported to inhibit inducible nitric oxide synthase (inos)-catalyzed no production in cell cultures. in the present study, to find the optimal chemical structures and to elucidate their action mechanisms, 41 synthetic chalcones having the substituent(s) on a-and b-rings were prepared and their effects on inos-catalyzed no production were evaluated using lps-treated raw 264.7 cells. among the tested compounds, 2-methoxy-3,4-dichlorochalcone (ch15), 2-hydroxy-6-methoxychalcone (ch29), 2-hydroxy-3-bromo-6-methoxychalcone (ch31) and 2-hydroxy-4,6-dimethoxychalcone (ch35) potently inhibited no production (ic 50 s, 7.0 -9.9 mm). the favorable chemical structures were found to be a methoxyl substitution in a-ring at adjacent position (2 or 6) to carbonyl moiety with/without 2-(or 6-)hydroxyl group and 3-halogen substitution in b-ring. when the cellular action mechanisms of ch15, ch31 and ch35 were further examined, it was revealed that ch15 and ch31 clearly down-regulated inos expression while ch35 did not. moreover, ch15 and ch31 were proved to suppress the nuclear transcription factor-kb activation. from the results, it is suggested that certain chalcone derivatives potently inhibit inos-catalyzed no production by the different cellular mechanisms, inos down-regulation or inos inhibition, depending on their chemical structures. these chalcone derivatives may be possibly used as lead compounds for developing new anti-inflammatory agents. an oligomeric stilbene alpha-viniferin (avf) was isolated from root of carex humilis (cyperaceae) as an inhibitor of cyclooxygenase (cox)-2 activity by bioassayguided fractionation. the avf was later found to downregulate lipopolysaccharide (lps)-induced cox-2 expression as well as to inhibit nuclear factor (nf)-kb activation, in addition to its inhibitory effect on cox-2 activity. furthermore, the compound exhibited antiarthritic effect in vivo. avf is a trimer of resveratrol and contains benzofuran moieties in its central part. starting from benzofuran and its related chemicals, 2cyclohexylimino-6-methyl-6,7-dihydro-5h-benzo [1, 3] oxathiol-4-one (lyr-64) was discovered to inhibit lpsinduced nf-kb transcriptional activity in macrophages raw 264.7. the lyr-64 reduced lps-induced dna binding activity and nuclear translocation of nf-kb as well as inhibited lps-induced degradation and phosphorylation of inhibitory kb (ikb) protein. these results suggest that lyr-64 could suppress lps signaling molecule, putatively ikb kinase (ikk) complex, upstream ikb degradation in nf-kb activating pathway. lyr-64 inhibited in vitro kinase activity, gst-ikb phosphorylation, of wild type ikkbeta or a constitutively active ikkbeta mutant (c/a, cys-179 to ala) but did not affect that of another constitutively active ikkbeta mutant (ss/ee, ser-177 and 181 to glu). therefore, lyr-64 could inhibit lps-induced nf-kb activating pathway by targeting ser-177 and/or 181 residues on the activation domain of ikkbeta. as pharmacological actions, lyr-64 prevented nf-kb-dependent expression of inducible nitric oxide synthase, cox-2, or inflammatory cytokines at the transcription level in lps-stimulated macrophages raw 264.7. furthermore, lyr-64 protected lpsinduced septic shock in vivo. faculty of medicine, institute of pharmacology, ljubljana, slovenia a part of anti-inflammatory action of antidepressants can arise from their effect on histamine elimination from the side of inflammation. in mammals histamine is mainly degraded by two enzymes: histamine-n-methyltransferase (hnmt) and diamine oxidase (dao). the aim of present investigation is to establish whether antidepressants amitriptyline and sertraline can affect histamine metabolism. their effects on enzyme activity and mrna expression were studied in guinea pig tissues. plasma and tissue homogenates were incubated with saline (control) and different antidepressant concentrations. specific enzymatic activities of dao and hnmt were determined by radiometric assay. in addition, guinea pigs were treated with saline or amitriptyline (4 mg/kg, ip), afterwards dao and hnmt mrnas were detected by pcr in different tissues. results showed thatamitriptyline, 100 nm, 50, 100 and 500 mm, increased guinea pig plasma dao activity by 5, 7, 17 and 11 %, respectively, while sertraline increased it at 30 mm (by 15 %). at higher concentrations (100 and 500 mm) sertraline decreased dao activity. in the guinea pig tissues hnmt activity changes were found only when incubated with amitriptyline; sertraline had no effect. at 10 and 50 nm amitriptyline the activity of hnmt increased by 20 and 9 %, respectively. in animals treated with amitriptyline an induction of dao and hnmt mrna expression was noticed in several tissues. our results suggest that in guinea pigs due to higher histamine metabolism antiinflammatory effects can be expected at lower concentrations of antidepressants. the effect might be the opposite with higher amitriptyline concentrations. steven hefeneider(1,2), c macarthur (1), d trune (1), s mccoy (2) (1) oregon health and science university, portland, oregon, usa (2) targeted gene delivery, inc., portland, oregon, usa engagement of toll-like receptors (tlrs) by bacterial components such as lps and dna initiates inflammation.the current study examines a novel anti-inflammatory peptide, termed p13, for treatment of inflammation induced by either lps or bacteria.peptide p13 was derived from an immunoregulatory protein of vaccinia virus, and interferes with tlr signaling.in this study we examined the efficacy of p13 to limit inflammation in a mouse model of sepsis and a model of middle ear inflammation, termed acute otitis media (aom).we demonstrate in the sepsis model, that in vivo treatment of mice with p13 inhibited lps-induced production of serum inflammatory mediators.moreover, p13 treatment, administered after initiation of inflammation, significantly increased survival of mice injected with lps.in the aom model, peptide p13 significantly reduced in vivo middle ear inflammation and fluid accumulation initiated by h. influenza.assessment of route of administration and delayed treatment studies demonstrated the efficacy of peptide p13.simultaneous injection of bacteria and peptide p13 resulted in a significant reduction in fluid accumulation, infiltrating cells, and tympanic membrane thickness.fluid accumulation within the eustachian tubes was also significantly reduced following p13 treatment.-subcutaneous and oral administrations of p13, but not intravenous administration, were also efficacious in reducing inflammation. administration of p13 after initiation of an ongoing inflammatory response was effective at reducing inflammation and fluid development.taken together, these results demonstrate the therapeutic potential of peptide p13 to limit an inflammatory response and suggest a possible new treatment strategy for bacterial-induced inflammation. (1), c zhou(2), y zhang(2), m sun(2), x wan (1), h yu(2), x yang(2), rd ye (3), j-k shen (1) formyl peptide receptor-like 1 (fprl1) is a structural homologue of fpr, which binds chemotactic peptides of as small as 3 amino acids (e.g., fmet-leu-phe, fmlf) and activates potent bactericidal functions in neutrophils. in comparison, fprl1 ligands include peptides of 6-104 amino acids, such as trp-lys-tyr-met-val-[d]met (wkymvm) and other synthetic peptides. to determine the core peptide sequence required for fprl1 activation, we prepared various analogues based on wkymvm and evaluated their bioactivities in an fprl1-transfected cell line. although substitution of d-met6 resulted in loss of activity, removal of val5 together with d-met6 produced a peptide that retained most of the bioactivities of the parent peptide. the resulting peptide, wkym, represents a core structure for an fprl1 ligand. further substitution of lys2 with nle slightly improved the potency of the tetrapeptide, which becomes a dual agonist for both fprl1 and fpr. based on these structure-activity studies, we propose a model in which the modified tetrapeptide trp-nle-tyr-met (wnleym) binds to fprl1 through aromatic interactions involving the side chains of trp1 and tyr3, hydrophobic interaction of nle2, and the thio-based hydrogen bonding of met4, with the respective residues in fprl1 which have not been identified. the identification of the core sequence of a potent peptide agonist provides a structural basis for future design of peptidomimetics as potential therapeutic agents for fprl1-related disorders.there is a growing awareness of the interaction of food constituents with the immune system. the present study aims to evaluate immunomodulatory effects of two of these nutritional components, i.e. glycine and lactoferrin. mice orally supplemented with glycine, lactoferrin or a combination were injected intradermal (in the ear) with zymosan. ear swelling, as a measure for inflammation, as well as il-1, tnf-a and il-6 levels in the ear and the number of tnf-a producing spleen cells were analyzed.-glycine and lactoferrin were able to decrease the zymosan induced inflammatory response locally (decreased ear swelling and pro-inflammatory cytokine levels) as well as systemically (reduced number of tnf-a producing spleen cells).glycine effects (20, 50 and 100 mg/mouse/day) were concentration dependent whereas for lactoferrin only the lowest doses (0.1 and 1 mg/mouse/ day) inhibited the inflammatory response significantly. surprisingly higher doses of lactoferrin (5 and 25 mg/ mouse/day) failed to influence the inflammatory reaction. a combination of both nutrients (lactoferrin 0.1mg/ mouse/day in combination with glycine 20 or 50 mg/ mouse/day) inhibited the zymosan induced ear swelling synergistically. additionally an additive effect of both components was seen on the number of tnf-a producing spleen cells. the present data show anti-inflammatory activity of glycine and lactoferrin using the zymosan induced inflammation model.moreover a combination of both components demonstrated a synergistic effect on inflammation of the skin and an additive effect on the number of tnf-a producing spleen cells. (1), p sambrook(1), k fukudome(2), m xue (1) (1) university of sydney, st leonards, nsw, australia (2) saga medical school, saga, japan objectives: to investigate the i) expression of endothelial protein c receptor (epcr) in synovial membrane and peripheral blood monocytes from patients with rheumatoid arthritis (ra) and osteoarthritis (oa) and ii) role of epcr and its ligand, activated protein c (apc), on the function of monocytes from ra patients.methods: epcr, cd68 and pc/apc in synovial tissues were detected by immunostaining and in situ pcr. monocytes were isolated from peripheral blood of patients with ra and treated with apc, lipopolysaccharide (lps), and/or epcr blocking antibody, rcr252. cells and supernatants were collected to analyze the expression/activation of epcr, nuclear factor nf-kb and tumour necrosis factor tnf-a.results: epcr was expressed by both oa and ra synovial tissues but was markedly increased in ra synovium. epcr was colocalized with pc/apc mostly on cd68 positive cells in synovium. in ra monocytes, apc upregulated epcr expression reduced monocyte chemoattractant protein-1-induced chemotaxis of monocytes by approximately 50%. apc also completely suppressed lps-stimulated nf-kb activation and attenuated tnf-a protein by more than 40% in ra monocytes. the inhibitory effects of apc were reversed by rcr252, indicating that epcr modulates the inhibitory effects of apc.conclusions: our results demonstrate for the first time that epcr is expressed by synovial tissues, particularly in ra, where it co-localizes with pc/apc on monocytes/ macrophages. in addition, apc inhibits the migration and activation of ra monocytes via epcr. these inhibitory effects on ra monocytes suggest that pc pathway may have a beneficial therapeutic effect in ra. key: cord-003532-lcgeingz authors: nan title: 39th international symposium on intensive care and emergency medicine: brussels, belgium, 19-22 march 2019 date: 2019-03-19 journal: crit care doi: 10.1186/s13054-019-2358-0 sha: doc_id: 3532 cord_uid: lcgeingz nan introduction: increasing evidence supports a central role for "immunosuppression" in sepsis. it is necessary to develop biomarkers of immune dysfunction that could help to identify patients at risk of poor outcomes [1] . the decreased expression of human leucocyte antigen (hla)-dra is proposed as a major feature of immunodepression and its persistent decrease is associated with mortality in sepsis [2] . in a previous study, we evidenced that fcer1a (fc fragment of ige receptor ia) is the gene showing the lowest expression levels of the entire transcriptome in sepsis [3] . here we studied the association between fcer1a expression and mortality in infected surgical patients. methods: fcer1a and hla-dra expression levels were quantified by droplet digital pcr in blood of 257 infected surgical patients. 26 patients died within 28 days (10.11%). spearman test was used to evaluate the association between gene expression and the sequential organ failure assessment (sofa) score. areas under receiver operating curves (auroc) were used to determine the gene expression cut-off values predicting mortality. kaplan-meier survival curves were obtained and differences in survival between groups were evaluated using the log rank test. cox regression was employed to assess mortality risk at 28 days. results: gene expression levels of fcer1a and hla-dra correlated inversely with patients' severity (r: -0.5 p<0.001; r: -0.3, p<0.001 respectively). both genes showed significant aurocs to predict survival, but fcer1a showed the best accuracy (fig. 1) . patients with introduction: severe pulmonary and renal conditions such as acute respiratory distress syndrome (ards), respiratory failure, and deterioration in kidney function often occur in patients with nosocomial pneumonia (np). the emergence and course of infection is genetically determined, hence host genetic landscape may influence an ability to resist infection. methods: variants for genotyping were selected using the phewas catalog which presents genotypic data for 13835 caucasian patients, 1358 phenotypes and 3144 single nucleotide polymorphisms (snps) with p < 0.05 [1] . snps with the lowest p-values for phenotypes with both, respiratory and renal manifestations were selected: intergenic variants rs7130588 and rs4980785, rs347344 (edil3) and rs2470893 (cyp1a1). cyp1a1 gene was associated with pneumonia and ards in our previous investigations, so we included in our analysis three sites of cyp1a1 gene (rs2606345, rs4646903 and rs1048943) studied on a smaller sample. genotyping was performed on 7 sites for a sample results: allele rs2606345-g of the cyp1a1 gene was protective against ards and an increase in creatinine level (fig. 1) . the rs7130588-g allele was associated with lung complications and with the development of severe respiratory insufficiency (fig. 2) . conclusions: the snps rs2606345 and rs7130588 can influence the aggravation of pulmonary and renal symptoms through genetically mediated response to infection. introduction: an uncontrolled inflammatory response plays a major role in the sepsis related organ dysfunction. mesenchymal stem cells(mscs) can improve survival of sepsis experimental models by modulating the inflammatory response. macrophages have been considered as important immune effector cells and their polarization imbalance aggravates the disordered inflammation reaction. the project aims to identify the effects of mscs on macrophages polarization against dysregulated inflammatory response. methods: raw264.7 cells were plated in the lower chambers of transwell system in the presence or absence of lipopolysaccharide (lps). then, mscs were seeded in the upper chambers and incubation for different time. finally, transforming growth factor beta (tgfβ) receptor (tgf-βr) inhibitor was added in transwell system. the phenotype of raw264.7 cells were analyzed by flow cytometry, the levels of inflammatory cytokines were detected by enzyme-linked immunosorbent assay (elisa). results: our data showed that lps increased the level of interleukin (il)-6 in raw264.7 cells (p<0.001) (fig. 1 ). in line with il-6 expression, lps induced the expression of m1 macrophage (p<0.001). moreover, lps stimulated raw264.7 cells co-culture with mscs in transwell system, mscs inhibited the expression of il-6 and m1 macrophages, while increased m2 macrophages (p<0.001). compared with lps group, the concentration of tgf-β was obviously increased in mscs treatment groups (p<0.001), furthermore, there were no significantly difference between mscs directed and indicted groups. more significantly, tgf-βr inhibitor abolished the impact of mscs on lps stimulated raw264.7 cells (p<0.001) (fig. 2) . conclusions: mscs polarized m1 macrophages into m2 macrophages and decreased pro-inflammatory cytokine levels by paracrining tgf-β. introduction: sepsis is dysregulated response to an infection, which can lead to progressive microcirculatory dysfunction, release of reactive oxygen intermediates (roi) and life-threatening organ dysfunction. our aim was to investigate the relationship between organ damage -characterized by the sequential organ failure assessment (sofa) scores, microcirculatory failure and roi production, in a large animal model of experimental sepsis. methods: fecal peritonitis was induced in anesthetized minipigs (n=28; 0.5g/kg autfeces containing 5-9 x10 6 cfu bacteria i.p.), control animals (n=9) received sterile saline i.p. invasive hemodynamic monitoring and blood gas analyses were performed between 16-24 hrs, the signs for failure of circulatory, respiratory and urinary systems were evaluated in accordance with the sofa score. the microcirculatory perfusion rate in the sublingual region was measured by orthogonal polarization spectral imaging technique (cytoscan a/r). the leukocyte-origin roi production was determined by lucigenine (mostly o 2 -. ) and luminol-based (h 2 o 2 ) chemiluminescence methods. results: between 16-24 hrs after induction the sofa score indicated moderate organ failure in 19 animals (m: 1.9; 25p: 1.5, 75p: 2.9) and the change was statistically significantly higher in 9 pigs, suggesting severe organ dysfunction (m: 4.1; 25p: 3.5, 75p: 5.2). the microcirculation was significantly deteriorated in all cases, independently of sofa score data. the h 2 o 2 production was significantly lower in septic animals as compared to controls, while the lucigenine enhanced roi production correlated with the sofa score-indicated moderate and severe organ dysfunction. conclusions: sublingual microcirculatory parameters are not correlating with the severity of sofa score-indicated organ dysfunction in abdominal sepsis. the measurement of roi production of the whole blood seems to be better biomarker for the detection of the progression of events from moderate to severe organ damages. introduction: the purpose of this study was to characterize differences in sepsis management in patients with and without left ventricular (lv) dysfunction. septic patients with lv dysfunction have higher mortality, and limited guidance exists for sepsis management of patients with lv dysfunction. the possibility exists that the cornerstones of sepsis management may contribute to these poor outcomes. methods: a retrospective chart review was conducted from may 2016 -january 2018 at two centers. adult patients who had a diagnosis of sepsis, were treated with vasopressors for > 3 hours, and had an echocardiogram within 12 months were included. patients were divided into two groups: reduced ejection fraction (ef) of < 40% and preserved ef defined as ef ≥40%. information about patient outcomes and sepsis management were collected. the primary outcome was the need for mechanical ventilation (mv). categorical and continuous data were analyzed using the chi-squared and mann-whitney u tests, respectively. the irb has approved this project. results: a total of 37 patients with ef < 40% and 42 patients with ef ≥40% were included. no significant differences in fluid management, vasoactive agent maximum rate or duration, or steroid use were observed. net fluid balance between low and preserved ef was positive 4.6 liters vs. 5.1 liters (p = 0.814), respectively. the number of patients that needed mv was higher in the low ef cohort (86% vs. 57%, p = 0.004), and this cohort had fewer mv-free days (20, iqr 0-25 vs. 24 (iqr 0 -28), p=0.064. conclusions: no significant differences were observed with regard to sepsis management, reflecting current guidelines. the significantly increased need for mv is a provocative result. a potential mechanism is the inability of a patient with reduced lv dysfunction to maintain appropriate cardiac and respiratory function in the face of fluid overload. prospective analysis of the role of fluid balance in septic patients with lv dysfunction is warranted. introduction: the relationship between myocardial injury and systemic inflammation in sepsis response is not well understood [1] . it´s proposed to evaluate the association between myocardial injury biomarkers, high-sensitive troponin t (hs-ctnt) and n-terminal pro-brain natriuretic peptide (nt-probnp), with inflammatory mediators (il-6, il-1β , il-8, il-10, il-12 / il-23p40, il17a, il-21 and tnf-α ) and biomarkers, c protein reactive (cpr) and procalcitonin (pct), in septic patients methods: this was a prospective cohort study performed in three intensive care units, from september 2007 to september 2010 enrolling patients with sepsis (infection associated with organ dysfunction), and septic shock (hypotension refractory by fluids infusion requiring vasopressor). blood samples were collected up to 48h after the development of first organ dysfunction (d0) and on the 7th day after inclusion in the study (d7) results: ninety-five patients were enrolled, with median age 64 years (interquatile?48-78), apache ii: median 19 (14-22), sofa: median 8 (5-10); 24.2% were admitted in icu with sepsis and 75.8% with septic shock. hospital mortality was 34.7%. in d0, nt-probnp correlated with il-8 (r = 0.495, p <0.001) and il-10 (r = 0.471, p <0.001). in d7, hs-ctnt and nt-probnp correlated with pct (r = 0.446, p < 0.001 and r = 0.495, p < 0.001; respectively). nt-probnp d0 was higher in nonsurvivors than in survivors on mortality in seventh day (p = 0.029) and in-hospital mortality (p = 0.030). hs-ctnt d7 (p = 0.030) and nt-probnp d7 (p <0.001) were significantly higher in non-survivors on in-hospital mortality. nt-probnp d7 (or 9.28; ic95% 2.05-41.94, p=0,004) and hs-ctnt d7 (or 10,93; ic95% 2.139 -55.795, p=0,04) were independently associated with in-hospital mortality conclusions: nt-probnp plasma levels at d0 correlated with il-8 and il-10, and both nt-probnp and hs-ctnt at d7 correlated with pct. in addition, nt-probnp has been shown to be an important predictor of mortality introduction: heparin-binding protein (hbp) acts proinflammatory on immune cells and induces vascular leakage through cytoskeletal rearrangement and cell contraction in the endothelium and is a promising novel prognostic biomarker in sepsis and septic shock. however, studies on repeated measures of hbp are lacking. our objective was to describe the kinetics of plasma hbp during septic shock and correlate it to hemodynamic parameters. methods: we included patients with septic shock (sepsis-3) on admission to helsingborg hospital's intensive care unit (icu) during september 2016 to february 2018. patients were sampled from icu admission and every 4 hours for 72 hours or until death or icu discharge. the plasma samples were analyzed for hbp and converted using the natural log (lnhbp) for normality. lnhbp was then evaluated against mean arterial pressure (map) as primary analysis and against systemic vascular resistance index (svri) as a secondary analysis, using mixed-effects linear regression models, treating patient id as a random intercept and adjusting for hemodynamic parameters. results: a total of 22 patients were included with median age 67 years, 9 females (41%), 7 surgical admissions (32%), median sofa-score 12 points on day one and 6 deaths from all causes within 90 days (27%). plasma hbp ranged from 0 to 932 ng/ml with a median of 47 ng/ml (lnhbp range 1.6 to 6.8, median: 3.9 ). an increase lnhbp was significantly associated with a decrease in map (coef. -2.58 mmhg, 95% ci: -0.62 to -4.55, p=0.010, n=22), when adjusting for heart rate (hr), noradrenaline (na), vasopressin (vp), dobutamine (dbt) and levosimendan (ls). in a secondary subgroup analysis, an increase in lnhbp was also significantly associated with a decrease in svri (coef. -94.2 dyne*s*cm-5*m-2, 95% ci: -1.3 to -187.1, p=0.047, n=13), when adjusting for map, hr, na, vp, dbt, ls and cardiac index. conclusions: repeated measures of plasma hbp during septic shock were correlated with important hemodynamic parameters in this small pilot study. introduction: mid-regional pro-adrenomedullin (mr-proadm) comes from the synthesis of the hormone adrenomedullin (adm), which is overexpressed during inflammation and progression from sepsis to septic shock. thus, mr-proadm can be a useful biomarker for the clinical management of septic patients [1] . the aim of our study was to understand the ability of mr-proadm to predict 30-day (30-d) mortality and to find a correlation between mr-proadm and sequential organ failure assessment (sofa) score in the first 24 hours from intensive care unit (icu) admission. methods: we evaluated 28 consecutive septic shock patients according to 2016 sepsis iii definitions. clinical data from the medical records included demographics, comorbidities, laboratories, microbiology and biomarker levels. whole blood samples for biomarker profiling were collected at 24, 72 and 120 hours from icu admission. mr-proadm measurement was detected in edta plasma using a sandwich immunoassay by trace® (time resolved amplified cryptate emission) technology (kryptor thermo fischer scientific brahms). results: overall 30-d mortality rate was 50.0%. mr-proadm [odds ratio (or) = 1.195], sofa score (or = 2.174) and lactate (lac) levels (or = 1.956) in the first 24 hours were associated with 30-d mortality in univariate logistic analysis (p value < 0.05, table 1 ). 30-d mortality rate was not associated with procalcitonin (pct) levels (or = 1.002). further linear regression analysis showed significant correlation between mr-proadm and sofa score at 24 hours from icu admission (p value<0.001, fig. 1 , table 2 ). conclusions: mr-proadm demonstrated superior accuracy to predict 30-d mortality compared to pct levels and is directly linked to sofa score at 24 hours from admission. mr-proadm may aid early identification of poor prognosis septic patients who could benefit a more intensive management. introduction: study of the expression of cell free dna (cfdna) in the search for new biomarkers for infection, sepsis and septic shock. methods: the population studied was all patients included in the sepsis protocol from march 2017 to january 2018, hospitalized patients of a federal public hospital. plasma samples were collected for quantification of cfdna, which after centrifugation were stored at -80°c and then thawed and analyzed by fluorescence using a varioskan flash fluorometer). cfdna values were expressed as ng/ml. the patients were divided into 2 groups: infection and sepsis/septic shock. we analyzed mortality, sequential organ failure assessment score (sofa score), qsofa (quick sofa), comorbidities, cfdna and laboratory parameters of 111 patients. results: among the 111 patients, 28% were classified as infection and 72% sepsis/septic shock. overall lethality was 33%, infection 9.7%, and sepsis/septic shock 42.5% (p<0.001). the mean of cfdna, sofa and lactate was higher according to the classification of infection and sepsis/septic shock: cfdna (159.4±117.3 and 282.7±358.6, p=0.006), sofa (1.9±2.1 and 6.6±4.3, p<0.001), qsofa (positive in 25% and 75%, lactate (1.6±0.8 and 3.8±3.5, p<0.001). we analyzed leukocytes, creatinine, crp (c reactive protein), inr (international normalized ratio), as predictors of severity and only crp showed no association with disease severity (p=0.84). levels of cfdna and qsofa showed worse prognostic utility as a predictor of sepsis / septic shock when compared to lactate and sofa: or 1.00 (95% ci 0.41-2.45), p=0.98 for cfdna, or 2.4 (95% ci 1.37-4.21), p=0.002 for sofa and or 2.00 (95% ci 0.94-4.28), p=0.072 for lactate. negelkerke r square was 0,633 for cfdna. in addition, area under the curve for cfdna mortality was 0.60 (95% ci 0.46-0.73) and sofa 0.81 ci 95% 0.19-0.91). conclusions: our study suggests that cfdna and qsofa have worse prognostic accuracy when compared to lactate and sofa, variables already used in clinical practice and easily measured. introduction: the aim of this study is to develop a "molecular equivalent" to sequential organ failure assessment (sofa) score, which could identify organ failure in an easier, faster and more objective manner, based on the evaluation of lipocalin-2 (lcn2/ngal) expression levels by using droplet digital pcr (ddpcr). sepsis has been classically defined as the exuberant, harmful, pro-inflammatory response to infection. this concept is changing [1] and the presence of a life-threatening organ dysfunction caused by a dysregulated host response to infection is now considered a central event in the pathogenesis of sepsis [2] . methods: lcn2 expression levels were quantified by ddpcr in blood of a total of 257 surgical patients with a diagnosis of infection. spearman analysis was used to evaluate if lcn2 correlated in a significant manner with sofa score. area under the receiver operating curve (auroc) analysis and multivariate regression analysis were employed to test the ability of lcn2 to identify organ failure and mortality risk. results: spearman analysis showed that there was a positive, significant correlation between lcn2 expression levels and sofa score (fig. 1) . aurocs analysis showed that lcn2 presents a good diagnostic accuracy to detect organ failure and mortality risk (fig 2) . in the multivariate regression analysis, patients showing lcn2 expression levels over the optimal operating points (oops) identified in the aurocs showed a higher risk of developing organ failure (table 1) and a higher mortality risk (table 2) . conclusions: quantifying lcn2 expression levels by ddpcr is a promising approach to improve organ failure detection and mortality risk in surgical patients with infection. introduction: sepsis is an inflammatory state due to an exacerbated immune response against infection. in cancer patients, sepsis presents a 10-fold higher mortality than in general population and leads to longer intensive care unit (icu) and hospital lengths of stay. it has been shown that reduced levels of circulating immunoglobulins (ig) might be a surrogate marker of unfavorable outcome in sepsis [1] . the aim of this study was to evaluate the association between ig levels in plasma and 60-day mortality rate in cancer patients with septic shock. methods: from december 2017 to november 2018, we conducted a prospective study in the intensive care unit (icu) of cancer institute of state of sao paulo, an 84-bed icu linked to university of sao paulo. patients ≥18 years old with cancer and septic shock were enrolled. descriptive statistics were computed for demographic and outcome variables. laboratory data and ig levels were collected at icu admission and at days 1, 2 and 3. a multivariate analysis was performed to evaluate predictors of 60-day mortality. results: a total of 190 patients were included in the study. the 30-day and 60-day mortality were 40.5% and 45.3%, respectively. no significant differences in igm and igg levels were observed between survivors and non-survivors. in both groups, the median igm levels were low and the median igg levels were normal. in the multivariate analysis for 60-day mortality, a favorable status performance measured by the eastern cooperative oncology group (ecog) was associated with better survival; metastatic disease, higher sequential organ failure assessment (sofa) score at admission and higher levels of initial lactate were associated with increased mortality. conclusions: low levels of serum endogenous immunoglobulins are not predictors of 60-day mortality in cancer patients with septic shock. introduction: cytovale has developed a rapid biophysical assay of the host immune response which can serve as a rapid and reliable indicator of sepsis. neutrophils and monocytes undergo characteristic structural and morphologic changes in response to infection. one type of response is the generation of neutrophil extracellular traps (nets), these have been proposed as potential mediators for widespread tissue damage. during netosis there is a fundamental reorganization of a cell's chromatin structurea signal that we have shown is sensitively measured by the cytovale cytometer. we hypothesized that quantification of plasticity (deformability) of leukocytes in the peripheral blood provides an early indicator of sepsis. the cytovale assay uses microfluidic cytometry to measure the plasticity of up to 100,000 white blood cells from edta-anticoagulated, peripherally-collected whole blood and provides a result in 5 minutes. methods: in two prospective studies conducted in two academic medical centers in baton rouge, la, the cytovale test was performed on peripheral blood samples obtained from 500 patients who presented to the emergency department with signs or symptoms suggestive of infection. the two studies included high acuity patients (400 patient study) and low acuity patients (100 patient study). an adjudicated reference diagnosis of sepsis or no sepsis was established for each subject, using consensus definitions, by review of the complete medical records. results: the receiver operator curve (roc) performance of the cytovale assay for both studies demonstrated an area under the curve (auc) greater than 0.85 (fig. 1) . conclusions: measurement of neutrophil and monocyte plasticity by a novel assay provides an accurate and rapid indication of sepsis in patients who present to an emergency room with signs or symptoms of infection. plasma hepatocyte growth factor in sepsis and its association with mortality: a prospective observational study introduction: sepsis and septic shock are commonly associated with endothelial cell injury. hepatocyte growth factor (hgf) is a multifunctional protein involved in endothelial cell injury and plays a pivotal role in sepsis. this study assesses its correlation with relevant endothelial cell injury parameters and prognostic value in patients with sepsis. methods: a prospective, observational cohort study was conducted in patients with sepsis admitted to the department of critical care medicine at the zhongda hospital from november 2017 to march 2018. the plasma hgf level was collected on the first 24h after admission (day 1) and day 3, then was measured by enzyme-linked immunosorbent assay. the primary endpoint was defined as all-cause 28-day mortality. furthermore, we analyzed the correlation of hgf with relevant endothelial cell injury markers. results: eighty-six patients admitted with sepsis were included. hgf levels of non-survivors were elevated upon day 1 (1940.62 ± 74.66pg/ml vs. 1635.61 ± 47.49pg/ml; p = 0.002) and day 3 (1824.82 ± 137.52pg/ml vs. 1309.77 ± 83.49pg/ml; p = 0.001) compared with that in survivors, and showed a strong correlation with von willebrand factor (r = 0.45, p <0.0001), lactate (r = 0.35, p = 0.0011), pulmonary vascular permeability index (r = 0.38, p = 0.0241), first 24 h fluid administration (r = 0.38, p <0.0001) and sequential organ failure assessment score (r = 0.40, p = 0.0001) (fig. 1) . plasma levels were able to discriminate prognostic significantly on day 1(auc: 0.72, 95%ci: 0.60-0.84) and day 3 (auc: 0.77, 95%ci: 0.63-0.91) (fig. 2) . conclusions: hgf levels are associated with sepsis and are correlated with established markers of endothelial cell injury. elevated hgf level in sepsis patients is a predictor of mortality. methods: adult patients with septic shock by the sepsis-3 classification due to lung infection or primary bacteremia or acute cholangitis are screened using two consecutive measurements of ferritin and of hla-dr/cd14 co-expression for mals (ferritin above 4,420 ng/ml) or immunosuppression (hla-dr/cd14 less than 30%) and randomized into immunotherapy with either anakinra (targeting mals) or recombinant ifnγ (targeting immunosuppression) and into placebo treatment. main exclusion criteria are primary and secondary immunodeficiencies and solid and hematologic malignancies. results: 101 patients have been screened so far. most common infections are community-acquired pneumonia (41.6%), hospitalacquired pneumonia (26.7%) and primary bacteremia (12.9%). mean +/-sd sofa score is 12.6 +/-2.9 and charlson's comorbidity index 5.21 +/-2.44; 25 patients have mals (24.8%); two immunosuppression (2%); the majority remain unclassified for immune state. conclusions: current screening suggests greater frequency of mals than recognized so far in a setting of septic shock due to lung infection or primary bacteremia or acute cholangitis. development of an algorithm to predict mortality in patients with sepsis and coagulopathy d hoppensteadt 1 , a walborn 2 , m rondina 3 , j fareed 1 study was to develop an equation incorporating biomarker levels at icu admission to predict mortality in patients with sepsis, to test the hypothesis that using a combination of biomarkers of multiple systems would improve predictive value. methods: plasma samples were collected from 103 patients with sepsis at the time of icu admission. biomarker levels were measured using commercially available, elisa methods. clinical data, including the isth dic score, sofa score, and apache ii score were also collected. 28-day mortality was used as the primary endpoint. stepwise linear regression modeling was performed to generate a predictive equation for mortality. results: differences in biomarker levels between survivors were quantified and using the mann-whitney test and the area under the receiver operating curve (auc) was used to describe predictive ability. significant differences (p<0.05) were observed between survivors and non-survivors for pai-1 (auc=0.70), procalcitonin (auc=0.77), hmgb-1 (auc=0.67), il-6 (auc=0.70), il-8 (auc=0.70), protein c (auc=0.71), angiopoietin-2 (auc=0.76), endocan (auc=0.58), and platelet factor 4 (auc=0.70). a predictive equation for mortality was generated using stepwise linear regression modeling. this model incorporated procalcitonin, vegf, the il-6:il-10 ratio, endocan, and pf4, and demonstrated a better predictive value for patient outcome than any individual biomarker (auc=0.87). conclusions: the use of a mathematical modeling approach resulted in the development of a predictive equation for sepsis-associated mortality with performance than any individual biomarker or clinical scoring system. furthermore, this equation incorporated biomarkers representative of multiple physiological systems that are involved in the pathogenesis of sepsis. the effects of biomarker clearances as markers of improvement of severity in abdominal septic shock during blood purification t taniguchi 1 , k sato 2 , m okajima 2 introduction: sepsis associated coagulopathy (sac) is commonly seen in patients which leads to dysfunctional hemostasis. the purpose of this study is to determine the thrombin generation potential of baseline blood samples obtained from sac patients and demonstrate their relevance to thrombin generation markers. methods: baseline citrated blood samples were prospectively collected from 49 patients with sac at the university of utah clinic. citrated normal controls (n=50) were obtained from george king biomedical (overland park, ks). thrombin generation studies were carried out using a flourogenic substrate method. tat and f1.2 were measured using elisa methods (seimens, indianapolis, in). functional antithrombin levels were measured using a chromogenic substrate method. results: the peak thrombin levels were lower (82 ± 40nm) in the dic patients in comparison to higher levels observed in the normal plasma (133 ± 10nm). the auc was lower (561 ± 280) in the dic group in comparison to the normals (624 ± 18). the dic group showed much longer lag time (4.1 ± 2.1) in comparison to the normal group (2.1 ± 2.2). wide variations in the results were observed in these parameters in the dic group. the f1.2 levels in the dic group were much higher (570±48 pmol) in comparison to the normal (210 ± 25 pmol). the tat levels also increased in the dic group (27.9 ± 5.1 ng/ml) in comparison to the normal (2.8 ± 0.8 ng/ml). the functional antithrombin levels were decreased in the dic group (64 ± 11%). conclusions: these results validate that thrombin generation such as f1.2 and tat are elevated in patients with dic. however thrombin generation parameters are significantly decreased in this group in comparison to normals. this may be due to the consumption of prothrombin due to the activation of the coagulation system. the decreased functional at levels observed in the dic group are due to the formation of the complex between generated thrombin and antithrombin. introduction: sepsis-associated disseminated intravascular coagulation (dic) is a complex clinical scenario involving derangement of many processes, including hemostasis. assessment of markers including inflammation, endothelial function, and endogenous anticoagulants may provide insight into dic pathophysiology and lead to improved methods for assessment of patient condition and response to treatment. methods: citrated plasma samples were collected from 102 patients with sepsis and suspected dic at icu admission and on days 4 and 8. dic score was determined using the isth scoring algorithm (e.g. platelet count, pt/inr, fibrinogen and d-dimer). cd 40 ligand (cd40l), plasminogen inhibitor 1 (pai-1), nucleosomes, procalcitonin (pct), microparticle tissue factor (mp-tf) and prothrombin 1.2 (f1. 2) were measured using commercially available elisa kits. protein c activity was measured using a clot-based assay. interleukin 6 (il-6), interleukin 8 (il-8), interleukin 10 (il-10), tumor necrosis factor alpha (tnfα), and monocyte chemoattractant protein (mcp-1) were measured using biochip technology. results: significant differences in levels of protein c (p=0.009), pct (p=0.0005), il-6 (p=0.019), il-8 (p=0.0149), pai-1 (p=0.015), were observed between survivors and non-survivors. significant variation of protein c (p=0.002), nucleosomes (p=0.05), pct (p<0.0001), il-6 (p=0.001), il-8 (p=0.003), il-10 (p=0.011), tnfα (p=0.021) and mcp-1 (p=0.021) were observed based on severity of dic score. conclusions: markers from multiple systems perturbed in dic were associated with mortality, suggesting that while these systems may not be routinely evaluated in the normal course of patient care, dysfunction of these systems contributes significantly to mortality. in addition, numerous inflammatory cytokines showed an association with dic score. this suggests that the measurement of additional markers in sepsis-associated dic may be of value in the prediction of mortality and may be helpful in guiding treatment for these patients. introduction: the endotoxin activity assay (eaa) is a rapid immunodiagnostic test based on chemiluminescence. it was approved by the fda in 2003 as a diagnostic reagent for risk assessment of severe sepsis in the icu. ascertaining endotoxin levels in the bloodstream is important in targeting patients and determining the appropriate timing for initiation of treatment. it has high sensitivity and specificity for endotoxin, and is considered to be useful in predicting clinical symptoms and determining prognosis. the usefulness of the eaa has yet to be fully clarified. methods: a total of 142 patients admitted to the icu between january 2014 and june 2018 with suspected sepsis or sepsis were enrolled. the eaa was conducted within 24 hr after admission. patient characteristics were determined, together with levels of il-6, procalcitonin, presepsin, and pao2/fio2. thereafter, the patients were classified into 5 groups depending on their eaa value: 1) < 0.2; 2) from ≤ 0.2 to < 0.4; 3) from ≤ 0.4 to < 0.6; 4) from ≤ 0.6 to < 0.9; and 5) ≤0.9). the transition of various markers was also examined. the spearman rank correlation, wilcoxon rank sum test, and a nonrepeated anova were used for the statistical analysis. a p-value of < 0.05 was considered statistically significant. the eaa values showed a positive correlation with both the apache ii (r=0.48) and sofa scores (r=0.56)(p<0.01), although that with the latter was stronger. a significant correlation was also observed with levels of procalcitonin (r=0.45) and presepsin (r=0.51 early diagnosis is important to allow early intervention. the current clinical methods are insufficient for early detection. we hypothesized that intraperitoneal microdialysis allows detection of peritonitis prior to changes in standard clinical parameters in a pig model. methods: bacterial peritonitis was induced in 5 pigs by bowel perforation and intraperitoneal fecal instillation, one pig underwent sham surgery. intraperitoneal microdialysis catheters were placed in each abdominal quadrant. the observation time was 10 hours. results: in peritonitis pigs the intraperitoneal lactate increased during the first two hours and remained elevated throughout the observation time (table 1) , whereas the arterial lactate remained within reference range (<1.6 mm). intraperitoneal glucose decreased significantly. hemodynamics were hardly influenced during the first two hours, and decreased thereafter. sham surgery did not influence in any of the parameters. conclusions: a rapid and pronounced increase in intraperitoneal lactate and decrease in intraperitoneal glucose was observed after instillation of intraabdominal feces. systemic lactate increase was absent, and the hemodynamic response was delayed. postoperative intraperitoneal microdialysis is applicable in detecting peritonitis earlier than standard clinical monitoring and should be evaluated in a clinical study in order to explore if early intervention based on md data will reduce icu length of stay, morbidity and mortality. introduction: procalcitonin (pct) is a serum biomarker suggested by the surviving sepsis campaign to aid in determination of the appropriate duration of therapy in septic patients. trauma patients have a high prevalence of septic complications, often difficult to distinguish from inflammatory response. pct values typically declined after 72h from trauma and increased only during secondary systemic bacterial infections. the aims of the study are to evaluate reliability and usefulness of pct serum concentration in trauma. methods: we retrospectively analyzed data from 40 trauma patients admitted to icu at bufalini hospital -cesena, from july 2017 to august 2018. we collected data about antimicrobial therapy, injury severity score (iss), first arterial lactate in emergency room, sofa score and sepsis severity. plasma pct concentration was measured using an automate analyzer (modular e-brahms) on 1st day of antimicrobial therapy and every 48h hours. antimicrobial therapy was stopped according to a local protocol; however medical judgment was considered the overriding point for therapeutic decision. results: median iss of patients was 33.5, inter quartile range (iqr) 13.5. pct mean concentration at the starting of antimicrobial treatment was 13.07 μg/l (d.s 40.1), median 0.72 (iqr 10.13). no significative correlation (spearman´s rho test) was found between pct at day 1 of antimicrobial therapy and iss (rho -0.186), between first arterial lactate in er and pct (rho 0.158). daily course of pct was not related to distance from trauma (rho -0.116). in 21 of 40 patients (52.2 %) pct measurement led physician to save days of antimicrobial therapy compared with standard clinical practice. we couldn´t find any cut off value. conclusions: our experience suggests that pct could help physician to optimize duration of antimicrobial therapy in trauma patients. no standard approach can be recommended at present. introduction: long duration of antimicrobial treatment may predispose to colonization and subsequent infections by multidrugresistant organisms (mdro) and clostridium difficile. progress (clinicaltrials.gov registration nct03333304) is an on-going trial aiming to use pct for the restraining of this calamity. methods: adult patients with sepsis by the sepsis-3 classification and any of five infections (pneumonia community-acquired; hospital-acquired or ventilator-associated; acute pyelonephritis; primary bacteremia) are randomized to pct-guided treatment or standard of care (soc) treatment. in the pct arm antibiotics are discontinued when pct on or after day 5 is decreased by more than 80% of the baseline or remains below 0.5 ng/ml; in the soc arm antibiotics are discontinued at the discretion of the attending physician. patients are followed for six months. primary endpoint is the rate of infections by mdro and/or c.difficile or death. serial stool samples are cultured for mdro and screened for glutamate dehydrogenase antigen and toxins of c.difficile. results: 201 patients have been enrolled so far. mean ± sd sofa score is 4.4 ± 2.4. most common diagnoses are community-acquired the progress trial is the first trial assessing the probable benefit from pct guidance to reduce ecological sequelae from long-term antibiotic exposure. analysis of baseline patient characteristics indicates that progress is a real-world trial so that results can have major clinical impact. prospective multi-site validation of 11-gene host response signature for influenza diagnosis s thair 1 , s schaffert 1 , m shojaei 2 , t sweeney 3 there are no blood-based diagnostics able to identify influenza infection and distinguish it from other infections. we have previously described a blood-based 11-gene influenza meta-signature (ims) score to differentiate influenza from bacterial and other viral respiratory infections. methods: we prospectively validated the ims in a multi-site validation study by recruiting 654 individuals (608 patients with suspected influenza, 46 healthy controls) in 10 community or hospital clinics across australia. we assayed the ims and 15 genes from viral genome of 3 influenza strains to generate the blood flu score (bfs) as a measure of viremia using nanostring from whole blood rna. results: using clinically determined phenotypes, the ims score distinguished patients with influenza from healthy (auc=0.95), non-infected (auc=0.83), bacterial (auc=0.88), other viruses (auc=0.77) ( figure 1a) . interestingly, probes of bfs were found in all phenotypic groups (non-infected, bacterial, and other viral infections) to varying degrees, and positively correlate with the ims score (r=0.53). ims aurocs improve when the bfs is used to inform the phenotypic groups: healthy (auc=0.92), non-infected (auc=0.90), bacterial (auc=0.93), other viruses (auc=0.88) ( figure 1b ). patients who were clinically influenza negative but had a high ims and bfs were admitted less often, yet had~4-fold higher mortality than those who were clinically influenza negative with low ims and no bfs (table 1) . conclusions: collectively, our prospective multi-center validation of the ims demonstrates its potential in diagnosis of influenza infections. introduction: previous findings of our group suggest that patients with gram-negative hospital-acquired severe sepsis have better prognosis when sepsis is developing after recent multiple trauma through stimulation of favorable interleukin (il)-10 responses [1] . under a similar rationale, we investigated if preceding osteomyelitis may affect experimental osteomyelitis. methods: sham or experimental osteomyelitis was induced in 32 male new zealand white rabbits after drilling a hole at the upper metaphysis of the left tibia and implementing diluent or 5log10 of staphylococcus aureus using foreign body. after three weeks, the foreign body was removed and experimental pyelonephritis or sham surgery was induced after ligation of the right pelvo-ureteral junction and instillation of 6log10 of escherichia coli in the renal pelvis. survival was recorded and circulating mononuclear cells were isolated and stimulated for the production of tumour necrosis factor-alpha (tnfa) and il-10. at death or sacrifice, tissue outgrowth and myeloperoxidase (mpo) were measured. results: four sham-operated rabbits (s), 16 rabbits subject to sham surgery and then pyelonephritis (sp) and 12 rabbits subject to osteomyelitis and then pyelonephritis (op) were studied. survival after 14 days of group sp was 56.3% and of group op 100% (log-rank 6.59; p: 0.010). lab findings are shown in figure 1 . il-10 production was blunted. negative correlation between e. coli outgrowth and tissue mpo was found at the right kidney of the op group (rs: -0.767, p: 0.016) but not of the sp group (rs: -0.318, p: 0.340). conclusions: preceding staphylococcal osteomyelitis provides survival benefit to subsequent experimental osteomyelitis through downregulation of innate immune responses leading to efficient phagocytosis. introduction: activation of neutrophils is a mandatory step and a sensitive marker of a systemic inflammatory response syndrome (sirs) which is closely related to development of multiple organ failure. the search for drugs that can prevent sirs and reduce mortality in critically ill patients remains significant. the aim of this study was to study the anti-inflammatory effect of the synthetic analogue of leu-enkephalin (dalargin) on human neutrophils. methods: the study was conducted on isolated from the blood of healthy donors neutrophils. their activation was assessed by fluorescent antibodies to markers of degranulation cd11b and cd66b (sd11b-fitc and cd66b-alexafluor647 (bd biosciences, usa). as inductors of inflammation lipopolysaccharide (lps) and the peptide formyl met-leu-pro (fmlp) were used. 100mkm fmlp and dalargin in concentrations of 50 and 100 μ g / ml were added to neutrophils at a concentration of 4 ppm / ml and incubated for 30 min at 37°c; then antibodies were added and incubated for 30 min on ice; then fluorescence was assessed by flow cyto flow meter beckman-coulter fc 500. non-parametric criteria were used; data were presented as a median and 25%-75% interquartile intervals. the statistical significance was estimated using mann-whitney test. the difference was considered statistically significant at p<0.05 results: synthetic analogue of leu-enkephalin in various concentrations has an anti-inflammatory effect on both intact and preactivated with bacterial components neutrophils, reducing their activation and degranulation in a dose-dependent manner (figs. 1, 2) . conclusions: synthetic analogue of leu-enkephalin prevents neutrophil activation by bacterial compounds. this has a potential of translation into clinical practice for sepsis treatment. introduction: the endothelin system plays important roles in circulatory regulation through vasoconstrictor et-a and et-b2 receptors and vasodilator et-b1 receptors (etar; etbr, respectively). tissue hypoxia during the progression of sepsis is associated with microcirculatory and mitochondrial disturbances. our aim was to investigate the possible influence of etar antagonist, etbr agonist or combined treatments on oxygen dynamics, microcirculatory and mitochondrial respiration parameters in experimental sepsis. methods: male sprague-dawley rats (n=8/group) were subjected to faecal peritonitis (0.6 g/kg faeces ip) or sham-operation. septic animals were treated with sterile saline solution, or received the etar antagonist etr-p1/fl peptide (100 nmol/kg iv), etbr agonist irl-1620 (0.55 nmol/kg iv) or same doses as combination therapy, 22 hr after sepsis induction. invasive hemodynamic monitoring and blood gas analyses were performed during a 90-min observational window. introduction: sepsis often induces immunosuppression, which is associated with high mortality rates. nivolumab is a human igg-4 antibody directed against the programmed cell death 1 (pd-1) immunecheckpoint inhibitor, which disrupts pd-1-mediated signaling and restores antitumor immunity. nivolumab is an approved anti-cancer drug that may have the potential to improve sepsis-induced immunosuppression. methods: this multicenter, open-label study investigated the safety, pharmacokinetics and pharmacodynamics of a single intravenous infusion of 480 or 960 mg nivolumab in japanese patients with immunosuppressive sepsis (lymphocytes ≤ 1100 /μl). the dosing of nivolumab was set using the predicted steady state concentration of nivolumab at 3 mg/kg every 2 weeks (q2w), which was the approved dosage for cancer patients at the time of planning. results: five and eight patients were assigned to the 480 and 960 mg groups, respectively. the mean (standard deviation) peak serum drug concentration in the 480 mg group was comparable to the predicted median concentration (90% pi [prediction (figures 1 and 2 ). adverse events (aes) were observed in four patients in each group. drug related-aes were observed in only one patient in the 480 mg group (table 2) . no deaths related to nivolumab occurred. conclusions: a single dose of 960 mg nivolumab appeared to be well tolerated and sufficient to maintain nivolumab blood concentration in patients with sepsis. results suggest both 480 and 960 mg nivolumab therapy could improve relevant immune indices. introduction: the systemic inflammatory response syndrome (sirs) accompanies tissue trauma and infection and, when severe or dysregulated, contributes to multiple organ failure and critical illness. observational studies in man and animal have shown that low-dose acetyl-salicylic acid promotes resolution of inflammation and might attenuate excessive inflammation by increasing the synthesis of specialised pro-resolving lipid mediators (spms). methods: we randomly assigned patients with sirs who were expected to stay in icu for more than 48 hours to receive enteral aspirin (200 mg per day) or placebo for 7 days or until death or discharge from the icu, whichever came first. the primary outcome was il-6 serum concentration at 48h after randomisation. the secondary outcomes included safety and feasibility outcomes. in one center, additional blood samples were taken during the first three days for exploratory analysis of spms using reversed-phase highperformance liquid chromatography -tandem mass spectrometry (rp-hplc-ms/ms). results: from march 2015 through december 2017 a total of 48 patients across four general icus in australia underwent randomization (table 1) . compared to placebo patients, il-6 serum concentration after 48h in aspirin-treated patients was not significantly lower (40 [16-166] pg/ml vs 44 [7.4-85] pg/ml; p=0.66). there were no significant differences for control vs. aspirin-treated patients in the change of pro-resolving/anti-inflammatory lipids between the time points (figure 1, 2 ). there were no between-group differences with respect to icu or hospital mortality, number of bleeding episodes or requirements for red cell transfusions (table 2) . conclusions: in patients admitted to the icu with sirs, low-dose aspirin did not result in a decreased concentration of inflammatory biomarkers compared with placebo. introduction: sepsis is associated with excessive ros production, nf-kb, inos and inflammatory mediators overexpression. vitamin c is a cellular antioxidant, it increases enos and decreases nf-kb; it has several immune-enhancing effects and is crucial for endogenous vasopressors synthesis. vitamin c reserves in sepsis are often as poor as in scurvy [1] . in recent studies, intravenous high vitamin c dose seems to reduce organ failure and improve outcome in septic shock. methods: we treated all septic shock patients admitted to our icu in 7 months (from 3/2018 to 9/2018) with intravenous vitamin c 1.5g/ 6h and thiamine 200 mg/12h (for its synergistic effects) [2] as adjunctive therapy for 4 consecutive days and we compared data to septic shock patients admitted in the previous 7 months period. we enrolled 24 patients: 13 received vitamins supplementation, 11 standard of care. we analysed 28-days mortality, sofa at 48 and 96 hours, pct variation from baseline in first 5 days, vasoactive therapy length and daf 28 (days alive and free from vasopressors, mechanical ventilation and rrt in 28 days follow up). patients with end stage kidney disease were ruled out. we analysed data with mann-whitney and wilcoxon tests. results: vit c group showed lower 28-days mortality (23% vs 54.5%: ns); sofa improvement at 48 (-2.4±1.5 vs -0.9±1.9: p=0.01) and 96 hours (-4.2±2 vs -1.9±2: p<0.01) was higher in vit c group; vit c patients had faster pct reduction without statistical significance. mean vasoactive therapy length was quite similar. daf was 16.5 (±9.1) days in vit c group and 9.3 (±8.9) in controls (p=0.03). 3 control patients needed rrt, none in vit c group. conclusions: despite small study size, we found that vit c has positive effects on survival and improves sofa score (fig.1) and daf (fig.2 ) in septic shock. no vit c patient developed oxalate nephropathy nor worsened renal function. introduction: toxin-producing gram-positive organisms cause some of the most severe forms of septic shock [1, 2] . adjunctive therapies such as intravenous immunoglobulins (ivig) have been proposed for these patients [3, 4] . however, at patient presentation, the presence of a toxin-producing organism is most often unknown. methods: we reviewed the use of ivig in our patients requiring extracorporeal membrane oxygenation (ecmo) in a 2-year period between february 2016 and march 2018. results: in 44% (15/34) of the patients that received ivig for presumed toxin-mediated shock, group a streptococcus or panton-valentine leukocidin producing s. aureus was isolated, but the clinical characteristics of these 15 patients were not significantly different from the ones with other final diagnoses, except for a predisposing influenza infection and the presence of an often very high procalcitonin level. these 34 patients were extremely unwell at presentation with a sofa score of 15 ± 3, high lactate levels (8.6 ± 5.8 mmol/l) and need for vasopressors (equivalent norepinephrine dose of 0.93 ± 0.62 μ g/kg/min). they had very high inflammatory parameters with a procalcitonin ≥ 100 ng/ml in more than half of patients (18/34). ivig use in these patients was generally safe, with only 1 possible transfusion reaction. the mortality of 35% (12/34) was lower than predicted based on the sofa scores. conclusions: ivig administration can be considered in a selected group of patients presenting with acute and very severe septic shock, as part of a multimodal approach [5] . introduction: extra corporeal treatments are used in septic patients to decrease the inflammatory mediators, but definitive conclusions are lacking . more over in many studies the effect of aki isn't evaluated and this may be an important bias. . the aim of this study is to evaluate in septic patients with aki: 1the effect of the adsorbing membrane oxiris on the immunological response 2-the different response in survivors and non survivors methods: from our local data base we analyzed retrospectively 50 septic shock patients with aki (kdigo classification) submitted to crrt with the adsorbing membrane oxiris (baxter, usa ) . at basal time ( t0 ) and at the end of the treatment ( t1 ) we evaluated the following variables: il 6 il 10 procalcitonin endotoxin (eaa). all data are expressed as mean ±sd or median and iqr. student t test or mann-whitney was used to compare values changes. p < 0.05 was considered statistically significant. results: thirty patients with sepsis /septic shock and aki were enrolled in this study. 10 patients had aki 3, 15 patients aki 2, 5 patients aki 1. the duration of treatment was 38± 10 hours. 20 patients had citrate as anticoagulation and 10 heparine continous ev. at table 1 are shown the main results of this study in all the patients. survivors vs non survivors had a significant decrease of il 6, procalcitonin and eaa. conclusions: data of this study confirm on clinical ground previous study "in vitro" [1] that the adsorbing membrane oxiris has important immunological effect during septic shock with aki. this must be confirmed in a rct. introduction: sepsis is common and often fatal, representing a major public health problem. hemoadsorption (cytosorb) therapy aims to reduce cytokines and stabilise the overall immune response in septic shock patients. methods: a prospective, multi-centre, investigator initiated study to evaluate hemoadsorption (cytosorb) therapy in septic shock patients admitted to a tertiary icu's in india during 2016 to 2018. all centres followed a common protocol and received ethics committee approval. results: a total of 45 patients were administered cytosorb in addition to standard of care. a total of 26 patients (62%) survived out of 45 patients. among survival group, 17 patients (71%) were administered cytosorb within 48 hours of icu admission resulting in significant reduction in sepsis scores, apache ii (24.42 vs 19.33) and sofa (12.46 vs 8.71) post cytosorb therapy. also there was reduction in inflammatory markers like cytokines il6 in most of the patients. all patients in survivor group showed a significant improvement in map (69.2 vs 76.8) and reduction in vasopressors (epinephrine 0.3 to 0.03 mcg/kg/min, nor-epinephrine 0.34 to 0.04 mcg/kg/min) after cytosorb therapy. no device related adverse effect was observed in any of the patients. among the non-survivor group, (16 patients, 38%) we observed that cytosorb was administered after 48 hours of icu admission. although a few patients showed improvement in sofa score, majority did not show a significant improvement with map (68.05 vs 60.4 mm of hg) and required increased demand in vasopressors. conclusions: in this multi-centered prospective iis study, we could observe clinical benefits of hemoadsorption (cytosorb) therapy in septic shock patients if the therapy was initiated early. larger randomised study are required to establish the above clinical benefits in larger patient population. a single centre experience with hemoadsorption (cytosorb) in varied causes of sepsis and mods y mehta 1 , c mehta 1 , a kumar 1 , j george 2 , a gupta 3 , s nanda 1 , g kochar 1 , a raizada 3 introduction: sepsis and the multiorgan failure is a leading cause of mortality in the intensive care unit. promising new therapies continue to be investigated for the management of septic shock. we tried to evaluate a novel hemoadsorption therapy (cytosorb) through a retrospective evaluation of patient's data in our centre. we used it as an adjuvant therapy in our patients with sepsis due to varied causes. methods: we retrospectively analysed data of 100 introduction: septic shock is a life-threatening multiple organ dysfunction that has high morbidity and mortality in critically ill patients, due to a dysregulated host response to infection. the aim of this study was to evaluate the efficacy of therapeutic cytokine removal (cytosorb®) in the management of patients with septic shock. methods: we retrospectively analyzed patients admitted to icu with septic shock between june 2015 and november 2017. patients included in the study were diagnosed according to the third international consensus definitions for sepsis and septic shock (sepsis-3), received maximal supportive care including continuous veno-venous hemodiafiltration (cvvhdf) for acute kidney injury and cytosorb® haemoadsorption column was added to return limb of the cvvhdf circuit. demographic data, procalcitonin and leukocyte levels before and after therapeutic cytokine removal and duration of cytosorb® haemoadsorption column application and apache ii scores were recorded. results: the mean age of 48 patients included in the study was 54 ±16.4 years (74% male) and the mean body mass index was 23.1 ±5.9. the mean apache ii score was 21.5 with an expected and actual mortality rates of 40% and 25%, respectively. 18% of the patients were admitted with sepsis and 82% of them with septic shock. 37.5% (n=18) of the cases were solid organ transplant recipients. cvvhdf was applied in all patients during therapeutic cytokine removal. treatment was combined with ecmo in 8 patients. while the mean duration of cvvhdf was 102.1 hours, the duration of cytosorb® haemoadsorption column application was 24.1±13.4 hours. procalcitonin (16.5 ± 25ng/ml vs 25±34ng/ml) and leucocyte levels (14048±10490/ mm3 vs 9278±8693 mm3) after therapeutic cytokine removal were found significantly lower than the pretreatment values (respectively p=0.0013, p=0.006). conclusions: therapeutic cytokine removal applied with cvvhdf in septic shock patients have positive contributions to biochemical parameters and provide survival advantage. introduction: recent studies have focused on demonstrating the potential benefits of immunomodulation in the management of septic patients. the aim of our study was to assess the effects of a hemoadsorption column (cytosorb®) in critical ill septic patients. methods: after ethical approval was obtained, we prospectively included 39 patients admitted to the general icu of fundeni clinical institute. three consecutive sessions of renal replacement therapy (continuous venovenous hemodiafiltration) in combination with cytosorb® were applied after icu admission. clinical (heart rate, arterial pressure, temperature, glasgow coma scale) and paraclinical data (pao2, serum bilirubin and creatinine, platelet count, white blood cell count, ph, c-reactive protein and procalcitonine), vasopressor support and need for mechanical ventilation were recorded before and after the three sessions. results: the mean age in the study group was 57±15 years. median number of organ dysfunction at the time of icu admission was 4 [1] [2] [3] [4] [5] and the mean sofa score was 10.0±3.7. the use of cytosorb® was associated with a non-significant increase in pao2/fio2 ratio from 216±80 to 248±94 (p=0,278) and creatinine levels from 1.8±1.3 to 1.6 ±1.2 mg/dl (p=0.685). although we observed a non-significant increase in c-reactive protein levels from 136±66 mg/l to 144±72 mg/ l (p=0.577), we noted a significant decrease in procalcitonine levels from a median of 20.0 [2.2, 100 .0] ng/dl to a median of 9.1 [0.7, 55.2] ng/dl (p=0.041). a significant decrease in platelet count was also noted from 135384±99263 /mm3 to 78470±61624 /mm3 (p=0.002). mean sofa score decreased non-significantly from 10.0±3.7 to 9.1 ±4.1 (p=0.533). conclusions: the use of cytosorb was associated with a slight nonsignificant improvement in organ function and a decrease of procalcitonine levels. thrombocytopenia remains one of the most important complications of renal replacement therapy. introduction: circulating cell-free neutrophil extracellular traps (nets) would induce a microcirculatory disturbance of sepsis. the removal of nets remnants from the circulation could reduce nets-dependent tissue injury. to address this issue, we evaluated the effect of hemoperfusion with a polymyxin b cartridge (pmx-dhp; toray, japan), which was originally developed for the treatment in patients with gram-negative bacterial infection, on circulating cell-free nets in patients with septic shock and in phorbol myristate acetate (pma)-stimulated neutrophils obtained from healthy volunteer. methods: ex vivo closed loop hemoperfusion was performed through a circuit formed by connecting the small pmx module to a tube and a peristalsis pump. whole blood from healthy volunteers incubated with or without pma or from septic shock patients were applied to circuit and perfused. blood was collected at 0, 1 and 2 hr after perfusion. circulating cell-free nets were assessed by myeloperoxidase (mpo)-, neutrophil elastase (ne)-, and cell free (cf)-dna. results: plasma mpo-dna, ne-dna and cf-dna levels were significantly increased at 2 hr after pma stimulation when compared with plasma levels without pma. when either blood from septic shock patients or pma-stimulated neutrophils obtained from volunteers were applied to circuit, circulating mpo-dna, ne-dna and cf-dna were significantly reduced in perfusion with pmx filter than in perfusion without pmx filter at times 1 and 2 hr. conclusions: in the ex vivo experiments, mpo-dna, ne-dna and cf-dna were found to decrease after ex vivo perfusion through pmx filters. selective removal of circulating components of nets may improve the remote organ damage in patients with septic shock. a retrospective study of septic shock patients who were treated with direct hemoperfusion with polymyxin b-immobilized fibers based on the levels of endotoxin activity assay s sekine, h imaizumi, i saiki, a okita, h uchino tokyo medical university, anesthesiology/icu, tokyo, japan critical care 2019, 23(suppl 2):p047 introduction: the purpose of this study was to evaluate the outcomes for septic shock patients with direct hemoperfusion with polymyxin b-immobilized fibers (pmx-dhp) and endotoxin activity assay (eaa). methods: according to the levels of eaa, 41patients were classified for three groups (low group (gl); eaa <0.4, intermediate group (gm); eaa >0.4 or eaa <0.6, high group (gh); eaa >0.6). in order to evaluate the severity of illness, acute physiology and chronic health eva-luationii (apache ii) score, the sequential organ failure assessment (sofa) score, catecholamine index (cai) were recorded. and the presence of pmx-dhp treatments were also recorded. blood samples were obtained to measure eaa levels, inflammatory markers (procalcitonin (pct), c-reactive protein (crp), and white blood cell count (wbc)), serum lactate level as an indicator of tissue hypoxia, and for blood culture. apache ii score, sofa score, cai, inflammatory markers, serum lactate levels (lac) and blood culture results were examined for diagnosis of septic shock and prognosis of 30-days mortality. each values were also compared to eaa levels. results: 41 septic shock patients were included (gl/ gm/ gh: 12/ 13/ 16). in gh, apache ii and sofa score was significantly higher than that in gl (p< 0.05). eaa levels were significantly increased in gramnegative bacteremia patients compared to the patients with grampositive bacteremia or fungemia. there was no relationship between eaa levels and other inflammation markers, cai, and lac. in gm, 30days mortality in patient with pmx-dhp treatments was lower than that of without pmx-dhp treatments (0.14 (1/7) vs 0.5 (3/6), p=0.27). in gh, 30-days mortality in patient with pmx-dhp treatments was same as that of without pmx-dhp treatments (0.5 (3/6) vs 0.5 (5/10), p=1.0). conclusions: these results of this study suggest pmx-dhp treatment may improve the outcome of septic shock patients with intermediate eaa levels. introduction: numerous inconclusive randomized clinical trials (rcts) in sepsis in the past years suggest a need to re-think trial design to improve resource allocation and facilitate policy adoption decisions. the inclass study (clinicaltrials.gov nct: 03345992) is an ongoing rct evaluating clarithromycin as an immune modulator in high-risk septic patients with clinical and cost-effectiveness outcomes. we aim to compare the original one-shot trial with an alternative sequential design that balances trial costs and value of information. methods: adult patients with sepsis, respiratory failure and total sofa score of at least 7, are randomized to receive intravenous clarithromycin or placebo adjunctive to standard-of-care therapy. for the cost-effectiveness study, efficacy is measured in quality-adjusted life years (qalys) by eq-5d-3l questionnaire at 90 days. the endpoint is the incremental net monetary benefit (inmb) of clarithromycin compared to placebo, defined as wtp x (increment in qaly) -(increment in costs), where wtp is willingness to pay per qaly gained. fixed and variable costs of trial execution (including administrative, insurance, supplies, tests) are calculated; hospitalization cost is extracted from patient records; medical care beyond day 28 is recorded; cost of adoption in the general population is estimated. previous data from rcts using clarithromycin are used to form a prior belief about the inmb. known incidence of sepsis with respiratory failure allows estimation of the population to benefit from trial decision. a bayesian model is used to determine the sequential design that maximizes trial value. results: we will compare the performance of the sequential trial design with the one-shot design of inclass trial in terms of sample size, cost, social-welfare, and probability of correctly identifying the best treatment. conclusions: in this protocol we validate a bayesian model for sequential clinical trials and assess the benefits for the patient population and health care system. the effect on the outcome of critically ill patients with catecholamine resistant septic shock and acute renal failure through implementation of adsorption therapy g schittek 1 introduction: cytosorb-adsorption has been described as an effective way for hemodynamic stabilisation in septic shock [1] . aim of this study was to examine whether the adsorption-therapy could influence patient-outcome with catecholamine resistant septic shock (crss) and acute renal failure(arv). furhtermore we tried to identify clinical constellations that would predict an effective use of adsorbers [ 7, 34] . initial il-6 in patients with catecholamine-reduction through adsorption was non-significantly different to those with no reduction (2376 ng/l [838, 5000] vs. 5000 ng/l [3488, 5000]). mortality did not differ significantly between the groups (71% vs 79%). length of intensive care unit stay (los) did differ significantly (13 days [4, 24] vs 22 days [19, 29] ). conclusions: il-6 can be reduced with adsorption. patients with catecholamine-reduction did not differ in regard to their initial il-6. los was shorter for patients treated with adsorption. according to our experience adsorption can be taken into consideration when crss is beginning. introduction: in our intensive care unit (icu), we have already started expanded application to the contact precautions. applied patients are; 1) emergency admission, 2) patients who had already had bacteria* that are required to contact precautions, 3) scheduled surgical patients with prolonged icu stay, although we have not yet decided the started period of expanded application exactly. *detected bacteria(db);mrsa, cd, mdrp, esbl, pseudomonas a, pisp, prsp, vrsa. the aim of this study was to determine the adequate starting period of expanded application to the contact precautions in the scheduled surgical patients in the mixed icu. methods: we performed retrospective observational study on 4221 patients who were admitted to our icu after planed surgery from may 2013 to dec. 2017. we detected the patients who acquired bd newly and investigated the relation to the length of icu stay. the relationship between detection rate and categorized date was also analyzed using logistic regression adjusted for age, gender, apache2, and sofa score. using youden´s index and roc curve, we also calculated cutoff point of the duration of icu stay related to detection rate. finally, we made the logistic regression model of each cutoff day(day1 to 7) and compared odds ratio(or) and auc of each models using stata. results: category day 2 or more, especially day 4 or more had significantly higher detection rate of db compared to day 1 ( results: pao2/fio2 was lower than 240 mmhg in 171 (67%) patients. compared to patients in group 2, patients in group 1 were less severely ill at admission but presented a higher sofa and cpis score and a greater incidence of ards and shock at pneumonia onset (fig 1) . 117 (69%) patients in group 1 had a microbiological diagnosis of pneumonia, compared to 71 patients (85%) in group 2 (p=0.007). pao2/fio2 ≤240 mmhg was associated with less probability of having microbiological diagnosis of pneumonia (or 0.40, 95% ci 0.21 to 0.79, p=0.008). when adjusted for other variables significantly associated with positive microbiology, pao2/fio2 ≤240 mmhg remained significantly associated with less probability of a microbiological diagnosis (adjusted or 0.34, 95% ci 0.12 to 0.94, p=0.038). hospital mortality was significantly higher in patients in group 1 compared to group 2 (42% vs 29%, p=0.044). however, no difference was found in non-response to treatment, icu and hospital stay, icu mortality (table 1) and 90-days survival (fig 2) . conclusions: a significant higher number of patients with vap didn't have a definitive etiological diagnosis when using the proposed threshold criteria of pao2/fio2 ≤240 mmhg. pao2/fio2 ratio does not seem a good predictor of etiology in patients with vap. introduction: immunological dysfunction is common in critically ill patients but the optimal method to measure it and its clinical significance are unknown. levels of tumor necrosis factor alpha (tnf-α) after ex-vivo whole blood stimulation with lipopolysaccharide has been proposed as a possible method to quantitate immunological function. we hypothesized that patients with a lower post-stimulation tnf-α level would have increased rates of nosocomial infections (nis) and worse clinical outcomes. methods: a secondary analysis of a phase 2 randomized, multicentre, double-blinded placebo controlled trial [1] . there were no differences in allocation groups; all the patients were analyzed as one cohort. on enrolment, whole blood was incubated with lps ex-vivo and tnf-α level was measured. patients were grouped in tertiles according to delta and peak tnf-α level. the primary outcome was the development of nis; secondary outcomes included 90-day mortality. results: data was available for 201 patients. baseline characteristics and outcomes are reported in tables 1 and 2 . patients in the highest tertile for post lps stimulation delta tnf-α compared to the lowest tertile were younger, had a lower acuity of illness and had lower baseline tnf-α. when grouped according to peak post-stimulation tnf-α levels, patients in the highest tertile had higher serum tnf-α at baseline. both comparisons showed no difference between nis and clinical outcomes between tertiles. in multi-variate analysis peak or delta tnf-α were not associated with the occurrence of nis. conclusions: admission ex-vivo stimulated tnf-a level is not associated with the occurrence of nis or clinical outcomes. further study is required to evaluate the ability of this assay to quantify immune function over the course of critical illness. results: sanitary and epidemiological examination revealed the connection between infection and intravenous infusion of dexamethasone performed concurrently with chemotherapy. in 5 patients fever with chills and hypertension developed within 2 hours after infusion of the infected drug; empirical intravenous antibiotic therapy started immediately after collecting blood culture. in 6 patients fever appeared after 2-4 days outpatiently, so they received antibiotics per os. all these patients had permanent vascular access, and bsi was detected either the next chemotherapy course when fever reappeared (3 pts) while using vascular access, or as a result of a specific examination (3 pts). in all cases empirical antibiotic therapy started on the first day of fever, drug correction was performed in 6 patients according to results of bacteriological research. septic shock developed in 1 patient, pneumonia in 3 patients. permanent vascular access was preserved only in 1 case. all patients were cured and continued to receive antitumor treatment. conclusions: detection of more than 1 case of b. cenocepacia bsi should be the reason for sanitary and epidemiological examination. a favorable outcome of bsi treatment is associated with the early start of antibiotic therapy and its correction after microbiological examination. emerging conclusions: implementation of asp in hospital allows to decrease incidence of eskape-bacteremia and candidemia, which may lead to improved clinical outcomes in icu's patients (fig 1) . association of multi-drug resistant (mdr), extended-drug resistant (xdr) and pan-drug resistant (pdr) gram negative bacteria and mortality in an intensive care unit(icu) s chatterjee 1 , s sinha 1 , a bhakta 2 , t bera 3 , t chatterjee 3 , s introduction: colistin-resistant klebsiella pneumoniae (cr-kp) is increasingly reported around the world. it is worrying to note emergence of resistance to last line of defence against mdr gram negative infections in regions endemic to carbapenem resistance. we report the first outbreak of cr-kp co-producing carbapenemases in an adult intensive care unit (icu) from south india. methods: retrospective analysis of all patients with carbapenem resistant klebsiella pneumoniae blood stream infection (bsi) was done between january 2017 and december 2017. microbiological and clinical variables along with outcomes were analysed. results: seven patients had cr-kp with no prior exposure to colistin. all seven were modified hodge test (mht) negative making probability of blakpc unlikely. in resource limited setting, analysis beyond mht could only be performed for cr-kp samples. 2/7 samples belonging to cr-kp isolates produced the blandm-1 whilst 5/7 cr-kp isolates did not produce either blakpc or blandm carbapenemases prompting hypothesis of blaoxa-48 or blavim as the causative factor. compared to carbapenem resistance only group, cr-kp group had higher apache ii, icu length of stay and mechanical ventilation duration. 28 day mortality was noted to be 56.3% for carbapenem resistant and 43% for cr-kp groups. aggressive infection control measures were undertaken with successful containment of cr-kp strains along with reduction in overall bsi. conclusions: infection control measures form the backbone of patient care in centres showing endemicity for carbapenem resistant klebsiella to prevent colistin resistance and also to reduce occurrence of overall blood stream infections. rapid diagnosis of carbapenem resistance: experience of a tertiary care cancer center with multiplex pcr s mukherjee tata medical center, critical care medicine, kolkata, india critical care 2019, 23(suppl 2):p074 introduction: sepsis due to carbapenem resistant organisms has high mortality; inappropriate empirical antibiotic is one of the main causes of this poor outcome. on the contrary, "too much" broad spectrum empiric antibiotics will increase drug resistance, even in community, because of selection pressure. so, early diagnosis of resistance pattern (carbapenemase genes) is crucial. aim of this study is to compare rapid diagnostic test like polymerase chain reaction (pcr) with conventional culture sensitivity (c/s) to identify carbapenem resistance. methods: this is a prospective observational study done in tata medical center, kolkata, india. real time multiplex pcr technique has been developed "in house" in our microbiology lab and can identify ndm, ndm1, kpc, oxa -23, oxa -48, oxa -58 & vim carbapenemase genes. blood cultures were sent as per clinical & laboratory diagnosis of sepsis in icu patients. culture positive samples had been used for conventional c/s by vitek 2 system along with pcr study to identify carbapenemase genes. result of pcr technique was been compared with conventional c/s method. results: multiplex pcr results were available within 3-4 hours of positive blood culture compared to conventional c/s method that takes 2 -3 days. among 392 positive blood cultures, 146 samples were positive for carbapenemase genes. most common gene identified was oxa -48 (43%), followed by ndm1 (25%). our pcr technique has very high sensitivity, specificity, positive & negative predictive value (99.35%, 94.65%, 93.1% & 99.43% respectively) while comparing with final c/s report by vitek 2 system (table 1) . there was only one false negative diagnosis for carbapenem resistance. conclusions: real time multiplex pcr for carbapenemase gene can be helpful for early diagnosis of carbapenem resistance and can help us to choose / modify antibiotics or to use 'targeted therapy'. it is more practical to "rule -in" infection rather than "rule -out" by this technique. carbapenemase producing enterobacteriaceae colonization in an icu: risk factors and clinical outcomes m miranda, jp baptista, j janeiro, p martins centro hospitalar e universitário de coimbra, intensive care unit, coimbra, portugal critical care 2019, 23(suppl 2):p075 introduction: carbapenemase-producing enterobacteriaceae (cpe) colonization has been increasingly reported in intensive care units (icus) since their first identification more than 20 years ago. colonization with cpe seems to constitute a risk factor for mortality. the aim of our study was to identify associated risk factors and clinical outcomes among patients with fecal colonization by cpe admitted to a portuguese tertiary hospital icu. methods: a 2-year retrospective study was performed in patients with previous unknown cpe status (colonization or infection), admitted to our icu. rectal swabs were performed and analyzed using real-time polymerase chain reaction testing. clinical records were reviewed to obtain demographic and clinical data. results: of 903 patients admitted, 38 (4.2%) harbored cpe, 15 (39.5%) were colonized at admission and 23 (60.5%) acquired cpe colonization during icu stay. the most frequent carbapenemase genes detected were kpc (87.5%) and vim (12.5%). cpe carriers had high rates of hospitalization (previous or ongoing), invasive procedures (mainly intraabdominal surgery), malignancy (hematopoietic or solid tumor), introduction: gram-negative pathogens-particularly pseudomonas aeruginosa and enterobacteriaceae-predominate in nosocomial pneumonia (np) and ciai both. these infections are becoming difficult to treat with available treatment options due to growing antimicrobial resistance in india. ceftazidimeavibactam has in-vitro activity against gram-negative organisms producing class a, class c and some class d beta-lactamases. we carried out a qualitative analysis to assess the safety and efficacy outcomes of the indian population cohorts involved in the re-prove and reclaim trials. methods: in line with the global reprove protocol, indian patients enrolled in the study with np, were randomly assigned (1:1) to 2000 mg ceftazidime and 500 mg avibactam or 1000 mg meropenem. in the reclaim study, indian patients with a diagnosis of ciai were enrolled in the study and were randomly assigned (1:1) to receive either ceftazidime-avibactam (2000 mg of ceftazidime and 500 mg of avibactam) followed by metronidazole (500 mg); or meropenem (1000 mg). the primary efficacy outcome measure in the reprove and reclaim studies was clinical cure rate of caz-avi compared with that of meropenem at toc (test-of-cure) visit in pre-defined analysis sets. in both studies, non-inferiority was concluded if the lower limit of the twosided 95% ci for the treatment difference was greater than -12·5% in the primary analysis sets. as the indian subset study was not statistically powered to detect a difference in the subgroup, we descriptively analysed the efficacy results in the indian population and compared them with the overall results in the global trial. in addition, the study also analysed the safety of caz-avi in the indian patients by monitoring the number and severity of adverse events. introduction: early administration of effective intravenous antimicrobials is recommended for the management of the patients with sepsis. although meropenem (mepm) is one of the first-line drugs in patients with sepsis because of its broad spectrum, the optimal dose in the critical care settings especially during continuous renal replacement therapy (crrt) has not been established since therapeutic drug monitoring of mepm has not been popular. methods: eighteen critically ill patients who received crrt were enrolled in this study. one gram of mepm was administered over 1 hour, every 12 hours, and blood samples at 1, 2, 6, 9 and 12 hours after administration were collected on day 1, 2 and 5. all samples were stored at -80°c until analysis. the measurement of the blood concentration of mepm was performed using high performance liquid chromatography with ultraviolet detection (hplc-uv introduction: meningitis is one of the complications of severe traumatic brain injury, and it is often associated with encephalitis (incidence from 1.3-4.8% to 10-20%). the aim of the investigation was to study the dynamics of the concentration of meropenem in serum and cerebrospinal fluid (csf) with intravenous and intrathecal administration of meropenem. methods: in eight patients with bacterial meningoencephalitis blood serum and csf were studied prior to the administration of meropenem and 5-10 min, 1, 2.5 and 5 hrs after it. antibiotic regimen: 2000 mg of vancomycin (1000 mg bid) and meropenem (2000 mg tid diluted in 100 ml of saline iv + 20 mg bid diluted in 5 ml of saline bolus slowly intrathecally). meropenem infusion was carried out for 30 minutes, 5 mins after it 5 ml of blood and 1 ml of csf were sampled. prior to antibiotics administration blood and csf were taken for microbiological examination. to determine the concentration of antibiotics iquid chromatography/mass spectrometry was used. the samples were analyzed on an agilent 1260 infinity liquid chromatograph coupled to a sciex qtrap 6500 mass detector (sciex, us introduction: the prophylactic use of probiotics has emerged as a promising alternative to current strategies viewing to control nosocomial infections in a critically-ill setting. however, their beneficial role in vap prevention remains inconclusive. our aim was to delineate the efficacy of probiotics for both vap prophylaxis and restriction of icu-acquired infections in multi-trauma patients. methods: randomized, placebo-controlled study enrolling 58 multitrauma patients, requiring mechanical ventilation for >10 days. participants were randomly assigned to receive either probiotic (n=28) or placebo (n=30) treatment. a four-probiotic formula was applied and each patient received two capsules per day from day1 to day15 post icu admission. the content of one capsule was given as an aqueous suspension by nasogastric tube, while the other one was spread to the oropharynx after being mixed up with water-based lubricant. the follow-up period was 30 days, while icu stay and mortality were also assessed. ], while no difference in 30-day mortality rate was identified between groups (10.7% probiotics vs 6.7% placebo). conclusions: the prophylactic administration of probiotics exerted a positive effect on the incidence of vap or other icu-acquired infections and icu stay in a critically-ill subpopulation being notorious for its high susceptibility to infections, namely multi-trauma patients. use of a c-reactive protein-based protocol to guide the duration of antibiotic therapy in critically ill patients: a randomized controlled trial i borges 1 introduction: the rational use of antibiotics is one of the main strategies to limit the development of bacterial resistance. in this study we aimed to evaluate the effectiveness of a c reactive protein (crp) based protocol in reducing antibiotic treatment time in critically ill patients. methods: an open randomized clinical trial was conducted in two adult intensive care units of a university hospital in brazil (clini-caltrials.gov: nct02987790). patients were randomly allocated to: i) intervention -duration of antibiotic therapy guided by crp levels, and ii) control -duration of therapy based on best in the intention to treat analysis, the median (q1-q3) duration of antibiotic therapy for the index infection episode was 7.0 (5.0-8.8) days in the crp group and 7.0 (7.0-11.3) days in the control group (p=0.011). in the cumulative suspension curve of antibiotics, a significant difference in the exposure time between the two groups was identified, with less exposure in the crp group (p=0.007). in the pre-specified per protocol analysis, with 59 patients allocated in each group, the median duration of antibiotics was 6.0 (5.0-8.0) days in the crp group and 7.0 (7.0-10.0) days in the control group (p=0.011). mortality and relapse rates were similar between groups. conclusions: daily levels of crp may aid in reducing the time of antibiotic therapy in critically ill patients, even in a scenario of judicious use of these drugs. introduction: the macrophage activation syndrome (mas) or hemophagocytic lymphohistiocytosis(hlh) is a life threatening complication characterized by pancytopenia, liver failure, coagulopathy and neurologic symptoms and is thought to be caused by the activation and uncontrolled proliferation of t lymphocytes and well differentiated macrophages, leading to widespread hemophagocytosis and cytokine overproduction [1, 2] .the etiology is unknown, but is considered to have an infectious trigger.the aim of our study is to evaluate the impact of hlh in our 9 beds infectious diseases icu, during 83 months period (2012-2018). methods: a retrospective study based on electronic databases, including all patients admitted in our icu, that have matched at least 5 out of 8 criteria for hlh diagnosis (1):fever; hepatosplenomegaly; >2 cytopenia (hb <9 g/dl, plt</dl, pmn -n<1000/mmc); hypertriglyceridemia>265mg/dl, fibrinogen<150mg/dl; hemophagocytosis-bone marrow, spleen, and/or lymphnodes; nk activity reduced/ absent; ferritin level>500ui/l; cd 25>2400. we have evaluated the etiology established with cultures, serology, and molecular methods, treatment with corticosteroids, iv immunoglobuline, cyclosporine, etoposide and outcome (2) . results: 2112 patients were admitted to icu, 15 patients(0.71%) met the criteria for hlh. the average length of stay in icu was 15 days; 6 patients died (40%) without relation with the followed treatment. conclusions: hlh is not a rare condition in infectious diseases icu. the etiology is more frequent established compared with literature data. treatment (corticosteroids, immunoglobuline, cyclosporine, etoposide) is not associated with increased survival forecasting hemorrhagic shock using patterns of physiologic response to routine pre-operative blood draws introduction: irreversible hemorrhagic shock (ihs), a critical condition associated with significant blood loss and poor response to fluid resuscitation, can induce multiple organ failures and rapid death [1] . determining the patients who are likely to develop ihs in surgeries could greatly help preoperative assessment of patient outcomes and allocation of clinical resources. methods: machine learning model of ihs is developed and validated via porcine induced bleed experiment. 36 healthy sedated yorkshire pigs first had one 20ml rapid blood draw during a stable period, and then were bled at 20ml/min to mean arterial pressure (map) of 30 mmhg. 10 subjects had ihs defined as map<20mmhg. arterial, central venous and airway pressures collected at 250 hz during the blood draw [ fig 1] were used to extract characteristic sequential patterns using graphs of temporal constraints (gtc) methodology [2] , and a decision forest (df) model was trained on these patterns to determine subjects at high risk of impending ihs. results: in a leave-one-subject-out cross-validation, our method confidently identifies 30% (95% ci [15.6%, 44.4%]) of the subjects who are likely to experience ihs when subject to substantial bleeding, while only giving on average 1 false alarm in 10,000 such predictions. this method outperforms logistic regression and random forest models trained on statistically featurized data [tab 1, fig 2] . conclusions: our results suggest that by leveraging sequential patterns in hemodynamic waveform data observed in preoperative blood draws, it is possible to predict who are prone to develop ihs resulting from blood loss in the course of surgery. future work includes validating the proposed method on data collected from human subjects, and developing a clinically useful screening tool with our investigations. work partially funded by nih gm117622. introduction: the h 2 s and oxytocin(oxy) systems are reported to interact with one another [1] . h 2 s plays a major role in the hypothalamic control of oxy release during hemorrhage [2] . there is scarce information about oxy receptor(oxyr) expression in the brain in general and what is there is ambivalent. oxyr has been immunohistochemically(ihc) detected in the human hypothalamus but not in the hippocampus, in contrast to rodents [3] , which underscores the need for additional characterization in relevant animal models. thus the aim of this study is to map the expression of the oxy and h 2 s systems in the porcine brain in a clinically relevant model of hemorrhagic shock (hs). methods: anesthesized atherosclerotic pigs (n=9) underwent 3h of hs (map 40+/-5mmhg) [4] , followed by 72h resuscitation. ihc detection of oxy, oxyr, the h2s producing enzymes cystathionine-γ -lyase (cse) and cystathionine-β -synthase(cbs) was performed on formalin fixed brain paraffin sections. results: oxy, oxyr, cse and cbs were localized in the porcine brain. proteins were differentially expressed in the hypothalamus (fig 2) , parietal cortex and cerebellum (fig 1) . cell types positively identified were: magnocellular neurons of the hypothalamus, cerebellar purkinje cells and granular neurons, and hippocampal pyramidal and granular neurons of the dentate fascia. arteries and microvasculature were also positive for oxyr and cse. conclusions: our results confirm the presence of oxy and oxyr in the hypothalamus similarly to the human brain. novel findings were: oxyr in the cerebellum and cse expression in the hypothalamus and cerebellum. the coexpression of oxyr and cse may link and help better understand neurochemical systems and physiological coping in hemorrhagic shock. funding: crc1149 introduction: septic shock is one of the main causes of intensive care unit (icu) admission, leading to mortality up to 50% of patients. acute kidney injury (aki) frequently occurs and is associated to great morbidity and mortality. hemodynamic optimization may reduce the incidence of aki, but the use of vasopressors to increase mean arterial pressure (map) could have deleterious effect on renal perfusion. we aimed at investigating the effect of map and norepinephrine (ne) on the incidence of aki in septic shock patients methods: retrospective study based on prospectively collected data on digital medical records (digistat) at our icu. introduction: in patients with distributive shock, increasing mean arterial pressure (map) to a target of >65 mmhg can improve tissue perfusion. patients unable to achieve the target map of >65 mmhg despite adequate fluid resuscitation as well as catecholamines and vasopressin standard care (sc), may benefit from the noncatecholamine vasopressor angiotensin ii to increase map. this posthoc analysis examined whether patients from the athos-3 study with a baseline (bl) map <65 mmhg and treated with sc plus either angiotensin ii (ang ii) or placebo achieved a map of >65 mmhg for 3 consecutive hours, without increasing the dose of sc therapy. methods: patients were assigned in a 1:1 ratio to receive ang ii or placebo, plus sc. randomization was stratified according to map (<65 or >65 mmhg) at screening. in patients with bl map <65 mmhg, we evaluated whether patients achieved a map of >65 mmhg for the first 3 hours after initiation (map measurements taken at hours 1, 2, and 3), without an increase in the dose of sc. results: among 321 treated patients, 102 had bl map <65 mmhg (ang ii, 52; placebo, 50). median bl map (iqr) was 61 (57-63) and 62 (59-64) mmhg for placebo and ang ii groups, respectively. patients with bl map <65 mmhg who were treated with ang ii were more likely to achieve map ≥65 mmhg for 3 consecutive hours after initiation without an increase in sc dose (67%, 95%ci 53-80), compared with placebo-treated patients (24%, 95%ci 13-38, or=6.52, p<0.0001). conclusions: in this post-hoc analysis of patients with bl map <65 mmhg, patients receiving ang ii plus sc were significantly more likely to achieve a map >65 mmhg for the first 3 consecutive hours after initiation than patients receiving sc only. this suggests that administering ang ii may help patients with catecholamine-resistant distributive shock to achieve the consensus standard target map. norepinephrine synergistically increases the efficacy of volume expansion on venous return in septic shock i adda, c lai, jl teboul, l guerin, f gavelli, c richard, x monnet hôpitaux universitaires paris-sud, hôpital de bicêtre, aphp, service de médecine intensive-réanimation, le kremlin-bicêtre, france critical care 2019, 23(suppl 2):p093 introduction: through reduction in venous capacitance, norepinephrine (ne) increases the mean systemic pressure (psm) and increases cardiac preload. this effect may be added to the ones of fluids when both are administered in septic shock. nevertheless, it could be imagined that ne potentiates in a synergetic way the efficacy of volume expansion on venous return by reducing venous capacitance, reducing the distribution volume of fluids and enhancing the induced increase in stressed blood volume. the purpose of this study was to test if the increase in psm induced by a preload challenge were enhanced by ne. methods: this prospective study had included 30 septic shock adults. to reversibly reproduce a volume expansion and preload increase at different doses of ne, we mimicked fluid infusion through a passive leg raising (plr). in patients in which the decrease of ne was planned, we estimated psm (using respiratory occlusions) at baseline and during a plr test (plr high ). the dose of ne was then decreased and psm was estimated again before and during a second plr (plr low ). . the increase in cardiac index induced by plr low was significantly greater than that induced by plr high (p<0.001). δ psmhigh -δ psmlow was moderately correlated with the diastolic arterial pressure at baseline-high (p=0.03, r=0.41) and with the ne-induced change in mean arterial pressure (p=0.004, r=0.57). conclusions: ne enhances the increase in psm induced by a plr, which mimics a fluid infusion. this suggests that it may potentiate the effects of fluid in a synergetic way in septic shock patients. this may decrease the amount of administered fluids and contribute to decrease the cumulative fluid balance. introduction: arginine vasopressin (avp) can be used in addition to norepinephrine (ne) for ne-resistant septic shock. however, a subgroup who will response to avp is unknown. the purpose of this study was to determine factors which could predict the response to avp in patients with ne-resistant hypotension. methods: this was a single-center, retrospective analysis of patients who administered avp for ne-resistant hypotension in our intensive care units (icus). eligible patients were adult patients who administered avp in addition to ne due to hypotension (mean arterial pressure (map) < 65) in our icus between august 2014 and december 2017. we divided all patients into two groups by response to avp; responders and non-responders. the responders were defined as an increase of map ≥ 10 mmhg at 1h after avp initiation. we conducted univariate and multivariate logistic regression analysis to evaluate the effect of variables on avp response. results: a total of 163 patients were included; 107 responders (66%), 56 non-responders (34%). there was no significant difference for map at the time of avp initiation (51 vs 52 mmhg; p = 0.40), initiation dose of avp (0.028 vs 0.031 u/min; p = 0.67), and dose of ne at the time of avp initiation (0.18 vs 0.18 μ g/kg/min; p = 0.95). map at 1h after avp initiation was significantly higher in responders than non-responders (74 vs 55 mmhg; p < 0.01). responders were older (69 vs 66; p = 0.02) and had lower heart rate (hr) (99 vs. 108; p = 0.01) and lactate (3.2 vs. 4.8 mmol/l; p = 0.02) at the time of avp initiation. the multivariate logistic analysis revealed that hr ≤ 103 (or 2.56, 95% ci 1.29-5.10, p < 0.01), lactate ≤ 3 (or 3.11, 95% ci 1.56-6.19, p < 0.01) and age ≥ 63 (or 2.16, 95% ci 1.05-4.45, p = 0.04) were significantly associated with the response to avp. conclusions: hr, lactate levels and age before avp initiation can predict the response to avp in icu patients with ne-resistant hypotension. the maximum norepinephrine dosage of initial 24 hours predicts early death in septic shock d kasugai 1 , a hirakawa 2 , n jinguji 3 , k uenishi 3 1 nagoya university gtaduate school of medicine, department of emergency and critical care, nagoya, aichi, japan; 2 fujita health university, department of disaster and traumatology, fujita health university, toyoake, japan; 3 fujita health university hospital, department of emergency and general internal medicine, fujita health university hospital, toyoake, japan critical care 2019, 23(suppl 2):p095 introduction: the mortality of septic shock refractory to norepinephrine remains high. to improve the management of this subgroup, the knowledge of early indicator is needed. we hypothesize that maximum norepinephrine dosage on the initial day of treatment is useful to predict early death in septic shock. methods: in this retrospective single-center observational study, septic shock patients admitted to the emergency intensive care unit (icu) of an academic medical center between april 2011 and march 2017 were included. cardiac arrest before icu admission and those with do-not-resuscitate orders before admission were excluded. the maximum dosage of norepinephrine initial 24 hours of icu admission (md24) was used to assess 7-day mortality. results: one-hundred-fifty-two patients were included in this study. median sofa score was 11 (9-13), and median md 24 was 0.24 (0.16-0.38) mcg/kg/min. vasopressin and steroid were administered in 58 (38 %) and 67 (44 %) cases. nineteen patients (13 %) died within a week. non-survivors had higher md24, higher sofa score, and higher rate of vasopressin use. the higher md24 predicted 7-day mortality (area under curve 0.797, threshold 0.60 mcg/kg/min, sensitivity 50 %, specificity 91%). after adjustment of inverse probability of treatment weighing method using propensity scoring, md24 higher than 0.6 mcg/kg/min was independently associated with 7-day mortality (or: 9.96, 95 %ci: 2.56-38.8, p < 0.001). conclusions: the maximum dosage of norepinephrine higher than 0.6 mcg/kg/min initial 24 hours was significantly associated with 7day mortality in septic shock, and may be useful in the selection of higher severity subgroup. the impact of norepinephrine on right ventricular function and pulmonary haemodynamics in patients with septic shock -a strain echocardiography study k dalla sahlgrenska university hospital mölndal, göteborg, sweden critical care 2019, 23(suppl 2):p096 introduction: septic shock is characterized by myocardial depression and severe vasoplegia. right ventricle performance could be impaired in sepsis. the effects of norepinephrine on rv performance and afterload in septic shock are not immediately evident. the aim of the present study was to investigate the effects of norepinephrine on rv systolic function, rv afterload and pulmonary haemodynamics. methods: eleven, volume-resuscitated and mechanically ventilated patients with norepinephrine-dependent septic shock were included. infusion of norepinephrine was randomly and sequentially titrated to target mean arterial pressures (map) of 60, 75 and 90 mmhg. at each target map, strain-and conventional echocardiographic were performed. the pulmonary haemodynamic variables were measured by using a pulmonary artery thermodilution catheter. the rv afterload was assessed by calculating the effective pulmonary arterial elastance (epa) and pulmonary vascular resistance index (pvri). results: the norepinephrine-induced elevation of map increased central venous pressure (38%, p<0.001), stroke volume index (7%, p<001), mean pulmonary artery pressure (19%, p<0.001) and rv stroke work (20%, p=0.045), while neither pulmonary vascular resistance index nor epa was affected. increasing doses of norepinephrine improved rv free wall strain from -19% to -25% (32%, p=0.003), tricuspid annular plane systolic excursion (22%, p=0.010) and tricuspid annular systolic velocity (17%, p=0.029). there was a trend for an increase in cardiac index assessed by both thermodilution (p=0.079) and echocardiography (p=0.054). conclusions: the rv function was improved by increasing doses of norepinephrine, as assessed both by strain-and conventional echocardiography. this is explained by an increase of rv preload. pulmonary vascular resistance is not affected by increased doses of norepinephrine. peripheral perfusion versus lactate-targeted fluid resuscitation in septic shock: the andromeda shock physiology study. preliminary report g hernandez 1 , r castro 1 , l alegría 1 , s bravo 1 , d soto 1 , e valenzuela 1 , m vera 1 , v oviedo 1 , c santis 2 , g ferri 2 , m cid 2 , b astudillo 2 , p riquelme 2 , r pairumani 2 , g ospinatascón table 1 . conclusions: this preliminary results suggest that using crt as a target for fr in septic shock appears to be feasible, and not associated with impairment of tissue perfusion-related parameters as compared to lactate-targeted fr. grant fondecyt chile 1170043 introduction: shock patients often become resistant to catecholamines which often require the addition of a non-catecholamine vasopressor. preclinical studies suggest that in the presence of aadrenoceptor antagonism, the renin-angiotensin aldosterone system exerts the major vasopressor influence. we sought to determine the effects of angii or lypressin (lyp [porcine vasopressin]) on blood pressure in a norepinephrine (ne)-resistant hypotension pig model. methods: phentolamine (phn), a reversible α-blocker that antagonizes the vasoconstriction by ne, was continuously infused to induce hypotension. after ne-resistant hypotension was established, lyp or angii was then co-infused with phn. mean arterial pressure (map) and heart rate were continuously recorded (fig. 1) . results: as shown in fig. 2 conclusions: in a background of α-adrenoceptor blockade, at clinically comparable doses, the vasopressor effect of ang ii was maintained while those of ne and lyp were attenuated. these data suggest that the blood pressure effect of vasopressin-like peptides may require a functioning α-adrenoceptor. patients with shock who are resistant to increasing doses of catecholamines may also have vasopressin resistance potentially making angiotensin ii a preferred vasopressor for these patients. introduction: resuscitative endovascular balloon occlusion of the aorta (reboa) has been increasingly used for the management of both traumatic and non-traumatic hemorrhagic shock. however, there is limited evidence for its use in gastrointestinal bleeding (gib), especially in the icu setting. we successfully treated a patient with massive gib using reboa in the icu. we will discuss the difficulty performing the procedure and its countermeasure. methods: a case report. results: an 83-year-old woman was transferred to our hospital with shock. coffee grounds material was found in a nasogastric aspirate after intubation and upper gastrointestinal endoscopy identified a pulsating large duodenum ulcer without active bleeding, for which an elective procedure was planned. she was admitted to our icu, responded to initial resuscitation, and thereafter extubated. her systolic blood pressure (sbp) suddenly dropped to 40mmhg with massive hematochezia at that night, and did not increase despite resuscitation with blood products, crystalloid and norepinephrine. to buy time until measures for stop bleeding, we planned to place reboa in the icu. following the placement of a sheath in the left femoral artery, we tried to place a 7 fr intra-aortic balloon occlusion catheter, which unintentionally and repeatedly went into the right common iliac artery because her left femoral artery was tortuous. after compressing the right lower abdomen, we managed to introduce reboa in zone 1. it took approximately 60 minutes to successfully place the catheter. the patient's sbp increased immediately after the balloon inflation and bleeding was endoscopically controlled. introduction: the natural components of the pomegranate fruit may provide additional benefits for endothelial function and microcirculation. we hypothesized that chronic supplementation with pomegranate extract might improve glycocalyx properties and microcirculation during anaerobic condition. methods: eighteen healthy and physically active male volunteers aged 22-28 years were recruited randomly to the pomegranate and control groups (9 in each group). the pomegranate group was supplemented with pomegranate extract for two weeks. at the beginning and end of the experiment, the participants completed a high intensity sprint interval cycling-exercise (anaerobic exercise) protocol. the systemic hemodynamics, microcirculation flow and density parameters, glycocalyx markers, and lactate and glucose levels were evaluated before and after the two exercise bouts. results: no significant differences in the microcirculation or glycocalyx were found over the course of the study. the lactate levels were significantly higher in both groups after the first and repeated exercise bouts, and were significantly higher in the pomegranate group relative to the control group after the repeated bout: 13.2 (11.9-14.8) vs. 10.3 (9.3-12.7) mmol/l, p = 0.017. conclusions: chronic supplementation with pomegranate extract has no impact on changes to the microcirculation and glycocalyx during anaerobic exercise, although an unexplained increase in blood lactate concentration was observed. introduction: extracorporeal membrane oxygenation in adults in accompanied by high mortality. our ability to predict who will benefit from ecmo based on currently available clinical and laboratory measures is limited. the advent of single cell sequencing approaches has created the opportunity to identify cell populations and pathophysiological pathways that are associated with mortality without bias from a priori cell type classifications. identification of such cell populations would provide both an important prognostic markers and key insight into immune response mechanisms and therefore a possibility for advanced drug matching that may impact clinical response to ecmo in these patients. methods: whole genome transcriptomic profiles were generated from a total of 40,935 peripheral blood monocytes obtained from 37 patients at the time of cannulation for ecmo (fig 1) . differential gene expression analysis was performed with the monocle package for the r statistical analysis framework. time-to-event data were analyzed in a survival analysis with a log-rank test for differences. results: genes encoding several members of the heat shock family of proteins were up-regulated in cells from non-survivors. notably, these genes were expressed by a small fraction of cells (2.4% on average). nevertheless, the proportion of cells expressing these genes was a significant predictor of survival to 30 days (p = 0.02 by log rank test), with a particularly pronounced effect in the first 5 days after initiation of ecmo support (fig 2) . conclusions: the proportion of cells expressing genes encoding members of the heat shock proteins is predictive of survival on ecmo. majority of pt (43%) had no known predisposing conditions, followed by immobility (21%) and cancer (15%). in ecg analysis tachycardia and v1-v3 t wave inversion were the most common findings whereas hypoxemia± hypocapnia were the most prominent features in abg analysis. 8 pt (10%) had bleeding complications (none intracranial), 3 (3.7%) during rtpa, 5 (6.3%) in the first 36h and only 3 pt required transfusion. mortality rate was 12%: 6% directly due to pe (all during cpr) and 6% due to late complications (newly diagnosed cancer and infections). conclusions: in our experience, fibrinolytic therapy is safe and effective but in submassive pe should be applied after thorough assessment of risks and benefits on individual basis aiming to patient tailored precision medicine. [2] trials evaluated the role of levosimendan in preventing low cardiac output syndrome in patients undergoing cardiac surgery. the studies were similar in their design and recruited patients with preoperatively low lvef undergoing either isolated cabg or valve surgery combined with cabg (table 1 ). in both, a 24-hour levosimendan infusion was started at induction of anesthesia. neither study met the primary efficacy composite enpoints, but both showed a clear tendency for better outcome in patients undergoing a cabg compared to a valve procedure. we are currently evaluating the solidity of a co-analysis based on shared end-points. we are planning a shared analysed of the data related to the cabg settings and analyze the aggregated mortality data for both studies at 1 and 3 months by cochran-mantel-haenszel odds ratio. data from individual studies would be analysed as fixed effect and breslow-day test was used to evaluate homogeneity of the odds ratios results: in the placebo groups of the two studies, the mortality is similar; 7.9% (22/279) in levo-cts and 7.3% (9/123) in licorn, corroborating the working hypothesis that the two studies can be coanalysed. in a preliminary combined analysis (fig 1) , 90-day mortality was 7.7% (31/402) in the placebo group and 2.9% (12/407) in the levosimendan group. odds ratio was significantly in favor of levosimendan (0.36; 95% confidence interval 0.18-0.72; p=0.0026, fig. 1 ) conclusions: the levo-cts and licorn trials can be co-analysed in their sub-setting of patients requiring isolated cabg surgery for mortality at 1 and 3 months. a preliminary analysis on mortality reinforce the hypothesis that, in isolated cabg surgery, levosimendan lowers post-operative mortality significantly both at 1 and 3 months, when started at the induction of anesthesia introduction: emergency medical system (ems) -based st elevation myocardial infarction (stemi) networks allows not only stemi diagnosis in the pre-hospital phase but also reduces treatment delays; treat your fatal complications and the immediate activation of the catheterization laboratory. the aim of study was to investigate the effect of out-of-hospital by mobile intensive care (micu) versus hospital beginning treatment in hospitalization length and survival of patients with stemi diagnosis introduction: contrast induced nephropathy (cin) is a complex acute renal failure syndrome, which can occur after primary percutaneous coronary intervention (pci) and is an important cause of morbidity and mortality in this subgroup of patients. the aim of our study was to establish the incidence and predictors of cin after primary pci. we performed a retrospective analysis of stemi patients treated with primary pci in the period from january until september of 2017. cin was defined as an absolute increase in baseline serum creatinine of ≥0.5 mg/dl (44 μmol/l) or >25% relative rise within 72 hours after primary pci. we analyzed demographic characteristics, risk factors, clinical status at hospital admission, laboratory parameters, left ventricle ejection fraction and data regarding pci procedure. results: the study included 729 patients, with an average age of 62.33 ± 11.78 years, 66.1% of the patients were males. an average of 175.67 ± 91.77ml of contrast medium per patient was utilized. cin developed in 11(1.5%) patients and overall intra-hospital mortality was 8.4 %. in multivariate analysis, the independent predictors of cin were age>75 years ( introduction: left main coronary artery (lmca) disease is a disease of the main coronary branch that gives more than 80% of blood supply to the left ventricle, it carries high mortality without surgical intervention; [1] however the influence of lmca surgery on morbidity icu measures needs to be explored. we aim to determine whether lmca is definitive risk factor for prolonged icu stay as a primary outcome and whether lmca is definitive risk factor for early morbidity methods: retrospective descriptive study with purposive sampling analyzing 398 patients underwent isolated coronary artery bypass surgeries (cabg). patients were divided into 2 groups those with lmca disease as group 1 (75 patients) and those with coronary arty disease requiring surgery but without lmca disease as group 2 (324 patients) then we will correlate with icu outcome parameters including icu stay length, postoperative atrial fibrillation, acute kidney injury, re-exploration, perioperative myocardial infarction, post operative bleeding and early mortality. results: patients with lms had significantly higher diabetes prevalence (43.3% vs 29%, p=0.001). however, we did not find a statistical significant difference regarding icu stay, or other morbidity and mortality outcome measures conclusions: diabetes was more prevalent in patients with lms. the latter group showed similar outcome as those without lms in this study these findings may help in guiding decision making for future practice and stratifying the patients care. introduction: multimorbidity in patients admitted for acute myocardial infarction [ami] is associated with higher risk for in-hospital mortality and adverse clinical outcomes. we investigated to what extent an increasing number of comorbidities affects the age-stratified excess risk of death and other clinical outcomes among patients with myocardial infarction. methods: we analyzed nationwide administrative data of 174`803 admissions for an acute myocardial infarction between 2006 and 2016. we calculated multivariate regression models to study the association of four comorbidities (chronic kidney disease [ckd], diabetes mellitus, heart failure [hf], and atrial fibrillation) and excess risk of in-hospital mortality, length of hospital stay [los] , and 30-day readmission and stratified the analysis for different age categories. results: the incidence of admissions for ami increased continuously during the observed decade without an increase in in-hospital mortality, los, and 30-day readmission. among admitted patients with ami, there was a stepwise increase in risk for adverse outcomes for each comorbidity. compared to patients with no comorbidity, patients with 4 comorbidities had 6-fold increased risk for mortality (adjusted odds ratio [or] 6.6, 95% confidence interval [ci] 5.6 to 7.7) and a similar risk for readmission (or 1.0, ci 0.9 to 1.2). the los was 5.3 days (ci 5.1 to 5.5) in patients with no comorbidity and increased by 2.5 days (ci 2.4 to 2.5) with each additional comorbidity. these associations were stronger in younger compared to older patients. ckd was the strongest predictor of in-hospital mortality and los, while hf was the strongest predictor of 30-day readmission. conclusions: this study of nationwide admitted patients with ami found a stepwise increase in the risk for adverse outcome with increasing number of comorbidities, particularly in the younger patient population. younger, multimorbid patients may thus have the largest benefits from multidisciplinary treatments. introduction: certified cardiac arrest centers, sophisticated post cardiac arrest care and prehospital ecls teams aim to increase survivor rates with a preferable neurological outcome after cardiac arrest. centers also provide emergency ecls and ecls pick ups for cardiogenic shock patients before arresting. few data answer the question of the long-term quality of life after ecls therapy. methods: in a retrospective single center register we included patients after emergency ecls (ecpr and cardiogenic shock) between 10/2010 and 10/2017 discharged alive and performed a follow-up after 2 years on average at 6/2018. in our center criteria to initiate ecls therapy in cardiogenic shock or under cardiac arrest are an observed collaps, shockable rhythm, absence of frailty and severe comorbidities. all patients were requested to take part in a telephone interview. thus, we analyzed survival, cpc scores and sf36 scores. results: 97 patients with hospital survival after ecls were screened. 44% (n=43) had survived until 6/2018; 38 patients were not accessible; 16 had ceased. 33 survivors (mean±sd; min-max; 53±17; 19-78 years, 8 women) answered sf36 questionaires 29±15; 7-64 months after ecls (45% cardiogenic shock, 55% ecpr with shockable rhythm in 89%). the participantsć pc scores were in median 1. the results of the sf36 were physical functioning 84±21, physical role functioning 84±28, bodily pain 92±17, general health 68±23, vitality 65±26, social role functioning 83±28, emotional role functioning 96±14 and mental health 81±22. survivors who did not take part at the sf36 had a cpc score of in median 2 (n=10, 5 personally signed refusals, 3 language barriers, 2 vegetative states). conclusions: after emergency ecls therapy and hospital survival 44% of our patients survived the following 2 years up to over 5 years with a preferable neurological outcome and a general mentally and physically satisfactory quality of life. a vague outcome in 39% limits the results of our study. introduction: successful weaning from va-ecmo requires the restoration of a sufficient cardiac function to ensure an adequate tissue perfusion. skin blood flow (sbf) is among the first to deteriorate during circulatory shock and the last to be restored after resuscitation. sbf would be a good predictor of successful weaning from va-ecmo. methods: patients with va-ecmo, who required a first weaning attempt, were included. weaning procedure (wp) was performed by a reduction of va-ecmo blood flow to 1 l/min for 10 minutes. the weaning criterion was an aortic velocity-time integral (vti) > 10 cm. successful weaning from va-ecmo was defined as hemodynamic stabilization and without the need to increase the vasopressor dose during the next 24 hours. sbf, assessed by skin laser doppler (peri-flux5000, perimed, right index finger); perfusion unit: pu), together with global hemodynamic parameters were obtained before and after 3 min of weaning. receiver operating characteristic curves (roc) were generated to assess the ability and reliability of baseline parameters to predict a successful weaning. results: we studied 22 wps in patients with va-ecmo for pulmonary embolism (n = 2), post cardiotomy (n = 3), acute coronary syndrome (n = 14), myocarditis (n = 3). these were successful (sw) in 12 and unsuccessful (nsw) in 10. at baseline, hemodynamic variables, lactate, ecmo blood flow were similar in both groups (table1). sbf was greater in sw than nsw patients (table1). during wp, ci rose from baseline and was similar in sw and nsw (p=0.1) ( table 2 ). vtis were higher in sw than nsw (13 (12-14) vs 8 (6-8), respectively, p=0.03). sbf decreased in sw and remained low in nsw (table 2) . from the roc curves analyses, baseline sbf had the highest area under the roc curve with a cut off ≥ 34 pu (sensitivity 83%, specificity 92%) (figure1). conclusions: sbf is a good predictor of successful weaning from va-ecmo introduction: postoperative cognitive dysfunction (pocd) is defined as a temporarily decline in cognition associated with surgery. long-term pocd (3 months after surgery) occurs in 10-30% of cardiac patients and is associated with a higher morbidity and mortality. endo-cabg is a new minimally invasive endoscopic coronary artery bypass grafting (cabg) technique that requires retrograde arterial perfusion which may be associated with a higher incidence of neurological complications. the aim of this study is to assess the incidence of pocd after endo-cabg. methods: sixty consecutive patients undergoing an endo-cabg were enrolled. pocd was assessed following the recommendations of the "1995 statement of consensus on assessment of neurobehavioral outcomes after cardiac surgery". a comparative group of 60 patients undergoing percutaneous coronary intervention (pci) and a control group of 60 healthy volunteers were also enrolled. additional tests included the digit span test and digit symbol-coding test. patients were tested at baseline and at 3month follow-up. pocd is defined as a reliable change index (rci) ≤ -1.645 (significance level 5%), or z-score ≤ -1.645 in at least two different tests. results: after enrolling 60 patients in each group, respectively 46 in the endo-cabg-group, 44 in the pci-group and 48 healthy controls were analysed. patients suffering from a cva within three months after their procedure were automatically classified as having pocd (pci: n= 1; endo-cabg: n= 1). the total incidence of pocd was not different between groups (pci: n= 7; endo-cabg: n= 6, p=0.732). conclusions: our results suggest that the risk of pocd after endo-cabg is low and comparable with the risk of pocd after pci. introduction: rhabdomyolysis ( rml) post aortic surgery probably affects the renal outcome adversely [1, 2] . there is no robust data regarding the same in literature. methods: retrospective single center data review; prior approval from institutional review board. patients were divided to two groups group 1-with rml ( ck above cut off levels 1050 u/litre) and group 2 without rml. the determinants of rml and the impact of the same on outcome; predominantly renal function was evaluated. chi-square tests are performed for categorical variables whereas, student t tests (un-paired ) are performed with continuous variables. correlation is performed between creatine kinase and creatinine rise. p value 0.05 (two tailed) is considered for statistical significant level. results: out of 33 patients, 21 patients (63.64%) developed rhabdomyolysis ( group rml) and 12 did not( group non rml). demographic and intraoperative factors had no significant impact on the incidence of rml. there was a significantly higher incidence of renal complications including new postoperative dialysis in the rml group. other morbidity parameters were also higher in the rml group. conclusions: there is high prevalence of rml after aortic dissection surgery -identification of risk factor and early intervention might help to mitigate the severity of renal failure introduction: we investigate whether central venous pressure (cvp) pressure waveform signal can be informative in detection of slow bleeding in post-surgical patients. we apply a novel machine learning method to analyze cvp datasets to characterize bleeding in a porcine model of fixed rate blood loss. methods: thirty-eight pigs were anesthetized, instrumented with catheters, kept stable for 30 minutes, and bled at a constant rate of 20ml/min to mean arterial pressure of 30 mmhg. cvp waveforms were extracted from inspiration and expiration phases of respiration and statistically featurized. the proposed machine learning method, canonical least squares (cls) clustering, identifies correlation structures that differ between subsets of observations. we extend it to supervised classification. both clustering and classification methods yield human-interpretable models that reflect distinctive patterns of correlations within cvp waveforms. results: we conducted three experiments to discover structure in the physiological response to bleeding. first, we clustered respiration cycles with full knowledge of blood loss. the color-coded cluster assignments are shown in the figure 1 . they are consistent with escalation of bleeding. second, we deployed clustering on only cvp features without blood loss. temporal structure was complemented with some subject-specific clusters (fig 2) . third, we ran cls classification to decide whether an observation came from before or after the onset of bleeding (performance shown in the results: over the last decade, the number of patients with hlhs who underwent norwood 1 has increased. interstage mortality has decreased, and is currently 30-40%. significant morbidity was not seen at a rate higher than in the international literature. discharge planning, and community access to allied health professional services remained a concern. conclusions: the paediatric congenital cardiac surgical service in the united arab emirates is relatively new (compared to some services around the world). interstage mortality in hlhs is improving as a result of programme development, surgical progress and postoperative care. in the interstage period, there is currently no home monitoring programme in place. some patients were found to have had very extended hospital admissions. improved community support may reduce interstage mortality further, as well as improve the social situation of many of these patients. postoperative complications were observed in 112(10.9%) patients. we lined out the prevalence of cardiac complications, such as heart failure and rhythm disturbances, observed in 87 (8.5%) and 53 (5.1%) patients respectively. hospital mortality rate was 2.8% (29/1020). the cause of mortality in all cases was acute heart failure, due to the initial severity of the disease, and in 11(1.07%) cases an acute myocardial infarction was diagnosed. duration of postoperative period was 7.8 ± 0.9 days. conclusions: off-pump coronary artery bypass grafting can be safely performed with relatively low incidence of mortality and postoperative morbidity. prognostic value of mid-regional pro-adrenomedullin and midregional pro-atrial natriuretic peptide as predictors of multiple organ dysfunction development and icu length of stay after cardiac surgery with cardiopulmonary bypass in adults introduction: one of the most harmful complications after cardiac surgery with cardiopulmonary bypass is a syndrome of multiple organ dysfunction (mods). we consider that mid-regional proadrenomedullin (mr-proadm) and mid-regional pro-atrial natriuretic peptide (mr-proanp) plasma concentrations can be used as predictors of mods development and los in icu. methods: thirty six adult patients (mean age 60 years, 27 male) with cardiovascular diseases undervent cardiac surgery with cardiopulmonary bypass (heart valve(s) replacement -20 (55.6%) patients, aorta and it`s branch surgery -14 (38.9%) patients, valvular surgery and coronary artery grafting -2 (5.5%) patients). nyha heart failure class ii was in 3 (8.3%) patients, iiiin 27 (75%) patients, ivin 6 (16.7%) patients. in the dynamics levels of mr-proadm and mr-proanp were measured in the venous blood with the kryptor compact plus analyzer (thermo fisher scientific, germany) before 1 day and on the 1st and 6th days after surgery. all patients were divided into subgroups according to the lengths of stay in the icu and the development of mod in the postoperative period. the data are shown as median and 25th and 75th percentiles. the data were compared by mann-whitney u-test, pvalue of <0.05 was considered statistically significant. results: levels of mr-proanp did not significantly change at the study stages and did not have a significant difference between subgroups. the levels of mr-proadm increased in the first postoperative day and remained elevated for 6 days. this increase was significantly higher in subgroups of increased los in icu and with mods. the data are shown in the table 1 . conclusions: mr-proadm can be used as predictor of mods and los in the icu for adult patients underwent cardiac surgery with cardiopulmonary bypass. introduction: prolonged intensive care unit (icu) stay after cardiac surgery is associated with increased mortality and cost .the aim of this study was to investigate factors influencing prolonged icu stay. methods: consecutive patients who underwent cardiac surgery from june 2012 to october 2018 in our cardiothoracic department, were retrospectively investigated. group a consisted of pts with prolonged stay defined as more than 3 days and group b the rest of the cohort. the following characteristics and perioperative factors were compared between the groups: smoking, diabetes, copd, redo(re-operation), ejection fraction (ef)<50%, emergent procedure, cardiopulmonary bypass time (cpb)>120 min, low cardiac output syndrome (lcos), acute kidney injury(kdigo) and mortalitychi square test was used for the statistical analysis. introduction: hemorrhagic complications of extracorporeal membrane oxygenation (ecmo) pose a major morbidity and mortality. optimal anticoagulation strategies balancing risks of bleeding and thrombosis in children are poorly understood. we aimed to identify factors associated with non-surgical bleeding in the first 12 ecmo hours. methods: we evaluated all pediatric (<18 yrs) post-cardiotomy patients requiring ecmo between dec 2002-july 2017 stratifying them by presence/absence of surgical bleeding. non-surgical bleeding was defined as chest tube output >3cc/kg/hr during the first 12-hours not requiring reoperation. patient characteristics and coagulation parameters at various time points after ecmo initiation were compared between groups, and receiver operator characteristic (roc) curves were constructed to identify models and thresholds with optimal predictive performance. figure 1 . conclusions: deranged coagulation parameters, particularly kaolin rtime may predict non-operative bleeding in pediatric ecmo patients. these findings may guide therapeutic anticoagulation while avoiding hemorrhagic sequelae in at risk patients. introduction: elevated cardiac troponin (ctn) level in patients (pts) admitted in the intensive care unit (icu) is multifactorial and has been associated with a worse prognosis. the aim of the study was to review the frequency and the main cause of ctn elevation and to calculate a discriminating index. methods: we retrospectively assessed all pts admitted in our eightbed general icu during a 6-month period with at least one measurement of ctn during their icu stay. we recorded clinical characteristics, the level of ctn on admission, the maximum ctn during icu stay and the possible causes of elevation. variables are expressed as mean ± sd or as median and interquartile ratio (ir), according to the normality of their distribution. student´s ô test or the mann whitney u tests were used to compare the group of elevated ctn with the group of normal ctn. the prognostic performance of elevated ctn was evaluated by the receiver operating characteristics (roc) curve. statistical analysis was performed using spss version 21.0 (spss, inc., chicago, illinois). results: in 84 out of 92 pts that ctn was measured at least once, abnormal levels (>15.6 pg/ml) were found in 58 (69%) of them, and the maximum ctn value was 633 (1718.25) pg/ml. the clinical characteristics of the pts are depicted in table 1 . sepsis was the main cause of troponin elevation, which complicated by acute kidney injury (aki) in 20 pts (34%). maximum ctn, aki and the difference of maximum -admission ctn (äctn) differed significantly between pts who survived and pts who died (p=0.029 and 0.001, respectively). the area under the curve (auc) was 0.753 and the optimal prognostic cut-off value of äctn was 57 pg/ml with a sensitivity of 0.656 and a specificity of 0.833 conclusions: raised cardiac troponin values is a frequent finding in icu pts and sepsis is the driving cause. aki and the difference between maximum and admission ctn measurements differ significantly between pts who survive and pts who die. an elevation of ctn during icu hospitalization >57 pg/ml seems to be a threshold indicating poor prognosis regarding both mortality and aki. the prognostic role of nt-pro-bnp in septic patients with elevated troponin t level introduction: sepsis is frequently accompanied with release of cardiac troponin t (tnt) and nt-pro-bnp, but the clinical significance of this myocardial injury and cardiac dysfunction remains unclear [1] . tnt is known to be an independent predictor of mortality, whereas the prognostic role of nt-pro-bnp is uncertain. methods: here, we report data of va-ecmo-patients, treated with dobutamine, levosimendan, suprarenin or no inotropic agens, in respect of 30-day survival. all data were collected retrospectively (10/ 2010 to 10/2018) at a single center, all patients with a survival below 24 hours were excluded. while treatment of va-ecmo patients is strongly guided by standard operation procedures at our institution, no recommendation on positive inotropic therapy could be made. results: a total of 232 va-ecmo patients were evaluated, of which 47 patients were treated with levosimendan within 48 hours after cannulation. 30day survival in the whole cohort was 41.9%. a total of 99 patients did not receive any positive inotropic therapy at 24 hours after implantation (survival 48.0%). survival was best in the levosimendan plus dobutamine group 50%, followed by dobutamine mono-therapy 46.2% and levosimendan mono 41.2%. survival with suprarenin mono was 33.3%, suprarenin plus levosimendan 25.0% and suprarenin plus dobutamine 18,8%. pooling data, we found no evidence that levosimendan and/or dobutamine (survival 45.8%, n=83, p=0.882) improves survival over no inotropic therapy (fig 1) . therapy with any combination including suprarenin however resulted in poor survival (27.7%, n=65, p=0.009). adjustment for lactate levels or ecpr did not change the results. conclusions: this retrospective analysis of 232 va-ecmo patients shows no evidence that early inotropic therapy improves outcomes in va-ecmo patients. this conclusion is obviously biased by retrospective design. until randomized data are available, suprarenin however should be avoided. survey of non-resuscitation fluids in septic shock a linden-sonderso 1 introduction: positive fluid balance is associated with poor outcome in septic shock. the objective of the present study was to characterize non-resuscitation fluids in early septic shock. methods: consecutive patients >18 years of age were screened for inclusion criteria during a 4-month period in 8 icus in sweden and in canada. inclusion criteria were septic shock per sepsis-3 definition within 24 hrs of icu admission. a maximum of 30 patients per center were included. type, indication and volume of non-resuscitation fluids were recorded during the first 5 days of admission. fluids other than colloids, blood products and crystalloids given at rate > 5ml/kg/h were considered to be non-resuscitation fluids. the study was registered on clini-caltrials.gov (nct03438097). data are presented as median (interquartile range). results: a total of 201 patients were included between march 1st and june 30th 2018 (see table 1 for demographics). patients received 7886 (4051-12670) milliliters (ml) of non-resuscitation fluids introduction: we aimed to ascertain the extent and make-up of fluid overload in critically ill patients and to identify whether delivery of more concentrated medications could reduce this. positive fluid balance is associated with increased mortality [1] . a recent study has shown that the predominant component of fluid overload was from iv medications and maintenance fluid [2] . methods: we reviewed 20 sequential patients admitted to our icu with an apache ii score of greater than 15 and a length of stay (los) greater than 72 hours. the patients' electronic admission summary was interrogated to establish: length of stay (los) fluid balance at 72 hours, total volume administered as iv medications, total volume administered as maintenance fluid and total fluid administered introduction: in children less than 30 kilograms, maintenance fluids are routinely added to the resuscitation requirements calculated using parkland's or other formulae. the contribution of this component for fluid resuscitation in children can add a significant quantity to total estimated fluid requirements. for example, in a child who is 10 kilograms with a 25% burn, the maintenance fluid requirement is 1000 mls per 24 hours and the resuscitation component per parkland's will be 4 x 10 x 25%=1000 mls. hence, the maintenance requirement can exceed the resuscitation requirement in this child if the burn surface area is less than a 25 % burn. the contribution of maintenance fluids to the total fluid requirements in small children with thermal injuries is under-recognised and not frequently studied. methods: to understand the contribution of maintenance fluids to the total fluid requirements in children less than 30 kilograms who need resuscitation for thermal injuries of different sizes, we numerically simulated 1. children who had similar weights but different burn sizes and 2. children with similar burn size but different weights. the results are as shown in fig introduction: accurate quantification of fluid in resuscitation of thermal injuries is important for benchmarking, comparing and improving outcomes. in adults, it is usually expressed as mls/kg/%tbsa. in children, maintenance fluids are added to the resuscitation requirements. this is kept constant and the resuscitation component is titrated to meet pre-defined end points-usually urine output. maintenance fluids are not uniformly stratified across the weight ranges. we propose that quantification of fluids in mls/ kg/%tbsa in children does not accurately capture fluid needs for resuscitation due to the maintenance component of the fluid requirement. methods: we conducted this retrospective study in children admitted to a single-center burns intensive care unit (bicu) between january 2010 and december 2014. children ≤30 kilograms with tbsa ≥15% admitted within 8 hours of their injury were included. oe (observed to expected ratio) and fluid in mls/kg/% tbsa were calculated as shown in figure 1 . results: there were 33 children in the cohort with half requiring invasive mechanical ventilation in the bitu and nearly a quarter requiring inotropic support. the demographic details are as shown in table 1 . the oe ratio at the end of 24 hours in the cohort was 1.01 (0.68-1.36). the total fluid given was 6.9 (6.1, 8) mls/kg/ % tbsa. the titrated resuscitation component was 4.2 (3.7, 5.6) mls/kg/tbsa. total fluid (which included the maintenance fluid) had a poor correlation with oe ratio r2=0.34 (fig 2) . exclusion of the maintenance fluid had a better correlation with the oe ratio r2=0.75 conclusions: to capture differences in the titratable resuscitation component rather than differences in the maintenance requirements, fluid should be quantified in children by excluding the maintenance component when expressed as mls/kg/%tbsa. dynamic arterial elastance for predicting mean arterial pressure responsiveness after fluid challenges in acute respiratory distress syndrome patients p luetrakool 1 , s morakul 1 , v tangsujaritvijit introduction: dynamic arterial elastance (eadyn; pulse pressure variation/stroke volume variation; ppv/svv) is a dynamic parameter of arterial load that can be continuously monitored. previous study proposed that eadyn was able to predict mean arterial pressure (map) responsiveness after fluid challenge [1] [2] [3] [4] [5] . the objective of this study was to assess whether the eadyn was able to predict map responsiveness in acute respiratory distress syndrome (ards) patients ventilated with low tidal volume. methods: we performed a prospective study of diagnostic test accuracy in adult ards patients with acute circulatory failure and fluid responsiveness. all patients are continuously monitored blood pressure via arterial line connected with flotrac® transducer and vigileo® monitor. once the attending physicians decided to load intravenous fluid, we recorded ppv/svv and also other hemodynamic parameters before and after fluid bolus. map responsiveness was defined as an increase in map ≥ 10% from baseline after fluid challenge. results: twenty-three events were included. nine events (39.13%) were map-responsive. cardiac output, heart rate and stroke volume were similar in both map-responder and map-nonresponder group. baseline map, diastolic blood pressure (dbp) and pulse pressure (pp) were significantly different after fluid challenge in map-responder group. eadyn of preinfusion phase was failed to predict map conclusions: one of the arterial load parameters such as eadyn derived from non-calibrated pulse contour analysis method was unable to predict map responsiveness in ards patients with low tidal volume ventilation. the our aim is to test the hypothesis that in fr septic shock patients, fluid load will determine a significant increase in pmsf but not in cvp. we prospectively included all mechanically ventilated patients with diagnosis of septic shock with invasive hemodynamic monitoring (transpulmonary thermodilution volumeview-ev1000 ed-wards©). we collected hemodynamic and metabolic data and pmsf with the inspiratory holds technique, before and after a fluid challenge (fc) of 500 ml of ringer lactate in 10 minutes). fr was defined as an increase in cardiac output (co)>15%. results: 13 measures were obtained in 11 patients. in 8 case we observed fr. we found a significant increase in pmsf after a fc (mean difference(md) 16.9±21.5 mmhg, p=.015). cvp increased significantly (md 2.5±2.4 mmhg, p=.002). pmsf increased significantly in non-fr (md 27±10mmhg, p=.013) but not in fr while cvp was higher after fc only in fr (md 2.4±2.1mmhg, p=.008). venous return gradient (pmsf-cvp) globally increased after fc (md 14±22 mmhg, p=.03), but only in non-fr such increase was significant (md 24±12 mmhg, p=.03). no correlation was found between the variation co and venous return gradient. we did not find any improvement in metabolic parameters after the fluid challenge. conclusions: pmsf and combined cvp variations do not correlate with fr in our cohort of septic shock patients. inspiratory holds may not be adequate to infer pmsf in such context. further studies are warranted to investigate the effect of fc on pmsf in this field. evaluation of pre-load dependence over time in patients with septic shock i douglas 1 , p alapat 2 , k corl 3 , m exline 4 , l forni 5 , a holder 6 , d kaufman 7 , a khan 8 , m levy 3 , g martin 9 , j sahatjian 10 , w self 9 , e seeley 9 , j weingarten 9 , m williams 9 , c winterbottom 11 , d hansell 12 is an effective method to predict fluid responsiveness (fr) or cardiac response to preload expansion. we have previously shown that fluid responsiveness is a dynamic state, changing frequently over a 72 hour monitoring period. methods: fresh is a currently enrolling prospective randomized controlled study, evaluating the incidence of fr and patient centered outcomes in critically ill patients with sepsis or septic shock (nct02837731). patients randomized to plr guided resuscitation were evaluated every 6-12 hours over the first 72 hours of care and classified as fr if the sv increased > 10% when measured with non-invasive bioreactance (starling sv, cheetah medical). the time of first fr was noted. results: a total of 608 plr assessments were performed in 86 patients over a 72 hour monitoring period. 56 % were female, and the average age was 61 years. plrs were evaluated over time, with time 0 representing initial fluid resuscitation ( figure 1 ). when individual subjects were evaluated over time, 100% of subjects who became fr only after 24 hours showed evidence of lv/rv dysfunction ( figure 2 ). conclusions: fluid responsiveness or preload dependence frequently changes for septic shock patients over the first 72 hours of care. evidence suggests it is beneficial to periodically perform an assessment of preload responsiveness to guide fluid administration, as preload dependence is a dynamic and changing state. preload dependence provides additional information beyond fluid responsiveness. those patients who remain primarily fluid non-responsive (preload independent) are more likely to demonstrate echo confirmed lv/rv dysfunction, as the delay in return to cardiac function may be related to underlying cardiac deficits. further evaluation may be indicated in preload independent patients. introduction: hydroxyethyl starch (hes), a synthetic colloid, has been used as a volume expander, and is associated with renal impairment in patients with sepsis. however, a small dose of hes (6%, 130/0.4) has sometimes been used in acute ischemic stroke. therefore, we investigated whether a small dose of hes was linked with renal deterioration in patients with acute ischemic stroke. methods: a consecutive 524 patients with acute ischemic stroke within 7 days from onset were included between january 2012 and may 2016 (fig 1) . we collected admission serum creatinine (scr), estimated glomerular filtration rate (egfr), and renal function was assessed using kdigo definition of acute kidney injury on hospital days 5 to 9 as to patient's hospitalization period. is crucial for venous return and volaemic status, and as such it is a useful parameter in physiology and clinical settings alike. we tested whether: near infra-red spectroscopy (nirs) could be effective at measuring msfp both in healthy individuals and in conditions with a rise in interstitial pressures; after an occlusion pressure is relieved, the decrease in venular blood volume could allow calculation of τ (time constant) and thus venous resistances (rv). in order to verify these hypotheses we used a forearm nirs probe on healthy individuals at rest and during different degrees of maximal voluntary contraction (mvc). methods: 10 healthy subjects volunteered in the study that took place at sant'andrea hospital in rome (italy). all subjects had venular pressures and volumes assessed via a nirs probe positioned on the forearm using a pressure-cuff in steps of 5 mmhg from 0 to 50 mmhg, at rest and at 10% and 20% mvc. for each patient msfp, unstressed volume (vu) and stressed volume (vs) were measured. a temporary 30 mmhg occlusion was obtained and volume time course was calculated upon release, to derive τ . results: p-v relationship was found to have a 3-slopes shape reflecting venular network changes. we measured vu, vs, and obtained msfp values of 6.74 ± 1.83 mmhg, p<0.001; during exercise no changes in vu and vs were noted but msfp values rose; value was found to be 2.9 ± 0.1 sec at rest and 0.9 ± 0.03 sec after exercise, reflecting a reduction in rv. conclusions: nirs measurements on healthy subject may have implications in the clinical assessment of critical care patients where changes in interstitial pressure are possible. introduction: in the pathogenesis of multiple organ dysfunction syndrome (mods) important role plays the development of hepatic dysfunction. a known method for assessing hepatic blood flow is reohepatography (rhg). however, it requires the analysis of a large number of parameters of the rheogram curve. the aim of this study was to develop a method for assessing arterial hepatic blood flow based on the rhg in patients with mods after abdominal surgery. methods: 55 patients in the department of anesthesiology and intensive care unit were included in a prospective study (36 men and 19 women, age 60.1 ± 16.1 years, weight 78.5 ± 13.9 kg.). all patients were divided into two groups: group 1 -patients after orthopedic and trauma surgery (n = 28), group 2 -patients after abdominal surgery with mods (n = 27). patients in the groups did not have statistical differences by sex, age, body weight, height. rhg was carried out using the "reo-spectr" (russian federation). we have compared the rhg indicators between the groups ( table 1) . we have developed a method for assessing hepatic arterial blood flow, which consists in determining the area under the arterial part of rhg curve using the simpson's rule. its normal values range from 43.2 mω *s to 49.0 mω *s. the method is non-invasive, can be applied at the patient´s bed. its advantage is simplicity, it can be used for rapid diagnosis and monitoring the effectiveness of treatment. area under the rhg curve in the group 1 were 46.1 ± 8.1 mω *s and 20.0 ± 16.8 mω *s in the group 2 (p <0.05). conclusions: patients after abdominal surgery with mods have impaired hepatic blood flow, which may be associated with liver pathology caused by main surgical disease (obstructive jaundice) and hemodynamic disorders caused by acute cardiovascular failure. the method we developed allows us to determine disorders of hepatic arterial blood flow in the early stages before signs of liver dysfunction appear. comparison of pulse oximetry hemoglobin with laboratory measurement of arterial and centralresults: 80 patients: 54% male, median 68years (59-73); p:f ratio 174 (132-208); peep 10 (7-12); apache iii 80.5 (26); median ventilation time 6 days (3-9). fair agreement was seen in subjective assessment vs objective measures with binary assessment of rv size and function. ordinal data analysis showed poor agreement with rvfws ( figure 1 ) and rv dimensions. if onestep disagreement was allowed the agreement was good ( table 1 , 2). significant overestimation of severity of abnormalities was seen comparing subjective assessment with rv eda and tapse, s' and fac. there was no difference in agreement values when accounting for clinician echo experience, perceived expertise (at level of cardiologist) or type of qualifications. conclusions: relatively low levels of agreement were seen with subjective assessment vs objective measures of rv size and function assessed by echo. it seems prudent to avoid subjective rv assessment in isolation and a combination of objective and subjective measures should be used. introduction: even short periods of hypotension are associated with increased morbidity and mortality. using high-density numerical physiologic data, we developed a machine learning (ml) model to predict hypotension episodes, and further characterized risk trajectories leading to hypotension. methods: a subset of subjects with 1/60hz physiological data was extracted from mimic2, a richly annotated multigranular database. hypotension was defined as >5 measurements of systolic blood pressure ≤ 90 mmhg and mean arterial pressure ≤ 60 mmhg, within a 10-minute window. derived features using raw measurements of heart rate, respiratory rate, oxygen saturation, and blood pressure were computed. random forest (rf), k-nearest neighbors (knn), and logistic regression models were trained with 10-fold cross validation to predict instantaneous risk of hypotension using features extracted from the data leading to the first episode of hypotension (cases) or icu discharge in subjects never experiencing hypotension (controls). for a given subject, risk trajectory was computed from the collation of instantaneous risks. results: from a source population of 2808 subjects, 442 subjects met our definition of hypotension, and 724 subjects without hypotension comprised the control group. 204 features were generated from the four vital signs. the area under the curve (auc) for random forest classifier was 0.829, out-performing logistic regression (auc 0.826) or k-nearest neighbors (auc 0.783) (fig 1) . risk trajectories analysis showed average controls risk scores <0.3 (<30% risk of future hypotension), while the hypotension group had a rising risk score (0.45 to 0.7) in the 8 hours leading to the first hypotension episode, and significantly higher scores leading into subsequent episodes (fig 2) . conclusions: hypotension episodes can be predicted from vital sign time series using supervised ml. subjects developed hypotension have an increased risk compared to controls at least 8 hours prior to the episode. introduction: in critically ill patients or in patients undergoing major surgery, monitoring of co is recommended [1] [2] [3] . less-invasive advanced hemodynamic monitoring with pwa is increasingly used in perioperative and critical care medicine. in this study, we evaluate the measurement performance of an uncalibrated pulse wave analysis (pwa) device (mostcareup, vygon, ecouen, france) compared with cardiac output (co) assessment by pulmonary artery thermodilution (patd) in patients after cardiac surgery. methods: in patients after cardiac surgery, we performed seven sets of patd measurements to assess patd-co. simultaneously, we recorded the pwa-co and compared it to the corresponding patd-co. to describe the agreement between pwa-co and patd-co we used bland-altman analysis showing the mean of the differences and 95%-limits of agreement and calculated the percentage error. results: we included 17 patients in the analysis. the bias between pwa-co and patd-co was 0.01 l*min-1. upper and lower 95% limits of agreement were +1.40 l*min-1 and -1.38 l*min-1. the percentage error was 28.1%. conclusions: pwa-co estimated with using the mostcareup device shows good agreement with pulmonary artery thermodilutionderived co in patients after cardiac surgery. introduction: non-invasive continuous blood pressure monitoring devices have been investigated, however, these devices did not have sufficient accuracy and precision. we developed a continuous monitor using the photoplethysmographic technique and tested the accuracy and precision of this system to ensure it was comparable to conventional continuous monitoring methods used for critically ill patients. methods: the study device was developed to measure blood pressure, pulse rate, respiratory rate, and oxygen saturation, continuously with a single sensor using the photoplethysmographic technique. patients who were monitored with arterial pressure lines in the icu were enrolled. the physiological parameters were measured continuously for 30 minutes at 5-minute intervals using the study device and the conventional methods. the primary outcome variable was blood pressure. results: pearson fs correlation coefficient between the conventional method and photoplethysmography device were 0.993 for systolic blood pressure, 0.985 for diastolic blood pressure, 0.998 for mean blood pressure, 0.996 for pulse rate, 0.995 for respiratory rate, and 0.963 for oxygen saturation. percent errors for systolic, diastolic and mean blood pressures were 2.4% and 6.7% and 6.5%, respectively. percent errors for pulse rate, respiratory rate and oxygen saturation were 3.4%, 5.6% and 1.4%, respectively. conclusions: the non-invasive, continuous, multi-parameter monitoring device presented high level of agreement with the invasive arterial blood pressure monitoring, along with sufficient accuracy and precision in the measurements of pulse rate, respiratory rate, and oxygen saturation. conclusions: stroke volume measurement using bioreactance technique had strong correlation with odm while pwtt had moderate correlation. both devices had small bias with wide limits of agreement and percentage error compared with odm. therefore, these devices are not interchangeable with odm. however, using trends in stroke volume to guide treatment might still be acceptable. introduction: hemorrhage is the most common cause of trauma deaths and the most frequent complication of major surgery. it is difficult to identify until profound blood loss has already occurred. we aim at detecting hemorrhage early and reliably using waveform vital sign data routinely collected before, during, and after surgery. methods: we use waveform vital sign data collected at 250 hz during a controlled transition from a stable (non-bleeding) to a fixed bleeding state of 93 pigs. these vital signs include airway, arterial, central venous and pulmonary arterial pressures, venous oxygen saturation (svo2), pulse oximetry pleth and ecg heartrate, continuous co, and stroke volume variation (lidco). we used gated recurrent units (gru), long short-term memory (lstm) and dilated, causal, one-dimensional convolutional neural (table 1) . however, outside of the very low fpr range (cf. rocs in fig. 1 and 2), our models appear inferior to a referenced random forest (rf) classifier. conclusions: our work demonstrates the applicability of deep learning models to diagnose hemorrhage based on raw, waveform vital signs. future work will address why the rf classifier can address the greater homogeneity of subjects when they bleed compared to an apparently wide dispersion of their statuses when being stable. this work is partially supported by nih gm117622. can myocardial perfusion imaging with echo contrast help recognise type 1 acute myocardial infarction in the critically ill? introduction: many instances of significant bleeding may not occur in highly monitored environment, contribution in the delay in recognition and intervention. we therefore proposed a noninvasive monitoring for early bleeding detection using photoplethysmography (ppg). methods: fifty-two yorkshire pigs were anesthetized, stabilized and bled to hemorrhagic shock, and their invasive arterial blood pressure (abp), and ppg data were collected [1] . time series of vital signs were divided into data frames of 1 minute updated every 30 seconds and beat to beat features were computed. the final feature matrix contained 18 abp features and 85 ppg features. a supervised machine-learning framework using least absolute shrinkage and selection operator regularized logistic regression model was constructed to score the probabilities for hemorrhage of each data frame. data in stabilization was set as negative and data in bleeding was set as positive. model performance was evaluated by receiver operating characteristic (roc) area under the curve (auc) with leave-one-out cross validation, and its precision was assessed with activity monitoring operative characteristic (amoc). results: two different models were proposed using abp and ppg features separately. figure 1 showed the ppg model could classify the hemorrhage with auc = 0.89, where the auc of abp model was 0.91. figure 2 showed the ppg model could detect the hemorrhage on average 15.5 minutes (equals to 320 ml blood loss) if the false alarm rate of 1/100 was tolerated, whereas the average detection time of abp model were 12.5 minutes at same threshold of false alarm rate. conclusions: we proposed a novel non-invasive bleeding detection approach using ppg signals only. this method potentially can improve the identification of hemorrhage with in patients and environments where invasive monitoring is unavailable. table 1 , catheter and procedure characteristics are shown in table 2 . the median angle of bed position was 26°. no patients were positioned in neutral or tp. all procedures were successful with a mean of 1.3 punctures per patient, and a maximum of 3. the median procedure time was 12.5 minutes. no major complications occurred in any of our patients. conclusions: central venous catheterisation in moderate upright position is feasible and can be done safely when using realtime ultrasound by well-trained physicians. we recommend performing clinical assessment and pre-procedural ultrasound to choose the optimal puncture site and position in order to attain an optimal ultrasound visualisation of the vessel and patient comfort. methods: a retrospective analysis of 10 patients presenting to tertiary-care emergency department who required cvc for vasopressor administration was carried out. all central venous cannulation into the right brachiocephalic vein was performed with ultrasound guidance using the high frequency linear probe. right brachiocephalic vein was visualised in its long axis. the needle was positioned just beside the centre of ultrasound probe 15 degrees below the coronal plane and 10 degrees angle to the ultrasound probe and advanced just behind the clavicle. results: the mean puncture time taken to perform this procedure, calculated from the needle piercing the skin until to the aspiration of blood from the brachiocephalic vein through the needle, was 23 ± 6.8s. no procedure-related complications were detected. conclusions: the oblique needle trajectory of right brachiocephalic vein cvc in adult is feasible and able to visualised well the anatomical structure, hence avoid complications. introduction: central venous cannulation, a routine procedure on intensive care units, is associated with a low complication rate. as a consequence, the routine use of chest x-ray (cxr) or ultrasound (us) to assess these complications is under discussion. our aim was to identify risk factors for central venous catheter (cvc) placement associated complications that can help decide whether or not follow-up using cxr and/or us is indicated. methods: multicenter prospective, observational study. consecutive critically ill adult patients who underwent cvc placement. either the internal jugular vein or subclavian vein was cannulated. complication rates were determined. predicting factors were obtained through a questionnaire filled in by physicians after placing a cvc. if the questionnaire was incomplete or data was missing, analyses were performed using the available data. patient characteristics were duplicated if a patient recieved more than one cvc. outcomes were iatrogenic pneumothorax and malposition. pneumothorax was detected using us, whereas cxr was used to determine cvc malposition. table 1 . usguidance, insertion site, and setting were predictive for complications. the overall cvc placement associated complication rate is low and multiple risk factors associated with the occurrence complications were identified. a complication rate this low, strongly suggests that routine post-procedural diagnostics is superfluous. therefore, we suggest, provided that uneventful execution of the procedure is assured, post-procedural diagnostics are only necessary in selected cases with (multiple) risk factors. introduction: the use of ultrasound for subclavian vein cannulation (scv) has developed poorly due to the difficulty of visualizing this vein via the classical infraclavicular approach. we explored the feasibility of ultrasound-guided subclavian vein catheterization via a supraclavicular approach methods: prospective study conducted over six-month period in intensive care unit. after approval of the ethics committee, we included patients over 18 years of age and requiring central venous access. exclusion criteria were: hemostasis disorders, puncture area infections and cervico-thoracic vascular malformations the procedure consisted of catheterization of the vsc with a supraclavicular approach under ultrasound guidance using an ultrasound in plane approach (fig 1 and 2 ). data collection included clinical and ultrasound data: scv depth, diameter and length, catheterization time, number of needle redirection, cannulation success and complications. results: thirty four patients were included. age: 57 ± 15 (mean ± sd), 60% of whom were male. the success rate of scv catheterization was 97% (one failure). the depth of the scv was 11 ± 4.5 mm and its diameter was 11 ± 3.5 mm. the puncturable length of the scv was 33 ± 7mm and the puncture angle was 36 ± 15°. the time required to obtain an adequate ultrasound image was 25 ± 9 seconds. the interval between the beginning of the puncture and the insertion of the guidewire into the vein was 42 ± 29 sec. the total catheterization time was 69 ± 33 seconds. the number of needle redirection 0.8 +/-1.2 redirects. the quality of the ultrasound image was excellent or good in 87.5% of cases. an arterial puncture was observed in two patients conclusions: this preliminary study demonstrated the feasibility of the subclavian vein cannulation via the supraclavicular approach. more study are required to confirm its safety and to compare this approach to the infraclavicular acces using ultrasound. introduction: lung ultrasound b-lines, a comet-like reverberation artefacts arising from water-thickened interlobular septa, indicate extravascular lung water which is a key variable in heart failure management and prognosis. aim of this study is to measure the correlation between lung ultrasound b-lines and nyha functional classification. methods: this is a 6 months prospective study on congestive heart failure patients conducted in 2 urban emergency departments in malaysia. following enrolment, patients had their functional capacity categorised based on nyha classification, followed by point of care ultrasound (pocus) lung scan using a 12mhz linear probe. the scanning was performed by trained emergency physicians. the longitudinal scan done at the recommended 6 zones of both left and right lungs and the total number of b-lines identified were summed up as the comet score. comet score of 0, 1, 2 and 3 were categorised based on amount of blines of less than 5, 5-15, 15-30 and more than 30 b-lines respectively. results: hundred and twenty-two patients were analysed (69 males(56.6%) and 53 females(43.4%)) ranging from 24 to 88 years old. comet score of 1,2 and 3 were found to be statistically significant with presence of paroxysmal nocturnal dyspnoea, elevated jugular venous pressure, lung crackles, bilateral pitting oedema and chest radiographic findings. a moderate correlation between nyha classes with comet score 1,2 and 3 (rs= 0.77 (p<0.01)) was documented. conclusions: our study demonstrated a moderate correlation between nyha classes and lung ultrasound b-lines. lung ultrasound may be a potential tool to objectively determine the functional capacity in patients with congestive heart failure and monitor its changes in response to treatment and disease progression. the introduction: point of care ultrasound (pocus) is a tool of increasing utility in the management of the critically ill patient. guidelines exist for training and accreditation in pocus [1, 2] however the widespread use of pocus has been hampered by a lack of mentors. online communication with end-to-end security, such as whatsapp ™ are increasingly used in medicine as a communication aid [3] . some individuals are using such communications to share pocus images for review-the overall sentiment around these tools is unknown. methods: an online survey of pocus users was conducted via twitter ™. the question was "in situations where an expert opinion on an ultrasound is not immediately available, is it acceptable to get an expert review via an online medium such as whatsapp, and would you be happy to be that expert?" results: 304 votes were received. voters were a mix of pocus users from the usa, europe, and australia. 58% said the medium was acceptable, and that they would be happy to provide expertise. 34% voted "no", with 8% voting "other" (fig 1) . conclusions: in this international survey of pocus users, 58% were happy to provide and receive mentorship using remote software such as whatsapp. distance mentorship for pocus training should be explored. [2] . a description of the development and refinement of insight -a feasibility and clinical effectiveness randomized controlled trial. methods: a modified delphi exercise was used to select the most beneficial ultrasound windows and imaging questions to ask for each window in scheduled inter-professional ultrasound. 260 nurses, 46 doctors and 6 physiotherapists from critical care were given the same information regarding potential utility of each window. the windows and associated questions were individually ranked; each window and question tested against three further criteria; and filtered by ease of training to level 1 standard; clinical usefulness; time of practical delivery and applicability across an inter-professional group. results: the modified delphi exercises and prioritization exercise ranked ease of adoption by training; feasibility within the time frame and clinical usefulness to develop a core insight scan of 5 domains, each with set binary questions (tables 1 and 2 ) conclusions: we have developed a research intervention that will allow us to test the effectiveness of inter-professional scheduled whole body assessment of critically ill patients by ultrasound. we now plan to conduct a clinical effectiveness trial with an internal pilot to confirm feasibility. to search for optimal pressing time, the plots from the color sensor during nail bed compression were analyzed. we found two phases in the color sensor plots. in the initial part of compression, the plots changes rapidly (rapid phase) and then the slope of plots reduces (slow phase). the pressure release during the rapid phase could destabilize the measurement. the longest period of the rapid phase was 1.9999 s among all the study subjects. thus, a pressing time of 2 s seems to be needed to obtain stable crt measurements. conclusions: on our study for the investigation of standard pressing time and strength for crt measurements, pressing the nail bed with 3-7 n and 2 s appears to be optimal. detection of pancreas ischemia with microdialysis and co2sensors in a porcine model introduction: pancreas transplantation is associated with a high rate of early graft thrombosis. current postoperative monitoring lack tools for early detection of ischemia, which could precipitate a graft-saving intervention. we are currently exploring the possibility of ischemia detection with microdialysis and co 2 -sensors in the organ tissue or on the surface in a porcine model. methods: in anesthetized pigs, co 2 -sensors and microdialysis catheters are inserted into the parenchyma or attached to the surface of the pancreas. pco 2 is measured continuously and lactate is sampled with microdialysis every 15 min. ischemia is induced by sequential arterial and venous occlusions for 45 minutes, with 120 minutes of reperfusion in between. results: pco 2 increased and decreased in response to ischemia and reperfusion within minutes. lactate increased and decreased with the same pattern, but with a considerable delay as compared to pco 2 . an example is depicted in figure 1 . the values are presented in introduction: reliable automated handheld vital microscopy (hvm) image sequence analysis is a prerequisite for use of sublingual microcirculation measurements at the point-of care according to the current consensus statement. we aim to validate a recently developed advanced computer vision algorithm [1] versus manual analysis in a wide spectrum of populations and contexts. methods: our collaborators were invited to contribute raw data of published or ongoing institutional review board approved work. inclusion criteria were use of the cytocam hvm device, manual analysis with the ava software, and image quality as independently assessed by massey score of <10 in >50% of recordings in a random subset of each study. 233 subjects from 11 studies were included, covering clinical and experimental populations, major shock forms and interventions to recruit the microcirculation (table 1) . results: 2,599,710 red blood cells were tracked by the algorithm across 150,163 frames in 1462 measurements in real time. a good to excellent correlation was found between algorithm-determined and manual capillary density (p<0.0001, r 0.6-0.9, figure 1 ). capillary perfusion was classified using space-time diagram derived red blood cell velocity (rbcv), yielding good correlation with manual analysis for functional capillary density und proportion of perfused vessels. microcirculatory alterations during disease and interventions were equally detected by the algorithm and manual analysis. change in flow short of severe abnormality was reflected in absolute rbcv but not microcirculatory flow index. conclusions: we demonstrate the validity of automated software for hvm image sequence analysis across broad populations, disease conditions and interventions. thus, microcirculatory assessment at the bedside may finally complement point-of-care evaluation of disease severity and treatment response in critically ill patients and during surgery. introduction: in 2016, naumann et al introduced the poem score as a real-time, point-of-care score to assess sublingual microcirculation [1] . our study aimed to determine the reproducibility of the poem score. methods: two expert operators used a sidestream darkfield (sdf) videomicroscope (cytocam, braedius, netherlands) to separately acquire four high-quality video clips and assign a poem score to each image in 20 adult mechanically ventilated patients. each operator was blinded to the other's images and analysis. video clip scores and acquisition times were recorded. results: of the 20 patients enrolled in this study, 45% (n=9) required vasopressors. we categorized poem scores 4-5 as "normal" and poem scores 1-3 as "impaired." (fig 1) . with only one instance of interrater disagreement (i.e., a single image scored as 3 versus 4), cohen's kappa (0.86) confirmed a strong correlation between interpreters. the mean time to complete a study session was 9 minutes. conclusions: the present inability to quickly characterize the quality of sublingual microcirculation as either normal or impaired at the point of care limits real-world clinical application of this resuscitative endpoint. the rapidly obtained poem score appears to be reproducible between bedside interpreters. future studies should assess the effect of poem score-guided resuscitation. . sublingual microcirculatory images were obtained using a cytocam-idf device (braedius medical, huizen, the netherlands) and analyzed using standardized published recommendations. results: the median age of participants was 32 years. we found no significant difference in proportions of hemodynamic responders before and after marathon (64% vs 55%, p=0.819). also we did not find differences between plr induced changes of total vessel density (tvd) and proportion of perfused vessels (ppv) of small vessels before and after marathon. correlations between changes of sroke volume and changes of tvd or ppv of small vessels during plr were not significant. conclusions: marathon running did not change microcirculatory responsiveness. introduction: clinical measurement of mitochondrial oxygen tension (mitopo 2 ) has become available with the comet system [1] . a question with any novel technique is whether it is feasible to use in clinical practice and provides additional information. in elective cardiac surgery patients we measured cutaneous mitopo 2 and tissue oxygenation (sto 2 ). methods: institutional research board approved observational study in patients undergoing cardiopulmonary bypass (cpb). mitopo 2 measurements were performed on the left upper arm (comet, photonics healthcare b.v.) by oxygen-dependent delayed fluorescence of 5aminolevulinic acid (ala)-induced protoporphyrin ix [2] . priming of the skin was done with ala (alacare, photonamic gmbh) applied the evening before surgery. sto 2 measurements (invos, medtronic) were done in close proximity to the comet sensor. results: at the time of writing 28 of 40 patients were enrolled and mitopo 2 measurements were feasible in this clinical setting. mitopo 2 appeared sensitive with a high dynamic range. for example, highdose vasopressor therapy decreased mitopo 2 and blood transfusion increased a low mitopo 2 but not a high mitopo 2 . in the example in figure 1 , mitopo 2 is clearly dependent on cpb flow and the restored cardiac circulation is able to maintain good cutaneous oxygenation after cpb even before returning of cellsaver blood. sto 2 had the tendency to provide relatively stable values within a small bandwidth and little response to even major hemodynamic changes. conclusions: mitopo 2 shows the effect of interventions on mitochondrial oxygenation and provides additional information compared to standard monitoring and sto 2 . introduction: traumatic asphyxia is a rare condition in which breathing and venous return is impaired due to a strong compression to the upper abdomen or chest region, and induces swelling, purplish red appearance, and petechiae around the face and neck. to our knowledge, there are no reports describing details of traumatic asphyxia including the clinical course and the therapeutic reactivity from cardiac arrest. we focused on cardiac arrest among all traumatic asphyxia patients treated at our hospital, and investigated their clinical features and therapeutic reactivity. methods: sixteen cases of traumatic asphyxia involved with our hospital between april 2007 and march 2018 were reviewed by using the pre-hospital activity record, medical record, and hyogo prefectural inspection record. these patients were divided into three groups. the first group had already cardiac arrest at the time of rescue from the trapped place (group a; 6 cases). the second group became cardiac arrest after the rescue (group b; 5 cases). the third group did not experience cardiac arrest (group c; 5 cases). results: all cases had abnormal findings in skin or conjunctiva (table 1) . total mortality rate reached 56%, but among 11 cases of group a and b who resulted in cardiac arrest, there were 10 cases with injury severity score 16 or more and abbreviated injury scale in the chest 3 or more. they had pneumothorax, flail chest, pericardial hematoma. seven of them restored spontaneous circulation, and two cases achieved neurologically full recovery. conclusions: there are some cases of traumatic asphyxia whose therapeutic reactivity is very good even after cardiac arrest, so it is important not to spare efforts for life support in such cases. rhythm and 75% witnessed arrest, five hundred ten (25%) patients had a good functional outcome at 6-months. physiological derangements were each negatively associated with outcome in bivariate analysis at the p <0.001 level. a summary score of physiological derangements was included with potential confounders in the final regression model, and was independently associated with outcome with the chance of a good outcome decreasing by 46% for each increase of one physiologic derangement (95% ci 0.46-0.63). conclusions: uncorrected physiological derangements are independently and cumulatively associated with worse outcome after cardiac arrest. although causality cannot be established, it is reasonable to consider that the correction of physiological parameters may be an important step in the chain of survival after resuscitation. characteristics introduction: glan clwyd hospital (gch) was recently designated one of three cardiac arrest centres for wales. it has offered a 24/7 percutaneous coronary angiography (pci) service to a geographically dispersed north wales population of approximately 690,000 since june 2017. prior to this, urgent coronary angiography was available on a more limited basis to patients requiring pci. the aim of this study was to investigate factors associated with hospital mortality after critical care admission following cardiac arrest. methods: retrospective review of the ward watcher critical care database at gch to identify patients who had undergone cpr in the 24 hours prior to critical care admission in 2013-18. patients likely to have sustained ooha of cardiac aetiology (ooha-c) were identified from primary and secondary diagnoses and free text entry. data were subsequently analysed using excel and spss. the project was registered as a service evaluation with gch audit department. results: there were 190 cardiac arrest admissions over this period, increasing from 25 in 2013-14 to 69 in 2017-18. of these 122 were ooha, of which 103 were considered ooha-c. although ooha-c hospital mortality appeared to decrease over the time period (89%% to 56%), this was not statistically significant (p=0.149). factors associated with survival to hospital discharge are presented in the tables below. on logistic regression, only pci and low ph within the first 24 hours of critical care remained statistically significant (p=0.027 and p<0.001 respectively). conclusions: although we have been unable to make a distinction between patients presenting following stemi and nstemi, and appreciating a potential influence of selection bias, the significant association between pci and survival to hospital discharge supports the introduction of clinical pathways enabling pci access following ooha-c [1] . chest radiography. [1] here, we aimed to derive and validate rules to estimate p_max.lv using anteroposterior chest radiography (ches-t_ap), which is performed for critically-ill patients urgently needing determination of personalised p_max.lv. methods: a retrospective, cross-sectional study was performed with non-cardiac arrest adults who underwent chest_ap and computed tomography (ct) within 1 h (derivation:validation=3:2). on chest_ap, we defined cd (cardiac diameter), rb (distance from right cardiac border to midline) and ch (cardiac height, from carina to uppermost point of left hemi-diaphragm) (fig 1, 2) . [1] setting p_zero (0, 0) at the midpoint of xiphisternal joint and designating leftward and upward directions as positive on x and y axes, we located p_max.lv (x_max.lv, y_max.lv). the coefficients of the following mathematically-inferred rules were sought: x_max.lv=a0*cd-rb; y_max.lv=ß0*ch+γ . (a0: mean of (x_max.lv+rb)/cd; ß0, γ : representative coefficient and constant of linear regression model, respectively 1) . conclusions: evaluable echocardiographic records were reached in most of the patients. etco2 positively correlated with all parameters under consideration, while the strongest correlation was found between cimax and etco2. therefore, cimax is a candidate parameter for real-time monitoring of haemodynamic efficacy of chest compressions during cpr. introduction: the uk resuscitation council has set out guidelines for management of patients post cardiac arrest [1] . this is in line with european resuscitation council guideline. we set out to find if we are following the guideline. methods: we did a retrospective audit over the course of 2 years looking at the data of 24 patients who had in hospital and/or out of hospital cardiac arrest and after the return of spontaneous circulation were admitted to the intensive care unit (icu). we focused on whether the care they received was as per the standards set by the uk resuscitation council. results: we had 11 in the hospital and 13 out of hospital cardiac arrests; 11 patients had less than 10 minutes of cpr, 9 had more than 10 minutes cpr and 4 patients the data was not recorded; 16 patients needed more than 61 minutes to reach from the site of arrest to the icu. the partial pressure of carbon dioxide was >5.5 kpa in 11 patients at two or more occasions. target map was not documented in 9 patients; blood sugar target was not documented in 15 patients and was not maintained within limits in 5 patients. target temperature was not documented in 10 patients. the withdrawal of treatment was not delayed for 72 hours in 1 patient out of 24. in 4 patients neurological tests were not documented. multimodal assessment tools were not used in 1 patient. electroencephalography and serum neuron specific enolase were not used to diagnose brain deaths as they were not available at our trust. 11 patients were discharged, 12 died in the icu and 1 died in hospital after discharge from icu. conclusions: the audit reflected our local practice and showed that our mortality was in line with the acceptable limits; poor documentation of plan of care which posed problems in analyzing the care that these patients received; some of the parameters were not being maintained as set by uk resuscitation guideline. introduction: high-quality chest compressions (cc) with minimized interruptions are one of the most essential prerequisites for an optimal outcome of resuscitation. therapy of reversible causes of cardiac arrest often requires intra-hospital transportation (iht) during ongoing cpr. the present study investigated cc quality during transportation depending on the position of the provider. methods: 20 paramedics were enrolled into a manikin study with four groups: a reference group with the provider kneeling beside manikin on the floor (group 1), and 3 groups performing cc during a simulated iht of 100 meters: walking next to the bed (group 2), kneeling beside the patient in bed (group 3, fig. 1 ) or squatting above the patient in bed (group 4, figure 2 ). indicators of cc quality were measured as defined in the erc guidelines 2015 (pressure point and depth, compression frequency, complete relief, sufficient pressure depth) [1] . all 20 paramedics performed cc during each scenario (group 1-4). results: there were no statistical differences in quality of cc between groups 1, 3 and 4. notably, group 2 performed significantly worse in respect to the proportion of cc with correct pressure point (p = 0.044 vs group 1), correct cc depth (p=0.004 vs. group 1, p=0.035 vs. group 3, p=0.006 vs. group 4). the results are shown in table 1 . conclusions: carrying out guideline-compliant cc [1] during iht is feasible with multiple provider positions. based on the present results, kneeling or squatting position next to the patient ( figure 1 and 2) is recommended, whereas "walking next to the bed" while performing cc should be avoided. methods: a retrospective review of clinical notes was undertaken for patients admitted to icu following return of spontaneous circulation but whom remained comatose. this audit encompassed three-month periods before and after introduction of the care bundle in october 2017. audit standards were assigned from target parameters documented in the bundle and reflected guidance from the cheshire and merseyside critical care network. results: 39 patients were included in our audit; 15 admitted prior to and 24 admitted following implementation of the care bundle. in patients whom targeted temperature management was indicated, improved adherence to thermoregulation between 34-36°c was observed (50 vs 71%). significant improvements were since in the observance to target values for oxygen saturation (0 vs 70.8%, p=0.0023) and mean arterial pressure (20 vs 95.8%, p<0.0001) following the introduction of the care bundle. improved observance of ventilation targets was also seen; maintenance of p a co 2 >4.5 kpa (40 vs 75%, p=0.0441) and tidal volumes <8 ml/kg ideal body weight (0 to 37.5%, p=0.0069). conclusions: the introduction of a post-cardiac arrest care bundle in our icu has improved care by providing discrete physiological targets to guide nursing staff and standardising management between clinicians. variations in care are associated with poorer patient outcomes [2] and introduction of this bundle has reduced disparities in practice. array of cardiac diseases and reported survival rate is low in spite of advances in resuscitation and ems services. methods: single-centre retrospective study analyzed outcomes of 42 ohca patients admitted to cardiac icu between 2010.-2015. we studied demographic data, initial rhythm, type of cpr, comorbidities and various post admission diagnostic findings in order to identify their impact on survival. results: ohca comprised 0,5% of all admissions. mean los was 8.24 days (0-64). mean age was 65,4y (29-90), m: f ratio 29:13 and bystander cpr was performed in only 19% ohca patients. the most common initial rhythm was vf (45.2%), followed by vt (16.6%), pea was found in 2,9% and asystole in 1.9% of pt more than half of pt received adrenalin (55%) and defibrillation (57%) and only 17% required a temporary pacemaker. 38% of pt had an ecg consistent with mi after rosc, 19% underwent coronary angiography resulting in pci in 75% of cases. in 5 pt (12%) therapeutic hypothermia protocol was performed. most ohca pt had hypertension (60%) and hyperlipidaemia (69%) as the most common risk factors followed by cardiomyopathy (33%), diabetes (29%) and cad (21%). only 9% had a preexisting significant valvular disease and the rest were extracardial comorbidities: chronic renal disease (17%), copd (9%) and cerebrovascular disease (9%). 14 patients survived (33%) and gcs on admission was the only significant impact factor on survival along with comorbidities (mean gsc was 6 in survivors vs. 3 in deceased). interestingly, age, initial rhythm, troponin i level, ph and therapeutic hypothermia had no impact on survival. conclusions: our data demonstrate the importance of early onsite resuscitation as the most important factor of neuroprotection and outcome and puts an emphasis on the importance of cpr education for layman population. prediction of acute coronary ischaemia and angiographic findings in patients with out-of-hospital cardiac arrest j higny 1 , a guédès 2 , c hanet 2 , v dangoisse 2 , l gabriel 2 , j jamart 3 introduction: coronary artery disease (cad) is the leading cause of out-of-hospital cardiac arrest (ohca). however, diagnosis of acute coronary ischaemia (aci) remains challenging, particularly in patients without st-segment elevation on the post-resuscitation ecg. in this regard, a consensus statement recommends the implementation of a work-up strategy in the emergency room (er) to exclude noncoronary causes of collapse within 2 hours. methods: retrospective single-centre study performed on 64 consecutive patients with resuscitated ohca who underwent a diagnostic coronary angiography (ca). we present data on coronary angiograms for patients who underwent cardiac catheterization after resuscitation. afterwards, we sought to identify parameters associated with aci. results: st-segment elevation was noted in 29 patients (45%). stsegment depression or t-wave abnormalities were noted in 35 patients (55%). invasive coronary strategy allowed to identify an acute culprit lesion in 46 cases (72%). 29 patients with st-segment elevation underwent an immediate angioplasty for an acute coronary occlusion. 17 patients without st-segment elevation underwent an ad hoc percutaneous coronary intervention for a critical lesion. stable cad was found in 9 cases (14%) and a normal angiogram was found in only 9 cases (14%) (figure 1 ). conclusions: aci was the leading precipitant of collapse. stsegment elevation was highly predictive of coronary occlusion. in addition, a culprit coronary lesion was identified in nearly 50% of patients undergoing ca despite the lack of stsegment elevation. finally, our findings suggest that the identification of risk criteria may help to improve the recognition of aci after ohca. the prediction of outcome for in-hospital cardiac arrest (pihca) score e piscator 1 , k göransson 1 , s forsberg 1 , m bottai 2 , m ebell 3 , j herlitz 4 , t djärv 1 figure. predictive value for classification into <3% likelihood of favorable neurologic survival was 97.4%. false classification into < 3% likelihood of favorable neurologic survival was 0.57%. the phica score has potential to be used as an aid for objective prearrest assessment of the chance of favorable neurologic survival after ihca, as part of decision making for a dnar order. introduction: prognosis of survival in patients with cardiac arrest remains poor. during and after cardiopulmonary resuscitation, pathophysiological disturbances in relation with a cytokine storm, are described as "post-resuscitation" disease like a combination of cardiogenic and vasodilatory shocks. veno-arterial extracorporeal membrane oxygenation (va ecmo) allows to restore adequate perfusion but little is known about its effect on left ventricular (lv) function and about the role of cytokines. methods: this study was performed in an experimental model of cardiac arrest performed in 3 groups of 3 anesthetized and mechanically ventilated pigs. cardiac arrest was obtained by application of electrical current to epicardium inducing ventricular fibrillation. after a no-flow period of 5 minutes, medical resuscitation with catecholamines and vasopressors was performed in "control" group while va ecmo was started in "ecmo" group and va ecmo in combination with cytosorb (extracorporeal blood purification therapy designed to reduce excessive levels of inflammatory mediators such as cytokines) was started in "ecmo-cyto" group. lv function was assessed with transthoracic echocardiography and arterial pressure with aortic pressure catheter. results: hemodynamic stability was obtained after 22 ± 6 and 24 ± 7 minutes in ecmo and ecmo-cyto groups, respectively. no return of spontaneous circulation was observed in control group. at 15 minutes following cardiac arrest, lv area fractional change on short axis was normalized in ecmo and ecmo-cyto groups (31± 3 and 34 ± 4 %, respectively). vasopressor requirements were significantly lower in ecmo-cyto group than in ecmo group. conclusions: after cardiac arrest (no-flow) of 5 minutes duration, va ecmo allowed complete lv recovery and hemodynamic stability within 30 minutes of "post-resuscitation" disease. cytosorb added to va ecmo could contribute to reduce post-resuscitation vasodilatation. impact of rapid response car system on ecmo in out-of-hospital cardiac arrest: a retrospective cohort study m nasu 1 , r sato 2 , k takahashi introduction: extracorporeal life support (ecls) has been reported to be more effective than conventional cardio-pulmonary resuscitation (cpr). in ecls, a shorter time from arrival to implantation of extracorporeal membrane oxygenation (ecmo; door-to-ecmo) time has been reported to be associated with better survival rates. this study aimed to examine the impact of the physician-based emergency medical services (p-ems) using a rapid response car (rrc) on door-to-ecmo time in patients with out-of-hospital cardiac arrest (ohca to study the interest and the educational contribution in the short and medium term of medical simulation compared to a classical training. methods: cohort, prospective, observational, single-center, randomized study with control group including 30 residents (20 in anesthesia resuscitation and 10 in emergency medicine). all benefited from a theoretical training with a reminder of the latest recommendations on the management of cardiac arrest and anaphylactic shock. they were randomized into 2 groups and received practical training on a high-fidelity simulator for the management of either cardiac arrest (acc group) or anaphylactic shock (ca group). each group was evaluated at 6 weeks (t0) and at 6 months on two scenarios: refractory ventricular fibrillation (fv) scored on 20 points and grade 3 anaphylactic reaction (ra3) scored on 30 points. each group served as the control group for the pathology in which they did not receive specific simulator training. the results are expressed on average with their standard deviations with "p" <0.05. introduction: simulation is a tool for improving the quality and safety of care, and its recognized as an essential method of evidence-based education. emergency medicine is a discipline in which there is a constant concern for the safety of patients. the emergency physician is often called upon to take charge of critical situations that use knowledge, know-how and knowledge as skills that must be mastered and whose theoretical learning alone is insufficient. methods: it´s a prospective study including residents in emergency medicine performing their specialty courses in emergency services and emergency medical assistance in the region of sousse from january to june 2018. they were randomized into two groups: the one benefiting from a traditional education and the other from an education based on simulation sessions. the chosen scenario was the management of a cardiac arrest. a pre-test and a post-test were performed in both groups. results: we included 30 emergency residents who did not receive specialized training in the management of cardiac arrest, there was a female predominance with an average age of 27, there was no significant difference regarding the pretest between the two groups with 10.08 there was no significant difference with respect to the pre-test score between the two groups 10.08 ± 2.7 / 20 for the control group versus 10.34 ± 3.3 / 20 for the simulation group. there was a significant progression after the course with an average posttest score of 13.87 ± 1.8 in the simulation group while this score was 11.94 ± 2.3 in the control group with a statistically significant difference (p <0.001). conclusions: simulation learning has led to a better acquisition of cognitive knowledge by learners. the simulation is not intended to replace bed-based teaching, nor theoretical or faculty teaching, but it is an essential complement . in tunisia, the simulation must continue its current integration in the initial and continuous training of doctors. introduction: recent studies have shown that obesity and its related metabolic dysfunction exacerbates outcomes of ischemic brain injuries in some brain areas, such as the hippocampus and cerebral cortex when subjected to transient global cerebral ischemia (tgci). however, the impact of obesity in the striatum after tgci has not yet been addressed. the objective of this study was to investigate the effects of obesity on tgci-induced neuronal damage and inflammation in the striatum and to examine the role of mtor which is involved in the pathogenesis of metabolic and neurological diseases. methods: gerbils were fed with a normal diet (nd) or high-fat diet (hfd) for 12 weeks and then subjected to 5 min of tgci. hfd-fed gerbils showed the significant increase in body weight, blood glucose level, serum triglycerides, total cholesterol, and low-density lipoprotein cholesterol without affecting food intake. results: in hfd-fed gerbils, neuronal loss occurred in the dorsolateral striatum 2 days after tgci and increased neuronal loss were observed cholesterol days after tgci; however, no neuronal loss was the in ndfed gerbils after tgci, as assessed by neuronal nuclear antigen immunohistochemistry and fluoro-jade b histofluorescence staining. the hfd-fed gerbils also showed severe activated microglia and further increased immunoreactivities and protein levels of tumor necrosis factor-alpha, interukin-1beta, mammalian target of rapamycin (mtor) and phosphorylated-mtor in the striatum during pre-and postischemic conditions compared with the nd-fed gerbils. in addition, we found that treatment with rapamycin, a mtor inhibitor, in the hfd-fed gerbils significantly attenuated hfd-induced striatal neuronal death without changing physiological parameters. conclusions: these findings reveal that chronic hfd-induced obesity results in severe neuroinflammation and significant increase of mtor activation, which could contribute to neuronal death in the stratum following tgci. abnormal mtor activation might play a key role. associations between partial pressure of oxygen and neurological outcome in out-of-hospital cardiac arrest patients introduction: exposure to hyperoxemia and hypoxemia is common in out-of-hospital cardiac arrest (ohca) patients following return of spontaneous circulation (rosc) but its effects on neurological outcome are uncertain and study results are inconsistent. methods: exploratory post-hoc substudy of the target temperature management (ttm) trial [1] , including 939 patients after ohca with rosc. the association between serial arterial partial pressures of oxygen (pao 2 ) during 37 hours following rosc and neurological outcome at 6 months, evaluated by cerebral performance category (cpc), dichotomized to good (cpc 1-2) and poor (cpc 3-5), was investigated. in our analyses, we tested the association of hyperoxemia pao 2 > 40 kpa and hypoxemia pao 2 < 8 kpa, time weighted mean pao 2 , (twm-pao 2 ) (fig 1) , maximum pao 2 difference (δ pao 2 ) and gradually increasing pao 2 levels (13.3 -53.3 kpa) with poor neurological outcome. a subsequent analysis investigated the association between pao 2 and a biomarker of brain injury, peak serum tau levels. results: 869 patients were eligible for analysis. 300 patients (35%) were exposed to hyperoxemia or hypoxemia after rosc (table 1) . our analyses did not reveal a significant association between hyperoxemia, hypoxemia, twm-pao 2 exposure or δ pao 2 and poor neurological outcome at 6-month follow-up after correction for co-variates (all analyses p= 0.146 -0.847) (fig 2) . we were not able to define a pao 2 level associated with the onset of poor neurological outcome. peak serum tau levels at either 48 or 72 hours after rosc were not associated with pao 2 . conclusions: hyperoxemia or hypoxemia exposure occurred in one third of the patients during the first 37 hours of hospitalization and was not significantly associated with poor neurological outcome after 6 months or with the peak s-tau levels at either 48 or 72 hours after rosc. introduction: cerebral hypoperfusion may aggravate the developing neurological damage after cardiac arrest. near-infrared spectroscopy (nirs) provides information on cerebral oxygenation but its clinical relevance during post-resuscitation care is undefined. we wanted to assess the possible association between cerebral oxygenation and clinical outcome after out-of-hospital cardiac arrest (ohca). methods: we performed a post hoc analysis of a randomised clinical trial (comacare) where both moderate hyperoxia and high-normal arterial carbon dioxide tension (paco 2 ) increased regional cerebral oxygen saturation (rso 2 ) as compared with normoxia and low-normal paco 2 , respectively. rso 2 was measured from 118 ohca patients with nirs during the first 36 h of intensive care and neurological outcome was assessed using the cerebral performance category (cpc) scale at 6 months after cardiac arrest. we calculated the median rso 2 for patients with good (cpc 1-2) and poor (cpc 3-5) outcome and compared the results using the mann-whitney u test. we compared the rso 2 over time with outcome using a generalised mixed model. finally, we added median rso 2 to a binary logistic regression model to control for the effects of possible confounding factors. results: the median (interquartile range [iqr]) rso 2 during the first 36 h of intensive care was 70.0% (63.5-77.0%) in patients with good outcome compared to 71.8% (63.3-74.0%) in patients with poor outcome, p = 0.943. we did not find significant association between rso 2 over time and neurological outcome ( figure 1 ). in the binary logistic regression model rso 2 was not a statistically significant predictor of good outcome (or 0.99, 95% ci 0.94-1.04, p = 0.635). conclusions: we did not find any association between cerebral oxygenation during the first 36 h of post-resuscitation intensive care and neurological outcome at 6 months after cardiac arrest. fig. 2 introduction: near-infrared spectroscopy (nirs) provides a noninvasive means to assess cerebral oxygenation during postresuscitation care but its clinical value is unclear. we determined the possible association between cerebral oxygenation and the magnitude of brain injury assessed with neuron-specific enolase (nse) serum concentration at 48 h after out-of-hospital cardiac arrest (ohca). methods: we performed a post hoc analysis of a randomised clinical trial (comacare) comparing two different levels of carbon dioxide, oxygen and arterial pressure after ohca and successful resuscitation. we measured rso 2 continuously with nirs from 118 patients during the first 36 h of intensive care. we determined the nse concentrations at 48 h after cardiac arrest from serum samples using an electrochemiluminescent immunoassay kit. the samples were tested for haemolysis and all samples with a haemolysis index > 500 mg of free haemoglobin per litre (n = 2) were excluded from the analyses. we calculated the median rso 2 for all patients and used a scatterplot and spearman's rank-order correlation to assess the possible relationship between median rso 2 and nse at 48 h. in addition, we compared the nse concentrations at 48 h after cardiac arrest in patients with good (cerebral performance category scale [cpc] 1-2) and poor (cpc 3-5) neurological outcome at 6 months using the mann-whitney u test. results: we did not find significant correlation between median rso 2 and serum nse concentration at 48 h after cardiac arrest, rs = -0.08, p = 0.392 (figure 1 ). the median (iqr) nse concentration at 48 h was 17.5 (13.4-25.0) μg/l and 35.2 (22.6-95.8) μg/l in patients with good and poor outcome, respectively, p < 0.001. conclusions: we did not find any association between cerebral oxygenation during the first 36 h of post-resuscitation intensive care and nse serum concentrations at 48 h after cardiac arrest. the association between lactate, cerebral oxygenation and brain damage in post-cardiac arrest patients introduction: patients admitted to the intensive care unit (icu) after being successfully resuscitated from a cardiac arrest (ca) have a large cerebral penumbra at risk for secondary ischemic damage in case of suboptimal brain oxygenation. therefore, resuscitation during icu stay should be guided by parameters that adequately predict cerebral hypoxia. the value of lactate as resuscitation parameter may be questioned in post-ca patients since the brain critically depends on aerobic metabolism. we aimed to investigate the relationship between arterial lactate, cerebral cortex tissue oxygenation (scto2) by near infrared spectroscopy (foresight) and unfavorable neurological outcome at 180days (cpc score 3-5) methods: subanalysis from the neuroprotect post-ca trial. lactate values and scto2 were recorded hourly in 102 post-ca patients during 24hours ttm33 and subsequent rewarming. results: in total 3290 paired lactate/ scto2 measurements were analysed. we found no correlation between paired lactate and scto² (fig.1) . moreover, temporary trends in lactate did not correlate with corresponding trends in scto2 during the same one-hour time interval (r²=0.003) (fig 2) . if lactate values above 2.0 mmol/l are considered to be abnormal, lactate could not adequately detect clinical important brain ischemia (scto2 < 60%): sensitivity 62% and specificity 53% (table 1 , 2). nevertheless, time weighted lactate at 6h (or 1.38; p 0.01), 12h (or 1.38, p 0.01), 24h (or 1.53; p 0.003) and 36h (or 1.60; p 0.005) were inversely correlated with unfavorable neurological outcome at 180days (fig 1, 2) . conclusions: although lactate was a marker of prognosis in post-ca patients, it should not be used to guide resuscitation since lactate values were not correlated with scto2 and changes in lactate do not correspond with changes in scto2 during the same time interval. simplified introduction: the aim of the study was to investigate whether simplified continuous eeg monitoring (ceeg) [1] post-cardiac arrest can be reliably interpreted by icu physicians after a short structured training, and whether acceptable interrater agreement compared to an eeg-expert can be achieved. methods: five icu physicians received training in interpretation of simplified ceeg (fig 1) consisting of lectures, hands-on ceeginterpretation, and a video tutorial -total training duration 1 day. the icu physicians then interpreted 71 simplified ceeg recordings. basic eeg background patterns and presence of epileptiform discharges or seizure activity were assessed on 5-grade rank-ordered scales based on a standardized eeg terminology [2] . an experienced eeg-expert was used as reference. results: there was substantial agreement (κ 0.69) for eeg background patterns and moderate agreement (κ 0.43) for epileptiform discharges between icu physicians and the eeg-expert. sensitivity for detecting seizure activity by the icu physicians was limited (50%), but with high specificity (87%). among icu physicians interrater agreement was substantial (κ 0.63) for eeg background pattern and moderate (κ 0.54) for epileptiform discharges. conclusions: after a one-day educational effort clinically relevant agreement was achieved for basic eeg background patterns after cardiac arrest. assessment of epileptiform patterns was less reliable, but bedside screening by the icu physician may still be clinically useful for early detection of seizures. interpretation of simplified ceeg requires awareness of its limitations and support from an eeg-expert when clinically indicated. introduction: hypoxic-ischemic injury on head computed tomography (ct), which manifests with varying degrees of cerebral edema and loss of gray-white matter differentiation, is a poor prognostic sign after resuscitated out-of-hospital cardiac arrest that may influence early clinical decision-making. agreement among physicians on the presence of hypoxic-ischemic injury on early head ct is unknown. methods: we recruited 10 faculty physician participants (2 emergency medicine, 3 critical care, 3 neurocritical care, and 2 general radiology; average 8.2 years of practice) across 3 academic medical centers each with >100 admissions for resuscitated out-of-hospital cardiac arrest each year. participants, blinded to clinical context, reviewed 20 unique head cts obtained within 2 hours of cardiac arrest that were randomly selected from a local registry. a blinded neuroradiologist also reviewed all scans (gold standard). participants determined if hypoxic-ischemic injury was present on each ct, and agreement was determined using multi-and dual-rater kappa statistics with 95% confidence intervals. results: overall agreement among physicians regarding the presence of hypoxic-ischemic injury on head ct was fair (kappa 0.34; 95% ci, 0.19-0.49) with agreement consistent across most specialties (table 1) . when compared to the neuroradiologist, individual physician agreement ranged widely, from poor (kappa 0.12) to substantial (kappa 0.68), with 6 of 10 physicians having fair or worse agreement compared to the gold standard interpretation. conclusions: the finding of hypoxic-ischemic injury on early head ct after cardiac arrest had high interobserver variability as interpreted by acute care physicians and general radiologists. pending the development of objective diagnostic criteria, clinicians should bear in mind the subjectivity and subtlety of cerebral edema or loss of graywhite matter differentiation soon after return of spontaneous circulation in these patients. figure 1 ). baseline characteristics and differences between the wlst and no-wlst groups are shown in table 1 . utilization of neuro-prognostication tests is shown in table 2 . while ct and eeg were commonly employed, ssep and mri were used less frequently. basic multimodal neuroprognostication (arbitrarily defined as at least one ct or mri, plus eeg, plus ssep) was performed only in 34.1% of all patients undergoing wlst but the rate increased significantly over six years (p<0.001) and was higher in the time period after 2015, compared to the one prior to 2015 ( figure 2 ). this association remained significant after adjustment for confounders such as age, arrest rhythm, downtime, targeted temperature management, apache ii score and organ failure in a logistic regression model (p=0.004). in an institution with access to a wide range of imaging and neurophysiology tests, mri and ssep remained underutilized but the rate of basic multimodal neuro-prognostication increased significantly over the study period, especially in the period after 2015. introduction: although multiple reports using animal models have confirmed that melatonin appears to promote neuroprotective effects following ischemia/reperfusion-induced brain injury, the relationship between its protective effects and the activation of autophagy in cerebellar purkinje cells following the asphyxial cardiac arrest and cardiopulmonary resuscitation (ca/cpr) remains unclear. methods: rats used in this study were randomly assigned to 6 groups as follows; vehicle-treated sham-operated group, vehicletreated asphyxial ca/cpr-operated group, melatonin-treated shamoperated group, melatonin-treated asphyxial ca/cpr-operated group, melatonin plus (+) 4p-pdot (the mt2 melatonin receptor antagonist)-treated sham-operated group and melatonin+4p-pdot-treated asphyxial ca/cpr-operated group. results: our results demonstrate that melatonin (20 mg/kg, ip, 1 time before ca and 4 times after ca) significantly improved the survival rates and neurological deficits compared with the vehicle-treated asphyxial ca/cpr rats (survival rates ≥ 40% vs 10%). we also demonstrate that melatonin exhibited the protective effect against asphyxial ca/cpr-induced purkinje cell death. the protective effect of melatonin in the purkinje cell death following asphyxial ca/cpr paralleled a dramatic reduction in superoxide anion radical (o2·-), intense enhancements of cuzn superoxide dismutase (sod1) and mnsod (sod2) expressions, as well as a remarkable attenuation of autophagic activation (lc3 and beclin-1), which is mt2 melatonin receptor-associated. furthermore, the protective effect of melatonin was notably reversed by treatment with 4p-pdot. conclusions: this study shows that melatonin conferred neuroprotection against asphyxial ca/cpr-induced cerebellar purkinje cell death by inhibiting autophagic activation by reducing expressions of ros, while increasing of antioxidative enzymes, and suggests that mt2 is involved in the neuroprotective effect of melatonin in cerebellar purkinje cell death induced by asphyxial ca/cpr. introduction: fucoidan is a sulfated polysaccharide derived from brown algae and possesses various beneficial activities, such as antiinflammatory and antioxidant properties. previous studies have shown that fucoidan displays protective effect against ischemiareperfusion injury in some organs. however, few studies have been reported regarding the protective effect of fucoidan against cerebral ischemic injury and its related mechanisms. methods: therefore, in this study, we examined the neuroprotective effect of fucoidan against cerebral ischemic injury, as well as underlying mechanisms using a gerbil model of transient global cerebral ischemia (tgci) which shows loss of pyramidal neurons in the hippocampal cornu ammonis 1 (ca1) area. fucoidan (25 and 50 mg/kg) was intraperitoneally administered once daily for 3 days before tgci. results: pretreatment with 50 mg/kg of fucoidan, not 25 mg/kg fucoidan, attenuated tgci-induced hyperactivity and protected ca1 pyramidal neurons from ischemic injury following tgci. in addition, pretreatment with 50 mg/kg of fucoidan inhibited activations of resident astrocytes and microglia in the ischemic ca1 area. furthermore, pretreatment with 50 mg/kg of fucoidan significantly reduced the increased 4-hydroxy-2-noneal and superoxide anion radical production in the ischemic ca1 area after tgci and significantly increased expressions of superoxide dismutase 1 (sod1) and sod2 in the ca1 pyramidal neurons compared with the vehicle-treated-group. we found that treatment with diethyldithiocarbamate (an inhibitor of sods) to the fucoidan-treated-group notably abolished the fucoidanmediated neuroprotection in the ischemic ca1 area following tgci. conclusions: these results indicate that fucoidan can effectively protect neurons from tgci-induced ischemic injury through attenuation of activated resident glial cells and reduction of oxidative stress following increasing sods. thus, we strongly suggest that fucoidan can be used as a useful preventive agent in cerebral ischemia. the effects of cold fluids for induction of therapeutic hypothermia on reaching target temperature and complications-a sub-study of the tth48 study a holm 1 , m skrifvars 2 , fs taccone ). there was no difference in early bleeding incidences (fig 1) . during late observation, ttm patients had fewer minor bleeding (55.2% vs. 100%) and more intracranial bleeding (23.1% vs. 0%; fig 2) . adjusted calculated risk ratio for major bleeding (including intracranial) for ttm was 0.24 (95%ci 0.10-0.54) at baseline and 1.45 (95%ci 1.18-1.77) over time. conclusions: bleeding complications were common. although the risk ratio for major bleeding increased over time in ttm patients, residual and unmeasured confounding in addition to selection and detection bias may limit the clinical relevance of this finding. methods: patients with neurological deficit > 10 by nhiss were included. the t°of the brain was recorded non-invasively using radiothermometer rtm-01-res (russia). we measured t°in 18 symmetric regions of left & right hemispheres, calculated the average t°of brain, fig. 1 (abstract p213) . temperature of patients given and not given pre-icu fluids (table 1) . conclusions: observed moderate brain t°heterogenecity in hp, marked increase brain t°heterogenecity in is & sharp decline of t°h eterogenecity in cci. supposedly, correcting the impairment of cerebral tb (increase or decrease t°) through physical (selective cerebral hypothermia, magnetic stimulation etc.) or pharmacological (sedation) can contribute to positive therapeutic results in is & cci. nonivasive radiothermometry of the brain can be an objective method of patients' condition evaluation & their rehabilitation potential. introduction: basilar artery stroke has a multitude of different presentations and may not be captured on plain computed tomography (ct). it can progress to severe disability, locked in syndrome and death [1] . with the advent of thrombolytic and endovascular therapies, prompt diagnosis can change the outcome. we present a case of basilar artery stroke, which was heralded by tongue spasticity and dysarthria, indicative of pseudobulbar palsy. methods: case reviewed with consent. a literature search was conducted using pubmed and medline. results: a 53-year-old presented with pulmonary oedema and hypertension. he was transferred to our intensive care unit for treatment of a suspected anaphylaxis. his marked lingual swelling was associated with dysarthria. glyceryl-trinitrate and labetalol infusions were started for hypertension. he developed left sided weakness and deteriorated over several days to the point that he could only move his right foot (table 1) . magnetic resonance imaging (mri) showed midbrain ischaemia and angiogram showed no flow in the basilar artery (fig 1,2) . conclusions: common presenting features of basilar artery occlusion include dysarthria, vertigo, vomiting, headache and motor defects; these may evolve gradually or be intermittent [1, 2] . presentation with pseudobulbar palsy is described in early literature [2] . delayed recognition of the stroke led to aggressive treatment of hypertension, potentially compromising perfusion to the penumbral area [2, 3] . this case highlights the need for a wide index of suspicion with posterior strokes. consent: informed consent to publish has been obtained from the patient prognosis is related to gcs < or = 8 on admission (p = 0.003) and to malignant cerebral edema (p = 0.004). conclusions: our study has shown some predictive factors closely related to mortality and morbidity in patients with acute ischemic stroke. gcs at admittance < or = 8 and onset of malignant cerebral edema lead to a worst prognosis at discharge from nicu. coherence analysis of cerebral oxygenation using multichannel functional near-infrared spectroscopy evaluates cerebral perfusion in hemodynamic stroke tj kim 1 table 1 ). in addition, severe stroke patients were more likely to have higher phase coherence in interval iii (p =0.078). conclusions: our results demonstrated that the higher phase coherence of oxyhb in myogenic signal, which was originated locally from smooth muscle cells in brain was related to impaired cerebral perfusion. this suggests that monitoring cerebral oxygenation using fnirs could be a useful noninvasive measuring tool for evaluating impaired cerebral autoregulation in stroke patients. is esmolol associated with worse outcome at the acute phase of ischemic stroke that receives thrombolysis? introduction: ischemic stroke patients experienced frequent early neurological deterioration (end) events. since ischemic stroke has also been shown as inflammatory disease, the neutrophil-tolymphocyte ratio (nlr) may associated with end events. however, the direct study regarding this association has not been addressed. poor grade sah, use of vasopressors, mechanical ventilation, intracranial pressure monitoring, external ventricular drainage, blood transfusions and renal replacement therapy were all more frequent among nonsurvivors (all p<0.001). mortality was also higher with initial lactate above 2 mmol/l, in those admitted to public hospitals and when admission to icu was delayed more than 24 hours after ictus. after adjusting for common predictors (age, gender and wfns) saps 3 non-neuro, sofa non-neuro, early vasopressor use and admission to a public hospital were independently associated with hospital mortality. moreover, the area under the curve for prediction of mortality with saps3, sofa and wfns was 0.86 ( figure 1 ). hospital, austria. the association of intensity and duration of intracranial hypertension episodes with 12-month glasgow outcome score (gos) was visualized using the methodology introduced by güiza et al. [1] . results: in both cohorts, it could be demonstrated that the combination of duration and intensity defined the tolerance to intracranial hypertension, and that a semi-exponential curve separated episodes associated with better outcomes from those associated with worse outcomes. the association with worse outcomes occurred at a lower pressure-time burden than what has been previously observed in patients with tbi. nevertheless, the percentage of monitoring time spent by every patient in the zone associated with poor gos was independently associated with worse 12-month neurological outcome, even after correcting for age and fisher score ( introduction: apnea test is an essential component in the clinical determination of brain death, but it may incur a significant risk of complications such as hypotension, hypoxia and even cardiac arrest [1] . we analyzed the risk factors associated with failed apnea test during brain death assessment in order to predict and avoid these adverse events. methods: medical records of apnea tests performed for brain-dead donor between january 2009 and january 2016 in our institution, were reviewed retrospectively. age, gender, etiology of brain death, use of catecholamine and results of arterial bleed gas analysis (abga), systolic/diastolic blood pressure (sbp/dbp), mean arterial pressure (map) and central venous pressure (cvp) prior to apnea test initiation were collected as variables. a-a gradient and pao2/fio2 were calculated for more precise assessment of the respiratory system. in total, 267 cases were divided into a group which was completed apnea test and the other which was failed the test. introduction: tunisia has already suffered recurrent outbreaks since 1997. 2018 outbreak started relatively earlier this year. we were interpellated by the frequency of neuroinvasive presentation of the disease. methods: we report a case series of 11 patients presented to icu with niwnd. results: we report 11 cases of niwnd with different severe presentations overlapping neurological manifestation including encephalitis (n=8/11), meningitis (n=10/11) and flaccid paralysis (n=8/11). almost all patients live in the locality of sousse. six patients presented a long course of isolated fever before developing neurological signs. cerebrospinal fluid was consistent with encephalitis within the 11 patients. cerebromedullar mri identified brain lesions (n=8/10), myelitis (n=1/10) and polyradiculoneuritis (n=1/10).three patients had electromyography for flaccid paralysis showed diffuse axonal polyneuropathy with motoneuron involvement. ten cases had a positive wnv igm antibody and nine had a positive wnv igg antibody in serum. urine polymerase chain reaction was positive for wnv in 8/10 patients. ten patients were mechanically ventilated. all patients were managed symptomatically. two received high doses of methylprednisolone for 3 days, one patient received polyclonal immunoglobulin intravenous and one patient had plasmapheresis. two patients died consecutive to brainstem lesions. two patients recovered significantly and discharged with no complications. five other patients evolved to persistent flaccid paralysis with a minimal consciousness state and weaning difficulties requiring tracheostomy. the last remaining patient is still evolving. conclusions: modification of the regional climatic conditions accounted probably for the early 2018 outbreak of niwnd. this initial case series displays the severity and the poor outcomes of niwnd with higher incidence compared to past epidemics. noninvasive estimation of intracranial pressure with transcranial doppler: a prospective multicenter validation study c robba 1 , c fig. 1 ], mean bias was -3.24 mmhg (limits of agreement are ± 2 sd 24.6 mmhg). 7.5% measures were outside the limit of agreement in the overall population. however, when icp was high, 43% of measures were out of the limit of agreement. the auc [ fig. 2 introduction: surgical treatment of aortic aneurysm needs extracorporeal circulation (ecc), aorta clamp and hypothermia, and it is often related to poor systemic perfusion and blood flow velocity. one of the main concerns of intensive care team is to prevent secondary neurological injury after long time without blood flow pulsatility, such as brain edema and seizure. the most common parameters for neuromonitoring would be intracranial pressure and eeg, however, for non-neurological patients this information is unusual and prevents optimal management. methods: we aimed to assess brain compliance and neurological condition of icu patients on immediate post-operative recovery of bentall-de bono procedure and/or other aortic aneurysm surgical treatment using a novel non-invasive intracranial pressure (icp) device. this device uses mechanical displacement sensor capturing extracranial continuous volumetric variation of the skull and this information proportionally reflects intracranial dynamic [1] . results: twenty patients were included in this study. ecc mean time was 111 minutes for 19 patients and only one did not need it. eleven presented altered icp curves with poor brain compliance (p2/p1 ratio > 1.0) assessed by icp curve morphology analysis. volemic optimization and neuroprotective measures were taken based on this icp information for acute case management. among these patients with altered icp curves, eight were discharged from icu with good clinical condition and glasgow coma scale of 15. overall mortality rate was six out of twenty (30%) and three of these had altered icp curves. conclusions: brain monitoring of cardiovascular post-operative patients is important to prevent secondary neurological complications and can be a helpful tool for neuroprotective acute management on icu. the technique supplies electrical current to muscle, combined with passive cycling. prior to a clinical trial, we first investigated the effects of one session of fes in healthy volunteers. methods: healthy male volunteers (n=15) were recruited. the participants had their postural sway assessed on a pressure sensitive board, and measurement of maximal inspiratory pressure (mip). ultrasounds were taken assessing thickness of the quadriceps and rectus abdominis. they performed 20 minutes of supine passive cycling, with fes supplying the lower limbs and abdomen. after a 1 minute rest, the tests were repeated. a further 14 participants performed just the initial baseline tests, to help assess muscular factors affecting balance and sway. results: the current needed for palpable contraction was significantly correlated to weight in the abdomen (r=0.79, p<0.001) and quadriceps (r=0.82, p<0.001). current required to stimulate the abdominal muscles was also correlated to depth of the subcutaneous fat layer (r=0.85, p<0.001) and echogenicity of the muscle (r=0.65, p=0.012). pre-cycling, left and right vastus lateralis thickness inversely correlated to postural sway in the antero-posterior (r=-0.638, p<0.001) plane. compared to pre-cycling, postural sway in the antero-posterior and lateral planes increased significantly after cycling. there was a significant decrease in mip after cycling and greater reductions in mip were found in participants who had thinner rectus abdomni. conclusions: sway at baseline is related to quadriceps thickness, which atrophies during critical illness, and could worsen balance. mip is reduced during fes and the severity of reduction is related to the thickness of the abdominal wall muscles at baseline, suggesting that fes can fatigue the diaphragm and abdominal muscles. in awake healthy volunteers, fes is a safe, comfortable technique. introduction: in most cases postoperative cognitive dysfunction (pocd) is transient, but still some patients suffer from persistent cognitive impairment which is associated with increased length of hospital stay, early withdrawal from labor market and higher mortality. available data on the prevalence of pocd after cardiac surgery is very diverse from 20% to 90% upon discharge and up 20% 3 months after surgery. we aimed to investigate the prevalence of short-term and long-term pocd after off-pump coronary artery bypass grafting (cabg) surgery. methods: psychometric testing was performed in 230 (mean age 63.5±8.2) patients before, 10 days and 6 months after the surgery. we used following tests to assess cognitive capacity: auditory verbal learning test (avlt), digit span test (dst), digit-letter substitution test (dlst), stroop's test and trail making test (tmt). a decline in comparison to preoperative test results for 20% or more in two or more tests was declared as pocd. results: the prevalence of pocd after 10 days was 31.7% (73 patients) and 9.1% (21 patients) after 6 months. when comparing patients who developed pocd with those who did not we found the former were older (69.2±8.7 vs 61.1±10.3 years; p<0.001), had lower education level (13.7±2.1 vs 10.6± 2.4 years; p<0.05) and had longer surgery duration (250.4±12.2 vs 232.1±15.9 minutes; p<0.05). the most affected cognitive domains were long term memory (avlt) and executive function (tmt) and least affectedworking memory (dst) and selective attention (stroop's test). conclusions: in our prospective study the prevalence of long-term pocd after cardiac surgery was slightly less (9.1%) in comparison to available data (from 9% to 20%). it might be due differences in psychometric testing and interpretation of its results among authors. advanced age, low cognitive reserve and long duration surgeries are linked with higher incidences of pocd. introduction: postoperative cognitive dysfunction (pocd) is a common and widely described phenomenon in surgical patients. advanced age, major surgery, certain general anesthetics, genetic factors, sleep deprivation and other factors were described as contributing factors to pocd. the hospital stay itself is a major 'social' trauma for patients; social isolation, sleep deprivation and changes in daily regimen may effect neurocognitive behavior of patients. in this trial we tried to assess the link between pocd and the length of hospital stay in cardiac surgery patients. methods: 94 patients who underwent 'off-pump' coronary artery bypass grafting (cabg) surgery selected for this trial. neuropsychological testing was performed prior to the operation and upon discharge. we used auditory verbal learning test (avlt), digit span test (dst), digit-letter substitution test (dlst), stroop test and trail making test (tmt). a 20% or more decline in two or more tests in comparison to preoperative test results was declared as pocd. patients were allocated into two groups according to the length of hospital stay: the short-stay group (group 1) included patients (n=36) who were discharged on the 8th day after surgery or earlier and the long-stay (group 2) group consisted of patients (n=58) who were discharged on the 9th day after surgery or later. patients received similar anesthesia, postoperative care and were operated by the same surgical team. reasons for prolonged duration of hospital stay were mainly surgical. results: 11 patients (30.6%) in group 1 and 21 patients (36.2%) in group 2 had pocd upon discharge (p<0.05). mean length of hospital stay were 7±1.2 and 10±1.4 days in group 1 and group 2 patients respectively (p<0.05). conclusions: prolonged length of hospital stay increased the prevalence of pocd in our trial. studies with various types of surgical procedures and larger patient populations needed to further understand the effect of length of hospital stay to pocd. the influence of multiple trauma with head trauma on posttraumatic meningitis: a nation-wide study with hospital-based trauma registry in japan introduction: posttraumatic meningitis is one of severe complications and results in increased mortality and longer hospital stay among head trauma patients. however, it remains unclear whether there is a difference in the incidence of post-traumatic meningitis due to single traumatic brain injury (tbi) and multiple trauma including head injury. methods: this study was a retrospective observational study during 12 years we included trauma patients registered in japanese trauma data bank whose head ais score was >3 in this study. multivariable logistic regression analysis was used to assess potential factors associated with posttraumatic meningitis such as csf fistula, skull base fracture, type of injury that divided into single tbi and multiple trauma. introduction: the aim of this study was to determine if regional cerebral oxygenation (rsco2) can be used as an indicator of tissue perfusion in icu patients with tbi [1, 2] , and to determine the prognostic value of cerebral oxygenation rsco2 in survival prediction. methods: patients were enrolled retrospectively from january 2012 through july 2018 in the icu of derince kocaeli training hospital. 250 patients with trauma patients and traumatic braine injury patients who were admitted to the icu from the emergency room were included in the study. the sedation levels of the patients were followed up with bis. the rsco2, bis was taken as well as blood lactate level, mean arterial blood pressure and cardiac output at baseline time, 6, 12, 24, 48 and 72 hours. results: no significant difference was also detected between the value of rsco2 in all patients . it was average sco2 (right) 60.54 ±4.8 and average rsco2 (left) 55.63± 5.4. conclusions: cerebral regional oxygen saturation might be helpful as one of the perfusion parameters in patients with tbi but it could have no prognostic value in mortality prediction. however, further studies with larger sample size are still needed to validate these results. introduction: tbi in elderly is an increasingly cause of admission in icu. data regarding management and prognosis of these patients are lacking. validated prognostic models refer to younger patients and do not adequately consider the influence of pre-injury functional status, which often compromises with aging. frailty has been defined as a state age-related of increased vulnerability and decline in autonomy of daily life activity. aim of the study is to evaluate the impact of frailty on outcome in tbi elderly patients. methods: moderate and severe tbi patients >65years, admitted in neuroicu from january 2017 to may 2018, were prospectively enrolled. data of age, comorbidity, glasgow coma scale (gcs), pupils' reactivity, ct scan characteristics, neurosurgical intervention and gose (extended glasgow outcome scale) at 6-months were collected. frailty status was measured by clinical frailty scale (cfs) [1] and patients were divided as frail (cfs>4) and not frail (cfs<4). bad outcome was defined as gose<4. results: 22(37%) of the 60 studied patients were frail. frailty was not related to age. frail patients had more comorbidities and worse pupils' reactivity at admission (table 1) . other variables did not differ between groups. in univariate analysis neurological diseases, gcs, tsah (traumatic subarachnoid haemorrhage), compressed/absent basal cisterns, non-reactive pupils and cfs were significantly associated to bad outcome. in multivariate analysis only gcs and cfs remained associated to bad outcome ( table 2) . conclusions: pre-injury frailty is strongly associated to outcome in tbi elderly patients. the age of the patients was 48.4±15.6 years. patients were operated on for intracranial traumatic (39 cases) and non-traumatic hematomas (5), brain tumors (4) and the need for plastic of postoperative skull defects (6). general endotracheal total intravenous anesthesia with fentanyl, propofol, rocuronium, or tracrium was used. after tracheal intubation, 4-12 nerves were blocked (e.g., supraorbital, supratrochlear, zygomaticotemporal, auriculotemporal, great auricular, greater and lesser occipital nerves), depending on the surgical site. 0.75-1.0% ropivacaine was used. for blockade of one nerve used 0.5-2.0 ml of local anesthetic. fentanyl was applied on section of a periosteum, dura matter and at inefficiency of blockade of nerves. anesthesiology monitoring included hr, ecg, spo2, nib, respiratory parameters, eeg (csi), body temperature, blood glucose and lactate levels. in 2 and 10-12 hours post-surgery, the intensity of pain was ranked by alert patients using vas. results: the volume of local anesthetic for blockade in one patient was 7.7± 1.7 ml. in 5 (9.3%) from 54 patients, an additional fentanyl injection was required to skin incision due to an increase in blood pressure and heart rate by 25% of the baseline values, and an increase in csi until 80 un. patients available to productive contact in 2 hours post-surgery ranked the pain by vas at 1 (0;2) point, and in 10-12 hours post-surgery ranked it at 2 (1;3) p. conclusions: at patients with craniotomies scalpe-block with lowvolumes of a ropivacaine showed high efficiency (90.7%). were transferred to hospital ward or 53 (13.1%) to the center of intensive nursing care; 2 (0.5%) went to the surgical recovery room. acute renal failure, hypernatremia and hyperphosphatemia were independent predictors of mortality as described in table 2 . conclusions: hypernatremia and hyperphosphatemia were independent predictors of mortality in critically ill patients. introduction: the strong ion difference (sid) is essential for the assessment of acid-base equilibrium, thus requiring an accurate measurement of plasma electrolytes. currently there is no gold standard for electrolyte measurements and sid computation. differences in electrolyte values obtained with point-of-care (poc) and central laboratory (lab) analyzers have been reported [1, 2] . in previous studies [3, 4] we have shown that changes in pco2 induce electrolyte shifts from red blood cells to plasma (and vice versa), yielding variations in sid. aim of the present in-vitro study was to induce sid changes through acute changes in pco2 and compare values of electrolytes and sid obtained with poc and lab techniques. methods: blood samples from 10 healthy volunteers were tonometered (equilibrator, rna medical) with 3 gas mixtures at fractions of co2 (fco2) of 2, 12, and 20%. electrolytes were measured quasisimultaneously with a poc analyzer (abl800 flex, radiometer) and a routine lab method (cobas 8000 ise, roche). for both techniques a simplified sid was computed as sodium + potassiumchloride. results: bland-altman analysis of sid calculated with poc and lab showed a proportional bias (slope = 0.64, r2 = 0.55, p <0.0001), indicating a variable agreement between methods according to the average sid value (fig.1) . sid values measured with poc and lab at different fco 2 differed significantly (p<0.001, fig.2) . a similar discrepancy was observed for chloride (p <0.001, fig.2 ), while sodium (p=0.439) and potassium (p=0.086) were similar. conclusions: sid measured with poc and lab differed significantly, mainly due to a variable discrepancy in chloride. our findings suggest that our poc analyzer is superior to the lab in measuring electrolytes and thus compute sid. introduction: this study evaluated the safety of half dose insulin (hdi) versus standard dose insulin (sdi) for the treatment of hyperkalemia in a medical intensive care unit (micu) population with renal insufficiency. recent emergency medicine data demonstrated a lower incidence of hypoglycemia in patients with renal insufficiency when hdi was used for the treatment of hyperkalemia [1] . there is limited data describing the safety of hdi in a micu population with renal insufficiency. methods: this was a retrospective, chart review of patients admitted to the micu with a diagnosis of aki and/or ckd stage 3-5 with a serum potassium ≥ 5.8 meq/l from january 2013 to september 2018. sdi is defined as 10 units of regular iv insulin and hdi as 5 units. the primary outcome was the incidence of hypoglycemia within 12 hours of insulin administration. secondary outcomes included severe hypoglycemia and change of serum potassium after insulin administration. results: a total of 265 patients were screened and 98 were included for analysis. the incidence of hypoglycemia occurred in 6/ 45 patients (13.3%) and 6/53 patients (11.3%) who received sdi and hdi, respectively. one patient in the sdi group and two patients in the hdi group developed severe hypoglycemia. the mean decrease in serum potassium after insulin administration was 1.0 meq/l in both groups. patients in the hdi group who were re-dosed with 5 units of regular insulin did not have any hypoglycemic events. conclusions: in a micu population with renal insufficiency, sdi and hdi regimens appear safe and effective for the treatment of hyperkalemia. introduction: sepsis and septic shock are common causes of admission in the intensive care unit with a high mortality rate [1, 2] . hence, electrolyte disturbances are common in this group of patients. acute hypernatremia is one of the multiple features of homeostasis disturbances and available data in the literature suggest that its incidence can reach 47% [3, 4] . (fig 1, 2) . the main source of sepsis was pneumonia with 37 affected patients (41.57%). conclusions: hypernatremia is significantly associated with higher mortality in septic patients. 2 (abstract p246) . the outcome versus the sodium levels higher in the group 2 -80% vs 39.1% (p=0.031). there were no significant differences between the groups in length of stay in the icu. in group 1, there was an increase of serum phosphorus level and in the group 2the tendency to decrease. however, statistically significant differences were obtained only on the 2nd day after surgery 1.74±0.84 mmol/l (group 1) vs 0.88±0.26 mmol/l (group 2) (p=0.01). the roc curve was constructed to assess the predictive significance of serum phosphorus levels (fig. 1) . auc was 0.687; 95% ci 0.574-0.799; p=0.002; sensitivity 55.2%, specificity 83.9%. the kaplan-meier survival analysis (fig. 2) introduction: the rate of extubation failure might be higher in obese patients than in non-obese patients. effect of obesity on mortality is controversial [1, 2] (obesity paradox). several pathophysiological changes contribute to an increase of respiratory complications [1] . we sought to identify incidence of extubation failure in obese and non-obese patients. methods: the primary endpoint of this post-hoc analysis of a prospective, observational, multicenter study [3] performed in 26 intensive care units was extubation failure, defined as the need for reintubation within 48 hours following extubation. only patients with body mass index (bmi) recorded were included. results: between december 1, 2013 and may 1, 2015, among the 1427 patients with bmi available undergoing extubation, 301 obese patients (21%) and 1126 non-obese patients (79%) were enrolled. extubation-failure rate was 7.6% (23/301) in obese patients, and 11.0% (124/1126) in non-obese patients (p=0.09). delay of reintubation did not differ between obese and nonobese patients (figure 1 ). length of intubation > 8 days was significantly more frequent in obese patients (84/301, 29%) than in non-obese patients (195/1126, 18%, p<0.0001). precautions to anticipate extubation failure were more often taken in obese patients (153/301, 51%) than in non-obese patients (426/700, 38%, p<0.0001). spontaneous breathing trial (sbt) characteristics differed between obese and non-obese patients (table 1) . physiotherapy was more often used in obese patients (173/301, 57%) than in non-obese patients (556/1126, 49%, p=0.01). conclusions: incidence of extubation failure did not differ between obese and non-obese patients. in obese patients, clinicians anticipate more a possible extubation failure, delaying the moment of extubation, performing more physiotherapy and providing an optimal sbt. introduction: in the acute phase of critical illness, growth hormone (gh) resistance develops, reflected by increased gh and decreased insulin-like growth factor-i (igf-i), mimicking fasting in health. the epanic rct observed fewer complications such as muscle weakness and faster recovery with accepting a macronutrient deficit in the first icu week, as compared with early full feeding [1, 2] . we characterized its impact on the gh axis in relation to the risk of acquiring muscle weakness. methods: in this epanic rct sub-analysis, for 564 matched patients per group, and all patients assessed for muscle weakness (n=600), serum gh, igf-i, igf binding protein 3 (igfbp3) and igfbp1 were measured upon icu admission and at day 4 or the last icu day for patients with shorter icu stay (d4/ld). for 10 matched patients per group, gh was quantified every 10 min between 9 pm and 6 am, and deconvolved to estimate gh secretion. groups were compared with wilcoxon test or repeated-measures anova. associations between changes from baseline to d4/ld and muscle weakness were assessed with logistic regression analysis, adjusted for baseline risk factors, baseline hormone concentrations and randomization. results: in the fully fed group gh, igf-i and igfbp3 increased, whereas igfbp1 decreased from admission to d4/ld (all p<0.001). accepting an early macronutrient deficit prevented the rise in gh and igf-i and the decrease in igfbp1 (all p<0.005) but did not affect igfbp3, whereas basal, but not pulsatile, gh secretion was lowered (p=0.005). a stronger rise in gh and igf-i was independently associated with a lower risk of acquiring muscle weakness (or (95%ci) per ng/ml change 0.88 (0.81-0.96) for gh; 0.98 (0.97-0.99) for igf-i). conclusions: accepting an early macronutrient deficit suppressed basal gh secretion and reduced igf-i bioavailability during critical illness, which may counteract its protection against muscle weakness. introduction: aim of the study was to relate hypokalemia (hypok) and hypoglycemia as diabetic ketoacidosis (dka) treatment complications and precocious insulin interruption also use of sodium bicarbonate with length of stay (los) in intensive care unit (icu). methods: analysis of retrospective cohort study data of 100 patient (pt) treated for dka at icu of hospital kaunas clinics of lithuanian university of health sciences during 2012-2018 has been carried out. serum kalemia, glycaemia; rate of episodes of hypok, hypoglycaemia and precocious insulin interruption; use of sodium bicarbonate, in relation with los in icu were analysed. spss 23.0 was used for statistic calculations. traits evaluated as significant at p<0.05. results: at the beginning of dka treatment hypok (3.1±0.3 mmol/l) was recorded in 53/100 (53%) pt. due to disregarding of blood ph (6.8 -7.3 (7.1 ± 0.1) kalemia was falsely misinterpreted as "normo-" or "hyperkalemia" 3.5 -6.1 (4.8 ± 0.8mmol/l) in 49 of 63 (78%) pt, as normo-and hyperkalemia thus not treated and complicated by hypok additionally in 20/49 (41%) pt. in hypok los in icu was 52.2 ± 31.3 vs 31.7 ± 18.2 h, p<0.05. insulin use has caused hypoglycaemia (1.2 -3.3 (2.5 ± 0.7 mmol/l)) in 20/100 (20%) pt, los in icu 62.1 ± 40.3 vs 37.7 ± 21.4 h, p<0.05. insulin use was interrupted in case of normo -and hypoglycaemia with still persisting ketoacidosis in 34/ 100 (34%) pt, los in icu was found to be 56.2 ± 32.1 vs 35.5 ± 22.5 h, p<0.05. sodium bicarbonate was given for symptomatic treatment of acidosis during the first 10 h of dka in 21/100 (21%) pt with stable hemodynamic: hco3buffer has increased (4.2 ± 3.1-7.5 ± 3.0 mmol/l), p<0.05, but ketoacidosis has still persisted, los in icu was 54.8 ± 30.0 vs 39.3 ± 26.5 h, p<0.05. conclusions: hypok (53%), hypoglycemia (21%), precocious interruption of insulin use (34%) have prolonged los in icu almost twice. symptomatic treatment of ketoacidosis with sodium bicarbonate (1/5 pt) didn't control it and has prolonged los in icu. introduction: cystathionine-γ -lyase (cse), a regulator of glucocorticoid (gc)-induced gluconeogenesis [1] , correlates with endogenous glucose production in septic shock [2] . the hyperglycemic stress response to noradrenaline (noa) is mediated by the kidney [3] and less pronounced with low cse [4] . gc receptor (gr)-mediated gene expression is differentially regulated: the gr monomer is considered to repress inflammation, and gc side effects are attributed to the gr dimer; recent reports challenge this view [5] . gc-induced gluconeogenic gene expression is reduced in gr dimerization deficient (grdim) mice [6] . the aim of this study is to investigate renal cse expression and systemic metabolism in grdim and grwt mice in a resuscitated model of lps-induced endotoxic shock. methods: anesthetized grdim (n=10) and grwt (n=9) mice were surgically instrumented, monitored, resuscitated and challenged with lps. noa was administered to maintain map and 13 c 6 glucose was continuously infused. 6h after lps, cse expression was determined via immunohistochemistry of formalin-fixed paraffin sections (n=8 p.gr.). results: grdim required 2.5-fold more noa than grwt and had 1.5fold higher glucose and 1.9-fold higher lactate 6h after lps. this was concomitant with elevated endogenous glucose production (2-fold), 10% lower glucose oxidation and 1.8-fold higher renal cse expression in grdim. conclusions: increased cse expression together with higher glucose production (confirming [2, 4] ) and glucose levels in grdim mice suggest an association that may link cse to gc signaling. the higher noa administration in grdim mice could contribute to these effects. introduction: to achieve safe glycemic control in critically ill patients frequent blood glucose (bg) measurements and according titration of insulin infusion rates are required. automated systems can help to reduce increased workload associated with diabetes management. this bi-centric pilot study combined for the first time an intraarterial glucose sensor with a decision support system for insulin dosing (sgcplus system) in critically ill patients with hyperglycemia. methods: twenty-two patients (3 females, 19 males, 12 with preexisting diabetes mellitus, age 65.7 ± 12.7 years, bmi 27.3 ± 3.5 kg/ m2, creatinine level 1.4 ± 1.2 mg/dl, saps (simplified acute physiology score) 64.6 ± 18.3, tiss-28 (therapeutic intervention scoring system) 39.2 ± 5.5 who were equipped with an arterial line and required iv insulin therapy were managed by the sgcplus system during their medical treatment at the intensive care unit. results: 1845 sgcplus-based bg determinations were performed and 0.8 ± 0.8 sensor calibrations per day were required. sensor glucose readings correlated well with reference bg (figure 1 ). mean treatment duration was 6.0 ± 3.8 days. time to target was 100 ± 178 min (80-150 mg/dl) and 135 ± 267 min (100-160 mg/dl). mean blood glucose was 142 ± 32 mg/dl with seven blood glucose values <70mg/dl. mean daily insulin dose was 62 ± 38 u and mean daily carbohydrate intake 148 ± 50 g /day (enteral nutrition) and 114 ± 50 g/day (parenteral nutrition). acceptance of sgcplus suggestions was high (>90%). the novel intraarterial glucose sensor demonstrated to be highly accurate. the sgcplus system can be safely applied in critically ill patients with hyperglycemia and enables good glycemic control. introduction: we aimed to assess the effect of frailty as assessed by clinical frailty scale (cfs) and karnofsky performance score (kps) on critical care (cc) and hospital mortality in this group at a nonspecialist tertiary critical care unit. methods: patients admitted to critical care were identified from our electronic database by screening for liver disease or cirrhosis in the admission diagnoses. those with an aetiology of liver disease other than alcoholic liver disease (ald) were excluded. data was collected on patient demographics, length of stay, status at discharge from critical care and hospital and cfs. kps was also calculated where sufficient in-formation was available in the medical record. data was analysed using logistic regression multivariate analysis with stata 14 software. [1] . results: tg13 diagnosis criteria and severity grading criteria for acute cholangitis and acute cholecystitis were judged from numerous validation studies as useful indicators in clinical practice and adopted as tg18 diagnostic criteria and severity grading without any modification. provide initial treatment, such as sufficient fluid replacement, electrolyte compensation, and intravenous administration of analgesics and full-dose antimicrobial agents, as soon as a diagnosis has been made. in new flowchart for the treatment of acute cholecystitis (ac) in the tg18, grade iii ac was indicated for gallbladder drainage, but some grade iii ac can be treated by laparoscopic cholecystectomy (lap-c) at advanced centers with specialized surgeons experienced in this procedure and for patients that satisfy certain strict criteria. we also redefine the management bundles for acute cholangitis and cholecystitis. introduction: 13 c-acetate breath tests provide a non-invasive assessment of gastric emptying [1] and could, hence, be used to judge tolerance to enteral nutrition. result values like t50 (time for 50% absorption) correlate with scintigraphic measurements. the data evaluation is based on model equations like the β -exponential function (bex) [1] . it considers a mono-phasic breath gas response. this may not be the case during critical illness, which could reduce precision too low for a reliable personalized assessment [2] . methods: we recently developed an evaluation of irregular gastric emptying patterns, which separates absorption from post-absorptive distribution and retention of tracer and from the terminal respiratory release of the oxidized tracer [3] . using breath test data of 31 icu patients (mean saps2 36 +/-14) the precision of this approach was compared with a bex analysis to explore how often an extended analysis is warranted and whether it improves the reliability of estimates. results: 15 patients had a release profile consisting of series of peaks with a periodicity of 60-90 min. a first dominant peak carries about 95% of the released moiety, as reported [4] for controls. for these patients the precision in t50 for the bex approach was 70 +/-30% of that observed for the new approach. for the other patients, the secondary peaks had a similar periodicity but were more pronounced, indicating persisting peristaltis, which has been linked to tolerance to enteral nutrition [4] . the bex approach achieved a precision of 40 +/-27 % relative to the new one, challenging its applicability for these patients. introduction: clinical scoring systems used to prognosticate the severity of acute pancreatitis (ap), such as apache ii, are cumbersome and usually require 24 hours or more after presentation to become accurate, at which time the window for early therapeutic intervention has likely passed. sirs at presentation is sensitive but poorly specific for severe ap. we postulated that sirs and accompanying hypoxemia would specify at presentation patients with ap who have severe inflammation and are at risk for clinically severe disease. methods: patients with ap who had sirs and hypoxemia at presentation were enrolled in an open-label study evaluating the safety and efficacy of cm4620-ie, a calcium release-activated calcium (crac) channel inhibitor (nct03401190). hypoxemia was defined as an estimated pao2 <75 mm hg calculated using a log-linear equation and the spo2 on room air at the time of presentation. a contrastenhanced computed tomography (cect) was performed at presentation and a cbc with differential, d-dimer and crp were analyzed daily. the cect was read by a blinded central reader who assessed the degree of inflammation using the balthazar scoring system (table 1) . results: 13 patients, seven men and six women, have been randomized in the study. the mean estimated pao2 at presentation was 74 mm hg. 10 patients had 2 sirs criteria present and the other 3 patients had 3 sirs criteria present. the median value for age was 53.5 (iqr 48-56), initial neutrophil-lymphocyte ratio (nlr) 10.6 (6. 3 introduction: to investigate whether circulating immune profiles were able to serve as early biomarkers in predicting persistent organ failure (pof methods: thirty-nine patients with predicted severe acute pancreatitis (psap) and 9 healthy control subjects were prospectively enrolled in our study. we measured the expression of monocytic human leukocyte antigen-dr (mhla-dr), the proportions of dendritic cells (dc) and its subtypes (including myeloid dendritic cell (mdc) and plasmacytoid dendritic cell (pdc)), the different cytokineproducing cd4+ t helper (th) cells and regular t (treg) cells. plasma crp and several inflammatory mediators levels were measured by elisa. results: compared with healthy controls, there is a significant decrease in the expression of mhla-dr, the frequencies of total circulating dcs and its subsets, and percentage of th1 cells in patients with psap. however, we found significantly higher frequencies of th17 cells, higher proportion of treg cells than healthy subjects. of interest, we observed that there was a significant decrease in the positive percentage and mean fluorescence intensity (mfi) of mhla-dr, the proportions of total dcs and pdc, and th1 cells in patients with pof compared with transient organ failure (tof). besides, there is a significantly higher frequency of th17 cells in pof than those in tof. area under the receiver-operating characteristic curve analysis showed that disease severity scores had a moderate discriminative power for predicting pof in patients with psap. more importantly, the expression of mhla-dr and the percentage of dcs and pdc had a significantly higher auroc and thus, better predictive ability than disease severity in patients with psap. conclusions: circulating immune profile show multiple aberrations in patients with psap who have developed pof. both the expression of mhla-dr and the percentage of total dc and pdc may be early good biomarkers for predicting risk of pof in patients with psap. introduction: pancreatic fistula (popf) due to anastomosis insufficiency is a common (12-30%) complication after pancreaticoduodenectomy and often discovered with delay, causing severe morbidity, icu stay and deaths. microdialysis (md) catheters have been shown to detect inflammation and ischemia in several postoperative conditions and organs. the aim was to investigate if md catheter monitoring could facilitate earlier detection of popf than current standard of care. methods: in a prospective, observational study 35 patients (44 to 88 years) were investigated. a md catheter was fixed to the pancreaticojejunal anastomosis. samples for analysis of glucose, lactate, pyruvate and glycerol were acquired hourly during the first 24 hours, then every 2-4 hours to discharge. popf was defined according to the international study group of pancreatic fistula 2016 update definition. results: patients who developed popf (n=7) had significantly higher glycerol levels (p<0.01) in microdialysate than did patients without popf (n=28) during the first 24 h. thereafter, the difference diminished. a glycerol concentration >400 μmol/l during the first 12 h detected patients who later developed popf with a sensitivity of 100 % and a specificity of 92%. lactate and lactate to pyruvate ratio were significantly higher (p<0.05) and glucose was significantly lower (p<0.05) in patients with popf from about 24 h. fig. 1 shows microdialysis measurements in patients with (red lines) and without (blue lines) popf. conclusions: a high level of glycerol in microdialysate is an early (first 12 hours) indicator of popf. glucose, lactate and lactate to pyruvate ratio are indicators of peritonitis caused by the leakage. thus, md monitoring detects popf several days earlier than current methods and may play an important clinical tool in the future. we are currently conducting a rct to explore if md monitoring will improve prognosis in these patients the phenomenon of total impaired of metabolic activity of gut microbiota in critically ill septic patients introduction: during a critical condition, dramatic disturbances occur not only in the change of species diversity, but in gut microbiota metabolism as well, that might lead to nonreversible breakdowns of host homeostasis and death [1] . metabolic activity of microbes can be assessed by the measurement of the levels of aromatic microbial metabolite (amm) in blood serum, which are associated with the severity and mortality of icu patients. critically ill patients are characterized by the totally different sfs profile than in healthy people, particularly by the absence of phpa; but dominated by p-hphaa and p-hphla [2] . the purpose of our study is to assess the gut metabolic activity via amm in sepsis. methods: in this study 40 simultaneously serum and fecal samples (sfs) were taken from icu patients: 14 -with sepsis, 21-chronic critical ill (cci) patients and control -5 sfs from healthy people. after liquid-liquid extraction from serum and fecal samples, 9 phenylcarboxylic acids (amm) were measured using gc/ms (thermo scientific). results: the sum of the level of 9 most relevant amm in serum samples were higher in patients with sepsis (median -4.7 μm) than in cci patients (1.1 μm) and healthy people (1.3 μm). at the same time the opposite pattern was observed in the fecal samples -2.4, 31.9 and 68.7 μm, respectively. the ratios of sums amm gut/serum were higher in healthy people than icu patients (fig. 1) introduction: the aim of this study is to describe the characteristic of bioelectric impedance vector analysis (biva) and muscular ultrasound during the first week after admission in the icu, and their correlation with indices of metabolic support. biva is a commonly used approach for body composition measurements [1] . muscular ultrasound represents a valid tool to provide qualitative and quantitative details about muscle disease [2] . methods: consecutive patients admitted to icu and expected to require mechanical ventilation for at least 72 hours were enrolled in the study. within the first 24 hours of icu admission (t1), patients were evaluated with muscular ultrasonography comprehensive of diaphragm thickness (dth) and rectus femoris cross-sectional area (csa). at the same time, biva and biochemical analysis. all the same measures were repeated at day 3 (t3) and 7 (t7) (figure 1 (table 1) . dividing the patients in two groups based on prealbumine changes (t3 vs t1: increase, anabolic vs decrease, catabolic), those in which prealbumine increased had a higher reduction in muscle mass ( figure 2 ). conclusions: this study showed how the pa tends to be reduced in the first week of icu stay. it is correlated with a concomitant introduction: the modified nutrition risk in critically ill (mnutric) has been developed in order to identify critically ill patients who may receive benefit from nutrition support [1] . several evidences showed the association between the mnutric score and clinical outcomes [2, 3] , however there are no data in thai critically ill patients. the purpose of this study was to find the association between mnu-tric score and 28-day mortality in medical intensive care unit (icu) patients, ramathibodi hospital. methods: we retrospectively reviewed the medical patient records from june 2016 to january 2017. a mnutric score of each patient was calculated to evaluate the risk of malnutrition. statistical analysis of the association between mnutric score and 28-day mortality, length of stay in icu and hospital were performed. results: a total of 78 critically ill patients were included in the study. the 28-day mortality was 53.06% in patients with high mnutric score (5-9) and 13.79% in patients with low mnutric score (0-4). modified nutric score was significantly correlated with 28 day mortality (r = 0.390, p<0.001), length of stay in icu (r = 0.394, p<0.001) and length of stay in hospital(r = 0.414, p<0.001). in the receiver operating characteristic (roc) curve analysis, the auc of mnutric score and 28-day mortality was 0.788 (95% confidence interval (ci), 0.686-0.889) (fig 1) . optimal cut-off value of 6 showed sensitivity of 66.7% and specificity of 77.1% in mortality prediction (youden's index, 0.438). additionally, patients who received adequate nutrition supplement within 7 days was 87.20% for calorie and 64.10% for protein. there was no association between nutrition support and 28-day mortality. conclusions: in thai medical intensive care population, the mnutric score was associated with 28-day mortality in critically ill patients. fig. 1 (abstract p264) . within the first 24 hours of icu admission (t1), patients will be evaluated with muscular ultrasonography comprehensive of diaphragm thickness and rectus femoris (medial vastus) cross-sectional area. at the same time, anthropometric measure will be collected (such as body height, ideal body weight, real body weight declared, right arm circumference) as well as biva measure (xc, r, pa, lean body weight and % of extracellular body weight) and biochemical analysis (inclusive albumin, pre-albumin, blood count, lymphocyte count, magnesium, phosphorus, reticulocytes, renal and hepatic function test). the day after, the fluid balance will be calculated as well as the nitrogen balance. all the same measures will be repeated at day 3 (t3) and 7 days (t7) introduction: ultrasonography is an essential imaging modality in critical care to diagnose and guide for therapeutic management of shock, multiple organ failure, etc. enteral tube feed intolerance occurs frequently in hospitalized patients and more so in critically ill patients. in present study, we consider that nursing staff may be able to use bedside ultrasound as an alternative to standard aspiration protocol or radiographic studies to assess gastric volume and nasogastric (ng) tube in patients with enteral feed intolerance. methods: in present prospective, single-center study, we performed ultrasound residual stomach volume and ng tube placement assessments of adult critically ill patients (figure 1 ) compared to standard protocol of stomach volume assessment (routine daily shift 60-ml syringe aspirations) and ng (nasogastric) tube placement verified by abdominal x ray. we used an abdominal (linear ultrasound transducer) probe (4-10 mhz). the residual volume was calculated according to formula: gv (ml) = 27 +14.6 x right-lateral csa-1.28 x age). results: hundred simultaneous double (ten critically ill patients) ultrasound measurements sessions were performed by nursing staff of our intensive care (icu) (fig 1) . double simultaneous measurements of the ultrasound assessments were compared to standard nurse icu protocol for assessment of residual volume of stomach. the new ultrasound assessment method demonstrated excellent intra-class reliability (icc-0.96 (0.95-0.98, p<0.001) and strong correlation with standard residual volume assessment method (icc-0.82 (0.7-0.89, p<0.001). ng tube placement was successfully verified by ultrasound measurements in all ten critically ill patients and, thereafter, confirmed by abdominal x-rays. conclusions: preliminary results of our study demonstrated good correlations between both methods of ng tube placement and residual stomach volume: standard icu nurse protocol and ultrasound assessment. evaluating the documentation of nasogastric tube insertion and adherence to safety checking l roberts 1 introduction: enteral feeding into a misplaced nasogastric (ng) tube is recognised by the national patient safety agency as a never event. ng tubes are commonly indicated in level 2/3 patients, thus we set out to evaluate current practice in critical care. the aim was to evaluate: documentation of insertion, adherence to safety guidance pertaining to checking safe use, chest x-ray interpretation. methods: this prospective cohort study was based on inpatients in critical care who had insertion of ng tubes over four weeks; there were 57 insertions. data was analysed from patients' medical notes and the hospital's imaging system. results: 65% of insertions were documented using proformas. 97.1% of proforma documentations included 4 or more details: type of tube, tube length at the nostril, nex measurement, aspirate adequacy, chest x-ray adequacy, whether it was safe to feed. only 13.6% of hand-written documentations included 4 or more details. 51% of initial aspirates were obtained on insertion, of these, 57% had an appropriate ph between 1 and 5.5. this led to 74% of patients having chest x-rays to confirm initial placement of the ng tube. only 54% of chest x-rays adequately satisfied the four criteria. written documentation in medical notes stating if it was safe to feed was completed in 67% of cases. conclusions: we found that proformas ensure a higher level of detail and uniformity in the documentation of ng tube insertions. there was a high incidence of chest x-rays performed to confirm correct placement of tubes due to difficulties in obtaining aspirates and failure to follow guidelines. a need for a uniform, ward-specific proforma on ng tube insertion has been identified, as well as a teaching session on chest x-ray interpretation and on techniques to aid obtaining aspirates. we have established critical care's shortcomings in ng tube insertion documentation and tube safety checking. introduction: pressure ulcers(pu) are considered as important types of public health problems, due to high mortality and cost. we aimed to investigate the efficiency of curcumin and fish oil on prevention and treatment of pu using a feasible mice model. methods: 28 mice were randomly divided into control(group 1), curcumin(group 2), fish oil(group 3), curcumin and fish oil(group 4) groups. 5mm skin bridge between two 1000 gauss magnets was formed on the back of mice, followed by 3 ischemia reperfusion cycles as 12 hours of rest after 12 hours of magnet placement [1] . a single dose of curcumin and fish oil was injected intraperitoneally. tissue samples had taken 10th day of first compression, rates of pu, inflammation, reepithelisation, neovascularisation and granulation were examined histopathologically. the data analyzed by pearson chi-square test. results: third degree pu were observed in all groups.there was no significant difference between groups in terms of inflammation.the formation of reepithelisation showed a significant difference between groups.partial reepithelisation ratios in group 3 and group 4 was elevated.there was significant difference between groups in terms of neovascularisation, the highest rate as 75% was observed in group 4.formation of granulation was observed at maximum rate as 46.2% at group 3. conclusions: depending on positive results of curcumin, fish oil, cur-cumin+fish oil on wound healing it may be advised to use them in treatment of acute pu.after similar rate of pu with control group we consider that it should be beneficial to evaluate the effect of these therapies with more studies by changing the mode of administration, time of initiation and duration of therapy. introduction: inflammation is a key driver of malnutrition during acute illness and has different metabolic effects including insulin resistance and reduction of appetite. whether inflammation influences the response to nutritional therapy in patients with disease-related malnutrition remains undefined. we examined whether the effect of nutritional support on the risk of mortality differs based on the inflammatory status of patients. methods: this is a secondary analysis of a multicentre trial in eight swiss hospitals, where patients with a nutritional risk score (nrs) of ≥3 upon hospital admission were randomly assigned to receive protocol-guided individualized nutritional support according to nutrition guidelines (intervention group) or a control group. the inflammatory status was defined based on admission crp levels as low inflammation (cpr <10 mg/dl), moderate inflammation (crp 10-100 mg/dl) and high inflammation (crp >100 mg/dl). results: we included a total of 1,950 patients of which 27.33%, 45.85% and 26.82% had low, moderate and high inflammation levels on admission. while overall there was a significant reduction in 30day mortality associated with nutritional support (adjusted or in the overall cohort 0.65, 95%ci 0.46 -0.91), the subgroup of patients with high inflammation did not show reduced mortality (adjusted or 1.36, 95%ci 0.76 -2.41, p for interaction = 0.005). there was no difference in other secondary endpoints when stratified based on inflammation. nutritional support did not affect crp levels over time (kinetics). conclusions: this secondary analysis of a multicentre randomized trial provides evidence, that the inflammatory status of patients influences their response to nutritional support. these findings may help to better individualize nutritional therapy based on patients initial presentation. introduction: low plasma glutamine levels have been associated with unfavourable outcomes in critically ill patients. this study aimed to measure plasma glutamine levels in critically ill patients and to correlate glutamine levels with biomarkers and severity of illness. methods: we enrolled critically ill patients admitted to three icus in south africa, excluding those receiving glutamine supplementation prior to admission. we collected clinical, biochemical and dietary data. plasma glutamine levels were determined within 24 hours of admission, using liquid chromatography mass spectrometry and categorized as low (<420 μmol/l), normal (420-700 μmol/l) and high (>700 μmol/l). results: of the 330 patients (average age 47.42±16.56 years, 56% male), 68% were mechanically ventilated, with a mean apache ii score of 18.57±8.55 and a mean sofa score of 7.05±3.78. plasma glutamine levels were low in 58.5% (median plasma glutamine of 382.15 μmol/l). baseline plasma glutamine correlated inversely with crp (r=-0.287, p<0.001) and serum urea (r=-0.124, p<0.026), and positively with serum bilirubin (r=0.262, p<0.001) and serum alt (r=0.119, p=0.032). significantly more patients with low admission glutamine levels required mechanical ventilation (chi2=12.65, p<0.001) and had higher apache scores (p=0.003), higher sofa scores (p=0.003), higher crp values (p<0.001), higher serum urea (p=0.008), higher serum creatinine (p=0.023), lower serum albumin (p<0.001) and lower bilirubin levels (p=0.054). using multiple logistic regression analysis, apache score (odds ratio, [or] 1.032, p=0.018), sofa score (or 1.077, p=0.016) and crp (or 1.006, p<0.001) were significant predictors of low plasma glutamine levels. roc curve analysis revealed a crp threshold value of 87.95mg/l to be indicative of low plasma glutamine levels (auc 0.7, p<0.001). conclusions: 58.5% of critically ill patients had low plasma glutamine levels on admission to icu. this was associated with increased disease severity and higher crp. introduction: the east of england deanery operational delivery network in the united kingdom came together as a group of intensive care units to comply an evidence-based care bundle. one of the branches of this care bundle is on parenteral nutrition and states: 'parenteral nutrition should not be given to adequately nourished, critically ill patients in the first seven days of an icu stay.' this is based on evidence [1] [2] [3] that showed that 'in patients who are adequately nourished prior to icu admission, parental nutrition initiated within the first seven days has been associated with harm, or at best no benefit, in terms of survival and length of stay in icu.´the objective of this second cycle was to assess whether or not we are adhering to the guidelines, last year we were failing to hit targets and after some action i reassessed how we performed in the year 2017 compared to 2016. methods: a retrospective audit of the whole year of 2017 for all patients admitted to icu who had parenteral nutrition started at any point during their stay. results: there is a significant improvement in the percentage of patients who are being started incorrectly on tpn before 7 days (36% compared to 69%) (fig 1, 2) . i also found a total reduction in the number of patients prescribed tpn, a reduction in the number of bags being used and a reduction in length of hospital stays. conclusions: as we have recently switched over to an electronic icu programme for all documentation and prescriptions, as part of our plan and act in the pdsa cycle we are organising for several things to be put in place on the new system on prescription: pharmacy authorisation, links to guidelines and alert/justification boxes. i will do a further cycle in another year. jg and mpc contributed equally. introduction: recent rcts revealed clinical benefit of early macronutrient restriction in critical illness, which may be explained by enhanced autophagy, an evolutionary conserved process for intracellular damage elimination [1] . however, in the absence of specific and safe autophagy-activating drugs, enhancing autophagy through prolonged starvation may produce harmful side effects. a fasting-mimicking diet (fmd) may activate autophagy while avoiding harm of prolonged starvation, which also improved biomarkers of age-related diseases in an experimental study [2] . we evaluated if short-term interruption of continuous feeding can induce a metabolic fasting response in prolonged critically ill patients. methods: in a randomized cross-over design, 70 prolonged critically ill patients receiving artificial feeding were randomized to be fasted for 12 hours, followed by 12 hours full enteral and/or parenteral feeding, or vice versa. patients were included at day 8 in icu and blood glucose was maintained in the normal range. at the start and after 12 and 24 hours, we quantified total bilirubin, urea, insulin-like growth factor-i (igf-i) and beta-hydroxybutyrate (boh) in arterial blood. insulin requirements were extracted from patient files. changes over time were analyzed by repeated-measures anova after square root transformation. results: as compared to 12 hours of full feeding, 12 hours of fasting decreased bilirubin (-0.23±0.06mg/dl; p=0.001) and igf-i (-13.94 ±1.62ng/ml; p<0.0001), and increased boh (+0.47±0.07mmol/l; p<0.0001), without affecting urea concentrations (fig 1) . fasting reduced insulin requirements (-1.16±0.14iu/hour; p<0.0001). conclusions: short-term fasting induces a metabolic fasting response in prolonged critically ill patients, which provides perspectives for the design of a fmd, aimed at activating autophagy and ultimately at improving outcome of critically ill patients. introduction: recent evidence has led to changed feeding guidelines for critically ill patients, with a shift towards lower feeding targets during the acute phase [1] . when micronutrients are not provided separately, prolonged hypocaloric feeding could induce micronutrient deficiencies and increase risk of refeeding syndrome once full feeding is restarted, which are both potentially lethal complications [2] . since there is limited evidence how to optimize micronutrient provision in order to avoid deficiencies, we hypothesized that there is a great variation in current practice. methods: within the men section of the european society of intensive care medicine (esicm), we designed a questionnaire to gain insight in the current practice of micronutrient administration. in 3 email blasts, invitations were sent to all esicm members, with currently more than 300 respondents. the survey will be closed at december 10, 2018. results: first, we will describe demographic characteristics of the respondents, including geographical location, icu and hospital type, and function. second, we will describe some aspects of the current practice of micronutrient administration. we will identify the proportion of respondents having a protocol, on which evidence such protocol is based and whether it takes into account the stability and daylight sensitivity of micronutrients. next, bearing refeeding syndrome in mind, we will identify whether there are respondents who never measure and/or separately administer micronutrients and phosphate. finally, we will make a top 5 of the most measured and most supplemented micronutrients. conclusions: this survey will deliver more insight in the current practice of micronutrient provision across different types of icus and may identify areas for future research. furthermore, we will evaluate whether there is need to increase awareness for refeeding syndrome. introduction: large gastric residual volumes (grvs) have been used as surrogate markers of delayed gastric motility to define enteral feeding intolerance (efi). recent studies have challenged the definition of efi. study objectives: 1) investigate the potential relationship between grvs and clinically outcomes, 2) develop an algorithm for early identification of patients at increased risk of mortality due to efi. methods: a retrospective study of inpatient encounters from electronic health record charts within the dascena clinical database. 4,018 patients were included in the study; 295 patients had efi. eight vital signs (diastolic/systolic bp, heart rate, temperature, respiratory rate, grv, glasgow coma scale, and feeding rate) and their trends were input to the classifier. machine learning classifiers were created using the xgboost gradient boosted tree method with 3-fold cross validation. results: rate of change in grv (δ grv) was measured over a 5-day period, beginning at the time of efi onset (figure 1a) . figure 1b shows a high likelihood of mortality for patients with none or modest grv reduction. patients with an increase in grv over the five-day period after efi onset had the highest mortality likelihood. a stratification algorithm was developed to identify efi patients who died inhospital despite grv reduction at 1, 12, and 24 hours in advance of efi onset. area under the receiver operating characteristic (auroc) curves demonstrated high sensitivity and specificity of algorithm predictions of in-hospital death up to 24 hours in advance of efi onset (table 1) . conclusions: the analysis suggests an association between grv and mortality, especially in patients with persistent grv increase over the 5-day period after efi onset and the potential of algorithmic models to predict efi development. prospective validation of these fig. 1 (abstract p274) . changes in metabolic markers of fasting over time for both randomization groups algorithms may assist in clinical trial design to develop treatments for patients at highest risk of experiencing serious outcomes due to efi. a quality improvement project to improve the daily calorific target delivery via the enteral route in critically ill patients in a mixed surgical and medical intensive care unit (icu) b johnston, d long, r wenstone royal liverpool and broadgreen university hospital trust, critical care, liverpool, united kingdom critical care 2019, 23(suppl 2):p277 introduction: 'iatrogenic underfeeding' is widespread with the calo-ries study reporting only 10%-30% of prescribed daily kcal was actually delivered to patients [1] . in the present project, quality improvement methodology was utilised with the aim of delivering greater calories by implementing 24-hour volume-based feeding and allowing increased feeding rates for, 'catch up' of missed daily feed volume. methods: baseline data assessing the percentage of daily kcal delivered to ventilated patients was collected in september 2017. data was presented and new intervention guidelines agreed based upon the pepup protocol [2] . nurse champions were identified and were responsible for cascade training of the pepup protocol. educational tools to help determine daily calorific requirement and volume of feed required were provided. repeat data was collected at 6 months (cycle 2) after pepup implementation. results: ten patients were included in cycle 1. during cycle 1 the percentage of kcal achieved via enteral feeding was 45%. following intervention this increased to 82% (p<0.001) during cycle 2. this increased further to 95.3% of daily kcal when calories obtained from propofol were included. conclusions: a 24-hour volume-based feeding regimen is a simple and cost-effective method of improving enteral feeding targets. through the use of quality improvement methodology, we demonstrated that this approach is achievable. the success of this project has led to the adoption of the protocol in other icu units in a regional critical care network. effect of non-nutritional calories on the calory/protein ratio in icu patients s jakob, j takala university hospital bern, dept of intensive care medicine, bern, switzerland critical care 2019, 23(suppl 2):p278 introduction: nutritional diets are composed to match the needs of critically ill patients. while effective calory needs can be measured or calculated, the needs of proteins are more controversial. we aimed to calculate non-nutritional calories and assess how they influence the ratio of calories to protein delivered to the patients. methods: in this retrospective analysis, nutritional and nonnutritional calories and protein delivery were calculated in 220 consecutive icu patients receiving enteral nutrition in 2016. introduction: marked protein catabolism is common in neurocritical patients. optimal nutritional monitoring and protein nutritional adequacy could be associated with outcome in neurointensive care unit (ncu) patients. we aimed to evaluate the impact of monitoring and optimal support of protein using nitrogen balance on outcome in neurocritical patients. methods: a consecutive 69 patients who were admitted to ncu were included between july 2017 and february 2018. nitrogen balance was calculated using excreted urine urea nitrogen during icu admission. follow-up nitrogen balance monitoring was performed in 33 patients. we divided patients into two groups based on the results of nitrogen balance (positive balance and negative balance). moreover, we evaluated improvement of nitrogen balance in 33 patients. we assessed the outcome as length of stay in hospital, length of stay in ncu, and in-hospital mortality. we compared the clinical characteristics and outcome according to nitrogen balance. results: among the included patients (age, 58.1; and male. 59.4%), 53 (76.8%) patients had negative nitrogen balance. the negative balance group was more likely to have lower glasgow coma scale (gcs), longer length of stay in hospital, and longer length of stay in ncu. in 33 patients with follow-up nitrogen balance monitoring, improvement of nitrogen balance group had lower in-hospital mortality (10.5% vs. 50.0%, p = 0.019), and received adequate protein intake (1.8 g/kg/day vs. 1.1 g/kg/day, p = 0.001) compared to no change group (table 1) . there was no significant difference in baseline nitrogen balance, baseline body mass index, and gcs between two groups. conclusions: this study demonstrated that critical illness patients in ncu are underfeeding using nitrogen balance, however, adequate provision of protein was associated improvement of nitrogen balance and outcome. this suggests that adequate nutrition monitoring and support could be an important factor for prognosis in neurocritical patients. increased protein delivery within a hypocaloric protocol may be associated with lower 30-day mortality in critically ill patients introduction: to test the hypothesis, using real world evidence that increasing protein delivery and decreasing carbohydrates (cho) may improve clinical outcomes. methods: retrospective analysis of existing electronic medical records (emr) of patients admitted to the intensive care units (icu) at the geisinger health system. logistic regression analysis was used to determine correlation between protein delivered (which was proportional to the concentration of protein in the formula utilized) and clinical outcomes. results: 2000 medical encounters for a total number of 12,321 icu days were collected and analyzed. average age was 62.2 years (55.2% male) and 68.1% were obese and overweight. primary diagnoses included sepsis or septic shock, acute and/or chronic respiratory failure (or illness), cardiovascular diseases, stroke and cerebrovascular diseases among others. median hospital los was 13.6 days, 6.9 days in the icu, median days of invasive mechanical ventilation of 4. 30-day readmission rate among patients discharged alive was 19.3%. patients in the high protein group received lower amounts of chos (data not shown). unadjusted 30-day post-discharge mortality was inversely proportional to the amount of protein delivered (table 1) . conclusions: a significant improvement in mortality is observed with increased protein delivery while decreasing carbohydrate loads. prospective randomized trials are warranted to establish causality. introduction: acute kidney injury (aki) is associated with high mortality. the risk increases with severity of aki. our aim was to identify risk factors for development and subsequent progression of aki in critically ill patients. methods: we analysed 2525 patients without end-stage renal disease who were admitted to the icu in a tertiary care centre between january 2014 to december 2016 and did not have aki on admission. we identified risk factors for development and non-recovery of aki as defined by the kdigo criteria. results: the incidence of new aki in 7 days was 33% (aki i 42%, aki ii 35%, aki iii 23%). multivariate analysis revealed bmi, sofa score, chronic kidney disease (ckd) and cumulative fluid balance as independent risk factors for development of aki. among patients who developed aki in icu, 69% had full renal recovery, 8% partial recovery and 23% had no recovery of renal function by day 7. aki patients without renal recovery in 7 days had significantly higher hospital mortality (60%) compared to the other groups. independent risk factors for non-recovery of renal function were ckd, mechanical ventilation, diuretic use and extreme fluid balance before and after first day of aki. (table 1) the association between cumulative fluid balance before aki and 48 hours after aki with risk of aki non-recovery are shown in figure 1 and 2. conclusions: aki is common and mortality is highest in those who do not recover renal function. cumulative fluid accumulation impacts chances of aki development and progression. (table 2 ). all were in r2. 5/8 (63%) of those with an admission ck>10000 had aki 2 or 3. all 12 (15%) patients who required crrt for aki associated with rm were at risk for aki regardless of initial ck: vascular surgery (4/12), multi-organ dysfunction (7/12), and/or pre-existing renal disease (3/12). conclusions: raised ck is common in icu but its cause is multi factorial thus an isolated measure >5000 does not require immediate high output treatment for rm aki. aki is more common in patients who have more than 1 ck>5000 on 2 sequential days or those whose first ck was >10000 as rm may be contributing. a single ck>5000 in patients with a clear reason to develop rm should also start treatment. surgical outcomes of end-stage kidney disease patients who underwent major surgery p petchmak 1 , y wongmahisorn 1 , k trongtrakul 2 introduction: acute kidney injury (aki) occurs in more than 40% of successfully resuscitated out-of-hospital cardiac arrest patients treated with targeted temperature management (ttm) [1] . the effect of the duration of cooling on aki has not been well studied. in this post-hoc analysis of the tth48 randomized controlled trial that compared 24 vs 48-hours of ttm (33°c) after cardiac arrest [2] , we studied the impact of ttm length on the development of aki. fig. 1 . duration of ttm had a significant impact on the development of creatinine values during the first 7 days in the icu, p<0.05. this was primarily driven by an increase in creatinine during rewarming on day 2 for the 24hour and day 3 for the 48-hour group (fig 2) . conclusions: in a trial of 24 vs 48 hours of ttm after out-of-hospital cardiac arrest, the length of ttm did not affect the incidence of aki. fig. 2 (abstract p287) . creatinine over time patients [1] , but there are no published data on longer-term renal outcomes in adult patients. the purpose of this study was to assess longer-term trends in serum creatinine in this cohort. methods: a retrospective study was conducted of all patients admitted to an adult regional referral centre for ecmo at a uk university hospital between 2010 and 2016. those who survived for >12 months were included. demographics, baseline serum creatinine, presence of aki during icu admission, and serum creatinine at hospital discharge were determined. serum creatinine and dependence on renal replacement therapy (rrt) were assessed at 6 and 12 months post ecmo. results: 13 patients had a complete (or near-complete) data-set available. the mean age was 45.5 years, 38% of whom were male. 8/13 had aki during their critical care admission. none were dependent on rrt at 6 or 12 months post ecmo. most patients had lower serum creatinine results at hospital discharge compared to their pre-hospitalisation baseline, but creatinine concentrations at 6 and 12 months post ecmo tended to be higher than at hospital discharge ( figure 1) . conclusions: in this cohort of ecmo patients who were discharged from hospital alive, serum creatinine tended to be lower at hospital discharge compared to baseline and rose again in the following 12 months. decreased creatinine production due to deconditioning and muscle wasting may offer a biological rationale for the lower creatinine results at hospital discharge [2] . therefore, caution should be exercised in the use of serum creatinine at hospital discharge to assess renal dysfunction -further research is warranted. introduction: aki complicates more than half of icu admissions [1, 2] and is associated with development of chronic kidney disease (ckd), need for renal replacement therapy (rrt) and increased mortality [3] . we prospectively evaluated all icu admissions during a one-year period in order to determine incidence, etiology and timing of aki as well relevant clinical outcomes. methods: prospective observational study of all patients admitted from jan to dec 2017 to a multidisciplinary icu in greece. patients with end-stage renal disease and anticipated icu stay less than 24 hrs were excluded. aki diagnosis and classification was based on kdigo criteria [4] . lowest creatinine level within 3 months before admission or first creatinine after icu admission served as reference. (fig 1) . conclusions: although aki alert does not include urine output criterion or aki risk factors, it remains a helpful tool to point out patients with aki. education and diagnostic algorithms are still needed to early diagnose and treat aki patients. influence of severity of illness on urinary neutrophil gelatinaseassociated lipocalin in critically ill patients: a prospective observational study c mitaka, c ishibashi, i kawagoe, d satoh, e inada untendo university, anesthesiology and pain medicine, tokyo, japan critical care 2019, 23(suppl 2):p291 introduction: neutrophil gelatinase-associated lipocalin (ngal) is a diagnostic marker for acute kidney injury (aki). ngal expression is highly induced not only in kidney injury, but also in epithelial inflammation of intestine, bacterial infection, and cancer. however, the relationship between ungal and severity of critically ill patients has not been well understood. the purpose of this study was to elucidate whether ungal is associated with severity of illness and organ failure in critically ill patients. methods: we prospectively enrolled patients with sepsis (n=34) and patients who underwent esophagectomy with gastric reconstruction for esophageal cancer (n=39). sepsis was defined according to sepsis-3. ungal levels were measured on icu day 1, 2, 3, 4 and 7. ungal levels and aki rate in patients with sepsis were compared with those in patients who underwent esophagectomy. aki was defined according to kdigo. acute physiology and chronic health evaluation (apache) ii score and sequential organ failure assessment (sofa) score were calculated. results: median ungal level (413 ng/mg creatinine) was significantly higher in patients with sepsis than that (26 ng/mg creatinine) in patients who underwent esophagectomy on day 1. median apache ii score and median sofa score in patients with sepsis were significantly higher than those in patients who underwent esophagectomy. four patients with sepsis developed aki, and 3 out of them underwent continuous renal replacement therapy, whereas no patients who underwent esophagectomy developed aki. ungal levels were positively correlated with apache ii score and sofa score in patients with sepsis. ungal levels were remarkably elevated (> 4000 ng/mg creatinine) in urinary tract infection (n=3), loops enteritis (n=1), and obstructive jaundice due to cholangiocarcinoma (n=1). conclusions: these findings suggest that ungal level is associated with severity of illness and organ failure in patients with sepsis. ungal levels might be influenced by severity of illness and inflammation. to assess the quality of the course 15 us renal images had to be evaluated in "post-renal obstruction" (p-ro) or "no p-ro". the rate of correctness (roc farius ) was determined. in 2018 we, once again, contacted the students to attend a web-based online "follow-up". this online survey was created with "google formular". 15 new and unknown us images were presented and rated in "p-ro" or "no p-ro" (roc fup introduction: septic-induced kidney injury worsen the patient's prognosis [1] . renal resistance index (rri) is correlated with an increased mortality in septic patients [2] . the aim of this study was to describe the evolution of rri in a rat sepsis model. methods: the local ethics committee approved the study (apa-fis#9385-2016113016181432). sepsis was induced in 3-month-old male rats by caecal ligation and puncture (clp) [3] . the rri was assessed before and 24h after clp by pulse doppler on the left renal artery (rri=(peak systolic velocityend diastolic peak)/ peak systolic values expressed as % per column. abbreviations in alphabetical order: aki acute kidney injury; akin acute kidney injury network definition; ckd chronic kidney disease. there were statistical differences between subgroups with and without aki for the subgroups of patients with previous ckd (p = 0.010*), sepsis at admission (p = 0.031**), hypotension (p= 0.003***) fig. 1 (abstract p290b) . target comparing accuracy and precision of aki alert and actual aki diagnoses velocity) (fig 1) . rri were compared by a paired wilcoxon test (r software v.3.3.4). a p value < 0.05 was considered significant. results: 14 rats were included. 24 hours after sepsis induction, all rats were in septic shock with cardiac dysfunction. the rri increased after sepsis induction compared to baseline (0.61 ± 0.03 vs 0.66 ± 0.02, p<0.05) and mean renal artery velocity decreased (11.8 ± 1.41 vs 7.7 ± 0.94, p<0.05) (fig 2) . systolic and diastolic peaks velocity of the renal artery were unchanged. conclusions: sepsis induced changes in rri and mean velocity on the left renal artery whereas no changes in systolic or diastolic velocities were seen. these results are consistent with available clinical datas. the rri could be an additional tool to assess renal failure in septic rats. further studies are needed to confirm the validity of this marker during sepsis. kidney failure is one of the most common organ dysfunction during sepsis. the rri could be an additional tool in small animals to assess the effects of potential therapeutic targets on renal function induced by sepsis. (fig 2) . the egfr improved more with the heparin group (53% vs 48%; p=0.21) (fig 1) . interruptions of the filter circuit were as expected less with the citrate group (144mins vs 45mins; p=0.17). finally, inotropic requirements increased following therapy interruptions, more so with patients receiving citrate (7.2% vs 14.5%; p=0.4). conclusions: our analysis suggests that using citrate anticoagulation for rrt results in a monitoring cost saving of approximately £9 per 24 hours, alongside the other conferred savings previously reported. furthermore, results demonstrate the efficacies of both systems are similar in the initial 24 hours, although there is a suggestion that heparin systems improves renal parameters more quickly. finally, interruptions and 'filter downtime' caused an increase in the patient's inotropic requirements, however results suggestive that this is greater in the citrate group. mmol/l respectively. demographic characteristics of the study group and the main parameters of the procedure were presented in fig 1. conclusions: regional citrate is a safe and effective anticoagulation method for crrt in children, when it is applied following a protocol. it significantly prolongs circuit survival time and thereby should increase crrt efficiency. we did not find any serious adverse effects of regional citrate anticoagulation. -5) , deceased at 1 year n=67 (14%). the mdrd trend is more indicative than creatinine of decline of renal function in the post operative period (fig 1) . crrt was used in 14.4% (69 pts) and was associated to a greater los and mortality (fig 2) . preoperative bilirubin, bun and creatinine are among the greatest risk factors for its use ( table 2 . at 1 year follow up n=6 pts (1.5%) were on hemodialysis. conclusions: aki requiring crrt in after lt is associated with higher mortality and los. identify patients at risk and adopt preventive strategies in the perioperative period is mandatory. introduction: we developed a new co2 removal system, which has a high efficiency of co2 removal at a low blood flow. to evaluate this system, we conducted in vivo studies using experimental swine model. methods: six anesthetized and mechanically ventilated healthy swine were connected to the new system which is comprised of acid infusion, membrane lung, continuous hemodiafiltration and alkaline infusion. in vivo experiments consist of four protocols of one hour; baseline= hemodiafiltration only (no o2 gas flow of membrane lung); membrane lung = "baseline" plus o2 gas flow of membrane lung; "acid infusion" = "membrane lung" plus continuous acid infusion; "final protocol" = "acid infusion" plus continuous alkaline infusion. we provided an interval period of one hour between each protocol. we changed the respiratory rate of the mechanical ventilation to maintain pco2 at 50-55 mmhg during the experiment. results: the amount of co2 eliminated by the membrane lung (vco2ml) significantly increased by 1.6 times in the acid infusion protocol and our final protocol compared to the conventional membrane lung protocol, while there was statistically no significant difference observed in the levels of ph, hco3-, and base excess between each study protocol. minute ventilation in the "final protocol" significantly decreased by 0.5 times compared with the hemodiafiltration only protocol (p <0.0001), the membrane lung (p=0.0006) and acid infusion protocol (p=0.0017). we developed a novel ecco2r system which efficiently removed co2 and is easy-to-setup to permit clinical application. this new system significantly reduced minute ventilation, while maintaining acid-base balance within the normal range. further studies are needed for the clinical application of this easy setup system comprising of the materials typically used in a clinical setting. , and psychomotor agitation (10%) while the most common symptoms of hypertensive emergency were chest pain (30.4%), dyspnea (28.6%) and neurological deficit (29%). clinical manifestations of hypertensive emergency were cerebral infarction (26.4%), acute pulmonary edema (24.8%), hypertensive encephalopathy (20.6%), acute coronary syndromes (20.4%), cerebral hemorrhage (4,.5%), congestive heart failure (12%), aortic dissection (0.8%), preeclampsia and eclampsia (2.6%). conclusions: hypertensive urgencies were significantly more common than emergencies (78.1% vs. 21.9%, p<0.0001). there was no statistically significant difference in the number of patients with hypertensive urgency and emergency in relation to age, gender, duration of hypertension, except for the 60-69 age group, where urgency was statistically significantly higher (p=0.0204). introduction: emergency department (ed) crowding is a major public health concern. it delays treatment and possible icu admission, which can negatively affect patient outcomes. the aim of this study was to investigate whether ed to icu time (ed-icu time) is associated with icu and hospital mortality. methods: we conducted an observational cohort study using data from the dutch nice registry. adult patients admitted to the icu directly from the ed in 6 academic centers, between 2009 and 2016, were eligible for inclusion. for these patients nice data were retrospectively extended with ed admission date and time. ed-icu time was divided in quintiles. the data were analyzed using a logistic regression model. we estimated crude and adjusted (for disease severity; apache iv probability) odds ratios of mortality for ed-icu time. in addition, we assessed whether the apache iv probability (divided into quartiles) modified the effect of ed-icu time on mortality. results: a total of 14,787 patients were included. baseline characteristics are shown in table 1 . the median ed-icu time was 2.0 [iqr 1.3-3.3] hours. icu and hospital mortality were 18.1 and 22.2%, respectively. the crude data showed that an increased ed-icu time was associated with a decreased icu and hospital mortality (both p<0.001, figure 1a ). however, after adjustment for disease severity, an increased ed-icu time was independently associated with increased hospital mortality (p<0.002, figure 1b ). figure 2 shows that only in the sickest patients (apache iv probability >60.9%), the association between increased ed-icu time and hospital mortality was significant (p=0.019, figure 2d ). we found similar results with respect to icu mortality. conclusions: this study shows that a prolonged ed-icu time is associated with increased icu and hospital mortality in patients with higher apache iv probabilities. strategies aiming at rapid identification and transfer of the sickest patients to the icu might reduce inhospital mortality. reliability and validity of the salomon algorithm: 5-year experience of nurse telephone triage for out-of-hours primary care calls e brasseur, a gilbert, a ghuysen, v d´orio chu liege, emergency departement, liège, belgium critical care 2019, 23(suppl 2):p308 introduction: due to the persistent primary care physicians (pcp) shortage and their substantial increased workload, the organization of pcp calls during out-of-hours periods has been under debate. the salomon (système algorithmique liégeois d'orientation pour la médecine omnipraticienne nocturne) algorithm is an original nursing telephone triage tool allowing to dispatch patients to the best level of care according to their conditions [1] . we aimed to test its reliability and validity under real life conditions. methods: this was a 5-year retrospective study. out-of-hours pc calls were triaged into 4 categories according to the level of care needed: emergency medical services (amu), emergency department visit (maph), urgent pcp visit (upcp), delayed pcp visit (dpcp). data recorded included patients' triage category, resources and potential redirections. more precisely, patients included into the upcp + dpcp cohort were classified under-triaged if they had to be redirected to an emergency department. patients from the amu+maph cohort were considered over-triaged if they did not spend at least 3 resources, 1 emergency specific treatment or any hospitalization. results: 10207 calls were actually triaged using the salomon tool, of which 19.1% were classified as amu, 15.7% as maph, 62.8% as upcp and 2.1% as dpcp (fig 1) . as concerns the amu+maph cohort, the triage was appropriate in 85.3% of the calls, with an over-triage rate of 14.7%. as concerns the upcp + dpcp cohort, 97.1% of the calls were accurately triaged and only 2.9% were under-triaged. sal-omon sensitivity reached 93.9% and its specificity 92.5%. these results indicate that salomon algorithm is a reliable and valid nurse telephone triage tool that has the potential to improve the organization of pcp out-of-hours work. introduction: inappropriate visits to the emergency department (ed), such as patients manageable by a primary care physician (pcp), have been reported to play some role in the ed crowding [1] . indeed, non-urgent patients directly managed by pcps could reduce ed workload [2] . triage and diversion to alternative care facilities, eventually co-located within the ed, could offer a solution [3] provided fig. 1 (abstract p308) . distribution of different calls, their triage using the salomon algorithm and the inappropriate triages (over and undertriages) based on the preselected criteria the availability of a reliable triage tool for their early identification. we created a new triage algorithm, persee (protocoles d'evaluation pour la réorientation vers un service efficient extrahospitalier) and tested its feasibility, performance and safety. methods: after initial evaluation with a 5-level ed triage scale [4] , ambulatory self-referred patients classified as level 3 or below benefited from a simulated triage with persee identifying 2 categories of patients: ed ambulatory patients and primary care (pc) treatable patients. we collected patients data and resources. patients requiring less than 3 resources, no specific emergency treatment and no hospitalization were considered as manageable in a pc facility. results: 1999 patients were included in the study of whom 66.9% were self-referred (fig 1) . among those self-referrals, 58.6% were triaged as level 3 or below. 38.9% patients were triaged as ambulatory patients of whom 10% were as pc treatable. we noted a redirection rate of 10% of the global visits or 15% of the self-referrals, an error rate of 7%, a sensitivity of 24.6% and specificity of 97.6%. conclusions: using advanced ed triage algorithm in addition to classical ed triage might offer interesting perspectives to safely divert self-referrals to pc facilities and, potentially, reduce ed workload. introduction: generally, prehospital medical provider should minimize staying prehospital scene to reach the patient to definitive care as soon as possible in prehospital medical activity. in addition, some textbook and report saids that medical provider minimize the number of procedure or limit minimum requirement procedure because unnecessary procedure may extend the staying time in prehospital scene. however, there are few studies evaluating this hypothesis and that this "extension is significant or not. therefore, we perform this study. methods: we evaluated the operated air ambulance(doctor-heli) case from 1st april 2015 to 31st march 2018, in gifu university hospital using our mission record. we evaluated about time from landing to ready for taking off(activity time), operation doctor, mission category (i.e. trauma), number of procedure in the each activity and work load. we only focused on prehospital care and exclude transportation from hospital to hospital . in addition, we exclude the case which are not suitable for analysis. results: 1299 cases were operated in these period. 511 cases were suitable for analysis. average activity time in prehospital scene was14.32±6.09. there was weak correlation between the number of procedure and activity time. (r=0.3452) the length of the activity time did not depend on mission category. if the doctor perform 6 and over procedures, staying time was 7minutes longer, this was significantly longer than that of under 5 and under procedures. conclusions: we confirmed that we have to minimize the number of procedure or limit minimum requirement procedure in prehospital scene. and our result suggest we may have to limit appropriate number of procedures. introduction: organ failure is a critical condition, but the prevalence is largely unknown among unselected emergency department (ed) patients. knowledge of demographics and risk factors could improve identification, quality of treatment, and thereby improve the prognosis. the aim was to describe prevalence and all-cause mortality of organ failure upon arrival to the ed. methods: this was a cohort-study at the ed at odense university hospital, denmark, from april 1, 2012 to march 31, 2015. we included all adult patients, except minor trauma. organ failure was defined as a modified sofa-score > 2 within six possible organ systems: cerebral, circulatory, renal, respiratory, hepatic, and coagulation. the first recorded vital, and laboratory values were extracted from the electronic patient files. primary outcome was prevalence of organ failure; secondary outcomes were 0-7-day and 8-365-day mortality. results: of 70,399 contacts 52.1% were female and median age 62 (iqr 42-77) years. the prevalence of new organ failure was 11.8%, individual organ failures; respiratory 4.9%, circulatory 3.0%, cerebral 2.3%, renal 1.7%, hepatic 1.4%, and coagulation 0.6%. the 0-7-day and 8-365-day all-cause mortality was 11.7% (95% ci: 10.9-12.6) and 19.3% (95% ci: 18.2-20.4), respectively, if the patient had new organ failures at first contact in the observation period, compared to 1.4% (95% ci: 1.3-1.5) and 6.6% (95% ci: 6.4-6.9) for patients without. seven-day mortality ranged from hepatic failure, 5.6% (95% ci: 4.0-7.5) to cerebral failure, 33.3% (95% ci: 30.4-36.2), and the 8-365-day mortality from cerebral failure, 13.2% (95% ci: 11.2-15.4 to renal failure, 26.7% (95% ci: 23.6-29.9). conclusions: new organ failure is frequent and serious, with a prevalence of 11.8% and a one-year mortality of 31% with wide variation according to type of organ failure. results: we proceeded to a descriptive study that showed that 39% of patients were male and 62% of them were female with a sex ratio of 0.62.the average age of patients was 34 years old and ranged between 15 and 90 years old.we found that 59 patients of our population had medical background, dominated by diabetes in 12 cases, high blood pressure in 8 cases and asthma in 6 cases.the results also showed that 29.5% of patients had a history of abdominal surgery while 13% of them had history of other types of surgery.the patients were oriented according to their severity level as following: 21% care unit of emergency department, 1.5% close monitoring room .the vaspi score was ranged between 1 and 10 with an average of 4±2. it was higher than 5 in 32.5% of cases.the results of physical examination found an isolated pain in 67,5% of cases, a reactionnal pain syndrom in15% of cases, a peritoneal syndrome in 12% of cases and an occlusive syndrome in 7% of cases.the final diagnosis was mostly represented by the following causes: 45.5% of gastroenteritis 11.5% of constipation and 9% of ulcer disease.the final orientation of patients according to the diagnosis led to hospitalization in 21% of cases and to outpatient clinic in13% of cases while 66% of them did not need any more care. conclusions: appropriate diagnostic evaluation and decision for or against hospitalization is a challenge in the patient who comes to the emergency department with acute abdominal pain it need an adequate evaluation and management. introduction: we assessed patients' impressions of a selfadministrated automated history-taking device (tablet) to gather information concerning emergency department (ed) patients prior to physicians' contact. the quality of communication was compared with the traditional history-taking. methods: the algorithm content was developed by two emergency physicians and two emergency nurses through an iterative process. item-content validity index (i-cvi) was measured by five experts rating the relevance of each item (from 1: not relevant to 4: highly relevant) [1] . next, quality control was realized by research team. to assess the feasibility, we used a computerized randomization. low acuity, ambulatory adult patients presenting to the ed were assigned either to a control group (cg, n=65) beneficiating form a traditional history-taking process or to the experimental group (eg, n=66) assigned to use the tablet with further history-taking by the ed physician. communication was analyzed by the health communication assessment tool [2] and satisfaction assessed by questionnaires. results: after two rounds, validity was excellent for each item (i-cvi > 0.8). the universal agreement method was of 0.9. refusals (n=11) to participate were analyzed: they fear using an electronic device or the experimentation. content satisfaction revealed that 93% of patients understood the questions. 94% of patients indicated that the device was easy to hold and use. medical communication was not affected by the device (p=0.15). we noticed that, among the subsections, physicians significantly introduced themselves better in the eg (p=0.04). conclusions: in this feasibility study, patients were highly satisfied. the use of a self-administrated automated history-taking device does not generate miscommunications and allow physicians better introduce themselves. . a positive point we have established is the possibility for the detorsion of a twisted retention ovarian cyst after its transvaginal aspiration. we used this method only in cases when the onset of torsion did not exceed 4 hours. 28.5% of all emergency conditions associated with retention cysts were recurred by conservative therapy, and 50.4% of patients with the retention cysts rupture were successfully treated in this way. conservative management is possible in the case of a small loss of blood (up to 150.0-200.0 ml), hemodynamic stability and the absence of signs of continuing bleeding. the detorsion and resection of the cyst when torsion is not more than 180°and even longer than 4 hours, in most cases did not reveal necrosis in the appendages. conclusions: improvement of organs of preservation and reproduction in women. criteria for admission to an intensive care unit of a tertiary hospital: analysis of the decisions of the outreach intensivist and 28 day in-hospital mortality introduction: the aim of this study was the analysis of icu admission criteria and evaluation of in-hospital mortality of patients assessed by our critical care outreach team. criteria for admission to the icu should be defined to identify the patients most likely to benefit from icu admission. this triage process is complex, associated with several factors, including clinical characteristics of the patients, but also subjective factors because it depends on the judgment of the intensivist who decides whether to admit or not the patient and is obviously conditioned to the structure and size of the icu. methods: the outreach intensivist records the patient observation in a form with 5 questions (reversibility of acute illness, objective of admission in icu, comorbidities, functional reserve and intuitive prognosis of the doctor). analysis of 6 months (january 1 through june 30, 2018) of admission decisions in icu, mean delay, icu mortality, and 28 day in-hospital mortality (28hm). results: the intervention of the intensivist in "outreach" was requested on 855 occasions. the main places of observation were the emergency room (40.7%) and the wards (40.4%). the 28hm increased with the degree of comorbidity decompensation. functional reserve also influenced 28hm, reaching 42.2% in partially dependent patients and 63.4% in totally dependent patients. there was agreement between the mortality and the physician´s intuitive prognosis in 71% of the cases. conclusions: a larger sample is needed to draw sustainable conclusions, however, the evaluation algorithm correlated well with hospital mortality. decompensated comorbidities and low functional reserve have a negative impact on prognosis, regardless of acute disease. there was agreement between mortality and the physician´s intuitive prognosis. electrochemical methods for diagnosing the severity of patients with multiple trauma introduction: multiple trauma is one of the leading causes of death worldwide [1] . timely diagnosis and treatment is crucial in this state. one of the promising areas is the use of new electrochemical methods they are simple, flexible, efficient and of low cost. among these methods, attention is paid to the measurement of open circuit potential (ocp) of the platinum electrode and cyclic voltammetry (cva). the ocp is a reflection of the balance of pro-and antioxidants in the body, and the amount of electricity (q) determined by cva is proportional to the antioxidant activity of the biological environment. methods: a total of 51 patients with severe multiple trauma (38.7 ±13.5 y.o., 33 men and 18 women) were enrolled; apacheii 17.4±7.2; iss 40.9±8.3; blood loss 2356±997 ml. blood plasma was collected from patients. measurement of the ocp was carried out according to [2] , cva analysis -according to the original method on a platinum working electrode. results: a shift in the ocp towards more positive potential values (fig. 1) , while the antioxidant activity of blood plasma decreased (fig. 2) . a more significant change of ocp, as compared to the q values, may indicate not only a deficiency in the components of the antioxidant defense system of the body, but also an increase in the concentration of prooxidants (e.g., reactive oxygen species), which are involved in oxidative stress. who underwent surgical fixation). information was collected from tarn, icnarc and surgical team databases. our primary outcome was itu resource utilisation (itu los and mechanical ventilation days). our secondary outcomes were morbidity and mortality (hospital los, infection burden, inotrope use and death before discharge). data was collected and analysed in microsoft excel and r. results: 691 patients were included (group 1 = 246, group 2 = 414, group 3 = 31). mortality was significantly higher when comparing the post 2014 groups undergoing conservative (12%, 51/414) vs. surgical fixation (0%, 0/31), p-value = 0.026. regarding potential temporal changes, there was no significant difference in mortality between the non surgical groups; pre-2014 (group 1: 28/218) and post 2014 (group 2), p-value 0.683. group 3 patients did spend more time mechanically ventilated (p-value 0.019) and used more antimicrobials (p-value 0.032) ( table 1) . conclusions: patients undergoing surgical rib fixation at the rlh had significantly improved mortality with more days spent mechanically ventilated. pilot study on ultrasound evaluation of epiglottis thickness in normal adult a osman 1 introduction: as the prevalence of epiglottitis is decreasing due to immunization, the difficulty in early detection remained. the aim of this study is to determine the thickness of epiglottis in normal adult with the utilization of bedside ultrasound. methods: this was a prospective observational study of convenience selection among healthy staff in emergency department, university malaya medical centre. the identification and measurement of epiglottis were performed using a 10mhz linear transducer by trained emergency physicians and registrars in em. subjects were scanned in either standing or upright seated position with the neck neutral or mildly extended. the epiglottis, thyroid cartilage and vocal cord were visualized and the epiglottis anteroposterior(ap) diameter was measured. difference in categorical parameters were analyzed by independent-sample t-test. the relationship between height, weight and epiglottic size was analyzed using pearson's correlation. results: fifty-six subjects were analyzed with 25 males and 31 females age ranging from 18 to 50 years old. the epiglottis ap diameter ranged from 0.15cm to 0.23cm, with average of 0.18cm. there was significant difference in epiglottic ap diameter between male (m=0.19cm, sd=0.19) and female (m=0.17cm, sd=0.12; t(54)=5.27, p=<0.001, twotailed). moderate positive correlation between height and epiglottic ap diameter (r=0.49) and weight (r=0.33) was documented. conclusions: our study demonstrated the identification and visualization of epiglottis was feasible and easy with the use of bedside upper airway ultrasonography. there was a little variation in the ap diameter of epiglottis in adults. indoor vs. outdoor occurrence in mortality of accidental hypothermia in japan y fujimoto 1 , t matsuyama 2 , k takashina introduction: the impact of location of accidental hypothermia (ah) occurrence has not been sufficiently investigated so far. thus we aimed to evaluate the differences between indoor and outdoor occurrence about baselines, occurrence place, mortality, and length of icu stay and hospital stay. methods: this was a multicenter retrospective study of patients with a body temperature ≤35°c taken to the emergency department of 12 hospitals in japan between april 2011 and march 2016. we divided the included patients into the following two group according to the location of occurrence of ah (indoor versus outdoor). the primary outcome of this study was in-hospital death. secondary outcomes were the length of icu stay, and hospital stay. results: a total of 537 patients were enrolled in our hypothermia database. there were 119 and 418 patients with the outdoor and indoor occurrence. the indoor group was older (71 versus 81.5 years-old, p<0.001) and worse in adl than the outdoor group. the proportion of in-hospital death was higher in the indoor group than the outdoor group (28.2% [118/418] versus 10.9% [13/119], p<0.001). the multivariable logistic regression analysis demonstrated that adjusted odds ratio of the indoor group over the outdoor group was 2.48 (95%ci; 1.18 to 5.17) ( table 1) . as for secondary outcomes, both of the length of icu stay and hospital stay in survivors were longer in the indoor group than the outdoor group. conclusions: our multicenter study indicated that indoor occurrence hypothermia accounts for about 78% of the total in this study, and the proportion of in-hospital death was higher in the indoor group. we have to raise an alert over the indoor onset accidental hypothermia and need to take countermeasures for prevention and early recognition of ah in indoor location. conclusions: during acute asthmatic attack, arterial hyperlactatemia is frequently present at ed arrival. nevertheless, the plasma lactate level was no significant difference between ed admission and 2 hr after treatment. the introduction: this is a case series of traumatic aortic injury (tai) which was diagnosed by transesophageal echocardiography (tee) in the emergency department. the number of patients with blunt thoracic aorta injury arriving at emergency department is on the rise and survival rate is time-dependent on early diagnosis. tee offers several advantages over transthorasic echocardiography (tte) including reliability, continuous image acquisition and superior image quality. methods: all trauma patients who presented to emergency department from 1st january 2017 until 30th november 2018 at hospital raja permaisuri bainun, perak, malaysia with suspected tai were evaluated with transesophageal echocardiography. over the 2 years period, tee was performed in 30 patients. 6 patients had positive findings suggestive of tai. results: the first case was an old lady who presented after a deceleration injury in a car accident. tee was performed due to hemodynamic instability and found an intimal flap along the ascending aorta. the second case, a stanford type a (figure 1) , was complicated with pericardial tamponade. the intimal flap was visualised from the aortic arch extending to the descending aorta by tee. the third case was a case of intramural haematoma involving distal aortic arch extending to the descending aorta which survived until corrective surgery. in the fourth case, tee revealed a motion artefact which mimicked an intimal flap in the ascending aorta. in the fifth case, tee showed intimal flap at aortic isthmus which was not detected by tte. in the last case, a traumatic aortic dissection was complicated by aortic regurgitation (figure 2) . conclusions: tee can be a useful point of care tool use by emergency and critical care physicians for early diagnosis of blunt traumatic aorta injury. introduction: reboa is an endovascular intervention intended to preserve central perfusion in the context of shock due to noncompressible torso haemorrhage. more so, it is less invasive than the traditional approach of resuscitative thoracotomy (rt) and aortic crossclamping. though its use dates back to the korean war, it has not been widely adopted in trauma management, as evidence demonstrating clear benefit compared with conventional rt is lacking [1] . we aimed to evaluate feasibility, outcomes and complications after reboa for haemorrhagic shock and traumatic cardiac arrest. methods: we performed a systematic literature review, searching scopus and pubmed databases using relevant terms (july 2018). we included studies enrolling patients with haemorrhagic shock or cardiac arrest after civilian trauma who had undergone reboa and reported hospital mortality (our primary outcome). abstract-only studies and single-patient case reports were excluded. we collated and analysed data using review manager v5.3. the newcastle-ottawa scale was used to assess risk of bias. results: sixteen in-hospital studies met inclusion criteria (n=2034). ten were case series and six were cohort studies comparing reboa outcomes with those of rt. there were wide differences between studies' inclusion criteria, case-mix (including cardiac arrest), injury severity, insertion details, and reported outcomes. overall hospital mortality post-reboa was 61.4%. meta-analysis of cohort studies indicated notably lower mortality in patients undergoing reboa (or 0.41, 0.32-0.54) than rt with low statistical heterogeneity between studies (i 2 = 0%), shown in fig 1. conclusions: whilst our findings are limited by methodological differences and biases in the included studies, almost 40% of patients undergoing reboa for haemorrhagic shock and/or cardiac arrest survived to discharge. furthermore, reboa appeared to offer a consistent mortality benefit compared with rt. introduction: trauma related coagulopathy remains a primary contributor to mortality on battlefields and in civilian trauma centres. fibrinogen is considered to be the first to drop below critical level and correspondingly compromised coagulation process. however, it is unclear if fibrinogen concentrate at a very early stage is feasible and effective to prevent from coagulopathy. methods: a total of 66 acutely injured patients in austria, germany and czech republic were screened and enrolled in this controlled, prospective randomized placebo controlled double blinded multicentre and multinational trial. upon the completion of randomization, fibrinogen concentrate (50 mg/kg, fgtw©, lfb france) or placebo was reconstituted and given to the patients at the scene or during helicopter transportation from the scene to nearby hospitals. blood samples were taken at baseline (scene of accident before study drug administration), at the emergency room, three hours, nine hours and twentyfour hours after admission to the hospital as well as after three and seven days after admission, for measurements of blood gases and coagulation, together with clinical data and outcome records. results: the demographic and injury characteristics and the estimated blood loss, iss, and gcs at the scene were similar in both groups. in the placebo group, fibrinogen concentration dropped from 200 mg/dl at injury site to 160 mg/dl () at er admission and clot stability reduced from 13.5 mm (4, 21 mm) to 10 mm (p=0.013) (fig 1) . fibrinogen concentrate administration prevented the drop of fibrinogen level (baseline of 170 mg/dl to 210 mg/dl and improved clot stability from 13 mm at baseline to 16 mm at er. conclusions: pre-hospital administration of fibrinogen concentrate in traumatic bleeding patients is feasible and effective in preventing the development of coagulopathy. data from this study support the use of fibrinogen to prevent trauma related coagulopathy. fibrinogen concentrate vs cryoprecipitate in pseudomyxoma peritonei surgery: results from a prospective, randomised, controlled phase 2 study results: the per-protocol set included 43 pts (hfc, n=21; cryo, n=22). the mean total intraoperative dose of hfc was 6.5 g vs 4.1 pools of cryo (containing approx 8.8 g of fibrinogen). median duration of surgery was 7.7 h. overall haemostatic efficacy of hfc was non-inferior to cryo and was rated excellent or good for 100% of pts receiving hfc and cryo, with similar blood loss. intraoperatively, only red blood cells were transfused (median: 1 unit). intraoperative efficacy is shown in table 1 . infusions were initiated 0.4 h earlier with hfc than cryo due to faster product availability. preemptive hfc led to a greater mean increase vs cryo in fibtem a20 ( figure 1 ) and plasma fibrinogen (figure 2 ). there were 6 serious adverse events (saes) in the hfc group and 17 in the cryo group, including 7 thromboembolic events (tees; 2 deep vein thromboses, 5 pulmonary embolisms). no aes or saes were deemed related to the study drug. conclusions: hfc was efficacious for treatment of bleeding in pts undergoing surgery for pmp. no related aes and no tees occurred in pts treated with hfc. fig. 1 (abstract p326) . fib mcf t1 to t7 with 95% ci fig. 1 (abstract p327) . fibtem a20 prior to and following the preemptive dose of hfc/cryoprecipitate introduction: patients in the intensive care unit often suffer from thrombocytopenia. in dealing with this problem, we need to figure out not only the cause of thrombocytopenia but also the risk of bleeding. however, there is no reliable method for evaluating bleeding risk. methods: in this preliminary study, four thrombocytopenic patients who required platelet transfusion before undergoing invasive procedure were enrolled. written informed consent was obtained from all patients for participation in the study. bleeding was graded using the who bleeding scale. thrombogenic activity was evaluated using total thrombus-formation analysis system (t-tas), rotational thromboelastometry (rotem), and multiplate impedance aggregometry. for t-tas analysis, we prepared a novel microchip, named hd chip, which is suited for analyzing low platelet samples rather than those with normal platelet counts. , key patient groups in which it was wasted and the use of standard laboratory tests (slts) to guide its use. the purpose was to assess the potential benefit a point of care viscoelastic haemostatic assay (vha) could have on ffp transfusion and waste. the national blood transfusion committee and nhs blood and transplant committee have published data showing that up to 25% of ffp is transfused inappropriately [1] . methods: blood bank data was obtained evaluating haemorrhaging patients in whom ffp was requested across a nine-month period in 2018. patient bleeds were categorised by speciality. the mean time ffp dispensed and wasted was recorded, as were timings of slt requests. where available, the inr result was recorded. results: 66 patients were identified. 74 transfusions were requested. table 1 shows that the highest transfusion requirements are for acute medical emergencies and major trauma. 70% of transfusion were surgical specialities, it would be expected that these patients would have anaesthetic or critical care input. 242 units were wasted. acute medical emergencies wasted the highest amount of ffp (82 units). table 2 demonstrates that 29.7% of transfusions had an inr available one hour prior to ffp being dispensed. conclusions: we conclude that use of slts to guide ffp transfusion is low. this suggests transfusion decisions are being made clinically. a point of care vha could give treating physicians better access to timely haemostatic data. introduction: we developed the process for the out-of-hospital packed red blood cells (prbc) transfusion in the hems of castilla-la mancha clm according to criteria of medical indications, security, monitoring and tracking. haemorrhage is a preventable cause of death among population suffering accidents or bleeding injuries in regions with low population density where health services should reach people in remote areas. hems of clm is the first out-ofhospital emergency service in spain that provides prbc transfusion there where the accident takes place. this program has been developed jointly between hematologists of the center for transfusions ct and the hems team. methods: observational retrospective study with data collected from june 2014 to august 2018. the medical helicopter was provided with two prbc o rh(d) negative (fig 1) . shock index was selected as indication for transfusion. to achieve feasibility and preservation of the prbc it was established a prospective monitoring and microbiological culture for both groups: case group for the prbc kept in the hems and control group in the hospital (fig 2) . controls and comparison of hematologic analysis were performed immediately and 35 days after collection. statistics used spss 23.0 (signification p<0.05). results: 138 prbc were evaluated, 67 case -71 control. analyses were tested days 1 and 35 after collection. hemolysis was not observed. all cultures were negative. results obtained of the prbc after 35 days transported in the hems related to monitoring parameters were not different than those observed on prbc conserved in the ct. 35 prbc were transfused to 28 patients in out-of-hospital assistance. neither post-transfusional reactions or undesirable events have been registered. prbc units are changed every 30 days. conclusions: the process designed (collection, conservation, tracking and tests) to make prbc available in the medical helicopter has demonstrated to keep the standard conditions and properties to be transfused in critically ill patients out-of-hospital. outcomes in patients with a haematological malignancy admitted to a general intensive care unit a corner east sussex healthcare nhs trust, intensive care, eastbourne, united kingdom critical care 2019, 23(suppl 2):p331 introduction: recent published data have challenged the view that critically ill patients with a haematological malignancy have a poor prognosis [1] . reports have largely originated from tertiary centres. the aim of this audit was to evaluate the intensive care unit (icu), in hospital and one year mortality for a cohort of patients admitted to a mixed medical and surgical icu in a district general hospital. methods: details were obtained for all patients with a haematological malignancy admitted to eastbourne and hastings icu between march 2012 and august 2017. patient characteristics, type of malignancy, reason for admission, degree of organ support and survival rates at icu discharge, hospital discharge and 1 year postadmission were collected. results: 60 patients, 65% male, were identified. median (interquartile range, iqr) age was 68 (60-73) years. 55% had neutropenia. the commonest malignancies were acute leukaemia 43%, lymphoma 27% and myeloma 17%. reasons for admission were respiratory 48%, cardiac 18% and renal 10%. organ supports used were noradrenaline 68%, intubation and mechanical ventilation 42%, renal replacement therapy (rrt) 32% and dobutamine 10%. overall survival rates are shown in figure 1 . 7 patients were discharged from hospital following a period of mechanical ventilation. for these patients, median (range) age was 64 (26-72) years. all were male. median (iqr) time in hospital prior to admission was 0 (0-2) days, 7/7 patients required vasoactive support, 3/7 required rrt, median icu length of stay was 12 (3-25) days. 3/7 were admitted following surgery for an unrelated condition. to date, only 1/7 patient has survived 5 years post icu admission. conclusions: although survival rates were disappointing, particularly in those patients requiring mechanical ventilation, selected patients have the potential for a good outcome. these results outcomes have been presented to our haematology department to aid patient counselling. analyses. cox regression was used for the survival analysis. organ failure was defined as the occurrence of renal failure based on acute kidney injury network (akin)-creatinine or need for; vasopressors, invasive ventilation or continuous renal replacement therapy (crrt) the first 28 days after admission. length of stay was only analysed in survivors. results: the study included 3585 unique patients. prolonged aptt was associated with mortality with a 95% confidence interval (ci) of hazard ratio 1.001-1.010. prolonged aptt correlated also with the occurrence of renal failure and the need for vasopressor and crrt with 95% ci of odds ratio (or) 1.009-1.028, 1.009-1.033 and 1.009-1.029 (fig 2) . increased pt-inr was associated with the need for vasopressors and invasive ventilation with 95% ci of or 1.138-2.056 and 1.151-1.871. both aptt and pt-inr correlated with length of stay with 95% ci of or 1.007-1.020 and 1.034-1.438. conclusions: activated partial thromboplastin time on admission to the icu is independently associated with mortality. both aptt and pt-inr are independently associated with length of stay and the need of organ support. all regression models were adjusted for saps 3 score which means that aptt prolongation and pt-inr increase on admission represent morbidity that is not accounted for in saps 3. introduction: the goal was to assess if daily venous thromboembolism (vte) assessment was being done in our critical care (cc) unit, and if not, what changes could be made. a mortality review showed the need for a dynamic vte assessment in cc patients, who are subject to daily changes influencing vte risk. a daily risk assessment was introduced, and a 'tab' on our clinical information system, metavision(r)(mv) was created. recently published national institute for health and care excellence guidelines on vte risk assessment in cc provided us cause to assess our compliance [1] . methods: data was collected from mv. review of daily vte assessment was made and a percentage completion of daily vteassessments was calculated per patient.interventions were done using standard improvement methods through pdsa cycles. results: baseline data, of 53 patients, was collected in july,2018.compliance with daily vte assessment was 51%. the results were presented at the clinical governance forum(cgf), and posters were displayed in cc. the second cycle, of 32 patients, was collected in october. compliance had increased to 68%.following discussion from presenting results at the cgf, the vte tool was appropriately modified.the responsibility of vte assessment was also shifted to becoming more shared, including all clinical staff, rather than mainly consultants. the third cycle, of 30 patients, was collected in november. compliance had increased to 80%.introducing a nursing care bundle with vte is in progress. conclusions: despite the identification of a risk in our clinical practice and the development of an appropriate it tool to facilitate improved practice, the advent of new national guidance revealed poor compliance with agreed standards. this shows the difficulties with achieving practice change in complex multiprofessional clinical environments. a sustained effort is required focusing on dissemination and engagement across the whole team. introduction: we describe the changes in anti factor xa (afxa) activity, thrombin generation and thromboelastography (teg) in critically ill patients with and without acute kidney injury (aki) following routine administration of tinzaparin as part of venous thromboembolism (vte) prophylaxis. methods: pilot prospective observational study. 18 patients divided into those with and without aki were administered tinzaparin by subcutaneous injection as per established local guidelines. 6 patients who did not receive tinzaparin were recruited as a 'control'. plasma afxa activity and thrombin generation were measured at intervals over a 24 hour period. teg parameters were collected at t0 and t24. results: afxa activity: results are shown in figure 1 . 3/18 patients failed to achieve a prophylactic afxa level of >0.2 at any point. 14/18 patients achieved a level of >0.2 however in all cases this was at the lower end of the prophylactic range and was achieved for only a short time (median 1.5 hours). 1/18 achieved a level of >0.2 for the whole 24h period. there was no difference between the aki and no aki groups. endogenous thrombin generation: there is no significant difference in thrombin generation between the aki and no aki groups. there is a significant decrease in thrombin generation between 0h and 5h (p<0.01) and a significant increase between 5h and 24h (p<0.01) (figure 2 ). there is no significant difference between 0h and 24h (p=0.90). teg: all teg parameters for all patients were within normal range conclusions: standard vte prophylactic dose tinzaparin rarely achieves an afxa range that has been suggested for vte prophylaxis. however, as assessed by thrombin generation, a hypo-coagulable state is generated in response to lmwh. there is no difference between critically ill patients with or without aki that would suggest the need for dose reduction in this context. 2 (abstract p334) . thrombin generation at 0h, 5h and 24h. t0 = time of tinzaparin administration, with the sample taken just prior to administration. patients from aki group shown with dotted line and from no aki shown with solid line 12% which takes the third place between cpb-associated complications . current data demonstrates the importance of researching of changes in haemostatic system in paediatric patiens after cpb. provided below data is an intermediate result of our research. methods: 39 patients in age up to 11 mohth 29 days (median age -5,5 months, youngest age -2 days after birth, oldest -11 months 29 days), who underwent cardiac surgery with cpb to treat congenital heart diseases, were enrolled in this study. all patients were divided into two groups: 1stwithout tc, 2ndwith tc. protein c (pc) and fibrin-monomer (fm) plasma levels were assessed in there points: before surgery, 24-hours and 72 hours after surgery. thrombotic cases were provided by doppler ultrasound or mri. results: thrombotic complications were diagnosed in 7 chidren (18%). between all tc ischemic strokes were diagnosed in 57% (4 cases), arterial thrombosis in 29% (2 cases), intracardiac thrombus in 14 % (1 cases). in group with tc fm-mean values in points 1,2 and 3 respectively were 9.62; 37 and 108 mcg/ml, meamwhile in group without thrombosis -7.5; 36.04 and 9.25 mcg/ml .pc-mean value in 1st groupwere 50; 56 and 76%, in the 2nd group -49; 52 and 39% respectively in the points 1, 2 and 3. statistically significant differences between groups in 3rd point (p<0.05) and correlation between pc and fm (r=-0.93; p<0.05) were detected. conclusions: cpb causes hypercoagulation with increasing of pc consumtion and fm level. moreover, cp associated with a high risk of tc on the 3rd day after cardiac surgery. further studies to investigate prognostic values of fm and pc in thrombosis are required. these studies would help to asses fm and pc as markers of tc and possibility of pc-prescribing for prevention and treatment of these complications. introduction: thrombocytopenia is a common condition in critically ill patients and an independent predictor of mortality. the relevance of a supranormal platelet count remains unclear. septic patients with disseminated intravascular coagulation (dic) are also known to have a high mortality, but the influence of sepsis on mortality rates in coagulopathic patients is less well characterised. our objectives were to: 1) evaluate mortality amongst patients with sepsis and nonsepsis associated dic. 2) assess incidence of dic during the first 7 days of admission. 3) assess the relationship between platelet count and mortality. methods: records of 935 adult critical care patients admitted to the royal liverpool university hospital between 2008-2014 were retrospectively reviewed. the presence of sepsis (using the definition of sirs with infection), coagulopathy, degree of thrombocytopenia and 28 day mortality were noted. modified isth dic score was used to define dic. results: the overall mortality rate was 19%. 522 patients were identified as having sepsis (56%) and 413 non septic patients (44%). mortality rates of patients with sepsis were significantly higher than without sepsis (71% vs 29% respectively, p<0.0001). in patients with dic, their dic scores tended to be 'positive' for the first 4 days of admission. fibrin-related markers were often not available for dic scoring. mortality rates amongst patients with sepsis-associated dic were greater than patients with non-sepsis related dic. thrombocytopenia severity was associated with mortality, and patients with platelets above the upper limit of normal had lower mortality rates (11% when platelets > 400x10^9/l, 30% when platelets <50x10^9/l). conclusions: sepsis-associated coagulopathy is associated with a higher mortality rate than non-sepsis associated coagulopathy. supranormal platelet counts may be associated with a mortality benefit. introduction: deep vein thrombosis (dvt) is a major problem in icu and affects overall lethality. dvt is widespread complication in icu, especially in elderly patients, when early activisation may not be achieved. aim of this study is comparison of haemostatic potential and analgesia methods of elderly patients who underwent major urological surgery during their stay in icu. methods: a cross-sectional study was employed. participants were ≥70 y.o., underwent major urological surgery, have had normal initial hemocoagulation data (thromboelastography was performed to all of them), had received analgesia with epidural catheter or iv by opioids use and were treated in icu >3 days due to non-coagulopathy states, were included. data were collected from october 2017 till october 2018. the patients were examined with thromboelastograph "mednord" for thromboelastogramm (teg) and with esaote usg for thrombi occurrence in lower limb deep veins. the anticoagulants were prescribed under the esa guidelines 2017. results: participants (n=30) were divided in two groups -non-opioid analgesia with epidural catheter (n=12) and opioid analgesia (n=18). we received moderate decrease in anticoagulants dosage to the patients with epidural analgesia with the same teg goals compared to the patients with opioid analgesia. other factors as comorbidities may provoke dvt events, but was not evaluated in this study. the dvt events were monitored by expert with the use of usg to locate thrombi in the vein. conclusions: use of epidural catheter analgesia provides moderate decrease of anticoagulants dosage compared to opioid analgesia patients; however strict control of teg data must be presented. comorbidity need to be monitored for early detection and prevention of dvt events. introduction: patients with morbid obesity (mo) have a high risk of thromboembolic events. in patients with a bmi >35, the hypercoagulable state is due to impairment of all parts of the blood coagulation as well as anticoagulation mechanisms by obesity. methods: the hemostasis system was studied in 100 patients with a bmi> 35 kg/m2 with various pathologies that were admitted to icu. all patients were divided into 2 groups depending on the type of therapy: 1 group (n=50) received monotherapy with enoxaparin sodium 0.1% 0.2 ml sc 2 times a day every 12 h; group 2 (n=50) received combination therapy with enoxaparin sodium 0.1% 0.2 ml sc 2 times a day every 12 h and pentoxifylline 100 mg 2 times a day every 12 h. to study the hemostasis system, we used lpteg immediately after hospitalization, on 1, 3, 5 days. results: in both groups, prior to treatment: contact coagulation intensity (icc) was increased by 23.57%, intensity of coagulation drive (icd) -by more than 32.68%, clot maximum density (ma) -by 74.52%, index of retraction and clot lysis (ircl) -91.18% above normal. patients of the 1st group: icc increased by 12.62%, icd was close to normal values, ma increased by 18.63%, ircl was increased by 31.17%. patients of the 2nd group on the 5th day: icc decreased by 15.22% compared with the norm; the coagulation and fibrinolysis parameters were close to normal values and the decrease in fibrinolysis activity reaches to normal. conclusions: combined therapy of thromboembolic complications in patients with obesity sodium enoxaparin sodium and pentoxifylline is more effective than enoxaparin sodium monotherapy because it affects all parts of the hemostatic system. introduction: a laryngeal injury secondary to blunt neck trauma can lead to life-threatening upper airway obstruction [1, 2] . ultrasound enables us to identify important sonoanatomy of the upper airway [3] . the purpose of this report is to discuss role of pocus airway in blunt neck trauma and to determine airway management based on standard schaefer 5 subgroups classification. methods: three cases of blunt neck trauma presented to our centre with either subtle or significant clinical signs and symptoms. standard airway management was performed prior to pocus airway using 15mhz linear transducer and it findings were later compared to flexible fibreoptic laryngoscopy and computed tomography (ct). results: pocus airway had identified one out of 3 cases to have schaefer 2 and the remaining as schaefer 3. all pocus airway findings were confirmed with flexible fibreoptic laryngoscopy and ct scan (figs 1, 2) . based on schaefer, supportive care and early steroid administration are advisable for group 1 and 2. for groups 3 to 5, immediate open surgical repair is deemed necessary due to extension of injuries.all cases were intubated using glidescope.all including those presented with schaefer 3 were managed conservatively and discharge well with proper follow-up. conclusions: upper airway ultrasound is a valuable, non-invasive and portable for evaluation of airway management even in anatomy distorted by pathology or trauma. an organised approach using pocus airway as an adjunct can expedite care and prevent early and long term complications in facilities without flexible laryngoscope and ct. introduction: high-flow nasal oxygen (hfno) and helmet noninvasive ventilation (hniv) are increasingly used for the early management of acute hypoxemic respiratory failure (ahrf). we compared the physiological effects of hfno and hniv during ahrf. methods: in this randomized cross-over study, we enrolled patients with acute-onset (<7 days), non-cardiogenic respiratory distress (respiratory rate>25/min), pulmonary infiltrates at the chest-x-ray and hypoxemia (spo2<90% while breathing on room air). all patients received hniv (peep 10 cmh2o, pressure support adjusted to achieve a peak inspiratory flow of 100 l/min) and hfno (flow 50 l/min) for one hour each, in a randomized cross-over manner. at the end of each period, arterial blood gases, inspiratory effort (esophageal pressure) and respiratory rate were recorded. self-assessment of dyspnea and device-related discomfort ( [3] [4] [5] [6] [7] ). conclusions: as compared to hfno among critically ill patients with ahrf, hniv ameliorates oxygenation, limits inspiratory effort and relieves dyspnea, without affecting paco2, respiratory rate and comfort. introduction: pre-intubation hypoxemia is a predictor of negative patient outcomes including in-hospital mortality. while successful first intubation attempt is also an important factor of patient outcomes, little is known about whether physicians achieve successful first intubation attempt for the hypoxemic patients in the emergency department (ed). the aim of this study is to investigate the first-pass success for patients with pre-intubation hypoxemia in the ed. methods: this is an analysis of the data from the second japanese emergency airway network study (jean-2 study)a multicenter, prospective, observational study of 15 eds in japan. we included all patients who underwent intubation in the ed from 2012 through 2018. we excluded patients 1) aged <18 years and 2) patients who underwent intubation for cardiac arrest. we grouped pre-intubation hypoxemia as follows: non-hypoxemia (oxygen saturation [spo2], ≥97%), moderate-hypoxemia (spo2, 96%-90%), and severehypoxemia (spo2, <90%). primary outcome was the first-pass success rate. to demonstrate the association between pre-intubation hypoxemia and the first-pass success in the real-world setting, we fit two unadjusted logistic regression models 1) using grouped preintubation hypoxemia as a categorical variable and 2) using the preintubation spo2 as a continuous variable. results: among 10,144 patients who underwent intubation in the ed (capture rate, 97%), 5,463 patients were eligible for the analysis. compared to the non-hypoxemia, the first-pass success rate was low in moderate-hypoxemia (72% vs 67%; or=0.77 [95%ci, 0.67-0.89]) and severe-hypoxemia (72% vs 69%, or=0.85 [95%ci, 0.72-0.99]). additionally, there was a linear association between pre-spo2 and lower first-pass success rate (or for the success, per one pre-spo2 decrease, 0.99 [95%ci, 0.98-0.99]). conclusions: based on the large, multicenter data, the first-pass success rate was low in hypoxemic patients compared to nonhypoxemic patients in the ed. introduction of rapid-sequence induction guideline to reduce drug-associated hypotension in critically unwell patients introduction: the aim of this project was to assess whether the introduction of a rapid sequence induction (rsi) agent guideline changed drug choice and the incidence of peri-intubation vasopressor use at st john's hospital, livingston. it is well documented that emergency airway management in the critically ill can be a source of significant morbidity and mortality [1, 2] and the choice of induction agent matters [3] . methods: an rsi agent guideline was instituted for all critically ill patients being intubated in icu and the ed [ figure 1 ]. following this, we set up an intubation registry to collect data from all intubation events. this data was then compared to a previous audit of intubations completed in 2012. results: the choice of agent used pre-and post-intervention are summarized in figure 2 . forty-five intubation events were included in the initial audit in 2012, of which, 7 (16%) required vasopressor support immediately following intubation. of the 41 intubation events following the guideline's introduction, 2 (5%) required vasopressors. ketamine use changed from 0% to 51%, propofol use from 43% to 29% and midazolam from 14% to 0%. thirty-eight of these intubation events (93%) were compliant with the guideline. conclusions: the introduction of the rsi guideline dramatically affected the choice of induction agent and reduced the incidence of significant hypotension requiring vasopressors (5% versus 16%). overall compliance with the guideline was excellent (93%). introduction: the purpose is to test the feasibility of using the i-gel® device for airway maintenance during bronchoscopic-guided percutaneous dilatational tracheostomy (pdt). usually pdt is accomplished via the tracheal tube. failure to position the endotracheal tube correctly can result in further complications during the procedure. the alternative implies extubation and reinsertion of an i-gel® airway device. methods: the pdt was performed using the blue dolphin method in 5 patients in intensive care unit. before undertaking bronchoscopicguided percutaneous dilatational tracheostomy (pdt), the patient's tracheal tube (et) was exchanged for i-gel®, as a ventilatory device for airway maintenance. the insertion of the i-gel®, the quality of ventilation, the blood gas values, the view of the tracheal puncture site, and the view of the balloon dilatation were rated as follows: very good (1), good (2), barely acceptable (3), poor (4), and very poor (5) [1] . results: the i-gel® successfully maintained the airway and allowed adequate ventilation during percutaneous tracheostomy in all patients. the ratings were 1 or 2 in 100% of cases with regards to ventilation and to blood gas analysis, for identification of relevant structures and tracheal puncture site, and for the view inside the trachea during pdt. conclusions: the i-gel® successfully maintained the airway and allowed adequate ventilation during percutaneous tracheostomy in all patients. the ratings were 1 or 2 in 100% of cases with regards to ventilation and to blood gas analysis, for identification of relevant structures and tracheal puncture site, and for the view inside the trachea during pdt. no damages to the bronchoscope, reports of gastric aspiration or technical problems were detected. the bronchoscopic view obtained via an i-gel® seems to be better than that obtained through an endotracheal tube (et) or through traditional laryngeal mask [1] . introduction: the purpose of this study was to investigate the efficiency of nasal airway inserted in the oral airway (on airway) in securing the airway patency during mask ventilation [1] (fig 1) . methods: fifty eight patients undergoing general anesthesia were randomly assigned to either oral airway group (group o) or on airway group (group n). in both group, 2 mg/kg of propofol was infused intravenously and mask ventilation was performed in the sniffing position without head extension or jaw thrust. the patients were ventilated with a volume-controlled ventilator with o2 flow of 10 l/min, tidal volume of 10 ml/kg (ibw), and respiratory rate of 10 /min. before the start of mask ventilation, airway was placed in the oral cavity. oral airway was used in group o and on airway was used in group n. peak inspiratory pressure (pip), tidal volume and etco2 were compared between the two groups. the location of airway tip was graded by fiberoptic bronchoscope as; 0: airway obstructed by tongue, 1: epiglottis visible, 2: airway touches epiglottis tip, 3: airway passes beyond epiglottis tip [2] . methods: a prospective uncontrolled observational study in 2017-18 in 4 ukrainian hospitals. 10 sma-pts from 6-18 mo were involved. all pts. ready for extubation: afebrile, no infiltrations on chest x-ray, normal wbc. however, each sma-pts. failed sbt (t-tube or psv). we evaluated: extubation success (no reintubation in 48 hours), icu los, one year survival. three pts. were excluded: two pts. by staff decision, 1 family have choosen tracheostomy. 7 sma-pts. included. a cuff leakage test performed -with a negative, dexamethazone 1mg iv was administered. after extubation niv was started by ventilogik ls in st mode via nasal mask giraffe. the epap and ipap settings were titrated to reach the chest excursion and target levels of spo2 (92-96%) and etco2 (40-45 mmhg). a sputum was draining by mechanical insufflation-excuflation (mie) and aspirator results: all pts, were extubated successful. the mean icu los was 8.5 days (7-10 days), one year survival rate was 100%, respiratory failure fully compensated by niv, there was no icu admission. every sma-pts. are in good condition, gaining weight introduction: aerosol delivery has previously been assessed during simulated adult hfnt, delivered by various stand-alone humidification systems [1] . the objective of this study was to evaluate aerosol delivery during simulated hfnt delivered by a mechanical ventilator, across three clinically relevant gas flow rates. methods: 2ml of 2 mg/ml salbutamol was nebulised using an aerogen solo nebuliser (aerogen, ireland). an adult head model was connected to a breathing simulator (asl5000, ingmar, us), vt 500 ml, bpm 15 and i: e, 1:1 (fig 1) . hfnt was supplied via the servo-u ventilator (maquet, getinge, sweden), using the integrated nebulisation option. tracheal dose was recorded at two nebuliser positions; a (after the humidification chamber) or b (before of the cannula), at three gas flow rates (10lpm, 30lpm and 60lpm) (n=3). the mass of drug captured on a filter placed distal to the trachea (tracheal dose) was quantified using uv spectroscopy at 276 nm. results: presented in table 1 . conclusions: to our knowledge, this is the first study to successfully demonstrate aerosol delivery during simulated hfnt, delivered by a mechanical ventilator. increasing gas flow rate was associated with a reduced tracheal dose (p= <0.0001). at 10lpm, a significantly greater tracheal dose was observed when the nebuliser was positioned before the nasal cannula (p= <0.0001). at 60lpm, a greater tracheal dose was yielded when the nebuliser was positioned after the humidifier (p= <0.0001). introduction: tracheotomies are often performed in critically ill patients who are in need of prolonged mechanical ventilation and respiratory care. our aim was to evaluate the possible effect of percutaneous and surgical tracheotomies on thyroid hormone levels. methods: eighty seven adult patients were included in our study from january 2017 to september 2018. patients were in need of prolonged mechanical ventilation and tracheotomies were performed after consent was taken. we have excluded patients with preexisting thyroid diseases. forty five patients were undergone percutaneous tracheotomies and forty two patients were undergone for surgical. thirty eight female patients and forty nine male, age range 18-85. we studied tsh, t3 and ft4 serum levels using chemiluminescence immunoassay method before either procedure and 2 hours post each procedure.: statistical analysis was performed using spss 15. significance was estimated at the level of p< 0.005 results: tsh levels were increased in surgical group compared to percutaneous group at 2 hours post procedure but the difference was not found statistically significant (p> 0.005). the rise in post operative levels of t3 compared to preoperative was found statistically significant for surgical tracheotomy group (p< 0.005).elevated ft4 levels for both groups have shown statistically significant difference between preoperative and postoperative period for the surgical tracheotomy group (p< 0.005) conclusions: we analyzed the effect of surgical versus percutaneous tracheotomy on thyroid hormones and it was found that both introduction: insertion of a tracheostomy for weaning purposes is associated with prolonged critical length of stay (los) and several adverse patient outcomes [1] . previous work has suggested that protocolised weaning may reduce weaning times [2] . we aimed to assess the impact of protocolised weaning on los following introduction of a standardised weaning protocol in 2017. conclusions: introduction of a standardised weaning protocol for patients with a tracheostomy in our unit has had a beneficial effect on several patient outcomes, notably duration of weaning and length of critical care admission. introduction: delirium is a relatively frequent neurologic complication in liver transplantation (lt) recipients, which is an important cause of increased morbidity, mortality, extended icu stay, and increased cost of medical care. extubation of the endotracheal tube at an appropriate timing is an essential part of intensive care after lt, suggested to improve graft perfusion and systemic oxygenation, and thus decrease intensive care unit (icu) stay and positively affect prognosis. the aim of this study was to compare the incidence of delirium between early and late extubation groups after lt. methods: medical records from 247 patients who received lt from january 2010 to july 2017 in a single university hospital were retrospectively reviewed. patients were divided into 2 groups: those who underwent early extubation after lt (group e, n = 52) and those who underwent extubation within few hours of icu admission after surgery (group c, n = 195). the data of patients´demographics, perioperative management, and postoperative complications were collected. early extubation was defined as performing extubation in the operating room after lt. a propensity score matching analysis was performed to minimize the effects of selection bias. results: postoperative delirium occurred in 4/52 (7.69%) in group e and 30/195 (15.38%) in group c, respectively (p = 0.15). after propensity score matching, there was no difference in icu stay (p = 0.96), time to discharge after surgery (p = 0.12), and incidence of delirium between groups (p = 1.00). conclusions: although this study is retrospective in nature, limited by small sample size, early extubation did not affect the incidence of delirium after lt. further prospective studies on this area are required. weight estimation and its impact on mechanical ventilation settings in queen elizabeth hospital intensive care unit a nasr, a iasniuk, a roshdy queen elizabeth hospital, icu, london, united kingdom critical care 2019, 23(suppl 2):p353 introduction: documented weight in the intensive care unit (icu) can be the total, ideal, adjusted or predicted body weight (pbw). lung protective ventilation depends on tidal volume (vt) delivery which is based on accurate calculation of patients´weight [1] . the weight is most probably documented on admission to the icu using estimation or one of many available equations. the aim of this study is to assess the documented versus the pbw and its impact on tidal volume delivery for mechanically ventilated patients in queen elizabeth hospital icu. methods: data was collected prospectively from all ventilated patients over a period of 2 weeks in june 2018. vt delivered in the first hour was calculated for each patient. documented body weight and height of each patient was obtained from the nursing chart. pbw was calculated and compared with the documented weight. the difference in vt attributable to the difference in weight has been subsequently calculated. results: 29 ventilated patients were included (17 males). the mean tidal volume delivered according to the documented body weight was 7.16 ml/kg versus 8.28 ml/kg based on pbw. vt more than 8 ml/ kg was delivered in 31% of patients based on documented weight versus 48% when correcting the weight according to the pbw equation. conclusions: inaccuracy in documenting weight on patients´admission to the icu is a potential cause of delivering unsafe tidal volume [2] . the harm can extend to drug dosage, nutrition provision and renal replacement therapy. introduction: ventilator-associated pneumonia (vap) is the leading cause of death among mechanically ventilated critically ill patients [1] . chest radiography (cxr) is essential in the diagnosis of vap. in the past decade lung ultrasonography has proven to be a valuable tool in the diagnosis and monitoring of lung diseases. the aim of the study is to assess sensitivity and correlation between cxr, lung ultrasound and clinical pulmonary infection score (cpis). methods: in this retrospective, non-randomized study seven patients with proved vap were enrolled. in all patients cpis and lung ultrasound score (lus) [2] were assessed. comparison of patients that had lus≥18 and cpis≥6 points was performed. the correlation between lus and cxr was done using the pearson model. results: we found significant difference between positive cxr patients with lus≥18 and cpis≥6 (100% vs 57%, p<0.05). there is a very high correlation between cxr and lus. these results render lung ultrasound as a highly sensitive tool in the diagnosis of vap. conclusions: our study shows that lung ultrasonography could be used as a reliable supplementary method in the diagnosis of vap. the benefits of lung ultrasound include the ability to perform it at the patient´s bed without need for transportation, no radiation exposure and repeatability. the high correlation between cxr and lung ultrasound makes echography a valuable adjunct in the diagnosis of vap. color introduction: it is difficult to differentiate between pneumonia and atelectasis as cause of lung consolidation in intensive care unit patients. tools like the clinical pulmonary infection score are of little help (sensitivity 60% and specificity 59% for detecting pneumonia) [1] . the objective of this study was to determine the accuracy of ultrasound assessed vascular flow within the consolidation to distinguish these causes. methods: adult patients with pulmonary symptoms and lung consolidation on lung ultrasound that were scheduled for chest-ct were included. vascular flow was analyzed with color doppler imaging (flow velocity scale was chosen at 0.25m/sec.). the final diagnosis made by the treating physician was regarded as the gold standard. results: 20 patients were included of which nine (45%) were diagnosed with pneumonia. vascular flow in the consolidation was present in seven (78%) out of nine patients with pneumonia, compared to three out of 11 (27%) patients with atelectasis (p = 0.07). the diagnostic accuracy in differentiating between pneumonia and atelectasis was 75%. the sensitivity and specificity were 78% and 73% respectively. the positive predictive value was 70% while the negative predictive value was 80%. conclusions: vascular flow in lung consolidations assessed by lung ultrasound in icu patients aids in differentiating between pneumonia and atelectasis. it outperforms the frequently used clinical pulmonary infection score. methods: three intubated patients for various causes of respiratory distress undergoing mechanical ventilation were subjected to tee. at the level of mid-esophagus, the descending aorta short-axis view (0°) the imaging plane is directed through the transverse axis of the descending aorta. sector depth was increased to image the left pleural space beneath the aorta. for the right lung, the tee is rotated to the right at the level of atria until lung is seen or until the image of the liver is seen and the probe was withdrawn until the right lung is seen. recruitment manoeuvres were performed after identifying pbl atelectasis. atelectatic lungs were visually observed to open up during and after the recruitment manoeuvres. results: the time to acquire the image of pbl atelectasis from the time of insertion by tee is short. the images of posterior lung and the effect of lung recruitments is successfully viewed (fig 1) . no immediate complication seen. conclusions: tee provides an excellent view of pbl atelectasis and able to directly monitor the success and failures of recruitment manoeuvres. introduction: high respiratory driving pressure (δ prs) is strongly associated with increased risk of lung injury and increased mortality during mechanical ventilation. δ prs consists of the pressure required to distend the lung the transpulmonary driving pressure (δ pl) and the pressure required to distend the chest wall. δ pl is the pressure that increases the risk of lung injury. data on δ pl is limited because its measurement requires an esophageal catheter. we aimed to assess changes in δ prs and δ pl during proportional assist ventilation (pav+) at different experimental conditions. methods: we retrospectively analyzed patients ventilated with pav+ who had esophageal pressure measurements before and after dead space or chest load addition. we calculated end-inspiratory plateau pressure (pplateau), δ prs, respiratory system compliance (crs) and δ pl during occluded breaths in pav+ (figure 1 ). data were compared with wilcoxon signed rank test and p value<0.05 was considered significant. results: 16 patients were analyzed. dead space increase (9 patients) did not affect the studied parameters. chest load (7 patients) significantly increased pplateau (p=0.03) and δ prs (p=0.02) and decreased crs (p=0.02) but δ pl remained the same (p=0.4). median (iqr) changes were 9.5 ml/cmh2o (7.7-13.2) for crs, 2.9 cmh2o (2.4-5.1) introduction: particle flow in exhaled air from mechanically ventilated patient's mirrors the opening and closing of small airways and can be detect by optical particle counter [1] . we hypothesized that this particle flow is affected by cardiac function. methods: exhaled air from 20 mechanically ventilated patients was analyzed using a customized optical particle counter pexa, figure 1 . introduction: we assessed the diagnostic accuracy of mechanical power (mp) and driving pressure (dp) alone and combined with stress index (si) to identify ventilator settings likely to produce ventilator induced lung injury caused by tidal hyperinflation [1] [2] [3] . methods: secondary analysis of a previous database of ards patients [1] . computerized tomography markers of tidal hyperinflation (were used as a "reference standard". analysis of the area under the receiver-operating characteristics curve (auc) was used using a two-fold cross-validation. results: in a cluster of 44 patients, a "training set" of 28 not hyperinflated patients was compared with a "validation set" of 14 hyperinflated patients. (figure 1-2) . conclusions: si seems to be more accurate than mp and dp in identifying tidal hyperinflation in patients with ards. specificity and sensibility were not improved combining si with mp or dp. the introduction: the pao 2 /fio 2 (p/f) ratio is widely used to assess the severity of lung injury. conceptually, the p/f ratio should be independent of the fio 2 and solely depend on the pulmonary condition. however, effect of fio 2 modulation on the p/f ratio has not been well characterized in ventilated intensive care (icu) patients. the purpose of the present study was to investigate the relationship between fio 2 and the p/f ratio in icu patients on mechanical ventilation. methods: in a prospective, interventional study 10 patients with a swan ganz catheter in situ were included. the p/f ratio was calculated at fio 2 levels ranging from 0.21 to 1.0 with 10 minute intervals. during the study other ventilator settings were not modulated. to understand the physiological effects of fio 2 modulation on gas exchange and hemodynamics, mixed venous oxygen saturation and cardiac output were assessed. shunt fraction was calculated as described by west [1] . results: patient characteristics and ventilator settings are reported in table 1 . all patients were admitted to the icu after elective cardiac surgery. modulation of fio 2 did have a significant effect on the p/f ratio, following a u-shaped pattern (p < 0.05) (figure 1 ). the shunt fraction varied with altering fio 2 levels, also exhibiting a u-shaped pattern (p < 0.05) (figure 2 ). cardiac output was not affected by fio 2 . conclusions: in contrast to current thinking, the p/f ratio varied substantially with altering fio 2 levels in mechanically ventilated icu patients. this is an important novel physiological observation. in addition, it demonstrates that the assessment of the severity of respiratory failure by using the p/f ratio should be standardized to a fixed fio 2 level. conclusions: in patients undergoing prolonged mechanical ventilation, we must take into account all the factors that may affect our patients. the assessment of diaphragmatic dysfunction is key to preventing weaning failure. an optimal level of consciousness as well as a good management of secretions are key to a successful weaning. prognostic value of the minute ventilation to co 2 production ratio as a marker of ventilatory inefficiency in the icu r lopez 1 , r pérez 1 , á salazar 1 , i caviedes 2 , j graf 1 introduction: ventilatory inefficiency for co2 clearance may provide better severity stratification in acute respiratory failure than oxygenation [1] . ventilatory inefficiency (vi) is best assessed by the bohr-enghoff physiological dead space [2] . we recently reported that the minute ventilation to co2 production ratio (ve/vco2), a simplified vi index from exercise testing that obviates the paco2 measurement, correlates better than other vi indices to physiological dead space in mechanically ventilated patients [3] . here we report the prognostic performance of this index using a survival analysis. mean±sem ve/vco2 was higher in patients who died than those who survived (58±8 vs 43±1, p<0.001, figure 1 ). we found a ve/ vco2 cutoff value of 45. mortality was higher in patients with high-ve/vco2 (≥45) as compared to those with low-ve/vco2 (21% vs 4%, p=0.021) with an odds ratio of 6.7 [95%-ci 1.2-35.8]. cumulative mortality was higher in the high-ve/vco2 than in the low-ve/vco2 group (log-rank p=0.015, figure 2 ). conclusions: in this unselected cohort of mechanically ventilated patients an early high ve/vco2 ratio was associated to 28-days mortality. the ve/vco2 ratio may be a simple and non-invasive vi index with prognostic value in this population. introduction: sodium thiosulfate (sts) is a clinically relevant and safe hydrogen sulfide donor that improved acute lung injury (ali) and brain ischemia/reperfusion injury in previous studies [1, 2] . methods: in a prospective, controlled, randomized, and doubleblinded trial, twenty adult, anesthetized, mechanically ventilated and surgically instrumented swine with preexisting coronary artery disease [3] underwent 3 h of hemorrhagic shock (hs; removal of 30% of the calculated blood volume and subsequent titration of mean arterial pressure to 40mmhg). post-shock resuscitation (72h) comprised re-transfusion of shed blood, crystalloids, and norepinephrine. animals were randomly assigned to "placebo" or "sts" (0.1g·kg -1 ·h -1 for 24h). before, at the end of and every 24h after shock, hemodynamics, blood gases, and lung function were recorded. results: survival rates did not differ between groups. sts-infusion attenuated the hs-induced impairment of lung mechanics and pulmonary gas exchange (table 1 ,2), resulting in a significantly higher horovitz/peep-ratio ( figure 1 ). conclusions: sts during acute resuscitation from hs may protect comorbid swine against hs-induced ali. introduction: alveolar epithelial cell (aec) death is a main mechanism of severe respiratory failure in acute respiratory distress syndrome (ards). classically, cell death is classified into necrosis or apoptosis. recent studies have reported that not only apoptosis but also certain types of necrosis are molecularly regulated and that these regulated necrosis can be therapeutic targets for various diseases. however, the relative contribution of necrosis and apoptosis to aec death in ards has not been elucidated. our study aimed to elucidate which type of cell death is dominant in aec death and to evaluate whether the regulated necrosis is involved in lps-induced experimental ards. methods: we established ards model by instilling 25μ g of lps intratracheally to mice. to estimate the relative proportion of apoptosis and necrosis in aec death, we measured cytokeratin18 m65 level (total cell death marker) and m30 level (apoptosis maker) in bronchoalveolar lavage fluid (balf) by elisa, and quantified propidium iodide-positive necrotic cells and tunel-positive apoptotic cells in the lung sections. moreover, we performed pathway enrichment analysis of gene expression data from pcr array to evaluate whether regulated necrosis pathway is associated with the ards model. results: both m65 and m30 levels were increased in the ards mice. the m30/m65 ratio (an indicator of the proportion of apoptosis to total cell death) in the ards mice was significantly lower than that of healthy controls. moreover, the number of propidium iodidepositive necrotic cells was significantly higher than that of tunelpositive apoptotic cells in ards mice. in the pathway enrichment analysis, the necroptosis pathway, a regulated necrosis pathway, was associated with lps-induced experimental ards. conclusions: aec necrosis is more dominant than apoptosis in lpsinduced ards model. moreover, necroptosis may contribute to ards pathogenesis. aec necrosis including necroptosis is a potential therapeutic target for ards. clinical ards diagnosis is not associated with a unique circulating neutrophil cell surface phenotype t craven 1 , s duncan 1 , s johnston 2 , c haslett 1 , k dhaliwal 1 , t introduction: acute respiratory distress syndrome (ards) is a form of non-cardiogenic oedema due to alveolar injury secondary to an inflammatory process. the clinical diagnosis is defined by the berlin criteria but this may not reflect the underlying biological process. the activated neutrophil is central to the pathogenesis of ards, characterised by altered cell surface markers. methods: three cohorts of seven participants were recruited. the first cohort suffered from mild, moderate or severe ards as defined by the berlin criteria [1] . the second cohort was composed of ventilated patients on the intensive care unit with acute inflammatory lung disease (diagnosis of clinical suspicion) but did not meet the berlin criteria for ards. a third cohort was composed of age and sex matched healthy volunteers. procurement of human tissue was approved by a regional ethics committee (14/ss/1074 or 08/s1103/38 or amrec: 15-hv-013) and with the informed consent of the participant or their personal legal representative. patients were excluded if aged under 16 or over 80 years of age, were expected to survive for less than 24 hours, if the attending physician refused, due to the absence of suitable indwelling vascular catheter, if the haemoglobin concentration was below 6.5 g/dl, or if the patient was enrolled in a trial of novel anti-inflammatory agent. whole blood (lysed erytocytes) underwent flow cytometry to determine cd11b, 63, 66b, 88, 62l and 16. results: a description of the enrolled cohorts can be found in table 1 . there were no significant differences between the mechanically ventilated, critically ill cohorts for any cell surface molecule in the multiplicity adjusted p values (fig 1) . the results support the conjecture that clinical diagnostic criteria should not be used as a surrogate to stratify patients according to biological changes, with implications for the testing of biological therapies. introduction: aim of the present study was to compare the global and regional diagnostic accuracy of lung ultrasound (lus) compared to lung computed tomography (ct) scan in patients with the acute respiratory distress syndrome (ards). ards is characterized by a diffuse, inhomogeneous, inflammatory pulmonary edema. lung ct scan is the reference imaging technique, but requires transportation outside the intensive care and exposes patients to x-rays. lung ultrasound (lus) is a promising, inexpensive, radiation-free, tool for bedside imaging. methods: lung ct scan and lus were performed at peep 5 cmh2o. lus was performed using a standardized assessment of 6 regions per hemithorax: superior and inferior; anterior, lateral and posterior. each region was classified for the presence of normally aerated, alveolar-interstitial syndrome, consolidation regions and pleural effusion. agreement between the two techniques was calculated, and diagnostic parameters were assessed for lus using lung ct as a reference. both a global and a regional analysis were performed. results: thirty-two sedated and paralyzed ards patients (age 65±14 years, bmi 25.9±6.5 kg/m2 and pao2/fio2 139±47) were enrolled. global agreement between lus and ct was 0.811±0.032. the overall sensitivity and specificity of lus are shown in table 1 . similar results were found with regional analysis (anterior/lateral/posterior lung regions is a common practice in our icu. during the interruption eit belt was positioned. when the presence of spontaneous breathing activity was evident by clinical assessment and ventilator traces analysis, nmba were administered to reach full paralysis, in accordance with the treating physician. eit tracing were analyzed offline and the change in eeli after nmba bolus, as compared to before nmba administration, was measured. respiratory mechanics and arterial blood gas (abg) data were collected results: we enrolled 5 ards patients, undergoing controlled mechanical ventilation with muscle paralysis. baseline respiratory mechanics and abg data are shown in table 1 . in 4 out of 5 patient the bolus of nmba led to an increase of eeli. in 1 case, the nmb administration led to no changes in eeli. the mean change in eeli was 159 ±152ml conclusions: in our small population of ards patients, the administration of a bolus of nmba after the regain of spontaneous breathing activity led to an increase in eeli in 4 out of 5 patients. further study are needed to 1) correlate this increase to global and regional respiratory system compliance and 2) correlate this increase to the time needed to wean the patient from nmba introduction: to analyze the use of the orthostatic board as an auxiliary device for the treatment of severe ards by assessing its risks and benefits. methods: we selected 91 patients, 43 females and 48 males, hospitalized in a neurological icu, between june 2014 and july 2018, in a physiotherapeutic follow-up with diagnosis of severe ards. the patients were submitted to orthotics assisted for 40 to 60 minutes and monitored hr, pam, fr, sato2 at 30°and 60°of inclination and the pao2 / fio2 ratio after the procedure. the mean number of sessions per patient was 6.6. all patients were undergoing anticoagulation in rass -5, in the treatment of the cause of ards. the mean time of mechanical ventilation was 8.5 days. results: among the patients selected, 36.3% presented tachycardia above 115 bpm, requiring intervention in 12.1% and interruption of the procedure in 6.6%. pam arterial hypotension <65 mmhg was observed in 34.1%, requiring intervention (increase of vasopressor dose and / or change of plank angulation) in 22% and interruption of the procedure in 14.3%. hypoxemia sato2 <92% was observed in 8.8%, without interruption, but an improvement in pao2 / fio2 was observed in only 95.6% of the patients. conclusions: assisted orthostatism as an auxiliary device for the treatment of severe ards was shown to be an alternative, with improvement of pao2 / fio2 in 95.6% of the patients, safe and without significant hemodynamic repercussions that could lead to interruption of the procedure. introduction: the eolia trial found that vvecmo compared to conventional mechanical ventilation (cmv) did not improve mortality in patients with severe ards [1] . the cmv strategy consisted of airway pressures below 30cmh 2 o. in patients with severe ards higher airway pressures are required to maintain lung aeration. grasso et al. measured the transpulmonary pressure (p l ) in patients with severe ards and increased peep until p l was 25cmh 2 o, accepting airway pressures above 30cmh 2 o. fifty percent of patients responded to an increase in airway pressure and did not require vvecmo [2] . we hypothesized that a p l guided open lung concept (olc) improves oxygenation and prevents conversion to vvecmo in patients with severe ards. methods: a retrospective study was conducted in a tertiary referral icu. the records of patients referred to our icu for advanced medical care were reviewed. inclusion criteria were severe ards according to the berlin definition and the eolia trial inclusion criteria for vvecmo. results: mechanical ventilation was limited to a p l of <25cmh 2 o instead of plateau pressures below 30cmh 2 o. the p l guided olc resulted in an increase in p/f ratio and none of the patients required vvecmo. during the first 6 hours peak airway pressure was increased, but was reduced within 24 hours while peep was maintained ( fig. 1 ). at 72 hours both peak airway pressures and peep were reduced to baseline values while p/f ratio remained stable. only one patient (12.5%) died of disseminated invasive aspergillosis. conclusions: the p l guided olc improved oxygenation and none of the patients required vvecmo. these findings support a ventilation strategy guided by transpulmonary pressures instead of plateau pressures in patients with severe ards. introduction: the mortality benefit conferred by early prone positioning in the treatment of acute respiratory distress syndrome (ards) has been well established. we also know that aprv improves oxygenation, and more recently has been shown to reduce ventilator dependent days and icu length of stay [1, 2] . however, controlled ventilation remains the mainstay mode of ventilation used during prone position. literature looking at combined aprv and prone positioning is scarce. we aim to explore and report our institutional experience with respect to feasibility and outcomes in combining aprv and prone positioning, and perform a literature review in this area. methods: we undertook a single-centre retrospective cohort study within a surgical icu of a tertiary hospital in singapore between jan 2013 -oct 2017. patients with ards who received combined prone positioning and aprv were reviewed retrospectively. a literature review of patients with ards who received combined intervention was also performed. results: 5 adult patients aged 21-77 years old diagnosed with ards received a combination of aprv and prone positioning for a duration of 16-45 h ( table 1 ). all the patients tolerated aprv with prone positioning well. our patients saw an improvement of p:f ratio ranging from 59-225 upon completion of combination therapy. 3 out of 5 patients were extubated within 72 hours of turning supine, 1 was weaned to tracheostomy mask after 14 days and 1 died while on the ventilator. only 1 case report and 1 randomized clinical trial were found on this topic upon literature review, which corroborated our findings. conclusions: in our experience, aprv is a practical and feasible alternative mode of ventilation that can be employed in the prone position, yielding significant p:f ratio improvements. the synergistic effects on improving oxygenation herald potential, especially in the subset of severe ards patients with refractory hypoxemia, where extracorporeal membrane oxygenation is unsuitable or unavailable. introduction: the recirculation during veno-venous extracorporeal membrane oxygenation (vv ecmo) had been a drawback, which could limit sufficient oxygenation. purpose of this study is to compare the short-term oxygenation in acute respiratory distress syndrome (ards) patients under vv ecmo according to their cannula configurations, especially in the national environment of the absence of newly developed double-lumen, single cannula. introduction: vv-ecmo is most commonly used in severe potentially reversible respiratory failure. this report looks at two patients in whom vv-ecmo was used to facilitate surgical airway stenting. methods: case 1-a 56-year-old with recurrent respiratory arrests, on a background of neurofibromatosis type 1 and kyphoscoliosis. he had complex airway pathology, including, airway neurofibromas and granulation tissue, tracheobronchomalacia, severe kyphoscoliosis and a permanent tracheostomy tube. rigid bronchoscopy was performed and following debridement of granulation tissue, a trouser-leg stent was deployed. case 2-a 75-year-old with progressive stridor due to recurrence of a malignant melanoma, which was causing mid-lower tracheal compression. three tracheal stents were deployed via a rigid bronchoscope. in both cases, percutaneous bi-femoral vv-ecmo was established prior to general anaesthesia and decannulation took place the following day. results: in these cases, vv-ecmo provided stable extracorporeal gas exchange without conventional tracheal intubation. cardiopulmonary bypass and veno-arterial ecmo have been described in patients at risk of compression of the heart and distal airway [1] . however, if the major threat is airway collapse, vv-ecmo can provide cardio-respiratory support without the problems associated with arterial cannulation and with lower anticoagulation requirements. introduction: ecco2r facilitates the use of low tidal volumes during protective or ultraprotective mechanical ventilation when managing patients with acute respiratory distress syndrome (ards); however, the rate of ecco2r required to avoid hypercapnia remains unclear. methods: we determined ecco2r requirements to maintain arterial partial pressure of carbon dioxide or co2 (paco2) at clinically desirable levels in ventilated ards patients using a six-compartment mathematical model of co2 and oxygen (o2) biochemistry [1] and whole-body transport [2] with the addition of an ecco2r device for extracorporeal veno-venous removal of co2. the model assumes steady state conditions and is comprehensive from both biochemical and physiological perspectives. o2 consumption and co2 production rates were assumed proportional to predicted body weight (pbw) and adjusted to achieve pao2 and paco2 levels at a tidal volume of 7.6 ml/(kg of pbw) as reported in lung safe [3] . clinically desirable paco2 levels during mechanical ventilation were targeted at 46 mm hg for a ventilation frequency of 20.8/min as previously reported [3] . results: model simulated paco2 levels without and with an ecco2r device at various tidal volumes are tabulated in tables 1 and 2 , respectively. table 1 shows a substantial increase in paco2 at a tidal volume of 6 ml/(kg of pbw) that is more pronounced when further reducing the tidal volume. additional simulations showed that predicted ecco2r rates were significantly influenced by ventilation frequency. conclusions: the current mathematical model predicts that ecco2r rates that achieve clinically acceptable paco2 levels at tidal volumes of 5-6 ml/(kg of pbw) can likely be achieved with current technologies; achieving such paco2 levels with ultraprotective tidal volumes of 3-4 ml/(kg of pbw) may be challenging. figure 1a ). pulmonary infections for each subtype of immunosuppression are shown in figure 1b . conclusions: ards vv-ecmo patients with underlying immunosuppression have higher mortality rates and higher rates of ecmo weaning failure. immunosuppressed patients suffer from a different spectrum of pulmonary infections in comparison to not immunosuppressed patients. introduction: acute asthma attack in children is a life-threatening emergency that requires urgent medical intervention. in the present study, we aim to clarify the effect of non-invasive ventilation (niv) on the heart rate (hr), respiratory rate (rr), and fraction of inspired oxygen (fio2) in children with acute severe asthma (asa) who failed to respond to standard medical treatment; and to evaluate the associated complications and length of stay (los) at the pediatric intensive care unit (picu). methods: this is a retrospective descriptive study of prospectively collected data. it was carried at the picu of a tertiary university hospital, saudi arabia. the study included children ≤14 years old with asa admitted to the picu from november 2011 to november 2015 and required niv. outcome measures include the effect of niv on the hr, rr, fio2, and los. the study included 118 children with asa and 52 (44%) of them required niv. of those 52 patients, 12 (23%) were excluded due to incomplete data, and 40 (34%) patients were included in the final analysis. they were 22 (55%) male and 18 (45%) female with a mean age of 66 months and a median pediatric index of mortality 2 (pim2) score of 6.3%. of them, 28 (70%) had moderate asthma scores (≥5-9) and 12 (30%) had severe asthma scores (≥9). the median duration of niv was 18 hours and the median los in the picu was three days. at 6 hours, only rr showed a significant decrease compared to initiation of niv (p-value <0.001) (fig 1) ; while hr, rr, and fio2 were significantly improved at 24 hours from initiation of niv (p-value <0.001) (fig 2) . conclusions: non-invasive ventilation, in association with standard medical treatment, was associated with clinical improvement in children with asa not responding to standard medical treatment alone. niv was not associated with significant complications or side effects. neurally adjusted ventilatory assist (nava) is a partial support ventilatory mode which triggers and tailors the level of assistance delivered by the ventilator to the electrical activity of the diaphragm. the objective of this study was to compare nava and pressure support ventilation (psv) in patients who were difficult to wean. methods: a total of 99 difficult-to-wean patients who were able to sustained psv in the critical care medicine unit (icu) of the zhongda hospital, southeast university were enrolled in the study (fig 1) . patients were classified according to the reason for weaning failure and were randomly assigned to receive nava or psv during weaning ( table 1 ). the primary outcome was the duration of weaning. secondary outcomes included the proportion of successful weaning and patient-ventilator asynchrony. results: there were 17% (8/47) and 33% (17/52) patients in the psv and in the nava group never weaned from mechanical ventilation (p = 0.073). the duration of weaning was significantly shorter in the nava group [2.4 (1.1-5.3) days], than in that in the psv group [4.1(1.1-7.7) days] (p = 0.041). the proportion of patients with successful weaning was 70% (n=33/47) in nava group which was much higher than that in psv group (48%, n=25/52) ( table 2) . compared with psv, nava improved the rate of successful weaning in patients with single reason (74% vs. 49%, p = 0.019) but not in patients with multiple reasons for difficult weaning (50% vs. 45%, p = 0.656). nava decreased ineffective efforts and improved the trigger and cycling-off delays when compared with psv. mortality was similar in the two groups (fig 2) . in patients who were difficult to wean, nava decreased duration of weaning and increased the probability of successful weaning. nava which improved patient-ventilator asynchrony, is safe, feasible and effective over a prolonged period of time during weaning. conclusions: only mrc score is independently associated with sbt failure and difficult or prolonged weaning. hgs is also associated with these two outcomes related to mv weaning and may serve as a simple tool to identify icuamw. introduction: there is evidence to support that in patients with hypoxemic respiratory failure (ahrf) under non invasive ventilation (niv), high tidal volume (tv) and high respiratory rate (rr) are associated with niv failure and possibly poor prognosis. we postulated that high minute ventilation (mv); or tv x rr; is associated with mortality in ahrf, when niv is initiated. methods: single-center, prospective and observational study. we included consecutives ahrf adults requiring niv. ahrf was defined as acute dyspnea with new pulmonary infiltrates on chest radiography and paco2 below or equal to 45mmhg. we registered demographic and clinical parameters (including rr, mv, arterial blood gases, heart rate and blood pressure) at baseline and after 6 hours of first session of niv, apache ii score, diagnosis, need for intubation and icu mortality. we performed a multivariate analysis to assess independent factors associated with mortality and roc .000) and (auc = 0.7; p =0.019), respectively for mortality, future exacerbations and readmissions. the optimal cut-off point for the 6mwt ratio to predict mortality was 0.23 and to predict future exacerbations and readmissions was 0.4. the 6mwt ratio performed at icu discharge reveals interesting discriminative properties to predict early mortality, future exacerbations and readmissions in ae/copd patients. diffuse alveolar haemorrhage in an intensive care unit -search and you will find m matias 1 , e ribeiro 2 , j baptista 3 , p martins 3 introduction: the incidence of diaphragmatic ruptures after thoracoabdominal traumas is 0.8-5% [1] and up to 30% diaphragmatic hernias present late [2] when there is a complication. we report two cases of delayed traumatic diaphragm rupture to highlight the diagnostic difficulties. methods: case 1 (image 1) presented left diaphragmatic hernia containing the stomach, spleen, bowel and pancreas. the patient reported a motor vehicle accident dating 4 months. he had thoracoabdominal trauma with several broken ribs on the left side. he then reported occasional pain in his left shoulder and occasional dyspnoea. case 2 (image 2) showed right diaphragmatic hernia containing right hemicolon, right hepatic lobe and gallbladder, he reported occasional dyspnoea and recent right chest pain. he had a 25 years car accident in which three ribs broke on the right side. results: almost 88% of the patients with delayed diaphragmatic rupture presented with complications between 9 and 12 months after trauma, singh [3] reported a diaphragmatic rupture presenting 50 years after the traumatic event. the physical examination is often not helpful. conclusions: those cases emphasizes on the delayed presentation, patients may be asymptomatic or produce only mild, nonspecific symptoms, such as vague abdominal pain, chest pain or recurrent dyspnoea for months or years. the best tool to guide the clinician toward the appropriate diagnosis is a high index of suspicion whenever there is a history of high velocity trauma, regardless of how remote. factors associated with asynchronies in pressure support ventilation (psv), a bench study introduction: critically ill patients frequently have increased risk of ocular surface disorders (osds) due to poor eyelid closure and reduced tear production due to sedation during mechanical ventilation. we conducted a study to look at the incidence of osds in our icu with the current eye care practices and the impact of a protocolised eye care on the incidence and outcome and to determine the correlation of risk factors with the incidence of osds methods: this study was done in our mixed medical surgical icu. it had a prospective cohort design and was done as before and after study in two phases (phase i and phase ii). in phase i existing eye care practices were continued. in phase ii protocolised eye care was implemented and incidence of osds was noted in both phases. introduction: both fentanyl and morphine are known as opioid analgesics, which blocks the brain from receiving pain signals, the route of administration and the adverse effects affect their use. we compare the efficacy of intranasal fentanyl versus intravenous morphine adults population presenting to an emergency department (ed) with acute post traumatic severe pain. methods: we conducted a prospective, randomized, double-blind, placebo-controlled, clinical trial in a tertiary emergency department between october 2016 and june 2017. adults with severe post traumatic was included to receive either active intravenous morphine (3 mg immediately and then 1 mg every 3 min if persistence of severe pain maximum 10 mg) and intranasal placebo or active intranasal concentrated fentanyl (2 μ g /kg maximum 200 μ g) and intravenous placebo. exclusion criteria: significant head injury, allergy to opiates, nasal blockage, or inability to perform pain scoring, pain scores were rated by using a digital scale at 0, 5, 10, and 30 minutes. routine clinical observations and adverse events were recorded. conclusions: iscs were related to k over-use in our bicu. burnt patients are at risk of hepatic injury [2] , but k related hepatic injury likely occurred. its not clearly understood mechanisms may involve a cumulative dose effect. although involvement of concomitant medications is being investigated, k restriction policy seemed to contain hepatic disorders. introduction: in november 2016, our institution switched from using alfentanil to fentanyl for analgesia and sedation in adult patients receiving ecmo. there is no published evidence comparing the clinical use of alfentanil vs fentanyl for sedation in ecmo patients, although some reported increased fentanyl sequestration into the circuit [1] . for these reasons, we conducted a retrospective observational study to explore whether there were any significant differences in patient outcome or adjunctive sedation before and after the switch. methods: outcome data and total daily doses of alfentanil or fentanyl as well as adjunctive sedation/analgesia for each patient where obtained from our clinical information system (philips icca®). data was included from ecmo patients who were sedated with alfentanil or fentanyl from 1/1/16 to 31/10/2017 until ecmo decannulation. patients not requiring either opiate or who were switched between the two during ecmo therapy were excluded. all medicines prescribed for the management of sedation or agitation were included. for each patient an average total daily dose of each drug, was calculated. data was analysed using stata®. results: both groups were found to be statistically equivalent for mode of ecmo, age, apache 2 score and charlson score (p=0.202) except for bmi (p=0.007). no difference in patient outcomes were found between groups (table 1) . patients in the alfentanil group were found to have received significantly higher median average total daily dose of quetiapine and midazolam (table 2) . conclusions: no differences in patient outcomes were found between patients sedated with alfentanil compared to fentanyl. we introduction: the european society of intensive care medicine consensus statement recommends that for comatose survivors of cardiac arrest 12 hours without sedation is the minimum acceptable before neurological assessment. they highlighted the need to investigate the pharmacokinetics of opioid drugs in post-cardiac arrest patients, especially those treated with controlled temperature [1] . methods: following approval by research ethics committee, we measured the blood concentration of fentanyl in 24 post-cardiac arrest patients treated with ttm following cessation of continuous infusion. the fentanyl was discontinued when the patients were rewarmed to a temperature of 36.5 degrees celsius and a blood sample taken 12 hours later. the blood was analysed using a commercial elisa kit (neogen corporation). using the total dose of fentanyl administered, the half-life of fentanyl was calculated for each patient. patient physiological data, cyp3a4 and abcb1 polymorphism and drug history were compared with half-life. results: the median fentanyl concentration at 12 hours was 0.82 mcg/l with a very wide range (0.07-8.29 mcg/l). the results for calculated half lives are shown in figure 1 . there was no correlation between fentanyl level and bmi, illness severity (saps ll), creatinine clearance, transaminase or lactate level. there was no correlation between co-administration of drugs of metabolised by the cyp3a4 and abcb1 enzyme systems or genotype. conclusions: there is marked variation in the concentration of fentanyl at 12 hours in patients managed with ttm following cessation of fentanyl infusion. the calculated clearance of fentanyl in some patients is greater than 48 hours and a 12 hour cut off is not safe. introduction: objective of this study was to compare the effects of three analgesic regimens, one opioid and two multimodal ones, on cardiovascular stability and pain intensity in patients undergoing elective surgery under general endotracheal anesthesia during the 24 h postoperative period. methods: sixty elderly patients, asa ii, undergoing elective knee sugary were assigned to receive 1) morphine 5 or 10 mg iv q6h, depending on body weight, and paracetamol 1 g iv q6h (mp group), or multimodal nerve block: 2) femoral nerve block, single shot (fnb group) or 3) fascia iliaca compartment nerve block single shot (ficnb group). measurement of pain intensity was performed with numerical introduction: opioids are frequently used in the intensive care unit (icu) to relieve pain and facilitate tolerance of life-support technologies. when discontinued abruptly, patients may develop a cluster of symptoms known as opioid-associated iatrogenic withdrawal syndrome (oiws). this phenomenon is poorly described in critically ill adults although it is associated with unfavourable outcomes, such as prolonged icu stay. the objective of this study was to describe the signs and symptoms of oiws in adult icu patients. methods: a prospective observational study was conducted in two tertiary care centres in patients requiring mechanical ventilation and regular opioids for more than 72 hours. after an opioid dose reduction of at least 10%, patients were assessed daily for signs and symptoms of withdrawal using a standardized form. concomitantly, the presence of oiws was assessed daily by a physician using modified dsm-5 criteria. all physician evaluations were blinded and performed independently. inter-rater reliability for dsm-5 evaluations was assessed with the kappa coefficient. results: a total of 1128 patients were screened and twenty-nine enrolled. the majority were male (72.4%) with a median age of 65. the median apache ii score was 27. withdrawal occurred in 20.7% of patient within a median of three days (iqr 3 to 9 days) from opioid weaning. according to investigator assessment, restlessness, agitation, anxiety, hallucinations, insomnia/sleep disturbance, mydriasis and elevated blood pressure were more prevalent in oiws-positive patients. dsm-5 evaluations identified dysphoric mood, muscle aches, lacrimation/rhinorrhea, pupillary dilation/piloerection/sweating, diarrhea and yawning more frequently in oiws-positive patients. the kappa coefficient showed good agreement (0.64). conclusions: oiws in critically ill adults presents with a large spectrum of signs and symptoms that occur within a median of three days from onset of opioid weaning. further studies are needed to confirm these preliminary findings. withdrawal reactions after discontinuation or rate reduction of fentanyl infusion in ventilated critically ill adults s taesotikul introduction: propofol is a well-known sedative, commonly used in intensive care units (icu s), that on rare occasions has been reported to cause green urine and has also been associated with pink or transient white urine discoloration. it can cause several adverse effects, such as low blood pressure, pain on injection, apnea, hypertriglyceridemia and when administered in high doses it may lead to the "propofol infusion syndrome". methods: we present two examples of interesting urine discolorations observed unexpectedly in our icu in patients under propofol sedation requiring mechanical ventilation. results: dark green urine discoloration as presented in fig.1 is the result of a phenolic metabolite of propofol that is produced in the liver and is subsequently excreted in the urine, thus changing its color. it is considered a reversible phenomenon that resolves after propofol discontinuation.respectively, pink urine discoloration as presented in fig.2 can also be the result of propofol infusion. the increase in urine excretion of uric acid caused by propofol, in combination with a low urinary ph can lead to the formation of uric acid crystals and turn the urine pink. discontinuation of propofol and urine alkalization can reverse the phenomenon. conclusions: green or pink urine discoloration due to propofol is generally a benign, reversible condition. its presence should not compel the physician in charge to perform unnecessary testing, although other causes of discoloration should be considered. as far as green urine discoloration is concerned, other factors such as drugs, dyes, certain nutritional supplements or even a pseudomonas urinary tract infection may be at fault. on the other hand, pink urine syndrome due to propofol infusion seems to be even rarer. although its presentation is not alarming, it may well increase the risk of uric acid lithiasis, a fact that the physician in charge should always keep in mind. conclusions: hepatic changes related to propofol are frequently observed and should be systematically monitored to ensure patient safety. fig. 1 (abstract p405) . dark green urine discoloration introduction: clevidipine (clev) and propofol (prop) are lipid-based medications used in the intensive care unit (icu) for hypertension and sedation, respectively. no data exists regarding potential adverse effects of concurrent therapy with this combination. this study aims to evaluate the incidence of hypertriglyceridemia (htg) and pancreatitis in icu patients using concurrent clev and prop. methods: this was a single-center, retrospective chart review in patients utilizing clev and prop concurrently from february 2015 to november 2018. patients were included if they were 18 years and older, on clev and prop concurrently for at least 24 hours with no more than 2 hours of interruption at a time, had at least one triglyceride (tg) level during concurrent therapy, and admitted to the medical or surgical icu. the incidence of htg (defined as tg equal to or greater than 500 mg/dl) and pancreatitis (provider assessment based on 2013 american college of gastroenterology guidelines) was evaluated. patients with and without htg were compared to identify risk factors for the development of htg. results: of 145 patients screened, 30 patients were included which comprised 36 observations. the incidence of htg was 13.9% with no patients developing pancreatitis. patients with htg had a higher median age compared to without htg (64.5 vs. 38), p=0.015. in patients with htg the median dose of clev and prop were 16 mg/h and 42.3 mcg/kg/min, respectively, which was higher but not statistically significant when compared to patients without htg. cumulative lipid load (g/kg/d) was non-significantly higher in patients with htg (2.6 vs. 1.9), p=0.599. conclusions: the incidence of htg was comparable to what is cited in literature for prop alone. patients with htg were older, had higher median clev and prop doses, and a larger cumulative lipid load compared to patients without htg. introduction: the 2013 society of critical care medicine guidelines for pain, agitation and delirium suggested use of nonbenzodiazepine sedatives like dexmedetomidine which is associated with a reduced duration of mechanical ventilation, shorter length of hospital stay and a lower incidence of delirium [1] . enteral clonidine represents a potentially less costly alternative for agitated patients with prolonged dexmedetomidine infusion. limited literature exists examining this transition for management of agitation [2] . methods: the critical care management initiated an action plan on the transition of patients with prolonged dexmedetomidine infusion to oral clonidine. a protocol was prepared with clinical pharmacist's assistance. risk factors were assessed and inclusion criteria were applied as per protocol. dexmedetomidine infusion rate was reduced gradually with oral clonidine administration in selected patients. other rescue managements were implemented as per protocol. oral clonidine was then tapered down by reducing frequency of administration over few days. results: post intervention data in 2017 showed significant decrease of dispensed doses and cost of the injections compared to 2016. the annual cost saving was 24% equating to 61,249 usd (table 1, figure 1 ). conclusions: transitioning to clonidine may be safe and less costly method of managing agitated critically ill patients on prolonged dexmedetomidine infusion. more studies are needed to evaluate the efficacy and safety of this practice. incidence of dexmedetomidine associated fever at a level 1 trauma center na beaupre, jt jancik hennepin county medical center, pharmacy department, minneapolis, united states critical care 2019, 23(suppl 2):p409 introduction: we evaluated the incidence of dexmedetomidine associated fever (daf) in a level 1 trauma center's medical intensive care unit (micu). hypotension and bradycardia are the most commonly reported adverse effects associated with dexmedetomidine (dex) infusion. case reports suggest dex can cause fevers and the clinical trials that led to the approval of dex demonstrated fever rate to be 4-5% [1] . methods: this was a single-center, retrospective chart review of patients admitted to the micu at hennepin county medical center between march and july of 2018 that were started on a dex infusion. patients were included if they were 18 years and older, on a dex infusion for at least 12 hours, and had temperature data available. fever was defined as >38.3c and other causes of fever including infections, medications, withdrawal, recent surgery, thromboembolic disease, thyroid disorders and seizures were excluded from analysis. results: of the 246 patients screened, 120 were included. the mean age was 50 years and 63.3% were males. of all the patients included, the mean change in temperature after initiation of dex infusion was +1.1c from baseline. the mean initial dose was 0.3 mcg/kg/hr. four of 120 patients (3.3%) had a daf. of those that had a daf, the median initial dose was 0.3 mcg/kg/hr; the median time of infusion was 43.5 hours; and the median cumulative dose was 0.57 mcg/kg/hr. the median time to fever after initiation of dex was 7 hours, with a range of 5 to 20 hours. the median time to fever cessation after discontinuation of dex was 3 hours. conclusions: in our population, the incidence of dexmedetomidine associated fever was relatively rare at 3.3% and similar to current literature rates. the results obtained showed a statistically significant fact that fewer points on the test, from 11 to 20 points, received older patients who underwent an urgent surgical procedure, over 60 years of age, of which 27% . also statistically significant data were obtained that patients who used a higher amount of sedatives during emergency surgery, 47% had a worse test result than under 20 points due to increased preoperative anxiety. the older population is more susceptible to postoperative delirium, especially in emergency surgery situations, which they carry, unpreparedness for surgery, increased use of medication for fig. 1 (abstract p411) . flowchart of enrolled patients calm, unpredictability of the duration of surgery, and therefore anesthesia as well the use of anticholinergics, which is sometimes impossible to avoid in operative procedures such as gall bladder surgery. the results of the study suggest that in cases of emergency surgery, the use of protocols for postoperative delirium should be planned regularly to prevent or at least mitigate the clinical picture of delirium that can lead to complications postoperatively. introduction: delirium is a serious and often underestimated condition with implications for morbidity, mortality and healthcare costs. as it presents in a wide range of settings from admission to discharge, early prediction and risk assessment are essential. e-pre-deliric is a delirium prediction score which has been validated in itu patients but not in other populations, and we conducted a quality improvement project using this score to assess its utility in other settings. methods: data was gathered from three patient categories: those undergoing elective surgery (es), admissions to the emergency observation unit (eou) in the a&e, and patients with fractured neck of femur (nof). clinical notes were reviewed to collect data to calculate e-pre-deliric score at admission, along with a number of other clinical variables including incidence of delirium, and statistical analysis performed. results: a total of 103 patients were included, with 52 in the es group, 32 in the eou group, and 19 in the nof group respectively, with an overall average e-pre-deliric score of 17.5%. es had a 5.4% average e-pre-deliric score, a mean age of 56 and no cases of delirium. the eou group had an average age of 63, a 20.5% average e-pre-deliric score and no incidence of delirium. the nof group had a mean age of 84 and an average e-pre-deliric score calculated on admission of 45.3%. this was the only group in which 5 patients developed delirium. a 50% cut off was demonstrated to be the most accurate to predict delirium in this population with a sensitivity of 0.80 and a specificity of 0.78. conclusions: despite the limitation of a small sample size, this project has shown that e-pre-deliric score could be a useful tool to predict patients at high risk of delirium in a non-itu setting, with a 50% cut off in hip fracture patients. further investigation should be conducted into the potential use of e-pre-deliric in non-itu patients. comparison of long-term mortality between patients with and without delirium during admission in medical intensive care units in a university hospital n kongpolprom king chulalongkorn memorial hospital, pulmonary unit, bangkok, thailand critical care 2019, 23(suppl 2):p415 that delirium is linked with preoperatory comorbidities. the complexity of surgery has a big influence on the development of delirium, especially in the cases of aortic dissection. delirium was associated with intraoperatory blood transfusions. finally, our data point to a bridge between postoperatory electrolytic disturbances, as well as inflammation as factors potentially triggering delirium onset. introduction: we did a retrospective case note study of mortality due to sepsis of our unit over three months as observational study in which we noted the causes of deaths, origin of sepsis, organism, patient characteristics and icnarc physiology scores and icnarc h2015 model predicted risk of acute hospital mortality percentage. methods: icnarc data base was used to gather the data and coding was used to identify the patients with sepsis for three months. patients mortality attributed to sepsis were identified from mortality list.causes of death were noted from patients notes and death certificates.cyber lab was used to access the data and case note were ordered for review.patients characteristics were noted including dnacpr orders and treatment withdrawal orders. scores (apache scores, icnarc physiology scores, icnarc h2015 predicted risk models of acute hospital mortality percentage) were noted. results: mortality percentage was found to be 17% as per codig which was reduced to 12% as 5% deaths were attributed to other causes. 44% patient had dnacpr in first 24hrs. average length of stay was 5.58 days with median of 2.53 days.median age was 66yrs in surviving age group and 78years in other. icnarc physiology score 23 with predicted risk of 56.8%. commonest cause was found pneumonia 49% followed by urine tract infection. 20% patients were with no source identification. conclusions: conclusion was made that we do need to improve the coding as significant percentage was mentioned as sepsis as cause of death where clinicians differed. pneumonia was found to be the commonest killer in critical care followed by urine tract infection. it was pointed to be useful to carry out further audit targeting pneumonia .review of icnarc case mix program, development of icnarc physiology score, which provides excellent local use with downside of lacking international comparison was done also. introduction: hospitals vary widely in the quality of care they provide for septic patients. since many septic patients present to their nearest hospital, local variations in care quality may lead to geographic disparities in access to optimal sepsis care. we sought to better understand geographic access to high quality sepsis care, taking advantage of publicly reported data on sepsis management and outcomes in a large us state. methods: we performed a cross-sectional analysis of geographic access to high quality sepsis care, taking advantage of a 2013 new york state initiative that mandates public reporting of sepsis quality data to the state government. we linked these data to the locations of hospitals in new york state from the us centers for medicare and medicaid services and population data from the us census bureau for 2016. we defined hospital sepsis performance using self-reported risk-adjusted mortality rates (ramr) and defined high-performing hospitals as those with a ramr <20%, which represents the lower end of short-term mortality typically observed in sepsis. we used arcgis to generate drive-time estimates and assess population access to high performing acute care hospitals for sepsis care. results: 161 hospitals publicly reported treating 47,081 cases of sepsis from a population of 19,569,040 persons. overall access to an acute care hospital was excellent at the 45-minute drive threshold (99.3%), good at the 30-minute threshold (95.6%), and marginal at the 15-minute threshold (84.4%). we classified 46 hospitals (28.6%) as high-performing based on a ramr <20%. high-performing hospitals reported 12,666 (26.9%) of the total sepsis cases. high-performing hospitals were geographically dispersed across the state, although population access diminished substantially with increasing drive times (92.6% at 45-minutes, 83.5% at 30-minutes, and 59.8% at 15minutes; figure 1 ). conclusions: one in six people do not have timely access to a high performing hospital for sepsis care using a 30-minute threshold. [1] . this poses a significant safety risk. a previous study found that the implementation of a multidisciplinary medication safety group in intensive care increased reporting of errors and near misses [2] . the purpose of our work was to set up a multidisciplinary group to provide a forum to review and improve medication safety at all stages of the process. here we discuss some of the initiatives and outcomes implemented in the last 12 months. methods: ccmsg was formed in 2013, under the leadership of the critical care pharmacy team, with representation from medical and nursing disciplines. the group meet fortnightly to analyse trends in medication errors, implement changes to local practice and review outcomes to improve patient safety. the cohesive, multidisciplinary nature of the group allows medication safety initiatives to be delivered in the most effective way. results: on average, ccmsg reviewed 21 medication errors per month. the most common high risk drug classes involved are seen in table 1 . medication safety initiatives implemented were based on these trends and included writing guidelines and policies, bedside education, teaching and training, informatics optimisation and operational changes. examples are seen in table 2 . conclusions: initiation of a ccmsg provides a cohesive approach to facilitate the implementation of targeted safety initiatives, which are proven to reduce some of the most common medication errors in critical care. in addition, these often result in optimisation of operational and financial inefficiencies. introduction: cis/hospital electronic medical records downtime can cause major disruptions to workflow, patient care, key communication and information continuity [1] . here we describe the consequences of deploying a business continuity plan (bcp) designed to support a critical care clinical informatics system (cis) failure, during an 8-hour unplanned downtime in a large central london icu. the institutional bcp was developed through an iterative process based on cis provider recommendations and internal workflow knowledge. it consisted of a web offline chart (woc) that is accessible at every computer connected to the network (in the event of a cis server fault), and via hard copy from designated back up computers connected to a printer (in the event of whole network loss). operational and clinical consequences were recorded during informal and formal debrief of the informatics team. the decision making around´drop-to-paper´was reviewed. -the bcp permitted´drop-to-paper´, service continuity and controlled uptime -patchy network loss and lack of a general institutional bcp delayed initial system failure diagnosis (network vs primary server); reduced reliability of´read-only´data and delayedd rop-to-paper-day-to-night handover during downtime led to loss ofḿ emory´of key patient data/events, and should have accelerated decision to´drop-to-paper-transfer of prescriptions was time consuming, distracting (occupied cis team) and prone to error conclusions: previous end-to-end testing of the bcp had not identified many of the observations and recommendations that came from the analysis of an actual period of unplanned downtime. we recommend sharing of similar experiences and scheduled high-fidelity simulated downtime in other institutions to replicate real world conditions, particularly in a critical care setting. .001) were predictors of icu transfer. we developed a simple score to predicting icu transfer from previous variables and performed analysis of auc of roc, which was compared to that of apa-che ii. the result showed the auc of roc of a new score was slightly higher than the apache ii, namely 0.797 vs. 0.720 respectively. conclusions: the immunocompromised patients take two times higher risk than the immunocompetent ones regarding icu transfer. the other risk factors are lower gcs, lower sbp, and higher rr. a newly developed score may be a promising tool for predicting and triaging site of care in patients who require imcu admission. introduction: this research aims to explore the role of situation awareness in the decision-making of patient discharge from the intensive care unit (icu). the discharge of these patients is a complex and, moreover, a challenging transition of care. readmissions are undesirable given the association with a more extended hospital stay and a possible chance of higher mortality. little is known on how the decision-making process takes place and accordingly, the role of situation awareness of patient discharge from the icu. in order to improve the quality of care of patient discharge from the icu, further research is necessary. methods: this research concerns a qualitative study in which various health care providers, working in an icu adults of a large teaching hospital, were interviewed. through purposive sampling, six nurses, two physician assistants, two intensivists and a physiotherapist were included. on the obtained data a thematic analysis was applied, based on the principles of the grounded theory. results: the discharge decision of icu patients seems mainly based on the team´s situation awareness, with the initiating role of the intensivist and the guiding role of the nurse. furthermore, there is an additional role for the physician-assistant and a consultative role for physiotherapy in the process of the decisionmaking. worries of patients and family seem not to affect the decision-making directly. in the decision-making process, the well-being of the patients and the possibility to provide the most suitable and best possible care were central. organizational factors, such as an urgent demand for icu beds do count but seem not to push the decision to transfer patients from the icu to the regular hospital ward. conclusions: the decision to dismiss icu patients is a complex process with different disciplines and a variety of factors involved. obtained knowledge and insights into the role of situation awareness provide starting points for improving the quality of the discharge process of icu patients. conclusions: despite the fact that older people was more severe illnes, and similar frequency of respiratory failure, the use of mechanical ventilation, the use of central venous catheter and arterial catheter was less frequent. the addition of a simulation fellow within the intensive care team and introduction of in situ simulation n bhalla, d hepburn, g phillips royal gwent hospital, intensive care unit, newport, united kingdom critical care 2019, 23(suppl 2):p429 introduction: traditionally, simulation based medical education has been carried out in off site simulation centres, however, we trialled the addition of a simulation fellow, within our intensive care team, to run an in situ simulation (iss) program on our intensive care unit over a 3 month period. methods: our multi-disciplinary iss program, led by a simulation fellow, incorporated participants, observers and facilitators including doctors (junior trainees up to consultants of varying medical specialties), nursing staff, healthcare support workers, operating department practitioners, physiotherapists and medical students. we ran simulated emergency scenarios and technical skills sessions. with every scenario, we collected data on participant and observer feedback using the world health organisation participant feedback form and conducted a satisfaction survey at the end of our trial period. results: our results, highlighted in table 1 , show participants found iss led by a simulation fellow realistic, well structured and organised. it was useful for testing and understanding our response systems, fig. 2 (abstract p426) . patient journey of group 2: those patients discharged home 2 days after step down from critical care identifying strengths and gaps and establishing individual roles/functions within emergencies; overall leaving us feeling better prepared for critical care emergencies. from our satisfaction survey, 100% of participants found the simulation fellow a useful addition to the intensive care team and expressed the need for more in situ simulation. conclusions: the addition of a simulation fellow allowed for numerous disciplines within the critical care team to be involved in challenging emergency scenarios (fig 1, 2) , with the additional realism of being on the intensive care unit playing the role they would in real life; as well as having opportunity for spontaneous discussion and learning. from this they reported great benefit and satisfaction. following our initial success with this program, we plan to have a simulation fellow as an ongoing role within our critical care team. impact of multidisciplinary team in readmission in a brazilian cardiac intensive care unit c bosso 1 , p introduction: the aim of this study is to determine the importance of the multidisciplinary team at readmission rates in a cardiac intensive care unit (cicu). methods: retrospective study with analysis of 2945 patients in a cicu of a medium size brazilian hospital. the years of 2012 and 2013 represent the reduced team (physician, nurse and physiotherapist) and 2015 and 2016 the complete multidisciplinary team (additional presence of phonoaudiologist, psychologist, pharmacist, dentist and nutritional professional). the risk of mortality was determined by saps3 score. in order to compare the teams, it was utilized odd ratio of a logistical sample to the discrete data, and t-student test to the continuous data. the data analysis was executed from the software rstudio (1.1.456), and the significance level adopted was 5%. results: the number of patients was of n=2945 (1422 from the reduced team and 1523 from the multidisciplinary team). the age, sex and bmi didn`t present significant difference between groups. the average age of the sample was 68±13 years old (p=0.13). the male sex represented 58% (p=0.93), and the bmi was around 29.2±23.3 (p=0.12). the main diagnoses were similar in both groups -coronary angiography with stent (16%), unstable angina and non st elevation myocardial infarction (9%). table 1 shows the average, standard deviation, p-value to t-student test to saps 3 score and lengh of stay (days), according to both reduced and multidisciplinary teams. table 2 exposes the mortality rate and readmission for both teams. the figure shows the odds ratio and its ic 95% to the comparison of the mortality, readmission, 24 hours readmission and 48 hours readmission rates between the teams. conclusions: the multidisciplinary team performance reduced the number of hospital readmissions in 24 and 48 hours in a cicu. methods: during the initial audit 24 hours' worth of waste from one itu bed was manually divided into the categories above. results: based on these figures it was estimated that a saving of £1745 per year would be made (£87.25 per bed space) over the course of a year should domestic waste bins be placed across the 20 bed icu/hdu. a business case was made, and every bay had a domestic waste bin installed with poster signs for explanation.the reaudit in which all domestic waste across the unit was weighed produced an even greater figure of a saving of £205 per bed space (£4100) per year. conclusions: introducing a domestic waste bin may save approximately £200 per year per bed. in a typical itu such as lewisham (10 itu beds/10 hdu beds) that may mean a saving of £4000 per year (with 100% capacity). there are also environmental benefits, burning of plastics releases harmful dioxins. the authors wish to make intensive care units and indeed all areas of the hospital aware of the cost and environmental impact associated with disposing of waste in incorrect categories. we hope that our quality improvement project demonstrates how easily money may be saved and environmental footprint reduced. association between resilience and level of experience in intensive care doctors in india j gopaldas, a siyal manipal hospital, bangalore, critical care medicine, bangalore, india critical care 2019, 23(suppl 2):p433 introduction: attrition of doctors in intensive care unit (icu) is one of the highest amongst all medical specialities globally, and is strongly associated with stress and burn out syndrome (bos). factors that contribute to bos are low pre-morbid resilience and low level of icu experience. studies from india have shown high levels of stress in intensive care doctors (>40%), but there are no published studies measuring pre-morbid resilience and risk of burnout in relation to years of experience amongst icu doctors. our main aim was to measure cross sectional resilience levels in icu doctors compared between those with less than 5 years of experience to those with 5 years or more. a secondary aim was to assess the impact of other factors that may contribute to low scores. methods: an anonymised survey was conducted involving 125 doctors in icus across 6 different states in india, using the connor-davidson resilience scale (cd-risc 25), which is validated in indian population. results: a statistically significant correlation was found between low levels of resilience in icu doctors with under 5 years of experience 1.456) , and the significance level adopted was 5%. a logistic regression model was used to test the difference between the mortality and readmission rates in <90 and ≥ 90 groups, which enabled the calculation of odds ratios. chi-square test was used to evaluate categorical variables and t-student test to some quantitative variables. the roc curve was constructed to verify the sensitivity of prediction of mortality through different saps 3 scores. results: among the <90 and ≥ 90 groups, respectively 59% and 37% was male (p = 0.0001). mean weight of the> 90 years was 76 ± 16 kg and <90 years was 61 ± 11 (p <0.0001). odds values indicated a significant difference only for the mortality rate, which was more than double among ≥ 90. readmissions in any time, 24h and 48h as well the mortality is shown in table 1 and odds in figure 1 . there was a significant difference in saps 3 points between groups ( table 2 ). the ≥ 90 group presented an average of 10 points higher on the severity scale when compared with those in the <90 group. there was no significant difference in lengh of stay. the highest amount provided by saps3 scores was 63% and a specificity of 86% for hospital mortality not group <90 years. in ≥ 90 group the highest sensitivity was 53% and the specificity was 84%. roc curve for saps3 is shown in figure 2 . conclusions: the extremely elderly patients of a cicu is more severe, with higher mortality and have the same lengh of stay and readmission rates. introduction: the purpose was to assess the prevalence and impact of non-urgent interruptions (nui) within critical care (cc).a root cause analysis of a never event in our cc discussed nui as a contributory factor, paralleled by learning from serious incidents.the negative impact of nui is well evidenced, resulting in delayed task completion, increased stress, and affecting patient safety. methods: any nui during a consultant ward round (cwr) or invasive procedure (ip), not relating directly to the current clinical episode, was included. qualitative data was collected by a survey, assessing the cc multidisciplinary teams(mdt) perception of nui. results: one third of reviews during the cwr, and 40%of ips, had a nui. adverse effects included prescription omissions, delayed cwr, near-miss with a cvc, and failed picc insertion. overall, 86% of staff considered nui a problem; 95% had experienced nui that led to distraction in train of thought. 45% felt that nui had led to an error: 83% of doctors, versus 20% of nurses. 86% overall felt nui contributed to stress at work. reasons for interruptions included: feeling overloaded, needing to resolve concerns before forgetting/being distracted, unable to prioritise, and to shift responsibility.lack of leadership or clinical supervision providing a point of contact for problems during shifts was mentioned as contributory. senior staff raised that whilst attempts have been made to level hierarchy, allowing a voice for all to express concerns contributes to interruptions. potential solutions included awareness on impact of nui, jobs book,´sterile cockpitd uring ips, and increased clinical supervision during shifts. conclusions: we have demonstrated the prevalence and consequences of nui within cc is significant.the impact on staff is significant, both for contribution to errors and also the negative impact on stress in the workplace. identified potential solution will be implemented. the impact of an education package on the knowledge, skills and self-rated confidence of medical and nursing staff managing airway & tracheostomy/laryngectomy emergencies in critical care l o´connor 1 , k rimmer 2 , c welsh methods: the factors affecting the delivery of intensive care was elucidated by a comprehensive review of the intensive care literature. a further understanding of intensive care delivery in south africa was obtained by "making sense of the mess" with eight workshops and 20 interviews using a systems approach. systemic intervention served as the meta-methodology and methods and techniques from interactive planning, critical systems heuristics, soft systems methodology and the viable system model were employed. results: making sense of the mess emphasised the complexity of intensive care delivery, on both a situational and a cognitive level. it became clear that a single methodology would not suffice, but that a pluralist methodology was required to guide improvement in intensive care delivery. based on this understanding, nine principles were formulated to guide the development of a framework. systemic intervention was again used as the meta-methodology. interactive planning was identified as the key methodology, incorporating methods and techniques used in the making sense of the mess phase to build a systemic framework for the improvement of intensive care delivery. embedded in the proposed framework are matters relating to systemicity, complexity, flexibility, empowerment, and transformation of intensive care delivery. the proposed framework allows for multiple-perspectives, including that of marginalised stakeholders, the mitigation of multivested interests and power relationships (fig 1) . it is both flexible and adaptable to promote learning about the complex problems of intensive care delivery and it accommodates the strengths of various relevant approaches to complex problem solving. conclusions: the proposed framework aims to facilitate sustainable improvement of intensive care delivery and to ensure the "just-use" of resources to foster distributive justice. the perioperative management of adult renal transplantation across the united kingdom: a survey of practice c morkane 1 , j fabes 2 , n banga 3 , p berry 4 , c kirwan 5 introduction: there is a limited evidence base to guide perioperative management of patients undergoing renal transplantation and no national consensus in the uk. we developed an electronic survey to provide an overview of uk-wide renal transplant perioperative practice and determine the need for future guidelines on patient management. methods: a 29-question survey was developed to encompass the entire renal transplant perioperative pathway with input from clinicians with expertise from renal transplant surgery, anaesthesia, nephrology and intensive care. the survey was sent to lead renal anaesthetists at each of the 23 transplant centres across the uk. results: twenty-two centres (96%) returned complete responses. there was limited evidence of guideline-based approaches to preoperative work-up, with marked variety in modality of preoperative cardiorespiratory function testing performed. questions regarding intraoperative fluid management (fig 1) , blood pressure targets and vasopressor administration (fig 2) identified a broad range of practice. of note, the routine use of goal-directed fluid therapy based on cardiac-output estimation was reported in six (27%) centres whilst nine centres (41%) continue to target a specific central venous pressure (cvp) intra-operatively. a dedicated renal ward was the most common postoperative destination for renal transplant recipients (62% of centres), whilst a renal or transplant-specific hdu provided postoperative care in 8 (38%) centres. the need for care in an icu setting was decided on a case-by-case basis. conclusions: this questionnaire highlighted a high degree of heterogeneity in current uk practice as regards the perioperative management of renal transplant recipients. development of evidence-based national consensus guidelines to standardise the perioperative care of these patients is recommended. fig. 1 (abstract p437) . framework for the improvement of intensive care delivery introduction: postoperative care of high risk patients in the icu used to be considered the gold standard of care in terms of reducing perioperative mortality [1] . new evidence comes to question this practice [2] . the primary objective of our study was to detect any benefit of postoperative icu care after elective surgery in terms of patient's outcome, length of hospital stay, complications and cost. methods: a 6-month retrospective analysis of high perioperative risk patients who were about to be subjected into an elective operation were included into the study. subsequently they were allocated into two groups. group i patients were those admitted into the icu for postoperative care while those admitted into the standard ward consisted group ii. demographic data, length of hospital stay, outcome, need of mechanical ventilation, complications and total cost were recorded. results: a total of 78 patients were recorded, 39 in each group. there was no statistical difference regarding the demographic data between the two study groups. seven patients died before hospital discharge (5 in group i and 3 in group ii, p>0.005). there was no impact of icu admission on length of hospital stay (p=0.03) which is primarily affected by the need of mechanical ventilation (p=0.04) and reoperation (p<0.05). the total cost and the postoperative cost of hospital care did not statistically differ among study groups. conclusions: according to our study the need of postoperative care of high risk patients in the icu is rather questionable in terms of perioperative mortality, length of hospital stay and cost of care. introduction: tivap is a preferred vascular access device for patients with solid tumors and radiological-guided insertion is a standard of care. however, many hospitals have no access to interventional radiology service. our study aimed to determine whether it is safe to place tivaps in icu for immediate administration of chemotherapy. methods: we analysed prospectively maintained database of our department and collected data for adult pts with tivaps implanted between 02/2008 and 10/2018. the median age was 55 (range 18-82) years, 59% were women. all procedures were performed by 3 trained physicians with experience in ultrasound (us). puncture technique was used and tip location was controlled with electrocardiographic (ecg) and us with subsequent chest x-ray confirmation. pts were followed up for at least 30 days after the procedure for complications, functioning of tivap and surgical wound healing. results: all 277 tivaps were successfully implanted in 276 pts. infraclavicular route was used in 231 cases (83.3%). difficulties with indwelling guide wire were observed in 11 (4.76%) pts but did not precluded implantation. placement complications included pneumothorax (n = 3), catheter malposition (n = 3) and artery bleeding (n = 1). these complications required additional therapy but were managed successfully and resolved without consequences. in the rest 46 cases internal jugular vein (jv) was used. complications were not observed. ecg and us navigation provided optimal tip location control in these situations. surgical wound healed after 10-14 days and chemotherapy initiation did not affect healing. all tivaps had adequate functioning 30 days after placement. conclusions: it is feasible to implant tivaps in icu. these devices can be used on the implantation day without jeopardizing patient safety. jv catheterization seems to be optimal approach and us navigation and ecg are sufficient methods for placement control. introduction: there is increasing use of clinical information systems to improve patient safety and quality of care in critical care. with all these systems, a rigorous business continuity access (bca) plan needs to be in place so patient safety is not compromised [1] and ensure continuity of care. here we evaluate the types of medication errors that occurred during a period of unscheduled downtime; potential contributory factors [2] and the number of errors involving critical medicines [3] were analysed. methods: during the unscheduled downtime, all prescribing and administration of medicines were transferred to a paper based system using the patients' web offline chart (woc -philips healthcare). pharmacists at the time double checked the paper charts that were transcribed, to mitigate errors but this was not consistent due to the timing of the event. we retrospectively compared the paper drug charts against the electronic prescriptions and noted all errors for 47 patients. results: in total 26 medication errors were identified & 1 allergy omission ( table 1) . pharmacists double checked 68% of the paper charts. conclusions: our data highlights the risks associated with unscheduled electronic patient management system downtime and the heterogeneity of the types of errors & potential contributory factors. it underscores the need for robust local bca plan implementation, critical review of the woc document and regular staff training around potential unscheduled system downtime. introduction: the transfer of patient care (toc) between the intensive care unit (icu) and hospital ward is associated with a high risk of medical errors [1] .according to uk national data between 30-70% of patients have an error or unintentional medication change made when moving between care settings [2] . currently different prescribing systems without interoperability are used between icu areas & ward settings in our institution, resulting in medications needing to be re-prescribed on transfer. we aimed to evaluate the time delay in medication re-prescribing, number of unintentional omissions of drug doses and reasons, as well as percentage of critical medicines [3] omitted in the first 24h following discharge. methods: over a 2 month period, 79 discharged patients (50% of all discharges) from two icu units were included. the icu discharge letter which contained the medication list on transfer was compared against the ward based electronic drug chart to identify all unintentional omitted medication doses during the first 24 hours. the starting time point was when the patient physically left icu. results: 13/79 (16%) of patients had their medication prescribed more than 4 hours post discharge. there were a total of 269/1,145 (23%) unintentional omitted doses (table 1) . of these 104/269 (39%) were considered critical medicines ( table 2) . conclusions: this data confirms the risk associated with toc especially around medicines. the need of interoperable electronic prescribing systems is one solution and could improve patient safety by streamlining the process. introduction: staff perceptions of safety may contribute to workforce stress and be organisationally important [1] . this study explored the feasibility of capturing perceptions of safety with a bedside professional reported (bpr) shift safety score, and explored relationships between bpr and measures of staffing and workload. methods: uk health research authority approval was obtained (id249248). data were collected for 29 consecutive days at imperial college healthcare trust (70 general critical care beds on 3 sites).the bpr asked all icu staff to rate each shift as "safe, unsafe, or very unsafe". responses were described and correlated with data on organisational staffing (care hours per patient day chppd) and nursing intensity (total number of organs in failure/ total number of nurses). results: a total of 2836 bpr scores were recorded (response rate 57%). we noted heterogenous responses between sites and days, and within shifts, only 14 % of shifts were unanimously rated. whilst 34% of shifts were rated by staff as "unsafe" or "very unsafe", organisational metrics recorded only 5% as 'unsafe'. we did not find a correlation between measures of staffing (chppd) and perceptions of safety ( figure 1 ). preliminary analyses suggest that staff perceptions of safety are not well correlated with nursing intensity (figure 2 ), although these numbers commonly inform staffing metrics. conclusions: completing the bpr tool was feasible and acceptable to staff. responses showed variations in perceptions of safety and a gap between organisational metrics and individual perceptions. introduction: delivery of intensive care (icu) is complex because of multiple stakeholders with varied perspectives and conflicting goals that interact and are interdependent. to inform the development of a framework for the improvement of icu delivery in south africa, it was essential to first understand icu delivery or "make sense of the mess". a systemic approach such as systems thinking is required to holistically explore and understand the complexity of icu. no methodology is perfect and methodological pluralism as proposed by systemic intervention, a systems thinking approach, was used for a more flexible and responsive intervention. the methods used was the making sense of the mess phase of interactive planning, stakeholder analysis as describe by critical systems heuristics, rich pictures from soft systems thinking and viable systems model diagnosis. making sense of the mess was done in 2 phases: first the mess was formulated with rich pictures generated in 8 workshops and 20 interviews. the discussions of the rich pictures by the respective stakeholders were transcribed and analysed using braun and clark's thematic analysis. secondly, based on the data generated from phase 1 a diagnosis of the viability of the icu system was made. results: the data from the 2 phases were very rich and complex and 6 themes emerged (figure 1 ). these themes were interdependent and resulted in disorganised icu delivery with limited opportunities for learning to improve icu delivery with dichotomies that existed at various levels of icu. it was a problem to present the complex data in the traditional linear manner due to the interdependence of the themes. the analysis is presented as 6 stories, a known approach in the complexity discipline, where the themes of the analyses are portrayed. the making-sense-of-the-mess phase confirmed the complexity of icu delivery, at both a situational and a cognitive level and with this understanding a framework for the improvement of icu delivery could be developed. introduction: improving prescribing practice involves changing prescriber behaviour. education is assumed to change behaviour but other approaches may be more effective (figure 1 ) [1] . changes to the presentation of information and the configuration of choices have potential to rectify common prescribing errors through subtle 'nudges' [2] . the implementation of clinical information systems (cis), including electronic prescribing, provides an opportunity to deploy strategies such as standard orders, dose limits, and product level prescribing. with an infinite number of configuration options available, clinical leaders need to know which interventions are most effective. we evaluated several of these strategies in a before and after observation study methods: interventions, utilising cis nudges, were chosen to improve four areas of prescribing practice in a tertiary critical care unit using methods matched to the top 4 levels of the hierarchy. data were collected for 2 months before and after interventions to map changes in compliance with a pre-defined standard except for the standardisation intervention where 4 months' data were collected due to low prescription numbers. no education on changes was given during the baseline data collection so any change in performance after the go-live date is entirely attributable to the intervention. results: the change in performance for each level ranks the intervention levels in the order (highest first) forced function, automation and standardisation ( table 1 ). the use of point of prescribing reminders was not associated with a significant difference in performance. conclusions: the effectiveness of intervention levels seen in practice is consistent with that of the model. further studies could be undertaken to strengthen these conclusions but in the meantime the approach to changing practice using cis nudges should focus on standardisation or above. introduction: intensive care unit (icu) sound pressure levels (spl) are persistently above world health organisation recommendations for clinical areas [1] . this may impact patient recovery. standard spl monitoring records single values for each 24h period (laeq24). we hypothesise this reporting rate is unsuitable for icu. methods: we measured spl october 2016 -may 2018, logging frequency (hz), spl (db), and loudness (perception of sound) every second [2] . the resulting dataset was of a size that conventional statistics programs would require computational resources not easily obtainable on standard university commodity hardware. we processed the full dataset without sampling by using distributed task dispatching, parallelism and scheduling of a cluster computing framework (apache spark). we created a system consisting of a single workstation (6 cores; 32gb ram) running ubuntu 18.04 lts, oracle java 1.8, apache spark 2.3, scala 2.11, r core 3.5, r studio 3.5 and sparklyr 0.8.4. we utilised the sparklyr library in r studio to run arbitrary r code using the dplyr library. we analysed aggregate data in r core & used ggplot (v3) to create visuals. results: we achieved more complex analysis than standard spl reporting with relatively modest computing resources. specifically we identified lower spl peaks in the early hours & loudness levels considerably higher than parallel spl. conclusions: simple laeq24 do not facilitate reflection on practice thus impetus for change is limited. loudness data highlight the patient experience of spl in the icu is more intrusive than laeq24 indicates due to high sensitivity to sounds~2-4khz, a common frequency range for alarms. higher fidelity increases understanding of spl which can lead to targeted interventions to reduce patient disturbance. introduction: survivors of critical illness face significant long term impairments in mental and physical function. early mobilisation (em) in the intensive care unit has been suggested to improve functional outcomes and reduce delirium in the icu. we hypothesized that implementing a protocol for em in the icu would improve mobilisation rates while remaining safe. methods: design: prospective non-blinded observational cohort study, based on a quality improvement project. data was collected conclusions: only 6 of 10 variables in boyd criteria were significant associated with morbidity or mortality. the physiologic score and operative score were significant higher in the patient on mortality and morbidity after sicu admission. effects of structural hospital characteristics on risk-adjusted hospital mortality in patients with severe sepsisanalysis of german national administrative data d schwarzkopf 1 introduction: the quick sequential organ failure assessment (qsofa) score is a simple tool used to identify severe patients with infection. as this score is calculated from three variables that can be measured at the scene of trauma-systolic blood pressure, respiratory rate and consciousness-the prehospital qsofa score may also be a good predictor of mortality in trauma patients. so we evaluated the discriminative ability of the prehospital qsofa score in patients with trauma for in-hospital mortality. methods: this is a retrospective multicenter study using the data from nationwide trauma registry in japan. we included 42722 patients with trauma aged ≥18 years old transferred to hospitals from scene. primary outcome is in-hospital mortality. results: the mean age was 59.4±21.5 years old and 27069 patients (63%) were male. in-hospital mortality occurred in 2612 patients (6%). in-hospital mortality in each qsofa score was 105/11783(0.9%), 941/17839(5%), 1280/11132(12%) and 286/1968(15%) in qsofa score 0, 1, 2 and 3, respectively (p<0.0001 for trend). area under receiver operating characteristics curve (auroc) of the aqsofa score for inhospital mortality was 0.70(95% confidence interval 0.69-0.71). if we use the cutoff ≥1, sensitivity and specificity of the qsofa score were 0.96 and 0.29. conclusions: in patients with trauma, the prehospital qsofa score was strongly associated with in-hospital mortality. we can identify patients with very low risk of death by using the cutoff ≥1 of the prehospital qsofa score. introduction: only one prospective study is available of the validation of the diagnostic and prognostic role of qsofa (quick sofa score) in the emergency department (ed). a prospective study was conducted in greek eds. methods: the prompt study (clinicaltrials.gov nct03295825) run in the ed of six hospitals in greece among patients with suspected infection and presence of at least one of fever, hypothermia, tachycardia, tachypnea and chills. clinical data were collected and the 28-day outcome was recorded. sepsis was defined by the sepsis-3 criteria. results: the sensitivity and the specificity of at least 2 signs of qsofa for the diagnosis of sepsis was 78.4% and 96.8% respectively and for the prognosis of 28-day mortality 45.7% and 94.2% respectively. the odds ratio for 28-day mortality when qsofa was equal to or more than 2 was 59.67 among patients with charlson's comorbidity index (cci) equal to or less than 2; this was 7.45 among patients with cci more than 2 (p: 0.038 between the two ors by the breslow-day's test; p: 0.040 by the tarone's test). conclusions: data validated the sensitivity of qsofa for the diagnosis of sepsis. cci was an independent predictor of severity. qsofa could better predict unfavorable outcome among patients with low cci. comparative accuracy between two sepsis severity scores in predicting hospital mortality among sepsis patients admitted to intensive care unit n sathaporn, b khwannimit prince of songkla university, internal medicine, hat yai, thailand critical care 2019, 23(suppl 2):p455 introduction: recently, the new york sepsis severity score (nysss) was developed to predict hospital mortality in sepsis patients. the aim of this study was to compare the accuracy of nysss with the sepsis severity score (sss) and other standard severity scores for predicting hospital mortality in sepsis patients. methods: a retrospective analysis was conducted in a medical intensive care unit of a tertiary university hospital. the performance of severity scores was evaluated by discrimination, calibration, and overall performance. the primary outcome was in-hospital mortality. results: overall 1,680 sepsis patients were enrolled, 895 patients (53.3%) were classified to septic shock by sepsis-3 definition. hospital mortality rate was 44.4%. the nysss predicted hospital mortality 34.6+/-21.5%, which underestimated prediction with smr 1.28 (95%ci 1.19-1.38) . however, the sss predicted hospital mortality 47 +/-22.2%, which slightly overestimated mortality prediction with smr 0.94 (95%ci 0.88-1.01). the nysss had the moderate discrimination with an auc of 0.772 (95% ci 0.750-0.794), in contrast to the sss presented good discrimination with an auc of 0.889 (95%ci 0.873-0.904). the auc of sss was statistically higher than that of nysss (p<0.0001). nevertheless the apache iv and saps ii showed the best discrimination with auc of 0.937. the auc of the nysss and sss was significant lower than that of apache ii, iii, iv, saps ii and saps 3 ( figure 1 ). the calibration of all severity scores was poor with the hosmer-lemeshow goodness-of-fit h test < 0.05. the nysss was the lowest overall performance with brier score 0.201. the apache iv present the best overall performance with brier scores 0.107. conclusions: the sss indicated better discrimination and overall performance than the nysss. however the calibration of both sepsis severity scores and another severity score were poor. furthermore, specific severity score for sepsis mortality prediction needs to be modified or customized to improve the performance. introduction: metabolic markers, especially lactate, have been shown to predict mortality in acutely unwell patients. we hypothesised that early changes in metabolic markers over time would better predict mortality and length of stay, with patients who correct their metabolic derangement having lower risk of death and reduced length of stay (los). methods: single centre, retrospective cohort study in a 31 bed icu. we included all patients who had an arterial measurement of lactate, paco2, base excess (be) and ph on admission and at 6 hours after admission to icu between 01/01/2016 and 31/12/2017. the 'clearance' of these markers was calculated using the equation ((value at admissionvalue at 6 hours)/value at admission). clearance calculations only included those patients with deranged results on admission (lactate>2mmol/l, be<-2mmol/l, ph<7.35, paco2>6.0kpa). roc analysis was used to predict in-hospital mortality and length of stay, using both the initial admission values, and using the clearance value, as well as icnarc and apache ii scores for comparison. if a patient was admitted twice in the time period, only the first admission was included. results: 1506 patients were included (sex ratio 1.5, mean age 61.6). table 1 ). none of the values tested had a auc greater than 0.6 for predicting length of stay. conclusions: the clearances of metabolic markers over the initial 6 hours after icu admission does not provide better prognostic information than the value at admission. initial lactate level was the best predictor of mortality, but compared poorly to icnarc score. metabolic markers do not accurately predict length of stay. 8.0-22.8) vs 9.1 (iiq 9.6-9.9), p=0.005]. the other hemogram parameters did not differ between groups (table 1) . when adjusted for severity score, in patients submitted to emergent surgery, the mpv value was still independently associated with mortality (or 1.146 ci 1.057-1.243, p=0.042), and its roc curve (auc) was 0.861 to mortality (figure 1 ). conclusions: mpv is a cheap and easily accessible marker which can add prognostic value in this specific population. in the future, we will validate it in a larger cohort of cancer pts admitted to intensive care. haematological malignancy in critical care: outcomes and risk factors c denny 1 introduction: about 7% of patients admitted to hospital with a haematological malignancy will become critically ill [1] . life expectancy in these patients is poor with a 6 month mortality of 60% or more in specialist units [2] . in contrast, patients without critical illness can expect a 5 year survival rate exceeding 60% for many cancers. this disparity results in differences of opinion on the best strategy for such patients among haematologists and critical care physicians. we conducted a local quality improvement project to quantify mortality and risk factors in critically ill patients with a haematological malignancy in our hospital. methods: patients admitted to the critical care unit of broomfield hospital, a district general hospital with tertiary specialist services, from january 2011 to december 2017 with haematological malignancy were included in the analysis. patients in remission for more than 20 years and patients admitted following elective surgery were excluded from analysis. death in critical care or in hospital after critical care discharge were the primary outcomes. mortality was correlated with demographic data using simple statistical measures and regression analysis. results: 93 patients were included in the analysis. overall mortality was 45%(n=42). survivors tended to be younger (65 vs 71 years) but had similar clinical frailty scores. early critical care admission (within 24 hours) was associated with better survival (72.5 vs 35.7%). nonsurvivors had a greater incidence of sepsis and respiratory failure, and required more ventilatory and vasopressor support. mortality was higher in patients requiring more than one organ support. conclusions: the overall mortality in our data is lesser than previously published data but supports the conclusion that mortality is determined primarily by the number of organs supported with the effects of malignancy playing a secondary role. (figure 1 ). increasing levels of frailty were associated with increasing risks of death at 1 year (p<0.001) (figure 2 ). frailty significantly increased 1-year mortality hazards in unadjusted analyses (hr 1.96; 95%ci; 1.41-2.72; p<0.001) and covariate-adjusted analyses (hr 1.41; 95%ci 1.00-1.98; p=0.0497) ( table 2) . conclusions: frailty was common and associated with greater age, more severe illness and female gender. frailty was significantly associated with heightened mortality risks in both unadjusted and covariateadjusted analyses. frailty scoring may encapsulate variables affecting mortality which are omitted in current predictive systems, making it a promising risk stratification and decision-making tool in icu. fig. 1 (abstract p461) . unadjusted survival curves stratified by frailty status. frail patients were statistically significantly less likely to survive to 1 year plateau at day 4=18, delta peak=2 and hpr=0.34. were assigned respectively a point value of 1, 1, and 2 to these predictors based on their beta coefficient in the predictive model. the score yielded a roc-auc: (auc=0.79; 95%ci, [0.72-0.86]; p=0.000). using the validation data set (n=104), the score had an roc-auc=0.8 and similar estimated probabilities for mortality. conclusions: the paw-mps seems to demonstrate interesting discriminative properties to predict mortality. what is the role of the pulmonary embolism severity index (pesi) and rv/lv ratio as clinical risk assessment tools for patients undergoing ultrasound-assisted catheter-directed thrombolysis (uacdt)? introduction: to evaluate if the pulmonary embolism severity index (pesi) score correlates with rv/lv ratio, biomarkers of cardiac injury, fibrinogen and length of stay(los). also to evaluate the correlation between rv/lv ratio with biomarkers of cardiac injury, fibrinogen and los for patients who underwent uacdt. methods: a retrospective review of patients with sub-massive pulmonary embolism (pe) who underwent ultrasound-assisted catheterdirected thrombolysis (uacdt) was performed. pesi score, rv/lv ratio, length of stay(los), fibrinogen levels, troponin levesl, and brain natriuretic peptide(bnp) levels, were calculated and collected prior to uacdt. spearman's rank correlation coefficient was calculated for all non-parametric variables. results: 31 patients, 17 males and 14 females, were included in the study. the mean (±sd) age was 54±17 years. the mean pesi score was 93±38. mean rv/lv ratio was 1.34±0.35. a significant correlation between the rv/lv ratio and both fibrinogen and troponin level (p=0.0013, p=0.0167) was noted. no significant correlation existed between pesi score and rv/lv (p=0.85). no significant correlation existed between both rv/lv ratio and pesi score with length of stay (p=0.1649) after uacdt. there were no noted mortality or complications. conclusions: pesi score is used as a prognostic factor for the patients with pe, however, our study shows that pesi score does not correlate with rv/lv ratio or length of stay after the uacdt. there was inverse correlation between rv/lv ratio and fibrinogen. there was also positive correlation between rv/lv ratio and troponin for patients with and without heart failure. according to our data, there may be limited use of pesi score and rv/lv ratio for risk stratification of pe patients undergoing uacdt. introduction: conventional scores for prediction of risk and outcome, such as sapsii and sofa, have not been validated for patients admitted to level ii critical care units (intermediate level or imcus). we compared the performance of sapsii and sofa scores with the intermediate care unit severity score (imcuss) in a general population admitted to imcu. methods: we conducted a prospective observational cohort study in a 31-bed level ii-iii icu from a university-affiliated hospital, during a three-month period. we applied sapsii, sofa day one and imcuss to all patients admitted during that period. primary outcome was a composite of hospital mortality and need to increase level of care. additionally, we tested the relevance of each variable within each score to predict the outcome. results: we included 108 patients with a mean age of 61.7±18.6 years. 63 patients were considered "step-down" (transferred from our level iii beds), and the remaining originated from the emergency conclusions: 12 months after completion, the primary care management intervention had no effect on mental health-related quality of life and physical function among survivors of sepsis. increase in ptsd symptoms in the control group may suggest a possible protective effect of the intervention. introduction: critically ill patients and their families are often confronted with an overwhelming amount of clinical information shortly after hospital admission. their reliance on internet resources for additional information is increasing, particularly for unfamiliar medical terminology. yet, little is known about whether these online resources meet the recommended reading level and complexity appropriate for the average reader. methods: an online search of 40 websites containing four common critical care diagnoses in the icu (respiratory failure, renal failure, sepsis and delirium) was performed. a total of 6 readability formulas were used. the flesch-kincaid grade reading level (grl) and flesch reading ease (fre) were used in the final analysis. document complexity was evaluated using the pmose/ikirsch formula. results: websites on respiratory failure were written at the 13th grl with fre of 29.6. renal failure resources had a 9th grl with fre of 54.7. sepsis websites had an 8th grl with fre of 48.3. delirium websites had a 12th grl with fre of 31.6. when comparing website types (government, non-profit and private), anova showed a difference in fre across all 3 groups and government websites had a conclusions: online resources used by intensive care unit patients and families tend to be written at higher than the recommended 6th grl, with government sites better meeting this target than nonprofit and private organizations. online resources should be improved to lower this unfortunate barrier to patient education. introduction: the recent enactment of the data protection act 2018, the general data protection regulations, and a series of data breaches in the healthcare sector, have renewed interest in how our patients' information is collected, used and shared. the complex framework of laws and regulations governing the use and disclosure of personal data may lead to professional and financial consequences if information is disclosed inappropriately. disclosures to the police when they concern incapacitous patients are particularly challenging, as the disclosure may have no direct benefit to the patient and may cause the patient considerable harm. methods: we have reviewed the relevant laws and regulations to identify the circumstances in which doctors must release information regarding incapacitous patients to the police. the laws and regulations are examined to identify the extent of the disclosure required, and any requirements for the disclosure to be lawful. we have also identified laws which confer a power to disclose information about incapacitous patients, and the circumstances in which these powers can be used. results: in conjunction with a local police constabulary we have developed an information request form which makes it easier for those requesting and disclosing information to understand the legal basis of the disclosure. we have also developed guidelines to allow practitioners to understand where a disclosure is obligatory or discretionary. conclusions: the next stage of the project is to audit disclosures of information in the intensive care unit, and identify whether information is being released lawfully and following the correct procedure. introduction: family members are affected both physically and psychologically when their relative is admitted to icu. there is limited knowledge describing their experiences and structured interventions that might support them during their relative's critical illness. the aim of this review is to describe published literature on the needs and experiences of relatives of adult critically ill patients and interventions to improve family satisfaction and psychological well-being. methods: design: scoping review. standardised processes of study identification, data extraction on study design, sample size, sample characteristics and outcomes measured (figure 1) . results: from 469 references, 43 studies were identified for inclusion four key themes were identified: 1) different perspectives on meeting family needs 2) family satisfaction with icu care 3) factors impacting on family health and well-being and capacity to cope 4) psychosocial interventions conclusions: family members of patients in icu experience unmet information and assurance needs which impacts on their physical and mental health. structured written as well as oral information show some effect in improving satisfaction and reducing psychological burden. icu's who are able to support interventions based on meeting family information needs, in addition to reducing psychological burden and increasing satisfaction will enable each family to provide more support to their relative within the icu. introduction: unmet informational needs lead to dissatisfaction with care and psychological distress. identifying interventions to help meet specific needs is a crucial and necessary step in providing family centred care in icu. we aimed to implement and evaluate the impact of delivering a structured communication strategy on levels of anxiety, uncertainty and satisfaction with care and decision making in families of critically ill adults. methods: a quasi experimental study with pre and post test design. a convenience sample of 52 family members were recruited from july 2016 to february 2018. the intervention group (n=26) received both oral and printed information to guide them in preparing for a structured family meeting. the control group (n=26) received usual fig. 1 (abstract p476) . article selection process for scoping review routine care and existing family informational support. anxiety, uncertainty and family satisfaction were measured in the two groups on icu admission and icu discharge. results: mean anxiety, uncertainty and satisfaction with care and decision making scores pre and post intervention were compared. there were no significant differences in mean anxiety, uncertainty or satisfaction scores between the two groups before the intervention (p>0.05). mean scores on anxiety (45.5vs 46.9), and uncertainty (72.4 vs 76.7) were lower post intervention, but not significantly so ( figure 1&2 ). total satisfaction, satisfaction with care and satisfaction with decision making mean scores were similar in both groups before and after the intervention (p.0.05). conclusions: providing relatives with a combination of targeted written and oral information delivered by nursing and medical staff reduced anxiety and uncertainty with this reduction being evident through to discharge from icu. although not statistically significant, there was what may be seen as a suggestion of a clinically significant drop in anxiety and uncertainty following the intervention introduction: clinical studies in intensive care unit (icu) patients are warranted in order to improve healthcare. the aim of this study was to analyse barriers and challenges in the process of achieving informed consent from icu patients. methods: we analysed patients considered for inclusion in a prospective observational study of venous thromboembolism in the icu, i.e. the norwegian intensive care unit dalteparin effect (norides) study. data were collected from the screening log, consent forms and associated research notes of the norides study. results: we observed that 58 of 279 (20%) eligible patients according to inclusion and exclusion criteria were omitted from the nor-ides study due to barriers and challenges in the process of receiving informed consent. 21 were categorized as psychiatric diseases consisting of known psychosis or recent suicide attempt, 19 likely or actual treatment withdrawals and 18 due to language barriers among non-norwegians. among the 70 patients included in the norides study, 29 (41%) consents were from patients and 41 (59%) obtained from their next of kind. from the patient consents, 11 (38%) consents were oral and 18 (62%) were written. 6 patients were physically unable to sign, and 7 patients did not recognize their own signature. the study further pointed at some specific challenges in the process of consent, herein questionable competence to give consent, failure to remember being asked/included, inability to separate research from treatment etc. there were also difficulties in evaluating who was next of kin and how to reach them. conclusions: barriers and challenges in obtaining informed consent from icu patients led to exclusion of one fifth of the eligible patients in our study. informed consent directly from patients was obtained from less than half of the included patients. obstacles in the process of achieving informed consent were practical, medical, ethical and/or legal. determinants of end-of-life decision-making in the intensive care unit p eiben, c brathwaite-shirley, s canestrini king´s college hospital nhs foundation trust, london, united kingdom critical care 2019, 23(suppl 2):p479 introduction: although the majority of intensive care unit (icu) deaths follow the decision to forgo life sustaining treatment (lst), variability in patterns is commonly observed [1, 2] . we reviewed end of life (eol) practice at our institution in order to explore: (i) patient characteristics affecting eol decision-making, (ii) communication among surrogate decision-makers, and (iii) eol management. methods: we retrospectively analyzed data from consecutive patients who died in our ten-bed icu over 16 months (study period). patient demographics, apache ii, functional status, diagnosis on admission, icu length of stay (los) were collected; family/next-of-kin (nok) involvement and rationale for lst limitation were recorded ( conclusions: our analysis shows that in our institution eol deliberations follow a shared decision-making process. lack of family/nok involvement and incomplete documentation was exceptional. the significant difference in los between w-group and nw-group, in the face of similar apache ii, warrants further investigation. vae calculator rheumatology review 2. van der jagt m. crit care consensus on circulatory shock and hemodynamic monitoring. task force of the european society of intensive care medicine cardiac output monitoring: how to choose the optimal method for the individual patient perioperative cardiovascular monitoring of highrisk patients: a consensus of 12 guidelines for nutrition support therapy in the adult critically ill patient references 1. nice guideline for aki: prevention, detection and management 2 serial creatinine results pre-and post ecmo references 1. polit et al. research in nursing & health reference 1. sherliker et al national blood transfusion committee, nhs blood and transplant arch otolaryngol head neck surg fig. 1 (abstract p343). rsi agent guideline references 1. nuckton tj nejm 2018 icm 2012 baseline characteristics reference elso guidelines for cardiopulmonary extracorporeal life support s3-leitlinie invasive beatmung und einsatz extrakorporaler verfahren bei akuter respiratorischer insuffizienz 1.auflage p387 handgrip strength does not predict spontaneous breathing trial failure or difficult or prolonged weaning of critically ill patients g friedman 1 total burn care introduction: we aimed to evaluate safety and efficacy of light sedation with dexmedetomidine (dex-ls) in acute brain injury (abi) patients. methods: retrospective analysis on icu patients with traumatic/medical abi, out of the neuroprotection window and undergoing dex-ls. data of pre-infusion and infusion periods were compared. results: 101 patients (age 51±24, males 83.2%) were included. traurespectively. conclusions: dex-ls among icu patients affected by abi turned out to be feasible and safe. it enabled discontinuation from mv and maintenance of spontaneous breathing in the majority of cases 88%) delirious patients and 93 of 114 (81.58%) non-delirious patients could be discharged from the hospital. we evaluated the 2-year mortality in the hospital survivors. results: totally, 153 patients participated in our study. the majority of them (55.1%) were male with the median age of 64[45, 76.25] years and the median apache ii score on the first day of icu admission of 13 risk of delirium was associated with preoperatory euroscore ii (p=0.028) and history of previous cardiac surgery (p=0.042). moreover, in the intraoperatory period the risk of delirium was associated with red blood cell transfusion, intervention for aortic dissection (p=0.013), hypothermic circulatory arrest (hca) with anterograde cerebral perfusion (acp) (p=0.036) (table 1). in the postoperatory period risk of delirium was associated with levels of creatinine clearance (p=0.035) and c-reactive protein (crp) (p=0.029). conclusions: delirium is relatively frequent in the cardiac surgical icu patient journey of group 1: those patients discharged directly home from critical care unit poor compliance with co-signing in icca (66%, n=161) compared to paper (93%, n=183) (figure 2) and the reported difficulty in co-signing (8%, n=85) reveals significant usability concerns and potential safety issues. 40% (n=85) found icca intuitive, though 16% (n=85) found navigating the interface difficult and reported concerns with losing saved work (19%, n=85). conclusions: this study highlights important usability issues that may impact staff satisfaction 4th national audit project of the royal college of anaesthetists and the difficult airway society. major complications of airway management references 1. guidelines for provision for intensive care services (gpics), version 2 medicines optimisation: the safe and effective use of medicines reducing harm from omitted and delayed medicines. a tool to support local implementation p444 understanding the delivery of intensive care in south p448 mobilising ventilated patients early with interdisciplinary teams (move it) singapore general hospital, department of respiratory and critical care p451 validation of boyd criteria and possum-score on mortality and morbidity in general surgical intensive care unit k chittawatanarat, y chatsrisuwan faculty of medicine pts with central nervous system neoplasms or submitted to elective surgeries were excluded. descriptive analysis and χ test, pearson´s, wilcoxon rank-sum, uni and multivariate logistic regressions were used when appropriate. results: from a total of 105 pts identified, 57.1% (n= 60) were admitted after emergent surgery and 42.9% (n=45) for medical reasons. global icu mortality was 25.7% (n=25). in comparison to survivors, the patients that died had a similar age were recorded data regarding demographics, clinical variables, paw (at admission and at day 4), high pressure ratio (hpr = number of days with high pressures: peak ≥40 and/or plateau ≥30; and/or driving pressure ≥14; and/or auto-peep ≥6; divided by los), trends of paw (paw at day 4 -paw at admission) and outcomes. the patients were divided into two groups: a construction group (n=200) and a validation group(n=104). the paw-mps was developed and validated by analyzing in a multivariate regression model the different paw ±0.1; pco2, 49±21 mmhg paw were respectively for peak, plateau, driving, and auto-peep at admission: 32 01±10, 20.7±7, 13.6±5 and three independent mortality risk factors were identified centro hospitalar do porto p469 five-year mortality and morbidity impact of prolonged icu stay n van aerde 1 , g hermans laboratory of cellular and molecular medicine we investigated differences in mortality and morbidity after short (<8 days) and prolonged (≥8 days) icu-stay. methods: prospective, 5-year follow-up study of former epanicpatients (clinicaltrials.gov:nct00512122, n=4640). mortality was assessed in all. for morbidity analyses, all long-stay and a random sample (30%) of short-stay survivors were contacted. primary outcomes were total and post-28-day 5-year mortality in multivariable cox regression analysis, icu-risk factors comprised hypoglycaemia, corticosteroids, nmba, benzodiazepines, mechanical ventilation, new dialysis, new infection, liver dysfunction, whereas clonidine may be protective. among 276 long-and 398 short-stay 5-year survivors hgf, 6mwd and pf sf-36 were lower in long-stayers 6mwd: 85% (95%ci:69%-101%) vs 94% (95%ci:76%-105%) multivariable regression identified associations with benzodiazepines (hgf and pf-sf36), vasopressors (pf-sf36) and opioids (6mwd) ptsd related symptoms were accessed with the post traumatic stress syndrome 14 questions inventory (ptss-14) at the post icu follow up clinic, six months after the acute stress event. the post icu consultation was carry out by an icu doctor and an icu nurse. exclusion criteria: previous severe psiquiatric disorders, not able to respond the questionnaire medical 67%, surgical 24% and trauma 9%. 60 patients (57%) were on imv and the median ventilation days was 7. ptsd scores ranged from 14 to 70. delusional memories were conclusions: in this study the rate of ptsd was lower 2.8% and related with a lower saps ii and the presence of memories of the icu stay. no relation was found with delusional memories, imv or superior icu length of stay. patients with lower illness severity and without imv, should be elective to the follow up-clinics. p471 long-term effects of a sepsis aftercare intervention k schmidt 1 united states; 4 jena university hospital patras general university hospital, intensive care unit, patras, greece; 4 patras general university hospital, division of infectious diseases results: 60 (41.6%) patients were readmitted within 72 hours and 84 (58.4%) in 4 to 60 days. the two groups didn't differ in age, gender, charlson comorbidity index and length of stay on both admissions. elective surgery was the most common type of admission (34.7%) followed by medical (28.4%), emergency surgery (27%) and trauma (9.7%). the mean time to readmission in the late group was 17.9 (±17.8) days. patients in the late group had higher apache ii score on their first and second admission, (17.3±5.5 vs 14.2±6.1; p=0.008) and (18.7±6.9 vs 14.4±6.7; p=0.002) respectively. respiratory insufficiency was the most common cause of readmission in both groups followed by sepsis and cardiac arrest. finally in the early group p480 introduction: in intensive care units, perceived inappropriate treatments (pit) have been associated with negative impact on caregivers univariate analysis revealed that burn-out, pit and intention to leave were greater in units where nurses´teams included no activity in the icu, compared to "shared" work in icu and idtcu. in multivariate analysis, perception of non beneficial treatment of patients with life support witholding was associated with: bad collaboration with other units p481 profile of intensive care unit (icu) patients on whom life-sustaining medical treatment were withdrawn or withheld s chatterjee 1 variables collected-age, sex, apa-che iv score, diagnostic-category and co-morbidities. primary outcomes were icu and hospital mortality. secondary outcomes included icu and hospital length of stay(los) female sex, n (%) 34 (46.6%) diagnosis on admission: medical, n (%) rrt at time of wlst, n (%) 28 (38.4%) dnr order, n (%) 61 (83.6%) organ donation services involved, n (%) 26 (35.6%) introduction: high flow nasal cannula(hfnc) is a new modality in respiratory failure management [1] . this study objectively held to compare the physiological outcomes in the non-invasive ventilation(niv) treatment of cardiogenic acute pulmonary oedema(apo) patient in the emergency department(ed) delivered by helmet cpap(hcpap) and hfnc. methods: single-centre randomized controlled trial on patients presenting with cardiogenic apo. primary endpoint was a heart rate reduction.secondary endpoints included: improvement in subjective dyspnoea scales, respiratory rate, blood oxygenation, intubation rate and 28 days mortality rate. results: 188 patients were enrolled and randomized (94 patients to hcpap; 94 to hfnc) ( 89 to 271.83±73.63). intubation rate was lower in hcpap (6.9% for hcpap versus 10.48% for hfnc) and 28 days mortality rate is lower in hcpap (9.6% for hcpap versus 14.9% for hfnc). conclusions: both hcpap and hfnc significantly improved patient condition in patient presenting to the ed with cardiogenic apo. however, hcpap was better than hfnc in improving physiology outcomes, lower intubation rate and mortality rate in patient introduction: the aim of the study was to compare the confusing assessment method of the intensive care unit (cam-icu) and the nursing delirium scoring scale (nu-desc) for assessment of delirium in the icu. furthermore we wanted to test the interpersonal variation of the nu-desc. delirium is proved to be associated with increased mortality [1] . nu-desc is an observational five-item scale that does not require patient participation and is adapted to the fluctuating nature of delirium. each item can be scored from 0 to 2. delirium is defined with a score > 2. the nu-desc has recently been translated into danish (nu-desc dk) but has not been validated.methods: icu patients, who met the inclusion-criteria for the cam-icu were scored with both cam-icu and nu-desc dk. patients were scored of two independent nurses at approximately the same time every day.results: a total of 24 patients were enrolled, and 54 comparisons between cam-icu and nu-desc dk were registered ( figure 1 ).there was agreement between nu-desc and cam-icu in 46 of registrations (hereof 44 registrations were delirium negative). in interpersonal variation, 14 registrations were made. the conclusion was identical in 86% of registrations, but only 57% agreed in all 5 scoring-scale items (all negative).conclusions: a high agreement between nu-desc and cam-icu was found however the comparison was based on predominately patients with negative delirium score. the interpersonal variation of nu-desc scoring was substantial. a future validation of the nu-desc dk as a screening tool in the icu requires thorough training and instructions to minimize interpersonal variation. introduction: an increasing number of patients are being discharged directly home from critical care units and this is currently viewed as a negative quality indicator [1] . the purpose of this audit was to characterise a cohort of patients who can be safely discharged directly home from adult critical care at st thomas´hospital (sth). methods: retrospective observational study of two groups of patients; 1) those discharged directly home from critical care, 2) those discharged within two days of step down to a ward from critical care (admissions 1st june-31st october 2017). the clinical notes of these patients were reviewed via online systems. results: baseline demographics of the 58 patients in group 1 and 90 patients in group 2 were similar (mean age of 51 years, versus 46 years, p=0.34); average length of stay in critical care was also similar (2.9 days versus 2.5 days respectively p= 0.72). in group 1, 24 of 168 icu days were after considered fit for step down versus 40 of 222 days in group 2, p=0.32 (fig 1, 2) . in group 1, drug related presentations were more common (27% versus 13% p=0.03), fewer patients had specialist follow up post discharge (36% versus 87%, p<0.001). in group 1, 7 patients (12%) were readmitted within 28 days, 3 to critical care. in group 2, 7 patients (8%) were readmitted, 1 to critical care (p=0.38 and 0.14 respectively); none of these readmissions were felt to have been preventable.conclusions: there is a cohort of patients suitable for discharge directly home from critical care who did not spend significantly longer in icu awaiting discharge than those who were stepped down to the ward. identifying these patients early, potentially by their diagnosis, and creating a pathway including access to specialist follow up clinic could allow prompt discharge directly from critical care, thus improving patient satisfaction and reducing hospital-acquired morbidity healthcare costs [1] . the evaluation of the usability of a critical care information system ( introduction: critical care information systems (ccis) support clinical processes by storing and managing data, but poor usability can lead to staff dissatisfaction and increased workload, promoting workarounds that may compromise patient safety [1] . the purpose of the study was to evaluate the usability of a philips intellispace critical care and anaesthesia (icca) ccis, recently implemented in 51 beds across three critical care units of a large uk teaching hospital. methods: a prospective, mixed method observational study conducted in may 2018, comprising of (1) an audit assessing the ease of linking bedside devices to icca, (2) an audit assessing the usability of co-signing medications in icca compared with a non-icca paper factors that commonly drive workforce metrics may not correlate with staff perceptions of safety. the bpr is a pragmatic, staff driven, tool to augment other measures of safety and is applicable to various icu settings. further research is needed to explore staff perceptions in order to understand the importance of this organisationally, and for staff stress. ventilator-free duration in icu, central venous catheter duration, urinary catheter duration, rates of deep vein thrombosis (dvt) and stress ulcer prophylaxis, rates of de-escalation antibiotic therapy, dvt prophylaxis duration, stress ulcer prophylaxis duration, icu and hospital mortality, 28-day mortality, rate of central venous catheter infection, length of stay in icu and hospital between two groups were analyzed. results: rate and duration of dvt prophylaxis in the intervention group were 81.3% and 3(2,6) days respectively, in the control group were 67.7% and 5(3,10) days, the differences between two groups were statistically significant(p<0.05) ( table 1 ). there were no differences in ventilator-free duration in icu, central venous catheter duration, urinary catheter duration, rate of stress ulcer prophylaxis, rates of de-escalation antibiotic therapy, stress ulcer prophylaxis duration, icu and hospital mortality, 28-day mortality, rate of central venous catheter(cvc) infection, length of stay in icu and hospital between two groups ( table 2) . conclusions: electronic checklist in ward rounds can increase the rate of dvt prophylaxis and reduce the duration, but it cannot improve the prognosis of critically ill patients. introduction: the goal of the project "i see you" is family-centeredcare based on family meetings that improve the experience of the patient´s family members during hospitalization in the icu. the meetings focus on relaying information, raising knowledge and addressing the social and emotional needs of families. providing support along with information was found to be the strongest predictor of family satisfaction and could lead to improve cooperation between family and staff [1] .methods: meetings and questionnaire: family meetings consist of a multidisciplinary team, a group facilitator and combined with a multimedia presentation about the unit and equipment. in addition, they focus on social and emotional needs: managing daily routine, sharing problems, fears and anxieties and more. at the end of the session a questionnaire was given to assess the impact of the intervention. sharing data: at the end of the first quarter, the data from meeting was summarized and sent to the staff alongside tools for effective communication.results: the project began in february 2018. to date, 162 family members of 74 patients have attended the sessions. the topics discussed by the participants include: contact with the patient, prevention of infections, procedures, visits, conversations with doctors, medical confidentiality; guardianship; tracheotomy and social issues (fig 1) . a sample of questionnaires was transferred to 57 participants report satisfaction at a very high level.conclusions: the meeting received a very positive feedback from the participants. the project has achieved its goals and therefore it has been decided to be continued.introduction: possum score and boyd criteria are used to predict the outcome for high risk surgical patients. the aim of this study was to validation of these two measurement tools on mortality and morbidity in a university-based surgical intensive care unit (sicu) in thailand.methods: nine hundred and fifty two patients were enrolled onto this prospective review. all patients who had been admitted to sicu in a university-based hospital were included. all patients were collected for boyd criteria and possum score and outcomes and morbidity during sicu admission and discharge. introduction: aromatic microbial metabolites (amm), such as phenyllactic (phla), p-hydroxyphenylacetic (p-hphaa), and phydroxyphenyllactic (p-hphla) are involved in the pathogenesis of septic shock and are associated with mortality [1] . according to previous studies, amm have a high prognostic value in patients with abdominal infection [2, 3] . we hypothesize that amm have the prognostic value in patients with pneumonia in icu. methods: data of patients with community-acquired pneumonia was obtained on admission to icu. the levels of amm (phla, p-hphla and p-hphaa) were measured in blood serum using gas chromatography with flame ionization detector and compared in 2 groups of patients: with favorable and with lethal outcome (mann-whitney utest). spearman's correlations between amm and clinical and laboratory data were calculated. using method of logistic regression and roc analysis, we measured the prognostic value of amm. (table 1) . it was revealed, that some amm have similar prognostic characteristics in comparison with sofa and curb-65 scales; high level of amm is associated with high risk of death (roc-analysis fig. 1) .conclusions: serum concentrations of amm can be used as independent and practical criteria for the assessing of prognosis in patients with infection in icu. introduction: frailty in the critically ill is associated with increased morbidity and mortality but the optimal timing of frailty assessment, how to best measure frailty, reasons for adverse outcomes and how critical illness impacts frailty are unknown [1] . in preparation for a multi-center study designed to address these knowledge gaps, we conducted a pilot study whose aim was to assess feasibility as determined by recruitment rates, ability to assess frailty at icu admission and hospital discharge, ability to measure icu and hospital processes of care and ability to conduct 6-month assessments. conclusions: a multi-center study is feasible but follow-up losses due to mortality and inability to return for assessment will require sample size adjustment. frailty characterization is method dependent, can be done on hospital discharge but varies with time of assessment. these findings will need to be confirmed in our larger study currently in progress. introduction: given the ageing of the world´s population, the demands of critical care resources for elderly patients has increased during the past decade. however, little is known about quality of life and outcomes of elderly icu survivors. the aim of the study is to assess outcomes of elderly icu survivors at least 6 months after discharge: quality of life and mortality. methods: it is a retrospective study performed in a medical adult icu between january 2016 to december 2017. the study included all elderly survivors ( ≥65 years) after icu admission. outcomes were assessed by telephone interviews at least 6 months after icu discharge. the primary outcome was assessing the quality of life after icu stay, measured by euro qol 5d questionnaire. the eq-5d descriptive system contains five dimensions (mobility, self-care, usual activities, pain and discomfort, and anxiety and depression). for each dimension, there are five levels (no problems, slight problems, moderate problems, severe problems and unable to/extreme problems figure 1 . conclusions: most elderly survivors patients showed a good health related quality of life using the euroqol 5d-5l after icu discharge. fig. 1 (abstract p468) . quality of life (euroqol 5d) scores after icu discharge introduction: sepsis survivors face mental and physical sequelae even years after discharge from the intensive care unit (icu). effects of a primary care management intervention in sepsis aftercare were tested. exploratory analyses suggest better functional outcomes within the intervention group compared to the control group at six and 12 months after icu discharge. longer term effects of the intervention have not been reported. methods: a randomized controlled trial was conducted, enrolling 291 patients who survived sepsis (including septic shock), recruited from nine german icus. participants were randomized to usual care (n=143) or to a 12-months intervention (n=148). the intervention included training of patients and their primary care physicians (pcp) in evidence-based post-sepsis care, case management provided by trained nurses and clinical decision support for pcps by consulting physicians. usual care was provided by pcps in the control group. the primary outcome of the trial was the change in mental healthrelated quality at 6-months after icu discharge. secondary outcomes included measures of mental and physical health. data were collected by telephone interviews using validated questionnaires at the 24-months follow-up (12 months after the 1-year intervention).results: 186 [63.9%, 98 intervention, 88 control] of 291 patients completed the 24-months follow-up. unlike the intervention group, the control group showed a significant increase of posttraumatic symptoms (diff. ptss-10 to baseline, mean (sd) 3.7(11.8) control vs.-0.7(12.1) intervention; p=0.016). there were no significant differences in the mcs and all other secondary outcomes between intervention and control group.introduction: survivors of sepsis often show symptoms of posttraumatic stress disorder (ptsd). only few studies report on courses of more than 12 month after discharge from the icu. the aim of this study was to identify predictors for changes in ptsd symptoms over time up to 24 month. methods: follow-up data of the smooth triala rct to evaluate a primary care management intervention on sepsis survivorswere analyzed. included patients were surveyed by phone for ptsdsymptoms at one, 6, 12 and 24 months after discharge from icu using the post-traumatic-stress-scale (ptss-10). scores changes between follow-up periods were analyzed using latent-change scores in structural equation models. predictors were clinical and sociodemographic baseline characteristics as well as physical, cognitive and functional sepsis sequelae assessed by validated questionnaires.results: 291 patients were included of which 186 participated in the 24 month follow-up. a decrease of ptsd symptoms between 6 and 12 months was predicted by higher education (b=-0.32, p=0.04), while higher pain intensity at one month predicted an increase (b=0.01, p=0.041). increasing ptsd symptoms between 12 and 24 months were predicted by reporting more than two traumatic memories at one month (b=0.44, p=0.004), more sleep problems (b=0.22, p=0.08) and worse cognitive performance at 6 months (b=-0.03, p=0.043) as well as more neuropathic symptoms at 12 months (b=0.04, p=0.015).conclusions: sepsis patients that suffer from physical, cognitive and functional impairments after icu discharge may be at increased risk for developing late-onset ptsd. these predictors need to be replicated by future studies. early versus late readmission to the intensive care unit: a ten-year retrospective study v karamouzos 1 , n ntoulias 2 , d aretha 3 , a solomou 3 , c sklavou 3 , d logothetis 3 , t vrettos 3 , m papadimitriou-olivgieris 4 , d velissaris 5 , f fligou 3conclusions: icu patients whose life-sustaining treatment was withdrawn or withheld had higher illness-severity scores, were older, had longer icu los and higher mortality than those in active-treatment group. healthcare introduction: caring for the critically ill patient is a complex task and becomes tougher when a death process takes place. a number of needs and coping strategies emerge from the healthcare providers before these issues but are mostly displayed out of individual skills and intuition. if those approaches are unappropriate and the needs are not met, patients' death process may be burdensome for caregivers. this could affect the quality of care for patients and families during the whole end-of-life care process. the aim of our study was to explore the different needs and coping strategies used by icu healthcare providers when facing patients in the dying process. methods: qualitative and collective case study. ten semi-structured interviews were conducted in icu personnel (3 physicians and 7 nursing professionals). a thematic analysis was done using nvivo 11 software. local ethics committee approved the study. results: respondents were 70% women, had 36.7 ± 8.2 years-old and 11.1 ± 8.2 years of icu experience. main needs identified in icu healthcare providers refer to a lack of tools for doing emotional containment when delivering bad news to families, handling personal mourning, the need to perceive consistency regarding end-of-life care management across the icu team, and a wish of having regular training from a psychologist. main identified coping strategies included closing rituals, finding quiet spaces to spend time, and asking for counselling with more expert colleagues. a need for systematic, although basic training on these issues from qualified professionals is demanded. conclusions: usually, basic needs from patients and families in the process of dying are well addressed, but healthcare providers' needs are underrecognized and coping strategies mostly unknown. visibilization of those needs and basic but formal training in emotional containment, self-care and coping strategies are greatly desired. introduction: in the intensive care unit (icu), patients often exhibit cognitive impairments that prevent them from participating in decisions related to therapeutic options at the end of life. consequently, their families are often asked to speak for them when difficult decisions must be made. the main of this study was to determine the frequence in wich family want to share in end of life decisions and factors associated with this desire.methods: a prospective study was conducted in one mixed icu in montevideo. relatives of patients were invited to participate in this study after 72 hours in the icu and completed a survey that included the hospital anxiety and depression scale. results: we analized 135 relatives from 96 patients hospitalized in the intensive care unit. the relationship with the patient was as follows: 23% spouses, 23% siblings, 21% grown children, 14% parents, and 19% other family members and friends. of them, 41.5% reported a desire to share in end of life decisions. anxiety and depression symtoms were present in 60% and 41% respectively. factors asociated with the desire of involvment in end of life decisions by bivariate analysis were: female sex (48% vs 29%, p=0.03), presence of anxiety (51% vs 28%, p=0.008) and patient ecog 2-4 (70% vs 34%, p=0.05). multivariate analysis shows that the presence of anxiety is the only independent factor associated with the desire to participate in end of life decisions (or 2.20, ic 95% 1.02-4.73; p=0.04). conclusions: have a loved one in icu is often associated with anxiety and depression after 72 hours of admission. only 41% of the relatives want to participate in end of life decisions. the presence of anxiety is independently associated with the want to share in decisions making process. introduction: intensive care aims to treat failure of vital organ systems. sometimes, a patient's condition is of such a degree that intensive care is no longer beneficial, and decisions to withdraw or withhold intensive care are made. this means that life-sustaining treatments are terminated or not initiated. we aimed to identify variables that are independent factors for the decision to withdraw or withhold intensive care. methods: registry study using extracted data from a national quality registry the swedish intensive care registry (sir) 2014-2016. data are delivered to the registry by nurses and doctors daily, during each patients' stay in the intensive care unit (icu). a total of 97,095 intensive care cases reported to the sir from 2014-2016. results: data regarding each patient´s age, sex, diagnoses, condition at admission (expressed as simplified acute physiology score version 3, saps 3), comorbidities and registered decisions to withdraw or withhold intensive care were analyzed. of the 97,095 cases reported, 41.9% were women and 58.1% men, and 47.1% were 61-80 years old. a total of 15.4% received a decision to withdraw or withhold intensive care, accounting for 16.2% of all women and 14.8 % of all men, p<0.001. independent variables associated with increased odds of receiving a decision to withdraw or withhold intensive care were older age, worse condition at admission, and female sex. female sex was associated with an increased odds of receiving a decision to withdraw or withhold intensive care by 18% (ci 13.4-23.0%) after adjustments for condition at admission and age. conclusions: older age, worse condition at admission and female sex was found to be independent variables associated with an increased odds to receive a decision to withdraw or withhold intensive care.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-004879-pgyzluwp authors: nan title: programmed cell death date: 1994 journal: experientia doi: 10.1007/bf02033112 sha: doc_id: 4879 cord_uid: pgyzluwp nan it is widely held that all developmental cell death is of a single type (apoptosis) and that neuronal death is primarily for adjusting the number of neurons in a population to the size of their target field through competition between equals for target-derived factors. we shall draw on our research and on that of others to criticize these views and replace them by the following. at least three types of neuronal death occur, only one of which resembles apoptosis; a neuron can choose between several self-destruct mechanisms depending on the cause of its death. the purpose of the death is to regulate connectivity, not neuron number. competitors for trophic factors are unequal, and many losers have made axonal targeting errors. a neuron's survival and differentiation depend on multiple anterograde and retrograde signals. activity affects retrograde signals and some but not all anterograde ones. the pattern of activity is more important than the overall amount. in rodents, the period of naturally occuring cell death of motoneurons is followed by a period of supersensitivity to axonal injury. thus, in newborn rodents lesion of the facial nerve leads to a rapid degeneration of the injured motoneurons. we have tested whether overexpression, in rive, of the bcl-2 proto-oncogene was capable of preventing death of axotomized motoneurons. to address this question we used transgenic mice whose motoneurons overexpress the bcl-2 protein. one of the two facial nerves of newborn mice was transected on the 2nd-3rd post-natal day. seven days after the lesion, the morphology of the facial nuclei was analyzed. in control mice, and when compared to the intact nucleus, 70 to 80 % of axotomized motoneurons had disappeared. in contrast, in the transgenic animals, the number of motoneurons on the lesioned side remained unchanged when compared to the eontralateral nucleus. furthermore, their axons remained visible up to the distal lesion site. these experiments show that, in rive, motoneurons overexpressing the bcl-2 protein survive after axotomy, and suggest that, in rive, bcl-2 protect neurons from experimentally induced cell death and could be a target for treatment of motoneurons degenerative diseases. messmer s., mattenberger l., sager y., blatter-garin m-c., pometta d., kate a., james r.w. drpt de mrdeeine, drpt. de pharmacologie, div. de neurophysiologie clinique, facult6 de mrdecine, gen~ve. clusterin is a widely expressed glycoprotein, highly conserved across species. numerous functions have been postulated for this protein. the most important are roles in lipid transport, as elusterin is associated with apolipoprotein ai in hdl, complement regulation and tissue remodelling, in particular during cell death and differentiation. using cultures of rat spinal cord neurones (90% neurons and 5-10% non-neuronal cells), we have studied the expression of clusterin and ape e in glutamate-induced neuronal cell death to examine potential roles in lipid management. up-regulation of the two proteins was observed. clusterin and ape e appear in the conditioned medium respectively 15h and 7.5h after incubation with glutamate. control studies, in the presence of a noncompetitive nmda receptor agonist showed the secretion of clusterin and ape e to be diminished by >60%. no up-regulation of either protein was observed in complementary studies with exclusively non-neuronal cell cultures. the cellular origin of the 2 secreted proteins is presently under investigation. programmed cell death and tissue remodelling are consequences of hormonally induced restructuring of the rat ventral prostate after castration and the rat mammary gland after weaning. we used the "differential display"-method (liang and pardee, 1992, science 257:967) to detect and isolate edna fragments whose corresponding rnas are regulated either coincidentally, or in an organ specific fashion during mammary gland involution and postcastrational prostate regression. partial sequencing of 12 clones revealed high, but not absolute homology of 5 fragments with sequences, previously characterized in different biological contexts. these five encode functions which could be anticipated to be important for cell growth and/or programmed cell death, we are presently investigating the functions of several of these transcripts in cell culture and in rive. antisense oligos are being employed in vivo to determine whether these genes contribute to the phenotype of programmed cell death. b epitopes derived from the envelope gp52 glycoprotein (ep3) or from the viral superantigen of mmtv have been incorporated into inert or live vaccines. the inert vaccine consists of purified chimeric proteins which contain the b epitopes alone or fused to multimeric promiscuous t helper epitopes from tetanus toxin. mice were immunized subcutaneously with these chimeric proteins. the live vaccine consists of an avirulent strain of salmonella typhimurium which expresses the mmtv epitopes in the form of chimeric proteins fused to the nucleocapsid protein of hepatitis b virus. this vaccine is given to mice in one oral dose. the level, duration and isotype of the immune response generated by each vaccine have been measured and compared. the level of protection has been investigated by systemically challenging immunized mice with the relzovims. a reduced binding of oxytocin (ot) occurs with aging in some, but not all, areas of the rat brain (arsenijevic et al., experientia 1993, 49, a75) . the candate putamen showed the most impressive loss of ot receptors. two other regions, the hypothalamic ventromedial nucleus (vmh) and the islands of caueja (icj) had also an important deficit of ot binding sites. on the other hand, these two regions were known to be sensitive to sex steroids. in the present work, we treated from 20 month old rats during one month with testosterone propionate (2 #g/kg s.c., once every 3 days) dissolved in oil. three rats of the same age injected with oil only served as controls. we labelled ot receptors throughout the brain of old rats using a 125i-labelled ligand specific for ot receptors. analysis of autoradiograms by an image analyzer revealed that the testosterone treatment increased ot binding sites in the vmh, in the icj, and, to a lesser extent, in the bed nucleus of the stria terminalis, a region also sensitive to sex steroids, by contrast, in the caudate putamen, the disappearance of ot receptors was not compensated. in conclusion, the decrease of ot receptors occurring in vmh and icj with aging can be reversed by administration of gonadal steroids. in contrast, the loss of ot receptors in the striatum appears to depend on another mecanism. vasopressin (avp) receptors are expressed transiently in the facial nucleus during development (tribollet et el., 1991, dev. brain res., 58, 13-24) . avp may therefore play a role in the maturation of neuromuscular connexions in the neonate rat, and possibly in the restanration of these connexions after nerve lesion in the adult. in order to investigate the latter proposition, we have sectionned the facial nerve in adult rats and used quantitative autoradiography to look at avp binding sites in the facial nucleus at various postoperative times. we observed a massive and transient increase of avp binding sites on the operated side. the number of facial avp binding sites reaches a maximum about one week after nerve section, remains stable during 2-3 weeks, then begin to decrease towards control level. the induction of avp receptors is markedly delayed if the proximal stump of the nerve is ligated. to assess whether other motor nuclei would also react to axotomy by up-regulating the expression of avp receptors, we have sectionned the hypoglossal nerve and the sciatic nerve. in both cases, the binding of avp receptor ligand increases massively in the respective motor nuclei, with a time-course similar to that found in the facial nucleus. altogether, our data suggest that central avp could be involved in the process of nerve regeneration. cytotoxic t-cell mediated apoptosis schaerer,e, karapetian,o.,adrian,m. and tschopp,j. inst.de biochimie, univ.de lausanne, 1066 epalinges. an apoptotic cell death mechanism is used by cytolytic t cells (ctl) to lyse appropriate target cells. ctl harbor cytoplasmic storage compartments, containing the lytic protein perforin and serineproteases (granzymes), whose content is released upon target cell interaction. we show that these granules are multivesieular bodies and that degranulation releases these intragranular vesicles (igv) having granzymes, t-cell receptor and yet undefined proteins associated. isolated igvs and perforin induce dna breakdown in target cells within 20 minutes. microscopic analysis demonstrates that igv specifically interact with target cell via the t-cell receptor and that their contents is taken up by the target cell. already 15 min. after interaction, 3 distinct igv proteins are found in the nucleus of the target cell.one of the molecules has been identified to be granzyme a, previously reported to be involved in apoptosis. we propose that lymphocytes transfer apoptosisinducing proteins to the nucleus of the target cells using vesicles as vehicles for delivery. cytotoxic t cells kill their targets by a mechanism involving membranolysis and dna degradation (apoptosis). recently, two sets of proteins have been proposed as dna breakdown-inducing molecules in t cells: granzyme a, b and tia-i. in this study, we cloned and further characterized the tia-i mouse homologue. aa sequence comparison with the human tia-1 showed an overall identity of 93%. devoid of a signal peptide, tia is yet localized to cytotoxic granules, probably targeted via a gly-tyr-motif. as tia-i, its mouse homolcgue contains three rnabinding domains. expression of tia during development shows a very strong signal in the brain and weaker signals in thymus, heart and other organs. during embryonic development several structures that contribute to organogenesis form transiently and are later eliminated by apoptosis. this pattern of tia expression could indicate its involvement in apoptosis. prostate involution occurs after castration in rats and is associated with the death by apoptosis of a large fraction of the epithelial cells. we have isolated several genes from a prostate involution bacteriophage lambda library using differential screening methods. among these clones, one d~monstrated an especially strong signal when used as a probe against northern blots of prostate mlhna obtained before, and at different times after castration. this gene is down-regulated after castration by 40-fold within 5 days. intramuscular injection of a testosterone depot resulted in complete restoration of expression within 24 hours. upon sequencing it became apparent that this clone has a high degree of homology to a known ndah dehydrogenase encoded in mitochondrial dna. the clone failed to hybridize to any transcripts from rat organs other than prostate. we are now in the process of isolating the htm~n hc~olog to this gene for use as a biomarker in study of benign hyperplasia and developing carcinoma. this gene is a possible indicator for testosterone-independent cell populations or of cells lacking ftl~ctional testosterone receptor. during the first three postnatal weeks the rat lung undergoes the last two developmental stages, the phase of alveolarization and the phase of microvascular maturation. the latter involves a decrease of the connective tissue mass in the alveolar septa and a merging of the two capillary layers to a single one. speculating that programmed cell death may play a role during this remodeling, we searched for the presence of apoptotie cells in rat lungs between days 10 and 24. lung paraffin sections were treated with y-terminal transferase, digoxigenin-dutp, and anti-digoxigeninfluorescein-f(ab)-fragments, and the number of fluorescent nuclei was compared between sections at different days. while the number of apoptotie ceils was low until the end of the second week and at day 24, we observed an about eight fold increase of fluorescent nuclei towards the end of the third week. we conclude that programmed cell death is involved in the structural maturation of the lung. brunner, a., wallrapp, ch., pollack, i, twardzik, t. and schneuwly, s. lehrstuhl genetik, biozentrum universit~t w~rzburg, mutants in the giant lens (g/l) gene show a strong disturbance in ommatidial development. in the absence of any gene product, additional phetoreceptors, cone cells and pigment cells develop. opposite effects can be seen in flies in which the gene product of the giant lens gene can be ectopically expressed by heat shock. a second very typical phenotype is the disturbance of photoreceptor axon guidance. molecular analysis of gil shows that it encodes a secreted protein of 444aa containing three evolutionary conserved cystein-motives very similar to egf-like repeats. we propose that gil functions as a secreted signal, most likely a lateral inhibitor for the development of specific cell fates and that gil, either directly or indirectly, is involved in targeting photoreceptor axons into the brain. the decrease in cellularity during scar establishment is mediated through apoptosis desmouliere, a., redard, m., darby, i., and g. gabbiani department of pathology, cmu, 1 rue michel server, 1211 gen~ve 4 dudng the healing of an open wound, granulation tissue formation is characterized by replication and accumulation of fibroblastic cells, many of which acquire morphological and biochemical features of smooth muscle cells and have been named myofibroblasts (sch0rch et el., histology for pathologists, t992). as the wound evolves into a scar, there is an important decrease in ceuuladty, including disappearance of myofibroblasts. the question adses as to which process is responsible for myofibroblast disappearance. during a previous investigation on the expression of (z-smooth muscle actin in myofibroblasts, we have obsewed that in late phases of wound healing, many of myofibroblasts show signs of apoptosis end suggested that this type of cell death is responsible for the disappearance of myofibroblasts (darby et al., lab. invest. 63:21, 1990) . we have tested this hypothesis by means of electron microscopy and morphometry and by in situ end-labeling of fragmented dna (wijsman et al., j. histochem. cytochem. 41:7, t 993) . our results show that the number of apoptotic cells increases as the wound closes and suggest that this may be the mechanism for the disappearance of myofibroblasts as well as for the evolution of granulation tissue into a scar. (supported by the swiss national science foundation, grant n~ s01-16 r. jaggl, a. marti and b. jehn. universit~t bern, akef, tiefenaustr. 120, 3004 bern at weaning the mammary gland undergoes a reductive remodelling process (involution) which is associated with the cessation of milk protein gene expression and apoptosis of milk-produclng epithelial cells. this process can be reversed by returning the pups to the mother within 1 day. elevated nuclear protein kinase a (pka) activity was observed from one day post-lactation, paralleled by increased c-los, junb, ]und and to a lesser extent c-]un mrna levels. ap-1 dna binding activity was transiently induced and the ap-1 complex was shown to consist principally of cfos/jund. oct-1 dna binding activity and oct-1 protein were gradually lost from the gland over the first four days of involution, whereas oct-1 m_rna levels remained unchanged. comparing nuclear extracts from normal mammary glands with nuclear extracts from glands which had been cleared of all epithelial cells three weeks after birth revealed that pka activation, ap-1 induction and oct-1 inactivation are all dependent on the presence of the epithelial compartment. the increased fos/jtm expression and the inactivation of oct-1 may be consequences of the increased pka activity. when involution is reversed, both, pica activity and ap-1 dna binding activity (and fos andjun mrna levels) are reduced to basal levels. our data suggests a role for pka and ap-1 on progranlmed cell death of manlnmry epithelial ceils. bcl-2~ does not require membrane attachment for its survival activity c. borner*, i. martinout, c. mattmann*, m. irmler*, e. sch&rrer*, j.-c. martinou-j-, and j. tschopp*. * institute of biochemistry, university of lausanne, 1066 epalinges, 1 institute of molecular biology, glaxo inc.,1228 plan los ouates. 8cl-2(z is a mitochondrial or perinuclear-associated oncoprotein that prolongs the life span of a variety of cell types by interfering with programmed cell death. how it exerts this activity is unknown but it is believed that membrane attachment is required. to identify critical regions in bcl-2o~ for subcellular localization and survival activity, we created by site-directed mutagenesis, various mutations in regions which are most conserved between the different bcl-2 species. we show here that membrane attachment is not required for the survival activity of bcl-2o< a truncation mutant of bcl-2(z lacking the last 33 amino acids (t3) including the hydrophobic domain is soluble, yet fully active in blocking apoptosis of sympathetic neurons induced by ngf deprivation or l929 fibroblasts induced by tnfc~ treatment. we further provide evidence for a putative functional region in bcl-2 which lies in the conserved domains 4 and 5 upstream of the hydrophobic cooh terminal tail. the breakdown of nuclear dna is considered to be a hallmark of apoptosis. we previously identified the perinuclear membrane localized dnase i as the endonuclease involved in the formation of oligonucleosomal-sized fragments (dna ladder). it is not clear how the nuclease is activated and has access to the dna. we show that in thymocytes induced to undergo apoptosis, lamin breakdown preceded dna laddering. by transfeeting hela cells with a constitutively active cdc2 mutant, nuclear envelope breakdown and typical apoptotic features (ehromatin condensation) were observed. moreover, co-transfection with cdc2 mutant and dnase i led to dna degradation. we propose that apoptosis can be induced by wrongly timed and hence abortive mitosis leading to uncontrolled nuclear membrane disintegration. s02-01 s02-04 platelet-derived growth factor (pdgf) is thought to play an active role in fibrosing diseases. bronchiolitis obliterans-organizing pneumonia (boop) is a condition characterized by intraluminal proliferation of connective tissue inside distal air spaces. to evaluate pdgf expression in boop we performed immunohistoehemistry on lung biopsies from 20 patients and 10 controls free of fibrosis. sedal sections were stained with an antibody against either pdgf or the monoeyte/macrophage marker cd68, in both groups the pdgf ~9 cells were essentially tissue macrophages. using point counting to measure volume fraction (vv) , pdgf-pesitive cells represented 4.65+1.63% (mean+sd) of the volume occupied by lung tissue in the boop cases, and 2,12+0.65% in the controls (!0<0,001). similarily, 10.73+4.69% of the lung tissue was occupied by cd68 e~ macrophages in the boop cases, compared to 5.37:~3.73% in the controls (p<o.005). a positive linear correlation was found between the v v of pdgf ~ cells and the v v of cd68@ cells on the 30 cases (r=g.50 p<0.0o5). we conclude that boop is characterized by a recruitment of tissue macrophages and that a significant proportion of them express pdgf. as pogf can also induce the macrophage chemo-attractant mcp-1, we speculate that maerophages are involved in a positive feed back regulation, and may play a pivotal role in the pathogenesis of boop. barrier lesions in hydrostatic pulmonary edema d.wu, h.bachofen, u.gyger and e.r. weibel department of anatomy, university of bern, ch 3012, bern pulmonary edema induced in isolated perfused rabbit lungs by moderate elevation of perfusion pressure (14 tort ) results in the formation of characteristic bleb-like extensions of the thin cytoplasmic leaflet of alveolar epithelium ( bachofen et al. am rev resp dis. vo1.147. pp989-996,1993) . in this study, we show by means of electron microscopy and morphometry that the frequency of the blebs correlate with the distribution of alveolar edema fluid. we find that 5% of the epithelial surface is involved in the formation of blebs. three-dimensional reconstruction showed that blebs had near-spherical shape and that their opening toward the interstitium is irregular. these finding demonstrate a great plasticity of epithelial cell extensions when subjected to moderate pressure stress. at high pressure the cell linings demonstrate clear breaks. two major roles have been defined for no: cellcell communication mediated by the stimulation of cgmp synthesis, and cytotoxicity by direct interaction of the free radical no with cellular target. to investigate these effects in human epithelia, which constitute a major source of no in man, we introduced the murine inos sequence into sv40-t immortalized human bronchial epithelial cells (beas-2b). inos activity was higher than 100 pmol citrulline/min/ mg prot in the first passages of inos transfected cells, but it decreased by subculturing. no inos activity was detected in cells transfected with control vector. in inos transfected cells, no stimulated a cgmp synthesis and c-los expression which could be inhibited by addition of lnma. thus, in human epithelial cells no is able to significantly alter signal transduction pathway. partial pneumoneetomy, i.e. the resection of lung tissue, is known to initiate compensatory growth in the remaining lobes. under appropriate conditions this results in a complete restoration of lung parenchymal structure and function. unfortunately the molecular mechanisms controlling the onset of this compensatory growth are poorly understood. the aim of our study was to find and eharacterise genes which are up or down regulated during this time. for isolating such genes, we chose the differential display approach (p.liang and b.pardee 1992, science 257:p 967). therein a 3'end fragment of a subset of reverse transcribed mrnas is amplified by the polymerase chain reaction. the fragments can be separated on a dna sequencing gel. genes differentially expressed in pneumonectomised and sham-treated rats show different patterns on these gels. the fragments of such genes can be isolated from the gel and cloned into vectors in order to provide tools to characterise their gene. a set of such genes, some up-regulated and others downregulated following pneumonectomy, have been isolated, partially sequenced and characterised. time periods. our new plasma marker consisting of highly concentrated gold nanospheres was infused via a femoral vein catheter into the right atrium of anaesthetised and ventilated rabbits. five or 10 seconds after start of infusion, the blood flow was stopped and the lung was fixed by instillation. silver enhanced paraffin sections revealed labeled and unlabeled blood vessels. electron-microscopic sections showed various plasma gold particle concentration. morphometric analysis of electron micrographs showed an increase of the portion of marked capillaries from shorter to longer circulation times. based on our results, we conclude the existance of inhomogeneous plasma flow velocities within the pulmonary microvascular bed. (supported by snsf grant 31-30946.91) in schizosaccharomyces pombe pairing of meiotic chromosomes is not accompanied by the formation of a tripartite synaptonemal complex (sc), a structure which connects the homologs in most other eukaryotes. meiotic nuclei revealed novel structures ("linear elements") that might be related to the axial elements ($c precursors) of other organisms and function in meiotic chromosome pairing, recombination, and segregation (baler et al. (1993) jcb 121:241). to get further insight into the behavlour of meiotic chromosomes, we performed fluorescence in situ hybridizations on spread nuclei with probes from different chromosomal regions. interestingly, all centromeres and telomeres become clustered during meiotic prophase. the centl:omeres are paired already before meiosis, whereas the telomeres initiate pairing specifically during meiotic prophase, followed by pairing of interstitial regions. painting of whole chromosomes revealed that the homelogs occupy together a specific territory in the nucleus. an expression vector containing the vai gene read by rna polymerase ill, injected into xenopus zygotes, yields high levels of rna in virtually all embryonic cells. anti sense fina is produced from a segment of the xhoxla gene inserted in opposite orientation into the vai gene. immunological staining of whole mount embryos shows that target mrna level and xhoxla protein expression of xhoxla protein is diminished in about half of the antisense treated individuals. accordingly, nearly half of the embryos develop typical malformations in regions where the xitoxla gene is normally expressed. somites el the anterior trunk are heavily disorganized and mesodermal denvalives surrounding the intestinal tract are reduced or missing. antisense inhibition, through production of rna in situ from expression vectors, thus represents a useful tool to study the functional role of zygotic genes in xenopus development. cloning and characterization of the mouse homolog to s.cerevisiae sep1 encoding a dna strand exchange and homologous pairing protein vladimir bashkirov and wolf-dietrich heyer. institute of general microbiology, university of bern, baltzer-strasse 4, 3012 bern. homologous pairing and dna strand exchange are the central steps postulated in the current models of genetic recombination. to date no mammalian gene encoding a homologous pairing protein has been cloned. to get insight into the mechanism of recombination in higher eukaryotes we were interested to clone such genes. protein sequence comparison of the s.cerevisiae homologous pairing protein, sepi, and its homolog from s.pombe, p140 ex~ revealed several conserved stretches of amino acids in their nh2-terminal regions. using fully degenerate primers and mouse testes edna as a template for the polymerase-chain-reaction a product with the expected length has been amplified. this 360 bp dna was shown to encode a putative polypeptide with high degree of homology to both proteins from the two yeasts. the mouse pcr-product was used as a hybridization probe for screening a 1 gtlo mouse testis cdna library. after several rounds of successive screening a total of about 4 kb of mouse edna (msep1) was cloned and sequenced. the putative translation product revealed high homology throughout the entire sequence to its s.pombe and s.cerevisiae counterparts, 38 % and 41% of amino acid identity, respectively. the msep1 sequence was shown to be unique in the genome and the chromosomal gone is likely to contain many introns. we want to establish a functional full length edna clone of msepi and characterize the gone and protein product. we have earlier developed a cell free system to study recombinational repair of dna double strand breaks and deletions. the method allowed us to purify and characterize a high molecular weight multiprotein complex (rc-1), which catalyzes dna recombination. a dna polymerase and dna ligase as well as other enzymatic activities copurify with rc-1.we have commenced an investigation of homologous dna recombination in nuclear extracts which were prepared fzom normal and mutated mouse thymocytes. the scid mutation in mice causes an aberrant v(d)j joining reaction, an elevated sensitivity to ionizing radiation and a defect in recombinational repair. moreover, the normal mouse thymocytes were sorted into the cd4/cd4 double negative (dn), double positive (dp), and single positive (sp) subclasses and their nuclear extracts, and extracts from rag-2 -/-mice, were tested. only the scid mice and the cd4/cd8 sp thymocyte extracts were inactive in the recombinational repair reaction. this indicates a development stage specific activity and a scid specific defect of recombinational repair in thymocytes. restoration of the recombination activity was observed in thymocyte extracts derived from scid, which express a wansgene for the t-cell receptor i~ chain. a protein could be purified from normal thymus cells to near homogeneity which specifically rescues the recombination activity in scid extracts. this protein, srsp ~cid recombination stimulatory ~otein), migrates as a single band of app. 7 2 kda in sds polyacrylamide gel electrophoresis. the ade6-m26 mutation in the ade6 gene of the fission yeast schizosaecharomyces pombe gives rise to a hot spot for meiotic homologous recombination. to address whether transcription plays a role in m26 activity, the effect of the deletion of the ade6 promoter in combination with either the m26 and m375 mutations on recombination frequency was studied at the tetrad level. deletion in eis of the promoter abolishes m26 hot spot activity relative to the m375 control deletion strain. it is possible that deletion of the promoter has eliminated a sequence necessary for m26 hot spot activity, rather than lack of transcription being responsible for the loss of m26 activity. the role of transcription in m26 hot spot activity is currently being examined. we are analysing meiosis in a temperature sensitive top2 mutant. complementary temperature-shift experiments showed that topoisomerase ii is required for both meiotic divisions; early meiotic events are not impaired. in a meiotic time course, dapi staining revealed that the cells are blocked before the first meiotic division. interestingly, in meiotic spreads we found that the spindle pole body (spb) cycle is not affected by the block: the final arrest point showed 1 nucleus with 4 completely separated spbs. we want to analyse the number of spbs and spindle formation with immunofluorescence. we are planning to analyse the course of meiosis in a top2 rec7 double mutant (rec7 is recombination deficient) to see if topoisomerase ii activity is necessary for separation of recombined chromosomes. analysis of meiosis in a top2 rec8 double mutant (rec8 is recombination deficient and does not form linear elements) could give us a clue about the function of the linear elements. genetic recombination is able to induce cancer and to mediate its further progression. in cells from cancer predisposed individuals, mitotic recombination can lead to loss of heterozygous tumor suppressor genes as is e.g. observed in the hereditary form of retinoblastoma. one mechanism for gene loss is inter-or intrachromosomal recombination involving short duplicated sequences. our aim is to identify xenobiotics that are able to induce mitotic recombination and to elucidate the involved molecular mechanisms. for that purpose we have constructed transgenic d. melanogaster (1). rn.). cell lines. cells were stably transfeeted with an extrachromosomal dna element providing a putative recombination substrate+ "l%e constructed element contained two ta'uncated fragments of the neomycin resistance gene (nee r) interrupted by a hygremycin resistance marker (hygr). the hyg r insert was flanked on both sides by identical 352 bp fragments of nee r, different d. m. promoters shown to be constitutively active in various d. m. tissues were inserted 5' to both e. coil genes. restoration of a functional nee r gone may occur by deletion of the hyg r after recombination between the homologous regions. stably transfected schneider line 2 cells were obtained and preliminary results concerning chemical induction of genetic rearrangements will be shown. fleck, 0., sch~r, p. and kohli, j., institute of general microbiology, baltzerstr. 4, 3012 bern to detect mismatch-binding proteins of s. pombe we performed band shift assays with radiolabeled oligonucleotides containing a defined mismatch. we found two distinct mismatch-binding activities. one shows high affinity to most of the mismatches, but is absent for c/c. this protein might belong to the general mismatch-repair system, which is thought to be related to the bacterial muts dependent pathway. the second activity preferentially binds to those mismatches containing a cytosine and therefore represents a novel type of mismatch-binding protein. baur m., sch~r p. and kohli j., institute of general microbiology, baltzerstrasse 4, ch-3012 bern homologs of the salmonella typhimurium (mutl, s and 59 and streptococcus pneumoniae (hexa, b) genes involved in mismatch repair have been identified in several distantly related organisms. so far no mismatch repair gone is known in schizosaccharomyces pombe. in order to get more insight into the ~chanisals of mismatch repair in s. pombe we started the cloning of a mutl homolog. degenerated oligonucleotide primers based on conserved regions of mutl homologs were used in the polymerase chain reaction (fcr) to amplify a corresponding dna fragment from s. pombe. a dna fragment of about 220 bp was amplified whose deduced amino acid sequence shared a high degree of homology with paiql of saccharomyces cerevisiae and mutl, hexb. with the help of this dna fragment the gone was isolated from a genomic dna library by colony hybridization. sequence analysis of this mlhl gene (mlh = mut~ homolog) shows an orf of the expected size with three putative introns. while the deduced amino acid sequence of the 5' region shares strong homology with the procaryotic genes mutl, hexb and the eucaryotic gene pmsi, the amino acid sequenoe of the 3' region shows homology only to pmsi and hexb. the central region of the protein seems to be conserved only weakly. parisi s., and kohli j., institute of general microbiology, university of bern, ch-3012 bern rec7 and rec8 are supposed to be genes with major functions in meiotic recombination since mutations in these genes reduce the frequency of meiotic intragenic recembination ~1000 fold. these genes have been isolated and sequenced but no enzymological activity could be assigned to the protein. a first step toward enzymological and biochemical characterization of rec7 and rec8 is to make beth protein products available in sufficient quantities and in reasonable pure form. for this purpose both genes will be cloned in a s. /x~nbe over-expression system using the strong and inducible nmtl promoter.in parallel both genes will also be expressed in e. coli as gst fusion proteins in order to produce polyclonal antibodies in rats and rabbits. these antibodies can then be used for protein purification in s. pombe as well as for immunofluorescence experiments. induction of expression in e. coli produces a band of the expected size of 56kda for rec7 and of 73kda for rec8. to remove the gst carrier, the rec7 and rec8 fusion proteins can successfully be cleaved by thrombin or factor xa respectively. production of antibodies is in progress. d6bbeling, u., nobi, r,, berchtold, m.w. and kuenzle, c.c. institut fdr veterinarbiochemie, universit6t zurich, wintertharerstrasse 190, normally, only pre b-and pre t-cells can rearrange their immunoglobulin genes by v(d)j recombination, but when other cell types are transfected with the rag-i and rag-2 genes they also become able to perform v(d)j recombination. by transiently transfeeting nih 3t3 fibroblasts with both intact rag genes we found da recombination rate of 0.06%. no recombination was detected, when we transfected the intact rag-1 gene with a mutant rag-2 gene, which lacks 89 c-terminal amino acids, or the intact rag-2 gene with a rag-i gene containing a deletion in the topoisomerase iz homology region. these experiments show that these two regions of the rag genes are essential for v(d)j recombination. when the l4 fibroblast cell line, which has been rendered recombination competent by stable transfection with the rag genes was overtransfected with both wild type rag genes, we found in contrary to an expected increase of v(d)j recombination a reduction by a factor of 2-2.5. this decrease was not observed with the mutant rag genes.this result indicates, that there exists an optimal intracellular rag concentration for efficient v(d)j recombination. m. fischer, a. sailer, h. blieler, t. riilicke*, a. aguzzi +, p. autenried and c. weissmann; 1nstitut fiir molekularbiologie l universitfit ziirich, h6nggerberg, 8093 ziirich, *biologisches zentrallabor and + lnstitut fiir neuropathologie , universitdtsspital ziirich, 8091 zfirich, switzerland the infectious agent of transmissible spongiform encephalopathies such as creutzfeldt-jakob disease in man or scruple in sheep is thought to be prpsc, a posttranslationally modified form of the normal host protein prpc we have shown that mice devoid of prpc (prn-polo) are completely resistant to scrapie. here we report that also heterozygous prn-p o/+ mice with reduced levels of prp c show enhanced resistance to scrapie, as manifested by a long delay in the onset and slow progression of clinical disease. conversely, prn-po/o animals overexpressing prp transgeues exhibit shortened incubation times and accelerated progression of the disease. propagation of the scruple agent is completely abolished in prnpolo animals. high titers of infectivity, about 108 logld50 units/g of brain, are detected in prn-po/+ mice 33 weeks after inoculation (the earliest time point measured), 24 weeks or more before the animals die of scrapie. in contrast, wildtype animals survive no more than 16 weeks after reaching similar titers of infectivity. a practical implication of our findings is that moderate inhibition of prpc synthesis could mitigate disease progression in spongiform encephalopathies. the ruvb protein has been shown, biochemically and genetically, to be involved in the process of homologous recombination in e. coli. one of its functions is to promote branch migration within holliday junctions formed during the process of recombination. to investigate the mechanism by which ruvb promotes branch migration we have used conventional electronmicroscopy, cryo-electron microscopy, scanning transmission electronmicroscopy, and image analysis to study the interaction of ruvb protein with dna. we observed that ruvb forms multimeric rings on dsdna which encircle the dna. we also observed that such rings tend to localize at the holliday junctions. we propose that ruvb rings can actively move along dna using the energy of atp hydrolysis.this movement continues upon encountering a holliday junction, thus forcing branch migration along the dna. gschwind m. and huber g., pharma division, preclinical research, in some families with early onset aizheimer's disease mutations on the 8-app gene have been identified which cosegregate with the disease. so far all these pathogenic mutations have been identified on exons 16 and 17 in humans framing the sequence of the 8-amyloid itself.a genomic clone was isolated from mouse strain c57 black/6 containing the coding regions of the last three exons (16) (17) (18) according to the human sequence. sequencing of all three exons including the large 3' non coding region showed a 100% homology to the previously described balb/c mouse cdna. exon-intron boundaries were determined at the same positions as in human and rabbit genome and the flanking regions in the introns are also very similar, not only is the homology between the human and mouse f$-app very high (97.6%) but also the genomic organisation of their genes is nearly identical. therefore homologous recombination can be applied to introduce molecular 8-app changes and to study their functional consequences on the protein level. we systematically investigated the ability of reca protein to push the dna strand exchange reaction through heterologous regions. we observed that reca can push the strand exchange through substantial heteroiogy (up to 150 bp) when the heterologous insert was placed on the so called proximal end of the linear duplex. in the case of the medial beterology the strand exchange reaction could overcome heterologies of up to about 50 bp. heterology at the distal end was most difficult for reca to resolve, and already 22 bp long insert was completely blocking the progress of the strand exchange. these results, together with earlier electron microscopy studies, allow to propose a molecular mechanism by which reca can exchange strands between partially heterohigous dna molecules. institut for veterin~r-virologie, universit~t bern, ch-3012 bern bovine viral diarrhae virus is a positive-stranded rna virus of the pestivirus group. two biotypes, cytopathic and non-cytopathic in cell culture, can be distinguished. all cytopathic strains characterized so far carry insertions in their genomes. they probably result from a strand-switching activity of the viral rna polymerase during viral replication. to study the switching capacity of the viral enzyme, rna isolated from cells infected with two different strains of bovine viral diarrhea virus were analysed for homologous recombination in an rt-pcr assay. performing the assay we found that fragmented rna present during reverse transcription, the reverse transcriptase enzyme itself and viral rna present in the pcr reaction result in the production of artificial recombination during the assay. it was not possible to detect a homologous recombination activity of the viral polymerase above the background recombination frequency of the rt-pcr assay. our results strongly question the use of the rt-pcr assay for the study of recombination of rna viruses. transpositional rearrangements mediated by insertion sequence (is) elements represent an important source of genetic variation within populations 1. we analyze dna rearrangements at the level of the complete genome by rflp, using 7 is elements as probes for hybridisation to southern blots samples taken at different time points from 12 independent cuttures of e coli b grown by serial transfer for 10'000 generations 2 and stored at -70~ are used to study structure and evolution of genetic diversity within populations. the seven is elements contribute differently to genetic variation and lead to a succession of mutants affecting the genetic structure of the population. we now search for correlations between rflp patterns and the increase of relative fitness over time observed previously 2 naas t., 81o~ m, fitch w.m and arber w, (1993) the complete dna sequence of the locusta migratoria mitochondrial genome has been derived from four cloned mtdna fragments. the sequence is 15.2 kb in length, contains 13 peptide coding sequences, and genes for 22 trnas and two rrnas. the organisation of the genome shows a strong resemblance to drosophila, the gene order and orientation being the same except for the positions two trnas. this contrasts with the only other non-dipteran insect mtdna to have been sequenced, apls mellifera, which shows differences in the positions of ii trnas. this finding is discussed in relation to the at content of the different genomes. the sequences of the three mtdna genomes are also compared directly and the results related to existing knowledge concerning the evolution of insects. udem, new jersey medical school, newark, nj 07103-2714, usa nonsegmented negative strand rna viruses are so far not amenable to reverse genetics. the genomes must associate with nucleocapsid (n), phosphoprotein and polymerase proteins to form a functional rnp. as a step towards reverse genetics of measles virus (mv), plasmid vectors were constructed allowing synthesis of rnas corresponding precisely to a mv genome in which all coding and intercistronic regions are replaced by the chloram-phenicol acetyl transferase (cat) coding region, transfection of these negative polarity transcripts into mv infected hela or 293 cell lines gave rise to cat activity which could be serially transferred and massively amplified together with progeny helper virus in cell cultures. both expected mv/cat chimeric mrna and replication product were identified in the progeny. preliminary experiments indicate that only rnas in which the total number of nucleotides is a multiple of six replicate and transcribe efficiently, consistent with the data on natural and modified copyback di rna of sendal virus (calain and roux, j. virol. 67 4822 (1993) ). precise end to end encapsidation of rna, each n protein contacting six nucleotides, might be required. the embryonic tissue specificity of the human cytomegalovirus immediate-early (hcmv-ie) promoter/enhancer (-522 to +13) was examined by l~-galactosidase staining of transgenic mouse embryos carrying hcmv-ie regulated lacz genes. embryos of three transgemc lines were analyzed between 8.5 -13.5 dpc. for all lines, hcmv-ie transcription was primarily confined to subsets of neural and vascular cells. however, extents of transgene expression along the embryonic primary axis were slightly and greatly restricted in two of the lines relative to the third line at all stages analyzed. these differences most likely represent integration site-dependent chromatin constraints that affect levels of hcmv-ie-directed lxanscripfion. together, the three lines can be taken to define a graded potential for hcmv-ie transcription along the anteriorposterior axis of the embryo with greatest permissivity in mid-axial structures which include the dorsal neural tube at the level of the hindbraln and upper cervical spinal cord, the inner ear and vestibular acoustic ganglia, branchial arteries, aortic sac, and lung buds. the cell typespecificity and axially graded permissivity for hcmv-ie transcription in the mouse embryo provide a basis for understanding the pathology and relative frequencies of different types of birth defects caused by congenital hcmv infection in humans. stauffer, y., 0fford, e. and beard, p., swiss institute for experimental cancer research (isrec), ch-i066 epalinges, switzerland. hpvi8 is associated with genital carcinoma. like other human papillomaviruses, hpvi8 cannot be easily propagated in cell culture, because the viral growth cycle is dependent on the differentiation of its host cell, the keratinocyte. moreover, very little viral capsid protein is found in vivo in infected tissues. the hpvi8 l1 and l2 genes code for the major and minor capsid proteins, respectively. to obtain the viral capsid proteins we made constructs with the l1 and l2 genes for expression in e. coli (the pqe system) and in recombinant vaccinie virus (a modification of the phgs-i system) infected cells. the l1 and l2 proteins expressed in e. cell contained a histidine tag encoded by the vector. this allowed us to purify the proteins on a sickelaffinity column in order to raise antibodies. these will permit us to detect the presence of capsid proteins expressed from recombinant vaccinia viruses. we expect the l1 and l2 proteins expressed together from the vaccinia-based vector to selfassemble to form virus-like particles. to papain-like proteinases. the role of these putative catalytic residues in the activity of the proteinase was studied by site-directed mutagenesis. a minimal proteinase has also been constructed by n-and c-terminal deletion to determine the boundaries of the proteolytic domain, sequence analysis of the c-terminal cleavage product should indicate the site of cleavage. cell entry, uncoat~ng and nuclear transport of adenovirus z . u. f. greber and a. helenius. yale university school of medicine, new haven, ct, u.s.a. to enter cells, viruses must achieve three major goals: transfer their genome across the plasma membrane into the cytosol, target the genome to the replication site and decondense it for replication. adenovirus, an icosahedral nonenveloped particle with a double stranded genomic dna and ii to 15 structural proteins~ enters into human kb cells by a sequential disassembly program during receptor-mediated endocytosi~, acid-dependent penetration into the cytosol, and targeting to the nucleus. during internalization, the fibers, primarily responsible for virus binding to the cell surface, are detached. in early endosomes, the capsid-stabilizing proteins ilia and viii are released. fenton base, a vertexassociated protein implicated in penetration across the endosomal membrane, is removed in endosomes, if penetration is blocked with lysosomotroplc reagents. in the absence of inhibitors, a large fraction of penton base stays associated with the cytoplasmic capsid. the viral dna is disconnected from the shell after proteolysis of the internal protein vi~ a capsid and dna binding component. removal of protein ix, a capsid binding factor, further destabilizes the coat, and the remaining 'stripped-down' virus particles associate with nuclear pore complexes to release their dna-genomes into the nucleoplasm. this stepwise dissociation program is thought to provide the high efficiencies of the entry process needed to successfully deliver the viral dna across multiple barriers to the cell nucleus, the site of replication. ch. schmidt and w. arber; biozentrum der universit~t basel, abt. mikrobiologie, klingelbergstr. 70, ch-4056 basel bacteriophage p1, carried as a single copy plasrnid in e. col~, is widely known for its horizontal gene transfer ability (transduction) in enterobscteria. as a temperate phage, it can lysogenize its host and thereby provides it with accessory functions (restriction of foreign dna by ecop1, functions expressed by transposons carried on the p1 genome or integrative suppression). these properties reflect both symbiotic and evolutionary functions of pi. p1 regulates expression from its genes by two different mechanisms: trans(:ription of early regulatory genes is repressed by the phage encoded repressors c1 and bof, whereas transcription of its morphogenetic genes from p1 specific late promoters is induced by the early expressed gpl0. by site-directed mutagenesis the late promoter ps, composed of the -22 inverted repeat aagttactt and the -10 box, was functionally characterized. p5 and its derivatives compare well with several other late promoters of pt with regard to their sequence dependent strengths: the strong promoters ps, pdar and lpr2b all share the perfect inverted repeat around position -22, whereas the weaker promoters lpr91, lpr82a, lpr82b and lpr83 contain mismatches in their inverted repeats or the inverted repeat is positioned mere upstream relative to the transcription initiation site. homologies. however, the phages formed two immunity groups. the results may suggest a common genetic trait providing a selective advantage to lysogenic aa. by using a dna substrate with defined gap size we found that human immunodeficiency virus type 1 reverse transcriptase (hiv~rt) was able to perform strand displacement dna synthesis. this activity was not affected first by calf thymus proliferating cell nuclear antigen and replication factor c and second by escherichia coli single-stranded dna binding protein~ which together allow dna polymerase ~ to perform strand displacement dna syrlthesis (podust , v. and h~bscher, u. (1993) nucl. acids res. 21, . 3'-azido-2',3'-dideoxy-thymidine tripbosphate inhibited displacement completely indicating that dna synthesis is required for this reaction. the hiv-rt p66 polypeptide alone could perform limited strand displacement dna synthesis, whereas the hiv-rt p51 polydeptide was completely inactive, likely due to its inability to replicate extensively on a mi3 dna template, on the other hand the hiv-rt psl polypeptide e~hanced the strand displacement activity of the hiv-rt d68 subunit at a molar ratio of 4:1 mainly by chasing short products into longer ones. furthermore kinetic experiments after complementation of hiv=rt p66 with kiv-rt psl indicated that hiv-rt psl can restore rate and extent of strand displacement activity by hiv-rt p66 compared to the hiv-rt heterodimer d66/d51, suggesting a function of the 51 kda polypeptide, the mouse mammary tumor virus proviral dna contains an open reading frame in the 3' long terminal repeat which can code for a 36 kda polypeptide with a putative transmembrane sequence and five n-linked glycosylation sites. this gene is known to code for a superantigen which deletes a specific subset el co4 + t-lymphocytes in vivo, the superantigen encoded by the exogenous mouse mammary tumor virus of the gr strain acts specifically on v[314 bearing t-cells. we produced recombinant vaccinia viruses to express either the complete or a truncated orf protein after infection of primate cells in culture. the complete err gene in mammalian cells leads to the production of a 47 kda protein which is specifically detected with an anti-orf-peptide antiserum. the 47 kda protein can be labeled with d-[2-3h]mannose and its synthesis is inhibited by tunicamycin, an n-linked glycosylation inhibitor, indicating that it is a glycoprotein. the truncated orf protein beginning at the second atg of the open reading frame is also modified, but the c-terminal half of orf, starting at the fifth atg (orf5), has the expected size of the non modified polypeptide. pulse-chase experiments indicate that the 47 kda orf g}ycoprotein has a short half-life of about 1.5-2 hours in this system whereas the orf5 truncated protein is even less stable; its half-life is about 20 minutes. the cellular localization of the orf protein and the role it could play in vivo are currently under investigation. conclusion was based on a restriction fragment length polymorphism (rflp) with probes of is-elements. the mutants displayed different growth behavior. the evolutionary stability of 32 of these mutants was further tested by competing a mixture of them for ten days. polymorphism was maintained during this competition but two fitter mutants took over 60% of the population. implications of these results will be discussed in terms of generation and selection of genetic variants for the adaptation of microbial population. mouse mammary tumor virus (mmtv) is produced in the mammary gland of infected females and transmitted by the milk to suckling new-boms. the passage of mmtv from the gut to the mammary gland is still poorly understood, the nature of the tissue tropism of mmtv has not been elucidated, a single receptor present on mammary epithelial and lymphoid cells might serve for the entry of the virus. a cell-type specific entry mechanism could in part be responsible for this phenomenon. alternatively different surlace molecules might be involved in its uptake. we have studied the cell susceptibility to mmtv using a sensitive method of detection of newly acquired exogenous proviral dna sequences, prior to viral integration and transcription. thereby we confirm that mmtv infects mouse, rat and monkey cell lines of different tissue origin in vitro demonstrating the lack of a strict host range specificity in tissue culture as previously described. we have also observed the infection by mmtv of various human cell lines. furthermore various infection susceptibilities are observed for cell lines of a same species. r. cattaneo, p. spielhofer, k. kaelin, m.a. billeter mo/eku/arbio/ogie /, universit~t zorich, hsnggerberg, 8093 zorich subacute sclerosing panencephalitis (sspe), a rare, lethal disease is caused by clonally selected defective measles viruses (dmvs) characteristically mutated. this emerged from sequencing of five mv genes from sspe cases and by functional tests of the envelope glycoproteins hemagglutinin (h) and fusion protein if). these expressed h and f variants migrate poorly to the cell surface but maintain some cell fusion activity whereas the envelope associated matrix protein (m) appears dispensable. thus, the spread of dmv genomes through the brain seems to occur by membrane fusion at local cellular contacts, essentially hidden from the massive immune responses. in more than half of sspe cases, the propagated dmvs contain m genes in which up to 50% of u residues are replaced by cs. such "biased hypermutations" are likely due to the simultaneous conversion of many a residues to inosine in double stranded rna regions accidentally formed, mediated by an ubiquitously occurring adenosine deaminase. similar events at less spectacular degrees have now been observed in functional genes of several negative strand rna viruses. thus, this enzyme might be an important evolutionary driving force for mononegavirates. pull, i., wieland, s., nieder~st, b., keist, r., walter, e., and blum, h.e. department of medicine, university hospital z'tirich infection by hbv-positive organ donors remains a serious problem in transplant surgery. here we present a case of a 58-year-old woman who received a heart transplant from a hepatitis b surface antigen (hbsag) positive donor. the patient was negative for all hbv markers prior to transplantation. two months after transplantation she became hbsag positive and died from subacute liver failure about seven months later. cloning and sequencing of hbv dna revealed several hbv mutants in the serum of the recipient, each with a 108 bp in-frame deletion in the pre-s 1 region. transfection studies in huh-7 cells with the predominant pre-s 1 deletion mutant showed a higher replication competence compared to wild-type hbv. sequence analysis of pcr products from serum of the donor showed mainly wild-type hbv dna in the pre-s region without the pre-s 1 deletion. these data demonstrate that mutant viruses accumulate in immunesuppressed patients. specific mutants may play a critical role in the severe or fatal clinical course of hbv-infection in transplanted patients. in non-neuronal cells herpes simplex virus (hsv) establishes a productive lyric infection starting with transcription of its immediate early (ie) genes by corecruitment of a viral protein (vpi6) and a cellular factor (oct-l) onto characteristic oetamer-like sequence motifs ftaatgarat) in the promotors of the ie genes. in neurons, however, hsv establishes latent infections whereby transcription of the ie genes is prevented and the lyric program aborted. our efforts are focused towards determining which neuronal factors are involved in the regulation of hsv ie gene transcription and whether they may be involved in hsv latency or reactivation in neurons. in electrophoretic mobility shift assays (emsa) using mouse brain nuclear extracts we found that neuronal oct factors (noct-2, noet-3 and noct-4) bind to taatgarat sequences from the viral icp0 and icp4 promoters. an additional brain protein also binds to these sequences but not to a consensus octamer sequence (atgcaaat) as do neuronal oct factors. emsa assays with mutated taatgarat probes show that the taat sequence is crucial for this binding and we have tentatively named this brain protein taat-1. we are currently testing the noct factors in in vivo and in vitro transcription assays to study their involvement in hsv ie promoter regulation. biochemical and functional analysis of a vaccinia virus recombinant containing two copies of the p37k gene. schmutz, c., and wittek, r. institut de biologie animate, univei-sit~ de lausanne, ch-1015 lausanne. the most abundant protein of the vaccinia virus envelope is the palmitated 37k protein. this protein is required for envelopment and subsequent release of virus from infected cells. the amount of extracellular enveloped virus (eev) produced varies considerably depending on the virus strain and host cell line but remains in any case low (<30% of the virus production). if p37k is a limiting factor for envelopment, its overexpression might be a taeans to increase the production of eev. for this purpose a vaccinia virus recombinant containing a second copy of the p37k gene was enginee,'ed. western blot analysis clearly showed increased levels of this protein. titration assays revealed slightly lower yields of both intracellular naked virus (inv) and extracellular enveloped virus (eeu in ceus infected with recombinant virus. unexpectedly, with recombinant virus, the ratio of eev versus inv was significantly lower when compared to wild-type virus infection, despite the higher level of p37k expression. we are currently testing whether this is a consequence of a lower production, or a reduced infectivity of eev particles in cells infected with recombinant virus. hanspeter stalder, christian hertig and ernst peterhans institut f~r veterin~r-virologie, universit-st bern, ch-3012 bern bovine viral diarrhoea virus (bvdv) causes acute transient infection in animals exposed to virus postnataly and persistent infection in animals infected in utero in the early phase of gestation. the latter is associated with specific immunotolerance to the infecting viral strain. animals infected in utero may be born normal and remain healthy despite the persisting infection with a noncytopathic biotype of bvdv. mutation to the cytopathic biotype, or superinfection with an antigenically similar cytopathic biotype, results in lethal mucosal disease. we have pcr amplified and sequenced parts of the region coding for the surface glycoprotein e2 and p80, a nonstructural protein associated with enzymatic activities. over a time of one year, virus obtained from an immunotolerant persistently infected animal remained identical in its nucleotide sequence in the analyzed regions. contrasting with this remarkable evolutionary stasis over some 600-800 virus generations is the heterogeneity of viral isolates obtained from different persistently infected animals. we propose that the muitilineage distribution of bvdv is the result of antigenic drift in the population of acutely infected animals, combined with evolutionary stasis in immunotolerant animals. in 20 to 30% of naturally infected goats, the sevedty of disease in infected animals correlates with the antibody titers to the viral envelope protein (enw) and especially to the gp 38 transmembrane podion (tm). in order to analyze linear b epitopes of env, we produced a random library of caev env polypeptides fused to ~-galactosidase and expressed in e. coli. the complete env gene obtained from an infectious clone of caev-co was subcloned in a puc18 plasmid and dnase digested, random fragments of 200 bp were inseded in ~,gtl 1 dna and expressed in e. coil y1090. as the first step of b epitope mapping and in order to identify immunodominant group epitopes we used high titer sera from naturally infected swiss goats to screen the library. six immunoreactive clones were obtained and subsequently sequenced. all six mapped to the tm region of env. a fine mapping of these immunoreactive regions was performed using pepscan technology and elisa with synthetic peptides. four epitopes could be identified including the eysteine loop corresponding to an immunodominant epitope in other lentiviruses. these peptides were used in vitro to test the reactivity of sera from naturally and experimentally caev-infected goats and in rive to modulate the immune response of goats to tm. the minute virus of mice (mvm) has a 5kb linear single stranded dna genome. at both termini, short selfcomplementary sequences are folded into stable hairpin structures. the 5' (right) hairpin is a single stem structure with only three unpaired bases within the stem. these unpaired form a bubble structure. we examined the requirement in mvm lytic infection for the bubble structure in the 5' terminal hairpin of mvmp. mvmp rf dna containing the entire 3' extremity and extending to the hha i site downstream of the axis of symmetry of the 5' extremity was cloned into pgem-szf(+). this construct contains more than half of the 5' palindrome, however it lacks the sequence information required to form a bubble. murine fibroblast a9 cells were transfected with this dna and the 5' terminal sequence of resulting virus was analysed. a bubble was seen to be generated, however the sequence of nucleotides contributing to the bubble was of n0n-viral, plasmid origin. the nucleotides in question were removed from the original plasmid construct and the experiment repeated. virus was obtained as before. sequence analysis of the 5' end indicated that both flip and flop forms were present. the corresponding 5' hairpin deduced from these two forms contains no unpaired bases at the position where the bubble is normally located in wild type mvmp. the immediate early lie) gene promoters of herpes simplex virus type-1 share a similar sequence which in some cases overlaps with a binding site for octamer binding proteins. this sequence confers responsiveness to a viral transaetivator, vp16. on these promoters, vp16 combines with multiple cellular proteins to form an activating complex. we set up a reconstituted in vitro system consisting of hela nuclear extract and recombinant oct-1/pou domain protein (without the activation domain of oct-1 ) and vp16 where we showed that the activation of ie genes is not mediated by oct-i itself. rather, oct-1 serves to recruit vp16 that has no intrinsic dna binding capacity. vp16, however, possesses a stong acidic c terminal activation domain of -80 amino acids which is indispensable for the in vivo function of the protein. as this and other acidic activation domains possess only a relatively weak activating capability when we test them in in vitro transcription assays we are interested in investigating the role of this acidic activation domain in vitro i.e. under condition of cell free extracts and on naked template dna. we are using recombinant oct-1/pou domain protein and recombinant vp16 that has been truncated at the c terminus. additionally, we are also testing different truncations of vp16 in vitro that have been produced in cos cells. furthermore, we are also trying to see whether these different truncations already affect the assembly of the actvating multiprotein complex. we have studied the kinetics of synthesis of transcripts initiated from the vaecinia virus 7.5 kd gene early promoter. unexpectedly, after a first burst of rna synthesis early in infection, the promoter showed a second peak of activity in the late phase.reactivation of transcription was neither dependent on the presence of the sequences upstream of the early promoter, nor on the location of the promoter in the genome. reactivation seems to be dependent on virus assembly since it is prevented by rifampicin, that specifically inhibits an early step of viral morpho-g~nesis. analysis of several other early genes showed that reactivation is not unique to the 7.5 kd early promoter. analyzing, by in situ hybridization using digoxigenin-labelled riboprobes, the expression of the viral genome relative to that of an array of cytokines including tnf-(z; ifn-% il-i~, il-2, il-8 and gm-csf. initial experiments suggest that viral genome expression is restricted in the inflammatory infiltrates whereas certain cytokines, particularly tnf-oq are strongly expressed in the synovial membranes. overall, the results point to a prominent role of macrophage activation in the maintenance of chronic inflammation and possibly also in the development of arthritic lesions. the core protein of the hepatitis b virus (hbv) interacts with the viral rna pregenome and the reverse-transcriptase.jdna-polymerase to form a core particle which allows for replication of the viral genome. deletion analysis of the core protein revealed, that the arg-rich carboxyterminal domain is required for rna encapsidation and productive viral dna synthesis. we demonstrate, that the duck hepatitis b virus (dhbv) core protein can be derided into two domains comprising amino acids 1-67 (n-ter) and 67-262 (c-ter), respectively. the co-expression of these two core subdomains leads to the formation of replication competent core particles. furthermore, we demonstrate that the carboxy-terminal domain of dhbv core (c-ter) can be functionally replaced by the corresponding domain of the human hbv. the complete hbv core protein, however, does not form replication competent core particles with the dhbv pregenome and rt. these results imply that the carboxy-terminal part of the hbv core proteins define an exchangeable, functionally conserved domain. influenza ha mediates the adhesion and penetration of host cell membranes. acylation of three conserved cysteine residues in the membrane spanning region of several ha subtypes has been shown. the biological significance of this hydrophobic modification remains to be elucidated. a possible role for the membrane fusing activity has been controversially discussed in the past. removal of covalently bound fatty acid from protein by incubation with hydroxylamine is a commonly applied method. as we shall show for a number of different envelope viruses with acylated glycoproteins, this treatment leads to quite different consequences in a functional seiase depending on the virus used. as an alternative approach to study the biological significance of palmitoylation we expressed wild-type influenza ha and an hamutant lacking the fatty acid binding sites using the baculovirns system. both wild type and fa--mutant ha, after expression in insect cells bind to red blood cells, induce hemolysis, and fuse efficiently with fluoreseently labeled erythrocyte ghosts. hydroxylumine treatment of both recombinant wt-and mutant-ha leads to a loss of hemolysis and of fnsion activity. at least in the ease of fa--i-ia its inhibitory action must be related to some other effect than fatty acid cleavage. these results indicate that hydroxylamine treatment may not always be suitable for the generation of fatty acid free glycoproteius or virus particles for functional studies. gesemann and j.g. nicholls, biocenter univ. basel, 4056 basel, switzerland depolarization of leech neurons leads to growth cone collapse and neurite retraction. this response to depolarization is influenced by the substrata on which the cells are growing and is mediated by the influx of calcium (s. grumbacher-reinert and nicholls, 1992, j. exp. biol. 167:1-14; neely, 1993 , i. neurosci. 13:1292 -1301 . the morphologieal changes induced by depolarization of leech neurons growing on leech extracellular matrix extract is accompanied by a loss of microfilaments in the growth cones. leech neurons growing on a different substrate, the plant lectin coneanavalin a (coma), did not retract their neudtes or lose rnierofilaments, but continued to grow after depolarization. we attribute this to the reduced calcium influx during depolarization of neurons on cona (ross et al., 1988, pnas 85:4075-4078) . increasing the intraeellular calcium concentration by incubating neurons with the calcium-ionophore a23187, led to cessation of motility and disruption of microfilaments but not microtubutes in growth cones on coma. to investigate the mechanism by which calcium affects the organization of microfilaments we stained neurons with antibodies against gelsolin, a protein that severs microfilaments when it is activated with calcium. we have observed that these antibodies colocalize with microfilaments in leech growth cones, we are now analyzing the nature of this antigen that cross-reacts with the anti-gelsolin antibodies with the goal of establishing its potential role in the calcium-induced disruption of microfilaments in leech neuronal growth cones. this work was supported by a grant from the swiss nationalfond no. 3127814.89 to j.g. nicholls. activation of metabotropic glutamate receptors (mglurs) was studied in voltage-clamped ca3 cells in rat hippocampal slice cultures using the whole-cell pateh-clamp configuration. in saline containing 16 mm k +, 1s,3r-acpd (50 ~tm), a mglur agonist induced an inward current associated with an increase in membrane conductance. the reversal potential, assessed with a voltage ramp protocol from -80 to -60 mv and calculated by linear regression, was -15.7 â�¢ 18.7 mv, and was shifted to -53.4 â�¢ 6.4 mv when the concentration of external monovalent cations was halved. these results indicate that the conductance underlying this current is selective for cations (mainly k + and na+). the steady-state i/v curve for the 1s,3r-acpd-induced inward current showed a slight inward rectification. in most of the cells, this inward current was followed (or overlapped) by an outward current concomitant with a decrease in a k + conductance (ere v = -51.2 ~ 5.6 mv in [k+]o = 16 mm and shifted to -74.0 :t: 2.8 mv in [i4+]o = 8 ram). this k + current was voltage-independent in the membrane potential range of-120 to -20 mv. similar results were obtained with methacholine, a cholinergic muscarinic agonist. the non-specific cationic current, seen only in the presence of elevated extracellular k + concentration, could play an important role during intense neuronal activity. the c-myc protein has been implicated in the regulation of cell growth and differentiation, c-myc specifically interacts with max which in turn forms heterodimers with mad and mxil and homodimera with itself. we present evidence that this network o! proteins is regulated both at the level of relative protein concentration as well as by post-translational mechanisms. in the hematopoietic cell lines u-937, ml-1 and hl-6o e-myc mrna and protein are rapidly downregulated after induction o| differentiation whereas max expression is not significantly altered. in contrast the expression of mad is induced with properties similar to immediate early genes, taxi1 is induced to a lesser degree and with slower kinetics, in an effort to study the regulation of o-myc we have mapped all the major in vivo phosphorylation sites in both c-mye and max. two sites in the n-terminal transactivation domain are important for the regulation of the transforming potential el c-myc both in established cell lines as well as in primary rat embryo tibroblasts. however, no difference in the transactivating potential of corresponding mutants was observed. phosphorylatien of max at sites close to the basic region increases both on-and off-rates of either myc-max hetero-as well as max homodimers to dna. brain research institute, university of z~rich, ch-8029 z~rich, switzerland gaba b and adenosine receptors act via pertussis toxinsensitive g-proteins of the gi/g o class to mediate longlasting inhibition in the cns. this class of g-protein is negatively coupled to the enzyme adenylyl eyclase. our aim was to determine whether the inhibition of adenylyl nyclase mediated by these receptors has functional consequences for electrical signaling in hippocampal neurons. the calciumdependent k + current, i~, underlying adaptation of cell firing, was used as an electrophysiological bioassay for adenylyl cyclase activity, as it is very sensitive to intracellular levels of camp. accordingly, the ~-adrenergic agoniat iaoproterenol (5-500 nm), that activates g,-linked receptors, reduced the amplitude of i~ by 51.5 â�¢ 3.6% in voltage-clamped ca3 pyramidal cells in ttx (n=20). reduction of imm by isoproterenol was curtailed to 27.3 â�¢ 2.9%, (p < 0.001) in the presence of baclofen (i0 ~m), acting at gaba b receptors, or adenosine (50 nm). thus, the inhibition of adenylyl cyclase activity due to activation of gaba a and adenosine receptors can modulate responses to other neuretransmitters by an interaction occurring at the level of intracellular signal transduction. furthermore, stimulation of these receptors will tend to enhance i~, suppressing repetitive cell firing. this represents a further mechanism which will result in prolonged inhibition of hippocampal neurons. a study of the possible modulation by nitric oxide (no) of endogenous dopamine (da) release was performed in bovine retina in vitro. isolated half retinas were incubated in kkg at 37 ~ for 3 successive 30-rain incubations, and da was measured in the incubation medium by hplc with ec detection. when retinas were incubated in the presence of the no-generating compound hydroxylamine (300 ttm), a decrease in either basal or k +-(50 ram) stimulated da release was observed during the 2 nd 30-rain period. tested under similar conditions, dibutyryl cgmp (1 mm) was ineffective. the no synthase inhibitor l-ng-nitro-arginine methyl ester (l-name, 100 gm) increased basal da release during the 1 st and 2 nd incubation, whereas it did not affects the k+-stimulated da release. in addition, its effect were potentiated in calcium-free medium. finally, we have also shown in cell-free experiments that retinal no synthase activity was totally calcium-dependent and blocked by l-name. taken together, these results suggest that endogenous no regulates da release via a cgmp independent mechanism. division of endocrinology, university hospital, ch-1211 geneva 14 while the stimulation of aldosterone biosynthesis by angiotensin ii (angii) in bovine glomerulosa cells clearly depends upon a sustained influx of ca 2+, the pathway for ca 2 ⧠entry is not yet accurately defined. at least two mechanisms seem to be involved: 1) the opening of voltage-operated ca z ⧠channels, and 2) the stimulation of a "capacitative" ca 2+ entry regulated by intraocllular ca 2 ⧠stores; however the relative contribution of these pathways to the stimulation of steroidogenesis needs to be determined. two pharmacological tools were used: nieardipine, a blocker of to and l-type ca 2+ channels, and thapsigargin, which releases ca ~+ and therefore induces a capacitative ca ~+ influx. nicardipine (1 lam) completely blocked the cytosolic ca 2+ response to k +, which exclusively activates voltage-operated ca 2. channels, but only partially (22 %) the response to angli. in the presence of nicardipine, angll was unable to stimulate a ca 2+ influx aiter depletion of ca 2 ⧠stores with thapsigargin, demonstrating that angli principally stimulates a capacitative ca 2+ entry pathway. in addition, nicardipine completely blocked the steroidogenic response to k +, but only 30% of the response to angll. thapsigargin-induced aldosterone formation, as the response to angll, was markedly potentiated by low concentrations of k +. in conclusion, this study shows that in bovine glomerulosa cells, angli activates a sustained ca 2+ influx, principally through a capacitativc pathway, leading to aldostcrone formation. this finding could partially explain the poor efficiency of dihydropyridine ca 2 ⧠antagonists for preventing aldosterone secretion. harald holder and john m. lucocq*. institute of anatomy, university of berne, ch 3012 berne *depanment of anatomy and physiology, university of dundee, uk dundee dd1 4hn. subjection of hela cells to 45~ for 30 minutes leads to a complex activation pattern of map-kinases as revealed with renaturadon gels. exponentially growing hela cells show activallon of a myelin basic protein (mbp) phospborylating kinase migrating with an apparent molecular mass of around 60kda upon heat shock induction. in contrast, in cells arrested in go by serum starvation for 24h, essentially three different proteins phosphorylafing mbp are activated subsequent to heat shock treatment. these kinases migrate with apparent molecular masses of around 32kda, 44kda and 55kda. western blot analysis with antibodies recognizing the human boraologues of erkl and erk2 revealed that considerable pools of erkl and erk2 show retarded elec/rophnretic migration behavioar indicating that they are phosphorylated and probably activated in heat subjected and growth-arrested hela cells. down-regulation of protein kinase c (pkc) by a prolonged treatment with 12-o-tetradecanoyl-phothol-13-acetate (tpa) did neither change the major activation pattern observed upon separation of cell lysates on renaturation gels nor that one observed on western blots decorated with anti-erkl or anti-erk2 antibodies. these results suggest that heat shock induced activation of at least erkl, erk2 and the 55kda mbp-kinase is not mediated via activation of pkc. this work was supported by swiss nsf grant no. 31-27636.89. effects of dominant negative glucocorticoid receptor mutants in cell cultures and transgenic animals stefano brenz verca, stefan schneider, sandro rusconi, institut fiir molekularbiologie h der universitat zf~rich, winterthurerstr. 190, switzerland the glueocorticoid receptor (gr) is involved in a large number of developmental and biochemical processes. recently, we obtained a trans-dominant negative gr mutant (tdn) -called gr[aia] -by a frame shift of the cag-repeat of the rat gr (see poster by hug et al.) . the gr[ala]-mutant shows normal llgand-binding and dna-binding but cannot stimulate transcription (lanz et al., steroids; in press) . modulatable versions of the tdn-mutant have also been created by substituting the hbd of the grmutant with the hbd of sex-hormone receptors. these constructs have only been tested so far in transient cotransfection assays. therefore, we are currently trying to express them permanently in cell lines (rat hepatoma cell line fto-2b) in order to verify whether they can affect the transcription level of a known endogenous gr-dependent target-gene, like the tyrosine aminotransferase (tat) gene or a permanently transformed mtv-iacz reporter. so far no rapid test-system has been described, that allows the quantitative analysis of such effects in transient expression assays. we are about to set up such a rapid and generally applicable transient test-system. in the future we plan to express dominant negative gr mutants in a tissuespecific manner in transgenic mice. it will be particularly interesting to investigate their effects on the immune system or on liver functions. these studies investigated growth cone responses m brief local encounters with surface molecules implicated in neuronal pathfinding using chick drg neurons. we tested two interrelated hypotheses: 1) discrete points of information (guideposts) override sustained surface information and dominate growth cone behavior and 2) guidance molecules provide such information by activating second messengers instead of simply increase adhesivity. the potential guidance molecule lamialn was covalentiy coupled to polystyrene beads in order to mimic guidepost cells. encounters with laminin model guideposts significantly altered the behavior and morphology of growth cones advancing on fibronestin or polylysine. growth cones steered towards theses beads in a saltatory medea once a long lasting contact with a single filopodium had been established. subsequent to a pause at the bead, growth cones advanced with a significantly increased growth rate for the next llmin. the average increase in rate stimulated by bead contact was 5 times the basal rate on polylysine and 2.5 times that on fibronectin. positioning of multiple model guideposts along a path and within filopodial reach maintained this bead stimulated rate of outgrowth and directed growth cone advance. the laminin induced pattern of events was totally blocked in the presence of either h7 or sphingosine, two pkc inhibitors, or neomycin, a plc inhibitor. neither of these inhibitors affected neurite outgrowth on the substrata alone. our results demonstrate the necessity of filopodial contacts, the importance of spatially restricted stimuli and the activation of transducing pathways as key mechanisms in neuronal pathfinding. mechanisms of glucocorticoid receptor desactlvation by homopolymeric alanines martin hug, manuela h&fferer and sandro rusconi, institut fiir molekularbiologie 11 der universitat uz zart'ch, winterthurerstrasse 190, 8057 ziirich, switzerland the rat glucocorticoid receptor (gr) edna contains in its y-portion a (cag-repeat that encodes a siring of 22 gin residues interrupted by 1 arg. a mutant with the frame-shifted repeat which codes now for poly-ala residues (called gr[aia]) is completely trans-inactive, is more cytoplasmitally located in absence of ligund and exhibits dominant-negative properties in presence of ligand (lanz et al., steroids, in press) . we generated gr recombinants with increasing numbers of cag-triplets in three possible reading frames (oligo-gln, -ser or -ala). the gr-oligo[ala]~-~.23 mutants showed an activity comparable to wild type gr, whereas the groligo[ala]>_25 were inactive. the severity of the effect of oligo a.la su'ings seems to be dependent on the position of the repeat along the primary sequence (e.g. progressive decrease of the effects by moving toward more earboxy-position). we have also examined the effects of the oligo[ala]21, 23, 25, 27, 31 on gal4 chimeric factors and found that the inhibition by ala repeats is probably factor-specific. our results made us speculate that possible differences in post-translational modifications may be at the root of the negative effects by the ala repeats. we believe that these effects are due to transient association of the nascent gr[aia] mutant proteins with intraceuular membranes. these hypotheses are currently tested. the activation of steroidogenesls in calcium-clamped bovine glomerulosa cells and its modulation by angiotensin ii and camp, python cp, laban op, rossier mf, vallotton mb and capponi am division of endocrinology, university hospital, geneva, switzedand. the ca2+-messenger system plays a crucial role in the regulation of steroid secretion in adrenal zona glomerulosa cells, as it is known to mediate the action of angiotensin ii and potassium ion. in the present study, we used intact isolated glomerulosa cells in which the cytosolic free ca 2+ concentration ([ca2+]c) was clamped at various levels with the ca2+-ionophore, ionomycin, in order to locate the sites of action of ca 2+, to determine their sensitivity towards ca 2+ and the modulation by other physiological factors. by measuring simultaneously steroid synthesis and [ca2+]c, we show that ca2+-clamping (50-860 nm) induced a concentration-dependent increase in the production of both pregnenolone (506___70 % of the basal level) and aldosterone (255+27 % of the basal level, ec50 = 273 nm). by contrast, ca 2+ did not influence the conversion of 1 t-deoxycorticosterone into aldosterone at concentrations up to 600 rim, although at higher concentrations we observed a modest but significant inhibition. in addition, ca2+-clamping did not affect the formation of pregnenolone from a freely diffusible analogue of cholesterol, 25hydroxycholesterol, indicating that ca 2+ acts at a step upstream of cholesterol side-chain cleavage. both angiotensin ii and a membrane-permeant surrogate of camp, dibutyryl cyclic amp, potentiated the steroidogenic response to increases in [ca2+]c . in summary, these studies reveal an exquisite sensitivity of the glomerulosa cell steroidogenic pathway toward very small physiological changes in [ca2+]c. radtke, rainer heuchel, oleg georgiev, gerlinde stark#, michel aguet# & walter schaffner institut fiir molekularbiologie 11, universilat ziirlch, 8057 z'6rich # lnstitut rir molehdarbiologie i, universitiit ziirich, 8093 ztirich we have previously described and cloned a factor (mtf-1) that binds specifically to metal-responsive dna sequence elements in the enhancer/promoter region of metallothionein genes. mtf-1 is a protein of 72.5 kda that contains six zinc fingers and multiple domains for transcriptional activation. here we report the disruption of both alleles of the mtf-1 gene in mouse embryonic stem ceils by homologous recombination. the resulting null mutant cell line fails to produce detectable mounts of mtf-1. moreover, due to the loss of mtf-1 the endogenous metalinthionein-i gene is silent, showing that mtf-i is required for both basal and metal-induced transcription. accordingly, reporter genes containing either synthetic or natural metal-reslxmsive promoters were not transcribed after transfection into the null mutant ceils. cotramsfection of the mtf-i expression vector rescued both basal and heavy metal-induced transcription. these results demonstrate that mtf-1 is essential for normal metallothionein gene regulation. rat pheochromocytoma cells (pc12) were differentiated with ngf for 3-4 days, a time sufficient for the formation of growth cones and elongation of neurites. addition of the phosphatase inhibitor calyculin a (cl-a) (0.2.50.0 nm) led to a concentrationdependent collapse of growth cones and retraction of the neurites within 15 minutes. after the retraction, the cell bodies showed the appearance of a grape-like domain opposite to the cell nucleus. they recovered to normal shape within about 30-60 minutes if the inhibitor was washed out. the neurite retraction was further analysed using both low-light level fluorescence video-microscopy and fura-2 single cell microfluorimetry. the onset of retraction started in the central part of the growth cone prior to a complete collapse of filopodia. the basal intracellular free calcium concentration ([ca2+] i) remained constant during neurite retraction. as a measure for the viability of cl-a-treated cells, agonist-isduced responses of [ca2+] i were analysed. ca2+ entry across voltage-activated ca 2-~ channels was inhibited by 50% after 30 minute treatment with cl-a, while the atp-induced ca2+ entry via ca2*-permeable cation channels was not significantly reduced. however, the onset of the atp-induced rise in [ca2+]i started at the grape-like domain. the speed of the ca 2+ wave across the cell body was slower than in control cells. the addition of 0.5 gm okadaie acid, a different type of phosphatase inhibitor, caused no cell shape change or neurite retraction within 24 hours. taken the specificity of the two phesphatase inhibiters into account, the observed effects seem to be induced by inhibition of a ppl-class phosphatase. myosin in nematocytes of hydra michel nakano & robert p. stidwill dept. zoology, university of ztirich, switzerland the functions of nonmuscle myosins during cell locomotion are not clear. several models have been presented reaching from very active participation of the molecules in different parts of cells to only minor or no roles at all. there is increasing evidence that the functions of myosin ii (capable of forming bipolar filaments) are quite distinct from the ones of myosin i and generally that the different members of the growing family of myosin-like proteins may not all have identieal functions. we have attempted to demonstrate the occurrence and determine pattern of myosins in tissue of hydra and especially in migrating nematoeytes mainly for two reasons. first, nematocytes are fairly rapidly moving cells which can be studied in vitro and, second, the question is of interest from a evolutionary point of view. we have utilized several antibodies against vertebrate and invertebrate myosins for immunofluoreseenee (confocal laser scanning microscopy) and western blotting studies. in addition to these results findings on the screening of a ~.zap c-dna library are presented. the jaki protein tyrosine kinase is a member of a family of intracellular kinases characterized by having two kinase catalytic domains: a bona fide tyrosine kinase domain and an amino terminally positioned kinase-related domain. recently it has been shown that jaki and the other members of this family (jak2, tyk2) are intimately involved in cytokine and growth factor signalling. the presence of two putative nuclear localizing sequences in the amino terminus of the protein prompted us to examine the subcellular localization of this protein. immunohistochemical and biochemical analysis revealed that a part of jaki was localized to the nucleolus. metabolic labelling showed that (a) the jaki protein partitioned rapidly into the nucleus and (b) the nuclear jaki became smaller with time. the size difference was not due to either co-translational glycosylation or phospherylation. these results suggest that part of the jaki protein is rapidly translocated to the nucleoli where it becomes processed. revealed that this clone is expressed predominantly in spleen, mammary gland and thymus as two transcripts, a major species of approximately 5kb and a minor species of approximately 4kb. nucleotide sequence analysis revealed a 84% homology to the porcine syk gene. the syk gene is expressed as a major 3.0kb transcript and has been reported to be lymphoid specific. due to the discrepancy of transcript size and number, we suggest that our cdna clone represents a novel member of this family of ptks. we are currently isolating a full length sequence from a mouse spleen cdna library. in addition, we want to characterize which cell(s) in the mammary gland are responsible for the observed expression. expression and function of g-protein isoforms were studied in differenflaring (6c8) and nondifferentiaflng ((33) subclones of red-1 rauscher murine erythroleukemia cells. erythroid differentiaflon induced by the combined action of erythropoietin (epo) and dmso was associated with a selective loss (>70%) of immunoreactive gai3. simultaneously, a iruncated form of g~2 accumulated in the cytosol, while the membrane concentrations of gai2, gets and gaq/11 remained unchanged. nondifferenfiating (33 cells contained sigrtificanfly higher levels of most get subunit isoforms that remained unchanged upon epo/dmso treamaent. changes in adenylyl cyclase activity and in intracellular ca ion levels ([ca]i) resulting from activation of g-protein-coupled receptors were monitored for differentiation-dependent effects on transmembrane signaling. adp-mediated changes of [ca]i in native and differenflaflng 6c8 cells were consistent with p2t receptor coupling to gai3. thrombin receptors seemed to couple preferentially to gai in g3 cells and to gaq in 6c8 cells. our results document substantial alterations in g-protein-mediated signaling during erythroid differentiation. to investigate the involvement of protein tyrosine kinases (ptks) in the growth control of the mammary gland, we have used pcr based cloning to identify ptks expressed during normal mammary gland development. this approach led to the isolation of three members of the eph-related family of receptor ptks: myk-;, a novel member expressed predominantly in lung, heart and mammary gland; m-eck, the murine homologue of the human eck gene and mek5, the murine homologue of the chicken cek5. all three ptks were expressed in a distinct and narrow window of mammary gland development: puberty and estrus cycle. no expression was found either in late pregnancy or in the end differentiated state of lactation. 0vet-expression was found in the undifferentiated and invasive tumors of transgenic mice expressing the h-ras oncogene. in contrast, no elevated expression of all three genes was detected in the well differentiated, non-invasive mammary tumors characteristic of c-myc expressing transgenic mice. these results suggest that members of this family of ptks are involved in the control of proliferation of the mammary gland but are incompatible with its differentiation. np-tcii is a lymphocyte specific transcription factor (lattion a.-l. et el., mol. cell. biol., 1992, 12:5217-5227 a nucleotide receptor that mediates phosphoinositide and phosphatidylcholine hydrolysis by phospholipases c and d, respectively. here we report that atp and utp potently stimulate mesangial cell proliferation. both nucleotides stimulate phosphorylation and activation of map kinase and the up-stream map kinase kinase. activation of map kinase by both nucleotides is dose-dependently attenuated by the p2 receptor antagonist suramin. stimulation of protein kinase c (pkc) by phorbol ester increased map kinase phosphorylation and activation, and downregulation of pkc partially inhibited atp-and utp-induced map-kinase activation. inhibitors of pkc which display a selectivity for the ca2+-dependent isoenzymes (c~, [3, 7) , as compared to the ca2+-independent isoenzymes (5, e, ~, rl) such as staurosporine and the specific pkc inhibitor cgp 41251 did not inhibit nucleotide-stimulated map kinase phosphorylation, thus implicating the involvement of a ca2+-independent pkc isoform. in conclusion, these data suggest, that atp and utp trigger the activation of the map kinase signalling cascade in mesangial cells and this may be responsible for the potent mitogenic acitivity of both nucleotides. subcellular ion-gradients in ca signaling p. lipp & e. niggli, department of physiology, univexsity of bern recent experimental evidence has indicated an important role for submicroscopic concentration gradients during ca-signaling in various cell types. in heart muscle cells influx of ca via l-type ca channels triggers ca release from the sarcoplasmic reticulum (sr) and links electrical excitation to mechanical contraction (ec-coupling). we used a ratiometric confocal method (fluo-3 and fura-red) to follow the intraceuular ca-concentration while ha-and ca-currents (inn, ica) were elicited in the whole cell mode of the patch-clamp technique. both, icand in, were able to trigger ca release from the sr. kinetic differences between ini and ic -induced ca-transients, li-substitution experiments and the existence of a residual inn-induced ca transient in the absence of sr function suggested that these ca signals were mediated by the ha-ca exchange running in the ca influx mode. control experiments showed that ins-induced ca transients did not result from spurious activation l-type ca channels. the significant increase of the na concentration required to activate the ha-ca exchange can only occur provided cytosolic diffusion of na is restricted in the vicinity of the exchangers. this limited diffusion results in significant ha-gradients in the subsarcolemmal space. in conclusion, i~iinduced ca influx may represent a safety factor for ec-coupling while ic, ensures sustained ca-release and is the source for refilling the sr with ca. supported by snf. the regenerative ca-induced ca release (cicr) mechanism is an important amplifier in cardiac signal transducilon. the cicr contributes to the ca transient responsible for mechanical activity, but also generates ca waves propagating within the cytosol. we investigated subcellular ca signals in neonatal rat and adult guinea-pig ventricular myocytes using ratiometric confocal microscopy with a mixture of two fluorescent ca indicators. individual resting myocytes exhibited spontaneous ca release events from the sarcoplasmic reticulurn (sr) that were attributable to three distinct patterns: i) local ca release events without propagation; ll) planar ca waves propagating across the cell; iii) spiral ca waves spinning around a nucleus or shifdng along a subcellular region without entering it. transitions between these patterns were frequently observed, suggesting that a particular release pattern is not the property of an individual cell but reflects the functional state of the sr. the existence of focal release and refractory zones within a single cell indicates that the sr is not uniform on the subcellular level. the sr seems to he a network of elements with distinct functional properties. a suhcellular variability in the positive feedback of the cicr mechanism can account for the observed features of ca signaling. the degree of positive feedback exhibited by a single sr element may depend on its ca content. to characterise the intracellular signalling and regulatory properties of the cloned parathyroid hormone (pth) receptor we have stably transfected two renal epithelial cell lines with cdna's encoding the pth receptor (provided by dr g. segre). the proximal tubular, porcine (llc pk1 [clone 4]), and the distal tubular, madine darby canine (mdck) cell lines were transfected since the culture of either cell line on permeant filter supports leads to the development of polarised epithelial cell layers that mimic the epithelial organisation in vivo. transfection of the cloned pth receptor into each of these cell lines allowed us to examine, independently, the functional properties of the receptors expressed at the apical and/or the basolateral cell surface. while the basolateral application of pth produces a large increase in camp production in both cell lines, the relative efficacy of an apical application of pth differs between the cell types. the transfected llc pk1 cells exhibit a greater response to apical pth compared to the transfected mdck cells. pth-mediated regulation of intrinsic phosphate transport was also examined in polarised, transfected mdck cells. neither apical or basolateral application of pth affects intrinsic apical or basolateral ha-dependent or phosphate transport activities. similar experiments are being performed with the transfected llc pk1 cells. a. and preiss, a. biozentrum der universit&t basel, ch 4056 basel hairless, a recessive larval lethal mutation, causes dominant defects in sensory organs of adult drosophila flies.manifold genetic interactions indicate that hairless antagonizes neurogenic gene function suggesting an involvment also in neurogenesis. in order to gain insight into its function we have cloned the hairless gene which encodes an extremely basic, very serine rich protein of ii0 kd calculated molecular weight. no significant homologies to proteins of known function could be identified. therefore, we are concentrating by various means on a structure -function analysis of the hairless protein. firstly, we are testing a series of hairless deletion constructs for rescue capacity and generation of anti-hairless phenotypes after overexpression compared with the wild type gene. secondly, we are analysing hairless protein distribution throughout development, also at the subcellular level. heat induced hairless protein runs at ~ 150 kd, possibly due to phosphorylation which might be intrinsic to hairless function, a hypothesis we are currently scrutinizing. thy-1 and cd26 surface glycoproteins are both capable of delivering proliferative signals to t cells upon cross-linking with appropriate antibodies. the tyrosine phosphatase cd45 is a key regulator of t cell proliferation as it controls the activity of src family kinases p56/~k and p59fy n. associations of thy-1 with cd45, and of cd26 with cd45 have been reported after chemical cross-linking of intact cells and it is likely that such associations constitute part of the structural basis for the t-cell stimulating capacity of thy-1 and cd26 accessory molecules. we report that thy-1, cd26 and cd45 can be specifically crosslinked at the cell-surface and isolated as multimolecular complexes containing a 78 kda protein that is recognized by an anti-bip (grp78) antibody. in vitro kinase assays show the selective association of p56 ick and p59fy n with thy-1 and cd45 contained in multimolecular complexes. the multimolecular structure we describe could representa transducing unit incorporating surface receptors and regulators of tyrosine phosphorylation that are stabilized through interaction with the bip protein in the plasma membrane. structure-function-analysis of hairless, a gene involved in drosophila nervous system development maier, d., functional coupling of ppars and trs ijpenberg, a., wahli, w., desvergne, b., institut de biologie animale, 1015 lausanne we are interested in a possible functional coupling of thyroid hormone (tr) and peroxisome proliferator-activated receptors (pfar). to this end, we performed cotransfection experiments in nih 3t3 cells using cat reporter constructs containing the promoter of either the t3 responsive malic enzyme (me) gene, or the ppar regulated acyl coa oxidase (aco) gene. the me reporter construct showed very moderate response to the activated mouse ppar (mppar), whereas combination of try1 and unactivated mfpar potentialized the t3 induction. a putative ppar response element was identified within this promoter and will be further investigated. the aco reporter constructs were not responsive to trs. in constrast, a tr and t3 dependent inhibition of the fpar mediated activation was observed. the mechanism of functional coupling of these receptors, which may possibly involve the 9-cisretinoic acid receptor rxr, will be further investigated by molecular analysis. unliganded steroid receptors form a complex with hsp90 via their hormone binding domain (hbd). the receptor activation involves a hormone-induced release of hsp90. we genetically addressed the role of hsp90 in budding yeast by analyzing three kinds of hsp82 (the essential yeast homologue of hsp90) mutants. these mutations affect either the quantity of the protein (low versus normal levels), its integrity (deletion analysis) or its nature (hsp82 homologues from other species). hsp82 mutant are examined for their ability to complement a hsp82 deletion mutant. moreover, they are tested for functional interaction with a hbd. interestingly, a viable deletion of a charged region, suspected to be involved in hsp82-hbd interaction, only slightly interferes with hormonal activation of both estrogen or glucocorticoid receptors. furthermore, a short deletion in the last third of hsp82 leads to the production of a dominant negative mutant which prevents ceil growth at elevated temperature. the ability of various hsp82 homologues to palliate or not the absence of the yeast protein allows us to define, using chimeras, the regions of the protein which are necessary and sufficient to support cell life. zhang,h.,reynaud, s. and spohr,g. d~partement de biologie cellulaire, sciences iii, 30, quai ernest-ansermet, ch-1211 gen&ve 4 id cdnas expressed during early xenopus development have been isolated and sequenced. the encoded protein is homologous to the proteins described in higher vertebrates. northern blot analysis reveals that transcription starts soon after mid blastula and decreases to some extent after tailbud-stage. lower levels of transcription are detected also in adult frog tissues such as liver and heart. microinjection into oocytes of in vitro-synthesized myodor/and id-mrna, along with a cat reporter gene whose expression is under the controll of an a3-actin promoter supplemented with four e-boxes shows that myo d function is repressed by id. to investigate the importance of negative regulatory sequences we have isolated and characterised the id promoter. as a strategy for identifying cis-regulatory elements, chimeric genes consisting of different 5' elements of the promoter were attached to the cat coding sequences and microinjected into embryos. we are studying the tissue distribution of the rat peroxisome proliferators activated receptor (ppar~), a member of the nuclear hormone receptors superfamily, during development and in adults. high levels of ppar~ mrnas are detected in adult liver, kidney and heart. muscle, stomach, and intestine show moderate amounts of the ppar~, whereas brain, testis and lung present a very low expression. during postnatal development, we observe a continuous increase of the ppar~ expression in the kidney, in contrast to a steady level in the liver. anjard, c., groux, b ., gamboni, s. and reymond, c.d., institut d'histologie, unil, rue du bugnon 9, 1005 lausanne. the camp dependent protein kinase (capk) from dictyostelium is composed of a single catalytic (c) and a single regulatory (r) subunit. the c subunit we characterised, pkac is about twice the size of its mammalian counterpart. we showed that it possesses all properties of a catalytic subunit. it associates with the r subunit in absence of camp, it copurifies with capk activity and an increased activity is observed in cells over-expressing pkac. furthermore, we dissected its role during development and cell differentiation. overexpressing pkac under cell type specific promoters allowed to show the requirement for its activity during spore differentiation. pkac seems to play a more complex role in prestalk cells. we are now dissecting the functional regions of the pkac protein, making use of the known tertiary structure of the c subunits of mammalian enzymes. dna binding properties and stimulation of gene transcription of baculovirus expressed xppar~. hihi, a.k, mermod, n., medin, j., ozato, k. and wahli, w., inst. de biol. animale, uni lausanne, ch-1015 lausanne. lab. of mol. growth reg., nih, bethesda, md, 20892 usa. the peroxisome proliferators activated receptors (ppars) are members of the steroid/thyroid nuclear receptor superfamily. so far, they have been found in amphibians, rodents and humans. in xenopus laevis, 3 subtypes (xppara,~.y} have been isolated. in order to study the mode of activity of the ~ form, the xppar~ gene product was overexpressed using a baculovirus expression system. the nuclear protein obtained has the correct molecular weight of 46 kd. gel shift assays showed that nuclear extracts containing the recombinant protein are able to specifically bind the 'ppar response element' which is a direct repeat of the core aggtca motif. this dna binding activity is potentiated by another nuclear receptor, the mouse rxr~ and is inhibited by phosphatase, suggesting a role for phosphorylation in ppar binding to dna. a functional approach, using an in vitro transcription system, was chosen to define ppar and rxr action on transcription stimulation, as well as the role of their respective activators. arachidonic acid (0.5 -20 ram) induces various patterns of ca2+i signal in bronchial smooth muscle cells, as revealed by single cell dynamic video imaging of fura-2 loaded cells. most frequently, ca2+i oscillations were observed, although other patterns (a single ca2+i transient; a single peak followed by a sustained elevated level, or a sustained ca2*i elevation solely) were occasionally seen. the amplitude of ca2*i oscillations was related to the concentration of arachidonic acid. the frequency histogramm was noticeably shifted to higher frequencies by nordihydroguaiaretic acid (ndga, 0.2 rtm, a lipoxygenase inhibitor), whereas it was markedly shifted to lower frequencies by indomethacin (50 pm, a cyclooxygenase inhibitor) and by 5, 8, 11, 14-eicosatetraynoic acid (etya, 3 ~tm, a lipoxygenase and cyclooxygenase inhibitor). these observations suggest that: (1) arachidonic acid per se elicits ca2+ i oscillations in these ceils; (2) cyelooxygenase activity products modulate the frequency of these oscillations. promoter analyses of a growth factor regulated gene t. triib, m. kalousek, r. kessler, and r. klemenz dept. of pathology, university hospital, 8091 ziirich stimulation of quiescent cells to enter the cell cycle results in altered gene expression. some of the first genes to be induced (immediate early (i.e.) genes) encode transcription factors which are thought to pass on the mitotic signal to the delayed early (d.e.) genes. we have analysed the promoter elements required for growth factor mediated induction of the d.e. mouse gene t1 which encodes a secreted glycoprotein of the immunoglobulin superfamily. a 448 bp region located between 3.6 and 4.0 kb upstream of the transcription start site is sufficient for growth factor mediated inducibility. it contains an ap-1 binding site which is absolutely required for t1 gene expression. it is surrounded by 3 e-boxes. at least 2 of them are essential for efficient gene induction. thus the i.e. proteins encoded by the fos and jun gene family which form the ap-1 complex can activate the d.e. gene t1 in collaboration with helixloop-helix transcription factors binding to the e-boxes. the increase of radiolabelled inositol phosphates ([3h]ips) production in agonist-stimulated human airway smooth muscle cells (asmc) was determined by hplc. in order to observe the role of calcium, pkc and pla2 on plc activity during agonist stimulation, the effects of pharmacological agents (able to alter calcium, pkc and pla2) were tested on ip production elicited by a 5 sec stimulation with histamine. the inhibitor of pkc staurosporine increased, while pma (an activator) decreased the histamine-stimulated production of [3hi 1,4,5-ip3 and of its degradation products. an inhibition was also observed with the two pla2 inhibitors, quinacrine and p-bromophenacyl bromide. in calciumfree buffer, no difference in the ip increase was shown, but an inhibition was observed when thapsigargin, an inhibitor of the ca 2+, mg 2+-atpasc of the sarcoplasmic reticulum, was added in the same conditions. the calcium ionophore ionomycin increased the lp production in presence of external calcium, whereas it completely blocked the stimulation in calcium-free buffer. the results suggest that plc activity, in human asmc, is modulated by a positive feedback of calcium and by a negative feedback of pkc and pla2. regulation of hormone-stimulated calcium signals m. boolman, afrc, laboratory of molecular signalling, dept. zoology, cambridge, uk stimulation of cells with hormones that activate inositol 1,4,5-trisphosphate (insp~) formation, leads to an increase in the intracellular ca 2. concentration ([ca2+]~). these hormone-stimulated [ca2 ⧠signals have a complex temporal and spatial regulation, with the [ca2*]~ increase often taking the form of a series of repetitive spikes or oscillations, arising from a steady basal level. the spatial counterpart of a [ca2+]~ spike is a wave, where [ca2 ⧠is first elevated in a localised region of a cell, and then spreads throughout the cell in a regenerative manner. the [ca~*]~ spike frequency is modulated by several environmental factors including hormone concentration, extracellular calcium and thiol reagents. current evidence suggests that a cell can interpret these complex [ca2+]~ changes to regulate a diverse range of cellular activities. although many of the biochemical steps between hormonereceptor activation and the [ca2 ⧠response are known, the precise mechanism generating these complex [ca2+]~ signals is unclear. in order to understand this mechanism more clearly, we have investigated the regulation of insp~-mediated ca ~ ⧠release from intracellular stores in intact cells. our current data suggests that under certain conditions, the insp3-sensitive intracellular stores behave as functionally discrete units, and that during a ca 2 ⧠spike these stores become functionally coupled by the diffusion of ca z+ from one store to the next, triggering a regenerative ca 2+ release. phenylarsenoxide as a tool for identifying proteins involved in the secretory process wiedemann. c., schaefer, t.,*gitler, c., burger, m. m. friedrich-miescher institut, p.o.box 2543, ch-4002 basel "department of membrane research and biophysics, weizmann institute of science, rehovot 76110, israel phenylarsenoxide (pao) at 20btm blocks exocytosis in chromaffm cells of the bovine adrenal medulla. this inhibition can be reversed by small dithiois, such as dithiothrcitol or 2,3~limercaptopropanol. because trivalent arsenicals interact specifically with dithiol4containing proteins, we conclude that dithiol-proteins do play an important role in regulated secretion in chromalym cells. to identify dithiol-proteins putatively involved in the secretory process, we compare pao-binding proteins from chromaffm and pci2 cells and from fibroblasts as well as from sulx:ellular fractions by 2-dimensional gelelectrophoresis. the proteins are identified by radioactive pao bound to the dithiol in a complex that witltslands sds-page. affinity chromatography on pao-scpharose with various protein fractions is used to purify the dithiol-proteins of interest. enzymatic activity, e.g. kinase or phosphatase activity, can be measured in the eluates of such a column to give further hints at the possible function of dithiol-proteins. rac-pks (~elated to pka_ and pkcc protein kinnses) represent a serine/threonine kinase family whose catalytic domain is closely related to those of protein kinases a and c. they contain a ph (pleckstrin homology) domain n-terminal to the catalytic domain. in addition, rac-pk~ was shown to be a proto-oncogene. in order to investigate the role of rac-pk in cellular signallimg we undertook genetic and biochemical analysis of the drosophila homolog (drac-pk). the drac-pk gene encodes multiple classes of transcripts. antisera raised against the drac-pk recombinant protein specifically recognize 3 distinct molecular weight forms (68, 85 and 120 kda). all forms are expressed throughout development and are both maternally and zygotically regulated. the drac-pk gene is localized at 89b4-i0 region of the third chromosome, betwee~ the pannie~ and the stubble genes. universit6 de lausanne, rue du bugnon 9, 1005 lausanne when spinal meninges homogenates were incubated with reduced glutathione (gsh), a cofactor of prostaglandin (pg) biosynthesis, [14c] arachidonate (aa) was bioconverted into a major and a minor product which comigrated on tlc with pge2 and pgd 2 respectively. in absence of gsh: 1) pgd2 could not be detected; 2) the level of pge2 was dramatically reduced but surprisingly counterbalanced by accumulation of an unusual [i4c] aa metabolite which exhibits particular traits of migration on tlc and of retention time on hplc. this aa metabolite: 1) does not correspond to a degradation product of pge2; 2) crossreacts with pge 2 antibody; 3) fits the conditions required for a 15 hydroperoxy pge2 (chemical degradation into pge2 by hydroperoxyde reducing reagents such as snc12 or gsh-hemin). it is therefore inferred that depletion of gsh favours accumulation of hydroperoxyprostaglandins which would participate to oxidative injury. the ecdysteroid receptor (ecr) and ultraspiracle (usp) from both, chironomus tentans and drosoplu'la melanogaster, were expressed in e. coil as fusion proteins with glutathione-s-trsusferase. the identity of the expressed recombinant proteins was confirmed by western blot analysis. the drosophila ecr binds to its cognate hormone response element as a heterodimer with usp as a partner. we now want to compare the capabilities of the two receptor partners from both insect species in regard to dna binding and heterodimerization by means of gel mobility shift assays. the use of naturally occurring andartificial hormone response elements will allow to define a hormone response element consensus sequence for ecr-usp heterodimers and, if existing, for ecr and usp homodimers, respectively. for other members of this receptor family it was shown that the dna binding domain and adjacent sequences alone sufficiently defines response element specificity for both homo-and heterodimers. the dna binding domains of ecr and usp from the two species show a high similarity (92% and 85% identity, respectively). the corresponding sequences were selected for overexpression in e. coli. in combination with immtmoprecipimtion of receptor-dna complexes and pcr amplification steps, the expressed proteins will be used for the detection of naturally occurring hormone response elements within genomic dna. university of fribourg, ch-1700 fribourg. various agonist-induced cell responses in neutrophils and fibroblasts, such as chemotaxis and cytoskeletal rearrangements, have been shown to parallel the synthesis of ptdins(3,4,5)p 3. but the importance of this rise is not clear. we show here that wortmannin blocks pdgf-induced production of ptdins(3,4,5)p 3 in human foreskin fibroblasts with an ic50 of about 5 nm. a similar inhibition was observed in in vitro assays (ics0=lnm) with pi 3-kinase ii~nunoprecipitated by antibodies directed against the 85 kd subunit (p85). on the other hand, wortmannin did not affect pdgf-mediated phosphorylation of p85, indicating the correct interaction of p85 with the pdgf-~ receptor. the pl10/p85 complex of the pi 3-kinase remained intact in the presence of ~m concentrations of wortmannln. these results are consistent with a direct, specific inhibition of pi 3kinase by wortmannin at concentrations relevant for its previously reported effects on cellular responses. when stimulated with pdgf, human foreskin fibroblasts form circular membrane ruffles rich in filamentous actin. the fact that wortmannin inhibits these pdgf-mediated aotin rearrangements suggests the need of pi 3-kinase activity as a signal for this cell response. caicineurin is a calcium/calmodulin-regulated protein phosphatase which is comprised of a catalytic subunit a and regulatory subunit b. although calcineurin was discovered in brain, the calmodulin stimulated protein phosphatase which has caicineurin like structure has been described in other tissues. the protein structure show that the calcineurin b is very conserved in different tissues and species, using anti-calcineurin b monocicnal antiserum, the tissue extracts from rat have been explored. immunoreactivity was obverved in all tissues, although its intensity was different. the highest intensity was found in brain. spleen, heart and thymus had an intermediate level intensity. the results were supported by the quantitative measurement of calcineurin. the caicineurin concentration was 10 to 50 fold higher in brain than that in other tissues. furthermore, the distribution of the calcineurin activity in rat tissue was studied using dephosphorylaticn assay of calctneurin. the highest caicineurin activity was found in brain too, but was only 5 to 15 fold more than that in other tissues. the specific activity of calcineurin was different in tissues. these results indicate the caicineudn activity might be regulated in tissue specific fashion. burst and cytoskeleton arcaro a. and wymann m.p., institute of biochemistry, university of fribourg, ch-1700 fribourg chemoattractants like n-formyl-met-leu-phe (fmlp) stimulate in neutrophils a rapid and transient increase in the levels of ptdins(3,4,5)p3 (pip3) which is due to the activation of ptdlns(3)-kinase. pip 3 has been proposed to be a second messenger molecule, but its cellular targets are presently unknown.here we report that pretreatment of human neutrophils with wortmannin inhibits the fmlp-etimulated production of pip 3 in a dose-dependent manner (ic50 = 5 nm). furthermore, ptdins(3)-kinase activity immunoprecipitated from resting cells is totally abolished by i0 nm wortmannin (ic50 = 1 nm). these results show a potent and direct effect of wortmannin on ptdins(3)-kinase. wortmannin also inhibits the respiratory burst of neutrophils at nanomolar concentrations, which suggests that pip3 is involved in the signalling pathway controlling activation of the nadphoxidase.when pretreated with wortmannin and stimulated with fmlp, neutrophils display oscillatory changes in factin content that correlate with oscillations in cell ~hape. this, and the inhibition of ptdins(3)-kinase, suggest a modulatory role for pip 3 in cytoskeletal rearrangements.we are currently investigating the effects of pip 3 on the functions of gelsolinl a protein that is proposed to mediate actin polymerization and can be regulated by polyphosphoinositides and ca 2+. matthias gstaiger, walter schaffner and christopher hovens institut f'tir molekularbiologie ill der universitat z'tirich, ch 8057 ziirich the octamer motif atgcaaat plays a central role in mediating the activity of a number of both lymphoid specific and ubiquitous promoters and in the immunoglubulin heavy chain intronic enhancer. the activity of thils motif in lymphoid cells is presumably mediated by the lymphoid cellspecific factor oct-2a. when ectopically expressed in non-lymphoid ceils, oct-2a is not sufficient to mediate enhancer activity of a multimerized 50 bp enhancer core element derived from the immunoglobulin heavy chain intronie enhancer, a segment, which is however highly active in b-cells. this and other findings support the supposition that additional b-cell specific factor(s) can account for the specificity and extent of the octdependent transcription observed in lymphoid cells. to further our understanding of the molecular mechanisms involved in mediating lymphoid-specific expression, we have employed the yeast two hybrid system to identify human proteins capable of physically associating with human oet-2a. to this end, a oct-2a expressing yeast strain has been constructed which bears two integrated reporter genes, his3 and lacz under the control of the octamer motif. transformation of this strain with a cdna library has allowed us to isolate clones interacting with oct-2a. we are currently analysing these candidates to determine the veracity of these interactions in higher enkaryotic cell background. in general, untransformed nuclear hormone receptors are predominantly confined to either the nucleus or the cytoplasm of a cell. receptors of the latter class are translocated to the nucleus upon interaction with ligand. our studies of the use of the ecdysteroid receptor complex from insects, consisting of a heterodimer formed between ecr and usp, as a tool for target gene activation in vertebrate cells have revealed a novel mechanism of nuclear translocation of ecr that does not depend on hormone action. after transient transfection of respective expression plasmids into hela or cv-1 cells, the subeellular distribution of the individual receptor types was analyzed by indirect immanofluorescence staining. while ecr alone was detected in both cellular compartments, nucleus and cytoplasm, usp was predominantly found in the nucleus of either host cell. cooxpression of both ecr and usp resulted in an efficient transloeation of ecr into the nucleus. usp appears to trap ecr in the nucleus, presumably by the formation of a heterodimeric complex. thus, heterodimer formation of ecr and usp not only appears to be required for high affinity binding to cognate hormone response dements (and subsequently for transactivation of an adjacent target gene) and for binding to ecdysteroid but in addition for an enhanced translocafion of ecr into the nucleus. activation of rodent macrophages with cytokines or bacteria is accompanied by the induction of a ca2+-independent nitric oxide (no) synthase which plays a key role in antimicrobial and antitumoral activity. firm evidence for expression of a similar enzyme in other mammals or in man has been lacking. we now show that bovine bone marrow-derived macrophages produce nitrite in an l-arginine-dependent manner upon stimulation with heat-killed grampositive or gram-negative bacteria. homologous interferon-7 and tumor necrosis factor-~ induced little no 2-production, but primed macrophages for enhanced n0 2-production induced by salmonella dublin or lps. this is one of the first demonstrations of no 2-production by nonrodent macrophages and encourages a search for an involvement of reactive nitrogen species in the killing of parasites or tumor cells by macrophages from mammals other than rodents. lectins are used in many analytical methods to study the carbohydrate structure of glycoproteins. we report here a technique which combines the high resolution of two-dimensional gel electrophoresis (2-dpage) to separate plasma proteins, the specificity of lectins to bind carbohydrates and the sensitivity of chemiluminescence. this method is compared with that of a commonly used colorimetrio reaction. since the reference plasma protein map obtained by 2-d page is available (i), the technique described here allows a quick and specific identification of plasma glycoproteins. therefore any ma j or glycosylation modification which may happen in some diseases can be detected. (i) electrophoresis 1992; 13:707-714. this work was supported in part by fcar (quebec, canada). in our laboratory we use genetic and biochemical approaches to study translation initiation, in particular the initiation factor eif-4a in yeast. this factor from higher eukaryofic cells is known as an rna dependent atpase and functions as a rna helicase together with another initiation factor, elf-4b. it belongs to a family of putative rna helicases, the d-e-a-d proteins. to find new genes involved in translation initiation, suppressors of a temperature sensitive eif-4a mutant were isolated. one of the suppressors (stm1) is a single copy gene which encodes a protein resembling the human translation initiation factor eif-4b. disruption of the gene is not lethal to the cell, but affects growth. polysome analysis of strains with a deleted stm1 gene have shown that this new gene is also involved in translation initiation. it carries six repeated sequences of 25 amino acids of unknown function and has -like the human eif-4b -a rna binding domain. the second suppressor (stm2) is also a novel gene. it encodes a large protein which shows no homology to other proteins present in the data libraries. it contains high portion of charged amino acids and is rich in glutamines and serines. its function in translation initiation is currently under investigation. tobacco translationini~ationfactore~4aisencoded by a large anddiverse genefamily karl brander, therese mandel, isabelle lutziger, george owttrim and cris kuhlemeier, institute of plant physiology, university of berne, altenbergrain 21, ch-3013 berne using heterologous probes we isolated cdnas coding for translation initiation factor eif-4a from tobacco, eif-4a is encoded by a large multigene family of which at least nine members are expressed in leaves and most if not all other organs of the tobacco plant. while screening genomic libraries we found several additional genes. two of them code for genes highly homologous with eif-4a throughout the coding region, but with changes in the gkt, ptrela and dead-box motifs. a third gene is expressed exclusively in the developing male gametophyte. this eif-4a-related gene may have a role in the well-documented regulation of translation in pollen. in order to study the function of these genes we have begun altering their expression in transgenic tobacco plants. seauence reauirements for a non-aug protein initiaf~jon non-aug initiation codons are still infrequent and were initially observed in animal viruses, but a number of cellular examples have now also been reported. several years ago we reported the use of an acg in the p/c mrna of sendal virus (sen) to initiate the non-structurar c' protein. we recently described a c' protein in the human parainfluenza virus type 1 (hpiv1), which is initiated from a gug. in both cases, the c' protein is an n-terminally ebngated form of the c protein which is initiated at the second aug, in the +1 reading frame relative to the first aug. this aug is used to initiate the p protein, an essential component of the viral rna polymerase. unlike the acg in sen, the gug in hplvt is clearly not a weak start codon since c' is the most abundant protein expressed from this mrna both in rive and in vitro. it appears that not any upstream gug in an optimal context will function as a start codon. for instance, the p gene of hpiv3 cannot express a c' protein (the orf is closed by stop codons) but it does contain a gug in an optimal context upstream of the p protein aug, which could encode a p' protein. nevertheless, we have been unable to demonstrate that this gug functions as a start cedon. with the use of chimeric mrnas between hpivl and 3, we have shown that positions +5 and +6 (the first g of the gug triplet being +1) are the major determinants of the efficiency of gug as an initiaton codon for protein synthesis. we are currently investigating the possibility that these nucleotides may increase the initiation rate of weak non-augs such as the acg of sen. biological activity of the heterodimeric subunit srp9/14 of the signal recognition particle is retained in 2 fusion proteins fabrice bovia & katharina strub, d6partement de biologie cellulaire, universit6 de gen~ve, ch-1211 gen~ve 4 the targeting of secretory proteins to the membrane of the endoplasmie reticulum is mediated by a cytoplasmic ribonucleoparticle, the signal recognition particle. it is composed of 6 proteins and f rna molecule (srp rna). the 2 smallest proteins srp9 and srp14 are required for the translational control function of srp. it effects a specific arrest in the biosynthesis of er-targeted proteins. these 2 polypeptides bind to the srp rna exclusively as a heterodimer (srp9/14). we have constructed single-chain variants of this dimer using a short peptide linker of 17 aa to connect the c-terminus of the 14 kd subunit to the n-terminus of the 9 kd subunit (14-9) and vice versa (9-14). we found that both proteins bind srp rna with a similar efficiency as the wild type protein. glutaraldehyde cross-linking experiments and sedimentation analysis indicate that the 2 fusion proteins fold into a similar structure as srp9/14 and bind srp rna as a monomer. furthermore, they can functionally replace srp9/14 in the elongation arrest and translocation assay. since the 2 fusion proteins are circular permutations of each other, we can conclude from this results that n-and c-termini of both proteins have no essential role in folding, rna-binding and in mediating their biological activity. furthermore, the possibility to express an oligomeric protein as a single polypeptide facilitates the analysis of its functions as well as its structure. a24 s06-08 studer, r. and hunziker, p. e., 8iochemisches institut der universit~t z0rich, ch-8057 z0rich metallothioneins (mts) are small cys-rich proteins which are inducible by a variety of agents such as bivalent transition-metal ions, tumor promoters, cytokines and hormones. the biological role of mts is still unclear. initially postulated to serve in the sequestration of the toxic heavy metals such as cadmium and mercury, they are now believed to play a major role in the cellular metabolism of essential metals such as zinc and copper. to explore this function further we have now established a method to quantify basal mt production in several human cell lines. thus, cellular isomt contents were monitored by h.pj.c, and the rate of synthesis was followed by the incorporation of ~ss-cysteine. the results show that maximum mt synthesis and accumulation occurs when the cells enter the exponential growth phase. with increasing celldensity the mt content decreases progressively until confluence is reached. in correlation with preliminary measurements our results suggest a close relationship between the mt content, the cell growth rate and the intracellular abundance of zinc. vincent bernard, adrian etter, heinz tobler and fritz mtiller. institute of zoology, university of fribourg, 1700 fribourg (switzerland). the nematode ascaris lumbricoides undergoes chromatin diminution which causes a loss of dna in all somatic ceils. we have identified a ribosomal protein (rp) gene (coding for alep1 = ascaris lumbrieoides eliminated protein 1) which is located within the germqine specific material. alep1 shows strong homologies with the ribosomal protein s19 (rps19). the transcription of its gene takes place only in the germ-line. 2d-gel electrophoresis on germ-line and somatic purified ribosome fractions shows that the alep1 protein (rps19) is present only in the germ-line ribosome. does the alep1 germ-line specific protein have a homologue in the somatic ribosomes? we have cloned a homologue of alep1 from ascaris somatic tissue named rps19', we are currently studying the expression pattern of this rps 19'. these results indicate the existence of a developmentally regulated ribosome heterogeneity between the germ-line and somatic ceils. what is the function of rps 19? since ascaris is not suitable for genetic analyses, we isolated rps19 from the nematode caenorhabditis elegans. the c. elegans rps19 cdna shows only 60% homology at the nucleic acid level. rps19 is located on the c. elegans chromosome iv. the identification of a rps19 mutant in c. elegans will indicate its function. favre, d, l, pavlovic, j2 and michel, m.r. t 1 institute of medical microbiology, university of beme, ch-3010 berne. 2 institute for immunology and virology, ch-8006 ziirich we have successfully purified the recombinant c (recc) protein from spodoptera frngiperda (sf9) insect cells which were infected with acnpv-c (a). the recc protein retained the biological activities of native c protein obtained from wild type sfv (b). our results suggest that the c protein is responsible for the cellular shut-off of host protein synthesis by inhibiting the initiation of translation. we are currently investigating the molecular mechanisms involved in this shut-off. tif3 is a new eukaryotic initiation factor which shows 26% identity with the sequence of mammalian translation initiation factor eif-4b. the tif3 gene is not essential for growth; however, its disruption results in a slow growth and coldsensitive phenotype. in vitro translation of total yeast rna in an extract from a tif3 gene-disrupted strain is reduced as compared to a wild-type extract. the translational defect is more pronounced at lower temperatures and can be corrected by the addition of purified yeast tif3,or mammalian eif-4b. in vivo translation of ~galactosidase reporter mrnas with varying degree of rna secondary structure in the 5' leader region in a tif3 gene-disrupted strain show preferential inhibition of translation of mrna with more stable secondary structure. chloroplast protein synthesis requires the import of elongation factors ef-tu (tuf-gene) and ef-g (fusgene). it seems that the expression of both genes is light regulated. we recently cloned and sequenced a chloroplast specific nuclear fus gene in soybean (biochim.biophys acta 1174 : 191-194, 1993 . further analysis revealed that a second very similar (sequence, anatomy) nuclear fus-2 gene exists which, however, shows strong sequence diversion in the 3'trailer region. several cdna clones were partially sequenced but sofar only transcripts from the fusi gene were retrieved. we currently study the promoter regions of either gene to test possible differential expression of fus genes. comparative mapping and sequencing studies of the two genes in the 3"-region indicate that a major dna insertion occurred distal to the coding part of both fus genes what may also influence gene expression. the rna-binding domains in the 9kd and 14kd subunits of the signal recognition part~clehavea putative~-h~licalstructure. nazarena buiand katharina strub dpt de biologie cellulalre, science ill, universit4gen~ve the signal recognition particle (sr2), a cytoplasmic ribonucleoprotein is important for targeting nascent secretory proteins to the endoplasmic reticulum. srp9and srpl4are constituents of srpandare, together with the alu sequences of srp rna, required for the elongation arrest activity of the particle. srp9 and srpi4 form a heterodimer in the absence of the rna and bind the rnaexclusively as a heterodimer. the primary sequence of srp9 and srpi4 revealed that both proteins are highly basic and lack structural similarities to other characterized rna-binding proteins. we have therefore been interested in characterizing the molecular nature of rna-protein interactions. the analysis of n-and c-terminal deletion mutants of the proteins have shown that both proteins contribute to the formation of an rnabinding pocket, the rna-binding and dimerization domains in both proteins, are found in regions that have a predicted ~~helical structure. in sp~pi4, this u-helical region is located betwen aa 72and i00 at the c-terminus; it contains the rna binding and dimerization domains adjacent to each other. our results support the model that the hydrophobic face of the putative u-helix is implicatedinprotein-protein interactions and the positively charged face in rna-protein interaction. in sp~pg, the two rna-binding domains found n-and c~ terminally, also have a predicted amphipatic s-helical structure. it has been proposed that the decline in protein synthesis observed in aging organisms may result from a decrease in the protein synthesis elongation factor ef-la. john shepherd's finding that transgenic drosophila melanogaster carrying an additional copy of the ef-la gene have an extended lifespan further indicated that the ef-la gene may play an important role in determining longevity (shepherd et al., 1989) . in order to test this hypothesis, we have quantitated ef-la mrna, ef-la protein, as well as catalytic ef-la activity in john's flies. young and aging ef-hx transgenic (ef) and control lines ((2) were examined. because the the additional ef-lc~ copy is under the control of the hsp70 promoter, flies were aged either at 25~ or at 29~ the results show that transgenic ef flies do not express more ef-lct than the control flies, neither at the rna nor at the protein level. the question arises, whether the lransgene can be induced at all. this was tested by doing rnase protection assays. transgenlc message can be detected only in ef flies heat shocked at 37~ but not in ef flies grown at 29~ nor in control flies at 37~ from this experiment we conclude that the transgene is indeed inducible, but is not expressed in detectable amounts at 29~ we conclude that the difference in the lifespan does not result from the overexpression of the ef-la transgene. phenobarbital (pb) induces the expression of liver enzymes involved in the metabolism of drugs and other foreign compounds (xenobiotics). these enzymes include several cytochromes p450, nadph:p450 reductase, aldeyde dehydrogenase, epoxide hydrolase, udp-glucoronyltransferases and glutathione-s-transferases. many other proteins not yet recognized may however be induced by pb. our first approach therefore is aimed at identifying gene products which respond to pb treatment. as model systems we are using either chicken embryos (induction in ovo) or primary cultures of chicken embryo hepatocytes (induction in culture) (althaus et al., jbc 254 pp 2148 , 1979 . to differentially display mrna of induced vs non-induced cells we are using a rt-pcr technique with sets of random primers. electrophoretic separation of amplification products allows to identify mrna species which are induced (or decreased) following treatment. data obtained from these experiments and sequence information revealed from extracted pcr products indicate that pb treatment results in the transcriptional avtivation (or repression) of a variety of so far unknown genes. antithrombin -binding heparan sulfates from ovarian granulosa cells hosseini g., de agostini, a., clinique de st6rilit6, hcug, gen~ve. at the time of ovulation, several serine proteases of the plasminogen activator and coagulation cascades are activated in the ovary. these proteolytic activities are tightly controlled in time and space, and the heparin-activated serpins plaminogen-aetivator-inhibitor-1, protease nexin-1 and antithrombin (at) are present in the follicular environment. we have observed that granulosa cells produce a subset of heparan sulfate proteoglycans that bind and activate at. these at-binding heparan sulfates (ahspgs) endow the vascular endothelium with antithrombotic properties. we are now examining the role of ahspgs in the extravascular compartment formed by the ovarian follicle. using 125i-at binding assays, we have detected ahspgs on granulosa cell surfaces and culture media, in amounts comparable to those found for endothelial ceils. after 48h of stimulation by the gonadotropin fsh (1-100 ng/ml) granulosa cells increased the amounts of ahspgs they released in culture media by 2-6-fold, while keeping their cell surfaceassociated ahspgs constant. analysis of 35s-labelled hspgs revealed that heparan sulfates constitute about 40 % of the surface-associated and 10% of the soluble granulosa cell glycosamineglyeans. finally, using 125i-at autoradiography on rat ovary cryosections, we have localized ahspgs on the granulosa cell layers of large antral follicles, while ahspgs could not be detected in smaller follicles. these data suggest that granulosa cell ahspgs could be critically located to modulate proteolytic activities in the changing environment of the growing follicle. division, cibabasel, switzerland. the development of chelators which can mobilise excess storage iron (fe) and permit its excretion is necessary in the treatment of fe overloaded diseases. although oral administration of such a pharmaceutical is highly desirable, no orally active fe chelator is so far in regular clinical use. searches for orally active and safe fe chelators require appropriate animal models of fe overload in order to evaluate the potential of these drugs. an animal model is particularly useful for testing the capacity of new fe chelating ce~oounds in enhancing the mobilisation of clinically relevant storage iron. iron loading in animals was achieved by dietary supplementation with carbonyl fe, fe fumarate and 3,5,5-trimethylhexanoyl ferrocene (tmh-ferrocene). liver fe concentrations were increased 6-21 times above normal levels. tmh-ferrocene was the most efficient compound to induce stable liver fe overload. the orally given con'pounds induced a predominantly hepatocellular iron distribution and was found distributed among reticuloendothelial cells only at a later stage. this pattern of liver fe storage is similar to that observed in the early stages of primazy ha6~ochromatosis and late stage of transfusional hae~ochromatosis. eeeently, we and ethers have cloned and characterized novel transcription factors called peroxisome proliferator-actirated receptors (ppak). they belong to the superfamily of nuclear hormone receptors as the steroid, thyroid and retinoid hormone receptors. ppars are activated, beside xenobiotic peroxisome proliferators, by natural fatty acids and induce, together with the receptor for 9-cis retinoic acld (rxr), the transcription of genes involved in the ~-and ~-oxidation of fatty acids. ppar and rxr bind as heterodimers to the ppar response element (ppre), consisting of a direct repeat of aggtca-iike sequences with one intervening nucleotide. now, we show by gel retardation analysis and transient transfection assays that ppar/rxr heteredimers also bind to and transactivate gene transcription through estrogen response elements (ere). the ere is a palindrome with aggtca half-sites separated by three nucleotides. the selectivity and specificity of the transactivation through an ere was demonstrated by the testing of various related palindromic and inverted palindromic repeats of aggtca sequences, which were all inactive. the possible activation of estrogen responsive genes by fatty acids via ppars in vivo is discussed especially with regard to a debated role of fatty acids in breast cancer. the purpose of this study was to evaluate whether intermittent doxorubicin (dox) treatment in vitro selects for multidrug resistance in rpmi 8226 myeloma cells and, if so, to determine the underlying mechanism(s). cells were exposed to constant doses of dox for 4 days alternating with growth in dox-free medium for 17 days. after > 9 months of treatment, the cells developed an atypical mdr phenotype with 4-to 6-fold resistance to topo ii poisons. cross-resistance to vincristine and taxotere was < 2-fold, sensitivity to cisplatin and melphalan remained normal. efflux of mdr1 drugs was incresed with no effect of verapamit; subcellular dox distribution was normal. expression of topo ii a was found to be reduced by around 50 and 60-70% at the rna and protein level, respectively. minimum mdrt rna overexpression was detected when using rt-pcr; p-glycoprotein was not detectable. mrp rna expression was increased by 60-90% relative to the wild type cells without detectable p190. this data suggests that atypical mdr might play a role in acquired dox resistance in human myeloma. mucosal surfaces cover a vast area in the human body. they satisfy its need to communicate with its environment, e.g. in terms of nutrient uptake and sexual reproduction. however, at the same time they offer a vulnerable gate for invasion by pathogenic microorganisms. for immunoprotection of these regions, large amounts of iga antibodies are produced in the bloodstream and transported across the epithelial barrier to the lumen by means of polymeric ig receptor. there the receptor is cleaved, and its extracellular portion remains associated as secretory component (sc) to the secretory iga complex. in the context of a project aiming to mass production of secretory iga as a passive vaccine, we evaluated a series of eukaryotic expression systems for production of sc. the cdna encoding polymeric ig receptor was engineered in a way that only its extracellular domains corresponding to sc were expressed in yeast, in insect cells infected with recombinant baculovirus, and in mammalian cells infected with recombinant vaccinia virus. furthermore, a c-terminal poly-histidine tag was introduced for simple purification of the products. we will show optimization of expression levels for each of the systems as well as a quantitative and qualitative comparison of the products. recombinant vaccinia viruses (rvv) that express gone products from other organisms has become a popular and widely used laboratory expression system. our current interest is directed toward the production of secretory component (sc), a subunit of secretory iga antibody complex involved in mucosal immunity. toward this goal, we constructed rvv governing sc transcription under the control of two viral-based and the t7 bacteriophage promoters. expression of the protein was assessed in several mammalian cell lines, such as human tk-, human hela (grown in suspension or as a monolayer) and monkey cv-i. optimization of infection parameters and culture conditions were also performed. institut de biologic animale de runiversit~ de lausanne protection of mucosal surfaces is mediated by secretory iga ant/bodies (alga) constituted of dimeric iga and secretory component (sc) . toward the aim of reconstituting slga complexes in vitro and using them for passive oral immunization, sc has been produced in yeast, insect and mammalian expression systems. the gone encoding sc has been engineered so that a) the c-terminus of the protein carries a 6xhis tag and b) the newly synthesized polypeptide is released in the culture medium. culture conditions have been established to permit recovery of sc in serum-free medium. purification procedures based on nickel chelate and classical chromatographies have been optimized to yield sc under native conditions. finally, in vitro reassociation with purified dimeric igas obtained from hybddomas has been used to assess biological activity of recombinant sc. using a magnet-assisted subtraction-hybridisation technique (mast), 17 cdna clones were isolated that are expressed in nqrm~l l~nq %issue but n__d_i expressed in the corresoondina nsclc tissue, ii of the 17 edna clones are highly homologous to edna sequences for the following proteins: pulmonary surfactant proteins sp-a and sp-b; receptor for advanced glycosylated endproducts of proteins (rage); calmodulin-like protein; natural killer gone 5 (nkg5); matrix gla protein (mgp); glutamine synthetase; ubiquitin; vascular smooth muscle alpha-actin; cytoskeletal beta-actin; vimentin. the remaining 6 edna clones may represent parts of still unknown genes. the mast edna clones were derived from a patient who developed squamous adenocarcinoma. northern blot analysis of normal/tumour rna from three other nsclc patients showed that the lack of gone expression was independent of nsclc type and stage. enzymatic methylation of cytosines in mammalian dna is believed to play a fundamental role in basic gene regulation. methylation in the vertebrate genome is restricted to cpg dinucleotides at the c-5 position of cytosine. in vitro studies have shown that methylation can drastically influence the dna structure. cpg islands remain unmethylated in normal cells, whereas several immortalised, transformed cell lines contain partially methylated cpg islands. transcription of genes can be inhibited by methylation of cpg's. this modification contributes e.g. to the stable repression of genes on the inactivated x chromosome of females. the collagen vi genes show typical cpg islands in their promoter region and they were shown to be down regulated at the transcdptional level in src or myc transformed cells. given these facts we dee~ded to investigate the effects of methylation in the collagen vi gwene. e could demonstrate that the promoter activity of (x2(vi) chicken collagen gone was strongly diminished by methylation in vitro. furthermore dna methylation completely prevented binding of a novel transcription factor to the promoter, whereas binding of sp1 was not affected. to show evidence of methylation in vivo, we are currently iocalising the exact positions of methylated cpg's in normal and transformed cells by genomic sequencing. alterations of putative tumour suppressor genes in human non-small cell lung carcinoma (nsclc). r. shipman and c.u. ludwig, molecular oncology, lab 405 (zlf), basel, ch-4031 chromosomal subregion llp13 displayed non-random allelic loss in 61 nsclcs. llp13 contains the genetic loci for catalase (cat), the wilms' tumour gene (wti) and the follicle-stimulating hormone ~-chain (fsh~). 76% of the nsclcs were deleted at cat suggesting that loss of a gene(s) near cat may be involved in the progression of nsclc. yac clones containing the cat locus are being prepared for use in a direct selection procedure for cdnas encoded at this region. although 38% of the nsclcs were deleted at wti, no mutations were detected in the remaining wti allele. wti expression was subsequently demonstrated in fetal lung, fetal hematopoietic tissue, normal adult lung and nsclc. allelic deletion at 17p13, immuno-histochemical and sequence analysis of the p53 gene were also examined in our nsclcs. these analyses support the contention that aberrant expression of the p53 gene is a pivotal event in the progression of nsclc. $08-11 recently, a number of advances have been made which enable the experimentalist to study the effects of low levels of ionizing radiation. the results suggest a threshold to radiation damage exists and that above a critical level, recovery mechanisms in the cell are induced and the biological response falls. thus low levels of radiation cause greater than expected levels of biological response. this has been demonstrated in studies of mutations, cell survival and cancer induction. because the low dose-rates and the low doses per fraction result in the total doses being small, the magnitude of the biological effects induced is insufficient to warrant alarm. identification of genes associated with the revertion of the malignant phenotype of a rhabdomyosarcoma cell line. michele genini, sabina solinas toldo*, ruedi fries* and beat w. schafer, dept. of pediatrics, university of ziirich, steinwiesstr. 75, 8032 ziirich, and *institut for nutztierwissenschaften, eth ztifich, 8092 ztirich. the human rhabdomyosarcoma cell line rd is tumorigenic and differentiates very poorly despite the expression of the myogenic factors myf3 and myf4. therefore it can be regarded as natural mutant. to identify genes which are involved in suppression of tumorigenicity or enhance differentiation we applied two different approaches. since the development of human embryonal rhabdomyosarcoma has been associated with abnormalities of chromosome 11 band p15, in the first approach we transferred a normal human chromosome 11 into rd ceils via microcell fusion. the microcell hybrids did not show a higher degree of differentiation. suppression of tumorigenicity was observed in one clone but is probably independent of chromosome 11, as it was shown by chromosome 11 specific painting. ' in the second approach we applied a subtractive hybridization procedure between primary myoblasts and rd cells to find genes specific for the normal phenotype. these genes will be tested in vivo for induction of differentiation and/or tumorsuppression. deficient synthesis of the gpi anchor is the molecular basis of idiopathic paroxysmal nocturnal hemoglobinuria ("pnh") and a similar phenotype can accompany aplastie anemia cpnh-iike"). the population expressing cd48, a gpi-anehored membrane glycoprotein, was quantified by flow cytometry in samples from patient p.g. (pnh) and patient m.m. (pnh-like) and amounted to 90% (p.g.) and 78% (m.m.), respectively. t lymphocytes from both patients were isolated, expanded and cloned. from both patients, clones expressing the cd48 marker (cd48+) and cd48 deficient clones (cd48-) were obtained. takeda et al. (cell 73, 1993, 703-11) showed that in some (cd59-) bcell clones, a deletion of the p1g-a gene product is responsible for the expression of the pnh-phenotype. pcr analysis of t cell mrna from extracts of both cd48+ and cd48-clones of patient m.m. (pnh-like) using pig-a specific primers revealed a band pattern from which can he concluded that no deletion is present in the pig-a gene product. this result suggests that cd48-t lymphocytes of the pnh-like patient express a normal pig-a gene product; a disorder in regulation of pig-a expression or an unrelated biosynthetic defect may explain the cd48phenotype in these t cells. further work is aimed at defining the differences in biosynthetic defects of pnh and pnh-like cd48-t cell clones. supported by grant 31-30757-91 of the snsf to egb. w@ have recently employed an experimental approach to turn the function of "nuclear messengers" such as c-fos (and c-jun) on and off at will in polarized me/nmary epithelial cells. to this end we used constructs encoding the c-fos (and c-jun) genes fused to the hormone-binding domain of the human estrogen receptor, designated c-foser (and c-juner), we could show that short-term activation (30 mins.) of c-foser by estradiole (e2) led to the disruption of epithelial cell polarity within 24 hours, as characterized by the expression of apical and basolateral marker proteins. during the next 3-5 days, however, the cells fully regained their polarized phenotype. conversely, long-term activation (24-48 hours) caused the irreversible loss of epithelial cell polarity, leading ultimately to an epithelial-fibroblastoid cell transition. these cells underwent dramatic changes in gene expression including the complete loss or reduction of epithelial markers such as e-cadherin/uvomorulin, zo-i and cytokeratins, and the de novo expression of mesenchymal proteins like vimentin, type i collagen and fibronectin. expression of the v-ha-ras oncogene (acting upstream of c-fos) did not markedly influence epithelial cell organization when the cells were grown on plastic. however, when cultured within type i collagen gels in the presence of serum, or when injected into the mammary glands of nude mice, rapid cell growth and a clear epithelial-fibreblastoid cell transition was observed. growth properties determine the organ colonization ability of a metastatic murine melanoma cell line. the ability of metastatic tumor cells to specifically colonize distant organ sites strongly depends on the interaction of the metastatic cells with the local organ environment. we have selected a b16 routine melanoma cell fine (b16-ls9) which has an increased ability to colonize the liver, and compared its behavior with either its parental (b16-f1) or inng-specific (b16-f10) cell line. we have found that adhesion in vitro and organ lodgement in vivo were not different for ls9 and f10. in contrast, b16-ls9 grew at slower rates than the other lines both in vitro and in vivo after subcutaneous or intra-foot-pad injection. analysis of tyrosine-phosphorylated proteins indicated a higher phosphorylation activity in b16-ls9 cells, which might suggest an altered regulation of some kinase(s) in this cell line. intercellular contact with hepatocytes in vitro, or the liver in vivo could restore an efficient growth of b16-ls9, enabling thus these cells to colonize this organ much better than others. hepatocytes or liver plasma membrane (p.m.) extracts conserved the growth stimulatory activity towards b16-ls9 ceils. chromatographic fractionations of these extracts showed that a major growth sfimulatory protein in this fiver p.m. fraction turns out to be ttansferrin, which in the liver appears to exist in at least two different mature forms. protein import into mitochondria requires atp in two locations: outside the organelle, and in the matrix space. depletion of matrix atp arrests translocation of precursors across the inner membrane, but does not prevent precursors from crossing the outer membrane. as a result, proteins that reach the inner membrane or intermembrane space without passing through the matrix are often sorted normally in atp-depleted mitochondria. external atp is used to maintain precursors in a translocationcompetent state. however, some precursors can fold and still remain translocation-competent, and import of these precursors is independent of external atp. with the folded cytochrome b2 precursor, it is matrix atp rather than external atp that drives translocation across the outer membrane. thus matrix atp generates a pulling force that can drive both the translocation and the unfolding of precursor proteins. mitochondrial hsp70 probably functions as the atp-dependent import motor. arsenijcvic d, dulloo ag & girardicr l, dpt of physiology, cmu. geneva, ch. the relationship between body weight, daily energy expenditure (assessed by indirect calorimetry), and immune status (determined by lymphocyte proliferation tests) was investigated in swiss webster mice maintaining a stable body weight several weeks after infection with toxoplasma gondii(me49). following an initial period of weight loss (infection-induced cachexia), the surviving mice could be differentiated into two main groups: (i) those showing a partial regain in body weight (the infected gainers) and eventually stabilizing al a mean body weight 25% below a non-infected control group, and (if) those showing no weight regain (the infected non-gainers) and eventually stabilizing at 45% below control levels. immune function was found to be markedly suppressed in the infected non-gainers, whereas it was normal (and similar to control levels) in the group of infecled gainers. daily energy expenditure (and food intake), after normalisation for differences in body weight, was found to be the same in both infected gainers and non-gainers, but lower by 15% when compared to controls (p<0.01); thereby indicating that the infected groups were able to increase their overall efficiency of food utilization in response to the low food inlake. in conclusion, this study conducted during a weight stable phase of chronic infection shows a clear-cut association between the inability to regain body weight and impaired immune function. in contrast, no relationship was found between metabolic rate and body weight nor between metabolic tale and immune function, but the chronically infected mice exhibited the same phenomenon of enhanced metabolic efficiency characteristic of chronic underfeeding per se. identification and functional analysis of chaperonin 10, the groes homolog of yeast mitochondria. biozentrum, university of basel, klingelbergstrasse 70, ch-4056 basel, switzerland. phone ++41-61-2672171, fax ++41-61-2672148. the mitochondrial chaperonin system consists of chaperonin 60 (cpn60; also termed hsp60), which is homologous to e. cull gruel, and chaperonin 10 (cpnlo), which is homologous to e. coil groes. we have used a functional assay to identify cpnl0 of yeast mitochondria. when dimeric rubisco is denatured and allowed to bind to yeast cpn60, subsequent refolding of the enzyme is strictly dependent upon yeast cpnl0. in the presence of mgatp, yeast cpn60 and yeast epnl0 form a stable complex that can be isolated by gel filtration. the atpase activity of hsp60 is not inhibited by complex formation with cpnl0. a different result is obtained with the e, coil system, where groes is able to completely inhibit the atpase activity of gruel (1). to test the in vivo role of cpnl0, we have cloned, sequenced and disrupted the corresponding nuclear gene cpnio. this gene encodes a protein of 11,372 da that is imported into the mitochondrial matrix without detectable cleavage. haploid cells lacking a functional copy of cpnio fail to grow at temperatures between 23 ~ c and 37 ~ c (2). (1) rospert, s. et al. (1993) proc. natl. acad. sci. 90, in press. (2) rospert, s. et al. (1993) febs lett., in press. characterization of yeast mitochondrial heat shock protein 70 , l. bolliger, b. s. glick and g. schatz, biocenter, university of basel, 4056 basel. mitochondrial hsp70 (mhsp70) is thought to use the energy of atp hydrolysis to pull precursor proteins across the mitochondrial membranes. to facilitate the purification of yeast mhsp70, we used the cloned gene (a gift of dr. n. morishima) to modify mhsp70 with a six-histidine tag at its c-terminus. this construct supports growth of yeast cells lacking wild-type mhsp70. we developed a purification procedure that allows us to recover this tagged protein with a purity of about 95%. experiments are in progress to characterize the nucleotidedependent interaction of purified mhsp70 with unfolded proteins. in addition, we are looking for partner proteins that may modulate the action of mhspt0. we have copurified a protein of about 20 kd together with mhsp70. the 20 kd protein could be released from mhsp70 by atp or adp. when tryptic fragments of this protein were subjected to microsequencing, one fragment showed strong homology to the grpe protein of e.coli. the transmembrane electrical potential of mitochondria (awmit) can be assessed in single hepatocytes by using epifluorescence microscopy. for this end the rhodamine 123 fluorescence of a mitochondria-rich (fr) and a mitochondria-poor region (fp) are measured. the ratio of these signals depends on the awmit according to a modified nernst equation (reber b .f.x., somogyi r. & stucki j.w. (1990) , bba, 1018, 190-193) . to calibrate this method the cell membrane was permeabifized for monovalent cations with nystatin and gramicidin. then the cytosolie k + was replaced by na + from a k+-free bath solution. subsequent addition of valinomycin made the mitochondrial membrane permeable for k + ions. by the addition of different k + concentrations to the bath, the awmit could be clamped to defined values. the relation between the ratio fr/fp and the awmit could be well fitted to our modified nernst equation. however, the calibration curve flattens out at potentials higher than about 110 inv. therefore the detection limit of changes of a~mit within the physiological range is about 10-20mv. maximal stimulation of the na+/k + atpase by addition of nystatin to the na + containing bath medium caused a drop of awmit of about 20inv. subsequent addition of ouabain resulted in an almost complete reversal of this drop. this shows that changes in atp consumption can be assessed via changes of audmit. schneiter ph., di vetta v., j6quier e. and tappy l. institut de physiologie de l'universitg bugnon 7,1005 lausanne. the metabolic effects of an exercise performed in the fasting or fed state were studied in 8 subjects over a 8 hour period in a respiratory chamber. a mixed meal containing 13c labeled carbohydrates was ingested at 9:30 am and an exercise session (walking at 5 km/h 45 rain, slope 10 %) was performed at 8:15 am (fasting) or 11 am (postprandial). net carbohydrate (net cho ox) and lipid oxidation (indirect calorimetry) and oxidation of exogenous carbohydrates (exo cho ox, breath 13co2) were measured. glycogen breakdown was calculated as net cho ox -cho exo ox, and glycogen synthesis as cho in -cho exo ox. energy expenditure over 8 hours was similar but net cho ox was 16 % lower, lipid ox 61% higher and exo cho ox 39 % lower when exercise was performed in fasting vs fed state; moreover, glycogen synthesis was increased by 40 % (p<0.0001), while glycogen breakdown was increased by 12 % (p=0.06). in conclusion, a 45 min exercise in the fasting vs fed state a) increases utilization of endogenous lipids, b) enhances muscle glycogen turnover over a 8 hour period. hormonal regulation of e-abl tyroslne kinase by fusion to a steroid binding domain t. mattioni, o. 8chini hooft van huijsduijnen, p.k. jackson and d. picard d4partement de bielogie cellulaire, universit4 de geneve, ch-1211 geneve 4 the activities of a wide variety of proteins can be subjected to hormonal control by fusion to the hormone binding domain of steroid receptors. we show that this strategy can also be applied to a tyrosine kinase. in particular, transforming derivatives of c-abl become hormone-inducible oncogenes by fusion to a steroid binding domain. it is noteworthy that the hormone-inducible transformation of nih3t3 cells is completely reversible upon removal of the specific ugand. reversion experiments reveal another facet of abl: its ability to inhibit cell proliferation. 48 hours after hormone depletion, cells are not only morphologically reverted, but the cell cycle appears to be blocked in g1. a cytostatic effect of abl overexpression has been reported by several groups but, until today, it could only be examined by comparing different abl derivatives or the same derivatives in different cellular contexts. in contrast, our hormone-regulable system has the advantage that the two distinct functions of abl (transformation versus growth inhibition) can be studied using the same derivative expresed in the same cell line. we are using the hormone inducible system to investigate the mechanisms regulating the transtorming and the cytostatic effects of abl in a variety of cell lines. jiewu yang and herbert zuber institute for molecularbiology and biophysic, eth, 8093 z'tirich enzymes from thermophilic organisms show higher thermostability and temperature optima than those of mesophilic organisms. it has been shown that these properties are based on a specific structure of the protein. the primary structure of thermophilic (b. stearothermophilus) and mesophilic(b, megaterium) triosephosphate isomerase(tim) have been determined by cloning and sequencing of the tim genes. comparison of the primary structures of the thermophilic and rnesophilic enzyme showed specific amino acid substitutions, particularly in the c~-helices of the tim barrel, which could play a critical role with respect to thermostability. we have consmacted mutants by exchanging (x-helices between tim of b. stearothermophilus and b. megaterium. helix h1, h2 and h7 of tim from b. megaterium, corresponding to amino acid residues 13-36, 44-55. and 220-227 respectively, have been exchanged. the properties (thermostability and temperature optimum) of the hybrid-mutants were compared with the wild type enzymes. in a comparative investigation of structural and functional parameters related to the oxidative substrate metabolism in skeletal muscle cells of dogs and goats, a close relationship was found between intracellular lipid stores and mitochondria. as revealed in serial sections with subsequent em analysis, each lipid droplet was in direct contact to one or several mitochondria. quantitatively, 23% of the lipid droplet surface in goats and 40% in dogs were in direct contact to outer mitochondrial membranes or -in other terms -1% of outer mitochondrial membranes in goats and 2% in dogs were in direct contact to lipid deposits. these findings were in good agreement with the physiological data showing a 2 times higher oxidation rate of fatty acids drawn from intracellular stores for dogs than for goats. human muscle adapts to altered load with changes in the expression of enzymes involved in energy metabolism. endurance exercise in humans is known to increase the oxidative capacity of skeletal muscles (hoppeler, int. j. sports med. (1986), 7, 187) . at the ultrastructurai level, a higher mitochondriai content contributes to the higher oxidative capacity of skeletal muscles. little is known about the molecular mechanisms that lead to such adaptations in humans. the expression of mitochondriai components involves complex regulation of both mitochondriai and nuclear encoded genes. we have developed a pcr approach to quantify dna and rna from human skeletal muscle cryostat sections and addressed the question, whether the structural adaptations in endurance trained athletes are accompanied by concomitant changes in respective rna steady state levels. preliminary data indicate a higher concentration of coxiv and coxl (1.8 and 1.6 times, respectively) in trained subjects. no differences were observed for mtdna, despite a two fold higher mitochonddal content. human skeletal muscle contains three main fiber types which are based on the myosin heavy chain composition of the individual fiber, the isoforms of other contractile proteins within a given fiber type can vary leading to a continuum of different phenotypes. we are studying the fiber type specific expression of the mrnas of myosin alkali light clmins (mlc) using in situ hybridization, especially the rarer slow form mlc lsa. differences were found between shoulder muscles and leg muscles: m. deltoideus yielded stronger signals for mlc lsa and mlc lsb in the least glycolytic type i fibers (ia), weak but detectable signals for mlc lsb and mlc lf/f mrna in more glycolytic type i fibers (ib) and strong signals for mlc lf/3f in type ii fibers. in m. vast. lat. mlc lsb was strongly expressed in type ia as well as ib fibers, mlc lsa mrna was only found in some ia fibers. mlc isa has been shown to be expressed at a particular time point during muscle development and regeneration. in a biopsy of a becker dystrophy patient, we found very strong signals in some small fibers displaying the morphology of regeneralmg fibers. thus this probe could be useful to dete~t regenerative processes in pathological muscle samples. the aim of this study was to validate the complementarity of two new non-invasive methods for the assessment of autonomic regulations. reactions to a moderale exercise (75 w on a cycloergometer) were compared in cardiac transplant recipients (ctr) who modulate their heart rate (hr) by means of circulating calecholarnines, their heart being donervated, and in normal subjects where such exercise level doesn't stimulate the sympathetic system. the methods used were: 1) the spectral analysis of hr variability where the high frequency component (hf, above 0.15 t4z, related to the respiratory frequency) is a marker of the vagal activity, the low frequency component (lf; at about 0.1 hz) depends on both sympathetic and parasympathetic activities and the lf/rf ratio indicates changes in the sympatbo-vagal balance; and 2) a pure sympathetic index obtained from the amplitude of the t-wave of the ecg (twa) which is decreased by adrenergic stimulation of the myocardium. our results indicate that in both groups hr was increased by the exercise. in ctr, a net decrease in twa (-39_+ 10%, n=6) was measured. contrasting with this, in normal subjects, no change in twa (+2_+12%, n=6) but a decrease in beth hf (-67_+13%, n=7) and lf (-85_+5%, n=7) without change in lf/hf ratio (-19+_.26%, n=7) were observed. these results indicate that ctr subjects increase their hr by an adrenergic stimulation, whereas the normal subjects regulate their hr for such moderate exercise only by redudng their vagal tone. there was a significant relationship (r=0.79, p<0.0005) between ps and the ree. the slope of the regression line indicated a net cost of ps of 3.6 kcal/g. we conclude that obesity in children is associated with an absolute increase in whole-body protein turnover consistent with the increased ffm and ree. the "glucose paradox" in the anoxic and reoxygenated embryonic myocardium raddatz, e., tran, l., rochat, a.-c., kucera, p. and de ribaupierre, y. institut de physiologie de l'universitd, ch-1005 lausanne. the hearts of 4-day-old chick embryos explanted in vitro react rapidly, reversibly and reproducibly to anoxia and reoxygenation. this work deals with the effects of exogenous glucose on the mechanical activity of the hearts submitted to strictly controlled normoxia-anoxia-normoxia transitions. the anoxic periods lasted from 10 to 60 seconds. instantaneous heart rate, amplitude of contraction, velocities of contraction and relaxation of the ventricle wall were determined optically. in presence of 8 and 20 mmol/1 of glucose 1) the arrest of cardiac activity under anoxia was delayed by 60 and 110 %, respectively, 2) the duration of the postanoxic cardioplegia was reduced and 3) recovery was faster. paradoxally, these concentrations of glucose induced arrhythmias both during anoxia and reoxygenation. the incidences of such arrhythmias increased with the duration of anoxia. the absence of glucose or blockade of glycolysis suppressed arrhythmias. image analysis showed that the observed myocardial dysfunctions originated in the atrium and were sometimes associated with disturbances of propagation. the microinjeetion of okadaie acid into incompetent oocytas leads to the release of the prophase block with entry into m-phase. these g2/m-like alterations include chromatin condensation, germinal vesicle breakdown (gvbd), microtubules reorganization and formation of an abnormal (often multipolar) spindle of meiosis i, with chromosomes not aligned on the metaphase plate. the polar body is never extruded. moreover okadaic acid microiujection induces the emergence of phosphorylated cytolasmic foci as detected by the monoclonal antibody mpm-2, known to react with mitotic phosphoproteins associated with centrosomes. the kinetics of gvbd following mieroinjection is extremdy retarded (24-48hrs) when compared to spontaneous maturation (30ran-lhr) and depends on the oocyte diameter. interestingly, when inc~ompetent oocytes are cultured during 48hrs and then microinjeeted with okadaic acid meiosis resumes within a short delay (3-rhrs). mpm-2 epitopes are present on ecntrosomes only after okadaic acid injection. following gvbd one of the step depending on protein synthesis, the expression of tissue type plasminogen activator, is triggered. indeed oa-injected incompetent oocytes produce small amounts of this activator. altogether these results argue for an effect of okadaic acid favoring entry into mphase including microtubule organization, centrosomes phosphorylatiou and the recruitment of one of the masked mrnas (tpa). st~rillt~, ncb~, c~-121l g~. ~t4rs-inse~, monf~llier, france. meiotic reinitiation of the mouse oocyte is caracterized by a slow entry into metaphas~ i, b6~jinnin9 with germinal ve~ir break@own (gvbd) and ending with spindle formation. it is accompanied by a cascade of protein kinases and phosphatases increasir~g protein phosphorylation. the activation of histone hi kinase (maturation promoting factor, mpf) and of the 1342 hapk have been compared during spontaneous or okedalc acid (oa)-induced rhetoric reinitiation. in spontaneously maturing oocytes, hist6ne hi kinase activity increases before gvbd (2x) and is a~sociated with the disappearance of the upper (tyrosine phosphorylated) migrating form of p34 cdc2, following gvbd, histone hi klnase activity culminates (8x) in metaphase i. activation by phesphorylation of p42 mapk occurs after gvfld. in contrast, no increase of histone hi kinase is detec~cable in oocytes induced to reinitiate meiosis by a transient exposure %o oa, neither before gvbo nor during the following 7 hours of culture. the upper migrating form of p34 cdc2 is present for 8 houra. the activation of p42 mapk b~lins before gvbd. furthermore, when oa is applied on coclrces microinjected with p13 sucl, neither iocrease of histone hi kinase activity nor p34 cdc2 dephosphorylation are observed although gvbd is induced; p42 mapk is activated. altogether these results suggest that gvbd may or may not be associated with a detectable activation of histene hi kinase, depending on the experimental conditions. activation of ps4 cdc2 and p42 mapk are separable events. we propose that, in the mouse c~yte, oa might be able to aetivaf8 an alternative pathw~ leading to gvbd that is cdc2-independent and that involves p42 mapk activation ensuing mpf-independent phosphorylations. s 10-04 urich, k., 4002 basel, switzerland transformation of cells by polyomavirus is mediated by middle-t antigen. this viral protein is able to form complexes with src family tyrosine kinases (e.g. c-src), phosphatidylinositol-3-kinase (pi 3-k) and phosphatase 2a (pp2a). c-src associated with middle-t is constitutively dephosphorylated at tyrosione 527, a site negatively regulating the activity of this enzyme. this results in high tyrosine kinase activity in interphase and mitotic polyoma-transformed cells. middle-t is transiently hyperphosphorylated during mitosis as reflected by an increase in the apparent lvl, on sds acrylamide gels. two putative phosphorylation sites for a cyclin-dependent kinase present in middle-t, threonine 160 and threonine 291, are also phosphorylated by purified p34 ~a~ in vitro. we constructed mutant middle-t genes where individual phosphorylation sites were converted to alanine residues. mutants lacking threonine 160 were still able to associate with all cellular enzymes described above yet defective for gell transformation. interestingly, the defect of this mutation could be compensated by additional changes in the middle-t protein sequence introduced further downstream in a domain suspected to play a role in the targeting of this protein to intracellular membranes. $10-05 schmidt, s., fa~khauser, c., and viesturs, s. isrec, 1066 epalin~es, switzerland little is understood of the mechanisms which regulate cytokinesis, or how it is integrated with mitosis. we have cloned a number of genes from the fission yeast s. po~be which are required for the initiation of septation and are characterising them. defects in the cdc7 and cdcll genes result in continued nuclear division without cytokinesis, while cdcl6 mutants undergo multiple rounds of septum formation without cell cleavage. our analysis suggests that phosphorylation will play an important role in the regulation of cytokinesis and its integration with mitosis. a functional cdcl6 product is required for maintenance of p34 r activity in mitosis and the primary sequence of the cdc7 protein indicates that it encodes a protein kinase. data concerning the role of the cdc7, cdcll and cdcl6 genes will be presented. rfc was originally identified in human cell extracts as one of the cellular factors essential for the replication of sv40 origin-containing dna in vitro. it is a five subunit protein with one large subunit of 100-i40 kda and four small subunits of 36-40 kda. rfc binds specifically to primertemplate structures at the 3'-end of the primer. together with proliferating cell nuclear antigen (pcna) it serves as a primer recognition factor for dna polymerase ~ and ~. to show the invobement of rfc in cellular dna replication we are currently cloning s. cerevisiae rfc. the amino acid sequences of a number of tryptic peptides have been obtained. this information was used to amplify parts of the s. cerevisiae genes by per and to screen a genomic library with these probes. three genes were cloned so far. the large subunit, rfc1, codes for a 95 kda polypeptide that is 36% identical to the large subunit of human rfc (lirfc). the sequences for two of the small subunits were also obtained. rfc2, coding for a 40 kda polypeptide, is 50% identical to the hrfc 37 kda subunit. rfc3, coding for a 38 kda polypeptide, is 51% identical to the hrfc 36 kda subunit. there is also considerable similarity between the different subtmits. rfcj, rfc2 and rfc3, as well as the hrfc subunits, have consensus sequences for a atp/gtp binding site. all three genes are essential in s. cerevisiae. p53 is a major tumor suppressor gene which spefically interferes with the onset of e phase. we have therefore cxa~ined the possibility that p53 modulates the normal functions of enzymes and proteins directly involved in dna replication. we have shown that human wtp53 protein binds physically to calf thymus single stranded dna binding protein a, rp-a, we have also found that p53 blocks dna helicases in a typical strand displacement assay. this may reflect the fact that p53 is able to reanneal complementary dna single strands. since both wt and some mutant forms of p53 share this function, they are unlikely to be related to the p53 tumor suppressor activity. to our surprise, among the helicases which were tested we determined that the p53 inhibition could be released by rp-a in the case of cellular but not in the case of viral dna helicases. additionally, we found that p53 can partially inhibit dna polymerase ~ and s holoenzymes on singly primed mi3 dna. this inhibition, however, was only evident when e. coli ssb was substituted for rp-a. our data thus show that p53 exerts an inhibitory effect on several enzymes involved in dna replication and dna repair. the p53-rp-a interaction may function to protect replication enzymes from inhibition by p53 under certain physiological conditions. catalytic subunit showed that pol ~ is the most conserved replicative pol (cullmarm g., hindges r., berehtold m.w. and htibscher u., gene, in press). we synthesized pcr primers and cloned three fragments spanning the whole catalytic subunit. the resulting pcr products, called n-term, middle and c-term have a size of 1600, 1570 and 1280 bp, respectively. the primer for the n-term and middle fragments contained a histidin tag in order to purify the recombinant proteins by using a ni-nta column. these fragments were cloned into the expression vector pgemex174. because of the natural termination codon in the c-term fragment, the histidin tag was placed directly in the vector in front of the fusion protein, generating a new vector, prhh1s. all three fragments were expressed in e.coli. the fusion proteins could be purified in a single step and were used to raise polyclonal antibodies. finally, the entire pol 8 sequence was joined together and could be expressed. data on the characterisation of the antibodies and the proteins will be shown. hsp 70 from calf thymus can influence dna polymerase under heat shock conditions. we have generated antibodies against dnak protein, the hap70 homolog from escherichia coil, and used them for detecting of hsp70 protein during its purification from calf thymus tissue. the apparently purified protein has a molecular weight of 70kda, and has been recognized by antibodies against dnak and eucaryotic hsp72/73, it possesses a very week atpase activity, which can be stimulated by different poly-and oligopeptide substrates. results will be presented showing the influence of hsp70 on dna polymerase r from calf thymus under heat shock conditions. identification of a vertebrate cdc2 mutant which is unable to complete the g1/s transition. nicole sr and viestars simanis. chemin boveresses 155, 1066 epalinges,.isrec. s. pombe strains have been constructed which should permit the generic and molecular analysis of the events which occur in late gi when cells become committed to the mitotic cell division cycle and the initiation of dna synthesis. a number of mutants of the chicken cdc2 gene were eonsuueted during the course of analysis of phosphorylation sites some of which lead to the interesting phenotype of cold sensitivity when expressed in a cdc2 null background, when the only source of p34 cde2 is the chicken homologue of the gene, expressed from a muldcopy plasmid. in order to create a genetically tractable strain, the wild-type chicken cdc2 gone and one mutant into the s. pombe genome to create a strain in which cell cycle progression is dependent upon the chicken cdc2 gene. analysis of this strain indicates gnat the cells block predominantly before replication of dna. in order to determine whether the cells were arrested before or after the execution of the start control their ability to conjugate at the arrest point was assessed. consistent with a block before the traverse of start and contmim~ent to s-phase, cells were able to conjugate with high efficiency. further support for the view that this mutant is defective only for the g1-s transition is provided by the observation that it is able to complement a cdc2a2l mutation in trans. this mutant is defective only for (32 function and not traverse of start. tl~e double mutant is viable at the restrictive temperature of either of tile parents alld is no longer cold sensitive. further work will concentrate on cloning genes involved in the traverse of gi into s phase in fission yeast. (masson and kreis, 1993, j. cell biol. 123:357) where it is localized on a subset of microtubulea. when caco-2 cells are grown to confluency and become polarized, e-map-115 expression levels increase concomitant with a higher microtubule stability. transfection of fibroblastic cells (veto), which do not contain any detectable e-map-115, with cdna encoding this protein, results in stabilization of microtubules to nocodazole treatment. these data suggest that e-map-115 may be involved in the stabilization and reorganization of microtubules in polarizing epithelial cells. since e-map-115 is a stabilizing protein, its interaction with microtubulen should presumably be modified at the onset of mitosis to allow disassembly of the interphase microtubule network and formation of the mitotic spindle. indeed, immunolabeling for e-map-t 15 varies during mitosis. in early prophase, no e-map-115 can be detected on the microtubules of the forming mitotic spindle. the protein is observed on the spindle microtubules of metaphase cells and staining becomes bright on the new interphase microtubules of telophase cells. analysis of the protein in cells blocked in mitosis by low concentrations of nocodazole indicates that e-map-115 is hyperphosphorylated. this hyperphosphorylated form of e-map-115 is no longer able to co-sediment with microtubules in in vitro microtubule-binding assays. phosphopeptide map and phosphoamino acid analysis show the existence of novel phosphorylation sites in e-map-115 during mitosis compared to the protein during interphase, we are currently characterizing the kinases involved in the modulation of e-map-115 activity. little is known about the specific functions of calcium-binding proteins in epithelial cells ~md in particular in tumoral cells of epithelial origin. calretinin (cr) and parvalbumin (pv) are supposed to be involved in cell proliferation. we observed the endogenous expression of cr in several cell lines from human colon adenoearcinomas, and we obtained stably transfected cells for pv in a human ovarian adenocarcinoma cell line. we studied the cell cycle in correlation with the endogenous or ectopic expression of cr and pv, respeetiveiy. g1 and mitotic celts are strongly labelled with the cr-antiserum 7696, while pv immunoreactivity is dominant in interphasic, but not in mitotic cells. in cr expressing cells, the cell cycle seems to be normal and the rate of proliferation to be proportional to the percentage of positive cells. the cell cycle is inhibited at mitosis after the addition of cr antisense oligo nucleotides to the culture medium. the cell cycle is blocked in gi/s and g2 in the cells eetopically expressing pv. the results support the hypothesis that these two calcium.binding proteins, cr and pv, could intervene at different moments and probably with different mechanisms on the mechanism of the cell cycle in the tumoral cells studied. production of polyclonal antibodies against calretinin cr-22k gander, j.-ch., gotzos, v., cello, m.r. and schwaller, 8. institute of histology and gen. embryol., university; 1700-fribourg a mrna for an alternatively spliced form of calretinin, ealretinin-22k (cr-22k) was detected in widr cells (a cell line from a human adenocarcinoma). we therefore decided to produce antibodies specific for this protein. it is directed against the 14 amino-acids localized at the c-terminus of the truncated protein which are unique for this protein and not present in full-length calretinin. two different approaches have been chosen to obtain a specific antiserum. we have produced a chimeric protein containing the cterminal region of cr-22k and the (h)6(nanp)19 polypeptide as carrier. the fusion protein was expressed in e. coil and purified on a nickel chelate column. as a second method, a synthetic peptide (corresponding to the last 15 amino acids of cr-22k) was crosslinked with the carrier protein klh (keyhole limpet hemocyanin). the antigens were used to immunize two rabbits. after the first boost, the 2 sera were tested. we have observed that both antisera recognize besides the protein used for immunization also cr-22k (expressed in e. coil) in a western blot specifically. no other protein bands of either e.coli extracts or from eytosolic extracts of widr cells crossreact with the 2 antibodies. both antisera detected the protein cr-22k in immunohistochemieal stainings of widr cells confirming the presence of cr-22k in widr cells. the tctp (p23) is a growth related tumour protein which is expressed under translational control. homologous acidic proteins have been found in higher plants and in saccharomyces cerevisiae (ykl312) . the striking conservation of this polypeptide between these very divergent organisms suggests some important but still, unknown cellular function. to address this question, a null mutation was constructed in yeast, by deleting almost all the ykl312 structural gone. the deleted strain shows no detectable phenotype. therefore, we conclude that, in yeast, this gene is dispensable. the protein ykl 312has been overproduced in e. coil and injected to rabbits to raise antibodies. we are now characterizing the ykl312 expression at rna and protein level. ellenrieder,c., forrer,p., kosovsky,j., rischle, m., nueller, r. and jaussi, r., institut f~r medizinische radiobiologie, paul scherrer institut & universitgt zh, ch-5232 villigen radiation therapy of tumors could be improved by influencing cellular radiation sensitivity. yeast strains with defective cell cycle checkpoint genes (e.g. radl, rad3, radg) show increased radiation sensitivity. cross-hybridization of a probe from the tad9 gene on human mrna northern blots yielded a single prominent signal. experiments to clone the corresponding 4 kb cdna are underway. we have cloned a hamster cdna encoding the homologue of human cdk2. probably, this cdna encodes the p40 protein from hamster whose phosphorylation responds to radiation checkpoint signals and who does not bind to p13 sucl beads. treatment of cells with cdk2 antisense oligonucleotides is hoped to modulate the radiation sensitivity. proliferation of smooth muscle cells (smcs) is a major event in vascular development and atheromatous plaque formation. in order to characterize smc replicative potential, newborn rats have been injected with 3h-thymidine (3h-tdr) during their first week of life when most of smcs were proliferating; then, 3h-tdr labelling was evaluated in adult rats. depending on the number of mitosis that smcs had accomplished after the first week of life, four different smc subpopulations could be defined indicating that rat aortic smcs are heterogeneous in their replicative activity. 5-bromo-2'-deoxyuridine (brdu) incorporation after balloon induced endothelial denudation of rat aorta in adult rats showed that smcs entering into the cell cycle were mainly devoid of 3h-tdr labelling, this category could derive from two distinct smc subpopulations: smcs which were arrested in their proliferation before or just after birth or smcs which had actively replicated during development. thus, one (or two) subpopulation(s) of rat aortic smcs, characterized by a particular replicative activity during development, is (are) selectively activated after balloon induced endothelial denudation. the situation in vivo of dermal fibroblasts embedded within the connective tissue can be emulated in vitro by growing the cells in collagen gels. we have investigated how fibroblasts cultivated within a matrix react upon wounding of this dermal equivalent. human skin-derived fibroblasts (kd-cells) growing within attached collagen matrices were monitored over a period of 8 days. fluorescently labeled cytoskeletal elements (tubulin, vimentin, acfin) and fibronectin served as phenotypica/ markers. the 3d-arrangement of fibroblast microtubules, intermediate filaments and microfilaments, as well as of fibronectin was visualized by clsm and optical sectioning. cells at the wound margin, adjacent to the wound, and in non-wounded controls, up to a depth of about 100gm were analyzed. upon wounding, i.e. upon excision of a 1 mm 0 "punch biopsy" from the center of the collagen matrix, fibroblasts in the wound region oriented towards the matrix defect. layer-like structures of increased cell density evolved over time at the wound margin. the tissue defect was covered by fibroblasts, reminescent of the situation in vivo. global yeast genome expression analyzed by 2-d page after tctp homolog gene (ykl312) disruption. sanchez a translationally controlled tumor protein(tctp) was identified and microsequenced from a human liver sample. previous publications described the presence of a gene related to tctp in plants, mouse erythroleukemia and ascetic tumor, human mammary carcinoma and more recently in the yeast. tctp has no known function but its high degree of homology from plants to human underlines its probable crucial role in cell function. in order to study multifactorial chan~es in protein expression as a function of the ykl312 gene disruption m the yeast, we used high resolution two-dimensional gel electrophoresis combined with an efficient computer system (melanie). two groups of gels (ykl312 +(n=10) and ykl312 -(n=l 0)) were analyzed, compared and classified using correspondence analysis. there was no significant difference between the two populations of gels according to either qualitative and quantitative spots expression except for two spots which completely disappeared. the first one corresponded to ykl312 gsne product. the second one corresponded to a 28 kda peptide with an isoelectric point of 4.6. this ykl312 associated peptide still needs to be characterized and linked to the yeast genome. demyelinating and neurodegenerative diseases are associated with altered cellular responses including processes mediated by receptor protein tyrosine kinases. using a pcr cdna cloning approach we have identified a novel member of the eck/elk/eph genefamily in myelinating serum-free brain cell cultures. alternatively spliced cdnas encoding complete open reading frames for putative transmembrane proteins have been isolated from both rat and human brain. a recombinant protein derived from the rat cdna was demonstrated to have protein tyrosine kinase activity. an affinity purified antiserum to this protein was used to identify a glycoprotein of 120 kd molecular weight in brain cultures, and developping and adult brain tissue. the protein expression is most prominent during embryonal development and during the first three weeks of postnatal life. by in-situ mrna hybridisation the transcripts were found predominantly in restricted neuronal populations. in order to investigate systematically the effects of geometrically defhied abrupt tissue expansion on the characteristics of impulse propagation, we constructed expanding cellular structures in vitro using patterned monolayer cultures of neonatal rat heart cells (photolithographically patterned substrates.) the preparations, which consisted of narrow cell strands (40-80 tim wide) inserting into large reetangular cell areas (side lengths >1500 ~m), were stained with the voltage-sensitive dye di-g-anepps and were imaged onto a 12 x 12 photodiode matrix array (maximal spatial resolution 151.tin x 151am in the object plane). while impulse propagation was uniform in linear cell strands (average conduction velocity 0.35 m/s), a transient decrease in conduction velocity hi the transitional region was invariably observed in the suddenly expanded structures. this decrease was accompanied by marked distortions of the local action potential shapes due to bidirectional electrotonic interactions across the transition: upstream from the expansion, the repolarization phase was interrupted, a few ms after its inception, by a small secondary depolarization ("spike and dome" configuration), while, downstream, the action potential upstrokes were preceded by prominent prepotentials. in those cases where conduction failed at the tlansition, action potential durations upstream were dramatically abbreviated. these findings showed that phenomena similar to those recorded in intact purkinje-fiber-ventricular preparations can be observed in cell ensembles in vitro having comparable geometlins but consisting of cells with uniform active membrane properties. supported by swiss nsf grant 823a-028424 (sr) and usphs grant ns 16824 03ms). it is a noncollagenous glycoprotein with a molecular mass of 148 kda consisting of three identical subunits. cmp is selectively extracted with edta-containing buffer what indicates a divalent cation dependent anchorage of the protein in cartilage matrix. electron microscopy revealed the presence of three compact globular subdomains and sequence analysis indicated the presence of a coiled-coil c~-helical assembly domain formed at the c-terminal end of each subunit. the trimeric structure was retained after complete reduction under native conditions which reveals that the subunit structure of cmp is not only stabilized by disulfide bridges but also by the coiled-coil assembly domain. tissue distribution studies in mouse revealed that cmp is selectively expressed in some but not all cartilages. adult rat cardiomyoeytes (arc) in culture degenerate the myofibrillar apparatus and after attachment to the substrate they grow and reassemble new rnyofibrils. the appearance of new, well organized rnyofibrils can be observerd in several distinct regions. they appear close to the membrane proximal to the culture substrate and in the perinuclear region close to the substrate. previous studies have shown that embryonic cultured cells assemble new myofibrils which insert in adherens junctions at sites of cell-cell contacts and in sub-sarcolemmal adhesions plaques (saps) at sites of cell-substrate contacts. this saps are thought to function as the nucleation sites for myofibril formation. we have investigated the formation of this saps in cultured adult rat cardiomyocytes. using confocal light microscopy we can show that the interaction between rnyofibrils and membrans occurs close to the membrane proxirnal to the substrate and that this saps are flanking both nascent myofibrils and sfls, the latter structures being believed to serve as scaffold for myofibrillogenesis, additionally we have used electron microscopy to investigate the formation of this structures at higher resolution. $11-05 muscle satellite cells (sc) are quiescent myoblasts, ready to be activated and to regenerate new muscle fibers in case of muscle damage. in vitro, single human muscle sc, cultivated as clones, give rise in proliferating conditions to two subpopttiations of cells, cells expressing both ~-striated muscle actin and desmin (c~-sr+dsm+), and cells expressing desmin alone (dsm+). in culture conditions promoting differentiation, a clone .typically gives rise to a fusing progeny, yielding c~-sr+dsm + myotubes, and to non fusing myoblasts (nfmb). the latter are dsm +, a phenotype similar to quiescent sc found in situ. in order to determine whether nfmb are the in vitro equivalent of in situ quiescent sc, this first generation of nfmb were selectively collected and subcultured. in proliferating conditions, nfmb were able to resume proliferation and to give rise to a progeny with both c~-sr+dsm + and dsm + cells. when cultured in differentiating conditions, nfmb formed ct-sr+dsm + myntubes, and a new generation of dsm + nfmb. when nfmb of the second generation were again selectively collected and subculture& they resumed proliferation and ~re again able to yield both myetubes and a third generation of nfmb. nfmb of the first generation were also cultivated as clones, and 5 to 25% of them were able to resume extensive proliferation, yielding in their progeny both c~-sr+dsm + and dsm + cells, which differentiated into myotubes as well as nfmb. these results strongly suggest that sc have the stem cell property, of selfrenewal, yielding in their progeny both cells ready to differentiate into muscle and ceils similar to themselves. extra cellular matrix (ec~ regulates the expression of b-casein in cultured mouse mammary epithelial cells. we have developed a functional ~ epithelial cell strain, which expresses high level of milk proteins, forms alveolar-like structures when plated onto a reconstituted basement membrane and secretes casein unidirectior~lly into a it~aen. we have further shown that fflv~dependent regulation of b-casein occurs mainly at the transcriptional level and that 5' sequences play an inloortant role in this regulation. we have located a 160bp transcriptional enhancer (bcei) within the 5' flanking region of the b-casein gene. using functional assays, we show that bcei contains responsive elements for ~ and prolactin-dependent regulation. bcei placed upstream of a truncated inactive b-casein promoter reconstitutes a promoter even more potent than the intact prcmoter, which contains bcei in its natural context more than 1.5kb upstream. this small fusion promoter reconstitutes the nonaal regulation pattern. we show that bcei mediates f/24 dependent regulation even when linked to a het~rologous viral promoter. purified satellite cells (sc) were obtained from human skeletal muscle biopsies following cell sorting by flow eytometry. a chemiluminescent assay for acetylnholine (ach) revealed the presence of 1,3 nmoles/weu of 100.000 sc. in elonal cultures of proliferating sc, this intracellular level of ach was 0.l nmoles/well. when cells were cultured in the presence of an esterase inhibitor (10 ixm phospholine), the ach amount was enhanced 2 fold. conversely, cultivating the cells in presence of a potent inhibitor of choline acetyl transferase (2 gm bromoach), gave a 50% decrease of ach content finally, the release of ach was detected in the supernatant of cultured sc, following a 15 min incubation, and the measured level of ach was 3 pmoles/well/min. the presence of an ach-like compound in myogenic cells in both freshly isolated and in proliferating sc suggests that sc may contain ach in situ. in additiou, the fact that ach was present in the extracellular medium suggests that ach can be released spontaneously by the cultured sc. type xii collagen is an extracellular matrix protein associated with collagen fibrils in vivo. as observed for several other extracellular matrix proteins, the synthesis of type xii collagen by chick fibroblasts cultured in 0.1% fcs is stimulated by tgf-i~i. two splice variants of type xii collagen are known, with subunits of either 220 kda or 350 kda. by using antibodies specific for the large (350 kda) form of type xii collagen, we could show a more restricted distribution of the large variant compared to the smaller form in the embryo. while only the large variant carries chondroitin sulfate, both variants are identical in their collagenous domain. it is thought that via this domain, type xii collagen binds to collagen type i fibrils. we demonstrate here that type xii collagen can bind to collagen i fibrils also in vitro. by neutralizing acid soluble collagen type i, we could coprecipitate type xii collagen together with newly formed collagen type i fibrils. this interaction occurs in physiological saline but is highly salt dependent. in further studies we investigated wether 35s-methionine labeled type xii collagen treated with chondroitinase abc, ct-chymotrypsin, or collagenase can still be precipitated together with type i fibrils. in addition, we found that type xii collagen also affects the rate of type i collagen fibril formation. h.u. keller department of pathology, university of bern, ch-3010 bern locomoting blebbing cells colchicine (10-sm) can induce locomotion associated with blebbing in walker carcinosarcoma cells. blebs expand at a rate (about 2~m/sec) which is much faster than known rates of actin elongation. this suggest that the membrane is directly pushed forward by forces other than actin elongation, possibly by hydrostatic pressure. this interpretation is suggested by the observation that there is significant f-actin staining all along the cell membrane but not with the cytoplasmatic content the blebs. blebbing is suppressed by 0.5 m sorbitol. the finding that cytochalasin d suppresses blebbing shows that actin polymerization is nevertheless indirectly instrumental in bleb formation, possibly by generating a high intracellular pressure. a tentative model explaining locomotion of blebbing cells is presented. osteopetrosis encompasses a family of diseases with different causes and the common phenotype of impaired bone resorption. the osteopetrotic mouse mutant oo/oo is deficient in csf-1, the growth factor for the cells of the mononuclear phagocytic system. the phenotype of oo/ou mice is characterized by a low number of peritoneal macrophages and peripheral monocytes, and a virtual absence of osleoclasts, leading to the osteopetrotic phenotype. injections of csf-1 into op/oo mice reversed the osteopetrotic phenotype, proving the requirement for csf-1 during the process of osteoclastogenesis. by in situ hybridization, expression of csf-1 was demonstrated during bone development. osteoclast precursors and mature osteoclasts were identified as putative target cells for the growth factor, since these cells express csf-1 receptors, which is encoded by the proto-oncogene c-fm~. subsequently, specific binding of csf-1 to osteoclasts was shown. expression of c-fms parallels the expression of csf-1 in bone both in time and place, suggesting a local action of this growth factor in osteoclast formation, but also in the modulation of osteoclastic activity. different transcripts, raised by alternative splicing of a commmon nuclear rna precursor, have been found to encode a secreted and a membrane associated forms of csf-i. the secreted form can be modified by attachment of a glycosaminoglycan side chain, which serves as an anchor to integrate the peptide into the extracellular matrix. investigation of the role the different csf-1 forms play during recruitment of osteoclasts and regulation of their activity will provide further insights into the mechanisms governing these processes. comparative sequence alignments of known voltage-gated k + channels revealed two conserved cysteine residues in the putative transmembrane segments $2 and $6. if the cysteines were connected by a disttifide bridge, it would put a structural consllaint on possible channel models by placing $2 next to $6. we used site-directed mutagenesis to study systematically the potential roles of these invariant cysteines. fourteen of 17 substitutions in $2 (c232), and 7 of 19 substitutions in $6 (c393) maintained channel function in xenopus oocytes microinjected with mutant crna. therefore, the conserved cysteines are not essential for k + channel expression in xenopus oocytes. however, electrophysiological characterization of the cysteine replacement mutants revealed distinct roles for the two cysteines. inactivation, deactivation, and ion permeation did not considerably change in $2 mutants. in $6, in contrast, cysteine replacement by leucine, asparagine, glycine and valine accelerated inactivation and deactivation kinetics substantially, whereas serine and threonine showed opposite effects. furthermore, the voltage dependence of deactivation was differently affected. in summary, it appears that the side-chain at position 393 in $6 of kv2.1 is involved in channel gating by participating in the transitions from the open to the closed and inactivated state of the channel. (supported by grants from the nih and mda to rhj, and from the snf and the swiss foundation for medical-biological grants to rdz.) the segment between $5 and $6 of voltage-gatedk + channels, called h5 or p region, has been implicated to form part of the ion conduction pathway. little is known about the conforroation of this region although various models have been proposed including a j3 barrel-like structure formed by four adjacent antiparanel j3 sheets. to gain insight into the secondary structure of this region, we used cysteine substitution mutagenesis and sulfhydryl-specific, membrane-impermeant reagents to probe the accessibility of amino acid side chains in and around the p region of kv2.1 (drki). twemy-eighi positions from k356 to t383 were each mutated to a cysteine. after expression of mutant k + channels in xenopus oocytes, cysteine side chain accessibilities were probed by superfusion with ch3so2sch2ch2nme3 + (mtset), mtset can form a mixed disulfide with an accessible cysteine, and this may lead to current reduction if the covalently modified side chain is in or close to the ion conduction pathway. thus far, we have identified four mutations that showed k + current reduction after superfusion with 3 mm mtset. the mutants p361c, i379c, y380c, and k382c showed 87%, 99%, 39%, and 21% reduction in current amplitude, respectively. in coutrast, mutants $363c, a367c, t368c behaved like wild type kv2.1 with less than 5% current reduction. our results suggest that the side chains of p361,1379, y380, and k382 are directly accessible from the extracellular environment. taken together, these residues may, therefore, face the lumen of the ion channel pore. furthermore, the degrees of inhibition are in agreement with a model in which the ion conduction pathway narrows from residue k382 to y380 to i379. (supported by grants from nih and mda to rhj, and from the snf and the swiss foundation for medical-biological grants to rdz.) motejlek k., h,,iuselmann r., and liig:her b.; pharmakologisches institut der universitiit ziirich, winterthmtr. 190, ch-8057 zlirich comparison of 5' flanking sequences of three gabaa-receptor subunit genes (atl, ~, 8) revealed a novel conserved purine sequence dement with the consensus sequence gagaggggagaoga gagag(gg/aa)g. this element is present once each in the (zl and 72 subunit gene promoters and in seven tandem copies in the 8 gene promoter. a novel dna binding activity (bsf1) was identified that binds to various versions of this purine element. bsf1 was found ordy in brain but not in any other tissues tested. furthermore, the factor is distinct of beta, another brain-specific dna binding .protein with a purine-rich dna recognition sequence. the expression of bsf1 during differentiation of eerebeuar granule cells in vitro correlates with the expression of gabaa-receptor subunit genes. this correlation suggests a role for bsf1 as a transcriptional activator of neuronal genes. therefore, bsf1 may be important for cell ty~specific regulation of gabaa-receptor gone expression. we also found that bsf1 binds to wheat germ agglutinin, suggesting that this protein is glycosilated. taking advantage of this property, we are in the course of purifying bsf1, the question, whether bsf1 is indeed a transcription factor is being investigated in vivo and in vitro. to produce a simple method of estimating the free magnesium concentration ([mgzq) in solution, we have manufactured dip cast mg 2+ macroelectrodes using the neutral mg carrier eth 7025.we have used the elecmides to measure the dissociation constant (k~) for mgatp in a background solutions mimicking the intracellniar milieu. for the mcasmement of the big atp dissociation constant, the background solution contained 2 mmol/l n%atp. this solution was titrated with mgci 2 and the changes in [mg ~] above 0.05 retool/1 monitored with the maeroelectrode. during the titration the ph was maintained constant. fitting such titration curves with the standard hyperbolic binding equation, after correction for zero drift, gave the following mean+sd values for the ka (pmol/1 at 37~ ph 6.4, 103.3-+-25,3 (n=9); ph 7, 59.7:k-27.4 (n=6) and ph 7.4, 30.g~15.4 (n=7). elevation of the [naq to 50 m~l/1 and reducing the [kq to 1(30 mmol/l was without effect at ph 6.4 (n=6). reducing the temperature to 25~ increased the k a at 7.4 to 49.0:k21.6 (n= 5). mg =+ macroelcctrodes provide an easy way of measuring [mg z ⧠in solution. [mg ~] can also be measured in a background solution containing i mn~i/l ca, although the electrode response is reduced. interference by protein to date prevents their use in plasma. similarily to the block by zn 2+ and ca 2+, protons only partially block the channel. the ability for all mutated channels to. conduct na + current, and the partial block induced hy zn 2+, ca 2+ or h + indicate that y401 is not located deep in the pore but rather in the extracellular mouth of the channel. progesterone modulates the activity of glycine receptor expressed in colliculli neurons of neonatal rats. maury, k. & bertrand, d. dpt of physiology, cmu, 1211 geneva 11. steroids are potent narcotics and have been shown to act on ligand-gated channels activated by gaba or nicotine. however, little is known about their possible actions on other receptor types. for instance, while it was demonstrated that progesterone inhibits glycine receptors expressed by spinal cord neurons isolated from chick embryos, nothing has yet been reported for brain receptors. in this study, we have determined the properties of glycine channels expressed by brain neurons of neonatal rats using the whole-cell voltage-clamp technique. in inferior colliculi neurons, glycine elicited little-desensitizing inward currents which reversed around -40 mv. these currents were blocked by strychnine in the nanomolar range. surprisingly, however, we found that progesterone, in the micromolar range, potentiated the glycine evoked currents. these results, contrasting those obtained in chick spinal cord neurons, suggest that progesterone enhances or antagonizes glycine currents depending on the subtype of the receptor. the mammalian auditory organ (organ of corti) relies on two groups of sensory cell for its normal hearing: inner hair cell & outer hair cell. ihcs are mainly innervated by afferent fibers while the ohcs essentially receive efferent fibers. the ohcs possess unique electromofile properties which are thought to enhance the motion the basilar membrane and to refine the mapping of the sound frequency. we found, using whole ceil recordings, that 5-10% of the fleshly dissociated ohcs displays fast inward currents. the magnitude of peak currents which can be as large as 2na vary from cell to cell. further experiments have revealed that this current is sensitive to ttx and strongly reduced when extracellular sodium is replaced by choline. its kinetic is relatively slow compared to that of sodium current of typelspiral ganglion cell, and has a more negative inactivation. other voltage activated currents and ligand-gated currents are under inverstigation. these results will provide further insights in the hearing transduction mechanism. the formation and the properties of homotypic and heterotypic gap junctions were studied using two types of insect cells, c6/36 and sf9. two single cells were pushed against each other and the subsequent de novo formation of gap junction channels was assessed by means of the dual voltage-clamp method (bukauskas and weingart: pfl~g. arch. 423:152, 1993 it was symmetrical for homotypic junctions. the vj-sensitivity was less pronounced in sf9 cells. hence, the relationship for heterotypic junctions was asymmetrical. all pairs examined revealed a s-shaped relationship between gj(ss) and v, which was virtually superimposable (gj(ss) declined upon depolarization). each channel contains a vm-gate and two ~-gates. the vj-gates are operated independently. they close when their intracellular aspect is made positive. shaped curve compatible with a two-state boltzmann process. half maximal inactivation was reached at -77 mv and +66 mv, implying an asymmetrical gating behavior. in case of an asymmetrical protocol (~ and vewas stepped simultaneous, but in opposite direction), the q-dependent asymmetry disappeared (half maximal inactivation at -59 mv and +60 mv). the asymmetry in gj-gating, attributable to the pulse protocol, was also reflected in the time constants of ij inactivation (r177 cerebellar granule cell cultures were used to study the regulation of the nmda receptor expression. we have shown previously that chronic membrane depolarisation (25 him k +) or treatment with nmda {i00 ~m) promotes the functional expression of nmda receptors, as assessed by nmda-evoked 45ca2+ influx. we have now developed a rnase protection procedure for the quantitative determination of the mrna levels of the nmda receptor subunits known so far (nr-i,2a,2b,2c,2d). the growth conditions mentioned above failed to alter the mrna levels of the different subunits. at the protein level, nmda receptors were evaluated quantitatively by the labeling pattern obtained by the new photoaffinity ligand 125i-cgp55802a. the intensity of the phctolabeled bands, thought to represent nr-i and nr-2a subunits, was increased by treatment with high k + or nmda compared with control cultures. this result suggest an increase in receptor protein by high k + or nmda treatment. thus, posttranscriptional mechanisms seem to play an essential role in the modulation of the nmda receptor activity. the gene encoding the pore-forming subunit of the rat epithelial na + channel (~renac) was cloned recently and shown to belong to a novel gene family coding for cation channels (nature 361, p467-470 (1993) . transport of na + ions through these channels is the rate-limiting step of na + absorption and thereby controls osmotic balance of body fluids and secretions. in order to study the implication of ~renac in regulating sodium balance, blood volume and blood pressure and therefore its involvement in hypertension, we expressed ~renac under the control of the human cmv promoter in transgenic mice. several lines of mice showing expression in different tissues were obtained. progress in the analysis of these mice will be discussed. activation of metabotropic glutamate receptors increases the excitability of neurones in the lateral septum of the rat. to elucidate the mechanism(s) of this action, we used whole-cell patch-clamp recordings from coronal brain slices of young animals. in voltageclamped cells, the selective agonlst (is,3r)-acpd, at 1-50 /~m, had the following effects, a) it evoked a sustained inward current, which was resistant to ttx and to cadmi~am and which persisted in neurones loaded with bapta, a calcium chelator. this current displayed inward rectification and was reduced, or suppressed, when extracellular sodium was partially replaced with n-methyl-d-glucamine. b) it elicited a ttx-insensitive, voltage-dependent inward current, which activated at -50/-40 mv and which reversed at aound 0 mv. this current was suppressed in a low-calcium/high-magnesium perfusion solution and was undetectable in the presence of intracelhilar bapta. we suggest that acpd exerts a dual action on lateral septal neurones. it causes a steady depolarization by generating a sodium-dependent current and it triggers transient plateau potentials by inducing a calcium-dependent cationic current. interaction between these currents results in a powerful, self-reinforcing neuronal excitation. we have investigated the effects of protein kinase c (pkc) activators (4[~-pma, oag) and of phosphatase inhibitors (okadalc acid, calyculin a) on voltage-gated ca 2+ and k + channels in ngf-differentiated pc12 ceils. whole-cell ba 2+ and k + currents were recorded with the patch-clamp technique. by using specific ca 2+ channel blockers (co-conotoxin (cgtx), isradipine) we found 3 types of ba 2+ currents (iba): a) a ~cgtx-sensitive iba; b) an isradipine-sensitive iba; and c) a t0-cgtx plus isradipineresistant iba. 4[~-pma and oag specifically down-modulated the isradipine-sensitive iba. the inhibition of iba was prevented by staurosporine and pkc (19-31) (2 pk inhibitors). the delayed rectifier k + current was unaffected by pkc activators. applied externally, okadaic acid and calyculin a inhibited the total iba by affecting several components of the ba 2+ currents. however, the 0~-cgtx plus isradipine-resistant iba was unaffected by okadaic acid. in conclusion, our results suggest a differential modulation of voltage-gated ca 2+ and k + channels by the pkc signalling pathway in ngf-differentiated pc 12 cells. agrin, a protein isolated from basal lamina extracts of the electric organ of the marine ray, is thought to mediate the motor neuron-induced aggregation of acetylcholine receptors (achrs) in muscle fibers at their neuromuscular junction. recent cloning in the marine ray, rat and chick has revealed that agrin can be alternatively spliced at two sites in its c-terminal half. site a encodes either 0 or 4 amino adds and site b encodes either 0, 8,11 or 19 (8+11) amino acids. studies of agrin mrna expression indicate that early in synaptogenesis, chick motor neurons contain high levels of a4b19. in contrast, non-neuronal cells and muscle calls contain a4b0 and aob0 mrna. in the adult ray, electric lobe motor neurons that innervate the electric organ contain a4b8, whereas agrin mrna in the electric organ again lacks both sites (mcmahan et al. (1992) , curr. up, cell biol. 4:869-874). to investigate the functional properties of agrin isoforms in more detail, we have now compared their specific activity to aggregate achrs on cultured chick myotubes. heterologous expression of the c-terminal half in cos-7 and 293 cells revealed that chick agrin isoforms a4b8 and a4b19 were highly active, while a4bll was up to 40 fold lower in activity. no activity could be detected for the a4b0 and aob0 isoforms. in agreement with these findings the a4b8 isoform of the marine ray also showed high achr-clustering activity. therefore, we conclude that motor neurons throughout development synthesize highly active agrin isoforms while the postsynaptic target ceils do not play a primary inductive role in the formation of synapses. we have used whole-cell patch-clamp recordings and hypothalamic slices in order to characterize the effect of n-methyl-d-aspartate (nmda) on suprachiasmatic neurones. in ceils clamped at or near their resting membrane potential, nmda (50-100 #m) generated an inward current of 10-60 pa, which was insensitive to ttx and which reversed at about 0 inv. the nmda current-voltage (i-v) relation contained a region of negative slope conductance. the nmda current was reduced or suppressed by d(-)-2-amino-5-phosphonopentanoic acid (d-aps) or by mk-801. it was potentiated by reducing the extracellular magnesium concentration from 1 to 0.01 ram, or by adding glycine (10 /~m) to the perfusion solution. in a majority of neurones, lowering the extracellular calcium concentration from 2 to 0.01 mm caused a 1.5to 4-fold enhancement of the nmda current. i-v relations indicate that in the low-calcium solution, the region of negative slope conductance was attenuated. this effect was due to extraceuular calcium, since it persisted in neurones loaded with the calcium chelator bapta. we conclude that nmda channels present in suprachiasmatic neurones may be modulated by extraeellular calcium. institut, and abt. pharmakologie*, biozentrum, basel. during innervation of skeletal muscle, the myonuclei underlying the synapse begin to express acetylcholine receptor (achr} ~-subunit gene and they remain activated after the nerve is removed. our experiments show that this is due to a factor in the synaptic portion of the muscle fibre basal lamina (bl). one candidate for this factor is agrin, which regulates ache clustering in the synaptic membrane: i) unlike in untreated muscle, the level of s at synapses was strongly reduced in cultured rat muscle fibres after proteolysis of synaptic bl. 2) conversely, when rat myotubes were cultured on bl isolated from adult muscle, they preferentially expressed achr accumulations and ~-mrna at sites where they contacted the synaptic portions of the isolated bl. 3) when various substrates were impregnated locally with agrin a4big, an isoform expressed by motor neurones in fetal spinal cord, it locally induced in the myotubes the expression of s-mrna. art increase of functional nicotinic acetylcholine receptors precedes the fusion of cultured human muscle satellite cells. r. m. kranse, c.-r. bader and l. berrtheim. department of physiology and division of clinical neurophysiohigy, university medical center, 1211 geneva 4, switzerland nicotinic acetylcholine receptors (nactlr) are expressed on embryonic myoblast and acb may play a role in mechanism of fusion. our study focuses on the appearance of functional nachr in freshly isolated and cultured satellite cells (sc) from normal human muscle biopsies. sc cart be conditioned to either proliferate or fuse to form myotubes depending upon culture media. presence of ach-activated current was investigated using the whole-cell and single.channel patch-clamp technique. in freshly isolated sc (whose properties should be close to the quiescent in vivo sc/no nachr were observed (n=16). in the proliferating state, 54% (n=35) of the satellite ceils (which are also called myoblasts) displayed a small ach-activated current (10 pa/pf) and, as expected, after fusion of salellite cells into myotabes, 100% of the ceils displayed an ach-activated current (n=6; 26 pa/pf). to examine, whether the increased expression of nachr precedes sc fusion, sc were cultured in a medium which promotes fusion, but were prevented from fusing by keeping them at a low density. in this culture condition, 93% of the sc (n=15) displayed an ach-activated current and, in addition, these cells expressed a 4 times higher ach-current density. (40 pa/pf) than the proliferating sc. we also observed that, in high density cultures, a small population of sc do not fuse even atter 3 months in the medium promoting fusion. these non-fusing myoblasts (nfmb) had no nachr. our results suggest that the appearance of nachr may be related to the process of sc fusion as i) quiescent sc in vivo do not express nachll ii) nachr expression increases in conditions promoting fusion and iii) no nachr were observed in nfmb. the latter cells may be equivalent of quiescent sc in vivo. $12-20 we have investigated the sensitivity of neuronal nicotinic acetylcholine receptors (nachrs) of known subunit composition to various cholinergic antagonists. neuronal nachrs were expressed in xenopus oocytes after injection of pairwise combinations of (x2 or c~3 with either 1~2 or 1~4 crnas. the two-electrodes voltage-clamp technique was used to measure currents induced by rapid application of low concentrations of acetylcholine (ach) together with increasing concentrations of antagonists. the response of ct31~4 neuronal nachrs to ach was halfmaximally inhibited by following antagonist concentrations (ic50): hexamethonium (0.3 i.tm), mecamylamine (0.2 p.m), pentolinium (0.2 i.tm) and trimetaphan (1.2 ~tm). with (x3132 nachrs the ic50s of the same antagonists were about 10 times higher. finally, (+)-tubocurarine was a conventional competitive inhibitor of ach in ~3132 and t~2~2 nachrs. in contrast, low concentrations of (+)-tubocurarine increased the response to low ach concentrations in a3(34 and t~2~4 nachrs. these results further underline the importance of [l subunits in determining the functional properties of neuronal nachrs. supported by the hochschnlstiftung and nf grant 31.31018.91 to abc. previously, the role of the transglial tubular system was shown for the squid giant axon: in na + free (tris) solutions the series resistance increased in correlation with a decrease of the tubular opening density (tod). in the crayfish giant axon, which show similar tubules, we correlate the change in tod, measured in freeze-fracture preparations, witl] the conduction velocity. the control to'd was estimated to be 16 per #ms. after 30 and 75 rain tris-inkubation the tod decreased to 10,4 and 1,5, respectively. this reaction was reversible when these axons were reincubated in na~-c41ntaining solution. after 120 rain reincubatiou the tod was 11.8 per tam'z. to determine the influence of decreased tod on the impulse conduction velocity, the nerve was incubated for 30 min in tris solution followed by reincubation. impulse propagation then recovered in two phases. during an initial fast phase with a short time constant of 1-2 rain na + diffused back into the periaxonai space and allowed impulses to occur with a low conduction velocity. in the second phase with a long time constant of 7 min, conduction velocity recovered slowly to normal values. this time course was comparable to the recovery of tod, as estimated from recent freeze-fracture preparations after short reincubation. these results indicate that the normal tod is a necessary condition for the normal excitability of the axon and determines its conduction velocity. most spinal cord neurons respond to the inhibitory action of gaba and glycine, suggesting co-expression of gaba a-and glycine receptors 4n individual ceils. while glycine-receptors are exclusively found in post-synaptic densities, the cellular localization of gaba areceptors has not yet been characterized. in this study, the distribution of gabaa-receptors in the spinal cord was analyzed with an antiserum recognizing the (xl-subunit. double-and tripleimmunofluorescence staining were employed to identify synaptic receptors apposed to gabaergic terminals (immunoreactive for glutamic acid decarboxylase) and to assess their co-localization with glycine-receptors (visualized with an antibody to the 93 kda protein gephyrin). staining for the gabaa-receptor ctl-subunit decorated the soma and dendrites of numerous neurons in laminae iii-viii and x, revealing their morphology in clear detail. by contrast, laminae 1i and ix contained little immunoreactivity for these gabaa-receptors. most gabaa-receptor-positive cells also exhibited a prominent glycine-receptor immunoreactivity. both types of receptors had very similar distribution patterns and were frequently co-localized in sites apposed to gabaergic boutons. these results indicate that gaba aand glycine-receptors may co-exist within single post-synaptic densities, suggesting a possible synergism between gaba and glycine neurotransmission in spinal cord neurons. prenatal benzodiazepine (bdz) exposure changes both behavioral and neuiochemical parameters of developing iats. these changes are not a consequence of remaining bdz in brain tissue, but rather indicate alterations in bdz receptol binding and function. since the bdz binding site is located on the gaba~ receptor complex, it is possible that prenatal bdz treatment influences the expression of gaba~ receptor subunits. to evaluate effects of pienatal bdz exposure on mrnas expression specific for several gaba~. receptor subunits (~i, ~5, ~ & y2), we treated plegnant rats with diazepam (l.25mg/kg/d; s.c.) from gestational day 14 to 20. we then analyzed specific mrna levels in offspring at four diffeient developmental stages (gd20, pns, pni5 & adult) by in situ hybridization. pleliminary data flom optical density measurements indicate a deciease in expression of mrna in particular for ~-subunit of gab~ receptors in different brain reglons of plenatally diazepam-treated zats. processing of sensory information within the auditory system has been well characterized using histological and eleetrophysiologlcal techniques. however, relatively little is known about the neurotransmitters involved. the present study was performed to elucidate the possible role of excitatory (eaa] and inhibitory (i.e., gaba) amino acids in neurotransmission within the primary auditory cortex (all the main afferent to the ai, the ipsilatera} medial geniculate body (mgb), was electrically stimulated and evoked responses were recorded intracellularly in the ai region in halothane anesthetized cats. a seven-barrelled iontophoresis pipette glued alongside the recording electrode allowed localized application of eaaergic and gabaergic compounds onto the recorded neuron. in most ai neurons, mgb stimulation evoked a short latency, short duration epsp followed by a long-lasting ipsp. pattern, duration, and amplitude of synaptic potentia{s were highly variable and strongly dependent on stimulation intensity, reduction of gabaa receptor-mediated inhibition by iontophoretic application of either bicuculline or sr95531 markedly enhanced mgb stimulation-evoked epsps. only the late component of this enhanced epsp was reversibly blocked by the competitive nmda receptor antagonistl ap7 at currents which selectively blocked neuronal excitation induced by iontophoretic application of nmda, our results demonstrate that in the eat 1) nmda receptors in ai are activated following mgb stimulation and that 2) a dominant gaba~ receptor mediated inhibition usually masks this nmda receptor activation under our experimental conditions, a highly purified and specific cell wall degrading endo-l,5-u-l-arabinanase was isolated from an a. niger pectinase preparation by low and medium (fplc) pressure column chromatographic separation methods. the isolated enzyme was most active on linear 1,5-~-l-arabinans (-90 i.u./mg), whereas branched arabinans from sugar beets were degraded to a lesser extent (-14 i.u./mg). the enzyme was shown to be electrophoretically pure after silver staining (sds-page) and its identity was confirmed through specific binding to an antiserum directed against endo-l,5-~-l-arabinanase. the major physico-chemical characteristics of the enzyme were the following: mr 42'000, iep ~ 3.0, ph optimum 4.8, temperature optimum 55~ ph stability 3.5-8.0, temperature stability s 45oc, km = 0.205 mg/ml, vmax = 17.7.10 -3 ~unol/min. zn and hg showed potent inhibitory effects. 3.2) is a specific enzyme of the glyoxylate cycle, which plays a key role in the initiation of gluconeogenesis in germination oilseeds, this pathway is localized in glyoxysomes, single membrane organelles which are converted into peroxisomes when lipid reserves are depleted. the glyoxylate cycle enzymes have been the subjects of numerous conflicting reports typically addressing the problems of their synthesis (on free or bound ribosomes), targetting and suborganelar localization (membrane bound or matrix proteins). recent biochemical studies have shown that ms from germinating soybean cotyledons is an interconvertible enzyme (membrane bound, aggregated or soluble fomls) which is significantly affected by its ionic environment (henry et al., 1992, plant science 82:21-27) . microscopy studies have now been carried out using immunofluorescence staining or immunogold-silver staining for light microscopy, and immunogold labelling for electron microscopy. all results consistently characterize ms as a glyoxysomal mawix enzyme. chorismate mutase (ec 5.4.99.5) catalyzes the first step in that branch of the shikimate pathway which leads to the aromatic amino acids phenylalanine and tyrosine. we have isolated a cdna for this enzyme from the higher plant arabidopsis thaliana by complementing a yeast strain (aro7) with a cdna library from a. this is the first chorismate mutase cdna isolated from a plant. the a. thaliana chorismate mutase expressed in yeast revealed allosteric control by the three aromatic amino acids as previously described for plastidic chorismate mutase isozymes. an attachment of rubisco to chloroplast membranes under cu++-stress has been described for wheat (mehta et al., j. biol. chem. 267, 2810 -2816 . the displacement to the membranes has been discussed as a possible step in the degradation of this predominant stromal protein, e.g. during leaf senescence. in our recent experiments it became evident that such interactions are not restricted to rubisco. several other stroreal, and even a peroxisomal enzyme (glycolate oxidase), were also detected in the membrane fraction after 6 to 30 hours of oxidative stress induced in wheat seedlings by a treatment with 10 mm cuso 4. the velocity and the degree of membrane attachment varied for different enzymes. based on these results it was interesting to check the solubility of rubiseo and its previously described 45 kd fragment which accumulates during the incubation of bean leaf discs under oxygen deficiency (hildbrand and feller, experientia 47, a67) . neither rubisco nor the breakdown product were found to accumulate in the membrane fraction. thus, at present, the steps involved in rubiseo catabolism cannot be generalized. different mechanisms depending on the metabolic situation and on environmental conditions should be considered. overexpression of the four parsley phenylalanine ammonia-lyase (pal) isoenzymes in e. coli. characterization of the enzymes and kinetic analysis of the inhibition by 2-aminoindan-2-phosphonic acid. christoph appert, j/irg schmid, jerzy zorn* and nikolaus amrhein institute of plant sciences, eth-z/jrich, 8092 zijrich. *technical university wroclaw, 50-370 wroclaw, poland. all four phenylalanine ammonia-lyase (ec 4.3.1.5) isoenzymes of parsley (petroselinum crispum nym.) were expressed as glutathione s-transferase fusion proteins in e. coil after affinity purification, the glutathione stransferase moieties were cleaved off with factor xa and the phenylalanine ammonia-lyases were characterized. the proteins form enzymatically active tetramers, even as fusion proteins. the four isoenzymes have comparable km values for l-phenylalanine (15gm to 24.5/.tm), similar temperature (58~ and ph optima (8.5). the aminooxy-and phosphonic-analogues of l-phenylalanine are competitive inhibitors of the enzymes. 2-aminoindan-2-phosphonic acid (j. zo{a & n. amrhein, liebigs ann. chem. 1992,625) was found to be a potent slow-binding inhibitor of these and other phenylalanine ammonnia-lyases, both in vivo and in vitro. chlorophyll a fluorescence has been used to compare the drought resistance of two varieties of solanum tuherosum (avr/i)c-12st-19 and sibtema). fast fluorescence rise kinetics were measured during the first second of illumination with a time resolution of l0 ms and 1z bits in fluorescence intensity. the rise kinetics shows the typical steps called fo -j -i -p. with this values different indexes have been defind with the goal to compare the behaviour of these two varieties of potato. salicylic acid (sa) was proposed as a putative signalling molecule for the induction of systemic acquired resistance (sar) in infected plants. we studied its biosynthesis as follows. cotyledons of cucumber plants were injected with a solution containing pseudomonas lachr~ans and fed with radioactive sa precursors by injection of 14c-benzoic acid (14c-ba) or 14cphenylalanine (14c-phe). alternatively, leaf 1 of cucumber plants were inoculated with tobacco necrosis virus (tnv) and leaf disks were then vacuum-infiltrated using 14c-ba or 14c-phe. free and bound 14c-phenolies were quantified by hplc. incorporation of 14c into sa was found in the two plant-pathogen systems. i~c-ba gave mostly 14c-sa and an unknown polar compound. 14c-phe gave also some 14c-sa, in addition to 14c-labelled lignin precursors like ferulic and p-coumaric acids. the biosynthetic pathway of sa from phe through cinnamic acid and ba will be discussed. after infection with a necrotizing pathogen, systemic acquired resistance (sar) is often observed in plants. in cucumber, salicylic acid (sa) increases in the lower infected leaf as well as in the upper uninfected leaves. sa was also found in the phloem sap of infected cucumber plants and was proposed as a signalling molecule for the induction of sar. we intend to clarify whether sa is translocated from the site of infection to the upper leaf, or wether it is made in the phloem tissue upon the action of a primary systemic signal. one cotyledon of cucumber plants was first infected with pseudomonas lachrymans and then fed with 14c-sa, 14c-benzoic acid (ba) or 14c-phenylalanine. after different times, cotyledons and first leaves were collected and the radioactive ~henolics were analyzed by hplc. in some cases, 4c-sa was found in the first leaf. in addition, two unknown radioactive compounds were detected in leaf 1 after treatment with 14c-sa and 14c-ba respectively. we will discuss signalling processes for the induction of sar in cucumber. one modern approach of creating virus resistant grapevine plants is the introduction of resistance genes into existing grapevine varieties by genetic engineering. the goal of this work is to use the coat protein (cp) mediated strategy to induce resistance to nepovirus in vitis spp. as a first step, several chimeric nepovirus cp genes, were constructed by addition of various promoter regions upstream of the gflv and armv cp regions. their ability of conferring resistance to nepovirus infection in transgenic nicotiana benthamiana and n. tabacum plants will be presented. the best constructions will be used to transform several grapevine varieties in order to create nepovirus resistant grapevine plants. $13-12 the bctalains axe a class of natural pigments found only in plants of the order caryophyllales and in some fungi. the first step in betalain biosynthesis is the conversion of tyrosine to dopa. subsequently , dopa is transformed to betalamic acid, the bctalain chromophorr through the action of dopa-4,5-dioxygenase. we have purified and characterised a copper-containing enzyme of -38kda from amanita muscaria pileus that catalyses the hydroxylation of tyrosine to dopa. considering that the enzymatic activity was restricted to the coloured parts of the mushroom, we postulate that it is involved in betalain biosynthesis. the enzyme featured a broad substrate specificity, and also oxidised the diphenols to their corresponding ortho-quinones, a reaction typical for tyrosinases. the implications of our findings on betalain biosynthesis axe discussed. chlorophyll a fluorescence was measured under steady state conditions of pea and tomato leaves adapted to low light intensity (30 wm-2s -i) at different temperatures (havaux and strasser, z. naturforsch. 45c, 1133 -1141 , 1990 ). when leaves were exposed for a short period (i0 min) to heat (25 to 45~ in darkness, the level of variable fluorescence decreased. when the heat stress was imposed in presence of low light, the variable fluorescence was much less affected and virtually no effect of heat treatment was observed until 37~ this protecting mechanism by low light was absent in algae and mostly absent in submerged water plants but fully present in free floating plants on the water surface. we conclude that a mechanism has been developed for higher land plants during evolution which protects the plants against heat damage on warmer days. caryophyllales, e.g. portulaca grandiflora, and in a few fungal species, e.g. amanita muscaria. we analysed the similarity of a key enzyme of the pathway, dopa-4,5-dioxygenase, in plants and fungi both at the protein and the dna level. antibodies against purified dopa-4,5-dioxygenase from the fungus a. muscaria crossreacted with protein from p. grandiflora petals and two cdna clones were obtained from this plant. their similarity with fungal sequences and with other dioxygenases is discussed. kinetics of prollne hydroxylauonj intrucellnlur transport and c-terminal processing of the tobacco vacuolar cbltlnase. ernst frcydl, thomas boiler and jean-marc neuhaus botenisehes instimt, abt. pflanzenphysiologie, hehoistxasse 1, ch-4056 basel the vacuolar chitinase a of tobacco (nicotlana tabacum) is syndietlzed as a preproprotein with ma n-teaminal signal peptide which causes its synthesis in the er mad a c-termlnal extension, which has been idcmified as the vacuolar targeting peptide (vtp) (1) and whleh is cleaved off from the maybe chitinase. it has recently been shown that mature chltin:~e a contains several hydi'oxyprolines within the short peptide spacer that links the n-terminal cysteine-rieh chitln-bindlng domain to the catalytic domain (2). we received specific antibodies against mature chitinasr (a kind gift from dr. f.meins. f/vii) and raised antihodiea against a synthetic vtp. we performed pulse-chase experiments and cell [ractlonation with 1) stably transformed tobacco plants expressing chitinase a or mutants lacking either the chitin-binding domain and spacer (chitah) or the vacuolar targeting poptid 9 (chitavtp) and 2) transient expression of the same conswacts in nicotiana plumbaginifolia protoplasts. in both systems, proline hydroxylatino in chltinase a was detectable after 30 vain. and complete after 90-120 rain. the ehltinase intermediate forms were detected in the mlcrosomal and soluble fractions for up to 90-120 rain. the mature chitinase continuously accumulated only in the soluble fraction. in both systems chitah showed the same kinetics of intraceilular transport and processing. whereas the transport of cbitavtp seemed more rapid.these results indicate that in vivo cbitinase a is synthetized as a proprotein that is than modified by proline hydroxylation. this second intermedinte form is then transported to the oolgi apparatus and sorted to the vacuole. c-terminal processing occurs late in this pathway or in the vacuole. infection of bean roots with the soil-borne phytopathogenic fungus fusarium solani f.sp. phaseoli leads to a rapid increase of chitinase activity (~five-fold). part of this increase in enzyme activity is due to transcriptional activation of the basic class i chitinase isoenzymes. semi-native polyacrylamide gels stained for chitinase activity using the substrate glycol-chitin revealed the appearence of three additional chitinase isoenzymes that are absent from uninfected control roots. conventional protein purification techniques showed that fusarium solani infected bean roots contain two basic and two acidic chitinase isoenzyrnes. their physiochemical properties and possible biological function will be discussed. proteasomes are multicatalytic protease complexes that function as a major nonlysosomal proteolytic system. they exhibit multiple endopeptidase activities that promote the intracellular turnover of abnormal polypeptides and short-lived regulatory proteins. moss proteasome has been purified from protonemata by successive chromatography on macroprep q, bio-gel a-1.5, bio-gel a-5 and ha. the molecular mass was estimated to be 1,300 kd by gel filtration. gel electrophoresis of proteasome under nondenaturing condition gave a single band and two-dimensional gel electrophoresis identified the moss proteasome to be composed of 14 different subunits with molecular weight in the range 22-35 kd. electron microscopy in aqueous solution showed that it is a cylindrical particle composed of four stacked rings with a diameter of 9 nm and a height of 14 rim. end-on projection established a 7-fold symmetry of the outer disks. substrate specificity of proteasome indicates that it contains endopeptidase activity against substrates bearing hydrophobic, basic, acidic and glycine residues immediately preceding the cleavage site. antibodies raised against moss proteasome reacted with proteasomes of other eukaryotes, including human. $14-01 hunger r.e., hess m.w., laissue j,a,, mueller c. the nod (non obese diabetic) mouse, a widely used animal model for insulin dependent diabetes mellitus, shows in addition to mononuclear cell infiltration of the islets of langerhans, massive cellular infiltrates of the submandibular and lacrimal glands with considerable tissue damage. several lines of evidence suggest that both insulitis and sialadenitis represent autoimmune disorders, to investigate a possible role of tumor necrosis faetor-a (tnf-c0 and activated cytotoxic cells (nk cells, t cells) in the inflammatory process, tissue sections of nod mice were hybridized with radiolabeled rna probes specific for the detection of mrna for tnf-r and the serine proteases granzyme a and b (gra and gr8) and perforin which are expressed in activated cytotoxic cells. tnf-e expressing cells were mainly located in infiltrates and are absent in non affected glands, whereas gra, grb and perforin expressing cells were distributed over the whole section with highest densities in the zones adjacent to parenchyma. the finding that activated cytotoxic cells are present in early stage of disease development indicates that cell mediated cytotoxicity may play a crucial role in initial tissue destruction. the role of tnf-a in autoimmune diseases is not known yet. the appearance, however, of cells expressing this gene in the infiltrate indicates a possible role of this cytokine in the progression of the inflammatory reaction, e.g. by increasing the lymphocyte traffic to this organ. we further demonstrated that the induction of inos was at the transcriptional level. in order to understand the underlying mechanisms we characterized the promoter region of the rat nos gene. a 9enomic rat library was screened using a cdna-fragment coding for the if-end of the inos cdna (nucleotides 1-817). a 1,8 kb hinc-ii fragment containing the 5"-flanking region of the inos gene was characterized by dna-sequencing. the transcriptional start site was determined by primer extension. sequencing analysis revealed a multitude of possible cis-acting elements homologues to consensus sequences for the binding of different transcription factors including ifn~' response element (ire), nuclear factor-kb (nf-~b), tumor necrosis factor response element (tnf-re), 7f-activated sites (gas) and one x-box. nf-kb, a transcription factor involved in signaling and immediate early gene activation during inflammatory processes was induced by treatment of mesangial cells with 2 nm of il-113. this was shown by the appearence in nuclear extracts of the dna binding activity of nf-~b using electrophoretic mobility shift assays (emsas) with a 32p-labeled ~r dna probe. pyrrolidinedithiocarbamate (pdtq), an inhibitor of nf-kb, suppressed the expression of inos mrna in mesangial cells without affecting the dibutyryl-camp-triggered increases in nos mrna levels. this data suggest that the il-i ~ signalling pathway responsible for nos expression requires nf-k8 activation and is definetly different from the signalling cascade directed by camp. recent findings indicate that the multifunctional cytokine il-6, which plays a key role in irm-nune and inflammatory responses, also exerts specific effects in the central nervous system (cns). for example, il-6 promotes neuronal survival, induces differentiation and modulates neurotrophin production. using the very sensitive technique of reverse transcription combined with polymerase chain reaction the developmental profile of il-6 and its receptor mrnas was analyzed in various rat brain regions. our results indicate that both genes are expressed in a region-specific manner and are developmentally regulated. these findings support the hypothesis that il-6 is involved in differentiation and maintenance of neuronal subpopulation in the cns. interleuldn 2 (il-2) expression is strictly dependent on a signal transmitted by the t cell receptor upon antigen stimulation. this signal can be mimicked in vitro with phorbol esters and calcium ionophores. we have found that stimulation with ca-ionophore alone induces expression of il-2 mrna, but does not induce secretion of il-2. in polysome gradient fractionation of cells stimulated with phorbol esters and ionophore, the fractions containing il-2 rrtrna are found at the bottom of the gradient, bound to polysomes. however, in ionophorestimulated cells the fractions containing il-2 mrna are clearly shifted to the top of the gradient, and therefore are not lzanslated. this effect is specific for il-2, as other mrna's like 1~2-microglobulin are not regulated. this data suggests that il-2 gene is translationally regulated. in addition, in vitro experiments have shown that the lack of translation of il-2 mrna in ionophore-stimulated cells is due, most likely, to the presence of a translational regulator that specifically inhibits translation of il-2 mrna. assuming the presence of a translational regulator that specifically represses il-2 mrna translation, we can predict that, if the represser is in excess, even upon a second stimulus that is by itself able to induce secretion of il-2, there will be no translation of il-2 mrna. the resuhs of such an experiment confirmed our prediction. polysome gradients showed that after ionophore stimulation, il-2 mrna gets "stacked" with one ribosome, and no further ribosome loading takes place. if malaria is highly endemic, every febrile child is a presumptive malaria case, and there is no differential diagnosis based on clinical, immunological or parasitological grounds. we evaluated the diagnostic usefulness of interleukin-2 receptor (il-2r), tumor necrosis factor-receptors 55 (tnf-r55) and 75 (tnf-r75) . sera came from tanzanian pediatric fever patients and controls, all enrolled in the kilombero malaria project. we found that all 3 receptors marked pediatric fever episodes, whereas stability of observed levels was highest for tnf-r75 and lowest for tnf-r55. tnf-r55 levels reflected severity of the fever attack. regardless of the presence of a febrile illness, tnf-r75 levels were strongly associated with parasite density. immunological markers can be applied in rapid pre-and post-intervention morbidity assessments, validation of health interviews and monitoring of convalescence. we have recently shown that human eosinophils produce mrna for interlenkin (il)-8 when stimulated with calcium ionophom (eur. j. immunol. 23 (1993), 956). we now present evidence that human eosinophils contain preformed il-8 protein although they do not express mrna for il-8 as measured by rt-pcr. il-8 protein expression was determined using specific anti-human il-8 monoclonal antibodies by western blotting, facs analysis and immunohistochemistry. moreover, preformed il-8 was released into supernatant by 99% pure eosinophils after priming with gm-csf or il-5 and subsequent 25-min stimulation with paf, rantes, ionomycin or pma, however not with il-1 or il-2, as measured by elisa. this observation implies that activation of protein kinase c and/or inwacellular calcium mobilization am necessary events for il-8 release in human eosinophils. the determined amounts of preformed il-8 protein was higher in patients with asthma compared to normal individuals suggesting a role for eosinophil derived il-8 in asthma. since the eosinophil is the predominant cell in the asthmatic airways, we determined 1l-8 concentrations in bronchoalveolar lavage (bal) fluids from normal individuals and asthmatic patients. indeed, il-8 concentrations in bals from both groups were similar to those seen in the supematants released by activated eosinophils. the role for 1i-,-8 in asthma remains to be determined. to study the effect of canthaxanthin in vitro, the hydrophobic compound was introduced in vivo into chick high density lipoproteins (hdl) by canthaxanthin feeding. the effects of hdl and hdl associated canthaxanthin on two types of cultures have been tested: flat sedimented cell cultures of embryonic chick neuronal retina, retinal figment epithelium (rpe), brain and meninges, and in reaggregate cell cultures of the neuronal retina. at high canthaxanthin concentrations the formation of colored, birefringent entities were induced in the neuronal retina in vitro. in line with human data, parameters of cellular function and cell differentiation remained unaffected by canthaxanthin at these concentrations. by contrast, fidl was found to induce various cell type dependent effects. proliferation of meninges cells was decreased at low hdl concentrations as concluded from.light microscopic examination and indicated by protein content, and lysosomal and mitochondrial activity of the cultures (ic50:15-30 mg hdl apoprotein/l medium). these parameters, however, were increased in brain cell cultures, while they remained unaffected in cell cultures of neuronal retina and rpe. concentrations above 0.3 g. hdl apoprotein/l caused a reduction of glial cell differentiation. $14-08 tnf-c~ had close-dependent antiproliferative activity on hormone-dependent human breast cancer cells that represent an early stage of the disease, whereas hormone-independent cells representing a late stage were not affected. however, in the presence of 1000 u/ml interferon ?(inf), high concentrations of tnf-c((lnm) were able to inhibit the growth of hormone-independent cells. using specific monoclonal antibodies in ftow cytometry, no significant changes of either the p75 or the p55 tnf receptors were observed upon inf treatment of hormone-independent cells. this indicates that the increased responsiveness of these cells for tnf-c~ is not due to a change in available tnf receptors. the affinity of the receptors for tnf-r are one order of magnitude different for the two cell types. inf treatment shifted the ecs0 of hormone-independent cells towards the ecs0 of hormone-dependent cells. thus, the basis for the increased tnf-sensitivity of hormone-independent cells in the presence of inf might be the interconversion of tnf receptors from low-to high-affinity. the adhesion of tumor cells to endothelium is the first step in the processus of metastasis, and is frequently dependent of cytokinemediated activation of endothelial cells. cytokines are also involved in nitric oxide (no) production by various cells. these experiments were designed to evaluate no production during tumor cell adhesion to endothelium. rat brain-derived endothelial and rat colon carcinoma cell lines were used during these experiments. no was evaluated with the griess reagent. activation of endothelial cells with cytokines (tnf-d, and ifn-~ ), only slightly increased (10-20 %) the adhesion of tumors cells to endothelium. activation of endothelial cells with tnf-ol and ifn-~, induced a 4-8 fold increase of no production. however, addition of tumor cells to the endothelial monolayer, either directly or in a semi-permeable membrane, decreased the cytokine-induced no production. these results indicate that no production is modulated during the proeessus of adhesion of tumor cells to endothelium. is induced in rat mesangial cells by inflammatory cytokines such as interleukin 1 ]3 (il-1 ~) and requires bh 4 as a cotactor. 2,4-diamino-6-hydroxypyrimidine (dahp), a selective inhibitor of gtp cyclohydrolase i, the rate-limiting enzyme for bh 4 synthesis, potently suppressed il-i~induced nos activity, measured as nitrite production. inhibition of no synthesis by dahp was reversed by sepiapterin, which provides bh 4 via a salvage pathway. sepiapterin dose-dependently augmented il-1 i],-stimulated no synthesis, indicating that availability of bh 4 limits the production of no in cytokine-stimulated mesangial cells. n-acetylserotonin, an inhibitor of the bh 4 synthetic enzyme sepiapterin reductase, completely abolished il-lj~-induced no formation whereas methotrexate, which inhibits the pterin salvage pathway, displayed only a moderate inhibitory effect, thus suggesting that mesangial cells predominantly synthesize bh 4 tom gtp. in conclusion, these data demonstrate that bn 4 synthesis is an absolute requirement for cytokine induction of nos in mesangial cells. inhibition of bh 4 synthesis may provide new therapeutic approaches to the treatment of pathological conditions mediated by no. -1 [3) can induce a macrophage-type of nitric oxide synthase (inos). northern-blot analyses of inos mrna-levels and nuclear run-on experiments of control and il-1 ~ stimulated mesangial ceils revealed that the induction of inos was at the transcripuonal level. here we demonstrate that platelet-derived growth factor bb (pdgf-bb) can suppress the il-113 dependent inducuon of the inos gene. nortllern-blot analyses of cellular rna isolated from mesangial ceils that were coincubated with il-1 [3 (2nm) and pdgf-bb (10 ng/ml, 100 ng/ml and 300 ng/ml) revealed a dosedependent suppression of inos mrna-levels. using run-on experiments with nuclei from mesangial ceils after stimulation with il-i~ (2nm) and pdgf-bb (100 ng/ml) we demonstrate that the inhibition occurs at the transcriptional level. basic fibroblast growth factor (bfgf), on the other hand, potentiates the il-1 dependent stimulation of inos. coincubation of mesangial cells with il-113 (2nm) and bfgf (3ng/m[, 10ng/m[, 30ng/m[, 100ng/m0 dose-dependently superinduces inos mrna levels as shown by northern-blot analyses of total cellular rna form control and stimulated cells. run-on experiments with nuclei from mesangial cells stimulated with il-113 (2nm) and bfge (100ng/ml) revealed an enhanced transcriptional activity of the inos gene. thus, pdgf-bb and bfgf differentaity modulate the il-i~ dependent expression of the inos gene and are therefore an excellent model system to study the crosstalk between signal transductjon pathways. we report that ifnyr-/-mice have an increased resistance to lipopolysaccharide-induced toxicity (lps). lps-induced lymphopenia, thrombocytopenia and weight loss seen in wild type mice were attenuated in ifntr-/-mice. ifntr-/mice survived in the d-galactosamine-lps model when conditions were 100% lethal for wild type mice, correlating with serum tnf levels of up to 10 fold higher in wild type mice. bone marrow and splenic macrophages from ifnyr-/-mice had a 3 to 5 fold decreased lpsbinding capacity, with serum from these mice lowering macrophage lpsbinding by a further 50%. thus, depressed tnf synthesis, diminished expression of lps receptors and low plasma lps-binding capacity in the mutant mice likely combine to manifest in the resistant phenotype of ifny r-/mice to endotoxin. in a complementary approach we have screened the 5' portion (-7.8 kb/+4.1 kb) of the gene for tissue specific and/or inducible dnasei hypersensitive sites. in chromatin from fibroblasts or kidney epithelial cells this segment of the il2ra gene does not contain any dnasei hypersensitive sites. in contrast, in resting t-and b-lymphocytes, as well as in activated t cells, two sites (dhi, -0.05 kb; dh3, -5.8 kb) were found. a third site (dh2, -1.35 kb) is detectable in t cells that have been activated with concanavalin a and il2. this site maps to the same position as cisacting regulatory sequences that are required for the response of the il2ra gene to signals from the il2 receptor. interleukin-6 (il-6) production in cultured human dermal fibroblasts can be stimulated by interleukin-i (il-i} added to the culture medium. absence of fetal calf serum and of growth factors (insulin, egf, t3) reduced the stimulation by more than 50 %. egf and t3 and even more choleratoxin increased the il-6 production in absence of fetal calf serum. the effect was dose dependent. 2% human ab serum substituted for i0 % fetal calf serum. pretreatment of the cultures with serum or growth factors prior to stimulation with il 1 had a stimulatory effect (serum, t3, egf) or an inhibitory effect (hydrocortisone, choleratoxin). our results suggest that the il-i signal transduction to il-6 is modulated by serum components and growth factors as well as through c-amp produced by choleratoxin. interleukin-6 (il-6) is produced by a variety of cells and plays a central role in host defense mechanisms. its function include induction of il-2 and il-2 receptor expression, proliferation and differentiation in t-cells. in cocultures of human dermal fibroblasts and allogenic human t-cells a cell number dependent 10 to 100 fold increase of the il-6 production could be demonstrated after 24h and 48h. conditioned medium from tcells and from fibroblasts failed to induce il-6 secretion in fibroblast and t-cell cultures, respectively. no up-regulation of il-6 production was found in cocultures of fibroblasts with tcells killed by ethanol and in t-cell culture incubated with recombinant hll-lc~. after separation of the cells il-6 production in fibroblast and t-cells returned to precoculture levels. our results indicate that cell-cell contact more than soluble factors must be involved in the up-regulation of il-6 synthesis in cocultures of t-cells and fibroblast. using cocultures of autologes human t-cells and fibroblasts, we are currently investigating whether the cell contact essential for the il-6 production depend on immunolcical interactions of t-cells and fibroblasts. rapid growth of tumor often results in oxygen and nutritional deprivation leading to extensive necrosis, which is one of the characteristics of glinblastomas (gbl). neoplastic cells surrounding necrotic areas often present a peculiar arrangement of cells called "pseudo-palisading" -as a hallmark of this entity, together with abundant neovascularization. a special biological milieu is probably formed in these palisading areas by certain unknown cytokines and/or growth factors induced by the necrotic process. plate et al. (1992) reported the expression of vascular endothelial growth factor (vegf) in the palisading cells. we previously demonstrated that gbl cells produce il-8, which is known to be an angiogenic factor. the aim of this study is first to assess if il-8 is expressed in the palisading ceils, and second to determine if hypoxia can trigger il-8 gene expression. in rive observation using in siru hybridization and immunohistuchemistry showed that il-8 is highly expressed specifically in the palisading cells. we also demonstrated the mrna expression of il-8 receptor in the endothelial cells adjacent to necrotic area. to simulate the in vivo condition, two in-vitro models are currently developed to determine if il-8 is induced by hypoxic insult on cells. first, gbl cell lines are cultured in the absence of oxygen and il-8 expression is assessed by northern blot analysis. preliminary results have demonstrated the induction of il-8 by hypoxia. secondly gbl cells are grown in spheroids until development of central necrosis. the il-8 expression will be assessed by in situ hybridization combined with immunohistochemistry to determine if the ceils surrounding necrosis express il-8. it has been proposed by taniguchi and his colleagues that activation of the interferon (ifn)-i3 gene by virus or double-stranded rna involves induction and modification of irf-1. overexpression of irf-1 can induce expression of the ifn-b gene in certain cells. a role of irf-1 in the activation of ifn-a genes has also been proposed. furthermore, irf-1 has been claimed to play the role of a tumor suppressor gene. we have generated mice in which both irf-1 alleles were disrupted and found them to develop normally. no spontaneous tumors have been observed so far. injection of poly(i)-poly(c) resulted in the same levels oflfn in blood of wildtype and null mice, and equal ifn-ct mrna levels in spleen. induction of wild type and irf ~ embryo fibroblasts (mefs) with virus resulted in the same levels of ifn-a and ifn-i] mrna respectively. in the case of polyo)-poly(c) induction, the levels of both 1fn mrnas were higher in wild type than in mutant cells; this difference was abolished if the cells were first primed with type i ifn. we conclude that irf-1 does not play an essential role in the induction oflfn genes. mitsuhiro tada, annie-claire diserens, isabelle desbaillets, marie-france hamon, nicolas de tribolet, erwin van meir; chuv, lausanne there is increasing evidence that a variety of cytokines produced by glioblastoma (gbl) cells modulate the tumor growth by affecting the host's immune response and by stimulating neovascularization. cytokines such as il-i, il-6, il-8, mcp-i, il-10 and tgf-132, affect each other's production and receptor system and seem to form a cytokine network. among them, il-1 may play a pivotal role, since il-i can induce or increase the production of other cytokines (il-6,il-8,mcp-i,tgf-132) and modulate their receptor expression. to test this hypothesis, we investigated co-expression of cytokines and their receptors in 15 gbl cell lines and 15 gbl tissues using rt-pcr, northern blot analysis, immunnhistnchemistry and elisa quantification. a majority of the gbl cell lines and gbl tissues expressed il-i~, il-113, type i and type ii receptors, indicating the presence of an il-1 autocrine loop. il-1 stimulated growth of some of the cell lines. a positive correlation of il-6 and il-8 expressions with the presence of an il-i autocrine loop in the cell lines was found. immunohistochemical studies showed the in rive co-expression of the il-i family and the secondary cytokines (il-6, il-8) in gbls. antisense oligonucleotide to il-lc~ and/or il-113 partly suppressed this secondary cytokine expression. these results demonstrate that the il-1 autocrine loop plays a central role in the formation of the cytokine network in gbls. double-stranded rna-dependent protein kinase (pkr) is induced by type i interferon (ifn) and is implicated in the establishment of the antiviral state. upon activation, pkr phosphorylates the eukaryotic initiation factor eif-2, thereby throttling protein synthesis. it was suggested that pkr may be essential for the induction of ifn-13 gene expression by both virus and double-stranded rna and for the activation of at least some ifninducible genes. recently it was proposed that pkr is a tumor suppressor gene because 3t3 ceils overexpressing inactive human pkr gave rise to tumors in nude mice (koromilas et al., 1992; meurs et al., 1993) . we have produced homozygous pkr knockout (pkr ~176 mice and found that they develop normally and have no striking phenotype. induction of ifn-c~ and ifn-13 gene transcription by virus was unimpaired in fibroblasts derived from pkr ~ mice, as was the induction of several ifn-stimulated genes. so far, no spontaneous tumor formation was observed. pkr o/o embryonal stem ceils showed no increased malignancy in nude mice as compared to their wild type counterparts. we conclude that pkr does not play an essential role in viral induction of the ifn genes and that its tumor suppressing effect, if any, is redundant. tgf-13 plays an important role as a negative regulator of hepatocyte proliferation in liver regeneration. exposure of rodents to nongenotoxic carcinogens like cyproterone acetate (cpa), thioacetamide (ta) and phenobarbital (pb) causes abnormal hepatocyte proliferation and liver tumors after long term treatment. this suggests that these chemicals have either a direct mitotic activity or impair the negative regulatory system for growth. therefore the inhibitory activity of tgf-6 on dna-synthesis induced whith cpa was investigated in cultured rat hepatocytes. after a 48h exposure to cpa, a dose dependent increase in dna synthesis (3h-tdr incorporation) was observed. the maximum effect was attained with 12 pm cpa (up to 4.3 fold) which was stronger than with 10 ng/ml egf (up to 2.8 fold). tgf-81 and tgf-132 inhibited this response dose dependently (idso = 0.1 ng/ml) and reduced it to control levels at 1 ng/ml. these findings show that the tgf-6 mediated pathway for negative growth control in hepatocytes is still responding after cpa treatment. mouse mxl protein is an interferon (ifn) induced gtpase with intrinsic antiviral activity against influenza a viruses. it accumulates in the cell nucleus and inhibits the transcription of influenza virus genes, recently, we have shown that mxl exerts its inhibitory activity by interfering with the function of the viral polymerase subunit p82. pb2 is responsible for recruiting 5" ends of cellular mrnas as primers and for the elongation of nascent viral transcripts. to elucidate which pb2 dependent step of viral mrna synthesis is the target of mxl action, we examined the activity of recombinant mxl protein in an in vitro transcription system of influenza virus. first, we expressed wildtype and mutant mxl proteins, carrying a hlstidinetag at their n-terminus, in e. coil and purified them under nondenaturing conditions by ni-chelate affinity chromatography. mxl efficiently inhibited viral transcription in vitro, while mutant mxl proteins, lacking antiviral activity in vivo ,were inactive. moreover, the use of capped synthetic rna primers revealed, that mxl almost completely abrogated the elongation of viral transcripts, whereas the the cap-binding and endonuclease activity of pb2 was not affected. a50 $14-28 neutralization rates of tnf-a from eight animal species by a monoclonal antibody against human tnf: implication for receptor action? we have recently developed a sensitive bioassay for measuring porcine tnf-a based on homologous pk(15) cells. this bioassay is 100-to 1000-fold more sensitive than the widely used l929 bioassay, also, human tnf-a and human tnf-8 were detected with a slightly higher sensitivity in the new test compared to the l929 bioassay, we now show that also canine, ovine, bovine, equine and caprine tnf-a can be detected with the pk(15) bioassay. we tested a murine monoclonal anti-human tnf-a antibody for its capacity to neutralize tnf-a from eight different animal species. the action of this antibody revealed that neutralization of tnf bioactivity is a complex phenomenon, indicating species-specific differences in the interaction with tnf receptors. to study this differential neutralization, we are cloning the porcine tnf receptors for expression in a heterologous cell system. administration.of high dose r-tnf(~ in combination with ifn 7 in isolation and perfusion of the limbs in melanoma patients has shown to be very promising with a response rate greater that 80%. this observation prompted us to investigate the possible expression of the tnf receptors in melanoma cells using the monoclonal antibodies utr-1 specific for the tnf type a (55 kda) receptor and htr-9 specific for the tnf type b (75 kda) receptor. flow cytometric analysis of cultured melanoma cells showed the presence at low level of the tnf type a and to a slightly higher level of the type b receptor. similar results were obtained in vivo by immunohistochemistry on fresh tumor raateriai, treatmem of melanoma cells in culture with the-amp induced up to a 2 fold increase in the number of type a tnf receptors with only a minimal change in the number of type b receptors. this increased expression of tnf receptors is conflrmed by direct binding experiments using 125i-labeled tnftx. the number of cpm's bound on dbc amp treated cells was about 0.5-3 fold higher than on untreated control calls, likewise incubation of melanoma cell lines with ifny increased the specific binding of subsequently added 125i-labeled tnfct. from flow cytometric analysis using the two anti-tnf receptor antibodies it became evident that flint was able to increase the expression of both type a and b receptors depending on the cell line used. characterization of a murine tumor necrosis factor (x-lacz reporter construct tumor necrosis factor ot (tnfcq is a prominent mediator of a variety of different pathologies such as endotoxic shock syndrom and rheumatoid arthritis. it also plays a crucial role in host defence against bacterial and viral infection. up to now, only few data about signal pathways leading to tnfo~ induction are available. an easy to handle tnfc~ -lacz reporter assay will represent a useful tool to study signal transduction on the one hand and provide data about compounds with potential tnfo~ inducible activity on the other hand. in order to develop a routine macrophage cell line capable to easily report tnfc~ induction, different tnfe-lacz reporter constructs were assembled. data about functionality of the different constructs in routine macrophages will be presented in the context of tnfot expression. cloning of a novel protein binding to lymphokine, fos and myc mrna with unexpected enzyme activity j. nakagawa, h-p. waldner, s. meyer-monard, and ch. moroni inst. medizinische mikrobiolegie, univ. basel an au-rich sequence in the 3' untranslated region of lymphokines, c-los, and c-myc proto-oncogene mrna is responsible for their rapid decay. we have purified a 32 kd protein by an auuua affinity column from human brain. partial amino acid sequence was obtained and the corresponding edna has been cloned. unexpectedly the edna sequence exhibited significant homology to enoyl coa hydratase and bacterially expressed recombinant protein indeed showed the activity. while rat hydratase did not show rna binding activity, the recombinant protein bound to cfos, c-myc, auuua cluster of il-3 mrna, and adeno virus iv, but not to mutated ad-iv, nor irrelvant transcript. the binding was competed out by poly u. interestingly the protein seems to be processed from a larger precursor. we suspect that the protein plays a role in mrna turnover and perhaps in oncogenesis. institute for medical microbiology, university of basel in t cells, the immunsuppressive agent cea is known to inhibit ca 2+ dependent induction of lymphokine mrna transcription. in contrast, the immunsuppressive drug rapamycin inhibits the lymphokine induced proliferation. we have analysed the effect of these drugs on a v-h-ras induced autocrine mast cell tumor line which expresses il-3 constitutively. csa and rapa inhibited autocrine growth by different mechanisms,, because added il-3 could antagonize inhibition by csa, but not by rapa. whereas csa acted by downregulation of il-3 mrna expression, rapa blocked il-3 induced proliferation. cea also inhibited il-3 superinduction by ca-ionophore, whereas rapa did not. nuclear run on assay indicated that the mechanism is posttranscriptional. therefore, we have introduced il-3 transgenes with and without the au-rich region in the 3' utr of the mrna into a tumor line. in contrast to the wild type, the expression of the mutant construct was insensitive to csa. since the mutant lacks sequences known to regulate rapid mrna decay, we suggest that csa affects the regulation of il-3 mrna stability. some non-hematopoietic cell lines produce both scf and its receptor, the c-kit protein (c-kit). testing the hypothesis that scf may be involved in secondary growth of nb bone marrow metastasis, we found and previously communicated (beck d. et al, proc aacr, 34, 52, 1993) a low or absent expression of c-kit in nb cells. the mrna and surface expressions of scf gene in nb cell lines grown in chemically-defined medium were evaluated by northern blot analysis and flow cytometry. the scf antigenic concentrations in culture supernatants were determined by elisa and growth-inhibition experiments performed by pre-incubating nb cells with anti-scf antibodies prior to 3h-thymldine uptake assay. the scf mrna was expressed in 4 of 5 tested lines but no membrane-bound antigen could be detected on those cells. detectable levels of scf in the supernatants were found in 5/7 lines (range : 39-96 pg/ml). blocking experiments resulted in a decrease of dna synthesis in i out of 7 lines only. from these and previous results, we conclude that scf is synthesized and released by many nb cells in vitro but the functional ~ole of it remains undetermined since scf does not appear to be usually involved in autocrine or paracrine growth stimulation of nb cells (supported by swiss fnrs grant 3200-031005). expression of the v-h-ras oncogene in the il-3 dependent pb-3c mast cells leads to the generation of two classes of il-3 autocrine tumors in vivo. autocrine il-3 expression in class-1 tumors results from increased il-3 mrna stability due to the loss of negative trans-acting posttranscriptional control. this defect can be corrected by somatic cell fusion to the nontumorigenic parental pb-3c resulting in downregulation of oncogenic il-3 expression and concomitant tumor suppression. expression of the v-h-ras oncogene remains unaffected in class-1 hybrids. in contrast, class-2 tumors are characterized by transcriptional activation of the il-3 gene due to the insertion of an endogenous retroviral element (intracisternal a-particle) which cannot be overcome by ceil fusion (see hirsch et al, 1993, j.exp.medicine 178, 403-411) . although v-h-ras is required for generation of either class of tumors, expression of anti-sense ras constructs inhibited only the proliferation of class-i tumor cells. experiments are underway to characterize the target and the nature of the class-1 alteration. nitric oxide (no) promotes vasorelaxatlon of renal vessels in vitro. the purpose of the present study was to assess the role of no in regulating renal hemodynamics in vivo. renal blood flow (rbf) and glomerular filtration rate (gfr) were studied in anesthetized mechanicallyventilated newborn rabbits both before and after the administration of a no synthesis inhibitor, ng-nitro-larginine methyl ester (l-name), at a dose of 50 pg/kg followed by 10 jzg/kg x rain. such a dose of l-name did not modify mean arterial pressure or pulse rate. by contrast, the renal vascular resistance increased by 31 + 9% (p < 0.005) while rbf decreased by 20-+6% (p < 0.02). gfr and urine flow rate remained constant. the overall results suggest that in normal conditions no may play a role in decreasing the renal vascular resistance of the immature kidney, without altering systemic blood pressure. interleukin-4 (il-4) is a t-cell derived lymphokine which, in addition to its effects on the immune system, is also able to suppress the growth of certain tumor cells. il-4 reduced the growth rate of four human colon tumor cell lines up to 70% in a dose-dependent manner. three other cell lines showed no significant alteration of 3h-thymidine incorporation or colony formation in the presence of 100 u/ml il-4. responder and nonresponder tumors were immunopositive for il-4 receptor (il-4r) expression in flowcytometric analysis, using monoclonal antibodies raised against recombinant il-4r and bound biotinyated il-4. responsive tumors expressed more receptors. 11-4 binds with high affinity to a 130 kda transmembrane receptor (il-4r), through which the extracellular signal has been shown to be transduced. coimmunoprecipitation and chemical crosslinking studies have enabled us to demonstrate il-4r target proteins. tyrosine phosphorylation of various proteins between 70 and 170 kda appears to be initiated during il-4mediated signaling events in colon tumor cells. the cloning of human il-4r target proteins will contribute to the understanding of the il-4 signal transduction pathway at the initial stage. the renal effects of endothelin-1 were investigated in 16 anesthetized and meehanleally-ventilated newborn rabbits. each animal acted as its own control. in 8 newborn rabbits, a bolus injection of 5 nmol.kg "1 of endothelin-1 caused an initial fall in mean blood pressure (mbp) followed by a gradual but significant increase in mbp that lasted for 45 rain. the dramatic increase in renal vascular resistance (+ 28 -+ 4%) induced by endothelin led to a fall in glomerular filtration rate (-12 -+ 4%) and renal blood flow (-16 _+ 3%). in spite of the reduction of gfr and rbf, urine flow and sodium excretion rates increased significantly (+ 20 _+ 5% and + 49 _+ 9%, respectively). in 8 additional newborn rabbits, a bolus injection of lnmol.kgaof endothelin-1 -a dose that usually induces marked renal and systemic vasoconstriction in adult models -did not affect systemic or renal hemodynamics. in conclusion, endothelin induces renal and systemic vasoconstriction, and affects water and sodium homeostasis during the neonatal period. however, these effects occur under higher doses than those used in adult animals, possibly reflecting receptor immaturity and/or interference of high levels of counteracting hormones. ontogeny of the endocrine pancreas in xenopus laevis c, maake 1 , w. hanke 2 and m. reine~ke 1 , 1 institute of anatomy, university of z0rich, switzerland and department of zoology ii, university of kaflsruhe, f.r.g. the pancreatic islets of anurans show insulin (ins), glucagon (gluc), somatostatin (som) and pancreatic potypeptide (pp) containing cells. since only limited information exists on the ontogeny and on the presence of insulin-like growth factor 1 (igf-1), we studied the development of the gastro-entero-pancreatic (gep) system in xenopus laevis using immunohistochemical techniques. singular ins-immunoreactive (-ir) cells were observed in the pancreas as early as on stage 41. at stage 43, the first pp-ir cells were found in the gep system followed by som-ir and gluc-ir cells. the first pancreatic islets consisting of ins-ir and gluc-ir cells occurred around stage 50. between stage 54 and 60, i.e. after the onset of metamorphosis, the endocrine cells decreased in number and the islets partly disintegrated. starting with stage 61, the islets reformed and the amount of endocrine cells increased reaching the adult status at stage 63. around stage 52, igf-l-immunoreactions appeared. igf-1immunoreactivity was found in pp-ir and gluc-ir cells but not in ins-ir and som-ir cells. it is assumed that islet-derived igf-1 may p~ay an important role during metamorphosis in xenopus. t. cathomen and r. cattaneo, institut for molekularbiologie i, universit~t zorich, hfnggerberg, ch-8093 zorich the signals used for synthesis of the measles virus fusion (f) protein are poorly understood: a long (>570 bases) untranslated region is followed by three in frame augs at codons 1,4 and 15. f is a type i transmembrane protein with a postulated signal sequence of 31 amino acids. we confirmed that the 46 aminoterminal residues contain a signal peptide by transfering them to another protein. with the other envelope protein hemagglutinin, f protein induces virus-cell and cell-cell fusion. mutagenesis of the three augs indicates that proteins initiated at codon 1, 4 or 15 are all functional, but show slightly different fusion efficiencies. forms translated from aug 1 or 4 appear larger in electrophoresis than forms initiated at aug 15, suggesting that two different signal peptide cleavage sites are used. we are currently investigating whether both f protein forms are incorporated in viral particles. the production of protein isoforms with staggered amino-termini is common in cytoplasmic and type ii transmembrane proteins, but signal sequences usually preclude a similar organization of type i transmembrane proteins. beguin p., beggah a.t., rossier b.c., jaisser f. and geering k. institut de pharmacologie et de toxicologie de l'universit6, ch-1005 lausanne recent experimental evidence suggests that na,k-atpase might be a candidate for regulatory phosphorylation by protein kinases a and c (pka and pkc). to verify this hypothesis, we have mutated several serine and threonine residues in consensus phosphorylation sequences of the ~subunit of bufo marinus na, k-atpase. the mutants were expressed in x~nopus ooeytes and phosphorylation was studied in oocyte homogenates upon stimulation of oocyte, pka and pkc. our results indicate that a unique phosphorylation site for pka is located at serine 943 in the c-terminus of the ~-subunit but its phosphorylation can only be revealed in the presence of detergent. on the other hand, mutations of several serine and/or threonine residues located in consensus sequences for pkc phosphorylation did not or only partially abollsh phosphorylation by pkc. in particular, mutations close to the catalytic phosphorylation site of the ~-subunit influenced phosphorylation by pkc, suggesting that either this region contains a real phosphorylation site or is part of a conformational domain necessary for phosphorylation by pkc. the heat-stable antigen (hsa or mouse cd24) gene is differentially regulated but has a housekeeping promoter. roland h. wenger*, georges kshler and peter j. nielsen. max planck institut f(jr immunbiologie, st0beweg 51, d-79108 freiburg i. bsg. "present address: physiologisches institut der universit&t zi.)rich, winterthurerstrasse 190, ch-8057 z0rich, expression of the gpi-anchored murine glycoprotein heat-stable antigen (hsa) shows tissue-specific as well as developmental regulation. during the maturation of several hematopoietic lineages, hsa expression is generally high in immature precursor ceils and low or absent in terminally differentiated cells. we present evidence suggesting that this regulation of the hsa gene (cd24a) occurs at the transcriptional level. in addition, sequence and methylation analysis of the cd24a promoter revealed characteristics of both "housekeeping" and tissue-specific promoters, including a methylation-free, hpall tiny fragment (htf) island, multiple putative sp1 and ap-2 consensus binding sites and a tata box. functional analysis of a 0.6 kb dna fragment containing these elements fused to the cat reporter gone in transient transfection experiments showed activity in both hsa expressing and non-expressing cell lines with a strength similar to that of the hsv tk promoter. large fragments from the flanking region of the cd24a promoter did not influence the ubiquitous nature of this promoter, finally, the cd24a, cd24b and cd24c genes were mapped to mouse chromosomes 10, 8 and 14 respectively. we have cloned, sequenced and characterized the gone for the m__itochondriat nadh-cytochrome b5 reductase (mcri) of saccharomyces cerevisiae. surprisingly, this gone encodes two mitochondrial isoforms of the flavoenzyme: a 34 kda integral protein exposed on the outer surface of outer mitochondrial membrane (mcrlp34), and a 32 kda soluble protein of the intermembrane space (mcrlp32). the smaller intermembrane space isoform is generated from the larger form by the action of the inner membrane protease i (impi) on the outer surface of the inner membrane. this is the first demonstration that a single gone encodes proteins which are located in different compartments of the same organelle. with special interest in any with a mitochondrial localization. degenerate primers were designed on the basis of high homology between members of the abc family, and pcr was performed on yeast genomic dna. ten dna fragments bearing significant homology to the abc family were amplified, nine of which were previously unidentified. disruptions of five of the corresponding genes were performed. disruption of one gene led to markedly impaired growth on rich medium and cessation of growth on minimal medium. the gene was cloned and found to encode a protein of 694 amino acids, a "half-transporter" which likely forms a dimer. ibi~tlnofluorescence on yeast expressing the gene cmyc-tagged at the c-terminus reveals co-localization of the protein with porin and with mitochondrial dna, visualized by dapi staining. nycodenz-purified mitochondria are enriched for the protein by immunoblotting. additionally,the c-myc epitope is protease-protected in mitoplasts, indicating an inner membrane sub-localization and a probable orientation with the c-terminus in the matrix. the gene, termed atmi for abc transporter of mitochondria, thus encodes the first member of the large abc transporter superfamily to be localized to this organelle. present work is aimed at revealing the substrate of this transporter. cycle in cardiac myocyte. the molecular study of this protein has been difficult even after its over-expression was made possible by the cloning of the corresponding cdna. the chief problem is the lack of convenient domains which could be used in the purification of the molecule. we have previously reported the efficient expression of the cardiac na+/ca 2+ exchanger in mammalian cell lines using the t7 rna polymerase-hybrid vaccinia virus system. we have now constructed the recombinant virus for the exchanger with a 6xhis-tag at the c-terminal. this tag has enabled the purification of the protein through a ni-nta agarose column. the expressed tagged protein induced na-dependent ca uptake which was not different from that in intact cells, i.e., cells in which the exchanger did not have the tag, the most important aspects of the purification produced and those of the reconstitution of the expressed protein will be described. $15-08 the neural cell adhesion molecule l1 is involved in the formation and specification of cell contacts in the central and peripheral nervous system during development, and thus may also play a role in synaptic plasticity of the adult central nervous system, using extracellular recordings of evoked field potentials in the rat hippocampal slice, we found that locally applied polyclonal anti-l1 and fusion proteins containing the ig-domains i-vi of l1 specifically block the stabilization of ltp in the ca1 region. baseline synaptic transmission was not affected by the presence of the anti-l1 antibodies. the anti-ll-induced effect was dependent on the antibody concentration and anti-l1 antibodies had no effect on previously established ltp. ltp was also reduced by an antiserum against the recombinantly expressed ig-domains i-vi of l1, whereas controt antibodies against liver cell membranes, which bind to neuronal membranes in the hippocampus, exhibited no effect on ltp. the contribution of l1 to stabilization of ltp within the ca1 synapses suggests that members of the ig-supertamily of cell adhesion molecules are involved in the structural modifications which are associated with synaptic plasticity in the mammalian central nervous system. an increasing number of surface proteins are described as being attached to the membrane by a glycosyl-phosphatidylinositol (gp1) anchor instead of the conventional transembranous hydropbobic domain. they are initially synthesized with a coohterminal polypeptide extension which is cleaved in the e.r. and replaced by the preformed glycolipid. this processing event is directed by a signal, located in the c-terminal region of the protein, which requires a group of three small amino acids positioned 10-12 residues upstream of an hydrophobic c terminal sequence. we previously transformed the transmembfanous glycoprotein d of herpes simplex type i into a gpi-anchored molecule by deleting its cytoplasmic domain and thus creating a molecule, gdww63, which meets the requirements for anchoring via a gpi structure. we now demonstrate, using gpi-deficient cell lines and biochemical studies, that a hybrid protein consisting of the thy-i ectoplasmic domain and of the c-terminal region of gdww63 can be alternative[y expressed in a gpi-anchored or in a transmembranous form. service de p6diatrie, chuv, ch-1oll lausanne proteolipid protein (plp) is a major myelin protein in the central nervous system (cns). a number of x-linked dysmyelinating disorders have been identified as mutations in the pip gene. we have identified a novel plp mutation in paralytic tremor (it) rabbit, characterized by body tremor, spastic limb paresis and hypomyelination in the cns. reduced levels of plp protein and its mrnas were observed in the cns as well as in the pns. plp sequence analysis revealed a single nucleotide change in exon 2 which results in the substitution of a histidine by a glutamine at position 36. histidine 36 is localized on the boundary between the first transmembrane region and the intracellular part of the protein. therefore, its position can be crucial for the efficient plp interaction with other proteins and lipids, and correct incorporation of the plp molecule into the membrane. histidine 36 is also part of a short fragment of ten amino acids which is conserved in all species studied. the same amino acid is altered in another hypomyelinated mutant, shaking pup, stressing its functional importance. furthermore, the pt mutation affects the plp "premyelin" functions, including oligodendrocyte maturation. equilibrium constants for octamer dissociation as well as dissociation rates in vitro increased in correlation to the number of charged residues eliminated, e.g. mutant 4-7, in which all four charged residues in the n-terminal heptapeptide are substituted with uncharged amino acids, showed a 100-fold higher equilibrium constant for octamer dissociation and a 145-fold increased dissociation rate compared to wild type. this strongly suggests that the charged amino acids in the n-termlnal heptapeptide of mib-ck, and therefore an ionic interaction, play an important role in forming the oetameric molecule. gangliosides and neutral glycosphingolipids (gsls) cell surface molecules are involved in a variety of biological processes and are assumed to play an important role in the mechanisms of hiv entry into lymphoid and epithelial cells. the total lipid-bound sialic acid (lbsa) content and the composition of gangliosides and gsls were investigated on pbmn cells from hiv+ve and normal donors. lbsa level was quantified by a fluorimetric assay, while gangliosides and gsls were assayed with high performance thin layer chromatography (hf'tlc) after multistep lipid extraction of 100 0013 x g membrane fractions. in normal pbmn cells the lbsa content accounted for 2.6/~g/108 cells or 20 nmol/mg protein whilst in pbmn cells from hiv+ donors the lbsa level decreased to 1.6 ,ug/108 cells or 13 nmol/mg protein. the qaantitation of neutral gsls and gangliosides was carried out by scanning spots revealed on hptlc plates and the integrated areas computed with a dedicated software. our findings showed that the most relevant ganglioside present in normal pbmnc was gm3 (65-80 % of total gangliosides) followed by small amounts of od3 (5 -15 %) and other acidic glycosphingolipids. neutral gsls included gl1, gl2, gl3 and gl4-hexosylceramide. hiv+ pbmn cells were characterized by a decrease of neutral glycosphingolipids as well as gm3 content which represented only 55 % of the ganglioside fraction, indicating an alteration in the glycolipid composition in the plasma membrane of infected cells. the neuropeptide y (npy) is one of the most abundant peptides of the mammalian brain. upon stimulation of cell surface receptors, npy generates diverse physiologic responses. npy acts on the cardiovascular system. it is a vasoconstrictor by itself and in addition, it potentiates the action of vasoactive substances such as angiotensin ii or norepinephrine. npy also inhibits glucose induced insulin secretion and is a potent inducer of appetite. to facilitate the development of npy antagonists we are investigating the interaction of npy with its cell surface receptor.we first constructed a series of mutants in which negatively charged residues present in putative extracellular domains of the y1 receptor were systematically replaced by alanines. the mutant cdnas were transiently expressed in hela ceils using a vaccinia virus derived expression system and the abi}ity of the encoded proteins to bind npy was evaluated. the capacity of the mutant proteins to be recruited to the cell surface was assessed by confocal microscopy. we identified initially 4 spacially clustered extracellular acidic residues of the human y1 npy receptor essential for npy binding. subsequently, we mutated 36 additional residues. based on these data a detailed computer model of the interactions between npy and its receptor was built. functional activity to energy metabolism. l. pellerin and p.j. magistretti. institut de physiologie, universit6 de lausanne, switzerland. astrocytes play a central role in brain energy metabolism. for example glucose, the exclusive brain energy substrate, is avidly taken up by astrocytes (pnas 90:4042, 1993) . application of glutamate (glu), the main excitatory neurotransmitter, stimulates in a concentration-dependent manner 3h-2-deoxyglucose (2-19(3) uptake by astrocytes in primary culture with an ec50 of ~ 100 ~m. the effect is not receptor-mediated since it can not be reproduced by glutamatergic agonists such as (nmda, kainate, quisqualate, t-acpd) nor prevented by antagonists (apv, cnqx, ap3). instead, it involves glutamate uptake since the effect is blocked by dl-threo-j3hydroxyaspartate, a potent inhibitor of the glu transporter. replacement of na + in the medium by choline also prevents the effect, suggesting a na+ driven process. finally ouabain, a selective inhibitor of the na+/k + atpase, completely prevented the effect of glu. these observations suggest that when glutamatergic synapses are active, glutamate, taken up by astrocytes, stimulates 2-dg uptake by astrocytes whose end-feet are known to surround capillaries, i.e. the source of glucose. they also provide a simple explanation for the mechanism linking functional activity to energy metabolism. cloning and molecular characterization of the mouse astrocyte glycogen synthase. g. pellegri, c. rossier, s. van berchem, p.j. magistre~i and j.-l. martin. institut de physiologie, universit6 de lausanne, switzerland. in the brain glycogen is predominantly localized in astrocytes, although its presence has been detected in certain large neurons, in ependymal and choroid plexus cells. glycogen is synthesized by the enzyme glycogen synthase (glys) from udp-glucose. out of the different glycogen synthase isozymes, only the muscle and the liver forms of glys have been cloned. we have isolated and characterized from a mouse astrocyte ~.zapii cdna library, a cdna clone encoding the mouse cerebral cortical astrocyte glys. the 3.5 kb clone isolated contains an open reading frame of 2214 bp encoding a protein of 738 amino acids. the coding region of the mouse astrocyte glys cdna shares 87% and 86% identity with the nudeotide sequences of the human muscle and the rabbit muscle giys cdna respectively. the cellular distribution of the glys mrna studied by northern blot shows the presence of a single transcript of 4 kb in cultures of astrocytes and, to a lesser degree, of neurons. the distribution of glycogen phosphorylase, which was also examined by northern blot shows the presence of a 4.2 kb transcript both in cultured astrocytes and neurons with however higher levels expressed in astrocytes. the functional molecular size of the vacuolar h+-pyrophosphatase (h +-ppase ec 3.6.1.1) from maize (zea mays l.) roots was estimated by radiation inactivation, both for substrate hydrolysis and for proton transport. light microsomal vesicles enriched in tonoplast membranes were prepared from young (2 days) maize roots using sucrose step gradients. these vesicles were still competent for ppi-dependent proton transport after freeze-drying and rehydration of the membranes. frozen native or freeze-dried samples were irradiated with 6~ for various periods of time. after thawing the samples, the activities of glucose-6phosphate dehydrogenase (added as an internal standard) and h+-ppase (hydrolysis of pyrophosphate (ppi) and ppi-dependent proton transport) were tested. by applying target theory, the functional size for hydrolysis was found to be around mr 155 000 when the native or freeze-dried samples were irradiated. no ppi-dependent proton transport activity was measurable after irradiation of the native samples. on the contrary, the freeze-dried membranes were still pumping protons after irradiation and a target size of around mr 250 000 was measured for the transport activity. these experiments suggest that the native h+-ppase from maize roots may exist as a dimer (at least) of the mr 80 800 catalytic subunit. $15-20 the na,k-pump moves 3 na + out and 2 k + ions into the cells. the domains of the protein involved in ion translocation have not been determined. we have studied the role of the n-terminal region in the na + translocation by measuring the kinetics of charge movements under na/na exchange conditions in wild type (wt) and two n-terminally truncated forms of the bufo ~ subunit with deletions of the 31 (t31) and 40 (t40) first amino acids expressed in xenopus oocytes. we have developped a new technique to perform fast (< i ms) voltage clamp on whole oocyte. the membrane on one side of the oocyte is permeabilized by digitonin allowing to measure current flowing across the other half. we observed presteady state ouabain-sensitive currents similar to that reported by other techniques (j.gen. physiol. 101:117,1993) . the estimated forward charge translocation rate was lower in both n-truncated mutants (wt 430 â�¢ 14, t32 215 z 8, t41 124 z 3 s-l). the midpoint potential of the charge translocation (best fit to boltzman equation) was -79 , -45, and -28 mv in the wt, t31 and t40 groups, respectively. the highly charged nterminus of the q subunit has a significant role in the forward translocation of na + ions. deletion mutants of human small intestinal lactase-phlorizin hydrolase (lph) were constructed to determine the role of protein subdomains in the intracellular transport of lph. the mutant lph1646mact (236 aa deletions), which contains the membrane anchor (ma) and the cytoplasmic tail (ct), was transported to the cell surface in a fashion similar to wild type lph. by contrast, lph1646 lacking the transmembrane domain persisted as a mannose-rich polypeptidc. this suggests that the membrane anchor and/or cytoplasmic tail are implicated in the er-egress of lph. another mutant, lph1559mact (323 aa deletions), was not efficiently transported to the golgi apparatus. epitope mapping with monoclonai antibodies as well as protease-sens{tivity assays provided an evidence that the tertiary structure of lph1646mact is very similar to that lph1559mact. in view of the variations in the proportions of the complex glycosylated molecules of the two mutants we conclude that the domain between lph1646mact and lphi559mact may play an essential role along the secretory pathway of lph. containing tyrosine. hussein y. naim, and michael g. roth; dept, of biocherni~try, ut-southwestem medical center, at dallas, dallas,texas-usa we have investigated an artificial internalization signal created by site-directed mutagenesis of the short cytoplasmic sequence of the influenza virus hemagglutinin (ha). mutation of cysteine 543 to tyrosine converted ha from a protein essentially excluded from coated pits to one that internalized at rate similar to some cellular endocytic receptors. to determine whether or not the ha-y543 signal is a degenerate form of the internalization signal found in proteins such as the transferrin receptor and the mannose-6-phosphate/igfii receptor, we have mutated amino acid positions in the ha-y543 shown to be important for internalization of the two receptors. we have changed amino acids on either side of y543 to those that either fit, or break the pattern of aromatic and hydrophobic residues proposed as consensus sequence. our results indicate that the ha-y543 mutant contains a sub-optimum sequence for a tyrosine-based internalization signal similar to those found in the receptors for transferrin, ldl, and mannose-6-phosphate/igfil. however, amino acids with side chains having different chemical properties functioned well in positions that are important for the internalization signal, indicating that the consensus sequence for this signal is more variable than previously proposed. n-linked glycesylation is an essential protein modification occuring in all eukaryotic cells. core-oligosaccharides are transferred by the enzyme n-oligosaccharyl-transferase frc~ the donor glc man gicnac -dolichol to selected asparagine 9 2 . resldues of t~e nascent polypeptlde chazn. the donor is build up in a stepwise fashion at the er-membraneby the products of the alg-genes (asparagine-linked-~lycosylafion) mutants defective in this pathway are available, but often lack a phenotype suitable for isolation of the corresponding genes. we found that double mutants of alg5 or alg8 with a wbpl mutation (defective oligosaecharyl-transferase) are severely growth retarded at 30oc. the double mutant phenotype allowed the isolation of both alg5 and alg8 genes which have been refractory to cloning so far. both genes encode potential trarsmenbrane proteins and are able to complement the biochemical defects of the corresponding alg5 or alg8 mutants. the na,k-atpase produces the driving force for the regulated transport of na across epithelial ceils. it is composed of an c~-subunit which carries the catalytic and ouabain binding sites and a b-subunit. to test the role of the usubunit in transport regulation, we transfected a6 cells with the b. marinus al-subunit (txl-tbm). this subunit confers a 300-fold higher resistance to ouabain (k i = 50/~m) compared to the endogenous pump. taking advantage of this difference, stable transfectants were selected with ouabain. expression of the cd-tbm subunit was visualized by western blotting. approximately half of the positive cell lines showed a high electrical resistance similar to untransfected cells, when cultured on filters. na-transport activity, measured as isc, was lower even in the absence of ouabain, but the relative increase in response to aldosterone was maintained. functional pumps containing the exogenous or endogenous al-subunit were quantified by equilibrium ouabain binding. interestingly, cells cultured on plastic dishes had a large fraction of low affinity binding sites (txi-tbm), while most sites were of the high affinity type when the cells were grown on filters. we conclude that the ouabain-resistant na,k-atpase cd-subunit of b. marinus can be expressed in a6 cells and used as a selective marker. however, the results suggest that the expression of functional ouabain-resistant pumps containing the al-tbm subunit could be influenced by the degree of cellular differentiation. in infected cells fully functional na/pi-cotransport was found in a time dependent manner; up to a 50-fold stimulation of na/p;-cotransport compared to uninfected cells was observed aft6r 3-4 days. transport of phosphate by infected cells was highly dependent on the presence of sodium, exhibited a kmtd~ of around 0.16 mm and was dependent on extraceuular i~h'. in agreement with expressed transport of phosphate, infected cells produced an immunoreactive polypeptide of 66 kda that represents a non-glycosylated form of the 80 to 90 kda mature na/p;-cotransporter. we conclud$ that the protein napi-2/rat when expressed in sf9 cells exhibits na/p-eotransport with similar kinetic characteristics as observdd in prox ma tubular apical na/pcotransport. therefore the napi-2 protein as expressed h sf9 cells can be used for further structural and functional studies. talin interacts in vitro with actin, vinculin, integrins and bilayers of acidic phospholipids, and nucleates actin filament growth at the bilayer surface. it is therefore believed to be a key protein mediating aetin-membrane linkage. we have analyzed functional properties of proteolytic fragments of purified platelet talin. using limited proteolysis two fragments of 47 and 190 kd were generated. preliminary results, obtained using viscometry and f-aetin-nucleating assays, suggest that the 190 kd fragment contains the actin binding site. we also assessed the lipid-binding capacity of the fragments. when a mixture of the two fragments was centrifuged in the presence of large multilamellar liposomes containing phosphatidylserine, only the 47 kd fragment, but not the 190 kd domain, co-sedimented with the liposomes, suggesting the presence of a specific lipid-binding site in the 47 kd domain. interestingly, this fragment contains the n-terminus of talin which shows homologies to the membrane-binding regions of protein 4.1. we are now analyzing membrane-binding properties of talin domains in more detail using hydrophobic photolabelling. recent studies suggest a role of the neural cell adhesion molecules l1 and ncam in mechanisms of memory formation (doyle e, et al, j. neurochem. 59:1570 -1573 , 1992 scholey a.b. et al, neuroscience 55:499-509, 1993; lijthi a. et al. unpublished data and see usgeb 94) . in the present study we analyzed the effect of chronic intraventricular infusion of polyclonal antibodies against l1 (anti-u) or ncam (anti-ncam) on the performance of male wistar rats during the acquisition and retention of a spatial learning task. briefly, rats had to remember the position of a submerged escape platform in a water tank (morris water-maze). when the escape platform was removed after acquisition training (= retention test), the performance of animals chronically injected with anti-l1 showed an impaired search pattern when compared with that of salineqnjected animals (p>0.05, mann-whitney). whereas control animals spent up to 45% of their time searching for the platform in the correct (= training) quadrant, the time anti-l1 animals swam in the correct quadrant was closer to chance level (31%). although monitoring penetration of antkncam into the hippocampus was almost as efficient as that of anti-l1, the performance of the anti-ncam-group was highly variable and did not differ from the performance of controls. these results agree with recent findings on the involvement of neural cell adhesion molecules in other forms of learning. we have isolated cdnas from mouse and from xenopus encoding the ~-subunit of the gastric h,k-atpase, which is expressed in the parietal cells of the stomach mucosa. the gastric h,k-atpase transports h + into the stomach lumen with the counter transport of k +, all at the expense of atp hydrolysis. when xenopus oocytes are injected with crna encoding ~h,k from either species, as well as a gastric ~h,k subunit, the two subunit proteins are synthesized, assemble, and form functional pumps located in the plasma membrane as demonstrated by 86rb+ uptake. the sensitivity to the gastric h,k-atpase inhibitor sch28080 was determined, with the ki for beth species found to be in the low ~m range. spodoptera frugiperda ceils are often used to overexpress eucaryotle proteins using baculovirus vectors. the atomic force microscope (afm) is a device which allows the observer to obtain high resolution images of biological samples immersed in a fluid medium; this instrument has therefore the potential to visualize overexpressed proteins on the surface of living cells. however, the interpretation of the images is sometimes difficult and requires a good knowledge of the specimen topography, i.e. the "normal" plasma membrane of the cell used for the overexpressisn. on our poster we present afm images of the plasma membrane of both fixed and living spodoptera frugiperda ceils. in additon, we discuss the possibility of achieving high resolution in the imaging of dynamic changes which affect /iving systems. the deduced protein sequence consists of 295 amino acids with about 55% amino acid sequence identicy when compared to mammalian ~h,k-subunits. data from other species and from the structurally similar na,k-atpase has shown that the ~-subunit is necessary for the formation of a functional pump, which consists of an ~/~ heterodimer. following co-injection of xenopus oocytes with crna for both the ~-and ~subunits, we have observed by rb + uptake h,k-atpase activity in the plasma membrane. we are presently studying the role of the ~-subunit in the maturation and function of the h,k-atpase. the atomic force microscope (afm) is a new type of microscope which allows visualisation of inorganic and organic samples with a very high resolution. a sharp tip fixed at the end of a cantilever is used as a topographic sensor. by scanning the sample with this device, a computer records the minute deformations of the cantilever and computes the topography of the sample. the instrument allows also the measurement of the interaction between the tip and the sample as a function of the distance. the knowledge of these interactions allows us to compute several mechanical properties like indentation, elasticity and stiffness. on this poster we report the results of these measurements on cells (sfg, c131), bacteria (e. coli, b. subtilis) and proteins (actin). in addition we discuss the use of the afm as a sensor for probing the mechanical properties of biological systems at a nanometric scale. neonatal thymectomy of balb/c mice induces aig. we have cloned the ~-and ~subunits of the balb/c h,k-atpase and expressed the proton pump in crna injected oocytes. both and ~ proteins are made, form functional pumps, and are immunoprecipated using auto-immune sera. the ~-subunit can also be in~unoprecipated independent of the ~-subunit when oocytes are injected with ~ crna alone. balb/c mice will be immunized with oocyte membranes containing either the ~/~ or ~ antigens in order to study the role of each h,k-atpase subunit in triggering aig. the prion protein is expressed in both astrocytes and oligodendrocytes of the neonatal brain and is down-regulated in adulthood. m. moser, colello, r, , pott, u, and b. oesch institut for hirnforschung der universit~t zorich, august forel str. 1, 8029 zorich the infectious agent causing transmissible spongiform encephalopathies consists largely or entirely of prp sc, a modified isoform o| normal prp (prpc). prpc is most abundant in the brain. the development of the disease strictly depends on the presence of prpc. and incubation times are shortened by overexpression of prpc. the normal function of prpe is not known and its cellular iocalisation in the cns is poorly characterised, except for its evident presence in neurons. here we describe the expression of prp c mrna in both oligodendrocytes and astrocytes during neonatal development of hamster and rat. in adult animals, expression is down-regulated in gila but not in neurons. our findings provide a link to the previously described accumulation of prpsc in astrocytes and the apparently faster course of prion disease in neonatal hamster. our results argue for a major involvement of non-neuronal cell types in prion diseases. such structures we raised mab against membrane protein fractions. a ~lab against a vitronectin receptor containing fraction was found, which if applied in vitro to bi6fi mouse melanoma cells, influenced growth, adhesion and morphology. ~tnen bi6fi cells prior to tail vein injection were treated, 2 different outcomes were observed. short treatment (lh) resulted in 85% inhibition of lung colonization whereas long-ter~ treatment ( 2weeks) resulted in 10-15 times more lung colonies. a candidate molecule likely involved in these observed effects could be identified as a 150 kda cell surface protein. sequence information from peptides revealed in several cases 100 % homology to mouse centrosomin a, i.e.a 32 kda cytosolic protein involved in the organization of the spindle apparatus. these data suggest that we may have found a protein which may participate in a link between the extra-cellular space and the microtubular system. we are currently trying to clone this 150 kda protein from a bi6fi c-dna library. wahlberg, j.m., and spiess, m. dept.biochemistry,biozentrum, university of basel. signals for correct basolateral sorting of subunit hi of the human asialoglycoprotein receptor are located in the cytoplasmic tail, involving a tyrosine at position 5. removal of this residue results in nonpolarized transport of the protein to the surface in mdckii-celis. a deletion mutant of hi, lacking 30 of the normal 40 residues of the cytoplasmic tail, including the tyrosine, has been found not to be transported to the cell surface, but to be accumulated intracellularly. the mutant protein is complex glycosylated and sialylated, indicative of passage into or through the trans-golgi. immunofluorescence analyses show defined juxtanuclear, tubular structures, which do not colocalize with markers for er, early endosomes or lysosomes, but which show similar staining pattern as markers for late endosomes and tgn. to define the determinants responsible for the intracellular accumulation a series of deletions and fusions have been constructed. the results suggest that the length of the cytoplasmic domain is one important determinant for missorting of hi. salerr~ n., medilansld, j., pellegrinelli, n. and eder-colli, l. departement of pharmacology, cmu, 1211 geneva 4 in chollnergic neurons the enzyme choline acetyltransferase (chat) exists as cytosolic(80-90%) and membrane-bound activity (10-20%}. are these two activities encoded by a single mrna? is membranebound enzyme preferentially associated with a given cellular membrane? in order to investigate these questions we isolated a fulllength cdna (4.2 kb) encoding drosophila chat and used it to transfect xenopus oocytes, rat fibroblasts and human neuroblastoma cell lines. triton x-114 fractionation of transfected cells showed that hydrophilic and amphiphilic chat activity were produced. the sp act. of hydrophflic enzyme reached 2, 0.8 and 0.25 ~mol ach/h/rng protein respectively in oocytes, fibroblasts and neuroblastoma whereas the sp. act. of amphiphilic enzyme was 1.8, 0.4 and 0.125 hinol ach/h/mg protein, respectively. amphiphilic chat was less sensitive to inhibition by the product acetylcholine and to heat denaturation than was hydrophilic enzyme. similar results were observed for the native drosophila chat. amphiphillc enzyme sedimentation was affected by the type of detergent present in linear density gradients of sucrose. transfected oocytes were subjected to subfractionation. besides a large amount of soluble chat activity, a significant proportion of enzyme was found associated with a fraction enriched in endoplasmic reticulum (er). preliminary results using transfected mammalian cells indicate that apart cytosolic chat, plasma mernbranes+golgi enriched fractions as well as er-enriched fraction contain chat activity. we have characterized a drosophila melanogaster gene encoding a very hydrophobic protein. the amino acid sequence shows considerable homology (33-42% identity) to neurotransmitter transporters. the gene is, however, not expressed in neural tissue, but in the male germline. transcription and translation occurs in early spermatocytes. antibody staining data suggest the following pathway of the protein during spermatogenesis: the protein is synthesized in early spermatocytes and is transported into the nucleus during prophase of rt i. at meiosis it associates with the mitochondria and it stays associated while they fuse to the nebenkern. during individualization it is stripped off in the cystic bulge. inspection of the putative protein sequence shows that it possesses alle the necessary targeting signals to allow its transfer from the cytoplasm through the nucleus into the mitochondria: three nuclear targeting signals, a mitochondrial import signal and, moreover, two pest-sequences for rapid protein degradation are present. we shall present a, currently tested, working hypothesis. in hg-treated toad skins, water permeability is high or low depending on whether the major anion of the inner bathing medium is so~ or ci. we report here the results of experiments designed to determine if, in skins bathed with so4-ringer, net water flow (j~,) could be diminished by agents added to the outer medium. toad skins were mounted inbetween glass chambers and exposed to a standard osmotic gradient of 200 mosm. jw was continuously monitored by automatic, volumetric techniques. exposure to lmm hgclz or chzcihg (external side) led to the typical anion-induced j,, changes. re-exposure to hg caused a mild (10%), although significant (n=8, p<0.001), fall in jw. since cuso4 is a sh-reagent like hg, we looked at the effects of cuso4 (lmm). there was a 68% reduction of j, (n=7, p<0.001), an effect totally reversible by simple washing of the skins. likewise, addition of dithiothreitol (dtt, 5mm) in the presence of cu, caused a 90% reversal of the cu effect ; there was no change in jw if dtt was given before cu, or if both agents were added simultaneously. finally, in hg-treated, glutaraldehyde-fixed skins, lmm and 5ram cuso4 caused 51% and 92% jw decrements, respectively, that were totally reversible. in conclusion, cu causes a dtt-sensitive inhibition of jr, in hgtreated skins. further work is needed to establish if the cu effect is exerted on sh-groups of apical aquapories of toad skin. $15-44 the amino acid sequences of the pc-645 e-subunits and other cryptomonad biliprotein ~-subunits pose the most intriguing question about the phylogenetic origin of these unique chromopeptides. cryptomonad pc-645 ~-subunits seem not to be closely related to the known phycobiliproteins, linker polypeptides (lpps), bpe or any other light-harvesting chl polypeptides from the thylakoid. for most of them the number of identical amino acid residues was 15%-20%. sequence alignment of c-pe associated lpps with t-subunits from cyanobacteria and red algae and the cryptophytan ~-subunits however show that the identity between c-pe associated lpps and cyanobacterial x-subunits is still significant, whereas it reaches the limit of significance between cyanobacteril and eucaryotic x-subunits. nevertheless it has to be assumed that x-subunit derive from cyanobacterial c-pe associated lpps as well as cryptomonad ~-subunits may derive from t-subunits. whereas the the phycobiliprotein ~-and 8subunits remained structurally conservative during evolution (e.g. -70% identity between cyanobacterial and red algal pe-subunits) the lpps and t-subunits show drastical changes in their chromophore binding domains. $15-45 functional and evolutionary relationships of the components of the primary events in photosynthesis have been traced using amino acid sequence comparison. basically, the question was posed regarding the interrelationship of the apoproteins of different photosynthetic organisms which gather light and/or separate charges across membranes. do their protein-chemical structure and organisation decipher us certain clues and parts of the path by which they have been selected and derived from a possible primordial reaction centre polypeptide? within that context the light-harvesting-and energy transfer systems of purple bacteria offered as ideal model components. a data set of more than 70 apoprotein sequences has pointed to some keystructural elements which have been partially verified as such by limited proteolysis and genetic engineering. a careful inspection of the determined bacterial antenna-structures revealed some remarkable similarities to eukaryotic antenna and reaction centre apoproteins indicating possible basic structural motifs for complexing pigment molecules, like chlorophylls or bacteriochlorophylls in very distant representatives of phototrophs. a number of glycoproteins of the nervous and/or immune system that are involved in cellular recognition and/or adhesion share the common lz/hnk-1 carbohydrate structure. in a large proportion of patients with peripheral demyelinating neuropathy associated with paraproteinemic gammopathy (ppn), monoclonal igm antibodies (m-igm) react with the lz/hnk-1 determinant of mag and p0. we plan to test the hypothesis that anti-mag m-igm autoantibodies may alter the expression of hnk-1 bearing myelin proteins such as mag, p0, mog, ncam or pmp22. by using monoclonal antibodies to myelin proteins on tissue sections, preliminary results indicate that mbp protein content seems to be strongly downregulated, whereas galc and $100 protein do not appear to be affected by the loss of myelin in ppn nerves. gfap protein expression appear to be upregulated in demyelinating biopsy specimens from ppn patients with anti-mag m-igm gammopathy. in conclusion, the expression of some l2/hnk-1 negative proteins might be disregulated by the presence of circulating anti-mag m-igm in the serum of ppn patients. we have isolated a human liver na+-taurocholate cotransporting polypeptide (ntcp) by screening a human liver edna library with a rat edna probe derived from ntcp (pnas 88, 10629, 1991) . the cdna codes for a protein of 349 amino acids which shows 77% amino acid homology with the rat ntcp. expression of ntcp in x. laevis oocytes resulted in na+-dependent bile acid uptake which exhibited saturability with an apparent km for taurocholate of 6 ~m. ntcp mediated taurocholate uptake into oocytes was inhibited by all major bile acid derivatives, bumetanide and bromosulphophthalein. southern blot analysis of genomic dna from a panel of human/hamster somatic cell hybrids mapped the human ntcp-gene to chromosome 14. in conclusion, these studies supplement the previously suggested selective mammalian distribution of the ntcp gene family and provide the possibility for direct molecular characterization of human bile acid transporter genes. diverse cell surface molecules of the nervous system play an important role in specifying ceil interactions during development. with the aid of a polyclonal antibody against l1, a 1.skb edna clone closely related to l1 (ch-l1) was isolated from a ~. gtl 1 library derived from poly(a)* l~qa of day 8 mouse brain. using this edna as probe the remaining cdi'ia 5' sequences were cloned out of a plasmid library constructed from postnatal day 6-14 mouse brain poly (a)+ p, na. two independent full length clones of 4.2 and 4.4 kb encompassing the entire coding region of chl] were isolated. these represent putative alternatively spliced mp, nas. the deduced primary structure of chl1 reveals that, like the cell adhesion molecules (cams) mouse l1, chicken ng-cam, and nr-cam (bravo), chl1 is composed of six ig-like domains, a transmembrane domain, and a short cytoplasmatic region. in contrast to the other l1 family members, only four and a half instead of five fibronectin type hi-like repeals are found in chl1. the fibronectin type [h-like repeats were expressed in e. col[ and the protein fragment was used for immunization. as observed for li, ng-cam, and nr-cam, chl1 is susceptible to proteolytic cleavage. bands of 185kd, 165kd, 125kd, and 50kd could be detected bywestern blot analysis. by in situ hybridization, a predominant expression by neurons was found in the adult mouse brain. western blot analysis showed expression of chl1 protein in brain and heart, but not lung, kidney and intestine. in liver, a 50 kd immunoreactive band was found. the relationship of chl1 to molecules known to be involved in cell adhesion and neurite outgrowth, combined with its pattern of expression, suggests a role for this molecule in cell interactions dndng neural development. work from this laboratory revealed that in hg-treated, glutaraldehydefixed toad bladders, the water permeability coefficient (pf) is high in sod-ringer and low in ci-ringer ; in all these studies the osmolality of the serosal medium was 220mosm. as an attempt to determine the mechanism(s) underlying such pf changes, experiments were carried out with bladders whose serosal surface was exposed to [so-, hypo-or hyperosmotic media ; the outer surface was always exposed to a hyposmotic solution (22 costa). net water flows were measured continuously with automatic, volumetric techniques. after exposure to 1 mm chzcihg (10', outer medium) followed by fixation with 0.25% glutaraldehyde (5', serosal medium), the bladders were thoroughly washed. in sod-ringer, pf (/~m/sec) was as follows (n=6) we wanted to determine the molecular weight (mw) of ntcp and its topology in the plasma membrane (pm) of rat liver. a rabbit antiserum generated against the c-terminus of ntcp was used to determine its mw on western blots and its subcellular distribution in liver and in hepatocytes by immunofluorescene (if). site directed mutagenesis and deletion experiments were used to determine the n-linked glycosylation sites. ntcp encodes a 50 kd protein in rat liver pm vesicles. the basolateral localization of ntcp in liver was confirmed by if. ntop could only be immunostained in permeabilized hepatocytes suggesting an intracellular localization of the cterminus, cnda constructs showed the ntcp is glycosylated at positions 5 and ii. the data show a selective basolateral expression of ntcp in rat liver and they suggest that the two ends of ntcp are located on opposite sides of the pm. zymogen granule membranes (zgm) are known to contain proteins with nucleoside phosphatase activity, but their exact nature and function are unknown. the activities require ca ++ and mg ++ and are sensitive to atpase inhibitors but not to inhibitors of na/k-atpase activity. the aim of this study was to isolate individual proteins of pig pancreatic zgm, to attribute enzymatic activity to individual proteins and to characterize them with various substrates (gtp, atp, amp etc.), inhibitors (amp-cp, amp-cpp, gtpgs etc.) and lectins (maa, dsa, gna etc.). we have subjected highly purified zgm solubilized in chaps to ief on immobiline dry strips | (pharmacia) with s ph range 3--10.5 (ist dim.). these strips were then electrophoresed in a polyacrylamide gradient gel containing 0.2% chaps and tris/hci at ph 9.5 (2nd dim.). nucleoside phosphatase activity was detected histochemically in the 2d gel by incubation with substrate in the presence of lead nitrate. focussed strips were also subjected to sds-page for comparison. several proteins showed strong activity to gtp, atp and itp. the role of these phosphatases are discussed in the context of the role of gtp-binding proteins and the de-/phosphorylating events on the zgm in intracellular targeting and exocytosis. the disease-specific isoform of the priori protein (prp sc ) is an essential part of the infectious particle causing scrapie or bse. prp sc differs from prp of normal animals (prp c ) by its relative protease resistance. the physical nature of this difference is still unknown. here we analyzed the protease-resistance of prp sc quantitatively. prp sc was rendered completely protease-sensitive at alkaline ph or in guanidinium thiocyanate (>1.5 m). denaturation in 2m guanidinium thiocxanate (gdnscn) completely abolished protease resistance of prp bc within 15 min while denaturation in 6 m urea showed a much slower time course. denaturation cuwes were used to calculate the gibbs free energy (as d ) in dependence of gdnscn or urea at different concentrations. the linear relationship between agd and the denaturant concentration is suggestive of a two state model involving the conformational change of a single protein domain. this is also reflected in the small number of side chains (11,6) additionally exposed to the solvent upon conversion of prp sc to its proteasesensitive isoform. our results suggest that minor rearrangements of the structure of prp sc are sufficient to abolish its protease resistance. of physiology, university of zurich, winterthursrstr. 190, ch-8057 zt~rich in contrast to mammalian kidneys where inorganic phosphate (pi) is reabsorbed unidirectionally, flounder renal epithelial cells both re.absorb and secrete pi. in order to investigate on a molecular level the cellular pi handling we have cloned a re,absorptive nalp i transport system from flounder renal tubules. based on homology with the cloned na/p i cotransporter from rat we have isolated a edna clone (flounder napi-ii, 2424 bp) encoding for a protein of 637 amino acids. in the eight transmembrane spanning domains the protein is 78% identical with the corresponding system from rat kidney, in the hydrophilic regions only 30%. expression of in vitro transcribed rna in xenopus oocytes specifically stimulated na-dependent pi transport. binding properties of na and pi as well as the ph-dependency were similar to the ones found in mammalian systems (rat, human, ok cells). by northern analysis three transcripts, 1.9, 2.7, and 4.2 kb in length, could be detected. rna from flounder renal tubules contained all of the transcripts, intestinal rna only the two lower bands, and non-epithelial tissue from kidney expressed only the 1.9 kb transcript. mammalian systems share only the 2.7 kb transcript but not the related species. the close functional relationship of flounder napi-ii with the known najp i transport systems and the pronounced differences in primary structure provide the basis for structure function analysis of na/p i cotransport. the amyloid 13/a4 protein precursor (app) is a transmembrane protein expressed in different isoforms throughout the organism. our work has consisted in a detailed study of the structure and biological function of app. specifically, we have shown that the secreted form (sapp) of app-695 is involved in the growth regulation offibroblast% and also stimulates neurite extension in b 103 neurnblastoma cell line. functional mapping studies have shown that the domain responsible for both the growth-regulating and the neurotrophic activities of sapp-695 is the rerms site, arg-328 to ser-332. the presence ofsapp binding sites on the surface of b 103 ceils (through the active rerms site) indicate that the neurotrophic activity of sapp is mediated by a receptor, and the subsequent activation of a signal transduction mechanism. in addition, we have shown that the rerms site is also involved in the neuronal survival properties of sapp-695. finally, we have initiated an in vivo study of app biological function, and shown that a peptide representing the neurotrophic domain ofsapp-695 can strengthen memory retention in rat through stabilization of synaptic structures in the frontal cortex. this work was supported by grants from the swiss national science foundation (823a-028366), and nih (ag 05131). we previously showed that in smooth muscle cells, the (~i-ar is rapidly phosphorymted following exposure to agonist. to understand this biochemical event, we investigated the phosphorylation of the c(1b adrenergic receptor stably expressed in rat1 fibroblasts (3.3 pmol/mg prot.). the =(1b-ar was solubilized from 32p-labeled rat cells and immunoprecipitated with antibodies raised against the n-terminus part of the receptor. treatment of ceils with 100 ~.m epinephrine showed a rapid and significant increase in receptor phosphorylation above basal. treatment of cells with a specific pkc inhibitor (ro-318220) did not affect agonist.promoted phosphorylation while it completely abolished phorbol esterinduced receptor phosphorylation. this strongly suggests that pkc is not involved in agonist-induced c{1b-ar phosphorylation. the elb-ar contains several putative phosphorylation sites in both its third intracellular loop and c-terminus tail. to investigate the role of these domains, a trunoated receptor (lacking its last 147 amino-acids) was constructed and stably expressed in rat cells (1.3 pmol/mg prot). this receptor mutant displayed similar ligand-binding properties and abirity to activate phospholipase c as compared to the wild-type receptor. phosphorylation experiments revealed that this mutant is not at all phosphorylated, neither by agonist nor by phorbol esters. agonistinduced decrease in cell surface receptors, measured by binding of the antagonist 3h-prazosin at 4~ was however similar for both the wild-type and mutant receptor. this suggests that loss of phosphorylation sites does not affect receptor internalization. human intestinal lactase-phlorizin hydrolase (lph) is synthesized as a precursor molecule (pro-lph), which undergoes proteolytic processing during maturation. lph expressed in cos-1 cells was enzymatically active, the electrophoretic properties of lph-species synthesized by transfected cos-1 cells correspond to those in organ cultured human intestinal explants, in caco-2 ceils and in transfected mock ceils. they included the pro-lph and a proteolytically processed form, which had similar electrophoretic properties to the mature enzyme. the appearance of the putative mature form was inhibited by brefeldin a, ammonium chloride and chloroquine. in addition, only complex glycosylated pro-lph (pro-lphc) was inserted into the cell surface membrane. these data indicate that in cos-1 cells pro-lph is inserted into the cell membrane, is internalyzed and enters lysosomes where proteolytic events lead to the appearance of a mature-like enzyme. $15-59 assembly and transport of paba peptide hydrolase alpha and beta subunits in cos-1 cells eldedng, j.a., gr0nberg, j. and sterchi, e., institut for biochemie und molekularbiologie, universit&t bern. paba peptide hydrolase (pph) (ec 3.4.24.18), a metalloendopeptidase from epithelial cells of human small intestinal mucosa, consists of two subunits, ~ and 13, which form homodimers and oligomers, cdna cloning has revealed homologies with developmentally important proteins from different species and has led to the definition of a new family of metalloendopeptidases, the astacin family (j. biol. chem. 266, 21381, 1991) . here, we report on the expression of the cdnas of pphcz and pphj3 in cos-1 cells. when expressed on its own, pph(z is synthesized in a core glycosylated form only, suggesting that it is transport-incompetent and not able to exit the er. immune-fluorescence studies have confirmed that pph~ is not expressed on the surface of cos-1 cells. pphl3, on the other hand, matures and is transported to the cell surface. when the two subunits are coexpressed, ppho~ can also be detected on the cell surface, suggesting that a heterooligomeric assembly is necessary for the surface expression of pphtz. truncated forms of both pphc~ and pphi~ could be detected in the medium of transfected cos-1 cells. in our previous work, we demonstrated by immunocytochemical reaction that sensory neurons expressed nuclear triindothyronin receptors (nt3r) in embryonic as well as adult rats. in contrast, in sciatic nerve, schwann cells exhibited only transient nt3r immunoreactivity from e17 to postnatal day 10. in the present work, we attempt to demonstrate by radioautography that the detected nt3r are effectively the binding sites of [125i] labeled triiodothyronin. cryostat sections of dorsal root ganglia (drg) or sciatic nerve were incubated with 0.1 nm of l, 3,5,3'-[t25i]triiodothyronin 2200 ci/m mol (nen), while the controls were incubated with a large excess (llxm) of unlabeled trfiodothyronin (t3). in aduit rat drg, radioautographs revealed that silver grains were exclusively restricted to neuronal cell bodies, while the schwann cells ensheathing the axons were free of significant label. in newborn rat sciatic nerve, silver grains accumulated over schwann cells; in contrast in adult rat sciatic nerve, schwann ceils were free of label. in control drg or sciatic nerve sections incubated in large exceas of unlabeled "1"3, all the radioautographs exhibited only scattered silver grains. in conclusion, our results show that sensory neurons and schwann cells are able to express functional nt3r which bind thyroid hormones (snf 31-33671-92). characterization of the maturepreeursor glycophospholipid for glycosylphophatidylinositoi anchors of saccharomyces cerevisiae. many attempts to isolate the mature yeast precursor gpi glycophospholipid ready to be attached to newly translated proteins for gpi-anehoring have failed although such precursors were previously identified in mammalian cells and protozoa. here we show that metabolically labeled glycolipids of the expected structure can be observed if the incorporation, their accumulation and their extraction are optimized. two very polar, (3h)-mannose-labeled glyeolipids named cpi and cp2 were identified and purified. they were structurally characterized using phnspholipase treatments, partial deacylation with methanolie ammonia, hydrofluoric acid dephosphorylation, nitrous acid deamination, acetolysis, exoglycosidase treatments and combinations thereof to produce labeled fragments which could be analyzed by paper chromatography or thin layer chromatography. cp1 and cp2 both contain the identical core oligosaccharide mangl,2(x->po4-6)manal,2mamxl,6(y->)mana-glcn-lansitol, x and y being hydrofluoric acid-sensitive substituents (most likely ethanolamine and phospheethanolamine, respectively). the inositol is acylated although no acyllaositol is present on protein-bound yeast gpi-anchors. cp1 and cp2 can also be observed after lableling with (3h)myo-inositol, the lipid moieties of cp1 and cp2 can be completely removed by mild alcaline hydrolysis altough the protein-bound gpi-anchors made by the pmi210 cells under identical labeling conditions are completely mild base resistant. this finding reinforces the notion that the ceramide moiety typically found on the majority of yeast gpi-proteins are added only after addition of the gpi precursor glycolipid to proteins. the distributions of the mannose-6-phosphat e/igfll-recept or and a2,6slalyltransferase in hepg2 cells: no evidence for co-localization by confocal laser scanning microscopy. the organization of the trans golgi apparatus (ga) with respect to (recycling) man-6-p/igfii receptor (mpr) and (resident) c~2,6sialyltransferase (st) has been investigated by double immunofluorescence laser scanning confocal microscopy in hepg2 cells. they were chosen because biochemical evidence suggested sialylation of mpr upon recycling (duncan & kornfeld, j. cell biol. 1988, 106, 617-28) implicating colocalization of both proteins. in the steady state st was confined to the ga whereas total (endogenous) mpr was localized to coarse vesicles without evidence for colocalization by confoeal microscopy. endocytosis of surface-labeled mpr was monitored in presence and absence of brefeldin a (bfa): without bfa, early endosomes (2' of warming up) showed no, late endosomes (20') little if any colocalization with st. in presence of bfa, the st-compartment disappeared whereas late endosomes persisted with some tubular emanations. under similar conditions, in "absence of bfa, endogenous mpr concentrated in the ga region suggesting colocalization with st. in presence of bfa the mpr compartment formed a redculum whereas the st compamnem disappeared. in conclusion, within the limits of the techniques used, exogenous mpr was not detectable in the st compartment whereas endogenons mpr concentrated in the ga region but obvious co-localization was exceptional. supported by grant 31-30757.91 of the snsf to egb. cultures of dissociated striatal neurons prepared from fetal rats were grown in the presence of neurotrophin-4/5 (nt-4/5) as well as the other known neurotrophins, ngf, bdnf and nt-3. we found that acute administration of nt-4/5 to seven days old cultures stimulates the hydrolysis of phosphatidyllnositol, an event involved in neurotrophin signal transduction. growth of stdatal cultures in presence of nt-4/5 resulted in increased cell survival as indicated by elevations in total cell number, protein content and number of viable cells. nt-4/5 exposure increased gaba uptake and staining intensity in gaba immunocytochemistry in these cultures indicating atrophic action on gabaergic neurons. to further identify responsive cell populations we analyzed for calretinin, a calcium binding protein known to colocalize with gaba in a number of neuronal cells. nt-4/5 strongly increased the number of calretinin positive cells in cultures prepared from rats of embryonic day 15, as we]l as calretinin levels determined by western blot analysis. when cultures were prepared from embryonic day 18 rats, nt-4/5 very strongly increased the morphological differentiation of calretinin positive cells. while all effects produced by nt-4/5 were mimicked by bdnf with similar potency, nt-3 was found to be only marginauy effective, our findings identify nt-4/5 as a potent neurotrophie factor for striatal gabaergic neurons. fusion of cells and organelles is a general patho-biological phenomenon: muscle cells, transport vesicles, langhans cells and virus induced fusion from within (ffwi, during replication) and without (ffwo, by the particles). in vivo and after virus isolation only little fusion can be seen, only after cultivation fusing hsv can be detected; a selective pressure must be released. 6 syn loci are known on the genome; the syn 3 locus is located inside of the cell in the carboxy terminal part of glycoprotein b, all other fusion domains are outside the ceil. fusion inducing mutations are in aa 816, 853, 854, 856, 857 and 872 and were correlated to fusion from within and without as well as to selective cyclosporin a effects. a model is presented for the 3 syn locus. -by recombination the fusion capacity could be transfected to fusion negative viruses. -hsv penetrates the cellmembrane by fusion by 2 ligand-recepter interactions. ffwo+-strains penetrate at 0~ cell/cell fusion was analysed and found to need also 2 receptor-ligand interactions if induced by syn 3 mutations. timing analysis of fusion events and by certain inhibitors allow further conclusions. quenching of lhcii isolated from dark-adapted (lhcii-d) or preilluminated (lhcii-l) rye leaves was similar in both preparations, but reversibility of this process within two hours of darkness was slower in lhcii-d. no differences in fluorescence quenching and full reversibility was observed for both lhcii preparations in reverse micellar solution. xanthophyll/chl concentrations in lhcii were consi-derably decreased under this conditions. excitation difference spectra (before and after illumination) of lhcii in asolectin liposomes showed typical changes of energy transfer between carotenoids and chl. a model is proposed according the xanthophyll cycle controls reversibility of light-driven excitation quenching as well as the xa~a~q~_x~tentof ~,,~nchi~g i/~ lhc~z. myelinogenesis is a complex, developmentally regulated process involving coordinate expression of myellnafion-related proteins. the myelin forming cells are the oligodendrocytes in the central nervous system (cns) and the schwann cells in the peripheral nervous system (pns). we differentially screened a rat postnatal day 16 (p16) spinal cord edna library with probes derived from normal (plus probe) and from myelin-free (minus probe) p16 spinal cord mrnas. we succeded in the isolation of several novel edna clones whose corresponding mrnas were expressed either selectively by oligodendroeytes or by oligodendroeytes and sehwann cells. here we describe one of the edna clones (tentatively denominated ns 2) whose mrna is specifically expressed by oligodandroeytes and schwann cells. the 3.5 kd transcript is expressed at highest level during myellnation and at much lower levels in the adult as it is known for other myelin-specific mrnas. sequence analysis of the full length edna sequence showed a 50% identity to the rat liver udpglucuronosyltransferase family, involved in the detoxificafion system. the 541 amino acid open reading frame encodes a 62 kd protein with a putative signal peptide and two transmembrane domains, whether this ns 2 protein is involved in a detoxification reaction or in glycosilation of proteins and lipids with glueuronie acid is under investigation. neuroblastoma (n8) are sympathetic tumors of childhood characterized by mycn amplification in advanced stages. cd44 standard (cd44s) and splice variants (cd44v) represent a group of surface glycoproteins whose expression has been recently linked to metastasis. we analysed the expression of cd44s and cd44v on n8 tumors and cell lines by immunohistology and northern blot. results showed high levels of cd44s on 33/44 nb, 6/10 n8 cell lines and 4/4 ganglioneuromas (mature, benign sympathetic tumors). lack of cd44s staining was observed on stage iv, mycn amplified tumors only. in cell lines, expression was not detected on cells with a neuronal phenotype while high levels of cd44s were expressed by cells with a glial differentiation, independently of mycn amplification. up-regulation of cd44s was obtained with agents that induce a glial differentiation, while neuronal differentiation by retinoic acid was not accompanied by a change in cd44s expression. no gd44v were detected on tumors or cell lines. we conclude that cd44s expression is down-regulated in a subset of nb ceils and tumors and that mycn product and additional mechanisms responsible for control of differentiation are involved in this regulation. the glycoprotein gc of herpesviruses is known to be non-essential for virus replication. non-essential herpesvirus proteins are suggested to perform "luxury functions" which may influence the outcome of an infection. we are aiming to analyse the function(s) of ibc specified by bovine herpesvirus 1 (bhv1), bovine herpesvirus 5 hv5) and caprine herpesvirus 1 (caphv1). these viruses are closely related but differ in their pathogenic potential. in a first step we have cloned and sequenced the individual genes. the nucleotide and the deduced amino acid sequence homologies of two 8hv1 reference strains was found to be >98%. the sequence homologies between bhv1 and bhv5 were >75% and those between bhv1 and caphvl were >65%. comparisons on the amino acid sequence level revealed that the differences were most prominent in the n-terminal parts, whereby the potential signal sequence region might be concerned. the putative glycosylation sites, however, were not affected, and the transmembrane sequences were similar in size and location. in order to characterize the significance of these differences, we are constructing gc-deletion mutants and recombinant viruses carrying a foreign gc gene. the in vitro and in vivo behaviour of these viruses will be compared to the wildtype virus. $15-67 o. moullet and j.-l. dreyer, instimt de biochimie, unlversit6 de fribeurg, ch-1700 fribourg camp has been recently shown to promote a cell survival response by retarding apoptosis (berridge, m.v& el. (1993) exp. hematol. 21,269-276) . on the other hand quinones are widely distributed substances of often potential toxicological significance. we have tested the effects of quinones on adenylate cyclase. the enzyme is rapidly inhibited by pbenzoquinone (ic50=40-45 ktm) or dicnlorophenol-indopbennl (/6'50=200 ilm), but 2-substituted quinones are inactive. the lc50's are decreased 3-10 times after membrane soinbilization but are not affected by gtp, gdp or analogues nor by cholera and pertussis toxins. the inhibition is not mediated by a g-protein or by the activation of a defined receptor. quinone inhibition stoichiometrically competes with forskolin activation of adenylate cyclase, equimolar concentrations of beth quinone and forskolin restoring the enzyme activity to its basal value. the cytotoxicity of benzoquinone, tested in vivo on hep-g2, a human bepatocellular carcinoma cell line, could be prevented with forskolin. in plasma membranes quinones tightly bind to only one major and two minor proteins that were purified by page electrophoresis under native conditions. together these observations indicate that quinone action can be attributed to targetting to a limited number of proteins at tile plasma membrane in a highly selective way. by affecting adenylate eyclase, a modulation of the camp pool induces apoptosis as a result of the cytotoxicity via. t.congolense is an african,unicellular hemoflagellate, pathogenic for domestic animals such as cattle.within the host bloodstream,parasites adhere to the wall of the microvasculature,eausing severe organ damage.we have set up and characterized an in vitro assay in order to study the metabolic requirements,reeeptor-ligand interactions and the ultrastructure of this adhesion phenomenon.our findings suggest that adhesion is an active process requiring metabolic energy on part of the parasite, but is independent of the target cells.we also show that t.congolense possess a leetin-like domain localized at specific sites along the surface of the anterior end of the flagellum, which interacts with specific carbohydrate receptors, probably sialic acid and/or n-aeetylglueosamine residues,on the endothelial cell surface. this suggests that adhesion is likely to be mediated by a trypanosomal surface component distinct from the variable surface glycoproteins(vsgs). we also suggest that the cytoskeletal protein aetin is involved in this process. local immune response based upon intestinal parasitespecific t and b cells to giardia lamblia may be impomrant in parasite clearance. experimental infections qf mice (cr:nih:s) with g. lamblia (clone gem-h7 which e~presses a major 72kd antigen on its surface recognized b~ mab g10/4) have demonstrated that the parasite undergoes surface antigenic variation in rive. experimental i~fection of immu/%odeficient mice (nu/nu mice and sci~d mice) and oontrol nu/+ littermates provided insights into associated immunological mechanisms: dominant surfade epitope-specific slga plays a major role in modulatin6] surface antigenic variation, and thymus-dependent t-lyn~phecytes in the induction of selfcure. athymio nu/nu mioe (zu. icr-strain) were reconstituted with immune peyer:~ patch lymphocytes obtained from self-healed littermat~ nu/+ mice. intestinal b-cells from these nu/+ mice @s well as from reconstituted athymic nude mice synthesized in vitro parasite-specific iga. this iga showed a predominant immunoblet reaction with the major surface antigen (a mr 72,000 polypep-tide) characterizing the giardia clone in question. nu/nu mice reconstituted wit~ immune cells acquired the potential to decrease significantly their intestinal parasite mass. thus the hypothesis on the causative role of intestinal iga in tl~ control of intestinal g. lamblia infection has found further experimental support. stephan jentsch, heinz tobler and fritz m011er, institute of zoology, university of fribourg, p4rolles, "1700 fribourg the process of chromatin diminution in the parasitic nematode ascaris lumbricoides leads to the formation of somatic cells that contain less dna than the germ-line cells. during this process, which occurs between the third and the fifth embryonic cleavage divisions in five different blastomeres, the termini of the chromosomes are cut off and eventually degrade in the cytoplasm. to analyze this process at the molecular level we constructed a xzap somatic telomere library. the analysis of three cloned telomeres and their corresponding germ-line genomic regions revealed that chromosomal breakage takes always place within short specific chromosomal regions (cbrs) and is followed by the addition of the telomeric sequences (ttaggc)n to the breakage sites. the different cbrs do not crosshybridize to each other. a comparative nucleotide sequence analysis is now being performed to screen for the existence of conserved sequence motifs. in a parallel approach we analyze the chromatin structure of the cbrs and their flanking regions in developmental stages before and during the elimination process, putative recognition or regulation sequences important for the elimination process might be recognized as dnasel hypersensitive sites. once such sequences will be identified, it should be interesting to establish their role in the elimination process and to look for specific binding factors. chromatin diminution takes place in all presomatie ceils of the early embryo of ascaris lumbricoides and is followed by the addition of many repeats of the telomeric sequence ttaggc. chromosomal fragmentation is developmentally regulated and occurs within specific chromosomal regions (cbrs). one of these cbrs (cbr1) was analyzed in detail. agene located close to the cbr1 encodes a putative gtp-binding protein, whose promoter region is located within a distance of only 2 kb from the telomeric ttaggc repeats of the corresponding somatic chromosome. a homologous gene was isolated from c. elegans. most interestingly, the c. elegans gtp-binding protein gene is also located at a telomeric position, namely at the end of chromosome v. in ascaris, transcripts of this gene are present in all developmental stages, though they seem to be stronger expressed in oocytes and early embryos than in later developmental stages and somatic tissues. a high degree of sequence conservation of these genes between the two nematode species and man (a corresponding partial cdna clone was identified in a human heart cdna library) suggests an important biological function. currently, we are using genetic methods to determine the possible function of this gene in c. elegans and to test whether its telomeric localization in both nematode species has any functional importance. it encodes a nuclear protein which is associated with she chromatin together with other probeins of the poiyoomb group. the mechanism of gene regulation caused by pelycomb seems to be similar to that of the modifier genes of the position variegation effect which also encode structural proteins of the heterochromatin. one of these proteins is hpi which shares with polycomb a region of similarity at the amino terminus called the ~omatin ~rganisation m~difier domain (chromo domain). the particular function of the chromo domain is not yet understood, but it seems to be important for the protein-protein interactions in chromatin associated complexes. furthermore, chromobox cdntai~ing genes were found in several species, suggesting that they are widespread in the animal and plant kingdoms. here we show chat ~. clemens contains at least one chromobox containing gene (cdna: cm08h7). length and szrusture of the putative ~. elegs~ chromo domain protein are similar to those of the chromo domain containing prozeins of other species. the expression pattern of cm08h7 is s:udied with antibodies raised against the putative chromo domain protein and by injecting a 5-ga! fusion construct inns the germ line of ~. eleman$. chromatin diminution in the parasitic nematode ascaris lumbricoides is a complex molecular mechanism, involving cleavage of the chromosomes at specific breakage regions, degradation of the eliminated chromatin and new telomere formation in all presomatic cells during the early embryonic development. the aim of the present project is to reproduce this dna elimination process in vitro, step by step or all in one, by using cell-free extracts of a. lumbricoides eggs. therefore, 4-8 cell extracts were established and their quality was assessed by the ability to transcribe 5s rrna genes (pol iii) and sl rna genes (pol li). faithful extracts were then tested for cleavage activity on dna substrates derived from different chromosomal breakage regions. finally, preliminary results revealed a possible non processiv rnase sensitive telomerase activity in the in vitro extracts, capable of adding specific nucleotide sequences to an oligonucleotide primer containing four repeats of the telomeric sequence traggc. as de novo synthesis of several kilobases of somatic telomere is likely to require a strong telomerase activity, we assume that this a. lumbricoides in vitro system is a good tool to isolate the telomerase protein and its corresponding gene or other implicated factors, s16-07 university of berne, ch 3010 berne a common feature of all mycobacteria -whether pathogenic or not -is their richness and variety of lipids. among numerous lipids widely distributed, pathogenic mycobacteria exhibit a group of sulfated surface lipids thought to be determinants of virulence. therefore, we studied sulfolipid-bioslrnthesis in pathogenic and apathogenic (opportunistic) mycobacteria species by monitoring the kinetics of [35s]sulfate incorporation into cellular lipids, pathogenic mycobaeterla (two virulent m. tuberculosis strains) showed a marked sulfolipid-synthesis with highest rates during exponential growth, while apathogenic ones (an avirulent m. tuberculosis strain and an opportunistic species) manifested negligible incorporation of the label. the partial characterization of the [35s]-sulfated lipids of pathogenic mycobacteria by thin layer chromatographic separation, combined with autoradiographic detection, revealed the presence of several radiolabeled lipid components of different polarities and with altering expressionpatterns during growth. these results strengthen the hypothesis, that sulfolipids are involved in the pathogenesis of mycobacterial disease. maiada (1,2) . some features of human pf maiada are observed in mudne malaria, although the pattern of sequestration changes due to the different plasmodial species involved, in mice, we showed by immunohistocbemistry that the expression of vcam-1 and icam-1 is upregulated in brain, lung and heart of p/asmodium bergheiinfected baib/c and also in lps-pdmed 8cid mice. in $gid mice injected with labeled human erythrocytes (e), a higher rate of sequestration to brain was observed with pfe than with uninfected e. lps-pdming increased the retention of pfe in the brain and lungs significantly (t test), but decreased it in the spleen. we conclude that pfe express surface structures which allow them to adhere to the vasculature of the brain more efficiently than uninfected e. lps increases the number of adhesive molecules on the host endothelium. interestingly, sequestration in the 8cid mouse resembles that of pfe in man. f. roth, f. riniker, k. schopfer and t. burkart inst. med. microbiology, univ. of berne, ch 3010 berne it has recently been shown that bacterial lipids cancarry antigenic determinants. the study of antibody specificity to such lipid epitopes in vitro turns out to be difficult since hydrophobic antigens have to react with watersoluble molecules. we have developed a solide phase lipid antigen immunoassay (laia) that allows to quantify the antibody reactivities with an array of lipid antigens derived from mycobacterial cells. defined amounts of extracted lipids were immobilized as equidistant lines on a nitrocellulose sheet. small strips of nitrocellulose, each carrying the same sets of lipids were incubated with different human sera. bound antibodies were then reacted with [125-i]-iodine labelled antihuman antibodies and visualized by autoradiography. the reaction was quantified by densitometry. assay conditions were optimized for antigen and antibody concentrations and appropriate incubation times. in addition, the effects of different ingredients (salts, proteins, detergent) on antigen immobilization and epitope accessability were studied. the presented laia-system is rapid, sensitive and reproducible. it allows to compare the specificities of different bacterial lipid antigens and to quantify their reactivity with serumantibodies. the atomic force microscope is a very convenient tool for studying hard and flat samples with atomic resolution. biological objects, usually soft and rough, are sometimes difficult to image using this technique. in contrast bacteria, which are too small to be observed with high resolution using the optical microscope, are hard enough to be observed with an afm at a relative good level of magnification. this kind of microorganisms can be prepared for afm imaging using very rapid and simple techniques. this method could be a convenient tool to observe the effect of bioactive substances such antibiotics. we show in this poster an exemple with the action of penicillin on b. subtilis. within the family trypanosomatidae, leishmania is a protozoan pathogen producing a broad spectrum of human disease. its life cycle is divided in two stages. promastigote is the flagellated form which colonizes the gut of the sandfly vector, and amastigote is the non-flagellated form that is specialized for growth and survival in the vertebrate host macrophage. to understand gene regulation during the transition between the promasttgote and the amastigote stage, we were interested in the characterization of stageregulated genes. we isolated a gene, sw3, from leishmania major whose mrna is more abundant in amastigote compared to promastigote. structural analysis of this gene showed that an additional polypeptide could be encoded by the opposite strand of sw3. we confn'med the presence of an open reading frame on the opposite strand by in vitro translation of the sw3 antisense rna. in vivo, an amastigote sw3 antisense transcript and the corresponding polypeptide were also detected suggesting that the sw3 is transcribed in both directions and that stage specific transcripts and polypeptides could be produced. analysis of other gene segments tend to indicate that an intensive usage of the leishmania genome to produce various polypeptides could be a general phenomenon in trypanosomatidae. the four variants and / or posttranslational modifications of histone h1 of procyclic trypanosoma brucei brucei (protozoa, kinetoplastida) were purified by hplc reversed phase chromatograpy and characterized by their amino acid composition and partial amino acid sequence. differences in sequence of up to 45% as compared to histone h1 of higher eukaryotes exist. alkaline phospatase digestion and analysis of h1 by means of hpce was used to demonstrate the presence of phosphorylated modifications. the unique biochemical properties of h1 of t. b. brucei contribute to the structural differences seen in the chromatin of the parasite as compared to that of the higher eukaryote host. larvae of the parasitic wasp chelonus inanitus (braconidae, hymenoptera) develop inside embryonic and larval stages of the host spodoptera littoralis (noctuidae, lepidoptera). parasitism induces in the host a precocious onset of metamorphosis and developmental arrest in the precocious prepupa. the wasp injects, along with the egg, pdv and venom into the host egg. injection experiments with pdv and venom revealed that pdv play a crucial role in altering host development and in suppression of the host's immune system. the genome of the pdv of c. inanitus consists of at least i0 segments of double-stranded circular dna ranging in size from 7-30 kb. we analyzed the expression of viral genes in the course of parasitization with cdna made from mrna at various developmental stages of s.littoralis. we also investigated the localization of the expressed viral genes on the various segments. in addition, these bacteria were isolated and cultured from brain tissue. to identify the infectious agent we used, among other techniques, an atomic force microscope (afm). when the. isolation fluids derived from the cortex of three alzheimer's cases were examined with afm and scanning electron microscopes, we observed bacteria whose size and morphology fit to the order of spirochaetales. these images are presented and discussed on our poster. ~ottstein bruno, institute of parasitology, ch-berne. %lveolar echinococcosis ae is due to infection with the metacestode (larval stage) of echinococcus multilocularls. an immunodominant antigen em2 of e. multilocularis was characterized by mab-gii, respective studies revealed, that (a) antigen expression metacestode stage-sdeci-fic and (b} that expression was restricted to the laminated layer. the em2-antigen i s a lectin-binding carbohydrate linked to a lipid residue. the "em2positive" laminated layer proved to be a crucial factor in protecting the parasite from host immune-effector mechanisms: only e. multilocularis larval structures exp ressing em2 were able to induce secondary alveolar chinococcosis in rodents. subsequent experimental studies in resistant (c57bl/10) versus susceptible (c57bl/ 6j and akr) mouse strains showed that resistance was as-sociated with synthesis of anti-em2 igg 3 and igg i. sus-ceptibility of c57bl/6j-mice was associated anti-em2 s and no igg 3 . the parasite-specific in vitro lym-~hoproliferative i~une response remained stationary ~ith time after infection in c57bl/10 and decreased in r and aki< mice. day 30 p.i. cd4 ⧠-lymphoblast cells dominated in all meuse strains; day 90 p.i. an in-creased nu/0ber of cd4-/cd8 + and cd4+/cd8 + cells were observed. only susceptible mice exhibited a significantly ~eereased response of lymph node cells to cona-stimula-~ien with time p.i. in conclusion, subtypes of parasitespecific cellular and humoral host immune responses seem leishmania species are protozoan parasites causing a spectrum of clinical manifestations in man ranging from cutaneous lesions to morbidity and death. the insect stage (promastigote) and intracellular stage (amastigote) differ in terms of morphology, physiology, antigenicity and gone expression. it is clear that the amastigote stage is uniquely adapted for survival within the inimical environment of the macrophage phagolysosome and that mechanisms regulating these adaptations must be called into play during the early stages of infection when the parasite undergoes transformation. in an effort to elucidate these regulatory mechanisms, we have constructed a "subtracted" cdna library which is specifically enriched for parasite transcripts expressed during the first 7 hours of infection ("switch" genes). several clones have been characterized by sequence, northern, and southern analysis. three clones, sw3, sw44, and sw20, are expressed at a several-fold greater level in the amastigote stage and potentially encode proteins which interact with nucleic acids. sw3 predicted protein is homologous with histone h1 proteins and sw44 and sw20 potentially encode ribosomal proteins. analyses of transcripts from this library are yielding clues not only to mechanisms underlying the regulation of parasite transformation, but also insights into post-transcriptional and translational control of gene expression in leishmania. $16-17 microbiology, university of geneva, medical school, c.m.u., 9 avenue de champel, 1211 geneva 4. a viral rna representing the sequence of a defective interfering rna, naturally selected for efficient replication, i.e. only containing the replication promoter, was cloned under the control of the t7 rna polymerase promoter. the expressed 17 transcript was then replicated in rive by supplying in trans the replication functions, equally expressed from plasmids. this system, completely accessible to genetic manipulations, allowed us to define a basic rule directing sendal virus replication, the "rule of six" (calain and roux, j.virol. 67 : 4822-4830, ig93). a second defective rna, representing a shorter version of the viral rna, in that it contains not only the replication but also the transcription promoters, was then cloned and replicated in the same system, although with lower efficiency. the replication of this "transcribing" viral rna was found to obey the "rule of six" as well. replicationitranscdpfion promoters were then achieved to define the cissequence requirements needed for efficient replication or for replication /transcdption. results obtained with these chimerical viral rnas will be presented and their contdbufion to the comprehension of the paramyxovirus replication and transcription processes bovine t-cells that become infected with the intracellular parasite theileria parva undergo lymphoblastoid transformation and acquire the ability to proliferate continuously. they cease to require specific antigenic stimulation, become independent of exogenous growth factors and cause tumors in nude mice. the il2 and il2r genes are constitutively expressed and the transcriptional activator nfwd3 which regulates these two genes is continuously activated. these processes are all strictly dependent on the presence of the parasite in the host cell cytoplasm and cease upon the specific killing of the parasite by the theilericidal drug bw720c. we have shown that the presence of the parasite results in the activation of the protein tyrosine kinases fyn and ick, which can participate in early t cell receptor signalling, suggesting that the parasite may use this signal transducti0n pathway to induce continuous proliferation. cysteine proteinases of leishmania as targets for chemotherapy. jacques bouvier*t and judy sakanari*. *department of pathology, ucsf school of medicine, san francisco, ca, and tanimal health, ciba-geigy, ch1566 st. aubin, switzerland. the overall goal of the study is to apply the structure-based approach to develop lead compounds and design novel enzyme inhibitors as potential therapeutic agents against leishmaniases. in this regard, we have identified and characterized the cysteine proteinases of leishmania major, and showed that they consist of excellent targets for drug design. we have purified a membrane-boand cysteine pmteinase not previously described in other trypanosomatids or protozoans and obtained amino acid sequence information from the purified enzyme. in addition, we have also characterized and purified the soluble cysteine proteinases of the parasite. consequently, we have isolated two genes encoding the soluble and membrane proteinases. the soluble cysteine proteinase gene occurs in multiple copies of 2.9 kb with flanking regions of 1.2 and 2.2 kb and is localized on one chromosome of approximately 440 kb. the membranebound enzyme gene is 3.1 kb and occurs as a single copy on a chromosome of approximately 1100 kb. a three dimensional computer model of both genes wiu be generated and enable us to scan the vast chemical database for new lead componds. we akeady have identified several cysteine proteinase inhibitors that are effective compounds in killing both promastigote and amstigote stages of the parasite in the micromolar range in vitro without affecting the host cells. dollenn~ier, g., cassinotti, p. and weitz, m., institute for clinical microbiology and irananology, 9000 st .gallen/swit zerland hav is a ~r of the picornaviridae. its rna gencrne rf~y be divided into 3 coding regions (pi, p2 and p3), qhe p3 region contains a protease 3cp ro that generates mature proteins by cis-cleavage of the initial polyprotein precursor. catalytic activity of 3cp r~ has been d6~aonstrated in a vacciniavirus/t7 ~qa-polymerase hybrid expression system. (balouter aided ali~ts of hav 3cp r~ sequence with that of other picornaviruses imply that amino acid residues his-44 and/or h) the complete hav open reading frame, hav specific expression products were identified by radioj_rmmnoprecipitation and lyy sds-page. the analysis with an antibody specific for putative nonstructural protein 2b revealed a 27,5 kda peptide. this is in contrast to its predicted size (12 kda). however, analysis with anti-(putative)2a antiserum confirmed an upstream location of the 2b n-terminus. the new 2bpeptide was also identified in lyeates of hav infected mrc-5 cells. hence, recombinantly expressed hav polyprotein underwent authentic processing and the predicted p2 organization has to be modified. we are interested in the development of animal models for one of the two major pathological changes found in the brains of patients with aizheirnar~s disease, the formation of fleuroitbrillary tangles, a main component of which is abnormally phosphorylaled tau-protaln. (tau itself is a protoin associated with mlcrotubuli.) two fundamental questions may be answered with our models: first of nit we woald like to reproduce the formation of tangles, and secondly we hope to answer the question, whether the formation of these pathological structures is sufficient for the bevalopment of senile dementia ot the alzheimer type (sdat). at present there are only a tow putative candidate genes known which may be in-voned in the hyperphosphor~lation ot tau in v/vo, one of which is the map-kinase erk2. we have cloned this candidate kinase from rat txaln rna via an rt-pcr approach and expressed the cona under,the co.rat of tour different promoters in transgenis mice. these promoters were tested for maximal expression. a similar approach was chosen to express the human taut0 isofonn in the brain ot transganic mice. we have stated to analyze different tau an~ erk2 expressing lines, northern blot analysis has revealed high levels of expression for all transgenes (up to fnetoid {n comparison to endocjenous levels). the neuronal spectficry of the transgenio erk2expression was c~nfirmsd by in situ hybridization analysis. ir~ addition several tauexpi'esalog tines were analyzed in wealernblots to determine the relative amount of htau= protein in the brain. sections of the brain were stained with tau-sp~c~c ardib~iies to identify the neurons expressing tau. from intemrosses of tau-with erk2-expressing lines, we have already identified double-positive mice which we currer~y analyze by immunohistocbemistry and in western nots to examine the phosphorylaiton status of tau. we are well aware of the possibility that only animals trans~enic for both tau and erk 2 might beveiop tang{es, whereas single transgenics mlsht not. pairs of individual neurons were recorded with sharp electrodes or in whole-cell configuratio~ in hippocampal slice cultures of rat. synaptic connections were studied between pre-and postsynaptie cells in ca3 and ca1 hippneampal fields by eliciting single aetinn potential in the presynaptic cell. monosynapfic excitatory connections were highly probable between ca3 and ca1 pyramidal ceils (p=0.76, n=82) with almost no feed-forward inhibition (p=0.01). by contrast, disynaptic ipsps, blocked by cnqx or bieuculline were observed with a very high probability (9--0.6) between ca3 pyramidal cells. monosynaptic excitation was found ha 56% of cases between ca3 neurons (n=43) but only in 27% of cases (n=ll) between two ca1 cells. monosynaptie ipsps with very short lateneies (<lms) and blocked by bicuculline, but not by cnqx, were recorded within area ca3 (5/6) and within cai (2/3), but not between ca3 and cat fields (2/2). probability of transmitlzr release, studied in recordings where failures could be unambiguously distinguished from spontaneous or small psps, was found to be close to 100% at both excitatory and inhibitory synapses. specific inhibitors of presynaptie release at excitatory and hahibitory terminals (i.e. adenosine and opioid peptides, respectively) gradually reduced the anaplitude of the psps indicating that the release at excitatory and inhibitory unitary synapses is multiquantal. the cns of a neonatal opossum, monodelphis domestic& shows profuse growth of fibers across a lesion. as in other mammals, such repair is not seen in adults. experiments have been made to define the age of the animal and stage of development at which repair ceases to occur. the entire cns was removed from animals aged 3-6 days or 11-14 days after birth. the spinal cord was crushed and growth of fibers assessed 5 days later by electrical recording of impulses conducted through the crush. repair after lesions to spinal cords of younger animals occurred in 38% of the trials (45 animals, aged 3-6 days). in nervous systems from older animals (11-14 days after birth) the success rate was low (only 4 out of 38 preparations). similarly, the carbocyanine dye dil labeled numerous fibers in spinal cords of younger animals (12 out of 21), whereas only one preparation out of 15 showed scant outgrowth into lesions in the older animals. our results suggest that there is a critical period for neuronal repair; at about 12 days nerve fibers lose the ability to grow across lesions. it is an advantage that at this later stage the nervous system is still small enough to survive in vitro. features correlated with these changes are oligodendrocyte differentiation and myelin formation. with immunofluorescent staining as well as electron microscopy myelination becomes apparent at 8 days after birth. we are now using time lapse-videomicroscopy to analyze molecular mechanisms that could play a part in promoting or preventing repair. supported by grants to j.g.n from the swiss nationalfonds (31-3626292) and from the internat. res. inst. for paraplegia. i. impulse conduction in axons has integrative functions. the dorsal root ganglion (drq) of the embryonic rat put into culture forms a monolayer and is thus accessible to eonventionalmieroelectrode technique, which allows testing the above hypothesis. we have chosen a dual approach by correlating ext~rimental el~ctrophysiological data from the chlture with results obtained from a compartrhental computer model. ii. the safety factor for conduction was found to be low. thus failures of ap invasion of the drg cell soma could occur at sites of impedance mismatch when a second stimulus felt into the relative refractory period of the first or when the axon was repetitively stimulated. the electrotonic resiuues (e.rs) of the failed aps had discrete amplitude levels, suggesting that the failures were always caused at the same site along the axon. these sites of low safety factor were found to be the branch point in the unipglar drg cell and the entrance of the stem piece into the soma in both cell types, the bipolar as well as the unipolar. a systematic comparison of 15oth cell types showed that the ap conduction through the latter is more reliable.'the length of this stem piece is inversely' correlated to the delay caused at the branch point, as tile electrical load of the soma is more efficiently masked by a long stem piece. the dendrites of motoneurons (and subsequently many other neurons) have been assumed to be electrically passive since the work of coombs, ecc]es and fatt in the 1950's. however, direct evidence has been impossible to come by until recently with the advent of appropriate dyes and equipment for measuring very small and very fast optical signals. we examined the propagation of action potentials, both spontaneous and evoked, in the dendritic trees of spinal cord neurons using a 12 â�¢ 1 2 array of photodiodes and a ccd camera as weft as conventional microelectrode techniques. organotypic slice cultures of embryonic rat spinal cord were stained with a voltage-sensitive dye (di-8-anepps) or a calcium dye (fluo-3) and recordings were made from motoneurons identified by their location and morphology. the results indicate that there is in fact an increase in calcium in the dendrites that accompanies action potentials whether synaptically evoked or evoked by current injection at the soma. spikes in the dendrites up to 1 60 i~m from the soma are much larger than could be explained by models assuming passive dendritic trees. work is currently underway to test the possibility that sodium conductances might contribute to the propagation of action potentials in the dendrites. cat auditory cortex contains multiple cochlear representations in both hemispheres that are interconnected through the corpus callosum (cc). the distribution of auditory callosal axons was studied on the mediosagittal level after injections of biocytin at electrophysiologically identified locations. single injections were done in ai (2 cats), aaf, aii and paf; multiple injections in ai & aaf in one cat. an additional cat received 1 injection in sii. the mediosagittal image of cc was subdivided into 30 segments along a rostro-caudal axis. labelled axons crossing the midline were counted in coronal or horizontal sections. axons from auditory fields were found in the posterior 2/3 and those from the somatosensory cortex in the anterior third of cc. axons from a discrete injection (ca imm in diameter) spread over as much as i/5 of cc. the rostrocaudal distribution of axons at the midline reflected roughly the rostrocaudal position of the field of origin, but apparently not the tonotopic order of a given field. the major efferent projections of substantia nigra pars reticulata (snr) convey information to the thalamus, to the tegmentum, to the superior colliculus, and to the spinal cord. such a range of targets suggests a highly organized activity within snr and this study investigates the temporal organization of spike trains. in the portion of snr between +2.7 and +4.5 mm interaural levels, we recorded 40 single units along 6 electrode tracks in 4 young female wistar rats. most units (n=32) fired in a non-regular poisson process, although only a small number (7/32) was characterized by an average burst size with more than 3 spikes. crosscorrelograrns were computed for 25 pairs of units simultaneously recorded from the same electrode and for 26 pairs simultaneously recorded from distinct electrodes. most crosscorrelograms (27/51) showed a symmetrical peak centered on time zero, which indicates a tendency to synchronous firing. when units were recorded from the same electrode tip we observed that 1/8 to 1/40 of their spikes were synchronous. about half of the significant interactions were characterized by a relatively long duration (>250 ms) and are likely to correspond to a different mechanism of synchronization. these results support the hypothesis that snr cells receive common afferences of two different types and their fine temporally organized activity yields coordinated activity between the target structures. the neuronal microtubule-associated protein map2 binds to microtubules via a domain containing 3 or 4 repeats of an 18 amino acid sequence. we have previously shown that when cultured nonneuronal cells are transfected with map2 their microtubules become stiffer and are then capable of supporting process outgrowth when their cortical actin cytoskeleton is depolymerised. we hypothesise that this stiffening effect of map2 is caused by the 18 amino acid sequences binding to neighbouring tubulin subunits in the walls of the microtubules thus reducing their freedom of movement relative to one another. to investigate this further we have created map2 mutants in which 1 or 2 repeats of the tubulin-binding motif were deleted. these were tested by transfection and expression in cultured cells. the results confirm that the number of repeats affects the stability, stiffness and the cytoplasmic arrangement of cellular microtubules. we were previously able to demonstrate the presence ot kinin-like immunoreactive structures in the mouse brain. in order to characterize them, we have now performed a donble-immunofluorescence study using both polyclonai auti-srudykinin (bk) and mouse monocloual anti-neeron-specificenolase (nse) antibodies. the latter is considered to be a specific neuronal marker. after fixation with bonin-hollande solution, the brains were removed, embedded in paraffin and serially sectioned at 10 am. the sections were incubated with the antibodies, which were visualized by an indirect immunofluorescence technique using fitc for nse and by an avidin-biotin technique using texas red for bk. double-immunofluorescent cells were found in the thalamus ventralis, in the nucleus cechleuris ventralis and in the nucleus mesencephaficns nervi trigemini (v). bk-positive cells in the nucleus principalis v were nse-negative ih spite of their typical neuronal aspect. the other bk-positive structures (pia, ependyma, hnic~es) also were alwa~ nse-negatlve. most of the nsepbsifive bk-negative cells were seen in the cortex. the neuronal nature of many kinin-like immunoreacfive structures in the mouse brain is thus ascertained. in contrast to the amphibian optic nerve (on), on of adult mammals is unable to regenerate after injury. astrocytes disappear from and macrophages accumulate at the lesioned site (blaugrund et al., j. neurocytol, 118, 105, 1992) , we have followed the distribution of astrocytes and microglial cells in xenopus tadpole ons over 10 d after mechanical crush. astrocytes were stained for cytokeratin and vimentin, and microglial cells for griffonia isolectin b4. soon after crush, immunoreactivity for intermediate filament proteins was strongly increased. astroeytic processes extended into the lesion from both proximal and distal stump. at ,5 d, astrocytes had crossed the injured region. in the distal stump, some of them contained vacuoles indicating phagocytic activity. while in the normal on microglial cells were ramified and scarce, 2.5 d after crush they were roundish and vacuolized, and scarce within the lesion but numerous distal to it. at 10 d, their number was decreased and most of them had reacquired ramifications. conclusively, in the tadpole on, astrocytes are virtually never absent from the lesion site but rather extend rapidly into it. astrocytes may thus provide a guiding support for outgrowing axons, microglial cells that are frequent in the distal stump, do not accumulate at the lesion. the behaviour of the two cell types differs profoundly from that in mammals and is likely to be one of the factors that may contribute to successful neuronal regeneration of the amphibian on. ci4mence, j. f. 1, ranieri, j. p.2, aebischer, p.2, and sigrist, h. 1, 1institute of biochemistry, university of berne, ch-3012 berne; 2division autonome de recherche chirurgicale, chuv, ch-1011 lausanne. small peptidic sequences of laminin (yigsr, ikvav) are known to promote attachment and/or neurite outgrowth of various neuronal cell lines (pc12, nb2, ng108-15). new and established heterobifunctional photo crosslinkers were used to immobilize these peptides in a topically defined fashion onto biocompatible surfaces such as hydroxylated fluorinated ethylene propylene (fep-oh) and pely(vinymlcohol) (pva) . n-[m[(3-trifluoromethyl) diazirine-3-yl]phenyl]-4-maleimido-butyramide (mad) was thermochemically linked to the synthetic peptide cdpgyigsr via its n-terminal cysteine, leaving the bioactive part (yigsr) free for cell receptor interaction. mad-cdpgyigsr was radioactively labeled by reductive methylation and purified by reversed phase hplc. patterned (20 and 300 #m) peptide immobilization was attained by photocoupling onto pva and fep-oh and visualised by autoradiography. light-structured biomaterial surfaces with molecularly oriented nerve growth promoting peptides were investigated for spatially controlled neuronal cell attachment and differentiation. it is striking to observe that these inhibitory or stimulatory effects were corroborated by binduced decrease or increase in 2-deoxyglucose uptake, respectively. this gives b a putative role in the limbic regulation. the sheet-like processes of oligodendrocytes wrap themselves spirally around central axons, thus formtng the myelin sheath. a single oligodendrocyte may donate internodal segments of myelin to each of 20-30 or more adjacent axons. it is not known, if one oligodendrocyte picks out axons randomly or if it prefers axons with a certain diameter or with a certain chemical specificity. we have studied this last possibility by combining intracellular injection of a fiuorochrome, and immunohistological detection of calciumbinding proteins (cabp's), markers for the axons ef certain subpopulafions of nerve ceils. lucifer yellow was injected into oligodendrocytes of the optic nerve, of living rat brain slices. the oligodendrocytes were identified by their electrophysiologieal properties. slices containing the iniected cells were immunostained /or parvalbumin or calretinin using second antibodies labelled with texas red. using co.focal laser-scanning microscopy combined with a three*dimensional reconstruction programm, the relationship between oligodendrocyte processes and axons positive for one of the cabp's was determined. for ultrastructural confirmation, single, lucifer-yellow injected, oligodendrocytes were photoconverted, and the cabp's-positive axor~ were revealed by gold-labelled antibodies. the few oligodendrocytes injected show the classic morphology, that is of a small cell body and few processes running parallel to the course of the axons. the proximity between glial processes and positive axons can be easily appreciated in confocal laser-scanning images. a preferential assodation of oligodendrocyte processes with parvatbumin-positive axons has not as yet been found. thus, the confirmation of the hypothesis that oligodendrocytes choose their axonal targets according to chemical cues awaits further work including the injection of a lar~er number of these cells. to initiate a molecular genetic analysis of brain development in insects, we are characterizing the mechanisms of embryonic brain development in the fly drosophila. early in embryogenesis, a small number of molecularly diverse brain neuroblasts generates the neurons of the brain. subsequently, pioneering brain neurons initiate axonal outgrowth along glialbound brain compartments. these pioneering neurons establish the primary brain tracts. the pioneering brain axons express the cell-cell adhesion molecule fasciclin i dynamically during outgrowth and initial fasciculation; a subset of the pioneering axons expresses fasciclin ii. the axonal framework of the embryonic brain tracts is used for outgrowth and fasciculation by subsequently differentiating axons and, thus, prefigures the tracts of the mature brain. a comparison of early brain development in drosophila and in vertebrates reveals common mechanisms for brain development. supported by the swiss nsf. institute ot histology, university of fribourg, p~rolles, ch-1700 fribourg calretinin (cr), an ef-hand ca2+-binding protein, is expressed in cajai-retzlus cells during development of the rat cerebral cortex. these cells are located in the marginal zone and are the ear{iest cortical neurons to be generated. they have been consldered as unusual on account of their morphology and their fate during corticogenesis. the functional significance of cajai-retzius cells and their destiny in adulthood is still controversial. in order to approach these questions we have applied organotypic culturing methods. slices of neocortex from brains of 0-6 days-old rat pups were cultured for 10-21 days applying the interphase technique (stoppini etal, j. neurosci. methods 37, 1991) or the roller-tube technique (g&hwiler, j. neurosci. methods 4, i981) . the fixed cultures were immunolabe0ed with an antibody against cr. the obtained results showed that cr positive structures develop in vitro like in vivo. however, in organotyp[c cultures their distribution corresponded to a more mature stage than the situation on the day of the explantations. many cr-positive cells were seen in layer l they were horizontally oriented or had a pyriform shape. based on their morphology these ceils were identified as cajai-retzius cells. orpositive cells were numerous in layer i1-l[i and showed mostly a bipolar morphology. some muripo[ar cells could also be observed. a fine net of crpositive fibers and puncta was found in layers [ and iv. the demonstration that cajai-retzius celts are detectable in these cultures will allow us to follow the fate of these cells dynamically and [nte~ene in their function by applying exogenous factors. cervical primary afferent input to vestibule-spinal neurones projecting to the dorsal horn: a double labelling study in the rat. wheat germ agglutinin-horseradish peroxidase (wga-hrp) was injected by pressure into cervical spinal ganglia c2 or c3. in the same experiments fluorogold (fg) was iontophorefically injected into the ipsilateral dorsal horn of different cervical spinal cord segments. anterogradely labelled fibers (wga-hrp) were present mainly in the external cuneate nucleus, but also to a lesser extent in the caudal part of the medial and descending vestibular nuclei (mvn, dvn) . the fg injections, restricted to the cervical dorsal horn, revealed retrogradely labelled cells in the central part of the caudal mvn. a double exposure (fluorescent and polarisadon optics) under the microscope showed primary afferent fibers surrounding like baskets retrogradely labelled fg cells. the projection from cervical ganglia cells to the mvn represents a pathway for direct afferent information from neck receptors (in particular muscles) to vestibular nuclei and supplies the mvn with afferent information which could enable the vestibulospinal nenrones, projecting to the dorsal horn, to influence the local information processing. in the sciatic nerve, all myelinated and non-myelinated axons were snap-ir. in peripheral tissues, all nerve endings were positive. the preterminal schwann cells of motor axons were also snap-25-ir. after sciatic nerve section, many myelinating sehwann cells also expressed snap-25-ir. in conclusion, snap-25 is expressed by neurons in the pns. it seems to be also expressed by preterminal schwann cells and by myelinating schwann cells during regeneration. $17-19 repair of completely transected spinal cord of neonatal opossum in culture h.a. vischer, dept. pharmacology, 8iocenter, uni. of basel, switzerland woodward et al. (j. exp. biol. 1993 , 1993 have shown that fibers grow rapidly and profusely across a lesion made by crushing the spinal cord of the neonatal opossum monodelphis domestica. in such experiments careful controls must be made to establish that fibers were in fact broken by the lesion. tests have now been made to determine whether similar repair can occur after a more drastic lesion involving complete transection of the cord. after dissection of the entire gns from animals aged 3-8 days, the cord was cut into two with scissors at the c5 -c7 level. the two-ends were glued together with matrigel on a sylgard mold, which encompassed and held the cns. the rostral end was labeled with the fluorescent carbocyanine dye dil in order to visualize growing fibers. in 33 preparations fibers grew over the cut into the separated part of the spinal cord. such growth could extend to 500 ixm. fibers grew straight, branched and multidirectionally, or in fascicles. in another set of experiments cords were combined from different animals of the same or different ages. again, fibers could grow across the cut but they did so less frequently. these results set the stage for investigating fiber growth after rotating the two ends at a defined angle and for analyzing factors that influence the direction of growth. map la is a microtubule associated protein of 360 kd. in rat brain two monoclonal antibodies, a and bw6, recognized map la in neurons and their axonal and dendritic processes. in mouse cerebellum, monoclonal a recognized purkinje ceils and granule cells uniformly, as already described for rat brain. in contrast, monoclonal bw6 stained selectively some dendrites of purkinje cells, forming bands in the molecular layer. we compared this striation with that obtained with a monoclonal antibody, anti-zebrin ii, which recognized aldolase c. in the mouse vermis, zebrin stained in a striated pattern, but complementary to bw6. these results demonstrate that map la is present in forms which are differentially distributed in the mouse cerebellum. these observations may be explained either by differences in metabolic states of neurons, or by differences in their regional functions, or by differences in the regional stability of microtubules. this work was supported by grant no 31-33447.92 from the swiss national science foundation. serum free aggregating brain cell cultures prepared from 16 day old fetal rat telencephalon are used as a model to study brain development. in these cultures it is possible to distinguish ontogenetic events such as cell proliferation, differentiation and myelination, in the present study we examined synaptogenesis by analyzing the expression of synaptic proteins such as synaptophysin, snap-25, synapsin i, and brain spectrin. different developmental stages were analyzed by western blotting. immunoreactivity for synaptic proteins was detectable already at day 12 in culture, suggesting that the first synapses are already present at this early stage. the expression of synaptic proteins strongly increased between day 20 and day 30 in culture, reflecting a period of intense synaptogenesis. treatment of the culture with an elevated concentration of kci (30mm) increased the expression of synaptic proteins, suggesting a stimulation of synaptogenesis. these findings demonstrate the utility of this approach to study brain synaptogenesis, and possibly synaptic plasticity. drenhaus, u.i gunten, a.v., rager, g. ; institute of anatomy, university of ch-1700 freiburg the retina of tupaia comprises three types of ganglion cells (rgcs) analogous to x-, y-, and w-cells found in other mammals. these cells differ in size and in their distribution pattern: large rgcs are frequent in temporal, small rgcs in nasal retina. as the fiber diameter correlates with the size of its respective rgc, it can serve as a parameter for the topographic representation of rgcs in the nerve. to study this we analyzed the optic nerves of three tupaia. using the electron microscope we recorded the density, diameter, and the position of fibers in the nerves. we found, that fibers with the largest diameter are located dorso-temporally and close to the center of the nerve. they are surrounded by zones of smaller fibers. the diameter decreases gradually towards the periphery and is smallest in the ventro-nasal region of the nerve. since the fiber diameter distribution corresponds to that of the size of rgc-types in the retina, we assume that the topographic relationships in the nerve and the retina are similar. (supported by s 17-23 stoppini luc, s. duport, l. parisl, c. oropes~, departement of pharmacology, cmu, 1211 geneva 4 the micro environment of the central nervous system is important for neuronal function. in this context, the blood-brain (bbb) provides and maintains the extracellular medium that is compatible with normal neuronal and synaptic activity. due to the difficulties inherent using whole animal as an experimental model to study permeability and metabolic processes at the cellular level, major efforts have been engaged in recent years, in order to bring up suitable /n vitro models. we are presently developping an in vitro bbb by interfacing a coculture of endothelial cells monolayem and nervous organotypic cultures. the main idea of this study is based on the premise that the complex intercellular interactions between the different cell types pertaining to the nervous tissue and the neighbeurlng endothelial cell is of fundamental importance to promote a realistic bbb system. we expect that erganotypic culture of nervous tissue, combined to endothelial cell monolayers, will initiate and maintain a bbb phenomena/n vilro. we will test more specifically the hypothesis that neuronal activities [spontaneous or elicited) will influence gila] cell response which will in turn modify endothelial properties. preliminary results clearely show a good nervous tissue and endothelial cell survival. tight junction llke structures could be identified using freezefracture or conventional transmission electron microscopic thechniques. {work supported by chemodyne gent~ve ). the process of myelination is a prerequisite for the proper function of the brain since it enables rapid saltatory conduction of axons. in the central nervous system (cns) myelination is performed by oligodendrocytes. a differential screening approach was used to isolate new rat cdna clones that are expressed in this glial cell type. here we report the further characterization of two of these novel cdnas which appear to be expressed specifically in oligodendrocytes. the two characterized cdna clones (tentatively called cns1 and cop-25.1) share an approximately 420 bp region of sequence identity which suggests that they are dedved from the same gene by alternative splicing, within this area a putative open reading frame is located. we demonstrated by northern blot analysis and in situ hybridization that the two mrnas of the novel gene are expressed exclusively in oligodendrocytes. however, the mrnas show a different specific spatial localization within the cell. we compared mrna expression of myelin basic protein (mbp) and proteolipid protein (plp), with that of cns1 and cop-25.1 in brain and optic nerve during development. it appeared that the mrnas of the novel gene were delayed significantly as compared to mbp and plp mrnas. streit. phyaiologisches institut, btihlplatz 5, 3012 bern in organotypic slice cultures of the embryonic spinal cord, rhythmic spontaneous activity arises when the inhibitory synaptie transmission is blocked. the rhythmic activity consists of bursts of regular oscillations of activity at 4 -5 hz. this study investigates the origin of the regular oscillations. when stimulated electrically at 5hz, the synaptic transmission in the cultures showed strong depression by about 50% on average. the synaptie depression was highly variable among individual connections and tended do be less pronounced in frequently used connections. when random depression ratios with an average of 50% at 5hz were implemented in a computer model consisting of 100 randomly connected excitatory cells, regular oscillations of activity did not arise, even in the presence of synchronous pacemaker cells. however, when individual depression ratios were adapted according to the rate of activity of individual connections, regular oscillations at 4 -5 hz arose in the presence of few unsynchronized pacemakers. this finding suggests that the regular oscillations in the cultures originate in a network formed by the plasticity of synaptic depression. maier a., schlumpf m., beer h.f., sehubiger p.a. and lichtensteiger w., inst. of pharmacology, univ. of zurich, the ontogeny of expression of dopamine d 1 receptor mrna in the rat brain and binding to the receptor was studied at 12 time points from gestational day (gd) 13 to postnatal day (pn) 60 by quantitative receptor autoradiography and in situ hybridization.long evans rat pups and fetuses of time pregnant rats were frozen and sectioned coronally and sagitally at i0 um. d 1 recepto~o~inding , determined with the selective antagonist ~ji-sch 23982 was first noted on gd 18 in the developing striatum, basal ganglia and the olfactory tubercle, reaching adult levels at around pn 14. the expression of d i receptor ~na was studied by using a mixture of 3-~js-labelled oligonu-specific cleotides. on gd 13 messages were noted in the developing stiatum, olfactory tubercle and retina. this study shows a good regional correlation between the development of receptor binding and mrna, however, mrna precedes detectable binding to the receptor by several days. earlier work had shown that q~ replicase forms complexes with q~, plus strand rna via interactions at two internal binding sites, the s-and the m-site, while binding of the 3'-end is mediated by host factor. in contrast, the 3'-end of the minus strand appeared to be directly accessible by replicase. we have prepared a series of plus and minus strand variant rnas containing either internal deletions or terminal deletions extending from the 5'-end. the template activities of the variants were determined by single-round replication assays. for the plus strand we find that while deletion of the s-site remains without effect (as expected from previous results), deletion of the m-site reduces template activity to 25 % or less compared to wild-type rna; the residual activity shows a decreased dependence on the presence of host factor. the results agree with an important role of the m-site interaction both with replicase and with host factor for the formation of productive initiation complexes. the template activity of the minus strand was unexpectedly found to be strongly dependent on the presence of a segment between nt 76 and 210 from the 5'-end. this shows that recognition of the minus strand by rep(icase does not only involve interactions near the 3'-end but requires a previously unknown structural feature near the 5'-end of this template. $18-02 karsten graning and angela kr&mer d~partement de biologie cellulaire, universite de gen~ve, 30, quai emest-ansermet, 1211 gen~ve 4 splicing of nuclear pre-mrnas takes place within multicomponent complexes (spliceosomes) that are assembled by interactions between the pre-mrna, snrnps and protein splicing factors. we have purified two splicing factors that function in the formation of the pre-splicing complex. sf3a consist of three subunits of 60, 66 and 120 kda and corresponds to three proteins present in the functional 17s but not in the 12s u2 snrnp. it binds to the 12s u2 snrnp only in the presence of sf3b, suggesting a two step assembly pathway of 17s u2snrnp: binding of sf3b to the 12s u2 snrnp generates a 15s intermediate particle that is converted to the active 17s u2 snrnp by the subsequent association of sf3a. comparison of proteins enriched in the sf3b fraction with polypeptides found in the purified 15s u2 snrnp suggests that sf3b comprises at least five of the other 17s u2 snrnp specific proteins and together with sf3a promotes the u2 snrnp/pre-mrna interaction. by edna cloning, two of the subunits of sf3a have been identified as homologs of well characterized yeast proteins that function at the same stage of spliceosome assembly in yeast. splicing factor sf1, a heat-stable 75-kd protein, functions early during spliceosome assembly. tryptic peptides of sf1 were sequenced and cdna libraries were screened with degenerate oligonucleotides. the information obtained by comparing the sequences of three overlapping cdnas suggests the existence of at least three mrnas that could be derived from a common pre-mrna by alternative splicing. in agreement with this assumption mrnas of the expected sizes that are differentially expressed depending on cell line or tissue are detected by northern blotting. in theory, three polypeptides could be generated that differ by the presence or absence of 7 internal amino acids and in their ctermini. the deduced amino acid sequence is rich in prolines and contains several possible phosphorylation sites and a putative leucine zipper. proline-rich domains, which are also present in splicing factors psf and sap62, as well as leucine zippers have been found in transcription factors and could mediate protein/protein contacts, suggesting that sf1 could function during splicing by interaction with other spliceesomal proteins. pre-mrna splicing is catalyzed by a multicomponent complex (the spliceosome) that consists of small nuclear ribonucleoprotein particles (snrnps) and non-snrnp protein factors. the spliceosome is assembled in a stepwise fashion on the pre-mrna. chromatographic fractionation of hela cell nuclear extracts and subsequent reconstitution of splicing in vitro has allowed the separation and isolation of snrnps and several protein factors. sf4 triggers the transition between splicing complex b, which contains all spliceosomal snrnps but is not competent for catalysis, and complex c, the active spliceosome. this transition involves a conformational change that leads to altered base pairing interactions between snrnas and pre-mrna. the purification of sf4 has been impeded by its low abundance; however, a correlation between sf4 activity and a polypeptide of 50 kd could be established in the most purified fractions. we are currently investigating whether this protein represents sf4. interestingly an atp-dependent rna-helicase cofractionates with sf4 through several purification steps. whether or not this activity is relevant for the splicing process is under investigation. selection of iron-responsive element rnas with high affinity for iron regulatory factor henderson, b.r., menotti, e., bonnard, c. , and kith_n, l.c., isrec, ch-i066 epalinges iron regulatory factor (irf) post-transcriptionally regulates iron homeostasis via binding to mrna iron-responsive elements (ires). the ire loop sequence (5'-cagugn-3') and "bulge" cytosine are phylogenetically conserved. we prepared a pool of 16,384 ire molecules randomized at these 7 nucleotide positions, and employed in vitro selection to identify optimal sequences which bind human irf. we define two major classes of high affinity rna ligand, the optimal loop sequences of each are 5'-cagugn-3' (wild-type) and 5'-uaguan-3'. this novel finding predicts base-pairing within the loop between positions i and 5. all nucleotide substitutions in the "bulge" or at loop positions 2,3 and 4 decreased binding by 36% to 99%. in addition, binding specificity of rat irf differed with that of the related rat ire-binding protein, irf b. in vitro analysis of ire-like stem-loops identified by computer search has not yet revealed any new ire-containing genes. a73 $18-08 3' processing of histone pre-mrnas: a phylogenetic comparison reto kohler, petra duda and daniel schiimperli. zoologisehes institut der universtit/it bern, abteihing fiir entwicldungsbiologie, baltzerstr. 4, ch-3012 bern, switzerland. we are using a phylogenetic approach to study the biochemical reaction by which animal histone pm-mrnas are processed at their 3' ends. in mammals, the efficiency and specificity of this reaction is known to depend absolutely on the u7 snrna which interacts with conserved spacer sequences downstream of the proc~sing site. a highly conserved hairpin element which precedes the cleavage site serves as a binding site for an additional processing factor, but its importanea for efficient processing varies greatly between different historic genes and between extracts of different mammalian cell lines. to determine whether the relative importance of the hairpin and spacer dements have been conserved during evolution, we analysed the processing of histone genes from three different animal phyla in vitro, i.e. vertebrates (mouse), arthropods (drosophila melanogaster) and nematodes (c. elegans). in the mouse system, processing was strongly dependent on the presence of a mouse spacer element. in nuclear extracts of drosophila ke cells, processing occurred exclusively when a drosophila spacer element was present in the rna. processing efficiency was not reduced by foreign (mouse, c. elegans) hairpins. in whole cell extracts of ascaris lumbricoides embryos, an inverted situation was observed. 3' processing products were only produced by rnas that included an upstream segment derived from a c. elegans histone gene, irrespective of the spacer sequences present. these surprising results correlate with certain unique features in the c. elegans upstream element. we are currently in the process of cloning ascaris lumbricoides histone genes, which will allow us to verify our results in a homologous system. in xenopus laevls ooeytes, wild type mouse u7 rna gets assembled into functional u7 snrnps, both when transcr~ed from an injected gene or when injected as/n vitro synthesized rna. if the sm binding site of u7 rna is converted into the canonical sm sequence (u7 sm opt), the rna assembles into a particle which accumulates more efficiently in the nucleus but wbach is nonfunctional. this u7 sm off particle inhibits the function of preformed endogenous u7 snrnps, most likely by nonproductive binding to histone pre-mrnas, histone pre-mrna processing can be demonstrated in c/s if u7 rna is placed 3' of hlstone pre-mrna and injected into oocytes. only u7 sm wt sequences are active in this c/s processing. three proteins can be photoaffinity cross]inked to u7 sm wt rna, the common snrnp proteins g and b/b' and a u7-specific protein of 40 kd (50 kd in mouse). these crosstinks map to closely spaced positions within the sm binding site with the u7-specific crosslink being located most 3'. u7 sm opt rna does not become crosslinked to the u7specific protein and is also more tightly associated with sm proteins. we now investigate the in vitro assembly of u7 snrnps using these characterized u7 rnas and a preparation of highly enriched snrnp proteins. u7 sm wt and u7 sm opt rnas associate with common sm proteins in this system, but the interaction with the u7-speciflc protein could not be demonstrated. the double-stranded (ds)rna modifying enzyme, which can convert adenine to inosine, was first identified in xenopus laevis, but has since then been detected in different species and in mammalian cells. the enzyme is a specific adenine deaminase which uses dsrna as substrate and recently, it has been postulated to be responsible for a specific mrna editing reaction. we have purified this enzyme to homogeneity, approximately 400,000 fold from calf thymus whole cell extracts. the protein was purified primarily by ion exchange chromatography over six columns, with the final step being chromatography on a dsrna affinity column. the purified protein has a molecular weight of 115 kd and is the only protein present when enzymatically active fractions are visualized on an sds-polyacrylamide gel stained with silver. the dsrna modifying enzyme is a very low abundant protein. we are in the process of further characterizing the purified enzyme and of cloning cdnas coding for it. detailed mutational analysis of histone rna 3' end formation carmen spycher, andr6 furger, adrian streit, daniel albrecht, d. schiimperli, zoologlsehes iustitut der universtit/it bern, abteilung thr entwicklungsbiologie. baltzerstr. 4, ch-3012 bern, switzerland histone rna 3' ends are formed by cndonucleolytic cleavage resulting in poly(a)" mrnas with a highly conserved 3'-terminal hairpin loop structure. for the mouse h4-12 gene major and minor processing sites are located 3 and 5 nucleotides downstream of the hairpin, respectively. for the cleavage reaction, the u7 snrnp interacts with the spacer element of histone pre-mrna by base-pairing through the 5' end of its rna moiety, u7 rna. we have observed that this base-pairing potential extends further than previously recognised in either direction. we have made site-directed rnutagenesis in fltis potential hybrid region and around the processing site. rna processing and complex formation with the u7 surnp were analysed by incubation in nuclear extract from mouse k 21 cells. our results indicate that base-pairing with u7 rna both in the classical spacer element and downstream of it are very important for histone rna processing. in contrast, sequences upstream of the spacer element do not seem to be involved in base-pairing with u7 rna, but mutations in this region may affect processing by other mechanisms. systematic mutations of the highly conserved four nucleofides immediately following the hairpin show no qualitative or quantitative effects in the processing reaction. alteration of nucleotides 5 to 7 around the minor processing site, however, result in a dramatic decrease in processing efficiency and alter the specificity of the cleavage site. in further experiments we will introduce specific nuclcetidc modifications at the two processing sites, which should allow us to characterize potential cleavage factors by chemical crosslinking. evolution callaerts p., glardon s., j.,oosli f., quiring r. and gehring w. cell biology, biozentmm, basel. the pax genes encode a family of dna binding factors which share the paired-box and play an important role in the control of development. they can be subdivided into four different classes which differ in the presence or absence of other conserved sequence motifs coding for a paired type homeobox or an octapeptide. the pax-6 gene, which contains a paired-box and a homeobox, has been cloned from humans, mice, rats and zebra fish. recently, we have cloned the pax-6 homologue of drosophila melanogaster. the five genes share a very high degree of sequence identity both in the paired-domain and the homeodomain. similar to its mammalian counterparts, the drosophila gene is expressed in the eye primordia and the nervous system. in addition, mutations in pax-6 affect eye development: aniridia in humans, small eye in mouse and rat, and probably eyeless in drosophila. this would imply that the pax-6 homologues share a conserved function in eye morphogeuesis in both vertebrates and invertebrates. furthermore, this suggests that pax-6 may have been present in a common ancestor. therefore, we are now trying to isolate pax-6 homologues from other, more primitive organisms by means of the polymerase chain reaction, and to determine whether they are implicated in eye development. putative candidates have been identified from a number of species and are being characterized. their sequence similarity and some evolutionary implications will be discussed. keegan liam and gehrin~ walter j., biozentrum, university of basel, klingelbergstr. 70, ch-4056 basel, switzerland using an enhancer map screen we have identified two genes that may be direct targets of antennapedia involved in forming the adult leg. spalt major is repressed in the leg imaginal disc and tea, shirt is activated by antennapedia. we wish to determine whether the control of these genes by antennapedia is direct, spalt major is expressed in a ring in the antennal disc and is repressed by antennapedia in the leg disc. the antennal ring is repressed by ectopie antennapedia expression that transforms the antenna to a leg. we are defining an enhancer at spalt major that is required for antennal ring expression and repression by antennapedia. we are using various approaches to show that antennapedia binds to the antennal ring enhancer at spalt major. these include polytene chromosome banding studies using antibodies to antennapedia as well as in vitro dna-binding and genetic experiments. in search for novel regulators potentially involved in myogenesis, we identified a homeobox of the paired type in human muscle. subsequent cdna cloning revealed that we had cloned the full length coding region of human pax7. our human sequence extends the known mouse edna both on the 5' and the 3' end. as a first step in order to test for a functional role of pax7 in myogenesis, we analyzed its expression pattern during chicken development. transcripts are present in the neural tube and in the dermamyotome of the developing somites. therefore, the pattern of expression of pax7 is conserved between mouse and chicken. next, we assessed the expression of pax7 in different mouse and human cell lines. we found pax7 transcripts specifically in myogenic cells and not in any other cell types. interestingly, pax7 is already present at the myoblast stage. moreover, 10ti/2 fibroblasts converted to myoblasts by either transfection of myod or treatment with 5-azacytidine expressed pax7, whereas the parental cells did not. while myod was able to activate pax7 in 10t1/2 cells, expression of pax7 was not sufficient to induce the myogenic phenotype. nevertheless, our expression data are consistent with a role of pax7 during the mesodermal commitment to the myogenic lineage. the segmental organization of the drosophila head is achieved by a flow of positional information from maternal gene products to the zygotic gap genes. recent genetic analysis has identified three new gap genes involved in head development. one of these genes is the empty spiracles (ems) gene. mutations in eras cause severe defects in the head and the filzk~rper at the posterior end are missing. the eros gene encodes a putative transcription factor with a homeodomain. the eros rna is expressed in two phases of embryonic development. first expression is seen at the blastoderm stage in a single anterior band, correlating with its function as an anterior gap gene. later during embryogenesis eros is expressed in the posterior spiracles as well as lateral regions of each segment where the tracheal pits form and lateral neuroblasts originate. using g-gal fusions we could identify at least five different regulatory elements in the eros promotor region responsible for tissue specific expression of the gene. a 300 bp element responsible for the early expression which is dependent on the maternal gene bicoid, the key gene of the anterior system, and the terminal gene tailless was identified and studied in detail. this element contains two bicoid and two tailless binding sites. the effects of muations in these binding sites on eros expression will be discussed.this analysis should allow us to elucidate the molecular mechanisms by which the morphogen bicoid regulates subordinate target genes like ems in the drosophila embryo. the pax-6 gene of the mouse encodes a transcription factor with both a paired-and a homeodomain. pax-6 is affected by several allelic mutations in the small eye (sey) gene. sey mutations cause a reduction of the eyes in heterozygotes, and homozygotes lack both eyes and nasal openings and die as newborns. the corresponding mutation aniridia in humans affects eye and cranlofacial development in a similar manner. we have isolated a pax-6 homolog from drosophila (dpax-6), which shares more than 90% sequence identity in the paired-and the homeodomain with the mammalian genes. also outside of these domains we find considerable sequence conservation arguing for a true pax-6 orthologue. the drosophila gene spans ~16 kb and is expressed in the brain and the ventral nervous system of the embryo, and in the eye imagjnal discs and the brain of the larva. the gene was mapped to the eyeless (ey) locus. mutations at the ey locus cause either the partial or complete absence of the compound eyes or embryonic lethality. in one ey allele we have identified a transposable element in the first intron of dpax-6, which affects dpax-6 transcription in the eye discs, suggesting that dpax-6 is encoded by the ey gene. this implies that pax-6 (sey) in the mouse is homologous to ey in drosophila. thus, despite of the different modes of eye morphogenesis, the same gene seems to control eye development in insects and vertebrates. the drosophila homolog of the vertebrate serum response factor (srf) was isolated by low stringency-hybridization. nucleotide sequence analysis revealed that the drosophila srf homolog (dsrf) codes for a protein which displays 93% of sequence identity with human srf in the mads domain, the region required for dna binding, dimerization and interaction with accessory factors. the dsrf gene is expressed during several phases of embryonic development. both the rna and the protein are maternally provided to the egg and slowly decrease in their levels during gastrulation. after germ band retraction, high levels of zygotic expression were observed in a distinct subset of peripheral tracheal cells distributed throughout the embryo. many of these cells are at the tip of tracheal branches and are in direct contact with the target tissues these branches tracheate. the dsrf gene was mapped to position 60c on the second chromosome, and overlapping deficiencies which remove the gene were identified. analysis of tracheal development in embryos carrying these deletions revealed a degeneration of most of the major branches of the tracheal system. although the initial migration of tracheal cells was not affected in those deficiency embryos, many tracheal cells appeared not to maintain their formerly correct position and continued to migrate. thus, the dsrf gene might play a role in the proper formation and maintenance of the major branches of the trachea. s 19-08 our experiments would be expected to provide insight into such problems as the evolution and function of transcription factors with multiple dna binding domains. ideally, we would be able to mimic "gene splitting" which occurred during evolution. the poan gene plays an important role during drosophila neurogenesis. it is expressed in specific subsets of sensory mother ceils (smcs) and neuroblasts. the smcs that express poxn differentiate into polyinnervated external sense (p-es) organs. in the absence ofpoxn, smcs differentiate into mono-innervated rather than poly-innervated external sense organs. as the poxn gene contains a paired-box, it probably encodes a transcription factor. since the function ofpoxn in the central nervous system is not known and no po~n mutants are available, an ems mutagenesis screen was initiated to isolate such mutants. based on the assumption thatpoxn null alleles are lethal, we screened for lethals uncovered by the deficiency df(2r)wmg, which includes poxn, but survive over the deficiencies df(2r)xte-18 and df(2r)kl-32 flankingpoxn. in a further test, such lethals are screened for the absence of embryonic p-es organs. ifpoxn mutants are not lethal, they will escape this screen. therefore, we are inducing local hopping of p-elements that have inserted in the vicinity of poxn. the offspring of flies that display an altered eye phenotype are screened for p-element insertion into poxn. we hope that these approaches will generate mutants that will be crucial for future studies ofpoxn function in neurogenesis. chromatin structures and transcription of rdna in yeast saccharomyces eerevisiae reinhard dammaun, renzo lucchini, thee keller and jest m. sogo; institute of cell biology, eth-honggerberg, ch-8093 ziirich the chromatin structure of yeast ribosomal dna was analyzed in rive by cross-linking intact cells with psoralen. we found that in exponentially growing cultures the regions coding for the 35s rrna precursor fall into two distinct classes. one class was highly accessible to psoralen and associated with nascent rnas, characteristic for transcriptionally active rrna genes devoid of nucleosomes, whereas the other class showed a cross-linking pattern indistinguishable from that of bulk chromatin and was interpreted to represent the inactive rrna gene copies. by cross-linking the same strain growing in complex or minimal medium, we have shown that yeast ceils can modulate the proportion of active (non-nueleosomal) and inactive (nucleosomal) rrna gene copies in response to variations in environmental conditions which suggests that yeast can regulate rrna synthesis by varying the number of active gene copies, in contrast to the vertebrate ceils studied so far. whereas intergenie spacers flanking inactive rrna gene copies are packaged in a regular uucleosomal array, spacers flanking active genes show an unusual cross-linking pattern suggesting a complex interaction of regulatory factors and histories with dna. $20-04 r. felleisen & b. gottstein; institute of parasitology, university bern, bern, switzerland most patients suffering from alveolar echinococcosis (ae) respond to infection with a marked igg synthesis directed against e.multilocularis metacestode antigen ii/3, which represents a novel member of the family of cytovillin related proteins. although the respective gene basically is also present and expressed in e.granulosus, most of the cystic echinococcosis (ce) patients do not recognize the antigen. this phenomenon was tackled by generating cdna derived from full length ii/3 genes from both echinococcus species, performed by rt-pcr. sequence analysis revealed a very high degree of conservation of the primary sequence (98.6% homology), cdna fragments were expressed as recombinant proteins and were comparatively assessed in elisa respective to antibody-binding characteristics. sera from patients suffering from ce were showing no significant differences in reactivity with the antigens derived from both species. therefore, parameters others than minor differences in the primary sequence seem to be responsible for the lack of antigen recognition with respect to ce. $20-02 patrick rigoni, sandro rusconi, institut f molekularbiologie h der universitat zarich, winterthurerstr. 190, 8057 z~rich, switzerland. trinuclcotide repeats are often found in the coding sequence of transcriptional regulators in both mammals and yeast, however, their function is yet unclear and data collected in our laboratory on the gheocorticoid receptor even suggest that they may not have any, and that their presence is probably the result of a selfish replicative behavior. nonetheless, we believe that these motifs can be used as tags to identify flexible protein domains typical of modular proteins such as transcription factors. another kind or repeat (coding for a poly-glutamine/alanine) has been discovered in the yeast regulators gall1 and ssn6. from northern blots, we know that such caggcn repeats are present also in higher eukaryotes and we want to isolate them by constructing an enriched edna library using a 5' race protocol. so far, no mammalian protein bearing the motif glrdala has been characterized. among the positive clones we hope to find the homologous or paralogous of gall1 and ssn6 that have escaped so far conventional cloning techniques. meanwhile, we have been testing the effect of coexpressed yeast gall 1 on different reporter-activator combinations in mammalian cells. preliminary results show that galll but not the mutant form galllp can inhibit the activation properties of some (not all) gal4 chimeras. we are also producing antibodies directed against oligo-ala and oligo-gln and oligo-gln/ala motifs in order to better characterize natural proteins containing these repeats. we are interested in the early development of c. elegans, particularly in the contribution of genes whose homologues in vertebrates and d. melanogaster mediate positional infolzzations during development. therefore, we are working on ceh-13 which belongs to a cluster of at least 4 homeobox containing genes. this cluster is considered to be equivalent to the homeotie cluster of drosophila and to the hox clusters in vertebrates. by generating ~-galactosidase transgenie lines, we have observed that ceh-13 is expressed very early during embryogenesis, embryonic expression appears to be restricted to the posterior half of the embryo, which is rather surprising, since the ceh-13 hemologues in drosophila and vertebrates are anterior-specific. during larval stages, ceh-13 is expressed in neuronal cells. these results are now in the process of being confirmed by immunolocalization of the ceh-13 protein product with a polyclonal antiserum. in order to clarify the function of ceh-13 in the c. elegans development, we have isolated a ceh-13 mutant from a tcl insertion library, in collaboration with r. h. a. plasterk from amsterdam. from this worm, which looks wt, we are now trying to obtain a deletion derivative. previous studies have shown that bovine herpesvirus 1 (shv-1) infected cell pratein (bicp) 0 acts -depending on the promoter -as a strong transactivator or as a repressor in transient expression assays. the 81cp0 polypeptide contains a cysteine-rich zinc finger domain (c3hc4) which is conserved in a number of viral and cellular regulatory proteins including the 8icp0 homologs icr3 of herpes simplex virus type 1 and protein 61 of varicella zoster virus. this type of zinc finger (the so-called ring tinge0 was shown to bind zinc ions but functional requirement for zinc has not yet been demonstrated, a gap which we aimed to fill with the present study. transient expression assays were performed in oocytes which had been microinjected with thionein to chelate and deplete the intracellular pool of zinc. 81cp0-induced cat activity of a promoter-cat construct was 72-fold higher than the basal cat activity of the reporter plasmid, in the presence of thionein, bicp0-induced transaotivatlon was reduced 54old. with a set of control experiments we excluded that thionein might affect transcription and protein synthesis in general, to our knowledge this is the first demonstration of zinc-dependence for a member of the ring finger family. we have identified by pcr and edna cloning five pou genes expressed during early embryogenesis of zebrafislx four of these genes show extended homology to the brn-1 class of pou genes. preliminary evidence suggests that the brn-1like pou genes overlap with most of their expression domains.the gene studied in most detail so far (zp-50) is first expressed on the ventral side of the future fore-and midbrain. slightly later, expression is also found in rhombomeres 3 and 5 in the hindbraln. these rbombomeres were identified by the specific expression of the krox-20 marker using a novel in situ hybridization protocol which allows the simultaneous detection of two different transcripts using different color substrates. in the 24 hours old embryo a complex expression pattern is found involving a variety of brain structures and the spinal cord. the distribution of the transcript suggests that zp-50 is mostly expressed in glia cells. another pou gene we have identified (gp-9) defines a novel class of pou proteins. as a maternal mp, na it is initially ubiquitously present. after gastrnlation its transcript is found most notably in the developing hindbrain in rhombomeres 2 and 4 for which so far no good molecular markers were known. we are investigating the roles the identified pou genes may have in zebrafish hindbrain segmentation. we are currently attempting to manipulate gene expression in developing embryos by injection of rna or by the production of transgenic fish. we are also screening the zebrafish genome for new developmental marker genes. progress about these experiments will be presented. $20-07 willimann, t. and trueb, b. to gain insight into the regulatory mechanisms of collagen vi synthesis we have characterized the cis-acting elements of the chicken ctl(vl) collagen promoter. footprinting experiments with nuclear extracts from chicken embryos revealed three distinct elements, designated a, b and c, that were protected from dnase i digestion. the nuclear proteins that interact with the three sites were identified by gel retardation assays in combination with the use of various oligonucleotide competitiors as well as specific antibodies raised against well-characterized transcription factors. site a was found to be a target for transcriptional activator apf, whereas sites b and c were shown to be recognized each by two distinct nuclear proteins which belong to the spl multigene family. to address the question whether the three sites alone are able to direct transcription, a minipromoter construct was created in which the sequences of sites a, b and c were placed in front of a reporter gene. after transfection into chicken fibroblasts, this construct exhibited a high relative promoter activity when compared to a large genomic fragment containing the basic ctl(vi) collagen promoter. thus, the three sites are sufficient to induce transcription of this gene. octamer transcription factors (oct or otf) are a subset of the pou family of transcription factors which regulate expression of cellular and viral genes by binding to the octamer sequence atgcaaat. neurons and astroglial cells harbour, in addition to the ubiquitous oct-i factor, brain specific factors termed n-oct 2, 3, 4 and 5. in the present study we determined the chromosomal localization of the gene encoding the n-oct 3 transcription factor and characterized the structure of isolated n-oct 3 genomie clones. the chromosomal mapping was performed by analysis of somatic cell hybrid panels and radioactive and fluorescence in situ hybridization of human metaphase chromosomes. it is interesting that the 5' end of the n-oct 3 coding sequence contains repetitive cag and ggc residues. several disorders have been discovered to be related to expansion of trinucleotide repeats. we are presently investigating if the n-oct 3 cag or ggc clusters are hot spots for such mutation mechanisms and if so, which diseases could be associated with it. two dna regions upstream of the distal glucocorticoid receptor binding site interact with nuclear proteins that are tissue-specific (region a) or ubiquitous (region c). the characteristics of the dna-protein interactions have been studied in vitro. these sequences, in different combinations with the natural mmtv promoter, were tested in transfection assays of fibrcblast or mammary cell lines. we show that they are able to modulate the level of glucocorticoid-stimulated transcription. the proximal region of the mmtv promoter has binding sites for the transcription factors ctf/nf-i and oct-1. p~asmids with a deletion or mutations in the oct-1 site were tested in stable transfections, that are most representative of the state of proviral dna with respect to both number of integrated dna templates and chromatin organization. we show that the oct-1 site is important for the basal level of promoter activity. the data further indicate that the functional outcome may depend beth on the relative ratio of factors to dna templates and on the relative affinity of binding sites, as determined by oligonucleotide competition footprinting. besides the fact that telomeres represent specialised structures important for chromosome stability, the particular significance of cloned c. elegans telomeres is that they will contribute to the completion of the physical mapping of chromosomes and that they provide one set of elements for the construction of artificial c. elegans chromosomes. the cloning of c. elegans telomeres, however, is impeded by the fact that tandem repeats of the sequence ttaggc are not only located at the ends of the c. elegans chromosomes, but also at many internal chromosomal sites. therefore, we have developed a special protocol for the construction of a c. elegans endlibrary in 2~ zap. the resulting telomeric library was screened for recombinants containing ttaggc repeats and yielded about 70 positive clones. so far, 30 were analysed by partial sequencing and twelve of them satisfy all criteria required for telomeric clones. their ttaggc tandem repeats are located at the expected position, namely just adjacent to the blunt end cloning site in the polylinker. furthermore, the g-rich strand of the repeats is oriented 5' to 3' towards the end of the fragment, thus corresponding exactly to the defined orientation of eukaryotic telomeric sequences. finally, hybridisation data with one of these putative telomeric clones show that a subtelomeric fragment hybridises to bal31-sensitive fragments on a southern blot with c. elegans dna, indicating that this clone represents a c. elegans telomere. $20-09 to investigate whether chromatin structure or transcription can interfere with replication, derivatives of the yeast trp1ars1 minichromosome were constructed that contain either the ded1 or pet56 promoter firing against the origin of replication ars1. while the pet56 constructs transformed yeast at high frequencies and were maintained as high copy number minichromosomes, the ded1 constructs transformed at low frequency and the constructs were integrated in the genome suggesting that ars function was impaired. insertion of the h4-ars, a second origin of replication, rescued a ded1 construct as a minichromosome (yrpdh3).the chromatin structure mapped at low and high resolution of the ars1 region in yrpdh3 was indistinguishable to that of the pet56construct yrpft61. ananlysis of rna showed that transcription was going through ars1 in yrpdh3 and in yrpft61, but the levels of transcripts in yrpdh3 were much higher. we conclude that transcription through are1 interferes with replication and prevents extrachromosomal maintenance. dna sequence of a repeated dna segment on circular and linear plasmids of borrelia burgdorferi w.r. z%ckert and j. meyer, abt. pzmom, zahn&rztliches institut der universit~t basel a plasmid-associated repeated dna segment of b. burgdorferi strain b31 was cloned. at least two copies of this segment appear to be present on the 29 kb circular plasmid, whereas one copy is carried on the 50 kb linear plasntid of this strain. dna sequence analysis revealed a region containing a novel 906 bp putative open reading frame (orfl) on the 50 kb linear plasmid, orfl displayed extensive sequence homology (95%) to putative open reading frames present on the clones obtained from the 29 kb circular plasmid. heterogeneity is mainly caused by 3rd-base-wobbllng. flanking sequences share 85-95% homology, including the 5' end of an additional putative open reading frame irmaediately downstream of orfl. whether orfl and/or the related sequences are being transcribed and yield, in the case of orfl, an approximately 36.5 kd, lysine-rich polypeptide, is yet unknown. the repeated sequence seems to be specific for b. burgdorferi sensu lato and therefore may be useful in nucleic acidbased diagnostics of lyme disease. $20-16 similar to type ib oculocutaneous albinism in humans, overall production of pigment is greatly reduced in dark eyed albino and only obvious in eyes. we have studied the molecular basis of the c 44h mutation and show that expression of the tyrosinese gene is not affected. after sequencing tyrosinase cdna isolated from c44h/c44h homozygotes we uncovered a single base alteration from wild type leading to a serine to isoleucine exchange. the importance of this mutation was demonstrated by generating transgenic mice containing a mutated tyrosinase minigene. this showed that the single base change is sufficient to severely depress pigment production in transgenic mice. we therefore conclude that the point mutation is responsible and sufficient to generate the dark eyed albino phenotype. members of the pou-family of transcription factors are involved in developmental and differentiation processes. using a pcr-based cloning strategy with degenerated oligonucleotides we identified several pougenes from the lactating and involuting mouse mammary gland. three of these cdna clones which contained as yet unknown sequences were chosen for further investigations: clone 5.80 which has a high homology to pit-l, clone 5.54 which is related to the brn-3 gene and clone 5.48 which belongs to the class iii of pou-proteins. the expression levels were analyzed in different mouse organs and in several developmental stages of the mammary gland, including the postlactational process of involution. because of the low abundance of the specific transcripts we used a semiquantitative, reverse transcriptase-mediated pcr assay to investigate the mrna levels of the three novel pou-family members. distinct and specific expression patterns for all three members were obtained in the different investigated organs. interestingly, the expression of the pit-1 related cdna clone 5,80 was elevated in all developmental stages of the lactogenic-hormone dependent mouse mammary gland and in sceletal muscle, whereas its expression was low in other organs. repetitive sequences in dna can allow ectopic (outof-register) homologous recombination, which is often undesirable and can even lead to disease in humans. to prevent this, long homologies should often be interrupted during evolution. accelerated mutation of repetitive sequences by so-called ripping may be one of the mechanisms used to accomplish this in fungi. we are investigating if introns in vertebrate genes have an undiscovered role as interrupters of homology within and/or between genes, in addition to their established role in exon shuffling. by inserting homology-poor introns in an otherwise homology-rich region the genome could prevent ectopic homologous recombination. such a mechanism could be especially useful where there is an advantage in encoding highly repetitive protein sequences. it appears that within the collagen gene family there is indeed a correlation between repetitiveness and intron density. the influence of prenatal diazepam exposume (i,25 mg/kg/d, s.c.) from gestational day 14 to 20 on the development of the ~-opioid binding sites in striaturn, nucleus accumbens and midbrain of pn 14, pn 28 and 8 week old male and female rats was studied. 8 week old prenatally diazepam exposed male rats showed a significant decrease of k-opioid binding sites in the nucleus accumbens. a sex-dependent difference in k-opioid binding site densities could also be detected between pn 28 prenatally diazepam exposed male and female rats. we now investigate by in situ hybridisation whether changes in the level of mrna encoding fo~ prodynorphin, the precttrsor for various endogenous ~-opioid agonists, could be responsible for the decrease of ~-opioid binding sites in the nucleus accumbens of 8 week old prenatally diazepam exposed male ~ats. $21-04 distribution and morphology of nitric oxide-positive neurons in the marmoset cerebral cortex during pre-and postnatal development j.p. hornung* and j. schultz** *institute of anatomy, university of lausanne, 1005 lausanne, and **university of fdbourg, 1700 fribourg nitric oxide, synthesized by the enzyme nitdc oxide synthase (nos), is a neuroactive substance and a limited number of neurons were shown to express this enzyme either by histochemistry or by immunocytochemistry. both techniques were performed on brains of marmosets with ages ranging from 6 weeks before birth to adult. the morphology and distribution of nos-positive neurons were analyzed in all lobes of the cerebral cortex. the same pattern was revealed by histochemistry or immunocytochemistry. three populations of nos-positive neurons were revealed. first, a transient population of neurons in the molecular layer of the embryonic cortex, many having the morphology of the retzjus-cajal cells. a second population was made of large multipolar neurons in the deep layers of the cortex and the underlying white matter, which persisted from embryonic to adult life. a third, permanent, population was made of small, weakly reactive neurons in the supragranular layers appearing postnatally, this study demonstrates that no can exert its actions in the cerebral cortex through several pathways at different developmental stages. trimethyltin (tmt), a well-known neurotoxicant, was used to study the sensitivity of the different brain cell types (i.e. neurons, astrocytes, oligodendrocytes and microglial cells) to a neurotoxic insult. aggregating brain cell cultures of fetal rat telencephalon were treated during 10 days with tmt at different concentrations, ranging from 10 -10 m to 10 -6 m. microglia were found to be the most sensitive cell type, since already at 10-10m of tmt an increased number and clustering of griffonia simplicifolia-positive ceils could be observed. at 10 -8 m of tmt astrocytes showed increased staining for glial fibrillary acidic protein, characteristic of gliosis. neurons and oligodendrocytes appeared to be the least sensitive cells, since a decrease in the activity of cell type-specific enzymes was observed only at 10 -6 m of tmt. these results show that microglial cells and astrocytes respond to a toxic insult before any neuronal changes could be detected, and that the microglial reaction may be the first target of tmt. this reaction could then trigger the astrocytic response. vif is widely distributed in brain with suggested functions related to development. vip expression was studied during rat brain development using hybridization histochemistry with a 48met probe. vip mrna was found in thalamic nuclei on el7, later recognized as the ventrolateral and reticular nuclei after further maturation during prenatal period, vip mrna was found in hypothalamus, especially suprachiasmatic nucleus on el9 and its expression matured over the next days. few cortical neurons contained vip mrna on e21, they continuously increased in number and signal intensity over the perinatal period. vip gradually matured in the first three postnatal weeks and adult-like patterns were found on p 22, when cerebral cortex, ventrolateral and reticular thalamic nuclei, hypothalamus, esp. suprachiasmatic nucleus, were the regions with most prominent vip expression. these results demonstrate the relatively late appearance of vip gene expression in the rat forebrain, as compared with peptides like srif and cck, not suggesting a major role in earlv brain maturation. in primary cultures of mouse cerebral cortical astrocytes, a rapid glycogenolysis followed by a massive glycogen resynthesis ( six-to ten-fold over basal levels after 9 hr) are induced by vasoactive intestinal peptide (vip) or noradrenaline (na). both actions of these neurotransmitters are mediated by camp. since the induction of glycogen resynthesis triggered by vip or na is abolished by inhibition of transcription and translation, we applied the 2-d gel electrophoresis technique to search for astrocytic proteins induced by vip or na. the comparison of 35s-labeled proteins from primary astrocyte cultures treated or not with vip 1 ~tm revealed two newly synthesized proteins: a 36 kda protein (pi=5) and a 23/24 kda protein (pi=6). only the latter is visible on a silver stained 2-d gel, which is a prerequisite to consider its purification and microsequencing. induction of the 23/24 kda protein by vip was time-dependent with a maximum at -12 hr. preliminary results indicate a similar induction by na and isoproterenol. in humans, the central analgesic effect of tramadol (t) is only weakly reversed by the ~t opioid antagonist naloxone (nx) (br j clin pharmacol 1993; 35:73p). t analgesia may thus result from an action on monoaminergic pathways as suggested by in vitro and animal data. we therefore investigated the effect of ct2-adrenoeeptor antagonism by yohimbine (y) on t analgesia. according to a randomised double-blind crossover and placebo (p) controlled design, healthy volunteers (n=10) received t (100mg orally), followed (+ 3 h) by y (0.1mg/kg intravenously), and y + nx (0.8mg intravenously). analgesia was assessed over 8 h by subjective pain threshold (numerical scale -ns-) and objective pain threshold (rill nociceptive reflex -riii-) monitoring. peak analgesic effect was observed at ca. 3.25 h (riii +8.8 +semi.6 ma and ns +8.4 +1.8) vs p (rih -0.3 +1.2 and ns +2.3 +1.6, p<0.05) and lasted ca. 6 h. y reversed t analgesic effects during ca. 1 h (r/ii -2.9 +1.8, p<0.05 and ns +2.8 +1.5, p<0.05), whereas y + nx abolished t effects thoughout the study period (rill -1.9 4-1.1 and ns +0.6 +1.3, p<0.05), y alone tended to lower pain thresholds (rill -4.1 +1.5 and ns -1.9 +1.4, p>0.05 anova). yohimbine, an r antagonist, reverses tramadol effects, thus pointing to the major role of monoaminergic modulation in tramadol anfinociception. a monoclonal antibody (in-l)was raised against neurite growth inhibitory proteins present in rat cns myelin (caroniand schwab, neuron 1: 85, 1988) . in-1 neutralizes the inhibitory ettect of cns tissue in vitro and in vivo, thus permitting regeneration of certain lesioned cns fiber systems. we have used this antibody to immunostain cryostat sections of the nervous system of the rat. we found that in-1 stains white matter and myehnated fiber tracts in the cns. the staining pattern corresponds to that of the known myelin specific antigens mbp (myelin basic protein), mag (myelin associated glycoprotein) and mog (myelin oligodendrocyte glycoprotein). the staining of in-1 in the cns is found in regions of the adult nervous system which have been shown to be inhibitory for axonal growth. interestingly, the regions which show a high abundance of in-1 antigens are low in staining for gap-43, a marker for growth and plasticity in the developing and adult brain. in contrast, gap-43 is strongly expressed in unmyelinated fiber tracts or nuclei. prevention of oligodendrocyte development in rat spinal cord resulted in suppression of in-1 expression and elevated gap-43 levels. this suggests that inhibitory myelin proteins may negatively influence plasticity in the rat cns. to date, the existence of anp and npy in the adrenal medulla has been shown only for a few mammalian and anuran species. thus the present immunohistochemical investigation attempts to localize anp-like and npy-like peptides n the adrena gland of representative mammals, birds, reptiles, amphibia and bony fish by the use of antisera specific for mammalian anp and npy. furthermore, the catecholamine-containing cells were characterized by antisera against enzymes of catecholamine synthesis. anp-and npy-immunoreactivies were detected in adrenal chromaffin cells of all vertebrates studied while no immunoreactivities were observed in the adrenal cortex or in its homolog, the interrenal. the representatives of the different vertebrate classes exhibited marked differences in the proportions of anp-immunoreactive and npy-immunoreactive cells, in the presence of anp-and npy-immunoreactivities in noradrenalineand/or adrenaline-containing cells and in the amount of of cells containing both anp-and npy-immunoreactivities. our results give evidence for a long phylogenetie history of adrenal anp-and npy-like peptides. thus, they stress the physiological impact of these peptides as adrenal paracrine and/or endocrine hormones. human neuronal growth inhibitory factor (gie) is involved in the regulation of neuronal growth and is deficient in the brains of alzheimer's disease victims. this brain-specific metalloprotein consists of 68 amino acids out of which 20 are cys and has been found to contain copper and zinc. proteins with similar primary structure were isolated from bovine and equine brain. the amino acid sequence of these proteins shows about 70 % homology to those of mammalian metallothioneins (mt), with two conserved inserts of 1 thr and a glutamic acid rich hexapeptide in the n-and c-terminal regions, respectively. in contrast to mts, which usually contain only zn(i1), the presence of cu(i) and zn(ii) (6-7 moles total) in all gif isolations suggests the functional importance of both metals ions. to spectroscopically probe the native zn-binding sites, the zn(ii) ions were replaced by cd(ii). both bovine and equine cd/cu-gif derivatives exhibit similar absorption and cd spectra with features characteristic of cd-thiolate clusters. the release of 3h-arachidonic acid evoked by glutamate was characterized in cerebral cortical neurons in primary culture grown under conditions that prevent glial cell proliferation. in these cultures, 99% of the total ceils are immunocytochemicauy characterized as being positive for specific neuronal markers such as neuron-specific enolase and neurofilament. specific markers for astrocytes, oligodendrocytes or microglia were not detected. glutamate induces a concentration-dependent release of 3h-arachidonic acid with a ec50 of 3 pm. the pharmacological profile of this glutamate response shows the involvement of two receptor types: the nmda and the ampa ionotropic receptors. the glutamate-evoked release of 3h-arachidonic acid is phsensitive. when the incubation buffer is alkalinized from 7.25 to 7.65, both basal and glutamate evoked release of 3h-arachidonic acid are enhanced. the glutamate response is also enhanced as intracellular ph is alkalinized by pharmacological treatments: such as inhibition of c1-/hco3-antiport by dids. these results further stress the role of intracellular ph in glutamate-mediated neurotransmission. $21-09 multiceli culture system for the study of drug kinetics wechsler d., *schittny j.c. and honegger u. e., depts. of pharmacology and *anatomy, university of bern, ch-3010 bern a cell culture system was developed for the investigation of cellular drug kinetics in cultures with up to 4 cell types. it was used for the study of uptake, competitive uptake, release and redistribution of drugs. individual cell types (human skin fibroblasts, rat astrocytoma cells (c6) and rat hybfidoma ceils (oligodendrocyte x astrocytoma, roc)) were separately grown to confluency on glass circle sectors. in the same petri dish 4 sectors in various combinations were exposed to media containing radiolabelled chlorpromazine (cpz). single and repetitive uptake and release of cpz were measured in each cell type after individual exposure or exposure in any combination of cell types: in 2 hour competitive uptake studies fibreblasts reached 1.7 and 2.6 times the concentrations of c6-and roc-cells, :respectively. in redistribution experiments the exchange of cpz from preloaded to unloaded cells was tested. the exchange rate was dependent on cell type and loading period. it decreased with increasing time of exposure suggesting the formation of more stable cpz stores. we have previously demonstrated that certain neurotransmitters such as vip, noradrenaline (na) and adenosine promote glycogenolysis in mouse cerebral cortical astrocyte cultures (brain res. 563: 227-233, 1991) . the question that we then addressed was the nature of the metabolic substrate released by astrocytes following glycogenolysis. we therefore determined the presence of various metabolic substrated released from astrocytes under static conditions and in the superfusate of a laminar flow system developed in our laboratory. neither glucose nor any intermediate of the tficarboxylic acid cycle could account for the decrease in glycogen stores; astrocytes however were shown to release great quantities of pyruvate and lactate at a rate of 50 nmol/mg protein/minute. noradrenaline but not vip promotes a significant increase (42%) in lactate release. the effect of noradrenaline is mimicked by isoproterenol and inhibited by propranolol, suggesting a i~adrenergic mediation. when astrocytes are incubated in an isotonic solution containing no energy suhstrate, the glycogen stores are rapidly depleted and lactate, but not glucose, is released in the medium. in conclusion, lactate appears to play a major role in cerebral energy metabolism homeostasis, as substrate released by astrocytes to prey!de energy fuel for neurones and other neighboring cells. it is known so far that calcium-binding proteins (cabps: calbindin, pan, albumin and ca/retinin) are expressed in some cells of the pineal gland of laboratory mammals. the exact phenotype of these cells is unknown. nevertheless, according to recent studies, it seems that some of them are ganglion cells. therefore we studied the pineal gland of different species (gerbils, rats, goats, cows and humans) using calretiain (cr) antibody which is considered as marker for neurons (andressen & al, cell & tissue rss, 27t:181, 1993) , the cr-expressing cells were found in all oar species: in gerbils, rats, cows and humans they were scattered throughout the parenchyrna, whereas in goats they lined mostly the pericapillary spaces. according to our findings, it seems that most of the reactive ceils are rather neuron-like elements since morphological criteria for true neurons (axosomatic synapses, nissl bodies, bundles of neurofilaments) are missing. however one cannot exclude that among these cells some are intrapineal ganglion cells, because of ranvier's nodes found along the processes of some crpositive cells. thus, it is possible that these cells are involved in the modulatory control mechanism of the pineal neurohumoral activity and/er by accumulation of intracellular calcium in the formation of pincal calcifications. besides the excitatory amino acid transmitter glutamate, its sulfur containing analogue homocystcic acid (hca) could play a role in excitatory processes in the central nervous system. hca is present in and released from nervous tissue and is a potent neuronal excitant, predominantly activating n-methyl-d-aspartate receptors (do et al., 1992) . these properties are suggestive of a classic synaptic neurotrausmitter role. on the other hand, hca seems to be localized not in neurones but in glial cells (grandes et al., 1991; tschopp et al. 1992) . the efflux of hca is temporally delayed following activation of specific pathways (klancnik et at., 1992) ; these latter observations are not in accordance with the classic concepts of synaptically-mediated neurotransmission. a laminar flow superfnsian system of monolayer cultures was developed and the released materials from mouse cortical astrocytes, previously preincubated with [35s]-methioulne, were investigated. during application of either noradrenaline or the b-adrenergie agonlst isoproterenol, the extracellalar level of [35s]-methianine was decreased and that of labelled hca was increased. this effect was not observed with the -adrenergic agonist methoxamine, and could be blocked by the ll-adrenergic antagonist propanolol these results, combined with the delayed hca release, the glial localisatian of hca and its neuroactivity, strongly suggest a potential role of hca as a gliotransmitter, modulated by noradrenaline inputs. vasoactive intestinal peptide (vip) induces long lasting metabolic effects in the mouse cns. we have investigated a shorter neuromodulatory action of vip on the synaptic responses in coronal slices of adult mouse cingulate cortex by means of intracelhilar current clamp recordings. electrical stimulation of the underlying white matter evoked a multiphasic synaptic potential consisting of early epsp~ early ipsp, late epsp and late ipsp mediated by non-nmda, nmda, gaba a and gaba b receptors, respectively. vip (0.1 ~d~l) superfused for 5 rain. had no effect, whereas concentrations over 0.2 ixm selectively and reversibly depressed the nmda-medlated responses in 12/16 cells. in the presence of cnqx (10 [tm) and biencuuine (10 ~tm), the isolated nmda-mediated late epsp was still attenuated by 0.3 ~m vip in 8/10 cells (mean:-42% al~er 30 rain.). in 3 other cells vip induced a spreading depression (sd) and a strong, reversals inhibition of the late epsp (mean:-64 % after 10 min.).these results indicate that in the absence of gabaergic inhibition vip can induce two types of effects in the mouse cingulate cortex, a prolonged decrease of the nmdamediated syaaptic response and a striking sd. interaction between vip and glutamate on c-fos mrna expression. |.-l. martin, d. gasser and p.] . magistretti. institut de physiologie, universit4 de lausanne, lausanne, switzerland. the regulation of c-los mrna expression by vip and glutamate was examined in primary cultures of neurons originating from the mouse cerebral cortex. in primary cultures of cortical neurons, vip increases camp levels and stimulates c-los mrna expression. interestingly vip stimulation is completely blocked by mk-801, an nmda receptor antagonist but not by cnqx or ap3, which antagonize ampa/kainate and metabotropic receptors respectively, c-los expression stimulated by glutamate shares the same pharmacology. these results suggest an involvement of endogenously released glutamate in the stimulation of c-fos mrna expression evoked by vip. furthermore, when added together, vip and glutamate interact synergistically to increase cfos mrna levels. consistent with the pharmacology of vip and glutamate, the synergistic interaction between vii' and glutamate on c-fos mrna expression is also inhibited by mk-801. interestingly, this synergism is completely inhibited by h-89, a protein kinase a inhibitor, whereas staurosporin and kn-62 which block protein kinases c and ca++/calmodulin dependent respectively exhibit smaller inhibitory effects. presynaptic proteins involved in neurotransmitter release (i.e. synapsin, b-50) and some postsynaptie gaba a receptor subunits possess phosphorylation sites for camp-dependent protein kinase (pka) and/or protein kinase c (pkc). the functional consequences of phosphorylation by these kinases is not known. we have studied the action of specific activators of pkc and pka, phorbol ester (pdbu) and forskolin, respectively, on gabaergic synaptic transmission in area ca3 of hippocampal slice cultures. pdbu significantly enhanced the amplitude of evoked monosynaptic ipscs (in cnqx and ap5), and both pdbu and forskolin increased the frequency of spontaneous miniature ipscs (m[pscs) in the presence of cnqx, ap5 and ttx. this effect by pdbu was antagonized by staurosporin, a protein kinase inhibitor. pdbu and forskolin did not change the amplitude or decay of mipscs, suggesting that only presynaptic kinases are involved. furthermore, pdbu enhanced mlpsc frequency by a comparable amount in the presence of the ca 2+ channel blocker cd 2+, indicating that pdbu did not enhance gaba release from presynaptic endings by facilitating ca 2+ influx into axon terminals. valiunas, v., sc~ilinsky fluri, g. and weingart, r. arachidonic acid (aa) reversibly impairs the gap junction conductance (g~) in cardiac cells. now, we examined whether aaacts directly or via its catabolites. experiments were performed on pairs of neonatal rat myocytes using a dual voltage-clamp method. we used blockers which interfere with enzymes of various catabolic pathways. pretreatment with i0 ~mpoca (blocks carnitine acyltransferase i) did not prevent the decline in g9 by i0 ~m aa; i.e. intermediates of p-oxidation are not involved. similarly, preexposure to i0 ~m indomethacin (blocks the cyclooxygenase pathway) did not prevent aa from uncoupling; this rules out an involvement of prostaglandins and thromboxanes. exposure to 10 ~mndga (blocks the 5-1ipoxygenase pathway) per se produced a decrease in gj. likewise, exposure to i0 ~m etya (blocks the 15-1ipoxygenase pathway) also impaired gj. these effects cannot result from accumulation of endogenous aa because preexposure to 50 ~m 4bpb (blocks phospholipase a 2) did not prevent ndga-or etya-induced uncoupling. exposure to 75 ~m skf 525-a (blocks the epoxygenase pathway) also produced a decrease in gj. hence, ndga, etya and skf 525-a, compounds with different biochemical actions and of different chemical structure, act on g~ either directly or via an unknown mechanism. $21-19 potential changes associated with electrical activity in nonmyelinated nerve fibres a. robert and p. jirounek d~partement de pharmacologie, cmu, 1211 gen~ve 4 simuheneous measurements of the extracellular concentration of k + ([k+]e), and of the concomitant changes in the membrane potential (era) were measured in the rabbit vagus nerve during and after a period of activity. during a 15 hz stimulation there was a large increase in the [k+]e , accompagned by a change in era, which roughly followed the depolarization expected from the increase in [k+] e . at the end of the stimulation, although the [k+]e was still elevated, the nerve developed a posttetanic hyperpolarization (pth). the pth was generated by the electrogenicity of the na+-k + pump, but its amplitude and time-course were strongly affected by two depolarizing currents: (i) an inward ba2+-sensitive current of k + and (ii) an outward current of ci-. we hypothesize that during activity and during the early phase of recovery there was a ba2+-sensitive uptake of potassium by the schwann cells. the ci" current, by short-circuiting the na+-k ⧠pump, contributes to the control of the e m, and by this way to the driving force for the inward k ⧠current. overdose of ifosfamide has ooo/red in a canc~c patient (25g i.v. iy0/24 hours with 20 g as ~utectarfe). urir~ collece{on was obtaj/3ed in 24 hour ~ fcr t~o days. c~ was measured ~ a gas liquid d~romatogr~ method of the u5~ ~ (s~@pl. v, pag.2627-262g) ~sir~ a ~e~s ar~ ni~, sensitive ths~mi(~3ic d~. ~ u~s p~mt ~ ~ ~ ~ ~ ~ 1500 m~/24h and 207 r~/24h at day one ard day res~ec~vely. 9~ _h=~=mmtly ~ has also bsm foa~ in mti~s ~ mgh e~e ~ ~ (~/o~e). w~n ~ ~s am/nist~ to rats (i~ m~/~ ~c 200 m~f~ intraperit~neally), no ~ omild be detected in urine within 24 ht~r~ after drug administl-4(cicn. from these ~ data we conclude that c~ is extensi~_ly metabolized in rats and ~ mscbard_sm of ~ el ~mlnaticn in pati~rfc t=cj_ne m~cjlr[c reflect a flmicticrsl der~,~t of c~ m~t~0olisn in man to ifo~=~de ~tion. there are numerous reports of improved memory functions in rats following a dietary supplementation with choline. in some cases, this improvement can be related to enhanced cholinergic transmission (mack et al., behav neurosci., 103, 1234 -1241 , 1989 ). but it is not clear whether there is a general improvement in memory or whether specific aspects of learning and memory are affected. this work was aimed at analysing the effects of perinatal cholinergic supplementation on the development of spatial abilities and upon adult performance. choline supplementation (3.5 g./l. in 0.02 m saccharine solution in tap water) was maintained during two weeks before birth. additional supplementation was maintained from the 5th to the 1oth week postnatal. spatial learning capacities were studied at the ages of 26, 65 or 80 days in a cimular swimming pool (morris place navigation task) and at the age of of 7 months on a homing board. adult and aged rats were tested on a radial maze. selective aspects of the performance were markedly improved. this treatment had no specific effect upon spatial memory per se, but, instead, affected learning abilities by promoting efficient behavioural strategies hypothetically based on active representation and anticipation processes. the effects of the treatment remained evident up to 6 months following the supplementation. barcelona, e-08193 bellaterra females of both lines (n=b/grp) were injected sc daily between days 15-20 of pregnancy with vehicle (v), i(di) or 3(d3) mg/kg diazepam, or not(c). in these studies(a,b) their 5-7 mo old offspring were subjected to a 50-run session in a shuttlebox(a:12 males, 6 females of each line/condition) or a 6min session in a hexagonal tunnel maze with strategically-placed barriers and a lit central arena(b_:6-7 males of each line/condition). a: the freezing behavior of ld3 rats was reduced ~s lc, this behavior reverting more to escapes in males & avoidances in females, although v had an effect in the latter also. no within-line differences in inter-trial responses were seen, but pre-session activity was increased in female lvs and ldis, returning to lc levels for ld3s. no differences existed among the h groups. b: whereas h maze activity exceeded that of l, no wtthin-line differences appeared. hd3s entered the lit arena less than hcs or hvs, indicating an increased timidity after pnd, and a trend in the same direction existed for ldi/ld3 vs lv. two groups of abstinent female smokers, performing either a fixed rate or a subject paced version of a rapid information processing task were compared. both groups participated in two test sessions (in which the task was performed twice) to compare two different types of cigarettes which were smoked before and during the second task trial: a) the subject's habitual cigarette with a nicotine yield of at least 0.7 mg/cigarette and b) a nearly nicotine free test cigarette with a tar yield comparable to that of the habitual cigarette. reaction times to correct detections (series of three odd or even digits in a pseudorandom sequence of single digits) were longer with the subject paced version and not affected by the type of cigarette but shorter with the fixed rate version and even more decreased with the smoking of the habitual cigarette. on the other hand, the pre-to posttreatment increase in the subject paced processing rate was greater with the habitual than the test cigarette. it was concluded that the two task versions assess different cognitive functions. whereas the fixed rate version primarily assesses vigilance performance and seems to be more suitable to measure changes in reaction time, the subject paced version is more sensitive to changes in speed capabilities in rapid information processing. heart rate, physical activity, and cigarette consumption were continuously recorded under field conditions, whereas subjective craving was assessed six times per day and saliva cotinine was measured once daily. abstinence, habitual cigarettes (with a nicotine yield of at least 0.7 mg/cigarette), and nearly nicotine free test cigarettes but with a tar yield comparable to that of the habitual cigarettes were compare~) in a sample of twelve female smokers for two days each. whereas physical activity was similar for the three conditions, heart rate was highest with the habitual cigarettes, lowest on abstinence days and in between with the test cigarettes. cigarette consumption was similar for both types of cigarettes and subjective craving was higher on abstinence than on smoking days but no differentiation between the two cigarette types was obtained. saliva cotinine values were highest on the days with the habitual cigarettes but lower and similar with the test cigarettes and on abstinence days. it was concluded that heart rate and saliva cotinine depended only on the amount of nicotine absorbed, whereas subjective craving was reduced by smoking independently of the actual nicotine yield of the cigarette. (ptps) is the rate limiting enzyme in the synthesis of bh, in man, a cofactor for several hydroxylases involved in catecholamine and serotonin biosynthesis. the human and rat liver cdnas encoding the 16 kda subunit of ptps were expressed and the recombinant enzymes purified to homogeneity. the apparent k~ for the substrate, pi and heat stability of the re-combinant enzymes were similar to the native enzymes. the rat enzyme was crystallized and the x-ray structure showed a hexameric native form arranged as a sandwich of two trimers. three histidine, a glutamic acid, and -also shown by site-directed mutagenesis -a cysteine residue characterize the active center of the enzyme thought to be mg2+-dependent. according to the proposed ligands we replaced mg 2* by fe 2 ⧠or mn ~+ and observed an enhanced activity up to 100%. m. neerman-arbez, v. cirnlli and p. halban. laboratoires/eantet. centre m4dical universitaire, 1211 gen~ve 4. pci(3) and pc2, members of the mammalian family of pro-protein convertases homologous to the yeast kex 2, are both expressed in pancreatic islets of langerhans and are thought to be responsible for the conversion of proinsulin to insulin and c-peptide in beta cells. however, the insulin secreting beta ceils are not the only ceils present in these complex microorgans, prompting us to evaluate the expression of pc1 and pc2 in islet beta and non-beta cells. rat islet cells were sorted by autofluorescence activated flow cytometry to separate beta cells from non-beta ceils and conversion cndoprotease levels were analysed by western blotting. in beta cells, pc1 levels were higher than pc2 (pc1/pc2=2.6+0.2) whereas the opposite was found for non-beta cells (pc1/fc2=0.05â�¢ confirming that pc1 is important for proinsulin conversion while suggesting a role for pc2 in the conversion of proglucagon, prosomatostatin and propancreatic polypeptide. transformed murine cell lines (insulin-producing beta-tc and glucagonproducing alpha-tc) were found to faithfully reflect the primary rat cells in terms of their pc1/pc2 ratios. finally, post-translational modification of the convertases themselves was found to differ between cell-types, in particular, a precursor 75 kd form of pc2 accumulated in beta cells whereas only the fully processed 67 kd form was detected in the non-beta cells. the kringle (2+3) domains (k2+3) were successfully expressed in e. coil a n-terminal hexahistidine tag was fused to insure the isolation of k2+3 by affinity chromatography on a ni-2+-ntaagarose-column. to be able to remove the hexahistidine tag a factor xa cleavage site was introduced between the 6 histidine residues and the n-terminus of the kringle structures. the correct arrangement of the disulfid bonds was determined by amino acid-and sequence analysis. the lysine binding site was proven by affinity chromatography on iysine-bio-gel, as previously shown for k2. the dissociation constant of k2+3 was measured by intrinsic fluorescence titration with 6-aminohexanoic acid. a replication protein a copurifying dna helicsse from calf thymus anthl georgakl, narendra tuteja, blrglt sturzenegger and ulrich hobscher department of veterinary biochemistry university of z0rich-lrchel winterthurerstrasse f 90 ch-8057 z0rich, switzerland a dna helicase from calf thymus copurified with replication protein a through several steps of purification including deae-sephacel, hydroxyapatite and single stranded dna cellulose. it is finally separated from replication protein a on fplc mono q where the dna helicase elutes after replication protein a. we named this new calf thymus enzyme dna helicase f. characterization of the helicase f by affinity labeling with [a32p]atp indicated that the enzyme has a catalytic subunit of 72 kda. gel filtration experiments suggested that dna helicase f can exist both in a monomeric and an oligomedc form. the enzyme unwinds in the 5' ->3' direction in relation to the dna strand it binds. all eight deoxyribonucleoside-and ribonucleosidetriphosphates could serve as an energy source. testing a variety of dnndna substrates indicated that the dna heitcase f preferentially unwinds very short substrates and is slightly stimulated by a single stranded 3'-tail replication protein a allowed the dna helicase f to unwind longer dna substrates up to 400 bases suggesting that copurification of replication protein a with the dna helicase might be of functional relevance. 2,4,5,2',4',5'-hexachlorobiphenyl (6-cb) shows limited elimination and extensive storage in adipose tissue in vivo (lipophilie unmetabolizable compound) while chlorpromazine (cpz) is known for its lysosomal storage. uptake and release of 6-cb and cpz were studied in 3t3-preadipocytes (p) and 3t3-adipocytes (a). 3t3-cells were cultivated in dulbecco's minimal essential medium supplement with 10% fetal calf serum and grown to eonfluency. differentiation was stimulated by dexamethason and bezafibrate exposure for 48h. radiolabelled 6-cb (20~tm) or cpz (5~tm) was added to the medium. following a single dose 70-80% of cpz were taken up within an hour into p and a. in contrast, 6-cb reached an intracellular plateau of 30-50% of the dose after lh in p whereas dependent on the triglyceride content a accumulated up to 95% of the dose. 6-cb uptake into a was temperature dependent and not saturable with repetitive daily doses up to 1 week. 6-cb uptake into p was fully reversible while the release from a was limited to 10% of the accumulated drug. in contrast, the release of cpz was higher from p than from a as a consequence of the different lysosomal activities of the respective cells. the use of 3t3 cells allowed to show a remarkable difference in cellular handling of neutral and basic lipophilic drugs and may be helpful to further elucidate basic mechanisms involved. chronic granulomatous disease: an attempt to reconstitute the function of the x-linked form of the disease. chronic granulomatous disease (cgd) is a group of congenital immunodeficiencies that predispose patients to recurrent severe bacterial and fungal infections. the cause of the disease is lack or impairment of 02-production in phagocytic cells due to mutations in any of the four components of the superoxide producing system. approximately 70% of all cgd patients suffer from an x-linked form of the disease where the phagocyte oxidase gp91phox is affected. this project concentrates on the reconstitution of the deficient gene in phagocytic patient cells. expression of recombinant gp91 was assayed in vitro and in vivo. attempts to "tag" gp91 with a foreign epitope were unfortunately so far unsuccessful. the target cells for gene reconstitution are non-dividing monocytes. thus, we have chosen an adenovirus over other viral systems because of: a) its ability to infect nondividing cells, b) its genetic stability and, c) lack of human adenovirus related malignancies or side-effects. we constructed two recombinant adenoviruses: one recombinant expresses a lacz gene (control), another the gp9 lphox gene. we are trying the expression of recombinants adeno/lacz and adeno/gp91 in normal and patient-derived cells (for example ebvtransformed normal and patient b-cell lines), as well as the reconstitution of nadph oxidase function in peripheral blood monocytes of cgd patients. kinetics of molecular chaperone action schmid, d., gehring, h., *baici, a. and christen, p., biochemisches institut der universit&t z~rich, ch-8057 zorich; *rheumaklinik, universit&tsspital, ch-8091 z%rich molecular chaperones of the hsp70 type transiently sequester unfolded segments of proteins and promote their correct folding. target peptides labelled with an environmentally sensitive fluorophore allowed to follow their binding to the molecular chaperone dnak of escherichia coli in real-time. the two-step process is characterized by relaxation times z~ = 27 s and z2 = 200 s at 2 ~m dnak and 0.i ~m ligand at 25~ in the presence of atp, the formation of the complex is greatly accelerated and follows a single-exponential process with z : 0.4 s. the rate of dissociation of the complex was even more increased than that of association resulting in a decreased net affinity for ligands in the presence of atp. the binding-release cycle of dnak thus occurs in the time range of polypeptide chain elongation and folding and is too fast to be stoichiometrically coupled to the atpase activity of the chaperone (ko~ ~ = 0.13 min i at 30"c). supported by the olga mayenfisch stiftung, z%rich, and the hartmann m~ller-stiftung, z~rich. comparison of the activities of native bovine seminal ribonuclease and that secreted from e. coil schein, ch* (siat, technopark, pfingstweidstr. 30, ch8005 ziirich) and haugg, m (lab. org. chemistry, e.tm., . seminal ribonuclease (bs-rnase) differs from the pancreatic rnase a in that it forms a cysteine-linked dimer and has a relatively higher specific activity in the cleavage of double-stranded (ds-) rna substrates. we expressed bs-rn in a secretion system similar to that described previously (biochem. j. 283:377-344, 1992) using the signal sequence of routine pancreatic ribonuelease. about i mg bs-rn was isolated per liter culture supernatant. human and bovine recombinant interfcron-~ (ifn-r) inhibit the degradation of ss-and ds-rna by bovine pancreatic rnase while they activate the activity of bs-rn on the same substrates (febs letters, 270:229-332, 1990) . recombinant bovine or routine pancreatic rnase (produced in our secretion system) are inhibited by ifn-y, while the recombinant bs-rn or a cysteine-linked dimer of rnase a is also activated by ifn-y. ifn-y activates bs-rn even in the presence of inhibitory concentrations (0.1-1 ram) of mononueleotides. thus the mode of activation cannot be attributed to alleviation of product inhibition. kinetic studies using 300 bp ds-rna as substrate suggest that ifn-~ interacts directly with the bs-rn to increase the rate of highmolecular weight producffsubstrate release, as the apparent ks increases proportionately to the increase in kcat. significantly smaller movements were observed in the simulation with the liganded enzyme. the structural differences between the protein in the crystal and the ensuing calculated structures might arise from the absence of lattice forces in solution. evolutionary relationships among pyridoxal-5'-p-dependent amino acid decarboxylases sandmeier, e., hale, t.i. and christen, p. biochemisches institut der universit~t zgrich, 8057 z~rich. the pyridoxal-5'-p (plp)-dependent amino acid decarboxylases (de) can be subdivided into four apparently unrelated groups. group i is represented by glycine dc, group ii comprises glutamate, histidine, tyrosine and aromatic-l-amino acid dc, group iii procaryotic ornithine and lysine dc as well as the procaryotic biodegradative type of arginine dc, group iv eucaryotic ornithine and arginine dc as well as the procaryotic biosynthetic type of arginine dc and diaminopimelate dc. (n-l) profile analysis, a more stringent application of profile analysis [gribskov et al. (1990) methods enzymol. 183, 146-159] established the homology among the enzymes of each group. a search with the profile of group ii indicated a distant relationship with aminotransferases and thus with the ~ family of plp-dependent enzymes. no evidence was obtained that groups i, iii and iv are related with other plp-dependent enzymes or any other protein in the database. apparently, the amino acid dc are of multiple evolutionary origin, in some cases even if they have the same substrate specificity. a. lo russo, a.c. passaquin, u.t. r~eg~, ecole de pharmacie, %hiv. lausanne, c~-1015 lallsaraqe drug-induced local vasoccmstricticn appears to be respmnsible for the hypertensive side effect of the imn/nosti~pressant cyclosporin a (csa). we have therefore ex~rnir~ the [ca 2+] i increase caused by the vasoccr~trictor hormcme [ar~]vascpressin (avp) in vascular ameoth ~uscle cells (vsmc) treated with csa. [ca2+] i levels were m~nitored either with a fluoresc~qce analyser using fura 2 or by 45ca 2+ efflux. pretreatment of v~mc with csa increased the avp induced rise in [ca2+]i. a leftward and upwsxd shift of the ccncentraticm-respmnse curve of the avp-induced rise in 45ca2+ efflux was also observed after csa treatxr~%t. ilzg/cticn of csa dote~tiaticn w-as detectable after 3 rain, took 1 to 2 h to become fully established and was reversed after washout. csa potentiaticm was cc~centraticn-deperdent with a threshold at 10 -7 m. %]]is potentiating effect of csa may be the underlyin~ cause for csa-ind/ced hypert~3sicn. 92) barja, f. 1 and turian, g. i, ilab. gen. microbiology, 2dept. biochemistry, 3station exp. zoology, university of geneva, 30 quai ernest-ansermet, 1211 geneva 4 the actin has been found from the simplest forms of cell to the highly evolved ones. in higher organisms it is transcribed by multigene families but in yeast and several filamentous fungi there appears to be only one actin gene. it was therefore of interest to study the expression of actin gene in n. crassa in which three actin isoforms have been characterized (barja et al., 1991, fems left. 77:19-24) . after isolating genemic dna from wild type n. crassa a pcr was performed with primers corresponding to actin consensus sequences and an amplified fragment of the gene (verified by sequencing) was obtained. this fragment was 'used as a probe in a southern to assess the number of aetin gene(s). our results suggest that n. crassa has alsc only one actin gene. the blocking effect of the n-terminal decapeptide of msmooth muscle actin (sma) (ac.eeedstalvc) on the monoclonal antibody anti-asm-1 was compared with that of synthetic peptides modified by changing the n-terminal acetyl group or by substituting a single amino acid in position 1 to 5. using immunofluorescence or immunoblotting techniques, anti-c~sm-1 activity was abolished after incubation with the native peptide and peptides modified in position 1 (only when e~a) or 5. on immunoblots running bsa crosslinked peptides on denatured gels, only the natural peptide and peptides with substitution in position 1 or 5 were detected by the antibody. our results indicate that ac.(e)eed is the epifope for anti-c~sm-l. binding of anti-~sm-1 to sma increased actin polymedzation by decreasing the critical concentration. as control this action was not exerted on skeletal actin. this is the first example of the role of a precise n-terminal sequence in the polymerization of a single actin isoform. double immunofluorescence for msma and total actin on cultured aortic sm cells microinjected with the native peptide showed a selective disappearance of msma staining suggesting that this peptide traps a protein involved in msma polymerization. work is in progress to search for the putative actin-binding protein(s) physiologically interacting with the n-terminal sequence of o~-sma. ( within mitochondria, the mechanism of selective protein degradation is poorly understood. while the bulk of mitochondrial proteins are long-lived, some are degraded rapidly. the selective degradation of mitochondrial proteins is likely to be mediated by proteases similar to those found in bacteria; this is based on the hypothesis that mitochondria have evolved from bacterial endosymbionts, in conjunction with the finding that mammalian mitochondria contain an atp-dependent proteolytic activity similar to that in bacteria. one atp-dependent protease from bacteria, lon, is of particular interest because it is involved in regulated protein turnover. degenerate oligonucleotide primers corresponding to two regions of the bacterial lon gene were used to pcr amplify a 1 kb fragment from yeast dna that encoded an open reading frame with striking similarity to the bacterial lon protease. by screening a yeast cdna library, we isolated a 3.6 kb clone potentially encoding a protein of 1133 amino acids. haploids bearing a disruptedlon gene were unable to respire or to grow on ethanol/glycerol. fractionation of rnjtochondrial matrix from wild-type cells revealed a peak of aip-dependent proteolytic activity which was absent in the disruptant. furthermore, we have identified matrix proteins that are degraded with a half-time of ~60 min in intact wild-type ceils but are stable in the ion disruptants. electron microscopy of lon disruptants revealed the presence of many electron dense bodies in the mitochondrial matrix which are not found in lon + cells. hydrolysis of high molekular weight kin1nogen by plasma kallikrein benner, s. and duffer, h., laboratory for organic chemistry, eth h6nggerberg, ch-8093 z/inch high molecular weight kininogen (hmwk) is cleaved by the serineprotease plasma kailikrein to generate the hypotensive peptide bradykinin in human blood. we established a measuring system for the time course of the hydrolysis of hmwk in its native and deglycosylated form and in the presence or absence of c~-inhibitor. analysis of the bradykinin release and the yielded protein fragments lead to the three following results: 1. the hydrolysis did not follow plane michaelis-menten kinetics due to a hmwk-fragment operating as a competitive inhibitor. 2. addition of the ci-inhibitor stopped the hmwk-hydrolysis immediately, which is in contrast to the physiological role of plasma kallikrein in blood. 3. so far we have no evidence that the high content of o-glycosylated side-chains of the hmwk-light chain has an effect on the kinin-liberation as proposed in literature. ( tetrahydrobiopterin (bh4) is the essential cofactor for the hepatic phenylalaoine hydroxylase, but also for tyrosine and tryptophan hydroxylases that are responsible for the production of nettrofransmitter precttrsors. furthermore, bh4 is required for the deavage of giyceryl ethers aztd is i~vo[ved in the biosynthesis of nitric oxide by the nitric oxide synthase. a vazia~t type of hyperphenylalani~emia is caused by a deficiency of bh4. the most frequent form of this cofactor deficiency is due to lack of 6-pyruvoyl-tetrahydropterin synthase (ptps) activity. the human cdna for ptps was isolated, and the recombinant protein was found to be active when expressed in e. coil, thus allowing us to perform hmctional t e,3ting of mutant proteins. primary skin fibroblasts derived from ~everal ptps deficient patieilts were investigated for the molecular nature of this autosomal recessive disorder. all fibroblasts had f1"ps mrna expressed, and subsequent cdna sequence analysis revealed different mutatkons in the coding region of ptps (codon replacemenls, deletions, or nonseme codons) responsible for a lowered or no enzyme activity. in order to potentially correct ptps activity (and restore bi-i 4 biosynthesis) we are establishing a recombhtant rel~oviral-mediated gene transfer system to efficiently introduce the wild-type human ptps-cdna in these fibroblasts. the thylakoid membrane (tm) contains i0 molecular species of phosphatidylglycerol (pg). the relative amount of these species depends on the plant species and growth conditions. using phospholipase a2 (pla2) at 0~ to digest preferentially pg in the outer monolayer of spinach tm, we have shown previously that this monolayer was enriched in pg (ca 70~). the purpose of this investigation was to determine the transmembrane distribution of each pg molecular species. to this aim, we have studied the hydrolysis kinetics of each species in the presence of pla 2 in spinach and squash tm containing different relative proportion of molecular species. the hydrolysis rate and extent of the molecular species depended on the unsaturation degree of the fatty acid at the sn-i position as well as on the presence or absence of 16:1(3t) at the sn-2 position. for instance, the hydrolysis rate of pg ig:3/16:l(3t) was greater than that of pg 16:0/16:0. these results will be discussed in terms of the thylakoid transmembrane distribution of each pg molecular species. (supported by the snsf 3100-33693.92). from previous electrophysiological evidence, we concluded that metal ions (hgz+,cd2+,ag +) at low concentrations (i0-6m) increase rapidly (10-60 s) cation (na + , k + ) conductance at the apical membrane of epithelial cells. we investigated here the effects of these metals on [ca2+]i in mdck-i cultured cells, for a potential correlation between functional membrane changes and [ca2+]i . metals (10-4-10 -6 m) were added at the apical side of the epithelium, and [ca2+]i was measured with fura-2. hg 2+ induced a rapid (peak 5-10 s) and marked increase in [ca2+]i, whereas cd 2+ entailed a slower (peak 2-5 min) and marked response. the time-course of effects for both metals was similar to the electrophysiological changes. in contrast, the pattern of effects of ag + on [ca2+]i differed markedly from that on apical membrane conductance. thus, the effects of metals on [ca2+]i are conspicuous but not tightly associated with changes in membrane conductance. c. bulliard and l-l. dreyer, institut de biochimie, universit6 de fribourg, ch-1700 fribourg trans-plasma membrane oxydoreductases (pmo) play a significant role in energy transduction and post-receptor signal transmission, but the enzymes have not been characterized so far. we have achieved the purification of pmo from rat brain synaptic vesicles and from synaptic plasma membranes. the membrane enzymes were extracted by 1% lubrol xl tm, precipitated with ammonium sulfate (between 40 and 70% saturation) and purified by means of hydrophobic chromatography on butyl-agarose and gel f'fltration on superose-12, followed by page under native conditions. specific enzymatic staining of native page gels yields two homogenous diaphoraselike activities, pmo-i and pmo-ii . sds-page of the enzymes showed that pmo-i is made of two subunits of 52 kda and 40 kda whereas pmo-ii consists of a single sub-unit of 52 kda. the 52 ki)a subunits from pmo-i and pmo-[i are probably identical from their respective properties. partial n-terminal sequencing (13 aa) of the 40 kda subunit from pmo-i showed 84% homology with rat brain fructose-l,6-bisphophate aldolase. these data indicate that pmo is closely associated with the glycolytic enzyme that co-purifies in all steps under suitable conditions. the biochemical functions of pmo's remain to be established. ferredoxin:thioredoxin reductase (ftr) is the key enzyme in the ferredoxin/thioredoxin system, the light-dependent regulatory system in oxygenic photosynthesis. it is a nucleus encoded ironsulfur protein, composed of two dissimilar subunits, one of which is the catalytic subunit containing a redox-active disulfide bridge functional in the reduction of thioredoxins and a 4fe-4s cluster. using pcr we have isolated and characterized a cdna clone of 738 bp from spinach and a clone of 911 bp from corn coding for the catalytic subunits including the transit peptides. the first 31 (spinach) resp. 38 (corn) amino acids after the start exhibit typical features of chloroplast transit peptides. following the transit peptides are 113 (spinach) or 114 (corn) ami[~o acids representing the catalytic subunits. the primary structures are highly conserved between these two species with 91% similarity and 81% homology. seven of the eight cys residues found in the spinach subunit and important for the catalytic activity are at conserved positions. (snf 31-28811.90 and 31-37725.93) $23-20 r. zurbriggen and j.-l. dreyer, institut de biochimie. univarsit6 de fribourg, ch-1700 fribourg most mammalian cells display transplasma membrane oxidoreductase activity (pmo). the enzymes use intracellniar reduced pyridine nucleoticle to reduce an unspecific extracellular electron accepter as substrate. in order to set up a methodology for purifying, characterizing and quantifying pmo's, the plasma membrane from a neuroblastoma cell line, nb41a3, has been biotinylated. subsequently the biotinylated proteins were purified by immanoprecipitation with avidin, antiavidin-antibodies and insoluble protein a. the protein recovery of an immunopurified membrane preparation was <0.15% of the protein content in the cell extract. specific pmo activity was increased 15 to 20 fold compared to the activity in whole cells. this approach demonstrates the presence o1' pmo within the cell plasma membrane. this activity accounts for about one third of the total cellular diaphorase activity and cannot be attributed to an increased perraeabilization of the plasma membrane induced upon biotinylation nor to intracelluiar activity from lysed cells. pmo also promotes cell growth at low substrate concentrations (0.1-ll.tm). native gel electrophoresis of iarinobiotinylated and affinity purified plasma membrane extracts displays two diaphorase-positive bands, indicating that a homogeneous cell population may express several pmo activities at the plasma membrane. fluvastatin (fs) is a new hmg-coa reductase inhibitor. its affinity for 3 major human drug metabolizing p450 monooxygenases (cyp2c9, cyp2d6 and cyp3a4) was determined in liver microsomes. fs showed low affinity (ki > 50 ~tm) for cyp2d6 and cyp3a4 whereas cyp2c9 was selectively and competitively inhibited (ki = 0.1 ~tm). the potential for in vivo fs interactions with cyp2c9 substrates was therefore investigated in 14 volunteers using dicinfenac (df) as a probe (25 mg orally) on days 0, 1 and 8. df and 4'-ho-df kinetics were determined by hplc/uv. df cmax increased over time (0.28 [sd 0.12], 0.38 [0.20] and 0.45 [0.4] mg/l on day 0, 1 and 8, resp.). oral clearance was reduced on days 1 and 8 (14 % and 15 %, resp.). a time dependent decrease in urinary metabolic ratio (4'-ho-df/df) was noted (1.07 [0.34], 0.90 [0,23] and 0.70 [0.18] on day 0, 1 and 8, resp. (p < 0.0001)) only over the first 4 hours. fluvastatin therefore exhibits a two-phase interaction in vivo: an early transient and a late cumulative inhibition. the transient phase matches inhibition profiles predicted by quantitative models integrating in rive pharmacokinetics and in vitro inhibition data (q-dips). simple competitive inhibition by fs cannot explain the late phase. other mechanisms (i.e. fs or metabolite cumulation, mi complexation) are hypothesized. in some situations fs is expected to cause interactions with cyp2c9 substrates, which are partially predicted by quantitative modeling of in vitro data (snsf project n o 32-36600.92). 1211 gen~ve, switzerland. previously, a parvalbumin mutant, pvf~02w, was constructed with a unique reporter trp in the middle of the hydrophobie center. in the present study three new parvalbumin mutants, derived from pvft~w and containing alterations essential for ca2+-binding in either one, pv.câ�¢ and pv_m, or both, pv-co~, of the two ca2+-binding sites, were analyzed in order to study the metal binding properties of individual ca~+-binding domains and their contributions to the structure and stability of the protein. both, pv_ct, and pv.~, bind i ca ~ ⧠with affinity constants, kc,, of 1.1,107 and 3.2-106 m "~ in the absence of mg 2+. mg 2+ shifts the ca~+-binding isotherms of pv.co to higher free [ca z+] according to the competition equation with km~ = 2 9 103 m t. pv.m~ is much less effected by mg z* (kmg = 80 m "t) and can be considered as possessing a ca2+-specific site. nevertheless, in the absence of ca 2 ⧠both, pv.co and pv~, bind i mole of mg 2+ with much higher affinity than predicted from the competition studies. pv_cd;~ binds neither ca 2. nor mg 2+. trp-fluorimetry and uv-difference spectroscopy revealed that the ca~*-loaded conformations of pv_co, pv.ef and pvr~o~w are similar, with the trp-102 deeply buried in the hydrophobie core. however, striking differences between the mutants are found in the metal-free and mg~+-loaded forms. the conformation of pv~t~;~ is not affected by ca 2~ or mg z*. our results indicate that destroying the ca2+-binding of the ef loop, but not of the cd loop, strongly destabilizes the protein in the absence of ca z ⧠. biochemlsches institut der unlversitfit zfirich, switzerland, ch-8057. the sea urchin, strongylocentrotus purpuratus, metallothionein gene, mta, was constructed and expressed under the control of a heat shock promoter in a protease-deficient strain of e, coll. the nascent recombinant protein was stabilised by the addition of exogenous cd. the cd-containing protein was precipitated with ethanol and subsequently purified to homogeneity by gel permeation and ionexchange chromatography. the purified protein was identified as the desired gene product by tryptic peptide map analysis, sequence analysis and mass spectroscopy. typical yields of protein were 2mg per litre of culture. the presence of 7 cd ions per molecule was confirmed by the sh/cd ratio and by the existence of 7 h3cd nmr resonances. the apparent association constants of sea urchin mt with cd, which has a charge of -8, were found to be approximately 15 times weaker than for rabbit mt, at ph 7. (mt) are small metalloproteins displaying as their main feature clusters of d i~ metal ions bound to the thiolate ligands of the abundantly occurring cysteine residues (cys). from a set of over 85 sequences a general multialignment of 62 class i mt has been obtained on the basis of the needleman & wunsch algorithm. all cys are conserved in the man%mals and over 83% of them in other vertebrates and invertebrates. on the basis of similarity/identity scores we can isolate groups of sequences. evolutionary relationships between species and groups have been calculated with the maximum parsimony method of felsenstein and with the distance matrix method of fitch and margoliash. the divergences observed among the mammalian mts indicate a partition into four distinct subforms which all may have arisen before the separation of the species in evolution and which in part may differ in function. $23-28 bourque, a., singh, a., dykeman, a., macmahon*, a., and foster*, w. atlantic veterinary college, charlottetown, canada, and *reproductive toxicology section, environmental health centre, tunney's pasture, ottawa, canada. hexachlorobenzene (hcb) is a known environmental pollutant that is produced as an industrial by-product of certain chemicals. when administered in high dosage, hcb induces alterations in the rhesus monkey ovary. a purpose of the study was to determine a no observable adverse effect level (noael) for the compound. twenty, cynomolgus monkeys, 6-13 years old were placed in five groups. hcb was given orally in concentrations of 0.01, 0.1, 1.0, and 10 mg/kg b.w. in gelatin capsules, daily, for 13 weeks. one group of animals fed glucose only, served as the control. ovary specimens fixed in 2% buffered glutaraldehyde were prepared by conventional methods for transmission electron microscopy. ultrastructural alterations were revealed in the developing ova and follicular cells in all the ovaries from hcb-treated animals in a dose-related manner. clinical chemistry was unaffected by hcb. a noael for this reproductive toxicant is yet to be determined. a.b. was a recipient of the nserc undergraduate student award. an acidic haem-and zinc-containing protein has been isolated from bovine brain. native and sds-polyacrylamide-gel-electrophoresis reveal a monomeric protein with an apparent molecular weight of 47,2 kda. the absence of co-staining with tetramethylbenzidine implies that the haem group is non-covalently bound. the electronic absorption spectrum, with a soret-band absorption maximum at 412 nm and c~-and i~-bands at 540 and 575 nm, respectively, is similar to that of fe(ll)-myoglobin, consistent with the presence of haemiron in the ferrous oxidation state. in air the protein was easily oxidized to the fe(lll) form with a subsequent shift of the soretband maximum to 405 nm. atomic absorption measurements indicate approximately 1 mole of zinc and 1 mole of iron per mole of protein. the amino acid composition is similar to that of indoleamine-2,3-dioxygenase, although no such enzymic activity has been detected. in this study, we looked for this substance and its metabolites in young and old bovine lenses, because of their possible role in cataract formation. the substances were detected by hplc analysis. the kynurenine aminotransferase (kat) activity was determinated by the method of tobes (methods enzymol 1987~ 142:217) . the fluorescent substance formed from 3-hk was characterized by thin layer chromatography followed by reaction with rtinhydrin, uv and fluorescence spectrometry, and atom bombardment for molecular mass determination. 3-hk at concentrations of 0.08-+0.01, 0.2_+0.04, and 1_+0.4 btg/g of tissue was detected in iris/ciliary body, retina, and calf lens respectively. this substance is deaminated by kat. the activity of this enzyme was 2.6_+ 0.3, 3.7_+0.2, and 9.6+_2 ~tmol/g of tissue/hour in retina, iris/ciliary body, and lens respectively of old bovine eyes, but it was not found in calf eyes. the deamination of 3-hk resulted in the formation of a fluorescent substance which was identified as oxo-xanthurenic acid (oxa) with a molecular mass of 205 daltons. the accumulation of oxa acid possibly interacting with lens proteins could induce cataract formation. snf 32-36058.92, emdo, and hartmann-mtiller. the goal of covalent immobilization of oligonucleotide capture probes to solid supports has been accomplished by light-dependent, photolinker polymer mediated immobilization strategies. synthetic oligonucleotides were immobilized by facile layer coating procedures. the procedure does not require functionalization of the oligonucleotide probe or the matedal surface. oligonucleotides were linked to polystyrene with a biopolymer which was multiply substituted by photoactivatable diazirines. capture probe photoimmobilization (350 nm irradiation) was strictly light and diazirine dependent. using radiolabeled oligonucleotides as capture probes and polystyrene as material surface, the light dependent coupling efficiency was 40%. nonspecific oligonucleotide binding was below 2 %. photojmmobilized oligonucleotides retained the ability to form stable complexes with complementary dna strands, and target oligonueleotides of varying length were captured as well as dna fragments produced by pcr techniques. indoleamine 2,3-dioxygenase (ido), a superoxide radical scavenger, is present in transparent human lenses and produces 3-hydroxykynurenine, a uv-b filter. the synthesis of 3-hydroxykynurenine by ido and its degradation by kynurenine aminotransferase (kat) were measured in transparent lenses and cataracts. post-mortem eyes of elderly persons between 72 and 84 years of age with transparent lenses were obtained from the eye-bank of the department. fresh cataracts were obtained from cataract surgery patients. ido activity was measured by the method of yamazaki et al (biochem j 1985; 230:635) adapted for hplc. kat activity was measured by the method of tobes (methods enzymol 1987; 142:217) . ido activity found in post-mortem transparent lenses (cortex and nucleus) was 0.7_+0.3 nmol/mg protein/hour. in cataracts a low 1do activity of 0.3_+0.2 nmol/mg protein/hour was found in the cortex but not in the nucleus. kat activity, found in the eight cataracts examined, was 1.6_+0.9 nmol/mg protein/hour. inhibition of ido activity and induction of kat activity seems to be involved in human cataract formation snf 32-36058.92, emdo, and hartmarm-m011er photolinker polymer mediated covalent immobilization of antibodies and f(ab') fragments has been achieved by newly established lightdependent coupling procedures. anti alpha-l-fetoprotein monoclonal antibodies and f(ab') fragments derived from anti prostate specific antigen monoclonal antibodies were covalently linked to microplates. diazirine derivatized bovine serum albumin served as multifunctional light activatable linking agent. both immunoreagents, monoclonal antibodies and f(ab') fragments remained immunologically active after 350 nm irradiation (irradiance 0.7 mw cm -2 for 20 minutes). covalency of antibody binding was inferred from i) the photoreagent dependence, ii) the light dependence of the immobilization process, and iii) the reversibility of immunocomplexation after acid treatment. $23-33 two ~ of extracelluiar elecirical resistance in v~kwricular myocardil~ fleischhauer j.c., kl~ber a.g., dept. of physiol. bern in myocardial tissue, the electrical resistive properties of the extracellular space are important for local current flow during e~citation and therefore influence impulse conduction and the magnitude of the extracellular electrical field. to assess the r~tlti~t nature of the resistance of the extracellular space, electrical cable analysis was applied on an arterially perf~-sed rabbit papillary mascle. ~he resistivity of the intravascular space was changed (70 to 220~i~n) by variation of hematocrit frcrn 0 to 60% in the perfusate. in order to change the voltm~ and hence the electrical resistance of the interstitial space, colloidosmotic pressure in the perfusate was varied by changing dextran {m r 70000) concentration (i0 to 80g/l). altering the interstitial space volume had a marked influence on the extracellular resistance (ro) , conduction velocity and the magnitude of the extracellular field. variations of the resistive properties of the intravascular space had no significant influence on ro, conduction velocity and the magnitude of the extraeellular electrical field. our results suggest that the microvascular tree is insulated from the interstitial space and local electrical current flow during excitation in heart muscle is confined to the narrow interstitial clefts. s04-25, s 15-69 s05-18, s05-19 s 11-05, s11-06 bchini hooft van huijsduijnen s08-12 s05-30 s15-45 s08-13 s08-05, s08-06 s01-08 s15-67, $23-19 s13-03 nook, ek s01-15, s10-15 s05-02, s05-05 s 15-03, s15-29, s15-30 s19-03, s19-04, $19-05 s16-02, $16-15 s12-22 s15-48 s05-24 s03-01, s03-02, s03-03 s03-01 lrmler s05-28, s09-06 s03-07, s03-10, s03-11 s18-05 lipp, p. s05-26 so 1-02 s15-15, s15-16, $21-03, $21-11, $21-12, $21-14 $23-29,$23-30 s01-17 s 15-29, s15-30 s12-12, s12-24 molar s15-19 s08-05, s08-06 e s09-06, s09-07 s15-03, s15-29, s15-30 e. s03-05 s05-09, s05-10, s05-51 s05-52 s10-16 s08-14 s04-23, s04-29, s05-13, s05-53, $20-15 s10-16, s10-19 s09-01, s09-02 s15-68, s 17-24 stalder h. s03-19 s10-21 s09-09, s13-10, s13-13 s10-21 teheesen s01-10, s01-11, s01-18 s15-16 van dillewyn s05-32, s05-37, s05-38 s08-05, s08-06 s05-18, s05-19 huber d., ojha m. and turian g. laboratory of general microbiology, university of geneva, sciences iii, 30 quai ernest-ansermet, 1211 geneva 4, switzerland. ca2+-dependent protease in allomyces is predominantly localized in the apical region of the exponentially growing hyphae (huber and ojha, submitted) . many cytoskeletal proteins like actin and tubulin are also mainly localized in this growing region (heath, l~91). it is known that the ca2+-dependent protease proteolyzes a number of cytoskele~ ton proteins in order to regulate the plasticity of the cell (mellgren, 1987) . we have studied the proteolysis of some cytoskeletal proteins by this protease purified from the aquatic fungus allomyces erbuscula. actin, tubulin and desmin, were found to be rapidly proteolyzed in vitro at a high substrate to enzyme ratio. immunofluorescence studies of proteolyais made in situ in exponentially growing hyphae showed modification of apieally localized cytoskeletal proteins. this colocalization of the ca2+-dependent protease and cytoskeletal proteins in the same apical region is inferred in relation to hyphal elongation. jbiological databases grow exponentially. in order to provide access, as much data las needed and as little volume as possible shall be returned upon a query. the icurrentiy available dna and protein sequence databases are updated in a sophisticated network of procedures and expose a considerable amount of redundancy. research is performed on optimizing the creation of non-redundant data sets. the resulting data are disttibuted in switzerland, or made accessible to researchers who cannot utilize local resources. transparent access to the swiss resources, as well as paneuropean services, is provided with a job scheduling system which was developed in basel, the hierarchical access system for sequence libraries in europe ("hassle"), and various other computer programs which allow comprehensive browsing such as "gopher" and "www". training courses to use the resource effectively are running throughout the year. key: cord-015024-2xzc0uc5 authors: nan title: esicm 2010 wednesday sessions 13 october 2010 date: 2010-08-31 journal: intensive care med doi: 10.1007/s00134-010-2001-7 sha: doc_id: 15024 cord_uid: 2xzc0uc5 nan power spectrums for vt and eadi are shown in fig. 1 (ps and nava) for a typical patient. the enlarged section highlights how changes in eadi are highly synchronized with nava ventilation, but less so for ps. table 1 ) and complications of mechanical ventilation ( table 2 ) did not differ significantly between the two studied groups. introduction. high tidal volumes in mechanically ventilated patients with ards lead to baro/bio-trauma and increase mortality. also, it was recently shown that ventilation with high tidal volumes is a risk factor for ''acquired ards'' in a medical population. objective. we evaluated the impact of high tidal volumes after cardiac surgery. method. we analysed the prospectively recorded data of 3,434 consecutive patients who underwent cardiac surgery from 2002 to 2005. we predefined 3 groups of patients based on the tidal volume delivered immediately after surgery: (1) low: 7-9.9, (2) ''traditional'': 10-12.9, (3) high: above 13 ml/kg of predicted body weight (pbw). we assessed the risk factors for organ dysfunction (prolonged mechanical ventilation, hypoxemia, hemodynamic failure and renal failure) by univariate and multivariate analysis, including the initial tidal volume in the models. mean tidal volume/actual weight and tidal volume/pbw was 9.2 ± 1.3 and 11.1 ± 1.5 in men (p \ 0.0001), 9.1 ± 1.4 and 12.5 ± 2.2 in women (p \ 0.0001). 411 patients (12%) were ventilated with low tidal volumes, 2,194 (63.9%) with ''traditional'' tv and 829 (24.1%) with high tv. the mean body mass index in the 3 groups was 23.8 ± 4.0, 27.0 ± 4.1 and 31.5 ± 5.4 respectively (p \ 0.0001). with increasing bmi, the tidal volume/ actual weight decreased while the tidal volume/pbw increased (figure) . the percentage of women was 12.2, 20.8 and 52.2% respectively for low, ''traditional'' and high tv (p \ 0.0001). high tidal volumes were associated with prolonged intubation ([48 h) (1.5 vs. conclusion. traditional and very high tidal volumes are associated with prolonged mechanical ventilation and organ dysfunction after cardiac surgery and use of high tidal volumes is an independent risk factor. ''prophylactic'' protective ventilatory strategy should be provided in this population with inflammatory state at risk to develop ventilator induced pulmonary edema. women and patients with high bmi are more at risk to be ventilated with injurious tidal volumes. introduction. evidence shows that clinicians' non-technical skills (behavioural and cognitive skills) have a significant impact on teamworking, patient safety, efficiency of care provided and potentially patient outcomes (1) . such skills are key for cardiac arrest teams (cats), which are multi-professional (anaesthetists, physicians and nurses) and normally function under high pressure. to date, most tools to assess nontechnical skills in healthcare have focused on surgery (2) and anaesthesia (3) . no validated, robust tools are currently available for assessing non-technical skills in cats. objectives. to develop and validate an observational skill-based clinical assessment tool for resuscitation (oscar). this should be psychometrically robust for use in both training and assessment contexts. methods. oscar was based on a well-validated tool for surgery (otas) (4) and was developed in phases. six behaviours were included in the assessment: communication, cooperation, coordination, monitoring, leadership and decision-making. observable behavioural exemplars were derived for each one of these behaviours across the three cat subteams-anaesthetists, physicians and nurses (phase 1). quantitative expert consensus methodology was employed to assess content and face validity and observability of the exemplars (phase 2). two clinician observers used oscar to blindly rate eight cats performance in a series of simulated cardiac arrests. psychometric analyses of these ratings were used to determine observable behaviour applicability, internal consistency, and inter-rater reliability (phase 3). . 15 of 18 oscar behaviours demonstrated high internal consistency (cronbach a = 0.736-0.970). psychometric analyses dictated removal of three behavioural exemplars (two in anaesthetic group; one in physician group) to significantly improve internal consistency. inter-rater reliability was also high (inter-observer pearson r = 0.661-0.911, all p \ 0.005). inter-observer reliability analyses revealed a learning curve between the two observers, with significant reduction in scoring discrepancies from the first to the eighth observed resuscitations. conclusions. oscar is a psychometrically robust (reliable, content-and face-valid) tool for the assessment of teamworking skills in cardiac arrest events. the tool is feasible to use and can be employed for both training and assessment purposes. introduction. different educational methodologies are used to teach basic skills in emergency medicine. high-fidelity patient simulation offers an ideal venue for presentation of critical events that can be managed by medical students without risk to a patient. therefore full scale simulation training could be superior to paper case based seminary rounds to achieve these specific educational objectives. objectives. the aim was to compare simulation to a standard education measured by multiple choice questionnaire. after written informed consent and approval of the institutional research ethics board 160 fifth year medical students were included in the survey. they took part in the compulsory emergency medicine curriculum of charité universitätsmedizin berlin. the students completed a basic multiple question tests on day 1 including 25 questions concerning the topic of ''acute coronary syndrome'' (acs). on day 2 for the topic ''acs'' half the group was assigned a 45 min session simulation training while half the group was assigned a 45 min session paper case training. on day 3 groups were reversed and the topic ''aic'' was taught in either simulation training or paper case seminary round. the test of day 1 was repeated after each training sessions. results of the tests were evaluated using spss(tm) 18. the mann whitney u test was used to show any significant differences in reaching educational objectives in the test (a \ 0.05 was considered significant). there was an even distribution of men and women among the two groups. the test results showed no significant difference between the two groups on day 1. on day two for the topic ''acs'' the group with simulation training achieved significantly better test results. for the topic ''acs'' on day 3 there was no difference while students received further training in acs not using a high fidelity simulator. the results were not linked to specific teachers. introduction. rapid sequence induction (rsi) involves loss of spontaneous breathing and mandates airway control. steps to reduce adverse incidents include adherence to minimum monitoring standards, appropriate drug selection, access to difficult airway equipment and presence of skilled anaesthetists. there is substantial evidence that appropriate monitoring reduces risk by detecting the consequences of errors, and by giving early warning of patient deterioration. objectives. to assess conduct of emergency anaesthesia (monitoring and drugs) for critically ill patients not in an operating theatre (or) administered by intensive care doctors. methods. prospective analysis of rsi for critically ill patients in a uk nhs acute hospital over 1 month. or based practice was excluded. reason for anaesthesia, location, drugs administered, monitoring modalities, adverse events and access to airway equipment were recorded. results. data from 26 patient episodes were collected: predominantly in the emergency department (38%) and intensive care unit (27%) for respiratory failure (46%), reduced consciousness (19%) and to facilitate investigations (19%) . the most common induction agent was propofol (52%); thiopentone (26%) and etomidate (11%) were less frequently used. suxamethonium (73%) was preferred for initial neuromuscular blockade. during induction most doctors used pulse oximetry, electrocardiography and blood pressure monitoring. only 24% used capnography. no doctor used minimum monitoring to association of anaesthetists of great britain and ireland (aagbi) standards. 1 rescue airway equipment immediately available is shown in fig. 1 . complications occurred in 12 cases (fig. 2) . patients that had a hypotensive episode during induction all had thiopentone or propofol used as induction agents. 15% of patients had a period of desaturation, and 23% required more than one attempt for successful intubation. in cases with complications, rescue airway equipment was unavailable in[60 and 75% did not achieve uk minimum monitoring standards. conclusions. shortcomings during emergency anaesthesia were recorded including monitoring, access to rescue airways and physiological disturbance. procedural guidelines and training are to be developed for emergency anaesthesia; access to capnography and alternative airway equipment will be assured. these issues are unlikely to be unique to our trust and assessment of practice is recommended. introduction. critical care echocardiography (cce) is performed and interpreted by the intensivist at the bedside to establish diagnoses and guide the management of patients with circulatory or respiratory failure in the icu. competence in basic and advanced cce has been recently defined [1] , but no curriculum to reach the required cognitive and technical skills has yet been elaborated. objectives. to assess the efficacy of a limited, tailored training program for noncardiologist residents without experience in ultrasound to reach competence in basic cce. methods. six noncardiologist residents (anaesthesiology: n = 5, pneumology: n = 1) without previous experience in ultrasound participated to the study during two 3-month periods. the curriculum consisted in 4 h of didactics, 2 h of interactive clinical cases and 6 h of tutored hands-on. color doppler mapping was excluded from the training. after completion of the training program, all eligible patients underwent subsequently a transthoracic echocardiography (tte) performed in random order by a recently trained resident and an experienced intensivist with expertise in cce who was used as a reference. in each patient, the resident and the experienced intensivist answered binary ''rule in, rule out'' clinical questions covered by basic cce [1] : global left ventricular (lv) size and systolic function (eye-ball evaluation of ejection fraction), homogeneous or heterogeneous lv contraction pattern, global right ventricular (rv) size and systolic function, identification of pericardial fluid and tamponade, and assessment of both the size and respiratory variations of the ivc. in case of undetermined interpretation, the corresponding clinical question was considered not addressed. the agreement between responses to clinical questions provided by the two investigators who independently interpreted the tte study at bedside was used as an indicator of effectiveness of the tested curriculum. proportion of graduates to work within a ''critical care'' setting. the level of support available to trainees may vary with local resources but risk management and national guidelines stipulate that close supervision is provided to junior doctors in high stake decisions and procedures until deemed competent at the relevant tasks 1 . furthermore, substantial ongoing reduction in working hours places further limitations on training; both majors can impact adversely on junior doctors service output and experience. a modified delphi method was used 2 years ago to design a task focused single-day course on the theoretical basis of critical care and provide lab-based training in delphi identified high risk procedures and interventions 2 . objectives. assess the impact of the course on the following: trainee confidence and the start of the ''novice'' critical care post trainee performance in comparison to peers perceived educational benefit from their training post compared to peers methods. 30 junior doctors attending the course were enrolled in the study and matched for graduation year and medical school to 30 junior doctors who did not attend similar training prior to commencing their post. data was collected through anonymous standardized forms on the day of the course, first day of the job, end of week 1, week 6 and 4 months into the post. trainee confidence and self perceived competence were assessed on a ten point scale. in addition, trainees were requested to maintain a log of 3 interventions: 1-ultrasound guided central venous catheter insertion, 2-arterial catheter insertion, 3-ventilation problem solving. candidates attending the course demonstrated greater confidence at multiple points within their post as well as higher performance, satisfaction and educational value scores. conclusions. critical care trainees benefit from a task focused orientation to the fundamentals of critical care before commencing first post in this setting. . 100 enrolled 50 patients in each group. no differences in age and gender. incidence of vap-study group 8.8% compared to control group 28.8% p value 0.004. vap per 1,000 hospital days: control-29.5% compared to study-10.2% p value 0.06; average days in icu control-14.97 compared to study-11.8, p value 0.151; average ventilated days, control-12.68 compared to 8.9, p value 0.044; average antibiotic use in days control-9.7 compared to study-7.2, p value 0.413. introduction. nosocomial infections are the most common in-hospital complications with high morbidity and mortality. educating healthcare professionals is an important prevention measure. objective. to analyze the impact of a nurse consultant team on nosocomial infections prevention in the icu, the improvement in prevention knowledge of the nurse staff, and its impact in the application of the prevention measures in the daily practice. methodology: the nurse referent team was constituted by 8 nurses. the study subjects were all the staff icu nurses and all the patients admitted during pre and post-intervention phases. the study was conducted in our medical-surgical icu (16 beds) in 3 phases: 1 pre-interventional (15/01/09-15/02/09) observational. record of the accomplishment of 12 cdc recommended variables about mechanical ventilation associated pneumonia (vap) and catheter related bloodstream infection (cr-bsi) prevention measures. 2 interventional (1/03/09-1/10/09) eight educational meetings with the nurses staff groups to teach the most important aspects of the nosocomial infections prevention. before and after lectures every nurses answered an anonymous questionnaire about their knowledge in those subjects. a poster with the most important reminders was place in every icu patient room. 3 post-interventional (15/01/10-15/02/10) observational. new record of the same 12 cdc pre-interventional variables. we compared the accomplishment of these variables before and after the interventional phase as well as the number of correct questionnaire answers. statistics were made with spss software. results. during the interventional phase 70% of the staff nurses attended the educational meetings. the number of correct answers increased significantly after the conference (77.1 vs. 55.2% p \ 0.0001). regarding to the daily practice, we observed a significant increase in the accomplishment in most of the variables (see table below), while in 3 of them no improve was observed and in 2 the improvement was not statistically significant. during the study period we observed a decrease in the incidence of vap (6.7-14.9 episodes/1,000 mv days) and cr-bsi (2. introduction. glucose variability has been found to be associated with mortality in critically ill patients, independent of mean glucose concentration [1] . objectives. the aim of this analysis was to assess the impact of real time continuous glucose monitoring (cgm) on glucose variability in critically ill patients receiving intensive insulin therapy (iit). methods. this is the post-hoc analysis of a prospective, randomized, controlled trial [2] . data of 124 patients admitted to the icu either receiving iit according to a real time cgm system (guardian ò , medtronic, northridge, ca, usa) (n = 63) or according to an algorithm (n = 61) with selective arterial blood glucose measurements (simultaneously blinded cgm) for 72 h were analysed. insulin infusion rates were guided according to the same algorithm in both groups. mean glucose and standard deviation, as a marker of glucose variability, were calculated for the first 24 h (glumean1, glusd1) and for the whole study period (glu sd ). statistical comparison of parameters between study groups and between icu survivors (n = 94) and non-survivors (n = 30) was performed using student's t test. results. the variability of sensor glucose during the entire study period was comparable between the real time cgm group and controls (21.51 ± 1.10 vs. 23 introduction. in the gastrointestinal tract, the gut flora which comprises several hundred grams of bacteria is crucially involved in host homeostasis through their metabolic, trophic, and protective activities. however, the immediate changes in the gut flora in critical illness following severe insults are unknown. objectives. to investigate the changes in the gut flora at an early phase of severe insult in critically ill patients. methods. fifteen patients who experienced a sudden and severe insult including trauma, out-of hospital cardiac arrest, and cerebral vascular disease were studied, along with 12 healthy volunteers as the control group. two fecal samples were acquired from the subjects by swabs of the rectum within 6 h after admission to the emergency room (day 0). samples were serially collected from patients on day 1, 3, 5, 7, 10, and 14. samples were collected from control subjects. results. total bacterial counts, especially various obligate anaerobes and total lactobacillus, significantly decreased in comparison to those of the control subjects on day 0. in addition, on day 0, the total organic acid levels of the patients were significantly lower than those of the control subjects; particularly acetic acid, propionic acid, and butyric acid. the levels of these acids remained low throughout the 14 days period of study. the total bacterial counts did not recover to normal levels during the 14 day study period. obligate anaerobe counts of the patients did not improve until day 14. total lactobacillus counts were low on day 0 and increased gradually thereafter, but did not attain the levels found in controls. the counts of pathogens (enterococcus and pseudomonas) increased during the study period. conclusions. gut flora in critically ill patients can change drastically immediately after a severe insult, and may not recover for up to 14 days. at the same time, the number of harmful bacteria can increase. total bacteria 10.1 (9.8-10.4) 7.5 (6.7-8.0) .001 obligate anaerobes clostridium cocades group 9.4 (9.0-9.7) 6.1 (5.4-7.0) .001 clostridium leptam subgroup 9.2 (9.0-9.7) 6.5 (6.1-7.3) .001 bacteroides fragilis group 9.4 (9.0-9.8) 6 .8 (5.9-7.6) .001 bifidobacterium 9.2 (8.9-9.4) 6.0 (\5.0-6.9) .001 atopobium cluster 9.0 (8.5-9.5) 6.0 (\5.1-7.1) .001 results. mean serum 25(oh)d level was 18.9 ± 10.9 ng/ml. by current definitions the majority of patients (62.5%) were vitamin d deficient (\20 ng/ml) and 24.9% were vitamin d insufficient (c20 and \30 ng/dl). normal 25(oh)d levels ([30 ng/ml) were present in 12.6%. table 1 provides information on clinical and laboratory findings in the three 25(oh)d groups. both lower 25(oh)d tertiles were associated with increased hospital mortality after adjustment for age, sex and saps ii. for 330 patients both 25(oh)d and pth levels were available. adjusting the cox regression analysis also for pth and dialysis status increased the hr for hospital mortality to 2.3 (1.1-4.7) and 2.1 (0.99 and 4.4) for the two lower 25(oh)d tertiles. in addition tertiles of pth and serum calcium levels suggested higher mortality rates for patients in the highest pth (p = 0.09) and those in the lowest calcium tertile (p = 0.12). our results demonstrate that independent of baseline saps ii, age and sex, critically ill patients with low 25(oh)d levels seem to be at increased risk for hospital mortality. whether a rapid correction of vitamin d status may be beneficial in the icu setting remains to be further explored in randomized controlled trials. • the autonomic storm after brain death must be early diagnosed and treated with a standardized protocol including hormone therapy introduction. the use of filling pressures of the right atrium and left atrium is normal in the monitoring of critically ill patients undergoing mechanical ventilation. this monitoring is done through an invasive catheter placed in the superior vena cava and pulmonary artery, which is not free of complications. the ability to make measurements of these parameters in a non invasive way, makes the echocardiography an useful and essential tool when monitoring critically ill patients objectives. we focus the study on validate the reliability of noninvasive measurements by echocardiography and invasive measurement catheters of filling pressures methods. we conducted a prospective observational study relating the filling pressures, between central venous pressure (cvp) with the diameter of the inferior vena cava and left atrial pressures with the values of the ratio e/e 0 . the filling pressure variables were only discriminated as high or low. low values were accepted when invasive measurement of cvp was \6 and \4 mmhg in the lap; and by echocardiography when the diameter of the ivc was\12 mm and the ratio e/e 0 \8. high values were accepted when the measurement of cvp was higher than 10 and 8 mmhg in lap and in echocardiography when the diameter of the ivc [16 mm and the ratio e/e 0 [15. we collected data from 38 patients in the immediate postoperative period, under mechanical ventilation (vt 8-9 ml/kg, fio 2 50%, peep 3), sinus rhythm, good cardiac function and without postoperative drug support. all of them had a central venous line and right atrium catheter as habitual monitoring of postoperative cardiac patients. we performed an echocardiography when the patient presented hypotension, with low values of cvp and lap, and we repeated the measurements after the infusion of the habitual fluid protocol (500 ml hes 6% in 30-45 min). the data we record were: diameter of ivc and ratio e/e 0 by echo and cvp and lap values by invasive catheters. 23rd esicm annual congress -barcelona, spain -9-13 october 2010 s333 introduction. an attenuated cardio-hemodynamic response to dobutamine is associated with a poor outcome in established human sepsis [1, 2] . establishing a sensitive method to identify early cardiac dysfunction in both experimental and human sepsis would be a useful tool to explore timesensitive mechanisms further. objectives. to assess myocardial responsiveness to dobutamine in early sepsis. methods. all procedures were in accordance with uk home office laboratory animal legislation. under isoflurane anaesthesia, male adult wistar rats underwent left common carotid and right internal jugular venous cannulation for blood sampling/continuous bp monitoring and fluid administration respectively. rats received either 1.8 ml caecal slurry (sepsis; n = 8) or 1.8 ml saline (sham; n = 8) ip, before fluid resuscitation (0.9% saline 10 ml/kg/h) and conscious monitoring was commenced. after 4 h, rats were re-anaesthetized with isoflurane and transthoracic echocardiography was performed. stroke volume was optimised with saline boluses prior to an incremental dobutamine infusion (1.25-20 mcg/kg/ min). data are presented as mean (sd); analyzed with 2-way anova and post-hoc tukey test. results. figure 1 summarizes hemodynamic changes after sepsis, fluid resuscitation and dobutamine infusion. baseline parameters were similar after echocardiography-guided fluid resuscitation, with contractility and stroke volume restored in septic rats to sham values. septic rats demonstrated an enhanced chronotropic response to dobutamine compared to sham (p \ 0.002). both peak velocity and cardiac output were attenuated by c25% in sepsis (p \ 0.0001). in sepsis, baseline map was higher but neither sham nor septic maps were affected by dobutamine infusion. conclusions. dobutamine stress echocardiography is a sensitive, reproducible, dynamic physiological probe that reveals early cardiac dysfunction in septic rats with apparently similar baseline cardiovascular physiology. introduction. the evaluation of right ventricular (rv) function is clinically useful in patients with acute respiratory distress syndrome (ards) because the presence of rv failure has large prognosis implications. the purpose of the current study was to compare right ventricular myocardial strain imaging parameters with conventional echocardiographic indices evaluating right ventricular function during ards. objectives. we hypothesized that peak systolic strain would be more sensitive than conventional echocardiographic parameters in detecting subclinical right ventricular systolic dysfunction in patients with ards. methods. in total, 10 patients with ards and with normal right ventricle function assessed by two dimensional echocardiography and 10 age matched subjects under mechanical ventilation without heart or pulmonary disease were included in the present study. conventional echocardiography parameters for rv function assessment like rv fractional area change (rvfa) or the tricuspid annular plane systolic excursion (tapse) were measured and compared to tissue doppler imaging parameters with strain value obtained from the right ventricle free wall. . strain values were reduced in the rv free wall of the patients with ards compared with the control group (23.45% ± 3.8 vs. 31.8% ± 2.8 p = 0.005) moreover no significant difference was observed in conventional two dimensional parameters evaluating rv systolic function between these two groups of patients. in patients with ards a significant relationship was shown between peak systolic strain at basal free wall and arterial carbon dioxide tension (rho = -0.79 p = 0.017) and with the end inspiratory pressure (rho = -0.67 p = 0.04). conclusions. during the ards, doppler tissue imaging parameters can determine rv dysfunction that is complementary to conventional echocardiographic indices and is correlated with respiratory parameters. on doppler tissue imaging, patients with ards exhibit abnormal rv systolic function even in patients with normal rv function assessed with conventional echocardiographic parameters. objectives. studying the effect of olv on rv outflow impedance during inspiration and expiration using transesophageal echo-doppler in a trial to differentiate the rv consequence of increasing lung volume from those secondary to increasing airway pressure during mechanical ventilation. methods. thirty stable patients on mechanical ventilation because of different causes were enrolled prospectively in this single center, cross sectional clinical study. each patient was firstly subjected to conventional ventilation (cv) with volume controlled ventilation, followed by open lung concept (olc) ventilation by switching to pressure controlled mode, then recruitment maneuver applied until pao2/fio2 [375 torr. hemodynamic (mean arterial pressure ''map'', central venous pressure ''cvp'' and heart rate ''hr'') and respiratory (total and intrinsic peep, peak, plateau and mean airway pressure and total and dynamic lung compliance) measurements were recorded before, 20 min after a steady state of cv and 20 min after a steady state of olc ventilation. also, transesophageal echo doppler was performed at end of inspiration and end of expiration to calculate the mean acceleration (ac mean ), as a marker of the rv outflow impedance, 20 min after a steady state of cv and 20 min after a steady state of olc ventilation. results. during inspiration, ac mean was significantly lower during cv compared to olc ventilation (p value .001). inspiration didn't cause a significant decrease in acmean compared with expiration during olv (p value.001) but did do so during cv. in comparison to baseline and cv, olc ventilation was associated with a statistically significant higher cvp (p value .001 for both), higher total quasi-static lung compliance (p value .001 for both) and dynamic lung compliance (p value 0.001 for both). moreover, pao 2 /fio 2 ratio of olv was significantly higher than in baseline and cv (p value .001 for both). conclusions. olc ventilation does not change rv afterload during inspiration and expiration as rv afterload appears primarily mediated through the tidal volume. moreover, olc ventilation provide a more stable hemodynamic condition and better oxygenation and lung dynamics. introduction. among indices provided by the analysis of aortic blood flow through esophageal doppler, mean acceleration (acc) is supposed to reflect the left ventricular (lv) systolic function, but this has been poorly validated. in particular, acc could be influenced by loading conditions of the lv. objectives. to test whether acc actually behaves as an indicator of lv systolic function by testing if 1. it increased with inotropic stimulation, 2. it was not altered by fluid loading, 3 . it correlated with the echographic lv ejection fraction (lvef) and it reliably tracked the changes in lvef during therapeutic intervention. in 37 patients with cute circulatory failure (sapsii 59 ± 18, age 64 ± 10 years, 26 receiving norepinephrine), we administered either a volume expansion (500 ml saline over 20 min in 24 patients) or dobutamine (5 lg/kg/min in 13 patients). we simultaneously measured acc (cardioq, deltex medical) and lvef at baseline and after therapeutic intervention. results. volume expansion significantly altered neither lvef (from 56 ± 12 to 58 ± 12%) nor acc (from 9.5 ± 3.2 to 9.9 ± 3.1 cm/s 2 ) while dobutamine infusion significantly increased lvef by 27 ± 15% and acc by 44 ± 27%. considering the 74 acc/lvef pairs of measurements, an acc \8.2 cm/s 2 predicted a lvef b 45% with a sensitivity of 77% (95% ci [56-91%]) and a specificity of 68% (95% ci [52-80%]). the changes in lvef and in acc during fluid and dobutamine administration were significantly correlated (r = 0.64, p \ 0.05). conclusions. acc fulfilled the criteria required from a clinical indicator of lv global systolic function. a given value of acc allowed detecting a low lvef with a modest accuracy. by contrast, the treatment-induced relative changes in acc were reliable for tracking the treatment-induced relative changes in lvef. objectives. to compare the relationship between systolic or diastolic dysfunction at icu admission and the incidence of cardiologic complications and mortality at sixth months. methods. prospective study of forty consecutive patients diagnosed of acute myocardial infarction (ami) (23 nstemi, 17 stemi) who were admitted in the icu of university hospital puerto real (cadiz, spain) from 1st may 2009 to 30th september 2009. studied variables: age, gender, type of ami (nstemi, stemi), left ventricular ejection fraction (lvef) by biplanar simpson's rule, diastolic function (ratio e/e 0 of the mitral annulus included), incidence of cardiac complications (acute pulmonary oedema, atrial fibrillation with hemodynamic instability and cardiogenic shock) and mortality at sixth month. echocardiographic studies were performed with a ge vivid 7 pro(r) by an intensivist who had performed up to 500 doppler studies in critical patients. all studies were remeasured by a second observer in an echocardiographic workstation with no statistical difference in measured velocities. patients were classified according to their lvef in (a) preserved ([55%), (b) mildly depressed (45-55%), (c) moderately depressed (30-45%) and (d) severely depressed (\30%); and according to their e/e 0 ratio in (a) normal e/e 0 ratio (\10) and (b) elevated e/e 0 ratio (c 10). the results were statistically analysed with chi-square test and odds ratio calculus. results. diastolic dysfunction measured with e/e 0 ratio was associated with high incidence of cardiac complications (chi 2 test cl 95% p \ 0.001, or 24). systolic dysfunction measured by lvef was also associated with more complications but with less strength of statistical association (chi 2 test cl 95% p \ 0.05, or 8. 25 ). there were no significative statistical difference between lvef and e/e 0 ratio in mortality at sixth month. conclusions. in our study, diastolic and systolic dysfunctions in patients with ami at icu admission were associated with high incidence of cardiac complications, with more strength of statistical association in patients with diastolic dysfunction. the small sample volume didn't allow us obtaining significative statistical differences in mortality at sixth months. a new method has been developed to assess global end-diastolic volume (gedv) and extravascular lung water (evlw) from a transpulmonary thermodilution curve. our goal was to compare this new method to the established method currently in clinical use, over a wide range up to extreme pathophysiological conditions. objectives and methods. 11 anesthetized and mechanically ventilated pigs (90-110 kg) were instrumented with a central venous catheter and a right (5f pulsiocath, pulsion, munich, germany) and a left (5f volumeview, edwards lifesciences, irvine, ca) thermodilution femoral arterial catheter. the right femoral catheter was connected to a picco2 monitor (pulsion) and used to measure cop, gedvp and evlwp using the old method based on the equation: gedv = cop 9 (mtt -dst). the left femoral catheter was connected to the new ev1000 monitor (edwards) and used to measure coe, gedve and evlwe using the new method based on the equation: gedve = f (s2/s1) 9 coe 9 mtt, where s1 and s2 are respectively the maximum up-and down-slopes of the dilution curve, respectively. 137 measurements were done during inotropic stimulation (dobu), during hemmorhage (hypo), during fluid overload (hyper), and after inducing oleic acid-acute lung injury (ali). overall, cop and coe ranged from 3.1 to 15.4 and from 3.4 to 15.1 l/min, respectively. cop and coe were closely correlated (r 2 = 0.99), mean bias (± sd) was 0.18 ± 0.29 l/min and %error was 7%. gedvp and gedve ranged from 701 to 1,629 and from 774 to 1,645 ml. gedvp and gedve were closely correlated (r 2 = 0.79), mean bias was -11 ± 78 ml and %error was 14%. evlwp and evlwe ranged from 507 to 2,379 and from 495 to 2,222 ml. evlwp and evlwe were closely correlated (r 2 = 0.97), mean bias was -5 ± 72 ml and %error was 15%. parameters over the study period are presented in the table (*p \ 0.05 intervention vs. base or hyper). introduction. fluid resuscitation is a major therapy in icu. various mechanisms are involved in the regulation of the microcirculation and the macrocirculation. objectives. the goal of this study is to assess the sublingual microcirculatory changes in response to fluid challenge in preload-responsive and non preload-responsive patients. after approval by our local institutional review board, 18 patients in surgical icu have been included in an observational study. each patient was monitored by an arterial catheter and an oesophageal doppler. the decision of fluid infusion was taken by the physician in charge of the patient. preload-responsive patients were defined by variations in cardiac index (ci) c 15%. sublingual microcirculation videos were obtained using the orthogonal polarized spectral (ops) imaging technology. functional capillary density (fcd, cm cm -2 ) and microcirculatory flow index (mfi) were collected. the macrocirculatory and microcirculatory measurements were obtained before, during and after the infusion of 500 ml of saline. five sublingual sites were recorded before and after the fluid resuscitation. the ventilator settings and sedative and vasoactive drugs infusion rates were kept constant throughout the procedure. results. patients were admitted in icu for acute brain trauma (n = 5), hemorrhagic shock (n = 4), septic shock (n = 4), acute brain hemorrhage (n = 3) and acute pancreatitis (n = 2). the average age of the patient was 44 ± 17. the mean values of ci and mean arterial pressure (map) before the fluid therapy were respectively 2.7 ± 1.2 l/min/m 2 and 80 ± 15 mmhg. nine patients responded to fluid infusion (ci c 15%.). about the microcirculation, there was no significant difference between responders (r) and non-responders (nr) concerning the variations of mfi (0. introduction. passive leg raising (plr) was shown to discriminate hemodynamically unstable patients who will benefit from subsequent fluid administration or not. 1 concerned by the possibility of harmful hypotension starting the plr maneuver from a 45°semirecumbent position, in a previous study, we found that raising patients' legs from a supine position, we were not able to predict fluid responsiveness in a heterogeneous cohort of medical intensive care unit (icu) patients. 2 objectives. to investigate whether starting plr maneuver from a 45°semirecumbent position would better predict volume responsiveness without harmful hypotension in spontaneously breathing critically ill medical icu patients. methods. fluid responsiveness was tested in 27 consecutive patients (14 sepsis, 4 respiratory failure, 5 heart failure, 4 others) with a mean arterial pressure (map) \60 mmhg and/or a cardiac index (ci) \2.4 l/min/m 2 . heart rate (hr), mean arterial pressure (map), global end-diastolic volume index (gedvi), cardiac index (ci) and stroke volume index (svi) were recorded using the picco method. patients were stable in a semirecumbent (45°) position when first measurements were taken (baseline 1). for the plr maneuver, patient's bed was tilt to have the lower limbs raised to a 45°angle while the patient's trunk was then in a supine position. changes after 2 min were recorded. the patient was then brought into a supine position, and heamodynamic measurements were recorded when stable (baseline 2). thereafter, 500 ml of 0.9% nacl were administered over 15 min. positive predictive values (ppv) and negative predictive values (npv) of the plr maneuver were calculated using a cut-off value of 15 % increase for ci and svi and 10 % increase for map. results. patients' median age was 60 (29-82) years and their saps score 59 (16-90). all patients received vasopressors and/or inotropes. baseline hemodynamics and changes after plr and fluid challenge are shown in table 1 . results are given as median (range); n/a = not available, *p \ 0.05 versus baseline. ppv and npv for ci were 50 and 86%, for svi 20 and 77% and for map 12 and 80%, respectively. conclusions. in our hands, plr was not useful identifying fluid responders in this heterogenous population of severely ill medical icu patients, the starting semirecumbent position being associated with a potentially harmful decrease in map. however, it was helpful to detect patients who will not benefit (or even suffer harm) from further fluid administration. recently, some studies suggested that an impaired diastolic function is a predictive factor of mortality in patient with shock. it is not already known whether fluid infusion could improve diastolic function. objectives. the aim of the study was to determine the impact of rapid fluid infusion on diastolic function. after acceptance by the local ethic committee, 94 icu patients were prospectively included. volume expansion (ve) by 500 ml of saline was performed by the intensivist in charge. transthoracic doppler echocardiography was performed before and after fluid infusion. stroke volume (sv), early diastolic transmitral velocity (e), early diastolic mitral annular velocity (ea) and e/ea ratio (reflect of lv filling pressure) were studied. patients were divided in 2 groups according to their sv' increase: responders (r) (those who increased their sv by at least 15 %) and non-responders (nr). wilcoxon rank sum test was performed to compare data before and after ve. data are presented in median (iqr) results. fifty-three (56 %) patients were r and 41 (44%) were nr. in the overall population, ea increased significantly with ve [from 12.8 (4.5) to 13.9 (5.3) cm/s, p = 0.002]. in the r group ea increased significantly [from 12.6 (4.1) to 14.6 (4.9) cm/s, p = 0.01] and e/ea did not change significantly [from 6.1 (2.1) to 6.3 (2. 3), p = 0.4]. however in the nr group, ea did not change significantly [from 12 (5) to 12.7 (6.6) cm/s, p = 0.47] while e/ea increased significantly [from 6.7 (2.8) to 8.5 (3.9) cm/s, p = 0.008]. conclusions. according to these results, adequate fluid infusion seemed to enhance lv relaxation without increasing lv filling pressure while inadequate fluid infusion did not affect relaxation but increased lv filling pressure. objectives. the aim of our study is to compare the rapid variation of co measured by vigileo-flotrac ò with doppler-echocardiography which is considered as a reference method. during the 36 first hours of hospitalisation, we studied 16 mechanically ventilated patients receiving norepinephrine who underwent arterial pressure monitoring via a radial artery catheter. the flotrac ò pressure sensor and the vigileo ò monitor were connected to the arterial line. at each fluid expansion or norepinephrine dose modification a transthoracic doppler-echocardiography was performed and co was calculated. variations for co measured by each method were compared. results are presented as median (iqr). linear regression and the bland-altman method were used for statistical analysis. methods. for the in vitro experiments blood of 20 healthy donors was incubated (in the ratio 5:1) with one of the following solutions: ringer solution, ringer-lactate solution, modified gelatin (gelofusin); hydroxyethyl starch (hes) 130/0.42. after incubation, the following parameters of erythrocyte aggregation were measured: t1 and t2-characteristic times of spontaneous erythrocyte aggregation; b-hydrodynamic strength of aggregates; i 2.5 -index of strength of the largest aggregates at shear rate 2.5 s -1 . rbc deformability at various shear stresses was determined by ektacytometry. in vivo study on 60 patients with trauma treated randomly with either only crystalloids (group 1; n = 20), or crystalloids + hes 130/0.42 (group 2; n = 20) or crystalloids + gelofusin (group 3; n = 20) over 7 days, the same parameters as in vitro study were determined at day 1-7. twenty healthy men and women were included as controls. for statistical analysis the statistical package spss version 15.0 was used. statistical significance was considered at p \ 0.05. in vitro study in the final analysis effects of different colloids on rbc aggregation and deformability were considered as increasing impact (:), decreasing impact (;) and no impact (-) ( table 1) . in vivo study significant microrheological disturbances were detected at day 1 after admission. deformability index was lower in patients compared with controls (0.39 ± 0.012 vs. 0.44 ± 0.016; p = 0.017). simultaneously, the patients showed erythrocytes hyperaggregation compared with control (;t1, ;t2; :i 2.5 , :b). in the first group (crystalloids) described violations persisted throughout the study time. in group 2 (crystalloids + hes), the deformability was higher than in the 1st group, from 3 days till the end of the study, attaining the normal range, and also higher than in the third group (crystalloids + gelofusin). in the third group, deformability index was not significantly different from group 1. according aggregatometrical data in the first group hyperaggregation syndrome remained the entire period of observation. hes adding (group 2) decelerated aggregate formation (:t1, :t2; ;i 2.5 ). in contrast, modified gelatin adding enhanced erythrocyte aggregation (;t1 :i 2.5 , :b). conclusions. crystalloid solutions are not able to improve microrheological parameters. hes 130/0.42 increases rbc's deformability and reduced rbc's aggregability. gelofusin increases erythrocyte aggregation and no effect on deformability. introduction. trauma patients often require norepinephrine (ne) infusion and fluid challenge to keep normal blood pressure values. the reliability of dynamic predictors of fluid responsiveness during vasopressors therapy is under debate. we investigated the impact of norepinephrine (ne) infusion changes on pulse pressure variation (ppv) assessed with the mostcare system (vytech health, laboratoires pharmaceutiques vygon, ecouen, france) in intensive care unit patients. this device is a pulse contour method that provides cardiac output and fluid responsiveness variables and does not need any kind of calibration or preloaded data. methods. 15 trauma patients (8 female, 7 male, mean age 53 ± 22) admitted to a 13-bed university hospital medico-surgical icu were prospectively enrolled. inclusion criteria were: mechanically ventilated patients (tidal volume [8 ml/kg and constant respiratory rate); invasive arterial blood pressure monitoring; ne infusion. ppv values were recorded continuously during three different haemodynamic states: at baseline (t1), 10 min after a 0.05 lg/kg/min ne increase (t2), 10 min after a further 0.05 lg/kg/min ne increase (t3), 10 min following the reduction of ne to t2 dosage (t2 0 ) and 10 min after setting ne to baseline value (t1 0 ). during the study neither fluid challenge nor other vasoactive/inotropic drug changes were done. anova test was applied. results. see data in table 1 . at t1 ne mean dosage was 0.12 lg/kg/min (range 0.05-0.2 lg/kg/min). the mean ppv was: at t1 10.69 ± 6.9%, at t2 8.78 ± 5.9%, at t3 7.77 ± 6.1%, at t2 0 8.73 ± 5.5%, at t1 0 12.07 ± 8.2% (p \ 0.05). conclusions. our findings demonstrated that ppv was significantly affected by changes in ne: the higher the ne dosage the lower the ppv. changes in arterial tone due to ne infusion can impair ppv reliability in assessing fluid responsiveness in trauma patients. introduction. in mechanically ventilated patients respiratory variation in the arterial pulse pressure (dpp) is a reliable predictor of fluid responsiveness 1 . respiratory variation of pulse oximetry plethysmographic waveforms correlate to dpp 2 and can be calculated automatically in real time (heart-lung index [hli ò ] from hamilton medical). this prospective study evaluates the relationship between dpp and hli ò to predict fluid responsiveness. mechanically ventilated patients were investigated; all connected to an hamilton g5 ventilator and ventilated in adaptive support ventilation (asv), paralyzed and none had severe cardiac dysrhythmia. 15 were eligible for fluid expansion. dpp, hli ò (obtained from a finger probe pulse oxymeter integrated to the ventilator) and cardiac index (ci from transthoracic echo-doppler), were obtained before and after fluid expansion (8 ml/kg of hea over 20 min). ci-responders were defined by 15% increase from baseline. results. 10 out of the 15 patients were ci-responders and had significantly higher hli ò before volume expansion (21% ± 6 vs. 9% ± 4, p \ 0.02). before fluid expansion hli ò was correlated with dpp (r 2 = 0.68, p \ 0.01, fig. 1 ). hli and dpp were significantly correlated with change in ic induced by fluid expansion (r 2 = 0.53 and r 2 = 0.73, respectively). objectives. the primary end point of this study was to evaluate the rvd of the ivc in icu patients with spontaneous breathing. methods. icu patient with spontaneous breathing and signs of hypoperfusion (oliguria, mottles, serum lactate level [2 mmol/l) were eligible after the approval of the local ethics committee. we excluded patients with acute heart failure with pulmonary edema, moribund and arrhythmic patients. the trans thoracic echocardiographic (tte) evaluation was done by 4 confirmed intensivists (level [2 in echocardiography). the aortic diameter measured at the lv outflow chamber and the tvi were measured. the vena cava inferior diameters at inspiration and at expiration were measured on the sub costal view. the rvd of the ivc was defined as the (maximal ivc diameter -minimal ivc diameter)/maximal ivc diameter. these measures were realized at t0, before fluid challenge, and after a fluid challenge of 500 ml of hes 6% (130.9/1) over 30 min (t30). patients with an increase of tvi of more than 15% were considered as responders to the fluid challenge. the measures of tvi and of the rvd of the ivc were validated by an experimented intensivist and echographist (level 3) after blinding the patient' name and of the times of measurement. roc curves were constructed, and the cut off was determined as the closest point of the roc curve to the ideal point (sensibility = specificity = 1). the values are expressed as median and extremes. objectives. our objective was to test whether non invasive assessment by trans thoracic echocardiography of sub aortic velocity time index (vti) variation after a low volume of fluid infusion (100 ml of hydroxy ethyl starch, hes) can predict fluid responsiveness. methods. sub aortic vti was measured by transthoracic echocardiography before fluid infusion (baseline) in 39 sedated patients with acute circulatory failure and low tidal volume mechanical ventilation in whom volume expansion was planned. then, vti was recorded after 100 ml of fluid infusion over 1 min, and after an additional infusion of 400 ml of hes over 1 min. we measured the variation of vti after 100 ml of fluid (dvti 100 ) for each patient. receiver operating characteristic (roc) curves were generated for dvti 100 in all patients. when available, roc curves were also generated for pulse pressure variation (ppv) and central venous pressure (cvp). , volumes (gedvi) and variabilities (svv, ppv) have been suggested to predict volume responsiveness (vr). the final classification of a patient as ''volume responsive'' is usually made by a volume challenge (vc) with an infusion of a pre-defined amount of fluid over a certain time. among many variations of vcs, the infusion of 7 ml crystalloid over 30 min is one of the most established. despite superior predictive capabilities of svv, ppv and gedi compared to cvp and pawp in a number of studies, they fail to predict vr in a substantial number of patients. furthermore, the use of these parameters is limited due to femoral access of the cvc (gedi; cvp) or the absence of controlled ventilation and/or sinus rhythm (svv, ppv). repeated ''exploratory'' vcs with 7 ml/kg might result in volume overload in some patients. objectives. therefore, we investigated the usefulness of a ''small vc'' with 3.5 ml/kg crystalloid over 15 min compared to a standard vc with 7 ml/kg over 30 min. in 48 patients equipped with picco hemodynamic monitoring we performed a 30 min vc with 7 ml/kg of crystalloid. during the vc transpulmonary thermodilution (td) was performed at 0, 15 and 30 min to obtain td-derived ci (ci td ). additionally pulse contour ci (ci pc ) was recorded in intervals of 5 min. introduction. the prevalence of obesity, defined as a body mass index (bmi) c30 kg/ m 2 , reaches epidemic proportions. it is not only a risk factor for health problems, but also exacerbates illness progression. consequently, the number of obese patients on the intensive care unit (icu) has increased enormously. caring for obese patients can be quite challenging due to the weight and size of this person. the extent of and specific problems associated to the care of obese icu patients are unknown. the aim of this study is to identify and quantify problems nurses face in caring for obese patients on the icu. this study was performed on the icu at the radboud university nijmegen medical centre and contained two parts. in the first part a selection was made of 44 obese patients admitted between 2004 and 2009; these patients were matched with 44 normal weight patients (bmi 18.5-24.9 kg/m 2 ). patients were matched on gender, age, length of icu stay and apache-ii score. all patient files were screened for the presence and intensity of problems in caring for these patients. in the second part 25 nurses were asked in a survey to share their experiences in caring for obese patients. they were asked about the nature, frequency and intensity of the problems they faced. in total, 94 problems were identified in the 88 screened patient files. seventy-two problems (76.6%) occurred in care for obese patients and 23 (23.4%) in care for normal weight patients. in both groups, most of the problems were related to activities of daily living (adl) such as (re)positioning in bed, transfers and personal care. surprisingly, the intensity of the problems was similar in both groups. most of the problems were moderate (hardly to solve by one person) or severe (only to solve with two persons or special equipment). moderate problems occurred in 32.3% of normal weight patients and in 33.3% of obese patients; severe problems 29.0 and 31.9%, respectively. this result was also confirmed by the survey. the nurses qualified most of the problems they were asked about as moderate or severe, and the frequency of the experienced problems was much higher. from the files it appeared that in 31.8% of the obese patients nurses had adl problems. strikingly, in the survey nurses reported that they frequently (72.0%) or even always (28.0%) experienced adl problems in obese patients. nurses reported and experienced more problems in daily care for obese icu patients compared to normal weight icu patients. although the intensity of the problems with obese patients did not differ from normal weight patients, the frequency in which they occur was much higher. differences between reported problems and the survey suggest an underestimation of problems that can be solved by performing a prospective study. nevertheless, based on these results, and taking into account that obesity will increase in the future, we recommend anticipating to the needs of the nurses whenever possible. introduction. worldwide the number of obese patients (bmi [ 40) is increasing rapidly (1); this also includes patients admitted to the intensive care units (icu). this raises special demands on the staff, the surroundings and the equipment (2) . often the obese patient is not mobilised according to the clinical standard this causes complications to breathing, circulation and skin etc. furthermore the length of stay in the icu increases and the mortality rises. objectives. the aim of this study therefore was to make clinical guidelines and recommendations for mobilisation of the obese icu patient based on evidence. this will increase the knowledge and importance of mobilisation between staff and on longer term improves the daily average number of mobilisations performed with these patients. a secondary aim is that increased knowledge on this topic will improve the interdisciplinary work between the different professions based on the same overall aim. a systematic review of the literature concerning mobilisation of the obese icu patients was made in the year 2009-2010. the study is still work in progress analysing the literature to make guidelines and recommendations based on evidence. furthermore evidencebased education of special trained staff in mobilisation has been conducted in january/ february to improve their knowledge of the impacts mobilisation has on the respiration, circulation and skincare etc. the education was planned to aiming at a interdisciplinary audience. results. the preliminary results shows that it is more difficult to care for and mobilise the obese icu patient, because there is lack of space, non-availability of the correct equipment, too few available staff members and a significant negative attitudes among the staff towards the obese patient. recommendations are made within airway, breathing, circulation, nutrition, pain, equipment and patient experience according to the procedure of mobilisation of the obese icu patient. the recommendation was implemented in the already performed education and resulted in a changed attitude among the participant and improved the status of mobilisation in the daily prioritization. this knowledge was obtained in the evaluationinterview conducted approximately one month after the seminar. conclusions. according to the literature mobilisation of the obese icu patient needs special attention towards a safe clinical practise based on evidence with focus on both the patient and the staff. special attention towards this group of patient is created by performing evidence based research resulting in clinical guidelines that has to be implemented through theoretical and practical education on an interdisciplinary level. nurses are constantly exposed to the pain and suffering of those in their care 3 . the primary aim of this study was to investigate the risk of secondary traumatic stress/compassion fatigue (sts/cf-the trauma suffered by the helping professional) and burnout (bo-emotional exhaustion, depersonalization, and reduced sense of personal accomplishment), and the potential for compassion satisfaction (cs-the fulfillment from helping others and positive collegial relationships) among nurses working in icu. an additional goal was to test the relationship of these three constructs to each other. (1) . the use of closed suction circuits has been suggested beneficial as a prophylactic measure (2) . objectives. the aim of this study was to compare the incidence of vap and the occurrence of desaturation during suction using either oss or css. we also investigated contamination of the closed suction circuit and the occurrence of adverse events. methods. css were a new product in our clinic. all staff underwent a user course supervised by the manufacturer of the closed circuit. after this, data were collected during four periods in 2009, 2 month css followed by 2 months oss which was repeated twice. during the summer period css were used without any data collection and then followed by two periods of css and oss. all mechanical ventilated patients were consecutively included. a culture of deep endotracheal aspirate and a blind microbiology brush was taken in association with the intubation, after 72 h and every monday. after changing css and in case of extubation, the tip of the catheter was sent for culture. demographic data were retrieved from the hospital database. data were analyzed with descriptive methods. results. the incidences of vap were higher in the css group (table 1) . both suction systems showed almost no desaturation during and after suctioning. positive cultures were obtained in 55% of all the retrieved css catheters. the microbiological flora resembled the species found in the airway cultures. there were no inter patient contamination and neither did the bronchoscopy frequency differ between oss and css patients. in the css group six adverse events were seen; three tube occlusion and three incidences with secretion clogging. conclusions. the use of a css did not prevent vap, in our study. there were no benefit with css other than maybe to protect the staff and our finding of positive culture in 55% of the cases is in line with earlier studies. objectives. the aim of this study was to determine which intensive care patients the nurses defined as 'difficult' and their experiences in coping with such patients. the study was carried out as a qualitative design with 18 voluntary nurses employed in five intensive care units of a research and training hospital. the data were collected using demographic characteristics form and a semi-structured interview form. interviews with nurses were made individually and face to face. the data were evaluated by using colaizzi's phenomenological data analysis method. as a result of data analysis into two categories and two themes were identified. the categories were (1) difficult patient definition of the nurses, (2) the effect of difficult patients on their care, and (3) how the nurses are affected and cope with difficult patients. the nurses listed their reasons for defining some persons as difficult as difficult physical care of the patients, and the difficulty in communicating due to dementia, agitation, alzheimer's disease or the patient's personal characteristics. the nurses said that they found taking care of patients they found difficult physically and psychologically demanding. they used methods such as finding out the patient's problem and taking appropriate measures, increasing communication with the patient and providing explanations, trying to obtain spiritual satisfaction and transferring the patient's care to another nurse when communication problems were impossible to overcome. intensive care nurses have difficulty in caring for and communicating with some intensive care patients due to the characteristics of the disease, physical/psychological factors and personal characteristics. we found that nurses continued the care of these ''difficult'' patients by focusing on solving their problems, transferring the care to another nurse when necessary or by trying to obtain spiritual satisfaction. methods. teams of three delirium experts visited ten icu's in the the netherlands in which the cam-icu was incorporated in daily practice, twice. these teams consisted of two consultants in either psychiatry, clinical geriatrics or neurology, and either a research-physician (mmjve) or a research-nurse (mvdb). based on cognitive testing, inspection of the files and dsm-iv criteria for delirium, the teams classified patients as awake and not delirious, or delirious or comatose. this classification served as gold standard to which the cam-icu as performed by the bed-side nurses was compared. a simple 2 9 2 table was used to calculate the sensitivity and specificity. results. 15 delirium experts performed 333 assessments. 181 (54%) of these patients were assessable for delirium, 126 (38%) patients were excluded because the level of consciousness was too low, and 27 (8%) patients were non-assessable due to other reasons. overall, we found a sensitivity of 47% (95% ci 35-58%) and a specificity of 98% (95% ci 92-99%). the strengths of this study include the large numbers, the multicentre design, the extensive evaluations by teams of various delirium experts and the independent assessments of delirium experts and bed-side nurses. a limitations is the time interval between the expert assessment and the administration of the cam-icu (mean 125 min; standard deviation 118 min). there were striking differences in implementation strategies of the cam-icu between the centres. tables 1, 2 . 23rd esicm annual congress -barcelona, spain -9-13 october 2010 s341 introduction. presence of expiratory ineffective efforts in mechanically ventilated patients is a common problem associated with increased duration of mechanical ventilation, length of stay and also a higher cost and mortality. nowadays, identification and categorization of expiratory asynchronies can only be done at the bedside with the continuous observation of the ventilator interface. nurses must be skilled to understand non appropriate situations of anomalous patient-ventilator interactions. objectives. we tested the hypothesis that after specific training nurses would acquire enough skills to detect expiratory efforts as intensive care expert physicians would do. training phase: nurses were provided with selected bibliography on patient ventilator interaction and afterwards trained by intensivists with expertise on mechanical ventilation (2 h/day during 20 days) on airway pressure, flow and volume waveforms identification and eye interpretation of early and late ineffective expiratory efforts during expiration. validation phase: airflow and airway pressure waveforms were obtained from 8 different icu mechanically ventilated patients using and acquisition and processing biomedical signal software (better care ò ). one thousand and seven breaths were randomly selected from a total of 2,608,800 breaths. subsequently, selected breaths were blindly analyzed by 2 trained nurses and 3 intensivists to identify ineffective expiratory efforts. introduction. several publications indicate that manual hyperinflation is a widely used measure in the icu, but more important is the fact that there is no uniformity in the implementation of this measure. this is also on my ward. in literature there are a number of reasons given to start manual hyperinflation: abolish mucus retension, improve oxygenation and removal of atelectasis. the positive effects are improved compliance, improved oxygenation and a decrease in the number of vap's (ventilator associated pneumonia). the negative effects are a decrease in cardiac output due to high peak pressures, an increased risk of baro-/volutrauma and the risk of giving too much tidal volumes. the risk of barotrauma increase with pressures above 40 cmh 2 o. other side effects include the development of a pneumothorax and increased icp (intra cranial pressure). objectives. creating more awareness of the procedure with lower peak pressures as a result. methods. through literature review, clinical courses and the introduction of a pressure gauge achieve greater uniformity and awareness of the procedure. we used a flow analyzer of imt medical, a laptop with flowlab software version 4.1.1 and an artificial lung to demonstrate how much pressure and volume is generated during manual hyperinflation. conclusions. compliance with bts guidelines could be improved. unsurprisingly co-morbidities were frequent, but did not seem to affect outcome. use of a pneumonia severity assessment tool was sub-optimal, however mean curb-65 score didn't correlate with that recommended to prompt critical care assessment. apart from functional status, we are unable currently to identify any factors in this age group which can be used to guide critical care admission decision making. conclusions. in our study the incidence of complicated pneumonia was 316/10,000 patients admitted in picu. in necrotizing pneumonias the blood cultures were more positive than in non-necrotizing patients. although the surgical approach in necrotizing pneumonia is controversial, it resulted in a insignificantly lower mortality rate, comparing with non-necrotizing pneumonias. background. community-acquired pneumonia (cap) of mixed etiology has frequently been described in the literature, but its clinical significance remains unknown. the aim of this study was to describe the prevalence, clinical characteristics, and outcome of severe cap of mixed etiology in icu patients. a 5-year prospective study was conducted on consecutive patients with severe cap admitted to icu in whom an extensive microbiological investigation was performed. results. 316 patients were included. a single pathogen was detected in 128 (40.5%) cases, while two or more pathogens in 38 (12.0%) cases. the most frequent pathogens' combinations were those of two bacteria (28.9%) and bacterium plus virus (21.1%). compared with patients with monomicrobial pneumonia, patients with mixed pneumonia were older, had higher severity score (psi) and were more likely to have previous chronic pulmonary disease (see table 1 below). moreover, mixed cap patients showed similar clinical and analytical data at admission but increases in the frequency of respiratory distress and in length of stay and a trend to higher orotracheal intubation and mortality rates. a mixed etiology was detected in 12% of cases with cap requiring icu hospitalization and was associated with older age and increased severity. despite similar radiological features (n of involved lobes, pleural effusion) at admission, cap with mixed etiology showed a trend to worse clinical course and outcomes than monomicrobial pneumonia. objectives. to assess the incidence and aetiology of pneumonia in a mixed medicalsurgical icu, in order to develop local epidemiologically guided protocols to reduce antibiotic resistance selection in patients with pneumonia. methods. retrospective observational study on prospectively collected data in a mixed medical-surgical icu of a secondary care italian hospital. at our institution, epidemiological data on infections and data on antibiotic use are recorded since 1992; in 2005 a new electronic recording of icu infections was introduced. type of infection, germ characteristics, clinician diagnosis and antibiotic use were prospectively collected in an electronic database and retrospectively reviewed. antibiotic exposure index was calculated as each antibiotic total amount administered divided by its defined daily dose times total days of admission. between 2005 and 2009 a total of 690 patients were admitted to our icu. pneumonia was the commonest infectious disease at admission (148 cases, 21% of patients), and the commonest infectious complication during icu stay (38 new occurrences, 21% of total pneumonia patients). table 1 shows major epidemiological findings in the study population. the incidence of acquired pneumonia was remarkable: 9.7 cases every 1,000 days of mechanical ventilation. the most frequent isolated organisms were s. aureus (18 patients) and p. aeruginosa (12 patients). methicillin-resistant s. aureus (mrsa) accounted for 61% of pneumonia caused by s. aureus, and its prevalence matched closely the exposure index to vancomycin. such a high incidence of mrsa is consistent with other records in mediterranean countries. carbapenem-resistant p. aeruginosa was somewhat less of a problem (33% of pneumonia by p. aeruginosa), and was not apparently associated with antibiotic exposure, at least within the unit. conclusions. in our retrospective observational study we found a high incidence of pneumonia at our institution, as well as a high percentage of mrsa, the latter with strong relationship with exposure to vancomycin. new protocols for infection containment and antibiotic usage are urgently needed. introduction. community-acquired pneumonia (cap) carries a high morbidity and mortality. a major problem is the insufficient monitoring of cap by standard chest radiography, as the evaluation depends highly on the observer and the extent of pulmonary infiltration cannot be assessed properly (1). objectives. the aim of our study was to compare the process of inflammation in cap measured by alveolar nitric oxide (no)-analysis (2) in exhaled breath and the extent of the inflammatory infiltration by electrical impedance tomography (eit) (3) in spontaneously breathing patients. after approval of the local ethic committee and obtained written informed consent 24 patients with cap were included in the study. all patients showed an acute pulmonary infiltration in chest x-ray, pulmonary symptoms (coughing, shortness of breath), positive findings in auscultation, leukocytosis, elevated crp and a pneumonia severity index c4. no analyses (analyser cld88sp, eco medics, dürnten, switzerland) were performed at t0 (up to 24 h after admission), t1 (2 days after admission) and t2 (4 days after admission. eit measurements (eit evaluation kit, dräger medical, lübeck, germany) were performed at t0 and t2 and inhomogeneity of ventilation was assessed by offline analysis. all measurements were made at beside in sitting position. data were compared by t test and regression analysis. results. there was no significant correlation between the alveolar no concentration and the extent of inhomogeneity of the local infiltration measured by eit. also during the study the time course of the inhomogeneity index was not correlated with change in exhaled no. the right/left distribution of the pulmonary infiltration in the chest x-ray and the eit measurement showed a positive correlation (p \ 0.0001; r 2 = 0.69). conclusions. pulmonary regional infiltration in cap measured by eit can not predict the actual alveolar process of inflammation in the lung. nevertheless the monitoring devices give additional information to better evaluate the time course of inflammation and the dimension of the respiratory dysfunction in diseased lung. organizing pneumonia (op) presenting as acute respiratory failure (arf) is a relatively rare disease, and was only previously specifically reported in 2 small series [1, 2] , with mortality up to 90%. these studies were performed before the publication of international consensus classification of idiopathic interstitial pneumonias in 2001 [4] . objectives. to compare clinical features and prognosis of patients with op with those of patients presenting diffuse alveolar damage (dad), during arf. design: retrospective monocentric study in a university hospital conducted during an 11 yr-period. to determine predictors of niv failure in patients who were intubated for respiratory failure and extubated directly to niv. methods. this is a retrospective analysis of prospectively collected data from january 2008 to dec 2009. patients with respiratory failure were mechanically ventilated in a university hospital's medical intensive care unit (icu) and subsequently extubated to niv. physiological and biochemical parameters, using arterial blood gas measurements, were collected at the end of the spontaneous breathing trial and 1 h after the application of niv. failure of niv was defined as respiratory failure requiring re-intubation within 48 h. out of 175 patients, 78.9% were successfully extubated to niv. success rates were 70.6% in patients with chronic obstructive pulmonary disease (copd) and 80.9% in other patients (p = 0.24). patients who failed niv were more tachypnoeic, acidaemic and hypercapnic pre-niv, and more tachycardic, hypotensive, acidaemic, hypercapnic and hypoxaemic post-niv (p all.05). on logistic regression analysis, three physiologic parameters predicted niv failure: pre-niv respiratory rate (or 1.57, 95% ci 1.07-2.31 per 5 breaths increase), post-niv heart rate (or 1.28, 95% ci 1.08-1.52 per 5 beats increase) and post-niv systolic blood pressure (or 1.15, 95% ci 1.02-1.29 per 5 mmhg decrease). conclusions. physiologic parameters, including the respiratory rate pre-niv, and heart rate and systolic blood pressure post-niv, independently predict niv failure post-extubation. these parameters should be taken into account in the decision to extubate directly to niv. introduction. discontinuation of mechanical ventilation in critically ill patients is a challenging task and involves a careful weighting of the benefits of early extubation and the risks of premature spontaneous breathing trial (sbt). only a few studies have explored indices derived from both heart rate and breathing pattern variability analysis for the estimation of weaning readiness. objectives. to investigate heart rate (hr) and respiratory rate (rr) complexity in patients with weaning failure or success, using both linear and nonlinear techniques from signal processing theory. methods. forty-two surgical patients were enrolled in the study. there were 24 who passed and 18 who failed a weaning trial. signals were analyzed for 10 min during two phases: despite of passing the protocol the decision to extubate was postponed in some patients. to gain insight on the physicians reasons for continuing mechanical ventilation after passing the wean screen protocol. a wean screen protocol was introduced at a mixed medical (neuro-)surgical icu of a teaching hospital in december 2009 to april 2010. ventilation practitioners assessed ventilated patients and recorded the physicians reasons for continuing mechanical ventilation despite of passing the wean screen protocol. . 223 patients were ventilated in this period. 135 daily screens were performed, 111 screens were successful. only 24 passed wean screens resulted in extubation. the rate of extubation was 22%. 78% screens did not lead to liberation from mechanical ventilation. the extubation rate does not correspond with the findings of the abc trial with an extubation rate of 54%. table 1 shows the physicians' reasons to continuing mechanical ventilation. it should be noted that all patients with an unsafe airway were patients with a glasgow coma scale (gcs) of b 8 [intracerebral haemorrhage (68%), cerebral infections (13%), post-cpr encephalopathy (13%) and severe brain injury (6%)]. we accomplished a reduction in the use of sedatives (-12% midazolam and -35% propofol) and morphine (-27%) ( table 1 ). the amount of time spend on ventilators decreased, albeit not significantly (p = 0.902). this was probably due to the vap-ventilatorbundle (introduced last year), the heterogeneity of our cohort and the already short mv-duration. . non-invasive ventilation (niv) has been utilized in selected patients with hypoxemic arf to avert endotracheal intubation, which is related to life-threatening complications. niv has been also proposed to facilitate weaning and extubation in patients with hypercapnic arf. so far, no controlled randomized study has investigated the potential role of niv in weaning patients with hypoxemic arf. objectives. we designed this pilot study to assess safety and feasibility of niv to wean hypoxemic arf patients. twenty mechanically ventilated patients with hypoxemic arf were randomized to receive early extubation followed by niv application via helmet (helmet group) or conventional weaning through the endotracheal tube (tube group). primary outcomes were the duration of invasive mechanical ventilation and the adherence to the study protocol. secondary outcomes were protocol failure (i.e. need for re-intubation), icu and hospital mortality, rate of tracheotomy, duration of continuous intravenous sedation, weaning time, and septic complications. table 1 . weaning through helmet by niv application following early extubation was safe and feasible. overall the adherence to the study design was 90%. in addition, in the helmet group, there was a significant reduction in the rate of tracheotomy and a trend toward a lower rate of protocol failure, and fewer days on invasive ventilation. there was no difference with respect to days of continuous sedation, icu and hospital mortality, weaning time and septic complications. (1) . delirium is a common occurence on the icu and is associated with increased length of stay (los) and poor outcomes (2) . objectives. we developed a combined daily sedation hold, delirium management, and weaning (sdw) protocol and implemented this to reduce icu los and improve outcomes. methods. a sdw protocol was implemented in 2009. we prospectively audited all patients from january to march 2010. delirium was measured using the icdsc. data was analysed using graphpad statistical software. results. 109 consecutive patients were analysed. the incidence of delirium was 35% (38 pts). of these, 63% (24) had risk factors for delirium. there was no difference in onset of delirium between sexes, age, type of admission, or severity of illness. however, in patients with delirium, duration of mechanical ventilation (mv) and icu los were significantly longer and there was a trend towards increased hospital los ( conclusions. measuring the linear dependence of variables through time by k 2 and ø may be used to determine non-linear behavior between the variables of the emmv. non-linear behavior during weaning perhaps indicates the dependency of, either the resistance or compliance of the respiratory system, on the ventilatory support (i.e. pi). accordingly, k 2 and ø, estimated at the frequency interval form 6 to 1 (h) -1 , can provide information concerning to the dynamics of the respiratory system that can be used as a complement to determine the suitability of the mv withdrawal. objectives. to study the potential superiority of aprv on cmv in a subgroup of patients with severe ards. methods. retrospective observational study on 39 patients severe ards who were admitted between july 2008 and january 2010 to mafraq hospital icu in uae. the diagnosis of ards was based on presence of bilateral infiltrates in cxr and p/f ratio of less than 200 in absence of evidence of elevated left atrial pressure. all patients were managed according to ardsnet guidelines using low tidal volume cmv and iv steroids. criteria for transition to airway pressure release ventilation (aprv) included failure to wean down fio2 below 60% after 24 h, hemodynamic instability due to high peep, and failure to maintain plateau airway pressure below 30 cmh2o. initial settings of aprv were ph 26, pl 2, th 5, and tl 0.5 with titration of fio 2 as required keeping pao 2 more than 60 mmhg. we compared the outcome of cmv and aprv groups with special concern to the duration of mechanical ventilation, requirement for tracheostomy, and survival to icu discharge. twenty four male and 15 females were included in the study with a mean age of 42 years (± 24). fourteen out of them fulfilled the criteria and were shifted to aprv within 24 h of initiating mechanical ventilation. ten out of 14 (70%) patients in the aprv arm survived to icu discharge versus 16 out of 24 (67%) patients in cmv group (p 0.45). survivors in aprv group spent significantly shorter periods of mechanical ventilation compared to survivors in cmv group (9.6 vs. 12.1 days p 0.03). while 8 out of 16 (50%) survivors in cmv required tracheostomy for prolonged intubation or recurrent lavage, only 2 out of 10 (20%) survivors in aprv group required tracheostomy tube placement (p 0.02). we concluded that aprv can be effectively used as rescue measure of ventilation in patients with severe ards. although our study does not show any mortality benefit of using aprv over cmv, there was a shorter ventilation days and icu stay using aprv. we strongly recommend further studies to investigate the probability of using aprv as initial mode of ventilation in this subset of patients. weaning from mechanical ventilation is a common daily procedure when caring for critically ill patients, and a lifesaving practice on which nurses are taking an increasing role with the introduction of nurse-led protocols. the literature supports that nurse-led protocols facilitate weaning and increase nurses' input in decision-making. on the other hand, decision-making is a complex function affected by the nature of the task, the decision environment and the characteristics of the decision maker. although the cognitive process of clinical decision-making has been investigated with many different methodologies, little is known about the decision environment and its impact on decisions' during the weaning process. objectives. this paper aims to address one of the factors of the clinical environment and its impact on the decisions when discontinuing mechanical ventilation. methods. this paper is part of a large comparative ethnographic study looking at nurses' input during the weaning process of mechanically ventilated patients. participant observation of critical care nurses took place in an 12-bedded icu in greece and an 18-bedded icu in scotland for 6 months each to examine nurses' involvement in the decisions made. in-depth semi-structured interviews with the nurses followed focusing on how nurses perceived their participation in the decisions made. data from field notes and interview transcripts were analysed thematically using the qualitative data analysis software nvivo, version 8. inter-personal and inter-professional relationships were considered revealing influences of nurses' input in decision-making. clinicians' personality played a significant role in their involvement in decisions, whereas trust and appreciation, the sense of support and the sense of accountability were also considerable dynamics of inter-professional relationships and predisposed decision-making. clinical decision-making is a multi-dynamic process specifically in complex clinical long-term situations such as weaning. aspects of the decision environment, such as the interprofessional relationships should be acknowledged when introducing methods to enhance nurses' role in teamwork and collaborative decision-making in order to improve the weaning process of ventilated patients and their outcome. objectives. the objective of our study was to analyze the temporal trends and outcomes of two cohorts of patients ventilated with psv and pav+. a cohort of 16 consecutive patients who were ventilated with pav+ and another cohort of 15 consecutive patients who were ventilated with psv were compared. all patients had the same inclusion criteria (gas exchange, ventilatory mechanics, peep level, resolution/stabilization of the cause leading to invasive mv and appropriate level of consciousness). both modes were adjusted to predefined clinical criteria (psv to reach a respiratory rate about 25 bpm and pav+ to reach a physiological inspiratory effort introduction. presence of expiratory asynchronies (ea) (ineffective efforts, cough and continued contraction of inspiratory muscles) is a common problem associated with increased duration of mechanical ventilation, longer stay, higher costs and increased mortality. because of the lack of systems that automatically detect and report ea, their identification is currently done by examining ventilator interface at the bedside or by applying dedicated algorithms in investigational conditions. validate the accuracy of linear mathematical algorithms to automatically detect ea built in a new computerized system that grabs and process data from different bedside icu monitors and mechanical ventilators. observational and prospective study in a general icu of 16 beds. two beds were equipped with a software (better care ò ), a technological platform responsible for data acquisition and synchronization, processing, storing-as non static and processable dicom objects-and also for integrating all this data with health information systems. by using the better care ò platform, a total of 2,600,000 breaths from 8 consecutive adult patients were collected with at least 24 h of mechanical ventilation. algorithm #1: the ea algorithm consisted in a mathematical analysis of the airflow and airway pressure waveform variations during expiration not followed by a mechanical breath. algorithm #2: designed to select 1,000 breaths out of the total number. this algorithm sorted and classified the breaths by the percentage of deviation from the expected expiratory curve. the result was 1,000 breaths covering most of the shapes the expiratory curve could have. five expert attendant physicians independently analyzed the 1,000 selected breaths and classified them as ea or not. the ea algorithm processed the same 1,000 selected breaths and assigned a percentage to each one, according to the variation in the shape and direction of the expiratory airflow and airway pressure curves. the expert criterion against the ea algorithm scores was used to construct a logistic regression model. we calculated sensitivity, specificity, positive predictive value and negative predictive value. the predictive performance of ea algorithm was evaluated using roc curves. optimal sensitivity and specificity were achieved by setting the cut-off point at a ea algorithm score of 42%. a variation in the shape and direction of the expiratory airflow and airway pressure curves [42% compared to the theoretical curve identified an ea with a sensitivity of 91.5%, specificity of 91.7%, a positive predictive power of 80.3% and a negative predictive power of 96.7%. introduction. near-infrared spectroscopy (nirs) in combination with a vascular occlusion test (vot) has been proposed to assess and identify metabolic and microcirculatory alterations during sepsis and shock in critically ill patients. however, to automatize repeated measurements at the bedside, this technique can potentially cause discomfort to the patient. vascular arterial occlusion performed in the finger may be a more attractive method to execute repeated measurements at the bedside because of more tolerability from the patient. we have previously showed in healthy volunteers that nirs can be used on finger to assess the sto2 response to vot and that 5 min was an adequate occlusion time to provide the best curve fit for nirs dynamic variables 1 . objectives. we aimed to investigate whether sto2 response to vot obtained from the finger could predict conventional sto2 response measurements obtained from the thenar of critically ill patients. parameters of sto2 response were measured with an inspectra spectrometer model 650 (hutchinson technology inc.) equipped with a 15-mm or a 5-mm probe. the 15mm probe was placed over the thenar eminence and the 5-mm probe was place over the ventral face of the middle finger. we performed in each patient a series of two vascular occlusion tests (vot): one on the finger (5 min) followed by one on the arm (3 min). the measurements were obtained within 24 h of intensive care admission and every 24 h thereafter until day 3. vot-derived sto2 traces were analyzed for baseline, ischemic (rdecsto2, %/min) and reperfusion (rincsto2, %/s) parameters. we performed 63 paired of nirs measurements in 25 critically ill patients (age 62 ± 15; 16 m/9f). although sto2 did not differ significantly between thenar and finger (76% ± 11 vs. 79% ± 12; p = 0.31), rincsto2 and rdecsto2 were statistically lower in the finger (1.5%/s ± 0.7 vs. 2.5%/s ± 0.8, p = 0.001; 3.1%/min ± 1.0 vs. 11%/min ± 3.3; p = 0.001). we performed bivariate linear model with correlated errors in which sto2 outcomes on thenar and on finger were treated as responses. the correlation was significant for sto2 and rincsto2, but not for rdecsto2 (table 1) . furthermore, mixed model analysis showed that thenar-sto2 as dependent variable could be significantly predicted by finger-sto2 parameters with estimation coefficient (± se) of 0.7 ± 0.06 (p = 0.0001), 0.9 ± 0.09 (p = 0.0001) and 0.98 ± 0.3 (p = 0.0061) for sto2, rincsto2 and rdecsto2, respectively. correlation of sto2 response: finger vs. thenar a prospective randomized clinical trial performed in icu's of an university and teaching hospital during a 2.5 year period, involving 120 septic and non-septic patients, randomized (after stratification) to hemodynamic monitoring, by picco tm or pac with both techniques allowing cardiac output and central/mixed venous o 2 saturation monitoring. methods. hemodynamic management was guided by extravascular lung water index (evlwi) and global end-diastolic volume index (gedvi) in the picco tm group and by the pulmonary capillary wedge pressure (pcwp) in the pac group for 3 consecutive days. primary outcome measures were ventilator-free days (vfd), for which the study was powered, and lengths of stay in icu and hospital. secondary measures were the course of cardiorespiratory parameters, fluid and vasopressor requirements, lactate levels, organ functions and mortality. in the study period, 72 septic and 48 non-septic patients were included. 60 patients received a picco tm and 60 a pac catheter. monitoring arms were comparable at baseline, although sepsis differed from non-sepsis in hemodynamics and severity of lung injury. premorbidity was greater in non-septic patients. the fluid infusions and balances did not differ between monitoring arms, except at t = 24 h when the picco tm group had a more positive balance (p = 0.044). cardiac index and central venous o 2 saturation increased more in the course of time in the picco tm than in the pac group. the decrease in norepinephrine requirements strongly tended to favor the picco tm group (p = 0.06). the course of lactate levels and organ failure did not differ between monitoring arms. vfd did not differ among monitoring arms. picco tm monitoring was associated with relatively fewer mechanical ventilation and icu days in sepsis but more in non-sepsis (after day 28). the changes in respiratory parameters, sofa and number of catheter-related complications did not differ among the arms of the study. overall, 19 patients (32%) died in the picco tm group before day 28 and 21 (35%) in the pac group (p = 0.85). conclusion. hemodynamic management guided by picco tm monitoring is safe and results in better tissue oxygenation than guidance by pac, without inducing pulmonary overhydration, in septic and non-septic, critically ill patients. this was associated with fewer mechanical ventilation and icu days in patients with sepsis but more days in patients with non-sepsis (after day 28), partly attributable to greater cardiovascular premorbidity in the latter. the major primary and secondary endpoints, vfd and mortality, were not affected. introduction. non-invasive evaluation of endothelial function may be easily accomplished by ultrasound assessment of flow-mediated vasodilation (fmd) of the brachial artery, but this technique has not been fully explored in septic patients. objectives. this prospective study aims to investigate the role o fmd analysis on intra hospital prognosis of patients with severe sepsis and septic shock. adult patients admitted to the intensive care unit with a diagnosis of severe sepsis or septic shock (\24 h of duration) were consecutively included. fmd of the brachial artery was measured upon admission and after 24 and 72 h using a high-frequency linear transducer (7.5-10 mhz) according to internationally accepted protocols. a group of apparently health subjects paired for gender and age was used as controls for fmd analysis. patients were followed up to discharge or death. we studied 42 adult patients 9 mean age 51 ± 19 years, 26 females, 79% on vasopressors with sepsis predominantly of abdominal or respiratory etiology (75%). apache ii risk score was 23 ± 7 and intra hospital mortality rate was 33%. fmd was similar in patients with or without use of vasopressors at baseline (p = 0.56). fmd in septic patients was significantly lower than in health controls (1.5 ± 7 vs. 6 ± 4%; p \ 0.001). we observed that survivors depicted a gradual improvement on endothelial function, so that 72 h after sepsis onset fmd was significantly lower in nonsurvivors (-3.3 ± 10 vs. 5.2 ± 4%; p \ 0.05; time-group interaction p value = 0.03). conclusions. brachial fmd is altered in septic patients with hemodynamic instability and its improvement may be an early marker of favorable prognosis. introduction. change in pulse pressure variation (dpp) and respiratory variation of the pulse oxymetry plethysmogram (pop) may predict the hemodynamic effect of peep in mechanically ventilated patients [1, 2] . reported comparisons [3, 4] between pop variations (popv) and co or dpp are based on selection of 3-5 consecutive breaths (dpp3b) during a ''stable'' period of pop. recently, a fully automatic ventilation mode (intellivent ò , hamilton medical, switzerland) that incorporates an automatic and continuous popv calculation (hli ò ) using a dedicated algorithm has been developed. the present study was designed to compare dpp3b, dpp calculated with the algorithm as hli ò (dppalg) and hli ò. . . 11 sedated icu patients ventilated with hamilton medical s1 ventilator (with integrated pulse oxymetry (po)) were included (age = 66 ± 13 years, saps ii = 54 ± 24, no arrhythmia, norepinephrine: 1.0 ± 1.2 mg/h in 6 patients, map = 74 ± 10 mmhg, vt = 8.4 ± 0.6 ml/kg). waveforms of po from a finger sensor and of blood pressure from a radial catheter were recorded for 1-2 h. from the waveforms, breath by breath (using respiratory flow signal), without pre-selection of stable periods and using known formula [1] dpp3b (averaging 3 breaths without any filtering), dppalg and hli ò were automatically obtained (matlab ò ). dpp3b was compared to dppalg (2600 pairs) using mann-whitney t test. 18287 pairs of hli ò and dppalg values (see fig. 1 below) were compared using linear regression and bland-altman method. a dppalg threshold value of 13% was used to generate hli ò roc curves. results. dpp3b and dppalg were significantly correlated (r 2 = 0.99, p \ 0.001), but standard deviation of dpp3b were higher than the standard deviation of dppalg (1.2 ± 0.4 vs. 0.2 ± 0.1%, p \ 0.001). dppalg and hli ò were correlated (r 2 = 0.73, p \ 0.001), mean difference was 2 ± 4%. hli ò above 13% predicted dppalg above 13% with a sensitivity of 95% and specificity of 82% (roc: 0.96). conclusions. dpp3b should be interpreted with caution due to the high variance of this index. in real conditions and during long time monitoring dppalg and hli ò are in acceptable agreement and hli ò may help estimating continuously the hemodynamic effects of ventilation. introduction. transthoracic echocardiography (tte) is supposed not to be useful in ventilated patients (pt). echocardiography is usually performed transesophageally in ventilated pt and is thought to be independent of the examiner's skills. we want to demonstrate that tte in ventilated pt could be learned even by medical students with reasonable results and that tte could add useful informations for interpretation of the hemodynamic status. objectives. in a prospective observational study 42 consecutive patients (pt) were enrolled in a 9-bed medical intensive care unit of a university hospital. inclusion criteria was septic shock according to actual guidelines. transthoracic echocardiography (acuson cv70, siemens, germany) was performed by a medical student in each subject on day 1, day 7 and survival was reported on day 28. tte-examination was reduced to an apical 4-chamber view for interpretation of left ventricular global function and calculation of left ventricular ejection fraction (ef) with the simpson method and to a subcostal view in order to examine the diameter of the inferior caval vein (ivc) and to rule out pericardial effusion. each examination was digitally recorded and was interpreted by an experienced cardiologist. every single pt was mechanically ventilated. cardiac output (co) was measured with the transpulmonary thermodilutional technique (picco-catheter, pulsion, germany). the insertion of the picco-catheter took place due to an individual physician's decision. crp was measured as an parameter of inflammation. results. 42 pt, mean age 71 years ± 7.22, 27 male (64%), 23 pt with known coronary artery disease (55%), 2 pt with known dilated cardiomyopathy (5%). mean apache ii-score 35.9 ± 6.8. 22 pt died within 28 days (52%). picco-catheter was inserted in 21 pt (50%). tte could be successfully performed in 38 pt (91%). the following values are expressed as mean values ± sd, student's t test, p \ 0.05 denotes statistic significance. ef on day 1 43.6% ± 16.2, ef on day 7 42.9% ± 15.7, p = 0.04. ivc on day 1 20.3 mm ± 4.5, ivc on day 7 20.2 mm ± 4.5, p = ns. co on day 1 5.5 l/min ± 3.4, co on day 7 4.0 l/min ± 3.4, p = 0.05. crp on day 1 19.82 mg/dl ± 10.4, crp on day 7 9.91 mg/dl±, p\ 0.0001. pericardial effusion in no pt. in older pt coronary artery disease is common and ef is at the start of septic shock severely diminished. ef decreased slightly in the early course of septic shock, may be as an expression of septic cardiomyopathy. the ivc diameter did not change and may not be useful as a predictor of preload in ventilated pt. co decreased over time as the hyperdynamic circulation in septic shock is getting normalised. tte adds useful hemodynamic information and should be performed in each ventilated pt. tte could be performed in almost each ventilated pt and is easily learned even by medical students. (1, 2) , which can often be caused by anaemia. in current guidelines the transfusion trigger is haemoglobin (hb) \ 7 g/dl, but there is no recommendation for scvo 2 (3). objectives. the aim of this retrospective study was to evaluate the change in scvo 2 before and after transfusion and to reveal whether co 2 -gap reflects it. methods. over a 6 month period hb, scvo 2 , co 2 -gap and o 2 -extraction ratio (o 2 er) were recorded before and after transfusion. data are presented as median [interquartile range], for statistical analysis wilcoxon, mann-whitney tests and pearson correlation were used as appropriate. results. out of 128 transfusion events the scvo 2 was measured in 50 cases. after transfusion hb increased significantly: 7.7 [7.1-8.2]-9.0 [7.9-9.7] g/dl, p \ 0.001. the median scvo 2 was 71 %, therefore two groups were created: ''low'' (scvo 2 \ 71 %, n = 27); ''high'' (scvo 2 c71%; n = 23). hb increased significantly in both groups (p \ 0.001), but scvo 2 conclusions. in the high-group the low hb levels did not cause oxygen debt, as after transfusion hb increased significantly but scvo 2 did not, and o 2 er and co 2 -gap were within the normal range. our results give further support that not only the hb level should serve as a transfusion trigger, but measures of oxygen debt such as scvo 2 and co 2 -gap should also be considered, hence unnecessary transfusions could be avoided. introduction. intellivent ò is a fully closed loop ventilation designed to keep the patient within target ranges of etco2 and spo2. the system includes an automatic adjustment of peep and fio2 following the ardsnetwork tables [1] . if required peep is changed by 1 cmh 2 o every 6 min with a maximal possible value set by the user or depending on an automatic and continuous calculation of the respiratory variations of the plethysmogram from an integrated pulse oxymeter (hli ò ), i.e. the higher the hli ò the lower the maximal peep allowed by the system. the present study was designed to estimate whether changes in peep are reflected in hli ò changes. in 17 sedated icu patients ventilated for 120 min in fully closed loop ventilation with intellivent ò (hamilton medical s1 ventilator), 34 episodes of significant changes in peep (c 4 cmh 2 o) were selected and hli ò values within 5 min before and after peep changes were collected. statistics were done using sigmastats with p \ 0.05 as significant. results. changes in peep and in hli ò are shown in the table 1 15 ± 3 cmh2o 9 ± 4 cmh2o 17 ± 12% 9 ± 6% p \ 0.001 the correlation between change in peep and change in hli ò is shown on the fig. 1 . conclusions. based on these preliminary data changes in peep are reflected hli ò changes and may help estimating continuously the hemodynamic effects of ventilation. objectives. we have tested a 3 axis accelerometer sensor for detection of regional left ventricular ischemia. in 12 pigs a 3-axis accelerometer was sutured to the left ventricular (lv) apical region in left descending coronary artery (lad) supply area accelerometer x-axis measured longitudinal-, y-axis circumferential-and z-axis radial epicardial motions. epicardial displacements were calculated from the acceleration signals and systolic displacements within 150 ms after peak r on ecg was measured. lad was occluded for 60 s to induce regional lv dysfunction. myocardial circumferential strain (shortening) measured by echocardiography in the lv apical anterior region was used to confirm ischemia. the ecg st-segment in lead ii was also monitored. data are presented as mean ± se. early systolic displacement at baseline was 11 ± 4 mm, 12 ± 2 mm and 3 ± 2 in circumferential, longitudinal and radial directions, respectively. lad occlusion induced akinesia in circumferential (0 ± 3 mm, p \ 0.001) and radial (0 ± 1 mm, p = 0.01) directions, whereas longitudinal displacement changed less to 10 ± 2 mm (p = 0.280). ischemia was confirmed by echocardiography strain, showing lengthening in systole (p \ 0.001). no significant changes were observer in the ecg st-segment during coronary occlusion (p = 0.341). introduction. there is increasing evidence to suggest perioperative complications are predictive of long term survival and that reducing them may improve survival rates 1 . goal directed therapy has been shown to reduce mortality and morbidity perioperatively, with those unable to increase oxygen delivery perioperatively having demonstrably worse outcomes. the advent of non invasive tissue oxygenation monitors using near infrared spectroscopy has allowed further study of oxygen flux during goal directed therapy. objectives. to observe changes in tissue oxygenation during an 8 h oxygen delivery targeted post surgical optimisation program and provide long term mortality followup of a surgical cohort of high risk patients. methods. 40 patients undergoing high risk surgery and postoperative optimisation (targeting of oxygen delivery index of [600 ml/min/m 2 ) on the tensive care unit at a london teaching hospital were enrolled. each patient underwent a protocolised haemodynamic optimisation protocol as per our standard unit policy for 8 h with consecutive recordings of tissue oxygenation at the thenar eminence using an inspectra 325 monitor. additional variables relating to global and tissue perfusion were measured concurrently. patients were followed up for survival status at 3.5 years using routinely available information held within our hospital records. in hospital mortality was 17.5% (n = 7), whilst at 3.5 years this had increased to 50% (n = 20). there was no significant difference between apii scores 11 (4) versus 9.5 (4), age 61.95 ± 16.23 versus 65.8 ± 16.35 or operation type for survivors and non-survivors at 3.5 years respectively. significant differences between groups were found however for admission and mid optimisation protocol (4 h) hr and sto2 (see table 1 there were no significant differences in measured variables for 30 day mortality. conclusions. there appears to be a statistical and clinical difference in hr and tissue oxygenation between the long term survivors of high risk surgery who undergo monitored postoperative goal directed optimisation. introduction. bronchoscopic bronchoalveolar lavage (b-bal) is today the gold standard for sampling of inflammatory markers in the distal airways. nonbronchoscopic bronchoalveolar lavage (n-bal) by ordinary suction catheter has been investigated as a more easily accessible method for alveolar sampling in the setting of acute respiratory distress syndrome (ards). the results, however, were disappointing, probably due to more proximal sampling by the n-bal. to investigate wether n-bal by a catheter with physical properties similar to those of the bronchoscope is comparable to b-bal. methods. b-bal and n-bal by cook's airway exchange catheter was performed with 3 9 30 ml normal saline on opposite sides 15 min apart at nine different occasions on 5 anesthetized and intubated pigs. the volume of the recovered lavage was noted, after which the fluid was analyzed for albumin, total cell count, viability and differential cell count. statistical analysis was performed using wilcoxon's rank-sum test. results. n-bal yielded significantly higher albumin content than b-bal (20.1 ± 8.7 vs. 11.7 ± 3.4 mg/l, p = 0.027). in all other measurements there were no significant differences between n-bal and b-bal (recovered volume 52. objectives. we hypothesized that collagen synthesis and degradation are disturbed in acute respiratory failure. in the finnali-study we defined acute respiratory failure as need of noninvasive and/or invasive ventilatory support for more than 6 h (1). after informed consent we collected blood samples for serum procollagen propeptides i and iii (pinp, piiinp) and ictp levels at study admission, day 2, 7 and 21. patients with all four blood samples were included in this substudy. multiple organ dysfunction (mod) was defined as two or more individual organ sofa scores of 3-4 at any day during the first week. results. the study population comprised 68 of 958 finnali patients (1). the mean (sd, range) age was 60 years (15, 20-86) and the majority were male 74%. on admission the mean sapsii score was 45 (15, 2-83). 16 patients (24%) developed mod during the first 7 days. over time piiinp/pinp-ratio first increased and then decreased to baseline by day 21 while pinp/ictp-ratio decreased and then decreased to baseline by day 21(p \ 0.001 and p = 0.006, respectively) ( fig. 1 ). there were no statistical differences in the ratios between patients with or without mod. conclusions. we found that in patients with acute respiratory failure the balance of collagen synthesis was towards degradation of type i collagen and production of collagen type iii. 3 ± 43 ng/ml in the ards group, and significantly higher than the 53.8 ± 37.0 ng/ml in the ali (not ards) group. the difference in hmgb1 values in the early stage between the group that died up to the by 30th day and the surviving group was not significant, but the hmgb1 values were significantly higher in the group that died until the 60th day and 90th day than in the survival group. it was concluded that differences in hmgb1 values in the early stage after the onset of ali (not ards)/ards are useful as outcome determining factors after 30 days of onset. an inverse correlation was observed between the hmgb1 values and lung oxygenation, suggesting the possibility that hmbg1 is involved in the development of respiratory failure. s. shibata 1 , g. takahashi 2 , n. shioya 3 , s. endo 3 1 akita city hospital, anesthesiology, akita, japan, 2 iwate medical university, emergency medicine, morioka, japan, 3 iwate medical university, critical care medicine, morioka, japan sivelestat sodium hydrate (sivelestat) is a selective polymorphonuclear leukocyte elastase (pmn-e) inhibitor and has also been shown to be effective for pulmonary disorders associated with sirs in clinical patients. blood levels of inflammatory cytokines have been shown to be decreased in patients treated with sivelestat. however, since patients with sirs have already received other drugs, it remains indefinite whether or not sivelestat might suppress the production of cytokines. moreover, it is difficult to clarify any cells releasing cytokines. in the experiment using cells isolated from the blood, intercellular mutual actions and cytokine networks were blocked and the experiment failed to faithfully reproduce the in-vivo condition. objectives. the possibility of sivelestat suppressing the production of cytokines from granulocytes and monocytes was assessed by intracellular cytokine staining using the whole blood culture method and flow cytometry to faithfully reproduce the in-vivo condition. methods. blood samples were collected from 9 healthy volunteers. a vehicle (control group), lipopolysaccharide (lps; lps group), or lps + sivelestat (sivelestat group) was added to the whole blood, followed by the addition of a protein transport inhibitor in each group. after incubation, they were subjected to staining of the cytokines retained in the cells by the addition of an anti-interleukin 8 (il-8) or anti-tumor necrosis factor a (tnf-a) antibody and analysis by flow cytometry. the data were analyzed by the kolmogorov-smirnov test. values obtained [d/s(n)] result from the comparison of the fluorescence histograms of each sample with a control one. addition of sivelestat at low concentrations (1 and 10 lg/ml) significantly (p \ 0.01) suppressed the production of il-8 from granulocytes induced by a low concentration (1 ng/ml) of lps. on the other hand, the granulocytic production of tnf-a induced by a high concentration of lps (10 ng/ml) was significantly (p \ 0.01) suppressed by treatment with sivelestat at high concentrations (10 and 100 lg/ml). with regard to the monocytic production of tnf-a and il-8 induced by lps, there was no significant suppression of either tnf-a or il-8 production by sivelestat. conclusions. sivelestat, a neutrophil elastase inhibitor, suppressed granulocytic production of il-8 and tnf-a, suggesting the potential usefulness of sivelestat for the treatment of various morbid conditions involving il-8 and tnf-a in their onset. introduction. coagulation, fibrinolysis and extravascular fibrin deposition are the hallmarks of the pathogenesis of acute lung injury (ali). pai-1 has a central role in antagonizing fibrinolysis by decreasing the plasminogen turnover to plasmin. pai-1 has been suggested as a clinical severity marker of ali. in previous studies it was associated with higher mortality and morbidity in the critically ill. upar is a cell surface receptor activating the serine protease upa. increased expression of upar is found in various stages, including inflammation, tissue remodelling and malignancies, indicating poor prognosis. pai-1 antagonizes the proteolytic activities of upa and plasmin. objectives. we sought to evaluate the prognostic value of supar and pai-1 for 90-day mortality of patients with acute respiratory failure (arf). the finnali-study patients needed invasive or non-invasive ventilation for more than 6 h (1). blood samples were collected from patients at baseline and on day 2 after baseline. healthy volunteers were also analyzed. sera were frozen at -80°c until analyses. concentrations of supar and pai-1 in blood serum were measured by enzyme linked immunosorbent assay (elisa). data are presented as median (iqr). the prognostic value of supar and pai-1 for 90-day mortality was determined with roc analysis. in the critically ill, supar and pai-1 were 12.7 (10.7-14.7) ng/ml and 2.33 introduction. acute lung injury is a common disease in intensive care, associated to various septic or inflammatory diseases. inflammation is part of the defense mechanisms of innate immunity, occurring after tissue injury. objectives. the aim of the project was to decipher the transcriptional changes occurring after the onset of an inflammatory injury by intravenous injection of oleic acid. experimental study of the lung transcriptome after oleic acid injection. thirtysix c57bl/6 j mice, aged of 7 weeks, were sacrificed at 1h, 1h30, 3h, 4h, 18h and 24h after physiological serum or oleic acid injection (20 ll) in the caudal vein. left and right lung were separated for mrna extraction and pathological examination. labelled cdna were hybridized on cdna nylon microarray (tagc, marseilles, france) and raw data were extracted from scanned images with bzscan2 software. raw data were normalized with the quantile method, and supervised analysis was conducted with significance analysis of microarray algorithm within the r statistical suite and bioconductor libraries. after the administration of oleic acid, the mice were tachypneic and prostrated. all survived during the 24 first hours. the pathological analysis of lung tissue revealed an early inflitration of the lung tissue by polynuclear cells, as well as a pulmonary edema. these alterations were not observed after 4h. the time course analysis of transcriptional lung data identified a thousand genes which expression is modulated after injury. hierarchical clustering identified 3 major groups of genes. the first one (365 genes) is composed of genes transiently up-regulated between 1h and 3h after oleic acid injection. th second group (366 genes) is composed of genes expressed between 1h30 and 4h. the third group (366 genes) is composed of genes expressed at the later time points (18h-24h). the functional annotation linked these signatures with keywords related to pro-inflammatory response, vascular endothelium modification and lipid metabolism, respectively. rt-pcr analysis of pro-(tnf, il6) and anti-inflammatory (il4, il10) markers related the pro-inflammatory phase to the earlier time points (1h-1h30) and the anti-inflammatory phase to the late points (after 4h). conclusions. oleic acid injection in mice induced a transient acute lung injury. this is confirmed by clinical, pathological and transcriptional modifications. the modulation of gene expression after the oleic acid injection revealed an early pro-inflammatory response, followed by an anti-inflammatory response and lipid metabolism modificiations. this model could now be used to describe the specific modulation occuring during pulmonary infection and critical injuries like acute respiratory distress syndrome. introduction. ventilator associated lung injury (vali) is influenced by tidal volumes, airway pressure and cyclic opening of alveoli during mechanical ventilation. preserved spontaneous breathing during partial ventilatory support may be protective, but it is not known whether the transpulmonary pressure generated by spontaneous breathing has the same effect on vali as if generated by the ventilator. to determine whether hemodynamics, respirtory function and vali are influenced by the amount of support provided by pressure support ventilation. after approval from the institutional animal care committee, acute lung injury was induced in 50 anesthetized sd rats by acid aspiration. ten animals each were then ventilated with positive end-expiratory pressure 5 cmh2o in pressure control (pc), pressureregulated assist control (ac) or pressure support mode with 100% (ps100), 60% (ps60) or 20% (ps20) pressure support of initial distending pressure needed to maintain tidal volume. pc animals were paralyzed. after 4 h animals were killed and vali determined. results. there were no differences in baseline characteristics. acute lung injury was characterized by a decrease of the p/f ratio from 447 ± 75 to 235 ± 90 mmhg and of the dynamic compliance from 0.5 ± 0.2 to 0.28 ± 0.1 ml/cmh 2 o. conclusions. compared to controlled ventilation, preserved spontaneous breathing activity improved hemodynamic stability, respiratory function and lung edema clearance. the reduction in pressure support did not lead to reduced tidal volume, but transpulmonary pressure was preserved by muscular activity of the chest wall. no difference was observed between full or 60% of pressure support, but further reduction in pressure support resulted in increased wet-dry ratio. objectives. we studied the effects of metabolic acidosis on enzymatic and non-enzymatic no-production in hypoxic and hyperoxic lung regions in a pig model. eighteen healthy anesthetized pigs were separately ventilated with hypoxic gas to the left lower lobe (lll) and hyperoxic gas to the rest of the lung. six pigs received hcl infusion (hcl group), six pigs received n w -nitro-l-arginine methyl ester (l-name) and hcl (l-name + hcl group) and six pigs received buffered ringer's solution (control group). no concentration in exhaled air (eno), no synthase (nos) activity in lung tissue, and regional pulmonary blood flow were measured. results. metabolic acidosis, induced by infusion of hcl, decreased the relative perfusion to the hypoxic lll (q lll /q t ) from 7 (±3) to 3 (±1)% in the hcl group (p \ 0.01), and from 4 (±1) to 1 (±1)% in the l-name + hcl group (p \ 0.05), without any measurable significant changes in eno from hypoxic or hyperoxic lung regions there were no significant differences between the hcl and control groups for ca 2+ -dependent or ca 2+ -independent nos activity in hypoxic or hyperoxic lung regions. metabolic acidosis augmented the hypoxic pulmonary vasoconstriction, without any changes in pulmonary enzymatic or non-enzymatic no-production. when acidosis was induced during ongoing nos-blockade, the perfusion of hypoxic lung regions was almost abolished, indicating acidosis-induced pulmonary vasoconstriction was not no dependent. assessing and monitoring biomarkers in acute lung injury (ali) may improve knowledge of its pathogenesis, early recognition, and management and predict remote organ injury and multiple organ failure. objectives. early consents for research are difficult to obtain in patients with or at risk of ali because of the emotional burden of the severity and sudden onset of the disease. however, study samples may be obtained from left-over clinical blood draws, which are readily available if processed adequately. the aim of this study was to compare fresh and ''waste'' blood samples prospectively in a series of consecutive critically ill patients. the hypothesis is that ''waste'' blood samples if appropriately processed provides accurate and reliable results comparable to the gold-standard, which is immediate collection and processing of fresh blood samples. prospective study comparing biomarkers of epithelial injury (srage) and inflammation (20 different cytokines/chemokines) in critically ill patients measured on fresh blood or waste blood, kept at 4 degrees celsius for 24 h. an automated system performed a daily screening of adults in the icu with an increased risk for ali (lung injury prediction score, lips) within 12 h of admission and/or on recognition of the diagnosis of ali, using the american-european consensus conference criteria. risks factors for ali include pneumonia, sepsis, pancreatitis, shock, aspiration, high risk surgery and high risk trauma. irb approved the protocol and written consent was obtained from patients or their surrogates. statistical measurements were performed using the bland-altman analysis for correlation between fresh and waste blood sample data. between may and december 2009, 30 patients were enrolled. one patient was excluded due to lack of sample. samples were obtained either at one time point (n = 25) or two, on consecutive days (n = 13). female/male patient ratio was 12/17. seven of the 29 patients had ali. twenty two patients had risk for ali with a median lips score of 4 (iqr 3.5-4.5). sepsis was the most common risk factor, present in 23 patients. in-hospital mortality was 28% (8/29). the bland-altman plot (mean bias ± se, limits of agreement) showed good correlation for il-1ra (-9 ± 14.3 pg/ml, -38.3 to 20.4 pg/ml), il-6 (0.9 ± 2.9 pg/ml, -5 to 6.8 pg/ml), il-8 (-0.3 ± 0.3 pg/ml, -0.9 to 0.3 pg/ml), il-12 (p40) (-0.2 ± 0.3 pg/ml, -0.7 to 0.4 pg/ml), mcp-1(-0.2 ± 2 pg/ml, -4.2 to 3.9 pg/ml) and srage (-24 ± 53 pg/ ml, -130 to 83 pg/ml) between fresh blood and ''waste'' blood samples. in patients with ali, properly stored blood, drawn for clinical purposes, can be processed within 24 h for research purposes. however, the stability of each biomarker of interest needs to be individually validated before using stored blood introduction. pulmonary surfactant inactivation following acute lung injury might promote alveolar derecruitment and reduce the airspace available for ventilation, making the lung more prone to ventilation-induced lung injury (vili). our aim was to test the potential for a protective effect of exogenous surfactant treatment in a model of acid aspiration and vili. methods. 17 male c57/bl6 mice were anesthetized, mechanically ventilated (vt 15 ml/ kg; rr 130/min; peep 2 ± 0.2 cmh 2 o; fio 2 0.5) and immediately subjected to intrabronchial (right) instillation of 1.5 ml/kg hcl 0.1 m. mechanical ventilation went on for 420 min. 180 min after the acid instillation, 9 mice were treated with exogenous surfactant (80 mg of phospholipids/ml) given as bolus of 1 ml/kg in the right bronchus (surf group). we measured oxygenation, lung compliance (measured every 60 min throughout the experiment), macrophage inflammatory protein (mip) 2 in broncho-alveolar lavage (bal) fluid. . pao 2 at the end of the experiment was significantly higher in the surf than in control group (383 ± 163 vs. 198 ± 104 mmhg, p \ 0.05). although surfactant bolus caused a reduction in lung compliance measured 10 and 60 min after treatment, in the surf group compliance restored to 98 ± 7% of the post injury level, while it decreased in control group to 88 ± 8% (p \ 0.05). there were no differences between groups in the dosage of mip-2 in bal neither in right or left lung. conclusions. exogenous surfactant treatment improved lung function in a murine model of two hit lung injury. grant acknowledgment. introduction. ventilator induced lung injury significantly contributes to the mortality in patients with acute respiratory distress syndrome, the most severe form of acute lung injury. understanding the molecular basis for response to cyclic stretch and its derangement during high volume ventilation is of high priority. objectives. to identify specific molecular regulators involved in the development of ventilator induced lung injury. we undertook a comparative examination of cis-regulatory sequences involved in the coordinated expression of cyclic stretch responsive genes using microarray analysis. analysis of stretched vs. non-stretched cells identified significant enrichment for genes containing binding sites for the transcription factor atf3 (activating transcription factor 3). to determine the role of atf3 in vivo, we compared the response of atf3 gene deficient mice to wild type litter mates in an in vivo model of ventilator induced lung injury. results. atf3 deficiency results in increased sensitivity to mechanical ventilation alone or in conjunction with inhaled lipopolysaccharide (10 mg/kg) as determined by assessment of lung and bronchoalveolar lavage cell infiltration and pro-inflammatory mediator release, pulmonary edema and indices of tissue injury. the expression of genes containing an atf3 cis-regulatory region was significantly altered in gene deficient animals. atf3 protein expression and nuclear translocation is increased after mechanical ventilation. conclusions. atf3 deficiency confers increased sensitivity to mechanical ventilation alone or in combination with inhaled endotoxin. in our model, atf3 acts to ''counterbalance'' cyclic stretch and high volume-induced inflammation, limiting its potential to cause additional lung injury and consequently protecting animals from injurious cyclic stretch. objectives. our aim was to evaluate the role of the alveolar macrophages in a murine model of ali, by selective depletion of this type of cells from the air space achieved by clodronate administration. mice were treated (it) with 100 ll of clodronate (clo)-or pbs (pbs)-liposomes. after 24 h mice were anesthetized and ventilated (vt 8-10 ml/kg, rr 140 min -1 , fio 2 0.21); in order to induce lung injury 2 ml/kg of hcl (0.1 m) or air bolus (sham group) was instilled in the right bronchus. mice were ventilated for 10 min, and extubated after awakening. 24 h after injury, animals were sacrificed and broncho-alveolar lavage (bal) and blood gas analysis (fio 2 = 0.21) were performed. . 24 h after lung injury animals with alveolar macrophages depletion, showed a better oxygenation versus pbs-treated group. however, recruitment of neutrophils in bal was not statistically different between clo_hcl and pbs_hcl group. results. high levels of oc were found in patients treated by 150 mg of ot bid. oc levels ranged from 2,507 to 4,551 ng/ml in these patients. concentrations of oc were five-to tenfold higher than concentrations reported in healthy volunteers. lesser levels were found in patients treated by 75 mg of ot bid. nevertheless, the patient with the moderate renal failure seemed to accumulate oc (levels ranged from 906 to 1470 ng/ml) whereas concentrations reported in the patient with a normal renal clearance were below (152-349 ng/ml). conclusions. ecmo seemed not to have any influence on oc concentrations while renal insufficiency seemed to be the parameter leading to oc accumulation. as ic50 was very low and reached even with usual dosage, increasing ot dose to 150 mg bid appeared to be unnecessary. objectives. aim of our study was to evaluate the effect of nursing care on patients undergoing venous-venous ecmo for acute respiratory distress syndrome (ards). methods. we recorded physiological and ecmo parameters (heart rate, arterial blood pressure, mixed venous saturation (svo 2 ), arterial oxygen saturation (spo 2 ), body temperature and extracorporeal blood flow (bf)) before and during daily nursing in 5 patients undergoing vv-ecmo for several days (each patient was followed on average for 4.6 days, 23 cases in total). arterial blood gases were also collected before and after nursing care. daily nursing was performed following defined steps (sponge bath, oral hygiene, change position of endotracheal tube, elevation with scooping stretcher for sheets replacement and back hygiene, dressing replacement) in agreement with a standard protocol in use in our department. (expressed as mean ± standard deviation). all patients were affected by ards h1n1-related. patients were sedated with propofol (182 ± 68 mg/h) or midazolam (7.9 ± 1.8 mg/h) plus an opioid drug (fentanyl 175 ± 45 mcg/h or remifentanil 0.2 ± 0.01 mcg/kg/min or sufentanil 0.32 ± 0.09 mcg/kg/min). ramsey score before nursing was 5.4 ± 0.9. in 11 cases patients were paralysed. in table 1 we summarized the adverse events observed during nursing care, divided into hypertensive or tachycardic episodes, blood oxygen desaturation, reduction in svo 2 or reduction in bf. forty-nine sedative bolus were administered during nursing (mean request for each patient: 2.1 ± 1.9), always after an episode of hypertension or tachycardia (most frequently during elevation with scooping stretcher and changing position of endotracheal tube). although in 13 cases preventive bolus of sedation were administered before nursing, in 8 of those cases (61%), additional bolus were required. we found an inverse correlation between bf and the increase in heart rate, drop in arterial saturation and svo2. despite active warming, we observed a drop of 0.31 ± 0.15°c (p \ 0.01) in body temperature. nursing care may have a significant impact on physiologic parameters of patients during vv-ecmo. tachycardia, hypertension and reduction in oxygenation were commonly recorded and were not prevented by pre-nursing bolus of sedation but were attenuated in patients with higher bf. introduction. prone position has been used in cases of ards with refractory hypoxemia but some physiological effects are still unknown. prone position could increase intraabdominal pressure (iap) and could lead to acute renal failure (arf). acute kidney injury in icu is associated with increased mortality. objectives. the aim of this study was to determine whether prone position could increase intraabdominal pressure and possibly promote arf. we studied all adult ards patients who were ventilated using the protective strategy defined by ards network criteria and who needed prone position to improve oxygenation. we collected respiratory data (ventilator parameters and gas exchange) and hemodynamic variables (heart rate, systolic, diastolic and mean arterial pressure). iap was measured using the abdo-pressure tm bladder transducer following world society of acute compartment syndrome recommendations. abdominal perfusion pressure was calculated as mean arterial pressure minus iap. main renal parameters were: filtration gradient (fg), creatinine clearance, fractional excretion of sodium (fena) and urea (feurea). patients were classified according to rifle score after each manoeuvre. all data were recorded in prone and in supine position at least once per day. results. the study included 18 patients (14 male) admitted to a medical-surgical icu over a one-year period. their mean age was 47.3 ± 17.8 and length of icu stay was 23 ± 18 days. all 18 patients had primary ards and 9 had received nephrotoxics. icu mortality reached 50%. we recorded at least 3 manoeuvres per patient (a, b, c). prone positioning improved pafio 2 ratio from 101.5 ± 5 to 150 ± 72 (p = 0.027). iap showed a small increase from 6.1 ± 2.7 to 8.9 ± 2.9 mmhg (a; p = 0.004), from 8.4 ± 3.4 to 11.2 ± 3.7 mmhg (b; p = 0.038) and from 7.6 ± 1.3 to 9.9 ± 0.8 mmhg (c; p = 0.001). there were no statistically significant changes in hemodynamic parameters or abdominal perfusion pressure. renal function parameters (fg, creatinine clearance, fena and feurea) showed no modification after each prone positioning. in contrast, when patients were classified according to rifle score, we observed a trend towards worsening, though this was not statistically significant. conclusions. prone positioning improved arterial oxygenation in primary ards patients and was associated with an increase in iap. however, creatinine clearance and glomerular filtration remained unchanged. percutaneous extracorporeal life support system (p-ecls) including ecmo becomes widely used in medical and surgical emergent situation, such as refractory cardiogenic shock, cardiac arrest and acute respiratory failure. patients requires highly specialized intensive care and monitoring system. we reviewed our ecls experience and tried to analyze the clinical outcomes, factors for survival and frequently faced problems during management for improving weaning and survival rate (medical vs. surgical patients). introduction. in spite of the huge efforts spent over the last years, conventional treatment of acute hypoxemic respiratory failure (ahrf) is often inadequate and alternative procedures must be instituted. icus skillful in extracorporeal membrane oxygenation (ecmo), as recently shown [1] , may improve survival of these patients. since 1989 we developed a treatment algorithm for ahrf which encomprises: (1) low flow venous-venous ecmo (lf-ecmo) consisting in a relatively low initial blood flow (bf, 2-2.5 l/min) to maximize extracorporeal co 2 removal while providing partial oxygenation (if needed, bf can be increased up to 4.5-5 l/min to keep arterial po2 above 45 mmhg); (2) femoral-femoral percutaneous cannulation with 21-25 fr cannulas to allow free movements of the neck and increase patient's tolerance; (3) early institution of spontaneous assisted ventilation (sb) and weaning from sedation and mechanical ventilation (mv) while on ecmo. objectives. to review our last 10 years lf-ecmo activity. methods. study period was january 2000-2010. lf-ecmo entry criteria were: potentially reversible acute hypoxemic respiratory failure, lis c3, no evidence of intracranial bleeding and no absolute contra-indications to heparinization. ecmo was performed with different type of heparin coated hollow-fiber artificial lungs. . we treated 31 patients (mean ± sd, 37 ± 18.5 years old, 68% males, bmi 27 ± 6, sofa 7.5 ± 3, oi 38 ± 14). 48% of these patients were placed on ecmo at other hospitals and transported to our icu by a dedicated ecmo team. ventilation days before ecmo were 12 ± 16 (range 1-69). before ecmo vt/kg was 6 ± 1.5 and rr was 33 ± 12: after ecmo beginning vt/kg was unchanged while rr decreased to 11 ± 4 (p\ 0.01). ecmo was set at bf 2.5 ± 1 l/min, gf 3.3 ± 1.5 l/min, fio2 0.98 ± 0. introduction. ventilating patients with acute lung injury (ali) in supine position potentially leads to an impaired pulmonary gas exchange. prone position (pp) is an attractive means to improve ventilation-perfusion (v/q) ratio [1, 2] but has several contraindications and showed no improvement in survival so far [3] . another therapeutical option is an upright position, which is easy to perform and has theoretical advantages over pp: the upward shift of the abdominal compartment is less pronounced, thus increasing thoracoabdominal compliance [2] . 4 however, to date regimes of an upright position did not tilt patients more than 45° [4] . 4 objectives. we hypothesised that a 60°standing position (sp) during mechanical ventilation may improve respiratory function. furthermore, we aimed to determine the feasibility of a sp for 2 h during mechanical ventilation. we studied 30 adult patients, receiving mechanical ventilation for more than 48 h in the intensive care unit of an university hospital. after recording baseline data, patients were placed in a 60°sp with the body entirely straight. further data sets were recorded during 2 h in sp, and after patients position was readjusted to supine position. functional residual capacity (frc) increased immediately after reaching sp (p \ 0.001) and remained elevated after repositioning to supine position. pao 2 /fio 2 ratio and compliance decreased initially during sp, but increased (p \ 0.05) after patients were retransferred to supine position. haemodynamic variables remained stable under a moderate increase of doses of catecholamines during the study period. conclusions. changes in respiratory function during sp are probably explained by a downward shift of the diaphragm due to gravitational forces 2 leading to an increased frc but not altering v/q ratio as demonstrated by the pao 2 /fio 2 ratio. after reaching the initial supine position the opening of the lung proved by the elevated frc is the predominant effect now associated with an increase in oxygenation as reflected by the pao 2 /fio 2 ratio due to an optimised v/q ratio. our results are confirmed in a subgroup analysis for 9 patients meeting ali criteria. ventilating patients in sp may be a new therapeutical approach to improve respiratory function in patients with ali. (1) . there are several clinical trials investigating the efficacy of the free radical scavenger n-acetylcysteine (nac) in ards, but its advantage remains uncertain. objectives. critically appraise and summarize all randomized clinical trials involving intravenous nac administration in adult patients suffering from ards. we included trials involving participants with ards according to the american-european consensus conference criteria (2) regardless of the underlying cause, and where one of the groups was treated with intravenous n-acetylcysteine in bolus intravenous doses or as continuous infusion, or combination of the two, and the other group was given placebo or standard treatment. conclusions. the main finding of this meta-analysis is that intravenous nac is ineffective in reducing mortality, length of stay or duration of mechanical ventilation in ards. we also found that late administration of nac may be associated with adverse outcome. the mechanism of this potentially deleterious effect remains unclear, but dosing and timing of nac appear to be critical issues. objective. to evaluate if extubation during ecls is harmful or beneficial. a 28-year-old woman was admitted to our intensive care unit (icu) after removal of a left ventricular assist device. this device was implanted as bridge to recovery for postpartum cardiomyopathy and ventricular function seemed to have recovered sufficiently. however, shortly after icu admittance she developed massive left and right ventricular failure. therefore a centrally cannulated veno-arterial ecls (maquet permanent life support) was implanted as a bridge to transplant. four days later she was extubated while on full ecls support, in order to reduce the risk of ventilator associated pneumonia. while on ecls, the patient was mobilized, practiced with an ergometer and chatted with her family. three days later the patient underwent cardiac transplantation. the postoperative period was characterized by temporary pulmonary failure, due to the combination of lung edema and atelectasis. eventually she made a full recovery. discussion. ecls provides a valuable means as bridge to transplantation, bridge to bridge or bridge to recovery. with the increasing use of ecls for circulatory failure, debate about the necessity of mechanical ventilation during this treatment ensues. ecls is usually applied under deep sedation and controlled mechanical ventilation. discontinuation of sedation possibly prevents intensive care acquired weakness. extubation during ecls may provide better pulmonary perfusion due to negative intra-thoracic pressure. furthermore, the awake and extubated patient is able to mobilize and exercise which may reduce the risk of atelectasis and ventilator associated pneumonia. our patient however developed pulmonary edema and atelectasis after discontinuation of ecls. the edema was probably a consequence of reperfusion injury, due to severely decreased pulmonary flow while on ecls. an absent ventilatory drive while on ecls may have led to hypoventilation while the patient was extubated, resulting in atelectasis. an extensive medline search resulted in one other case report describing an extubated patient on ecls. 1 intermittent non-invasive positive pressure ventilation was used to prevent atelectasis, but the patient developed pneumonia after 30 days of ecls. our patient was successfully extubated while on ecls. however, we conclude that there is insufficient evidence to recommend or oppose extubation of patients on ecls for circulatory failure. severe ards and refractory hypoxemia were defined with a pao2/ fraction of inspired oxygen (fio2) ratio of b100, or uncompensated hypercapnea with a ph of \7.20 despite receiving optimal conventional treatment. the ecmo can be used as a rescue treatment in these case. objectives. evaluation of severe ards treated with extracorporeal oxygenation (ecmo). all these ards were due to bacterial pneumonia or h1n1 influenza. over the last year (december 2009-january 2010), the recourse to extracorporeal oxygenation (ecmo) was used in ten patients with severe ards and severe hypoxemia. two groups were defined: bacterial pneumonia with ards (bp group, n = 5), and h1n1 influenza with ards (h1n1 group, n = 5). all ecmos were implanted at the bedside to facilitate intra-hospital or inter-hospital transfer, because of severe hypoxemia or hemodynamic instability making impossible patient mobilization before ecmo. results. data sets of 44 patients of 47 consecutive patients treated with ecmo were complete and included into analyses. we had no clinical or radiological evidence for thrombosis or clotting within ecmo-circuit with a target-ptt of 35 s. one patient with systemic aspergillosis died because of intracranial hemorrhage. one ecmo circuit had to be replaced due to insufficient oxygenator function after 10 days. further data are presented in tables 1 and 2 . conclusions. in this retrospective analysis of 44 patients who underwent ecmotreatment, ac with low-dose heparin (target-ptt of 35 s) was safe and without any observation of macroscopic thrombosis or clotting within the circuit. transfusion requirements and intracranial hemorrhage were low as compared with previous reports [1, 2] . therefore our data suggest that it is possible and safe using ecmo-therapy with low-dose heparin. introduction. in response to h1n1 pandemy, italy and lombardy created a national and a regional icu network, respectively, for treatment of ards patients. our hospital policlinico san matteo of pavia participated with a team for inter-hospital ecmo implantation and subsequent patient transport. objectives. description of the pavia ecmo team and activity analysis. methods. our team is composed by a cardiac surgeon, two intensivists, a perfusionist, an icu nurse, two emergency rescue technicians and a driver. all necessary aids for implantation and intensive care are ranged in three trolleys and three transport bags. equipments are firmly mounted on a two-level steel bridge connected to a spinal board. a portable ultrasonograph is also available. the ecmo team was alerted by the national call center. each mission used two ambulances, and in one case the ambulances were embarked on a hercules c130 j. from 31 october to 31 december 2009, four patients were implanted and transported, three suffering from h1n1 influenza (including a 190-kg body weight patient) and one from acute mitral valve rupture. all patients, already mechanically ventilated with maximal support, had veno-venous ecmo implanted by femoro-femoral percutaneous cannulation. the median mission duration was of 7.25 h (range 6-17 h). all patients were transported to our icu, where the median ecmo duration was of 8 days (range 6-10 days). no major managing issue occurred during the ecmo missions, and patient hospital survival was of 100%. a multispecialist team with good knowledge of ecmo can provide an effective support in severe respiratory failure, with ecmo implantation in peripheral hospitals and subsequent patient transport, thus realizing a fast and safe continuum between phone call activation and admittance to the reference center. introduction. when patients with sever respiratory failure are treated with v-v ecmo the right heart sometimes fails. this is a serious complication with a high mortality. in our unit these patients have been converted to v-a ecmo, although it is not fully agreed upon in the ecmo community due to previously depressing results. objectives. to evaluate the results of conversion to v-a from v-v ecmo in case of right heart failure. retrospective analyses of all patients with severe respiratory failure, treated between 1987 and 2009 at the karolinska ecmo centre. patients who were converted to v-a ecmo due to right ventricular failure were evaluated. a total of 214 patients (78 adults, 57 peadiatric, 79 neonatal) were treated on v-v ecmo for severe respiratory failure. of them 50 (33 adults, 9 peadiatric, 8 neonatal) needed conversion to v-a ecmo due to right ventricular heart failure demonstrated clinically by multiorgan failure and verified by echo cardiography. the survival after conversion to v-a ecmo was 10/33 (30%) in the adult age group, 7/9 (78%) in the peadiatric age group and 2/8 (25%) among the neonates. conclusions. given the high risk of fatality if not treated, conversion to v-a from v-v ecmo should be considered when the right ventricle fails. patients on v-v ecmo with right ventricle heart failure have very bad prognosis. it is concluded from the present results that conversion to v-a ecmo can save some of these patients. cardiac surgery and regional hemodynamics: objectives. to test whether tapse and right ventricular systolic (sm) and diastolic (em and am) tissue doppler imaging velocities are related with pulmonary artery systolic pressure (pasp) and length of the weaning process in mechanically ventilated patients with acute heart failure (ahf). methods. rv fractional area change (rvfac), left ventricular ejection fraction (lvef), pasp, tapse, sm, em, am rv tdi velocities, early diastolic mitral e wave and e 0 maximal tdi velocities of the mitral annulus at the lateral wall were obtained at admission by doppler echocardiography in a cohort of 32 patients with ahf, presented with pulmonary oedema, who required positive-pressure ventilation for more than 48 h in the intensive care unit (icu). echo-derived measures were compared between patients with and without pulmonary hypertension, whereas their association with duration of mechanical ventilation and length of the weaning process was tested with multivariate linear and logistic regression analysis. and increased e/e 0 ratio (10.8 ± 1.21 vs. 7.6 ± 0.54, p \ 0.001) compared with subjects with normal pasp (n = 10). these variables were negatively associated with duration of mechanical ventilation (r 2 = 0.55, beta slope = -0.89 for tapse, r 2 = 0.52, beta = -0.57 for sm, r 2 = 0.45, beta = -0.27 for em/am, p \ 0.001) and were proven to successfully discriminate patients with (n = 12) and without (n = 20) prolonged weaning ([7 days of weaning after the first spontaneous breathing trial failure, p \ 0.001 for all comparisons). conclusions. we suggest that in critically ill patients with ahf presented with pulmonary oedema, low tapse and rv tdi velocities upon admission are associated with pulmonary hypertension and prolonged length of the weaning process. objectives. the aim of the study was to study changes in cerebral blood flow (cbf), as determined by tcd, during the early postoperative course of cvs and to correlate such changes with post-operative nc. we studied 72 patients undergoing extracorporeal circulation cvs (coronary by-pass, valve replacement or both) between march 2007 and march 2008. cbf was assessed by measuring bilateral mca flow velocities by tcd before and 10, 24 and 48 h after cvs. changes c20% between consecutive tcd results were considered significant. demographic and clinical variables, co morbidities, euroscore, sofa, type and duration of surgery and type and severity of nc were also recorded. patients were assigned to 3 groups according to cbf changes from baseline: a) changes b20%; b) cbf increases c20%, c) cbf decreases c20%. nc were classified as major (stroke, tia and coma) and minor (delirium, encephalopathy, transient cognitive impairment). we used descriptive statistics and inference by v 2 , anova and pearson's correlation. of the 72 patients, 20 were excluded (2 early post-operative death and 18 due to technical difficulties or incomplete tcd recordings). of 52 evaluable patients, 17 (33%) had no cbf changes (group a), 12 (22%) had increases c20% (group b) and 23 (45%) had decreases c20% after cvs (group c). a positive correlation was found between cbf changes and duration of circulatory arrest (p \ 0.02), maximum sofa score (p \ 0.001), respiratory dysfunction (p \ 0.016) and duration of mechanical ventilation (p \ 0.001). neurological complications occurred in 16 patients (31%), of which 4 (25%) were major and 12 were minor (75% introduction. the sole monitoring of macrohemodynamic variables is not always sufficient in the early detection of tissue hypoperfusion, especially in cardiac surgical patients that frequently present with microcirculatory derangements. near infrared spectroscopy (nirs) is an easily applicable non invasive technique that has been used to provide an estimate of tissue oxygenation at the bed side. objective. the aim of our study was to evaluate the effect on outcome of guiding hemodynamic therapy and specifically inotrope titration in cardiac surgical patients postoperatively with nirs. methods. patients operated on with cardiopulmonary bypass were assigned, after stratified randomization (gender, euroscore-cutoff of 6), to an intervention (ig) and a control group (cg). postoperatively, following cardiac intensive care (cicu) admission, after initial resuscitation according to cicu protocol, sto 2 (%) was measured in patients of the ig in 3 muscle sites: thenar, masseter and deltoid. if it was less than 80% in 2/3 sites, dobutamine was administered in incremental doses (2.5 lg/kg/min), with the sto 2 (%) measured every half hour. the interventional period began upon cicu admission and lasted for 6 h, after which both groups were treated according to cicu protocol. primary outcome measured was the oxygen consumption rate at the end of the 6 h intervention period as assessed with nirs vascular occlusion technique. . 18 patients were included in the study (8 in the intervention group and 10 in the control group). the 2 groups did not differ statistically significantly regarding age, euroscore, and macrohemodynamic variables postoperatively (with the exception of cvp). microcirculatory parameters upon admission to the cicu also did not differ, excluding masseter sto 2 (%). the oxygen consumption rate and the reperfusion rate increased in the 6 h study period in both groups, without differing statistically significantly between the groups at any time point (cg oxygen consumption rate 20.1 ± 12.4 upon cicu admission and 32.8 ± 15.5 6 h later, ig 15.4 ± 6.6 and 19.7 ± 7.7 respectively) (cg reperfusion rate 297 ± 210 upon cicu admission and 592 ± 254 6 h later and ig 201 ± 119 and 304 ± 163 respectively). as far as outcome parameters were concerned, the 2 groups did not differ statistically significantly in the total hours and total dose of vasopressors ± inotropes received, in the hours of mechanical ventilation, in the duration of cicu or hospital stay, and in sofa scores the days following the operation. conclusion. nirs guided titration of inotropes did not lead to a greater improvement in the microcirculation 6 h postoperatively, or to a better outcome. the limited power of the study prevents definite conclusions on the role of nirs in hemodynamic therapy in cardiac surgery patients. objectives. to estimate the prevalence of pulmonary embolism among mv patients in icu and its association to deep vein thrombosis (dvt). in a monocentric prospective observational study, we included all the patients requiring mechanical ventilation with no previously diagnosed pe, who underwent a thoracoabdominal ct contrast scanner for any medical reason. we used a modified protocol for pe diagnosis with a 64-multidetector row ct scan read by two independent radiologists. the association with a dvt was explored by performing venous compression ultrasound of four limbs. objectives. the aim of this animal study was to evaluate the effect of intraabdominal hypertension on left ventricular diastolic function. after approval by an institutional animal care committee, 7 rabbits were anesthetised before mechanical ventilation. an intraperitoneal infusion of 1.5% glycine solution was used to increase intraabdominal pressure to 20 mmhg. the right common carotid artery was catheterised in the neck in order to introduce a millar mikro-tip catheter (millar instruments inc., houston, usa) into the left ventricle. heart rate, arterial pressure, central venous pressure, oesophageal pressure and intraabdominal pressure were measured. the s time constant of relaxation which is considered as best index of relaxation was calculated using the derivative method (1). all haemodynamic measurements were registered at baseline and after inducing intraabdominal hypertension. data are presented as mean (iqr) and were compared using a wilcoxon rank sum test. results. heart rate (from 202 ± 100 to 166 ± 100 beat/min, p = 0.6), mean arterial pressure (from 60 ± 29 to 50 ± 34 mmhg, p = 0.20) and dp/dt max (from 3,590 ± 255 to 2,111 ± 197 mmhg/s, p = 0.14) were not significantly modified by intraabdominal hypertension. however, the s time constant of relaxation increased significantly (from 16 ± 3 to 43 ± 18 ms; p = 0.05). conclusions. in this animal model, intraabdominal hypertension impairs left ventricular relaxation. these changes in the condition of the microcirculation have been related to the degree of organ dysfunction and thus patient outcome ie hospital length of stay. near infrared spectroscopy (nirs) is an easily applicable non invasive technique that has been used to provide an estimate of tissue oxygenation at the bed side. objectives. the aim of our observational study was to examine whether impaired tissue oxygenation as assessed with nirs immediately postoperatively correlates with hospital length of stay. patients undergoing a planned cardiac surgical procedure on cpb were included in the study. patients' thenar tissue oxygenation (sto 2 %) was assessed with nirs postoperatively in the cardiac intensive care unit (cicu). results. 22 patients undergoing cardiac surgery on cpb (13 male/9 female) (age: 64 ± 14 years, euroscore: 5.7 ± 3; mean ± sd) were enrolled in the study. patients length of stay was 8.5 (3-26); median(range). the haemodynamic parameters of our patients upon admission to the cicu were: map 84 ± 12 mmhg, cvp 9 ± 4 mmhg, pcwp 12 ± 4 mmhg, mpap 24 ± 5 mmhg, ci 2.6 ± 1.1 l/min/m2, svr 1407 ± 501 dyne x s/ cm5, pvr 239 ± 106 dyne x s/cm5, hr 97 ± 15 bpm, hb 11.7 ± 1.7 g/dl, lactate 2.8 ± 1.8 mg/dl; (all variables expressed as mean ± sd). upon admission to the cicu all patients were mechanical ventilated, under vasopressor ± inotrope support and their central temperature was 36.9 ± 0.68; mean ± sd. the thenar sto 2 % was 80 ± 11; mean ± sd. thenar sto 2 % correlated statistically significantly with hospital length of stay (r = 0.53, p = 0.11). discussion. tissue oxygenation as assessed with nirs reflects the balance between regional oxygen delivery in relation to oxygen utilization. an elevated sto 2 in the presence of normal macrohemodynamics may reflect impaired oxygen consumption and thus an impaired microcirculation. conclusion. patients with impaired tissue oxygenation immediately postoperatively have a longer hospital length of stay. further studies are needed to confirm these results and to investigate the potential benefit from incorporating this information regarding tissue oxygenation in the treatment algorithm. objectives. the goal of this study was to compare two different sedative agents for implantation of crt-ds related to incidence of adverse events and patient's satisfaction. methods. the study included forty-two, asa iii-iv patients, undergoing transvenous implantation of crt-ds under local infiltrative anesthesia with 20 to 30 ml of 1% lidocaine. intraoperative sedation was established with intermittent boluses of midazolam (1-5 mg) to achieve desirable level of sedation. before the induction of ventricular fibrillation in order to test the defibrillator function of the crt-d device, patients received an additional bolus of either propofol (1.5-2.5 mg kg -1 , p group, n = 20) or etomidate (0.1-0.25 mg . kg -1 , e group, n = 22) targeting bis values in the range 50-60. the incidence of apnea, hypotension, nausea, myoclonus, pain at injection site, allergic reactions as well as patient's satisfaction with anesthesia described as feel of well being were registered and compared between groups. results. in 23 subjects (56%) no complications were recorded. myoclonus was registered in 5 patients from e group (23%) and in none from p group (p \ 0.05). no patients receiving etomidate reported pain at injection site compared to 4 patients (20%) receiving propofol (p \ 0.05). there was no significant difference in incidence of apnea between two groups (15 vs. 9%, p = 0.30). two patients in p group (10%) and 1 in e group (4.5%) became hypotensive after delivering the hypnotic agent (p = 0.36). also, there was no statistically significant difference between groups considering the frequency of nausea (0% vs.9%, p = 0.26). all the patients whom propofol had been delivered (100%) reported feel of well being and only four of them filed the same after etomidate (18%) (p \ 0.01). no allergic reactions and major adverse events were registered. conclusions. implantation of crt-ds and its testing can be successfully performed with administration of both propofol and etomidate as a safe procedure with low per operative morbidity and shorter complication rates. still, treating with propofol tends to be more satisfactory for the patients. introduction. ultra-short-acting b 1 selective adrenergic antagonists are now widely used to control tachycardia and tachyarrhythmia perioperatively. among them, landiolol, a new ultra-short-acting b 1 -blocker, has been reported to exert a more potent negative chronotropic effect with little effect on blood pressure than esmolol (1). however, detailed mechanisms underlying different cardiovascular actions are still unknown. objectives. in this study we evaluated direct effects of landiolol on cardiac performance and single cell electrophysiology in comparison to those of esmolol. methods. the present study composed of two parts. the first part of the study used isolated guinea-pig hearts which were perfused in the langendorff mode at constant flow with oxygenated tyrode solution at 37°c. the coronary perfusion pressure (cpp) was continuously monitored throughout the experiment, and intrinsic heart rate (hr) and isovolumetric left ventricular contraction were measured with a thin saline-filled balloon inserted into the left ventricle. the second part of the study was to measure action potentials and ionic currents in ventricular myocytes isolated enzymatically from guinea-pig hearts. comparison of data was conducted by repeated-measure anova with post hoc test (bonferroni's correction). conclusions. esmolol had a more potent negative inotropic effect than landiolol. this effect is, at least in part, derived from shortening of apd. in addition, increase of the coronary resistance would facilitate the negative chronotropic action of esmolol in vivo. conclusions. nma moderates hpv in the conscious spontaneously breathing beagle, but not to the same degree as acz. as compared to acz, the additional methyl-group in nma may impair its capability in vivo to act on a non-ca acz-sensitive cellular receptor or channel or that both, ca-dependent and ca-independent actions of acz yield a greater effect. introduction. tee with bubble test is considered as the ''gold standard'' method to detect a pfo with right to left shunt. tcd is a non-invasive method which has been shown to be as accurate as tee for pfo detection. we conducted a multicenter trial to estimate the prevalence of pfo, the influence of the size of the heart chambers on the prevalence of pfo and the accuracy of tcd as a non invasive method for pfo detection in mechanically ventilated icu patients. one hundred icu patients (74 m and 26 f) under mechanical ventilation who needed a tee study for hemodynamic assessment were included in the study. in each patient, the presence of a pfo was detected by tee and tcd. three bubble tests with agitated haemacel ò were performed by each method, with tee probe at 608 and 908 rotation and with tcd the gate of pulse wave doppler (pwd) at the m 1 segment of the middle cerebral artery (mca). patients without temporal acoustic window to perform tcd were excluded from the study. the size of pfo was classified as grade i, ii and iii according to the number of microbubbles passing from the right to the left atrium and the number of hits (high intensity transient signals) detected with pwd in the mca (grade i: \6 microbubbles or hits, grage ii: [6 and \25 and grade iii: more than 25 microbubbles or hits). for each patient included in the study we measured and correlated the presence of pfo with the tidal volume (v t ), the plateau pressure (p plat ), the compliance of the respiratory system (c rs ) and the size of the right (rv) and left (lv) ventricle. results. mean p a o 2 /fio 2 was 198 (min 50, max 350), mean c rs was 39 ml/cmh 2 o (min 11, max 72), mean v t was 574 ml (min 260, max 980) and mean p plat was 22 cmh 2 o (min 14, max 37). the prevalence of pfo detected with tee was 28% and with tcd 48%. there was no pfo detected with tee and missed by tcd. tcd was more sensitive than tee in detecting pfo of grade i (7 with tee, 17 with tcd) and ii (6 with tee, 16 with tcd), while for grade iii the two techniques had equal sensitivity (15 with tee, 15 with tcd). no correlation was found between p plat , c rs , v t and the presence of pfo. on the contrary, a strong correlation was found between rv dilatation and the presence of pfo (p \ 0.001). conclusions. the prevalence of pfo detected by tcd is very high in mechanically ventilated icu patients and this may have important clinical implications. tcd is more sensitive than tee in detecting a small pfo. the presence of rv dilatation increases the prevalence of pfo. objectives. the aim of our study was to identify in mechanically ventilated patients for ali/ards the prevalence of pfo and to evaluate the factors that may influence the prevalence of pfo. methods. two groups of mv patients, one with ali/ards and one without respiratory failure (rf), were enrolled in the study. all patients underwent a tee study for hemodynamic assessment. in each patient three consecutive bubble tests with agitated haemacel ò were performed at 60 0 and 90 0 rotation of the tee probe. the bubble test was performed through a central line in the inferior or superior vena cava (ivc, svc). a pfo was diagnosed by the presence of microbubbles in the left atrium within five cardiac cycles following the injection. furthermore, in ali/ards patients in whom a pfo was not detected at baseline mv, three consecutive bubble tests during recruitment maneuver at 45 cmh 2 o for 20 s were performed. the compliance of the respiratory system (c rs ), blood gas exchange and the ventilatory settings (p plat , v t ) were recorded in both groups. o, respectively. the presence of rv dilatation was a strong predictor for the fo opening (p \ 0.01); on the contrary, no statistical significant difference was found between the site of injection (svc vs. ivc), the c rs , v t , and p plat and the presence or absence of a pfo. a high prevalence of pfo was found in ali/ards patients. rv dilatation seems to be the reason of this high prevalence. rv dilation may be due to the lower c rs and higher p plat of the ards patients. introduction. the clinical evaluation of arterial tone is mainly based on the calculation of total systemic vascular resistance (tsvr). however, given the pulsatile nature of arterial flow, this parameter provides an inadequate assessment of vascular tone. another approach proposed would take account of changes in pulse pressure and blood flow, relationship known as arterial elastance (ea). so, for a given stroke volume, the blood pressure generated in the circulatory system will depend on ea (1). to assess the ability of the dynamic arterial elastance (ea dyn ), defined as the relationship between pulse pressure variation (ppv) and stroke volume variation (vvs), to predict the hemodynamic response in mean arterial pressure (map) to a increase in stroke volume (sv) in hypotensive preload-dependent patients with acute circulatory failure. we performed a prospective clinical study in a 17-bed multidisciplinary intensive care unit, including 21 patients with controlled mechanical ventilation and monitored with the vigileo ò monitor, for whom the decision to give fluids was taken due to the presence of circulatory, including arterial hypotension (map b 65 mmhg or systolic arterial pressure \90 mmhg), and preserved preload-responsiveness condition, defined as svv c10%. dynamic arterial elastance (vpp/vvs ratio), arterial pulse pressure to sv ratio, map/sv ratio, tsvr and map were compared to predict a map increase c15% after volume expansion (map-responders). results. at baseline, only ea dyn was significantly different between map-responders and nonresponders. ve-induced increase in map was strongly correlated with baseline ea dyn (r 2 = 0.82, p \ 0.0001) and changes in ea dyn after ve (r 2 = 0.77; p \ 0.0001). the only predictor of map increase was ea dyn (auc 0.98 ± 0.03; 95% c.i.: 0.8-1). a baseline ea dyn value [0.89 predicted an increase c15% in map after fluid administration with a sensitivity of 92.9% (95% c.i.: 66.1-99.8%) and a specificity of 100% (95% c.i.: 59-100%). conclusions. dynamic assessment of arterial elastance by pvv to svv ratio during controlled mechanical ventilation could be used to predict mean arterial pressure increase after volume loading in hypotensive preload-dependent patients. severe sepsis is one of the major reasons for intensive care unit (icu) admission and leading causes of mortality. some of these score systems have been customized for patients such as apache ii, apache iii, sasp ii and mods. this study is to assess the validity of mortality prediction systems in severe septic patients. objectives. the aim of this study was to compare and evaluate four severity scoring systems in intensive care unit (icu), including apache ii, apache iii, sasp ii and mods in severe septic patient. methods. fifty-six severe septic patients were divided into two groups. one was survival group and the other was non-survival group. besides general data, the continuous surveillance of apache ii, apache iii, sasp ii and mods were recorded by 1st, 3rd and 7th day. results. compared with survival group, mods was significant difference in non-survival group only in 1st day (7.9 ± 3.1 vs. 10.1 ± 2.9, p \ 0.05) but apache ii, apache iii and sasp ii were significant difference through 1st, 3rd and 7th day(p \ 0.05). in seven-day comparison, p value of apache iii in non-survival group was the minimum (p = 0.000) and p value of mods was the maximum (p = 0.008). in optimal survival evaluation, it seemed that apache iii was the best (apache iii [ apache ii = saspii [ mods). conclusions. in order to evaluate the critical condition and prognosis of severe septic patients, apache iii was the best and apache ii and sasp ii were followed and mods was the worst. objectives. to assess compliance with the cem standards for management of severe sepsis across three ed sites in the west midlands. methods. data was collected retrospectively over 3 months. patients presenting to the ed within this period were assessed for likelihood of severe sepsis by the diagnostic code given to each patient upon leaving the ed. data was analysed using a scanned copy of the ed clerking. patients' notes were assessed for sirs criteria and signs of new infection. if these criteria were met, and organ dysfunction was present, they were included in the audit. results. 255 patients with severe sepsis were identified. of these 17% were documented as septic by ed staff. the cem standards of care were received in 41% of patients with a documented diagnosis of severe sepsis in the ed, and 23% of patients overall. 89% of patients received the 'treatment' aspects of care: oxygen, iv antibiotics (with blood culture) and iv fluids. 71% of severely septic patients had no documented consideration of icu referral. conclusions. early recognition of severe sepsis in the ed led to greater performance in meeting the cem standards. although 100% of patients received observations and 89% received the treatment interventions, we performed poorly in meeting the remaining cem standards. the trust has developed a severe sepsis proforma which incorporates the cem standards to accurately record the completion of each intervention. a sepsis course for staff has been launched trust wide, and a formal referral process to icu for all severely septic patients is being implemented. objectives. to observe association of body temperature (bt) and antipyretic use with mortality in the critically-ill. a prospective multi-national, multi-center observational study. consecutive patients whose icu stay were expected to be more than 48 h were recruited from 15 centers in japan and 10 centers in korea. patient's bt was prospectively recorded every 4 h until patient's death, discharge from the icu or up to 28 days. information including patient's clinical characteristics at admission, presence of infection, and use of steroids, extracorporeal circuit, and antipyretics were recorded. (1). while blood culture results take time, treatment for bloodstream infection should be provided swiftly, usually before results are available (2) . prior treatment with antimicrobials increases the chances of false negative results. haste, poor technique and alteration in commensal flora may increase the chances of falsely identifying pathogens. objectives. we have investigated the utility of blood culture tests in our general critical care unit over 1 year in terms of results yielded and actions prompted. methods. the indication for blood culture was clinician's discretion. all critical care sourced blood cultures for the period oct 2008 to sept 2009 were reviewed from the microbiology laboratory database. blood culture specimens were collected in bact/alert ò bottles (biomerieux, durham, nc, usa 27704). notes review was made of the positive blood culture episodes to determine actions after the results were known. consideration was given to the source of the blood sample: clean stab versus from an intravascular device. categoric data was analysed using the chi-squared test and p value of 0.05 was accepted as significant. objectives. we hypothesized that in the emergency department of our hospital many patients with sepsis are not recognized as such. methods. in a retrospective design, patients of an age of 18 years and older who were admitted to the emergency department during a period of 3 months between january-april 2008 and diagnosed as having an infection were included. the diagnose infection was made on admission by the emergency department nurse. the included patients were either classified as having sepsis or not having sepsis, according to the sirs criteria. conclusions. h1n1 infection was associated with significant morbidity and mortality. it occurred mainly in young pts with co-morbidities and was associated with severe hypoxemia, a trigger for prolonged mechanical ventilation and frequent use of lung rescue therapies. a significant delay in hospital admission and start of antiviral therapy should also be noted. admission to administration time difference between cycles was 1.26 h, with a mean reduction of 1.04 h between clinician assessment and prescription time in cycle two. we identified delays against the standard after both cycles of the audit. we demonstrated that the method of prescription should be taken into consideration when prescribing antibiotics in patients with suspected sepsis. there are a multitude of factors that could contribute to a reduction in the clinician assessment to prescription time, which may be investigated in further audits. conclusions. despite high levels of resistance among psa and ab from these icus, cfr for most carbapenem dosing regimens were above the reported susceptibility. doripenem provided greater cfr than meropenem, which was superior to imipenem against these isolates. while higher doses combined with prolonged infusions significantly improved cfr against psa, alternative therapeutic strategies will be required to address these highly resistant ab. grant acknowledgment. the passport study is supported by a grant from janssen-ortho-mcneil. introduction. drug interactions are common, and the effects of these interactions can range from innocuous to deadly. critically ill patients often receive a variety of potent drugs, including antimicrobials, making this population extremely susceptible to drug-drug interactions. therefore, physicians must be familiar not only with the antimicrobial drugs capable of producing adverse drug events, but also their potential drug-drug interactions. there are scarce data about the incidence of these types of drug interactions and the how frequently it might cause adverse events. objectives. the purpose of this study is to evaluate the incidence of potential drug interactions involving antimicrobials and the possibility to cause adverse events. the clinical pharmacist has prospectively analyzed icu prescriptions between january 2009 and december 2009 with the purpose to identify potential drug-drug interactions involving antimicrobials. the screening was done with the relief from a software (epocrates rx ò drug reference). the interactions detected were classified in eight groups according to the affected system (neurological, cardiovascular, gastrointestinal, renal, endocrine, hematological, musculoskeletal and others) and through the type of interaction (pharmacokinetic, pharmacodynamic and others). we have identified the most common potential effects, the medications involved and have observed the incidence of adverse drug events. results. the icu admitted 347 patients during the study period. we have analyzed 661 physician orders with 8209 prescribed items. we have identified 871 antimicrobial drug interactions (71 different interactions) which compound 20% of the total drug interactions (n = 4349). the cardiovascular system and the pharmacokinetic interaction were the most potentially affected (38%; 42%). the most common medications involved were: fluconazole (24%), clarithromycin (23%), levofloxacin (12%); linezolid (11%). the clinical pharmacist has made an intervention regarding medication safety in 3% (n = 27) and the acceptance rate by the medical icu staff was 74%. we have not been able to identify any adverse drug event caused by drug interaction even with our active search and the spontaneous reports. however, sub notification must be taken into consideration. conclusions. clinicians should be aware of potential drug-drug interactions when making therapy selections for critically ill patients. antimicrobial drugs are susceptible to interact with other drugs, which may increase the risk of adverse drug events. the clinical pharmacist interventions may improve clinical outcomes by optimizing medication use, monitoring potentially preventable adverse drug events and promoting information about this important issue to the icu multi-professional team. introduction. cefazolin is one of the most frequently administered antimicrobial agent for prophylaxis in ''clean'' surgery. its broad spectrum against gram + micro-organisms and its pharmacological characteristics make it an easy-to-use choice to prevent infections caused by staphylococcus aureus and coagulase-negative stapylococci. objectives. the aim of this study is the evaluation of the plasma concentrations of cefazolin administered as a prophylactic antimicrobial agent during cardiac surgery with cpb. adequate cefazolin plasma levels can maintain a tissue concentration high enough to prevent the risk of developing post-operative infections. after obtaining ethical committee approval and personal written consent, two groups of patients were enrolled in this prospective study. the first group, 12 patients, received cefazolin, 2 g, 30-60 min before skin incision and 1 g adjunctive dose after 4 h. then, three 2 g doses were administered every 8 h. in the second group of 12 patients the adjunctive 1 g cefazolin dose was given at the beginning of the cpb. blood samples were collected immediately before the first dose and every hour for the whole time of surgery, and, only in the second group, after surgery, at 12th, 18th and 24th hour. plasma cefazolin concentration was determined with a biological radial diffusion assay. results. plasma cefazolin was constantly higher than the mic90 of the most involved micro-organisms (according to clsi). in the first group, cefazolin concentration suddenly decreased after starting cpb. the 1 g adjunctive dose immediately restored it. the earlier administration of this dose in the second group prevented this sudden fall. plasma cefazolin was maintained at effective inhibitory levels for the whole time of surgery in all patients ([32 mcg/ml). during the postoperative period cefazolin decreased slowly, but inhibitory plasma levels were always maintained. the rate of cefazolin clearance was found equal to the creatinine clearance in all patients. perioperative plasma cefazolin concentration conclusions. the administration of cefazolin 2 g every 8 h can guarantee effective inhibitory plasma concentrations during surgery and during the first 24 h after surgery. cpb causes a sudden fall in cefazolin plasma levels. this can be avoided administering an adjunctive 1 g dose immediately before starting cpb. objectives. vancomycin dose regimen was adjusted based on trough plasma levels in burn patients that were distributed according to the extension total burn surface area (tbsa); also pharmacokinetics changes were compared. methods. twenty seven adult burn patients of both sexes, requiring antimicrobial therapy with vancomycin for the control of sepsis were investigated. pharmacotherapeutic follow up was performed in a serial of periods (112 observations) for all patients investigated by collection of blood samples, 1 ml each from the venous catheter as follows: 1st blood sample collection, 2 h after the beginning of drug 1 h infusion and a 2nd sample blood collection at the trough, immediately before the next dose. if necessary, additional sample blood collections were performed based on the laboratorial data for patients any time, for dose adjustment purpose and optimization of drug therapy. vancomycin plasma concentrations were determined by highperformance liquid chromatography. plasma curve decay was plotted, and pharmacokinetics was analyzed by one-compartment open model against the reference data reported. results. burn patients receiving the empiric dose regimen showed trough plasma level lower than the minimum effective concentration, consequently dose adjustment was required. vancomycin adjusted dose regimen showed statistical significance differences according to tbsa (p \ 0.05) as follows for daily dose normalized to body weight and expressed by mean ± sd: 25.5 ± 11.9 mg/kg/day were required for patients with tbsa below 20%, 32.0 ± 6.4 mg/kg/day for tbsa 20-40% and 34.8 ± 12.2 mg/kg/day were required for tbsa above 40%. relevant changes on pharmacokinetics were observed by drug plasma clearance increased according the increase of tbsa (p \ 0.05), while the apparent volume of distribution and also the biological half-life remained unchanged. additionally, a weak correlation was observed between vancomycin plasma clearance and creatinine clearance (r 2 = 0.36; p = 0.0004), probably due to the contribution of the extra-renal clearance on total drug elimination. on the basis of data obtained in the present study and to prevent therapeutic failure and also to reduce the risk of bacterial resistance, dose adjustment in burn patients is recommendable based on vancomycin plasma monitoring and also on the extension of total burn surface area. introduction. the importance of early antibiotic therapy has been recently demonstrated. regarding a rapidly increasing number of obese patients, appropriate drug dosage in these patients is an important challenge of critical care since it has been shown that not only early start of antibiotics but also correct target concentrations decrease mortality. vancomycin is administered according to body weight (bw). nevertheless, little is known about the percentage of obese patients achieving pre-defined target serum levels within 24 h after initiation of vancomycin therapy compared to patients with normal bw. objectives. therefore, it was the aim of our study to analyze the appropriateness of serum vancomycin levels in patients with a bw between 40 and 150 kg. vancomycin is almost entirely excreted by the glomerulus and may be responsible for nephrotoxicity [1] . however, there is a lack of definitive evidence linking concentrations to either outcome or toxicity [2] . few reports exist comparing intermittent dosing and continuous infusion. ingram [3] suggested that whilst associated with a slower deterioration in renal function, there was no difference in the prevalence of nephrotoxicity. similarly, hutschala [4] demonstrated worsening creatinine in 149 patients following cardiac surgery with both intermittent and continuous infusion but infusion tended to be less nephrotoxic despite receiving higher doses. we wish also to report our experiences with vancomycin infusion in critically ill cardiac patients. methods. we examined retrospective data from 2,512 patients treated with vancomycin. we perform adjusted and un-adjusted analysis using sofa on the day of starting vancomycin and total dose received. to assess the differences in either an initial pulmonary or non pulmonary presentation. methods. prospective, observational, multi-center study conducted in intensive care (icu). we reviewed demographic and clinical data for all pandemic h1n1 influenza a infections reported in the esicm h1n1 registry. results. 512 patients were screened from the registry. 330 patients with completed data entry for pulmonary and non pulmonary with outcomes were identified and analysed. all patients had either suspected, probable or confirmed pandemic h1n1 influenza a infection and were being cared for in an icu. 53% of the patients were male with a median age of 43 (iqr 32-55) years. the admission mean saps3 score was 54 ± 16 and the apache ii score was 21 ± 9. 33% of the patients subsequently received non invasive ventilation and 72% received invasive mechanical ventilation. the icu mortality rate was 30%. the hospital mortality was 32%. 77% of patients presented with a pulmonary presentation. 73% of these were admitted with ards and/or bacterial pneumonia and 22% with an acute bronchospastic exacerbation. 23% of patients were admitted to the icu with a non pulmonary presentation. the main reasons for admission in these patients were: cardiovascular instability (33%), altered level of consciousness (20%), renal failure (11%) and acute coronary syndromes (5%). patients with a pulmonary presentation were older, had a increased history of asthma or copd and were more likely to be ventilated. they had a higher mortality rate in the icu. non pulmonary presentations were more likely to suffer from chronic renal impairment. a total of 512 episodes of pandemic influenza a (h1n1)v infections in critical care setting were analyzed: 169 with bacterial pneumonia (94 males and 73 females) and 343 with wheezing or viral pneumonia (182 males and 161 females). the mean age was 45 (± 17) years in patients with bacterial pneumonia and 40 (± 17) in patients viral pneumonia. the mean apache ii score was 25 (± 9) and 20 (± 9), with a corresponding probability of death of 37 (± 25)% and 24 (± 21)%. comorbidities were common, but without significant differences between the two groups (only exceptions pregnancy-more prevalent in patients without bacterial pneumonia-and dialysis dependence-more prevalent in patients with bacterial pneumonia). at icu admission shock and acute renal failure were more common in patients with bacterial pneumonia. in patients without pneumonia; severe hypoxia and ards did not presented significant differences between groups. aims. evaluation if an isocaloric beginning of artificial nutrition in critically ill medical patients is associated with increased nutritional related side effects compared to a hypocaloric start. methods. 110 critically ill medical patients with an expected need for artificial nutrition of [5 days were included into this prospective, randomized clinical study. artificial nutrition was started either isocalorically right from the beginning (group a; n = 55) or hypocalorically (50% of the energy demands) followed by a stepwise increase over the next 2 days (day 2: 75%); day 3: 100%) (group b; n = 55). nutrition related side-effects were defined as the occurrence of hyperglycemia, hyperlactatemia, hypertriacylglycerolemia, upper digestive intolerance, cholestasis, or diarrhea as well as disturbances of serum electrolytes and were assessed on a daily basis. patients were randomized to receive either an artificial nutrition started isocalorically (group a) or hypocalorically followed by a stepwise increase (group b). of the 150 patients, 100 patients completed the study (group a: n = 55; group b: n = 55). the calculated, cumulative energy requirements of patients of group a and b were 9,058 ± 1,236 and 9,125 ± 1,569 kcal, respectively (p = ns). patients of group a received 76 ± 17% and patients of group b 69 ± 12% of the calculated energy requirements (p \ 0.01). the incidence of nutritional related side effects was not different comparing both groups, except for hypophosphatemia, which was more pronounced in group a. additionally, exogenous phosphate needs were higher in patients of group a. the number of interruptions of the artificial nutrition did not differ between groups. conclusions. an isocaloric start of artificial nutrition provided more energy during the first 5 days of their icu stay than a hypocaloric beginning. there was no difference in the number of interruptions and in the incidence of nutritional related side effects, except hypophosphatemia suggesting the presence of refeeding syndrome. in studies carried on to demonstrate positive effects of glutamine (gln) that has innumerable biological features, the main point of discussion isn't whether gln has positive effects in sepsis but rather the effect difference between different administration routes. only enteral (en.) or parenteral (pn.) administration was analyzed in this respect and no studies on combined administration were performed. the primary endpoint in this study was to analyze the effects of administration of en. and pn. gln together or separately on intestinal mucosa + immune system in the experimental sepsis model. for this purpose villus atrophy, bacterial growth in blood and tissue, levels of blood gln, tnfa and il10 were examined. the secondary endpoint was to evaluate the different administration models in terms of cost. wistar, adult female rats were used. they were fed standard. sepsis was developed in 4 groups (all rats) by injection of intraperitoneal(ip.) 1 ml (5 9 10 7 cfu/ml) e. coli. grup c (n = 12):en./pn. isotonic saline (1 ml/day; 2 ml/d); grup e (en., n = 10):en. gln (0.5 g kg -1 day -1 ) + pn. saline (2 ml/d); grup p (pn., n = 11):pn. gln (0.4 g kg -1 day -1 gln) + en. saline (1 ml/d); group ep (en. ± pn., n = 11):pn. gln (0.3 g kg -1 day -1 ) ala-gln = 0.2 g kg -1 day -1 gln) + en. gln (0.3 g kg -1 day -1 ); were administered. feeding of rats began 4 h (h) after administration of ip e. coli. blood gln (with spectrophotometer), tnfa and il10 concentrations(with elisa) were examined at the start (baseline levels) and at 24-96 h after the experiment started. samples of tissue from mesenteric lymph node, liver, lung, blood and small intestine were collected. ala-gln = 0.27 g kg. . rates of reproduction of the strain administered were found lower for group ep than group c (p \ 0.05). rates of villus atrophy in ileum of group ep, p and e were lower than group c (p \ 0.05).plasma gln levels were found lower in groups ep and p at 24 h, and higher at 96 h than other groups (p \ 0.05). when plasma gln levels at 96 h were compared with their baseline levels, significant increases were detected in groups ep and p and significant decreases were detected in groups c and e (p \ 0.01). serum tnfa and il10 levels were found lower for groups ep and p at 24 and 96 h when compared between groups (p \ 0.05). when serum tnfa and il10 levels at 96 h were compared with their baseline levels, more distinctive increases were detected in groups c and e than other groups (p \ 0.01). significant positive correlation was determined between tnfa and il10 levels at 24 h (p \ 0.01) and 96 h (p \ 0.01). cost of simultaneous administration of en. and pn. gln was higher than en. administration but close to pn. administration at these doses. methods. medline and embase were searched. hand citation review of retrieved guidelines and systematic reviews was undertaken and academic and industry experts were contacted. only methodologically sound randomised controlled trials (rcts) were eligible for inclusion in the primary analysis. the primary analysis was conducted on clinically meaningful patient oriented outcomes, which included mortality, functional status and quality of life. secondary analyses considered vomiting/regurgitation, pneumonia, bacteremia, sepsis and multiple organ dysfunction syndrome. meta-analysis was conducted using the peto analytic method, which is known to minimize bias in the presence of sparse events. the impact of heterogeneity was assessed using the i 2 metric. results. 4,217 unique abstracts were identified, resulting in the retrieval of 701 papers for detailed eligibility review. four rcts were identified to be on topic however one rct reported excessive loss to follow-up such that an intention to treat analysis could not be conducted. analysis based on the three methodologically sound rcts demonstrated the provision of early en was associated with a significant reduction in mortality (or = 0.20, 95% confidence interval 0.04 to 0.91, i 2 = 0). no other outcomes could be pooled. sensitivity analysis including all four on-topic rcts (or = 0.26, p = 0.04, i 2 = 0), and a simulation analysis conducted using a different analytical method. (or exact = 0.14, 95% ci 0.01 to 0.97), confirmed the presence of a mortality reduction. conclusions. although the detection of a statistically significant reduction in mortality is promising, overall trial size was small. the results of this meta-analysis should be confirmed by the conduct of a large multi-center trial. reference(s). results. the mean ibp was 10.7 ± 4.1 and mean igp was 11.6 ± 4.1. correlation between the ibp and igp was significant however moderate (r2 = 0.51). analysis according to bland and altman showed a bias and precision of 0.8 and 2.7 mmhg respectively, however the limits of agreement (la) were large and ranged from -4.5 to 6.1 mmhg. the median grv proto was 80 ml (0-1,050) and median grv classic was also 80 ml (0-1,250). correlation between the 2 methods was excellent (r2 = 0.89). analysis according to bland and altman showed a bias and precision of -0.8 and 52.3 ml respectively and the limits of agreement (la) ranged from -103 to 102 mmhg. the median drainage time and return times were 5 min (0.5-15) and 2.5 min (0-21) for grv proto compared to 2 min (0.1-10) and 1 min (0-8) for grv classic. a preliminary cost effectiveness analysis shows that the price of measuring grv with the classic method ranges from 3.84€ to 24.18€ per day, depending on the grv size. price of measuring grv with the gastro pv system is independent of grv size and is estimated at 9.49€ per day. the gastro pv system if priced at 8.5€ could become cost effective at grv of 100 cc and more. conclusions. the interim results of an ongoing multicentre pilot study show that the gastro pv is a good alternative to the standard method for measuring grv. because the nurse can perform other tasks during drainage and return of the grv, and the fact that the system remains closed during measurement, this could be a major step forward in standardisation of grv measurement. furthermore it allows screening for intra-abdominal hypertension via igp estimation. acknowledgment. the gastro pv devices were provided by holtech medical, free of charge. introduction. the importance of early enteral feeding of the critically ill patient has been well documented. it is the more physiological approach, which is associated with lower rates of infectious complications. early enteral nutrition within 24 h is recommended by the espen guidelines on enteral nutrition. a recent meta-analysis revealed that mortality and the incidence of pneumonia were significantly reduced in patients with enteral nutrition within 24 h. parenteral nutrition may be associated with higher mortality. objectives. evaluation of a new technique for the placement of postpyloric feeding tubes by intensive care physicians. methods. prospective cohort study in 27 critically ill patients subjected to transnasal endoscopy and intubation of the pylorus. attending intensive care physicians were trained in the handling of the new endoscope for transnasal gastroenteroscopy for 2 days. a jejunal feeding tube was advanced via the instrument channel and the correct position assessed by contrast radiography. primary outcome measure was successful postpyloric placement of the tube. secondary outcome measures were time needed for the placement, complications like bleeding and formation of loops and the score of the placement difficulty graded from 1 (easy) to 4 (difficult). data are given as mean values and standard deviation. out of 34 attempted jejunal tube placements, 28 tubes (82%) were placed correctly in the jejunum. the duration of the procedure was 28 ± 12 min. the difficulty of the tube placement was judged as follows: grade 1: 17 patients, grade 2: 8 patients, grade 3: 7 patients, grade 4: 2 patients. in 3 cases, the tube position was incorrect, and in another 3 cases, the procedure had to be aborted. only in one patient, bleeding occurred that required no further treatment. conclusions. fast and reliable transnasal insertion of postpyloric feeding tubes can be accomplished by trained intensive care physicians at the bedside using the presented procedure. this new technique may facilitate early initiation of enteral feeding in intensive care patients. grant acknowledgment. the authors acknowledge the support of pentax, hamburg, germany, who provided the endoscope used in the study and of fresenius kabi, bad homburg, germany who provided the feeding tubes. a well-nourished condition before prolonged endotoxemia results in a better ability to adapt to endotoxin-induced metabolic deterioration of arginine-nitric oxide metabolism than does reduced caloric intake before endotoxemia (1). the role of individual organs in the arginine-citrulline metabolism during malnutrition and sepsis is unknown and may be key to direct future interventions. to study the effects of reduced caloric intake and endotoxemia on the citrulline-arginine metabolism in the gut-liver-kidney axis. organ arginine-nitric oxide metabolism was measured by using a primedconstant stable-isotope infusion of [15n2]arginine and [13c-2h2]citrulline during 4 conditions; a 7-day reduced caloric intake feed regimen (starv; n = 9), normal control feed regimen (co; n = 9), endotoxemia alone (ce) and reduced caloric intake and endotoxemia (re) in. catheters for blood sampling were placed in the abdominal aorta, which, in combination with the catheters in the portal, hepatic and renal veins, served for metabolic measurements across the portal-drained viscera, liver and the kidneys, respectively. results. interestingly, re animals had similar citrulline appearance from the gut (331 ± 36 nmol/kg/min) compared to control and animals during ce, but higher in endotoxemia alone (102 ± 14, p \ 0.01). this was related to a significantly higher no production from the gut in the re group (1,644 ± 153 vs. 720 ± 41, p \ 0.01). in the kidney arginine appearance from citrulline decreased significantly during re compared the control animals (3 ± 7 vs. 224 ± 30 nmol/kg/min, p \ 0.01). in contrast, the liver disposed more arginine in the re group compared to the other conditions, while no production was not higher. conclusions. despite reduced caloric intake prior to endotoxemia, the gut remains capable of increasing release of citrulline, although the capability of the kidney for the de novo production of arginine is severely compromised. metabolic control of the citrullinearginine metabolism in the gut-liver-kidney axis should focus on increasing de novo arginine production from citrulline. objectives. the aim of this study was to measure duodeno-caecal transit times of enteral feed in this patient group using a scintigraphic technique. a prospective observational study was performed in 16 mechanically ventilated critically ill patients (12 m, age 49 ± 17 yr, bmi 25 ± 5 kg/m 2 , icu admission day 5 ± 3, apache ii on study 20 ± 7; mean ± sd) and 6 healthy subjects (3 m, age 24 ± 9 year, bmi 24 ± 45 kg/m 2 ). after a 6 h fast a 100 ml enteral feed (ensure 1 kcal/min), labelled with 20 mbq 99m tc-sulphur colloid, was infused into the distal duodenum over 6 min. dynamic anterior scintigraphic images were recorded in 3 min frames for 240 min and the time of first appearance of activity in the caecum was recorded by two blinded operators (kj, ar). data were assessed using mann whitney u test and are presented as median (iqr). introduction. erythromycin, a macrolide antibiotic is widely used as a prokinetic agent in intensive care unit (icu) despite the lack of data supporting its prolonged effectiveness in enteral nutrition (en) intolerant critically ill patients. to evaluate impact on clinical outcome of erythromycin prescription as prokinetic agent in icu. all patients consecutively admitted from january 2008 through december 2009 mechanically ventilated for more than 5 days and receiving en were included in an observational cohort study. en intolerance was defined clinically as a 8-hourly gastric residual volume (grv) c150 ml or vomiting. successful en was defined as a grv\150 ml with a feeding rate c40 ml/h. erythromycin prescription was left to practician appreciation. objectives. this study aims at evaluating the relationship between diarrhoea and en in icu patients. methods. during 1 month, the days with and without diarrhoea (c 3 liquid stools/day) and the characteristics of nutritional support of all patients staying in our icu were recorded. patients staying \24 h or presenting an intestinal stomy were excluded. we compared, between days with and without diarrhoea, total energy coverage and energy coverage by en as % of needs, en energy intake and en volume for each patient. needs were estimated as 25-30 kcal/kg body weight for women and men, respectively. the relationship between antibiotics, laxative treatment and diarrhoea was also analysed. results are presented as mean ± sd. comparisons were made by mann-whitney test. the risk of diarrhoea with en was calculated by odds ratio and confidence intervals (ci). the study included 1133 days of hospitalisation of 171 patients (60 ± 16 years, bmi 26 ± 5 kg/m 2 , sofa score at admission 6 ± 6). en was present in 85 days of diarrhoea and 581 days without diarrhoea. determining the small bowel function is of great concern in icu patients, because a malfunctioning small bowel may predispose to malnutrition and may increase the risk of sirs. a recently developed test, the citrulline generation test (cgt), measures the enterocytes' capability to convert glutamine into citrulline. the production of citrulline exclusively takes place in functioning enterocytes, therefore this conversion represents small bowel function. objectives. we aimed to define the cgt reference values in 16 'stable' icu-patients to assess small bowel function. secondly, we wanted to compare four different cgt methods; enteral and iv administration of dipeptiven and measurement of citrulline in both arterial and venous samples. we performed the cgt on 16 stable icu-patients, defined as having respiratory failure but not dependent on vasopressors. they had a normal renal function and were able to tolerate enteral nutrition. a 5 h fast was followed by administration of 20 g of glutamine-alanine (dipeptiven ò ) either intravenously or enterally, randomly determined. the next day the same test was performed by using the other route. after each administration of dipeptiven, citrulline levels, both arterial and venous, were measured at fixed time points using reverse-phase high performance liquid chromatography (hplc). results. nine females and 7 males were admitted to the icu with either a medical (11) or a surgical (5) diagnosis. they had a mean (± sd) age and bmi of 60.9 ± 10 years and 26.7 ± 7, kg/m 2 respectively. their median apache ii score was 24.5 (iqr = 19.5-26.8). on the day the cgt was performed their median sofa score was 4.0 (iqr = 3.0-4.8 early post-pyloric feeding has been shown to improve clinical outcomes [1] . commonly used methods for placing a nasojejunal tube (njt) are blind, endoscopic or fluoroscopic placement. the later two methods are relatively invasive, expensive and can cause delay to feeding, whereas blind placement is often unsuccessful. electromagnetic sensor guided njt insertion is a bedside technique able to confirm successful placement without the need for abdominal x-ray. the system incorporates a liquid crystal display and a receiver unit. the receiver is placed over the patient's xiphoid process and picks up the signal from an electromagnetic transmitter located at the tip of the feeding tube. the screen provides a visual aid to enable the operators to trace the route of the tube tip and identify its' location according to anatomical markers. objectives. we were interested to determine the suitability of electromagnetic sensor guided njt insertion especially in relation to success rate and procedure time. methods. fifty patients were referred for electromagnetic njt insertion on units at the leeds teaching hospitals. insertion time was measured from oesophageal visualisation until post-pyloric placement. various positional manoeuvres were employed along with administration of sedatives, prokinetics and air insufflation when applicable. all insertions were carried out by experienced investigators. all njt insertions were confirmed by abdominal x-ray. data collection included patient demographics, hospitalisation and procedural information. results. forty male and 10 female patients, mean age 40 (range 1-81 years), bmi mean 25 (14-33), had attempted electromagnetic njt placement. patients had been hospitalised for a median of 12 days (1-180). indication for njt insertion was either large aspirate and/or reflux (86%). seventy six percent of patients had an artificial airway and 50% of patients were receiving sedation. forty six percent of patients received metoclopramide and 58% air insufflation. thirty six percent of patients were moved into either left or right lateral position. successful post-pyloric placement was achieved in 90% of patients confirmed by additional abdominal x-ray. procedural time varied from 2 to 180 min (mean 23). two of the placement failures were due to patient intolerance. conclusions. bedside electromagnetic guided njt placement technique is an acceptable method of placing post pyloric feeding tubes with a high success rate. gastrointestinal failure (gif) score has been suggested (1). the gif score defines gi failure as the occurrence of feeding intolerance (fi) and intra-abdominal hypertension (iah) simultaneously. to compare the outcome of patients with primary vs. secondary gif. methods. all consecutive, mechanically ventilated (mv) patients treated for at least 24 h during january 2008 to december 2009 in two icus were studied. gif was defined as gif score equal or above 3 points according to the gif score (1). 3 points = fi and iah simultaneously; 4 points = abdominal compartment syndrome (acs). fi was defined as the need to stop enteral feeding for any clinical reason (vomiting, high gastric residuals, bowel distension etc). iah was defined as mean intra-abdominal pressure (iap) c12 mmhg on any day. acs was defined as iap [ 20 mmhg with the new onset organ failure. when gif developed in a patient with primary pathology in abdomino-pelvic region it was classified as primary gif, when occurred without previous pathology in abdomino-pelvic region it was taken as secondary. objectives. in this study the biochemical quality and prion safety of the pharmaceutically licensed plasma octaplaslg ò was evaluated. the prion reduction factor achieved by western blot was confirmed by animal studies. eighteen consecutive batches of octaplaslg ò (octapharma ppgmbh, vienna, austria) were tested on global coagulation parameters, fibrinogen levels, activities of coagulation factors and protease inhibitors, activation markers, as well as von willebrand factor multimers. in parallel studies, plasma pool was spiked with exogenous spike material, derived from brains of hamsters infected with hamster-adapted scrapie 263 k, and a down-scale of the octaplaslg ò manufacturing process was performed. the prp sc reduction factor for the resin was investigated in both western blot and hamster bioassay studies. a reduction factor of c3.1 log10 prp sc was found for this process step by western blotting. the outcome of the hamster bioassay confirmed that the high level of removal prp sc seen during octaplaslg ò manufacturing was equivalent to a removal of infectivity (3.0 log10). in octa-plaslg ò , a parallel reduction of the s/d virus inactivation step led to significantly higher activities of plasmin inhibitor. our studies demonstrated that the same amounts of prp sc and prion infectivity bind rapidly and with a very high affinity to the chromatography resin. octaplaslg ò has the same clinical safety and efficacy profile compared to that demonstrated by octaplas ò over the last 18 years, except for the increased safety margin in terms of prion disease transmission and the possible effect of a significantly increased plasmin inhibitor activity. uniplas ò is a second generation solvent/detergent (s/d) treated, coagulation active plasma for infusion produced with an implemented prion removal step. it was developed as an alternative to the blood group specific s/d plasma products, octaplaslg ò and octaplas ò , in order to obtain an universally applicable (i.e. blood group independent) plasma that can be used without taking into account the blood group of the recipient. due to an initially controlled, optimal mixing of plasma of different blood groups prior to s/d treatment, in uniplas ò , the blood group specific antibodies (anti-a and anti-b of both igm and igg type) are neutralised and/or removed by free a and/or b substances and red blood cells (rbcs) to a clinical acceptable level with very limited or no complement activation. objectives. in this study an extensive biochemical characterisation of the first uniplas ò validation batches was performed. methods. three batches of uniplas ò were produced by octapharmappgmbh (vienna) under production conditions in [2008] [2009] . uniplas ò batches were tested on all important coagulation factors, protease inhibitors, activation markers, adamts13 and factor h levels, as well as von willebrand factor multimers. in addition, anti-a and anti-b titres of igm-and igg-type were investigated. finally, complement activation products, as well as key components of the complement system, were measured. results. in uniplas ò batches, all coagulation factor activities were higher than 0.7 iu/ml and all protease inhibitor activities, including protein s and plasmin inhibitor, were higher than 0.5 iu/ml. uniplas ò contained standardised levels of adamts13 and factor h, within the normal ranges for single-donor freshfrozen plasma. there was no activation of fvii obtained during manufacturing, thrombin-antithrombin (tat)-complex, prothrombin fragments (f1 + 2) and d-dimer levels were within the normal ranges. anti-a and anti-b titres were within the uniplas ò specification, i.e. anti-a igm and anti-b igm\1:8 as well as anti-a igg and anti-b igg \1:32, respectively. uniplas ò did not contain an increased amount of immune complexes and the manufacturing of uniplas ò associated with more complement activation than the one seen for octaplaslg ò . conclusions. the present study confirmed that uniplas ò displays the same high quality and clinical efficacy as the s/d treated blood group specific plasma octaplaslg ò , but with the additional advantage in being a blood group independent universally applicable plasma. most pts received more fluids than calculated by parkland formula (7 ± 7.5 ml/kg 9 %tbsa). interestingly, nonsurvivors received less (3.9 ± 4.1 vs. 8.3 ± 8.2 ml/kg 9 %tbsa). gastric decompression, ascites drainage and the implementation of a stool protocol with rectal enemas (18 interventions in 12 pts) was able to remove 2.2 ± 1.3 l of body fluids and this was related to a significant decrease in iap and cvp and an improvement in oxygenation and urine output ( conclusions. pris is a difficult condition to diagnose and routine monitoring of the adverse effects of high-dose propofol remains sub-optimal. hypothermia has been reported to alter propofol pharmacokinetics 3 and we propose that active cooling may increase the risk of developing pris. this may be particularly relevant in patients with tbi who are on high doses of propofol to control icp in addition to concomitantly administered catecholamines to maintain cerebral perfusion pressure. we recommend that further research is required in this area in view of the increasing use of induced hypothermia in icu. objectives. to compare differences in fluid resuscitation based on direct or indirect admissions to the london burns unit. methods. 67 admissions to the burns unit with [20% burned surface area (%bsa) were identified over 5 years. 16 were excluded from analysis due to palliation or death within the first 8 h. 12 sets of notes were randomly selected for analysis of fluid balance in the first 24 h period of fluid resuscitation after the burn injury. results. mean (sd) time from burn injury to arrival at the burns unit was lower for patients transferred direct to the burns hospital rather than via another hospital (117.9 ± 75.27 vs. 372.5 ± 255.7 min p = 0.032). mean (sd) error in burn size estimation was lower for patients initially treated by burns specialists versus non-burns specialists (4 ± 3.6 vs. 10.6 ± 4.3%, p = 0.016). all patients were resuscitated according to the parkland formula calculated at one of 2, 3 or 4 ml/kg/%bsa. the mean (sd) actual fluid volume differed from the target by 30.06% (±40.43%); the lower the calculated fluid target, the greater the error between actual and planned resuscitation volumes; there was no difference in accuracy of fluid resuscitation at 24 h between patients initially managed by burns specialists versus non-burns specialists (44.14 ± 49.04 vs. 10.34 ± 7.70% respectively, p = 0.163). conclusions. burned patients transferred directly to specialist burns care receive a faster and more accurate assessment of their burn injury. despite this, we found no difference in fluid targeting errors at 24 h, though this may reflect corrective fluid management on arrival at the specialist centre. echocardiography is an useful and minimally invasive tool that allows to know the heart filling pressures, also it has proven highly accurate in predicting the response to volume in critically ill patients. we try to determinate the response to fluid infusion by static variables as cvp or lap, comparing with the variation of ivc. methods. an observational prospective study with 18 patients undergoing coronary cardiac surgery (7 patients were excluded by a no presenting a good echo views), in the postoperative period under mechanical ventilation (vt 8 ml/kg, fio2 50%, peep 3). we performed an echocardiography if the patient presented hypotension, just before the habitual fluid load protocol were started (500 ml hes 6% in 30-45 min). we collected data before and after the infusion, and determine the responsiveness to volume if the cardiac output increased more than 10%. data in the report included invasive cvp and lap, and echo measures, ratio e/e', diameter and variations of inferior vena cava (ivc) and variations of stroke volume by echocardiography (ølvot x vti lvot) and with vigileoò system. . the correlation between low values of cvp/lap and volume response was poor, the relationship between cvp below 6 mmhg with increased cardiac output had a correlation (pearson correlation -0.06) with a significance (2-tailed) 0.981, and the relationship between lap \4 mmhg and an increase in cardiac output had a correlation (pearson correlation 0.229) with a significance of (2-tailed) 0.362. the measurement of the variation of the inferior vena cava, led us to calculated a cutoff point more sensitive to determine which patients were responders to volume. through the roc curves (sensitivity/specificity), with the area under the curve of 82.5% (se = 11.2%) and with a confidence interval of 95% (p significance of 0.002), resulted in a 12% variations of ivc with a sensibility of 100% and specificity of 42% (younden's index of 42.9%). the same calculation, based on kraemer's quality indices (qi) gave us a 13% of variation in ivc, with a w = 0.2 specificity rather than sensitivity (qi 0.58), and with a w = 0.8 sensitivity rather than specificity (qi 0.61) objectives. to ascertain whether postoperative hypothermia is linked to high or low risk surgical patients. we conducted a prospective systematic analysis looking at the incidence of postoperative hypothermia in adults who underwent general anaesthesia. children age \18, pregnant women and patients undergoing regional anaesthesia were excluded from the survey. to identify the current level of doctors' knowledge on perioperative fluid management. methods. the survey was conducted at george eliot hospital, nuneaton, uk in may 2009. questionnaires consisting of ten multiple-choice questions on basic sciences and clinical scenarios were devised by a consultant anaesthetist. these were personally distributed to doctors of all grades working in anaesthetics and the surgical specialties. doctors were asked to complete the questionnaire within 5 min. of the 66 questionnaires distributed, 52 were completed. results. the mean questionnaire score varied between specialties from 87% in the anaesthetics department to 64% for doctors in surgical specialties. the mean score of registrars and fy1 doctors in surgical specialties was found to be 72 and 65% respectively. the overall mean score was 74%. of all doctors surveyed, the daily maintenance water requirement was known by only 52%, 62% knew the daily maintenance sodium requirement and 75% knew that of potassium. the electrolyte contents of 0.9% sodium chloride and hartmann's solution was answered correctly by 65% and 63% respectively. there is a significant deficiency in doctors' knowledge on perioperative fluid management. more emphasis on optimal perioperative fluid management is required in undergraduate and postgraduate training. increased awareness of the british consensus guidelines on intravenous fluid therapy for adult surgical patients would aid training. based on this survey, a regional online survey of junior doctors is planned to further identify gaps in perioperative fluid management training. optimal fluid management could also help to reduce prolonged hospital stay which can result from fluid-related complications. objectives. to evaluate dynamic echocardiographic parameters as predictors of volume responsiveness in surgical patients. methods. 25 patients were included in the study after laparotomy surgery performed on the same day (4 breathing spontaneously and 21 mechanically ventilated in volume controlled mode with tidal volume of 10 ml/kg). a fluid challenge was performed in spontaneously breathing patients by passive leg raising and infusing saline (7 ml/kg). echocardiographic analysis of respiratory changes of inferior vena cava diameter (ddivc) and aortic blood flow (dabf) was performed in all patients. a threshold of 18% for ddivc was used for classifying patients as volume responders or non-responders. age, sex, gender, bmi, cvp, iap, map, left ventricular ejection fraction, left ventricular systolic and diastolic area, and stroke volume in all patients, as well as itbvi, ci, ppv and svv in 16 patients were measured. a positive correlation with ddivc was established for itbvi (r = 0.49, p = 0.05), iap (r = 0.47, p = 0.01) and ef (r = 0.38, p = 0.05). a positive correlation with dabf was not established for any variable measured. 16 patients (64%) were classified as volume responders and 9 (36%) as non-responders. responders had overall higher iap than non-responders (13.45 ± 4.99 mmhg vs. 9.82 ± 3.33 mmhg respectively, p = 0.04). respiratory changes of ivc diameter showed positive correlation with itbvi. so, conclusions about itbvi could be indirectly made from ddivc values in patients who are not being invasively monitored. ppv and svv did not show positive correlation with itbvi. surprisingly, we confirmed a positive correlation between ddivc and iap. we detected 9 patients with high iap, while all the volume responders had overall higher iap. although further investigations are needed to establish how longer duration of high iap may influence ddivc, it seems that ddivc is a good parameter of volume responsiveness during first 24 h after laparotomy surgery. unlike from other studies, we could not establish a positive correlation between dabf and any variable measured. these studies were performed in hypovolemic septic patients, so this could be the reason for such different results. more studies are needed in a larger set of patients undergoing laparotomy surgery to evaluate dabf. introduction. fluid optimization after major cardiac surgery was shown to improve patients postoperative outcome significantly. several hemodynamic parameters were proposed for the guidance of therapy but never compared in a head to head trial. objectives. in this prospective randomized trial patients scheduled for elective cardiac surgery underwent early goal directed fluid therapy guided either by stroke volume variation (svv) or by oxygen delivery index (do2i). we hypothesized that while svv is easier to obtain it will not be inferior to do2i in outcome parameters. methods. following ethics committee approval and signing of a written informed consent, 84 patients were randomized in two groups to undergo either fluid optimization guided by do2i or svv in the first 8 postoperative hours in the icu following elective cardiac surgery (cabg). following a standardized egt protocol the parameters were collected by using hemodynamic monitoring based on a pulse contour analysis and a transpulmonary lithium dilution (lidco plus, lidco,uk). we compared amount and type of volume infused, need and amount of inotropic or vasopressor substances, time spent on ventilator, los in the icu and postoperative complications. statistics were evaluated by using a t test for unpaired samples. table 1 . compared to the do2i group fluid optimization using svv showed reduced ventilator times (p = 0.043) and less complications (p = 0.004) in the first 80 days after surgery. no differences between the groups were detected concerning the type and amount of volume infused, need for inotropes or vasopressors or the los in hospital conclusions. while svv is less invasive, cheaper and easier to be obtained than do2 outcome was at least not inferior and even showed improvements in postoperative cardiac surgery patients. 23rd esicm annual congress -barcelona, spain -9-13 october 2010 s377 introduction. over the years, there have been concerns over incompatibility of transfused blood with various intravenous fluids during blood transfusion, especially related to increased levels of haemolysis. it is often impractical, particularly in an emergency situation, to flush through a giving set with a so-called ''safe'' fluid prior to and after delivering blood. we wanted to investigate whether this is actually necessary and whether the usual fluids used in the perioperative period really do cause any demonstrable alteration in the composition of transfused blood. objectives. the purpose of this study was to expose packed red cells to a variety of different intravenous fluids commonly used during the perioperative period and to measure a number of parameters in the blood following their contact with each different fluid, including a blood film to examine for clumping of cells or haemolysis. a unit of a positive blood was passed through blood giving sets which were primed with various intravenous fluids. after adequate mixing of blood with fluids, samples were collected for full blood count, urea and electrolytes and blood films. one millilitre of mixed blood was taken in each bottle at a time. the intravenous fluids used in this study were normal saline, hartmann's solution, 5% dextrose, 10% dextrose, starch and gelatin. there was no significant rise in blood parameters suggestive of haemolysis. the potassium and ldh levels were not significantly different with various fluids. the haemoglobin and haematocrit levels were also comparable to one another. there was no demonstrable changes in blood parameters suggestive of haemolysis, nor were there any change in electrolyte values. this suggests that all of the fluids investigated during this study would be suitable to be used via the same giving set before and after the transfusion of pack red cells. objectives. to assess the compliance with the national guidelines in avoiding inadvertent peri-operative hypothermia in an acute district general hospital in england. we prospectively studied our local practice on maintaining normothermia in 165 consecutive adult surgical patients {61 men, mean age 57.4 years, 112 patients with asa grade 2 (67.7%), 60 emergency surgical patients (36.4%), 49 patients with significant cardiac disease 29.7%}. we used a questionnaire that was filled pre-operatively by anesthetic nurses, intra-operatively by anesthesiologists, and post-operatively by recovery nurses. patients were recruited from the following surgical subspecialties: general surgery (34%), gynecology (24%), trauma (22%), breast surgery (9%) and orthopedics (7%). day surgery patients were excluded. peri-operative hypothermia was defined as temperature \36°c as per the nice guidelines. results. less than half of our patients (47.9%, n = 79) had their temperature measured preoperatively, on whom incidence of hypothermia was 25.3% (n = 20). only one of these patients was warmed prior to induction. patients requiring emergency surgery and those with asa grade 2 had increased incidence of preoperative hypothermia (30.8% and 28.6% respectively, p \ 0.05). based on nice guidelines, 164 patients needed intraoperative forced air warming but only 64 (38.8%) patients received it. intraoperative temperature measurement was made on 111 patients, of whom 28.8% (n = 32) were hypothermic. incidence of intraoperative hypothermia was high in surgical procedures lasting longer than 30 min (p \ 0.05) but was not affected by the use of regional anesthetic techniques. 161 patients had their temperature measured on arrival to recovery of whom 46 (26.6%) were hypothermic. 38 patients (23.6%) had their temperature measured every 15 min (nice recommendation) and the mean time interval for temperature measurement in recovery was 26 min. 10 patients were still hypothermic on leaving recovery. conclusions. majority of our surgical patients did not receive adequate perioperative care on maintaining normothermia. consequently, the incidence of hypothermia was significant pre-, intra-and post-operatively. we are currently analyzing the data to investigate the effect of hypothermia on duration of recovery stay, length of hospitalization and mortality in our patients. we completed a double-blind randomized trial in patients undergoing cardiac surgery in which we compared fluid resuscitation with a hydroxyethyl starch (hes, 10% 250 mw pentastarch) and saline. use of hes resulted in markedly less use of catecholamines the morning after surgery. an underlying design principle was that assessment of cardiac index (ci) is essential for a proper fluid protocol. in this analysis we examine that supposition. all subjects had pulmonary artery catheters. patients were consented preoperatively, but randomized post operatively to receive up to 4 blinded 250 ml boluses for predefined hemodynamic targets; ci \2.2 l/min/m 2 , blood pressure (bp) set by admitting team, cvp \3 mmhg, or urine output \20 ml/h. hemodynamic measurements were made before and after each bolus. after the 4 study boluses, only saline was used. results. 237 patients received fluids, 119 hes and 118 saline. there were 727 study boluses, 348 hes and 379 saline. of these, 99 boluses (14%) could not be assessed for this hemodynamic analysis (but were still used for the primary outcome) because of protocol violation or missing data. of the rest, 235 (37%) of boluses were given for a low ci; in 33 bp and 11 cvp were also low so that ci was the only trigger in 30%. a low bp was a trigger in 290 (46%). low cvp was the trigger in 95 (15%). only 50 hes and 65 saline patients required the maximum allowed 4 blinded boluses. at the 4th bolus, low ci was the trigger for 8 (16%) of hes but 20 (31%) of saline patients. there were 511 that could be evaluated for hemodynamic response based on four possible outcomes of cvp and ci. objectives. the aim of our study was to evaluate the predictive value of cvp with regard to gedi, and to correlate these parameters to cardiac index (ci). conclusions. volume depletion according to gedi was found in more than half the patients. the predictive values of cvp with regard to volume depletion were low gedi and its changes significantly correlated to ci and its changes, which was not observed for cvp. therefore, gedi appears to be more appropriate for volume management during mayor liver resections. introduction. regional anticoagulation with citrate is an effective and established anticoagulation strategy during crrt in critically ill patients, especially in surgical patients with a high risk of bleeding and in case of a heparin-induced thrombocytopenia (1). however, citrate crrt could be associated with major metabolic derangements such as metabolic alkalosis, hypocalcemia, hypernatremia and citrate toxicity. objectives. the aim of our study was to investigate efficacy, safety and metabolic stability during citrate crrt in critically ill patients with acute kidney injury. methods. the retrospective study was performed in a mixed surgical and trauma icu in a university hospital. patient charts were reviewed for demographic data, the period and dosage of citrate crrt and metabolic parameters. reasons of admission, comorbidities and severity of illness were also evaluated. citrate crrt was performed using commercially available equipment and fluid solutions (multifiltrate ò with integrated ci-ca ò -system; fresenius medical care; germany). to maintain stable metabolic and haemodynamic conditions we used an internal standard protocol for citrate crrt. statistical analysis was performed using descriptive methods (mean, median and standard deviation) and a mann-whitney u test where appropriate. p \ 0.05 was regarded as statistically significant. conclusions. although minor metabolic imbalances were observed, none led to a termination of citrate crrt and all of them could be managed by adjustments of blood flow and dialysate rates according to a preset protocol. our findings suggest citrate crrt to be a safe and effective strategy for crrt even in patients with hepatic dysfunction. nevertheless, metabolic parameters need to be monitored regularly to avoid severe metabolic derangements. introduction. the liver is central to ammonia metabolism, being the main site of urea cycle enzyme pathways. in acute liver failure (alf) and decompensated chronic liver disease (cld) ammonia dysmetabolism results in hyperammonaemia, thought to be of central importance in the pathogenesis of hepatic encephalopathy and, in alf cerebral oedema [1] . continuous renal replacement therapy (crrt), commonly used in critically ill patients may be an effective method of clearing ammonia. little is known of the efficacy such techniques have on ammonia clearance. objectives. to quantify the clearance of ammonia using an aquarius haemofilter (ahf) using different renal replacement doses and techniques. methods. patients with a circulating ammonia level[100 lmol/l due to commence crrt were enrolled. the ahf was programmed to run in either pre-or post-dilution modes at a blood flow rate of 200 ms/min using a 1.2 or 1.9 m 2 filter depending on the crrt ultrafiltration (uf) dose, which included 35, 60 or 90 ml/kg/h (adjusted for ideal body weight). 2 ml of blood and effluent fluid were collected, on ice into lithium/heparin and serum separation tubes, from pre and post filter access points and effluent tubing to calculate urea and ammonia clearance using the cordoba formula [2] . delta whole body ammonia clearance was determined by measuring arterial ammonia at 0 and 60 min. ammonia measurements were performed using a pocketchem ò blood ammonia bedside testing machine. results. 20 patients (9 alf and 11 cld) were recruited (mean age 45 years, sd (14), with mean arterial ammonia 123 lmol/l, sd (49). 60 min whole-body ammonia clearance was -12 lmol/l, p = 0.016, paired t test). ammonia and urea clearance were correlated (r = 0.748, p = 0.020); uf rate correlated negatively with filtrate ammonia (r = -0.496, p = 0.007) and positively with ammonia clearance (r = 0.515, p = 0.005). filter ammonia clearance was not dependent on filter size for the standard blood flow rate. pre or post dilution modes did not affect ammonia clearance (p = 0.100, student's t test). a constant filter size and blood flow rate achieved ammonia clearance of 39 ml/min/m 2 for 35 ml/kg/h, 60 ml/min/ m 2 for 60 ml/kg/h and 52 ml/min/m 2 for 90 ml/kg/h (p = 0.008, one way anova). conclusions. 60 ml/kg/h based on ideal body weight appears to be the optimum dose of crrt for ammonia clearance when using a blood flow rate 200 ml/min and a 1.2 m 2 filter. filter and delta whole body ammonia clearance may be increased further using the combination of a higher dose (90 ml/kg/h) with a larger filter size and higher blood flow rates. introduction. malnutrition is common in intensive care following the catabolic state induced by critical illness. patients who progress from enteral nutrition back to oral feeding are usually in an energy deficit. espen guidelines recommend increasing calorie delivery during the recovery period to cover this anabolic phase. oral nutritional supplements (ons) are widely used to facilitate calorie delivery within the hospital setting however the effectiveness of this strategy is dependent on patient compliance with the products. compliance among the elderly ward-based population has been considered (1) however that of intensive care patients has not been reported. to evaluate compliance to ons in a mixed medical and surgical adult intensive care unit (icu) in a district general hospital. prospective observational study was conducted over a 2 month period with data compiled from fluid chart analysis and discussions with nursing staff. all adult icu patients prescribed, or offered without prescription, an ons were included until the point they were discharged to the ward. the supplements studied, resource ò energy, 2.0 fibre, fruit and dessert (nestlé nutrition), were selected based on their availability within the trust. patients were offered a choice of flavour. results. data was collected and analysed for 51 patient days. a total of 122 supplements were prescribed. of the prescribed supplements, 42.6% were offered to patients and 26.6% consumed. 20% were offered the same at nursing discretion based on clinical need and 39.8% were consumed. resource ò energy was the most frequently prescribed and offered product (76.2 and 59.2% respectively). most common flavours selected by patients were strawberry and vanilla. resource 2.0 fibre was better tolerated (91.7%) than resource energy, resource fruit and resource dessert (73.9, 70.8 and 50.0% respectively). across all products the best tolerated flavours were apricot, chocolate and coffee (100%). the highest calorie supplement, resource ò 2.0 fibre, resulted in the best compliance in both tested flavours. compliance with ons demonstrated here is higher than previous studies (1) partly attributable to one-on-one nursing of icu patients enabling active encouragement with feeding. nursing staff discretion had better uptake than routine prescription of ons. however, difficulties with ons still remain. interestingly in our study the highest calorie density supplement was tolerated the best and thus giving the most benefit to the patient. despite the difficulties associated with ons uptake we would recommend its regular use on icu with a drive towards the highest calorie supplements being offered. introduction. cirrhosis is a chronic disease and the patient's quality of life is affected in a negative way due to the problems like ascites, jaundice, nutrition deficiency, fatigue, activity intolerance, itching, pain, insomnia, anxiety, hopelessness, work loss and depression. objectives. the aim of this study is to examine the changes in patient's lives that diagnosed with cirrhosis of the liver disease owing to the symptoms they experienced. methods. this research is a qualitative study that has been carried out with 13 inpatients diagnosed with liver cirrhosis in the gastroenterology clinic of a teaching and research hospital. average age of patients was 54 (ranging 39-70). descriptive characteristics form and semi-structured interview form were used in the data collection. interviews with patients have been performed individually and face to face. the data were evaluated by using colaizzi's phenomenological data analysis method. as a result of the data analysis, three categories and six themes were identified. categories include: (i) problems of symptoms related to the physical limitations (ii) psychosocial issues. patients suffer mostly from fatigue and malaise (12 patients), while those in the later stages suffer from, additionally, physical ailments caused by acid. inability to sleep due to anxiety and increase in tendency to sleep in advanced stages have been identified after being diagnosed. the majority of patients were identified to have undergone an anxiety besides having a fatal disease due to concern for the future, being forced to quit the job and being affected by the experiences of the patients in advanced stages. it also has been discovered that the patients had experienced social isolation because of fatigue and weakness in particular. as the result of this study it has been determined that patients with cirrhosis have mainly problems of fatigue, weakness, sleep disorders, anxiety and associated problems. 23rd esicm annual congress -barcelona, spain -9-13 october 2010 s379 [1] . while in patients with acute liver failure, elevation of arterial ammonia levels has been linked to cerebral complications and increased mortality, the role of arterial ammonia in hh patients is unknown. our study aims at evaluating arterial ammonia levels in patients with hh. furthermore, we wanted to elucidate the potential consequences of high ammonia levels in these patients. arterial ammonia levels were measured and documented in 72 hh patients without liver cirrhosis who were admitted to the medical icu. icu mortality and overall 28day-survival were documented. cox regression was performed to describe the impact of ammonia levels on mortality. mann-whitney test was used for comparison of metric variables. results. overall median arterial peak ammonia level in our patients was 65 lmol/l (41.9-100.6 lmol/l), whereas median arterial peak ammonia value was significantly higher in icu non-survivors compared to survivors (77 (53-119.2) vs. 52.9 (36.4-71.1); p \ 0.01). saps ii and sofa score were significantly higher in icu non-survivors (p \ 0.01 and p \ 0.05, respectively). cox regression revealed that arterial peak ammonia levels were significantly associated with higher 28-day-mortality (p \ 0.01), even after adjustment for saps ii. median arterial peak ammonia levels in patients with verified brain edema were significantly higher than in patients without (130.7 lmol/l (92-349.7 lmol/l) vs. 70.6 lmol/l (43.2-81.7 lmol/l); p \ 0.05) after exclusion of patients following cardiopulmonary resuscitation with consecutive hypoxic brain damage. our results suggest that increased levels of ammonia are associated with high mortality and can lead to brain edema in patients with hh. 53% of patients had a diagnosis of sepsis and 21% of patients were admitted under the neurosurgical team, the latter of which may have contributed to the relatively low anticoagulant use of 54%. systemic heparinisation was the sole anticoagulant used, but compliance with local protocols was poor with 48% of appts below the therapeutic range and 46% of infusions commenced at the wrong rate. 68% of filter changes were due to clotting and mean filter life was 32 h. despite this, dose delivery was acceptable, with 84% of prescribed dose delivered. conclusions. as previously reported 1 , our demographic data confirm the relatively poor outcome of patients needing crrt. we have identified areas where care for these patients could be optimised and endeavour to do this locally via improved protocol design and an ongoing educational programme. many of the components of crrt could be incorporated into care bundles, but certain aspects of treatment remain controversial 3 which may be a barrier to their adoption. given the high numbers of neurosurgical patients in our unit, consideration should be given to the use of regional anticoagulation such as citrate. introduction and objectives. accurate prognostic indicators of patient survival in an intensive care unit (icu) help guide clinical decision making. factors known to portend poor prognosis in acutely ill cirrhotics in icu include the need for mechanical ventilation, development of shock, renal failure and sequential increase in the number of failing organs. while serum lactate is now an established marker of survival and/or the need for transplantation in fulminant liver failure, its impact on critically ill cirrhotics is less well known. methods. we retrospectively studied 133 consecutive acutely ill cirrhotics admitted to the icu between 2005 and 08 at the royal free hospital, a tertiary referral centre in liver diseases and transplantation. data were collected on demographic variables, aetiology of liver disease, liverspecific prognostic scores [child-turcotte-pugh (ctp), model for end-stage liver disease (meld), united kingdom model for end-stage liver disease (ukeld)], and acute illness scores [acute physiological score and chronic health evaluation (apache ii), sequential organ failure assessment score (sofa) ]. in addition, serum lactate levels at 0, 24 and 48 h were also recorded. multivariable logistic regression analysis was performed, and the discrimination ability of each of the above-mentioned scoring models in predicting icu and hospital survival of these patients was evaluated using the area under the receiver operating characteristic (roc) curve. conclusions. one third of lt recipients present a documented bacterial infection within 1 year after surgery. we found a high prevalence of ciprofloxacin resistance and a low incidence of s.aureus witch was often resistant to methicillin. non fermentative gram negative bacilli represent 18% of the pathogens and should be taken in account for treatment of the most severe patients. extracorporeal liver support therapy is in its infancy but is valued as a detoxification treatment option for patients with cirrhosis who have rapid worsening of their liver function. we report the use of prometheus ò , a new extracorporeal liver support system allowing the removal of protein bound and water soluble toxins by fractionated plasma separation and absorption (fpsa) in a patient with wilson's disease (wd) who developed rapid worsening of their liver function. a 26-year-old female patient, diagnosed with wd since the age of 17, was initially treated in an irregular pattern with penicillamine. therapy was discontinued. now, 2 years later, she developed acute decompensated liver failure with hepatic encephalopathy with a meld 29. liver transplantation (lt) was the treatment option for this patient. but, in this case, the rapid and adverse evolution of the liver failure with renal failure and the unknown waiting time for a emergency liver donor in our country led us to use the extracorporeal liver support therapy. after 5 h 30 min of therapy we reduced the amount of bilirrubin for less than a half, we increase the urinary output and next day the patient went to liver transplant, stable, with a renal function improved. conclusions. acute liver failure due to wd is most of the time fatal without emergency lt. this case report highlights discontinuation of chelants treatment in a patient with wd. as the patient progressed to decompensated liver cirrhosis with encephalopathy, lt was the only treatment option but while we don't get a donnor, we can use, for a short period of time, an extracorporeal liver support therapy as a very useful bridge. results from two studies presented at the recent easl 2010 congress have shown that treatment with extracorporeal devices may not confer a survival advantage for severe liver failure patients, despite positive dialysis effects. however, results among a small sub-group of patients show promise like severely ill patients with hepatorenal syndrome type 1 or a meld score over 30. (1) . metoclopramide is used to stimulate the upper gi tract and seems to have no effect on colonic motility. objectives. the aim of this in vitro study was to compare the prokinetic potency of those substances. a tissue bath with guinea pig colonic segments fixed on a polyacrylic tray allows the evaluation of the transit time (tt), the time necessary for a wooden pellet to perambulate. a decrease of the tt reflects stimulation, and an increase inhibition of peristalsis. after stable peristalsis activity the effect of increasing concentrations of prucalopride, neostigmine or metoclopramide on tt were evaluated. dose response curves were constructed, two way anova (sigma stat) was used for statistics, p values b 0.05 were considered to be significant. effect of prucalopride and neostigmine on motility results. prucalopride stimulates normal peristalsis in vitro only in the highest tested concentration of 3 lm (p \ 0.05). neostigmine's prokinetic effect was limited to a small concentrations range (0.1 lm, p \ 0.05), the concentration of 0.03 lm had a moderate, but not statistically significant prokinetic effect and the highest tested concentration (1 lm) lead to a complete block of peristalsis (fig. 1) . metoclopramide, as expected, was devoid of any effect on colonic motility. conclusions. this experimental setting is a reliable method to evaluate the effect of different substances on colonic motility in vitro. prucalopride's prokinetic activity is concentrations dependent and limited. neostigmine is well known to improve colonic motility, but it seems imperative that the drug's effective dose range be use-higher concentrations have inhibitory effect on peristalsis. objectives. robotic radical prostatectomy involves extreme changes in patient position and often associated with a longer operative time than other commonly performed laparoscopic procedures. this review discusses the anesthetic considerations in robotic radical prostatectomy while analyzing potential risk factors related to pulmonary complications. we retrospectively reviewed the medical records of all the patients who had undergone robotic radical prostatectomy at our institution. among the total patients of 80, aged 49 to 82 years, 58 patients were capable of spontaneous respiration at the end of surgery (group i) whereas 22 patients needed assist ventilation (group ii). the demographic characteristics, coexisting diseases, anesthesia and operation time, anesthetic agents, the amounts of blood loss, infused fluid and transfused blood products were compared between the groups. results. the mean age of the patients was 67.2 ± 7.3 years. the mean operation times were 445.3 ± 210.3 min (range, 235-1250 min). age, body mass index (bmi) and asa status did not differ significantly between the two groups, whereas operation time, the amount of blood loss and the incidence of transfusion were significantly higher in the group ii. although 3 patients with subcutaneous emphysema and atelectasis needed prolonged ventilator care for 48 h, the incidence of atelectasis and subcutaneous emphysema was similar between the groups. conclusions. prolonged laparoscopic surgery in a steep trendelenburg position has a high possibility of postoperative respiratory insufficiency and the possible contributing factor is a long operation time. objectives. we examined the frequency of postoperative cough reflex and its effect on postoperative clinical outcome retrospectively. we examined the patients who admitted into the icu after the esophagectomy with lymphadenectomy during the period from september, 1999, to february, 2010. in addition to usual criteria for extubation we removed their tracheal tube if the cough reflex was identified when one milliliter of half saline was distilled into their trachea. if the cough reflex was absent until 7 days after the operation the patient underwent tracheostomy and after that they weaned from the ventilator. results. there were 125 patients (f/m 24/101), and their mean age was 58.5 ± 7.8. cough reflex were confirmed by seventh postoperative day in 95 patients (76%) but residual 30 patients underwent tracheostomy because of absence of cough reflex ( introduction. the technique of laparoscopic cholecystectomy carried with carbon dioxide pneumoperitoneum may lead to adverse events in mechanical, hemodynamic and respiratory systems as a consequence of physiopathological changes such as increased intraabdominal pressure. _ it may cause hypoxemia, hypercapnia, hemodynamic instability and impairment of oxygenation. decreased functional residual capacity, ventilation/perfusion imbalance and sympathetic stimulation effects of co2 that is absorbed from peritoneum are basic problems. in perioperative period, application of mechanical ventilation and anesthesia should be reviewed because of these physiopathological mechanisms. in this study, we aimed to investigate the effects of 5 cmh 2 o peep application on etco2, minute ventilation and arterial oxygenation during laparoscopic cholecystectomy operations. for this reason, the study included total 40 patients and they were randomly divided into two groups. same anesthetic protocol was applied in both groups. for general anesthesia induction; 1 mg/kg dose of fentanyl, 2 mg/kg dose of propofol were administered. following this procedure endotracheal intubation was applied with 0.15 mg/kg dose of cisatracurium. patients received %50 o2-%50 n2o (mixture with equal amounts) with 0.8-1.2 mac end-tidal sevoflurane for anesthesia maintenance. before co 2 insufflation, respiratory parameters were recorded on the respiratory apparatus adjusting etco2 32-36 mmhg, respiration rate 12/min., inspiration/expiration rate 1:2, vt: 8-10 ml/kg. patients were ventilated by volume controlled mechanical ventilation. heart beats, mean arterial blood pressure and peripheric o2 saturation (spo2), etco2, minute ventilation(v) and peak airway pressure(p _ ip) values of all patients were recorded just before insufflation (t0). after recording, 5 cmh 2 o peep was applied to the first group (group 1). peep wasn't applied to the 2nd group (group 2). these parameters were repeated in 5 periods such as 5 (t1) and 30 (t2) minutes after insufflation, preexsufflation (t3) and postexsufflation (t4) in both groups. before insufflation, respiration rate (12/min) and etco2 (32-36 mmhg) values were adjusted as planned in both groups and minute ventilation was also adjusted. at the same time, total insufflated amount of co2 for distending abdomen was recorded. arterial blood gas analyses were made just before induction (while patients were breathing normal room air, t0), 30 min after induction (t2) and just before the end of the operation (t4). in our study, we found that minute ventilation to stabilize etco2 32-36 mmhg was significantly increased in group 2 in which peep was not applied (p \ 0.01). none enhancement was needed in minute ventilation in group 1 and arterial oxygenation was significantly increased in group 1 (p \ 0.01). aside from the cholesterol lowering effects of statins, as a class of drugs they have been shown to exert anti-inflammatory effects and have the potential to be therapeutic in neuroinflammatory disorders 3 . we tested the hypothesis that atorvastatin improves memory retrieval post unilateral nephrectomy in a murine model. methods. c57/bl6 mice were randomly allocated into 4 groups (n = 8-10/group): control plus placebo, control plus atorvastatin, nephrectomy plus placebo and nephrectomy plus atorvastatin. animals were given either a placebo (0.4 ml normal saline) or 250 lg in 0.4 ml normal saline of atorvastatin by gavage once a day for 5 days. on day 4 all animals underwent fear conditioning training using a conditional stimulus of a 70 db tone and an unconditional stimulus of a 0.70 ma electric shock. on day 5 the surgical animals underwent unilateral nephrectomy, whilst the control animals received no surgery. at post-surgical day 3 all animals were tested for hippocampal dependent memory retrieval using the fear conditioning paradigm, with freezing response to the 70 db tone as a marker of memory retrieval. all animals were then terminated. results. surgery evoked a reduction in hippocampal dependent memory retrieval in the nephrectomy plus placebo group as measured by % freezing time (mean ± sd: 40 ± 16) when compared to the control plus placebo group (70 ± 19; p \ 0.01); a situation mimicking pocd. this change was obviated in the nephrectomy plus atorvastatin group (59 ± 16; p [ 0.05 vs. control plus placebo). conclusions. our data suggested that atorvastatin has the potential to improve postoperative cognitive performance in a murine model of pocd. the proven safety of the drug along with its already widespread use and cost effectiveness would permit rapid instigation of a human randomized controlled trial to explore efficacy in the clinical setting. a. puxty 1 , r. docking 1 1 glasgow royal infirmary, department of anasethetics, glasgow, uk hypotension in the post-operative period is common but guidelines recommend its prevention/treatment [1] . epidurals are common practice following major surgery in many institutions and can prevent pulmonary complications [2] but have also been associated with falls in blood pressure when compared to other analgesic techniques [3] . fluids therapy is a common intervention for hypotension but fluid overload has been associated with worse outcomes in surgical patients [4] . we decided to audit the incidence and management of hypotension in the surgical high dependency unit of a large tertiary referral hospital. to determine the incidence and management of hypotension in the surgical high dependency unit in pancreatic, upper gi and lower gi patients. we prospectively looked at 48 patients who underwent major upper gi, lower gi or pancreatic surgery involving epidural analgesia. the first 24 h of care from onset of anaesthesia was closely looked at with regards to fluid management, epidural management and actions taken on episodes of hypotension or severe hypotension (defined as systolic blood pressure of \90 and \80 respectively). each episode of hypotension was looked at to determine the actions taken at that point. of the 48 patients looked at, 17 were major pancreatic, 17 lower gi and 14 upper gi patients. 40 (83%) had at least one episode of hypotension, with 11 (23%) having at least one episode of severe hypotension. mean fluid in during the first 24 h was 6523 ml, with a mean fluid balance of 4133 ml. there was no difference between the doses of epidural local anaesthetic in 24 h between the hypotensive and non hypotensive groups (p = 0.275). management of hypotensive episodes was variable, but the most common intervention at episode one was fluid bolus (80%) and discontinuation of epidural was most common at episode two (39%). use of vasopressors for hypotension was very low with only two infusions being started altogether. conclusions. hypotension is very common in our high dependency unit. fluid balance in our patients was far more positive that we had expected. management of hypotension was variable. we plan to institute a protocol for hypotension and fluid administration to determine if improvements can be made. objectives. to identify predictive factors associated with the need for relaparotomy in patients with ssp. adult ssp patients undergoing laparotomy between 2004 and 2009 included within a single-center peritonitis registry (perit) were collected. patients subjected to relaparotomy were studied. we excluded patients with severe peritonitis secondary to appendicitis. apache ii and sofa score at icu admission after the initial laparotomy were recorded. variables with a p value.1 in a bivariate analysis were included in a multivariate logistic regression for further analysis of predictors for need for re-laparotomy. results. two-hundred forty-seven patients were obtained from perit registry. a total of 212 patients with spp were included in the analysis. eighty seven patients (41%) required relaparotomy. median number of re-laparotomies was 3. most spp were associated to colon (n = 94, 44.3%), small intestine (n = 83, 39.2%) and biliary tract (n = 33, 15.6%) perforations. cultures were positive in 74.5% of first laparotomy: gram negative bacteria were isolated in 53.3%, gram positive bacteria in 16.5% and fungi in 4.7%. hospital mortality was 17% (n = 36). multivariate analysis is described in the table 1 . conclusions. in obese patients scheduled for surgery, the previous use of cpap has not shown an improvement in blood gas parameters. the use of cpap in the hours before and immediately after surgery has not been associated with better postoperative oxygenation. combined icu-surgery dpt. action in these cases seem to contribute to better patient outcomes. objectives. we set out to quantify the intensive care workload and changes to that workload over the first 4 years following the transfer of a specialist bariatric service to our hospital. a prospectively collected bariatric surgical database was cross-referenced to the itu database (ward-watcher) to identify admissions to the 10-bedded critical care unit of all patients who had undergone any bariatric procedure. for each patient identified; demographics, reason for admission, level of support, length of stay and outcome were recorded. data were grouped into 12-month periods for trend analysis. research in emergency situations and especially in resuscitation field raises important ethical and regulatory issues. the globalization of the resuscitation science through multicentric trials for example highlights the need for a more consistent approach to regulatory aspects to enable the science to grow while protecting human rights. objectives. the purpose of this analysis is to compare the different regulations approaches in emergency research in north america (canada, usa) and in europe (european directive, france). conclusions. this analysis emphasizes the lack of international standardization of regulatory measures and ethical decisions. however some countries like the us seem to advance in the democratic process by mandating additional regulatory measures (community consultation, public disclosure to the communities) prior to initiation of clinical investigation; nonetheless, there is little evidence of their effectiveness. many challenges are raised. firstly, the variability in regulations, and consequently in local board's assessments, is problematic, pleading for international regulations. secondly, the current heterogeneous ethical review process and demanding unsubstantiated regulatory measures poses a risk to all when it is not evidence based and it is applied inconsistently between countries, within a country and worse at the level of each individual hospital review board. it puts the investigator at risk for unnecessary criticism and the community at risk as it is unknown if we truly consult or inform our target communities about waiver of consent research through our current ethical and regulatory processes. globalization and evaluation of the ethical and regulatory processes are urgently needed; regulatory community has to work towards a standardized evidence-based process upon which to base regulatory decisions. introduction. in research outside the intensive care field it is known that a high score for the psychological factor ''perceived hopelessness'' experienced by healthy individuals increases risk of death several fold. objectives. the aim of this study was to examine if the score of the psychological factor ''perceived hopelessness'' may predict long term mortality (mean or high perceived hopelessness score) when assessed post icu care in former icu patients. methods. prospective, multicenter study in three mixed icu's in sweden. questionnaires, including the 2-item hopelessness scale, demographic data and previous illnesses, were sent 6 months after discharge to all former adult icu patients who thereafter were followed for another 3 years. a reference group of 6093 individuals from the uptake area of the hospitals served as controls. results. 980 (59%) patients returned the questionnaires. the icu patients reported significantly higher mean scores in perceived hopelessness score compared with the general population, 2.5 (sd 2.2) compared with 2.1 (sd 2.1) (p \ 0.001), and 40% (n = 392) of the icu patients perceived a mean or high hopelessness score compared with 36% of the general population (p \ 0.001). the icu patients who died during the follow-up period reported a significantly higher perceived hopelessness score (n = 123) 3.7 (sd 2.5) (p \ 0.001) as compared with those who survived up to 3 years after discharge (n = 857) 2.4 (sd 2.1). in a logistic regression model the long term mortality for the icu group was found to be affected by: pre-existing disease [odds ratio (or):2.2], age (or: 1.04) and perceived hopelessness score (or: 1.18). the new and interesting finding of this study is that icu patients score higher on ''perceived hopelessness'' than a control population and this increase is predictive for the post icu mortality. furthermore, the size of this effect is significant and only exceeded by pre-existing disease and age. we performed a retrospective observational study to evaluate what proportion of met calls was associated with lomt issues. to estimate the proportion of met reviews involving patients with a not-forresuscitation (nfr) order and the timing of met calls in relation to admission and death or discharge from hospital. to compare the patient characteristics and outcome for met calls associated or not associated with lomt issues. we obtained hospital research ethics committee approval. we performed a retrospective observational study involving five-year (august 2005-april 2010) in a single tertiary australian hospital. we obtained information on demographics, on the met review and hospital outcome. lomt included nfr orders, not for met orders and palliative care plans. results. we analysed 4829 met reviews in 3629 patients. table 1 and fig. 1 summarize major findings for overall population and the two subgroups of patients with or without lomt. patients with lomt care plan were older, more likely to have medical diagnoses, were reviewed later during their hospital stay and closer to their hospital discharge or death. fewer lomt patients were admitted to icu. hospital length of stay was shorter, mortality in lomt care patients was double that of non-lomt patients. however, more 50% of patients with lomt were discharged alive from the hospital. conclusions. more than one third of met activations deal with lomt issues. although the mortality of these patients is high, a large proportion survives to hospital discharge. evaluation of the patient experience in intensive care (icu) frequently depends on reports from surrogates such as relatives. there is a concern regarding the validity of the surrogate opinion which might not represent the values of the incapacitated patient and treatment decisions therefore maybe biased [1] . others have found that there is a strong preference within a population for utilizing relatives as surrogate decision-makers in the event of admission to icu and this attitude is not influenced by ethnicity, religion or education level [2] . objectives. the objective was to measure the ability of the relative to answer on behalf of the patient. a further wish was to determine the validity of their surrogate responses. a retrospective study, which surveyed relatives of patients who had died within a critical care service during a 2-year period (2005, 2006) . the 31 item questionnaire allowed for the collection of quantitative and qualitative data with respect for each item to overcome the limitations of the quantitative format which may not be sensitive to all the issues which can surround the provision of end-of-life care [3] . for 3 items, relatives were asked specifically to grade their capacity to represent the patient. results. quantitative data from the 3 items designed to test the relatives' perception of their ability to act as surrogates indicates that relatives considered they could respond to these items for 46% (average) of instances. when the relative did answer on the patient's behalf, the level of concordance between the surrogate (relative) and the patient's perceived opinion was 60% suggesting that when the relative is willing to act as surrogate the response is likely to have validity. (table 1) . results from the qualitative data indicates that the low (46%) level of willingness to answer these questionnaire items reflected a reluctance to answer on behalf of a sedated or ventilated patient, rather than an inherent inability to represent the patient. conclusions. the response rate to the 3 items vindicates concerns regarding the ability of relatives to represent the patient in icu settings and supports a need for further study. where the relative is willing to act as surrogate, concordance does exist. qualitative data clarified quantitative results and was instrumental in promoting a better understanding of the concerns of relatives who have a family member admitted to icu. . the majority of patients that died in icu were provided some kind of therapy restriction. an important conflict strains between clinical practise, bioethical principle and jurisdiction laws; the solution of this conflict is more and more urgent. therapy restriction has also important economical aspects since the number and cost of available treatments constantly increase. our survey studied therapy restriction procedures in hungary for the first time. in 2007 we performed a survey with questionnaire among intensive care physicians. questionnaires were sent out electronically to 743 registered members of the hungarian society of anaesthesiology and intensive care. respecting anonymity we have statistically evaluated 103 replies (14%) with t test and anova. we grouped intensive care physicians based on gender, years spent in work, religion and type of department they were working, and we compared data from these groups. intensive care physicians generally make their decisions alone, based on the patient's long-term life prospects and physical status (3.75/5 points). they are slightly influenced by the opinion of the patient (2.57), the relatives (2.14) and other medical personnel (2.37). if the physician sees any chance of recovery but the patient or relative requests treatment restriction then 67.3% of physicians that completed the forms would continue therapy against the will of the patient or relative. only 27.7% would accept the patient's/relative's opinion and autonomy in such a case and would stop therapy. in fact 28.3% of physicians would make their decisions without considering or even against the opinion of patient if they think therapy is useless. if there is no chance of recovery despite medical treatment 3% of physicians stop the treatment, 8.1% would continue it without informing the patient or the relatives, 17.2% informs the relatives but continues useless treatment irrespective of the will of the patient or relative. having analyzed the groups we found two significant differences. in case of useless treatment physicians working in university hospitals more often choose treatment restriction without informing relatives (p \ 0.001) then those working in non-university hospitals. physicians who declare themselves as atheist rather choose the continuation of treatment without informing relatives (p = 0.007). conclusions. the hungarian practise of end of life decisions among intensive care specialists is paternalistic, physicians make their decisions alone, do not consider the requests of the patient or relatives. our goal is to strengthen patient autonomy and to support their opinion by training icu physicians. on the other hand it is inevitable to define what useless medical treatment exactly is and to introduce this category in medical ethics and also in jurisdiction practise. objectives. to determine the frequency and processes of eol care at our centre. between october 2007 and december 2009, 150/388 (39%) patients staying in the icu for more than 1 day, underwent some form of eol care in the icu. icu staff notified investigators whenever an eol decision was made. we recorded demographic details, documentation of the eol care process in the case notes, and interviewed icu staff to determine the eol care processes involved. results. 97 patients (65%) were male, 53 (35%) were females. mean age was 42.4 ± 2 years. icu stay was 6.7 ± 6.3 days, admission apache ii score was 18.1 ± 7.8 which increased to 24.9 ± 9.4 on the day of eol care decision. 33% patients had metastatic cancer. reasons for initiating eol care were refractory acute illness in 53%, advanced cancer in 51%, brain death in 11%, and lack of finances in 11%. eol discussions were initiated by the family in 11%, and by the icu medical team in 89% patients. 16 families wanted to take the patient home to die. the icu consultant was involved in all discussions with the family, the primary consultants in 91% and primary team residents in 79%. nurses were involved in only 3 patients. agreement on eolc was reached after 1 discussion in 70%, 2 discussions in 22%, and 3 discussions in 8% of cases. documentation of the eol care process was not done in 51% cases. withholding of life support (wh) was practised in 110/150 patients (73%) and withdrawal of life support (wd) in 27%. intubation was withheld in 14.5% patients, cardiopulmonary resuscitation in 50%, inotropes in 76% and dialysis in 8%. regarding wd, only 3/40 patients were extubated and the ventilator withdrawn in another 7/40 patients. inotropes were withdrawn in 20 patients (50%). reduction of fio2 0.21 without discontinuing mechanical ventilation was the commonest mode of wd, in 35 patients (88%). all patients received morphine infusions during lols/wols. family members were present by the bedside in 61% cases. conclusions. wh is preferred over wd. documentation of the eol process does not occur in a significant proportion of cases. nurses are rarely involved in the eol care decision making process. legal issues may be barriers to good eolc in our icu, and perhaps in india. objectives. to know the point of view of the staff is essential to understand their beliefs, attitudes and decisions. brazilian private general icu with 23 beds. the following items were analyzed: profile of the interviewed; their opinion about end of life questions: fear of death, fear of experience pain before death, the best place to die, advanced directives, decision-making process, therapeutic withhold of mechanical ventilation, nutrition, fluid management, antibiotics, vasoactives drugs, sedation and analgesia in patients which death is imminent and irreversible. results. about 84.41% of our icu team answered the research (n = 65). the mean age is 32.61 years (sd 6.74), 58.50% of female, 58.50% married, 50.85% protestants and 38.50% catholics and icu professional experience of 6.75 years (sd 4.65). using a visual analog scale (0, no fear to 10, the worst fear possible) the team pointed 5.96 as their fear of death; the fear of suffering pain before death was 8.17. for 44.60% of the responders, the best way to die would be with their lovely ones, no matter if at home or at hospital. only 9.20% would prefer to die an icu. the majority of the team (88.25%) would share the eol decision-making process with the family instead only by the medical staff (52.30%). about 73.10% would leave an advanced directive with their therapeutic preferences like do not resuscitation orders. the icu team agreed on the withdrawal of vasoactives drugs (41.50%), antibiotics (57.00%), nutrition (18.50%) and mechanical ventilation (1.53%) in patients out of treatment. our results showed the staff vision about their own death and their opinion about the end-of-life care issues. in developing country as brazil there is a still gap between everyday practice and the current legislation. fortunately, the debate about eol issues has increased in last years. the end-of-life discussions and decisions should begin by respect to points of view of all involved: patients, family, medical staffs with a legal support of the society's beliefs and expectations. prospective observational study conducted in 8 greek multidisciplinary icus. we studied all consecutive icu patients who died, excluding those who stayed in the icu \48 h or were diagnosed with brain death. 306 patients comprised the study population [mean age 64 ± 17 (sd) years, mean apache ii score on admission 21 ± 7]. results. of patients studied, 41% received full support including unsuccessful cardiopulmonary resuscitation (cpr). 48% died after withholding of cpr, 8% after withholding of other treatment modalities besides cpr, and 3% after withdrawal of treatment. patients in whom therapy was limited had a longer hospital (p = 0.01) and icu (p \ 0.01) stay, a lower admission gcs score (p \ 0.01), a higher apache ii score 24 h prior to death (p \ 0.01), and were more likely to be admitted with a neurological diagnosis (p \ 0.01). patients who received full support were more likely to be admitted with either a cardiovascular (p = 0.02) or trauma diagnosis (p = 0.05), and to be surgical rather than medical (p = 0.05). the most important factors affecting the physician's decision to provide full support were reversibility of illness and prognostic uncertainty; the physician's religious beliefs and legal concerns had minimal impact. the main factors guiding the decision to limit therapy were unresponsiveness to treatment already provided, prognosis of underlying chronic disease, and prognosis of acute disorder; old age was not a determinant, while economic cost and lack of icu beds seem to play no role. relatives' participation in decision-making occurred in 20% of cases and was more frequent when a decision to provide full support was made (p \ 0.01). the principal reason for not discussing end-of-life dilemmas with relatives was the fact that the family was thought not to understand (60%) advance directives were rare (1%). icus. however, in a large majority of cases, it involves the withholding of cpr only. withholding of other therapies besides cpr and withdrawal of support are infrequent. physician has a dominant role in decision-making. objectives. the primary objective of this study is to determine the prevalence of inappropriate or non-beneficial care in icu patients as perceived by their icu healthcare providers, as well as the reasons for this perception. second, we want to determine which factors are associated with the perception of inappropriate care. a descriptive survey design is used. a single-day cross-sectional evaluation of perceptions of inappropriate care among 2,327 icu healthcare providers in 85 icu centres in 10 european countries will take place on may 11th 2010. questionnaires will be administered to icu healthcare providers (nurses, head nurses, junior and senior icu physicians) providing bedside care to adult icu patients on that particular day. in this study, inappropriate care is defined as a patient care situation that is similar to one or more of seven scenarios. these scenarios were created based on the literature and a multidisciplinary conference attended by experts in intensive care, geriatrics, and palliative care. . the cross-sectional study will take place on may 11th 2010. preliminary results will be given at the esicm conference. we have designed a one-day cross-sectional study to record inappropriate or non-beneficial care in european icu's. results will be available for the esicm conference. grant since the introduction of the mental capacity act in the uk in 2007, the impact within research in the intensive care environment has not been elucidated. since many of the patients are incapacitated and therefore unable to consent, it is now stipulated by the ethics committee that the researcher must make reasonable attempts to identify a consultee, failing this, nominate a person unrelated to the research project to be consulted. in order to comply with the mental capacity act, retrospective consent must be obtained, once the patient regains capacity. objectives. the aim of the study was to highlight the difficulties in obtaining retrospective consent, evaluate the methods used and demonstrate the adaptations made to increase retrospective consents. methods. this explorative analysis investigated the process of obtaining consent in 246 patients enrolled in an observational study on critically ill patients. consent was obtained on admission if the patient had capacity. assent from the patient's next of kin or a legal professional representative was obtained before enrolment in patients who lacked capacity. after discharge from icu, a member of the research team re-visited these patients to explain their involvement in the research, its purpose, procedures, implications and any further participation required by the subject. at this point, the patient could consent or withdraw from the study. if the patient decided to withdraw from the study, all data collected and samples stored were destroyed. the researcher visited the patient for a minimum of two visits; firstly to explain the study; secondly to establish if the patient has retained the information and to gain retrospective consent. results. 246 patients were recruited within the time period of which 44 (17.7%) died. in 65 (26.2%), consent was obtained on admission as the patients had capacity, 36 (14.6%) were discharged prior to obtaining retrospective consent, 10 (4%) lacked capacity on the researcher's visits, and 1 patient (0.4%) withdrew from the study. 90 patients (36.3%) were successfully consented retrospectively. overall, the researchers performed 221 visits to obtain from the 137 patients for whom retrospective consent was required. conclusions. the process of recruiting patients who lack capacity within the intensive care unit is challenging and time consuming. stipulations set by the ethics committee to seek retrospective consent once the patient has regained capacity, has a major impact on research staff time and finances. detailed recommendations as well as guidelines how to assess capacity in the post-icu patient and how the assessment of capacity has to be applied to intensive care research are needed to fully comply with ethical and legal requirements. objectives. we wanted to know if patients expressed to surrogate decision makers, after icu discharge, specific resuscitation directives, and we have investigated any factors related to the patients and their illness or care process that might be associated with this. we reviewed patients admitted in the icu between december 2008 and may 2009. a random sample of 60 survivor patients has been defined. seven patients were excluded (3 for language barrier, 2 died, 2 were no more reachable). fifty three patients took part in semistructured interview at 6-12 month post icu discharge. the questionnaire discussed in detail the aspects of advance directives. patients had also completed a quality of life questionnaire (euroqol 5d), and we calculated the eq-5d visual analog scale. we reviewed medical records in icu data base: age, gender, length of stay, saps ii, bmi, length of ventilator support and central venous catheterization as well as prescription of transfusion, hemodialysis or adrenergic agonist. multivariate logistic regression was practiced to investigate any factor associated to expression of specific resuscitation directives after icu discharge. after icu discharge, 60% of interviewed survivors expressed specific resuscitation directives to an appropriate identified surrogate (written ''living will'' or oral statement). eq-5d visual analog scale was 58 ± 19. on multivariate regression analysis, only one studied variable was significantly associated to the post-icu expression of specific resuscitation directives: age (odds ratio = 0.94, z = -2.1, p = 0.035). conclusions. after icu discharge, a majority of our patients expressed to surrogate decision makers specific resuscitation directives, especially the younger patients. our findings suggest that surviving to icu is an opportunity to specify oral or written directive, and both may help to illuminate future decision making from the patient's perspective. objectives. to explore the issues around eolc provision for cancer patients in a critical care unit through family, professional and patient experiences. to explore how a diagnosis of cancer impacts upon eolc provision for critically ill patients. a heideggerian phenomenological interview approach was undertaken, in order to gain personal experiences. families of those patients who died after decisions to forgo lifesustaining treatment (dflsts) were interviewed. patients who were seriously critically ill (apache ii [ 25 or had received cpr) who experienced critical care were also interviewed, since patients' views about eolc provision are very rarely explored. doctors and nurses also contribute their vision for, and experiences of, eolc in a cancer critical care unit. thirty seven participants were interviewed. tensions between treating families versus treating patients impacted on timeliness of eolc. achieving a good death was possible through caring activities that made best use of technology to prevent prolonged dying. decision-making and eolc could be difficult to separate out which, in turn, affects prospects for eolc. three main themes included: dual prognostication; the meaning of decision-making; and care practices at eol: choreographing a good death. these themes outlined the essence of moving along a continuum toward patients' deaths and the impact that had on opportunities for care and a good death. conclusions. cancer affected the trajectory in unexpected ways. the trajectory could be very quick, especially in unexpected death and some newly diagnosed cancers. even in the face of a life-limiting and serious disease like cancer, death could be unexpected. the rapidity of trajectory related to cancer diagnosis, prognosis, withdrawal and patient demise significantly impacted on the potential for, and timing of, eolc. a sentiment of moving on from historical practices around critical care for cancer patients, and related poor prognoses, was overwhelmingly agreed on but important caveats in cancer prognostication remains. conclusions. these data suggest that oscillation settings of 5 and 10 hz provided more optimal pef/pif ratio ([1.1). our data also suggests that airway clearance using hfcwo may facilitate improved gas exchange in mechanically ventilated patients. further study is required to confirm these results grant acknowledgment. partial funding support in the form of devices was provided by hill-rom inc. a. esquinas 1 , m. folgado 2 , j. serrano 3 1 hospital morales meseguer, intensive care unit, murcia, spain, 2 hospital virgen de la concha, zamora, spain, 3 hospital reina sofia córdoba, intensive care unit, cordoba, spain objectives: we hypothesized that the use of intrapulmonary percussive ventilation (ipv) could effect hypercapnia/acidosis and airway secretions control during treatment with noninvasive mechanical ventilation (nippv) in exacerbations of copd associated with bronchial secretions. prospective multicenter study. the study was performed in the medical icu of 3 spanish university hospitals members of the spanish ipv working group. we enrolled copd exacerbation patients with secretions and the need for nippv in icu. criteria of exacerbations of copd are: a respiratory frequency c25/min, a pao 2 [ 45 mmhg and ph b 7.35. we define two ipv strategies as complementary treatment during nippv to evaluate the effects of ipv. strategy group i: nippv at first line and combination of ipv in early periods without nippv in spontaneous breathing and ph c7.35. strategy group ii: first line of ipv with mouthpiece/face mask and oxygenation previous to the application of nimv with ph \ 7.35. in both groups daily sessions ipv were applied by for 30 min/3 day by mouthpiece or face mask during stay in icu. nippv was applied with bipap ventilator (respironics) and face mask with bipap mode. cardiopulmonary monitoring, clinical and arterial blood gases were evaluated. therapy was considered as successful when patients did not need nippv support and clinical and arterial blood gases returned to baseline. results. 65 patients with copd exacerbation were admitted in icu for nimv, age 70 ± 12 years, male (90%) 15 were excluded for severe hypoxemia (pao 2 :fio 2 b 200) associated with pneumonia (5/15) and cardiac insufficiency (10/15). fifty patients were enrolled in the study. -up tilt-table rehabilitation better than sitting in a chair for ventilated adults in intensive care in terms of improving lung function? j. manners 1 , a. thomas 1 , s. boot 1 , g. mandersloot 1 1 barts and the london school of medicine and dentistry, london, uk physiotherapy intervention is a fundamental part of the patient stay in an intensive care unit (icu) and treatment is often aimed at maintaining/improving respiratory function. physiotherapists use the upright posture to elicit these improvements and sitting in a chair and standing with a tilt-table are commonly used interventions. to date there are no published reports comparing the efficacy of these interventions in ventilated subjects. • to compare the effects of these two positioning techniques employed with icu patients. • to measure changes in respiratory rate, tidal volume and minute volume during these positioning interventions. • to measure functional residual capacity during positioning interventions. • to measure the change in metabolic demand during positioning interventions. methods. convenience sampling of ventilated subjects meeting the inclusion criteria was employed. subjects acted as their own controls undergoing sitting in a trauma chair and standing on a tilt table at 70 degrees in random order on the same day. respiratory rate (rr), tidal volume (v t ), ventilation (v e ) and oxygen consumption (vo 2 ) were measured at minute intervals during baseline and intervention for 10 min. functional residual capacity (frc) was measured once at rest and following each intervention. measurements were recorded using the ''e-covx'' module for the ''ge carestation ventilator''. results. 10 subjects were recruited. no adverse events occurred during interventions. significant increases from baseline rr (p \ 0.0001), v e (p \ 0.0001) and vo 2 (p = 0.009) occurred during the tilt table intervention. there was an increase in frc during tilting of 0.6 l which failed to reach significance. significant increases from baseline rr (p \ 0.0001), vo 2 (p = 0.024) and a decrease in v t (p = 0.015) occurred with the chair intervention. conclusions. these interventions are safe in a critical care population. increased muscular activity associated with upright interventions elicited expected elevations in vo 2. the tilt-table produced an increase in v e driven by an increased rr at the expense of v t . v e was not elevated during chair sitting despite an increased vo 2 and was accompanied by an unexpected decrease in v t. introduction. uk guidelines about rehabilitation after critical illness highlight the need for outcome measures to determine patient progress and efficacy of treatment [1] . there is no consensus about the most appropriate measures of patient function. the austoms [2] tool was designed by therapists in australia to measure activity and function across nine scales assessing structural and functional difficulties and ability to perform activities. scales are split into four domains (impairment, activity limitation, participation restriction and distress/wellbeing) and scored from 0 to 5 with 0.5 intervals allowed. acceptable inter-rater variation is defined as an absolute difference of 0.5. austoms has not been appraised in patients recovering from critical illness. objectives. to prospectively determine the inter-rater reliability of the austoms physiotherapy scales in adult patients who had undergone cardiothoracic surgery and required critical care admission for over 5 days. methods. the therapy (physiotherapy and occupational therapy) team underwent a 1 h teaching session using the austoms handbook prior to commencing the trial. austoms was then used over eight consecutive weeks during the weekly therapy goal setting meeting. each week a patient was selected to be scored using the most appropriate functional scales. the clinical history was presented to the team by the therapist leading the patient's care. therapists were then asked to independently score patients across the four domains for each scale. reasons underlying differences in scores were explored by group discussion. the difference between the 10th and 90th centiles of the initial scores was calculated for each domain as a measure of inter-rater variability. results. 6-8 therapists were present at each meeting. respiratory function and musculoskeletal movement related function were the most common scales used. the mean difference between 10th and 90th centiles was greater than 0.35 (± 0.21) for all domains. none of the scales/domains showed consistent inter-rater reliability over the 8 week period. overall the activity limitation domains of each scale showed the least inter-rater variance of scores. clinical experience of therapist did not appear to influence scores. conclusions. the austoms outcome measure showed poor inter-rater reliability when evaluated over an 8 week period on our intensive care unit. further work is ongoing to evaluate the ability of austoms to reveal changes over time when scored by therapists. introduction. uk guidelines on the rehabilitation of patients after critical illness highlight the importance of establishing and reviewing individualised rehabilitation goals for all patients that are at risk of developing physical and non-physical morbidity [1] . our institution's practice is to create objective goals that are smart-specific, measurable, achievable, realistic and timed [2] . objectives. the aim of this audit was to prospectively collect data regarding the setting of rehabilitation goals in a group of patients admitted to a cardiothoracic intensive care unit. methods. all consecutive patients admitted under the intensive care team in november 2009 were included. data regarding the timings of initial physiotherapy assessment, goal setting, and concomitant sedation were collected using a structured questionnaire completed by the treating physiotherapist. results. 30 patients were admitted under the critical care team. 29 patients were assessed by a physiotherapist within 24 h of admission. of these 29, 21 had smart goals set within a median of 7 days of initial assessment (range 1-29 days). there was a correlation between level of consciousness and the number of days taken to set goals. patients who were fully conscious or drowsy on initial contact (n = 11) had a smart goal set in a median of 3 days. by contrast patients who were sedated/paralysed on initial assessment (n = 18) had goals set in a median of 12 days. initial goal setting did not include other therapists or the family. goals fell in to 5 categories, range of movement, hoisting out to chair for periods of time, sitting on the edge of the bed, transferring out to the chair by standing and mobility goals-i.e. walking set distances. the maximal interval between reviews of the patients' goals was 7 days. most patients had smart goals defined and regularly reviewed. however, despite physiotherapy assessment within 24 h of admission, there was often a delay in setting these objective goals. the need for continuous sedation acted as a barrier to explicit setting of goals. the results emphasised the need to improve patient and family/carer involvement with initial goal setting in order to be compliant with uk standards. objectives. investigation of ems effects on muscle strength and exploration of issues in relation to handgrip dynamometry in icu patients. one hundred seventy two consecutive patients with apache ii score c13, were randomly assigned to the ems (n = 86, age: 62 ± 18 years, apache ii: 18 ± 5) or the control (n = 86, age: 59 ± 18 years, apache ii: 18 ± 5) group. ems sessions applied daily in muscles of both lower extremities. the strength evaluation of various muscle groups of the upper and lower extremities was made clinically upon awakening with the mrc scale, ranging from 0 to 5 (normal strength) for each group. the same scale was also employed in the diagnosis of cipnm (mrc \ 48/60). a subgroup of these patients also performed handgrip dynamometry. results. fifty seven patients (ems: 28, control: 29) were finally evaluated. ems patients scored higher than controls (p b 0.05) in wrist flexion, knee extension, ankle dorsiflexion and right side hip extension, while they tended to perform higher in all other muscle groups (p: 0.20-0.10) ( table 1) . grant acknowledgment. this project has been co-financed by e.u. and the greek ministry of development. background. secretion removal is major aim of respiratory physiotherapy in intensive care. manual hyperinflation provides a tidal volume to the lungs that is greater than baseline. it is effective in secretion clearance and is frequently used [1, 2] . there is a limited evidence that addressed the effects of combining rib-cage compression and suctioning on oxygenation, ventilation, and airway-secretion removal in mechanically ventilated patients [3] . objectives. the aim of this study was to investigate the effects of manual hyperinflation administered in combination with expiratory rib-cage compression on lung compliance, gas exchange, and secretion clearance in mechanically ventilated patients. methods. twenty-two intubated, mechanically ventilated, and hemodynamically stable patients were studied. the patients received manual hyperinflation, with or without expiratory rib-cage compression, with a minimum 3-h interval between the two interventions. manual hyperinflation with or without expiratory rib-cage compression was performed for 5 min before endotracheal suctioning. respiratory mechanics and hemodynamic variables were measured 5 min before (baseline) and then 5 and 20 min after the interventions. arterial blood gases were determined 5 min before (baseline) and 20 min after the interventions. secretion clearance was measured as sputum weight. the two measurements were obtained on the same day. results. no significant differences were observed in gas exchange and secretion clearance between the two interventions (p [ 0.05). in each case, static lung compliance and tidal volume improved significantly at 5 min post-intervention (p \ 0.01), whereas at 20 min postintervention, only static lung compliance had improved significantly above baseline (p \ 0.01). our results suggest that the addition of expiratory rib-cage compression to manual hyperinflation does not improve lung compliance, gas exchange, or secretion clearance in mechanically ventilated critically ill patients. recently, there has been an interest in mobilization of acutely ill patients who are in an intensive care unit (icu). in the literature, the major safety issues while mobilizing critically ill patients has been outlined. cardiac reserve [(cr) (% of age predicted maximal heart rate)] and respiratory reserve [(rr), ratio of partial pressure of oxygen in arterial blood to the inspired fraction of oxygen (pao 2 /fio 2 )] are the important factors that can affect the ability to tolerate the mobilization. patient who has rr more than 300 and cr lower than 50% is considered to have sufficient reserve to tolerate mobilization [1, 2] . objectives. the aim of this study was to compare the effects of mobilization on respiratory and hemodynamic parameters in patients with sufficient and insufficient respiratory and/or cardiac reserve. mobilization events are divided into two groups (sufficient, insufficient) according to the pre-mobilization cr (sufficient, \50%; unsufficient, [50%) and rr (sufficient, [300; insufficient, \300). heart rate (hr), systolic/diastolic/mean arterial blood pressure (sbp, dbp, mabp), respiratory rate (rsr) and percutaneous oxygen saturation (spo 2 ) were recorded from the monitor. respiratory and hemodynamic parameters were collected just prior to the mobilization, just after the completion of the mobilization when the patient had been returned the supine position and 5 min of the recovery period and compared between the groups. a total of 87 abdominal surgery patients (47 male, 40 female) received 113 mobilization treatments in icu. the mean age was 60.1 years, mean body mass index (bmi) was 24.9 kg/m 2 , mean apache ii score was 20.5 and mean icu stay was 7.3 days. mobilization events included 88 (78%) sitting on the edge of the bed, 12 (11%) standing, 13 (11%) walking to chair and sitting in the chair. 85% (96) of mobilization events had insufficient rr and 15% (17) of mobilization events had sufficient rr. 74.3% (84) of mobilization events had insufficient cr and 25.7% (29) of mobilization events had sufficient cr. all respiratory and hemodynamic parameters were found similar in sufficient rr and insufficient rr group at all stages of the mobilizations (p [ 0.05). spo 2 was higher, while hr and rsr was lower at all stages in sufficient cr group compared to insufficient cr group (p \ 0.05). resting hr and cr may affect the safety of mobilization, for this reason it is important to consider respiratory and hemodynamic parameters prior to and while mobilizing the icu patients. introduction. obesity is a chronic disease and a major health problem. obesity in critically ill patients is associated with a prolonged duration of mechanical ventilation and intensive care unit (icu) length of stay [1] . objectives. the aim of this study was to investigate the effects of mobilization on respiratory and hemodynamic parameters in the critically ill obese patients. [ 30.00 kg/m 2 )] were included as soon as their cardiorespiratory stability allowed mobilization protocol. mobilization was defined as sitting in the bed, sitting on the edge of the bed, standing, walking to chair and sitting in the chair. heart rate (hr), systolic/diastolic/mean arterial blood pressure (sbp/dbp/mabp), respiratory rate (rr) and percutaneous oxygen saturation (spo 2 ) were recorded from the monitor. respiratory and hemodynamic parameters were collected just prior to the mobilization (supine position), just after the completion of the mobilization when the patient had been returned the supine position and 5 min of the recovery period. all parameters were compared with initial values. the ratio of partial pressure of oxygen in arterial blood to the inspired fraction of oxygen (pao 2 /fio 2 ) was calculated from the arterial blood gas samples before and after the mobilization. introduction. the use of respiratory therapy for patients with a variety of lung disease is a standard in medical care [1] , including in the intensive care unit (icu) setting [2] . in this context, it is widely accepted the routine use of physical therapy in several situations in the intensive care, such as the care of critically ill patients not requiring ventilatory support, assistance during the postoperative recovery and the assistance to critically ill patients requiring ventilatory support [3] . at present definitive recommendations cannot be made regarding the use of respiratory physiotherapy for decreasing relevant clinical outcomes in critical ill patients requiring mechanical ventilation. objectives. this study aimed to determine the impact of providing chest physiotherapy on the duration of mechanical ventilation, intensive care length of stay, intensive care and hospital mortality in mechanically ventilated patients. single-centre, randomized, controlled trial in a university hospital general intensive care unit (icu). were included in the study 139 patients aged more than 18 years, admitted to the icu needing mechanical ventilation for longer than 48 h. physiotherapists provide group intervention (p) with the intensity and frequency of therapy they felt appropriate based on their assessment of the likely treatment benefit. control patients (group c) only received suctioning, decubitus care and general mobilization. results. primary outcomes were icu and hospital mortality regardless of the cause of death. secondary outcomes were length of icu and hospital stay, length of mechanical ventilation, weaning and extubation failure. patients in the p group more frequently achieved parameters to start weaning, but there were no significant differences between p and c groups on weaning and extubation failure, length of mechanical ventilation and length of icu stay. there was fewer hospital, but not icu, mortality in the p group. conclusions. we demonstrated that respiratory physiotherapy decrease hospital mortality and suggest that this effect was, in part, secondary to the effect of the intervention on weaning from mechanical ventilation. introduction. critical illness can cause diverse cerebral dysfunctions ranging from unconsciousness to minor cognitive impairments (mci). severe cerebral dysfunction, as delirium, is known to affect outcome after critical illness but it is uncertain whether minor impairments affect mortality or morbidity [1] . objectives. the primary aim of this study was to estimate the incidence of mci in a group of general icu survivors immediately after icu stay and three and 12 months after discharge. secondary we wanted to explore if type of cerebral dysfunction after icu discharge affected mortality and morbidity. methods. patients admitted to our general icu were included prospectively. we included 80 patients. 11/80 (14%) were delerious and 14/80 (18%) were not delerious but had mmse \ 24 after icu stay. of the 55 patients with mmse c24, 28 were possible to classify as having mci or not. 18/28 (63%, 95% ci: 45-83%) were found to have a mci after icu discharge. on 3 and 12 months these numbers were respectively: 11% (95% ci: 0-20%) and 10% (95% ci: -1 to 21%) there was an increased risk of both death and being institutionalised at both 3 and 12 months regarding delirious patients and patients with mmse \ 24 compared to patients with mmse [ 24. no such differences were found regarding patients with or without mci. (tables 1 and 2) . conclusions. the incidence of mci after critical illness is high on discharge but drops on 3 and 12 months after. severe cognitive impairments affect mortality and morbidity, but minor cognitive impairments do not. objectives. this study analyzes mid-term survival and risk factors associated with survival of patients undergoing cardiac surgery in son dureta hospital. methods. 1938 patients were consecutively operated from november 2002 to december 2007. patients who were discharged alive from hospital were followed until december 2008. we did kaplan-meier survival analysis and logistic regression study of variables associated with mid term mortality. results. in-hospital mortality was 1.96% (95% ci: 1.36-2.60%). information was available on 1,844 (97%) of 1,900 patients who survived until hospital discharge. at the end of the follow-up period, observed mortality was 6.5% (ci 95%: 5.4-7.7%). survival probability at 1, 3 and 5 years of follow-up was 98, 94 and 90%, respectively. the mean time of follow-up was 3.2 years (range 0.01-6.06). patients c70 years showed a lower survival rate than patients \70 years of age (log rank \ 0.0001). age c70 years, history of severe ventricular dysfunction (ef \30%), diabetes mellitus, preoperative anemia and hospital stay were independently associated with mid-term mortality. conclusions. mid-term survival of patients alive after hospital discharge was very satisfactory. mid-term mortality varied according to age and several preoperative chronic diseases. a closed-ended questionnaire was developed by the nurse congress commission of the société de réanimation de langue française (srlf). an invitation to complete it online was sent by email to caregivers registered on the srlf push-list. results were analyzed by icu or by respondent. results. 731 caregivers working in 263 icus completed the questionnaire (65% were nurses, 24% were doctors, 9% were nurse's aides, 86% worked in adult icus and 14% in pediatric icus). 7% of adult icus (n = 222) had unrestricted policy but 58% had a visiting time of less than 4 h per day. at the opposite, 59% of pediatric icus (n = 41) had unrestricted policies. 63% of the respondents working in icus with a visiting time \4 h per day considered very useful or essential to enlarge visiting periods but 29% of them considered this enlargement as unhelpful. at the opposite, 9% of the respondents working in icus with unrestricted policy found very useful or essential to reduce visiting periods. 81% of caregivers working in icus with unrestricted policy but only 35% of caregivers working in other icus thought that an unrestricted policy was able to improve often or systematically the relations with families. moreover, only 8% of caregivers working in icus with unrestricted policy but 46% of caregivers working in other icus thought that an unrestricted policy disturbs the organization of care. 96% of respondents found very useful or essential to give information in a dedicated room whereas it was often or systematically done in only 52% of icus. identically, 97% of respondents found very useful or essential to give information to proxies with the patient's nurse whereas it was often or systematically done in only 54% of icus. some cares were often or systematically programmed for family participation in 56% of pediatric icus but in only 0.5% of adult icus. indeed, proxies often or systematically participated in nursing in 66% of pediatric icus but never in adult icus. at the opposite, proxies often or systematically participated in tracheal aspirations in only 5% of pediatric icus and in 0.1% of adult icus. conclusions. more than half of respondent's adult icus are closed but caregivers working in icus with unrestricted policy perceive it favorably. some improvements are also expected by caregivers on the use of dedicated rooms for information and on the participation of nurses in meetings with families. finally, participation of families to care is not a practice of french adult icu caregivers. methods. included: patients with dysfunction of two or more organs in the first 24 h, admitted and discharged from icu during 2008. excluded: neurocritical and politrauma patients. contact 1 year following discharge; questions were asked concerning symptoms related to a period in intensive care that presented following discharge and which were not present prior to admission. in the case that the patient was not contacted, the next of kin was asked. results. 154 patients included. general characteristics during admission to icu: 63% male; age 60.2 ± 17.5 years; sofa* 7 ± 2.8; apache** ii 16.8 ± 6.4; apache** iv 57 ± 18.8; length of stay in icu: 5.1 ± 28.4 days; 42.9% were on invasive mechanical ventilation and 17.5% on non-invasive mechanical ventilation. data collection was carried out over 18 ± 4.4 months, on average 19 months (range: 12-26 months). 18.2% (28 patients) had died at the time of contact. the person contacted was the patient in 33.3% of the cases, the spouse in 13.7% and immediate family (patient's parent/child/sibling) in 43.1% of the cases. 46.6% had difficulty sleeping following discharge from icu with an average time since discharge of 10.5 ± 8.9 months; 51.1% suffered feelings of sadness and difficulty in finding enjoyment which had persisted for 8.7 ± 8.1 months; 30.3% had experienced difficulty in concentrating over an average of 9.3 ± 8.8 months; 28.2% had suffered some form of memory loss after discharge over an average period of 8.9 ± 8.6 months; 76.5% presented with asthenia over an average of 10.8 ± 8.3 months; 64.7% had arthromyalgia over a period of 12.6 ± 8.4 months; 35.3% had experienced changes in appetite over an average of 6.6 ± 7 months; 43.3% had changes in intestinal habit over an average of 7.78 ± 9 months; of which 64.3% had diarrhoea, 30.4% constipation, and 5.4% both symptoms; 18.8% presented with headache over a period of 12.9 ± 9.2 months; 15.2% had tremors, that had not previously been present, over an average of 12 ± 7.3 months; 12.8% had experienced reduced vision, over an average period of 16.5 ± 7.1 months; 10.7% presented with speech/ language problems, over an average period of 14.6 ± 8.6 months; 14.5% exhibited newly presenting changes in micturition, over 16 ± 7.7 months. another less frequently occurring symptom was loss of hearing (1.3%). conclusions. severely ill patients that are admitted to icu frequently present with ''residual'' symptomatology following discharge, most notably arthromyalgia and asthenia. many of these conditions persist for months. intensive care unit (icu) readmission rates range from 3 to 12%, in spite of initial recovery from critical illness. previous researches report that the revised acute physiology and chronic healthy evaluation (apache ii) score at either admission or discharge is an important predictor for readmission after icu discharge. however, there are a few papers concerning the association of discharge apache ii score with readmission after discharge from surgical intensive care unit. objective. we compared the ability of the discharge apache ii score with that of the admission apache ii score in predicting readmission, especially early readmission within 48 h, after discharge from icu. conclusion. this study showed that both discharge apache ii score and admission apache ii score are useful predictors for readmission after icu discharge, but discharge apache ii score is only independent factor in predicting early readmission within 48 h after icu discharge. introduction. health related quality of life (hrqol) is decreased in former icu patients. in research outside the intensive care field it is well known that the psychosocial factors, coping strategies and perceived hopelessness affect hrqol. however, the influence of coping and hopelessness on hrqol after intensive care is unknown. objective. the aim of this study was to examine how coping strategies and perceived hopelessness among former icu patients compares to corresponding in a reference group. we also evaluated the effect of coping and hopelessness and icu related factors on hrqol. methods. prospective, multicenter study in three mixed icu's in sweden. patient demographics, length of stay, apache ii score, reason for admission and time on ventilator were collected for all adult patients. questionnaires, including the coping instrument pearling-schooler mastery scale (pms), the 2-item hopelessness scale, sf-36, demographic data and previous illnesses were sent 6 months after discharge from hospital to the patients. the reference group (n = 6,093) was a random selection of persons from the same catchment area as the study patients. . 780 (59%) icu-patients, 18-74 years, returned the questionnaires. the patients reported significantly lower mean scores in coping 13.6 (sd 4.6, p \ 0.001) and higher perceived hopelessness 2. conclusions. this study indicates that coping strategies and perceived hopelessness are important for the hrqol of previous icu patients. however, the magnitude of these effects are smaller than that of pre-existing diseases. introduction. mortality on a medical intensive care unit (icu) is estimated to occur in about 25% of patients. its association with age, severity of illness and comorbidities is well established. for other diseases like coronary artery disease it has been shown that pre-existing depression is a risk factor for worse outcome. the role of depression regarding the outcome of icu patients has not been investigated so far. we studied the association between pre-existing depression and mortality in medical icu patients and present preliminary data of this ongoing study. objectives. assessment of a possible association between mortality of icu patients and prevalent depressive mood at time of icu admission. the primary endpoint was 28-day mortality. methods. prospective cohort study. all patients admitted to a medical 8-bed icu in a university hospital, older than 18 years, were eligible. postoperative patients and patients who had an expected length of stay below 48 h (survey) were excluded. patients whose cognitive function allowed appropriate comprehension and response answered the hospital anxiety and depression scale (had). prevalent depressive mood at admission was defined by a score c8 in the depression dimension. all other patients were assessed by observer rating by next-ofkin. in this case the hammond scale, a validated instrument for observer rating of depressive mood (cut-off c4), and a modified version of the had for observer rating (cut-off c10) were used. in addition apache ii, saps ii, sofa, age, sex, comorbidities, reason for admission, length of icu stay and ventilator days were recorded. . by now 173 patients had complete follow up data. of these patients 53 (31%) were classified to have depressive mood at icu admission. in total 46 patients had died by day 28 (27%). the 28-day mortality was 40% (21/53) in patients with depressive mood and 21% (25/120) in patients without (p = 0.01). patients with and without depressive mood did not differ with respect to age, sex, apache ii, saps ii or sofa score at admission. multiple logistic regression analysis with 28-day mortality as the dependent variable revealed that prevalent depressive mood at the time of icu admission was an independent risk factor for mortality (table 1) . conclusions. pre-existing depressive mood is an independent risk factor for mortality in medical icu patients. introduction. some classical post-icu discharge predictors of death are described, such as age, severity of disease and level of nursing care [1] . besides these factors, some laboratorial data at icu discharge are potential predictors of post-icu death. objectives. the aim of this study was to investigate whether standard base excess (sbe), ph, lactate, hemoglobin level, creatinine, platelets, leukocytes and albumin at the icu discharge as well as the 25% decrease on c-reactive protein concentrations (crp [ 25%) from the day pre-icu discharge to the day of icu discharge may be useful predictors of in-hospital outcome. patients discharged from the icu after at least 72 h of stay were retrieved from our prospective collected data base. a multivariate analysis was performed using a backward-lr binary logistic model taking in-hospital death as a dependent variable and the cited data as independent variables. results. 396 patients were retrieved. the average age was 49 ± 19 years old, mean apache ii score was 17 ± 8, and the main causes of admission were septic syndromes and respiratory failure. the in-hospital mortality after icu discharge was 18%. the icu length of stay was 9 ± 9 days. at the time of icu discharge ph was 7.41 ± 0.04, sbe was -0.3 ± 4.0 mmol/l, lactate was 1.9 ± 1.0 mmol/l, hemoglobin 9.6 ± 2.0, creatinine was 1.3 ± 1.46 g/dl, albumin was 2.68 ± 0.64 g/dl, platelets was 280,645 ± 166,100/mm 3 , leukocytes was 10,795 ± 8,769 cells/mm 3 and the number of patients who lowered crp at least 25% were 159 (40% conclusions. this study demonstrated that sbe, lactate, hemoglobin and albumin concentrations on the day of icu discharge are independent predictors of in-hospital mortality. moreover, the reduction on crp levels above 25% in the last 24 h of icu stay is a strong predictor of better in-hospital clinical outcome. we suggest that these variables together with the clinical judgment may be taken into account on the icu discharge decision process. readmissions to the intensive care unit (icu) are usually associated with increased morbidity and mortality, and they may evidence the quality of patients' care. the risk for icu readmission varies across studies, and is generally analyzed just before icu discharge, leading to deviation of icu team and patients' daily goals. early prediction may improve the care for patients in risk for icu readmission, and help developing mechanisms for its prevention. objectives. to analyse risk factors for readmission in intensive care unit looking at the first 24 h data after unit admission. methods. the first intensive care unit admission of patients was analyzed from january to december 2009 in a medical-surgical unit. readmission to the unit was considered those during the same hospital stay or within 3 months after intensive care unit discharge. deaths during the first admission were excluded. demographic data, acute illness and comorbidity prognostic scores, and use of mechanical ventilation were submitted to uni and multivariate analysis for readmission. numeric variables were expressed as median or percentage. conclusions. age, medical admission, sofa score and respiratory-and/or sepsisrelated admission are early associated with increased icu readmission risk. objectives. the aim of this study was to examine patient perceived hrqol in former icu patients that die in the period from 6 month up to 3 years after discharge from intensive care unit and the hospital. methods. prospective, multicenter study in three mixed icu's in sweden. questionnaires, including hrqol (sf-36), demographic data and previous illnesses, were sent out six, 12, 24 and 36 months after discharge to all former adult icu patients. data for this study were only collected among those dying before the 36 months post-icu follow-up. of the 980 patients who returned the questionnaires 123 (12.6%) died, 31 (25.2%) between 6 and 12 months, 55 (44.7%) between 12 and 24 months, and 37 (30.1%) between 24 and 36 months. the most frequent admission diagnoses were respiratory problems n = 36 (29.3%) and gastrointestinal diseases n = 33 (26.8%). examining hrqol in the former icu patients the following observations were made: (see fig. 1 ). a pronounced and quantitatively large decrease in hrqol is seen for the surviving patients with pre-existing disease as compare to the previously healthy survivors. although already at a very low value further decreases in hrqol for the patients dying before 3 years post icu is significantly less as compared to the icu patients with pre-existing disease that survives. the decrease is mainly in physical function, role physical function and role emotional function (marked in the figure). conclusions. yes, health related quality of life is extensively affected, mainly in the dimensions physical function, role physical function and role emotional function. importantly, in these two affected physical dimensions a shorter time to death increases such a decrease. the finding further stresses the importance of pre-existing diseases for the final hrqol outcome of former icu patients. introduction. despite initial recovery from critical illness requiring icu admission, many patients remain at risk of subsequent deterioration and death [1] . recent studies have shown readmission rates ranging between 4 and 7% [2] ; this population had mortality rates six times higher and were eleven times more likely to die in hospital [1] . 1. to calculate the readmission rate in our mixed icu unit over a 6 months period 2. to identify risk factors associated with readmission into the icu 3. to study the outcomes of these readmissions methods. a retrospective observational study, data was collected from an icu computer database (metavision) and analysed manually results. the total number of admissions in this period was 365, average patient age was 57 ± 17 with 52.4% being males. readmissions constituted 10.5% of the total admissions with 23.8% of those readmitted within 24 h of their initial discharge. 40% of the initial discharges from the unit were made out of hours i.e. unplanned, presumably due to heavy demand on beds. readmissions were particularly associated with patients discharged to surgical wards 23.8% and the hepatobiliary hdu 28.6%, the latter might reflect the proportion of that particular patients population received. 52.4% of the readmissions required to stay 72 h or less in icu. the overall mortality of the patients requiring more than one admission in this 6 months period was 57.1%. there is an urgent need for expanding icu services in our hospital, i.e. extra beds, staff, outreach teams, etc in addition to investing in nursing capacity building especially in surgical wards. we agree with others studies that compared with the general population, icu survivors report lower hrqol. moreover, a relationship between several factors like sepsis, renal failure, sofa (first and second day score), critical illness polyneuropathy, mechanical ventilation, sedation time, previous psychiatric history and blood products transfusions were found in our study population. conclusions. according to our data, subclavian vein was the most common insertion site used, especially as 2nd and 3rd placement and was related with the lower incidence of becteremia episodes. although the risk of placing a cvc for inflection complications is against the risk for mechanical complications, we have to improve our cvc policy, preferring the subclavian or the jugular site of insertion, in order to minimize the infection risk for a nontunneled cvc. objectives. objectives for this study were to determinate the frequency and the risk factors associated with bos. secondarily, we searched several variables as civil status, age, sex, work seniority as potential risk factors. inclusion criteria were to work in critical care unit (ccu) the hospital clínico universidad de chile (hcuch). this unit included 2 subunits: intensive care unit (icu), middle care unit (mcu). the mbi ò instrument was applied between april to july of 2009. all staff of ccu were asked to response the instrument. as previously reported, bos was defined with high ee, high dp and low pa. risk of bos was anything of the three dimensions positive for bos. we gave information on specifics objectives and the schedule of a future intervention programme. for analysis, comparisons were made based on student t test, chi-square test with yates corrections or fisher exact test as corresponded. for all tests we used confidence interval 95% with p \ 0.05. a total of 111 mbi ò tests that included all sub-units in ccu. this is a 56% of all personal working in the ccu. bos was found in 8.26% of cases. women (75%), unmarried (75%), with an average of age 31.9 years old. (23-47 years old) and with a work seniority younger than 5 years (50%). ee is high (31.5%), for nurse and paramedical personals. dp was 22.5 and 31.5% to middle level, for nurse and medical doctor, and low pa in 34% for paramedical personal, with longer work seniority (more than 10 years). risk factors were female gender, unmarried status, childless, middle aged (23-47 years old) and recent start in the job (stay younger than 5 years). introduction. burnout is a prolonged response to chronic emotional and interpersonal stressors on the job, and is defined by three dimensions: exhaustion, cynism (depersonalization), and inefficacy. icu physicians are exposed to several stress factors and are particularly predisposed to this syndrome. to describe the prevalence of burnout syndrome among intensivists and its relation to their quality of life. methods. an epidemiological cross-sectional survey conducted to evaluate all adult icu physicians in salvador, ba (brazil), from october to december 2006. the quality of life and burnout syndrome were evaluated respectively by the whoqol-bref instrument and the maslach burnout inventory (mbi). burnout was classified into low, moderate and high levels for the three studied dimensions, according the mbi classification, and it was defined by the presence of a high level in at least one dimension. the quality of life was evaluated in four domains: physical, psychological, social relationships and environment, graduated from 0 to 100, with higher scores denoting higher quality-of-life. [1] ) has been successfully used to measure nursing workload on an intensive care unit over a 24-h period. in contrast to intensive care, the nursing care workload on mc is not evenly spread over a twenty four period, but tends to vary between shifts. objectives. the aims of this pilot study were (1) to assess the fitness of nas as an accurate reflection of nursing workload on an mc unit. (2) to determine the nursing work load, per patient, per 8 h shift. prior to the commencement of the study all thirty one nurses taking part received instruction in the content and registration of nas. at the end of each 8 h shift, each nurse retrospectively scored their patient(s) using nas. this consists of a check list containing twenty three items giving a possible score between 0 and 177, where 177 equates to 1.77 full time equivalent (fte) intensive care nurse. the nas were entered in to a database and the average scores, per patient, per shift were calculated. three hundred patients were retrospectively scored over a 2-month period in october and november 2009. not all patients were scored on all three shifts as some patients had been transferred out of the unit before shift end. in addition any incorrectly completed forms were discarded and excluded from the study. methods. this multicenter pilot study included 14 doctors working at (pediatric) intensive care units (icu). subjects were randomly assigned to two groups: one was first tested during day, then during night, while the other was tested in reverse order. the d2 test of attention [3] was used to assess attentional performance. total performance (tn-f) score, standardized for age and level of education, was used to express attentional performance. subjective, 1-to-10 scores were gathered in two questionnaires. results. figure 1 displays standardized total performance scores of 14 doctors. measured attentional performance showed high intra-and interpersonal variability and did not differ between both shifts (p [ 0.9). in contrast, doctors expected alertness to be decreased (7.5 ± 1.7 and 6.1 ± 1.7 (mean ± sd) on subjective 1-to-10 scale during day and night shifts, respectively; p \ 0.02) and the chance of making errors to increase (from 3.3 ± 1.7 to 6.2 ± 1.3 (mean ± sd); p \ 0.01) during night shifts. conclusions. physicians working at icu are aware of the risk of making errors during night shifts. however, we showed that doctors perform equally during night and daytime when confronted with a short-time challenging task. consequently, a discrepancy between measured attentional performance and expected alertness was observed. these results suggest nocturnal alertness might be comparable to daytime during short-lasting tasks that elicit a high level of stress and motivation (e.g. testing, medical emergency). further research is needed to elucidate if longlasting (routine) tasks reflect decreased sustained attention and contribute to medical errors. we studied 297 physicians, the majority of whom were male (70%). mean age and time since graduation were 34.2 and 9 years, respectively. high levels of emotional exhaustion, depersonalization, and reduced personal accomplishment were found in 47.5, 24.6, and 28.3%, respectively. prevalence of burnout syndrome, defined as a high score in at least one dimension, was 63.3%, while prevalence was 7.4% for all three dimensions. in conclusion, burnout syndrome was common in this sample of icu physicians. aims. our goal was to assess the physician's opinion about potential competencies of a triage nurse. a representative cross sectional study design was applied with self-fill-in questionnaire about physician's attitude related to skills of triage nurses. the questionnaires were distributed between september and november 2007 in 24 (out of 31) eds. in this survey 159 physicians' questionnaires were processed. chi-square and student-t test was used for comparison of variables. p values less than 0.05 were considered statistically significant. results. 79.2% of physician would support the special training of triage nurse. 55.3% of physician suggests that the nurses use the patient's physical examination regularly in eds. the full time (ft) emergency physician significantly would reduce the basic competencies of nursing (e.g. dressing, feeding of patient, p = 0.008, and p \ 0.001, respectively) than parttime (pt) emergency physicians. significantly greater part of the ft physician would widen the competency of triage nurses in the field of physical examination of nervous system (p \ 0.001) and cardiovascular system (p = 0.005) than the pt physician. conclusion. hungarian emergency physician would widen the competency of triage nurse, but only half of physician would like to that nurses apply physical patient examination in practice. the full time physician would give more competencies for triage nurse than part time ones, but the final field of competency will be depended on other factors. healthcare-associated infections (hcai) are estimated to affect 1.4 million people worldwide, causing longer hospital stay, increasing hospital costs and excess mortality [1] . hand hygiene represents the single most effective way to prevent healthcareassociated infections. compliance with hand hygiene amongst healthcare workers (hcw) has been demonstrated to be quite low at 40% [2] . to quantify the degree of compliance to hand hygiene norms in the icu and to assess the short term success of strategies to improve hand hygiene compliance. setting. 34 bedded medical-surgical icu in a tertiary care centre. design. prospective observational. method. unobtrusive observer (single person). observed over sessions of 1 h. the compliance was calculated as :number of times the staff performed hand hygiene/number of hand hygiene opportunities. the number of hand hygiene opportunities was based on the who tools [3] : before touching a patient, before clean/aseptic procedures, after body fluid exposure risk, after touching a patient and after touching patient surroundings. introduction. icu delirium represents a form of brain dysfunction that in many cohorts has been diagnosed in 60-85% of patients receiving mechanical ventilation. delirium is a common but complex clinical syndrome characterized by disturbed consciousness, cognitive function or perception, which has an acute onset and fluctuating course and is associated with poor outcomes. and yet, it can be diagnosed and treated. in the uk, reporting of delirium is generally considered to be poor. in light of updated nice guidelines on delirium due out this year, specialist clinical assessment will soon become gold standard as a means of diagnosing and reducing the prevalence of this condition in the icu setting. nice recommends that cam-icu (confusion assessment method) be used by healthcare professionals who are trained and competent in the diagnosis of delirium. on our 16-bed unit, we are currently implementing cam-icu assessments to be performed twice daily (at the commencement of each nursing shift) as well as rass (richmond agitation and sedation scale) scoring on an hourly basis for all patients. objectives. to implement training of all our icu nursing staff in the use of cam-icu and rass scoring. to periodically validate and reinforce earlier training, so as to improve assessment and reporting of delirium. methods. our 'delirium group' comprising both nursing and medical staff, taught cam-icu and rass to 48 staff members using multimedia presentations in small groups and/or individual teaching sessions over 6 weeks. scoringofcam-icu andrasswassubsequentlyauditedon3occasions post training. discrepancies were discussed and post-audit retraining provided where necessary. results. the following audit and validation data were generated on our unit as documented in table 1 . no statistical analysis was undertaken. we anticipate focusing on the challenges encountered and strategies used in managing this change in our icu practice. methods. the factors causing resistance to change based on multisource data. qualitative technical methods were used: brainstorming and focal groups. the data collection elaboration was created by the collaboration of icu nurse, quality department nurse and external reviewers. finally, the main factors were classified in different categories. each category was scored by 1 to 5 according to gravity and prevention possibility. finally, priority was given to more serious and easier prevention problems. results. the most serious problems for icu professional was the historical factors. the easier solution problems were ''the lack of information'' and all evaluators were agree with it. we arranged the factors in order to the next classification (tables 1 and 2 ). discussion. all investigators were agreed with the low importance of problems with payments and low prevention probability of low organisational flexibility, so they were agreed on not to work about them. the icu professionals were more pessimistic and have lower confidence in prevention possibilities but they showed more confidence about the capacity to learn new skills. they weren't worried about resistance to do experimental things. probably, historic factors play an important role in this pessimistic attitude. on the other side, quality and safety experts have more experience in prevention programs and they put all their trust in its. after doing the analysis, we chose the ''lack of information problem'' to plan prevention activities. we consider it is a serious and real problem but at the same time, easy of prevent. conclusions. the implementation of the patient safety program in the icu means a real cultural change. the priority analysis could help to plan strategies in order to avoid the program failure. objectives. we concerned about whether medical personnel could recognize management of the cuff of artificial airway or not. we asked to doctors and nurses working in intensive care unit of konyang university hospital, daejeon, republic of korea. we asked 12 questions with 18 contents of questionnaire that was composed of methods of set initially, maintenance and appropriate pressure of cuff. results. 96 of medical personnel replied to us. most of them had worked in intensive care unit, so they had placed of artificial airway. 86.4% of them used manometer to adjust the cuff. we could find that nurses had more cognition compared to doctors for it (94 vs. 76%). only 28.5% of doctors described pressure of the cuff in medical record. 89 of medical personnel replied that they knew the appropriate range of cuff pressure. 37% (32/89) of them replied that the range of cuff pressure was kept with 25-30 mmhg and 34% (30/89) was 20-25 mmhg. 70% of nurses in the icu knew that range of cuff pressure was 15-25 mmhg. most of them knew complications of high and low pressure of the cuff. 69.8% of medical personnel monitored the cuff balloon during receiving mechanical ventilation and they used manometer to adjust it. 78% of nurses knew that the cuff should be adjusted continuously, but 58% of doctors did. interval measuring the cuff pressure was 38% of once a day, 38% of three times a day, 17% of more than four times a day conclusions. most of the medical personnel knew to keep appropriate cuff balloon to prevent various complications of artificial airway. they had insufficient cognition about maintaining the cuff balloon and appropriate level of cuff pressure. that was more prominent in doctors than nurses interhospital transfer is occasionally required as a consequence of limited therapeutic options or because of a need for a higher intensity of medical care that cannot be given in rural intensive care units. along with the potential benefit for the to be transferred patient, transport may also lead to hemodynamic and pulmonary deterioration. in order to minimize additional risk of interhospital transport of critically ill patients, a mobile intensive care unit with a specialized retrieval team was established in our university hospital-based intensive care unit. from march 2009, transport of the critically ill patients in our adherence region are performed by micu. objectives. in this prospective audit adverse events and patient stability during micu transfers were assessed and compared to our previous data on transfers performed by standard ambulance [1] . results. 74 interhospital transfers over a 10-month period were evaluated. systolic blood pressure, glucose and haemoglobin were significantly different at arrival compared to departure, although never significant values for major deterioration were reached. an increase of total number of variables beyond threshold at arrival was found in 38% of patients, 32 percent exhibited a decrease of one or more variables beyond threshold and thirty percent showed an equal number of trespassed thresholds. there was no correlation between the patients status at arrival and the duration of transfer or severity of disease. icu mortality was 28%. compared to standard ambulance transfers of icu patients performed in 2005, there were far less adverse events: 12.5 vs. 34%, which in the current study were merely caused by technical (and not medical) problems. although mean apache ii score was significantly higher, patients transferred by micu showed less deterioration in pulmonary parameters during transfer than patients transferred by standard ambulance. conclusion. transfer by micu imposes less risk to critically ill patients compared to transfer performed by standard ambulance and has therefore resulted in an improvement of quality of interhospital transport of icu patients. introduction. previous studies in adult intensive care units (icus) reported rates of pre-mortem to post-mortem discrepancies ranging between 7 and 32% depending on the population studied. and, most of them were retrospective studies, which included small number of patients. to compare clinical and pathological diagnoses and to determine the types of errors in a large and multidisciplinary icu-patient population. we conducted a prospective study of all consecutive autopsies performed on patients who died in the icu of the hospital universitario de getafe, madrid, spain, between january 1982 and december 2007. the diagnostic errors were classified in two categories: class i errors that were major misdiagnoses with direct impact on therapy, and class ii diagnostic errors which comprised major unexpected findings that probably would not have changed therapy. conclusions. this study found significant discrepancies between clinical diagnoses before death and post-mortem findings. this reinforces the importance of the post-mortem examination in detecting otherwise unexpected diagnoses and improving the quality of care of critically ill patients. introduction. unplanned extubation is associated to a high risk of reintubation end correlates with increased risk of nosocomial pneumonia. on the other hand, reintubation significantly increases morbidity and mortality in critical ill patients, increasing the incidence of ventilator associated pneumonia (vap) rate and makes the airway management risky. objectives. the aim of our study was to test the rate of unplanned extubation as well as the reintubation rate in our icu, in order to evaluate the efficiency of our airway and weaning time protocols. methods. during a nearly 4 year's period, 216 patients admitted to the icu, mean age: 61.8 years, mean apache ii score: 18.5, mean los: 13.5 days, with predicted and actual mortality: 31.1 and 22.47% respectively. from these, 191 were intubated and included retrospectively in our study. 109 patients were extubated, while the others either underwent bedside percutaneous tracheostomy or died. we concerned that the number of days of mechanical ventilation were about equal to the number of days of intubation. reintubation was defined as the need to reintubate during the first 48 h after extubation. we recorded four episodes of unplanned extubation. three of them caused by malfunction of the tube due to secretions and airway obstruction and one of them was undesired extubation caused by the patient himself. the total number of days of intubation was 2,111, mean ± sd: 11.06 ± 11.87, min: 1, max: 51 days. therefore the rate of unplanned extubation was 1.897%, while the standard limit is below 15 %. the total number of reintubations was 19, while the total number of scheduled extubations was 109. therefore, the reintubation rate was 17.43%, while the standard limit is below 12%. conclusions.the recorded rate of unplanned extubation was low in our icu patients, below the acceptable limit, assuming that our sedation and airway management policy is effective. on the other hand, the recorded rate of reintubation was high in our study, above the acceptable limit. although a low rate of reintubation might indicate excessively long mechanical ventilation times, this did not recorded to our study. nevertheless, our data suggest that we have to improve further our weaning time protocols, making the extubation procedure safer, and avoiding risk factors for vap. . pvs such as inappropriate enrollment of patients with a contraindication to the study treatment may lead to excess harm in the active intervention group [1] and failure to deliver the study intervention according to the study protocol may underestimate true treatment efficacy [2] . full reporting of pvs may aid in the interpretation of rct results however there are no published reviews on this topic [3] . objectives. to determine reporting rates for key types of pvs and to investigate study characteristics that may be related to reporting. publications were excluded because they were subgroup or economic analyses of a previously published rct [6] , not a rct [5] , not published in the target journal [4] , systematic reviews [2] , or other reason [5] . median trial size was 700 participants (range: 20 to 162,387). 13/80 (16%) of rcts were single centre, 58/80 (72%) were industry funded and 21/80 (26%) reported negative findings. overall 71/80 (89%) of rcts reported some form of pv, these included: 51/80 (63%) patient compliance; 40/80 (50%) discontinuation of study intervention due to safety; 32/80 (40%) study intervention-related researcher error; 16/80 (20%) inappropriate enrollment and; 8/80 (10%) technical errors in randomisation. multi-centre rcts may be more likely to report study intervention-related researcher errors (44% of multi-centre trials vs. 15% of single centre trials, p = 0.06). academic trials were less likely to report discontinuation of study intervention due to patient safety (38% of academic trials vs. 80% of industry trials, p = 0.002) and were less likely to report technical errors in randomization (5% of academic trials vs. 23% of industry trials, p = 0.03). conclusions. multi-centre trials are accepted to be organizationally complex. on-site education may be required to reduce errors in study intervention delivery attributable to the research team. it is possible the apparent excess harm attributable to industry trials is a reporting artifact however, if it is real, it must be addressed. additional research is required to investigate patient safety-related pvs and technical randomization errors, which may be lower in academic trials. to determine the occurrence of harmless incidents and ae related to physician's competences in icus, disclosing their potential risk factors. conclusions. this prospective study was essential to identify the proportion of our icu admissions affected by md-inc and md-ae, disclosing their nature. our md-ae rates, affecting more than 40% of admissions, were higher than those described in prior general studies, including not only icus. among the detected md-ae, hypoglycemic episodes not related to insulin administration predominated, indicating important deficiencies regarding nutritional support. severity on admission and length of stay were important risk factors for the occurrence of at least one md-ae. a systematic measurement and analysis of unintended events (ue) have been recommended for patient safety and improvement of quality of care in critically ill patients. however, a spontaneous reporting system may be inefficacious in intensive care unit (icu) because of a poor data collection, particularly by physicians staff. objectives. the aim of this study was to evaluate the reliability of a staff spontaneous event report by comparison with events collected by an external observer in a surgical intensive care unit (icu). to facilitate the reporting and the analysis, we identified a series of events with a serial number and a colour code related to their for each of the following 5 macro-phases: icu bed booking, admission procedures, patient stay, discharge and emergency procedures. a specific structured form including ue's code and colour, date and hour of the event and type of patient has been prepared and proposed to staff 1-week for each month after a proper phase of education. the report was voluntary and anonymous and the data collected during the morning shift from september to december 2009 have been compared to those collected from an external observer. in the studied period, healthcare staff reported 53 ues: 51% collected by nurses, 47% occurred during the morning shifts and 67% were classified as moderate or severe. the rate of ue in the morning shift was 12 ues per 100 patient days. the external observer identified 55 events in 8 morning shifts with an incidence of 76 ues per 100 patient days. the violation of isolation rules for patient with multi-drug resistant bacteria infection both by icu staff and surgical consultant was the ue observed more frequently by the staff (26%) and by the external observer (35%). conclusions. the above data indicated that: 1. in our icu the incidence of ue is very high, particularly for compliance to isolation of infected patients and 2. the spontaneous reporting system under-estimated largely the real incidence of ues. introduction. importance of renal assessing in intensive care unit (icu) patients is unquestionable for a correct drug dosing, fluid requirements or decisions for renal replacement therapies. serum creatinine (sc) is a very common biochemical parameter in clinical practice for assessment of renal function. many equations have been designed to estimate creatinine clearance based on sc, but their capacities for providing a correct estimate of glomerular filtration rate (gfr) are suboptimal. this is even worse in critically ill patients due to malnutrition and/or immobilization. in clinical practice, despite its limitations, 24 h-urine creatinine clearance (crcl 24h ) is used as a reference method to determine gfr. data show that cystatin-c could be promising as an endogenous filtration marker in icu settings. objectives. to assess in a medical icu population whether the arnal-dade formula of cystatin-c clearance (cc) developed from serum cystatin-c (scc) shows better predictive performance of gfr than sc-based formulae, as regards to patients' renal function: crcl 24h c60 ml/min 1.73 m 2 or crcl 24h \60 ml/min 1.73 m 2 . results. all formulae showed notable bias from the reference method. interestingly, all equations based on sc-values clearly overestimated crcl 24h (cg: 19.6%; mdrd: 1.1%; fv-mdrd: 27.5%), whereas cc showed underestimation of these crcl 24h (cc: -17.5%). in the crcl 24h c60 ml/min 1.73 m 2 group (n c60 = 146; 89 patients), cc showed the best correlation indexes (cc-crcl 24h ; r 2 = 0.296, r = 0.544), the second most biased (-16.7%) and the worst precision (24.9%). in this group, mdrd was the least biased (-2.4%) and the most precise (21.3%). in the crcl 24h \60 ml/min 1.73 m 2 group (n \ 60 = 95; 54 patients), cc was the worst correlated with crcl 24h (r 2 = 0.214, r = 0.462), in contrast to mdrd (r 2 = 0.299, r = 0.547). in terms of precision, mdrd showed again better results than cc: 33.5% vs. 38.9%, respectively. conclusions. in our icu population, cc did not demonstrate a clear improvement on the remainder sc-based formulae in either of the two groups according to crcl 24h . however, in a patient with high mdrd values and suspicion of low gfr, cc could be useful as guidance before obtaining the definitive confirmation by crcl 24h . introduction. there are well established and robust techniques for measuring and categorizing renal function in people with chronic kidney disease (ckd). a number of rapid bedside estimates of renal function have been devised incorporating routine daily measurements, such as serum creatinine, in combination with demographic data (e.g. cockroft-gault, the mdrd series). the addition of serum cystatin c measurements to some equations may also improve accuracy of estimation. the current and accepted categorical classification of acute kidney injury (aki: akin/rifle) has been useful epidemiologically but does not provide a continuously variable measure of severity of aki which would be valuable for both clinical management and research. objectives. previously published abstracts have suggested a role for egfr in describing renal function in the critically ill but a more comprehensive analysis was needed. methods. 37 (21 male) (mean age 72 range 38-90) critically ill patients with aki were recruited. a 4 h creatinine clearance ( 4 crcl) (previously validated as a measure of renal function in critically ill patients) was measured and simultaneous blood sampling was done for creatinine, urea, albumin and cystatin c. various equations used to estimate gfr were compared to 4 crcl with regression and bland-altman analysis. all patients had a 4 crcl of\60 ml min per 1.73 m 2 introduction. epithelial-mesenchymal transition (emt), a key process in tissue development and repair, has also been identified as a major mechanism in fibrogenesis. the cytokine tgfb has been shown to induce transformation of epithelial cells into matrixforming and smooth muscle actin (sma)-expressing myofibroblast (mf) via emt. the other prerequisite is an injury-induced loss of intercellular contact, including adherens junctions (ajs). the classical experimental method to induce aj disruption is the uncoupling of e-cadherin-mediated contacts by low calcium medium (lcm). this concept has been termed as the two-hit model of emt (1). b-catenin, a scaffold protein of the aj, released by cell contact injury, can act as a transcription factor and has been shown to facilitate emt. however, the mechanism whereby cell contact injury promotes emt is not understood. our recent studies have shown that smad3, one of the main signal transducers of the tgfb pathway is a strong inhibitor of epithelial sma expression, by interfering with myocardinrelated transcription factor (mrtf) [2] . the latter is the main driver of the sma promoter, through it association with serum response factor (srf). intriguingly, b-catenin can bind to smad3. to clarify the mechanisms whereby aj injury promotes sma expression. methods. ajs were manipulated in kidney tubular cells, either by sirna-mediated downregulation of e-cadherin, b-catenin or through chemical uncoupling of ajs by lcm. protein expression was detected by western blotting and immunofluorescence microscopy, proteinprotein interactions were monitored by co-immunoprecipitation, and the activity of the sma promoter was determined by luciferase reporter assays. knockdown of e-cadherin promoted b-catenin translocation to the nucleus and induced a threefold rise in the tgfb-triggered sma expression. conversely, silencing of b-catenin strongly suppressed the two-hit (tgfb + lcm)-induced activation of the sma promoter, and inhibited sma protein and mrna expression by 50%. the same stimuli induced strong association of b-catenin with smad3. transfection of cells with a b-catenin expression vector dose-dependently prevented the inhibitory action of smad3 on the mrtfinduced activation of sma promoter. moreover the active (myogenic) mrtf-srf complex was restored, as b-catenin preempted smad3's inhibitory effect on the complex. these studies define a novel mechanism whereby epithelial injury activates the myogenic program, a central process in organ fibrosis. our results imply that b-catenin, liberated from the injured ajs, facilitates the activation of the myogenic program by preventing or mitigating the inhibitory action of smad3 on mrtf. these hitherto unknown interactions among smad3, b-catenin and mrtf represent novel targets to lessen fibrogenesis. introduction. in intensive care unit (icu) patients, kidney function is monitored by the creatinine clearance (crcl). it can be measured by two methods. urinary crcl (ucrcl) is directly measured, using the urinary and serum creatinine. but commonly crcl is estimated from serum creatinine (scr) alone, as estimated glomerular filtration rate (egfr); using equations validated in chronic kidney diseases. there is paucity of literature on validation and comparison of these methods in icu (hoste) . objectives. we compared 4-h timed ucrcl and egfr in the newly admitted critically ill. we also sought to ascertain the incidence of high crcl and the agreement between 2 methods in this subgroup. conclusion. the use of rifle criteria gives a high incidence of aki in the icu setting. in this unselected population of critically ill pts, cysc seems to be superior to cre in predicting pts who will develop aki and will need rrt during their hospitalization in the icu. early identification of high risk patients may allow potentially beneficial therapies to be initiated early in the disease process, before irreversible injury occurs. introduction. the contrast-induced nephropathy (cin) is consider to be the most frecuence reason of acute renal failure in hospitalized patients. they are defined by a fixed increase (0.5 mg/dl) o a 25% rise serum creatinine level after to be exposed 24 h to the contrast. the main complications are kidney and cardiac problems and this will lead to longer hospitalization and increased mortality. objectives. to compare cin occurrence after a injecting a iso-osmolar contrast (ioc, idixanol) or a low-osmolar contrast (loc, iohexol) to a group of patients submitted to coronary angiography, with o without percutaneous coronary intervention (pci). to establish unrelated cin markers and to evaluate the efficiency of the kidney protection protocol used in our hospital. conclusions. the loc was associated to a greater number of cin than ioc. patients who developed cin were significantly longer hospitalized. the use of point giving system that includes cin's predictors like dm, hematocrit \39%, ami, and treatment with diuretics helps us to classify cin risk and use a correct kidney protection protocol. introduction. the incidence of acute renal failure in the intensive care unit (icu) is around 50% of cases and is related to increase in mortality in patients who required dialysis as far as 80%. early detection of acute kidney injury (aki), after damage is not on set could be crucial to develop therapeutic strategies to modify the course of injury. blood and urinary concentrations of ngal are early biomarkers of aki 1 ; to date, little information exists regarding ngal usefulness in critically ill patients. objectives. to analyze: 1. the capacity of urine ngal (ungal) to predict akievaluated by rifle score-in critically ill patients and, 2. the ungal values in patients with sirs, sepsis or septic shock. methods. ngal was measured in urine sample by an automatic analyzer device (architect ci4100 ò ; abbott diagnostics) at admission and 48 h later in patients admitted to a general icu. patients were classified both by rifle score at admission and 24 and 48 h later and by ungal concentrations at admission. to the later classification, the cut-point for aki prediction was obtained by roc curve analysis. ungal values at admission were compared in patients with sirs, severe sepsis or septic shock. clinicians were blinded to ngal results. the study included 76 consecutively-admitted patients (29 female) with mean age 59.6 ± 17.6 years, and length of icu stay of 10.4 ± 10 days. fifty-four sirs, 6 severe sepsis and 16 septic shock. thirteen patients developed rifle f score, 9 of them at icu admission; extracorporeal renal therapies were required in 6 cases. when patients were classified according to their rifle score at 24 h of admission, ungal values at admission were: 26 (10-81) ng/ml in 48 patients with rifle 0, 163 (49-998) ng/ml in 5 with rifle r, 357 (151-596) ng/ml in 8 with rifle i and 384 (31-539) ng/ml in 10 with rifle f (p = 0.02). five patients were excluded, three died before 24 h with ungal 816 (17-1,029) ng/ml and two were discharged before 24 h with ungal 182 (13-352) ng/ml. the area under roc curve of ungal at admission for aki prediction was 0.84 (95% confidence interval 0.74-0.92, p \ 0.0001), with an optimal cutoff value of 114 ng/ml with 69% sensitivity and 84% specificity. forty-seven patients have ungal b 114 ng/ml. ungal concentrations at admission were 30 (10-132) ng/ml in 54 patients with sirs, 36 (10-235) ng/ml in 6 patients with severe sepsis and 400 (126-592) ng/ml in 16 patients with septic shock (p = 0.001). conclusions. urine ngal concentrations measured at icu admission appeared as a useful predictor of aki in critically ill patients; in addition, ungal concentrations showed an increasing pattern from sirs to severe sepsis and septic shock. 23rd esicm annual congress -barcelona, spain -9-13 october 2010 s403 introduction. two previous studies using the rifle criteria in intensive care patients have found the incidence of acute kidney injury (aki) to be 36 and 67%. however, these studies used calculated basal value of creatinine in a considerable proportion of their patients, which is a possible source of error. objectives. the aim of this study was to investigate the incidence and severity of acute kidney injury in intensive care patients using true baseline creatinine values. objectives. the aim of this study was to define the status of hcy and b vitamins at admission and 7 days of icu stay in critically ill patients, and to evaluate its relationship between them. a prospective study was done on critically ill consecutive patients with inclusion criteria: c18 years old, sirs and apache ii [15. hcy, b12 and folic plasma levels were measured by enzymoimmunoassay and enzymatic method. for b1, b2 and b6 in erythrocyte. permission was obtained from an institutional ethical committee and written informed consent was asked. results. at 0 and 7 days of icu stay 50 and 53% of patients were b 1 deficient, respectively. 12 and 20% were b 12 deficient on both times, respectively. folic levels show significant differences between 0 and 7 days of icu stay. we found association between b 2 vitamin and hcy at admission and 7 days. no differences were found between 0 and 7 days hcy values. introduction. cytochrome p450 3a (cyp3a), the most abundantly expressed cytochrome p450 enzymes in liver, are responsible for the metabolism of over 50% of drugs used across several therapeutic classes. in adults, cyp3a is represented primarily by the major isoform, cyp3a4, and a polymorphically expressed isoform, cyp3a5. individuals with at least one wild-type cyp3a5*1 allele synthesise functionally active enzyme while homozygotes for the *3 allele are functional non-expressers of the enzyme. the presence of functional cyp3a5 increases the hepatic metabolism of cyp3a substrates such as tacrolimus. ckd is known to reduce the hepatic metabolism of drugs via the cyp3a enzyme system and we have shown, recently, that aki has a similar effect and that the length of time with aki is the most important variable. we hypothesise that expression of functional cyp3a5 may reduce the impact of aki on hepatic drug metabolism as has been shown to be the case for drug interactions with the imidazole antifungals. methods. 72 (45 male) (mean age 72 range 23-90) critically ill patients with no aki and varying degrees of severity of aki were recruited. midazolam concentration was measured 4 h after intravenous administration as a probe-drug for hepatic cyp3a 4/5 enzyme activity (t 4 [midazolam] ). this is a validated method for testing cyp3a activity in critically ill patients. patients were excluded if they were on any known cyp3a4/5 inhibitors. results. two patients with severe aki had unexpectedly high t 4 [midazolam] . figure 1 demonstrates the following: without a cyp3a5*1 allele, the rate of midazolam metabolism increased with duration of aki (r 2 = 0.24; p \ 0.0001) (solid line). patients who had at least one *1 allele (dashed line) were protected from the inhibitory effect that aki has on hepatic drug metabolism (significant difference between the correlation lines p = 0.011). if the two major outliers are removed (dotted grey line) from the *3/*3 group (r 2 = 0.23; p \ 0.001), the correlation lines remain statistically different (p = 0.048). conclusions. the presence of an allele which codes for functional cyp3a5 protects critically ill patients from the inhibitory effect of aki on the hepatic metabolism of midazolam. thyroxine replacement therapy has become commonplace in the management of organ donors to reverse hemodynamic instability and homeostasis, yet the pharmacokinetics of thyroxine are unknown in this patient population [1, 2] . since t3 is only available in oral form, we studied the pharmacokinetics of oral versus intravenous t4 to determine if oral administration is suitable. objectives. (1) to study the pharmacokinetics of oral versus iv t4 therapy; (2) to determine if oral thryoxine therapy is suitable. with ethics approval and signed consent from the substitute decision maker, 34 patients who were determined to be neurologically dead and consented for organ donation, were randomized to receive either an oral or intravenous dose of t4 (2 mcg/kg). all patients received an oral and iv preparation; one of which was a placebo. this study was also double blinded and randomization occurred in blocks of 4-6. free serum levels of t4 and t3 were measured hourly until the time of organ procurement. the area under the curves (auc) were determined and compared using. results. there were 15 patients (13 males) in the oral versus 17 patients (8 males) in the iv group, with an average age of 61 ± 14 vs. 53 ± 17, respectively. there was no significant difference at baseline or 6 h between groups for hemodynamic variables, free t4, free t3 or tsh levels. the only exception was map where it was higher at baseline in the oral group and there was a significant increase at 6 h in the iv but not the oral group (77-96 vs. 91-89 in the oral). the auc for t4 was greater for the iv group (301 pmol/l/12 h) compared to the oral group (274 pmol/l/12 h). there was no statistically significant difference in any of the levels from 0 to 12 h between the oral and iv groups. oral bioavailability of t4 was 91%. conclusions. administration of iv t4 resulted in a slightly greater auc compared to oral administration. however, oral bioavailability of t4 in our population was very high, at 91%. t3 is currently the recommended thyroid replacement in neurologically dead organ donors. however, intravenous t3 is unavailable in many jurisdictions. iv t4 has been used as a substitute. our study shows that in this select population, oral bioavailability is high suggesting that oral t4 may be a reasonable alternative. further work is needed to determine whether there was a difference in the number and rate of organ retrieval in the oral versus intravenous groups. introduction. specific characteristics of metabolic derangements occurring in critical illness is domination of developing catabolic state particularly in acute necrotizing pancreatitis. as a result, we faced such a problem as developing a clinically apparent protein-calorie deficiency which is resistant to standard nutritional support. the treatment of acute necrotizing pancreatitis in chronic abuse patients is difficult to handle for the clinician and should include sufficient energoplastic supply. objectives. in our research we aimed to assess the efficacy of adding of ornithineaspartate complex in carbohydrate metabolism in chronic abuse patients with acute necrotizing pancreatitis. methods. 56 comparable chronic abuse patients with acute necrotizing pancreatitis (control group n = 28, mean age 45.1 ± 8.5; ornithine group n = 28, mean age 44.0 ± 8.1) received early parenteral nutrition from the moment of admission to hospital with universal system ''three-in-one''. ornithine group also received ornithine-aspartate complex by parenteral administration (40 g/day). on the second day the patients were admitted parenteral nutrition and tube feeding 24 h/day. the volume of parenteral nutrition was gradually decreasing. biochemical and metabolic endpoints were measured at baseline and on 6th day (nitrogen balance, amino acids spectrum, plasma whole protein, transferring concentrations, glucose and insulin levels) at the clinical laboratory in all patients metabolic disturbances with protein status and carbohydrate metabolism shifts were revealed. dynamic of the whole protein, albumin/protein ratio and nitrous balance in both group showed similar tendency of metabolic improvement. dynamic of essential and nonessential amino acids concentration remained normal showing adequate energoplastic supply in both groups. glutamine concentration in ornithine group remained stable and even increased by the 6th day of nutritional support, while in control group glutamine concentration was decreasing, and by the 6th day of nutritional support it was below normal values. in ornithine group higher levels of endogenous insulin at normal values of glucose and faster fisher index improvement were detected. conclusions. administration ornithine-aspartate complex in therapy of acute necrotizing pancreatitis in chronic abuse patients, probably, may influence on disease outcome. in ornithine group duration of delirium tremens causes was 4 ± 1 days versus control group (6 ± 1 days). restoration of metabolic activities confirms adequate nutritional support in both groups but ornithine-aspartate complex adding provides faster improvement of protein and carbohydrate metabolism. objectives. this study was designed to evaluate the nutrition indexes including serum prealbumin level as prognostic indicators of patient recovery in critically ill patients with comparing severity scoring systems. we selected 59 patients over 16 years old, supplied with total parenteral nutrition (tpn) for more than 7 days in surgical intensive care unit, ajou university hospital, suwon, korea. the serum prealbumin, albumin levels and total lymphocyte count were measured at the first, 3rd, 5, 7, 10, 14th days of nutrition support care by tpn. we checked apache (acute physiology and chronic health evaluation) ii score, saps (simplified acute physiology score), mods (multiple organ dysfunction score) and sofa (sequential organ failure assessment) score of patients. results. there were 43 male patients and 16 female patients with mean age 54.3 years. the mean day of sicu staying was 13.2 days. we compared two groups; survivor group (n = 49) and non-survivor group (n = 10). there were significant statistical differences in icu staying days (p = 0.021), apache ii score (p \ 0.001), saps (p \ 0.001), mods (p = 0.007) and sofa score (p = 0.005) between two groups. however, serum prealbumin level (p = 0.370), albumin level (p = 0.444) and total lymphocyte level (p = 0.584) did not showed significant difference between two groups. receiver operating characteristic curve showed low accuracy of serum prealbumin level as a prognostic factor (area = 0.559). prealbumin level showed correlation with albumin (r = 0.561), however did not show correlation with apache ii (r = -0.151), saps (r = -0.056), sofa (r = -0.056) and mods (r = -0.076). conclusions. nutrition indexes including prealbumin did not correlated with clinical outcome of critically ill patients. introduction. physical function is impaired following critical illness [1] . anaemia is a common complication of critical illness and has the potential to influence physical function [2] . it is not known whether anaemia affects the physical components of quality of life, the ability to carry out the activities of daily living (aodl) or the actual physical function of patients during recovery from critical illness. to determine the physical quality of life, ability to perform activities of daily living and actual physical function in a cohort of icu survivors dichotomised on the presence of anaemia at 3 months following icu discharge. one other organ failure were recruited from a general icu population. patients with a preexisting haematological condition were excluded. baseline and characteristics of icu stay were recorded. the patients were assessed with the sf-36 quality of life questionnaire (pcs), the frenchay activities index (fai) of aodl recalled for pre-morbid status and at 3 and 6 months, and the 6 min walk test (6 mwt) for actual physical function at 3 and 6 months following discharge from icu. organotopic measures of haemaglobin, creatinine, serum c-reactive protein and albumin concentration were also recorded. the results were dichotomised on the presence of anaemia at 3 months for statistical analysis. baseline characteristics were compared with student's t test. a 2 way anova was performed on the pcs and fai score as well as comparisons with t test between each time-point. the distance walked as part of the 6 mwt was compared with mann-whitney u test. patients who remained anaemic at 3 months were older, had a longer icu stay and had a greater requirement for inotropes during their icu stay. the pcs score of quality of life and the fai score was significantly impaired in both groups during follow up, but there was no effect of anaemia. the results of the t tests showed that there was a significant difference between the groups at 6 months for pcs but not for fai scores. the distances walked were severely impaired compared to the normal population (170 and 228 m at 3 and 6 months for anaemic group and 240 and 358 m for non-anaemic) in both groups was not significantly different between the two groups. the non-anaemic group did increase the distance walked significantly from 3 to 6 months. there was no difference between albumin, crp and creatinine concentrations between the groups. methods. this experiment was divided into two procedures. the first procedure is to choose two kinds of cell strains, including jurkat cell strain (comes from leukemia) and ccrf-cem cell strain (comes from acute lymphocyte leukemia).we cultivate this two kinds of cell strains to mature stage, then inoculate every kind of cell strain into four culture dishes, two culture dishes was stimulated by 1 lg/ml lipopolysaccharide(study group), and the other two culture dishes serve as blank control(not stimulated by 1 lg/ml lipopolysaccharide). eight hours later, we extracted the microrna in each culture dish. the second procedure is to use the technique of gene microarray to analysis the difference expressions of microrna. in the context of a high altitude expedition human subjects can safely be submitted to prolonged hypoxia and the resulting changes in mitochondrial function can be explored in a controlled fashion. the effect of hypoxia on immune cells-key players in the pathophysiology of sepsis-is of particular interest. to measure mitochondrial function of monocytes during prolonged hypobaric hypoxia. methods. serial blood samples were collected and oxygen saturation was measured in twelve climbers before and throughout a high altitude climbing expedition to pik lenin (7,134 m). measurements were performed at 440 m (baseline) and at the altitudes of 3,600 m (day 1), 4500 m (day 4) and 5,400 m (day 9) above sea level. pure monocytes were isolated by the use of an antibody-antigen mediated immunomagnetic cell isolation procedure and lysed for determination of activities of mitochondrial enzymes cytochrome c oxidase and citrate synthase. repeated measurements anova followed by least significant difference (lsd) post hoc test were used to compare results on different altitudes. mean oxygen saturation was 91 ± 3% on 3,600 m, and decreased to 86 ± 3% on 4,500 m and 81 ± 5% on 5,400 m (p = 0.001). we observed an increase in citrate synthase activity on all altitudes compared to baseline levels (p = 0.003). compared to the baseline, prolonged hypobaric hypoxia induced an increase in the mitochondrial respiratory chain enzyme cytochrome c oxidase enzymatic activity only at 4,500 m (p = 0.003). normalization of cytochrome c oxidase enzymatic activity by citrate synthase activity (relative enzymatic activity) yielded a decrease in relative cytochrome c oxidase enzymatic activity during hypoxia on 3,600 and 5,400 m (fig. 1) . expressing cytochrome c oxidase enzymatic activities as a ratio to citrate synthase is intended to act as a safeguard for potential differences in mitochondrial enrichment. conclusions. the data demonstrates that prolonged hypobaric hypoxia leads to a decrease in relative cytochrome c oxidase activity. this is due to an increase in citrate synthase activity as a marker enzyme for the mitochondrial matrix representing mass and/or number of mitochondria which is not counterbalanced by a corresponding increase of cytochrome c oxidase activity. results. glycocalyx degradation was increased in the lps-treated animals (0.3 lm, p \ 0.05) compared to controls. intracellular tissue no concentrations were two-to threefold higher in the lps-treated mice compared to controls (liver, kidney, heart, gut). the number of infiltrating mpo-positive cells increased significantly during endotoxemia. levels of both plasma arg and cit were significantly lower in lps-challenged mice than in controls, whereas plasma ornithine levels were significantly higher. conclusions. in this new developed murine sepsis model, the prolonged infusion of lps resulted in increased glycocalyx degradation and associate endothelial leakage. the enhanced no levels correlated with decreased plasma levels of arg and cit. our murine model with prolonged infusion appears applicable as a model for the human clinical situation, enabling adequate investigation of the influences of the arg-no metabolism on endothelial dysfunction in sepsis. critical illness polyneuromyopathy is a muscular weakness occurring in intensive care unit. one of the major risk factor is sepsis. an early decrease in membrane excitability was described [1] but corresponding mechanisms are imperfectly known. tnfa is released in the first time of sepsis and could be involved in the physiopathology. objectives. the aim of our study was to investigate tnfa effects on muscular voltage gated sodium channels (nav) in an in vitro model. early effects of tnfa on nav were analysed by macro-patch clamp on muscular fibers isolated from rat peroneus longus. measurements were performed on control fibers and after addition of tnfa at concentrations ranging from 2.5 to 100 ng.ml -1 . the effects of chelerythrine, a specific inhibitor of protein-kinase c (pkc), were also tested. experimentations were realised in a laboratory with permission of experimental research on animals and under the supervision of an authorized person (no 29-028). tnfa produced a concentration-dependant inhibition of nav currents (fig. 1) . maximal inhibition (75% of control current) was observed with concentrations from 50 ng ml -1 and above. this decrease was fast: 40% of maximum inhibition was observed in less than 5 min. moreover, chelerythrine inhibited tnfa action on nav. conclusions. in our experimental model, tnfa induce a rapid and concentration dependant decrease of muscular nav currents like observed in chronic sepsis [2] . as this effect is too quick to be a transcriptional one, and as it is blocked by chelerythrine, it can be assumed that tnfa action is mediated by a nav phosphorylation secondary to pkc activation. in conclusion we evidenced that tnfa reduce muscle excitability in the early stages of sepsis. further studies are needed to obtain a precise description of tnfa mechanisms. may also contribute to cell signaling and regulation of the immune response. nad(p)h oxidase in leukocytes and the vascular wall is a major regulated source of o 2 . we hypothesized that mice deficient in the p47phox (ko) component of nad(p)h oxidase would have less pulmonary inflammation than wild type (wt). we treated wt or ko mice with iv saline or lps and assessed lung injury by: 1. wet-dry-weight ratio; 2. leak of evans blue (eb) labeled albumin; and 3. histological score for edema. we used myeloperoxidase activity to indicate neutrophil (pmn) accumulation in lungs, and measured accumulation of macrophages and neutrophils in bronchial alveolar lavage (bal). apoptosis was assessed by tunnel staining. we also expression of icam-1, an adhesion molecule, and nitric oxide synthase (nos) enzymes, enos and inos (western and northern analysis) as well as nitrotyrosine formation. results. lung injury was increased in both groups. surprisingly there was greater eb leak in ko than wt at 2 h and a greater edema score at 4 and 8 h. pmn and macrophage accumulation in bal were the same in both groups at 2 h but greater in ko mice at 8 h. myeloperoxidase activity was similar at 2 h post lps in ko and wt indicating that similar accumulation of pmn in the lungs. apoptosis was increased in both groups at 4 h, but resolved in wt at 24 h and persisted in ko. nitrotyrosine was increased in both groups but appeared higher in ko. expression of enos and inos increased in both groups but was greater in ko than wt. conclusions. in contrast to our prediction, lung injury was greater in p47phox ko mice which indicates that this complex is not essential for lung injury. however, the injury was more severe and prolonged in ko mice indicating that o 2 may regulate the inflammatory response. introduction. septic shock remains the main cause of mortality in the icu, thus a persistent challenge. recently, dna and mrna analysis by microchip and gene expression by real time pcr highlighted 2 proteins s100a8, s100a9 and their complex, known as the calgranulins, as potential key prognostic markers for this disease: those two proteins, whose expression seems to be restrained to phagocytes cells are newly recognized components in sepsis-induced inflammation. moreover, they were shown to be at significantly higher concentrations in the plasma of septic shock patients that were going to die. in the contrary, those who were to survive saw their plasmatic concentration decrease, all severity scores in between the 2 population being the same. objectives. the aim of this study was to determine the repartition of these proteins in immune cells, their intracellular variation, at baseline and after cell activation and finally to understand the relation between their intracellular and extracellular expression. we used an in vitro model close to the immuno-inflammatory aggression that is septic shock. we stimulated in vitro for 1, 3 and 6 h whole blood from healthy volunteers using agonists found in the inflammatory storm that is septic shock (lps, fmlp, gmcsf, ifng). we also induced death cell, either using an apoptotic agonist, or by necrosis technics. we then analysed the intracellular variation of the calgranulins using flow cytometry technics. the extracellular quantification was made using elisa methods. all the statistic analysis were made using a mann-whitney test. we showed in this work for the first time that the intracellular repartition of the calgranulins is different depending on the type of cell: the complexe is the main form in the monocyte cytoplasma, whereas s100a9 is the main intracellular form of the pmn. this repartition remains after cell activation. we also checked the absence of calgranulins in lymphocytes. after cell activation we showed that intracellular s100a8, s100a9 and s100a8a9 increased, but at different levels depending on the cell and the agonist used. extracellular s100a8 also raised after cell stimulation, but the concentration found were very low compare to those found in the plasma of septic shock patients. conclusions. together, these results suggest a different regulation depending on the form of the protein and of the cell and thus of proper distinct function of each monomer and of the complex. in the limits of our model the increased concentrations found in the plasma of patients with a septic shock can't be explained by immune cell activation. objectives. although there is no specific antidote for these potent toxins, drugs like penicillin g and silibinin have been used with conflicting evidence. we successfully managed two patients with mushroom poisoning by using silibinin and nac. methods. two members of a family, a mother 38 years old, and her son 15 years old were admitted to our icu 36 h after the ingestion of wild mushrooms. they presented with abdominal cramps, vomiting, profuse diarrhea ([20/day), myalgias, confusion and agitation. the clinical examination showed severe dehydration, tachycardia, oliguria with grade i-ii hepatic encephalopathy. laboratory exams revealed elevation of liver enzymes sgpt: 1106/880 u/l, sgot: 1884/930 u/l. coagulation parameters were as following: prothrombin time 15.5 00 /15.1 00 , factor v \55%/60%, factor vii \50/55%. high ammonia levels were noted, reaching 360 and 400 ng/dl, respectively. metabolic acidosis was also present with mild renal dysfunction. the ultrasound performed in both patients showed hepatosplenomegaly. aggressive fluid and electrolyte replacement started upon admission. silibinin was given at a dose of 20 mg/kg/day intravenously, in four divided doses, for three consecutive days, while nac was given as a continuous infusion at a dose of 150 mg/kg for the first hour, 50 mg/kg for the next 4 h, and thereafter 100 mg/kg/day for the following four days. hepatic encephalopathy, mild jaundice and renal dysfunction resolved within 48 h, and liver function tests returned to normal within 4 days. the patients recovered fully and were discharged to a medical ward. recent experimental and clinical studies have shown a strong protective and antioxidant effect against hepatic cell injury in amanita toxicity by the administration of nac and silibinin, either as monotherapy or as a combination therapy. although further clinical research is required to confirm their efficacy in reducing mortality and transplantation rate, nac has been used in our icu in hepatic dysfunction of different etiologies with promising results. we have recently shown that in patients with lactic acidosis due to metformin intoxication (serum drug level = 61 ± 25 lg/ml; therapeutic level is b4 lg/ml) systemic oxygen consumption (vo 2 ) can be abnormally low despite a preserved global oxygen delivery (do 2 ) (1). the study, however, suffered from being retrospective. objectives. to prospectively clarify whether metformin primarily impairs vo 2 . methods. eight sedated, paralyzed and mechanically ventilated pigs received a continuous i.v. infusion of metformin, at a rate of 1.6 g/h. the amount of metformin administered to each animal ranged from 4 and 8 g. the experiment always finished 9 h after the initiation of drug infusion. use of sedative and neuromuscular blocking drugs, as well as ventilatory setting, were always kept constant. serum metformin concentration was measured at the end of the experiment, using high performance liquid chromatography (hplc). arterial ph, lactatemia, vo 2 (indirect calorimetry) and do 2 (computed from cardiac output measured by pulmonary artery thermodilution) were recorded hourly. data are presented as mean ± sd. statistical testing was performed using the one-way repeated measure anova and the linear regression analysis. metformin infusion produced toxic serum drug levels (77 ± 30 lg/ml; n = 5). arterial ph drop from 7.63 ± 0.05 (prior to infusion) to 6.96 ± 0.26 (end of the experiment) (n = 8; p \ 0.001) and lactatemia rose from 1 ± 0 to 17 ± 7 mmol/l (n = 8, p \ 0.001). vo 2 progressively decreased (from 117 ± 37 to 66 ± 30 ml/min; n = 8, p \ 0.001) while do 2 did not significantly change over time (from 274 ± 74 to 239 ± 66 ml/min; n = 7, p = 0.39). the decrease in vo 2 was proportional to the dose of metformin administered (r 2 0.62; n = 8, p = 0.02) and to the serum drug level reached by the end of the experiment (r 2 0.69; n = 5, p = 0.08). conclusions. lactic acidosis develops during metformin intoxication in the presence of a diminished vo 2 but in the absence of any clear evidence of inadequate do 2 . this finding suggests that impaired oxygen utilization, rather than availability, may have a role in the pathogenesis of metformin-induced lactic acidosis. : 180 min) . death was consequent to multiorgan failure, anoxic encephalopathy or capillary leak syndrome if ecls was performed under cardiac massage. four patients presented with documented brain death, allowing organ donation in 2 cases. among these patients, the heart of one flecainide-poisoned patient was successfully transplanted, after normalization of ecg and myocardial function as well as toxicant elimination under ecls. prognostic factors in ecls-treated poisoned patients were as follows: qrs enlargement on admission (p = 0.009), saps ii score on admission (p = 0.005), ecls performance under massage (p = 0.008), arterial ph (p \ 0.001), lactate concentration (10.7 [6.6-19.6] versus 15.0 mmol/l [6.2-29.5], p = 0.003), as well as red cell (p = 0.008), fresh plasma (p = 0.003), and platelet (p = 0.03) transfusions within the first 24 h. conclusions. to our knowledge, this is the larger series of ecls-treated poisoned patients ever reported. ecls appears to be an efficient salvage technique in case of refractory toxic cardiac failure or arrest, with a 28% survival rate. our series clearly demonstrate that toxic refractory cardiac failure remains the best indication with a 46% survival rate. objectives. aim of the study was to investigate the incidence of infections in patients treated with hypothermia while receiving sdd. in this retrospective case control study 35 patients treated with prolonged hypothermia (cases) were identified and 169 patients with severe brain injury were included (controls). propensity score matching was performed to correct for differences in baseline characteristics and clinical parameters. primary outcome was the incidence of infection. the secondary endpoints were the micro-organisms isolated from surveillance cultures and during infection. the demographic and clinical data indicated that the cases and controls were well matched. the length of stay in the icu and duration of mechanical ventilation were comparable between the groups. the overall risk of infection during icu stay was 20% in the hypothermia groups versus 34.4% in the normothermia group (p = 0.388). pneumonia was diagnosed in 11.4% of patients in both groups (p = 1.000). the incidence of meningitis, wound infection, bacteremia, and urinary tract infection was low and comparable between the groups. staphylococcus aureus was most frequently identified as the causative infectious microorganism in both the hypothermia (14.3%) and normothermia (36.3%) group (p = 0.375), followed by coagulase negative staphylococci (14.3% in the hypothermia and 27.3% in the normothermia patients, p = 0.625) gram-negative bacteria were isolated from the surveillance cultures in 51.4% of patients treated with hypothermia and 31.4% of patients in the control group (p = 0.143). colonization of the rectum with gram-negative bacteria was significantly more frequent in patients treated with hypothermia compared with normothermia (48.6 vs. 20.0% respectively, p = 0.041). in contrast, colonization of the upper gastrointestinal tract and sputum was comparable between the groups with an incidence of 14.3% in the hypothermia patients versus 11.4% in the normothermia patients (p = 1.0). use of sdd mitigates the increased risk of infection in patients treated with hypothermia. based on the surveillance cultures, it seems that oropharyngeal decontamination is the most effective part of the sdd regimen in the prevention of pneumonia. introduction. prognostic scores specific for critical patients were developed in order to predict mortality based on physiologic and laboratorial variables. on the other hand, specific scores for burn patients are calculated taking into consideration inhalation injury, age and total burned surface area (tbsa), among others. however, scores utilized in general icu have not been evaluated in burn patients. objectives. therefore, the aim of the present work was to validate apache ii, saps 3 as well as initial sofa in a population of patients with massive burn. these scores were compared to some specific burn patient scores, including absi (abbreviated burn severity index) and estimates of the probability of death. retrospective study employing data collected prospectively from may 2005 to february 2010 (58 months) at an icu specialized in burn patients at a teaching hospital which is considered a reference centre in trauma care. all patients admitted during this period were included. one hundred and fifty-four consecutive patients were studied (male: 73%; female: 27%), with averaged age of 37.7 ± 15.9 years and a hospital stay of 33.8 ± 28.4 days. mortality rate of our sample was 43.5%. incidence of inhalation injury was 45% and total burn surface area (tbsa) was the following: 35.5% of patients had 20% or less; 36.1% had 20-40% of tbsa whereas 28.4% showed 40% or more. area under curve of receiver operating characteristic (roc) of evaluated indexes is displayed on table 1 . computerized head tomography is routinely performed as a diagnostic tool after the occurrence of neurologic deterioration in the icu adult patients. however, the ct findings in this setting are rarely reported. we hypothesized that the analysis of a series of cranial cts would help to understand the neurologic conditions of the critically ill patients and improve their management. objectives. to analyze, over a three-month period, the head ct scans performed in the adult icu in the albert einstein hospital in são paulo, brazil. methods. all cranial cts performed in the icu patients during the studied period were analyzed by two radiologists from the albert einstein hospital staff from may 1st to august 31st, 2009, according to a pre-established protocol: 1. presence of acute cerebral ischemia; 2. presence of previous cerebral ischemia; 3 . presence of acute cerebral hemorrhage; 4. presence of cerebral edema; 5. cerebral aneurisms; 6. cerebral tumors and 7. normal cerebral tomography. we studied 69 ct scans from 34 (49.2%) males and 35 (50.8%) females, mean age 64.43 ± 21.7 years. the head ct findings were the following: (1) presence of acute cerebral ischemia = 10 (14.4%); (2) presence of previous cerebral ischemia = 17 (24.6%); (3) presence of acute cerebral hemorrhage = 27 (39.1%); (4) presence of cerebral edema = 7 (10.1%); (5) cerebral aneurisms = 2 (2.8%); (6) cerebral tumors = 2 (2.8%) and (7) c16 years c 18, abc (assessment blood consumption) cp: c2 and ets (emergency transfusion score) cp: c3, c4 years c6. these scales handle the following combinations of variables for calculation: age, sex, type of admission, mechanism, blood pressure, focussed assessment for the sonography of trauma, hemoglobin, orthopedic or pelvic trauma, heart rate. mt was defined as the transfusion of 10 units or more of packed red blood cells in the first 24 h. we study the sensitivity (s), specificity (sp), positive and negative predictive value (ppv, npv), likelihood ratios positive and negative (lhr+ , lhr-) and area under the receiver operating characteristic curve (auroc) of different scales for the predictive power of tm validated in the literature. 568 patients were available for analysis (77.6% men, iss 30 ± 13, blunt trauma 93.8% objectives. we measured patient-reported outcome following surgical management with dc using a quality of life instrument. methods. survivors discharged between 3 and 18 months after severe tat were contacted after obtaining approval by our institutional irb. we excluded patients with neurotrauma. we applied self-response version euroqol questionnaire (eq-5d) and visual analog scale (eq-vas: 0 (worst health)-100 (best health). euroqol it is based on a descriptive system that defines health in terms of 5 dimensions: mobility, self-care, usualactivities, pain/discomfort and anxiety/depression. each dimension has 3 levels of response: no problems (level 1), some problems (level 2) severe problems (level 3). results. thirty four patients were contacted. mean ± sd age was 31.8 ± 11.6 yrs, male were 88.2% and penetrating trauma occurred in 79.4%. mean ± sd in severity scores were: ati 24.4 ± 9.6, iss 28.1 ± 8.5 and apache ii 20 ± 6. the median time from discharge was 12 months (iqr 6-15 months). the eq-5d dimensions in which the largest proportion of patients reported severe problems were usual-activities (work, study) and pain/discomfort 14.7% and 5.9% respectively as shown in the conclusions. survivors of severe trauma and dc, reported acceptable quality of life with minimal limitations with social functioning. a prospective study should assess quality of life in these patients from hospital discharge and systematically over time. introduction. brain tumors surgery is one of the main causes of admittance to the nicu. it is important to know the risk factors associated to hospital mortality of patients admitted to nicu due to this reason. to identify perioperative factors associated to higher hospital mortality in a series of patients admitted to nicu immediately after a bt elective resection. methods. data of 101 patients operated for bt elective resection and consecutively admitted to nicu at imss umae 1 bajío were prospectively obtained. nicu bt database includes 269 perioperative items. we divided the series in two groups: surviving and deceased patients. then, we analyzed the perioperative behavior differences between both groups. either student's t test or chi-square test was used, as it corresponded, for the analysis of differences observed between both groups. values of p lower than 0.05 were considered significant. results. the hospital mortality observed in this series of 101 patients was 8.9% (9/101). data of the nine variables showing significant differences between surviving and deceased patients groups are shown in table 1 . even if hypoxic brain injury has been reported as the strongest factor affecting the poor outcome of near-drowning patients, little has been known about prognostic factors affecting the outcomes of those patients receiving mechanical ventilation. to define prognostic factors affecting the outcomes of patients mechanically ventilated after near-drowning. , white blood cell counts (or, 1.00; 95% ci, 1.00-1.00; p = 0.001), serum creatinine (or, 0.19; 95% ci, 0.06-0.67; p = 0.010), and serum lactic acid (or, 0.95; 95% ci, 0.93-0.98, p = 0.002) were associated with favorable outcomes, respectively. however, only higher body temperature as a clinical parameter and the level of serum lactic acid as a laboratory parameter were significant predictors of favorable outcomes in multivariate analyses; the or were 2.87 (95% ci, 1.01-8.14; p = 0.048) and 0.96 (95% ci, 0.93-0.99; p = 0.011), respectively. conclusions. initial body temperature and the level of serum lactic acid were two most important clinical and laboratory prognostic factor in nearly drowned patients. the outcomes were not affected by the degree of initial hypoxemia. to determine the use of automated external defibrillators (aed) and manual defibrillators deployed in the various hospital wards (unmonitored areas) in a university hospital. a prospective study was performed according to utstein style of all cardiac arrests occurred in the hospital during the first 6 months after the implantation of a new protocol of care for hospital cardiac arrest. because of this plan automated external defibrillators were located for hospital wards and common service areas (radiology areas, outpatients, …) where one would expect a lower incidence of cardiac arrests, according to the risk map elaborated previously. in areas of greatest risk manual defibrillators previously existed. all resuscitation attempts in these areas were analyzed, excluding the emergency department because of a separate protocol against the rest of the hospital. special attention was given to the use of aeds by wards staff before the arrival of resuscitation team. also a comprehensive volunteer training program was designed, but it began after the analyzed period was finished. results. during the first 6 months we collected a total of 20 pcr in hospital wards and public areas, with a median age of 71 years and predominantly male (12 patients). the most common origin was respiratory (13 patients) followed by cardiac (4 patients). the most frequent rhythm detected was non-shockable (17 patients), only in 2 was shockable and unknown in 1. before the resuscitation team arrival only two patients had been manually defibrillated and were never used the new aeds. conclusions. the aeds provided in the hospital were completely useless in the first months after placement, probably due to the lack of a comprehensive training plan associated to the population goal. methods. descriptive longitudinal study. 208 patients were studied by encephalic death, as potential donors of organs, alerted to the network of regional transplant 5 (cdtot), by units of intensive care, for 36 months, in barranquilla's city. it was applied qualifying each of the variables in agreement to the vital opposing signs and biochemical tests brought in this moment. . 78.3% of the subjects were male; the average of age was 37.34 years (±sd: 16.24). the values of blood sugar, sodium, osmolaridad, tonicidad, po2, fc, pam, and glasgow, determined a score of 22, qualification that there had patient with encephalic death with the scale mbcm, as a test of certainty of the scale to diagnose encephalic death in total absence of reflections of stem. conclusions. there is recommended the application of mbcm's scale to every neurological patient by diagnosis of encephalic death in proof of certainty, in absence of others. by the high specificity of the already demonstrated scale there is recommended that scores lower than 19 they should restate the qualification. a score of 22 is an encephalic death in absence of reflections of stem. grant acknowledgment. clínica general del norte-cdtot introduction. prospective analysis of tracheostomies performed in patients admitted to a neurotrauma icu, the reasons for its implementation, and intraoperative complications in the first week. methods. all patients admitted to the icu of neurotrauma, which underwent a tracheostomy after admission. data were collected: affiliation, cause of admission, average stay, cause for realization of tracheostomy, tracheostomy time delay from its indication, place of performance of the procedure (icu or operating room), perioperative complications (event at transfer to operating room or during surgery: hypoxia, hypotension, arrhythmia, bleeding, premature extubation, false cannulation, cardiac arrest, pneumothorax or death), and postoperative complications in the first week (bleeding, difficulty in changing cannula, stomal infection, pneumothorax, death). introduction. the s-100b protein is a brain-specific protein release from astroglial cells into the circulation after traumatic brain injury (tbi). researches indicate that the s-100b serum level could be a useful indicator of tbi severity, however there is not evidence enough about the role of s-100b in nonsevere head trauma. the hypothesis that s-100b is a useful screening tool to detect brain injury in patients with a normal level of consciousness after a head trauma was tested. a total of 115 patients with the diagnosis of mild tbi without decrease of consciousness (according to the gcs) with at least one neurological symptom or finding like amnesia, headache, dizziness, convulsion and vomits, were prospectively included. we recorded the clinical data on admission and a blood sample before 6 h after tbi, for s-100b inmunoluminescence analysis. a routine cranial computed tomography scan (ct) was obtained within 24 h after the injury (categorized in normal or pathological). the diagnostic properties of s-100b serum levels.105 lg/l, for prediction of intracranial lesions revealed by ct were tested with receiver operating characteristic (roc) analysis. seventy of the patients (60.9%) were men, with a mean (sd) age of 49.07 (20.65) years (range, 14-92 years). a total of 11 patients (9.6%) had intracranial lesions. serum s-100b levels were significantly higher in patients with intracranial lesions than in the remaining patients. the average value of the protein in patients without intracranial lesion was 0.315 lg/l with a ci 95% (0.265-0.365 lg/l), and in those with pathological findings in ct was 0.601 lg/l with a ci 95% (0.403-0.798 lg/l). significant differences were found between levels of s100b protein and the presence of pathological findings in the ct (p = 0.001) (fig. 1) . the roc curve analysis showed that s100b protein is a useful tool to discriminate the presence of intracranial injury in ct (auc, 0.76, 95% ci, 0.685-0.907, p \ 0.001). s100b analyses with a cut-off level of 0.105 lg/l showed a sensitivity 100% but a specificity 19.2%. we evaluated different cut off values and in our series, the best cut off of the s100b protein is at 0.253 lg/l with a sensitivity of 100% and specificity 58%. (fig. 2) conclusion. determination of serum protein s-100b is a useful biochemical indicator of brain damage in head trauma. our results show that an increase in the cut-off point of s-100b to 0.253 lg/l increases its accuracy in the prediction of the existence of macroscopical lesions. key words. protein s-100b, brain injury, minor head trauma, cranial computed tomography. critically ill patients with systemic inflammatory response syndrome frequently suffer muscle weakness due to critical illness myopathy (cim) and polyneuropathy (cip). several in vitro studies have shown that the cause of muscle weakness is a loss of membrane excitability accompanied by membrane depolarization [1] . objectives. we investigated membrane polarization and excitability parameters in muscle and motor nerve in vivo within the first week after intensive care unit (icu) admission. methods. the study was approved by our local ethics committee. patients with sofa scores c8 on 3 consecutive days underwent nerve conduction studies including direct muscle stimulation to categorize patients as icu-control, cim-(dmcmap \3 mv) and/or cippatients (reduced snap amplitude) within the first 8 days after icu admission. to assess excitability parameters we recorded stimulus-response behaviour, threshold electrotonus, current-threshold relationship and recovery cycle from abductor pollicis brevis muscle following stimulation of the median nerve [3] . data are shown as median and 25%/75% percentile. conclusions. we describe for the first time that critically ill patients in general show muscle-and nerve membrane depolarization, whereas patients later suffering from muscle weakness due to cim or cim/cip feature additionally reduced membrane excitability. this suggests that membrane depolarization in critically ill patients is caused by energy failure leading to dysfunction of the na-k pump, the motor of membrane repolarisation-whereas reduced membrane excitability in cim or cim/cip needs an additional dysfunction of voltage gated sodium channels for example occurring in the presence of endotoxins [2] . in intensive care patients with central nervous system (cns) disease, the systemic inflammatory response syndrome (sirs) criteria are often unreliable as a basis for identifying the inflammatory process. even with the presence of some infection they could be signs of the diencephalons-catabolic syndrome. diencephalons-catabolic syndrome like sirs constitutes of hyperthermia over 38°c, tachypnea of over 20 per minute, tachycardia, and arterial hypertension. thus, sirs symptoms may occur after antibacterial treatment even if there is no infection or inflammation. we suggest a more precise method which could help to avoid the excessive antibacterial therapy and to control it in patients with cns disease-a procalcitonin test. objectives. reduce the use of wide specter antibiotics makes the control over antibacterial therapy in patients with cns diseases more precise; reduce the number of complications related to unnecessarily long antibacterial treatment. after obtaining the informed concern, in our investigation we included 30 patients with different neurological disorders, who had recently transferred neurosurgical operations. all of them demonstrated sirs symptoms on different postoperative terms. when sirs symptoms occurred, we checked the level of procalcitonin in the patient's serum by a semi quantitative method on a disposable brahms pct-q system. the procalcitonin level was determined against a color scale. procalcitonin level over 0.5 ng/ml (16 patients) considered a sign of infection and in such cases we prescribed antibacterial treatment 2,000 mg of selenase for 5-7 days. if the test result was negative (14 patients) we repeated it in 24 h and in cases with the same results, no antibacterial treatment was administered even if there were sirs symptoms. if pct-q test was negative patients were sedated (fentanyl 0.8-1.5 lg/kg/h and clonidine 0.2-0.5 lg/kg/h) to achieve autonomic stability and attenuate clinical manifestation of sirs. we had not observed any cases of sepsis in both groups of patients. by mince of pct, we had managed to reduce the quantity of wide specter antibiotics, used in neurosurgical patients for 34.7%. conclusions. procalcitonin test in neurosurgical clinic let us determine the necessity of antibacterial treatment reduce the use of wide specter antibiotics, medical costs and prevent the forming of polyresistant infection. l. combe 1 , r. appleton 1 , c. gilhooly 1 , j. kinsella 1 1 university of glasgow, department of anaesthesia and critical care, glasgow, uk intensive care unit-acquired weakness (icuaw) is increasingly recognised as a common complication of critical illness with potentially prolonged debilitating sequelae. the estimated incidence is 46% in patients with sepsis, multi-organ failure or prolonged mechanical ventilation [1] and suggested risk factors include: the systemic inflammatory response syndrome (sirs), sepsis, higher severity of illness, hyperglycaemia, renal replacement therapy and parenteral nutrition. objectives. the aims of this study were to determine the incidence, risk factors and outcomes for patients diagnosed with icuaw in glasgow royal infirmary's (gri) icu. the study was undertaken in two parts, firstly as a case-control study [matched for age (within 5 years), sex and admission apache ii score (within 5 points)] and secondly by comparing identified cases of icuaw to a 3-month cross-sectional sample (1/10/09-31/12/09, 112 patients) of gri's icu patients. data for both parts of the study was obtained from two electronic databases, wardwatcher and carevue. carevue was searched to identify patients with icuaw and wardwatcher was used to identify the controls. data collected included: patient and illness characteristics, severity of illness scoring, organ support and treatments provided, laboratory results and outcomes. minitab software was used for statistical analysis. conclusions. the incidence of icuaw was very low, we hypothesise this to be explained by the absence of systematic evaluation of patients for icuaw. the risk factors and outcomes for icuaw were consistent with some of the published literature. prospective study is now planned to systematically evaluate this condition. with increasing age, comorbidity, and socioeconomic deprivation being associated with higher risk pregnancies, there comes a potential higher risk of complications. neurological and neurosurgical complications, which can be particularly devastating during the peripartum period, include those due to medical conditions of pregnancy (hypertensive disease, sepsis, thromboembolic disease, hypoxic-ischaemic brain injury), iatrogenic complications secondary to anaesthetic or obstetric interventions, incidental illness or injury (pharmacological alterations, trauma, tumour), and deliberate self-harm and violence. objectives. to ascertain the frequency of neurocritical care admissions in the west of scotland, the nature of the admission diagnoses, the impact they have on our service (length of stay), and maternal and foetal outcome. methods. using the scottish intensive care society audit group wardwatcher patient database, female patients aged 14-50 years old who were admitted to the neurocritical care unit were identified (january 2008-december 2009). we manually reviewed the electronic admission note for each of these women in order to gain diagnoses; a targeted case note analysis ensued. within the 24 month study period there were a total of 915 admissions to neurocritical care, of whom 164 fulfilled the age and gender criteria; 6 admissions (0.66% of total) were for neurological complications in the peripartum period. the age range was 18 to 45 years (median 31 years). three women (50%) were intrapartum (20-36 weeks gestation) at the time of their admission, and three were postpartum (1 day-7 months). half of admissions were due to incidental illness or injury, a third to pregnancyrelated medical complications, and one case was iatrogenic in nature. length of stay in icu was 1 to 12 days (median 4.3 days). one patient sustained a residual facial nerve weakness and deafness. conclusions. this survey provided insight into the incidence and nature of pregnancyrelated pathology requiring acute referral to a regional neurosciences centre. as highlighted in other surveys, there may be many more peripartum patients with neurological complications who are cared for in general critical care units, and do not require admission to a tertiary referral centre [1] . further work is underway to ascertain the true numbers of neurological complications of pregnancy countrywide. our approach represents a paradigm for the continuing audit of pregnancy-related critical care resource use in scotland. introduction. hypertonic saline has an osmotic effect on the brain because of its high tonicity and ability to effectively remain outside the blood-brain barrier. there may be a minimal benefit in restoring cerebral blood flow, which is thought to be mitigated through local effects of hypertonic saline on cerebral microvasculature. most comparisons with mannitol suggest almost equal efficacy in reducing icp but not compared their effects on eeg. objectives. we aimed to compare the effects of 20% mannitol, 3% or 7% hypertonic saline on hemodynamic parameters, intracranial pressure and electroencephalography in experimental head trauma. bilateral craniotomy were carried out in the parietal region and head trauma was applied for all rabbits. the rabbits were randomly divided into four groups. in group i rabbits were only observed. in group ii: 20% mannitol, in group iii: 3% hypertonic saline and in group iv: 7% hypertonic saline was administered intravenously to achieve similar osmolar load. electroencephalography, mean arterial pressure, heart rate, intracranial pressure were recorded before trauma and 5 and 60 min after trauma. results. increased intracranial pressure was significantly decreased by mannitol, 3 and 7% hypertonic saline solutions at the end of study (p \ 0.05). but intracranial pressure values of mannitol and 7% hypertonic saline groups were lower than the other groups (p \ 0.05). the electroencephalography scores decreased after trauma in all groups (p \ 0.05). at end of the study, 3 and 7% hypertonic saline groups had similar electroencephalography scores with pretrauma scores (p [ 0.05). the mean arterial pressure and heart rates increased after trauma in all groups (p \ 0.05). mean arterial pressure values were found lower only in mannitol group at end of the study (p \ 0.05). our study showed that when used in intracranial hypertension treatment, 7% hypertonic saline solution is as effective as mannitol, and preserves hemodynamic parameters, and normalizes traumatic electroencephalography abnormalities better than mannitol. objectives. to identify the causes of new onset seizures in patient admitted in medical icu. methods. all the patient admitted in icu and who had new onset seizures were evaluated. the patients were evaluated for metabolic profile. imaging (ct/mri) was done whenever needed. patients with preexisting seizure history were excluded from study. . 44 (29 males, 15 females) patients, who had first seizure during hospitalization in icu were included. 43 patients had generalised and one had focal seizures. 28 patients had metabolic abnormalities. 13 (46.42%) had evidence of hepatic encephalopathy. 3 (11.36%) had only hepatic encephalopathy while rest had associated uremia, hyponatraemia, hypophosphatemia and hypomagnesemia. out of 10 patients, who had renal failure, 4 had evidence of uremia while rest had associated hyponatraemia or hypophosphatemia. only one patient had evidence of hypocalcemia. imaging was done in 16 patients. 14 (30%) had abnormal ct scan results. 4 (9.09%) had intracranial hemorrhage, 4 (9.09%) had infarct, 2 (4.55%) had brain metastasis, 2 had evidence of hydrocephalus and one each had evidence of extradural hemorrhage and tuberculoma. csf analysis was done in 7 (15.9%) patients. 3 (6.8%) had evidence of tuberculosis and 1 (2.27%) had evidence of pyogenic infection. to study the role of various investigations and ct in evaluating these patients. all patients admitted with new onset seizures within 72 h prior to presentation were included. all the patients were questioned and an attempt was made to assign an electroclinical syndrome to seizure. patients were evaluated for metabolic profile, neuroimaging. csf examination was done in those who had persistently altered mental status, infectious symptoms and fever. results. 110 patients were admitted (1.03% of total patients who came to emergency) with history of new onset seizures. 71.8% patients were diagnosed to have acute symptomatic seizures and were placed in ilae category 4.13 and three patients were placed in ilae category of remote symptomatic seizures. the cause of seizures was established in 82 (74.5%) patients and remained unestablished in 28 (25.5%) patients. 14 (12.7%) patients were diagnosed to have neurocysticercosis. other important causes were acute infarct, uremia, hyponatremia, hypernatremia, viral encephalitis, post partum eclampsia, pyogenic and tubercular meningitis. alcohol withdrawal seizures were seen in 3.6% patients. metabolic derangements were seen in 19 (17.3%) patients. computed tomography was done in 88 patients and 50% had abnormal findings. mri was done in 13 patients and 12 had abnormalities. conclusions. neurocysticercosis was found to be most common cause of seizure activity in our part of country. though metabolic derangement can cause significant proportion of new onset seizure patients routine imaging of brain should be performed in patients with new onset seizures. work environment and organisational issues: 1335-1347 1335 subjective and objective research into the working conditions and their effect on the health and safety of people working in icu, focusing mainly on the natural factors of temperature, humidity, ventilation, lighting and noise (part 1) n. karachalios 1 , e.c. katsilaki 1 , d. sfyras 1 1 general hospital of lamia, icu, lamia, greece the aim of the project is the subjective and objective investigation of the conditions of work and the relation repercussions on the health and safety of people working in the icu, focusing mainly on the natural factors that are likely to cause the sick building syndrome. for this purpose a protocol of research in two phases has been planned. the first included objective measurements, with the use of suitable equipment, of the natural factors of temperature, humidity, ventilation, lighting and noise. the second phase included the subjective estimation of the working people about their own health and conditions of their work, in the particular area of the hospital with the use of substantiated anonymous questionnaire. after the subjective and objective study and analysis of 32 questionnaires and 55 measurements of natural factors, we found that the medium temperature of the icu was 24°c. the mean relative humidity of the icu was 38% (highest 38.3% and lowest 37.8%). the mean ventilation rate of the icu was 17 m 3 /h (highest 28.8 and lowest \7.2 m 3 /h). the mean sound pressure was 64.8 db (highest 75 and lowest 63.5 db). the average lighting was 331.5 lux (160 lux lowest and 598 lux highest). the objective data seem to keep pace with the subjective opinions of the working people, as they were impressed in the questionnaires of subjective estimate. the objective data were compared with the subjective. the results of the research were also compared with data from the existing bibliography and current legislation, leading to a line of conclusions. (1) insufficient and bad quality ventilation. (2) the existing temperature of the environment contributes to the appearance of sick building syndrome. (3) the working environment is noisy. (4) the environment of work has problematic or insufficient lighting. (5) the icu under study is a building area which can be characterized as ''sick'' if immediate action is not taken. background. up to 75% of critical care nurses test positive for (symptoms of) post traumatic stress disorder (ptsd) [1, 2, 3] . it is assumed that these symptoms are caused by professional involvement in life-threatening events [3] . in a sample of intensive care nurses, we investigated which work related incidents were perceived as most distressing. method. in interviews, 12 nurses (75% female) were asked to memorize and tell about their most traumatic work related event. all interviews were recorded. after verbatim transcription, the 'most critical events' were extracted and categorized bij two independent psychologists. . none of the nurses reported major life-threatening events such as trauma-related injuries, massive bleeding or seeing patients die as their 'most critical incident'. conclusion. not the major life-threatening events but relatively 'normal work related events' under unusual circumstances are mentioned as most critical by nurses. in contrast to major life-threatening events, these 'normal events' are usually underestimated by colleagues, and thus potentially compromise peer-support. a care bundle refers to evidence based interventions and information grouped together to improve outcomes and consistency of provided care [1, 2] . at the icus charge nurses and intensivists as shift leaders are responsible for daily management of unit activities. several immediately made decisions by shift leaders are made under time pressure and high information load with inadequate information. though we have evidence of structure and process based factors such as material and human resources, admission and discharge decisions or bed utilization, the support for information transfer and integration is poor in organizational decision-making concerning these factors. objectives. to identify immediate information needs of charge nurses and intensivists during the management of daily activities at the icu and evaluate how necessary this information is for their decision-making. from september 2009 to november 2009, all charge nurses (n = 515) and intensivists (n = 223) of 17 university affiliated icus providing comprehensive care in finland were surveyed with an on-line questionnaire using 122 statements. the questionnaire was developed based on our previous observation study and statements of our survey regarded information needs related to the icu care activities. a rating scale from 0 to 10 (completely unnecessary-absolutely necessary) was used to assess the necessity of the information. for each statement, a response with mean 7 or over was regarded as necessary information for immediate decisions. results. the response rate was 47.97% (charge nurses 50.1%, intensivists 43.1%). the working experience varied from 0 to 35 years (mean 13.6, sd 7.9). over 50% of respondents worked as a shift leader once a week or more often. 72 statements of 122 were valued as a necessary (mean [7 or more) for immediate decision-making. absolutely necessary information (mean [9 or more) for immediate decision-making were assessed related to the 11 statements. these statements concerned isolations, mechanical ventilation, admissions and discharges, special treatments, patient's condition, and scheduled dates or times for surgery or other procedures. conclusions. both icu charge nurses and intensivists identified several information needs that are crucial for immediate decision-making during the whole icu care process. information needs of the shift leaders differed and they were strongly connected to the needs of one's professional requirements. an integrated overview and summarization of immediately needed information-a care bundle for organizational decision-making-at the icus is highly needed for icu shift leaders. the common interests of both professionals, charge nurses and intensivists, should be emphasized when new technology-based systems are developed. background. the nursing shortage is an international problem that is expected to worsen in the coming years. studies show that one of the main reasons nurses leave the profession is their dissatisfaction with their work environment. structural empowerment and nurse-physician collaboration are two elements of the nurses' work environment that are potentially related to one another according to kanter's theory (1977) . in addition, a nurse's clinical specialization has been found to influence perceptions related to these two concepts. to examine the level of perceived structural empowerment, the perceptions of nurse-physician collaboration and the relationship between these two variables, among intensive care unit (icu) nurses and general ward nurses in israel, and to compare the groups. a descriptive, correlational, comparative study design was used on a sample of 84 icu nurses and 88 nurses from internal medicine and general surgery wards in a large university hospital in israel (response rate 79%). a three section, self administered questionnaire was used to measure the study variables: the condition of work effectiveness scale-ii (cweq-ii), the collaboration with medical staff scale (cmss) and demographic-professional background. results: perceived structural empowerment was found to be moderate (m = 19.41, sd = 3.66, range = 6-30). nurses tended to agree that there was nurse-physician collaboration (m = 2.69, sd = 0.55, range = 1-4, 1 = strongly disagree, 4 = strongly agree). a correlation was found between structural empowerment and the nurse-physician collaboration (r = .52, p \ 0.01). a significant difference was found between icu nurses and general ward nurses on their perceptions of nurse-physician collaboration (t (170) = -3.39, p \ 0.01; general wards: m = 2.83, icu: m = 2.55). no significant differences were found between nurse specialization on perceived level of structural empowerment. conclusion. nurses in this study tended to agree that there was nurse-physician collaboration on their unit/ward. nurses who perceived themselves as having a higher level of structural empowerment, felt that there was a higher level of nurse-physician collaboration. general ward nurses had more positive perceptions about nurse-physician collaboration on their ward as compared to icu nurses. no difference was found between the two groups on the level of structural empowerment. recommendation. the findings of this study can be used as the basis for the design of interventions, aimed at enhancing structural empowerment and nurse-physician collaboration, in order to improve nurses' work environment, as one of strategy to decrease the nursing shortage. further study of additional hospitals in the country is also recommended. teams have expanded and in some hospitals 24 h cover has been instituted. researchers are questioning the validity of outreach services and its impact on patient outcomes. as cco has been viewed as the panacea to all problems, data collection and analysis is fundamental in proving its financial and clinical benefits. objectives. this comparative study aims to evaluate retrospective data from 1 month in 2006 and 1 month in 2010. data does not encapsulate patient outcomes; it will compare frequency of referrals and interventions. this data provides an indication to the extent cco has participated in the care of the acutely ill over a given time period. methods. data was collected from the 4d medicus database collating intervention data. analysis occurred using 13 key interventions using excel software conclusions. whilst the validity of services has been questioned, the data itself indicates that more patients are referred and frequency of interventions has increased. various system changes occurred during this time period such as a change of mews trigger scores, the advent of 24 h cco and courses such as alert and survive sepsis were introduced into the basic training of staff. it must be noted that the intention in the uk for cco was a service that empowered staff through education to undertake this care themselves; therefore the increase in interventions could indicate that the educational approach hasn't made progress. although the study compares 13 interventions, an increase in the type of interventions was also noted such as ward based cco supervised cpap and establishing a picc line service. therefore this highlights the changing application of interventions. further analysis is required to look at the appropriate skills required for the delivery of safe care to the acutely ill in the ward environment. whilst ward staff are increasingly under resourced, both in skills and manpower, cco do provide the skills, knowledge and time to meet the shortfall in safe timely care. introduction. working as a critical care nurse involves situations where teamwork is essential and rapid, effective communication is of importance [1] . the education to become a specialist icu nurse gives skills and knowledge to manage patients who are critically ill with rapidly changing conditions [2] . experimental research is one way of contributing to the acquisition of such knowledge. to describe how icu nurses may contribute and perform in the experimental research process, an environment usually unfamiliar to them. we describe our experiences with regard to clinical contribution and our subjective evaluation of involvement in animal experimental research. method. three icu nurses in a swedish hospital were asked to participate in a research project investigating myocardial metabolism in porcine models of shock. the tasks were anaesthesia and pain management, assisting with catheter insertion and haemodynamic monitoring the pigs during the process results. although the situation was new, the nursing role and function in the team were at once similar and different to the daily work situation in the icu. one major skill learnt was the rigour of experimental measurements and sources of error, which is sometimes neglected in clinical care. being able to observe changes due to shock in a controlled setting, we improved our ability to critically 'think ahead' in anticipation of clinical deterioration [3] . our first-hand experiences at the animal experimental laboratory allayed many anxieties and misconceptions with this type of research. conclusions. the critical care environment demands skills such as the ability to accurately define and change priorities rapidly, good communication and teamwork [4] . we believe that the experimental research setting is one way of enhancing this ability. in these units patients condition may change rapidly and they may need close inspection as well as emergency response. early warning scoring (ews) system may make early recognition of and response to bad condition possible by observation based on systematic parameters. ews was developed as a simple scoring system to be used at ward level utilizing routine observations taken by nursing staff. ews is based on five physiological parameters; systolic blood pressure, pulse rate, respiratory rate, temperature and avpu score (alert; reacts to voice; reacts to pain; unresponsive). objectives. the aim of this study was to evaluate ews among patients admitted to pacu. methods. ews parameters were recorded four times from 167 patients after their admission to pacu. the first record was taken during the first admission to pacu (ews 1), the second (ews 2) after 15 min, the third after 30 (ews 3) and the fourth record after 60 min. the correlation between variables like differences of four ews, patients age, the asa score, duration of operation were statistically examined. early treatment and recognition of sepsis is a stated aim of the surviving sepsis campaign [1] but in busy clinical environments the delivery of antibiotics and fluids can often be delayed. we describe the implementation of an audit proforma, based on the survivesepsis.org [2] resuscitation bundle, as a tool to deliver six aspects of management within 1 h of recognition sepsis. 1. improve the early recognition and treatment of sepsis in acute medical patients. 2. provide a sustainable change in the management of septic patients 3. improve mortality and length of hospital stay methods. the proforma consist of six treatment management steps, based on the survivesepsis.org ''septic six'': 1 oxygen, 2 blood cultures, 3 antibiotics, 4 lactate, 5 iv fluids, 6 strict fluid management. it is triggered by patients satisfying two or more of the systemic inflammatory response syndrome criteria. all management steps should be implemented within 1 h of the trigger time stated on the form. the forms are collected and analysed every month and the results are displayed for staff working on the medical admissions unit and accident and emergency. a total of 239 forms have been collected, 30% diagnosed with severe sepsis. the progress on all six parameters is shown below. over the initial seven month period we have demonstrated a sustained improvement in the rapid delivery of all six of the management parameters. introduction. the early goal-directed resuscitation has been shown to improve survival in patients presenting with septic shock. a recent systematic review demonstrated the inability of central venous pressure (cvp) to predict the hemodynamic response to fluids infusion, and it should not be used to make clinical decisions regarding fluid management in critical patients. the clinical implication of this fact in septic shock is not well-known. objectives. the aim of this study is to determine if the resuscitation with fluids guided by cvp has clinical implications in patients with septic shock. post-hoc analysis of a patients' cohort with septic shock admitted in the medical intensive care unit since june 2007 to june 2009. all of them were treated on basis of a bundle for severe sepsis management. chi-square analysis was used to compare categorical data. continuous data were compared using student's t test. we used multiple logistic regression model to assess the association between the independent variable and mortality, after adjustment for possible confusing factors (we considered variable to be confounding if the estimate of the coefficient changed by more than 10%). eighty-five patients were studied. 66% were male. their average age was 57 ± 17 and 40% had previous chronic diseases. severity scores: apache ii 24 ± 8, sofa 10 ± 4 and 76% of patients had multiorganic dysfunction. infectious focus was respiratory in 48%. cvp mean was 12 ± 4 mmhg, scvo2 73 ± 8% and the mean amount of fluids provided was 2840 ± 844 cc. 71% of patients needed mechanical ventilation. hospital-stay middle was 28 days (1-160) and 10 days in icu conclusions. in our patients' cohort with septic shock treated under the basis of the early goal-directed resuscitation, the volume of fluids infused was associated independently with mortality. a lower fluid administration in the resuscitation probably could be caused by the early reach of a high central venous pressure. blinding of study interventions is necessary to prevent bias in randomized controlled trials (rct). since normal saline and 5% albumin are packaged in bags and bottles, respectively and they have different color and texture, a blinding procedure is necessary to ensure the fluids appear identical for comparative rcts. objectives. to describe the blinding procedure and evaluate sterility and stability involved in the transfer and storage of study fluids in the precise pilot rct. a standard operating procedure for concealment, meeting pharmacy guidelines and good manufacturing practices was developed by the manufacturing pharmacist at the coordinating centre and used by all participating sites. fluids were transferred with aseptic technique into identical 500 ml bottles under a sterile hood by the pharmacy or transfusion medicine technician then covered with an opaque wrapping. average time to transfer of study fluids from their original packaging was recorded to understand labor involved with creating each study fluid package. yellow intravenous tubing was manufactured to also conceal the fluid color. six blinded bottles of normal saline and 5% albumin from the participating centers were stored at room temperature for at least 3 months. cultures of the fluids using blood culture media and/or endotoxin levels (measured by commercial assay) were obtained to document sterility of the study fluids. protein electrophoresis was used to assess albumin stability. results. transfer of the study fluids was the responsibility of the research pharmacist/ technician and blood bank at 3 and 3 sites, respectively. average time to transfer 20 containers of normal saline and 5% albumin into bottles was 65 ± 29 and 77 ± 19 min, respectively. sterility (culture negative and/or endotoxin undetectable) of study fluids was confirmed from all bottles of normal saline and albumin that underwent testing. protein electrophoresis of albumin samples showed a single band suggesting no degradation of albumin during transfer and storage. conclusions. the standardized blinding procedure developed for transfer of study fluids in this pilot rct confirmed sterility and stability of our study fluids for 3 months. these data are important when considering the length of allowable storage time for these study fluids. due to the resources and time involved with the transfer of these fluids for individual sites, this transfer method needs to be incorporated into budgeting and may not be feasible in the context of a large rct. grant acknowledgment. the precise pilot rct was funded by a grant from canadian blood services. covidien, singapore, singapore, 18 yong loo lin school of medicine, national university of singapore, biostatistics unit, singapore, singapore introduction. the surviving sepsis campaign recommends a 6-h resuscitation bundle and a 24-h management bundle to improve outcomes in severe sepsis. compliance with and relevance of these recommendations to asian intensive care units (icus) are unknown. objectives. the primary objective of the present study was to assess the compliance of asian icus and hospitals to these bundles. the secondary objectives were to evaluate the impact of compliance on mortality, and the organisational characteristics of asian hospitals which are associated with higher compliance. methods. this was a prospective observational study of patients with severe sepsis who were admitted to the participating icus in july 2009. we recorded the organisational characteristics of participating centres, the patients' baseline characteristics, and the achievement of targets within the resuscitation and management bundles. results. sixteen countries and 150 icus participated, enrolling 1285 patients. hospital mortality was 44.9%. achievement rates for the bundle targets were: lactate measurement, 39.8%; blood cultures, 62.5%; broad-spectrum antibiotics, 64.0%; fluids ± vasopressors, 81.4%; central venous pressure, 39.7%; central or mixed venous oxygen saturation, 10.8%; low-dose steroids, 55.8%; drotrecogin alfa, 3.2%; glucose control, 27.1%; lung-protective ventilation, 11.7%. compliance rates for the entire resuscitation and management bundles were 7.6 and 3.5% respectively. on logistic regression analysis, achievement of the targets for blood cultures, antibiotics, and central venous pressure independently predicted decreased mortality. high-income countries, university hospitals, icus with an accredited fellowship programme, and surgical icus were more likely to be compliant to the resuscitation bundle. conclusions. compliance to the resuscitation and management bundles is generally poor across asia. given the resource limitations in asia, the most appropriate strategy to improve outcomes in severe sepsis may be to concentrate on ensuring early administration of antibiotics after blood cultures, and appropriate fluid therapy. cerebral oxygen desaturation predicts cognitive decline and longer hospital stay after cardiac surgery monitoring brain oxygen saturation during coronary bypass surgery: a randomized, prospective study the work is supported by departmental sources. 1032 clinical features and prognosis of organizing pneumonia pre-senting as acute respiratory failure in icu reference(s). 1. webster nr. ventilation in the prone position prone position in acute respiratory distress syndrome effect of prone positioning on the survival of patients with acute respiratory failure acute effects of upright position on gas exchange in patients with acute respiratory distress syndrome this study was funded by arjo international ag, florenzstrasse 1d metabolic acidosis and fatal myocardial failure after propofol infusion in children: five case reports longterm propofol infusion and cardiac failure in adult head-injured patients mild hypothermia alters propofol pharmacokinetics and increases the duration of action of atracurium intermittent haemodialysis versus crrt for arf in the intensive care unit dialysis dose in acute kidney injury: no time for therapeutic nihilism cirrhotics admitted to icu, and when added to the liver-specific scores of meld or ukeld, improves their respective predictive value intensive care, london, uk, 3 royal free hospital epidural anesthesia, hypotension and changes in intravascular volume intraoperative fluid restriction improves outcome after major elective gastrointestinal surgery surrogate designation: can we trust our relatives? does chest physical therapy work? physiotherapy in intensive care: towards an evidence-based practice fisioterapia no paciente sob ventilação mecânica this research was supported by grants from the following brazilian funding agencies/programs: cnpq, capes, fapesc and unesc readmission to surgical intensive care increases severity-adjusted patient mortality physiological scoring systems and audit predicting death and readmission after intensive care discharge a case-control study of patients readmitted to the intensive care unit severity of illness and risk of readmission to intensive care: a meta-analysis a comparison of admission and worst 24-h acute physiology and chronic health evaluation ii scores in predicting hospital mortality: a retrospective cohort study learning from the past to inform the future-a survey of consultant nurses in emergency care assessing emergency nursing competence post-traumatic stress among swedish ambulance personel levels of mental health problems among uk emergency ambulance workers partial and full ptsd in brazilian ambulance workers: prevalence and impact on health and on quality of life ambulance personnel and critical incidents impact of accident and emergency work on mental health and emotional well being 3 artemis health institute, director, critical care, pulmonology and sleep medicine, gurgaon, india, 4 artemis health institute, nursing, gurgaon, india reference(s) the australian incident monitoring study in intensive care: aims-icu. the development and evaluation of an incident reporting system in intensive care adverse events in critical ill patients ministry of health and social policy 1254 communication: a key factor in the patient safety? anemia of the critically ill: acute anemia of chronic disease impact of allogenic packed red blood cell transfusion on nosocomial infection rates in the critically ill patient high dose recombinant human erythropoietin stimulates reticulocyte production in patients with multiple organ dysfunction syndrome: the journal of trauma: injury, infection and critical ca to the staff of the critical care department, faculty of medicine injury severity and quality of life: whose perspective is important? quality of life and persisting symptoms in intensive care unit survivors: implications for care after discharge variations in health-related quality of life in critical patients funded in part by fogarty international center nih grant no. 1 d43 tw007560-01 and clinical research institute-fundacion valle del lili glasgow coma score, use of mechanical ventilation and vasoactive agents, and the occurrence of severe sepsis (according to bone's criteria-1992). the causes of admission were divided as: ischemic stroke, hemorrhagic stroke, subarachnoid hemorrhage, status epilepticus, traumatic brain injury, elective neurosurgeries, and miscellanea. the foci of infection, microbiological data and bacteremia were analyzed from septic patients. numeric data were expressed as median and interquartiles, while categorical data were calculated as percentage. univariate and multivariate (logistic regression) analysis was carried out to point factors associated with hospital mortality. results. we included 661 patients, with median age 65 years (iq range 50-78) and 54% were male 30%) patients, while it occurred during icu stay on 110 (70%) patients. hospital mortality was associated with age, the admission cause (higher for hemorrhagic stroke, traumatic brain injury and status epilepticus), apache ii score, glasgow coma score and severe sepsis on the univariate analysis cnpq 1317 perioperative factors associated to higher mortality in patients admitted to the neurological intensive care unit (nicu) immediately after brain tumor (bt) resection saldívar 2 1 umae 1 (high-specialty medical unit no 1) el bajío, imss and nicu, hraeb (high-specialty regional hospital of el bajío) anaesthesiology and intensiv care medizin anaesthesiology and intensive care unit charité universitätsmedizin-berlin, department for anesthesiology and intensive care medicine after approval of the local ethics committee, the pdr icg was measured within 24 h post injury (day 0) using the non-invasive limon system (pulsion medical systems of pdr icg to supranormal values higher sofa scores were indirectly associated with lower pdr icg values, particularly for sofa scores[8. when patients were grouped by icu length of stay (\11, c11 days, corresponding to the mean icu los of the german trauma registry), logistic regression analysis identified pdr icg consumables were provided by pulsion medical systems influence of apoe polymorphism on cognitive and behavioural outcome in moderate and severe traumatic brain injury genetic variation of the apoe promoter and outcome after head injury effects of apolipoprotein e genotype on outcome after ischaemic stroke, intracerebral haemorrhage and subarachnoid haemorrhage the association between apoe « 4, age and outcome after head injury: a prospective cohort study decreased cerebrospinal fluid apolipoprotein e after subarachnoid hemorrhage correlation with injury severity and clinical outcome « association of ventilation rates and co2 concentrations with health and other responses in commercial and industrial buildings « sensitivity to noise, personality hardiness, and noise-induced stress in critical care nurses recommended lighting level for offices » the chartered institution of « sick building syndrome, sensation of dryness and thermal comfort in relation to room temperature in an office building: need for individual control of temperature silent misery: most severe critical incidents post traumatic stress disorder in the emergency room: exploration of a cognitive model trauma exposure and post-traumatic stress disorder in intensive care unit personnel increased prevalence of post-traumatic stress disorder symptoms in critical care nurses drivers of quality in health services: different worldviews of clinicians and policy managers revealed systems thinking, system dynamics the fifth discipline: the art and practice of the learning organisation the development of system dynamics as a methodology for system description and qualitative analysis finnish funding agency for technology and innovation nursing activities score tradução para o português e validação de um instrumento de medida de carga de trabalho de enfermagem em unidads de terapia intensiva: nursing activities score (nas) nursing activities score in the intensive care unit: analysis of the related factors 1341 the self-perceived health between medical-surgical and crit-ical care nurses in hungary deutsch 1 , i. boncz 1 , a. sebestyen 3 , a. olah 1 1 university of pecs faculty of health sciences a longitudinal study design was used to explore the self perceived health of inhospital nurses in acute care settings (surgery, casualty, internal medicine, intensive, coronary care, emergency room) in two hungarian factors predicting team climate, and its relationship with quality of care in general practice nurse working conditions, organizational climate, and intent to leave in icus: an instrumental variable approach critical care nurses' work environments: a baseline status report quality of practice in an intensive care unit (icu): a mini-ethnographic case study vasps/intv 2010). medicinska fakulteten, lunds universitet critical thinking and clinical decision making in critical care nursing assessing and developing critical-thinking skills in the intensive care unit gulhane military medical academy, haydarpasa training hospital, istanbul, turkey, 3 gulhane military medical academy technology as a catalyst to transforming nursing care devices and desire: gender, technology and american nursing surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock division of pulmonary and critical care medicine, seoul, republic of korea, 3 peking union medical college hospital, department of critical care medicine mai hospital, intensive care department, hanoi, viet nam, 10 king saud bin abdulaziz university for health sciences, king abdulaziz medical city, intensive care department 12 dr soetomo general hospital, department of intensive care republic of china, 14 ripas hospital, intensive care unit surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock the surviving sepsis campaign: results of an international guideline-based performance improvement program targeting severe sepsis great differences in compli-ance with surviving sepsis campaign bundles surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock delayed diagnosis is associated with increased morbidity, mortality and cost in the icu. as the mortality rate of severe sepsis remains unacceptably high, a group of international expert developed guidelines in 2004, termed the surviving sepsis campaign (ssc). the ssc group has introduced the ''sepsis care bundles surviving sepsis campaign guidelines for severe sepsis and septic shock implementation of a bundle of quality indicators for the early management of severe sepsis and septic shock is associated with decreased mortality improving outcomes for severe sepsis and septic shock: tools for early identification of at-risk patients and treatment protocol implementation observational, prospective follow-up. patients who were admitted into the intensive care unit in university hospital complex a coruña (chuac) during the months of hospital mortality was 30 surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock early goal-directed therapy in the treatment of severe sepsis and septic shock associated with decreased mortality translating research to clinical practice: a 1-year experience with implementing early goal-directed therapy for septic shock in the emergency department improvement in process of care and outcome after a multicenter severe sepsis educational program in spain duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock delta co2 (pvco2-paco2) as a prognostic factor in septic shock septic shock using the new device inspectra 650: relation to macro-and microhemodynamic and outcome c. luengo 1,2 , f. vallée 1 , c. damoisel 1 , m. resche-rigon among the techniques assessing microperfusion, near infrared spectroscopy (nirs) gained interest. more than baseline sto 2 values, the reperfusion slope after a vascular occlusion test (vot) nirs parameters, especially the reperfusion slope scvo 2 or svo 2 ); metabolic (ph, base excess and lactate) parameters were collected. microperfusion data consisted in: nirs (baseline sto 2 , occlusion and reperfusion slopes (%/s), automated software); skin laser doppler microflow (baseline flow (tpu), peak flow (tpu) and slope during reperfusion (tpu/s), measured during and after a 3 min vot. survivors (s) and non-survivors ] differed between s and ns at day 1. macro-hemodynamic and metabolic data did not differ between s and ns plan quadriennal ea3509 svo 2 does not predict fluid responsiveness in critically ill septic patients supported by msm 0021620819 research grant: replacement of and support to some vital organs years) were studied. apache ii and sofa score at study entry were 21 (range: 5-32) and 8 (range: 1-16) respectively. the septic syndrome was due to sepsis (n = 12), severe sepsis (n = 3) or septic shock (n = 39). sites of infection included the lung reference(s). 1. ungerstedt u: microdialysis: principles and applications for studies in animals and man the pathophysiology and treatment of sepsis management of sepsis surviving sepsis campaign guidelines for management of severe sepsis and septic shock relation between muscle na + k + atpase activity and raised lactate concentrations in septic shock: a prospective study long-term continuous glucose monitoring with microdialysis in ambulatory insulin-dependent diabetic patients whether it is worth to correct acidemia by infusion of alkaline solutions is a matter of discussion. there are a number of evidences against the use of alkalinization therapy with respect to the benefits of reversing ph and the side effects of sodium bicarbonate infusion [1]. nonetheless, as recently shown by means of an on line survey, 67% of critical care physicians administer base to patients with lactic acidosis mmol/l), animals were randomized to 60 min of: a) sustained lactic acid infusion, a + b) sustained infusion + sodium bicarbonate, o) transient infusion, b) transient infusion + sodium bicarbonate. in the transient infusion (group o and b), at randomization lactic acid was replaced with normal saline. acid-base status and lactate levels were measured over time. in a number of animals phosphofructokinase (pfk) enzyme's activity was also measured. results. following lactic acid infusion blood lactate rose unnecessary use of alkali perturb acid-base status and lactate metabolism potentially overcoming metabolic adaptive strategies. reference(s). 1. boyd jh, walley kr. is there a role for sodium bicarbonatein treting lactic acidosis from shock? use of base in the treatment of acute severe organic acidosis by nephrologists and critical care physicians: results of an online survey strong ions gap (sig) quantifies unmeasured blood anions and it is calculated by the difference between strong cations and strong anions (all of them, dissociated in blood plasma) retrospective, observational study of all patients with septic shock as defined by the american-european consensus, admitted to the icu from arterial blood gases, albumin, lactate and electrolytes were obtained at admittance and 24 h later; apache and sofa score, central venous saturation and lactate comparison of acid base models for prediction of hospital mortality following trauma forty-five sepsis patients [median age, 62 (iqr, 47-37) years; admission saps ii, 45 (33-53) pts; severest multiple organ dysfunction syndrome score interaction of vasopressin infusion, corticosteroid treatment, and mortality of septic shock comparing two different arginine vasopressin doses in advanced vasodilatory shock: a randomized, controlled, open-label trial lambert 1 1 university of leicester, division of anaesthesia, leicester, uk blood samples were taken: at induction of anaesthesia, at 3 and 18-24 h post-cpb. neutrophils were isolated, mrna extracted, dna cleaned and reverse transcribed supported by a grant from the association of anaesthetists of great britain and ireland, and the british journal of anaesthesia/royal college of anaesthetists secretoneurin (sn), a neuropeptide, is specifically expressed in endocrine elevated nucleosome levels in systemic inflammation and sepsis extracellular histones are major mediators of death in sepsis 23rd esicm asymmetric and symmetric dimethylarginines (adma, sdma) are protein-breakdown markers; both compete with arginine for cellular transport and are excreted in urine. moreover adma, sdma, their ratio (marker of adma catabolism), arginine, interleukin-6(il-6), tumor-necrosis-factor-a (tnf-a), c-reactive-protein(crp) on day 1, 3, 6, 9, 12 and at discharge in 72 consecutive severely-septic patients were measured sdma were higher than normal, adma/sdma ratio was halved, arginine was low. adma was related to total sofa and arginine, inversely related to il-6 and crp; sdma was related to saps ii, sofa, blood urea, creatinine, arginine. adma/sdma ratio was inversely in 14 non-survivors, creatinine, il-6, tnf-a, crp and adma were stable, sdma increased, adma/sdma ratio remained low figure: time course of adma and sdma blood levels (mean ± standard error) during icu stay and the last icu day protein-hmgb-1 levels as predictors of outcome in patients with sepsis and septic shock hmgb1 as a predictor of organ dysfunction and outcome in patients with severe sepsis early low dcs counts may be correlated to disease severity and could predict fatal outcome. however, little is known about dc number in other shock than septic. objectives. to evaluate and compare the circulating dcs number in patients with severe sepsis, septic or cardiogenic shock. methods. in a prospective multicentric study (3 icu), consecutive immunocompetent patients with severe sepsis (ss), septic shock, cardiogenic shock were included. peripheral blood dc counts, measured by flow cytometry, were evaluated and compared between the three populations at admission and 24 h later. correlation to disease severity evaluated by clinical scores and day 28 mortality was studied. results. 100 patients were included (age 61 ± 14 years, 56 male, sofa d0 10.5 ± 3.9, saps ii 61 ± 18): 49 septic shock, 16 severe sepsis and 22 cardiogenic shock. mortality at d28 was respectively 43, 14 and 59%. 13 patients presented a sepsis associated to cardiogenic shock. at baseline and at day 1, a dramatic diminution in the numbers of total dcs either myeloid (mdcs) or plasmacytoid (pdcs), was observed in sepsis (severe sepsis or septic shock) compared to cardiogenic shock patients. no difference was seen between severe sepsis and septic shock patients (fig. 1). we did not observe any correlation between the number of total dcs at admission or at day 1 and severity of illness scores dc reduced number is a valuable marker of severe sepsis in shock and is not affected by hemodynamic changes. it could not be used as a prognostic marker in severe septic patients. 1382 preliminary results from a prospective study assessing the relationship between standard laboratory coagulation and global tests of clot-formation using thromboelastography in patients with fulminant hepatic failure v the routine use of international normalized ratio (inr) to establish the coagulation status in patients with fulminant hepatic failure (fhf) may be misleading. anecdotally, fhf patients, despite a significantly deranged inr, may display a normal or even hypercoagulable state, as recently shown, albeit in an extracorporeal setting, with frequently clotted circuits, despite raised pt we prospectively studied coagulation, demographic, survival and outcome measures of fhf patients (defined by de-novo liver failure, coagulopathy-inr [1.5, and encephalopathy) admitted to the royal free hospital liver and/or intensive care unit(s) (icu), a tertiary referral centre in liver diseases and transplantation we present the standard clotting tests and teg results from 10 (of a required 20) patients currently enrolled, demonstrating variable degrees of encephalopathy and coagulopathy effect of norepinephrine on cardiac output and preload in septic shock patients apparent heterogeneity in splanchnic vascular response to norepinephrine during sepsis aggressive use of high-dose norepinephrine in the treatment of septic shock norepinephrine requirement is not an independent variable to predict outcome in severe septic shock patients aim. the aim of this study was to measure the level of ptsd among hungarian ambulance workers, and explore factors which can influence it.sample and methods. 411 hungarian ambulance workers were involved to this crosssectional study (147 ambulance drivers, 173 ambulance nurses, and 91 ambulance team leaders: medical doctors and ambulance officers). self filling questionnaire were used for data collection, including briere's trauma symptom checklist, and socio-demographic questions. chi square test, independent t test and variance analysis were used for comparison of variables.results. the average ptsd-points of ambulance workers was 20. there was significant association between level of ptsd and gender: women's average 23, men's average 18 ptsd-points (p = 0.049). there were no correlations between level of ptsd and type of settlement, location of ambulance station and level of education. those who would need psychological support (p = 0.01), and those who had psychologically traumatic experiences in the last 2 years have significantly higher ptsd-points (p = 0.01).conclusions. hungarian ambulance workers are exposed with many effects which can lead ptsd. professional psychological support is needed in order to cope with ptsd successfully.the results were presented and discussed in our weekly meeting on patient safety and healthcare for all icu personnel. by the end of this year all the recommendations will be implemented in our icu.conclusions. we improved the safety and quality of in hospital transportation of icu patients by performing a prospective risk analysis. bow-tie is a good instrument to identify health care risks. to determine the incidence of phrenic neuropathy associated with the catheterization of internal jugular and subclavian veins, without ultrasound support, in patients admitted to an icu. a prospective study was performed by following patients admitted in the icu between october 2008 and may 2009. a normal neurography of both right and left phrenic nerves at the moment of their admission was the main inclusion criteria. after this baseline study, a new neurography was repeated weekly (chen and resman method, sinergy medelec), during their stay and at the moment of being discharge from icu. simultaneously, all vascular subclavian and internal jugular vein catheterization were registered. a final neurography and a fluoroscopy study were performed after being discharged from hospital. results. 40 patients were included and two hundred and ten neurographies of both right and left phrenic nerves were performed. 14 patients did not receive any vascular punctures in the cervical region during the follow up period, acting as control group. 26 patients underwent a total of 48 vascular catheterization, 43 in subclavian vein (89.6%) and 5 in internal jugular vein (10.4%). a phrenic neuropathy was diagnosed in 4 patients. this represented an incidence of 15% (4/26) of phrenic neuropathy per patient and 8% (4/48) related to subclavian and internal jugular vein catheterization. in relation to patients without phrenic nerve injury who underwent subclavian and internal jugular vein catheterization, patients affected of phrenic neuropathy had longer mechanical ventilation time (33 ± 36 days vs. 19 ± 13, p = 0.3) and longer average stay time in icu (49.7 ± 35 days vs. 25 ± 14, p = 0.08), although these differences have not statistical significance. we did not find significantly differences related to age (53 ± 9 vs. 55 ± 18, p = 0.2) and apache ii index (16 ± 3.5 vs. 15.8 ± 5.8, p = 0.4) between both groups (wilcoxon two-sample test). we performed a control neurography of 2 case patients after being discharged from hospital. we checked the cmap phrenic nerve reappearance after 3 weeks and 6 months of being diagnosed its neuropathy, respectively. conclusions. we found an incidence of phrenic neuropathy of 15% per patient and 8% related to subclavian and internal jugular vein catheterization, during the follow-up period. the time of reappearance of phrenic cmaps after being detected its neuropathy points to a neuroapraxia or partial axonotmesis as pathogenic type of injury.discussion. phrenic neuropathy has to be considered in cases of difficult weaning of unclear etiology. the catheterization of subclavian and internal jugular veins should be recommended employing ultrasound support. p. merino 1 , m.c. martin-delgado 2 , j. alvarez 3 , i. gutiérrez-cía 4 , á . alonso-ovies 3 , syrec 1 hospital can misses, icu, ibiza, spain, 2 isde, á rea de salud, madrid, spain, 3 hospital de fuenlabrada, icu, madrid, spain, 4 hospital clínico universitario, icu, zaragoza, spain introduction. syrec project aims to improve icu patient safety. the project includes an epidemiological study. we present the main results.objectives. to estimate the near miss (nm) and adverse events (ae) rate in spanish intensive care units (icus). we study the incidence and nature. finally, we classify and analyze its severity.methods. multicenter prospective observational cohort study. inclusion criteria: patients admitted to the 79 participant icus during the 24-h observation period. during this period, nm and ae detected and reported inside and outside icu were included. only outside icus were considered when its were the reason for admission. we evaluate the kind of incident, severity and preventability. data collection studied under the distribution of frequencies.results. 1,017 patients were included. 1,424 incidents were reported in 591 patients, 943 were nm and 481 ae. risk: the median risk of nm was 73% versus ae 40%. 22.1 incidents per patient admitted. incidence rate: the incident rate median was 5.89 per 100 patients per hour icu stay, the nm of 3.47 per 100 patients per hour icu stay and that of ae, 2.04 per 100 patients time of stay in icu. the 66% of the incidents reported have been nm and 34% ae. this incidents causing temporary damage in the 29.50% of occasions and in the 4.28% permanent damage, compromised the patient's life or contributed to death. classification of incidents (table 1) . conclusions. our study shows a high individual risk. our icus services present a highrisk environment. therefore we have to go into the developement of epidemiological studies depth, in order to create further strategies supporting patient safety. restore cardiovascular performance in severe lactic acidotic rats a. kimmoun 1 , n. sennoun 1 , n. ducrocq 1 , b. levy 1,2 1 inserm u961, groupe choc, vandoeuvre-lès-nancy, france, 2 chu nancy brabois, intensive care unit, vandoeuvre-lès-nancy, france introduction. lactic acidosis during shock is responsible for myocardial failure, vascular hyporesponsiveness and a decrease in sensitivity to vasopressor agents. sodium bicarbonate is a proposed treatment to correct acidosis, although with deleterious cardiovascular effects. indeed, hypocalcemia and hypercapnia, both powerful myocardial depressants, are the main side effects of the administration of this therapy [1] . objectives. already studied in experimental models of isolated lactic acidosis, the cardiovascular effects of sodium bicarbonate administration have never been explored after correction for hypocalcemia and hypercapnia. methods. we therefore compared, in a rat model of severe lactic acidosis (ph \ 7.2, hyperlactatemia[3 mmol/l) induced by a state of controlled hemorrhagic shock, the cardiovascular effects of: (1) standard resuscitation plus administration of sodium bicarbonate with correction for calcemia and paco 2 (''adapt'' group, n = 5); (2) standard resuscitation plus administration of sodium bicarbonate without correction for paco 2 and calcium (''nonadpat'' group, n = 5);(3) standard resuscitation; (''stand'' group, n = 5); (4) standard resuscitation plus calcium administration (''calc'' group, n = 5). evaluation at steady and shock state, 60 min and 120 min was focused in vivo on arterial gas and myocardial contractility (emax) by conductance catheter. ex vivo vasoreactivity was tested on mesenteric arteries (300 lm) by myography. sodium intakes were equivalent between groups. results. our model displayed a profound acidosis from 7.31 to 7.18 ± 0.01 (p = 0.02) and hyperlactatemia from 2.8 ± 0.4 to 9.2 ± 0.6 mmol/l (p \ 0.01). emax decreased from 2.5 ± 0.33 to 0.54 ± 0.06 mmhg/ll p = 0.01. in the adapt group, at 60 min, ph was normalized at 7.36 ± 0.01 (p = 0.04). furthermore, emax was enhanced at 332 ± 43% (p \ 0.001) (stand: 145 ± 26%, nonadapt: 94 ± 18%, calc: 234 ± 52%). the cumulative dose of infused norepinephrine was significantly lower in the adapt group 145 ± 26 lg/kg compared to other groups (stand: 344 ± 10 lg/kg, nonadapt: 438 ± 10 lg/kg, calc: 423 ± 103 lg/kg, p = 0.02). ex vivo mesenteric vasoreactivity in the adapt group was normalized (graph 1).mesenteric vasoreactivity to phenylephrine conclusions. in severe lactic acidosis, infusion of sodium bicarbonate after correction of its side effects improves myocardial function and vasoreactivity. [1] . the prevalence and significance of 25-hydroxyvitamin d deficiency in the intensive care unit have not been fully determined. a recent study of an unselected group of itu patients [2] has suggested low itu admission 25-hydroxyvitamin d levels are common. objectives/hypotheses to be tested. royal free hospital intensive care unit patients exhibit low circulating levels of 25-hydroxyvitamin d. circulating levels of 25-hydroxyvitamin d decrease further during the course of hospital admission. admission circulating levels of 25-hydroxyvitamin d affect itu morbidity and mortality methods. all itu admissions were assessed within 72 h of presentation and patients who were deemed to have the potential to require admission for at least 1 week were included. demographic and clinical data were obtained in a prospective manner. results were recorded from samples obtained at admission, 7 days and 28 days. standard itu nutrition protocols were used. no interventions were performed. results. clinical and outcome data were obtained for 40 patients. no significant differences between apache 2, saps 2 or apache 3 scores for survivor and non-survivor groups at either itu or hospital discharge were noted. 60 further patients await complete data analysis. 0% (0 of 39 for whom results were available) achieved an adequate ([75 nmol/l) circulating 25hydroxyvitamin d level. 3 patients (7.7%) demonstrated levels within the insufficient range (50-75 nmol/l). 2 patients (5.1%) did not have any detectable 25-hydroxyvitamin d. the remaining 34 patients (87.2%) were either in the deficient (41.0%, 25-50 nmol/l) or severely deficient (46.2%, 10-25 nmol/l) ranges. admission 25-hydroxyvitamin d levels in survivors and non-survivors were compared at itu and hospital discharge. no significant differences between the four groups (p [ 0.05, anova) were observed, indicating that in this data set, admission 25-hydroxyvitamin d levels do not appear to alter or determine clinical course. mean 25-hydroxyvitamin d levels were compared at admission, day 7 and at day 28. no significant differences between the three groups (p [ 0.05, anova) were identified. no significant differences between the mean 25-hydroxyvitamin d levels of the survivors and non-survivors at day 7 or day 28 were apparent (small numbers). admission [1, 2] and patients undergoing surgical procedures [3] . patients with neurological illness can receive significant quantities of ns, chosen primarily for its iso-osmolar properties. objectives. ns is commonly used as maintenance and resuscitation fluid by the anaesthetist, and as intravascular flushes by the radiologist during prolonged interventional neuroradiological (inr) procedures. this pilot feasibility study aimed to ascertain the effect of ns infusion on acid-base measurements in patients undergoing inr procedures under propofol-remifentanil anaesthesia. methods. we collated routine electrolyte, albumin and acid-base data of 5 patients who underwent coil/glue embolisations of intracranial aneurysms and vascular malformations, both before and after the procedure. base excess (be) was partitioned into the effects of sodium chloride difference (na-cl), albumin, lactate and unmeasured anions (uma), using the stewart-fencl-story approach [4] . all values are reported as medians (ranges objectives. to investigate the erythropoietic response to hight dose of a weekly schedule of recombinant human erythropoietin (rhuepo) in critically ill anaemic septic patients. a total of 60 patients admitted to the intensive care unite (icu) were enrolled in this study, patients were randomized to receive either rhuepo or not, 30 patient did to form the rhuepo group, 30 did not to form the control group.results. the epo treated group of patients showed significant increase in reticulocyte count compared with baseline p \ 0.001, as well as with the control group p \ 0.006. the epo treated group exhibited also a significant increases in hb concentration compared with baseline p \ 0.001 as well as the control group .0 3. all patients in the control group received rbc blood transfusion 100%, while only 83.33% of the epo group did. the epo treated group showed significant decreases in their apache ii score during the study period compared with baseline p \ 0.001 as well as with the control group p \ 0.05. the epo treated group showed no significant difference in their sofa score compared with baseline p \ 0.923, however the control group exhibited continuous and significant increase in their sofa score throughout the study period compared with their baseline p \ 0.003, there was no significant difference in the final outcome recovery, mortality or morbidity p .337, p \ 0.286 respectively.conclusions. the administration of rhuepo to critically ill anaemic septic patients is effective in raising their reticulocytic counts, hb concentrations and in reducing the total number of units of rbcs they require. in addition there was a trend toward better in hospital clinical course, increased recovery and decreased mortality in the rhuepo group.conclusions. anaemia is common following critical illness but does not appear to affect the physical aspects of recovery during medium term rehabilitation. this may be due to an overwhelming degree of symptom burden from other complications of critical illness impairing physical function to such a degree that the effects of anaemia are negligible in the medium term. although decreases in number and function has mainly been described in skeletal muscle, also other organs seem to be affected and it has been hypothesized that mitochondrial dysfunction might be involved in the development of organ failure. to study the effect of plasma of patients with septic shock on mitochondrial function in vitro to potentially later on identify a central factor affecting mitochondria in all tissues during sepsis and leading to multiple organ failure.methods. after sacrificing 6-8 week old sprague-dawley rats, mitochondria from soleus muscle were isolated through homogenization and a series of centrifugations. mitochondrial function was assessed by measuring of oxygen consumption, using an oxygraph containing a clarke-electrode, after addition of adp. before these measurements, mitochondria were incubated with plasma from septic patients or healthy volunteers, respectively, for 30 min. in our second series, the mitochondria were incubated with different concentrations of il-6, tnf-a or buffer. respiration rates were measured in the presence of adp (state 3; a measure for the oxidative capacity to produce atp) and without the presence of adp (state 4; a measure for the amount of uncoupling). respiratory control ratio (rcr; a measure for the respiratory efficiency of the mitochondria) was calculated by dividing state 3 by state 4 activity. all measurements were related to citrate synthase activity to compensate for the amount of mitochondria. statistical differences between the groups were analyzed using a student's t test.results. adp dependent (state 3) respiration was 98% higher and rcr 165% higher in the mitochondria incubated with plasma from the septic patients compared to those incubated with plasma from healthy volunteers (table) . there were no significant differences between the groups incubated with preservation buffer or the different cytokines (table) . introduction. microvascular fluid loss from the intravascular to the interstitial space generates tissue edema and is one of the major challenges in emergency and intensive care medicine. isolation of interstitial fluid (if) from skin makes it possible to study the microcirculation and proteins in this environment both during normal as well as pathophysiological conditions such as acute inflammation.objectives. by studying bio-markers from proteomic analysis by mass spectrometry in an inflammation model, we wanted to find proteomes that could be important in explaining inflammation. we have applied a recently described centrifugation method in a porcine model and compared it with implanted wicks. in nine anesthesized piglets we compared the methods and evaluated the if, by overhydrating the pigs with 3000 ml of acetated ringer's solution for 1 h, and thereafter continuously supplemented for 1 h according to fluid losses. if was isolated from implanted dry wicks, wet wicks and by centrifugation of excised skin. the methods were evaluated by the ability to reflect overhydration and to show the expected composition of plasma proteins in if by use of hplc. the if was also processed further with mass spectrometry to find possible tissue degradation or inflammation due to overhydration. statistics: by spss v 13.0 and graphpad instat (version 3.05). significance level: p = 0.05. colloid osmotic pressure in if was significantly lowered after overhydration for all the tree methods. wet wicks p = 0.05, dry wicks p = 0.001, skin samples p = 0.05. hplc of if collected with centrifugation after overhydration, identified peaks representing molecules smaller than albumin. mass spectrometry of the same if identified several proteins associated with inflammation: alpha-1-antichymotrypsin and lumican, the latter a protein identified as a modulator of inflammation. we have introduced a new centrifugation method for isolation of if from the skin of pigs. by further analysis of if isolated by centrifugation we were able to distinguish proteins found only in the if of the pigs overhydrated with ringer's acetate. these proteins could be associated with an inflammatory condition in the skin caused by massive overhydration, again causing tissue degradation. identification and validation of proteomic biomarkers can be a useful tool in future treatment of inflammation in general, and in sepsis in special. objectives. to define the pattern of change in metabolites by mrs in experimental sepsis. male sd rats (weight 325-375 g) underwent cecal ligation and puncture or sham procedure (n = 8 per group), and 24 h after surgery were euthanized. pulmonary tissue was extracted for magic angle mrs (hr-mas) and processing by the r metabonomic package. a supervised statistical analysis of main components (mc) was performed on the processed spectra.results. the mc analysis discriminated both group (septic and nonseptic) indicating a different metabolite profile. in addition, the analysis of mc loading revealed 4 displacement positions in the discrimination between groups with a variation in the signal intensity of 50%.conclusions. metabolomic analysis of pulmonary tissue by mrs is a potentially useful technique for the detection of biomarkers in sepsis.grant acknowledgment. introduction. cd14 + cd16 + neutrophils are a key subset of phagocytes associated with severe bacterial sepsis [1] . their characteristics, and potential neuro-immunomodulation, have not been explored in humans neutrophils exposed to septic plasma from icu patients. to assess the effect of adrenergic/cholinergic neurotransmitter molecules on human neutrophil adhesion and activation markers following exposure to human septic plasma. with irb approval, neutrophils were isolated from 5 healthy volunteers (ficoll density gradient separation) and incubated for 24 h with either plasma from healthy volunteers or septic patients plus pathophysiological concentrations of epinephrine (e), norepinephrine (ne) or acetylcholine (ach) and nicotine (nic) to assess potential parasympathetic-related neuro-immunomodulation. flow cytometry (dako cyan) measured expression on neutrophils of cd11, cd14, cd16 antibody markers and viability. median values are shown; analyzed by anova.results. neutrophils were unaffected by ne, e, ach or nic after incubation with plasma from healthy volunteers. after incubation with septic plasma, marked neutrophil activation occurred (p = 0.02). however, nic reduced cd14 + cd16 + activation (* fig. a ) by 95% (median (35-97%; 25th-75th centiles); p = 0.01). nic also attenuated cd11 expression, suggesting reduced neutrophil adhesion (* fig. b) . neutrophil viability was similar across drug and plasma treatments. conclusions. these preliminary data suggest that nicotine attenuates both the activation and adhesion of human neutrophils exposed to human septic plasma, but does not affect viability. objectives. the aim of this study was to evaluate the potential impact of lag between sepsis initiation and start of treatment on mitochondrial respiration. methods. 24 animals [40.1 ± 4.1 kg] were randomized (n = 6/group) to a control group (group i) and three groups resuscitated at 6 (group ii), 12 (group iii), and 24 (group iv) hours, respectively, after fecal peritonitis induction. fecal peritonitis was induced with instillation of 2.0 g/kg of autologous feces via intra-peritoneal drain. resuscitation was performed according to the ssc and esicm sepsis guidelines for 48 h. respiration of permeabilized skeletal muscle fibers and their isolated mitochondria was assessed at baseline and after 6, 12, 24, 48 and 72 h, when applicable, or before death occurred, if earlier. at the end of the experiment, also isolated brain, hepatic and myocardial mitochondrial respiration was measured using high resolution respirometry (oxygraph-2 k, oroboros instruments, innsbruck, austria). results. mortality (33%, each) and organ dysfunction was highest in groups iii and iv. in these two groups, different pattern of changes of skeletal muscle mitochondrial complex i-dependent respiratory control ratio (rcr) were observed (table 1) . no significant differences between groups were observed for complex i-and ii-dependent rcr values of hepatic, myocardial and brain mitochondrial respiration (fig. 1 ). there were no significant differences between the groups for any of the complexes in permeabilized skeletal muscle fibers mitochondrial respiration (data not shown). conclusions. despite the high mortality observed in groups resuscitated at later time points after induction of sepsis, end organ mitochondrial function assessed using physiological substrates was preserved. despite significant changes in skeletal muscle mitochondrial respiration efficiency in the two groups with the highest mortality, our findings do not support the view that mitochondrial dysfunction plays a major role in the pathogenesis of multiorgan dysfunction in experimental sepsis. grant acknowledgment. swiss national fund, nr: 3200-061988; stiftung für die forschung in anästhesiologie und intensivmedizin. adipose tissue is an endocrine organ which produces signalling proteins involved in inflammation and glucose homeostasis [1] . one of these proteins, adiponectin, promotes glucose utilisation and fatty acid oxidation and thus improves insulin sensitivity via its two receptors, adipor1 and adipor2 [2] . adiponectin expression has been shown to be reduced in type ii diabetes, obesity and endotoxaemia [2, 3] . adiponectin also exhibits antiinflammatory properties [4] . in this study, we have examined whether adiponectin and its receptor gene expression changes in murine adipocytes stimulated by lps. methods. 3t3--l1 adipocytes were grown in culture media (dmem with 10% fetal calf serum) until confluent. pre adipocytes were differentiated with the addition of 10 mg/ml insulin, 1 mm dexamethasone and 100 mm ibmx. media was changed every 48 h. cells were treated on day 12 with 100 ng/ml, 1 or 10 mcg/ml lps (escherichia coli, sigma-aldrich). cells were harvested at 4 and 24 h. mrna levels were determined by rt pcr in a 12.5 ll reaction volume consisting of 12.5 ng of reverse transcribed cdna mixed with optimal concentrations of primers and probe and qpcr tm core kit (eurogentec, uk) in 96-well plates on a mx3005p detector. results. cell response to lps was confirmed using il6 as a reference gene. expression of adiponectin mrna was significantly reduced in cells treated with 10 lg/ml lps harvested at 4 h (7.1 fold p = 0.002). there were no changes in cells treated with lower concentrations of lps. there were no changes at 24 h. r1 gene expression was significantly reduced following treatment with 100 ng/ml lps at 4 h (1.5 fold p = 0.04), but treatment with higher concentrations did not change expression. there were no changes at 24 h. r2 expression levels were significantly reduced at 4 h in the 1 and the 10 mcg/ml groups (2.6 fold p = 0.02 and 4 fold p = 0.01) respectively. there were no changes at 24 h. discussion. our results add to the evidence that changes occur in the adiponectin system during inflammation. in this model, we observed rapid reduction (at 4 h) in adiponectin at high dose lps, r1 at low dose lps and r2 at medium and high doses. there were no changes in expression levels at 24 h. this suggests that a rapid change in the adiponectin system may occur in response to lps but this change is not maintained at 24 h. in a previous study, our group has shown reduced adiponectin gene expression in adipose tissue depots in lps induced endotoxaemia [3] . it is interesting that different concentrations of lps induce different changes within the adiponectin system. further studies are needed to elucidate whether reductions in both adiponectin and its receptor may contribute to the inflammatory changes and hyperglycaemia commonly observed during sepsis including all co poisoned patients treated with hyperbaric oxygen. following parameters were seized: age, sex, date of admission, sofa, the source of the intoxication, the gravity co score, the initial clinical examination (realized by first aid), biology, the rate of hbco, the murray score and the rate of complication. results. 405 patients were included in the study. the sex ratio was 61%, the mean age was 42 ± 18 years and the global mortality was 5, 9%. among the 405 patients 29% were poisoned by smoke (s group), 60% by pure co (c group) and 11% by exhaust fumes. more than 30% of the exhaust fumes victims were suicide origin. this characteristic is associated with neurological impairment induce by ingested drugs. then, their neurological status is impossible to link to the co poisoning. we have therefore decided to exclude this group. the sofa score was higher in the s group compared with the c group (0.38-2.17; p \ 0.0001). a co score equal to 4 was present in 33 versus 1% respectively in s versus c group (p \ 0.001). in the under group of patients having a co score at 3, 0% (0/101) of co poisoned patients versus 18.4% (7/38) of smoke poisoned patients were ventilated (p \ 0.001). these patients were intubated either during transport or in the intensive care and none of them received hydroxycobalamine during the first aid (before intubation). the laboratory data showed in the s group a higher lactates level (2.2 vs. 3.9 mmol/l; p = 0.005) and lower initial pao 2 /f i o 2 ratio (365 vs. 306; p = 0.002). nine percent of the s group present a murray score at 4 versus 0% for c group (p \ 0.0001). pneumonia, shock and death were significantly more frequent in the s group (respectively 8.6 vs. 0.8%, p \ 0.0005; 11.2 vs. 0.8%, p \ 0.0001; and 17.9 vs. 1.2%, p \ 0.0001)conclusions. as expected the smoke poisoned group has a higher mortality than pure co group (mortality 17% vs. overall mortality 5.9%). at equivalent co gravity score, mortality and complications are always more frequent in the smoke poisoned group. the smoke poisoned group has a high risk of degradation. those patients require specific monitoring and support and probably early administration of hydroxycobalamine. hypothesis. at administration and maintaining higher plasma levels of at can reduce the need for inotropes in burn shock patients. we performed a retrospective cohort study of burn shock patients admitted to a single tertiary care center over 7 years period. patients were eligible for inclusion if they were received fluid resuscitation with ringer's solution and colloid according to clinical guidelines. data were abstracted including demographic, burn injury characteristics, resuscitation fluid volume, the type of colloid and the average of plasma at levels within 72 h after burn injury. administration of fresh frozen plasma and/or recombinant human at was defined as at administration. the decisions of at administration and inotropic support (dopamine or dobutamine) were made by the attending intensivists. primary outcome measure was the need for inotropes within 72 h after burn injury. cox regression model was used to estimate the risk reduction by at administration and average of at levels. [1] . argon, another member of the noble gas family has been reported previously to have a neuroprotective property [2] . the aim of this study was to investigate whether it attenuates neuronal injury in a rat model of neonatal asphyxia. methods. seven-day-old postnatal sd rats underwent right common carotid artery ligation and then recover with their dim for 1 h. thereafter, they were exposed to 8% o 2 balanced with nitrogen for 90 min. after 2 h, they were treated with 70% argon or 70% nitrogen (positive control group) for 90 min. the cohort pups without intervention served as naïve control. they were perfused 7 days later and their brains were sectioned and stained with 0.5% cresyl violet. microphotographs were taken from ca area of the hippocampus near -3.6 bregma relative to adult brain at 40 9 magnification. healthy cells were counted in a blind manner and their mean value was used for data analysis. results. the thickness of healthy layers in the right ca area of the positive control group was remarkably reduced compared with other groups (fig. 1 ). quantitative analysis revealed that argon treatment significantly increased healthy cell numbers in the right ca area of hippocampus from 36.53 ± 1.201 in the positive controls to 54.37 ± 1.162 (p \ 0.01) (fig. 2 ). grant acknowledgment. this study was supported by a grant from action medical research, uk. objectives. our objective was to study the mechanisms of death following high-dose citalopram administration in rats. experimental study in sprague dawley rats with intraperitoneal (ip) citalopram administration; determination of the median lethal dose (mld)using the dixon and bruce upand-down method; clinical descriptive study of citalopram-induced features and measurement of alterations in respiratory pattern (arterial blood gases and plethysmography) and biological parameters including blood lactate (scout ò , ekf diagnostic), plasma and platelet serotonin concentrations (high-liquid performance chromatography-fluorometry); determination of the preventive activity on seizures and death of diazepam, cyproheptadine, and propranolol pretreatments with the determination of their minimal effective dose; comparisons using anova for repeated measurements followed by bonferroni post-test.results. citalopram ip-mld was determined as 102 mg/kg in rats. seizures were significantly increased in rats receiving 80 and 120% of citalopram mld versus controls (p \ 0.01 and p \ 0.05, respectively), while death rate was only significantly increased in rats treated with 120% of citalopram mld (p \ 0.001). significant decrease in body temperature was observed after 90 min in rats treated with doses[60% mld in comparison to controls (p \ 0.05). occurrence of serotonin behavioural syndrome was comparable in all groups. citalopram administration did not result in significant hypoxemia, hypercapnia, and lactate elevation, thus not supporting the hypothesis of the occurence of any significant deleterious cardiovascular effect in citalopraminduced toxicity. however, a significant moderate increase in the inspiratory time (p \ 0.05) accompanied with an expiratory braking was observed. a significant decrease in platelet serotonin and increase in plasma serotonin concentrations were measured (p \ 0.05). pre-treatment with diazepam (1.77 mg/kg) and cyproheptadine (17.1 mg/kg) of rats receiving a lethal citalopram dose prevented seizures and death, while propranolol was ineffective.conclusions. citalopram respiratory toxicity remains mild, while deaths result from seizures probably related to serotonin toxicity. our observations may be helpful to better understand and manage human citalopram poisonings. objectives. to define the population pharmacokinetics (pk) of phenytoin in the critically ill, in addition to risk factors for sub-therapeutic dosing.methods. free and total ptn concentrations were measured in serum by means of high performance liquid chromatography following microfiltration, two to three times in the first 24 h after a loading dose. population pk modelling, including intra and interindividual variability, were determined using nonmem (r) . in the netherlands the use of diazepam is advised as first line treatment although evidence is not established and mainly provided through case-reports [1] . to compare the effect of diazepam on mortality in (hydroxy) chloroquine intoxication to standard therapy. we performed an extensive medline search (1950-april 2010) with a manual reference search of identified papers. (hydroxy) chloroquine intoxication studies and case reports in english, dutch or french were evaluated. patients older than 15 years with severe intoxications, based on measured concentrations or life-threatening symptoms, were included. pooled relative risk (rr) for mortality with corresponding 95% confidence interval (ci) were calculated by means of a fisher exact test. our results were compared with two retrospective and one prospective study.results. there were 32 case reports identified from which 25 case reports met our inclusion criteria. thirteen patients received diazepam of whom two died, compared to twelve patients who did not get diazepam of whom one died. statistical analysis demonstrated that treatment with diazepam was not associated with a lower mortality rate (rr: 1.1 ci 0.8-1.5; p = 1.0).although pooling of case reports is debatable, these results were comparable to the retrospective and prospective studies that didn't show any benefit from diazepam in chloroquine intoxication [2, 3, 4] . the positive effect of diazepam may have been underestimated, due to the fact that it has been given only as rescue therapy.conclusions. based on our analysis there is a lack of evidence concerning any antidotal effect of diazepam. good supportive treatment is pivotal. if the clinical manifestations of (hydroxy) chloroquine intoxications require sedation or treatment of seizures, diazepam is a good choice based on its pharmacological profile. a prospective study which compares diazepam to sedativa with similar pharmacokinetic and dynamic profile is required to prove that diazepam has any antidotal effect. introduction. brain is one of the first organs affected in sepsis and evaluation of brain function is difficult since patients are under sedation. it has been shown that mitochondrial dysfunction may play a significant role in the pathogenesis of septic encephalopathy. here we investigated inflammatory and metabolic parameters in a model of polymicrobial sepsis in mouse. methods. sepsis was induced by intraperitoneal injection of feces. animal received imipenem 6 h after the procedure. control animals received intraperitoneal saline and imipenem after 6 h. blood cytokines and serum lactate were measured. the animals were sacrificed by cervical dislocation. brain slices of 400 mcm were used to measure oxygen consumption and glucose uptake.results. interleukin 6, mip 1a and interleukin 1b significantly raised in the first 6 h after sepsis induction (p = 0.001; p = 0.0017; p = 0.0001 respectively). in 24 h only mip 1a was significant higher (p = 0.0001). lactate was elevated 6 and 24 h after sepsis induction (p \ 0.01 and p \ 0.001 respectively). oxygen consumption increased after 6 h of sepsis and drops under control values 24 h after the induction of sepsis. glucose uptake, measured by the nbdg fluorescence, was higher after 6 h (p = 0.0475) and 24 h after sepsis induction.conclusion. in a murine model of abdominal sepsis, inflammatory markers, lactate production, and brain glucose uptake increased and were parallel to alterations in the mitochondrial oxygen metabolism. introduction. the royal bournemouth hospital has one of the highest out-of-hospital cardiac arrest admission rates in the uk. in 2005, following ilcor/aha guidelines [1] , a cooling protocol was developed for patients with return of spontaneous circulation after advanced life support for ventricular fibrillation or pulseless ventricular tachycardia. in preparation for potential new ilcor/aha guidelines in 2010, the prospective database of outcomes for these patients was analysed.objectives. to evaluate the outcomes of therapeutic hypothermia for patients with return of spontaneous circulation following cardiac arrest. outcome data from our prospective registry of cooled patients are summarised.results. sixty-three patients were cooled in 4 years (median age 68 years; mode 76; range 19-88 years). 84% survived to itu discharge and 70% to hospital discharge. 98% of these were discharged home (14% to a rehabilitation hospital before home and one patient to a long term care facility). ninety-five percent of survivors were alive at 6 months and 80% alive at 1 year with seven status results still pending. median itu length of stay was 3.5 days (range 1-13). six patients required temporary percutaneous tracheostomies for airway protection and weaning from ventilation. median duration from itu to hospital discharge was 16 days (range 0-62).conclusions. this series is large by comparison to other uk centres. survival to hospital discharge, at 6 months and 1 year were better than other published results. although neurological outcomes were not formally assessed, we believe that the capacity to discharge home is a desirable patient outcome and represents the beneficial neurological effect of our cooling protocol. selection bias will have undoubtedly affected our results. however the age of our patients was higher than in published trials and in other reports is considered an adverse outcome predictor. our data would not support restricting induced hypothermia on the basis of age alone. we consider the itu and hospital lengths of stay required to discharge these patients to be long. these data were not reported in original trials. discharges may obviously be delayed for non-clinical reasons. this aside, neurological recovery progresses for months after cardiac arrest and discharge home may still prove possible if time is allowed. however, post-itu resource implications should be considered when introducing a cooling protocol. introduction. acute ischaemic stroke (ais) is the third largest cause of mortality and the leading cause of chronic disability in the industrialized world. in some parts of europe and the united states 26-43% of patients with ais may be admitted to a neurological intensive care unit (icu) for supportive therapy with 8-10% receiving mechanical ventilation [1, 2] . there are currently no agreed uk criteria for the admission of ais patients to critical care.objectives. to review the incidence and outcome of ais in our tertiary icu over the last five years. november 2005 and november 2009. ais was classified as thrombo-occlusive or embolic. subarachnoid haemorrhage and primary intracerebral haemorrhage were excluded. demographic and outcome data were recorded and compared against a mean value of all icu admissions.results. ais comprised 0.8% of icu admissions during the study period. demographic data is presented in table 1 as mean ± standard deviation or median (interquartile range) as appropriate. in 2008 4% (11/272) of hospital ais admissions were admitted to icu. 14 patients had surgical procedures including 8 decompressive craniectomies. 27% of survivors had a discharge gcs of 15/15. mortality for unselected medical admissions over the study period was 36%. there are differences of significance in the mortality according to the age, classified by age groups with an age cut off of 80 years (\80 years 38.6 vs. c80 years 40%, p \ 0.01). apart from the gcs, the rest of the variables analyzed in the ich score are not of significance; supra and infratentorial, presence of intraventricular blood neither on the divided volume over or under 30 cc although, in the latter, a p \ 0.1 can be observed and if we only analyze the supraventricular, it comes out as significant. other analyzed data are the time of the surgery, which is not significant, the need for mechanical ventilation, which is (42.9 vs. 15.8%, p \ 0.05), and the days of ventilation with a mortality clearly higher on those patients with\5 days of ventilation (83%) and on those of shorter stay (lesser then 5 days 79%).conclusions. let be remarked that the samples have been taken from patients admitted in the intensive care unit, losing a possible sample of less serious patients, and with a higher level of consciousness, what might explain why supra or infratentorial location and the volume don't come out as forecasting factors, since its likely that there are many small infratentorial outside the intensive care unit. we highlight also that the high mortality in the first few days can be caused by those patients who are admitted as donors, developing an encephalic death in the first days, conditioning also the data regarding the mortality on fewer days with ventilation. the finish up, we have to point out the fact that the presence of previous hypertension during the treatment might be a bad forecasting factor that should be deeper studied. to determine whether a delay exists between the time of diagnosis of intracranial haemorrhage and the time of reversal of anticoagulation, in patients presenting within our region. following approval by all audit and haematology departments a 6 month retrospective analysis was performed. we reviewed consecutive patients who received reversal of anticoagulation with pcc and vitamin k having presenting with intracranial haemorrhage whilst on warfarin. time of diagnosis was obtained from the time of scan and time of pcc issue was obtained from the blood bank database. case note analysis was performed to obtain further information.results. 47 patients were identified, 12 in the neurosurgical centre and 35 in peripheral hospitals. the median time from scan to issue of pcc was 84 min. 14 patients were reversed within 60 min and 14 patients waited longer than 120 min to have pcc issued. no adverse thromboembolic events were encountered.conclusions. avoidable delay exists between ich diagnosis and pcc issue. pcc could be stored in the emergency department and a stat dose administered immediately after diagnosis facilitating rapid correction of inr. repeat audit will be required to assess safety and efficacy. objectives. the aim of this study was to compare the functional ability and muscle strength between these two groups of patients. twenty-nine patients were evaluated (m:24, f:5) (age: 55 ± 17 years).the diagnosis of critical illness polyneuromyopathy was based on muscle strength measurement according to the medical research council (mrc) of muscle strength methodology. nine patients were diagnosed with critical illness polyneuromyopathy during their icu stay (mrc \48/60).the patients were evaluated with mrc and hand-grip dynamometry (hgd) every 10 days until their discharge from the hospital. the fim scale (functional independence measure) was used to evaluate the functional ability (18-126).the first evaluation was done at the discharge from the hospital and the second one 8 ± 1 months afterwards.results. the patients who developed critical illness polyneuromyopathy had statistically significantly lower mrc (39 ± 12 vs. 57 ± 2, p \ 0.001) and hgd at icu discharge (left 8 ± 5 kg vs. 23 ± 8, and right 9 ± 7 kg vs. 27 ± 9, p \ 0.001) compared to those who did not. the muscle strength as assessed with the mrc 10 days after icu discharge had statistically significantly lower (40 ± 11 vs. 56 ± 3, p \ 0.001), just as the second hgd evaluation (left 11 ± 7 kg vs. 25 ± 8 and right 9 ± 7 vs. 21 ± 8 kg, p \ 0.05).compared to those who did not develop critical illness polyneuromyopathy, the patients who did, had statistically lower fim values during their discharge from the hospital (49 ± 26 vs. 77 ± 30, p \ 0.05)and 8 months afterwards (90 ± 27 vs 125 ± 1, p \ 0.05).conclusions. the patients who developed critical illness polyneuromyopathy had significantly inferior muscle strength at their discharge from the icu. these patients also had lower functional ability. this functional ability remained defected even 8 months after their discharge from the hospital. these initial findings are suggestive that the appearance of critical illness polyneuromyopathy affects the patients mobility after their discharge either from the icu or from the hospital and persists for several months after icu discharge. further studies are needed to evaluate the effect of this impairment on the quality of life of these patients and also to evaluate therapeutic tools for critical illness polyneuromyopathy. introduction. this poster presents a qualitative system dynamics (sd) analysis of the factors which influence the care of acutely unwell ward patients in new zealand. this systems thinking approach is commonly used in organisational research and offers a way to make sense of complex relationships between variables. this approach has previously been used in health care to demonstrate differences in mental models between policy makers and clinicians (cavana et al., 1999) . since the factors which influence the care of acutely unwell ward patients are complex and multi faceted the qualitative sd method becomes an ideal analytic approach (e.g. see wolstenholme and coyle, 1983; senge, 1990; vennix, 1996; or maani and cavana, 2007) .objectives. the aim of this study was to examine the factors which influence the care of acutely unwell ward patients from an organisational perspective. key objectives were to determine the enablers and barriers to care from a nursing, medical and managerial (at ward and executive level) perspective.methods. using a multiple case study approach in four wards in two new zealand hospitals, focus groups and one to one interviews were conducted with key stakeholders identified as nurses, doctors and managers. initial coding of the data generated themes. these themes were then clustered to provide variables which were mapped to generate separate causal loop diagrams (clds) for each of the stakeholder groups to provide the basis for analysis. the clds were compared for characteristics and world views. preliminary results demonstrate a difference between clinical and managerial staff in characteristics and world view regarding the factors which affect the care of acutely unwell ward patients.conclusions. the qualitative sd approach has offered a novel and helpful way to make some sense of the complexity associated with caring for acutely unwell ward patients. organizational responses that may improve care delivery to these patients should be based on frank and open discussions between staff at all levels to ensure a shared mental model as the basis for change. objectives. the aim of the study is to explain the nursing in the technologicallyadvanced intensive care units. in this phenomenologically-designed study, a face-to-face in-depth interview was performed with 20 nurses, who were experienced for 1-22 years in the intensive care unit of cardiovascular surgery clinics. during the interviews, a semi-structured form was used. data were analysed using colaizzi's method of data analysis. the study was approved by the ethics committee of the institution.results. according to the nurses, nursing in technologically-advanced environment has three stages. these stages constituted three themes of the study: technology shock (first stage), understanding the technology-supported care (second stage), competency in technological environment (last stage). in the first stage, the nurses focus on themselves and technology; perceive the environment as frightening and complex. in the second stage, nurses gain control on technology, feel themselves safe and recognize their responsibility. in the last stage, the nurses experience anxiety related to their accountability. this anxiety may be motivating but also may be wearisome.conclusions. the nurses passes through three stages in a technologically-advanced environment. helping nurses to pass through these three stages appropriately will increase the contribution of technology to the patient care, more utilization of technology by nurses and more job satisfaction. unexpectedly, the compliance rate with the recommendations was significantly better over night. although the number of nurses is constant in the 24 h, the number of doctors is lower and less differentiated in the night shift. in an attempt to find an explanation for these findings we looked at the patient flow and time span until the first medical observation in the different time periods and we found that over night admissions (between 00:00 a.m. and 08:00 a.m.) corresponded only to 10% of all admissions and were seen sooner, which might explain our findings. a. objectives. the purpose of the study was to assess whether the completion of the sepsis resuscitation bundle within the first 6 h after icu admission, but beyond the specific time limit of the various bundle interventions, is related to an improvement in survival in patients with severe sepsis/septic shock. this was a single-center prospective observational study of patients admitted to the medical-surgical icu of an urban tertiary care teaching hospital with severe sepsis/septic shock. patients were recruited from june 2005 to november 2009. we assessed the compliance with the different tasks included in the 6-h resuscitation bundle. furthermore, we ascertained within the first 6 h after icu admission the compliance with those tasks not carried out within their specific time limits; we have called this variable ''bundle improvement at the icu''. results were stratified by the number of tasks of the bundle completed before admission at the icu, and the lag time between the beginning of severe sepsis and admission to the icu. these late completed tasks at the icu were related to hospital mortality by a cox regression model. objectives. the aims of this study were to assess the compliance rate with 6 h bundle as defined in the surviving the sepsis campaign guidelines 2008 in patients diagnosed with sepsis regardless of severity and whether compliance affects the rate of mortality and/or hospital stay. we conducted a prospective observational study. we randomly recruited 145 adult patients from acute admissions unit and intensive care in an acute district general hospital in england who met the diagnostic criteria for sepsis. for each patient, compliance with sepsis care bundle was obtained from medical notes. the following components of the 6 h sepsis bundle were assessed: obtaining blood cultures, initiating antibiotic therapy, measuring serum lactate and in the event of septic shock administration of fluid therapy. conclusions. long and unacceptable delays in admission to iccu were identified despite evidence of significant organ dysfunction in many of these patients. with all bundle elements being met for only 1 patient it is apparent that evidence based endpoints aimed at reducing mortality from severe sepsis are not being met despite all the bundle elements being practically deliverable. poor compliance with taking blood cultures prior to antibiotic administration and lack of scvo 2 measurement are areas requiring particular attention. further work is recommended to identify potential contributing factors to non-compliance. introduction. international guidelines recommend that cardiac output measurement is required in addition to arterial pressure monitoring in patients with persistent shock after initial therapy [1] . nevertheless, these recommendations are not supported by any comparison of arterial pressure and cardiac output for monitoring the effects of the most current treatments like fluid therapy. objectives. to evaluate in which extent monitoring the haemodynamic effects of a standardized fluid challenge with the sole arterial pressure could help for detecting the fluidinduced changes in cardiac index (ci). in 228 critically ill patients with acute circulatory failure deemed at receiving a 500-ml saline infusion over 20 min, we measured the systolic (sap), diastolic (dap), mean (map) and pulse (pp) arterial pressure and transpulmonary thermodilution ci before and after volume expansion.results. volume expansion significantly increased ci, sap, dap, map and pp by 24 ± 25%, 15 ± 19%, 9 ± 16%, 13 ± 17% and 21 ± 29%, respectively. the fluid-induced changes in pp, sap and map were significantly correlated with the fluid-induced changes in ci (r = 0.56, 0.55 and 0.37, respectively). the changes (in %) in pp were significantly related to the changes (in %) in stroke volume for all quartiles but with different coefficients of correlation: r = 0.39 for the 1st quartile (36-53 years), r = 0.44 for the 2nd quartile conclusions. pp and sap were the best arterial pressure values for detecting the fluidinduced changes in ci. using the sole pp for assessing fluid responsiveness led to a non negligible proportion of false negative cases. this supports the recommendation that when a precise monitoring of fluid resuscitation is required, like in refractory shock, a direct assessment of cardiac output is required. objectives. aim of our study is to show that it is possible to reduce high catecholamines in previous improper volume resuscitated patients by forced volume resuscitation combined with active dose reduction and generate the hypothesis of an avoidable catecholamine induced circulation injury. introduction. the sialic acid content of the red blood cell (rbc) membrane decreases early in sepsis [1] , and this alters the rbc shape and metabolism [2] . an increased ratio of the rbc proteins band 3/alpha spectrin was observed in a mouse model of septic shock, suggesting a possible alteration of the rbc membrane integral/peripheral proteins ratio [3] . as there are interspecies differences in membrane composition, these observations need confirmation in humans. we studied rbcs from patients with (n = 15) and without (n = 9) sepsis at icu admission and on day 3 in the septic patients. exclusion criteria were recent rbc transfusion, hematologic diseases, cirrhosis and diabetes mellitus. procedures included screening for rbc membrane protein alterations by cryohemolysis test and separation of the rbc membrane and skeletal proteins by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate [4] . comparison between groups was made by the student's t test or the mann-whitney test. a p value .05 was considered as statistically significant.results. the hemogram, including reticulocyte count was similar in septic and non-septic patients at icu admission. no significant difference was observed for cryohemolysis test results and the amount of the rbc proteins (table 1) . objectives. our purpose was to compare a new method (patrol fr 08-02110) with the reference method (randox tm ) during cbp. patients scheduled for coronary artery bypass (cb) and aortic valve replacement (avr) under cbp were enrolled after written informed consent in this protocol approved by local ethics committee. anesthesia protocol was standardized with systematic use of tranexamic acid. three blood samples were harvested: t1 = induction; t2 = 30 min. after cross aorta clamping; t3 = 24 h after induction. the patrol method was performed after serum exposition to a photosensibilizer agent then to a laser irradiation leading to the formation of free radicals. oxidation by those free radicals of a fluorometric sensor allowed an indirect measure of tas. this measurement in arbitrary unit (au) corresponded to area under curve compared to a control value from a pool serum. a value higher than 1 indicated a lower capacity for the given serum to neutralize free radicals whereas a lower value indicated a higher capacity. the same sample allowed tas determination (lmol/l) with randox tm method. results were expressed as absolute numbers, mean ± sd. tas were compared with anova test; p \ 0.05 was significant.results. the seven patients (5 male, 2 female; 71 ± 10 years old) enrolled underwent cardiac surgery (4 cb and 3 avr) without any problem. there was no variation in tas determination with the randox tm method: t1: 1.39 ± 0.13; t2: 1.41 ± 0.26; t3: 1.33 ± 0.15 lmol/l. conversely a two fold significant increase was measured during cpb with the patrol method: ti: 1.12 ± 0.31; t2: 2.21 ± 0.60*; t3: 0.99 ± 0.34 au. *p \ 0.001 versus t1.conclusions. oxidative stress due to overwhelming release of reactive nitrogen/oxygen species (rn/os) is held largely responsible for sepsis-induced organ failure and mortality [2] . up-front and/or ongoing distortion of the pro-oxidant/anti-oxidant balance is likely to play an important role in this situation and in ischemia-reperfusion. therefore the patrol test which appeared to be more sensible than the randox tm method could a good tool in these cases and for evaluation of new anti-oxidant treatments in critical care medicine. these results have to be confirmed in a larger population. introduction. sepsis is the leading cause of death in critically ill patients. despite attempts to improve standardized strategies in resuscitation and treatment of sepsis, the morbidity and mortality remain unacceptably high. early diagnosis and stratification of the severity of sepsis is the key to start timely the appropriate treatment. sepsis is the systemic inflammatory response syndrome to infection; it can lead to hypoperfusion and organ dysfunction and at the cellular level to aerobic mitochondrial dysfunction. lactate is the product of anaerobic metabolism and thus may serve as a prognostic factor in this subset of patients.objectives. the authors propose to test the association of the first serum lactate at hospital admission with shock and icu mortality in patients with community-acquired severe sepsis. during the study period 1,223 patients were admitted in the unit, of those 300 (25%) had severe community-acquired severe sepsis (cass). crude icu mortality rate among cass was 33%. considering the model previously described in methods and when the variables were adjusted only gender, age, saps ii, severity of sepsis and serum lactate were retained in the final model for icu mortality and saps ii nad serum lactate for shock (see table 1 ). a first blood lactate level was independently associated with shock and icu mortality in patients community-acquired severe sepsis admitted in intensive care. objectives. the objective of this study was to test whether svo 2 can predict fluid responsiveness in these patients. we studied 65 patients who were monitored with a pulmonary artery catheter for severe sepsis and septic shock. hemodynamic measurements were obtained before (baseline values) and after a fluid challenge with colloids or crystalloids. responders were defined as those with a[10% increase in cardiac index (ci). no additional interventions were performed during the test. student's t test and linear correlation were used for the statistical analysis.results. mean patient age was 70 ± 2 years and the mean sofa score 10 ± 2. mean arterial pressure was 71 ± 9 mmhg, cardiac index 3.0 ± 0.8 l/min/m 2 , pulmonary artery balloon-occluded pressure 13 ± 3 mmhg, and heart rate 104 ± 19 bpm. thirty-four patients (52%) responded to the fluid challenge. responders and non-responders had similar baseline svo 2 (67 ± 9 vs. 67 ± 8%, p = 0.98). baseline svo 2 was[70% in 13 responders (38%) and in 11 non-responders (35%). there was no correlation between changes in ci (%dci) and the baseline svo 2 (fig. 1) . sepsis is a disorder of microcirculation [1, 2] . although the pathogenesis of microvascular dysfunction in sepsis is extremely complex, neutrophil activation and their interaction with endothelial cells are considered central features of sepsis-induced microcirculatory alterations. to our knowledge, however, no study evaluated the microvascular pattern of septic patients with chemotherapy-induced severe leukocytes depletion.objectives. to assess early microcirculatory response to sepsis in patients with and without drug-induced neutropenia.methods. demographic and hemodynamic variables together with sublingual microcirculation recording (ops-sdf videomicroscopy) were collected in four groups of subjects: septic shock (ss, n = 9), septic shock in neutropenic patients (nss, n = 8), neutropenia without inflammation (neutr, n = 7) and healthy controls (crtl, n = 13). except for controls, all measurements were repeated after complete resolution of septic shock and/or neutropenia (tp2). collected video-files were processed using appropriate software tool and semi-quantitatively evaluated (functional capillary density, fcd (cm/cm 2 ); mean flow index, mfi [1] ) [3] . conclusions. microvascular derangements in sepsis did not differ between non-neutropenic and neutropenic patients. surprisingly, neutropenia per se without measurable systemic inflammation was also associated with alterations of the sublingual microcirculation. although we cannot exclude the role of residual neutrophils, our data could indicate that leukocytes are not the only and exclusive modulators of septic microvascular dysfunction. in addition, the role and mechanisms of microvascular changes associated with chemotherapyinduced neutropenia warrants further investigation. multiple organ failure is a leading cause of death in critically ill patients. improvements in outcome will most rely on our capacity to measure rapidly accessible biomarkers.objectives. to investigate if the time sequence of reactive oxygen metabolites (roms) production with sofa score could be prognostic for outcome. the study included 33 critically ill patients (from september to december 2009) who had roms measured (hydroperoxides) during icu stay, when the diagnostic criteria for sepsis (observed n = 14), severe sepsis (observed n = 11) and septic shock (observed n = 68) were present, 4-5 days and 3 weeks after the diagnosis (samples n = 93); on the same days, the sofa score was calculated. the plasma roms values were assayed by a diacron-italia kit, applied to an automatic instrument (olimpus au 640). statistical analysis was performed used mann-whitney test and the linear regression analysis. the roms values and sofa score were inversely correlated (r 2 = 0.64 for sepsis; r 2 = 0.42 for severe sepsis; r 2 = 0.37 for septic shock). the droms (the difference between the first and the last measurement of roms levels in each individual patient) was significantly different between survivors and non-survivors. clinical characteristics of the patients are presented in table 1 . values are presented as median and interquartile rangers. a p value .05 was considered as statistically significant.conclusions. the plasma roms values decreased when the critically conditions rapidly evolved towards organ failures with higher sofa. to explore: (a) stress neuropeptides (acth, cortisol, prolactin, neuropeptide y (npy) and substance p (sp)) in critically ill subjects and controls, (b) potential association between levels of stress neuropeptides, disease severity and pain. a prospective correlational study, with repeated measurements and cross-sectional comparisons. fifty-three critically ill patients with diverse primary diagnoses and 53-age and gender-matched healthy controls were studied for 8 days. serum neuropeptides were quantified by elisa (npy, sp) and chemiluminescence immunoassays (acth, cortisol, prolactin). pain levels were assessed by payen and puntillo scales. clinical severity was quantified by multiorgan failure scoring system (mof) and the multiple organ dysfunction score (mods). results. we observed: (a) statistically significant differences between critically ill and control subjects in regard with cortisol (p \ 0.0001), npy (p \ 0.0001) and sp (p \ 0.05) levels throughout the study. specifically, cortisol levels were higher and npy and sp levels were lower in patients compared to controls, (b) significant bivariate associations between stress neuropeptides (p \ 0.04), (c) statistically significant associations between acth and pain intensity levels assessed by payen (r = 0.391, p = 0.018) and puntillo (r = 0.509, p = 0.002) scales. there was also a constant but not statistically significant (p = 0.1) trend for lower sp levels in patients receiving opioids than in controls. moreover, npy levels were significantly lower in patients receiving analgesia (p = 0.042), (d) lower acth and cortisol levels in survivors (p \ 0.05) (e) at the day of least severity, a significant association between sp levels and mof was observed (r = 0.29, p \ 0.05).conclusions. (a) despite the fact that npy and sp are stress neuropeptides, their levels appear to be decreased in mods patients. it is worth-exploring whether critical illness may be a state of suppressed activity of some neuropeptides, (b) the observed association between stress neuropeptide levels and survival in critical illness needs to be explore further, (c) bedside measurement of selected neuropeptides in the future may provide an estimation of pain in uncommunicative patients.hence, the study of stress neuropeptides may provide new insight for the management of the critically ill. objectives. the objective of this study was to compare septic and non-septic inflammatory process in critically ill patients with respect to paraoxonase 1 activity, lipid profile and lipid peroxidation markers. methods. analyzed were serum paraoxonase 1 activity, lipid profile, oxidized low density lipoproteins and conjugated dienes in critically patients with sepsis n = 30), age/sex/ap-acheii matched critically ill controls with non-septic sirs (n = 15) and age/sex matched outpatient controls without inflammation (n = 30).results. the activity of pon 1 was lower in septic patients (96.7 ± 47.4 u/ml) as well as in patients with non-septic sirs (107.8 ± 39.8 u/ml) compared to healthy controls (158 ± 40.5 u/ml). the decrease in paraoxonase 1 activity, high density lipoprotein cholesterol and apolipoprotein a-1 concentrations was closely followed by the counter increase of serum amyloid a in both groups of patients. there was no difference in paraoxonase 1 activity between septic and non-septic critically ill patients. the concentration oxidized low density lipoproteins and conjugated dienes as markers of lipid peroxidation, were raised in both septic and non-septic sirs critically ill patients as compared with healthy controls. however there was no difference between both critically ill patient groups.conclusions. the decreased activity of paraoxonase 1 in negative correlation with lipid peroxidation markers offers a potentially useful nonspecific marker of inflammation in critically ill patients.grant acknowledgment. objectives. in the present study, we studied the short-term and direct effects of ivig with sepsis.methods. 16 patients was investigated. following the administration of 5 g of ivig for 1 h, we took blood samples immediately following ivig treatment and at 1 h after ivig treatment. blood samples taken at 1 h and just prior to ivig administration were used as controls. while there was no difference between 1 h before and just prior to ivig treatment, statistically significant decreases were observed in the levels of il-6 after the administration of ivig. no significant changes were observed in the levels of tumor necrosis factor-a and high mobility group box-1.changes in serum tnfa, il-6, hmgb1 we confirmed the results of previous animal studies. while we reported that the administration of ivig directly reduces the levels of il-6 in patients with sepsis, a further prospective study of the ant-cytokine effects following ivig treatment will be conducted in the near future. objectives. to investigate the levels of nucleosome in septic patients and to determine whether nucleosome could serve as a biomarker for sepsis. sixty-four consecutive patients who were newly admitted in surgical intensive care unit at two university hospitals were enrolled in this study. whole blood samples were drawn within 12 h of admission and on the third, fifth and seventh days. a last blood sample was drawn after recovery at icu discharge in survivors or at imminent death in the cases of non-survivors. plasma levels of nucleosome as well as cytokines il-6 and il-10 were detected by means of enzyme linked immunosorbent assay. . fifty patients were diagnosed as sepsis and the other fourteen patients were classified as controls. plasma levels of nucleosome were significantly higher in septic patients than in controls (two-way anova, p \ 0.05), while the levels of il-6 and il-10 were comparable between septic patients and controls. the septic patients presented the highest levels of nucleosome on the admission day, which was significantly different from the admission levels of nucleosome in controls (4.16 ± 3.48 vs. 1.61 ± 1.36, p \ 0.05). the plasma levels of nucleosome between survivors and nonsurvivors showed no statistical significance.conclusions. plasma levels of nucleosome may serve as a valuable biomarker for sepsis.introduction. high mobility group box protein 1 (hmgb-1) is a cytokine that can mediate inflammatory response in different conditions included rheumatoid arthritis, infections, sepsis and septic shock. hmgb-1 released by activated macrophages/monocytes acts as a late mediator of sepsis. studies have shown that serum hmgb-1 concentrations were elevated in patients with severe sepsis.objectives. in the present study, we evaluated the role of the hmgb-1 levels at the time of admission at the intensive care unit (icu) as predictor of outcome in patients with sepsis and septic shock.methods. forty-four patients admitted to the icu with sepsis and septic shock was recruited. serum samples were obtained at the time of admission for the determination of hmgb-1 levels. the results were correlated with the origin of sepsis, severity, organ dysfunction, requirements of mechanical ventilation and vasoactives, days at the icu, comorbidities and mortality at the icu and 28 days after admission. twenty-six patients were male (68.2 vs. 31.8%). septic shock was present in 28 patients (63.6%). the mortality rate at the icu was 31.8% (n = 14) and 38.6% (n = 17) at day 28th. hmgb-1 levels were 34.7 ng/ml ± 39.02 (1.34-230.2 ng/ml). hmgb-1 levels were significantly higher in non-survivors at the icu than in survivors (56.81 ng/ml ± 58.69 vs. 24.38 ± 19.18, p \ 0.05). higher levels of hmgb-1 in serum at the admission were correlated with a higher mortality rate in the icu (p \ 0.05) but not at day 28th (p = 0.07). these levels were not correlated with days at the icu, requirements of vasoactives, mechanical ventilation, and apache score.conclusions. the determination of hmgb-1 levels at admission at the icu in patients with sepsis and septic shock is a good predictor of worse outcome and lethality.introduction. recent experimental and clinical data (1, 2) support the hypothesis that costimulatory molecules, such as cd40, play an essential role in the innate immune response during sepsis. expression of cd40 on the surface of monocytes could represent an important pathway in the modulation of the production of several key inflammatory mediators.objectives. to investigate whether the expression of cd40 molecule on the surface of plasma monocytes differs among the various stages of sepsis. a total of 32 participants (12 icu patients with sepsis, 14 icu patients with septic shock and 6 healthy controls) were included in the study (male patients 73.1%, mean age 46.6 ± 16.8 years). inclusion criteria: icu patients on mechanical ventilation with first episode of sepsis or septic shock during current hospitalization. exclusion criteria: immunosuppression, neoplasia, autoimmune disease, cardiovascular disease. age, gender and comorbid conditions were recorded. a blood sample for quantification of cd40 expression was obtained at the time of enrollment (day 1), and on the fifth day after the onset of sepsis; measurement was made on the same day. cd40 expression on the surface of plasma monocytes (on days 1 and 5) was assessed by flow cytometric analysis. statistical analysis: kruskal-wallis test to identify difference of cd40 expression among the 3 groups was performed. post-hoc analysis was made by mann-whitney u test between independent groups, using bonferroni correction for multiple comparisons. roc curve analysis was used to determine the accuracy of cd40 in identifying patients with sepsis or septic shock. patients with sepsis had significantly higher levels of cd40 (day 1) compared with healthy controls subjects (0.86 ± 0.41 vs. 0.34 ± 0.07, p b 0.006). on the contrary, patients with septic shock did not show any significant difference compared with controls. a roc curve analysis for cd40 (day 1) (auc = 0.72, p b 0.05), revealed that a cut-off value of 0.53 could predict patients with sepsis with a sensitivity of 91% and a specificity of 57%.conclusions. upregulation of cd40 expression may reflect a protective phenomenon during sepsis. on the contrary, low cd40 expression could represent impaired immune function associated with more severe disease. in order to increase the cardiac output in the septic shock patients, according to surviving sepsis campaign team, norepinephrine (ne) or dopamine administration was recommended. the both agents increase the sympathetic tone which antagonize against parasympathetic activity used for gastrointestinal motility (involved gastric emptying). then, it is raised a question whether ne delayed the gastric emptying or not.objectives. this study was aimed to evaluate the gastric emptying in the septic shock patients with norepinephrine. a prospective observational study involved 54 adult septic shock patients, who received ne continuously in icu sardjito general hospital (yogyakarta, indonesia). patients with any head pathologies (trauma, surgical procedures for tumor or bleeding), any gastrointestinal or abdominal pathologies (diarrhea, trauma, surgical procedures for cancer, peritonitis, ileus etc.), and administrations of metochlopramide or alinamin were excluded. nutrition fluids (100 ml) was given passively via nasogastric tube, then after 60 min the tube was aspirated. the volumes of aspirates were recorded in % as a gastric residue. once measurement was done with time randomly for every patient. at the measurement time were recorded the dose of ne and the vital signs.results. the gastric residues were 41.76 ± 28.11% (17 patients), 45.46 ± 25.27% (13 patients) and 13.57 ± 8.52% (7 patients) for the doses of ne of 0.1, 0.2 and 0.3 lg/kg b.w./ min respectively. at the ne doses of 0.4, 0.5 and 0.6 lg/kg b.w./min, all of the gastric residues were zero (17 patients). the correlation between the ne doses and the gastric rescues was statistically significant (p: 0.01). the mean arterial pressures (map) were 84.16 ± 18.01 mmhg (ranges from 58 to 120 mmhg. there was no significantly correlation between map and the gastric residues.conclusions. the gastric emptying in the septic shock patients was not disturbed by administration of ne. introduction. anemia is a frequently encountered problem on the intensive care unit. several factor lead to anemia, among which are traumatic blood loss and the drawing of blood for routine laboratory tests. it's not known how this may affect innate immunity. hepcidin is a central regulator of iron homeostasis. it is induced in response to iron and inflammation and reduced in response to anemia and hypoxia. the suppression of hepcidin leads to the internalization and degradation of the iron exporter ferroportin on intestinal cells and macrophages, leading to the uptake of iron from the gut and the release of iron from the macrophages from the reticulo-endothelial system (res). these cells are central to the innate immune response and the altered iron status of these cells due to suppression of hepcidin may affect the inflammatory response of these cells. we tested the hypothesis that phlebotomy in human volunteers would lead to a suppression of the innate immune response. this abstract provides data of a pilot study carried out in 3 subjects. to investigate the effect of phlebotomy on the innate immune response of whole blood in human volunteers.methods. three volunteers were subjected to the letting of 400 ml of blood by phlebotomy. blood for the determination of hemoglobin and iron parameters, leucocyte count and differential, and hepcidin-25 was drawn at day 0, 4 and 7 after phlebotomy. further whole blood stimulation was carried out at each time point by adding 0.5 ml heparin anticoagulated whole blood to a prepared tube containing endotoxin, pam3cis or rpmi as a control. final concentrations of lps and p3c were 10 ng/ml and 1 lg/ml respectively. these tubes were incubated at 37°c for 24 h and centrifuged for 10 min at 4,000 g. the supernatant was frozen at -80 until the measurement of tnf-alfa and il-6 by elisa. cytokine production was corrected for the number of monocytes present. data are expressed as mean ± sem. hemoglobine decreased from 8.5 ± 0.3 mmol/l at baseline to 8.0 ± 06 at day 4. it returned to normal at day 7. there were no apparent changes in serum iron levels. there was however a clear decrease in serum ferritin levels from 162 ± 48 at baseline to 109 ? 46 at day 7. leucocyte count and differentiation did not show any significant changes. hepcidin was clearly suppressed from 4 to day 7 after phlebotomy (from 6 ± 1 to 2 ± 1). tnf-alfa production dropped from 81 to 47 ng/10 7 monocytes at day 7. il-6 production dropped from 327 to 195 ng/10 7 monocytes. hepcidin levels correlated well with cytokine production (r 2 0.53 for tnf-alfa, r 2 0.51 for il-6).conclusions. phlebotomy leads to suppression of the innate immune response in whole blood. this could be a result of the intracellular decrease of iron in immune cells due to the systemic suppression of hepcidin. these findings are relevant to critical care patients that are subject to the repeated drawing of blood while their immune system is often compromised. introduction. hypothermia and hyperthermia occur in many pathological states presenting to the emergency department. both these processes are known to significantly impair coagulation pathways but as yet there is little evidence to show what affect they have on the evolving clot structure. previous studies have attempted to determine the effect of temperature on whole blood coagulation using techniques such as thromboelastometry (teg) but its ability to provide meaningful outcomes in terms of clot quality and structure remains elusive. recent studies have highlighted the potential of a new technique, gel point (gp) and fractal dimension (d f ), as a functional biomarker in haemostasis. to explore both the changes in coagulation pathways and their associated effect on clot structure and quality based on the new biomarkers, gp and df. following full ethical approval, 30 healthy whole blood samples were obtained from individuals and tested at temperatures of 27°c (n -6), 31°c (n -6), 35°c (n -6), 37°c (n -6), 41°c (n -6). an oscillatory shear technique [1] using an ar-g2 instrument (ta instruments) was applied to each sample. the gp, which indicates the formation of the fibrin network, was obtained for each sample using the chambon-winter gel point criterion [2] . this method provides the basis from which d f can be determined [3] to interpret the structural properties of the clot network. the results were compared with the standard teg analysis. firstly, results showed a significant progressive change in the clot structure by this new biomarker across the whole temperature range (27-43°c). secondly, it also highlighted a significant and meaningful correlation between coagulation pathway change (time to gp, tgp) and the eventual clot outcome (fractal dimension). the tgp of the incipient clot was prolonged and the corresponding d f decreased with reduced temperature values. although, the changes in the coagulation pathway of the teg (r time) and the rheometer (tgp) correlated, the new biomarker, d f , provided additional structural data on the fibrin network formed and highlighted the relationship between coagulation pathway changes and the eventual fibrin clot structure.conclusions. in this study, we describe and quantify for the first time how temperature affects the coagulation pathways and how this impacts on the fibrin clot network, morphology and strength by using the new biomarkers, gp and d f . the potential of these new biomarkers in determining the effects of temperature change in critical illness and injury needs to be evaluated clinically. key: cord-006860-a3b8hyyr authors: nan title: 40th annual meeting of the gth (gesellschaft für thromboseund hämostaseforschung) date: 1996 journal: ann hematol doi: 10.1007/bf00641048 sha: doc_id: 6860 cord_uid: a3b8hyyr nan the variable molecular weight (mw) of vwf is due to differences in the number of subunits comprising the protein. it is assumed that endothelial cells secrete large polymeric forms of vwf and that smaller species arise from proteolytic cleavage. vwf has two main properties: it stabilizes factor viii protecting it from inactivation by activated protein c or factor xa, and it mediates platelet adhesion to subendothelium of the damaged blood vessel wall. each vwf subunit contains binding sites for collagen and for platelet giycoproteins gp ib and gp iib/i~a. multiple interactions of the multivalent vwf lead to extremely strong binding of platelets to subendothelial surface, that is capable of resisting high wall shear rate in the circulating blood. only the largest multimers are hemostatically active. lack of the largest vwf multimers was observed in patients with yon wiuebrand disease type 2a. unusually large molecular forms of vwf were found in patients with thrombotic thrombocytopenlc purpura. proteolytic enzyme(s) may be involved in the physiologic regulation of the polymeric size of vwf and thus play an important role in the pathogenesis of vwf abnormalities in some patients with congenital or acquired disorders of hemostasis. we have purified (-10,000-fold) from human plasma a vwf degrading proteas¢ using affinity chromatography and gel filtration. the proteolytic activity was associated with a high mw protein (mr -300 kd). vwf was resistant against the protease in a physiologic buffer but became degraded at low salt concentration or in the presence of 1 m urea. proteolytic activity had a ph optimum at g-9 and was not inhibited by serine protease inhibitors or sulfl~ydryl reagents. inhibition by chelating agents was best reversed by barium ions, the observed properties of the vwf degrading enzyme differ from those of all hitherto described pretenses. analysis of cleaved vwf showed that the peptide bond 842tyr-g43met had been cleaved -the same bond that has been proposed to be cleaved in rive. the endothelium releases the vasodilator nitric oxide (no) and the vasoconstrictor endothelin (et)-i. no is formed from l-arginine via the activity of constitutive nitric oxide synthase (cnos or enos). an inducible form of nos (inos) is activated by cytokines. no activates guanylyl cyclase in vascular smooth muscle and platelets, leading to the formation of cgmp which induces relaxation or platelet inhibition, respectively. in vessels, no is responsible for endothelium-dependent relaxations; in vivo it exerts a vasodilator tone which can be enhanced by shear forces and receptor-operated agonists such as acetylcholine, bradykinin, thrombin, atp and adp. infusion of no-inhibitors in vivo leads to vasoconstriction and increases in blood pressure and oral administration to hypertension in the rat. within the endothelium, no inhibits et gene expression and release of the peptide via cgmp. hence, no-induced hypertension is associated with increased plasma et levels. et, a 21-amino acid peptide, has potent vasoconstrictor properties via eta-and in part etb-raceptors on vascular smooth muscle. in endothelial cells, et activates etb-receptors linked to no and prostacyclin formation. under basal conditions, little et is formed, but is increased by thrombin, angiotensin ii, arginine vasopressin, cytokines and ox-ldl. et antagonists have been developed and allow to study the effects of et in vivo. et and no most likely play an important role in disease states such as hypertension, atherosclerosis, coronary artery disease, heart failure, pulmonary hypertension and subarachnoid hemorrhage. clinical trials to further define their role in these disease states are now under way. in summary, the endothelium is an important regulator of vascular tone and structure in vitro and in vivo. in disease states, their interaction is imbalanced leading to enhanced vasoconstriction, thrombus formation and structural changes of the blood vessel wall. pharmacological tools aiming to inhibit those changes are now being developed. j.m. harlan, r.k. w/nn, s. sharar, and n. vedder universit3, of washington, seattle, washington ischemia-reperfusion injury has been implicated in the pathogenesis of a wide variety of efinical disorders. [n preclinical models, tissue damage .clearly occurs during ischemia, but, parado.,dcally, may be exacerbated during reperfusion. this reperfusion injury appears to involve activation of the intlammato~, cascade with generation of complement 'components, lipoxygenase products, and chemokines as proximal mediators and neutrophils as final effectors of vascular and tissue damage. we have examined the role of leukocyte adhesion in reperfusion injury in two models -the rabbit ear as a model of isolated organ injury and hemorrhagic shock and resuscitation in the rabbit and primate as a model of traumatic shock and multiple organ failure. data regarding the efficacy, timing, and safety of leukocyte adhesion blockade using selectin-or integrindirected reagents in these models w/ll be presented. the current status of anti-adhesion therapy in other pre-clinical models and early clinical trials will be re~4ewed. an amidolytic assay for the determination of activated protein c (apc)-resistant factor va (fva) has been developed. this assay measures the cofactor activity of fva in diluted plasma samples via the rate of thrombin formation. the apc response is calculated from two fv determinations: one performed in the presence (apc-fv) and one in the absence of recombinant apc. the apc-fv activity is expressed as percentage of the initial fv activity and indicates the sensitivity of fva to apc. normal ranges were established by analysing plasma samples of 100 healthy individuals and an apc-fv activity above 60% was found to be indicative for apc-resistance (apc-r). in a control group of 34 patients the apc-r assay gave abnormal results in 15 patients. dna analysis confirmed heterozygous fv r506q mutation in all 15 patients and confirmed the non-carrier status in all of the 19 patients yielding normal results. an aptt-based apc-r assay performed on the same group of patients showed abnormal results in two of the non-carrier patients. one of these patients was diagnosed as positive for lupus anticoagulant, whereas the reason for the wrong positive result in the second patient remains unclear. eleven patients were analyzed before start of oral anticoagulation and during oral anticoagulant treatment. comparison of the assay results demonstrate a correlation of 96% indicating that the assay is independent of the activities of vitamin k-dependent clotting factors. the apc-r amidolytic assays allows specific and sensitive detection of fva-resistant to apc. the assay is performable in plasma samples of all persons in whom the diagnosis of apc-r may be indicated. in patients treated with oral anticoagulants or showing other clotting abnormalities affecting the aptt the apc-r amidolytic assay is helpful to establish the diagnosis of apc-r. dept of pediatrics, university hospitals kiel and mtinster, germany resistance to activated protein c (apcr), in the majority of cases associated with the arg 506 gin point mutation in the factor v gene is present in more than 50 % of patients < 60 years of age with unexplained thrombophilia. to determine to what exteut this relativdy common gene mutation affects the risk of thromboembolie events in infants and children, its occurrence was investigated in a population of children with unexplained venous or arterial thromboernbotism: thrombosis of the central nervous system (cns, n=4), vena portae (n=4), deep vein thrombosis (n=4), vena caval occlusion (n=4), neonatal renal venous thrombosis (rvi'; n--3), neonatal stroke (n=14), stroke (n=3), arteria femoralis ocdusion (n=2). four ont of these 38 patients showed a positive history of unexplained familial thrombophilia. apcr was measured in an activated thromboplastin time (afit) according to dahlbtick. the results were expressed as apc-ratios: clotting time obtained using the apc/caci2 -solution divided by dotting time obtained with cac12. concerning the special properties of the childhood hemostatic system, infants and children with apcr < 2 were considered to be apc-resistent only when the results were confirmed in a 1: l 1 dilution with factor v deficient plasma (instrumentation laboratory munich, germany). plasma of 268 healthy children served as controls. the arg 506 gin mutation of the factor v gene was assayed by amplification of the dna samples by pcr followed by digestion of the amplified products with the restriction enzyme mul i. results were confirmed by sscp -analysis or by direct sequencing of dna from patients with apcr. consistent with the ap'it based method 10 out of 19 children with venous (v) thrombosis and eight out of 19 patients with arterial (a) vascular insults showed the common factor v mutation. additional coagulation defects (autithrombin, protein c type i, enhanced antiphospbolipid igg, enhanced lipoprotein (a)) were found in 30% (v) and 28% (a). furthermore, we diagnosed exogenlc reasons (septicemia, postpastal asphyxia, fetopathia diabetica, central line and steroid/asparaginase administration) in six out of 10 (v) and three out of 8 (a) children with thrombosis and apcr. all four patients with a positive family history of thrombophilia (mothers only !) showed the common factor v mutation arg 506 gin. in the control group the prevalence of apcr was 5.1%. the high incidence of additional exengenic factors in children with apcr confirm literature data of previously described inherited coagulation disorders during infancy and childhood: an acquired risk of thromboembolic disorders masks the coagulation deficiency in the majority of patients with an inherited prethrombotic state. furthermore, the incidence of 42 % apc resistant children with arterial insults in this study challenge the view that apcr is associated with venous but not with arterial thrombo-st8. 12 activated protein c resistance and plasminogen deficiency in a family with thrombophilia m, zttger 1 , f. demarmels biasiutti 1, ch. mannhalter 2, m. furlan 1 , b.~e 1 1httmatologisches zentrallabor der universit~t, inselspital, ch-3010 bern 2klinisches institut fur medizinische und ehemische labordiagnostik, universit~t wien, a-1090 wien several hereditary defects of the proteins regulating blood coagulation have been associated with familial thrombophilia. since the recent discovery of activated protein c (apc) resistance due to the factor v rf06q mutation as a highly prevalent hereditary risk factor for venous thromboembolism (tel, evidence is accumulating that familial thrombophilia may be due to a combination of genetic defects. thus, protein c-or protein s-deficient patients having suffered from te seem to be more likely to carry the factor v r506q mutation than expected from its allelic frequency in the population. we report a family (see figure) in which plasminogen deficiency (0.60 u/ml) had been found in the propositus having had twice postoperative deep vein thrombosis (dvt) at ages of 29 and 31 yrs, respectively, as well as in 5 family members (0.53-0.69 u/ml). out of these 5 plasminogen deficient individuals, only the propositus' daughter had suffered from recurrent dvt at age <20yrs. reinvestigation of this family in 1995 showed factor v r506q mutation in the propositus, his daughter, an asymptomatic sister and a brother with postoperative pulmonary embolism (pe). his father had had postoperative pe; he is deceased and could not be examined. ~, [] plasminogen deficiency ~ ,rll factor v rf06q mutation /~ propositus ~¢ bistory of dvt and/or pe e superficial phlebitis j2~,,~ not investigated "1" deceased even though this family is small for establishing unequivocal association of te with known defects, the two most severely affected individuals with recurrent te at ages <30yrs had combined plasminogen deficiency and apc resistance whereas those with isolated plasminogen deficiency were asymptomatic. these data support the concept of multigenie interactions leading to familial thrombophilia. resistance to activated protein c (apc resistance) is the most common dsk factor for venous thrombosis (vt). in most cases apc resistance is caused by a single point mutation at position arg 506 in the factor v gene (factor v leiden). while ample data in hetarozygous patients have been published, reports in homozygous patients are limited. we studied 29 patients (12 males [m] , 17 females it]) in whom a homozygous mutation had been verified by dna analysis. the median age at the time of the study was 38.8 years (y) (range 9-83 y). twenty-five patients had experienced vt (10 m, 15 f). four patients were discovered dunng family studies and were asymptomatic, three were children (between 9 and 13 y) and one patient was a 62 y old man. in males the first thrombosis occurred at a median age of 38 y (range 21-82 y), in females this was at a significantly younger median age of 26 y (range 17-49 y). twelve of the 15 symptomatic females had taken oral contraceptives (oc, estregen content 0.02-0.ling) for 6 to 150 months (median 71 m) pdor to thrombosis. in 2 women vt occurred during pregnancy, in 1 female it was precipitated by hormone replacement therapy. in contrast, in 8,<10 males the thrombosis happened spontaneously, in 2 males it followed surgery. the sites of thrombosis were dvt in 9 males and 14 females, dvt and pulmonary embolism (pe) in 4 females and 1 male, dvt and caval vein thrombosis in 1 female and superficial thrombophlebitis in 4 males and 1 female. eight females had at least one pregnancy, in total 11 children and 5 abortions. two had thrombotic events during pregnancy and 2 after delivery. all homozygous patients showed apc ratios between 1.12 and 1.68 (mean 1.39 + 0.19). conclusion: patients with homozygous fv leiden have similar clinical symptoms as patients with deficiencies of antithrombin-, protein c or protein s deficiency. however, in contrast to these defects a very high dsk dudng oral contraceptive medication leading to an ealier manifestation in females can be observed. several synthetic (efegatran, argatroban, inogatran and napsagatran) and recombinant (hirudin, peg-hirudin and hirulog) antithrombin agents are in different stages of nlinical development for cardiovascular and thrombotic indications. while the specificity of these agents for thrombin is a concern, little has been done to study the effects of these agents on other serine proteases involved in coagulation and fibrinolytic processes. fihrinolytic compromise by site directed thrombin inhibitors has been reported recently (thromb res 74(3): 193-205, 1994) . while these agents have been shown to inhibit plasmin and related enzymes, little or no informatienentheir effects onthe generation and functionality of apc is available. sinceapc plays an increasingly important role both as an antienagulant enzyme, by inhibiting factors v and viii, end a pro-fibrinelytic enzyme, by stimulating the release of t-pa fi'om endothelial sites, an inhibition of apc may result in both pro-coagulant state and fibrinolytic deficit. representative thrombin inhibitors (dup 714 -a prototype boronic acid peptide derivative, efega~an, argatroban, hirulog, hirudin and feg-hirudin) have been compared for their ability to inhibit apc (american red cross). these bionhemically defined studies in which the remaining activity of apc after incubation with a thrombin inhibitor was determined speclrophotometrically with a ehromogenic substrate (s-2288, pharmacia, franklin, oh) , demonstrated that dup 714 and efegatran inhibit apc in a coneentrafinn dependent manner (ic~0=0.75 and 8,4 gm resp~tively), hirnlog inhibits apc weakly (60 p2¢i produced only 25% inhibition), while argatroban and ~ have no anti-apc activities, while hirulog, hirudin and argatroban produced no direct enti-apc activities, it is conceivable that they may inhibit thrombomodnlin-bound thrombin and thus prevent activation of protein c, resulting in a functional apc deficit and failure to improve clinical outcomes despite higher dosage. while initially it was thought that sole targeting of thrombin will provide monospacifin anticoagulant agents devoid of some of the adverse effects observed with heparin, the recent clinical trials clearly suggest that thrombin is not the only determinant of thrombogenesis. furthermore, potent antithrombin agents such as hirudin, hirulog and peptides, indirectly inhibit the generation of apc, by compromising thrombomodulin-bound thrornhin and such agents as efegalran and dup 714 also produce direct apc inhibition. endogenous inhibition of formed ape by thrombin inhibitors may therefore compromise the feedback regulatory funetiens of apc and may lead to thrombotic amplification in fully enticoagulated patients. these studies warrant prcelinlcal assessment of thrombin inhibitors to evaluate their relative inhibitory effects on apc. poor anticoagulant response to activated protein c (apc resistance) causes a significant portion of deep vein thrombosis (dvt) whereas its association with coronary artery disease (cad) and myocardial infarction (md is still controversial. therefore, we investigated 346 recently hospitalised patients suffering from cad with or without previous mi. the cad was proven by coronary angiography. apc resistance was analysed by using the ap'l-i'-based assay coatest apc resistance (chromogenix). eleven patients showed an apc sensitivity index below 2.0 viewed as apc resistance. using pcr technology, the factor v mutation causing apc resistance 11691 g "-) a) has been shown in nine of these eleven patients. this represents 2.6 % (9/346), compared to 8,5 % found in healthy blood donors (8/94). one homozygous carrier (male, age 76) was identified (apc sensitivity index 1.4) who suffered from dvt at age 59. recent angiography demonstrated diffuse cad, no thrombotic events were reported in his family. in contrast, multiple thrombotic manifestations (dvt, mi, stroke) occurred in the relatives of four heterozygous patients. we conclude that the prevalence of apc resistance is rather low in patients with cad. nevertheless, the natural history of coronary manifestation of apc resistance seems to vary, probably depending on the presence and severity of cardiovascular risk factors. resitanee to activated protein c (apc resistance) is the most cormnon hereditary cause of thrombophilia and significantly linked to factor v leiden pcr based methods are used to identify the crucial point mutation in the factor v g(me. we designed primers in order to identify factor v leiden by allele-specific pcr amplification. amplification specificity for factor v was ensured by the 3'primer fv1001, located at the introng/intronl0 border of the g~ae. one sense and two antisense primers were used in ~vo separate primer mixes specific for factor v arts06 (wildtypo) or factor v otn506 (factor v leiden) yielding 235bp products each. in each pcr reaction a pair of primers amplifying a fragment of the human growth hormone gene was included, fimctioning as an internal positive amplification control (429bp pcrfragment). after an initial denaturation step 10/.tl samples (100rig genomic dna) were subjected to 10 two-temperature cycles fouowed by 20 threetemperature cycles. for visualisation 8p3 of the amplification product were run on a 2% agarose gel presmined with ethidittm bromide. the presence or absence of specific pcr amplification allowed defiu/te allele assignment without the need for any postamplifieation specificity step. the in~ernal positive control primers it~cate a sucessf-u/pcr amplification, allowing the assignment of homozygosity. in a prospective study 126 p~e.ients with tlaromboembolic events were analysed using this technique and enmpared with pcr -rflp according to bertina et al. the concordance between these techniques was 100 %. in 27 patients a heterozygous factor v ohas06 mutation was detected, whereas one pa6ent with recurrent thrombcembolism was homoz-ygous. no false-positive or false-negative results worn observed in the homozygous as well as hcterozygous samples. in addition in 15 samples identified to carry the point mutation by al/ele-specifin pcr anxplification automatic :~equencing confirmed the heterozygous or homozygous point mutation. due to its time-and cost-saving features allele-specific amplification should be considered for screening of factor v leiden. background: an initial intravenous course of tmfractionated heparin ~ljasted on the basis of the activated partial thromboplastin time is the currmt standard treatment for most patients with venous thrombosis. low-molecularweight hqmrin pre~a~tious can be administered subcutaneously, once or twice daily, without laboratory monitoring. we compared the relative effic.~y and safety of low-molecular weight heparin versus anfractionated heparin for the initial treatment of deep venous thrombosis. methods: english-language reports of randomized trials vtta'e identified th~ a medline search (1984 through 1995) and a complementary extensive manual search. reasons for exclusion from the analysis were no hepada dosage adjustments, the lack of um of obj~tive tests for deep venous thrombosis, dos~ranging studies that used higher doses of low-molecularweight heparin than are ctareatly in use, and the failure to provide blind endpoint ~sossmeat. we assessed the incidence of symptomatic recurrent vinous thromboembolic disease, the incidence of clinically ii~t bleeding and mortality. results: twelve of the 21 identified trials satisfied the predetermined criteria. the relative risk reductions for symptomatic thromboembolic complicatious, clinically ~t bleeding, and mortality varied firom 20.60% aad were all statistically significantly in favor of low-molecular-wedght hqtmrin. coadusions: low-mol~ular--weight hoparim administered subcutaneously in fixed doses adjusted for body weight aml without laboratory nmaitori~ =re more effective and safea" tlum adjn~_-dose standard h~. sauce low molec~dar weight hqmrim vary in o~apositiou =ad pharma~ogical im)fil~ the benefits of each ~ shodd ~tabllsbcd separately. unfraetionated hcparin (uh) and low molecular weight heparin (lmwh) are widely used for the prevention and treatment of thrombotic disorders. uh and lmwh induce platelet aggregation in vitro. rgd peptides compete with fibrinogm for the binding to the glycoprotein receptor (gp lib-ilia) of platelets and inhibit platelet aggregation. to inhibit the heparin-indueed platelet ~tion and prolong the half-life in blood of rgd peptides, we linked ac-rgdv-ssggs-ahx-yk eovalently to lmwh in a ratio 1:1. the peptide is composed of three regions: a. rgd-gives the specificity for the receptor gp lib-ilia; b..ssggs-ahx-is the spacer between carrier and ligand, which should facilitate the intnraetion between the conjugate and the gp lib-ills receptor; c. -yk arc functional antino acids for iodination (y) and for covalent attachment (k) to the cattier lmwh. the aggregation achieved with different concentrations of lmwh, lmwh-eonjugate and lmwh/rgd-peptide mixture in a ratio 1:1 was mea.~ared after 20 rain.; maximum aggregation after platelet activation with 10 pm adp was set equal to 100%. platelet aggregation in normal human plateletrich eitrated plasma (prp; 220 000/p.l) was induced by lmwh in a dose ~ndent manner. heparin can induce antbodies which interact with platelets and endothelial cells. this causes thrombocytopenia and thromboembolic complications. hitpatients do need effective parenteral anticoagulation. we treated 82 patients (30m,52fm), median age 61 years (18-84) with laboratory prooven hit (hipa-test) with recombinant (r-)hirudin. as these patients had been preseleeted by their immunological response during heparin treatment and the treatment duration of the study was longer than in any other study using thlrudin, all patient samples were investigated for anti-r-hirudin antibodies himdin antibodies were screened by a sandwich elisa using r-hirudin fixed to the solid phase as antigen. all plasma samples were screened for anti-hirudin antibodies of the igg class, but solar only a suset of samples for lge anti-r-himdin antibodies. 38 of 82 patients (46.3%) developed anti-hirudin antibodies of the igg class. anti-hirudin antibodies were not detectable not before 6 days of r-hirudin administration solar no ige anti-hirudin antibodies were found. none of the patients devdoped thromboeytopenia or allergic symptoms. however, in a subset of patients the anti-hirudin antibodies enhanced the anticoagulatory effect of r-hirudin. in 5 patients the hirudin dosage had to be decreased by 2-3 fold to maintain a stable aptt level, in 3 patients, despite stable r-hirudin maintenance dose the aptt increased to values >100 see.. during the study 4 patients with anti-hirudin antibodies had to be reexposed to a second course of r-hirudin for parenteral anticoaguhtion none of these patients developed any allergic reaction. in conclusion we found a high proportion of anti-hirudin antibodies in hatpatients treated with r-hirudin for more than 7 days. these antibodies seem to have minor clinical relevance in regard to allergic reactions. however, one has to consider that these antibodies may influence the pharmacokinetics of rhimdin and thereby enhance its antieoagulatory potency. therefore, aptt must be monitored closely in patients receiying r-hirudin for more than 5 days a major concern in the use of hirndin, the most potent and specific thrombin inhibitor, is the risk of bleeding associated with the potential effect of this drug on hemostasis, particularly when the antithrombotic therapy is combined with invasivo procedures, fibrinolytic treatment, or patient's predisposition to abnormal bleeding. thus, availability of an antagonist to hirudin would be essential for instant neutralization of the antithrombotie action. however, thueh a hirudin antagonist is unknown in nature. to prepare an antagonist to hirudin, a mutant derivative of human prothrombin, in which active site aspartate at position 419 is replaced by an asparagine, has been designed, expressed in recombinant chinese hamster ovary cells, and purified to homogeneity. d419n-prothrombin was converted to the related molecules d419n-meizothrombin and d419n-thrombin by limited proteolysis by e. carinatue venom and o. scvutellatus venom, respectively. both d419n-thrombin and d419nmeizothrombin exhibited no thrombin activity and titration resulted no detection of the active site. however, binding to solid phase immobilized hirudin and fluorescence studies confirmed that the binding to the most specific thrombin inhibitor, hirudin, was conserved in both proteins, hi vitro examinations showed that d419n-thrombin and d419nmeizothrombin bind to immobilized hirudin, neutralize hirudin as well as in the purified system and in human blood plasma and re-activate the thrombin-hirudin complex. animal model studies confirmed that d419nthrombin and d419n-meizothromi.,in act as hirudin antagonist in blood cireulatlon without detectable effects on the coagulation system. while i.v. injections of hirudin in mice resulted in an increase in partial thromboplastin time, thrombin time and anti-thrombin potential, additional injections of d419n-thrombin and d419n-meizothrombin resulted in a normalization of these coagulation parameters. elevation of plasma homocysteine is a hereditary disorder of methionine metabolism associated with a high risk of arterial vascular disease. however, as yet relatively little attention has been directed towards the association between hyperhomocysteinemia and juvenile venous thromboembolism (vte). consequently the aim of our study was to evaluate the prevalence of hyperhomocysteinemia (hyper-hcys) and juvenile vte. patients: 85 patients (29 men, median age 42 ys; 56 women, median age 35 ys) who had at least one verified episode of vte before the age of 45 ys were investigated in regard to their total plasma hcys levels. none of the patients had renal or liver dysfunction or evidence of any autoimmune or neoplastic disease. methods: plasma total homocysteine levels were determined by hplc with fluorescence detection. hyperhomocysteinemia was defined as hcys levels exceeding the upper limit of the normal range obtained in our laboratory from 80 healthy control subjects (40 males, median age 25 ys, hcys 95% ci: 2.02-5.67 pmol/l; 40 females, median age 27.5 ys, hcys 95% ci: 2.99-5.40 ,gruel/l). resuits: 13 out of 85 patients had hyper-hcys, giving a prevalence of 15.3 %. of these 13 patients, 9 were male and 4 female, indicating that the relation between elevated plasma hcys levels and vte may not be as strong in woman as in men. discussion: according to previous reports, our study shows that there is a high prevalence of hyper-hcys in patients with juvenile vte. however, the mechanisms by which hyper-hcys can provoke vte and whether hcys is an exclusive risk factor or if it contributes to other existing predispositions, possibly working as a trigger factor is unknown yet. some authors suggest hcys-iaduced effect on factor v activation or inhibition of thrombomodalin-dependent protein c activation. in addition an influence on thrombocyte aggregation has been postulated. conclusion: measurement of hcys levels may be useful in the evaluation of patients with a history of juvenile venous thromboembolism and could be clinically important as hyper-hcys is easily corrected by vitamin supplementation. detailed determination of the pathogenesis of vte in patients with hyper-hcys should be the aim of further investigations. a deficiency of one of the coagulation inhibitors antithrombin (at), protein c (pc) or protein s (ps) and resistance to activated protein c (apc resistance) are established risk factors for venous thromboembolism (vte). in the majority of patients with apc resistance, the .tug 506 gin mutation (factor v leiden) is present. whereas deficiencies of one of the coagulation inhibitors are rare in the normal population, the allele frequency of factor v leiden is 2-7% in western europe. heterozygous individuals have a 3-7fold, homozygous an 80fold increased risk for vte the typical clinical features of all abnormalities are deep vein thrombosis, pulmonary embolism, superficial vein thrombosis and thrombosis at unusual sites, like mesenteric vein thrombosis or cerebral vein thrombosis. the thrombotic risk is low during childhood, but increases considerably after the 13th year of age. a retrospective study in adult patients out of families with a symptomatic deficiency of at, pc or ps revealed that around 30% of surgical interventions and traumas of the lower extremities were complicated by vte. therefore, these patients should receive thrombosis prophylaxis al~er surgery and trauma if their age is higher than 13 years. pregnancy is associated with a very high risk for vte in individuals with at deficiency and prophylaxis should be initiated already in the first trimester. after delivery, thrombosis prophylaxis is adviced for all females known to have an abnormality. oc increase the risk, especially in at deficient and in homozygous factor v leiden females and are therefore contraindicated in these individuals. oc do also increase the risk for vte in patients heterozygous for factor v leiden and females known to have this abnormality should be discouraged from taking oc or should at least be informed on their increased risk. university hospital-', jerusalem, israel, hospital bcan.iou ~. paris, france, increased frequency of thrombocmbolie events have been observed iu patients with b-thalassemia. our findings of shortened platelet survival and enhanced urinary excretion ofthmmboxanc a: metabolitcs (blood 77:1749 (blood 77: , 1991 suggested an increased platclet activation in tbese patients. we also fouud that isolated thalassemie rbc enhance prothronlbin activation, suggesting an increased membrane exposure of procoagulant phospholipids i.e, phasphatidylserine (am j. hematol. 44:33, 1993) . we now show that annoxin v, which has a high specificity and affinity for anionic phospholipids inhibits pmthrombm activation by factor xa, by binding to thalassemic rbc (ic~, = 0.32 nm). kerckhoff-klinik, bad nauheim ~, medizinische poliklinik bonn 2, institut for immunologie und transfusionsmedizin universit~ll greifswald a antibody-mediated intravascular platelet activation is believed to be the basis for both arterial and venous thrombosis in patients with hat. while the development of arterial thrombosis can explained sufficiently by intravascular platelet activation, it is a matter of discussion whether additional risk factors are involved in the pathogenesis of hat-related venous thrombosis. since resistance to activated protein c (apc) is the most common inherited risk factor for venous thrombosis described the frequency of apc resistance among a population of 68 hat patients has been studied. hat was diagnosed using the heparin-induced platelet aggregation assay and confirmed by the ~4c-serotoninrelease test. the diagnosis of apc resistance was established by two functional assays and genetic analysis. at time of diagnosis of hat, 38 patients showed venous thromboembolic complications. among these, 24 were found positive for apc-resistance. pulmonary embolism was diagnosed in 18 hat patients, 14 of them were apc resistance positive. none of the 18 hat patients who showed exclusively thrombocytopenia were apc resistance positive. early oral anticoagulation (oa) was initiated in 7 patients after the diagnosis of hat has been established. six of these patients developed serious thrombotic complications including skin necrosis. these results demonstrate that apc resistance is an additional and common risk factor for the development of hat-related venous thrombosis. early initiation of oa during an acute episode of hat dramatically increases the risk of thrombosis. therefore, oa in hat patients should be initiated only after platelet counts have been returned to baseline levels and effective parenteral anticoagulation is achieved. controlled trials for primary and secondary prevention of stroke g. de (3aetano, c. cerletti and v. bertel~ consorzio mario negri sud, santa maria imbaro, italy this presentation will review the antithrombotic treatments to prevent ischemic stroke that have been evaluated in controlled clinical trials. in two studies of aspirin therapy for pdmary prevention in male physicians there was no reduction in the incidence of stroke, while that of first myocardial infarction was significantly lowered. similar results were obtained in a prospective study in a large cohort of women taking aspirin daily. the incidence of vascular death was not modified by aspirin in any of these trials. this is possibly due .to an excess of strokes associated to aspirin treatment: indeed the four vascular events avoided in 1000 us physicians under aspirin prevention for five years would result from five myocardial infarction and one vascular death avoided and two additional strokes occurred. oral anticoagulant therapy decreases the relative risk of stroke in patients with non valvular atrial fibrillation. warfarin appears to be superior to aspirin, but the latter drug is a useful alternative when long-term anticoagulant therapy cannot be administered. a metanalysis of about 150 trials and over 100,000 patients with different vascular diseases treated with aspirin (at different doses) and/or other platelet inhibitors showed 25% overall reduction of vascular events including stroke. the optimal dose of aspirin for secondary stroke prevention could not be established. in patients with previous minor strokes or tia there was 22% reduction of vascular events and 23% of non fatal strokes. the avoidance of nine strokes of any cause among the 38 expected in 1000 patients at risk would result from the sum of 10 ischemic events avoided and a haemorrhagic one occurred in excess. ticlopidine was reported to reduce the risk of stroke in two large tdals (one in patients with major stroke), but there is no evidence that it is better or safer than aspirin. we compared the effect of the direct specific thrombin inhibitors, napsagatran (na) and rec. hirudin (rh) with unfractionated heparin (uh) on the further growth of preformed thrombi. as a model of thrombogenesls, an annular perfusion chamber exposing rabbit aortic subendothelium was perfused with native rabbit blood at an arterial wall shear rate (650/s). fibrin and platelet thrombi were allowed to form during a 10min perfusion period after which the test agents were given iv as a bolus and a continuous infusion (3 and 10pg/kg/min, n=6) and the perfusion continued for 20min. the control groups were perfused for 10 or 30 rain (n=6). fibrin deposited and platelet thrombi formed on subendothelium were evaluated by microscopic morphometry. the % surface coverage with fibrin was not reduced in the drug-treated groups since fibrin deposition was similar in the 10 and 30 min control groups (39+8% and 335:6%, respectively, mean:l:sem). platelet thrombus area (ta) in the control groups increased from 24+7pm2/pm after 10min to 97+32pm2/lim after 30rain perfusion. na at 1011g/kg/min reduced ta by 94% to values (6+_2ptm2/ ~m) lower than those of the 10min control group whereas rh at this dose reduced ta by 70% (30-j:141.tm2/i.tm). uh at both doses was ineffective. these findings show that in contrast to uh the direct thrombin inhibitors na and rh inhibit the growth of preexisting thrombi. these results could be explained by the higher potency of na and rh as compared to uh for inhibiting clotbound thrombin (gast et al., blood coagul fibrinol 1994, 5.' 879-87) and suggest that thrombus-bound thrombin is an important modulator of platelet thrombus growth and/or stability in this thrombosis model. platelet adhesion -the initial event of thrombosis -is believed to be completely prevented by intact endothelium. we challenged this theory by superfusing intact human umbilical vein endothelial monolayers with activated human platelet rich plasma utilizing the stagnation point flow adhesio-aggregometer (spaa). the spaa provides flow mediated contact of platelets with the superfused surface. heparinized (3.5 -4.0 u/ml) platelet rich plasma (prp) was obtained from healthy volunteers and activated by addition of adenosine diphosphate (adp 2"10 -6 m). platelet deposition was recorded on-line by video as well as by measuring scattered light. fixed samples were examined by phase contrast and electron microscopy, inhibition experiments were performed with either the tetrapeptide rgds, the non-peptide gpiib/llla-inhibitor ro-43-8857 or a monoclonal antibody directed against the gpilb/llla complex. stimulation with adp prompted platelets to adhere to intact endothelium single or as microaggregates of a diameter of up to 100 micrometer. adhesion was dependent upon convective transport resulting in platelet collision with the endothelial monolayer. infusion of rgds or ro-43-8857 into the flowing, adp-stimulated prp completely prevented platelet adhesion to the endothelium as well as subsequent aggregation. when the inhibitor inflow was stopped while adp stimulation persisted, adhesion and aggregation occurred immediately. re-establishing the inflow of the inhibitors -with still continued adp stimulation -led to disintegration of the adhering aggregates. when prp preincubated with the monoclonal antibody against gpllb/llla was superfused, platelet adhesion to the endothelium and aggregation were irreversibly blocked. our results suggest that convective transport and stimulation of platelets are prerequisites to overcome endothelial thromboresistance and that subsequent platelet adhesion to the endothelium is mediated via the platelet gpilb/llla receptor complex. prevent thrombus formation affer ptca i.p. 8tepanova t, g.v. bashkov 2, l.p.kapralova, 2 s.p. domogatsky ~ cardiology research center t and national haematology scientific cettter 2 russian academy of medical sciences, moscow, russia percutaneous transluminal coronary angioplasty (ptca) results in atheroselerotie plague rapture, vascular wall damage and thrombogenic collagen exposure. subendothelial collagen type i-lll is a very ~rong agonist of platelet-dependent thrombus formation in arteries. the anlithrombotic action of rabbit polyclonal antibodies to rat collagen type i-ill and their chemically synthetized conjugate with monoclonals to human recombinant two-chain/one-chain urokinase type plasminogen activator (rtcu-pa/rseu-pa), cross reacting with rat tcu-pa/scu-pa was studied both an in vifro and in vivo. anticollagen antibodies and bispecific conjugate inhibited human platelet adhesion, aggregation and formation of thrombi-like ~ructures induced by rat collagen immobilized with the polystiroi surface in a condition mimics the high shear rate in the large elastic-type arteries. the short-term treatment of the collagen-soaked silk thread by the collagen antibodies suppressed the platelet-dependent thrombus formation in the arterio-venous shunt in rats by 56_+4 % (p<0.001) as well as by the bispecific conjugate (44_+4%, p<0.001). the treatment of collagen-adsorbed conjugate by rtcu-pa did not increase the autithrombotic effect of bifunctional antibodies. the present date suggest, that the local administration of the anticollagen antibodies at the site of atherosclerotic plague rapture may tm the efficient tool for prophylaxis of platelet-dependent thrombus formation in arteries after ptca. increased levels of certain hemostatic factors have been shown to be related to an increased risk of cardiovascular events. hypercoagulability is suggested to predispose to arterial thrombosis and thereby to participate in atherogcncsis. we therefore assessed fibrinogen, prothrombin fragment 1+2 (fi+2) and yon willebrand factor (vwf) antigen in 80 consecutive patients (aged 59+5 years) with known coronary artery disease (cad) who all underwent coronary angiography. the extent of coronary artery disease was quantified according to modified criteria of the american heart association (total, proximal and distal "score"). furthermore the intima-media thickness (imt) was determined in the carotid and femoral arteries by standardized ultrasonographie measurement, vwf antigen was found to correlate positively with the total and proximal coronary score (r=029, p<0.05 and r=o.36, p< 0.01). while fi+2 showed no correlation with the coronary scores, it was significantly correlated with the imt values in the carotid arteries (r=0.27. p< 0.05). after differentiating tertiles of the parameters patients belonging to the upper tertile of fi+2 concentrations had significantly higher imt values of the carotid and femoral arteries (0.81_+0.11 mm vs. 0.72+_0.13 mm in the lower tertile, p<0.05:1.38_+0.44 mm vs. i. 15-+0.25 ram, p=0.05) whereas in patients belonging to the upper tertile of vwf antigen concentrations the proximal coronary artery score was significantly higher (!.71-+0.59 vs. 1.31+ 0.92 in the lower fertile, p<0.01). fro correlation of fibrinogen concentrations and extent of cad or imt values of the carotid and femoral arteries could be demonstrated. in conclusion procoagnlatory mechanisms as indicated by elevated concentrations of yon willehrand factor antigen and fi+2 may be contributing factors in atherogenesis. we have previously shown that pgei is a potent inhibitor of pdgf-ioducod proliferation of vascniar smooth muscle cells (vscm) and inhibits dna replication by a camp-related mechanism (grol~er et al, 1994) . the present study investigates of whether or not this aatimitogeni¢ activity of pget can be amplified by trapidil, a compound that has been shown recently to inhibit the incidence of restenosis of hmnan coronary arteries subsequenmt to ptca (maresta et al. 1994) , vsmc were prepared from coronary arteries of adult bovine hearts, passagod and kept under standard tissue culture conditions. cells of passage 4-9 wore incubated in serumfree medium for 24h in the presence of indomethacin (3 p.m). addition of pdgf-bb (10 ng/ml) under these conditions stimulated dna-replication as assessed from 'hthymidin lncm'poration, by 3.-4laid above control level. trapidil at 10 idvl caused a minor reduction of pdgf-induced mitogenesis whereas 10t) of the compound resulted in a marked reduction of dna replication by 69% (p < 0.05, n = 4). pgei at 0.3 nm diminished the incorporation rate by t 1% while the simultaneous administration of both pged and trapidil (100 idyll caused a significantly stronger response as seen from n reduction of ~h-thymidine incorporation rate by 82% (p < 0.05, n = 4). as a possible mechanism of action, trapidil might have inhibited phosphodiesterases. to establish this, we measured the camp-depcudont proteinkiaasc (pk) a activity in cell homogenates. trapfdil increased the basal fka-activity from 19% to 31% of the maximum response while the response to pget (10 am) amounted to 55%. coincubation of pgei with trapidil caused a 65% stimulation of pka activity, sugesting a small though detectable inhibition of vscm phosphodiesterases by trapidil at anttmitogenic concentrations. essentially similar results wore obtained when thrombin was used as the mitogenic agent. the data demonstrate a significant antimitogenic effect of trapidil at p.molar concentrations that are in the range of plasma levels after therapeutic administration of the compound in rive. at these concentratrations, pget induced inhibition of mitogenesis is markedly enhanced by trapidil. inc. i~enna, and ~cenlral itematnlogy laboroto~. , university hospital of bern pibrinogen (fg), yon willebrand factor antigen (vwf) and tissue-type plasminogeu activator antigen (l-pal have recently been shown in be independent risk factors for subsequent coronary events in patients with angina pectoris (nejm 1995; 332:635) although paul antigen has also been proposed as a risk factor, conclusive dam showing its predictive value is still lacking. furthermore, we have recently shown in a study investigating 200 survivors of myocardial infarction that not only are fg, t-pa and pai-i significantly increased in these patients when compared to a heahhy conlro[ group, but pci activity is also elevated (7hrornb. tfaemost. 1995;73:1137 abst.) , hi order to obtain cut.off points for the individual parameters, frequency histogram plotl; were transformed into straight line cumulative frequency (probit) plots (thromb i/aemost. 1995;74:718) . the cut-off valu~ for the four parameters were determined as follows: fg at 2.7 g/l, t-pa at 8.7 ng/ml, pal-i at 25 ng/ml and pc[ at 126% of a normal pooled plasma. utilising there cut.off points it was then possible to determine the accumulative discriminatow effectiveness of the parameters. when fg w;qs employed alone as the discriminatow factor, it was observed that 47% (941200) of the coronary heart dir, ease (chd) group eilher had the cul..off value or were below it aud 29% (29199) of the normal group were above the cut-off value, thus, resulting in 47% false ne$atives and 29% false positives. when a second additional risk factor, t-pa wa_~ introduced, the number of false negatives dropped to 21% [i.e. 79% (158/200) had two, risk factors elevated] and the number of false positives to 13% to investigate whether a third parameter could discriminate further, pai-i antigen was used to analyse the rcnudning false positives and negatives. an additional 4% could be detected, resuhing in 83% of the chd group having three risk factors elevated. similarly, the number of normal aubjecta with three parameters elevated dropped by 4% to 9% furthermore. when a fourth parameter was introduced, namely pci, it was round to discriminate a further 8% in the chd group, thereby increasing tile di~riminalion to 91%. the number of false positives dropped to 4%, additionally, determination of pci increased the discrimination of patienta having had multiple infarctions from 88°/= when thrce parameters were mcasured to 100%. from these results it can be concloded that determination of fibrinogen levels alone is not sumcicnt to separate patients from controls as t-pa adds significant discrimination. pai-i antigen which correlated strongly with t-pa did not significantly increase the discriminatory potential of both fg and i-pa. however, by employing pci as a fourth paramctcr, virtually complete separation between the chd and normal groups as well as rurthcr recoguitiou of' patients having had multiple infarctions could be obtained. to test the hypothesis that oral contraceptives (oc) enhance exercise-induced activation of blood coagulation we examined 11 women (27 + 3 (sd) years, bmi 20.4 + 2.0 kg/m 2, vozm.. 49 + 7 ml/kg/min) without oc between day 18 and 22 of the menstrual cycle and 10 women (24 + 2 (sd) years, bmi 20,6 ± 1,6 kg/m', vo2max 47 + 7 ml/kg/min) taking oc (150 mg desogestrel and 30 mg ethinylestradiol) between day 7 and 21 of drug intake. prothrombin fragment 1 +2 (ptf1 + 2) and fibrtnopeptide a (fpa) were measured before and after running for one hour on a treadmill at a speed corresponding to the anaerobic threshold. mean heart rate [191 ± 10 vs. 196 ± 12 min 1) and mean plasma lactate (3.3 ± 1.6 vs. 3.1 + 1.2 mmot/i) wera comparable during exercise between control and oc group, respectively. results for markers of thrombin and fibrin formation were: ptf1 +2 (nmol/i) fpa (ng/ml) control before 0,66 ± 0.19 1.0 + 0.2 after 0.73 + 0.23 2.2 + 1.2" oc before 0.82 + 0.31 1.0 + 0.2 after 1.11 + 0.48* + 5.8 -+ 6.0* + * p < 0.05 vs. baseline, + p < 0.05 between groups. we conclude that oral contraception with 150 mg desogestrel and 30 mg ethinylestradiol enhances exercise-induced thrombin and fibrin formation, our data suggest that exercise testing might be useful for evaluating the risk of thrombosis associated with different compositions of oc. a. haushofer +, wm. halbmayer +, j. radek +, m. dittel *, r. spiel *, h prachar *, j. mtczoch *, m. fischer + + zentraltaboratorium mit thrombose-und c~rinnungsambulanz -krankenhaus lai~: * 4. medizinische ab[eilung mtt ka~liolo$i¢ -krankenhaus lainz und ludwig bottzmann-lnstitut ftlr herzinfarktforsohung, wien fifty-one patients (age 61.6 ± 9.2a; 34 m / 171) implanted with 74 coronary stems 03 palm~-schatz, 27 gianturco-roubin, 14 micro stcnts) received a now antithrombotic treatment using a combination of ti¢lopidine (tic) 2 ×250 mg/d for 28 days and acetyl salicylic acid (asa) zoo mg/d for long-term treatment. patients (pat) only received 15000 tu standard hepartn as i.v. bolus immediately before stent implantation (day l ). side effects and changes in hematological (day i to 7, 14. 28 and 35 [= without t[cil liver and kidney parameters (day 7, 14, 28, 35) were monitored. thirty-eight pat (75%) came for the controls to our del~rtment and were additionally monitored by thromboelastograpy (teg) and bleeding time (bt) (day 2g and 35). the other pat were monitored externally, side effects were reported. thrombin geucration after stenting was monitored from day i to 7 by prothrombin fragment 1+2 (f 1+2) and thrombin-antithrombin-lll-comptex (tat). "k" of the "leg decreased (day 28 vs 35; p< 0.0l ). bt prolongation was negatively correlated with the bodysurf ace area (tic+asa: p< 0.05, asa: p< 0.0 l) and showed a reduction after withdrawal of tic (2 l0 sec, 180/ 300 so: [median, quartiles] vs. 120 sec, 881157 sec; p< 0.ix)01). f 1+2 and tat of day i (blood collection: 0, 2, 4, 6 h after intervention, f 1+2:0.84 nmol/i, 0.68/i.07 nmol/l: tat: 2.6 pg/1, 2.0/4.6 ilg/1) were lower compared to day 2 to 7 (f i +2: 1.31 nmol/l, ].08/i .62 nmol/l; tat: 4.8 pg/i, 3.2/7.2 ijg/1; p< 0.0001). tic scorns not to be a strong thrombin generation inhibitor. during stenting one pat (i.9%) sustained a non penetrating mci and one (1.9%) an ischaemic stroke. tic+asa were very effective, only with one pat (1.9%) stent thrombosis (acute) occurred. side effects: 8/15.7% gastrointestinal (one lead to hospitalization), 5/9.8% hematomas at the needle site in the groin (one surgical intervention), 5/9.8% leucopcnias (one agranulozytosis with hospitalization), 3/5.9% allergic skin reactions and 2/3.9% increased liver enzymes (got, gpt, "pgt, alkaline phosphatase; > 2 × of the j. ). with one pat with gastrointestinal disturbances and skin reactions tic had to be withdrawn and treatment was changed to oral anticoagulatlon + asa. one pat showed a combination of skin reactions, gastrointestinal distufl~aneas and on day 28 a heavy reaction of the liver enzymes ( j. after 5 weeks). a decrease of the white blood count (day 1:7.19 gh, 6.03/8.2 g/l, day 28:5.46 g/l, 4.63/6.47 g/l; p< 0.000 i) could be observed. the safety of the therapy with tic+asa should be elucidated and extensively discussed. the serpins c1 esterase inhibitor (cllnh), antithrombin iii (atiii), alantitrypsin (slat), and a2-antiplasmin (azap) are known inhibitors of coagulation factor xla (fxla). although initial studies suggested al at to be the main inhibitor of fxla, we recently demonstrated cllnh to be a predominant inhibitor of fxla in vitro in human plasma (wuillemin et el., blood 1995; 85:1517) . the present study was performed to investigate the plasma elimination kinetics of human fxla-fxla inhibitor complexes injected in rats. the amounts of complexes remaining in circulation were measured using elisas. the plasma tl/2 of clearance was 98 min for fxla-alat complexes, whereas it was 19, 1 8, and 1 5 min for fxla-cllnh, fxla-a2ap, and fxla-atiii complexes, respectively. thus, due to this different plasma tl/2, preferentially fxla-alat complexes may be detected in clinical samples. this was indeed shown in plasma samples from thirteen children with meningococcal septic shock (mss), a clinical syndrome which is complicated by activation of coagulation, fibrinolytic, and complement systems. fxla-fxla inhibitor complexes were assessed upon admittance to the intensive care unit. fxla-a 1at complexes were elevated in all patients, fxla-c11nh complexes in nine, fxla-atiii complexes in one patient, and no elevated fxla-a2ap complexes were found. we conclude from this study that, (1) although c1 inh is the predominant fxla inhibitor, fxla-alat complexes may be the best parameter to assess activation of fxi in clinical samples, (2) measuring fxla-fxla inhibitor complexes in patient samples may not help to clarify the relative contribution of the individual serpins to inactivation of fxla in rive, and (3) fxl is activated in patients with meningococcal septic shock. dudng the coagulation of plasma about 20 % of the (x2ap present is covalently crosslinked to fibrin by factor xiila (aoki und sakata 1980, thomb. res. 19: 149-155) . we investigated the binding of azap by factor xiila to soluble fibrin (desaabb-fibdno) whose polymerization was inhibited by an isolated fibrin ddomain named d=,,, (haverkate and tiemann 1977, thromb. res. 10: 803-812) . d==. is known to have an intact fibrin-polymerization site and is able to block the prolongation of the fibrin protofibrils at an early stage depending on its concentration. lateral association to fibrin fibers does not take place, since the inhibited protofibnls formed at the conditions used here do not reach a sufficient length (williams et el. 1981, biochem. j. 197: 661-668; hantgan et al. 1983, ann. n. y. acad sci. 408: 344-365) . material and method: soluble desaabb-fibrino was prepared by incubation of (lztl)-fibrinogen (0.48 mg/ml), d=1o (1.91 mg/ml; molar ratio d==o to fibrin 14:1) and 0.4 u/ml thmmbin for 45 min. then q2sl)-c~2ap (16 p.g/ml), faktor ×ill (2 ulml) and ca 2) (5 mmol/i) were added. the crosslinking reaction was stopped at different times of factor xiila-incubation by adding of urea/edtasolution. the suspension was analysed by ultracentrifugation on gradients containing saccharose, urea and sos. re~ultl: the elution profiles of the ultrecentifugation-gradients show the formation of cmsslinked fibrin oligomers of increasing size depending on the time of factor xiila-action. the crosslinked fibrin polymers contained about 80% of the fibrin initially added. although factor xiila acted well, crosslinking of azap in the fibrin oligomers could not be observed. conclusl0n: as we already demonstrated (kelach et el. 1994, ann, hematol. 70 (suppll) : a 60) the crosslinking of azap to fibnn clots depends on the structure of the fibdn network, especially on the degree of lateral association of the fibrin pmtofibdla. in desaabb-fibrino no lateral association of fibrin protofibnls takes place under the conditions chosen here. thus it is consistent with our theory that we did not observe any binding of aiap to the fibrin oligomers of desaabb-fibrino. human pci is a non-specific serpin that inhibits several proteases of the coagulation and fibrinolytic systems as well as tissue kallikrein and the sperm protease acrosin. it is synthesized in many organs including liver, pancreas, and testis. the physiological role of pci has not been defined yet. recently, we have cloned and sequenced the mouse pci gene (zechmeister-machhart etal., manuscript in prep.) . this enabled us to study pci gone expression in murino tissues using mouse pci edna and crna probes. by northern blot analysis, mouse pci tar.ha was exclusively found in the reproductive tract (testis, seminal vesicle, ovary), all other organs analyzed -including the liver were negative for pci mkna, indicating that in the mouse pci is not a plasma protein. to determine which cells of the reproductive tract synthesize pci, cellular localization was assessed by in situ hybridization of mouse testis and ovary sections. in testis, pci mrna was present in the spermstogonia layer and in leydig cells, while sertoli cells and peritubular myoid cells were negative. these results are consistent with the immunohistological localization of human pc1 (laurell et al,, 1992) . in the mouse ovary, stroma cells of the medulla and around the follicles were positive for pci mrna. no pci expression was detected in theca or granulosa cells. we also studied the regulation of mouse pci gone expression by steroid hormones in vivo. [n mature male mice castration caused an increase in pci mrna in seminal vesicles, which was reversible upon the administration of testosterone. in tissues of intact adult male and female mice, pci mrna levels decreased after injection of human chorionic gonedotropin (hcg), while in castrated male mice, hco had no effect on seminal vesicle pci mrna. progesterone and 17-b estradiol decreased ovarian pci mrna levels in immature female mice. these data suggest direct down-regulation of mouse pci by sex steroids. the different tissue specific pci-geoe expression in men and mice furthermore indicates a different biological role of this serpin in the two species. ctr. transgene technology, leuven "[' 1-tissue factor ('it) is a 47 kda glycoprotein mainly known a the primary cellular initiator of blood coagulation. whether tf expression may also play a role in development is unknown, but the lack of spontaneous viable mutations of the tf gene in rive leads to the speculation that its absence may not be compatible with normal embryonic development. to determine the significance of "if in ontogenesis, the pattern of tf expression in mouse development was examined and compared to the 'if distribution in human postlmplantation embryos and fetuses of corresponding gestational age. at early embryonic period of both murine (6.5 and 7.5 pc) and human (stage 5) development there is a strong tf expression in both ectodermal and entodermal cells. "if decoration was seen during ontogenetic development in tissues such as epidermis, myocardium, bronchial epithelium, and hepatocytes, which express "if in the adult organism. surprisingly, during renal development and in adult organism tf expression differs between men and mice. in humans maturing stage glomerali were "if positive whereas in mice glomeruli were negative and instead epithelia of tubular segments were tf positive. in ncuroepithelial cells there was a striking 'if expression indicating a possible role of'if in neumlation. moreover, there was a robust tf expression in tissues such as skeletal muscle, and pancreas, which do not express in adult. in contrast to tf, its physiologic ligand factor vii was not expressed in early stages of human embryogenesis, but was detectable in fetal liver, the temporal and spatial pattern of tf expression during murine and human development support the hypothesis, that 'if serves as an important mo~hojzenic factor darinz embrvozenesis. to serve as an anticoagulant, protein c (pc) must be activated by a complex formed between the enzyme thrombin (t) and its cofactor thrombomodulin (tm). therefore, downregulation of endothelial cell surface expressed tm, for example, triggered by an inflammatory stimulus, could become a critical factor in effective pc activation. in order to develop a recombinant (r) pc mutant which can be activated independently of the tkm-complex, a peptide sequence including p1-7 in the activation peptide of pc was modified to be identical to the factor xa (fxa)-cleavage site in prothrombin. the mutant was expressed in hu 293 cells, purified and its anticoagulant properties characterized. using purified fxa the mutant showed activation rates between 0.02 and 0.13 nmlmin at pc concentrations between 97 and 770 nm, while the rpc wild type was insensitive for fxa activation. the activation reaction is calcium-dependent reaching maximal activation rates at a calcium concentration of 3 mm and was enhanced to 3.8-fold by addition of anionic phospholipids (pl). in contrast to the wild type pc the rpc mutant was insensitive for activation by the t/i-m complex. addition of the mutant to normal human plasma induces a prolongation of tissue-factor and p-it-based clotting assays. using normal human plasma as a source for fxa the the activation rates of the mutant were found 5-fold higher than in the purified system if tissue factor was used to generate fxa. in conclusion, our data demonstrate that the rpc mutant is effectively activated by fxa in a purified as well as in a plasma system. interestingly, the activation rates are enhanced in the presence of pl and normal human plasma. fudher studies should clarify the potential use of this mutant as a novel anticoagulant. thrombln plays a pivotal role in thrombotic events. the time course of thrombln concentration in blood or plasma after activation is of special interest to answer a variety of questions. with a chromogenic assay developed by hemker et el. [thromb. haemostas, 70, 617, 1993] it became possible to measure the generation of thrombin in activated plasma continuously. inhibitors of clotting enzymes which are to be developed as anticoagulants should be able to inhibit thrombin generation or to immediately block generated thrombin. we have used a test based on hemker's thrombin generation assay to elucidate which potency and specificity an inhibitor of factor xa needs to efficiently block thrombin generation in human plasma. thrombin generation after extrinsic (tissue factor) or intrinsic (ellagic acid) activation was followed using the chromogenic substrate h-~ala-gly-arg-pna (pentapharm ltd.). a series of synthetic low molecular weight inhibitors as well as naturally occurring inhibitors of factor xa with different potency were investigated. because of the inhibition of activated factor x the generation of thrombin in plasma is delayed and the amount of the generated thrombin is reduced. the concentrations which cause a 50 % inhibition of thrombin generation (icso) correlate with the k~ values of the inhibitors. low molecular weight inhibitors with k~ values of about 20 nmol/i inhibit the generation of thrombin after extrinsic activation with icso in micromolar range. after activation of the intrinsic pathway tenfold lower concentrations are effective. the strongest inhibitory activity after extrinsic as well as intrinsic activation is shown by recombinant tick anticoagulant peptide (r-tap) with ic~o of 0.049 pmol/i (axtdqsic) and 0.037 pmo/i (intdnsic). in the compadson of synthetic low molecular weight inhibitors of thrombin end factor xa which have similar k= values for the inhibition of the respective enzyme (lowest i<1 20 nmol/i), factor xa inhibitors are less effective tn the thrombin generation assay. in contrast, the highly potent xa inhibitor r-tap shows a stronger inhibition of thrombin generation than the tight binding thrombin inhibitor hirudin. background: resistance to degradation of coagulation factor v by activated protein c is associated with a point mutation in which adenine is substituted for guanine at nucleotide 1691 in the gene coding for factor v. to date this specifc mutation appears to be the most common inherited abnormality which predisposes patients to venous thrombosis. for this reason a reliable, fast and automatable system for the diagnosis of the described point mutation is required. the conventional methods used to identify the mutation are based on allele-specific restriction enzyme site analysis or direct sequencing. these methods have disadvantages for a large scale dna diagnosis, which include the need for electrophoresis or a high cost and time consumption. methods: an alternative strategy of dna diagnosis, the allele-specific oligonucleotide ligation assay, was adapted for the diagnosis of tile point mutation of factor v. following pcr amplification of the target dna, tile procedure was performed completely automatically on a robotic workstation with an integrated elisa reader using a 96-well microtiter plate. allelespecific restriction enzyme site analysis was performed to confirm the genotypes. results: in 10 patients with the mutation and in 20 individuals without the mutation the genotypes determined with the conventional allele-specific restriction enzyme site analysis were in 100% concordance with the elisabased oligonucleotide ligation assay. discussion: the pck-oligonucleotide ligation assay applied as automated detection system for the identification of the coagul;mon factor v point mutation allows tile rapid, reliable, and large scale analysis of patients at risk for thrombosis. resistance to the asticoa=m~lant activity of activated protein c (apc resistance) has emerged as the most con'anon inherited thrombophilic state. patients lreterozygous for factor v leiden are more likely to suffer from thromboembolie events than controls. this risk is even more pronounced in homozygotes. due to the low sensitivity and speeifity of most coagulation tests some investigators suggest to examine patients for the presence of factor v leiden mutation by pcr-based methods. re e~tly we presented an aptt-based functional test (acceleria inactivation test ait): 1:20 diluted plasma (50bi) is mixed with factor v deficient plasma (50~tl) and aptt reagent (501.d), incubated at 37°c and then coagulation is induced by caci2 and a.pc (50~d). using a standard curve, the clotting time (see) is transferred in per cent accelerin inactivation (%ai). using this test, the widely used apc-ratio as well as pcr-based factor v leiden detection (confirmed by direct sequencing) we prospectively studied 172 consecutive patients with thromboembolic events. patients without the factor v mntation eonsitently showed more flazm 50% al with the exception of one patient with severe factor deficiencies (including factor v) due to hepatic failure and heterozygous for factor v-leiden resulting in 62*/. ai, there was a complete concordance between the pcrbased method and dysaseelerinemia detected by ait. due to these result a specifity and sensitivity of ait above 95 % was calculated. furthermore, a clear discrimination could be obsoved beween 34 heterozygotes (20%<ai<50%) and 8 (incl. 5 samples obtained from jeaa) homozygotes (ai<20%). in contrast a.pc-ratio resulted in a great overlap between patients homozygous for the wildtype , hcterozgous or homozygous for factor v leiden. in addition we confirmed thc di.~eulties nsing the apc-rauo for patients trader anticoagulation, or with different types of other d~rombophilie defects such as lupus anticoagulant or protein s deficiency. due to the time and cost-saving featttres the/kit should be considered for the rapid detection of apc resistance without the use of pcr-based techniques. in summary we found, well in accordance with previous reports, that apcr due to the fv-leiden mutation is • highly prevalent defect in vte pts. pts with the fv-leiden mutation were eharacterised by a higher incidence of vte's without recognised prothrombotic risk factors and more frequent positive family history; no higher incidence of recurrent vte's or other prothrombotie coagulation defects was found; the age at initial manifestation of thrombophitia was comparable. acquired a recently identified mechanism for famdial thrombophilia is characterized by a poor anticoagulant response to actavated protein c (apc), it is now welt recoginzed to be due to a genetic defect in the factor v gene, which results in the syn-,thesis of an abnormal factor v molecule and to be a frequent risk factor for thromb.osis, however apc resistenee can also be found in certain acquired conditions. since pregnancy ~s known to be associated with a hypercongulable state resulting in a prevalance of thrombosis up to 1% and thromboembolic events, we determined the prevalence of apc resistance during the course of pregnancy at the city hoslfital ruesselsbeim. germany. blood samples from pregnant woman were drawn at mmester 1, 2, 3 and at term. besides the routine coagulatton screening, protein c and viii:c, the apc resistance were evaluated. a modified aptt method utilizing human plasma fracnonated apc (amencan red cross. bethesda, maryland, ~usa) was used to assay apc resistance. this method relies on the determination of two aptt's in the patient plasma, one in the absence of apc and one in the presence of a fixed amount of apc. clotting tune was determined after mcubation using the aptt reagent ( we investigated the sensitivity of various species towards activated, human protein c (apc) and found an impaired response in the order monkey < horse < pig < dog < rabbit. we assume that this effect is mainly caused by differences in the apc cleavage region of factor v, as recently described for a factor v mutation in bumat~s: factor v-leiden. this was corroborated by the finding that addition of human factor v similar to the factor v added with the animal plasma had only minor influence on test outcome. coagulation assays dependent on apc, such as for protein c and protein s determination, in the standard apc assay, the more factor v-leiden specific apc assay and in the pcat, a new screening assay for the whole protein c system, are based on the inactivation of factor va via apc. thus, in all these assays, 5% of rabbit plasma added to human plasma mimicked a apc response as observed for heterozygous carriers of factor v-leiden and 20% as for homozygous carriers. we exploited this factor v-leiden like behaviour to prepare plasma standards for calibration of apc dependent assays by mixing rabbit plasma with human plasma~ as reference for these standards the 0% value was assigned to the clotting time in the absence of apc, while the 100% value was assigned to the clotting time in presence of apc as obtained with a human, normal plasma pool. then these calibrated standards were used for establishing reference curves. on automated coagulation analyzers, the results obtained were automatically reported in '% of normal' or '% sensitivity'• this not only eases evaluation, more importantly, this procedure also considerably improves the comparability of results obtained with different reagents and instruments. in order to establish the methods for activated protein c resistance and the faktor v leiden-mutation we investigated 204 healthy individuals (128 women, 76 men, median age 37 ys) using the assay "contest ® apc-resistance" (chromogenix, sweden). the mean apc-ratio was 2.65, the cut-off level for low response to apc was calculated as 2.15, 46/128 women were oral-contraceptive users. they showed a slight decreased apc-ratio (mean 2.46). 24 of the 204 individuals (11,8%) had a poor response to apc (mean ratio 1.94) as a apc-resistance. for detecting the factor v gene mutation we a used non commercial own modified easy to handle pcr-based test. in 14 of the 204 individuals (6.9%) the factor v-leiden mutation could be found as heterozygotes. in this group, the apc-ratio was significant lower (mean 1.87) as in the group with apc-resistanee and without deteetiun of the factor v mutation (mean 2.04). nevertheless in one individual with beterozygote factor v-leiden, the apc-ratio was found in the normal range (2.24) , also in a few individuals with very iow apc-ratio the factor v mutation could not he detected. finally this data suggests a high prevalence of apc-resistance and factor v-leiden mutation in the population of the southwest of germany. resistance to activated protein c (apc) has been reported to interfere in clotting assays for protein c (pc) and protein s (ps) from various manufacturers, resulting in decreased values or even apparent deficiency (type ii') of pc. or is. in our laboratory, when using functional, avit-based assay kits for pc., is, and apc sensitivity from behring (marburg, germany) on an automatic turbidimctric coagulation analyzer (fibrintiraer a, behring), the interference in the is assay has been observed in a very pronounced manner soon after the introduction of apc sensitivity testing into the standard laboratory procedure for patients with thrumbophilia. yet we were not aware of an interferenca caused by apc resistance in the pc test. we therefore evaluated our results for pc eed ps with respnet to apc sensitivity status in a retrospective analysis. exclusion criteria were: oral anticoagulant therapy with vitamin k antagonists, impaired liver function, borderline result in the apc sensitivity test, known analytical intedercnc* other than apc resistance (e.g. elevated factor viii:c). resu/ts: apparant pc activity was slightly (12 %) lower and apparent ps activity was grossly (45 %) lower in ape resistent patients than in patients with normal apc sensitivity: there was no case with a presumed du~l defect of both at~ rnsistaac¢ and pc deficiency. in addition, none of the few patients diagnosed with pc deficiency before the introduction of the apc sensitivity assay turned out to ix: apc resistent. much more of a problem is the known sensitivity of the pc test to an elevated factor viii concentration which is quite a common finding among hospital patients but rarely known in an individual case. by aeccelerating the clotting process, elevated factor viii can cause apparent pc deficiency that can not be confirmed in a chromogunie pc assay. ,clearly, when combined with apc resistance, this problem is exspacted to be more severe. on the other hand, the comhination of both apc resistance and apparent is deficiency was found on a regular basis with many decreased and some bordeflinc values for is but only few in the lower normal range and virtually none in the upper non~al range. also, several #eats previously diagnosed with is deficiency turned out to be pc resistent, and the diagnosis had to be corrected, but dual defects cannot be ruled out. conclusion: for, the interpretation of pc and/or 1:"3 values from clotting assays the apc sensitivity status must be considered. when apc resistance and an apparent deficiency of pc or is is found, a type i deficiency may be confirmed or ruled out using antigen assays, but at present, there seems to be no reliable way other than genetic analysis to address the question of dual defects with presumed type ii deficiency, especially in the case of ps. therefore, there is a clear need for analytical improvements and the development of functional assays that are less sensitive to interference by apc resistance. the most common inherited risk factor for venous thrombosis is the resistance to activated protein c (apc). the main reason for this resistance is a point mutation in the gene of dotting factor v (f v). a nueleotide exchange leads to a different amino acid sequence in the protein and thus to the loss of one of the cleavage sides which is important for the inactivation of the procoagulatory form off v. the imbalance in the coagalation cascade leads to a strong risk for venous thrombosis, which is thought to be inoreaced by factor 7 to 10 for heterocygotes and about 80 for homocygote individuals. the prevalence in patients with a history of thrombosis is about 20%, among apcresistant patients as high as 80%. prevalence-off v ,leiden" is reported to be 2% of the normal population in leiden/netherlands and as high as 15% in a certain city in the south of sweden. due to the obviously different geographic distribution and the highly increased risk of venous thrombosis in carriers of the mutation investigated the distribution of this mutation in the normal population of different regions in germany. first attempts to assess the prevalence off v mutation by molecular methods (polymerase chain reaction followed by restriction fi'agment length polymorphism analysis) revealed that in the area of wiirzburg 7.7% of blood donors carry the mutation, which is significantly different (fischer t-test, level of significance 0.05%) from published data observed in freiburg (3.4%). homburg/saar and harmover revealed 5.8% and 8.5%, respectively, and were not significantly different from wfwzborg. further investigations of blood somples fi:om other regions should give an overview of the geographic distribution of this mutation in germany and might finally help to draw a map era regional risk of venous thrombosis. the regular apc resistance test is not applicable to plasma from orally anticoagulated (oac) or heparinized patients due to decreased levels of vitamin k-dependent clotting factors and to thrombin inhibition by antithrombin, respectively. the former limitation is overcome by prediluting samples 1-1-4 with fv-defident plasma. for this purpose a special quality of fv-deficient plasma has been prepared, which also allows analysis of heparinized plasmas. this fv-deficient plasma has now been evaluated with the coatest apc resistance assay. a complete ape ratio discrimination for the fv q506 mutation is obtained for plasmas from oac and heparin patients as well as from non-treated individuals. ratios above 2.2 are obtained in normal genotype plasmas, whereas heterozygotes for the mutation yield ratios in the range 1.6-2.0. the basal apt times for 15 oacplasmas with widely spread inr-values were reestablished into the normal range. .the stability of the reconstituted fv-deficient plasma was found to be' at least 3 hours at room tempetature, indicating its suitability for .routin.e u.~. f .u~ .e~mv~., the influence of preanalytical procedures is rmm~ single or double cenuifugation as well as freezing of samples do not alter the apc ratios to any significant extent. our results prove the usefulness of the modified coatest apc resistance kit method as a reliable, simple and rapid tool when screening for the presence of the fv q506 mutation. hereditary resistance against the anticoagulatory action of activated protein c (apc-resistance, apcr) which was first described by dahlb~ick et al. (pnas, 90:1004 , 1993 was identified as a possible new thrombophilic factor in a high percentage (17-60%) of young adults with thrombotic events. a single missense mutation (r506q) due to a g/a transition (g1691a) in exon 10 of the factor v gene (bertina et al., nature, 369:64, 1994) is tightly linked with apcr and is regarded as the causative molecular defect. identification of this mutation by pcr-based methods is easy to perform and avoids preanalytie and analytic errors in the eoagulometric assay for apcr. since the impact of this mutation in children with thromboembolic disease has not been determined to date, we initiated a multi-center prevalence study on two pediatric populations, with and without thromboembolie events. we compared 125 pediatric patients with thrombosis, divided into 3 different age groups (0 to <0,5 years; >0,5 to <10 years; >10 to <18 years) with a normal population of 159 children. the mutation g1691a was found with an unexpected high prevalenee of 12% in our normal controls. however, the prevalence was significantly higher in the age groups: 0 to< 0,5 years (25%) and >10 to <18 years (30%). in patients between >0,5 to <10 years the overall prevalence was similar to the control (13%). however in patients of this age with spontaneous thrombosis apcr was also a significant risk factor (29%). our results emphasize the impact of apcr for thrombogenesis in children. however, the significance is agedependent and does possibly reflect the different physiology of haemostasis in our three age groups. activated protein c (apc)-resistanec is a newly reeognised risk factor for thrombosis. in at least 90% of the cases it is caused by a single point mutation in the factor v gene (g->a at nucleotide 1691), which predicts replacement of arginin 506 with ghitamin. one of the apc cleavage sites in factor va is located c-terminal of arginin 506, and mutated factor va (factor v leiden) is resistant to apc-mediated inactivation. from epidemiologic studies it is known, that this abnormality can be found in about one third of patients with thrombosis. apc-resistance is a major basis for venous thromboembolism and is prevalent in about 5.10% of the general caucasian population. recurrent spontaneous abortion (rsa) affects 1-5% of couples and represents a major concern for reproductive medicine. in spite of extensive endocrine, genetic, serologic and anatomic evaluation some 30-40% of rsa women remain unexplained. a frequent morphologic finding in placentae of aborted pregnancies is an increase of fibrin deposition within the intervitlous space. because of these findings we studied the prevalence of apc-resistance in women with rsa (more than 3 miscarriages) of unknown origin. in 2 of 35 cases we found a pathologic apc-resistance, both patients had a history of recurrent thrombosis and were heterozygous for factor v leiden. the prevalance of apc-resistance is 5,7% and thus equals the prevalence in the general population. our data do not support the hypothesis that apc-resistanee is a risk factor for recurrent spontaneous abortion. h~matologisches zentrallabor der universit~t, inselspital, 3010 bern resistance to activated protein c (apc) due to the mutation 506 arg --~ (]in of factor v (factor v leiden mutation) is the most frequent hereditary thrombophilic defect known today, with a prevalence of 20 -50 % in patients with idiopathic venous thromboembolism and of about 3 -5 % in the general population. with an allele frequency of 2 % the expected number of homozygous individuals is about 4 in 10000. homozygous and heterozygons individuals differ considerably with respect to the relative risk of thrombosis (80 -fold versus 7 -fold) as well as to the age of the first thrombotic event (31 versus 44 years). deficiency of the vitamin k dependent protein s (p$), an important cofactor of apc, is another hereditary thrombophilia which is, however, much rarer than apc resistance with a prevalence of 5 to 8 % in patients with venous thromboembolism. factor v leiden mutation as well as ps deficiency are associated with impaired anticoagulatory activity of apc, which is most pronounced in case of the combination of the two defects. the combination of ps deficiency (with an assumed prevalence similar to that of pc deficiency) with heterozygous or homozygous apc resistance can be expected with a probability of 1: ~ 5000 or 1: ~ 500000, respectively. it is well known that ps levels decrease towards the low normal or even subnormal range during pregnancy. moreovar, there is increasing evidence that the sensitivity of plasma to the antieoagulatory effect of apc decreases during pregnancy resulting in an acquired apc resistance. these pregnancy associated effects art obviously much more relevant in case of preexisting ps deficiency or apc resistance and should contribute to the elevated thrombotic risk during pregnancy in a subject with either of the two defects, and even more so for a woman who suffers from both defects. we describe a young woman with a combination of homozygens apc resistance ( apc ratio 1.10, normal range: 2.02 -3.73), pronounced ps deficiency (free ps 0.ll u/i, total ps 0.30 u/i, normal range: 0.55 -1.20 u/1 and 0.65 -lag u/i, respectively) and, moreover, impaired fibrinolysis (no change of euglobulin -lysis time after 10 rain venous occlusion) who developed deep vein thrombosis after cesarean section in her first pregnancy. examination of her familiy showed heterozygous apc resistance in her asymptomatie father (apc -ratio 1.99) , combination of heterozygous apc resistance (apc -ratio 1.60) and ps deficiency (free ps 0.30 u/i, total ps 0.58 u/i) in her nsymptomatic mother and no defect in her sister. considering the fact that the mother was still thrombosis free at the age of 49 one may assume that the thrombosis risk in the proposita was mainly influenced by the homozygnsity for apc resistance. s. ehrenforth, m. adam, b. zwinge, i. scharrer university hospital, dept. of angiology, frankfurt a.m., germany introduction: apc resistance has been shown to be the most commonly inherited defect which constitutes a risk factor for venous thrombosis (vt). however, most of the present epidemiological studies concerning apc-r prevalence in thrombophilia were derived from results of tests conducted onplasmas collected under various conditions. this may influence the great differences reported on the prevalence of apc-r among these patients. for example, it has been shown that freezing of plasma specimens prior to analysis of apc-r causes a significant decrease in the assay results.the aim of our study was to evaluate the influence of eentrifugation conditions on the results obtained with the chromogenic apc-r assay. patients and methods: blood was collected from 31 patients (t9 women, 12 men; fv gent.type: 13 r/r 506, 14 r/q 506, 4 q/q 506) through veinpuncture into trisodmm ciwat (1:9). platelet-rieh and platelet-poor plasma was obtained by immediately centrifugation at 4"c for 10, 20, 30, 40, 50, 60 rain at 1000, 2000, 3000, 4000, 5000 and 6000 rpm. additional, pnp obtained from 14 healthy individuals (7 male, 7 female without hormonal trealraent) was prepared equally. apc-response was determined within one hour after centrifugation using the coatest apc resistance kit from chromogenix. results: for both, pnp and sin/gle plasma samples, we observed continuous higher af'c-ratios after increasing cenwifugation intensity. for example, an increase from 1000 to 6000 rpm resulted m an increased apcratio from 2.7 to 3.26 (20 min), from 2.88 to 3.326 (60 rain) respectively. even though less distinctive, similar results were observed concerning the duration ol eentrifugation: when the duration was increased from 10 to 60 minutes we observed a continuous increase in apc-ratio, for example from 2.74 to 3.12 when using 2000 rpm and from 3.09 to 3.36 when using 4000 rpm. the decrease of the ratio after low eentrifugation is the eonse-9nence of the shortening of affft in the presence of apc, without a signhcant influence of basal al:rl~ without apc. conclusion: our results demonstrate that centrifugation conditions are important to consider for the interpretation of apc-r results. supporting our observations, recent studies from sidelmann et al. have shown that an increase in plasma platelet concentration, low eentrifugation respectively, causes a signficant decrease in the apc-response. however, so far the mechanism responsible for the significant effect of both on apc-r assay results is unknown. although technically simple, the biochemical cemplexitiy inherent in the chromogenic apc-r assay necessitates a standardized plasma handling procedure to secure a reproducible determination of apc-il compapjson of different assays for determination of apc-resistance with the geno'fyping factor v (arg -> glu) g. siegert*, s. gehrisch*, e. runge**. r. naumann**, r. kn6fler*** *institute of clinical chemistry, **clinic of internal medicine, *** childrens hospital resistance to apc diagnosed on the basis of prolongated clotting time in the aptt assay" is now considered a major cause of thrombophilia. in the majority apc resistance is ~ted with a point mutation in factor v molecule (arg506glu), but both are not synonym. protongated baseline aptt is a limitation of the assay. following the determination is not possible in risk groups of patients (factor)ill deficiency, lupus anticoagulan0 and in patients under anticoagulant therapy. in these causes a dilution of plasma in factor v deficient plasma is recommended. the immunochrom assay is based on the inactivation of factor villa by apc. the aim of the study was to compare different functional apc response assays with the result of the dna analysis. apc response was tested in 100 healthy probands, 81 thro~ patients and 16 family members using the lmmtmochrem assay, the contest (chromogeaix) and the contest with 1+ 4 dilution of the plasma in native factor v deficient plasma (immune). the dna analysis was performed as described by bertina. one patiem was homozygoas for factor v mutstion~ a hetemzygous result was obtained in 4 members of the control group, in 28 patients and in 6 family members. in all cases with factor v mutation the ratio of the immunochrom assay was lower than the laboratory own value, independent on anticoagulant therapy. pathological ratios in this assay were also obtained in one member of a family" with high thrombotic incidence (dna arg/arg) and in 17 patients under anticoagulant therapy ( two of this patients are one cloned twins). in the contest a ai~ response was diagnosed in all cases with factor v mutation without anticoagulant therapy and in 40 % of heterozygous patients under anticoaglant therapy. results of the test using the dilution in factor v deficient plasma showed a good agreement vath the results of the dna analysis but the method is obviously only sensitive for the factor v mutation. the reason for pathogical ratios in the lrnmunechrem assay in wildtype patients is unclear. the majority of this patients is treated with anticoagulants, a comparison with the contest is not possible. interestingly in one patient under heparin and low ratio in the immunochrom assay' after reduction of hepann the ratio of the coatest was also low. it seems necessary to investigate in which distance to the thrombotic events the apc resistance should be tested. following pathological ratios in ftmctional apc assays must be discussed: high levels of factor viii and or v wiuebrand antigen (acute phase reactien), other mutations in factor v and viii. the factor v dilution assay should be replaced by the dna analysis. due to their differing compositions, the "sensitivities" of various aptf reagealts differ not only with respect to factor depletions, heparin and fibrin-fibrinogen degradation products, but also with regard to pathological inhibitors. for lupus anticoagulants this means that "lupus-sensitive" reagents can be delineated from "lupus-insensitive" reagents. with a "lupus-insensitive" ai~ reagent there is no or only slight prolongation of the aptt in the plasma under investigation, whereas with a "lupus-sensitive" reagent marked prolongation is observed. for the meaninof~l use of aptr reagents it is necessary to know the extent to which they are influenced by lupus anticoagulants. the following 14 apti' reagents were tested: • ptt-reageaz, p'rta, ptta liquid, ptt-la, pti'-lt (boehringer/stago) • pathromtin, pathromfin sl, necthroratin (behring) • platelin s, piatehn excel ls (organon tekinka) • actin-fs, actin-fsl (dade) • aptt silica lye, aptt silica liquid (instntmentation laboratory) the material for investigation consisted of 20 plasmas from patients with lupus anticoagulants. a confirmatory test (lupus anticoagulant test, immune) was positive for all of the patients. measurements were made using the sta coagulation analyser (boehringer/stago). it can be seen from the results that in some instances very different prolongations were obtained in identical plasmas by using differing aptt reagents. low susceptibility to lupus anticoagulants was shown by actirt fs (dade), ptt-reagenz (bcehrlnger) and neothromfin (behring). high susceptibility was shown by platetin excel ls (organon teknika), ptt-la and pti'-lt (boehringer/stago). lupus anticoaguhant screening with the aptt reaction is promising when two aptr reagents differing as greatly as possible in their lupus anticoagulant sensitivity are used. the resistance to the anticoagulant response of activated protein c (apc) is a major cause of venous thrombosis. apcresistance is due to a single mutation in factor v gene, which predicts replacement of arg 560 in the apc-cleavage site with gln (factor v leiden mutation). in contrast to other known genetic risk factors for thrombosis, this factor v 1691 g-a mutation has a high prevalence in the common population of western europe (average 4-5 %). we have determined the prevalence of the factor v 1691 g-a mutation in a population of 700 probands of north-eastern part of germany. the mutation was found in 7 %. (heterozygoty were found in 48 subjects 1 person was homozygous.) the results are compared with our studies of populations from argentine and poland. me analysed the factor v 1691 g-a mutation in 71 patients with thrombosis from germany and hungary. this mutation has been found in about 60 % of these patients. in contrast, the frequency of this mutation was strongly reduced in a group of 47 patients with thrombosis and pulmonary embolism of argentine (3 heterozygotes in 47 patients; 6 %). the results of these different populations will be described and discussed. past medical history: venous thromboembolic events (re) at 18, 19 and 2i years; intermittent oral anticoagulation (oac) without te's. diagnosis of autoimmune disorder with elevated antinuelear-antibody-fiters and positive lupus-anticoagulant test. no other relevant illnesses; family history uneventful. two weeks prior to the referral to us -acute febrile illness with nausea, diarrhea, abdominal pain; hospitalisation, treatment with iv antibiotics and anticoagulation with fraetionated heparin; development of extensive deep vein thrombosis (dvt) of the right leg; initiation of full-dose unfractionated heparin; decline of platelet count from 121 to a nadir of 21 g/l; referral to our department. on admission an extensive coagulation screen yielded the following results (n/normal, t/elevated, i/reduced, +/positive, -/negative): pt t, aptt t, tr n, factor ii, v, viii n, factor vii, ix, xi, xii /,, fibrinogan t, atiii n, protein c, s *, activated protein c sensitivity ratio 1.92 ($), fv-leidenmutation pcr -, fibrinolytic system n, tat t, ft÷2 t, lupus anticoagulant +, heparin induced platelet antibodies +; no diagnosis of a specific autoimmuna disorder could be made. an immunosuppressive therapy with corticosteroids and anticoagulation with recombinant hirudin were init'~at~; no p~ogr~zsion of the dvt oeeured and normalisation of the platelet count was observed. during follow-up under oac ) and low-dose corticosteroids, the patient was well, the pathologic coagulat;.on results, including lupusanticoagulant and activated protein c resistance, have returned to normal; no further te's have been observed. in summary we present a case of a complex coagulation disorder as part of an autoimmune process, resulting in a clinically manifest prothrombotie dysbalance including lupus anticoagulant, acquired resistance against activated protein c and heparin induced thrombocytopenia (type ii), entering complete remission under combined immunosuppressive and anticoagulant therapy. in the last 30 years, a vast number of simplified analytical procedures have been developed for the diagnosis of haemostatic disorders. today the detection method have evolved from the mechanical hooking method or ball coagulometry to optical systems, which additionally can utilise chromogenic substrates or immunological methods. in these systems the clotting time is derived from algorithms (e.g. threshold or maximum of the first or second integral). we studied 50 healthy subjects, aged 21 to 65 years and 118 patients, aged 9 to 93 years using a new aptt reagent (pathromtin $l). the results were compared with those obtained with a routinely used reagent (pathromtin). the reference range, factor-, heparin-and lupus anticoagulant sensitivity were determined. analysis was performed using the behring fibrintimer a (bfa) with optomechanical clot detection, the behring coagulation timer (bct) with op-"dcal clot detection by threshold and the dw test and dw confirm for lupus anticoagulant diagnostic. our results showed that the new pathromtin sl reagent met the demands for a higher factor and lupus anticoagulant sensitivity. it is highly suitable for monitoring heparin therapy and gave comparable results with the optical and the optomechanical analyser systems, hence reagent c~n be used for both systems. restenosis following percutaneous transluminal angioplasty (pta) continous to be a major clinical problem. neoinfimal hyperplasia, being the major undedying cause, can not be sufficiently avoided. vadous plasmatic coagulation and fibrinolytic factors, have been associated with artedal restenosis. anticardiolipin antibodies (act_) have been established as dsk factors for venous or arterial thrombosis. methods: in a cohort of 75 patients (53 men and 22 women, age 68±13 years) undergoing pta of a peripheral artery we prospectively evaluated whether acl could influence 6 months restenosis rate. patients were clinically examined before, 3 and 6 months after pta. noninvasive grading of artedal stenosis was done by duplex scanning of jet peak velocities. restenosis was arbitrarily defined as more than 50% occlusion of the lumen at the site of dilatation 6 months after successful intervention. laboratory investigation at the same time included acl and other known atherosklerosis risk markers, such as fibdnogen (fbg), yon willebrand factor (vwf), homocystein (hcy), c-reactive protein (crp). thrombin generation markers, such as thrombin-antithrombin iii complexes and prothrombin fragments 1+2, as well as thrombomodulin (fm) as an endothelial activation marker, were also measured. results: 31/75 (41.3%) patients were considered to have developed restenosis after 6 months. 9/75 (12%) patients were found to have positive igg-(19-35 gpl) and/or igm-acl (14-103 mpl) at all three measurements. 1/75 was negative before but seroconverted (igm) 3 months after pta. 2/10 (20%) acl-positive and 29165 (44.6%) acl-negative developed restenesis at 6 months (chi-square p-value=0.14). all above mentioned coagulation parameters did not differ between acl-positive and -negative patients, measured before or 6 months after pta. some of them are shown below (values before pta): fbg ( basilar artery stenosis is a rare event in young children. risk factors are head or neck trauma with consecutive dissection of the vertebral artery, cardiac diseases or hypercoagulability. elevated lipoprotein (a) (lp(a)) serum levels in adults can mediate atherosclerosis. in addition, lp(a) might interfere with fibrinolysis. here we report on a 3 year old boy , who presented with acute brain stem symptoms. history revealed neither trauma nor infectious disease. conventional and mr angiography showed stenosis of basilar artery without ischemic lesions. laboratory findings were normal in routine blood and csf tests. global coagulation parameters as well as procoagulant and anticoagulant factors were normal. cardiac and autoimmune disease could be ruled out. lp(a) serum levels were significantly elevated to 104 mg/dl (normal range <30 mg/dl). analysis of other family members revealed a hereditary hyperlipoproteinemia (a) which might explain family history of an increased incidence of myocardial infarction and cve in elderly family members. clinically the patient recovered completely from brain stem symptoms after heparinization and subsequent oral anticoagulation with phenprocoumon. however, radiological signs of basilar artery stenosis were progredient. in a recently developed specific test, an elevated anti-phosphatidylserin antibody titer was detected one year after primary diagnosis. in conclusion, this is the first report on a child with stenosis of the basilar artery and elevated levels of lp (a). it is unclear, whether apa contributed to the onset of basilar artery stenosis or developed secondary due to endothelial defects after thrombosis and anticoagulation. apa, however, might increase the risk of further thrombotic events in this patient. in 110 patients with thrombotic events respectively patients with systemic lupus erythematodes antioardiolipin antibodies (aca) aund lupus anticoagulant (la) were ~ea~ured. for aca detecting we use the assays from elias for igg-and ig~}-antibodies. we use as sensitive methods for detecting la in our laboratory the testkits from diagnostlca stago (staclot la with hexagonal array of phospholipids, ,ptt-la a very sensitive pttmethod and staclot p~p-a platelet neutralization procedure) and the ptt from organon teknik~ (platelin excel ls with two incubation times, 1 and 10 minutes). i"~e results of this tests were compared with three new or~e on german market: specktin apot (aktlvated plasma clotting time), specktin aptt (aptt wlth purified soy extract) and 8pecktin la (phospholipid preparation in concentrations between 10 and 500 ~g/ml); all wak chemie. traditional aptt reagents were developed for the sensitive detection of factor vib an ix as a cause of hemorhage. high sensitivity against lupus anticoagulants, which also prolong aptt, was not required for this purpose, with increasing recognition of the importance of antiphospholipid antibodies as a risk factor for thrombembolism, more sensitive reagents were designed, which now reliable detect this condition. using such reagents as a screening test in a general hospital makes it necessary to distinguish both conditions quickly. we here report an algorhythm, by which we use an inhibitor (lupus anticoagulant) sensitive (sta aptt, boehringer) and an inhibitor insensitive reagent (actin fs, dade) to distinguish anticoagulants and factor deficiencies as a cause of prolonged aptt. citrate plasma from 33 patients with various diseases showed an unexpectedly abnormal inhibitor sensitive aptt (>40s). 13 plasmas with factor deficiencies remained abnormal with the insensitive aptt reagent. a regular correction of their defect occured on mixing with normal plasma. by measurement of single coagulation factors five patients with contact factor xii deficiency were found. this condition is associated with thrombosis and very rarly with bleeding. three patients with factor xi deficiency and two patient with factor ix deficiency were also identified. antiplatelets, of any kind, permits a secondary prevention of myocard ischemic lesions. there is no general consensus regarding secondary prevention of cerebral ischemic lesions. aspirin remains the most common substance, ticlopidlne also brings about prevention, but with important secondary effects. european stroke prevention study i has demonstrated that the combination of antiplatelets, in particular aspirin/dipyridamole (persantln), is also very active. to collect more information, esps 2 was organized and 6602 patients receiving either a placebo,either 50 mg aspirin,either 400 mg sustained release form of dipyridamole (persantin (r)), or the combination aspirin/dipyrldamole, were recruited. it ended march 31st 1995 with the following conclusions: i-aspirin, 50 mg a day, brings about a significant secondary reduction of stroke (18.z %), after a two year follow-up. notwithstanding the low dose of aspirin, haemorrhages remain important. 2-dipyridamole, at 400 mg a day, brings about a significant reduction of stroke (i6.3~), similar to that of aspirin. one could thus substitute 50 mg aspirin by 400 mg dipyridamole. 3-the combination of 50 mg aspirin and 400 mg dipyridamole brings about a significantly greater reduction of stroke (37.0 ~). esps 2 revealed that a low dosage of aspirin is active, that dipyridamole alone is also active, but that the combination of both gives far better results. the study of the primary end-points,the study of the survival curves, the factorial statistical analysis and the pairwise comparison analysis, led to these conclusions. the conclusions drawn from esp£ 2 underline that the combination aspirin/dipyridamole is a privileged choice for cerebral ischemia, the state of activation of circulating platelets in acute cerebral ischemia is controversial. activation of platelets on single cell level can be assessed by determining the shape change or the expression of antigens such as p-selectin (cd62). shape change is an early and rapidly reversible event in platelet activation whereas p-selectin is irreversibly expressed on the platelet surface upon stimulation. methods: we investigated 20 untreated patients within one day after cerebral ischemia, 20 patients months after stroke treated with warfarin, and 21 age and sex matched control subjects without vascular risk factors. venous blood was collected into a fixation solution blocking the metabolic processes in platelets within milliseconds. we determined the fraction of resting discoid platelets by phase contrast microscopy. the expression of p-selectin was measured by flowcytometry. results: the fraction of platelets expressing p-selectin was higher in patients with acute cerebral ischemia (7.8_+4.7%) than in control subjects (1.9_+1.1%; p<0.001, u-test). patients with stroke (n=15, 7.8+4.5%) and patients with transient ischemic attack (tia; n=5, 7.6-+6.1%) had similar values. patients months after stroke still had higher values (4.3+9,3 %, p<0.05) than control subjects. the rate of discoid platelets was not different between patients with acute ischemia (n=17, 85.6-+8.8 %), patients months after stroke (n=19, 85.7-+5.1%) and control subjects (n=21, 86.7_+5.8 %). platelet count was not significantly different between groups. conclusion: the elevated proportion of platelets expressing pselectin indicates strong platelet activation in acute cerebral ischemia and in a majority of patients months after stroke. assessment of pselectin revealed a higher sensitivity for platelet activation after stroke or tia than analysing the reversible shape change. further studies have to clarify if monitoring of platelet activation by flowcytometry is helpful as a prognostic tool and to evaluate therapeutic strategies after stroke. vascular smooth muscle cell (smc) proliferation and migration into neointima are the hallmarks of atherogenesis. the complexity of these processes and their concerted action and interaction of molecules are yet to be fully elucidated, one crucial molecule seems to be the urokinase-type plasminogen activator receptor (upar) recently also assigned as cd87 antigen, upar serves a dual function: (1) it directs upa proteolytic activity to a special location on the cell surface and (2) induces cellular signals leading to various phenotypic changes. we have investigated the signal-transducing capacity of upar in human smcs and provide here a molecular explanation for uparrelated cellular events. activation of these cells with upa (even with inactivated catalytic center) results in the induction of tyrosine phosphorylation, suggesting modulation of upar-associated protein tyrosine kinases (ptks) upon ligand binding. we obtained patterns of tyrosine-phosphorylated proteins with molecular masses of ~ 55-65 and 35-40 kd. using antibodies against different types of ptks as well as immunoprecipitation-and immunoblotting techniques the ptks involved in the upar-signalling complex were identified to be members of the src-ptk family. the cotocalization of upar and ptks at the cell surface of smcs was further confirmed by confocal microscopy studies. we conclude that the upar-ptk complex is most likely involved in this signal transduction pathway that provides the coordinated action of extracellular proteolysis, adhesion, and cell activation, which is required for cell migration. this mechanism may be crucial for the progression of atherosclerotic plaques. activation markers of haemostasis have been found elevated in relation to diabetic vascular lesions. simultaneous pancreas-and kidney transplantation (pkt) in type i diabetes has been shown to improve diabetic complications and long term survival. we measured haemostatic vascular risk factors and activation markers in plasma of 18 patients after successful pki', 17 patients after pkt and rejection of the pancreas graft and 5 patients after pkt and rejection of the renal graft. blood samples were taken during routine ambulatory visits, patients were free of any ongoing acute disorder or transplant rejection and under continuous immunosuppressive medication. despite individually adjusted insulin therapy hba1 plasma levels increased after pancreas rejection (5,37 vs 7,i2, p<0.001). platelet counts and plasma levels of fibrinogen, f1+2 fragment, tat-, app-complex and-fibrin monomer were found significantly elevated as compared to diabetic controls but not significantly different with respect to complete or partial successful pkt. one major reason of the increased activation state of haemostasis may be cyclosporin treatment given to all patients, t-pa and pal i plasma levels were within the normal range and significantly correlated to plasma triglyceddes (r.0.049; p<0.005). d-dimer plasma levels were significantly lowered after pancreas rejection (772(375) vs 324(232) nglml; mean(sem) p<0.005), which might reflect impaired fibrin degradation related to increased glycosylation of fibrinolytic factors. in conclusion, despite the marked improvement of glucose and lipid metabolism, plasma markers of activation of coagulation and flbrinolysis are not decreased to normal after simultaneous pancreas and kidney transplantation. according to the investigations of fowler et al. and pepe et al. the probability of an ards occurring with one risk factor is 5-8%, and in the presence of several risk factors, 25%. goris et al. and johnson et al. determined the level of severity with the aid of a fixed scale: the injury severity score. all these investigations are however not to be interpreted as typical following coronary surgery. these investigations demonstrated that the kallikrein and factor xii systems are of great importance as intraoperative risk factors. here the factor xii system plays a major role with direct or indirect activation of the kauikrein-kinin system with the splitting products alpha-factor xiia and bfactor xha respectively. all ards scores take the pmn-elastase into account. if the pmn-elastase values (1000 pg/l) are constantly high postoperatively then lung complications are to be expected. patients developing an ards displayed significantly lower alpha2-macroglobulin values. patients who developed a highly significantly raised kallikrein-like acdvity (>60 u/i) after the beginning of bypass and showed constantly high values during ecc are difficult to keep under control due to the blood pressure behaviour. the platelet pal also shows a significant rise and intraoperatively runs analogous to platelet factor 4, only antiparailel, since it attacks the endothelium. we were able to show that pai-1 is suitable as an indirect marker for a possibly developing restenosis. 85% of the patients investigated with lowered pai-1 values in the postoperative phase did not develop a restenosis. however, with patients showing significantly rising pa[-1 values from the 1 st. to 3rd. postoperative day 50% of all the cases had a restenosis. a further risk factor in this respect are significantly raised fibrinogen levels which lay over 800% at the end of surgery. if these fibrinogen values do not fall from the 1st. postoperative day onwards a raised risk of thrombosis must be reckoned with in the absence of therapeutic intervention. the following parameters represented haemostaseological risk parameters with significant behaviour within the framework of this study: 1) regards the blood pressure behaviour, the kallikrein-like activity (>60 u/i); 2) with regards to the lung complications, aipha2-macroglobulin and pmn-elastase (>900 g/i); 3) and final/y as a possible marker for a developing restenosis pai-1 and fibrinogen (>800%). resulting from numerous clinical studies homocysteinemia is found to be an almost independent risk factor of atherosclerosis including thrombotic complications as well as of venous thromboembolism. experimental investigations on the underlying mechanisms suggest endothelial cell damage accompartied by the development of an atherogenic and thrombogenic potential, increased platelet reactivity, oxidative modification of ldl, and enhanced affinity of lp(a) for fibrin. to our knowledge no results are published on the influence of homoeysteine on leukoeytes although these cells are deeply involved in pathological events within the vasculature. therefore, as a first approach different functional parameters of human polymorphonuclear leukozytes (pmnl) were followed under incubation with 60, 300, and 600 i.tm (final concentration) dl-homocysteine (hc) in isolated fractions or whole blood, respectively: l) spontaneous mobility of pmnl, measured as migration distance into micropore filters in a modified boyden-chamber, is found to be significantly enhanced by the two smaller hc concentrations. 2) chemotaxis induced by 0.1 i.tm formylmethionylleueylphenylaianine (tmlp) shows no significant differences. 3)monitoring of chemiluminescence signals (autolumat lb953, berthold) is complicated as hc influences the luminol-mediated indicator reaction. adjusting appropriate conditions the following results are obtained: spontaneous chemiluminescence and that induced by zymosane, tmlp, and the ca2+-ionophore a23187 are entranced by the two higher hc concentrations. there are, however, differences between the blood donors as a minority does not respond to hc in repeated measurements. with phorbol 12-myristate 13 acetate the signal is diminished by hc in all cases and with all concentrations. 4) phagoeytosis induced by zymosane (microscopic evaluation) as well as by opsonized e. coil (cytoflowmetric evaluation) is significantly increased by the two higher hc concentrations. conclusion: the activation of human pmnl is enhanced with respect to the majority of investigated stimuli by hc in concentrations reached under pathophysiological condititions. the effect of pysical exercise on hemostatic parameters was studied in 12 patients (male, mean age: 55 [range 36-68] yrs) with angiographically documented coronary artery disease (cad) and in 11 controls (male, 53 [43-62] yrs) both participating in an 1 hour group exercise session for cardiac rehabilitation. in each group relevant arteriosclerotic lesions in carotid, abdominal and leg arteries were excluded by doppler ultrasound examinations. patients were all under 6-blocking agents and aspirin. plasma levels of prothrombin fragment 1+2 (ptfi+2) and fibrinopeptide a (fpa) reflecting formation of thrombin and fibrin, respectively, were measured at rest and immediately after 1 hour of exercise consisting of jogging, light gymnastics and ball games. training intensity in both groups was comparable as indicated by the mean heart rate during exercise corresponding in patients to 80+6% (mean-+sd) and in controls to 79-+5% of the maximal heart rate previously determined on a bicycle ergometer. baseline values for ptf1 +2 were significantly lower in oatients (0.67-+0.15 nmol/i; mean-+sd) than in controls (1.01-+0.21; p<0.001 i. after exercise we found an increase of ptf1 +2 in controls to 1.14-+0.24 nmol/i (p<0.001) while in patients ptfi+2 remained unchanged (0.67-+ 0.14 after). accordingly, exercise induced r se of fpa was more pronounced in controls (from 0.62-+0.24 to 1.60-+0.62ng/mt; p<0.001) than in patients (from 0.63-+0.26 to 1.20+0.46ng/ml; p<0.00t). we conclude that in terms of thrombin and fibrin generation exercise training does not exert detrimental effects on hemostasis in patients with cad. lower baseline values and lack of exercise induced increases of ptf1 +2 in patients with cad might be attributed to medication with aspirin and/or b-blocking agents. periodontitis marginalis (pm) is an inflammatory oral disease that is caused by gram-negative bacteria and that has a high incidence in the second half of the life. clinical signs of pm are gingival bleeding, periodontal pockets, alveolar bone destruction and loss of teeth. recent epidemiologlcal studies have provided some evidence for an association between pm and atherosclerosis. in the present paper we will summarise some of the results that we have obtained in studies on patients with pm as well as on patients with hypercholesterolaemia (hc) and atherosclerosis. pm was frequently found to be associated with hc (90 % in rapidly progressive pm) and increased reactivity of peripheral blood neutrophils and platelets (e.g. generation of oxygen radicals and paf-induced aggregation). patients with hc and atherosclerosis had a higher frequency of severe pm when compared with data on the community periodontal health. the severity of pm was higher in patients with plasma cholesterol levels _> 6.5 mm when compared to those with plasma cholesterol < 6.5 mm. in patients with coronary atherosclerosis the severity of pm was significantly correlated with plasma cholesterol level, systolic blood pressure and the number of diseased coronary arteries. these results provide further evidence for an association between pm, hc and atherosclerosis. it can be speculated that hc is not only a risk factor for atheroscterosis but also a risk factor for pm and acts by increasing the reactivity of neutrophils and platelets. on the other hand, pm as a mild chronic inflammation could promote the development of atherosclerosis due to effects of endotoxins on vessel wall, blood cells and haemostatic factors. it has been also speculated that phagocyting leukocytes in the inflamed periodontal tissues could contribute to oxidative modification of ldl. so far, there is no evidence that atherosclerosis may contribute to the pathogenesis of pro protein z (pz) is a vitamin k dependant plasma protein synthesized in the liver. it promotes the association of thrombin with phosphorlipid surfaces. recently it has been shown that a deficiency of pz may lead to a bleeding tendency. in patients undergoing chronic hemodialysis, disorders of hemostasis are common. to examine if plasma levels of pz are altered in patients with end stage renal disease we determined pz in plasma of patients at the beginning of hemodialysis treatment. the results were compared with a group of 50 healthy controls. the difference of pz levels in plasma of patients with end stage renal disease with the control group was not significant. control group was 2900 + 487 ng/ml and in patient group was 3133 + -1394 ng/ml. one patient with marked bleeding tendency after hemodialysis pz was 1387 ng/ml. we concluded that patients with bleeding disorders pz determination may be helpful. the normal range of actin fs was reinvestigated in a multicentric approach. a protocol was developed which requests from each center to assess the aptt with one common and one variable lot of actin fs in 50 samples of suspected normals. inclusion and exclusion criteria based upon the results of clotting assays, liver enzymes and clinical data were defined. results: a total of 1056 results was obtained. the majority of centers in this study used the electra 1000 or 1600 c (mla). results for the electra group (n = 6) showed a precision for the common lot of actin fs with a common lot of a three level control from 1.5 % (level 1) to 1.1% (level 3) with an excellent accuracy between the 6 centers. clotting times with the variable lots of actin fs were very similar. the results from normals, however, showed a somewhat higher dispersion using the common lot of actin fs. 4 of 6 centers had almost identical mean values (range 26.6 to 27.2 sue) whereas one reported shorter and one longer clotting times (25.2 and 29.7 sec). results with the variable lots gave almost identical results as the common one. a total of 556 results of all lots gave a normal range of 23.5 to 31.7 (5 -95 % percentiles) on electra. mean values on acl (n = 200) were 26.3, on bct, 27.65 sec, on amga coagulometric, 29.4 sec, on amga turbidimetric, 26.6 sec (n = 100 each). all centers used sarstedt monovettes with 3.2 sodium citrate. discussion: the results of this study demonstrate the lot to lot consistency of all lots of reagents included in this study since the common and variable lots showed very consistent results. interestingly in the large group of electra users the normal ranges showed some differences, though the controls in all centers were almost identical. this confirms the recommendation that a normal range as stated from the manufacturer should be used for orientation only and that each laboratory should assess its own range. direct acting anticoagulant agents such as hirudin (r-h), argatroban (arg), efegatran (efe) and peg-hirudin (ph), represent specific and potent inhibitors of thrombin. blood samples collected in r-h (10 ~g/ml), arg (50 ~tg/ml), efe (25 ~tg/ml) and ph (10 ~tg/ml) do not clot for extended periods (>24 hours), thus allowing for the collection of plasma for analytical purposes. unlike heparin, these agents do not require any plasma cofactor for their anticoagulant effect. in contrast to citrate, oxalate, edta and heparin, these antithrombin agents do not alter the electrolyte or protein composition of blood. thus, blood collected in these agents may provide a physiologically intact (native) sample for clinical laboratory profiling. we have used all of these agents to prepare whole blood and plasma samples for various diuical laboratory measuroments. plasma samples collected with these agents are obviously not suitable for global clotting tests (pt, aptt, thrombin time, fibrinogen); however, these agents are optimal anticoagulants for the collection of samples for the molecular markers of hemostatie activation, such as fibrinogen/fibrin related degradation products, prothrombin fragment, protease cleavage products, tfpi, tnf and other protein mediators. electrolytes, blood gases, enzymes and protein profiling can also be satisfactorily measured on blood samples collected with these agents. antithrombin anticoagelatad blood used fur hematologic analysis showed equivalent blood count and differential results as that obtained with edta blood. unlike other anticoagulants, these agents do not interfere in the cell staining process. washed blood cells can also be prepared using antithrombin aents supplemented buffers for morphologic and fuuctional studies. thrombin inhibitors such as hirudin have also been used for flow cytometry and image analysis of blood cells and tissue exudates. our observations suggest that these anticoagulants can be used as suitable anticoagulants for clinical laboratory blood sampling. these agents can also be used as a flush anticoagnlant fur most automated instruments as these exhibit superior anticoagulant properties to heparin. furthermore, the hematologic parameters obtained in antithrombin anticeagnlated blood may be physiologically more relevant than those determined on blood collected in edta, citrate or heparin. antithrombin ul determination is one of the most popular method for in vitro diagnostic of number of different disorders. human fhrombin a~nity purified on heparin-rnodified silica-based sorbents was used for level of antithrombine lu determination by abilgaard method in blood of 40 patients with pregnancy pathology, acute leukemia, thrombocytopenia and anemia. it was founded, that antithrombin level is decreased to 50-60% of normal values in case of pregnancy pathology, to <50% -in case of acute leukemia and thrombocytopenia, to s0% -in case of anemia. obtained results show the strong relationships between named disorders and patient antifhrombin iii level. therefore anfifhrombine iii estimation may be used as simple and quick method for preliminary diagnosis of above named disorders. bm coasys 110 is a complete automated analyzer system for coagulation tests. it is well suited for routine coagulation testing in random access in a medium throughput laboratory environment. analytical performance and practicability were tested by a common evaluation program in five hospital laboratories. within run and day to day cv's were below 5 % in different samples (controls, patients) . comparison in different therapeutic ranges confirms the declaration of the isi-value for calculating inr-values. normal values for coagulation tests with results in pdmary units were checked in 390 samples and confirmed. due to the optical measuring principle of the bm coasys 110 there was a little tendency to shorter times with the thrombin reagent. in conclusion the performance of coagulation tests with the bm coasys 110 was rated as well or better compared to existing systems in the laboratories with advantages due to short timed familiarizing and easy handling. flexibility and stability of the system permit optimal integration and innovation into the w0rkflow of the routine laboratory. the purified thrombin and antithrombin iii (at iii) have a great interest in the clinical diagnostic and treatment practice, so their isolation methods are very important. molecules of these proteins have some fragments replying for interaction with native glycosaminoglycan, heparin. this interaction is used for isolation and purification of thrombin and at ul from native materials, blood plasma or its fractional products. we have done comparative studying these proteins purification on heparin sorbenfs, which contain heparin immobilized on sificagel, modified by glycidooxipropyl, gamma-aminopropyl and tosyl chloride groups, or on cellulose: heparin-epoxy-silica (1), heparin-gammapropyl-silica (2), heparjn-tozylsilica (3), and heparin-cellulose (4). we founded that thrombin binds with all sorbents, while at iii doesn't binds with sorbents 2 and 3. there wasn't any difference between silica and cellulose sorbents in thrombin desorbfion by t m naci. at iii binds more stronger with heparin-ceuulose t[~,~n with silica sorbents but specific activity and purity degree were approximately the same on both kinds of sorbents. thrombin specific activity and purity degree were approximately twice higher on sorbents 2 and 3 in comparison with sorbents ! and 4 (2250-3000 nih units/mg versus 1260-t350 nih unlts/mg). therefore, sorbents 2 and 3 can be used for isolation and purification of thrombin and sorbents t and 4 can be used for isolation and puriiication of at ii1. we used these sorbents for large scale purification of named proteins. purified thrombin was used for production of diagnostic kits for anfithrombine iii, fibrinogen, fibrin/fibrinogen degradation products and thrombin time determination. after an aerobic or anaerobic physical exercise various alterations of the hemostatic system were detected. numerous investigations of the hemostatic system exist of running and of bicycle ergometer exercise but not of swimming. young volunteers (n=54; median age 25 years) were investigeted~ there was an aerobic exercise (achieved heart rate 130 --10/min, lactate < 4 mmol; n= 27) and an anaerobic exercise (achieved heart rate 150 ~ lo/min, lactate > 4 mmol/1; n= 27). in both groups there was a significant shortening of the ptt. under anaerobic conditions hematocrit and quick significantly increased. factor viii activity rose significantly in both groups. indicating plasmatic clotting activation there was a significant increase in molecular markers tat and f1+2 only under anaerobic conditions (tat from 2,5 to 5,4 pg/1; f1+2 from 0,87 to 0,93 nmol/1). indicating activation of fibrinolysis t-pa activity increased significantly in the anaerobic group (from 0,1 to 0,4 iu/ml) but not in the other group. this findings indicate that there is e balance in the hemostatic system by activation of clotting as well as of fibrinolytic system in young volunteers during exercise by swimming dependend on the degree of exercise load. membranes as well as compact, porous disks are successfully used for fast analytical separations of biopolymers. as far as capacity, speed and performance of separation are concerned, the supports are as effective as other recently developed fast media for the separation of biopolymers °). so far, technical difficulties have prevented the proper scaling-up of the processes and the use of membranes and compact disks for preparative separations. in this report, the use of a compact tube made of poly(glycidyl methacrylate) for fast preparative separations of proteins is shown as a possible solution of these problems. the units have yielded excellent results, regarding performance and speed of separation as well as capacity. the application of compact tubes made of poly(glycidyl methacrylate) for the preparative isolation of the coagulation factors viii and ix from human plasma shows that this method can even be used for the separation of very sensitive biopolymers. in terms of yield and purity of the isolated proteins, this method was comparable to preparative column chromatography. the period of time required for separation was five times shorter than with corresponding column chromatographical methods. our measurements showed an excellent correlation of the two systems (r=0,99). the maximum amplitudes on the roteg were on average 2.2% higher than on the hteg, corresponding to a slightly lower reverse momentum of the measuring system in comparison to the hteg. we report first results out of the evaluation of sta compact (boehringer mannheim/diagnostica stack)). sta compact is designed for automated analyses of routine and special coagulation (chronometfic, photometric [405 nm] and turbidimetric [540 run]) tests. in addition, it does measure ,,derived" fihrinogen. tests as follows were evaluated: prothrombin time (pt), partial thromboplastin time (aptt), fibrinogen (clauss method), thrombin time, at iii (chromogen), hepato quick, as well as the factors ii, v, vii, x, and viii. results: within run cvs of the clotting tests were below 2% (calculated on the basis of seconds) in most cases, day to day cvs below 4% (not measured for factors, yet). at iii yielded within run cvs below 3% in the decision range. measuring ranges: at iii: 20-140 %; fibrinogen: 1.3-9.0 g/l (plasma -dilution 1/20), after rerun with other dilutions: from 0.3 g/1 (dilution: 1/5) to 18 g[l (dilution: 1/40). method comparisons, using sta as reference, yielded slopes close to 1.00 and negligible intercepts. throughput: with routine clotting tests about 100 tests/h, in a sample selective access mode. we conclude, that sta compact allows precise measurement of routine and special coagulation tests. it is also a reliable system for photometric tests and well suited for intermediate workloads as well as stat analyses. we did evaluate ptt lt, a new liquid, silica based ptt reagent. special attention was given to reference interval and heparin sensitivity. the new reagent is well suited for the measurement of intrinsic clotting factors and is reported to have high sensitivity for lupus anticoagulants (higher sensitivity than sta aptt [boehringer mannheim = bm]). it is stable for 4 days in the cooled compartment of the sta analyzer. methods: all experiments were made on sta. for comparison, we used three other ptt reagents (a lab. routine, silica based aptt, as well as sta aptt and sta ptt kaolin from bm). in addition, thrombin time (3 u/ml thrombin, sta thrombin reagent) and heparin (chromogenic xa test, rotachrom heparin) were measured. results: within mn imprecision (n=21) was below 0.7 % cv in the normal range and in two controls (mean values: 35 s, 76 s), and 1.4 % in a heparin plasma (mean: 81 s). between day imprecision (d=10) was below 3% in two controls ( mean values: 34 s and 58 s). the upper limit of the reference range is 40 s (97.5 th perc., median: 32 s; 90 patients with normal coagulation status [routine aptt, fib., pt], median age: 54 years); almost identical reference ranges were obtained with sta aptt and the routine ptt reagent, while sta ptt kaolin showed significantly lower values (97.5th perc.: 33 s, median 28 s). method comparison study: good agreement using plasmas from patients without heparin: (y= 0a + 0.98 x, n= 198, range of(x) from 25 to 79 s, r = 0.97; x = sta aptt). the median values from 54 patients under high dose heparin were: routine ptt: 81 s, sta aptt: 75 s, ptt -lt: 82 s0 sta ptt kaolin 54 s, thrombin time 37 s and heparin 0.5 iu/ml in conclusion, results of the new reagent compare well to our routine ptt and to sta aptt system reagent. it allows sensitive monitoring of high dose heparin therapy and is well suited for detecting abnormalities of the intrinsic clotting factor pathway. is a standard technique since many years. the interpretation of the thrombelastograms has been widely based on phenomenologic observations, while there is a lack of exact information concerning the coagulation mechanisms leading to the teg amplitude (a're~). the aa'ec is a measure for the mechanical stiffness of the clot and depends on: a) fibrin formation and adequate polymerisatiun of a 3-dlmensional network: measurements with nonrecalcified citrated blood activated with adp or epinephrine (both n=10) did not show any clot formation in the teg this relies on the need for a mechanical coupling between the teg pin and cup over a distance of 1 mm, which is accomplished by the fibrin network therefore, teg can only be performed under thrombin formation and thus under thrombin-activation of the platelets in the sample. factors, which inhibit platelet aggregation but don't limit thrombin-activation of platelets, cannot be monitored by teg. b) the attachment of the dot on the surface of the teg pin and cup. according to recent literature we suggest that the attachment of the clot in the teg relies exclusively on fibrinogen/fibrin adsorption to the surfaces of the pin and cup. interruption of this attachment can result in lower amplitudes or the so-called ,,stairway" phenomenon. we could show a complete interruption of the clot attachment by dipping the pin for one second in 30% albumine solution (n=10). c) the fibrinogen concentration (fg) and platelet count (pc) of the sample. in 50 volunteers we found only a poor correlation of the maximum amplitude (ma) with fg alone (r=0.40) or pc respectively (r=0.50), while there was a very good nonlinear correlation to the product of fg and pc. we suggest that the fibrin network forms the main structure of the clot while the thrombocytes enhance its stiffness in a concentration-dependent manner. this effect of the ptatelets can be completely reversed by gplibfllla antagonists. d) adequate coagulation activation: in nonactivated teg even small amounts of inhibitors can lead to a significant reduction of the ateg. conclusion: alterations in teg measurements can be judged more properly when the underlying mechanisms are understood. the consideration of the limitations of the method allows a more specific interpretation of the results. as a response on a customer request we did investigate the sample stability of blood samples for the aptt. the study was set up in a way that simulated the conditions of a large private laboratory in which the samples arrive several hours after blood collection. blood was drawn from 10 donors into 3.2 % sodium citrate and mixed well before it was divided into several aliquots which were kept at room temperature. the aliquots were centrifuged after ~ 0,5, 11, 23 and 29 h after venopuncture and the plasma was analyzed immediately with 3 different reagents on electra 1000. results: there was a clear difference in the change of the apttover time with these reagents. also f viii (determined with a chromogenic assay with complete and standardized activation) change considerably. 2 reagent a: ellagic acid, plant phospholipid, reagent b: sulfatide/kaolin, phopholipids, reagent c: ¢llagi¢ acid, plant and rabbit brain phospholipids the increase of aptt was apparently not a function of the decrease of fviii because the in vitro f viii sensitivity of reagent b. was inferior to reagent a though reagent b showed more prolongation of the aptt than reagent a. reagent c, however, showed only minor changes in the aptt. discussion: these data show that the sample stabifity of the aptt is reagent dependent and that it is not simply a function off viii sensitivity. other factors such as the buffer system but also the sensitivitiy towards other factors than f viii seem to contribute. a comparison of the technical principle of the roteg coagulation analyser and conventional thrombelastographic systems an. calatzis, p. fritzsche. al calatzis, +m. kling, +r. hipp, a. stemberger institute for experimental surgery and +institute of anesthesiology yechnische universit~t m0nchen thrombelastography (teg) was introduced by hartert in 1948 as a method for continous registration of the coagulation process. in 1995 we presented the roteg coagulation analyser, using a newly developed technical method. in teg systems according to i/artert the sample (blood or plasma) is placed in a cup which is alternately rotated to the right and left by 4,75 °. a cylindrical pin, which is suspended freely on a torsion wire, is lowered into the blood. when coagulation starts, the clot begins to transfer the rotation of the cup to the pin against the reverse momentum of the torsion wire. the angle of the pin is electromagnetically detected, transformed to the teg amplitude and continously recorded. in the roteg the pin is attached to a short axis, which is guided by a ball beating. thus all possible movement is limited to rpotation (r_oteg). the cup is stationary, and the pin is rotated alternately by 5 ° to the right and left by a feather system. when a clot is formed, it attaches to the surfaces of the pin and cup and starts preventing their relative movements against the reverse momentum of the feather. here the reduction of the rotation of the pin, which is detected optically, is tranformed to the teg amplitude. as can be shown by theoretical analysis and by control measurements, the roteg provides the same measuring capabilities as conventional teg systems. the main advantage is the solid guiding of the measuring system, which makes the roteg easily transportable and less susceptible to shock or vibration during measurement. yhrombelastography (teg) is a standard monitoring procedure for evaluation of coagulation. usually only nonactivated native blood teg measurements (nateg) are performed, which leads to a) a long time interval until coagulation and fibrinolysis parameters are available b) very high susceptibility of the measurement to inhibitors like heparin, which disturbes the judgement of other components of coagulation, c) unspecific results. our aim was to develop a coagulation monitoring system based on teg providing fast and specific information on the different components of coagulation. methods: the following measurements are performed in paralel using disposable pins/cups (haemoscope): a) extrinsic activated teg (exteg): 3551al whole blood (wb) + 5~tl innovin (recombinant thromboplastin reagent, dade). b) intrinsic activated teg (integ): 3551al wb + 5~tl kaolin (suspension 5g/l, behring). c) aprotinin teg (apteg): exteg + 20 kie aprotinin (trasylol, bayer). d) heparinase teg (hepteg) as decribed in (1). results: exteg and integ provide information on the extrinsic/intrinsic system within 5-10 min and information on the platelet/fibrinogen status within 10-20 min. because of the addition of potent activators integ and exteg can be performed when inhibitors like heparin are present in the circulation. fibrinolysis effects can be seen on exteg and integ and by comparison of exteg and apteg (apteg: invitro-fibrinolysis inhibition by aprotinin). if fibrinolysis is detected by exteg or integ and aprotinin-susceptibility is verified by apteg, aprotinin therapy will be initiated. heparin effects are revealed by hepteg. discussion: by the comparison of parallel teg measurements which have been differently activated, specific and fast information on the different aspects of the clinical coagulation status is provided. the presented tests can be easily performed bedside and only a small specimen of whole blood is needed (0,4-1,8 ml). introduction: a severely prolonged aptt (333s; normal: 40~os) was observed during preoperative screening for a planned splenectomy in a 71-year-old man with an 8 year history of osteomyelofibrosis. fellewing neer-normal~atien (71s) of the ap'ci" after 10 rain preincubation in a kaolin based aptt assay, pk deficiency was suspected and studies were performed to further investigate the nature of the pk deficiency as well as the mechanism underlying the normalization of the prolonged aptt by increasing the preincubation time. methods: the apl-r assay was peal'armed using kaolin/inesithin. high molecular weight kininogen clotting activitiy (hk:c), fxii:c end pk:c were measured by an aptt based assay using neothromtin ® (behnng) and 1 rain (pk:c) or 4 min (hk:c, fxli:c) preincubation. pk amidolytic activity (pk:am) was assayed using cosset pk ~ (chromogenix) and pk antigen (pk:ag) by quantitative immunoblotting. fxll and hk proteolysis dunng activation of plasma by kaolin (10mg/ml at 37=c) or ds (12.5~tglml at 4=c) was demonstrated by immunotilotting assays of fxii and hk following sds-page. assay pk:c pk:am pk:a~i fxfi: the propositus had pk:c<5%, pk:am=5% and pi~ag<2.5% as compared to normal pooled human p(asma (nhp). his son and two daughters had pk:c-50% and normal aptt values, incubation of the propositus' plasma with ds did not result in fxii or hk cleavage within 180 rain, whereas jn nhp detectable f×ii and hk proteolysis occurred after 5 rain and complete proteolysis was observed after -120 rain. in contrast, kaolin activation of propositus' plasma led to slow activation of fxii after 10 rain, presumably by autoactivation, and to fxlla-induced hk proteolysis. near-normalization of the propositus' aptt by prolongation of the preincubation time paralleled fxii autoactivation as evidenced by immunobletting. we describe a propositus with severely prolonged aptt due to hereditary, crm negative pk deficiency suffering from omf. activation with a particulate suspension of kaolin led to slow fxii autoactivation and hk proteolysis, whereas ds in solution did not induce fxii or hk cleavage. fxii autoactivation seems to be responsible for the normalization of the prolonged aptt in pk deficiency after prolonged preincubation times. in our study we compared a conventional bag with silicone tubing (a) for blood donation with 2 new ones (]3 from biotrans and c from baxter) with a newly developed y-shaped adapter. this adapter is integrated into the tubing and therefore provides the advantage for drawing blood samples in a closed system. the 3 systems were identical in amount and content of anticoagulant, i. e. 63 ml of cpd per bag resulting in approximately 14% of the final whole blood volume. the purpose of the study was to determine whether the different tubings can influence the quality of plasma products conceming the blood coagulation system. in plasma samples we measured several factors of the procoagnlatory and fibrinolytic systems. intralndividual control eitrated (.135 m) blood samples were initially drawn from the contralateral cubital vein from the same male donor (34 in each group). in all bag samples we found small but significantly higher levels of the global test parameters ap'it and ti" compared to controls, indicating a higher amount of anticoagulant. pt, however, revealed no differences, thus suggesting that factor activities were not altered (statistics according to mann-whimey). increase of procoagulatory activity measured as tat complexes showed elevated levels in bags a and c whereas prothrombin fragments fl+2 decreased only in a. conceming the fibrinolytic system, plasminogen a~tivators and pai-1 values were diminished in all three systems 03 < a < c) compared to controls. d-dimers were lowest in a followed by slightly higher values in c, controls and b. fibrin monomers did not reveal any significant differences: a < c < controls < b. in summary, the quality, of the 3 different blood sampling devices was comparable to the intraindividual controls as to factor activities measured by global tests. the activation of the procoagulatory and fibrinotytic systems was slightly but in most cases significantly higher in the two new devices than in the conventional one. all values, however, obtained from the plasma samples did not exceed the normal range of healthy blood donors. therefore we concluded that the two new closed blood drawing systems are favorable for blood donating procedures. in 20 patients with acute myocardial infarction (ami) and thromholytie therapy (13 patients with rt-pa, 6 patients with streptokinase and one with heparln) with ck, myoglobin and ekg criterions the patients were divided in two groups (reperfusion/no fellow two hours after starting the thrombolytic therapy) . blood samples were taken before, 30 rain, i h, 2 h, 4 h, 8 h,12 h after lysis and than every day till day 10. because of the central role of factor xii in activation of coagulation, fibrinolysis, kallikreln-kinin-system and complement cascade we investlgate the role of factor xila initiated by ami and the relation of factor xiia to the thrombolytie agent and reoeclusion rate. for the investigatlens we take the kits from shield diagnostics (xiia), behring diagnostica (c~-inactivator, pl~nogen, ~-antip]~n~n, pap), chromogefiilx ab (prekallikrein) and di~nostica stage (vile). the results: there is an increase of factor xiia immediately after starting the fihrinolysis (max. 30 rain after starting); the increase 5/i independently of the thrombolytie agent. parallel to factor xiia raises factor viia without significant changes of c1-1naotor and prekalllkrein. that means: activation of xiia and fibrinolytic pathway leads to relatively mild c.hanges in kallikrein system, hut to significant activation of extrinsic system by vila-tissue factor. in some patients is an additional rise in the system xiia -viia, when the fibrinolytic system is already in the normal range. there will need further investigations to define the risk of reocclusion as a result of activation of faktor viia by faktor >li ia. autoimmune thrombocytopenic purpura (aitp) is a frequent complication of chronic lymphocytic leukemia (cll] which developes on different stages of the disease and needs special treatment measure. mechanism of autoimmune disorders in cll remains uncleared. we investigated immunologic phenotype of blood lymphoid cells in 22 patients suffering from cll with aitp. in these patients we did not observe disorders in expression of b-lineage markers as compared with cll patients without immune complications (13 patients). but in the 1st group of the patients the greater number of b-celts expressed markers of activation. according to ig heavy chain expression, the lymphocytes in most cases of cll complicated by aitp had more mature phenotype. in all patients with k phenofype of cll lymphocytes we found immune disorders. the development of aitp was accompanied by lowered level of t cells and changed dis'flibution of their immunoreguiatory subsets: diminished number of cdz~cells and increased one of cd~'÷lymphocytes. the results of our investigations undirectly proved that malignant b-cells in cll are involved in production of autoantibodies against blood cells. dysbalance in t-cell system with functional disturbances of immunoregulation are significant in development of autoimmune complications in cll a24 in women with severe fvii deficency (<10%) hypermenorrhagia may cause life threatening blood loss. therefore, hysterectomy at a young age is reported frequently in the literature. a 12 year old girl without history for a bleeding disorder was transfered with hypermenorrhagia. the initial laboratory data revealed an abnormal quick-test of 30% due to fvll of 9,0%, normal platelet count and hemoglobin level of 7,2 g/dl. antifibrinolytic therapy (tranexamic acid 4x15mg/kg bw/d) and lynestrenol substitution were started to reduce the hemorrrhage. despite treatment the daily blood loss increased to a maximum of 290ml. therefore, substitution therapy with recombinant fvila (rfvila) (novonordisk) was started at a dose of 15 ilg/kg bw q 6 h. subsequently blood loss decreased to 30ml/d, but even with an increasing dose of rfvlla up to 35 i~g/kg bwq 4h (fvil activity max. 7400% 10 min after injection) and additional hormonal support with a lh-fsh-anatgonist some hemorrhage remained. a short .course of methergin was stopped due to severe pain. ultrasound of the uterus revealed a hypertrophic endometrium causing the persistent bleeding. it decreased slowly over several weeks and hemorrhage stopped completely after 40 d. the total rfvlla dose administered was 118 rag. no side effects were observed. no transfusions of blood products were necessary. currently, menstrual cycle is suppressed by estriosuccinate. conclusion: due to close cooperation with a specialised gynecologist, hypermenorrhagia was controlled and in this woman with severe fvll deficiency hysterectomy was avoided. in three male members aged between 27 and 52 years of a family suffering from inherited bleeding disorders the diagnosis of protein z deficiency was established. plasma protein z evaluated by elisa (asserachrom protein z, diagnostica stago, france) ranged between 200 and 300 ng/ml. the patients mostly suffered from moderate bleeding complications like prolonged bleeding secondary to trauma or invasive measures and also spontaneous hematuria. previous laboratory investigations revealed variable platelet function deficiencies and transitory boderline decrease of von-willebrand factor. spontaneous bleedings were rarely recognized, however, they occured more frequently when analgetics were taken. bleeding complications showed good response to hemostyptic measures and antifibrinolytic therapy. the use of pcc containing a high level of protein z in these patients is restrained to severe bleeding disorders or major surgery. defibrotide is a mammalian polydeoxyribonucleotide derived anti-ischemic and antithrombotic drug (crinos s.p.a., v"flla guardia, italy). while the drug is known to produce polytherapeutic effects owing to its multicomponent nature, the exact mechanisms of its anti-ischemic effects remain unknown at this time. since defibrotide is found to be effective in ischemic disorders such as paod, vod related occlusive disorders and related rnicroangiopathic conditions, we studied the effect of this drug on the contraction of dog and pig arterial strip/rings obtained from various sites. in vitro supplementation ofdefibrotide to the organ bath containing control dog and pig arterial rings did not modulate the serotonin and thromboxane (generated) contraction, however, tissues obtained from dogs treated with 10 mg/kg defibrotide iv exhibited a profound desensitization to the agonist induced contractile process. the time course of these effects was found to be much larger than the plasma half-life of defibrotide. this presentation will provide additional data on the effect of defibrotide on the contraction of vascular smooth muscles as a possible explanation for the anti-ischemic effects of defibrotide. a. wehmeier, a. popescu, w. schneider klinik for h,~matologie, onkologie und klinische immunologie der heinrich-heine-universit&t d0sseldorf in chronic myeloid leukemia (cml), evolution of blast crisis is the limiting factor of survival. however, as in other chronic myeloproliferative disorders, bleeding and thrombotic complications are a major source of morbidity but their incidence has rarely been analysed in larger patient groups. we retrospectively evaluated 182 patients with cml during chronic phase (170 cases), accelerated disease (58 cases), and blast cdsis (72 cases), and determined the incidence of thrombohemorrhagic complications in relation to the stage of the disease. in chronic phase, 28 patients had bleeding complications (8.4%/patient year) and 15 patients thrombotic episodes (4%/patient year). the incidence of bleeding increased significantly in accelerated disease (18 patients, 51.2%/patient year) and blast crisis (37 patients, 347%/patient year), and many patients had repeated complications. contrary to our expectations, the incidence of thrombotic complications also increased to 10.2%/patient year in accelerated phase and 39.8% /patient year in blast crisis, tn chronic phase, 3 patients died because of bleeding events. in accelerated phase, 5 patients died due to bleeding and 1 patient due to thrombotic complications. in blast crisis, bleeding was associated with 21 deaths, and pulmonary embolism with 2 deaths. analysis of the cause of thrombohemorrhagic complications revealed that in chronic phase, bleeding was often associated with uncontrolled busutfan therapy, whereas in blast crisis, severe bleeding occurred mainly when platelet counts were low and peripheral blasts increased. however, there was no obvious explanation for thrombotic complications. we conclude that bleeding and thrombotic complications are a major source of morbidity and mortality also in cml, and that the incidence of such complications increase in advanced stages of the disease. klinik for innere medizin °, klinikum schwerin patients suffering from primary or secondary amyloidosis may occasionally acquire a coagulation disorder characterised by isolated factor x deficiency. we report on a 60-years-old man who presented with lower gastrointestinal bleeding and prolonged prothrombin time (quick 50 %). amyloidosis was suspected and proven using biopsy of the rectum and histological analysis. in addition, a monoclonal gammopathy of undetermined significance was diagnosed by immunofixation (light chain, type x). detailed investigation of the prolonged prothrombin time led to the discovery of a pronounced factor x deficiency (residual activity 4 %). inhibitors of coagulation factors could not be demonstrated. the treatment of the patient consisted of red blood cell transfusion and infusion of prothrombin complex concentrates. due to the extremely rapid clearance of infused factor x, no increase of its activity was observed. chemotherapy of the monoclonal gammopathy was initiated (melphalan/ prednisone). over the following six months the frequency of major bleeding episodes gradually decreased. however, subclinical occult bleeding continued. the factor x activity was repeatedly found between 10 and 12 %. we support the suggestion from literature data that clinically relevant bleeding episodes are likely to occur in patients with amyloidosis-associated factor x deficiency if the residual activity is below 10 %. sepsis and septic shock is a disease entity which is characterized by inflammatory reactions (sirs), coagulation abnormalities (dic), organ failure (mof) and severe hemodynamic alteration frequently leading to death in a shock. the aim of our studies was to investigate the efficacy of antithrombin iii (kybernin ®) on ~he outcome of septic shock in a pig endotoxemic model. pigs, in this model respond to lps with elevated tnflevels, decreased leukocytes and platelets counts, increased tat and fibrin monomer levels, hypotension and in increase of the pulmonary arterial pressure (pap), indicating impaired lung function. a total number of 13 male castrated juvenile domestic pigs (25 -30 kg) were anaesthetized, ventilated mechanically and infused with saimonella abortus equi lipopolysaccharide (s. equ-lps) over three hours (0.5 ~g/kg * h). a swan-ganz-catheter was inserted into the pulmonary artery to measure the pap. animals were allocated to two groups,, the treatment group (n = 7) received antithrombin iii (at iii) according to the following regimen: 250 u/kg (t = 60 -30, i. v. infusion), 125 u/kg (i. v. bolus, t = 0) and 250 u/kg (t = 180 -240 rain, i. v. infusion). the placebo group ( n = 6) received the appropriate amount of human serum albumin: 50 -25 -50 mg/kg (same schedule as with at iii). main objective was defined as the mortality rate at six hours a_~er s. equ-lps infusion. whereas in the placebo group 4 out of 6 animal died (mortality rate: 66 %) all at iii-treated pigs survived the observation period of 6 hours (p < 0.05, x2-test). the at iii group was shown to have a lower pap than the control group, especially the second peak of hypertension was abolished by at iii. it is therefore concluded that at iii should be a useful tool for the treatment of severe sepsis and septic shock. in a nationwide monthly survey all childrens hospitals in germany (esped) were asked to clinical and therapeutical informations about children suffering from pmi. during july 94 till june 95 299 children were registered. from these, 87 had either ecchymoses and/or necroses related to an increased mordibity and mortality (20%), whereas 212 showed no bleeding signs except for petechiae. of these children one died. the therapeutic interventions concerning hemostasis are listed according to the defined two risk ~oups. from the patients with ecchymoses or necroses, 13/87 received combination therapy (compared to 5/212 with petechiae or no bleeding sign) of at iii, heparin and/or plasma. only t child received protein c concentrate. the data show that children with low risk did in part receive higher doses of heparin and/or at iii concentrate than did high risk patients, whereas plasma therapy was adjusted to severity of eoagnlopathy. furthermore, the wide range of given therapeutics allows no information about the different medications. therefore, controlled studies with respect to the different therapeutic interventions in children with high risk pmi is desirable. a fully automated procedure for the reptilase time assay y. schmitt (1) and h.j. kolde (2) (1) institute for laboratory medicine, st~dtisches klinikum, darmstadt, frg, (2) dade diagnostics, unterschlei6heim the reptilase time assay is a relatively simple technique for the detection of fibrinogen degradation products and fibrinogen deficiency or abnormality. the procedure is performed with citrated plasma and batroxobin reagent, a snake venom enzyme from bothrops atrox. this enzyme cleaves fibrinogen by releasing fibdno peptide a only but not fibdno peptide b. in contrast to the physiological enzyme thrombin that is readily neutralized by antithrombin iii and hepadn batroxobin is not inactivated by physiological inhibitors. at present this assay is mainly performed manually or on mechanical instruments. we have adapted this assay to the electra 1000 fully automated coagulation analyzer (medical laboratory automation, pleasantville, n.y.) using the thrombin clotting time procedure in the instrument software with batroxobin reagent (dade diathe clot formation is registrated turbidimetrically and the dotting time is pdnted. the within run precision (n= 10) of this procedure was tested with two plasmas from the daily routine and was between 2.8 and 3.4 %. in 25 normal samples we found clotting times from 10.5 to 12.8 sec. in 30 samples with liver disease (confirmed by pseudochlinesterase < 2000 u/ml) or on thrombolysis therapy with streptokinase or urokinase the fully automated assay on the electra was compared to the semiautomatic method using a kc 10 coagulometer (amelung, lemgo, germany) based on a rolling metal ball pdnciple and magnetic endpoint detection. the two assays agreed very well with a correlation coefficient of r = 0,948 and a regression line according to passing and bablok of y = 1.0 x + 1.7. these data show that the reptilase time can be performed with good precision and with good correlation to the manual technique on mechanical instruments on the electra 1000. introduction: disseminated intravasal coagulation (dic), due to a massive activation of the coagulation system, is frequently observed in intensive care patients suffering from severe underlying diseases. laboratory diagnosis of dic is based on different coagulation tests, but unfortunately the routine haemostaseological parameters react with latency in the course of acute dic objective: in four cases from a cohort of 43 patients with severe sepsis and dic we analysed special haemostaseological parameters (tat, f1-t2, d-dimers, human-leucocyte-elastese (file), catepsin g and heparin cofactor ii (hc ii)) and correlated them with a mof-score in order to test their predictability on the prognosis of these patients. results: all patients were substituted with at iii concentrate. l,1 the investigated patients median time of treatment with at iii concentrate was 8 (6-9) days and median time of dic-duration was 6 (4-8) days. none of the presented patients died during observation period. all analysed parameters, except d-dimers, showed a sufficient correlation with the evaluated mof-score (tat: r= 0,78; f1-f2: r= 0,84; hle: r= 0,71; catepsin g: r=-0,75; hc ii: 1"=-0,88). the d-dimers did not correlate with the mof-score, which is probably due to the delayed reactive hyperfibrinolysis in the course of dic. furthermore, the decrease of the tat-complexes, f1-f2, hle and catepsin g levels were followed by an increase of at hi and hc ii activity. conclusion: in general the analysed activation markers and coagulation parameters are sufficiently to describe the ongoing process of the dic. the hyperfibrinolytic activity of dic is sufficiently represented by the d-dimer test, but is of defered reactivity in the course of dic. unfortunately these parameters are not established in the routine monitoring of dic on intensive care units and therefore further studies are needed to investigate the practicability and reliability in the daily routine monitoring. we have previously reported that notoginsenoside r1 (ng-r1) has an effect on counteracting lipopolysaccharide (lps) induced upregulation of plasminogen activator inhibitor-1 and tissue factor expression in cultured human umbilical vein endothelial ceils in vitro and in mice in vivo [fibrinolysis 1994;8:(suppl 1)119]. in this study we investigated the effect of ng-r1 on prevention of lps induced lethal toxicity in mice. because mice are relatively resistant to lps when applied as a single agent, we sensitized them by simultaneous treatment with d-galactosamlne. the 80% lethality induced by lps (1.5 mg/mouse) plus d-galactosamine (8 mg/mouse) in c3hs-ie mice was reduced to 16% by simultaneous administration of ng-r1 (1.5 mg/mouse) with lps/galactosamine (p<0.05 by x 2 test). ng-r1 also significantly delayed lps/galactosamine induced lethal toxicity from 12 hours to 30 hours with all animals surviving beyond 30 hours. because lethality induced by lps involves the synergistic effect of multiple effector molecules such as tumor necrosis factor (tnf)-ct, interleukin (il)-i, interferon 3' etc., we also investigated the effect of ng-r1 on lps induced tnf-ct production from leukocytes in cultured human whole blood cells (hwbcs) ex vivo. the production of tnf--ct induced by lps (1 ng/ml for 24 hours) in the supernatant of hwbcs was inhibited by 46% and 22% respectively, when the cells were incubated 1 ng/ml or 10 ng/ml lps together with 100 i~g/ml ng-r1, respectively (tnf-ct concentration, 1 ng/ml lps treated cells: 297+192 pg/ml, i ng/ml lps plus 100 l.tg/ml ng-ri treated cells: 162+137 pg/ml, p<0.01; 10 ng/ml lps treated cells: 3094_+487 pg/ml, 10 ng/ml lps plus 100 pg/ml ng-r1 treated cells 2423+713 pg/ml, /'=-0.02). the present results suggest that ng-r1 can prevent the onset of lps toxicity as well as the lps induction of cytokines. therefor ng-ri may be effective in preventing the effects of septic shock in gram-negative infections. to elucidate the mechanisms by which coagulation is initiated in septic patients in vivo, coagulation measurements were prospectively evaluated in patients with severe chemotherapyinduced neutropenia. this group of patients was chosen because of their high risk of developing severe septic complications, thus allowing serial prospective coagulation testing prior to and during evolving sepsis or septic shock. 62 patients with febrile infectious events were accrued to the study. of these, 13 patients progressed to severe sepsis and an additional 13 patients to septic shock. at onset of fever, factor (f) vlla activity, f vii antigen and antithrombin iii (at iii) activity decreased from normal baseline revels and were significantly lower in the group of patients who progressed to septic shock compared to those that developed severe sepsis (medians: 0.3 versus 1.4 ng/ml, 21 versus 86 u/dl and 45 versus 95%; p < 0.001 ). the decrease of these variables in septic shock was accompanied by an increase in a marker of thrombin generation like prothrombin fragment 1 + 2 (medians: 3.6 versus 1.4 rim; p=o.05). these differences were sustained throughout the septic episode (p < 0.0001 ). f vlla and at ill levels of <0.8 ng/ml and <70%, respectively, at onset of fever predicted a lethal outcome with a sensitivity of 100 and 85%, and a specificity of 75 and 85%, respectively. in contrast, fxila-alpha antigen levels were not different between both groups at onset of fever and were only marginally higher further during the course of septic shock (p=o.001). thus, septic shock in neutropenia is associated with significant coagulation activation, presumably driven by the tissue factor pathway rather than the contact system. furthermore, in septicemia both f vlla and at iii measurements are sensitive markers of an unfavourable prognosis. hemostatic parameters in sepsis patients treated with anti-tnfct monoclonal antibodies c. salat 1, p. boekstegers 2, e. holler 1,3, b. reinhardt i, r. pihusch 1, k. werdan 2, m. kaul 4, t. beinert 2, e. hiller 1 med. klinik iii 1 und i 2, klinikum grosshadern der ludwig-maximilians-universitat monchen, h~imatologikum der gsf 3, knoll ag ludwigshafen 4 tumor necrosis factor et (tnfc~) is a central mediator in the pathogenesis of sepsis and septic shock. as administration of anti-tnfct monoclonal antibodies was able to protect animals from an otherwise lethal endotoxin challenge clinical studies were initiated in patients with sepis. tnfct exerts a procoagulant effect, e.g. by enhancing pai-i and activating thrombin as indicated by an increase in tat and pf 1/2 levels. therefore it may be involved in disseminated intravascular coagulation in sepsis. we determined tat, pf 1/2, d-dimers, tpa, upa, pai-i and vwf levels in 30 patients with sepsis or septic shock. 14 patients received the anti-tnfa monoclonal antibody mak 195f (knoll ag, ludwigshafen), whereas 16 patients served as controls. we found a significantly lower level ofupa in anti-tnfc~ treated patients. since the difference existed before onset of treatment it can not be attributed to tnfot antagonisation. all other parameters investigated did not differ significantly between the two groups throughout the study period. failure to detect modulation of hemostasis by anti-tnf~ might be explained by delayed initiation of treatment in clinical sepsis. in animal experiments it has been observed that the antibody prevented lethal endotoxin effects when given prophylactically or 30 minutes after endotoxin challenge, but not when it was administered 2.5 hours later. in addition, beneficial clinical and hemostatic effects of tnfet antagonisation might be observed only in subgroups of patients with hyperinflammatory sepsis. larger studies addressing this point are under way. protease receptors for thrombin and trypsin have been described for different cell lines. we investigated the ability of trypsin to activate human umbilical vein endothelial cells (huvec). cell activation was measured by the increase of intracellular free ca 2* (caff) with help of microscope fiuorometry (fura-2) and by the von willebrand factor release measured by a sandwich elisa. incubation of huvec with thrombin (1u/ml) or trypsin (10nm) showed a 2-10 fold increase of c~ff. a subsequent homologous stimulation after 80 s lead to a 2-5 fold lower concentration of ca~ 2÷ compared to the first stimulation. therefore cells have been desensitised by the first stimulation. inhibition of the proteolytical activity of trypsin by soybean trypsin inhibitor was followed by failure of trypsin inducing an increase of ca~ 2÷ concentration. in cross stimulation experiments with thrombin and trypsin, we could demonstrate, that cells first stimulated with thrombin showed a second maximal response by subsequent stimulation with trypsin. the same effect was measured with first stimulus trypsin and second stimulus thrombin. trypsin and thrombin induced a release of von willebrand factor (2-5 fold in comparison to unstimulated cells). we found a vwf release dependent on the concentration of trypsin similar to thrombin. an electrophoretic analysis of the released von willebrand factor showed a different multimeric composition of vwf between trypsin and thrombin stimulation. these results indicate, that there might be a protease receptor on huvec for trypsin being different from the thrombin receptor. clinical and laboratory findings of coagulopathy were investigated by an 1-year-survey to 320 children's hospitals. 291 meningococcal infections were evaluable. severe disease (characterized by need for mechanical ventilation, dialysis and/or catecholamines) was seen in 42 of these children; 29 of those survived and 13 died. clinical signs of severe coagulopathy were seen in 83 children: ecchymoses (n = 73) and skin necrosis (n = 36) were associated with increased mortality (16% and 20%, resp., compared to 4.5% overall mortality). five of 29 surviving children with skin necroses required surgical interventions (skin transplantation and/or amputations). petechiae were frequent (n = 156) and as isolated finding not related to severe disease or fatal outcome (6% mortaliy). platelet counts at admission were lower in non-survivors (10th-90th percentile: 30 -450.000/gl, median: 139.000/i.tl) than in survivors (10th-90th percentile: 140 -480.000/i.tl, median: 242.000/gl). at iii values showed no difference between survivors and non-survivors. protein c was available in few patients (n =14): in this subgroup, protein c was lowered in patients with limited disease (10th-90th percentile: 20 -105%, median: 48%) as well as severe disease (10th-90th percentile: 30 -75%, median: 60%). in conclusion, the findings "ecehymoses" and "skin necroses" were related to fatal outcome and therefore included in a prognostic score for severity of meningncoccal disease. the influence of irradiation on pai-i and vwf levels in human umbilical vein endothelial cell cultures k. fragiadaki, c. salat, r. pihusch, b. reinhardt, m penovici, e. hiller med. klinik iii, klinikum grosshadern der ludwig-maximilians-universitat monchen an elevation of pai-1 in bone marrow transplant recipients developing veno-occlusive disease (vod) of the liver has been described earlier. endothelial cell damage due to the preparative myeloablative radioehemotherapy is supposed to be an important step in the pathogenesis of the disease, which is characterized by an obstruction of small intrahepatic venules. in order to investigate a possible role of irradiation we studied the influence of several doses (0, 5, 15, 30 gy) on pai-1 and vwf levels in the supematant of human umbilical vein endothelial cell cultures (huvec). pai-1 antigen and vwf were determined by enzyme immunoassays. whereas pai-1 and vwf levels remained unchanged alter irradiation with 5 gy and in control cultures, a rise was observed one day after irradiation with 15 gy (mean day 0"-)day +1) in pai-1 (100,0% --)171,2 %) and vwf (100%--)159,7%) levels. the increase was more pronounced and reached levels of statistical significance after a dose of 30 cry (pai-1 100%--) 278,7% and vwf 100%--)168%). both pai-1 and vwf levels decreased on day 2 after irradiation with 15 and 30 gy. our results indicate that irradiation induces an increase of pal-1 and vwf in endothelial cells. nevertheless, this effect was observed only in doses above those ones used during conditioning when patients receive 3x4 gy. additional factors seem to be of significance. cytokines like tnfo~ enhance pai-1 and vwf in endothelial cell cultures and are known to be elevated in bmt-associated complications. it can be speculated that irradiation in concert with these factors may contribute to the development of veno-occlusive disease. disseminated intravascular coagulation is characterized by high consumption of coagulation factors, systemic elevation of fibrinolysis by tpa and concomitant elevation of pai-i secreted from inflamed endothelial cells. in an attempt to investigate the contribution of inflammatory cytokines, endothelial cells lines of microvascular origin were stimulated in vitro and pal-1 antigen was measured 2h, 4h and 24h after stimulation. in contrast to results published from experiments performed with macrovascular human umbilical vein cells (huve), our results obtained with 3 different microvascular endothelia isolated from skin, solid tumor tissue and bone marrow revealed that inflammatory cytokines reduced pal-1 antigen levels. in addition to tnf-a (25ng/ml) and lps (10pg/ml), we found that il-10 (100 u/ml) and gm-csf (100 u/rot) also reduced pai-i levels within the first 2h of incubation (from 120ng/ml to 80-110 ng/mll and the effect was even more pronounced after 4h and 24h (from 380 ng/ml to 250 ng/ml). il-1 (10 u/ml) and lps (10 pg/l) also reduced constitutive levels of pal-1 but the effect occured later than 4h after addition of the stimulator. the strongest synergistic effect was demonstrated with gm-csf plus il-1 resulting in pal-1 suppression of 50% after 2h and 30% after 24h. in contrast, g-csf (300 u/ml) induced the immediate (120 to 140 ng/ml after 2h and 380 to 420 ng/ml after 24h) upregulation of pal-1 antigen. stimulation of pat-1 levels was also observed with tgf-i~ (10 pg/ml), however not earlier than 18h of incubation. interestingly, both stimulatory cytokines, ie. g-csf and tgf-13, alone were able to counteract the decrease of pat-1 antigen by tnf-a but only a combination of g-csf plus tgf-g neutralized the effect by il-1. results indicate that inflammatory cytokines regulate pal-1 fibrinolysis in a synergistic and antagonistic fashion. we established the culture of human brain microvascular endothelial cells (hbmec) in order to investigate the pathophysiology of hu~man cerebral malada, which is still associated with a high mortality rate. it is widely accepted that among the reasons for the fatal outcome of cerebral malaria, the interaction of endothelial cells with cytokines and paras lites with subsequent changes in haemostaseological parameters is involved. the human microvascular endothelium may therefore play a deci §ive role in the pathophysiology of cerebral malaria. ery throcytes containing later stages of p. falciparum specifically bind to capillary ec in vivo (sequestration). tnf-cq il-1 and il-6 are considerably elevated in severe malaria. coagulation factors such as tissue factor and von willebrand factor are affected by malada suggesting the involvement of the hbmec in cerebral malada. so far, research on the involvement of the hbmec has been performed on ec cultured from human umblilical veins (huvec). the relevance of this model may be questioned on t, ,he grounds that the capillary endothelium probably plays a greater role than the endothelium of the large vessels. besides, some propertie.$ of the endothelioum seem to vary, upon the organ of origi/n. for the~ reasons, our laboratory has established the hbmec as a model to study the pathophysiology of human cerebral malaria. to demonstrate the relevance of this model in the context of malaria, hbmec were challenged with sera from different patients with severe p. falciparum malaria and with serum from a healthy donor. we can demonstrate that in cells challenged with malaria patient sera icam-1 and substance p were upregulated. on the other hand cells challenged with serum from a healthy donor expressed neither icam-1 nor substance p. these results strongly suggest the relevance of this model for vessel involvement in malaria. both, histamine and serotonin have been described as potent stimulators of yon willebrand factor (vwf) release from human umbilical vein endothelial cells (huvec). we performed experiments to differentiate the receptors for histamine and serotonin induced vwf release. absolutely unexpected we don't found any significant vwf release after the addition of serotonin to huvec or human artery endothelial cells (huaec) in concentrations from 0.1 ijm to 50 pm. in the case of histamine (0.1 pm -50 pm) we measured a vwf release 2-5 fold compared to unstimulated cells. this release was in the same order of magnitude as the release induced with 11u thrombin. to verify these results we measured the effect of histamine and serotonin on the intracellular ca 2÷ concentration (ca~ 2÷) in huvec and huaec. cells were labelled with fura-2 and the change in fluorescence after agonist addition was measured with a microscope fluorometer. using the same agonist concentrations as above we found an 5-10 fold increase of caj 2. with histamine or thrombin but no effect by addition of serotonin. this results indicate a similar activation of human endothelial cells by histamine and thrombin and that serotonin don't stimulate endothelial vwf release or increase of cay. activation and/or dysfunction of the endothelium can be triggered by cytokines (e.g. interleukin-2, tumor necrosis factor-alpha) or bacterial substances (e.g. endotoxins) and may contribute to shock and multi organ failure. pal-l and tm were assessed as parameters of activated endothelium following bsct in three to four days intervals from start of conditioning therapy through day +35. data were compared to the occurrence of sepsis, veno-occlusive disease (vod), capillary leakage syndrome (cls) and graftversus-host-disease (gvhd). patients with neither complication served as controls. no *days after stem cell tranplantation pai-1 and tm were increased in all patients with sepsis, cls~ vod and/or gvhd. pai-1 peaked at days 14 to 18 and the increase was highest in sepsis and lowest in cls. the increase in tm values was somewhat delayed (day +24) and was highest in vod and cls and lowest in gvhd. pai-1 and tm are sensitive markers of endothelial activation in sepsis, vod, cls, and/or gvhd, but they do not allow a differention between these complications. endothelin (et) is the most potent vasoconstrictor. it is known that et plasma concentration is correlated with a poor prognosis in patients with non ischemic cardiomyopathy (cm). the contribution of the heart to the production of et is still unknown. to investigate the pathogenetic mechanism in patients without coronary artery disease (cad), we examined 13 patients with hypertension ( . pulmonary capillary wedge pressure (pcwp) was measured in all patients. et and its precursor big-endothelin (bet) were determined at rest and after pharmacological stimulation with dipyridamole (0.5 mg/kg body weight), that increases coronary blood flow by factor 2 -4 on a non endothelial pathway. cardiac coronary et and bet concentrations were determined from the arterial blood samples, obtained from the aorta, and simultaneously from the coronary sinus (venous blood). blood samples were collected into ice chilled vacutainer tubes and stored after centrifugation at -70 *c. et and bet were analysed after extraction by a sepal< c 18 cartridge by radio immuno assay technique (immundiagnostik). it is concluded that et is increased with elevated filling pressures of the heart in patients with cm. it is not produced in considerable quantity by the heart neither at rest nor at increased blood flow. there4ore the lung has to be considered as the major organ for the production of et and bet in patients without cad. to characterize the incompatibility of blood with foreign surfaces valide in vitro methods especially in testing of platelet function are neceessary. it seems to be effective to use test systems which can also be helpful lateron in the clinic when foreign surfaces (e.g. venous catheters) are used and evaluated in so called phase-4-studies. we studied the influence of 21 reference polymers under standardized and controlled flow conditions on platelets in citrated blood specimen of healthy blood donors.the following tests were performed pre and post platelet-pol)aner contact: decrease of platelet count, platelet aggregation (wu-gmtemeyer index), analysis of platelet spreading capacity on standardized plastic surfaces by using a visual microscopic evaluation according to breddin and bfirck (1963) and an interactive computer-aided system (ibas, kontron gmbh, manchen, frg) by digitalizing the morphological picture of the platelet slides and area detection with a resolution of 512x512 pixels. results: platelet counts showed significant differences pre and post polymer contact, the wu-grotemeyer index demonstrated platelet activation only by blood contact with large volumes of polymeric material whereas both visual and computer-assisted evaluation of platelet spreading ability revealed a marked shift in the different classes of platelets: platelet activation results in a decrease of large structural elements and an increase of elements with spider threads. (pre contact (n=1000): 27:~-6 large forms of platelets, 700~-39 small forms and 275:l-41 spider forms; post contact (n=1000): 6-+-5 large forms, 510a:56 small forms and 484±58 platelets with spider threads). in some series there were significant differences between visual and computer-aided evaluation in the detection of small and spider forms. however, the relative increase of these nonspread spider forms could be stated with beth methods (wilcoxon test). we therefore conclude, that platelet morphometry with both methods is a sensitive and reliable ex vivo method to evaluate platelet interactions with artificial surfaces and can also be used lateron in phase-4-studies in patients. however, the ibas-system requires further maprovement in hard-and so,ware to reduce the high expenditure of this method. despite for the most part standardised methods such as hypothermia, cardioplegia the perioperative myocardial infartion rate is still high at approx. 6%. in cardiovascular surgery it is well known that various cardioplegic solutions are employed for myocardial protection during the ischemic phase. in order to evaluate the possible influence of these solutions we selected two of the most commonly used cardioplegic solutions for investigation in a randomised double-blind study: htk (group 1) and st. thomas (group 2). after randomisation each group consisted of 20 patients who had to undergo aortocoronary bypass surgery. aim of the investigation was to establish possible varying cellular changes during the reperfusion phase or in the early operative phase in order to be better able to apply reinforcing clinical measures. in the context of this study the classical enzyme-diagnostical methods ck,ck-mb and ldh as most useful, however not as convincing. still, we have in the meanwhile been able to show that the cardiac muscle troponin t proves a particularly sensitive parameter regards differentiated ischemic damage to the myocardium. ~his we were able to conflrm in extensive preliminary trials. cardiac troponin t was registered with a one-step lmmunoassay using two highly specific monoclonal antibodies directly via two different epitopes of cardiac troponin t. simultaneously the corresponding pre-and postoperative ecg was registered. further, within this context we investigated parameters that indicate cellular damage, such as platelet factor 4 (pf4), t-pa, interleukin-6 and pmn-elastase. in the reperfusion phase in group 2 there is a significant rise in tmponin t while in group 1 these values remain practically unchanged up to the 1st. postoperative day. of special importance is interleucin 6 since according to most recent studies the release of this substance leads to platelet activation via the arachidonic acid metabolism. this pathway must, further, be regarded within the context of free radical formation. on the 1st. postoperative day the 11 6 values in group 2 are significantly higher. the effects of membrane damage is also observed via pf4 and the pmn-elastase to be different in both groups. on the basis of this study we arrive at the conclusion that the htkcardioplegia is essentially less damaging than that of the st. thomas solution. (2) r. hetzer (2) (1) department of hematology and oncology, vimhow klinikum, humboldt university, berlin, germany (2) we investigated the influence of two different vad systems on these hemostatic changes. vads were implanted in 18 patients [11 bi-vad (berlin heart), 7 left vad (novacor n 100)] with end-stage heart disease who were awaiting heart transplantation. the following hemostatic parameters were measured during the first 51 days of bddging or until heart transplantation: thrombin-antithrembin iii (tat) complexes, prekallikrein, factor (f) xll, plasminogen, or2 -antiplasmin, and i?,thremboglobulin. results: during the first week of bridging, significantly higher tat levels were observed in novacor patients compared to berlin heart patients. prekallikrein activity levels were significantly lower in the berlin heart patients in the early bridging period. all other parameters were comparable in both groups throughout the entire observation period. differences in hemostatic parameters became apparent only in the early bridging period with more enhanced pmthrombin activation in the novacor group and more prominent contact activation in the berlin heart group. avoidance of the transmission of viral infections and saving in the use of blood products encouraged the use of apparatwe intraoperative autetransfusion techniques. patients and methods: arer randomization apparative intraoperative autotransfusion was performed in 5x7 patients during elective hip surgery using i-iaemonetics cell saver ill, haemonetics cell saver v, electromedics elmd, haemolite 3 and fresenius continuous autotransfnsion system (cats). at defined tmaes we detenmned a lab panel (clinical chemistry, lipids, proteolytic capacity, hemolysis, coagulation panel) at 9 determination points in the reservoir, the retransfused blood and in the patient. results: no significant differences concerning proteolytic capacity, prothrombin time, platelets, lipids, electrolytes. increased hemolysis (p<0.01) in the hcs iii group vs. the other groups (lo rain. after application of the retransfnsed blood). low heparin concentrations of retransfused blood in the hcs iii group( 0.32+-0.3 u/ml) vs. high concentrations in the cats group (0.47 +-0.3;p--0.01). parameters of thrombin generation were elevated in the hcs iii group vs. the other groups (p=0.02). conclusions: the use of 5 different apparative autotransfnsion systems dunng elective hip surgery results in dysturbances of hemocompatibility. the activation of the coagulation system during the collection and filtering is partly influenced by the elimination kinetics and the dose regime of heparin. however intraoperative autotransfusion must be roan~ged very carefully and possibly adverse effects of perioperadve heparin peak levels have to be considered. little information is available on the management of patients with factor viii deficiency who require cardiac surgery. we report the case of a 54 year old man with factor viii deficiency and combined severe aortic stenosis and incompetence and mitral incompetence who underwent a double valve replacement at our institution. he had a history of several bleeding episodes following minor surgery. previous factor viii levels were between 8 and 26%. using standard cardiopulmonary bypass, a double valve replacement with a 23 and 29 mm bileaflet prosthesis in aortic and mitral position, respectively, was performed. a high dose aprotinin regime was used (5.5 x 10 a iu). three doses of factor viii concentrate were given in the perioperative period, totalung 7000 1u until the 1st postoperative day. repeated measurements of the factor viii level were performed. the postoperative chest tube drainage was 350 rot. until the 4th postoperative day an additional dose of 3000 iu of factor viii was given to maintain a level of at least 30%. the obligatory anticoagulation was achieved initially with heparin i.v. in therapeutic dosage. due to a persistent 3rd degree av block a permanent pacemaker was inserted with additional 2000 iu of factor viii. on the 17th postoperative day warfarin was commenced aiming for an inr of 3.0 -3.5. the patient was discharged home therearer. he was trained to monitor his inr with a coagu chek device. no bleeding episode occurred during the first 3 months follow up. open heart surgery can be performed safely in patients with factor viii deficiency with the use of factor viii concentrates and monitoring of factor viii levels. coating of biomaterials was developed using synthetic polymers with incorporated anticoagulants. stents were coated with a thin layer consisting of a polylactide polymer containing peg-hirudin and a stable prostacyclin analogue. these materials were tested with a ,,human shunt model" using nonant/coagulated blood of healthy volunteers. within minutes uncoated stents were covered by fibrin and aggregated platelets, which could be seen macroscopically and by scanning electron microscopy; coated stents were free from coaguiation plugs. this observations were supported by analysis of coagulatiuon activation markers. unlike coated stents, uncoated stents revealed high levels (>detection limit) of tat complexes and prothrombin fragments (f1-2). in a series of experiments stents were tested in sheep. in 16 sheep stents (coated/uncoated patmaz-schatz stents) were ptaced by conventional techniques in the left anterior descending artery. anticoagulant therapy consisted of a heparin bolus and intravenously given aspirin before stent implantation. no ant/coagulation was given thereafter. existing data show hyperplasia in the area of uncoated stents which was reduced around coated stents (this study will be finished in january 1996). this coating technique with incorporated anticoagulants reduces thrombogenicity during the early and late phase of biomaterial implantation. studies concerning catheters, vascular prosthesis and oxygenators are in progress. the mechanical circulatory support (mcs) is a therapy for patients (pts) with endstage cardiac insufficiency. during mcs thrombeembolic events, due to the surface thrombogenicity of the implanted device, are feared complications. activated blood platelcts play a major role in this context. therefore, patient's platelet morphology was investigated. during the period of mcs, using the novacor left ventricular assist system n100, blood samples of 8 pts were observed by means of scanning electron microscopy (sem). blood was collected preoperatively and after implantation daily during the first week as well as weekly for the first 3 months. samples were drawn via an 18gauge cannula into caeodylic-acid buffered glutaraldehyde and platelets were prepared for morphological investigations. platelet alterations were classified as non activated, activated and aggregated, based on "shape change" morphology. additionally, the common blood coagulation parameters were evaluated. preoperatively, 15.0 + 4.6 % of activated platelets were found. within the first postoperative week, the mean level of activated platelets raised to 32.8 + 8.0 % (p<0.05). comparing short-(<30 days) vs. long-term (>30 days) mcs, a significant difference of activated ptatelets (overall mean values) could be seen (24.3 +_ 3.3 % vs. 34.8 _+ 3.4 %, p=0.004). during mcs a correlation between hemolysis and platelet aggregates, as well as the values of activated dotting time and activated platelets were observed. also, specific platelet deformations and damages appeared during mcs, which could not be found preoperatively. all pts with mcs showed alterations of their platelet morphology induced by the activation of the implanted synthetic material. with regard to the postoperative antithrombotic therapy, these observations should be taken into consideration. during extracorporeal circulation (ecc) the blood and its compenents are exposed to artificial surfaces and inflammatory respenses are activated, especially the complement, coagulation, fibrinolytic and kallikrein systems. furthermore leukocyte activation occurs and platelet function is impaired. these humoral and cellular systemic responses are known as the "pustperfusion syndrome" with clinical symptomes like lenkocytosis, increased capillary perraeability, accumulation of interstitial fluid and organ dysfunction. the impertance and even perhaps the existence of the damaging effects of cpb have been widely debated in the literature over the past 30 years. many efforts have been made to reduce traumatizing factors, e.g. the use of membrane instead of bubble oxygenators. recently, heparin-coated equipmen~ and tubings have been proposed to avoid excessive contact activation during cpb, the here presented study was designed to assess changes in coagulation and flbrinolytie activity in 20 patients undergoing cpb. in this regard we investigated coagulation parameters like fibrinogen, antithrombin, pmthrombin-fragments fl+2, thrombin-anthhmmhin complex, tissue-factor, fibrin-monomeres and parameters of the fibrinolytic system like tissue-plasminogen-activator, plasminantiplasmin-complex, d-dimers and plasminogen-activator inhibitor before, during and after cpb. the activation of the complement cascade was followed by measuring the concentration of c5a, c4 and c3c. the results demonstrate distinct alterations in above mentioned parameters. in spite of a high dose hepariulzation (act>450s) combined with an antifibrinolytic tw, atment an activation of the coagulation system was observed immediately after the onset of cpb followed by an activation of the fibrinolytic system. therefore further efforts should be done to develop new anticoagulatory regiments and improve the biocompatibility of materials used for cpb. during cardiopulmonary bypass blood is exposed to nonphysiologic conditions. the contact with artificial surfaces and mechanical stress result in a periopemtive response which includes activation of the complement, coagulation, fibrinolytic and kallikrein system, activation of nentrophils with degranulation and pmtease enzyme release, oxygen radical production and the synthesis of various proinflammatory cytokines. this so-called "pest-pump intlammatory response" has been linked to respiratory distress syndrome, renal failure and neurologic injmy. our goal was to investigate the time course of eytokine levels and the activation of leukozytes and platelets and to quantitate leucocyte subpepulatioas in 20 patients undergoing cpb. at different time points, pre, during and pest cpb, we determined the levels of interleukin (il) 113, il-2, il-4, il-6, il-8, il-10, tumor necrosis factor ¢z (tnf-a) and interferon "1' (ifn'--/) using elisa-techulques. lymphozyte subpepulations were characterized by flow cytometry and specific monoclonal antibodies against cd3 (pan t-cell marker), cd4 (surface antigen on t-helper cells), cd19 (surface antigen on b-cells), monocytes were determined by cd14 and platelets by cd41 (act. gpilb/llla) and cd42b (gp ib). single cell activation was analyzed using markers against cd25 (il-2 receptor), cd126 (il-6 receptor), hla-dr (mhc class ii), cd71 (transferrin receptor) and cd69 (activation inducer molecule), platelet activation was monitored with an antibody against cd62 (gmp-140). preliminary results revealed distinct increases in r,-6, il-8, and il-io following cpb whereas tnf-a and ifn--/levels were not significantly influenced. fttnhermore, activation of particular cell populations was observed. finally, our investigations should contribute to a better understanding of the complex humeral and cellular respenses induced by cpb and thus might help to develop new strategies to circumvent the negative impacts of cpb. optimal adjustment of anticoagulation in machine plasmapheresis is important for the quality of the prepared fresh frozen plasma (ffp) as well as for the safety of the donation. in the present study the suitability of prothrombin fragment ( ft+2 ) in the assessment of anticoagulation during plasmapheresis was investigated. matarlal and methods: 75 plasmapheresis procedures were performed on 25 donors (10 ~, 15o" ) using 3 different plasmapheresis machines (a 200, baxter; mcs 3p, haemonetics; pph 900, electromedics/medtronic). acid citrate dextrose formula a (acd-a) in a ratio to whole blood of 8 : 92 was used for anticoagulation. the concentration of fi+2 in the donor's blood was measured before and after plasmapheresis and in the prepared ffp. the actual acd-a volume used was also registered. results: there was a significant rise of the ft+2 -concentration in the donors blood after plasmapheresis with each of the three automatons: a 200:1.32 vs 1.14, p < 0.05; mcs 3p: 1.26 vs 0.98, p < 0.05; pph 900:1.20 vs 1.05, p < 0.05. the ffp prepared with each machine showed the following f~+2concentrations: 0.91± 0.18, 1.0:2 ± 0.17 and 0.93 ± 0.11 respectively. the difference between the groups was not significant. the elevation of the ft+2 -concentration in the donor's blood showed a negative correlation with the volume of the acd-a used. during 6 of the 75 procedures technical problems occurred (inadequate venous acces, occlusion of the citrate tube, reduced whole blood flow). after these procedures there was a marked elevation of f~+2 in the donors blood (2.74 ± 0.53), accompanied by an elevated f~+2 -concentration in the prepared ffp's. conclusion: these data show that ft+2 is a suitable parameter for the assessment of anticoagulation during plasmapheresis. several epidemiologic studies demonstrated that fibrinogen is an independent cardiovascular risk factor and should be considered for screening programs. prothrombin time derived fibrinogen (df) measurement combines the advantage of an established highly reproducible automated method with no additional reagents, except for calibration. several studies showed that the df values correspond well with the clanss method except in cases such as thrombolytic therapy in which the df results are higher. however, no results exist whether in patients with coronary heart disease with fibrinogen as a risk factor the df values are also comparable to the established clausss method. the aim of our study was to compare df values to clauss method results in cardiac patients, especially in patients before and after coronary bypass grafting (cab(]). measurements of df were performed on an acl 3000 (il) using the pt-fibrinogen-hs reagent. fibrinogen clanss method was done on the acl using fibrinogen c reagent (il) and on a kc4 (amelung) with fibrinogen a reagent (boehringer maanheim). for calibration we used the calibration plasma half volume (it.) with the fihrinogen concentration proposed by the manufacturer. plasma samples were obtained from 24 patients at admission before cabg and postoperatively up to 1 week, and from 23 healthy persons (staff). within assay imprecisious using normal and abnormal controls (il) were comparable with both methods showing cvs between 1.99 and 4.22 %. in normal healthy persons the medians of the df and the clanss method run on the acl were very similar (296 vs 302 rag/all), whereas kc4 values were about 10% lower (268 md/dl). in cabg patients at admission we found the same differences as in normals with the clanss method (acl: 363 vs kc4: 337rag/all), however the df values were siginficantly higher (median 418mg/dl). if we took a cutoff value of 320 mg/dl, as suggested by the results from the northwick park heart study, we would categorize into the high risk group 21 out of 24 patients using the df method, 20 with the clanss-acl method and 16 with the clanss-kc4 method, i.e. nearly 30% more patients were classified in the high risk group using the df method. postoperative samples showed the expected increases due to the acute phase response with the same magnitude of differences. because of its rapidity and reproducibility the df method is well suited for routine measurements, however, standardization remains an urgent task in order to avoid misinterpretation of results. for fibdnogen measurements in clinical laboratories, the two most widely used methods are the clotting time method according to clauss (cfib) and the sn called "derived" fibrinogen method (dfib) implemented in optical coagulometera with the fibrinogen concentration being derived flora the optical density of the fibrin clot in a standard prothrnmbin time (pt) assay. it is well known that under certain circumstances, e.g. in the presence of fibrin(ogen) degradation products (fdp), there is a discrepancy between the two methods with higher values for dfib than for cfib. yet the opposite discrepancy, i.e. fibrinogen values derived from the optical density of the clot grossly lower than values from dotting time assays, seems to be very rare and is poorly understood so far. the patient (male, 26 years) had ingested the esterase inhibitor parathion (e605) in an attempt o f suizide and was treated with high doses of atmpin. he had no clinical signs or history or family history of bleeding or thrombotic disorders. except for a very low pseudocholinesterase activity, all laboratory results were normal ineinding pt, afft, thrombin time, and factor xiii. pt and aptt did nnt differ between an optical coagulometer (electra 1000c, mla) and a mechanical one (kc..4, amelung). there was no evidence of disorders known to interfere with hemostasis like paraproteinemia or dyslipldemia. however, in all 7 blood samples received for dotting tests during a period of 7 days the macroscopic appearance of the fibrin clot was quite unusual (only slightly turbid/almost transparent) and there was a striking discrepancy between a very low or low dfib on the electra (pt reagent: thromboplastin is, dade) and a normal or high cfib (kc4; thrombin reagent, dade). on admission, values were 57 mgml (derived) vs. 275 mgldl (clauss). cfib rose to s42 mg]dl with dfib at 155 mg/dl in the last sample on day 7. ~ al! samples dfib was about 20 % (ls-23) of cf[b. when the patient's plasma was added m normal pooled plasma it caused, in a dose-dependent manner, values lower than predicted for dfib and values slightly higher than predicted for cfib. in the absence of data from additional (e.g. immunologic) methods the following principal possibilities (and combinations) have to be considered: 1) normal fibrinogen concentration and clot formation rate, but abnormal optical properties of the clot (cfib correct, dfib falsely tow); 2) normal optical properties of the clot, but accelerated clot formation and very low fibrinogen concentration (dfib correct, cfib falsely high). in either case, the molecular basis could be: a) a genetic or acquired molecular abnnrmality of fibrin/fibfinogen; b) an interfering substance. direct effects of the loxic agent parathion and/or the antidot drug atropin are not likely to be the cause since other patients, often with more severe parathion inmxicatian requiring higher doses of atmpin, showed normal optical density of the clot. we hope to perform a more in depth investigation of this abnormality in the future, including various methods, reagents, and instruments for fibrinogen measurement, a survey of the patient "s family, and studies of the molecular nature of the phenomenon. increased fibrinogen is known to be an independent predictor of subseqtmnt acut~ coronary syndromes. however. a multitude of methods for fibrinogen determination is available. there is a lack of standardisation among fibrinogen assays. in a family cohort study (patients'with combined hyperlipidaemia and f or hypemricaemia) fibrinogen was determined in plasma samples from 340 family members using a functional and an immunochemical assay. the fimctional assay according to clauss was performed on the analyser ca 5000 using the test fibrinogen a from boehringer. the immmmephelometric assay was performed on ~e behring nephelometer system using the reagent and standard from behring. a good similarity between both assays was obtained at low and high flbrinogen levels as well as in samples with increased c-reactive protein (crp). values obtained by both assays correlated similar with total cholesterol, ldl--cbelesterol and apolipeprntein b. the ratio functional fibrinagen / immlmochemial fibrinogen showed no dependence on cholesterol, t-pa, v wiuebrand factor and crp. release of two fibrinopeptides a from fibrinogen generates desaa-fibrin monomer, which rapidly aggregates, forming fibrin complexes. fibrin monomers can be detected in plasma samples after chemical desaggregation of fibrin complexes using thiocyanate by monoclonal antibody binding to the alpha-chain neo-n-termini generated by fibrinopeptide release. although postulated, an intermediate of fibrin formation, carrying one fibrinopeptide a and one fibrin alpha-chain neo-n-terminus has so far escaped analytical procedures. we have employed a monoctonal antibody specific for fibrin alpha-chain neo-n-terminus, mab 2b5, attached to magnetic microparticles, for isolation of fibrin-related material from plasma samples of patients with elevated soluble fibrin. the material was desorbed by sds-urea buffer and subjected to sds-page and immunoblotting. immunostaining with panspecific anti-fibrinogen and anti-fdp-e antisera showed a range of bands corresponding to fibrin monomers, and fibrin derivatives containing the fibrin e-domain. lmmunostaining with monoclonal anti-fibrinopeptide a antibody resulted in a doublet band corresponding in size to fibrin monomer. similar results were obtained with polyclonal antisera against fibrinopeptide a. for a more quantitative approach, desa-fibrin monomer was detected by an elisa procedure using mab 2b5 as capture and monoclonal anti-fibrinopeptide a antibody as tag. a sample with extremely high level of desaa-fibrin monomer, determined by elisa (enzymun®-test fm) was used for calibration, since reference material is not available. a correlation of r=o.g4 was found between desaa-fibrin monomer and relative desa-fibrin monomer levels. detection of desa-fibrin monomer required sample pretreatment with thiocyanate for desaggregadon of fibrin complexes. from these preliminary data it appears that desa-fibrin monomer accounts for a fairly constant proportion of soluble fibrin and is a polymerizing species. fibrinogen has been shown to be a major cardiovascular risk factor. especially for epidemiological studies, exact quantitation of fibrinogen in clinical plasma samples is of great imporance. fibrinogen levels are generally measured by clotting assay according to clauss, or by determination of derived fibrinogen values upon photometric measurement of prothrombin time (derfbg). the clotting assay has been shown to be influenced by high levels of soluble fibrin derivatives. the pt-derived fibrinogen levels appear rather convenient in clinical routine, since no additional reagents are needed. we have compared the clauss assay and derfbg with a turbidimetric fibrinogen assay using snake venom protease for fibrinopeptide release, performed in photometric autoanalyzers. d-direct antigen was measured in parallel using tinaqaant d-dimer lpia. results were correlated with total fibrinopeptide a release by thrombin, measured by elisa. a total of 484 samples were included, of which 29 samples (6 %) were recorded as above measurung range by derfbg. these samples encompassed a range of 5.90-10.40 g/l and 5.21-12.37 g/l in clauss, and turbidimetric assay, respectively. the range of values measured by derfbg assay was 0.72-9.14 g/i, corresponding to 0.26-11.00 gll and 0.24-10.48 g/1 in the clauss and turbidimetric assay, respectively. the correlation of derfbg with the clauss assay was re0.91, correlation with turbidimetric assay was r=0.92 for the values actually detected. the correlation between clauss and turbidimetric assay was r=0.93 for all values. there was no dependency of test results or inter-test variation upon d-direct. correlation graphs displayed a decreased test response of clauss assay in the high concentration range, resulting in an underestimation of fibrinogen concentration. the derfbg assay, in contrast, showed normal range values in samples from patients with fibrinotytic treatment and low fibrinogen levels in the other assays. correlation with fibrinopeptide a release was r=0.88 for clauss assay, r=0.89 for turbidimetric assay, and r=0.82 for derfbg. for clinical routine, derfbg appears to be applicable for all samples between 1.00 and 5.00 g/l with exclusion of samples from patients with fibrinolytic treatment or endogeneous hyperfibrinolysis. other samples may be analyzed by clotting assay or turbidimetric assay, although the latter appears to be more suited for measurement of high range samples. for inhibition of pk is 0.067 pmol/l the antifibrinolytic activity of the inhibitors was determined by measuring the lysis of radiolabelled human plasma clots• the compounds which inhibit plasmin and pk influence remarkably the streptokinase-induced clot lysis but not lysis induced by uk and tpa. surprisingly, inhibitors of uk and tpa do not influence clot lysis induced by uk or tpa. the structure-activity relationships for inhibition of ptasmin, uk, tpa and pk could help in the design of more potent inhibitors of fibrinotytic enzymes. uk inhibitors are of interest for the development of anti-invasiveness drugs, while plasmin/pk inhibitors could be prototypes of a "synthetic aprotinin". in the ecat angina pectoris study t-pa antigen was an indepcndem risk factor of subsequent acute coronary syndromes. pat indicates the risk bat depends on other known risk factors. it should be tested in 183 members of a family cohort study (patients with combined hyperlipidaemia and / or hyperuricaemia), if the active pal antigen or the whole pai antigen showed a stronger relation to t-pa and metabolic variables. the active pall antigen was determined using elisa actibind pat-1 (technoclone / lmmuno) , the whole pai-i antigen was measured using the f_lisa pat-1 (technoclone i immuno). t-pa activity was determined with the coaset t-pa from chromogenix, the tintelize tpa from biopool was the used test for determination of t-pa antigen. the active pat antigen showed a stronger correlation to t-pa activity and t-pa antigen than the whole pal antigen. circulating t-pa activity was influenced predominantly by the active pal antigen. both pat antigens were correlated in similar manner with metabolic variables, lipoproteins and b/vii. table: correlations of active and whole pal antigen (** p < 0,001) active pal antigen whole pal antigen active pat antigen 1,000 0,851 ** whole pal antigen 0,851 ** 1,000 t-pa activity -0,594 ** -0,492 ** bpa antigen 0,604 ** 0,497 ** body mass index 0,502 ** 0,462 ** triglycerides 0,452 ** 0,441 ** total cholesterol 0,252 ** 0,255 ** ldl-cholesterol 0,263 ** 0,264 ** hdl-cholesterol -0,357 ** -0,355 ** apolipoprotein b 0,428 ** 0,402 ** apolipoprotein a i -0,233 ** -0,211 ** the lower relationship of the whole pat antigen to t-pa is obviously caused by patient samples with high levels of whole pat antigen in contrast to normal values of active pat as well'as of t-pa. possibly, a high ratio of whole pai antigen / active pat antigen is caused by a raise of latent pal the main form of pat in the platelets. the clinical importance of an increased ratio whole pal antigen / active pal antigen remains under investigation. the cyclic antibiotics-polypeptides bacifracin a, bacilliquin from boci/lu~, licheniformis and gramicidin s from bocil/us brevis, var. (3. b., were used for investigation. we studied their influence on the fibrinoly~c and coagulation activity in vitro• me~hods. to solution of human plasmin (thrombin). containing 0.2 mg of protein (1 nih unff)/ml, the analyses' solution of antibiotics (0.1-8,0 mg) was added. then we defined the tlbrinolytlc activity of the mixes using azofibrin lysis, and fhrombin activity was determined according to the speed of fibrin clots formation from fibrinogen solution. results. in following table are submifled the results received in our laboratory {we also offer results of antibiotics influence on urokinase activity): ki, mm ki --the constant of inhibition. n. d. ~ in studied lirnils the inhibitor's activity was not observed. ---the inhibitor's activity was not define. i. --the inhibitor% activity was observed but ki not determined. +, +% +++ --effect of inhibffion (in rela*iive indexes). conc/us/on.~ the results received by us testify to the necessity of cautious approach to the use of antibiofics-polypeptides for various sorts of therapy in view of their possible influence on fibrinolytic and coagulation actlvlfy, of the organism. these results were used for preparation in our laboratory of biospeciflc sorbents containing c-ramicidin a, bacil}iquirt and gramicidin s.as ligands, they can reversibly bind thrombin, plasmin {plosminogen) and urokinase directly from crude exkacts. the enzymes are selectively eluted without substantial losses of specific activity in e yield of 60-90%. there is a great body of rather contradictory informations dealing with fibrinolysis in liver.. cirrhosis, which can be accelerated, normal or reduced, depending on the type of cirrhosis and investigation techniques (clot-lysis, fibrinolytic component measurements). our previous finding was, that in vitro plasma-clot lysis, induced by exogeneously added tpa or streptokinase proved to be reduced, and this had a good correlation with severity of the disease and the elevation of plasmatic yon willebrand factor levels. in vitro clo~/-[ lysis tests, induced by tpa were performed in 41 patients with alcoholic liver cirrhosis, utilising a microplate light-scattering assessment method. the tests were repeated using the same plasma samples in each patients with a microplate which was covered by cultured endothelial-cell monolayer (umbilical vein, huvec}. clot lysis speed proved to be 1.5-2 times slower with huvec milieu in the control group, while in the cirrhotic patients this inhibition was stronger and resulted in 5-fold reduction of lysis speed. our results suggest, that cirrhotic plasma is able to accelerate the release of fibrinolytic inhibitors from cultured endothelial cells, which phenomenon may also contribute to the complex alterations of in vivo fibrinolysis in cirrhotic patients. deep vein thrombosis (dvt) is a systemic disease with prolonged clinical manifectation. anticoagulation therapy in dvt is not completely effective. thrombolytic therapy may give rise to a systemic lytic state, the fibrinospesific agents (scu-pa and t-pa) have short half-lives in the circulation. we investigated the potency of the acylated plasminogen streptokinase activator complex (gbpg-sk) to deep vein clot dissolution as compared to well known sk and apsac both in v~tro and in vivo in the model of venous thrombosis in artherio-venous shunt in rats. it was shown in in vitro study that fibrinolytic activity of plasminogen activators mainly depends on their stability in plasma. stability studies carried out by incubating sk and pg-sk activator complexes in plasma with euglobulin precipitation . total fibrinolytic activity was measured by the fibrin plate method. gbpg-sk possessed the greatest stability in human plasma than apsac or sk because of its prolonged inactivation period (the deacylation half-life for gbpg-sk was 230 :e 21 rain in contrast with 73 -~ 6 min for apsac). the stability degree of two acylated thrombolytics (gbpg-sk and apsac) was in order to inverse proportion of their first order rate deacylation constants (2.9 • 10 -4 and 6.0 • 10-s sec-1 respectively). the fibrinolytic potency of sk, apsac and gbpg-sk was measured by 1251-labeled fibrin clot lysis in plasma and in vivo by lysis of the preliminary formed 1251-labeled fibrin clot inserted into the jugular vein. fibrinolytjc activity of acylated plasminogen activators gradually increased in time. under sk administration, the clot lysis came to the end by 2 hours while apsac and gbpg-sk haven't lost their activity for 5 -6 hours. gbpg-sk possessed significantly more prolonged fibrinolytic activity than apsac, the acyl-enzymes did not significantly influence on plasminogen,,.~2-anfiplasmin and fibrinogen levels in plasma according to their activity specific to fibrin-bound plasminogen. in opposite, sk produced a significant depletion of plasminogen, ~-2antiplasmin and fibdnogen levels in plasma. it seems, on the basis on in vitro and an animal experimentation, than apsac with its moderately fast deacylation rate is more suitable for rapid thrombolytic effect, but gbpg-sk with its slow deacylation rate is suitable for deep vein thrombosis, when the rapid thrombolysis is less critical. it's well known that the complete lysis of thrombi usually isn't observed at the thrombolytic therapy. at present study we have attempted to quantify the possible mechanism of fibrinolysis inhibition during the thrombolysis. 125i-labelled partially cross-linked fibrin clots of different volume (0.1-0.35 ml) were immersed in tris-hcl buffer (3 ml) containing plasmin (5-100 nm) at 37°c. the lysis rate was detected by counting of soluble fibrin degradation products (fdp). at all the eases lysis slowed down and stopped in 3 hs though clots dissolved up to only 60-85%. no irreversible inlaibition of plasmin caused by denaturation occur as was judged by the measurement of fibrinolytic activity at the diluted samples. however the increase of fdp concentration in surrounding buffer led to the reversible inhibition of fibrinolytic activity of plasmin up to 5% of baseline. the sds-page analysis under non-reduced conditions shown the acoumulation of high-molecular weight fdp at the surrounding buffer. the inhibition phenomenon could be connected with the specific binding of plasrnin with soluble fdp having exposed lysine residues and the subsequent removal of enzyme from fibrin surface. unexpectedly since the heterogeneous character of occurred reactions tile change of the clots surface area during lysis didn't affect the fibrinolysis kinetics in all the concentration intervals. to estimate the kinetic parameters the kinetic curves were linear in the coordinates [p1/t (l/t*ln(isl°{(lslo.lpi)). the obtained parameters were following: keat=l.36 min-l,km=l.33 ixm,kp=0.12 ~tm. the clinical trials have shown that fdp concentrations at the thrombolytic therapy of deep venous thrombosis and acute myocardial infarction usually was approximately in the range 0.05-0.2 ~tm. therefore the described phenomenon of fibrinolysis inhibition by formed fdp may take place during thrombolytic therapy. al. calatzis, an. calatzis, +m. klmg, +l. mielke, +r. hipp, a. stemberger institute for experimental surgery and +institute of anesthesiology technische universit~.t monchen thrombelastography (teg) is an established method for the detection of fibrinolysis. fibfinolysis is usually determined when the teg amplitude decreases by more than 15% atter the maximum amplitude is reached. this takes a considerable amount of time (more than 30 minutes). our approach bases on the understanding of fibrinolysis as a process which runs in paraue[ to coagulation and is not exclusively subsidiary to it. the effect of fibrinolysis on the growing clot in the teg is shown by the comparison of two parallely performed teg measurements: exteg: teg measurement with standardised activation of the extrinsic system. apteg: exteg with in-vitro-fibrinolysis inhibition via aprotinin. exteg-reagent (ex): 1:2 dilution of innovin (recombinant thromboplastin reagent, dade) with aqua dest. apteg-reagent (ap): 5 parts innovin, 2 parts trasy[ol (aprotinin, bayer, i0.000 kie/ml), 3 parts aqua dest. test procedure: l0 p1 ex or ap + 300 ~l citrated blood (cb) + 50 lal cacl2-solution 0,15 m. the only difference of the two reagents is the addition of 20 kie aprotinin in the apteg, leading to an in-vitro fibrinolysis inhibition. the usage of disposable pins and cups (haemoscope, illinois, usa/e.m.s., vienna) is recommended for ensuring standardised conditions for both measurements. results and discussion: when there is a better clot formation in the apteg (corresponding to a lower so-cafled k-value) than in the exteg, fibdnolysis can be suspected. this technique requires only commercially available reagents and is easy to perform on conventional teg systems. due to the standardised coagulation activation with a thromboplastin reagent, fibrinolysis can be detected also when inhibitors like heparin are present in the circulation. according to our experience using this technique during liver transplantation, clinical relevant fibrinolysis can be detected as described in less than l0 minutes. many thromboembolic (massive pulmonary embolism, proximal deepvein thrombosis, etc.) and coronary diseases (infarction, acute phase, etc.) require fibrinolytic therapy to early recanalizafion. the application of the well-known or new thrombolytic agents needs the use of specific, simple and reproducible methods for the determination of fibdnolyfic activity. we suggest new methods for measuring the blood plasma concentrations of plasmin, plasminogen, antiplasmins, and urine urokinase activity. these methods involve the employment of chromogenic substrafe azofibrin (human fibrin, covalently labeled with p-diazobenzenesulfonic acid). method~. 0.2 ml of studied solution was added to 0,8 ml of azofibrin suspension in certain buffer (5-10 mg/ml) and the mixture incubated at 37 oc for 10-60 rain. after the end of incubation the mixture was filtered, the volume of solution brought up to 4 ml by 0.02 m naoh and the optical density was determined at 440 nm. resuffs. azofibrin can be used for quantitative determination of proteinases activity in search of new fibrinolytic means. for comparison the results of our studies fibrinolytic activity of some proteinases with the use of azofibrin are presented: activity. with an increase of pal and ldl-and a decrease of hdl-cholesterol concentrations k is concluded that the increased cardiovascular risk in diabetes meilitus was partly caused by a down regulation of the fibrinolytic system, increase of erythrocyte aggregation and plasma viscosity. also disturbances of lipid metabolism an abnormal whr seems to be of an additional atherogenous factor in dm. plasma concentrations of thrombin-anfithrombin-iii (tat), alpha-2antiplasmin-plasmin (app) complexes and ddimer were investigated in 50 patients treated with thrombolytic therapy for acute myocardial infarction (ami) either with streptokinase (n=24), urokinase (n=16) or recombinant t-pa (rt-pa, n=10). all patients received an intravenous heparin bolus of 5,000 iu on admission, which was followed at once by an infusion of 1,000 iu/hr for the next three days titrated to maintain the partial thromboplastin time at twice control value. tat, pap and ddimer were measured by enzyme immunoassay on admission, 1, 2, 4, 6, 8, 12, 24 hours and on day 3 and 7 after admission. groups did not differ significantly in regard to age, sex, delay and infarct location. on admission, no marker differed significantly between groups. thereafter, tat levels increased significantly exclusively in rtpa treated group. from 2 to 6 hours after admission, tat were significantly higher in rtpa treated patients than in streptokinase and urokinase treated group (p<0.02). however, during continous heparin infusion, which was started immediately after stop of thrombolytic therapy, in each group tat concentrations decreased below admission values. app were significantly higher only 1 hour after admission in the rt-pa group (p=0.03). ddimer did not differ signifieanfly between groups. our results demonstrate, that rtpa induces a hypercoagulable state, which may contribute to reocclusion after successful reopening of the infarctrelated coronary artery. the significant tat decrease during continous heparin infusion supports the concomitant use of thrombin inhibitors as adjunctive therapy with thrombolytlc treatment for ami. thus, in acute myocardial infarction patients, thrombin generation is markedly influenced by the thrombolytic agent used and concomitant heparin therapy. endothelium derived relaxing factor-no (edrf-no) plays a major role in regulation of vascular tonicity and also exerts platelet inhibitory action~ however, due to the chemical nature of edrf-no few is known about its production and activity as a general index or marker of vascular function in human diseases. one way to achieve this can be measurement of nitrate/nitrite excretion in the urine, which seems to reflect vascular edrf-no production. in this report a self-developed elisa method is described, which was used for this perpose. nitrate/nitrite urinary exretion proved to significantly decreased in insulin dependent and in non-insulin dependent diabetes mellitus as well after a comparison of the excretion values to other markers of angiopathy (yon willebrand factod soluble thrombomodulin, beta -thromboglobulin) it seems to be acceptable, that urinary nitrate/nitrite excretion can be a useful indicate of diabetic vascular disorders. two major concerns still accompany the application of prothrombin complex concentrates (pcc). viral safety has to be guaranteed and therefore several measures for virus inactivation or elimination are taken during the manufacturing process. the inherent risk of thrombo-embolic side effects has to be considered. to minimize these risks and to achieve good clinical efficiency the quality criteria for pcc's are under pending discussion. it is generally accepted that a modem pcc-preparation should contain all of the four coagulation factors in a well balanced proportion and that it should also contain protein c and protein s. additionally, the concentration of activated coagulation factors should be kept at a minimum. a present pcc-produedon process mainly consists of a qae-sephadex extraction of cryopeer plasma followed by a solvent/detergent virus inactivation step. further purification is achieved by subsequent chromatography on deae-sephamse. the aim of this study was to improve product quality by avoiding f viiactivation without implementing major changes to the production process. at the same time, a second virus eliminating step was added to the production process. it could be shown that speeding up the chromatographical process by switching the deae-sepharose-chromatography from a classical axial column to a radial chromatography resulted in a significant reduction of f viia-genemtion. mainly the reduction of contact time, resulting from the highest possible flow rates, leads to the wanted effect. the relation between f vii/f viia was 10 : 1 or more. in order to investigate the feasibility of virus filtration the eluate of the deae-sepharose column was filtered through a virus removing ultipor vffilter. the analysis of the solution before and after fillration showed that the filtration had no influence on coagulation factors activity, protein content, proteolytic activity etc. preliminary studies showed significant virus reduction values. in the past few years the problem of expediency of the treatment aimed at developing immunological tolerance in hemophil;a patients by way of complete removal of inhibitor with high doses of factor viii has been discussed in literature. we observed 121 patients with hemophilia. inhibitors to factor viii:c were revealed in 32.7 % of patients with hemophilia a and fo factor ix --in 1.6 % of patients with hemophilia b. the level of an inhibitor was not higher than 87 befhesda u/ml, that is those patients were not regarded as "high responders". a high incidence of inhibifors in young patients [from 7 to 26 years of age, 51.9 %) compared with older patients (from 27 to 40 years of age, 11.2 %) testifies to the probability of inhibitors development during treatment with modern concentrated preparation of factor viii, ix. inhibitor development in patients (40.5 %] in the course of antihemophilic concentrates transfusions is an evidence of alloimmunization of patients with proteins. the investigations show that in the course of transfusion therapy patients develop secondary immunodeficiency due to chronic antigenic stimulation of immune system with high doses of allogenic proteins. against the background of immunodeficiency patients with hemophilia develop complications of immune character: infections complications --53.9 %, aufoimmune processes --44.9 %, secondary tumours --1.2 %. plasmapheresis is the most rational method of removing inhibitor in patients with low level of inhibitor ("low responders", < 10 bu/ ml) and in patients with mean response. thus it should be noted that the treatment of patients aimed at developing immunological tolerance is not only expensive and economically unprofitable but also not indifferent fo the organism. in a recent multicenter study 73 previously untreated patientens (pups) with severe hemophilia a were treated with a recombinant factor viii concentrate (rfviii, recombinate©). during fviii treatment 21 (29%) developed inhibitors, 6 high titer (>5 bethesda units (bu)/ml), 4 low titer (<5 bu/ml) and 11 transient inhibitors. plasma samples from before treatment and during treatment but before inhibitor occurrence were available in 12 inhibitor patients. these plasma samples were analyzed by a highly sensitive immunoprecipitation (ip) assay for the presence of anti-tviii antibodies. in 9 (66%) a significant increase of anti-fv]]i antibodies was seen indicating the development of a clinical relevant inhibitor titer. this immune response occurred after 2 to 17 (median 5) exposure days (ed). in the same period only 3 out of 15 inhibitor patients showed a decreased in vivo recovery. in 16 pups who developed no inhibitors plasma samples from the entire treatment period were available. an immune response to rfviii treatment was seen in 7 pups after 2 to 43 ed (median 24 ed). the immune response was later and less pronounced in comparison to inhibitor pups before inhibitor occurrence. with the ip method the detection of an early immune response is possible which might be predictive for a later inhibitor development. the inclusion of the lip method should be considered for future multicenter pup studies. in the past anaphylactie reactions to plasma and plasma components have been a common complication of replacement therapy in patients with hemophilia a and b. we report on 3 severe bleeding episodes in 2 patients with hemophilia a and b, respectively. both patients had a history of life threatening anaphylactic reactions after exposure to different plasma derived clotting factor concentrations including intermediate purity factor viii-and factor ix-concentrate, respectively. high purity factor concentrates were tolerated well without any allergic side effects. a 67 years old patient with a moderate form of hemophilia a (f viii 4 %) had a history of severe immediate reactions with skin manifestations and bronchospasm after exposure to fresh frozen plasma, ctyoprecipitate and 3 different plasma derived factor viii-concentrates of intermediate purity. in all episodes pretreatment with corticosteroids and antihistamines was unsuccessfull in avoiding severe bronchospasm. replacement therapy with two different recombinant factor viii concentrates was tolerated well without any side effects. a 12 years old haemophiita b patient developed hypersensitivity reactions to prophylactic factor ix substitution, which could be overcome by using a factor ix .concentrate with improved purity. a recent recurrence of hypersensitmty under this treatment was finally overcome by the use of highly purified (monoclonal antibodies) factor ix concentrate. we conclude from these findings that high purity of factor concentrates, possibly due to the absence of soluble hla-antigens, are advantageous in patients disposed to allergic reactions. introduction: antibody formation against factor (f) viii remains one of the most severe complications of repeatedly transfused patients with haemophilia a. as reported previously in our study about the incidence of fviii inhibitors, we have observed a high incidence of fviii inhibitors among our haemophilia a patients. it is still not clear why certain haemophiliacs develop antibodies and others do not. a number of previous studies suggest that there is a genetic predisposition for the fviii inhibitor development. thus, the purpose of our study was to examine, if there is a correlation between fviii antibody-formation and genetically determined histoeompatibility antigen (hla) patterns in our haemophiliacs. patients and methods: hla-class i (a, b, c) and hla-class ii (dr, dq) typing was carried out for 51 respectively 44 multi-transfused paediatric haemophilia a patients (fviii:c activity < 5%), including 22 who had developed an antibody to fviii: 19 were high responders (> 5 bu), 3 were low responders (< 5 bu). hla-typing has been performed by a standurcl two-stage microlymphoc~.ftotoxicity procedure (drk frankfurt) using antisera with defiend hla-specifity (biotest diagnostica). results: we found an under-representation of hla-a2 in fviii inhibitor patients when compared with the subgroup without inhibitor. in regard to the hla-b and hla-c antigen frequencies there are no apparent differences between the groups. among the class ii antigens there were higher frequencies of dr1, drw52 and dqwl in the non-inhibitor group. however, the reduction in hla-a1, hla-cw5, hla-dqw3 respectively hla-dr4 frequency for inhibitor patients as reported previously could not be confirmed in our study. conclusion: so far it remains unclear if there is a significant association of a certain hla allels with the development of fviii antibodies. recombinant factor sq (r-viii sq, pharmacia) is a b-domain-deleted recombinant factor viii. it is formulated without albumin (hsa). the product has been shown to have in vitro and in vivo biochemical characteristics similar to a plasma derived full-length protein (p-viii). the international clinical trial programme was initiated in march 1993. pharmacokinetic studies have shown that the b-deleted r-viii sq should be given according to the same dosage principles as a full length p-viii. at present, the product is being tested in previously treated patients (ptps) and untreated patients (pups) with severe haemophilia a (viii:c < 2 %), both during long-term treatment (on demand therapy or prophylaxis) as well as during surgery. the long-term study in previously treated patients in germany was started in january 1994. thirteen patients have been included in 8 centers. all patients are still on treatment with r-viii sq, most of them receiving prophylactic treatment. global treatment efficacy has in general been considered excellent or good. no serious clinical adverse events related to the study product have been reported, nor have any inhibiting antibodies to factor viii or antibodies to mouse-lgg or cho-cell components developed in the patients. further results such as data on efficacy, half-life, recovery and safety will be presented in detail at the meeting. nowadays it is not sufficient to regard hemophilia only as hemorrhagic diafhesis of coagulation genesis, caused by deficiency or molecular anomalies of coagulation factor, without taking into account the immunity state. on examination of 125 patients (pts) (hemophilia a --110 pts, hemophilia b --11 pts, willebrandt's disease u 4 pfs) the development of immune complications was revealed in 34.4 %. chronic persistent hepatitis (3.2 %), chronic active hepatitis (3.2 %), herpes simplex (1.2 %), chlamidiosis (1.2 %), bacterial infection (7.2 %} were regarded as infectious complications. bacterial infections have a routine course due to preserved phagocytic function of neufrophils. and viral infections, whose ability to resistance is connected with t -cell link immunity, take on a chronic persistent course, mechanism of the development of autoimmune processes (autoimmune thrombocytopenic purpura --2.4 % of pts, immunocomplex disease --4.9 % of pts, the appearance of immune inhibifors --34.4 % of pts} is connected with the impairment of immunological surveillance over b -cells aufoimmune clones as a result of dysbalance in the system of t -lymphocyfes immunoregulatory subpopulations. lymphadenopathy and splenomegaly (4.9%) develop due fo benign proliferation of lymphoid tissue as a result of impairment of regulatory function of t -lymphocytes system, or they may be an evidence of virus infection. we observed one episode of acute leukemia. immune complications in hemophilia patients develop against the background of secondary immunodeficiency caused by chronic antigenic stimulation of patients' immune system with high doses of allogenic proteins, which plasma preparations contain. in immune complications hemophilia patients develop hemorrhages, whose pathogenesis is quite different from that caused by coagulation factor, so it should be taken into account in the course of treatment. control of hemophilia therapy classically was based on four parameters: life span expectancy of patients, orthopedic status (normal zero), pettersson score and social integration. oren, however, these parameters described an irreversible status with permanent damage particularly of the joints, especially when patients were grown-up. in order to establish risk-adapted therapy protocols to prevent hemophllic osteoarthropathies, quality control programs have to he set-up that allow for early adjustment of dosage and substitution frequency. here bleeding frequency is one the main parameters, being a clear hint for the possible development of a target joint. since 1988 we have established a computer database (haemopat) that contains data from all patients treated in our center. tables and graphs allow for early detection of increased bleeding tendency in a given joint, and accordingly for adjustment of therapy. the results of 8 years of measuring reasons of joint damage and not documenting the orthopathies as such will be demonstrated. parallelly a new program (haemopat win 1.0) will he introduced allowing for easier handling of data and their evaluation. this program will be used as of december 1995. in combination with a substitution calender to be filled in by all patients, in which factor concentrates, lot numbers, dosage, and date of administration will he constantly recorded, this program will extend our existing database in order to follow closely clinical and orthopedic parameters of each patient, and consequently acts as strict control of therapy quality. additionally, it provides sufficient data to fulfil any documentation needs, requested by medical authorities. the program will be available for all those interested free of charge. 2) kinderklinik der westf. wilhelms univ. mttuster 3-6) biotest pharma gmbh, dreieich haemoctin® sdh; the fviii sdh (sdh = solvent detergent and dry heat = 100 °c, 30 rain) from biotest pharma is a high purity (specific activity ~ 100) fviii concentrate manufactured from large human plasma pools. virus validation studies have shown virus inactivation/reduction (log 10) during the manufacturing process for lipid coated vints~ such as: h]v-1 > 16.2; psr > 16.8; vsv > 14,5; bvdv > 15.7; hcv > 4.5* and non enveloped vimsas such as: parvo** = 2.7; reo > 5.3*** and hav > 13.9. more than 50 hemophilia a patients (ptps = previously treated patients), baseline fviii activity < 1%, were included in an international drug monitoring study to follow their fviii inhibitur status. the hemophilia centers included were three centers from hungaria (helm pal children hospital and the national inst. of haematology, budapest and regional blood transfusion center, debrecen) and four centers from germany (two from berlin, one fraukfurffmain and one monster). patients were enrolled in the drug monitoring beginning aug. 1993. at the entry none of the patients had a detectable inhibitor. at the end of sept. 1995 there were no side effects or adverse events in connection with the use of haemuetin®. before the haemoctin drug monitoring study, the patients were treated with cryoprecipitate, or purified fviii products. inhibitor testing was done on patients plasma samples using the bethesda method. repeated fviii recovery determination at one time (between 12 to 24 hrs) after haemoctin® application demonstrated the expected recovery and normal half life time. none of the hemophilia a patients, treated with haemuetin® sdh developed a clinical relevant inhibitor. at the beginning of the stud)', the clinical efficacy of haemuetin® was studied in 16 hemophilia a patients and shown to give an in vivo recovery of 71 + 15 % by one stage assay and 77 + 17 % by a chromogenic assay. t ½ values were 13 + 2.8 and 12.7 + 3.2 hrs respectively. the study for the clinical efficacy of haemoctin® sdh was repeated in a group of 6 patients approximately two years later. although cd4 lymphocyte counts are known as reasonable predictors of prognosis in hiv infection, the cd4 count is not in all cases an infallible indicator of prognosis. therefore several serological markers are used to predict disease outcome, including beta-2 microglobulin (132m), immunoglobulin a (iga), lymphocyte counts (lymph) and others. in this study we followed a cohort of 23 haemophiliacs (19 with haemophilia a, 4 with haemophilia b) and 2 patients with severe von willebrands disease over a period of 28 months (mean, range: 22-34). testing for l~2m, igg, iga, igm, cd4 and cd8 cell counts (abs. and relat.), cd4/cd8 ratio, and absolute resp. relative leucocyte and lymphocyte counts was performed at least 3 times a year. at the same time clinical examinations and review of history were undertaken. mean of laboratory tests for every quarter of a year and significant changes during time of observation were calculated and correlated with clinical data. 1-4 5-8 9-12 13-16 17-20 21-24 cd41 440+956 344+924 278+925 302.-1:240 273.+.220 166+125 cd8 ~ 1165+474 1171+523 1236+1187 1017+439 930±412 873+478 1~2m z 2.0+0.6 3.0+1.0 3.0+1.2 3.0+1.1 3.5±1.0 3.5±1.3 lymph ~ 1.98+0.6 1.83+0.6 1.72+0.7 1.67±0.6 1.46±0.6 1.31±0.5 means/pl ± standard deviation means mg/l ± standard deviation during time ef observation we found significant changes of cd4 (abs. and relat.), abs. cd8 counts, cd4/cd8 ratio, f~2m, leucocytes and lymphocytes. the abs. cd4 and cd8 counts correlated clearly with lymphocytes und leucocytes counts but not with 1~2m. the prognostic value of the tested parameters is discussed by calculation of correlations with clinical data, anti-retroviral treatment and treatment of haemophilia. the availability of high purity factor concentrates has recently encouraged clinicians to use perioperative continuous infusion of fviii or fix to prevent or reduce bleeding in patients with haemophilia. in conliast to repeated highdose bolus injections, the continuous infusion trealment regime maintains constant coagulation factor activity at a level necessary for hemostasis, reducing the total cost of treatment by about 20% and preventing possible side effects of bolus doses. the new application mode, however, requires stable products which tolerate slow passage through an infusion device. our objective was to test in vitro the fviii concentrate immunate (stim plus) and the fix concentrate immi.ynine (stim plus) at room temperature, under conditions of long-term contact with polypropylene tubing in an infusion pump. infusion rates were chosen to mimic clinical situation. the control samples were not infused through the pump but were otherwise treated identically. test samples were drawn before and at 1, 4, 8, 24 and 48 hours after the onset of each infusion run. fviii (one-stage, two-stage and chromogenic assay) and fix (one-stage) activity were measured using immuno reagents. presence of activated factors were measured by napt'i', while flla, fxa, plasmin and pre-kallikrein activator were detected with specific chromogenic substrates. the data showed equivalent results between test and control samples with no loss of fviii or fix activity. the potencies of both immunate (stim plus) and immunine (stim plus) remained within 100 + 20% of labeued values within 48 hours after onset of infusion. in conclusion, immunate (stim plus) and immunine (st1m plus) are suitable for contiuous infusion when using automatic infusion device within applied test criteria. in htanans, circulating half-lives of asparaginase enzymes from e. coli and erwinia chrysanthemi vary within a wide range. moreover, half-lives differ not only among different e. coli strains but also among commercial e. coli preparations. to investigate the possible influence of two different sources of e. coil asparagmase (asn) preparations on the fibfinolytic system of leukemic children a prospective randomized study was performed correlating asn pharmacokiuetics (asn activity, asparagine depletion) with fibrinolytic parameters (plasminogen (plas), o.2-antiphismin (ct2ap), tissue-type plasminogen activator (t-pa), tissue type plasminogen activator inhibitor 1 (pal 1), d -i)imer (i)-d)). together with prednisono, vincristine and an anthracycline 20 children received i0000 iu-/m 2 asn medae r (originally purchased: kyowa hakko, kyogo japan) and 20 children 10000 iu/m 2 crasintin r (bayer, leverkusen, germany). blood samples for pharmacokinetic and coagulation analysis were drawn before the first asn administration and every third day whilst on medication. the results are shown in the 0.05 asn activity shows a negative correlation (spearman: rho/p) to plas (-,637/0.0003) and ct2ap (-, 751/0.0001). a positive correlation was found between asn activity and d -dimer formation (0.475/0.01). t-pa and pal 1 showed no relationship to asn activity. all children showed complete aspamgiue depletion at a detection limit of 0.1 um during the course of asn admiatstration. two thrombotic events occurred in the kyowa group, one of the distinctions between the two e. coli asn preparations administered ill this stndy is the absence of cystine in the kyowa asn, which also has a lower isoelectric point and a longer half-life than the bayer type a asn. with respect to this observations this may lead to longer inhibition of protein synthesis, which then may be the cause of a bigher rate of side effects. along with studies on asn pharmacokinoties dose recommendations need to be tailored to the specific asn preparation employed to ensure optimal antineoplastic efficacy while minimizing the hazard of complications. different types of coagulopathy in hepatic veno-occlusive disease (vod) and capillary leakage syn-drome (cls) after bone marrow transplantation w. ntimberger, s. eckhof-donovan, st. burdaeh and u. g6bel department for pediatric hematology and oncology, heinrich heine university medical center, diisseldoff, germany it is generally accepted, that cls, coagulation activation and refractoriness to platelet transfusions are part of the syndrome of hepatic vod. we assessed patients with either vod or cls or both vod and cls, in order to analyze the influence of either syndrome on different aspects of hemostasis. vod was diagnosed according to jones et al. [transplantation 44 (1987) 778]. diagnosis of cls was >_3% increase of body weight in the past 24 hours and non-responsiveness to furosemide [niirnberger et al., ann hematol 17 (1993) 67] . patients with vod, cls or both were compared to control patients without either diagnosis. eight patients suffered from both vod and cls, 5 patients only from vod, and 8 only from cls. 61 patients had neither syndrome and served as control population. activation of the coagulation system was assessed by increase of tat-complexes and/or increased consumption of at iil the hemostasis patterns were as follows: no. introduction: lung cancer goes along with coagulation activation and increased thromboembolic risk. acute phase reaction in cancer patients leads to elevated levels of c4b-binding protein (c4b-bp) followed by a shift from free to c4b-bp-bound protein s. we tried to find out whether there is a correlation between alterations of c4b-bp, protein c protein s system and interleukin 6 (il-6), which is one of the most potent inducers of hepatic acute phase reaction. patients: i. 25 patients with lung cancer; 2. control group: 11 patients in complete remission after lung cancer. methods: clotting methods: protein c and s activity; elisa tests: protein c antigen, tat-complexes, prothrombin fragments f i+2, il-6. electroimmuno-diffusion (laurell): free and total protein s, c4b-bp. results: tat-complexes and f i+2 were elevated in cancer patients. c4b-bp levels were slighthly increased (129±19 % of n.), protein s activity was 89±33 % of n. (control group: 108±23 % of n.). il-6 in lung cancer patients was 37.2252.9 pg/l (control: 27.2±8.2 pg/l). conclusion: one source of the hypercoagulable state in lung cancer patients is decreased protein s activity due to elevated c4b-bp levels. this is probably caused by hepatic acute phase reaction which is triggered by increased il-6 levels. these plasma levels correlate with levels of the tumor marker ca 125 and with the stage of the disease but correlations with patient outcome (disease recurrence and overall survival) have not previously been shown. plasma levels of d -dimer and ca 125 (determined by sandwich elisa assays} were measured prior to treatment in 36 women with figo stage t to iii ovarian cancer and correlated with tumor stage, relapse and overall survival over a mean follow -up period of 28 months (range 16 to 40 months). levels in 71 healthy women and 27 patients with benign ovarian disease served as controls. the occurrence of deep vein thrombosis in the cancer patients was also determined by impedance plethysmography that, when positive , was confirmed by contrast venography. preoperative d -dimer and ca i25 levels in ovarian cancer patients were statistically signfficantly higher than in controls. preoperative cut off values were calculated for the prediction of cancer relapse and survival for both measurements. d -dimer levels above a cut off level of 2060 ng/ml were statistically significantly associated with the rate of relapse but ca 125 levels were not. deep venous thrombosis occurred in 33 % of cases but there was no difference between properafive levels of d -dimer in patients who subsequently did versus did not develop deep vein thrombosis. high levels of d -dimer are associated with more advanced disease and with poor prognosis in patients with ovarian cancer. the high levels of d -dimer are a biologic feature of the malignancy itself that may be attributable, at least in part, to increased conversion of fibrinogen to fibrin in the tumor bed with subsequent degradation of fibrin by the fibrinolytic mechanism. thus d -dimer levels may serve as a marker for overall tumor burden as well as "disease activity". a high incidence of deep vein thrombosis exists in the course of the disease in ovarian cancer patients but preoperative levels of d -dimer are not predictive of this occurence. yon tempelhoff georg -friedrich, michael dietrich, dirk schneider, lothar heilmann. dept. obstet. gynecol. city hospital of ruesselsheim. -germany. an increase of plasminogen activator inhibitor activity (pai act.) in the plasma of cancer patients has been recently discribed. we have longitudinally investigated pai act. in 136 patients with primary breast cancer and compared the results with the outcome of malignancy. patients with untreated primary breast cancer and without proof of metastasis (t 1-4 n0-2 m0) were eligible for this study. in all patients coagulation tests including fibrinogen {method according to clauss), d -dimer (elisa} and pal act. (upa dependent inhibition test) were performed prior to primary operation, 6 months thereafter and at the time of cancer relapse. seventy -two healthy women and 43 patients with benign breast disease served as controls. during a mean follow -up of 32 + 16 months 34 patients (25 %) developed cancer recurrence and 13 (9.6 %) patients died. in all cancer patients preoperative levels of fibrinogen and pai act. were significantly higher compared to healthy women and to patients with benign breast disease. preoperatively only pal act. was significantly higher in patients with vs. without cancer recurrence (4.52 _+ 1.67 u/ml vs. 3.4 1 + 1.55 u/ml; p = 0.002). in patients with later recurrence pai a~t. significantly dropped 6 months after operation (p = 0.02) and was again significantly increased at the time of cancer recurrence (4.90 _+ 2.89; p = 0.001). a preoperative cut off value (calculated via cox model) of pai act. above 3.52 u/ml was significantly associated with the rate of relapse (tog rank: p = 0.0005) and in 70 % of patients who died of cancer preoperative pai act. were also above this cut off. impaired fibrinolysis in patients with breast cancer is significantly associated with the outcome of cancer. a monoclonal heparin antibody (mab) has been raised against native heparin using a heparin-bovine serum albumin conjugate prepared by reductive amination. for further analyses tyramine, which was covalently bound to low molecular mass heparin by endp0int attachment (malsch r et al: anal biochem 1994; 217: 255-264) , was labeled with 125-iodine at the aryl residue. the tracer antibody complex was immunoprecipitated by goat anti-mouse immunoglobuline igg. the mab recognized specifically intact heparin and heparin fractions. the lower detection limit of heparin preparations was 100 ng/ml. no cross reactivity of the mab occurred with other glycosaminoglycans such as heparan sulfate, dermatan sulfate, chondroitin sulfate a and c. oversulfated heparin showed lower affinity to the antibody hl.18 than 2-0-and 6-0-desulfated beparin. the method established for the purification of the mab was ammonium sulfate precipitation with followed dialysis. sds-page and high pressure capillary electrophoresis prooved the high purity of the received antibody. the biological activity of mab was tested by the chromogenic assay $2222 and remained stabile while purified. in conclusion, the present abstract describes an purified igg 1 monoclonal antibody directed against heparin and heparin fractions, which can be used for biological measurements. the concentration of heparin and dermatan sulfate in biological fluids is usually measured using radiolabeling. for this purpose aromatic compounds are usually used to insert radioactive iodine labeling at the saccharide backbone of the glycosaminoglycan. we developed methods for the specific labeling of hepann and dermatan sulfate at the terminal residue. tyramine was bound by reductive amination to the 2,5 anhydromannitosyl end of heparin, produced by nitrous acid degradation and confirmed by 13c.nm r spectroscopy. (anal biochem 217: 254-264, 1994) this method was also used to produce a low molecular mass dermatan sulfate (lmmd)derivative after partial deacatylation. in order to choose the proper method for evaluating the specific anticoagulant activity in the row of chitosan polysulphate (cp) samples with different degrees of pol3~merization and sulphation we applied to pharmaeapea article (a~) when assessing the ability of direct anticoagulants to depress the coagulability of recalcificated sheep blood (using the 3rd international heparin standard), and to measuring such acti¢ity as per pharmacokinetic model (a2). the model admits the "kinetics of cp elimination be linear in ease of intravenous injection to rabbits, as it is observed in heparin: ct=co exp(-i~ x t), where ct is cp concentration at the time moment t; co is cp concentration at the moment of injection; i~ is the elimination constant. besides, it is assumed that there is a linear approximation of the anticoagulant effect on the dose, which finally makes it possible to calculate the specific actidty a2 : t=kt ct + tin, where t is the time of clot formation at different tlme intervals after of cp injection; t~, is the time of clot formation prior to cp injection. t value was assessed in two tests: in blood coagulation time (bct) and in activated partial thromboplastin time (aptt). no correlation was observed between a1 and a2. at the same time the values of ifm and the period of semieliminatinn (tvz) with the use of the original method that were obtained with the help of the quantitative determination of cp in rabbit's blood taken at different time intervals after injection, showed a close correlation (1"=0,94 p<0,05) between the same parameters, obtained with the help of the of the pharmacokinetic model in bct test. thus, experimentally it was proved that the assumption of the linear elimination and the effect-dose dependence was true, which is necessary for a2 calculation. we recommend to use intravenous injection of the samples to animals with further assessment of the results according to the pliarmacokinetic model to calculate the specific anticoagulant activity in the row of chemically related potential direct anticoagulants. in this investigation we compared the biological activity of a low-molecular-heparm (lmw-heparin, mono embolcx®) after intravenous, subcutaneous and oral application in rats. sprague-dawly rats were anaesthetized by ketamine/diazepam and the blood samples were taken from the retro orbital sinuus. 150 axa u/kg body weight of the lmw-heparin were injected intravenously and subcutaneously to 10 rats each. between 3 minutes and 10 hours after injection serial blood samples were taken. 200 mg/kg (20.000 axa u/kg) body weight of the lmw-heparin were applicated orally using a stomach tube. blood samples were taken between 1 and 24 hours after oral application. the antifactor xa and antithrombin activities of the plasma samples were measured, using ehromogenic assays and the substances s 2222 and s 2238 (kabi vitmm). after i.v. injection the maximum axa and alia activities were 2.8 axa u/ml and 0.8 aiia u/nil respectively. after s.c. application the antifactor xa activity of the lmw-heparin showed a maximum of 0.5 axa u/ml atter 120 minutes. the antithrombin activity exhibited an eatiier maximum activity of 0.2 alia u/nil 60 minutes after injection. after the oral application no increase of the axa or alia activities was measured. the lmw-heparin has a high antifaetor xa and antithrombin activity after i.v. and s.c. injection. after oral application no activity of the lmw-heparin was measurable. these results implicate that fractionated heparin is not absorbed after oral application or is inactivated in the gastrointestinal tract. to improve the activity after oral application modified hepatins have to be synthesized. in an in vitro study the effect of various heparin derivatives (calciparin, fraxiparin, cy 222, cy 231, astenose, hexasaccharide, ssh 14) on thrombin-and adp-induced platelet aggregation as well as on adpmediated platelet activation in whole blood was investigated. all heparin derivatives caused a concentration-dependent inhibition of thrombin-induced aggregation of washed platelets. calciparin and astenose were found to be the most effective compounds with ic5o values of 0.67 and 1.2 p, mol/l, resp.; higher concentrations (5-30 times) were required for the other compounds. furthermore, the heparin derivatives were studied with regard to their potentiating effect on adp-induced platelet aggregation. in a concenwation range from 1 to 10 u/nil calciparin, fraxiparin, cy 222 and astenose led to a potentiation of the adpinduced aggregation whereas cy 231, hexasaccharide and ssh 14 did not show this effect. the increase in aggregation was associated with an increase in thromboxane a2 lbrmation. in addition, the effect of calciparin, fraxiparin, cy 222 and astenose on adp-induced platelet activation in whole blood was investigated by flow cytometric analysis using monoelonal antibodies to platelet surface receptors opiiia (cd-61) and p-selectin (cd-62). at concentrations that caused a maximum potentiation of adp-induced platelet aggregation these substances led to a strong increase of adp-mediated activation of platelets in whole blood. the effect was most pronounced when the blood was anticoagulated with calciparin and astenose, resp. in conclusion, the results suggest that the aggregation-promoting effect of heparin derivatives included in this study is dependent on the molecular weight and the degree of sulfation and is in part due to the generation of thromboxane. heparins are negatively charged polysaccharides and bind protamine forming a stable complex. here we report on the properties of microbeads (4.5 pro) coated by protamine. protamine chloride (0.16 ijm) was covalently bound to 0.5 mg paramagnetic tosyl..activated microbeads m-450 (dynal). the covalent binding of protamine was from 1.0 to 13,0 mg/g beads. protamine-dynabeads were produced in a phosphate buffer at different ph (7,0; 7,5; 8,0 and 8,5). the protamine-dynabeads produced ph 7.5 showed the best properties for flow cytometry analysis. in saline solution they bound lmm-heparin-tyramine-fitc (lmmh-tyr-fitc) dose dependently from 0.001 to 2 u/ml, whereas in plasma and blood they bound lmmh-tyr-fitc from 0.05 to 2 u/ml. dependent on the binding protocol, the microbeads also bind proteins unspecifically, i.e bovine serum albumine and protamine to a lower extent.the adsorbed proteins, however do not bind lmmh-tyr-fitc dose dependently. the saturation of the proteins on the beads was determined as their relative fluorescence intensity (rfi). in saline solution the saturation was measured at 380 rfi, in human plasma at 325 rfi and in whole blood at 252 rfi. using flow cytometry erythrocyctes, lymphocytes, monocytes and granulocytes were not bound to protamine dynabeads. these data demonstrate that protamine-dynabeads can be used to measure the concentration of lmmh-tyr-fitc in saline solution, plasma and blood because they do not bind to human blood cells. the present study was designed to investigate the anticoagulant action of inhaled low molecular weight (lmw)-heparin in healthy volunteers. 3,000 iu (group t), 9,000 iu (group 2), 27,000 iu (group 3) or 54,000 iu (group 4) lmw-heparin were given to 20 healthy volunteers each at 4 weeks intervals. in group 1 tissue iactor pathway inhibitor (tfpi) antigen and activity, 82222 chromogenic factor xa assay, heptest, aptt and thrombin clotting time (tot) remained unchanged during the 10 days observation period. in group 2 tfpi antigen and activity, aptt, tct and the $2222 method remained uneffected. heptest coagulation times were 18.7 + 2.0 before, 26.1 + 5.2 sec. 6 hrs and to 20.5 + 1.9 sec. 24 hrs after inhalation. in group 3 tfpi antigen increased from 74.1 + 13.9 to 80.5 + 14.2 ng/ml 3 hrs after inhalation. tfpi activity remained unchanged. $2222 method increased from 0.01 to 0.08 + 0.06 iu/ml 6 hrs after inhalation. heptest coagulation values were prolonged up to 42 _+ 7.6 s ec after 6 hrs and returned to normal within 72 hrs after inhalation. aptt and tct remained unchanged. after inhalation of 54,000 iu lmw-heparin, the following changes were observed: tfpi antigen increased to 103 +_. 17.9 ng/ml and normalized within 24 hrs. -i'fpi activity increased to 1.14 _+ 0.23 u 3 hrs after inhalation and was normal after 24 hrs. antifactor xa activity, as measured by s2222 method, increased to 0.343 + 0.196 u/ml after 6 hrs and was normal after 72 hrs. heptest coagulation values increased to 77.5 + 11.8 sec 6 hrs after inhalation and normalized after 144 hrs. aptt and tct did not change throughout the observation period. the data demonstrate a resorption of lmw-heparin by intrapulmonary route in man. no side effects were observed. recently we developed a tritium-labelled arachidonic acid ([3h]aa) release test with high sensitivity to membrane-toxic agents. the assay performed in u937 cells is intended to evaluate ehemicals, drugs and biomatefials with regard to their eytomembrane toxicity [kloeking et at. (1994) , toxicology in vitro 8, 775-777]. local irritation reactions are described in patients receiving therapeurieat dosages of lmw heparin. this fact prompted us to examine the following lmw hepafins and heparinoids for their membrane toxicity in u937 cells: reviparin-sodium, enoxaparine-sodium, mueopolysccharide polysulphate (mps), pentosan polysulfate sodium (pps), polysulfated bis-lactobionic acid amide derivatives lw10082 (aprosulate) and lw10086. for this purpose, [31--1]aa labelled u937 ceils were incubated with different concentrations of lmw heparins and heparinoids at 37°c for 1 hour. compared with untreated cells, the [~h]aa release of cells treated with 5 mg of the drugs was two times higher with reviparin sodium, three rimes higher with bis-lactobionic acid amide lw10086, five times higher with pentosan polysulfate, 20 times higher with ertoxaparine-sodlum, but it was equal to the control with mucopolysaccharide polysulphate. the rate of araehidonic acid release in response to a test chemical may therefore be used to assess the membrane-toxic effect of this substance and to predict its the inflammatory potential in the skin. semi-synthetic glyensaminoglycans (gags) with antithrombotic properties can be prepared from the e. coli k5 polysaecharide by coupled chemical and enzymatic methods. the molecular weight of these semi-synthetic gags can be adjusted to obtain products mimicking the molecular profile of a low molecular weight hepatm. in order to compare the biochemical and pharmacologic properties of a semi-synthetic gag (sr 80486a, sanofi/choay) with a commereiany available low molecular weight heparin, fraxiparine (sanofi, paris, france), valid biooheanical and pharmacologic methods were used. the molecular profile of this agent as determined by hplc exhibited a comparable distribution profile (mr=5.7 kda) in comparison to fraxiparine (ma=5.1 kda) . the anticoagulant properties of sr 80486a were comparable to fraxiparine in the aptt and heptest. however, in the usp assay, this agent showed slightly weaker activity. sr 80486a also exhibi~d comparable affinity to atffl and hcii. in comparison to fraxiparine, it produced a much weaker response in the hit screening system. in~ viv0 studies, sr 80486a preduecd strong dose-dependent antithrombotic actions in both the iv and sc studies in the rabbit jugular vein stasis thrombosis model (ed50=i 5-60 gg/kg). additionally, it also produced antithrombotic aefiorts in a rat jugular vein clamping model. the hemorrhagic effects of this agent were comparable to those of fraxipafine as measured in a rabbit ear blood loss model. intravenous administration of sr 80486a also revealed a comparable pharmaeokinetie behavior to fraxiparine. no abnomaiitias of the clinical chemistry (change in liver enzymes) and hematology profile (thrombocytopenia and lencecytosis, etc.) were noted in primates. at a dosage of i and 2.5 mg/kg iv, this agent also caused a release of functional tfpi which was comparable to the observed responses of other low molecular weight heparins. these studies suggest that sr 80486a is capable of producing similar pharmacologic effects as other low molecular weight heparms, however, additional optimization studies are required for demonslrating product equivalence. limited information on the comparative pharmacoldnetics of low molecular weight heparin (lmwh) is available on the data obtained from aptt, heptest, anti-xa and antmia assays. since these drugs are currently used for therapeutic indications using relatively high dosages and intravenous administration. aptt, heptest and antmia test may be valuable in the assessment of their effects. in order to investigate the relative pharmacokinetics of lmwh using apt'i', heptest, anti-xa and anti-iia methods, certoparin (sandoz, basel, switzerland) was administered to individual groups of healthy male volunteers (52-70 kg) via intravenous (30 mg) and subcutaneous (50 nag) routes in a crossover study. blood samples were drawn at 0, 5, 15, 30, 60, 90, 180, 360, 540 and 720 minutes. using a baseline pool plasma obtained from the same volunteers, calibration curves for each of the individual tests were constructed to extrapolate circulating levels of certoparin. a non-compartmental model using trapezoidal technique was used to obtain pharmacokinetic parameters such as t 1/2, vd, and clsys. in the intravenous studies, the t 1/2 was found to be dosedependent for aptt, heptest, anti-xa and antm]a. the auc, however, was significantly different for each test and was dose-dependent following the order: aptt<heptest<anti-xa<anti-iia. in the subcutaneous studies, 1the t 1/2 was both test and dose-dependent. the auc was also dose-dependent and followed the order: anti-xa>heptest>aptt>antmia. the clsys of the antma was much faster in comparison to the other tests. the clsys of the aptt and heptest was independent of dose. however, anti-xa clsys by this route was lower than other tests. the apparent vd followed the order aptt>antmia>heptest>anti-xa. the bioavailability of the certoparin as measured by various tests ranged from 81-119%. these studies suggest that beside providing pharmacokinetic data, aptt, heptest and anti-iia assays may provide useful data on thier safety and efficacy at high dosages. the immunological type of heparin associated thrombocytopenla (hat ii) is a severe complication of heparin treatment and is associated with arterial and venous thrombosis. only patients with absolute thrombocytopenia have prompted suspicion of hat in clinical practice. we report on a 44 year old male, who developed thromboembolic episodes after coronary angiography like reinfarction and thrombotic episodes of a. brachialis. fibrinolytic therapy combined with i.v. unffactionated heparin treatment was the therapy of choice and was followed by severe fua~er thromboembolic adverse effects. besides an impaired fibrinolytic response and elevated antiphospholipid anitbodies, we diagnosed hat type ii in hipa and elisa (stago-boehringer, marmheim). this special patient had platelet counts within a normal range, when developing the thromboembolic episodes. it appears that the normal platelet count during the thromboembolic episodes reflect a relative thrombocytopenia. from a clinical point of view we recommend the use of a lab panel to exclude hat type ii in patients with thromboembolic episodes under therapy with fractionated or unfractionated hepafin. platelet counts within a normal range are no absolute exclusion criterion for hat ii. low molecular weight heparins (lmwhs) are now commonly used for the prophylaxis of post-surgical thromboembolic complications. in this indication, lmwhs are administered as a single or twice a day subcutaneous regimen. usually these agents are administered at 30-40 mg total dose which is equal to 3000-4000 anti-xa (axa) iu. newer methods such as ehromogenic substrate based axa methods and the heptest clotting time can be used to determine the effects of lmwhs during the initial phases of prophylactic therapy. this may be useful in the elderly and weight compromised patients where a fixed dosage may not be optimal and may produce bleeding effects. similarly in the overweight patients, a fixed dose may not be efficacious. thus, monitoring of lmwhs in these patients may be useful in the optimization of their therapy. lmwhs are also used in the treatment of deep vein thrombosis using both intravenous and subcutaneous protocols. high dosages of up to 200 mg sc/day and infusions of up to 30 axa iu/kg/hr have been administered. in these conditions, the monitoring of the circulating lmwh levels may be useful in optimizing the dosage. we have modified the aca heparin (do_pont merck, wilmington, de) method to measure the lmwh levels in the plasma of patients treated with both the prophylactic and therapeutic dosage. owing to the required turnaround time, simple operation and reliable results, this method was found to be of value in the monitoring of these agents. this presentation provides an overview of the clinical application of various lmwhs with particular reference to the need of monitoring for their effects to optimize the clinical outcome. a double-blind, multicentric, controlled trial was performed in order to compare the antithrombotic efficacy and safety of single daily doses of 5000 ie anti-xa of low molecular weight heparin (lmwh) sandoz (certoparin) and 5000 ie unfractionated heparin (ufh) tid. in 288 patients undergoing elective total hip replacement blood samples were drawn before the first subcutaneous injection of lmwh or ufh resp., two hours after administration on the first and 7th postop, day and on the last day of prophylaxis (day 10-14), anti-xaactivity was measured by chromogenic substrate assay, heptest and aptt by clotting assays and tissue factor pathway inhibitor (tfpi) and heparin-pf4-antibodies by elisa techiques. as expected, the anti-xa-activity and the heptest values were significantly higher in the lmwh-group at all time points after administration of the drugs; the mean values of heptest were 35 sec in the ufh-and 75 sec in the lmwh-group respectively, the aptt was not different in both groups. at the end of prophylaxis positive antibodies to heparin-pf4complexes were detected ~n both groups; this however was not correlated with clinical thrombocytopenia. a detailed correlation between patients with deep vein thrombosis (dvt) and positive antibodies has still to be done (all patients were screened for asymptomatic dvt between day 10-14 by bilateral phlebography. tfpi was markedly increased in the lmwh-and only slightly elevated in the ufh-group; the differences are statistically significant. summarizing it can be concluded that antibodies to heparin-pf4complexes may occur without clinical symptoms of hepafin-induced thrombocytopenia type ii and that tfpi may play a sigificant role for the antithrombotic efficacy of ufh and lmwh. unfractienated heparin represents one of the most severe and frequent causes of drug-induced thrombooytopenia. heparin-indueed thrombocytopeala (hit) occurring early in therapy is often mild and serf-limited, appearing to be caused by a direct aggregant effect of heparin on platelets (hit type i). hit type ii, however, is immune-related an may result in absolute thrombocytopenia (platelet count <i50/~l) or a relative decrease in platelet count (?_50% reduction from baseline values). unlike most other immune thrombecytopenias in which bleeding complications often predominate, hit type ii may results in severe arterial and/or venous thromboses. the management of patients with hit type ii and thrombosis is often difficult. heparin must be stopped immediately. antithrombotic, fibfinolytic, or antiplatelet agents have been used in these situations, as have anticoagulants other than unfraetionated hepafirl plasmapheresis, used in other immune-related disorders, has also been occasionally reported. at our institution this intervention appears to provide significant elininal benefit to hit patients. several recent reports have noted that the heparin-dependent antibodies in hit type ii are directed against a complex of heparin bound to platelet factor 4 (pf4) and an elisa-based test system for hepafin-pf4 eomplexinduced antibodies (hpia; stago) has been develuped~ we report our experience in 3 patients with hit type ii in whom plasmapheresis was instituted and the antibody levels in blood were measured pre-and post-treatment to identify wkether circulating antibody titers decreased thus providing some explanation for the clinical benefit. the patients developed profound absolute thrombocytopenia (range, 3-21 k/~tl) and positive heparin-induced platetet aggregation studies following treamlent with uofractionated heparin. thromh0cytopenia induced by hepariu (hat-syndrome) typically appears several days after initiation of heparin therapy. this phenomenon is caused by antibodies of the lgg type which are induced by a heparin-pf 4-complex and which also activate platelets. in a recent study (n engl j med 1995; 332:1330-5) the hat-syndrom was diagnosed in 2.7 % (9 of 332) patients who had received unfractionated heparin (ufh) and in none of those 333 patients who had been treated with low-molecular-weight heparin (lmwh). 8 out of the 9 patients treated with ufh presented with at least one thrombotic event (7 times a venous, once an arterial event). the incidence of heparin-dependent igg antibodies (7.8 %) was higher in the ufh treated group than in patients treated with lmwh (2.2 %). our patient was a male caucasian white, 62 years of age, otherwise in good clinical condition with no severe diseases in his pmh. both parents, however, had suffered several thrombotic episodes. the patient was admitted to the hospital in september "95 with clinical signs of a deep vein thrombosis (dvt) of the right leg. the diagnosis was phiebographically confirmed. after having given informed consent the patient was treated with 5 cycles of r-tpa (loco-regional application) and 3 cycles of uhsk as part of a clinical trial (uhsk vs. rtpa, boehringer/m). lysis was successful with complete recanalisation of the veins. after completion of lysis the patient was further anticoagulated with coumarin overlapped by heparin (ufh). as a complication the patient suffered from a retroperitoneal and an upper gi4ract bleeding from esophagitis grade ii to hi while being on coumarin prophylaxis. therefore the patient was put on lmwh and then discharged with this prophylaxis. after less than two weeks the patient presented again to the hospital with a relapse of dvt in the same leg. phiebographically nearly all deep veins were occluded to a higher degree than before, i. e. also with concomitant pelvic vein affection and with hea~ 3, swelling of the whole leg. blood count showed a marked decrease of platelets to 27/nl. the platelet aggregation test demonstrated a hat-syndrome which was confirmed by the hipa-assay. the heparinoid orgaran® was not reactive in the assay. having seen a previous bleeding episode in this patient, we did not see an indication for another b'sis therapy. therefore we decided to put the patient on an intermediate dose of coumarin overlapped by hiradin (hat study, behring) instead of orgaran®, since hirudin allows a much more precise dose adjustment. after 10 days the patient was discharged with no further complications. risk factors for dvt have not been detected until now. to our knowledge this patient is one of the first cases of hat-syndrome induced by lmveh. under routine conditions aptt is the most frequently used test method for the surveillance of patients undergoing hepann therapy. in literature a 1,5 fold to 2,5 fold prolongation of the initial value is recommended for individual therapy a~ixatian. in contrast to the control of the enmarin therapy by means of prothrombin time measurement the individual hupafin sensitivity of the ~ reagents of this standard value hardly receives notice. commercially marketed ~ reagents differ from one another method and concentration of the activator as well as their phosphelipids. when choosing a reagent the user must only take into consideration the sensitivity against its factors, heparin and lupus but also the adalxability of the reagent towards the given measurement system ( software variability towards incubation time, sodimentalion problems in reagents with high kaolin concentration). reagents offering a high heparin sensitivity already are showing a prolongation of the coagulation time at low heparin dosage (0.1 -0.3 u/ml). in the therapeutic range (0.4 -0.8 u/ml) very often no linear relationship between prolongation of the aptr and heparin concentration level is found_ therefore very often coagulation times above the routine measuring range (180 s) can be observed in this range. moreover, various medical measuring devices differ in their ability to recogniso the retarded entry of coagulation. coagulation limes with different apq't reagents in correlation with the heparin concentration level can be found in the adjoined table. significantiy prolonged coagulation ti.~es of aptt lead to a reduction of the hel~ dose in clinical practice. due to the discrepancy between heparin concentmon level and the prolongation of the afrt in reagents with a non-linear heparin sensitivity a reduction of heparin dosage leads to an inmt~cient anticeagulalion and the risk of further thrombosis. therefore the producers should be asked to include precise directions on the heparin sensitivity of the aptt reagent in the therapeutic range in order to enable an optimal surveillance of the heparin therapy. a coordination between clinic and laboratory should be compulsory for the choosing of the aptt reagent. introduction: hat is a rare but sometimes fatal complication of therapy with unfractionated (ufh) and low molecular weight beparins (lmwh). we report on our experience with the clinical coume and management in 12 patients (pts). in all cases a severe decline of platelet count and in 11 cases a positive hipa test (= heparin-induced platelet antibodies) were documented. patients, clinical course and outcome: 8 female and 4 male patients (median age 68 years, range 22-78) were analysed in this retrospective study. 9 pts were referred from outside hospitals. all pts had been treated with ufh (5 pts additionally with lmwh) for prophylaxis (n = 9. in 2 pts pedoperatively) or treatment (n = 3) of venous thromboembolism (v'te). in 11 pts at least one vte occured during heparin therapy, in all cases prior to hat-diagnosis (vte: n = 13; putmonary embolism: n = 6; artenal embolism n = 1; thrombosis of sinus transversus n = 1). hat was suspected 16.5 days (median; range 12 -48) after first heparin application. hepadn therapy was discontinued immediately, at that time the median plateiet count was 35 g/i (range 10 -117). 9 pts were treated with orgaran r, followed by recombinant hirudin (r-hirudin, behdngwerke ag: clinical study) in 4 cases; 3 pts were treated exclusively with r-hirudin. plat~let count normalized in 9 pts within 3 days (median; range 1-10). one patient in the orgaran" group died on recurrent pulmonary embolism. 11 pts recovered without any further thromboembolic complication. conclusion: in our small series of 10ts hat was complicated with vte in 11/12 cases. in many pts hat was diagnosed strikingly late. for early diagnosis careful monitonng of platelet counts are mandatory dunng hepadn therapy, both orgaran ~ and r-hirudin seem to be safe and effective in preventing further thromboembolism, p 179 to prospectively evaluate coagulation or fibrinolytic activation in children long-term anticoagulation with lmwh was administered a longitudinal study was perfomed. within a 30 months period 48 children and adolescents (6 -19 years) suffering from malignant bone tumors (ewing's sarcoma or osteosarcoma) received enoxaparin (clexane r/rhone-poulenc rorer, france) for primary propylams. the majority of patients received 0.6 mg/kg bw enoxaparin 12 hours before major surgery (limb salavage). in addition, ten neonates and children received enoxaparin in the same dosage for secondary prophylaxis. in both groups therapy was adjusted to 0.2 -0.4 iu/ml anti xa activity. median (range) duration of therapy was performed for l0 (6 -24) weeks. before ('1"1), one (t2) and 3 weeks (t3) within the context of the present study an investigation on the physiology of clotting was conducted in three groups each consisting of 12 patients, who had to undergo a total end-prosthetic hip implantation (teph) for the first time. criterion for allocation to a particular group was type of intraoperative transfusion the patient received : infusion of whole blood, erythrocyte concentrate or fresh frozen plasma. at eight fixed times the activity or concentration of the components of the kallikrein-kinin and inhibitory systems was determined. the statistic evaluation ensued with the spss software package. for all three patient collectives an activation of both systems was to be observed with the teph-implantation whereby there were no statistically striking differences between the three groups. thus in the case of the kallikrein-like activity an intraoperative rise of over 37% was registered, while at the same point in time there was only a 4% reduction in the prekallikrein activity. postoperatively a 10% rise in the prekallikrein activity was to be observed. as a reserve inhibitor alpha 2-macroglobalin showed no significant change in activity during the period of observation whereas the acute phase proteins alpha 1-proteinase inhibitor and cl-esterase-inhibitor yielded a postoperative rise of 135% and up to 100%. the beta-factor xllainhibition showed a rise of 121% during the reconvalescent phase. the 65 year old female caucasian patient presented here, was admitted to a nearby district hospital in december '94 with the acute symptoms of pulmonary embolism. the source of the embolus could not be detected. high dose heparin was administered. pmi-i revealed a coronary artery disease and a peripheral artery occlusive disease of the left lower leg, yet blood pressure values of the lower ex~emities were only slightly below brachial pressures. the first episode of pulmonary embolism was followed by a second one two weeks later for which the patient received heparin again. following this treatment the platelet count decreased significantly to 10/ni. with an acute thrombosis of the lower caval vein and with leriche's syndrome, i. e. occlusion of the abdominal antra from the bifurcation down to the ileofemoral arteries bilaterally, the patient was transferred to our hospital. on initial examination we saw a patient in no acute distress, no dyspnea, blood pressure 120/80 mmhg, pulse 94/rain, no peripheral edema or disturbances of sensitivity. neither femoral, popliteal, nor pedic pulses were palpable on either side. a heparin associated thrombopenia was detected with the platelet aggregation assay and confirmed by the heparin induced platelet aggregation assay (hipaa). the autoantibodies targeting the underlying pf4-heparin complex were determined later by an elisa method in serum samples of the patient (asserachrom hia, diagnostica st,ago). a temporary anth&~r filter system was inserted transbmchially into the lower eaval vein and placed jnst above the thrombus. in the following days, 2 cycles of lysis therapy with 9 mil u streptokinase were administered taking six hours for each cycle. heparin was given in the mean time via the filter system in therapeutic dosages in order to prolong values 1.5 to 2 times the individual base line level. at the end of the second cycle, pulses were palpable inguinally, two hours later both popliteal and the right pedic pulse were present. the left leg showed better microperfusion than before. computer tumoglaphy did not reveal an)-thrombotic material in the descending aorta and the iliac arteries. for continuous anticoagulation after lysis we used the heparinoid orgaran® to overlap the period until full anticoagulation was achieved by coumarin. the heparinoid was tested before us non-reactlve in the hipaa. with this prophylaxis the platelet count increased to normal values. hat ii related thrombosis is the most severe complication of heparin therapy, especially in patients with pre-existing thromoembolic disorders (dvt, dic). we present to our knowledge the first case of hat ii during thrombolytic therapy (it) with simultaneous hepatin administration. at admission a 16 year old girl showed complete occlusion of the pelvic veins of the right leg (r common and external iliac v., common and proximal superficial femoral v.). as in children no prospective studies on the thrornbolysis of dvt with rt-pa are available, we started tt according to the frankfurt/m. regimen: rt-pa (actilyse) bolus injection 0.4 nag/kg bw followed by continuous infusion of 1 mg/kg bw/d; simultaneous low dose heparin 100 -200 u/kg bw/d. doppler ultrasonographie control on day nine after the beginning of tr revealed a further distal growth of the thrombus into the popliteal vein. at the same time at ir was decreased to 70% and there was a dramatic drop in platelet count to <50% of the baseline value (216 to 86 gpt/1). the diagnosis hat ]i was confirmed by positive hipa-test. the only heparin preparation not cross-reacting with the antibody was the lmw heparinoid orgaran. heparin therefore was replaced by orgaran: bolus injection 1,500 u/h followed by infusion of 200 u/h. rt-pa-therapy was not discontinued. at ri substitution was performed at levels <70%. under orgaran administration platelet count rose from 77 to 239 gpt/1 within 5 days. on day 12 complete reperfusion was achieved and rt-pa was discontinued ,on day 14. the patient was discharged from the hospital on prophylaxis with phenprocomnon in good general condition at admission thrombophilia parameters (protein c and s, apc) were within normal range. heparin induced thrombocgopenia (hit) is a serious but rare side effect of treatment with unfractionated heparin. patients with hit present with a rapid decrease in thrombocyte count and occurence of venous or arterial thrombosis under heparin treatment. patients with arterial occlusive disease who need surgical intervention are of high risk to develop rethrombosis especially during the first few postoperative days while they are under i.v. heparin-treatment. in our study we investigated prospectively all patients who were admitted to a surgical unit because of arterial occlusive disease and received surgical intervention. 15 patients were screened on the 3rd and 6th postoperative day for heparin-antiplatelet 4-antibodies, decrease in platelet count and thromboembolic complications. 3 patients developed antibodies against heparin-platelet factor 4. one of the 3 patients developed a venous thrombosis in the operated leg in combination with a decrease in thrombocyte count. we conclude that heparin-platelet factor 4-antibodies are often produced in patients under i.v. heparin treatment. only few of these patients actually suffer from clinically s3anptomatic hit. medizinische universit~itsklinik, institut far klinische biochemie und pathobiochemie, d-97080 wtirzburg, germany the important physiological and pathophysiological role of platetets in health and disease is well established. platelets are also the primary target of many vasoactive hormones, factors and drugs. in addition, platelets provide a very important model system for studying agonist-and antagonist-mediated signal transduction events (1,2). platelet activation by agonists is associated with shape change, extension of filopodia, generation of intracellular messengers, secondary agonists and activation of adhesion receptors and integrins including the fibrinogen receptor gp iib/iiia. activation of protein tyrosine ldnases and calcium-dependent protein kinases appear to be of critical importance for the mechanism of platelet activation. in contrast, platelet inhibition by vasoactive factors and drugs is often mediated by the camp and/or cgmp signal transduction cascades. recent data form our laboratory suggest that the focal adhesion protein vasp, a major target of the intracellular no/cgmp and prostacyclin/camp effector systems, is an important link between signal transduction pathways and elements controlling cell motility (3-6). the implications of the recent advances in platelet signal transduction research for understanding the physiology, pathophysiology and pharmacology of human platelets will be discussed. domain. however, in vivo truncated "i'po is much less potent than full length tpo due to its rapid clearance. this suggest that the c-terminal glycosylated domain acts to stabilize circulating tpo. tpo stimulates both proliferation of progenitor megakaryocytes and their maturation to platelet producing megakaryocytes in vitro. in vivo tpo induces dramatic increa~ses in megakaryocyte number and platelet production in animals, indicating that tpo regulates both thrombopoiesis and megakaryocytopoiesis and is the primary regulator of physiological platelet production. this later notion is supported by the phenotype of ti~ and cmpl knockout mice in which platelet levels are only 10-20% of normal while other blood lineages are unaffected. in animal models of myetosuppresion ,and marrow transplant, tpo reduces the platelet nadir seen in the~se models and significantly accelerates platelet recovery, tpo also has profound effects on red cell recovery in these models. these results suggest that tpo will be clinically u~ful for the treatment of thrombocytopenia in caucer patients who undergo chemotherapy or bone marrow transplant. the haparg-study ( .~a.emostatic parameters as risk factors in healthy volunteers) was sponsored by the german ministry of research and started in 1984. a previous study ( pard, intern. angiology 5, 181-195,1986 ) had shown that spontaneously enhanced platelet aggregation as measured with the pat-ill-test and a high fibrinogen level were significant risk factors for new vascular occlusions in diabetic patients. it was the aim of the haparg study to establish if spontaneous plateletsggregation and other hemostatic variables are independent risk factors for vascular occlusions also in healthy volunteers. employees of hschst ag, frankfurt aged 40-65 years and personnel of the university of mainz, aged 30-60 y. entered this prospective study. besides anamnestic data on smoking, hypertension and diabetes blood pressure, the ankle/arm doppler -index and an ecg were recorded and serum cholesterol, hdl, ldl, triglycerides, uric acid and glucose were measured. 1885 men and 988 women entered the study and were followed for 4-6 years. during this period 53 vascular occlusions ocoured ( 36 coronary and 9 cerebral events and 8 peripheral vascular occlusions). only three of these events occured in women. besides age and gender as expected risk factors the multivariate logistic stepwise regression analysis revealed as significant risk factors slightly elevated levels of blood glucose (p=0.001), smoking (p=0.0013), an elevated diastolic blood pressure (p=0,018),followed by spontaneous aggregation (p = 0.017) and higher hdl-levels (p=0.034) as significant risk factors for men. higher fibrinogen levels just missed significance in this analysis (p=0.059). none of the other hemostatic parameters showed any significant correlation with the vascular events.to our knowledge this has been the first prospective trial in a large population of healthy individuals in which a platelet function parameter has been studied together with other possible risk factors. spontaneously enhanced platelet aggregation seems to be an independent risk factor and may be an indicator of an ongoing active atherosclerotic process. mild bleeding symptoms -petechia, prolonged bleeding after tooth extraction -were observed in a patient with normal coagulation tests but slightly disturbed phtelet function: decreased aggregation in response to adp, pal: and thrombin receptor-derived peptide (trap-6). platelet count, agglutination with both ristocetin and botrocetin and thrombininduced aggregation were in the normal range. an abnormal membrane glycoprotein detected in electrophoretic studies of the patient's platelet proteins was shown to be related to gp llxx by immunoblotting. the variant gp differed even from the largest normal polymorphic gp iba form a by an increase in molecular mass, platelets of the patient contain the variant and normal c form in equal amounts. the quantity of gp ib as detected by flow cytometry is normal. lmmunoblotting studies of proteolytic fragments of the abnormal gp indicated that the defect is located in the c-terminal part of the glycocalicin fragment. measurement of the length of glycocalicin molecules after glycerol-spraying rotary shadowing electron microscopy demonstrated two populations of molecules in the patient sample: a normal on with an average size of 470 angstr6m and a fraction with an average size 0f650 angstr6n~ preliminary results of molecular analysis of the gp iba gene in the patient confirm the localization of the genetic defect by protein studies. in summary, the patient is heterozygous for a hitherto undescdbed molecular variant ofgp ~a. barnes and co-workers have shown that simple triple-helical collagenlike peptides, comprising basically a repeat gly-pro-hyp structure, are highly platelet reactive in polymeric form. they additionally have shown that the activity does not involve gpla/lla (integdn c~z~l). platelets adhere under static conditions to these fibrillar peptides mainly in a bivalent-cation independent manner; the bivalent cation dependent adhesion can be inhibited by an antibody against gpflb/llla (integfin m~d33). however, platelets of a patient with type i glanzmann's thrombasthenia secreted their ¢z-granule-and dense body content just like normal controls while activated with the triple-helical gly-pro-hyp peptide. the lack of an essential involvement of cd36 was shown with cd36deficient platelets, which respond to the peptides as readily as normal platelets when fibdnogen binding and cd62 and cd63 expression were measured. to study the involvement of the yon willebrand factor (vwf) platetets from a patient with severe type 3 vwd (vwf not detectable in platelets and plasma) were investigated. the vwf deficient ptatelets responded quite normally to the fibritlar collagen-like peptide. addition of pudfied vwf to the vwf-deficient platelets before adding the paptide raised the platelet respond slightly we conclude that neither ~3~ integrin, nor m~ integdn, nor cd36, nor vwf are the pdmary platelet receptor for the collagen-like peptide. there is evidence that native low density lipoprotein (ldl) can be oxidatively modified in vivo and the oxidized ldl may contribute to thrombogenesis and atherogenesis by damaging endothelial cells, eliciting vasocontractiola and stimulating blood platelets. by means of biochemical methods, electron microscopy, reflection contrast microscopy and computer image analysis, we find that oxidized ldl, compared to native ldl, affects platelets at least in the following five aspects. 1) oxidized ldl alters platelet functional morphology in a time and dose dependent manner. 2) oxidized ldl induces secretion of serotonin from both morphological resting platelets and shape changed platelets. 3) oxidized ldl advances, accelerates and enlarges platelet adhesion. 4) oxidized ldl causes cytodamage in platelets and cultured endothelial cells. 5) oxidized ldl stimulates platelets and endothelium, which leads to a formation of microthrombi on the monolayer of human umbilical vein endothelial cells. further experiments show that oxidized ldl inhibits the activity of ca2+-atpase in purified plasma membranes of platelets, and therefore increases the cytosolic calcium level in platelets. these results clearly indicate that calcium signaling pathway is intimately involved in the mechanism of platelet activation induced by oxidized ldl. since oxidized ldl, a relevant platelet agonist, has been confirmed to be present in vivo, further studies on its role in platelet activation and plateletendothelium-interaction can help for better understanding thrombogenesis and atherogenesis. recantly physical parameters such as sheer stress, hypo-or byperoxic conditions and hyperthermia have been shown to modulate endothelial cell dependent fibrinolysis. in this study we investigated whether yet another physie~ stimulus namely hypothermia might have an impact on the expression of components of the fibdnolytic system of human endothelial cells in culture. confluent cultures of human umbilical vein endothelial cells (hlivecs) were exposed to gc ° for 10, 20, 40, 80, or 160 rain, respectively. control cultures were exposed to 37°c for the same time periods. thereafter medium was removed, fresh medium was added and the cells were incubated for 24 hours at 37°c conditioned media (cm) were harvested and plasminogen activator inhibitor-i (pal-i) antigen was determined by a specific elisa. our data showed that huvecs responded to exposure to 8°c with a time-dependent increase of pai-i in cm. maximum induction of pal-! antigen was seen after 20 minutes exposure to 8°c (pal-! antigen: 754_+.83 ng/105 cells for treated cells; 4202_17 ng/105 cells for control cells). the inereese in pal-1 antigen was also reflected on the level of specific mrna synthesis as evidenced by northern blotting which showed a twofold increase in pai-i specific mrna in huvecs exposed to 8°c as compared to control cells. in conclusion, our data gives evidence that hypothermic stress like several other forms of physiological or pathological stress, results in activation of endothelial ceils as evidenced by an increased expression of pai-i. our findings provide ~rther evidence that such activation of endothelial cells can be induced not only by biochemical mediators but also by physical stimuli. nitric oxide (no) is formed from intracellular l-arginine by two no synthase (nos) isoforms: a ca+ +-dependent constitutive form (cnos) and a ca+÷-independent inducible form (inos) which is expressed after challenge of cells with immunologic or inflammatory stimuli. endothelial cell derived no is an important signaling molecule inducing smooth muscle cell relaxation and platelet inhibition, however its role in cell proliferation is poorly understood. the present study investigates the pattern of nos isoforms and no production in actively proliferating low density human umbilical vein endothelial cells (ld-huvec) and in high density monolayers of quiescent cells (hd-huvec). ld-huvec but not hd-huvec expressed inos (measured as ca ++independent citrulline formation from l-arginine in cell lysates) with a maximal activity of 30-40 pmoles/min/mg protein, cnos (ca +÷dependent citrulline formation) was demonstrated in hd-huvec (0.02-0.2 pmoles/min/mg) but not measurable in ld-huvec lysates. lmmunoprecipitation of cell lysate with a mab against murine macrophage inos decreased inos activity of supernatant by 70% in ld-huvec. immunoprecipitation with an anti-ecnos mab did not modify inos activity in ld-huvec but abolished cnos activity in hd-huvec. no release (measured as nitrite + nitrate production in an 18 h incubation in the presence of 1 mm l-arginine) was 1.9 nmol/mg cell protein in the supernatant of hd-huvec whereas it was t0fold higher with ld-huvec. measurement of intracellular cgmp after stimulation of cells with i-2 #m ionomycin (a sensitive index of enos activity) showed a 400-800% increase of basal levels in hd-huvec but only 50-150% increase in ld-huvec. on the contrary, sodium nitroprusside-induced cgmp increase was similar in non-confluent and confluent cells. the expression of inos and the high no production in ld-huvec suggest a role of endogenous no in mediating cell proliferation. indeed, the nos inhibitors l-nmma and l-canavanine inhibited [~h]thymidine incorporation in ld-huvec by 60%. on the ++ nt no r uctlon ar x r d m other hand enos and ca -depende p od " e e p esse " differentiated huvec and correspond to the ability of the cells to regulate vascular tone and platelet activation. the processes of replication, maturation and motility of progenitor cells of different lineages in the bone marrow are tightly regulated, particulady by various cellular contacts and their modulation involving pericellular proteolysis. while mononuclear phagocytes and related cell lines are well characterized for surface localized plasminogen activation by receptor bound urokinase, megakaryocytes are poorly described in this respect. in the present study the expression of mrna of urokinase receptor in megakaryoblastic cell lines chrf-280, dami and meg01 was found at a low basal level and increased 5-10 fold after pma-treatment. urokinase receptor mrna levels remained differentiation dependent elevated for up to 5 days. functional analysis of upar expression on the cell surface was documented immunologically by facs-anatysis and on the functional level by direct binding of radio-labeled amine-terminal fragment of urokjnase and mirrawed the increase in urakinase recptar mrna level. as expected, antibody and ligand binding was sensitive towards treatment with phosphatidyl-inositol-specific phospholipase c, since the urokinase receptor belongs to the group of gpi-anchored receptors. in cultered megakaryocytes derived from bone marrow urokinase receptor mrna was demonstrated by rt-pcr, as well. these results indicate a differentiation-dependent increase of urokinase receptor expression on cells of megakaryoblastic cell lineage suggesting the participation of urokinase dependent events during maturation process and intravasation of these platelet precurors. regulation of am gene expression may be coupled to oxidative stress through redox sensitive transcriptional regulatory factors. the aim of this study was to investigate the influence of selected antioxidants on tnfa (100 u/ml, 6 h)-induced surface expression of elam-i, icam-i and vcam-i in human umbilical vein endothelial cells. am expression was determined by quantitative flow cytometry and immunohistochemistry (alkaline phosphatase anti-alkaline phosphatase method). the following antioxidants were tested: pyrrolidine dithiocarbamate (pdtc), n-acetylcysteine (nac), glutathionemonoethyl ester (gshmee), probucol and the estradiol-derived scavestrogens j824, j861, j901. additionally, 17fi-estradiol was examined. the used concentrations were 1-5 mm for nac and gshmee and 1-100 ~m for all other substances. according to their influence on tnf-induced am expression, the tested antioxidants can be divided into three groups: 1) pdtc, nac and gshmee inhibit the expression of all three am, 2) j861 and j824 reduce vcam-i and icam-i and increase elam-i, 3) j901 and probucol show no effect. 17fl-estradiol elicits a potentiation of tnfa-induced expression of all three am. tnfa-induced vcam-i expression was most effectively inhibited by ptdc and j861 (100 em: 68% and 78% inhibition, respectively). the best inhibitors of icam-i induction were pdtc (100 ~m: 51% inhibition) and j824 (100 t~m: 47 % inhibition). j824 and j861 most effectively increased the elam-t induction (100/~m: 85 % and 62 %, respectively). the results confirm that antioxidant-sensitive mechanisms play a role in tnfct-induced am upregulation. it is suggested that icam-1-and vcam-l-expression is regulated differently from elam-i. the diversity of antioxidant effects might be due to distinct levels of redox control of transcription with an antioxidant-inhibited and an antioxidant-promoted step. heamophiliacs who continue to produce tilers of f.v11i inhibitors no higher than 5 to 10 bethesda units (bu) when given continuous f,viii replacement therapy are classified as low responders. studies carried out on low responders to assess the effect of immune tole~lcc therapy flit) in these patients. it was found that elimination ofiahibitors in low responders could be achieved using f.vlii at 20 to too u/kg given every 2 to 3 days over a period of 6 weeks. we consider that 1tt is justified in this group of patients. prevents rebooster effects and reduces elimimfion time of f.viii inhibitors. it also reduces the enhanced mortality in patients with inhibitors and has been shown to be cost-effective. in view of the improved prognosis, we recontmend starting itt in low respor, ders soon after the diagnosis has been made in order to minimize f.vill exposure before isfi.tiating el'. itr in low responders has a better success rate an.,d requires shorter u'eatment than itt in high responders. w.schramm, med.klin~, innenstadt, lmu, mfinchen following replacement therapie~ in hemphilm t_be developmem of inhibimrs to the p*ficat's deficient factor is the most serious sequelae. the incidence of inhibitors varies between 15 and 35 % of persons with hemophilia a. inhibitor develop, mostly in paticm~ with sovere bemoph/lia a, early in life and afmr few exposure days (9 -12 days). using the bethesdamethod the inh~itom can be derided in low (< 5 bu) and high msponder (>5 bu) hemopb~iacs with high fitcrs have ~ually serious ~ problems. they are resistent to mg,,flary replacement therapy, the ~ goal in the treawnent is to control severn acum bleedin~ and to eradicate the inlu'bitor perrmnanfly and to induce tolea'ance. in the tream'tmt of acute blcedings in patients with hlhibitors factor viii inhibitor bypassing ag~ts like activated prothro~ complex concenuxtes (feiba) or prothrombin complex concentrates (pcc) arc mostly used. the meehani~n of aefiou of theses concentrates is net fully investigated. their effect is usually related to the high coment of activated clotting factors ~d phosphoupids. since some years acdwated recombinant factor vii (f vii a) is used to treat patients with inl'dbitocs successfully in several clinical situations including surgery. in addition porcine factor vii1 is widely used in particular in the uk for the treatment of factor v].ii inhibitor patients and could demonstrade good clir3cal results, in case of life threatening bleedings a temporary reducfic~ of inhihitors could be. ~hieved by using extem*,ivc plasma exchange (protein a adsorption) and immune suppression with cyclophosphamid (~alm6 protocol). follow~g the first description by h. bmc~'~mn some modifications for tlm induction of irmnune tolerance in hemophilia a patients have ~en propet'ed. these schedule, can be derided into high, intemxxfime and low dosage roglrmms di:ffea'jng in the dosage of factor viii infused. successful rates about 70 to go % can be obtained with ~ and high dose regimens. but is has to be co~sidered that the~ expensive trea.t~nt regimens have a great physical and p .syc.hosocial impact to the benx~-li~s and thch" farm~e& the different immu~ mler-a.~ze mg~-'~ predominantly used in high rcsponder inhibitor. most of the patients with low concentrations of inhibitors cm be managed with factor viii in increased dosage. this is in agreement with the consensus recorrnr~rdadons for u'eatlncnt hemophiliacs in germany fi'orn 1994. before vitamin k(vk) prophylaxis was generally accepted in japan, the incidence of infantile vk deficiency was 1:4000 both idiopathic and secondary types. since 1981, 4 nationwide surveys have been conducted. the current incidence rate is now about one-tenth that in early 1980. however, in a small number of eases, vk deficiency oceured despite prophylactic administration during the neonatal period. in order to clarify the absorption,excretion and transplacental transport of vk in the perinatal period,following studies were carried out. t)hepaplastintest(normotest) were performed on 65 women in the last stage of pregnancy and each coagulation factor was estimated as well. 2)correlations were made between mothers'and babies'hepaplastin test values. 3)transplacental transport of vk 2 was studied. the general activity of vk dependent factors in pregnant women was much higher than in non pregnant women. as far as the correlation between mothers'venous blood during delivery and cordvenous blood is concerned, in the group of mothers with hepaplastin test value of less than 120% of the normal adult value, the value of the hepaplastintest was less than 30 % of normal adult value in the cord venous blood° we also demonstrated that vk passed through the placenta but only in small qualities. 61hiv-negative patients (median age 4]yrs, 13-70}, formerly treated with non-virusinactivated coagulation products, underwent hepatologic examination, including afp screening and sonography. 31.suffer from severe, 20 from moderate or mild haemophilia a or b, 10 from other severe coagulation factor deficiencies. 52 had been treated with products of the swiss red cross (src) only (28 with small pool cryoprecipitate}, 4 with foreign products only, 5 with both src and foreign products. treatment intensity was variable with>20'000 iu/yr in 26,< 20'000 in ]6, < 1 treatment episode/yr in ]0, a total of only 1-3 treatment courses in 6 patients. 3 afibrinogenemic patients had prophylactic replacement therapy. hcv serology was positive in 56/6] patients (92%), in 47 with detectable hcv rna (77%). the 5 persons who escaped hcv infection, with normal alt-levels and without sonographic alterations, had low intensity treatment with small pool src preparations only. alt-levels were elevated in 33/56 anti-hcv positive patients (59%). 26/56 had abnormal sonographic findings (46%). there was a clear correlation between elevated alt-levels and abnormal sonographies: of 33 patients with elevated alt 23 had abnormal sonography, of 23 with normal alt 3 had abnormal sonography. 8 patients had liver cirrhosis (6 with clinically overt hepatopathy), 4 (4/56 = 1%!) with hepatocellu]ar carcinoma (hcc) with elevated afp-leveis. of these 4 patients 2 had intraarterial embolization with ]ipiodol-epirubicin; in 2 patients hcc diagnosis was made in a late stage. i patient with advanced liver cirrhosis underwent successful liver transplantation. 2 of the 6 patients with hepatopathy had severe haemophilia with temporary high alcohol intake, 4 had mild coagulatlon disorder with few treatment episodes. possible precipitating factors were coinfection with hbv, high alcohol consumation and first exposure to hcv contaminated blood products in an advanced age, but not intensive replacement therapy. very similar results for f vlll and vwf. since the factor viii level is kept steady above the level where there is an increased risk of haemorrhage, continuous infusion is haemostatically safer and more efficacious than bolus injections, another advantage is a progressive decrease of clearence during the first days after surgery which leads to a substantial reduction of factor concentrate consumption by avoiding the innecessary peaks of bolus injections. 22 children with severe form of haemophilia a undergoing elective surgery received continuous infusions with different plasma-derlved and recombinant f viii concentrates. before surgery, patients got bolus injections to raise the factor viii levels to more than 80 %. during continuous infusion factor viii levels were measured two to three times a day and the infusion rate of 4 to 5 iu/kg/h could be reduced on the second or third day to 2-3 iu/kg/h. the clinical efficacy was excellent with no bleeding events. in 5 children with vwd also undergoing elective surgery continuous infusions with humate pr were performed in the same way. no bleeding events were observed in these patients. none of the patients developed postoperative wound infections. the overall doses of f vtll concentrate 'were about 20-30 % lower than those required during replacement therapy with bolus doses. lg8 factor x frankfurt i : molecular and functional characterisation of a hereditary factor x defect (gla +25 lys) huhmann i., holler b., krinninger b., turecek p.l, richter g., scharrer i., forberg e., watzke h. univ. klinik f0r inhere med.i, abteilung for h~tmatologie und h~mostaseologie, w~en; immuno-ag, wien ; klinikum der j.w. goethe-univ. frankfurt am main, abt. f. angiologie. factor x (fx) is a vitamin k-dependent plasma protein which is activated either by fvila/tissue factor or ixaniila. fxa is the main enzyme for conversion of prothrombin to thrombin. the congenital fx-deficiency (stuart -prower-defect) being inherited as an autosomal recessive trait subsequently leads to bleeding diasthesis of varying severity. our propositus is a 24 year old patient presenting a mild bleeding tendency. his p'fi (36 sec) is within the normal range, the pt ( 73% of normal) is slightly reduced. the factor x antigen level is reduced to 55% of normal. molecular charactedsation of the genetic defect was performed by amplification of the eight exerts and exonintron junctions by pcr and subsequent direct sequencing of the products. in comparison to the normal sequence we could determine a single mismatch within exon ii resulting in the substitution of +25 gla (gaa) by lys (aaa). the mutation abolishes a naturally occuring mboll site in the dna sequence of exon ii. the status of the fx encoding alleles was determined in the propositus, his mother and one of his brothers by amplification of exon ii and restriction digest with mboll. these family members were heterozygous with respect to the mutation in exert ii. fx was isolated from plasma of the propositus by monoq ion exchange chromatography. performing clotting assays with purified fx frankfurt i we determined an activity of 89% of normal fx upon activation with rw, 77% upon intdnsic activation (aptt) and 81% upon extrinsic activation (pt). this compares well with the results obtained from the patient plasma ( pt 56%, ptt 55% and rw 57% of normal) when the reduced fx-ag-level of the plasma (55%) is taken into account. we therefore conclude that the substitution of gla +25 to lys results in a fx molecule which is severely defective in both the intrinsic and extrinsic pathway of blood coagulation. bleeding after cardiothoracic surgery is still a frequent, important and sometimes life-threatening complication. thus, the aim of this study was to examine routine parameters of hemostasis and their predictive values for severe bleedings. this prospective study included patients undergoing cardiopulmonary bypass surgery. blood samples were drawn preoperatively as well as 0, 6, 18 hours and 2, 3, 4, 5, 6, 7, 8 days after surgery. blood loss from drains, transfusion of blood products and other important clinical data were monitored apart from platelet count, hematocrit, thrombin time, thromboplastin time, aptt and levels of fibrinogen, atiii and c-reactive protein; soluble fibrin (sf) was measured via protamine sulfate aggregability and total fibrin(ogen) degradation products (ftdp) by an elisa from organon teknika. n= 109 patients were examined (age: 64__+9 y). they lost 750+__460 ml blood (mean+sd) into the drains within the first 18 hours after end of surgery. a severe bleeding was defined to exist, if the blood loss exceeded this range (> 1200 ml within 18 h). fibrin(ogen) split products proved to be a useful parameter in predicting the risk of severe bleedings : ftdp levels exceeding 12 mg/i at end of surgery (n = 105) had a negative predictive value of 94%, positive predictive value of 60%, specificity of 96% and a diagnostic efficacy of 91%. in contrast, soluble fibrin which correlated well with fibrinopeptidea (r>0.91, n= 20) did not correlate neither with degradation products nor with bleeding complications (n = 109). this observation does not match to the correspondence of sf with organ dysfunction during dic: sf reached a neg.predictive value near 95% and a diagnostic efficacy of >70% (pat. without antifibrinolytic drugs), which complies to findings from bredbacka (1994). other parameters were less predictive than ftdp and sf. therefore, further examinations are necessary to determine the value of soluble fibrin for a risk prediction of bleeding complications or dic. a differentiation of splits products deriving from either fibrinogen, fibrin or xl-fibrin will provide further insights into fibrin(ogen) metabolism. heparin induced thrombocytopenia represents a multicomponent syndrome associated with the use of heparin and related drugs resulting in not only thrombocytopenia, but also arterial thrombosis of varying magnitude. the initial diagnosis ofthis syndrome is usually made by clinical observation and a drop in platelet count. conventional diagnostic methods include platelet aggregation responses to patient's serum and ~4c serotonin release in response to patient's serum, aggregation/agglutination of patient's platdets in response to heparins and the detection of patients anti-heparin platelet factor 4 (hpf4-ab) ned-antibodies by using elisa methodology. several other individualized methods are also used to demonstrate platelet activation. to test the diagnostic validity of the platelet aggregation (pa) 14c serotonin release (sr) and the relevance of hpf~-ab 340 serum samples collected from patients with clinically eunfwmed eases of lilt syndrome were compared in parallel in various assay systems. the diagnostic efficacy of these tests varied from 60-74% with the pa test providing better results than others. when the pa test was compared with serotonin release, a poor correlation was noted (r=0.47). in contrast, the correlation between the pa and hp4-ab was somewhat better (r=0.66). in another study, blood samples collected from 50 patients treated with ahigh dose low molecular weight beparin for two weeks (80 mg o.d.) were tested. 20 of these patients showed a high titre of hpf4.ab without any decrease of platelet count. none of these patients were found to be positive in the 14c serotonin release assay. a third study included blood samples from dvt patients administered with iv heparin infusion, high dose sc lmw heparin (certoparin) and iv lmw heparin for the management of dvt. none of these patient groups (20-34) exhibited any hit responses, hmvever, the incidence of high hpf4-ab titre was found to be 53% in heparin, 36% in patients with lmw heparin iv and 26% in lmw heparin sc groups. pa and sr studies revealed 8% and 12% false positive ~ respeetively. these studies clearty suggest that the currently available ~ for laboratory diagnosis of hit syndrome are of limited value, and caution should be exercised in the interpretation of the results obtained with these tests. heparin-induced thrombocytopenia (hit) is one of the major severe side effects during treatment with heparin. in postoperative medicine clinical studies demonstrated the prevalence of hit with unfractionated over fractionated heparins. few data are available from the non-ope1"ative medicine and from patients without thmmboembolism before heparinization. in a controlled prospective randomized study the safety and efficacy of low-dose heparin was compared with a lowmolecular-weight (lmw) heparin over 10 days in bedridden medical inpatients (haemostasis, in press). 1968 patients were randomized and controlled for the development of thrombocytopenia. thrombocytopenia was defined as a platelet count below 40.000lid at day 10. 4 patients developed thrombocytopenia in the heparin group and no patient in the lmwheparin group (p<0.05). none of the patients with thrombocytopenia developed a thromboembolic complication. in a second prospective case control study 90 patients with side effects on anticoagulants were treated with lmw-heparin once daily subcutaneously for a period of 1 month to 9 years. platelet count was performed every 1 to 3 months. none of these patients developed thrombocytopenia during heparinization with lmw-heparin. it is concluded that hit is a very rare complication in nonoperated bedridden medical patients. a decrease of platetet count may occur in about 0.5% of patients receiving low-dose heparin. the incidence of hit with thrombosis during low-dose heparin and of hit during lmw-heparin in non-operated patients is manyfold lower and remains to be determined. terminology: instead of the term "hemorrhagic disease of the newborn (hdn)" the term vkdb should be used, since neonatal bleeding is often not due to vkdeficieacy and vkdb may occur after the neonatal period (i.e. after 4 weeks). definition: vkdb is a bleeding disorder caused by reduced activity of vkdependent coagulation factors which responds to vk. diagnnsis: in a bleeding infant a prolonged pt (inr > 3.5) together with normal fibrinogen and platelet count is almost diagnostic of vkdb. the diagnosis is proven, if vk shortens the pt (after only 30-60 minutes) and/or stops bleeding. classification: classification by age of onset into early (< 24 h~. classic fdav 2-7) and lale form (> i week <6 months), and by etiology into idionathic and ~ec0nd~'y. in secondary vkdb in addition to breast feeding other factors can be demonstrated, such as poor intake or absorption of vk and increased consumption of vk. vk-prophylaxis: benefits: oral and intramuscular (i.m) vk (one dose of i nag) prevents equally well the classic form of vkdb. lm. vk appears to be more effective in preventing the late form (times 15-> 50). the protection achieved by single oral prophylaxis (times 3-5) is improved by triple oral vk (times 15-30). risks: because of poten[ial ri~l~ associated with extremely high levels of vk and the possibility of injection injury, i.m. vk has been questioned as the prophylaxis of choice for normal neonates. since vk is involved not only in coagulation but 'also in carboxviation with multiple effects, excessive deviations from the low physiologic concentrations, which prevail in the fully breast-fed healthy mature infant should be avoided. proposal: repeated (daily or weekly) small oral doses of vk are closer to physiologic conditions than single i.m. bolus doses, which expose neonates to excessively high vk levels. the incidence of intracranial vkdb can be reduced if the grave significance of warning signs is recognized (i.e, icterns, failure to thrive, feeding problems, minor bleeding, disease with cholostasis). whether or not the more reliable absorption of the new mixed mieellar (mm~ nrenaral~i0n of vk can reduce the protective oral dose of vk-.prophylaxis has to be evaluated. before vitamin k(vk) prophylaxis was generally accepted in japan, the incidence of infantile vk deficiency was 1:4000 both idiopathic and secondary types. since 1981, 4 nationwide surveys have been conducted. the current incidence rate is now about one-tenth that in early 1980. however, in a small number of cases, vk deficiency occured despite prophylactic administration during the neonatal period. in order to clarify the absorption,excretion and transplacentel transport of vk in the perlnatal period,followlng studies were carried out. 1)hepaplastlntest(normotest) were performed on 65 women in the last stage of pregnancy and each coagulation factor was estimated as well. 2)correlatlons were made between mothers'and babies'hepaplastin test values. 3)transplacental transport of vk 2 was studied. the general activity of vk dependent factors in pregnant women was much higher than in non pregnant women. as far as the correlation between mothers'venous blood during delivery and cordvenous blood is concerned, in the group of mothers with hepaplastln test value of less than 120% of the normal adult value, the value of the bepaplastlntest was less than 30 % of normal adult value in the cord venous blood. we also demonstrated that vk passed through the placenta but only in small qualities. the point mutation g to a at nt 449 in exon v of the factor x gene (gin 102 to lys) has previously been found in two independent kindreds with fx deficiency. it occured in both families in an heterozygote state and was associated with two other genetic defect in the fx gene. we have identified another familiy in which this mutation occurs in a homozygote state. in this family the mutation is associated with the previously reported mutation gla 14 to lys which also occurs in a homozygote state. the pt and ptt of the proposita and her siter are markedly prolonged. the fx activity is reduced to <1% in the extrinsic system, to 30% in the intrinsic system and to 18 % after activation with rvv. the fx antigen is reduced to 20%. the coagulation profile of this family thus is identical with that of fx vorarlberg despite the fact that the fx vorarlberg kindred is only heterozygous for the mutation glal02 to lys. haplotype analysis could not rule out consanquinity with the fx vorarlberg kindred. these data suggest that the mutation at nt 449 which leads to a fairly dramatic amino acid change from glu to lys would indeed represent a polymorphism. to further address this question we cloned the fx gene in an expression vector (pcep 4) for transient expression in the human embryonic kidney cell line 293 and introduced the mutation at nt 449 by site directed mutagenesis. hereditary deficiency of factor ixa, a key enzyme in blood coagulation, causes hemophilia b, a severe x-chromosomelinked bleeding disorder; clinical studies have identified nearly 500 deleterious variants. the x-ray structure of porcine factor ixa shows the atomic origins of the disease, while the spatial distribution of mutation sites suggests a structural model for fx activation by phospholipid-bound flxa and cofactor villa. the 3.0 a resolution diffraction data clearly show the structures of the serine proteinase module and the two preceding epidermal growth factor (egf)-like modules; the n-terminal gla module is partially disordered. the catalytic module, with covalent inhibitor d-phe-pro-arg chloromethyl ketone, most closely resembles fxa but differs significantly at several positions. particularly noteworthy is the strained conformation of glu-388, a residue strictly conserved in known fixa sequences but conserved as gly among other trypsin-like serine proteinase. flexibility apparent in electron density together with modelling studies suggests that this may cause incomplete active site formation, even after zymogen activation, and hence the low catalytic activity of fixa. most hemophilic mutation sites of surface fix residues occur on the concave surface of the bent molecule and suggest a plausible model for the membrane-bound ternary flxa-fvilla-fx complex structure: the stabilizing fvilla interactions force the catalytic modules together, completing flxa active site formation and catalytic enhancement. factor x frankfurt i molecular and functional characterisation of a hereditary factor x defect (gla +25 ---, lys) huhmann i., holler b., krinninger b., turecek pi., richter g., scharrer i., forberg e., watzke h.. univ. klinik ftlr innere medi, abteilung for h~matoiogie und hamostaseologie, w~en; immuno-ag, wien ; klinikum der j.w. goethe-univ. frankfurt am main, abt. f. angiologie. factor x (fx) is a vitamin k-dependent plasma protein which is activated either by fvila/tissue factor or ixaniila. fxa is the main enzyme for conversion of prothrembin to thrombin. the congenital f×-deficiency (stuart -prower-defect) being inherited as an autosomal recessive trait subsequently leads to bleeding diasthesis of varying severity. our propositus is a 24 year old patient presenting a mild bleeding tendency. his ptt (36 sec) is within the normal range, the pt ( 73% of normal) is slightly reduced. the factor x antigen level is reduced to 55% of normal. molecular characterisauon of the genetic defect was performed by amplification of the eight exons and exonintron junctions by pcr and subsequent direct sequencing of the products. in comparison to the normal sequence we could determine a single mismatch within exon ii resulting in the substitution of +25 gla (gaa) by lys (aaa). the mutation abolishes a naturally occuring mboti site in the dna sequence of exon ii. the status of the fx encoding alleles was determined in the propositus, his mother and one of his brothers by amplification of exon ii and restriction digest with mboll. these family members were heterozygous with respect to the mutation in exon i1. fx was isolated from plasma of the propositus by monoq ion exchange chromatogrephy. performing clotting assays with purified fx frankfurt i we determined an activity of 89% of normal fx upon activation with rw, 77% upon intrinsic activation (aptt) and 81% upon extrinsic activation (pt). this compares well with the results obtained from the patient plasma ( pt 56%, ptt 55% and rw 57% of normal) when the reduced fx-ag-level of the plasma (55%) is taken into account_ we therefore conclude that the substitution of gla +25 to lys results in a fx molecule which is severely defective ip both the intrinsic and extrinsic pathway of blood coagulation. bleeding after cardioth~)racic surgery is still a frequent, important and sometimes life-threatening complication. thus, the aim of this study was to examine routine parameters of hemostasis and their predictive values for severe bleedings. this prospective study included patients undergoing cardlopulmonary bypass surgery. blood samples were drawn preoperatively as well as 0, 6, 18 hours and 2, 3, 4, 5, 6, 7, 8 days after surgery. blood loss from drains, transfusion of blood products and other important clinical data were monitored apart from platelet count, hematocrit, throm. bin time, thromboplastin time, aptt and levels of fibrinogen, atiii and c-reactive protein; soluble fibrin (sf) was measured via protamine sulfate aggregability and total fibrin(ogen) degradation products (ftdp) by an elisa from organon teknika. n= 109 patients were examined (age: 64+9 y). they lost 750+__460 ml blood (mean_+sd) into the drains within the first 18 hours after end of sur. gory. a severe bleeding was defined to exist, if the blood loss exceeded this range (> 1200 ml within 18 h). fibrin(ogen) split products proved to be a useful parameter in predicting the risk of severe bleedings : ftdp levels exceeding 12 mg/i at end of surgery (n = 105) had a negative predictive value of 94%, positive predictive value of 60%, specificily of 96% and a diagnostic efficacy of 91%. in contrast, soluble fibrin which correlated well with fibrinopeptide a (r>0.91, n= 20) did not correlate neither with degradation products nor with bleeding complications (n= 109). this observation does not match to the correspondence of sf with organ dysfunction during dic: sf reached a neg.predictive value near 95% and a diagnostic efficacy of >70% (pat. without antifibrinolytic drugs), which complies to findings from bredbacka (1994). other paramelers were less predictive than ftdp and sf. therefore, further examinations are necessary to determine the value of soluble fibdn for a risk prediction of bleeding complications or dic. a differentiation of splits products deriving from either fibrinogen, fibrin or xl-fibrln will provide further insighls into fibrin(ogen) metabolism. this study was conducted as a randomized parallel -group clinical trial comparing the safety and efficacy of a low molecular weight heparin {lmwh} -monoembolex sandoz and unfractionated standard heparin glfh) for the perioperative prevention of venous thromboembolie disease (dvt) following major surgms' in patients with gynecologic malignancy.. three hundred and twenty 4 women (six drop outsl werr randomized and received either 3 times daily 5000 [l" s.c. ul.'i-i (sandoz nuemberg germany] (n = 164) or once a day t5000 i~'v'. units s.c. monoembolex (n = 160) plus two placebo injections. heparin therapy was started the morning before opcrati(m and continued until the 7th postoperative day. up to the 10th poatop, day the incidence of dvt was 6.25 % (n = 10; incl. 7 pulmona~ embolisms pe) in the lmwh group and 6.10 % (n = 10; incl. 4 pe} in the ufh group. the overall incidence of clinically hemorrhagic wound complications was significantly decreased in the lmwh group 16.3 % (n = 2hi compared to the ufh group 26.8 % {n = 44; p < 0.0051. the incidence of major hemorrhagic episodes was 9.4 % in = 151 in the lmwh group and 14.0 %/n = 23) in the ufh group. this difference was not statisticauy significant. one case of fatal pe was observed in the lmwh -treated group. five women deaths in the lmwh group were observed during the study and 3 in the ufh group. this study demonstrates that the perioperative treaunent of low molecular weight heparins is more safety than standard heparins in gynecologic -oncologic patients undergoing major surge .ry. however, the incidence of thromboembohc complications is simmilar in both treatment regimes. to explore the effect of targeting an antithrombin to the surface of a thrombus, recombinant hirudin (hir) was covalently linked to the fab' fragment of fibrin-specific monoclonal antibody 59d8 (fab) resulting in a stable conjugate (hir-fab). in vitro, hir-fab was 9times more efficient than hir alone in inhibiting fibrin deposition on experimental clot surfaces in human or baboon plasma (p<0.01). to validate these results in vivo, hir-fab was compared to hir in a baboon model. the deposition of ill-in-labeled platelets onto a segment of dacron vascular graft present in an extracorporeal arteriovenous shunt was measured. blood flow rate was 40 ml/min. one hour local infusions of 4500 atu of either hir-fab or hir resulted in deposition of 0.16 x 109 and 2.17 x 109 plate!ets, respectively. equieffective dosages were 2000 atu hir-fab and 9000 atu hir resulting in deposition of 1.06 x 109 and 0.93 x 109 platelets, respectively. based on full dose response curves (n = 14), hir-fab was found to be > 4.5-fold more potent (based on activity) than hir. because of the small total amounts of antithrombins used and the short duration of these experiments, no significant systemic effects were observed. thus, fibrin-targeted recombinant hirudin prevents platelet deposition and thrombus formation more effectively than uncoupled hirudin in vitro and in an in vivo primate model. triabin, a 17 kda protein from the saliva of the assassin bug triatoma pallidipennis, is a new specific thrombin inhibitor (1). tt does not block the catalytic center but interferes with the anionbinding exosite of thrombin. the recombinant protein was produced with the baculovirus/insect cell system and used to study the inhibitory effect of triabin on thrombin-induced responses of human blood platelets and blood vessels. aggregation of platelets in tyrode's solution was measured turbidimetrically at 37°c. for the studies on blood vessels rings (2-3 mm) from small porcine pulmonary arteries were placed in organ baths for isometric tension recording. the integrity of the endothelium was assessed by the relaxant response to bradykinin. like hirudin, triabin inhibited the thrombin (0.1 u/ml)-induced aggregation of washed human platelets at nanomolar concentrations (ec50 = 2.6 nmol/l); whereas the adp-and collagen-induced aggregation were not suppressed. in pgf2c~-precontracted porcine pulmonary arteries, the thrombin (0.2 u/ml)-induced endothelium-dependent relaxation was inhibited by triabin in the same concentration range as found for inhibition of platelet aggregation. higher concentrations of triabin were required fo affect the contractile response of endothelium-denuded porcine pulmonary arteries to thrombin (1 u/ml). in all these assays, the inhibitory potency of triabin was dependent on the thrombin concentration used. these studies suggest that the new anion-binding exosite thrombin inhibitor triabin is one of the most potent inhibitors of the thrombin-mediated cellular effects. dept. of medicine, university hospital benjamin franklin, free university of berlin, dept. of medicine and dept. of surgery, heinrich-heine:university dusseldod after standardized training in home prothrombine estimation using the coaguchek system, 150 consecutive patients (p) who had st. jude medical aortic or mitral valve implantation were allocated to two random arms; 75 p were asked to control the inr themselves every third day. in the remaining 75 p anticoagulation was managed by the home physician without recommending an interval for these controls. all 150 p were monitored during the education period to a target therapeutic range of inr 3.5-4.0. p were asked to contact their home physician immediately if the inr was measured 0.5 below or above the target range (inr-corrider 3.0-4.5). all p had out-patient re-examinations every three months. thrombotic, thromboembelic and hemorrhagic complications were documented by the p using special documentation cards. the following findings were documented during the follow-up period: 4.49 0.9 0 the results of this randomized study demonstrate a significant improvement in the management of oral anticeagulation by home prothrombine estimation. significant (p<0.001) more inr measurements were found inside the target therapeutic range. moreover. bleeding and thromboembolic complications could be reduced (p = 0.038) in the study group with home prothrombine estimation. life-threatening thromboembolic and hemorrhagic complications were not observed in p who were on home prothrembine estimation, while three such events (2.72 %/year) were documented in group a. local vascular injury following ptca exposes circulating platelets to prodmmbogenic stimuli. by binding to platelet gp iiblliia fibrinogen crosslinks platelets, which represents the final common pathway of platelet aggregation. fradafiban (bibu52zw) is a non-peptide compound with effective, reversible inhibitory effects on fibrinogen binding to gp iib/ii/a on human platelets. in the first double-blinded, prospective phase ii study three escalating doses of bibu52zw as a continuous 24 h-i.v, infusion were tested in comparison to placebo in 65 patients with stable angina pectoris undergoing elective ptca. the mean receptor occupancy with 20rag, 40ms and 60ms per hour were 71.974, 84.5 % and 87.9% at 24 hours, respectively. as compared to placebo breeding time was significantly prolonged (7 vs 20rain) during fi-adaiiban infusion with a weak dose-dependency. platelet aggregation in platetet rich plasma ex vivo with collagen (2.0 and 4.0 gg/ml), adp (2.5 and 5.0 gmol/ml) or ca-ionophor a 23187 (2.5 and 5.0gg/ml) was significantly and dose-dependently inhibited as compared to placebo. using the two upper doses of fradafiban, we observed major bleeding complications in 8 patients requiring blood transfusions or vascular surreal repair. in these patients, too, maximal antiplatelet effects could be documented. these data sugest that bibu52zw is an effective fibrinogen receptor antagonist in patients. the requirement of ad hoc receptor occupancy determination or platelet function monitoring for safe and effective clinical use should be evaluated. in a placebo controlled interaction study 9 healthy volunteers were randomized to receive either a 24 hour infusion of peg-hirudin ( 0.02 mg/kg/h) after an i.v, bolus of 0.2 mg/kg + placebo, or 325 mg/day acetylsalicylic acid (asa) for three days followed by a placebo infusion or the peg-hirudin infusion + asa. each volunteer received all three treaments. there was a washout period of at least 14 days between the infusions. at short intervals aptt, activated clotting time (act), ecadntime (ect), alia-activity using the chromogenic substrate 2238, collagen-induced aggregation, platelet adhesion and platelet induced thrombin gene,ration time (pitt) were measured, bleeding time (simplate) was studied before drug administration, on day three before the infusion and 6 hours after start of the infusion.the infusion of peg-hirudin after 4 and 8 hours led to a mean hirudin plasma level of 1.8 pg/ml. asa markedly inhibited collagen induced aggregation as expected. the mean bleeding time was prolonged under the influence of peg-hirudin from 5.2 to 6.22 min, after asa from 5.8 -18.2 min and after the combination of peg-hirudin + asa from 5.4 -33.7 min. in each volunteer the bleeding time was longer under the combination than after asa alone. in two volunteers receiving peg-hirudin + asa the bleeding time measurement was stopped after 60 rain. none of the coagulation parameters or platelet function tests correlated with the prolongation of the bleeding time. however the bleeding time was excessively prolonged in those volunteers who had a marked prolongation under asa alone.the combination of hirudin at a higher dosage with asa probably is associated with a relative high risk of bleeding. either the hirudin dosage should be reduced if the combination seems feasabie or asa should be given after the end of hirudin treatment. fibrinogen with the sta/stago and the mla/dade systems correlated well, but neither system correlated well with the acl/il system. at iii, protein c, protein s, and anti-xa heparin assays using stago reagents performed as expected for normals and low abnormals on the sta. factor levels on the sta/stago system were less sensitive than factor levels obtained with the dade reagents on the mla or fibrometer. using the sta/stago system, thrombin time results correlated well with the aptt and heparin levels. the thrombin time was not associated with additional manipulation for assay preparation, nor any cross-contamination of reagent or sample, since on the sta reagents do not come into contact with tubing. the sta was not sensitive to hemolytic, icteric or lipernic samples for clotting assays artd showed the same sensitivity as the mla for chromogenic assays. the overall data comparisons, high throughput, minimal operator intervention for reagent/assay change and ease of operation warrant further evaluation of the sta hemostasis analyzer. a. wehmeier, d. s6hngen, c. rieth klinik for h#,matologie, onkologie und klinische immunologie der heinrich-heine-universit~it d0sseldorf hirudin selectively inhibits thrombin by direct interaction. because the effect of hirudin is independent of antithrombin iii and other factors, it seems an attractive alternative to current anticoagulants. however, it is uncertain whether hirudin influences plateletassociated thrombotic disorders and how it compares with conventional and lmw heparin. we investigated the effect of 2 recombinant hirudin preparations (rhein biotech, dt3sseldorf) on platelet function tests: in vitro bleeding time, adhesion to glass beads, aggregation in platelet-rich plasma and whole blood. hirudin was used in concentrations of 0.1-100 i.tg/mi, and was compared to trisodium citrate (0.38%), conventional heparin (50 iu/ml) or lmw heparin (fraxiparin, 500 iu/ml). both recombinant hirudins showed normal activity in thrombin neutralization tests, and prolongation of thrombin time and aptt. however, in vitro bleeding time was not prolonged by hirudin, but was more than doubled by addition of conventional and lmw heparins. platelet retention to glass bead columns was reduced by hirudin in a dose-dependent manner to about 40% but was more effectively reduced by both heparin preparations and citrate. hirudin had an inhibitory effect on p!atelet aggregation in prp induced by thrombin, collagen, and predominantly epinephrine but not adp and ristocetin. in whole blood, a small effect could only be observed with hirudin concentrations of >25 ~g/ml as compared to citrateanticoagulated blood. in summary, thrombin inhibition by recombinant hirudin has little effect on in vitro platelet function tests in comparison to heparins and calcium depletion. the role of endothelin (et), prostaglandins and the coagulation system in the pathogenesis of acute renal failure is still to be defined. in anaesthesized pigs the effects of i.v. infusion of et (3 /~g/kg) alone (group 1, n=6) and after pretreatment with the potent thrombin-inhibitor hirudin (0,5 mg/kg)(group 2, n=6) on haemodynamics, coagulation parameters (factor viii, antithrombin iii, precallicrein, fibrin monomers, aptt) and prostaglandins were investigated. plasma renin activity (pra)-, creatinine clearance-, urine volume-measurement and blood gas analysis were performed hourly. et-infusion caused an initial bp-reduction and marked hr-reduction followed by a transient bp-elevation and hr-reduction. activation of platelets can be directly measured by flow cytometry using monoclunal antibodies. in an in vitro study the effect of the thrombin inkibitors argatroban, efegatran, dup 714, recombinant hirudin and peghirudin on platelet activation induced by various agonists was studied in whole blood. blood was drawn from normal human volunteers using the double syringe technique without use of a tourniquet to avoid autoaggregatiun of platelets. for anticoagulation of blood the thrombin inhibitors mentioned above were used at a final concentration of 10 ~tg/ml each. blood samples were then incubated at 37°c either with saline, r-tissue factor (rtf), arachidonic acid (aa), adenosine diphosphate (adp) or collagen. at definite times (1, 2.5, 5, 10 rain) aliquots were taken and after various steps of fixative procedure the percentage of platelet activation was measured by means of fluorescent monoclonal antibodies to platelet surface receptors gpiiia (cd-61) and p-selectin (cd-62). the agunists used induced a platelet activation of 37.4 + 15.2 % (rtf), 65.1 + 12.1% (aa), 19.3 + 7.4 % (adp) and 27.1 + 12.8 % (collagen). flow cytometric analysis showed that all thrombin inlaibitors studied caused a nearly complete inhibition of r-tissue factor-mediated platelet activation. in contrast, after induction of platelet activation with the other agonists an increased percent cd-62 expression was found showing a strong platelet activation with a maximum at the same times as in non-anticoagulated blood. in conclusion, the results show that in whole blood thrombin inhibitors are effective in preventing platelet activation induced by r-tissue factor. the formation of active serine proteases including thrombin may be effectively inldbked by these agents. the observations further suggest that, while thrombin inkibitors may control serine proteases, these agents do not inhibit the activation ofplatelets mediated by other agonists. this work was supported by the grant bmft 07nbl01. animal experimental studies on the pharmacokinetics of peg-hirudin e. bucha, a. kossmehl, g. nowak max-pianck-gesellschaft e. v., arbeitsgruppe "pharmakologische h~imostaseologie", jena hirudin, when complexed with polyethylene glycol (peg), increases its molecular weight from 7 to 17 kda, thereby preventing extravasation of this drug. peg-hirudin is distributed almost only in the intravascular blood space. in addition, its increased molecular weight retards the renal elimination. the elimination half-life of hirudin in rats (58 + 12 min, as determined) is increased five-fold (244 ± 18 min). with the same hirudin dose applied, the blood level of hirudin is increased 19-fold, measured in the 13-elimination phase. in the urine of rats, 2 -3% of the hirudin activity were recovered following hirudin administration, but 48% could be detected after peg-hirudin had been applied. after subcutaneous administration of peg-hirudin, the trnaxwalue is reached at 380 rain (r-hirudin: 65 min); the cmax-value is increased 3-fold, compared to that of r-hirudin (2.5 pg/ml). 24 hours later, still one fifth of the maximum concentration (cma,) is present in the blood, and the renal elimination is still retarded. in the urine of rats, 12% of the hirudin activity applied were recovered in the 24-h urine sample. with intact renal function, following subcutaneous administration, peghirudin is abte to produce a constant blood level of hirudin over a long pedod. thrombin inkibitors such as r-hirudin (rh), argatroban (a), efegatran (e), and peghiradin (ph) are currently undergoing extensive clinical trials in such cardiovascular indications as ptca, ami, and treatment of unstable angina. a rapid assessment of the anticoagulant actions of these agents is, therefore, crucial to assure their efficacy and safety. currently, act and aptt are used to measure the anticoagulant effect of these agents. we have utilized a dry reagent technology based on the motion of paramagnetic iron oxide particles (plop) to measure the antithrombin effects of various thrombin inhibitors (cv diagnostics, raleight, nc). the heparin monitoring card has been modified to measure antithrombin agents in various anticoagulant ranges for (a)0 (e), (rh), and (ph). blood samples drawn from patients treated with (a) and (rh) have been evaluated and concentrations of these agents have been calculated using an external calibration curve. in the in vitro setting, citrated whole blood or citrated frozen plasma can be used to evaluate the anticoagulant effects of these agents. the results obtained are comparable to the act which is conventionally used for the monitoring of these agents. both (rh) and ( period. we would like to present a case of heparin-induced-thrombocytopenia (hit) in a 47 years old woman who underwend open heart surgery. she suffered from a combined aortic valve disease and leading stenosis. laboratory analysis showed constant low platelet counts (50/nl) without heparin application, so that an idiopathic thrombocytopenlc purpura was suspected. but platelets also decreased after heparin application. heparin-antibodies were found in the heparin induced platelet activation assay (hipaa). treatment with corticosteroids and immunoglobulines, respectively, showed no improvement but the patient unfortunately developed a pneumonia with legionetla pneumophila. therefore, the only suitable anticoagulant for the necessary aortic valve replacement was hirudin: a bolus injection of r-hirudin of 0,75 mg/kg b.w. was administered 10 min. bevore start of the extracorporal circulation (ecc), the heart-lung machine (hlm) was primed with 6 mg r-hirudin and another bolus of 5 mg of r-hirudin was administered. additionally 10 mg of r-hirudin was applicated to the cell-saver-reservoir. during the period of ecc ecarin clotting time and aptt values were taken every ten minutes for monitoring r-hirudin concentration. the postoperative anticoagulation was performed with a constant infusion of r-hirudin starting eight hours after the end of ecc and monitored by aptt. due to mechanical aortic valve the further anticoagulation was performed with phenprocoumon, starting 3 days postop. the therapy with hirudin showed no side-effects. hirudin, threrefore seems to be a suitable anticoagulant in patients with high risk for bleeding complications like this. doses fi:om 2-30 mg/kg gave similar post-op blood loss measurements without s dnseresponse (4-15 oc/kg) (less blood oozing than a historical heparin control but equivalent post-op blood loss; 10 q-3 ec/kg). doses >30 mg/kg showed more intra-op blood loss than the lowe~ doses, but equal post-.op blood loss. the bleeding time test was less elevated than for heparin. platelet counts and hematoerit did not vary except for hemodihition on pump. liver enzymes did not vary significantly pre-op to post. act values showed arg was eliminated (dose-dependently) by 1 hour post-op. dogs were hemodymamieally stable during the peri-operative period, and overall gave predictable responses to arg (as opposed to variable responses to heparin). in a substudy it was demonstrated that hypothermia did not affect the activity of arg, nor did varioos formnlations. this dose finding study strongly suggests that arg may be a safe and effective alternative to heparin for patients undergoing cpb. this is particularly important for the growing population of patients with hit who require cardiac surgery, for which no anticoagulant alternative is presently available. three recent clinical tdals with r-hirudin (timi 9, gusto 2 and hit) have shown that the risk of severe haemorrhagic side effects was strongly associated with high aptt-levels. the large intedndividual variability of the aptt and the lack of a linear dose-effect ratio, however, limits its value for reliable monitoring of the anticoagulant effect of hirudin since even severe overdosage due to impaired renal elimination may not be detected with this assay. we have therefore evaluated the ecadn clotting time (ect) as descdbed by nowak and bucha (thromb. haemost. 1993; 69: 1306) under conditions which allow conclusions on its reliability in the clinical situation.for this, citrated venous blood obtained from healthy volunteers, patients with unstable angina pectoris, and patients treated with marcumar was supplemented with different concentrations of peghirudin. measurements of aptt and ect were made in duplicate. in contrast to the aptt, the ect showed a close, linear relationship with peg-hirudin plasma concentrations in the range of 100 and 10 000 ng/ml. the lineadty of this relationship was not affected by the presence of unfractionated or low molecular weight hepadns in concentrations of up to 10 pg/ml. the ect was not affected by fibdnogen concentrations 60% below normal. a somewhat higher slope but no change in linearity was found in plasma from marcumar-patients with quick-values between 20 and 32%. no significant differences were found between values measured in citrated blood or plasma or using different coagulation timers. the most potent thrombin inhibitor containing a benzamidine moiety is napap (k i = 6 nmol/i). unfortunately, the pharmacokinetic properties (fast elimination by hepatic uptake and biliary excretion, poor enteral absorption) are unsuitable for the use of napap as an oral anticoagulant. the application of choice of a synthetic thrombin inhibitor would be the oral one, therefore, we looked for other lead structures. with the nc~-arylsulfonylated piperazides of 3-amidinophenylalanine we found a new group of derivatives which inhibit thrombin with ki-values in the nanomolar range. the piperazides exert anticoagulant activities with high selectivity, leaving activated protein c and components of the fibdnolytic system unaffected. in rats, the piperazides are rapidly eliminated from the circulation (tl/2 ~ 10 min) upon i.v. administration, too. after oral administration, the systemic bioavailability is low. upon intraduodenal administration of high doses widely varying blood levels were seen, depending on the mode of administration. to cladfy the importance of a possible hepatic first pass effect we studied in more detail the pharmacokinetics of the n~-(2naphthylsulfonyl)-3-amidinophenylalanine n'-acetylpiperazide in rats using hplc-analysis. like other benzamidines the piperazide is excreted via the bile to a high extent. enteral absorption rates of about 20 % are found after blocking the hepatic uptake and biliary excretion. hence, a hepatic first pass effect appears to be the main reason for low systemic bioavailability after orallenteral administration. at the same time, fast elimination from the circulation by hepatic uptake is the main problem for maintaining effective blood levels with benzamidines. therefore, the elucidation of the structural elements influencing the absorption and elimination processes of these types of inhibitors is necessary. the piperazides of 3-amidinophenylalanine bear the possibility to easily introduce a wide variety of substituents on the second nitrogen of the piperazine moiety. a 69-year-old female patient with diabetic nephropathy increasingly developed signs of allergisation combined with dyspnea, erythema, pruritus, and circulatory insufficiency two months after start of heparin-anticoagulated haemodialysis und initial surgical application of a double lumen venous catheter. in addition, growing thrombocytopenia was observed involving a drop in platelets by 50%, compared to the initial values. the haemodialytic efficiency was reduced by massive thrombosis of the dialyzer and subsequent repeated interruption of treatment. at the end of may 1995 heparin antibodies were detected and the hat diagnosis was confirmed. immediately afterwards, haemodialysis treatment was continued, applying hirudin as anticoagulant. using steam-stedlised haemophan dialyzers and 0.14 mg/kg r-hirudin (iketon, italy), the minimum therapeutic blood level of hirudin (0.4 pg/ml whole blood) was reached. this provided therapeutically relevant blood level conditions during a 4.5h haemodialysis. more than 80 regular haemodialyses were run without problems. in all hirudin-anticoagulated haemodialysis treatments the ecarin clotting time was used as the method of choice for bedside blood level and dosage control. after the 34th haemodialysis, the frequency was reduced from 3(4) to 2 haemodialyses a week. accordingly, the hirudin dose was increased to 0.2 mg/kg. the creatinine clearance increased continuously from initially 2.6 to 10.4 ml/min after the 13th week of hirudin-anticoagulated haemodialysis. platelet count and haemodialytic efficiency normalized. we could demonstrate that the regular use of hirudin as anticoagulant along with dialyzers impermeable to hirudin enables very good results in haemodialysis treatment in heparin-associated thrombocytopenia, hirudin is suited for use as anticoagulant in problem patients with hepadn-induced allergy when combined with a drug monitoring method fit for bedside use. capillary electrophoresis methods provide a fast measurement of proteins. thus we developed for pharmacokinetic measurements of r-hirudin and peg-hirudin capillary electrophoresis methods. for the measurement of r-hirudin we used fused silica capillary and a borate buffer. this buffer was used to detect r-hirudin, but could not be used to measure peg-hirudin. for simultaneous measurement we used a neutral capillary to prevent protein absorption to the capillary wall. the buffer was a 20 mm tricine buffer (ph = 8.0 field strength 500 v/cm). it resolved r-hirudin from peg-hirudin at 214 nm using reverse polarity. a linear correlation between the peak area and the concentration was found between 80 pgtml and 10 mg/ml for hirudin (r 2 = 0.99) and between 2,5 and 10 mg/ml for peg-hirudin (r2= 0.99) was found by coinspiking of human plasma and urine with r-hirudin and peghirudin the two proteins were completely resolved. a linear correlation between the peak area and the concentration was found. the method separates r-hirudin from peg-hirudin and may be applied to biological systems to measure the concentration of r-hirudin. triabin is a thrombin inhibitor from the saliva oft. pallidipennis structurally unrelated to any protease inhibitor known and which probably functions by an interaction with the anionbinding exosite of thrombin. we used sf9 insect cells infected with recombinant baculovirus to produce sufficient triabin for a detailed biochemical characterization. the activity of the protein purified from cell lysates was assessed in a fibrinogen clotting assay and was found to be similar to that of the natural protein. a 4-fold prolongation of thrombin-clotting time and aptt was achieved with 22 nm and 600 nm triabin, respectively. a kinetic analysis of the thrombin-catalyzed fibrinopeptide a release from fibrinogen showed that triabin is a tight-binding inhibitor. using the graphical method of dixon, the ki was determined to be 3 pm. introduction: thrombocytopenia is a common adverse effect of heparin therapy, in type ii hit platelet decrease induces severe complications. we here present two special cases of type ii hit. case report i: a 66 year old male patient with dvt of the left leg was treated with therapeutic doses of heparin. from the first to the 12th day of therapy, platelet count decreased from 122000 to 54000/ui. hit was confirmed by hipa-test, heparin therapy was s~opped and treatment with the heparinoid orgaran n was started. during the following days, arterial thromboses in the right a. femoralis occurred. several thrombe~tomies were not successful and although orgaran ~" was stopped because of suspected crossreactivity, amputation of the right leg could not be avoided. during the following days under hirudin-treatment platelet count normalized and no further complications occurred. case report 2: a 74 year old female patients suffering from hip fracture was treated by surgery with tep-operation and received prophylactic heparin treatment. after 6 days, platelet count decreased from initially 170000 to 8000/ul and dvt of the right leg was diagnosed. on the same day, severe bleeding into the left leg was observed and hemoglobin concentration was diminished to 7.8 g% (before surgery 16.0 g%). hit was confirmed by hipa te~t, heparin was stopped and treatment with orgaran started. thrombocyte count normalized and no further complications occured. conclusion: hit type ii can cause severe bleeding as well as thromboembolic complications. because of possible cross-reactivity between heparin and orgaran~,, hirudin should be given in hit patients. currently thrombin time (ti), aptt, activated clotting time (act) or anti ila -activity (alia), measured by a chromogenic substrate test are used to monitor hirudin treatment or prophylaxis. the "13" responds very sensitive to hirudin plasma levels end thus requires variable thrombin concentrations. aptt appears to be more adequate, however, it shows large interindividual variations and does not respond sensitive enough to higher hirudin concentrations. act is a simple whole blood clotting assay, but it is strongly influenced by the blood collection technique. the ecadn clotting time (ect) is a new clotting assay, recently described by nowak and bucha (thromb.haemost 1993, 69,1306) . it measures the clotting time of citrated blood or plasma after prothrombin activation by ecarin, a snake venom of echis carinatus. ec.t shows a linear dependence on different hirudin concentrations over a wide concentration range ( e.g. 0.1-5 pg/ml). in a clinical interaction study healthy volunteers were administered hirudin, asa or both. 15 male volunteers received an i.v. infusion of peg-hirudin (0.02 mg/kg/h) for 24 hours after an initial i.v. bolus of 0.2 mg/kg to compare the sensitivity and reliability of ect with aptt, l-r end act. the act was measured on the hemochron 801, usa, ect on a fibrin timer, aptf using the aptt lyophylized silica reagent by il and alia on an acl (il-milan) with the chromogenic substrate 2238. all tests were performed in duplicate. ect was more sensitive to different hirudin concentrations than aptt, or act. the ect results were better correlated with the alia-activity than ap'l"r and act. the lower detection range for ect is 0.05 pg/ml hirudin. ect is a very sensitive, simple and reliable test for the monitoring of hirudin treatment and prophylaxis. recombinant and synthetic inhibitors of thrombin such as hirudin, efegatran and argatroban are currently in various phases of clinical trials in several surgical and medical indications. the therapeutic effects of these agents are usually monitored by aptt whereas in cardiovascular indications, cefite act and hemotech® act are used. the reliability of both aptt and the act tests in predieting the safety of various thrombin inhibitors has been heavily debated. furthermore, some of these inkibiturs are administered simultaneously to heperinized or coumadinized patients and the obtained aptt and act results do not lady refleot the effects of these agents. fcafin is a snake venom derived fi'om echis carinatus which converts prothrombin into mesothrombin, targeting the arg~3-ile tm bond between the a and b chains of prothrombm. while thrombin inhibitors are capable of inhibiting mesothrombin, atiii/beparin complex does not have any effect. using purified ecarin, nowak and bucha (1993 thromb haemost 69:1306) proposed to assay hirudin. since thrombin inhibitors exhibit similar mechanisms of thrombin inhibition, ecarin clotting time (ect) was evaluated to test its diagnostic efficacy in various experimental and clinical settings. lyphilized eoarin was obtained from knoll ag, ludwigshafen, germany). concentration dependent clotting times for himdin, efegatran and argatroban were obtained in a range of 0-15 p.g/ml. all of the antithrombin agents produced a concentration dependent prolongation of ect and showed va~angpotendies inthe order ofefegatran> argatreban> hirudin, on a gravimetriebasis. on a molar basis, the anticoagulant order of potency was found to be hirudin> afegatran> argatroban. utilizing the ect, the effect of these inhibitors on patients undergoing bolus or infusion therapy, resulting in a concentration level of ~ 10 gt g/rnt, have been measured. unlike such global tests as pt and aptt, patients receiving simultaneous heparin or oral anticeagulants can be monitored for antithrombin specific prolongation ofthe ect. plasma samples from heparinized (aptt 45-90 sec) or coumadinized ('pt 15-25 see) patients, supplemented with argatroban or hiredin did not show any differences m the ect. a medified ecarm act comparable to the celite act has also been developed. initial results demonstrate that this test is not affected by aprotinin, heparin and reduction of the prothrorabin complexes in the inr range of 1.5 -3.0. these results indicate that ecarin based clotting times provide slx~etlie ~lts of circulating levels of thrombin inhibitors, which can provide reliable information to optimize their safe(y and efficacy. r-hirudin is a highly potent and selective inhibitor of the serine proteinase thrombin. after intravenous administration, r-hirudin is eliminated exclusively with the urine. its plasma half-life is very short, 1-2h. peg-hirudin is a derivative produced by coupling polyethylene glycol (peg) to a specially designed recombinant hirudin mutein. peg-coupling results in a considerable prolongation of the plasma half-life of peg-hirudin, compared to r-hirudin. after intravenous administration of r-hirudin into rats, a very small amount of ,,hirudin-like" activity (2 -4% of applied activity) was recovered in the urine. in contrast, after peg-hirudin had been administered, more than 30% of the applied activity could be recovered in rat urine. these results suggest differences in the renal metabolism of peg-hirudin and r-hirudin. within the scope of pharmacokinetie studies in rats we investigated the appearance of biologically active metabolites of peg-hirudin after kidney passage in urine. affinity chromatography on immobilised thrombin was used as a quick and gentle method in searching for biologically active hirudin metabolites in rat urine. but it had to be completed by anion-exchange and/or reversed-phase chromatography to ensure that all active metabolites were detected. the isolated biologically active metabolites were purified by reversed-phase hplc and were biochemieally characterized. in previously reported studies we found a hlrud n derivative consisting of the amino acids 1-50 as the main metabolite in rat urine following intravenous administration of r-hirudin. this metabolite was not detected in the urine after administration of peg-hirudin, confirming the suggestion of a different renal metabolism. carrageenans are high molecular weight sulfated polygalactans of plant origin (derived from red algae) with anticoagulant properties. in previous studies we investigated the anticoagulant activity of lambda-carrageenan, a highly sulfated type of carrageonans. unlike heparin, lambda-earrageenan exerts its anticoagulant activity primarily through direct inhibition of the serine proteinase thrombin. only a part of its antithrombin activity is indirectly mediated through antithrombin iii. to investigate relations between molecular weight and biological activities, tambda-carrageenan has been hydrolysed and fractionated. the molecular weight has been determined with the aid of size exclusion hplc using dextrans as molecular weight standards. the degree of sulfation has been determined by anion-exchange hplc. we have obtained low molecular weight lambdaearrageenans ranging from 10,000 dalton to 100,000 dalton with degrees of sulfation of 13 -17% and 33 -38%. the anticoagulant and antithrombin activity of low molecular weight carrageenans have been determined using coagulation assays and purified systems, and we have compared their activities with those of heparin and other sulfated polysaecharides. further, we have investigated the ability of lambda-carrageenan and its low molecular weight derivatives to inhibit the activity of human blood phagocytes. the activity has been determined by measuring the cellular chemiluminescence in a mieroplate himinometer using a himinol-dependent assay and zymosan as phagocytosis activating agent. we have used an assay in human whole blood and assays with isolated human mononuclear and polymorphnuclear cells. the anticoagulant activity and also the ability of carrageenans to inhibit the activity of human macrophages decrease with decreasing molecular weight and decreasing degree of sulfation. the natural ocouring, yellow pigment curcumin is the major component of tumeric and is commonly used as a spice and food-coloring agent. since curcumin has been reported to have anti-tumorpromoting, antithrombotic and anti-inflammatory properties, we studied, whether curcumin acts on the transcription factors ap-l(jun/fos) and nf-~:b in cultured endothelial cells (ec). when ec were cultured in the presence of curcumin, electrophoretic mobility shift assays (emsa) demonstrated, that binding of endogenous ap-1 to its dna recognition motif was suppressed. inhibition was due to direct interactions of curcumin with the dna-binding motif for ap-i. enhanced ap-1 binding, induced after tnfa stimulation of ec, was decreased in cells pretreated with curcumin. this resulted in reduced transcription and expression of tissue factor, known to be controlled by ap-f and nf-~b. nuclear run on assays proofed, that curcumin directly reduced the tnfa mediated transcription of genes, regulated by ap-1, as tf, endothelin-1 and c-jun. thus, curcumin did not only suppress apl(jun/fos)-binding, but also inhibited tnfa induced jun transcription, transient transfections with tissue factor promotor plasmids confirmed, that inhibition by curcumin was dependent on intact ap-i sites. beside its effect on ap-l-binding, curcumin reduced the radical dependent activation of nf-kb due to its antioxidant properties, however, this inhibition was indirect and less prominent. the relevance of the in vitro data was confirmed in vivo in mice bearing meth-a-sarcoma. when mice received curcumin before tnfa was injected, tumors showed reduced ap-1 activation. simultanously fibrin/fibrinogen deposition decreased, most probably due to reduced tissue factor expression. thus, curcumin inhibits ap-t activation and expression of endothelial genes controlled by ap-t in vitro and in vivo. (jung, 1991) . additionally, haemorhenlogical parameters (plasma viscosity, erythrocyte aggregation) were measured. in all patients aptt, bleeding time, platelet adhesiveness, von wiuebrand f~ctor and factor viii concentration and activity were determined. the patients with von willebrand disease showed characteristic morphological changes of capillary geometry. tortuosity of nailfold capillaries was markedly increased as well as the diameter of capillariez on the arterial and venous side. plasma viscosity was significantly low. multiple parameter analysis concerning to galen and gambino (1983) and using the parameters ,,plasma viscosity below 1.25 mpas", ,,torquation index higher than 5", ,,erythrocyte column diameter bigger than 16,9 gin" showed a positive predictive value of 100 %. capillary diameter and capillary tortuosity have a positive predictive value of 91,2 %. additionally, a reduction of the vasomotorie reserve and/or a decreased erythrocyte velocity in the capillaries below the reference range was found in most of the yon willebrand patients. it was quite remarkable, that 16 of 40 of the yon willebrand patients showed significant capillary bleedings. these findings confirm some former observations (e.g. o'brian 1950) and preliminary reports of our group (koscielny 1994). polymerase chain reaction (pcr)-based quantitation of mrna transcripts is an important tool in the investigation of the underlying molecular defects in inherited platelet disorders, such as the bernard-soulier syndrome. however, for the exact quantitation of mrna a number of methological requirements has to be met. first, a standard (s) mrna must be synthesized which is able to undergo the same processing as the target wild type (wt) mrna. secondly, the quantitation step following the pcr must differentially recognize standard and target dna, and thirdly, the assay must be precise with respect to both inter-and intraassay variability. in order to satisfy these requirements we constructed a s-gpib mrna which is identical to the wt-gpib mrna except a 13 bp long primer recognition site at its 5" end allowing differentiation between the pcr amplified wt-or s-gpib cdna through incorporation of a fluorescein or biotin labelled 5" primer. both standard and w[ gpib mrna showed identical amplification kinetics in the pcr reaction. the amplified dna was quantified using an dna binding assay. in this assay binding of amplified dna to gcn4 fusionprotein-coated microtiterplates is measured. since the gcn4 binding motif is incorporated into the wt-and s-gpib cdna through an identical 3" primer, competition between s-and wt-cdna during amplification has been analyzed. at a given concentration of 250 nm of gcn4. primer no competition between the sdna and wt-dna for the primer was observed during 25 pcr cycles. the sensitivity limit of the assay performed in this way was 250 amol wt-gpib~, dna, and intraassay variability reached from 1.66% to 6.72% calculated for 100 fmol and 5 fmol dna, respectively. to sum up, combination of rt-pcr with the amplified dna binding assay and usage of an internal standard mrna allows sensitive and accurate quantitation of gpiba mrna in human platelets. since upa and thrombin are main conrtibutors to the process of proliferation and migration of vascular smooth muscle cells (vsmc), which is part of the pathogenesis of atherosclerosis. we are currently assessing the role of spatial expression of upa and thrombin receptor (tr) on cells with human carotid artery plaques (n=10). we have used a double immunolabeling approach, combining anti-upa and anfi-tr antibodies. to identify the different cell types, we used the following antibodies: anti a-smooth muscle actin (a-sma) for smooth muscle cells, ulex europaeus agglutinin i (uea i) for endothelial cells, inflammation cell cocktail (cd68+cd45) for monocyte/macrophage and lymphocytes and an anti-proliferation cell nuclear antigen antibody (pcna) to stain proliferating cells. in the carotid atherosclerotic plaques, upa immunostaining was distributed focally, preferentially in the fibrous cap and some cells of the foam cell rich region (fcrr). it was present in distinct patterns: cytoplasmic staining. tr staining was distributed similar to upa staining. with double staining combining anti upa antibodies with anti-tr antibodies, cellular co-localisation of both upa and tr was demonstrated. these cells were identified as smooth muscle cells by -sma. inflammatory cells were mainly localized within the fcrr, they only stained for upa. in conclusion: our data demonstrates that upa and tr are coexpressed in vsmcs in human carotid artery atherosclerotic plaque tissue. we therefore conclude, that the mitogenic activity of upa is associated with the thrombin signalling pathway. in the proficiency test of the ,,deutsche gesellschaft flir klinische chemie" (dgkc) 1/95, 5 lyophilised plasma samples (immuno ag) were sent to participants: a normal plasma and 4 plasmas from persons under oral anticoagulation (oac-plasmas. inr 1.9 to 3.7). the participants (n=552) returned the pt times obtained and in most cases (n=355) also the isi value for the thromboplastin used (isi of pack insert). the inr was calculated using the pt of normal plasma and the isi of pack insert (method i). two additional methods for inr calculation were compared with method i. according to the concept of calibrated plasmas (houbouyan et al., t993), a calibration curve was constructed using the normal plasma and the 30ac-plasmas. the inr calculated using the pt •fn•rma¿ plasma and the laboratory-specific isi value given as 1/slope of the calibration curve (method ii) or was read off directly (method hi). for inr values, calculated by the 3 methods from the participants data (n=355), outlier elimination (2sd, iterative) was performed. the inr mean values for all 3 calculation models remain in a narrow range. using calibrated plasmas (method 1i and m), less outlier were eliminated and cv's obtained were smaller than using the conventional procedure ( i ). obviously, the inr inherited problems, such as accurate isi value, pt value of normal plasma and instrument/laboratory influences on isi, can be reduced using calibrated oac-plasmas. practical approach and educational considera-tions of home prothrombin time estimation a. bernardo, a. bernardo, c. halhuber herz-kreislauf-klinik, bad berleburg, germany specific training is necessary for the patient to achieve reliable and reproducible results in prolhrombin time measurement. the training scheme is based in many respects on experience with similar training courses for home control and management of diabetes and asthma. the education program is divided into a theoretical and a practical part. the theory part has group sessions of twenty patients of a time. the practical course is reduced to a maximum of five patients. the sessions are conducted by a medical doctor and by specialized medicaf/technical assistants. on average eight hours of theoretical education and two hours of practical training are sufficient. the contents of the theoretical lessons are: • need for anticoagulation after heart valve replacement, • potential interaction between anticoagulants and other medication, • accurate recording of the measured prothrombin time results, • techniques of prospective determination of the necessary amount of anticoagulant, • calculation of the individual doses, • potential pitfalls and mistakes, • corrections in case of over-and under-dosage, • early recognition of thromboembelic and/or bleeding complications. an alternative is a full-day intensive course which can be held during the weekend. our recently reported (1) observation that oral anticoagulant treatment causes an increase of heparin cofactor ii (hc ii) activity in plasma is now confirmed by a more extensive study. in 43 thrombophilic patients who were on vitamin k antagonist therapy (marcumar r) we found a median hc ii level of 142 % as compared to 119 % for 72 thrombophilic patients without any therapy (p < 0.002" ) and 104 % for 59 healthy controls (p < 0.001" ). moreover we observed that the increase of hc ii level was significantly correlated with increasing inr-values (r = 0.63, p < 0.001). follow-up observations on some patients showed, however, clear differences in the levels of hc ii activity after onset of vitamin k antagonist therapy. thus, some patients responded rapidly with a significant increase in activity ("strong responders") while others showed only slight changes ("weak responders"). in conclusion, the determination of hc ii activity may result in an improved estimation of the risk of bleeding, especially in high intensity treated patients (inr > 3.5). after intracoronary stent implantation an aggressive oral anticoagulation (oac) therapy is mandatory. to find out whether coagulation activation occurs after coronary stent implantation during high dose oac therapy markers of plasmatic coagulation and d-dimer were measured. patients 5 male patients (average age 57 years) were examined. blood samples were taken before and right after stent implantation and during the following week. patients got 30 mg phenprocoumon during the first three days and additionally heparin and acetylsalicylic acid (asa) were given. methods ptz, aptt, tz, protein c, tat-complexes, fi+2 and d-dimer were measured. results d-dimer levels increased steadily between day 0 and day 7. tatcomplexes showed a slight increase from day 0 (2.4 bg/i) to day 3 (15.3 ~tg/i). on day 7 tat levels were down again (2.0 p,g/l). fl+2 (day 0:1.0 ng/ml) also showed a slight increase on day 3 (1.3 ng/ml). protein c decreased steadily from day 0 (108%) to day 7 (15%). conclusion during the initial phase of oac therapy a coagulation activation is reported but no significant elevation of tat or fl+2 was found. this result shows that additional heparin and asa therapy was sufficient to avoid systemic coagulation activation. the increase of d-direct should be interpreted as a si~=m of local fibrinolytic reaction due to stent implantation. three methods for the determination of prothrombin time from capillary blood in patients under oral anticoagulation have been investigated. two methods were run on coaguchek® monitors (boehringer mannheim) from capillary whole blood. after fingerpuncture the first drop of blood was applied to the well of a coaguchek® test strip directly from the finger-tip, whereas the second drop was sucked into a non-anticoagulated plastic capillary (hirschmann) and immediately applied to the test strip -and vice versa to eliminate any influence of first and second drop of blood. the third method was hepato quick (boehringer mannheim) which was determined out of citrated capillary blood from an earlappuncture. 66 specimen of patients under oral anticoagulation were investigated. the method comparisons between each of the coaguchek® methods and the laboratory method show good results and the correlation between the coaguchek® methods is excellent. mean differences to the lab methods are -0.1 inr in both cases. no mean deviation was detectable between the coaguchek® methods. scattering of coaguchek® versus hepato quick was +/-0.6 inr in the range 1 to 4 inr except for three outliers and one patient with fluctuating results in the lab method which could not be resolved. introduction: haemorrhagic coumarin skin necrosis is a severe complication during initial phase of oral anticoagulant therapy. histological examination shows thrombotic occlusion of small vessels, but little is known concerning the pathophysiologic background of the bleeding component. recently, we described protein z deficiency in patients with bleeding complications of otherwise unknown origin. thus, we were prompted to measure protein z in patients with coumarin skin necrosis. patients: 4 patients (i man, 4 women; age: 35±10 years) suffering from haemorrhagic coumarin skin necrosis were examined. all patients had normal liver protein synthesis function, none was under oral anticoagulant treatment during this study. method: protein z antigen test, diagnostika stago, france. results: 4 out of the 5 patients examined had diminished protein z levels ( 700, 820, 1080, 1700 ug/l) in comparison to normals (2900 ug/l). in one of our patients, protein z was normal (3020 ug/l). conclusion: low protein z levels are additional risk factors for haemorrhagic coumarin skin necrosis. oral anticoagulant therapy is the treatment of choice in patients with need for long-term anticoagulation. since oral anticoagulants interfere with the function of vitamin k, it is not clear whether stable oral anticoagulation can be achieved in patients with need for continous substitution of fat-soluble vitamins including vitamin k. we report about a 59-year-old man who had experienced progressive hypertrophic obstructive cardiomyopathy over the preceeding 21 years. atrial fibrillation has been first diagnosed 18 years ago. latter on, recurrent ischemic attacks and embolism of the right arteria iliaca occurred. in 1993 the patient received extirpation of the ileum and subtotal amputation of the jejunum because of mesenteric infarction. the resulting short bowel syndrome requires continous substitution of fat-soluble vitamins. since vitamin k free preparations of fat-soluble vitamins for parenteral use are not available, prophylaxis of thrombosis has been performed with unfractionated hepadn. as a consequence of the longterm treatment with hepadn the patient developed severe osteoporosis. therefore, the decission 1:o discontinuate heparin therapy and initiate oral anticoagulation has been made. because of its shorter halflife warfarin (coumadin) was used instead of dicoumarol. over a 4 weeks lasting induction phase inr values were controlled daily. a dosage regime starting with '10 mg warfarin at the day of vitamin application (day 1) followed by 3.75 mg on day 2 and 1.25 mg on days 3, 5, and 6, respectively, was found to be optimal to maintain inr values within the target range (inr: 2.0-3.0). in order to minimize the risk of hemorrhage the vitamin administration was changed to the subcutaneous route. during an observation period of 6 months neither any bleeding or thrombotic complications nor a vitamin deficiency occurred. these data indicate that stable oral anticoagulation can be achieved despite extreme variation of vitamin k plasma levels. portable monitors for home monitoring of inr are well established for adults on oral anticoagulants. patient's compliance is improved as well as long term outcome. experience concerning accuracy of the procedure in children is limited. 32 inr determinations were performed in parallel from venous and capillaryblood samples of an infant on phenprocoumon, starting at the age of 4 months. the coaguchek® monitor from boehringer mannheim was used. choosing an arbitrary range of agreement of ,qnr 0.5 for both determinations, 81% of the measurements were within the defined range. 5/6 outliers were due to low inr resulting from difficulties in capillary blood sampling. the degree of agreement increased when the procedure was performed at least once a week. in conclusion: inr determination with a portable monitor may be helpful in home monitoring oral an.ticoagulant therapy in young children. a dose adjustment should be done only on the base of inr determination of venous blood -if it is considered the gold standard -to avoid over-anticoagulation. a stable anticoagulation is one of the most difficult tasks in attending patients with heart-valve-prosthesis. if prothrombin times are out of the therapeutic range, the risk of bleeding or thromboembolism increases disproportionately. for this reason any improvement in anticoagulant control and/or management can have far reaching consequences in decreasing complications, in extending longevity and in improving quality of life. for the first time a clinical trial was started in 1986 and continues until today at the cardiac rehabilitation center bad berleburg, germany with patients mainly after heart valve replacement. the patients were trained to measure their own prothrombin time and to adjust their own dosage of the oral anticoagulant. within six years 600 patients were trained: 216 patients could be followed up with regard to their selfdetermined prothrombin times. the results were within the therapeutic range in 83.1% of the measurements (n=14.812) taken by the patients themselves. on average, the patients who determine their prothrombin time themselves did so at a weekly interval. neither major bleeding nor thromboembolic complications could be observed in the 205 patient-years of home prothrombin estimation. it is to be hoped that the usual rate of complications can be reduced when patients determine their prothrombin time themselves at a close interval, resulting in more constant values in the therapeutic range and slight corrections of the anticoagulant dose. home prothrombin estimation promises better quality of life and has a considerable potential to achieve this goal. circulating plasma thrombomodulin (tm) is a novel endothelial cell marker, which may reflect endothelial injury. tm acts as thrombin receptor which neutralises the fibrin-forming effect of thrombin, and also accelerates the formation of the anticoagulant protein c/s pathway. tm therefore belongs to the anticoagulant defence system against thrombosis. increased tm levels have been described in various diseases such as ards, thromboemboembolic diseases, ttp, diabetes, le and cml reflecting alterations of the vascular system at the endothelial level. to find out to what extent cardiac catheterisation imtates vascular endothelium, tm concentrations (stago, asnieres, france: x 10 3 iu/ml) were investigated prospectively in 58 infants and children (three days -16 years). blood samples were drawn before the intervention, immediately at the end and 24 h later, snap frozen (-70 °c) and investigated serially in dublicate six weeks -3 months later. the results (median and range values) are shown in the enhanced tm concentrations immedately after the operative intervention, followed by normalisation within 24 h, indicates that cardiac catheterisation in pediatric patients rather leads to a short lasting irritation of the vascalar endothelium than to severe irreversible endothelial damage. recently in an al=wl" based method dahlb~ick et al described in vitro resistance to the anticoagulant effect of activated protein c (apc) in thrombophilic adult patients. apcr is in the majority of cases associated with the arg 506 gin point mutation in the factor v gene. concerning the special properties of the neonatal hemostatic system (low vitamin k dependent coagulation factors, physiological prolongation of the pt and aptf) we adjusted this ap'it based method (chromogenix, m~,lndal, sweden) to neonatal requirements: apcr was measured in 120 healthy infants according to dahlb~ck. the results were expressed as apc-ratios: clotting time obtained in a 1:1, 1:5 and 1:11 dilution with factor v deficient plasma (instrumentation laboratory munich. germany) using the apc/caci2solution divided by clotting time obtained with cac12 in the same i:1, 1:5 and 1:11 dilution. in addition, plasma of 24 neonates with septicaemia were investigated and data of 18 infants aged birth -three months with arg 506 gin +/-were shown. the arg 506 gin mutation of the factor v gene was assayed by amplification of the dna samples by pcr followed by digestion of the amplified products with the restriction enzyme mnl i. results were confirmed by sscp -analysis or by direct sequencing of dna from patients with apcr. results are shown in the 1.6(1.4-1.95) neonates and infants were considered to be apcr when the aptt ratio was < or = 2. concerning the special properties of the neonatal hemostatic system, our data show concordance with the pcr method in neonates and infants only, when the aptt based method was performed in the i: 11 plasma dilution. case report: we report on an 8-year old boy with severe hemophilia b and frequent screaming at night. eeg showed spike wave activity, starting from the temporal lobe, but generalizing within seconds. complex partial seizures were diagnosed and therapy with carbamazepine was initiated. as no improvement was seen nmr was performed. this revealed lesions within the right frontal cortex. higher doses of carbamazepine were not succcssfull as was therapy with phenytoin and pfimidone respectevely. the patient is now treated with carbamazepine and valproate. he still suffers from one short seizure per day. because of his seizures we started prophylactic replacement therapy with 600 i.e. factor ix twice per week. discussion: in 1992 wilson et al. first detected brain abnormalities in 25 of 124 children and adolescents with hemophilia a or b who were negative for immunodeficieney virus (1). the most common findings (14/25 patients) were small, focal, nonhemorrhagic white matter lesions of high signal intensity on t2weighed images. similar lesions have been reported in children with sickle cell cerebral infarction (2) . only three of these 14 patients had seizures, all of those having a documented history of intracranial hemorrhage. our patient has similar lesions as those described by wilson et al. but no history of intracranial hemorrhage is documented. even if tuberous sclerosis might be a differential diagnosis, we think that the abnormalities are related to hemophili a or its treatment, because the patient has no further signs of this disorder. conclusions: 1. in patients with hemophilia and seizures nmr might be useful as a high sensitive method for the detection of gray and white matter changes. 2. further studies should be initiated to determine the prevalence of pathological conditions in the brain of hemophiliac patients. disseminated intravascular coagulation (dic) is a rare, but foudroyant disease occuring in gram-negative sepsis like meningococcal septicemia. despite the avallibility of potent antibiotics, mortality in mertingococcal disease remains high ( about 10 % ), rising to 40 % in patients presenting with severe shock and consecutive dic. as the clinical course and the severity of manifestations of systemic meningococcal infections varies there is a need for early diagnosis of the infection and stage of coagulopathy in order to reduce the high mortality rate. few and rapidly available parameters are needed to classify the wide spectrum of clinical and laboratory findings in patients with dic. the parameters include partial thmmboplastin time, pmthmmbin time, plasma levels of fibrinogen, fibrin monomers and dimers, fibrin degradation products and the thrombocyte count. monitoring the course of hemostaseologicai findings in 26 pediatric patients with systemic meningococeal infections we observed a change of coagulation parameters as early as in the first stages of the infection: a prolongation of partial thromboplastin time to an average of 69. 1 sec (range 22 -150 sec, norreal 30 -45 sec), a decrease of prothrombin time to 45.7 % (range 13 -71 %, normal 70 -100 %) and of antithrombin iii to an average level of 16. 8 u/ml (normal 20 -29 u/ml ) was found 1 to 4 (-6) hours after admission. the consecutive development of hemostaseological parameters mentioned above permitted to define the stage of coagulopathy and thus to induce a stage related therapy. primary treatment consisted in control of shock by liquid substitution, compensation of metabolic acidosis, correction of clotting disorders ( at iii and heparin in stage of pre-dic ; at iii and fresh frozen plasma in case of advanced dic ) and treatment with g-lactam antibiotics ( e. g. cefotaxime or ceftriaxone ). an early assessment of the coagulation disorders in meningococcal disease can be based on few coagulation parameters, thus an appropriate treatment may be arranged to prevenl the patient from a fatal outcome of meningococcai septicemia and protect him from the development of a waterhouse-friderichsen-syndrome. this study was designed to prospectivdy evaluate coagulation and flbrinolyfie activation in 60 children (neonate -16 years) during cardiac catheterisation with low dose flush heparin (10 iu/ml saline). aptt (instrumentation laboratory: see), anti xa activity (xa; chromogenix: iu/ml), prothrombin fragment ft.2 (f1.2; behring werkc marburg: nmol/l) and d -dimer formation (d-d; bnhring werke/vhrburg: ug/l) were investigated before (t1), at the end (t2) and 24 h after cardiac catheterisation (t3). in addition, to evaluate the influence of inherited thrombophilia in all patients resistance to activated protein c (apcr), protein c, protein s and antithrombin were investigated. during catheterisation median (range) hepadn was administered in a total dose of 60 (17-206) iu/kg bw. in addition infants < 6 months of age (arterial catheterisatiun only) or patients with known thrombophilia received 300 -400 iu/kg hepafin for fmther 24 hours. the results (median and range) are shown in the ft.2 was sigificanfly elevated above the pediatric boundary immediately after the intervcation and nearly reached baseline values 24 h later. in contrast no cfinically relevant fibrinolytic activation was seen: d -dimer formation increased within the pediatric boundary immediately after the catheter and returned to basdine levels 24 h later. three children showed resitance to apc. tn one child stroke occurred before. not knowing the result of apcr in the remaining two patients only one neonate received further prophylactic heparin. the third neonate without heparin prophylaxis suffered from venous occlusion within two days after the intervenfon~ in addition, no protein c, protein s or antithrombin deficiencies were found. although administration of low dose flush heparinisation during cardiac cathetefisation could not prevent short -term coagulation activation, no thrombotic events occurred in children without inherited thrombophilia. if fnrther prophylactic hepariuisation in children with a~r, protein c, protein s or antithrombin deficiencies may prevent vascular occlusion requires a more intensive study. a.sandvoss, w.eberl, m.b0rchert introduction: capillary leakage, edema and hypovolemia are common complications in preterm infants especially if birth weigth is below 1.500 g. septicemia, asphyxia and immaturity seem to be most important risk factors. to determine the influence of c 1-esterase inhibitor (cilna) in preventing contact phase and complement activation we investigated c11na concentrations in normal and symptomatic preterm infants. methods: activity of cilna were measured by chromogenic substrate method (behringwerke), cilna concentration with radial immunodiffusion (behringwerke,germany). results: cllna-activity in asymptomatic preterm infants (n= 14) was 65+/-15% of normal at birth. healthy newborns showed activities of 80+/-20%. cilna reached normal adult values 2-4 days after birth. preterm infants with respiratory distress syndrome(n = 14) showed lower activity on day 2-5, patients with additional septicemia (n=15) had decreasing c1 ina-activities in the first three days of life. individual course of cllna-activity and thrombocyte count correlated in the group with irds with and without septicemia. in children with capillary leakage onset of diuresis went parallel with raising cllna-activity. markers of contact phase (f xlla) and complement activation (c 5al were investigated in single cases and evidence for involvement of both systems was found. conclusion: contact activation and complement system play an important role in capillary leakage in preterm infants. cilna regulates both systems. activity of cilna correlates with clinical course, substitution therapy is possible and may improve outcome of these critical ill patients. antiphospholipid antibodies (apa) interfere with hemostasis probably by inhibition of protein c or prothrombinase complex. thereby, apa might lead to thrombosis or increased bleeding. however, incidence and clinical importance of apa has not yet been investigated in children. therefore, we assayed plasma samples of 220 children, aged 0,1 to 19 years (mean 7 years) by elisa detecting igg-and lgm-antibodies directed against eardiolipin, phosphatidyl serine and phosphatidic acid. in patients with increased bleeding, thrombophilia or prolonged clotting tests a detailed coagulation analysis was performed. according to their diagnosis children were devided into 5 groups: i. autoimmune diseases, ii. infections, iii. metabolic diseases, iv. other diseases, v. healthy children. results: apa were found in 69/220 patients. in the respective groups we demonstrated apa in the following proportions: 1. lgg-isotype: activitiy of c1 esterase inhibitor (c11na) is reduced in preterm infants especially if birth weigth is below 1.500 g and respiratory distress syndrome and/or septicemia is present. capillary leakage with generalized edema, hypovolemia and hypotension is resulting in imbalance between inhibition and activation of contact phase and complement system. iln four patients we investigated seven courses of substitution ;with commercial c1 esterase inhibitor preparation (berinertr,behringwerke), case reports are given. all patients had clinical symptoms of capillary leakage, all had septicemia accompanied by either respiratory distress, disiseminated intravascular coagulation or mutiple organ failure. jefficiacy of substitution therapy is dose related, supranormal iactivities of cilna are necessary, reflecting raised consumption of inhibitor in ongoing disease. clinical effects on diuresis, catecholamine need and especially on thrombocyte counts are demonstrated. or arterial thromboembolic event in children e. lenz, c. heller, w. schr6ter*, w. kreuz johann w. goethe-universit~itskinderklinlk, frankfurt a. main, germany * georg-augast-universit/itskinderidinik, g/3ttingen, germany venous thrombosis as well as arterial thrombo-occlusive events are rarely observed in childhood, but can lead to life-threatening situations and longterm sequelae in these patients. after the initial stage of treatment (thrembolysis or thrombectomy) the pediatrician has to decide how to efficiently prevent re-thrombosis in the individual patient. anticoagulation after venous thrombosis is generauy recommended for 6 months after the event; if an underlying thrombophilic condition has been detected in the patient anticoagulation has to be considered lifelong. when evaluating antithrombotic therapies for children it is of importance to consider whether the anticoagulatory effect is mainly necessary in the venous or arterial vessel system. the hemorrhagic risk and side effects of the different anticoagulatory preparations have to be taken into account, especially when treating small children. only limited experiences exist concerning the suitability of the preparations for long-term anticoagulation in children and general recommendations on the ideal dosage in pediatric patients are still missing. we want to disscuss different types of anticoagulants (such as coumarins, unfractionated heparin, low molecular weight heparin (lmwh) and inhibitors of platelet aggregation) their mode of action, their suitability for pediatric patients and their side effects and relevance of these side effects especially in children. from the experience in our own pediatric patients, we would like to report on the indications, which can be given to administer these different preparations, the dosage regimen we recommend and the laboratory tests to monitor save and efficient re-occlusion prophylaxis in our patients. in this context we would like to present our data on 8 patients with either thrombosis or arterial infarction due to a thrombophilic condition, who had all contraindicatioas to oral anticoagulation by coumarins. because prophylaxis for re-thrombosis was mandatory in these patients, lmwh was given for long-term anticoagulation in a dally subcutaneous dosage of 100-150 anti-xa u/kgbw. monitoring was done by anti-xa-test (0,4-0,8 anti-xa u/ml). under this regimen none of the patients developed re-thrombosis or bleeding complications. alopecia was seen as a side-effect. this study was designed to prospectively evaluate coagulation and fihrinolytic activation after cardiopulmonary bypass with aprotinin (2x17000 u/kg bw) in 42 infants and children aged 0.1 -15 years, and to correlate these findings to the clinical outcome. prothrombin fragment f 1.2 (f1.2; behring werke marburg: nmol/l), antithrombin-serinesterase -complex (atm; stago: ng/ml), d -dimer formation (d-d; behring werke marburg: ug/l), tissue-type-plasminogen activator ag (t-pa; chromogenix: ng/ml), plasminogen activator inhibitor 1 antigen (pai; chromogenix: ng/ml) and cl-inhibitor (c1; behring werke marburg: x 10-3 g/l) were investigated before the operation (t1), at the end of the operation (t2), and on postoperative days 1 (t3), 4-6 (t4) and 7-9 (t5), respectively. the results are shown in the table (median and median absolut deviation): t1 t2 t3 t4 "1"5 nv fi.2 0.9 +/-0.5 1.7 +/-0.9 1.4+/-1 1.8+/-0.8 1.6+/-0. the platelet (pl) function defect induced by thrombolytic agents has been attributed either to the degradation of pl surface receptors or to the anti-aggregatory effect of fgdps. in contrast to other plasminogen activators scu-pa is intimately inked with pl: they can rapidly incorporate exogenous seu-pa, release it upon stimulation and bind the proenzyme. recently we have reported that exposure of prp to recombinant scu-pa (2.5-t00 um) in timed interval 1-30 min resulted in dose-dependent inhibition of pl aggregation. timecourse changes of the process were followed by the biexpotential kinetics: a rapid initial inhibition during the first 3-5 rain with the moderate suppression of pl aggregation in the 30 min period. when tcu-pa (25-100 nm) was exposed to prp in the same conditions dose-and time-dependent inhibition of pl aggregation was also observed. since the effect was obtained no earlier than t0 min after exposure of tcu-pa to prp, and the threshold dose was higher. comparable inhibition of pl aggregation was obtained with 25nm of scu-pa versus 100nm of tcu-pa and the llbrinogen depletion by the end of the 30 min period was 2% and 30% respectively. it's likely that tcu-pa and its precursor have different mechanisms of action on the pl aggregatory function. in a recent study we have shown that recombinant rscu-pa inhibits platelet (pl) aggregation in prp. to exclude the possible influence of rscu-pa/plasma interfere on this process the aggregation of washed pls was under the investigation. pls were washed according to modified mustard's method, suspended in buffer and adjusted to 250,109/1. the resuspended pls were exposed to 5-100 nm of rscu-pa for 30 min at 370(;. at time points 3, 5, 15 and 30 min the aggregation with 0.6 iu/ml of thrombin was measured. it was found that the exposure of pls to rscu-pa (20-100 nm) for 3 man resulted in marked inhibition of their aggregation. since after 15-30 man of incubation with 20-50 nm of rscu-pa the inhibitory effect on pl aggregation became less pronounce or even disappeared. when 5 nm of rseu-pa was used the inhibition of pl aggregation became significant only by 15 rain of exposure period and didn't change for 30 man of investigation. the observed results may be cormeeted with uptake of rscu-pa by pls from surrounding buffer as well as with individual variations of pl response to the same concentration of rscu-pa. loss of glycosylation may result in a reduced platelet (p) survival and perhaps altered function. we analyzed the structural and functional effect of specific deglycosylation (combinations of n/o-glycosidase and neuraminidase treatment) of p and isolated p gpib. washed and formaldehyde-fixed p were digested as follows: 1) with neuraminidase (0.125u/ml) + o-glycosidase (3.1mu/ml) + n-glycosidase (1.25u/ml), 2) with neuraminidase alone (0.2u/ml), 3) with n-glycosidase (2u/rnl) and 4) with neuraminldase (0.2u/ml) + o-glycosidase (5mu/ml). all reactions were performed in the presence of protease inhibitors (pmsf, leupeptin, sbti), after washing x2 the p and identically treated controls were analyzed by flowcytometry with the antibodies 6di (mab: a-gpib), 7i-l2 0vlab: a-gpiiia), and the lectins wheat germ agglutinatinln (wga, for neunac) and peanut agglutinin (pna, for [3dgal(1-3)-galnac) which confirmed effective and specific deglycosylation by the respective enzymes (but gave only minor differences with 6di and 7h2). the botrocetin (13) and ristocetin (r)induced agglutinations showed arer treatment 1) (all enzymes) a full inhibition of r-induced agglutination but only a mildly reduced b-induced agglutination (70% of normal). treatment 2 and 3 (neuraminidase alone, and n-glycosidase alone) affected both agglutinations only mildly (70-80% of normal).treatrnent 4) (o-deglycosylation) however showed a major inhibition of r-agglutination down to 30%, while b-agglutination interestingly was almost fully retained. the results of the rotary shadowing electron microscopy of purified gpib suggested a collapse of the normally stretched, glycosylated, gplb, not only after the treatment with all three glycosidases, but also .after o-deglycosylation alone. we conclude that oglycosylation is most important for ristocetin-induced platelet-von willebrand factor-interaction and responsible for the typical stretched shape. the phenomenon of in vitro platelet aggregation and consequent pseudothrombocytopenia (ptcp) in the presence of calciumchelatization by na-edta and sodium-citrate was studied in blood samples of a patient. initial platelet counts electronically measured were 20000/ul blood anticoagulated with na-edta and sodium-citrate. normal platelet counts were found in heparin-anticoagulated blood and in capillary blood. immunoglobulines of the igg and igm subclass were identified in the patients plasma. by incubation of the patient's serum with platelets of healthy individuals, platelet-clumping occurred in the presence of na-edta and sodium-citrate but not in the presence of heparin. the platelet membrane glycoproteins (gp) hb/llia, ix and iiia/vnr g-chain were involved in the antigen antibody reaction as demonstrated by specific antibodies and flow-cytometry. on platelet surface permanent calcium-exchange and -replacement is dependent on external calcium concentration. calcium depletion induced by calcium chelators as na-edta and sodium-citrate might conformationally change platelet surfaces and induce formation of neoantigens. the decrease of gp llb/illa platelet surface antigen to 10% (normal >75%) indicated the important role of the gp iib/iiia receptor at ptcp. the saliva of tdatoma pallidipennis, a triatomine bug, was found to contain a protein called "pallidipin", that specifically inhibits collageninduced platelet aggregation but not adhesion or shape change. to investigate the mechanism of action of recombinant pallidipin the influence on platelet fibdnogen binding after activation by collagen type i in different concentrations was measured by flow cytometry. the same concentrations of pallidipin that inhibited the couagen-induced platelet aggregation completely did not cause any inhibitory effect on fibdnogen-binding in the prp from the same donor measured contemporaryly. collagen type i-induced platelet aggregation of cd36-deficient platelets from two different unrelated blood donors was inhibited by the same concentration of pallidipin that inhibited aggregation of control platelets. there was no inhibition of collagen-induced fibdnogen-binding in the cd36-deficient platelets as well. pallidipin did not cause inhibition of collagen-induced membrane expression of cd62 and cd63 of control and cd36-deflcient platetets as measured by flow cytometry. however eadier studies had shown an inhibition of collagen-induced atp and {3tg secretion by pallidipin. therefore we compared the effect of pallidipin in unstirred and stirred prp samples. while pallidipin had no effect in unstirred samples it showed strong inhibition of ptg secretion in stirred samples. we therefore conclude that pallidipin does not act on collagen-induced aggregation through cd36 and that the inhibition is a post fibdnogenbinding event. pallidipin does not influence the first steps in secretion, which are independent from cytoskeleton and platelet-platelet contact, but inhibits the following steps. 17-hydroxy-wortmannin does not inhibit the transport of 1nm-gold labelled fibrinogen in resting platelets. e. morgenstem, b. kehrel and k.j. clemetson medical biology, saarland univ., homburg, germany, haemostasis research, univ. muenster, germany and theedor-kocher-lnstitut, univ. bern, switzerland. wortmannin, an inhibitor of phosphoinositide 3-kinase and of myosin light chain kinase blocks reactions of the activated platelet. to obtain informations about the role of the contractile cytoskeleton in receptor-mediated transport of resting platelets, the effect of 17-hydroxy-wodmannin (hw) on the endocytosis of fibrinogen from the surface of resting platelets was studied. gel filtered platelets (gfp) were incubated for 10 min at 37°c with hw (3x10-6m) or with iloprost. controls and gfp preincubated with hw or ilopmst were incubated with 1.4nm-gold labelled fibrinogen molecules (fg-au; final concentration 40p.g/ml) at 37°c. the experiments were stopped after 5 or 30 min by rapid freezing. after freeze substitution in acetone with 4% osmiumtetroxide, sedal sections were prepared. the sections were examined after incubation with ascorbic acid (5% in h20) for 30 rain at 20°c (to reduce metallic osmium) and silver-enhancement using danscher's (1981) method (to visualize the fg-au). examination of adp stimulated platelets in the presence of 40fg/ml fg-au shows that the ligand is able to mediate aggregation. the examination reveals, that fg-au was present in a low density on the platelet surface, in higher density in the surface connected system (scs), in coated pits and vesicles and separated smooth vesicles (representing endosomes?) as well as in the matrix of alpha-granules. after 30rain, the number of labeled granules was increasing. labels on the surface and on the mentioned cytoplasmic membranes were observed during the whole period of incubation. hw or iloprost did not alter the resting gfp and the mentioned qualitative ultrastructural findings in both preparations did not show differences to the controls. we conclude from the results with hwthat the regular contractile function of the cytoskeleton is not necessary to transport the fg-au in resting platelets. methods: edta anticoagulated whole blood was incubated with thiazole orange and analyzed with a flow cytometer. young platelets were defined by having a high fluorescence from thiazole orange (normalized to platelet size). platelets were also incubated with fluorescent antibodies to gpib, gp lib/ilia and gmp-140 (two colour method). results: surface expression of gpib was the same in young and older platelets. results for gp lib/ilia and gmp-140 (in resting and activated platelets) will be presented. conclusion: young platelets can easily be detected using thiazole orange and flow cytometry. there is no differential expression for gpib. further results will be presented. the influence of erythrocyte and thrombocyte content on the release of atp by different agents in whole blood specimens was tested. the measurement had been performed in the lumi-aggregometer using the principle of the luciferin-luciferase reaction. altogether 39 blood samples were diluted gradually before induction of the release reaction by arachidonic acid (1,25 mmol/i final concentration), adp (30 ijmol/i) and collagen (1,0 and 5,0 tjg/ml). the peak of the obtained curves was transformed into percent values of the maximal deflection by the undiluted sample (= peak in relation) and into atp concentrations (= absolute peak) after testing the atp standard in parallel for each dilution step separately. the peak in relation increases by increasing dilution with all inducers. it was identic with the atp standard and with collagen, somewhat lower with arachidonic acid and much higher by adp. a luminescence-optical effect may influence all these results. the absolute peak decreases by dilution under arachidonic acid and collagen as it was expected by the decreasing thrombocyte content of the samples. under induction by adp no decrease of the absolute peaks by increasing dilution of the samples was abserved. this can be explained only by liberation of atp from the erythrocytes. the atp standard is essential for the quantification of the release reaction. adp doesn't suit for it. collagen with a final concentration of 1 pg/ml was proven as the best inducer. platelet aggregation induced by several agents has been photometrically investigated in disc shaped rotating cuvettes coated with vessel wall tissues obtained from human umbilical cord, either endothelium or smooth muscle cells or extracellular matrix or combinations of them. in addition, effects of endothelium incubated with several cytokines on platelet aggregation have been studied. endothelial cells strongly inhibited aggregation depending on their cell count and the concentration of the inducer. smooth muscle cells showed the same effect but very less marked. in presence of extracellnlar matrix spontaneous aggregation occured. endothelium could inhibit this spontaneous aggregation when present in the same cuvette, smooth muscle cell could not. incubation of endothelium with several cytokines increased its anti-thombotic properties. for example, at a platelet count of 3x105/id in the prp, 10 -6 m adp led to maximal aggregation in uncoated cuvettes, in presence of 5,5x106 endothelial cells aggregation was completely abolished, in presence of 2,75x10 "6 cells aggregation was decreased to 40%. smooth muscle cells diminished the aggregation effect of 0,1 nih thrombin to 67% when only one side of the cuvette was coated and to 63% when both sides were coated. endothelium could not inhibit aggregation induced by 2,5 x 10 -6 m adp but endothelium incubated with 500 u/ml tnf-a or 30 u/ml intedeukin-lfl or lmm l-nitro-arginin for 24 h did completely inhibit aggregation. platelets become sticky and adhere to surfaces or to another without contracting and secreting. during maturation of megakaryocytes finally platelets lost their genomic nuclear message. only mitochondrial dna of platelets can be identified. we focused our attention on the impact of mitochondrial dna and the mitochondrial transscriptive mechausisms during platelet activation in normals. materials and methods: leucocyte free (nagentte chamber, flow cytometric analysis) platelet rich plasma or platelet concentrates a_~er hemapheresis were filtered by pall 100 leucocyte filters. the influence of different anticoagulants (commercially available sarstedt tubes containing citrate, heparim edta and 500 atu/ml hirudin wacker) was examined. activation was due to a 60 nun. hemapheresis procedure ( 3-5fold increase of cd 62, cd 63) and ex rive stmaulation due to 4 niy u/ml thrombin, 0.025 m cac12 or combmatious. the guanidiurn method for total rna preparation were used according to t. brown: current protocols in molecular biology 4.21-4.9.14,1991. different primers of mitochondrial genome (e.g. cytochrome b and atpase) were prepared using pcr and mitochondrial transscription was examined using northern-blot-technique. results: 1., there is less activation of mitochondrias using hirudin anticoagnlation, but a 2fold increase of mitochoindrial rna content in heparinized samples. 2., stimulation with thrombin leas to an increase to 5.5 e-l0 rna btg/platelet, compared to 4.7 -4.8 e-10 rna ~tg/platelet under unstimulated conditions.. conclusion: there is evidence for the importance of platelets mitochondrial dna and mitochondrinl transsefiption in regulation of cytosceleton and platelet activation. thrombospondin-1 (tsp-1) is a large homotrimeric glycoprotein originally identified as a platelet alpha-granule component. the investigation of its putative role in a variety of pathophysiologies like haemostatic disturbance, malignancy and wound healing requires specific laboratory reagents. monoclonal antibodies are one of the most powerful of these reagents. therefore, we purified human tsp-1 from thrombin-stimulated platelets using affinity chromatography to generate monoclonal antibodies in mice. a subclass igg 1 monoclonal antibody designated 48.42 was purified from ascitic fluid and further characterised. western blot experiments demonstrated that this antibody reacted only with the unreduced molecule whereas the tsp-1 subunit chain was not recognised. no cross-reactivities with human fibrinogen, fibronectin, vitronectin and von willebrand factor were found. preliminary results indicate that the monoclonal antibody 48.42 can be used to investigate tsp-1 function in several assays including immunocytochemistry and cell adhesion as has been demonstrated for hl-60 cells. in addition, a sandwich enzyme immunoassay was developed using goat-antihuman tsp-1 igg and derivatised monoclonal antibody 48.42 (peroxidase, biotin) as a sensitive method for detection of tsp-1 in human body fluids. in the following study the expression of the platelet antigen (cd62p) and the leukocyte antigen (cdllb) were measured in whole blood, in addition to platelet-leukoeyte adhesion (rosette formation) by means of multicolour fluorescent labelling (cd45, cd14, cd42a). the measurements were carded out both in freshly drawn whole blood which had been antieoagulated with different agents, and in stirred samples of whole blood under controlled conditions (37°c, 1000 rpm, different stirring times). the results are presented as the percent positive events in each gate (platelets, leukocytes -pmnl, monocytes, lymphocytes and rosettes -plateletpositive events in the pmnl, monocyte and lymphocyte gates), whose mean fluorescence is given in addition to an index comprising the product of the percent positive events and their mean fluorescence. stirring (max 15 rain) induced an increase of cd62p on the platelet surface of ca. 10%, without any change in the mean fluorescence. under these conditions increased cdllb on pmnl and monoeytes could be detected. an increase in the rosette formation could also be measured (greater index), in that the percent of monocytes which were platelet-positive increased with no change in the mean fluorescence of the positive events, whereas pmnl showed an increased mean fluorescence, but not an increased number, of platelet-positive events. the time-dependent changes in rosette formation on stirring could be further increased by addition of adp. these results show that it is possible to measure rosette formation, and also the influence of effector agents (inhibitors or activators of platelets or leukocytes) on rosette formation, in whole blood using flow eytometry. 17 itp patients undergoing splenectomy were observed after 1-30 years following operation and divided into 2 groups. first group consisted of 8 patients with normal platelets count and absence of haemorrhagic syndrome. second group was formed of 9 itp-patienfs with episodes of thrombocytopenia recovery following certain time period after splenectomy. in the aim to study the cellular immunity there were carried out immunophenotypical investigations of blood samples using immunofluorescence method with monoclonal antibodies application. the increase of b-cells, expressing cd22, cd37, hla-dr-antigen has been revealed in the 2nd group. quantity of srfc, cd3 +, cd5 + cells in the blood of recovered patients was lower than in patients of the first group. this group was also characterized by statistically significantly increased level of cd4 + cells while the cd4/cd8 ratio was equal to 1.0 :i: 0.3 % (0,5 + 0,1% in patients of the second group, respectively, p>o,05}. also the relatively high expression of activating antigens in patients with thrombocytopenia recovery after splenectomy was stated. among infectious complications in all patients observed were predominantly found various types of throat infection, mainly with unsatisfactory treatment possibilities. we have observed the opsi-syndrome in 2 patients, being featured with marked tiredness, breath loss, intolerance of hard physical working, diminished ability to maintain physical activity. extracellular matrix (ecm) produced by human endothelial cells closely resembles the vascular subendothellal basal lamina in its organization and chemical composition. thus it contains collagens, fibroneetin, von witlebrand factor, thrombospondin, fibrinogen, vitronectin, laminin and heparin-sulphate. platelets carry different receptors on their membrane surface with specific binding capacities for one or more of these extracellular matrix proteins, such as glycoprotein (gp) iibiiia, gp ib/ix and gpiiib. incubation of platelets with ecm results in platelet adhesion, degranulation, prostaglandin synthesis and aggregation. we studied patients whose platelets showed either a receptor defect in gpiibiiia or gpiiib or a storage pool disease. adhesion experiments were performed using siliconised glass, collagen coated surfaces, immobilized fibrinogen as well as human subendothelial matrix. platelet adhesion of patients with thrombasthenia glanzmann (receptor defect of gpiibiiia) resulted in a total lack of binding to silieonised glass and immobilized fibfinogen. adhesion to collagen was almost normal in spite of the fact that only single platelets sticked to the surface and no microaggregates were observed. the adhesion to ecm was diminished and also no aggregates were detected. patients with a receptor defect in gpiiib showed normal platelet adhesion to siliconised glass and immobilized fibrinogen but binding to collagen and ecm was markedly reduced, while platelets with a storage pool defect sticked to siliconised glass but failed to adhere to ecm. by centrifugation of citrate blood (250 x g, 10 min) erythrocytes and leucocytes go to the bottom, whereas plasma and thrombocytes stream in the upper part of the probe. so the thrombocyte count doubbles in the platelet rich plasma in contrast to the platelet count in the whole blood volume. if the thrombocytes are more or less activated, they adhaere on erythrocytes, leucocytes or aggregate end are not able to stream upwards. the quotient between thrombocyte counts in prp and whole blood is a measure for thrombocyte activation. we chequed the value of this screening in different groups of patients with arterial occlusions disease (aod), chronical venous disease (cvd), diabetes mellitus (dm] and in healthy control persons (control). variation coefficient of the method is 3.7 (prp) and 4.4 (tc) respectively (coulter counter). differences to the control group are significant. changes in the patient groups in dispensaires follow up 5 years are also significant. nicardipin -induced immunthrombocytopenia p. eichler 1, c. hinrichs 2 , g greinacher l i.institut fur immunologic und transfusionsmedizin, ernst-moritz-arndt-universitat greifswald, 2. deister-s0ntel-klinik, bad m0nder drug-dependent immune-thrombocytopenias are a rare but clinically important variant of immune-thrombocytopenias. patients are at risk to suffer from severe bleeding complications. especially in patients receiving multiple drugs, diagnosis of drug-dependent immune-thromboeytopenia is often difficult. we report the case of a 71 year old male patient who received allopurinol, captopril, digitoxin, furosemid, and nieardipin. the patient presented with hematomas (pit. count < 10 g/l) and later developed bone marrow dysplasia. in an elisa using whole platelets and patient serum, a weak reactivity in the presence of furosemid, but a stronger reactivity in the presence of nicardipin (antagonil, ciba-geigy) could be demonstrated. the reaction pattern is given in the the enzyme-immunological determination of soluble fibrin (sf) proved to be highly sensitive and specific. this sf-elisa detected fibrin hacking fibrinopeptide a (fpa) via the monoclonal antibody 2t35 specific for the neoepitope generated on the aa-chain after the split of fpa. lill et al. recently introduced a new assay modification which utilizes the same antibody as the old one but takes advantage of a pretreatment of plasma specimens with kscn. this strong chaotropic ion is used to dissociate the various fibrin complexes possibly hiding fibrin epitopes. it was the aim of this study, therefore, to compare the two sf-elisa modifications (with and without kscn-pretreatment of specimens) . in order to examine the dynamics of thrombin-induced fibrin(ogen) metabolism we made course observations in patients with a certain form of septicemia. both assay modifications detected fibrin(ogen) derivatives which differed considerably in kinetics (n= 160 samples from 10 courses). the former sf-elisa (no kscn) correlated well with prothrombin fragments, thrombin-antithrombin !11 -complexes and with the release of fibrinopeptide a ( r > 0.96, n= 151). results of the new sf-elisa with kscn pretreatment of patients' plasma, however, correlated conspiciously well with d-dimer levels (r > 0.94) but distinctly less with the markers of thrombin generation (-0.12 < r < 0.29). this good correlation with d-dimer levels was unaccountable since the d-dimer maximum occured significantly later than the peak of markers of thrombin generation (p < 0.05). therefore, kscnpretreatment of fibrin specimens seems to lead to a change in the specificity of the fibrin assay despite usage of the same catching antibody. different half-iifes of differently composed fibrin complexes should be considered in trying to explain the findings. nevertheless, the results of the former assay without kscn-treatment correlated much better with the well-known dynamics of thrombin-induced fibrin generation during hemostasis activation than the data from the new assay modification. consequently, further examinations are necessary to specify the effect of kscn on soluble fibrin complexes and the resulting assay specificity. a rapid assay for the determination of the primary hemostasis potential (php) of whole blood has been developed (kundu et al, 1995) from the original method of kratzer and born. the new system employs a disposable test cartridge which holds the sample (citrated whole blood) and all components for the tests at the same time. the test procedure is very simple. the cartridge is loaded with -500 p.l citrated whole blood and is inserted into the platelet function analyzer (pfa 100aaw). the test is started automatically after a preincubation phase of 2.5 rain. the reaction starts with the contact of the whole blood and the capillary which is connected with a collagerdephinephrin coated membrane with a small aperture inside the test cartridge. under constant negative pressure the sample is aspirated and through the contact ofplatelets and vwf with collagen adherence and aggregation begins. the adhesion and aggregation process leads to the formation of a platelet plug which obstructs the flow through the aperture. the result of the php is reported as closure time (ct). additional parameters such as bleeding volumes are possible as well. first results show good reproducibility, normal values in the range of up to 150 sec. and a good discrimination of healthy donors from patients with congenital or acquired platelet dysfunctions. the system detects aspirin induced thrombocyte function defects and von willebrand disease. in ease of an abnormal result in the collagerdepinephrin system a second type of cartridge with a collagerdadp coating can be employed. in the majority of cases aspirin induced dysfunctions are normalized and could thus detect aspirin use. the proposed system may be a valuable tool for routine assessment of the primary hemostasis potential in a routine citrate blood sample laboratory. inducing mental stress in 20 young healthy male volunteers aged 20 to 40 ),ears with no previous history of thmmbophilia or a hemorrhagic diathesis was performed by a first time parachute descent from an altitude of 1000 meters. the purpose of this investigation was to find out whether there are any changes in the corpuscular and plasmatic fractions of peripheral blood. we were especially interested in elucidating changes in the procoagulatory and/or fibrinolysis systems. venous blood samples were obtained directly before and directly after the jump. flight time from the departure of the airplane to the landing of the parachutists was approximately 20 minutes. the maximum time that elapsed between the two blood withdrawals were 45 minutes. in a preliminary study with different voinnteem, certain fluid imbalances had been observed. absolute numbers of leukoeytes (6.9 vs. 9. l/n0, erythrocytes (4.6 vs. 5.1/pl), and platelets (246 vs.276/nl) significantly increased (p < .001), as well as the hemoglobin concentration from 145 to 156 g/l (p < .018). even though fluid imbalances before and after the jump had practically been excluded by measuring nearly identical hematoerit values (.41 vs..42), we noticed a marked drop in aptr (27 vs. 23 sec) and a significant increase in factor viii ~tivity. as a direct stress response, we found a rise in fibrinogen concentration (2.4 vs. 2.8 g/l) which is one of the shortest acting acute phase proteins. concerning reactive fibrinolysis, d-dimers showed an increase in concentration from 115 lag/l to still normal values of 192 lag/l, which was not significant due to low numbers of values (p = .086). we observed similar changes in fibrin monomers and prothrombin fragments fl+2. from other investigations on the kinetics of the activation of the procoagulatory system we know that maximum activil7 is not reached until 24 hours after initiation of activation.these investigations studied perioperative changes in different kind of operations which served as a control group concerning the degrees of tissue damage and resulting coagulation disturbances. to better understand these phenomena we plan to induce mental stress in a laboratoq' environment to further exclude unknow~a influences on the mechanisms which can activate the procoagulatory and fibrinolytic systems. triodena (t) 30/40/30 ug ee, 50/70/100 ug gestodene) were tested for their effect on hemostatic parameters. three groups (n=20) of healthy female volunteers were treated for 6 months with one of these oc. blood was taken before treatment (day 24-28 of pretreatment cycle, 0) and on days 18-22 of the 3 ~ (i) and 62 (ii) treatment cycle. indications of an activation of blood coagulation and fibrinolysis were detected as the plasma levels of prothrombin fragment f i+2 and of fibrin split product d-dimer and plasmin antiplasmin complexes were found elevated during treatment. the following main regulatory components of blood coagulation, activators and inhibitors, were investigated: factor vii antigen fviiag, fvii clotting activity fviie, circulating activated factor vii cfviia and antithrombin 3 at3 activity, total protein s antigen tps-ag, free protein s antigen fps-ag, protein s activity psact, circulating thrombomodulin etm fviiag, fviie and cfviia significantly increased during treatment; cfviia: 0: c 32.4 mu/ml a prethrombotic condition characterized by elevated levels of circulating soluble fibrin has been claimed to be a predisposing factor for accumulation of coronary thrombotic material in acute myocardial infarction. the present study includes 161 patients with clinical suspicion of myocardial infarction. blood samples were drawn by the primary care physician, upon arrival in the hospital, and after 2, 6, 12, and 24 hours of hospital stay. patients with myocardial infarction were identified by typical course in 12 lead ecg, and upon sequential determination of troponine t, myoglobin, ck, and ck-mb. patients with primary cpr were excluded from evaluation. soluble fibrin was measured by enzymun®-test fm (boehringer mannheim). patients with acute myocardial infarction display soluble fibrin levels within the normal range (< 5 ~tg/ml) during the initial two hours after onset of symptoms. there was no significant difference between patients with myocardial infarction and patients with coronary heart disease without myocardial infarction. slightly elevated levels were found in patients with atrial fibrillation, reflecting intracardiac fibrin formation. in patients without fibrinolytie treatment, a slight increase of soluble fibrin levels with a maximum after approximately 8 hours is observed. most patients with fibrinolytic treatment display a considerable increase in soluble fibrin, with maximum levels immediately after infusion of the fibrinolytic agent. four patients with pulmonary embolism showed soluble fibrin levels in the range of 40-300 [.tg/ml, which remained in the same range during the entire observation period. in conclusion, circulating soluble fibrin is not increased in patients with acute myocardial infarction and does not appear to be a predictor of acute coronary events. high levels of soluble fibrin in patients with fibrinolytic therapy may reflect release of fibrin from thrombotic material, but also de novo generation of fibrin due to release of active thrombin from thrombi not necessarily located in the coronary vessels. detection of elevated levels of soluble fibrin in patients with acute chest pain should result in careful examination for signs of pulmonary embolism or aortic aneurysm. the possibility to determine activated coagulation factors opens the question if data provide evidence of an activated coagulation or fibrinolysis and if this has a prospective value. we investigated patients with confirmed thrombosis, postsurgical septieaemia and also after liver transplantation. in all patients factor viia, xii, xiia and also the fibrinolytic parameters t-pa, pai-1, pap, plasminogen and a2-ap were determined. in addition, f1+2 and apc-resistance with heterocygote factor v-leiden-mutation and confirmed thrombosis. we found increased factor viia which showed partly also an increased fl+2. patients with other pathological results such as a reduced t-pa and/or increased pai-1 showed a low incidence of elevations in factor vii or f1+2. the activation of factor xii seems to be of minor importance in patients with thrombosis. a different picture is found in septic and transplanted patients. obviously factor xii-activation is of major importance in this group. a deterioration of the clinical symptoms is correlated with an increased factor xiia which is paralleled by a decrease of factor xiiactivity. the investigation of fibrinolysis parameters such as pai-1 and pap demonstrate a fibrinolytic disturbance of the balance. statistically significant are differences in septicaemic patients both in the surgical and in the internistical group in contrast to polytrauma patients. in patients with liver transplantations significant changes are apparently related to rejection of the transplanted organ together with a deterioration of the clinical picture. the possibility to detect activated coagulation factors may be a tool to detect changes in the hemostasis system at an early stage and to use this for an improved therapy. control of long-term oral anticoagulation is usually performed by serial determinations of the prothrombin time. however, the assessment of effective anticoagulation versus the potential risk of bleeding complication is difficult to achieve. molecular markers of blood coagulation activation might add valuable information in individual cases. we investigated 48 patients with thromboembolic manifestations (deep vein thrombosis n=22, pulmonary embolism n= 13, myocardial infarction n= 13) for one year beginning with admission to the hospital. tat, prothrombin fragments f 1 +2, d-dirner and fibrin monomer concentrations were analysed. all markers were significantly increased at the time of initiation of anticoagulant therapy thus reflecting a prethrombotic situation. patients suffering from venous thromboembolism demonstrated higher concentrations of tat and f 1 +2 in comparison to myocardial infarction (34.6 vs 12.3 pg/1, p=o.009; 2.8 vs 1.3 nmol/i, p=0.0025). f 1 +2, tat and d-dimer concentrations decreased gradually over the first 14 days of anticoagulant therapy reaching values within the established normal ranges in all cases. f 1 +2 and tat concentrations reflect the activity of the coagulation system during long-term anticoagulation whereas analysis of fibrin monomer yielded partly controversial results. we conclude that f 1 + 2 and tat appear to be superior to fibrin monomer for the individual control of oral anticoagulant therapy. the influence of thyroid failure on haemostasis is controversial. mainly hypoceagulable states have been described in clinically overt hypothyroidism. since hypothyroidism has been associated with an increased risk of atherosclerosis, we studied a wide range of haemostatic factors in untreated female patients with subclinical (b, n=42, age 59+13) or overt (c, n=8, age 55-zcj) hypothyroidism, as well as in hypothyroid women under 1"4 treatment (d, n=8, age 57+9) and euthyroid controls (a, n=80, age 50+14). simple screening tests (prothrombin time, activated partial thromboplastin time, fibdnogen), procoagulant factors (fvii, fviii, von willebrand factor), coagulation inhibitors (antithrombin ill, hepadn cofactor ii, protein c, protein s) and fibdnolytic factors (plasminogen, antiplasmin, plasminogen activator inhibitor, tissue plasminogen activator) were measured. results factor vii activity (vii:c), factor vii antigen (vii:ag) and their ratio were found increased in hypothyroid patients. factor viii activity showed the same tendency, whereas von willebrand factor ramained unchanged, as did all other parameters with exception of free protein s, which declined in overt hypothyroidism and in t4 treated subjects. these differences tended to diminish after exclusion of 26 women with estrogen replacement therapy for menopause, but the ratio vii:cnii:ag, as well as fvii:c still remained significantly higher in hypothyroid patients. conclusions: subclinical and overt hypothyroidism are associated with significantly higher levels of factor vii:c and vii:ag. the disproportionate increase in vii:c compared to vll:ag, as shown by their ratio, might reflect the presence of activated factor vii (vila), which in turn indicates a hypercoagulable state. this pattern becomes more pronounced with the concomitant estrogen replacement after menopause. exocytosis following platelet activation leads to translocation of cd62p (p-selectin), cd63, and thrombospondin, from cytoplasmic granules to the cell surface membrane, where these molecules, serving as activation markers, can be detected by flow cytometry. we here report detectability of these molecules preformedprior to platelet activation -inside the cytoplasm of resting platelets. two different methods are compared, i. e. using either methanol or the fix&perm kit (an der grub) for cell membrane permeabilization. in addition, interleukin(il)-ice is shown to be present in platelet cytoplasm after methanol treatment, but not after permeabilization using fix&perm. whenever cell surface positivity for a specific marker coincides with intracellular presence, blocking of the surface membrane sites prior to membrane permeabilization is required in order to obtain fluorescence intensity attributable to cytoplasmic staining. our data demonstrate the feasibility of the methods presented for the detection of intracellular platelet molecules. this technique should also provide a means for estimating the relative quantity of intracellular platelet antigens, provided the permeabilization procedure does not lead to antigen leakage or destruction. physical exercise activates the clotting as well as the fibrinolytic system as indicated in numerous investigations of exercise by running and by bicycle ergometer but not by swimming. the positive effect of an endurance training in coronary sport groups is induced also by influences on the hemostatic system. the influences are suppression of the clotting activation by the acute exercise and by an increased fibrinolysis response. different hemostatic parameters, therefore, were analyzed before and after swimming of male coronary patients (n=33; median ag~ 61 years, achieved heart rate: 68/min). indicating plasmatic clotting activation there was a significant increase in molecular markers tat and f1+2 among the coronary patients (tat from 2,1 to 3,4 pg/1; fi+2 from 0,92 to 1,1 nmol/1). the degree of clotting activation among the coronary patients was less than that observed in a group of young volunteers in a former investigation. this must be explained by existence of the coronary heart disease or by the higher age in the patient group. indicating an activation of fibrinolysis t-pa activity increased significantly in coronary patients (from 0,14 to 0,5 iu/ml) resulting in an unchanged balance between coagulation and fibrinolysis. from this findings of the hemostatic systems no increased risk of the coronary patients by swimming can be derived. a prerequisite, however, are precautions l±ke to devoid exercise in the anaerobic range, exclusion of major heart failure and of cardiac arrhythmias before begirming of the swim training. the principle of the fontan operation consists in anastomosing the right atrium to the pulmonary arteria, thus bypassing the right ventricle and using the only functional single ventricle as a pump for the systemic circulation. there are only few data about the influence of the changes in hemodynamics on coagulation and fibrinolysis. we investigated the coagulation system in 20 children and young adults aged 4 to 21 years in a general examination 4 to 61 months after fontan procedure. besides other abnormalities of the coagulation system, there were significantly increased values for the thrombin-antithrombin-iii-complex (tat) in 12 patients (60%). as a marker for an activation of the fibdnolytic system we found elevated plasmin-alpha2-antiplasmin-(pap-) levels in 14 patients (70%). less frequently, the concentrations for the prothrombin-fragments 1 and 2 (f1 and 2) (7 patients, 35%) or the d-dimer (2 patients, 10%) were increased. we didn't find significant differences in a clot-lysis-assay between fontanoperated patients and an age-matched control group. there was no significant correlation between activation of coagulation and clinical situation or diameter of the pulmonary arteria. whether the present data can help to estimate the risk for a thrombo-embolic complication following fontan procedure, still has to be investigated. the results of the clot-lysis-assay suggest, that for lysis of thrombi the same dose of rt-pa should be used as for other patients. a 2nd generation functional protein s assay p. van dreden* and e. adema** * serbio, gennevilliers france, ** boehringer mannheim, tutzing germany a second generation protein s test was developed with improved sensitivity to protein s and better reagent stability. the test result was found to be unaffected by apc-resistence (10 patients, heterozygote for the mutation with a apti' + apc ratio between 1.4 and 1.9), heparin up to 2 iu/ml and f viii activity between 1 and 250%. in the test, diluted sample is mixed with protein s deficient plasma, activated factor v, activated protein c, phospholipids and an intrinsic pathway activator. this mixture is incubated for 3 minutes. during this time, the activated protein c inactivates part of the f va. the extend of f va inactivation depends on the protein s concentration. after 3 minutes caci2 is added and the time untill clot formation is measured. the clotting time is a linear function of the protein s concentration between 10 and 140% protein s. for the three preproduction lots the difference in dotting time between 10 and 100% protein s was 43-54 seconds. this compares to 30-40 seconds typically obtained with the old test. within run precision (n= i0 on sta) is cv= 2 -7% on the basis of protein s. day to day precision (n=10 on sta) was found to be cv= 4 -11%, again calculated on the basis of protein s concentration. the cv of 11% was obtained for an avk plasma with 13% protein s; it corresponds to a standard deviation of only 1.5% in protein s. the insensitivity to interferences, in particular apc-resistence and better precision and stability are expected to improve the quality/reliability of a protein s determination. in this study we evaluated the use of hormonal contraception on the parameters protein c, protein s and pal. samples from 71 women with, without hormonal contraception and in menopause were assayed by coagulometric (protein s clotting test (behdngwerke, marburg, frg) or chromogenic methods (protein c activity test and pal reagent from behringwerke, marburg, frg) in double determination and were compared with the reference ranges. in addition thromboplastin time (thromborel s reagent) and fibrinogen (multifibrin) from behringwerke, marburg, frg, and aptt (actin fs reagent from dade corp., unterschlei6heim, frg) were determined. in women using hormonal contraceptives (p<0,01) and in menopause (p<0,05) protein s activity was significantly reduced compared to other women (<45 years) while protein c acitivity did not change. in menopausal women a higher susceptibility to thrombosis was supported by an increase of aptt (p<0,05) and fibronogen (p<0,01). while there was no change for pal, plasminogen was significantly lower in women using hormonal contraceptives and in menopause (p<0,05). we could not observe a higher turnover of coagulation and fibdnolytjc system with hormonal contraception. noteworthy was the occurence of low (<200 mg/dl) and borderline fibrinogen (max. 220 mg/dl) in 40,9% of women res. in 22,8% of women (together with borderline aptt) who had an individuell risk for arterial disease. protein s protein c fibdno~en aptt plasminog~ without hcc 109,1-+13,6 78,3-+14,1 255,0-+14,0 36, [2] [3] [4] [5] 4 24, [0] [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] 2 with hcc 85, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] 2 78, 5±12, 0 253, 8.+24, 1 35, [2] [3] [4] 8 14, 9 menopause 90, 3+97, 6 87, 4±41, 4 307, 05:57, 6 39, [8] [9] [10] 0 12, 6 hcc= hormonal contraception hemostatic parameters in a patient undergoing bone marrow and subsequent liver transplantation due to veno-occlusive disease c. salat 1, , e. holler t,3, hi. kolbl, 3, b. reinhardt l, r. pihusch 1, p. g0hring 2, s. poley 2, e. hiller 1 l=med. klinik iii, 2 = institut flit klin. chemie, klinikum grosshadern der ludwig-maximilians-universit~tt mfinchen, 3=h~tmatologikum der gsf a 40 year old patient suffering from all received allogeneic bone marrow transplantation (bmt). after an uncomplicated early posttransplant period the patient was dismissed after 4 weeks. a bilirubin rise with subsequent liver failure was observed during the following weeks. according to biopsy proven hepatic veno-occlusive disease (vod) liver transplantation was performed on day 79. unfortunately the patient died on day 140 due to aspergillosis. we monitored levels of protein c (pc) and s (ps) as well as pall during the pre-and posttranspiant period. pal1 level was normal (<43 ng/ml) during the first 4 weeks after bmt but increased with the manifestation of vod (317.5 ng/ml on day 47). it reached its peak immediately before liver transplantation (547.6 ng/ml) and returned to normal levels within the next few days. pc levels which were normal before bmt decreased prior to clinical diagnosis of vod and were normal after liver transplantation. ps levels lay within the normal range at all timepoints. vwf was elevated before bmt (240%) and remained relatively stable during the whole investigatonal period ranging from 170 to 260%. it is assumed that vod is initiated by an endothelial cell injury -possibly due to radiochemotherapy -and subsequent hypercoagulability. our results indicate that the "endothelial cell marker" vwf is not helpful in predicting vod. the kinetics of the investigated parameters underline the significance of pc and pai-1 as described by others and our group earlier, whereas ps does not seem to play a role in the pathogenesis of vod. the budd-chiari syndrome (bcs) is characterized by hepatic venous outflow obstruction that may be caused by the precipitation of a thrombus. it frequently coseggregates with other major diseases like myoloproliferative diseases or defects in the haemostatic system (antiprotein c and protein s deficiencies e.g.). only recently, the factor v leiden mutation (fvlm) has also been associated with bcs. we hypothesized that defects in the thrombo-modelling associated anticoagulant pathways (tmaap) are a major risk factor for the precipitation of bcs. we screened our cohort of 27 patients (pts) with bcs for the presence of defects in the tmaap and identified 3 pts with protein s deficiency (psd). these pts were screened for the three point mutations in exon 1 (codon-25; ins t), exon 15 (codon 636; a-->t) and in intron 10 (g-->a + 5) of the ps alpha-gene that have been demonstrated by bertina et al to coseggregate with psd. restriction enzyme analysis and confirmation-sensitive gel electrophoresis for the detection of single-base differences in doublestranded pcr-products were employed. all living family members of the indicator pts were also screened for heterogeneties in the three point mutation as described. no single abnormality in these genes despite presence of pbd in those family members was found. in addition, pts and family members were also screened for fvlm. one pt and two of his family members, in addition to psd, were subject to fvlm. the other two lots and their family members were not subject to fvlm. in contrast to the first family, despite psd, those two pts suffered from morbus crohn and acute myeloid leukaemia as risk factors for bcs. we conclude: psd is one major risk factor for the precipitation of bcs. to precipitate this disease, one additional risk factor is required. psd may be caused by genomic defects in the protein s gene other than those described by bertina. only a few publications describe a thromboembolic disease due to dramatically reduced protein s levels being associated with viral or bacterial infections, autoimmune mechanisms are suspected but the aetiopathogenesis is still under discussion. we report on a 5 year old boy who developed purpura fulminans of the left leg during varicella infection. on the fourth day of infection the disease started with pain and haemorrhagic efflorescence localized at the left taft. on admission the boy suffered from a purpura fulminans with central necrosis measuring 15x8 era. suspecting a hereditary thrombophilic disease we started therapy with protein c concentrate and recombinant tissue type plasminogen activator. the fellowing coagulation investigation showed a severe deficiency of protein s (total protein s-antigen < 5 u/ml, free antigen not measurable) in combination with factor v leiden mutation. other thrombophilie and coagulation parameters did not show deviation from normal range. after 4 weeks we saw a slight improvement of the total protein s antigen up to 50 u/ml. the free protein s antigen was still undetectable. during the following weeks the patient recovered slowly and the protein s activity and antigen normalized. because of skin necrosis thromboembolie prophylaxis was initiated with low molecular weight heparin (fragmin®, 100 ie/kgbw/die) and continued for 6 months. under this therapy there were no further thromboembolic events. these results suggested an autoimmune protein s deficiency in a patient suffering from chickenpo×. an analyses of autoantibodies at the time of diagnosis showed a slight increase of the antieardiolipin antibodies (igg 16,1 iu/ml, igm 15,1 iu/ml) which normalized during hospitalisation. we suspect an antibody to protein s probably caused by similar presented viral antigens. we suppose that autoimmune mechanism during different infections in combination with a heterzygous apc-resistance may be a potential risk factor for developing thrombotic disease. in the central nervous system mrna encoding for prothrombin and thrombin receptor is present and astroglial cells in culture process and secrete thrombin. moreover, effects of thrombin on brain cells including change of neudte outgrowth and astrocyte shape are described, but the molecular mechanisms are unclear. we investigated the effects of human <z-thrombin and a six amino acid thrombin receptor activating peptide (trap-6, sfllrn) on [ca2+]i, phosphoinositide hydrolysis and protein kinase c activation in the rat glioma c 6 cell line which is a widely used in vitro model for studying glial functions. stimulation of c 6 cells with both c{-thrombin and trap-6 resulted in transient [ca2+]i mobilization, [3h]inositol phosphate response and activation of the pkc isoenzymes c{,i],7,5, and s. these results suggest that ~-thrombin and trap-6 activate at least partially the same intraceltular signaling pathways in rat glioma c 6 cells which is an evidence for involvement of "tethered ligand" receptor in thrombin induced signaling in glioma c 6 cells. further investigations in this field are in progress including both receptor binding studies and more detailed analysis of thrombin receptor mediated signaling network and the cellular response of thrombin induced transmembranal signal transduction pathways in c 6 glioma. thrombin is a potent mitogen in vascular smooth muscle cells (smc). the mitogenic effect of bovine thrombin and human thrombin receptor-activating peptide (hutrap-14) was investigated in bovine coronary artery smooth muscle cells. confluent cells (passage 4-8) were incubated for24 h in serum-free medium with thrombin (10 nm) or trap (10-100 pm). the mitogenic response was assessed using [sh]thymidine incorporation. results demonstrated that thrombin induced a three-to fourfold increase in [3h]thymidine incorporation compared to the control, thus being as active as the potent mitogen pdgf-bb (20 ng/ml). the mitogenic effect of thrombin was dependent on its proteolytic activity. when the active site of thrombin was irreversibly inhibited by d-phe-pro-arg-chloromethylketone (ppack) no increase in [°h]thymidine incorporation was observed even at the high concentration of 250 nm ppackthrombin. thrombin-induced [3h]thymidine incorporation was significantly reduced by the reversible direct thrombin inhibitor nc£-(2naphthylsulfonylglycyl)-4-amidinophenylalanine piperidide (napap, lpm) . in contrast to thrombin trap-14 at a concentration up to 100 pm did not increase [3h]thymidine incorporation in the absence or presence of the aminopeptidase inhibitor amastatin (10 ijm). in order to demonstrate whether hutrap caused a stimulation of the receptor and signal transduction in bovine smc the activation of protein kinase c (pkc) was measured as translocation of the cytosolic to the membrane bound fraction. it was found that both thrombin (10 nm) and trap (10 pm) induced a translocation of pkc after 20 rain. in conclusion, thrembin and trap activated pkc in coronary smooth muscle cells but only thrombin acted as a mitogen. endothelial cells once stimulated with thrombin are resistant to subsequent stimulation directly after the first. but after a recovery period of about 90 min the cells are sensitised again for activation by thrombin. in our experiments this resensitisation couldn't be inhibited by actinomycin d or cycloheximide nor by the phosphatase inhibitor ocadaic acid. therefore an intracellularly pool of thrombin receptors has been proposed possibly co-localised with golgi vesicles. brefeldin a (bfa) has been used extensively to investigate the intracellular sorting of proteins because of its dramatic alteration of the structural and functional organisation of the golgi apparatus. because of this we examined the effects of bfa on the regeneration of the thrombin receptor response in human umbilical vein and artery cells. the vwf release from weibei-patade vesicles was used as physiological parameter of activation of thrombin receptors by thrombin. the addition of bfa (20 pg/ml -20 ng/ml) to endothelial cells blocked the receptor response regeneration, whereas the first stimulation was not influenced by bfa at the concentrations used. these results give further evidence that the proposed storage pool of thrombin receptors is iocalised in vesicles of the golgi apparatus. randomized comparison of double bolus reteplase (r-pa) and front-loaded alteplase (rt-pa) in patients with acute myocardial infarction (rapid ii) c. bode, rw. smalling, j. kalbfleisch, g. berg, dj. odenheimer, e. bshm, wd weaver and the rapid ii investigators. med. klinik iii, university of heidelberg, germany the study was undertaken to assess the efficacy of a double bolus regimen of 10 + 10 megaunits of reteplase, a recently developed deletion mutant of tissue-type plasminogen activator, relative to front-loaded (gusto-regimen) alteplase. there was no age limit and patients were recruited up to 12 hrs after onset of symptoms. the primary endpoint "patency at 90 minutes" was centrally assessed in a blinded fashion. infarct related coronary artery patency (timi grade 2 or 3) and complete patency (timi grade 3) at 90 min for reteplase vs. alteplase were 83.4% vs. 73.3% (p=0.03) and 59.9% vs. 45.2% (p=0.01), respectively. at 60 minutes, the incidence of both, patency and complete patency was also significantly higher in retep ase vs. aiteplase treated patients: 81.8% vs. 66.1% (p=0.03). and 51.2% vs. 37.4% (p=0.01). reteplase treated patients required fewer additional coronary interventions within the first 6 hrs of treatment (13.3% vs. 26.5%, p=0.01). there were no significant differences between reteplase vs. alteplase regimens in 35 day mortality (4.1% vs. 8.4%), severe bleedings (12.4% vs 9.7%) • or hemorrhagic stroke (1.2% vs. 1.8%). reteplase, when given as a double bolus of 10+10 mu to patients with acute myocardial infarction, achieves significantly higher rates of early patency and requires fewer acute coronary interventions than front-loaded alteplase without an apparent increased risk of complications. lysis therapy with uhsk of deep vein thrombosis (dvt) of the lower extremity commoniy results in a relatively high patency rate. in our patients, patency was achieved in approx. 70-80%. these results were supported by several studies. in dvt of the pelvic veins, however, this kind of aggressive therapy is excluded due to the risk of iatrogenically induced pulmonary embolism. on the other hand, the need for an effective therapy is evident because of the usually occtnring complete occlusion of the pelvic veins and the following severe clinical symptoms 0phlegnmsia cerulca dolens etc.). until september "95, 44 consecutive patients (16 males, mean age 41 ranging from 18 to 63 years) with dvt of pelvic veins were included in this study. after clinical and phiebographic/computer tomographic (ct) diagnosis, a temporary filter system was placed intravenously either by a wambmchial (34 pat.) access route, lransfemorally (6 pat_) or wansjngularly (4 pat.) under mdiological control. the open filter was positioned above the thrembus in the lower caval vein: 26 times an antbeor system, 11 times an anglocor and 10 times a cook filter were used. in the following three days up to 3 cycles of lysis therapy with 9 mli u of streptokinase were administered, each cycle lasted six hours. heparin was given simultanously via the filter system in therapeutic dosages in order to prolong alrit values 1.5 to 2 times the individual base line level. effective lysis was controlled by a second phlebography/ct, and the filter system removed through the introducer sheath valve. after lysis therapy all patients were put on coumarin with overlapping heparinization. successful lysis (complete recaanlisation without remaining thrombi and lysis of more than 50 per cent of the original thrombus) could be achieved in 62% (29 patients). in two cases a fatal pulmonary embolism occurred. 5 times great parts of a thrombus were captured by the filter and could be removed with the system. due to an insufficient boparin therapy we detected a newly developed thrombosis of the lower cavul vein twice after lysis. this complication could be resolved by additional administration of streptokinase. in nearly all cases we found extensive hematomas on the catheter insertion sites. 4 patients with transfemoral applications had to be transfitsed with red packed cells due to retroperitoneal hematoma. one filter system was broken after use. lysis of dvt of the pelvic veins with uhsk and with the protection of a removable temporary filter system situated in the lower caval vein enables us to carry out an effective therapy. the filter systems, however, have to be ameliorated in order to better prevent fatal pulmonary embolism. heparin should be administered at least in the remainder of the day of lysis. the insertion site should be the enbital vein because compression for bleeding complication is easily feasible at this location. we report about seven of our patients (pts.) with bcs treated by fibrinolytic therapy (5 female, 2 male, age 27, 6 years + 8). only one case was of unknown origin while in four pts. myeloproliferative syndromes, in one protein c deficiency and in another one morbus behcet was detected in five pts. chronical and in two acute bcs was diagnosed. as complications were found: in three pts• thrombosis of vena iv.) cava, in one ofv. porta and v. lienalis, in one more of v• porta and v• cava. we treated just one patient with urokinase (3 x 800.000 iu/d for 18 days). four pts. were treated with rt-pa in concentrations of 25 to 50 mg/d (for a period of up to 14 days) and continuous heparin infusion. high doses of rt-pa (80 to 120 mg/d for one or four days) were given in one case of acute and one of chronic bcs. partial (after high-dose rt-pa nearly complete) recanalisation of thrombotic hepaticel veins was only achieved in the two acute cases of bcs. no significant recanalisation was proven in hepatical veins of chronic bcs. three times complete fibrinolysis of v. cava thrombosis (two after highdose therapy) and one partial lysis could be attained. in one patient partial lysis of v. porta and in another one of v. lienalis was found. furthermore complete recanatisation of v. porta followed local rt-pa lysis during surgical intervention. in our two pts. with acute bcs significant improvement of hepatical venous flow could be measured after fibrinolytic therapy• in chronic bcs hepatical venous flow did not improve while the complication of v. cava thrombosis could eventually be treated successfully. probably due to fibrinolytic therapy one case of esophageal varix bleeding occurred. the benefit of flbrinolytic therapy of bcs seems to be influenced by the dur~ion of the disease• according to our results fibrinolytic therapy should be considered in cases of acute bcs as well as in the above mentioned thrombotic complications• recent studies suggest that transport of thrombolytic agents through the clot can play a major role in determining the reperfusion time needed for thrombolysis. as the mechanisms of this phenomena are rather unclear we studied fibrin (0.4-1.5 ~tm) clot lysis by plasmin (30 nm) presented outside or inside the clot in purified system (tris-hc1 buffer, 37°c). the clots was formed by addition of cac12 (30 mm), thromboplastin (1 u/ml) and human thrombin (6 nih/ml)t the lysis degree was determined either by counting of radiolabelled fibrin degradation products or by time of total lysis. obtained kinetic parameters of this reaction: kest=99.4 rain -1, km=i.9 ~tm (plasmin inside clot); kcat=l.36 rain -1, km=i.3 lzm (plasmin outside clot). this data indicate that eatalytie efficiency of plasmin from inside the clot is 50-fold higher than from outside as measured from kcat/km. furthermore we studied plasma clot (0.25 ml) lysis in original plasma (3 ml) containing sk (5-100 nm) and seu-pa (5-100 rim) in the final concentrations from inside or outside the clot. the results are presented in the the similar results have been got when the fibrin clots were formed with batroxobin instead of thrombin. it may be concluded that fibfinolysis is a surface-dependent process. it's likely that the higher level of plasminogen activators in blood would prevent thrombus formation, while physiological clots would stay resistant. for the improvement of the thrombolytic therapy in children more clinical data are needed. therefore we report on our experiences with rt-pa presenting the clinical course in 11 patients (age range: 7 days to 16 years) with venous thrombosis (n=9) and purpura fulminans by meningococcosis (n=2). the patients were treated with rt-pa (actilyse r) for 10,5 hours to 13 days. the venous thromboses were localized in lilac/femoral veins (n=4), brachiocephalic/jugular/subclavian veins (n=3) and the cava superior vein (n=l). central venous catheters were causative in 2 cases. the dosage range was between 0.2 to 0.5 mg/kg for the initial dose and 0.8 to 2.0 mg/kg/d for the subsequent continous infusion. ten patients received simultaneously heparin. complete clot dissolution occured in 6 cases, partial clot dissolution in 1 patient. in 4 patients with a history of the first clinical signs of thrombosis of 3 weeks or more the lysis was not successful. concerning the side effects only small bleeding episodes on former venipuncture sites were observed in 5 patients. systemic effects such as the decrease of the plasma flbrinogen concentration were rare too. in conclusion, the thrombelysis with rt-pa presents an effective and safe therapy in children at: the rt-pa dosage used. in case of a history of the thrombotic event of more than 3 weeks a thrombotytic therapy should not be carried out. d.dimer is widely used in clinical situations associated with thrombotic diseases. the most popular application is the exclusion diagnosis of deep-veinthrombosis and pulmonary-embolism and the assay is performed in emergency. d.dimer is also frequently measured for evaluating the thrombotic risk in the post-operative period. quantitative, flexible and sensitive assays are required. we have developed a new automatic method which works on the coagulation walk-away instrument, sta. this assay was developed with two monocloual antibodies specific for d.dimer coated on microlatex particles and it uses an immuno-turbidimetric technology. absorbance is measured at 540 nm and is a direct relationship of d.dimer concentrations. the assay works with 50 ~tl undiluted plasma and is performed in less than 10 minutes. the dynamic range is from 0.2 to 16 [~g/ml and no prozone effect is observed up to concentrations higher than 100 ~tg/ml. the detection threshold is of 0.2 [lg/ml. the intra-assay reproducibility is below 5% and the inter-assay reproducibility is below 6%. recovery studies of purified d.dimer added to normal plasma were between 94 and 105%. no interference of heparin, bflirubine, lipids or of rheumatoid factor up to concentrations of 100 u/ml is observed. there is no effect of high fibrinogen concentrations (> 10 g/l). when compared with conventional elisa techniques (asserachrom ddi), the assay demonstrated a correlation coefficient of 0.97 on 131 samples from normal individuals and hospitalised patients with elevated d.dimer concentrations. slope was of 0.97 and intercept was of -0.07. this new assay offers a full flexibility for individual testing as the calibration curve is stable for at least one week on the instrument. it is then well adapted for all the applications of d.dimer measurements in coagulation laboratories. 16 children between an age of 3 days and 11 months ( median 6 weeks ) with thrombotic or embolic occlusion of major vessels were treated with rt-pa for thrombolysis. the affected vessels were both sided renal veins or one sided renal vein and v. cava inf. in 8 cases, the v. cava superior in 3, the v. cava inf. plus renal veins plus aorta in 1, the left ventdcle in 1, the aorta in 1, the a. femoralis in 1 and the v. portae in 1 case. 10 out of 16 occlusions were associated with an indwelling catheter. underlying dieseases were sepsis (4), prematudty (3), vitiurn (2), asphyxia (1), short bowel syndrome (1), hus (1), diabetes (1), cmv (1), exsiccosis (1) and m. hirschsprung (1). thrombolysis was performed with an bolus of rt-pa (0.1-0.2 mg/kg) followed by continuous infusion (0.8-2.4 (-9) mg/kg/24h, median 1.8 mg/kg/24h). low dose hepadn (100 ie/kg/24h) was given dudng full dose hepadn (aptt 1,5-2 times normal) after the thrombolysis. in 5 pts. rt-pa was administered locally through the catheter and in 11 cases systemically. in 13 patients the vessels could be recanalised completely, in 2 partially, in 1 patient the therapy had to be discontinued. in 2 vessels a reocclusion occurred. bleedings were noted in three patients, all from recent venous puncture sites. the results encouraged us to start a multi-canter trial which has been approved by the ethical committee and is open for recrural. the aim is to compare efficacy and safety of rt-pa with urokinase, the only recommended standard in the management of critical major vessel obstruction in newborns and infants. the design is a randomised, notblinded trial with a cross-over option after three days in cases without success. study end points are recanalisations, major bleedings and number of cross-overs. inclusion criteria are age under 1 year, lifethreatening vessel obstruction, age of thrombus up to 10 days, no precaeding fibdnolytic therapy. exclusion cdteda are cerebral hemorrage, pedventricular leukomalacia, surgery dudng the last 7 days and cns injuries during the last 2 months. although our knowledge on inherited thrombotic coagulation disorders has greatly expanded within the last years, there are still man}, patients with recurrent venous thrombosis in whom no obvious predasposition can be identified.thus we decided to include also so-called rare defects associated with thrombosis in our routine thrombophilia screening programme, such as fxii deficiency. fxii is an important element m the intrinsic pathway of fibrinolysis and there is evidence for an insufficient fibrinolytic activity in fxii deficient pts..up to date only few and controversial data exist about the frequency of fxii deficiency in pts. with thrombophilia. cons~uently the aim of our study was to evaluate the association between fxii deficiency and juvenile venous thrombosis in a great population. patients and methods: 1554 pts. (851 female, 703 male, aged i to 61 ys, median age 38.2 ys) with venous thromboembolism before the age of 45 ys were studied. one-stage clotting activity assay of fxii (fxii:c) was performed on acl using fxii deficient plasma from instrumentation laborato~. fxii antigen concentration (fxii:ag) was measured by electroimmundiffusion using reagents from behfingwerke, enzym research respectively. the normal ranges are tl~. routine reference values obtained m our labratory from 80 healthy subjects (40 males, 40 females, median age 26.2 ys); 95% range: fxii:c 53-135%, fxii:ag 57-132%). results: 122/1554 pts.were classified as fxi1 deficient (f 60, m 62), giving a prevalence of 7.8%. severe fxii deficiencies with fxii:c below 1% were observed in 7 pts..ll5 pts= proved to have moderate fxii deficiency with fxihc ranging lrom 2 to 51% and fxii:ag ranging from 1 to 53%. in none of them inherited deficiencies of other well established thrombophila risk factors could be detected. none of the fxii deficient pts. had positive lupus anticoagulant tests. familial fxii deficiency was found m 9 cases. discussion and conclusion: the precedences of fxii deficiency amongpts, with venous thromboembolism was previously described to be 7.5-10%. supporting these data, we have shown a praevalence of fxii deficiency of 7.8 %. in comparison to the frequency of other well established thrombophila risk factors we consequently have observed a relatively high prevalence of fxii deficiency m our study group.these data, from the largest such study reported, strongly indicate that fxii deficiency may not be a rare deficiency and may be more frequently associated with thrombosis than currently suspected. we describe a family with an exceptionally rare, i.e. plasminogen, deficiency, combined with subnormal activities of coagulation factor xii (hageman factor). the first thromboembolic event, pulmonary embolism in the proposita was diagnosed at age 35. since that time, 'spontaneous' venous thromboembolic events verified by phlebography and perfusion/ventilation lung scan recurred once every year despite oral coumarin therapy, whose intensity varied over an exceptionally wide range despite tight control the patient was repeatedly given succesful thrombolytic therapy with streptokinase or recombinant tissue plasminogen activator. her plasma plasminogen chromogenic activity was 51-59 % compared to a normal plasma pool (reference range 70-130 %), plasminogen antigen was diminished to the same extent. the patient's factor xii exhibited only 28-55 % activity in a factor-deficient plasma assay as compared to a normal plasma pool. other known risk factors for recurrent venous thromboembolism were not present : no evidence of malignancy, no obvious precipitating events, normal values of antithrombin iii, protein c, protein s, fihrinogen, thrombin time, platelets, lupus-like anticoagulant, aptt prolongation after addition of activated protein c. the proposita's mother had died at age 60 from pulmonary embolisnt no coagulation studies are available. the proposita's sister was first diagnosed deep leg vein thrombosis at age 17, since that time recurrent episodes of venous thromboembolism have been diagnosed also in an other hospital. this sister's plasminogen activity was 120%, but factor xii activity was reduced to 55 %. three brothers of the proposita were examined, too, all in their 3rd decade of life. none of them recalled symptoms of or treatment for thromboembolic disease. in one brother, factor xii activity was normal (100-105 %), but plasminogen only about 50 %. in the 2nd brother, factor xii was very variable (64, 42 and 94 %), plasminogen was in the lower normal range, in the 3rd brother, factor xii was about 50 % (repeatedly), plasminogen was normal. current knowledge about the risk of thromboembolism with both enzymes is limited, the optimal management remains controversial. msrgit serbsn,maria cucuruz,dan madras,carmen petrescu, natalie rosiu,rodica costa iii rd psediatric clintc,universtt v of medicine, the unsatisfactory efficiency of entihepetitis b vaccination in our haemsphiliscs suggested the control of the immune status in 52 hiv negative patients,by establishing through flowcitomstrie with monoclonsl antibodies the lymphocyte subsets (cd3,cd4,cds,cd&/cd8 ratio and cd19) and by seric tmmunoglobulins levels; the immunological parameters have been correlated with the serological markers of hepatitis infections (hay, hbv,hcv ebd hdv) as well as on dependence with the treatment (blood,plasma,crysprecipitate,fector viii/ix concentrate) and the quantity of their consumption (ui/k9 weight/yesr).the interpretation of the results pointed out • significant lower level of cd3,cd& (p<o,o01) st the group of hsemophiliscs treated with blood,plasma and cryopraclpitate; sdditionsly,in this group of patients it was found s significant drop of the cd19 counts (p< 0,001) without an immunoglobulin-deficiency. the same behaviour was found at the group of haemophiliacs carriers of hb~ and hcv markers.the superpositisn of hepatitis infections on the treatment with blood and native blood products in most of the cases has let unanswered the question related to the mechanism of the immunodeficiency: the hepatitis b or c infections or the negative impact of the itarative important alloantigenlc load ? illumination with l~tm methylenblue (mb) is used for inactivation of enveloped viruses in human plasma. via a photooxidative mechanism viral structures as well as plasmaproteins especially fibrinogen can be damaged. fibrinogen is an important mediator in the cell-cell interaction of platelets. the four rgd sequences (arg-gly-asp) of fibrinogen can be recognized by the activated fibrinogenreceptor gphb/iiia . after binding to gpiib/iiia one molecule fibdnogen can link two platelets. the aim of this study was to investigate the influence of mb treatment on the fibrinogen activity in human plasma. furthermore we were interested to demonstrate if altered fibrinogen inhibit platelet aggregation by blocking the gpiib/]iia receptor. human plasma was treated with 1-50~m mb. after illumination fibdnogen was isolated by fl-alanin precipitation. purified fibdnogen was analysed by one-and twodimensional gelelectrophoresis. influence of modified fibrinogen on platetet aggregation was measured with gelfiltrated platelets. possible receptorblockade were examined in an 125j-fibrinogen competition assay. densitometric scanning of reduced sds-pages showed a decrease of the a-subunit as well as an increase of high molecular aggregates with increased mb concentrations. this effect was not detectable for fibrinogen isolated from plasma with 1bm mb. this results were confirmed by two-dimensional gelelectrophoresis. ibm mb treatment showed no receptor blockade in aggrement with normal platelet aggregation after stimulation with adp and fibrinogen. proposed validation of a quantitive quality-assured pcr method f. dorner, j. eibl, g. zerlauth immuno ag we have developed a highly sensitive and reliable quantitative method, based on the polymerase chain reaction (pcr) and on the reverse transcriptase polymerase chain reaction (rt-pcr), to screen for the nucleic acids of human immunodeficiency virus type 1 (hiv-i), hepatitis b virus (hbv) and hepatitis c virus (hcv) in plasma and plasma products. the detection of pcr products is carried out by laser induced fluorescence, the procedure is therefore termed "laser induced fluorescence pcr" (lif-pcr). the procedure allows the precise quantitation of genome equivalents due to the use of two calibrators that are co-amplified by the same set of primers as the target template in one and the same test tube. a high degree of reliability is achieved by co-precipitation of the extract, co-amplification and co-determination of the calibrators together with the genome equivalents to be determined. in this report we present validation data for the lif-pcr and discuss them in the light of the recently published ich-guidelines on the validation of analytical procedures. taking into account the pecularitles of the replacement therapy of haemcphiliacs in romania the study aimed at evaluating the dimension and profile of blood-born infections in these patients; 142 hsemophlllacs,elasslfled in 3 groups: a-ulth no exposure to blood or bloodproducts, b-treated ~ith factor-concentrates and c-ulth plasma,cryoprecipitate + factor-concentrates in their therapy have been investigated (elisa method) for serological markers of hepatitis a,b,c end d, cvtomegalovirus (cmv) and hiv i and hiu 9 infection. the infections profile of our haemophiliacs~hes some peculsrities. the hepatitis infection is the most extended,lnvolving more than 75% of the patients. the frequency is for hepatitis -bz,z~ , u -"/~,~ , g -bb,7% end o -24% : the multiple infection was diagnosed in more than 50% of cases. the prevalence uas significantly hlgher in the group c. we studied blood count and relevant coagulation system parameters of autologous blood donors. indications for autologous blood donation were divided in 4 categories: a group of diverse (a, n=13), reduction mammoplasty (b, n:5), prostatectomy (c, n=io) and total hip replacement (d, n=74). we performed bleod count (bc), thromboplastin time (tt), partial thromboplastin time (aptt), fibrinogen (fib), f vhi act, vwf rco, vwf ag, f xiii, at iii, d-dimer-fragments (dd) and fibrinmonomers (fm). all parameters were analyzed as initial value, bc, tt, aptt, fib, f xiii and at iii were measured additionally at each donation. the percentage of initial pathologic data was (1) were included in the study. the inclusion criterittm was: an endegen(y.~ red cell reserve (ercr) of less than 400 ml in men and less than 540 ml in women. ercr is the volume of blood that can be withdrawn up to tim hematocrit of 0.34. the selected dosages of rhepo (200, 400 and 800 iu/kg body weight biweekly over 4 weeks) were based on the calculated blood volume and the ercr. a unit of whole blood, separated into an autologens red cell concentrate and one unit of fresh frozen plasma, was collected when ]mnoglobin concentration was at least 11.5g/100 ml (or hematocrit was at least 0.34) up to an autologons predeposit of 800 ml red cell volume (rcv). additionaly, platelet concentrations and serum ferritin levels were measured~ results: at the initial clinical examination the ercr was 333 ml in women and 307 in men. this resulted in a weekly dosage of 2x32.000 iu rlaepo in women and 2x25.000 iu rhepo in men. 7 patients donated 5 red cell each (858 ml rcv on an average) within 19.1 days. the targeted rcv could not be reached in 3 patients because the operation was reschednled. the collection from one patient had to be aborted because of angina pcetoris complains. tic following side-cffczts occurred: episodic hypcrtejlsion (2 tim~), chest pain (5), fafigne (8), inflnenza-lihe syndrome (3). platelet count increased from initially 203 4-68 x 10'//d (i00 %) during therapy to a maximum level of 260 + 93 10v~ (128 %) without any clinical signs of thrombeembolism. in the control group (autologous blood donation weekly 3 or 4 times without erythrupoiedn stimulation, n=12) platelets increased from initially 230 __+ 73 10'/fi (100%) to 255 + 71 10v#i (110%) i1]aximlzm colldttsions: the adlninist/'ation of lhepo in the above described manner to autologous donors undergoing heart surgery is well tolerated. it allows to collect a sufficient number of autologous blood units and the preparation of a predeposit at short notice. essential thrombocythemia is a chronic myeloproliferative disease with a benign clinical course that is mainly complicated by microcirculatory or thrombotic vascular disorders. it is at present uncertain which patients need (lifelong) therapy and whether cytoreduction is necessary. we retrospectively evaluated vascular complications in 128 patients (33 men, 95 women) with a median age of 61 years in relation to therapy. three major treatment groups were analysed: 17 patients treated with acetylsalicylic acid (ass) alone, 29 patients given alkylating agents, and 62 patients treated with interferon alpha (ifn). there was no significant number of patients treated with hydroxyurea. ifn-treated patients were younger (median age 54 years) than patients in the other groups, but platelet counts before therapy were similar (median 0.84-1.13 x 1012/i). before therapy, 47% of patients treated with ass, 45% treated with alkylating drugs, and 32% treated with ifn had bleeding events, microcirculatory or thrombotic complications. dudng therapy, median platelet count was 0.9 x 1012/i in patients on ass, 0.68 x 1012/i in patients treated with alkylants, and 0.48 x 1012/i in ifn-teated patients. the rate of patients with complications during therapy was 18% (9.5%/patient year) for ass, 14% (6.3%/patient year) for patients on alkylating drugs, and 15% (6.2°/dpatient year) for ifn-treated patients. we conclude that all three treatment regimes seemed to have a beneficial effect on the incidence of vascular complications in essential thrombocythemia. the results give a rough estimate of complication rates to be expected and may form the basis for randomized clinical trials. adhesion of activated platelets to leukocyte* has been shown to be mediated by at least two molecules on the platelet surface: p-selectin (cd62) and fibrinogen (fg) bound to the gp iib/iiia complex. interaction of platelets with teukocytes via cd62 is believed to stimulate the production of reactive oxygen species (ros) in leukocyte*. the role of platetet-bound fg as a signalling molecule in platelet-leukocyte interaction is controversially discussed: stimulatory as well as inhibitory effects on leukocyte ros production has been described. in the present study we measured ros production in undiluted samples of citrated whole blood (wb) as well as in suspensions of erythrocytes and leukocytes in platelet-rich plasma (platelet-rich wb) or platelet-poor plasma (ptatelet-poor wb). ros production was determined by measuring luminol-enhanced chemiluminescence (cl). stirring wb samples at 1,000 rpm for 10 min resulted in an activation of leukocyte* (increase of cl, upregulation of cd1 lb) and platelets (platelet aggregation, upregulation of cd62). stirring-induced cl was significantly higher in platelet-poor wb when compared to platelet-rich wb. higher cl in plateletpoor wb than in platelet-rich wb was also found when adp (moderate stimulation of platelets and leukocyte*) or fmlp (strong stimulation of leukocyte*) but not when pma (strong platelet and leukocyte stimulation) was added. dextran sulphate that is known to block cd62-mediated interaction ofplatelets with leukocyte* caused a dose-dependent inhibition of stirring-induced cl in wb samples. in contrast, when binding of fg to the platelet surface was blocked by rgds or the fg receptor antagonist gr144053, stirring-induced cl, but not cl in unstirred samples, was significantly enhanced. gr144053 also reduced adhesion of platelets to neutrophils, but did not affect cd62 exposure. the results indicate that interaction of platelets with leukocyte* in whole blood via cd62 and fg may have opposite effects on leukocyte ros production: stimulation by cd62 and inhibition by platelet-bound fibrinogen. late reocclusive processes represent one of the most commonly seen serious adverse events after coronary angioplasty. the pathophysiology of both of these processes is multifunctional and represens complex time dependent pathophysiologic events involving both the humeral and cellular processes. to test the effects of low molecular weight heparin (certoparin, sandoz ag, numberg germany) against late occlusive processes, four groups (groups a-d) of rats (7-10) were anesthetized and the external common carotid arteries were exposed. all groups received a 200 u/kg of unfraetionated heparin through the femoral vein. a 2f balloon emboleetomy catheter was inserted into the left carotid artery and passed throug the left common carotid artery leading to the aortic arck vascdar injury was induced by repeatedly (x3) inflating the catheter balloon and withdrawing it to the bifurcation. following this procedure, two hours after, equlgravimetric amounts of various agents were administered via sc route to each of the individual groups. group a was administered with certoparin od for 21 days. group b received a lower low molecular weight heprin (mr 3.4 kda). group c received unfraetionated heparin and group d served as a control. on day 21, perfusion fixation was performed on all animals. the injured and contralateral carotid arteries were excised for histelogic examinatior~ in comparison to the saline control, varying degrees of the inhibition of myointimal proliferation following arterial injury was noted in all groups. however, the degree of inhibition followed the order: heparin<certopann<llmwh_ in contrast to heparin treated group (c), the other groups a, b, and d did not exhibit any hemorrhagic lesions. however, degree of perivascular fibrosis, hemorrhage and inflammation were observed at the infusion site. in comparison to heparin, certoparin and its lower low molecular weight derivatives exhibit better efficacy in reducing balloon angioplasty induced restenosis. the observed efficacy of these agents may be due to better bioavailability and sustained duration of action. in the present paper the binding of heparin to granulocytes of human and animal species was analyzed and compared with the binding to monocytes and lymphocytes. a low-molecularmass heparin (lmmh) was covalently bound to tyramine (tyr) and subsequently tagged with fluorescein-5-isothiocyanate (fitc) by endpoint attachmenc. binding to leukocytes was quantified using flow cytometry analysis. the leukocyte populations were identified by their light scatter properties in man by cd4, cd13, and cd14 antibodies, in mice by cd 4 and gr 1 antibodies, and in rat by cd 45, ed 9, and rp 3 antibodies. granulocytes, monocytes and lymphocytes of all species bound dose-dependently lmm-heparin. the binding of lmmh-tyr-fitc ~to granulocytes was higher in human, rat, rabbit, dog, and pig as compared to monocytes and lymphocytes. mouse and sheep granulocytes did not bind more heparin than monocytes or lymphocytes. binding of lmmh-tyr-fitc was reversible in the presence of unlabeled heparin or lmmh. more than 99% of human, rat, rabbit, dog and sheep granulocyte populations could be'distinguished from monocytes and lymphocytes by their fluorescence intensity after incubation with lmmh-tyr-fitc. this separation was not obtained for mouse and pig granulocytes. the data present evidence of a specific heparin binding to granulocytes of many species and indicate the significance of fluorescent labeled lmm-heparin for biological investigations. background and purpose: ptca is an effective treatment for restoring coronary artery pat~cy; however, acute thrombotic rencclusion is a drawback of the procedure. currem prevention of rencclusion relies on antiplatelet agmts (aspirin) and antithrombin agents 0aeparin). in the present study we examined the time course of changes in blood coagulation and fibrinolytic function following ptca in patients with ischaemic heart disease. 20 patients were randomly assigned to receive either standard therapy (250 nag aspirin p.o.; 5.000 iu hepafin i.e. bolus, and 5-10.000 iu heparin intra-proeadurally)(n=10), or adjunctive therapy (aspirin, hepann; 100 mg sodium pmtosan polysulphate, spp intra-cortmarily and subcutaneously) (n=10). the sensitive markers of thrombin activation (thrombin-amithrombin iii complex ('rat), prothrombin fragmem f 1+2 (pl+2), d-dimer) and of fibrinolytic activation (plasmin-antiplasmin complex (pap), tissue type plasminogan activator (t-pa) and plasminogen activator inhibitor (pal)-i activities) were measured before, immediately after, and 6 hs after ptca. results: we found elevated mean levels of tat and fl+2 in both groups during the procedure, while plasma coneentrafiuns of d-dimer and pap remained in normal range. activity of pai-i was not elevated compared to normal comrol. the t-pa activity obtained immediately post-ptca showed significant increase in patients given spp. primary angiologic success was achieved in 80% of both groups. one patient in each group had recurrent ischaemia and emergency interventions (angioplasty, stent, bypass surgery) were performed. one patient receiving adjunctive therapy died in cardiogenic shock within 24hs arer ptca. major bleeding was observed in one patient given spp. conclusion: no significant intracoronary haemostatic activation occurred in the majority of patients during and up to 6hs after ptca. treatment with spp was associated with enhanced fibrinolytic activity. the benefits and risks of adding spp to heparm and aspirin should be evaluated by further studies, in july 19,¢1 the paul ehdich institute became responsible for quality and safety of bt~d and blood products. since ~his time, batch m~se of ppsb and factor ix prapamtions were carded out regularly, whereas ihe obligatop i testlng of all other products derived frem pooled plasma will be starling in janualy 1996. the european pharmacopoeia does .or contain directions for the quality control of ppsb preparations. therefore, ifua ~ coneenning factor ix clotting concentrates are applied, ircludmg the non activated ptt and the classical tkrombln test these m~ods are not su'rlable for reliable exclusion of ituombogenk~b/ ,of pp,sfl, because vlla actlvry is not d~. a hllhedo unsolved problem is the high sensitivity of file classical thrombin test often not correlating with ~ data of thrombogenlci~. in ordm td es~abl~ an effective quality conlrd protocol, different methods for the qual~ative detennlna~on of ila, vlla. ixa, and xa (chrornogenic, fluorege~c clolfing reactions) wera instaued. in agreement with the [deralure, considerable differences were found among the ppsb pr~ons registered in germany. cedain amounts of activated do~ng racers may be well tole~ted in redp/ents. ~no are in a steady state condition. however, in severely ill patienls, eg. post operatively, tissue factor may be expressed which pofenllatesthe dsk of ~aclor vlta. in the case of a certain ppsb preparation hlgh ¢onceatratio~ of acthrated cto~ng factors, especially ~vated factor vii, cotmlaled with fatal comlffcal~ns, evaluating ~ expe~ concenlralbn limits for adjvated prothrombin co~np~.x factors as prerequisite for ~e batch r~ease have to be applied. aprotinin is used as a hemostatic agent in cardiopulmonary bypass surgery and other indications where hemostatic compromise results in hemorrhagic manifestations. the mechanism by which this agent produces hemostatic effects is not known, however, inhibition of serine proteasas such as plasmin, kallikrein and dastase and modulataion of platelet receptors have been considered as possible target sites. to determine the effect of aprotinin on tissue factor mediated activation of human platelets, whole blood samples were drawn at baseline and 24 hours aider aspirin (650 mg po) ingestion (n=5). blood samples were supplemented with saline and aprotinin (40 ug/ml) and tissue factor activation was studied at 200 pg/ral. platelet activation was investigated using gtycoprotein ffla and gmp 140 specific antibodies on a flow cytometer (epic coulter). tissue factor was found to produce the activation of both the baseline and asp' "mnized platelets. aprotinirt augmented the tissue factor activation of platelets in both the control and aspirin supplemented groups. however, the relative increase in the aspifinized group was much monger (25% of the baseline). these results suggest that aprotinin augments the tissue factor mediated activation of platelets in both the normal and aspirinized individuals. while these effects may result in hemostatic restoration in aspirin compromised patients, in normal individuals the clinical implications of this effect remain to be clarified. , 10-20 (group 2) or >20 years (group 3) duration. anticoagulated whole blood was incubated with fluorescent antibodies to gpib and gmp-140 (two colour method) and analyzed with a flow cytometer. thrombomodulin, f1+2, protein s, 13-thromboglobulin were measured according to standard procedures. results: surface expression of gmp-140 was not different in groups 1 to 3, however, there was a tendency to higher acitvation in group 1 (<10 years iddm). results for thrombomodulin, f1+2, protein s, 13-thromboglobulin will also be presented. conclusion: though it did not reach statistical significance, platelet acitvation seems to be more important during early diabetes. this wilt be correlated with endothelial and plasmatic activation markers. in our clinic four patients with hiv-related thrombocytopenia were treated with a lot of gammagard (93f21abllf), which later turned out to be hcv contaminated. before infusion all patients were negative for hcv antibodies and hcv rna. 2 to 8 months after infusion 2/4 patients, who suffered from arc at the time of hcv infection with cd4 counts >100/pl, seroconverted, whereas in the two other patients, who suffered from aids with cd4 counts below 100/pl, there was no seroconversion. in all cases hcv rna was found. genotyping with inno-lipa (innogenetice) showed hcv genotype l(b) in all patients. liver enzymes and hcv rna copies were measured repeatedly over a period of one year after infection. the 2 patients with arc showed a strong increase of hcv rna titre during the first 3 to 4 months after infection, followed by a rapid decrease within the next months. in the patients with aids hcv rna copies increased moderately within the first 4 to 6 months, followed by a slow decrease. elevation of liver enzymes was mild in the aids patients and seems to be independent from the hcv rna titre. in the arc patients liver enzymes changed parallel to hcv rna titers with a delay of 2 to 3 months. the course of hiv infection was only slightly influenced by the acute hepatitis c as measured by cd4 counts, i%2microglobulin and hiv rna copies. introduction:mechanisms underlying ischemia/reperfusion injury have been thoumughly investigated in experimental models. leucocytes appear to play a main role through production of cytokines and overexpresssion of adhesion molecules. in experimental animals, administration of monocional antibodies (mab) recognizing cd18 can reduce organ injury following ischemia/repedusion. no data, however, have been reported concerning clinical ischemia situations. patients and methods:we investigated expression of cdt8, cd1 la, cdf l b and cd1 lc in granulocytes, monocytes and lymphocytes from peripheral blood of five patients undergoing elective hand surgery. the tourniquet was applied on the upper arm and heparinized samples from cubital veins were obtained before and at the end of ischemia. control samples were drawn from the nonischemic contralataral arm with the same timing, duration ot ischemia ranged between sixty and one hundred minutes (80~16). whole blood samples were incubated with specific, fluorochmme labelled antibodies and analyzed by fluorocytometry (facscan, becton dickinson, san jose, ca). mean fluorescence intensity (mfi), quantitatively reflecting surface expression of the indicated markers was evaluated for the individual cell populations. data were compared by the paired student's t-test, p<0,05 was evaluated as significant. results:mfi for all markers was comparable in all cell populations in samples obtained before ischemia from both arms. in contrast, expression of cd18 was significantly enhanced in granulocytes (321_+50 vs. 189_+38), monocytes (653-+54 vs. 426+122) and lymphocytes (299_+45 vs. 228-+36) from samples derived from the ischamic arm, as compared with the nonischemic arm, as measured at end of ischemia. at the same time, an increase of cdf lb on granulocytes (500~_342 vs. 213+150) and monocytes (533+359 vs.237-+206) but not on lymphocytes was found, no modifications of cdlta and cdttc expression could be observed. there was no correlation between duration of ischemia and quantitative expression of these markers, conclusions:our data indicate that relatively short ischemia periods induce an increased expression of ~2" integrins adhesion molecules on leucocytes. these results suggest, at close similarity with findings from expodmental models, that overexpression of adhesion molecules might play an important role in the induction of ischemia/reperfusion injury, in humans. in patients suffering from chronic inflammatory bowel diseases, such as morbus crohn and colitis ulcerosa, we observe massive, sometimes barely staunchable bleedings. hereby, the deficiency of coagulation factors, especially of factor xiii in plasma is established. ttowever the influence of factor xiii on the pathomechanism of the underlying disease is still under discussion. therefore we studied the f xiii content in the intestinal mucosa. an immunohistochemicat method was developed using commercially available antibodies against f xiii subunit-a, the detection of mucosal factor xiii depends on the amount of chromogen bound to the antibody-horseradish-peroxidase complex. with this method, it is possible to locate but not to quantify f xili in the intestinal tissue. therefore we developed an elisa-metbod in homogenized intestinal tissue, using commercially available antibodies. its precision was validated using a standard curve with commercially available factor xiii preparations (fibrogemmin®). the detection limit of this method is > 0.05 i.u. f xiii/ml of tissue solution. freezed dried intestinal tissue (lmg) was homogenized in 1 ml buffer using a potter. specimens of the large bowel revealed f xiii values of 0,21 + 0,0038 i.u. (x __+ sd), tissue solution. with this method it is possible to quantify tissue-bound faxtor xiii. studies are in progress to elucidate the content of f xiii in the intestine of patient's suffering from infammatory bowel diseases in order to contribute data to the pathomechanisms of f xiii deficiency. in a previous double-blind, controlled trial we were able to show that aprotinin administration has significantly contributed to reduce periand postoperative bleeding complications without increasing the risk of thromboembotic complications. the question arises whether this beneficial effect may be associated with its effects on intraoperative fibrinolysis. therefore, 20 patients were treated with or without aprotinin (2 million kiu loading dose over 15 minutes followed by 500,000 kiu per hour), and citrated blood samples were obtained at the following time points: before operation, after induction of the anesthesia, at the beginning of operation, intraoperatively when the femur shaft was implanted, and 24 hours postoperatively. the determinations of plasmin/antiplasmin-complexes, d-dimers, thrombin/antithrombin iii-complexes, and prothrombinfragments 1 +2 were performed by means of test kits from behring, germany (enzygnostrpap micro, enzygnost r d-dimer testkit, enzygnost r tat micro and enzygnost r f 1 +2 respectively). -all markers of activated fibrinolysis and blood coagulation were significantly increased in the groups with and without aprotinin treatment, the highest activities to be seen when the femur shaft was implanted. however, the values of pap and d-directs of the aprotinin group were below the values of the control group until the end of operation. the markers of activated coagulation showed the opposite effect, however the differences between the two groups were not significant. as expected, the aptt was significantly prolonged in the aprotiningroup. the aprotinin treatment was also associated with a significantly lower blood loss in these patients. -concluding it can be said it is not clear whether the blood saving effect of aprotinin may be exclusively attributed to its antiplasmin activity since the differences of the fibrinolysis parameters were not statistically significant. further blood samples should be analysed between the implantation of the femur shaft and the end of operation. in our laboratory large amounts of human prothrombin are required (30-50 mg/week). as we try to produce meizothrombin and meizothrombin-des-fragment-1 from human prothrombin and to apply it as an antidote for hirudin, the classical adsorption to barium sulphate or aluminum hydroxide from human plasma cannot be used. commercially available human prothrombin is expensive and of an unacceptable quality for our applications. in most of these batches we found small amounts of factor x and prothrombin activation products. we now developed a procedure to isolate prothrombin from "prothrombin complex concentrates" (ppsb-250-bulk, drk-blutspendedienst nds.). the concentrate also contains fac-tor vii, factor ix, and factor x. the prothrombin had to be separated from these factors. the concentrate we used contained amounts of other proteins and activation products of prothrombin (e.g. prethrombin-1) as well. for the preparation of prothrombin from ppsb we used anion exchangechromatography (resource-q ®) on an fplc ®. we applied dissolved ppsb directly or after buffer exchange on sephadex g-25 onto the column at room temperature. the prothrombin was eluted with an naci-gradient in trisodium citrate buffer, ph 7.0. the buffer conditions are similar to the conditions used in the preparation of ppsb. the quality of the prothrombin so obtained was sufficient for most of our experiments. a second purification step on ion-exchange resulted in a 99% pure product devoid of contaminating factor activities and activation intermediates as examined with coomassie and silver stained sds-page electrophoresis and assays for factor x. this prothrombin contained full enzymatic activity and its activation by specific snake venom prothrombin activators showed the known activation products. we are now able to isolate the amounts of pure prothrombin required for preclinical investigations. most of the commercially available lmwhs such as enoxaparin, fraxiparin, and fragrnin are prepared by chemical methods which can result in desulfation and other chemical modifications of the internal structure leading to differences in the pharmacologic effects. on the other hand, tiactionated lmwhs retain their native characteristics and are structurally similar to heparin. in addition, the oligosaocharide sequence responsible for atiii binding is not modified. physical methods such as gamma irradiation (~co) have been used to fi'agment sulfated glycosaminoglyeans yielding fragraents without chemical modifications (deambrosi et at. in : biomedical and biotechnological advances in industrial polysaccharides, pp. 45-53). utilizing this technique, depolymerized heparius exhibiting different molecular weights can be obtained. this communication reports on the biochemical and pharmacologic effects of several such depolymerized heparins to demonstrate the molecular weight dependence on biologic activity. fragments exhibiting molecular weights of 5, 7, 8, and 9 kda were prepared by exposing concentrated heparin solutions to a rectilinear gamma ray beam at intermittent doses of 2.5 to 25 mrad under controlled temperatures. unlike the chemically depolymerized heparins, these fractions did not exhibit any decrease in charge density or atiii affinity. in routine assays for heparin, a clear cut molecular weight dependance on the anticoagulant and antiprotease actions was observed. on a gravimetric basis, these agents produce superior antithrombotic actions in comparison to chemically depolymerized derivatives. these studies suggest that gamma irradiation can be used to prepare lmwhs which retain their molecular integrity and therefore may prove to exhibit a more comparable biologic profile to hepari~ futthermore, lmwhs produced by gamma irradiation lack the usual double bond fommtion which requires the use of additives which can alter the product profile. university hospital, dept. of angiology, frankfurt a.m., germany introduction: thromboembolic disease constitutes a major clinical problem and among others a defective fibrinolytic system has been suggested as a predisposing factor for the development of thrombosis. the plasma fibrinolytic system can be impaired by inherited deficiencies of plasminogen defective release from the wessel wall tissue plasminogen activator (t-p'a) or by high ptusma levels of regulatory proteins, such as plasmino-8en. activator inhibilors (pal). the aim ....... of the present study w~s to eshmate the prevalence of decreased fibnnolyl~c actwlty m young pls. with thrombophilia. patients: a great population of 884 pts. (fenmle 478, male 406; age 21-61 ys median 39.8 ys) with venous thromtx~emolism before the age of 45 years were investigated in regard to their plasma fibrinolytie system. in none of them well established thrombophilia risk factors could be identified previously. methods: plasminogen ~behdngwerke), pai-1 activity (ehromogenic assay, biopool), pal-i anugen coneentration (elisa, biopool), t-pa activity (chromogenic assay, biopool) and antigen concentration (elisa, biopool) were measured before and after venous oeclusion.vo was performed z 12 month after the last thromboembolic epi~xle. 24 healthy subjects (median age 24.7 ys) served as controls. results." 24 pts.(2.7%) were classified as plasminogen deficiencies (activity and antigen). 142 pts.(16%) had significantly elevated levels of pal activity (up to 120 u/ml) and pal antigen (up to 90 ng/ml). none of the pts. with high pal levels had laboratory signs of acute phase reaction. low t-pa activity could be demonstrated and confirmed in 121 pts., aecordingto a prevalence of 13.6% (range: 0-2.7 u/ml; reference limils: 2.8 -21.8 u/ml). however, there was a significant negative correlation between t-pa activity and pal values. in 67 pts. (55.4%) the low t-pa activity was associated with increased pal levels whereas the t-pa antigen concentration was normal. a parallel reduction of t-pa activity and t-pa antigen (range: 0.35-3.5 ng/ml; reference limits: 3.6 -21.0 ng/ml) were determined repeatedly in 54 pts. (f 23, m 31, median age 39 ys). thus, the prevalence of a defective t-pa release was 6.1% in our study group. conclusion." in comparison to the frequency of inherited deficiencies of other well established thrombophila risk factors we have observed a relativel~ high prevalence of diminished t-pa activity, elevation of pal respectively in our study group. our data strongly indicate that besides t-pa and pal acuvity, antigen concentration for both parameters should be determined in pts. with thrombophilia. the antithrombotic and anticoagulant effect of the supersulfated low molecular weight heparin ssh 14 was studied after i.v. and s.c. administration in rats. thrombus formation in the jugular vein was induced by i.v. injection of activated human serum and following stasis for 20 rain and was assessed by a thrombus score ranging from 0 (no thrombus formation) until 3 (complete thrombus formation). ssh t4 injected either 10 min (i.v.) or 30 rain (s.c.) before thrombus induction caused a dose-dependent antithrombotic effect in a range from 0.25 to 2 mg/kg i.v. and 1 to 4 mg/kg s.c. there were clear differences in the antithromboric effectiveness between female and male animals, i.e, in female rats antithrombotically effective doses were lower than in male rats (edh0 after i.v. injection in females 0.35 mg/kg, in males 0.9 mg/kg). the sex differences were confirmed in studies on the time course of the antithrombotic effect. after i.v. injection of fully effective doses (2 mg/kg i.v. and 4 mg/kg s.c., resp.) the antithrombotic effect disappeared after 8 h in female or after 4 h in male rats. for studies on the anticoagulant action blood was drawn from the femoral artery and after centrifugation global clotting assays were performed in plasma. similar to its antithrombotic action ssh 14 also caused doseand sex-dependent anticoagulant effects. the most sensitive assays were the aptt and the heptest; thrombin time and prothrombin time were less or not influenced by ssh 14. in conclusion, ssh 14 was found to be an effective anticoagulant and antithrombotic agent in experimental studies in rats. at present there is no explanation for the clear sex differences found in this species. venous thromboembolic disease is the most frequent complication in patients undergoing total knee replacement therapy. patients and methods: after informed consent 3x30 patients were included in an open randomized clinical study and the incidence of venous thromboembolisrn was examined using different regimes for heparin prophylaxis (30 patients received fraxiparin 36 rag once daily, 30 patients clexane 40 once daily and 30 patients 7500 u calciparin twice daily). there were no differences between the groups concerning age, sex, body weight, risk factors, surgeons, decrease in hemoglobin~ and requirements for blood products. pre surgery, day 1, day 5-7 phiebograms were performed and also tat, dimers, fl+2 prothrombin fragments were examined. results: 1., dvt in 26 patients (28.9%). dvt in 5/30 patients under calciparm prophylaxis, 8/30 patients under fraxiparin and 13/30 patients under clexane treatment. 2., low speciflty (3.4%) of dimers and tat (24%) for detecting a dvt in these special patients undergoing knee replacement therapy, elevated fi+2 fragments in the dvt group at ti and t2 vs the patients without dvt (t1 dvt: 3.24+-1.8 vs. 1.6+-0.3 -p= 0.0042). 3, only 8/26 patients (31%) with dvt had clinical signs of thrombosis. conclusions: 1., there is an increase of thrombin gneeration measured by tat and dimers after knee replacement therapy. there are further studies with more patients necessary to confirm that fl+2 prothrombin fragments can discriminate between patients with and without dvt from a clinician's point of view. 2., phlebographicauy confimled dvt in almost 30% of our patients demonstrate the high thromboembolic risk in these patients. von willebrand's disease (vwd) type 2 is characterized by absence of high molecular weight muitimers. qualitative changes in the structure of the molecule might be associated with enhanced binding of von willebrand factor (vwf) to platelet glycoprotein lb. therefore in some patients vwd type 2 is associated with severe thrombocytopenia. here, we report on a 9 year old boy who presented with severe purpura and platelet counts about 20000/gl at the age of 2 years. thrombocytopenia did not respond to corticosteroids. a normalized platelet count of short duration was observed after high-dose immunoglobulins. in addition, increase of platelets was seen after anti-d treatment. thus, although platelet associated antibodies were not detected, thrombocytopenia seemed to be caused by an autoimmune mechanism. despite platelet counts above 50000/gl, the patient experienced severe bleedings with a significant decrease of hemoglobin levels. therefore, he needed several transfusions. coagulation analysis revealed vwd. application of ddavp lead to a normalization of partial thromboplastin time (ptt) and an increase of factor viii with subsequent cessation of bleeding symptoms. recently, vwd was typed 2 by lack of high molecular weight multimers. in conclusion, we report a case with vwd type 2 responding to ddavp. however it is unclear, whether thrombocytopenia is part of the vwd type 2 or of autoimmune origin. since autoimmune antibodies have not been detected, the effect of immunoglobulin treatment might be explained by blockade of enhanced binding of vwf to glycoprotein lb. von willebrad disease (vwd) with a prevalence of 0,8% (ruggeri 1994, rodeignere 1987) seems to be the most frequent inherited hemostatic disorder. • the diagnostic criteria for vwd are clinical picture, family hostory, laboratory findings: bleeding time, partial tromboplastine time (ptt), level of factor viii:e, vwf, vwf:ag, ristocetin induced platelets aggregation (ripa) and multim~-analysis.the diagnosis ofvwd is occasionally difficult, especially in early childhood because the laboratory data may vary due to time of investigation, as well as abnormalities may not be present in all sub-types the aim of this study was the evaluation of diagnostic approach to vwd in childhood and diagnostic reliability of all available laboratory tests. all previously mentioned laboratory tests have been done on our own material (51 child who satisfied all criteria for vwd, 23 boys and 28 girls, 1-9 years old) except mulfimer analysis which was unavailable in some cases. majority of laboratory tests proved to be highly specific and necessary for diagnosis. however, the diagnostic reliability of fviii:c and adhesion of platelets is much lower in mild cases in comparison to total sample, while ptt is an unvaiied test. the most specific screening test for vwd is vwf which diagnostic reliability is almost 1,00. the optimal strategy to establish general diagnosis of mild forms ofvwd is use of vwf and vwf:ag plus ripa if necessary and multimer analysis to classify variant types. we report on a new multimeric structural defect of vwf detected in a german family (two sisters and their three children): all members of the family who presented to our outpatient clinic had an increased spontanous bleeding tendency (moderate or strong hematoma, epistaxis, menorrhagia). prolonged bleeding could be observed after surgical procedures (adenotomia, tooth extraction) and after trauma (laceration). wound heeling was impaired in two cases. clotting assays showed slightly prolonged apti" and a mild decrease of f viii:c, vwf:ag and vwf:rcof levels. collagen binding activity was within normal ranges. bleeding time (simplate i) was slightly prolonged. the analysis of the multimeric structure in plasma showed quantitative and qualitative abnormalities: all multimers were detectable; the structure of vwf was reproducably abnormal in all family members so that the defect must be caused genetically. the thmmbocytic vwf showed neither qualitative nor quantitative alterations. minirin@ (ddavp) was administered as a test dose of 0,3 ~tg/kg bw in 100 ml 0,9% nacl-solution i.v. to evaluate efficacy and tolerance: clotting assays showed normalization of a_vrt, f viii:c, vwf:ag, vwf:rcof in plasma and shortening of bleeding time in three cases. an insufficient rise of vwf:ag and vwf:rcof levels could be observed in one case. one patient had no rise of f vm:c but a corrected bleeding time. multimeric analysis showed no structural change. the administration of ddavp was well tolcrated in all cases. the existance of all multimers in plasma and the normal collagen binding activity suggest that the structural abnormalities of vwf in this family does not cause functional defects so that the defect could be classified as a type i vwd. the response to ddavp was only partially effective. mild von willebrand disease (vwd) is far the most frequent congenital bleeding tendency. its diagnosis is very helpful in pre-operative check-up in order to avoid bleeding complications during surgery. following post-operative periods or monitoring the management of haemorrhagic episodes in vwd patients is also strongly recommended. current methods involve complex technologies, are time consuming and require large series. these assays lack the expected flexibility for rapid individual testing in patients. a new and flexible assay which works on the fully automatic walk-away coagulation instrument, sta, has been developed for these applications (liatest vwf). the technology is an immuno-turbidimetric method using mierolatex particles coated with rabbit polyelonal antibodies specific for vwf. the assay has a dynamic range from 2 to 420% yon willebrand factor (vwf) concentration, it works with a 2 fold dilution of tested plasma (50 td) and it offers a calibration established with the nibsc international standard. the total assay time is of less than 10 minutes and the detection threshold is of 2% there is no prozone effect up to concentrations higher than 1,000% vwf. intra-assay reproducibility is < 4% and inter-assay one < 5%. in dilution studies a mean recovery of 98% was obtained. in a study on 55 plasma samples from norma~ individuals, patients with high vwf concentrations, and vwd, comparison with the elisa technique demonstrated a correlation coefficient of 0.997 with a slope of 0.978 and an intercept of 3.30. in the low assay range too, a good agreement was obtained with the elisa. we conclude that liatest vwf is a reliable, flexible, sensitive, and rapid automated assay which fits well the vw'f assay applications in coagulation laboratories. fibrinolysis, the process during which the active enzyme plasmin is generated in a regulated and localised way, is -in a classical understanding -responsible for the dissolution of blood clots formed in a vessel. for this activity, t-pa is generally assumed to be the most important plasminogen activator and its activity, is regulated by enzyme kinetic mechanisms dependent on the presence of fibrin. with this background t-pa is used for thrembolytic therapy with great success. however, data from t-pa knockout mice indicate that t-pa might not be responsible for inhibiting the spontaneous development of intravsacular thrombi but only for dissolution of fibrin formed upon a coagulation challenge. in contrast, u-pa, generally assumed to be important for extravascular proteolytie activity on activated or tumour cells, seems to lead to the development of spontaneous fibrin formation in a mouse knockout model. on the other hand, the major plasminogea activator inhibitor pal-i seems not only to regulate intravascular fibrinolysis but seems to also be important for the progression of vascular diseases (neointima formation is e.g. increased in a pai-1 knockout model, but increased levels of pai-1 seem to predict reocclusion after angioplasty). in addition to their functioning as enzymes and inhibitors, components of the fibrinolytic system seem also to be involved in signalling processes in tumour and other cells. the u-pa/u-pa-receptor system could be shown to function as a chemotactic system and to elicit a migratory and mitogenle response in monoeytes and tumour ceils as well as in vascular cells. for such a response activation of tyrosine kinases of the sre-family might be responsible in some cell lines, but other signal transduction pathways e.g. involving caveolae and the starprotein can not be excluded. there seems to be a further important role of components of the fibrinolytic system which involves serine protease inhibitors (serpins): serpins have homologies to hormone binding proteins and cleavage of serpins by their target enzymes not only leads to inactivation of the enzyme but also to a possible release of bound hormones from the serpins. from these data clearly the relevance of any regulation of the fibrinolytie, system depends on the specific function of the system to be dealt with. in addition to "fibrin binding", "receptor mediated" and "genetic control" (e.g. 4g vs. 5g in the pai-i promotor) also "signal transduction" and "hormone delivery" are distinct functions of the system with specific regulation. plasmatic for both, healthy persons as well as for patients with angina pectoris it could be shown that increased values of plasma fibrinogen, factor viic and vwf:ag are significantly associated with the risk to suffer an acute myocardial infarction or cardiac sudden death. the same holds for tpa:ag. however, a group analysis in quintiles reveals that particularly low tpa:ag values are connected with a particularly low coronary risk. unexpectedly also the acute phase protein crp is positively associated with increased coronary risk. for clinical purposes these factors have already been included into coronary risk scores in order to improve the individual risk prediction in combination with lipids and other risk factors. the assessment of the pathophysiological significance of these observations remains at dispute. 4 pathways are discussed: 1. the assumption that increased plasma values of those factors indicate increased coagulation activity could so far not be established in prospective studies. 2. both vwf:ag and tpa:ag are produced in endothelial cells. an increase of their plasma level could therefore indicate increased endothelial cell functions which accompanies progressive atheromatosis. the risk association of the two acute phase proteins crp and fibrinogen could be interpreted analogously. 3. first prospective studies favour the assumption of a genetic determination to an increased production of coagulation proteins in persons at particular coronary risk. it could also be shown that there is a certain dependance of the gene-polymorphism for co-and 13-fibrinogen chains from the coronary risk. 4. even slightly elevated concentrations of fibrinogen and/or vwf:ag may influence the quality of a coronary thrombus both by increased physical stability and by reduced fibrinolytic lysibility. this could mean that an early coronary clot under these conditions could more readily develop to a stable, occlusive thrombus. a newborn with pronounced bleeding tendency had a prothrombin (prth) deficiency below 2.8% in a clotting assay. both parents had activities of 71% and 69%, respectively. however, the immunological determination ofprth by elisa revealed normal concentrations in all family members (93%-101%). furthermore, thrombin generation as investigated by a chromogenic assay using ecarin for activation of prth was normal as well. activation of prth by fxa was investigated by reealcificafion of the plasma samples and further analyzed for prth and its derivatives produced. although clotting times still were different, finally, normal levels of fl+2 and tat were generated as determined by elisa. western blot analysis using polyclonal (rabbit) antibodies to prth and a monclonal antibody specific to human thrombin, revealed different patterns of prth degradation products. tat was only weakly visible in the serum of the mother and nearly absent in the child.the mobility of prothrombin and thrombin was different compared to normals indicating a lower molecular weight. after reduction of disulfide bridges a higher molecular weight of thrombin was observed compared to normals indicating an insufficient cleavage ofprth and formation ofprethrombin 2. these observations let suggest that prothrombin marburg is a deletion mutant lacking the cleavage region arg 320-ile321. upon cleavage by factor xa only prethrombin 2 is formed under liberation of fl+2. this prethrombin 2 is able to cleave chromogenic substrates in the ecarin assay. probably, prethrombin 2 forms a complex with atiii which is detected by elisa, but unstable under denaturing conditions as in the western blot. as a major complication of haemophilia a treatment, up to 30% of the severely affected patients develop antibodies to substituted factor viii. investigating 133 patients and considering the data of further 231 patients of the haemophilia database, we could show, that risk of inhibitor developement depends on the patient's mutation type. patients with more severe gene defects, like intron 22 inversions, stop mutations or large deletions had a risk of about 35% for inhibitor developement, which was about 7 times higher than for missense mutations or small deletions. besides an influence of mutation type, we investigated other parameters e. g. immune response genes (i-ila-genotype) and clinical aspects (treatment onset and frequency, type of concentrate) that might also affect inhibitor formation. to exclude any effect of mutation type, we focussed on 72 patients with an intron 22 inversion. hla-typing showed that some t-ila-alleles (dqb0602, bt) occurred more otten and others (dqa103, dqb603, dr13, c2) less frequent in inhibitor patients. treatment onset, frequency and type of concentrate apparently do not affect inhibitor incidence. the results presented here, prove that inhibitor development is considerably influenced by the mutation type. this supports the hypothesis that patients with severe molecular defects have no endogenous factor viii protein and that substituted factor viii represents a foreign protein, leading to an immune response, e. g. the production of alloantibodies. in addition, the immune response seems to be modified by the hla-genotype. however oar findings (in terms of genotype and treatment parameters) can only explain part of the inhibitor pathogenesis. it is still unsolved why substituted factor viii does not lead to a recognizable immune response in 2/3 of the patients with severe molecular factor viii gene defects. consequently other factors, probably concerning the antenatal phase, must be involved. viia in the treatment of patients with inhibitors against factor viii or ix: a german/swiss/austrian multi~center trial d. ellbriiek*, i. scharrer**, j. dethling***, and the rfviia study group *section h~mostaseology, university ulm **dept. of angiology, jwg-university hospital frankfurt a.m. ***novo nordisk, mainz administration of activated recombinant factor vii (rfviia) can by-pass the fvnlwlx pathway and offers an alternative treatment for patients with antibodies (inhibitors) against these factors. from november 1994 to october 1995, a total of 25 bleeding episodes and 10 surgical interventions in 18 patients were treated with rfviia in a phase iiib multicenter trial. diagnosis was hemophilia a (n = 15) or b (n=l) with inhibitor, and acquired inhibitor against factor viii (n=2). various serious bleeds, from complicated joint and gingival bleeds to lifethreatening psoas bleeds, have been treated. operations have been tooth extractions, radiosynovectomy, implantation and explantadon of porth-acaths and one adenotomy. dose regimen was 90-120/zg/kg bw every two to three hours until clinical improvement, with subsequent dose reduction. results: for bleeding episodes, response to rfviia after 24 hours was effective in 72%, partially effective in 12 9"0, ineffective in 129"o and not evaluable in 1 (4%) of the patients. two of the three treatment failures were associated with very long dosage intervals of rfviia. the third patient was in a critical situation with artificial high pressure respiration and polytransfusion because of a hematothorax, and suffered a terminal intracerebral bleed. the efficacy of rfviia for surgery was very good. response to treatment was independent of antibody titer. no signs of dic or activation of coagulation were noted. conduslon: in our experience, rfviia is an efficient and safe treatment for inhibitor patients with acute bleeding episodes. it should be investigated, whether rfviia can be an alternative treatment also for the hometreatment situation. successful immunetolerance therapy of f vih-inhibitor in children after changing from high to intermediate purity f vih concentrate w. kreuz, j. joseph-$teiner, d. mentzer, g. auerswald*, t. beeg, s. becker zentrum der kinderheilkunde, j. w. goethe-universit~itj frankfurt am main *professor hess kinderklinik bremen introduction: inhibitor to f viii is the most severe complication in treatment of patients with haemophilia a. the incidence of f viii inhibitors is estimated to range between 15-33%. several authors reported that the immunetolerance therapy (itr) of f viii-inhibitors can be induced with high dose f viii concentrate. objective: this presentation will show data of four children with haemophilia a and f viii inhibitor (high responder), who had an unsuccessful lit with high dose f viii concentrate (high purity) in the first step. f viii concentrate was changed to an intermediate purity product (haemate hs®) in the subsequent course of h't. all patients received bleeding prophylaxis with an activated-prothrombin-complex-concentrate (feiba®). results: median age was 13 (9-18) months, when the inhibitor was first detected. in all four patients the f viii inhibitor titre increased under immunetolerance treatment with f viii concentrate (high purity) in the first step of therapy. after changing the f viii concentrate (intermediate purity) the inhibitor titres decreased continuously after a rebooster effect to 0be within months. median duration of f viii inhibitor elimination time (until first testing of 0 be) was 3 (2-5) months. in all patients the f viii inhibitor was successfully eliminated. until now all patients are under prophylactic treatment with f viii concentrate and had no positive inhibitor testing since. median observation time since the first testing of 0 be is 14 (4-60) months. conclusion: different studies concerning immunetolerance treatment have been successful with f viii concentrates of different purity. according to our experience in these four presented patients, we assume that probably not the purity of the f viii concentrate is important for the induction of immunetoleranee, rather than the type of f viii presentation in the used concentrate. the used preparation (haemate hs®) is a f viii concentrate with high concentration of vwf, which is known to be important for the protection of f viii against degradation by proteases. this may be a mechanism for a prolonged antigen presentation to the immunesystem and thus may have a positive impact on the outcome ot itr. long scale trials are needed to prove the above assumptions. thrombasthenia glanzmann is a disease affecting platelet function because of a partial or total lack of glycoprotein (gp) ilbllla expression or a modification of this complex. since the receptor dysfunction goes along with reduced or absent platelet aggregation and adhesion, it causes bleeding complications in case of injury. here we report about a 60 years old women, who suffered since early childhood from a severe bleeding disorder. life threating bleeding complications occured after tooth extraction and after abdominal surgery. analysis of the patients platelets revealed normal values for the platelet count, whereas their volume showed to be increased (11 fl). clot retraction was diminished to 17%. platelet adhesion to siliconised glass and human subendothelial matrix was reduced, as was the spreading of the platelets. adp (i#m) induced platelet aggregation was inhibited, while collagen-, ristocetin-and thrombin-induced aggregation showed to be normal. cross immunelectrophoresis resulted in an atypical peak of gpiibllla with reduced electrophoretic mobility. in the electroimmunoassay according to laurell 14% of gpiibllla was detected. moreover we observed a markedly diminished 125j-fibrinogen binding. sequence analysis of the gpiib and gpiila cdna after pcr amplification unraveled a g2508 --, a transition in gpiib, substituting gly805 --* glu. the structure/function relationship of this mutation has still to be investigated. we report two new abnormal fibrinogen variants, denoted as bem iv and milano xi, both having an exchange of arginine to histidine in position 16 of the ac~-chain. routine coagulation studies revealed prolonged thrombin and reptilase clotting times, low plasma fibrinogen concentrations determined by a functional assay but normal fibrinogen levels measured by the immunological assay. the onset of turbidity increase following addition ofthrombin to purified fibrinogen was markedly delayed in both variants. release of fibrinopeptide b by thrombin, measured by reversed phase hplc, was normal whereas only one half amount of normal fibrinopeptide a was released. in addition to normal fibrinopeptide a, an abnormal fibrinopeptide a* was cleaved from both dysfunctional fibrinogens. the structural defect was determined by asymmetric pcr and direct sequencing of a gene fragment coding for the nh2-terminus of the aachain. both variants were found to be heterozygous for the transition g to a at nucleotide position 1203, leading to the substitution actl6 arg-->his, resulting in a delayed fibrin polymerization. the simple assay permits detection of the most common amino acid substitutions occuring in the nh2-terminus of the ac~-chain of the functionally abnormal fibrinogen variants. protein c inhibitor (pci) a member of the serpin family is also known as plasminogen activator-3 (pal-3). pci was first described as a component of human plasma, regulating the activity of activated protein c and other sedne proteases of the human coagulation and fibdnolysis system. since then pci was found to be present in extra-plasmatic systems also. high concentrations of pci were detected in human seminal plasma suggesting a role for pci in human fertility. significant concentrations of pci mrna and antigen were located in lysosomes of proximal tubular kidney cells suggesting an intracellular function for pci in this environment. in this study we present evidence that pci is also present in human pancreas. rna from human pancreas was reverse transcribed and pcr amplified. the resulting pci cdna was identical with pci cdna from human liver. ~p labeled antisense rna probes used in in situ hybridization experiments with human pancreas tissue sections showed that pci rna was located in the acinar ceils. pancreatic fluid was analyzed by sds-page and immunoblotting. using monospecific antibodies directed against human plasma pci, a 57 000 mw protein band was observed which comigrated with purified human plasma pci. our results show that pancreas cells contain a significant concentration of pci mrna. this message is localized in the secretory acinar cells. therefore we conclude that pci antigen found in pancreatic fluid is likely to originate in the pancreas. the role of pancreatic pci is unknown at present. however, since thrombosis and systemic hypercoagulable states are known complications of pancreatic diseases our results and in vitro experiments by others showing that pci can inhibit pancreatic enzymes such as chymotrypsin and trypsin indicate that pci may be part of the inhibitor potential which protects pancreatic tissue from auto degradation. these inhibitors normally prevent the release of active pancreatic proteases into the vasculature or microcirculation where destabilization of the coagulation balance and subsequent thrombus formation could occur. institute for clinical chemistry and laboratory diagnostics and *clinic for cardiology, universi w of duesseldorf p-selectin (cd 62p, the former granule membrane protein 140 or gmp 140) is an integrated membrane protein of platelets and endothelial cells. under inactivated conditions it is stored in the alpha granules of platelets and in the weibei-palade bodies of endothelial cells. endothelial cells covering atherosclerotic plaques show an increased expression of p-selectin. 13-thromboglobulin (13-tg), which is also expressed from the alpha granules of platelets during adhesion or aggregation, is regarded as a marker of platelet activation in vivo. coronary thrombosis plays a central role in the pathogenesis of acute coronary syndromes. we therefore analysed cd 62p and 13-tg in acute coronary syndromes, healthy subjects (hs, n=l i), patients with stable angina pectoris (sap, n=20), unstable angina pectoris (uap, n=l 2) and acute myocardial infarction (ami, n= 12). plasma samples were obtained by using ctad vacutainer tubes (0.109 m na~-citrate, theophylline, adenosine dipyridamole). patients with cad showed significantly increased plasma concentrations of cd 62p (hs: 98+20 versus sap: 133+38ng/ml, p<0.05; versus uap: 128+28 ng/ml, p<0.01; versus ami: 144+72 ng/ml, p<0.05) independent of the severity of clinical symptoms. in comparison only patients with ami showed significant higher 8-tg concentrations compared with hs (hs: 30+20 versus ami: 39+14ng/ml, p<0.05). although the cd 62p plasma concentrations showed no relationship to the clinical severity, hence there was a positive correlation between cd 62p (r=0.47; p< 0.001; n=55) to the severity of cad classified as i, 2, 3 vessel disease. it is concluded that elevated cd 62p concentrations are correlated with the severity of cardiovascular disease. cd 62p is not suitable for differential diagnosis of acute coronary syndromes, because it is elevated independently of the clinical status of the patients. the involvement of platelets in the pathogenesis of acute myocardial infarction may be indicated by the increased 13-tg concentrations. iklinik nr herz-, thorax-und herznahe gef&schirurgie und 2institut x~tr klinische chemie und laberatodumsmedizin der universint regensburg an increased blood loss following surgery with extracorporeal circulation (ecc) contributes to the morbidity and mortality. postoperative haemorrhage following ecc has been related to a platelet function defect and the activation of the blood dotting and fibrinulytic system. we investigated platelet surface antigen expression and parameters indicating activation of the clotting and fibrinolytic cascade to assess the predictive potential of these variables for increased blood loss after ecc. g0 patients referred for coronary bypass gra~ing with no history of a bleeding disorder and normal routine clotting tests were included. on the day prior to surge~ and immediately upon arrival on the intensive care unit blood samples were drawn. the surface expression of glycoprotein (gp) lib-ilia, gp lb, and p-selectin was meamred with and without in vitro stimulation with adenosine diphosphate (adp) using whole blood flow cytomet~y. platelet counts and platelet factor 4 (pf4), as well as, routine clotting tests were performed. activation of the clotting and fibrinolytic system were judged from thrombin-antithrombin-iii complex fiat), fibrinogen fig), d-dimers (dd), cc2-antiplasmin (tz2a), prothrombin fragment 1+2 (fl+2),and tissue plasm~ activator (t-pa). blood loss fxom chest tubes was measured hourly until removal of drains. following ecc the levels of pf4, tat, dd, o~2a, fl+2, and dd were sigulticnatly increased (p<0.0001) compared to baseline values. gp iib-iila, gp ib, p-selectin, platelet count, and fg were significantly reduced (p<0.0001). analysis of variance (anova) revealed that postoperative values of gp ib (p<0.0001), dd (p<o.05), fg (p<0.01),and platelet count (10<0.05) had a significant influence on blood loss. none of the preoperative data was significantly associated with increased blood loss. a subgroup of patients who bled more than the mean + 1 sd (14 cases) was formed_ in logistic regression analysis gp ib expression (p=0.01) and dd (p=0.007) were of value in identifying bleeders. the sensitivity of the equation was 56%, the stx~ificity 97% and the positive predictive value 82%. although the reasons for an increased blood loss following ecc are multifactorial, we were able to identify a small number parameters with a significant infl~nco. the combination of the expression of yon willebrand receptor on the platelet su_rfaco and d-dimer levels in plasma was successful in predicling an increased blood loss following heart surgery. this may be helpful to define indications for platelet transfusion and flesh frozen plasma. the most widely distributed receptor for the fc-region of igg is the fcreceptor iia (fcyriia). for this receptor a functionally relevant polymorphism of amino acids argmhis is known. we previously showed hit-antibodies to bind to platelets via feyriia and now whether clinical manifestation of hit is associated with the fe'/riia polymorphism. therefore, we developed a rapid two step allelle-specific pcr (asp) method for genotyping the foyriia. with control samples the asp was compared with the time consuming and complicated allele-specific oligonucleofide hybridization technique, the phenotype expression of fctriia (using the moabs iv.3 and 41h16) and the monocyte stimulation assay. complete concordance between the assays was found. we used the asp to determine the allelic distribution of the inst. physiol. chem., marburg; *)univ. of virginia, charlottesville, the functional heterogeneity of blood platelets is clinically significant and may contribute to the pathogenesis of cardiovascular and atherosclerotic diseases l). in order to understand the biochemical basis of the functional heterogeneity of platelets, we have investigated the signal transducing pathway of platelet activation a) in terms of guanylate cyclase and phosphodiesterase activities and b) at the level of protein phosphorylation and the receptor-effector coupling via g i-and gs-proteins in functional heterogeneous blood platelet subpopulations separated according to their densities. furthermore, we analyzed the adp-ribosylation of rhoa, which is a lowmolecular weight gtp-binding protein and is thought to be involved in the platelet "shape change" reaction and aggregation. aggregation was enhanced in low-density platelets (sp i) and less inhibited when cgmp was increased by no, compared to high-density platelets (spiii). sp i possessed a lower basal level of cgmp and exhibited a smaller increase in cgmp after stimulation with no. cyclic amp-dependent phosphodiesterase activity was higher in sp i compared to spiii, whereas cgmp-dependent phosphodiesterase activity was similar in sp i and sp iii. sp i, when compared to sp iii platelets, showed a higher degree of prephosphorylation of proteins in the range of 47, 32-34, and 20-22 kda. after thrombin stimulation, the phosphorylation of these proteins was similar in the subpopulations. pertussis toxin,induced adp-ribosylation of the 40-41 kda gi-protein was highest in sp i, whereas cholera toxin induced adpribosylation of the gs-protein and botulinum c3 exoenzyme-induced adpfibosylation of rhoa were highest in sp iii. the findings suggest that a) the higher aggregability in sp i compared to sp iii may be caused by a poorer ability of guanylate cyclase to form cgmp and a higher activitiy of campdependent phosphodiesterase and that b) the adp-ribosylation of g-protein subunits and rhea may contribute to the functional heterogeneity in the subpopulation leading to the greater ability of sp i for aggregation. 1) opper et al., atherosclerosis, 113, 211-217, 1995. causes of accelerated atherogenesis in non-insulin-dependent diabetes mellitus (niddm), known to be associated with increased plasma activity of plasminogen activator inhibitor type 1 (pal-l) as well as with increased plasma concentrations of proinsulin and insulin, remain enigmatic. this study was designed to determine whether proinsulin and insulin increase pal-1 in vivo thereby potentially accelerating atherogenesis. to simulate hyper(pro)insulinemia seen with niddm, we infused equimolar concentrations of insulin (1 iu/kg), proinsulin, c-peptide, or placebo, all endotoxin-free, i.v. over 1 h in 53 conscious rabbits maintained euglycemic. plasma pal-1 did not increase with placebo (n=16) or c-peptide (n=4), but did with proinsulin and insulin peaking in 3 h at 3.5-and 3.7-fold (n=16 and n=17, p<0.o02 for each) in parallel with pal-1 protein quantified by reverse fibrin autography. the increases were specific as reflected by unchanged activities of o~-antiplasmin and t-pa. pat-1 gene expression (pal-1 mrna) increased with proinsulin and insulin by 2.1-and 2.1-fold in 3 h in aorta, 1.9-and 2.4-fold in liver (p<0.05 for each), but not in heart, lung, spleen, or kidney and returned to baseline in 24 h (n=4 each). in situ hybridization localized the increased pal-1 mrna in aorta to endothelium (n=3 each). thus, both proinsulin and insulin directly augment gene expression of pal-1 in arterial endothelium in vivo, thereby increasing plasma pal-1 activity, attenuating endogenous fibrinolysis, and potentially accelerating atherogenesis. the sarco-endoplasmic-retikulum ca+÷atpases (serca) that sequester calcium into intracellular stores, represent the most important mechanism to maintain cytosolic calcium levels low and to return to a resting level after activation. recent studies in platelets and rat endothelial cells have identified two forms belonging to the serca 2b and serca 3 types. the monoclonal antibody pl/im 430 raised to human platelet intracellular membranes has been shown to recognise the serca 3 ÷+ isoform and to inhibit ca sequestration in human platelets. the aim of this study was to identify ca atpase distribution in human umbilical vein endothelial cells (huvec) and human polymorphonuclear leukocytes (pmnl). polyclonal antibodies with known specificity towards serca 2b (r 2/88) and serca 3 (89/3) in addition to pl/im 430 were used in histochemical studies of permeabilised cells and in western blotting experiments using cell lysates and mixed membranes. using the alkaline phosphatase anti-alkaline phosphatase (apaap) method r 2/88 and 89/3 reacted strongly with proteins in permeabilised huvec and pmnl, however pl/im 430 showed good staining only with pmnl. in western blotting experiments r 2/88 recognised proteins with molecular sizes of 100 kda (known to be the parent ca++atpase) and 70 kda (which may represent antibody recognised degradation product), in both huvec and pmnl. 89/3 ÷+ recognised bands at 100 kda (parent ca atpase), 74 kda and 46 kda in both huvec and pmnl membranes. in agreement with histochemical results pl/im 430 recognised proteins (100, 74 and 46 45 + + kda) only in pmnl membranes. in ca transport studies pl/im 430 ÷+ inhibited ca uptake in membranes prepared from pmnl but not in huvec. the distinct reaction of pl/im 430 with pmnl and huvec suggests the possible presence of distinct sub-isoforms of serca 3 with the pmnl and platelet proteins being similar and different to the serca 3 isoform present in huvec. microvascular endothelial cell (ec) lines with a limited life span of 2-3 months in vitro were established from human bone marrow, skin or solid tumor tissue. homogeneity of the lines was verified by flow cytometric analysis and cytokine expression patterns. the cultured ec constitutively expressed hla-class i antigens, cd44, procollagen type i, cd62e, cd34, and receptors for granulocyte colony stimulating factor (g-csf). endothelial cells constitutively produced il-6 and il-8 as well as tpa and pai-i as determined by elisa. in the presence of 5 iu/ml of exogeneous il-la, g-csf was secreted at high amounts (lng/ml) and gm-csf at low amounts (15pg/ml) in confluent monolayers and during over night incubation. these ec potently bound to isolated neutrophilic granulocytes. when granulocytes were stimulated by either 10~ 4 or 10 .7 m formyl-methionyl-leucyl-phenylanaline (fmlp) to produce oxygen radicals, the preincubation with a single cell suspension of ec caused oxygen radical scavenging. a ratio of 1 : 100 ec to neutrophilic granulocytes resulted in a 50 __+ 10% inhibition of fmlp-induced and luminol-enhanced chemiluminescence in vitro. this effect was probably due to the constitutive nitrogen oxide (no) production by ec. unexpectedly, preincubation of ec with 50 iu/ml of interferon-y (ifn-y) to increase no-secretion did not alter the extent of the oxygen scavenging effect measured at different ratios of ec to neutrophilic granulocytes. according to the surface antigen expression pattern, identical ifn-f treatment upregulated adhesion receptors on ec. concomitantly, binding of neutrophils to ec was significantly increased. thus, the net oxygen-scavenging effect by ec appears to be stable under the condition of ifn-y stimulation which indicates maintenance of ec function and effective neutralizition of non-specific oxygen radical damage. moreover, the established test system can be useful to investigate the selective contribution of other inflammatory cytokines, adhesion molecules, macrophages and platelets on ec function in vitro. atherosclerosis is characterized by intimal migration and proliferation of smooth muscle cells (smcs), by macrophage infiltration and extracellular matrix remodeling. circumstantial evidence suggests that urokinasetype plasminogen activator (upa) may be relevant in these processes. this study examines upa expression and upa antigen localization in human coronary arteries with ,carious degrees of atherosclerotic lesions. representative segments of macroscopically normal areas (mnas), early lesions (els) and fibrous plaques (fps) were processed in parallel for in situ hybddization and immunohistochemical analysis. in mnas, upa was detected both in the luminal endothelium and in celocalization with ~-actin-positive smcs throughout the tunica media. in els, upa mrna and antigen were substantially reduced in the luminal endothelium. however, cellular upa expression was detected in the subendothelial area in colocalization with tissue-infiltrating cd68positive macrophages. in the deeper intimal and in the medial layers strong upa expression was observed in association with ~-actinpositive smcs, with most of the upa-positive cells being located in areas with a high proliferative activity as detected by pcna staining. in fps, upa mrna was widely expressed in macrophage-containing areas within the fibrous cap and at the periphery of the necrotic core of the lesions. in addition, upa antigen was identified in acellular areas of the necrotic core regions. compared to the diseased intima of these lesions, the adjacent media stained only weakly for upa. in conclusion, this study demonstrates an enhanced expression and synthesis of upa by intimal smcs and macrophages in advanced atherosclerotic lesions of human coronary artedes these findings suggest a role for upa in the progression of the coronary atherosclerosis. adenosine produced by vascular cells, platelets and other tissues has been proposed to be a vasodilator, inhibits platelet aggregation, stimulates proliferation and migration of endothelial cells as well as exhibits antiinflammatory effects. adenosine of endothelial origin intra-and extracellularly formed and highly concentrated at the luminal surface may constitute an important antiaggregatory mechanism, which is part of the well-known antithrombogenicity of an intact endothelial lining. it was the aim of this study to investigate whether adenosine could influence the fibfinolytic potential of endothelial cells. when confluent human umbilical vein endothelial cells were incubated with adenosine (0.01-10mm) a significant dose dependent decrease in plasminogen activator inhibitor type-1 (pai-i) antigen production by this cells was seen as compared to control. this effect by adenosine was also time dependent and seen after 2 hours of incubation with adenosine. as determined by elisa, pai-i antigen in conditioned media of endothelial cells incubated with 10mm adenosine decreased to 57% as compared to control. pai-i antigen in extracelullar matrix of these cells was also reduced by incubation with adenosine. these results were also reflected on the level of specific mrna as determined by northern blotting. pai-i mrna decreased in endothelial cells after incubation for 4 hours with adenosine (10mm) to 77% (2.2kb) and 85% (3.2kb) of control. in conclusion our data demonstrate that adenosine can downregulate the expression of pai-1 in cultured endothelial cells thereby increasing the profibrinolytic capacity of these cells. this effect of adenosine might contribute to its antithrombotic potential in addtion to its known antiaggregatory effect. sowohl die effizienz als such die komplikationen einer antikoagulantien-dauerbehandlung sind in erster linie yon der gore und engmaschigkeit der gednnungskontrellen abh~ngig seit 1986 wird die schulung zur gerinnungs-selbstkontrolte in der herz-kreistauf-klinik bad bedebarg in gruppen eingeobt. die theoretische und praktische anleitung erfolgt in 8 stunden durch eine in der patientenschuiung erfahrene arzthelfedn (oder krankenschwested-pfleger) und einen arzt. die praktische schulung wird durch einen patientennahen theoretischen tell erg~nzt. verglichen mit konventionell ~berwachten oral antikoagulierten patienten (ca. 50% der tpz-werte liegen im theapeutischen bereich) erreichten 216 quickwert-selbstbestimmer, daf$ 83,1% yon 14 812 tpz-werten im therapeutischen bereich lagen (tabelle 1 ). schwerwiegende thromboernbolische ereignisse oder blutungskomplikatienen wurden nicht beobachtet. durchschnittlich kontrolliert der quickwert-selbstbestimmer seine gerineungswerte w6chentlich, itmgstens im zweiwochen-abstand. ob sich blutungs-und thremboembolierisiko langfristig senken lassen bei gleichze~iger verbesserung yon prognose und lebensqualit~it, bedarf des nachweises im rahmen einer randomisierten, kontrollierten und prospektiven studie. tabelle i verteilun~l der selbstbestimrnten gednnur ~swerte zeitraum 1986 zeitraum -1988 zeitraum 1986 zeitraum -1990 zeitraum 1986 zeitraum -1992 to evaluate the potential benefit of an optimized oral anticoagulation management with inr measurements performed by the patients themselves, two prospective randomized studies have been completed. the first study (n=60) was performed to evaluate the accuracy of inr self-testing (inr-st) compared with standard laboratory controls as well as the improvement of anticoagutation monitoring by inr-st, e.g. the percentage of measurements found inside the target therapeutic inr-range. the 231 parallel measurements revealed a high accuracy of inr-st with a 4.5% median difference between parallel controls of single measurements. in addition, inr-st resulted in a significant increase of measurements inside the target therapeutic range (from 54 to 80%) in the entire group. however, inr-st performed every 4 weeks did not result in a significant improvement, while selfcontrols every week (from 44 to 84%) and every 2 days (from 51 to 84%) did. in a second study (150 patients) the incidence of bleeding and thromboembolic complications were evaluated in patients who either had standard anticoagulation measurements o[ performed inr-st. during a median follow-up of 1.5 years in the group with standard oral anticoagulation management an incidence for bleedings of 10.9% per year and for thromboembolic complications of 3.6% per year was found, while in the group of patients with inr-st bteedings occured with an incidence of 4.5% per year and thromboembolisms with 0.9% per year. it can be concluded that inr-st can be performed with high accuracy by the patients themselves. inr-st results in more frequent controls of the inr and consequently in significantly longer time periods where the patients are inside the optimal target therapeutic inr range. the more accurate anticoagulation management resulted in our study group in a significantly lower incidence of thromboembolic and hemorrhagic complications. the cell biology of tissue factor (thromboplastin) h. prydz, the biotechnology centre of oslo, norway tissue factor (tf,thromboplastin) is the most potent trigger of blood clotting knuwn.lts presence in tissue extracts has been known since before the turn of the century.erwin chargaff (of dna fame) first described that tf consisted of two parts (a protein and a lipid moiety), a fact that was independently rediscovered in the early sixties by deutsch and lechner in vienna and by myself in oslo. in 1973 tf was first purified and shown to be an integral membrane protein of mr about 50 kda (this was a slight overestimate,recent studies indicate 46-48 kda). in 1987 four groupes independently cloned tf edna, later also genomic dna. the structure of tf suggested that it was a membrane spanning receptor, with an extracellular part of about 219 amino acids folding into two domains remotely like the domains of the ig-superfamiliy, and more specifically the eytokine receptors. tf is synthesized to a varying degree in many tissues, but not normally in tissues in contact with blood. however, during acute inflammatory situations and under the influence of a number of agents (immune cnmplexcs, lectins, cytuklnes,bacterial components, complement components etc) monucytes and vascular endothelial cells are induced to synthesize tf. another possible way tu get tf into contact with blood is thruugh trauma, surgical ur otherwise. the fact that tf is inducible has created a lot uf interest in its cell biology. we reported recently that binding of factor viia tu tf un the cell surface elicited a marked increase in intracenular ca spikes, thus rendering the final proof that tf not only luoks like a receptor but really is one, since all the three essential criteria are fulfilled: tf has a transmembrane segment and a cytoplasmic tail tf has a high specificity/high affinity ligaud (factor vii and factor x) tf induces an intracellular signal upon ligand binding recent results regarding tf as a receptor and regarding its intracellular traffic will be reported. both f.ix and f.x are activated by the tf/f.viia complex. we have used a tf-initiated model system to examine the roles played by f.ixa and f.xa in initiating coagulation. the in vitro model contained a cellular tf source; plasma concentrations of f.ii, v, viii, ix and x, tissue factor pathway inhibitor and antithrombin iii. we tested the ability of exogenously added f.ixa or f.xa to substitute for tf/viia activity in initiating platelet activation and thrombin generation. f.xa was 10 times more potent than f.ixa in initiating platelet activation, but f.ixa was more potent in promoting iia generation. in the presence of tf/viia, zymogen f.x was required both for platelet activation and iia generation, while f.ix was only required for iia generation. thus, tf/viia-activated f.ixa and f.xa have distinct physiological roles. the main role of f.xa is to activate platelets by producing an initial small amount of iia. the process of platelet activation is much more efficient if this initial iia is produced on the tf-bearing surface. f.ixa, by contrast, enhances iia generation on platelets by providing f.xa for platelet surface prothrombinase assembly. we conclude that activation of both f.ix and f.x by tf/viia is essential for efficient initiation of coagulation. furthermore, f.x activated on the surface of a tf-bearing cell isnot functionally equi.valent to f.x activated on the platelet surface. therefore, f.x activation by tf/viia does not compensate for a lack of f.ix or viii in hemophilia because the f.xa activated by tf/viia is located on the wrong surface for efficient thrombin generation. our results emphasize that the location, of an activated coagulation factor is a critical determinant of its role in hemostasis. the x-ray crystallographic structure of the fviia-tf complex located the areas within fviia in contact with tf to the first egflike and the protease domains. high-affinity binding of fviia to tf depends on ca2+ and previous studies suggest that ca2+ binding to a site in the protease domain is required. however, using surface plasrnon resonance we have shown that removal of the n-terminal gla domain and hydrophobic stack (hs) from fviia results in a decreased affinity for tf. in search for the mechanism by which regions in fvua not in direct contact with tf affect the affinity, various techniques have been employed. a plausible model is one in which the gla domain and hs optimize the interaction with tf by affecting regions that mediate the association with tf. using gel filtration we demonstrated that the hydrodynamic radii of des(1-38)-fviia and, especially, fviia decreased upon ca2+ binding, whereas des(1-44)-fviia was virtually unaffected. this suggested that the gla domain and the hs interact in a ca2+-dependent manner with other regions in fviia, presumably the protease domain, making fviia more globular. using cd spectroscopy, we could detect ca2+-induced changes in the secondary structure of fviia, all assignable to an increased helical content of the gla domain. the amidolytic activity of fviia in the absence of tf has a ca2+ dependency that suggests involvement of the gla domain in obtaining full activity. this activity was very sensitive to guanidine hydrochloride (c50<0.5 m) and its quenching corresponded to unfolding of the ca2+-loaded gla domain as measured by changes in the trp fluorescence of fviia. based on these results it is conceivable that the gla domain and perhaps the hs of fviia interacts with the protease domain and that this globular form mediates the initial contact with "it. several studies have indicated a profound role for fvii(a) in arterial thrombogenesis. in the present study we quantified the inhibitory effect of dansyl-glutamyl-glycyl-arginyl-chloromethyl ketone recombinant fviia (degr-rfviia) on acute thrombus formation at medium-sized artery blood flow conditions characterized by a wall shear rate of 650 s -1. native human blood was drawn directly from an antecubital vein by a pump into a heparin coated mixing device where degr-rfviia (0.9 -880 nmol/l final plasma concentration) or buffer were mixed homogeneously with the flowing blood. subsequently, the blood entered a parallel-plate perfusion chamber in which a thrombogenic surface consisting of immobilized tf and phospholipids was positioned. fibrin deposition, platelet-fibrin adhesion and platetet thrombus volume triggered by this surface were measured by morphometry. degr-rfviia inhibited these thrombus parameters in a dose dependent manner with ic50 values between 0.5 and 0.7 nmol/l the plasma levels of thrombin-antithrombin m complexes were inhibited at an ic50 of 0.6 nmot/l since the concentration of fvii and fviia in normal plasma is about 10 and 0.1 nmol/l, respectively, theic50 values indicate that degr-rfviia is a very efficient inhibitor of thrombus formation in this model. thus, inhibition of the extrinsic pathway of coagulation may represent and entirely new and effective arterial anti-thrombotic approach. the anticoagulant agent from medicinal leeches, hirudin, is presently gaining popularity as an antithrombotic drug. the design of recombinant forms of the minipretein and their availability led to the clinical use of this naturally occurring thrombin inhibitor. extensive studies in experimental animals have shown that due to its pronounced and specific antithrombin effect hirudin is an antithrombotic agent of high quality. apart from its anticoagulant effect, hirudin is pharmacodynamically inert and is well tolerated in vivo. brain abnormalities in male children and adoleescents with hemophilia: detection with mr imaging mri of sickle cell cerebral infarction in addition, portions of the preparations were incubated with 1.4nm-gold labelled fibrinegen molecules (fg-au; final concentration 4gp_g/ml) for 10 see at 37°c. the expenments were stopped al~er 3 or 10 rain by glutaraldehyde fixation. serial sections were prepared and examined after silver-enhancement using danscher's (1981) method to visualize the fg-au. hwdid not alter the resting gfp. after .3,rain, activated hw-platelets changed their shape moderately but in contrast to the controls did not aggregate. moreover, the conatdcting cytoskeleton that centralizes platelet organelles was not formed. fg-au was found on the surface of the control platelets, in high density in the contact spaces between the aggregated platelets and internalized in the surface connected system. only a very small number of hwtreated cells had fg-au on their surface and showed internalization. 10 rain after adp most of the platelets in both preparations had retumod to a discospherecytic shape it is confirmed that hw inhibits the platetet aggregation and in addition, 1) the formation of the contractile cytnskeleton as well as 2) the binding and interalization of the used ligand fibrinogen santoso 2 l.institute for immunology and transfusion medicine, emst-moritz-arndt-university greifswald; 2. institute for transplant vasculopathy in cardiac allografts is the leading limitation to long-term survival of cardiac transplant recipients. activation of the coagulation mechanism by adherent monocytes expressing tissue factor (tf) is expected to be involved in the development of transplant atherosclerosis. in this in vitro study the effect of the immunosuppressant cyclosporine a (csa) on monocyte tf expression and the regulation of the tf gene transcription was analysed. csa was shown to inhibit the induction of monocyte procoagulant activity (pca) by decreasing lipopolysaccharide (lps)-induced tissue factor (tf) expression in monocytes. csa exerted a dosedependent inhibitory effect on monocyte tf expression at concentrations (1,5 txmol/l csa: 50 % inhibition) not decreasing the cellular protein synthetic rate. addition of csa during lpsstimulation prevented activation of the nuclear factor (nf) ~b as demonstrated by electrophoretic mobility shift assay. western blot analysis confirmed reduced nuclear translocation of nf-~b in the presence of csa. inhibition of the lps-induced nf-~b activation by csa resulted in a reduced tf gene transcription as demonstrated by rt-pcr-analysis. thus, cyclosporine a prevents tf expression by inhibiting the induction of tf gene transcription in activated monocytes. csa may therefore provide a therapeutical tool acting not only as a direct immunomodulating agent, but also by influencing local coagulation activation. the n-glyean pattern of recombinant human coagulation factors ii (rf-i~ and ix (rf-ix), derived from both transfected chinese hamster ovary (cho) cells and african green monkey (vero) cells, and produced at industrial scale were analyzed by binding to carbohydrate specific lectins and were compared with the glycan structure of human plasma-derived coagulation factors. human plasma-derived coagulation factors 1i (hpf-i1) and ix (hpf-ix) exhibited complex-type glycan structures with carbohydrate chains capped with c~(2-6)sialic acid. terminal galactose-b(1-4)-n-acetylglucosamine units were detected in hpf-ix. both cho cell-derived rf-]i and rf-ix exhibited complot-type glycosylafion and contained ~2-3)-sialio acid in addition to terminal galactose-b(1-4)-n-acetylglucosamine. vero cell-derived if-ix exhibited a complex-type glycan structure sinailar to that of cho cell derived rf-ix. in contrast, rf-li produced by vero cells exhibited a glycan microheterogeneity composed of hybrid-type glycosylation containing "highmarmose" structures and complex-type glycosylation containing et(2-3)-sialie acid. galaetose-b(1-4)-n-acetylglucosamine structures and a low concentration of ct(2-6)-sialic acid were detected in both micro-heterogeneity fractions of veto cell-derived rf-li. although different in the'tr carbohydrate structures, coagulation factors ii and ix obtained recombinantly from both transformed cho-cells and vero-cells exhibited coagulation activities comparable to the plasma-derived proteins. exposure of tissue factor (tf) to flowing blood, as a consequence of vascular injury, leads to tf/f.viia-initiated coagulation which may play a key role in triggering intravascular thrombosis. in order to evaluate the potential advantages of tf pathway blockade over existing therapy, the antithrombotic and the anticoagulant .effects of a monoclonal anti-rabbit tf antibody (ap-1) and heparin were compared in a rabbit arterial thrombosis model. thrombosis was induced by a local injury to the carotid artery and the recurrence of thrombus formation was followed by monitoring the cyclic flow variations (cfv) of the arterial blood flow. rabbits received intravenously either ap-1, heparin or vehicle, and cfv were monitored on line for 120 min. activated partial thromboplastin time (aptt), and activated clotting time (act) were measured at the end of the experiment control ap-i heparin <lmg/kg* 0.2mg/kg/hr 0.8mg/kg/hr intraabdominallretroperitoneal, cns) on a named-patient basis. later also mild/moderate bleeds, predominantly joint bleeds were included.in 80-90% of the bleeds novoseven was found to be hemostatically efficacious. also in major surgery (hip-and knee-replacement including a bilateral total condylar arthroplasty, herniotomy) hemostatic efficacy has been found to be 94% since rfvlla is proteotytically active only in complex with tf the risk for systemic activation of the coagulation system should be minimal. also very few side-effects have been seen.doses between 75-120 tjg/kg have been used, which initially have to be given at 2 hours intervals. to achieve satisfactory hemostasis both an initial dose high enough to induce immediate hemostasis and an appropriate dose interval ensuring a plasma level of fvli:c well above the hemostatic lower level for a period of time, the length of which is dependent on the type of bleeding, are important. accordingly, the highel the initial dose the more robust with regard to dose interval will the treatment be. acute myocardial ischemia is usually triggered by coronary thrombosis. heparin (hep), along with aspirin, is commonly used for the treatment of unstable angina (ua) and myocardial infarction (mi). hirudin (hiq is a potent antitbrombin agent which may offer therapeutic advantages over standard hep for the treatment ua and mi. the oasis phase i pilot study conducted in 24 canadian centres, has evaluated the safety, feasibility and efficacy of hir (hbw 023, behringwerke ag, marburg) compared to hep in 601 patients with mi or suspected non q-wave mi. patients (95 % received aspirin) were randomized to a 72 hr infusion of either hep (5000 u bolus followed by 1200 u/hr; n=253), or low dose hir (0.2 mg/kg bolus then 0.10 mg/kg/hr; n=175) or medium dose lair (0.4 ms/ks bolus then 0.15 mg/kg/hr; n=173). at seven days of follow-up the incidence of major bleeds was 0.4 % in the heparin group, 0.6 % low dose hit, 1.2 % medium dose lair (p=ns), minor bleeds were 10.3 %, 17.1% and 19.1% respectively (p---0.008 hep vs med dose); there were no hemorrhagic strokes. the incidence of the primary outcome of cardiovascular death, new mi and severe recurrent ischemia (refractory or severe angina) was 17.0 %, 15.4 % and 8.7 % respectively (p---0.014 hep vs reed dose). there was also a trend towards lower rates of coronary artery bypass surgery in reed dose hir compared to hep. in a 200 patient substudy, levels of thrombin antithrombin complex, fibfinopeptide a and d-dimer were all suppressed to a greater extent by mr compared to hep. these results indicate that hirudin may be a more effective agent for the treatment of acute coronary syndromes, but more information on safety and efficacy is needed from larger randomized trials. full clinical and coagulation resuks will be presented. hat is caused by an immunologic response to heparin. affected patients need effective parenteral anticoagulation. we treated 82 patients (30m,52fm), median age 61 years (18-84) with laboratory confirmed hat (hipa-test) with r-hirudin (behringwcrke ag, marburg). treatment regimens were: ai acute hat: 0.4 mg/kg b.w., continuous infusion 0a5 mg/kg b.w/h (51 patients); a2 acute hat with thrombolysis: bolus 0.2 mg/kg b.w., continuous infusion 0.1 mg/kg b.w./h (5 patients); b: proph, treatment: continuous infusion 0. i mg/kg b.w.pa (18 patients); c: during cardio-pulmunary bypass. primary objective was: increase in platelet counts or maintenance of normal baseline values during effective anticoagulation. secondary objectives were the incidence of new thromboembolic complications (tecs), major hemorrhage, limb amputations and deaths. results were compared with a historical control of 120 ix-at patients, all confirmed by the hipa test, but treated before rhirudin was available (danaparoid, marcumar, no anticoagulation). primary efficacy analysis: respunders ai: 74%o, a2: 60%, b: 44%, c: 50%, total: 65%. all adverse events beside bleeding complications had been judged to be caused by the underlying disease and not directly related to r-hirndin. proportion of patients with event in the obse~wation period; death: a1 5.9%, a2 0%, b 16.774, c 0%, total: 7.3%, during rhirudin treatment: 1.2%; new tec: ai 5.9%, a2 0%, b 27.8%, c 0%, total 9.8%, during rhirudin: 2.4%; limb amputation: ai 3.9%, a2 0%, b 5.6°/0, c 0%, total: 3.7%, during rbirudin: 3.7% eaapoiat we conclude, that r-hirndin is an effective anticoagulant in hat typa ii patients allowing rapid re, coved" of platelet counts. compared to a historical control the r-hirudin group had a strongly reduced incidence of combined endpuint of mortality, limb amputation and new tecs. major bleedings occurred slightly more frequent than in the historical control. however, the dosage used in regimen b might have been too low, as flds group revealed an effective anticoagulatiun and increase in platelet counts in only 43% and concomitantly had the higl)est incidence of new tecs. key: cord-000083-3p81yr4n authors: nan title: poster exhibition date: 2009-01-31 journal: hepatol int doi: 10.1007/s12072-009-9123-4 sha: doc_id: 83 cord_uid: 3p81yr4n nan background: biliary atresia (ba) is one of the most common causes of neonatal cholestasis and the most frequent hepatic cause of death in early childhood. the incidence rate of ba is higher in asian countries, occurring in approximately 1 of 8,000 (asian countries ) to 1 of 18,000 (european countries) live births. early identification and prompt intervention is very important. to im prove the early diagnosis, we used proteomic technology to screen serum biomarker for ba. methods: two-dimensional electrophoresis (2-de) and matrix-assisted laser desorption /ionization time-of-flight mass spectrometry (maldi-tof-ms) were employed to screen serum biomarkers specific to ba sera from idiopathic neonatal hepatitis. after pretreatment including albumin and immunoglobulin (igg ) depletion, sera were subjected to 2-de and there after image analysis. the differentially expressed protein spots were identified by maldi-tof-ms. result: from optimized 2-de gel images, thirty-four spots were differentially expressed and identified by maldi-tof-ms to be eight proteins. overall, kininogen 1 variant was under expressed and alpha-1-b-glycoprotein, leucine-rich alpha-2-glycoprotein 1, 'sp40,40', a1bg protein, vitamin d-binding protein/group specific component , apolipoproteina-, aqgv 3103 were over expressed in ba group com pared to idiopathic neonatal hepatitis. conclusion: 2-de based serum proteome analysis can be useful in detecting protein expression alteration and new discovered biomarkers might be an aid in the diagnosis of ba, though further validation is needed. s. somani 1 , a. somani 2 , a. jain 3 , v. dixit 3 1 suvidha, 2 navjeevan hospital, 3 ims, bhu, varanasi, india background: a variety of autoreactive antibodies are detected in patients with chronic liver disease. this prospective, nonrandomized study was undertaken to evaluate the nature & prevalence of various autoantibodies in patients with chronic liver disease of diverse etiologies. methods: study population included 53 patients (75% males), who met defined criteria for chronic liver disease. detailed clinical, laboratory and sonographic evaluation was done. sera were tested for asma, anti-lkm type1, ama, apa, ana, by standard methods. p<0.05 was considered significant. results: among various etiologies for chronic liver disease, hepatitis b was most common (28%), followed by alcohol (19%), autoimmune hepatitis in 15%, hepatitis c (6%) and miscellaneous (2%). 30% of patients were labeled as cryptogenic after detailed investigations. ana (>1/80) was positive in 100% of definite aih, 33% of hcv related cld but at titer of >1/40, 66.6% of hcv related cld & 60% of probable aih were found positive. asma (>1/40) was positive in 6% of hbv related cld, 10% of alcohol related cld, 33% of definite aih, 40% of probable aih, 33% of hcv related cld but asma in titer of >1/80 was positive only in 33% oh definite aih. apa was detected in 12.5% of cryptogenic cld, 13.3% of hbv related cld & 20% of alcohol & probable aih related cld each. ama was detected in 1% of cryptogenic, hbv, aih (definite) & hcv related cld each, and 2% of alcohol related cld & 100% of pbc. conclusions: apart from aih there is high prevalence of ana & sma in hcv related cld while other antibodies has low prevalence in non-aih related clds. this study also suggests that prevalence of various autoantibodies should be borne in mind while considering the diagnosis of cld especially of mixed etiology. conclusions: oa infusion did not lower ammonia levels or improve survival. results: the mortality (50.0%) of patients in lamivudine group with meld score from 30 to 40 was lower than that (86.1%) of control group ( 2 =23.319, p=0.000). univariate analysis showed that mortality was significantly related to age (p=0.005), meld score (p=0.009), treatment method (p=0.000), pretreatment hbv dna load (p=0.000), the decline of hbv dna load during therapy (p=0.006) and encephalopathy (p=0.007). in multivariate analysis, in patients with meld scores 30-40, treatment method (p=0.004), pretreatment hbv dna load (p=0.009), decline of hbv dna load during therapy (p=0.014) and encephalopathy (p=0.019) were independent predictors of mortality; for meld scores above 40, only meld score (p=0.015) was independent predictive. conclusions: lamivudine treatment significantly decreases the 3 month's mortality of patients with meld score 30-40, and a low viral load pre-treatment and quick decline of hbv dna load are good predictors for the survival of lamivudine treatment. background/aims: early identification of patients with fulminant hepatic failure (fhf) who need a liver transplantation is very important. to construct a prediction model for early diagnosis and prognosis of fhf, we studied dynamics of metabolic profiles using a d-galactosamine/lipopolysaccharide (galn/lps)-treated mouse model. methods: balb/c mice were used to construct fhf model and sacrificed for blood collection at 4, 5, and 6 hour after treatment, respectively. levels of plasma metabolites were quantified using gas chromatography/time-of-flight mass spectrometry and data were processed using partial least squares discriminant analysis (pls-da). results: distinct clustering differences were observed 5 and 6 h after treatment between survival and dead groups. at 5 h, plasma levels of some metabolites differed significantly between survival, dead and control groups. ketogenesis and the tca cycle were inhibited in both survival and dead groups, but in dead group, the urea cycle was also inhibited and glycolysis was elevated. pls-da indicated that principal component weighting was greatest for plasma levels of phosphate, -hydroxybutyrate, urea, glucose and lactate. the y-predicted scatter plot in pls model assigned samples to survival or dead groups using an apriori cutoff of 0.10 with 100% sensitivity and specificity. similar results were observed in 11 fhf patients with different outcomes. pe012 association between polymorphisms in the interleukin-10 gene promoter and hepatitis b-related acute liver failure conclusions: the pls model based on metabonomics analysis can be used to predict outcomes well, and plasma levels of phosphate, -hydroxybutyrate, urea, glucose and lactate may constitute a set of markers for early diagnosis and prognosis of fhf. il-10 promoter are associated with the susceptibility to hepatitis b-related alf in the chinese population. il-10 a-592c may be a regulatory polymorphism that affects gene regulation. hepatocyte cell death in aclf: mechanism and significance -an immunohistochemical study. p. sakhuja 1 , a. rastogi 2 , s. s hissar 1 , a. singh 1 , a. kumar 2 , r. gondal 1 , s.k. sarin 1 1 gb pant hospital, 2 institute of liver and biliary sciences, new delhi, india background: acute on chronic liver failure (aclf) is defined as acute hepatic insult complicated within 4 weeks by ascites and/or encephalopathy in a patient with previously diagnosed or undiagnosed chronic liver disease. caspases play an essential role in apoptosis. cox-2 is an inducible immediate early gene responsible for the release of prostaglandins during inflammatory response. we studied the immunohistochemical expression of cox-2 and caspase-1 in liver tissue to assess their role in pathophysiology and in predicting outcome of aclf. method: a retrospective analysis of 50 liver biopsies with clinical diagnosis of aclf was undertaken. patients were divided into two groups a and d based on clinical outcome (alive/died respectively). immunohistochemical analysis for cox-2 and caspase was performed on 39 and 36 cases respectively and scored from 0-8 as per intensity and distribution. score 6-8 indicated high intensity with focal to diffuse distribution, and was considered significant. results: etiology of acute liver failure was viral or alcoholic. increased expression of caspase was observed in 10/21 cases in group d and none of the cases in group a(n=15) (p=0.001). increased expression of cox-2 was observed in 4/21 cases in group d and none of the cases in group a (n=18) (p=0.052). conclusion: increased immunoreactivity of caspase in liver biopsies of patients of aclf may indicate worse prognosis and its important role in the pathophysiology of aclf. immunostaining for caspase is useful for assessment of prognosis and possibility of anti-apoptotic and anti-fibrotic therapies in future. conclusion: hhgf expression vector (pcmv-hhgf) has been successfully constructed and repeated hydrodynamic injections can promote sustained and high expression of hhgf in vivo. j.h. kim 1 , k.w. kim 1 1 asan medical center, seoul, korea background: splenic artery embolization (sae) is performed to increase hepatic arterial flow or to decrease portal venous flow in recipients of liver transplantation (lt). thus, the purpose of this study was to estimate sae effect on the basis of changes in caliber of related vessels and splenic volume on pre-sae and serial post-sae ct scans in lt recipients. methods: between 2003 and 2007, among 73 lt recipients who underwent sae and serial follow-up ct, 43 with no compounding factor that may obscure sae effect were included in this study. they underwent ct before and after (1week, 1month, and 1year) sae. a radiologist retrospectively measured diameters of ca, cha, sa, sv and splenic volume on serial ct scans. their diameters and splenic volume on each ct were compared with those on the prior and pre-sae ct. the difference was compared using repeated-measures anova tests. results: cas decreased between 1week and 1month after sae (p<.05), but were stable before 1week and after 1 month. chas increased within 1week (p<.05) but decreased between 1week and 1month (p<.05) and remained stable after 1month. compared with pre-sae ct, chas were larger for 1month after sae. sas continuously decreased for 1year (p<.05). svs decreased for 1 month (p<.05) and remained stable after 1 month. compared with pre-sae ct, sas and svs were smaller from 1week after sae and on. splenic volume continuously decreased for 1year except a period between 1week and 1month. conclusion: the increase of hepatic arterial flow persists for 1month after sae, but returns to baseline thereafter. the decrease of portal flow may lasts for at least 1year after sae. poster exhibition -cholangioca and other liver neoplasm poster session, hall 5b background: now, rfa has becoming an important practice of hcc therapy. in this study, we evaluated whether rfa therapy for metastatic liver tumor has a beneficial effect on patients' survival. methods: forty six patients were treated by rfa for metastatic liver tumor from july 2001 through february 2008 in our hospital, of the 46, 33 patients were metastasis either form colon or stomach cancer. these 33 patients were analyzed in this investigation. cumulative survival rate from initial rfa therapy was calculated by kaplan-meier method. predictive factors for survival were identified using cox proportional hazard regression model. results: the mean age of the 33 patients were 64. 6 (range, 40-79) . the mean size of the tumor is 28mm (range,8-70mm) and the numbers of tumor foci are 2.7 nodules range,1 18. the survival rates of patients treated by rfa were 49.5% at 3 years and 31.8% at 5 years in colon cancer, 15.6% at 3 years and 15.6% at 5 years in gastric cancer. in this series of 33 patients, primary cancer: colon (p=0.002 odds ratio 0.132 95%ci 0.037-0.473), younger patients ( 64) (p=0.041 odds ratio 0.312 95%ci 0.102-0.955) and multiagent chemotherapy (p=0.012 odds ratio 0.223 95% ci 0.069-0.723) were significantly correlated with better survival. conclusion: the survival of patients treated by rfa for metastatic colon cancers had better survival than those of gastric cancers. in addition, good indication of rfa is for metastatic colon cancers, younger patients and has to be treated by multiagent chemotherapies. utility of contrast enhanced ultrasonography with sonazoid in radiofrequency ablation (rfa) for liver metastasis e. goto 1 , s. shiina 1 , r. tateishi 1 , r. masuzaki 1 , k. enooku 1 , t. sato 1 , j. imamura 1 , t. goto 1 , y. sugioka 2 , h. ikeda 1,2 , h. yoshida 1 , m. omata 1 1 department of gastroenterology, university of tokyo, 2 department of clinical laboratory, tokyo, japan background & aims: contrast enhanced ultrasonography (ceus) with sonazoid is effective for liver metastasis because enhance defect in kupffer imaging is well delineated. the aim of this study is to investigate the detection ability of ceus and the utility of sonazoid in rfa for metastasis liver tumors. material & methods: from january 2007 to december 2007, a total of 346 liver metastatic nodules in 87 patients (62 colon cancer, 13 breast cancer, 3 gastric cancer, 3 islet cell tumor, and 6 others) admitted to receive rfa were studied. the detection ability of liver metastasis was compared between ceus and conventional us using enhanced ct as reference standard. the mean numbers of treatment session of rfa were compared between patient treated with ceus assistance and historical controls matched for size and number of tumors. results: the detection rate was 78.6% with conventional us and 96.4% with ceus (p=0.0004). 83 nodules in 25 patients were not detected by conventional us and detected after injection of sonazoid. in addition, 12 nodules in 2 patients were detected not by ct but only by ceus. the mean number of session was 1.5±0.5 as compared to 2.0±1.0 in the historical controls (p<0.001). conclusions: ceus with sonazoid is useful for detection of liver metastasis. sonazoid is an excellent supportive agent in rfa of liver metastasis. background/aims: carcinogenesis of intrahepatic cholangiocarcinoma (icc)-associated liver fluke infection accumulated genetic and epigenetic alterations. cholangiocarcinoma cell line (kku-m213) is adenosquamous carcinoma which rare variants and not commonly found in icc. however, interactions of liver fluke-associated icc proceed to genetic alterations in adenosquamous carcinoma that have been not elucidated. objectives: to analyze the whole genome-wide genetic alterations in kku-m213 using microarray comparative genomic hybridization. methods: dna of kku-m213 and matched-sex reference were differentially labeled with fluorescence dries (cy3 and cy5) and mixed together with cot-1 dna. the mixture was hybridized on array with spotting 2,464 human bacterial artificial chromosomal (bac) clones in triplicate and mapped these directly onto human genome sequence. the genetic alterations were classified the dna copy-number variations according to the intensities of log2 ratio (cy3/cy5) as dna copy-number loss/gain and deletion/amplification. results: the whole genomic alterations in kku-m213, which revealed a variety of chromosomal aberrations with a part and/or entire chromosomal gain and loss. chromosomal amplifications were detected on 4q13.1, 4q21.1, 4q21.2, 5p tel, and 5p15.3, whereas homozygous deletions were detected on 1q23, 1q25, 1q31, 1q32-41, 1q32.2, 1q41, 1q43, 5q15-5q21, 8p22-8p23, 9p24, 10q11.2, 10q11.2-10q2.1, 10q11.2, 10q21.1 and 20q13.3. conclusions: the whole genome-wide genetic alterations were characterized which previously not defined in adenosquamous carcinoma. this recent advance tool is usefulness for discovering novel cancer-related gene (oncogene/tumor suppressor gene) and substitutes in in vivo experiment for functional testing of candidate gene involving liver fluke-associated icc carcinogenesis. artificial chromosomal (bac) clones in triplicate and mapped these directly onto human genome sequence. the genetic alterations were classified the dna copy-number variations according to the intensities of log2 ratio (cy3/cy5) as dna copy-number loss/gain and deletion/amplification. results: the whole genomic alterations in kku-m213, which revealed a variety of chromosomal aberrations with a part and/or entire chromosomal gain and loss. chromosomal amplifications were detected on 4q13. 1, 4q21.1, 4q21.2, 5p tel, and 5p15.3 , whereas homozygous deletions were detected on 1q23, 1q25, 1q31, 1q32-41, 1q32.2, 1q41, 1q43, 5q15-5q21, 8p22-8p23, 9p24, 10q11.2, 10q11.2-10q2.1, 10q11.2, 10q21.1 and 20q13.3 . conclusions: the whole genome-wide genetic alterations were characterized which previously not defined in adenosquamous carcinoma. this recent advance tool is usefulness for discovering novel cancer-related gene (oncogene/tumor suppressor gene) and substitutes in in vivo experiment for functional testing of candidate gene involving liver fluke-associated icc carcinogenesis. acknowledgements: this work was supported by faculty of medicine, kku, thailand (grant no. i51117 background/aims: we studied the clinical efficacy of arterial chemoinfusion therapy through an implanted port system for patients with intrahepatic cholangiocarcinoma (icc). thirty patients with unresectable icc or intrahepatic recurrence of icc after surgery were studied. comparison was made between patients who received arterial chemoinfusion therapy through an implanted port system with adriacin and lecithin-added lipiodol emulsion in 5 patients and 5-fluorouracil (5-fu) in 7 patients. eighteen patients were treated without port system. results: disease was stable in 5 patients with adriacin and lecithin-added lipiodol emulsion and in 3 patients with 5-fu. disease was progressed in 4 patients with 5-fu. the mean survival period was 20.8 months in patients with adriacin and lecithin-added lipiodol emulsion, 9.3 months in patients with 5-fu, and 10.5 months in patients without port system (p=0.02, p=0.04). conclusion: arterial chemoinfusion therapy through an implanted port system is useful for patients with intrahepatic recurrence of icc after surgery. pe038 s. kaur 1 , t. kaur 1 1 department of biophysics, panjab university, chandigarh. india background: a number of dietary factors have been involved in the pathogenesis of cholelithiasis. cholesterol overfeeding is the primary means of inducing supersaturated bile and cholesterol gallstones in animal models.aim of the study was to investigate the rate of epithelial cell death and proliferation in gallbladder during gallstones formation. methods: balb/c mice was divided into two groups control in this group animals were fed normal chow diet, high fat diet group in this group (2% cholesterol,0.5% sodium cholate, 5% butter fat and 15% coconut oil) mixed with chow diet was fed to the mice for 4 weeks. cell apoptosis and proliferation was assayed in gallbladder epithelial cells. histological analysis of gallbladder sections were done with hematoxylin and eosin staning. results: mice fed high fat diet had apoptotic as well as necrotic epithelial cells. rate of proliferation was enhanced after 24 and 48 hrs in mice fed high fat diet group as compared to the control group. the histopathological section of control gallbladder has normal morphology whereas gallbladder wall thickness was markedly increased; epithelial cells appeared more elongated in mice fed high fat diet. conclusion: results obtain show that high fat diet markedly induced biliary epithelial cell proliferation and biliary epithelial cell apoptosis. it has been determined that when there is an injurious stimulus that leads to apoptosis, it is later followed by reparative proliferation and when there is no injurious stimulus, apoptosis occurs late in the course as part of remodeling. background: obstructive jaundice can be caused by malignancy. the treatment can be drainage by biliary stenting. in advanced malignant jaundice, the stent placement is often difficult. objective: to evaluate the success rate of malignant obstructive jaundice evaluation of ercp and success rate of stent placement. methods: retrospective study based on data of ercp from october 2004 until july 2008. results: we evaluated 139 patients who has done ercp examination, 131 (94,2 %) patients have clinical diagnosis of obstructive jaundice. there were 73 (55,7%) male and 58 (44,3%) female, age range 20 -84 (median age was 51). there were no malignancy in 66 (50,4 %) patients; malignancy in 48 (36,6%) patients and 17 (13%) patients need further evaluation.. from 114 patients, 57 (50 %) patients attempted to have stent placement, 50 (43,9 %) patients do not and 7 (6,1 %) patients have no data. we done descriptive study on 57 patients attempted to have stent placement, 32 (56,1 %) patients succeed in stent placement whereas 25 (43,9 %) failed. malignancy was showed to be a factor of stent failure (malignancy: 23 fail and 10 success (30,3 %) vs non malignancy: 2 fail and 22 success (91,7%)). conclusion: ercp can identify the cause of obstructive jaundice in 87 % patients. the success rate of stent placement was 56,1 %. the success rate of biliary stenting in malignant obstructive jaundice was 30,3 % whereas in non-malignant cases was 91,7 %. papillary carcinoma was the most frequent cause of malignant obstructive jaundice. background: in hydatid disease of the liver cystobiliary fisula (cbf) constitutes an entity characterized by the occurrence of a life-threatening cholangitis with increased morbidity. aim: to study the different diagnostic and therapeutic aspects of cystobiliary fistula in hydatid disease of the liver. patients and methods: fourteen patients with complicated cysts were divided into 2 groups; group a: nine patients presented with cholangitis, and group b: five patients had history of jaundice. in all patients, the diagnosis of cbf was confirmed by erc (endoscopic retrograde cholangiography). preoperative endoscopic sphincterotomy (es) was done in group a with retrieval of hydatid daughter cysts. seven patients (subgroup a1) were subsequently submitted to surgery entailing endocystectomy in 5 and hepatic resection in two. the remaining 2 patients in group a (subgroup a2), were managed by endoscopic therapy only. patients of group b (n=5), were not submitted to preoperative es and were subsequently managed by hepatic resection in one patient and endocystectomy in four. results: there was no mortality in the studied group. postoperative bile leak occurred in four cases in group b. in contrast, none of the patients who were submitted to preoperative es (subgroup a1) had bile leak. all patients received albendazole treatment. conclusion: erc is important in confirming the diagnosis of cbf. also, therapeutic erc has a place in the treatment algorithm of cbf as it was found to be a safe and a reliable therapeutic alternative especially in high risk patients for surgery. v. singh 1 , g. singh 1 , g.r. verma 1 , v. gupta 1 , s. ghosh 1 , r. gupta 1 , r. kapoor 1 , n. sharma 1 , a. bhalla 1 , s.k. mahi 1 1 background: endoscopic palliation in malignant hilar biliary obstruction requires ercp. however, contrast injection leads to cholangitis. recently, contrast-free metal stenting with or without mrcp has shown encouraging results. however, mrcp and metal stents are costly. there have been no reports on the use of air cholangiography in these patients. methods: we prospectively studied the role of air cholangiogaphy assisted unilateral plastic stenting in these patients. results: ten patients with unresectable malignant hilar biliary obstruction were studied. air cholangiography detected type ii obstruction in 8 and type i in 2 patients which is similar to mrcp. all patients underwent unilateral plastic stenting. a successful endoscopic drainange was achieved in 100% patients. cholanngitis occurred in none and there was no 30-day mortality. no major complications were observed. conclusion: air cohlangiography assisted plastic stenting in these patients is a safe and effective method of palliation. however, it requires a larger study. introduction: a description of igg4-related sclerosing cholangitis (igg4-sc) without pancreatic lesion has recently been reported. in addition to imaging, diagnosis relies on findings of elevated serum igg4 and immunodetection of invading igg4-positive cells. here we report a case of igg4-sc with only slight common bile duct abnormalities and normal pancreatic findings. case study: a 65-year-old man suffering from cephalalgia, general malaise and muscle ache was admitted to our hospital. his blood examinations on admission revealed eosinophilia, mild anemia, liver dysfunction and an igg level of 2820 mg/dl (igg4 374 mg/dl). although ercp did not reveal typical stenosis or irregularities of the bile duct wall, visualization of peripheral bile ducts was slightly impaired. echography revealed thickening of the intrahepatic bile duct and gallbladder walls as well as adenopathy. due to a gradual increase in pleural effusion and a progression of anemia, oxygenation was begun on the seventh day of illness. based on the combination of eosinophilia, elevated serum igg4 levels, image findings and a negative result for helminth, igg4-sc was suspected. liver biopsy was performed on the ninth day of illness and steroid therapy was initiated, after which symptoms and laboratory findings improved. the igg4-positive plasmocytic infiltrate present around the portal region at the time of biopsy disappeared within eight months of treatment. summary: this case displayed two unusual features that are not generally observed with igg4-sc: complications due to hemolytic anemia, and destruction of the peripheral bile duct with little damage to the common bile duct. introduction: various systemic diseases have been reported to be associated with igg4. although steroids are effective in the treatment of igg4-related diseases, there are some reports on relapses with their treatment, and cases are often difficult to differentiate from malignant diseases. we encountered a case of autoimmune pancreatitis with sclerosing cholangitis (aip-sc), in whom ca19-9 was elevated with episodes of exacerbation and an elevated serum igg4 concentration. igg4 staining was also useful for the diagnosis. case study: an 81-year-old woman noticed tumors beneath the bilateral jaw and was found to have an elevated level of ca19-9 (304) seven years previously. her left submandibular gland was removed and diagnosed as sclerosing sialadenitis. four years previously, she was diagnosed as having diabetes mellitus complicated by a recurrence of ca19-9 (419) elevation and liver dysfunction. cholangiocarcinoma was suspected based on ercp, but was not confirmed by histologic findings of bile duct biopsy. elevated igg4 and other test results established the diagnosis of aip-sc, so steroid therapy was initiated, after which symptoms and laboratory findings improved. this recurrence of ca19-9 elevation (634) was diagnosed as a relapse of aip-sc based on an increased igg4 level and histologic findings. summary: some papers have reported that igg4-positive cells are found in liver tissue in this disease, but such cells were not detected in the liver specimens in our case. this might be because intra-liver sites may have differed in the degree of morbidity, and long-term steroid therapy might have suppressed inflammation in the liver tissue. s. kaur 1 , t. kaur 1 1 department of biophysics, panjab university, chandigarh, india background: cholelithiasis, a gallstone disease is major cause of morbidity affecting millions of people throughout the world. aim of the present study was to investigate the predisposing factors that lead to the formation of gallstones. methods: the study was carried out on gallstones, bile and serum of patients. gallstones and bile were divided into three groups' cholesterol, pigmented and mixed gallstones. blood of the patients was divided into two groups with gallstones and without gallstones patients. trace elements and various biochemical estimations were carried out. clinical history of the gallstones patients was recorded from the hospital records. results: trace elements analysis in bile and gallstones showed that calcium is the main element in all the three types of stones. iron was the main element in mixed gallstones. in pigmented gallstones magnesium and zinc were the major trace elements. liver function tests and lipid peroxidation levels in sera were significantly increase whereas, antioxidant enzymes concentrations in sera were significantly decreased in patients with gallstones. clinical history of the gallstones revealed the cases had jaundice, diabetes mellitus and estrogen replacement therapy respectively. conclusion: results suggest that trace elements in gallstones and bile as well as clinical history of patients with chronic cholelithiasis could be the underlying factor in the pathogenesis of gallstones. the concentration of products derived from the free radicals reactions increases with degree of inflammation. such a condition increases risk of bile saturation which would further contribute to the progress of gallstones formation. background and aims: diseases of the biliary tree and gallbladder are being described with increasing frequency among patients with the acquired immunodeficiency syndrome (aids).therefore there is a need to do a research about the risk factors of gallbladder diseases in hiv/aids patients. so it can be useful to clinicians to predict the possibility of a patient having gallbladder disease and consider the options of further plans. the aim of this study was to find the prevalence and varieties of gallbladder diseases in hiv/aids patients. methods: a cross sectional study was performed in patients with hiv/aids who visited ciptomangunkusumo hospital, jakarta. the risk factors (route of transmision,cd4,arv,hepatitis) and clinical presentations were studied.ultrasonography examinations were performed to detect gallbladder annormalities. results: 68 patients with hiv/aids match the study criteria. there were gallbladder abnormalities in 22 (32.4%) subjects, which 19 (27.9%) had acalculous cholecystitis and 3 (4.4%) had cholecystitis with cholelithiasis. on bivariate analysis, there was a significant association between abdominal pain, jaundice and the use of arv to gallbladder abnormalities (p = 0.000; 0.000; 0.004; 0.012). however, there was no association between age, sex, transmision route of hiv, hepatitis and cd4 to gallbladder abnormalities. conclusion: hiv/aids patients are susceptible to opportunistic gallbladder infection. acalculous cholecystitis is the most frequently encountered gallbladder abnormalities of hiv/aids patients in this study. poster exhibition -hbv poster session, hall 5b long-term stopping therapy t.b. trung 1 , p.h. phiet 1 1 university medical center, hochiminh city, vietnam background: among the approved nucleos(t)ide analogues therapies for chronic hepatitis b, lamivudine was used widely, sometime inappropriate in practice due to high safe and low price but lamivudine is associated with the highest rate of drug resistance. objectives: the aim of the study was to determine the ymdd variants after long-term stopping treatment in lamivudine-resistant patients using more sensitive technique. methods: 16 blood samples from lamivudine resistant patients were collected after long-term stopping therapy. the ymdd variants are detected using technique pcr restriction fragment length polymorphism (pcr-rflp) at hcmc university medical center results: after stopping lamivudine treatment 25 months (6-72 months) ymdd mutants were detected in 14 (87,5%) of 16 patients. among them 13 (92.9%) had the most important m204v/i mutant, 1(7 1%) had accompanying l180m mutant. it means that once drug resistant mutants have been selected, they are archived for the long time even if treatment is stopped. many of patients have the features characterized for the patients in immune tolerance phase (young age, hbeag positive, normal alt). the treatment of this group is not strongly recommended due to low efficacy and high risk of drug resistance. conclusion: the most important m204v/i mutant was still detected with significant portion of the virus population after long-term stopping therapy in lamivudine resistant patients. the options of retreatment for this patients when necessary are limited due to cross-resistance. the management of chronic hepatitis b should be followed strickly the recommendations of specialized association to avoid this problem. background/aim: whether liver stiffness measurement (lsm) using transient elastography is reliable to assess liver fibrosis in the settings of severe acute exacerbation of chronic hepatitis b (chb) is uncertain. methods: we prospectively recruited consecutive patients with severe acute exacerbation of chb (alanine aminotransferase or alt >10x upper limit of normal). the relationship of alt levels and lsm were serially assessed and liver biopsy was performed after alt normalization. results: eleven patients (10 male, median age 43 years) were followed up for 25 weeks; 9 patients received anti-viral therapy. overall, lsm was positively correlated with alt levels (r=0.67, p<0.001). at initial presentation, the median serum alt and lsm was 1136 (581-2210) iu/l and 26.3 (11. 1-33. 3) kpa. a progressive reduction in lsm was observed during subsequent visits in parallel with the reduction of alt levels. even after the normalization of alt at week 12, lsm of 9 patients continued to drop at week 25. at the last visit, the median alt was 27 (11-52) iu/l and lsm was 7.7 (4.7-10.8) kpa. among the 5 patients who had liver biopsy performed at week 25, 4 patients had f2 fibrosis (lsm 5.7-8.1 kpa) and 1 patient had f3 fibrosis (lsm 8.6 kpa). conclusions: lsm using transient elastography may misdiagnose liver cirrhosis in patients suffering from severe acute exacerbation of chronic hepatitis b. lsm should be assessed after normalization of alt levels in order to accurately assess the degree of fibrosis. h.c. lai 1 , s.w. lai 1 , k.f. liao 1 , c.s. liu 1 , t. lin 1 , c.c. lin 1 1 china medical university hospital, taichung, taiwan background: in 2007, chronic liver disease was the seventh leading cause of death in taiwan. hepatitis b and hepatitis c are two major causes of chronic liver disease in taiwan. the purpose was to investigate the seroepidemiology of hepatitis b surface antigen (hbsag) and hepatitis c virus (hcv) antibody in taiwan. method: this was a hospital-based cross-sectional study. we analyzed viral hepatitis data from 2695 subjects who received health checkups at one medical center in taichung from 2003 to 2004. all subjects were divided into three age groups, including 20-39, 40-64 and 65. this study emphasized the prevalence of hbsag and hcv antibody by gender and age. the statistical analysis was performed by t test and 2 . result: there were 1526 men (56.6%) and 1169 women (43.4%). the mean age was 49.2 (standard deviation 12.2, range 20-84). the overall prevalence of hbsag was 14.7%, with statistically significant difference(ssd) between gender (17.4% for men vs 11.2% for women, p <0.001). the prevalence of hbsag was decreased with age in men, with ssd (p <0.001), and also decreased in women, without ssd (p =0.08). the overall prevalence of hcv antibody was 5.2%, without ssd between gender (4.8% for men vs 5.7% for women, p =0.272). the prevalence of hcv antibody was increased with age both in men and in women, with ssd (p <0.001). conclusion: we hope this study can provide the epidemiological data for further studies of hepatitis b and hepatitis c virus infection in taiwan. s.m. wu 1 , x. zhou 2 1 wuhan medical treatment center, 2 center for gene diagnosis, zhongnan hospital, wuhan university, china e-selectin is revealed to facilitate leukocyte adhension to the endothelium and migration into inflamed tissue in inflammatory diseases. chronic hepatitis b virus infection is regarded as a chronic inflammatory process. to examine the possible involvement of e-selectin in the etiology of chronic hbv infection, we analyzed two polymorphisms of e-selectin and determined the plasma souble e-selectin levels in patients with chronic hbv infection and controls. the frequency of c allele of the a561c polymorphism was significantly increased in patients with lc campared with controls. no significant positive association was observed between the g98t polymorphism and chronic hbv infection. but in patients with lc, divided according to the child-pugh classification, the frequency of t allele was of significant difference between child'class a and class b plus c. plasma levels of soluble e-selectin were significantly increased in patients with chronic hepatitis and liver cirrhosiscompared with controls. in the liver cirrhosis group, levels of se-selectin were significantly decreased from child' class a to class c. in each group, patients with c allele of the a561c polymorphism showed higher soluble e-selectin levels than those with a allele. this is the first report describing the association between e-selectin polymorphisms and hbv-related hepatic fibrosis. our data showed the a561c polymorphism of e-selectin gene is associated with disease progression in patients with hbv infection and controls the expression of plasma soluble levels, the g98t polymorphism may be related to fibrotic severity in patients with liver cirhosis. background: chronic hepatitis b (chb) patients with high serum hbv-dna and normal serum alanine aminotransferase (alt) levels might be considered for treatment if histopathological findings show fibrosis stage 2 or more. however, to our knowledge there is no recommendation with regard to the therapeutic agents for this group of patients. objective: this study was aimed to evaluate the efficacy of nucleoside analogues (entecavir or telbivudine) in treating chronic hepatitis b patients with high serum hbv-dna and normal serum alt levels. patients and method: this was an open-label study in chb patients with high level serum hbv-dna levels between january 2007 and october 2008. patients were included if they showed normal serum alanine aminotransferase (alt) level at two measurements within a 3-month interval and had fibrosis stage > 2 on liver biopsy specimens. patients were treated with entecavir 0.5 mg/day or telbivudine 600 mg/day. the primary endpoint was the reduction or undetectable of serum hbv-dna at 24 week and 48 week of treatment, while the secondary endpoint was hepatitis b e antigen (hbeag) seroconversion. results: during a 2-year period, 37 chb patients with high level serum hbv-dna with normal alt two times with 3 months interval underwent a liver biopsy. twenty-eight (75.7%) of 37 pts showed fibrosis stage 2 on histological findings (metavir score). twelve of these 28 patients received nucleoside analogues, 7 (58.3%) of them were men. patients' median age was 42 (range: 24-52) years. there were 5 patients with stage-2, 6 patients with stage-3 and 1 patient with stage-4 fibrosis. eleven (91.7%) patients had genotype b virus. at baseline, the mean serum alt level was 32 + 11.8 u/l and mean hbv-dna level was 2.48 x 10 6 iu/ml, ranging from 1.23 x 10 3 to 2.4 x 10 7 iu/ml. six patients received entecavir and the other six received telbivudine therapy. undetectable hbv-dna was achieved by 9 (75.0%) patients at week-24 and 2 (16.7%) patients at week-48 of treatment. one patient who had the highest hbv-dna level had viral load reduction to 1.6 x 10 4 iu/ml at week-48 of treatment. two out of 5 patients with positive hbeag achieved hbeag seroconversion at week-48 of treatment. conclusion: this preliminary study has shown that nucleoside analogues might be considered in the treatment for chronic hepatitis b patients with high serum hbv-dna and normal serum aminotransferases levels. j. chen 1 , x.j.. wu 1 , y. wang 1 , g.q. wang 1 1 department of infectious diseases, peking university first hospital, beijing, china background: the dysfunction of t cells may represent a mechanism of hepatitis b virus (hbv) persistence. programmed death-1 (pd-1) and its ligands, pd-l1/pd-l2, are new members of cd28/b7 family, as co-stimulatory molecules expressing on t cells and antigen present cells (apcs). their engaging can downregulate the t cells function, including proliferation, cytokines secretion and cytotoxicity. in periphery blood, pd-1 was upreguated on virus specific-t cells, leading to the impairment of t cells. blocking the pd-1/pd-l can improve the function of t cells. methods and patients: 21 patients with chronic hepatitis b (chb) were treated by pegylated ifn -2b (pegintron from schering-plough, once a week, 0.5 or 1 g/kg/weight). the periphery blood were taken at 0 weeks, 4 weeks, 8 weeks, and 12 weeks. periphery blood mononuclear cells (pbmc) were isolated from fresh heparinized blood by ficoll-hypaque (density: 1.077g/l) density gradient centrifugation. then the cells were incubated with apc-conjugated anti-pd-1 antibodies. the pd-1 expression on lymphocytes was detected by flow cytometry (fcm). results: the pd-1 expression on lymphocytes at 0 weeks was 14.47±5.8%, at 4 weeks was 9.68±3.75%, at 8 weeks was 6.95±2.39%, at 12 weeks was 6.08±1.31% (p<0.05). conclusion: treatment with ifn -2b can downregulate the pd-1 expression on lymphocytes and may partially restore the function of t cells. to investigate the effects of nucleoside analogs therapy in hepatitis b related acute-on-chronic liver failure, we treated 55 hbv related acute-on-chronic liver failure patients with entecavir. as control, the remaining 74 were not treated with nucleoside analogues. results show the survival rate of entecavir therapy group has no significantly difference with none-treated group (p>0.05). although entecavir greatly reduced hbv replication during different therapy times (p<0.001), the meld score and liver function (alt, albumin, bilirubin, prothrombin time) had no significant changes (p>0.05). further more, we analyzed the meld score and liver function in different hbv-dna level patients .no significantly difference was observed (p>0.05). there is no significant correlation between hbv-dna level and meld score in different therapy times (p>0.05).the hbv-dna level between patients with over 3 months and less than 3 months survival patients showed no significant difference either (p>0.05). however, meld score and some parameters of liver function (albumin, bilirubin, prothrombin time) showed significant difference (p<0.05). these results suggest hbv-dna loading may not be a direct factor to increased liver injury and suppression of hbv replication may not reduce the severity of liver failure in hbv related acute-on-chronic hepatitis. s. firdoos 1 , u. adeeb 1 , a. mehmood 1 , m. gill 1 1 islamabad specialists clinic, islamabd, pakistan background: before the availability of etv, it was common to use adv for treatment of chronic hepatitis b patients. primary nonresponse and suboptimal response is a common problem with adv treatment. methods: we wanted to study the outcomes of entacavir therapy in this subset of patients. study was conducted between april 2007 to april 2008. we enrolled 30 chb patients who had non response to 12-24 weeks of 10 mg adv therapy. non response and suboptimal response was defined as non dimunition of at least one log of hbvdna from baseline after 12 weeks of therapy and persistence of 3 log10 after 24 weeks of therapy respectively.they were switched to 1mg entacavir before breakfast daily for at least 12 months.they had serial alt cbc and hbvdna measured every 12 weeks. results: out of 30 patients 20 male and 10 were female. only 8 patients were hbeag(+).mean hbvdna level prior to adv exposure was 6.5 log copies/ml.mean duration of exposure to adv was 26 weeks.5 patients lost to f/u.we did intention to treat analysis. 15 out 30 (50%) patient has, undetectable level of hbvdna after 12 weeks of therapy labelled as group 1.6 out of 30 (20%) had hbvdna level reduced by a mean of 3 log 10 copies/ml labelled as group 2.on week 24 treatment analysis all 15 patients from group 1 was hbvdna undetectable, 2 additional patients from group 2 had undetectable hbvdna. conclusion: entacavir therapy results in rapid suppression of hbvdna levels in majority of patients with primary nonresponse or partial non response to adv therapy. background: except for serum alt level, baseline factors predictive of therapeutic response to lamivudine in patients with hbeag-positive chronic hepatitis b remain largely unknown. we thus studied the influence of pre-therapy viral factors on end-of-treatment responses to lamivudine therapy. methods: a total of 116 treatment-naïve hbeag carriers who had pre-therapy serum alt level> 5xuln and received lamivudine for 18 months reimbursed by the national health insurance were prospectively enrolled. hbeag seroclearance and combined hbeag seroclearance, alt normalization as well as undetectable hbv dna at the end of therapy were defined as primary and secondary endpoint, respectively. the pre-therapy viral factors including viral load, genotype, precore stop codon (pc)/ basal core promoter (bcp) status, and pre-s deletion were determined to correlate with therapeutic endpoints. results: the frequency of patients with detectable pc mutation (g1896a), bcp mutation (a1762t/g1764a), and pre-s deletion at baseline was 22.4%, 21.6%, and 12.1%, respectively. after completing 18-month lamivudine therapy, overall hbeag seroclearance rate was 56.0%. patients with hbeag seroclearance had a higher prevalence of baseline pc mutation than those without (30.8% vs, 11.8%, p= .015). by multivariate analysis, the odds ratio of patients with pc mutation to develop hbeag seroclearance was 3.33 (p= .024). in addition, the presence of pc mutation also correlated with the combined response. conclusions: for hbeag-positive chronic hepatitis b patients with serum alt> 5xuln, pc mutation could predict a higher hbeag seroclearance rate at the end of 18-month lamivudine therapy. the efficacy of adefovir dipivoxil against all patterns of lamivudine resistant hepatitis b d.j. kim 1 , y.d. park 1 , y.g. kwon 2 , h.g seo 1 1 daegu fatima hospital, 2 kunngpook national university hospital, daegu, korea background: our aim was to evaluate the efficacy of adefovir dipivoxil (adv) and determine patient-dependent or laboratoroy variables that are predictive of hbeag loss and ivr for hepatitis b patients resistant to lamiduvine. also we evaluated the activity of adv against all patterns of lamivudine-resistant hbv. method: 179 hbv-infected patients with lamivudine resitance received adv for 6 months. quantitative hbv dna, hbeag/anti hbeag, alt was checked every 3-6months. the hbv polymerase of 161 patients were sequenced for baseline samples to determine the presence of lamivudine resistance mutations. result: there is no significant difference in all patterens of hbv mutation about hbv dna reduction at 24w, 48w, 72w. there is no significant difference in all patterens of hbv mutation about alt normalization at 24w, 48w, 72w. conclusion: adefovir dipivoxil demonstrated similar potent anti-hbv efficacy regardless of the different patterns of lamivudine-resistant hbv mutations. g. novelli 1 , m. rossi 1 , v. morabito 1 , f. pugliese 1 , p. berloco 1 1 la sapienza university, rome, italy background: hepatitis b (hbv)-related end-stage liver disease is one of the most common indication for liver transplantation (lt). a number of patients dying while on the waiting list or removed because of being too ill is progressively increasing. we valued the possibility to improve the model end-stage liver disease (meld) of patients awaiting liver transplantation using a albumin dialysis: molecular adsorbent recirculating system (mars). methods: we treated 34 patients (19 male and 15 female) with a mean age 49.5. inclusion criteria: serum bilirubine > 15mg/dl, meld 25, inr > 2.1, encephalopathy grade ii. all patients were treated with mars mean 9±2.5 hr cycles and mean 9 treatments (range 3-15). all patients received standard medical treatment in addition to mars dialysis. the patient survival was valued at six months. results: we obtained a significant change of cytokines levels as interlukine 6 (p< 0.02) and tumor necrosis factor alfa (p<0.01) in association with an improvement of kidney, hepatic and hemodynamic parameters. at the end of mars treatments we observed a significant reduction of meld score (p<0.003). the results of meld show a rebound effect between the end of treatment and the follow up at six months without returning at starting values (p<0.005). twenty patients lived and 14 dead for clinical complications. conclusion: the improved meld score with mars gave patients on lt waiting list more time of survival, thus allowing them more opportunity for liver transplantation. entecavir for treatment of lamivudine-refractory patients chronic hepatitis b h.t. dat 1 , p.t.t. thuy 1 1 medic medical centre, ho chi minh, vietnam lamivudine treatment is associated with frequent development of resistant hepatitis b virus. this incidence especially is higher in longer time of treatment and loss of treatment benefit. entercavir is a new antiviral agent shown its high efficacy even in cases of mutations with lamivudine resistance. in this study, we evaluate the efficacy, the safety of entercavir in treatment of lamivudine-refractory patients chronic hepatitis b. sixty chronic hepatitis b patients with evidence of lamivudine resistance were randomly divided into two groups in proportion of 3:1. group i (n=45) used entecavir 1mg/day, group ii (n=15) used lamivudine 100mg/day. treatment time was 48 weeks. histology, alt, hbvdna were evaluated in the end of the treatment. age, sex, alt, hbvdna, genotype, hbeag were analyzed to evaluate their influences to the treatment. the results have showed hbvdna<2000 copies/ml in entecavir group 37.78% vs. 0% lamivudine group (p<0.01). hbvdna negative in entecavir group was 17.77% and incidence of seroconversion of hbeag was 8.82%. alt was normal in entecavir group 77.77% vs. 26.66% in lamivudine group (p<0.001).histologic improvement in entecavir group was 37.77% vs.6.66% in lamivudine group (p<0.05). patients with hbeag negative, genotype b, low viral load were shown better results. entecavir was shown to be efficacious in treatment for chronic hepatitis b patients experienced with lamivudine resistance. entercavir is safe, with almost no side effects. factors such as hbeag negative, genotype b, low viral load seems to be better in response to treatment. recurrence or mutation of entecavir resistance should be studied further in future. j.m. kim 1 , s.k. hwang 1 , b.h. choe 1 1 department of pediatrics, kyungpook national university hospital, daegu, korea backgrounds: by analyzing the characteristics of children with chronic hepatitis b who have lost hbsag by long-term lamivudine treatment, the selection of target patients could be relevantly predictable in the treatment of chronic hepatitis b in children. methods: a total of 75 hbeag positive children (< 18 y-o) were recruited who have visited kyungpook national university hospital from mar. 30, 1999 to may 8, 2008 . they were treated with lamivudine for at least 6 months. hbeag seroconversion occurred during lamivudine treatment in 49 out of 75 children. they were divided into hbsag clearance and non-clearance group. parameters influencing treatment results were analyzed according to hbsag loss. result: thirteen out of the 49 (26.5%) patients with hbeag seroconversion were classified as hbsag clearance group, while 36 (73.5%) as non-clearance group after lamivudine treatment. twenty five of 49 patients with hbeag seroconversion were under 6 years old, in 10 (10/25, 40%) of whom hbsag loss occurred as well. twenty four of 49 patients were over 6 years old, in 3 (3/24, 12.5%) hbsag loss occurred, that showed significantly difference (p-value= 0.029, or: 4.667, ci: 1.094-19.902) compared to younger group. age was significantly lower in hbsag clearance group (5.1±4.3 years) than non-clearance group (8.2±5.0 years) (p=0.043), but no difference was observed in other parameters. anti-hbs appeared in 12 patients. conclusion: in the treatment of hbeag positive chronic hepatitis b with lamivudine, age was significantly lower in hbsag clearance group than non-clearance group. background: dysfunction of t cells may represent a mechanism of hepatitis b virus (hbv) persistence. programmed death-1 (pd-1) and its ligands, pd-l1/pd-l2, are members of cd28/b7 family, was reported to transfer inhibitory signal, leading to the dysfunction of t cell. background: hepatitis b viral mutants can emerge in patients as a result of selection pressure from either immune response or treatment options. mutations of hbsag allow mutant virus to propagate in the presence of a neutralizing immune response, while wild-type virus in reduced to undetectable levels. methods: immunohistochemical analysis of tissue samples from 56 patients with chronic hepatitis b (chb), 12 acute hepatitis b (ahb) patients and 10 health controls was performed. results: pd-1 was positively expressed on lymphocytes infiltrating the portal area.pd-l1 expression was the same as pd-1,also expressed in interlobular.pd-l2 expressed on kupffer cells and dendritic cells.pd-1-,pd-l1-,and pd-l2-positive cells express index of chb patients were much more than that of health controls and ahb patients(p 0.05).between groups in chb,the expression rate increase with the disease progression (p 0.05). methods: 58 chronic hepatitis b patients with both positive for hbsag and hbsab were studied.serological markers of hbv were detected by elisa and microparticle enzyme immunoassay. hbv dna levels were determined by fluorescent quantitative pcr, s gene fragments were directly sequenced, liver function was analyzed by automatic biochemistry analyzer au400. correlation test was conducted to evaluate their dependablity. conclusion: overexpression of pd-1 and pd-l within liver might be involved in inhibiting the immune response and be a mechanism of chronicity in hbv infection. results: the level of hbsag and hbsab was 254.4±68.3 s/n and 39.4±38.1 miu, respectively. hbv dna was detectable in 46 patients. fifty-one mutations of s gene were detected in 38 patients, and the relating amino acid substitution was at the sites of 36, 39, 47, 63, 77, 89, 90, 115, 126, 129, 139 and 154. eight (15.7%) out of 51 mutations were located at the "a" determinant region in 14 patients, while no mutation was found at the sites of 124, 137 and 147. however, the mutation did not affect hbv replication. hbv dna was positive correlated with hbeag. conclusions: change in hbsag antigenicity due to s gene resulted in concurrent hbsag and hbsab. the existence of hbsab did not affect hbv replication. the damage of liver failure in those patients was slight. background: hbv infection is common in bangladesh. we often encounter young patients incidentally detected with hbeag negative chronic hepatitis b (chb) in our clinical practice. however the characteristics of these patients is yet to be studied in this country. the aim of this study was to study the characteristics of young bangladeshis incidentally detected with hbeag negative chb. methods: we did percutaneous liver biopsies of 36 chb patients aged between 8 to 20 years. they were all hbeag negative with persistently normal or raised serum alt values. we did pre-core mutation (pcm) study in 4 patients who were randomly selected. results: 56% patients had significant necro-inflammation (hai-ni >3), while significant fibrosis (hai-f >2) was seen in 17.6%. serum alt (cut off 42 u/l) was raised in 38.2%, while high hbv dna load (>10 5 copies/ml) was observed only in 26.5%. pcm was negative in all 4. conclusion: although chb patients between 10-20 years of age are supposed to be in immune clearance phase, which is characterized by low hbv dna and hbeag positivity, the study shows that hbeag negative chb is an entity that can also be seen in this age group and a significant percentage of such patients may have considerable hepatic involvement. this challenges our current concept about immune clearance state of hbv infection, although much larger study is needed to draw any specific conclusion. background: hbv infection is common in bangladesh, but characteristics of young patients incidentally detected with chronic hepatitis b is yet to be studied in this country. methods: we did percutaneous liver biopsies of 88 chb patients aged between 8 to 20 years. results: significant necro-inflammation (hai-ni >3) was seen in 79.6% patients with hbeag positive and 56% patients with hbeag negative chb, while significant fibrosis (hai-f >2) was seen in 20.3% and 17.6% patients in these two groups respectively. serum alt (cut off 42 u/l) was raised in 37% hbeag positive and 38.2% hbeag negative patients, while in these two groups 87% and 26.5% patients respectively had high hbv dna load (>10 5 copies/ml). conclusion: hbeag negative chb is an entity that can also be seen in young population. a significant percentage of both hbeag positive and negative patients may have considerable hepatic involvement. profile of hbeag +ve chronic hbv infection in bangladesh m. mahtab 1 , s. rahman 1 , f. akbar 2 , f. karim 1 , a. shrestha 1 , m. khan 1 , m. kamal 1 1 bangabandhu sheikh mujib medical university, 2 toshiba general hospital, dhaka, bangladesh background: inactive hbv carriers constitute the major reservoir of hbv. present management guidelines provide inadequate treatment modalities. they are recommended for regular check-up; treatment is only recommended when patients exhibit evidence of liver damage. this is due to lack of information about their extent of liver damage. aim of this study was to assess extent of liver damage in hbeag +ve patients, unaware of their infection. methods: in this retrospective study, records of 206 hbeag +ve chb patients from our pool of 561 chb patients were reviewed. they were tested for hbsag, hbeag, hbv dna, anti-hcv and serum alt. all underwent per-cutaneous liver biopsy. results: 78.2% (161/206) patients were males and 21.8% (45/206) females. they were between 8-45 years of age. alt was raised >2times unl in 17% (35/206). 92. 2% (190/206) patients had high hbv dna (>10 5 copies/ml), while low hbv dna (<10 5 copies/ml) was seen in 7. 8% (16/206) . in high hbv dna group, significant necro-inflemmation (hai-ni >7) was seen in 48. 9% (93/190) and significant fibrosis (hai-ni >3) in 24. 7% (47/190) . figures were 37.5% (6/16) and 31.3% (5/16) respectively in low viral load group. none tested positive for hcv infection. conclusion: study indicates that machinery should be developed to characterize undetected hbv carriers in developing countries by conducting multi-center clinical studies. we have shown that considerable number of patients, unaware of their hbv infection, suffer from progressive liver damage. the overall strategy of management of chronic hbv infection should also be revisited. high viral load does not necessarily represent significant liver damage in patients with chronic hbv infection in bangladesh m. mahtab 1 , s. rahman 1 , f. akbar 2 , f. karim 1 , a. shrestha 1 , m. khan 1 , m. kamal 1 1 bangabandhu sheikh mujib medical university, 2 toshiba general hospital, dhaka, bangladesh background: in general, it is assumed that patients with chronic hepatitis b virus (hbv) infection with high viral load exhibit increased liver damages. treatment guidelines also emphasize on reducing viral load. these observations were mainly accumulated from developed countries. >80% chronic hbv carriers live in the developing nations, but little is known about relationship between hbv viral load and extent of liver damage in these countries. in this study, we addressed this issue. methods: in this retrospective study we reviewed records of 306 chb patients from our pool of 561 patients. all had high hbv dna (>10 5 copies/ml). 62. 1% (190/306) were hbeag +ve and 37.9% (116/306) hbeag -ve. they were alsotested for anti-hcv and serum alt. all underwent per-cutaneous liver biopsy. results: 51.1% (97/190 ) hbeag +ve patients with high hbv dna had non-significant hepatic necro-inflammation (hai-ni <7); this figure was 53.4% (62/116) in hbeag -ve patients. non-significant hepatic fibrosis (hai-f <3) was observed in 75. 2% (143/190) and 69.8% (81/116) in hbeag +ve and -ve patients respectively. none tested positive for hcv. conclusion: correlation doew not exist between viral load and liver damage in chb in bangladesh. many with both hbeag +ve and -ve chb with high hbv dna do not have significant hepatic necro-inflammation and fibrosis. further study may be needed to find out influence of other factors on liver damages in chb in bangladesh. most of these patients have not been characterized and treatment modalities have not been defined for them. background/aims: expression of intrahepatic hepatitis b core antigen (hbcag) is related to the immunopathogenesis of hepatitis b virus (hbv) infection. the role of hbv genotype and basal core promoter (bcp) mutation in expression of hbcag was investigated. methods: seventy hbeag-positive chronic hepatitis patients (genotype b in 52 and c in 18; bcp t1762/a1764 mutation in 16) were enrolled. clinical, virologic and histologic features were compared with regard to localization and expression of intrahepatic hbcag. the effects of hbv genotype and bcp t1762/a1764 mutation on the expression of hbcag were further evaluated by in vitro assays. results: cytoplasmic, mixed cytoplasmic/nuclear, and nuclear localization of intrahepatic hbcag were found in 38 (56.7%), 25 (37.3%) and 4 (6.0%), respectively. fifty-eight (80.6%) of these patients expressed a high level of hbcag. in multivariate analysis, cytoplasmic localization of hbcag correlated only with low serum viral load (p=0.045) and bcp mutation (p=0.04). high expression level of hbcag also correlated with high serum viral load (p=0.015) and bcp wild-type sequence (p=0.037). in vitro assays supported that hbv bcp mutant had lower subcellular expression of hbcag compared with bcp wild-type strain. conclusions: hbv bcp mutation and viral load but not genotype contributes to the expression of intrahepatic hbcag. hepatitis b virus (hbv) genotypes show distinct geographical distributions and virological and clinical differences. in some of genotypes, specific substitutions and mutations have been described in association with hepatitis b e (hbe) protein expression and viral replication. in this study, genetic characteristics of hbv genotype e (hbv/e) were investigated using clinical samples obtained from 12 hepatitis b e antigen (hbeag)-positive, and 11 anti-hbe-positive asymptomatic carriers (ascs) in west-africa. full-genome analysis of isolated hbv strains revealed strong association between precore (pc) mutation and hbeag to anti-hbe seroconversion. furthermore, using 53 partial genome sequences, correlation among hbeag/anti-hbe status, viral load and key mutations were analyzed. the data showed that pc mutation is associated with hbeag seroconversion and enhanced viral replication efficiency. comparison between hbv/e and hbv/d strains reveals these two genotypes to have an identical sequence in their core-promoter-upstream and basic core promoter (curs/bcp) regions. it has been known from the previous phylogenetic studies, that hbv/d and hbv/e cluster together in trees reconstructed on x and precore/core orfs. in addition, this study, demonstrates that in spite of the high sequence similarity of curs/bcp region, the seroconversion-related mutation patterns are different between hbv/e and hbv/d in asc. further studies are needed to clarify the clinical significance of the regulatory sequence similarity between hbv/e and hbv/d. necro-inflammation and fibrosis p. siddappa 1 , p. kar 1 , b. das 2 , r. gondal 1 , m. asim 1 1 maulana azad medical college, 2 icpo, new delhi, india background: chronic hepatitis b(chb) is an important cause of morbidity and mortality. methods: pilot study involving 30 patients of chb, were equally randomized to receive either adefovir or lamivudine for 6 months. quantification of serum and hepatic hbv dna levels by real time pcr and liver biopsy done at start and end of 6 months. results: after 6 months there was significant and comparable reduction in serum and hepatic hbv dna viral load and liver biopsy showed significant reductions in hai scores in both the groups. serum alt which was elevated to 2 or more times normalized in both the groups. in the adefovir group 2 patients became hbeag negative and 2 patients who were hbeag negative at the start of therapy remained so. in the lamivudine group one patient became hbeac negative and 2 patients who were negative at the start of therapy remained so. in the adefovir group 4 patients became hbv dna (qualitative test) and in the lamivudine group 2 patients became hbv dna negative. there was strong correlation between serum and hepatic hbv dna levels both before and after the completion of therapy. conclusion: both the drugs bring about biochemical, histological and serological improvement with significant reduction in viral load in serum liver after 6 months without complete clearance of virus. there was not enough evidence to show therapeutic advantage of one drug over the other. the serum and hepatic hbv dna levels correlate well with eachother before and after treatment. aim: assessing efficacy and safety of treatment of chronic hepatitis b in children with pegylated ifn. materials and methods: 13 children (9 boys and 4 girls) aged 11-17 years with chb treated with peg-ifn alfa-2a, 100 g/m 2 /week during 48 weeks, 5 hbeag-positive and 8 hbeag-negative children, 4 previously treated with recombinant interferon. no child had liver disease greater than grade 2, stage 2. serum hbv dna was quantified at baseline, tw 4, ("rvr") tw 24, tw 48 (etr) and w 72 (svr) with rt pcr method (roche taqman). alt activity, haematology and adverse events were monitored. results: after 4 weeks treatment median hbv dna level decreased from 7.8x10 3 iu/ml at baseline to 1.43x10 2 iu/ml (p<0.01). "rvr" -undetectable hbv dna at tw4 was observed in 6/13 children and associated with lower pretreatment alt levels <25 iu and pretreatment viral load <750 iu/ml. all children with "rvr" were hbeag-negative pretreatment. at tw 24 and tw 48 seven children including all with "rvr" had undetectable hbv dna. 5 children achieved svr (undetectable serum hbv dna in w 72), among them 3 with "rvr". in 2/6 children with "rvr" hbsag disappearance was observed since tw 48. leukopenia was reported in 7 children, thrombocytopenia in 3. no adverse events were observed following dose modifications. conclusions: 1. peg-ifnalfa-2a is a good therapeutic option for children with chb, in particular with hbeag-negative chb 2. low pretreatment viral load and "rvr" seem to be predictive factors of efficient therapy. control by investigating the sanitizing modes among appliances used in the public service places (psp) and hbsag among appliances and practitioners worked in those places. methods: 63 beauty parlors, barber shops and bathing centers selected by stratified randomization sampling, 682 workers were investigated in questionnaire. the hbsag in appliances of psp and employee was detected by ria. results: the rate of hbsag among appliances of psp was 2.13%. the rate of hbsag in large-, medium-and small-sized appliances was 0.63%, 2.67% and 3.70%. the rate of hbsag has different( 2=6.68 p 0.05). the rate of hbsag among appliances of beauty parlors, barbering shops and footbath inns was 2.97%, 0.61% and 3.42%. different appliances had different rate of hbsag, such as the rate of acne needle and the forceps was 5.13% and 4.17%. the positive of hbsag amongworkers in psp was 7.13%. the rate of hbsag among workers in large-, medium-and small-sized psp was 7.34%, 8.33% and 2.94%. the rate of hbsag among workers in beauty parlors, barbering shops, footbath inns and bathing centers was 9.01%, 6.37%, 4.35% and 7.29%. the hbsag rate among workers was different in different works, the rate was higher in tattoo workers (13.33%), pedicures workers (12.68%), massagists (8.03%). conclusions: it is important to enhance the sanitizing management in psp and improve workers kap) of hepb. and we should promote health education to enhance the knowledge of hepatitis b control and build up supervision consciousness. background: integration of hepatitis b virus (hbv) dna into host chromosomes is often found in chronic liver disease and hepatocellular carcinoma, which is likely an early event of hbv-related carcinogenesis. however, the molecular mechanism of integration remains unclear. here we describe a potential mechanism of hbv integration and identify that ku70 and ku80, the gatekeepers of non-homologous end-joining (nhej) repair pathway, can serve as targets for anti-hepatitis virus integration. methods: using i-sce endonuclease-based system, we induced a dna double-strand break (dsb) in human hepatoma cell line huh-7. the cells were then incubated with serum from patients with chronic hbv infection. pcr amplification and direct sequencing were used to detect the inserted sequence in the site of dsb. finally, we employed taqman-based real-time pcr assay to quantify the integrated hbv dna and evaluate the effects of shrna on hbv integration. result: when huh-7 were exposed to viral serum and incubated for several days, hbv dna was detected in integrated form at the exact site of dna damage. furthermore, small interference rna (sirna) targeted against gatekeeper genes for nhej can down-regulate nhej repair and even the frequency of hbv integration. conclusion: thus, this project provided us with the first direct evidence that dna double-strand breaks are potential targets for hbv integration. the study has also shown that shrnas targeted against gatekeeper genes for nhej can regulate the frequency of hbv integration. objective: to screen proteins of human pancreas cdna library interacting with hbsag protein. methods: the library was amplifed, purified and evaluated, and then the puried library plasmids were transformed into yeast strain y187. the reconstructed plasmid pgbkt7-hbsag was transformed into yeast strain ah109 and screened on the nutrient deficiency medium sd/-trp. the transformed ah109 mated with y187 containing the library plasmid. the diploid yeast cells were plated on nutrient deficiency medium sd/-trp/-leu/-his/-ade and sd/-trp/-leu/-his/-ade containing x--gal for selecting. the plasmids in diploid yeast cells were extracted and electrotransformed into e.coli dh5 . the plasmids in dh5 were extracted, sequenced and analyzed by bioinformatic methods. results: sixteen proteins interacting with hbsag were founded. conclusions: these results show that hbsag protein may be related with metabolism of glucose and lipid. comparison of the sensitivity and specificity of the elecsys ® hbsag ii assay with other available assays in china for detection of hbsag j.d. jia 1 , l. wei 2 , x.x. zhang 3 , y.l. mao 4 , l.l. wang 5 , z.l. gao 6 , j.l. hou 7 , j. zhang 8 , w. melchior 9 , w. van der helm 9, 10 1 beijing friendship hospital, beijing, china, 2 beijing people hospital, beijing, china, 3 ruijin hospital, shanghai, china, 4 beijing 302 hospital, beijing, china, 5 west china hospital, chengdu, china, guangzhou, china, 7 guangzhou nanfang hospital, guangzhou, china, 8 shanghai public health clinical centre, china, 9 roche diagnostics ltd, rotkreuz, switzerland, 10 conclusions: in this patient population the prevalence of hbsag positive and anti-hcv were much higher than reported in community studies. genotypes 1 and 6 accounted for most of hcv. these very high rates of viral hepatitis in a hospital setting challenge to healthcare providers in terms of patient management as well as caregiver's prevention. hepatitis b is one of the major diseases of mankind that kills about one million persons each year in the world. accoring to primary study about 3% of iranian population is chronic hbv carriers. among iranian cirrhotics, 70-84% has evidence of exposure to hbv and 51-56% is carriers. because increase demand of blood transfusion, high blood dependent patients and long term window period of hbv infection, any controlling hbv infection program in blood donors can enhance the blood safety and public health. pe078 in this descriptive study included all the blood donors that referred to dezful blood transfusion center during 2005-2008. all the blood donors screened for hbs ag by using enzyme immuno assay and repeatedly reactive (r.r) samples confirmed by hbc-ab or confirmatory (neutralization) tests. the data analyzed by using spss 11.5. we found that in the first year 1.052 % were repeatedly reactive and 1.045 % confirmed. the results for other years as the followed: 0.782 %(r.r) and 0.770 % confirmed and in the last 0.725 % (r.r) and 0.700 % confirmed. the repeated blood donors increase in this period (42.11 %, 50.15 % and 51.68 % respectively). aim: we aimed to evaluate the cost-effectiveness of telbivudine versus entecavir with reference to lamivudine by roadmap model. methods: decision analysis model was used to study the incremental cost-effectiveness ratios (icer), i.e. the additional cost (in usd) required to achieve undetectable hbv dna or hbeag seroconversion for a patient at 2 years in america and hong kong. entecavir was used as a continuous monotherapy. lamivudine and telbivudine would be shifted to entecavir if hbv dna was detectable at month 6 and continued otherwise with drug resistance treated by add-on adefovir. weighted event rates based on previous reports were estimated for analysis. according our study, although the prevalence was higher than other region in our province, the hbv prevalence showed good decrease after stablishment strategies such as of repeated blood donor recruitment , improvement the donor selection and other educational programs . good following up those strategies to enhance the blood safety recommended. results: telbivudine was generally cheaper than entecavir to achieve an incremental case of undetectable hbv dna from lamivudine at 2 years. entecavir was least effective and most costly for hbeag seroconversion. conclusions: telbivudine is a cost-effective alternative to entecavir particularly when its cost is low in hong kong. h. tang 1 , g.l. zhang 1 , y.x. li 1 , r.q. tian 1 , m. liu 1 , x. li 1 1 tianjin life science research center, tianjin medical university, tianjin 300070, china micrornas (mirnas) are single-stranded noncoding rnas of 18 to 25 nucleotides that play critical roles in a wide spectrum of biological processes. we investigated whether the mirnas-silencing machinery influences hbv replication or antigen expression. on the basis of elisa and mtt, the effect of 328 mirnas on the hbsag expression and cell proliferation was examined. three micrornas efficiently inhibited hbsag expression without significant effect on the proliferation of hepg2 2.2.15 cells compared to lacz control. subsequently, bioinformatics analysis were used to predict targets for the three mirnas, and the prediction results were conformed by cdna microarray analysis. the target region in hbv genome and the 3'utr region of one cellular gene were identified by fluorescent reporter assay, semi-quantitative rt-pcr and western blot. the results demontrated that mirna may play an important role in replication and gene expression of hbv. hepatitis b virus (hbv) infection is a global public health problem, which plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis and hepatocellular carcinoma. although considerable progress has been made, the pathogenesis of hbv infection is still elusive. there's an urgent need to elucidate the mechanisms of hbv-host interactions, to discover novel biomarkers for diagnosis and prognosis and to develop therapeutic targets for anti-hbv treatment. herein, we applied a two-dimensional gel electrophoresis and maldi-tof/ms based comparative proteomics approach to globally analyze the host response to hbv by using an inducible hbv-producing cell line hepad38. of the 23 differentially expressed proteins identified, glucose regulated protein 78 (grp78) was one of the most striking proteins elevated by hbv replication, which was confirmed by real-time pcr and western blotting. knockdown of grp78 expression by rna interference resulted in a significant increase of both intracellular and extracellular hbv virions in hbv-transfected hepg2 cells. reversely, grp78 overexpression led to hbv suppression. the expression levels of hepatitis b surface antigen (hbsag) and hepatitis b e antigen (hbeag) were determined by enzyme linked immunosorbent assay (elisa). immunofluoresce further revealed a positive correlation between the expression levels of grp78 and hbsag in both hbv-transfected hepg2 cells and hbv-infected human liver tissues. altogether, these data demonstrate for the first time that grp78 is an endogenous anti-viral factor in hbv-transfected hepg2 cells and may serve as a potential prognostic indicator of viral status in anti-hbv therapies. background/aims: to evaluate the predictors of response to long-term treatment of adefovir dipivoxil (adv) in patients with emerging lamivudine (lam)-resistant hepatitis b e antigen (hbeag)-positive chronic hepatitis b (chb) patients. methods: one-hundred-thirty-four lam-resistant hbeag-positive chb patients were treated with adv for a median of 28.0 months (range, 18 -54 months), following lam therapy for a median of 25.5 months (range, 5 -66 months). 65 patients (48.5%) were switched from lam to adv monotherapy, 43 (32.1%) were switched to adv with 1 month of lam overlap therapy, and 26 (19.4%) were switched to adv with 3 months of lam overlap therapy. the influence of baseline parameters on treatment response to adv in patients with lam-resistant hbeag positive chb was analyzed. result: during the follow-up period, 28 (20.9%) of 134 patients achieved complete response, defined as normalization of alt level, negative hbv dna by a digene hybrid capture assay and achievement of hbeag loss. sixteen (11.9%) patients achieved hbeag seroconversion. twenty-eight (20.9%) patients developed adv-related mutations during adv treatment. in multivariate analysis, virological response at 3 months (or=9.70, 95% ci: 32.63-35.78, p=0.001), defined as serum hbv dna levels less than 5 log10 copies/ml or a reduction in serum hbv dna levels greater than 2 log10 copies/ml after 3 months of adv therapy, independently predicted complete response. conclusions: virological response at 3 months was the strongest predictor of adv response in lam-resistant hbeag-positive chb patients. background/aims: to explore the effects of hbv dna level hbv genotype/subgenotype on the pathogenesis of severe liver diseases in chongqing. methods hbv dna level was analyzed in patients with severe liver diseases in retrospect,and hbv genotype/subgenotype hbv dna level and hbeag were determined in patients with hepatocellular carcinoma (hcc,n=78 ), liver cirrhosis(lc, n=58),chronic hepatitis b(chb, n=20) and acute on chronic liver failure(aclf, n=42). results hbv level from high to low with chb were lc, aclf and hcc in turn(p 0.05). hbv genotype was mainly genotype b.the rate of genotype b and c were 51. 3% and 47.4 respectively in hcc patients, 60.3% and 39.7 in lc patients, 55% and 40 in chb patients, 55% and 40 in aclf patients. the percentage of genotype b/c in aclf patients was higher in compared with other groups. but the distribution of hbv genotype among groups was not statistically different(p 0.05).subgenotypes of genotype b were almost ba but one. subgenotypes of genotype c were mainly ce in chongqing area, and there was no statistical difference among the 4 groups (p 0.05). conclusion: hbv dna level seems not to be a determining factor at end point of severe liver disease. both genotype b and c of hbv can lead to severe liver diseases, and there are more mixed infections by different genotypes in aclf. the efficacy of switching to entecavir (etv) monotherapy in japanese lamivudine (lvd)-experienced patients. background: this study aims to determine the efficacy of switching to 0.5mg etv daily in chronic hepatitis b (chb) patients previously treated with lvd. method: retrospective analysis of chb patients (n=134) previously on 100mg lvd daily and switched to 0.5mg etv daily. results: lvd-experienced patients were divided into three groups based on hbv viral load at time of switching to etv (<2.6 log10 copies/ml; 2.6-5.0 log10 copies/ml and >5.0 log10 copies/ml). detection of lvd-resistant virus at the time of switching was higher in the group with hbv dna 2.6 log10 copies/ml (76% in both 2.6-5.0 and >5.0 log10 copies/ml groups versus 23% in <2.6log10 copies/ml group) and was higher in patients treated with lvd for 3 years (52% versus 24% for patients on <1 year of lvd). a year after switching to 0.5mg etv daily, hbv dna undetectable rates were 100% (42/42), 94% (17/18) and 43% (6/14) for <2.6, 2.6-5.0 and >5.0 log10 copies/ml groups, respectively. alt normalization occurred in more than 90% patients at the end of the first year of switching to etv for all three patient groups. only one patient in the 2.6-5.0 log10 copies/ml group, who had lvd-resistant mutants at the time of switching, developed etv resistance during follow-up. conclusion: switching from lvd to etv maintains or improves viral suppression and alt normalization, especially in patients with viral load <5.0 log10 copies/ml. background/aims: we investigated the association between on-treatment hbsag decline and sustained response in patients treated with pegasys±lamivudine. methods: hbsag levels were measured retrospectively pre-treatment and at weeks 12, 24, 48 and 72 using the abbott architect hbsag assay in sera from 542 patients (83% asian) treated with pegasys alone (180 g qw; n=271) or combined with lamivudine (100mg qd; n=271) alone for 48 weeks as part of a large multinational trial. response was measured 6 months post treatment. results: more patients treated with combination therapy had >1 log decline in hbsag from baseline to week 48 ( figure) . hbsag level <1500 iu/ml at week 12 was associated with higher rates of response to pegasys±lamivudine 6 months post treatment ( figure) . data comparing hbsag and hbv dna as on-treatment predictors of response will be presented. conclusion: on-treatment hbsag monitoring may be useful for predicting response in patients treated with pegasys. y. wakui 1 , j. inoue 1 , y. ueno 1 , t. shimosegawa 1 1 division of gastroenterology, tohoku university graduate school of medicine, sendai, japan background/aim: chronic hepatitis b patients are clinically treated with nucleot(s)ide analogues and ifn-. nucleot(s)ide analogues have problems including drug resistance in continuous treatment, and ifn-has disadvantages of limited effectiveness and side effects. therefore, novel antiviral drugs are still needed. in this study, the suppressive effect to the replication of hbv was examined in vitro by using bezafibrate and rosiglitazone, which are ligands of peroxisome proliferator activated receptor (ppar) and , respectively. methods: the cytotoxicity of bezafibrate and rosiglitazone to hepg2 cells was examined with mts assay, and the concentration of 50% cytotoxicity (cc50) was calculated. hepg2 cells were transiently transfected with the plasmid containing 1.3-fold hbv genome of a genotype b strain. after 24 hours of transfection, rosiglitazone and bezafibrate was added to the cells. using the medium at day 3 after the addition of drugs, hbv dna was quantified with real-time pcr. results: the cc50 of bezafibrate and rosiglitazone in hepg2 cells were 250 m and 150 m, respectively. the amount of hbv-dna in the medium was decreased when the density of bezafibrate was over 200 m, but the density demonstrated considerable cytotoxicity. in contrast, rosiglitazone of 5 m, which showed no cytotoxicit, decreased the amount of hbv dna. the 50% effective concentration (ec50) was calculated to be 9.8 m. conclusions: in this study, it was suggested that the replication of hbv was inhibited by rosiglitazone of the density without cytotoxicity. the mechanism is uncertain and being investigated now. q. zheng 1 1 center for liver diseases, the first affiliated hospital, fujian medical university, fuzhou, p. r. china background: the objective of this study was to evaluate the early virologic response for prediction of achievement of hbeag seroconversion and hepatitis b virus (hbv) dna negativity after two years of lamivudine treatment in chronic hepatitis b (chb) patients. methods: in this retrospective study, adult patients with chronic hepatitis b (255 hbeag-positive and 122 hbeag-negative) were treated with lamivudine (100 mg/day), and followed-up up to 72 months. response and resistance to the treatment were assessed during the treatments with lamivudine. results: it was found that gender, age, baseline levels of alt and hbv dna, serum hbv dna at week 24 (p =0.019 , or = 0.442) were closely related to the achievement of hbeag seroconversion, undetectable hbv dna level and emergence of drug resistance after 2 years of lamivudine treatment. hbeag positive patients with baseline serum hbv dna in 10 6 -10 8 copies/ml and serum hbv dna 10 3 copies/ml at week 24 showed high response rate of alt normalization rate (91.7%), undetectable hbv dna rate (84.5%), hbeag seroconversion rate (55.2%), as well as low drug resistance rate (25.4%) after 2 years of treatment. similarly, hbeag negative patients with serum hbv dna 10 3 copies/ml at week 24 could achieve high 2-year response rate of alt normalization rate (72.41%), undetectable hbv dna rate (82.3%), and low drug resistance rate (15.5%). conclusion: serum hbv dna 10 3 copies/ml at 24-week provide the best prediction of 2-year lamivudine treatment response. background/aims: unlike oral antivirals, a finite course of (peg)interferon can induce sustained post-treatment response in patients with chronic hepatitis b (chb), with increasing rates of hbsag clearance observed in patients who respond during post-treatment follow-up. hbsag clearance is considered to be the closest outcome to a cure, being associated with improved histological outcome, reduced incidence of hcc and increased survival. methods: in a randomised multinational study, patients (hbeag-negative) received 180µg pegasys+placebo (n=177); 180µg pegasys+100mg lamivudine (n=179); or 100mg lamivudine (n=181) for 48 weeks, and were assessed 6 months post-treatment. from this initial study, 230 of those who had received pegasys±lamivudine and 85 patients who had received lamivudine monotherapy participated in a long-term observational study to investigate post-treatment response. hbsag clearance at yearly post-treatment follow-up visits up to 5 years post-treatment was analysed. results: hbsag clearance in patients treated with pegasys±lamivudine increased post-treatment (3% at 1 year to 6%, 8%, 11% and 12.2% at years 2, 3, 4 and 5). at year 5, 28 pegasys-treated patients (12.2%) had cleared hbsag compared with 3 (3.5%) of lamivudine-treated patients (p=0.022). 16/28 pegasys-treated patients had anti-hbsag (hbsag seroconversion). detailed analysis of the 5-year follow-up data will be presented. conclusion: the ability of a finite course of pegasys to induce sustained response with increasing hbsag clearance rates in responders during post-treatment follow-up supports its use as first-line therapy in hbeag-negative patients with chb. background/aims: recent studies suggest that quantification of hbsag levels early during treatment can be used to predict post-treatment response to pegasys. elecsys ® hbsag ii (roche) is a sensitive assay for the detection of hbsag. this assay can be used for the quantification of hbsag levels using a simple dilution algorithm. we compared results obtained using the elecsys ® hbsag ii method with those of a commonly used quantification assay. methods: hbsag levels obtained using the elecsys ® hbsag ii assay were compared with those obtained using the abbott architect ® hbsag assay for a total of 40 samples from patients infected with hbv genotypes a (n=8), c (n=1), d (n=29) and f (n=2). samples were diluted 1:100 in diluent provided by the manufacturer. samples with hbsag levels >250 iu/ml were retested at a final dilution of 1:1000. samples with hbsag levels <0.05 iu/ml were retested undiluted. results: overall, hbsag levels measured with the two assays correlated well (r 2 =0.9718) over a wide range (3-3x10 5 iu/ml). discrepancies in hbsag levels >±20% were reported for a minority of the samples (n=15), mainly distributed evenly above and below the ideal line (n=11). in the four low titre (range 3-9x10 3 iu/ml) samples with greatest discrepancy elecsys ® underestimated values (in two cases by >50%). conclusion: the elecsys ® hbsag ii assay provides a simple and reliable means for determining hbsag levels. this simple assay format could be used to provide useful information during on-treatment monitoring of hbsag levels in patients with chronic hepatitis b undergoing therapy. conclusions: hbv/a has been increasing in chb patients in japan as the consequence of ahb, spreading in the younger generation through promiscuous sexual contacts, thrust by an inclination of hbv/a to induce chronic hepatitis. the spread of hbv/a infection in japan should be prevented by universal vaccination programs. introduction: chronic viral hepatitis is common in end-stage renal disease (esrd), from endemic hepatitis b (chb) and nosocomial hepatitis c (chc). reduced outcomes post-renal transplant were reported thus chb and chc cirrhosis became contraindications to listing. however, these predated effective anti-viral therapies. we reviewed outcomes of patients with chronic viral hepatitis following assessment for renal transplantation. methods: prospective database of esrd patients with viral hepatitis referred for renal transplantation was reviewed. results: 110 patients were assessed. 23 patients underwent kidney transplantation. two were cirrhotic and had liver/kidney transplantation; both died within 6 months. 21 were non-cirrhotics, of whom 20 are alive. 14/20 have functioning allografts; predictors were normal alt and low viral load. of the 87 non-transplanted, 31 had cirrhosis; 17/31 received anti-virals. mortality was 35% -7 liver-related (3 hepatoma,1 bacterial peritonitis,3 sepsis -5 inactive cirrhosis); 4 non-liver related (2 cerebral,1 haemorrhage,1 renal -3 inactive cirrhosis). 12/20 surviving cirrhotics received anti-virals. in non-transplanted non-cirrhotics, mortality was 18%; 80% of survivors had inactive disease. 23 chb patients received lamivudine; 11 adefovir (lamivudine resistance). 11 chc patients received ifn-based therapy. conclusion: excellent outcomes are achieved in esrd patients with chb/chc post-renal transplant, in absence of cirrhosis. normal alt/non-detectable viral load can predict graft function. however, cirrhosis is associated with high mortality on dialysis whereas non-cirrhotics with inactive disease do well. the role of kidney transplantation in cirrhotics with suppressed viral replication needs to be reassessed. the truncated hbc149 interferes with replication of hepatitis b virus j.c. han 1,2 , x.b. pan 1,2 , l. wei 1,2 , k. deng 1,2 1 institute of hepatology, 2 peking university people's hospital, china hepatitis b virus (hbv) capsids play an important role in production of progeny virus and other elements of the virus life cycle. misdirection of capsid assembly and formation of aberrant particles may be an effective approach to interfere with virus replication. hbv capsids can be assembled in vivo and vitro from the dimeric hbv core protein (hbcag). the interaction of single and dimeric hbcag with some truncated hbcag is verified in vitro. the truncated hbcag consists of the first 149 amino acids and lacks the c-terminal, 34-residue rna-binding domain. method: we transiently transfected hepg2.2.15 with pcdna3.1hbc149 by fugene6.after 48 and 96h, hbvdna, hbeag and hbsag in culture supernatant were detected and cell subjected to southern blot and immunofluorescence analysis. result: the level of hbsag and hbeag had gentle change, we found that hbvdna decreased at 96h after transfection( 10 3 10 4 copies/ml p<0.01) ,but replication intermediates obviously decreased from 48h. some positive signal of hbcag located around the nuclear and conglomerated in cytoplasm compared to the control. conclusion: the truncated hbc149 can inhibit replication of hepatitis b virus. misdirection of capsid assembly and formation of aberrant particles could be an important cause. y.p. li 1 , r.c. li 1 1 objective: to assess the long-term efficacy of recombinant yeast derived hepatitis b vaccine in infant s born to hbsag and hbeag carrier mother. methods: a total of 273 neonates born to hbsag, hbeag both positive mothers were vaccinated with 5 ,5 ,5 g doses of recombinant yeast derived hepatitis b vaccine by 0 ,1 , and 6 months schedule. they were all followed for 129 years after the primary vaccination. results: twelve infant s (6.63 %) become hbsag positively conversed in 9 year after primary vaccination ,and the positive rate of hbsag in 1-9 year was 0.72 %-6.8 % ,17.18 % of child in no/ lowly respond become hbsag positively. at the ninth year, the positive rates of anti-hbs were 60 % above. anti-hbs positive rates and immunity level were higher at 3-5 year old by repetition immunity than others. conclusion: the recombinant yeast derived hepatitis b vaccine have good immunogenecity and long-term protective efficacy to hbv interruption of perinatal transmission , a booster dose seems necessary in aged 3-4 years to the mother with hbsag and hbeag.it is high risk tobecome hbsag positively in the baby of norespones to hepatitis b vaccine. chb patient group-initiated programme to improve awareness, adherence and treatment outcomes in asia pacific n. leung 1 1 founding chairperson of asiahep background: worldwide, over 400 million people live with chronic hepatitis b (chb); 300 million in asia pacific. regional survey data from 1,500 patients in 10 countries showed a lack of knowledge and understanding of chb, its severity and impact on quality of life. this initiative aims to coordinate patient groups in the region and devise programmes to improve knowledge and healthcare outcomes. methods: the patient groups met in hong kong in may 2008 and identified common needs to: (1) improve educational resources; (2) raise awareness; (3) increase diagnostic yield; and (4) enhance treatment compliance through education about the need for sustained viral suppression to reduce long-term complications. results: a patient engagement programme was developed for people with newly diagnosed or known chb. the programme comprises: -detailed information about chb -a health-tracking tool for self-monitoring of blood tests and treatment progress -detailed information for carers/family -a patient-physician communications video (including role-play) -mobile phone text messages providing advice and compliance/appointment reminders conclusion: this programme was developed to address the needs of patients and clinicians. improved knowledge and long-term support, particularly for patients on antiviral medication, is expected to improve quality of life. the programme encourages clinicians and patients to develop enduring therapeutic partnerships to promote optimal outcomes. acknowledgement: the chb patient group meetings and the patient engagement programme are supported by an unrestricted educational grant from glaxosmithkline. serum hbv rna level reflects the potency of nucleos(t)ide analogue y.w. huang 1,2 , k. chayama 3,4 , m. tsuge 3,4 , s. takahashi 3,4 , t. hatakeyama 3,4 , m.y. lai 2,5 , h.l. you 1 , j.t. hu 1 , c.j. liu 2,5 , p.j. chen 2,5 , d.s. chen 2,5 , s.s. yang 1 , j.h. kao 5, 6 1 liver unit, cathay general hospital medical center, 2 background and aims: serum hbv rna is detectable in patients treated with lamivudine (lmv) or entecavir (etv) (hatakeyama, 2007 and huang, 2008) . the aim of this study was to determine the clinical significance of serum hbv rna levels in patients treated with nucleos(t)ide analogues of different potency. methods: serum hbv rna was serially determined in 20 patients treated with nucleos(t)ide analogues for 48 to 52 weeks (9 with adefovir (adv), 5 with lmv, and 6 with etv). serum hbv rna was quantified by reverse transcription of hbv nucleic acid extract with subsequent real-time pcr. results: hbv rna was detectable in 11 patients as follows: 2 of 9 in adv (22%), 3 of 5 in lmv (60%), and 6 of 6 in etv (100%) (p = 0.002). mean log serum hbvdna levels at baseline were 10.1 ± 0.6 for adv, 6.6 ± 2.0 for lmv, and 9.5 ± 0.9 for etv, which were comparable between less potent adv and most potent etv (p = 0.14). during antiviral therapy, peak log hbv rna level of patients with etv was significantly higher than that of those with adv or lmv (3.2 ± 0.9 vs. background: in the phase iii clinical trials, clevudine 30mg for 6 months showed potent antiviral activity along with a marked post-treatment antiviral effect. the objective of this study is to compare the anti-hbv activity of combination of clevudine and vaccine over clevudine alone in chronic hepatitis b (chb) patients in a randomised way. methods: the patients are received clevudine for 24 weeks and then combination of clevudine and vaccine for another 24 weeks or clevudine alone for 48 weeks. eligible patients were treatment-naïve hbeag(+) chb patients with hbv dna levels 500,000 copies/ml. the primary endpoint is the proportion of patients with hbeag loss. preliminary results are presented here. results: thirty-one patients have completed week 24 visits and from them, 15 patients (11 in clevudine alone and 4 in combination group) have completed week 36 visits. at week 24, 26% of patients had hbeag loss. at week 36, 46% in clevudine alone and 25% in combination group (3 months on combination after clevudine monotherapy) had hbeag loss. at week 24, 63% of patients had negative hbv dna by amplicor pcr (<300 copies/ml). at week 36, all of patients in both groups had negative hbv dna by pcr and 73% in clevudine alone and 80% in combination group had normal alt. conclusion: clevudine demonstrated good serologic response as well as significant viral suppression and alt normalization. with this data, we conclude that combination therapy of clevudine and vaccine for short period does not show the superiority over clevudine alone. background/aims: to determine the reasonable number of clones for hbv quasispecies analysis. methods: 16 chronic hepatitis b patients were enrolled with hbvdna levels range from 4~10 log copies/ml. hbvdna was extracted. hbv reverse transcriptase (rt) gene encompassing the overlapping surface s gene was amplified by polymerase chain reaction, then cloned and sequenced. ten positive clones for each sample were sequenced in the first group, and then additional ten positive clones were sequenced in other groups until up to thirty clones. the characteristics of hbv quasispecies including shannon entropy and genetic distance were calculated. results: the shannon entropy and genetic distance of 10 clones group was higher than those of 20 and 30 clones group, either in rt gene or in s gene (p<0.01). while the shannon entropy and genetic distance of 20 clones group showed on difference with those of 30 clones group, neither in rt gene nor in s gene (p>0.05). the number of different quasispecies detected in 30 clones group was higher than that of 20 and 10 clones group (p<0.01). the shannon entropy and genetic distance in three different clones group had no correlation with hbv dna levels (p>0.05). conclusion: although the number of different quasispecies detected was increased with the augmentation of clone number, the quasispecies characteristic didn't changed significantly when the clone number more than 20. the information contained in 20 clones per sample could well represent the quasispecies characteristics. the clone number was not necessary modulated according to different hbv dna levels. background: recent studies reported that basal core promoter mutation (a1762t and g1764a) was associated with more aggressive progression of liver disease from inactive carrier to active hepatitis, and eventually to liver cirrhosis and hcc. but the effect of the double mutations on the activity of enhancer ii/basal core promoter is still uncertain. objectives: to evaluate the influence of nt1762 a/t and nt1764 g/a mutations on hbv enhancer ii/basal core promoter activity. methods: the pcr fragments of hbv enhancer ii/basal core promoter (nt1601 to nt1815) from the serum-derived genotype b hbv dnas of one hbv carrier aged 66 and one hbv related hepatocellular carcinoma patient aged 26 were introduced into the pgl3-basic-vector from promega via restriction sites of xho i and hind iii. the nt1762 a to t and t to a, the nt1764 g to a and a to g mutations were carried out by genetailor site-directed mutagenesis system from invitrogen. the promoter activity was evaluated by comparing firefly luciferase measurement with renilla luciferase as the internal control using the dual-luciferase reporter assay system from promega. results: the luciferase reporter assay results indicated that the 1762 t to a combined with 1764 a to g mutations increase (p<0.001) while the 1762 a to t combined with 1764 g to a mutations decrease (p<0.05) the hbv enhancer ii/basal core promoter activity significantly. conclusions: associated with increased risk of hepatocellular carcinoma, a1762t and g1764a double mutations of hepatitis b virus reduce the enhancer ii/basal core promoter activity. background/aims: a substantial proportion of chronic hepatitis b (chb) patients with mildly elevated alanine aminotransferase (alt) have significant fibrosis. we evaluated the factors associated with significant fibrosis and clinical outcomes in these patients. methods: one hundred five chb patients with alt less than two times the upper limit of normal underwent liver biopsy. multiple clinical, biochemical and virologic variables were evaluated to determine the predictors of significant fibrosis and progressive liver disease. results: there were 27 patients in the low normal alt group, 21 in the high normal alt group, 37 in the low elevated alt group, and 20 in the high elevated alt group. fifty eight patients (55.2%) had significant fibrosis ( stage 3) and 35 (21.0%) had significant inflammation ( grade 3). the age, platelet count and grade of inflammation were factors associated with significant fibrosis. progressive liver disease was observed in 23 (27.4%) of the 84 followed-up patients. the stage of fibrosis, alt group and antiviral therapy were significant predictive factors for progressive liver disease. conclusion: liver biopsies should be recommended in patients over 35 years with mildly elevated alt levels, and antiviral therapy should be considered in patients with significant fibrosis to prevent progressive liver disease. background: four nucleos(t)ide analogues (nas) are currently approved for the treatment of hbv infection in china. however, long-term benefits are limited by the emergence of drug-resistant viruses. methods: patients accepted the examination based on physician's instruction. hbv reverse transcriptase gene was amplified from serum via nested pcr and sequenced directly. results: well-recognized drug-resistant mutations were detected in 567 of 2,000 patients. in patients receiving na monotherapy, corresponding drug-resistant mutations were detected in 214/381 for lamivudine (lam), 35/333 receiving adefovir (adv), 0/57 for entecavir (etv), and 9/17 for telbivudine (l-dt). the mutations were detected in 272/615 patients receiving 31 kinds of sequential/combined usages of the 4 nas. m204i (32%), m204v+l180m v173l (32%), and m204i+l180m (21%) were identified as major mutant patterns of lam monotherapy. n236t a181 substitution was the dominant adv-resistant mutation. t184 substitution was the dominant etv-resistant mutation always accompanied with lam-resistant mutation. l-dt-resistant mutation was m204i l180m exclusively. adv-resistant mutation was frequently seen in lam-resistant patients receiving adv sequential therapy rather than those receiving adv add-on therapy. controversial lam/adv-resistant mutations including a181t, v214a, q215s and i233v were detected in some patients singly or with the well-recognized drug-resistant mutations. interestingly, the drug-resistant mutations were also observed in a few of patients naïve to nas. conclusions: the exploration of hbv drug-resistant mutation profile in large clinical samples furthers our understanding of hbv drug-resistant status in china with implications for administrating anti-hbv therapy more reasonably. toll-like receptor (tlr) 2, tlr4 and cd4+cd25+cd127low/-regulatory t cells correlate with hepatitis b virus infection y. zhang 1 , j.q. lian 1 , c.x. huang 1 , x. wei 1 , j.p. wang 1 , p.z. wang 1 , x.f. bai 1 1 center of infectious diseases, tangdu hospital, the 4th military medical university, xi'an, china background: tlrs play a crucial role in sensing and initiating innate antiviral response and tregs actively suppress immune response, contributing to viral persistence and chronic tissue damage. in this study, we determined tlr2 and 4 expression and treg frequency, as well as their function in the effect of hbv infection. methods: tlr2 and tlr4 expression on monocytes and circulating cd4 + cd25 + cd127 low/-tregs were determined by flow cytometry in 16 ahb, 42 chb, 22 asc and 20 nc. spearman correlation was performed to investigate associated variables on treg or tlrs. pbmcs were stimulated with hbeag or hbcag and the tlrs profile was examined. result: tlr2 expressions were up-regulated in chb and asc, while tlr4 were increasingly expressed in ahb and asc. treg frequency in chb was significantly higher than that in nc. in chb, the increased tlr2 negatively correlated with hbv dna loads and treg frequency negatively correlated with tlr4 expressions. tlr2 was up-regulated after hbeag stimulation in both nc and chb. conclusion: increased tregs may be associated with chb and there might be possible interactions between hbeag, tlr signaling and the innate immune response, which may partially explain the mechanism of hbv infection induced immuno-tolerance. (6.7±1.35) . hbv-dna was quantitatively determined by polymerase chain reaction (pcr) technique, and hbv genotype was determined by pcr microwave gene chip technique. antiviral efficacy was assessed using measuring the following scales: the alt normalization rate, hbv-dna negative conversion rate and the hbeag/anti-hbe seroconversion rate. results: among 55 serum specimen, hbv genotype distribution was 38 genotype c, 14 genotype b, and 3 genotype non-b or c respectively. in genotype b, alt normalization rate was 78.57%(11 cases), hbv-dna negative conversion rate was 35.71%(5 cases) and the hbeag/anti-hbe seroconversion rate was 14.28%(2 cases). in genotype c, alt normalization rate was 76.31% (29 cases), hbv-dna negative conversion rate was 47.37%(18 cases) and the hbeag/anti-hbe seroconversion rate was 18.42%(7 cases). the efficacy of adefovir dipiroxil showed no significant differences between genotype b and c in the treatment of chronic hepatitis b p>0.05 . conclusion: adefovir dipiroxil is an effective antiviral drug. hbv genotype is irrelevant to the antiviral efficacy of adefovir dipiroxil in treatment of patients with chronic hepatitis b. the effect of anti-hbv drugs on albumin and bilirubin levels, and platelet count h. yoshida 1 , h. taniguchi 1 , r. nagano 1 , k. sakitani 1 , e. seki 1 , t. serizawa 1 , y. ito 1 , h. mizuno 1 , y. mitsuno 1 , r. nakata 1 , m. omata 2 1 japanese red cross medical center, 2 university of tokyo, japan background/aim: we assessed the efficacy of anti-hbv drugs on the liver function. methods: patients with hbv-related disease followed at our center between 2005 and 2007 were enrolled. lamivudine (100mg), lamivudine (100mg) +adefovir (10mg), or entecavir (0.5mg) was administered to the patients with detectable hbv dna and elevated alt. liver function (alt, alb, and t.bil) and platelet count were observed. alt, alb, t.bil, and platelet count of treated group at pretreatment, year 1, and year 2 were compared with untreated group. results: eighty six patients with positive hbsag were enrolled between jan 2005 and dec 2007. seven patients (2 acute infection, 1 overlap infection with hcv, 7 lost of follow up) were excluded. in total 76 patients were followed up for a median follow up of 26 (range 2-39) months. of 76 patients, 32 received anti-viral treatment. twenty one patients were treated with lamivudine, 4 with lamivudine+adefovir, and 7 with entecavir. the mean of levels of pre-treatment-year1-year2 were alt:129-37-43 (u/l), alb:4.0-4.4-4.4 (g/dl), t.bil:1.0-0.8-0.7 (mg/dl), and plt:14.3-15.4-16.0 (x10 4 mcl) respectively. markers of untreated group (n=44) (at baseline-year1-year2) were alt:33-40-35 (u/l), , t. bil:0.8-0.8-0.9 (mg/dl), and plt:18.7-18.0-18.5 (x10 4 mcl) respectively. although all of four markers in treated group were significantly worse than untreated group at baseline, all of four markers did not showed significant difference from untreated group at year2. conclusion: treatment with anti-hbv drugs showed the efficacy not only transaminase levels, but also on albumin, bilirubin, and platelet count improvement-improvement of "hepatic reserve" which is valuable for prevention of cirrhosis. background: currently, hbeag-negative chronic hepatitis b(chb) is increasing. but there are still controversial on the treatment of hbeag-negative chb with alt 2×uln. we have investigated the clinical efficacy of nucleotide analogues(nas) in the treatment of hbeag-negative chb with alt 2×uln. methods: the data of patients who were treated by nas for more than 2 years and with alt 1 2×uln (n=87) , alt 1×uln(n=43) and alt 2×uln(n=88) were collected, and 12w and 24w virologic response, 48w and 96w complete response, virologic breakthrough and clinical resistance were analyzed. results: compared with the base line, hbv dna level in all three groups were significantly decreased (p 0.01), and there was no significant difference between alt 1 2×uln group and alt 2×uln group. the viral load was significant decreased in alt 1×uln group at 12w, 24w and 36w (p<0.05). virologic response at 12w and 24w complete response at 48w and 96w was 71.3%, 82.8% , 82.8% and 86.2% respectively in alt 1 2×uln group and was 51.2%, 60.5%, 65.1% and 74.4% respectively in alt 1×uln group. there was no significant difference between alt 1 2×uln group and alt 2×uln group. virologic response at 12w and 24w and complete response at 48w were significant decreased (p<0.05) in alt 1×uln group. there was no significant difference among the three groups in virologic breakthrough and clinical resistance. conclusion: hbv replication can be satisfactory inhibited by nas in hbeag-negative chb patients with alt 2×uln, which suggests that in these patients the indication of alt is different from hbeag-positive patients. quantitative hbeag assay as a predictive factor of hbeag seroconversion induced by peg-ifn -2a therapy to hbeag-positive chronic hepatitis b y.y. zhu 1 , j. dong 1 , y.t. chen 1 , j. chen 1 , j.j. jiang 1 1 liver diseases research center, the first affiliated hospital of fujian medical university, fuzhou, fujian, china rp, 350005 background: to find predictive factor for hbeag seroconversion in the treatment of hbeag-positive chronic hepatitis b (chb) by peg-ifn -2a. methods: hbeag-positive chb patients were given peg-ifn -2a treatment for 48 weeks. clinical data were collected every 3 months. receiver operator characteristic (roc) curve was employed to calculate positive predictive value (ppv), negative predictive value (npv), sensitivity and specificity. results: sixty-five patients completed peg-ifn -2a therapy. among them, 17 (26.15%) were found hbeag seroconversion and 19 (29.23%) were found hbeag loss at cessation of therapy. none of age, gender, alt level and hbv dna load at baseline had relationship with hbeag seroconversion. hbeag level of baseline was correlated to hbeag seroconversion, with p value as 0.003 (table 1) . according to roc curve, supposed auc as 0.705 and p value as 0.042, the ppv, npv, sensitivity and specificity of hbeag level as 61 at 12 week were 0.361, 0.862, 0.765 and 0.521, respectively. supposed auc as 0.828 and p value as 0.001, the ppv, npv, sensitivity and specificity of hbeag level as 15 at 24 week were 0.452, 0.912, 0.824 and 0.646, respectively. the hbeag level (s/co) and decreased degree (percentage) at 12 week and 24 week were significant related to hbeag seroconversion (table 2) . conclusion hbeag level at baseline and at 12th and 24th week and its decreased degree (percentage) during the treatment course could be used as predictive factor for hbeag seroconversion. background: it is well documented that perinatal transmission is the major cause of chronic hbv infection in china. the aim of this study was to evaluate the efficacy of interruption of hbv intrauterine infection with hepatitis b immunoglobulin (hbig) in pregnant women with hbeag positive. methods:: a prospective randomized controlled trial was adopted. each subject in the trial group (28 cases) was given 200 iu hbig intramuscularly every 4 weeks from 28-week of gestation, while each subject in the control group (24 cases) received placebo in the same way. the cord blood of newborns were collected for detecting hbsag, hbeag and hbv-dna. results: for newborns, hbeag positive rate in trial group was 21.4%(6/28).hbeag positive rate in control group was 79.2%(19/24). there was significant difference in hbeag positive rate of newborns between the two groups( p < 0.01, rr = 0.27). hbv-dna positive rate in trial group was 25%(7/28). hbv-dna positive rate in control group was 83.3%(20/24). there was significant difference in hbv-dna positive rate of newborns between the two groups( p < 0.01, rr = 0.30). hbv-dna load of 7 cases of newborns in trial group was lower than that of their mothers(t = 28,p = 0.02). there was no significant difference in hbv-dna load between women and their newborns after delivery in control group (t = 81.5,p > 0.1). conclusion: it is effective and safe to prevent hbv intrauterine infection with hbig from the 28(th) wk in pregnant women with hbeag positive. ), especially, the cirrhosis and hcc cases obviously more in both hbeag and anti-hbe patients are negative than hbeag-negative but anti-hbe positive patients (.36.7% vs 26.7%; 7.9%vs5.5%, p ) .the prevale nce of pre-core g1896a mutate have no significant difference regardless of hbv serum marker status or the state of illness. conclusion: recent 5 years the hbeag-nagative chronic hepatitis b patients are gradually increasing in yunnan province. while the hbeag disappear but no anti-hbe serum transfer, and the virus still active replication -it may be a crucial phase determined the diseases outcome, which should be pay more attention by physicians. the clinical significant of pre-core g1896a mutate remain unknow. efficacy of interferon for chronic hepatitis b patients with normal or paranormal alt z. liu 1 , j.z. guo 1 , y.j. lin 1 , y.j. zhang 1 , z.w. lang 1 1 beijing ditan hospital, beijing, china background: we reported interferon treatment for 4 cases with normal or paranormal alt but in which liver histologic exam showed g2-4 and/or s3-4. methods: 4 patients were male with an average age of 28 years.mean alt was 46.7 iu/l and hbv dna level was 3~5 log10copies/ml. two patients were hbeag positive; one patient was both negative for hbeag and anti-hbe and one patient was anti-hbe positive. liver biopsy showed g2~g4 and s3~s4 respectively. 3 patients were treated with ifn-alpha, liver biopsy was repeated after 1 year.only one patient had received combination therapy with ifn and adefovir after 10 months treated ifn monotherapy and liver biopsy was taken after 1.5 years. results: all patients got normal alt after 1 year treatment. hbv dna was undetectable in 3 patients. 2 patients with initial positive hbeag cleared. but 3 patients still were anti-hbeag negative.liver biopsy showed change fromg4 s3-4 to g2 s2-3 in 1 patient; fromg2 s3 to g3 s4 in 1 patient and no change in the other 2 patients. conclusions: though alt and hbv dna improved after 1 year treatment, histological improvement is not satisfying. 1 patient's improvement in liver histology may be due to seroconversion before treatment and adding adefovir after 10 months of interferon therapy. after 8 months of combination therapy we did liver biopsy again. the other 3 patients were hbeag negative, but hbeab were also negative, liver biopsy was taken 1 year later without combination of nucleoside analogs. evaluation of long term efficacy of hepatitis b vaccination r.c. li 1 , j. gong 1 , j.y. yang 1 1 objective: to evaluate the long term effectiveness of preventive hbv infection and to monitor the incidence of hepatitis b in children to see possible impact on the program of long an that was launched in 1986. methods: (1) set up a surveillance systemof hepatitis ,to evaluate the possible impact on incidence of hepatitis b. (2) to serologically evaluate the effect of the program, a stratified random sampling of 1000 subjects in 1987 birth cohorts was recruited for long term follow up at the age 1-20 years. (3) cross-sectional seroepidemiolgical survey was carried out in the county in 1985 before the program and 20 years later. hbsag , anti-hbs and anti-hbc were tested by ria. results: the average coverage of hepatitis b vaccine was 89. 1 %. at 20 years after vaccination, the seropositivity for hbsag in population of 1-20 years has decreased from 16. 0 % to 2. 3 %, the annual effectiveness was 89. 7 %. hbv accumulated infection rate was 3. 5 %, protective rate was 96. 2 %. the incidence of acute hepatitis b was 1. 60 per 100,000 in population aged 1 -20 years , it decreased by 91.3 % as compared with the incidence of 18.38 per 100,000 in same age group in 1985 -1987. conclusion: mass hepatitis b vaccination program in long an county has proved to be effective in control of hbv chronic infection and incidence of acute hepatitis b. background and aims: although the evolution of viral quasispecies may be related to the pathological condition of disease, little is known about this in hepatitis b virus (hbv), especially during hbeag seroconversion. methods: nucleotide sequences of hbv precore/core genes from 5 time points were analyzed in four cohorts of chronic hepatitis b, interferon-induced seroconverters (is, n=9), interferon non-responders (in, n=9), spontaneous seroconverters (ss, n=9) and non-seroconverters (sn, n=9), followed during 60 months on average. only patients with genotype c were used. viral diversity was then estimated after nucleotide genetic distance was assessed and phylogenetic trees were constructed. results: analysis of 1800 nucleotide sequences showed that the nucleotide genetic distance of seroconverters (is and ss; 9x10 -3 substitutions/site and 8.5x10 -3 subsititutions/site, respectively) was similar to that of non-seroconverters (in and sn; both 7x10 -3 substitutions/site) before seroconversion. compared to that of nonseroconverters (in and sn; substitutions/site and 7.5x10 -3 substitutions/site, respectively) the viral diversity of seroconverters (is and ss; 13x10 -3 substitutions/site and 12x10 -3 substitutions/site, respectively) was significantly higher after seroconversion (p<0.05) and it was higher after seroconversion in seroconverters compared with that berore seroconversion (p<0.05) while it almost didn't change in non-seroconverters irrespective seroconversion. phylogenetic trees also showed that complex trees appeared in secoconverters and relatively simple in nonseroconverters. conclusions: the distinctly higher viral diversity after seroconversion in hbeag seroconverters could be related to increased hbv-specific t-cell responses and escape mutant which arise from stronger selective pressure caused by host immune activity. adefovir dipivoxil 10mg (adv) resistance at 5 yrs in chinese hbeag+ve chronic hepatitis b (chb) j.l. hou 1 , y.z. wang 2 , x.q. zhou 3 , j.q. niu 4 , y.m. wang 5 , h. wang 6 , y.m. mao 7 , k.f. barker 8 1 nanfang hospital, guangzhou, prc, 2 jinan infectious disease hospital, jinan, prc, 3 ruijin hospital, shanghai, prc, 4 1st hospital of jilin university, changchun, prc, 5 xinan hospital, chongqing, prc, 6 people's hospital, beijing, prc, 7 renji hospital, shanghai, prc, 8 glaxosmithkine r&d, london, uk background: long term adv provides clinical and histological improvement in chb, but may lead to emergence of treatment associated resistant mutations. we report on adv resistance data from chinese hbeag positive subjects treated for 5 years. methods: 480 hbeag positive chb subjects were randomized in an initial 52 weeks controlled adv study (with a 12 weeks placebo period in half of patients) and then offered open label adv treatment for a further 208 weeks. a total of 474, 456, 443, 421 and 390 subjects completed the 1 st , 2 nd , 3 rd ,4 th and 5 th yr, respectively. at the end of each year samples were analysed from those subjects with protocol-defined hbv dna breakthrough for the rtn236t or rta181v adv mutations associated with resistance. sera from subjects with breakthrough were analysed at all subsequent yearly timepoints whenever possible. results: at the end of the 1 st yr, none of the 45 subjects with hbv dna breakthrough had either mutation. sera were available for analysis from 137, 199, 228 and 187 subjects with viral breakthrough at the end of the 2 nd , 3 rd , 4 th and 5 th yr, respectively, with new mutations identified in 6, 20, 24 and 20 subjects at the same timepoints. of the cumulative 70 subjects at the 5 th yr analysis 31 had rtn236t, 29 had rta181v, and 10 had both mutations. conclusion: treatment with adv in chinese hbeag positive chb subjects for up to 5yrs resulted in a cumulative rate of 14.6% (70/480) adv resistance-associated mutations with hbv dna breakthrough. background and aims: to evaluate the predictive significance of rapid virologic response (rvr) for achieving an end-of-treatment virologic response (er) or hbeag seroconvertion and the predicting indicator of nonresponse (nr). methods: 205 patients with chronic hepatitis b were treated with adv and prospectively observed to 48 weeks. we assessed the values of virus load reduction at weeks 4, 8, and 12 weeks to predict the er and hbeag seroconversion. the association between less reduction of viral load at 12 and 24 weeks and nonresponse was also analyzed. results: of 146 etv-treated patients enrolled in etv-901, 98 met criteria for inclusion into 5 year etv treatment analyses. the proportion of patients achieving efficacy endpoints through 5 years of etv therapy is presented the table. results: after 48 weeks of therapy, serum hbv dna levels decreased with a median 4.07±2.47 log10 copies/ml. twenty-three(11.2%) of patients had er. twenty-six (12.7%) patients achieved hbeag seroconversion. hbv dna <4 log 10 copies/ml at week 4 predict both er and hbeag seroconversion. hbv dna> 4log10 copies/ml at 4 weeks but decline to <4log10 copies/ml at 12 weeks or 24 weeks both can predict er and hbeag seroconversion. less than 2 log 10 hbv dna reductions at 24 weeks might predict nr. conclusions: the majority of patients experienced durable serum hbv dna suppression (94%) and alt normalization (80%) after 5 years etv therapy. conclusions: the virologic response within 4 weeks could be useful for prediction of er and hbeag seroconversion of adefovir therapy. failing to evr might not predict nr. objectives: to determine the accuracy of hbcigm in diagnosing ahb and the correlation between hbcigm and liver inflammation (alt), bilirubin & biosynthetic functions (albumin,pt). result: a total of 233 patients were included: in 40 patients, adv was added on lam (add-on therapy), and in 193 patients, lam was switched to adv (switch therapy). during 16.5 months of follow-up, 25 patients developed adv resistance (rta181v and/or rtn236t) and all had undergone switch therapy. the cumulative probability of adv resistance at the 24th month was 17.5%. although add-on therapy induced no adv resistance, it failed to show significant superiority over switch therapy (p=0.220). in multivariable analysis, female (odds ratio [or], 2.75; 95% confidence interval [ci], 1.22-6.17; p=0.014), liver cirrhosis (or, 2.39; 95% ci, 1.07-5.33; p=0.033), and age >45 yr (or, 3.31; 95% ci, 1.14-9.66; p=0.028) were independent risk factors of adv resistance. methods: a retrospective cross-sectional study involving patients with hbcigm positivity between june 2006-december 2007, satisfying the definition for ahb and chbf,and fulfilling the exclusion criteria was performed. hbcigm test were done by using microparticle enzyme immunoassay (meia) and results were expressed as an index value.hbcigm positivity was defined as index value of >1.00 results: 74 patients were positive for hbcigm and 60 fulfilled the criteria(33 ahb, 27 chbf).hbcigm was significantly higher in ahb compared with chbf(median 3.46 vs 1.70;p <000.5).the hbcigm arbitary index value of 2.76 was highly sensitive(97%) and specific(85%) in diagnosing ahb with high accuracy(auroc 0.964;95% ci:0.925-1.003;p<0.0005).among patients in both groups, there was a weak, but significant negative correlation between hbcigm and pt above control(r = -0.309,p =0.016).however, among patients with chbf,the negative correlation between hbcigm and pt above control was moderately strong(r = -0.557,p =0.003).there was also a weak, but significant positive correlation between hbcigm and albumin in with chbf(r = +0.434,p =0.024). conclusion: adv add-on therapy developed no adv resistance during the observation period. therefore, add-on therapy is recommended to lam-resistant chb patients with genotype c who have any risk factors for development of adv resistance: female, liver cirrhosis, and age >45 yr. hbcigm-hepatitis b core igm antibody ahb-acute hepatitis b,chbf-chronic hepatitis b flare alt-alanine transaminase,pt-prothrombin time pe117 detection of emerging drug resistance mutations associated with major approved hbv antivirals using a novel line probe assay (lipa). j. doutreloigne 1 , f. shapiro 1 , r. maertens 1 , e. van assche 1 , e. sablon 1 1 hepatitis diagnostics unit, innogenetics nv, belgium background: in study etv-022, etv demonstrated superior virologic, histologic and biochemical benefit compared to lamivudine (lvd). this study (etv-022/-901) presents efficacy and safety results for patients who received 5 years continuous etv treatment. background/aims: an increasing number of antiviral drugs are being used to treat chronic hepatitis b virus (hbv)-infected patients. however, induced viral escape mutants -some potentially cross-resistant -lead to viral non-responsiveness and treatment failure. effective treatment strategies must therefore take possible drug resistance (dr) into account with respect to monitoring and selection of alternative drugs. we evaluated the use of an updated inno-lipa hbv dr v2+v3 reverse hybridization assay versus sequence analysis to detect resistance mutations. methods: the study evaluates etv-treated nucleoside-naïve hbeag (+) patients who completed etv-022 and enrolled into etv-901 with a treatment gap 35 days. the proportion of patients with hbv dna <300copies/ml, alt normalization, hbeag loss or hbeag seroconversion was evaluated at week 240. background: etv resulted in improved liver histology compared to lvd at 1 year. histologic data for patients on etv for a median of 6 years is evaluated. methods: 273 clinical samples (from untreated hbv patients or treated with different antivirals; hbv genotypes a-h) were tested for mutations with the lipa assay and sequencing. for lipa, samples were extracted with the qiaamp® dna blood mini kit (qiagen), and then tested on the lipa strips. sequencing-derived reference data were subjected to phylogenetic analysis (kodon version 3.10 applied maths, neighbour joining, with kimura-2 parameter). sequential samples from 9 patients were evaluated as well. methods: etv-treated patients completing etv-022 or etv-027 received etv (1.0mg daily) in etv-901. primary endpoints included 2-point decrease in knodell necroinflammatory score, no worsening of knodell fibrosis score and improvement in ishak fibrosis score (ifs) ( 1-point decrease) vs. baseline. secondary endpoints included proportions with hbv dna<300 copies/ml, alt normalization, and ifs normalization in patients with advanced fibrosis/cirrhosis. results: quasi-perfect concordance (> 99.6%) was obtained between the two assays for the samples tested. no indeterminate results were observed. for one sample, lipa provided additional information (wild-type/mutant mix), whereas sequencing showed only wild type. for sequential samples, lipa was clearly able to detect emerging treatment-resistance mutations associated with viral breakthrough. results: etv treatment led to significant histological improvements and improved ifs in 96% (55/57) and 88% (50/57) of patients respectively. of 10 patients with baseline fibrosis/cirrhosis (ifs 4), all demonstrated 1-point improvement in ifs (median change of -3). conclusions: lipa accurately detects the complex quasispecies nature of hbv and can help unravel the dynamics of emerging hbv resistance during treatment with different antiviral drugs. like its predecessor, it is useful for the monitoring and early detection of drug resistance. conclusions: long-term etv therapy in nucleoside-naïve chb patients results in durable virologic suppression, continued histologic improvement and regression of fibrosis/cirrhosis. precore (pc, g1896a) and basal core promoter (bcp, a1762t and g1764a) mutations of hbv are important for predicting the risk of hepatocellular carcinoma (hcc). we developed a new mass spectrometry-based assay using restriction fragment mass polymorphism (rfmp) to detect a1896 and t1762/a1764 mutations, and applied it to analyze their clinical significance in type b liver diseases (n=336), including 35 hccs, 118 liver cirrhosis (lc), 157 chronic hepatitis b (chb), and 25 hbsag-positive with low level viremia (inactive hbsag carrer, ihc). we devided patients into 4 major groups according to the presence of wild (w) or mutant (m) genes in bcp/pc regions; w/w, w/m, m/w and m/m gene types. each proportion was 9.5%, 3.6%, 41.4% and 23.5%, respectively. mixed infection (x) was also found as minority; 4 w/x, 28 m/x, 15 x/w, 4 x/m and 13 x/x. disease distributions (hcc, lc, chb and ihc) in each group were as follows; [w/w (n=32)] 3.1%-6.3%-90.6%-0; [w/m (n=12)] 0-16.7%-58.3%-25%; [m/w (n=139)] 9.4%-42.5%-46%-2.2%; [m/m (n=79)] 11.4%-45.5%-20.1%-13.9%. these results suggest that, in korea where only genotype c has been identified, bcp dual mutation is predominant (>73.2%), while bcp wild alone is only 13.1%. especially, a1896 mutation alone without bcp mutation (w/m type) is uncommon, while bcp mutation alone without a1896 mutation (m/w type) is most common. it might be suggested that prognosis of wild type in bcp and pc region (w/w type) is much better than that of m/w or m/m types. background/aims: entecavir is a potent inhibitor of hbv dna polymerase, which has been shown to be safe and effective for the treatment of chronic hepatitis b (chb) patients. the aim of this study was to evaluate the virologic, biochemical, and serologic responses of entecavir through 1 year in chb patients. methods: from 2007 may to 2008 october, we reviewed 82 patients (mean age 46±12 years, male:female=57:25) who were diagnosed as chb patients (hbeag (+) 64). forty-seven patients (57.3%) had been treated with 0.5 mg of entecavir and 35 (42.7%) with 1 mg of entecavir, respectively. mean follow-up period was 25±17 weeks. hbv dna was quantified by bdna assay with a lower limit of detection of 141,500 copies/ml. results: median hbv dna levels before therapy was 7.64 log10 copies/ml and the median decreases from baseline in hbv dna were -4.19, -4.31, -5.09, -5.31, and -5.59 log10 copies/ml at 4 (n=39, p<0.001), 12 (n=62, p<0.001), 24 (n=42, p<0.001), 36 (n=22, p<0.001), and 52 (n=15, p =0.053) weeks of follow-up, respectively. at baseline, overall median alt was 106 iu/l and the proportions of patients with normal alt were 13%, 48%, 56%, 71%, 67%, and 69% at baseline (n=82), 4 (n=42), 12 (n=63), 24 (n=44), 36 (n=24), and 52 weeks (n=16) after entecavir therapy, respectively. thirteen cases (15.9%) of hbeag seroconversion were noted. background: hepatitis b virus (hbv) infection is a major risk factor for the progression of liver diseases. because its clinical course varies, it is difficult to detect the predictive factor for the prognosis of patients with hbv infection. the aim of the present study was to determine the risk factors for the occurrence of hcc. methods: a total of 620 patients who tested positive for hepatitis b surface antigen and were referred to chiba university hospital between february 1985 and march 2008 were included in the study, and their following characteristics were analyzed: age, gender, the status of hbeag, alt, hbv-dna level, and plt. result: hcc was detected in 30 cases during the follow-up period (5.4 ± 5.1 years). multivariate analysis revealed that age [compared with young patients: odds ratio (or) = 1.07, 95% confidence interval (ci) = 1.03-1.11] and plt level (compared with patients with low plt level: or = 0.99, 95% ci = 0.98-0.99) were the predictive factors for hcc occurrence. in patients with age more than 35 years, the hbv-dna level (compared with <5.0 log copies/ml: or = 3.29, 95% ci = 1.40-11.5) and plt level (or = 0.99, 95% ci = 0.98-0.99) were the predictive factors for hcc occurrence. conclusion: advanced age and low plt level were the risk factors for hcc occurrence in patients with hbv infection irrespective of the plt level at baseline. in patients with age more than 35 years, viral load was also a risk factor for hcc. results: before treated by lps, the total mapk p38 level of pbmcs have no significant difference among the healthy control group, different stage groups with hbv infection, however, after treated with lps, the phosphorylated mapk p38 (ptpy180/182) in healthy control group are significant elevate than hbv infected groups(120.0±52 vs 80.2±84.8, p<0.05). in the two groups which hbsag, hbv dna are positive, alanine aminotransferase elevate than normal and hbsag positive, but hbv dna lower under the detect limited level, after treated by lps the ptpy180/182 although lower than healthy control yet, but significant elevated than themselves before treated by lps(86.6±38.6 vs117.8±21.3, 63.3±24.7 vs 93.1±21.8; p<0.05).otherwise, in the group of both hbsag and hbv dna are positive, but alt is normal, before and after treated by lps, the level of ptpy180/182 have no significant difference. conclusion: mapk p38 is a important signal transduction pathway which involving in inflammation and immune response, especially, mapk p38 activated up-regulate the ifn-gamma mrna. according to the result shown, we propose a hypothesis, hbv infection and virus active replication inhibit the mapk p38 activated, consequent on host immunotolerance and hbv persistence, thus, mapk p38 may be as a potential therapeutic target to break immnotolerance and establish host anti-viral states. van der helm 10 1 siriraj hospital, bangkok, thailand, 2 ramathibodi hospital, bangkok, thailand, 3 phyathai hospital, bangkok, thailand, 4 singapore general hospital, singapore, 5 national university hospital, singapore, 6 changi general hospital, singapore, 7 cheil general hospital, korea, 8 hwasun jeonnam university hospital, korea, 9 st mary hospital, korea, 10 roche diagnostics ltd, rotkreuz, switzerland background/aims: hepatitis b virus (hbv) surface antigen (hbsag) is one of the most important markers for diagnosis of acute and hbv infection. high sensitivity of hbsag assays can reduce the diagnostic window during course of disease. in addition, the presence of hbv mutants may be affected by the performance of the hbsag kit. therefore, the technical performance of the elecsys ® hbsag ii assay was explored, using samples (including recombinant mutants), at multiple sites in three countries. methods: nine hbsag screening centers in thailand, korea and singapore compared the sensitivity of elecsys ® hbsag ii assay with that of their routine testing procedure -abbott architect ® (7 centers), abbott axsym ® (1 center) and bayer advia ® centaur hbsag assays (1 center) using preselected seroconversion panels (n=5), recombinant hbv mutant panels (n=13) and routine clinical practice samples (n=1,863). results: the sensitivity of elecsys ® in seroconversion samples was equivalent to the architect ® assay, but more sensitive than the axsym ® and advia ® centaur assays (26 vs 23 and 24 vs 18 positive bleeds, respectively). there was concordance between the elecsys ® and architect assay results with respect to potentially cross-reactive samples (99.74%). the elecsys ® and architect ® assays detected all recombinant mutant samples, whilst axsym ® and advia ® centaur failed to detect three and nine samples, respectively. conclusion: elecsys ® hbsag ii assay was not only highly sensitive and specific when compared with established hbsag screening assays, but also reliably detected hbsag mutants. therefore, this attractive assay is suitable for hbv diagnosis and assessing safety of blood products. background: recent studies have shown a higher rate of adefovir-resistant mutation in lamivudine-resistant chronic hepatitis b (chb) patients treated with switch-to therapy than those treated with add-on therapy. we compared the clinical efficacy of adefovir monothrapy and lamivudine-adefovir combination therapy in lamivudine-resistant chb. methods: a prospective cohort study was performed in 49 patients with lamivudine-adefovir combination therapy and 77 patients with adefovir monotherapy for lamivudine-resistant chb over 18 months. result: biochemical response was achieved in 41 patients (83.7%) treated with combination therapy and in 77 patients (92.8%) treated with monotherapy (p=0.101). virologic response was observed in 19 patients (38.8%) in combination therapy and in 26 patients (31.3%) in monotherapy (p=0.383) and treatment periods for virologic response was significantly shorter in patients with combination therapy than in monotherapy (8.9±4.8 months vs. 13.9±5.0 month, p=0.001). cumulative rate of virologic response was significant higher in patients with combination therapy than monotherapy (p=0.033). hbeag loss was found in 6 patients (16.2%) in combination therapy and 17 patients (20.5%) in monotherapy (p=0.485). biochemical breakthrough was found in 17 patients (20.5%) with monotherapy significantly more frequent than 3 patients (6.1%) with combination therapy (p=0.026). genotypic resistance to adefovir was developed in 1 patient (2.0%) in combination therapy and 9 patients (10.8%) in monotherapy conclusion: to achieve a complete virological response and reduce the risk of adefovir-resistant mutants in lamivudine-resistant chb patients, adefovir in combination with lamivudine is preferable. background/aims: adefovir dipivoxil (adv) effectively inhibits both wild-type and lamivudine (lam)-resistat chonic hepatitis b virus (chb) replication. the aims of this study were to determine the factorts associated with antiviral effect of adv in lam-resistant chb. methods: one hundred-eighteen lam-resistant chb patient (74.6% hbeag-positive) were treated with adv plus lam (n=96) or adv monotherapy (n=22) for a mean of 33.0 months. restriction-fragment mass polymorphism analysis was used for detection ymdd and adv mutants. results: fifty-eight patients (49.2%) achieved complete response(cr) defined as hbv-dna levels <2000 copies/ml and alt normalization. twenty-eight patients (23.7%) achieved initial vilologic response(ivr) defined as hbv-dna levels <10 4 copies/ml within the first 6 month of treatment. 47 (53.4%) of 88 hbeag-positive patients exhibited hbeag loss and 31% seroconverted to anti-hbe ab. five (4.23%) patients developed adv-related mutations. factors associated with ivr were pretreatment level of alt (p=0.00), ast (p=0.00), pretreatment hbv dna level (p=0.03), hbeag negativity (p=0.01) and hbeab positivity (p=0.00). factors associated with cr were ivr (p=0.00), hbeab positivity (p=0.01), pretreatment level of alt (p=0.01), ast (p=0.02) and y-glutamyl transferase (p=0.04). age, sex, presence of liver cirrhosis, pretreatment hbv dna level and the type of ymdd mutants were not related to an cr during adv treatment. conclusions: adv therapy achieved cr in more than 49% of lam-resistant chb. factors associated with cr were ivr, hbeab positive status, high base line alt, ast, ggt levels. q.j. sheng 1 , y. ding 1 , x.g. dou 1 1 department of infectious disease, shengjing hospital affiliated to china medical university, shenyang, 110004, china objectives: to study a kinetics of hepatitis b virus during 12-week and 24-week of treatment with enticavir (ent); to compare the detecting results of hbvdna levels from different detection reagents. methods: thirty-seven cases of chronic hbv infections were selected randomly, treated with daily dose of ent 0.5-1.0mg (0.5mg for nucleoside-naïve patients, 1.0mg for lamivuding-refractory patients). evaluation indexes: serum hbvdna, hbv serological markers, and liver function tests. hbvdna levels were measured by pcr assay, using both domestic reagents and roche cobas amplicor,. the lower limits of measure level of hbvdna were 1000 copies/ml and 300 copies/ml, respectively. results: mean baseline of hbvdna was 5.24log10copies/ml for detection using domestic reagents and 5.70log10copies/ml for that using roche cobas amplicor, (p>0.05). the ratios of cases with undetectable (<1000 copies/ml) hbvdna at week-12 and week-24 were 46.2% and 72.9%, respectively. the ratios of cases with undetectable (<300 copies/ml) hbvdna at week-24 were 54.5%. among the cases whose hbvdna were lower than 1000 copies/ml(using domestic reagents), the ratio of hbvdna lower than 300 copies/ml(using roche cobas amplicor) was 94.7%. conclusions: ent can suppress hbv dna rapidly no matter the patients with alt elevation or not. there is a concordance on hbvdna levels detection between domestic reagents and roche cobas amplicor. background the study on the effect of nucleoside analogue therapy on the quantity of hepatocellular cccdna and tdna and sera hbv dna, hbsag to probe reliable marks for evaluation of therapy endpoint. methods the quantity of hepatocellular cccdna and tdna and sera hbvdna were assayed by fq-pcr, and sera hbsag by elisa in 4 chb patients over 2 years nucleoside analogue therapy satisfied the china criteria of therapy endpoint (therapy group)and 12 chb patients without antiviral therapy and sera hbvdna<10 3 copies/ml(control group). results: the quantity of hepatocellular cccdna, tdna and sera hbvdna, hbsag in therapy group were lower than that of control group,but low level hepatocellular cccdna in therapy group could be detected. conclusion: long term nucleoside analogue therapy may consume hepatocellular cccdna with decreasing of hepatocellular tdna and sera hbvdna, hbsag; although the patients have satisfied the china criteria of therapy endpoint, low level of hepatocellular hbvcccdna were detected, cessation of therapy may cause relapse. peptides that lead nuclear entry of nucleocapsid of hepatitis b virus in hepg2.2.15 cells x.b. pan 1,2 , l. wei 1,2 , j.c. han 1,2 , k. deng 1,2 1 peking university hepatology institute, 2 peking university people's hospital background: the nuclear entry of nucleocapsid is a key step for the hbv life cycle and the formation of covalently closed circled dna (cccdna). it has been supposed that the carboxyl-terminal arginine-rich domain of the core protein contains a signal for nuclear localization (nls). whereas hbcag was primarily distributed in cytoplasm and no marked cccdna was detected in hepg2.2.15 cells. methods: we designed peptides containing a cell-penetrating sequence (rrrrrrr) and a nucleocapsid binding sequence (gsllgrmkga) with/without a classic nuclear localization sequence (pkkkrkv) and these sequences were linked by a soft linker acp. hepg2.2.15 cells were treated with the peptides at levels of 0 m, 25 m and 50 m for 4 days. results: compared with that of control cells, the results showed hbv dna levels in culture medium decreased at least 2 log10 both in 50 m of peptide rrrrrrracpgsllgrmkga treatment group and rrrrrrracpgsllgrmkgaacppkkkrkv treatment group; whereas hbsag and hbeag increased at 1.34+0.21 folds and 2.15+0.37 folds respectively. the signal strength of cytoplasmic hbcag increased at about 2.5-fold in both groups. in rrrrrrracpgsllgrmkgaacppkkkrkv treatment group, nuclear hbcag increased about 3.2-fold and obvious cccdna signal was detected by southern blot. conclusion: our results implied that the nls of core protein likely does not expose to surface of nucleocapsid in hepg2.2.15 cells, the artificial peptide containning nls binds to the nucleocapsid and leads nuclear entry of nucleocapsids and then facilitates the formation of cccdna. our study presents a tool for study on cccdna formation and nuclear entry of nucleocapsid. b. tang 1 , j. xia 1 , y.m. wang 2 , h.f. wang 1 1 liver failure treatment and research center, 302rd military hospital, 2 the dept. aims: to establish a reliable real-time fluorescence quantitative (rfq) pcr method to quantify hbv cccdna, basing on lightcycler system and taqman probe. methods: hbv genotypes a-g were aligned to obtain a conserved sequence, crossing rcdna gap, which was used to design cccdna primers and taqman-mgb probe. also another pair of primers for quantify hbv total dna (tdna) was designed, utilizing the same probe. to increasing specificity, we added plasmid-safe atp-dependent dnase (psad) digestion step just before cccdna pcr amplification. a standard curve from 9 standard plasmid samples, from 2.0×10 9 to 2.0×10 1 copies, was created to examine our system. 50 hbv cccdna samples with known-amount were quantified by creating standard curve from 5 standard samples. results: the standard curve had clear log-phase and excellent parallelism, which means nice and equal amplification efficiency in all reaction capillarys. the slope (regression coefficient) of standard curve was -3.141, mean square error was 0.0990 and regression coefficient was -1.0. all of these key indexes measured up. in 50 tests, we got right results if the starting templates of cccdna copies were between 10 2~1 0 9 copies. the range was superior to commercial hbv kits. by quantifying 16 samples containing different amounts of cccdna and rcdna, digested or undigested, psad digestion would eliminate 10 7 rcdna molecules, leaving 10 2 cccdna molecules untouched. the test specificity was maintained up to 1:100,000 ratio of cccdna:rcdna. conclusions: the rfq-pcr based on lightcycler system for hbv cccdna quantification is reliable, sensitive, with high specificity and low cost. background: to study the changes of toll-like receptor (tlr)3 on dendritic cells derived from peripheral blood mononuclear cells(modc) and its role in the pathogenesis of chronic hepatitis b(chb) and chronic severe hepatitis b(cshb). methods: the expressions of tlr3 on 10000 modc were stimulated by poly i:c, and then were determined by flow cytometry in 20 healthy controls, 28 patients with chb and 30 patients with cshb.the level of interferon ifn-was determined by elisa. the differences of expression of tlr3 on modc and serum ifn-among the three groups of study subjects were determined by student-t test.the correlation between tlr3 and ifn-were determined by linear correalation test. results: the values of mean fluorescence intensity(mfi) of tlr3 on modc of the healthy controls, patients with chb and cshb were 1593.00±349.65,1369.56±287.08,and 1203.96±192.40.the serum ifn-(pg/l) of respective groups was 172.66±37.96,107.98±31.15 and 72.06±29.58. there was a gradual decrease of these values from the group of healthy controls to the group of patients with chb and cshb .significant positive correlations between tlr3 and serum ifn-were found. conclusion: tlr3 may have a role in the pathogenesis of chb and cshb. background/aim: to evaluate the efficacy and safety of entecavir treatment in patients with hbeag-positive chronic hepatitis b who had not previously received a nucleoside analogue. methods: fifty-five patients received 48-week entecavir 0.5mg/d therapy. serum hbv dna load was measured with quantitative real-time-pcr. alanine aminotransferase (alt) activity, hbeag, anti-hbe-antibodies, hbv dna level in serum were evaluated at baseline, week 2, 12, 24 and 48 during therapy. evaluation of safety and tolerance was based on clinical adverse events and laboratory analyses. results: hbv dna levels declined sharply by around 3 log10 copies/ml during the first two weeks, with a highly significant reduction (p<0.0001) at week 2 and thereafter, as compared to those at baseline; 31%, 51% and 78% of the patients had undetectable serum hbv dna levels at week 12, 24 and 48 respectively. highly significantly decreasing serum alt (p<0.0001) occurred during the first 2 weeks of the study. at week 48, alt levels were normalized in 84% of the patients. hbeag seroconversion (hbeag negative, hbeab positive) was achieved in 7.3% and 14.5% of patients by 24 and 48week. at the end of 24 th and 48 th weeks, complete response (alt normalization and hbv dna and hbeag loss) was observed in 11% and 15%, respectively. there was no evidence of drug resistance or adverse effect in chb patients treated for up to 48 weeks. conclusion: entecavir treatment through 48 weeks was well tolerated and resulted in continued benefit for patients with hbeag-positive chronic hepatitis b. aim: to assess the associations of single nucleotide polymorphisms of the mxa gene promoter and sustained treatment response of chronic hepatitis b or c patients with interferon treatment by meta-analysis of individual dataset from all studies published till date. methods: to clarify the impact of mxa gene promoter polymorphisms on sustained treatment response of chronic hepatitis b or c patients with interferon treatment, we performed a meta-analysis of the published data from eight studies comparing the frequencies of mxa gene promoter polymorphisms at nt -88 g/g, -88 g/t, -88 t/t and nt -123 c/c, -123 c/a , -123 a/a alleles in individuals with interferon treatment. as we identified the heterogeneity between studies, summary statistical data were calculated based on a random-effect model. results: the sustained treatment response rate was higher in patients with the nt -88 g/t and nt -123 c/aalleles in the mxa promoter snp . the meta-analyses yielded summary estimatesodds ratio (or) were 2.07 [95%ci (1.58, 2.7), p <0.00001] and 1.9 [95%ci (1.32, 2.73), p =0.0006] of the nt -88 g/t and nt -123 c/a alleles, respectively. conclusion: mxa gene promoter polymorphisms at nt -88 g/t and nt -123 c/a may be useful as a marker to predict the sustained treatment response of chronic hepatitis b or c patients with interferon treatment, and further investigation regarding their real significance is warranted in a large series of patients. background: to determine whether hbv with the same characteristics causes dissimilar mutations in different hosts. methods: full-length hbv genome was amplified and linked with pmd t18 vector. positive clones were selected by double-restriction endonuclease digestion (ecori and hindiii) and pcr. twenty seven clones were randomly selected from an asymptomatic mother [at two time points: 602 (1 d) and 6022 (6 mo)] and her son [602 (s)]. bioeditor, clustal x and mega software were used to perform phylogenetic and mutational analysis. potential immune epitopes were determined by the stabilized matrix method (smm), smm-align method and emini surface accessibility prediction. result: all of the 27 sequences were genotype c, the inner-divergence for the mother and son was 0%-0.8%.13 specific nucleotides differed from the other pubished genotype c isolates were co-exist in the mother and her son. aa 1-11 deletion in pres1 was the dominant mutation in the mother (14/18). the 1762t/1764a double mutation existed in all clones of the mother, 3 of them were also coupled with g1896a mutation, but none were found in the son. 17 bp deletion starting at nucleotide 2330 was the major mutation (5/9) in the son, which caused seven potential hla class i epitopes and one b cell epitope deletion, and produced a presumptive new start codon, downstream from the original one of the p gene. conclusion: the son was infected hbv from his mother, and discrepant mutation occurred in the mother and her son during infection. background/aims: nucleos(t)ide analogues have been recognized as an effective treatment for chronic hepatitis b. this randomized, double-blind trial compared the efficacy and safety of telbivudine and lamivudine after 104-week therapy in patients with compensated chronic hepatitis b in taiwan. methods: we analyzed 114 taiwanese patients from globe trial receiving telbivudine 600mg (n=58) or lamivudine 100mg (n=56) once daily for 104 weeks. the primary efficacy endpoint was therapeutic response with serum hbv dna <5 log10 copies/ml and either hepatitis b e antigen (hbeag) loss or alanine aminotransferase (alt) normalization. results: the therapeutic response at week 104 was 74.8% in telbivudine group versus 50% in lamivudine group (p=0.005). more patients with telbivudine achieved nondetectable serum hbv dna (<300 copies/ml) (p=0.024) and alt normalization (p=0.021) at the end of treatment. the cumulative resistant rate was significantly lower in those with telbivudine treatment (p=0.0032). the rate of hbeag seroconversion was comparable in both groups (p=0.407). although a lower percentage of patients in lamivudine group (83.9%) reported adverse events than those in telbivudine group (89.7%), the difference was not significant. conclusions: telbivudine demonstrates a significantly greater efficacy and a lower resistant rate than lamivudine in treatment of chronic hepatitis b in taiwan. background: tumor necrosis factor-(tnf-) plays a pivotal role in the viral clearance and host immune response to hbv, and the capacity for tnf-production in individuals is influenced by a major genetic component. the studies of tnf--308 gene promoter polymorphism in chronic hbv infection have reported apparently conflicting results. objective: to derive a more precise estimation of the relationship between the polymorphism of tnf--308 gene promoter and chronic hbv infection. method: meta-analysis was done of 22 case-control studies in relation to tnf--308 gene promoter, involving a total of 4338 chronic hbv infection cases and 3013 controls. the pooled odds ratios (ors) for the risk associated with the genotypes of ga, aa, and ga+aa (a-allele carriers) compared with the gg genotype were calculated. results: overall meta-analysis indicated that -308a heterozygotes (ga) had 22% decreased risk of developing chb with a borderline significance (or = 0.78; 95% ci: 0.60-1.02; p = 0.065). for the -308a allele homozygotes (aa) and carriers (ga+aa), the pooled ors both indicated a significantly decreased risk of chb (or = 0.39; 95% ci: 0.21-0.73; p = 0.003; and or = 0.74; 95% ci: 0.57-0.96; p = 0.026, respectively) ( table 1 ). in the subgroup analyses by ethnicity, significantly decreased risks were associated with -308 variant genotypes (ga and aa) in mongoloid populations in all genetic models. however, no significant associations were found in caucasoid. conclusion: the meta-analysis suggests that the tnf--308a allele is a low-penetrant protective factor for chronic hbv infection, especially in mongoloid. aim: to define the potential role of pd-1/pd-lpathway in different hbv infection status; we examined the expression of pd-1on cd8+ t cells in pbmc of patients with chb and aehb infection. methods: the pd-1 level on cd8+ t lymphocytes and the number of hbv specific cd8+ t lymphocytes in patients and healthy controls were analyzed by flow cytometry. pcr was used to measure the serum hbvdna levels. results: the level of pd-1 expression on cd8+ t cells in chb patients was higher than that in aehb patients and healthy individuals. compared to aehb patients, lower frequency of hbv-specific cd8+ t cells was detected in chb patients. there was an inverse correlation between the strength of hbv-specific cd8+ t-cell response and the level of pd-1 expression. besides, there was a significant positive correlation between hbv viral load and the percentage of pd-1 expression on cd8+ t cells in chb and aehb subjects. however, pd-1 expression was not associated with alt levels. conclusion: our results confirm previous reports that hbv specific cd8+ t-cell response in the peripheral blood is more intense in patients with aehb than in chb with persistent viral infection. moreover, there is a negative correlation between the level of pd-1 and the intensity of virus specific cd8+ t cell response. observed in 10.9% of patients with a mean age of 51.8±12.9. the ethnic composition was 53.1% chinese; 27.3% malay; 14% indigenous sabahans; 2.9% indigenous sarawakians; 1.8% indians and 0.8% others. chinese patients were on average, older (mean 45.6±14.5 years), indians patients had higher mean alanine transaminase and indigenous sarawakian patients had the highest rate of cirrhosis (p<0.0001). during the study period, 20.7% of patients were on treatment and they were significantly older than those who were not on treatment (mean age 44.6 ± 14.5 vs 40.7 ± 14.3). lamivudine was the first agent used in 86.9% of cases. conclusions: in malaysia, chb remains a public health issue and significantly afflicts males in the productive age groups and of chinese ethnicity. the observed differences among ethnic groups could point to different disease severity which needs to be addressed in the local treatment guideline and policy. background/aims: liver stiffness measurement (lsm) has been validated for predicting fibrosis stage in patients with chronic hepatitis c. however, studies on lsm for chronic hepatitis b (chb) are few, and the relationship between histologic findings and liver stiffness needs to be further elucidated. this study was conducted to assess the association of histologic activity on liver stiffness in addition to fibrosis in patients with chb methods: thirty three patients who had taken liver biopsy and lsm at korea university ansan hospital between march 2008 and october 2008 were enrolled. necroinflammatory activity and fibrosis stage were assessed by metavir system. activity, fibrosis, and the sum of both score were included for the correlation analysis with lsm results: among 33 patients, 29 (87.9%) were male, and median values were as follows: age, 42 (20~54); ast, 49 iu/l (19~627); alt, 62 iu/l (8~778); total bilirubin, 1.03 mg/dl (0.4~2.39); lsm, 10.1 kpa (4.2~43.5). fibrosis stages were f1 in 4 (12.1%), f2 in 9 (27.3%), f3 in 12 (36.4%), and f4 in 8 (24.2%) patients. spearman correlation coefficient with lsm were 0.457 (p=0.008) for activity, 0.694 (p<0.001) for fibrosis stage, and 0.724 (p<0.001) for the sum of activity and fibrosis. in linear regression analysis, only the sum of activity and fibrosis remained to be significant. conclusions: not only fibrosis but also activity was an important factor for determining lsm for chb. it would be more appropriate to consider both activity and fibrosis for interpretation of lsm in patients with chb background: hbeag seroconversion is a key goal of chb therapy. hbeag kinetics may predict hbeag seroconversion during treatment. we aim to develop a robust hbeag quantitative method as the value of hbeag quantitation is undefined and data is limited. methods: we evaluated two commercially available qualitative hbeag assays (abbott architect, siemens centaur) for their linear range and validated them against paul-ehrlich institute (pei) standards. hbeag levels were determined from samples of untreated and telbivudine-treated chb patients. results: as a pre-requisite for quantitative use, the linear range for the architect (0.5-43 peiu/ml) and centaur (0.05>5 peiu/ml) assays were defined. architect was selected for further investigation. hbeag levels of 44 untreated patients (mean hbv-dna 9.8 log10 copies/ml, mean alt 166.7 iu/ml) varied from 0.4 to 1073.5 peiu/ml (median 161.7 peiu/ml). in 23 patients (mean hbv-dna 9.9 log10 copies/ml, alt 166 iu/ml) treated with telbivudine for 12 weeks, baseline hbeag levels varied from 1.6 to 664 peiu/ml (median 150.7 peiu/ml). after 12 weeks of telbivudine treatment, median hbeag level was 4.4 peiu/ml, with 76% decline from baseline (median decline 95.1 peiu/ml, range 1.4-657.6 peiu/ml). individual hbeag decline from baseline varied but occurred in all patients and was not correlated to baseline or decline from baseline hbvdna. conclusion: hbeag quantitation is feasible and robust with architect hbeag assay. hbeag decline occurred in all telbivudine-treated patients, and was not correlated to hbvdna. whether the magnitude of hbeag decline is predictive of future hbeag seroconversion merits further investigation. experience from the combined globe (nv-02b-007/cldt600a2302) and 015 (nv-02b-015) study clinical safety database. c. avila 1 , r. laeufle 2 , w.b. bao 3 1 novartis pharma ag, fabrikstrasse 6, basel, switzerland, 2 novartis pharma ag, basel, switzerland, 3 novartis pharma, east hanover, us background: creatine phosphokinase (ck) is a commonly used marker of muscle damage and is elevated by many factors (e.g. exercise, injury, drugs). normal ck levels are affected by muscle mass and elevated levels are described during the natural course of chb. 22% of patients in the globe study had pretreatment grade 1-4 ck elevations. methods: we reviewed data from this combined study clinical safety database, and describe the experience of ck elevation and its relationship to adverse event reports of muscle related symptoms. results: the frequency of new onset of grade 3-4 ck elevations in telbivudine-treated patients (combined database itt population) was 1.8% (15/847), 6.3% (53/838), 3.2% (27/826) and 5.1% (40/786) from weeks 0-24, 24-52, 52-76 and 76-104 respectively. the frequency of grade 3-4 ck elevations for all patients from week 0-104 was 12.6% (107/847). the majority of grade 3-4 ck elevations were asymptomatic, rarely resulted in discontinuation or interruption, spontaneously declined within 1 or 2 visits and were not associated with more frequent muscle-related adverse events. cumulative data from this combined database showed no relationship of the degree of increased ck to acute or persistent muscle disease. conclusion: ck elevations are associated with hbv disease and were also common during the globe and 015 trials and were not predictive of the development of muscle related symptoms. onset of muscle-related symptoms should prompt clinical and treatment review, including concomitant medications. backgrounds: leptin plays a crucial role in the regulation of energy balance and body weight control by activating the long form of the leptin receptor (ob-rl). epidemiologic studies showed that obesity is one of the factors associated with hbv related hepatocellular carcinoma. methods: huh7 cells were transiently transfected with 1.3 copies of hbv-replicon plasmid. after 48h, cells were harvested and total rna of the cells were extracted and reverse-transcribed into cdna. long form and short form leptin receptor (ob-rl, ob-rs) mrna transcription levels were assayed by real-time pcr respectively. and mrna transcription levels and protein expression of ob-rl and ob-rs in hepg2.2.15 cells were also detected. results: after transfected by 1.3 copies hbv-replicon plasmid, the mrna transcription level of ob-rl was inhibited significantly (**p<0.01), but the mrna transcription level of ob-rs did not change, and the ob-rl protein expression was reduced. in hepg2.2.15 cells, the mrna transcription level of ob-rl was also significantly lower than the mrna transcription level of ob-rl in hepg2 cells, while the mrna transcription of ob-rs in hepg2.2.15 and hepg2 cells didn't show significant difference. besides, the protein expression level of ob-rl in hepg2.2.15 was also lower than it in hepg2 cells. conclusion: hbv replication down-regulated the expression of long form leptin receptor in cell cultures, which could in part explain the clinical observation of obesity in association with development of serious sequelae in hbv infections. the results of entevavir treatment in patients with chronic hepatitis b s. kose 1 , g. akkoclu 1 , m. turkeri 1 , a. gozaydin 1 1 the ministery of health tepecik training and research hospital, izmir purpose: we evaluated the short and long term effectiveness of entecavir. patients and methods: those patients had received diagnosis of chronic hepatitis b. their pretreatment transaminases, hbsag, anti-hbs, hbeag, anti-hbe, hbv-dna were checked and a liver biopsy and a resistance test for lamivudine (lam) and adefovir (adv) were performed. a total of 44 patients who were taking entecavir for at least 24 weeks were included in the study. findings: the biochemical and virologic response were observed in 88.8 % at 3 and 6 months and in 75 % at 12 months. in 9 hbeag positive patients who had received therapy previously, the biochemical response was observed in 88.8 % at 3 and 6 months and in 100 % at 12 months. the virologic response in 77.7 % at 3, in 88.8 % at 6, and 100 % at 12 months. posttreatment hbeag seroconversion did not develop. in 9 hbeag negative patients the biochemical and the virologic responses were observed in 88.8 % at 3 months and 100 % 6 and 12 months, respectively. in 17 hbeag negative patients had received therapy previously, the biochemical response was observed in 76.4 % at 3, in 100 % at 6 and 12 months. the virologic response in 94.1 % at 3, in 100 % at 6 and at 12 months. conclusion: in our study, a higher therapy-response rate was achieved, especially in hbeag negative patients. in hbeag positive patients biochemical and virologic response rates were high. background/summary: patients with liver disease are known to have a higher prevalence of glucose intolerance. preliminary studies suggest that viruses can be an additional risk factor for the development of diabetes mellitus. individuals with type ii diabetes have an increased prevalence of cirrhosis, and a proportion of patients with acute and chronic liver disease develop diabetes mellitus. there is now emerging epidemiological data to suggest that hepatitis c virus (hcv) infection may also contribute to the development of diabetes reported to be higher than expected compared with the general population. while these investigations suggest an epidemiological association between hcv infection and diabetes, large controlled studies are required to observe association between hbv infection and diabetes. the present study was designed to study the relative proportion of diabetes mellitus in patients suffering from hepatitis b virus (hbv) infection. background: in easl 2008, we reported significant liver disease among hbeag negative patients with serum hbv-dna level <4log10copies/ml (i.e. 2.0x3log10iu/ml) and alanine aminotransferase (alt) <55iu/l. this reflects fluctuating nature of these levels. the aim of this retrospective study is to demonstrate the frequency of fluctuation among this group of chb patients. methods: clinical records of hbeag negative treatment naïve chb patients with at least one serum hbv-dna <4log10copies/ml were reviewed. results: there were 194 (62.5% male, median age 48.0 years) chb patients with negative hbeag and hbv-dna <4log10 copies/ml (roche cobas amplicor pcr assay, lod<300copies/ml). 32 had serial hbv-dna measurements within 2 years; 7 of them (21.9%) had increase serum hbv-dna level by >1log10 copies/ml; 2 patients had associated serum alt elevation from normal (normal range <55 iu/l), 4 had persistent normal alt, and one had persistently raised alt. 5 (15.6%) had serum hbv-dna level decrease by >1log10 copies/ml. another 20 patients had hbv-dna levels fluctuating within 1log10 copies/ml. 159 hbeag negative patients with single hbv-dna measurement showing <4log10 copies/ml had serial serum alt measurements within 3 years. 35 (22.0%) patients had intermittent / persistently raised alt; while 124 (78.0%) patients had persistently normal alt. conclusions: hbeag negative chb is common among chinese. serial serum hbv-dna and alt measurements are necessary to detect fluctuating levels and progressive liver disease that may require antiviral therapy. background: to determine the best vaccination strategy, a model that reflects the country-specific infection profile is needed. methods: a model was built in order to obtain the age-specific infection frequency q(t) for neonates(n), infants(i), children(c), and adults(a). the infected group can either become hbsag(+) or anti-hbs(+)* based on f(t). q(t) can be found from p(t) = [q(t) x (1 -f(t)) x crs + q(t) x f(t) x (1 -cras)], where p(t) represents the proportion of the late anti-hbs(+)** group and crs/cras denote natural conversion of hbsag/anti-hbs. to test the model, cross-sectional serologic marker data in korea were used. because f(t), crs, and cras were known(f(n)=0.1, f(i)=0.5, f(c)=0.8, f(a)=0.95, crs=0.015, cras=0.02), in order to determine q(t), only p(t) values were needed, which were evaluated from logistic modeling using the glm() function of s-plus. results: the infection frequencies during neonate, infant, children, and adult periods in non-vaccinees were 15.4%, 33.3%, 16.6%, and 34.7%, respectively. each group's likelihood of infection compared to adults was then: neonates 170.2 times more likely, infants 33.5 times, and children 0.9 times, making a strong case for neonatal and infantile vaccination for the studied region. conclusions: the hbv infection model can be used for determining the most cost-effective strategy for hb vaccination in nations where longitudinal data are not available. and where longitudinal data are available, it can be used to determine the appropriate time of transition of vaccination strategy to maintain cost-effectiveness. the effect of telbivudine on peripheral blood regulatory t cells and its significance in patients with chronic hepatitis b x.c. pan 1 , f. yang 1 , m. chen 1 1 objective: to investigate the effect of telbivudine on peripheral blood regulatory t cells and its significance in patients with chronic hepatitis b. methods: 36 patients with hbeag positive chronic hepatitis b were recruited and receiving telbivudine treatment for 9 months. before and during 3 6 9 months of treatment , flow cytometry was used to detect the proportion of peripheral blood tregs; real-time pcr was used to detect the levels of hbv dna in surum, markers of hepatitis b virus infection were detected by elisa assay and levels of alanine aminotransferase in serum were measured. results: the proportion of peripheral blood tregs in patients with chb was significantly higher than that in healthy controls and decreased over 6 or 9 months of treatment to a level comparable to that of healthy controls. after 3 months of treatment, the rate of alt normalization in patients which the proportion of peripheral blood tregs was unreduced was significantly lower than that in patients which the proportion of peripheral blood tregs was reduced (p<0.01). 3, 6 or 9 months of telbivudine treatment resulted in negative hbeag in 4(11%) patients, 7(19%) patients or 9(25%) patients respectively. within 9 months of treatment, 7 (19%) patients seroconverted from hbeag to anti-hbe , in which the proportion of peripheral blood tregs had decreased to a level comparable to that of healthy controls over 3 or 6 months of treatment. conclusion: during antiviral treatment with subsequent reduction of the viral load or alt levels, the proportion of tregs decreased to a level similar to that of normal healthy controls. in addition, seroconversion from hbeag to anti-hbe was prone to be established in patients which the proportion of tregs decreased quickly at the early phase of antiviral treatment with telbivudine. background: in china a part of patients with alt <1.5×uln and hbv dna >10 5 copies/ml will advance into hepatic cirrhosis even hepatoma. so these patients should not only be monitored but also be treated. this study was made to determine the safety and efficacy of combining therapy of pegylated interferon alpha 2a (peg-ifn -2a) and entecavir in treating naive patients with alt <1.5×uln and hbv dna>10 5 copies/ml. methods: nine patients with hbsag positive over 6 months and alt<1.5×uln hbv dna >10 5 copies/ml were taken as research subjects. before treatment,liver biopsy was used to assess histological damage. patients were treated with peg-ifn -2a 180 g /week for 48 weeks, and in the first 12 weeks entecavir 0.5 mg/day was applied, then it was stopped. results: 1 liver biopsy showed that 7 patients had mild inflammation. 2 after 12 weeks' treatment , hbv dna level in all patients decreased to less than 10 4 copies/ml, and after 24 weeks' treatment(12 weeks after entecavir was stopped)hbv dna in all patients was less than 10 3 copies/ml. 3 normal alt was seen in all patients after 12 weeks' treatment and 24 weeks' treatment. 4 none of the patients had peripheral neuropathy with combining treatment. conclusions: 1. bulk of patients with alt <1.5×uln and hbv dna>10 5 copies/ml had mild inflammation and need treatment. 2 combing treatment of peg-ifn and entecavir was safe and effective to this group. 3 it proved that it was safe for patients to stop treatment with entecavir after short time use. background & aims: quantification of serum hbv dna levels is important to monitor viral replication in chronic hepatitis b (chb) patients. both abbott realtime hbv and roche cobas amplicor hbv monitor are updated fully automatic commercial assays for hbv dna quantification. the aim of this study is to compare the performance of these two assays on the hbv dna quantification in chb patients. methods: serial serum samples from 30 chb patients were collected at the baseline and at days 4, 7, 10 and 14 and weeks 3, 4, 8 and 12 after the commencement of therapy. genotype was determined by sequence alignment. abbott and roche assays were employed for hbv dna extraction and quantification according to the instructions of manufactories. results: hbv dna quantification results of abbott assay was significantly correlated with those of roche assay (r=0. 972, p<0.0001). for genotype c, the difference in hbv dna levels [median (range): 1.22 (-0.16-2.36) log units] measured by these two assays was significantly higher than that for genotype b [0.66 (-0.52-1.63) log units, p<0.0001]. moreover, the difference in serum hbv dna levels after 12 weeks antiviral treatment [1.18 (-0.21-1.76) log units] measured by these two assays was significantly higher than that in baseline serum hbv dna levels [0.68 (-0.12-1.24) log units, p<0.0001]. conclusion: the quantification results of abbott realtime hbv showed a good correlation with those of roche cobas amplicor hbv. but the performances of these two assays have significant difference in the quantifications of serum hbv dna levels in genotype c patients and in patients after 12 weeks antiviral therapy. background: guidelines suggest hepatitis b virus (hbv) vaccination to all hepatitis c virus (hcv) infected patients and healthcare workers. we attempted to find out hbv vaccination status in our hcv infected population, and healthcare workers. methods: prospective survey of 100 consecutive hcv infected patients and also doctors and paramedical staff in our hospital. results: major sources of viral infection in study patients (58 males; average age 40 years -range 18 to 65 yrs) were reused syringes (38 pts). twenty had a household member infected with hcv. twenty were co-infected with hbv. eighty five of hcv infected patients were not vaccinated.against hbv. twenty five of them (29%) had financial reasons and 45 patietns (52%) had lack of awareness. out of 30 doctors, 12 and 15 did not know about their hbv and hcv status respectively, but none was known to have either of these infections. four (13%) were not vaccinated against hbv. out of 29 paramedical staff, 1 was hcv positive, 11 each were unaware of their hbv and hcv status, and remaining were negative for these markers. thirteen of them (44%) were not vaccinated against hbv. conclusion: thirty eight percent hcv infected patients were infected by reuse of syringes. eighty five percent were not vaccinated against hbv, out of which 52% had no awareness about it, whereas 29% could not financially afford it. a significant number of paramedical staff and some doctors were also not vaccinated background: early prediction of efficacy could decrease unnecessary interferon exposure of patients with chronic hepatitis b. methods: a multi-center clinical study. patients were injected interferon alpha 2b 5 million iu subcutaneously every other day for 24 weeks and 24-week follow-up was followed. results: 53 patients (44 male) were enrolled, 27.5 ±9.1 years old. 24 hours after administration, hepatitis b virus (hbv) load decreased significantly (6.96±1.03log copies/ml, p<0.05) from baseline (8.00±0.93 log copies/ml). hbv load was 4.94±1.47 and 5.25±1.58 log copies/ml at week 24 and 48, respectively. at the two points upwards, complete response rate was 4.2%(2/48) and 7.9%(3/38), partial response rate was 35.4%(17/48) and 34.2%(13/38), respectively. at week 4 and 12, hbv dna levels of complete responder and partial responder were lower than those of non-responder (p<0.05) at week 24. at baseline, on hour 12, day 2, 3, week 2, 4 and 12, hbv dna levels of complete responder were lower than those of non-responder at week 48 (p<0.05). multiple linear regression showed that baseline hbv dna was the independent variables to predict the response at week 24 and 48. conclusions: interferon alpha 2b was effective in treating patients with hbeag positive chronic hepatitis b. it could decrease the hbv dna level rapidly. early hbv dna levels were predictive to response at the end of treatment and follow-up. baseline hbv dna level was the independent predictor of the response at the end of treatment and follow-up. aim: to investigate features of pd-1 expression on peripheral tcells and pd-l1 expression in liver in chronic hepatitis b (chb) patients in immune clearance phase. methods: pd-1 expression on total peripheral t cells were evaluated by using flow cytometry. immunostaining was performed according to the envision chemmate methods. the degree of pd-l1 expression was scored and assessed according to the percentage and staining intensity of positive cells. results: compared to health control, the percentage of total peripheral t cells expressed pd-1 was elevated in chb with repeatedly increasing alt level. no specific association between the percentage of pd-1 positive and the mean fluorescence intensity mfi of pd-1 expression on total t cells with serum viral load were found. but alt level was correlated with the mfi of pd-1 expression on total cd8+t cells significantly. pd-l1 is up-regulated on hepatocytes by viral infection, and high expressed in fibrosis section. conclusion: the mfi of pd-1 on cd8+t cells plays important role in regulating the immune-host interaction in chb in immune clearance phase. and pd-1 expression on t cells is correlated with high immune inflammatory refection. aim: to study the quantity, characteristic of hbv-specific t-cell and the extent of liver damage in chronic hepatitis b (chb) patients with different hbeag status. methods: 103 chb patients were enrolled and divided into two groups according to the hbeag status, and the liver damage index were analyzed. the frequency and foxp3 expression of cd4 + cd25 + regulatory t cells (treg) were measured, as well as the frequency and phenotypic molecules expression of hbv-pentamer+ t-cell. hbv specific t-cell responses including cellular proliferation and ifn-production, with or without anti-pd-l1 and/or anti-ctla-4 blocking, were also observed. results: the demographic characters, serum alt, ast levels, the frequency and foxp3 expression of cd4 + cd25 + treg were similar, while the serum hbv dna levels were higher in hbeag+ patients (p <0.05). the liver necroinflammation was comparatively more severe in hbeag-patients (p =0.056), but the median percentage of liver cirrhosis was much higher in hbeag+ patients (p <0.05). the difference of hbv-specific t-cell frequency was not significant between two groups, while the expression levels of pd-1 and ctla-4 on hbv-specific cd8 t cells were significantly higher in hbeag+ patients (p both <0.05). combined using of anti-pd-l1 and anti-ctla-4 mab significantly increased the cellular proliferation in either hbeag+ or hbeag-patients, but only markedly enhanced the ifnproduction in hbeag+ patients. conclusion: hbeag persistency could probably induce higher expression of pd-1 and ctla-4 on the hbv-specific t cells and result in t-cell impairment, high hbv dna load and high percentage of liver cirrhosis in hbeag+ chb patients. hepatocyte apoptosis in patients with chronic hepatitis b y. liu 1 , k. wang 1 1 background: to investigate the relationship between hepatocyte apoptosis and the level of inducible nitric oxide synthase (inos) in hepatic tissue in the patients with chronic hepatitis b chb . methods: we observed 37 cases with chb and 10 normal controls. transferase-mediated-utp-biotin nick-end labling ( tunel) technique was used to detect apoptosis cells and immunohistochemical staining were also performed to investigate the expression of inducible nitric oxide synthase (inos) in biopsy samples .the serum level of alt hbv-dna grading of necroinflammatory activity and staging of fibrosis were also assessed. results: hepatocytes in all chb liver tissues were positively stained by tunel in various degree. in contrast, control tissues did not show dna fragmentation. a significant correlation was seen between apoptosis index (ai) and necroinflammatory grading ((r=0.404, p=0.015) and serum inos level r=0.465, p=0.004 . it did not correlate with fibrosis stage and serum alanine aminotransferase level. conclusion: the oxidative stress.in patients with chb may reflected the apoptosis of hepatocyte. apoptosis involves in liver injury of chb,but with no significant correlation to serum level of alt. objectives: to investigate the genotype-dependent development of lamivudine resistance in hepatitis b virus (hbv). methods: 215 patients with chronic hepatitis b who had been treated with lamviudine for more than 1year, and become lamviudine resistance were analysed for the hbv genotypes and cumulative rate of rt region mutant with standard dna sequencing technology. results: among the 215 patients, 60 patients were infected with hbv genotype b (hbv/b)(27.9%), and 155 with genotype c (hbv/c)(72.1%). in the hbv/b patients, 16/60(26.7%) were of subtype ba, and 44/60(73.3%) were of of subtype bj. the cumulative type and ymdd mutation rates in patients with genotype c were showed as l180m+m204v (67/155,43.2%) > l180m+m204i (52/155,33.5%) > m204i (36/155, 23.3%), while in patients with genotype b as l180m+m204v 36/60(60.0%) > m204i(24/60, 40.0%), none of l180m+m204i. conclusions: our results indicated that in patients with lamivudine resistance, hbv genotype c (hbv/c) were higher than genotype b (hbv/b). in both genotypes the combined mutations (180+204 sites) were found more than the single 204 site, showed some significance for monitoring lamivudine resistance. background & aims: il-35, a novel identified inhibitory cytokine specifically produced by regulatory t cells (tregs), is an ebi3-il-12 heterodimer encoded by epstein-barr-virus-induced gene 3 (ebi3) and interleukin-12 alpha (il12 ). the aim of the study is to determine the expression levels of il-35 in peripheral blood mononuclear cells (pbmcs) of chronic hepatitis b (chb) patients in different phases. methods: a total of 36 treatment naïve chb patients, including 17 in immune-tolerant phase [group 1, alt: 21 (12-51) u/l, serum hbv dna: 2.48 x 10 9 (6.30 x 10 6 -1.20 x 10 10 ) copies/ml] and 19 in immune-clearance phase [group 2, alt: 221 (71-1530) u/l, serum hbv dna: 5.10 x 10 8 (1.62 x 10 6 -1.77 x 10 10 ) copies/ml] were enrolled in the study. the relative mrna expression levels of ebi3, il12 and foxp3 were determined by semi-quantitative pcr. results: the significant correlations were observed between the expression of ebi3 and il12 (r=0.661, p<0.001), ebi3 and foxp3 (r=0.388, p<0.05), il12 and foxp3 (r=0.431, p<0.01). the relative expression levels of ebi3 and il12 in pbmcs were significantly higher in group 2 when compared with those in group 1 (1.46 ± 0.23 vs 0.80 ± 0.10 and 1.26 ± 0.19 vs 0.65 ± 0.09, p<0.05, respectively). furthermore, the relative expression levels of ebi3 and il12 in group 2 were significantly correlated with alt levels (r=0.473, r=0.474, p< 0.05, respectively), but not with serum hbv dna levels. conclusions: the expression levels of il-35 in pbmcs were significantly higher in chb patients in immune-clearance phase than that in immune-tolerant phase. increased il-35 expression levels were associated with liver injury. background: there are a number of oral antivirals approved for chronic hepatitis b. lamivudine, the first oral nucleoside analog, is associated with increased rates of drug resistance with prolonged use--from 20% at one year to 50% at three years. therefore, an alternative or add-on treatment is necessary. adefovir, an oral nucleotide analog, is used either in combination with lamuvudine or as monotherapy in lamivudine-resistant chronic hepatitis b. we did a meta-analysis to compare the efficacy of adefovir in combination with lamivudine versus adefovir alone in the treatment of lamivudine-resistant chronic hepatitis b infection. methods: a comprehensive literature search was performed using the following databases: medline, cochrane, and embase. a total of 3 randomized controlled trials were retrieved and analyzed. outcomes measured were virologic response, biochemical response and resistance rates. results: meta-analysis on virologic response showed that combination treatment with adefovir and lamivudine is as effective as adefovir monotherapy (or 1.04, 95% ci 0.50-2.15, p=0.92). likewise, in terms of biochemical response, both regimens were equally effective (or 1.06, 95% ci 0.47-2.40, p=0.90). one study showed statistically significant increase in adefovir resistance rate in the monotherapy arm compared to combination arm (p= 0.0182) after the first year of therapy. conclusion: in patients with lamivudine-resistant chronic hepatitis b infection and compensated liver disease, adding adefovir to lamivudine is as effective as switching to adefovir alone in terms of virologic and biochemical response. r. safadi 1 , q. xie 2 , y.g. chen 3 , y.k. yin 4 , l. wei 5 , s.g. hwang 6 south korea, 7 bnai zion medical center, haifa, israel, 8 beijing friendship hospital, beijing, china, 9 novartis pharma ag, basel , switzerland background: ldt produces greater viral suppression than lam. we investigated whether patients receiving lam can benefit from switching to ldt. methods: hbeag positive and negative persistently viraemic patients (median hbv dna 5.0 (ldt), 5.3 (lam) log 10 copies/ml) and lam treated for 3-12 months, were randomized to either switch to ldt or continue lam. we report the benefit of ldt switch assessed by primary treatment failure (tf, <1 log hbv dna decline) and viral breakthrough (vb, >1 log above nadir). results: 17% (21/122) of the ldt switch and 15% (18/124) continuing lam patients had pre-existing m204 mutations at screening. tf was 5% (ldt) versus 21% (lam, p<0.05). in patients with >24 weeks prior lam treatment, tf was 10% (ldt) versus 41% (lam). 83% ldt tf (5/6) was associated with resistance at screening versus 56% lam tf. in ldt switch with <24 weeks prior lam, no ldt tf occurred versus 12% lam. in hbeag positive, tf occurred in 6% (ldt) versus 29% (lam). among hbeag positive with >24 weeks prior lam treatemtn, vb was 19% (ldt) versus 44% (lam, p<0.05). differences were not significant for hbeag positive with >24 weeks lam or for hbeag negative regardless of duration of prior lam treatment. conclusions: early switch to ldt is associated with better virological outcomes in these patients. persistent viraemia for >6 months on lam treatment is associated with a high risk of tf and vb. for these patients, genotypic analysis is recommended prior to screening. objective: the aim of this study is to evaluate the proper endpoint in the treatment of chronic hepatitis b with antivirals by investigating the viral rebound ratio after one year's nucleosides or (three months) sustained treatment with lamivudine, adefovir, entecavir, or interferon when viral response and seroconversion response have been finished . methods: eag positive chronic hepatitis b naïve patients with alanine aminotransferase (alt) more than 2 uln were assigned to receive 100 mg of lamivudine, 10mg of adeforvir, or 0.5mg of entecavir once daily, respectively. patients in the interferon group were administrated with 5,000,000 iu of 2a interferon on every other day, and the therapeutic duration lasted for another three months after eag-ab seroconversion appeared. hbv dna and eag-ab in the serum were tested during the off-treatment period of 12 months. results: thirty four patients in lamivudine group of 148 cases got eag-ab seroconversion after treatment with 20±5 months of average duration, and the viral rebound ratios in the off -treatment 6 an 12 months follow up period were 20.6 7/34 and 40.1 15/34 , respectively. in adeforvir group were 19 4/21 and 33. 7/21 . in enticavir group were 20 3/15 and 46.7 7/15 . in interferon group was 16.2 6/37 in the off-treatment 6 months follow up period. conclusions: we conclude that eag-ab seroconversion in the treatment of eag positive chronic hepatitis b patients is the goal but not an endpoint of therapy physicians should aim at. to gain everlasting effect, longer duration of treatment may be needed. background: universal hepatitis b(hb) vaccination of hbsag negative people (especially infants) is widely recommended and practised. objective: to assess whether there is robust evidence of protective efficacy to back such practice. methods: this cochrane review included randomised trials identified from six databases through detailed electronic searches. trials comparing hb vaccine versus placebo/another vaccine, in hbsag negative persons were included without any restrictions. the primary outcome was hb infection (developing hbsag or anti-hbc). robustness of evidence was assessed through comparison of available-case analysis versus intention-to-treat(itt) analysis using four different models: (i)assuming unfavourable event for all missing data, (ii)assuming favourable outcome for all missing data, (iii)best-case-scenario and (iv)worst-case-scenario results: twelve trials were eligible among 2964 citations; all were methodologically poor (high risk of bias). data from four trials could be included in meta-analysis. efficacy of vaccination varied with the type of data analysis. available-case analysis suggested efficacy in reducing risk of developing hbsag (rr=0.17;95%ci=0.09-0.31;n=1341) and anti-hbc (rr=0.42;95%ci=0.31-0.57;n=1235). itt analysis results varied depending on the model chosen (table) , but liberal approaches suggested high efficacy, whereas conservative approaches did not. the available evidence on efficacy of hb vaccination in hbsag negative people is not robust; there are serious limitations in quality and quantity. background: open-label rollover study (etv-060) assessed histologic improvement in chb patients on at least 3 years etv therapy. methods: 100% nucleoside-naïve patients and 98% lamivudine (lvd)-refractory patients from etv-053 and etv-052 studies, respectively, entered etv-060 study and received etv at 0.5/1mg for greater than 96 weeks. improvement in knodell necroinflammatory (ni) score and knodell fibrosis score at weeks 48 and 148 were studied. results: at week 148, 95% of nucleoside-na ve patients and 56% of lvd-refractory patients achieved hbv dna <400copies/ml. furthermore, 95% of nucleoside-na ve patients and 93% of lvd-refractory patients had normalized alt levels. mean platelet counts in both naïve and lvd-refractory patients were improved at weeks 48 and 148 compared with baseline. conclusions: naïve and lvd-refractory chb patients showed significant improvement in liver histology after 3 year etv therapy, and improved dna and serum alt levels. results: 84.7 percent of patients were younger than 30 years old, 15.3 percent were older than 30 in this study. 48.0% patients' mothers were hbsag positive. high levels of serum hbv dna were founded in all patients, >10 7 copies/ml were 78.6%. only 5 cases (5.1%) whose liver inflammation grade were g0, the rest patients were mild inflammation, in which g1 were 64 cases (65.3%), g2 were 29 (29.6%); there were 56 patients (57.1%) had no signifecant liver fibrosis, the rest 42 cases (42.9%) had different fibrosis, among those s1 were 23 cases (23.5% , s2 were 14 14.3% , s3 were 5 5.1% , none of patients had cirrhosis. the fibrosis stages of higher alt level were markedly severer than lower alt in patients with normal alt p < 0.01 . conclusions: most of patients with chronic hepatitis b virus in immune tolerant phase present mild inflammation in liver, part of them have already appeared fibrosis, so some patients determinated by clinics are actually not in immune tolerant phase. although alt testing are in the normal range, but the possibility of liver fibrosis is increased in patients with relative higher alt level, so liver pathology should be recommended to judge illness correctly. background/aims: hepatitis b virus infection (hbv) is a global health problem. in bangladesh, 5-7% of people are hbsag positive. this study was carried out to evaluate the efficacy and safety of peginterferon alfa-2a in chronic hepatitis b patients. methods: a total of 60 patients with chronic hepatitis b, 32 (53.3%) were hbeag positive (group a) while 28(46.7%) were hbeag negative (group b) were included in this study after meeting the following criteria: age 18 to 60 years, hbsag positive for more than 6 months, serum hbv-dna was >5 log(10) copies/ml and alt more than two times the upper normal limit. they were given peginterferon alfa-2a (180 microgram once weekly) for 24 weeks and followed for an additional 24 weeks. results: after 24 weeks of follow-up, the percentage of patients with normalization of alanine aminotransferase levels or hbvdna levels below 20,000 copies per milliliter was significantly higher in hbeag positive patients (59 percent and 43 percent, respectively) than among hbeag negative patients (45 percent and 33 percent). loss of hepatitis b surface antigen occurred in 3 patients in group a, as compared with 1 patients in the group b (p<0.01). adverse events including pyrexia, fatigue, myalgia, headache and haematologic abnormalities were similar in both groups. conclusions: patients with hbeag positive chronic hepatitis b had significantly higher rates of response, sustained for 24 weeks after the cessation of therapy, with peginterferon alfa-2a. background: the effect of hepatitis b vaccination on individuals with isolated anti-hbc in endemic areas is not clear. we investigated the prevalence of individuals positive for anti-hbc only and their antibody response after hepatitis b vaccination in a single healthcare center. methods: the study included 1,812 healthcare workers. after screening for hbsag and anti-hbs, the individuals negative for both hbsag and anti-hbs were examined for anti-hbc and were vaccinated with a recombinant hepatitis b vaccine at 0, 1, and 6 months. the serum anti-hbs level was measured after the vaccination. results: of the subjects, 334 (220 females) were negative for both hbsag and anti-hbs. forty (2.2%) subjects had isolated anti-hbc, including more males (60.0% vs. 30.6%) and older people (45.7±8.2 vs. 37.3±8.5 years), compared with individuals negative for all of the viral markers. the anti-hbs seroconversion rate and anamnestic response in the individuals with isolated anti-hbc after the first vaccine injection were 60% and 27.5%, respectively. in the 294 persons who were negative for all hepatitis b viral markers, the seroconversion rate after the first vaccination was 52.6%. the anti-hbs seroconversion rate did not differ between the isolated anti-hbc positive individuals and those negative for all hepatitis b markers (89.5% vs. 96.6%) after the full course of vaccination. conclusions: serum hbsag and anti-hbs tests are sufficient for screening before hepatitis b vaccination, especially in healthcare workers. objective: to understand the quantity and distribution of cd83 + mature dendritic cells in patients with hepatitis b virus in immune tolerant phase. methods: there were 30 immune tolerant phase patients with hepatitis b virus infection (fibrosis stages were s0), 10 immune clerance phase patients, 10 non-active status patients and 5 healthy controls involved in our research. the quantity and distribution of cd83 + mature dendritic cells in liver were determined by immunohistochemical staining. result: the liver inflammation grades were between g1-g2 in patients who in inmmune tolerant phase and non-active status, moreover, patients in immune clerance phase were between g2-g4. there were a small amount of cd83 + dendritic cells in healthy liver tissue, scattered in portal areas and hepatic lobules. the quantity and distribution of cd83 + dendritic cells in patients who in inmmune tolerant phase and non-active status were similar to the healthy, and the quantity were no difference among them p 0.05 .the number of cd83 + cells in patients of immune clerance phase was significant increased compared with other groups, there were differences among them p 0.05 , the cd83 + cells mainly distributed in portal areas infiltrated with inflammatory cells and hepatic lobules with inflammatory necrosis. conclusion: cd83 + mature dendritic cells are involved in liver immune response in patients of inmmune clerance phase, is likely to related to hepatitis b virus clearance. lack sufficient mature dendritic cells may be one of the mechanisms of immune tolerance. background: local hospitals provide obstetric services including antenatal care to women normally living in the mainland china, whose prevalence of hepatitis b carrier is unknown. objectives: compare prevalence of hbv carrier of pregnant women from the mainland china with local counterparts and discuss the implications of results. materials and methods: antenatal serological results were retrieved from corporate laboratory information system databases. pregnant women from the mainland china were identified by a specific set of temporary-allocated identity number during january 2007 -october 2008. results: 7491 pregnant local residents and 1397 pregnant women from the mainland china underwent antenatal serological tests for hepatitis b surface antigen. positive hepatitis b surface antigen results were more frequent in pregnant women from the mainland china (12.7%) than in local pregnant women (8.17%) (p<0.001). discussion: because infected pregnant women can transmit the hepatitis b virus to the infant at delivery, specific management could entail maternal medication, injection of hepatitis b immune globulin to the infant at birth and immunization later on. however, early repatriation to the mainland china, which is common, will make completion of immunization program difficult. these babies will be at a higher risk to be infected by hbv, particularly when breast-fed by hbv carriers. their return to hong kong later will dilute the effects of local immunization program. the volume of work derived from the provision of obstetric services to women from the mainland chinese is larger with regard to medication, counseling and immunization for babies born to hbv carriers. immunosuppressive or anticancer therapy k. hirano 1 , t. kodani 1 , s. sato 1 , y. narita 1 , t. kikuchi 1 , t. genda 1 , k. iijima 1 , k. ogawa 1 , t. ichida 1 1 background/aim: we compared the prevention of hbv reactivation in (hbsag)-positive patients with hbsag-negative patients who were positive for antibody to (anti-hbc) and/or (anti-hbs) undergoing immunosuppressive, anticancer or molecular target therapy. methods: from sep 2004 to nov 2008 hbsag-positive patients and 22 anti-hbc and/or anti-hbs-positive patients were enrolled in this study. we compared with 2 groups about background disease, age, blood examination, and nucleoside analogues. results: in hbsag-positive patients mean age were 56.6±10.2 years old, median ast levels were 30 (18-706) iu/l, and median alt levels were 36 (13-544) iu/l for 8 (47%) haematological disease and 9 (53%) collagenosis disease. in anti-hbc and/or anti-hbs-positive patients mean age were 71.3±9.6 years old, median ast levels were 20 (9-106) iu/l, median alt levels were 16.5 (5-247) iu/l for 17 (77%) haematological disease and 5 (23%) collagenosis disease. serum hbv-dna levels >5.0 log copies/ml were 8 (47%), 2.6~5.0 were 7 (41%), <2.6 were 2 (12%) in hbsag-positive patients, and serum hbv-dna levels <2.6 were all cases in anti-hbc and/or anti-hbs-positive patients. 14 (82%) of hbsag-positive patints received nucleoside analogues (7 lam and 7 etv), and 12 (55%) of anti-hbc and/or anti-hbs-positive patients received nucleoside analogues (8 lam and 4 etv). mean duration of treatment for 14.2 months in hbsag-positive patients, and for 4.1 months in anti-hbc and/or anti-hbs-positive patients, the resistance virus occurred to 3 (75%) of 4 hbsag-positive patients treated with lam for collagenosis disease more than two years. conclusion: when hb carriers of collagenaous disease undergoing immunosuppressive therapy required the nucleoside analogues more than two years, we recommended treatment to prevent hbv reactivation with etv. background: adefovir dipivoxil is used for the initial treatment of chronic hepatitis b or rescue treatment of lamivudine-resistant chronic hepatitis b, and exhibits excellent antiviral activity. however, the presence of resistance to adefovir dipivoxil was more frequently in lamivudine-resistant chronic hepatitis b patients than in lamivudine-naïve patients during adefovir dipivoxil monotherapy. but the rate of adefovir resistance related mutations is little known in lamivudine-resistant patients before adefovir dipivoxil treatment. the aim of this study was to investigate the rate of adefovir resistance-related mutations in polymerase gene of hepatitis b virus in lamivudine-resistant patients not treated with adefovir dipivoxil. methods: the existence of adefovir resistance-related mutations was examined in 240 lamivudine-resistant chronic hepatitis b patients with breakthrough hepatitis and 100 antiviral-naïve chronic hepatitis b patients. both polymerase chain reaction restriction fragment length polymorphism (pcr-rflp) and directly sequencing of pcr product were used to detect resistant viruses. results: rta181t mutants were detected in only two sera of lamivudine-resistant patients, while none in the antiviral-naïve chronic hepatitis b patients. there was no rtn236t detected in the two groups. conclusion: our results suggest that the rta181 mutant virus were present in a few lamivudine-resistant chronic hepatitis b patients before they have been treated with adefovir dipivoxil, but the rtn236t mutant was not detected in any of the two groups. the rate of adefovir resistance-related mutations in polymerase gene of hepatitis b virus was low in such lamivudine-resistant patients before adefovir dipivoxil treatment. objective: in this study, we tried to detect and identify the special protein of hbv related chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. to find new opinion on the developing of chronic liver disease. methods: the sera of health adult, hbv related chronic hepatitis, liver cirrhosis and hepatocellular carcinoma were respectively detected by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (seldi-tof-ms). the arrays of every group were analysised by clustering analysis and to establish disease predictive model. then the sample was eluted with different ph tris, trypsinization on-chip, mass determination and peptide database comparison. results: according cm10 chip we find 18 protein with obviously deviation (p<0.05) among hbv related chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. clustering analysis for the data from seldi-tof-ms confirmed 42 differentially expressed proteins. then we developed disease predictive mathematic models ( decision tree model, dt model ) with average validity up to 73.9 . the 5805 da protein peak was identified to be chondroitin sulfate synthase 2 (chss2), which is a potential molecule involved in the pathologic process and a potential serum marker for the hbv related hepatic diseases as well. conclusions: our results suggest that seldi-tof-ms is a usefull technique for differential expressed proteins screening and analysis in hbv related chronic liver disease. chss2 may be useful during the developing of hbv related chronic liver disease. backgound: clevudine is a new nucleoside analogue with potent antiviral activity in chronic hepatitis b patients. however, the efficacy and safety of clevudine in cirrhotic patients are not well recognized. this study was conducted to evaluate the early virologic and biochemical response rate as well as safety of clevudine in cirrhotic patients with chronic hbv infection. methods: 46 patients with chronic hbv infection who visited korea university ansan hospital and guro hospital between may 2007 and may 2008 were included. 24 patients had chronic hepatitis b (group a) and 22 had liver cirrhosis (group b). early virologic response was defined as hbv dna less than 200 iu/ml at week 12. early biochemical response was defined to be normalization of alt (<45 iu/l) at week 12. result: pretreatment hbv dna levels were higher in group a compared with group b (8.06 log iu/ml vs 7.09 log iu/ml, p=0.06). pretreatment alt levels were not significantly different between the two groups (166 iu/l vs 139 iu/l, p=0.725). the rate of early virologic response was significantly higher in group b compared with groups a (72.7% vs 33%, p=0.01). the rate of early biochemical response were not significantly different in both groups (75% vs 72.7%, p=0.725). conclusion: clevudine is considered to be safe and effective in cirrhotic patients with chronic hbv infection as well as chronic hepatitis b patients. long term safety and efficacy need to be evaluated in the future. objective: the aim of this study was to evaluate the role of nucleos(t)ide analogues against hbv reactivation in immunosuppression. methods: non-active hbsag carriers suffering from cancer, autoimmune diseases and needing the treatment of immunosuppressants or cytotoxic chemotherapy were enrolled in the study. the outpatients or in-patients from april 2007 to july 2008 were enrolled. the nucleos(t)ide analogues were used in cancer patients 1-2 weeks before chemotherapy, and the duration lasted 6-12 months according to patients' compliance after completion of chemotherapy. patients with other diseases used nucleos(t)ide analogues in 1-3 months before using glucocorticoids or other immunosuppressive agents, and continued to use for 6-12 months after accomplishing the course of immunosuppressant treatment. the characheristics and clinical manifestations about hbv reactivation were investigated. results: of the thirty two patients in prospective group, twenty two patients suffered from cancer, eight patients suffered from idiopathic thrombocytopenic purpura, two patients suffered from chronic nephritis. the amount of hbv dna was detected in the first, third, sixth and 12th month after the use of nucleos(t)ide analogues. after chemotherapy or immunosuppressant treatment, only 9.4% (3 / 32) of them suffered from hbv reactivation, which presented with hbv dna positive and abnormal liver function. conclusion: non-active hbsag carriers would appear potential incidence of hbv reactivation during use of chemotherapy or immunosuppressant. nucleos(t)ide analogues could be used in early phase as prophylaxis for reactivation of hepatitis b in immunosuppression and to improve clinical prognosis. background: hbv therapies are evolving toward combination antivirals. this study evaluated the combination of clevudine (clv), a potent nucleoside analog, with tenofovir dipivoxil (tdf). methods: a phase i, single-arm, multi-dose study in 18 healthy adult volunteers to evaluate pharmacokinetic and safety interactions between clv and tdf. subjects received 21 days of clv 30mg followed by 7 days of clv 30mg +tdf 300mg. pk profiles were obtained on days 1, 21 and 28. clv auc and cmax were compared on days 21 and 28. day 28 tenofovir pk was compared to historical data. safety assessments were conducted throughout. results: 18 subjects were enrolled (13m/5f);17 completed the study. the mean (range) age was 37y (22-49) and body mass index (kg/m2) was 27.6 (22.9-31.9). 22 aes were reported by 6 subjects, with 10 aes reported during clv-only dosing and 12 aes reported during clv+tdf dosing. aes included nausea (2) and pharyngolaryngeal pain (2). the majority of the aes were mild. there were no clinically significant changes in ecgs or laboratory parameters. comparisons of clv auc and cmax on day 21 and 28 revealed no significant impact of tdf upon the plasma clv exposure (d28/d21 auc ratio=0.98, d28/d21cmax ratio=0.89). there is no significant effect of clv on tenofovir when comparing auc and cmax of tdf to historical values. conclusion: safety and pharmacokinetic results demonstrate that clv and tdf may be safely co-administered, supporting the further study of this drug combination for the treatment of chronic hbv infection. s. kuznecovs 1,2 , i. kuznecovs 1 , k. jegina 2 , g. kuznecova 1 1 background: dolichyl (dol), the main lipid intermediator of dolichyl phosphate cycle (dpc) has been reported to be elevated in urine of patients with multidrug resistance in cancer. drug resistance poses a major threat to nucleoside analogue-based therapies for chronic hbv infection. methods: with focus on a risk predictor for susceptibility to the development of hbv drug resistance the present study was carried out to estimate urinary levels of dol in chronic hbv infection. the samples obtained every week before and during the course of treatment from 42 patients with hbv. the occurrence of exacerbations of chronic hbv were registered for 2 years. dol in urine was assayed by hplc method. results: the normal amounts of dol in healthy persons urine (n=1500) are 6,0 + 10,0 mkg/mmol creatine. during the period of observation 36 (86%) of patients treated with nucleoside analogue-based therapies were diagnosed with exacerbations due to resistance of hepatitis b virus to antiviral drugs. from this group of hbv patients 35 (98%) have had elevated urinal dol excreation (45,8±5,2 g/ml vs . 8,2±1,9 g/ml, p<0.0001) in more than 3 months of observation. conclusion: there is a reason to suggest that elevated urinal dol detected in patients with exacerbations during hbv treatment may evidence of possible defect of host mechanism of drug resistance development to nucleoside analogue-based therapies. the interest drawn to the employment of dol as a predictor for exacerbation of chronic hbv is explained by the role of dpc in p-glycoprotein regulation in human hepatocytes. background: a significant proportion chb patients treated with adv have a suboptimal response, increasing the risk of disease progression and development of resistance. we report clinical results from patients who either failed or relapsed following adv therapy and were subsequently switched to etv. methods: study etv-079 was a randomized, open-label study comparing antiviral efficacy of etv (0.5mg/day) vs adv (10mg/day) in nucleoside-naïve hbeag-positive patients. after up to 96 weeks of treatment in etv-079, 24 patients treated with adv (13 suboptimal responders) rolled over into study etv-901 (1.0mg/day). hbv dna viral suppression and safety was evaluated during 48 weeks of etv treatment. results: at entry to etv-901, the median hbv dna was 5.72log10 copies/ml. median exposure to etv (1.0mg) in etv-901 was 46 weeks and 18 patients currently remain on study therapy. at week 24, the mean reduction in hbv dna was 4.5log10 copies/ml and 8/16 (50%) reached hbv dna levels <300 copies/ml. nine patients have achieved week 48 and all have achieved hbv dna <10 4 copies/ml and 8/9(89%) had hbv dna levels <300 copies/ml. no patients experienced virologic breakthrough on etv. the safety profile of etv in adv-treated patients remained consistent with the previously reported experience. conclusions: the majority of patients who were suboptimal responders or virologic rebounders following adv treatment in study etv-079, experienced rapid reductions in hbv dna levels when switched to etv. hbv dna levels continued to decline to undetectable levels with 48 weeks of etv treatment. y. li 1,2 , t. han 1 1 tianjin third central hospital, 2 aim:to quantify hepatitis b virus (hbv) total dna and covalently closed circular dna (cccdna) in liver biopsies and sera which from chronic hepatitis b(chb) liver cirrhosis of hepatitis b(lc) and hepatitis b relevance hepatocellular carcinoma(hcc) patients, and analysis hbv replication under the circumstances of different diseases. methods:total hbv dna and cccdna in serum and liver biopsy samples were measured in 21 chb 23 lc and 25hcc patients by the real-time pcr assay. results: the levels of total hbv dna in serum,intrahepatic total hbv dna, intrahepatic cccdna, as well as the proportion of intrahepatic cccdna in total hbv dna decreased progressively in chb,lc and hcc ,moreover chb had significantly higher levels of total hbv dna in serum and liver biopsy samples than lc (log [total serum hbv dna] p = 0.024;log [total intrahepatic hbv dna] p = 0.034); chb and lc had significantly higher levels of intrahepatic cccdna and the proportion of intrahepatic cccdna in total hbv dna than hcc(p <0.01); cccdna couldn't be detect in all patients'serum. in chb ,the levels of serum's total hbv dna,intrahepatic total hbv dna and cccdna in hbeag-positive group had significantly higher than the hbeag-negative group(p <0.01) ,but in lc only intrahepatic total hbv dna had statistical difference between hbeag-positive and negative group (p=0.026) , no statistical difference between hbeag-positive and negative group in hcc. conclusions: the replication activity of hepatitis b virus in chb,lc were higher than hcc, hbv reproduction reduced significantly in hcc. duplication of hbv in lc was lower than chb but had no statistical difference. the levels of hbv reproduce in hbeag-positive group was higher than hbeag-negative group of all three desease. background: clevudine is a pyrimidine analogue with potent and sustained antiviral activity against hbv in the 24 week therapy. the present study assessed the efficacy and viral resistance of 48 week clevudine therapy in patients with chronic hepatitis b. method: a total of 42 patients (26 hbeag positive and 16 hbeag negative) who were received clevudine 30 mg once daily for 48 weeks were included in this analysis. serum hbv dna was quantified by real time pcr assay. result: at week 48, median reductions of serum hbv dna from baseline were 4.98 log10 iu/ml (5.00 log10 iu/ml for hbeag positive and 4.95 log10 iu/ml for hbeag negative) and 76.2% of patients showed undetectable serum hbv dna (<60 iu/ml) (65.4% for hbeag positive and 93.8% for hbeag negative). the normalization of alt levels (<35 iu/l) was achieved in 81.0% (88.5% for hbeag positive and 68.8% for hbeag negative). 15.4% of hbeag positive patients showed hbeag loss or seroconversion. hbv dna negativity at week 48 was associated with hbeag negativity (p =0.037), hbv dna <2,000 iu/ml at weeks 4 and 24 (p =0.019 and 0.001, respectively). two hbeag positive patients showed viral breakthrough with m204i mutation during 48 week. conclusion: clevudine therapy in patients with chronic hepatitis b showed potent virologic responses at week 48, especially in those with hbeag negativity and complete early virologic response (hbv dna <2,000 iu/ml at weeks 4 and 24). but clevudine resistance can occur in hbeag positive patients. background: serum alanine aminotransferase (alt) activity, the variable most commonly measured to assess hepatic disease, fails to identify many patients with hepatic injury. current standards for "normal" alt level were defined by using populations that included persons with subclinical liver disease. there is no study regarding normal level of alt and its modulating factors in healthy thai people. objective: to definitions of normal ranges for serum alt level in thai people. design: prospective observational study setting: phramongkutklao hospital and army institute of pathology pramongkutklao medical center(a.i.p.), bangkok, thailand participants: 200 persons who were first-time blood donors from august through december 2007 were negative for anti-hepatitis c virus(hcv), negative hbsag(hbv), and had no contraindications to donation. measurements: univariate and multivariate analyses examined associations between clinical and laboratory factors and alt levels. normal ranges for alt were computed from the population at lowest risk for liver disease. results: serum alt activity was independently related to body mass index, age, alcoholic consumption and to laboratory indicators of abnormal lipid or carbohydrate metabolism. normal ranges for serum alt level in thai people upper limits for men 26 u/l and for women 21.67 u/l. conclusion: in men serum alt is strongly associated with body mass index, age, alcoholic consumption and to laboratory indicators of abnormal lipid or carbohydrate metabolism. the normal range of alt should be defined for male and female separately. background: the open-label rollover study etv-060 was conducted after etv phase ii clinical study etv-047 for nucleoside-na ve adult chb patients in japan. in this analysis, we report etv long-term efficacy and safety in patients who were switched from 24-week lvd treatment to etv therapy. methods: ninety-seven percent (33/34) of lvd-treated patients from etv-047 were rolled over into etv-060 treated with 0.5mg of etv. thirty patients completed 96 weeks of etv therapy and were evaluated for hbv dna level, alt normalization, hbeag seroconversion, resistance and safety. results: comparing to baseline before switching to etv, after 96 week of etv treatment, the proportion of patients achieving undetectable hbv dna (<400 copies/ml) increased from 21% to 90%. increases were also observed for alt normalization (81% to 90%) and hbeag seroconversion (10% to 19%). three patients had detectable hbv dna at week 96 after etv treatment and samples from two were tested for resistance. neither demonstrated substitutions associated with etv or lvd resistance. five patients had grade 3-4 laboratory abnormalities, including increased ast/alt and increased lipase levels. conclusions: switching patients from lvd therapy to etv resulted in increased proportions of patients achieving hbv dna suppression, alt normalization and hbeag seroconversion, with no evidence of etv resistance. etv was well tolerated during treatment. backgrounds/aims: hepatitis b virus (hbv) reactivation in patients undergoing chemotherapy hampers an adequate administration of cytotoxic agents and even causes an treatment failure. prophylaxis failure occasionally results from viral breakthrough or withdrawal flare. the aims of this study were to identify predictors of anti-viral prophylaxis failure and to determine the optimal strategy for anti-viral prophylaxis. methods: cancer patients with positive hbsag who underwent cytotoxic chemotherapy in a tertiary medical center from january 2005 to june 2008 were included. prophylactic lamivudine was started with initiation of chemotherapy, continued during the chemotherapy, and discontinued within 6 months after the completion of chemotherapy. all patients were followed up even after withdrawal of lamivudine. results: 115 patients were enrolled. twenty-nine patients (23.7%) had hematologic malignancies and eighty-six (76.3%) had solid tumors. median follow-up duration was 15.9 months and twenty-six patients (22.6%) experienced the prophylaxis failure: viral breakthrough (11 patients, 9.6%), withdrawal flare (15 patients, 13.0%). ymdd mutation developed in four patients. withdrawal flare occurred at a median 2.5 months after discontinuation of lamivudine. using log-rank test and cox multi-variate analysis, our results showed that the type of underlying malignancies (hr 2.46, 95% ci, 1.11-5.43; p=0.026) and baseline hbv dna titer (hr 3.91, 95% ci, 1.63-9.39; p=0.002) were significant independent risk factors for antiviral prophylaxis failure. conclusion: cancer patients with high viral load of hbv and hematologic malignancies may need more prolonged and potent anti-viral prophylaxis to avoid interruption or delay of chemotherapy. back ground: the usefulness of hepatitis b virus (hbv) dna and hbv core-related antigen (hbcrag) was evaluated for timing hepatitis flare after viral breakthrough or withdrawal of antiviral treatment in chronic hepatitis b. method: a total of 32 events of hbv reactivation due to withdrawal of lamivudine (lam) or emergence of mutants resistant to lam or adefovir dipivoxil (adv) virus were analyzed in 25 patients with chronic hepatitis b (20 men, median age 56 years [range: 30-66]). they were followed monthly for serum alt, hbv dna and hbcrag before, during and after the treatment. result: high alt flare (alt > 100 iu/ml) after viral breakthrough or withdrawal was related with baseline hbeag positivity (p=0.016), hbcrag level at hbv dna elevation (p=0.038) and duration from hbcrag elevation to salvage therapy (p=0.044). in multivariate analysis, hbcrag > 4.0 log u/ml (or 22.9, 95%c.i. 1.7-304.1, p=0.018) and salvage therapy after 8 weeks from hbcrag elevation (or 10.6, 95%c.i. 1.6-71.4, p=0 .015) were selected as related factor with high alt flare. after appearance of resistant-virus or withdrawal of lam or adv therapy, hbv dna re-elevated without increase of hbcrag, then hbcrag elevated with hbv dna. re-elevations of alt occurred in 27 of the 32 (84%) events. in 23 of the 27 (85%) events, alt re-elevated within 8 weeks from the start of hbcrag increase. conclusion: hbcrag was useful for timing the re-elevation of alt after hbv dna re-elevation induced by drug-resistant virus or withdrawal of lam or adv therapy. background and objectives: the aim of the study was to observe the efficacy of a patient's therapy for switching lamivudine + placebo to adefovir dipivoxil (adv), and modeling the viral dynamics. methods: the studied object was a chinese chb patient with lamivudine mutation. used the lvd + placebo for 12 weeks' therapy. then switched adv for another 95 weeks. after that stopping the treatment and following up for 24 weeks. based on our modified basic virus infection model, we introduce a personalized model consisting of four variables: x, y, v, e , representing uninfected cells, infected cells, free virus, and ctl cells, respectively. results: selected the model parameters, the simulation data of hbv dnas of our model are good in agreement with the clinical ones. observe that after 24 weeks' treatment cessation, the benefit (hbv dna < 250 copies/ml) for suppressing hbv replication can still be kept. numerical simulation show that if the patient's immune functions can be kept after therapy stops, it needs 9 years to replace all infected cells by normal ones. conclusion: for lvd mutation patients, lvd+ placebo to avd therapy scheme may help patients to suppressing hbv replication. further researches are promising. acknowledgments: this work is jointly supported by the nnsf of china background: g-a-1896 pre-core mutants (p-c-mt) cause hbeag-negative chronic hepatitis (chb) in genotype d infected mediterranean adults. we studied their emergence during chronic hbv infection in children. methods: eighty consecutive hbsag carriers (50/30 males/females, age 11y, range 0.2-17y) with vertical (66%) or intra-familial (16%) transmissions were followed-up for 12.5y (range 1-25 y). hbv genotype and hbeag status were determined at the admission, hbeag/anti-hbe every 2 years thereafter. during the follow-up, hbv-dna was measured in 185 sera (1-7 sera/patient) (cobas-amplicor, roche); p-c populations were characterized by direct sequencing (ds), by oligo-hybridization (oha) and allele-specific-pcr (as-pcr) with 30%, 10% and 0.1% sensitivities, respectively. results: seven children were genotype a and 73 d; 70 (87.5%) were hbeag-positive. fifty-five (78.6%) underwent hbeag/anti-hbe seroconversion (median age 13y, range 1.3-27y). baseline hbv-dna (cp/ml) was lower in seroconverters (7.9+1.9 vs 9,4+0,6, anova p=0.012). ds/oha p-c-mt were 4.2% at the admission and 45.8% after follow-up; as-pcr p-c-mt 33.3% and 50% respectively. after seroconversion 47 (85.5%) became inactive carriers, 6 (14.5%) lost hbsag (5 genotype d/p-c-wt); 8 p-c-mt had chb. hbv-dna (cp/ml) was lower in 31 p-c-wt than in 14 p-c-mt inactive carriers (3.42+1.05 vs 4.58+0.91; anova p=0.007). conclusions: in genotype-d infected children p-c-mt is selected progressively after hbeag/anti-hbe seroconversion to become predominant in hbeag-negative chb. early and efficacious immune control of hbv replication avoids p-c-mt selection and leads to hbsag loss. , a, , d, k, u, m, n , k1 ,k2 and k3. here k1, k2, and k3 are the rate of ctl production and dead, killing virus, respectively. results: the patients with hbv dna levels less than 1000 copies/ml were reported in 17.5% (7/40). a patient whose hbv dna levels were higher than 40000 copies/ml can keep treatment benefits even stopping the therapy for over ten weeks. the simulation data of our model are in agreement with the patient's hbv dna data. our simulation also shows that it needs to spend about 18 years for clearing all infected cells. conclusion: the simulation result implies that some chinese patients may need long term's therapy to clear all infected cells. patients' ctls assays are needed to confirm the effectiveness of the personalized modeling, and help doctors to decide whether stop the drug treatment even patients' hbv dnas are higher than undetectable levels. background/aim: recent reports have shown that programmed death 1(pd-1) expression is associated with t cell exhaustion and persistent viral infection. we studied longitudinally 28 chronic hepatitis b(chb) patients undergoing treatment with nucleos(t)ide analogues or pegylated interferon-(peg-ifn-) in 12-16 weeks to determine the relationship between pd-1 expression levels on t cells and early reduction of viral load induced by treatment. methods: our investigations were focused on three points: baseline (time point 1, t1), treatment weeks 4-6(time point 2, t2) and treatment weeks 12-16(time point 3, t3). pd-1 expression on total cd4 and cd8 t cells in chb patients during antiviral therapy was detected by flow cytometry. serum hepatitis b virus (hbv)-dna load was measured by real time polymerase chain reaction. results: between t1 and t2, pd-1 expression on total cd8 (p<0.01) and cd4 t cells (p<0.01) dropped concurrently with treatment-induced hbv-dna decline(p<0.01). between t2 and t3, however, only the hbv-dna levels reduced significantly (p<0.01). conclusion: early suppression of hbv replication induced by antiviral treatment results in a significant decrease in pd-1 expression on total cd8 and cd4 t cells in chb patients. c. zhao 1,2 , w. zhang 2,3 , x.c. tian 1 , c.y. fang 2,3 , h.j. lu 2,3 , p.y. yang 2,3 , y.m. wen 1,2 background/aims: hepatitis b virus (hbv) is still regarded as one of the major causes of chronic hepatitis, cirrhosis and hepatocellular carcinoma worldwide. the interactions between hepatitis b surface antigen (hbsag) and host cells still remain largely unknown and need to be explored in detail. methods: differential protein expression profiles of hepg2-s-g2( stably expressing hbsag cell line) and hepg2-neo-f4 ( control cell line) were compared using two dimensional gel based differential proteomic approach. cell proliferation assay and survival assay were used for further studies on the candidate protein. results: compared with the control down regulation of 44 proteins and up regulation of 38 proteins were found in hepg2-s-g2 cell. all these regulated proteins were identified by ms/ms and could be fell into several categories including metabolism-associated, immune-response-related, protein modification, signal transduction and others. among them, a group of proteins in putative pathways associated with apoptosis were found out and discussed, including glucose-regulated protein 78kd (grp78/bip), heterogeneous nuclear ribonucleoprotein (hnrnp), far upstream element-binding protein (fusebp), rho gdp dissociation inhibitor (gdi), cystatin b and some scaffold proteins. grp78, an important chaperone protein involved in multiple functions in host cells, was consistently decreased in hepg2-s-g2 and in huh7 cell transiently transfected with hbsag expression plasmid. decreased crp78 inducing by hbsag or blockage of rnai consistently led to the less resistance to staurosporine-induced cell death. conclusions: these results revealed a possible pathogenesis induced by hbsag via grp78. background/aim: to evaluate the efficacy of adefovir dipivoxil alone and in combination with lamivudine in treating patients with lamivudine-refractory hbeag-positive chronic hepatitis b. methods: eighty-five hbeag-positive patients who had received lamivudine treatment for various periods and had a lamivudine-resistant liver function abnormality, documented ymdd mutations and persistent viremia were randomized to adefovir dipivoxil 10 mg, lamivudine 100 mg, or addition of adefovir dipivoxil to ongoing lamivudine daily. the primary efficacy measure was virological response. the secondary efficacy measure was serological response (hbeag loss rate and hbeag seroconversion rate) and alt normalization rate. results: after 24 weeks of therapy, mean reduction of hbv-dna level, the percentage of patients with hbv-dna lower than 5 log10 copies/ml and the percentage of patients with hbv-dna level decrease of more than 2 log10 copies/ml in patients of adefovir dipivoxil/lamivudine and adefovir dipivoxil monotherapy groups were significantly higher than those in patients of lamivudine group (2.58, 2.21 log copies/ml vs. 1.02 log copies/ml, 92.3%, 88.5% vs. 33.6%, 76.9%, 75.8% vs. 28.8%; p < 0.001, respectively). at the end of 52 weeks, mean reduction from baseline in serum hbv-dna level at was 0.08, 4.25, and 4.12 log10 copies/ml in the lamivudine, adefovir dipivoxil/lamivudine, and adefovir dipivoxil groups, respectively. alt normalization rates were significant hihger in adefovir dipivoxil/lamivudine and adefovir dipivoxil recipients than those in lamivudine recipients (62%, 55% vs. 8%, p < 0.001, respectively). a similar pattern was observed in hbeag loss among three groups. conclusions: adefovir dipivoxil is an effective treatment option for patients with lamivudine-refractory hbeag-positive chronic hepatitis b. aim: to find the prevalence of hbv virologic flare as defined by hbv dna viral load of > 2,000 iu/ml in inactive chronic hepatitis b (hbv dna less than 2,000 iu/ml), hbeag-negative patients who have not received any treatment and to identify if there are any predictors that can predict virologic flare. methods: we retrospectively analyzed medical records of the patients who have attended hepatitis clinic, siriraj hospital from january 1, 2002 to february 28, 2008 . the patients were eligible if they were naïve to any treatment and hbv dna less than 2000 iu/ml at entry. co-infection with hiv and/or hepatitis c virus were excluded. hbv dna measurement determine by roche amplicor ® (detection limit of 60 iu/ml). hbv virologic flare was defined as hbv dna more than 2000 iu/ml during follow up period. result: there were 84 patients with mean follow up time was 598 days with annual prevalence of hbv virologic flare of 12.8, 4.8, and 4.2% for the first, second and third year of follow up, respectively. initial hbv dna level was the only predictor that can predict reactivation. no patients with hbv dna at entry below detection limit developed flare and the patients with hbv dna above 740 iu/ml had 22 times higher chance to develop flare during follow up. conclusion: hbv dna flare is not uncommon in inactive chronic hepatitis b patients. most of the virologic flares occur in the first year. the most important predictor or virologic flare is higher hbv dna at beginning. background: multi-drug resistant hbv developed with multiple antiviral agents. there existed difficulty in dealing with multi-drug resistant hbv. methods: retrospective analysis of 23 consecutive patients who exhibited chronic hepatitis b associated with multiple drug-resistant mutations to lamivudine and adefovir during antiviral treatment. multiple drug-resistant mutations were detected in those patients by dna direct sequencing. result: before multiple drug-resistant hbv emerged, 20 patients accept sequential antiviral therapy, 3 patients accept na monotherapies. there were 16 cases of rta181t/v rtm204v/i mutation, 2 cases of m204v/i +n236t mutation, 4 cases of a181t/v+m204v/i +n236t mutation, 1 case of l180m+a181t/v mutation. 14 cases received rescue therapy of interfronand hbv dna level of 8 cases decreased; other 9 cases received combination treatment and hbv dna level of 4 cases decreased. conclusion: the main reason of multiple drug-resistant mutations was sequential antiviral therapy. another reason may be pre-exist drug-resistant mutation before nucleoside or nucleotide analogue treatment. de novo combination of antiviral agents should be recommended. combination therapy directed against mutants resistant to each treatment may not be adequate in suppressing multi-drug resistant hbv. interfron may be one choice for hbv of multiple drug-resistant mutations. background: to furnish basis for an accurate evaluation of hbeag negative chronic hepatitis b (e chb), the present study studies the clinical features and hepatic pathology, and analyzes the relation between the data and the grade and stage of hepatic pathology in e chb. methods: a study is performed in 120 chinese e chb patients (106 men and 14 women; mean age sd, 34.3 9.4 years). the relationship between the clinical features and the grade and stage of hepatic pathology was analyzed by spearman's rank correlation test or kruskal-wallis test by applying stata 7.0 software. result: negative correlation is shown between the grade and leucocyte count methods: 80 patients with hbeag-positive compensated chb with hbv dna >6 log10 copies/ml, serum alt 2 x uln were divided two groups:one treated with telbivudine and the other treated with entecavir. results: baseline characteristics were well balanced between treatment groups. at 12wk of the treatment the hbv dna undetectable rates of hbeag-poitive patients in the telbivudine group and the entecavir were respectively 50 52.5 (p 0.05), the rates of hbeag negative were 10 0 respectively, the rates of hbeag seroconversion were 20 5 respectively; at 24wk of the treatment the hbv dna undetectable rates of hbeag-poitive patients in the telbivudine group and the entecavir were respectively 80 70 (p 0.05), the total rates of hbeag negative were 20 15 respectively, the total rates of hbeag seroconversion were 27.5 17.5 respectively(p 0.05).no adverse reactions were found in both groups conclusion: there was no significant difference in hbv dna undetectable rates between two nucleotide analogs in short-term (24 weeks).the telbivudine group has better effect in hbeag seroconversion rate than the entecavir group in early stage,but no statistical significance. j.w. song 1,2 , z. xin 1 , j.x. tang 1 , l. yao 1 , b. wu 1 1 sun yat-sen university, 2 zhuhai sinochips biosci. co.,ltd., china background: to develop a equipment free, and can be widely used in clinical practice biosensor-based microarray for hepatitis b virus pre-c/bcp mutation assay. methods: a thin film optical biosensor were applied for amplification the microarray signal in situ. and hbv sites 1762, 1764,1768,1770 and 1896 were selected as the targets and the microarray were be fabricated. the 5 mutated plasmids contained 1762, 1764,1768,1770 and 1896 sites and 30 hbv sera were be tested in our study and all the plasmids and sera pcr products were be assayed by really time pcr and sequencing. results: the biosensor based microarray signal can be easily record by digital camera or even by the naked eyes and the detection signal for positive discriminated from negative were sharply contrasted as whole yes or no and it looks be significantly superior to classical microarray technique; 2. the sensitivity of the detection limitation of sera hbv load is 2x10e3 copies/ml with 95% reproducibility. the concordance index of 50 times negative and mutated plasmids were 98%(kappa=0.932). 3. 30 sera samples of hbv>10e4 load and 20 sera of hbv negative tested by pcr fragment sequencing were showing very good agreement between sequencing with our biosensor based microarray and the concordance index kappa was 0.7619. conclusion: our biosensor-based microarray for pre-c/bcp mutation assay were a both sensitive and accurate method. and its advantages of equipment free, sharply contracted signal of positive vs negative and easily be perform in testing were make it be a promised assay for clinical application. objective: to investigate the frequencies of cd4 + cd25 + regulatory t cells in the cord blood of fetuses whose mothers are patients with chronic hepatitis b, we assayed the differences among hbsag-positive and healthy subjects by flow cytometry. the results might offer some experimental evidence to explain the high rates of hbv persistent infection in vertical transmission of hbv from hbv-infected mothers. methods: 12 newborns born from hbsag positive mothers were recruited , 10 healthy subjects being used as a control group. the cord blood and peripheral blood of mothers were collected respectively .frequencies of cd4 + cd25 + regulatory t cells in the cord blood of fetuses whose mothers are patients with chronic hepatitis b were analyzed by flow cytometric analysis. result: the number of cd4 + cd25 + regulatory t cells/pbmcs in the cord blood of newborns born from hbsag positive mothers 4.49 ±1.18 significantly exceeded that in normal controls 2.26 ±0.97 ,p 0.001 ;and newborns born from hbsag positive mothers presented a much higher fraction of cord blood cd4 + cd25 + /cd4 + 9.62 ±2.34 than those in normal controls 7.93 ±1.43 ,p p=0.0025 p 0.05 . conclusions: the results indicate that the proportion of cd4 + cd25 + regulatory t cells in hbsag positive mother cord blood was higher than those of healthy cord blood. objective: to study the clinical features of chronic severe hepatitis b with negative hepatitis b e-antigen (hbeag) and positive hepatitis b e-antigen (hbeag) methods: a total of 353 in-patients with chronic severe hepatitis b were recruited into the study and divided into two groups according to the hbeag status. the serological chemistry data, hepatitis b virus (hbv) dna quantification data were detected, and morbility of cirrhosis, its complications and prognosis were also studied. results: of the 353 in-patients, 236(66.8%) patients were hbeag-negative. 117(33.2%) patients were hbeag-positive. the ratio of hbeag-positive patients was significantly higher than that of hbeag-positive patients (p<0.05).the average age of hbeag-negative patients was older than that of hbeag-positive patients (p=0.048). the serum hbv dna level of hbeag-negative patients was significantly lower than that of hbeag-positive patients (5.49±2.02) vs(6.64±1.41) log copies/ml (p<0.01).the ratio of patients who had a serum hbv dna level less than 5log copies/ml in hbeag-negative patients was significantly higher than that in hbeag-positive patients (41.8% vs 11.9% ,p=0.000). there was no significant difference in serological chemistry data, morbility of cirrhosis and its complications on infections, ascites, hepatoencephalopathy, gastrointestinal hemorrhage, as well as prognosis of the patients between those two groups. conclusions: the study suggested that serological chemistry data, morbility of complications and prognosis of the disease of hbeag-negative patients mimics that of hbeag-positive patients. the hbeag-negative patients had a higher level of age, while a lower level of serum hbv dna. to reduce the incidence of liver failure, more frequent monitoring and earlier antiviral therapy prone to be reasonable for chronic hepatitis b patients with negative hepatitis b e-antigen. background: the emergence of lam-resistant virus greatly limits the efficacy of therapy and induces the liver injury. the aims of this study were to assess the related factors of lam-resistant mutation in hbeag positive chb patients. methods: thirty-five patients carrying lam-resistant with hbeag positive were enrolled in this study. all of them underwent percutaneous liver biopsy, histological findings and had detectable viral load. age, viral load, levels of alt, types of mutation and hbv genotype was monitored. result: the median year of mutation found was 23months. 85.71% were genotype c and 14.29% were genotype b. the mutation of l80i, l80v, g173l, l180m, m204v and m204i were detected. the emergence rates were 34.3%(12/35), 25.7%(9/35), 17.1%(6/35), 60%(21/35), 57.1%(20/35), 54.3%(19/35) respectively. the rate of patients with two or three mutation were much more than one or four mutation. 62.9% patients were found to have significant histological findings, even 5 had established cirrhosis. two had no histological finding. one had rtl80i and rtm204i. the other had rtl80v, rtl180m and rtm204v. the number of resistant mutation has no significant finding with histological finding, basic alt level and basic viral load. conclusions: the emergence rate of l180m, m204v and m204i were higher than that of l80i, l80v, g173l in hbeag positive chb patients with lam-resistance. most of them have two or three lam-resistant mutation regardless of histological finding severity, level of basic alt and viral load. we must select the efficacious method to treat the patients with lam-resistant. objective: to investigate the therapeutic efficacy of foscarnet sodium in the treatment of patients with severe chronic hepatitis b. methods: forty four patients were randomly divided into foscarnet sodium treatment and placebo groups.each group consisted of 22 patients, 22 patients in foscarnet sodium group were treated with foscarnet sodium twice daily 3.0g given by intravenous infusions ,in addition to general therapy for 28 days.the other 22 cases were treated without any form of antiviral therapy as control.all patients were followed up for 6 months.the hbv markers, quantification of hbv-dna, serological chemistry data were measured at baseline , during therapy period and the end of follow-up period . results: clinical symptoms were improved in two groups patients, meanwhile alanine aminotransferase (alt) and total serum bilirubin (tbil) decreased. compare alt and tbil at the end of trentment, there were no significant differences between the two groups (p>0.05). in foscarnet sodium treatment group, the level of serum hbv-dna descreased from (6.993±0.898) log copies/ml to( 4.033±1.286) log copies/ml (p<0.05), the rate of hbv-dna descrease of more than two log was 81.1% 18/22 . in the control group, the level of serum hbv-dna descreased from( 7.068±0.938) log copies/ml to (5.188±1.926 )log copies/ml, the rate of hbv-dna descrease of more than two log was 45.4% 10/22 .a comparison of serum hbv-dna showed significant differences between the two groups(p<0.05) conclusion: foscarnet sodium administered can inhibit hbv replication in treating severe chronic hepatitis b.it can rapid lower the level of serum hbv-dna obviously.but the relapse rate was 47.0% in foscarnet sodium treated at the end of follow up period objective: evaluation of efficacy and safety of five years trail of entecavir for chronic hepatitis bpatients failed with lamivudine therapy in the chongqing area. methods: thirty-two eligible patients were enrolled who had documented lvd failure.in the double-blind phase,patients were randomized(4:1)to etv1.0mg/d (n=28)and placebo (n=4) for 12 weeks.in the open-lable phase ,patients received etv 1.0mg/d for 240 weeks.hbv-dna level,liver function tests,hbv serology and safety assessments were conducted. results: the mean reduction in hbv dna levels at week 12 was 4 05 logl0 copies ml in etv group compared to 0.08logl0 copies ml in placebo group(p< 0 05). the mean of hbv dna levels after 240 weeks of etv treatment decreased to 2.58logl0 copies m1 the proportion of hbv dna<3log10copy/ml raised from 0 at baseline to 6.25% at week 8,to 15.6% at week 24,to 50% at week 96,and raised to 57.14% at week 240.there were two patients with hbsag seroconversion and four patients with hbeag seroconversion at the end of study. the mean of alt became normal at week 12 and remained normal throughout week 240.there was one patient who had a severe adverse event during the trail. conclusion: the findings from this study demonstrated the antiviral activity and safety of etv in adults with chb who have failed lvd pe198 showing delayed response on t cells as increased on day 7.the mrna expression of il-5 and il-2 showed no response to hbv vaccine but highly regulated in tt after day 7(p=0.00,0.001).myd88andtraf 6(p=0.042)upregulated in hbv vaccine group followed by of ifn-(0.012)no change of ifn found in tt conclusions: i) hbv vaccine stimulates innate response by day 3 which potentiates further cascade,peripheral dendritic cells plays significant role in generating immune flare follows myd88 pathway and releases ifn-.ii) whereas t cells marjory involved in tt showed delayed immune response.iii) identification of key factors at different time points may prove to be a novel model to study the initial events after vaccination. objective: to compare th1/th2 cytokines' dynamic change and its clinical significance in hepatitis b e antigen-positive patients treated with telbivudine. methods: twelve hepatitis b e antigen-positive patients treated with telbivudine.the blood sample was collected at baseline, week 4, week 8, week 12, week 24 and week 48 and stored at -70c; serum il-2, il-4, il-6, il-10, tnf-and ifn-were tested at each time point by cytometric bead array (cba), compare th1/th2 cytokines' dynamic change at different time point in each group and compare th1/th2 cytokines' dynamic change cross four different groups: complete response, partial response, non-response and break through . results:the level of th1 type cytokines in complete response group are obviously higher than the group of partial response non-response and breakthrough,but the level of th2 type cytokines are lower than the group of partial response, non-response and breakthrough. conclusions: th1/th2 cytokines is essential for the regulation of the immune function of the body. after treated with telbivudine, the level of th1-type cytokines in the complete response group increased significantly, while the level of th2 cytokines declined trend. a. soamni 1 , s. somani 2 , a. jain 3 , v. dixit 3 1 navjeevan hospital, 2 suvidha, 3 background: chronic infection with hepatitis b virus causes spectrum of manifestations ranging from asymptomatic carrier state (often inactive with low replication) to the development of cirrhosis-related complications.the characterization of asymptomatic state has not been done in this part of the country, which forms important objective of present study. methods: 61 incidentally detected asymptomatic hepatitis b surface antigen positive (idahs) subjects having hbsag positivity for >6 month presenting to our liver clinic were enrolled after appropriate consent. detailed clinical, laboratory and sonographic evaluation was done. they were divided into two groups according to presence or absence of e antigen. group a -hbeag + (n=48) group b -hbeag -(n=13) results: most of our patients (49%) were young adults (21-30 years) with male to female ratio of 3.6:1. approximately half of our patients were detected during routine medical checkup, followed by family screening of contacts. most of our patients were asymptomatic, and fatigue was most common symptom found in 16%. all demographic and biochemical parameters other than ast & alt were comparable in both groups. among hbeag negative 48 (79%) subjects, hbv dna level >10 5 copies/ml was found in 31%. subjects with positive hbeag as compared to non-replicative infection (antihbe positive and hbv dna negative) had more frequent elevation of transaminase levels (62% versus 31%, p<0.05). antihbe antibody was positive in all hbeag negative subjects. mean age of seroconverted (antihbe positive) individuals was a decade older than hbeag positive. conclusion: from our study we can suggest that ongoing liver disease is present in approximately one-thirds of incidentally detected asymptomatic hepatitis surface antigen positive subjects previously referred to as carrier state. hbsag testing should be mandatory in all routine medical checkup and family and sexual contacts of index case should be screened. background and objectives: this research was carried out to determine the prevalence of hbcab among the hbsag negative first-time blood donors who had referred to khorramabad and borujerd centers for blood donation. materials and methods: this study was established on a descriptive cross-sectional basis in which hbsag test (elisa) was primarily performed on all of the donors having referred to khorramabad and borujerd blood centers; then, out of all those referred 1000 subjects, who were first-time and hbsag negative, were selected for furthur investigation. the information concerning age, gender, job, blood transfusion, and hbv vaccine injection was included in the questionnaire of the study. hbcab (total & igm) and hbsab tests were performed on the selected donors. data were collected and finally the prevalence rate of hbcab was determined. results: the results of the study showed that out of 1000 hbsag-negative first-time blood donors, only 47 were hbcab+, from which 27 were hbcab (total)+, and 3 were hbcab (igm)+. 18 were both hbsab+ and hbcab+, and 53 were seropositive only for hbsab. conclusions: it was demonstrated that the first-time blood donors who are seronegative for hbsag marker will be easily identified through hbcab test if they are in the so-called core window period of the virus. meanwhile, this group of donors have been implicated as high-risk for transfusiontransmitted hbv infection. so, detecting this marker will remarkably reduce the chance of latent cases of hbv infection and help promote blood safety. background: tumor necrosis factor-(tnf-) plays a pivotal role in the viral clearance and host immune response to hbv, and the capacity for tnf-production in individuals is influenced by a major genetic component. the studies of tnf--308 gene promoter polymorphism in chronic hbv infection have reported apparently conflicting results. objective: to derive a more precise estimation of the relationship between the polymorphism of tnf--308 gene promoter and chronic hbv infection. method: meta-analysis was done of 22 case-control studies in relation to tnf--308 gene promoter, involving a total of 4338 chronic hbv infection cases and 3013 controls. the pooled odds ratios (ors) for the risk associated with the genotypes of ga, aa, and ga+aa (a-allele carriers) compared with the gg genotype were calculated. results: overall meta-analysis indicated that -308a heterozygotes (ga) had 22% decreased risk of developing chb with a borderline significance (or = 0.78; 95% ci: 0.60-1.02; p = 0.065). for the -308a allele homozygotes (aa) and carriers (ga+aa), the pooled ors both indicated a significantly decreased risk of chb (or = 0.39; 95% ci: 0.21-0.73; p = 0.003; and or = 0.74; 95% ci: 0.57-0.96; p = 0.026, respectively) ( table 1 ). in the subgroup analyses by ethnicity, significantly decreased risks were associated with -308 variant genotypes (ga and aa) in mongoloid populations in all genetic models. however, no significant associations were found in caucasoid. conclusion: the meta-analysis suggests that the tnf--308a allele is a low-penetrant protective factor for chronic hbv infection, especially in mongoloid. method: 20 hbv transgenic mice were randomly divided into physiologic saline group and matrine injection group. another10 normal mice at the same species and age with hbv transgenic mice were regarded as the normal group. the mice in matrine injection group were administrated at dosage of 82.2 mgkg -1 d -1 by intraperitoneal for 30 days. the mice in physiologic saline control group and normal group were administrated normal saline with the same volume at same time. the contents of hbv dna in serum and liver were quantitated by pcr. and the spleens were separated for cultivating dendritic cells. the surface molecules of dendritic cells were tested by flow cytometry. ifn-mrna and tnf-mrna in liver were tested by rt-pcr. result: there was no significant difference of the serum hbv-dna level between physiologic saline and matrine injection groups. the content of serum hbv-dna after treatment showed a significant decrease in two groups. the content of serum hbv-dna in matrine injection dropped significantly as compared with that in the physiologic saline group. but there was no significant difference in the content of hbv-dna in liver between physiologic saline and matrine injection groups. the expression level of mhc-ii on dendritic and hepatic ifn-r mrna and tnf-a mrna showed a significant decrease in hbv transgenic mice than normal mice. in comparison with physiologic saline group the expression level of them in matrine injection group showed a significant increase. conclusion: matrine injection was effective on depressing hbv-dna in hbv transgenic mice. its antiviral action may be achieved through regulating mhc-ii on dcs surface and promoting the production of antiviral factor such as ifn-and tnf-. purpose: to stimulate non-specific immune response capacity as the main content of the study to explore the hbv-dna and non-specific immune responses in the relationship between the low response capability, methods: 190 cases of asymptomatic carriers, double-blind, randomized into mycobacterium fu 36, lamivudine and traditional chinese medicine for the treatment group, mycobacterium fu36 with traditional chinese and lamivudine with traditional chinese medicine were in the control group, a total of 12 weeks of treatment, follow-up six months after the termination of treatment. results: different treatment of hbv -dna effect of the existence of significant differences; p> 0.01, the performance of different types of asymptomatic carriers negative rate of hbv-dna there is a significant difference; p> 0.01, as well as the performance of the different types of asymptomatic carriers continued application a treatment plan presented hbv-dna rebound rate there is a significant difference; p> 0.01, conclusion: hbv-dna and non-specific immune responses in response to the lower capacity, anti-hbv therapy is not associated with non-specific immune response capacity or improve is the anti-hbv drugs alone can not solve the asymptomatic carriers in anti-hbv therapy where the cause of the problem, solve the asymptomatic carriers in the anti-hbv treatment although the need for anti-hbv drugs with non-specific immune activation synchronous drugs on the basis of the joint application , but the simultaneous combination of two drugs rather than as a result of hbeag and hbv-dna can hbeag-positive asymptomatic carriers receive hbv-dna negative effect of the results. background: adefovir dipivoxil (adefovir) effectively inhibits both wild-type and lamivudine (lam)-resistant hepatitis b virus (hbv) replication and resistance to this drug is infrequent compared with lam. in this study, we tried to identify factors affecting the emergence of resistant mutants after adefovir monotherapy in lam-resistant chronic hepatitis b (chb) patients. methods: the subjects were 87 chb patients with lam-resistance who had received adefovir for more than 12 months (range 12-39 months). the initial viral response (ivr) was defined as hbv dna <4.0 log copies/ml. the adefovir resistant mutant was assayed at baseline and every 6 months during adefovir administration. results: ivr was observed in 38% of patients. the cumulative emergence rates of adefovir resistance were 2.6% at 6 months, 10.4% at 1 year, 14.6% at 2 years and 21% at 3 years. in univariate analysis, factors contributing to the emergence of adefovir resistant virus were baseline hbv dna > 6 log copies/ml (p=0.003) and ivr (p<0.0001). the presence of precore mutation and type of ymdd mutants were not related. in multivariate analysis, only ivr was an independent factors affecting the emergence of adefovir resistant virus (p<0.0001). conclusion: ivr is a useful predictor for emergence of adefovir resistant mutants after adefovir monotherapy in lam-resistant chb patients. for ivr-negative patients, the change of therapeutic options such as add-on lam or switch to other drugs should be considered because of the high incidence of the emergence of adefovir resistant mutants. background: elevated hbv dna is strongly associated with the risk of disease progression. this study investigated the early viral suppression effects of etv and lvd in nucleoside-naïve chinese patients with active hbeag (+) chb. methods: this open-label study was conducted in 5 major hospitals in china. at study entry all patients had hbv dna levels 10 7 copies/ml, elevated alt (1.3-10xuln) and compensated liver function. patients received either 0.5mg etv or 100mg lvd daily. hbv dna measurements were taken at baseline and at weeks 2, 4, 12 and 24 during treatment, using roche cobas amplicor assay (llod 300 copies/ml). results: a total of 97 patients were enrolled; 42/50 etv patients and 40/47 lvd patients completed 24 weeks of treatment. at baseline, mean hbv dna levels were 8.45 0.81 in etv group and 7.67 1.76 log 10 copies/ml in lvd group (p<0.05). the mean change in hbv dna from baseline (log10 copies/ml) was -2. 96±0 ngo's/funding-agencies representative at apasl2009-conference need to address-this-issue. we ngo-representatives from developing-nations need exposure to research treatments used by european/american experts. do we all failed in addressing socio-economic issues of cancer-sufferers? we need to address these socio-economic issues of affected population in resource-poor-nations. background: a garlic derivative s-allylcysteine (sac) has anti-cancer effect in human prostate and colon cancers. we aimed to investigate the effect of sac and combination of chemo-drug on tumorigenesis and metastasis of liver cancer. methods: the orthotopic liver tumor model using a metastatic liver cancer cell line mhcc97l labeled with luciferase gene was applied. sac was given at day 7 after tumor implantation at 1mg/g/day, or 1mg/g/day combined with low dose cisplatin for 5 weeks. tumor growth and metastasis were monitored by xenogen in vivo imaging system. hepatic stellate cell (hsc) activation and tumor-associated macrophage (tam) in the tumor tissue were detected by -sma and ed1/ed2 staining. tumor micro-vessel density (mvd) and apoptosis were also analyzed. in vitro functional tests including proliferation assay, cell cycle analysis and apoptosis analysis were performed. results: tumor growth was inhibited by sac combined with cisplatin treatment at different time points accompanied by lower incidence of lung metastasis compared with other groups. the observation of xenogen ivis was confirmed by histopathological examination. the hsc activation by -sma staining in the liver tumors was suppressed by sac and cisplatin treatment accompanied with less tam infiltration. consistent with in vivo study, in vitro functional study also demonstrated that sac not only induced cell cycle arrest, apoptosis, and inhibited tumor cell proliferation, but also sensitized the anti-cancer effect of cisplatin. conclusion: sac treatment inhibited liver tumor growth and metastasis by inhibiting tumor cell proliferation, inducing apoptosis and sensitization of chemotherapy. background: anti-angiogenic therapy would be a promising approach against hepatocellular carcinoma (hcc). although a sorafenib has survival benefits in patients at advances stages of hcc, there seem to be several serious concerns to employ this agent for chemoprevention against hcc. branched-chain amino acid (bcaa) reportedly inhibits the incidence of hcc in patients with insulin resistance (ir). however, the possible mechanism is still obscure. the aim of the current study was to examine the effect of bcaa on hepatocarcinogenesis under the condition of ir, especially in conjunction with angiogenesis. methods: the effect of bcaa on the development of liver enzyme-altered pre-neoplastic lesions and angiogenesis in the obese diabetic otsuka long-evans tokushima fatty rats was examined. we also performed an in-vitro study to elucidate the possible mechanisms involved. result: treatment with bcaa markedly inhibited the glutathione-s-transferase placental form (gst-p)-positive pre-neoplastic lesions along with suppression of neovascularization in the liver. the hepatic expression of the vascular endothelial growth factor (vegf), a potent angiogenic factor, was also attenuated. bcaa treatment significantly suppressed the glucose-and insulin-induced in-vitro angiogenesis in the presence of vegf. these results indicate that bcaa exerted a chemopreventive effect under the condition of ir via suppression of vegf-mediated angiogenesis. conclusion: since bcaa is widely used in the clinical practice for patients with chronic liver diseases, this agent may represent a new strategy for chemoprevention against ir-based hcc in the future. background: krüppel-like factor 8 (klf8) is a member of transcription factors. whether and how klf8 signaling pathways contribute to hepatocellular carcinoma (hcc) development and progression is unknown. this study investigated role of klf8 in hepatocellular carcinoma cell line hcclm3 proliferation, invasiveness and epithelial to mesenchymal transition (emt). methods: the expression of klf8 in different liver cell lines was detected by quantitative real-time pcr and immunocytochemistry. we used small interfering rna (sirna) to down-regulate klf8 expression in hcclm3. the change of proliferation and invasive ability of klf8 down-regulated hcclm3 was investigated by mtt reduction assay and trans-well invasive assay respectively. the change of proliferation, invasiveness and emt related gene in klf8 down-regulated hcclm3 was evaluated by quantitative real-time pcr. result: klf8 protein expressed predominantly in the nuclei of cancer cells and its expression is positively correlated with metastatic potential of these cell lines. hcclm3 has the highest klf8 level. decreased klf8 expression can notably inhibit the proliferation (p<0.001, n=6), mobility and invasiveness of hcclm3 (p<0.001, n=8). we found that the mrna level of n-cadherin, fibronectin and vimentin is much higher than that of e-cadherin in hcclm3. the expression of cyclin d1, focal adhesion kinase (fak) and fibroblast markers including n-cadherin and fibronectin was obviously suppressed in klf8 down-regulated hcclm3. conclusion: klf8 plays an important role in the process of hcclm3 proliferation, invasiveness and emt. background: insulin resistance (ir) has shown to play an important role in the progression of chronic liver diseases, including liver fibrosis development and hepatocellular carcinoma. the aim of this study was to elucidate the possible mechanisms of ir on the liver fibrosis development and hepatocarcinogenesis using obese diabetic otsuka long-evans tokushima fatty (oletf) rats. methods: to induce liver fibrosis, 1.0ml/kg of pig serum was injected twice a week for 4 weeks in the oletf and leto rats. in the hepatocarcinogenesis model, glutathione-s-transferase placental form (gst-p)-positive pre-neoplastic lesions were induced by a single injection of 200mg/kg of diethyl nitrosamine (den). we also performed in-vitro studies to examine the mechanistic insights. results: the liver fibrosis development and gst-p-positive pre-neoplastic lesions were both markedly accelerated in oletf. in the fibrosis experiment, -smooth muscle actin-positive activated hepatic stellate cells (hsc) also increased in oletf along with augmentation of the hepatic collagen content and transforming growth factor-b1. in the den model, the neovascularization was up-regulated in oletf almost in parallel with the pre-neoplastic lesions development and a potent angiogenic factor, the vascular endothelial growth factor. our in-vitro study showed that both glucose and insulin stimulated the proliferation of the activated hsc and augmented the neovascularization. conclusion: these results indicated that the ir status directly accelerated the liver fibrosis development and hepatocarcinogenesis at least partly through the stimulation of activated hsc proliferation and hepatic neovascularization, respectively, in the rat. n. wakui 1 , t. ikehara 1 , r. takayama 1 , m. takahashi 1 , k. shiozawa 1 , h. nagai 1 , m. watanabe 1 , k. ishii 1 , k. iida 1 , y. igarashi 1 , y. sumino 1 1 case: a 68 years old man diagnosed with chronic hepatitis c regularly visited our hospital. in april of 2005, ultrasonography revealed a tumor 20mm in diameter in s2 of the liver and another tumor 15mm in diameter in s6/7 of the liver. the patient was hospitalized for further examination. computer tomography (ct) revealed that the tumor localized in s6/7 presented a pattern of hypervascular hepatocellular carcinoma (hcc). for the tumor localized in s2, the following were revealed. 1) contrast-enhanced ultrasound findings: a tumor vessel passed from outside the tumor to the center of the tumor in the early vascular phase, then radiated in a wheel-like shape at the center of the tumor; parenchymal phase perfused imaging in the area produced a similar imaging obtained from the area surrounding the liver. 2) ct: no tumor was detected. 3) spio-mri (t2 weighted imaging): iso-low intensity images were obtained. although these imaging findings indicated fnh, the patient was hcv positive. in order to disprove the possibility of hcc, a biopsy was performed on the tumor at s2 in the liver. the resulting diagnosis was well-differentiated hcc. discussion: until now, a characteristic finding of fnh has been spoke-like vasculariation, which is considered diagnostically quite important. however, some recent cases of hcc have been reported to present fnh-like vascularization. from now on, when evaluating a tumor that presents spoke-like vascularization underlining chronic hepatitis, the possibility of hcc should be considered and a close examination may be needed. chronic infection with hcv is problem .clinical management of chronic hcv depend on extent liver fibrosis .liver biopsy gold stander an invasive procedure responsible for severe complications and sample variability interpretation. serum biomarkers for inflammation/fibrosis investigated to wave liver biopsy. diagnostic accuracy panel of non-invasive serum biomarkers for hepatic fibrosis (fibrosure , apri score, forn's score) versus liver biopsy. 20 hcv patients subjected for: apri, forn's , fibrosure scores pcr quantitative hcv-rnaliver functions .lipid profile cbc . ultrasound guided liver biopsy. forns score; auroc (0.917) with 95% ci(0.791-1.042) for(fof1) vs. (f2f3f4) while(0.688)with 95% ci(0.464-0.91)for(fof1f2) vs.(f3f4). cutoff(>6.9)sensitivity for significant fibrosis(f2f3f4)and extensive fibrosis (f 3f4)were (100%) and with low specificity ,with accuracy(40%) and (20%)respectively.-apri score; auroc(0.792)with 95% ci( 0.568 -1.015)comparing(f0f1) vs.(f2f3f4)while was(0.875)with 95% ci(0.703 -1.047)for( f0f1f2)vs.(f3f4).cutoff(<0.5) had low sensitivity and specificity(100%)with accuracy(60%)for significant fibrosis and(80%)for extensive fibrosis.-fibrosure(fibro-acti test); showed best auroc(1.00)in different fibrotic stages with 95 % ci (1.00-1.00).cutoff(>0.59) sensitivity(50%)for significant fibrosis and(100%)for extensive fibrosis while specificity(100%)in all fibrotic stages. the ppv (100%)for significant and extensive fibrosis .npv and accuracy(75%, 80%)respectively for significant fibroses,while it was (100%) for extensive fibrosis respectively.significant correlation between liver biopsy and fibro-test(p0.002)and acti-test(p0.000).significant correlation between liver biopsy hepatitis activity score and apri (p 0.047)and forns score (p0.000). conclusion: forns score wasn't considered since does not discriminate between significant and extensive fibrosis. low sensitivity of apri prohibtes detection of minmal fibrosis and allow undetermined results. fibrosure classified all cases of chronic hcv sufficient to wave liver biopsy pe215 introduction: hcc is the 6 th common cancer. global increase of hepatitis b and c infection, the incidence of hcc steadily increasing. egypt seroprevalence of hcv in nile delta 20 -35%. afp had limited sensitivity 60% and specificity 90% for small hcc. gpc-3 oncofetal protein over expressed in hcc. evaluating validity of glypican-3 as early detector of hcc.:10 healthy controls and 40 hcv positive patients:10 patients chronic hepatitis c virus infection.10 patients compensated cirrhosis [child-pugh class a and b].10 patients decompensated cirrhosis [child-pugh class c]. 10 patients hcc. liver functions: alt, ast, bilirubin(t), albumin, gt.tumor markers: afp and gpc-3.viral markers: hcv antibodies, hbs ag and hbc ab. the median value of gpc-3 in hcc, dc, cc significantly higher than chronic hepatitis and control groups. no significant correlation between afp and gpc-3. auroc of afp 0.85 & auroc of gpc-3 0.84. the diagnostic sensitivity of afp (20 ng/ml) 70% with ppv 53.8%. the specificity85% with npv 91.9%. while the diagnostic sensitivity of gpc-3 (2 ng/ml) 100% with ppv 27%. the specificity 42.2% with npv 100%. combined serial approach of afp and gpc-3 improved specificity to 87.5%. conclusion:gpc-3 although a serological test for early detection of hcc, showed limited specificity, where detected in different stages of chronic liver disease,it is oncofetal protein produced by regenerating liver cells. the diagnostic signature approach for simultaneous determination of afp and gpc-3 improve prediction accuracy of hcc patients in those showing seronegativity to afp. results: patients with hcv infection (n=388) were significantly older (mean age, 69 years) than patients with dual virus (n=75, 65 years) and hbv infection (n=752; 60 years) (p< 0.0001). the male-to-female ratio for hbv, dual virus and hcv group was 5.2, 3.4 and 1.3, respectively (p< 0.0001). patients in the hbv group more often had higher total tumor volume (mean, 409 cm 3 ) than the dual virus group (244 cm 3 ) and hcv (168 cm 3 ) group (p< 0.0001). no significant differences of the severity of liver cirrhosis, performance status, cancer staging and tumor cell differentiation were noted among the three groups. patients in the hcv group had a significantly poor survival in comparison to the hbv group only in the subset of patients with small tumor volume (< 50 cm 3 ) in the cox proportional hazards model (relative risk: 1.44, p=0.041). conclusions: dual hbv and hcv virus infection does not accelerate the speed of hcc formation in patients with chronic hepatitis b, and appears to have a modified course of carcinogenesis pathway diverted away from the biological behavior of hbv and hcv infection. background: patients presenting with hcc is not infrequent in our clinical practice. the aetiology vary ranging from hbv, hcv, nash and alcohol. the aim of this study was to see the aetiology of hcc in bangladeshi patients. methods: in this retrospective study, records of 976 patients who attended our opd between july 2004 to august 2008 were reviewed. patients having hepatic sol and/or heterogeneous echotexture of liver on usg and/or ct scan were included. diagnosis of hcc was confirmed at usg guided fine needle aspiration cytology with or without elevated serum afp (>500 ng/ml). results: of the 976 patients, 75% (732/976) had hbv infection. hcv infection was diagnosed in 17% (165/976). nash was responsible for 5% (49/976) cases, alcohol in 1% (10/976), while in the rest 2% (20/976) cases no specific aetiology could be established. conclusion: the study shows that hbv is the commonest cause for hcc in bangladesh followed by hcv. background: the aim of this study was to determine whether the hepatitis b virus (hbv) dna viral load and antiviral therapy is associated with hepatocellular carcinoma (hcc) recurrence. methods: this retrospective study involved 93 patients who underwent hepatic resection or radiofrequency ablation for initial hcc curative treatment. the patients were divided into four groups. fifteen patients with low serum hbv dna levels ( 4 log10 copies/ml) at the time of initial hcc treatment received antiviral therapy (lamivudine, adefovir, dipivoxil, entecavir) before hcc appeared (pre antiviral therapy group; pre-tg). thirty-four had low serum hbv dna levels without antiviral therapy (low virus group; lvg). fourteen had high serum hbv dna levels and received antiviral therapy after hcc appeared (post antiviral therapy group; post-tg). thirty patients had high serum hbv dna levels without antiviral therapy (high virus group; hvg). results: the cumulative hcc recurrence rates at 3 years in the hvg, lvg, pre-tg, and post-tg groups were 68.9%, 42.2%, 44.3%, and 57.1%, respectively. there were significant differences in the hcc recurrence rates between the hvg and lvg groups (p = 0.016), and between the hvg and pre-tg groups (p = 0.013). the recurrence rate was lower, though not significantly, in the post-tg group than in the hvg group (p = 0.18). conclusions: not only hbv dna viral load but also antiviral therapy is associated with hcc recurrence. antiviral therapy before hcc appears is important for patients with high serum hbv dna levels to prevent hcc recurrence. background/aims: few reports have described methods for predicting prognosis in unresectable hepatocellular carcinoma (hcc) patients, especially those treated by repeated transcatheter arterial chemoembolization (tae). to determine risk factors for death and determine prognosis in patients treated with repeated-tae, we evaluated clinical data. methodology: we retrospectively analyzed clinical parameters of 224 unresectable hcc patients treated with repeated-tae from january 1997 to december 2007. tae was repeated when recurrence was diagnosed by tumor marker elevation and/or dynamic computed tomography findings. factors affecting survival were evaluated using multivariate analysis after univariate analysis. next, we combined the score for each significant factor into a single prognostic score, after which the results were compared with jis and clip score methods. results: multivariate analysis revealed that bilobular hcc, alpha-fetoprotein ( 400 ng/ml), tumor invasion of the portal vein, tumor size ( 10 cm), and albumin (<2.8 g/dl) were related to poor prognosis, using those 5 factors, we developed a new prognostic scoring system. the 50% survival period was 29.2 months for all subjects, while it was 54.6, 29.2, 15.0, 5.6, and 1.0 months for those with scores of 0, 1, 2, 3, and 4 or over, respectively (p<0.0001), using our new system. clip score was not useful to predict prognosis, while jis score was better. however, subjects with jis scores of 1 and 2 were difficult to differentiate. conclusion: our scoring system was easy to perform and the results showed that repeated-tae was effective for unresectable hcc with a score of 2 or less. local ablative therapies and intrahepatic pressure c. kawamoto 1 , a. yamauchi 1 , k. kaneko 1 , n. miyagi 1 , k. kani 1 , t. aoyama 1 , k. yakabi 1 1 saitama medical center, saitama medical university, japan background: some of the unexpected recurrence observed after radiofrequency ablation (rfa) might be caused by increased intratumoral pressure. the present study examined the relationship between local ablative therapies and intrahepatic pressure. methods: a. basic study: under general anesthesia, laparotomy was performed on 19 pigs. a leveen needle and a percutaneous ethanol injection (pei) needle were inserted into the liver and intrahepatic pressure was monitored using an invasive blood pressure monitor. ablation was performed as follows: 1. rfa. 1) single-step method: after fully deploying the electrode, the power was initially applied at 30 w, then increased in increments of 10 w/min until power roll-off. 2) multi-step method: the array was deployed in 8 steps. at each step, the power was fixed at 30 w until power roll-off. 2. pei. injection of ethanol (2 ml). b. clinical study: we examined the multi-step rfa and pei for hcc. under local anesthesia, intratumoral pressure was monitored. 1. rfa. 39 patients with a mean tumor size of 15.3±4.9 mm were studied. 2. pei. in 10 patients with a mean tumor size of 21.4±8.8 mm, 5 to 10 ml of ethanol was injected per session. results : a. basic study: the intrahepatic pressures were: single-step method, 154.5±30.9 mmhg; multi-step method, 24.1±18.2 mmhg; and pei, 12.0±8.5 mmhg. b. clinical study: intratumoral pressure was 39.5±27.9 mmhg for rfa and 12.2±9.0 mmhg for pei. conclusion: these results suggest that consideration of intrahepatic pressure is crucial in local ablative therapies. background: a late evening snack (les) is recommended for liver cirrhosis. however, no clinical study has evaluated the nutrition status and the effect of les in cirrhotic patients with hepatocellular carcinoma (hcc). we investigated the effect of les undergoing hepatic arterial infusion chemotherapy (haic) in patients with hcc. method: nineteen patients with hcc were enrolled. ten patients were les group, and nine were control group. in the les group, the patients received les supplementation with a branched-chain amino acid (bcaa)-enriched nutrient mixture. in the control group, the patients received ordinary food. there were no significant differences in relation to age, gender, etiology, child-pugh scores, tumor stage, clinical responses to haic between two groups. blood biochemical data, nutrition status using an indirect calorimeter were evaluated at before and at the end of chemotherapy. results: the non-protein respiratory quotient (nprq) and molar ratio of branched-chain amino acid to tyrosine (btr) were significantly improved in the les group but not in the control group. there were no significant differences in the area under the concentration curve for glucose between before and the end of chemotherapy in two groups. background & aims: hepatocellular carcinomas (hccs) often show hypoor mixed vascularity, and the prognosis of these relatively hypovascular hccs is not fully elucidated. cytokeratin (ck) expression profiles may also be useful prognostic indicators, and specifically ck19 may reflect metastastic potency in hccs. this study was to assess the prognostic implication of tumor vascularity and its relation to ck19 expression in hcc patients. methods: a total of 150 patients who underwent surgical resection for hcc were enrolled. tumor vascularity was evaluated according to arterial enhancement pattern on ct scans and ck19 expression was evaluated using tissue microarray methods. clinicopathologic data were analyzed using kaplan-meier and cox proportional hazard model. results: during follow-up period, 91 (60.7%) patients experienced tumor recurrence. forty-five patients (30%) had hypovascluar tumor at the time of diagnosis, and they showed significantly higher positivity for ck19 expression (p=0.001) and shorter disease-free survival (p=0.023) than patients with hypervascular hccs. in addition, recurred tumors in these patients showed more frequently hypovascular pattern than in patients with hypervascular hccs (p=0.001). hypovascularity at initial diagnosis and microvascualr invasion were independent poor prognostic factors predicting survival. following treatment of recurred hccs, hypovascular tumors showed poor response to transarterial chemoembolization (tace), which resulted in shorter overall survival than hypervascular tumors (p=0.057). conclusions: these results demonstrate that tumor hypovascularity in hccs is associated with positive ck19 expression, early tumor recurrence, poor tace response and poor survival. therefore, tumor vascularity may also be a prognostic indicator in hcc patients. background: hepatic stellate cells (hscs) transdifferentiate to become extracellular matrix-producing myofibroblasts during liver injury. myofibroblasts can also promote invasion and metastasis of hepatocellular carcinoma(hcc). in this study, we determine gene expression changes in two different models of hscs activation and investigate whether induction-activated hscs(ihscs) gene expression changes are different from culture-activated hscs(ahscs). methods: hscs were isolated by density centrifugation and exposed to conditioned medium from rat hcc cell lines c5f. twenty-seven thousands and one hundred gene expression between quiescent hscs(qhscs), ahscs and ihscs was analyzed by microarray and confirmed by real-time rt-pcr and western blot. results: sixteen hundreds and seventy-one probe sets were differentially expressed in ahscs, including genes that encode proinflammatory factors, adhesion molecules, cell surface receptors, signaling transduction and immune factors. seven hundreds and eleven probe sets were differentially expressed in ihscs. induction-activated hscs showed specific gene expression patterns including raf1, rac2, adam17, wnt6, mmp-9 and tnf, suggesting that hcc cells can specifically induce hscs activation. induction-activated hscs might play a important role in invasion and metastasis of hcc. conclusions: induction-activated hscs gene expression patterns are different from ahscs. culture-activated hscs does not properly regulate gene expression in hscs, suggesting that ihscs may be considered the model for the study of hscs biology in hcc. background: hepatocellular carcinoma (hcc) is a hypervascular tumor, and angiogenesis is important for tumor growth. ephrin receptors are related with vascular system development and the polymorphism of ephb1 in the carcinogenesis of digestive tract has been reported. our aim was to examine the polymorphsims of ephb 1 with the occurrence of hepatocelluar carcinoma in korean population. methods: genomic dna was extracted from 182 patients with hepatocellular carcinoma (hcc), 266 healthy subjects. ephb 1 polymorphism was determined by polymerase-chain reaction-based assays, and the association with hcc was investigated. results: with regard to ephb 1 polymorphism, a/a genotype at rs11926992, t/t genotype at rs7644369, a/a genotype at rs6776570, t/t genotype at rs3821502 and g/g genotype at rs6766459 were significantly associated with hcc but these were not associated with clinical characteristics of hcc. conclusions: five out of seven polymorphisms on ephb1 gene were statistically associated with hcc, in the korean population. therefore, more studies of ephb1 gene polymorphisms including various risk factors should be performed to use as genetic markers of hcc occurrence. background: we aimed to compare the results of hepatectomy for hcc in patients older than 70 years old with those for younger patients. methods: clinicopathological data and outcomes for 155 elderly patients and 333 younger patients with hcc who underwent hepatectomy between 1992 and 2007 were retrospectively compared. results: although postoperative delirium was more common in the elderly group, there were no significant differences between the 2 groups with regard to operative morbidity, hospital death, disease-free survival, and overall survival. the overall recurrence rate was significantly higher in the elderly patients with alcohol abuse than in younger patients with alcohol abuse. multivariate analysis revealed that preoperative alcohol abuse was a prognostic factor for elderly patients. conclusions: elderly patients with preoperative alcohol abuse should be followed closely, even after r0 surgery, because alcohol abuse is strongly correlated with postoperative recurrence and worse survival. background: little is known about the effect of transfusing fresh frozen plasma on the outcome after hepatectomy for hepatocellular carcinoma. methods: among 410 patients who underwent curative resection between 1992 and 2005, 180 patients had perioperative transfusion with whole blood or packed red blood cells and fresh frozen plasma (group a), while 46 patients were only transfused with packed red cells (group b), 43 patients were only transfused with fresh frozen plasma (group c), and 141 patients had no transfusion (group d). results: group c had significantly fewer postoperative complications and a shorter hospital stay than group a. preoperative coagulation was significantly worse in group c. survival was significantly better in groups c and d than in group a. conclusions: perioperative transfusion of fresh frozen plasma improves clotting factors without an adverse influence on the survival of patients with liver dysfunction undergoing resection of hepatocellular carcinoma. background: this study investigated risk factors for postoperative liver failure after resection of hepatocellular carcinoma to detect markers that could identify candidates for hepatectomy. methods: perioperative risk factors for liver failure after hepatectomy were analyzed in 191 patients with hepatocellular carcinoma. results: liver failure occurred postoperatively in 16 patients, 3 of whom died. the hyaluronate/gsa-rmax ratio was a risk factor for postoperative liver failure by univariate analysis and was the only risk factor according to multivariate analysis. all 3 patients who died had a hyaluronic acid/gsa-rmax ratio 500 mg min/dl. conclusions: to reduce postoperative liver failure, preoperative planning should employ various measures of the hepatic functional reserve, including tests of both parenchymal and nonparenchymal liver function. the hyaluronate/gsa-rmax ratio can predict liver failure after hepatectomy, and a ratio greater than 500 mg min/dl is a relative contraindication to liver resection. the patient was a 73-year old japanese man with chronic hepatitis c(ch-c) who achieved a sustained virological response(svr) to interferon(ifn) therapy. as a result the liver functions were normalized and the histological findings of the liver also improved. however, 13 years after svr, mild liver dysfunction was noticed along with a marked increase of tumor markers. several modalities revealed huge liver tumors about 13 cm in greatest diameter in the left lobe invading the bile ducts and another tumor about 3 cm diameter in segment v. we performed liver biopsy and confirmed that this tumor was well-differentiated hepatocellular carcinoma (hcc). only mild fibrosis development could be observed in the adjacent non-cancerous lesions. we successfully treated these tumors with transcatheter arterial chemoembolization and stereotactic radiosurgery. recent studies revealed that the risk of developing hcc still exists even after svr. since most of hcc that develop in patients with svr are usually detected within 5 years, several investigators speculate that hcc is already present but too small to be detected at the time of completion of ifn therapy. this speculation is not the case in our patient, since svr was achieved 13 years ago and no hcv-rna could be detected when hcc appeared. therefore, another possible mechanism should be considered. an annual follow-up with strict surveillance program for hcc should be performed for more than 10 years after the completion of ifn therapy. background/aims: in order to investigate the role and importance of oxidative stress as to carcinogenecity of hepatocellular carcinoma (hcc) we analyze the expression of 8-hydroxydeoxyguanosine (8-ohdg) in the liver tissue of the hcc patients with and without hepatitis viral marker. methods: patients undergoing hepatic resection for the first hcc from 1995 to 2004 were enrolled into the study. only the cases that took no alcohol or small amount of alcohol were enrolled. 24 cases were negative for hepatitis b surface antigen (hbsag) and antibody to hepatitis c virus (hcvab) (nbnc group). 24 were positive for hbsag and negative for hcvab (b group). 21 were positive for hcvab and negative for hbsag and antibody to hepatitis b core antigen (c group). staining with hematoxylin and eosin (h&e) and berlin-blue, and immunohistochemical staining for 8-ohdg were performed using the non cancerous liver regions. the degree of 8-ohdg immunostaining was expressed as the labeling index, which means the percentage of positive hepatocytes per 1000 hepatocytes. results: the labeling index of 8-ohdg for nbnc group is 19.3(±55.3), significantly lower (p=0.014) than that for b group 49.7(±89.5), and also lower (p=0.016) than that for viral group (b group and c group)(35.4±71.8). the labeling index of 8-ohdg had no correlation with grading, staging, fatty and iron deposit among all cases. conclusions: there is possibility that oxidative stress might not associate with the carcinogenesis of hcc in some cases without hepatitis viral infection. background: no effective chemopreventive agent has been approved against hepatocellular carcinoma (hcc) yet. since neovascularization plays a pivotal role in hcc, an angiostatic agent is considered as one of the promising approaches. recently, it has reported that vitamin k2 (vk) and angiotensin-converting enzyme inhibitor (ace-i) exert anti-angiogenic activity. the aim of the current study was to elucidate the combination effect of the clinically used vk and ace-i on cumulative recurrence after curative treatment, especially in consideration of neovascularization. methods: vk (menatetrenone; 45 mg/day) and/or ace-i (perindopril; 4 mg/day) were administered for 36 to 48 months after the curative therapy for hcc. the cumulative recurrence and several indices were analyzed. results: a 48-month follow-up revealed that the combination treatment with vk and ace-i markedly inhibited the cumulative recurrence of hcc in association with suppression of the serum level of vascular endothelial growth factor (vegf); a central angiogenic factor. the serum level of lectin-reactive a-fetoprotein was also suppressed almost in parallel with vegf. these beneficial effects were not observed with single treatment of vk or ace-i for 36 months. conclusions: the combination treatment of vk and ace-i may suppress the cumulative recurrence of hcc after the curative therapy, at least partly through suppression of the vegf-mediated neovascularization. aim: the aim of this study was to clarify the cilnicopathologic features and management of hepatocellular carcinoma (hcc) patients surviving more than 5 years after hepatectomy. materials & methods: retrospective study was carried out on 823 hcc patients who underwent curative hepatectomy between 1973 and 2002. clinicopathologic factors in 5-year survivors and patients who died within 5 years were compared. the prognostic factors affecting survival were examined among the 5-year survivors. results: there were 290 patients who survived for more than 5 years after initial hepatectomy, and 83 of those patients survived for more than 5 years after hcc recurrence. the overall 3-, 5-, 10-and 20-year survival rates were 57.4%, 40.9%, 17.2%, and 8.5% respectively. in multivariate analysis, absence of underlying cirrhosis, solitary tumor, alfa-fetoprotein less than 1000 ng/ml, and absence of microscopic vascular invasion were favorable independent factors associated with 5-year survival. negative hepatitis c virus antibody status was favorable independent factor associated with longer disease-free interval and survival after tumor recurrence. multimodal treatments such as repeat hepatectomy or percutaneous ablation led to improved survival after recurrence, compared with the survival after transarterial chemoembolization (p<.05). conclusions: the results suggest that patients without underlying cirrhosis who have a solitary hcc that does not demonstrate vascular invasion or high afp levels might survive for longer than 5 years after the initial hepatectomy. close follow-up and multimodal treatment could contribute to prolongation of survival in such patients, even if cancer recurrence occurs. the history of the use of carbon ion radiotherapy (cirt) for treating hepatocellular carcinoma (hcc) goes back to 1995, when clinical trials were initiated at the national institute of radiological sciences. we have already reported that cirt used for the treatment of hcc is safe and effective, and that it causes only minor liver damage. in a phase ii clinical trial, the local control and cumulative overall survival rates were 94 % and 35 % at 5 years, respectively. however, the patients with tumor adjacent to the gastrointestinal tract are thought to be ineligible for cirt because of the high risk of radiation injury of the digestive organs. in order to extend the indication of cirt, we have challenged the cirt for such patients under the use of spacers. a case was a 67-year-old female with 8cm tumor in segment 4. in radiological findings, the tumor revealed typical enhancement pattern for hcc, and was near the ec junction. she had been judged ineligible for hepatectomy because of the high retention rate of indocyanine green. she could undergo the 42.8 gye/2-fraction cirt after the placement of gore-tex soft tissue patch under the laparoscopic procedure. up to the present date, no adverse effect due to the spacer has been occurred, and an apparent anti-tumor effect has been observed. this method seems to have a promising efficacy for extension of the indication of cirt to the patients with tumors adjacent to the gastrointestinal tract. background: previously we reported that high ubiquitination was marker of human hepatocellular carcinoma. on the basis of these finding, we firstly analyzed the effect of bortezomib(proteasome inhibitor) on human hcc cell line. we also reported that hhm/dip1/gcip1 was early marker for human hepatocarcinogenesis. hhm was suggested to be a new tumor suppression gene, but the mechanism was not well confirmed. we analyzed change of hhm signal by bortemib. method and result: we used hcc cell line (huh7, hlf, hepg2) . the inhibitory effect of bortezomib was evaluated using mtt assay. 20nm bortezomib significantly inhibited proliferation of hcc cell line. the inhibitory effect by 20nm bortezomib was similar with 10 m cisplatin. on the other hand, bortezomib has no inhibit effect in isolated hepatocyte from rat. in this condition, we analyzed the expression of cyclin d1, phospho-rb and hhm in hcc cell line by western blot analysis. expression of cyclin d1, phospho-rb decreased, but hhm was increased with time. next we analyzed cell cycle by facs. bortezomib induced hcc cell line into cell cycle arrest in g2/m. the transcriptional activity of hhm was also activated by bortezomib administration using ptimer-promoter-hhm plasmid. conclusion: bortezomib has specific anti-proliferative effect on hepatocellular carcinoma. the induction of hhm by bortezomib might be related with cell cycle arrest. bortezomib will be a useful drug for hcc. neovascularization is required for carcinogenesis of non-alcoholic steatohepatitis: experimental and clinical study m. kitade 1 , h. yoshiji 1 , r. noguchi 1 , k. kaji 1 , t. namisaki 1 , y. aihara 1 , h. background/aim: non-alcoholic steatohepatitis (nash) may progress to liver cirrhosis, and finally hepatocellular carcinoma. recent study suggested that development of hepatic angiogenesis correlates the risk for hepatocarcinogenesis in liver cirrhosis patient. we therefore examined the role of angiogenesis in the hepatocarcinogenesis of nash in both experimental and human study. methods: as an experimental nash model, zucker (z) rats, which naturally develop leptin receptor mutations, and their lean littermate (l) rats were fed a choline-deficient, amino acid-defined (cdaa) diet. in human study, 11 patients with nash-related cirrhosis or pre-cirrhosis, regarded as high risk group of hepatocarcinogenesis, and 13 with simple fatty liver (fl) were enrolled and underwent clinico-pathological examinations. immunohistochemical analysis of 4-hydroxy-2-noneal (4-hne) and cd34 were employed for detection of reacrive oxidative stress (ros) and angiogenesis in the liver tissues, respectively. results: in experimental nash model, both groups showed marked steatohepatitis by feeding cdaa diet. in sharp contrast, the development of glutathione-s-transferase placental form (gst-p)-positive pre-neoplastic lesions and hcc could be observed only in the l-rats. the hepatic neovascularization was also significantly increased only in the l-rats. in human study, both nash and fl exerted a marked elevation of ros. in sharp contrast, significant development of hepatic neovascularization was observed only in nash, whereas almost no neovascularization could be observed in fl. conclusion: in conclusion, these results suggested that neovascularization might play a important role in hepatocarcinogenesis in nash. background: paternally expressed gene 10 (peg10), which was an imprinted gene with an active paternal allele but silent maternal allele, was highly expressed in a great majority of hepatocellular carcinoma(hcc). the aim of this study was to generate transgene mice expressing peg10 in the liver under the control of mouse albumin (alb) promoter and study the integration, transcription, expression of peg10 gene in the transgenic mice methods: the linearized 1716bp transgene fragments, which contained alb promoter and structural gene of peg10, were microinjected into fertilized eggs of mice. then manipulated embryos were transferred into the oviducts of pseudo-pregnant female mice. all the newborn mice were screened and identified by pcr detecting genomic dna in tail tissue. as the transgene was driven by the alb promoter, we examined its expression in the liver of transgenic mice by rt-pcr and western blotting. results: the transgene fragment was microinjected into the male pronucleus of 3741 fertilized oocytes. the injected eggs were implanted into oviducts of 94 pseudo-pregnant foster mothers, of which 22 mice became pregnant and give birth to 108 offspring. 65 of them died from unknown reason. among the 43 offspring, 3 were identified to carry peg10 cdna as demonstrated by pcr, and peg10 transgene could be expressed successfully in the liver of the established transgenic mice. the ratio of transgene integration were 6.97% (3/43) by pcr. conclusions: the peg10 transgenic mouse model should be valuable for studying the in viro function of this imprinted gene in hcc. background/aims: brivanib alaninate is the l-alanine ester prodrug of bms-540215, an oral selective dual inhibitor of vascular endothelial growth and fibroblast growth pathway receptors. it is being developed in treating hepatocellular carcinoma (hcc), a disease highly prevalent in asia-pacific region. this analysis investigated whether bms-540215 exposure was different between asian and non-asian subjects. methods: a population pharmacokinetic (ppk) model was developed with data collected in 125 subjects (78 non-asian, 47 asian) with advanced and metastatic solid tumors (including hcc) from 2 clinical studies. potential effects of the following covariates on model parameters were examined: age, gender, race, and baseline body weight. model-based simulation was performed to examine bms-540125 exposure in asian and non-asian patients following brivanib doses of 800 mg qd (phase iii dose). results: the ppk of bms-540215 was characterized by a 2-compartment model with first-order absorption and elimination. clearance was found to slightly increase with body weight (p<0.01). however, effects of age, gender and race on clearance were not statistically significant. the median of apparent clearance in asian was 9.5% lower than that of non-asians, which was adequately explained by 16% lower body weight in asians. there was substantial overlap in steady-state bms-540215 auc of asian and non-asian patients, simulated based on their observed body weight distributions in these patient groups. conclusions: bms-540215 pk can be adequately described by a linear 2-compartment model; exposures in asian and non-asian subjects are similar following brivanib doses of 800 mg qd. background/aims: hepatic resection is the standard treatment for hepatocellular carcinoma. in some patients with multiple hcc, one-block resection can not be feasible due to either the tumor location or the reserved liver function. in this study, we attempted to analyze the outcome of multiple-site resection or combined resection and rfa in patients with multiple hcc. the prognostic factors for postoperative survival were also investigated. methods: among 507 patients who received resection from january 1996 to august 2006, 58 patients had a radiologically detected multiple hcc. patients with multiple hcc were divided into: group a, patients treated with one-block resection (n=40) and group b, patients with multiple-site resection or combined resection and rfa (n=18). results: in group b, 6 received multiple-site resection and 12 underwent combined resection and rfa. the clinicopathological variables and postoperative complication rate were not significantly different between the two groups. the 5-year disease-free survival rates for group a and b were 24.1% and 18.3%, respectively (p=0.386). the overall survival rates were also not significantly different (36.9% vs. 39.4%, p=0.528). the multivariate analysis revealed that radiological tumor number 3, edmondsons-steiner grade (iii-iv) and indocyanine green retention rate at 15 minutes> 10% were adverse prognostic factors for overall survival. conclusions: active treatments including multiple-site resection and combined resection and rfa showed similar treatment outcomes compared with one-block resection in patients with multiple hcc. the prognosis after treatment was associated with tumor number, tumor grade and icg r15. background: nasopharyngeal carcinoma (npc) is endemic to southern china. mortalities are mostly associated with secondary metastases. novel treatments for npc metastases are thus urgently needed. we aim to test the efficacy of a physiologically stable gold compound, gold (iii) meso-tetraarylporphyrin 1a (gold-1a), in treating intrahepatic npc metastasis in athymic mice. methods: twenty million of c666-1 human npc cells were injected into the livers of athymic mice to induce primary tumors. gold-1a was administrated by intraperitoneal injection. survival times, tumor volumes and degrees of metastasis of the animals were evaluated. intratumoral microvessel density was determined by immunohistochemical staining for cd34. tube formation by ms1 mouse endothelial cells were conducted with an in vitro angiogenesis assay kit. gene expression level was determined by semi-quantitative reverse transcription-polymerase chain reaction. cell proliferation was performed by methylthiazolyldiphenyl-tetrazolium bromide assay. result: gold-1a prolonged the survival and inhibited intrahepatic and lung metastasis of the tumor-bearing animals. the compound induced tumor tissue necrosis and reduced tumor microvessel formation. in in vitro studies, gold-1a inhibited tube formation and proliferation of ms1 cells, and downregulated the expression of stanniocalcin 1 (stc1), which plays roles in angiogenesis. furthermore, our preliminary data showed that overexpression of stc1 in ms1 cells rescued cells from gold-1a-induced death. conclusion: gold-1a is a novel anticancer agent that prolongs survival of the npc metastases-bearing mice. it inhibits intrahepatic and lung metastasis in vivo and inhibits angiogenesis in vitro, in part via downregulation of stc1. tbx3 is a transcriptional repressor that is important for embryonic development. overexpression of tbx3 was found in a large variety of cancers, including breast cancer, ovary cancer, cervical cancer, lung cancer, bladder cancer and liver cancer. tbx3 promote carcinogenesis by bypass cellular senescence via suppression of p14 arf . our resent studies revealed that two key motifs composed of 7 + 3 residues are essential for its transcriptional repression. based on this finding, we designed a set of peptides to block its transcriptional repression activity and tested their antiviral effects. we found that tat-tagged peptides (taps) effectively transduced hepatoma hepg2 and bel7404 cells at almost 100% efficiency and inhibited cell growth in a dose dependent manner. further studies revealed that the tap treated cells underwent up-regulate apoptosis via suppression of p14 arf both at mrna and protein levels, demonstrating the potential of novel taps for anti-hcc treatment in the future. safety and long-term outcomes of radiofrequency ablation therapy in elderly and cirrhotic patients with hepatocellular carcinoma k. kakisaka 1 , h. kuroda 1 , k. kasai 1 , y. takikawa 1 , k. suzuki 1 1 iwate medical university background and purpose: a tendency of the aging in patients with hepatocellular carcinoma (hcc) is predominantly seen in japan. in fact, the mean age of patients with hcc in our institute in 1990 was 60.1 years old, while that in 1999 was 67.8 years old. it is not still remained whether the percutaneous radiofrequency ablation (rfa) therapy in elder patients with hcc is safety and equal in therapeutic usefulness compared to the non-elder patients with hcc. subjects and methods: two hundred six cirrhotic patients with hcc (376 tumor nodules) received rfa therapy curative intent since august, 2006 were enrolled. we divided all patients into two groups: over 65 years (elder group: n=135) and under 65 years (non-elder group: n=71), and compared the patient's characteristics, tumor factors and survival rate and causes of death in two groups. results: the characteristics of patients, tumor factors, cumulative survival rate and recurrence rate were not revealed in two groups. although in elder group two patients complicated aspiration pneumonia and respiratory depression due to sedation under rfa respectively, total occurrence rate of complications did not differ between two groups. conclusion: rfa therapy is safety and effective even in elder patients with hcc, although their care is necessary to prevent any complications which are often occurred during the rfa therapy. background and purpose: the aim of this study is to evaluate whether administration of the branched-chain amino acid (bcaa) enriched nutrient (namely, aminoleban en, ostuka pharmaceutical company, japan) might improve protein-energy malnutrition (pem) status and quality of life (qol) in cirrhotic patients with hcc receiving rfa therapy. subjects and methods: thirty-five cirrhotic patients with hcc who had received rfa therapy from october 2005 to october 2006 in our institute were randomized into two groups: diet with supplementation of aminoleban en (en group: 20 patients, 420 kcal/day) and diet only (control group; 15 patients). the total intakes of calories (30-35 kcal/kg) and protein (1.0-1.5 g/kg) were equal between tow groups. the primary end point was event-free survival rate (development of liver cancer, rupture of esophageal varices, or progression of hepatic failure) and second end points were serum albumin levels and the health-related qol by shortform-8 questionnaire (sf-8). results: total intakes of calories and protein were similar during the one year after rfa. no significant differences in event-free survival rate were seen between two groups. however, decreased serum albumin levels and one (general health perception) of domains in sf-8 were significantly improved in en group compared to the control group. conclusion: supplementation of bcaa-enriched nutrient may improve the impaired liver function and qol after rfa therapy. large scale prospective study should conduct to confirm these results near the future. backgrounds and aims to investigate the effects of selective cox-1 and cox-2 inhibitor on proliferation and apoptosis of hcc cell. methods hep3b and snu 387 cells were treated with ns-398 and sc-560. mtt assay, caspase 3/7 activity assay and tunel assay were performed. cox protein and mrna expression were measured by western blot and real time rt-pcr. results in hep3b cell line, cox-1, cox-2 (50, 100, 200 um) and combination (25+25, 50+50, 100+100 um) treatment after 24hr showed a significant dose dependent inhibitory effect on cell growth (p<0.05). cox-1, cox-2 (100 um) and combination (50+50, 100+100 um) treatment after 24hr significantly increased caspase 3/7 activity (p<0.05) and induced apoptosis (p <0.05). however, the combination treatment could not showed a additive effect to cox-1 or cox-2 inhibitor (p>0.05). in snu 387 cell line, cox-1 inhibitor and combination treatment showed a inhibitory effect on cell growth (p <0.05) similar to hep3b cell line but any of treatment could not induce apoptosis significantly (p >0.05). in cox protein and mrna expression, snu 387 cell line showed significant cox-1 predominency (p=0.037) but hep3b cell line showed cox-2 predominency (p=0.032). conclusions in hcc cells, no additive effect of the combination treatment of cox-1 and cox-2 inhibitors could be anticipated. the apoptosis inducing effect of cox inhibitor could be different between hcc cell lines. more studies for the mechanism of different response to cox inhibitor between cell lines is needed. background: the aim of this study was to determine the maximum tolerated dose and recommended dose of combination chemotherapy with mitoxantrone and uracil/tegafur (uft) (phase i part), and to clarify its efficacy (tumor response, overall survival, and progression free survival) and safety in patients with advanced hepatocellular carcinoma (hcc) at the recommended dose (phase ii part). methods: patients eligible for study had histologically confirmed, chemo-naïve advanced hcc, who were unsuitable for resection, local ablation therapy or transcatheter arterial chemoembolization. the therapy consisted of mitoxantrone dosages (6, 8 and 10 mg/m 2 /day) intravenously on day1 and oral administration of uft 300 mg/m 2 on day 1 through day 21. the treatment was repeated every four weeks if there was no evidence of tumor progression or unacceptable toxicity. results: a total of 25 patients were entered into the study. all had a good ecog performance status score of 0-1. in phase i part, dose limiting toxicities occurred in all three patients (two patients: grade 4 neutropenia, one patient: grade 3 creatinine elevation) given mitoxantrone at dosage of 10 mg/m 2 /day, and the recommended mitoxantrone dosage was 8 mg/m 2 /day. among 19 patients administered at the recommended dosage, one patient (5.3%) achieved a partial response, 8 patients (42.1%) had stable disease and 10 patients (52.6%) had progressive disease. one-year survival proportion, median survival and median progression free survival were 26.3%, 8.4 months and 2.5 months, respectively. the most common toxicities were grade 3-4 leucopenia (15.4%) and neutropenia(10.3%). conclusion: mitoxantrone 8 mg/m 2 with uft 300 mg/m 2 /day is recommended dose. this regimen is generally well tolerated, but appears to have little activity for advanced hcc. these findings do not support its use in practice, and further trials with this regimen in patients with advanced hcc are not recommended. the study assessed the benefits of 3-d reconstruction of spiral ct scans for the diagnosis of and surgical guidance to large liver tumors or tumors at the hepatic hilum. we retrospectively analyzed 33 cases of children with such tumors treated in past 5 years.the patients were examined by 3-d reconstruction using 64 slice spiral ct. in 28 cases, the volume of tissue removed exceeded 1/3 the entire volume of the liver. in 5 cases, the excised tissue represented less than 1/3 of the total liver volume, but the location of the tumor was adjacent to major hepatic vessels. pathological diagnoses included hepatoblastoma (n = 20), hepatocellular carcinoma (n = 4), mesenchymal hamartoma (n = 4), teratoma (n = 1) and adenoma (n = 4). all children had curative resections with tumor-free microscopic margins. 3-d ct imaging can provide high quality images and accurate location of the tumors. it could help the surgeon identify the tumor borders accurately and devise a safe surgical strategy. with its help the surgeon could identify vital hepatic blood vessels before operation, and can avoid massive hemorrhaging during operation. background: to investigate the association between c-509t polymorphism of transforming growth factor (tgf)-1 gene and hbv-related hepatocellular carcinoma (hcc). methods: patients with hbv infection (196 cases were hbv carriers, 379 cases were hcc) and 299 healthy volunteers were enrolled. the polymorphism of tgf-1 gene c-509t was identified by polymerase chain reaction-restriction fragment length polymorphism method. the concentrations of plasma tgf-1 were measured by enzyme linked immunosorbent assay (elisa). tgf-1 mrna expression was quantified by real-time pcr. a recombinant construct containing -509c>t variant as promoter and cat as reported gene was transfected into hepg2 cells. the reporter gene cat was detected with elisa. results: the ct genotype at position -509 of tgf-1 gene prevailed in all three groups, the frequency of genotype cc and allele c at -509 in hcc were significantly higher than those of the hbv carriers and controls. the plasma tgf-1 concentration among the three genotypes did not show any significant difference in three groups. however, both the tgf-1concentration and liver mrna levels were statistically higher in patients with cc genotype than in those with tt genotype in the hcc group. reporter gene cat was elevated when hepg2 were transfected with -509c-cat recombinant construct compared to that with -509t-cat one (p<0.05) conclusion: the presence of c allele at position -509 may play an important role in the development of hbv-related hcc through influencing tgf-1 expression both at mrna level and protein level. background: to assess diagnostic value of n-glycan markers in identifying hepatocelluar carcinoma (hcc) from liver fibrosis after hbv infection. methods: a total of 273 cases of hbv related liver fibrosis (n=128) and hcc (n=145) patients as well as matched healthy controls (n=120) were recruited. routine liver function and tumor markers were detected by automatic biochemistry or immunological analyzer. n-glycome of serum protein was profiled by dna sequencer-assisted fluorophore-assisted carbohydrate electrophoresis with a capillary electrophoresis-based abi3130 sequencer. results: the abuncance of a single agalacto biantennary glycan (ng1a2f, peak 4) was increased in liver fibrosis and decreased in hcc, while that of a branching triantennary glycan (na3fb, peak 9) was decreased in fibrosis and increased in hcc. the efficacy of the log ratio of above two n-glycan abundance [log (p9/4)] was similar to afp in differentiation hcc from fibrotic patients. with logistic regression analysis, the accuracy and sensitivity of the diagnostic model combining afp with n-glycan analysis(cscore b) were increased 7-10% compared to afp. log(p9/4) was even more powerful in monitoring the progresison of hcc with the specificity improved 16% and accuracy improved 8% compared to that of afp. besides, log(peak 9/4) was correlated well with other tumor markers and tnm stages. conclusions: the log ration of the abundance of a branching triantennary glycan (na3fb, peak 9) to a single agalacto biantennary glycan (ng1a2f, peak 4) and the model combining afp with n-glycome markers are promising in hcc diagnosis and progression monitoring. the low incidence of tumor seeding and post-procedure bleeding after radiofrequency ablation (rfa) of hepatic tumors has been attributed to the use of thermocoagulation of the tract, which results in necrosis, upon electrode withdrawal. however, different investigators use different techniques with no experimental evidence of the effectiveness of a particular technique. objective: we aimed to compare the necrotic zone produced using different electrode withdrawal techniques. methods: eighteen tract ablation zones were created in ex vivo porcine livers by withdrawing an internally-cooled rfa electrode (cool-tip radiofrequency system, valleylab) 1-2 mm/second using energy-dependent (20 vs.40 vs.60 vs.80 watts) and temperature-dependent (80 vs. 90 0 c) techniques. horizontal mathematical modeling suggests an impractical number of radiofrequency ablation (rfa) zones needed in order to ablate a medium-large hepatic tumor. however, overlapping rfa zones may increase the necrotic diameter disproportionately to that deduced from single ablation alone. objectives: to compare the necrotic diameter in single (group1), dual overlapping (group2) and dual non-overlapping (group3) ablation. methods: single (n=5) and dual (overlapping n=18; non-overlapping n=6) ablation zones were created in ex vivo porcine livers using cool-tip rfa electrodes. necrotic diameter was measured at the midpoint (maxd) of the single and the two distinct rfa zones of the dual ablation groups and compared with the necrotic diameter at the tip of the second ablation (maxd-o), corresponding to the point of overlap in group2. the rfa electrode was withdrawn 2.5 and 4.1 cm before re-ablating for group2 and group3, respectively. results: despite no difference in end-rfa temperature between 3 groups (group1=75.6+7.4cvs.group2=73.9+7.3cvs.group3=74.8+4.8c; p=0.873), maxd was significantly greater (p=0.011) in group2 (4.1+0.7cm) as compared to group1 (3.3+0.6cm) and group3 (3.3+0.4cm), with no difference between group1 and group3(p=1.00). further proof of synergism between two overlapping ablations is that the maxd-o in group2(4.3+0.6cm) was larger than maxd of group1 (p=0.042) and group3 (p=0.028), and was similar to maxd of group2 (p=1.00). conclusions: overlapping two rfa zones results in incremental increase in necrotic diameter compared to single and dual non-overlapping ablation. this may explain the discrepancy in the number of ablation zones needed between clinical and mathematical modeling studies. background: hepatocellular carcinoma (hcc) is the fourth most common cancer worldwide, main etiological factors being chronic infections with hepatitis b and c viruses. the present study was undertaken to evaluate the association of glutathione-s-transferase (gst) t1 and m1 null genotypes and microsomal epoxide hydrolas e(mephx) polymorphisms with hepatitis virus related hcc risk in indian population. subjects and methods: three groups of subjects were considered viz. control (n=169), chronic viral hepatitis (n=174) and hcc (n=63). pcr-rflp was used for this polymorphic study. genotype distributions between categories were compared using the 2 test; odds ratios (ors) and 95% ci were calculated to express the relative risk. results: presence of gstm1 null genotype significantly (p<0.05) decreased the risk for hcc development among chronic viral hepatitis subjects. however, gstt1 null genotype was associated with an increased risk for hcc by 2.23 and 1.42 times among control and hepatitis subjects respectively. in case of mephx, tyr113his and his113his genotypes significantly (p< 0.05) reduced the risk of hcc development in both viral hepatitis and control subjects. in case of mephx exon 4 genotypes, arg139arg imposed an approximate 2 fold risk for hcc development in the two groups. combination of heterozygous mutant genotypes at mephx exons 3 & 4 also imposed around 2 fold risk (non-significant) for hcc. conclusions: polymorphic forms of gst and mephx share an association with viral related hcc risk in indian population and should be further evaluated as the candidate genes to determine individual susceptibility for viral related hcc. background : the association between type 2 diabetes mellitus (dm2) and hepatocarcinoma (hcc) has been identified in the last ten years. methods: to clarify the temporal relationship between dm2 and hcc and the possible effects of antidiabetic therapy on hcc risk, we recruited 465 patients with hcc compared with 490 control subjects without liver diseases and 618 cirrhotic patients. results: prevalence of dm2 was 31.2% in hcc, 23.3% in cirrhotic and 12.7% in control group. in univariate and multivariate analysis, the odds ratio (or) for hcc in diabetic patients were respectively 3.12 (ci 2.2-4.4; p <0.001) and 2.2 (ci 1.2-4.4; p= 0.01). or in univariate analysis were higher in male than in female patients. in 84.9% of the patients dm2 pre-exists the diagnosis of hcc from a mean time of 141.5 months. moreover, the insulin treatment was more frequent in diabetic hcc patients than controls and we report an or for hcc of 2.99 (ci 1.34-6.65; p= 0.007) in patients treated with insulin or sulfonylureas, and an or of 0.33 (ci 0.1-0.7; p= 0.006) in patients treated with metformin. conclusion: our study confirms that male patients with type 2 diabetes mellitus have a significantly increased risk of hcc independently of other cofactor such as hbv, hcv and alcoholic abuse. dm2 is a pre-existing disease in most hcc patients and suggests that insulin and sulphonylurea treatments in dm2 are associated with an increased risk of hcc development, while metformin may have a protective effect. background & aims: over the last few years, techniques that allow systematic analysis of chromosome aberrations at a genome-wide level were applied to hcc. the purpose of this study is to apply gene loss expression profiling in the attempt to discover new related genomic regions not revealed by loh or cgh, and search the new tumor suppression genes for hcc. methods: primary hcc and corresponding non-tumor liver tissues were obtained from surgery. serologically, 13 cases were with hepatitis b virus infection and 12 cases were with hepatitis c virus infection. four non-viral infected tissues from four patients receiving surgical resection for hepatic adenoma or focal nodular hyperplasia.affymetrix genechip, u133a, was used to compare the loss and gained gene expression in liver needle biopsy samples (n=54). results: after adjusting by chromosome arm length, 16p, 17p, 19p, 19q and 22q showed higher gene loss-expression ratio (>= 10 loss / cm) in the comparison between normal samples and tumor samples; 1q, 2p and 4q showed higher gene loss-expression ratio in the comparison between tumor and non-tumor tissues. more than 50 genes showed different loss expression level in this study. for example, cd10 was loss expression in all non-tumor samples comparing to four normal samples. ficolin 3 and ficolin 2 were loss expression in hcc samples with hbv infection and with hcv infection, respectively. conclusion: our results revealed the potential tumor suppression genes and the genomic region they harbored. further study is needed to validate the observation. background/aims: hepatocellular carcinoma is common malignancy in human, accounting for 1 million deaths in the world annually. caspase 8, as an initiator caspase, is involved in the induction of apoptosis. survivin, a novel inhibitor of apoptosis is related to the ability to inhibit caspases and involved in critical steps of onset and progression of hcc with unfavorable prognosis. methods: to explore the possibility that the epigenetic alteration of caspase 8 and survivin genes is implicated in the development and progression of hcc, promoter methylation of two genes was analyzed in 73 cases of primary hcc by methylation specific pcr. the relationship between immunohistochemical expression of gene products and proliferative/apoptotic indices, and clinicopathologic parameters was also investigated. results: the methylation of caspase 8 (34.2%, 25/73) and survivin (32.9%, 24/73) demonstrated a negative correlation with immunohistochemical expression of capsase 8 (47.9%, 35/73) and survivin (43.8%, 32/73) (p=0.042 and p=0.001 respectively). methylation of caspase 8 and immunohistochemical expression of its gene product was significantly correlated with apoptosis (p=0.026 and p=0.032). survivin nuclear immunoreativity revealed significantly correlated with proliferative activity of tumor cells (p=0.001). by survivial analysis, the negative caspase 8 expression and positive survivin expression showed worse prognosis in hcc, that was statistically insignificant (p>0.05). conclusion: in conclusion, caspase 8 and survivin may contribute an important regulatory mechanism for tumor cell proliferation and apoptosis, and may be prognostic predictors in hcc. injection was recently reported to be effective against hcc with pvi, though the therapy is not always applicable for the patients with arterial abnormality. therefore we tried combination therapy of transcatheter arterial cisplatin embolization and radiation, and will report the effectiveness and toxicity of the therapy. methods: the combined therapy was conducted in 7 hcc patients with pvi. transcatheter arterial embolization with 1mg/kg cisplatin powder (ia call) was performed against intralobar lesions, followed by external radiation targeted for pvi (60gy in 2 gy fractions). the following variables were evaluated with the survival rate: gender, age, viral etiology, child's class, performance status, and location of pvi. results: one (16%) patient showed complete response and another two (33%) partial response. two (33%) showed no change, and one (16%) showed progress of disease. the survival rates at six months among overall patients were 85.7%. adverse events were limited to nausea and appetite loss. one of the patients with partial response underwent curative resection, and is still alive without any recurrence for 530 days. conclusions: the combination therapy of cisplatin embolization and radiation is safe, effective and also feasible to the patients with arterial abnormality. this therapy is suggested to be a useful alternative therapy for the patients with extensive pvi. recently, the injection port has been used for hepatic arterial infusion chemotherapy (hai) in japan. hai is usually used for the treatment of multifocal bilobar tumors of the liver or hccs combined with portal vein tumor thrombosis (pvtt), not amenable to tace. this study examined the efficacy and toxicity of repeated hepatic hai using lipiodol suspension mixed with cisplatin powder. methods: from april 2005 to september 2008, 20 patients with inoperable advanced hcc were enrolled in this study. all received cisplatin powder (50mg) and lipiodol (2ml) suspension, with an intervening 4 weeks interval. the drugs were delivered from an injection port. 13 patients had hcc with pvtt, and 7 had hcc without pvtt. 11 patients with liver function of child grade a, 7 of grade b, and 2 of grade c were enrolled. result: the mean number of hai given during the follow-up period was 4.1 times. we found complete response in 1 case, partial responses in 6, no change in 6, and progressive disease in 7. the overall response rate was 35.0%. the 1-year survival rate was 36.7% and the 2-year survival rate was 12.1%. although 3 patients had cisplatin-induced anaphylaxis, no severe adverse events (hepatic failure and renal failure) were observed. conclusion: chemo-lipiodolization using cisplatin powder delivered via an injection port provides some clinical benefits without severe adverse events in patients with far advanced hcc. background: recently, the antitumor efficacy of angiogenesis inhibitors is expected in the treatment to hepatocellular carcinoma. the gene expression relevant to the vascularization, which is a target of these inhibitors, has a difference according to each case and it is thought that it influences the therapeutic effect of them. however, there are still few reports of mrna expression of vascular endothelial growth factor (vegf) receptors in hcc. methods: the relative mrna level of vegf and its receptors (kdr and flt-1) was analyzed using quantitative rt-pcr in 51 patients with hcc. matched samples of hcc (t) and non-tumor liver tissue (nt) were obtained by fine needle (21gauge) biopsy. results: gene expression level of vegf and flt-1 was significantly higher in hcc than nt (vegf; p<0.001, flt-1; p<0.001). according to the clinicopathological findings, gene expression level of vegf and kdr in hcc was significantly high in hypervascular hcc compared to hypovascular hcc (vegf; p=0.002, kdr; p=0.042). additionally, flt-1 tended to be expressed higher in hypervascular hcc than hypovascular hcc (p=0.09). moreover, gene expression level of vegf, kdr and flt-1 tended to be higher in advanced-stage hcc than early-stage hcc. conclusion: not only vegf but kdr and flt-1 were highly expressed in hypervascular and advanced hcc. aims: fibrinogen-like protein 2/fibroleukin (fgl2) has been reported to play a vital role in the pathogenesis in mhv-3 (mouse hepatitis virus) induced fulminant and severe hepatitis, spontaneous abortion, allo-and xeno-graft rejection by mediating "immune coagulation". fgl2 functions as an immune coagulant with the ability to cleave prothrombin to thrombin directly. therefore, this study was designed to examine the role of fgl2 in tumor development. methods: tumor tissues from 133 patients with six types of distinct cancers and the animal tumor tissues from human hepatocellular carcinoma (hcc) model on nude mice (established from high metastasis hcc cell line mhcc97lm6) were obtained. results: hfgl2 was detected in tumor tissues from 127 out of 133 patients as well as tumor tissues collected from human hcc nude mice. hfgl2 was highly expressed both in cancer cells and interstitial inflammatory cells including macrophages, nk cells, and cd8 + t lymphocytes and vascular endothelial cells. hfgl2 mrna was localized in cells that expressed hfgl2 protein. fibrin (nogen) co-localization with hfgl2 expression was determined by dual immunohistochemical staining. in vitro, il-2 and ifn-increased hfgl2 mrna by 10-100 folds and protein expression in both thp-1 and huvec cell lines. one-stage clotting assays demonstrated thp-1 and huvec cells expressing hfgl2 had increased procoagulant activity following cytokines stimulation. conclusion: the hfg12 contributes to the hypercoagulability in cancer and may induce tumor angiogenesis and metastasis via cytokine induction. . the therapy was either terminated at the end of the first cycle in cases with progressive disease, or continued for at least 2 cycles, when responses to treatment were evaluated by eastern cooperative oncology group criteria. results: of 146 patients treated (male, 79%; median age, 64 years), 64% had child-pugh a, and 31% had b. 90% had either metastasis or vascular invasion. 71% had metastasis and 49% had vascular invasion. on the basis of independent assessment, three (2.1%) patients achieved a complete response, thirteen (8.9%) had a partial response, and 42 (28.8%) had stable disease. there was no grade 3/4 drug related toxicities. median overall survival was 6.9 months. conclusion: combination therapy of ifn +5-fu has modest efficacy in hcc. background: amt is a mixture of approved pharmaceuticals in low therapeutic doses (human insulin and chlorpheniramine) and herbal components (aqueous camomile extract). preclinical and phase i data in healthy volunteers showed a favourable safety profile for amt. this pilot study should examine efficacy and safety of amt in the patients with advanced hepatocellular carcinoma (hcc). methods: thirteen patients with advanced hcc (tnm stage iii-iv), who did not respond to existing therapy, were treated with i.m. amt at 0.066 ml/kg up to a maximum volume of 5 ml twice daily for 1-10 months. primary study objectives: clinical benefit response (cbr). secondary objectives: safety of amt, tumor response according to who-recist criteria, quality of life (qol) and iimmunomodulatory effects. the effects were evaluated by cytokine production of pbmcs before and after the treatment. results: there were no significant safety issues. four and 3 patients showed positive and stable responses for cbr, respectively. tumor response was 1 pr, 6 sd and 6 pd. even in the patients with pd, 2 and 1 patients showed positive and stable responses for cbr. qol data showed clear improvement. immune monitoring demonstrated effects of amt on the functional immune parameters in about half of patients. in the patients with pr, histological examination showed tumor necrosis and many lymphocytes including plasmacytes infiltrating in the tumor. conclusion: these results suggest that a promising rate of patients with advanced hcc respond clinically to the amt treatment without significant safety issue and amt has some immuno modulatory capacities. background/aims: dysplastic nodules are important due to premalignant potential. the aim of this study was to evaluate the electron microscopic findings of liver dysplastic nodule in patients with liver cirrhosis. methods: a total of 5 patients (mean age: 64±9 years old, male 4) with dysplastic nodules which suspected as malignant nodule (mean size 1.38±0.22 cm) was enrolled from 48 cases of liver cirrhosis undergone ultrasonography-guided biopsy from december 2006 to january 2008. the etiologies of liver cirrhosis were as follows; alcohol (1 patient), hepatitis b virus (2), and hepatitis c virus (2). results: hepatocytes showed rosette formation of regenerative hepatocyte or degeneration. the nucleus was round or oval shaped and the nucleus membrane was irregular. the nucleolus was prominent and clear, the mitochondria were crowded to one side in the cytoplasm with megamitochondria. glycogen granules and lipofuscin pigments were abundant. sinusoid formation was poorly developed and collagen fiber bundles were increased. the hepatocytes of rosette formation and bile ductules cell made of canal of hering, which was dilated and microvilli was decreased. the number of canal of hering was 7, which was composed of 1.7±0.8 with hepatocyte and 2.1±0.7 with bile ductule cell, respectively. there was no oval cell in the canal of hering, which was relatively well developed. schwann cells were clustered together in nerve plexus. therefore, these electron microscopic findings showed that dysplastic nodule was similar to early hepatocellular carcinoma. conclusions: this study showed that dysplastic nodule in liver cirrhosis is nearly identified to early hepatocellular carcinoma. dcp is an important risk factor for recurrence after radiofrequency ablation of single hepatocellular carcinoma -< 3cm in diameter r. kuromatsu 1 , a. takata 1 , n. fukushima 1 , s. sumie 1 , m. nakano 1 1 division of gastroenterology, department of medicine, kurume university school of medicine background and aims: the aim is to analyze the risk factors for local recurrence + intrahepatic metastasis after radiofrequency ablation (rfa) and hepatic resection (hr) for single hepatocellular carcinoma (hcc) < 3cm in diameter. methods: between 1999 and 2007, 219 patients with single nodule < 3cm in diameter and child-pugh grade a were treated by hr and rfa, and recurrence rate and survival rate using kaplan-meier method, and important risk factors for recurrence using cox's proportional-hazards regression model were analyzed. factors used for multivariate analyses were age, gender, viral marker, tumor diameter, afp, afp-l3, dcp, and platelet count. results: mean age was 66 years old, m/f ratio was 136/83, hr/rfa was 59/160, and mean observation period was 1194 days. five-year survival rates, and 2-year local recurrence-free + intrahepatic metastasis-free rates were not significant between hr group and rfa group (82/72%, 92/89%). in rfa group, the only independent risk factor for local recurrence-free + intrahepatic metastasis-free survival was dcp (p=0.0034). tumor diameter was not significant for recurrence. in hr group, there was no risk factor for recurrence. in pathological analyses of hr group, dcp had a tendency to associate with microvascular invasion (p=0.065). conclusions: rfa was effective for hcc < 3cm in diameter and dcp < 40 mau/ml. hepatic resection should be selected for single hcc with dcp > 40 mau/ml even though hcc <3cm in diameter. background: radiofrequency ablation (rfa) is now a common treatment for small hepatocellular carcinoma (hcc). however, critical complications after rfa such as rapid intrahepatic dissemination have been reported. in this study, we investigated the method how to estimate the malignant potential of small hcc by dynamic ct before rfa. methods and results: firstly, 61 hccs less than 3cm in diameter were analyzed. those tissues were classified into 4 groups as followed, small nodular type with indistinct margin (type e), simple nodular type (type 1), simple nodular type with extranodular growth (type 2), confluent multinodular type (type 3). in the type 2 and 3 groups, portal invasion over vp1 were observed more frequently than those in the type 1 and e groups. at the next step, these 61 hccs were classified into above-mentioned 4 types by two radiologists according to the shape of early stain or defect of delay phase of dynamic ct before operation. the accorded rate was 87% between those classifications. next, 45 patients, which had solitary hcc less than 3cm in diameter and treated with rfa, were classified into those 4 types by dynamic ct before rfa. the recurrence rate and prognosis of those patients were examined. in the type 2 and 3 groups, the recurrence rate was higher and significant worse prognosis was showed than those in type 1 and e group . conclusion: it was suggested that hcc with type 2 and 3 might process higher malignant potential and rfa should be carefully performed on those types of hcc. background/aims: hypoxia-inducible factor-1 (hif-1 ) is the central transcriptional factor in the cellular response related to various aspects of cancer biology, including proliferation, survival, angiogenesis, and extracellular matrix metabolism to hypoxia. il-8 became known to replace hif-1 functions in the other cancer cell lines. the aim of this study was to evaluate whether il-8 may induce angiogenic factors without hif-1 by inflammation signal of hypoxic condition. methods: hif-1 knockdown cell lines of hcc (huh7 and hepg2) were constructed by rna interference tools, and cultured under normoxia (21%o2, 24 hours) and hypoxia (1%o2, 24 hours) conditions. following transfection, the amounts of hif-1 , il-8, angiogenic factors and matrix metalloproteinase (mmp) were examined using rt-pcr and western blotting, respectively. results: the expression of hif-1 , angiogenic factors, mmp, il-8 was markedly enhanced in wild types that were cultured under hypoxia, and the hypoxic induction of angiogenic factors and mmp was partially blocked in hif-1 knockdown hcc cell lines. nf-b inhibitor suppressed angiogenic effects by blocking il-8 activity. conclusion: these data suggest that il-8 induced tumor angiogenic factors in hif-1 knockdown hcc cell lines. background there were some reports that liver caner related to the levele of sera hbvdna our research focused on the relationship between the quantity of hepatocellular hbv cccdna , tdna and liver cancer. methods the samples included the liver tissue of 24 chb patients (chb group) and the para-liver cancer tissue of 26 primary liver cancer patients (phc group) the quantity of hepatocellular hbvcccdna, tdna were assayed by fq-pcr in both groups. result: the quantity of hepatocellular hbv cccdna in chb group was 11.56±16.14 copys/cell higher than phc group(3.96±9.27 copys/cell), p=0.011; the quantity of hepatocellular hbv tdna in chb group was 57.38±91.64 copys/cell higher than that of phc group(35.07±93.03 copys/cell),p=0.13.; conclusion: the quantity of hepatocellular hbvcccdna, tdna can not be used as predictors of liver cancer for hepatitis b patients. hepatic cancer predominantly occurs in males. this is almost a commonsense to most of us. but the detailed mechanisms underlying such phenomenon are still not well-known. the average age of liver cancer patients are about 30-40 years old. so most female patients have udergone pregnancy at least one time. pregnancy is a very important event before or during the development of liver cancer in females. in this special period, not only sex hormones secrete in a strange manner, but also immune system functions in a special module which is very different from normal. so it is urgent to investigate the impact of process of pregancy on the development of hepatic cancer. in this study, 100 female sd rats are randomly divided into two groups: pregance group and controll group. rats in both groups are injected iv diethylnitrosomine(a chemical carcinogen). in pregnance group, rats are raised together with male rats in 3:1 ratio(3 female, 1 male) to make every rats undergo pregnancy. while in controll group, rats are coupled with spermaduct-ligated male rats. the size and amount of hepatic cancers in pregancy group are smaller and less than those in controll group. the survial rate is also significantly higer than that in controll group. we conclude that the process of pregnancy exerts an inhibitory role in the development of chemical induced hepatic cancer in rats. acknowledgement: this project was sponsed by the national natural science foundation of china. the number of the grant is 30600727: the use of alpha-fetoprotein measurement in detection of recurrent hepatocellular carcinoma after living donor liver transplantation n. yamashiki 1,2 , y. sugawara 1,3 , s. tamura 3 , r. tateishi 2 , h. yoshida 2 , j. kaneko 3 , y. matsui 3 , n. kokudo 1,3 , m. omata 2 1 organ transplantation service, 2 department of gastroenterology, university of tokyo, 3 department of surgery, university of tokyo background: the recurrent hepatocellular carcinoma (rhcc) after liver transplantation (lt) can occur in 10 to 20 % of transplant recipients despite with a careful patient selection. for the surveillance of rhcc, frequent measurement of alpha-fetoprotein (afp) and annual ct scan is commonly used. however, the usefulness of afp is not clear. we report the update of our experience using our surveillance protocol. methods: between 1996 and march 2008, 330 adult living donor lt were performed at the university of tokyo. among them, 100 recipients with hcc in their explanted liver were subjected to analysis. we used monthly measurement of afp and des-gamma carboxy prothrombin (dcp) with annual dynamic ct scan. results: 83 met milan criteria pre-operatively and 12 did not. 5 were incidental hcc. rhcc was experienced in 9 patients at 10 (2-24) months after lt. recurrence sites were graft (1), lung (2), bone (3), and multiple organs (3). rhcc was first suspected from elevation of tumor markers in 8; afp in 4, dcp in 2, and both afp and dcp in 2. rhcc was confirmed with ct scan (7) or mri (2) in 4 (0-6) months after the first sign of rhcc. when the cutoff level of afp>20ng/ml was used, the sensitivity and specificity for rhcc were 66% and 100%. six cases were treated surgically of which two achieving prolonged survival. conclusions: although the confirmation of the rhcc sites required multiple imaging studies, afp measurement was useful as for the first sign of rhcc. purpose: to evaluate the therapeutic effect of heated (60 c) lipiodol via hepatic artery administration in vx2 rabbit liver cancer model. materials and methods: thirty male new zealand white rabbits were randomly divided into 3 groups with 10 rabbits for each group. vx2 carcinoma cells were surgically implanted into the left liver lobe. the tumors were allowed to grow for 2 weeks, and studies were performed until the diameter of tumors detected by ultrasonograph reaching 2 to 3 cm. under the anaesthesia, transcatheter hepatic arterial embolization was performed and doxorubicin-lipiodol (37ºc) (1 ml), lipiodol (60ºc) (1 ml) and control (physiological saline (37ºc) (1 ml)) were injected into hepatic artery of the 3 different groups. one week later, the volume of tumor was measured by ultrasonograph again. the serum of all rabbits was collected before injection and at 4 and 7 days after injection and the level of aspartate aminotransferase (ast) was checked. the survival period of 3 groups of rabbits after treatment was also recorded. during the last course of their disease, the rabbits were given some analgetics to relieve suffering. results: the tumors' growth rate in lipiodol (60ºc) background/aims: hepatocellular carcinoma (hcc) is one of the male-dominant cancers, and hepatitis c virus (hcv) is one of the causes of hcc. it was reported that androgen receptor (ar) is expressed in hcc and its surrounding tissues. androgen signaling and ar may be involved in hepatocarcinogenesis. in this study, we investigated whether hcv interacts with androgen signaling in human hepatocytes. methods: hcv protein expression vectors were co-transfected with ar-expression vectors and ar-responsive element-driven reporter vector into immortalized human hepatocytes (ihhs) and human hepatoma cell lines. kinase inhibitors were used to examine the activation of the akt, mapk, and jak/stat3 pathways. real-time pcr and western blotting were performed. cell culture grown hcv (hcvcc) were also used, and angiogenesis was evaluated by tubule formation assays in human coronary microvascular endothelial cells in the presence of 5 -androgen-1 -ol-3-one. results: hcv enhances ar-responsive gene expression in the presence of androgen. hcv core protein has the strongest effects and induced ar activation associated with jak/stat signaling. hcvcc enhances vegf mrna expression and angiogenesis. conclusions: hcv core protein is an enhancer in androgen signaling and can be expected to play an important role in hcv-related hepatocarcinogenesis. background: to evaluate the therapeutic outcomes and the toxicity of the combination of arsenic trioxide and the chinese traditional jianpiliqi (jplq) formula in the treatment of advanced hepatocellular carcinoma (hcc). methods: patients with advanced hcc, not suitable for resection but with normal major organ functions, were enrolled to receive a therapeutic regimen consisting of intravenous arsenic trioxide (6 mg / m 2 ) administration from days 1-14, and an oral administration of jplq formula twice daily from days 1-28. each cycle was composed of 28 days and treatment could expand up to 4 cycles before evidences of intolerable toxicity or disease progression. result: one patient had partial response, one had minor response, 15 showed stable disease and 15 (46.9%) had disease progression. total disease control rate was 53.1%, median survival time was 5.8 months (2-23.9 ms), and time to progression was 4.2 months (1-16.9 months). the incidences of grade 1-3 abdominal distention and nausea/vomiting were 65.6% and 37.5%, respectively. increases in ggt occurred in 4 patients (2 grade 2, 1 grade 3, and 1 grade 4) and increases in serum creatinine in 2 patients (1 grade 3 and 1 grade 4), respectively. conclusion: compared with the single arsenic trioxide treatment reported in past literature, treatment by arsenic trioxide combined with jplq showed modestly higher anti-tumor activity and tolerable toxicity in patients with advanced hcc; its manageable toxicity and increased tumor response rate may offer a better treatment regimen, and deserve further investigation. aim: to investigate the effect of osteopontin (opn) expressions down-regulated by rna interference (rnai) on the invasion and metastasis of human hepatocelluar carcinoma (hcc). methods: hcc cell line (hcc-lm3) was transfected with the chemically synthesized small interfering rna (sirna) in study arm and with non-specific sirna in control arm. real-time pcr and western blotting were used to quantify the mrna and opn protein levels. the malignant phenotypes including cellular growth rates, colony formation and matrigel invasion activities of the hcc cell line were analyzed. results: in study arm opn mrna expressions decreased 75% and opn protein decreased 80% compared to those of blank arm. the number of formed colonies and migrating numbers of the cells in vitro decreased significantly (64.6% and 59.6% respectively) in study arm compared to these of blank controls (p<0.05). the parameters in the control arm did not differ from those of the blank arm (p>0.05). conclusion: the specific sirna was able to reduce opn expressions at both the mrna and protein levels and significantly diminished the invasiveness of hcc cells. methods: the expressions of mif and vegf in hcc and adjacent tissues were detected from 4 patients. specific sirna targeting mif gene was synthesized, and transfected into the hcc cell lines (plc and hepg2) in study group and non-specific sirna was used in controls. the mrna and protein expressions of mif and vegf were examined by pcr and western blot. results: mif and vegf mrnas were overexpressed in the hcc tissues compared with adjacent tissues (rq=8.91±1.85 and 3.00±0.86, p 0.01). the mrna and protein expressions of mif and vegf of hcc cell lines significantly decreased in study group compared with controls (p 0.01). vegf mrna levels decreased 75.6%±2.6%; 79.8%±1.2% in plc, and 73.6%±4.6%; 80.7%±2.2% in hepg2 cells when disposed with sirna 50nm and 100nm. vegf protein levels also significantly reduced in study group p 0.01 . conclusions mif and vegf mrnas were overexpressed in the hcc tissues in vivo, and mif sirna was able to knock down the expressions of mif and vegf in hcc cell lines in vitro. y.y. li, y.c. zhang, y.j. zhou, y.m. wei aim: to identify tumor-associated genes by constructing transcription profiles of pure hepatocellular carcinoma (hcc) tissues and normal liver tissues with the combination of laser capture microdissection and microarray. methods: hcc cells and normal liver cells from resection samples of 4 patients were laser capture microdissected. micro-rna was isolated from them for linear amplification then crna was tested with whole genome microarray. differentially expressed genes were screened. results: the quality control of this technique was satisfactory with rna integrity number>7, a260/a280 ratio for crna measurement=2.0~2.1 and good pictures for microarray. compared with normal liver tissues, hcc had 1361 differentially expressed genes, with 607 being up-regulated and 754 being down-regulated genes respectively. among the top ten ranked up-and down-regulated genes (total 20), 5 genes were known as hcc differentially expressed genes, 11, known as other tumors expressed genes previously. four unknown tumor related genes (depdc1b, aspm, fcn2 and bbox1) were detected in this study. conclusion: the combination with laser capture microdissection and microarray was effective in screening the differentially expressed genes of hcc. background/objective: young patients present with large hcc on initial presentation are not uncommon. our aim is to study the computed tomography(ct) imaging of hcc and the clinical features of this special group of patients. methods: 521 hcc patients had ct imaging of liver peformed in a three year period, 385 patients had ct imaging peformed at the time of initial hcc diagnosis in our centre and were selected. they were divided into three age groups: young patients with age </= 40 year-old(group 1), age 41 to 60(group 2), age >60(group 3) to study imaging and clinical factors. univariate and multivariate analysis by cox regression model done to look for prognostic predictive factors. results: infiltrative tumour in ct scans, symptomatic presentation, child's and tm staging are prognostic factors in hcc. conclusion: young hcc patients have larger infiltrative tumour in initial ct scans and more being symptomatic. age is not an independent prognostic factor. aim: to investigate the expression change of nk cells receptor nkg2d from human peripheral blood in patients with primary carcinoma of liver and study the relationship between nkg2d expression and cytotoxicity of nk cells. background/aims: lens culinaris agglutinin-reactive alpha-fetoprotein (afp-l3) is a specific protein produced by hepatocellular carcinoma(hcc), which is more valuable than afp in the diagnosis of hcc. aptamers are oligonucleotide ligands binding to target molecules sensitively and specifically, which are screened from a great capacity of synthetically oligonucleotide library by systematic evolution of ligands by exponential enrichment (selex). our aims were to select the aptamers against afp-l3 from a self-designed ssdna library for potential application in diagnosis of hcc. methods: a random ssdna library and its corresponding primers were designed and synthesized. aptamers against afp-l3 were selected by selex. individual aptamers were separated by polymerase chain reaction-single strand conformation polymorphism (pcr-sscp) analysis and characterized. results: a ssdna library of 78 nucleotides with 40 random nucleotides in middle were designed and used for the selection. the binding rate of library against afp-l3 was increased from 5.1% to 48.2% after 13 round selection. seven aptamers (s1 to s7) were isolated, and their sequences in random region and secondary structures were different from each other. all aptamers could bind afp-l3 in a different extent, and the dissociation constants of s4 and s7 are 650nmol/l and 132nmol/l. conclusions: aptamers for afp-l3 are successfully screened out and could bind afp-l3 specifically. methods: flow cytometry was used to determine the number of nk cells and the expression of nk cells receptor nkg2d from human peripheral blood in patients with 20 case primary carcinoma of liver 23 case hepatitis b cirrhosis 20 case hepatitis b and 20 healthy cases and enzyme mark instrument was used to detect cytotoxicity of nk cells in all cases. results: killing rate of nk cell for k562 cell,nkg2d expression level of nk cells, and the number of nk cells in the patients with primary carcinoma of liver decreased significantly p<0.05 compared with those in the healthy subjects and hepatitis b group ,and decreased a little compared with those in the hepatitis b cirrhosis (p>0.05).the activity of nk cells showed a obvious positive-correlation with the number of nk cell and expression level of nk cell receptor nkg2d. conclusion: the cytotoxicity of nk and the nkg2d expression of nk cells decreased significantly from human peripheral blood in patients with primary carcinoma of liver .the activity of nk cells is closely related to the nkg2d expression level of nk cells. enhancing the nkg2d expression level of nk cell may provide a new idea for adoptive immunotherapy of primary carcinoma of liver. and alpha-fetoprotein afp in serum and tissues for primary hepatic cancer(phc). methods: sixty-six phc and 32 cirrhotic patients were enrolled. in phc patients,male /female was 48:18, age was 55.4 ± 9.91.of them, 16 patients were defined as stage a-a. in cirrhotic patients, male /female was24:8, age was 53.3 ± 5.81. serumgpc3 was detected using elisa. serum afp was detected using electrochemiluminescence. the hepatic expressions of gpc3 and afp were measured using immunohistochemistry in 21 phc and 6 cirrhotic patients. results: the cutoff value of afp diagnosis for phc was 400 g / l or more, afp positive in phc patients was 28.8% (19/66); the cutoff value of gpc3 diagnosis for phc was 300ng / l or more, gpc3 positive was in 47.0 % (31/66), p = 0.031. in a-a stage phc patients,the positive of gpc3 and afp was 62.5% (10/16), 0(0/16), respectively,p = 0.000. in serum afp negative or positive patients, the positive of gpc3 was 46.7% (14/30) , 47.2% (17/36), respectively,p = 0.964. the relationship between gpc3 with age, sex, child-pugh grade, hbv infection, tumor size and metastasis were not observed.the positive expression of gpc3 and afp in hepatocellular carcinoma tissue was 85.7% (18/21), 4.8% (1 / 21), respectively, p = 0.000. neither gpc3 ,nor afp in the paracarcinomatous and cirrhotic tissue, was expressed. conclusions: diagnosis of glypican-3 protein for primary hepatic cancer is superior to afp.gpc3 can be regared as a early marker to diagnosis phc. objective: to investigate the effects and the possible mechanism of curcumin on the proliferation and the invasion of human hepatocellular carcinoma in vitro and in vivo. methods: hcclm3-rfp cell lines were maintained in dmem medium supplemented with 10% fetal bovine serum. the fluorescent areas of hcclm3-rfp were photographed daily and repeated in 4 consecutive days after curcumin treatment for obtaining cell growth curves. the cell morphologic changes were also observed. cell invasion experiment was performed with boyden chamber array. the rfp-expressing human hcc xenograft model in nude mice was established to study the anti-tumor effects of curcumin. the ctc was detected by facs. the expression of cyclind1 and mmp-2 was detected by sybr green real-time pcr. results: after incubation with 20 m, 10 m and 5 m curcumin respectively for 24, 48 and 72 hours, the growth of hcclm3-rfp was significantly inhibited and some morphologic changes were observed. the mean tumor size in nude mice treated with curcumin since day 21were significantly less than those of the control group(p 0.01). the mean metastasis area of lung and the number of ctc in curcumin group on day 35 were remarkably less than in the control group(p 0.01). the mrna levels of cyclin d1 p 0.01 and mmp-2 p 0.05 in curcumin group on day 35 were significantly lower than in the control group. conclusion: curcumin can inhibit the proliferation and invasion of hcclm3 cell line not only in vitro but also in vivo mainly by down-regulating the expression of cyclin d1 and mmp-2 in mrna levels. phosphorylated erk is a potential predictor of sensitivity to therapy with sorafenib in hepatocellular carcinoma -evidence from in vitro study z. zhang 1 , y.h. wang 1 1 background: sorafenib is the first agent that has demonstrated an improved overall survival benefit in advanced hepatocellular carcinoma (hcc) and thus sets the new standard for the first-line treatment of advanced hcc. however, it remains unresolved to predict the drug sensitivity in treating hcc with sorafenib. pretreatment perk level has been shown to be associated with favorable response to such therapy in a phase 2 preclinical study, indicating that perk may be a potential biomarker for treatment of hcc with sorafenib. methods: the effects of sorafenib and 5-fluorouracil on cell proliferation were evaluated by cell viability assay in four types of hcc cell line (smmc-7721, mhcc97-l, mhcc97-h and hcclm6), with different metastatic potential and basal perk expression. levels of perk expression were determined by immunocytochemical analysis and quantification, along with western blot analysis. correlation analysis was carried out between the ic50 values of drugs and mean optical density values of perk. results: the basal perk levels increased stepwise in cell lines in accordance with their metastatic potential. sorafenib inhibited erk phosphorylation at a concentration between 5 and 20 m dose-dependently, while no changes were observed after 5-fu treatment. correlation analysis between the ic50 values and mod values of perk revealed that the effects of sorafenib were significantly correlated with basal perk levels (spearman r=-0.8671, p=0.0003). on the other hand, the resistance to 5-fu were significantly associated with basal perk expression in these hcc cell lines (spearman r=0.7846, p=0.0009). conclusions: in this vitro study, perk was confirmed to be a useful biomarker predictive of sensitivity in treating hcc with sorafenib. the raf/mek/erk pathway may be involved in invasion, metastasis and drug resistance to traditional chemotherapy in hcc. background: to investigate the dynamic expression of igf-ii and igfbp-3 and its alteration of bcl-2 in hcc. methods: hcc models were induced with 2-faa on male sd rats. morphological changes of livers were observed and the dynamic changes of liver or serum igf-ii, igfbp-3, and bcl-2 were quantitatively analyzed by elisa. the expression and distribution of liver igf-ii were observed by immunohistochemistry. result: hepatocytes from granule-like degeneration to a typical hyperplasia to hcc and the progressing increasing of the levels of hepatic igf-ii after rats induced by 2-faa. the levels of igf-ii in hepatoma and sera were significantly higher than any of other groups. the positive relationship of igf-ii was found between liver and sera (p<0.01). the igfbp-3 levels in hepatoma were significantly lower than that in other groups (p<0.01) and the progressing increasing of the levels of hepatic bcl-2 expression during the course. the levels of bcl-2 in hepatoma tissues were significantly higher than those in normal and degeneration ones. the immunohistochemistry evidences indicated the positive expression and hepatocyte distribution of bcl-2 in rat hepatoma. conclusion: hepatic igf-ii, igfbp-3 and bcl-2 may participate in hepatocyte canceration and accelerate the occurrence and development of hcc. the expression of igf-ii and igfbp-3 could be useful molecular markers for early diagnosis and prognosis of hcc. background: this study was done to assess the etiological role of hepatitis b virus (hbv), hepatitis c virus (hcv) and aflatoxin b1(afb1) in development of hepatocellular carcinoma (hcc) in bangladesh. it was also investigated whether alpha-feto protein (afp) and protein induced by vitamin k absence or antagonist ii (pivka-ii) has any diagnostic advantage over each other methods: fifty five histologically proven hcc patients were tested for serological markers of hepatitis b and hepatitis c, and afb1-dna adduct. during the diagnosis, they were also investigated for liver function tests, afp pivka-ii. results: out of fifty five hcc patients, 41(74.5%) were found positive for serological markers of hbv, 21(38%) for hcv and 15(27%) for both. eight cases (14.5%) were negative for the markers of hbv and hcv. however, none had afb1-dna adduct above normal range. both pivka-ii and afp is strong marker for hcc with satisfactory level of sensitivity and specificity; but pivka-ii is more sensitive (92.7%) and afp is more specific (96%). conclusions: hbv and hcv is the major etiological agent responsible for the development of hcc in bangladesh. background: to investigate the influences on the malignant transformation of hepatocytes through the intervention of nf-b activation pathway. method: hcc models were induced with 2-faa on sd rats, thalidomide was administered intragastrically and rats were sacrificed fortnightly interval to the twelfth week. morphological changes were observed by he staining. nf-b expressions were detected by ihc. the relationship between nf-b expression and pathological characteristics in hcc and non-hcc were analyzed. results: rat hepatocytes showed vacuole-like denaturations at the early stages, then dysplastic nodules appeared at middle stage, and finally progressed to tubercles of cancerous nest, all of which were highly differentiated hcc. thalidomine can repress the morphologic change of liver cells. there were only punctiform denaturations at the early and middle stage; nodosity hyperplasy and minority atypical hyperplasia were found at the finally stage. the ihc results demonstrated that nf-b level was significantly higher than those in normal ones, and the nf-b level of livers in hcc was higher than those in thalidomide group. an increasing tendency of nf-b was found from normal to hcc. nf-b in hcc were significantly higher than those in nc. the nf-b levels with thalidomide intervence raised first and decreased later. nf-b expressions in hcc were higher than that in their non-cancerous tissues. no positive relationship presented between nf-b expression and histological differentiation grade or the number of tumor, and size of tumor. conclusion: decrease nf-b expression can inhibit hcc development and nf-b is expected to be a new molecular target of hcc therapy. method: the cellular distributions of vegf expression in hcc tissues were investigated by immunohistochemistry. the levels of total rna and vegf were quantitatively detected in hcc, their paracancerous, and distal cancerous tissues, respectively. simultaneitily, serum vegf were analyzed in patients with chronic liver diseases for clinical values. results: the positive expression showed palm-yellow or palm-brown granules and distributed in hepatocyte plasma of hccs. the incidence of vegf was 63.9% in hcc tissues, 78.3% in non-encapsulated hccs, and 90.9% in hccs with extrahepatic metastasis, respectively. no significant difference was found between hepatic vegf and hcc diameter or differentiation degree. the specific concentration (pg/mg liver) of vegf expression was significantly higher (p<0.01) in hcc than their paracancerous or distal cancerous tissues, respectively. the circulating vegf was abnormally elevated in hcc. if the cut off values was more than 280 pg/ml, the incidence of serum vegf was 88.4% in hcc, 14.3% in chronic hepatitis, and 10% in liver cirrhosis, respectively. the combined vegf and afp can increase positive rate up to 94.2% for hcc. conclusion: the vegf overexpression is a useful marker for vascular invasion and metastasis of liver tumors. background: hepatocellular carcinoma (hcc) represents a major health problem world wide. it accounts for 90% of all primary liver cancers and is the fifth most common malignancy (1). objectives: evaluation of radiofrequency thermal ablation versus transarterial hepatic chemoembolization with the effect of viscum (fraxini 2) on tumour recurrence. methods: 60 patients with hcc were enrolled in the study. group 1 include 30 patients and were treated with radiofrequency thermal ablation (15 patients of them received viscum 2 by subcutaneous route for 2 years). group 11 included 30 patients with hcc and were treated by tace (15 patients of them received viscum 2 subcutaneously for 2 years). results: group 1 patients showed total ablation in 70% with persistant inactivity during 2 years follow up. group 11 did not show significant difference from group 1 as regards relapse rate nor the performance status. complications as nausea, vomiting, fever, jaundice, and elevation of transaminases were significantly more encountered with tace. viscum 2 did not significantly arrest tumour recurrence. conclusion: non surgical patients with hcc can achieve curative treatment with radiofrequency with minmal side effects. tace is a palliative treatment option for large hcc. a new technique had been attempted to increase the field of radiofrequency ablation of expandable electrode needles in the treatment of hepatic neoplasms much larger than the routinely covered size of 5-7 cm according to the needle size overcoming the technical difficulties usually met with in the overlapping balls technique due to the hyperechoic focus that develops at the needle tip making reinsertion difficult and inaccurate. in this technique, two or three needles were inserted from the start into the mass with accurate estimation of the exact field of ablation of each needle trying to cover the whole extent of the mass before application of radiofrequency waves. 40 patients were included in the study, all presented with hepatic neoplastic mass lesion that range in size between 7 and 10 cm in its maximum diameter. all had a pretreatment helical (triphasic) ct study for accurate delineation of the whole extent and vascularity of the mass. two needles were sufficient to cover the whole extent of the mass in 23 patients (57%) while in the remaining 17 patients (43%) three needles were necessary. the procedure was done under general anathesia and ultra sound guidance, patients tolerated procedure well with smooth recovery. no major complications. follow up spiral (triphasic) ct was done 2 weeks after ablation revealed percentage of tumour necrosis of 90% or more in 30 patients (75%), 70-90% in 6 patients (15%) while in the remaining four patients (10%) the percentage was 50-70% necrosis. in conclusion this technique should be considered in the treatment of hepatic masses larger than the usual field of the needle. results: the median value of gpc-3 in hcc, dc, cc was significantly higher than chronic hepatitis and control groups. no significant correlation found between afp and gpc-3. auroc of afp was 0.85 & auroc of gpc-3 was 0.84. the diagnostic sensitivity of afp (20 ng/ml) was 70% with ppv 53.8%. the specificity was 85% with npv 91.9%. while the diagnostic sensitivity of gpc-3 (2 ng/ml) was 100% with ppv 27%. the specificity was 42.2% with npv 100%. combined serial approach of afp and gpc-3 improved the specificity to 87.5%. conclusion:gpc-3 although it is a serological test for early detection of hcc, it showed limited specificity, where it is detected in different stages of chronic liver disease, as it is an oncofetal protein produced by regenerating liver cells. the diagnostic signature approach for simultaneous determination of afp and gpc-3 may improve the prediction accuracy of hcc patients in those showing seronegativity to afp. outcome of inoperable hepatocellular carcinoma patients receiving transarterial chemoembolization: retrospective analysis in an asian regional hospital w.m. yip 1 , k.f. li 1 , k.k. li 1 , m.l. szeto 1 1 background: hepatocellular carcinoma (hcc) is a common cancer worldwide causing substantial mortality. although surgical resection is a form of curative treatment in hcc, only a minority of patients is suitable for this treatment and the postoperative recurrence remains high. transarterial chemoembolization (tace) is a treatment option for inoperable hcc and it was proven by randomized control trials that tace can prolong survival in selected patients. the aim of this study is to evaluate the survival and the prognostic factors in patients with advanced hcc treated by tace. methods: seventy four patients with inoperable hcc diagnosed from january 1998 to december 2003 were analyzed retrospectively in this study. only patients with unresectable hcc or who refused operation were included. patients with advanced cirrhosis, extrahepatic metastasis or previously treated hcc were excluded. multiple host, tumor and treatment variables were analyzed in order to evaluate the predictive factors of favorable response to treatment and better survival. results: the median survival of the study patients was 213.5 days. the cumulative survival rates at 1 year, 2 year and 3 year were 28.4%, 12.2% and 6.8% respectively. by multivariate analysis, superselective cannulation performed in tace (hazard ratio: 0.47, 95% ci: 0.23-0.95, p=0.034), embolization with gelfoam (hazard ratio: 0.30, 95% ci: 0.11-0.80, p=0.017), treatment interval more than 45days (hazard ratio: 0.33, 95% ci: 0.15-0.72, p=0.006), child-pugh grade b (hazard ratio: 5.62, 95% ci: 2.11-14.97, p=0.001), and pre-treatment serum fp level (hazard ratio: 2.93, 95% ci: 1.50-5.73, p=0.002) were independent predictors of survival. conclusions: survival of patients with inoperable hcc is still grave despite treatment. this study provided information in predicting the survival of patients with inoperable hepatocellular carcinoma treated by transarterial chemoembolization. result: age <65, total bilirubin (tb) <2.0mg/dl, albumin (alb) 3.5g/dl, prothrombin time (pt) 70%, platelet counts (plt) 10.0 10 4 /mm 3 , single nodule, and type of treatment (surgery or local ablation therapy) were linked to increased survival at univariate analysis of clip 0-1 hcc patients. of clip 2-6 hcc patients, tb <2.0mg/dl, alb 3.5g/dl, des-gamma-carboxy prothrombin (dcp) <100mau/ml, absence of vascular invasion, and type of treatment were correlated with survival. the following factors were related to survival by multivariate analysis: clip 0-1 hcc patients; age, alb, single nodule, and absence of vascular invasion, clip 2-6 hcc patients; age, tb, alb, alpha-fetoprotein (afp) <100ng/ml, dcp, absence of vascular invasion, and type of treatment. conclusion: age, albumin, vascular invasion were significant predictors of survival both clip 0-1 and clip 2-6 hcc patients. clip 0-1 hcc patients: single nodule; clip 2-6 hcc patients: lower levels of tumor markers and patients receiving promising treatment had a better chance of prolonged survival. the role of gross classification as the predictor of microvascular invasion in hepatocellular carcinoma. s. sumie 1 , r. kuromatsu 1 , k. okuda 1 , e. ando 1 , a. takata 1 , n. fukushima 1 , m. sata 1 1 background; the presence of microvascular invasion (mvi) as the risk factor in hepatocellular carcinoma (hcc) is controversial. the aim of this study was to determine the outcomes and predictive factors after hepatic resection for hcc with mvi. methods; one hundred and ten patients who underwent curative resection for hcc were included in this retrospective study. the risk factors of these patients for recurrence-free and disease-specific survival were investigated, and the clinicopathological factors predicting the presence of mvi were also evaluated. result; multivariate analysis showed that cirrhosis and mvi were identified as independent risk factors for recurrence-free survival. the 2-year recurrence-free survival rates for patients with and without mvi were 44.6% and 76.7%, respectively. multivariate analysis showed that the number of tumors, presence of mvi, and im were identified as independent predictors of disease-specific survival. the 5-year disease-specific survival rates for patients with and without mvi were 59.3% and 92.0%, respectively. by univariate analysis, mvi was significantly associated with greater tumor size, gross classification, histological grade, and intrahepatic micrometastasis (im). gross classification proved to be the only independent predictive factor for mvi by multiple logistic regression analysis. the gross classification could be evaluated by preoperative imaging diagnosis. conclusion; mvi is strongly associated with recurrence and survival in hcc patients after curative resection. furthermore, gross classification of hcc can be helpful in predicting the presence of mvi. background: hcc is a common cause of cancer morbidity and mortality. pxd101 is a novel, low molecular weight, histone deacetylase inhibitor. this phase i study aims to determine dose limiting toxicity (dlt) and maximum tolerated dose (mtd). methods: patient eligibilities include unresectable disease, ecog 2, adequate organ functions. pxd101 was given intravenously on day 1-5 every 3 weeks; dose levels were: 600 (level 1), 900 (level 2), 1200 (level 3) and 1400 mg/m 2 /day (level 4). dlts are defined as grade 4 hematological toxicity or grade 3/4 non-haematological toxicity during cycle 1 (according to nci ctc v3), or treatment delay >2 weeks. the mtd is defined as the dose below which > 2 of 3 or > 2 of 6 patients experiencing dlt. results: 18 patients were entered; level 1 (3), level 2 (3), level 3 (6) and level 4 (6). grade 3/4/5 toxicities in cycle 1 included: raised alt 1/0/0, diarrhea 1/0/0, abdominal distension 1/0/0, anaemia 1/0/0. a total of 56 cycles were administered; overall grade 3/4/5 toxicities: raised alt 1/0/0, bilirubinaemia 1/0/0; cardiac ischaemia 1/0/0; diarrhoea 1/0/0, abdominal distension 2/0/0, anaemia 2/0/0; variceal haemorrhage 1/0/0; hypercalcaemia 1/0/0; hyperkalaemia 0/1/0; hyponatraemia 1/1/0; infection 2/0/0; liver dysfunction 0/2/0; muscle weakness 1/0/0; abdominal pain 1/0/0; prolonged qtc 1/0/0; syncope 1/0/0; seizure 1/0/0. there were 3 sd and 15 pd. conclusion: at the maximum dose of 1400 mg/m 2 /day, mtd has not been reached. pxd101 is very well tolerated. sponsor: the division of cancer treatment and diagnosis, national cancer institute, usa. tumor thrombus (pvtt) is prone to be produced in the portal vein near the main tumor nodule for hepatocellular carcinoma (hcc) patients and its molecular mechanism is still unclear. in this study, we first established a hcc cell line named csqt-01 from resected tumor thrombus in portal vein in a patient with histopathologically proved to be a moderately differentiated hepatocellular carcinoma . this cell line was composed of polygonal shaped cells and its peaks of the chromosome number was 69 and 77. study on stem cell biology in this cell line suggests that cd133 cells represent about one fourth of the tumor cell population and cd133(+) cells possess a greater colony-forming efficiency, higher proliferative output, and greater ability to form tumor in vivo. with this cell line model and resected tumor thrombi specimen, we also studied the different expression of proteins in primary tumor and tumor thrombus and found 20 proteins expressed differentially between primary tumor and the pvtt. from these proteins, annexinv, prx , cycb were selected for further analysis to find potential biomarkers of pvtt in hepatocarcinogenesis. for clinical study, we recommended a new tumor thrombus type system ( type i iv) according to anatomic features of portal vein and tumor thrombus of hcc developing modes, then evaluate this type system to predict prognosis of hcc patients. the retrospective data of 406 hcc patients with pvtt underwent resection shows that the 1y, 2y, 3y overall survival rates were 44.7 , 26.3 and 19.7 for type i, 29.9 , 20.6 and 12.4 for type ii, 25.0 , 12.5 and 3.6 for type iii, 27.3 , 0 and 0 for type iv, respectively, suggests tumor thrombus type system may be helpful to determine treatments and prognosis of hcc patients with pvtt. polyprenol could decrease the risk of hepatocarcinogenesis in hbv g. kuznecova 1,2 , s. kuznecovs 1,2 , i. kuznecovs 1,2 background: over-expression of p-glycoprotein (pgp) is associated with liver cancer development from hbv . glycoprotein synthesis in malignant tissues is limited by dolichyl phosphate (dolp). the aim of the present study was to investigate the effect of polyprenol (pp) which provides a dolp substitute in regulation of n-glycosylation on pgp over-expression in the development of liver cancer in hbv infection. methods human hepatocytes, infected with hbv and human hepatocarcinoma hep 3b cell line were used. pgp was assessed by an immunohistochemical technique. dolp fractions were analysed by hplc methods. results it is confirmed that plasmatic membrans of hepatocytes cells contain 7,9 -9,4% of pgp (the total protein amount) as a resistance marker. hbv infected cells differ from normal hepatocytes in pgp content by 4-5 times and hep 3b cells differ by 10-12 times the study showed 5-fold dolp decrease in hbv infected cells and 10-fold dolp decrease in hep3b cells. the investigations demonstrate that the situation can be changed by treatment with dolp and pp. the dolp concentration in hbv infected hepatocytes was returned to the normal level. it is established that dolp in the concentration 10 -6 m aid 6-8-fold reducing pgp in membranes of hbv infected cells. background: metastasis is one of the most complicated and major pathological processes responsible for poor prognosis of hepatocellular carcinoma. snail was recently highlighted as a critical transcriptional factor for tumor metastasis. method & result: real time rt/pcr and western blot analysis demonstrated that snail mrna and protein, respectively, were induced by 12-otetradecanoylphorbol-13-acetate (tpa) in hepatoma cell hepg2. blockade of gene expression of snail by antisense oligodeoxynucleotide and/or sirna technique can prevent not only the tpa-triggered emt/cell migration and growth inhibition of hepg2 but also tpa-induced down-regulation of e-cadherin and up-regulation of p15ink4b. moreover, the tpa-triggered promoter activation of p15ink4b was also prevented. on the other hand, two of the hepg2 clone overexpressing snail, namely s7 and s15, had a scattered fibroblastic morphology and acquired higher motility than parental hepg2. also, the proportion of g0/g1 phase of s7 and s15 was higher than that of parental hepg2, consistent with the longer doubling time of both cells. semiquantitative rt/pcr analysis demonstrated a greatly elevated gene expression of snail accompanied with decreased e-cadherin and increased p15ink4b in both snail-overexpressing cells. on the transcriptional level, p15ink4b promoter activity was 2.6-fold higher in s7 as compared with parental hepg2. furthermore, electrophoretic mobility of dna fragments encompassing proximal p15ink4b promoter can be retarded by incubation of nuclear extract of s7. conclusion: our results demonstrated that snail play diverse trans-regulatory roles in hepg2. notably, we suggested that snail may upregulate p15ink4b gene expression by directly activating its promoter. a. schmitt-graeff 1 , r. fischer 1 , m. grosse-perdekamp 1 , o. skalli 2 1 universityhospital freiburg , 2 louisiana state university health sciences center, shreveport background/aims: synemin is an intermediate filaments (if) protein which affects the motility of several cell types by modulating the dynamic properties of alpha-actinin and f-actin. we have previously shown that synemin is expressed in resident hepatic stellate cells (hsc) and myofibroblasts (mf) in hepatic inflammation and fibrosis. in the present study we evaluated systematically the expression of synemin in a large cohort of western european hepatocellular carcinoma (hcc). methods: single and double immunolabelin for alpha-smooth muscle actin (sma), vimentin, cd31, cd34, cd68, cea, cd10, cellular retinol-binding protein1 (crbp-1) and synemin were performed on 75 paraffin-embedded hccs and 10 controls. results: synemin-positive hscs/ mfs were a hallmark of non-neoplastic fibrotic liver tissue at the border to the neoplastic lesion but were absent from normal controls. tumour cell plates of the trabecular and pseudoglandular types of hcc were covered by scattered synemin-positive cells outlining sinusoidal structures. a subpopulation of these cells showed features of pericytes while others resembled endothelium. this pattern correlated with the degree of differentiation and was not observed in poorly differentiated hccs which generally contained rare intratumoral mfs. conclusion: the presence of synemin-positive hscs/mfs in the vicinity of hccs suggests a possible contribution of mesenchymal cells to the promotion of liver carcinogenesis. since synemin expression is linked to motility, a migration of this cell type into the tumour and a differentiation in vascular mural cells may be implicated in sinusoidal remodeling and the expansion of the neoplastic population. a.s. butt 1 , a. ahmed 1 , s. hamid 1 , w. jafri 1 , h. ali shah 1 1 aim: to estimate the prevalence of viral marker negative hcc and to compare the clinical, biochemical, histological, radiological characteristics and initial treatment response among patients with viral marker negative and viral marker positive hcc. methods: medical records of patients diagnosed to have hcc visiting aga khan university hospital, karachi during january 1998 to december 2007 were reviewed. patients were divided in to nbnc-hcc(those who have negative hbsag and anti-hcv antibody)and viral hcc(those who have positive hbsag and anti-hcv antibody)group. results: out of 433 patients 68(15.7%) had nbnc-hcc. over all mean age was 57.48± 10.9 years and 69.5% were males. the proportion of hcc detected under surveillance was significantly smaller in nbnc-hcc group(p0.02). there was no difference in distribution of age, gender, bmi, child score, bilirubin, serum albumin, prothrombin time and alfa feto protein in both groups. however, patients with viral-hcc were found to be more thrombocytopenic(52.67±86.7vs.226.15±153.9,p<0.001) and had hepatopulmonary syndrome. on liver biopsy greater proportion of moderate to poorly differentiated hcc was observed in nbnc group(19.2%vs.7.9%,p<0.001). hcc measuring 5cm in diameter(60.3%vs.41.9%, p 0.01), non -solitary hcc(p0.038) and portal thromboses(p0.01) were strongly demonstrated in nbnc-hcc group. involvement of right hepatic lobe and extra hepatic tumor spread was greater in nbnc-hcc group but that difference was not statistically significant. out of 178 patients who underwent for liver transplantation(0.5%),tace(36.7%),resection(1%),ethanol ablation(2%) and chemotherapy(2%), poorer responses were observed in nbnc-hcc group (p 0.04). conclusion: hcc secondary to nbnc-cirrhosis is not uncommon. patients with nbnc-hcc tended not to be under surveillance that leads to diagnoses at more advanced stage and poor prognosis. background: hepatocellular carcinoma is a common malignancy in asia and is related to the high prevalence of chronic viral hepatitis. we examined the clinical features, treatments and survival rates in asian americans with hcc. methods: retrospective cohort study of 278 hcc patients who presented to the ucla liver cancer center in los angeles, california, usa from september 2000 to december 2007. results: two hundred and seventeen of 278 (78%) hcc patients were male, 58% and 30% had hbv and hcv infection respectively, and 73% had cirrhosis. hcc patients detected by surveillance had smaller tumor sizes, more within the milan and ucsf criteria, lower hcc tokyo system scores and had improved 1,3,5 year overall patient and disease free survival rates compared to hcc patients who presented with symptoms (p<0.01 to p<0.0001). by multivariate analysis, independent predictors of patient survival were tumor volumes greater than 5cm (hr1.48, p=0.04), afp per unit log10 increase (hr 1.12, p=0.02), hcc tokyo score per unit increase (hr 1.3, p<0.0001), liver transplantation (hr 0.14, p<0.0001), hepatic resection (hr 0.18, p<0.0001), rfa (hr 0.17, p<0.0001), tace (hr 0.44, p=0.0006), and hepatitis b infection (hr 0.71, p=0.03). factors associated with disease free survival were age per year increase (hr 1.01, p=0.03), meld per unit increase (hr 0.97, p=0.0049), liver transplantation (hr 0.23, p<0.0001), and hepatic resection (hr 0.39, p<0.0001). conclusion: hbv and hcv infection accounts for the majority of hcc in asian americans. hcc detected by surveillance resulted in treatments which improved overall patient and disease free survival. treatment of small ( 2cm) hcc tumours can be achieved by surgical resection and complete eradication always correlates with good patient's outcome, with low local recurrence and high survival rates. indeed, surveillance program for the early detection of small hcc tumour is imperative to facilitate curative treatment, and hence better survival. discovery of new blood-based biomarkers is obligatory and vimentin is a distinct novel small hcc tumour marker herein identified using proteomics. experimental design: a total of 76 liver tissues were evaluated by 2-de analysis. differentially expressed proteins were unequivocally identified by maldi-tof/tof and validation of the best candidate from protein to gene levels. indirect elisa assay was developed to detect soluble vimentin from 152 serum samples. results: vimentin was significantly over-expressed in small hcc tumours compared to non-malignant controls and maintained expression in >2cm tumours using 2-de analysis. blind verification displayed over-expression of vimentin in both transcripts and proteins levels. soluble vimentin was significantly detected at high level in small hcc as well as in overt hcc tumours. receiver operating characteristic analysis showed vimentin exhibited 40.91% sensitivity and 87.50% specificity in detecting small hcc at a cutoff of 245ng/ml. combined diagnostic performance of soluble vimentin and serum afp increases the detection sensitivity and specificity to 50.53% and 98.15%, respectively. conclusion: in this context, over-expression of vimentin is associated with the favourable 2cm sub-class of hcc thus may potentially be used as an effective serum-based diagnostic marker for cancer surveillance in high-risk cirrhotic patients. purpose: our recent comparative oncogenomic analysis in mouse model has identified yap (yes associated protein) as a novel oncogene in hcc. however, its clinical significance is unknown. in this study, we aimed to investigate the clinical values of yap as an independent prognostic marker in hcc. experimental design: a total of 177 hcc cases with retrospective clinicopathologic and follow-up data were recruited in this study. both tumor and adjacent non-tumor tissues were examined for immunoreactivity of yap expression by immunohistochemistry. clinicopathologic features and yap expression were investigated with pearson 2 test. hcc-specific disease free survival and overall survival with yap expression were analyzed by kaplan-meier curves and log-rank test. cox regression was used to test the independence and magnitude of the effects. results: yap was found over-expressed in hcc (62.1%) with nuclear expression pattern. positive yap immunoreactivity was significantly correlated with worse tumor differentiation grade (p=0.021) and high serum alpha-fetoprotein (afp) level >400 ng/ml (p<0.001). kaplan-meier plot and cox regression showed that yap was an independent predictor for hcc-specific disease free survival (hazard ratio, 1.653; 95% ci, 1.084-2.522; p=0.02) and overall survival (hazard ratio, 2.226; 95% ci, 1.312-3.778; p=0.003). conclusions: yap expression in hcc is correlated with tumor differentiation and serum afp level. it served as an independent prognostic marker for hcc. background: integrative analysis of global protein and mrna expression patterns could help researchers to understand cancer cell physiology without the need of any prior hypothesis. methods: we used a 2d-page approach to profile and compared the global protein expression profiles of hepatitis b virus-related hcc tissues, adjacent non-tumor liver tissues, normal liver tissues and hcc cell lines. subsequently, we established the bioinformatic tools for integrative analysis of gene expression and protein expression data. we compared the dysregulated protein list and the dysregulated gene lists obtained by meta-analysis of microarray gene expression data from 6 research centers in different countries. results: we identified 66 proteins dysregulated in hcc. hierarchical clustering analysis revealed that there was a progressive change of protein expression patterns from normal liver, adjacent non-tumor liver tissues, hcc tissue, then to hcc cell lines. according to the biological functions, the differential proteins could be classified into various groups, including heat shock protein, chaperone, kinase substrate, cell signaling, apoptosis regulation, transcription regulation, free-radical scavenger and metabolic enzyme. ontology analysis of the genes with consistent dysregulations at both mrna and protein levels identified specific pathways down-regulated during the progression of hcc. the inhibition of those pathways provides new insights in the hepatcarcinogensis and treatment strategies. results: in pre-s/surface regions, hcc patients had higher frequencies of pre-s deletions, amino acid substitutions at codon 4, 7, and 81 in pre-s1 genes, at the start codon in pre-s2 genes, and at codon 68 in surface genes. but they had a lower frequency of amino acid substitution at codon 2 in pre-s2 genes than those without hcc. in bcp/precore regions, hcc patients had higher frequencies of c or g1753, a1762/t1764, t1846, and a1899 than those without hcc. multivariate analysis showed that pre-s deletions, i68t in surface gene, t1762/a1764, and a1899 were independent factors for hcc. the hbv with a complex mutation pattern (pre-s deletion, t1762/a1764, and a1899) rather than a single mutation was associated with hcc. patients with combined mutations of t1762/a1764 and pre-s deletion, t1762/a1764 and a1899, pre-s deletions and a1899, and t1762/a1764, pre-s deletions and a1899 had a 7.81, 7.7, 7.0, and 16.88 fold increased risk of hcc, respectively, compared to patients with wild-type at both or three genomic regions. conclusions: pre-s deletions, i68t in surface gene, t1762/a1764, and a1899 were independent factors for hcc. combination of these viral mutations appeared increasing hcc risks. high peritumoral expression of placental growth factor in hepatocellular carcinoma is a poor factor for survival after curative resection h.x. xu 1 , x.d. zhu 1 , p.y. zhuang 1 , w.zhang 1 , h.chuan sun 1 1 background/aims: angiogenesis plays a significant role in the metastasis and recurrence of hepatocellular carcinoma (hcc). placental growth factor (plgf), which is one member of the vascular endothelial growth factor family, may have prognostic values in patients after curative resection of hcc. methods: expression of plgf was assessed by immunohistochemistry in tissue microarray containing paired peritumoral liver tissue and tumor from 105 patients underwent hepatectomy for histologically proved hcc. prognostic values of plgf and clinicopathological factors were evaluated. result: plgf staining was mainly on the cytoplasm of tumor cells or hepatocytes. the mean integrated optical densities of peritumoral and intratumoral density of plgf were 0.018±0.018 and 0.006±0.007 respectively. peritumoral plgf density was significantly higher than that in tumor (p<0.001), and this result was also validated in another cohort of 37 patients by quantitative real-time reverse transcription-pcr (p=0.007). intratumoral density of plgf was not correlated with common clinicopathological factors (eg, tnm stage, tumor size, microvascular invasion, intra-hepatic metastasis) or overall survival (os) (p=0.425) and time to recurrence (ttr) (p=0.419). however, peritumoral density of plgf, which was correlated with tumor size (p=0.003) and intrahepatic metastasis (p=0.004), was a prognostic factor for both os (p=0.015) and ttr (p=0.018). in multivariate analysis, peritumoral expression of plgf was also an independent prognostic factor for os (p=0.015, rr: 2.500 95% ci: 1.191-5.253 ) and ttr (p=0.045, rr: 2.013 95% ci: 1.014-3.996). conclusion: peritumoral expression of plgf in hcc patients is an independent risk factor for survival and recurrence, and may be a target of anti-angiogenic therapy in preventing post-operative recurrence. purpose: to further research rfa in combination with hepatic artery-portal vein chemotherapy and ethanol injection for treatment of advanced hepatocelluar carcinoma (hcc). methods: 25 cases were treated with transhepatic artery chemoembolization (tace) + radiofrequency ablation (rfa) + introportal vein chemotherapy (pvc) + percutaneous ethanol injection (pei) (four combined group) and this method was compared with 23 cases that were performed tace + pei (two combined group) . the serum level of afp was measured respectively after 2 and 6 months, ct scan and color doppler ultrasound were measured after treatment for six months. results: the serum level of afp declined in two groups after 2 months. for treatment after six months, afp in four combined groups was rose lower than two combined group (x 2 =5.06, p<0.05). ct and doppler ultrasound examination, four combined groups was superior than two combined groups to the control in tumor shrinkage (x 2 =8.29, p<0.01) and blood supply x 2 =6.81, p<0.01), relapse and mortality are also less. conclusions: rfa in combination with hepatic artery-portal vein chemotherapy and ethanol injection is a safe, effective combined method and has less complication in treatment of advanced hcc. poster exhibition -hcv poster session, hall 5b on the average, hepatitis c virus infects 1.2% of the population worldwide. in egypt, the prevalence rates reach 20% in some areas. ability of the virus to persist in about 75-85% of infected individuals is related to the virus higher mutation rate. six major hcv genotypes have been identified. genotype 4 seems to be confined to the middle east and central africa. extra hepatic syndromes have been reported in up to 1/3 of hcv patients. we aim in this study to determine the relationship between viral genotypes and specific extra hepatic haematological disease in patients with chronic hepatitis c. the study group included 60 selected patients with chronic hepatitis c having various haematological problems. we studied hepatitis c virus genotypes using rt-pcr. we found among 60 patients , 57 genotype 4 (95%) and 3 patients genotype 1a (5%).28 patients (46.66%) were diagnosed as chronic hepatitis c with associated thrombocytopenia, 16 patients (26.66%) were diagnosed mixed essential cryoglobulinemia(mec), 13 patients (21.66%) were diagnosed non-hodgkin's lymphoma, and 3 patients (5%) were aplastic anemia. positive serum cryoglobulins level was found in 20 patients (33.3%).no significant correlation was found between the level of viraemia and specific haematologic disease, biochemical liver markers or liver enzymes (p>0. 05). we did not find correlation between hcv genotype and specific extrahepatic haemological disorder in hcv infected patients. several environmental, genetic and immunological factors may contribute in disease progression. results: in the targeted area 111 shops of barbers were successfully interviewed and total 715 questionnaires were filled by both groups. the mean age were found in both groups of barbers (n=186) and clients (n=529), 28.47 years. the both groups showed that there are no any drugs which can protect us from diseases. both of the groups were not vaccinated for hepatitis b diseases. regarding the care providers the barbers replied that they prefer registered medical practitioners and the clients generally prefer the hakeems. those who knew hepatitis as liver disease, were 73 (39.2%), out of 186 barbers only 68 ( 36.6%) were knowing about hepatitis-b&c, when we enquired about routes of hbv& hcv transmission only (37%) replied correct routes of transmission in both groups. about hbv vaccination (49.7%) were aware, only (14.8%) were vaccinated against hbv. 60% barbers claimed for disinfection of instruments before shaving (88.9%) claimed for use of new blades. in the sero-surveillance the hbv found was very low and hcv became epidemic (5.7% -14.4 %) respectively. conclusion: the both groups need awareness for transmission. the use of new blade for the clients reduces the burden of hbv and hcv. the study highlights the roles of male sex, older age, and genotype 1b in the progression from chc infection to hcc. patients with higher hcv viral load potentially tend to develop hcc; however, hcc occurrence could be prevented using antiviral treatment. these two points need to be clarified further by a larger study population with longer follow-up period. an approach have recently been described that retroviral vectors encoding t cell receptor (tcr) genes are used to redirect the specificity of normal peripheral blood lymphocyte (pbl)-derived t cells to recognize the tumor antigens. the therapy in which t cells have been genetically modified with tcr genes to recognize hcv would represent a novel approach for the treatment of hcv infections and hcv-related malignancies. we have previously shown that hcv+ liver transplant patients that have received hla disparate liver allografts have hcv reactive t cells of host origin in their peripheral blood that are restricted by the donor hla molecules. initial studies indicate that the tcrs expressed by hcv reactive t cell clones from these patients have relatively high affinity for their ligands. we have cloned and expressed two tcrs which mediate recognition of the 1406-1415 and 1073-1081 epitopes from the hcv ns3 protein. the results indicate that these tcr transduced t cells can recognize the wild type epitopes, as well naturally occurring mutant variants of these epitopes. most importantly, the tcr transduced t cells could also recognize hcv+ hepatocellular carcinoma cells. these data suggest this high affinity hcv-specific tcr might have potential new immunotherapeutic implications. background: factors associated with svr in patients without an rvr remains unclear. methods: 200 hcv-1 (100 for 24 and 48 weeks, separately) and 150 hcv-2 (100 for 24 weeks, 50 for 16 weeks) patients were randomized to peginterferon-alpha-2a and ribavirin for analysis. results: multivariate analysis showed that treatment duration and a complete evr were the strongest independent factors associated with an svr. a higher svr rate and a lower relapse rate were observed in the standard regimen group than in the abbreviated group in patients who had a cevr (table 1). the best levels of viral loads in predicting cevr at week 4 were < 10 4 iu/ml (table 2) . conclusion: it was crucial to achieve a cevr with adequate treatment duration in patients who failed to achieve an rvr. our aim was to evaluate the impact of some biochemical, histological and viral factors on both evr and svr in patients with genotype 1 chronic hepatitis c (chc) treated with peginterferon plus ribavirin. patients and methods: we evaluated retrospectively 188 naïve patients with chc treated with peginterferon plus ribavirin at standard weight-based doses for 48 weeks. biopsies were assessed for inflammatory activity and fibrosis. steatosis was categorized by the proportion of hepatocytes per low-power field with fatty changes: >5%, >5-33%, 34-66%, >66%. biopsies were also assessed for stainable iron using the brissot scoring system. all patients were evaluated for metabolic syndrome (ms) using the ncep-atp iii criteria. results: evr was achieved in 115/188 pts (61.17%) while svr occurred in 82/115 (71.3%). after adjusting for sex and age, independent factors that negatively interfered with both evr and svr were: fibrosis score, steatosis, iron score, homa-ir index and viral load. after excluding the patients with ms criteria (n=32), evr was observed in 102/156 (65.4%) and svr in 82/102 (80.4%). factors that independently influenced both evr and svr were: fibrosis score, steatosis, iron score and viral load. conclusion: fibrosis, steatosis and iron scores, as well as viral load are independent parameters that can affect both evr and svr in genotype 1 chc patients, regardless the presence of ms. if ms is present, high homa-ir index can also additionally impair viral response. issue/argument: asia has rising cases of hepatitisb/c. alcohol/food-habits cause high prevalence in rural/tribal areas. lack of monitoring/follow-up complicates management. vaccines emerge as hope. clinical-trials of vaccines debated-issue. design of hepatitis-vaccine-trials in developing-countries complex ethical-issue. we focus on controversies identified in international/regional/local cme/pharma programs as vulnerability of volunteers to exploitation by foreign/local research-groups/funding-agencies. critical task is protect interests of vaccine-subjects in face of substantial-risks. determine if hepatitis-vaccine-volunteers will have access to treatment during trial. access to vaccine-trial-outcome. interaction with seniors 19 th apasl-congress from developed-countries will give voice to such burning-issues. methodology: researchers/pharmaceuticals/govt-policy planners need to develop forum to solve these problems. ngo's can play pivotal role. obligation on part of researchers to create mechanisms to offset anticipated risks of participation in controversial, risky vaccine-development. conclusion: counselling/right to withdraw from trial be made basic guideline. apart from monetary aspects unsuspected adverse reactions/deaths be properly evaluated/monitored. researchers need to evolve policy-guidelines to overcome barriers as variation in interpretation of essential ethical ideas, legal-system-differences, educational/economic-status. need to develop common consensus between research-community/pharma sector to reduce suffering of hepatitis-affected patients community. recommendations: researchers/ngos should come together at 19 th apasl-congress platform to form workgroup to settle these issues. we shall raise our this burning issue & present hepatitis-prevention-advocacy plan of our ngo graphically to apasl-2009 participants. results: 53% patients expressed that alternative-medicines-rx most important factor to cope with hepatitis. higher scores of qol (anova p < 0.001) correlated with alternative-medicines-rx. our ngo-initiative suggests that over 70% patients will need well trained specialist for home-based-care unit. conclusions: life-span/qol of hepatitis-sufferers depends on appropriate-palliative-care. ngo-personals should be trained in palliative-care-services. our data is being used for palliative care advocacy. field of spiritual/psycho-social/community support is fertile ground for further investigations. such use of complimentary indian medicinal plant extracts needs further evaluation in a large group in multicentre trial. treatment with adacolumn in patients with hepatitis c related who have undergone kidney transplantation: preliminary study g. novelli 1 1 la sapienza university introduction: patients who have undergone kidney transplant and suffer from hepatic c related (hcv) cannot be treated with standard therapy (peg-ifn combined with ribavirine) due to acute rejection risk. furthermore, immuno-suppressive therapy facilitates progression and infection and chronic hepatopathesis. monocytes and macrophages are known to produce extra-hepatic breeding sites and spread disease. our aim was to lower macrophages, granulocytes,monocytes, pro-inflammatory cells and viremia levels using an extra-corporeal device:adacolumn®(otzuka). methods: the adalcolumn filter is filled with 2mm. cellulose acetate beads immersed in sterile saline solution. these carriers absorb granulocytes and monocytes/macrophanges through fcr receptors. six patients were treated in our department. all patients were affected by virale genotype 1b. patients underwent five 1hour treatments for five consecutive days according to protocol. results: during treatment cycles and successive follow ups we observed a stabilization of kidney parameters and a non significant decrease in transaminase levels. at 3 rd month follow up we observed a significant decrease in plasma hcv-rna in 3 patients (p<0.01) associated with attenuation of inflammatory phase (p<0.2) and variations in immunomodulation. only one patient presented altered cd4+ and cd8+ where positive was observed at 3 rd month. in another patient, even though immunomodulation improved, there was no reduction in viremia. conclusions: considering the results this method should be used on a greater number of patients evaluating successive treatment times in case of viremia increase. background: patients who have undergone kidney transplant and suffer from hepatic c related (hcv) cannot be treated with standard therapy (peg-ifn combined with ribavirine) due to acute rejection risk. furthermore, immuno-suppressive therapy facilitates progression and infection and chronic hepatopathesis. monocytes and macrophages are known to produce extra-hepatic breeding sites and spread disease. our aim was to lower macrophages, granulocytes,monocytes, pro-inflammatory cells and viremia levels using an extra-corporeal device:adacolumn®(otzuka). methods: the adalcolumn filter is filled with 2mm. cellulose acetate beads immersed in sterile saline solution. these carriers absorb granulocytes and monocytes/macrophanges through fcr receptors. six patients were treated in our department. all patients were affected by virale genotype 1b. patients underwent five 1hour treatments for five consecutive days according to protocol. results: during treatment cycles and successive follow ups we observed a stabilization of kidney parameters and a non significant decrease in transaminase levels. at 3 rd month follow up we observed a significant decrease in plasma hcv-rna in 3 patients (p<0.01) associated with attenuation of inflammatory phase (p<0.2) and variations in immunomodulation. only one patient presented altered cd4+ and cd8+ where positive was observed at 3 rd month. in another patient, even though immunomodulation improved, there was no reduction in viremia. conclusions: the treatment was found to be safe without hemodynamic or infective complications. considering the results this method should be used on a greater number of patients evaluating successive treatment times in case of viremia increase. m. sharaf-eldin 1 , h. el batae 1 , n. abd el-ghaffar 2 , w. rasheed 2 1 tanta faculty of medicine , egypt., 2 national research centre, cairo, egypt aim: we aimed to characterize serum cytokine levels of interleukin-1 beta (il-1 ) and interleukin -6 (il-6) in hcv infected patients & in patients with hepatocellular carcinoma (hcc) in comparison to control group and their possible use as markers of disease progression. patients and methods: sixty patients were divided into three groups: group i: twenty hcv infected patients without cirrhotic changes. group ii: twenty hcv infected patients with liver cirrhosis (lc). group iii: twenty hcv infected patients with hcc and 20 healthy subjects as control group. all patients and control group were subjected to biochemical and serological tests, anti hcv, hcv (rt-pcr) and cytokines measurements of serum il-1 & serum il-6 levels. results: showed a high statistically significant elevated serum il-6 and il-1 levels in patients with chronic hcv infection in comparison to control group. highly statistically elevated levels of il-6 and il-1 in liver cirrhosis and higher levels were found in hcc group in comparison to control group. the levels of il-6 and il-1 increased significantly in hcv infected patients as the disease progress. conclusion: serum il-1 , and il-6 levels are elevated in patients with hepatitis c-related liver diseases, especially in lc and hcc patients. their levels reflect hepatic dysfunction better than liver inflammation parameters; accordingly, we may use serum il-1 and il-6 as markers for liver disease progression in hcv-infected patients instead of invasive techniques. atsushi tanaka 1 , naoko hanawa 1 , mitsuhiko aiso 1 , yoriyuki takamori 1 , hajime takikawa 1 1 teikyo university school of medicine background and aim: pegylated interferon (peg-ifn) therapy is not indicated for many cases with hcv-related cirrhosis due to various adverse effects. however, patients with hcv cirrhosis are at high risk for development of hepatocellular carcinoma (hcc). thus we have introduced low-dose peg-ifn treatment for patients for compensated hcv cirrhosis. patients and methods: selection criteria for low dose peg-ifn is 1) compensated hcv-related cirrhosis, and 2) either the elderly (>65) or presence of thrombocytopenia (<8.0x10 6 / l). we have treated patients who met these criteria with low-dose peg-ifn, consisting of either peg-2a 90 g/1 -2w or peg-2b 0.5 g/kg/w+ribavirin 200mg/d. [results] twenty patients with compensated hcv cirrhosis (all patients genotype 1b) have been treated with low-dose peg-ifn (peg-2a:9, peg -2b+rib:11 . the age, platelet counts (x10 6 / l), and alt (iu/l) of 20 patients at baseline were 64.1±8.3, 7.8±4.6, and 90.4±68.6 respectively. all patients were well tolerated. low-dose peg-ifn has been continued 59.6±43.8 weeks on average. although viral response was not detected, biological response (br), defined as maintenance of alt within normal range, was obtained in 12 patients (12/20=60%). of note, neither development of hcc nor decompensated cirrhosis was observed in these 12 br cases. by contrast, hcc and decompensation developed in 5 and 1 patients respectively among 8 patients who failed to achieve br. conclusion: low-dose peg-ifn treatment was safe and well tolerable, and could potentially prevent hcc or decompensation in patients with liver cirrhosis when br was obtained. aims: to study the efficacy of peginterferon and ribavirin in treating chronic hepatitis c (chc) with genotype 6a in hong kong chinese. methods: to assess sustained virological response (svr) (serum hcvrna< 500iu/ml) at 6-months follow-up. results: nine patients with genotype 6a chc (included from jan2003 to dec2007) received peginterferon and ribavirin. mean age: 50 (range 28-64). mean alt before treatment: 96 iu/l (range 29-173 iu/l). seven patients had liver biopsy performed, only one showed stage 3-4 fibrosis and others showed active hepatitis without advanced fibrosis. mean serum hcv-rna: 9.17x10 5 iu/ml(range 9.0x10 3 -3.3x10 6 iu/ml). six patients had received peginterferon alfa-2b (1.5mcg/kg/week), other 3 received peginterferon alfa-2a (135mcg/week). ribavirin dosage ranged from 600mg-1000mg/day depending on body weight and baseline haemoglobin. treatment durations were 48-52 weeks in 7 patients, 24-26 weeks in 2 patients as one showed rapid virologic response at week 4 and the other was intolerant to side effect of peginterferon. eight patients had early virologic response at week 12 and one had >2log drop of hcvrna. eight patients had end-of-treatment response. eight patients (88.9%) achieved svr at end of follow-up. two patients who received only 24-26 weeks of combination therapy also achieved svr. the one who failed to achieve svr was at older age of 60 and had advanced fibrosis. conclusions: the efficacy of pegylated interferon and ribavirin in treating chinese patients with chronic hepatitis c genotype 6a can achieve high sustained virologic response rate of 88.9%. t. bharati 1,2 , p. kar 2 , a. mohammad 2 , k. mariappan 1 , j. annamalai 1 , r. introduction: hcv is a recognized cause of hcc. information on hcv genotypes in hcc are scanty in india. methods: a total of 154 hcc cases from delhi, 96 hcc cases from madurai and 246 cases of chronic hepatitis without hcc were controls in the study. rt-pcr for hcv rna and genotyping were carried out in all the cases results: in group-i, hcv rna was positive in 26.58% hcc cases in which genotype 3 was found in 57.1% genotype 1 was observed in 26.2% hcc cases. whereas 16.6% cases remained nontypable. in group-ii, hcv rna was positive in 23.95% hcc cases, with genotype 3 in 30.4% cases, genotype 1 in 52.2% cases and genotype 4 in 4.34% cases. however, 13.04% cases remained nontypable. out of the 246 control cases, 187 were ch and 59 were cirrhosis. in ch group, hcv rna was positive in 22.45% cases in which, genotype 3 was detected in 71.4% cases whereas genotype 1 was observed in 21.4% cases . however 7.1% cases remained nontypable. in cirrhosis group, hcv rna was positive in 37.2%cases. genotype 3 was found in 59.1% cases. while genotype 1 was present in 31.8% cases and 9.1% cases remained nontypable. conclusion: genotype 3 in delhi and genotype 1 in madurai were predominant. in hcc cases. our study demonstrates that no particular hcv genotypes were associated with hcc and genotype did not appear to influence the development of hcv-associated hcc. background/aims: the standard treatment for chronic hepatitis c infected with hcv genotype-1 is a combination of pegylated interferon alfa and ribavirin for a 48weeks. it is unclear if 24weeks treatment is possible for patients showing a rapid virologic response (rvr) without compromising the sustained virologic response (svr) in korea. method: between june 2005 and july 2008, among patients chronically infected with the hcv genotype-1 (hcv-1) who were treated with pegylated interferon alfa subcutaneously once weekly plus ribavirin (weight-based), 20 consecutive paients who had low pretreatment viral load ( 1.5x10 6 copies/ml) and rvr were treated for 24weeks and then followed up for 24weeks. the hcv rna was quantitatively assessed pretreatment, at 12weeks of treatment and was qualitatively assessed at 4weeks of treatment, the end of treatment (24weeks), 24weeks after end of treatment. rvr was defined as undetectable hcv rna at the 4weeks. results: baseline characteristics of patients was as followed; age (30-65years:mean 45years), bmi (21-27kg/m²:mean 23.5kg/m²), hcv rna titer (0.3-1.4×10 6 copies/ml:mean 0.5×10 6 copies/ml), alt (5-751iu/l:median 77iu/l). among the 20 patients, all patients (100%) had sustained virologic response (svr). conclusions: hcv-1 infected patients with a low baseline hcv rna concentration ( 1.5×10 6 copies/ml) who had hcv rna negative at week 4 of treatment may be treatment for 24weeks without compromising sustained virlolgic response. however, an additional trial will be needed to optimize the treatment duration. background/aims: acute hepatitis c (ahc) has a high chronicity rate of up to 50~84% if it is not treated. although the good treatment response to pegylated interferon (peg-interferon) therapy has reported, there is not definite guideline to treat of ahc in korea yet. the aim of our study was to investigate the clinical course and treatment outcome of ahc in single center of korea. methods: we performed a retrospective analysis of 35 patients who were diagnosed with ahc during the period from may 1996 to december 2007. the diagnosis of ahc was based on seroconversion to anti-hcv antibody or the clinical and biochemical diagnostic criteria satisfactory to ahc and on the presence of hcv rna in first serum sample. the spontaneous resolution was defined as loss of hcv rna in serum for 6-month in untreated group, and in treatment group, the sustained virological response (svr) was defined as a index of treatment success. results : thirteen of thirty-five patients were treated, six of thirty-five were untreated and observed clinical course, and sixteen patients were not followed up after diagnosis. in treatment group, nine of thirteen (69%) acquired svr, and two of six (33%) showed spontaneous resolution in untreated group. ten of thirteen treatment patients used conventional interferon, and another three patients used peg-interferon. conclusion : compared with untreated group, there was higher svr rate in treatment group (33% vs. 69%). so early interferon treatment in acute hepatitis c should be considered. background: this study was conducted to identify predictors of thyroid dysfunction and to determine whether virologic factors or treatment response affect thyroid dysfunction development during peginterferon (pegifn) therapy in chronic hepatitis c patients. methods: sixty chronic hepatitis c patients treated with pegifn -2a or -2b in combination with ribavirin from 1st july 2004 to 30th july 2008 were included in this study. treatment responses were evaluated and thyroid functions were assessed every 4 weeks. results: seventeen patients (28.3%) experienced thyroid dysfunction during treatment, and that occurred more frequently in women and in patients with a lower body mass index (bmi). the proportion of patients with a high viral load (a serum hcv rna titer >750,000 iu/ml) was significantly higher in the thyroid dysfunction group rather than in the euthyroid group(88.2% vs. 48.8%, p=0.005). among patients with hcv genotype 1, the rate of sustained virologic response was lower, and relapse occurred more frequently in the thyroid dysfunction group than in the euthyroid function group during pegifn-based therapy(svr, p=0.024; relapse, p=0.017). the female gender and the high viral load were independent predictors of thyroid dysfunction in multivariable analyses (female, or 9.44, p=0.009; high hcv rna titer, or 8.40, p=0.030) . conclusion: the risk of thyroid dysfunction during pegifn therapy for chronic hepatitis c was found to be higher for women and for those with a low bmi and a high viral load. background: in peginterferon alpha2b (peg-ifn 2b) and ribavirin (rbv) combination therapy for 48weeks for patients with chronic hepatitis c, it is still difficult to predict which patients will achieve sustained viral response (svr) at the completion of this therapy. aim: to predict svr and non-svr (relapse) at the end of this combination therapy by determining changes of serum hyaluronic acid (ha) levels. methods: eighteen patients were enrolled and their serum ha levels were measured before therapy, and after the 1st, 2nd, 3rd, and last trimesters during therapy. results: eleven patients achieved svr and 7 became relapsers. all patients showed higher ha levels in the 1st trimester than the pretreatment levels. in the svr group, 3 of 11 (27.2%) patients in the 2nd, 5 of 10 (50.0%) in the 3rd, and 9 of 11 (81.8%) in the last trimester showed lower ha levels than the pretreatment levels. by contrast, in the relapser group, none in the 2nd, 1 of 6 (16.7%) in the 3rd, and 1 of 7 (14.3%) (p<0.05) in the last trimester showed lower ha levels than the pretreatment levels. this study revealed that as the 48-week therapy went on, ha levels were more likely to fall below the pretreatment levels by the last trimester in patients achieving svr. however, ha levels of relapsers tended to continuously be above the pretreatment levels. conclusion: determination of changes of serum ha levels during peg-ifn 2b and rbv therapy predicts svr and non-svr at the completion of this therapy. results: the most frequent lymphomas were with high malignancy (40%), intermediate (32.5%) and low degree (27.5%). cryopathy was negative (87.5%). the presence of viral markers was performed soon as possible after the nhl diagnosis, at the same time (52.5%) or during the first year of evolution (32.5%). the prevalence of the hcv infection was 15%, comparable to the one in the control patients group (22.68%), admitted in a gastroenterological clinic. on the other hand, this prevalence is significantly increased compared to the one in the general romanian population (4.9%). the patients with nhl and hcv infection belonged especially to the low and intermediate malignancy degrees; the survival was influenced by the malignancy degree and not by the presence of hcv infection. the prevalence of hbv infection in the tested patients was 2.5%, being lower than that of hcv infection (2.5% vs. 15%, p = 0.113) but comparable to the one in the general population (2.5% vs. 6.3%, p = 0.525). conclusions: the prevalence of hcv infection in the patients having nhl was 15%, comparable to the one in the control group, but significantly increased compared to the one in the general population, leaving open the issue of a causal relationship between hcv infection and nhl. iron hepatic overload and hepatitis c d. damian 1 , m. grigorescu 1 , m.d. grigorescu 1 , t. zaharie 1 1 third medical clinic, cluj napoca, romania aim: evaluation of the prevalence and the degree of iron loading and the relationships with the clinical, biological and morphological changes. method: 212 patients with chronic hepatitis c were included, to whom we tested the blood iron level. in order to evaluate the hepatic iron accumulation we performed the perls staining, using a qualitative analysis and a semiquantitative scoring system (deugnier). results: from a total of 212 patients, 16.5% presented increased blood iron level (p = 0.000). the evaluation of the liver iron loading was performed in 54 patients, some having normal blood iron level (n = 34) and others (n= 20) increased (p = 0.007). the stainable iron was observed in 27 patients. the iron loading was usually low, the deposits were observed mostly at the sinusoid cells and the hepatocyte and less in the portal spaces, usually as a pale staining or of small, nonmerging granules. the total iron score deugnier was low. the increased blood iron correlated with the alt and ggt levels, the necroinflamatory activity and fibrosis. no correlationships between stainable iron and increased blood iron. the presence of liver iron accumulation only correlated with the fibrosis degree. conclusions: of the 212 patients to whom we tested the blood iron, 16.5% had increased levels. the perls staining was positive in 50% of the patients. the iron loading was mainly low, with a more frequent distribution in the sinusoid cells and in the hepatocytes and correlated only with the stage of fibrosis. response patients whose hcv rna became negative at 12 -16 weeks t. ide 1 , t. arinaga 1 , k. ogata 1 , i. miyajima 1 , k. kuhara 1 , r. kuwahara 1 , m. background/aim: chronic hepatitis patients whose hcv rna became negative at 4 weeks of peg-interferon/ribavirin treatment achieved excellent svr(sustained viral response) rate of almost 90%. however, in patients whose rna became negative after 20 weeks, the svr rate is very low. since many patients became rna negative at 12-16 weeks, it is important to clarify the characteristics of the patients. material and method: among 234 patients, 41 became rna negative at 12-16 weeks and the therapy completed (total 48-60 weeks). the characteristics were analyzed by using sex, age, weight, bmi, alt, gtp, hemoglobin, platelet counts, ccr, hyaluronic acid, the mutation of hcv core region (aa70, 91) and interferon sensitivity determining region, adiponectine, home-ir, rna dynamics, dose and the treatment period. result: the svr rate was 75.6%(31/41). because all 10 patients of non svr were female, we compared these 10 patients and 15 female svr. the platelets counts were low in non svr (non svr 15.0 ± 2.8 (x10 4 /mm 3 ) vs svr 20.2 ± 4.8 (p<0.05)). the mean dose of ribavirin was lower (447 ± 126 mg/day) in non svr (p<0.05) than in svr (625 ± 127). conclusion: as for the characteristics of the patients whose hcv rna became negative at 12-16 weeks but became non svr, female, low platelet count and low dose of ribavirin were important factors. in the patients who received reduced ribavirin doses, the idea to increase the ribavirin dose and to maintain it are necessary. (ex, use 400mg and 600mg alternately) pe329 background: chronic hepatitis c virus (hcv) infection poses a challenge for a growing number of infected patients who exhibit disease complications, including cirrhosis, hepatocellular carcinoma, and liver failure in china. the combination treatment of peginterferon alpha (peg-ifn alpha) plus ribavirin (rbv) is recommended as a standard care for hcv infections, which can improves hepatic markers and eradicates the virus in about 50% of patients. however, a significant number of patients do not respond to therapy or relapse following treatment discontinuation. several viral, hepatic, and patient-related factors influence response to therapy. methods: in our clinical practice, a total of 77 interferon-naïve patients (61% male; median age 47 years) with chronic hepatitis c include 11 cirrhotic patients (no genotyping) received peg-ifn alpha-2a 180 mcg/week plus rbv 900-1200mg/day for 48 weeks and follow up 24 weeks. results: show that the patients have more rvr and evr rate (54% and 90.9% respectively). while the svr (undetectable hcv-rna 24 weeks after treatment completion) rate is only 51.5% in conclusion: comparing with the data of clinical trail, the rvr, evr and eotr were higher, while svr was the same in chinese patient with chronic hepatitis c patients received the combination therapy of peg-ifn plus rbv. the reason of high relapse was still unknow. although optimal duration of retreatment and benefits and safety of maintenance therapy have not been determined, an extended duration is likely needed, even for the patients who achieved evr. s. nakamoto 1 , f. imazeki 1 , k. background/aims: recently amino acid (aa) substitutions in hepatitis c virus (hcv) core region (double wild (dw); arginine at aa 70, leucine at aa 91) were reported to be associated with sustained virological response (svr) in a combination therapy of peginterferon and ribavirin. we evaluated the viral factors influencing treatment response. methods: nucleotide sequences of core region were determined directly in 104 patients with genotype 1 and high viral load ( 100 kiu/ml) treated with peginterferon-alpha 2b and ribavirin for 48 weeks. rapid virologcal response (rvr) was defined as more than 2 log decrease of hcv-rna during the first four weeks of therapy and early virological response (evr) as that during the first 12 weeks. svr was defined as negative hcv-rna 6 months after the end of treatment and non-virological response (nvr) as less than 2 log decrease of hcv-rna during the treatment. results: dw at aa 70 and 91 was shown in 17/44 (35%) patients with rvr and in 5/44 (11%) with non-rvr (p=0.003), in 25/72 (35%) with evr and in 1/28 (3.6%) with non-evr (p=0.001), in 16/37 (43%) with svr and in 6/44 (14%) with non-svr (p=0.003), and in 1/24 (4%) with nvr and in 25/79 (32%) with non-nvr (p= 0.005). in multiple logistic regression analysis, dw was significantly associated with rvr, evr, svr and nvr. conclusions: dw at aa 70 and 91 in hcv core region was closely associated with virological response in a combination therapy of peginterferon and ribavirin. medicine and hepatology, henry dunant hospital, athens, greece, 3 biometrics, ist gmbh, mannheim, germany, 4 background: among patients with chronic hcv treated with pegylated interferon and ribavirin, the highest sustained virologic response (svr) rates are achieved in patients with a rapid virological response (rvr). here we investigate how the time taken to become hcv rna undetectable influences the probability of relapse during untreated follow-up. methods: data from 569 patients treated for 48 weeks with peginterferon alfa-2a (40kd) 180µg/week plus ribavirin 1000/1200mg/day were included in the intent-to-treat analysis. response was classified as rvr, complete early virological response (cevr) slow responder and non-evr. results: there was a correlation between the time required to become hcv rna undetectable and the relapse rate after stopping treatment. patients with an rvr had the lowest relapse rate (4%); this increased among patients with slower responses. conclusion: there was an inverse correlation between the time taken to achieve a virologic response and the probability of relapse. background: rapid virologic response (rvr; hcv rna <50iu/ml) at week 4 of treatment with pegylated interferon plus ribavirin can be used to predict the probability of achieving an svr. patients with detectable hcv rna at week 4 have a lower probability of achieving an svr than those with an rvr; further subdivision of these patients may be useful in predicting outcomes. methods: we conducted a retrospective analysis including 569 genotype 1 patients treated for 48 weeks with peginterferon alfa-2a (40kd) 180 g/week and ribavirin 1000/1200 mg/day. patients were categorized as rvr and non-rvr. those without an rvr were further subdivided into detectable but unquantifiable, 3, 2, 1 or <1 log10 drop in hcv rna. the proportion of patients with undetectable hcv-rna at week 12 and achieving an svr was calculated within each category. results: rvr and non-rvr patients had an 88% and 43% rate of svr respectively. among non-rvr patients, rates of svr depended on the categorical response at week 4: detectable but unquantifiable hcv rna, 77%; 3 log10 drop in hcv rna, 61%; 2 log10 drop, 43%; 1 log10 drop, 27%; and <1 log10drop, 13%. independent of week 4 response, undetectable hcv rna at week 12 was also highly predictive of svr. conclusions: patients achieving an rvr have high rates of svr. among patients who do not achieve an rvr a more precise prediction of svr can be achieved by considering the extent of viral load reduction at week 4 and week 12. retrospective japanese validation study of fibrotest and actitest in patients with chronic hepatitis c. n. nagata 1 , t. mine 1 1 background: fibrotest (ft) and actitest (at) are biochemical markers of fibrosis and activity for use as a non-invasive alternative to liver biopsy in patients with chronic hepatitis c virus. the aim of this study was to perform a validation study the discordances between ft and at(ft/at) and liver biopsy in patients with chronic hepatitis c in japan. methods: 117 serum samples of chronic hepatitis c patients sended at -80°c at the biochemistry department of pitié salpetrière hospital were analysed between july and august 2007. ft/at components were assessed on thawed sera for 110 patients. 96 from 110 patients had liver biopsy at the moment of serum analysis. liver biopsy fibrosis and activity scores were assessed by a pathologist in japan according to metavir scoring system. for each individual test-ft/at the following statistical analysis were performed result: ft observed auroc for the diagnosis of advanced fibrosis was 0.81 and after adjustment according to the prevalence of different stages of fibrosis the auroc was 0.89. this difference could be explained by the non-homogenous distribution of different stages of fibrosis (low prevalence of extremes stages of fibrosis -f0 and f4-and high prevalence of adjacent intermediate stages -f1 and f2). the observed auroc of ft for the diagnosis of precirrhosis and cirrhosis was 0.86 and the observed auroc of at for the diagnosis of moderate to severe activity was 0.74 . conclusion: these results are similar to those observed in all independent validations worldwide. background: accurate monitoring of hcv-rna level throughout anti-hcv therapy is key factor for predicting sustained virological response (svr). real-time detection polymerase chain reaction (rtd-pcr) based methods are sensitive, have wide dynamic range of quantification and carryover contamination caused by classical pcr. aim: to compare rtd-pcr based assays; cobas ampliprep/cobas taqman (cap/ctm) and recently developed abbott realtime hcv for hcv rna quantification and measurements differences by 2 assays in different genotypes. methods: in total, 253 serum samples were used including, 135, 39, 24, 15, and 40 with genotypes 1b, 2a, 2b, 3a and 4 respectively were tested quantatively for hcv-rna by cap/ctm and abott realtime. results: good correlation between two assays as overall (r=0.96) with correlation coefficient (r) in genotypes 1b, 2a, 2b, 3a ranged between 0.99 to 0.98 and least in genotype 4 (r=0.78). mean differences between cap/ctm and abott realtime was significnat in genotypes 1b and 4. significantly hcv-rna genotype 4 underestimation by cap/ctm (4.3+0.9 log iu/ml) than abbott realtime (4.8+0.9 log iu/ml; p=0.01). in genotype 1b, significantly higher hcv-rna measurement by cap/ctm (5.7+1.7log iu/ml) than abbott realtime (5.0+1.4log iu/ml, p=0.001). two hcv genotype 4 samples showed measurement differences (cap/ctm minus abbott realtime) of -3.75 and -1.68 log iu/ml. studying genotype 4 sequences within 5 utr , target for cap/ctm rt-pcr amplification, revealed nucleotide polymorphisms at positions a107, a165, t203, a205, and a243. conclusion: different measurement efficiency by commonly used cap/ctm in different genotypes compared to abbott realtime. 6 new york, new york, usa, 7 vertex pharmaceuticals, 8 cambridge, ma, usa, 9 duke clinical research institute , 10 duke university, durham, nc, usa background: prove1 is a placebo-controlled study of 250 subjects with genotype 1 chronic hepatitis c randomized to 48 weeks of peginterferon-alfa-2a 180 ug/week (p) plus ribavirin 1000-1200 mg/d (r) (pr48, n=75), or 3 regimens of 750 mg q8h telaprevir (tvr) with pr: tvr/pr for 12wks followed by pr for 0wks (t12/pr12, n=17), 12wks (t12/pr24, n=79) or 36wks (t12/pr48, n=79). the impact of african american race (aa) and bridging fibrosis on sustained virologic response (svr) was examined. methods: subjects with cirrhosis were excluded from study. fibrosis was categorized as mild/minimal, portal, or bridging from biopsy within 2 years. itt analysis was performed. results: overall, svr was achieved by 41% of subjects in the pr48 group, 35% in t12/pr12 group, 61% in t12/pr24 group, and 68% in t12/pr48 group. subgroup analyses indicated svr was improved with tvr/pr (tvr/pr arms pooled) vs pr48 alone in aa subjects (44% (8/18) vs 11% (1/9)), and in subjects with bridging fibrosis (69% (22/32) vs. 26% (5/19)). adverse events leading to discontinuations were more frequent in the tvr/pr groups (21% vs. 10%). rashes, gastrointestinal events and anemia were more common in the t/pr arms, and rashes were more frequently severe (7% vs 1%). conclusions: tvr-based treatment for 24 or 48 weeks was associated with an increase in svr rates compared to pr48. subgroups with impaired response to standard peg-ifn/rbv therapy appeared to benefit from the addition of telaprevir. adverse events leading to discontinuation were more frequent in tvr-based regimens. background and aims: induction of type i ifns is a core issue in antiviral responses and must be tightly controlled. the protein kinase tbk1 is critically involved in virus-triggered type i ifn signaling. in previous studies, an alternatively spliced isoform of tbk1, termed tbk1s, was identified to be induced in both human and mouse cells. bound to rig-1, it is able to disrupt the interaction between rig-i and visa. this study was designed to observe the expression of tbk1s in hcv-infected patients. methods: total rna was extracted from samples of peripheral blood mononuclear cells obtained from 11 hcv patients, 9 hcv patients treated with ifn-/ribavirin and 9 healthy controls, and subjected to real -time pcr using the primer-probe sets for human tbk1s, tbk1 and ifn-genes. results: the tbk1s expression was significantly elevated in hcv-infected patients, while treatment of hcv-infected patients with ifn-/ribavirin resulted in down-regulation of tbk1s to the normal level. conclusions: the study strongly supports the idea that expression of tbk1s is correlated with hcv infection, and indicates that tbk1s may play an important role in the regulation of hcv infection.this work was supported by nsfc(30571643, 30672380, 30700702 background: infringement of iron metabolism is one of fibrosis progressing factors during diffuse liver diseases. the interrelation between the syndrome of iron overload (sio) and svr achievement is studied during chronic hcv infection treatment. methods: 68 patients with chronic hcv infection (genotyping: 1-34; 1+3-5; 3-22; 2-6; 4-2) are investigated. sio criteria: iron increase-more than 37 mkmol/l, ferritin -more than 200 mkmol/l, percent of transferriny saturation with iron (%tf) -more than 50%. results: sio revealed in 10 patients (14.7%): 5 patients -1 genotype (2 assotiative with a diabetes) and 5 patients-genotype 3 (3 -in combination with liver steatosis and obesity). venipuncture series were done up to getting ferritin referential parameter values before therapy beginning. rvr: sio -5 patients, normal metabolism -48; evr: 6 and 54, svr: 4 and 54 relatively. 5 nonresponding patients (sio) had steatosis and diabetes, hereditary hemochromatosis (c282y/h63d) is verified in 1 case. increase of ferritini values and %tf during therapy and positive hla-a3 and hla-b7 is registered in nonresponding patients. conclusions: sio in hla-a3, hla-b7 and c282y/h63d positive patients is independent predictor of nonresponse during peginterferon alpha-2a (40 kd)" ribavirin treatment. a.p. srivastava 1 , g. dogra 2 , s. sachdeva 1 , n. nigam 1 , a. chakravarty 2 1 dr. rml hospital, new delhi (india)-110001, 2 maulana azad medical college & associated hospitals, new delhi, india background: hepatitis c virus (hcv) has emerged as a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. genotyping and assessment of viral load in hcv patients are vital for designing therapeutic strategies. we aimed to determine the pattern of hcv genotypes and its association with viral load and biochemical profile. methods: 71 hcv rna positive patients were included in the present study attending the medical-opd and wards of dr rml hospital, a tertiary care hospital in new delhi during 2006-2008. hcv genotyping was carried out by restriction fragment length polymorphism (buoro et al 1999) followed by the type specific primers from the core region (ohno et al 1997) . viral load estimation was carried out by taqman real time pcr system using previously described method (martell et al 2000) . result: 67.6% of cases were having genotype 3 (3a, 3b, 3f & 3i) followed by genotyping 1 (1a & 1b) in 26.8% and genotype 2 in 5.6%. there was no statistical significant difference seen in the biochemical profile between the three groups of genotypes. genotype one was associated with a significantly higher viral load as compared to the genotypes three and two. parentral mode of transmission was accounted for the 68% of all the infected cases. conclusion: hcv genotypes 3 and 1 accounted for 94% of our cases. the genotype 1 is associated with higher degree of disease severity as assessed by viral load. also two unusual subtypes 3i and 3f were identified from this geographical region. a.p. srivastava 1 , g. dogra 2 , s sachdeva 1 , n. nigam 1 background: the development and resolution of an inflammatory process is regulated by a complex interplay among cytokines that have pro and anti-inflammatory effects. regulatory mechanisms that control the production of cytokines include genetic polymorphism in particular promoter/leader region. polymorphisms may directly or indirectly affect the binding of transcriptional factors, consequently increasing or decreasing the production of mrna, thus regulating cytokine production. we aimed to determine the polymorphism of tumor necrosis factor-alpha (tnf-alpha) and interleukin-10 (il-10) genes in chronic hepatitis c patients. methods: 40 hcv rna positive patients were included in the present study conducted during 2006-2008. 25 healthy controls were also included. genomic dna was extracted by using q1a amp dna blood kit protocol according to manufacture's instruction and desired fragment was amplified by using the primer's of vidigal et al 2002. result: genotyping of -308-promoter variant of tnf-alpha was performed by pcr. polymorphism in the tnf-alpha (g/g, g/a and a/a allele) was different between hcv patients and healthy controls. il-10 variants (c/t, c/c) were more frequent among hcv patients as compared to healthy controls. conclusion: genetic polymorphism analysis on il-10 promoter have indicated that distribution pattern of il-10 polymorphism was significantly different between controls and hcv patients. furthermore, polymorphism in promoter region of tnf-alpha (-308) was found, though the difference was not significant. since this is a preliminary study, we believe that our findings may stimulate further research on larger number of patients. introduction: the assessment of liver fibrosis provides useful information not only for diagnosis but also for therapeutic decision. although liver biopsy is the gold standard for fibrosis assessment, it is invasive and may have some risks, this has led to the development of non-invasive biochemical markers of liver fibrosis. fibro-test which have five parameters used for the quantitative assessment of liver fibrosis. our aim is to validate the performance of fibro-test in an independent cohort of patients with chronic hepatitis c genotype 4. methods: subjects were 50 patients with chronic hepatitis c genotype 4. all biopsies were scored using metavir system by two independent pathologists. fibro-test was done with (biopredictive, houilles, france) for the assessment of liver fibrosis. sensitivity, specificity, ppv and npv were measured for distinguishing between different degrees of severity of fibrosis. results: patients (45 male and 4 female) age ranged 21-56 years, liver biopsy showed 4% (f0), 40% (f1), 20 % (f2), 28% (f3), 8% (f4). the efficacy of fibrotest is 63.26%, sensitivity 83.3%, specificity 53%, positive predictive value 50% and negative predictive value 85%. conclusion: fibrofast has a low performance in assessment in fibrosis in chronic hepatitis c genotype 4. introduction: liver biopsy is the reference method for assessing liver fibrosis. however, it is invasive, costly and has some limitations. european liver fibrosis (elf) markers have shown to be accurate in assessing liver fibrosis in a range of chronic liver disorders. our aim is to test the performance of elf markers in an independent cohort of patients with chronic hepatitis c genotype 4. methods: subjects were 199 patients with chronic hepatitis c genotype 4. all biopsies were staged for fibrosis using metavir system by two independent pathologist. elf markers were done by (diagnostic & operations, england) and fibrosis scores were derived using the published elf algorithm. the area under the curve (auc) for receiver operator characteristic curves was measured along with sensitivity and specificity, positive (ppv) and negative (npv) predictive values for distinguishing between different stages. results: patients (179 male and 20 female), age was ranged 25-51 years, liver biopsy showed 2% (f0), 27% (f1), 34% (f2), 26% (f3) and 11% (f4). elf markers had no correlation with fibrosis score where r = -0.003, p = 0.963, aucs: 0.469, specificity 87.9 %, sensitivity only 9.3 %, ppv: only 6.03 %, npv: 61.5 % and efficacy 58.2%. conclusion: the performance of elf marker is low and can not be used for assessment of fibrosis in chronic hepatitis c genotype 4. background: the prove2 trial is a randomized, placebo-controlled study that assessed the safety and efficacy of 750mg q8h telaprevir (tvr) combined with 180 g/week peg -ifn alfa-2a (p) ± 1000-1200mg/day ribavirin (r) in chronic hcv genotype 1-infected treatment-naïve patients without cirrhosis. methods: overall, 323 patients received tvr + pr for 12 weeks (t12/pr12; n=82), tvr + pr for 12 weeks then pr for 12 weeks (t12/pr24; n=81), tvr + p for 12 weeks (t12/p12; n=78), or to pr for 48 weeks (pr48; n=82). primary endpoint: sustained virologic response (svr, undetectable hcv-rna 24 weeks post-treatment). results: baseline characteristics were well balanced across groups. numerically higher svr rates were observed in patients receiving t12/pr24 (69%; p=0.004 for difference vs. pr48) than t12/pr12 (60%), t12/p12 (36%) or pr48 (46%). relapse rates were lower in the t12/pr24 group (14%) than the t12/pr12 (30%), t12/p12 (48%) and pr48 (22%) groups. the relapse rate in patients receiving t12/pr24 with 4-week and 12-week undetectable hcv-rna was 7% (3/45). the aes occurring more frequently with the t/pr regimen were pruritus, rash, asthenia, nausea and anemia. in the t/pr arms, 12 patients discontinued due to rash, 2 discontinued due to pruritus, and 2 patients due to anemia. conclusion: these results showed that a telaprevir-based regimen led to significantly higher svr rates than pr, and indicate that this regimen could shorten the overall treatment duration from 48 weeks to 24 weeks for most patients infected with hcv genotype 1. a.c. cardoso 1 , c. stern 1 , r. moucari 1 , n. giuily 1 , p. bedossa 1 , p. marcellin 1 1 hopital beaujon background/aim: this study evaluated the effect of the response (svr) to therapy on fibrosis stage, as assessed by ls, in patients with advanced fibrosis (f3) or cirrhosis (f4). methods: hcv patients with f3 or f4 who received interferon-based treatment were studded. ls was assessed after treatment (median delay of 36 months, 2-206) in patients with or without svr. correlations between ls and clinical and treatment characteristics were analyzed. results: 114 patients were included: male gender (72%), mean age (54±9 years), diabetes (26%), mean bmi (26±6 kg/m2), genotype 1 (59%). 33% had svr. ls was performed 0-3, 3-6, >6 years following treatment. by linear regression, the median of the ls was independently associated with svr (p=0.005) and diabetes (p=0.008). svr patients had lower ls (8.4 kpa; range 3.3-45) than non svr patients (15.7 kpa; range 5.3-75) (p<0.001). among the svr patients the median ls was lower when the delay between ls and the end of treatment was longer (10.9,8.8,6. 3) (p=0.02). on the opposite, among the non-svr patients the median ls was not significantly different (p=0.57). the median of liver stiffness was higher in patients with diabetes (p=0.006). bmi and dyslipidemia did not influence the median of the ls. conclusion: in patients with advanced fibrosis or cirrhosis, ls was lower in patients with svr and decreased with time while it was higher and did not decrease in non-svr patients. ls could be important for assessment of fibrosis stage during the post-treatment follow-up. to study peripheral blood and intrahepatic natural killer (nk) cells in patients with chronic hepatitis c in relation to disease activity and severity of hepatic fibrosis. patients & methods: fifteen untreated patients with histologically-proven chronic hepatitis c, and 12 matched healthy subjects. the nk cells and natural killer t (nkt) cells were identified in fresh whole blood samples using two-color flow cytometric assay as cd3 -cd56 + and cd3 + cd56 + positive cells. immunohistochemical staining of liver biopsies taken from all patients was done using monoclonal antibody against cd56 for detection of nk cells and rabbit polyclonal antibody against smooth muscle actin (sma) for identification of activated hepatic stellate cells (hscs). results: patients with chronic hepatitis c showed significant decreases in the percentages of nk cells and nkt cells in peripheral blood. a negative correlation was found between serum hcv rna levels and the percentages of peripheral blood nk cells and the intensity of intrahepatic nk cells. the percentages of circulating nk cells and nkt cells and the intensity of intrahepatic nk cells were inversely correlated with the metavir fibrosis stage and the steatosis grade, and also with the intensity of intrahepatic activated hscs. conclusion: patients with chronic hepatitis c had significant deficiency in circulating nk and nkt cells as well as in intrahepatic nk cells. this may provide a possible mechanism for the suppression of innate immunity against hcv. background: hcv infection is the major cause of chronic hepatitis, cirrhosis and hepatocellular carcinoma. the virus is classified in six genotypes and more 50 subtypes, which are related distinct with antiviral therapies reply. in brazilian amazon, epidemiologist's studies in blood donors had pointed high frequency of genotype 1 (74%) followed by genotypes 3 (25%) and 2 (1%). however, epidemiological research in populations of risk to the infection still is scarce. aim: to determine hcv genotypic frequency in 116 blood donors, 55 patients with blood transfusions multiples, 57 patients in hemodialysis and 52 drugs users in the state of pará, brazilian amazon. methods: using real time pcr and nucleotide sequencing followed phylogenetic analysis had been gotten viral diagnosis and genotyping. results: in blood donors, hcv distribution was constituted by genotypes 1 (93.1%) and 3 (6.9%). in multitransfunded patients occurs maximum prevalence of genotype 1 (100%), probably reflect of genotype 1 specific transmission of blood donors population. on the other hand, in hemodialysis patients had been detected genotypes 1 (85.7%), 2 (3.6%) and 3 (10.7%), result of a bigger diversity of transmission routes (transfusional, interfamilial, nosocomial, etc) . in drug users occurs the biggest frequency of genotype 3 (38.1%) with prevalence of genotype 1 (61.9%), suggesting that the sharing of abuse machinery is allowing strains diffusion of genotype 3. conclusions: the genotype 1 possesses the biggest frequency in different population. moreover, through hcv genotypic frequency if it detached the contribution of transmission distinct routes indicated by previous epidemiologists researches. virol. 2007 ). we established real-time polymerase chain reaction (pcr) assays for the easy detection of these hcv mutations. methods: plasmids p-core-w, including wild type hcv core coding region (70r and 91l), and p-core-m, including mutant type hcv core (70q/h and 91m), were constructed by cloning and pcr-based mutagenesis for control vector of wild type core and that of mutant core, respectively. using serially diluted forms of these vectors, sybr green-based real-time pcr detections with mutation-specific primers were performed. results: analysis of known scalar concentrations of references indicated that the detection limits of these methods were at least 10 copies, 10 copies, 1000 copies, and 10 copies of 70-wild, 70-mutant, 91-wild, and 91-mutant, respectively. each primer could clearly distinguish the difference between p-core-w and p-core-m at the same copy numbers. concerning substitution 70, the ratios 100:1, 10:1, 1:1, 1:10, and 1:100 of p-core-w versus p-core-m could be distinguished. on the other hand, for substitution 91, the ratios 100:1, 10:1, 1:1, 1:10, 1:100, and 1:1000 could be distinguished, confirming the sensitivity and specificity of the assay. conclusions: this method could represent a useful alternative for the detection of genotype 1b hcv core amino acid substitutions 70 and 91 and be reliably applied for rapid screening. efficacy and tolerability of hcv treatment in asian patients according to age and genotype at a tertiary centre in western australia n. saroj 1 , n. kontorinis 1 , t. lorenzo 1 , m. marion 1 , s.l. chen 1 , w. cheng 1,2 1 royal perth hospital, 2 centre for international health, curtin introduction: race and ethnicity can influence efficacy and tolerability to treatment in hcv. the higher response rate in asians is thought to be associated with better adherence and tolerability. objectives: (1) to evaluate the adherence according to age and genotype (2) to assess the effect of age on treatment efficacy (3) background: hepatitis c virus (hcv) infection is a major health problem. there is huge regional variation in its prevalence and genotypic distribution. voluntary blood donors are thought to have somewhat lesser prevalence than the rest of the community. reliable statistics are not available for the entire country, particularly for the rural areas. it is important to know local situation and rationalize use of limited resources. methods: retrospective study of the records of patients attending the free liver clinic (flc) of our hospital located in a rural area of pakistan, and those screened for hcv infection prior to voluntary blood donation. results: patients at flc (324 out of 1638 [20%; males 65%] were found to have higher chances of being reactive for hcv antibodies as compared to voluntary blood donors (121/804 [14%]; p = 0.004; or 1.39 -95% ci = 1.11 -1.75). out of a total of 1022 hcv reactive patients, 904 (88%) were found to be positive on hcv rna testing. out of a total of 166 typeable genotypes, 125 (75%; 95% ci = 68.7 -81.9, estimated odds = 3.05) were infected with a single genotype, and only 7 patients (4%) were infected with genotype 1, either alone (n=4) or in combination with 3a. conclusions: one out of every 5 people tested in our flc is seropositive for hcv, and 14% of "healthy" voluntary blood donors have the same results. genotype 1 is very rare in our region. s.a. batool 1 , s.z. abbas 1 1 department of gastroenterology, muhammad hospital, mirpurkhas, pakistan background: hepatitis c viraus (hcv) infection is common in our region. data is not available on success rates of conventional interferon (inf) based products here. we attempted to find out the dominant genotype, and to determine the success rate of conventional inf-based treatment in eradicating hcv. methods: retrospective case series study of hcv infected patients' records treated with 14 different brands of inf. results: 320/1858 (17%) of all patients tested were positive for hcv antibodies. hcv-rna was tested by pcr for 1022 patients, of which 904 (88%) turned out to be positive. genotype type 3 was the dominant genotype -found in 118/168 (70%) patients. 101 men and 57 women were treated with various brands of inf with the same manufacturer's brand of ribavirin. the overall etr achieved was 100/142 (70%) -58/85 (68%) men and 42/57 (74%) women. 41/62 (66%) of genotype 3 achieved etr. there was no significant difference in average ages for those who achieved good etr and those who did not (39 years each). the etr achieved by different brands ranged from 48% to 91%. svr was achieved by 17/30 patients. conclusions: 17% of all people tested positive for hcv antibodies, of which about 88% had evidence of active hcv infection. etr achieved by different brands averaged 70%. this was 74% in female sex, although age did not appear to be a factor in determining a favourable etr. patients & methods: a total of 439 consecutive diabetic patients of either sex were evaluated for hcv and hbv infection by using enzyme linked immunosorbant assay (eliza-3) along with serum alt levels. on the basis of this test, the patients were divided into two groups, sero +ve and sero -ve. different variables were: age, sex, bmi, area of residence (rural or urban), type and duration of dm, smoking, literacy and alt. results: males 50.3% and females 49.7%. age ranged from 18 to 95. majority were married (98.4%), from rural area (70.4%), had type-2 dm (99.8%), normal weight (39.2%), normal alt(60.1%) and non-smokers (78.6%). seroprevalence for hcv, hbv and both were 25.1%, 1.8% and 1.6%. two groups were made, sero +ve and sero -ve. raised alt (59.2%) was significant (p<0.05) factor while all others variables were insignificant (p>0.05). conclusion: hbv and hcv infections are more prevalent in dm with increased alt levels. while hcv infection is more common than hbv in patients with dm. hepatitis c virus (hcv) envelope proteins (e1 and e2) mediate the entry of virus into host cells by binding to its cellular receptors and resulting in the fusion of the viral membrane with host cell membrane. the expression and secretion of biologically active envelope proteins in vitro have proven to be a difficult task due to the high degree of glycosylation and the existence of hydrophobic domains within these sequences. in order to obtain glycosylated, correctly-folded hcv envelop proteins in large quantities, we optimized the dna sequences of hcv envelop proteins by substituting the encoded sequence with human preferable codons and expressed them in human embryonic kidney (hek) 293 cells. both proteins were detected intracellularly, with a small portion secreted into supernatant. in order to enhance secretion, truncated forms of envelop proteins including e2 tm, e2384-661, e2484-661 were also expressed. both full-length and truncated forms of envelop proteins were glycosylated and expressed at high level. in addition, we also expressed the codon-optimized hcv receptors cd81 and claudin-1 in 293 cells. by comparing the expression level of codon-optimized sequences and the sequences that were obtained from cdna library by pcr, we found that codon-optimization enhance protein expression significantly in 293 cells. these results not only lay solid foundation for further research concerning the mechanism of hcv entry, including the optimal ph and right protein conformation for fusion, cell types that permit viral entry; but also potentiate a useful cell model for testing antiviral agents. background: prolactin (prl) is an immunoregulatory hormone secreted from lymphocytes, however, prl induction in relation to hepatitis c virus (hcv) infection has not been elucidated. methods: serum prl levels were measured in both 232 subjects of our hcv cohort study and 31 male patients of the hospital, who were chronically infected with hcv. furthermore, serum prl levels were compared in 27 male patients before and after interferon therapy. we measured expression of prl mrna level in pbmcs in 12 male patients, and also investigated prl mrna of pbmcs collected from 5 healthy men that stimulated by hcv produced by huh7.5 cells in vitro. result: serum prl levels were significantly higher in the hcv-infected subjects than in the controls (p< 0.01). they were significantly higher in hcv-infected male subjects than in the controls (p< 0.001). serum prl levels were significantly higher in male patients than in the controls (p<0.01). serum prl levels decreased significantly after interferon therapy in patients with sustained virological response to therapy (p<0.05). the levels of prl mrna in pbmcs derived from hcv-infected patients were significantly higher in 12 male patients than in the controls (p<0.001). conclusion: the high levels of prl expression are associated with hcv infection in carriers. background and objectives: hepatitis c virus (hcv) is a major cause of chronic liver hepatitis, cirrhosis, and hepatocellular carcinoma.current clinic standard therapy is interferon alpha (ifn-) combination with ribavirin, but this treatment is associated with adverse effects and often fails to induce a sustained response. until recently, development of a hcv cell culture system (hcvcc) provides a suitable tissue culture system to study the complete hcv life cycle. in this study, we tested the effect of ifn omega (ifn-)-a member of type interferon on hcv compared with ifnbased on hcv 1b replicon and hcvcc. methods: we compared ifn-and ifn-2a effects on hcv rna replication and protein expression, as measured by ribozyme protection assay and western blot. we also compared the intracellular protein level of phosphorylated signal transducer and activator of transcription 1 (p-stat1) treated with different interferon type and concentration with western blot analysis. results: hcv rna and protein level were inversely related with ifnconcentration and compared with ifn-2a, at the same concentration, the hcv rna and protein levels treated with ifn-were lower than that treated with ifn-2a p 0.05 .also based on the hcv rna analysis, ec50 of ifn-was 10 folds lower than ifn-2a. ifns increased intracellular p-stat1 level at a dose dependent manner and compared the same concentration of ifn-and ifn-2a, p-stat1 protein level was higher in ifn-treated group p 0.05 . conclusions: these results demonstrate distinct antiviral effect of ifncompared with ifn-2a and this difference maybe partly caused by the stronger stimulation of ifn receptor . outstanding antiviral activity of ifnmay be useful for developing new hcv treatment strategies. background & aim: hepatitis b virus (hbv) infection with undetectable levels of hepatitis b surface antigen (hbsag) is called an occult infection, which although has been described among subjects with chronic hepatitis c liver disease in the western world, it's prevalence and clinical significance are still ambiguous in the indian subcontinent. materials and methods: we investigated hbv-dna pcr in serum samples of 260 hbsag negative subjects with chronic hcv-related liver disease, and 70 apparently healthy volunteers negative for hbsag and anti-hcv as control. results: serum samples found positive by at least two independent pcr assays were considered hbv dna positive. hbv-dna was detected among 19 hcv-related chronic liver disease (cld) patients (7.3%), which was higher (p = 0.2) as compared with the control volunteers (4.3%). it was more frequent (37.5%) in 24 anti-hbs negative/anti-hbc positive patients than in 180 anti-hbs/anti-hbc positive (5 %, p < 0.05). hcv rna by qualitative pcr was significantly (p <0.001) higher in occult hbv compare to non-occult. hcv genotype 1b was predominantly associated with occult hbv (73%), especially among subjects with hepatocellular carcinoma (hcc) (p<0.05) as compared to non-occult hbv cases. though not significant, frequency of occult hbv infection was higher than healthy controls and hcv 1b genotype was significantly associated in patients with hcc. conclusion: this study suggests that in all hbv-endemic areas, the possibility of occult hbv in patients with hcv should be considered and hbv-dna should be performed. j. zhao 1,2 , w.d. cai 2 , l. chen 2 , y.x. gan 2 , m.l. he 1 , x.r. wang 1 1 background: besides hiv and syphilis, hepatitis c virus (hcv) is also rapidly spread among men who have sex with men (msm). this study was designed to identify the prevalence of these 3 sexual transmitted diseases in msms in shenzhen, china. methods: a cross sectional study was conducted by using time location sampling method from april to july, 2008. 831 msm participants (including 454 male sex workers) were recruited and finished guided self-administered questionnaires (or interviews if they have difficulty in reading or understanding) in 37 venue-date-time randomly selected from 48 active venues. results: results were analyzed using spss. 736 blood samples were collected for hiv, syphilis and hcv test. participated msms were between the age of 18 to 51 years (25.5±5.9) with a majority of 20-29 years (73.7%). most of them finished junior high school education (74.9%). 84.2% had high level of knowledge on modes of transmission and prevention. likewise, 56.0% msms have ever sold sex to men, 38.3% of them were self identified as gay, 35.1% as bisexual. 78.3% msms had multiple male sexual partners and 50.3% msms always used condom. 48.6% of them had sex with women in the past 6 month, and the condom use rate decline to 35.1% during both male and female sex. hiv positive rate is of 6.7% and syphilis for 18.3%, hcv is only found in 3 cases (0.4%). conclusions: a greater number of the participants have both male and female sex partners. this survey shows that hcv infection rate is still low among msms in shenzhen, although the hiv and syphilis rate is high and continuing increased in the past few years. the change of insulin sensitivity in hepatitis c patients with normal insulin sensitivity s.g. park 1 , y.k. cho 1 , j.w. lee 1 , j.w. yun 1 , h.j. kim 1 , w.k. jeon 1 , b.i. background: hepatitis c virus (hcv) infection is associated with a high prevalence of diabetes mellitus (dm). insulin resistance (ir) is known to play a crucial role in the development of dm in chronic hepatitis c (chc) patients. we prospectively investigated the change of insulin sensitivity in chc patients during 5-year period, and analyzed factors significantly associated with ir. methods: subjects consisted of 62 non-cirrhotic chc patients with normal alanine aminotransferase (alt) and normal insulin sensitivity (chc group), and healthy control group of 172 subjects matched by age, sex, body mass index and life styles. we compared initial baseline insulin sensitivity, metabolic parameters and incidence rate of ir at the end of follow up period in both groups. the change of insulin sensitivity and metabolic parameters and development of ir was analyzed, and factors associated with development of ir were evaluated. results: ir developed in 22.5% of 62 chc patients and 5.2% of 172 normal individuals (p<0.001). hcv infection per se and genotype 1 were independent risk factors of ir. initial fasting glucose 90-100 mg/dl, fasting insulin 10 uiu/ml, homa-ir 2.3-2.7 were significantly associated with development of ir in chc group. conclusions: hcv infection is independent risk factor of ir. even if chc patients with normal insulin sensitivity, careful monitoring for ir is necessary. prevalence of viral hepatitis c in latvia i. tolmane 1,2 , b. rozentale 1,2 , j. keiss 1 , f. arsa 1 1 sa infectology center of latvia, 2 riga stradin's university background and aim: viral hepatitis c (vhc) because of its prevalence and clinical course has become one of the most actual infectious diseases in the world. to date chronic hepatitis c affects over 170 million individuals worldwide. chronic vhc is a leading cause of cirrhosis and hepatocellular carcinoma. the aim of this study was to investigate how many residents of latvia, that are over 18 years of age have been exposed to vhc (anti-hcv prevalence) and how many are infected at the moment (hcv-rna prevalence). until now such research has not been performed in latvia. methods: from the register of general practitioners there were randomly selected 26 gp's from different regions of latvia, 60 persons over 18 years of age were selected out of each gp register and tested for anti-hcv with screening test (elisa). in case of positive result antibodies were confirmed with western-blot reaction and person was tested for hcv-rna (pcr). results: in total 1591 person was invited by general practitioners for the test and 1442 persons responded (response rate 90.6%). confirming test (western-blot) was positive in 34 participants and out of which hcv rna test was positive in 25 patients. conclusions: there are 2.4% of people exposed to hepatitis c virus in latvia and 1.73% are infected with hepatitis c virus, respectively, 1734 infected persons per 100 thousand individuals. genetic variation in the ikk/nf-b pathway and the live fibrosis progression in chronic hepatitis c r. sho 1 , k. ishii 1 , r. ishii 2 , h. watanabe 2 , k. sugahara 2 , y. nishise 2 , k. okumoto 2 , t. saito 2 , s. kawata 2 , a. fukao 1 1 department of public health, 2 department of gastroenterology, yamagata university faculty of medicine background/aims: i b kinase/nf-b (ikk/nf-b) signaling pathway is thought to play critical roles in liver inflammation and fibrogenesis. we carried out a haplotype-based association study to examine the contribution of common genetic variations in the genes encoding nf b inhibitor kinase alpha and beta (ikbka and ikbkb; the major components of ikk/nf-b pathway) to the progression of live fibrosis in chronic hepatitis c. methods: based upon the common single nucleotide polymorphisms (snps; minor allele frequency(maf) 0.05) and linkage disequilibrium (ld) information derived from the hapmap, we selected 5 and 3 tag snps from ikbka, and ikbkb, respectively, for genotyping. by using melting curve analysis, snps were genotyped in 217 chronic hepatitis c patients, including 80 patients with hepatocellular carcinoma. association between common genetic variations in ikbka/ikbka and platelet count (plt) was tested by both genotype-and haplotype-based approaches. results: we succeeded in genotyping a total of 8 tag snps that efficiently capture common variation across the 32 kb-block of ikbka and the 25 kb-block of ikbkb. for each of genes tested, 5 haplotypes were found in population studied. all snps were in hardy-weinberg equilibrium, but no significant association was observed between any single tag snp or haplotype and decreased plt in patients analyzed. conclusions: our data suggest that it is unlikely that polymorphisms within the ikbka and ikbkb genes are involved in the progression of live fibrosis in chronic hepatitis c. further studies on genetic variations in other nf-b-related genes in chronic hepatitis c are needed. background: hepatitis c virus infection is a major burden after liver transplantation. the effective treatment for patients who underwent liver transplantation has not been well established. management of these patients is the most challenging task. cyclophilins are essential host factors for hcv replication. we report here the efficacy of divided administration of ifn plus cyclosporine a in the treatment of chronic hepatitis c patients who failed peg-ifn or ifn combined ribavirin. patients and method: we prospectively included 59 patients (median age, 63) with genotype 1b and, failures to combination ifn plus ribavirin or combination pegylated ifn plus ribavirin. the present treatments consisted of an induction therapy, an intensified therapy and a maintenance therapy. the induction therapy comprised intravenous 1 mu ifn every 4 hours for the first 3 days, 1.5 mu ifn every 6 hours for the next 4 days and 2 mu ifn every 8 hours for the following 3 weeks, totaling 168 mu of ifn . the intensified therapy was induction therapy shortened to 2 weeks. the maintenance therapy comprised of pegylated ifn 2b and ribavirin. csa was given 4 times daily during the induction and the intensified therapies. ribavirin was given twice daily during the maintenance therapy. results: the end treatment response and sustained virological response rate of the present study were 73 % (43/59) and 59% (35/59), respectively. the relapse rate was 19 %(8/43). non-responders was 16 % (3/19). all adverse effects were completely reversible. the treatment protocol was well tolerable. conclusion: we concluded that our protocol should be effective in failures to the previous combination therapies. host factor targeting treatment will become a promising treatment option. cyclophilin targeting treatment is a promising new anti-hcv treatment k. inoue 1 , t. watanabe 1 , s. yoshiba 1 1 background: hepatitis c virus (hcv) is the most common cause of chronic liver disease. however, the efficacy of currently available treatments is limited. we recently reported the effects of combined interferon-/cyclosporin a treatment. cyclophilins are associated with hcv replication and bind cyclosporin a. which cyclophilins are closely associated with hcv replication remains controversial. in this study, several cyclophilins were found to be essential host factors for hcv replication and hcv replication was rescued by overexpression of cyclophilin a in the presence of cyclosporin a. methods: we evaluated the effect of cyclosporin a and its analogues on the replication of hcv in vitro using several types of hcv replicon. the gene expression of representative cyclophilins and pin-1 was knocked down using small interfering rna2 (sirna) to identify cyclophilins associated with hcv replication. the specificity of the effect of sirna was confirmed by western blot analysis. the effect of overexpression of cyclophilins on hcv replication in the presence of cycloporin a was also studied. results: cyclosporin a and its analogues suppressed hcv replication in a dose dependent manner. cyclophilin f, cyclophilin lc1 and cyclophilin lc2 as host factors which are closely associated with hcv replication, in addition to the previously reported cyclophilin a. knockdown of chclophilin b showed little effect on hcv rna replication. cyclophiln-dependent hcv replication varied among the three hcv replicon cell-lines used. overexpression of cyclophilin a rescued hcv replication in the presence of cyclosporin a. conclusions: these findings suggest several cyclophilins are essential host factors for hcv rna replication. thus potent cyclophilin inhibitors have the potential to be anti-hcv drugs. background/aims: hepatitis c virus (hcv) genotypes 1-6 have a worldwide distribution. types 1a and 1b are predominant in northern europe and north america, and in southern and eastern europe and japan, respectively. type 3 is endemic in south asia and is variably distributed in different countries. genotype 4 in egypt, genotype 5 in central and south america and genotype 6 is common in china, japan and south east asia. in pakistan 3a is the commonest genotype, which is associated with the most favorable outcome regarding end treatment response and sustained virological response after 24 weeks of therapy. the aim of this study is to find out hcv genotypes in newly diagnosed chronic hepatitis c patients. methods: this observational study was conducted in chronic hepatitis c patients. all patients had raised alt levels for last 06 months, had positive polymerase chain reaction (pcr) for hcv rna by real time method and liver biopsy was done in all patients under national program for prevention and control of hepatitis during year 2006 -2007. genotyping was done on roche genotyping kit. data was analyzed by spss 13.0 results: out of 164 patients, 85.9% (n=141) were genotype 3a. 6.1% (n=10) were genotype 3b. 3.0% (n=5) were genotype 1a. n= 01 had genotype 1b. 4.2% (n= 7) had mixed genotype (3a,3b/1a,1b,3a,3b). conclusion: majority (85.9%) of chronic hepatitis c patients were genotype 3a which is associated with favorable outcome after 24 weeks of interferon and ribavirin therapy and only 3.0% had genotype 1a in this cohort. s.t. zhou 1 , y. zhao 1 , f.j. zhang 1 background/aims: as human immunodeficiency virus (hiv) infected children who are receiving antiretroviral therapy (art) are living longer in china, comorbidities of hepatitis b virus (hbv) and hepatitis c virus (hcv) coinfection should be carefully considered when making management decisions. however, the coinfection rate of either hbv or hcv is unknown in hiv-infected children in china. we evaluated the seroprevalence of hbv and hcv in the china national pediatric art cohort of hiv-infected patients. methods: patients were selected from hiv infected children medically eligible for art who were enrolled into the china national pediatric art cohort since 2004. interviews, medical assessment, serology for hbsag, anti-hcv antibody, transaminase levels, and hiv serostatus and cd4 counts at baseline of patients were obtained. results: 42 of 763 hiv-infected children were hbsag seropositive (5.50%; 95%ci: 3.88%-7.12%), and 69 of 662 children were anti-hcv antibody seropositive (10.42%; 95%ci: 8.09%-12.75%). only age was associated with hbv coinfection. multivariate analysis revealed that children infected with hiv through contaminated blood or transfusion of blood products were 6.35 times more likely to be anti-hcv antibody positive than those infected with hiv through other routes. and children from central china provinces, henan, anhui, shanxi, and hubei were 2.5 times more likely to be hcv seropositive. conclusion: the high seroprevalence of hbv and hcv coinfection in hiv-infected children attending china national pediatric art cohort calls for routine screening for hepatitis viral coinfection and modification of the management of hiv-infected children in china. background: bms-790052 is a first-in class and highly selective hepatitis c virus (hcv) ns5a inhibitor with picomolar in vitro potency against genotypes 1a and 1b. in a sad study with healthy subjects, bms-790052 was safe, well-tolerated, and had a pharmacokinetic profile suggestive of once-daily dosing. methods: the objectives of this randomized, double blind, placebo-controlled, sad study were to evaluate the safety, tolerability, antiviral effect and pharmacokinetics of bms-790052 in patients with genotype 1 chronic hepatitis c (chc). treatment naïve or experienced patients were randomized to receive 1, 10, or 100 mg of bms-790052 or placebo. results: all bms-790052 single doses were well tolerated and had a safety profile similar to that of placebo. following oral administration, bms-790052 was readily absorbed with dose proportional exposures over the studied dose range. the mean terminal half-life of bms-790052 was approximately 12 hours. mean decline in hcv rna 24 hours after a single 1, 10 and 100 mg dose of bms-790052 was 1.8 log10 (range 0.18 to 3.0 log10), 3.2 log10 (range 2.9 to 4.0 log10) and 3.3 log10 (range 2.7 to 3.6 log10), respectively. the 100 mg dose resulted in a mean decline of 3.6 log10 (range 3.0 to 4.1 log10) 48 hours after dosing, which was maintained at 144 hours. conclusions: single doses of up to 100 mg of bms-790052 were safe and well tolerated in patients chronically infected with hcv genotype 1. bms-790052 produced a robust decline in hcv rna and has a pharmacokinetic profile that potentially supports once-daily dosing. background: the global infection rate of hcv is approximately 3%, and nearly 3.2% in china. only 42%-46% of patients with genotype 1b can achieve sustained virological response (svr) after antivirus therapy, nearly half of them experienced treatment failure. the study aimed to determine hcv-1b sequence evolution in patients experienced treatment failure during and after therapy, and further analyze relations between the mutations and treatment outcome. methods: 19 patients with genotype 1b accepted antiviral treatment of ifn plus ribavirin for 48 weeks, and long-term follow-up after therapy. 2 patients experienced treatment failure were further analyzed (one for relapser, another for nonresponder). sera were reserved at baseline, 12w, 48w and 4-year after therapy. hcv-rna was extracted. hcv full-length orf was amplified by rt-nested-pcr and sequencing. result: 9 of the 19 patients achieved svr (47.4%). from sequence alignments of relapser at baseline and 48w, we find that p7, ns5a and ns4a have higher mutation rate both in nucleotide and amino acid level (7.41% and 6.35%, 4.08% and 5.63%, 4.32% and 5.56%, respectively). but there is no significant difference in the alignments of 48w and 4-year after therapy, the mutation rate is lower. mutation rates of the non-responder among baseline, 12w, 48w and 4-year after therapy are very low. conclusion: antivirus effect is correlated with specific hcv sequences in chronic hepatitis c, mutations in hcv non-structure protein p7, ns5a and ns4a have important impacts on treatment outcome in ifn-based therapy. background: the results of antiviral therapy for hepatitis c (hcv) have improved recently with the use of peg-interferon (peg-ifn)/ribavirin therapy. however, age of patients are concerned because of side effects and safety. as we known, a few studies have targeted therapy in elder with chronic hcv. aim: we reviewed the results of interferon based antiviral therapy in the elderly with chronic hcv at our institution. methods: patients were defined as elderly if they were 65 years and elder who received therapy for hcv. the prescribed treatment duration, end of treatment response were mention. the data recorded included laboratory tests, adverse events (ae), dose modification, and withdrawal rate of therapy. results: 304 of chronic hcv patients treated with peg-ifn/ribavirin between nov 2004 and feb 2008. 50 patients were older than 65 years old. the mean age of the elder patients was 70.3 ± 4.8 years old. 21 were male and 29 were female. histological studies showed 18 with cirrhosis. almost all patients had experienced ae/side effects. the most common abnormalities were anemia and neutropenia. therapy was discontinued in 22% (11/50). the rate of dose modification was 46% (18/39) patients who received 24 weeks therapy. transaminases were normalized in 66% (33/50) after 24 weeks treatment and sustained in 66% (29/44) one year later. conclusion: the elder patients are more at risk of developing ae while on treatment. most patients should be discontinued or decreased dosage of medication. however, the elder patients with chronic hcv can be treated successfully. background: in the general population the incidence of interstitial lung disease is estimated to be 0.03% and has also been reported with the use of interferons. the higher reporting rate of ip in japan has created interest and warrants further investigation. methods: using both data from 12 randomized clinical trials (ex-japan) and the roche world-wide safety database (advent), the frequency of ip was estimated in patients treated with peginterferon alfa-2a ± ribavirin. ip was defined as: interstitial lung disease, alveolitis, pulmonary fibrosis, pneumonitis and pulmonary toxicity. results: one case of ip was reported among the 6180 patients included in the clinical trials (0.02%). in the advent database considering the estimated 926,000 patients with cumulative exposure to peginterferon alfa-2a (42,600 in japan and 883,400 us/row) the 228 reported cases of ip represent a rate of 0.02% with a proportional reporting ratio (prr) of 1.7 (p<0.0001). of these cases, 140 were reported in japan (prr 2.9; p<0.0001), 34 in the usa (prr 0.7; p=0.06) and 54 row (prr 1.2; p=0.11) representing reporting rates of 0.3% in japan and 0.01% in the usa and row. japanese patients with reported ip were older (66 versus 50-55 years) and were more likely to have been treated with peginterferon alfa-2a monotherapy (81% versus 21-39%). furthermore, the yearly incidence rate has remained unchanged. conclusions: the apparently higher rate of ip reported in japan may result from differences in patient demography, diagnostic criteria and treatment patterns. the overall incidence of ip remains low. background: hepatitis c virus (hcv) infection carries a significant risk for development of insulin resistance (ir) and/or diabetes (dm). recently, retinol-binding protein 4 (rbp4) has been reported as a protein contributing to ir. this study aimed to assess the different expression of serum rbp4 between chronic hcv infection (chc) patients and non-chc controls. methods: serum rbp4 was measured in 105 treatment-naïve chc patients and its correlation with the homeostasis model assessment of insulin resistance index (homa-ir), liver histology, virology and metabolic factors was investigated. patients were stratified into different stages of glucose tolerance by oral glucose tolerance test. another 100 sex-and age-matched non-chc adults served as the controls. results: the mean rbp4 level of controls tended to be higher than that of chc patients (32.46 ± 20.92 vs 25.48 ± 13.13 g/ml, p=0.07). the mean rbp4 level of 34 igt control-group subjects was 43.7 ± 24.0 g/ml, which was significantly higher than that of 34 ngt (24.4 ± 13.0 g/ml, p<0.001) and 32 dm controls (29.0 ± 19.5 g/ml, p<0.01). in contrast, the mean rbp4 level (22.2 ± 10.3 g/ml) of 32 dm/chc patients was not significantly different from that of ngt/chc (24.9 ± 10.5 g/ml, n=28) and igt /chc (28.1 ± 15.8 g/ml, n=45) patients. amongst chc patients, there was a significant decreasing linear trend of rbp4 dependent of both histological grading and staging progression, whilst a significant increment of homa-ir was found. conclusion: serum rbp4 is dysregulated in chc patients. introduction: sustained viral response (svr) in hepatitis c treatment with interferon alfa and ribavirin is affected by adherence and compliance due to severe myalgia, fatigue-anxiety and disturbed sleep. pregabalin, an orally effective gabasergic drug is not metabolized via cytochrome p450 and is used in fibromyalgia and fatigue-anxiety syndromes without hepatic toxicity.this study evaluates the addition of pregablin to standard agents in achieving svr by reducing side events. methods: thirty patients with chronic hepatitis c {mean age -46 years, male: female -2:1,genotype(g)1(n=29), g6 (n=1), fibrotic score f2-3 (n=24) and f4 (n=6), mean bmi > 29 kg/m 2 , initial viral load > 800,000 iu/ml} were randomized to pregablin100mg (n=15) or duloxetine 20mg (n=15) both orally daily with interferon alfa 2a 180mcg sq once a week and ribavirin 1200mg daily for 48 weeks. myalgia anxiety scale, modified quality of life score -evaluated at entry and tri-monthly. all were tested for rapid viral response, early viral response and end treatment viral response and svr. results: at the end of 48 weeks, in the pregablin arm, 14(97.3%) completed the therapy without interruption, one stopped due to excessive somnolence. duloxetine arm -10(66.6%) completed with interruptions, 4(22.2%) withdrew from the trial due to side events, one left the country. 9(62.2%) achieved svr in pregablin arm and 5(33.3%) with duloxetine. conclusions: pregablin may be considered with ifn and rbv for better adherence and compliance in achieving svr in treatment of chronic hepatitis c. larger randomized studies are needed to confirm the findings. in this study we extended this treatment approach to on treatment nonresponders (defined as having detectable hcv-rna after at least 24 weeks of soc). methods: so far, 5 pts. hcv-rna pos. after 24 weeks of soc (3 male, 2 female, genotype 1:4; genotype 3a:1, 3 with cirrhosis) participated in this protocol; 4 were treatment naïve pts, 1 relapser to two previous therapies (24 and 48 weeks). 20 mg/kg/d sil was given for 14 days, soc was continued. hcv-rna was quantified by taqman (roche diagnostics, usa) at monthly intervals on standard treatment and weekly after starting sil. results: all patients received at least 24 weeks of soc, at week 24 3 had a log drop < 3, two patients had detectable but unquantifiable hcv-rna (< 15 iu/ml). after 14 days of sil all 5 had undetectable hcv-rna, in one hcv-rna increased to 100 iu/ml and recived after a second course of sil. .all 5 patients are still on soc and are hcv-rna negative. conclusion: sil iv. is an effective "rescue treatment" for on treatment nonresponders to full dose of peginterferon/ribavirin combination therapy. poster exhibition -imaging modalities poster session, hall 5b background: levovist-enhanced ultrasonography using subtractions makes it possible to depict the perfusion of hyperechogenic nodules. our institution performs sonazoid-enhanced ultrasonography using a toshiba aplio80 that is set to a ps low images, as generally recommended. the resulting images, however, are difficult to evaluate the kind of staining image that is obtained from a hyperechogenic nodule. these staining images were then compared to advanced dynamic flow (adf) images of a hyperechogenic nodule recorded using levovist-enhanced ultrasonography. methods: the subjects were five nodules who had undergone sonazoid-enhanced ultrasonography. two patients had experienced a recurrence of hcc after tace, while three patients had a hyperechogenic nodule of hcc that had never been treated. one patient with hcc after tace was imaged at a ps low. the second patient with hcc after tace and the three patients with hcc showing a high echoic nodule, were imaged using adf. results: in the patients with hcc after tace, the remaining tumor was difficult to observe in both the vascular phase and the kupffer phase taken at a ps low. in the other patients, however, images taken using adf clearly showed the residual tumor. also, with regard to the findings from the perfused images obtained from the three patients with hyperechogenic nodules of hcc, the hcc was more easily detectable in the adf images than in those taken at a ps low. conclusion: hyperechogenic perfused nodules are easier to identify in images taken using adf than in images taken using ps low. y. komorizono 1 , t. shibatou 1 , k. sako 1 1 nanpuh hospital background: this study aimed to evaluate the usefulness of sonazoid enhanced radiofrequency ablation under real-time virtual sonography (rvs) guidance in a series of patients with hepatocellular carcinoma (hcc). method: twenty-five patients with a solitary hcc tumor measuring < = 2.5 cm in greatest dimension were enrolled in this study. eight patients received an initial treatment, seven also received an additional treatment for local recurrent tumors, and the remaining ten had distant recurrent tumors. all patients were easy to scan by multiple detector ct (mdct), but not by conventional ultrasound ( conclusions: the combination of the rvs system with sonazoid-enhanced us appears to have a high potential for use on patients that are difficult-to-scan by us examinations for percutaneous radiofrequency ablation. background & aims: contrast enhanced ultrasonography (ceus) with sonazoid can be expected to be useful not only for detection of tumor but also for us guided ablation therapy because kupffer imaging lasts for long time. the aim of this study is to investigate the usefulness of sonazoid in rfa for hcc. material & methods: a total of 716 hcc nodules in 316 patients admitted to receive rfa were studied. the detection ability of hcc was compared between ceus and conventional us using dynamic ct as reference standard. the effectiveness in the treatment was assessed by comparing the mean numbers of treatment session of rfa in patient treated with ceus assistance and that in historical controls matched for tumor and background conditions. results: the detection rate was 83.5% in conventional us and 93.2% in ceus (p=0.04). sixty-nine nodules in 52 patients were not detected by conventional us and detected after injection of sonazoid. the mean increase in detected tumor number with contrast enhanced us were well correlated with serum albumin level (p=0.016). ceus was not superior to conventional us in patients with low albumin level. the mean number of session was 1.33±0.45 as compared to 1.49±0.76 in the historical controls (p=0.0019). conclusions: ceus with sonazoid is useful for detection of tumor in patients with well-conserved hepatic reservoir. the decrease in the mean number of sessions compared to historical controls suggested that sonazoid is an excellent supportive agent in rfa treatment of hcc. direct measurement of peri-operative change in portal blood flow and pressure is difficult in human. in the present study, computational simulation of pre-and post-operative portal blood flow and pressure was performed using computational flow dynamic (cfd) software in patients with primary liver cancers. methods: patients with fibrotic or non-fibrotic livers were analyzed. according to preoperative md-ct, mesh models of portal branches were constructed. cfd software (fluent 6.2, fluent inc.) was employed for flow simulation. on the fluent 6.2, changes in flow dynamics in the remnant portal branches were simulated by virtual cutting of an interested portal branch. the simulation was also performed 14 days after the operation using dicom data obtained at that time. results: relative increase in blood flow in each remnant portal branch was not uniform throughout the liver in each patient. the sudden increase in portal pressure just after the virtual cutting of interested portal branch was almost normalized by day 14 in non-fibrotic liver according to the flow simulation, while the increase in fibrotic liver did not return to the pre-operative values by day 14. these results suggest that responsive dilatation of remnant portal branches and subsequent regional regeneration could normalize the sudden increase in portal pressure after surgery in non-fibrotic livers, while the mechanism is impaired in fibrotic livers. discussion: computational flow dynamic simulation is useful to analyze the differences in the peri-operative portal flow dynamics and liver regeneration between non-fibrotic and fibrotic livers. aim: to determine if roi analysis can characterize washout in hepatocellular carcinoma (hcc) better than visual analysis. methods: surgically proven hccs from a single institution were studied. the patients' gender, age, date of scan, date of surgery were recorded. 94 patients with pre-operative triphasic (n=67) and quadriphasic ct scans (n=27) were included. a representative section containing the lesion was selected for each case. the hu change between the precontrast and arterial (huabsolute hypervascularity) and the hu change between the peak attenuation and late portovenous phases (huabsolute washout) were recorded. cases were deemed positive if the hu change was more than the standard deviation (11 hu). this was compared against visual analysis to determine if our method would increase sensitivity of ct for hcc. results: the mean patient age was 63.7 years (range 19 to 84 years); there were 77 males and 17 females. the mean duration between surgery and the scan was 39.5 days (range 1 to 348 days). peak enhancement was seen in the early portal venous phase in 76.6% cases. the mean huabsolute washout was 23.8 hu (range -5 -54). roi analysis detected 86/94 cases (91.5%). this was 12.8% more than visual assessment, which detected 74/94 cases. this was statistically significant (p=0.014). conclusion: visual assessment of lesion density is subjective. quantitative measurement of lesion attenuation changes between scan phases is a simple and objective method that is more sensitive than visual assessment in determining lesion washout. background: abdominal ultrasonogram(usg) is a common available diagnostic tool to screen and follow up for hepatocellular carcinoma(hcc). but it has been reported that the specificity of ultrasonogram is high but the sensitivity of it is insufficient. we investigated the characteristics of hccs that was missed in the usg but was detected in the ct. methods: total 122 patients who were diagnosed with hcc between december, 2003 and february, 2008 , were enrolled and analysed retrospectively. all patients were performed with a usg prior to a spiral ct. the period between usg and spiral ct was limited within 1 month. we investigated age, gender, cause(hbv, hcv, alcohol), the size of hcc(the length of long diameter), stage(modified uicc), child-pugh grade, cirrhosis, tumor number, portal vein thrombosis, diffuse type of hcc, regenerative nodules(rns), and the tumor location at segement 8 as the possible related factors. results: the mean period between usg and spiral ct was 3.04±5.70 days. the diagnostic accuracy rate to hcc was 84.4%(103/122). there was no interobserver variation. in analysis of associated factors, there was no statistical significance in age, gender, cause(hbv, hcv, alcohol), stage(modified uicc), child-pugh grade, cirrhosis, portal vein thrombosis, diffuse type of hcc, regenerative nodules(rns) (p > 0.05). there was statistical correlation in the tumor size less than 2 cm, the solitary tumor and location at segement 8. (p > 0.05). conclusion: tumor size less than 2 cm, solitary lesion and location at segment 8 are significant factors to miss hccs in usg diagnosis. s. somani 1 , a. somani 2 , a. jain 3 , v. dixit 3 1 suvidha, 2 navjeevan hospital, 3 background: histopathological examination is required in the evaluation of various liver diseases for both diagnosis and prognosis. earlier blinded percutaneous liver biopsy was done commonly but now there are various studies suggesting that sonographic guided percutaneous liver biopsy could be more precise and safer. our aim was to compare the safety and diagnostic utility of sonographic guided versus blind percutaneous liver biopsy. methods: it was a retrospective single center study done between june 2003 and may 2007. trucut liver biopsy needle was used in all patients. demographic, clinical and histological characteristics between the two groups were evaluated. insufficient biopsy was defined as a sample with less than 6 portal spaces. we reviewed the type of complications and if hospitalization was required, or any mortality related to the procedure. results: out of 256 liver biopsies done in this period after excluding 16 patients we included 240 patients, 144 in group a(60%, blind approach) and 96 in group b (40%, sonographic guided approach). mean age was 38±12.4 years and male: female ratio was 1.6:1. biopsy was sufficient in 76% in group a and 94% in group b (p < 0.05). minor complications occurred in 58% in group a and 49% in group b which was not significant. major complications occurred in 2.8 % in group a and 1.2% in group b which was statistically significant. mortality was 1.2% in group a and 0.4% in group b which was statistically significant. conclusion: our study suggest that sonographic guided percutaneous liver biopsy is superior in the diagnosis of liver diseases in all aspects when compared to blind approach as it is more safe, has more diagnostic utility with significantly less complications and mortality. poster exhibition -liver fibrosis poster session, hall 5b background/aims: hmg-coa reductase inhibitors have been shown to reduce hepatic stellate cell proliferation and collagen production and decrease oxidative stress and hepatic vascular tone in cirrhotic patients. therfore, the aim of the present study was to examine whether the lipid lowering agents atorvastatin (ato) or rosuvastatin (ros) would prevent experimentally-induced acute or chronic hepatic damage in rats. methods: liver cirrhosis was induced by thioacetamide (taa, 200 mg/kg, i.p.) twice a week, for 12 weeks. acute damage was induced by two consecutive taa injections (200 mg/kg in a 24 h interval). rats were treated concurrently with taa only or taa and either ato or ros daily by nasogastric gavage. another group was treated with taa+pentoxifyline (ptx), an agent with known antifibrotic effect through a different mechanism and served as positive control. results: presented in the conclusions: the lipid lowering agents used in our study had no effect on the development of acute or chronic hepatic damage in rats or on oxidative stress induced by taa. purpose: the development of hepatic fibrosis in patients with chronic liver disease increases the risk of liver cancer. the present study was conducted to determine whether an easily performed myocardial examination technique can be applied to the assessment of hepatic fibrosis. strain rate imaging is a new method based on tissue doppler imaging (tdi). the usefulness of strain rate imaging in assessing the degree of hepatic fibrosis was evaluated. this time, it mede comparative study with fibroscan in 11 cases. methods: strain rate imaging was performed using a diagnostic ultrasound system (aplio tm , toshiba medical systems corporation, tochigi, japan) in a total of 47 subjects:25 in the chronic hepatitis group, 12 in the cirrhosis group, and 10 in the normal control group. tdi-q, the tissue doppler analysis software installed in the aplio system, was used for analysis. measurement was performed five times from the epigastrium, with the roi size set to 10 mm and the derivative pitch to 3 mm. results: (i): both scores were largely reproducible among the different laboratories. however, compared to the histological findings, the error ratio was 77% for all results calculated by fibrotest and actitest. (ii): calculated scores varied among f2 (9%), f3 (31%), f3-f4 (6%), and f4 (54%) (fibrotest), as well as a1/a2 (48%), a2 (9%), a2-a3 (5%), and a3 (38%) (actitest). results: the mean strain value was 0.156 in the chronic hepatitis group, 0.055 in the cirrhosis group, and 0.26 in the normal control group.the correlation was not thought to be fibroscan. conclusion: the results of the present study suggest that this noninvasive method permits quantitative assessment of the degree of hepatic fibrosis to be performed easily and in a short time. it is expected that the accuracy of the strain rate imaging method in determining the degree of hepatic fibrosis will be improved when it is used in combination with histological examination. conclusion: despite reproducibility of fibro-and actitest results among the six laboratories, large scale investigation (n=64) displayed increasing variability of the results depending on interlaboratory differences that were still in a quality controlled, analytically acceptable range. furthermore, calculated scores coincided with histological findings only in less than 25% of all cases. thus, the diagnostic accuracy of these tests seems low, if histology is accepted as gold standard. background: current knowledge attributes connective tissue growth factor (ctgf/ccn2) a crucial role in enhancing tgf-actions during hepatic fibrogenesis. recently, we demonstrated that caffeine leads to an upregulation of ppar in hepatocytes, thus sensitizing these cells to the well known inhibitory effect of 15-deoxy-12,14 -prostaglandin j2 (15-d-pgj2) on ctgf expression. however, upregulation of the receptor alone is not sufficient per se, its physiological ligand 15-d-pgj2 is required for exerting its inhibitory effect on ctgf synthesis. aim and methods: this study compares serum concentrations of 15-d-pgj2 in caucasian patients with fibrotic liver diseases (n=289), caucasian controls (n=136) and caucasian non-liver disease sick (n=307), as well as of chinese patients with hepatocellular carcinoma (n= 43) and chinese healthy controls (n=63) in order to characterize their suitability for therapeutic approaches with ppar inducing (i.e. ctgf inhibitory) drugs such as caffeine. results: presented data show that caucasian patients with ongoing hepatic fibrogenesis (mean 6.2 ± 5.9 µg/l) display impressingly higher serum concentrations of 15-d-pgj2 than healthy probands (mean 2.3 ± 1.0) and caucasian patients with non-liver disease (mean 2.7 ± 1.4 µg/l). similar results are found in chinese patients with fully developed hcc (mean 1.3 ± 0.7 µg/l) compared to chinese healthy controls (mean 0.4 ± 0.2 µg/l). we identified the predictors of tumor recurrence using cox-regression model. introduction: non-invasive, i.e. serum-based multiparametric panels of biomarkers have been proposed for the diagnostic assessment of liver fibrosis. aims/methods: (i) haptoglobin, alt, ggt, alpha 2-macroglobulin, apolipoprotein a1 and bilirubin in sera of 4 patients with histological proven fibrosis (f1-f4, a1-a3) were determined in 6 different quality-controlled laboratories. interlaboratory variations of the calculated fibrotest score for staging and actitest score for grading (both biopredictive tm ), and their error ratios compared to biopsy results were calculated. (ii) the variability of obtained fibrotest/actitest scores depending on 64 differential combinations of the allowed analyt-specific maximum/minimum permissible values as determined by the external quality control of the german association of laboratory medicine was determined and the frequency distribution of the results calculated. results: a total of 51 patients (mean age, 54.3±9.8 years; male, 78.4%) were included. median follow-up duration was 14.2 months (range, 5.6-37.3) and 20 patients (39.2%) experienced local tumor recurrence during the observational period. multivariable analyses showed that low p2/ms level (relative risk, 0.98; 95% confidence interval [ci], 0.96-0.99; p=0.035) and serum alpha-fetoprotein level >100 ng/ml (relative risk, 5.41; 95% ci, 1.59-18.18; p=0.007) were independent risk factors for tumor recurrence. patients with p2/ms level <45.0 revealed 3.79-fold (95% ci, 1.05-13.76; p=0.042) increase in the risk of recurrence after adjustment for serum alpha-fetoprotein level, as compared to those with p2/ms level >45.0. however, tumor size, child-pugh score, and hepatitis b virus dna level failed to significantly affect the time-to-recurrence. conclusion: our study suggests that lower p2/ms value, which means more severe liver fibrosis, is an independent predictor for hcc recurrence after rfa. background/aims: despite of its high prevalence, osteoporosis is an underestimated complication of liver cirrhosis. the aims of this study is to prove the prevalence of osteoporosis and osteopenia in patients with liver cirrhosis and to identify the principal risk factors associated. methods: the prevalence of osteoporosis and osteopenia was studied in patients with alcoholic or viral liver cirrhosis who were admitted to the institute of gastroenterology and hepatology, cnuh between march 2008 and september 2008. osteoporosis and osteopenia was evaluated by measuring their bone density using dual energy x-ray absorptiometry (dexa) at lumbar spine and femoral head. the variables taken into consideration were: sex, body mass index (bmi), presence of cholestasis, severity and duration of liver disease. results: total 45 patients (male 32 and female 13, respectively) were estimated for association of liver disease and osteoporosis. of these, 39 patients were estimated for bone density of lumbar spine and neck of femur by dual x-ray absorptiometry (dexa). morning blood samples were taken for hormonal and biochemical analysis from all patients. among 39 patients, 25 patients (64%) were found to have osteopenia or osteoporosis. there was no statistically significant correlation between age, bmi, severity and duration of liver disease, pth, vitamin d, alp and igf-1. conclusion: there is high prevalence rate of osteopenia or osteoporosis in liver cirrhosis. although the causes of osteopathy are heterogeneous, the early diagnosis and treatment of osteopathy in patients with liver cirrhosis is important. background: to build and to evaluate mathematical models for predicting liver fibrosis progression by using conventional laboratory indicators in chronic hepatitis b. methods: liver biopsy and routine laboratory tests were performed in 391 patients with chronic hepatitis b. using multiple logistic regression to analyze evidently relevant indicators, then the predicting models were built and analyzed by roc curve. results: after spearman analysis, factors such as age, platelet count(plt), international rate(inr), total bilirubin(tbil), albumin(alb), aspartate aminotransferase (ast), gamma glutamyltranspeptidase (ggt), total bile acid(tba) and cholinesterase(che) were found to be correlated with liver fibrosis p 0.01 . three models (s 2, s 3, s=4, respectively) were built by plt, inr, alb, ggt and che, which were independent predictors after multiple logistic regression analysis.finally, fibrosis score (fs) was calculated to predict different liver fibrosis stages. roc curve analysis revealed that the auc of fs was 0.784 in model1 (s 2), 0.768 in model2 (s 3) and 0.806 in model3(s=4) fig1 .the cut-off fs in model1 was at 7.09 with 67.4% sensitive, 79.3% specificity and the accuracy was 71.1%. the cut-off fs in model2 was at 5.67 with 75.0% sensitive, 67.7% specificity and the accuracy was 72.9%. the cut-off fs in model3 was at 3.65 with 71.4% sensitive, 78.5% specificity and the accuracy was 73.7%. conclusions: the predicting models, built by using conventional laboratory indicators, have fairly well value for diagnosing hepatic fibrosis or hepatocirrhosis in chronic hepatitis b. background: to investigate the effect of liver cirrhosis on the development of atherosclerosis in the rabbits chronically fed with high fat diet. methods: normal male new zealand white rabbits were randomly divided into four groups: a control group, a high fat diet group, a carbon tetrachloride (ccl4) group and a complex group. pathologic changes in ascending aortas and livers were observed. the levels of serum alanine aminotransferase alt , lipid, c-reactive protein (crp) were also determined. results: significant hepatic steatosis, inflammation and fibrosis could be observed in the three treatment groups; while atherosclerosis and typical arteriosclerotic plaques in ascending aortas could only be observed in the two high fat diet groups. compared with the control group, serum alt and lipid levels in ccl4 group were increased significantly (p<0.05), but no difference of arterial intima-media thickness (imt) and i/m ratio between these two groups. the levels of serum alt, lipid, crp and imt in two high fat diet groups were significantly increased compared with the control group (p<0.05). the level of serum alt in the complex group was significant higer than that in the high fat diet group, but the i/m ratio was just opposite (all p<0.05), and there was no difference of imt between the two groups. conclusions rabbits treated with ccl4 can elevate serum lipid levels, but can not induce atherosclerosis. though the activity of liver inflammation was aggravated in the complex model group, it has no effect on atherosclerosis possibly partly because of malnutrition. higher values of liver stiffness in males with mild chronic hepatitis c c. stern 1 , a.c. cardoso 1 , r. moucari 1 , a.d. pumpo 1 , n. giuily 1 , p. bedossa 1 , p. marcellin 1 1 hopital beaujon background/aim: liver stiffness (ls) measured by fibroscan (echosens) is a noninvasive method to assess liver fibrosis in patients with chronic liver diseases. we evaluated the impact of factors on ls results in mild chronic hepatitis c (chc). methods: chc patients with metavir fibrosis stage 1 at liver biopsy and a reliable ls exam were eligible. all patients had no prior antiviral treatment. the ls values were compared to clinical and biochemical data. results: 93 patients were included with the following characteristics: mean age 50 11, male gender (46%), mean bmi 23 2.7, median ls 5.8 kpa (3.2-21.8), diabetes (7%), genotype 1 (61%), metavir activity a1 (86%), a2 (10%), steatosis at biopsy 30% (92%), mean glucose 4.9 1, abnormal alt (78%), abnormal ggt (47%), homa (2 1.2). the ls values were associated wtih male gender (median 6.1 in males vs 5.2 in females) (p=0.04), bmi (p=0.03), alt (p=0.006), ggt (p=0.02) and glucose levels (p=0.04). no association was found between ls and activity stage (p=0.34) or steatosis (p=0.14). in the linear regression, the only factor independently associated with higher ls was gender (p=0.038). in men, higher ls was related to levels of alt (p=0.005), but not to necro-inflammation grade (p=0.4). in women, ls was not associated with alt levels, but with bmi (p=0.045) and ggt levels (p=0.035). conclusion: in patients with mild chc, liver stiffness values are higher in males. these results suggest that different cut-off for fibrosis stage 1 should be proposed according to gender. aims: to investigate the effects of shuanghu qinggan granule(sqg) on prevention and treatment of hepatic fibrosis induced by carbon tetrachloride in rats. methods: 60 sd rats were divided into 6 groups, normal control groupamodel groupb, sqg largec1, middlec2small dose groupsc3 and silymarin positive contrast groupd. the rats of bc1c2c3d were injected with carbon tetrachloride for 8 weeks. the rats of c1c2c3 were then administered with sqg for 8 weeks. the rats of d were then administered with silymarin for 8 weeks. results the liver structure of rats of b was severely damagedlarge amount of liver cells became obviously degeneratedand hepatic veins were clearly congested. the hepatic cells fatty degeneration and infiltration of inflammatory cells in rats of c1c2c3d reduced significantly. there was no fiber hyperplasia in liver tissues of rats of c1c2c3d. blood serum ha cp p levels in rats of b were significantly higher than those in ac1c2c3d. conclusion: sqg has remarkable therapeutic effects on rats with hepatic fibrosis induced by carbon tetrachloride, the higher the dosage of sqg was, the more effective the results would be. conclusions: none of sophisticated biomarkers had value in addition to readily available laboratory data for the prediction of significant fibrosis in hbeag positive patients. two markers out of 7 sophisticated biomarkers provide additional diagnostic information in hbeag negative patients. before new biomarkers are accepted, their superiority to routine laboratory data should be meticulously appraised. objective: to evaluate the efficiency and safety of "tinmax" hb-3 herbal compound (cpd) in treatment of hepatofibrosis and cirrhosis post chronic hepatitis b. methods: a double-blind randomized method was employed. 60 patients of hepatofibrosis or cirrhosis post hepatitis b were separated into study group ("tinmax" hb-3 group) and control group (natural vitamin group) by randomized method. the course was 52 weeks. patients visited once every 12 weeks and the last visit at 12 weeks after the cessation of treatment. part of patients had liver biopsy before and after treatment. before, during the course and at the end of therapy, clinical symptoms and physical signs were evaluated, hepatic function, and serum markers of hepatofibrosis (such as hyaluronate acid, laminin, serum type iii procollagen and collagen iv) were tested, and ultrasound evaluation was performed. results: 60 patients enrolled in the evaluation. 58 patients completed the evaluation according to the protocol. 20 patients had liver biopsy twice, 10 from the study group and 10 from the other one. at the end of therapy, the total effective rate of hepatofibrosis in histopathology is 74.13% in the study group, much higher than that of 21.95% in the control group (p<0.05). the total effective rate of serum markers of hepatofibrosis at the end of therapy in the study group was 70.10%, much higher than that of 30.24% in the control group (p<0.05). the total effective rate of non-invasion markers of hepatofibrosis at the end of therapy in the study group was 76.08%, much higher than that of 19.41% in the control group (p<0.05). the drugs of adverse event had not happened in both groups. conclusion: "tinmax" hb-3 herbal compound (cpd) is effective and safe in treatment of hepatofibrosis and cirrhosis post chronic hepatitis b. w.h. sha 1 , xiaohui zeng 1 , yuyuan li 1 1 gi department, first municipal hospital of guangzhou, guangzhou aim: to investigate the clinical value of serum indices for hepatic fibrosis in chronic liver diseases. methods: competitive radioimmunoassay was used to determine the serum level of collagen type ( c), laminin (ln) and hyaluronic acid(ha) in 193 patients with different severity degree of chronic liver diseases, and in 30 healthy subjects. results: the serum levels of c, ln, and ha in the patients with liver diseases increased to different extent, compared with those in the healthy subjects. of which the highest of c, ln, and ha were found in the patients with primary carcinoma of liver or hepatocirrhosis and the serum level of ha is highlight. the combination detection of serum c, ln, and ha is more valuable than single index. conclusion: joint detection of serum c , ln, and ha is of higher significance in clinical diagnosis and prognosis of hepatocirrhosis, and is also available for successive observation on the development of liver diseases. aims: to investigate the mechanism of fuzheng huayu decoction (fzhy) on hepatic stellate cells (hscs) activation relating to tgf-1 signal transduction pathway. methods: hscs were isolated from normal rats by in situ pronase/collagenase perfusion followed by density gradient centrifugation. at day 4 after isolation, cells were stimulated with 100pm tgf-1 for 24h, then incubated with 10% fzhy pharmacological serum or 10 m t r -i inhibitor (sb-431542) for 24h. protein expression of -sma, smad3 was assayed by immunofluorescent stain; total protein expression of -sma, t r -i, smad2/3 and nuclear expression of smad3 was analyzed by western blotting. results: fzhy pharmacological serum significantly decreased expression of -sma, t r -i, and inhibited smad3 nuclear expression and translocation in tgf-1 stimulated hscs. conclusions: fuzheng huayu decoction can prevent hscs activation through tgf-1 signaling transduction pathway in hscs, which may be the important molecular pharmacological mechanism of fuzheng huayu decoction action against liver fibrosis. background: fatty liver disease has become a health problem related to metabolic syndrome worldwide although its molecular pathogenesis has remained further studied and it is unclear whether advanced fibrosis induced by steatohepatitis will regress when diet is controlled. aim of this study is 1) to study the involvement of endoplasmic reticulum stress (er stress) in the occurrence of seatohepatitis and 2) to obtain the evidence of resolution of fibrosis by changing the diet. methods: non-alcoholic steatohepatitis with advanced fibrosis was produced in rats by giving methionine-choline-deficient diet (mcdd) for 10 weeks. methionine-choline-control diet (mccd) instead of mcdd was given for the last 2 weeks in an experimental group. fibrosis and inflammation was determined by several tissue stainings. gene expression related to fibrosis and inflammation was determined by immunoblotting and real-time pcr. expression of caspase-12, caspase-7, and glucose-regulated protein 78 was evaluated to clarify the presence of er stress aim against liver fibrosis relating to hypoxia and angiogenesis regulation. methods: the rats were divided into normal, model, sa-b and perin control group. rats in sa-b and perindopril group were administrated with sa-b and perindopril respectively. liver fibrosis was induced by ip dimethylnitrosamine (dmn) for 4w. fibrosis degree was observed by sirius red staining. col-i protein expression was analyzed by western blot; col-i , vcam-1, icam-1, hif-1 and vwf expression in liver tissue was checked by immunohistochemistry; gelatinase activities in liver tissue were detected by gelatin zymography and in situ flourescent zymography. result: compared to normal group, col-i, hif-1 , icam-1, results: 1) changing the diet from mcdd to mccd triggered the reduction in fat in hepatocytes, the decrease of inflammatory gene expression and oxidative stress, and the regression of fibrosis accompanied by the disappearance of activated stellate cells and macrophages. 2) immunohistochemistry, immunoblotting, and rt-pcr analysis all indicated the occurrence of er stress in steatohepatitis while it recovered immediately after changing the diet from mccd to mcdd. vwf protein expression and gelatinase activity in liver tissue were increased obviously in model group, while sa-b and perindopril treatment significantly decreased these protein expressions and gelatinase activity. conclusions: this simple experiment clearly shows that the changing diet from steatohepatitis-causing mcdd to mccd triggers the resolution of inflammatory and fibrotic reaction in the liver, suggesting that food intake is a very important factor for controlling the state of fat and pathology of the liver. er stress is involved in the process. background: liver fibrosis results from chronic damage to the liver in conjunction with the accumulation of extracellular matrix proteins, which is a characteristic of most types of chronic liver disease. under injury conditions, hepatic stellate cells (hscs) are activated to transdifferentiate into myofibroblasts, which are capable of secretion of many connective tissue elements, especially collagens i, iii, and iv. gynostemma pentaphyllum is a popular folk medicine that has been used for treatment of hepatitis in asia. gypenosides are the major saponins derived from g. pentaphyllum. in previous study, gypenosides have hepatoprotective and anti-fibrotic activities in rat chronic liver injury induced by ccl4, and anti-proliferative effect in rat isolated hscs. methods: in cultured hscs model, we detected type1 procollagen protein and mrna by western blot and rt-pcr. result: we found that g. pentaphyllum inhibited type1 procollagen protein expression in 66% at 48 hours. furthermore, g. pentaphyllum also inhibited type1 procollagen 1 and 2 mrna expression in 39% and 11% respectively. in addition to transcriptional inhibition, we found that g. pentaphyllum also enhanced the degradation rate of type1 procollagen protein. base on the effect of enhancing protein degradation, we used some protease inhibitors like ca-074 me, z-fa-fmk, aebsf, tpck and tlck to identify the potential target of g. pentaphyllum. on the other hand, in the ubiquitin-proteasome system analysis, we quantified the change of some target proteins of proteasome in the presence or absence of g. pentaphyllum. conclusion: g. pentaphyllum reduced type1 procollagen protein by inhibiting transcription and enhancing protein degradation. aim: excessive oxidative stress in diabetic patients has been implicated in the pathology and complication of liver. the present study was designed to examine whether ginger has a direct hepatoprotective effect in diabetic cases. methods: wistar strain albino rats were selected for this study. the rats were divided into 4 groups: (i) control, (ii) ginger treated (200mg/kg b.w. orally, 30 days) (iii) diabetic (50 mg/kg b.w., i.p.) and (iv) diabetic + ginger treatment. the lipid metabolic profiles such as total cholesterol, triglycerides, phospholipids and lipid peroxidation as stress markers and histopathological studies were carried out to assess the damage in hepatic tissue. results: ginger treated diabetic rats demonstrated significant reduction in glucose levels as compared to the nontreated diabetic animals. diabetic rats have shown increased total cholesterol, triglycerides, phospholipids and lipid peroxidation content in hepatic tissue compared to control, indicate prevailing of oxidative stress and alterations in fatty acid metabolism in these rats. further, degenerative changes of hepatic cells in diabetic group are minimized to nearness in structure by administration of ginger as evinced by histopathological examination. conclusion: we summarize that the hypolipidemic and antioxidant compounds present in ginger may be useful in delaying the complicated effects of diabetes. this results also reveal that ginger possess hepatoprotective properties in diabetic cases. anti-fibrotic action m. naime 1 , s. ali 1 1 hamdard university rhizomes of valeriana jatamansi (family, valerianaceae) have long been used in indian subcontinent by the traditional healers for the treatment of various diseases. this study provides experimental evidence suggesting the therapeutic effect of the crude extract of rhizomes on rat liver fibrosis, and demonstrates its antiproliferative role. crude extract (50% ethanolic) at a dose level of 800 mg/kg body weight was administered to rats to study the effect on biochemical and other markers of liver fibrosis. administration of the extract for 9 weeks could bring down elevated the levels of biochemical markers of liver injury, and modulate several other biochemical responses. morphology and hisopathological examination cooroborated with the biochemical changes, and indicated partial reversal of fibrosis. dpph assay confirmed the antioxidant property of the extract, which is suggested to be due to -ionone, -sitosterol and other chemical constituents. further, treatment could restore depleted glutathione level, inhibit lipid peroxidation, and inhibited elevated xanthine oxidase activity in fibrosis. the study also reports anti-tumour promotion activity of the extract as evident by a significant decrease in [ 3 h]-thymidine incorporation by hepatic dna in extract treated rats. results suggest that v. jatamansi extract has curative effect and can partially reverse biochemical and histological changes associated with liver fibrosis. with chronic hepatitis c c. wongjitrat 1 , s. chainuvati 1 , a. manuyakorn 1 , s. aroonparkmongkol 2 , t. tanwandee 1 1 mahidol university, 2 background: leptin is a peptide hormone that mainly regulates food intake, energy expenditure and reproductive function. leptin also releases from activated hepatic stellate cells and may have a role in regulation of fibrogenesis and inflammation. in human chronically infected by hcv, the role of leptin-associated fibrosis of the liver is still unclear. there is no data in thai patients chronically infected by hcv regarding leptin level and its correlation with hepatic histology and fibrosis.the purpose of this study was to evaluate the relationship between leptin level and severity of liver fibrosis in thai patients chronically infected by hcv. methods: sixty-six patients (31 men, 35 women) with chronic hcv infection and liver biopsy was done within 3 months were enrolled. fasting blood samples were obtained and serum leptin levels were measured by elisa. bmi, blood sugar, liver function test, lipid profile, hcv rna viral load and hcv genotype were also measured and related to histological findings. results: mean serum leptin levels were significantly higher in women than in male. there was a significantly correlation between serum leptin and bmi (r = 0.469, p < 0.001). leptin levels were not associated with hepatic fibrosis (r = 0.166, p = 0.183) and necroinflammation (r = 0.203, p = 0.102). steatosis was significantly associated with severe necroinflammation (r = 0.261, p = 0.034), but not fibrosis (r = 0.22, p = 0.076). conclusions: these findings failed to demonstrate correlation of serum leptin and hepatic fibrosis in thai patients chronically infected with hcv. background and aim: liver cirrhosis is one of the leading causes of mortality in our country as well as in our region. even though deterioration of glucose metabolism and existence of insulin resistance in liver cirrhhosis has been well documented in many studies, it is still unclear how insulin resistance mechanism develops. the aim of the present study is to assess insulin resistance, cytokines and crp levels in patients with liver cirrhosis and control subjects. in additon, we aimed to investigate the relation of insulin resistance in liver cirrhosis with such parameters as age, sex, etiology, child-pugh classification, spleen size, tnf-?, il-1?, il-2res, il-6, il-8, il-10, crp and hs-crp. material and method: a total of 79 patients with cirrhosis of different etilogy (49 male, 30 female) were included into the study. as controls, 50 (23 male and 27 female) subjects were taken. the two groups were compared with each other in terms of glucose, insulin, c-peptid, homa-ir, tnf-?, il-1?, il-2res, il-6, il-8, il-10, crp and hs-crp levels. in the second part of our study, the liver cirrhosis group was divided into two subgroups: patients with homa-ir value >2.7 as insulin resistance positive, and those with homa-ir value >2.7 as insulin resistance negative. these two groups, i.e. , homa-ir positive and homa-ir negative, were compared in terms of age, sex, etiology, child-pugh classification, spleen size, tnf-?, il-1?, il-2res, il-6, il-8, il-10, crp and hs-crp levels. results: in liver cirrhosis group, glucose, insulin, c-peptid, homa-ir, tnf-?, il-2res, il-6, crp and hs-crp levels were determined to be significantly higher than controls. between patients with homa-ir positive and negative, however, statistically no significant difference was found in terms of age, sex, etiology, child-pugh classification, spleen size, tnf-?, il-1?, il-2res, il-6, il-10, crp and hs-crp levels, but il-8 level was seen to be significantly low in patient homa-ir positive. conclusion: in patients with liver cirrhosis, the levels of glucose, insulin, c-peptid, homa-ir, tnf-?, il-2res, il-6, crp and hs-crp increase with respect to normal population. determination of increased homa-ir level in liver cirrhosis supports the view that insulin resistance develops in liver cirrhosis as reported in related studies. in the study, it was also determined that the mechanism of insulin resistance development occurs independent of age, sex, etiology, child-pugh classification, spleen size, tnf-?, il-1?, il-2res, il-6, il-10, crp and hs-crp levels. the determination of statistically lower level of il-8 in patients with homa-ir positive with respect to those with homa-ir negative does not indicate similarity with the studies carried out earlier. ) in patients (48.08%) than in controls(14%)in group i hla-b5 significantly increased in patients (60%)as compared to controls (14%) . in group ii hla -b5 significantly higher in patients (45.46%)than controls (14%) also hla-aw19 significantly higher (40.91%) in patients than controls (12.67%).in group iii hla-aw19 significantly increased in patients (46.67%) compared to controls.no significant association between hla antigens and cases with hbv or hcv infection. conclusion: the significantly high association of hla-aw19 and hla-b5 in patients with hepatic schistosomiasis as compared to normal controlstogether with the lack of any association with active intestinal schisto . antigens predispose to liver affection.individuals possessing hla-aw19 appear to be more prone to severeform of liver disease background: atp8b1 mutation is one of the factors that result in cholestasis and progress to chronic liver disease, but has never been reported in the mainland china before. the aim of this study was to elucidate the role of atp8b1 mutation in mainland chinese patients with progressive intrahepatic cholostasis and low ggt. methods: 24 children who presented with progressive intrahepatic cholostasis and low ggt were admitted in a tertiary pediatric hospital in eastern china. abcb11 gene was analyzed firstly to exclude bsep deficiency. afterwards, all the encoding exons and their flanking areas of atp8b1 gene were sequenced in the remaining 19 patients in whom only one or no mutations of abcb11 were found. results: 10 mutations of atp8b1 gene were found in 9 patients. i694n had been reported in taiwanese patients with pfic1, and the others were novel. p209t and ivs6+5t g were linkage and found in 4 of 9 patients, including 2 homozygote and 2 heterozygote. liver biopsy had been performed in 6 patients with atp8b1 mutations and 5 with abcb11 pe408 mutations. variety portal fibrosis was showed in 2 patients with atp8b1 mutations and 4 patients with abcb11 mutations. giant cell transformation was detected in one patient with atp8b1 mutations and 4 patients with abcb11 mutations. 1 laboratory of exercise biochemistry, taipei physical education college, jhongcheng rd., no. 101, sec.2, taipei city-11153, taiwan, roc background/aim: generation of reactive oxygen metabolites are depends on the consumption of oxygen and their cumulative effects may be different from lean to obese population. this study was designed to investigate the deleterious effects of oxidants on hepatic antioxidant defence system in lean and obese rats under hypoxic condition. methods: zucker rats lean (200±10gms) and obese (450±15gms) were divided into control and acute hypoxia groups. the acute hypoxia treatment was performed in a hypoxic chamber at 14% oxygen consumption. objectives: to compare the diagnostic value of morning urine copper to zinc (copper/zinc) ratio and 24 hour urinary copper excretion in wilson's disease (wd) children. results: in the results, acute hypoxia caused a significant (p<0.05) decrease in major antioxidant enzymes including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gsh-px), glutathione reductase (gr) and glutathione (gsh) content in lean groups when compare to their controls. the decrease in the activities of all antioxidant enzymes was also noticed in obese group with hypoxia treatment. however, this decrease was not significant in case of cat, gsh-px activities and gsh content. the mda levels (lipid peroxidation marker) were higher in obese rats compare to lean rats. methods: morning urine and 24 hour urine were collected from 96 patients over three years age who were hospitalized in a tertiary pediatric liver service. each patient was re-evaluated according to wd scoring system, and was assigned to one of the three groups: wd, suspecting wd, and non-wd. 24, 6, and 64 cases were assigned to wd, suspecting wd, and non-wd respectively. urine copper and zinc concentration was determined simultaneously by using inductively coupled plasma mass spectrometry. conclusions: the higher hepatic mda values observed in obese rats indicate that accumulation of free radicals may be more in obese rats thus leads to promote the lipids oxidation. from this study it is concluded that decrease of antioxidant enzymes (except gr) with acute hypoxia treatment were more in lean group compared to with that of obese group. results: the morning urine copper/zinc ratio and 24hr urinary copper excretion correlated well (r=0.758, p < 0.001). the median of morning urine copper/zinc ratio, 24hr urine copper/zinc ratio, 24h copper excretion, and 24h zinc urinary excretion were 0.370, 0.394, 87.1 and 398.7 in wd group, and 0.051, 0.061, 24.2 and 358.9 in the non-wd group respectively. the differences of morning urine copper/zinc ratio, 24hr urine copper/zinc ratio, and 24h copper excretion were significant (z-value -6.502, -6.020 and -6.208 respectively, all p values < 0.000 chd1l is a recently discovered oncogene localized at 1q21, one of the most frequently amplified chromosomal regions in hcc. herein, by yeast-two hybrid assay, we demonstrate that the anti-apoptotic ability of chd1l is associated with its interaction with nur77, a critical member of a p53-independent apoptotic pathway. as the first cellular protein identified to bind nur77, chd1l inhibits the nucleus-to-mitochondria translocation of nur77, and subsequently hinders the release of cytochrome c and the initiation of apoptosis ( figure 1 ). further study found that c-terminal macro domain of chd1l is responsible for the interaction with nur77, and a chd1l mutant lacking residues 600-897 failed to interact with nur77 and prevent nur77-mediated apoptosis. we also find that chd1l confers cellular chemoresistance to drugs that induce apoptosis via the nur77-mediated pathway, which may lead to the identification of new therapeutic targets for hcc treatment. background/aim: accumulation of oxidative damage to proteins, lipids and mitochondria could increase with advancing of age. the current study was aimed to test the hypothesis that swimming exercise training could revert the age dependent oxidative damages in liver. methods: sprague-dawley rats of young (3 months) and old (12 months) were divided into four groups; young control (n=5), young exercise (n=5), old control (n=5) and old exercise (n=5). 90 minutes of swimming exercise was given to the exercise group for a period of two weeks. results: the estimated antioxidant enzyme activities including, superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gsh-px) and glutathione reductase (gr) were decreased with age and significantly (p<0.05) increased with exercise training. however, elevated protein carbonyls and mda levels were noticed in old animals, which indicate that old liver had greater accumulated oxidative damages. the significant drop in protein carbonyl content and increase in mitochondrial succinate dehydrogenase (sdh) activity was observed with response to swim training in old rats. conclusions: this data implied that swim exercise training could revert the oxidative damages in liver. this was also proven by enhanced antioxidant enzyme status with response to exercise training in old rats. to sum-up these results it is cleared that age induced detrimental effects to the liver might be reversed by regular swimming exercise training in old rats. results: peg10 was expressed in l02/peg10 (fig.1) . peg10 accelerated the growth of l02. after treatment with 400mm h2o2 for 24 h, the inhibitory rate of l02/peg10 cells was 32.5%; the chromosomal condensation and ladder-like dna fragmentation were not observed (fig.2) . methods: hepg2 or hepg2.2.15 were co-cultured with jurkat cells, with blocking test by adding anti-pd-1 antibody. the pd-1 expression was detected by flow cytometry (fcm); cytokines in culture supernatant in blocking groups and controls were measured by enzyme-labeled immunosorbent assay (elisa); cytotoxic test of t cells were measured by methyl thiazolyl tetrazolium (mtt). conclusions: over-expression of peg10 can significantly promot l02 proliferation and ameliorate apoptosis-inducing effects of h2o2 on l02. results: the pd-1 expression on jurkat cells was induced by hepatoma cells, the expression rate were 16.17±6.5% (by hepg2) and 17.43±6.8% (by hepg2 2.2.15), respectively. the cytokines il-2 level (202.9±53.0 pg/ml), inf-level (88.6±4.6 pg/ml) and il-10 level (63.7±13.4 pg/ml) in culture supernatant of blocking groups were significant higher than that of controls (il-2, 102.9±53 pg/ml, inf-, 39.3±4.2 pg/ml and il-10, 34.6±13.7 pg/ml, respectively. p < 0.05). the cytotoxic test (od value) was markedly higher in blocking group (0.29±0.06) than that of control group (19±0.09 p<0.05) . conclusion: the pd-1 expression on lymphocytes can be induced by hepatoma cells, and cytokines expression and cytotoxic test were recovered by blocking pd-1/pd-l1 interaction. background: hedgehog (hh) pathway is well known as a positive regulator for tissue construction( during development) and reconstruction (in adults). our aim to observe the expression change of hh pathway on rat hepatic regeneration . materials and methods: adult male sprague-dawley rats underwent approximately 70% partial hepatectomy (ph) or sham operation (so). liver specimens were collected at 2, 6, 12, 24, 36, 48, 72 , and 168h after ph or so. hedgehog expression was determined in mrna level by rt-qpcr as well as in protein levels via immunohistochemical staining and western-blotting. results: so treatment did not induce remarkable changes in hedgehog expression; however, the level of transcript for hedgehog was significantly upregulated after ph. we found sonic hedgehog(shh )and glioblastoma (gli1-3) mrna expression in the regenerating liver arrive at its peak at as early as 24 h and returned to its physiologyical level 168 h later. it is similar to the change of proteins (shh and gli1) .as seen from immunohistochemistry experiments; shh protein was expressed uniquely in regenerating hepatocytes. similarly, ph induced over expression for shh protein occurred from 12 h with a peak level at 36 h after surgery. but gli protein mainly located in nucleus and no significantly changes in the phrase of liver regeneration. conclusion: hedgehog pathway may play a role in the activation of hepatic proliferation during liver regeneration induced by physiological stress or pathological states, such as ph. background: to investigate whether peg10, an imprinted gene with an active paternal but silent maternal allele, was involved in hydrogen peroxide (h2o2) induced cellular apoptosis. methods: peg10 gene was stable transfected into l02. cellular gene expression was determined by rt-pcr, western blot and immunocytochemistry. cell proliferation was analyzed by mtt. after treatment with different concentrations (50-400 mm) of h2o2, cell proliferation inhibition rate was measured by mtt. morphological changes of apoptotic cells were determined by hoechst33342 staining, dna fragmentation was observed by agarose gel electrophoresis. hua tang 1 , xiao-yan tang 1 , min liu 1 , xin li 1 1 tianjin life science research center, tianjin medical university, tianjin 300070, china we determined how afp modulates the proliferation of hepatoma cells. a recombinant adenovirus expressing sirna against afp (adv-afpsirna) was created and found that it reduced expression of afp specifically in hepatoma cells, and markedly inhibited the proliferation of hepatoma cells in vitro. local treatment using adv-afpsirna caused significant repression of the growth of hepatoma derived hepg2 cells in xenograft in nude mice. knockdown of afp resulted in an obvious delay in the g1/s transition of cell cycle, but did not affect apoptosis in hepg2 cells, as analyzed by flow cytometry and tunel assay. also, differential expressions of some genes related to the cell cycle, including skp2, cyclin d1, csk and ebag9 were identified by microarray and rt-pcr in hepg2 cells and hepg2 cells with knocked down afp. these results suggest that endogenous afp is a critical determinant of the growth of hepatoma cells. hematopoietic stem cell (cd 34+) therapy can improve liver function in patients with cirrhosis. these cells can be mobilized into peripheral blood using granulocyte colony stimulating factor (gcsf). this study was undertaken to assess feasibility and safety and of gcsf induced cd 34+ cell mobilization and its impact on liver function in patients with cirrhosis. patients with liver cirrhosis (cryptogenic or alcoholic with 6m abstinence) with cpt > 7 and < 12, and splenic diameter < 17 cm were included. gcsf injection was given for 5 days (5mcg/kg/dose). baseline & day-6 cd 34+ counts in peripheral blood were done by flow cytometry. follow up was weekly for 4 weeks and then monthly. cpt was compared at baseline and 6 months. 9 patients (median age 51 y, range 33-64 y, 8 males; etiology: 6 alcohol, 3 cryptogenic; median cpt 10, range 8-12) were included. cd 34 + cell counts at baseline and day 6 were 2(0-3) and 15 (13-41) respectively (median, range). side effects were fever in 8, allergic reaction in 1 and increase in splenic size in 1 (excluded). in follow up, 3 patients died (1, 3 & 4m after therapy, 1 after olt), 1 lost to follow-up. 4 patients showed improvement in 10 -7) at 6-month follow-up. gcsf treatment is safe and yields adequate cd 34+ cells in peripheral blood. in short term it results in improvement in liver function in patients with cirrhosis pe415 molecular cloning and transcriptional analysis of kctd9 gene promoter b. pi 1 , j.s. wang 1 , m.f. han 1 , y.y. zhou 1 , x.j. liu 1 , x.p. luo 2 , q. ning 1 1 department of infectious disease, tongji hospital, tongji medical college, huazhong university of science and technology, 2 department of pediatrics, tongji hospital, tongji medical college, huazhong university of science and technology aim: our previous work has shown that high expression of kctd9, a potassium channel associated gene, correlated with the disease severity of patients with severe chronic hepatitis b(schb). to further understand the gene transcription and regulation, kctd9 promoter was cloned and gene transcription was studied. methods: a full length of isolated promoter and series of 5' truncated promoter of kctd9 gene was subcloned into the luciferase report vector pgl2-basic to form the promoter-report constructs. the kctd9 promoter-report construct upstream of the luciferase report gene was cotransfected with constructs expressing hbv x,c and s protein respectively or stimulated with cytokines (il-2, ifn and tnf ) in 293 t cells to investigate kctd9 gene regulation upon both viral factors and host cytokines. result: a 759bp kctd9 segment upstream of atg translation start site was evidenced to contain potential regulative domains. an important regulation site located between -268bp and -81bp upstream of atg translation start site. based upon the luciferase activity assay, il-2 was able to upregulate the transcription of kctd9 whereas there was no effect from neither hbv viral proteins nor ifn and tnf . conclusion: here we first successfully cloned the full length promoter of kctd9. il-2 significantly enhanced the transcription of kctd9, a gene which has been shown to be involved in t cell activation and disease severity of schb from our group. this work was supported by nsfc(30571643, 30672380, 30700702) 1 1 istanbul university, cerrahpasa medical school, 2 marmara university, 3 okmeydani teaching hospital, 4 background: the treatment in chronic hepatitis c virus (hcv) is not highly effective, and cost, duration, and side effects are challenging. predicting favorable factors of response to treatment would make it possible to give it only responsive patients. recent studies report more conclusive results about the role of apoptosis in inflammation and fibrosis seen in chronic viral hepatitis. hepatocyte damage in hcv is mediated by cytotoxic t-cells. apoptosis primarily developed by the interaction between fas antigen on hepatocyte and fas ligand on t-cell corresponds to a main mechanism for hepatocyte damage. methods: in this study, we aimed to detect any relationship between apoptotic markers (fas, fas ligand, fas-associated death domain, caspases 3,8, and 9, insitu apoptosis) in liver biopsy taken before the treatment and response to the treatment of interferon+ribavirin. additionally, any relationship between these parameters and the other ones predicting the response to therapy including alt level, viral load, genotype, and gender were studied. results: the study includes the patients in 4 centers managing chronic hcv infection. all parameters were studied in 180 patients. study results revealed that histological activity index is correlated with cd95 staining density, caspase 8 intensiveness, and portal and parenchymal fas ligand scores. fibrosis is also seen to be correlated with the same parameters. apoptotic parameters of the responsive cases were not significantly different from unresponsive ones. conclusion: apoptotic parameters studied in the liver tissue is associated with inflammation and fibrosis, however these parameters may not predict the response to the treatment. s. gao 1 , d. xi 1 , j.w. guo 1 , c.l. zhu 1 , x.p. luo 2 , q. ning 1 1 department of infectious disease, tongji hospital, tongji medical college, huazhong university of science and technology, 2 department of pediatrics, tongji hospital, tongji medical college, huazhong university of science objective: this study was designed to explore the opportunity of microrna interference technique in the inhibitory application of human fgl2, human fas and tnfr1 expression. methods: the eukaryotic expression plasmids of human fgl2, fas and tnfri genes were constructed and have successfully expressed hfgl2, hfas and htnfri protein. mirna expression plasmids of hfgl2, hfas and htnfri complimentary to the sequence responsible for hfgl2, hfas and htnfri respectively were also constructed, meanwhile an irrelevant mirna plasmid was used as control. by respective cotransfection of p-hfgl2mirna and pcdna3.1-hfgl2, p-hfasmirna and pcdna3.0-hfas, p-htnfri mirna and pcdna3.0-htnfri expression construct into 293t cells, the inhibition of hfgl2, hfas and htnfri expression were analyzed by quatitative real time pcr and western blot. results: the experiments showed the significantly inhibitory effect of p-hfgl2mirna on hfgl2, p-hfasmirna on hfas and p-htnfri mirna on htnfri expression at 48h post-transfection both at rna level and at protein level, as well in 293t cell lines the inhibitory efficiency reached as high as 89.3% for hfgl2, 87.5% for hfas and 80% for htnfri, respectively. conclusions: the study demonstrated the constructs of p-hfgl2mirna, p-hfasmirna and p-htnfri mirna successfully interfered their target genes expression in vitro, which provides the foundation for further investigation of these constructs' application in vivo and further more as a therapeutic strategy for a targeting intervention in the diseases which the gene fgl2, fas and tnfri contribute to. this work was supported by nsfc30571643, 30672380, 30700702; 2005cb522901, 2007cb512900 pe418 influence of the id2 on the anti-tumor activity of histone deacetylase inhibitor in hepatocellular carcinoma cells r. tsunedomi 1,2 , s. harada 1 , n. iizuka 1 , m. oka 1 1 dept. of digestive surgery and surgical oncol., yamaguchi univ. , 2 research fellow of the japan society for the promotion of science for young scientists background: our recent study revealed that levels of the inhibitor of dna binding/differentiation 2 (id2) were associated with the progression of hcv-related hepatocellular carcinoma (hcc) and can affect susceptibility of hcc cells to histone deacetylase (hdac) inhibitors. we here aimed to investigate how and whether id2 expression affected on the anti-tumor activity of sodium butyrate (nab), one of hdac inhibitors. methods: two hcc cell lines, hle and huh-7, were used for gene targeting experiments. the id2 over-expressing and knockdown cells were subjected to mts assay to evaluate the susceptibility to nab. time-course of the expressional change of bcl-2 and bcl-xl genes after nab administration was measured by real-time rt-pcr. result: upregulation and downregulation of id2 levels in hcc cells resulted in decreased and increased susceptibility to nab, respectively. we observed that after nab administration, the id2 expression was induced gently, the bcl-2 expression was greatly increased immediately, and the bcl-xl expression was decreased to less than half once and then recovered. these increase and recovery of the expression of anti-apoptotic genes were inhibited in the id2 knockdown cells. in the id2 overexpressing cells, the bcl-2 expression was more upregulated than mock-transfected cells. conclusion: in hcc cells, id2 influences the susceptibility to the hdac inhibitor by regulating the expression of anti-apoptotic genes caused by the hdac inhibitor stimulus. we suggest that id2 could serve as a fascinating marker predictive of response to hdac inhibitors. the role of zinc finger protein 146 in liver cancer m.m.y. waye 1,2 , t.l. yeung 1,2 , j.l. zhu 1,2 1 the chinese university of hong kong, 2 croucher laboratory for human genomics background/aims: the aim of this research project is the characterization of a krüppel zinc finger protein, zinc finger protein 146 (znf146) using hepatocellular carcinoma as a disease model. zinc finger protein 146 (znf146), also named as only zinc fingers protein (ozf), is a 33kda nuclear zinc finger protein consisting solely of ten c2h2 zinc finger motifs of the krüppel type. like most of krüppel proteins, it is assumed to be the transcription factor and involved in the regulation of gene expression. the znf146 gene is amplified in 15-25% of pancreatic carcinomas and overexpressed in more than half of the tumors including liver cancer. it is thus proposed that overexpression of the znf146 may contribute to the development or progression of hepatocellular carcinoma. methods: we used flow cytometry, microarray, green fluorescent recombinant protein, rt-pcr site-directed mutagenesis, and transfection to study the effect of expression of znf146. results: our results shown that znf146 was over-expressed in two human hcc cell lines hepg2 and hep3b. expression profiles of znf146 over-expressing shown that genes related to the p53 tumor suppressor activity or dna damage, repair response and control were deregulated upon overexpression of znf146. conclusions: znf146 is possibly involved in liver carcinogenesis by affecting dna repair and cell cycle control upon induced dna damage. background: in the present study, we reported the establishment of a real-time monitor system for directly observing the catalytic, kinetic characteristics of dnazyme 10-23 in vitro cleavage on the target rna molecules as well as for rapid, accurate, high-throughout evaluation of varied dnazymes on their counterpart rna molecules. methods: dnazyme named dz-hcv-9 specific to hepatitis c virus (hcv) orf aug were designed and synthesized. dz-hcv-mis-9 with mismatched substrate-recognition domains, dz-hcv-mut-9 with mutant catalytic domains, antisense oligonucleotide ason and nonsense oligonucleotide nson were synthesized respectively as controls. a chimeric oligonucleotide of 29nt containing both rna and dna bases was designed and synthesized as the substrate: 5' fam-gt agaccgugcaccaugagcacgaaucct-bhq 3', corresponding to the330-354 nt (underline) of hcv genome(gi: 329873) the reporter fam/bhq was incorporated at the 5' and 3' end, respectively. under simulated physiological conditions (37 ), kinetic characterization of rna-cleaving dnazyme was analyzed in a real-time way. factors that influencing dnazyme cleavage were analyzed. results: dz-hcv-9 specific to hcv orf aug could cleave target rna at a•u site, a continuous change of fluorescence intensity was monitored. while the control oligonucleotides couldn't cleave rna, there were no change of fluorescence intensity. factors that influencing dnazyme cleavage concluded different substrate-recognition domain, mg 2+ concentration and ph. conclusion: a real-time monitoring system for kinetic characterization of rna-cleaving dnazyme was successfully established in the first time. the study on the apoptosis of hepatoma cells synergeticly induced by plasmid-mediated anti-angiogenesis and immunopotentiation therapy p.y. li 1 , q. zhang 1 , y. chang 1 , j.s. lin 1 , d.a. tian 1 1 background: angiogenesis is improtant to hepatoma and decreasing of host immunity promotes the development of tumor. we want to study the effect and mechanism of apoptosis of mice implanted hepatoma cells induced by eukaryotic plasmid-mediated anti-angiogenesis and immmunopotentiation therapy. methods: mouse endostatin eukaryotic plasmid (pseces) and mouse il-12 (interleukin 12) eukaryotic plasmid (pmil-12) were extracted and purified from e. coli. h22 hepatoma cells were inoculated into the leg muscle of mice, which was divided into four groups and injected with pseces, pmil-12, pseces+pmil-12 or pcdna3.1 naked plasmid dna respectively into implantation sites repeatedly. tumor formation and its weight was evaluated. tumor microvessel density, tumor infiltrating lymphocytes and apoptosis of tumor cells were assayed by cd31 staining, he staining and tunel assay respectively. results: inoculated mice received pseces, pmil-12 injection formed tumor slowly with less microvessel density, more tumor infiltrating lymphocytes in the latter and more tumor apoptosis cells in both groups compared with pcdna3.1 injection. there were much more tumor apoptosis cells in pseces+pmil-12 group (19.9±5.5 per 400× microscope field p<0.05) than any other single plasmid injection group (400× microscope field: pseces 11.3±4.1, pmil-12 14.6±3.2, pcdna3.1 1.4±1.3). conclusion: tumor cells of implanted hepatoma in mice could be synergeticly induced to apoptosis by eukaryotic plasmid-mediated anti-angiogenesis and immunotherapy through inhibiting tumor angiogenesis and promoting tumor lymphocytes to infiltrate, by which mice implanted hepatoma was inhibited. (20, 40, 80, 160, 200 ng/ml) in a serum-free medium for 24 h. cell proliferation was measured by brdu incorporation analysis, untreated wb-f344 cells were taken as controls. after treatment with wnt3a (160ng/ml) for 24 h, subcellular localization and protein expression of -catenin in wb-f344 cells treated and untreated with wnt3a were examined by immunofluorescence staining and western-blot analysis. cyclind1 mrna expression was determined by semi-quantitative reverse-transcript polymerase chain reaction (rt-pcr). mrna levels of some phenotypic markers (afp, ck-19, alb) and two hepatic nuclear factors were measured by rt-pcr. expressions of ck-19 and afp protein were detected by western-blot analysis. results: wnt3a promoted proliferation of wb-f344 cells. stimulation of wb-f344 cells with recombinant wnt3a resulted in accumulation of the transcriptional activator -catenin, together with its translocation into the nuclei, and up-regulated typical wnt target gene cyclind1. after 3 d of wnt3a treatment in the absence of serum, wb-f344 cells retained their bipotential to express several specific phenotypic markers of hepatocytes and cholangiocytes, such as afp, ck-19 following activation of the canonical wnt signaling pathway. conclusion: the canonical wnt signaling pathway promotes proliferation and self-renewal of rat hepatic oval cells. the expression level of bid and other pro-and anti-apoptotic proteins were detected by immunoblotting. results: hbx/51 showed the most sensitive towards dox treatment, and truncated bid (tbid) was also only detected in this cell line. the level of bax was also increased in hbx/51 cells. conclusions: the carboxy-terminal of hbx may enhance the processing of bid into tbid, which may contribute to increased sensitivity of the cell towards the dox treatment. cell homeostasis were performed with concentrations of oxysterol (3x10 -5 -10 -4 m) faraway from the physiological and/or pathological one (0.2 and 2x10 -7 m). in our study, we asked the effects of oxysterols (7k and 5'6s) on hepatoma cell lines homeostasis. to this purpose we used concentrations similar to those described in physiological or pathological conditions. sub-physiological (10 -9 m) to pathological (10 -7 m) oxysterol (7k and 5'6s) concentrations were used to stimulate hepg2 cells. a surprising pro-proliferative effect of 5'6s at sub-physiological (10 -9 m) concentration was observed. this behaviour was confirmed by the synergic increase of erk1/2 levels. facs analysis revealed an early progression of cells in s phase at the lowest concentration of 5'6s, while all the remaining concentrations of the two studied oxysterols induced a weakly accumulation of cells in g2/m phase. apoptosis was absent at all concentration used, except for the highest one (10 -5 m). at this point we asked if cells didn't undergo apoptosis but acquired a senescent profile. effectively, both 7k and 5'6s, at all concentration used (except for 10 -9 m), induced cell senescence (revealed by sa-ß-gal staining and sirt1 and p21 over-expression). in conclusion the two oxysterols analyzed have different and in same case opposite effects on hepatocellular line. the main effect is surely the senescence induction, but it is important to highlight the proproliferative effects of 5'6 secosterol at low concentration. mortalin, a member of hsp70 family protein, has been shown to play an important role in hepatocellular carcinoma (hcc). it has been reported that mortalin is binding to the c-terminal of p53, which acts as a safety guard and is a commonly mutated gene in hcc. in this study mortalin was silenced by specific shrna in plc/prf/5, a hcc cell line constitutively expressing p53ser249, and normal liver cells miha, and we found that suppression of mortalin can selectively trigger the mitochondria mediated apoptosis pathway by p53 dependent way in plc cells. tunel staining positive cells were only found in the plc cells mortalin knockdown group, and apoptosis associated protein, such as p53, bax, bcl-xl, cleaved-caspase 3, have been screened by western blot after transfection. quantitative-pcr data also showed that p53 mrna level are upregulated about 2 folds in mortalin knockdown group compared with the control groups in liver tumor cells. two p53 inhibitors, pft-and pft-, which can reverse this apoptosis was applied to demonstrate p53 dependent way. in summary, knockdown mortalin can selectively kill liver cancer cells through reactive apoptosis by sensitizing mutant p53 in plc cells, but had no effect on normal cells. the clinical application of this study suggested that motalin specific shrna might be a potential anti-cancer drug for hcc. background: nafld can proceed to nash and are at risk of cirrhosis and hcc. aim was to study profile of bangladeshi nafld patients. methods: 52 patients with nafld were included. of them 59.6% were males and 40.4% females. patients were between 12-60 years of age. they presented with dull right upper abdominal ache and/or incidental detection of raised alt/ast and/or fatty liver on ultrasonography. all tested negative for hepatitis b and c. none had history of alcohol. all underwent per-cutaneous liver biopsy for histopathology. they were also tested for dm, dyslipidaemia, insulin resistance, hypothyroidism and hepatitis c. their bmi and bp were recorded. results: 88.5% had nash. 63.0% of them were males and rest 37.0% females. 11.5% had nafl. of them 50% each were males and females. majority had nash. 47.8% were obese and 41.3% had dyslipidaemia. 28.3% had hypertension, 28.3% insulin resistance and 13% were diabetics. 6.5% had hypothyroidism. none had hepatitis c. alt was raised in 72% and ast in 40%. although all patients with nash did not have elevated alt, it was raised in majority, contrary to ast, which was normal in most. conclusion: majority nash patients in bangladesh are obese. other leading causes of nash include dyslipidaemia, hypertension and insulin resistance. some nash also had diabetes and hypothyroidism. this study also reveals that elevated alt in patients with nafld is suggestive of fibrosis, although normal serum alt does not exclude nash. the study further suggests that alt is superior to ast in predicting nash. background: non-alcoholic fatty liver disease is prevalent in obese patients. liver biopsy remains the best diagnostic tool for confirmation. we tried to find out the correlations of laparoscopic parameters with histology and laboratory data. besides, we also evaluated the effectiveness of laparoscopy in liver disease diagnosis. methods: in the period of one year and five months,126 morbidly obese patients submitted to laparoscopic bariatric surgeries at our institutions were prospectively studied. results: laparoscopic parameters of significant correlations with histologic steatosis, inflammation and fibrosis were summarized in table 1 . besides, important parameters with relationships to laboratory data were summarized in table 2 department of internal medicine, seoul national university hospital gangnam healthcare center, seoul, south korea, 2 department of internal medicine and liver research institute, seoul national university college of medicine, seoul, south korea background/aims: hepatic fibrosis is associated with poor prognosis in non-alcoholic fatty liver disease (nafld). recently, many non-invasive fibrosis markers have been studied to overcome the limitations of liver biopsy. among them, bard score and guha's simple panel are easy to use in clinical practice. in this study, we evaluated the efficacy of bard score and guha's simple panel as a noninvasive fibrosis marker in korean nafld patients. methods: data from 79 patients with biopsy-proven nafld in seoul national university hospital from 2000 to 2007 were used. bard score and guha's simple panel were calculated by using clinical and biochemical data and were compared with the histological fibrosis stages. results: stage 0 fibrosis were found in 67 patients, stage 1 in 4, stage 2 in 2, stage 3 in 1 and stage 4 in 5. the relationship between fibrosis stage and bard score ( = 0.43, p < 0.001) was statistically significant. all patients with advanced fibrosis (stage 3-4) had bard score greater than 2. mean values from original guha's simple panel for no fibrosis were not different between the patients with and without fibrosis. however, after adjusting coefficients by logistic regression analysis, the differences in mean values became statistical significant (p < 0.001). conclusions: our data suggest that bard score may be effective for detecting high risk patients for advanced fibrosis, and modification of coefficients within the guha's simple panel may be needed to use as a fibrosis marker in asian nafld patients. s.k. mohan 1 , s. subramaniam 2 , s. subramaniam 3 1 assistant professor, department of biochemistry, saveetha medical college & hospital, saveetha university, t.n, india., 2 consultant, department of biochemistry, apollo hospitals, chennai, t.n, india. , 3 department of biochemistry, apollo first med hospitals, chennai, t.n, india. background: non-alcoholic fatty liver disease (nafld) covers a spectrum of liver diseases from simple fatty infiltration to progressive fibrosis. non-alcoholic steato hepatitis (nash) is a severe form of nafld and progresses to the end stage of liver disease. it is becoming the leading cause for referral to liver clinics in most areas. the prevalence of nafld in indian population is estimated around 7 -13%. the nafld has the potential to progress to hepatocellular carcinoma or liver failure, both events that ultimately lead to early death. aim: to evaluate the combination of inter cellular adhesion molecule -1 (icam -1), adiponectin and type-iv collagen, a new biomarker profile for nash in patients with nafld. methods: 76 patients with nafld and age & sex matched 68 normal healthy individuals as controls were selected for this study. levels of serum icam -1, adiponectin, type-iv collagen, lipid profile and liver function test parameters were estimated in patients and compared with controls. results: serum icam -1 & type -iv collagen levels were significantly increased in patients with nash among the nafld patients compared to controls. the serum adiponectin levels were significantly reduced in patients with nash among the nafld patients compared to controls. compared to liver function test parameters and lipid profile levels, nash profile has got positive negative predictive value among the nafld patients. conclusion: in patients with nafld, nash profile test -a simple, noninvasive and reliable to predict the presence or absence of nash. background/aim: oxidative stress and cytokines plays an important role in the pathogenesis of nonalcoholic fatty liver disease (nafld). aim of study was to assess lipid peroxidation, serum levels of transforming growth factor-( tgf-) and tumor necrosis factor-( tnf-) in patients with nafld and compare it with patients of chronic viral hepatitis (cvh) and healthy controls (hc). methods: lipid peroxidation was studied by estimating plasma malondialdehyde (mda) levels as per the methodology described by buege and aust and tgf-& tnf-levels were measured by elisa kits (ray biotech, usa, & diaclone, uk) in the stored sera in 10 biopsy proven patients with nafld (m: 4, f: 6, mean age: 41.7 ± 11.0 yrs), 15 patients with cvh ( m:14, f:1, mean age: 33.7 ± 11.4 yrs) and 5 hc (m:5, mean age: 25.2 ± 2.7 yrs). results: there was no difference in mean plasma mda levels amongst patients with nafld (17.28 ± 3.6 mol/l), cvh (15.29 ± 2.3 mol/l) and hc (16.79 ± 1.2 mol/l). serum tgf-levels between nafld (0.56 ± 0.41 ng/ml) and cvh (0.52 ± 0.25 ng/ml) patients and hc (0.58 ± 0.57 ng/ml) were also comparable. though patients with cvh (17.2 ± 27.0 pg/ml) and nafld (7.5 ± 6.3 pg/ml) had higher levels of tnf-than hc (5.5 ± 1.1 pg/ml), the difference was not significant statistically. conclusion: lipid peroxidation, tgf-and tnf-need to be studied in a larger number of patients with nafld. background/aim: burnt out nonalcoholic fatty liver disease (nafld) may be responsible for cirrhosis and hepatocellular carcinoma (hcc) in the absence of other causes. aim of this study was to evaluate the surrogate markers of nafld in patients with cryptogenic cirrhosis (cc) and cryptogenic hcc (chcc). methods: sixty five patients with cc and 31 patients with chcc were analyzed for the presence of abnormal body mass index (bmi) and type 2 diabetes mellitus (dm aim: to investigate the relation of phosphatidylethanolamine n-methyltransferase pemt gene g175a single nucleotide polymorphism (snp) with the susceptibility to nonalcoholic fatty liver disease nafld . methods the genotypes and allele frequencies of pemt exon 8 snp g175a were analyzed by using pcr-rflp in 51 nafld patients and 50 controls. results: the g to a variation of the pemt gene g175a snp was significantly higher in nafld group compared with controls. the frequencies of gg ga and aa genotypes were 58.8 39.2 and 2.0 in nafld and 78.0 22.0 and 0.0% in controls (p=0.038 . the a allele of the pemt gene was significantly more frequent in nafld group (21.6%) than that (1.0%) in controls p=0.042 .there were significant differences in serum levels of cholesterol, triglyceride, hdl-c and ldl-c between gg and ga/aa genotypes p <0.05 . n. assy 1,2 , g. lipez 3 , s. korem 3 , m. grozovski 3 1 sieff hospital, safed, israel, 2 2technion institute, faculty of medicine, haifa, israel, 3 ort braude college, karmiel, israel background: previous studies reported increase in serum protein c and decrease in serum paraoxonase levels in patients with non alcoholic fatty liver diseases (nafld). conclusion people with pemt gene g175a snp were more susceptible to develop nafld aim: 1) determine whether there is a relationship between nafld, protein c and paraoxonase levels in quiescent and in regenerating rats fatty liver 2) determine the effect of isa on hepatic "protein c" and paraoxonase mrna. pe439 methods: forty-eight sd rats were treated with fructose enriched diet (fed), or fed with metformin (2 mg/kg/d), fed with rosiglitazone (3 mg/kg/d), or the combination of both drugs for 5 wks. 30% phx was performed at wk 5. protein c, paraoxonase mrna expressions, lipids, mda were measured before and 24 hours after phx. results: hepatic "protein c" mrna was higher in rats with fatty liver than control rats (+105%, p<0.01) whereas hepatic paraoxonase mrna was lower in rats with fatty liver than control rats (-28%, p<0.005). hepatic protein c and paraoxonase mrna increased in rats with fatty liver in regeneration (+116%, p< 0.01, and +15%, p<0.01 respectively). the combination of metformin and rosiglitazone decreased hepatic protein c expression at 24 hours after phx by -170% (p<0.001) and increase paraoxonase mrna by +50% (p<0.01). serum paraoxonase correlates with serum protein c (r=-0.2), mda (r=0.4), background: non-alcoholic steatohepatitis (nash) is a type of non-alcoholic fatty liver disease (nafld), and may progress to hepatic fibrosis and cirrhosis. the pathogenesis of nash remains unclear. the aim of this study was to explore the arginase change in the progress of steatohepatitis in rats. methods: male sd rats weighing 70-80g were obtained. twenty animals were randomly divided into two groups. in the model group, five animals were fed with high lipid forage that includes 3% cholesterol and 20% lard for 6 weeks, five were fed for 12 weeks, while the control group ate normal foods. the animals were sacrificed after 6 weeks. the animals were sacrificed after 6 weeks and 12 weeks. liver and blood serum were collected while the serum levels of alt, ast, tg and tc were measured. the pathology of liver was observed by he staining. western blot was used to investigate the expression of arginase in control and model group. tg (r=-0.23). conclusion: hepatic "protein c" mrna levels are high at baseline, up regulated during liver regeneration and decrease after treatment with (isa) whereas hepatic paraoxonase mrna levels are low at baseline, up regulated during liver regeneration and increase after treatment with isa. results: vacuolization were observed extensively in hepatic cells in the model group after 6 weeks and 12 weeks of high-fat diet. it is demonstrated that rats fed with high-cholesterol food are indeed fatty liver models. western blot showed that the level of arginase ii increase in the liver of model group rats as compared to the control group. furthermore, the level of arginase was higher in liver samples obtained from model rats that were 12 weeks on a fat diet as compared to rats that were only 6 weeks on the same diets. conclusion: the level of arginase ii was altered in the progress of non-alcoholic steatohepatitis in rats suggesting that arginase ii is putative biomarkers and may represent new targets in the development of therapeutic strategies against fatty liver disease hepatic fibrosis and cirrhosis. methods: c57bl6/j mice were fed with mcd diet to induce hepatic fibrosis and rosiglitazone was given in treated group. effect of rosiglitazone was assessed by comparison of the severity of hepatic fibrosis in liver sections, expression of mmp-2/9, timp-1/2 mrna and protein detected by rt-pcr and western blot respectively. the ethanolic extract of fructus schisandrae chinensis decreased hepatic triglyceride level in mice fed with a high fat/cholesterol diet results at week 8, fibrosing nash models showed severe hepatic steatosis, infiltration of inflammation and fibrosis, which is associated with down-regulated mmp-2/9 mrna and protein, up-regulated timp-1/2 mrna and protein. rosiglitazone significantly reduced mcd-induced fibrosis by induced mmp-2/9 expression and reduced timp-1/2 expression by activating ppar . s.y. pan 1 , z.l. yu 2 1 beijing university of chinese medicine, 2 hong kong baptist university effects of the ethanolic extract of fructus schisandrae chinensis (etfsc) on serum and liver lipid contents were investigated in mice fed with normal diet or high fat/cholesterol diet for 8 or 15 days. single dose of etfsc (1 or 5 g/kg/day, i.g.) increased the serum triglyceride (tg) level (40 and 142%, respectively), but decreased hepatic total cholesterol (tc) level (15 and 16%, respectively) in normal mice. the hypertriglyceridemia produced by etfsc was suppressed by the co-administration of fenofibrate. the induction of hypercholesterolemia by high fat/cholesterol diet caused significant increases in serum and hepatic tc levels (up to 165%) and hepatic tg levels (up to 528%) in mice. etfsc treatment (1 or 5 g/kg/day for 7 days, i.g.) significantly decreased the mouse hepatic tg level (by 35%) and slightly increased the hepatic index (by 8%). whereas fenofibrate treatment (0.1 g/kg/day for 7 days, i.g.) significantly lowered the hepatic tg level (by 61%), it significantly elevated the hepatic index (by 77%) in hypercholesterolemic mice. the results indicate that etfsc treatment can invariably decrease hepatic tg in hypercholesterolemic mice, suggesting its potential use for fatty liver treatment. aim: to investigate the influence of multiple gene polymorphisms in the susceptibility of nafld. methods: the data of single nucleotide polymorphisms (snps) in 201 nafld patients who had at least one of the genetic variations at the sites of tnf--238, adiponectin -45 and leptin-2548 were analyzed. the genotypes were determined by using pcr-rflp. our previous studies showed that the variations of these sites increased the susceptibility of nafld. results: the prevalence of nafld in adiponectin variation alone group (n=45) was 35.6%; in tnf-alone group (n=33) 42.4%; in leptin alone group (n=54) 35.2% (p>0.05). in comparison with the above groups with single snp, the prevalence of the groups with two gene variations of tnf-plus adiponectin (57.9%, n=19) increased significantly (p<0.05). however the prevalence of other two groups i.e. adiponectin plus leptin (34.6%, n=26) and tnf-plus leptin (40.0%, n=15) did not differed significantly from those of groups with single snp (p>0.05). the prevalence in the group with three gene variations (55.6%) differed significantly from all (p<0.05) except that of tnf-plus adiponectin group (p>0.05). the metabolic features of the nafld patients in the 7 groups mentioned above were not different significantly (p>0.05). conclusion: nafld is a polygenic disease. multiple gene polymorphisms may, but not always, increase the susceptibility of nafld. chronic hepatitis b patients with nonalcoholic fatty liver disease r.d. zheng 1 , c.r. xu 1 , j. chen 1 , b.f. chen 1 1 southeast hospital background: to investigate clinical pathological characteristic in hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease (nafld). methods: we measured fasting blood glucose, insulin, triglyceride, cholesterol, alanine aminotransferase (alt), aspartate aminotransferase (ast) in hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease (nafld). and we detected hepatitis b virus marker, hbv-dna, counted body mass index, insulin resistance index and observed pathological characteristic. all these patients with diagnosis were confirmed by clinical and pathological evidence. result : the body mass index, homeostatic model assessment (homa) of insulin resistance, fasting blood glucose, insulin, triglycerides, cholesterol, were significantly higher in hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease (nafld) than hbeag negative chronic hepatitis b patients. but the alanine aminotransferase (alt), aspartate aminotransferase (ast), hbv dna levels were significantly lower in hbeag negative chb patients with nafld than in hbeag negative chronic hepatitis b patients. histologic features in hbeag negative chronic hepatitis b(chb) patients with nonalcoholic fatty liver disease (nafld) are in zone 3 predominate macrovesicular steatosis and mild inflammatory infiltrate in portal region. conclusion: the hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease, whose hepatic steatosis changes are mainly caused by the metabolic factors. to carry out liver biopsy selectively for the patients with hbeag negative chronic hepatitis b having metabolic factors, which is helpful for early diagnosis in hbeag negative chronic hepatitis b (chb) patients with nonalcoholic fatty liver disease (nafld). aims: to investigate the preventive effect of cordyceps sinensis and its possible mechanism on apoptosis of nafld. methods: rats were randomly divided into basic diet group (b group), pathologic group (nash group) and cordyceps sinensis group(cs group).the latter two groups were administered with high-fat diet to establish nafld animal models. cs group were treated with cs at the 9th week after high fat diet. rats were sacrificed at the end of the 18th week. biochemical examination were used to detect superoxide dismutase (sod) of liver tissue. hepatocyte apoptosis was assessed in each group using the tunel assay and immunohistochemistry for activated bax bcl-2 caspase-3 and nf-kb p65. results: (1) compated with the b group, severe hepatosteatosis, inflammative necrosis and local fibrigenisis were showed in liver of nfsh group. sod lever was significantly decreased (p<0.01) and tunel-positive cells were significantly increased (p<0.01). immuunohistochemistry test demonstrated active bax caspase-3was increased (p<0.01) while no apparent change was observed in bcl-2. (2) in cs group, only diffusive steatosis but not inflammation or fibrosis was found. sod lever was increased than that of nash group (p<0.05). tunel-positive cells and active bax caspase-3 were significantly decreased (p<0.05 p<0.01) that those of nash group. bcl-2 and nf-kb p65 were increased (p<0.01) than those of nash group. conclusions: hepatocyte apoptosis is a prominent feature of nafld. cordyceps sinensis may be useful as an antiapoptosis theraphy in this syndrome through increasing activity of sod, decreasing express of bax and increasing express of bcl-2 and nf-kb p65. background: non-alcoholic steatohepatitis (nash) is a leading cause of chronic liver disease. insulin-sensitizing , anti-inflammatory and anti-fibrotic effect of thiazolidinediones support their use in the treatment of nash. we aimed to evaluate the efficacy of thiazolidinediones in the treatment of nash. methods: we have identified randomised clinical trials, evaluating the efficacy of thiazolidinediones versus placebo in nash, through medline, embase, ami, cochrane central register of controlled trials. data were abstracted from each study and disagreements were resolved by consensus. dichotomous outcomes were reported as relative risk with 95% confidence interval based on fixed-effects model. results: we included three trials, two evaluating pioglitazone and another rosiglitazone. a total of 171 patients were involved in the analysis. thiazolidinediones was noted to improve liver function tests. it was effective in the reduction of steatosis among patients with nash (rr 0.67, 95% ci 0.52-0.87). it was found to be beneficial in improving ballooning necrosis (rr 0.79, 95% ci 0.66-0.95). it was also found to improve lobular inflammation (rr 0.72, background: it is well known that the weight reduction is effective for alt normalization in patients with non-alcoholic fatty liver disease (nafld). the necessary condition for alt normalization is still unclear. to clarify the necessary and sufficient condition for alt normalization, we investigated the effects of body fat decrease in nafld patients by body composition analyzer. methods: forty-six nafld patients (23 male, 23 female, mean age 49.8±12.9 years old) with abnormal alt levels were evaluated. the volume of skeletal muscle, body fat and bmr were examined by using the body composition analyzer (in body 720; biospace co. ltd., tokyo japan). all patients were received an individualized diet consultation by dietician every 4 weeks for 6 months. daily energy was bmr (basal metabolic rate) x1.2 kcal and protein was 1.0-1.2g per ideal body weight. result: twenty-eight of 46 patients (60.9%) were achieved normal alt level. in alt normalized group, the body weight and fat loss were 3.6±2.3 kg, 3.0±1.5kg (2.8±1.7 %body fat) respectively. on the other hand, in cases with alt remained abnormal level, the body weight and fat loss were 0.5±1.5kg, 0.4±1.6kg (0.5±1.6 %body fat). conclusion: our results demonstrate that the fat loss of 3 kilograms or more was necessary to normalize alt level in nafld patients. a. somani 1 , s. somani 2 , a. jain 3 , v. dixit 3 1 navjeevan hospital, 2 suvidha, 3 background : nafld is often clustered within families and the causes include both genetic and environmental factors. family studies done thus far have been limited by small sample size. to examine the familial patterns , we performed a prospective study to see (a) whether nafld is more common in first degree relatives (b) genetically determined risk factors associated for clustering. methods: first degree relatives of histologically confirmed nafld patients and spouses (controls) were included after excluding other causes of fatty liver. those having raised transaminases >3 months or sonographic examination consistent with fatty liver, had undergone liver biopsy for histological confirmation. they were divided into three groups. group i patients group ii first degree relatives group iii spouses results: nafld was more prevalent among first degree relatives then spouses (37% and 17%, p<0.01). anthropometric measurements, systolic and diastolic blood pressure, lipid profile and liver function tests were comparable in three groups. homa-r was similar in group i and ii (p=0.073), but was significantly different in group i and iii (p= 0.0001) and group ii and iii (p=0.0001) respectively. metabolic syndrome was present in >70% of patients and were comparable in three groups except for fasting glucose > 110, which was present in 76%, 77% and 57% of patients in group i, ii and iii respectively. majority (>50%) of our patients among groups i, ii and iii were having only steatosis while nash was present in 20%, 13% and 14% of patients. a. somani 1 , v. dixit 2 , a. jain 2 , s. somani 3 1 navjeevan hospital, 2 ims, bhu, varanasi, 3 suvidha background: normal levels of alanine aminotransaminase (alt) have been demonstrated in nafld patients. alt levels are also modulated by age, gender, bmi, fasting glucose, and serum triglyceride levels. we performed a prospective study of patients with histologically confirmed nafld and having alt < 1.5 times and compared them with those having raised alt to determine (a) clinico-pathologic features of nafld patients with normal alt (b) to observe any differences between them. methods: patients with fatty liver on sonography had under gone biopsy for histological confirmation after excluding other causes of fatty liver. participants were divided into two groups (a) those having alt > 1.5 times normal (n=97) (b) those having normal alt (n=47) results: mean age was comparable with slight male predominance. there were significant differences in anthropometric measurements like bmi (p=0.0001) and whr (0.90±3.57 and 0.88±3.23, p=0.0071). mean bp, lipid profile, fasting glucose, insulin, and homa r were comparable. there were significant differences in both mean ast (50.2±5 and 37.4±3.21, p=0.0001) and alt (104±7.29 and 69.9±11.5, p=0.0001) levels. metabolic syndrome was present in >75% of patients and individual components were comparable except for increased waist circumference which was significantly more in those with raised alt (78.35% and 44.68%, p<0.001). majority of our patients were having only steatosis, while nash was present in (27.82% and 8.5%, p<0.05) of patients. conclusion: nafld can exist in patients with normal alt values. although more work is needed to determine who should be screened for nafld and how such individuals should be evaluated, this study is a step toward the identification and characterization of nafld patients with normal alt. we can suggest that patients having metabolic syndrome or insulin resistance, despite having normal alt, should be screened for nafld. also alt values should be adjusted for variables like bmi to appropriately screen nafld patients. background: scientific evidence has demonstrated that traditional chinese medical (tcm) approaches and products can be beneficial for managing non-alcoholic fatty disease (nafld), but few rigorous criteria of patterns of tcm therapy are available to guide practitioners in deciding the cam interventions. objectives: to evaluate criteria of patterns of tcm therapy for the management of nafld identified by biomedicine. methods: literature research, clinical epidemiological investigation and mathematical statistics were employed to make information collecting tables and to establish database. descriptive analysis, factor analysis, and cluster analysis were involved. results: (1) background/aim: serum uric acid level has been suggested to be associated with factors that contribute to the metabolic syndrome. the aim of this study was to investigate the association of serum uric acid level with nonalcoholic fatty liver disease (nafld). methods: a cross-sectional study was performed among the employees of zhenhai refining & chemical company ltd., ningbo, china. results: the study included 8925 subjects (6008 men) with a mean age of 43 years. the prevalence rate of nafld and hyperuricemia was 11.78% and 14.71%, respectively. nafld patients had significantly higher level of serum uric acid than controls (370.3 ± 86.6 vs. 321.1 ± 82.6 mol/l; p < 0.001). the prevalence rate of nafld was significantly higher in the subjects with hyperuricemia than those without hyperuricemia (24.75% vs. 9.54%; p < 0.001), and the prevalence rate increased along with serum uric acid levels (p value for trend < 0.001). multiple regression analysis showed that hyperuricemia was associated with increased risk for nafld (odds ratio [or]: 1.291, 95% confidence interval [ci]: 1.067 -1.564; p < 0.001). conclusion: serum uric acid level is significantly associated with nafld, and increased serum uric acid level is an independent risk factor for nafld. background: development of fatty liver is believed to be an early and reversible consequence of excessive alcohol consumption. however, the cellular and molecular events in the early development of alcoholic liver diseases (ald) and the contributory effects of a high fat diet are not fully understood. methods: this study was designed to quantify specific enzymatic and cytokinetic activity as well as the development of hepatic steatosis in a rat model of alchohol-induced liver injury without high fat diet. results: ethanol-fed rats exhibited high blood ethanol levels (0.85+0.31%) and significant increases in serum alt (67.7+ 13.4 unit/l), ast (136.3+ 60.2 unit/l), and alp (400+108.9 unit/l) when compared with control rats (p<0.01, respectively). histopathological examination found unevenly raised knodell scores (5.33+1.15 in the ethanol-fed livers vs. 0.33+0.58 in control), which were characterized by scattered hepatocyte ballooning, portal inflammation and collagen fiber deposition. however, typical steatosis lesions were absent. qpcr demonstrated up-regulation of genes in the ethanol-fed livers, including hepatocyte metabolism enzymes/receptor (adh1, p<0.05; cytochrome p450 2e1, cyp2e1, p<0.05; gsta2, p<0.01; ppar , p<0.05), and genes coding for pro-inflammatory cytokines (il-1 , p<0.01 vs. control livers; tnf-p<0.05; tgf -, p<0.05; rantes p<0.05), ecm components and proteinases (collagen-1, p<0.01; sma, p<0.01; mmp -9, p<0.05 and timp-1, p<0.05). conclusion: chronic administration of ethanol to rats without high fat diet productively induces alcohol hepatitis in the absence of fatty liver, suggesting that alcohol hepatitis may precede steatosis in the development of ald. the aim of the present study was to evaluate the changes of several cytokines associated with inflammatory liver disease and liver regeneration by molecular adsorbent recirculating system (mars) in 15 aclf patients versus 15 patients treated with medical standard therapy (smt) that presented alcoholic liver disease etiology and similar model end-stage liver disease (meld). methods: mars group: fifteen (10 male and 5 female) patients were treated with mars® (gambro). five patients were excluded by study.the number of mars applications was about 9, the length of applications was about 10h. smt group: fifteen patients (10 male and 5 female) were treated medical standard therapy such as prophylaxis against bacterial infections, albumin and fresh plamsa and judicious use of diuretics. three patients were excluded by the study. the patients were valued during 30 days from inclusion with a survival follow up a three months. results: mars group: we observed a significant changes in levels of il-6(p<0.01), il-1(p<0.002), il-8(p<0.04) and tnf-alfa (p<0.03) in association with improvement of hepatic growth factor (p<0.001). the patient's survival at three months was 50%. smt group: we observed only a significant changes in il-1(p<0.01) and tnf-alfa (p<0.2). the patient's survival at three months was 33%. conclusion: the mars liver support device has corrective effects on disturbed pathophysiology of aclf and may be used to enhance spontaneous recovery or as bridge to transplant. a study of protective effect of centella asiatica in 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (mptp)-induced liver injury n. haleagrahara 1 , s. chakravarthi 1 , p. kumar 1 1 international medical university, malaysia background: centella asiatica has been used for centuries as a medicinal herb for wound healing, memory enhancement, cancer, vitality, respiratory ailments, psoriasis and eczema, revitalizing connective tissue, burn and scar treatment, skin infections, arthritis, rheumatism, periodontal disease, varicose veins, hypertension, sedative, anti-stress, anti-anxiety, aphrodisiac, and as immune booster. results: ppc significantly reduced hepatocyte damage, hepatitis, and hepatic fibrosis, but did not affect steatosis. phosphorylation of apoptosis signal-regulating kinase 1, p38 mitogen-activated protein kinase, and protein kinase c, as well as activation of nuclear factor-kappa b, were markedly suppressed by ppc. these effects were likely a consequence of decreased oxidative stress through down-regulation of reactive oxygen species (ros)-generating enzymes, including cytochrome p450 2e1, acyl-coa oxidase, and nadph oxidases, in addition to restoration of ethanol-induced increases in toll-like receptor 4 and cd14. ppc also decreased the pro-apoptotic proteins bax and truncated bid, thus inactivating mitochondrial permeability transition. furthermore, ppc suppressed overexpression of transforming growth factor-1 and hepatic stellate cell activation, which retarded hepatic fibrogenesis. conclusion: ppc exhibited anti-inflammatory, anti-apoptotic, and anti-fibrotic effects on ald as a result of inhibition of alcohol-induced ros production. background: dysctamnus dasycarpus has used for the promotion of health in south korea. but, there were rare a report concerning the hepatotoxicity. we report cilinical features of liver injury by dysctamnus dasycarpus. method: eighteen patients diagnosed as acute toxic hepatitis by dysctamnus dasycarpus in chungnam national university hospital between january 2005 and arpil 2008 was enrolled. toxic hepatitis was diagnosed by rucam score ( 4). the medical records were reviewed, retrospectively. result: eleven patients (61%) were female and the mean age was 53.5. most common symptom was jaundice. initial laboratory findings were as follows(mean value): wbc 6126/ul, hemoglobin 13.4 g/dl, platelet 212×10 3 /l,alt 1375iu/l, total bilirubin 11.3 mg/dl, alkaline phosphatase 159 u/l, ggt 244u/l, prothrombin time(inr) 1.1. the mean hospitalization was 21.5days. peak laboratory findings were as follows: alt 1382iu/l, total bilirubin 15mg/dl. recovery time of each biochemical finding was as follows: alt 37days, total bilirubin 39.4 days. recovery rates of alt and total bilirubin were 27.8% and 38.9%, 89.9% and 89.9% at 4 weeks, 8 weeks, respectively. the main biochemical pattern of hepatotoxicity was hepatocellar (72.2%) type. prednisolone was prescribed in six patients. progressive anemia and thrombocytopenia were detected in one patient diagnosed as pure red cell aplasia. other one patient had prolonged jaundice (117 days). but, all patients had recovered without sequelae. conclusion: in south korea, liver injury by dysctamnus dasycarpus was more frequent in women. the main pattern of hepatotoxicity was hepatocelluar type. most patients had prolonged icteric phase and hospitalization. patients were recovered by supportive management after drug cessation or prednisolone therapy. in korea, traditional medicine that is based on the use of herbal medicine developed from a long time ago. however, clinical study of the herbal medicine is not conducted in a structured manner. we report three cases of toxic hepatitis caused by the intake of dictamnus albus. the first patient, a 44 year old woman was admitted due to nausea after ingestion of liquor containing dictamnus albus for 2 months. total bilirubin was 2.55 mg/dl ast/alt 774/1,424 iu/l on admission. liver biopsy observed hepatocyte necrosis and cholestasis. the elevated bilirubin and transaminase returned to normal 2 weeks later after cessation of dictamnus albus. the second patient, a 64 year old man was admitted due to jaundice after ingestion of boiling dictamnus albus for 3 months. total bilirubin was 11.37 mg/dl ast/alt 2,010/2,522 iu/l on admission. liver biopsy observed pericellular fibrosis and necrosis. the bilirubin decreased slowly compared to the transaminase and normalized 3 months later after cessation of dictamnus albus. the third patient, a 48 year old man was admitted due to jaundice after ingestion of liquor containing dictamnus ablus for 1 month. total bilirubin was 12.8 mg/dl ast/alt 1,097/1,869 iu/l the hepatocyte necrosis was observed by liver biopsy. the elevated bilirubin and transaminase levels normalized 1 month later after cessation of dictamnus albus. all patients had negative viral markers and non-specific ultrasonographic findings. the above mentioned three cases demonstrate that liver may have been damaged by dictamnus albus, which indicated clinical characteristics. background/aims: cmili poses a diagnostic challenge as no tests are available to confirm the causality. the aims of this study were 1) to evaluate clinical features and patterns of cmili and 2) to assess the likelihood of causality among patients with liver impairment and exposure to chinese medicine (cm) by a multidisciplinary approach. method: between 5/2005 and 8/2007, patients who had liver derangement and cm or proprietary cm exposure within six months managed in the united christian hospital were studied. clinical features and the cm were reviewed by a multidisciplinary team involving a hepatologist, a toxicologist and cm experts. literature search of relevant herbs in chinese and western journals were performed. cm samples or residue were sent to toxicology laboratory for analysis to look for any toxic constituents, adulterant or contaminant. the likelihood of causality was ranked by various experts independently and disagreements were settled by a consensus meeting. results: there were forty-six cases of suspected cmili, nineteen cases with alternative causes of liver diseases were excluded. twenty-seven cases of cmili proceeded to detailed analysis. median age of patients was 51 (21-76) with female predominance. the median duration of cm exposure to presentation was 20 (1-150) days. majority of them (82%) had hepatocellular liver injury pattern. one case of adulteration with nsaid and erroneous substitution of herb was identified respectively causality were classified as unlikely, possible, probable and highly probable in 3, 12, 8 and 3 patients respectively. conclusion: a multidisciplinary approach allows systemic evaluation of suspected cmili. mouse model i. nassar 1 , t. pasupati 1 , i. segarra 1 , j.p. judson 1 1 international medical university, kuala lumpur, malaysia background: imatinib, a selective tyrosine kinase inhibitor, exhibits drug interactions with other drugs that are metabolised via the cytochrome p450 pathway. acetaminophen, a widely used analgesic and anti-pyretic drug is also metabolised via p450 pathway. this study aimed to evaluate the nature of hepatotoxicity after co-administration of imatinib and acetaminophen in a preclinical mouse model. methods: four groups of male icr mice (30-35g) were used. the mice were administered either saline solution orally, imatinib 100 mg/kg orally (control), acetaminophen 700 mg/kg intraperitoneally (positive control) or co-administered imatinib 100 mg/kg and ip acetaminophen 700 mg/kg (study group). the mice (n=4 per group) were fasted overnight, dosed respectively and sacrificed at pre-determined time intervals of 15, 30 minutes, 1, 2, 4, 9 and 12 hours and liver samples obtained by dissection. h&e stained liver sections (3µm thick) were histopathologically analysed. results: the liver samples showed reversible cell damage like feathery degeneration, microvesicular fatty change, sinusoidal congestion and pyknosis, with both imatinib and acetaminophen, administered separately. the damage increased gradually with time, peaked at 2 hours and then resolved completely by 6 hours. liver samples showed irreversible damage (cytolysis, karyolysis and karyorrhexis) when both drugs were administered concurrently, the damage increased with time and had not resolved after 12 hours duration. conclusion: co-administration of acetaminophen and imatinib increased the hepatoxicity caused by acetaminophen and imatinib to become irreversible. this may be due to the fact that both drugs are metabolised by the cytochrome p450 pathway in the liver. background: a higher risk of antituberculosis drug (att) induced hepatotoxicity has been reported in indian subcontinent compared to the western counterparts. slow acetylator genotype of n-acetyltransferase2 (nat2) and ci genotype of cytochrome p4502e1 (cyp2e1) gene are two known risk factors associated with this disease. cyp2e1 gene encodes a rifampicin inducible enzyme which increases hepatotoxicity. therefore slow acetylation of isoniazid and simultaneous use of rifampicin may augment the toxicity of isoniazid. objectives: to analyze the allelic distribution of nat2 and cyp2e1 gene in patients of pulmonary tuberculosis who developed att induced hepatitis materials and methods: the study included cases of pulmonary tuberculosis (190) and att induced hepatitis (95). polymorphism of nat2 and cyp2e1 gene was studied by pcr-rflp method in both these groups. results: occurrence of att hepatotoxicity was 17.89%. there was a higher prevalence of slow acetylator genotype particularly nat2*5/*7 and nat2*6/*7 in patients with hepatotoxicity compared to patients without hepatotoxicity (72.09% vs 44.2%, p value < 0.05). no association of cyp2e1 rsai polymorphism could be considered with att hepatotoxicity. however, drai c/d genotype of cyp2e1 appears as a risk factor for predicting the occurrence of antituberculosis drug induced hepatitis (or 5.06, p value < 0.05). conclusion: the study demonstrates that patients with slow acetylator genotype particularly nat2*5/*7 and nat2*6/*7 and heterozygous mutant c/d genotype of cyp2e1 gene are predisposed to develop antituberculosis drug induced hepatotoxicity. regular monitoring of clinical and biochemical profile may be considered in these patients when they receive antituberculosis treatment. background: drug-induced liver injury is the most common adverse drug reaction. we often use two kinds of diagnostic scales to evaluate suspected patients. however, we still can't diagnose accurately without the direct drugs history and the pathological evidence. methods: twenty-seven drug-induced liver injury cases with liver biopsies from 2001 to 2008 were reviewed retrospectively by maria and japanese scale. result: there were 11.1% of cases with increasing eosinophils. herbs (29.63%) were the most common suspected drug and unknown drugs intake history (14.81%) were described in these cases. the high possibility and possibility were 25.93%, 29.63% by maria scale and 85.19%, 3.7% by japanese scale, respectively (p=0.015). conclusions: japanese scale seems more sensitive than maria scale in these cases. however, there are still some definite cases ignored as low possibility due to absence of obvious drug using history. early treatment and suspected drugs prohibition interferes the outcomes of the two diagnosis systems and lead to a false result. it is still a clinical challenge without strong drug using history or pathological evidence of liver biopsies to diagnose the drug-induced liver injury quickly and accurately. background: previous study suggested that oxidative stress may be an important mediator of methamphetamine-mediated tissue injury. the study was to examine the mechanism of antioxidant activity and methamphetamine-mediated liver injury. materials and methods: the 25 days old male sprague-dawley rats were subcutaneous injected daily with methamphetamine (10 mg/kg body weight) for 15, 30, 60 and 120 days. control group received equal volumes of vehicle. the liver tissues were extracted to measure the activities of sod, catalase, glutathion reductase (gr), and glutathione peroxidase (gpx), and the level of glutathione. western blot were used to measure the expression of rho and phosphor-ezrin-radixin-moesin (p-erm). results: compared with vehicle group, treated with methamphetamine for 15 and 30 days, the activities of liver sod, gpx, and catalase were significantly decreased. in 60 and 120 days group, the activities of antioxidant enzymes of methamphetamine-treated liver was not different from that of vehicle group. the levels of glutathione production also had the same trend. the activities of gpx and catalase on vehicle group gradually reduced following the days of treatment. however, administration of methamphetamine resulted to a lower activity of catalase through the treated days. there was no difference on the activity of gr between vehicle and methamphetamine group. the expression of rho and p-erm were also increased by methamphetamine treated for 30 days. conclusion: these results suggested the methamphetamine lead to liver remodeling via decreased antioxidant activity. finally, the situation of mechanism needs taking in advantage discussion. background: to observe intervening effects of preventive and theraptical treatment of radix sophorae tonkinensis's polysaccharides(rstp) on alpha-naphthylisotheganate(anit)-induced cholestasis in mice. methods: kunming mice intoxicated with anit 50mg/kg orally and treated with rstp 50mg/kg for 7 days before anit exposure and for 2 days after anit exposure respectively, the general condition,mortality rate and serum alt activity are obeverated. result: it was found that by preventive treatment the general condition and mortality rate were improved, serum alt activity reduced.by therapeutic treatment,the general condition deteriorated,mortality rate and serum alt activity increased. conclusion: the preventive treatment of rstp reduce the liver damage due to increasing the anti-stress ability such as the antioxidant capacity,its therapeutic treatment increase the injuried liver damage due to increasing the non-specific immune response and aggregating the preexisting liver inflammation. background: the product's instruction pointed out that in some patients polyphenolic acids' salt from salvia miltiorrhiza(ppas-sm) may lead to a temporary increase in serum alt activity.so we observe effects of ppas-sm on alpha-naphthylisotheganate(anit)-induced cholestasis in mice. methods: 24 hours after intoxicated with anit 50mg/kg orally, kunming mice were treated with ppas-sm 50, 25,10mg/kg/days for 2 days orally, then serum alt activity was measured. result: all doses of ppas-sm led to rise of serum alt activity in mice, most obvious in group of high dose.but the general situation and mortality rate did not increase significantly. conclusion: ppas-sm lead to rise of serum alt activity in mice with damaged liver.the auther suggests as a double-edged sword,the antioxidant ppas-sm may have a prooxidative effect in some condition too. *this project was supported by grants from shanghai municipal education commission under high school high-tech characteristic development programme (no smec finance (2005) 81) pe467 a. somani 1 , a. jain 2 , v. dixit 2 , s. somani 3 1 navjeevan hospital, 2 ims, bhu, varanasi, 3 suvidha introduction: hepatic encephalopathy, a complex neuropsychiatric syndrome secondary to acute liver failure, chronic parenchymal liver disease or portal-systemic shunting, may possibly develop through mediators of endotoxin and tumor necrosis factor-alpha (tnf-). several studies have shown that serum levels of (tnf-) are significantly elevated in patients with acute and chronic liver diseases, where these elevations are independent of the etiology of the underlying disease. it has been shown that plasma levels of tnf-correlate with the severity of hepatic encephalopathy (he) in fulminant hepatic failure. however, still there is very few published data regarding the relationship between serum levels of tnf-and the presence or severity of he in patients with chronic liver failure. methods: the aim of this study is to determine the relationship between serum levels of tnf-and clinical grades of he in patients with chronic liver failure. this prospective study included 149 consecutive male patients with alcoholic cirrhosis in various clinical grades of he (according to west haven criterion). detailed clinical, biochemical and sonographic examination was done in all patients. circulating levels of tnf-was measured using solid-phase elisa. results: the mean±sem values of serum tnf-at presentation in patients with mhe (n=37), grade 1 (n=17), grade 2 (n=41), grade 3 (n=44), and grade 4 (n=10) were 6.2±0.4, 9.5±0.6, 15±0.7, 26.3±1.7, and 46±5 .9 pg/ml, respectively. significant positive correlation was found between serum levels of tnf-and severity of he (correlation coefficient = 0.7). conclusion: from the present study we can suggest that there is significant relationship between tnf-and he in patients with alcoholic cirrhosis and it could be involved in its pathogenesis. background: acute hepatitis a (aha) is one of the most common infectious diseases and usually a self-limiting disease. although extrahepatic manifestations are not common, a few cases associated with acute renal failure (arf) have been reported. methods: we reviewed clinical features of aha patients complicated with arf (group a) and compared with non-complicated aha patients (group b). medical records of 191 patients with aha were reviewed between january 2003 and december 2007. we experienced 11 patients (5.8%) with arf associated aha. result: there were no differences between group a and group b in sex ratio and age. the peak value of alt (median: 6133 iu/l vs 1685 iu/l, p<0.001), alkaline phosphatase (median: 235 iu/l vs 201 iu/l, p=0.03), prothrombin time (inr, median 1.72 vs 1.09, p<0.001) was significantly higher in group a than b. nine patients (81.8%) recovered completely with hemodialysis (6 patients, 66.7%) and only conservative management (3 patients, 33.3%), while 1 patient underwent liver transplantation and 1 patient died due to fulminant hepatic failure. there were 4 patients who underwent kidney biopsy. two patients were diagnosed as acute tubular necrosis and 2 patient as acute interstitial nephritis and iga nephropathy. conclusion: aha patients with arf had higher alt and more prolonged prothrombin time. the prognoses were poorer than those without arf. however, arf patients with nonfulminant aha had a good prognosis with a proper treatment and should not be confused with hepatorenal syndrome. background/aims: to investigate the hev infection among different animals and people with special profession, and to analyse the genotype of hev isolated in this study. methods: serum and fecal samples were collected from various animals and people with special profession in the south suburbs of beijing. hev antigen and anti-hev antibody were detected by das-elisa. hev rna was extracted from fecal samples and amplified by rt-npcr. the nucleotide sequence homology and phylogenetics of hev strains isolated from swine were analysed. results: the anti-hev antibody positive rate of adult swine, cow, sheep and younger swine were 98.23% (222/226), 29.35% (54/184), 9.80% (20/204) and 60.73% (99/164), respectively. the hev antigen positive rates of adult swine, cow, sheep and younger swine were 2.65% (6/226), 4.35% (8/184), 1.45% (3/207) and 9.75% (16/164), respectively. the hev antigen and anti-hev antibody positive rate of professional group was 0.40% (1/247) and 42.51% (105/247) respectively. the hev rna positive rate of fecal samples from younger swine was 22.89 %(19/83). 16 of 19 samples were hev rna positive by pcr with primers of hev orf1 and orf2. the sequence analysis of the 16 samples showed that there were 2 groups designated as bj-1 (11/16) and bj-2 (5/16). the nucleotide homology of bj-1 and bj-2 was 99%. phylogenetic analysis of hev orf2 indicated that both of them belonged to genotype 4d. conclusion: phylogenetic analysis of hev orf2 indicated that hev isolated in the south suburbs of beijing belonged to genotype 4d. 6 bracops hospital brussels , 7 st. jan hospital, bruges , 8 chu brugmann, 9 chu sart tilman, liège , 10 gent university hospital , 11 zna middelheim, antwerp hepatitis delta virus is a subviral satellite requiring hepatitis b virus to propagate, usually leading to severe, chronic liver disease. as data on epidemiology and management practice of hdv infection in belgium are lacking, a retrospective and prospective, multi-centric questionnaire-based registry is performed in 2008. results of 32 patients are reported. background/ aims: hepatitis a is an acute infectious disease that is transmitted by fecal-oral root. because the incidence of hepatitis a has been increased in gwangju and chonnam province of korea recently, hepatitis a patients in chonnam national university hospital employees had been increased. so we investigated the seroprevalence of igg anti-hav in hospital empolyees less than 40 years old. methods: we analysed seroprevalence of anti-hav igg from 1,002 hospital employees (men: 190, women: 812) . serum alt and bilirubin at admission were 2,979 1,711 iu/l and 3.9 2.4 mg/dl, respectively. these levels were elevated up to 3,397 1,789 iu/l and 6.8 3.7 mg/dl, respectively. ana was positive in 81 patients (64.3%). age, duration from peak-alt day, duration from peak-bilirubin day, alt level, and peak-bilirubin level were not different between ana(-) patients and ana(+) patients. in the while, sex, duration from symptom-onset day, and bilirubin level, and peak-alt level were significantly different. in 51 (62%) of 81 patients with positive ana, ana was followed after 1 month and ana became negative in 38 patients (74.5%). among 13 patients with positive ana after 1 month, titer decreased from the baseline in 7 patients, showed no interval change in 4, and increased in 2. conclusions: positive ana result is not rare in patients with acute hepatitis a. it is considered that ana transiently appear during the course of acute hepatitis a and then, disappear with the improvement of acute hepatitis. (89), 2007(107). the clinical data such as sex, admission period, ast, alt, total bilirubin, prothrombin time, crp, alt normalization time did not show difference. just wbc and gtp were higher on 2008 group. the older age patients were more on 2008 group. the patients admitted mainly on april, may, june, july (84%) on 2008 while admitted even on past years. conclusion: acute hepatitis a ptients is increasing. it is occurring in older age people and mainly on specific period. the more concern to prevention should be needed. background: we analyzed the 5' non-translated region (5'ntr), non-structural proteins 2b and 2c of hepatitis a virus (hav) genome, whose mutations have previously been shown to be important for enhanced replication in cell culture systems, in order to align all of our data and examine whether genomic differences in hav are responsible for the range of clinical severities. methods: our accumulated hav strains of 5'ntr (nt 200 and 500), entire 2b and 2c from 25 japanese patients with sporadic hepatitis a, consisting of 7 patients with fulminant hepatitis (fh), 5 with severe acute hepatitis (ahs), and 13 with self-limited acute hepatitis (ah), in whom the sequences of all 3 regions were available, were subjected to phylogenetic analysis. results: fh patients had fewer nucleotide substitutions in 5'ntr, had a tendency to have more amino acid (aa) substitutions in 2b, and had fewer aa substitutions in 2c, than ah patients. four fh and 2 ahs with higher viral replication were located in the near parts of the phylogenetic trees, indicating the association between the severity of hepatitis a and genomic variations in 5'ntr, 2b and 2c of hav. conclusions: our study suggests that genetic variations in some parts of hav might cooperatively influence replication of the virus, and thereby affect virulence. viral factors should be considered and examined when discussing the mechanisms responsible for the severity of hepatitis a. aims: the incidence of acute viral hepatitis a in adults is increasing very much in south korea, 2008. the aim of this study was to the clinical features and course in daejoen and its surrounding area. methods: forty seven patients admitted as acute viral hepatitis a in chungnam national university hospital between january 2008 and june 2008 were enrolled. the medical records were reviewed, retrospectively. results: the mean age was 30.5. common occupations were company employee and studuents. most common symptom was jaundice. presumptive infection sources were raw fish or shellfish and raw meat. initial laboratory findings were as follows(mean value): wbc 6126/ul, hemoglobin 13.4g/dl, platelet 212×10 3 /l, ast 2268iu/l, alt 2755iu/l, total bilirubin 5.2mg/dl, alkaline phosphatase 207u/l, ggt 379u/l, prothrombin time(inr) 1.2. hospitalization was 21.5days. peak laboratory findings were as follows: alt 3181iu/l, total bilirubin 8.8mg/dl. leukopenia (<4000/ul) and thrombocjtopenia (<10×10 5 /l) were ocurred in sixteen and six patients, respectively. recovery time of each biochemical finding was as follows: alt 20.2 days, total bilirubin 26 days. recovery rates of altand total bilirubin were 74.4% and 74%, 88.5% and 92% at 4 weeks, 8 weeks after diagnosis, respectively. prolonged jaundice (115days) was detected in one patient. all patients were recovered by supportive management. conclusions: in south korea, acute viral hepatitis a was more prevalent in young adults, recenlty. presumptive infectious sources were raw fish or 1 guangxi center for disease prevention and control shellfsh and raw meat. if it can not change the food style that many korean enjoy raw seafood, vaccination for adults must be considered to prevent it. objective: to assess the safety and immunogenicity of a new inactivated hepatitis a vaccine (vero cell). pe479 methods: 1507 subjects were selected in gongcheng city of guangxi zhuang autonomous region, and the clinical trail was carried out according to the random, double-blind and parallel principle from january to august, 2005. after vaccination by 0, 6 schedule, adverse events of the subjects were observed, the seroeonversion rate and geometric mean titer (gmt) were tested by the competitive inhibition elisa. results: after immunization, the systemic and local reaction rates of adults were 8.80% and2.67%, which was no significantly statistical difference compared with control group, 12.41%and 4.41%; while the rates of children were 10.60and 2.28%%, and no significant statistical difference compared with control group, 10.71%and2.86%. one month after first dose of vaccination, the seroconversion rates of children and adults were 88.2% and 93.8%, and one month after second dose of vaccination, the rates were all 100%, the gmts of children and adults were 16447miu/m1 and 8555miu/ml, which was significant statistical difference in children compared with control group, 1946 miu/ml and 5881 miu/ml, respectively. methods: igg anti-hav was measured in a total of 3905 subjects under the age of 50, who visited hanyang university seoul and guri hospitals between january 2001 and may 2008. results: fig. 1 shows the relatively low positive rates of the antibody in ages of 11 to 30 and the lowest rates of 9.9% and 9.2% in the age group of 16 to 20, following the ages of 21 to 25 with rates of 12 background: some viruses encode proteins that affect their cap-independent internal ribosomal entry site (ires)-mediated translation and their replication. it was recently reported that hepatitis a virus (hav) proteases interact with intracellular dsrna-induced retinoic acid-inducible gene (rig-i)-mediated signaling, but it remained unknown whether hav proteins have any effects on hav ires-independent translation. in this study, we investigated the effects of 7 hav non-structural proteins on their ires-mediated translation using a reporter assay. pe480 methods: the bicistronic reporter constructs, termed psv40-hm175-ires, psv40-a1-ires, psv40-a2-ires, psv40-f1-ires, and psv40-f2-ires, contain the sv40 promoter that controls the expression of a bicistronic message coding for renilla and firefly luciferases separated by hav ires, and are derived from strain hm175, acute convalescent hepatitis clones a1, a2, fulminant hepatitis clones f1, f2, respectively. human hepatoma cell lines were co-transfected with psv40-hav-ires and each hav protein-expression vector. luciferase activity was determined 48 h after transfection. were from other countries within asia, africa, middle east, and eastern europe. patients of a wide age range were affected by hepatitis delta (mean age 46.0, median 47.0, range 19-79). 18 (40%) of 45 were co-infected with hcv. hepatitis b virus (hbv) dna was detectable in 43 (78%) patients and negative in 8 (15%) patients. all hepatitis delta patients were extracted from a prior study conducted by this collaboration. there were 1,191 chronic hbv carriers. 18 (1.51%) were hbv/ hcv/hdv infected. 32 (58%) of 55 patients carried a diagnosis of cirrhosis compared to 262 (22%) of 1191 chronic hbv patients. 13 (72%) hcv co-infected patients had evidence of cirrhosis while 4 (22%) patients did not. conclusion: individuals with hbv/hdv co-infection have higher rates of cirrhosis. individuals with hbv/hcv/hdv infection have rates of cirrhosis significantly higher than individuals with either chronic hbv infection or hbv/hdv co-infection. testing for hdv should be performed in all patients, especially those with advanced liver disease or high risk behavior. clinical characteristics were compared between the patients with significant endoscopic findings (group a) and without such findings (group b). peak ast and alt level were higher in group a (p<0.01). there were no statistical differences in age, gender, comorbidity, and etiology of acute hepatitis between group a and group b conclusion: significant endoscopic findings were found in considerable proportion of patients with acute hepatitis. severity of acute liver injury was associated with significant upper gastrointestinal endoscopic findings. in patients with severe acute hepatitis who complain of upper gastrointestinal symptom, esophago-gastro-duoenoscopy should be performed. background: in japan, hepatitis e virus (hev) testing is not allowed as routine one. to study the role of hev testing, we checked 22 sera of the patients diagnosed as etiology-obscure acute liver injury. methods: we have seen 54 cases of acute liver injury from january 2004 through december 2007 in our hospital and 25 cases of them were etiology-obscure. in 25 cases, 22 were retrospectively tested for hev-igm, hev-iga and hev-rna (rt-pcr) by direct sequence method on stored sera taken at the time of presentation. result: two of 22 cases (9.1 ) were positive for both hev-igm and hev-iga and one case was positive for hev-rna. in 54 cases of acute liver injury, the cause of virus was 31 cases (57.4%) and unknown was 8 cases (14.8%). hev was occupied in 3.7% in all cases and 6.5% in the cases caused by virus. one of the two cases had been misdiagnosed as "drug induced hepatitis". hev of genotype 3 was detected in one case and its nucleotide sequences of hev showed quite a high degree of similarity to the reported one at closed city in the same year. conclusion: hev is not rare in japan and the hev testing can reverse the diagnosis of acute liver injury. hev testing sould be used as routine one for acute liver injury. association of progesterone receptor gene with hepatitis e disease severity in pregnancy p.d. bose 1 , b. das 2 , a. kumar 1 , p. kar 1 1 maulana azad medical college, 2 background/aims: incidence of fulminant hepatic failure (fhf) in hepatitis e is high in pregnancy particularly during 3 rd trimester when there is an altered status of hormone and immunity. progesterone receptor (pr) up regulation provides fetal protection via immunosuppression but lower immune status in pregnancy may add to the disease severity. till now, no data is available whether pr can play any role in hepatitis e disease severity during pregnancy. progins, a haplotype of pr consisting of 320-bp insertion in intron g together with point mutations in exons 4 and 5 is associated with increased stability and higher transcriptional activity. the aim of the study is to analyze pr mutation (progins) and m rna expression in hepatitis e virus infected pregnant women with avh and fhf. methods: a total of 68 avh and 32 fhf cases were studied. blood and placental tissue were collected from the medicine and gynecology wards of lnjp hospital, new delhi. cases were screened for acute viral markers by commercially available elisa kit. extraction of dna from blood and rna from placental tissue was done by qiagen kit. mutation in pr was detected by pcr-rflp. semiquantitative rt-pcr for pr expression was performed in placental tissue using beta-actin as internal control. results: pr mutation (progins) was significantly more in fhf compared to avh (28.1% vs 10.3%, p value<0.05). protein expression was found higher in progins carriers. conclusion: progesterone receptor mutation (progins) may have a role in the hepatitis e disease severity in pregnant women. results: the hepatitis e was predominantly sporadic, some patients superinfected with other viral hepatitis, especially hepatitis b. in the old patients, jaundice lasted longer and the length of stay was longer, the incidence of complication was higher than the young men. the incidence of complication in the superinfected group was higher than the simple infection. the transaminase in the simple infection group was obviously raise than superinfected with liver cirrohsis. methods: liver sample were paraform-glutaral fixed, paraffin-embedded, sectioned and immunohistochemical stained, and positive samples were selected for histological analysis and rt-pcr detection. result: positive rate of hev immunohistochemistry ranged from 90% to 100% (fig. 1) . hepatocyte degeneration, scattered singled karyopyknosis, lymphocytic infiltrate, hyperplasia of bile canaliculus at the portal area and fibrous connective tissue hyperplasia been observed during histological analysis (fig. 2) , and two genotype 4 hev which closely related to many strain isolated from patients with sporadic acute hepatitis been detected. conclusion: the patients infected with hepatitis e of young men were frequently. jaundice lasted long in the old patients, the incidence of complication was higher in the superinfected men and the old men. conclusion: additional public-health concerns might be placed on pork safety and the risk of hev infection via the consumption of undercooked pork products. poster session, hall 5b aim: esophageal varices (ev) recurs frequently after endoscopic variceal ligation (evl) or endoscopic injection sclerotherapy (eis). we retrospectively investigated risk factors for early recurrence of ev after endoscopic treatment. methods: we treated 110 patients with ev, who had no past history of ev, at ehime prefectural central hospital from october 2005 to june 2008. of those, 78 (71%) were observed for at least 2 months after treatment and enrolled. we divided them into rupture cases at initial endoscopic treatment [(bleeding group; n=25 (32%)], and cases with preventive evl or eis performed [preventive group; n=53 (68%)]. all received periodic upper endoscopy examinations to confirm recurrence or no recurrence of ev. results: recurrence of ev occurred in 18 of all subjects and the average period after treatment was 6.5±3.0 months. the recurrence rate was significantly higher in the bleeding group (11/25) as compared to the preventive group (7/53) (p=0.0026). there was a significant relationship between recurrence of ev and hepatic reserve function (child-pugh a+b, c; 11/64, 7/14 respectively; p=0.0083). in logistic multi-variant analysis, ev rupture at initial treatment and child-pugh c were risk factors for recurrence. in contrast, age, sex, hepatocellular carcinoma, portal tumor thrombosis, continuous alcohol consumption, therapeutic modality (evl or eis), number of treatment sessions, and operator experience did not have a significant relationship with recurrence. conclusion: in cases with ev rupture at initial treatment or child-pugh c, the risk for early recurrence must be considered and patients carefully observed in follow-up examinations. endoscopic cyanoacrylate injection: less oil for less ectopic embolism c.z. li 1 , l.f. cheng 1 , z.q. wang 1 , f.c. cai 1 , q.y. huang 1 , e.q. linghu 1 1 general hospital of chinese pla background and aim: endoscopic injection sclerotherapy with n-butyl-2cyanoacrylate (nbca, histoacryl) has been reported to be effective for hemostasis of bleeding gastric varices, but occasionally the gel flows to other organs and causes ectopic embolism. the present study aimed to determine whether less amount of iodized oil preload in nbca injection helps in decreasing ectopic embolism. methods: from january 1997 to april 2006, 2 different methods of endoscopic nbca injection, "sandwich method" and "modified sandwich method" (in which iodized oil preload was minimized), were applied on 635 gv cases, to evaluate if decrease of iodized oil preload resulted in less ectopic embolism. results: altogether 5 cases of ectopic embolism occurred in the whole group (0.8%), including 3 cases of splenic infarction, 1 case of transient paralysis and 1 case of minor infarction of the lung. the modified sandwich method showed some superiority over original method in decreasing ectopic embolism (0/276 vs. 5/359, p=0.049). less cough during procedure was also found with the modified method (3/276 vs. 18/359, p=0.006). conclusions: less amount of iodized oil preload in endoscopic nbca injection is beneficial to decrease ectopic embolism. background: portal hypertension is closely associated with serious complications of liver cirrhosis which contribute to bad prognosis. hepatocellular carcinoma (hcc) and low serum sodium (sna) are manifestations of end-stage liver disease (esld) and are associated with poor survival in decompensated cirrhosis patients. therefore, we aimed to determine the relationship between hepatic venous pressure gradient (hvpg) and the development of hcc or low sna in decompensated alcoholic cirrhosis patients. methods: child-pugh scores, meld scores, and hvpg at baseline, and the development of low sna (sna <130 meq/l) or hcc during follow-up were analyzed prospectively in 170 patients with decompensated alcoholic liver cirrhosis. the predictive values of different risk factors for the progression to the esld were investigated by multivariate analysis and the kaplan-meier method results: twenty-four patients developed hcc during the follow-up period. in the multivariate analysis, only baseline hvpg>15mmhg was an independent predictive factor for the development of hcc (relative risk (rr)=1.128, p<0.05). those with hvpg >15 mmhg showed a significantly shorter time for the development of hcc on kaplan-meier analysis. twenty patients developed low sna during follow-up. initial hvpg was also an independent predictive value for the development of low sna in the multivariate analysis (rr=1.169, p<0.05). those with hvpg>15 mmhg also showed significantly shorter times for the development of low sna on kaplan-meier analysis. conclusions: in decompensated alcoholic cirrhosis, hvpg may be a useful predictive factor for the development of hcc and low sna, both of which are characteristic of esld and poor prognosis. the effectiveness of the treatment of octreotide on chylous ascites after liver cirrhosis d.x. zhou 1,2 , h.p. hu 1,2 background: octreotide is a crucial drug used for treating patients with chylous ascites; however, there have been few reports related to octreotide that are being used in cirrhotic patients. thus, this thesis is designed to determine the effects of octreotide on patients with chylous ascites after liver cirrhosis. methods: eight patients were diagnosed with chylous ascites, on the basis of laboratory findings on ascites samples, between january 2003 and may 2008. octreotide was given to the six patients, while the remaining two were treated as a control. all patients had persistent peritonea drainage with the quantity and quality of the drainage fluid observed once every other day. all the necessary care was individually given to the patients during the therapy results: all patients properly received combined therapy including low fat and sodium diet, and diuretic and peritoneal drainage. the volume of the peritoneal drainage was reduced to zero in one of the six patients who received octreotide therapy, while the other five had the drainage volumes decreased from 2000 ml to 50 ml with a clear appearance and negative qualitative analysis of chyle for those two patients who did not receive octreotide therapy, the conditions of peritoneal drainage seldom changed both from the qualitative and quantitative aspects. conclusion: octreotide, along with combined therapy, can rapidly relieve portal hypertension and reduce fat absorption from intestinal mucosa. it appears to be an effective therapy available for the treatment of chylous ascites caused by liver cirrhosis. albumin <38g/l were the best predictors of large varices. a model using these predictors in a validation cohort study is planned. background-aim: cirrhosis is associated with raised acute phase proteins (app), irrespective of infection. it is, however, unclear whether their values differ significantly or whether a particular app might be more indicative of infection, and these questions were addressed in our study. methods: we measured serum crp, fibrinogen, ferritin, haptoglobin, 2-microglobulin, c3, c4, and c1 inhibitor in 88 consecutive, cirrhotic patients, on admission. all patients were investigated according to a standard protocol for infection. child-pugh scores (cps) were calculated. results of app were expressed as means sem and compared with the mann-whitney test. results: 19 (21,6%) patients, median age 60 years, (cps: a=0; b=7; c=12), were diagnosed with infection (spontaneous bacterial peritonitis=7; pneumonia=5; septic shock=4; extensive cellulitis=1; listeria monocytogenes meningitis=1; viral infection=1), while 69 (78%) patients, median age 59 years, (cps: a=25; b=28; c=16), showed no infection. although most app values were raised, there was no statistically significant difference between patients with or without infection, or among different cps groups, except for crp, which was significantly more raised in patients with infection (p<0.01). this difference remained even after cps a cases in the non-infection group were excluded from analysis. interpretation: a significantly raised crp in cirrhosis would seem to be independent of cps staging and should prompt a thorough work up to exclude infection. by contrast, the discriminating power of all other app in the face of possible infection is negligible. the predictive value of crp towards infection is under investigation prospectively. although bleeding from ectopic varices such as duodenal, jejunal, ileal, colonic, and rectal varices is less common, it can also cause life-threatening problem, which is often difficult to diagnose and treat successfully. here we present a novel endoscopic approach for hemorrhagic rectal varices using endoscopic injection sclerotherapy with ligation (eisl). patients and methods: in 2000-2008, we performed endoscopic treatment in 215 patients with portal hypertensive varices. among those, four cases of hemorrhagic rectal varices were treated with the combined evl and sclerosing technique. the etiology of portal hypertension included oen idiopathic portal hypertension and three hcv cirrhosis. all patients had a history of prior abdominal surgery or endoscopic treatment for gastro-esophageal varices. results: hemostasis was obtained easily by the evl initially. furthermore, to avoid recurrent bleeding, the patients underwent endoscopic varicerography injection sclerotherapy (evis) using 5% ethanolamine oleate with iopamidol and the feeding vein was sclerosed successfully with no major complication occurred during the entire course of the treatment. conclusions: it is important to recognize the possibility of ectopic varices as a cause of gastro-intestinal haemorrhage especially in patients with a history of variceal therapy or abdominal surgery. the eisl technique is useful to control the initial and recurrent bleeding from rectal varices. t. hirano 1 , t. okada 1 , j. yamanaka 1 , y. iimuro 1 , n. kuroda 1 , k. oh 1 , y. yoshida 1 , j. fujimoto 1 1 aim: interferon (ifn) therapy is a powerful treatment for hcv-related hepatitis and is known to decrease the incidence of progression of hepatocellular carcinoma (hcc). however, thrombocytopenia is a common side effect of ifn treatment, often leads to discontinuance without insufficient therapeutic effect. in this study, we investigated the efficacy and safety of laparoscopic splenectomy ( ) in reversing thrombocytopenia in patients with hepatitis c cirrhosis and portal hypertension. patients and methods: out of 41 patients who underwent ls in our department during aug 2003 and december 2007, 13 patients associated with portal hypertension. among these patients, three patients had hcc, and they were simultaneously underwent partial hepatectomy after splenectomy. platelet count, operative time, blood loss, complications and length of stay were calculated. results: thirteen patients underwent laparoscopic splenectomy; their mean age was 59 years (range 20 to 68 years). six patients were child's class a and seven patients were class b. mean operative time was 178 minutes (range 97 to 255 minutes). blood loss was little, and none required transfusion with packed red cells. a hand-assisted laparoscopic technique was used in four cases (30.8%). average length of stay was 12.7 days. there have been no major complications during follow-up. platelet counts improved from a preoperative mean of 62000/ul (44000 to 108000) to 273000/ul (126000 to 469000) postoperatively. six patients are ongoing ifn treatment without remarkable thrombocytopenia. conclusion: laparoscopic splenectomy is safe and in patients with portal hypertension and thrombocytopenia. it may allows these patients by reversing thrombocytopenia. background: hepatic encephalopathy (he) is a significant cause of mortality in advanced cirrhosis patients. l-acyl-carnitine has been suggested as an alternative treatment for patients with he patients. to assess the clinical efficacy of acetyl-l-carnitine in the treatment of hepatic encephalopathy in cirrhotic patient, especially in diminishing the recurrence and reduction serum ammonia level. methods: we performed a randomized placebo-controlled, cross-over study. we administered acetyl-l-carnitine to group 1 during 3 months first then placebo during later 3 months, and administering acetyl-l-carnitine to group 2 alternatively. results: between january 2008 and february 2008, thirty two selected cirrhotic patients were enrolled in this study. following randomization, the patients were divided into two groups (group 1=14, group 2=18). during administering acetyl-l-carnitine period, serum ammonia level was decreased significantly in both groups significantly (p=0.005, vs. p=0.001 respectively). however, during administering placebo period, serum ammonia level changes were not significant. in group 1, the first recurrence cases of hepatic encephalopathy were more than group 2(group 1=5, group 2=2), and the first recurrences were occurred during first 3 months in all groups. conclusion: our study demonstrates that acetyl-l-carnitine administration reduced serum ammonia level, but not definitely diminishing the recurrence of hepatic encephalopathy. sodium (na + ) and water retention are the most common abnormalities in cirrhotic patients and the magnitude varies from patients to patients. aim: to assess the relationship between the meld score and urinary excretion of na+ in non-azotemic cirrhotic patients. methods: fifty four cirrhotic patients with ascites and normal serum creatinine (<1.5 mg/ml) were admitted and placed on a low sodium diet (4g/day), while all diuretics were withdrawn for 3 days. the electrolytes (na + , k + , na + / k + ) were measured in a random urine and both the volume and na + concentration of urine collected for 8 h after administration of furosemide 80 mg i.v. were determined. results: table. conclusions: the meld score was significantly correlated with the degree of impairment of urinary na+ excretion. the ratio of na + /k + in a random urine specimen and furosemide-induced na+ excretion reflect the degree of impaired natriuresis in non-azotemic cirrhotic patients with ascites. background: portal hypertensive gastropathy (phg) is common finding in patients with liver cirrhosis and portal hypertension. despite portal hypertension remains the crucial trigger for the development of phg, the relationship between portal hypertension and phg has not been widely investigated. methods: fifty-three cirrhotic patients (48 males, mean age 51 years) who were performed hepatic vein catheterization between november 2006 and august 2008 were prospectively included in this study. the degree of phg was assessed according to the third baveno international consensus workshop, and classified three degrees as no, mild and severe. the hepatic venous pressure gradient (hvpg=whvp-fhvp) measurements were performed by triplicate in each case, and results were given as arithmetic means of the three determinations. result: hvpg values did not differ between the patients without phg (13.28±4.72 mmhg) and those with phg (14.91±3.84, p=0.187), nor between those with mild (15.31±3.69 mmhg) or severe phg (14.27±4.12mmhg, p=0.323). the degree of phg and hvpg did not differ regarding the etiology of the cirrhosis(p=1.0, p=0.085) nor regarding the child pugh classification(p=0.085, p=0.738). no correlations were found between the degree of phg and child pugh score, age, with or without ascites, albumin, bilirubin, creatinine, meld score and the degree of gastroesophageal varices. conclusions: our data show that the presence and the severity of phg does not correlate with the degree of hvpg, and that correlate with esophageal varices in patients with liver cirrhosis. introduction: phlebosclerotic colitis is a rare form of ischemic colitis characterized by the thickening of the colonic wall due to fibrous degeneration of the submucosal layer and fibrotic sclerosis of the venous wall. there are a few reports those this entity might be related to portal hypertension with disturbed venous return from the colon and mesentery. case description: a 61-year old man with alcoholic liver cirrhosis presented with right lower abdominal pain/tenderness and bloody diarrhea. a colonoscopy revealed multiple circumferential ulcerations in the transverse colon and the scope could not get through the ascending colon due to luminal stenosis, showing histologic finding of ulcerative inflammation with inflammed granulation tissue. abdominal computed tomography demonstrated liver cirrhosis with splenomegaly, multiple portosystemic venous collaterals, diffuse vascular engorgement and the wall thickening of right proximal to mid ascending colon with increased density in the surrounding fatty tissue. a follow-up colonoscopy performed one month later showed still remained multiple ulcerations in the transverse colon and could not further advance to ascending colon. superior mesenteric angiography revealed no main branch occlusion but pooling at the venous phase on ascending colon. a right hemicolectomy was performed because of the colonic obstruction. gross findings on operation showed thickening of the cecum and ascending colon. microscopic examination showed fibrous thickening in the submucosa, abundant neurovascular bundles in the mesentery and several intravascular hyaline thrombi of the mesenteric vessels. here we report the first case of early stage of phlebosclerotic colitis in a cirrhotic patient in korea. spontaneous bacterial peritonitis (sbp) is one of the severe complications in advanced cirrhotic patients with a high mortality rate. although a more rapid diagnosis should lead to the better survival, it takes several days to detect the causal bacteria from ascitic fluid cultures. furthermore, despite the use of sensitive methods, ascitic fluid cultures were negative in more than 50% of patients with suggestive clinical manifestations of sbp. therefore, diagnosis of sbp is based on the polymorphonuclear leucocytes (pmn) cell count in the ascitic fluid. the hybrizep kit (fuso pharmaceutical industries, osaka, japan) detects the dna of bacteria that have been phagocytized in neutrophils and macrophages, using in-situ hybridization method within one day. here we present a case of the patient for whom the hybrizep kit was used to detect the causal pathogen of sbp. a 76-year-old man had been admitted for the treatment of ascites and esophageal varices. one week after the admission, he complained abdominal pain and fever. because the pmn cell count in ascites fulfilled the criteria of sbp (1141/mm 3 ), we started an empirical antibiotic therapy without waiting for a result of the culture, and his symptoms improved within a few days. on the following day of the onset, in situ hybridization showed the positive signals by the ek probe, which detected the genomeic dna of e.coli species. however, the ascitic fluid culture was negative. this case suggested that the hybrizep kit was useful for the rapid diagnosis of sbp with high sensitivity. background: it has not been known that the hemodynamic effect of a portal hypertension for splenomegaly or esophageal and gastric variceal formation. this study was performed to access the parameters of doppler ultrasonography associated with splenomegaly or varices in patients with cirrhosis. patients and methods: from may 2007 to may 2008, 144 cirrhotic patients were performed the doppler ultrasonography. 91 of these patients were accessed the severity of varices endoscopically. the three dimensional volume of spleen was measured from a length, width and thickness on sonography. results: the splenic volume (415.2ml vs 505.6ml, p=0.048) and blood flow of main portal vein (12.6cm/s vs 15.1cm/s, p=0.012) were statistically significant different in alcoholic (38/144) and non-alcoholic (105/144) cirrhosis groups. the splenic volume (600.1ml vs 384.4ml, p=0.001), damping index (0.52 vs 0.38, p=0.024), and blood flow of main portal vein(12.5cm/s vs 15.7cm/s, p=0.006) were statistically significant different in esophageal variceal groups (55/91) and non-esophageal variceal groups(36/91). the only splenic volume (669.4 ml vs 461.7 ml, p=0.004) were statistically significant different in gastric variceal groups (24/90) and non-gastric variceal groups (66/90). the hemodynamic parameters venous ammonia and cff at baseline and after one month of treatment with lactulose. mhe diagnosed by abnormal psychometry and/or p300erp.response defined by normalization of abnormal test parameters. results: mhe diagnosed in 35(58%) patients. of 35 patients 26 (74%) had both abnormal psychometry and p300erp whereas30 (86%) alone had abnormal psychometry, 31 (89%) had abnormal p300erp.cff was <39hz in 28(80%) patients. mhe recovered in 55% with treatment and cff >39hz was seen in 22(69%) of 32 patients. cff sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and diagnostic accuracy before and after treatment is shown in table. conclusions: critical flicker frequency is a simple and accurate test without any age or literacy dependence for the diagnosis and recovery of patients with mhe. background/aims: endoscopic injection of n-butyl-2-cyanoacrylate (histoacryl) is an effective treatment of varix bleeding. but nontarget embolizations and septicemia are unwanted complications. we evaluate the risk factors for complications. methods: thirty-three patients with esophageal or gastric varix bleeding received endoscopic histoacryl therapies (54 procedures). baseline varix size, ctp score were checked. serum leukocyte, blood culture and body temperatures were repeated checked within one week after procedure. average volume of histoacryl per each session was 1.6 ml, and dilution volume ratio of histoacryl/lipiodol was 1/1 or 1/2. results: average of ctp score was 8.0 ± 1.7. three cases of septicemia were correlated with ctp score rather than session frequency or injection volume. two cases of systemic embolizations (pulmonary and splenic arterial embolism) were correlated with high lipiodol dilution ratio (1/2) and lipiodol volume rather than histoacryl volume or ctp score. conclusion: ctp score, lipiodol volume and dilution ratio of histoacryl/lipiodol were significant risk factors for complications. detection of circulating toll-like receptor 2 and 4 and cd4+cd25+ regulatory t cells in patients with hbv-related liver cirrhosis x.q. wang 1 , y. zhang 1 , x.f. bai 1 , j.q. lian 1 1 background : to detect circulating cd4 + cd25 + regulatory t cells and toll-like receptor(tlr)2 and tlr4 expression on the peripheral blood mononuclear cells (pbmcs) of patients with hbv-related liver cirrhosis (lc), and to explore the correlation between them. methods: pbmcs isolated from 14 lc patients , 21 chronic hepatitis b (chb) patients and 16 normal controls(nc) were stained with fluorescent labeling anti-tlr2-pe, anti--tlr4-apc, anti--cd14-fitc monoclonal antibodies and anti-cd4-percp anti-cd25-fitc anti-cd127-pe. samples were collected and detected of three-color immunofluo rescence by flow cytometry. results: the expression of tlr2 and tlr4 were significantly up-regulated in patients with lc than those in the controls.the expression of tlr2 was significantly increased in patients with lc than those in patients with chb, but there were no differences of tlr4 expression between lc and chb.treg/cd4 + t cells were significantly increased in patients with chb than those in patients with nc and lc, but there were no differences between lc and nc. there were no correlation between the expression of tlr2,tlr4 and treg in patients with lc . the expression of tlr2 and tlr4 on pbmcs in patients with lc were positive correlation.the expression tlr4 and hbv dna level were negative correlation in patients with lc. conclusion: the expression of tlr2 and tlr4 were up-regulated on pbmcs in patients with lc. it seems to be expression of tlr2 and tlr4 invovlved in the pathogenesis of lc. evaluation of 13c-phenylalanine breath test for the measurement of hepatocyte function in patients with chronic liver disease z.j. bao 1 , d.k. qiu 2,3 , x. ma 2,3 , g.s. zhang 1 , y.q. huang 1 , z.p. fan 2,3 , s.m. yin 1 1 huadong hospital, fudan university, 2 renji hospital, shanghai jiao tong university school of medicine, 3 background: the objective is to investigate whether the 13c-phenylalanine breath test(pbt) would be useful for the evaluation of hepatic function in patients with chronic hepatitis b, liver cirrhosis and minimal hepatic encephalopathy (mhe). methods: l[1-13c] phenylalanine was administered orally in a dose of 100 mg to 80 patients with liver cirrhosis, 20 with chronic hepatitis b and 20 healthy subjects. the pbt was measured at 8 different time points (0, 10, 20, 30, 45, 60, 90, 120 min) to obtain the values of delta over baseline, percentage 13co2 exhalation rate and cumulative excretion (cum). the relationships of the cumulative excretion with the 13c-%dose/h and blood biochemical parameters were investigated. results: the 13c dose h -1 at 20 and 30 min combined with the cumulative excretion at 60 and 120 min showed correlations with the chronic liver diseases, especially child-pugh score and mhe or not. and the data showed correlations with serum albumin hemoglobin platelet and child-pugh score. prothrombin time, total and direct bilirubin were significantly increased, while serum albumin, hemoglobin and platelet, the cumulative excretion at 60 and 120 min values decreased by degrees in healthy controls, child-pugh a, b, and c patients (p<0.01). similar results of pbt were in the patients with and without mhe, while only prothrombin time prolonged and total bilirubin increased (p<0.05). conclusions: the pbt can be used as a non-invasive assay to evaluate hepatic function in patients with liver cirrhosis and mhe. the %13c dose h-1 at 20 min, %13c dose h-1 at 30 min and cumulative excretion at 60 min may be the key value for determination at a single time-point. branched chain amino acids in improving survival and decreasing risk of liver failure among cirrhotic patients: a meta-analysis h. flores 1 , e.l. ang 1 , n. iv estanislao 1 1 philippine general hospital background: the state of a patient's nutritional status greatly affects disease outcome. among cirrhotic patients, approximately 60-90% are in a state of protein-energy malnutrition. hence, adequate nutritional support is essential to improve their general medical condition and long term prognosis. several studies have shown than branched chain amino acids (bcaa) may be of benefit for this purpose. it is the aim of this study to evaluate the effectiveness of diet plus bcaa compared to diet alone in improving survival and in decreasing liver failure among cirrhotic patients. methods: pubmed, cochrane, and embase search was done for articles which compared the clinical effects of bcaa supplementation versus diet alone among patients with liver cirrhosis. the following free-text terms and mesh words were used -"branched chain amino acids", "amino acids, branched chain", "bcaa", "liver cirrhosis", "cirrhosis", "randomized controlled trials'" and "meta-analysis". after critical appraisal of the included studies, a random effects model using odds ratio was used to synthesize the results (revman 4.2). results: 3 rcts were included for analysis with a total study population of 836. combination of the studies showed a significant decrease in the risk of liver failure (or 0.45, 95% ci 0.25-0.82, p=0.009) and a trend towards benefit in improving survival (or 0.57, 95% ci 0.27-1.17, p=0.12). conclusions: the overall trend appears to show benefit in the use of branched chain amino acids for patients with cirrhosis with respect to liver failure and survival. background: the proxisome prolifrator-activated receptor gamma (ppar ) is a member of the nuclear hormone receptor superfamily that is involved in the control of inflammation, carcinogenesis and gastric ulcer. on the other hand, the frequency of gastrointestinal ulceration is higher in cirrhotic patients compared with the normal population. the present study was designed to investigate the effect of specific ppar ligand, pioglitazone, on the mucosal lesions induced by ethanol in cirrhotic rats and the possible involvement of nitric oxide in the pioglitazone effect. methods: cirrhosis was induced by surgical ligation of bile duct and sham-operated rats served as controls. both cirrhotic and sham rats were kept for 28 days after the operation. different groups of sham and cirrhotic animals received saline, or 5, 10 or 15 mg/kg pioglitazone, daily during last 5 days of the fourth week after the surgery. another 2 groups of bdl or 2 groups of sham rats received l-name, a non selective inhibitor of nitric oxide synthase, alone or along with 5 mg/kg pioglitazone for 5 days. on day 28, rats were killed 1 hour after ethanol administration and the area of gastric lesions was measured. results: the ethanol-induced gastric mucosal damage was significantly more sever in cirrhotic rats than sham-operated ones (p < 0.001). pretreatment with pioglitazone dose dependently attenuated gastric lesions induced by ethanol in both sham and cirrhotic rats, but this effect was more significant in cirrhotic ones. concurrent treatment of l-name and pioglitazone decreased the ulcer index in bdl rats more than the groups that received l-name or pioglitazone alone. conclusion: we conclude that chronic treatment with pioglitazone exerts a potent gastroprotective effect on the stomach ulcers of cirrhotic rats probably due to inhibition of nitric oxide synthase. inhibition of phosphodiesterase 5 -a novel therapeutic strategy for portal hypertension l. halverscheid 1 , p. deibert 2 , b. pannen 1 , r. schmidt 2 , m. roessle 2 , w. kreisel 2 1 university hospital duesseldorf, germany, 2 university hospital freiburg, germany introduction: the no-cyclic gmp system is a key factor in the regulation of splanchnic and hepatic blood flow and may be a target for medical treatment of portal hypertension. clinical data have shown that inhibitors of phosphodiesterase 5 (pde5) lower portal pressure in cirrhotics. methods: we monitored in rats the effects of the pde5 inhibitors vardenafil and sildenafil on systemic and hepatic hemodynamic parameters up to 60 minutes after the drug. the drugs were administered intravenously into the tail vein at 1 (group a), 10 (group b), and 100 g/kg body weight (group c). 0.9% nacl was the control. n = 7 for each group. results: the most prominent changes were observed in the vardenafil b group: mean arterial and portal venous pressure decreased (-9%, -8%), as well as portal venous, hepatic arterial, and systemic vascular resistance (-31%, -30%, -12%). portal venous and sinusoidal flow increased (+31%, +13%). in the vardenafil c and sildenafil b and c groups there was an increase of portal venous flow by 20-30%, an increase of sinusoidal flow by 12-30%, and a decrease of portal venous resistance by about 25%. there was a trend for reduction of portal venous pressure. conclusions: vardenafil and sildenafil influence portal hemodynamics in the rat. portal venous flow increases by 20-30%, portal venous resistance decreases by >25%. dependent on the dose, portal venous pressure decreases significantly. these data yield further evidence that pde5 inhibitors may be a novel therapeutic option for portal hypertension. groups of liver cirrhosis e. havrilyuk 1 1 lviv national medical university introduction: rupture of esophageal varicose resulting in posthemorrhagic anemia is a common life-threatening complication of liver cirrhosis. but it is not clear, why the other patients, having the same degree of sclerosis and histologic activity index, die from hepatocellular failure or other reasons. aims & methods: 3713 autopsy cases performed in lviv regional hospital in 2004-2007 were analyzed. screening of slides with liver tissue allow to select 580 cases (15,6%) with cirrhosis (complete and incomplete), which are examined in order to evaluate the frequency of lethal portal hypertension complications in the different etiologic groups of liver cirrhosis. results: according to the etiologic factor the following groups of liver cirrhosis were examined: alcoholic disease (49,8%), viral hepatitis (7,8%), nonalcoholic steatohepatitis (14,8%), secondary biliary cirrhosis (2,9%), cardiac sclerosis (0,9%), combined lesions (11,9%) and cryptogenic cirrhosis (11,9%). analysis shows that in 287 cases (49,5%) patients die from cirrhotic complications (hepatocellular failure, jaundice, portal hypertension) and only in 105 cases (18,3%) -from posthemorrhagic anemia caused by rupture of esophageal varicose. in the latter cases correlation between the etiologic types of cirrhosis is almost the same, as in the main group and only alcoholic lesions (60%) and biliary cirrhosis (6,7%) are more frequent. conclusion: analysis shows that development of lethal complications of portal hypertension can not be explained only by etiologic factor. probably additional stimuli are more important for morphogenetical variants of cirrhotic transformation. patients with viral cirrhosis k. mumtaz 1 , s. ahmed 1 , h. ali shah 1 , s. hamid 1 , w. jafri 1 background and aims: increased nitric oxide (no) production is incriminated in the pathogenesis of arterial vasodilation and hyperdynamic circulatory state in non cirrhotic models of portal hypertension (pht). we investigated the relative roles of constitutive nos (enos) and inducible nos (inos) isoforms in the development of rabbit models of endotoxemia induced portal hypertension (eipht) methods: eipht was induced by chronic injection of lipopolysaccharide via an indwelling cannula placed in the gastrosplenic vein of rabbit and maintained for 6 months. the concentration of no, expression of nos (enos and inos) mrna and protein was measured in eipht and sham operated control animals. results: rabbits with eipht compared with controls had raised portal pressure (in mmhg-14.34±1.78 vs 6.30±0.60;p<0.05;1mo; 14.91±0.56 vs 7.04±0.42; p<0.05, 3mo; 19.8±3.10, vs 10.2±4.80;p<0.05), arterial hypotension (in mmhg-65.40±3.2 vs 80.04±1.40, p<0.05, 1mo; 62.90±5.40 vs 76.05±2.60,p<0.05, 3mo; 65.85±2.50 vs 79.1±5.10, p<0.05,6mo), splenomegaly (in g-0.92±0.14 vs 0.60±0.13,1mo; 0.90±0.16 vs 0.62±0.04, 3mo; 0.97±0.12 vs 0.67±0.07, 6mo), normal liver functions and preserved hepatic architecture at 1,3 and 6 mo. serum levels of no2 as well as the no3 were significantly elevated in eipht rabbits as compared to the controls. the expression of enos, at the level of mrna, was significantly increased in eipht rabbits consistent with increased levels of expression of inos as compared to the controls. the enos but not inos protein expression was elevated in eipht than control rabbits. conclusion: vascular dysfunction in the splanchnic circulation during the development of endotoxemia induced portal hypertension is predominantly characterized by enos and partly by inos gene up-regulation. liver cirrhosis contributed to the immunocompromised status by shedding the membranous tnfrii t.n. lin 1 , c.h. chao 1 , i.s. sheen 1 , y.p. ho 1 , w.t. chen 1 , c.j. lin 1 , c.t. yeh 1 , c.y. lin 1 1 liver research unit, linkou medical center, chang gung memorial hospital, chang gung university, taoyuan, taiwan background & aim: patients with decompensated liver cirrhosis (dlc) were regarded as immunocompromised, reflected by high incidence of bacterial infection. paradoxically, the proinflammatory cytokine like tnfincreased significantly in patients with dlc even in the face of this immunocompromised status. on the other hand, regulatory t cell (treg cell) is believed to play an important role in inhibiting immune responses, including innate immune responses like blockade of tnf-effect through soluble tnfrii. here, we studied the role of treg cells and tnfrii in patients with decompensated liver cirrhosis. patients and methods: 33 healthy volunteers and 78 cirrhotic patients were enrolled. the percentage of treg cells were enumerated by flow and serum levels of il-10, tgf-and tnf-by elisa. results: the percentage of treg cells increased significantly in patients with dlc associated with increased serum levels of il-10 and tgf-. in addition, these treg cells were mainly memory type reflected as high cd45ro. furthermore, the tnfrii expression increased significantly on these treg cells of dlc. interestingly, these membranous tnfrii on treg cells could be shed-off. lastly, we found the serum soluble tnfrii concentration increased significantly in patients with dlc when compared with normal volunteers. conclusion: our results demonstrated memory treg cells with high tnfrii expression increased significantly in patients with decompensated liver cirrhosis that could possibly blocked the biological effect of tnf-by shedding membranous tnfrii and contributed to the immunocompromized status of dlc. background: portal pressure measured as hepatic venous pressure gradient (hvpg) correlates with severity of portal hypertension and the development of complications. hvpg measurement is invasive. recently, liver stiffness measurement has been shown to correlate with liver biopsy and helps predict outcome in chronic liver disease patients. this study was conducted with the aim to study the correlation between portal pressure as measured by hvpg and liver stiffness as measured by fibroscan among patients with portal hypertension due to various causes. methods: between august and september 2008, consecutive patients with portal hypertension were included and were subjected to hvpg measurement and fibroscan (echosens, france). results: of the 18 patients with portal hypertension, both hvpg and liver stiffness were measurable in 12[9 (75%) males; mean age 37.5(10.6) years]. the etiological distribution was hbv related cirrhosis in 3 patients, hcv cirrhosis in 3, cryptogenic cirrhosis in 2, alcoholic cirrhosis in 2, hbv and alcoholic cirrhosis in 1and primary extra-hepatic portal vein obstruction in1. the mean hvpg and liver stiffness of this group were 13.9 (5.1) mm hg and 26.6 (16.5) kpa respectively. there was a strong positive correlation between hvpg and liver stiffness [r = 0.708; p = 0.01]. conclusions: non-invasive measurement of liver stiffness correlates well with invasive measurement of portal pressure. liver stiffness measurement could be used as a prognostic indicator to predict the severity of portal hypertension. endpoints were rate of rebleeding and mortality till day 30 after inclusion and to see for any adverse events. results: the bleeding was stopped in all 15 patients (100%). rebleeding till day 30 was observed in 4 (26%) patients (2 each in group a and b). total 2 patients (13%) died (1 each in both groups) due to rebleeding. transfusion needs were higher in group a (4.2±2.8 versus 2.3±2.1, p<.05). serious adverse effects leading to treatment discontinuation were not seen in any patients in both groups. conclusion: prolonging terlipressin treatment did not confer any significant decrease of mortality or bleeding recurrence. however transfusion requirements were significantly decreased in patients receiving prolonged treatment. serious adverse effects leading to treatment discontinuation are rare. poster exhibition -miscellaneous poster session, hall 5b background: it is important to know the prevalence of atp7b gene mutations of different geographical areas to justify the local screening strategies for wilson disease (wd). materials: eleven unrelated lithuanian families, including 13 wd patients were tested. genomic dna was extracted from whole venous blood using a salt precipitation method. firstly, semi-nested pcr technique was used to detect the c.3207c>a (p.h1069q) mutation. patients not homozygous for c.3207c>a (p.h1069q) mutation were further analyzed. the 21 exons of the wd gene were amplified in a thermal cycler. direct sequencing of the amplified pcr products was performed by cycle sequencing using fluorescent dye terminators in an automatic sequencer. results: total of 13 wd patients (mean age 26.4 years; range 17-40; male/female, 3/10) presented with hepatic disorders and 16 their first degree relatives were studied. some of wd patients in addition to hepatic symptoms have had extrahepatic disorders (haemolytic anaemia 3; fanconi syndrome 1; neurophsychiatric and behavioural disorder 2). twelve of 13 (92.3%) wd patients have had c.3207c>a (p.h1069q) mutation, 6 of them in both chromosomes, 5 were presented as compound heterozygotes with additional c.3472 -82delggtttaaccat, c.3402delc or c.3122g>a (p.r1041q) mutation. for one patient with liver cirrhosis and psychiatric disorder no mutations were found. out of 16 first degree wd relatives 11 (68.7%) were heterozygous for c.3207c>a (p.h1069q) mutation. conclusion: c.3207c>a (p.h1069q) missense mutation is characteristic for lithuanian wd patients. even 92.3% of wd patients with hepatic presentation of the disease are homozygous or compound heterozygote for this mutation. background: diabetic dyslipidemia is a crucial problem of diabetic patients with inadequate control. we investigated the relationship between glutamic-pyruvic transaminase (gpt) and high-density lipoprotein cholesterol (hdl-c) in diabetic patients. methods: with informed consents, we recruited outpatients with diabetes at a hospital in rural area in taiwan in [2004] [2005] [2006] [2007] . anthropometric measures, blood tests and urine screening were examined in diabetic patients. results: overall, there were 1241 diabetic patients aged 19-91 years enrolled in this study and 660 (53.2%) of them had low hdl-c. diabetic patients with the highest quintile of gpt had higher average of body mass index (p<0.0001), diastolic blood pressure (p = 0.003), but lower average of hdl-c (p<0.0001) compared with diabetic patients with the lowest quintile of gpt. the prevalence of obesity (45.7% vs. 24.8%, p<0.0001) and low hdl-c (64.6% vs. 48.3%, p<0.0001) were higher in diabetic patients with highest quintile of gpt than in diabetic patients with lowest quintile of gpt. in the multivariate logistic regression, diabetic patients with highest quintile of gpt had higher odds ratio (or) of low hdl-c compared with diabetic patients with lowest quintile of gpt (or = 1.88, 95% confidence interval [ci] = 1.21-2.92). the corresponding or of low hdl-c in patients aged 70 years and older was 3.30 (95% ci = 1.15-9.47). conclusion: high gpt is one of factors associated with low hdl-c in diabetic patients. the effect of desferrioxamine as supplement to cefotaxime in the treatment of spontaneous bacterial peritonitis na. seda 1 , m. el-hamamsy 2 , r. el-wakil 3 , m. al azizi 4 1 ain shams specialized hospital, 2 faculty of pharmacy,ain shams university, 3 faculty of medicine,ain shams university, 4 faculty of pharmacy ,ain shams university background: oxidative damage lead to cell damage, organ dysfunction and death in sepsis. desferrioxamine (dfx), an antioxidant iron chelators. the aim was to assess the efficacy of desferrioxamine supplemented to cefotaxime in the treatment of spontaneous bacterial peritonitis (sbp) in cirrhotic patients. methods: thirty patients divided into two groups: group i (n=15) with sbp and receiving cefotaxime (1g iv every12 hours) alone and group ii (n=15) with sbp receiving cefotaxime (1g iv every 12 hours) with desferrioxamine (500mg im twice daily).all patient were monitored for seven days, their vital organs were screened and their ascitic fluid was assessed completely including microbiological investigations. results: the concomitant administration of desferrioxamine with cefotaxime significantly at (p<0.001)and(p<0.01) improved the therapeutic outcome and the cure rate after 5 days of treatment as compared to patients using cefotaxime only. conclusions: desferrioxamine can improve the therapeutic outcome by preventing iron-induced organ damage and inhibiting bacterial growth. oligella ureolytica is a gram-negative, nonfermenting rod that is infrequently recovered from clinical specimens and is most commonly isolated from the urine of patients with chronic indwelling urinary catheters or other urinary drainage systems. bacteremia due to this organism is an extraordinary finding. we describe here a case of oligella ureolytica being detected in the blood of a patient with decompensated cirrhosis. a 51-year-old male man was admitted to hospital with 9-month duration of debility, poor appetite and abdominal distension. decompensated cirrhosis was diagnosed based on clinical findings such as hepatic face, ascites, edema of lower limbs and icteric sclera. laboratory results showed positive serum anti-hcv and high serum hcv rna level. the patient received a therapeutic regimen of pegylated interferon alpha 2a plus ribavirin after being admitted to hospital. during hospital stay, a fever of 2-day duration with shivering occurred to the patient. three blood cultures were drawn, which all grew oligella ureolytica in pure culture. the organism was identified by the viteck 2 compact (biomerieux, france). additional tests for identification resulted positive for nitrate reduction and urea hydrolysis, strongly positive for phenylalaninedeaminase activity and showed no growth at 42.7c. tests for nitrite reduction and motility resulted negative. the organism was resistant to amikacin, cefoperazone, levofloxacin, piperacillin/tazobactam, trimethoprimsulfamethoxazole, aztreonam, cefotaxime, piperacillin and was susceptible to gentamicin, imipenem, meropenem, and netilmicin. a 14-day of combined therapeutic regimen with cefminox and isepamicin was administered to the patient. within 2 days, the patient became afebrile. background: endoscopic ultrasound (eus) is often performed in patients with unexplained liver tests to assess the gallbladder, bile ducts and pancreas. an unremarkable eus exam and negative hepatology workup often leads to a liver biopsy. eus may provide histopathologic evaluation of the liver in these cases under direct, real-time visualization. aim: to assess the feasibility and efficacy of eus guided core biopsy of the liver in a porcine model. methods: female pigs were used and live procedures were performed under general anesthesia. a linear echoendoscope was used and the liver identified endosonographically. transgastric core biopsies of the liver were obtained with a 19 gauge quick-core ultrasound biopsy needle (wilson-cook) and sent for histopathologic evaluation. live animals were euthanized at the end of the procedure and necropsy performed. results: core biopsies of the liver biopsy were obtained in 4 animals (1 cadaver and 3 live anesthetized). a total of thirteen needle passes were made (mean 3.25; range 2 -4 needle passes per animal) and a visible core of tissue obtained. the maximum length of liver tissue obtained was 10 mm and considered adequate for assessment as more than one such specimen could be obtained. microscopic evaluation confirmed liver tissue. no complications were noted. necropsy did not show any evidence of bleeding, perforation or damage to surrounding structures. conclusion: eus-guided liver biopsy is feasible and can be performed at the time of routine echoendoscopic exam in select patients undergoing eus examination for abnormal liver tests. background: as the common indexes, alt, ast and plt play an important role in disease diagnosis, treatment and prognosis. many researchers suggested that there was inflammatory changes and fibrosis in chronic hepatitis b and c patients whose alt level was persistently normal. a large sample investigation showed that the serum level of alt in healthy persons is lower than the normal reference value. this study re-evaluated the normal serum level of alt, ast and plt. methods: 3815 people were enrolled in the study between sep. and oct. 2007. the platelet count and serum alt and ast levels were measured. frequencies, one-sample kolmogorov-smirnov test and nonparametric tests were used to analyze the difference between age groups, male and female, glucose groups, cholesterol groups and triglyceride groups. result: in the five groups, there is significant difference in alt and ast levels between male and female. in group 1, the alt and ast levels showed a significant difference between different age groups, between different glucose groups and triglycide groups. in the three groups the plt level is significantly different between male and female, and the serum level in male is higher than female. there is significant difference between different age groups conclusion: the serum levels of alt, ast and plt are all significantly different between male and femal. there is significant difference between different genders and age groups for plt. the serum level of plt is higher than the reference value. background/aims: myeloproliferative disorders (mpd) (like polycythemia vera, essential thrombocythemia and primary myelofibrosis) are responsible for 50% cases of hepatic venous thrombosis (hvt) and 35% of portal venous thrombosis (pvt) in western series. latent form of mpd lacks the characteristic blood picture and may be classified as idiopathic thrombotic disorder. a point mutation at val617phe of janus kinase 2 tyrosine kinase gene (jak2 v617f mutation) occurs in high proportion of the patients with mpd. this non-invasive test with high positive predictive value is now considered to be essential for diagnosis of various mpd. this test may be useful in diagnosing latent form of mpd in splanchnic venous thrombosis methods: 232 patients with confirmed pyogenic liver abscesses admitted from 1995 to 2007 in our institution were included. there were 145 men and 87 women ranging in age from 19 to 91 years. the medical records were reviewed for clinical, laboratory and radiographic characteristics. results: among 232 patients, 81 (34.9%) experienced at least one complication. there were 67 pulmonary (pleural effusion, pneumonia, empyema) complications, 16 septic shock, 12 acute renal failure, 2 abscess rupture, 2 pseudomembranous colitis, and 2 pericardial effusion. the predictive factors for its complications were: systemic inflammatory response syndrome (sirs, 2 factors), thrombocytopenia ( 80,000/ml), hypoalbuminemia ( 3.0g/dl), elevated ast or alt (>200 iu/l), hyperbilirubinemia ( > 2.0 mg/dl), k. pneumonia, air within abscess cavity (p<0.05). conclusions: the incidence of complications in the pyogenic liver abscess was 34.9%. the various predictive factors of complication should be monitored carefully. further large scaled study should be warranted. background/aims: hepatic iron deposition is a common feature in chronic hepatitis c (ch-c), however, whether it could enhance the progression of fibrosis or not is controversial. the aim of this study was to evaluate the status and significance of hepatic iron deposition in the korean patients with chronic hepatitis c. methods: untreated, 78 ch-c patients who underwent liver biopsy were included. the hepatic iron was assessed by scheuer's scoring system, and activity, fibrosis, and steatosis were scored by a pathologist in a blind manner to the clinical features. clinical and laboratory data including serum iron indices, virological, biochemical results were analyzed to search for significant factors associated with hepatic iron deposition. results: hepatic iron staining was positive in 26(33%). among 26 patients with hepatic iron deposition, serum levels of ferritin (p=0.005) and -fetoprotein (p=0.002), and body mass index(bmi) (p=0.039) were significantly elevated. there was no significant association between the degree of hepatic iron deposition and fibrosis stage (p=0.321), although elevated levels of serum hyaluronic acid (p=0.049), -glutamyl transpeptidase (p=0.028), and prothrombin time (p=0.012) were associated with advanced fibrosis. conclusions: hepatic iron deposition in asian-pacific ch-c patients seemed to be neither frequent nor related to hepatic fibrosis, but related to obesity. therefore, phlebotomy might not commonly applicable to this area. further studies on the pathogenic role of iron in ch-c in asian-pacific countries are warranted. a late stage of progressive hepatic fibrosis characterized by distortion of the hepatic architecture, necrosis of hepatocytes and the formation of regenerative nodules contributes to cirrhosis. limitations like organ donors shortage, high cost, absence of proliferation in cultured hepatocytes, inherent risks of infection, rejection in xenogenic cells and other socio-economical complications emerges advanced regenerative human hepatic stem cells(hhpscs) transplantation. hhpscs are located in the ductal plates in fetal and canals of hering in adult livers [schmelzer et al.(2006) ]. hepatoblasts, in turn, give rise to the hepatocytic and biliary lineages, the hepatocytes and cholangiocytes [schmelzere etal(2006) ].hhpscs express cd326(epcam)marker. scjelzer etal demonstrated that during embryogenesis 90% of the epcam positive cells had hepatoblast phenotype. in animal study, on transplantation of freshly isolated hhpscs in scid mice results in mature liver tissue expressing human-specific proteins. recently, we(aleem etal 2008)have shown clinical improvement in study in patients with crigler-najjar syndrome, biliary atresia using hhpscs infusion. in the present study we transplanted hepatic progenitors to five subjects of end stage liver cirrhosis with meld score >30. hhpscs were sorted using macs with cd326 antibody microbeads and infused through hepatic artery via femoral artery catheterization, a safe procedure provided portal pressure to monitor cell infusion route in order to prevent vascular thrombosis. all the patients showed improvement clinical and functional biochemical parameters after first month of cell infusion. ascites was decreased and changed encephalopathy grade into normal level was observed. meld score system falling to normal level from >30 to <22 after infusion. 1 the aga khan university patients. the apri of 1.5 in combination with a cut-off ha of 300 ng/ml can best detect patients with moderate to severe fibrosis (stages 2-4). it has a ppv of 93.7%. also, for patients without moderate to severe fibrosis, the test is hardly ever positive (specificity of 98.9%). but the apri of 1.5 in combination with different ha as cut-off points is not possible to detect patients with no or mild fibrosis. objectives: terlipressin is used in esophageal variceal bleed (evb) along with endoscopic band ligation (ebl) for 3days (uc). due to its high cost, it was stopped <3days (sc) who could not afford & were stable after achieving hemostasis with ebl. we retrospectively assessed the efficacy of sc vs uc of terlipressin for control of evb and length of stay. conclusion: the apri of 1.5 in combination with ha 300 ng/ml as cut-off points to predict patients with moderate to severe fibrosis (stages 2-4) is an easy and accurate method. methods: patients with evb who had achieved hemostatsis with ebl from jan 2004-dec 2005 were included. all were managed on standard protocol on hospital variceal bleeding pathway. the course of terlipressin as sc or uc was based on patient's inability to afford the cost of hospitalization and terlipressin. the efficacy of terlipressin in the control of evb was defined based on baveno iii criteria. results: total of 117 patients were admitted during the study period. out of them, 66 received uc & 51 sc of terlipressin. the base line characteristics were comparable except younger age in sc. there were 2 re-bleed (3%) in uc and 1 (2%) in sc terlipressin group. the length of stay was shorter in sc group. (2.47±0.57 vs 6.15±2.92 days). conclusions: sc seems as effective as uc terlipressin in the control of evb after initial control of hemostatsis with ebl and may reduces the length of hospital stay. rcts are needed to assess this as all stable patients may not need to continue terlipressin for 72 hours. background/aims: to analyze the relationship between conventional laboratory results and death risk in patients with esophageal varices bleeding due to cirrhosis (cevb), and establish a simple model for timely predicting death rsik of the patients. outcome of patients with gastro-oesophageal bleeding in a tertiary center j. wat, w.h. li, m.t. cheung methods: the medical documents of cevb patients were reviewed retrospectively and the data were collected. univariate and multivariate logistic regressions were performed, in which the discharged results (survival or death) as dependent variable and the results of liver function, kidney function, serum electrolytes and blood cell analysis as independent variables. the multivariate regression equation was as the model for the prediction of patient outcome and its predictive performance was evaluated. objectives: to determine the rebleeding rate, mortality and long term survival in cirrhotic patients presented with acute gastro-oesophageal variceal bleeding. method: this is a retrospective review of adult patients who were admitted to our hospital with the diagnosis of acute gastro-oesophageal variceal bleeding for the first time regardless of their underlying causes for cirrhosis. the study period was from january 2000-october 2008. data were collected from our hospital computer system and records. results: in univariate regression, the significant positive variables for death outcome were dbil, akp, k, wbc and plt, and the significant negative variables were tp, ap, a/g, na, cland ca 2+ . the variables entered the multivariate regression are alt, tbil, dbil, gp, a/g, cr, na + , cl -, ca 2+ , wbc, hb, plt. the sensitivity, specificity and accuracy of the regression model for predicting death of cevb patients were 97.1%, 95.1% and 95.8%. results: a total of 172 patients were included in this study, with 122 male and 55 female. their mean age was 61.9. the initial failure rate in endoscopic haemostasis was 15.1%. the 5-day and 6-week mortality rates were 11.8% and 21.9 %, respectively. poor child's grading, multiple columns of oesophageal varices, high grade of varices, failed initial endoscopic haemostasis, presence of inoperable hcc, low platelet count on admission, and short duration from index bleed to rebleed were factors associating with increased risk of 6-week mortality (p < 0.05). mean duration from index bleed to first rebleed was 15.1 months. poor child's grading and presence of inoperable hcc were associated with both early or multiple rebleed (p <0.05). overall, 37.2% of our patients developed rebleed before their variceal eradication. 5-year survival in patients with child's a, b and c were 65%, 22%, and 10%, respectively (log rank test p 0.000). conclusions: the liver function, kidney function, serum electrolytes and blood cell analysis are generally independent factors for cevb patient death risk, especially dbil, a/g and ca 2+ . the established model shows a excellent predictive performance. an imbalance in plasma amino acids of advaced cirrhotic patients impairs the maturation of dendritic cells via mtor/s6k signaling pathway e. kakazu 1 , y. ueno 1 , y. kondo 1 , k. fukushima 1 , m. shina 1 , j. inoue 1 , k. tamai 1 , m. ninomiya 1 , t. shimosegawa 1 1 division of gastroenterology, tohoku university hospital conclusion: although endoscopic haemostasis is an effective treatment modality; rebleeding is still commonly seen among patients with poor child's grading and inoperable hcc. this will result in significant bleeding-related death and poor overall survival. further advancement in treatment strategies for this group of patients are required to improve their outcome and prognosis. background: we have demonstrated that extracellular branched-chain amino acids (bcaas), especially valine, regulate the maturation and function of monocyte-derived dendritic cells (j immunol. 179: 2007) . however, it is not clear whether an imbalance in plasma amino acids of advaced cirrhotic patients influence the function of dendritic cells (dcs). methods: we used human pbmcs and cd1c+dcs in this study. we made two mediums: a serum free culture medium consistent with the average concentration of the plasma amino acids from a healthy volunteer (n=100) was defined as the healthy control medium (hcm); whereas that from advanced cirrhotic patients (n=50) was defined as the advanced cirrhotic active hepatitis b replication is defined as hbeag + or hbv dna > fibrosis was scored according to the metavir system. alt levels were characterized as being normal, < 2 x normal, and > 2 x normal. conclusions: pe494 frequency of portal hypertensive gastropathy and its non-invasive predictors in patients with viral methods: medical record of all patients with cirrhosis due to hepatitis b and c who underwent for screening egd for varices in last 2 years was reviewed. phg was defined endoscopically by using mccormack classification. noninvasive markers such as spleen/platelets ratio, meld score and child score of all the patients who underwent for egd were recorded. results: out of 360 patients 226(62.77%) were males. out of 300(83.3%) patients who had phg, 136(45.3%), 93 (31%) and 71(23.7%) had mild, intermediate and severe phg respectively. higher proportion of esophageal varices (89.7%) was present among those who have phg (p<0.001).196(65.3%) with phg has child score of 7. meld score >15 and 15 were seen in 32.3% and 67.7% of patients with phg, respectively. platelet/spleen ratio was 916.08± 400 in patients with phg as compared to 1476 methods: retrospective analysis of cirrhotics undergoing surveillance endoscopy was undertaken assessing for oesophageal varices. clinical, biochemical and radiological indices were analysed. results: 81 cirrhotics underwent surveillance endoscopy during the study. childs pugh scoring (cps) to assess prognosis of liver disease showed cps a(58%), cps b(27%) and cps c(14%). 68% were male albumin (42g/l vs 35g/l significant factors on multivariate analysis were albumin (p=0.003) and platelet count (p=0.026) conclusion: in our cohort there were significant biechemical and radiological differences in differentiating patients with large varices (grade 2-4) on surveillance endoscopy aims and objectives: to study the frequency of ev in patients with cirrhosis due to viral etiology and its correlation with different non-invasive markers. methods: medical record of all patients with cirrhosis due to hepatitis b and c who underwent screening egd for varices in last 2 years was reviewed. ev were divided in two grades (small and large) as proposed in consensus development workshop. noninvasive markers such as spleen/platelets ratio, meld and child turcotte pugh (ctp) scores of all patients were recorded. results: out of 360 patients, 226 (62.77%) were males on multivariate analysis ctp score of 7 (or 2.06, p<0.01), meld score >15 (or1.63, p<0.05) and platelet/spleen ratio 900(or 2.35, p=0.005) were found as significant predictors of large ev. conclusion: the frequency of ev is high in viral cirrhosis patients on screening egd. meld score>15, ctp score 7 and spleen/platelets ratio 900 can be used as non-invasive predictors of large ev. pe519 treatment outcome and prognostic factors of spontaneous bacterial peritonitis and culture negative neutrocytic ascites in patients with hepatitis b virus-related thirty-seven (28.5%) patients had sbp while 91 (71.5%) had cnna. except the higher proportion of renal failure at admission in patients with sbp than cnna (32.4% vs. 7.5%, p=0.001), no significant difference in the clinical and laboratory data related to liver and renal function was observed. overall mortality during hospitalization was higher in patient with sbp than that of cnna evl was repeated every 2-weeks till varicial eradication.bb dose was titrated to achieve a resting heart-rate of 55bpm or a maximum dose 320mg/d or when side-effects began to appear.primary end-points were rebleed and death.secondary end-points were complications as a result of evlor beta-blocker,variceal recurrence afterevl,and decrease in variceal grade inbb limb. results: 71 patients (median age 14[range2-68] yrs, males65%) were included (evl arm[n=37]and bb arm[n=34]. median grade of varices was iii(range ii to iv) nitric oxide synthase isoforms play distinct roles in the evolution of hyperdynamic state in endotoxemia induced portal hypertension m.r. rizvi 1 , m. shahid institute of genomics and integrative biology, mall road, delhi 110 007, 4 department of physiology a seconderc was performed after two or more years to assess the progression of the disease. results: 79 ehpvo patients(median age 20[range 5-62]yr, males 67%) were studied.history of present or previous jaundice was present in 24%,ascites 18% and pain 9%. on erc,92% had portal biliopathy.the type of bile duct involvement was categorized as: type 3 b(51%) and type 1(42%).the pattern of involvement included indentations(49%) and dilatation and strictures(36%).20%of the patients had bile duct stone and 9% had history of cholangitis.the mdian bilirubin was1.1(range0.3-15.9) mg/dl and median serum alkaline phosphatase 171(range 42-617) iu/l.all patients were treated endoscopically by endoscopic stone extraction, dilations with/without stenting. 24(30%) patients underwent second erc after a median interval of 21(range 1-111)months.the type of involvement progressed,75% patients developed type-3 involvement compared to54% at the baseline (p=ns).indentations progressed to develop strictures, from46% to71%.the frequency of new bile duct stones per year was 4%(p= ns). conclusions: portal biliopathy is very common in ehpvo, often remaining asymptomatic.however,it is slowly progressive leading to development of biliary strictures objective: to investigate the mechanisms of angiotonin ii (angii)-induced ca (2+)-independent pathways mediated by rho kinase in hepatic stellate cells (hscs) various vasoactive drugs that reduce portal pressure are used in treatment of esophageal variceal bleeding alongwith endoscopic treatment. terlipressin use decreases both, recurrent bleeding and mortality. it is given usually for 3-5 days, nevertheless there is very little data comparing different time periods. our aim was to compare the efficacy and safety of 5-days versus 10 days of terlipressin treatment in bleeding esophageal varices. methods: out of 15 patients who presented with variceal bleeding, 8 were randomized to receive terlipressin 2 mg 8 hrly, i.v. daily for first 5 days and placebo for next 5 days (group a) and 7 to receive terlipressin 2 mg 8 hrly, i.v. daily for 10 days (group b). both groups were both age and sex matched. (svt) {consisting of hvt and pvt}. there is no such data from india a comparative study of male vs background: mucosal lesions are frequently observed.gender differences are expected due to food habits, nature of job, mobility related to work, consumption of alcohol, tobacco etc. material and methods: procedure done in 1395 m & 19(3.4%) f& duo 48(5.73%) m & 73%)f.varices (48) eso varices 31 (4.3%) m &10(1.8%) f & gastric varices 1 (.11%) m & 1(.11%) f .growth (17)eso growth 5 (.59%) m &1(.17%) f & stomach growth 9(1.07%)m & 1(.17%) f & laryngeal 1(.11%)m. eso monoliasis (7) 3(.35%) m & 4(.71%) f.polyp (8) eso polyp 4(.47%) m & 1(.17%) f & gastric polyp 1(.11%)m & 1(.17%) f & moniliasis and ulcers are more common in female lee 1 1 department of internal medicine, gyeongsang national university school of medicine background/aims: although the pyogenic liver abscess is a common intraabdominal inflammatory disease, this complications are not rare. however, reports dealing with this complications are not good enough and results are often variable. the aim of this study was to identify the predictive factors of complication in the pyogenic liver abscess. pe538 effects of saikosaponinsd (ssd) on expression of c-myc and pcna in experimental hepatocarcinoma of all rats were killed in the 18th week, then general conditions of rats were recorded, the serum alt akp ggt afu was detected and pathological examination was made. the expression of pcna and c-myc were tested by immunohistochemistry. results: he staining showed that rats were induced to hepatocellular carcinoma,the results of liver function in the 18th week displayed that alt akp ggt and afu of all groups were increased than that of normal control group conclusion: ssd can inhibit development of hepatoma induced by den, possibly by down-regulating the expression of pcna and c-myc protein lethal endotoxic shock was induced by single endotoxin (e.coli) 6mg/kg injection into abdominal cavity. ppc in 5% gs was given via tail vein at 1ml/100g (232.5mg/kg) 24h and 6h before endotoxin. rats' behavior and 24h survival were recorded, venous blood taken for ast and alt, liver preserved for he staining and liver/body weight ratio l/b and liver wet/dry weight ratio (w/d) calculated. intercellular adhesion molecule-1 (icam-1) expression in liver tissue was observed with 2-step immunohistochemistry assay. results: rats of ns and ppc group demonstrated similar normal activity, histology and other characters (p>0.05), while lps group showed sag and less water-intak and severe inflammation in liver including inflammatory cell accumulation, parenchymal cells edema and tissue exudation. p+l group turned tired but could drink water the role of insulin resistance, adipokine and cytokine pro-inflammatory in non-alcoholic fatty liver disease n. ratnasari 1,2 , s. anam 2 , p. bayupurnama 1,2 , s. maduseno 1,2 , s. nurdjanah 1, 2 1 dr sardjito general hospital yogyakarta indonesia, 2 background: non-alcoholic fatty liver disease (nafld) is a benign disease during 5-10 years period, with 67%-59% survival. nafld can progress to fibrosis, cirrhotic and cancer of the liver. the etiopathogenesis of nafld is still unknown, however genetic and environment factors are predicted. objective: to know the role of insulin resistance, adipokine and cytokine pro-inflammatory on nafld. methods: the cross sectional study was performed on general check-up population at dr. sardjito general hospital yogyakarta, indonesia. the study was begun from january 2007 until november 2007 at internal medicine outpatient department. inclusion criteria: adult, alcohol consumtion 20g/day, metabolic syndrome patients, and healthy subjects. exclusion criteria: the diseases with increasing liver enzymes (hbv, hcv, ischemic hepatitis, congestive liver), a "bright liver" on ultrasound examination (malnutrition, rapid weight decreased, post gut surgery on obesity patients, and drug induced). based on liver ultrasound subjects were devided into steatosis group and non-steatosis group. data were analyzed by t-test and non-parametrical test. results: 101 subjects that were enrolled the study 61 steatosis (60.4%) and 40 non-steatosis (39.6%) and the subjects who completed cytokine and adipokine examination were 48 steatosis and 30 non-steatosis. there were significantly different on homa -ir and adiponectin level in steatosis group (homa-ir 3.367±4.901 vs. 1.430±1.889, p=0.019; adiponectin 4.049±1.929 vs. 7.034±3.777, p=0.000). there were not significantly different on tnf-, il-6, leptin and visfatin level (p>0.05). conlusions: there were significantly different on homa-ir and adiponectin level in nafld patients compared non-nafld patients. background: to optimize management of nonalcoholic fatty liver disease (nafld), a simple screening tool is necessary. in this study, we aimed to devise a simple index that reflects the presence of nafld in the korean population. methods: a cross-sectional study was conducted on 10,724 health check-up subjects at a healthcare center (5,362 cases with nafld versus age-and sex-matched controls). study subjects were randomly assigned to a derivation cohort or a validation cohort. an index reflecting the presence of nafld was derived in the derivation cohort and validated in the validation cohort. results: multivariable analysis indicated that body-mass index (bmi), serum alanine aminotransferase (alt) to serum aspartate aminotransferase (ast) ratio, sex, and the presence of diabetes mellitus were independent predictors of nafld. using these variables, a formula was derived using a linear regression model: nafld index (nafldi) = 8×alt/ast ratio +bmi (+3, if female; +2, if diabetes mellitus).nafldi had an area under receiver-operating curve of 0.812 (95% confidence interval, 0.801-0.824). at a value <31.0, nafldi ruled out nafld with a sensitivity of 89.0% and a negative likelihood ratio of 0.22, and at a value >36.0, nafldi detected nafld with a specificity of 91.2% and a positive likelihood ratio of 5.42. in the validation cohort, the predictive power of nafldi was maintained at similar levels.aim: since nash could progress to liver cirrhosis and hepatocellular carcinoma, it is important to correctly diagnose between nash and simple steatosis (ss). the aim of this study was to determine the prevalence of nash among nafld patients and to clarify differences in clinical features between nash and ss. subjects and methods: thirty-one patients with nafld showing abnormalities in serum transaminase (ast and /or alt >40 iu/l) were enrolled (sex: male 11, female 20; mean age: 49.5 yrs, mean body mass index: 29.3), after obtaining informed consent. differential diagnosis between nash and ss was performed histologically according to the matteoni classification and clinical features were compared. results: among the patients with nafld, 81% and 19% were diagnosed with ss and nash, respectively. no significant differences in the sex, mean age and bmi were seen between nash and ss groups. the levels of ast, alt, homeostasis model assessment-insulin resistance (homa-ir) and hyaluronic acid were significantly elevated in nash patients compared to ss patients. no significant differences in serum levels of adiponectin, as well as the rates of occurrence of diabetes, hypertension and hyperlipidemia were observed between the two groups. conclusion: the prevalence of nash in nafld patients was about 20%. nash patients showed higher levels of serum transaminase, homa-ir and hyaluronic acid, compared to ss patients. a large-scale biochemical study is required to accurately diagnose nash patients and confirm these results. conclusion: nafldi was a simple, efficient screening tool for nafld that could be utilized for selecting individuals for liver ultrasonography and for determining the need for lifestyle modifications.pe432 aim: this study was conducted to evaluate the hepatoprotective effects of the centella asiatica extract in 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (mptp)-induced liver injury in rats. methods: sprague dawley rats were treated with alcohol extract of centella asiatica orally in two doses (20 and 40 mg/kg/day) for 3 months along with intraperitoneal injection of mptp (1 ml/kg). biochemical parameters such as serum total protein, albumin and marker enzymes were estimated. histopathological studies of liver were also carried out to confirm the biochemical changes.results & discussion: mptp -induced hepatotoxic effects were evident by a significant (p < 0.05) increase in the serum marker enzymes and a decrease in the total serum protein and albumin. administration of extract of centella asiatica effectively inhibited these changes in a dose-dependent manner; maximum effect was with 40 mg/kg. histopathological examination of liver tissue corroborated well with the biochemical changes. hepatic steatosis, hydropic degeneration and necrosis were observed in mptp-treated group, while there was a significant reduction in these changes in the treatment group. conclusion: centella asiatica extract exhibited hepatoprotective action against mptp induced liver injury. further optimization of tuberculosis chemotherapy requires a comprehensive evaluation of the effects of antitubercular drugs on metabolic processes in organism.wistar albino male rats, body weight (b.w.) of 160-200 g, were divided into three groups: group i received pyrazinamide per os at a dose of 1000 mg/kg b.w./day, whereas group ii received a dose of 2000 mg/kg b.w./day, in both groups it was given for 60 days; the control group was composed of intact animals. the contents of free amino acids were determined using an amino acid analyzer -881 (czech republic). the study of the effects of pyrazinamide administered in different doses on the liver contents of free amino acids showed the largest number of changes at a dose of 1000 mg/kg b.w./day. the content of free amino acids at the level of 16 amino acids and total sum of amino acids significantly differed from controls. part of these changes could be regarded as compensatory answer of organism to this drug action. further pyrazinamide dose increasing caused exhaustion of liver adaptive possibilities. the study of the influence of pyrazinamide on liver contents of free amino acids allows to fully estimate the effects of this substance on metabolic processes in this organ. moreover, the effect of pyrazinamide on the majority of free amino acids in the liver is dose-dependent. background: taiwan is an endemic area of hbv and hcv infection, chemotherapy for lymphoma patients who has been hbv infection, may induce serious clinical sequela due to reactivation of hbv. this study want to clarify the difference of the chemotherapy induced hepatitis between hbv and hcv carrier in lymphoma patients. methods: from july, 2003 to july 2006, 537 non-hepatocellular carcinoma patients were enrolled, 49 (12.5%) cases were lymphoma, in these lymphoma patients, 24(48.9%) cases have been hbv infected, and 22 (44.9%) cases have no hbv or hcv infected. 3 (0.6%) cases have been infected with hcv. all patients received chemotherapy with the regimen of chop or r-chop. liver function , viral markers, hbv dna, hcv rna were checked before and after chemotherapy. results: hepatitis happened in 16 (32.7%) lymphoma patients, 11 (45.8%) cases were in hbv infected patients, 3 (12%) cases were in non-hbv infected patients, 2 cases of hcv infected patients suffered from hepatitis. hbv infected hepatitis patients hbv dna elevated more than 2 log as before chemotherapy. non of the hcv infected patient has elevated of hcv rna after chemotherapy. the mortality rate in hbv infected patient is 33%. no mortality in hcv infected patients after chemotherapy. conclusions: 1.high rate of hbv reactivation and mortality in chemotherapeutic lymphoma patients who has been hbv infected. screening of hbv viral markers among candidates for cancer chemotherapy is mandatory, especially in lymphoma patients. large number and prospect study for chemotherapy induced hepatitis in hcv carrier are needed. background: excessive drinking leads to social, psychological, physical and other problems. this study investigated the epidemiology of ald and analyses the associated risk factors. methods: from 6,043 residents 3,815 blood samples were collected. alcohol consumption and the impact of alcohol on liver function, blood lipids, blood pressure and bmi and mcv have been evaluated. results: the drinking rate and average daily alcohol intake was 35.0% and 36.97±48.76g respectively. the total alcohol intake was 297.90±506.52kg and the average drinking age was 19.21±11.34 years. the average -gt, ast, alt, mcv, chol, tg, ldl-c, hdl-c and bp increased gradually with increase in alcohol intake. the population ald prevalence was 3.98%. the prevalence of ald among the drinking population and the alcoholic population was 11.76% and 44.17% respectively. conclusion: chol, -gt, ast, alt, and mcv were highly correlated with daily alcohol intake which closely related to the occurrence of ald. n. tanaka 1 , w. okiyama 1 , t. aoyama 1 background: alcoholic liver disease (ald) is one of the leading causes of cirrhosis and yet efficient therapeutic strategies are lacking. polyenephosphatidylcholine (ppc), a major component of essential phospholipids, prevented alcoholic liver fibrosis in baboons. however, its precise mechanism remains uncertain. we examined the effects of ppc on ald using peroxisome proliferator-activated receptor (ppara)-null mice treated with an ethanol-containing diet, which showed pathological features similar to human ald. methods: male ppara-null mice were pair-fed a lieber-decarli control or 4% ethanol-containing diet with or without ppc at a clinically comparable dose (30 mg/kg/day) for 6 months. o. parkash 1 , a. almas 1 , s.h. ali shah 1 , w. jafri 1 , s. hamid 1 , j. akhtar 1 1 aga khan university hospital karachi 1.hdv-hbv co-infection presents mostly as moderate to advanced liver disease. background: liver injury due to dengue infection is not uncommon. acute liver injury is a severe complicating factor in dengue, predisposing to life-threatening hemorrhage, dic and encephalopathy. results: there were no significant differences of demographic features and laboratory parameters such as peak serum alt, total bilirubin and creatinine between 2 groups. however, peak ast was higher in superinfected group than control group (median: 2,000 iu/l vs 731 iu/l, p=0.035,). additionally, the peak serum albumin levels, prothrombin time and platelet counts were lower in superinfected group than control group (median: 3.0 mg/dl vs 3.3 mg/dl, p=0.02, 51.8 % vs 87.2 %, p=0.027 and 103 x 10 3 /mm 3 vs 165 x 10 3 /mm 3 , p<0.001, respectively). of superinfected group, 9 patients were followed over 6 months after resolution of aha. interestingly, serum hbv-dna levels decreased significantly over 3 months following resolution of aha, then rebounded subsequently (median: -1.89, -1.85, -0.38, 0.58 and 1.10 log10 copies/ml at 1, 3, 6, 12 and 24 months, respectively).methods: the overlapping fragments of hev isolate swgx40 were amplified with reverse-transcription nested polymerase chain reaction (rt-npcr) and the 5' and 3' ends of viral genome were amplified with rapid amplification of cdna ends (race). the pcr products were cloned and sequenced. the phylogenetic analysis of swgx40 was performed.result: the genome of swgx40 consisted of 7,233 nucleotides, excluding the poly (a) tail of 36 residues. the genome contained three open-reading frames (orfs), orf-1 encoding 1705 amino acids, orf-2 encoding 674 amino acids and orf-3 encoding 114 amino acids. the full-length genomic sequencing showed that swgx40 strain shared similarity with all known hev genotype 1, 2 and 3 isolates by 73.4% to 76.5%, and with an identity of 83.1% to 91.2% among genotype 4 hev isolates, and a high nucleotide identity as 94% with chinese guangxi human strain lz-105.conclusions: acute hav super-infection may suppress hbv-dna replication in chronic hbv carriers and chronic hepatitis b, although the suppressive effect did not seem to sustain longer than 3 months. conclusion: the swine hev strain swgx40 was phylogenetically close to the human hev strain lz-105, both from the same region in south china. therefore it was concluded that hev sub-genotype 4b might have existed in south china at least for 6 years and now it was prevalent both in local human and swine, which also strongly supported the zoonosis hypothesis of hepatitis e. associated with splenic volume were damping index (r=0.213, p=0.022) and blood flow of portal vein (r=-0.314, p=0.001). conclusions: the splenomegaly in portal hypertension was more frequent in non-alcoholic groups, and associated with a damping index and a blood flow of portal vein. the measurement of blood flow of portal vein, damping index, and splenic volume by a doppler sonography can be helpful to predict the varices.z.q. zhang 1 , j. cao 1 , w. lu 1 , l.g. shi 1 1 objective: to appraise the clinical efficacy of simple non-invasive models of ast-to-alt ratio (aar), ast-to-platelet ratio index (apri), spleen-to-platelet ratio index (spri), age-platelet index (api), age-spleen-to-platelet ratio index (aspri) for predicting hepatitis b associated cirrhosis. methods: 138 patients and 32 patients were diagnosed pathologically as non-cirrhosis and cirrhosis, respectively. the simple non-invasive models were calculated as described originally. spss 13.0 was used for statistical analyses.background: to reveal the microrna (mirna) expression profile of the hepatic fibrosis inducing cells, rat hepatic stellate cells (hscs), during in vitro activation. results: the areas under roc curve of aar, apri, spri, api, aspri for predicting the cirrhosis were 0.81, 0.62, 0.71, 0.73, 0.74, respectively which were larger than those under the diagnosis reference line (p 0.000, 0.033, 0.000, 0.000, 0.000, respectively).methods: the hscs were isolated from male sd rats by in situ perfusion and density-gradient centrifugation. the quiescent and activated hscs, which were harvested at day 2 and 14, respectively, were then subjected to immunocytochemical staining (desmin and -sma), oil red o staining and quantitative rt-pcr (desmin, -sma, albumin, cd31, cd68 and cytokeratin-19). after extraction and labeling, the hy3-labeled cellular rna samples and hy5-labeled reference pool rna samples were mixed pair-wise and hybridized to the lna mercury microarray. differentially expressed mirnas were filtered and randomly verified by stem-loop rt followed by quantitative pcr.conclusion: all of the simple non-invasive models of aar, apri, spri, api, aspri can be used for predicting hepatitis b associated cirrhosis; and aar has the most practical efficacy for predicting hepatitis b associated cirrhosis. results: both the purity and the total activation of hscs were validated. global analysis of the mirna expression profile based on quiescent and activated hscs demonstrated 21 differentially expressed mirnas. among these, 12 mirnas were up-regulated more than 2-fold in activated hscs as compared to that in quiescent hscs, while 9 mirnas were less than the threshold level (0.5-fold) during the hsc activation. furthermore, the expression of mir-16, 15b, 122, 138, 143 and 140 had been proved. background/aims: only limited patients with chronic hepatitis b virus (hbv) infection will develop liver cirrhosis, and no effective methods to precisely predict ones who will develop cirrhosis. we try to establish a model to predict the patients with the risk of cirrhosis development basing on a clinical epidemiological factor survey. z.q. zhang 1 , w.y. bao 1 , w. lu 1 , l.g. shi 1 methods: cirrhosis patients with hbv markers (case group) and asymptomatic hbsag carriers (control group) were recruited and inquired by researchers with a specific designed questionnaire including 98 items. a multivariate logistic regression analysis were conducted to establish a predictive model, in which two third patients selected randomly as model sample and another 1/3 patients as validating sample, key factors screened out as variables. the predictive performance of the model was evaluated. objective: to explore the practical significance of the peripheral blood corpuscle counts for prediction of hepatitis b associated cirrhosis. methods: 122 and 31 male patients with chronic hepatitis b were pathologically diagnosed as non-cirrhosis and cirrhosis. peripheral blood corpuscle counts were measured by coulter ac•t diff hematology analyzer. results: red blood cell (rbc), platelet (plt), neutrophil (n) counts in cirrhosis were significantly lower than those in non-cirrhosis; and lymphocyte, mid-cell counts were similar to those in non-cirrhosis. the areas under the roc curves of rbc, plt, n counts for prediction of cirrhosis were 0.24, 0.32, 0.34 respectively; according the optimal cut-off determined by the roc curves, the sensitivity, specificity, positive predictive value, negative predictive value, accuracy of rbc, plt, n counts for prediction of cirrhosis were 0.55-0. method: pbmcs of 8 active chb patients under pyg-interferon treatment were analyzed for their th17, treg and pdc by flow cytometry. they were determined by cd4/il-17 for th-17, cd4/cd25/foxp3 for treg and cd123/cd86/cd14-for pdc. pbmc were collected every 4 weekly during the treatment until end of therapy (week 48) and every 12 weekly until the end of follow-up (week 72). alt was quantified at every time of the pbmc collection. ifn-gamma release cells were analyzed by elispot to hbv-core and s ag.background: alpha fetoprotein (afp) is a well-recognized tumor marker for hcc; elevated level of afp is found in at least 70% of hcc. other liver diseases such as cirrhosis and chronic hepatitis are also related with an elevated level of afp. the regulation of afp gene expression has been relatively less studied although the gene has been suggested to play a role in hcc development. in this study, we tried to identify genetic variations in afp gene and analyze its effect on serum afp level and possible hcc progression.results: in parallel with decline in alt for the first 12 weeks, we found decline in both pdc (r=0.71, p=0.03) and elispot (r=0.65, p=0.03 for hbv-core ag; r=0.48, p=0.05 for hbv-s ag). although there is trend that th-17 decline with treg decline, but they are not statistically significant differences in the same period. there is no significant difference between the svp and non-svp patients.methods: direct dna sequencing was carried out to sequence afp promoter and 500 bp upstream and downstream of afp coding regions in dna samples isolated from 99 hcc subjects and 105 controls respectively. for each samples serum afp levels were determined using commercially available elisa kits. conclusions: under pegyintron treatment, pdc, ifn-gamma change in the same trend of alt during 12 weeks of treatment. it implied that pdc take regulatory effects on ifn-gamma releasing cells and be very tightly related to alt. there wasn't a significant difference in both treg and th-17, which implies that treg and th17 might be of important cells in keeping the stability of the immune system.results: a total of 30 snps were detected in the afp genomic region analyzed, including 29 known snps and one novel snp. among the identified snps, the c>g nucleotide change in the position -250 bp upstream of afp transcriptional start site showed a significant association with hcc (p < 0.05) and a decrease in afp gene expression level. conclusion: our preliminary results indicated a possible association between serum afp expression and -205g allele. the identified snp is located in afp promoter region with possible binding sites for known transcription factors, such as tfiid, coup, apf and nfiii. -30°tail-suspension (ts) rats were used as the model to simulate the physiological effects of weightlessness. thirty-two wistar male rats were randomly divided into 4 groups: control for 7d (7d con), 14d con, ts for 7 d (ts7d) and ts14d. histopathological changes of testicle of the rat were observed by he stain. localization and expression of ar and hsp70 in testicle of rat were observed comparatively by means of immunohistochemistry, and the density of ar and hsp70 immunoreactivety in four groups were compared. results: signal molecules mrna level are shown in the table 1 (** p<0.01, * p<0.05). tnfa, il1, il6 and il8 were higher in group 1, 2 and 4 than those in group 5. ifna was higher in group 5 than that in other groups. there are no significant difference in infc, il2, and il4. there was a positive correlation between tnf and myd88 in group 2, tnf and nfkb in group1 and 2. results: obvious pathological lesions presented in testicle of ts7d and ts14d rat. germinal epithelium irregularity and malformed spermatozoa were found in seminiferous tubules. degeneration and necrosis of germinal epithelium appeared in testicle of ts7d and ts14d rat. ar immunoreactive cell density in the ts7d and ts14d groups were significantly decreased compared with the in-phase normal control groups ( p < 0. 01). while hsp70 immunoreactive cell density in the ts7d and ts14d rats were significantly increased than those of control rats( p < 0. 01), and in testicular interstice or extracellular there were very strong ehsp70 immunoreactive positive staining signals. the results indicate that ground simulated weightlessness induced by 7d-14d tail-suspension in rats can lead to the serious injury , depressed expression of ar and enhanced expression of hsp70 in testicle. despite the absence of any serologic marker of hbv recurrence, however, it remains unknown whether there is occult reinfection in the liver graft. we aimed to detect and quantify the presence of intrahepatic hbv dna in the liver grafts of patients who remain seronegative for hbsag for more than 1 year after liver transplantation. materials and methods: liver biopsy and blood samples were obtained from 31 patients who had been receiving nucleoside analogue prophylaxis alone and remained persistently seronegative for hbsag for at least 1 year (median 44.5 months, range 13.6 to 126.4 months) after liver transplantation for chronic hepatitis b. quantitative polymerase chain reaction was performed to detect and quantify total and covalently-closed circular (ccc) hbv dna in the liver (lowest detection limit, 10 copies/ml), serum and pbmc. direct sequencing was used for hbv quasispecies screening. results: liver biopsy was performed and intrahepatic hbv dna as measured by quantitative real-time pcr was detectable in 26 of 31 recipients. donors anti-hbc status before liver transplant was significantly related to the presence of intrahepatic hbv dna in the recipient's study biopsy (p=0.038). donor intrahepatic hbv cccdna levels correlated with recipient post-liver transplant intrahepatic hbv cccdna levels (p=0.004). hepatitis b virus sequencing results and phylogenetic analysis revealed that hbv reinfection in two recipients were of donor origin, four recipients were of recipient origin and four recipients were of both donor and recipient origins. conclusions: our findings demonstrate the presence of occult hbv reinfection with persistence of hbv dna in liver allografts despite long term nucleoside analogue prophylaxis after liver transplantation, suggesting the need to continue indefinite antiviral therapy. the use of liver grafts from anti-hbc-positive donors might increase the risk of occult hbv reinfection. both donor and recipient hbv dna could contribute to occult hbv reinfection in liver transplant recipients. aim: to construct one noninvasive assessment model consisting of routine laboratory data to predict both significant fibrosis and cirrhosis among patients with chronic hepatitis b(chb). methods: we have retrospective analyzed 137 consecutive patients with chronic hepatitis b who underwent percutaneous liver biopsy. we calculated sensitivity, specificity, positive predictive value(ppv), and negative predictive value(npv) of an apri 1.5 in combination with different hyaluronic acid(ha) cut-off points medium (acm). we stimulated pbmcs or dcs under hcm and acm, and evaluated the function. results: after adding the stimulants under hcm, the cd83 and cd86 expression of dcs from cirrhotic patients (lc) were lower than those from healthy contorols (hc). in both hc and lc, the cd83 and cd86 expression of dcs stimulated under acm was lower than that under hcm. the il-12 production in acm was lower than that in hcm. the expression of cd98, which is related to amino acid transport, was not different between hcm and acm. however, dcs cultured in acm expressed lower levels of phospho-p70 s6k than those cultured in hcm. finally, we ascertained that the ifn gamma production by pbmcs was significantly decreased under acm.conclusions: an imbalance in plasma amino acids of advanced cirrhotic patients suppresses the maturation of dcs via mtor/s6k signaling pathway. key: cord-022650-phsr10jp authors: nan title: abstracts tps date: 2018-08-14 journal: allergy doi: 10.1111/all.13539 sha: doc_id: 22650 cord_uid: phsr10jp nan (either in men or women) between metabolic syndrome and incident asthma. conclusion: this study confirmed the significance of obesity as a risk factor for incident asthma. moreover, obesity appeared to be a stronger risk factor than metabolic syndrome. 0638 | relationship between helminth infection, blood eosinophils and asthma symptoms in a rural community from the tropics peñaranda d; alvarez l; sierra n; lopez j; zakzuk j; caraballo l institute for immunological research. university of cartagena, cartagena, colombia background: immune response to helminths shares many features with the allergic response. in tropical regions where helminths are highly prevalent, asthma is still a major public health burden. large clinical cohorts suggest that high blood eosinophils (hbe=>400 cells/ mm 3 ) are associated with asthma exacerbations. however, the association between hbe and asthma severity in rural communities with prevalent helminthic infections is unclear. method: patients with wheezing symptoms in the last year living in a rural tropical community (santa catalina, colombia) where helminths are highly prevalent, were recruited for this study. blood eosinophils were assessed by complete blood count. parasitic infection was evaluated with two serial coprological exams (kato-katz method) and skin prick tests were conducted to determine reactivity to ascaris. results: seventy-three patients (mean age: 21; range: 2-64 years old) were recruited in this study. a. lumbricoides and t. trichuria active infection (47.9% and 16.4%, respectively) were not related to age or gender. a positive spt to ascaris extract, aba-1 and d. pteronyssinus was observed in 23%, 18.4% and 34.2%, respectively. mean eosinophil count was 621 cells/mm 3 ; 43.9% had hbe. rate of patients with at least one emergency department visit was 61.9% and hospitalization, 21.9%. blood eosinophil counts (as a continuous variable) were inversely associated with age (p = 0.03) and higher in helminth infection (p = 0.002). in crude univariate analysis, exacerbations (er and/or hospitalization) were associated with age (or: 0.96; 95% ci: 0.93-0.99, p < 0.01) and hbe (or: 2.9; 95%ci: 1.1-7.8, p = 0.04), but not with helminth infection. for a better definition of asthma, multivariate analysis done in those >7 years old indicated that hbe, helminth infection and positive ascaris spt were not associated with asthma exacerbations. conclusion: uncontrolled asthma is common in rural places of the tropics. since helminth infection influences eosinophilia, the clinical value of hbe to predict exacerbations is limited in helminth-endemic populations. castro mc 1 ; ferreira j 2 ; sarmento d 2 ; carvalho c 2 ; matos a 2 ; bicho m 2 1 chln-immunoallergy; lisbon medical school-genetic department, lisboa, portugal; 2 lisbon medical school-genetic department, lisboa, portugal background: the bioavailability of no and endothelial homeostasis depends on the functional polymorphism of 19-bp del/ins within intron-1 of dhfr (dihydrofolate reductase enzyme) (rs70991108) that could interfere in the regeneration of bh4 (tetrahydrobiopterin) from bh2 (7,8-dihydrobiopterin) and contributes to endothelial dysfunction in asthma. method: asthmatics (n = 123) compared with control group (n = 50).the polymorphism was analyzed by pcr. control of asthma assessed by (acq7 and paqlq). statistical analysis with spss 23.0 establishing a significance level of p < 0.05. results: there are 80 women and 43 males in asthmatics and 39 women and 11 males in controls (p = 0.137). in asthmatics: age ( x ± sd): 38.26 ± 19.24 ; and in control group: age ( x ± sd): 51.50 ± 13.34. the genotype frequencies in asthmatics are: dd (6.5%); id (66.7%); ii (26.8%); in control group: dd (12.0%); id (64.0%); ii (24.0%); there is no statistical difference between groups (p = 0.478). the allelic frequencies in asthmatics are: allele d (39.8%); allele i (60.2%); in control group: allele d (44%); allele i (56%); there is no statistical difference between groups (p = 0.476). the genotype frequencies in the uncontrolled asthmatics are: dd (0.0%); id (61.1%); ii (38.9%) ; in the controlled asthmatics are: dd (9.4%); id (68.2%); ii (22.4%); there is statistical difference between groups (p = 0.047). genotypes id and ii are more frequent in the uncontrolled asthmatics. the allelic frequencies in the uncontrolled asthmatics are: allele d (30.6%); allele i (69.4%); in the controlled asthmatics are: allele d (43.5%); allele i (56.5%); there is a trend to have differences between groups (p = 0.059). allele i is more frequent among uncontrolled asthmatics. the uncontrolled asthmatics are older than the controlled asthmatics (p < 0.001). there is no differences in gender distribution (p = 0.903). the genotype ii confers a risk of being uncontrolled asthmatic of 2.950 times when compared with controlled asthmatics and adjusted for age: or b : 2.950 [1.117-7.789 ]; p = 0.029. physiopathology and the emergence of evidence-based clinical guidelines. however, variation still exists among some diagnostic aspects of asthma in real life. it is unknown to what degree diagnosis is affected by the treating physician's medical specialty. results: a total of 62 gps, 239 pediatricians, 364 allergists, 161 pulmonologists and 34 otolaryngologists (orls) replied. although for general application of diagnostic clinical criteria all physicians rated similarly, in general accordance with the mag suggestions, a third of non-pulmonologist practitioners don't recognize chest discomfort as one of the clue symptoms of asthma, but they erroneously believe crackles are (p = 0.01). we found agreement in almost half of all physicians to erroneously believe that viral illness' induced wheezing in non atopic children predisposes asthma. conversely, 75-85% are aware that allergic sensitization predisposes to asthma. most specialists -except pulmonologists (p = 0.02)-incorrectly listed fev1 as the best parameter to identify airflow obstruction (ao) and fev1/fvc to assess ao severity. 20% of gps do not know peak expiratory flow (pef) measurements could be valuable, and 75% of all specialists are not aware that changes in pef can also be used to confirm ao reversibility. to classify asthma, only pulmonologists adequately considered the level of control in similar proportion than severity (81% and 83%, respectively), which is uniformly the preferred method by most other specialists. conclusion: although in general many clinical aspects of asthma diagnosis seem to be accurately assessed, there is a wide specialityspecific variation regarding some aspects of phenotyping and classification, diverging from mag's recommendations. as such, our results can help to detect knowledge-gaps and to guide the development of more focused specialty-specific learning tools to improve clinical impressions, process medical evidence, and apply it to patient care. 0652 | issues, continuous medical education on treatment of acute asthma, exercise induced asthma and asthma in pregnancy should include, per medical specialty background: to unify and improve the management of asthma, including asthma exacerbations, the mexican guideline on asthma (25.5%) of 51 employees who stated increased symptoms with flour exposure. among all workers 9 (5.5%) employees were diagnosed as asthma and 7 (4.3%) workers were diagnosed as ba. conclusion: wheat flour sensitivity is high among workers who are exposed to wheat flour, however the prevalence of ba is similar to the previous data in the literature. johnsen cr 1 ; callesen kt 2 ; jensen bm 2 ; poulsen lk 2 1 clinic of allergy, dept. of dermato-allergology, gentofte hospital, copenhagen, denmark; 2 laboratory of allergy, gentofte hospital, copenhagen, denmark background: enzymes are well known as sensitizers and causes of occupational allergy primarily in the industries producing and using the products. we present a case of occupational contact urticaria, rhino-conjunctivitis and asthma in a 32 year male chef who was using a transglutaminase enzyme powder obtained from fermentation of streptomyces mobaraense as meat glue in processing of fine culinary dishes. this transglutaminase has been used for protein food preparation in industrial settings since 1992 to improve the texture of protein rich foods such as surimi or ham. in this case it was used in small scale in a gastronomy restaurant kitchen spraying enzyme powder with a sieve over raw meat without any protective equipment in contrast to the producer's recommendation. the chef was also found allergic to dried, edible mushrooms also forming part of the meat dish prepared with the transglutaminase enzyme powder. in one occasion he experienced an oral reaction with itching and swelling of the mucosa in the mouth, stridor, angioedema of the face, and urticaria after ingestion of beef meat treated with transglutaminase and rolled in horn of plenty dried mushroom powder. no other symptoms of food allergy were reported but a known cat allergy was. background: formaldehyde and xylene are occupational skin and respiratory irritant and/or sensitizer, exposure to those may be associated with dermatitis, rhinitis and asthma. health care workers, as nurses, laboratory technicians, doctors could be exposed in different tasks in operating rooms, endoscopy and in pathology laboratory. we describe three cases of work-related rhinitis in technicians employed in the same unit of hospital pathology . first case: a woman of 38 years old underwent medical examination in our occupational allergy unit because allergy respiratory symptoms. she has been working for 8 years in pathology laboratory and was exposed to xylene and formaldehyde. she developed rhinitis, rhinosinusitis, hyposmia and cough with sputum after 5 years started work. she had negative skin prick test for common aeroallergens. lung function was normal with a fev1/fvc ratio of 80% of predict. blood cells count reveled 15% of eosinophils (980/mmc) with 6550 total leucocytes. second case: a woman of 40 years old was affected by moderate persistent allergic rhinitis with positive skin prick tests to house dust mite, dog and cat. in the last year rhinitis symptoms worsened in relation to work and improved during vacation. when she was exposed mostly to formaldehyde during shift at the end of it she usually experienced face skin and conjunctival erythema. she developed work-related symptoms after 14 years of exposure in the pathology unit. third case: a woman of 38 years old, who has been working for 14 years in the pathology unit and was exposed to formaldehyde and xylene, in the last year developed moderate-severe persistent rhinitis with hyposmia and chronic cough. she referred to otorhinolaryngologist and an irritant induced rhinitis was diagnosed. she had negative skin prick test for common allergens and normal lung function. results and conclusion: the workers experienced respiratory symptoms in relation to work exposure to formaldehyde and xylene. the suspected causal agents were monitored in the work environment and an exceeding of the recommended limit values was found. preventive measure were adopted with a reduction of exposure and symptoms improve only in the second and third case. challenge test with mannitol is considered to be more specific than test with methacholine. also, duration of procedure is shorter and safer. therefore the study aim was to compare the usefulness of these two tests in monitoring of sict. method: four bakery workers with suspicion of oa underwent single-blind, placebo-controlled sict with workplace allergens accompanied by evaluation of nsbhr with mannitol and methacholine before and after sict. clinical examination, spirometry, skin prick tests (spts) to common aeroallergens and occupational allergens, serum specific ige antibodies to occupational aeroallergens were also performed. results: positive spts results to occupational aeroallergens were found in all bakery workers, specific ige to flours were detected only in two subjects. three out of the four patients displayed positive sict reaction (in two cases early spirometric response). in all of these 3 patients, airway response to methacholine increased significantly. in the first two patients also airway reaction to mannitol was significant, whereas in one subject with early reaction there was no increase in nsbr after mannitol inhalation. the patient with negative sict results did not reveal any changes in nsbr before and after the test, neither to methacholine nor mannitol. 0668 | rice-induced occupational anaphylaxis and socio-economic impact-case report method: in this prospective study, a total of 240 students completed a self-administrated questionnaire that comprised different questions and gave information about the participants and their glove use, working habits, signs and symptoms related to these gloves, precautions taken to minimize it, etc. skin prick test is performed through commercial extract latex gloves (stallergenes), while patch test is prepared through latex gloves and adhesives. two types of gloves are used: gloves that contain latex and gloves without latex (vinyl gloves), which are used also as e negative control. results: questionnaire items and diagnostic tests revealed that one-fourth of subjects were suspicious for latex gloves hypersensitivity. their mean value for skin reactions like irritant or allergic dermatitis or contact urticaria was between 10% and 14%, while for other symptoms the mean value was under 5%. logistic regression analysis revealed an association between different questionnaire items and positive allergy tests among suspected cases and diagnosed cases of latex allergy. approximately 20% of people who work with laboratory animals experience some allergic symptoms and about 10% of animal technicians go on to develop serious symptoms of asthma. uk government guidelines state that employers must prevent or adequately control exposure of employees to animal allergens and should undertake monitoring to ensure that suitable controls remain effective. the most widely used monitoring method is personal iom filters. however, these need to be attached to a pump and carried by the technician which can be cumbersome and awkward. previous data has demonstrated that allergens from dust mite, cat, dog and pollen could be captured and quantified by a novel type of nasal filter. in this current study, we sought to assess the feasibility of using the nasal filters for the assessment of exposure to mouse allergen in a laboratory facility. method: technicians working in a laboratory animal facility were asked to wear the filters during normal routine work. for comparison, they were also asked to wear an iom filter for the same duration. allergen was extracted from nasal and iom filters by gentle rocking in pbs-tween for two hours. levels of the major mouse urinary protein (mus m 1) were quantified using our multiplex array technology, which is highly sensitive and allows for quantification of mus m 1 down to 0.01 ng/ml. results: significant levels of mus m 1 were detected in the nasal filter extracts and these levels correlated with the type of activity that was being performed by the technician, as well as the housing environment of the mice. levels were compared to the suggested 'safe' limit of allergen exposure of 5 ng/m 3 . we also found that the technicians grew accustomed to the nasal filters quickly and found them far more practical for every day monitoring that wearing the iom filter and pump. conclusion: these data indicate that nasal filters may be considered a simple and easily wearable method for monitoring laboratory animal allergen exposure. future studies are planned to assess the feasibility of wearing the filters for analysing exposure to other laboratory animal allergens from rat and guinea pig. havana university lower co 2 emission, water and feed consumption and limited waste production. insects are currently allowed both for human and animal feeding in some eu countries, including italy and the risk profile related to production and consumption of insects as food and feed, including risk of allergenicity, is currently under evaluation by efsa. both food and feed products derived from insects require multiple manipulations by the breeder and/or by the workers who transform the insect into the commercial products, thus the occupational exposure have to be considered too. the aim of this work is to evaluate the allergenicity of tenebrio molitor, one most used species for animal feeding. method: t. molitor proteins were extracted from intact dried larvae and from flour of dried larvae. the protein extracts were separated in one-dimensional electrophoresis conclusion: according to these results, the larva flour seems to be less immunoreactive than the intact counterpart, probably due to the processing that causes the degradation of protein bands over 50 kda. working in gastronomy is associated with exposure to many factors with an irritating and allergic potential influencing respiratory system. food products and organic dust are the source of inhaled allergens which may cause sensitization during apprenticeship. the study aim is a prospective observation of incidence of sensitization to selected environmental and occupational allergens among culinary school apprentices and identification of work-related allergic diseases in this group. method: the cohort comprised 374 apprentices. they were examined in the first and the second year of education. questionnaire and allergological tests [(skin prick test) spt to common and occupational allergens, ige level evaluation (total and specific for occupational allergens) and pulmonary tests] were performed]. results: the most frequent symptoms reported by examined apprentices were rhinitis (12.8%), conjunctivitis (7%), skin symptoms (6.7%), dyspnea (5.6%) and cough (2.7%). 10 subjects developed nasal symptoms during the second year of education, while in 6 cases the skin symptoms and in 4 subjects conjunctivitis appeared. in 7 cases the work-related symptoms were reported. the most frequent positive results of spts were obtained with dermatophagoides pteronyssinus 24.4%, dermatophagoides farinae 21.6%, grass pollens 18.6%. positive spt to rye and barley flour were found in respectively 2.2% and 1.4% apprentices. 4.1% of apprentices had specific ige to flours. the preliminary results indicate that work-related allergy symptoms and hypersensitivity to occupational allergens are rarely found among culinary school apprentices in the first years of education. the further observation will allow to evaluate the trends in incidence of allergy to occupational allergens, as well as the clinical presentation of allergy in that group. 0677 | how multifaceted the clinical presentation and etiology of allergic diseases could be? method: the study was done in children from west georgia randomly and on based of questionnaire of representative cohort. (2014) (2015) (2016) . the cohort was 1500 children, 1-16 years old, risk factors were studied by way of interviewing, clinical-laboratory dates. for assessing the risk factors, was used 'case control' method. the statistical processing of material was done with computer program sps/sv12. inclusion criteria for enrolment were: collectors of dust, gender, existence of moisture and mold consuming of tabasco, atopic dermatitis and seasonality. results: the groups, which we have studied, prevalence of acute respiratory viral infection was 61%, bronchitis −29.3%, allergic rhinitis 33.9%, atopic dermatitis 9.1%, food allergy 5.4%. the reliability was high (p < 0.05) in families with bronchial asthma compared with healthy population. bronchial asthma was detected in 5.7% of population. the hereditary load of allergic diseases in patients with bronchial asthma was 9.7% and in healthy cohort it was 5.7% (p < 0.001). conclusion: based on the results, we can conclude that, ecological factors and genetic predisposition significantly influences on prevalence of sensibilisation of house dust mite, molds and formation of bronchial asthma. as the genetic and environmental factors that act on an immune system are better elucidated and their roles established, the implementation of more enduring preventive efforts will be developed. however, at present, the best approach to the child at high risk for the development of allergies is to institute dietary and environmental control measures early to decrease sensitization, and to recognize and appropriately treat the evolving signs and symptoms of allergic disease. background: plantation of road-side avenue trees has become a major part of the urbanization programme in kolkata metropolis of india for megacity beautification and environmental management. due to evergreen habits, gulmohor (delonix regia) and chhatim (alstonia scholaris) are frequently selected for plantation programme to generate green belts. however, an increasing incidence of seasonal pollinosis was observed among the inhabitants living in close vicinity to these trees suggesting a possible link between the airborne pollen load and the concomitant respiratory hazards. this prompted us to investigate the allergens in the pollen of these two dominant avenue trees. method: aerobiological surveys were conducted at multiple sites of kolkata for a period of two years using seven-day volumetric burkard sampler to record the pollen concentration in the outdoor ambient air. clinical data and residual blood of pollinosis patients were collected from a public hospital. allergens were detected in the pollen proteome fractionated in 2d gel by ige-serology. the major igereactive proteins were partially purified by ammonium sulphate fractionation followed by ion-exchange chromatography. the allergenic activity of the fractions was tested by histamine release assay. results: a clear correlation was observed between the pollinosis related morbidity and the aeropollen load especially during the peak flowering period of these two trees. about 41% and 52% of the patients displayed positive spt response and ige-reactivity using pollen extracts of gulmohor and chhatim respectively. immunoproteomic analyses revealed the presence of 7-8 ige-reactive components in the 2d pollen proteome of these species. hierarchical cluster analysis with patient immunoblot data identified a 31 kda and a 25 kda protein as major allergens of gulmohor and chhatim respectively. the purified fractions containing each of these two major allergens induced histamine release from granulocytes within a range between 42 and 77%. method: immortalized human keratinocyte cell line (hacat) and primary normal human epidermal keratinocytes (nheks) were differentiated with calcium chloride for 1 and 3 days, respectively. following the differentiation, the cells were treated with il-4 (100 ng/ml), il-13 (100 ng/ml), and/or hcho (4 × 10^-4%) for 2 hours. the mrna expression of flg, ivl, lor, dsg1, dsg3, dsc1, dsc3, as well as tslp was analyzed using quantitative real-time pcr. results: hcho exposure decreased the mrna expressions of structural components (flg, ivl, and lor) and cell adhesion molecules (dsg1, dsg3, dsc1, and dsc3) in a short-period time of exposure (2 hours). we also found that hcho exposure significantly enhanced il-4-and/or il-13-induced tslp production in nheks as well as hacat. interestingly, exposure to hcho alone is enough to increase the tslp mrna expression in both cells. conclusion: our results suggest that hcho exposure might synergistically damage the skin barrier function with il-4 and il-13 by increasing tslp expression and decreasing structural components as well as cell adhesion molecules. 0685 | skin prick test reactivity to aeroallergens in adult allergy clinic in a tertiary hospital: a 12-year retrospective study results: five different human sera were screened for specific ige level against 29 different allergen sources using test methods of three different suppliers. the sensitivity of the three different methods can be arranged in the ascending order manufacturer a < manufacturer c < manufacturer b. with the test of manufacturer a, 45% of the measurements were below the detection limit (0.35 ku/l), with the test of manufacturer c, 16% of the measurements were below the detection limit, whereas the test of manufacturer b leads to values below the detection limit in 10% of the cases. in terms of variation coefficient, the test system of manufacturer c had the best performance. test systems of manufacturers a and b exhibited comparable variation coefficients, which were considerably higher than that of manufacturer c. conclusion: based on these test results, only the test of supplier c is recommendable for determination of levels of specific ige for diagnostics of allergic patients. with the test of manufacturer a, elevated levels of specific ige antibodies for many allergens cannot be detected due to the poor sensitivity of the test system. the test system of supplier b exhibits a good sensitivity but the coefficient of variation is rather high for a diagnostic test. this drawback could be circumvented by multiple determination of one test parameter. although this is an advisable strategy in general, the routine in diagnostic laboratories is incompatible with this approach, since throughput would decrease while costs would increase. this study is another good example for the need of the implementation of a characterized standard material with known values of sige, as demanded by wojtalewicz et al. method: cd203c and cd63 expression on basophils were monitored upon exposure of whole blood samples (<24 hours) to anti-ige and/or allergenic extracts. staining was conducted on exposed samples using dry room temperature stable antibody panels (dura innovations format) coated in 96 well plates, eliminating all antibody pipetting steps from the workflow. red blood cells were lysed and data was acquired (without further wash steps) on a cytoflex flow cytometer (beckman coulter). staining and lysing were automated using a biomek (beckman coulter). results: the described no-wash preparation protocol, already established for manual preparation mode in tubes, could be transconclusion: the hr-test was significantly less likely to be positive, if a patient suffered from monosymptomatic ae than in ae patients with concomitant urticaria. this could signify a higher likelihood of treatment response to antihistamine and other anti-allergic medication in the latter group. background: pathogenetic mechanisms of allergy are polymorphic. they include ige-dependent and ige-independent, allergen-specific granulocyte-mediated and lymphocytic reactions, as well as nonspecific hypersensitivity, which are realized through a variety of mediators: histamine, tryptase, etc. allergen bucal challenge mimics the natural situation and is useful for understanding the mechanisms of allergic airway inflammation and airway hyperresponsiveness (ahr).saliva used as a non-invasive readily available bio-sample for diagnosis instead of blood. biomarkers in saliva are associated with the pathogenesis and clinical outcome of allergic diseases method: aim: to examine mediators for ahr with buccal(mucosal) challenge tests. we examined 14 patients with allergic asthma(the history, positive skin prick test, serum specific ige) and 12 healthy volunteers. saliva were collected. then, both groups were subjected to buccal(mucosal) allergen challenge by a water-salt solution of the mite allergen dermatophagoides pteronyssinus. saliva was recollected in 30 minutes and 24 hours after the provocation. the level of myeloperoxidase, elastase, tryptase in saliva were determined by the elisa. that provocative test did not cause clinical symptoms development or reduction in nasal bronchial patency in any patient. results: in patients with allergopathology, an initially increased level of myeloperoxidase and tryptase in 30 minutes after the provocation, elastase increased in 24 hours (table 1) . tryptase in saliva after 30 minutes increased till 0.07 (0.04; 0.19) (me, pg/ml (lq;uq)), p = 0.0006. increased tryptase is presence of increased cellular inflammation, e.g. mast cells. its ige-dependent hypersensitivity, because there was the correlation between the elevated level of tryptase and positive prick tests. elevated levels of myeloperoxidase and elastase in saliva may be the criteria for the neutrophil hypersensitivity and ige-independent reactions. in healthy volunteers this increase was not observed. the identification of tryptase, myeloperoxidase, elastase can be used for diagnosis of types of ahr. tryptase is a mediator of early (immediate) response to allergen. increased myeloperoxidase and elastase indicates the involvement of the eosinophils and neutrophils in the oral mucous membrane in the allergic process. these mediators have additional roles in the late phase response. elevated levels of myeloperoxidase and elastase in saliva may be the criteria for the neutrophil hypersensitivity. conclusion: safe and acute in vitro methods allow to conduct early etiological diagnosis of allergy, which contributes to the effectiveness of therapy; the detection of polyvalent sensitization dictates the need for molecular diagnostics to single allergens, which has a higher prognostic level and the clinical significance of predicting the appropriateness and effectiveness of allergen-specific therapy; laboratory diagnostics of the allergy allows to reveal sensitization at the 0 (0, 0) 0 (0, 0) 0 (0, 7.28) *p = 0.002; **p = 0.008; ***p = 0.038. subclinical level, which increases early diagnosis and identify persons with a predisposition to allergy; the establishment of causal allerbackground: antibacterial chemicals like parabens and triclosan have been associated with allergic disease in children. parabens are also suspected to affect metabolic functions, possibly due to their weak endocrine disrupting properties. furthermore, a possible link has been suggested for eczema and adiposity, and thus, how body burden of chemical exposures affect both of these outcomes are of interest. we aimed to describe the association between exposure to parabens and eczema and body mass index (bmi) in an adult population in norway. method: urine biomarkers of butyl-, ethyl-, methyl-and propylparabens were quantified by mass-spectrometry in 496 adult participants (median age=29 years) from the rhinessa study in bergen, norway. linear regression models adjusted for gender, age and bmi (for eczema outcomes) and with clustering for siblings, were applied to model possible association between specific gravity standardized urine biomarker concentrations of parabens with bmi and eczema. results: propyl-(ppb) and methyl-parabens (mpb) were detected in 95% of the urine samples; ethyl (epb) in 62% and butyl (bpb) in 38% of the samples. in women, epb and bpb were detectable in 73% and 61%, respectively. participants with current eczema (15%) had lower level of several parabens compared to those without eczema (bpb for both genders; epb in women only and sum of all parabens in men only). body burden of epb (geometric mean (gm)) was 6.7 μg/l in women with current eczema compared to 22.3 μg/l in women without eczema (p = 0.03). body burden of parabens (mpb and epb) were inversely associated with obesity (bmi>30, (11.4%) ), as compared to normal range bmi (bmi=18. 5-25 (56.4%)) in both men and women. the concentration of mpb for obese women was gm=2213 μg/l compared to 11503 μg/l in women with normal range bmi. for men, the gm for mpb was 35.3 μg/l in obese compared to 134.4 μg/l in normal weight men (p = 0.03). conclusion: person with eczema or obesity had lower paraben levels in urine. we speculate that these chemicals might be stored in adipose tissue, and therefore excreted in urine in lower levels among the obese. eczema and obesity was not strongly associated in the current study. method: we retrospectively analysed medical records of 75 patients who were patch-tested with our dental screening series of 23 substances. adverse reactions to dental materials were suspected based on subjective complaints in the oral cavity and/or objective conditions of the oral mucosa. square plastic chambers on hypoallergenic tape were used. patch tests were applied to the upper back and removed by the patient after 48 hours. readings were performed 3 and 7 days after application (d3 and d7). results were evaluated according to the international contact dermatitis research group guidelines. positive patch test reactions fulfilled the criteria of at least a one plus (+) reaction on d3 and/or d7. the term »contact allergy« is usually used for such reactions. we prefer the term »contact sensitization«. clinical relevance of positive reactions to dental materials was not systematically assessed in this analysis. conclusion: we report a high frequency of positive reactions on d7 that were not seen on d3. this finding demonstrates the importance of an additional late patch test reading in patients with suspected contact allergy to dental materials. background: psoriasis is a chronic inflammatory skin disease. its etiopathogenesis is not exactly known. it is believed that the disease occurs in people with genetic tendency with the effect of a triggering factor. in some studies it is observed that contact dermatitis in psoriasis is increased with respect to normal population. for this reason it is proposed that allergen materials could trigger psoriasis. in this study it is aimed to determine contact allergy frequency in psoriasis cases using patch test. results: 62 of the cases were plaque, 5 of them were guttate, 10 of them were palmoplantar and 3 of them were inverse type. more positivity rate is observed in psoriasis cases (26.25%) than control (12%). the positively responsed materials with respect to decreasing number of patients are found as follows: nickel sulphate (16.25%), thimerosal (7.5%), peru balsam(5%), p-phenylenediamine(2.5%), colophony(2.5%), n-isopropyl-n-fenil-4-fenilendiamin(2.5%), mercaptobenzothiazole(2.5%), benzocaine(2.5%), most frequently plaque type and following guttate type positive responses are observed in evaluations with respect to clinical types. no statistical significance is found between patch test results and pasi values in psoriasis cases. conclusion: patients with psoriasis should be carefully evaluated. sometimes some materials may trigger psoriasis. the composition of the pigments that professional tattooists use are varied inorganic salts of metals or organic vegetable pigments. red tattoos, especially those that contain mercury, are the most common cause of late reactions. method: 35 year-old male patient with no previous allergy history known, who gets a tattoo on his right leg and develops within months, cutaneous erythema and pruritus on the same location as the tattoo. true test ® for skin allergy patch epicutaneous testing is performed. results: 48 and 96 hours reading: showed positiveness for mercury ++, with no late positive reactions after that. conclusion: as allergists we should be familiar with the different types of tattoos available, and know the possible cutaneous complications that each of these decorative techniques can present. it is our responsibility to be able to diagnose any complications at an early stage, establish the most appropriate treatment and, if possible, prevent them by informing the possible users. background: within otorhinolaryngological pathology chronic eczematous otitis externa is one of the most common, usually treated with topical medication successfully; however, there are cases in which the poor response to treatment, or the recurrence thereof, may be due to causes secondary to the medication itself, as observed in cases of allergic contact dermatitis caused by these drugs. case report: we present a 68-year-old male patient without relevant pathological antecedents or known allergies, who consulted the otorhinolaryngology service of our center for otorrhea of 15 days of evolution, bilateral external otitis is diagnosed and a topical otological combination is recommended (beclomethasone dipropionate 0.025% and clioquinol 1%, excipient: macrogol) with improvement. however, during the following 3 years the patient presents exacerbations and remissions of the condition, with negative or inconclusive microbiological studies. during all that time he was using the prior topical treatment and other combination treatments of topical antibiotics, corticosteroids and local antiseptics. more aggressive causes of external otitis such as malignant external otitis were ruled out. during the third year of follow-up, a clear relationship of exacerbations was observed with the use of the first combination of topical drugs, so it was decided to investigate allergic sensitization. material and methods: we perform patch tests using true test ® , standard spanish series (geidac -spanish group for investigation of contact allergy dermatitis), topical corticosteroid battery, antiseptic, as well as topical medications used by the patient. results: from the first reading on day two, positivity was observed for: mixture of quinolines ++ patient's otological combination ++ and chlorquinaldol ++; being confirmed in the reading at day four. eczematous external chronic otitis is diagnosed with allergic sensitization to quinolines (clioquinol, chlorquinaldol). we conclude that in the case of chronic external otitis, allergic contact dermatitis should also be investigated as a possible cause, and it is important to perform epicutaneous tests with the patient's own products to evaluate non-common or hidden allergens that may be relevant to their current pathology. most common causes of acd. it is important to distinguish local findings of infection from acd caused by topical antibiotic treatments. here we present a patient with acd with topical use of bacitracin and neomycin combination therapy due to recurrent blepharitis. an 11-year-old male patient presented with the complaints of itching, redness, swelling of the eyelids and facial edema. he had used various topical ophthalmic antibiotherapy and eye shampoo for 7 years due to recurrent blepharitis. five days ago, due to the redness of the eyelids, burning sensation in the eye, itching of the eyes; he was examined by an ophthalmologist. the eyelids and eyelashes were scaly and dry. the patient was treated with warm water soaked cloth dressing, mechanical eyelash cleaning and topical antibiotherapy (neomycin-simple combination therapy). after the second day of treatment, the patient's topical ophthalmic antibiotherapy was discontinued due to an augmentation of the redness in the eye0723 | contact allergy after exposure to ivy (hedera helix l) potent steroid treatment associated with systemic antihistamines, but with no improvement. on dermatological examination a small, well delineated, eczema-like plaque was noticed on a digital finger, as a new finding striking with her old burn scars. she denied any symptoms and was in good health condition. a 4 mm punch biopsy was performed and histological report established the diagnosis of squamous cell carcinoma. the patient was transferred to oncology department for further investigation and treatment. conclusion: early diagnosis and prompt surgical therapy are recommended to all patients with chronic wounds and scars who develop malignant transformation. *written informed consent for the publication of potentially identifiable personal details of patient (gender, age, illness, location) was obtained. **in relation to this presentation, i declare that there are no conflicts of interest. ertugrul a; hizli demirkale z; bostanci i dr sami ulus maternity and children training and research hospital, ankara, turkey introduction: the incidence of contact sensitization among adolescent has been increasing. nickel is one of the important causes of allergic contact dermatitis (acd) in this age group. increased exposure to nickel and deterioration of the skin barrier are among the important risk factors in children. the gold standard for diagnosis is skin patch test. we report here an adolescent patient who has allergic sensitization to nickel and cobalt. case: a 17-year-old female patient admitted in our clinic with a complaint of edema on her face. the patient had applied chickpea water to her face at least once a day for one week because of her acnes. her medical history revealed that she had experienced similar edema on her face after applications of clay mask one year ago. she was diagnosed with cellulitis and she had been treated with antibiotics for five days. on her physical examination, angioedema was observed on her face, especially on the glabellar region. eosinophilia was not found on her laboratory data. c-reactive protein (crp), c4 and c1 esterase inhibitor protein levels were also normal. the skin prick test was performed with aeroallergens, chickpea, lentil, bean and nuts, and no reaction had been observed. the patch test was performed with 'thin-layer-rapid-use-epicutaneous' (t.r.u.e) test and chickpea. the patient had positive reactions to nickel and cobalt. detailed questioning disclosed that the patient was preparing the chickpea water in a metal pot. result: chickpea water and clay mask contain varying amounts of nickel. it was thought that the edema of the patient is due to nickel allergic contact sensitization. an increased exposure to nickel and cobalt raises the frequency of sensitization. nickel allergy can cause different clinics ranging from localized lesions to systemic reactions. we want to emphasize that a detailed medical history and the patch test would enable clinicians to demonstrate hidden allergens and then make a correct diagnosis. case report: autoimmune progesterone dermatitis is a condition of hypersensitivity to progestogens. it is not an easy diagnosis given the variety of clinical presentations it may have, ranging from eczema, urticaria, erythema multiforme, folliculitis, to angioedema or even anaphylaxis. manifestations are cyclical, occurring when the levels of progesterone are higher, this is, at the luteal phase of the menstrual cycle, and disappear during menses, with the physiological decrease of the hormone. it can also be triggered by exposure to exogenous progestins. we report the case of a 49-year-old woman with a cyclical erythematous and violaceous rash related to the menstrual period. the symptoms typically began 4-6 days before the onset of menses and ended 1-2 days before. the diagnosis was based in the clinical history and intradermal skin tests: skin prick testing with levonorgestrel and medroxyprogesterone were negative, but the intradermal skin test with medroxyprogesterone was positive at a concentration of 50 mg/ml. we performed intradermal testing with the same concentration in three other women with no symptoms to exclude an irritative reaction, which were negative. autoimmune progesterone dermatitis is, perhaps, not so rare, but rather poorly recognized and reported, and thus, underdiagnosed. clinicians should be aware and include always this condition in the differential diagnosis, especially in cases of atypical or intractable skin eruptions. case report: a 46 year old male was referred to a community allergy clinic for assessment of chronic urticaria (cu). allergy assessments for foods, inhalant and inducible physical triggers revealed no association. an autoimmune workup followed, with treatment consisting of standard dose antihistamines (h1 and h2). blood work revealed a persistently low hemoglobin with low-normal ferritin. hematology consulted and followed attempted iron replacement to no avail. skin biopsy revealed neutrophilic rich urticaria with the presence of eosinophils. serum protein electrophoresis (spep) revealed a monoclonal gammopathy with elevated igm, felt to be of undetermined significance (mgus). c-reactive protein (crp) was consistently elevated (183, 256) in conjunction with anemia. rheumatology consulted and cleared of any evidence of vasculitis. hematology considered the anemia to be of chronic disease linked to cu. the cu was resistant to treatment including high dose antihistamibackground: isolated head and neck angioedema (ae) can be mediated by bradykinin (bk) or histamin (hi) . the objective of our study was to determine which etiology was most frequent in cases of death by asphyxiating ae in france. we sought all cases of death by isolated asphyxiating ae reported in france between 2000 and 2014 via death certificates and/or the national pharmacovigilance database. results: the overall mortality by asphyxiating ae for all causes was 0.36 / million inhabitants. the death rate of bkae per million inhabitants was 0.14 and lethality of 0.27 per thousand patients per year. the death rate of hiae per million inhabitants was 0.09 and lethality of 0.006 per thousand patients per year. we found a 45 times higher risk of death in case of bkae than hiae. conclusion: consequently, particularly severe episodes must be initially considered as bradykinin mediated and quickly reassess any first-line treatment that is inappropriate. case report: we present the case of a 72-year-old man who suffered recurrent abdominal pain since age of eight, leading to 3 unnecessary emergency surgical interventions and 5 endoscopies before hereditary angioedema due to c1 inhibitor deficiency (c1-inh-hae) was diagnosed at the age of 70. rare subcutaneous swellings were considered allergic reactions preventing proper diagnosis. family history, positive for recurrent abdominal pain and swellings was totally neglected until diagnosis of c1-inh-hae type i was established through appearance of severe oro-facial symptoms in the propositus' grandson. the diagnosis was suggested by the boy's mother, directed by educational materials available in the international hae patients' association website (www.haei.org). this report highlights and emphasizes the importance of accurately evaluated personal and family history to suspect condition that are scarcely known to the majority of physicians. highlights: diagnostic delay in hae and iatrogenic procedures are an underestimated problem, hiding undefined consequences, possibly destructing an entire lifetime. correct, publically available information provided by patients' associations raise awareness about the disease and could put the milestone of establishing correct diagnosis. de luque v 1 ; lara p 1 ; guardia p 1 ; jimenez ar 2 1 virgen macarena hospital, seville, spain; 2 hospital virgen macarena sevilla, seville, spain background: in the protocol for the study of patients who consult for recurrent acute angioedema with facial involvement, the contactant battery is included (epicutaneous test). we review the results in our patients with facial angioedema to evaluate the contactants to which these patients present sensitization, some of them coexisting with contact dermatitis clinic. we reviewed the patients referred to the clinic for recurrent acute angioedema with facial involvement and to whom a standard battery epicutaneous test was requested. in all these patients, other habitual triggers included in the diagnostic protocol (food, medications, autoimmune diseases, bradyinergic aea/complement deficit …) were ruled out. conclusion: it seems to be profitable to continue including in the diagnostic battery of patients who consult for aea with facial affectation, study of epicutaneous with standard battery. it is a small sample, but the data correlate with what has been published, being more frequent the sensitization to contactants in women and the contactant more frequently involved nickel sulphate. 0734 | ace inhibitor-related angioedema-the value of history taking background: angioedema is a well-recognized side effect of angiotensin-converting enzyme (ace) inhibitor therapy. although it occurs in <1% of the patients who take these drugs, it seems to be responsible for 40% of the episodes of angioedema. this entity is underdiagnosed and failure to recognize it leads to recurrence of episodes, with an impact on morbidity and increased risk of serious reactions. our objective is to analyze the clinical, therapeutic and orientation approach of patients diagnosed with ace inhibitor-related angioedema, evaluated at the outpatient consultation (oa) of immunoallergology (ia). a 3-year retrospective study was performed by analyzing the clinical files of all patients diagnosed with angioedema observed in oa of ia. the following variables were analyzed: gender; age; clinical data; evaluation in emergency department (ed); therapy in the episode; evolution and orientation. the chi-square test was used to study the association between categorical variables: "established therapy"/"disease evolution" and "place of reference"/"withdrawal of ace inhibitor". results: review of 62 cases of patients referred for angioedema. only in 29% the final diagnosis was "ace inhibitor-related angioedema". the mean age of the patients was 63.7 years and 67% were male. the location of angioedema occurred in the tongue in 33% and in the remaining sites (lip, hemiface, tongue and hemiface, tongue and lip) appeared in the same frequency, 17%. none of the patients had airway obstruction. during the episode of angioedema, 22% of patients were not referred to ed and the therapeutic approach was done with antihistamines in 75%. in patients who were referred to ed (78%), antihistamines and corticosteroids medications were administered in 85%. regarding the evolution, it was verified that the duration of the episode was independent of the established therapy (p > 0.05). regarding the place of reference, 60% of the patients were referred form hospital (ec or ed) and, in these, the ace inhibitor was suspended in 73%. in patients referred from general practitioners (40%), in none of them the ace inhibitor had been withdrawal. a causal association between the use of ace inhibitors and the episode of angioedema becomes crucial, since drug withdrawal is indicated. a reference for ai oa should be weighed. therapy with antihistamines and corticosteroids has no proven efficacy. hereditary angioedema (hae) seen by physicians belonging to the hae scientific committee of the aaaeic background: patients with c1-inh-hae frequently suffer from anxiety and stress. the impact of prophylactic treatment on anxiety and stress in c1-inh-hae patients is largely unknown. here, we analyzed data from the apex-1 study, a phase ii study that investigated the effects of the oral kallikrein inhibitor bcx7353. method: c1-inh-hae patients with a history of at least 2 hae attacks per month were randomized to receive four different doses (350, 250, 125, 62.5 mg) of bcx7353 or placebo for 28 days. the depression anxiety stress scale (dass) was administered at baseline and at day 29. the dass consists of three self-reported scales designed to measure the negative emotional states of anxiety and stress. subjects used a 4-point severity/frequency scale to rate the extent to which they have experienced each state. results: baseline dass total scores as well as anxiety and stress domain mean (sd) scores for the 125 mg treatment arm (n = 13) were 20.9 (23.7), 5.0 (5.9), and 8.9 (9.1) points respectively. placebo scores were generally similar or slightly lower at baseline than for the 125 mg treatment arm. the dass questionnaire data showed statistically significant improvements in total score vs. placebo at day 29 (−11.4 method: a two-phase mixed methods approach was used to develop the hae-rt tool including: phase 1: delphi study [hae specialists (n = 9) and national patient advocacy group members (n = 3)] was conducted to reach consensus (80% agreement) on predictor variables to include in the tool. phase 2: retrospective chart review was conducted to assess the predictive findings of the decided variables. a convenient patient sample presenting with angioedema (with and without hae) between january 2012-january 2017 were included in the study. results: nine of 12 invited experts (75%) participated in the delphi study. of 8 hae-specific predictive variables, 4 reached consensuses including: (i) recurrent angioedema; (ii) absence of urticaria; (iii) recurrent abdominal pain/swelling; (iv) lack of response to allergic therapy. the retrospective study included 85 patients (n = 46 with hae; n = 39 non-hae; overall 72% female). hae patients were significantly more likely to have a family history of hae (72% vs 0%; p < 0.0001); previous recurrent angioedema (96%; p < 0.009); present with no hives (91%; p < 0.036); previous recurrent abdominal pain (80%; p < 0.0001); and 2% responded to allergy treatments (p < 0.0001). a regression analysis categorized observed frequencies (actual patient outcomes from chart review) versus predicted (by model); plotted on a 2 by 2 table and calculated the sensitivity and specificity of the hae-rt which resulted in one hundred percent for both. conclusion: our study demonstrated that expert involvement led to the identification and prioritization of variables that when included an hae-rt tool, were associated with a high level of sensitivity and specificity when applied to known patients. the next step is to observe the effect of the hae-rt tool on patient care in the ed. method: evaluation of cardiovascular manifestation included morphology, serum level of troponin t, electrocardiography (ecg) and echocardiography. evaluation of pulmonary manifestation included spirometry, diffusing capacity of the lung for carbon monoxide (dlco) and evaluation for mastocytosis included bone marrow biopsy and serum total tryptase measurements. results: in the study there were 55 patients -35 women and 20 men between 21 and 71 years old (the average age was 45). there were 7 (12.73%) patients with mpcm, 4 (7.28%) with bmm, 42 (76.36%) with ism and 2 (3.64%) with ssm. the average level of serum tryptase was 45.1 μg/l (6.9-270) . troponin levels was within the normal range in all patients. one patient had lowered the ejection fraction (eflv=23%). no one patient had restriction. the average value of a forced lung capacity was 3.33 l (104%) and a total here, we describe a case series of twelve mis patients seen at our department over a 3-year period and report how many of these patients have sm. common phenotypical manifestations of acute hae1 episodes in this region, to review therapeutic challenges in a rural setting in comparison with world standards, and lastly to evaluate the socio-economic burden inflicted by the disease. method: a sample of 13 individuals from a total of 28. the exclusion criteria was the inability to attend booked appointments more than 3 times in 1 year (2017). the following methods were used: an interview to formulate a family tree identifying affected individuals in 4 contiguous generations, and review of the acute presentations in the past year (2017) . a questionnaire to obtain the relevant hae1 associated socio-economic burdens. a chart review to identify the therapeutic strategies in this region. c1inh levels, and complement c4 to confirm the diagnosis. results: polygamy as a local culture was found to be an important factor that perpetuated the genetic burden of the disease. c1inh and c4 levels confirmed hae1 in all participants individually. clinical features during acute attacks included swelling of extremities (100%), facial swelling (69%), neck swelling (23%), and laryngeal swelling (8%). therapeutic strategies for acute attacks included fresh frozen plasma or fresh dried plasma. danazol was used for prophylaxis. hae1 has had a significant negative impact upon the socioeconomic status of the affected individuals. conclusion: hae1 is a newly identified disorder in the broad spectrum of allergy medicine in kwazulu-natal. the diagnosis is simple to confirm but requires an initial high index of suspicion, and therapeutic management still poses a challenge in this region due to lack of resources. genetic counselling is of paramount importance during intervention since polygamy forms part of most cultures in this region. a support strategy is highly recommended in order to help alleviate the socio-economic burden posed by the disease in this region. the socio-economic burden secondary to hae1 (10 participants/5 identical questions each) question 1 (0-5 points) 3 question 2 (0-3 points) 2 question 3 (0-3 points) 1 question 4 (0-3 points) 1 question 5 (0-1 point) 1 method: a total of 172 medical faculty senior year students were included to study on a voluntary basis. students are divided into two groups. one group was given visual user guide that has not been modified, and a visual user guide on which we have modified to the other group ( figure 1 ). then they were asked to show how to use the inhaler spacer. results: the mean age of the volunteers was 25.3 ± 126 years and 104 (60.5%) were male. there were 82 students in the group without modification of the visual user guide and 90 students in the other group with modified the visual user guide. sixty-four per cent of the modified user guide group showed correct use of the inhaler spacer, while 15% of the unmodified group showed correct use (p = 0.001). the group that given modified visual user guide was more successful in all of the display steps of the inhaler spacer. conclusion: modification of the currently available visual user guide of inhaler spacer in our country will increase the correct usage rate. results: mean fev1, fvc, fev1/fvc z-score were 1.8 (1.1-3.9), 1.7 (0.9-3.7), 0.9 (0.8-0.9), respectively. restriction had 31 (57.4%) and obturation 3(5.3%) patients. fev1 (p < 0.001, r 2 = 0.37) and fvc (p = 0.001, r 2 = 0.38) decreased with age (%pv and z-score). background: children who were treated for leukemia are known to have developed long term impairment of lung function. the reasons that complication are only partially known. the aim of this study was to asses pulmonary function in children treated the lower dlco is the most frequent abnormality in childhood leukemic survivors. hsct and pulmonary infection (in particular cmv pneumonia) is a strong risk factor for impairment of dlco in children. clinical manifestation of dlco impairment is poor exercise tolerance. a screening for respiratory abnormalities in survivors following treatment for childhood haematologic malignancies, seems to be of significant importance. 0746 | phenotyping allergic respiratory diseases: an unsupervised classification using latent class analysis allergen groups was significantly associated to uawi (aor[95% ci]:2.1 [1.3-3.6] ), compared to uasi. results: 80.8% of br and 49% of py were women, median age was 32 years, 35% br and 52% py reported having more than four years of training. although they recognized the main symptoms of ar, 26% br and 100% py never asked whether the patient had a medical diagnosis of ar; 20.5% br and 100.0% py did not ask whether the symptoms occurred when close to animals or allergens; 55% br and 76% of py did not ask if the patient had a medical diagnosis of asthma; 59% br and 70% py did not ask if rhinitis worsens asthma symptoms and 51.3% br and 84.3% py did not ask whether symptoms of rhinitis interfere with their daily activities. results: there was a predominance of female (br: 73%, py 50.4%, uy: 70%) median age 31 years old, 124/235 worked in the community and 75/127 in the emergency departments, 34% of the br had more than 10 years of education, 67% from py had between 1 and 5 years, and 82% from uy had been graduated for less than 1 year. br/uy recognize the main symptoms of ar, however 67% of those from uy do not ask: if the patient has physician diagnosis of ar, 72% present shortness of breath, and 93% a medical diagnosis of asthma, 94% if rhinitis worsens asthma symptoms and 90% if symptoms of rhinitis interfere with the patient's daily activities. the prescribed treatment varied a lot, the intranasal corticosteroid use rate was: bd: 78%, pd: 92% and ud: 54%. 100% of doctors in py, 73.4% in br and 78% of uy never refer the patient to the specialist. 22.9% of pcd of br, 62% of py and 6.0% of uy are aware of aria guideline. conclusion: although ar is largely attended by pcp, recognition of symptoms and their impact on asthma, as well as the knowledge about aria guide is low and treatment is not always prescribed according to best practice. allergy education programs, with an emphasis on ar and aria guide, need to be directed to pcp in la for the better assistance of ar patients. 0749 | assessing knowledge of allergic rhinitis among final year medical and pharmacy students in croatia-curriculum change necessity? the two factor structured questionnaire was formed by the authors regarding the topics mentioned. t-test was used for statistical analysis. the global results were formed as composites of (1) ar general characteristics, (2) ar treatment approach, and (3) the participants' overall knowledge. of the 201 respondents, 143 (71.1%) were female and 58 (28.9%) were male (p < .0001). medical students had a median score of 6 of 10 correct answers on (1), 4 of 10 on (2), and 10 of 20 on (3), whereas pharmacy students had median score of 7 of 10 correct answers on (1), 4 of 10 on (2), and 10 of 20 on (3). there were no significant differences in knowledge between two student groups. the results indicate an inadequate level of knowledge of ar in both groups, especially regarding the therapy approach. since general practitioners and community pharmacists have a major role in providing treatment to patients suffering from ar, it is important to develop advanced knowledge on this topic during medical and pharmacy degree courses. despite a relatively small study population, it would be advisable to introduce change by improving the core curriculum regarding ar with more emphasis on treatment, but additional research on this topic is necessary. tan r 1 ; cvetkovski b 1 ; kritikos v 1 ; price d 2 ; yan k 3 ; smith p 4 ; bosnic-anticevich s 5 1 woolcock institute of medical research; university of sydney, sydney, australia; 2 observational pragmatic research institute pte ltd, singapore, singapore; 3 royal prince alfred hospital, sydney, australia; 4 clinical medicine, southport, australia; 5 griffith university, sydney, australia background: people with allergic rhinitis symptoms frequently selfselect over-the-counter medications from community pharmacies without seeking advice from a health care professional. this increases the incidence of complications due to delayed diagnosis and suboptimal treatment. this study aims to (i) compare the demographics, clinical characteristics and medication selected, between pharmacy customers who choose to self-select and those who interacted with a pharmacist when purchasing medication for allergic rhinitis symptoms, and (ii) identify the key factors associated with allergic rhinitis patients' medication self-selection behaviour. a cross-sectional observational study was conducted in a convenience sample of community pharmacies from the sydney metropolitan area. data were collected using a researcher administered questionnaire that included: demographics, pattern of allergic rhinitis symptoms, their impact on quality of life, factors triggering allergic rhinitis symptoms and medication(s) selected. logistic regression was used to identify key factors associated with participants' medication self-selection behaviour. results: of the 296 recruited participants, 202 were identified with allergic rhinitis, of which 67.8% were female, 54.5% were aged more than 40 years old, 64.9% had a diagnosis of allergic rhinitis, and 69.3% self-selected medication(s). significant differences were noted in allergic rhinitis symptoms, impact of allergic rhinitis on quality of life and medication(s) selected between participants who chose to self-select and those who interacted with a pharmacist. participants who experienced moderate-severe wheeze were 4 times more likely to self-select allergic rhinitis medication(s), and those who had allergic rhinitis symptoms impacting on their quality of life were 0.4 times less likely to self-select allergic rhinitis medication(s). conclusion: there is a high incidence of self-selection of over-thecounter treatments for allergic rhinitis symptoms in community pharmacy, with the majority of allergic rhinitis sufferers failing to seek pharmacist advice. this research identified predictors of medication self-selection behaviour in community pharmacy among people with allergic rhinitis, which can inform the design of tools/strategies and targeted interventions, aimed at improving pharmacist engagement and future practice in optimising allergic rhinitis management. the weir family health clinic, cork, ireland; 2 university college, cork, ireland background: allergic rhinitis is a common condition that is predominantly managed in primary care. the incidence of allergic rhinitis is increasing. it is frequently under diagnosed, misdiagnosed and mistreated. it has a significant impact on patients' health related quality of life and represents a huge cost both to healthcare systems and society. the aim of this study was to implement appropriate guidelines regarding the management of allergic rhinitis in primary care and evaluate the effect on patients' health related quality of life. method: patients with a history of allergic rhinitis were selected from three general practice bases in west cork, ireland and quality of life of patients was assessed initially in year one and followed up one year later in a general practice setting using the standardised rhinoconjunctivitis quality of life questionnaire (rqlq). allergic rhinitis and its impact on asthma (aria) guidelines and appropriate prescribing were implemented during this year and patient education and structured follow up was arranged in the intervention group. this was compared with the control group who received usual care. results: 93 valid responses were received, 34 from the control group and 59 from the intervention group. the study demonstrated a statistically significant difference in quality of life in the intervention group. in the adult intervention group the quality of life score decreased between 2015 and 2016 representing an improvement in their quality of life, (t = 4.13; df=56; p < 0.01). the difference in the score between the control and intervention groups in 2016 was also statistically significant.(t = 6.11; df=54; p < 0.01). the numbers in the adolescent groups and paediatric group also demonstrated an improvement in quality of life but the sample size was too small to demonstrate a statistically significant difference. conclusion: as the majority of patients rely on their general practitioners for treatment and diagnosis of allergic rhinitis, primary care represents an important area to target in the management of allergic rhinitis to improve patients' quality of life. the implementation of guidelines has been shown to improve patients' quality of life. this study demonstrates this care can be delivered in a primary care setting with an improvement in patients quality of life but substantial investment in education and resources available to primary care physicians is needed. 0752 | the predictive value of allergy tests in the diagnosis of peanut allergy in adults rey-garcia h; gunawardana n; wheeler k; scadding g; durham s; skypala i royal brompton hospital, london, united kingdom background: adults presenting with either new-onset symptoms attributed to peanuts or with early-onset peanut allergy, often wish to know whether they should continue to avoid peanuts. clinical history and standard tests may be sufficient to provide an answer, but for many the tests are inconclusive and an oral food challenge is required. this review was undertaken to determine the most accurate tests. conclusion: these data suggests that peanut spt and ara h 2 provide the most accurate prediction of the outcome of oral food challenge in adults. should components not be available, then spt would be the test of choice being more accurate in all aspects than sige. combining spt and sige improves the sensitivity and negative predictive value of spt alone. however, the best combination is spt and ara h 2, which increases the overall accuracy to 87%. further studies are needed before it can be determined whether peanut diagnostic tests can replace the oral food challenge in adult patients. method: retrospective chart review was carried out in a community allergy clinic. patients with rap, bloating and altered stools who underwent bt were characterized by age, gender and atopic status. a separate study to assess patients' outcome post-dietary counselling was carried out to determine impact on symptom management. results: thirty-four patients were assessed for fi from january 2014 to december 2017. female gender predominated (31/34, 91%) with an average age of 31 years at presentation. results of fi were positive in 15/34 (44%), borderline in 2/34 (6%) and negative in 17/ 34 (50%). the average age of patients with a positive, borderline and negative tests were 30, 20 and 34, respectively. of the patients who tested positive for fi, 5 (33.3%) had comorbid inhalant allergies alone, 1 (6.7%) had comorbid (unrelated) food allergies alone, 2 (13.3%) had inhalant and food (unrelated) allergies, and 7 (46.7%) were non-atopic. of the patients who tested negative for fi, 7 (41.1%) had comorbid inhalant allergies alone, 0 (0%) had comorbid (unrelated) food allergies alone, 1 (5.9%) had inhalant and food (unrelated) allergies, and 9 (53.0%) were non-atopic. conclusion: patients investigated for carbohydrate intolerance with rap, bloating and altered stools were predominantly female (91%). fi was confirmed in half. atopic status did not help differentiate between the fi positive or negative groups. results of a fod-map elimination diet are separately reported. conclusion: post-bt, 88% of patients reported symptom improvement. patients who implemented fructose or fodmap avoidance reported symptom improvement. one patient who tested negative for fi reported symptom improvement with a low fodmap diet. patients suspected as being fructose intolerant may benefit from a fructose restriction or fodmap diet, while awaiting bt confirmation. this form of dietary intervention may assist and shorten the natural history of non-specific chronic gi symptoms. inappropriate referrals to a uk paediatric tertiary allergy clinic demonstrate lack of allergy education and knowledge in primary care marriage de bristol royal hospital for children, bristol, united kingdom background: up to 8% of children have a food allergy. allergy has become an explanation for all manner of nebulous symptoms and self-diagnosis is common. sham allergy tests are easily available giving incorrect results and resulting in unnecessary, potentially harmful abstracts | 423 parentally-imposed dietary exclusions. there are 100 million allergyrelated google searches per year. the rising prevalence of perceived allergic disease has led to an increase in health service utilisation, including increased referrals to secondary care. clinic waiting lists are long and children with severe food allergies have to wait longer than necessary to be seen method: uk paediatric tertiary allergy clinic referrals were prospectively reviewed over three months. five inappropriate referrals deemed most reflective of poor knowledge in primary care were selected as case summaries to highlight this gap in knowledge. results: 1: schoolchild referred for investigation of allergic cause for a red, watery eye after splashing juice in her eye whilst cutting a kiwi, despite having a co-existent dendritic ulcer. 2: schoolchild referred for peanut allergy testing after inhaling a peanut and developing wheeze, with all respiratory symptoms resolving following peanut removal. 3: young child referred for peanut allergy testing after developing a rash on leg following skin contact with faeces 24 hours after ingestion of peanut butter. 4: teenage boy referred for investigation of likely peanut allergy despite eating peanut butter and tree nuts almost every day. the family were concerned he was allergic to peanut butter. 5: toddler referred for milk allergy investigation after developing urticaria lasting 24 hours 15 minutes after drinking a bottle of milk. the child had consumed cow's milk formula since birth, and continued to consume milk daily for a further five months following the episode of urticaria. conclusion: provision of allergy services in the uk is poor and lack of investment in allergy services has led to suboptimal recognition and management of food allergy in primary care. allergy education provision for primary care practitioners is inadequate and fails to empower healthcare professionals to discern between allergy requiring full investigation and management, parentally-diagnosed allergy or symptoms which clearly have no association with allergy. progress to improve primary care training for allergy needs to be optimised to prevent further unnecessary referrals and lengthening clinic waiting lists. background: in case of allergic reactions to food or insect venom, quick and adequate treatment, based on clear instruction for use of emergency medication and calling for help, is necessary. however, daily practice shows that patients do not use the prescribed emergency medication because they are afraid to use the epinephrine auto-injector or they do not know how to use it. information and instruction offered by a reliable app could be a useful aid. we aim to develop an app for adult patients and children older than 12 years with allergy to food or insect venom, which offers a step-wise approach to support patients, their relatives or acquaintances in case of an allergic reaction. method: first, the content of the app, including a step-wise approach to treat the allergic reaction has been determined, based on literature, a survey about needs of patients and on consultations of healthcare professionals. subsequently, a web-based prototype has been developed with an adult profile and children profile. the content and flow of this prototype was tested by the project group, as well as by selected healthcare professionals and patients and improved according to the test results. next, the revised prototype was submitted to representatives of patient and professional organizations for final approval. currently the procedure for ce approval is ongoing. finally, the app will be built and offered to the market for ios and android. results: a web-based prototype of the allergy app is available with two profiles: adult and older children. the app is useful for patients with a doctor's diagnosed allergy to food or insect venom, who received emergency medication and instructions to use prescribed medication in case of an allergic reaction. based on severity of complaints, the user is informed about the steps to treat the allergic reaction. in case of a moderate to severe reaction, the patient is advised to use an epinephrine auto-injector, to call the emergency number and, if prescribed, to use medication such as antihistamines, prednisone or inhaler. besides that, the app provides links to websites of expertise centres and patient organisations and includes instructions how to use the epinephrine auto-injector. conclusion: the allergy app will help patients, their relatives or acquaintances to adequately treat an allergic reaction to food or insect venom. involving patients and professionals in the development of the app will contribute to its acceptability and usability. 0757 | electronic documentation of drug allergies in a tertiary hospital in singapore: are we relying too much on it? choo kjl; garuna murthee k; naing cs singapore general hospital, singapore, singapore background: singapore has 26 hospitals shared between public and private healthcare system. its healthcare system, ranked #6 in the world by who in 2010 serves a multi-racial population of 5.2 million of which 9% are above 65 years old. drug allergy alert cards (medik awas) were started in 1970s by singapore medical association to improve patient safety. work on computerisation of drug allergy and medical alerts started in the 80s, a precursor to today's critical medical information system (cmis). cmis serves as a platform across all public hospitals in singapore. it promotes uniform reporting of drug allergy and notification of adverse drug events to the health science authority. yet, we found that there is a lack of awareness of one's own drug allergies. method: all patients admitted to ward 73 (general medicine ward) at singapore general hospital from 17 july to 31 oct 2017 were screen for any previously documented drug allergies. consenting patients who had previously documented drug allergies on cmis were interviewed to document their demographics, education level, current medications, knowledge of their own drug allergies and possession of a drug medication alert card. the answers were then compared with their electronic documentation of drug allergies for accuracy. we interviewed 192 patients aged 20-94 with documented drug allergies during the recruitment period. 74% had secondary school education or higher. the majority (76%) spoke english and (58.5%) mandarin. almost half had medical problems and are on long term medications (mean 5.17 medications); hypertension and diabetes being the top two common diseases. 48% of the patients could accurately relay their drug allergies; antibiotics and analgesia being the most labelled. only 24% had a drug allergy alert card while the rest both rely on the hospital's electronic documentation and/or their caregivers to record and relay their allergies to future prescribers. about 21% received prescription from multiple healthcare sites in both the public and private healthcare system. we found patients' knowledge of their own drug allergies dismal. the cmis electronic documentation provided a false sense of security. unfortunately, the cmis platform is not available to all private hospitals, increasing the risk of mis-prescription due to the lack of information. unless this is made available nationally, patients with drug allergies should be given some written documentation, either a letter or medik awas. how frequent are they and how are they treated? method: for this cross-sectional study, participants were recruited in the waiting rooms of local doctors in the rural bavarian forest region of southern germany (q1/2017). a paper questionnaire was handed out to the participants, asking for allergies (pollen, animal hair, bee and wasp venom, drugs, food, house dust mites, contact allergies and other allergies) and how or rather by whom (e.g. general practitioner, specialist, self-treatment) these allergies are treated. results: 718 participants with a mean age of 50.61 years (sd=15.15) and 59% women were included in this study. 38.2% indicated to have at least one allergy, including pollen allergy most frequently (17.4%). women had significantly more often at least one allergy than men (rr= 1.552; ci [1.243;1.937] ) and for almost all examined allergies a significant higher risk of disease. younger age groups indicated more often to have at least one allergy (18[.506;1.151 ]) seemed to be affected less. participants indicated most frequently that their allergy was treated by a general practitioner (37.5%), except of the 18-29-yearold young adults who indicated "no treatment" most frequently (28.2%). conclusion: there is a high self-reported prevalence of allergies in the examined rural bavarian region, that increases with decreasing age and is significantly higher among women. moreover, the data on the absence of an appropriated treatment for allergies is alarming. therefore, medical care needs to be improved in rural regions to lower the burden of allergies. 0759 | the prevalence of the burnout syndrome among medical professionals involved in allergology education programmes astafieva n 1 ; kobzev d 2 ; gamova i 1 ; perfilova i 1 ; udovichenko e 1 ; skuchaeva l 1 ; michailova i 1 1 ≥10) and rpa (low ≥0-11, subscales were calculated and analyzed. results: on average students demonstrated: moderate/high ee scores (24-26); moderate/high dp scores (9-12) and moderate rpa scores (17); higher rpa scores were common (41.4%) among junior students, which is also linked with their levels of engagement, and lower (28%) among senior students. junior specialist (starting specialization) had very low scores in all 3 subscales and expressed a very high motivation in their course and new profession. clinical allergologists with significant experience demonstrated moderate / high ee scores (22-26); low dp (1) (2) (3) (4) (5) and rpa (below 10) scores. high ee scores associated with pressures of service were compensated by a substantial loyalty to their profession and positive assessment of the outcomes of their work. clinical academics demonstrated the highest level of ee scores (29 + among 70 + % of the group) with low to moderate dp and rpa scores, the latter being associated with a loyalty to their profession. it was also possible to identify a correlation between engagement in research activities and lower rpa scores. conclusion: burnout is a complex and multifaceted phenomena, which requires further investigation. however, this research identified that students and junior specialists involved in allergology and clinical immunology programmes with higher levels of engagement and motivation to acquire new specialist knowledge had lower levels of ee, while loyalty to the profession and positive assessment of the outcomes of clinical and research work allows to compensate high levels of ee among experienced practitioners and clinical academics and to reduce burnout effects overall. 0760 | appeal (allergy to peanuts impacting emotions and life): the first pan-european study to evaluate the psychosocial burden of living with peanut allergy deutscher allergie-und asthmabund (daab)/german allergy and asthma association, mönchengladbach, germany; 8 food allergy italia, padua, italy; 9 aepnaa asociación española de personas con alergia a alimentos y látex, madrid, spain; 10 afpral association pour la prevention des allergies, paris, france; 11 asthma-allergy denmark, roskilde, denmark; 12 anaphylaxis campaign ireland, cork, ireland; 13 brainsell ltd, london, united kingdom; 14 aimmune therapeutics, london, united kingdom background: peanut allergy, one of the most common and rapidly growing food allergies, is most frequently a lifelong condition. current management is limited to avoidance and symptomatic treatment of allergic reactions when accidental exposures occur. peanut allergy can affect the quality of life (qol) of individuals and also that of parents/caregivers and family members. appeal was designed to assess the impact of peanut allergy on qol in peanut-allergic individuals and their parents/caregivers and families. method: the first, quantitative part of appeal is described here and consisted of a pan-european, cross-sectional online survey of approximately 30 minutes in length. the study was conducted in the uk, republic of ireland, france, spain, germany, italy, the netherlands and denmark. over 1800 participants were recruited via patient advocacy groups or directly through a specialist survey recruitment panel. ethics committee approval was obtained and all participants provided their informed consent. eligible participants were: (1) parents or caregivers of a child/adult with peanut allergy; (2) parents or caregivers responding as a proxy for a child aged under 18; (3) adults. all allergic subjects had self-reported diagnosed peanut allergy. after several screening questions, eligible participants answered a set of clinical questions about their (or their child's) allergies and other conditions, details on the peanut allergy diagnosis, contact with healthcare professionals, worst allergic reaction to date and use of emergency medicine. depending on whether they were an allergic adult, a parent responding on behalf of the child, or a parent/caregiver recounting their own experience, they then answered specific questions on restrictions on life choices, coping strategies and the impact of peanut allergy on feelings and emotions of families, friends and other people. sociodemographic questions completed the questionnaire. the results of the survey are being summarized using descriptive statistics and the data are being analysed on a pan-european level, by country, and according to the participant's perspective (parent, caregiver or individual). conclusion: this comprehensive, pan-european online survey has been specifically designed to uncover the psychosocial burden and effect on qol of peanut allergy in terms of individuals' lives and those of their families. results: 246 pediatric patients were included in the study. 65.9% (n = 162) were male. the median age of onset of symptoms (interquartile range) was 4 months (2-6).the median age (interquartile range) of diagnosis was 6 months (4-7). initial reactions associated with cmpa were observed in 95.8% of patients before 12 months old. patients' diagnoses were atopic dermatitis (39%), urticariaangioedema (24%), anaphylaxis (12.2%), proctocolitis (10.2%), atopic dermatitis and urticaria (8.9%), food protein induced enterocolitis (4.9%) and eosinophilic esophagitis (0.4% method: the study involved 147 patients, who are two years old (and above), diagnosed with cow milk allergy and are observed for at least six months in our hospital. socio-demographic features of the patients, their symptoms, symptom-start ages, age of diagnosis, clinical findings at diagnosis and during observation were recorded in data collection questionnaires. results: the samples were 91 (61.9%) boys. the average symptomstart age was observed to be 5 months , average diagnosis age 6 months (1-54) and average age of final check 36 months . when symptoms at entry were observed, 85.7% of the patients had dermal system, 25.2% gastrointestinal system, respiratory system disorders, and 15% were detected to have developed anaphylaxis. among the patients diagnosed with cow milk allergy, 31.3% showed food reaction to nutrients with lge agents, 51.7% to mixed types, and 17% to nutrients with non-lge agents. it was also observed that, in the end of 30.4 ± 19.8-month observations, sensitivity to cow milk was observed to continue in 34 (23.1%) of our patients. when tolerance improvement rates among the patients were compared, anaphylaxis (p < 0.001) during entry were observed to be influential in continued allergic state. 5 (3.4%) patients were able to consume yoghurt, 10 (6.8%) patients could consume dairy products and 19 (12.9%) patients could not consume dairy products. conclusion: in the end of our investigation, it was observed that 57 (50.5%) of the 113 patients developed cow milk tolerance before the age of 2. when the factors enabling the continuation of sensitivity in cow milk allergy were investigated, anaphylaxis during entry, entry specific lge and pasteurized milk antigen as well as high skinprick test results were detected to be significant. 0765 | initial lower threshold was a risk factor of severe adverse reaction during oral immunotherapy for cow's milk anaphylaxis results: before oit, median age was 5.7 years old, median threshold to induce initial reaction was 2.1 ml, to induce anaphylaxis was 14.6 ml of cm and median milk specific ige was 56.4 ku/l. twentyseven subjects (24%) dropped out from the protocol, 69 subjects 0766 | investigation of heat and matrix effect on milk proteins' allergenicity and the development of hypoallergenic food products reduce some milk proteins' allergenicity (ß-lactoglobulin) . in this project we aimed to investigate the effect of heat and matrix on different milk protein fractions through maillard reaction and eventually develop hypoallergenic food products that have milk protein with low reaction risk. method: milk cake matrix is prepared in different flour/sugar (f/s) ratio (2f/1s, 1f/1s, 0.5f/1s) and baked 30 minutes at 180°c. proteins that cake contains are separated using sds page and stained with coomassie blue to check total protein. in parallel specific proteins are detected by western blotting using pooled sera from patients with milk specific ige>60ku/l for incubation results: in normal milk cake recipe (2f/1s) ß-lactoglobulin bands are disappeared but casein bands did not differ in size. in order to investigate the matrix effect f/s ratio is changed and it is found that when this ratio decreases, with the affect of heat and maillard reaction, milk casein bands' intensities also decrease in sds gel coomassie staining. in western blot experiments it is also shown that milk specific ige bound weakly to casein bands in low f/s ratio cake (0.5f/1s) whereas in cakes that have high f/s (2f/1s) ratio it bound significantly higher. and ß-lactoglobulin proteins' structure and lower the milk specific ige bindings to milk proteins in low f/s ratio cake through maillard reaction. 0767 | extensively hydrolyzed formulas for the management of cow's milk protein allergy in infants: is extensive hydrolysis sufficient to guarantee success? method: to better understand the range of ehfs, we aimed to analyse samples of commercially available ehfs from 11 countries and various manufacturers, with a focus on suitability for cmpa management. samples were de-identified and coded for the analyses. molecular weight (mw) distribution of hydrolysates and residual proteins and peptide profiling were assessed with sds-page gel and size exclusion-high-performance liquid chromatography (se-hplc), as they reflect both the design of the formula and the quality management applied during production. osmolarity, nitrogen fractions, lactose content, total and free amino acids, β-lactoglobulin, and casein content were quantified and β-lactoglobulin residual allergenicity was assessed. results: peptide mw distribution displayed significant variation, with the percentage of peptides with mw >1.2 kda varying from 1% to 36%. mw distribution was shown to be positively correlated with β-lactoglobulin specific in vitro degranulation. twenty % of samples had non-measurable β-lactoglobulin content (smaller than or at the limit of quantification (loq): 0.010 mg/kg); however, 80% of samples had β-lactoglobulin content greater than the loq, with high variability from 0.020 to 36 mg/kg. surprisingly, even in samples featuring a high degree of hydrolysis, significant levels of residual β-lactoglobulin were quantified. conclusion: lack of consensus over the definition of 'extensively hydrolysed' is reflected in the wide range of degree of hydrolysis in commercially available ehfs, and can result in products that are mislabelled as 'extensively hydrolysed' and may be high-risk or even unsuitable for the management of cmpa. results of these analyses also highlight that degree of hydrolysis alone is not sensitive enough to characterise ehfs, and that whilst a high degree of hydrolysis is desirable, further quality control measures are essential to ensure clinically safe and suitable products. actionable guidelines to better define hypoallergenic formulas based on extensively hydrolysed milk proteins are warranted. background: assessing the effect of baked milk products on accelerating unheated milk tolerance in patients with cow's milk allergy. method: a randomized clinical trial was done on 84 patients (6 months-3 years old) divided randomly to case and control groups matched for age and sex. baked milk in form of muffin for 6 months followed by baked cheese in form of pizza for next 6 months was given to the patients in case group. skin prick test and serum ige (sige) levels (immunocap) of milk, casein and betalactoglobulin were measured before and after the study. the ones having milk sige less than 3 ku/l and being asymptomatic during the study underwent oral food challenge test for evaluating unheated milk tolerance. chualalongkorn university, bangkok, thailand; 8 kk women's and children's hospital, singapore, singapore; 9 university of the philippines, philippine general hospital, manila, philippines background: problems in recognising cow's milk allergy (cma) and lactose intolerance (li) in infancy may lead to a delayed or incorrect diagnosis, as well as inappropriate dietary interventions. method: between january and november 2017, a survey was conducted online in china, india, singapore, thailand, mexico, kuwait, united kingdom, australia, and paper-based in the philippines. the survey consisted of 12 multiple-choice questions on cma and li in infants aged under 12 months, two case scenarios (non-ige cma and anaphylaxis) and 10 questions on educational needs (likert scale [1] [2] [3] [4] [5] . data on the type of medical practitioner and clinical setting were collected. responses were summarised as percentages and categorised by country. results: 1663 responses were received from general practitioners (22.2%), paediatricians (39.7%), paediatric allergists (6.9%), paediatric gastroenterologists (11.2%) and other specialities (20.0%). there were significant misconceptions about the clinical importance of primary li in infancy. while primary li rarely manifests before 3 years of age, 73.7% of participants felt it was a significant clinical problem in the first year of life. regarding secondary li, 44% of respondents recommended lactose restriction for viral gastroenteritis, and 36% for cow's milk protein-induced enteropathy. while the management of ige cma was relatively well understood, there were greater knowledge gaps for non-ige cma. 59% of practitioners appropriately identified extensively hydrolysed formula (ehf) as first-line treatment of cma in formula-fed infants. however, the distinction between lactose-free and lactose-containing ehf appeared to be an area of uncertainty. in india, 34.4% used soy-based formulas as firstresults: patch tests were positive in 62 (79.5%) and negative in others. positivity to milk was seen in 29 patients (37.2%), to soy in 41 (52.6%), to egg white in 7/72 (9.7%), to wheat in 12/66 (18%), to potatoes 6/48 (12.5%), to corn (maize) in 3/38 (7.9%), to rice in 1/2, and to peanut in 3/37 (8.1%). patients were requested to withdraw the suspected food(s) from their diets during a 4 months period. preliminary follow-up data show the improvement of one or more symptom in 19/20 patients (gastroesophageal reflux in 10, appetite in 5, stool consistency in 1, respiratory symptoms in 10, pain in 3, eczema in 1). conclusion: patch tests are informative, easy to use tools in order to identify potential causes of common lasting symptoms in children with negative or weak rast results and introduce beneficial changes in the daily diet. longer follow-up is necessary in order to refine and assess the benefit of such strategy. background: currently in the us, 1 in 13 children suffer from food allergies. at present, there is no cure and strict avoidance of the relevant foods is the only way to prevent allergic reactions. elimination diets put infants and children at risk for nutritional deficiencies and impaired growth. we examined the role of the registered dietitian (rd) in advising patients and families of food allergic children. method: a retrospective review of clinical notes was performed for the first 13 consecutive children who required a dietetic consultation in a dedicated food allergy clinic. we examined common questions from parents that were addressed by the dietician during the consultation. results: patients were aged 10 months -9 years (median: 16 months) and were diagnosed with the following food allergies: cow's milk: 69.2%, egg: 62%, tree nut: 54%, peanut: 39%, wheat: 31%, soy: 31%, fruit/vegetable: 15%, legume: 8%, fish: 8%, sesame: 8%. the most common questions for the dietitian included: ways to meet nutritional needs following a prescribed allergen-restricted diet (31%), meeting vitamin d and calcium requirements on a milk protein-free diet (23%), suitable oral supplements and recommended serving sizes (14%), appropriate order of solid foods introduction in food protein-induced enterocolitis syndrome (fpies) (14%), cautionary food ingredient statements (7%), baked milk protein introduction (7%), cross-reactivity risk of milk protein with soy (7%) and crossreactivity of nuts in retail bakeries (7%). conclusion: parents of children with food allergies have multiple questions with regards to nutrition. dietetic input in the food allergy clinic addresses important issues for children and families including successful avoidance of allergen-containing foods while ensuring optimal nutrition, decreased exposure to high-risk situations and avoidance of allergen cross-contamination. 0773 | multicenter prospective study of a stepwise single dose oral food challenge of egg background: oral food challenges (ofcs) are necessary for allergy management. we previously reported that a low-dose ofc can avoid complete elimination, even if patients react to higher doses of causative foods. nevertheless, this approach has only been validated in a retrospective single-center trial. we have previously reported that the median time for initial symptom onset is 50 minutes for egg ofc using a single exposure. therefore, this study aimed to confirm the safety and effectiveness of a stepwise single-dose ofc in a multicenter, prospective study. who showed a positive reaction to low-dose ofc, only 1 patient (2%) showed a severe reaction: barking cough immediately improved with adrenaline inhalation. among 8 patients with a positive reaction to medium-dose ofc, none had a severe reaction. the median times to symptom onset were 60 and 50 minutes following low-dose and medium-dose ofc, respectively. patients in the three groups, divided according to threshold doses, differed significantly in sige levels against egg white and ovomucoid. conclusion: this multicenter prospective study confirmed that stepwise single-dose ofc to egg will help to clarify the severity of egg allergy, and will contribute to improved food allergy managemethod: the study design was a retrospective cohort study extracting data from the electronic chart of children older than 4 years who visited our out-patient clinic for egg or milk allergy and who underwent an oral food challenge test (ofc) twice within 24 months between november 2013 and december 2017. the patients were divided into five groups according to their treatment schedule, which consisted of those who: a) started from 1/10 of the first ofc reaction threshold and maintained 1/10 till the end of oit; b) started from 1/100 of the threshold and maintained 1/10; c) started from 1/10 000 of the threshold and maintained 1/10, d); conventional slow oit (started from just below the first ofc reaction and increased 1.2-1.5 times every few weeks); or e) continued elimination. we determined the presence or absence of an increase in threshold reacted to the allergen, any adverse events during oit, and food-specific ige reduction. results: the number of participants was 217 and their median age was 6 years. the number of patients in groups a, b, c, d, and e was 17, 51, 33, 95, and 21, respectively. the percentage of patients in groups a, b and c showing an increase in reaction threshold to the allergen was higher than that in group e (p < 0.05), and that in group b was higher than that in group d (p < 0.001). the number (percentage) for group a, b, c, d, and e was 12 (70.6%), 43 (84.3%), 23 (69.7%), 54 (56.9%), and 5 (23.8%), respectively. there was a significant difference in the frequency of adverse events during oit between group a-c and d, which was as follows: 5 (29.4%), 9 (17.6%), 8 (24.2%), and 67 (70.5%), for the respective groups (p < 0.0001). there was no significant difference in the percentage of patients showing a decrease in food-specific ige in each group. conclusion: the regimen starting from 1/100 of the ofc reaction threshold and maintaining the dose at 1/10 was safer and more effective for increasing the threshold reacted to the allergen than the 'conventional slow oit' regimen. elimination continuation was not effective for increasing the threshold reacted to the allergen. legumes allergy was presented in different clinical features; urticaria and angioedema in 32 (50%) patients, anaphylaxis in 23 (35.9%) patients, atopic dermatitis in 5 (7.8%) patients, eosinophilic esophagitis in 3 (7.8%) patients and as food-related enterocolitis in 1 (1.5%) patient. thirteen (35.1%) of the patients had asthma, 9 (24.3%) had allergic rhinitis. fourteen (58.3%) of the 24 patients with single legume allergy showed improvement. the patients who developed tolerance, of these 7 (29.1%) had peanut allergy, 4 (16.6%) had lentil allergy and 3 (12.5%) had chickpea allergy. two of 13 patients with multiple legumes allergies, it developed tolerance to all the legumes they are allergic. conclusion: peanut and lentils were the most frequent legumes that displayed allergic reactions in our study. in these patients the rate of allergy to non-legumes food is high. in patients who were allergic to single legumes, the symptoms were ameliorated in 58.3%. conclusion: cashew nut is a potent allergen and can cause quite severe reactions. avoidance of pistachio nut and other related allergens should be advised to patients after allergologic investigation. in the majority of the patients, presence of atopic dermatitis with food allergy is noteworthy. therefore, it would be useful to investigate these patients for cashew and other tree nut allergy before they present with a serious clinical reaction. and jellyfish sting. serum allergen-specific ige test was negative; skin prick test was positive for natto and pork. we performed an oral food challenge with natto, pork, crustaceans, and wheat, and she developed a general itchy rash after 7 hours of eating natto. h1blocker was administered and she recovered soon. however, the general itchy rash relapsed after 4 hours. hence, we intramuscularly injected epinephrine, h1-blocker, and steroids; then, her symptoms did not relapse. based on these findings, we inferred that anaphylaxis caused by natto could be associated with a jellyfish sting. discussion: although association between japanese fermented soybeans (natto) allergy and jellyfish sting has been previously reported, its anaphylaxis is a rare event. in this case, we suggest that anaphylaxis was caused by natto allergy, which was perhaps related to jellyfish sting. hence, further investigation is essential to elucidate the association between fermented soybeans allergy and jellyfish sting. introduction: non-celiac gluten sensitivity (ncgs) is a syndrome characterized by intestinal and extra intestinal symptoms related to the ingestion of gluten-containing food, in subjects that are not affected by either celiac disease (cd) or wheat allergy (wa).once the gluten-containing foodstuff is removed from the diet, the patients will have relief of their symptoms. case: a 6-year-old girl was referred by his general practitioner with history of occasional constipation and abdominal pain (especially after main meals and defecation), short stature and low weight. the growth indices were proper for her age till she was 5. then after there was a stunting. she had short stature and low weight. despite different types of supplementation, there was no improvement in growth indices, so she was referred to a pediatric endocrinologist for gh therapy. primary investigations and anti-ttg, iga, anti-ema all were normal. after a consultation with a pediatric gastroenterologist, a genetic study of hladq2 and 8 were done because of the highly suspicion of celiac disease. the results were also negative. at last she was referred to immunology-allergy clinic for evaluation of probable food allergy. ige level was checked and a prick test was performed which they were not indicative of any suggestive food allergy. because of the history of the abdominal pain and constipation which was more prominent after meals, negative results of genetic study, spt to wheat, and serologic markers, a gluten free diet was suggested for her with the suspicious of non celiac gluten sensitivity. a significant improvement in her symptoms was noticed within 2 weeks of starting gluten free diet. she has 2kg of weight gain and height improved from 116 cm to 120 cm in 4 months. she continued to improve on a gfd and when seen in the follow-up clinic 6 months later reported complete resolution of symptoms and another 2 cm and 1 kg gain in her height and weight. conclusion: non-celiac gluten sensitivity syndrome is a diagnosis made by excluding celiac disease and wheat allergy. it should be taken into consideration especially in patients who have the suspicious symptoms of celiac without supporting lab data, and also negative spt to wheat. the young man in question along with his parents were keen to proceed, so with some hesitation we proceeded to a hazelnut oral provocation challenge, having very carefully explained the risks of undertaking such a challenge. he successfully completed the challenge and experienced no allergic symptoms and is now able to have hazelnuts in his everyday diet. discussion: this young man wanted to confirm if indeed he was allergic to hazelnuts. not being hazelnut allergic would mean that he would be no longer allergic to any nut and would not have to take precautions prior eating products. positive results to both cor a 9 and cor a 14, hazelnut storage proteins are associated with the patient possibly experiencing systemic reactions, at a higher risk of experiencing anaphylaxis it they were to ingest hazelnut. these facts in conjunction with his specific ige to hazelnut would have prevented us from proceeding to challenge was it not for this young man's persistence that he wanted to proceed to challenge despite the risks. conclusion: appearances are deceptive, as this case demonstrates; allergen-specific ige and component testing can only predict the probability of an allergic reaction, the final test in the diagnostic process is the oral provocation challenge. the patient and his family were happy for me to share the above with other health care professionals. method: we present the case of a female of 29 years old diagnosed of acu with poor control of the symptoms at maximum doses of antihistamines. we decided to associate omalizumab treatment. the patient had a good control of the symptoms with omalizumab at dose of 300 mg/4 weeks, but in the 6th month she presented an erythematous, raised and pruritic lesion in the area of injection together with localized abdominal edema at 12 hours of the administration, with two weeks of evolution without symptomatic treatment. we decided to discontinue omalizumab alter a second episode with half doses. results: we performed a skin biopsy of the lesion and epicutaneous tests with the drug. immediate hypersensitivity tests were not taking due to the impossibility of stopping antihistamines. skin biopsy showed a perivascular lymphocytic inflammation of the superficial and deep dermis with frequent presence of perivascular and interstitial eosinophils, suggestive of a hypersensitivity reaction. results: nine months before presentation at our clinic, the patient had been hospitalized and treated with imipenem and tmp/smx for pulmonary nocardiosis. once discharged, she had been prescribed oral tmp/smx alone, according to antimicrobial susceptibility. at our first evaluation, the patient presented with fever, macular erythematous non-pruritic (vasculitic-like) skin lesions on the upper limbs, polyarthralgia and bilateral ankle arthritis. tmp/smx was transiently stopped. after four days, there was a dramatic improvement, with resolution of all signs and symptoms. she was tentatively diagnosed with a viral infection and thus tmp/smx was started again. however, after three days, the symptoms (fever, arthritis and skin lesions) recurred. laboratory investigations showed increased levels of inflammatory markers. complete blood count with differential, serum creatinine, urinary sediment, liver enzymes, rheumatoid factor, antinuclear antibody, c3, c4, immune complexes, serology for rickettsia, borrelia and coxiella were all negative. hence, tmp/smx was stopped again and cutaneous lesions, fever and arthritis resolved spontaneously in five days. conclusion: given the clinical course and the resolution after the withdrawal of tmp/smx, we diagnosed a sslr due to sulfonamides. to the best of our knowledge, this is the first case of sslr occurring after a nine-month therapy with tmp/smx and allergists/immunologists should be aware of the possibility of such a reaction even after months. case report: * we received written informed consent for publication of these clinical details and/or clinical images included in my abstract was obtained from the patient. drug rash with eosinophilia and systemic symptoms (dress) syndrome is a severe adverse cutaneous reaction that usually appears 2-6 weeks after treatment with the causative drug. this syndrome is characterized by severe dermal rash, fever, eosinophilia, and internal organ involvement, and clinically, diffuse maculopapular eruption, exfoliative dermatitis, and facial edema are often observed. we performed blood tests and laryngeal fiberscopy for the diagnosis of the patient. intradermal test with delayed reading and patch test were performed 2 months after the end of treatment. a 53-year-old man had begun treatment with carbamazepine for epilepsy. after 4 weeks of treatment, he observed skin rash with pruritus on both lower extremities, and after 6 weeks, his skin lesions had begun to spread over his whole body, and he complained of several new symptoms, including hoarseness, dyspnea at rest, and dysphagia. an examination revealed maculopapular rash, facial edema, and bilateral cervical lymphadenopathy. laryngeal fiberscopy revealed both arytenoid and epiglottic swelling. laboratory studies revealed eosinophil counts of 2100 /μl and increase in alanine aminotransferase level to 65 u/l. a diagnosis of dress syndrome was definite according to the regiscar group criteria. carbamazepine, the suspected culprit drug, was withdrawn, and systemic corticosteroid was initiated. the patient experienced rapid improvements in hoarseness, dyspnea, and dysphagia. after 5 days of treatment, laryngeal fiberscopy revealed complete resolution of both arytenoid and epiglottic swelling. to the best of our knowledge, our case is the first reported case of dress syndrome to manifest with laryngeal edema. case report: bortezomib (velcade ⓡ ), a targeted therapy works by blocking the action of proteasomes in side cells, is commonly used to treat newly diagnosed as well as relapsed/refractory myeloma. bortezomib has been reported to have gastrointestinal symptoms, peripheral neuropathy, neuropathic pain and thrombocytopenia as its most common side-effects. although several cases of skin lesion caused by bortezomib have been reported, severe cutaneous adverse reaction (scar) such as stevens-johnson syndrome (sjs) is very rare. we here report a case of bortezomib induced sjs. a 71-year-old female patient, who was diagnosed with multiple myeloma, received bortezomib and melphalan /dexamethasone therapy. after the 4th dose of bortezomib, she presented with fever and maculopapular skin rashes spreading from face to the trunk. erosive lesions in the oral mucosa and corneal ulceration with conjuntival injection were observed. she was diagnosed as sjs. the symptoms of sjs improved after bortezomib was discontinued and systemic steroids and intravenous immunoglobulin were administered. drug patch test was performed, the result was positive in bortezomib. this is the first case report of bortezomib induced sjs in this country, which was diagnosed by a patch test. although the scar by bortezomib is generally considered very rare, we suggest that clinicians be aware of potential adverse reactions, including sjs. case report: we report the case of a healthy 46-year-old woman with history of red erythematous macules in both hands, one hour after taking a fluconazole (150 mg) tab for a vaginal candidiasis. it faded spontaneously. she didn't recall if she had ever taken that medicine, but denied known drug allergies. although fde is primarily a clinical diagnosis, we conducted an oral challenge test with fluconazole (150 mg). two hours after intake of the drug the patient started complaints of pain and erythema in both hands and the challenge was stopped. two days after the challenge, she developed red painful erythematous macules on the same sites of the first episode. due to the specificity of the challenge, local patch testing was not performed. introduction: the classic form of a fixed drug eruption is one or more anular or oval erythematous patches as a result of systemic exposure to a drug. these skin lesions normally resolve with hyperpigmentation and may recur in the same location with re-exposure to the drug. other types of fixed drug eruptions have been described, being fixed drug urticaria a rare form of presentation. (1, 2) case report: in the last 2 years, a 48 year old woman has developed more than 15 episodes of a wheal in the right supraciliary region 5 minutes after taking 600 mg of oral ibuprofen. the symptoms resolved in less than 6 hours without treatment and without leaving residual lesion. after the last episode, she refers good tolerance to 1 g of oral paracetamol. she denies local traumas. she also refers mild spring rhinoconjunctivitis well controlled with antihistamine, and sneezing with house dust. a 37-year-old-m patient had psoriasis vulgaris for 15 years, and had been using methotrexate at intervals of 4 years. despite the addition of phototherapy, he underwent a new treatment with biological agent (antitumor-necrosis-factor; anti-tnf), since the disease control was insufficient. before anti-tnf, preventive treatment against latent tuberculosis (tb) activation was indicated with positivity in tuberculin skin test (20 mm). he was given inh 300 mg/day, and at the 20th day of treatment, desquamation, erythema, and subsequent exfoliation developed in his hands and foots dorsum. inh was withdrawn. in order to distinguish the lesions from psoriasis attack, skin biopsy was performed and reported as erythema multiforme-like dermatitis with no relation to psoriasis. the lesions were completely improved at 3 weeks of topical steroids, and inh was re-initiated at the same dose. a week after the initiation of the drug, skin lesions similar to previous reoccurred with more severity and progression from distal to proximal extremities. cell counts, renal and hepatic function tests, and hepatitis markers in blood were in normal limits. skin lesions were retracted after 4 weeks of topical steroids, and withdrawal of inh. there was positivity in skin patch test with inh at 72 hours. finally, for tb prevention an alternative drug rifampicin (10 mg/kg/day) was given, and the patient successfully completed with no adverse event. his psoriasis lesions were improved with anti-tnf which was started after 1 month of tb prevention with rifampicin. in these days which the use of biologic agents is increasingly widespread, inh use will be more prevalent than the past. even tough, it is effective and safe in most of the patients, its adverse event dermatitis may be a reason to withdraw in patients with dermatological diseases. in this case, diagnostic drug allergy evaluation should be performed to optimize the second-line treatment of tb infection, in addition to early withdrawal of the culprit drugs. background: around 50% of cancer patients will receive radiotherapy (ionizing radiations) as a treatment, either as a single therapy or as an adjuvant to chemotherapy and surgery. several side effects have been described due to radiotherapy, of which we can mention erythema multiforme and stevens johnson syndrome, but in lower prevalence. erythema multiforme can be described as an acute skin condition and may be present within a wide spectrum of severity. erythema multiforme minor represents a localized eruption of the skin with minimal or no mucosal involvement. the papules evolve into pathognomonic target or iris lesions that appear within a 72-hour period and begin on the extremities (see the following image). lesions remain in a fixed location for at least 7 days and then begin to heal. it is considered to be a type iv hypersensitivity reaction associated with certain infections, medications, and other various triggers precipitating factors and complex interactions may trigger the appearance of signs and symptoms. these include especially recurrent herpes simplex virus (hsv), epstein-barr virus (ebv), histoplasmosis, alcohol, systemic diseases and immunological factors. method: 69-year-old male diagnosed with prostate adenocarcinoma who underwent transurethral resection and was taking trinomia (ramipril, atorvastatin, acetyl-salicylic acid) after his 8th rte external radiotherapy session, he presented erythematous maculo-papular lesions in the suprapubic area with some vesicles. therefore, withdrawal of treatment was decided and the performance of a skin biopsy. 15 days later, regarding the improvement of the lesions, rte was continued, presenting incipient exacerbations of the lesions but it allowed us to end the cycle of treatment. results: skin biopsy results (anatomical pathology): basal keratinocytes, which blur the dermoepidermal interface, with lymphocyte exocytosis at this level, associated with isolated images of spongiosis. the dermis shows a superficial perivascular lymphocytic inflammatory infiltrate of moderate intensity. compatible with erythema multiforme. conclusion: radiotherapy is a technique of increasing use, so it is important to recognize the associated cutaneous lesions that appear less frequently and are sometimes underdiagnosed. diagnosis is both clinic and pathological and is usually late in most cases so it is vital to take into account this skin disease complication in order to be properly managed. including chinese herbal medicine is usually considered to be without any allergic and adverse reaction. method: visits were made to pharmacies in hong kong and luoyang, china and a martial art monastery/temple in dengfeng, china. some cam were found to have ingredients with potential allergic and adverse reaction. results: three cam, one from hong kong (1), one from shaolin martial art monastery/temple in dengfeng (2) and one from luoyang (3), china were found to contain chinese herbal medicine with potential allergic and adverse reaction. (1) cordyceps ling-zhi complex ingredients: cordyceps sinensis "caterpillar fungus", tremella fuciformis "snow fungus", ganoderma lucidum (ling-zhi) "reishi mushroom" and others 30 years old female patient who has ulcers in oral mucosa and purple, itchy lesions on her right hand palmar area, little finger, index finger, on her left hand palmar area, pollex finger. in her history, she has relapsing vaginal yeast and she hasn't any hypersensitivity reaction with fluconazole before 1 month ago she started to take fluconazole because of vaginal candidiasis. after using fluconazole she started to itch from described areas and dark redpurple eruptions appeared. she was prescribed oral methylprednisolone and topical pomade which included corticosteroid for four days but she didn't aware of fluconazole related drug reaction. lastly four days ago she took fluconazole and metronidazole for severe vaginal yeast. 12 hours later pruritus, same eruption appear on the same area, lip and tongue angioedema than she had dyspnea, dizziness, hypotension, arrhythmia and consciousness. she had admitted to the emergency department and performed adrenalin. after a day bullae and ulcers came into existence in her oral mucosa. in her blood analysis there was mild increase in white blood cell count (13.300 /mm 3 ), eosinophil count was normal (300 /mm 3 ), biochemistry parameters were in normal limits, crp and sedimentation rate were in normal limits, total ige was 439 iu/l. introduction: tuberculosis is a disease that most commonly affects the lungs, which is transmitted by the respiratory tract and drugs are the most important factor in the treatment. non-resistant tuberculosis infection is usually treated with hrze. in rare cases, a hypersensitivity reaction may develop against one or more of the drugs during treatment. case: a 37-year-old female patient was diagnosed with culturepositive pulmonary tuberculosis and hrez treatment was started by the related department. seven days later she referred to the policlinic with edema and itchy erythematous lesions which are common in her extremities, which developed after 6 hours of taking her medication. liver and kidney function tests and eosinophil count were normal. drug eruption was considered with current physical examination findings. the treatment was interrupted, short time systemic corticosteroids and antihistamine treatment started. desensitization planned. there was no feature in the prick and patch tests with drugs. desensitization was performed with isoniazid, no reaction was observed during the procedure. six hours after the procedure, the patient applied to the emergency department with painful edema and pruritic erythematous lesions in the extremities. desensitization procedures with rifampicin, ethambutol, pyrazinamide were performed without any problems after the lesions were regressed. isoniazid was withdrawn from the treatment protocol. outcome: we would like to present on this case that drug eruption may develop in the form of maculopapular rash after desensitization. this study aims to compare ethmoid mucosa and nasal polyp regarding density of tissue eosinophil and its sensitivity, specificity, and correlation with clinical characteristics for diagnosing ecrs. method: patients with crs with polyps scheduled for endoscopic sinus surgery were enrolled. specimens were collected from polyp apex, polyp pedicle and ethmoid mucosa. tissue eosinophil from these three sites in the same patient were compared. using eosinophilic mucin as a reference, sensitivity, and specificity of each site for diagnosing ecrs was assessed. correlations between tissue eosinophilia (defined as greater than 10/ hpf) and clinical characteristics of ecrs including asthma, serum eosinophilia, and eosinophilic mucin were analyzed using each site of specimens. results: thirty patients with crs with polyps were enrolled. polyp apex, polyp pedicle and ethmoid mucosa gave similar results regarding tissue eosinophilia in 16 patients (53.3%). eleven (36.7%) patients were ecrs (having tissue eosinophilia at all sites) and five (16.6%) were non ecrs (no tissue eosinophilia at any sites). median tissue eosinophil was significantly greater in polyp apex (84, and polyp pedicle (96, iqr: 80-320) than ethmoid mucosa (21, iqr: 10-220), p = 0.04. sensitivity of polyp apex, polyp pedicle and ethmoid mucosa for diagnosing ecrs were 100%, 60% and 80% respectively. specificity were 36%, 40% and 48% respectively. correlations between tissue eosinophilia and asthma were significant when assessing ethmoid mucosa (p = 0.04), and polyp pedicle (p = 0.05) but not polyp apex (p = 0.21). correlations with serum eosinophilia, and eosinophilic mucin were not significant (p > 0.05) when assessing any specimens. gov/pubmed/) was performed using the following key words: "obstructive sleep apnea syndrome"; "allergy rhinitis"; "hypoxia"; "intermittent hypoxia"; "fluctuating hypoxia"; "cyclic hypoxia"; and "hif-1α" results: osas may affect the prognosis of ar patients based on the following evidence: 1) ar is thought to be a cause of osas. 2) exposure to hypoxia could mediate immune activation in ar and affect the response to treatment. 3) hif-1α expression may be a risk factor for ar. 4) intermittent hypoxia can induce robust expression of hif-1α. conclusion: first, improvement of ventilation during sleep represents an efficient strategy for treating ar. therefore, continuous positive airway pressure or nasal surgery to resolve a nasal obstruction could be added to ar treatment. finally, medications that target hif-1α, such as digoxin, can be tested as adjuvant therapy. method: forty patients diagnosed with allergic rhinitis and olfactory dysfunction were recruited in current study in the group 1 and 2. patients of group 1 were administered with no treatment and patients administered with the traditional chinese acupuncture therapy were incorporated into the group 2. before the treatment, all of them underwent t&t olfactory testing, nasal sinus computer tomography scanning and visual analog scale (vas; 0-100), and repeated the assessment after four-week treatment. results: improved total t&t olfactory testing scoring averages and vas scoring averages was observed in eleven patients treated with traditional chinese acupuncture compared with four patients in the observation group. no side effect was found. no significant differences in olfaction recovery were found according to age, gender, or duration of disease between the two groups. the observation group underwent nasal endoscopic sinus surgery and the control group underwent external approach surgery, and the therapeutic effect of the two groups were investigated. results: the total effective rate was 94% in observation group and 74% in control group, the total effective rate of observation group is significantly higher than control group (p < 0.05). the recurrence rate was 3% in observation group and 24% in control group, the recurrence rate of observation group is significantly lower than control group (p < 0.05). complication occurrence rate of observation group was 6% which is significantly lower than control group 24% (p < 0.05). the therapeutic effects of endoscopic sinus surgery on chronic sinusitis in geriatric patients are better than conventional external approach surgery which is worth clinical application. results: (descriptive). the equick app is user-friendly even for vkc patients with sub-optimal reading ability. home use between clinic appointments allows responsive temporal data gathering of qol, symptoms, medication scores and impact of medical interventions. equick may be used in future as a research tool in gathering outcome data following interventions for vkc. ectoine, a substance deriving from halophilic micro-organisms, is a strong water structure forming solute exerting cell protective antiinflammatory and antiallergic properties. method: purpose of our study was to assess the efficacy of the preventive administration of 2% ectoine eye-drops (3 times a day for 6 months) to shorten the duration of vkc relapses (which begin, in our country, very early in spring and usually end in october), or to mitigate the attacks, which are only controlled by topical corticosteroids or cyclosporine resulting in an important burden of side-effects. in this retrospective study, we included 192 children of both sexes (148 males and 44 females), under the age of 10 years (mean age 7.8 years), affected by vkc from more than 3 years/seasons and treated for more than 4 months during a year, with cyclosporine eye-drops. these patients underwent, from february to september 2017, the additional-to-the-usual protocol treatment with 2% ectoine eyedrops. results: 8% of the included subjects astonishingly had no relapse of vkc, 38% needed topical cs or cyc treatment but it was started 2 months later compared to previous years, 29% needed the topical drugs 3 months later and 25% had a similar to previous years course (no ectoine efficacy). the treatment was well tolerated and only 1 child had to stop it because of local allergy to the eye-drops. the preventive administration of 2% ectoine eyedrops was able to stabilize and to delay vkc attacks in more than 50% of the selected patients showing the importance of anti-inflammatory and anti-allergic properties of this product. following international criteria we considered normal levels of vitamin d the levels between 50 nmol/l (20 ng/ml) and 125 nmol/l (50 ng/ml), a potential deficiency between 30 nmol/l and 50 nmol/l and a severe deficiency less than 30 nmol/l. results: 58.8% (30 children) of akc group patients presented vitamin d low levels, among them 18 children showed a potential deficiency and 12 a severe deficiency. 24.3% (18 subjects) of vkc patients suffered a deficiency in vitamin d which was mild in 13 and severe in 5 patients. 40.3% (25 children) of sac group showed a deficiency in vitamin d which was potential in 17 and severe in 8 subjects. conclusion: our study shows that in different forms of allergic conjunctivitis many children are suffering a vit. d deficiency and it can be supposed that a correlation between the severity of the allergic form and the level of vit. d deficiency exists. we recommend allergists and ophthalmologists to check vit. d levels in children suffering from allergic conjunctivitis because its deficiency is very common and many are unaware of it; in case of a vit. d insufficiency it is fundamental to give a vit. d suitable-to-the-case supplementation. method: 83 children (57 males and 26 females, mean age 7.3 ± 5 months) affected by vkc and allergic rhinitis from more than 2 years were treated with mometasone furoate nasal spray 1 spray bid × 2 weeks in a month, for 3 consecutive months as a co-seasonal treatment at the beginning of eye allergic symptoms. other systemic or topical treatments did not vary compared to the previous 2 years. results: a quick questionnaire administered to children and their care-givers showed that nasal symptoms regressed after a mean period of 9.2 days from their beginning but, impressively, in more than 30% of them, these patients did not show a vkc typical relapse along the 3 months of mometasone treatment, moreover the following summer period was milder in subjective ocular symptoms in more than 25% of the patients. our experience pointed out that incs adjunctive treatment was positively associated with a regression of eye and nose symptoms in children suffering from vkc, confirming previous literature data which concern milder forms (seasonal allergic conjunctivitis or allergic rhino-conjunctivitis) compared to the severe forms (like vkc) we analyzed in our work. one of the involved mechanisms of action can be the alleged effect on the reduction of substance p in tears; it is supposed to reflect the neuropeptides levels in ocular tissues. 0816 | patient response to mp-azeflu in an allergen exposure chamber onset of action (ooa) timing may impact treatment adherence. mp-azeflu, intranasal azelastine hydrochloride (aze) and fluticasone propionate (fp) in a single device, has proven to have greater efficacy and faster ooa than a combination of oral loratadine and intranasal fp (lora/infp), but the clinical relevance for patients is unclear. this single-center (ontario, canada), randomized, double-blind, double-dummy, three-period crossover trial examined by which extent mp-azeflu provides clinically relevant symptom improvements according to different efficacy parameters. method: ar symptoms were induced in asymptomatic, ragweedsensitive patients via ragweed pollen challenge in an environmental exposure chamber. patients received a single dose of mp-azeflu, lora/infp, or placebo and were monitored for 4 hours. symptoms were assessed using total nasal symptom score (tnss) and total ocular symptom score. responder analyses included the number of patients to achieve relevant response (rr) to therapy (30% or 50% reduction in tnss), time to rr (ie, first time point at which rr was reached), and minimal clinically important difference (mcid) in ooa. background: nasal allergen provocation test (napt) is a standardized diagnostic tool indicated in the diagnosis of allergic rhinitis, to design and monitoring allergen immunotherapy, and to study the pathophysiology of airway allergy. unfortunately, until now very few studies have evaluated its reproducibility and safety. in this study we wanted to analyse the safety and reproducibility of napt in a large group of rhinitis patients and healthy controls. unit until december 2017. a bilateral saline challenge followed by a bilateral napt were performed in symptoms-free individuals. the response was assessed by nasal-ocular symptoms and acoustic rhinometry. all subjects signed a written informed consent. the safety of napt was checked by the occurrence of extra-nasal/ ocular reactions (enor), severe adverse events (sae), and use of rescue medication (rm). enor was assessed by clinical symptoms, physical examination, cardiopulmonary auscultation, spirometry, and oxygen saturation. the reproducibility of napt was tested by comparison of the results in 2 or more sessions with≥1-month interval. background: nasal hyperreactivity (nhr) is self-reported by a majority of patients with allergic rhinitis (ar) and is likely mediated by neural-immune interactions. the combination of fluticasone propionate (fp) and azelastine (aze) hydrochloride administered in a single spray (mp-azeflu) has been shown to be superior to fp or aze alone in patients with seasonal ar (sar). we hypothesize mp-aze-flu may reduce neuro-immune mediators in ar with nhr. in a post hoc analysis of three pivotal studies of mp-azeflu, we analyzed the efficacy of mp-azeflu, fp, and aze in patients with ar with and without nonallergic triggers. method: in three randomized, double-blind, controlled trials, patients with sar were randomized 1:1:1:1 to mp-azeflu, fp, aze, or placebo (pbo). patients self-reported sensitivity to nonallergic triggers. change from baseline in total nasal symptom score (tnss) and treatment differences between active agents and pbo were calculated. results: across 3412 patients in three studies, mean age was 36.3 years and mean age at ar symptom onset was 15.6 years. overall, 89% reported ≥1 nonallergic trigger, which included sudden temperature/humidity change (72%), tobacco smoke (61%), perfumes/fragrances (57%), incense/candles (38%), and cleaning products (38%). change from baseline in tnss for patients with ar and nonallergic triggers was greater with mp-azeflu than with fp or aze (table) , and patients with nonallergic triggers improved slightly less than patients without nonallergic triggers in both the mp-azeflu and fp groups. background: in low-income countries (lics), assessment of phenotypes, prevalence and risk factors for allergy-related diseases (ards) using allergen-specific ige may be complicated by environmental exposures such as helminths. these exposures may also induce cross-reactive carbohydrate-specific ige profiles that could inhibit allergic effector responses. we sought to elucidate the molecular basis of ige sensitisation among individuals in uganda, using a component-resolved approach to ige measurement. we employed the isac ® allergen microarray to assess plasma ige reactivity to 112 purified natural and recombinant allergen components in participants of three studies: a trial of intensive versus standard anthelminthic treatment in the rural helminth-endemic lake victoria islands (n = 126), a parallel urban survey of allergy outcomes in a lower helminth exposure community (n = 60) and a study on asthma risk factors in children from the urban setting and from nearby rural schools (n = 100). data on sensitisation to crude allergen extracts were obtained by skin prick testing (spt) with cockroach and house dust mites (hdm), and by immunocap ige testing (cockroach, hdm, and peanut). results: the rural setting was characterised by high prevalence (≥34%) of sensitisation to crude extracts (immunocap ige>0.35 ku/ l) but low sensitisation to the major, established, allergenic components on the microarray (≤2%, ige>0.30 isu). however, sensitisation to cross-reactive carbohydrate determinant (ccd)-bearing components and venoms was more common in rural (up to 14%) versus urban (up to 8%) individuals, and was associated with helminth infection. urban individuals mounted higher responses to allergenic components of dust mites but responses to other components were similar between the two settings. sensitisation to allergenic components was higher among asthmatics and spt+ children but ccd sensitisation profiles were similar between asthmatics and nonasthmatics, and between spt+ and spt-school children. conclusion: we show that, in lics, ige to crude allergen extracts (detected in standard immunocap assays) reflects sensitisation to a myriad of environmental exposures (absent in more developed countries), such as ccds expressed by helminths, and may not accurately define ard phenotypes in this setting. however, our data does not seem to indicate that ccd-specific ige detected by isac ® microarray protects against ards. considered minor allergens. due to their sequence homology and conserved structure, they show a high cross-reactivity. the objectives were to study the ige/igg binding properties of polcalcin in relation to the calcium ions, and the ige cross-reactivity between purified polcalcin from olea europea (ole e 3) and two recombinant polcalcins (rphl p 7 and rbet v 4). method: ole e 3 was purified by immune-affinity chromatography using polyclonal antibodies anti-rche a 3. serum samples were obtained from 6 patients allergic to grasses recruited at hospital de guadalajara (spain), all of them positive to phl p 7 with sige values ranging from 12.1 to 92.6 ku/l. equal volumes of all sera were used to prepare a pool. calcium binding assay was performed either by addition or not, or depletion of ca 2+ . ole e 3 was incubated with 0.1 mm cacl 2 or with 1 mm egta ph 7.5 (ca 2+ chelator agent) at the same time as the antibody in immunoblot or elisa assays with the pool of sera or with anti-che a 3 polyclonal antibody. crossreactivity assay was performed by immunocap inhibition. aliquots of the pool of sera were previously incubated with amounts of ole e 3 ranging from 0.02 to 12.5 ng. the same dilution of the pool of sera without ole e 3 was used as a control. after 2 hours of incubation, sige (ku/l) binding to rbet v 4 or rphl p 7 was determined. results: a 9 kda protein was purified from the o. europea extract and identified by lc/ms-ms as ole e 3. in the calcium binding assay there were no differences between the samples with or without ca 2+ . however, the addition of egta to the reaction completely inhibited the binding of the polyclonal antibody by immunoblot and also produced a 32.3% reduction of ige binding by elisa. in the cross-reactivity assay, a 50% inhibition of ige binding was obtained with 2.8 ng of ole e 3 for rbet v 4 and 3.9 ng for rphl p 7. the maximum rate of achieved inhibition was 68.6% for rbet v 4 and 61.6% for rphl p 7. conclusion: native purified ole e 3 contains the ca 2+ necessary to bind to the specific antibodies and the depletion of ca 2+ inhibited this binding. high cross-reactivity of ole e 3 with rphl p 7 and rbet v 4 was demonstrated. 0826 | effect of glutathione-s-transferase pi on the cysteine protease activity of the house dust mite allergen der p 1 background: environmental proteases have been proposed to be involved in the pathogenesis of allergic disorders via different mechanisms, such as the disruption of epithelial tight junctions, the cleavage of surface proteins, the activation of damage and pathogen-associated molecular patterns receptors, and the alteration of redox status. der p 1 from house dust mite is one of the most clinically relevant indoor allergens worldwide, which exhibits cysteine protease activity and has been linked to allergenic rhinitis and asthma. however, it is unknown whether the host microenvironment could regulate der p 1 activity once it reaches the mucosal surface. glutathione-s-transferase pi (gstpi) is an anti-oxidant and detoxification enzyme. gstpi is the predominant gst in human lung epithelial cells, where it is expressed in high levels. polymorphic variants of gstpi have been associated to various inflammatory lung disorders such as allergic asthma. more recently, gstpi has been identified as a redox regulator through protein s-glutathionylation, a post-translational modification where glutathione (gsh) is conjugated to cysteine residues. method: this work aimed at determining if gstpi affects the cysteine-protease activity of der p 1, compared to gstmu -a different gst isoform-by using different in vitro approaches. results: we found that gstpi increased der p 1-activity, but not gstmu. our results suggested a potential role of gstpi in upregulating the protease activity of der p 1 allergen. however, the clinical implications of these findings in allergic airway diseases needs for further investigations. 0827 | cari p 1, a novel polygalacturonase allergen from papaya acting as respiratory and food sensitizer biswas sarkar m; sircar g; ghosh n; das ak; jana k; dasgupta a; gupta bhattacharya s bose institute, kolkata, india background: papaya was globally reported to elicit ige-mediated hypersensitivity. certain papaya sensitive patients with food allergic symptoms were found to experience recurrent respiratory distresses at peak flowering period of papaya even after quitting the consumption of papaya fruits. the immunoreactive protein present both in pollen and fruit proteome was detected by ige-serology and identified by mass spectrometry. one such allergen, designated as cari p 1 was cloned, and purified as recombinant protein. the ige-reactivity of rcari p 1 was examined by immunoblot using patient sera. the allergenic activity of rcari p 1 was evaluated by histamine release assay from ige-sensitized granulocytes. the aggregation and folding pattern of rcari p 1 was assessed by size exclusion chromatography and circular dichroism spectroscopy respectively. the presence of cari p 1 in papaya fruit was searched by igg-immunoblot using allergen-specific rabbit antisera. a mouse model of papaya allergy was established to study the role of rcari p 1in eliciting respiratory and food hypersensitivity. results: a 55 kda ige reactive protein commonly present in pollen and fruit proteome of papaya was identified as endopolygalacturonase. recombinant cari p 1 remained monomer and the cd-spectra revealed predominantly β-sheet characters. the melting curve of the allergen showed partial refolding from a fully denatured state indicating the possible presence of conformational ige-epitopes in addition to the linear ige-epitopes of food allergens. 7 out of 7 papaya allergic patients displayed ige reactivity to rcari p 1. rcari p 1 at 1 μg/ml, induced histamine release from challenged granulocytes within a range of 30% to 72% (i.e. 50 ± 9.2%; n = 4 patients). expression of cari p 1 was detected in the peel and pulp tissues of papaya fruits at two edible stages of fruit maturation. in mouse model, rcari p 1 exhibited a comparable level of eosinophil infiltration and goblet cell hyperplasia in lung and duodenum histology. conclusion: cari p 1 the first major allergen reported from papaya with a dual role in respiratory sensitization via pollen inhalation and sensitization of gut mucosa via fruit consumption. the recombinant allergen can be used as marker allergen for molecular diagnosis and immunotherapeutic management of papaya allergy. background: lipids can be potent stimulators of the immune system, and their role in allergy is highly investigated and debated. since many allergens bind lipids, one question that arises is the relative importance of the lipids versus the lipid-allergen complex in eliciting the immune response. also of interest is an evaluation of the importance of the allergen-lipid complex. in our characterization of the structure of the cockroach allergen bla g 1, we discovered that it could promiscuously bind a variety of lipids in a large central cavity. this suggested that bla g 1 could be used as a prototypical allergen and lipid delivery vehicle to test in various models of sensitization. method: cd spectroscopy. nmr spectroscopy. molecular modeling. we have developed an hplc procedure to strip the phospholipids derived from the e. coli-based expression system, and reconstitute the allergen with a variety of lipids. using cd spectroscopy and nmr, we have verified that the protein conformation is highly similar in the presence and absence of lipids. temperature dependent cd spectroscopy revealed that unloaded bla g 1 is the least stable, and the melting temperature increased with increasing fatty acid chain length up to c20. similar cd melting experiments revealed that bla g 1 could bind lipoteichoic acid (lta) from gram positive bacteria, but did not interact with lipopolysaccharide (lps) from gram negative bacteria. molecular modeling studies have suggested that the stoichiometry of phospholipid binding is likely 4 phospholipids per bla g 1 and give insight as to the different binding characteristics that would allow bla g 1 to bind lta but exclude conclusion: these biophysical studies will allow the design of bla g 1-lipid systems to test a variety of sensitization models. 0829 | sal k 7, a new allergen from salsola kali sola jp; pedreño y; fernández j; cerezo a; peñalver m probelte pharma, murcia, spain background: the polcalcin from salsola kali was identified and sequenced (genbank kt254655) and the recombinant protein was characterized as a minor allergen with a prevalence of 40% of patients with a spt positive to s. kali. the objective of this study was to purify the polcalcin from s. kali pollen and to include the allergen in the website for the systematic allergen nomenclature (www.allergen.org). method: the native polcalcin from s. kali (npsk) has been purified from pollen after a first step of protein extraction and then diverse chromatographic steps: a size exclusion chromatography to remove particles minor than 5 kda, an ionic exchange chromatography, a hydrophobic interaction chromatography and a final step of size exclusion chromatography to obtain the purified sample of polcalcin. the purity of the npsk has been determined by sds-page and the binding capacity to a specific polcalcin antibody from rabbit serum was tested by immunoblot. the specific antibody had previously been obtained by immunization with the recombinant polcalcin from s. kali. the allergenicity of the npsk has been assayed by immunoblot with a pool of sera of patients sensitized to s. kali. the identity of the purified npsk has been analyzed by peptide footprint in hplc-ms/ms after digestion with trypsin. all the information about the polcalcin from s. kali was sent to who/iuis allergen nomenclature sub-committee. the npsk showed a high purity in sds-page with a molecular weight of approximately 9 kda and this purified protein reacted with the specific polcalcin antibody from rabbit serum. the ige binding capacity of the npsk was confirmed by immunoblot using a pool of sera from patients sensitized to s. kali. the analysis of peptide footprint confirmed that the purified protein is a polcalcin. the who/iuis allergen nomenclature sub-committee included the polcalcin from s. kali in the website for the systematic allergen nomenclature as a new minor allergen named sal k 7. conclusion: the polcalcin from s. kali has been purified from pollen and tested for its ige binding. it is included in the website for the systematic allergen nomenclature as the new allergen sal k 7. background: alt a 1 protein is the major allergen from the fungus alternaria alternata and responsible for chronic asthma, yet little is known about its physiological role and immunological activity. our main purpose was to investigate the mechanism through which alt a 1 induces an allergic response in bronchial epithelium. method: although alt a 1 has a unique topology, we studied the structural relationship by in silico procedures consisting of three distinct structural alignment methods in order to understand its nature. the immunological properties of the allergen were investigated by using monocyte cell line thp1 and human peripheral blood mononuclear cells. results: its crystal structure has been recently reported and claimed to be exclusively in fungi without equivalent in the protein data bank. data obtained in silico show that this allergen shows some structural relationships with a number of other β-barrel proteins such as human lipocalin 2 (lcn2). besides, our experimental data demonstrate that alt a 1 is also able to interact with lcn2, human lipocalin. in this way, the results obtained from several immunological assays showed that alt a 1 is able to produce a response of the immune system through different immune innate receptor pathway inducing the th2 cytokines. background: increasing evidence of cross reactivity syndromes between pollen grains and fruits, with immediate or delayed reactions, has been reported. while some syndromes such as the birch pollen/apple syndrome are well documented, some other such as the cypress pollen/peach syndrome remain to be understood. for the latter, significant progress has recently been made with the discovery of a new allergen family, the gibberellin regulated proteins (grps), which has been shown to be responsible for the observed cross reactivity i.e. pru p 7 and bp14 (1, 2) for the peach and the cypress pollen respectively. grps are small cationic proteins with anti-microbial properties and have been shown to be over produced in response to a stress. herein, the case of a patient, born and raised in the south of france but currently living in paris, has been studied. this patient has been suffering since childhood from allergic rhinoconjunctivitis to cypress pollen and from some oral symptoms to peach and other fruits (including pomegranate). method: in addition to the clinical exploration and cutaneous tests, a very thorough biological characterization of the patient samples has been performed through various specific ige quantitation techniques, western blotting after one and two-dimensional gel electrophoresis and flow cytometry based basophil activation testing (bat). results: specific iges to cypress pollen, birch pollen, peach, orange and apple have been found. pr10 allergenic proteins are recognized by iges but no ltps. the presence of specific iges to cypress pollen bp14, peach peamaclein (pru p 7) and a cationic 14 kda protein from pomegranate has been shown through western blotting after gel electrophoresis separation of the protein extracts. the use of bat finally enabled to demonstrate that the basophils of this patient were, ex vivo, strongly activated with protein extracted from orange and cypress pollen and also with purified proteins such as bp14 and pru p 7. conclusion: these results unambiguously show that the cypress pollen grp, bp14, is clinically relevant, similarly to its homologous protein in peach, pru p 7. it can be proposed that these two allergens are at the basis of the observed cross-reactivity syndrome. the search for new cross-reactive allergenic grps in pollen, fruits or vegetables may enable to better understand other pollen/food associated syndromes that still remain unexplained. background: nine allergens of phleum pratense have been described until now (iuis database) and classified into groups based on their function and cross-reactivity. group 1 and 5 allergens are considered the most immunodominant, due both to their greater ige-binding capacity and the number of patients ige-reactive to them. previously published studies have estimated that group 1 is recognized by almost 95% of grass pollen-allergic patients, and group 5 by 80%. however, until now a comparative of the ability of these allergens to provoke an immune response has not been performed. the objective was to study the immunogenicity of the major allergens phl p 1 and phl p 5, by analyzing the ability of the recombinant forms (rphl p 1 and rphl p 5a) to induce a humoral immune response. method: five mice were immunized with the same amount of each recombinant protein: rphl p 1 and rphl p 5a (indoor biotechnologies) (60 μg plus two boosters of 30 μg). the specific igg antibodies produced by each mouse were tested against the recombinant proteins by direct elisa and the title of each of them was determined by optical density (o.d.). additionally, the recognition of both allergens in native and depigmented-polymerized (dpg-pol) extracts of p. pratense was studied by direct elisa using these generated antibodies. results: preimmune sera were negative. all mice produced antibodies against the corresponding recombinant protein. the immune response (sigg) was statistically significant higher in mice immunized with rphl p 5 than in those immunized with rphl p 1; it was needed 8 times more rphl p 1 serum than rphl p 5a serum to obtain the same o.d. values. the difference in responses was higher in the group of mice immunized with rphl p 1 than with rphl p 5a. differences in the recognition of phl p1 and phl p 5 in native and depigmented-polymerized extracts of phleum pratense was also observed. it was necessary 8 times more rphl p 1 serum to produce the same signal than rphl p 5a serum in native extract and it was necessary 4 times more rphl p 1 serum to produce the same signal than rphl p 5a serum in dpg-pol extract. conclusion: rphl p 5a is more immunogenic than rphl p 1, which was also probed with native and dpg-pol extracts. background: glioblastoma (gbm) is an incurable primary malignant brain tumour with a median life span of less than 15 months despite multimodal treatments. therefore, there is a serious need for the development of innovative medications. several epidemiological studies underlined an inverse correlation between pre-existing igemediated allergy and gbm risk, where having such an allergy decreased the odds of developing gbm by 20 to 40%. we aim to delineate the intrinsic immuno-biological and molecular mechanisms that can be responsible for these correlations, based on the hypothesis that allergies may promote a state of increased immuno-surveillance in the brain through the presence of immunological factors such as immunoglobulins, cytokines and cells involved in th2-driven allergic reactions. we consider that as the major immune cell type of the brain, microglia should be implicated in this beneficial association and may favour the elimination of the nascent tumour in brain parenchyma in an allergic context. we implemented a long term allergic airway inflammation by repeated nasal instillation of house dust mite (hdm) extract in a syngeneic orthotropic mouse model of gbm. we followed animal survival and the tumour growth by mri. in addition, we purified microglia from allergic vs non-allergic mice in order to assess their cytotoxic function against the gbm cell line ex vivo and their secretory capacities. finally, we investigated immunoglobulin reactivity against gbm antigens in the context of allergic reactions by reverse phase protein array (rppa). we demonstrated an increase of the animal survival that was correlated with a delayed tumour engraftment and a reduced tumour growth. these phenotypes were associated with functional modification of microglia from sensitized mice. indeed, these microglia showed a rise in the production of il-6 and tnf-a as well as an increase in cytotoxic functions against a gbm cell line ex vivo. in parallel, we observed an increase in serum igg1 reactivity against gbm antigens in mice sensitized with hdm compared to control mice. results: in 23 patients (48%) with cvid we recorded at least one temporary platelet count decrease below 150 × 10 9 /l compared to only 1 patient (10%) with xla (p = 0.024). more importantly in 18 patients (38%) with cvid this decrease was observed in a period longer than 6 months compared to 1 patient (10%) with xla (p = 0.077). in 10 patients (21%) with cvid we recorded at least one temporary platelet count decrease below 100 × 10 9 /l and only in 3 patients (6.5%) with cvid this decrease was observed in a period longer than 6 months. we did not record any platelet count decrease bellow 100 × 10 9 /l in patients with xla however the difference with cvid did not reach statistical significance. no thrombocyte count decrease bellow 50 × 10 9 /l was observed in either group. none of patients required immunosuppressive treatment for immune thrombocytopenia (itp). conclusion: although the statistical significance was documented only in temporary platelet count decrease below 150 × 10 9 /l it is obvious that numbers of thrombocytes commonly fluctuate in some patients with cvid. the mechanism leading to these temporary decreases is unclear. monitoring of complete blood count is a basic follow-up investigation in patients with cvid. introduction: wegener's granulomatosis (wg) is a systemic disease that may affect all organs, most frequently the ears, noses, throats, sinuses, lungs and kidneys. it is a rare autoimmune disease, also called granulomatosis with polyangiitis, and characterized by necrotizing granulomatous inflammation in small and medium sized blood vessels. anti-neutrophil cytoplasmic antibody against to proteinase 3(c-anca) is thought to be responsible for autoimmune inflammation. the coexistence of wg and common variable immunodeficiency (cvid) is extremely rare. in this report, we describe a patient with wg and cvid who was treated with immunosuppressive drugs and intravenous immunoglobin concomitantly. case report: a twenty-four-year-old male patient was referred to our clinic for immunological evaluation due to recurrent infections, fever of unknown origin and neutropenia. the patient had been diagnosed with wg and taking immunosuppressive therapy for three years. he had chronic renal failure due to wg and had also been on peritoneal dialysis for three years. serum igg, iga levels, peripheral blood cd19 + b cell percentage and absolute count of the patients were found to be low according to reference limits. he was diagnosed with cvid after excluding secondary reasons for hypogammaglobulinemia and he started to receive 600 mg/kg intravenous immunoglobulin (ivig) therapy once in a month. also, the treatment that consists of mycophenolate mofetil (mmf) and glucocorticoids was continued to decrease c-anca levels in serum. he has been accepted as a candidate for kidney transplantation, and prepare for this purpose. discussion: the management of the patient with cvid and wg may be complicated. it is considerably difficult and needs competency and courage. moreover, the cases similar to ours, are extremely rare. therefore, the authors should share their own experiences on cvid and discuss them by comparing the data obtained from other cases. background: leukocyte adhesion deficiencies (lads) are a group of three genetic disorders leading to defective leukocyte adhesion to the endothelium and as a consequence decreased leukocyte recruitment and immune defense. lad-i is caused by mutations in the gene encoding the ß2-integrin cd18 on chromosome 21.lad-iii is a rare primary immunodeficiency syndrome, characterized by homozygous mutations in the kindlin-3 gene (official symbol fermt3). we have aimed to evaluate our patients who were followed up with lad for the last 15 years, retrospectively. method: all data of the cases were obtained from the file records of age at diagnosis. results: seven patients from separate 6 families were included in the study. four patients were lad-iii and 3 patients were lad-i. the female to male rate was 2/5. the age of diagnosis is ranged from 16 days to 4 years. the median umbilical cord detachment was 21 days (7 -53 days groups: up to 6 times (6 people) and from 6 to 12 times (6 people). 10 healthy donors were examined as a control. flow cytofluorometry method was used to study peripheral blood and assess the parameters of innate and adaptive immunity results: it was found that at a frequency of edema up to 6 times a year there are changes in the t-system of adaptive immunity, which are shown by a decrease in the expression of late activation markers (cd3 + hladr+ 3.46 ± 0.60%, in control 8.04 ± 0.14%), an increase in the number of cd3 + cd8 + cytotoxic lymphocytes (0.60 ± 0.17x10 9 /l, in control 0.39 ± 0.01x10 9 /l) and as an increase in their functional activity (cd8 + gr+ 0.53 ± 0.02x10 9 /l, in control 0.16 ± 0.01x10 9 /l). the nature of disorders of cellular factors of the innate immunity is manifested by decrease in the adaptive resources of neutrophils (kstnbt 1.62 ± 0.13u.e., in control 2.15 ± 0.02u.e.). patients with hae with a frequency of edema up to 12 times a year, we observed the disorders of the humoral link of adaptive immunity, which consist in an increase in the number of circulating b lymphocytes (0.27 ± 0.11x10 9 /l, in control 0.11 ± 0.01x10 9 /l). in addition, with the strengthening of the hae clinic, changes in the system of innate immunity progressed very fast and consisted in increasing the amount (cd16 + 0.37 ± 0.05x10 9 /l, in control 0.21 ± 0.01x10 9 /l), and functional activity (cd16 + gr+ 0.32 ± 0.16x10 9 /l, in control 0.14 ± 0.0210 9 /l) of natural killer cells results: we included 261 children, mostly males (54%), aged between 1 month and 16 years. 37.5% of patients (n = 98/261) showed abnormal absolute results of lymphocyte count for age. we found more patients evaluated in the age group of 2 to 5 years (31.8%), followed by 5-10 years (24.5%), lymphopenia was found in 15.4% of patients. b lymphocyte deficiency was the most common pattern (37%) followed, in decreasing order, by low cd4, t cd3, tcd8 and nk. many patients have more than one affected population (12.6%) . some patients were affected in all three series (4.2%). the cd4 / cd8 ratio decreased in 28.7% of the patients. the majority of the children were males between the ages of 1 month and 16 years. 37.5% of patients showed abnormal absolute lymphocyte count for age. b-cell deficiency was the most common pattern followed, in decreasing order, by low cd4, t cd3, tcd8 and nk. many patients have more than one affected population. 0847 | indicators of the humoral immunity in the mechanical jaundice of benign genesis the aim of the investigation was to study the indices of humoral immunity in patients with benign mj, depending on the level of bilirubin. method: 62 patients with mj and 125 practically healthy volunteers were examined. patients with a level of bilirubin less than 60 μmol / l -9, with a bilirubin level of 60-200 μmol / l -37 and with a bilirubin level of more than 200 μmol / l -16 patients. the concentration of immunoglobulin classes a, m, e and g in serum was determined by enzyme immunoassay. the statistical significance of the differences was determined using the ranked mann-whitney test. the critical level of significance in checking statistical hypotheses was assumed to be p < 0.05. results: of the contacted dermatologists, 136 participated (53 women, 83 men; mean age 53.2 ± 8.5) which results in a response rate of 27.3%. the guideline compliant prescription rate of biologicals in patients with csu was 6.9%. the most prevalent barriers in the prescription were the high cost of the treatment (64.7%), low reimbursement for doctors (62.5%) and the fear of a recourse claim (52.9%). however, a lack of evidence or an insufficient efficiency were not concase report: eosinophil associated gastrointestinal disorders (egids) including eosinophilic colitis are commonly associated with atopy. aeroallergen sensitization may accompany food allergy in these patients. a case with eosinophilic colitis responsive to anti-ige monoclonal antibody (omalizumab) treatment is presented. an eleven-year-old boy had bloody diarrhea lasting nearly one month in autumn for last 3 years. this year diarrhea lasted more than 3 months. colonoscopic biopsy revealed lymphoplasmacytic inflammatory cells including eosinophils leading to a diagnosis of ulcerative colitis. corticosteroid and mesalazine treatment was started with a good clinical response. recurrence of diarrhea during corticosteroid dose reduction suggested corticosteroid dependent ulcerative colitis. eosinophilic/allergic colitis was an alternative diagnosis when seasonal recurrence, lack of weight loss, eosinophils in biopsy and high serum ige level were considered. colonoscopy done after cessation of therapy for one month, revealed exudative ulcerous lesions, lacerations, loss of haustration compatible with colitis (inflammatory/allergic?). presence of significant mucosa associated lymphoid tissue in biopsy supported any inflammatory, reactive process. he had recurrent bronchiolitis until age six and allergic rhinitis in spring for three years. total ige and mix aeroallergen specific ige were high (518 iu/ml, 56.1 kua/l), absolute eosinophil count was normal (210/mm3). food skin prick and patch tests were negative. he had positive skin reactions with dermatophagoides, grass and olea pollens (induration diameter: 9, 10, 6 mm, respectively). pulmonary function test was normal. he was considered as eosinophilic/allergic colitis and omalizumab was started according to manufacturer's dosing table (300 mg/ 2 weeks). rectal bleeding decreased after first dose and ceased after the second dose. early colonoscopy examination after 3rd month of therapy showed that exudations disappeared and haustrations became evident. microscopy revealed mild nonspecific colitis. few patients with eosinophilic colitis improved with omalizumab were reported before. ige-mediated processes are responsible from eosinophilic inflammation in egids, making anti-ige therapy as a promising treatment option. 0859 | design of liposomal carriers modified by glycoconjugates for liver cell delivery of nucleic acids used. the surface of liposomal nanoparticles can be modified to increase the selectivity of intracellular delivery. it is well known that asialoglycoprotein receptors of hepatocytes have a strong affinity to galactose carbohydrate. therefore, the aim of this study was to assess the effect of the modification of the liposome surface by glycoconjugates on the selectivity of intracellular transport of nucleic acids into the liver cells. method: liposomes based on ornornglu(c 16 ) 2 were chosen previously as the effective nucleic acid delivery system. we modified liposomes with novel lactose-based derivatives. every of four glycoconjugates was added to ornornglu(c 16 ) 2 in an amount of 5, 10 and 15%. as a result, 12 variants of modified liposomes were obtained. to determine the cytotoxicity, an mtt test was used. using luciferase test, the selectivity of penetration was evaluated on nonspecific 293t (human embryonic kidney) and specific hepg2 (human liver cells) cell lines. results: modified liposomal compositions ornornglu(c 16 ) 2 -4 + lacc 16 (5%) and ornornglu(c 16 ) 2 -4 + lacggg16 (15%) had the lowest cytotoxicity similar to that for unmodified ornornglu(c 16 ) 2 . the ic 50 , calculated based on the data of mtt test, was 0.14 and 0.26, vs. 0.16 mg/ml, respectively. ornornglu(c 16 ) 2 -4 + lacggg16 (15%) showed a 1.5-fold increase in transfection activity on the nonspecific 293t cells, compared to unmodified ornornglu(c 16 ) 2 , whereas the modification of ornornglu(c 16 ) 2 -4 + lacc16 (5%) resulted in a 6-fold decrease in transfection activity. however, the ability of these variants to penetrate the specific liver hepg2 cell was significantly higher by 15 and 25 times, respectively, than for unmodified ornornglu(c 16 ) 2 . results: the greatest inhibitory effect of sbfhd was observed in mdm infected with hiv-1 bal: 90% and 50% suppression of hiv replication was achieved at concentrations of 1.5 μg/ml and 0.4 μg/ ml, respectively. the activity in pbmc and dc was less pronounced (the respective ic90 values were 5.8 μg/ml and 19.7 μg/ml). studies in endometrial hec-1a cells demonstrated that sbfhd suppressed cd4-independent entry of hiv-1 (10 3 tcid 50 /ml) by 33%, 54%, and 98%, respectively, at 1, 10, and 100 μg/ml. the effect was also observed after increasing the dose of the virus. at 10 4 tcid 50 /ml, sbfhd suppressed hiv infection by 45% (10 μg/ml) and 97% (100 μg/ml). the cytotoxicity of sbfhd in this system was low. similar results were obtained with colorectal caco-2 cells. sbfhd exhibited no spermicidal activity at concentrations of up to 3 mg/ml. combining within a single microbicide two agents that target distinct steps of hiv life cycle will maximize its efficacy (via synergistic effects and/or interference with multiple stages of the transmission). we therefore explored the synergistic potential of combinations of sbfhd and azt, the classical nucleoside rt inhibitor. in these experiments, 50% suppression of hiv infection was reached at concentrations of sbfhd and azt, which were significantly lower than the respective ic50 values of each component (determined in parallel experiments). the synergistic effect was most pronounced for the combination of 0.1 μg/ml sbfhd (which is 60 times less than the ic50) and 0.16 nm azt (which is 45 times less than its ic50). cd80 expression was increased after the co-culture with reishi, shiitake and boletus mushrooms (c -5.6(1.4-9.2)%; pma -60.5 (27.2-70.9)%; )%; shiitake -9.6(4.3-15.5)%; boletus -16.6 (14.0-24.0)%). method: the study included 38 men (mean age 34 ± 5.9 years) before and immediately after staying in countries with a hot climate. results: the development of lymphopenia observed in the first week of observation. this was accompanied by a decrease in the number cd3 + lymphocytes expressing the markers of late activation (cd3 + hladr+ 4.8 ± 1.09x10 9 /л и 3.2 ± 2.04x10 9 /л). revealed significant decrease of cd4 + cd25 + foxp3 + regulatory cells in the first week after returning from the area of adverse climatic conditions, as well as a significant sustained decrease in the number cd3 + cd8 + hladr+(p < 0.05). change of the effector link of innate immunity was determined in significant reliable decrease in relative (cd16 + 13.2 ± 2% and 4.6 ± 2.1%, respectively, p < 0.05) and absolute (cd16 + 0.3 ± 0.02 x109/l and 0.1 ± 0.03%, respectively, p < 0.05) in the number of a population of natural killer cells in the first week of observation. in the context of acute stress marked a significant increase in relative and absolute numbers of b lymphocytes (8 ± 1.16% (0.15 ± 0.04 × 109/l) before a trip to countries with a hot climate and 19 ± 3.1% (0.4 ± 0.07 × 109/l) in the first week after returning, p < 0.05). the activity is the production of antibodies was not changed. (ast) which is the intramuscular injection of patients own serum, is a promising therapy with a substantial efficiency on ciu patients. in this study we aim to assess the efficacy of ast on chronic urticaria patients by dlqi questionnaire. method: this was a single-blind randomized clinical trial which evaluated the efficacy of autologous serum therapy compared to oral antihistamines in patients with ciu. 49 ciu patients received the ast. every session 5 cc of each patient's blood was centrifuged at the speed of 2000 rpm for 10 minutes and 2.5 cc of the serum was injected intramuscular into the patient's deltoid muscle weekly for 9 weeks. the control group consisted of 51 ciu patients took 10 mg of cetirizine daily for 9 weeks. patients answered the dlqi questionnaire at the first session of treatment as baseline and 7 weeks after the last session(week 16) as response to treatment. the mean baseline score of dlqi for ast group was conclusion: pharmacotherapeutic and inpatient costs for patients with prevalent ar and asthma were lower in those prescribed ait than in those not prescribed ait in all years, both with and without including the cost of ait itself. this indicates that treatment with ait is associated with lower cost burden for health services. background: immunotherapy with peptides rather than conventional whole allergens is being developed to improve the benefit/risk balance of subcutaneous immunotherapy (scit). lolium perenne peptides (lpp) demonstrated reduced allergenicity following ex-vivo analyses, allowing higher doses to be given over a shorter period to improve treatment adherence and compliance. such treatment resulted in significant reduction in symptoms and rescue medication intake during the grass pollen season. here we report the safety of lpp immunotherapy in adults. background: a new allergoid from alternaria alternata was characterized to determine its reduced allergenicity in vitro. the objective of this study was to determine the skin response to the allergoid and to evaluate the clinical tolerance of the immunotherapy with the allergoid product using a rush schedule. method: to assess the skin response (sr) two groups of patients were included: group 1 with patients sensitized to a. alternata and with respiratory disease caused by this mold; group 2 (control) with patients sensitized to others allergens and non-atopic patients. the sr was determined by spt using three concentrations of the allergoid: p1 (lowest concentration), p2 (four times higher than p1) and p3 (estimated to obtain a wheal area similar to histamine 10 mg/ml). in spt was also used a native extract of a. alternata (n) and histamine 10 mg/ml (h). all products were tested in duplicate in all patients and the sr was evaluated by comparing the median of the wheal area produced by different products. to evaluate the clinical tolerance to immunotherapy the patients of group 1 were treated with the allergoid product using a rush schedule consisting in a dose of 0.2 + 0.3 ml the first day and 0.5 ml after one month (maintenance dose). the clinical tolerance was determined as the percentage of adverse reactions (ar) to the treatment and the classification of ar was established according to eaaci. the number of patients included to evaluate the sr was 46 (group 1: 25; group 2: 21, 16 atopic and 5 non-atopic) with an average age of 34.8 (range . the spt data from group 1 were expressed as median and interquartile range of wheal area (mm 2 ): h: 19.58 (15.3-25.2); n: 22.71 (11.1-33.1); p1: 0.99 (0-5.7); p2: 6.44 (0-12.5); p3: 19.78 (12.0-30.4 ). it was determined that sr of allergoid was reduced in 87% respect to the native. the products n, p1, p2 and p3 did not produce any response in patients of group 2. to evaluate clinical tolerance, 21 patients of group 1 were treated with the allergoid product with a rush schedule and only two ar were registered (3.2% of doses). these were retarded local reactions with a wheal diameter higher than 5 cm. no systemic reactions were registered and all patients continued the treatment. the allergoid from a. alternata produces a significant reduced response to spt due to its reduced allergenicity. the treatment with an allergoid product in a rush schedule is safety and clinically well tolerated. background: in our study we aim to determine the more effective, the total cost of 3 years of patients using scit was 15665 tl per person whereas the total cost of 3 years of patients using slit was 15271 tl per person. when we compare the total cost data of both groups, we found that they are close to each other. while the greatest portion of the cost data of patients with scit treatment was direct costs associated with the treatment itself (72%); the remaining part of the total cost was indirect (16%) with non-medical expenses such as transportation (12%). in the slit group, direct costs including drug expenditures have a larger percentage (92%) and it was significantly more costly compared to the direct costs of the scit group (72%). transportation costs were found to be more costly in the scit group (12%) when compared to the slit group (4%). similarly loss of parent work days in the scit group(%16) was found to be significantly more expensive compared with slit group (4%). our study results show that slit is a similar treatment for clinically and laboratorially and has a similar efficacy to scit to reduce the patients' complaints and to the need for medication. for cost-effectiveness however medicines for treatment of scit are less costly; when long term total treatment costs are calculated slit and scit treatment are economically close treatments. the protein content of the new acd was 182.28 μg/mg and the protein profile in sds-page and sec-hplc confirmed the presence of proteins with high molecular weight and the absence of smaller proteins. the content of free lysine in acd, involved in glutaraldehyde modification, was reduced in 91.96% respect to ncd and it can be considered as the polymerization degree. regarding to the allergenic profile, through elisa inhibition was determined a reduction of 18 times in the capacity to bind ige of the proteins in acd respect to ncd, whilst the igg binding capacity was maintained. in immunoblot there was no reaction of acd proteins to specific ige from sera. the analysis by peptide footprint determined the presence of fel d 1 and others allergens in acd. the content of major allergen fel d 1 in acd was determined as 11.9 μg/mg. the new developed and characterized allergoid from cat dander has an excellent safety profile and will allow a safer immunotherapy to treat the allergy to felis domesticus. results: the protein content of the new aaa was 65.6 μg/mg and the protein profile in sds-page and sec-hplc confirmed the presence of proteins with high molecular weight and the absence of smaller proteins. the content of free lysine in aaa, involved in glutaraldehyde modification, was reduced more than 85% respect to naa and it can be considered as the polymerization degree. regarding to the allergenic profile, in immunoblot there was no reaction of aaa proteins to specific ige from sera and by elisa inhibition was determined a reduction of 91% in the capacity to bind ige of the proteins in aaa respect to naa. the igg binding capacity in aaa was maintained. the analysis by peptide footprint determined the presence of alt a 1 and others allergens in aaa. the content of major allergen alt a 1 in aaa was determined as 2.4 μg/mg. a. alternata shows an excellent safety profile and allows a safer immunotherapy to treat the allergy to this mold. she was an otherwise healthy woman: she took no drugs and she did not have any remarkable concomitant diseases. the distribution and appearance of the remaining body hair was normal and the hormonal level profiles (lh, fsh, estrogens, progesterone and testosterone) did not show any significant alteration according to her age. a 30 year old woman with allergic rhinitis underwent sq glutaraldehyde-modified ait to house dust mites (d pteronyssinus and g domesticus) without any incidences and complete tolerance to maintenance dose without local reactions during a 5 year period. two years after ait discontinuation, patient first experienced a local urticarial reaction with multiple hives at previous sq ait injection sites 40 minutes after 600 mg of ibuprofen intake. these symptoms recurred at least in seven occasions when patient was exposed to ibuprofen (in five) and metamizol (in two). results: case 1: dermatologist diagnoses localized hypertrichosis. case 2: a single blind, placebo controlled oral challenge (sbpcoc) with ibuprofen 600 mg was performed and elicited multiples hives in the circumscribed area in the arm where ait was conducted. subsequently, sbpcoc with aspirin was carried out showing the same reaction although a controlled challenge with celecoxib was negative. conclusion: local hypertrichosis is a very rare injection-disease associated with injected allergen vaccine treatment. we also firstly described a recall urticaria phenomenon after allergen immunotherapy which has been only elicited after different nsaids intake. results: there were included 47 patients, in five spanish hospitals. following aria 2010 guidelines, 95.7% of patients were diagnosed of persistent moderate/severe rhinitis. the mean age was 37.7 ± 11.8 years, being 59.6% female. moreover, 57.4% of the patients had concomitant mild/moderated asthma. the period between the diagnosis of rhino-conjunctivitis and the informed consent signing was 9.8 ± 7.5 years. according to international 2006 guidelines, eight systemic reactions were registered, representing 1.9% of the administered doses: five reactions grade 0, (described as nonspecific ocular pruritus, nasal herpes, general discomfort, localized non-specific pruritus plus nausea and non-specific pruritus in throat), a grade i reaction described as rhinoconjunctivitis and two reactions grade ii, registered as generalized urticaria and asthma. all reactions were classified of mild or moderate intensity and only two required symptomatic treatment. there were five clinically significant delayed local reactions, which were higher than 10 cm or involved modifications in next dose. regarding efficacy parameters, immunoglobulin titers between baseline and final visit according to specific igg and igg4 significantly increased. cutaneous reactivity also decreased significantly in the dose response skin prick test. results: 47 patients were included, 24 to accelerated and 23 to polymerized cluster group schedules. according to aria criteria, 89.4% of patients presented persistent moderate/severe rhinitis. the mean age was 31.1 ± 9.9 years, being 40.4% male. moreover, 61.7% had concomitant mild/moderated asthma. immunoglobulin titers method: the quantification of total proteins in the products was carried out by means of a colorimetric technique using the bradford reagent (sigma-aldrich™, us) in accordance with the manufacturer's instructions. the absorbances of each standard and samples were obtained in a scinco™ s-3100 spectrophotometer (seoul, korea) at 595 nm. all samples were analyzed in duplicate. the electrophoretic profile of the proteins in the tested allergens was obtained according to the procedure described by laemmli, under denaturing conditions in a polyacrylamide gel at 12.5% concentration and stained in silver. in each lane approximately 30 μg of total proteins were applied. commercial extracts of the main allergens marketed in mexico were obtained, rossel ® , alk ® , alerquin ® , alergomex ® , allerstan ® , ipi asac ® ; and they were assigned randomly with the numbers 1, 2, 3, 4, 5 and 6. results: the following protein concentrations were found in the various extracts analyzed: see table 1 conclusion: differences were found in the protein profiles anabackground: a new allergoid from cat dander was developed and characterized to determine its reduced allergenicity in a 95% and the maintenance of igg binding capacity. the objective of this study was to develop an immunogenicity assay in mice with the new allergoid and a native extract from cat dander. the study included 24 female balb/c mice separated in three groups of 8 mice each: group 1, immunized with a mold allergen extract (control); group 2, immunized with a native extract from cat dander with a fel d 1 content of 0.525 μg per dose; group 3, immunized with the new allergoid from cat dander with a fel d 1 content of 2.36 μg per dose. all mice were immunized four times by subcutaneous injections with a volume corresponding to 1/10 of the recommended human maintenance dose with an interval between injections of 2 weeks. one week after the last injection the mice were sacrificed and the serum was obtained. to determine the specific antibody title indirect elisa were performed using a cat dander extract as antigen, sera from mice as primary antibody and antimouse igg or igg1 as secondary antibody. elisa assays were performed using serial dilutions of sera or a simple dilution by duplicate to determine the specific antibody title as arbitrary units/ml (au/ml). the data were analyzed by one-way anova and tukey hsd test to compare the averages of specific antibodies in each group. results: the immunization with both the native extract and the allergoid from cat dander produces specific igg and igg1. regarding to igg, a higher title was observed in group 3 respect to group 2 in a curve obtained after elisa with serial dilutions of sera. the specific igg title obtained in terms of au/ml was 18.6 ± 2.6 in group 1, 105.0 ± 15.0 in group 2 and 139.9 ± 16.6 in group 3. concerning to igg1 the au/ml obtained was 5.7 ± 0.4 in group 1, 73.5 ± 16.8 in group 2 and 97.5 ± 18.8 in group 3. the increase of specific igg or igg1 in mice from group 3 respect to mice from group 2 and control group was statistically significant (p ˂ 0.01). the safety profile of the allergoid from cat dander allows a treatment with higher dose of allergens to produce a greater response to immunotherapy to induce formation of specific this was an open, multicenter clinical trial, in patients aged between 18 to 60 years with rhinoconjunctivitis with or without concomitant mild asthma sensitized to house dust mites (hdm). the aim was to evaluate the safety and tolerability of the vaccine. secondary endpoints included were: changes in immunoglobulin levels (specific ige, igg and igg4) versus d. pteronyssinus and d. farinae and changes in cutaneous reactivity. patients were under study treatment for 17 weeks: five for the induction phase (weekly injections) and 12 for the maintenance phase (monthly injections). results: 42 patients were included. there were 6 withdrawals from the trial; no one was related to treatment. the patients mean age was 33.6 years, being 50% female. 71.4% were diagnosed of persistent moderate/severe rhinitis according to aria guidelines and 31.0% presented concomitant mild asthma. regarding to safety results, 22 systemic adverse reactions were registered which corresponded to 7.9% from a total of 277 administered doses. the most of systemic reactions were grade i, (5.4%) described as rhinitis or urticaria, grade 0 or nonspecific (2.2%) and 1 reaction (0.3%), was grade ii. all of them were mild or moderate and only 4 needed treatment. among local reactions, 21 (7.6%) were clinically relevant late local reactions, meaning a wheal at injection site >10 cm and /or requiring a dose readjustment in the next administration; 6 (2.2%) were clinically relevant immediate local reactions meaning a wheal >5 cm. concerning the efficacy parameters, cutaneous reactivity at the final visit versus baseline was, in average, significantly decreased, and specific titers of igg and igg4 against tested hdm increased significantly at final visit. 162 patients completed the study. mean values in rqlq questionnaire (total score) decreased from 2.56 to 1.24 points (51.6% score reduction) in final visit, reflecting a statistically significant improvement (p < 0.01). annual episodes of rhinoconjunctivitis decreased from 13.7 to 9.7 (p < 0.01). 43.8% of patients improved from persistent to intermittent rhinoconjunctivitis (p < 0.01) and 46.9% from moderate/severe to mild intensity (aria) (p < 0.01). moreover, 16 .1% of asthmatic patients at baseline, did not have any bronchial symptoms after 1-year treatment (p < 0.01). mean value of treatment satisfaction was 7.2 (sd=1.8) and 7.2 (sd=1.7) for patients and physicians respectively. 0884 | evaluation of safety and tolerability of "allergovac poliplus" in polysensitized patients with allergic rhinitis-rhinoconjunctivitis with or without asthma: an observational prospective study (apolo) background: the objetive of this study was the safety and tolerance assessment of "allergovac poliplus" scit treatment, with 2 allergen combination-mixtures in polysensitized patients, as well as the evaluation of the clinical improvement and patients' satisfaction after treatment. method: this is a prospective observational clinical study. allergovac poliplus treatment is being administered in a "1-day" or in an abbreviated schedule. polysensitized patients (to pollens or mites), with rhinitis or rhinoconjunctivitis, with or without asthma, and between 5-60 years have been included. all adverse events are being recorded. visual analog scales (vass) are being used to evaluate clinical improvement, tolerance and satisfaction after treatment (10 months). results: a total of 147 patients have been included, with an averresults: in all groups prevailed severe forms of the disease and the phenotype of frequent exacerbations. groups were comparable in age composition and structure of severity. observations in the group of vaccinated pcv13 continue the dynamics of decreased dyspnea up to 1.66 (1.11;2.21) results: of a total of 631 pts under scait, 110 were excluded due to data unavailability, and 521 included (♀283 (54%), mean age 32 ± 13 years (minutes: 7 max 73 md30), age range [18-30] being most prevalent (40%). the most frequent diagnosis was rhinitis/rhinosinusitis (97%), followed by asthma (43%), diagnosis coexisting in abstracts | 479 214 pts (41%). other diagnosis such as conjunctivitis (24%), atopic eczema (15%) and food allergy (9%) were also found. mite sensitization occurred in 422 patients (81%) of which 199 (47%) were monosensitized. the pollen sensitization was verified in 288 (55%) with 88 monosensitized pts (31%). the double sensitization mitespollens was displayed in 189 (36%). sensitization to epithelia and fungi occurred respectively in 100 (19%) and 42 pts (8%). it was found that 20 pts (4%) presented sensitization to the 4 groups of allergens (mites, pollens, fungi, dander). an average of 58 ± 23 pts started this treatment per year. prescription included 10 laboratories with the following %: a-39.6; b-29.3; c-13.2; d-5.4; e-4.7; f-4.2; g-2.5; h-0.9; i-0.1; j-0.1. option for extract of physical modification (5%), physical-chemical (16%) and chemical (79%). table 1 shows the frequency of distribution of scait composition. conclusion: in this population sensitization to mites was predominant being the most prescribed scait followed thru sensitization to grasses with the respective scait. the majority of the population was polysensitized. however, in composition preference the choice of 1 group of allergens prevailed and only 5% had more than one sort of pollen and 4% pollen+mites. polysensitization is a reality, nonetheless the choice of ait composition should be guided thru scientific criteria and not through the availability of mixtures encouraged by laboratories. background: allergen immunotherapy (ait) has been proven to be an effective treatment of allergic diseases in numerous studies. however, its use in seniors remains limited and questionable, due to common comorbidities and limited evidence of efficacy and safety of ait in aging population. the aim of presented study was to assess the safety of ait in patients over 55 years of age undergoing subcutaneous immunotherapy (scit) and analyze the potential risk factors of adverse reactions in this population, compared to younger adults. we followed subcutaneous immunotherapy in a group of 1302 patients treated in the outpatient clinic of medical university of lodz, of whom 163 were aged 55 and older (118 between the age of 55-60, 31 aged 61-65 and 14 patients above the age of 65). we recorded detailed information of each administration and corresponding adverse reactions over the period of 2 years. we compiled results of our observations with patients' medical records to compile a database, which we then analyzed using statistical software. method: a total of 46 cases with seasonal allergic rhinitis undergoing pre-seasonal immunotherapy and 28 cases followed with conventional drug treatment were included in the study. immunotherapy and control groups were divided into monosensitized (only pollen) and polysensitized (at least 1 additional allergen except pollens) patient groups according to skin prick test reactivity. all patients were followed between march-september 2013 with symptom and medication scores, and visual analogue scale (vas). the quality of life was assessed using the mini-rqlq questionnaire. phleum pratense (phl p) specific ige and specific igg4 (uni-cap 100, phadia) measurements were performed before and after 7 weeks of immunotherapy in all patients. gramineae pollens were counted during the grass pollen seasons. results: mean age was 34.9 ± 10.6 and 34.2 ± 12 years, female/ male ratio was 29/17 and 17/11, the number of monosensitized/polysensitized patients were 37/9 and 20/8 in immunotherapy and control groups, respectively. in the immunotherapy group, june-july symptom scores, may-june-july-august vas scores and june combined symptom-medication scores were lower than the control group (p = 0.005). furthermore, improvements in activities-practical problems and other quality of life scores were significantly different between two groups (p < 0.05). in immunotherapy group, phl p specific ige and phl p specific igg4 levels measured after immunotherapy were significantly higher compared to those before immunotherapy (p < 0.001, p < 0.001, respectively). phl p specific igg4 levels measured after immunotherapy were also significantly higher in the immunotherapy group than in the control group (p < 0.001). there was no difference in terms of clinical and immunologic parameters in monosensitized and polysensitized patients (p > 0.05). conclusion: clinical improvement with pre-seasonal allergoid immunotherapy is accompanied by an important increase in specific igg4 blocking antibodies despite short-term injections. our findings show that pre-seasonal allergoid immunotherapy has similar clinical efficacy and b cell response in polysensitized subjects compared to monosensitized patients. 0891 | the safety trial of sequential sublingual immunotherapy with japanese cedar droplet and house dust mite tablet matsuoka t 1 ; kuroda y 1 ; igarashi s 1 ; fukano c 2 ; natsui k 2 ; ohashi-doi k 2 ; masuyama k 1 1 university of yamanashi, yamanashi, yamanashi, japan; 2 torii pharmaceutical co. ltd., tokyo, japan background: sublingual immunotherapy (slit) is recognized as the only treatment option with the potential to provide long-term posttreatment benefits. in japan, the prevalence of japanese cedar (jc) pollinosis is very high, about 30% of the population, of which the majority are co-sensitized to hdm. slit is now well established, safe and convenient treatment form for allergic disease, and recently, jc slit-droplet and hdm slit-tablet products were approved in japan for treatment of jc and hdm induced allergic rhinitis, respectively. however, the safety of sequential jc slit-droplet and hdm slittablet has not yet been investigated. therefore, we investigated the safety trial on slit combined with jc droplet and hdm tablet in allergic patients. method: eleven subjects with jc pollinosis and hdm rhinitis were enrolled. patients were treated once-daily with jc slit-drops for 4 weeks, followed by 24 weeks of sequential slit treatment where the jc slit-drops and the hdm slit-tablets were administered daily with a 5 minute interval (1st: jc-slit drops, 2nd: hdm slit-tablet). the primary endpoint was the frequency and severity of adverse events (aes) during sequential slit by common terminology criteria for adverse events (ctcae) v4.0 and slit grading system. serum antibodies were measured as the secondary endpoint. results: eleven patients were recruited. aes after jc slit-drops administration were found in 9 patients out of 11 cases (82%). aes after sequential slit were found in 8 patients out of 10 cases (80%). all aes were graded 1 or 2. no severe aes were observed during the study period. the levels of jc-and hdm-specific ige and igg4 in serum were increased during treatment. conclusion: sequential-administration of jc slit-drops and hdm slit-tablets was well tolerated by patients suffering from both jc pollinosis and hdm rhinitis. background: according to the ema guideline on the clinical development of products for specific immunotherapy products should be tested in phase ii at different doses in several study-arms to establish a dose-response relationship for clinical efficacy before confirmatory trials can be initiated. allergen exposure in an aec may be used as primary endpoint. the study was a single-center, randomized, double blind, placebo-controlled, phase ii trial, treatment duration 10 months. 168 grass pollen allergic patients (18-65 years of age) with seasonal rhinitis/rhinoconjunctivitis (arc) with (mild, gina i) or without concomitant asthma were randomized to three different dosages of a liquid phase iii study is in preparation. as part of an effort to prepare the analysis plan using the csms as primary endpoint, the grass pollen data of the european aeroallergen network (ean) was used to identify the window within the grass pollen season (gps) with optimal correlation between the grass pollen counts and the csms. method: ean currently includes information from more than 400 active and 300 historical pollen-monitoring stations in europe including 39 countries. the ean database used for analysis included grass pollen data collected during 2009-2016. the daily allergy symptoms and medication were recorded spontaneously using an app questionnaire on the subject's smart phone. the csms was re-calculated using the ean database, using the recorded symptom scores with estimation of the medication score using similar methods as recently published. the correlation between the daily grass pollen count and the daily csms was analyzed with a mixed effects model accounting for patient-specific correlations and symptom levels. conclusion: these results confirm a statistically significant correlation between grass pollen counts and the csms. importantly, these findings suggest that the optimal window to observe treatment effects after immunotherapy may be a short interval after start of the gps and during the peak gps, due to generally higher csms values. this provides sufficient basis to consider additional sensitivity analyses to evaluate the treatment effect of grass mata mpl scit on the primary csms endpoint during a shortened window after the start of the gps and to consider excluding the overlapping period between the bps and gps from the primary analysis. 0894 | combo-vas as a tool to assess efficacy of allergen immunotherapy ciprandi g 1 ; silvestri m 2 ; olcese r 2 ; tosca ma 2 1 ospedale policlinico san martino, genoa, italy; 2 istituto g. gaslini, genoa, italy background: allergen immunotherapy (ait) is at present the unique cure for respiratory and venom allergy. usually, ait lasts for some years, but its efficacy is longstanding. criteria for assessing ait efficacy are mainly based on symptom severity improvement and saving of symptomatic medications. in this regard, there are different score grading for both measuring symptom severity and drug use. visual analogue scale (vas) is a well-defined and validated method widely used in many diseases, including allergic disorders. vas is a psychometric tool measuring the patient's perception of symptoms, emotions, pain, drug use, etc. recently, it has been published an eaaci position paper concerning the recommendations for the standardization of clinical outcomes used in ait trials for allergic rhinoconjunctivitis, but it is complex. so we would propose a simpler way to measure ait efficacy by vas, in particular a combo-vas based on one vas for symptom and one for medications. results: globally 150 patients were retrospectively evaluated. all of them were treated with a 3-year ait course: 120 were defined as responders and 30 as non-responders. in responders group the combo-vas mean value was 14 (iqr 12-15) at baseline and 4 (iqr 3-6) after ait treatment. in non-responders group combo-vas mean value was 13 at baseline and 11 (iqr 9-12.5) at the end of ait. the difference was significant (p = 0.012). the d combo-vas was −66.67% in responder group and −10% in non-responders group (p < 0.0001). conclusion: combo-vas, i.e. the sum of vas for symptoms and medications, could be an easy and quick tool for assessing ait efficacy and reflects the patient's perception. therefore, it could be very fruitful in clinical practice. 0895 | rapid up-dosing in sublingual specific immunotherapy is safe, well-tolerated and effective in patients suffering from tree pollen allergic rhinitis background: an optimised up-dosing period of specific immunotherapy (sit) is desirable for better patient compliance because a long or complicated up-dosing scheme is sensitive to disruption. the aim of this study was to compare the safety, tolerability and effectiveness of an optimised up-dosing scheme with two preexisting schemes of sublingual sit (slit) in patients under standard medical care. method: this was a prospective, open, active controlled, multi-center non-interventional study in germany and austria to document the treatment of children and adults with allergic rhinoconjunctivitis and/or allergic asthma treated with a slit containing purified, aqueous extracts of birch, alder and hazel pollen. the investigators were free to select an up-dosing scheme for included patients: scheme a consisted of an up-dosing period of up to 12 days at the patient's home using three different solution strengths to reach the maximum dose; ultra-rush scheme b performed only with the highest solution strength at the physician's office within 2 hours, and the optimised scheme c which was initiated at the physician′s office and continued at home using exclusively the highest solution strength within 2 (long-term) or 4 (pre-seasonal) days. data on up-dosing and maintenance treatments were documented by physicians during 5 patient visits and by patient diaries. the study was approved by ethic committees, and all patients or parents gave their informed consent. results: in total, 164 patients aged 3-76 years were included into this study. scheme a was applied by 90 patients, 29 patients decided on regimen b, and 45 patients on the optimised scheme c. conclusion: one-day ur-scit conducted in an outpatient clinic was safe and well-tolerated in patients with ad sensitized to hdm. ur-scit can be a safe and useful option to start a subcutaneous allergen immunotherapy for ad. 0899 | factors affecting on adherence to allergen specific immunotherapy results: among 1162 enrolled patients, 228 (19.6%) patients failed to complete at least 3 years of ait, which were regarded to be nonadherent in this study. univariate analysis revealed that male, younger age group less than 40 years, cluster and ultra-rush schedules, atopic dermatitis, the absence of associated diseases, and follow up of other department were found to be associated with nonadherence to ait. in multivariate analysis, younger age group less than 40 years (or 2.31, 95% ci 1.55-3.45), cluster (2.37, 1.56-3.60) and ultra-rush schedules (6.03, 3.18-11.42) , and absence of follow up of other department (2.11, 1.31-3.40) were independently associated with non-adherence to ait. no association was found in gender, diagnosis of allergic diseases, kind of allergen extracts, and patients' distance from hospital. conclusion: various factors are related with ait non-adherence to interfere the effectiveness of immunotherapy. clinicians need to be aware of the factors associated with non-adherence to ait and consider them when choose to maximize ait adherence. 0900 | cost-effectiveness of allergen immunotherapy to grass in patients with allergic rhino-conjunctivitis and asthma background: allergen immunotherapy (ait) has been shown to reduce symptoms and medication use in subjects with rhino-conjunctivitis and asthma. however, long-term cost effectiveness of this therapy needs to be evaluated. our aim was to assess cost effective of ait, both subcutaneous immunotherapy (scit) and sublingual immunotherapy (slit), vs. pharmacotherapy alone in subjects with rhino-conjunctivitis, with or without allergic asthma, to grass pollens. method: a markov cohort state-transition model with a time horizon of 9 years was used to assess the costs and effects of 3-year ait in adults. relative efficacy of the treatments expressed as standardized mean difference was estimated using an indirect comparison on symptom and medication score extracted from available meta-analyses. the rhinitis symptom utility index was used as a proxy to estimate utility values for symptom score. the societal perspective, through the human capital technique, was used to estimate indirect costs, to represent the scenario of a country with nationalized medicine. data on drug and other medical costs were derived from published sources as well as ait duration and asthma occurrence. additional sensitivity analyses were performed to test the robustness of our results. results: in the base case analysis, using italy clinical practice patients with moderate-to severe allergic rhino-conjunctivitis (ss ranging from 6 to 15 points) and a mean age at entry of 21 years, both scit and slit were associated with increased cost but superior efficacy compared to pharmacotherapy alone. the results were most sensitive to variation in efficacy estimates and ait persistence rates. conclusion: this analysis suggests that ait is cost effective relative to pharmacotherapy alone. scit, despite significantly higher indirect cost burden, seems to be the most cost effective option. the results should be interpreted in the context of the data input and modelling assumption used. 0901 | ielisa as a tool to measure ige binding towards single modified peanut allergens background: immunotherapy has shown to be a potential treatment for food allergies but needs further research to improve safety. modification of peanut allergens to reduce their allergenicity is a promising approach to develop a safe and effective immunotherapy as shown by the successful completion of a first-in-human safety and tolerability study using hal-mpe1 in adult patients with peanut allergy (eudract 2013-004238-13) . in order to assess the impact of modification on individual peanut allergens and to assess its impact on ige binding by individual patient sera, we have developed peanut allergen-specific inhibition elisas. with this methodology we are able to identify patients with residual ige binding to modified peanut allergens. method: ige inhibition elisas (ielisas) were developed and performed to test ige binding towards purified ara h2 and ara h6 and their reduced and alkylated (modified) versions, using the individual responses of single patient sera. results: ara h6-specific ielisas showed that modification of ara h6 results in >95% reduction in ige-binding for all individual sera tested. ara h2-specific ielisas showed that modification of ara h2 also results in >95% reduced ige-binding for most of the sera, but some sera were identified which showed residual, 10%-20% ige binding to mara h 2. in some of the latter sera, the presence of ige binding to a linear hydroxyproline-containing peptide could be confirmed as a possible source for the residual ige binding to mara h2. we have developed a methodology to assess residual ige binding to modified peanut allergens. the sensitivity of the allergen-specific ielisas allowed us to discriminate between patient sera in which ige binding to mara h2 and to mara h6 was virtually completely absent and sera in which 10-20% residual ige binding to ara h2 was observed. the clinical importance of these observations is yet unknown. future clinical studies will need to reveal whether the patient-specific ige binding profiles to individual modified peanut allergens do correlate with the adverse events profile of immunotherapy with modified peanut extract. 0902 | design of a phase ii allergen immunotherapy study to determine the optimally effective and safe dose of subcutaneously administered tyrosine adsorbed modified grass allergen+mpl (mpl) adjuvants for the treatment of allergic rhinoconjunctivitis (arc) due to grass pollen. there is increasing evidence that the effectiveness of allergy immunotherapy to control arc symptoms is related to the cumulative allergen (or allergoid) dose administered. previously, two clinical studies have been conducted using a conjunctival provocation test (cpt) as primary efficacy measure for a similar scit mata mpl product for birch allergy [eudract 2012-004336-28 and 2015-000984-15] . these studies showed a 5.5 fold increase in cumulative dose to achieve~50% increase in efficacy, with a relative reduction in total symptom score (tss) of 32.3% compared to placebo and no safety signals of concern. the shape of the dose response curve was curvilinear, where this high dose almost reached plateau. method: this is a multi-center (~47 clinical study centers across europe), randomized, double-blind, placebo-controlled, parallel-group study in~440 adult patients with moderate to severe seasonal arc with or without mild asthma. a positive cpt is to be achieved at screening and verified prior to randomization. the primary outcome is the post-treatment tss following cpt. a wide range of cumulative dose regimens is used (5100, 14400, 27600 and 35600 su) applied over 6 weekly injections to establish the shape of the dose response to support dose selection for phase iii. the design of the current phase ii grass allergoid scit study will be discussed, including the rational of using 4 cumulative dose regimens and placebo and the pre-selected shapes of the dose response curves. in addition, the number of patients screened and randomized will be presented by country, gender and/or age category and screen failures will be categorized. conclusion: this phase ii study was initiated to establish the dose response of a grass mata mpl scit product, using cpt to measure the effect of a wide range of cumulative dose regimens. the achievement of its aim will be an important milestone in the development of an efficacious and safe state-of-the-art grass scit. conclusion: we observed that the specific nasal challenge with house dust mite generates an inflammatory response within the first hours, but we did not demonstrate any correlation with the response to immunotherapy after six months. 0905 | tolerability of a two week rush updosing with modified allergens in pollen allergic subjects in the day-to-day practice background: in two phase iv studies the tolerability of a subcutaneous rush up-dosing, using three injections in two weeks, has been tested and proven to be save in adults. in the course of a non-interventional study (nis) now the tolerability of this treatment scheme was tested in the day-to-day practice. conclusion: over 97% of the patients could reach the highest dose of 0.5 ml. the overall tolerability is very good. the data from daily practice confirm the data that were previously obtained in two phase iv studies. siges from 40 patients, evaluated during the 1st semester of 2017 at an outpatient clinic. all patients presented persistent moderatesevere allergic rhinitis, in pollen season and had not been submitted to it. all patients had positive spt for grasses (grass) and olive (olea). sige-tot for phleum pratense and olea europaea and some sige-crd (rphl p 1, rphl p 5, rphl p 7, rphl p 12, role1 and nole7) were determined. physicians were divided into 2 groups (group 1 if <10 years of practice and group 2 if≥10 years of practice) and were asked to choose which it to prescribe for each patient (none, only grass, only olive or both grass and olive), according to spt and sige results. results: fifteen physicians (60% with ≥10 years of practice) participated in the survey. considering only the sige-tot results, the it choice (group 1/2) was: no vaccine in 10%/3%; grass vaccine 50%/ 58%; olive vaccine 5%/10% and both grass and olive vaccines in 35%/30% of the patients (p = 0.42), the intergroup agreement was 70% (kappa 0.612). according to the sige-crd results the physicians chose (group1/2): no vaccine at 10%/13%; grass vaccine in 63%/60%; olive vaccine in 10%/5% and both vaccines in 18%/23% of patients however, data on control of allergic rhinitis (ar) after discontinuation of therapy are insufficient. the aim of our study was to assess sustained control of ar in three consecutive years after grass-pollen slit discontinuation. method: a total number of 35 patients [24 (68.57%) males; mean age 32 years, age range 15-56] well-controlled after a three-year course of slit with grass pollen extract were prospectively evaluated in three consecutive years after discontinuation of therapy. conclusion: a three-year course of grass-pollen slit seemed to have a long-term effect on control of symptoms in patients with ar. the authors declare no conflict of interest. results: all patients showed a positive sensitization profile by skin prick test to either betula and/or alnus. in 40% of patients this profile was furtherly confirmed with serum specific ige levels to betv1, (mean 13.56 ku/l). allergic symptoms in patients with birch/alnus pollen allergy after ingestion of certain food can result from crossreactivity of bet-v1-specific ige to homologous pathogenesis-related proteins, particularly the pr-10 protein. conclusion: within the allergy history we emphasize on focusing on sao symptoms as many patients under-recognize them. among the sensitization profile of these patients it is quite important to highlight cross reactivity between bet v1 and alnus. the other patient suffered from anaphylaxis(grade ii) induced by minimum amount of lettuce consumption without co-factors. both patients suffered from oral allergy syndrome to peach and allergic rhinitis. spts to foods and pollens were performed with commercial extracts, prick-through-prick with fresh plant foods, while specific ige was determined accordingly. ltp syndrome was defined as a sensitization to pru p 3 and symptoms elicited by at least 2 unrelated plant foods. co-factors were also investigated. results: the first patient was sensitized to lettuce, peanut, hazelnut, sunflower's seed, peach and banana, and plane tree, olive tree, grasses, parietaria and mugwort. sige to lettuce was 2.04kua/l, to pru p 3 was 20.8kua/l and total ige was 703.4u/ml. co-factors, such as exercise, were involved. the second patient was sensitized to peanut, walnut, hazelnut, almond, sunflower's seed, cashew, lettuce and peach, and, plane tree, olive tree, grasses, parietaria, mugwort and willow. sige to lettuce was 27.6kua/l, to pru p 3 was 37 and total ige was 1705.9ku/l. no co-factors were identified. background: garlic (allium sativum) is a vegetable that belongs to amaryllidaceae's family. hypersensitivity to garlic is not very common. it has been mainly reported in occupational allergy but it also may cause contact dermatitis, rhinoconjunctivitis, asthma, urticaria, gastrointestinal symptoms and anaphylaxis after its ingestion. some studies have identified alliin lyase, a 56 kda protein, as a major garlic allergen and it seems to be a heat-sensitive allergen. we report on a 9-month-old infant who presented, 15 minutes after an accidental ingestion of garlic sauce, generalized erythema and cough. she was still breastfeeding and she had never abstracts | 491 eaten garlic before (although the mother usually consumed garlic). the patient had never tasted other vegetables belonging to amarylidaceae's family either but zucchini, with good tolerance. we performed skin tests and specific ige (sige) to different vegetables. a raw garlic extract was also carried out and analysed in the patient by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page). results: prick by prick with garlic was positive (10 mm) and negative to onion, leek, asparagus, zucchini and saffron. skin prick tests to commercial extracts of mugwort, grass pollen, peach ltp and profilin were negative as well. specific ige to garlic was 3.15 ku/l (out from a total ige 32 ku/l) and 0 ku/l to onion and asparagus. sds-page immunoblotting assay with patient′s serum revealed ige reactivity with proteins of 6 kda and 8 kda. conclusion: we report a garlic ige-mediated anaphylaxis case in an infant with proteins of 6 and 8 kda as the relevant allergens. the mechanism of sensitization in the present case remains unclear. the authors hypothesized that breastfeeding, cutaneous contact or inhalation might be possible mechanisms involved. chong kw 1 ; saffari se 2 ; chan n 3 ; seah r 3 ; tan ch 3 ; goh sh 1 ; goh a 1 ; loh w 1 1 allergy service, department of paediatric medicine, kk women's and children's hospital, singapore, singapore; 2 centre for quantitative medicine, office of clinical sciences, duke-nus medical school, singapore, singapore; 3 yong loo lin school of medicine, national university of singapore, singapore, singapore background: the predictive decision points for both peanut skin prick test (spt) wheal size and serum ige concentrations, in peanutsensitized children, have not been evaluated in singapore. we aim to assess these for purposes of risk stratification and prediction of oral food challenge (ofc)s' outcomes by means of a retrospective chart review. results: the number of patients evaluated was 328, of which 269 had clinical diagnosis of peanut allergy based on recent immediate reaction to peanut (pa group) and 59 were tolerating peanuts regularly (pt group). the mean age of both groups were similar, 3.9 ± 3.2 and 3.7 ± 3.3 years in pa and pt groups respectively. there was a high prevalence of atopic diseases in both groups, with atopic dermatitis (75.8% in pa, 79.7% in pt), and other food allergies (55.4% in pa, 44.1% in pt). presence of rhinitis was statistically higher in the pa group compared to the pt group, with odds ratio of 2.52 (95% ci: 1.42-4.47) . a wheal size of ≥8 mm and a peanutspecific ige of ≥6 ku/l provided for a 95% positive predictive value. the larger the wheal size on spt, the higher the probability of a clinical reaction to peanuts. the results will help us in deriving preliminary cut-off values when conducting future prospective studies with ofcs in our peanut-sensitized cohort (whom had no prior peanut exposure), and to eventually reduce the need for expensive and potentially risky food challenges. 0913 | ginger: flavory, spicy …allergenic? a report of four patients with allergy to ginger background: ginger (zingiber officinale) belongs to the family zingiberoidae, along with cardamom and turmeric. the edible portion is the horizontal rhizome, and it is very appreciated for its aroma and spicy flavor. it also presents great interest for its therapeutic and culinary use. hypersensitivity to ginger is rare and has been scarcely reported. we report 4 cases (p1, p2, p3, p4) of adverse reactions to ginger after its ingestion and with good tolerance to cardamom and turmeric. method: skin prick tests (spt) to environmental allergens and prick-by-prick with ginger were carried out. total ige, and specific ige to ginger were also determined. a raw ginger extract was prepared. this extract was analyzed in all the patients by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page). background: food allergy is divided into 3 groups according to pathophysiology: ige-mediated, ige-and non-ige (mixed type), and non-ige (cellular type). however, in clinical practice, patients who fall under more than one group may be observed. method: patients who were diagnosed with food allergy at our clinic from january 2012 to december 2016 were included in the study. the medical files of patients were retrospectively evaluated, their symptoms and findings after consumption of foods were recorded, and they were categorized into 3 groups (ige-mediated type, non-ige type, and mixed type) according to their symptoms and findings. results: a total of 587 patients (63.5% male) with food allergies were included in the study. according to categorization via symptoms and findings, the distribution of patients was as follows: 140 (23.8%) ige-mediated type, 44 (7.5%) non-ige type, 195 (33.2%) mixed type. the remaining 208 (35%) patients were found to show various combinations of symptoms and findings that fit more than one group. in this study, we observed that food allergy symptoms and findings were distributed in a broad range which caused difficulties in the categorization of more than one-third of our patients. background: fish allergic patients suffer a lifetime of strict dietary restrictions. crocodile meat is a nutritious alternative choice in many countries around the world; however, it has recently been reported to also trigger severe allergic reactions. in these two case reports from 2017 pediatric patients were sensitised to the major fish allergen parvalbumin (pv), a potential cross-allergen. bony fish contain predominantly pvs of the β-lineage, which are the most common trigger of allergic reactions in fish allergic patients. in most other vertebrates, pvs of the α-lineage are most abundant, which have been reported as a causal allergen in frog, cartilaginous fish, and chicken allergies. we aimed to evaluate the allergenicity of crocodile meat in fish allergic children, with focus on pv. method: over 70 children with clinical history of ige-mediated fish allergy were identified, skin tested to commonly consumed fish species using commercial and in-house preparations, and serum samples were collected. a sub-cohort was skin tested to crocodile using heat-treated tail muscle tissue from saltwater crocodile (crocodylus porosus). extracted proteins and purified pvs were analysed by sds-page, immunoblotting, and mass spectrometry. serum from all fish allergic children was analysed for ige reactivity to the crocodile pv. this reactivity was compared to those of raw and heated crocodile protein extracts as well as protein extracts and purified pvs from frequently consumed fish species. results: more than 5 fish allergic children were positive on skin testing to crocodile (wheal size>3 mm), demonstrating its clinical reactivity. in vitro analyses revealed ige reactivity to crocodile pv in serum from more than half of all patients. two βand one α-crocodile-pv isoforms were identified. pvs constituted approximately 90% of total proteins in heated crocodile extracts, with β-pv (11 kda) being 10 times less abundant, but up to 500 times more ige-reactive than α-pv (13 kda). αand β-pvs from crocodilians, including alligators and crocodiles, share more than 96% and 74% of their amino acid sequence, respectively. conclusion: crocodile pv is a new allergen as per the iuis guidelines. fish allergic patients may be at risk of severe allergic reactions upon ingestion of crocodile meat due to strong ige cross-reactivity of β-pvs. this study suggests that fish allergic individuals and health professionals need to be aware of potential allergic reactions to meat from crocodilians, termed 'fish-crocodile syndrome'. background: we present a 39-year-old nonatopic woman that in december 2016 after eating a seafood paella with green pepper presented asthenia, nasal obstruction, incoercible vomiting and diarrhea. later she ate a grilled loin sandwich in a bar and she had the same symptoms (she asked the waiter at the bar and the chef had cooked her sandwich in the same pan where he had cooked green pepper just before). after that, she suffered from abdominal pain, nausea, abdominal distension without diarrhea two hours after she ate an omelet sandwich with certain flavor of green pepper. at present even the casual smell of pepper causes her nausea. the woman eats everything including spices and just avoids pepper. method: skin prick tests were performed using extracts from food (nuts, fish, mollusk, fruits, vegetables, legumes), aeroallergens (mites, abstracts | 493 pollens and epithelia) and purified proteins (pru p 3, profilin, polcalcin, alfalactoalbumin, betalactoglobuline, casein). we also performed prick by prick to raw and cooked green pepper. sds-page immunoblotting according to laemli under reducing conditions (with 2-mercaptoethanol) was performed to study the molecular mass of the ige-reactive proteins. extracts from green pepper and green pepper seed were used. the prick tests were all negative and the prick by prick test to raw and cooked green pepper was positive in both cases. blond was taken from all patients to specify the levels of allergenspecific ige against 112 allergen components of immunocap isac test, the result≥0.30 isu-e was assumed as positive. results: in the study group, in 12 patients (24%) specific antibodies against ltp were detected, the isu-e level range 0.3-34 average 5.26 isu-e on average, in patients with detected ltp ige was detected for 3.5 components belonging to the ltp, however the highest number 41% patients were detected ige only for 1 ltp. in 9 subjects (75% of respondents with detected ltp), ige was detected against art v 3 and this is the only ltp component whose occurrence was statistically significant (p = 0.044). in 6 patients ige was detected against pru p 3, jug r 3, pla a 3,; 5 patients ige to ara h 9; 4 patients cor a 8; 3 patients ole e 7, 2 patients tri a 14, and in 1 person para j 2. background: the birch pollen-associated oral allergy syndrome (oas), an ige-mediated local allergic reaction, is the most common manifestation of pollen-associated food allergies. its origin is explained by cross-reactions between birch pollen-and food-allergens belonging to the pathogenesis-related protein subfamily 10 (pr-10). [1] so far, there is no marker available for its detection and no standardized test established to evaluate objectively the subjective feelings experienced by oas. the diagnosis is based on a characteristic history and on detecting the sensitization to triggering allergens in skin prick test (spt) and laboratory examination. method: the aim of this study was to evaluate whether the food skin prick test could be a helpful marker in the diagnosis of a birch pollen-associated oas. for this exploratory study, data from 2016-2017 was collected retrospectively at the dermatological outpatient department of the ordensklinikum linz elisabethinen. patients with positive spt results for birch pollen were included. the variables age, gender, tree pollen-(birch, alder, hazel) and food-spt, laboratory tests (ige, bet v 1, bet v 2, gly m 4) and symptoms (oas, rhinoconjunctivitis allergica, atopic dermatitis, anaphylaxis) for statistical analysis. results: there was an association between food-spt and oas but also between the negative oas patients and food-spt (p = 0.9-0.1). all of the bet v 1 sensitized patients with positive gly m 4 results had also a positive food-spt result. conclusion: there was no evidence for a possible role of food-spt as helpful markers in the diagnosis of birch pollen associated oas. maybe gly m 4 could be a helpful marker, but more data are needed. bet v 1 seems to be the cause of birch-pollen associated oas, due to the dominant sensitization pattern to major allergens in austria. background: eggs are among the foods most frequently causing allergy. the most common one is hen egg, although we may consume other bird's eggs such as duck's, those of goose, quails and seagulls. clinical and serological crossreactivity between hen egg proteins and those of other birds eggs have been described. allergy to other species eggs are less frequent and are usually described in patients allergic to hen eggs. we report a case of food allergy after ingestion of duck egg in an adult patient without hen egg allergy. the patient was a 50 year-old man who had symptoms of generalized itching, swelling uvula, erythema neck and deglutition difficulty immediately after he ate eggs from duck and hen. he claimed to have eaten hen eggs almost daily without clinical symptoms and he had not previously ingested duck egg. he denied allergic reactions to any other food but did complain of seasonal allergic rhinoconjunctivitis in the spring. we performed skin prick test with extracts of egg and feathers, prick by prick test with cooked and fresh yolk and white from duck and hen egg and oral challenge with hen eggs. specific serum ige was measured to hen proteins and we carried out a western blot with the proteins of allergenic extracts from different eggs (white and yolk of quail, chicken, goose and duck) and an inhibition of western blot with ovalbumin as inhibitor allergen. results: skin test with extracts of eggs and feathers, cooked and fresh hen and duck eggs were positive. total serum ige was 29.2 ku/l. specific ige to hen's egg was class two for hen egg white, ovoalbumin and class one for yolk and ovomucoid. oral challenge with heated egg yolk negative and with egg white was positive. the patient's serum recognized mainly and intensely several proteins of white and egg yolk of quail and duck with a molecular mass around 45 to 50 kda respectively. on the other hand a protein around 45 and 50-52 kda was unique recognized in white eggs of hen and goose. the western blot inhibition revealed ovalbumin inhibited the protein recognized in the egg white but not egg yolk involved in. background: management of tree nut allergy is usually based on the avoidance of the suspected tree nut (tn), as well as peanuts and seeds, either because of the risk of cross-reactivity and/or contamination, or due to the clinical severity. objective: to assess the sensitization pattern and clinical reactivity patterns to different tn, peanut and sesame seeds (ss) in patients with a history of reaction to at least one of these foods. results: a total of 47 patients with confirmed nut allergy were included; 70% female, median age [interquartile range] of 28 years; 72% were atopic. the most frequently involved foods were walnuts (53%), hazelnuts (40%), almonds (32%) and peanuts (38%). anaphylaxis was the clinical presentation in 51% of the patients. in those with a history of reaction to only one nut (24), the most prevalent was peanut (42%). in the 23 patients that reacted to more than one nut, the most frequent combinations were walnut/hazelnut (15), walnut/almond (12) and almond/ hazelnut (10). of these patients, 5 tolerated other nuts. two had sesame seed allergy, one reacted only to ss. nine patients (19%) were sensitized to foods that they tolerated. fourteen (30%) patients were sensitized to ltp and 8 of them reacted to more than one nut. conclusion: these data concur with the existence of different sensitization profiles (primary, concomitant or cross-reactive), which may predict different clinical reactivity patterns and therefore, influence dietary recommendations. background: buckwheat (fagopyrum esculentum) is a polygonaceae weed, not a cereal, which is increasingly been consumed and used as an alternative food in the diet of celiac patients. despite its wide use, allergy to buckwheat has unfrequently been reported in our setting. method: two female patients, aged 37 and 58, suffered an immediate severe allergic reaction after eating a bread and a pancake containing buckwheat among its ingredients. the first patient presented generalized urticaria, palpebral angioedema and pharyngeal occupation. the second one showed those same symptoms, as well as abdominal pain, nausea, dyspnea, dizziness, hypotension and loss of consciousness. skin tests and specific ige determination to aeroallergens and food allergens were carried out, including prick-prick test with buckwheat and all other components contained in the food involved. buckwheat allergens were studied by sds-page and ige-immunoblotting of one of the patients. results: prick-prick tests yielded strongly positive results to the food itself and buckwheat, and negative to the remaining food components in both cases. cap was positive to buckwheat (11.30 ku/l and >100 ku/l, respectively). basal serum tryptase levels were normal. both patients were not sensitized to cereals, ltps, profilins or pr-10 proteins. for the first patient, the skin tests were negative for other foods, seeds and nuts. the cap was negative for ltps, profilins and storage proteins of peanut and other nuts. the second patient who had the most severe reaction, was also sensitized to hazelnut(cap 13.7 ku/l), pistachio (1.44ku/l), almond(0.66ku/l), walnut(29.7ku/l) and sesame(4.47ku/l), which were not included in the pancake. the buckwheat immunoblot of the first case, under non-denaturing conditions, revealed a ige-binding protein of 60-kda. ige-immunoblotting under reducing condition showed protein bands of 30, 20, 17 and 14 kda in the buckwheat extract. conclusion: two cases of anaphylaxis by buckwheat flour contained in two frequently consumed foods are presented. the absence of sensitization to ltps, together with the pattern of specific ige binding in the immunoblot in one of the cases suggests that the responsible allergen could correspond to a storage protein of buckwheat, without cross-reactivity with other seed and nut allergens. buckwheat must be taken into account as an unsuspected food allergen capable of causing severe allergic reactions. background: allergy to linseed (linum usitatissimum) has infrequently been reported despite of its wide use in bread and in a range of "health food" products. linseed contains potent allergens which have not yet been characterized. we studied three patients who presented allergic reaction after eating different foods which contain linseed. two of them (patient p1 and patient p2) had anaphylaxis and the third one (patient p3) had oral syndrome, abdominal pain and diarrhoea. p2 was also allergic to mustard and p3 to sesame seed. all of them tolerated the remaining seeds, nuts and food. baseline tryptase levels were in normal range in all patients. skin tests and specific ige determination to inhalants and food allergens were carried out. linseed allergens were studied by sds-page and ige-immunoblotting. skin tests: patient 1: prick tests were positive to pollens and linseed, and negative to ltp, profilin, nuts, seed and the remaining food; patient 2: prick tests were positive to linseed and mustard and negative to other food and inhalants; patient 3: prick tests were positive to linseed, pollens, sesame and nuts (peanut, hazelnut, almond, pistachio). we present three cases of severe allergic reactions to linseed. the pattern of specific ige binding in the immunoblot seems to lead to storage proteins as the responsible allergens. 0925 | pr-10 sensitization-looking it up in food allergy background: pr-10 protein group sensitization is found in patients with respiratory allergy, mainly in areas inhabited by trees of the betulacea or fagacea families. its role in food allergy is, however, more frequently described in the context of cross reactivity. our aim was to characterize the pattern of molecular sensitization of food allergic patients sensitized to pr-10 protein group with immu-nocap isac ® (isac). the remaining patients, with more severe reactions, were all co-sensitized to either ltp and/or storage proteins (sp). only 2 of the 7 patients without pfa were co-sensitized to ltp or sp versus 7 of 9 with pfa (p < 0.05). conclusion: pr-10 sensitization is rare in our population. approximately half of the patients had allergy to plant foods, but the majority were co-sensitized to ltp and/or sp. the few patients only sensitized to pr-10 had minor reactions. among the patients without plant food allergy, co-sensitization to ltp and sp was significantly less common. according to these results, in our population, pr-10 seems to be less relevant to food allergy, when compared to reported results from other european countries. gür çetinkaya p; uysal soyer ö; esenboga s; sahiner üm; sekerel be hacettepe medical school, ankara, turkey background: pistachio is a tree nut belonging to "anacardiacea" family, and constitutes 7% of tree nut allergies. this nut most often abstracts | 497 cross-reacts up to 95% with cashew which is located in the same family. pistachio allergy is mostly seen in iran, turkey, the united states, and china where this tree nut is frequently consumed. in this study, we analyzed age and cut-off values for development of tolerance to pistachio. method: children who had reported allergic reactions with pistachio, and who have not consumed pistachio, but had positive spt and/or specific ige levels were enrolled into the study. spt and specific ige levels were measured in all patients. oral provocation(op) tests with pistachio were performed by 82 patients, and 21 of them had positive test result. the median age of tolerance development was 60 months (iqr: 48.0-89.0 months). the most commonly involved systems during op tests were skin (90%, n = 19), gastrointestinal system (33%, n = 7), and lower respiratory tract (24%, n = 5). concomitant allergic diseases were atopic dermatitis (70%), asthma (40%) and allergic rhinitis (17%). there was a positive correlation between skin prick test diameter (spt) and specific ige levels (spige) (r = 0.331, p = 0.001). spt of≥8.25 mm to pistachio nut was found as highly predictive of clinical allergy (auc: 0.83, 95%ci: 0.73-0.94, p < 0.001). any relation was not determined between eosinophil, basophil counts, triptase levels, and op test positivity. conclusion: pistachio allergy is one of the frequently seen nut allergies in turkey which may cause serious allergic reactions including anaphylaxis. op test showed that tolerance was achieved by the median age of 60 months, and a cut-off level of 8.25 mm was best predictor for positive reaction. in an oral challenge test, the patient responded with generalized urticaria, swelling of the lips and difficulty breathing at a cumulative dose of 0.1 g peanut protein, however, without blood pressure drop. (grade 2, moderate symptoms). the patient was treated with anti-ige for 3 months (with 450 mg every 4 weeks sc). oral desensitization began with ingestion of 500 μg peanut, escalating to 500 mg, on the first day and escalating weekly doses of peanut from 500 mg to 4000 mg (8 peanuts). then anti-ige was discontinued while the patient ingested 8 peanuts every day. we have followed the patient's sensitivity to peanuts from before anti-ige treatment to after anti-ige washout with basophil testing. results: the patient completed desensitization without side effects, and continues to ingest 8 peanuts a day. basophil sensitivity was reduced 13-fold by anti-ige treatment, but returned to baseline levels after anti-ige washout. conclusion: anti-ige allows rapid desensitisation of peanut allergic subjects with peanut oral immunotherapy. in the majority of subjects, this desensitization is sustained after anti-ige is discontinued. additional studies will help clarify which patients would benefit most from this approach. the return of basophil response to pre-treatment levels suggests that the patient is desensitised, and depends on the daily ingestion of allergen. what do we (not) know? background: the use of biologic prosthesis is a well-established surgical procedure. acute and delayed complications may occur, but accurate epidemiologic data about allergic reaction to graft tissue is lacking. methods: a 9 years old boy referred to our unit for urticaria and gastrointestinal symptoms which developed a few years before. he received a biologic porcine vascular duct during a cardiovascular surgery at 20 days of life. at the age of 5 urticarial episodes occurred, and a diagnosis of beef allergy was made. after the exclusion of beef meat from the diet, most symptoms resolved, but the child began to complain about occasional episodes of vomit and diarrhoea. results: skin prick tests confirmed the beef meat sensitization and prick-prick test resulted positive against raw pork meat but not against cooked pork meat. in vitro tests demonstrated the presence of pork meat specific iges (14.1 kua/l). component-resolved diagnostic tests revealed allergy sensitization toward bos d 6 (bovine serum albumin, bsa; porcine serum albumin was not tested due to lack of a specific test). after accurate exclusion of pork meat from diet, a complete remission was achieved, and the diagnosis of pork meat allergy was confirmed. conclusion: bsa is a major beef allergen, responsible for the raw beef-cow milk cross-reactivity but with a scarce importance in milk allergy. it is highly homologous with human serum albumin and other mammalian serum albumins, including porcine albumin. porcine albumin is also highly homologous with cat serum albumin and therefore responsible for cross-reactivity in patients affected by cat-pork syndrome. we hypothesize that the implanted porcine tissue was the trigger for pork meat allergy in our patient, as this condition is exceptional in childhood and that our patient never owned a cat. few cases of pork allergy due to porcine tissue implantation have been reported so far. of interest, pig sensitization was recognized as a rare but possible cause of blood-culture negative endocarditis in patients with porcine bioprosthesis according with anamnesis, increased ige level against pork, tissue eosinophilia during autopsy. background: desensitization to foods is assuming a new paradigm in food allergy. this technique is becoming widespread especially for patients with egg and milk allergy, but the effect of desensitization on the consumption of similar but not identical foods is still uncertain. material and methods: we describe the case of a 16-year-old patient, with a history of chicken egg allergy, who had been successfully desensitized tolerating cooked and raw chicken eggs for a year. the patient came to the office after presenting an episode of anaphylaxis immediately after eating fried quail eggs. an immunological study was carried out. we performed skin prick test with chicken egg′s proteins (white, yolk, ovoalbumin and ovomucoid). an immunoblotting to detect specific ige to egg proteins was also performed. for this purpose, the following extracts were used: chicken egg white and yolk (commercial extract form alk) and quail egg white and yolk prepared following a similar procedure (extracted in 10% phosphate buffer (w/v)). conclusion: we present the case of a patient with specific allergy to quail egg white and yolk, probably through ovotransferin, but not to chicken egg. the desensitization against chicken eggs does not allow the consumption of eggs of other species of birds, such as quail eggs, and this indication must be made specifically to patients after a protocol of desensitization against chicken′s eggs. case report: seven years ago we presented the case of a 32year-old male, who suffered two acute episodes of oral pruritus, lip angioedema, epigastric pain, generalized urticaria and dizziness after ingesting lettuce. he previously tolerated salads (including lettuce). he was later diagnosed with non-ltp-dependent lettuce anaphylaxis and an aspartyl protease was identified as a new lettuce allergen with cross-reactivity with other members of the compositae family. since the diagnosis he has avoided lettuce and all the other compositae. we present the patient's curious outcome after 7 years of follow up. methods: total ige, basal tryptase, specific ige to lettuce by immu-nocap, prick-prick and oral challenge tests with lettuce and other compositae. sds-page, immunoblotting and molecular characterization of ige binding bands by mass spectrometry. skin prick tests and specific ige to lettuce were repeated on several occasions in the following years. after 7 years an oral challenge test with lettuce was carried out. results: prick-prick test was positive to fresh lettuce (11 × 15 mm) and to other compositae (raw endive, chicory, thistle, artichoke and chamomile). prick-prick test was negative with these boiled compositae. basal tryptase was 7.07 μg/l. total ige in serum was 189 ul/ml. specific ige by immunocap was positive to lettuce (7.35 ku/l) and negative to profilin, ltps and thaumatin. oral challenge test with endive was positive and negative with cooked compositae (artichokes and thistle). sds-page and immunoblotting detected an intensely binding ige band about 46 kda, common for endive and chicory, which was identified by mass spectrometry as an aspartyl protease. no bands were detected at ltp (9 kda) and profilin (16 kda) . in the first two years after the diagnosis, prick-prick test and specific ige to lettuce remained positive (cap 1.91 ku/l and 0.44 ku/l, respectively). after 7 years, prick-prick test and specific ige to lettuce became negative (cap 0.08 ku/l). with this findings a new oral challenge test with lettuce was carried out which result turned out negative. we report the first case of spontaneous tolerance to lettuce in a patient who previously presented lettuce anaphylaxis and identify an aspartyl protease as the causative allergen. introduction: eosinophilic esophagitis (eoe) is an emergent allergic inflammatory disease that is triggered by food allergens and characterized by progressive esophageal dysfunction. recently, it has been seen that eoe develops in up to 2.7% of patients with ige-mediated food allergy undergoing oral immunotherapy (oit). ingestion of baked milk and egg was associated with increased development of tolerance to regular milk and immunologic changes have been reported in subjects ingesting baked milk and egg, similar to those seen in food oral immunotherapy studies. case description: we present the case of a 13-year-old girl, with history of ige mediated cow′s milk allergy and rhino conjunctival and bronchial asthma symptoms. prick test against milk proteins showed: milk 6 mm, alpha-lactalbumin 6 mm, casein 3 mm and to beta-lactoglobulin: negative. total ige: 469 ku/l. specific ige to milk: 3, 15 alpha-lactalbumin: 3, 16 ku/l; casein: 2 ku/l and beta-lactoglobulin: 0.10 ku/l. we performed an oral food challenge with baked milk which was well tolerated. then, we performed an oral food challenge with fresh milk and she presented facial urticaria and pharyngeal pruritus. after 3 months eating baked milk every day, she had symptoms of dysphagia and esophageal food impaction. for this reason, we performed an esophagogastroduodenoscopy (egd) and biopsies which showed white exudates and vertical furrows. the histological study showed eosinophil count>20 per high-power field. eosinophilic esophagitis was diagnosed and she started treatment with esomeprazole 20 mg. the egd was repeated 8 weeks later with similar results in the biopsy. she was treated with a comprehensive diet free of cow′s milk proteins. after 8 weeks she was asymptomatic and endoscopy and biopsy findings were normal. we report the case of a cow′s milk allergic patient who developed eoe after introduction baked cow′s milk which apparently was tolerated in her diet. the avoidance proved efficacy in inducing the remission of eoe. method: we examined 87 bottle-feeding infants with food allergy aged from 1 to 12 months. serum vitamin levels were measured by immunoassay methods (retinol binding protein (rbp), vitamin b6, 25hydroxy vitamin d), biochemistry methods (vitamin c, vitamin e) and microbiology methods (vitamin b1, vitamin b2). as criteria for complete sufficiency standards adopted in the russian federation were used (the lower limit of normal levels: rbp -0.52 μmol/l; b6-8 ng/ml; 25-hydroxy vitamin d-15 ng/ml; vitamin c -0.4 mg/dl, vitamin e -0.8 mg/dl; vitamin b1 -28 μg/ml; vitamin b2 -6 ng/ ml). results: complete sufficiency were observed in 21 infants (25.9% cases), one vitamin deficiency in 33 infants (40.7%), two vitamins deficiency in 23 infants (28.4%), three and more vitamins deficiency in 6 infants (7.4%). should be used after vitamin status asses, mainly using monovitamin medication. results: patients were followed with the diagnoses of cma and asthma. age at the beginning of symptoms and start of oit were in the range of 3-6 months and 4-19 years, respectively. they had high total ige (139-843 iu/ml), milk spige (2.9-418 kua/l), casein spige background: food allergies may affect up to 6% of school-aged children. it has been shown that approximately 20% of all anaphylactic reactions caused by food allergy are firstly presented at (pre) school. therefore, it is of high importance that (pre)schools have a policy on food allergy management and the use of an epinephrine auto-injector (eai). to our knowledge, limited is known on the policies of food allergy management at (pre)schools. the aim of this study was to investigate the policy on food allergy management in preschools and primary schools in the northern part of the netherlands. results: we included 87 preschools and 110 primary schools in this study. we showed that 80.5% of the preschools and 73.6% of the primary schools had a child(ren) with food allergy. only 13.8% of the participating preschools and 27.2% of the primary schools had a policy on allergen avoidance and only 35.6% of the preschools and 35.8% of the primary schools had a policy for the use of an eai. the majority of the pre-and primary schools in the northern part of the netherlands have children with food allergy. however, only a limited number of (pre)schools do have written guidelines for food allergy management in (pre)schools. additionally, there is limited experience how to use an eai at (pre)schools. therefore, an evidence-based policy on food allergy management in (pre) schools is needed. background: food allergies are the most common cause of anaphylaxis in childhood. here we present 3 cases had anaphylaxis due to cow 's milk allergy, and treated with specific oral tolerance induction (soti̇) to cow' s milk. method: soti protocol was administered according to previously published by longo et al. skin prick test were performed according to standard methods with allergens. cow's milk specific ige was investigated with immunocap system. in all cases, the wheal size of the cow's milk in skin prick tests or the specific ige levels was higher than level of positive predictive value of 95%. results: case 1: a 7 years old male patient who had 3 anaphylaxis after milk consumption. soti treatment was started one year ago. there was no complications during the dose increasing phase. however, he had two episodes of anaphylaxis during the maintenance phase. in the final visit, we observed that he could drink 200 ml milk and could consume dairy products. case 2: eight-year-old male patient has an intensive care-of-hospitalization story due to anaphylaxis three times after milk consumption. his accordance to strict diet was bad, and had frequent asthma attack. anaphylaxis developed 2 times during the dose increasing phase in soti protocol. after soti treatment, he could consume 200 ml cow's milk and dairy products without problems. case 3: a 7-year-old male patient followed-up for asthma and cow's milk allergy. it was learned that anaphylaxis developed 2 times after milk consumption. he was fed in accordance with the milk-free diet. he has used fluticasone nebules, montelukast, mometasone nasal spray, and cetirizine. anaphylaxis developed 4 times during the dose increase phase of soti administration. there were many mild to moderate anaphylactic episodes during the maintaining phase. after the soti treatment, he could consume 150 ml milk and dairy products. background: cow's milk protein allergy (cmpa) is one of the most common food allergies in early childhood. small dietetic group sessions for parents of infant with non-ige mediated cmpa were held to meet increasing demands and reduce waiting times. parents were given information on cmpa, advice on weaning and milk reintroduction using a locally designed milk ladder. parents were also advised to contact the dietitians via telephone if they had further questions. we aim to evaluate the sustained effectiveness and patient satisfaction of the group sessions. method: parents and carers who attended the group dietetic sessions held between november 2015 and july 2016 were included in the survey. feedback were obtained via a self-designed questionnaire using a likert-type scale, rating several questions from 1 (least satisfied) to 5 (most satisfied). initial feedback was obtained directly after the session. we followed these patients up a year after the initial session via telephone and postal questionnaire. results: overall attendance rate of the 9 group sessions held was 58% (n = 40). during the initial survey, participants found the group session useful (mean score 4.7 out of 5) and felt more confident in managing cmpa (mean score 4.8). we successfully obtained follow up feedback from 13 participants. majority agreed that the group sessions have been informative (mean score 4.8). they also said they felt confident weaning their child on milk-free diet (mean score 4.8), and in reintroduction of cow's milk in diet (mean score 4.3). 30% (n = 4) said that they would have preferred an individual session. 38% (n = 5) have contacted the dieticians via telephone after the initial session, and 15% (n = 2) had requested individual consultations. 69% (n = 9) have attempted reintroduction of cow's milk in their child's diet using our local milk reintroduction guide. the mean age at first challenge was 14 months (age range 6 to 26 months), with average of two attempts. 31% (n = 4) have been successfully challenged and are managing well on a normal diet. we recognised the limitation in obtaining feedback via telephone and postal questionnaire, which resulted in the poor follow-up response rate. overall, parents felt more confident in managing cmpa and the positive responses were sustained a year on, highlighting the success of these group sessions. follow up opt-in sessions could be offered to provide additional support and allay parental anxiety in challenging their child with cow's milk at an appropriate age. results: goats and rabbits were immunized with specific allergoids, the allergoid-specific igg titer determined and sera pools produced. the allergoid reference material was comprehensively characterized. while ige reactivity of the allergoids was not detectable anymore, igg reactivity was maintained. allergoid-specific assay parameters as serum dilution, reference dilution and sample dilution factor to obtain at least six data points within the pseudo-linear range of the inhibition curve were determined. with these set parameters the evaluation of the analytical method was performed. the assay showed very good results in terms of linearity, accuracy, precision, reproducibility and robustness for all investigated allergoids as well as aluminum-adsorbed allergoid-preparations. conclusion: with the developed immunological inhibition assay, it is possible to determine the specific igg reactivity of allergoids in different preparations. the performance of the analytical method met all pre-defined acceptance criteria, which will be confirmed in the next step by a validation procedure according to ich-guidelines. case report: we present the case of a 32-year-old patient, diagnosed with rhinitis and asthma due to sensitization to pollens, as well as dyshidrosis and allergic contact dermatitis to cobalt. the patient presented a cutaneous pattern consisting of erythematous papules, some scaly, very pruritic, of initial appearance in the upper limbs 2-3 days after initiation of administration of specific immunotherapy extract (depigoid forte grasses, leti ® ). subsequently generalize lower limbs and neck. she presented them repeatedly and late after 2-3 days of the first 3 doses administered. she referred partial control of pruritus with oral antihistamines, without total resolution of lesions for weeks. immunotherapy was suspended persisting the skin lesions for 3 more weeks. according to the personal history of sensitization to metals, and the clinic presented in a temporal relationship with the use of an extract of immunotherapy with aluminum hydroxide, a study was requested with epicutaneous tests with aluminum hydroxide as well as with epicutaneous tests with immunotherapy extract. aluminium hydroxide and depìgoid forte grasses extract epicutaneous test were negative. in subsequent visits the patient reported that coinciding with the start of immunotherapy, presented at home and mainly in her bedroom a plague of cimex lectularius, popularly known as bedbugs, proving that they had been the cause of bites on their skin, and later skin reaction. patient reported that with the elimination of said pest the skin lesions disappeared. the administration of its immunotherapy extract was tolerated. cimex lectularius, commonly known as bed bugs, is a hemiptera insect of the family cimicidae. the clinical picture usually corresponds to multiple pruriginous lesions from the prurigo type, to multiple erythematous plaques, some infiltrated and others with a urticarial appearance, or even bullous. the lesions last for 3 to 6 weeks without treatment, and while the older ones heal, new ones may appear. in our patient it was not considered as an initial diagnosis, having considered immunotherapy as an etiological factor, but we must not forget that although in our country it is not a reason for frequent consultation, either due to underdiagnosis, because of the transitory nature of the pathology or because of scarce number of causative agents, it is important to consider insect bites in the differential diagnosis of dermatosis. di cara g; salvatori c; testa i; pacitto a; bizzarri i; isidori c; tarsia m; esposito s università degli studi di perugia-dipartimento di scienze chirurgiche e biomediche, perugia, italy background: house dust mites (hdm) are one of the most important allergens involved in childhood respiratory diseases, and the most frequently prescribed extract for sublingual immunotherapy in children (slit). despite the improvement of standardization methods for the production of slit, the differences in cultivation and purification processes used to produce raw materials for specific immunotherapy extracts may still impact on the final composition of mite allergen extracts. our study investigated the total protein and main allergen content of five commercial hdm sublingual immunotherapy extracts using sds-page and immunoblotting. recombinant allergens of group 1 and group 2 major allergens were used to test the immunogenicity of such extracts. method: hdm slit extracts were purchased from five italian suppliers (alk-abellò, allergy therapeutics, anallergo, lofarma, stallergenes). the protein composition of extracts was evaluated analysing equal volumes (15 ml/lane) by sds-page (12% separating gel) and subsequent immunoblotting. for identification of allergens in the extracts, western blot analyses were performed with rabbit monoclonal antibodies (raybiotech) against der p 1 and der p2. the total protein content in the five tested commercial extracts showed a relevant variability. the protein contents ranged from 32.9 to 44.2 μg/mg for what concerns der p1, while der p2 showed a greater variability, ranging from 4.2 to 14.6 μg/mg. sds-page showed a similar pattern of distribution in 3 of the tested extracts, which showed protein bands of comparable intensity, while 2 extracts showed a lower total protein count. extract 2 showed a higher intensity band corresponding to the molecular weight of tropomyosin. western-blotting showed a similar concentration of der p 1 in most extracts, while der p2 was more variable. conclusion: our analysis of five commercial extracts commonly used for sublingual specific immunotherapy against hdm showed important variations in term of total protein content. a less evident but still relevant difference was also evidenced when testing the major allergen content, with up to 25% variation in der p1 and up to a 3-fold variation in der p2 concentration. this differences, likely related to the different production and extraction methods, could still be responsible of a different immunological response in children who underwent slit. method: we evaluated 29 children who had completed their immunotherapy treatment. along with demographic data we were able to record skin prick test (spt) results and mrqlq at start and end of treatment. we only included patients who had completed pre and post treatment questionnaires in the study to allow a comparison. the scores were evaluated using a student t test. results: patients' starting age ranged from 6 to 17 years (mean 11 years). 29 of the 48 children had completed pre and post treatment questionnaires. all 29 had grass pollen allergy confirmed on spt at the start of treatment. 10 patients (34%) had isolated grass pollen allergy on spt and 19 (66%) had multiple allergies. mean start treatment score for all patients was 37 on mrqlq. mean score at end of treatment was 24, indicating a 35% reduction in total mrqlq score (p value <0.05). for those with multiple allergies the mean total mrqlq scores were 33 at start of treatment and 25 at end of treatment, indicating a 27% reduction (p value 0.16). for those with isolated grass pollen allergy scores were 44 at start of treatment and 23 at end of treatment indicating a 50% reduction (p value <0.05). conclusion: for children with uncontrolled symptoms of allergic rhinoconjunctivitis, grass pollen immunotherapy is associated with statistically significant improvement in quality of life. this improvement is most beneficial for patients with isolated grass-pollen sensitivity on spt. those with multiple aeroallergen sensitivities on spt did show an improvement (not statistically significant) post-treatment. grass-pollen immunotherapy is an effective treatment for rhinoconjunctivitis to offer patients in a rural dgh setting. background: allergic asthma is a common clinical refractory disease, most patients with asthma are accompanied by varying degrees of allergy. in clinical practice, treatment of this disease using specific immunotherapy has proven effective. in the current study, we examined the effectiveness of specific immunotherapy in a total of 99 patients admitted to our hospital from 2015 to 2016. method: to investigate the clinical efficacy of allergic asthma-specific immunotherapy. 99 patients were selected, of which 49 were males, aged 22 to 61 years, 50 females, aged between 23 to 64 years old, all patients were clinically diagnosed only as allergic asthma. the 99 patients were randomly divided into two groups, including the observation group containing 50 cases, the control group of 49 cases. all patients were first treated with conventional basic treatment. the observation group was subsequently treated with specific immunotherapy. both groups were followed-up and the treatment efficiency were analyzed. results: after treatment, both two groups of patients showed improvement, in the observation group, the effective rate was 93%, while for the control group, the effective rate was 75%. observation group showed significantly better outcomes than the control group. conclusion: in allergic asthma treatment, adding specific immunotherapy on the basis of routine treatment is beneficial and could be widely used in clinic. case report: atopic dermatitis(ad)is the most common itchy dermatosis that affects millions of children and adults. during recent years, diagnosis and treatment based on component resolved diagnostics (crd)is recommended. we report a 9-year-old boy with severe atopic dermatitis. he had positive family history of atopy. the atopic dermatitis was developed since infancy. he was referred to our clinic when he was 5 years old. he had generalized xerosis with ulcerative eczematous lesions on his neck, popliteal and antecubital areas. he had mild eosinophilia and his serum total ige level was 590 iu/ml. daily bleach bath, moisturizing agents, topical steroids and systemic antibiotics in addition to antihistamine were prescribed. he had multiple food and aeroallergen sensitization in skin prick test (spt). he started to eliminate some foods according to the spt results. he was suffering from recurrent relapse even after strict food avoidance; so treatment with cyclosporine was initiated for him, with partial response. crd showed sensitization to alternaria alternata (alt a1 specific ige:10.97 ku/l). allergen immunotherapy by alternaria alternata was started. after accomplishment of buildup phase, he had significant improvement and we were successful to taper and finally discontinue cyclosporine. now he is on maintenance phase of immunotherapy, his skin is in optimal condition only by hydration and moisturization. result: a 15-year-old thai girl is a known case of severe asthma since one year old. her asthma was uncontrolled asthma even treatment with high dose combination of inhaled corticosteroid and long acting beta agonist (ics/laba), montelukast and omalizumab. spirometry revealed the force expiratory volume in one second (fev1): 34% predicted, fev1/forced vital capacity (fvc): 48% predicted and 14% improvement of fev1 after salbutamol 400 ug inhalation. allergic sensitization showed specific ige to cat: 2.04 kua/l. slit with cat allergen started at the dose of 450 au per month and increased to 1500 au per month (scit dose is 1000 au per month) for three-year-and-six-month course. after slit, her asthma symptom improved significantly. she can exercise without exacerbation and plays sport at school. her last episode of asthma exacerbation was 1.5 year ago. her fev1 (% predicted) was improved from 34% predicted to 48% and the fev1 bronchodilator response decreased from 14% to 6%. conclusion: an improvement of pulmonary function and asthmatic symptoms of the presenting case would support the efficacy of slit of cat allergen in a patient with severe asthma. ra developed during pollen scit in this case might be related with immunomodulation effect of immunotherapy. background: we report a case of 20-year-old woman with allergic rhinoconjunctivitis and mild persistent asthma due to sensitisation to seasonal pollens and molds with bad clinical evolution and not response to conventional drug therapy. we decided to start subcutaneous allergen specific immunotherapy with alternaria extract in our immunotherapy unit in accordance with the guidelines of the european academy of allergology and clinical immunology (eaaci) and we used a cluster regimen. the immunotherapy was not well tolerated: the patient had two grade 2 systemic reactions with the first dose in two attempts. method: sensitisation was diagnosed through skin prick test with aeroallergens standard panel and serum specific ige by elisa. due to the bad tolerance to immunotherapy, molds molecular diagnosis by immunocap, study of the molecular weight to specific ige binding proteins by sds-page ige immunoblotting, and cross-reactivity study by means of immunoblotting-inhibition assay were performed. a. fumigatus extract was able to produce a total ige binding inhibition on the 38 kda band of a. alternata extract when ige immunoblotting assay was performed. conclusion: respiratory allergic disease due to alternaria is difficult to control, the use of subcutaneous specific immunotherapy could be of significant benefit. most of the allergic patients to a. alternata are sensitized to alt a1, major allergen from a. alternata. however, our patient is sensitized to a 38 kda alternaria protein due to cross-reactivity with a. fumigatus allergens, this sensitization could explain the bad tolerance to the alternaria immunotherapy. background: the association between natural pollen exposure, clinical symptoms as well as allergen-specific immune responses has not been investigated at a molecular level. our aim was to monitor the effect of seasonal birch and grass pollen exposure on clinical symptoms as well as specific b cell and t cell responses to defined allergen molecules in sensitized subjects during two consecutive years. method: grass pollen sensitized (n = 10) and birch pollen sensitized (n = 13) subjects were included in this study and were followed for two consecutive years (2013) (2014) . subjects were taking part in a clinical trial for the recombinant grass pollen vaccine (bm32) but did not receive immunotherapy for the allergen they were sensitized to. before, during and after the respective seasons ige and igg levels as well as t cell responses to the major birch pollen allergen bet v 1 and the major grass pollen allergens phl p 1, 2, 5 and 6 were measured. pollen counts were recorded throughout the year and patients kept a daily diary including symptom medication score (sms) and visual analogue scale (vas). results: we noted that ige levels specific for bet v 1 and the grass pollen allergens increased most in the seasons in which patients experienced the highest peak symptoms according to vas and sms but not depending on cumulative pollen counts. increases in allergen-specific t cell responses were observed in the pollen seasons as compared to shortly before the pollen seasons in the grass pollenallergic patients also in association with vas and sms but not in the birch pollen allergic subjects. no relevant changes of allergen-specific igg levels were observed during the two years observation in grass and birch pollen allergic patients. we found an association of increases of allergen-specific ige increases shortly after the pollen season with clinical symptoms in the pollen season as reflected by vas and sms which was not necessarily reflected by cumulative pollen counts in the season. these results may be important for the analysis of allergen-specific immunotherapy trials. background: the morbidity and mortality of severe asthma is much higher than that of mild to moderate asthma. this study was performed to understand the clinical characteristics of severe asthma in korea. results: data from the questionnaire showed that bronchial asthma was diagnosed before pregnancy only in 193 women (4.83%). 43 patients (1.1%) were diagnosed with chronic bronchitis at the pregestation stage. asthma attacks were experienced repeatedly during a lifetime in 4.2% of patients, 12.7% of patients noted long periods of dry cough at night, among them 8.1% had wheezing. the cold did not precede the wheezing breathing in 5.1% of patients. difficulty in breathing on waking was noted in 2.6% of patients, at night-4.3%. after examination, the diagnosis of asthma was confirmed in 14.65% of the respondents (586 people). symptoms of rhinitis are noted in 58% of women surveyed, 18% of rhinitis was allergic. before examination, the diagnosis of ar was only in 3.5% of patients. the incidence of symptoms of asthma and ar in pregnant women is significantly higher than the reported cases of these diseases, which leads to untimely initiation of treatment. method: a total of 117 nonsmoker asthmatic patients without concomitant pulmonary pathology are recruited to our study. all patients underwent spirometry tests, measurement of fraction of exhaled nitric oxide and sputum induction to asses sputum cell counts, demographic features and current medications were recorded. using the variables of age at onset, bmi, allergy status, fev1%, fev1/fvc, asthma severity and induced sputum cytology cluster analysis is performed. results: 4 clusters are identified. cluster1: (n = 43) early onset atopic asthma, consists of mild asthmatics with a good asthma control and lower bmi. cluster 2: (n = 14) severe atopic asthma, consists of lowest spirometry measurements with a least act scores. induced sputum cytology shows a neutrophilic character, while having also the highest percentage of eosinophils. cluster 3 (n = 27) late onset obese asthma, nonatopic asthmatics having high spirometry measurements, with a lower act scores. cluster4 (n = 33) nonatopic mild asthma, consists of patients with the best respiratory functions and least inflammation in means of lowest total ige, feno, sputum cell counts. conclusion: identification of asthma phenotypes in different countries will improve our understanding on the heterogeneity of the disease among the different geographies. results: results and discussion. in the course of analysis, obesity was more common in children with bronchial asthma −25% than in the comparison group-in 9.3%. obesity of the 1st degree was diagnosed in 10 patients of the main group, ii degree-in 8, and iii degree-5 and iv degree-in 2 patients e diagnosis of obesity, the sds indices of body mass index (bmi) were determined. obesitymore than +2.0 (i degree: sds bmi 2.0-2.5, ii degree: sds bmi 2.6-3.0, iii degree: sds bmi 3.1-3.9, iv degree: sds bmi ≥4.0). conclusion: thus, the results obtained indicate a high prevalence of constitutional-exogenous obesity in children with bronchial asthma and precedes the formation of the underlying disease e diagnosis of obesity, the sds indices of body mass index (bmi) were determined. obesity-more than +2.0 (i degree: sds bmi 2.0-2.5, ii degree: sds bmi 2.6-3.0, iii degree: sds bmi 3.1-3.9, iv degree: method: postal questionnaires were distributed to an unselected group of asthma patients (n = 190). healthy non-asthmatic volunteers were recruited amongst university and hospital co-workers (n = 53). the presence of self-reported nhr, the type of triggers evoking nasal symptoms, asthma phenotype, medication use and environmental factors were evaluated. results: 114 patients and 53 controls completed the questionnaire (responder rate of 60% and 100% respectively). nhr was reported in 71% of asthma patients and 22% in non-asthmatic controls (p < 0.0001), with changes in temperature being the most important inducer of nasal symptoms (74% of asthmatics), followed by strong odours (62%) and cigarette smoke (61%). interestingly, nhr was more prevalent in patients with severe (79%) compared to mild (50%) asthma symptoms (p = 0.015), and more prevalent in atopic (77%) compared to non-atopic (55%) asthmatics (p = 0.028). most asthma patients reported more than one trigger evoking nasal symptoms, with 44% of patients reporting 3 or more triggers evoking nasal symptoms. results: the mean score of cbcl questionnaire in case group with 28.12 ± 2.06 was significantly higher than in comparison with a control group with 17.33 ± 9.7 (p = 0.01). the mean scores of the subscales of social isolation (the case group: 5.83 vs control: 1.18, p = 0.006), anxiety-depression (4.99 vs 2, p = 0.22), intellectual problems (4.8 vs 2.3, p = 0.000), and aggressive behaviors (5.1 vs 2.3, p = 0.003) were significantly higher in children with asthma than in healthy children. the study showed a significant correlation between the mean duration of asthma and a general score of cbcl (p = 0.012, cc=0.8). moreover, there was also a significant correlation between asthma severity and cbcl scoring (p = 0/001, cc=0.03). conclusion: behavioral disorders in children with asthma are significantly more than healthy children. the duration of asthma and the severity of asthma, are related to and can predict behavioral disorders in children with asthma. background: assessment of asthma control is an integral part of the management of asthma. whilst asthma control test (act) is a commonly used questionnaire to assess symptom control, its utility in predicting long term risk of exacerbation has not been well studied. aim: to analyze the factors associated with uncontrolled asthma symptoms using act and its impact on predicting future exacerbation. method: severe asthma patients on at least step 4 background: most of the asthma-scoring tools detect the asthma severity from patients' symptoms but there is no scoring tool using parameters to define risk of asthma exacerbation. thus, this study use factor analysis to evaluate the relationship of parameters in childhood asthma. method: the descriptive study using factor analysis in asthmatic children aged less than 15 years old, who attended thammasat university, the center of excellence for allergy, asthma and pulmonary diseases, thailand. the participants or caregivers were inter the factors which have the major impact on asthma control are changing bed sheets less than once per month and using dust mite-proof bed sheets. this study is supporting non-pharmacological strategies but further studies are needed to create a more efficient asthmatic symptom checker. were not different between the controlled and uncontrolled group. the act score in the controlled group was significantly higher than the uncontrolled group (p < 0.001). the study showed that cigarette smoke is one of the significant factors that can trigger asthma exacerbation (p < 0.001) and mosquito repellent coil smoke is also significantly associated with asthma exacerbation (p < 0.03 background: experimental studies have demonstrated that tumor necrosis factor family member 14 (tnfsf14/light) plays an important role in airway remodeling. there is little data available concerning in vivo regulation of tnfsf14/light expression in humans. the aim of this study was to evaluate serum concentration of tnfsf14/ light in different subsets of asthmatic patients. the study was performed on 36 nonsmoking asthmatic patients (a), including 24 mild-moderate-severe asthmatics controlled on inhaled corticosteroids (aics) and 12 asthmatics evaluated twice during asthma exacerbation (aex) and during subsequent remission (arem). in addition age and sex matched 24 nonsmoking healthy controls were included (hc). serum tnfsf14/light concentration was evaluated using elisa method. in asthmatic patients lung function tests, exhaled nitric oxide concentration (feno), serum total ige concentration (t-ige), allergen-specific ige concentration (s-ige) and peripheral blood eosinophilia were evaluated. (250 + /-134 pg/ml) was significantly greater than that in hc (72 + / -32 pg/ml; p < 0.001). among all asthmatic patients studied the greatest tnfsf14/light serum concentration was demonstrated in aex (357 + /-79 pg/ml), which was significantly greater than that in aics (194 + /-123 pg/ml p < 0.001). during resolution of asthma exacerbation a significant decrease in serum tnfsf14/light concentration (118 + /-70 pg/ml; p < 0.001) was demonstrated. in arem the mean serum tnfsf14/light concentration was comparable to that seen in aics (p = 0.06) but was still significantly greater than in hc (p < 0.01). no significant correlation could be demonstrated between serum tnfsf14/light concentration and baseline lung function parameters, exhaled nitric oxide concentration, serum t-ige or s-ige concentration or peripheral blood eosinophilia. conclusion: enhanced production of tnfsf14/light seen in asthmatic patients, which is further upregulated during asthma exacerbations may play an important role in asthma pathogenesis. method: two models of aspergillus fumigatus-induced allergic airway inflammation were used in the study: long terms (4 weeks) and short terms (2 weeks background: chalcone 4 is identified as an inhibitor of the interaction between cxcr4 or cxcr7 and their ligand cxcl12. therefore it is called a neutraligand. the chemokine cxcl12, interacting with the cxc-receptor4 (cxcr4) can play a role in the progression and development of bronchial asthma. asthma is defined as a chronic disease characterized by episodes of obstructive events which affects about 300 million people over the world. the aim of this study is to approach the mechanism of the anti-inflammatory effect of the cxcl12 neutraligand chalcone 4 and also to assess its impact on the migration of dendritic cells in a murine model of allergic airway inflammation. method: chalcone 4 is administered intranasally to balb/c ovalbumin (ova) asthma mice and control groups as well. our results indicate that the cxcl12 neutraligand chalcone 4 can modify the inflammatory reaction in an airway allergic hypereosinophilia model. furthermore, found out that cxcl12 neutraligand chalcone 4 prevents dc migration to the airways and airway jnc ganglia during allergic airway inflammation. the detection of the cxcr4-cxcl12 pathway and its role in the pathophysiological actions of asthma offers a promising target for allergic diseases treatments. method: four groups of balb/c mice were defined: control and asthmatic, with and without treatment. asthmatic groups were sensioverexpression of ptgdr in pulmonary cells associated to a generalized increase of cytokine expression. conclusion: in a mouse model we confirmed the involvement of ptgdr in allergic asthma by the increase of its expression levels after ovalbumin sensitization. we also identified a reduction of ptgdr levels in response to dexamethasone treatment. the in vitro model suggests that ptgdr induces an inflammatory response, increasing the cytokines levels. 0979 | immune imbalance between transcription factor t-bet/gata3 and allergic asthma results: t-bet mrna expression of peripheral blood lymphocytes in patients with allergic asthma was lower than that of the normal control group, and the expression level of gata-3 mrna was higher than that of the normal control group (p < 0.05). the th1 percentage of peripheral blood lymphocyte subsets was lower than that of the normal control group (p < 0.05), the percentage of th2 cells was significantly higher than that of the normal control group (p < 0.05), and the changes in t-bet/gata3 expression and th1/th2 ratio was highly correlated. our objectives were to assess the changes of bmp4 and bmp7 serum levels in the response to allergen and methacholine challenge tests and the correlation between bmp4 and bmp7 serum levels and fev1 before and after allergen and methacholine challenge tests. method: study group consisted of 59 patients with asthma and 48 healthy volunteers. spirometry, skin prick tests, allergen and methacholine challenge tests were performed in compliance with eaaci, ers and ats guidelines. personalized clinic surveys including act ™ were performed. venous blood was collected before and after 1 hour, and 24 hours afterwards the provocation to edta-ke-filled test tubes. evaluation of bmp4 and bmp7 serum protein levels was performed using specific elisa immunoassay kits according to the manufacturer's protocol. results: the increase in bmp4 and bmp7 serum level 24 hours after provocation test correlates significantly with the concentration methacholine during provocation time (p < 0.05). bmp7 serum level before the provocation, 1 hour and 24 hours after provocation, correlates negatively with fev1 change (p < 0.05). the median bmp7 level 24 hours after provocation was significantly lower in patients with negative methacholine challenge test compared to the control group (p = 0.03). the median bmp4 level 24 hours after provocation was higher in patients with positive allergen provocation test than in patients with negative test results (p = 0.03). the bmp7 serum level 24 hours after positive methacholine test is lower and correlates inversely with fev1 change in every time point, which could indicate that serum level of bmp7 is a predictive factor of fev1 change. the higher bmp7 serum level, the lower fev1 change was observed. this could suggest the protective influence of bmp7 in patients with obstructive pulmonary disease, i.e. asthma. the higher bmp4 serum level 24 hours after positive allergen provocation test result shows that the bmp4 could be an indicator of the response to a specific trigger. background: inflammation and coagulation are closely linked events. thrombin is the key enzyme in coagulation system. besides its well-known functions in hemostasis, thrombin plays a role in inflammation. the aim of our study was to evaluate thrombin generation in children with mild asthma and demonstrate associations between thrombin levels and control of asthma. method: forty-two children with mild asthma and forty-nine healthy children included in the study. asthmatic children had no asthma exacerbation during the last 6 months. patients (n = 27) who had mild persistent asthma, were using either inhaled steroid or montelukast. all patients performed spirometry. thrombin levels were measured by thrombin generation test. thrombin peak levels, endogenous thrombin potential, thrombin lag time, time to thrombin peak and thrombin tail time were recorded. results: thrombin lag time was significantly longer in children with asthma (3.98 ± 1.2) compared to those in control group (3.29 ± 0.6) (p < 0.01). children with asthma also had longer thrombin tail time compared to control group (19.5 ± 8.9 vs 16.7 ± 2.9, p = 0.02). thrombin peak was inversely correlated with fef 25-75 (-0.41, p < 0.01). thrombin lag time was inversely correlated with fef 25-75 (-0.39, p < 0.01). thrombin generation parameters did not show difference according to asthma control treatment, asthma control scores and having atopy. conclusion: coagulation/anticoagulation balance is disturbed in mild asthma but this disturbance may not be as strong as to increase thrombin levels. factors increasing inflammation may cause an increase in lag time, and increase in inflammation and excessive fibrin deposition may contribute to airway narrowing. background: cytokines represent key mediators in the onset and persistence of inflammatory process, in both asthma and copd. il-31, which belongs to il-6 family, it might act in a similar way with il-6 at the beginning of the inflammatory process. its role in atopic skin diseases has already been demonstrated, but there are conflicting results related to its role in respiratory allergic diseases. the aim of the study was the evaluation of il-31 plasmatic level in patients with asthma and copd and the its correlation with clinical and lung function parameters. method: fifty consecutive patients with bronchoobstructive diseases were included in the study. thirty-two patients presented asthma and 18 patients had copd. the evaluation included: number of exacerbation in the last year, disease's severity, spirometry. plasmatic levels of il-31 and il-10 were determined in all patients. results: the mean age was higher in patients with copd dermatophagoides pteronyssinus [house duste mite (hdm), 100ug/ mouse] were administered oro-tracheally on days 0, 7, 14, 21, 28, 35, 42 and 49 . at was performed in a treadmill during 4 weeks in moderate intensity, from day 24 until day 52. results: at inhibited hdm-induced total cells (p < 0.001), eosinophils (p < 0.01), neutrophils (p < 0.01) and lymphocytes (p < 0.01) in bronchoalveolar lavage (bal), and eosinophils (p < 0.01), neutrophils (p < 0.01) and lymphocytes (p < 0.01) in peribronchial space. at also reduced bal levels of il-4 (p < 0.001), il-5 (p < 0.001), il-13 (p < 0.001), cxcl1 (p < 0.01), il-17 (p < 0.01), il-23 (p < 0.05), il-33 (p < 0.05), while increased il-10 (p < 0.05). airway collagen fibers (p < 0.01), elastic fibers p < 0.01) and mucin (p < 0.01) were also reduced by at. at also inhibited hdm-induced airway hyperresponsiveness (ahr) to methacholine 6.25 mg/ml (p < 0.01), 12.5 mg/ml (p < 0.01), 25 mg/ml (p < 0.01) and 50 mg/ml (p < 0.01). mechanistically, at reduced the expression of stat6 (p < 0.05), stat3 (p < 0.001), stat5 (p < 0.01) and jak2 (p < 0.001), similarly by peribronchial leukocytes and by airway epithelial cells. socs1 expression (p < 0.001) was upregulated in leukocytes and in airway epithelial cells, socs2 (p < 0.01) was upregulated in leukocytes and socs3 down-regulated in leukocytes (p < 0.05) and in airway epithelial cells (p < 0.001). conclusion: at reduces asthma phenotype which is followed by positive modulation of socs-jak-stat signaling in peribronchial leukocytes and in airway epithelial cells. rodolfo a 1 ; paciência i 2 ; rama t 1 ; leão l 1 ; silva d 3 ; rufo j 2 ; mendes f 4 ; padrão p 5 ; oliveira fernandes e 6 ; moreira p 7 ; delgado l 8 ; moreira a 9 porto, portugal; potentially irritating chemicals that may have a cutaneous drying side effect. this study aimed to evaluate if skin barrier function, as measured by transepidermal water loss (tewl), is affected by a training session in swimmers compared with football players. environment impact on the human respiratory health (clinicaltrials.gov identifier: nct03017976) and football players were invited to participate. due to the lack of prior information no sample size calculation was possible and all athletes that provided informed consent were included in the analysis (n = 58, 29 females, aged 12 to 21 years). tewl was measured using the tewameter ® tm 300 before, immediately after, and 30 minutes after a 2 hours training session. the probe was held on the dorsum of hand, the volar forearm and the antecubital flexure for 60 s. the average of two consecutive measurements was recorded. non-parametric statistic was used were appropriate. ethical approval was obtained from the university clinical research ethics committee and informed consent provided. results: mann-whitney u test showed significantly higher baseline median tewl level on football players hand's dorsum compared with swimmers, median (p25-p75) respectively 17.1 (14.7 to 21.7) and 13.7 (11.9 to 16.2); p = .001. friedman test revealed a significant effect of swimming on tewl on the hand's dorsum, volar forearm and antecubital flexure (p < .001) while football training affected only the hand's dorsum (p = .008). differences in changes after swimming and football training were significant only for tewl in volar forearm (p = .028). in conclusion, our exploratory findings do not provide support for a specific deleterious effect of swimming, compared with football training, on the training induced changes in tewl. background: exercise-induced bronchoconstriction (eib) is defined as transient, reversible airway narrowing occurring during or after exercise, is common among elite athletes and associated with epithelial damage. however, little is known about the existence of eib in young athletes. the goal of this study is to investigate the presence and to evaluate potential (bio)markers of eib in young high-school elite athletes in different sport disciplines versus age-matched control subjects. method: high-school selected elite athletes (12-13 years) from different sport disciplines: basketball (n = 26), football (n = 44) and swimming (n = 47) performing at least 12 hours of sport per week (median=13 h) and 17 control subjects (performing less than 6 hours of sport per week) were recruited. the eucapnic voluntary hyperventilation (evh) test was performed according to ats guidelines and adapted for this age group. lung function was measured before, immediately after and 5, 10, 15 minutes after the evh test. the test was considered positive if a maximal fall in fev1 of 10% was measured on at least one time point and exhaustion was excluded. a blood sample was obtained at baseline. sputum induction and skin prick test for the most common allergens were performed after the evh test. results: fifteen swimmers had a positive evh test (33.3%), which is higher than in basketball players (18.2%), football players (17.95%) and controls (17.65%). 40.4% of the swimmers were atopic which is also higher than in basketball players (23.1%), football players (31.8%) and controls (23.5%). serum clara cell secretory protein (cc16) levels are significantly higher in swimmers (7.6 ± 2.5 ng/ml) compared to indoor athletes (5.1 ± 2.2 ng/ml) and controls (5.2 ± 1.2 ng/ml). a significant positive correlation was found between the magnitude of maximal fall in conclusion: young elite swimmers have a higher prevalence of eib compared to basketball and football players. atopy and/or chlorine is a risk factor for the development of eib in young elite athletes. cc16 levels and sputum uric acid levels are increased in athletes compared to control subjects suggesting the presence of epithelial damage already at young age. this is especially observed in young elite swimmers, pointing to a probable role of exposure to chlorineby-products in combination with intensive exercise. results: data from 237 subjects (134 females, 56.5%) were analyzed. frast was positive in 97 (40.9%) patients (55 females, median age of 14 years (iqr 11-18)). in this group, 55 (56.7%) had a previous diagnosis of asthma, 27 (27.8%) practiced federated sports, 18 (18.6%) had smoke cigarette exposition and 3 (3.1%) had a bmi >30 kg/m 2 . 82.5% of patients showed a δfev1% >10% in the first 5 minutes after finishing the challenge. median fev1 reduction was 13.4% ) and 390 ml . frast was more frequently positive in patients with previous diagnosis of asthma (p < 0.01). there were no differences related to conclusion: frast is an important tool to diagnose exerciseinduced bronchospasm without asthma (eib wa ), as well as to diagnose asthma. in our study, frast was fundamental to access eib wa in 28% of patients with rsee, and confirmed asthma diagnosis in 53% of cases with previous asthma diagnosis and negative sbt. there was no difference in the prevalence of atopy between patients with positive and negative frast. patients older than 9 years-old presented higher δfev1% compared to younger patients (p = 0.04). higher levels of feno were observed in patients with positive frast (p = 0.032, p = 0.045), both in patients with and without previous diagnosis of asthma. a positive correlation was observed between feno levels and δfev1% in the whole sample (r = 027, p = 0.009); when these data were analyzed considering a previous diagnosis of asthma, only patients with this condition showed a positive correlation of feno and δfev1% (r = 0.29, p = 0.031). conclusion: our results evidenced that higher feno was associated with atopy and a positive frast, both in patients with and without previous diagnosis of asthma. higher feno seems to correlate with δfev1% in patients with previous diagnosis of asthma. background: specific immunotherapy is the casual treatment for allergic rhinitis. a 28 year old professional footballer suffer from severe allergic rhinitis since two years. during may, june and july his level of playing, concentration and durability decreased about 20%. patient was complaining of runny nose, nasal blockage, each eyes, sneezing, tearing. method: we did skin prick tests -which showed greatest allergy to grass pollen. we confirmed the allergy by specific ige and nasal provocation tests. spirometry was done-fev1 116%. the patient was qualified to undergo specific immunotherapy. however, because of his profession, it was hard to find a day without trainings to get the vaccine. after long discussion, patient decided to start specific immunotherapy-scit. results: the patient start the immunotherapy. he was attuning very irregularly, because of matches, injuries, trips, trainings, and lack of time. several times we had to call the patient to remind him about the immunotherapy. after one year of scit the patient felt big improvement. during grass pollen season he suffered from mild allergic symptoms, and just for few days. after next year of immunotherapy, the patient had no symptoms of allergic rhinitis during the grass pollen season. however, it was the reason for him, to stop sit, before 3rd year of immunotherapy. conclusion: such a treatment-specific immunotherapy-is a burdensome method for both, for professional athletes and doctors. such a patients need to be on special observation, and cooperation with trainers must be obtained, if we want to see results. to improve compliance we have to keep in touch with patients, to remind them about next visit. gherasim a 1 ; choual i 1 ; radu c 2 ; khayath n 2 ; beck n 1 ; jacob a 1 ; schoettel f 1 ; domis n 1 ; de blay f 2 1 alyatec, strasbourg, france; 2 hôpitaux universitaires de strasbourg, strasbourg, france background: late allergic response (lar) is a good asthma model. it has been shown, in individual challenge tests that mite allergen induces more frequently late allergic responses (lar) than cat allergen. the aim of this study is to compare the frequency of lar in asthmatic subjects allergic to mite with asthmatic subjects allergic to cat. method: 24 asthmatic subjects allergic to mite were compared to 21 subjects allergic to cat (gina 1 or 2). the subjects had prick tests≥5 mm compared to the negative controls and specific ige ≥ 0.70 ku/l. the dose selected for the mite and cat allergen was the airborne allergen concentration inducing the most frequently early asthmatic response (ear) (a 20% drop in fev1) and lar (a 15% drop in fev1). results: the frequency of lar with mite allergens was 66.6% and 20% with cat allergens (p = 0.007). the frequency of ear for mites was 78.2%; of 91.3% for ear or lar, and 50% for ear and lar. in contrast, with cat allergens, 55% of patients had an ear, 60% had ear or lar and 25% had an ear and lar. no significant differences was observed between cat and mite allergen regarding the severity and the time necessary to obtain an ear and lar. no significant differences was observed between cat and mite allergen regarding the severity and the time necessary to obtain an ear and lar. the frequency of lar in asthmatic subjects allergic to dust mite exposed in alyatec ® eec was higher than in asthmatics sensitized to cats. our results confirmed previous results with individual bronchial challenge. therefore, it appears that the mite model is more interesting in the study of asthma. exposure chamber in strasbourg (alyatec ® ) in asthmatic patients allergic to cat allergens gherasim a 1 ; choual i 1 ; radu c 2 ; khayath n 2 ; beck n 1 ; jacob a 1 ; schoettel f 1 ; domis n 1 ; de blay f 1 1 alyatec, strasbourg, france; 2 hôpitaux universitaires de strasbourg, strasbourg, france background: as recommended by the task force on environmental exposure chamber (eec), allergenic and non-allergenic exposure must be better controlled in eec. it is the aim of alyatec's eec. the aim of the study is to validate alyatec's eec by determining the concentration of fel d1 inducing 50% of early asthmatic response (ear) and/or late phase asthmatic response (lar) in subjects sensitized to cat. method: it was a randomized, double blind, cross-over study including group a:20 asthmatic subjects allergic to cat and group b:10 asthmatic subjects allergic to another allergen. all subjects were first exposed to placebo. group a was exposed to 2 fel d1 concentrations. the number and size of particles were recorded online during the exposure. group b was exposed to the concentration of fel d1 which fulfills the objective of the study. the mean age of subjects was 29 years (±8). for the 2 concentrations of fel d1, we obtained more than 50% ear and/or lar. the mean time necessary to obtain an ear was: 59.7 ± 8 minutes and 138.6 ± 90 minutes for the lar. the mean fall in fev1 during ear and lar was −29.22% and −17.64% respectively. we didn't observe any severe reaction. no subjects in group b experienced any symptoms during exposure. we have validated alyatec's eec in asthmatic subjects allergic to cat allergens. we also demonstrated its specificity. background: the best test and strategy for diagnosing asthma especially in those patients with negative bronchodilator reversibility tests still remains unclear. in this study we aimed to investigate the diagnostic yield of peak expiratory flow (pef) variability for the patients with symptoms suggesting asthma but negative bronchodilator reversibility tests. method: subjects referred to our outpatient clinic with suspicion of asthma were enrolled in this study. demographics and referral symptoms were recorded, asthma control test (act) scores and health related quality-of-life scores (aqlq, sf36) were calculated. monitoring of pef variability during 2-weeks and bronchial challenge test with methacholine (bpt) were analyzed. asthma was diagnosed by having pef variability ≥20% and/or positive bpt. results: thirty out of 50 enrolled patients were diagnosed as having asthma. when we compare asthmatic patients with nonspecific respiratory symptomatic subjects there were statistically-significant differences regarding to wheezing (p = 0.020), activity limitation (p = 0.058), total symptom score (p = 0.028) and basal fef (p = 0.050) in the favor of asthma cases. multiple logistic regression analysis revealed that lower basal fef 25-75 was an independent predictive factor of asthma diagnosis (p = 0.05). when the bpt positivity was assessed as gold standard for the diagnosis of asthma, the sensitivity and specificity of pef variability for different cut-offvalues (≥20%, >15% and >%10) were 61.5-83.3%, 88.5-62.5% ve 100-16.7%, respectively. conclusion: fef 25-75 is an important diagnostic parameter for asthma. although current guidelines recommend pef variability of 10% for the diagnosis of asthma in general, this cut off level may not be appropriate for this defined group of subjects. our results suggest to use a cutoff level of >15% while excluding asthma and ≥20% while confirming the diagnosis of asthma for patients with asthma suspicion but without shown reversibility. de barayazarra s background: in recent years obesity has been considered as a factor that contributes to the development of asthma, increases exacerbations and leads to poor control of it due to resistance to drugs to control this pathology. it is known that obesity produces chronic systemic inflammation; one of the markers that are affected is the levels of c-reactive protein (crp), which are increased. objective: evaluate, lung function, the use of medications to control asthma and systemic inflammation, after bariatric surgery. results: 15 obese asthmatic patients with surgery, 15 non-asthmatic obese patients with surgery, 16 obese asthmatic patients without surgery. a significant difference was found between the severity of obesity and forced expiratory volume in patients with asthma and without asthma of 1 second (fev1) before surgery with an average of 87.9% at the beginning of the study and 105.5% at 12 months (p:0.0004). in the non-operated group, fev 1 at the beginning was 69% and 83.04% at 12 months (p: 0.0018). the crp, before surgery in all operated patients had crp: 9, at 12 months after surgery they became negative, crp: 0 (p: 0.004). in obese asthmatics with surgery at the beginning, 100% used medication, and at 12 months only 20% in obese asthmatic patients without surgery, 93.75% used the medication at the beginning, at 12 months 62.5% (p:0.0006). method: 20 patients with asthma aged 18 to 80 and a predetermined positive methacholine pc20 were recruited and underwent a single challenge to cause bronchoconstriction of~20% comparing the outcome of the device with spirometry. the subjects were monitored at baseline, after a~20% fall in fev 1 and after bronchodilation back to baseline. the study protocol allowed for an interim analysis of the initial 8 subjects at which point the sensor was calibrated to optimise sensitivity. a further 12 subjects were studied using the optimised sensor. results: all 20 subjects successfully completed the study. the device was found to be straightforward to use by both operator and subject with no concerns regarding safety. the initial sensitivity of the device was found to be suboptimal in the first eight patients to reliably detect changes in lung function. after adjustment to the device the tests results of the remaining 12 subjects were analysed. 66.6% of subjects were female. the mean age of all subjects was 45.8 years. an average baseline fev 1 value of 2.575 (s.d. 0.881) was observed. changes in lung function were detected in 100% of subjects. a baseline value, drop in lung function and reversal were measured in 66% of subjects. the mean percentage drop observed in 66% of subjects using the investigational device was 13.04 %. the mean percentage increase observed using the investigational from drop to reversal was 25.36 %. the device (using ebc 1 ) was able to detect changes in lung function tracked using fev 1 . this provided proof of concept that the device could potentially be used to monitor lung function more effectively in the home than peak flow and supports further development to optimise the device and demonstrate functionality in clinical asthma. method: ninety four patients under 18 years of age seen in the allergy department due to common asthma symptoms (wheezing, dyspnea, cough, chest tightness) with normal spirometry and negative bronchodilator response, underwent mct during 2016 and 2017. the variables studied were: sex, age, body mass index (bmi), asthma symptoms, exercise symptoms, rhinoconjunctivitis, family history of atopy, sensitization to respiratory allergens, spirometric data and fractional exhaled nitric oxide (feno). results: of the total sample, half were women (51.06%) and the other half were males (48.94%). mean age was 11.8 years. bmi was normal in most of them (with an average of 19.55 kg/m 2 ). the most common symptom among the patients with positive mct was cough (79.8%), followed by dyspnea (62.76%), wheezing (34%) and chest tightness (11.7%). 43.6% had symptoms of asthma with exercise and 70.2% had rhinoconjunctivitis. 37.23% had a family history of atopy. 74.4% were sensitized to aeroallergens, mainly to pollens (grass and olive tree). 75.5% of the mct′s were positives, with a mean pc 20 of 1.89 mg/ml. 62.7% had a moderate-severe result (pc 20 ≤4 mg/ml), 7.4% mild (pc 20 4-6 mg/ml) and 5.3% bordering (pc 20 6-8 mg/ml). the mean feno was 19.62 ppb. conclusion: in our series, the completion of a test of hrb was decisive to confirm the diagnosis of asthma in most patients of a pediatric population with symptoms of suspicion (cough, mainly), normal spirometry and negative bronchodilator response (with normal feno in most of them). therefore, we consider it important to include in the routine clinical practice hrb tests in the pediatric population with suggestive symptoms of asthma, despite normal functional and/or inflammation tests. 1000 | cut-points of the ′control of allergic rhinitis and asthma test′ (carat) asthma subscale based on an international survey patients with asthma) in kashan, iran. the data collection tool was a questionnaire with 38 questions, designed to gather information on demographic asthma patients, the current use of mobile functionalities, and the willingness to use these functionalities to receive selfmanagement services, which was distributed among patients with informed consent. the collected data were analyzed by descriptive statistics method using spss software. results: the most use of patients from mobile phone functionalities was to receive information about asthma symptoms and allergens and irritants via mobile internet (42.1%). patients were most likely to use social networking (31.7%) in comparison with other mobile phone functionalities, to receive reminders about appointments and medication. the respondents were most likely to use social networks through mobile phone functionalities, to receive asthma self-management information (53.1%), to communicate with other patients (55.9%), to receive reminders about medication use, and to perform a peak flow meter test (52.4%) and to get an alert when the asthma is not controlled (53.1%). the findings show that asthma patients are currently using the internet search for educational information and they have a tendency to use social networks to receive asthma-related services. patients believe that mobile health is an appropriate intervention for providing educational information, reminders, and alerts and communication with other patients. 1002 | concordance between the determination of asthma control through the gina 2015 guidelines and the act questionnaire-results of the efimera study background: the exacerbation of asthma, progressive worsening of acute episodes, is one of the most frequent attending reasons at hospital emergency unit 1 . several factors causing poor control of asthma, such as inadequate therapy, have been described. in the present study, estimations of asthma severity by researchers were assessed by comparing the concordance between the assessment of asthma control through the gina 2015 guidelines and the act questionnaire method: cross-sectional observational study on the evaluation of factors related to treatment that influence the poor control of asthma was assessed through the gina guidelines and the act questionnaire. patients referred to a pneumologist or allergist by a primary care for the first time were evaluated. two variables were collected for the assessment of asthma control: one derived from the gina 2015 guidelines and another derived from the act questionnaire. regarding the gina assessment, researchers' evaluations guidelines were compared with the scoring calculated from the variables registered in the crd. both measures were compared in terms of sensitivity-specificity to determine their ability to classify patients. the patients included in this study (n = 1682) had a mean age of 45 ± 17 years, with a 64% of women and an average disease evolution of 14.9 ± 14.1. the control of asthma according to "gina results: pts were reasonably representative of those in sls asthma (at sls baseline: 43.8% male; mean age 48.4 yrs; mean asthma control test [act] score 16.5) . the most frequently reported symptoms during sls asthma for these pts were cough/ breathlessness, followed by wheeze, phlegm and chest tightness; breathlessness and wheeze were perceived as the biggest impactors on pts' lives. the aspects of daily life most impacted by asthma were reported as walking at a hurried pace, strenuous physical activity, and asthma-related frustration. since sls began, 50% of pts in this subset reported improvements in overall asthma (44% no change; 6% worsening). perceived changes in symptoms are shown (table) . most pts (66.0%) reported avoiding places with dust, smoke or fumes. most pts (57.5%) perceived no change in overall qol; 36.3% reported improvement. being an act responder during sls (total act score ≥20 or ≥3 change at end of sls) was associated with reported improvements in overall asthma symptoms, lower impact of asthma on qol, and higher perceived confidence/control in managing asthma. more pts (65.8%) in the ff/vi arm reported an overall improvement in asthma vs uc (34.3%); the most evident differences between treatment groups were for breathlessness, wheezing and chest tightness. improvements in confidence/control in managing asthma were reported by 53.8%/49.3% of pts (ff/vi) vs 31.3%/25.3% (uc). conclusion: breathlessness and wheezing were key symptoms in sls asthma and had the biggest impact on pts' daily lives. this patient-centred study enriches the findings of sls asthma. funding: gsk (study 204500) 1004 | asthma and copd treatment adherence and breach using tai questionnaire suarez-vergara m; fuentes-soltero f; garcia-nunez i; ignacio-garcia j background: adherence is defined as medication (inhalator) intake following the dosage and schedule prescribed. adherence mistakes are a public healthy problem according to the big morbi-mortality presented in patients with an incorrect intake. our aim is to evaluate the adherence level and fulfillment in patients with asthma or copd using tai (inhalators adherence test) questionnaire. method: patients with a diagnosis of persistent asthma or copd were selected. we used to size adherence and breach type the tai questionnaire. adherence is defined as good when patient′s test reaches 50 points, medium (46-49 points) and bad (less than 45 points). breach type is defined as erratic when points between questions 1 to 5 are less than 25, deliberate when questions 6 to 10 are less than 25, and unconscious when questions 11 and 12 are less than 4 points. a correct fulfillment is defined when questionnaire reaches 50 points plus 4 points of conscious fulfillment. results: fifty-five patients more than 14 years old (mean age 50.61 years and 50.9% males) were selected. a 67.27% of them were asthmatics, 30.9% copd and 1.81% a mix phenotype. a 21.81% presented a correct fulfillment with conscious fulfillment, and the other 78.18% presented good, medium or bad adherence with a breach type. according to adherence level, a medium adherence was defined in 16 patients (29.09%), with an erratic mistake in 8 patients (50%). bad adherence was seen in 21 patients (38.14%) , with the three breach types in 9 patients (40%). good adherence with unconscious breach type was defined in 6 patients (10.9%). conclusion: tai questionnaire confirms a good adherence and fulfillment in less than 30% patients. an erratic mistake is the most frequent breach type defined in our patients. educational protocols should be applied to improve adherence and fulfillment. 1005 | what is adhesion to treatment of asthmatic patients like in argentina according to the tai questionnaire? background: asthma is a chronic inflammatory disease of the airways, which requires an adequate treatment and control. the adhesion of a patient to an asthma treatment is a critical factor in order to achieve and maintain control. this adherence arises from a consensual agreement of the doctor-patient relationship; it is a complex multifactorial variable in which the variability in human behaviour in relation to its environment influences. background: the association between ambient pollen and asthma has been studied intensively with inconsistent results, attributed to differences in study population, geographic factors (geoclimatic features), data sources, measurement of pollen (different types of traps), and different outcome occurrence (hospitalizations or emergency department visits). we investigated the associations between daily sales of short-acting β2-agonists (saba) and outdoor pollen concentrations in the central france area. the relationship between daily changes in pollen concentrations and daily saba sales obtained from the social security database was analysed with generalized additive models, taking into account confounding factors such as air pollution, weather conditions, and day of the week. results: the daily saba sales (mean, sd) rose from 46. 5 (23.6) conclusion: this study indicates that outdoor pollens contribute to asthma morbidity in the general population. it confirms the highly allergenic role of fraxinus, betula and quercus pollens, but also shows a relatively unknown association between treated asthma and carpinus and platanus pollens, despite their counts being less than 1% of overall pollen concentration. results: of 15 437 asthma patients (mean age 47.8 years, female 62.6%), regular ocs use was identified for 223 patients (1.4%), periodic ocs use for 3054 patients (19.7%), and no ocs use for 12 160 patients (78.7%) 1-year post-index. regular ocs users had a greater mean age, were more often male, and had greater eosinophil counts, lower lung function, and greater prevalence of comorbidities than did the periodic and no ocs users (p < 0.001). total yearly cost was greatest for the regular ocs users (€ 5509), followed by periodic ocs users (€ 2892) and no ocs users (€ 2042) (p < 0.001). among regular ocs users, hospital admissions were the main cost driver (41.5% of total cost), while gp consultations were driving the total cost in periodic and no ocs users (48.0% and 52.9% of total cost, respectively). conclusion: in this sample of patients with asthma in sweden, the total yearly cost of health care resource utilization for a regular ocs user is twice as high as for a patient with no ocs use, demonstrating substantial economic and clinical burden in asthma patients on regular oral steroid treatment. method: children with physician-diagnosed asthma who attended to an outpatient pediatric allergy and asthma center were enrolled in the study along with control subjects. asthma severity and control status of the patients were evaluated according to recent gina guidelines. laboratory investigations including skin prick tests, complete blood counts with differential, total ige levels, serum periostin levels and pulmonary function tests were performed. results: a total of 158 children (125 with asthma and 33 age and sex-matched control subjects) with a median age of 10.2 years (range 5.9-17.0) were enrolled. asthma severity was mild in 41 (32.8%), moderate in 63 (50.4%) and severe in 21 (16.8%) children. children with asthma had significantly higher periostin levels than controls (53.1 ± 13.1 vs 43.0 ± 11.2 ng/ml; p < 0.001). the mean serum periostin levels of children with severe asthma (63.8 ± 10.8) were significantly higher than in children with moderate asthma (53.3 ± 12.7) and mild asthma (47.4 ± 11.1) (p < 0.001). serum periostin levels were found to be significantly correlated with asthma severity (spearman's rho [r]=.41, p < 0.001). analysis using roc curves identified the role of periostin levels in determining children with severe asthma (auc: 0.77, 95% ci: 0.67-0.87, p < 0.001]. conclusion: serum levels of periostin, a novel asthma biomarker, were higher in asthmatic children, and were associated with asthma severity. adam i 1 ; selevestru r 1 ; rogut v 2 ; sciuca s 1 background: nowadays, data from several epidemiological studies confirm the important role of fungi in respiratory disease in the indoor as well as in the outdoor environment. in general, exposure to fungi occurs via inhalation, skin contact, or ingestion. alternaria alternata is one of the most common fungi associated with presence asthma and persistence and severity of asthma. although exposure to a. alternata is also may represent a risk factor for development of asthma. in ukraine has been an increase in the number of the mold sensitized children for the last few years. at the same time we can see increasing frequency ba at the children of pre-school age. method: thirty five children aged 3-7 years with allergic rhinitis and high level of asthma predictive index (api) sensitized to a. alternata were included in a 2-year cohort study of the efficacy and safety of slit (diater laboratories, spain) using standardized sublingual extracts containing molds (alternaria alternata). treatment efficacy was analyzed using the score of symptoms such as difficulty in nasal breathing, rhinorrhea, sneezing, itching of the nasal mucosa (upper palate) and discharge from the nose and recurring wheezing. we also have analyzed the level api during the period investigation. symptoms were measured before starting treatment, and at 4, 12 and 24 months after starting immunotherapy. results: slit significantly reduced both symptoms and medication score: nasal symptoms (38% vs. control group) and the use of rescue medications (38% vs. control group), and improved fev1 (in children aged≥5 years). in the slit group, api decreased by 15% for the first year, by 31% for the second year. no patient had a systemic reaction during therapy. our results have shown that slit is an effective treatment in pediatric patients suffering from allergic rhinitis and high api with significantly improved clinical outcomes (less symptoms and less medication intake) in comparison with children treated with symptomatic drugs only. in this study, large and statistically significant differences in symptom and medication scores were demonstrated in patients receiving slit compared to control group. sublingual immunotherapy is effective for allergic rhinitis in children especially early age and is generally advantageous because of the convenient administration and safety profile and ensure prevention of developed ba. bednarek a background: the classification of asthma based on the severity of its clinical course has been recommended by gina since 2008. this division is useful for the patient's initial assessment when asthma is being diagnosed and essential decisions concerning an appropriate therapy are made. the objective of the work is to evaluate the influence of a clinical form of asthma on vaccine immunity in preschoolers following three years after the programme of mandatory vaccination has been realised. the study encompassed 172 preschool children (mean age of 5.22 ± 0.34 years old) with asthma being newly diagnosed, including 140 patients with mild asthma and 32 ones with moderate asthma, whose vaccine immunity (igg specific antibody titer) was assessed after the mandatory early childhood vaccines had been administered. monovalent vaccines (hbv+ipv+hib) along with a three-component combined vaccine (dtwp) were given to 86 children while a six-component vaccine (dtap+ipv+hib+hbv) was given to the remaining 86 children. the vaccine doses were consistent with the polish immunisation programme and manufacturers' recommendations. the elisa immunoenzymatic method was applied to assess titer of specific antibodies to diphtheria, tetanus, pertussis, poliomyelitis and h. influenza type b. the level of hbv antibodies was measured chemiluminescently. the immunity class for particular vaccinations was assessed according to the test manufacturers' instructions. results: children suffering from mild asthma had considerably more frequently vaccinations on time (p < 0.001) and the type of vaccines (monovalent, highly-combined) administered to them did not have a significant influence on a clinical form of asthma in the children examined (p > 0.6951). apart from the vaccines against hepatitis b and rubella where considerably more frequently a high antibody titer occurred in children with mild asthma, the titers of antibodies to other vaccines, namely diphtheria, tetanus, pertussis, hib and mumps, were not associated with a clinical form of asthma. the protective antibody titers in the children with asthma were found in 100% after vaccinating them against poliomyelitis (≥12u/ ml) and measles (≥300 ml u/ml). significantly higher current weight was solely found in the children with mild asthma (m = 20.80, sd=3.77; p < 0.05). conclusion: there are some clinical and cultural differences among the four southern chinese cities within the canton province. this study identifies potentially modifiable environmental and treatment factors associated with poor asthma control and qol for healthcare interventions. having a smoker in the family is independently associated with poor asthma control and qol. were classified into two groups (levocetirizine group (l) and montelukast group (m)) and we treated each group for another 10 week. to evaluate the therapeutic effectiveness, we used symptom score (ss) and ebc leukotriene e4 (lte4). ebc samples were collected with rtube. each parameter was checked at 0, 2, 12 week therapeutic period. results: most ar patient showed clinically improvement with 2 and 12 week fluticasone therapy (0 wk ss=6.6, 2 wk ss=3.2, 12 wk ss=1.9 p < 0.01 in l group; 0 wk ss=6.8, 2 wk ss=3.6, 12 wk ss=2.0 p < 0.01 in m group). lte4 levels of ar were higher than control (0 wk 77 vs. 12 pg/ml), and were reduced after 2 week flutimic were: md allergic rhinitis, wheezing apart from colds, eosinophilia ≥4%. outcome was defined as md asthma and at least 1 episode of asthma during the previous year or more than 3 episodes of wheezing during the 12 months regardless of asthma diagnosis. results: from a total of 144 of parents approached, 86 (58%) agreed to participate in a phone interview. 18 (21%) children were diagnosed with asthma. the age at the time of admission (mean, abstracts | 533 (sd)) was 27.0 (9.9), at the time of phone survey 77.6 (10.9) months, respectively. positive loose api at 2-3 years of age had sensitivity of 77.8%, specificity 75%, positive predictive value (ppv) 45.2%, negative predictive value (npv) 92.7%. positive stringent api at 2-3 years of age had sensitivity of 50%, specificity 91%, ppv 64.3%, npv 85%. background: asthma is the most common chronic airway disease in childhood, with a high unmet need for new treatments due to insufficient symptom control in a relevant percentage of patients. ethics and resource factors limit the feasibility of large, long pediatric trials required to assess outcomes such as exacerbations and symptoms. for diseases like asthma, where the disease process is largely similar in children and adults, with the same expected therapy outcome, the international council for harmonisation advise extrapolating adult data to those of a younger age, reducing unnecessary pediatric trials. here we assess the partial extrapolation used in the clinical development of tiotropium. phase 3 trials in adults (aged 18-75), adolescents (aged 12-17) and children (aged 6-11) with symptomatic severe (primotina-/pensie-tina-/vivatina-asthma) or moderate asthma (mezzotina-/rubatina-/ canotina-asthma), respectively. trials lasted 12-48 weeks, all with tiotropium respimat 5 μg add-on vs placebo as two puffs once daily. results: in adult trials, lung function, symptoms and exacerbation endpoints were evaluated in a confirmatory manner: tiotropium significantly improves lung function and asthma control, and reduces risk of exacerbation, vs placebo ( conclusion: based on similarities in disease profile and magnitude of treatment responses between age groups, it is reasonable to expect tiotropium add-on to produce clinically meaningful improvements in exacerbation and symptom endpoints in children and adolescents, as in adults. the robust tiotropium clinical program supports using a partial extrapolation to avoid overly long and large trials in pediatrics. 1019 | clinical state of treatment and examination during last 3 years before remission about asthmatic children in long-term remission cases method: remission cases (no symptom and no therapy) for 3 years of asthmatic children were studied. clinical background and treatment (drugs) was studied during last 3 years before remission annually. acetylcholine inhalation test by standard method was performed, and respiratory threshold of acetylcholine (rt-ach) was obtained. fev1%, and serum ige also examined. these data were compared before remission with 3 years after remission. results: mean age of 25 cases at 3 year before remission was 9.2 years old. male to female ratio was 1.3. severity of asthma was all mild type, and number of attack was 2 to 8 times in a year. there was no admitted case during this study. the long-term therapeutic drugs were leukotriene receptor antagonist (anti lt) in 20 cases, and/or inhaled corticosteroids (ics) in 12 cases, but 5 cases had no treatment for the control. geometric mean of rt-ach (after then: 3 years before and after remission) was 2900 μg/ml and 5800 μg/ml. the mean fev1% was 83% and 90%. geometric mean of serum ige level was 360 iu/l and 410 iu/l. complicated cases of atopic dermatitis decreased after remission, but the incidence of allergic rhinitis increased slightly. conclusion: characteristics of asthmatic children during last 3 years before remission were mild type, had several times of attack in a year, and the treatment was mainly anti lt and/or ics. fev1% was within normal range, and serum ige level was not changed after remission. rt-ach had the tendency to improve during 3 years before and after remission. these data is supposed that airway hyperresponsiveness is one of the indicators for quitting treatment. 1020 | clinical aspects of polyvalent mechanic bacterial lysate (pmbl) treatment in children with uncontrolled asthma our results indicate that long-term treatment with omalizumab in children can help to achieve better asthma control and reduce the amount doses of basic therapy. method: allergic rhinitis (ar) and allergic rhinoconjunctivitis (arc) diagnosed-patients' demographic information, accompanying-asthma, the allergic history of the family, the onset of symptoms, types of aeroallergens sensitivity were noted from patients' files in our hospital's pediatric allergy clinic. results: in this study, 675 patients were evaluated. the mean age of the patients were 11.65 ± 3.85 years and 61% (n = 412) were male. 452 (67%) patients had ar and 223 (33%) patients had arc. background: allergic rhinitis (ar) is a disease characterized by symptoms of nasal discharge/congestion, sneezing, and pruritus, and is caused by an ige-mediated immunological response to inhaled allergens. we aimed to evaluate pollen season and out of pollen season pulmonary function tests (sft) of patients with ar in our study. method: in our study, the demographic characteristics and aeroallergens were recorded from patients' files with ar diagnosed. in addition, pollen season and out of pollen season sfts were evaluated and compared. conclusion: in patients with ar, fev1 and fvc values are seen to be lower during the season even though there is no lower respiratory symptom. therefore, sfts of patients with ar should be evaluated during pollen season. results: among the clinical manifestations, the most common combination of allergic rhinitis (ar) and conjunctivitis (ac) is noted in 83.80% of adults and 55.11% of children, but in children aged 3-7, the combination of ar and ac is observed only in 48.40%, among 8-12 years old-51.25%, while in the remaining age groups it is encountered in more than 80%. higher percentage of isolated ar is also observed among young children-17.55%, and those of the results: allergic rhinitis was a main symptom in 69.8% of children with pollen-food sensitization. in all of them concomitant allergic disorders were noticed: bronchial asthma (77.7%), atopic dermatitis (77.8%). only in 26.7% temporal association between ingestion of pollen-related foods and nasal symptoms was observed (mainly apple and peanuts); occurring also outside the pollen period. the simultaneously sensitization to animal origin food allergens was stated in 63.3% of children with sar, but only in two of them milk and white egg proteins were an additional exacerbation factor of nasal symptoms. in 18.5% anaphylactic reactions to food allergens were registered. 36.7% of children were asymptomatic despite pollen-food sensitization. the statistically significant differences were noticed in comparison to the control group. conclusion: 1. allergic rhinitis in children, similar to adults, is a common manifestation of pollen-food syndrome and this type of sensitization should be taken into account regardless to age. 2. children with pollen-related food allergy have the predisposition to multiorgan clinical manifestation. 3. the lack of association of symptoms with plant-origin foods in the majority of cases and the asymptomatic course of food sensitization in more than one third of patients indicate the need for follow-up. 1026 | clinical benefit of the screening of suspected food allergen using multiple allergen simultaneous test in the patient with pollen-food allergy syndrome (pfas) background: the quantitative fluoresce enzyme immunoassay immunocap (ic) system has been widely used for detection of allergen-specific ige for the diagnosis of allergy. however, the system can only detect ige against a single allergen, the multiple antigen simultaneous tests has been developed such as the fluorescence enzyme immunoassay view allergy 39 (va) or chemiluminescent enzyme assay mast iv (ma) and both assay detect more than 39 allergen-specific ige. in this study we examined the diagnostic capability of these two systems for screening test in the patient with pfas. method: total number of participants are 37 (male/female: 20/17), aged 12.0 ± 3.9 (range 3~20) years old. all the patients showed oral allergy syndrome (oas) to rosaceae family plants (apple, peach) and/ or kiwi and/or banana, also showed tree pollen allergy. specific ige assay were performed using ic, ma or va. results of greater than class 1 were to be regarded as positive, and the concordance rates between the assays were assessed. results: the correlation of sensitivity between pr-10 (rbet v1, rmal d1, rpru p1, measured by ic) and specific ige to apple (measured by va), specific ige to peach (measured by ma) in 27 oas patients to rosaceae family plants were assessed. rbet v1, rmal d1, rpru p1 were found to be 96.0%, 96.3%, 92.6% positive measured by ic while the specific ige to apple (supposed to be including pr-10) were found to be 100% positive measured by va. on the other hand, the specific ige to peach (supposed to be including pr-10) were found to be only 33.3% positive measured by ma, this detection rate was lower than that of va (p < 0.0001). also, the correlation of sensitivity between pr-10 (ract d8, measured by ic) and specific ige to kiwi in 17 patients with oas to kiwi were assessed. ract d8 were found to be 76.5% positive measured by ic while the specific ige to kiwi (supposed to be including pr-10) were found to be 64.7% and 17.6% measured by va and ma, respectively (p < 0.005). additionally, all the 3 oas patients to banana found to be positive for the specific ige to banana measured by va, but only 1 patient was detected as positive measured by ma. conclusion: in this study, we found that va showed better agreement of sensitivity and specificity with ic compared to ma in the oas patients to rosaceae family plants, kiwi, or banana. therefore, it may be clinically useful for screening of allergen specific background: the hygiene hypothesis for autoimmune and allergic diseases, which exists nowadays, shows that human immune system is dependent on various environment factors. we consider the effects of humic substances (hs) to be important in understanding the hygiene hypothesis. due to urbanization, the amount of human interaction with hs found in soil has significantly dropped. the goal of our work was to study allergenic potential and antimicrobial activconclusion: hs appear to be exogenous immunocorrectors, and also to have an ability of suppressing propagation of allergic reactions and sensibilization, which leads to conclusion that they seem to play a major role in hygiene hypothesis. moreover, hs selectively interact with bacterial cell wall, and this effect could be used in order to create antimicrobial drugs based on hs. background: peach tree pollen has been identified as having relevant allergens (the third most prevalent after olive tree and grass pollen) in areas of high cultivars (murcia, east-spain). when analyzing molecular components in sensitized patients, along with pru p 3, we have identified other relevant inhalant allergens one of which was named pru p x. because pollen of different species share allergens and with plantderived food, we have also studied peach tree pollen sensitization in a non-exposed population (madrid, central-spain). the aim was to study the association between peach tree pollen and several panallergens, as well as the relevance of pru p x in our area (madrid). method: a total of 328 patients who came to our allergy unit in those patients with positive spt to at least one pollen we also performed peach tree pollen spt. if positive, we tested pru p 3, pho d 2, pho d 3 and pru p x. to study the clinical relevance of these findings, we also performed nasal provocation test (npt) with peach tree pollen and pru p x. results: a total of 57 patients were sensitized to peach tree pollen. from these, 33% had also positive spt to pru p 3 and none of them to pru p x. positive spt to polcalcin were found in the 33% of the cases and to profilin in the 13%. in 7 patients sensitized to peach tree pollen npt was performed being 4 cases positive to peach tree pollen and none to pru p x. conclusion: peach tree pollen sensitization in non-exposed patients with allergy to other pollens is high although primary sensitization is unlikely. these patients present clinical response when exposed to that pollen that needs further evaluation. in our study, one third of the patients were also sensitized to polcalcin and pru p 3 and none to pru p x. we have not found clinical response to this new inhalant allergen identified in highly exposed peach tree pollen population. results: the bet v 1 elisa 2.0-ep complete kit format (including pre-coated plates and all buffers and reagents) allowed for the consistent measurement of bet v 1 in birch pollen extracts within the same lab (intralab cv=5.5%) and between different labs (interlab cv=13.5%). the average recovery from matrix spiked samples (crs in birch pollen extracts) ranged from 75-112%, with an average recovery of 91% (n = 10). assay time was reduced from several days to two hours compared to the original method. the performance of the bet v 1 elisa 2.0-ep kit was comparable to that of the stallergenes greer candidate standard method and has been successfully cross-validated. this will enable allergen manufacturers and regulatory authorities to adopt a standard method for bet v 1 determination, which, ultimately, may be included in the european pharmacopoeia. the development of a certified elisa represents a major step forward in the standardization and quality control of allergen products. 1031 | an isoform of the ole e 7 allergen assembled by proteomics could explain the cross-reactivity with pollen and food nsltps results: a total of 457 peptides were obtained by de novo sequencing. ten of them allowed the completion of the full-length amino acid sequence of the allergen. after purification, role e 7 was obtained with a yield of 1.5 mg/l of cell culture. immunological assays confirmed that the recombinant isoform of ole e 7 shared most of the allergenic and antigenic properties of the natural allergen. moreover, we observed its implication in cross-reactivity with pollen extracts, and plant-derived food extracts. conclusion: these results suggest that the presence of this isoform in the olive pollen could explain the co-sensitization observed in some allergic patients between ole e 7 and nsltps from foodderived extracts and might be used for a more effective clinical diagnosis of olive pollen sensitized patients. background: penicillium oxalicum, one of the prevalent airborne fungi in india, was selected to detect its spores as potential source of allergens and also to identify and characterise its major ige-reactive component. the airborne spores of penicillium oxalicum was detected by andersen 2-stage air sampler at different parts of west bengal. the allergenic potency of p.oxalicum was tested by spt, elisa and immunoblotting. total protein was resolved in 1-d and 2-d gel electrophoresis and allergens were identified by 1-d and 2-d immunoblots. identification of major ige-reactive protein spots was made by mass spectrometry based maldi-tof-tof. major allergen was partially purified by ion exchange chromatography. results: aerobiological investigation clearly indicated the predominance of p. oxalicum spores (56 cfu m −3 ) in the air of west bengal, india. sensitivity of patients to spore antigens was highly correlated with rhinitis. in sds-page, 80 bands were detected with molecular weight range of 16-180 kda. the allergenic potency of spores was confirmed by skin-prick test, elisa and dot-blotting. eleven ige-reactive proteins were detected as allergens by 1-d and 2-d immunoblots, of which 43% patients were sensitized to 22 kda allergen. this 22 kda protein was found to be the major allergen which was further characterized by mass spectrometry based maldi-tof-tof. this major allergen (pi 6.08) was partially purified by ion exchange chromatography. the eleven allergens were identified from spore of penicillium oxalicum fungi for the first time from india. immuno-proteomic identification of major ige-reactive protein (22 kda background: airway epithelium (ae) is one of the largest cellular surfaces exposed to the environment. ae constitutes a physical barrier due to the presence of intercellular apical junctional complexes between neighboring cells. in the past years evidence indicates an association between epithelial airway dysfunctionality and allergic asthma. it is still unclear if an impaired epithelial barrier could be the cause of allergy development as opposed to the consequence. one of the most common comorbidities of asthma is house dust mite (hdm) allergy. it has been shown that hdm allergen der p 1 can disrupt the epithelial airway due to its protease action against cellular apical junction complexes damaging the epithelial monolayer. in the last decade, metabolomics has been successfully employed as a new approach to describe metabolic changes in biological systems. metabolomics focuses on describing and identifying small molecules to explain complex biological processes. we theorized that metabolomics could be used as a new tool to detect damage of epithelial barrier in vitro after der p 1 exposure. method: human cell line calu-3 cultured at air-liquid interphase (ali) was used as an in vitro model of bronchial epithelium. ali culture system allows establishing 2 different compartments, mimicking the conditions found in the human airways: a basolateral compartment in which basolateral surface of the cells is in contact with the culture medium, and an apical compartment where the apical cellsurface is exposed to air. after 7 days in ali, the cells were exposed to either der p 1 or pbs as a control in the apical side for 24 hours. then, apical and basolateral media were collected and processed for metabolomics analyses. results: metabolic profiles from samples were obtained, these were composed by 248 and 397 features for apical and basolateral media, respectively. of these, using mann-whitney unpaired test as statistical analysis, 108 and 15 features were found changed within the apical and basolateral compartments, respectively. specifically, in the apical compartment there were 104 signals significantly increased and 4 decreased after der p 1 exposure; whereas for the basolateral compartment, 11 signals were found to be significantly decreased and 4 increased after exposure. background: mites are one of the major causes of allergies. it is known that allergen concentration varies depending on the species of mites and the degree of allergy induction is different, but the difference in microbiota according to mite species is not known. in addition to allergen, endotoxin or bacterial dna, adjuvants of allergen derived from the microbiota in the mites, are also present in the feces. bacterial endotoxin is found in gram-negative bacteria, acting on tlr4 and acting as an adjuvant to allergies. method: three species of mites (d. farinae, d. pteronyssinus, and t. putrescentiae), known to cause allergies, are cultured in same condition(autoclaved media, 80%rh, 25°c)and analyzed for microbiota of each species. using the next generation sequencing that complements the existing sanger sequencing, we analyze the difference of microbiome according to the dust mite species and measure the level of endotoxin. method: six hundred and thirty five patients (51.8% males and 48.2% females, mean age 30.2 years old, range 3 to 82 years old) were included. all of them referred respiratory symptoms (rhinitis, conjunctivitis or bronchial asthma) and had skin prick tests positive with any pollen. patients were skin prick tested with a battery of common pollens in our area, including three species of chenopodiaceae: chenopodium album, salsola kali and salsola oppositifolia. results: three hundred and forty tree (54%) patients were sensitised to pollen of any chenopidaceae species: 255 (40.2%) to chenopodium album, 245 (38.6%) to salsola kali and 220 (34.6%) to salsola oppositifolia. the prevalence of skin sensitisation to pollen of salsola oppositifolia was 34.6% in the population studied and 64.1% in patients sensiresults: in patients aged 1-60 years of age in 80.5% of the cases ige reactivity was at least to one allergen tested. the majority of patients (more than 2/3) had a complex sensitization profile and reacted on average to more than 5 allergens. the highest frequency of sensitization in ukraine among patients who turned to the clinic among adults was found phl p1 (34.1%), amb a1(33.3%), fel d1 (33.3%), bet v1 (30.2%) and children (29.2%, 34.6%, 38.5%, 36.9%), respectively. when analyzing the results of tests for the source of the allergen, most often among house dust mites (hdm) allergens in adults and children is sensitization to fel d1 (35.9%), as well as to hdm: in adults (der f2-15.1% der p2 −15.1%, der f1-12.7%, der p1-11.9%) and in children (12.3%, 13.1%, 9.2%, 10 .8%), respectively. among fungal allergens the most common is sensitization to alt a1 and varies from 2.4% in adults to 26.9% in children. among pollen allergens in adults is sensitization to phl p1 (34.1%), amb a1 (33.3%), bet v1 (30.2%), cynd1 (23.8%), art v1 (22.2%), bet v2 (11.1%) and in children (29.2%, 34.6%, 36.9%, 19.2%, 16.9%, 21.5%), respectively. tests for food allergens in adults and children are more common on pr-10 proteins. in children, sensitization to milk and egg proteins is more common than in adults. conclusion: most patients who came to the clinic have a complex ige reactivity profile in which pollen sensitization predominates. among hdm allergens, more than 1/3 of the examined have sensitization to the cat's proteins. sensitization to mold alternaria alternata in children occurs 10 times more often than in adults. results: total 130 children were examined, aged 1-16 years (median 5 years). 72% children were sensible to two and more components 57.7%to 5 and more components. the frequency of sensitization to inhalation components was 65.4%, to food abstracts | 545 components-33.1%. among the most frequent inhalation components were feld 1-39%, betv1-37%, amba1-35%, phlp1-29%, alta1-27%, the sensitization to house dust mites (hdm) was most often observed to der p2-13%. however, the analysis of these protein by the level of isu showed that the highest levels were for der f1median 19 (iqr 9.4-47.0), whereas for fel d1-6.9 (iqr 2.6-15.0). among food allergens, sensitization was most commonly observed to pr-10 proteins -42%. children sensitized to pr-10 proteins were in most cases sensitized to -mal d1(28%), cor a1.0401(27%), pru p1 (22%).this co-sensitization was accompanied by a high correlation of isu levels among these components. sensitization to celery and kiwi was less common, the level of these proteins was also low. the frequency of sensitization to storage proteins was 38%, among which the highest level of isu was in ara h6 median 13 . sensitization to ltp proteins was detected in 22% of children, among which the most commonly detected pru p3 protein was 9.2%. the sensitization to profilins, which was evaluated at the level of bet v2, was found in 21% of children, but the levels of these proteins were not high. among the food products of animal origin, the most frequent was sensitization to egg component gal d2 −13.1%, however, isu levels were the highest to milk component bos d4 −9.8 (iqr 1.2-21.0). the most frequent causative inhalation allergens were epidermal allergens and weed pollen, however, the highest level of isu was to hdm and mould. among food allergens, the most commonly observed sensitization was to pr-10 proteins. hypereosinophilia of peripheral blood was observed in 87 children under study, which was 50% (4.7%). as a result of testing patients with a wide panel of allergens, 98% of the patients had diagnostic levels of antibodies to allergens siged1, 91.2%to allergens siged2. in 50% of cases, a significant level of antibodies to plantain allergens sige w8 was detected, 30.8% to dandelion allergens sige w9, 35.7% to evergreen trees sige t23, to maple sige t11 to 33.3%, to allergens of olive tree sige t9 -40%, to the banana allergens sige f92 -63.6%, to the egg protein sige f1 in 24.5%, in 34% to the milk allergens sige f2, to the food mixture sige f x5 -37.7%, to allergens of mold fungi mx2 −17.8%. among the leading household allergens were registered in the 1st group and in the 2nd group of the investigated children -d pteronyssinus (80.3%, 97.8%), and d. farinae results: the prevalence results are expressed in the table 1 . we have not observed any significant association in allergic rhinitis patients group with any ltp or pr-10 molecules. for atopic dermatitis only rara h 9 (or with 95% ci -10.2 (1.9-54.6) and njug r 3 (or with 95% ci -6.7 (1.6-29.5)) were associated significantly. for asthma, the most important molecules were rbet v 1, raln g 1, rcor a 1.0101, rcor a 1.0401, rmal d 1, rpru p 1 and rapi g 1 (p-values for or less than 0.05). conclusion: future studies focusing on the evaluation the association of cross-reactive molecules with allergy phenotype should be done. background: the fuzzy/green kiwifruit (actinidia deliciosa), widely grown commercially, contains various pulp allergenic molecules, including the major allergen cysteine protease actinidin. methods. this case report is about a 40-year-old male patient with house dust mite allergic persistent rhinitis and intermittent asthma, presenting a convincing history of anaphylaxis immediately after eating a kiwifruit on empty stomach, followed, a few months later, by a severe oral allergy syndrome after licking a slice of raw kiwi. previously, the patient ate kiwi without any problems and had no manifestations of pollen or latex allergy. skin prick testing was done with commercial allergen extracts, while prick-prick testing was performed with raw kiwifruit, avocado and banana. molecular approach consisted in assessment of serum specific ige to native extracts and molecular allergen components using patient-friendly allergen nanobead array multiplex test and singleplex capsule-enclosed activated cellulose solid phase fluorescence enzyme immunoassay. results. regarding kiwifruit allergy, the patient presented positive prick-prick tests with raw edible kiwifruit components: outer pericarp and inner pericarp (each 15 mm wheal) and columella/core (19 mm wheal) and negative with kiwifruit whole seeds, avocado and banana, and pollen extracts. serum specific ige to kiwifruit were detected (0.5 ku/l), but specific ige values were negative (≤0.01 fiu/ml) for actinidin act d 1, thaumatin act d 2, kiwellin act d 5, nsltp type 1 act d 10, bet v 1-like major latex/ripening-related protein act c 11, act c chitinase_iv, act d 9 cross-reactive profilins bet v 2 (birch pollen profilin) and hev b 8 (latex profilin), and also negative (<0.1 ku/ l) for pr-10 ract d 8. moreover, specific ige to avocado were nor found (≤0,01 fiu/ml). although ige against seed proteins cupin/11s globulin act d 12 and 2s albumin act d 13 were not determined, this was not considered of great importance since allergic symptoms were also induced by licking kiwi pulp, in which abundantly expressed actinidin enzymatically degrades seed storage proteins, and prick-prick test was negative to kiwifruit seeds. conclusion: in a patient with anaphylaxis to kiwifruit, positive skin tests to its pulp and detectable serum specific ige to actinidia deliciosa, a detailed molecular allergy diagnosis is necessary, including assessment for act d 3 glycoallergen or other molecules, not performed in this patient. 1049 | is pr-10 sensitization a portuguese phenomenon as well? background: bet v 1, a major allergen found in birch pollen, belongs to the pr-10 protein group. in our practice, some bet v 1 sensitized patients have been identified, residing in areas without this tree genus in its flora. our aim was to characterize a portuguese patient population with pr10 sensitization. method: a group of patients in whom immunocap isac ® (isac) study was performed, between january 2009 and june 2017, were analyzed. all subjects with one or more pr-10 sensitizations were selected, and their clinical records reviewed. a sequential sample of the last subjects (n = 80) who underwent isac study, was then used for comparison. results: out of 234 isac studies performed, only 16 were positive for pr-10 protein group. median age was 18.3 years, 68% (n = 11) were male. pr-10 sensitized individuals were more likely to live in portalegre district compared to the control group (7/16 vs 1/80; p < 0.001). 15 patients were positive for pr-10 family pollens (93.7%), frequently bet v 1 (n = 13), followed by aln g 1 (n = 10) and cor a 1 (n = 8). 14 out of the 16 patients were sensitized to pr-10 foods, mostly cor a 1.0401 (n = 13) and mal d 1 (n = 12). skin prick tests revealed birch as the main sensitizing pollen as well (14/ 16). moreover, only four patients were skin prick tested for fagaceae trees which were positive for oak (4), chestnut tree (3) and cork tree (1) . all patients were co-sensitized to other pollens, namely grass and all had respiratory allergy. nine patients were food allergic, although seven of them were co-sensitized to other cross reactive (ltp/profilin) or species specific proteins. conclusion: although pr-10 sensitization is known to be rare in our population, mostly alto alentejo inhabitants showed sensitization to this protein family in our sample, either by in vitro and/or in vivo methods. this phenomenon is consistent with the native plant species of this region, which should be taken into account when studying the allergic profile of these patients. in our sample, all pr-10 sensitized patients had respiratory allergy while this protein didn't seem to be relevant when it comes to food allergy. further studies are needed to characterize which plant species belonging to this protein family are more significant for our country's aerobiology context and to determine its clinical relevance. included. allergic asthma, rhinitis, conjunctivitis and eczema allergic symptoms were diagnosed. all patients were tested by immunocap with mugwort pollen extract and the natural components nart v 1, nart ar 2, nart v 3, and nart an 7. results: the positive frequency and sige levels of the four components in the artemisia allergic patients from southwestern china were significantly lower than that from the north. art v 1 and art an 7 were the highest recognized allergens, followed by art v 3 and art ar 2. patients from northern china were more likely to have abstracts | 549 asthma (50%) than patients from southwestern china (3%), and being sensitized to more than two allergens increased the risk of asthma. sensitization to art v 1, art v 3 and art an 7 played a significant role in the development of asthma. artemisia pollen allergic patients is helpful to assess the potential risk of asthma. conclusion: a small but significant part of the population react to ragweed pollen extract and are not identified as disease-positive by standard sige tests. there is a need for targeted tests towards a larger spectrum of allergen molecules. in ragweed allergic individuals, this allergy can be the main cause of overall sige levels and also of in vivo reactions (tested by spt). 1052 | molecular profile of pollen sensitization of tashkent residents with respiratory allergy background: in paediatric cohorts, a correlation between specific ige (sige) levels to house dust mite extract or allergen components and the occurrence of asthma has been shown. higher levels of sige to mite extract were associated with a higher risk of wheezing. moreover, asthmatic children recognized more allergens and had higher sige levels to nder p 1 as well as rder p 2, 5 and 23. we sought to investigate potential differences in sige levels or sensitization patterns between asthmatic and non-asthmatic patients in a mixed paediatric and adult house dust mite allergic cohort. method: total ige and specific ige against house dust mite extracts (dermatophagoides pteronyssinus and farinae) and allergen components (rder p 1, 2, 10, and 23) were determined in 190 house dust mite allergic patients. 35 patients had diagnosed asthma ("asthmatic", 54% females, mean age 27 ± 15 years, 31% younger than 18 years), whereas 155 had rhinitis (and conjunctivitis) without respiratory symptoms ("non-asthmatic", 54% females, mean age 28 ± 15 years, 29% younger than 18 years). results: total ige levels were markedly higher in asthmatic compared to non-asthmatic patients (315 vs. 144 ku/l, p = 0.022). positivity to rder p 1 (71 vs. 52%, p = 0.039) as well as rder p 10 (9 vs. 1%, p = 0.044) differed between both groups. specific ige levels to house dust mite extracts and allergen components (rder p 1, 2, 10, and 23) and positivity to rder p 2 and 23 did not differ between both groups. conclusion: in contrast to previously published data, sige levels to house dust mite extracts or allergen components were not statistically different between asthmatic and non-asthmatic patients in our mixed paediatric and adult house dust mite allergic cohort. only higher total ige levels and a higher reactivity to rder p 1 and 10 were found in asthmatic patients. however, larger studies are needed to confirm clinical relevance of these findings. results: prior treatments reported at baseline (bsl) included: 17.3% of pts were receiving 1 or more second-generation h 1 -ah at approved dose (recommended first-line), 23.9% were receiving them at increased dose (second-line); 8.5% were receiving omalizumab (third-line); 29.9% had no treatment. the majority of pts (78.2%) had uncontrolled csu (uct<12) at bsl (table) . treatment changes were most evident at the bsl visit, with an increase in pts receiving omalizumab (21.4%) and a decrease in those receiving no treatment (12.8%) vs. prior therapy. these changes were associated with improvements in rates of hives and/or angioedema, uct and qol scores at month 3, but only modest improvements thereafter (table) . a sub-analysis of 528 pts with uct<12 and who were receiving the approved (36.9%) or increased dose h 1 -ah (63.1%), revealed that few pts had recommended escalation from the approved to increased dose h 1 -ah (0.0-6.7%) or from increased dose h 1 -ah to omalizumab (0.0-11.1%) (table) . conclusion: poor physician adherence to guidelines was evident throughout aware. initial improvements in disease activity and qol plateaued after month 3, possibly owing to fewer changes to recommended therapies. greater physician adherence to guidelines is needed for better symptom control in pts with uncontrolled csu. results: we revealed that in russians urticaria is associated with rs20541*arg/gln genotype of the il13 gene (p = 0.02) and rs5743794*cc genotype of tlr6 (p = 0.0011) gene polymorphism. in tatars the association with disease development was shown for rs5743571*tt genotype of tlr1 gene snp (p = 0.0054). the rs5743794*c allele of tlr6 gene polymorphism is associated with acute and chronic forms of urticaria (p = 0.0209 and p = 0.0063, respectively) and rs2243250*c allele of il4 gene polymorphismwith acute urticaria (p = 0.0247). method: 100 csu patients from the urtica cohort (clinicalttrials.gov number: nct01940393) participated in the study. a questionnaire was carried out evaluating the triggers identified by the patients, the comorbidities and the treatments received. patients with a self-report of skin exacerbation by foods, nonsteroidal antiinflammatory drug (nsaid) or physical triggers were subjected to a controlled provocation test with the suspect food, medication or physical stimuli report by the patient. the levels of anti-tpo ige were measured during a period of clinical control and during two exacerbations in all patients. results: 30% of the patients had at less one inducible urticaria demonstrated by provocation tests (24% dermographism, 10% cold, 8% pressure). self-reported exacerbation for a food (60%) or medication (30%) were high, but positive provocation tests were low (1% and 14% respectively). 30 patients had (+) anti-tpo ige during the baseline period. among them, 60% presented a significant elevation of anti-tpo ige during at less one of the two exacerbations. 18.5% of patients (n = 13) with (−) anti-tpo ige, presented elevation of anti-tpo ige one of the two exacerbations. conclusion: foods, drugs and physical triggers must be verified by challenge tests to avoid unnecessary lifestyle restrictions in patients with csu, nevertheless self-report is usually greater than positive provocation tests. increase concentrations of anti-tpo ige seems to be implicated in urticaria exacerbations in some patients with csu. brzoza z 1 ; adamczyk k 2 ; wcislo-dziadecka d 3 ; zbiciak-nylec m 2 ; brzezinska-wcislo l 2 adipokines. the aim of the study was to evaluate the possible contribution of leptin to chronic spontaneous urticaria pathophysiology. the study included 48 chronic spontaneous urticaria patients and 41 healthy subjects. the leptin level in both examined groups was measured. results: no statistically significant difference in leptin level was determined between the studied subgroups. we are among the first to present the effects of exploration aimed at assessment of the possible role of adipokines in chronic spontaneous urticaria pathogenesis. in this study we did not prove any difference in leptin level. in our opinion it is valuable to perform further studies in this area. the microorganisms were inactivated with phenol, and the concentration was adjusted to 1 000 000 microbial cells/ml (labeled as a 1/ 1). dilutions 1/2 and 1/4 were made from the product labeled 1/1. the dot blot technique was used to detect the presence of specific ige to the different microbial antigens and controls (anti ige 1/1 and 2 fold dilution ½ and ¼). the dot blot images were processed with a documentation system (gel doc ez, bio-rad), and the different microbial antigens in different dilutions were compared with the positive anti-ige controls. results: all patients have specific anti ige to microbial antigens (see table below). the presence of microorganism-specific ige could explain, the relationship between the infections and / or microorganisms in ciu, as well, the urticaria control by omalizumab, even when it has not been detected ige sensitizations to common allergens. finally, these findings, showed that the bacterial allergy could be one line of research to understand the unresolved etiology of urticaria. background: dermographism is the most common form of inducible urticaria. it shows itself as hives made by scratching or rubbing on the surface where it has been produced and with the same morphology. the pathogenesis has not been clarified nor has it been associated until now with the sensitization to allergens. we have studied the relationship between the presence of dermographism and domestic mites sensitization. we have selected 95 patients older than 14 years old. all of them had symptoms compatible with dermographism at the moment of medical evaluation. at least one third of patients additionally showed rhinitis and/or asthma symptoms. we performed:: -skin prick tests with our basic neumoalergens (mites d. pteronyssinus y lepidoglyphus destructor, pollen, molds, dog, cat and horse dander, latex and anisakis simplex). -determination of specific ige levels for dermatophagoides pteronyssinus, lepidoglyphus destructor, and anisakis were measured in serum by using the immunocap (thermo fisher scientific). -blood count, serum immunoglobulins, antithyroid antibodies, serine tryptase and proteinogram. results: blood count, serum immunoglobulins, antithyroid antibodies, serine tryptase and proteinogram were normal. we divided patient in different groups. background: urticaria results from the appearance of pruritic papules and/or erythematous plaques caused by substances from mastocytes present in the skin, notably histamine. chronic urticaria is defined as flare-ups that occur at least two or three days per week over a six-week period. in addition, affected subjects are often prone to an atopic or auto-immune profile that promotes urticaria [1] . the association of polyphenols (ambora, green tea) and the soothing active ingredients slow down the itching biological process from the outset by reducing the release of pruritic mediators (e.g. histamine, cytokines, etc.) of immune cells such as mastocytes and lymphocytes, involved in urticaria. in this context, the purpose of the study was to evaluate the efficacy and the tolerance of an anti-pruritic spray containing the polyphenols and the soothing ingredient. the tested product aims to quickly calm the itching in subjects with chronic urticaria. results: on average, the product was applied 2.3 times per day with a significant decrease of 5d-pruritus scale (-35%) and sensations of itching (-58%) between d0 and d21. in terms of quality of life, a significant decrease of the skindex score was observed (-48%). the product soothed the pruritus within 60 seconds for all subjects and the anti-pruritic effect lasts at least 2 hours for 80% of subjects. the product also showed very good cosmetic properties and was well tolerated; no intolerance case was reported. showed near complete remission. in the week before omalizumab and for a few days after, her urticaria flared but on 3 of 4 weeks she was largely asymptomatic (uas7 0-5). after 3 years of successful treatment she reported an increase in csu activity. no trigger factors could be identified. add-on treatment with cyclosporine was refused, montelukast showed no, and prednisolone only transient benefit. over a period of 2 months wheals occurred almost daily and a maximal score of 42 was achieved on uas7. we replaced omalizumab with cyclosporine but this was subsequently discontinued due to side effects. 3 months later the patients' csu remained poorly controlled with up to 100 wheals occurring almost daily despite rupatadine 40 mg/d. due to the good initial response to omalizumab and lack of good treatment alternatives, a trial of re-treatment was considered. results: within 1 week of re-commencing omalizumab she once again achieved near complete remission of csu with uas7 ≤ 3 on 3 of 4 weeks. the mechanism of action of omalizumab and the development of resistance to it in csu, are incompletely understood. our case shows that some csu patients developing resistance to omalizumab may benefit from a subsequent trial of re-treatment, particularly if treatment alternatives are poorly tolerated. manipulate and store data by electronic means. this includes e-mail, sms text messaging, video chat and online social media as well as all the different computing devices that perform a wide range of communication and information functions. a rapid increase in the use icts in recent decades is an enormous contributing factor in the development of a number of novel clinical and public health intervention strategies. the aim of the present study is to assess the level of ict use and to examine patterns of preferences among patients with chronic urticaria (cu). method: we will conduct an anonymous multicentre cross-sectional study, starting from january 2018, to investigate the use of icts in patients with cu, using a questionnaire as a survey method. this questionnaire will assess the frequency of use of social media and icts in patients, and their preferences for receiving and asking disease-related information. the survey will consist of 20 items, evaluating demographical information, time with disease, medication currently used, and additional aspects of social network use. results: we will use a chi-squared test to assess the association between internet access or owning a cell or smartphone, and age, gender, type of urticaria, educational level and number of years since diagnosis. we will employ the same test to assess the association between the independent variables previously introduced and the frequency of use of each ict type (short messaging service [sms], facebook, twitter, youtube, email, internet, linkedin and skype) as well as agreement in receiving and seeking information (i.e. asking questions to the practitioner) through such icts. we will perform adjusted regression analyses between categories of age, gender, educational level, type of urticaria, years since diagnosis and the use and level of interest shown in communicating through icts. our aim is to report on remarkable findings from a registry of a large sample of patients, potentially providing clues for its approach and results: 285 patients with a median length of 14 months suffering from urticaria were registered, being 71% women; mean age 35.9 years. in 72% of patients no causal agent was identified. parasites were found in 9.3% and thyroid peroxidase antibodies in 8.9%, while autologous serum skin test was positive in 47% and igg to mycoplasma in 42% of evaluations. two thirds of patients reported wheals on uas7, with just 1/4 having concomitant angioedema. almost 2/3 reported significant affection on quality of life because of itch by cu-q2ol. just 14% of patients achieved total control on first anti-histamines provided, and less than half had good control of urticaria. cetirizine was the first choice in 44%, followed by fexofenadine (15%) and first generation anti-histamines (16%). method: cases at 18-65 years of age which were being followedup in our clinic with diagnosis of chronic urticaria and were not receiving any antihistaminic medication for last one month were included in the study. cu-q2ol, uas-7, psqi and psg results of the patients were evaluated. correlation of data with each other in regard to sleep disturbances was evaluated. results: 21 patients were included in the study. patients' mean total score in cu-q2ol was 36.25 ± 13.23. patients' mean uas-7 value was 16.71 ± 9.41. mean total psqi was 9.75 ± 3.92, the ratio of total scores ≥5 and those with poor quality of sleep was 87.5%. mean epworth sleepiness scale (ess) score was 9.71 ± 2.05, with total score ≥10 in 52.4%. in psg, mean apnea-hypopnea index (ahi) was 11.93 ± 12.6, with 44.4% of the patients having ahi ≥5. when patients having ahi<5 were compared with patients having ahi ≥5, no significant difference was determined in regard to total cu-q2ol score, mean score for questions concerning status of sleep, uas-7 and psqi. when correlation analysis was performed between cu-q2ol and total score for questions concerning status of sleep, a positive correlation was determined with psqi (p = 0.037). conclusion: it was demonstrated in our study that patients with chronic urticaria had poor quality of sleep and this disturbance was independent from ahi. omalizumab was discontinued due to absence of improvement in csu symptoms after three consecutive doses. the plasmapheresis without intravenous immunoglobulin replacement was initiated. results: the symptoms were relieved during the first procedure and the disease improved shortly thereafter. the following weeks the symptoms still occurred but with lower intensity and severity. (angioedema was gone). the second attempt with omalizumab was successful after this course (5 procedures of plasmapheresis). case report: rosacea is a chronic skin disorder associated with flushing, erythema, dryness, burning and stinging, and inflammatory papules and pustules. new treatments available or in development target the inflammatory and erythematous components of the disease. these agents include the selective alpha-2 receptor agonist brimonidine. allergic contact dermatitis to brimonidine is an unusual condition. in addition to this, urticarias due to brimonidine are rarely reported. we report on a 35-year-old woman who, immediately after apply a thin layer of brimonidine gel as preparation for a rosacea treatment on her face developed facial urticaria, which reverted in approximately four hours with systemic steroids. she had previously tolerated this product without any problems, but has not use it again ever since. skin prick-tests with brimonidine (0.03 mg/ml) and latex were realized in the patient. skin prick-tests with brimonidine were realized in eleven healthy control subjects. results: skin prick-tests with latex was negative in the patient. skin prick-tests with brimonidine were positive in the patient (9x5 mm). the prick-test with brimonidine was negative in teen healthy control subjects. we report on a case of immediate urticaria due to brimonidine and triggered by an immediate, probably ige-mediated, hypersensitivity mechanism. we highlight this case because it is the only case described in the literature with a positive prick-test. method: the study was in accordance with the helsinki declaration and was previously approved by the national comity of ethics. this was a one dose study conducted on 16 fasting young healthy volunteers, of which 11 were females and five males. the mean age was 21 ± 1 years old and the body weight 61. 13 + 9.44 background: cetirizine is a potent h1-receptor antagonist indicated in the treatment of allergic rhinitis and urticaria. cetirizine is widely used due to its potent antihistaminic effects in yielding strong and fast relief of itchy sensation, sneezy and rhinorrhea and its unlikely probability to manifest anticholinergic side effects in therapeutic doses. histamine flare and wheal inhibition by anti-h1 are widely used as a standard to test and compare the effect intensity and duration. our study aimed to test these effects of cetirizine in young healthy adults. method: this was a double-blind, single dose study in healthy young adults, previously approved by the national comity of ethics. eleven females and five males with a mean age 21 ± 1 years participated in this study. histamine skin pricks were tested before and after they received a tablet of 10 mg cetirizine as previously scheduled. twenty minutes after each test flare and wheal were drawn in a transparent paper which was then scanned and measured with a software. wilcoxon signed ranks test two-sided with significance at 5% level was used to analyze the differences. claims that the preparation relieves itch within 60 seconds of its application. we performed a simple study to verify this claim. we used irp in 20 consecutive subjects, 15 males, median age 12, range 6-49 years, whose workup implied ast. their preliminary diagnoses were "asthma" (6 subjects), "allergic rhinitis" (10 subjects), "atopic dermatitis" (2 subjects) and "food allergy" (2 subjects). all of them had refrained from systemic antihistamines for at least one week. standard skin prick tests (spt) were applied as appropriate, including histamine controls to assess the level of their skin sensitivity. subjects were asked to mark their sense of itch in the area of the skin to be tested on 100 mm visual-analogue scales (vas) starting from "0"-"no itch" to "100"-"unbearable itch". vas assessments were repeated 20 minutes after ast was done; then irp was applied according to the manufacturer's instructions, and the vas assessments were repeated after 60 seconds and 20 minutes. results: there vas assessments are shown in table format: table 1 irp did not affect the wheal and flare of the histamine control, nor did it abolish positive spt. no differences were outlined between subjects with different diagnoses. the commercially available itch relieving preparation not containing defined pharmacological antihistamine is effecting in relieving itch associated with allergen skin testing. before ast (1) 4.9 ± 2.6 vs (2) p < 0. represent the first-line treatment for osteoporosis-related mastocytosis. we report a case of sm with bone pain and with an area of osteolysis in the femur as first sign and symptom. we had to consider the risk of adverse reaction when we decided to treat the patient with bp, but the patient was under antihistaminic treatment and also we made a premedication to reduce the risk. the pk/pd model available was informed by data from 12 clinical trials. the pd endpoint data was available from two studies and used to characterize the effect of bilastine on wheal and flare. moreover, food effect had been characterized in 2 pk studies and the data was used to model the effect of food in the pk of bilastine. the pk parameters relative to the fed state were then used to simulate the temporal evolution of the wheal effect using the pk/ pd model. all analyses were conducted by nonlinear mixed effect modeling (nonmem v 7.2). using the pk model developed (food effect model) and the pk/pd model already available, monte-carlo simulations for plasma concentrations and pd over time were performed for both the fed and the fasting states. results: . a reduced bioavailability (f) and a slow absorption constant characterized the pk of bilastine when administered concomitantly with food (f = 77% relative to the fasting state and ka = 0.51 hour −1 , a 3-fold reduction compared to fasting conditions). the rest of the pk parameters remained unchanged. onset of action was 1 hour for bilastine both in fed and fasted conditions. maximum wheal inhibition occurred at 3.5 hours (fasted 78% and fed 77%). from 2 to 12 hours, the percentage reduction with bilastine for both fasted and fed was between 75% and 86% after the third day of treatment. a 50% inhibition in wheal effect was maintained during 23 hours for both conditions after the third day. the results of the simulations show that even if the pk is altered with food, the pd is maintained unchanged. conclusion: even if a significant food effect was described for bilastine at a pk level, the difference is not translated directly into the pd. therefore, the antihistaminic effect of bilastine remains unaffected by the concomitant administration with food. the results of these simulations will be further confirmed in a dedicated clinical trial. results: we also found no correlation between the different tgt parameters and other clinical and analytical parameters associated with uc (table 1) results: both cetirizine products have no differences in respect to the pharmacodynamic and pharmacokinetic parameters analyzed. the 95% confidence interval of the mean ratios of the auc 0-24 , auc 0-inf , cmax, auce 0-24 , and e max , between the test and the reference, were within the bioequivalence ranges (80%-125%) in both cases. no statistical difference was revealed when comparing the respective t max and te max too. the two cetirizine products tested were bioequivalent. the bioequivalence was evident even when tested with the pharmacodynamic parameters. there is strong evidence that supports the use of histamine skin prick test for the bioequivalence evaluation of different cetirizine products. 1089 | bradykinin-mediated angioedema associated with combination of angiotensinconverting enzyme and dipeptidyl peptidase iv inhibitors: a disproportionality analysis from the who database method: we performed a disproportionality analysis using data from the who pharmacovigilance database by a case-noncase study, until the 14/12/2017. we extracted all individual cases safety reports (icsrs) included in the high level term "angioedemas", according to the medical dictionary for regulatory activities classification. given the absence of term "bma", we selected only the icsrs of angioedema without associated symptoms evoking another underlying mechanism, such as histamine angioedema (e.g. pruritus, urticaria, rash, etc.). drug class exposure was "acei" and "dpp4i", considered suspect or concomitant, using the atc classification. we results: there was no correlation between mother's disorders such as periodontitis, rhinitis, diabetes etc. and the onset of ar (p > 0.05). a multivariate analysis showed, neonatal jaundice (p < 0.001), respiratory system infection (p < 0.001), diarrhea (p < 0.001), eczema (p < 0.001) in the first 6 months of life and home environmental factors (house decoration (p < 0.001), mold environment (p < 0.001), keeping flowers (p < 0.001), passive smoking (p < 0.001)) increased the risk of ar. besides, there was no significant difference in current height and birth weight of the participants between ar and control group. however, ar group had significantly lower current weight (p = 0.003) and age (p < 0.001) compared with the control group. paternal age and maternal age in the ar group were significantly higher than the control group (p < 0.01). conclusion: diseases in the first 6 months of life and home environmental factors increased the risk of sequential ar. the older parents increased the possibility of ar in the offspring. the data of general characteristics of participants were statistic analysis by z text analysis. *significance at p < 0.05. results: anosmia was more frequent in crs than in rhinitis (28.1% vs 3.9%, p < 0.001) and in crswnp than in crssnp (40.6% vs 13.4%, p < 0.001). lms was higher in crs than in rhinitis (8 [4-15] vs 0 [0-0], p < 0.001) and in crswnp than in crssnp (10 [6-18] vs 5 [1] [2] [3] [4] [5] [6] [7] , p < 0.001). in addition, lms was associated with loss of smell in patients with hyposmia (or = 2.66 [1.27, 5.53 clinics. patients were submitted to confirmatory exams including oral provocation test with aspirin. nasal polyps were removed by functional endoscopic sinus surgery and eosinophils in this tissue were quantitated. eosinophil counts in peripheral blood was obtained. serum periostin was measured by elisa and total ige was determined using immunocap. as control groups, 12 (9f/3m) patients with par and 23 healthy subjects (14f/9m) were selected. samples of nasal tissue and blood were collected from these subjects during elective surgery for correction of anatomical variations, and compared with the patients with aerd. results: ar symptoms were significantly improved in the treatment group compared with the control group (76.6% (36/47) vs 21.7% (10/46); p < 0.05). furthermore, the mean total vas score for patients in the treatment group was reduced from 6.21 ± 1.83 before treatment to 1.36 ± 1.51 after treatment (p < 0.05). moreover, the reduction in free ige levels was greater in the treatment group than in the control group. the results of this study suggest that the chinese herbal medicine ber may be effective for improving the symptoms of ar. a multicenter clinical trial is needed to confirm this finding. results: in patients with "eosinophilic" polypoid rhinosinusitis, mucociliary transport was 35.2 ± 0.80 minutes, ph 7.4 ± 0.01, suction-88.6 ± 6.5 minutes, excretory-57.9 ± 0.9 mlg and in patients with "neutrophilic" polypous rhinosinusitis, mucociliary transport was 34.5 ± 0.65 minutes, ph 7.3 ± 0.01, suction-76.2 ± 5.0 minutes, excretory-54.9 ± 0.8 ml. the study showed that disruption of the transport function, changing the concentration of hydrogen ions method: this prospective controlled study was carried out on crs patients underwent ess. patients participating in the study were divided into two groups-group 1: partial middle turbinectomy (n = 22) and group 2: partial middle turbinectomy and middle turbinate fenestration (n = 23). objective assessment of olfactory function using the university of pennsylvania smell identification test (upsit) and subjective assessment of symptom using visual analogue score (vas) were performed before and 3 months after surgery. results: there were significant improvement comparing postoperative and preoperative upsit in both group 1 (35.23 ± 2.96 vs 32.23 ± 2.54, p = 0.000) and group 2 (36.09 ± 2.35 vs 32.04 ± 2.64, p = 0.000). the vas were also significantly improved postoperatively compared to preoperatively in both group 1 (5.77 ± 0.75 vs 6.73 ± 1.08, p = 0.000) and group 2 (5.13 ± 0.81 vs 6.78 ± 1.28, p = 0.000). patients undergoing partial middle turbinectomy and middle turbinate fenestration were more likely to show improvements in upsit (4.00 ± 1.20 vs 3.00 ± 1.11, p = 0.014) and vas (1.59 ± 0.21 vs 0.95 ± 0.15, p = 0.000) compared to those with only partial middle turbinectomy. conclusion: partial middle turbinectomy and middle turbinate fenestration during ess is an effective method for improving postoperative olfactory function. 1109 | nasal irrigation for the alleviation of nasal symptoms in pregnant women with allergic rhinitis we sought to determine specific ige responses to bacterial pathogens in sera from cystic fibrosis patients and analyze their kinetic during disease course. genes, respectively. in contrast, most of healthy donors had normal homozygous genotype with tt-60.0 ± 8.9%(n = 18) and cc-56.6 ± 9.0%(n = 17) with low frequency of mutations; gg-16.6 ± 6.8%(n = 5) and tt-23.3 ± 7.7%(n = 7) and heterozygous genotype tg-23.3 ± 7.7%(n = 7) and ct-20.0 ± 7.3%(n = 6) for il-2 and il-4 genes, respectively. following a 2 month treatment, there was a significant reduction of cytokine levels in the il2-29.5 ± 0.5 and increased in the il4-16.6 ± 0.4, when compared to the beginning of therapy and after 2 months (p < 0.001) results: at baseline the 1st group had serum levels of il-2 (43.6 ± 0.8) pg/l; il-10 (34.8 ± 0.8) pg/l and ifn-γ (108.2 ± 1.1) pg/ l; 2nd group had il-2 (39.6 ± 1.5) pg/l; il-10 (38.6 ± 1.2) pg/l and ifn-γ (103.3 ± 1.4) pg/l vs il-2 (21.6 ± 0.8) pg/l; il-10 (50.2 ± 1.2) pg/l; ifn-γ (63.8 ± 2.2) pg/l in the control group. after 2 months, there was a significant decrease in pro-inflammatory cytokine levels in the 1st (il-2: 35 ± 0.9; ifn-γ: 75.6 ± 1.9) pg/l and 2nd group (il-2: 28.6 ± 1.3; ifn-γ: 66.7 ± 3) pg/l, respectively. conversely, il-10 increased in 1st and 2nd groups to 41.9 ± 0.9 pg/l and 46.0 ± 1.7 pg/l (p < 0.05). conclusion: prior to the study initiation patients with tuberculosis had higher il-2, ifn-γ and lower il-10 content than healthy controls. two-month chemotherapy produced significant reduction in proinflammatory cytokines and increase in anti-inflammatory il-10, with levels approaching those of healthy controls. thus, tuberculosis drugs appear to have the anti-inflammatory effect in tuberculosis patients, which was predictive of positive clinical outcome. 1114 | antibiotic resistance: ligands of innate immunity take the challenge in this work, we aimed to perform an ex vivo hrsv infection in precision-cut lung slices (pcls) from human, rhesus, and cynomolgus macaques, comparing whenever possible the response with the viral surrogate poly i:c. method: pcls containing airways were prepared from lung sections of human, rhesus, and cynomolgus macaques. the slices were inoculated with hrsv-a2 10 6 iu/ml, uv-inactivated hrsv, or vehicle control for 48 hours. macaque slices were also incubated with poly i:c 100 μg/ml with and without the immunosuppressive dexamethasone 50 μg/ml. viral replication, tissue viability, and immune response assays were assessed in supernatants, lysates, or slices. the inoculum infectivity of 10 6 iu/ml as well the uv-inactivation were confirmed by plaque-assay on hep-2 cells. immunofluorescence staining using a fitc-labeled anti-rsv showed the presence of infected macrophages in pcls, but not in mock infected samples. hrsv stimulation slightly decreased tissue viability, as seen by live/dead staining and ldh assay. the viral infection increased ip-10 production in pcls of human, rhesus, and cynomolgus macaques, reaching respectively 13.3, 3.4, and 1.7 fold-increase in comparison to the vehicle controls. poly i:c stimulation caused ip-10 response comparable to hrsv in rhesus and cynomolgus pcls. the ip-10 production ratio comparing hrsv/poly i:c was 1.1 in rhesus and 0.9 in cynomolgus pcls. conclusion: hrsv infects ex vivo pcls of human and non-human primates, inducing the release of the pro-inflammatory chemokine ip-10. this response is comparable to the viral surrogate poly i:c. in the future, these systems can be used to further investigate host response to hrsv, especially in the context of asthma development. however, a relatively small number of reports are related to the association of ebv with allergic diseases, in particular atopic ones. we found that among patients with activated ebv infection, polysensitization was found to be 2.2 times more frequent, chest syndrome was 1.32 times more common and hyper-ige syndrome occurred 1.5 times more frequently. in most of these patients, atopy was not detected in medical history. method: we evaluated the laboratory test results of five boys (45.4%) and six girls (54.6%), 11 children (6 with hbov and 5 with cov). their average age at the study time was 59.2 ± 45 months. nasal swab specimens were taken from these patients who admitted to our hospital with respiratory symptoms between 2015-2016. patients are recalled after an average of 21 ± 2.5 months. isaac questionnaire and skin prick test to common inhalated allergens were performed. results: only one patient had family history of atopy. forty percent of the patients with cov and 50% of the patients with hbov developed rhinitis. one patient with cov and one patient with hbov developed recurrent wheezing. one patient with cov developed atopic dermatitis. all skin prick tests were negative. it was noteworthy that 72.7% of the patients were passive smokers. conclusion: hbov and cov may be associated with rhinitis but there is a need for more patient groups for a clear result. rna_lig (ccg-agg-aug-cga-ggc-uug-uu) . to study chemotaxis in vitro, a boyden96 chamber was used -wellfiltrationplatemultiscreentm -mic with a pore size of 8 μm (millipore, usa). chemotaxis was studied in dynamics after 10, 60 minutes and a day using the above ligands. as control, rpmi-1640 medium without glutamine was used (paneco, russia). the statistical analysis was carried out using the computer statistical program biostat2009 conclusion: with all the data provided, a drug induced hypersensitivity was diagnosed. we present a case of immediate allergic reaction with eosinophilia due to carbapenems, with tolerance to other beta-lactams antibiotics. written informed consent of patient has been obtained in the two cases. discussion: the first case shows cutaneous immediate hypersensitivity response to infbeta1a. literature reports a few cases of urticaria and anaphylaxis but this is the first for the pegylated formulation. polyethylene glycol (peg) confers to a drug modified pharmacokinetics, solubility and immunogenicity. immediate reaction to peg (macrogol) have been described when combinated in vaccines or drug pils. dmf is a known cause of contact dermatitis related to footwear, wallets and furniture. flushig is a reported side effect of dms in ms managed with dose reduction. this case shows the possibility to immediate sensitization to dmf. as the armamentarium to treat ms now combines immunomodulatory and biologic drugs, the avaliability of diagnostic and desensitization protocols for hypersensitivity reactions must be keeped in mind. case report: drug rash with eosinophilia and systemic symptoms (dress) syndrome is an uncommon but serious hypersensitivity drug reaction, manifested with rash, fever, lymphadenopathy and visceral organ involvement. table) . drug withdrawal and prednisolone treatment leaded to attenuating of mentioned skin symptoms within 2 days, associated by occurrence of a exfoliative dermatitis. one week after admission, the patient developed fever that lasted for 4 days with enlarged lymph nodes on submandibular, paracervical, axillar and inguinal regions. a preventive antibiotic therapy is started and 2 weeks later, the lymph nodes were not palpable and the skin got the normal appearance. corticoid therapy is reduced gradually according to symptoms resolvement. case 2: a 56-year old woman presented to our department with a 6-day history of pruritic, macular rash, periorbital swelling, cheilitis and fever. she had started some weeks ago the allopurinol for asymptomatic hyperuricemia, had longer history for treatment of arterial hypertension and type-2 diabetes mellitus (olmersartan, nitrendipine, methyldopa, furosemide, regular and glargine insulin), and experienced nephrectomy and cholecystectomy. the patient was febrile, while blood tests revealed eosinophilia, increased seric creatinine/urea levels (due to nefrectomy), and severely-altered liver parameters (see table) . the allopurinol withdrawal, topical and systemic corticoid therapy, and the liver protectors attenuated serologic transaminases levels and patient's skin lesions within few days, followed by substantial improvement of laboratory findings one week after therapy start. the treatment dosage was gradually tapered and finally stopped within a period of 2 months in accordance with attenuating and complete resolvement of the clinical and laboratory abnormalities. our case demonstrated that dress syndrome is a severe drug reaction, but the immediate introduction of treatment and supportive measures can improve disease's outcome even after a temporary exacerbation or severe affection of internal organs. case report: a 40-years-old woman, diagnosed of ischemic cardiopathy, developed an anaphylactic shock 5 minutes after the administration of 2 ml sulphur hexafluoride intravenous during an echocardiogram. she was treated in emergency room with a total recovery. 2 months earlier, she had developed an extensive erythematous-maculopapular rash converging in plaques in relation with adhesive dressings which had been placed during a hospitalization due to thoracic pain. an allergic contact dermatitis was suspected and recommendations thereon were given. interestingly, an arteriogram with iodixanol (icm) was carried out one week before skin reaction with good immediate tolerance. methods: blood test: blood count and serum chemistry were done during both reactions to contrast media. serum tryptase level was not measured during the anaphylaxis, but its baseline level was quantified later. conclusions: we present a patient with a double sensitization to parenteral contrast media: an anaphylactic shock due to sulphur hexafluoride and an atypical delayed exanthema related to iodixanol, and diagnose was obtained with st in both cases. this is the first documented case with a positive immediate st to sulphur hexafluoride. with the culprit drugs mixed with 10% and 30% petrolatum resulted negative. patient was suspected to have behcet's disease, and consulted to rheumatology department. oral colchicum dispert twice a day was prescribed. afterwards, patient achieved to take oral amoxicillin-clavulanate for a week without any hypersensitivity; and has been following by oral colchicum dispert maintenance therapy since then. the reported patient had one anaphylactic perioperative reaction to morphine and another anaphylactic reaction to tramadol during her diagnostic investigation. remain the question if this patient had two allergic anaphylactic reactions with cross-reaction between morphine and tramadol, or two non-allergic anaphylaxis due to "hypersensitive" mast cells. case presentation: a 61-year-old female was diagnosed with rectal adenocarcinoma. one year after radical surgery, progression with pulmonary metastasis was shown. in first line of systemic therapy she received premedication with pantoprazole, metoclopramide, clemastine and dexamethasone, followed by cetuximab infusion. during first minutes of infusion, grade 4 anaphylactic reaction occurred. a reaction started with generalized pruritus, urticaria, rhinitis, followed by hypotension, bradycardia and loss of consciousness. she was treated with fluids, clemastine and methylprednisolone. next day she received same premedication followed by panitumumab. during first 30 minutes she had grade 1 reaction with generalized urticaria. the third day she had generalized urticaria 10 minutes after metoclopramide application. skin prick tests with cetuximab (5 mg/ml) were negative, but intradermal test were posibat response was highly positive for both cetuximab and alpha-gal, with comparable values and dose response curves. thus, we showed 70%, 63%, 58%, 35% and 4% of cd63 positive basophils for stimulation with cetuximab (500-0.1 μg/ml), and 67%, 40%, 17%, and 1% for stimulation with alpha-gal (33.3-0.033 ng/ml). bat response to panitumumab was negative (<5%; 500-0.1 μg/ml). drug provocation with panitumumab was negative and patient received treatment with panitumumab. in the operating theatre, the skin is disinfected using povidoneiodine and pupil dilation is carried out with tropicamide (showing no immediate reaction in the surgery). method: as we are dealing with a late cutaneous reaction, the study of the medicine involved is carried out by means of epicutaneous medicine testing. in order to do the study of aflibercept, we wore gowns, two sets of gloves, a mask, eye protection and in a containment hood in the outpatients hospital. the patient diagnosed himself with dermatitis when in contact with povidone-iodine and despite the fact that the cutaneous provocation was negative, it is known that when there is surgery involved, there needs to be moistness and occlusion for it to show up clinically. the application of this antiseptic seems to lose its irritation and allergic properties when it dries on the skin and therefore tends to give a negative result in these patients, but this does not mean that they are not allergic to this antiseptic. we report the case of a 60 year old man who experienced erythema and pruritus immediately after an intravenous injection of ranitidine and hyoscine butylbromide given for gastric pain treatment. results: spt and idt were performed for ranitidine (10 mg/ml and 0.01 mg/ml respectively) and hyoscine butylbromide (0.5 mg/ml and 0.005 mg/ml respectively) being exclusively positive for ranitidine at idt dose with a 15 × 25 mm papule (histamine control 23 × 35 mm). oral provocation test for hyoscine butylbromide was negative. bat for ranitidine and famotidine were carried out, being negative for both drugs. conclusion: skin tests for h2ra are the best option when studying a suspected reaction to h2ra and are also useful for assessing cross-reactivity between other h2ra. the sensitivity for bat in diagnosis of drug allergy is about 50%, and the specificity up to 93%, although these percentages make reference to the common drugs studied (beta-lactams, quinolones, pyrazolones, etc). specific studies for h2ra are still to be done. in our case we had a negative result for the bat test, although we proved ranitidine was responsible for the reaction. conclusion: gentamicin is an aminoglycoside antibiotic used systemically for septicemia and as prophylaxis during surgery. immediate type allergy (type i) to gentamicin is rarely reported. since 2016, approximately only five cases have been reported in literature. in our case, initial theories were pointed towards cefazolin as beta-lactams report a higher rate of allergic reactions. after an exhaustive allergological study, results disproved our initial theory indicating gentamicin as the responsible drug. giangrande n 1 ; bobadilla-gonzález p 1 ; garcía-menaya jm 1 ; cámara-hijón c 2 1 allergy department, infanta cristina university hospital, badajoz, spain; 2 clinical immunology department, san pedro de alcántara hospital, cáceres, spain background: polyethylene glycol (otherwise known as macrogol or peg) is a polymer with a wide application as an excipient, solvent and dispersing agent in food, cosmetic and pharmaceutical industry. it presents distinct length polymer chains with a molecular weight from 200 to 10 000 000 g/mol conferring them specific properties. macroglol with a molecular mass between 3500 and 4000 g/mol is commonly used as osmotic laxative previously to colon endoscopy and radiologic examinations. after the introduction, anaphylactic reactions to macroglol are rarely reported, considering it safe and well tolerated. we report on a 56-year-old man who, immediately after of the topical application of benzindamine in left inferior limb developed acute urticaria in this limb, which reverted in approximately 2 hours with systemic steroids. she had previously tolerated this product without any problems. skin prick-tests with benzindamine (0.06 mg/ml) and latex were realized in the patient. skin prick-tests with benzindamine were realized in eleven healthy control subjects. results: skin prick-test with latex was negative in the patient. skin prick-test with benzindamine was positive in the patient (6 × 5 mm). the prick-tests with benzindamine were negative in eleven healthy control subjects. we report on a case of contact urticaria due to benzindamine and triggered by an immediate, probably ige-mediated, hypersensitivity mechanism. some of the drug used in daily clinical practice can cause allergic contact urticaria and should therefore be borne in mind. background: the use of new oral anticoagulants which act as direct inhibitors of activated factor x is constantly increasing, due to lower rates of serious and fatal bleeding events than warfarin/acenocoumarol. rivaroxaban, the first commercialized drug in this group, is the most used for prevention of thromboembolic events. however, <10 cases of hypersensitivity reactions have been described so far, most of them delayed and severe. to present a case of delayed hypersensitivity to rivaroxaban, diagnosed by a positive ltt (lymphoblastic transformation test). a 79 year old woman with hypertension and chronic atrial fibrillation (af) was referred to our clinic for suspected drug allergy. she reported that 2 months before, for af she was started on oral amiodarone and rivaroxaban, presenting on the seventh day with both of them generalized erythema, pruritus, micropapular rash and facial angioedema. no oral or other mucosal were observed, neither pustules, vesicles or blisters. blood eosinophilia, enlarged lymph nodes, renal and hepatic injury were discarded in emergency, where the new drugs were discontinued and replaced by acenocoumarol. the rash subsided one week later, with oral antihistamines. before and after the episode the patient also has been taking losartan and hydrosalurethyl, with good tolerance. she denied other adverse reactions. in allergy department we performed skin prick tests and intradermal tests with amiodarone (0.05 mg/ml and 0.5 mg/ml) and rivaroxaban (0.1 mg/ml and 1 mg/ml), and a ltt with both drugs, 3 months after the reaction. background: patients with history of beta lactam allergy, often self-reported, are commonly encountered in the hospital setting. this frequently leads to increase use of broad spectrum and more expensive antibiotics that may be unnecessary or even less efficacious at times due to fear and concerns about potential disastrous outcomes. nonetheless, with increasing awareness, many patients are now being referred to allergy service for formal evaluation. we aim to look at patients who underwent evaluation for beta-lactam hypersensitivity and determine the number of patients that were successfully de-labelled. method: a retrospective analysis was conducted with patients referred for evaluation of questionable beta-lactam allergy to the allergy service in our institution from the years 2016-2017. initial evaluation process included a thorough history to determine the type of hypersensitivity reaction and suitability for further testing. patients underwent skin prick test (spt) and intradermal (idt) with either (a) both major and minor determinants of penicillin, benzyl penicillin, amoxicillin and ampicillin, and/or (b) the culprit drug itself. if skin testing was negative, oral or intravenous (iv) drug challenge was then performed after informed consent. clinical details and reactions were documented. patients were also contacted post challenge to ensure no delayed reaction had occurred. results: a total of 130 patients were evaluated for beta-lactam allergy in the 2 year period, of these 80 were females and 50 were males. 107 of the referred patients had presumed penicillin group allergy and 29 had cephalosporin group allergy (3 patients had both penicillin group+cephalosporin allergy). 95 cases (73%) were successfully de-labelled. beta-lactam allergy was confirmed in 26 patients (20%); identified by positive spt in two patients, positive idt in six patients and positive drug challenge in 18 patients (15 patients developed rash/urticaria, 1 had respiratory symptom and 2 patients developed anaphylaxis). 14 patients were referred before any drug allergy labelling was done, out of which 10 were confirmed not to have beta-lactam allergy. conclusion: in our study, 80% of patients were confirmed not to have true beta-lactam allergy. we were able to successfully remove beta-lactam allergy label from the electronic record for 73% of the patients. results: a total of 66% referred amoxicillin-clavulanic acid (ax-clv) as trigger for the hypersensitivity reactions (hrs), followed by ax (21%), penicillin (6%) and cephalosporins (5%). almost 80% of hrs were immediate (<60 minutes). positivity of skin tests was observed in 65% subjects, of bat in 61% and of rast in 68%. in conclusion: the label of penicillin allergy is quite often erroneous. this involves using of more expensive and less effective therapeutic alternatives, which also facilitate the emergence of multi-resistant micro-organisms. hence the importance of confirming the diagnosis of allergy. finally, we did not find differences in the study of penicillin allergy in patients older than 60 years compared with the general population. background: severe cutaneous delayed drug reactions (toxic epidermal necrolysis -ten-, stevens-johnson syndrome -sjs-, acute generalized exanthematous pustulosis -agepand drug reaction with eosinophilia and systemic symptoms/drug-induced hypersensitivity syndrome -dress/dihs-) among others, are a rare but potentially fatal complications of drug treatment. although its epidemiology has been described in different latitudes, it is unknown in latin america. our aim was to describe the epidemiological characteristics of severe cutaneous reactions to drugs in countries of latin america. method: an online questionnaire was designed to report new and old cases (since 2013). it was a modified and adapted version of enda questionnaire for drug allergy interesting group. sociodemographic data, type of reaction (ten, sjs, dress-dihs, agep), culprit drug (s), treatment, complications, mortality and sequelae, were described. three centers from colombia, one from argentina, one from brazil and one from paraguay were included. an excel database was created, in which cases were recorded and analyzed. results: thirty seven cases were reported. 24 (65%) were women. the median age was 47 years. 19 (51%) had dress/dihs, 6 (16%) ten, 3 (8%) sjs, 3 (8%) agep, 3 (8%) other not classified scars, and 1 (2.7%) overlapping ten/sjs. the main culprit drugs were aromatic anticonvulsants in 17 cases (46%), beta lactam antibiotics in 6 (16%), non-beta lactam antibiotics in 3 (8%) and allopurinol in 2 (5.4%). in 100% of the patients the suspect drug was withdrawn. thirty one patients (83.7%) received systemic corticosteroids. complications occurred in 17 cases (49%) and death in one patient (2.7%). seven patients (19%) had some type of sequelae. countries, dress/dihs was the most frequently reported clinical entity, and the anticonvulsants were the main triggers. complications were frequent, but mortality was low. 1155 | drug-induced cough: analysis of nationwide spontaneous reports in korea over ten years using who-adverse reaction terminology (who-art) indicative of cough. results: from 856 524 cases of spontaneously reported adverse drug event cases, a total of 9003 cases (4.5%) were identified as drug-induced cough. most cases occurred in adults (93.4% of the subjects) and females were more common than males (54.9% vs 45.1%). regarding severity, only 629 cases (7.0%) were classified as serious based on who criteria. the most common causative drug category was antineoplastic and immunomodulating agents (24.8%), followed by cardiovascular drugs (24.2%). the most common causative drugs were ace inhibitors including perindopril and ramipril. conclusion: in the nationwide spontaneous reports of adverse drug events, many cases of drug-induced cough have been reported so far. much attention is needed to find new causative drugs of cough in the future. background: allergological assessment to determine the mechanism of the perioperative reaction and to identify the agent responsible and recommendation of a range of drugs or agents likely for future surgery is essential, but it often poses a significant challenge. in this study, we analyze our experience in the investigation of adverse reactions during anesthesia in the last 4 years. method: a total of 15 patients who attended our allergy unit with suspected perioperative reactions between january 2014 and december 2017 were reviewed retrospectively. the severity of the perioperative allergic reactions was graded according to ring and messmer system. results: grade iii, ii and i reactions were observed in 9, 1 and 3 patients, respectively. in 2 patient we didn't know the reaction suffered. tryptase measurements were available for 11 patients. of those, 3 and 4 patients had elevated and normal levels respectively and suffered grade 3 reaction. ige mediated reactions was diagnosed in 9 patients (60%): 3 for ßlactam antibiotics (33.3%), 2 for patent blue (22.2%), 1 for neuromuscular blocking agents-nmbas (11.1%), 1 for latex (11.1%), 1 for colloids (11.1%) and 1 for ranitidine (11.1%) . cefazolin was the ß-lactam antibiotics causing the largest number of reactions. non-ige-mediated reactions was diagnosed in 6 patients (40%). the allergy tests were negative and tryptase levels were normal. conclusion: in our series, among the 9 patients who suffered allergy reactions during anaesthesia and the cause was subsequently identified, ß-lactam antibiotics were the most common causative agent (33.3%), followed by patent blue (22.2%), nmbas, latex, colloids and ranitidine (11.1% each agent). in contrast, data from other authors indicated that nmbas were the most common cause of anaphylaxis, followed by latex, hypnotics, antibiotics, plasma substitutes and opioids. these differences might be due to the small size of our study, which was limited to our centre over the last 4 years and thus may not be representative. diagnostic evaluation. all patients signed an informed consent. we made a retrospective analysis of their clinical records and excluded 11 patients whose records were missing or incomplete. it was analyzed each patient's medical history (focusing allergic disease) and clinical reaction to the suspect drugs. signals/symptoms at pcc were characterized. we also studied the variation of the dpt's results when it was performed after a pcc. aim: to define and quantify the ongoing pharmacy needs in sustaining a large drug allergy assessment program. method: a retrospective review of pharmacy files was used to identify and quantify the drugs and dosages most frequently used and to determine prescription trends within the allergy testing program over the last 3 years. results: initially, this reaction was thought to be a result of a drug allergy, but upon further review and the onset of fever, we determined that it met the diagnostic criteria of jhr. his twin brother was diagnosed with penicillin and betalactamic allergy. neutrophilia 83% was to be underlined in the blood test. after this, drug oral challenge with penicillin was performed, ruling out penicillin allergy. conclusion: it is not uncommon to confuse drug allergy with jhr. jhr should be an anticipated reaction to early doses of antibiotic treatment for treponemal diseases, such as syphilis. antibiotic treatment should be continued; it is not a warrant to stop treatment. clinicians should be aware and anticipate jhr as a potential complication to early doses of antibiotic for spirochetal diseases such as syphilis or lyme, leptospirosis. the patient was unresponsive in oral drug provocation tests with amoxicillin-clavulanic acid, clarithromycin and trimethoprim sulfamethoxazole for 6 months. the patient could use these drugs. results: chronic abacterial inflammation of the prostate gland was accompanied by a significant increase in concentration of slpi, il-8, tnf-α, il-17 in the seminal plasma and serum concentration, and a decrease in the concentration of il-6 and tgf-β1 compared to healthy men (p < 0.05). there was no statistically significant difference between slpi, il-8, tnf-α, il-23, il-17, and tgf-β1 in the ejaculate of patients with inflammatory and non-inflammatory forms of cap (p < 0.05). the concentration of il-6 in ejaculate of patients with inflammatory forms of cap was significantly greater than in patients with non-inflammatory form of cap (p = 0.01). the inflammatory and non-inflammatory forms of cap are pathologically similar with changes in the concentration of the studied cytokines except for il-6 in both forms with signs of inflammation. the terms "leukocytic" vs "non-leukocytic" chronic abacterial prostatitis are more correct than "inflammatory" and "non-inflammatory" when describing chronic abacterial prostatitis. results: the status of all patients after dc immunotherapy was evaluated as satisfactory. heart rate, blood pressure in patients remained within the age norm. skin had normal color without rash or peripheral edema. there were no local or systemic allergic reactions. the body temperature after the injection did not exceed 37°c. conclusion: these results show, for the first time, that among mastocytosis patients, besides the already known periodontal disease risk factors that include diabetes, age, osteoporosis and alcohol consumption, the bone marrow mast cell burden is also associated with increased periodontal disease severity. results: metformin at relatively low doses (1-10 μm) was shown to mildly suppress ige-mediated responses, including degranulation (34% reduction, p = 0.0135), tnf-α (23% reduction, p = 0.0139) and il-13 (38% reduction, p = 0.0015) secretions in bmmcs. importantly, metformin at the same doses potently inhibited mast cell responses in all parameters (100% reduction, p < 0.0001 for degranulation; 87% reduction, p < 0.0001 for tnf-α; 90% reduction, p < 0.0001 for il-13) in mast cells treated with an ahr ligand, 5,11-dihydroindolo[3,2-b]carbazole-6-carbaldehyde (ficz). mechanistically, its inhibitory effect was mediated through the suppression of ficz-induced mapk activation, intracellular calcium release and ros generation. metformin also blocked ahr-mediated pca in vivo (90% reduction, p < 0.0001). conclusion: metformin, a common anti-diabetic agent, was shown to exert inhibitory effect on ahr-mediated mast cell activation in vitro and in vivo, suggesting its potential utility as a newer form of therapy for asthma and allergic diseases; this is particularly relevant when considering the adverse effect of the exposure to environmental polycyclic aromatic hydrocarbons. gasser p 1 ; brigger d 1 ; zbären n 1 ; jardetzky t 2 ; pennington l 2 ; eggel a 1 results: in one of affected family members, we were able to identify the c.9886a>g mutation in the plasminogen (plg) gene that was recently described to be associated with hereditary angioedema. this mutation leads to a missense mutation with an amino acid exchange p.lys330glu in the 3rd kringle domain of plasminogen. there is no direct relationship between the earlier described cases with this mutation and the family we report here. in all affected members of the family, the symptoms manifested in early adulthood, with swelling of the face, the tongue and the larynx. the frequency of attacks was variable, between once in a year to once in a month. in one of the three family members, we found a slightly decreased level of coagulation factor xii and of plasminogen. icatibant proved to be very effective for the treatment of acute attacks in the affected family. the occurrence of the same c.9886a>g (p.lys330glu) mutation in the plg gene in many families with no or only unknown distant relationship suggests that the disease might have been inherited through the generations without being purged from the population. the mutated amino acid exchange appears to be significant for the function of plasmin or plasminogen. we found a decrease in plasma levels of coagulation factor xii and plasminogen, which may be beneficial markers for diagnosis and monitoring of this disease. several biomarkers are useful in the diagnosis (fibrin degradation products (fdps), d dimer (dd), and fragments of prothrombin 1 + 2). also, a correlation between the levels of biomarkers and activity phases of the disease has been detected. alterations in coagulation parameters have an etiopathogenic role in the ae attack, but have not been considered as biomarkers of activity phases. tgt is a global coagulation test which quantifies in vitro the ability of plasma to generate thrombin and estimates alterations in coagulation parameters. the objective is to assess the usefulness of the thrombin generation test (tgt) to characterize patients with hereditary angioedema (hae). method: seventeen hae patients from hospital la fe were recruited to obtain blood samples in remission and during ae attacks. none of them experienced thromboembolic events. plasma was collected in citrate tubes to obtain platelet rich plasma. hemostatic parameters were analyzed:. tgt was conducted using a calibrated automated thrombogram (cat) method and a fluoroskan ascent as a reader. results were analyzed via thrombinoscope v5. citrated plasma was incubated with calcium, tissue factor, phospholipids and a fluorogenic substrate. a thrombin generation curve is generated, obtaining parameters: latency time (lagtime), thrombin generation maximum speed (vo), maximum peak of thrombin generated (peak), time to generate the maximum peak of thrombin (ttpeak), total quantity of generated thrombin (etp), and the end time of thrombin generation (starttail). tgt parameters from 322 healthy donors were used as controls. results: thirty-eight samples were collected from seventeen hae patients (58.8% female). fifteen (39.5%) samples were collected during ae attacks. tgt parameters and fdps were significantly higher in hae patients compared with controls (p < 0.0001), although no significant differences were found in tgt between acute attacks and remission. a decrease trend in tgt is observed in ae attacks. fdps were increased during ae attacks, but normalized at remission periods. these results support the involvement of coagulation in the pathophysiology of hae, although no increase in prevalence of thrombosis is observed during acute attacks. method: the repeated measures design study included 108 patients in two groups: the slit group, 63 patients-67 follow-ups per allergen (p), and the vit group, 45 patients-54 p. the slit group had patients treated for hdm (41 p), and patients on pre-coseasonal pollen ait (grass 11 p, ragweed 10 p, birch 5 p). the vit group had 28 patients on rush protocol (18 for bee and 9 for wasp) and 18 patients on conventional protocol (9 bee, 2 wasp, and 7 for both). the ige and igg4 levels were measured by the immunocap method. the friedman test was used to compare data. results: when compared to placebo group, slit+vitamin d group therapy was more effective in the reduction of nasal symptoms (p = 0.04), asthma symptoms (p = 0.001) and combined symptommedication score (p = 0.001); there was no significant difference between groups in medication and ocular scores. we observed a significant improvement of fev1 (vitamin d group p = 0.014, placebo group p = 0.015) and fev1%vc levels (vitamin d group p = 0.004, placebo group p < 0.001), within both groups, between visits. feno results did not differentiate statistically significantly the study participants in terms of receiving slit along with vitamin d or placebo. significant increase in the percentage of cd4 + cd25 + foxp3 + and in tlr positive cells in children receiving slit+ vitamin d was observed compared to placebo group. increase in cd4 + cd25 + fox-p3 + induction, and in tlr positive cells recruitment were independently associated with better clinical effect of slit in children. conclusion: overall, ait with a high-polymerized ash pollen extract was well tolerated. as ash pollen are supposed to be an important allergen during spring time, it is recommended to include spt and npt with ash pollen in the test panel for allergological diagnostic. additionally, determination of ash pollen specific ige could be applied. furthermore, appropriate ait should be considered for ash pollen allergic patients. 1194a | impact of sublingual immunotherapy with a five-grass pollen tablet on grass pollen allergic rhinitis and asthma: a real-life, long-term analysis in france background: data on the fulfilment of prescriptions of symptomatic medications in patients with grass pollen allergy were analysed to evaluate the long-term effectiveness of sublingual immunotherapy (slit) on allergic rhinitis (ar) and asthma. method: by using data in the lifelink ™ treatment dynamics database (iqvia, paris, france), we compared two cohorts of patients with ar: a group treated with oralair® (stallergenes greer, antony, france) slit tablets (n = 617), and a matched control group having received symptomatic medications only (n = 10 990). oralair®'s effectiveness was assessed as the change in symptomatic medication fulfilments between the pre-index period (before the initiation of slit) and the follow-up period (after slit), and as the onset of asthma or the progression of pre-existing asthma (based on fulfilments of prescriptions for asthma medication). the number of fulfilments per year was calculated for each patient and each period. results: in line with prescribing guidelines, the mean duration of treatment with oralair® was 6.3 months per season for either 2 seasons or 3 seasons. the mean number of symptomatic medications for ar fulfilled per patient and per year in the pre-index period was 6.7 ± 5.9 in the slit tablet group and 9.6 ± 7.7 in the control group. in the follow-up period, this value fell for the slit tablet group (to 2.6 ± 4.2) but did not change significantly in the control group (8.6 ± 8.1). when considering individuals not taking any asthma medications in the pre-index period, asthma onset during the treatment period was observed in 12.1% of those in the slit tablet group and in 15.8% of those in the control group. the corresponding values for the follow-up period were 1.2% in the slit tablet group and 5.8% in the control group. when considering individuals already taking asthma medications in the pre-index period, the mean ± sd number of asthma medication fulfilments in the pre-index period was lower in the slit tablet group (4.7 ± 4.6) than in the control group (8.1 ± 7.9). the corresponding values for the treatment period were 4.1 ± 5.4 and 10.2 ± 9.5, respectively. in the follow-up period, the number of asthma medication fulfilments fell more in the slit tablet group (to 2.3 ± 4.1) than in the control group (7.2 ± 9.0). oralair® tablets have long-term effectiveness by relieving allergic rhinitis and slowing a progression to asthma. 1194b | a real-life, retrospective analysis evidencing slower long-term progression of asthma in grass pollen allergy patients treated with sublingual immunotherapy tablets 1195 | an examination of the reasons for treatment discontinuation and non-compliance to allergen immunotherapy background: allergic rhinitis (ar) patients treated with subcutaneous immunotherapy (scit) and sublingual immunotherapy (slit) may be non-compliant or discontinue treatment too early, which can negatively impact efficacy. therefore, understanding the reasons for non-compliance and treatment discontinuation is vital to help improve compliance, persistence and thus outcomes. this study reported reasons for treatment discontinuation to scit and slit and non-compliance to slit in patients with ar in published real-world studies. method: a literature review was conducted in embase, medline, ebm reviews, psycinfo and econlit (1998-2017) using key search terms for allergic rhinitis, scit, slit, non-compliance and non-persistence. across all studies,~20% of patients were non-compliant, and 1-year drop-out rates ranged from 23% to 74%. reasons for noncompliance and treatment discontinuation in this subset of patients were stratified according to the who dimensions for adherence (patient-related, treatment-related, or socio-economic). results: from the 428 publications identified, six studies reported reasons for non-compliance to slit (n = 3) or treatment discontinuation (n = 3) to scit or slit, and the results were grouped for analysis. the majority of patients cited treatment-related factors as the primary reason for discontinuation (66% for slit, 50% for scit). common reasons were a length of treatment for slit and frequency of injections for scit. 45% of patients discontinued scit due to patient-related factors such as travel to doctors and waiting time for administration. only 24% of slit patients discontinued due to patient-related factors. socio-economic reasons for discontinuation were low for both therapies (10% slit and 5% scit). conversely, for non-compliance to slit, socio-economic factors were the most frequently cited reasons (50%), and included taking time off work and financial concerns. conclusion: of patients who discontinued therapy, treatmentrelated factors were the most cited reasons for scit and slit, reflecting concerns with administration and treatment length. noncompliant slit patients cited socio-economic factors as common reasons for non-compliance, suggesting financial concerns over a long treatment course. differences in reasons for non-compliance and treatment discontinuation may be due to patients assigning differing importance for compliance (a day-to-day decision) compared to the long-term decision to discontinue treatment. results: 53% of patients had monosensitization to rbet v1 component. the rest 47% had combinations ige to rbet v1 and ige to one, two or even three minor allergens (35%, 10%, 2% accordingly). after 2 courses of slit by standardized pollen extracts symptoms of arc and pfas decreased in 90% and 63% patients accordingly. in group 54 patients with monosensitization to rbet v1: 49 patients had a reduction of arc (85% had 3-4 degree by ado); 41 patients had reduction of pfas. 3 patients hadn't finished treatment due to allergic reactions. among 26 patients with sensitization to rbet v1/v6: 24 patients had a reduction of arc (75% -3 to 4 degree by ado); 17 had reduction of pfas. 1 patient hadn't finished treatment due to allergic reactions. in 9 patients with sensitization to rbet v1/v2: 7 patients had a reduction of arc (57% -3 to 4 degree by ado); 3 had reduction of pfas. 10 patients with sensitization to rbet v1/v2/v6 showed the similar results: 10 patients had a reduction of arc (57% -3 to 4 degree by ado); 3 had reduction of pfas. 2 patients had sensitization to all cra, and only 1 patient who also received slit with grass allergens had reduction of arc only (2 degree by ado). as the result of the study it was identified that beneficial effect of slit is highest in patients with monosensitization to rbet v1. the increase of sige sensitization profiles to minor birch allergens caused less efficacy of slit treatment. dermatophagoides pteronyssinus immunotherapy is independent of sensitization to blomia tropicalis among children with allergic rhinitis and asthma method: 95 children (5-17 years old) with allergic rhinitis and asthma sensitised to both dp and bt received 3 years dp-scit. clinical symptom and medication scores, serum specific ige and specific igg 4 were evaluated during dp-scit. in order to investigate whether the treatment outcome was dependent on the sensitisation pattern between dp and bt, patients were further grouped into dp and bt co-sensitisation and cross-reaction, according to positive or negative ige against bt major allergen (btma) blo t 5 and blo t 21. btma+ group, with specific ige to either blo t 5 or blo t 21, was defined as the co-sensitized group; btma-group, with no detectable ige to both blo t 5 and blo t 21, was defined as the cross-reactive group in this study. results: all the recruited 95 patients completed 1 year of dp-scit, 74 (78%) patients completed 3 years of treatment. after 3 years of dp-scit, compared to baseline, all patients had significant reduction in symptom and medication scores. lung function (fev 1 ) was significantly improved as well. 65% of the patients were free of medication use and asthma symptoms, 3% of them were free of rhinitis symptom, and the fev 1 % in all patients were higher than 95% of predicted. dp-scit induced significant increases in dp and bt specific igg 4 . in 50% of patients, dp specific igg 4 increased more than 67 fold and bt specific igg 4 increased more than 2.5 fold. further investigation in btma groups showed moderate correlation (spearman r = 0.48, p = 0.004) between specific ige against dp and bt in the btma-group (n = 34), indicating specific ige cross-reactivity. no specific ige correlation (spearman r = 0.03, p = 0.82) was found in the btma+ group (n = 61) indicating co-sensitisation to both dp and bt. the two groups showed almost identical change in clinical responses. dp and bt specific igg4 significantly increased during dp-scit, no difference was found between the two btma groups. conclusion: dp-scit can induce specific igg4 cross-reacting with bt allergens. patients with specific ige sensitisations to both dp and bt may have clinical benefit from dp-scit treatment. moreover, the clinical benefit of scit was independent of ige cross-reactivity or co-sensitisation to dp and bt. method: we investigated 16 allergic rhinitis children who were basically sensitized to house dust mite and received house dust mite slit for 1 year and 6 months. among 16 patients, 9 patients were mono-sensitized to house dust mite (group 1) and 7 patients were poly-sensitized aside from house dust mite (group 2). we also assigned another 7 allergic rhinitis children who were only treated by medication as control group. nasal symptoms (rhinorrhea, sneezing, nasal obstruction, nasal itching, sleep disturbance) and anti-allergic medications use were assessed at every 6-month visit. results: the symptoms of allergic rhinitis started to improve after 6 months of slit and significantly improved after a year and a half in group 1 and group 2 compared with control group. there was no significant difference between group 1 and group 2. anti-allergic medication use in group 1 and group 2 significantly decreased after a year and a half compared with control group and there was no significant difference between group 1 and group 2. conclusion: house dust mite slit was more effective than treatment only by medication. the effect of house dust mite slit was similar between mono-sensitized and poly-sensitized allergic rhinitis children. house dust mite slit could also be recommended to polysensitized allergic rhinitis children. method: a prospective, randomized, double-blind, controlled, multicenter phase ii study was conducted with four different concentrations of cluster allergoid clustoid wiesenlieschgras (group 1: 2000 tu/ml; group 2: 10 000 tu/ml; group 2: 30 000 tu/ml; group 4: 50 000 tu/ml). out of 103 patients screened, 83 grass pollen allergic patients (18-59 years) were randomized. the cluster build-up phase was followed by four monthly maintenance injections of 0.5 ml. the efficacy was evaluated by the change of the threshold concentration step needed to induce a positive reaction in a titrated nasal provocation test (tnpt) before start and after end of the study (pre-post analysis). the safety profile was assessed for each treatment group by analyzing treatment-related adverse events. background: allergen immunotherapy relies on the consistent administration of allergen extract, therefore compliance to these treatments (subcutaneous immunotherapy (scit) and sublingual immunotherapy (slit) tablets and drops) is vital for efficacy. as scit is administered as an injection by a healthcare professional, and slit is self-administered, compliance to scit may be perceived as superior. therefore, a review of real-world studies investigating compliance to scit, slit-tablets or slit-drops was conducted. real-world studies, instead of clinical trials, were included in this review as they are more likely to reflect actual clinical practice and patient compliance. method: a literature review was conducted in embase, medline, ebm reviews, psycinfo and econlit (1998-2017) using key search terms for ar, scit, slit-tablets and slit-drops, and real-world compliance. compliance was reported according to ispor medication compliance and persistence work group definitions. results: from the 428 publications identified, eight studies (seven slit [one slit-tablets, two slit-drops, four unspecified], one both scit+slit-tablets) reported compliance rates and were included in the analysis. real-world compliance rates ranged from 80% to 81% for slit administration and 83% for scit administration. only one study compared compliance of slit to scit, with similar rates reported over three years (81% and 83% respectively). three studies reported "good" compliance (physician-reported or patients consuming >60% of allergen extract) to slit-drops or slit-tablets. the good compliance rates were higher for slit-drops (90%-99%) compared to slit-tablets (77%-80%). observed compliance to slit-drops or slittablets did not vary by country or geographical region. the percentage of patients defined as having "good" compliance to slit-tablets or slit-drops did not vary by study length or patient population. conclusion: whilst compliance to scit may be perceived as superior to slit-tablets and slit-drops, comparable compliance rates between scit, slit-tables and slit-drops were identified across real-world studies. differences between perception and real-world results may be explained by a lack of direct comparisons between scit and slit administration. limitations included discrepancies in definitions of compliance, as well as methodology between studies. however, these are common to reviews analysing compliance, regardless of therapy area. conclusion: in the allergic rhinitis patients, successful compliance for 3-year slit compared with control was approximately 52%. method: igg inhibition elisa: rabbit igg antibodies specific for grass allergen allergoids are pre-incubated with different concentrations of alum-adsorbed grass pollen allergoid. the mix is added to an allergoid coated microtiter plate. unbound igg will bind to the allergoid coat and is subsequently incubated with anti-igg hrp labeled conjugate and stained with tmb. results are expressed as percentage inhibition relative to the uninhibited value. the concentration of alum-adsorbed allergoid that is required to inhibit 50% igg is used as read-out. circular dichroism: far-uv cd spectra (190-260 nm) were recorded on a j-815 spectropolarimeter. a cuvette with a stirring compartment was used to keep the suspension homogeneous during measurement. results: the igg inhibition elisa assay is specific for grass pollen allergoids (not for other allergen allergoids), has a good inter-and intra-assay precision and is robust for assay variation. thermally stressed alum-adsorbed grass pollen allergoids were used to show that the igg inhibition assay can be used as a stability indicating method. severe thermal stressing resulted in a higher 50% inhibition value, indicating a loss of igg epitopes. furthermore, far-uv cd analyses showed that there is a close relation between the decreasing igg binding capacity (50% inhibition values) and the loss secondary protein structures by unfolding (cd-ratio 207/222 nm values). the igg inhibition assay was demonstrated to be a valuable method to determine the stability of alum-adsorbed grass pollen allergoid preparations. in addition, a relation was shown between the igg binding capacity and the change in secondary protein structures. 1205 | design of a pivotal phase iii trial of allergen specific immunotherapy (ait) using a high-dose house dust mite (hdm) allergoid in patients with allergic bronchial asthma method: 1038 male and female outpatients (age 12-65 years) asthmatics allergic to hdm are enrolled. during the baseline phase, the patient's minimal dose of ics required to achieve asthma control will be assessed. after the baseline period, approx. 446 patients will receive double-blind placebo-controlled treatment for approx. 8 months, followed by a 2nd period of 16 weeks to assess the minimal ics dose and further 2 months of observation for the assessment of asthma exacerbation. based on the results of the dose finding study regarding the efficacy endpoints and the safety profile, the optimal allergoid dose is considered to be 5400 pnu. results: competent authorities and ethic committees in all participating 12 eu countries, serbia, russia and ukraine approved the study design. the primary endpoint of the trial is the change in predefined dose steps of the minimal daily ics dose required to achieve asthma control after approximately 8 months of subcutaneous ait. all efficacy data will be determined using daily questionnaires and the acq6 by e-diary for 4 months from october to january. the aim of this clinical trial is to demonstrate efficacy and to evaluate safety of ait with an allergoid preparation of major allergens of dermatophagoides pteronyssinus in patients suffering from allergic bronchial asthma caused by house dust mites. asthma increases the burden of allergic disease and health care costs, especially when uncontrolled. with the development of a high-dose preparation we intend to treat asthmatic patients highly efficiently. romantowski j; jassem e; lata j; wasilewska e; chelminska m; specjalski k; niedoszytko m background: specific immunotherapy (sit) is the only causal treatment in patients allergic to airborne allergens. it has been proven to be widely effective in allergic populations, but individual patients vary in terms of response to the therapy. the aim of the study was to assess the factors that might affect the efficacy of sit. method: patients treated with sit for grass pollen or house dust mites were included. the efficacy of sit was assessed with the use of allergy control score (acs), performed before and at least after one year of sit. the following variables were assessed as potential risk factors for a poorer response to sit: age, gender, type of allergy, type of allergen, type of vaccine, type of sit and smoking history. background: specific immunotherapy (sit) is a suitable treatment option for asthma and allergic rhinitis (ar), but it is not commonly used in korea. in the achievement of the treatment, it is important that immunotherapy is applied with ideal dose and regular intervals and it is essential for the patient compliance. the aim of this study is to investigate evaluate compliance with immunotherapy protocols of patients who were treated with sit in clinic and their satisfaction of the treatment. we performed a multicenter, cross-sectional survey using a specially designed questionnaire that was given to allergy specialists and patient in korea. a member of the trained research group conducted face-to-face questionnaire interviews with each respondent. conclusion: this study shows that most patients are ar with asthma. in our study sit compliance and satisfy are found to be high in both groups. aim: to identify the frequency of regress claims in a dermatological setting and to assess its impact on general prescription behaviour and immunotherapy. method: all physicians of the psoriasis-praxisnetz süd-west e.v. (n = 222) were invited to participate in a web based questionnaire study on the topics of dermatology and medical law. the survey was separated into two sub-polls which were carried out after a first poll deciding whether the topic of medical law is of any interest for the dermatological practice. the topic of interest was located in the second poll. results: overall, 66 dermatologists participated in this study. most participants were form bavaria, baden-wuerttemberg or rhineland-palatinate and had more than 10 years of experience as a dermatologist. out of the 66 participating physicians 28.8% (n = 19) already experienced a previous regress claim. of these, 73.7% (n = 14) stated, that the experienced regress claim changed their prescription behaviour. half of these participants (n = 8) further stated, that the fear of a possible recourse affects their prescription behaviour, whereas only 4 out of the 47 other participants declared a possible influence. missing values excluded, this leads to a substantial hesitation in physicians who experienced a prior recourse (50.0% vs 16.7%). nevertheless, this seems not to affect the usage of allergen immunotherapy, as all 19 physicians who already experienced a regress claim, stated to use allergen immunotherapy. the fear of a possible regress can change physicians' prescription behaviour but does not seem to have an effect on the prescription of allergen immunotherapy. therefore, the topic should be addressed from another perspective such as providing trainings on relevant regulations for physicians who experienced a prior recourse claim. this approach could also improve patient centred care related to modern treatments. results: the sds were significantly reduced in patients subjected to slit (p < 0.01) 1 year after the onset it. vas also was significantly reduced (p < 0.05) with satisfied control of sar and the same time with translation from moderate-severe to mild-moderate sar, after slit. nbh was also significantly reduced (p < 0.05) 1 year after the onset slit. in patients receiving pht only, sds, vas and severity of sar did not change and significantly higher (p < 0.01) from the value obtained in the experimental group. nbh also remained unchanged and significantly higher (p < 0.05) then in experimental group. precoseasonal slit added to pht shows short-term beneficial clinical effects in polysensitized patients with sar and scuad phenotype. results: ten studies (248 children, 99 adults; median sample size, 28) met the inclusion criteria. the risk of bias was moderate to high in all but one studies. low strength evidence supports the assumption that ait is effective in reducing symptoms and medication use, with only 5 out of 10 studies reporting higher benefit in the ait group vs comparator group. subgroup analyses of studies sharing similar characteristics did not explain inconsistency. safety does not appear was not major concern for alternaria ait. conclusion: this is not enough strength of evidence to suggest that mold ait is efficacious for the treatment of respiratory allergies. high-quality studies with an adequate sample size are needed. abstracts | 627 1213 | tolerability of a two week rush updosing with modified trees, modified grasses or modified grasses/trees mixture in pollen allergic subjects in the day-to-day practice table) severe systemic reactions (grade iii and iv) did not occur. conclusion: rush immunotherapy is an effective therapeutic method for patients with allergic rhinitis. it seems that in cases requiring faster response to treatment, this immunotherapy can be considered as a substitute for conventional immunotherapy. 1217 | design of a pivotal phase iii allergen immunotherapy study to assess the efficacy and safety of subcutaneously administered tyrosine adsorbed modified birch allergen+mpl results: the design of this study, including sample size and primary and secondary endpoints, will be discussed based on prior experience gained in two dose finding studies. in addition, the number of patients screened and randomized will be presented by country, gender and/or age and screen failures will be categorized. results: the primary analysis showed an absolute difference in tcs between placebo and 12 du of 3.02 (40%, p < 0.0001). the odds of experiencing a severe day during the bps were approximately doubled in the placebo group compared to the 12 du group (or = 2.12, p < 0.0001) and the odds of experiencing a mild day were halved (or = 0.52, p < 0.0001). similar results were seen for the tree pollen season (tps), covering both alder, hazel and birch pollen seasons. the total rqlq score was improved for 12 du compared to placebo during the bps and tps (p < 0.05, except for the last week of the tps), with the most pronounced effects during week 2-5 of the bps (absolute difference: 0.47-0.58, p < 0.0001). treatment was well tolerated. the most frequent adverse reactions were mild or moderate local reactions related to the sublingual administration. no deaths were reported and no serious adverse events were assessed as related to the sq tree slit-tablet. conclusion: treatment with the sq tree slit-tablet improved arc symptoms and need for symptomatic treatment. the 12 du group had less "severe days" and more "mild days" during the pollen seasons. the quality of life was similarly improved. these findings substantiate the clinical relevance of the sq tree slit-tablet for patients with arc induced by pollen from the birch homologous group. nagaraju k 1 ; nagaraju k 2 ; katare s 3 ; kapatkar v 3 ; shah a 3 ; rathod r 3 results: total n = 57 subjects (mean age 4.6 ± 2.09 years, 61.4% males) completed entire study. the mean incidence of artis reduced from 7.7 ± 2.34 episodes at baseline to 1.4 ± 1.23 (p < 0.001), with 31.6% subjects not suffering from any episode. the mean duration of episodes reduced from 8.2 ± 3.58 to 2.5 ± 1.23 days (p < 0.001). 64% of episodes (vs 95% at baseline, p < 0.05) required antibiotics for mean duration of 2.3 ± 0.98 days (vs. 6.6 ± 1.76 days, p < 0.001). none of arti episodes in follow-up period required hospitalization as against 22.8% episodes, (mean duration 5 ± 2.06 days; p < 0.05) before pidotimod therapy. the number of school days lost & work days lost showed reduction of 3.6 ± 5.34 days(p < 0.001) & 0.1 ± 4.07 days(p = 0.871) respectively. the average expenses incurred in treatment of artis shows significant reduction of rs. 13 193 ± 1541 (p < 0.001). 11 adverse events were reported in 8 (14%) subjects, which were mild in nature. a statistically significant increase in absolute counts of t-& nk cells was seen in explorative assessment of immune markers. the study shows pidotimod to be well-tolerated effective therapy in reducing the incidence and severity of recurrent artis, thereby providing additional benefit of reduction in discomfort & healthcare cost due to recurrent artis. thus, pidotimod can be considered as potential therapeutic option for treatment of recurrent artis in children. martignago i 1 ; ridolo e 1 ; incorvaia c 2 1 department of medicine and surgery, university of parma, parma, italy; 2 cardiac/pulmonary rehabilitation, asst pini/cto, milan, italy background: two registered sublingual immunotherapy (slit) products are available to treat grass-pollen induced rhinoconjunctivitis, consisting of the 1-grass (phleum pratense) and the 5-grass pollen tablets. no study of direct comparison of the efficacy of the two products was performed. we report the case of a patient who was treated in different years with the 5-grass or the 1-grass tablets with contrasting efficacy. the patient was a 18-year old woman suffering from 3 years of grass pollen induced rhinoconjunctivitis. in 2015 slit was started with the 5-grass pollen tablets, but in 2016, due to unavailability of the product, slit was performed by the 1-grass pollen tablets. in the third year of treatment slit with the 5-grass pollen tablets was resumed. for the 5-grass tablets slit was initiated before the pollen season and stopped after 7 months of treatment, while for the 1-grass tablets the treated was prescribed to be continuous. the efficacy of slit was evaluated by symptom-medication scores as reported in diary cards by the patient during the month of may, when the grass pollen usually reach the higher concentration in the atmosphere in lombardy, where the patient lives. results: the mean symptom-medication score in the first year of treatment (5-grass tablets) was 4.35, compared with a mean score of 7.2 in the second year (1-grass tablets). the patient was unsatisfied of the symptoms control and asked to resume for the last year of slit the 5-grass tablets. the mean symptom-medication score in such year was 0.77. no clinically relevant adverse event was reported with any slit product. conclusion: based on the momentary unavailability of the 5-grass pollen tablet, it was possible to assess in a same patient the clinical outcome associated to either of the two registered slit products. a significantly different efficacy of slit with the 5-grass tablets compared with the 1-grass tablets was observed. 1222 | fusion proteins consisting of bet v 1 and phl p 5 form ige-reactive aggregates with reduced allergenic activity najafi n 1 ; hofer g 2 ; gattinger p 1 ; smiljkovic d 3 ; blatt k 3 ; selb r 4 ; stoecklinger a 5 ; keller w 2 ; valent p 3 ; niederberger v 4 ; thalhamer j 5 ; valenta r 6 ; flicker s 6 background: bet v 1 and phl p 5 representing major allergens in birch and grass pollen, occur as monomeric proteins with high allergenic activity as assessed by clinical provocation testing in patients. she did not suffer more hymenoptera stings after the last reaction. one bee sting several years before bst resulted in no reaction. she has no symptoms with honey, vegetable or other food ingestion. methods: total ige and specific ige were determined using inmu-nocap system (thermofisher, scientific inc). apis, vespula and polistes (hørsholm, denmark) were also performed. prick tests showed negative results for all extracts tested. intradermal skin tests were positive for apis at 1 μg/ml, but negative for vespula and polistes. our patient was diagnosed of anaphylaxis due to apis venom, thus bst was contraindicated and an epinephrine autoinjector was prescribed. she rejected hymenoptera venom immunotherapy. conclusion: to our knowledge, this is the first case of anaphylactic reaction after bee sting therapy. bee sting therapy should be considered a risk factor for anaphylaxis. patient reported good control of their disease, improved their quality of life, tolerating contact and exposure to numerous horses as well as contact with clothes of people who had been exposed. although ita is absolute contraindication on uncontrolled asthma with a degree of evidence ia, our case had only "transitory" con and immunocap among wasp allergen components-i3, i209, i211 were 88%; 0.5, 90%; 0.6 and 68%; 0.4 respectively and honey bee allergen components-i1, i208, i217 were 95%; 0.9, 93%; 0.7 and 97%; 0.9 respectively. agreement between polycheck and immuno-cap i1 and i3 allergen components were 85%; 0.7 and 66%; 0.3 respectively. agreement between polycheck and euroline i1 and i3 allergen component were 86%; 0.7 and 61%; 0.2 respectively. based on wasp and bee components in all three systems, sensitization pattern was analyzed. similar test results were found between euroline and immunocap systems. the comparative studies carried out showed a markedly higher compliance of results with the euroline tests compared to polycheck with the immunocap system. percent agreement was extremely high and kappa value was substantial or almost perfect in the case of bee venom allergy between euroline results: a total of 113 patients were included; 80 (71%) males, with a mean age of 38 (±15) years; 23 (21%) beekeepers, 25 (23%) were atopic, 4 (4%) had asthma, 14 (13%) rhinitis and 18 (16%) cardiovascular disease, and 14 of these patients were on ace/beta blockers. vit with honeybee was proposed in 73 (64%), wasp 38 (34%) and polistes 2 (2%). the mean duration of vit was 45 (±16) months. however, 23 completed less than 36 months. of the total, 28 patients (25%) were not treated with vit. eighty-eight patients (78%) participated in the telephone interview: 49 completed vit (56%), 15 were still on vit (17%) and 24 did not undergo vit (27%). of those who completed vit, 14 (29%) were restung and 3 went to the emergency department (er). twenty-four patients (36%) were stung while still on vit. of those never on vit, 12 (50%) were re-stung and 9 went to er. the severity of the reactions according to mueller of the patients who completed vit (mean follow-up time was 45 months (1-110 months)) and were stung again was: local reaction in 11 (79%), grade i in 1 (7%); grade iii in 1 (7%). one had a toxic reaction after multiple stings. in those who were stung during vit, 20 (87%) had local reactions, 1 (4%) grade i and 2 (9%) grade iii. of those who were not treated and were re-stung: 3 (25%) had grade i, 4 (33%) grade iii and 5 (42%) grade iv. in this series, the patients who did not undergo vit presented a greater number of systemic reactions when re-stung as well as more severe reactions (p < 0.01). conclusion: in this group with indication for vit, the reactions of the re-stings were less severe in the patients who had completed or who were on venom immunotherapy, as expected. three quarters of those who did not undergo treatment had severe anaphylactic reactions when they were stung again. this study reinforces the importance and the efficacy of immunotherapy in the treatment of hymenoptera venom allergy. method: this is a retrospective, descriptive study of cases diagmethod: data were issued from the reference centre in mastocytosis of toulouse university hospital. ms diagnosis was determined using world health organization diagnostic criteria. hymenoptera venom immunotherapy was performed with an ultra-rush protocol (table) . results: seven patients were included (4 women, 3 men), median age 47 years old. during the anaphylactic reaction, cutaneous signs missed in all cases. the reaction was most often severe: grade 2 (n = 1), grade 3 (n = 5), grade 4 (n = 1). three patients suffered from digestive symptoms and one from respiratory manifestations. basal tryptase in serum reached 10.1-41.7 μg/l. hymenoptera venom specific ige were low (0.11-1.51 kui/l) except for one patient (35.6 kui/l). ait was initiated with vespula venom in 3 patients, polistis in 1 patient, apis mellifera and vespula in 1 patient, vespula and polistis in 2 patients. no reaction was observed during ait. four restringing accidents led to increase the cumulative dose to 150 μg and 200 μg in 2 patients. in these patients, the diagnosis of mastocytosis was made due to the resting. conclusion: hymenoptera venom ait using ultra-rush protocol seems well tolerated in patients with systemic mastocytosis. specific studies are necessary to determine the real tolerance profile of this protocol. collaboration with reference centres for mastocytosis should be considered for all patients with mastocytosis associated to hymenoptera venom allergy. dose (μg/ml) results: see table. conclusion: there is a shift or immunomodulation in terms of sige to vespids. even in patients double sensitised who were receiving venom of only one of the vespids. albanesi m background: slit has been suggested as an alternative route for allergen-specific immunotherapy. aim of this study was to investigate allergen-specific antibody responses in birch pollen allergic children who had received slit for two years using recombinant allergens. method: children (n = 28; 5-12 y o) with respiratory symptoms of birch pollen and oral allergic syndromes (oas) were studied. ten children received slit with staloral, 10 were treated by slit with microgen, and 8 children received only symptomatic therapy (control group). sige and sigg levels to rbet v 1, rbet v 2, rbet v 4 were measured twice (before therapy started and after two years) using quantitative immunocap and a panel of more than 170 microarrayed allergens using immunocap isac technology. clinical efficacy of slit was evaluated by recording symptoms upon allergen contact and need of rescue medication. results: all 28 children were sensitized to the major birch pollen allergen, bet v 1 and one patient from each of the groups showed to bet v 2, no patient had sige to bet v 4. after two years of slit clinical improvement was observed in the slit patients. in the staloral group there were no respiratory symptoms in 3 patients and a decrease of symptom severity in the other 7 cases as well as a partial or complete tolerance to pr10 allergen-containing food in the 10 patients. microgen treatment had no influence on oas symptoms but decreased of pollinosis severity in 8 children. however, there were no statistically significant differences of bet v 1-specific levels measured before and after treatment in the slit and control groups (mann-whitney, p > 0.05). in this real-life study we found that birch allergic children who had been treated with slit showed a reduction of clinical symptoms but we did not find a significant induction of allergen-specific igg levels in the slit-treated group when compared with children who had only symptomatic treatment. conclusion: our study confirms the scarcity of food additives allergy. it also suggests that even when the diagnostic of allergy was excluded with a negative oral food challenge, families remain suspicious about industrials feeding products containing food additives. these results should reassure health professionals and parents who incriminate too frequently food dyes and conservators when a manifestation which mimics allergic reactions occurs. background: autumn/winter birth has been reported to be a risk factor of food allergy (fa) development. a putative mechanism is that dry/cold weather causes and exacerbates infant atopic dermatitis (ad), which is a major risk factor for food sensitization through inflamed/damaged skin. we investigated prevalence of fa among infants under well skin care in relation with seasons of birth (sob). we recruited full-term newborn infants without perinatal diseases at an obstetric/pediatric clinic. participants were followed up for skin status and food allergy symptoms until 12 months of age. sob were defined as spring (march-may), summer (june-august), autumn (september-november) and winter (december-february). ad was diagnosed based on the united kingdom working party's criteria. use of moisturizer (mo) and topical corticosteroids (tcs) was recorded. primary outcome was fa based on apparent immediate allergic reaction after ingestion of causative food. we classified infants who avoided any food because of sensitization or mother's anxiety as suspected fa. results: six hundred and thirty-one infants were screened for 12 month-period and 531 infants were enrolled in this study. of them, 277 infants were born in spring-summer (s-born) and 254 infants were born in autumn-winter (w-born). fa developed in 24 (4.5%) infants and 31 (5.8%) infants had suspected fa. there was no difference (p = 0.68) in prevalence of fa and suspected fa between s-born and w-born. multivariate analysis revealed ad at 2 and 7 months of age was a significant risk factor for fa with or=2.6 (95%: 1.1-6.0) and or=3.5 (95% ci: 1.4-8.9), respectively. prevalence of ad at 2 months of age was higher in w-born than s-born but prevalence promptly decreased thereafter and stayed low with early use of mo and tcs. prevalence of ad was rather higher at 7 months in s-born than w-born. results: 160 cases and 126 controls were included. the median age was 44 years, (q1-q3 33-53). men and women were almost equally represented (50.35% males). alcohol consumption associated with the intake of mammalian meat or innards as the trigger factor. the overall prevalence of a positive result of sige to α-gal was abstracts | 645 15.7% ic95% (11.5, 20 .0); cases _26.3% ic95% (19.4, 33.1) controls _2.4% ic95% (0.0, 5.1)_. among cases sige anti α-gal positivity rate ranged from 55.8% (rural), to 35.7% (half-urban) and 12.6% (urban). the rates of positivity were 69.5%, (northern) 1.4% (center) and 0% (mediterranean). a positive result of sige to α-gal was more frequently observed among men (24.31%) than women (7.04%) and associated with history of tick bites, practice of outdoor activities, pet's ownership and the antecedent of having eaten mammalian meats or innards previously to the development of symptoms background: a special challenge in the 21st century for allergists is allergy to food, which is considered "the second wave" of epidemics of allergic diseases. panallergens occur in unrelated organisms and perform a similar function in them. in their structure, they have highly conserved amino acid sequence regions and a similar three-dimensional structure, and thus meet the requirements for cross-recognition by ige. results: in 46 patients (92%) isac test has been shown to have specific ige for panallergen components. mostly, the presence of ige for pr-10 proteins has been shown in 41 patients. in 12 patients ige to ltp; 11 patients ige to ccd; 6 patients to profilin; 5 patients to tropomyosin; 4 patients to serum albumin, 1 person to tlp. an important aspect is undoubtedly the occurrence of simultaneous sensitization to several panallergens. analysis of data from the study group showed that isolated sensitization to one panallergen concerned only pr10 proteins (24 patients), tropomyosin (2 patients) and profilin (1 patient). in the remaining 19 patients, the analysis of the isac test results showed that two or more panallergens were allergic. in the study group, asige for the component responsible for the occurrence of real food allergy was detected in 13 (26%) patients. mostly, the presence of ige for jug r 2 has been shown in 9 patients. in the study group, panallergens were more likely to be responsible for food intolerance than specific food allergens. results: of 43 children, 14 children had peanut allergy only, 14 children had tree nut allergy only, and 15 children had both. the mean age was 3.3 ± 1.9 years in peanut allergy, 5.0 ± 3.6 years in tree nut allergy, and 4.2 ± 3.0 years in both. male to female ratio was significantly higher in tree nut allergy (78.6%) than peanut allergy (42.9%). among tree nut allergens identified, walnut (75.9%) was most frequent, followed by almond (55.2%), hazelnut (34.5%), pine nut (20.7%), chestnut (13.8%), cashew (6.9%), pistachio (3.4%), and macadamia (3.4%). mean serum total ige level was 888 kua/l in tree nut allergy and 1440 kua/l in peanut allergy. mean serum specific ige level to peanut, walnut, almond, hazelnut, and pine nut was 30.0, 21.9, 14.2, 22.4, 5.6, and 6 .3 kua/l, respectively. children with peanut allergy had higher rate of co-sensitization with soybean and higher soybean-specific ige levels than children with tree nut allergy. however, there was no difference in co-sensitization rate with tree pollen between peanut and tree nut allergy. children with peanut allergy showed significantly increased co-sensitization rate with egg white and wheat compared to children with tree nut allergy. a 42.8% of the children with peanut allergy and 28.6% of tree nut allergy showed co-sensitization with aeroallergens. a total of 75% of the children with peanut allergy showed decreased specific ige levels within 1-5 years. conclusion: prevalence of peanut and tree nut allergy is similar. tree nut allergy develops later than peanut allergy and more common in male. children with peanut allergy showed higher co-sensitization rate with soybean, egg white and wheat compared to children with tree nut allergy. 1257 | natural history of egg allergy in a large cohort of infants with food allergy shows its high prevalence but also its transient nature in a 36 months of follow-up background: the cohort of 80 infants (55 boys,25 girls,3-36 months) with the food allergy has been followed for 36 months. as more than 70% of infants manifested atopic dermatitis (ad), a condition closely linked to egg sensitisation, we focused our attention on egg allergy, following its natural history as well as a development of atopic march. method: the diagnosis of food allergy was based on a personal history, clinical examination, skin prick tests and/or atopy patch tests with native foods. laboratory tests were performed within 1 year of age the latest. the specific ige levels against food allergens were measured using immunocap or immulite. patients with ad were scored according to scorad system. the oral food challenges (ofcs) with cooked/baked egg were done in children at the age of 12 months except for children at risk of anaphylaxis. results: within the whole cohort the allergy to cow milk proteins was confirmed in 70 pts (87.5%), to egg in 35 pts (43.7%), to wheat in 8 pts(10%), to lentil in 3 pts (3.75%) to banana in 3 pts (3.75%), to soya in 2 pts (2.5%) and to potatoes in 1 pt (1.25%). in a cohort of 35 egg allergy patients we found out that: 83% of pts presented the early onset of allergy-up to 3 months of age, 80% of pts presented severe ad (scorad >50), 46% of pts showed cosensitisation to peanuts, 34% of pts had early sensitisation to inhaled allergens, and majority 54% of pts presented with early onset allergic rhinitis and/or asthma. we proved that egg allergy is closely linked with the early onset of allergy symptoms, with severe forms of ad, co-sensitization to peanuts, early sensitisation to inhaled allergens and an early onset of allergic rhinitis and/or asthma. we also proved that the egg allergy in infancy is transient. the tolerance to baked/cooked egg was achieved in about 75% of pts at the age of 3 years, unlike previously published results claiming the reach of tolerance in 75% of pts at the age of 7 years. in these patients we studied: sex, personal history, type of reaction they presented, time of onset of symptoms and food involved. results: out of a total of 1479 patients over 60 years of age (from 60 to 99 years old) who have been attended the consultation for the first time during these period, 70 patients (4.7%) ask about possible allergic food allergies. of these patients who came for possible allergic pathology, 28 patients (40%) presented positive results. these are the other item we have studied: 1. sex of patients: 55% of the patients are women. these patients because of that, it is important to remember that food allergy can also appear in old people. the food that is mainly involved in our population is fish and seafood. a much higher percentage than in other populations, probably due to the mediterranean diet of spain. the symptoms mainly involved are itching and skin lesion, which is the characteristic symptom of a mild allergic reaction. in our population, there was 3 patients with a anaphylactic shock, a much higher percentage than in other studies. the experience with this group of patients is still limited. more studies are needed to know better this patient profile. background: cow's milk allergy (cma) is the first atopic disease in children. diagnosis suspicion in the emergency room (er) is increasingly frequent, however, further assessment by an allergist is often difficult to schedule. therefore, screening for cma through a blood test (specific ige) while the infant is still in the er has gained momentum in recent years. we set out to analyse (a) symptoms which had led the emergency physician to prescribe specific ige, (b) the prevalence of confirmed cma among infants screened in the er, and (c) the long-term outcome of the screened infants. method: a retrospective study of medical records and laboratory results was performed. patients were infants under 6 months, without a previous diagnosis of cma, attending one of the two pediatric er of the university hospitals of marseille, france. allergy blood tests were specific ige to cow's milk extract (immuno-cap, thermofisher, sweden). in infants with specific ige to cow's milk extract of 0.12 kua/l or higher, ige directed to the main three individual proteins (casein, alpha lactalbumin et beta lactoglobulin) were also measured. results: 482 infants were included from december 2011 to june 2016. the sex ratio was 1.45. 40% of infants were atopic et 48% were currently or had been breastfed. the most prevalent symptoms were vomiting and reflux. one third of infants were hospitalized after the er visit. following the er visit, 25% of infants attended a specialized consultation with an allergist. 15% of infants with a follow-up visit were diagnosed with an ige mediated cma. 18 infants with cma developed further food allergies (egg, nuts, cashew…). it is difficult to diagnose it. the emergency pediatrician are increasingly confronted to infant with symptoms evoking cma. thus they prescribe sige and extensively hydrolysed proteins because they know that ige-positive infants can be ige-negative during the interval between the er visit and the follow-up one. after bad results interpretation of blood assay after er visit, cma was probably over diagnosed without prick test for ige positive allergy and no eviction/ reintroduction test for non ige. the lack of allergist is probably leading to over prescription of blood assay in er to diagnose cma and prolonged eviction of milk. results: a total of 96 patients were included (59% female) aged from 18 to 82yo with an average 33.9 ± 15 years. 77% of patients had history of atopic disease: 60% rhinitis, 30% asthma, 9% prior food allergy, 8% eczema, 6% drug allergy, 5% eosinophilic esophagitis (ee) and 3% chronic urticaria. mean serum total ige was 441.9 ui/ml. sensitization to aeroallergens was present in 63% of patients, the most common were dust mites (22.9%), pollen (15.6%) or both (25%). in 69(72%) patients, first symptoms of fa appeared ≥18 yo, with an average age of 35 ± 15.4 yo. in this group, 4 were diagnosed with ee, 1 with eosinophilic colitis and 2 with eosinophilic gastritis. from the remaining 62 patients, 20 had history of reaction with more than 1 food group (fg). cutaneous reactions were referred in 60% of patients followed by anaphylaxis (20%) and gastrointestinal symptoms (10%). the fg most commonly implied were: fresh fruits (n = 26), seafood (n = 19) and tree nuts (n = 6). fa diagnosis was confirmed in 66% of patients, the remaining had negative ofc. in 27(28%) patients, their symptoms started under 18 yo, with an average age of 11.5 ± 5yo. from this group, 10(44%) were diagnosed ee. from the remaining 17 patients, cutaneous complaints were the most frequent (47%) followed by gastrointestinal (35%) and respiratory symptoms (17%). the most common fg implied were: fresh fruits(n = 9), seafood(n = 4) and tree nuts(n = 3). only one anaphylaxis was referred. fa was confirmed in 82%, the remaining had negative ofc. in patients with history of anaphylaxis 13 of 14 had positive st and/ or sige; one had negative sp and sige, with ofc positive. the 401 blood donors were classified based on their clinical symptoms related to possible as contact via fish intake: allergic to as (1%), chronic urticaria (0.5%), unspecific dyspepsia (2.8%) and asymptomatic (95.5%). the prevalence of sensitization (anti-as ige >0.35 kua/l) were 12.7% (ic: 9.6-16.4%; mean 0.69 kua/l; median 0.03 kua/l) with a maximum value of 40.70 kua/l. raw fish consumption was the only variable associated with statistical significance (p < 0.05) to as sensitization (20.4% vs 9.5%, respectively). albacore and codfish were the most consumed species associated to seropositive results (15%), followed by hake (10%). coastal population (13.6% vs 9.3%), non-previously frozen fish consumption (13.7% vs 9.2%) and >3 times per week fish consumption (51.2%) were other seropositive associated factors. background: oral allergy syndrome (oas) is an ige-mediated allergy caused by raw fruits and vegetables in patients with pollen allergy, which is known as the most common food allergy in adults. however, there has been no nation-wide study on oral allergy syndrome in korea. the aim of this study is to investigate the prevalence and clinical manifestations of oas in korea. method: twenty two investigators from 19 hospitals and 2 private clinics participated in this study. the patients with allergic rhinoconjunctivitis and/or bronchial asthma with pollen allergy were enrolled to the survey. the questionnaires include demographics, a list of fruits and vegetables, and clinical manifestations of food allergy. pollen allergies were diagnosed by positive results of one or more pollen allergens including birch, alder, hazel, beech, oak, willow, poplar, bermuda, meadow, orchard, rye, timothy, mugwort, ragweed, hop japanese on allergy skin prick tests (allergen/histamine ratio ≥3+) and/or serum specific ige levels using multiple allergen simultaneous tests (mast ≥2+) or immunocap (≥0.35 ku/l). conclusion: this is the first nation-wide study for oas in korea. the prevalence of oas in korea was 41.7%, in which substantial proportion had anaphylaxis. these results will provide useful information for clinicians to apply in clinical practice. we conducted a self-administered, questionnaire-based survey in 2013-15 during the 18-month checkup. children were considered to have food allergies if they were diagnosed by a physician or if they had been instructed to avoid a causative food after medical examination by interview. we divided the year into three periods. the months of march-june were considered spring, july-october as summer/fall, and november-february as winter. while the season of onset for the 4095 boys occurred in 8.2%, 12.7%, and 15.3% in spring, summer/fall, and winter, respectively, it was 6.9%, 10.5%, and 12.2%, respectively, for the 3922 girls. thus, the onset rate was the highest in winter for both genders. in boys whose mothers did not consume folic acid (fol − ), the food allergy onset rate was significantly higher for boys whose mothers ate no eggs and for boys whose mothers ate 2-5 eggs per week than for those whose mothers ate eggs daily according to the dunnet multiple comparison test. however, no relationship was observed with egg intake if the mother had consumed folic acid (fol + ). on the basis of seasons, fol − and egg intake by mothers affected only children born in winter, with a significant difference in the dunnet multiple comparison. among mothers who did not eat eggs, fol + was 15.2% and fol − was 28.2%; for mothers who ate 2-5 eggs per week, fol + was 16.5% and fol − was 12.9%; and for mothers who ate eggs every day, fol + was 17.9% and folwas 4.9%. thus, consumption of folic acid seemingly annulated the effects of eating eggs. however, for girls, neither folic acid nor eating eggs had any effect on the onset rate. conclusion: since this effect varied according to the birth season, consumption of folic acid, a methyl group donor, appeared to affect the allergy onset in children. results: skin prick test with commercial extracts of tuna (7 mm), cod (5 mm), rooster (8 mm), hake (6 mm), salmon (4 mm), trout (7 mm) and anisakis (7 mm ige to shrimp, lobster, crab and mixed seafood were all undetectable. dermatophagoides pteronyssinus 10, to assess for tropomyosins was negative. outcome: the patient continues to react to both hdm and shrimp, despite undetectable ige levels to tropomyosin associated components. this is the only testing available in south africa currently and hence we are unable to look at other proteins. the relationship between tropomyosins in shellfish allergy and mite allergy has been well documented and investigated, but other allergens are now also being implicated in cross-reactions. we also established the level of ige specific to allergen components using the immunocap isac method. allergen-specific ige was not elevated to any shrimp allergens available in immunocap isac: n pen m1 (tropomyosin), n pen m2 (arginine kinase) and n pen m4 (calcium binding sarcoplasmic protein). the patient was diagnosed with a shrimp allergy. the molecular diagnostics used did not explain which allergen component is the patient allergic to. it is possible that the patient is allergic to hemocyanin, which can also cross-react with house dust mite allergens, but confirmation of this diagnosis requires further investiresults: the groups were identical in terms of the age and sex (table 1) . ara h 2 ige correlated (spearman test) with the cumulative protein dose (threshold dose) r = −0.439 (p = 0.023) but not with reaction severity r = 0.091 (p = 0.348), or the use of adrenaline r = 0.300 (p = 0.093). patients with ara h ige < 10 ku/l had higher threshold doses (644 vs 71 mg) than children whose ara h 2 ige was ≥10 ku/l (p = 0.016). there were no significant differences in severity of the reaction or in use of adrenaline (table 1) . the level of ara h 2 ige is relevant in predicting the threshold dose at peanut exposure. a low reaction threshold dose increases the risk of reaction at an accidental exposure leading potentially to a severe reaction. flaxseed allergy is uncommon and most of the cases reported involved anaphylaxis. cross reactivity has been described with other seeds. case report: a 9-year-old atopic girl diagnosed with egg allergy and rhinoconjunctivitis and asthma due to pollens. when she was eight, she presented two reactions consisting of conjunctival, periorbicular, malar erythema and abdominal pain after eating egg free french toasts cooked with flaxseed. she was treated with oral antihistamines. the allergic workup included prick-by-prick test with flaxseed which was positive and skin prick tests with mites, molds, cockroach, cat, dog, profilin, ltp and pollens with positive results for olive and grass pollens. the serum total immunoglobulin (ig) e was 476 u/l, and specific ige to flaxseed was 7.23 kua/l. the flaxseed extract was resolved with sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) and an ige immunoblotting was performed under nonreducing conditions. the patient's serum showed specific recognition of a 18-kda band in the immunoblot. proteins were identified using mass spectrometry (maldi-tof) that showed results highly consistent with conlinin, a 2s storage protein of flaxseed. we described for the first time a patient with allergy to flaxseed due to conlinin, a 2s storage protein of flaxseed. background: cyperus esculentus is an herbaceous plant that has edible tubers called tiger nuts. in spain, they are used mainly in the elaboration of the well-known "horchata" or tiger nut milk, which is obtained by macerating tiger nuts with water and sugar. tiger nut allergy has rarely been reported, despite of its widespread consumption. case report: we present the case of a 19-year-old male with a history of oral syndrome allergy with several fruits (peach, melon, banana, kiwi, apple, pear and plum) and seasonal allergic rhinoconjunctivitis due to grass pollen. he reported oral pruritus, vomiting and cutaneous itching in both arms and hands immediately after drinking tiger nut milk. he became asymptomatic without treatment after 3-4 hours. the allergic workup included skin prick tests with profilin, ltp, latex and fruits that were positive to melon and watermelon and negative to profilin, ltp, latex and the rest of the fruits. prick-by-prick tests with melon, banana, kiwi, apple, pear, tiger nut and tiger nut milk were positive. the serum total immunoglobulin (ig) e was 68.7 μg/l, and specific ige was negative to profilin, ltp, bet v1 and all the tested fruits. background: specific blood ige tests for food allergens are mainly used to confirm a suspect food allergy than to diagnose such an allergy. this is due to the low positive predictive value and the high negative predictive value they have. nevertheless, they are very helpful when they are interpreted in the context of medical history by an experienced allergist. we analyzed the prevalence of sige in children with suspected food allergies. we retrospectively analyzed the all the consecutive laboratory tests of sige for food allergens during the two-year period (2015) (2016) (2017) . tests were of children diagnosed or suspected to have food allergies. a quantitative immunoblot assay was used to measure the circulated 30 different sige. t-test, wilcoxon signed rank test, and chi-square were used to make comparisons. results: fifty-six (55.4%) men and 45 (44.6%) females, 3.4 + 3.2 years old with a maximum of 16 years and a minimum of 2 months old were part of 101 children whose tests were analyzed. one-third of the tests (30.7%) reveals more than one sige present and 55.4% of the tests resulted negative for the sige for the 30 allergens tested (table) . only 3 (3.0%) children have very high concentrations (>100 iu/ml) of egg whites sige, and 2 (2.0%) have egg yolk sige. concentrations of 79.27% of sige positive cases were 0.35-3.5 iu/ml. in our study more than half of the children suspected of food allergies resulted negative for sige for 30 most common food allergens. seventy-nine percent of the positive cases had relatively low sige. only a few positive cases have higher sige than 100 iu/ml. our data support the recommendation that sige couldn't be decisive in food allergic diagnosis, but they may help if they are interpreted cautiously. method: seven patients with a mean age of 61.4 years and female to male ratio of: 3:4, with a background history of hypertension treated with an acei presented with oro-pharyngeal irritation (itch, tingling), face and tongue angioedema and laryngeal constriction, on ingesting fresh fruits (cherries, apples, plums, peaches, apricots, strawberries, grapes), vegetables (parsnips) and/or nuts (peanuts, hazelnuts). two patients required admission to emergency department and three received adrenaline auto-injector. six out of seven patients underwent skin prick testing to common aeroallergens and the index foods. in five cases, immunocap/isac testing was undertaken. in one case the diagnosis was based on the history and in one other case it was based on history and skin prick tests. results: a diagnosis of pfas was confirmed in all patients through the clinical history, spt and/or specific ige serology to the offending food confirming predominant sensitisation pr-10 allergens. primary food allergy and spontaneous angioedema was excluded in all patients. in the cohort studied, the pfas symptoms were unusually severe. we therefore postulate that this was secondary to concurrent use of acei. the management of these patients to sensitization to a β-casein with high homology between only the first 3 milks. more precisely, the allergen candidate could be γcasein, which is derived from β-casein by proteolysis, whose abundance increases during cheese production from fresh milk, and which is absent in cow's milk. a lactoglobulin specific to buffalo's milk may also be responsible. in case of ewe's and goat's milk allergy without cow's milk allergy, sensitization to buffalo's milk should systemically be seeked out. we recommend inclusion of all mammalian milks in the list of the 14 mandatory allergens for declaration on food products. method: prick tests with fruit battery, ltp and profilin was made. analytical with blood count, immunoglobulins, triptasa, total ige and specific ige to banana, apple and orange. finally, open oral provocation with fruits was performed. the diagnostic key was given by the mother of the patient who attended consultations because the infant had erythema in the temporary zone after drinking sea water on the beach. associated with salivation, the patient presented erythema in the malar area lasting a few seconds many times. the prick tests with fruits, ltp and profilin were negative. hemogram: 100 eosinophils/μl, total ig e: 4.9 iu/ml, specific ige to fruits were negative. triptase: 5.1 μg/l. method: here we describe a variety of cases of nsltp allergy presenting to a tertiary allergy centre in the north west of england. results: nsltps have been found to be major allergens in various foods and they are likely to produce severe and systemic allergic reactions. this is reflected in the cases we present here. these proteins are highly cross-reactive due to extensive sequence homology and are panallergens. nsltps are remarkably heat stable and retains its allergenicity in processed foods. it is assumed that nsltps may sensitise both by inhalation and ingestion. an intriguing aspect in nsltp hypersensitivity is the extreme variability of its clinical expression. co-factors are often needed for the clinical expression of nsltp hypersensitivity. conclusion: patients regardless of where they are from, presenting with multiple severe/systemic food allergies need to be investigated for nsltp allergy. these patients require specific dietary advice on foods to avoid and a tailored management plan on how to deal with their allergic reactions. cow's milk as well as cow's milk products are tolerated. material and methods: skin prick tests with different sorts of milk, cheese and milk proteins were performed, specific ige antibodies were measured, a basophil activation test with cow's and goat's milk was performed and an oral provocation test (opt) with cow's milk, cow's milk cheese, raw milk and raw milk cheese was conducted. results: skin prick tests were positive for sheep's milk, goat's milk, goat's milk casein, feta, pecorino and parmesan cheese. elevated specific ige against goat's milk (12.7 ku/l) and sheep's milk (11.7 ku/ l) were detected. activation of basophil granulocytes after incubation of the patient's blood with cow's milk and goat's milk was measurable but also in the non-incubated control blood. all cow's milk products were tolerated in the opt. conclusion: despite consistent homologies between whey and casein proteins of mammals and high cross-reactivity between cow's, goat's and sheep's milk an isolated goat's and sheep's milk allergy with tolerance of cow's milk is possible. skin testing and specific ige help to distinguish from allergy against cow's milk proteins. diet counseling is possible after opt. introduction: salmon roe's allergy, without concomitant fish allergy, is rarely described in western countries. there are few studies on its allergenicity. objective: to report of a case of salmon roe allergy without concomitant fish allergy in a western country. case report: a 26-year-old male with house dust mite allergic rhinitis and asthma, describes for the 1st time, in 2015, an acute episode of dyspnoea, rhinorrhoea, ocular pruritus, epigastric pain and nausea, a few minutes after the ingestion of a sushi meal with rice, salmon, salmon roe, wasabi, soy and ginger. these complaints motivated observation in the emergency room, where it was still documented uvular oedema. he was prescribed intramuscular adrenaline, intravenous steroids and anti-histamines with complete symptoms resolution. the patient declares not had eaten other foods, taken any drugs including nsaid, been infected or practised exercise. skin prick tests with food extracts (salmon and other fish, shellfish, soy, rice, egg total, egg white, egg yolk, ovalbumin and ovomucoid) were negative. skin prick-prick tests were positive for salmon roe (17 × 10 mm) and negative for egg (white and yolk), ginger, salmon, flying fish roe (tobiko), sturgeon roe (caviar) and black scabbard fish roe. specific-ige (sige) to salmon roe extract was 0.28 kua/l (immu-nocap-phadia) and negative against extracts from salmon fish and other fish (<0.1 kua/l). sds-page immunoblotting with salmon roes extract showed a 20 kda-ige binding band, that may correspond to a lipovitelin. after the allergic reaction the patient have tolerated abstracts | 659 salmon fish and other fish roes (tobiko, caviar and black scabbard fish). no oral provocation test with salmon roes was performed given the severity of the reaction. conclusion: this report is an example of a severe allergic reaction to salmon roe without concomitant fish allergy, where the clinical history and the in vivo and in vitro tests were important to an accurate diagnosis. the authors believe this is the first report of a salmon roe anaphylaxis in our country and highlight the importance of this allergen in the western countries, given the increase of sushi consumption in these countries. case report: the prevalence of food allergy is increasing worldwide and consumer habits are changing. pomegranate (punica granatum) was commonly consumed in the mediterranean area but in the last few years became also popular in the different parts of europe. a 5-year-old boy was admitted to our emergency department suffering from allergic reaction within 10 minutes after consumption of pomegranate. he presented with skin pruritus, generalized urticaria and eyelid edema. symptoms resolved within an hour after oral intake of cetirizine. prick-to-prick test with pomegranate was positive (wheal diameter 13 × 7 mm). he had a history of anaphylaxis with egg (urticaria and wheezing) at the age of 9 month, meanwhile consumption of egg is well tolerated. an episode of anaphylaxis with unknown origin appeared at the age of 4 years (urticaria, wheezing and abdominal pain). skin prick tests with aeroallergens revealed birch pollen allergy and he was diagnosed with allergic rhinoconjunctivitis. dietary elimination of pomegranate was suggested and adrenaline auto-injector was provided. we have analyzed omalizumab effectiveness and safety in patients with csu from our database. clinical response was categorized as: no response, partial or complete response by using the urticaria activity score 7 (uas 7). furthermore, the dosage, administration frequency and any side effects were recorded. results: effectiveness: 23 out of 25 patients (92%) achieved complete response. 15 (65.2%) after a single dose of 300 mg (8 patients) or 150 mg (7); 5 patients (21.7%) after two doses of 300 mg (4 patients) or 150 mg (1) results: multi-ethnic adolescents accounted for approximately 1.2% of the total sample of adolescents. prevalence of asthma was significantly higher in multi-ethnic group than non multi-ethnic group. we examined if maternal or paternal foreign born status had a differential effect: in multi-ethnic family with foreign-born father, prevalence of asthma was significantly higher. parental region of country at birth had a significant influence on the prevalence of asthma. adjusted logistic regression analysis was used to determine risk factors for occurrence of allergic disease. residential area, perceived household economic status, parental region of country at birth, and body mass index (bmi) had a significant effect on prevalence of asthma. conclusion: population admixing appears to have significant effect on the prevalence of asthma. further study will be needed to clarify the effect of population admixing on prevalence of allergic disease. several studies have aimed to explore the possibility of mirs as biomarkers for various diseases. in our study we examined six different mirs, previously shown to be involved in eosinophil development and other immune responses, in serum from non-allergic and allergic asthmatics and healthy control subjects in order to determine their potential ability to be used as biomarkers for varying forms of asthma. method: serum from healthy individuals as well as age matched non-allergic asthmatics (naa) and allergic asthmatics (aa) were utilized. additionally, the naas and aas subjects had high eosinophila (≥0.4 × 10 9 cells/l) compared to healthy controls (≤0.1 × 10 9 cells/l) and eosinophil cationic protein (ecp) in serum was measured. asthmatic subjects were included irrespective of inhaled corticosteroid usage. rna was extracted from serum, reverse transcribed and subjected to qpcr analysis. expression changes in six candidate mirs, mir-126, -145, -146a, -155, -223, and -374, were investigated. results: two mirnas, mir-155 and mir-146a, were significantly upregulated in aas as compared to naa or healthy subjects. additionally, mir-223 was upregulated in naa, but not aa or healthy subjects. furthermore, the expression change observed in the aa mirs appeared to correlate with the use of inhaled corticosteroids, but not in the naa mirs. finally, mir-223 and mir-374 expression levels were altered based on the number of eosinophils, which correlated to ecp levels, in naa subjects. conclusion: using six mirs found in the literature to be involved in eosinophila or immune responses, we were able to detect expression changes in the serum of healthy and asthmatic individuals. moreover, were able to distinguish between healthy individuals, aas, and naas on inhaled corticosteroids or with differing eosinophil levels, leading to the possibility that these mirs may be valuable future biomarkers for asthma. background: some studies report that certain sensitization profiles may increase the risk of a more serious allergic respiratory disease. the aim of this study was to describe the sensitization patterns to major allergens of dust mites in our area and investigate the association of these patterns with a specific clinical picture. method: multicenter study performed in 3 hospitals for 4 months. we recruited 133 patients older than 5 years with rhinitis and/or bronchial asthma, with a history of allergy to dust mites and both skin test and specific ige to d. pteronyssinus, d. farinae or l. destructor positive. der p1 and der p2 were determined to all of them. we analyzed 133 patients with an average age of 28.25 years, 60.9% women, 9.7% smokers, 69.9% rhinitis and asthma, 17.3% only rhinitis and 12.8% only asthma. 94% of patients presented sensitization to d. pteronyssinus, 83.5% to d. farinae and 63.2% to l. destructor. the detected sensitization patterns were: both der p1/der p2 positive 73.9%; der p1 positive 8.4%, der p2 positive 11.8% and both der p1/der p2 negative 5.9%. it was observed that patients with higher specific ige levels had more severe forms of respiratory disease, with isolated asthma or associated with rhinitis. for d. pteronyssinus, d. farinae, der p1 and der p2 > 50ku/l there is a greater number of cases of asthma associated with rhinitis, while for l. destructor >3.5ku/l greater number of cases of asthma. no relationship was observed between a specific sensitization pattern and an increased risk of asthma. conclusion: 1. specific ige values greater than 50 ku/l for d. pteronyssinus, d. farinae, der p 1 and der p 2, were significantly associated with a higher probability of asthma and this association was significant for l. destructor>3.5 ku/l (class 3). 2. there are four well-defined sensitization patterns in our population that are influenced by geographic location, being the double sensitization to der p 1 and der p 2 the most prevalent and allowing the correct characterization of 94% of the cases. none of them increased the risk of asthma. kobori t 1 ; nagao m 1 ; ekenkrantz t 2 ; borres m 2 ; sjölander a 2 ; fujisawa t 1 1 national mie hospital, tsu-city, japan; 2 thermo fisher scientific, uppsala, sweden background: reliable biomarkers for diagnosis and management of asthma in young children are needed since pulmonary function test and exhaled no measurement, good biomarkers for asthma in older children and adults, are difficult to perform in young age. we have developed a sensitive and stable assay system to measure eosinophil-derived neurotoxin (edn), an eosinophil granule protein, that is released upon activation, and that may serve as a marker for eosinophilic inflammation also in young children. method: volunteer children from 0-6 years old were recruited and an isaac-based questionnaire was filled out by their caregivers. venous blood was obtained to measure serum and plasma edn and eosinophil count. edn was measured with a research assay developed on the immunocap ® platform. conclusion: blood edn may be a reliable biomarker for diagnosis of asthma in preschool children. conclusion: feno measurement as an add-on option in asthma management to identify asthma patients with th2 driven airway inflammation is less costly than the use of standard diagnostic methods. new biologics may have an additional impact on overall asthma treatment costs. our model demonstrates that incorporating feno measurement may help to optimize asthma medication and reduction in physician visits as well hospitalizations due to severe exacerbations. 1294 | peripheral airway inflammation assessed by fractional measurement of the exhaled breath temperature is a leading feature of asthma background: airway inflammation is considered to be a hallmark of asthma. the potential clinical benefits of assessing it non-invasively has led us to develop a method and device for measuring the temperature of the exhaled breath (ebt=exhaled breath temperature) reflecting the thermal state of the airway mucosa. studies have demonstrated that ebt is increased in asthma, proportionately to the level of control of the disease. in an attempt to further increase the usefulness of this approach, we have further developed a device to allow the assessment of the relative contribution of the central and peripheral airways (caw and paw). now we present the frebt data gathered from patients with suboptimal control of their asthma and from healthy subjects. method: in this cross-sectional study we included 120 volunteers: 71 patients with suboptimally controlled asthma of mild to moderate severity (median age 44, range 18-68 years, 31 men) and 49 nonsmoking subjects without respiratory disease (median age 49, range 18-70 years, 17 men). we measured the fractions corresponding to caw and paw sampled with a fast reacting inflatable balloon valve system operated by a computer during a single breathing cycle. it allows steering of the expired airflow through channels with sensitive temperature sensors. during an initial deep inhalation, the inspired volume is measured and the sequence of valve openings is adjusted so as to yield volumes of air characteristic of caw or paw during expiration. the ratios between [pawebt-cawebt] over the total ebt [%] measured during the same manoeuvre (fractional ebt, frebt) were calculated and compared between asthmatics and controls. results: there was high statistically significant difference between the frebt ratios of asthmatics and controls: 16.25 ± 0.51 (mean ± sem) vs 12.66 ± 0.28, p < 0.001. as the magnitude of the ratio depends on the difference between pawebt and cawebt, higher values of the frebt ratio point to bigger contribution of the peripheral lung tissues, presumably indicative of peripheral inflammation. multiple regression analysis with frebt ratio as dependent variable identified only asthma diagnosis as significant predictor (p < 0.001) and excluded all other anthropometric indices. conclusion: peripheral airway inflammation assessed by frebt measurement appears to be a leading characteristic in asthmatics compared to healthy subjects. method: we examined 142 outpatients (30% male, aged 18-82 year, mean age 54.7 years) with severe asthma according to ers/ ats (2014) definition treated with high dose of ics/laba± tiotropium, antileukotrienes and omalizumab. some patients (n = 21) had orally steroid-dependent asthma. they referred to our secondary care center by gps. pulmonary function tests were measured by dry spirometer (2120, vitalograph ltd., uk). skin prick tests or serum specific ige to common inhalant allergens (house dust mite, animal dander, pollen) were used to assess atopic status. results: seventy five percent (n = 107) of patients with severe asthma had fao in 50% of those was diagnosed concomitant copd. duration of asthma was 14.6 years in patient with reversible airway obstruction (rao) and 14.5 years in those with iao (p > 0.05). early (before age 12 years) onset of asthma was established in 13% of patients with rao and in 9% of patients with fao (p > 0.05). prevalence of atopy did not differ between both groups (81% vs 81%, p > 0.05) but total ige level in serum was higher in severe asthmatics with rao than fao (924 me/ml vs 330 me/ml respectively, p < 0.01). most of atopic patients with severe asthma both with fao (91%) and roa (83%, p > 0.05) were sensitized to house dust mites (d. pteronyssinus and d. farinae). hypersensitivity to pollen was diagnosed in 36% patients with foa and in 17% with rao (p > 0.05), to cat and dog dander in 49% and 25% respectively (p < 0.05). the majority (75%) of patients with severe asthma had fao. hypersensitivity to house dust mites was most common in severe atopic asthmatics with foa and roa where as sensitization to animal danger was associated with presence of foa. 1296 | loss of smell as a clinical marker of severe asthma and its association with upper airway inflammatory diseases background: asthma is frequently associated with rhinitis and chronic rhinosinusitis (crs) while severe asthma is more associated with crs with (crswnp) than without (crssnp) nasal polyps. loss of smell (los) is associated with crs, mainly with crswnp. we aimed to assess loss of smell as a clinical marker to discriminate crs from rhinitis and severe from non-severe asthma. method: in a cross-sectional multicentric study, asthmatic patients (n = 383) were evaluated by pulmonologists and ent specialists using gina, aria, and epos definitions. los was evaluated by severity [vas scale, 0-100 mm, median (iqr,inter-quartil range)] and by prevalence of anosmia (hyposmia vas >0-70 mm, anosmia vas>70 mm). results: los was present in 55.4% of asthmatics (hyposmia 41.5%, anosmia 14.9%). los was more severe [22 mm (0-75), p < 0.001] and anosmia more frequent (26.4%, p < 0.001) in severe persistent asthma than in moderate [10 mm (0-50); 11.4%] mild [0 mm (0-28); 10.7%], or intermittent [0 mm (0-45); 8.6%] asthma. in addition, los was more severe [38 mm (2-76) vs 0 mm (0-20), p < 0.001] and anosmia more frequent [28.1% vs 3.9%, p < 0.001] in crs than in rhinitis patients. in those asthmatic patients with crs, los was even more severe [50 mm vs 20 mm (0-56) p < 0.001] and anosmia more frequent (40.6% vs 13.4%, p < 0.001) in crswnp than in crssnp. conclusion: loss of smell and specially anosmia may clearly discriminate severe from non-severe asthma and crs (specially with np) from rhinitis alone in asthma patients. thus, los may be considered a significant clinical marker of severe asthma and its association with upper airway inflammatory diseases. 1297 | last station in the eosinophilic asthma with chronic rhinosinusitis and/or nasal polyposis march: eosinophilic asthma with radiological findings associated with blood eosinophilia yilmaz i 1 ; nazik bahçecioglu s 2 ; türk m 3 ; tutar n 1 ; oymak fs 2 ; gülmez i 3 1 erciyes university school of medicine, kayseri, turkey; 2 department of chest diseases, kayseri, turkey; 3 division of immunology and allergy, kayseri, turkey background: eosinophilic asthma with chronic rhinosinusitis and/or nasal polyposis (eacrs/np) is a subphenotype of adult-onset eosinophilic asthma. blood eosinophil levels are shown to be highly elevated in patients with ea-crs/np and have potential for tissue infiltration. we aimed to demonstrate the clinical features of the patients who have a blood eosinophil level above 10% and have thorax computed tomography findings due to blood eosinophilia. results: we identified 36 patients who met the above criteria. we defined this group as "eosinophilic asthma with chronic rhinosinusitis and/or nasal polyposis with radiological findings related to blood eosinophilia" (earr). the mean age was 44.9 ± 11 years and 64% was female. nasal polyps, aspirin exacerbated respiratory disease and atopy was present in 81%, 47% and 25% of the patients, abstracts | 665 respectively. the mean blood eosinophil count was 1828.6 cells/mm 3 (19%). the majority of earr patients had upper lobe dominant ground-glass opacities. the mean follow-up period was 3.2 ± 2.5 years. earr patients did not evolve into eosinophilic granulomatous polyangiitis in the follow-up. method: we aimed to identify features more probably associated with asthma in a unselected group of patients with diagnostic criteria for aco. we consecutively selected the first 10 consecutive patients with diagnostic criteria for aco. all patients were evaluated by accurate clinical history interview, assessment of asthma control test (act) and copd activity test (cat), lung function and exhaled nitric oxide (fe no ) measurements, sputum cytology, blood eosinophil count, serum total ige and periostin levels, methacholine and adenosine-mono-phosphate (amp) bronchial challenges. all these measures were repeated after an oral corticosteroid (ocs) trial of methylprednisolone 32 mg/day for 14 days. we defined 9 parameters that we expected improved after the ocs trial, and therefore considerable as markers of asthma: fev 1 , fef 25-75 , fev 1 /fvc, fe no , act, sputum and blood eosinophilia, methacholine and amp challenges. patients with improvement of at least 4 of these parameters after ocs trials were defined as "responders" to the treatment, and therefore more likely to be asthmatic than copd or aco. results: five (50%) patients were classified as responders and they were characterized by having basal higher fe no values (102.8 ± 34.2 vs 13.6 ± 3.5 ppb, p = 0.032), greater bronchial reversibility basal values of serum periostin and total ige, and blood eosinophils were higher in responders but without reaching the statistical significance. conclusion: fe no and the degree of bronchial reversibility (and possibly also the degree of response to an amp challenge) are reliable biomarkers to distinguish asthmatics among those with suspect aco. method: the preliminary case-control study included 52 obese persons with asthma who were matched for age and sex and 48 nonobese asthma subjects. non fasting serum levels of adiponectin, and leptin were measured by commercially available immune assay kits, and routine biochemical parameters were analyzed in both the study groups. the results show statistically significant lower levels of serum adiponectin and higher serum leptin levels in obese asthma subjects with respect to non-obese asthma patients (p < 0.001). moreover, an inverse correlation was also observed between serum adiponectin and serum leptin in obese asthma subjects (p < 0.05). our results indicate the association of these hormones might act as a significant predictor in the progression of asthma. moreover, the role of serum adipokines is promising and might potentially act as a meaningful drug target in the pathogenesis of asthma. background: overweight/obesity is known to be a possible factor for poor asthma control. the aim of the study is to determine the serum concentrations of leptin in atopic asthmatic patients and its relationship with body mass index (bmi), asthma severity defined by medical treatment and asthma control defined by the asthma control test (act). method: we randomly selected 33 adult patients previously diagnosed with allergic asthma based on gina (global initiative for asthma) guidelines, returning for follow-up to an outpatient allergy/ immunology clinic during november 2017. following an informed consent, the patients were asked to fill act, their bmi was recorded, and elisa blood assays for leptin were drawn. exploratory data analysis, spearman's correlation (95% ci by bootstrapping), and partial correlations were performed. results: female/male ratio was 24/9, mean bmi was 27.2 ± 4. conclusion: leptin was significantly associated with overweight/ obesity in asthmatic subjects, and showed higher values for women. leptin inverse correlation with act did not reach statistical significance, likely owing to underpowered estimates, in a small sample characterized by an elevated mean bmi and severe allergic asthma. background: immunoglobulin lowering may be associated with recurrent wheezing symptoms and clinic by increasing the tendency to viral respiratory tract infections. in this study, it was aimed to investigate the frequency of immunoglobulinemia in preschoolers with wheezing. the study was conducted between 01.01.2013 and 01.01.2016 between. university of health sciences, ankara child hospital, the children allergy and immunology clinic included patients who had been followed up and treated for at least one year with recurrent wheezing attacks within younger than 48 months. the immunoglobulin (g, a, m) values of the patients were retrospectively analyzed. immunoglobulin levels were determined to be normal and low according to age limits. the study included 585 patients (65.6% male, 34.4% female) under the age of 6 years with a mean age of 26.9 months. the mean follow-up period of the patients is 2.2 years. in 33.7% of these patients, at least one immunoglobulin was found to be low. none of these patients had any signs or symptoms of immunodeficiency. immunoglobulin a was low in 21% of the patients, immunoglobulin g in 18%, and immunoglobulin m in 7.5% of all patients. conclusion: immunoglobulin was found to be low in these patients when there was no immunodeficiency and preschool wheeze was diagnosed. this should be etiologically investigated as to whether if this is a special group in preschoolers with recurrent wheezing and hypogammaglobulinemia combination. method: asthma severe unit is formed by allergists, pneumologists, pediatricians and otorhinolaryngologists. hematologists and immunologists make specific collaborations. we present the partial results of our data collection, which include 75 patients with severe asthma according to ers/ats task force, selected by peripheral eosinophils >220 according to wagener et al. we followed them up to assess control for 1 year. we obtained cellularity in sputum using induced sputum technique. values of il of th2, th1, th17 pathway, periostin and ilc were not yet available. results: median age was 36 ± 24, feno 45 ± 34, exacerbations previous year 1.8 ± 2.4, act 17.7 ± 5, fev1 75% ± 21 and dose of inhaled corticoids (budesonide equivalent) 1235ug ± 618. most of the patients were sensitized (78%) and 15.9% were polysensitized. the most frequent sensitization was dust mites (81%). 27% had received immunotherapy of whom 35.3% with lack of response. not sensitized patients were older. sputum cell analysis of 54 patients was performed, 37% had sputum eosinophils>3%, mean sputum eosinophil value was 4.1 ± 4.7 and peripherally 518 ± 384. correlation among sputum and peripheral eosinophilia was 0.097 (p = 0.485). the peripherally eosinophil value >220 had a sensitivity of 80% and a specificity of 54% for the detection of sputum eosinophils >3%. no differences were observed in sputum cell count depending on allergic sensitization. 64% had an uncontrolled asthma. presence of polysensitization, rhinitis or polyposis were not statistically related with the control. different patterns were observed in function of cause of poor control: patients with obstructive pattern (fev1 < 80%) were older and received more inhaled treatment. patients with high rate of exacerbations had more sputum eosinophilia and neutrophilia. both groups had worse act and received more oral steroids. patients who received oral steroids were more often sensitized to fungi in some follow-up visits. not significant differences were observed in control according to the act. asthma had more sputum neutrophilia, were older, received higher inhaled steroids dose and had adult onset asthma. the only control variable related with sputum eosinophilia was exacerbation. fungi sensitization was more frequent among patients with oral steroids. method: a randomized, double blind, placebo controlled study with 80 patients from the de la salle university medical center with a mean age of 44 with partly or uncontrolled asthma. they were assigned to either cm glucan or placebo group for two months. act score and %fev1 postbronchodilator were assessed at the 1st visit, 4th week follow up, and 8th week follow up visits. an independent and paired t-test were used to determine mean changes in act scores and %fev1 between the groups. results: in the two treatment groups, those in the cm glucan group had a greater % fev1 mean change of −6.95 compared to placebo which had only −14.1, a mean difference of −7.15, and a trend toward significance with a t-test p value of 0.061. in terms of changes in act score, those in the cm glucan group had a mean change of 10.12 and 9.75 for placebo, a mean difference of 0.37 and was not significant at t test p value of 0.718. the result of the emanuel trial showed a trend of improvement among patients on both groups in terms of act score and %fev1 postbronchodilator. however, it was not statistically significant. 1306 | lung function improvements with tiotropium in patients across all ages: impact of episodes of asthma worsening during phase 3 trials vogelberg c 1 ; casale tb 2 ; bleecker er 3 ; goldstein s 4 ; szefler s 5 ; engel m 6 ; el azzi g 6 ; dewberry h 7 ; hamelmann e 8 method: post hoc analyses involved 6 phase iii, randomized, double-blind, placebo-controlled trials: 4 in patients aged 6-17 years (rubatina-/canotina-/vivatina-/pensietina-asthma) who received tiotropium (5 or 2.5 μg) or placebo, as two puffs once-daily via the respimat, as add-on to ics ± other controllers; and 2 in adults (pri-motina-asthma replicate trials) who received once-daily tiotropium 5 μg or placebo, as add-on to ics/laba ± other controllers. we analyzed change from baseline for peak fev 1(0-3h) and trough fev 1 at week 12 in vivatina-and pensietina-asthma, and week 24 in pri-motina-/rubatina-/canotina-asthma, comparing patients with and without episodes of asthma worsening during the trials. asthma worsening was defined as an episode of progressive increase in dayto-day asthma symptoms (recorded by patients and confirmed by the investigator) or a decrease of patient's best morning pef ≥30% from mean for ≥2 consecutive days. as a post hoc analysis, p values are nominal. results: there were no differences in baseline disease characteristics between those who experienced episodes of asthma worsening and those who did not, within specified age and asthma severity groups. placebo-adjusted lung function improvements were observed with tiotropium 5 μg in patients who experienced episodes of asthma worsening and those who did not during the trials (table 1) . there was some variability in subgroups with low numbers of patients. conclusion: once-daily tiotropium add-on had a similar efficacy in adult and pediatric patients with symptomatic asthma, irrespective of whether they experienced episodes of asthma worsening or not during the trials. these data support the broad efficacy of tiotropium and show largely consistent improvements in lung function even in patients who experience episodes of disease worsening. 1307 | cochrane review of the use of antibiotics for acute exacerbations of asthma method: we searched the cochrane airways trials register, trial registries and reference lists of primary studies. we extracted outcome data and assessed risk of bias in duplicate and used current cochrane methodology throughout. our primary outcomes were intensive care unit (itu) admission, duration of symptoms/exacerbation and adverse events. we included six studies, including a total of 681 adults and children. trials were of varied methodological quality and we were able to perform only limited meta-analysis. one study reported a single itu admission but no other studies reported admissions to itu. two studies investigating macrolides reported diary card symptom score and showed antibiotics improved symptoms (md −0.34, 95% ci −0.60 to −0.08). one study including 40 participants reported more symptom-free days in the macrolide group than usual care. one study of a penicillin including 69 participants reported asthma symptoms at hospital discharge; the between group difference was reported as non-significant. serious adverse events were rare; 10 events were reported across the three trials (n = 502). the pooled effect estimate for all adverse events from three studies was imprecise (or 0.99, 95% ci 0.69-1.43). no deaths were reported. conclusion: our results confirmed that omalizumab significantly improves disease control and is a safe add-on therapy. also in appropriate patients with controlled disease over time, efforts to stepdown other asthma medications will be appropriate. (45) aerd: aspirin exacerbated respiratory disease; sd: standard deviation. data are n (%), mean ± sd or n/n (%). c-act: asthma control test for children, fev1: forced expiratory volume in one second; fev1/fvc: the ratio of forced expiratory volume in one second to forced vital capacity, pef: peak expiratory flow; feno: fractional exhaled nitric oxide, vas: visual analogue scale *these included allergic rhinitis, asthma, eczema, atopic dermatitis, food allergy, etc. **there were 11, 13 and 18 missing data in treatment a, b, and c, respectively. this study examines the potential treatment effects of sq ® hdm slit-tablet on qol measured by sf-36v2 in people with aa and ar. the analyses are based on data from the mt-04 trial (eudract no. 2010-018621-19) and utilize data from the 12 sq-hdm treatment group (282 subjects) and the placebo group (277 subjects). throughout the trial, qol was measured at each of visit 3-13 via sf-36v2. this yielded psychometrically-based physical and mental health summary measures, as well as a sf-36v2 total score. according to trial design, the use of inhaled corticosteroid (ics) was reduced by 50% for a three months period (visit 10 and 11) and completely withdrawn for the last three months of the trial (visit 12 and 13). results: by estimating a simple regression on differences in sf-36v2 total score from baseline measurements (visit 3), a positive and statistically significant treatment effect on the overall qol of the 12 sq-hdm treatment compared to the placebo group in visit 9 and 10 was found. further analyses show that the qol improvements are mainly driven by increases in the general mental health score, which are carried through to visit 12. in particular, the mental health and role emotional domains show statistically significant improvements. the results show that the sq ® hdm slit-tablet improves qol measured by sf-36v2 in patients with hdm induced aa and that this effect is driven by improvements in the mental health domains. 1315 | impact of treatment prescription, adherence to treatment and use of inhalers in asthma control-results of the efimera study method: cross-sectional multicenter observational study conducted with patients who use any type of medication with inhaler devices. patients referred from primary care and seen by a pneumologist or allergist for the first time were evaluated. the following data was collected in a single visit: adequate prescription according to gina2015 guidelines (gina); specific and general treatment adherence using morisky-green questionnaire (mg) and inhaler adherence test (tai); disease control with asthma control test (act) and assessment of inhaler use technique were measured with the extended tai. results: patients included in this study (n = 1682) had a mean age of 45 ± 17 years, an average disease evolution of 14.9 ± 14.1 years, 64% of which were women. according to gina recommendations, 35.9% of patients have insufficient or inadequate prescription. when measured by the mg test the 68.5% of patients showed bad adherence, meanwhile measured by the tai test adherence was 76.8% measurements of inhaler use technique resulted in 17% of patients having one or more mistakes regardless of whether the device was a mdi or dpi. several factors showed to be related with bad asthma control: inadequate prescription (or: 8.05 [5.74-11.27 background: it is well known that the constant and prolonged tobacco smoking affects the natural history of asthma. vaping is the act of inhaling and exhaling the vapor produced by an electronic device called e-cigarette (e-cig), whose basic structure includes a power source and an atomizer. two types of vaping are the most popular ("mtl" and "cloud chasing"). we have created a web-survey with questions concerning epidemiological data, quality of life and symptoms worsening in asthmatic vapers. the survey has been advertised through various social networks and local press. 2403 people responded, including 437 asthmatics (18%). the asthmatics were: males 88%, under 18 3%, 18-25 years 36%, 26-30 years 19%, 31-65 years 42% and over 65 0%. 70% used ecig-only, 30% smoked and vaped together, 0%. those who preferred mtl-type of vape were 43% and "cloud chasing" were 57%. results: to the question: "has vaping ever worsened asthma symptoms?" 90% answered no, 10% yes. to the question: "as asthmatic, would you suggest to an asthmatic smoker to start vaping instead of smoking?" 1.3% answered no, 98.7% yes. to the question: "how much nicotine do your vaping liquids have?" 15% answered 0 mg/ml, 4% 1.5 mg/ml, 62% 3 mg/ml, 11% 6 mg/ ml, 6% 9 mg/ml, 1% 12 mg/ml and 0% 18 mg/ml. to the question: "do you take medications for your asthma?" 57% declared to use a drug as needed, 0% used a single drug daily, 16% used more than one drug daily and 27% declared "i don't take any asthma medication". we related (χ2 test) the worsening of asthma symptoms with the nicotine content (p = 0.313), the type of vaping (p = 0.305), the current therapy (p = 0.123) and we did not find a statistically significant correlation. vaping has undoubtedly shown an advantage in terms of improvement of symptoms compared to cigarette smoking (p = 0.016), in particular 82.5% subjects who smoke and vape did not have a worsening of symptoms, while 17.6% of them had a worsening. the vaper-only users who never worsened were 271 (90.6%) and 28 (9.4%) had a worsening. conclusion: despite the limits related to the online survey as a data source, e-cigs seem to be a useful tool in the pathway to quit smoking. in fact, 98% of the asthmatics who smoked traditional cigarettes would recommend switching to e-cig and 90% did not worse their asthma symptoms. background: despite the success of pharmacotherapy, more than half of patients with persistent bronchial asthma (ba) do not achieve disease control. in recent years, the issue of approaches to treatment based on the identification of phenotypes of the disease has been increasingly discussed. this approach becomes the key to optimizing therapy for asthma, allowing the personification of treatment. anti-ige-therapy using omalizumab is one of the most researched variants of phenotype-specific treatment. method: aim of our study was to investigate of the causes of uncontrolled predominantly atopic asthma, the frequency and effectiveness of the personalized therapy in real clinical practice. 54 patients with uncontrolled severe atopic asthma were examined in outpatient department of the city hospital during 2017. all patients underwent physical examination, pulmonary function testing, and total serum ige evaluation. results: 35% of patients had uncontrolled asthma due to inadequate basic therapy of the disease. the change in therapy allowed them to achieve control of the disease. obstructive sleep apnea syndrome (osas) was revealed in 11.1% of patients. these patients underwent cpap (continuous positive airway pressure) therapy. 13% of patients had gastroesophageal reflux disease (gerd). 25% of patients had an elevated level of serum ige level and needed anti-ige therapy. in 7.4% of cases, the initial serum ige level was more than 1500 iu/ml which was a contraindication to therapy of omalizumab. 8 patients received omalizumab therapy. this therapy led to relief of symptoms and decreased frequency of asthma exacerbations. results: it was found that the prevalence of obesity among the 367 patients with asthma and being treated in inpatient conditions in 2013-2015 was 44.9% of patients, which is comparable to the prevalence of obesity among the population in general. the data of the patients suffering from asthma and obesity treated both in inpatient and outpatient conditions, was analyzed and it is set that obesity does not affect the severity of the clinical course of asthma. it is shown that obesity does not affect the control of symptoms of asthma. thus, the control of asthma symptoms depends on timeliness of diagnosis, the adequacy and terms of appointment of basic asthma therapy, the presence, severity and adequate treatment of concomitant diseases, psychoemotional background of patients, their compliance and adherence to therapy. results: the causes of smoking in asthmatics were not significantly different from the control (p > 0.05). patients most often used smoking as "support for emotional stability". the motivation to smoking cessation was higher in the asthmatics group (58%) than in the control group. the main reason for smoking cessation was a deterioration in health -60%. the majority of smokers -85%, performed attempts for smoking cessation. low level of br was revealed in 90% asthmatics (27% non-smoking asthma patients and 18.5% of cases in the control group, p < 0.001), cf had low values and was lower in asthmatics group in compare to the control group (p < 0.05). the pac values correlated with the level of br: a low level was determined in 100% in smoking asthmatics, in 50% in nonsmokers with asthma and 7.4% in smokers of the control group obstructive sleep apnea (osa). all of the patients were on regular treatment with low dose inhaled corticosteroids for at 12 months and start treatment with continuous positive airway pressure (cpap) .to assess quality of life, we used asthma symptom control tools (asthma control test) .patients performed daily peak flow meter and spirometry (once a week) during period of 4 weeks after start using cpap. results: during the study, 47 of the followed patients had no exacerbation of asthma. four of patients during this period had exacerbation, due to upper airway infection so they were excluded from study. results of following showed that there was improvement in quality of life in all 47 patients included in study but there no statistically significant improvement in pulmonary function tests fpt. huang y 1 ; yao t 1 ; huang y 1 ; chiu c 1 ; tsai z 1 ; kao p 1 ; lu k 1 ; fang h 1 ; lin c 1 ; gau c 1 ; lee w 1 ; tsai h 2 results: the rate of preterm birth among the study subjects was 18.2%. the prevalence of physician-diagnosed rhinitis was 50.6%. there was no significant association between preterm birth and physician-diagnosed rhinitis (p = 0.43). when stratifying by atopy status, we found that preterm birth was associated with physiciandiagnosed rhinitis among children without atopy (adjusted or [aor] = 0.33, 95% ci = 0.12-0.93, p = 0.04), but not among children with atopy (p = 0.77). when further classifying by gender, greater protective effect of preterm birth on rhinitis was only found in boys without atopy (aor = 0.12, 95% ci = 0.03-0.56, p = 0.007). the results suggest that preterm birth may have a protective effect against the development of childhood rhinitis in our study population. the protective effect is only observed in boys without atopy. further investigations will be merited to confirm these findings and to investigate underlying mechanisms. background: folic acid supplementation (fas) during pregnancy has been suggested due to its protective effect against neural tube defects. at present the effect of fas during pregnancy on childhood rhinitis has remained unclear. we aimed to investigate the relationship between fas during pregnancy and childhood rhinitis. logistic regression analysis with covariate adjustment was performed. adjusted covariates included sex, age, number of older siblings, breast feeding duration, maternal smoking during pregnancy, maternal allergy, maternal education level, maternal age and socioeconomic status results: the prevalence of physician-diagnosed rhinitis was 55.3%. there is a significant association between fas and physician-diagnosed rhinitis (adjusted odds ratio [aor] = 2.07; 95% confidence interval [ci] = 1.25-3.43 for fas ≥3 months) compared to the group of never use. in the stratified analysis by atopy status, maternal fas during pregnancy was significantly associated with physician-diagnosed rhinitis in the atopic group (aor = 1.91, 95% ci = 1.07-3.40 for fas <3 months; and aor = 2.34, 95% ci = 1.19-4.58 for fas ≥3 months), but not in the non-atopic group. when further stratified by gender, significant association between maternal fas during pregnancy and physician-diagnosed rhinitis was only found in boys with atopy (aor = 3.23, 95% ci = 1.39-7.50 for fas <3 months; and aor = 4.02, 95% ci = 1.51-10.72 for fas ≥3 months). the results demonstrate that maternal folic acid supplementation during pregnancy might increase the risk of childhood rhinitis, especially among boys with atopy. further investigation will be needed to validate our findings and to understand potential underlying mechanisms. according to sequence data 13 from 16 detected adv (in all groups of patients) belongs to species f 41 type and 3 samples to species c 1 type (rei group). bv type 1 was identified in 3 strongly positive (ct ≤ 25) swab samples in ari group. conclusion: simultaneous testing of respiratory and stool samples together shown that at least 6.6%/7.9% of 151 study subjects had dual/mixed infections, respectively, including 11%/9.5% of 63 respiratory disease patients, 3.6%/7% of 56 gastroenteritis patients and 3.1%/6.2% of 32 patients with combined respiratory/enteric infections. we found no virus combination specific for different groups of patients. 1327 | neonatal respiratory supports and future asthma-like presentation in prematurity with bronchopulmonary dysplasia results: of all the tests analyzed 54.4% were males and 45.6% females with a mean age 5.7 ± 4.8 years old. half of the tests (49.2%) reveals positive specific-ige to more than one allergen and 38.6% (66) have no serum specific-ige for the tested allergens (table 1) . sixteen patients (9.4%) have very high concentrations (>100 iu/ml) of derm. pteronyssinus specific ige, 13 (7.6%) of derm. farina, 7 (4.1%) of rey pollen and 6 (3.5%) of oak and timothy grass pollen. further studies are needed in order to elucidate the effect of these cytokines on allergy development and protection. shinohara m 1 ; matsumoto k 2 1 department of pediatrics, ehime university hospital, toon, japan; 2 department of allergy and clinical immunology, national research institute for child health and development, tokyo, japan background: probiotics consumption during perinatal and postnatal periods reportedly reduces the risk of atopic dermatitis in the offspring, whereas such probiotics consumption did not affect ige levels or the risks of other allergic diseases; the precise mechanism how probiotics consumption reduces the risk of atopic dermatitis remains unknown. we hypothesized that probiotics consumption may reduce skin hypersensitivity to histamine. to test this hypothesis, we investigated whether perinatal/postnatal consumption of yogurt associates with skin hypersensitivity to histamine or not. method: this was a cross-sectional study enrolled 256 motherinfant (≥6-months-old) pairs. physician-diagnosed allergic diseases and food consumption, such as milk, fermented drinks, and yogurt, by mothers during the third trimester of pregnancy and by infants during the first 6 months of life were assessed using self-questionnaires. skin prick tests (spts) to saline and 1 mg/ml histamine were performed using bifurcated needles, and wheal sizes were measured 15 minutes after the puncture. the spt wheal sizes in infants with eczema/atopic dermatitis (n = 44) were significantly larger than those in infants without eczema/atopic dermatitis (n = 156; 4.6 ± 1.9 mm vs 3.8 ± 1.8 mm, respectively, p = 0.015), and thus these infants were excluded from the further analyses. the spt wheal sizes to histamine in infants with daily yogurt consumption during the first the aim of this study was to evaluate the prevalence and clinical relevance of sensitization to profilins in atopic patients with food allergy. the study was performed on a group of 43 children age 2-14 years with sensitization to at least one plant-derived food allergen (ige > 0.7 ku/l). the included patients had never been treated with allergen immunotherapy before the study. the presence of ige to recombinant (r) rbet v 2, rart v 4 and ramb a 8 in serum was evaluated using elisa method as previously described (jbc 2016; 291:15447) . in addition serum level of igg4 to rbet v 2, rart v 4 and ramb a 8 was also evaluated. results: sensitization to profilins was found in 8 out of 43 (18.6%) patients (p+). sensitization to all 3 studied profilins was demonstrated in each p+ patient. the remaining 35 children, with pollenfood sensitization, were not sensitized to any of the studied profilins and they served as a comparator group (p−). analysis of the clinical status revealed that asymptomatic patients in regard to plant-derived food hypersensitivity were found more frequently among p+ (75%) than p− (37.1; p < 0.01) patients. sensitization to profilin was associated with positive ige to the same food allergens as in the control group. clinical manifestation of pollen-food sensitization expressed as allergic rhinitis, bronchial asthma and atopic dermatitis was comparable between groups, except of oral allergy syndrome, which was not seen among p+ children. similarly, history of anaphylaxis to plant-derived foods was registered only among p− (11.4%) patients. interestingly, all patients with sensitization to profilins had also elevated level of serum igg4 against rbet v 2, rart v 4 and ramb a 8. results: no significant difference of physician-diagnosed eczema (p = 0.88) or current eczema (p = 0.82) was observed between children born full-term and preterm. after stratifying by atopy status, we found that children born preterm had a more than three-fold higher risk of having physician-diagnosed eczema (adjusted or (aor) = 3.92; 95% ci = 1.25-12.29; p = 0.02) and current eczema (aor = 3.16; 95% ci = 1.06-9.41, p = 0.04) than their counterpart in the non-atopic group. no statistical significance was observed for the association between preterm birth and eczema in the atopic group. no association between preterm birth and eczema was found when stratifying by gender. our results reveal that non-atopic children born preterm have a higher risk of developing eczema. the results suggest potential modifiable effect of atopy on the association between preterm birth and eczema. further studies with a larger sample size are needed to validate the findings in this study. background: there is a need for more knowledge about factors of importance for a successful transition from childhood to adulthood among adolescents with allergic disease and especially those with severe allergy. therefore the aim of this study was to describe experiences of living with severe allergy from the adolescents and their parent's perspective and thereby identify factors of importance for transition from pediatric to adult care. method: a qualitative study was performed based on six focus groups interviews, two with adolescents and four with their parents. in total 11 adolescents (age 10-16 years old) and 21 parents participated. the interview guide contained questions about experiences of living with severe allergy. the transcribed data was analysed using systematic text condensation. results: in total four themes were presented, two themes occurred in both the adolescent and the parent's focus groups, to be special and to be prepared. for two themes there was a difference between the adolescents and their parents. the theme, the importance of the parents, only occurred in data from the adolescents and the theme the meetings with health care only occurred in the parent's data. the adolescents felt that they had low priority in the class and several stated they were teased at school and their parents felt that focus on their child often was in a negative way. the adolescents described that they took responsibility for their diseases while their parents expressed a need to protect. the adolescents stated that one of the parents were always present or had been during the years, the reason being safety and security. only the parents mentioned experiences from healthcare. parents who described that they had continuity in healthcare meetings and where met by high competence and with a professional approach were more satisfied with the support from the health care. one factor that was felt to be important was whether the doctor involved the youth in the conversation or not. the teenagers in this study relied on their parents while also taking responsibility for their illness at the same time. parents, on the other hand, showed a tendency to overprotect their adolescents. for healthcare professionals it is important to involve the adolescents in the care to facilitate the transition. results: 50.9% of the children used antibiotics currently and 21.0% out of them used antibiotics ≥3 times yearly. current wheeze (w) was established in 6.5%, sleep-disturbing w in 3.6%, exerciseinduced w in 1.7%, dry night cough apart from a cold in 12.2%, and asthma in 2.3%. current antibiotics use ≥3 times yearly was positively associated with current w (aor: 13.37; 6.14-29.11; p < 0.001), sleep-disturbing w (aor: 7.87; 3.34-18.57; p < 0.001), exercise-induced w (aor: 5.44; 1.89-15.61; p = 0.002), dry night cough (aor: 3.80; 2.29-6.29; p < 0.001), and diagnosed asthma (aor: 5.68; 1.96-16.50; p = 0.001) while antibiotics use <3 times yearly was positively associated only with current w (p = 0.003) and dry night cough (p = 0.011). the results suggest an aggravating role of antibiotics use on asthma in school age thus further supporting the recommended restriction of antibiotics exposure. results: after questioning 31.6% 95% ci, 23.2-40.9 were suffering from respiratory diseases, having symptoms of chronic disease: cough-86.1%, wheezing-41.66%, tightness in the chest-27.7%. the risk factors (passive smoking, open fire house warming and no air conditioning) were commonly met in major cases at ill children rather than healthy ones (68.4% 95% ci, 59.1-76.8). as a result of studies made of the equal to 17.5 ± 0.3, comparative the end of lessons equal 19.2 ± 0.2 (p ≤ 0.05); air relative humidity varies during lessons equal with 62.9 ± 1.4 (norma toilet 30%-60%); co2 concentration exceeds allowable limits −0.3 ± 0.02 (mac −0.1%). conclusion: respiratory morbidity in high school examined has a tendency to increase. we noticed deviations from the hygienic norms: the indoor temperature and relative humidity was lower and the co 2 level was twice higher than the normal one. the "asthma ever" outcome was reported in 32 cohorts. 28 cohorts defined this as parental reported asthma (with or without specifying that it was doctor-diagnosed), 2 cohorts used gp records as the only source of diagnosis, and 2 used parental report or gp records. the "current asthma" outcome was reported in 40 cohorts. there was little consistency with how current asthma was defined or worded, with 23 different definitions used. the most common definition of current asthma, reported 16 times, was "asthma ever and either asthma symptoms in the last 12 months or asthma medication in the last 12 months". other criteria included in asthma definitions were bronchial hyper-responsiveness, reversible airway obstruction, positive exercise test, and asthma symptoms reported at a previous questionnaire. only one "current asthma" definition was based exclusively on prescription data: "dispensed two asthma medication during the past year". nine cohorts reported asthma outcomes without specifying how it was defined, and were categorized as "asthma unspecified". conclusion: "asthma ever" and "current asthma" are two main asthma outcomes used to define asthma in child cohort studies. definitions of asthma vary substantially across cohorts. case report: thereby we present two case reports of two children with impairment verbal communication as part of asd and allergic diseases. the first patient was a 3 year old boy with sneezing, rhinorrhea night cough and eye redness. he had been suffering for almost 2 years from the above mentioned symptoms. he had family history for atopic diseases and was for 7 month breastfed. specific ige revealed sensitization to birch, alder, hazel, oak, mugwort pollen and dog epithelia and dermatophagoides farinae. specific ige resulted positive for nuts and rye flour. the second patient was almost 3 year of age in the tame that he presented in our hospital. he cried and screamed all the time because of severe atopic dermatitis and typical symptoms such as itching all over the body and his impairment of verbal communication. specific sensitization showed sensibilization to egg white and egg yolk, to nuts, rye and wheat flour. the food specific ige leaded to positive results to alder, birch, hazel and oak pollen, but also to grasses, ragweed and mugwort. prick by prick test showed positivity to egg white and egg yolk. atopy patch test to pollens resulted negative. results: the first patient symptoms were well controlled after treatment with antileukotrienes. his verbal communication was also improved after a year or more. the second three year old patient after required a combination of specific treatment with antihistamines, corticosteroids, immunosuppressive drugs and diet recommendation. afterwards he had a reduced level of itching and anxiety but compared to other children he had a severe eczema. erythema multiforme (em) is an acute, immune-mediated, mucocutaneous condition that is most commonly caused by infection and drugs. it is characterized by targetoid lesions, sometimes accompanied by oral, genital or ocular mucosal erosions. there was no pediatric patient that had previously been reported in the literature with development of type 4 reaction after omalizumab treatment. we presented a case who developed em to omalizumab therapy. an 16 year-old female patient was admitted to pediatric allergy clinic with complaints of fever and rash. she had been diagnosed with chronic spontaneous urticaria (csu) 8 years ago and she was planned to treat with omalizumab (75 mg, subcutaneously every 2 week) because of the inadequate response of antihistamines at a medical center. her complete blood counts, liver, renal, thyroid function tests and serum c3,c4,c1 esterase inhibitor protein levels introduction: celiac disease is an autoimmune disease triggered by exposure to gluten in genetically predisposed individuals and characterized by chronic inflammation of the small intestine. chronic urticaria is a skin disease, characterized by the appearance of pruritic wheals with or without angioedema, whose underlying mechanism cannot be identified. objective: to report a sporadic case of an 11-year-old boy with chronic urticaria associated with celiac disease. methods: an 11-year-old boy(weight 31 kg, 3rd-10th percentiles) was admitted to our clinic with a 6-year history of chronic urticaria. during the first three years, he was under antihistamine treatment(of incremental doses)and occasionally received preparations of cortisone according to the eaaci guidelines. he was asymptomatic for 2 years until treatment was discontinued. eight months earlier, after a viral infection, a recurrence of urticaria, involving the trunk and extremities without angioedema was noted. subsequently, he was under antihistamine treatment with cetirizine but had an uas-7 score of 11. total laboratory investigations were performed. results: laboratory control was negative except for positive antibodies to celiac disease(anti-transglutaminase >200 u/ml, anti-endomysial, gliadin antibodies).further control with colonoscopy and biopsies (from duodenum and stomach) were obtained. the histopathological findings along with the clinical findings indicate celiac disease, type 3b marsch-oberhuber and grade b1 corazza-villanacci. in the past, similar cases have been reported. efforts have been made to associate chronic urticaria with celiac disease, although the mechanism remains unclarified. evidence suggests that the duration of gluten exposure, among otherwise asymptomatic patients with celiac disease, is related to the development of other autoimmune mechanisms. this can be explained by resolution of urticaria manifestations after the onset of gluten-free diet. in our case, three months after gluten-free diet, an improvement of urticaria with decreased uas-7 score of 5 was observed. conclusion: he specific case of subclinical diagnosis of celiac disease in a child with chronic resistant urticaria further reinforces the suggestion that screening for celiac disease should be included in the diagnostic approach of chronic urticaria. 1350 | allergy to gingival balm in an infant with cow's milk protein allergy we report a case of an infant with a diagnosis of cmpa with an allergic reaction to a gingival balm caused by the presence of cmp in its constitution. furthermore, it is important to reinforce that milk proteins were labeled in an unusual form which might increase the risk of misunderstanding. these findings illustrate the difficulty in implementing total avoidance of common food allergens as well as the need to improve their labeling, particularly in non-food products. bakiri ah results: after specific treatment with corticosteroids, antihistamines, emollient creams, disinfectants and antileukotriens he was feeling better, he was smiling again and wished to have the chance to play with his classmates again. conclusion: this case report shows an association between level of stress and risk for atopic dermatitis. as previously showed children with low educational level parents and boys with higher stress have increased risk of having severe atopic dermatitis as compared to "no stress" boys. so early treatment and diagnoses are key important factors improving the children`s social life. results: the data cover 69 immigrants (mean age 39.0, range 8-62) and 232 locals (mean age 40.8, range 9-80). a slight difference in male prevalence (30.4% vs 47%, p = 0.01), and pet possession (20.2% vs 41.8%, p = 0.01) were found between immigrants and locals, respectively. no differences were find in term of age and symptoms at presentation. the pattern of sensitization to the different allergens showed no statistically significant differences between migrants and controls. the rate of monosensitization resulted slightly higher in migrants (27.5%) than controls (21.1%). pollen-only sensitization was statistically higher among migrants than control (39.1% vs 22.4%, p < 0.01). monosensitization was more frequent among patients who have been living in italy for less than 4 years (52.6% vs 20%, p = 0.05). the opposite phenomena can be seen among polysensitized patients. conclusion: migrants are more frequently monosensitized than locals and tends to cluster towards either a pollen or dust mite sensitization. sensitization to house dust mite tends to appear early (< 4 years of stay). pollen or mixed sensitization is more frequent the longer the residence time. 1353 | allergenonline.org: update of comprehensive allergen and celiac protein searchable databases for risk assessment of novel food proteins goodman re 1 ; baumert jl 1 ; taylor sl 1 ; ebisawa m 2 ; ferreira f 3 ; bohle b 4 ; van ree r 5 ; kleine-tebbe j 6 ; abdelmoteleb m 1 ; koning f 7 ; amnuaycheewa p 8 conclusion: allergen and cd databases have been updated following a described review process. they can be used to identify proteins that might represent risks of food allergy or cd for affected consumers. han dh 1 ; lee jw 1 ; yim hj 1 ; ko yk 1 ; kim d 1 ; rhee c 2 1 seoul national university hospital, seoul, south korea; 2 seoul national university bundang hospital, seoul, south korea background: stress can change the immune response and aggravate various allergic diseases. we already demonstrated in previous allergic rhinitis cohort (arco) kids study that stress might be a risk factor for pediatric allergic rhinitis (ar). the aim of this arco study is to investigate relationship between stress intensity, symptoms severity and quality of life as well as allergic markers in adult ar patients. results: as stress intensity increased, the proportion of moderatesevere ar patients was significantly increased. ar patients in high stress group was likely to belong to moderate-severe group (or, 1.60; 95% ci, 1.01-2.50). global vas of ar symptom was 7.0 ± 1.9 in high stress group and 6.7 ± 2.0 in low stress group, respectively. the each 7 rqlq domain score was significantly higher in high stress group than in low stress group. total rqlq scores were 75.3 ± 35.5 in high stress group and 57.4 ± 36.0 in low stress group, respectively. however, as the level of stress increased, there were no significant changes in serum levels of allergic markers. our results suggest that stress may affect ar symptom severity and quality of life in ar patients. 1355 | skincare and synbiotics for the prevention of atopic dermatitis or food allergy in newborn infants: a 2 × 2 factorial randomized non-treatment controlled trial dissanayake e 1 ; tani y 2 ; sahara m 2 ; mitsuishi c 2 ; nagai k 3 ; sato y 4 ; suzuki y 5 ; nakano t 1 ; yamaide f 1 ; shimojo n 1 (n = 118). the skin care group was advised to apply an emollient 2-3 times/day especially on cheeks and peri-oral area. the synbiotics group consumed a mixture of fos (1 g) and bifidobacterium bifidu-mol6378 (7 × 10 9 )/day. the last group received both. emollient application was not prohibited in the no-intervention group. interventions were carried out from birth to 6 months of age. the development of ad was assessed at 1 month, 6 months and 9 months by a pediatrician and at 1 year by a questionnaire. ad was diagnosed using guidelines of the japanese society of dermatology. sensitization to food allergens was assessed by allergen-specific ige levels at 9 months of age. results: skin care and synbiotics, alone or in combination, did not prevent the development of ad at 1 year of age or the sensitization to food allergens at 9 months of age. conclusion: our data suggest that skin barrier protection using emollients may be insufficient to prevent the development of ad as other factors affecting skin barrier integrity and trans-epidermal water loss such as the method of skin washing may have an additional effect. the probiotic bacterial species used may also affect the outcome as lactobacilli have been shown to be more beneficial. more studies are required to confirm the effects of skin care and synbiotics on ad. results: in the population number of girls exceeded the one of boys (p < 0.001), especially within the age group from 4 to 15 years. questioning, for 12 months, symptoms of allergic rhinitis (rhinorrhea, sneezing, nose itch, nasal obstruction and eyes' itch) were identified in 19.5 (p < 0.05); symptoms of bronchial asthma (wheezing (14%), episodes of cough at night (8.3%), intolerance to physical load (2.5%), indoor and outdoor (13.6%), coughing and rales in response to stimulus (7.2%)) in 9.8% of the population; atopic dermatitis (dermatitis, itch, revelation in early age, involvement of large areas in early age, damage of extremities bending and stretching surfaces in adults)-5.7% (p < 0.01); food allergy-3.7% (p < 0.001) etc. at the second stage of clinical studies, on the basis of prick-testing, average ige, in our case, was 3-5 times greater than normal level. results of study of allergens showed sensibilization to domestic dust (d.f. and d.p.) (64, 43%) (p < 0.05). in 25.46% of cases there was stated sensibilization conditioned by cat and dog epidermal allergens results: among the 2624 women with available serum, 39.3% were sensitized of whom 12.5% were monosensitised, and 25.3% polysensitised (to two or more allergens). sensitisation to inhalant allergens dominated (38.9%), with grass being most common (25.5%). only 4.0% were sensitized to food allergens, most often to peanuts (2.6%), while among the 11.4% who reported ddfa, ige reactivity to foods were identified in 17.5%. compared to women with no asthma, women with dda (14.9%) were in a significantly higher background: regular exercise has been known as beneficial that it reduces the risk of chronic diseases including allergic diseases. however, little has known regarding the relationship between exercise and allergic diseases in korean adolescents. we analyzed the national data whether exercise is related to the prevalence of allergic diseases in the population of korean adolescents method: data from sixth korean national health and nutrition examination survey (2013-2014) that included 1272 adolescents from 12 to 18 years old was analyzed. we defined regular exercise according to physical activity guidelines for americans. multivariate regression analysis was performed to find whether lack of exercise could be a risk factor for allergic diseases. results: the prevalence of asthma, allergic rhinitis (ar) and atopic dermatitis (ad) were 1.3%, 9.3% and 5.2% in korean adolescents, respectively. after adjusting for factors, lack of exercise was not associated with asthma and ar, but was significantly related to ad in korean adolescents (adjusted odd ratio 3.254, 1.202-8.810 , results: it was found that over 43% of ch up to 14 y.o. having the ad within allergic disease (ads). the most significant symptom was a long-lasting itchy rash lasting for 6 month in 12.42 ± 0.56% of 1g and 9.23 ± 0.49% of 2g. the first morbidity of ad was noticed at the age of up to 2 y.o. among 56.29 ± 1.27%. at the age of ch 2-4 y.o. and older than 5 y.o. the skin ads onset was noticed for 31.12 ± 1.11% and 12.58 ± 0.57% accordingly. the ad sl was determined as follows: 52%moderate (mo), 12%severity (s), 36% were 13 ± 10 kua/l, 344 ± 132 iu/l respectively. skin prick tests were positive in 43.4% of the patients (61.7% multiple allergens). grass pollens (53%) and dermatophagoides (45.2%) were the most common allergens. average vitamin a and d levels were 469.8 ± 108 μg/l (257-832), 54.8 ± 21.3 (13-187) respectively. thirty percent of the patients vitamin d levels were mildly low, 8.6 percent was low. in control group 20% was mildly low, vitamin a levels was low in 6.7% of the patients. none of the children in control group had low vitamin a levels. we didn't find any statistical significant difference for both vitamin levels between patient and control groups. vitamin a deficiency was mostly found in asthma patients whereas vitamin d deficiency was mostly in allergic rhinitis and asthma groups. passive smoking and vitamin d deficiency was significantly related (p = 0.09). there wasn't any relation between asthma attacks and vitamin levels. conclusion: in conclusion vitamin a and d levels weren't found significantly related with allergic diseases but was found lower than control group. patients having chronic diseases are one of the population groups that are chronically exposed to drugs. this study aims at evaluate the impact of this factors in developing drug allergies in the medical staff. method: this was a cross-sectional study that included 639 nurses from the uhc "mother theresa" of tirana. they were asked to fill up a questionnaire where questions about chronic diseases and drug allergies were included. 92.18% were females and the mean age was 43.28 (+10.71) years old. relative risks with 95% ci were calculated for different groups. results: 40.69% (260) nurses reported to have at least a chronic disease. the most common non-atopic disease was hta followed by the groups of autoimmune and thyroid diseases. nurses who had one chronic disease have a rr of 1.82 (95% ci = 1.03-3.21, p < 0.05) to develop a drug disease higher than those who didn't had any chronic disease, and those who have more than one chronic disease have a rr of , p < 0.05) to develop a drug disease. the presence of chronic diseases can be a risk factor to develop a drug allergy probably through the increased risk to drug exposure. these patients may be exposed to drugs not only through therapy but also through hospitalizations and other forms of health care. lapeere h 1 ; oosterlinck p 2 ; vermeir p 3 ; vermeire i 2 ; coppens m 3 ; gevaert p 3 1 ghent university hospital/ghent university, ghent, belgium; 2 ghent university hospital, ghent, belgium; 3 ghent university hospital/ghent university, ghent, belgium background: the key to managing latex allergies in healthcare professionals and patients lies in correct recognition and appropriate action. 7.7 million people are employed in the health care sector. while there are no overall statistics on the prevalence of latex allergy in that work force, studies do indicate that 8%-12% of health care workers regularly exposed are sensitized, compared with 1%-6% of the general population. latex allergy is defined as an immune mediated reaction to latex products (e.g. balloons, contact dermatitis for gloves, condoms, surgical catheters); these encompass immediate and delayed hypersensitivity reactions. method: based on the experience of the belgian dutch pathway network, a 7-phase method to develop, implement, evaluate and continuously follow up a care pathway for latex allergy was designed and implemented. the purpose of the study was to develop and implementation of latex allergy clinical care pathways to provide all staff at ghent university hospital with appropriate knowledge and skills to identify and manage patients who have a known latex allergy or those at risk of developing latex allergy. results: care pathways, also known as clinical pathways, are used all over the world to implement and monitor patient-centered care processes in a transparent way. care pathways are defined as a complex intervention. 7 -phase method consists of: 1) screening phase; 2) project management phase; 3) diagnostic-and objectification phase; 4) development phase; 5) implementation phase; 6) evaluation phase and 7) continuous follow-up phase. this phased approach is based on the deming cycle, better known as the "plando-study-act" (pdsa)-cycle. conclusion: this method can offer support to multidisciplinary teams (re)designing and implementing safe, efficient, effective, person-centered, timely, equitable, continuous and integrated care processes. however, the method is no guarantee to success. the key to success is the collaboration and critical attitude of the entire multidisciplinary team when implementing pathways. background: cord blood ige (cb-ige) were considered to be a useful predictive tool for allergic symptoms especially in early childhood. there is only sparse knowledge about their importance for health in later life. the aim of our work was to determine the importance of cb-ige for allergic symptoms in young adults. we also studied the possible modifying factors for cb-ige concentration. results: resutls shown as daily mean, pollen grains/m³: table 1 . the daily means of pollen concentrations of cupressus arizonica, platanus acerifolia and plantago lanceolata in our area differs from other sites in madrid city. although cupressus arizonica and platanus acerifolia counting were lower, plantago lanceolata counts were higher, representing a relevant pollen in our area. the clinical relevance of these findings is under evaluation by our group. method: grass pollen counts were performed since 1979-2016 using a burkard 7 days spore trap located in our allergy center in madrid. the beginning of the algid period of pollination was considered the first of three consecutive days with more than 10 grains/m 3 and the end, the last day of three consecutive days with more than 10 grains/m 3 . madrid, barajas meteorological station data, was used. skin prick tests (pt) to grass pollen was also studied in comparison conclusion: total grass pollen concentration did not suffer any increase or decrease in its counts despite the dramatic increase of the temperature. an advance at the beginning and the end of the season was seen. these changes significantly correlate with the temperature increase during may and july. discrete decrease in the sensitization prevalence. since several years, the reference method to monitor the biological particles concentrations has been the hirst method: a volumetric pollen trap, located on the roof of building for background measurements, sucks continuously 10 l of air per minute, particles depositing by impaction on a coated tape. the tape is then analyzed by optical microscopy. the hirst method produces accurate but past data. nowadays, many researches are focused on the development on new devices to get real time information. method: rapid-e from plair sa is a device using red laser beam to determine the size and the shape of sucked particles and an ultraviolet ray to measure the fluorescence of these particles. the results: the correlation coefficients got between rapid-e and hirst trap are higher than 70% for most of calibrated pollens, this correlation reaching 86% for all pollen taxa: • plane 99% • pine 99% • birch 96% • oak 82% • plantain 75% • dactylus 73% • urticaceae 55% conclusion: new calibrations are planned for 2018 and a real time information will be set up. results: the quinquennial media concentrations since 1979-2016 were 3.866; 4.903; 6.610; 13.454; 9.501; 7.248;11.027 and 19.406 grains/m 3 . the quinquennial media temperatures were 14.2; 13.8; 14.3; 14.9; 14.2; 14.7; 14.9 and 15.8°c. increase of 1.4°c (r s = 0.9 p < 0.05). the beginning and the end of the actual season advanced 5 days respectively in regard to the period from 1979 to 1983. the annual prevalence of positive pt to platanus in 1979 was 2% an 52% in 1994. the quinquennial media from 1999 to 2016 was 50, 38, 26 and 29%. conclusion: platanus pollen counts had a dramatic increase that meaningfully correlates with the dramatic increase of the temperature. a discreet advance at the beginning and the end of the season was seen. these changes did not influence in a longer duration of the season. we observed a significant increase in platanus pollen sensitization prevalence whiting madrid pollinosis patients. results: the quinquennial media concentrations since 1979-2016 were 1963, 4813, 5455, 6948, 6985, 5976, 6727 and 9298 grains/m 3 . the quinquennial media temperatures were 14.2; 13.8; 14.3; 14.9; 14.2; 14.7; 14.9 and 15.8°c. increase of 1.4°c (r s = 0.9 p < 0.05). the actual season beginning advanced in 51 days and the end has results: over 60% of house dust samples collected between april and may from central european countries were found to contain bet v 1 allergen at levels well above the limit of detection of 0.0039 μg/g for elisa 2.0 ep kit and 0.001 μg/g on maria. samples were found to have much higher levels of bet v 1 allergen from midto-late april, particularly those that were collected in germany, belgium and hungary. samples taken from outside of the pollination season were tested and found to be negative for bet v 1. in conclusion, we found that bet v 1 allergen can be detected and quantified in house dust samples. these data suggest that household dust is a source of pollen allergen and could therefore be contributing to asthma and allergic rhinitis symptoms in individuals affected by pollen allergy. household dust may also be considered as a source of bet v 1 allergen which could contribute to allergic sensitization. 1383 | cupressaceae pollen in the atmosphere of alentejo: disruption of pollen grain during air transport spring, depending on the temperature. despite being considered moderately allergenic, it might be responsible for winter allergic outbreaks. as ornamental trees, they are found scattered throughout the territory but are more abundant in pockets of wild forest, outside alentejo. despite being more common in mountain, this pollen type is captured in considerable amounts in alentejo, portugal, where its aerobiological features and allergenic impacts are poorly characterized. the aim of this work is to characterize the aerobiology of cupressaceae pollen, to evaluate the effect the meteorological conditions and the source of this allergenic pollen type in the atmosphere of evora, alentejo. method: pollen were collected using a hirst type 7-day pollen trap and pollen was identified following standard methodology. background: allergic rhinitis caused by pollen is one of the most common allergic diseases. the presence of pollen in the air is currently centrally monitored at roof top levels, and not in the direct living environment of sensitized subjects. in the current project we aimed to develop a handheld pollen sampler, called pollensniffer, that can collect pollen in the living environment of the allergic subjects. as a first step this device was validated against the standard burkard pollen sampler and used to monitor local pollen concentrations at street level in the city of leiden. method: rooftop level pollen were monitored routinely by a hirst type pollen sampler (burkard, uk). the pollensniffer ( 1385 | does the allergy risk due to pollen exposure information is useful for the allergy sufferers? sindt c; oliver g; thibaudon m background: in france the information for the allergy sufferers is not made with pollen counts, which have not a real signification, but with the allergy risk due to pollen exposure. method: since more than 20 years, rnsa (réseau national de surveillance aérobiologique), the french aerobiology network, has measured the pollen exposure in the main cities of france, using background: the effect of environmental factors on allergic sensitizations is still unclear. rural areas vs cities have different exposure levels to pollutants and aeroallergens. these differences could give clues on the causes of higher allergic sensitization rates in children exposed to city air. method: two studies with children aged 5 years old were initialized to analyse the airborne drives of allergic sensitization: seal (günzburg, 350 children) and ae2r kids (munich, 200 children). capillary blood was collected and the parents filled in a questionary. sensitization rates were quantified using the immunocap ® isac sige array. pollen data were measured at both locations. results: in günzburg more children were sensitized to aeroallergens, however munich children showed significant higher sensitization to phl p 1 (p < 0.05), despite the lower concentration of pollen. in günzburg 66% children had no sensitization at all compared to 58% in munich. 80% of the children in munich spend at least 1 hour per day outside and 91% of the total have no animals at home. 23% felt symptoms of hay fever in the last 12 months, the majority between march and june, which correlated with the pollen flight. results: the total rate of atopy in crd patients was 36.1%, and asthma patients was the highest (45.9%). the positive rate of phadiatop in urban asthma patients (56.0%) was significantly higher than that in rural areas (25.0%, p < 0.05) and the phadiatop positive rate of office staff (62.5%) was significantly higher than that of outdoor workers (37.5%, p < 0.05). the total rate of atopy in copd patients was 32.7%, and in patients with acute exacerbation was 42.9%. beside, atopy is a risk factor for dyspnea (or = 1.22, p < 0.05), and the fvc levels in copd patients with atopy were significantly lower than those without (2.06 l vs 2.63 l, p < 0.05). optimal scaling analysis show that, there were a correlation between the tige and smoking coefficient (cronbach's alpha = 91.1%). in addition, the correlation between the level of tige and phadiatop sige was so strong in the patients with mild to moderate asthma (r s = 0.709, p < 0.001), but it was weak in severe asthma patients (r s = 0.486, p < 0.001), and up to 35.0% of the gold iii iv patients with low phadiatop level (≤10 ku/l) had a high level of tige (≥1000 ku/l) compared gold i ii (5.5%). conclusion: the rate of atopy in patients with crd is high, and atopy is an important factor affecting the process of crd. the patients with severe copd or asthma is likely to has high serum tige level but the level of common allergen sige is low, so the allergy screening strategy should be adjusted and we should pay attention to those patients, therefore, it is necessary to screen the sensitization situation of crd patients at first, and the results can guide the treatment, management and prevention of crd. background: due to a limited amount of epidemiological data [1] it has been thought that many severe allergic asthmatics in germany remain unidentified and are therefore not adequately treated. a pilot project demonstrated that more than 50% of patients, having been mean total ige (sd) was 366.4 (692.2) ku/l. 26.5% of the patients had no sensitization towards any of the specific iges tested, whereas 24% were positively tested on 5-10 allergens and further 23.5% showed sensitizations towards >10 allergens. conclusion: approximately 75% of 392 online recruited (severe) asthmatics had a total ige level of >30 ku/l and ≥1 sensitization (allergen-specific ige) towards atopic allergens. this further supports the high prevalence of atopy in asthma. results: 875 patients (mean age: 26 ± 11.9 years, range 2-64 years, m/f ratio: 0.89) who suffered from allergic rhinitis or allergic rhinoconjunctivitis enrolled in this study. highest rate of skin sensitivity was for weeds/grasses pollen including salsola kali, amaranthus retroflexus, chenopodium album and compositae family (74.3%, 62.5%, 50.9% and 39.3% respectively). among tree's pollen; ash (49%), walnut (46.9%) and mesquite (29.3%) were the most common. less than 20% of patients showed skin reactivity to indoor allergens and storage mites, mix of cockroaches and house dust were the most common (17.6%, 17.3% and 9.8% respectively). the results of current study confirmed the importance of weed/grass and trees pollen as the major source of allergic sensitization in our area. interestingly the rate of sensitization to indoor allergens was low which can be explained by geo-climatic situation. background: there are few studies of cutaneous sensitivity to gramineae in our region. mostly of them use allergens of foreign species. the study aims to estimate the prevalence of skin sensitivity to widespread grasses in our region. method: this is a retrospective observational study of 894 patients with seasonal allergic rhinitis. patients were studied using skin tests with pollens extracts from pooideae, chloridoideae and panicoideae grass species. results: the prevalence of positive reaction to pollen from pooideae subfamily was 86.8% (ic: 84.4%-88.9%). in turn, prevalence of allergy to panicoideae subfamily pollens was 69.6% (ic: 66.5%-72.5%) and positive reaction to chloridoideae subfamily reach 48.1% (ic: 44.8%-54.1%). cochran test suggests that prevalence in those three groups is different (χ2 = 319.11, p < 0.01). when comparing just the groups of allergens from pooideae and panicoideae differences are also significant (χ2 = 71.43, p < 0.01). in particular, 52.5% (ic: 49.2%-55.7%) of patients were allergic to paspalum notatum. regarding cross-reactivity between subfamilies, we find a no crosscorrelation between pooideae and panicoideae (χ2 = 2.197, p = 0.138). conclusion: in bahia blanca, patients with seasonal rhinitis are sensitive to pooideae, chloridoideae and panicoideae. paspalum notatum, belonging to panicoideae, has a significant prevalence, high reactivity and low cross-reactivity within the group of species studied. this last species is relevant because it is a native grass from the northwest region of our country, paraguay and the south of brazil. prevalence of grass positive skin tests in patients with seasonal rhinitis by species. allergen frequency percentage 95% ci results: correlation analyzes were performed between sige and spt and area results. the concentration with the highest correlation by diameter and area for blo t was 30 μg/ml and for der f of 400 μg/ml. in the case of der p the concentration with the highest correlation for the diameter was 30 μg/ml and for the area of 3 μg/ml. when evaluating the reproducibility of the results according to the area and the greater diameter of the spt, a strong agreement was observed for blo t in the concentrations of 3 μg/ml and 0.3 μg/ml. results: among the 94 patients, the majority of allergens-positive was t20, accounting for 60.6%, followed by f14 (59.6%), f13 (58.5%), ds1(57.4%) and ccd (56.4%). the prevalence of plant-related allergens (t20, w1, f14, f13, w6 and u80) in ccd-positive patients were significantly higher than those in ccd-negative patients (all results: with our new point-of-care methods using a selected recombinant protein e other markers, we were able to detect the disease early as 10 days post-infection and more than 95% of positive cases from chronic and low endemicity areas (which are characterized by hard to detect patients with extremely low parasite load, <10 eggs per gram of feces) were obtained. plus, chromatography poc-cca ® test was improved by our group with a urine concentration step that turned its sensibility from 6% to 56%. conclusion: monoclonal antibody and recombinant protein technologies allowed superior detection methods when comparing it to the conventional ones. in conclusion, data showed 100% of sensitivity of chronic patients and 98% of acute patients. marton c county hospital, oradea, romania background: allergic rhinitis is a disease that affects about a quarter of the population, a disease with an important negative impact on daily activities, both on learning and working ability, as well as spending leisure time or sleeping. in the western part of romania, the most popular and blamed allergen is ambrosia, in the late summer months. it is a plant of the compositae/asteraceae family, along with goldenrod, sunflower, dandelion, cocklebur, chamomile, wormwood, daisy, etc. allergen identification is important for applying prophylactic measures, but especially for determining the allergen to be desensitized. considering the possible cross-reactivity within the compositae plant family, as well as the possibility of co-sensitization, as well as the number of patients sensitized to these pollens, which is steadily increasing, i considered is necessary a broad screening for a more precise identification of the allergen and increase chances for a successful desensitization. method: the observational study includes 37 patients who presented on october for testing with standardized allergen extracts, as recommended. criteria for inclusion: patients with specific symptoms of rhinoconjunctivitis in august and september, with or without asthma symptoms, who returned for allergic prick test after the end of treatment. criteria for exclusion: patients who disagreed with cutaneous testing, who did not discontinue antihistamine treatment or who had been treated for other diseases with drugs that influence skin testing. background: in recent years, cationic liposomes are thought to be the most effective and non-toxical nucleic acids`transport system, so most of gene therapy drugs are developed on their base. however, lipoplexes are quickly captured by reticuloendothelial system cells after the injection and taken out of a blood stream. there are many modification methods of liposomal surface for liposomes with prolonged pharmacokinetic properties production. addition of hydrophilic polymers (peg) is seemed to be the most promising approach, that is able not only to create steric barrier on the particle`s surface and prevent the interaction with blood plasma lipoproteins, but also inhibits the protein adsorption, opsonisation and subsequent degradation in human body. the aim of this study is the evaluation of liposomal surface modification by hydrophilic polymers influence on nucleic acids`lipoplexes conjugation and on their physico-chemical and biological properties. method: liposomes preparation (including peg-modified liposomes), size determination by photon-correlation spectroscopy, examination of transfection efficacy by luciferase assay. (c16h33)2) were obtained. also the modified liposomes were produced by addition of 5% of peg (by mass) during thin lipid layer preparation step. the size distribution was analysed by photon-correlation spectroscopy. it was shown that peg addition does not increase the parconclusion: it can be noted that addition of peg can change the lipoplex formation but the cationic liposomes still remain an effective rna delivery system. and peg modification will be able to impart prolonged properties for the vehicle in bloodstream. foundation (grant № 17-74-10111). ory c4 may cause a cross reaction with fel d4 (cat), can f6 (dog), equ c1 (horse), mus m1 (mouse) and rat n1 (rat). february 2017 eight patients that were treated at our institution were diagnosed with rabbit allergy. results: all eight patients with the diagnosis of rabbit allergy presented with signs of upper respiratory involvement. two patients had itching teary eyes, watery nasal discharge and sneezing while feeding farm rabbits. one of those also presented with dyspnea. four patients developed problems whenever in contact with domestic rabbits. one patient developed allergic rhinoconjunctivitis whenever she was home-her parents own a rabbit, but while away in her college room she had no problems. another patient had dyspnea whenever visiting his girlfriend's house. she owned a rabbit. two patients developed asthma-like symptoms, one also presented with angioedema. the other two had developed allergic rhinoconjunctivitis. two patients have problems in contact with cats, one of them also with cows, however skin prick tests were also positive to rabbit. three out of eight patients developed allergic asthma with a positive methacholine test. six patients had a positive house dust mite prick test. all patients were diagnosed with a positive prick tests to rabbit allergens. all were treated with a nasal steroid and antihistaminesic. they were also advised to avoid contact with the animal. conclusion: domestic rabbit-induced asthma and/or allergic rhinoconjunctivitis is possible, however it is still rare in our environment. it is very important to always ask the patient about their pets in general, not just focusing on cats or dogs. only with a thorough examination and history we can find the true cause of the patient's allergy where pets play an important role. korea. changes of protein and major allergen concentration were measured over one year by bradford assay, two-site elsia, and sds-page after reconstitution of the lyophilized allergen extracts in various buffer (normal saline, 0.3% phenol saline, and 10 or 50% glycerol with saline) and stored at room temperature (rt, (18) (19) (20) (21) (22) (23) (24) (25) (26) or refrigerated (4°c). results: more than 90% of the initial protein concentration in all four extracts examined was detected over one year when 50% glycerol was added and refrigerated, whereas 57.9%-94.5% remained in the extracts at rt. the addition of 50% glycerol to the storage buffer was found to prevent protein degradation at rt. all four extracts were found to be stable when reconstituted in 50% glycerol. amb a 1, a major allergen of ragweed, was almost completely degraded in 9 weeks at rt when reconstituted in a buffer without 50% glycerol. however, 55.6%-92.8% of amb a 1 content was detected after one year of incubation at 4°c in all buffer conditions except 0.3% phenol. conclusion: addition of 50% glycerol as well as refrigeration was found to be the important to increase the shelf-life of allergen extracts from pollens of allergenic importance. results: this is the first genetic study of the bulgarian hae patients. genetic defects were identified in 10 hae families are: 3 nonsense, 2 splice-site defects, 2 frameshift mutations, 1 indel non frameshift, 1 missense, and 1 large deletion of exon 4. novel mutations, not previously reported in human gene mutation databases were discovered, and were predicted to be deleterious due to the expected effect on dna transcript and protein. descriptive statistics were used to summarize the eq-5d-y descriptive system responses and vas scores by treatment and visit. results: twelve patients with hae type i and a median (range) age of 10.0 (7) (8) (9) (10) (11) years were enrolled, 7 (58.3%) of whom were female. during bop, treatment with 500u c1 inh, and 1000u c1-inh, ≤33.3%, ≤22.2%, and none of the patients, respectively, reported having problems with mobility, self-care, doing usual activities, pain or discomfort, and feeling worried, sad or unhappy. the mean [sd] eq-5d vas scores increased from 78.3 (13. results: overall, the model-derived median exposure and peak concentration across all weight ranges in paediatric patients is predicted to be higher with 5 wb vs weight-based dosing (table) . the effect was most pronounced in patients aged 2-5 years, where the 5wb dosing achieved approximately 30% higher values than weight-based dosing for median auc 0-6 (1251 ng hour/ml vs 853 ng hour/ml, respectively) and c max values (777 ng/ml vs 529 ng/ml, respectively). the 5wb levels are closer to those in adults receiving 30 mg icatibant (median auc 0-6 2975 ng hour/ml; median c max 1254 ng/ ml) but never exceed them. results: samples from 124 patients were analysed. lanadelumab concentrations in plasma increased with higher doses and dosing frequencies. steady state was reached around week 10 (range week 8 to week 14 as evaluated by predose concentrations). at baseline, mean (sd) chmwk levels were 49. 9% (28.8), 47.7% (27.1), 53.7% (24.5) and 48.4% (30.0) for patients in the placebo and lanadelumab 150 mg q4 wks, 300 mg q4 wks and 300 mg q2 wks treatment arms, respectively. by week 14, mean (sd) chmwk levels decreased to 24.0% (13. 3), 28.7% (17.2), and 22.4% (11.5) following treatment with lanadelumab 150 mg q4 wks, 300 mg q4 wks, and 300 mg q2 wks, respectively, and remained reduced throughout the treatment period. conversely, chmwk levels remained elevated at 52.3% (28.1) at week 14 in patients who received placebo. patients in the placebo group had the highest attack rates over the 26-week treatment period (mean 2.46 attacks/month), whereas the rates were markedly lower in patients treated with lanadelumab 150 mg q4 wks (0.48 attacks/month), 300 mg q4 wks (0.60 attacks/month) and 300 mg q2 wks (0.31 attacks/month). dose-and frequency-dependent manner. exposure to lanadelumab was associated with decreased chmwk levels (indicating inhibition of plasma kallikrein activity) and lower hae attack rates, corroborating the efficacy findings and utility of chmwk as a bioactivity marker in the help study. with cyklokapron (tranexamic acid) since she was 9 years old and took the medication very irregularly due to lack of efficacy. one year before presentation at our clinic, she married and moved to vienna. we started a treatment with the bradykinin-2 receptorantagonist icatibant sc at the beginning of the menses and if needed a second time at the time of ovulation. she responded well until she got pregnant. during pregnancy, she developed weekly attacks with increasing severity. therefore, a weekly treatment with humanplasma-derived, pasteurized, nanofiltered c1-inhibitor (inh)-concentrate, 1000 units iv once a week was started and had to be increased to twice per week after one month of therapy due to increasing number and severity of attacks. results: with this treatment attack frequency and severity attenuated. in january 2016 she had a normal delivery at term and gave birth to an otherwise healthy son. treatment had to be continued during 1 year of lactation period and also thereafter due to persistent attack severity. conclusion: there are only limited data for the use of humanplasma-derived, pasteurized, nanofiltered c1-inh concentrate during pregnancy and lactation period. this case confirms the safety and efficacy of the named drug during these periods. wheals are yet classified. the best characterized stem from hereditary or acquired c1 inhibitor deficiency (c1-inh-hae and c1-inh-aae) . last year, the french angioedema network (creak) joined the registry of angioedema without wheals (cloud-r hae). here we present the contribution of the grenoble alpes university hospital (chuga) to this disease registry. the study population is composed of patients with a proved diagnosis of c1-inh-hae/aae. the following items are collected: patients' personal-demographic data, clinical/laboratory/genetic characteristics, major comorbidities, treatments (prophylaxis/acute attacks). data from existing registries at chuga are merged into cloud-r hae and missing data obtained at follow-up visits. as from cloud-r hae structure, patients can directly provide information on angioedema attacks and their treatment through a dedicated electronic app, web connection or paper support, which is then transferred into the registry at chuga. method: in a retrospective study, we included a total number of 48 patients, suffering from a chronic skin disease, whose lesions did not improve or even worsened under immunosuppressive treatment (18 chronic ulcers/pyoderma gangrenosum, 21 bullous autoimmune diseases, 9 skin lymphomas). ffpe tissue was examined for the presence of cmv dna by pcr. next, within the framework of a small prospective study (n = 29) we analyzed the seroprevalence of cmv as well as the presence of cmv dna in lesional skin in patients that had been diagnosed with a chronic skin disease and in whom longterm immunosuppressive therapy had been initiated. results: in the retrospective study cmv dna could only be detected in 1/18 chronic ulcers/pyoderma gangrenosum (5.6%), but not in bullous autoimmune diseases and skin lymphomas. 21/29 patients (72.4%) of the prospective study group were seropositive for anti-cmv-igg, as compared to 55/87 patients (63.2%) in an ageand sex-matched control group. anti-cmv-igm could be detected in method: in this study, we aimed at testing the diagnostic potential of skin function measurements in ss. sixteen patients with conformed diagnosis ss were enrolled in the study. skin fibrosis was assessed by conventional rss and involvement of inner organs and serum inflammation parameters were determined. four objective criteria, namely transepidermal water loss (tewl), corneometry, ph and elasticity, were assessed at nine predefined sites of the body. results were compared to patients with atopic dermatitis (n = 19) and acne vulgaris (n = 22). method: a multicenter prospective observational study was conducted to investigate the clinical significance of serum scca2 in children as a biomarker for ad. patients with ad younger than 16 years old and age-matched healthy children without any allergic disease were enrolled in this study. the severity of ad was evaluated using the objective scorad (o-scorad). the serum levels of scca2, tarc and total ige were also measured. results: in total, 176 patients with ad and 159 non-allergic healthy children were recruited. the serum levels of scca2 had the strongest significant correlation with o-scorad, compared with tarc and ige (r = 0.622, 0491 and 0.407, respectively). after standard treatment with topical steroids and emollients resulting in an improvement of symptoms, the serum levels of scca2 and tarc decreased significantly. the area under the curve (auc) for the roc curve was higher for scca2 (0.929) than for tarc (0.871) or ige (0.820). the difference in aucs between a single cut-off value and age-dependent cut-off values was not significant for scca2, compared with that for tarc (0.042 and 0.064, respectively). conclusion: scca2 is a more reliable biomarker than tarc for the diagnosis of ad and for determining the clinical severity of ad in children. challenges in predicting severity of atopic eczema patients results: before modeling, we checked for significant differences between patients and controls. these were detected for the levels of ccl17, ccl22, cxcl10, ige and ldh. next, we assessed whether single serum proteins already explain disease severity by calculating correlations. twelve of the proteins, namely gcsf, il-5, il-13, il-22, ccl22, il-1ra, cxcl8, ifng, ccl3, il-1ß, ccl17, and il-6, significantly correlate with severity (r 2 range: 0.3-0.45). finally, we built a model for the severity of ae based on all measured serum proteins. ten of the proteins are included in the best-fit model (adjusted r 2 =0.47). the overall correlation between original and predicted severity scores is high (r 2 =0.759) nevertheless the cross validation prediction error is substantial with 19%. conclusion: applied in daily practice, a prediction error of 19% translates to a possible miscalculation of 19 scorad points in both directions and therefore the model is of no practical use. aside from using model-based quality measures like cross validation prediction errors to infer the usefulness of predictive models, testing them in independent cohorts could validate these models. collaborations among scientists working on similar approaches would lead to an increase in statistical power and ideally to more robust models. only robust and validated models are going to have the chance to take the step forward from being a result of computational modeling to being applied in the clinical practice of assessing disease severity in patients. jargosch m 1 ; lauffer f 1 ; pätzold k 1 ; krause l 2 ; garzorz-stark n 1 ; schmidt-weber c 3 ; eyerich s 3 ; eyerich k 1 preclinical studies in cell cultures, mice, guinea pigs and rabbits, comprising sterility, cytotoxicity, systemic toxicity, skin irritation, delay contact sensitization and phototoxicity tests, demonstrated safety of this therapeutic agent. we next conducted a single-blinded, intra-individually controlled, 2phased clinical trial on patients with keloids. the aim was to determine the effects of 1-month therapy on keloid volume and symptoms of pain and itch. two similar keloids on each subject were selectedone was treated with once-daily, self-administered application of triamcinolone-loaded (0.015 mg/patch) microneedles for 4 weeks, while the other served as control with no intervention. outcome measures were (a) keloid volume using a 3-dimensional high-resolution (0.1 mm) scanner and (b) pain and itch scores on 0-10 numerical rating scales. evaluations were performed at baseline, 4 and 8 weeks. in phase 2 of the trial, the whole process was repeated using microneedles loaded with a higher dose of triamcinolone (0.1 mg/patch). case report: a 15-year-old girl was admitted to our department with a single round-shaped lesion in the popliteal fossa which spread to extremities, trunk and face and persisted for several weeks and then faded slowly to residual hyperpigmented patches. courses of antihistamines, antibiotics, cyclosporine a, fluconazole, hydroxychloroquine, prednisolone and topical steroids were ineffective. also patient has had history of itchy urticarial rash and angioedema since 5-year-old, suffered from the flares triggered by physical exertion, stress, cold air and water, spicy food, which resolved within 3-4 hours. the patient's father and 7-year-old brother also had chronic urticaria induced by the same stimuli. the physical examination revealed multiple pink-to-red non-scaly, non-pruritic papules coalescing into annular, arcuate, polycyclic plaques (4-5 cm) with central clearing, centrifugal spread, indu1448 | the role of humoral immunity in the pathogenesis of psoriasis results: we found significantly increased levels of iga in the serum of treatment-naïve psoriasis patients correlating with disease score. however, iga was only observed in dermal vessels of skin sections. we next performed in-depth analysis of peripheral b cell subsets using flow cytometry. among all investigated subsets, we only found a moderate positive correlation of cd138 + plasma cells with iga levels and disease score in untreated psoriasis patients. however, in the group of treated psoriasis patients, neither did iga levels drop nor did plasma cells correlate with iga levels and disease score, rather hinting at an epiphenomenal finding. confirming our hypothesis that psoriasis can develop in the absence of proper humoral immunity, we present a patient who suffered concomitantly from both psoriasis and a hereditary common variable immune defect (cvid). conclusion: here, we provide new insights in the immunology of psoriasis, demonstrating the clear dominance of t cells over shifts in b cell subsets. conclusion: allergic diseases show an increasing incidence in geriatric age. this is partly due to the growing emphasis on a more accurate and careful diagnosis of the aging population. we must also take into consideration the influence of other factors, besides comorbidities and therapeutic regimens in elderly that might affects the immune response, such as environmental pollution as well as food contamination and changing dietary habits of elderly such as easy access to exotic food. one of the challenges in the decades to come is recognizing and fulfilling the need for accurate and timely diagnostics of allergic manifestations in elderly patients, as important part of achieving the best possible quality of life for this growing age group. method: sixty-eight patients with various forms of psoriasis and 30 healthy subjects (healthy control group) were assessed after informed consent was obtained. all subjects were asked to complete a questionnaire including age, gender, duration of psoriasis, concomitant diseases and medications. in the group of patients psoriasis was with only skin involvement with skin plus joints involvement ranging from moderate to severe. psoriaticplaques were evaluated by a specialized medical team using the psoriasis area and severity index (pasi). all patients were seen by a dermatologist and clinical immunologist, who collected data considering the demographic, health status and any other relevant details. blood samples included serum levels of 25-hydroxycholecalciferol and tnf-α using an elisa kit (germany). method: 30% ethanolic extract of sp (sp etoh ) and its five major chemical constituents are prepared. to elucidate whether human orai1 modulated by sp etoh and its chemical constituents, conventional whole-cell patch clamp performed in horai1-overexpressing hek293t cell. we also assessed whether sp etoh and its constituents could inhibit mast cell degranulation and t cell activation. results: in jurkat t lymphocytes, we found that 3 mg/ml sp etoh inhibited orai1 current (i orai1 ) by 81.0 ± 11.1%, while one of its constituents (compound v (com v ); 100 μm) inhibited i oria1 by 48.9 ± 8.71%. investigation of human primary t cell proliferation induced by co-stimulation with antibodies to cluster of differentiation 3 and 28, and of rbl-2h3 mast cell degranulation following ige-antigen complex stimulation, revealed that 100 μm com v inhibited both t cell proliferation (by 34.8 ± 6.08%) and mast cell degranulation (by 36.7 ± 0.07%); these effects were concentrationdependent, and no cytotoxicity was observed. conclusion: considering that most regional plants have not been investigated chemically or pharmaceutically, they remain as untapped potential sources of topical agents for drugs and other application. our findings suggest that com v , which derived from sp etoh , represents a promising candidate compound for the development of therapeutic agents for the prevention and treatment of allergic diseases. results: the cohort consists of 14 caucasian patients. eight of them (57%) are women. the mean age (and range) at the clinical presentation of disease was 33 years (4-51 years). the mean age at diagnosis for men was 23 years and 48 years for women. all patients have a positive history of recurrent and/or persistent lip swelling, 3 of them (21%) report oral ulceration, 5 cases (36%) have history of previous or current facial palsy and 4 patients (28%) present tongue fissuring. concurrent cd has been diagnosed in one patient. biopsy reports were available for 10 patients (71%); in 6 cases (60%) non-caseating granulomas were seen. various therapeutic approaches have been described: intralesional corticosteroids had a good response in 6 patients, infliximab was partially effective in 3 cases; oral corticosteroids and/or methotrexate seem to cause a partial symptoms improvement. conclusion: this is the first attempt, in our knowledge, to (a) centralize all data of patients with ofg in a national registry with the aim of carrying out epidemiological data and (b) develop italian guidelines including a diagnostic-therapeutic flow chart, shared by the participating centers. the registry will guide the clinicians in the identification and management of the ofg patients, reducing the diagnostic delay and hopefully improving quality of life. case report: a 10-year-old girl without personal history of atopy, got a temporary tattoo with henna. after three days, she developed a local exudative, erythematous eruption with painful blisters lesions that followed the contours of the tattoo. she had neither fever nor other lesions. she was treated with topic methylprednisolone-gentamicin showing an important improvement 10 days after. as a liquenoid scar remained in tattoo area, trofolastin ® (centella asiatica, αtocopherol, hydrolysed collagen, elastin) patch was prescribed to be placed on the scar. forty-eight hours later, the patch was removed and was newly observed an exudative, erythematous and painful wound that required oral treatment with amoxicillin-clavulanic. after three days, the girl developed on a maculopapular, generalized and itching rash. she was treated with dexchlorpheniramine and methylprednisolone with a complete resolution in 4 days and she was referred to our allergy unit to be studied because of a suspicion of drug allergy to amoxicillin-clavulanic acid. an allergy workup was performed after obtaining an informed consent. case report: it may be sometimes difficult to find the causing allergen in allergic contact dermatitis. face is a region on which various materials contact. in this manuscript a woman case is presented who shows patch test positivity to her husband's shaving product. a 35 years old woman applied because of allergic contact dermatitis on her face. it is learnt that lesions have been continuing for a long time, occasionally getting well with corticosteroid creams; but continuing again. patch test was performed with european standard series and cosmetic products she was using. negative result was observed. following, patch test was performed for the products her husband was using. 2 positive results were obtained for the shaving cream of her husband was using. in detailed anamnesis, it is learnt that the lesions developed approximately 1 month after her husband started to use this cream. it is advised not to use this product to her husband. the disease did not repeat again. it should not be forgotten that cases with allergic contact dermatitis could get in touch with allergenic materials via individuals in close contact. gül ü akdeniz university faculty of medicine, department of dermatology, antalya, turkey case report: tnf-alpha plays role in etiopathogenesis of allergic contact dermatitis (acd). in mice which lack tnf-alpha, the response of late type hypersensitivity is spoiled. in addition, tnfalpha blocker are also used in some cases with acd. in this poster the results of european standard patch test is given in which acd is observed and tnf-alpha blocker are used without dermatological indication. 3 cases who use tnf-alpha blocker applied because of acd: there was lesion in one case on face, in other case on face and hand, and in the last case only on hand. european standard patch test was performed to patients who were continuing to use tnf-alpha blocker. in one case no positive response was observed, while in two cases positive response to more than one allergen were obtained. in conclusion, tnf-alpha blockages cannot suppress the response of delayed type hypersensitivity. 1459 | case of allergy to nickel on the background of its intake in food peredelskaya m case report: nickel is one of the most commonly used metals; it is used for the manufacture of jewelry, plates and dishes, and medical products. a patient n, 32 years old, female, complains of pruritic rash on the body skin with the itch intensity up to 8-9 points and the number of lesions more than 50. allergic background: for quite some time now the patient noted occasional eruptions on her skin after a contact with jewelry made of non-precious metals. previously patch skin tests with nickel showed a positive reaction. the patient sought emergency medical care with complaints of a number of itchy lesions erupted on her whole body during the last 24 hours. on admittance: state of moderate severity, the patient was emotionally labile, focused on her body sensations, tearful. on the skin of face, upper and lower extremities and torso a punctuate purpura with lesions up to 0.5 cm diameter, prone to confluent. a physical status was within normal limits. in order to control the itching, as well as to sedate the patient, antihistamines of the first generation were administrated parenterally; but the eruptions kept to progress and to intensify; lesions were spread throughout the whole body, merged in gigantic areas. system glucocorticosteroids therapy was administrated, with 120 mg of prednisolone, but then new lesions kept appearing in a large number, including after-meal rash. water, tea, bakery products, thin yoghurts did not impact the skin condition, whereas the intake of pasta, cereals, and similar products provoked intensifying of eruptions. the patient observation revealed a sharp increase in the rash after such manipulations as intravenous injections or blood sampling from the vein, the process spreading from the injection site to the entire arm. a detailed anamnesis of the disease: on the eve of the start of hives, the patient purchased a coffee machine (with metal nickel-plated parts) and started to use it. diagnosis: a systemic contact dermatitis. an allergy to nickel. the injection treatment was discontinued and a therapy with per oral gcs and antihistamines of the second generation was administrated. a recommendation was given to cook and to eat food using ceramic or wooden utensils. three days later marked positive dynamics of the skin process has been noted. the episode of systemic contact dermatitis has developed due to exposure to nickel from ingestion in food, as well as during the parenteral treatment. background: anaphylaxis reactions during anesthesia can have a mortality of 3%-6%. 2/3 of the anaphylaxis in the operating room are due to the use of neuromuscular blockers. rocuronium is frequently involved because is oftenly used. we present a case of a 41 years old man with an anaphylaxis shock due to the administration of rocuronium. method: 41 years old man with no personal history of interest that is going to undergo vertebral surgery. minutes after anesthetic induction with fentanyl, propofol and rocuronium he started with lowering of oxygen saturation. orotracheal intubation is performed and, with the suspicion of anaphylaxis shock, adrenaline, antihistamines and corticoids were administered. after 20 minutes without improvement, 500 mg of sugammadex was administered, given the possibility that the condition was secondary to the use of rocuronium. tryptasa level was 63. results: skin test to fentanyl, propofol, látex and rocuronium were done 6 weeks after and only rocuronium test was positive. conclusion: in summary, the occurrence of anaphylactic shock after neuromuscular blockers is widely described in medical literature. there are conflicting data about the use of sugammadex as coadjutant treatment in case of anaphylaxis due to the use of rocuronium. we believe is a good option when conventional treatment is not useful. case report: a 31-year-old woman with past history of allergic rhinitis and hypertension was admitted to the obstetrics service in labor of first child in april 2016. epidural anesthesia with ropivacain and sufentanil was administered. as there was no labor progression, eighteen hours later she was admitted to undergo cesarean section and epidural anesthesia was re-administered. metoclopramide, ampicilin and ranitidine were given intravenously. during ranitidine perfusion, the patient presented general cutaneous erythema and pruritus, tongue, lips and eyelids angioedema and dyspnea. perfusion was suspended and hydrocortisone and supplementary oxygen administered. she denied any type of previous adverse reaction to drugs and any symptoms with use of latex-containing material. allergic evaluation revealed negative latex skin prick test (spt) and negative penicillin, amoxicillin and ampicillin specific ige assay. skin prick and intradermal tests with sufentanil, ppl, mdm, amoxicillin and ampicilin were negative. oral amoxicilin and metoclopramide provocation challenge were negative. spt and subcutaneous provocation challenge with ropivacain were negative. spt with ranitidine was negative but skin intradermal test proved to be positive. the patient was taught to avoid histamine h2 receptor antagonists and use as a safe alternative proton pump inhibitors. conclusion: anaphylaxis during anesthesia is an unpredictable, severe, and rare reaction. the identification of responsible drugs is a complex task. we report a case in which a commonly used and generally safe drug caused a severe reaction, which demonstrated that even the least obvious culprit should not be disregarded. epidemiologic data suggest that the number of cases of chx allergy appears to be increasing. background: chlorhexidine is a synthetic chemical with excellent antiseptic and disinfectant quality frequently used in everyday products and medical devices. the prevalence of allergic reactions towards chlorhexidine is rare, though there is increasing evidence for its allergenic potential. in this case we report about a patient with serious perioperative anaphylaxis. next to multiple potential allergens that he was exposed to, a chlorhexidine containing lubrication gel has been used for urinary catheterisation. within minutes post-exposure, the patient developed generalized urticaria, bronchospasm, tachycardia and hypotension. material and methods: we performed skin prick tests and intradermal tests with all substances documented in the anaesthesia chart, further we analysed specific immunoglobulin e (sige) antibodies and performed oral provocation challenges for exclusion. results: in the skin tests all substances except for chlorhexidine (spt: 7 mm wheal diameter/31 mm erythema) were negative. a sensitization for chlorhexidine was further corroborated by chlorhexidine-specific ige antibody (4.08 ku/l) in the patient's serum. in addition, the challenges for the drugs without sensitization (cefuroxime, lidocaine) were tolerated. considering all potentially relevant allergens that the patient was exposed to and the proof of specific sensitization, we diagnosed an immediate-type allergy towards chlorhexidine. conclusion: with the ubiquitous use of chlorhexidine an increase in hypersensitivity reactions including immediate-type allergic reactions is observed. anaphylactic reactions are rare, but potentially life-threatening, the diagnosis is crucial. as a warning declaration in medical devices is missing, the diagnosis of chlorhexidine allergy might be easily under-recognized or misdiagnosed. unfortunately, until now validated provocation tests are not existent, but the evaluation of combined skin tests and sige is sensitive and specific. 1464 | an approach to incidence of death due to anaphylaxis in spain (1998 spain ( -2011 background: reports about death due to anaphylaxis are still scarce because of its rarity and limited information to few countries. also, data source analysis is usually not included. we report incidence of death due to anaphylaxis in spain using two databases. method: we used a hospital series of anaphylaxis deaths from the spanish hospital system and a series from the national institute of toxicology and forensic sciences (intcf) predominantly formed by extra-hospital deaths. deaths from the spanish hospital system were extracted using codes from icd-9-cm, related to anaphylaxis among all deaths occured in the 1998-2011 period. for extracting deaths due to anaphylaxis at the intcf in the same period, two allergist researchers identified these deaths among cases with suspicion of anaphylaxis cause. a regression logistic was run to discriminate the probability of anaphylaxis death belonging to each database. incidence rates were calculated for the different groups (age, sex) using the spanish population as the denominator. temporal trends were calculated from the hospital database using poisson regression models with the number of cases of anaphylaxis detected each year as the dependent variable, and age and sex as covariates. results: there were four positive predictors of fatal anaphylaxis after the logistic model (usual allergen, positive specific ige, suggestive symptoms and previous reaction to the same allergen case report: we were informed that a girl was admitted to the pediatric endocrinology department due to early breast development. she had been diagnosed as central precocious puberty (pp). later, triptorelin acetate (ta) therapy had been started monthly. within 20 minutes after first sc injection of ta at home, she had developed shortness of breath, decreased air entry, and coughing for ten minutes and lastly she had developed vomiting for 15 minutes. her symptoms were accompanied by a pruritic blanchable maculopapular rash on her ears, cheeks, lips, and eyelids approximately for two hours. although they had applied emergency department of the local hospital. based on the diagnosis of anaphylaxis she was immediately treated with adrenalin. she was subsequently hospitalized for possible recurrence and discharged next day without any further events. treatment with another preparation, leuprolide aseptate(la-lucrin), as an alternative treatment was started with premedication against anaphylaxis risk only at first time and the patient did not develop any reactions. the patient is still on this treatment with no complications. anaphylaxis is diagnosed in the presence of a detectable allergen accompanied by symptoms of two systems. our patient had symptoms of the three systems as described above, that is, dyspnea with coughing, hives, nausea, and vomiting. main treatment of anaphylaxis is the epinephrine use. early usage maximizes the likelihood of survival. diagnostic tests with culprit drug were not performed in our hospital if the patient had the anaphylactic drug reaction and grouped as "physician diagnosed anaphylaxis". there has been only one report regarding anaphylaxis to ta treatment in cpp in turkey. in the literature, anaphylactic reactions against ta have been reported only in few pediatric cases. gnrh analogues are important to ensure the physiological growth in precocious puberty. because anaphylaxis can be lethal, and gnrh analogues are similar structure; the present case suggests that one should bear in mind the possibility of anaphylaxis in all patients who receive gonadotropin-releasing hormone and anologs and monitor such patients carefully as needed. furthermore, we must provide sufficient information of adverse reactions, including anaphylaxis, to patients. hence, managements against anaphylactic shocks should be recognized and treatment should be given immediately. 1467 | an anaphylactic shock induced by the rocuronium anesthesia: a case report cabrera v; barrios j; callero a; gonzález ce; pérez e; martínez ja hospital universitario nuestra sra de la candelaria, santa cruz, spain background: the anesthetic act is a unique pharmacological situation, where the patient is exposed to a multitude of substances.among them, neuromuscular blocking agents are the leading cause of preanesthetic anaphylaxis, with a frequency of between 50%-70%.followed by latex in second place and antibiotics in third place. among the neuromuscular relaxants, most reactions are due to suxamethonium or succinyl-choline in 60.6%, followed by atracurium, rocuronium and verocuronium.the one that produces the least reactions is cisatracurium. method: a 61-year-old woman presented a type iii anaphylaxis of the brown classification during the anesthetic induction in a surgery scheduled for laparoscopic cholecystectomy. for which adrenaline, dexchlorpheniramine, hydrocortisone, ranitidine and sugammadex was administered and was transferred with orotracheal intubation to the anesthetic resuscitation room. due to good evolution of the patient, she was extubated within three hours. the drugs involved in the reaction were: rocuronium, amoxicillin-clavulanic, fentanyl, propofol, midazolam, lidocaine and atropine. there was a high suspicion by the anesthesiology and resuscitation service that the abstracts reaction could have been due to the neuromuscular relaxant used, in this case rocuronium, since the reaction was reversed with sugammadex. the patient had undergone surgeries under general anesthesia previously without incidents. a specific allergy study was performed with laboratory tests with tryptase, skin tests with drugs and basophil activation test for rocuronium, sugammadex-rocuronium mixture and cisatracurium. • serial measurement of serum tryptase: 12.9 u/l, 10.9 u/l y 3.42 u/l there is no activation of basophils for sugammadex-rocuronium mixture and cisatracurium. the patient is diagnosed with rocuronium allergy. sugammadex not only acts as an antidote to reverse the neuromuscular block against rocuronium, but also has antiallergic properties by inhibiting mast cells. as an alternative for future interventions, the patient can use cisatracurium, as the skin tests and the basophil activation test are negative. unal d yedikule chest disease, istanbul, turkey case report: tetracycline hydrochloride may rarely cause hypersensitivity reactions. (hrs). immediate type reactions are at the level of case presentations and anaphylaxis is reported. we report a patient with late onset anaphylaxis caused by tetracycline. a 47-year-old woman referred to our allergy outpatient clinic because of urticaria due to an antibiotic that she does not remember the name of. the patient reported that many years before she had presented urticaria on her arms and legs one hour after taking the drug. to confirm drug allergy invivo and invitro testing have to performed. for many drugs there was no validated skin test. for all that invitro tests are often less sensitive and more expensive. therefore single blind placebo controlled drug provocation tests (sbpcdpt) is the gold standard in the diagnosis of drug hypersensitivity reactions. we did not know which group of antibiotics were allergy to the patient. because the patient had history of asthma and atypical pneumonia we were performed the allergy tests with clarithromycin and she had tolerated. it was necessary to use tetracycline because of patient had vaginal infection. skin tests have not yet been validated for tetracyclines. for skin prick tests of tetracycline that is only available as tablet, not in a soluble form. therefore, the tablet was smashed and diluted with 0.9% nacl. it was also tested. 10 healthy controls to exclude irritation. because of skin prick tests with tetracycline negative. sbpcdpt was planned. sbpcdpt was performed by progressively increasing four divided doses at 30 minute intervals. two hours after last dose the patient experienced dyspnea, palpitations, and hypotension. as the reaction was considered to be anaphylaxis, she was given 0.5 mg of intramuscular epinephrine, intravenous 45 mg of pheniramine, and 40 mg of methylprednisolone. the reaction resolved within 3 hours. blood tryptase level was 14.9 ug/l taken at the 2nd hour of the reaction approximately 2 months after the anaphylaxis, serum tryptase level was 1.59 ug/l the serum tryptase level and the patient's clinic confirmed anaphylaxis due to tetracycline. we had proved late onset anaphylaxis due to tetracycline with the patient's clinic and serum tryptase level. anaphylaxis due to tetracycline is limited to case reports and small series but to our knowledge, there is no previous report of late onset tetracycline anaphylaxis. 1469 | case series of ige mediated anaphylactic shock due to polysorbate case 2: an 86-year-old male patient with hypertension, hypothyroidism and episodes of sustained monomorphic ventricular tachycardia (smvt), developed an anaphylactic shock after the administration of injectable amiodarone due to smvt. serum tryptase levels reached 34.8 μg/l during the reaction (baseline 6.59 μg/ l). skin tests were positive to injectable amiodarone (prick 50 mg/ ml, intradermal 0.5 mg/ml) and polysorbate 80 and 20 (prick-prick). skin prick-prick to amiodarone and dronedarone tablets were negative. the patient tolerated oral amiodarone. we report an anaphylactic reaction during the first intravenous administration of amiodarone in a female patient being treated for supraventricular tachycardia. bat was positive, suggesting a direct effect on basophil activation, as the patient was not previously exposed to the drug. 1472 | anaphylaxis during labor: don't forget to think of an amniotic fluid embolism case report: a 32-year old primigravida (40 weeks of gestational age) was admitted with signs of pre-eclampsia and labor was induced. benzylpenicillin and ropivacaine (epidural anesthesia) was administered >3 hours before the event. eighteen minutes after starting an infusion with oxytocin (15 ml/h) and a vaginal toucher, the patient developed a decreased level of consciousness, generalized edema/erythema and thoracic pain, followed within 3 minutes by fetal bradycardia and maternal collapse. after resuscitation, an urgent sectio was performed, and a baby girl was born. patient was extubated the same day. serum tryptase, 1 hours after the event, was 11.2 μg/l (basal tryptase level 3.4 μg/l). allergy workup demonstrated negative specific ige and skin tests for latex and chlorhexidine, negative skin and provocation testing for ropivacain. however, skin testing was hampered by dermographism: intradermal (idr) testing of benzylpenicillin (10 000 iu/ml, 1/1000-1/1) and oxytocin (10 ie/ml, 1/10 000-1/100) showed extensive erythema. idr testing of oxytocin in healthy volunteers showed pallor around the injection site (n = 3). intravenous provocation with benzylpenicillin was uneventful. a basophil activation test with oxytocin (patient and control) was negative. an additional bone marrow evaluation showed no evidence for mastocytosis. although clinical criteria for anaphylaxis were fulfilled, a diagnosis of an amniotic fluid embolism (afe) was concluded. no drugs were prohibited. patient gave consent for publication. conclusions: afe is one of the most devastating conditions in obstetrics, occurring typically during labor and delivery or immediately postpartum. the pathogenesis remains incompletely understood, however, it has been suggested that afe involves an anaphylactic reaction to fetal tissue exposure associated with breaches of the maternal-fetal physiological barrier, supported by transiently increased serum tryptase levels. the diagnosis is primarily clinical, and generally one of exclusion. no specific antemortem diagnostic tests are available to confirm afe. postmortem identification of fetal squames in the maternal pulmonary circulation gives final diagnosis. differential diagnosis includes drug-induced anaphylaxis or mastocytosis, which were ruled out in our case. method: the patient presented after hymenoptera stings dyspnoea, generalized erythema with pruritus, edema of the face that required emergency therapy in 3 episodes. results: an angio-ct was performed at the inferior limbs with optiray and 5 minutes after the end of the investigation, the patient presented an anaphylactic shock requiring admission to the icu for 4 days. conclusion: the patient's progression was slowly favorable. results: thirty seven cases were reported. 24 (65%) were women. the median age was 47 years. 19 (51%) had dress/dihs, 6 (16%) ten, 3 (8%) sjs, 3 (8%) agep, 3 (8%) other not classified scars, and 1 (2.7%) overlapping ten/sjs. in 100% of the patients the suspect drug was withdrawn. thirty one patients (83%) received systemic anti-inflammatory treatment. twenty six patients (70%) received intravenous (iv) corticosteroids alone, 3 (8%) iv corticosteroids plus ivig, 1 (2.7%) iv corticosteroids plus ivig, infliximab and colchicine, and 1 (2.7%) iv corticosteroids plus infliximab and cyclosporin. there were complications in 17 cases (49%), and death occurred in the patient with overlapping ten/sjs who had received corticosteroids plus immunoglobulin. in this study, our aim was to evaluate severe ihr to icm. method: we retrospectively analysed patient who consulted to our allergy unit between july 2011 and july 2016 reporting symptoms within 1 hour after icm administration. from a total of 109 patients, we selected eight that had suffered an anaphylactic reaction. a written informed consent had been obtained for diagnostic procedures. introduction: immediate type hypersensitivity reactions to pemetrexed have been reported as very rare case reports. as limited availability of alternative therapies in chemotherapeutic allergy, desensitization plays an important role in ensuring reuse of the culprit drug. we report a case of pemetrexed anaphylaxis and successful desensitization. case: 43 years old female patient with lung adenocarcinoma had been treated with cisplatin-pemetrexed as second-line therapy. during the 5th cycle within 5 minutes after the end of pemetrexed infusion she had chest pain, shortness of breath, cough, swallowing difficulty, erythema on face and body, nausea and vomiting. she was diagnosed as anaphylaxis and adrenaline was administered besides antihistamine and methylprednisolone. symptoms and findings of the patient were improved within 15 minutes. oncologists decelerated no suitable alternative therapy for the patient. although skin tests (prick test with 1/1 concentration, intradermal test with 1/10 000-1/10 concentration) were negative with pemetrexed, taking into account the severity of the reaction, pemetrexed desensitization was applied with the consent of the patient. no reaction was observed during the procedure result: desensitization is a successful and safe method of reusing the culprit drug. successful desensitization of pemetrexed with immediate type hypersensitivity reaction is described. the 28 years old man was admitted emergency department with fever, rash (maculo-papular) and pain in joints. it was the 9th day of taking of amoxicillin. the hematological abnormalities were revealed -eosinophilia, increased erythrocytes sedimentation rate. the level of serum ecp was 183 μg/l. the liver functional tests were increased too. hepatomegaly and cervical lymphadenopathy were observed. the patient was treated as a dress syndrome (infusion therapy, systemic steroids) and discharged after 2 weeks with improvement. all hematologic parameters were in normal limits. lymphadenopathies were resolved. the level of ecp was retaken -84 μg/l. patient was prescribed oral steroids till normalization of limits of ecp. it lasted 2 weeks after discharging. the serum level of ecp can play key role in the management of dress syndrome and in the making of diagnostic processes. until now, allergic or anaphylactic reactions to peg have been rarely reported. although patient with hypersensitivity to peg should avoid peg-containing drugs or products, patient who needs colonoscopy has few alternative bowel cleansing methods. no successful desensitization to peg has been reported to date. we report a case of successful desensitization and subsequent safe colonoscopic examination in patient with allergic reaction to peg. method: a 50-year-old woman developed generalized urticaria, pruritus, throat swelling, and shortness of breath immediately after taking a bowel preparation solution for colonoscopy. she had the first symptoms 3 years ago, and has had 2 more experiences so far. the symptoms appeared within 20-30 minutes of taking cleansing solution, and the endoscopy was no longer possible. seven years ago, she underwent endoscopy with no specific symptom. when the last symptom occurred a year ago, she was treated at emergency room because of severe dyspnea and dizziness. the patient came to our clinic for the proper diagnosis of allergy reaction and possible colonoscopic evaluation. objectives: to describe a successful desensitization to vedolizumab in one patient diagnosed with ulcerative colitis, refractory to infliximab and intolerant to azathioprine and sulfasalazine. methods: our patient was a 38 year old woman receiving treatment with intravenous vedolizumab (300 mg/cycle). cycles 1 and 2 were well tolerated, but in cycles 3, 4 and 6 she experienced hypotension and dyspnea, in spite of premedication with oral dexamethasone and metoclopramide. during cycle 6, she also showed facial angioedema, systemic urticarial reaction and oropharyngeal pruritus treated with methylprednisolone and ebastine. the results of prick (vedolizumab concentration 60 mg/ml) and intradermal skin tests (1:10 and 1:100) with vedolizumab were negative in our patient and in ten healthy controls. total ige level was 26.4 ui/ml and specific ige against dermatophagoides were positive, being negative for hamster epithelium and latex. since vedolizumab was the only therapeutic alternative, the patient was planned to undergo vedolizumab desensitization according to an 8-step protocol. patient informed consent was obtained previously. premedication consisting of ebastine, acetylsalicylic acid, montelukast and methylprednisolone one hour before desensitization was administered. desensitization protocol was performed with a total duration of 4 hours and 35 minutes and a total dose of 293 745 mg. dose steps were 0.015, 0.03, 0.3, 0.6, 1.2, 9, 18 and 264.6 mg. conclusions: our 8-step protocol desensitization to vedolizumab resulted safe and effective in our patient and it has allowed the continuation of treatment with vedolizumab for her ulcerative colitis. montelukast, anti h1 and h2 blockers were used for the pretreatment of desensitization. all procedures (skin and blood tests, desensitization) were carried out with the informed consent of the patient. we present an exceptional, non-immediate case of fever after cisplatin and etoposide infusion with positive skin test. case report: a 63-year-old man, recently diagnostic of lung cancer stadium iv, in first line of treatment with cisplatin and etoposide, started 2 hours after finishing the 2nd infusion: facial erythema that becomes generalized after 4-5 hours from infusion. twelve hours later, developed warmth sensation, shivering and fever (39°c) that persisted despite the use of several oral antipyretics treatment. infectious disease was discarded, so he was referred to our department in order to assess further administration of cisplatin and etoposide. methodology: skin testing was performed 30 days after the last reaction to minimize false-negative results, as follows (a) cisplatin prick test (1 mg/ml) and intradermal tests (0.1 mg/ml); (b) etoposide prick test (20 mg/ml) and intradermal tests (2 mg/ml); with histamine as the positive control and nacl-diluent as the negative control. the results of skin test were negative for immediate reading. but two hours later, intradermal test for cisplatin turned into red and itchy and 12 hours later, still associated a wheal. the patient was classified as high-risk (lung diseases, forced expiratory volume in 1 second <1 l) and underwent programmed inpatient desensitization according to the standardized birmingham women's hospital protocol. desensitization was performed in the medical intensive care unit. the patient received only standard oncology premedication. he tolerated the final dose of cisplatin with no breakthrough reactions followed by etoposide standard infusion. two additional desensitization procedures were performed, with no breakthrough reactions. therapy ended when the disease worsened. the importance of this case, lies in the fact that fever has not been described as a clinical hypersensitivity reaction for cisplatin but for oxaliplatin. although a non-immediate reaction at the 2nd infusion of cisplatin could scarcely suggest a hypersensitivity reaction, the positive skin test and successful desensitization with this drug, could suggest it. introduction: propylthiouracil is commonly used as the first treatment option in patients with hyperthyroidism. although it is generally a well-tolerated drug, it may lead to some side effects including liver damage, leucopenia and skin rash. among skin rash findings, urticaria is considerably common. nevertheless, in cases that developed urticaria, a rapid desensitization protocol specific to propylthiouracil has not been encountered. we represented a case in which we applied successful oral desensitization via a scheme in accordance with general desensitization principles in a case that developed propylthiouracil-induced urticaria. case report: propylthiouracil at a dose of 50 mg/day was initiated for a 36 year-old female patient with diagnosis of hyperthyroidism in internal diseases clinic. the patient developed widespread itching and swelling in the body 5-6 hours after she took the first dose of the drug. she had experienced a similar reaction with use of propylthiouracil in 2012. the patient who was breastfeeding a baby and did not have any treatment option other than propylthiouracil was referred to us with pre-diagnosis of drug allergy. the patient was thought to have propylthiouracil-induced hypersensitivity reaction and desensitization was planned. we prepared a desensitization scheme in accordance with general desensitization principles (table 1 ). in accordance with this prepared scheme, we successfully applied the desensitization protocol with propylthiouracil for the patient. the patient gave informed consent before testing and desensitization. results: spt was negative, but idt reaction was positive at 1:1000 method: we present a desensitization protocol to intravenous etoposide used in a 35-year-old male for non-hodgkin's lymphoma who was referred to the department of allergy at sotiria general hospital of athens. within 5 minutes after receiving the first dose of the drug, the patient complained for flushing, retrosternal pain, difficulty in breathing and weakness. the infusion was ceased immediately and the patient received proper treatment with gradual recovery of the symptoms. the next day, skin prick test (spt) and intradermal test (id) were performed with etoposide at dilution 1:10 (2 mg/ml). both of the tests, spt and id, were negative. histamine and nacl 0.9% were also used as positive and negative controls, respectively. a desensitization protocol of three-day cycle with intravenous etoposide was conducted. premedication for 3 days was administered including methylprednisolone, cetirizine, ranitidine, paracetamol and montelukast. results: the desensitization protocol of the first day consisted of 11 steps of rapid pulses administered at increasing infusion rates every 15 minutes, and 1 step of drip infusion at a final rate of 60 ml/hour (372 mg/232.5 ml) until completion of the infusion. the following 2 days, the patient received a modified rapid protocol consisting of the administration of the calculated dose of 400 mg in only one step of infusion rate of 60 ml/hour completing in only 4 hours and 15 minutes. the same protocol was applied in another three-day cycle with no adverse reactions. conclusions: hsrs to etoposide are rarely described in the literature. we propose a three-day modified rapid desensitization protocol to intravenous etoposide that could be particularly useful compared to other time-consuming desensitization protocols. case report: imatinib, a tyrosine kinase inhibitor, sometimes causes cutaneous reactions that can be of various severity. we present a case of a patient who was started on imatinib 400 mg daily and after 3 months developed diffuse mildly pruritic rash with some desquamation of palms of the hands. the dose of imatinib was reduced to 300 mg daily and therapy with prednisone 30 mg was started. after resolution of rash, the dose of prednisone was tapered to 10 mg daily, but the rash reappeared, although milder in intensity. the dose of prednisone was increased and levocetirizine added and rash resolved. prednisone was slowly discontinued and rash did not appear. in the case of reactions to imatinib the dose of drug can be reduced and short course of oral corticosteroid given. milder reactions can be treated with antihistamine or topical corticosteroid. therefore, when adverse skin reaction to imatinib occurs, induction of tolerance to this important drug should be attempted. method: the exosomes were collected from in vitro primary human sinonasal epithelia cell, which derived from three different groups (normal control, chronic rhinosinusitis and chronic rhinosinusitis with asthma). generation of exosomes in epithelia was confirmed by nanosight, tem and western blot. the proteins of exosomes were identified by proteomics analysis. the cellular proliferation and ciliogenesis were analyzed by cck8 and qpcr.the ciliary beat frequency was detected by sava system. we found that epithelial cellular exosomes from chronic rhinosinusitis and chronic rhinosinusitis with asthma could reduce the multiplication rate of normal epithelial cell at a certain concentration (≥10 μg/ml).we found that exosomes from chronic rhinosinusitis with or without asthma could interrupt the cellular ciliogenesis and ciliary beat frequency. using mass spectrometric analysis we demonstrated that the epithelial exosomes contained different proteins in different disease states. conclusion: our findings first identified that exosomes could be secreted by nasal epithelial cells. we also demonstrated exosomes from chronic rhinosinusitis with or without asthma could be a pathogenic factor in the remodeling of sinonasal mucosa. it could be considered as a significant biomarker for detecting the progress of chronic rhinosinusitis and a alternative therapy target. background: mucociliary transport (mct) is a major respiratory tract host defense mechanism and chronic exposure to allergen can deteriorate the these defense mechanism. the aim of this study was to investigate the effects common allergen (dp/df) on human nasal mucociliary transport in allergic rhinitis patients, and to determine the pathophysiology of ciliary beat frequency (cbf) during allergeninduced change method: allergic nasal mucosa cells of allergic rhinitis patients were exposed to common allergen (dp/df), and cbf was analyzed using an optical flow technique with the peak detection method results: the allergen(dp/df) exposed group showed a decreased cbf when compared to the control group. in the cytotoxicity assay, difference in survival rates was not found between the two groups. in the allergen(df/df)-exposed group, protein kinase c (pkc) activity was increased during a pkc activity assay. the broad pkc inhibitor, calphostin c abolished the allergen(dp/df)-induced decrease of cbf. the allergen-induced decrease of cbf was abolished by gf 109203x, a novel pkc (npkc) isoform inhibitor, whereas the decrease was not attenuated by g€o-6976, a specific inhibitor of conventional pkc (cpkc) isoform. conclusion: allergen may inhibit cbf via an npkc-dependent mechanism. therefore, we have confirmed that chronic exposure to allergen could decrease cbf by increasing pkc activity. method: ova-alum allergic rhinitis mouse model (ar model) and poly(i:c) induced il-17 dominant mouse model (neutrophil dominant model) were used. both mouse models were exposed to tio2 particles for 2 hours twice daily for 7 days, while the controls (n = 5) were not. sirius red staining for eosinophil infiltration, immunohistochemistry for neutrophil and il-17a, serum immunoglobulin (ig) g and e were assayed by using enzyme-linked immunosorbent assay. in addition, the expression of interleukin (il)-4, il-17, and interferon (ifn)-γ in the nasal mucosa and cervical lymph nodes was measured by immunohistochemistry, and real-time reverse transcription-polymerase chain reaction (rt-pcr), il-17 monoclonal antibody (secukinumab) was administered in vivo to evaluate il-17a dependency. results: tio2 exposure did not influence eosinophil infiltration in both ar and neutrophil dominant model. however, tio2 exposure increased neutrophil infiltration in both models and neutrophil infiltration was correlated with il-17 expression in the nasal mucosa. serum igg and ige levels were changed significantly in the tio2exposed group. th2 cytokines (il-4, il-5) and th1 cytokine, ifn-γ were not changed significantly in both models after tio2 exposure, however, il-17 were increased in tio2 exposure group. and these increased type 17 pathway and neutrophil infiltration were reversed after il-17 monoclonal antibody administration. conclusion: exposure to airborne tio2 induced neutrophil infiltration in the nasal mucosa. the type 17 response seems to play a dominant role in the nasal immune response following airborne tio2 exposure. 1501 | toll-like receptor 9 ligands increase type i interferon induced b-cell activating factor expression in chronic rhinosinusitis with nasal polyposis results: first: paf-r mrna expression was very low in fibroblasts from nm and np (data not shown). paf-r mrna expression was detected in whole sinonasal tissue, submerged and ali epithelial cell cultures from both controls nm and np. paf-r mrna was also detected in peripheral blood eosinophils. although no differences were found between nm and np tissues and cultures, paf-r mrna expression was significantly higher (p < 0.001) in eosinophils than in upper airway tissues and cells. second: protein paf-r was found expressed in whole tissue (predominantly in the epithelium and submucosal glands), submerged and ali epithelial cell cultures from both nm and np. peripheral blood eosinophils also showed paf-r protein expression. conclusion: both paf-r mrna and protein expression was found in sinonasal nm and np tissues (epithelium and submucosal glands) and in peripheral blood eosinophils. these findings suggest the paf/ paf-r system could play a pathophysiological role in crswnp through the modulation of structural and inflammatory cell functions. "this study was funded with a research grant from uriach group". background: allergic rhinitis (ar) is an increasingly more common nasal inflammatory disease in which an antigen such as pollen or dust mites triggers symptoms such as itching, sneezing, and rhinorrhea, which can lead to nasal obstruction. ar is mediated by thelper type 2 cells together with mast cells, eosinophils, and several inflammatory cytokines and chemokines. for example, recent abstracts | 757 research indicates that hypoxia-inducible factor 1α (hif-1α) is involved in the mechanism of ar development. the anti-heart failure drug digoxin has a specific inhibitory effect on hif-1α, and thus, the aim of the present research was to explore the anti-hypertensive effect and mechanism of digoxin in ar. method: an animal model of ovalbumin-induced ar was established in guinea pigs. the experimental group was treated with digoxin through the tail vein. for the comparison of symptoms between the experimental and control groups, the incidence of sneezing was recorded, and the eosinophilic interleukin il-4 and il-5 levels in nasal secretions were measured by enzyme-linked immunosorbent assays. western blotting and reverse transcription polymerase chain reaction analyses were conducted to evaluated hif-1α expression in guinea pig nasal mucosa. results: the ar symptoms of guinea pigs in the experimental group were significantly improved after administration of digoxin. specifically, the experimental group exhibited a significantly lower numbers of sneezing times(average 36.0 ± 1.10 vs 7.4 ± 0.78, p < 0.05) and lower il-4 and il-5 secretion levels (p < 0.01) compared with the control group. moreover, guinea pigs of the experimental group showed less severe nasal mucosa edema, lower hif-1α production, and reduced eosinophil infiltration in nasal mucosa compared with the control group. conclusion: the anti-heart failure drug digoxin may ameliorate the symptoms of ar by inhibiting hif-1α production. campo p 1 ; eguiluz i 1 ; bogas g 1 ; gomez f 1 ; ariza a 2 ; espino t 1 ; torres mj 1 ; rondon c 1 1 allergy unit-regional hospital of malaga-ibima, malaga, spain; 2 allergy laboratory_regional hospital of malaga-ibima, malaga, spain background: similarly to what has been described in allergic rhinitis, there is an important association of local allergic rhinitis (lar) with lower airway symptoms suggestive of asthma, being selfreported in 24.4% of lar patients after five years of follow-up, and increasing to 30.7% after 10 years. however, clinical suspicion alone it is not enough for asthma diagnosis and could overstate its real prevalence. the aim was to evaluate the real prevalence of asthma in lar patients based on validated objective methods. method: seventy-five patients (29 with lar, 20 with non-allergic rhinitis (nar), 18 with allergic rhinitis (ar)), and 8 healthy controls (hc) were included. all patients had perennial history of rhinitis and bronchial symptoms suggestive of mild-moderate asthma for at least two years. non-specific airways hyperresponsiveness (methacholine challenge test, using tidal breath method following ats guidelines) was performed in all subjects. results: subjects were mostly young females, non-smokers. median μg/day of inhaled corticosteroids (budesonide/equivalent dose) was similar in all groups. median fev1% in ar group (75.5%) was significantly lower compared to lar (90%, p = 0.002), nar (85%, p = 0.007) and hc (88%, p = 0.005). in the lar group, 15/29 (51.7%) had a positive methacholine, 12/20 (60%) in the nar, 15/18 (83.3%) in ar group and 0/8 (0%) in hc. patients with lar had a significant lower percentage of confirmed asthma than ar (p = 0.031) and similar to nar (p = 0.771). no differences were detected between ar vs nar (p = 0.155). conclusion: presence of objectively demonstrated asthma was lower in lar compared to ar, and with better lung function. conclusion: ambient air pollution influenced the hospital visit of patients with rhinitis, even, the level of pollutants, below the national standard. so2, o3, no2, and pm10 could increase an incidence of rhinitis and/or induce an aggravation of rhinitis symptoms. health care provider might expect upraising patients with rhinitis in the clinic with increase of air pollutants, even under the standard levels. results: during relapse of erosive oral lichen planus mononuclear cells obtained from peripheral blood of patients showed increased number of nk-cells cd3 + cd56 + in acute (15.5 ± 4.5%) and chronic (14.9 ± 6.3%) disease periods, and cd16 + grb cells in acute (18.1 ± 1.5%) and chronic (14.5 ± 2.5%) disease periods, p < 0.05. in patients with the non-erosive forms of olp there were cd3 + cd56 + and cd16 + grb cells in acute (8.9 ± 2.5%) and (7.3 ± 1.3%) and chronic disease (9.5 ± 2.2%) and (8.2 ± 3.1%), p < 0.05. the number of cd3 + cd56 + and cd16 + grb cells in the controls were (9.8 ± 2.1%) and (6.2 ± 5.4%), p < 0.05. conclusion: acute relapse of erosive oral lichen planus, unlike nonerosive forms, is characterized by increases in the number of cd3 + cd56 + and cd16 + grb cells. chronic disease in patients with erosive oral lichen planus showed a steady increase in the number of cd3 + cd56 + killer cells and cd16grb lymphocytes. bite", were observed in 6 patients (85%). typical hyper-and hypopigmentation were observed in six patients mainly on the fingers and the cheekbones. fibrosis of the skin of the fingers often leads to flexion contractions, which we observed in 6 patients. we were watching two-sided swelling of the fingers, but it was very pronounced in 5 patients (71%). 86% of our patients have impaired motility of the esophagus. accelerated esr and c-reactive protein were found in 5 patients as follows-2 intensively accelerated and 3 moderate. in our patients with positive ana, we observed 4 patients -at low titer 1:40 at 2 and titration 1:80 in 2 patients. the spectrum of ana found by us in raynaud's syndrome patients is closer to scleroderma than to lupus. we underline the importance of ana (57%) and anti-cc antibodies (27%) for the early diagnosis of raynaud's syndrome and scleroderma, which is also seen in our patients. anti-scl-70 antibodies were observed in 3 patients coinciding with other publications describing about 40% of the patients. low levels of complement were observed in 2 patients. low hemoglobin levels were observed in 1 patient, with no iron deficiency. conclusion: 1. we observed a typical fibrinoid necrosis and polymorphonuclear infiltration, and collagen accumulation in the walls of small and medium-sized blood vessels. results: statistically significant increase of il4 level (3.23 pg/ml [2.18; 5.02]; 3.42 pg/ml [2.8; 4.18] respectively) was determined in patients with uc both in acute stage and remission compared to controls (1.87 pg/ml [1.4; 3.2] , (p = 0.002; 0.009 respectively). statistically significant increase of il17a level (15 pg/ml [12.11; 23.38] ); 14.68 pg/ml [11.29; 17.19 ] respectively) was also observed in patients both in acute stage and remission compared to controls (7.36 pg/ml [5.18; 8 .06], p = 0.00007, p = 0.00029 respectively). besides statistically significant increase of ifnγ both in acute stage (176.15 pg/ml [65.15; 359.84] ) and remission (42. 6 pg/ml [29.4; 64.45 ]) compared to controls (16.5 pg/ml [12.3; 23 .2], p = 0.00107; 0.0118 respectively) was revealed. background: the presence of antinuclear antibodies (ana) is commonly associated with a broad spectrum of connective tissue diseases. low titres might be detected rarely also in healthy individuals, especially in higher age. an indirect immunofluorescence (iif) detection of ana antibodies on hep-2 cells is the most frequently used laboratory method in this respect. the method is quite reliable regarding sensitivity, however the specificity of this test is lower. we would appreciate a biomarker for clinical discrimination of anaother autoantibodies were tested in relation to basic diagnosis. results: a cohort of patients was divided into 6 groups according to main diagnosis: immunodeficiency, connective tissue diseases, bronchial asthma and allergic rhinitis, recurrent infectious diseases, gastrointestinal diseases, endocrinopathy and others and the last group was generated from healthy subjects. the presence of anti dfs70 antibodies was highest in the group of recurrent infections, mostly in females. in these subjects homogenous pattern of ana antibodies by iif was also detected quite often, probably induced by non-specific activation of immune system. on the other hand, in a group of connective tissue diseases, we have not found any anti dfs70 positive patient. the clinical impact of anti-dfs70 antibodies is not yet finally confirmed, but their low frequency in connective tissue diseases and presence in 5%-10% of healthy subject suggests their potential role as a new biomarker to be used as a negative predictive factor in non aard. confirmation of presence or absence of anti-dfs70 antibodies seems to be helpful to exclude potential diagnostic errors in iif ana positive patients. background: multiple sclerosis is a debilitating autoimmune and degenerative condition of the central nervous system, that predominantly affects young adults. both genetic and environmental factors are associated with increased risk for this disease. we propose that the effect of environmental factors, particularly latitude of childhood, is mediated through epigenetic mechanisms. specifically, we propose that unfavourable gene methylation predisposes individuals to multiple sclerosis, that this is set in childhood and adolescence, and transmitted from haematopoietic stem cells to progeny. method: cd34 + , cd14 + and cd56 + cell subsets were isolated from peripheral blood of healthy controls. libraries enriched for cpg islands and promoter regions were generated using modified reduced representation bisulfite sequencing and subjected to next generation sequencing. site specific methylation profiling of genome wide cpg islands, including ms susceptibility genes was conducted using methpipe software. results: genomic coverage was consistent with other published methylomes using modified reduced representation bisulfite sequencing. the methylation signature of peripheral blood derived subsets showed greater differences in methylation compared to buccal cells than with each other. individuals displayed differences in cd34 + methylomes, and these were recapitulated in the progeny cd56 + and cd14 + cells for those individuals. methylation of specific genes regions (e.g. prf1), were consistent with the known biological function of these genes and their potential contribution to ms risk. the vast majority of cpg islands interrogated show recapitulation of their methylation signature from cd34 + to progeny. however, individual differences and cell subset differences identified, likely reflect the known biological function of these genes in progeny cells. our preliminary results are consistent with the hypothesis that the epigenetic signature (that predisposes to ms risk) is set in childhood and adolescence. the physiological basis underlying the setting of this epigenetic signature is still to be elucidated, but may involve uv light and/or vitamin d, and may provide novel therapeutic targets, especially at a personalised level, for treatment of ms. background: auto-inflammatory diseases are rare disorders characterized by recurrent episodes of fever/inflammation affecting serosal surfaces, joints, eyes and skin without autoantibody production or an underlying infection. innate immunity is implicated in their pathogenesis and the underlying genetic defect has been identified in a fraction of the syndromes. during last years, the increased knowledge about auto-inflammatory diseases and the difficulty in their characterization aroused great interest to better understand these pathologies. the acidic soluble fraction of salivary proteome of patients and controls (hc) were analyzed by rp-hplc-esi-ms. 60 known salivary proteins (salivary acidic proline-rich phosphoproteins (aprps), histatins (hst), salivary cystatins s, sn and sa, statherin, p-b peptide, α-defensins 1-4, cystatins b, c, thymosin β-4, s100a7, s100a8, s100a9, and s100a12 proteins) and several derivatives (acetylated, glutathionylated, phosphorylated, and oxidized forms) were searched in the chromatographic profiles by xic (extracted ion current) procedure. 21 adult patients (mean age ± sd: 34.4 ± 10.1; 15 f, 6m) were enrolled and compared with 27 sex/age matched healthy controls (mean age ± sd: 33.4 ± 9.6; 18 f, 9m). patients are classified on the base of clinical manifestations as follows: 6 patients with fmf (mean age ± sd: 33 ± 7.9; 5 f, 1m), and 15 with unclassified fever syndrome (uc) (mean age ± sd: 34.9 ± 11.1; 10 f, 5m). results: fmf patients showed low levels of α-defensins 2, 3 and 4, this last was absent, with respect hc, and high levels of the glutathionylated proteoforms of cystatin b, and s100a9, and of antileukoproteinase (slpi). similar results were obtained on saliva of unclassified patients, which showed also levels of cystatin c higher than controls. interestingly, proteins and peptides typically secreted by salivary glands (cystatin c, histatins, statherin, aprps) were found more abundant in uc patients than in controls, and in some cases also than fmf patients (see table) . an evaluation of relative abundance of phosphorylation of phosphorylated proteins/peptides highlighted a significant hypophosphorylation of hst-1, prp-1 and prp-3 in uc patients with respect to controls, probably due to a less active fam20c kinase responsible for their phosphorylation conclusion: we show by a top-down proteomics approach a wide salivary modification, highlighting dysregulation in neutrophil-derived proteins and significant differences between fmm and uc patients. the control group consisted of 10 healthy donors aged 40-50 years. immunological methods of investigation included determination of membrane antigens b cells: cd3 − cd19 + cd45 + , cd19 + cd5 + cd45 + , cd19 + cd23 + cd45, cd19 + cd25 + cd45 + , cd19 + cd40 + cd45 + , cd19 + cd86 + cd45 + , cd3 + cd4 + cd40l + cd45 + , cd19 + cd45ra + cd27 + cd45 + , by flow cytometry. results: in the study subpopulation composition of lymphocytes in seropositive variant form of ra visceral a statistically significant increase in relative amount as b1-cells with immunophenotype cd19 + cd5 + cd45 + (0.6 ± 0.01% 0.12 ± 0.03%) and b2 lymphocytes with the phenotype cd19 + cd5 − cd3 − cd45 + (16.4 ± 1.2% and 7.9 ± 0.5%). in the analysis of the processes of maturation and differentiation of b2 cells detected statistically reliable increase of the relative number of mature cd19 + cd3 − cd45 + naïve b2 cells cd19 + cd45ra + cd27 number of mature cd19 + cd3 − cd45 + naïve b2 cells cd19 + cd45ra + cd27 − cd45 + (11.8 ± 0.9% and 5.7 ± 0.5%) compared to the control group. in the study of surface markers b2 lymphocytes revealed an increase of expression of costimulatory cd19 + cd40 + (19 ± 1.3% and 7.0 ± 0.42%) molecules and increasing the relative amount of cd40l 0.9 ± 0.2% (0.3 ± 0.05%) ligand on cd3 + cd4 + cd45 + subpopulation of t-lymphocytes. analysis of surface antigenic receptor b2 cells in the visceral form of ra showed an increased expression of early markers of cd19 + cd23 + cd45 + (2.9 ± 0.08% and 0.91 ± 0.1%), cd19 + cd25 + cd45 + (1.6 ± 0.4% 0.05 ± 0.01%) activation in comparison with the control group. case: a 44 year-old male patient who works as a dental technician with a history of lung silicosis and recurrent sinusitis applied to an orthopedics clinic for left hip pain and difficulty in walking. he has a history of keeping a dog during childhood. hip mri revealed a 5 × 3 cm sized mass on left iliac wing extended to gluteus muscle and subcutaneous tissue. incisional biopsy was reported as chronic granulomatous osteomyelitis. the lesion was considered as tuberculous abscess. despite anti-tuberculous (fourdrug regimen) treatment for one year, the lesion showed no regression. excisional biopsy was carried out by the same orthopedics clinic. chronic inflammatory reaction and fibrosis was considered to be due to cyst hydatid in the detailed evaluation. antiechinococcus igg and igm was performed with elisa and found positive. no other lesion was detected in lungs and liver. albendazole 400 mg twice a day was initiated and substantial regression observed after three months. atypical and sustained infections made us think of primary immunodeficiency disorders. immunoglobulin subgroups were as follows: iga: <1 mg/dl (70case description: 31 year-old male was firstly admitted to gastroenterologist due to intermittent diarrhea, abdominal pain and reactive lymphadenopathy. celiac disease was suspected as genetic test showed hla dq8 (hla-dqa1*03 and hla-dqb1*0302), histological evaluation of duodenum biopsy provided picture of lymphoid hyperplasia and marsh iiia variant. however, laboratory testing for celiac disease showed very low amount of antibodies against transglutaminase. gluten free diet for almost one year was ineffective as patient had a continuous problem of gaining weight due to chronic diarrhea. additional questioning revealed recurrent respiratory tract infections with a need of antibiotics more than two times/year during last decade. lymphocyte phenotyping by flow cytometry showed that cd3, cd4, cd8, cd19 are in normal ranges, but amounts of all immunoglobulins are low: igm <0.22 g/l, igg 2.53 g/l and iga 0.09 g/l. based on clinical symptoms and immunological evaluation diagnosis of cvid was confirmed, and replacement therapy with subcutaneous immunoglobulin (400 mg/kg/month) was initiated. after six months of treatment patient affirmed reduction of gastrointestinal symptoms; he gained 12 kg of weight, has no more infections and stable sufficient level of igg (7.9 g/l). conclusions: this clinical case shows the importance of immune testing for primary immunodeficiency in all subjects (despite age) with unusual symptoms of autoimmune and/or infectious disorders. cvid may have manifestation of various symptoms, which can lead to misdiagnosis, as well as inadequate treatment. results: in our sample, all patients who progressed to hypogammaglobulinemia were receiving lymphomas. there is no immunoglobulin dosage record prior to treatment. of the 9 cases, mean age was 52 years (4 men and 5 women), 2 lost follow-up, and 1 of them also presented neutropenia. seventeen patients who continued in followup required ivig replacement, due to infectious exacerbations, mainly pneumonia and sinusitis. the mean serum igg dosage at the time of onset of ivig replacement was 491 g/dl. the mean time between the first dose of rtm and the need for ivig replacement ranged from 2 to 8 years, with an average of 5 years. the iga dosage was used as a parameter for the recovery of hypogammaglobulinemia, and it was observed that only 1 of the 7 patients presented recovery of the condition up to the moment. conclusion: given the data, we considered the immunoglobulin dosage to be important before initiating rtm treatment and periodically, in order to indicate the replacement of ivig or igsc in a timely manner avoiding complications such as potentially serious infections. background: steinert's disease, also known as type 1 myotonic dystrophy (md1), is the most common dystrophy of the adult. it is inherited with an autosomal dominant mechanism. it causes myotonia, progressive muscles atrophy, muscular weakness, and problems at the heart's conduction tissue and at the respiratory muscles. in patients with myotonic dystrophy, hypogammaglobulinemia is frequently described. the associations and the pathogenesis between those affections are not totally clear, but it is recognized an increased catabolism of the immunoglobulin in these patients. in most of the cases, hypogammaglobulinemia affects only the igg class and does not become clinically manifest. however, replacement treatment is not always successful in these patients. we report the case of a patient with myotonic dystrophy and hypogammaglobulinemia. case report: a 44-years-old man with md 1 came to our attention for a history of recurrent infections of the upper respiratory tract and persistent infection by helicobacter pylori. at the laboratory tests, we documented low serum igg levels (449 mg/dl), normal igm and iga levels and protective antibodies against tetanus consisting with the diagnosis of hypogammaglobulinemia. due to the recurrent infections, he started replacement therapy with ivig (0.4 g/kg/ months), switched one year ago to facilitated subcutaneous ig (fscig) with achievement of protective serum igg levels (>600 mg/dl) and significantly reduction of infectious episodes. conclusion: hypogammaglobulinemia is frequently reported in patients with md1. in literature most of the cases described does not become clinically manifest, but in our case, the patient was symptomatic with recurrent infections. the replacement therapy with fscig showed both clinical effectiveness and safety. 1532 | real-world experience of a novel, highly purified 10% liquid iv human immunoglobulin for the treatment of antibody deficiencies guidelines for immunoglobulin use (july 2011). a highly purified 10% liquid iv human immunoglobulin (ig), with low levels of iga, anti-a and anti-b haemagglutinins, factors xia, xiia, kallikrein and aggregates (i10e) was recently approved for use in the uk. here i report our centre's experience in using this novel 10% i10e in three patients with antibody deficiencies. case presentations: a patient who presented in clinic with a first diagnosis of pid, was initiated on 10% i10e at 30 g infused every four weeks. after starting i10e, they experienced a decrease in the rate and frequency of infections, in line with expectations for igrt. a young patient on home therapy with a 20% subcutaneous ig for pid presented in clinic with low trough igg levels. non-compliance was identified as the cause of these low trough levels and therapy was switched to 10% i10e at 40 g infused every four weeks in a clinical setting. both the rate and severity of infections reduced and trough igg levels normalised. an older patient on igrt for sad was reviewed in clinic due to discontinuation of their current igrt product. they were switched to 10% i10e at 20 g infused every four weeks. the efficacy and tolerability of i10e was comparable to their previous therapy. a detailed analysis of patient, clinical and safety parameters associated with the initiation of 10% i10e will be presented, including infection rates, white cell counts, c-reactive protein levels, tolerability and infusion-related adverse events. conclusion: these cases highlight the real-world use of 10% i10e in two patients with pid and one patient with sad. they show that i10e was well-tolerated and efficacious in one treatment-naïve, and two previously-treated patients. method: prospective study of families with one or more members with c1-inh-hae followed in 7 hospitals in the northern area of spain. a cohort of 105 patients from 28 families with c1-inh-hae was evaluated for familiar diagnosis of c1-inh-hae one or several patients from the same family were chosen and were given a questionnaire to identify the total family members from the family branch affected by c1-inh-hae that had been already studied (members with diagnosis of c1-inh-hae and healthy members) and those that had not been previously studied. we also register the difficulties for obtaining these data. family members not previously studied and that consent to be contacted were asked for study of c1-inh-hae. for c1-inh-hae screening we use c4 blood levels results: we have studied 28 families with c1-inh-hae, 24 (87%) type 1 and 2 (7%) type ii; 13 families had all their known members already studied for c1-inh-hae (50%): 12 families have all their members studied (42.8%), 18 families have 90% or their known members studied (64.2%) and 10 families had less than 50% of their total known members studied. we have identified 85 members from 11 unrelated families that had not been previously studied for hae, 10 healthy, 4 had low c4 levels and had presented symptoms of hae; 1 had not presented symptoms of angioedema, had normal c4 levels, and low antigenic and functional c1-inh levels. difficulties for a complete family testing study have been: family dispersion, scarce or no family relationship, do not wish to know their possible pathology conclusion: it is crucial to insist on the study of the relatives of patients with hae. we propose to include a questionnaire to identify all patient's relatives at medical reviews of hae patients. case: a-4 year old boy was admitted to our clinic with the history of recurrent respiratory tract infections. his all immunoglobulins were low (igg <154 mg/dl, iga <25.4 mg/dl, igm <17.8 mg/dl) associated with the absence of b cells. his aunt cousin also had xlaa missense point mutation, c562c>t in exon 17 of the btk gene was identified in both affected cousins. the patient was commenced on regular ivig treatment every 3 weeks. at the age of 8, he suffered from intermittent fever attacks, abdominal pain and weight loss. tests for giardia lamblia, clostridium difficile and cryptosporidium, noro virus or parasites were negative. mr-enterography revealed intra-abdominal fluid and thickened walls of his jejunum and cecum. histopathological examination of the biopsy material obtained from terminal ileum, colon and cecum showed crohn disease. initially, he was treated with prednisolone and infliximab. because of the lack of response, infliximab treatment was switched to adalimumab. terminal ileum was resected to relieve obstruction complication. although he had been treated with adalimumab for 1 year, a significant improvement was not observed. vedolizumab (entyvio ™ ), is a humanized monoclonal antibody α4β7 integrin-receptor antagonist, was commenced. induction dosing was 300 mg infusions at 0, 2, and 6 weeks followed by a maintenance phase at 8week intervals. at the 6 month of the treatment, fever and abdominal pain attacks reduced, while his weight and oral intake increased. no side effects were observed. discussion: vedolizumab is effective for inducing and maintaining remission in adults with inflammatory bowel disease (ibd); however, there is limited pediatric data. this is the first immunocompromised child treated with vedolizumab. the symptoms of the patient receded and no side effect observed during 6 months of the treatment. results: louis-bar syndrome is a multisystem progressive disease with polymorphic manifestations which varies by age. the locomotor disability of these children is determined by neurological disorders, the exitus being caused by respiratory infectious and malignancies. the children involved in the study, had frequent episodes of respiratory infectious (bronchitis, pneumonia, atelectasis, empyema, lung abscess), ent infections (otitis, mastoiditis, sinusitis), chronic pulmonary disease (pulmonary fibrosis, bronchiectasis). index of death in this group is high (66.7%). in one of the boy, the pulmonary ct showed lymphadenopathy, later was confirmed non-hodgkin lymphoma, with subsequent death. another child died from pulmonary and systemic infectious complications. results: in this paper we present the clinical and morphological analysis of children with nezelof syndrome diagnosed post-mortem. clinically were predominantly the generalized intrauterine infections or their development in the postnatal period. at macroscopic examination all patients had thymic hypoplasia. later on the microscopic study of the thymus specimens determined dysplastic changes, defined by the presence of concentrically arranged epithelial cells. in all patients, was determined the total lack of hassall corpuscles and its predecessors. besides the above-mentioned modifications in all specimens, there was no cortico-medullary segregation. thymic parenchyma outside pseudorrhagia was made up of a reticular stroma with total lymphocyte depletion. conclusion: nezelof syndrome is a severe primary immunodeficiency associated with thymic dysplasia and alymphocytosis, which is manifested early with generalized infections and major risk of death in neonatal and infant. background: the study was aimed to evaluate the cytokine profile in nasal secretion and blood serum in patients with seasonal (sar) and perennial allergic rhinitis (par) with a potential for additional sensitization with microbial allergens. method: the inclusion criteria for ar were as follows: a diagnosis of ar for more than 2 years, the absence of nonallergic disorders of the nasopharynx, age of patients from 4 years to 60 years.control group: healthy volunteers at the age of 3-43 years without any allergic disorders at examination.in order to evaluate the innate and adaptive immunity, the cytokine profile of blood serum (il-4, il-10, and tgf-β) and nasal secretion (tslp, il-1β, tnf-α, and gm-csf) was determined. to determine tslp, tgf-β, il-10, and gm-csf concentrations, enzyme-linked immunosorbent assay kits were used (ebioscience, bender medsystems, r&d systems, mn, usa). we have noticed a significant correlation (r = 0 46, p = 0 014) between the tslp concentration in nasal secretion and as-ige level to staphilococcus aureus enterotoxin (allergen component m80) in patients with par. there was a significant correlation conclusion: staphylococcal superantigens might be one of the stimuli of local tslp hyperproduction by the epithelium. there was a significant correlation between gm-csf concentrations in nasal secretion and the intensity of sensitization to a staphylococcal enterotoxin (seb) in the patients with ar. seb is one of the polyclonal t cells activators, which may account for increased concentrations of cytokines such as gm-csf locally within the system of mucosal immunity. the patients with ar and additional high sensitization to ses demonstrated a higher tnf-α production profile due to macrophage and tcell activation by these toxins. 1547 | evaluation of circulating osteopontin level as potential biomarker of allergic asthma in patients with caucasian and south-east asian ethnicity background: osteopontin (opn) is a pleomorphic cytokine known to influence a wide range of immune cells; allergic asthma was previously associated with high circulating opn levels. in the present study, we aimed to verify if opn may qualify as biomarker of activated immune response in allergic patients belonging to two different ethnic groups: caucasians and south-east asians. method: serum opn levels were measured by elisa test (human osteopontin duoset, r&d systems) in a series of 121 italian adult patients affected by extrinsic asthma, allergic rhinitis, hymenoptera venom allergy, food allergy, allergic contact dermatitis and ige mediated hypersensitivity to beta lactams. 116 healthy subjects served as controls. 576 ethnic chinese subjects were recruited at the national university of singapore (nus) as cross-sectional cohort of an ongoing epidemiological study on the national prevalence of allergic diseases, and opn levels were detected by luminex (milliplex map, merck) and elisa assays (r&d systems). results: in the italian cohort, opn levels were significantly higher in cases compared to controls (p = 0.0010 by the mann-whitney test). statistically higher opn levels were found in asthma (p = 0.0269) and food allergy (p = 0.046) groups in comparison to controls. no significant differences were found (p = 0.597) between singaporeans with lifetime asthma and healthy controls, only the highest opn levels were heterogeneously found to correlate with asthma. however, a strong gender effect was shown, in both cases (p < 0.0001) and controls (p < 0.0001), with males presenting higher opn levels in comparison to females. consequently, we checked the mrna expression levels of opn gene (spp1) with illumina chips in whole blood of males and females, and no difference was found (p < 0.05). several experiments with western blots and different gel types were performed to verify if possible post-transcriptional/posttranslational modifications of opn could explain these findings. conclusion: opn seems to be a promising biomarker for current, active allergic asthma in caucasians even though technical difficulties, due to opn intrinsically disordered structure, the complex enzymatic metabolism, and the low circulating levels, significantly affect the experiments. further studies are needed to confirm these data. 1548 | mortality, intubation, and healthcare cost in patients with allergic bronchopulmonary aspergillosis in a hospital setting: a nationwide study fan x; luo y; yue b background: abpa is a complex hypersensitivity reaction to aspergillus fumigatus that colonize in airways, it is almost exclusively seen in patients with asthma or cystic fibrosis(cf). this study is to estimate hospitalization outcomes and healthcare cost of hospitalized patients with abpa. method: we conducted the study using data from national inpatient sample(nis) from 2010 to 2014. diagnosis were identified using icd-9-cm codes. hospitalization with a primary diagnosis of abpa and hospitalization with a primary diagnosis of acute respiratory failure/acute and chronic respiratory failure/respiratory distress/ asthma/cf and a secondary diagnosis of abpa were included. the study population was divided into groups including abpa with asthma, and abpa with cf. mortality and intubation rate were the primary outcomes; length of stay and total hospitalization cost(adjusted to cost in 2014 based on medical care cpi) were secondary outcomes. student t-test and chi-square were used for univariable analysis, linear and logistic regression were used for multivariable analysis. results: a total of 6308 hospitalizations with abpa were included, with 2896 hospitalizations with abpa and asthma, and 2793 hospitalizations with abpa and cf. the overall mortality rate was 0.82% (95% ci: 0.44%-1.50%), the mortality for abpa with asthma was 0.69% (95% ci: 0.26%-1.83%) and for abpa with cf was 0.56% (95% ci: 0.19%-1.63%). the overall intubation rate was 4.83% (95% ci: 3.77%-6.18%); the intubation rate for abpa with asthma was 5.51% (95% ci: 3.91%-7.72%) and 84.0% were early intubation (<3 days); the intubation rate for abpa with cf was 1.95% (95% ci: 1.13%-3.35%) and 36.6% were early intubation. the overall mean length of stay(los) was 8.7 (95% ci: 8.0-9.3) days, while the los for abpa with asthma was 5.6(95% ci: 5.2-6.1) days and the los for abpa with cf was 12.1 (95% ci: 11.1-13.0) days. the overall total cost was 147 million usd, the total cost for abpa with asthma was 33.4 million usd with a mean of 12 069, while the total cost for abpa with cf was 101 million usd with a mean of 38 005. conclusion: mortality among hospitalized patients with abpa is low<1%. intubation rate is relatively low, intubation, especially early intubation (<3 days), is more common in patients with asthma. although abpa is not a common disease in inpatient population, it does have a high health care cost and despite lower intubation rate, patients with abpa and cf generally have a longer hospital stay with a higher hospitalization cost. 1549 | frequent exacerbations of bronchitis with wheezing in adults: is it possible to predict and prevent asthma? case report: frequent episodes of bronchitis, accompanied by wheezing and dry with a prolonged duration in adults, the clinical course may be similar to bronchial asthma. the aim is to assess the risk of asthma in adult patients with 2 or more episodes of acute bronchitis per year, had a prolonged duration and accompanied by a dry wheezing. for 5 years in two regional clinical pulmonology centers were observed in 46 patients (19 men and 27 women) with average age 34 ± 6.8 years. each had at least 2 episodes of acute bronchitis per year, which was accompanied by prolonged cough and presence of wheezes. average number of acute episodes per year was 3.2 ± 0.8. in the course of the observation the patients were divided into 2 equal groups. the first group consisted of 23 persons treated in acute episodes of the disease symptomatic therapy, including inhaled β2-agonists short-acting short course. in the second group to the corresponding treatment added montelukast 10 mg per day lasting for 1 month. in all cases of exacerbation had a viral nature. held in the period of remission of allergic sensitization, the survey revealed. starting from the first year of follow-up all patients were vaccinated against influenza annually. by the end of the fifth year of observation in the first group in 5 cases was diagnosed of bronchial asthma-4 cases easy persistent asthma and 1 case moderate. the diagnosis was exhibited in accordance with the gina criteria. in the second group, the diagnosis of bronchial asthma were exposed to 1 patient (hazard ratio of 0.18). prospective observation suggests that the use of anti-inflammatory potential antileukotriene medicines in complex therapy of recurrent acute episodes of bronchitis accompanied by a dry wheezing in adults may be a factor preventing the development of asthma. for more conclusive results require more extensive research. background: chemokine receptors play an important role in regulating the migration of t lymphocytes, monocytes and neutrophils from the peripheral blood into inflamed tissue, such as lung. however, little is known about their expression on natural killer (nk) and natural killer t (nkt) cells in patients with chronic obstructive pulmonary disease (copd). therefore the aim of the study was to determine the chemokine receptor profile of peripheral blood nk and nkt cells of copd patients. method: for analysis of lymphocytes subtypes the flow cytometry method was used. the study population consisted of 57 smokers with copd, 12 healthy smokers and 12 healthy non-smokers. results: we observed an increase in blood nk cells expressing cxcr3 receptors in smokers with copd compared to healthy smokers (p = 0.003) and healthy non-smokers (p < 0.001). the percentage of nkt cells containing cxcr3 receptors was also significantly higher in blood of smokers with copd compared to healthy smokers (p = 0.021) and healthy non-smokers (p < 0.001). copd smokers had significantly higher proportion of ccr5 + nk cells than smokers without copd (p = 0.002) and healthy non-smokers (p < 0.001). increased proportion of blood nkt cells expressing ccr5 on their surface was observed in smoking copd patients compared to healthy smokers (p = 0.002) and healthy non-smokers (p < 0.001). there were no significant changes in the percentage of cxcr3 + and ccr5 + nk and nkt cells between healthy smokers and non-smokers. in addition, no differences were seen in the proportion of nk and nkt cells expressing cxcr4, cxcr6, ccr6 and ccr7 among all studied groups. method: 58 patients with copd in stable condition (gold stage 2) aged 40-70 years old, smoking history of ≥10 pack-years, were studied. bmi of patients were divided into 2 groups: obese (n = 31) (bmi-30.0-39.9 kg/m 2 ) and non-obese (n = 17) (bmi-18.5-24.9 kg/ m 2 ). ten subjects with normal lung function and bmi were the control group. the level of il-26 assessed in induced sputum (pg/ml) was measured using an elisa (raybiotech ® ). serum levels of crp were measured using the "vector-best" (russia federation). spirometry was performed according to american thoracic society and the european respiratory society (ats/ers) guidelines. results: obese copd patients had significantly increased concentrations of il-26 compared with healthy subjects and non-obese copd patients by 2.6 fold (139.0 ± 85.4 pg/ml vs 53.15 ± 18.5 pg/ ml) (p < 0.008) and 1.15 fold, (139.0 ± 85.4 pg/ml vs 120.6 ± 60.15 pg/ml)(p < 0.04), respectively. non-obese copd patients had higher levels of il-26 by 2.3 fold compared with healthy subjects (120.6 ± 60.15 pg/ml vs 53.15 ± 18.5 pg/ml) (p < 0.008). results: among these three groups, the level of cer in lung cancer patients (0.35 ± 0.10 g/l) was significantly higher than that in ild patients (0.31 ± 0.25 g/l) and healthy individuals (0.25 ± 0.04 g/l) (p < 0.05). meanwhile, the levels of c3 and c4 in healthy individuals, which are 1.70 ± 0.29 g/l and 0.27 ± 0.34 g/l respectively, were both significantly higher than that in lung cancer patients (c3: 1.04 ± 0.26 g/l, c4: 0.24 ± 0.09 g/l) and ild patients (c3: 0.97 ± 0.25 g/l, c4: 0.21 ± 0.09 g/l), (c3: p < 0.05, c4: p < 0.05). results from optimal scaling demonstrated that lung cancer was closely associated with immune factors including crp, cer, c3 and c4 (cronbach's alpha = 84.7%). conclusion: for ild patients, when the level of crp and cer is increased and the level of c3 and c4 is decreased simultaneously, the risk of the development of lung cancer should be considered for these patients. results: among pbmc subpopulations, endurance exercises impacted the number of nkt and activated t cells with nkt cell numbers greater in male bobsledders vs bullet shooting and biathlon (42.4% and 44.3%, respectively). the number of activated t cells (cd25 + ) was greater in bullet shooting and bobsleigh athletes vs the biathlon group (34.9% and 22.7%, respectively). in female athletes the number of cd25 + cells in the shooting and bobsled groups was greater by 46.1% and 25.5%, respectively vs the biathlon group (p < 0.05). increased il-10 occurred in bobsleds in comparison to bullet shooting and biathlon: 32% and 80% in male and 70.5% and 83% in female, respectively (p < 0.05). the concentration of il-18 in male bobsledders and biathlon was 30% and 34% greater, respectively, compared with bullet shooting (p < 0.05). serum concentrations of ifnγ in male as well as female athletes showed an increase of 41.6% and 39.5%, respectively vs biathletes (p < 0.05). increased il-4 occurred in the male biathlon group by 39.7% and 13%, respectively, vs the bullet shooting and bobsleigh athletes (p < 0.05). il-6 was increased in the male biathlon group, compared to bullet shooting and bobsledders by 76.7% and 70.3%, respectively (p < 0.05). conclusion: prolonged endurance exercises impacts secretion of pro-and anti-inflammatory cytokines in athletes of different sport specializations. concentrations of studied cytokines did not exceed reference values perhaps due to specialized sport nutrition, which may restore immune function during endurance exercises. background: oral immunotherapy (oit) is a promising therapeutic approach to treat food allergic patients. recently, we have shown that the use of a mixture of short-chain-and long-chain fructo-oligosaccharides (scfos/lcfos) improves the efficacy of oit in cow's milk and peanut allergic mice. however, concerns with regard to safety and long-term efficacy of oit remain and there is a need to identify novel biomarkers (panels) that predict, monitor and/or evaluate the effects of oit. here we present a method for the selection of candidate biomarkers by using the computational approaches bayesian networks (bn) and topological data analysis (tda). method: data were used from scfos/lcfos diet-supported oit studies performed in 2 independent cow's milk allergy (cma) and 2 independent peanut allergy (pna) experiments in mice. first, a subset of the data was used for learning the data structure and their interactions in terms of a bn. this bn was used to compare the key parameters in both experimental food allergy models. finally, the relations within the dataset in combination with the bn were explored to identify and rank candidate biomarkers for the effect of oit by applying tda. the bn was able to predict the efficacy of oit in the cma and in the pna model with 82% and 80% accuracy respectively, thereby identifying a set of 5 parameters (allergen-specific ige and igg1, body temperature, mmcp-1, earswelling) being key in the mechanisms involved in both scfos/lcfos-aided oit food allergy models. the tda zoomed in on the full set of 67 previously analyzed parameters and identified clusters of biomarkers closely linked to biologically relevant clinical symptoms but also unrelated and redundant parameters within the network. taken together, this enables the prioritization of candidate biomarkers. moreover, the tda indicated differences between pna and cma models in how the data are related to each other. here we provide promising bioinformatics methods to compare mechanistic features between two different food allergies and to determine the biological relevance of biomarker (panels) of oit for food allergy. we have shown that the key drivers that influence pna and cma are similar, but that these phenotypically similar diseases show mechanistic differences in their subnetworks. these new insights provide excellent starting points to generate new hypotheses to explain why cma has a different disease pattern than pna and to select biomarkers that are useful in future clinical studies. 1562 | functional and immunoreactive levels of igg4 correlate with clinical responses during the maintenance phase of house dust mite immunotherapy basophils were identified as ssc low cd193 high , and cd63 was used as an activation marker. reactivity was confirmed by anti-ige as a positive control. results: in 4 patients, basophil reactivity and sensitivity was comparable for grass pollen extract and recombinant phl p5, while phl p1 only caused a lower basophil activation. in one patient, recombinant phl p5 did not cause any basophil activation, while phl p1 elicited an even higher sensitivity and reactivity than grass pollen extract. conclusion: in 4 patients, basophil reactivity was comparable for grass pollen extract and recombinant phl p5, while phl p1 only caused a minor basophil activation. in one patient, recombinant phl p5 did not cause any basophil activation, while phl p1 elicited an even higher sensitivity and reactivity than grass pollen extract. reactivity of extract and the main sensitizing components correlated, while sensitivity did not. 1564 | in vitro assessment of hypersensitivity to allergen before and after allergen immunotherapy with whole blood basophil histamine release assay wbbhr assay in these patients was performed 7-10 days before and 7-10 days after ait. heparinized whole blood samples (8 ml) of each patient after substitution of plasma with pipes buffer were incubated one hour at 37°c with different concentrations of birch pollen extract (t3) in u-shape 96-well micro-titer plates. after incubation plates were centrifuged and supernatants from each well of the plate were directly analyzed for histamine content by reversedphase high performance liquid chromatography with electro-spray ionization mass-spectrometry (rp-hplc-esi-ms). results were expressed as ng/ml released histamine. sensitivity (limit of quantification) was 5-7 ng/ml. to compare results of histamine release in patients before and after ait data were calculated as area under the curve (auc) values. results: in contrast to pre-immunotherapy activity of blood basophils there were significant decreases in hr induced by t3 extract after ait. according to auc values all patients demonstrated decrease in hr after ait in compare to hr before ait in a range of 25%-65% demonstrating a decrease of hypersensitivity to birch allergens. analysis of basophil hr in patients received s.c. or s.l. the asthma and rhinoconjunctivitis symptom scores during and after pollination season decreased significantly and showed correlation with histamine release by t3. 1565 | immunotherapy with the recombinant b cell epitope-based grass pollen allergy vaccine bm32 induces a biphasic allergen-specific igg1 and igg4 response background: immunotherapy with the recombinant b cell epitopebased grass pollen allergy vaccine has been shown to reduce symptoms of grass pollen allergy in a multicenter, double-blind, placebocontrolled study. aim of this study was to investigate the levels and kinetics allergen-specific igg responses in a double-blind, placebocontrolled phase iib combined field and exposure chamber trial studying the effects of three, four and five pre-seasonal injections of bm32 as compared to placebo. method: a quantitative elisa assay based on purified human monoclonal allergen-specific igg 1 as well as igg 4 antibodies as standards was developed to measure allergen-specific igg 1 and igg 4 concentrations induced by ait with bm32. results: we found rises in levels of both tested allergen-specific igg subclasses in the actively but not placebo-treated patients. phl p 1-and phl p 5-specific igg 1 levels up to 83 μg/ml and 784 μg/ml, respectively and phl p 1-and phl p 5-specific igg 4 levels of up to 468 μg/ml and 1423 μg/ml, respectively were measured in bm32treated patients. five pre-seasonal injections induced the highest allergen-specific igg levels. interestingly, allergen-specific igg 1 and igg 4 antibodies showed a biphasic response with early rises of allergen-specific igg1 which declined quickly after the pollen season and a delayed but very sustained allergen-specific igg4 response. conclusion: treatment with bm32 induces a biphasic allergen-specific igg response consisting of an early igg1 and a sustained allergen-specific igg4 response which may be responsible for early and sustained protection against allergic symptoms. 1566 | t reg cd4 + cd25 high in peripheral blood in patient with grass pollen allergy during sublingual specific immunotherapy slit the aim of this study was to evaluate t reg cd4 + cd25 high in peripheral blood in patients with grass pollen allergy during sublingual immunotherapy slit. method: we examined 27 adult patients, aged 20-41, 13 female and 14 male. patients were qualified to slit after confirmation of allergy-by skin prick tests, specific ige and nasal provocation tests. we determined t reg cd4 + cd25 high from blood sampling of those patients (by flow cytometry method), before the slit, after reaching the maintenance dose, before the grass pollen season, during the grass pollen season, and after one year of slit. results: during slit the percentage of t reg cd4 + cd25 high increase after reaching the maintenance dose, then it decreased before and during the grass pollen season, and again increase after one year os slit. we observed, that in the group with significant improvement of symptoms, t regcd4 + cd25 high decreased during grass pollen season, comparing to group without clinical improvement. conclusion: slit as a method of immunotherapy influence on levels of t reg cd4 + cd25 high cells. the observed decreased levels of these cells during the grass pollen season might be consider as a prognosing marker of clinical improvement. 1567 | t reg cd4 + cd25 high from peripheral blood during subcutaneous specific immunotherapy (scit) for grass pollen hofman a; hofman j; hofman t centrum alergologii, poznan, poland background: specific immunotherapy is the only causal method for grass pollen allergic rhinitis. however, we don't observe in every patients satisfying clinical effects. because the treatment of allergic rhinitis takes 3-5 years, and is quite expensive, everyone is constantly looking for a perfect parameter, which may prognose the effectiveness of specific immunotherapy (sit). the aim of this study was to evaluate t reg cd4 + cd25 high lymphocytes from peripheral blood in patients during subcutaneous specific immunotherapy (scit) for grass pollen . method: we examined 99 adult patients (36 female, 63 male), age 18-60, who undergo scit for grass pollen allergy. we have done skin prick tests and specific ige in those patients. additionally, we confirmed the allergy by nasal provocation tests. we have determined lymphocytes t reg cd4 + cd25 high from blood sampling from those patients, with flow cytometry method: before immunotherapy, after reaching the maintenance dose of scit, before and during the grass pollen season, and after one year of scit. our control group was represented by 12 adult healthy volunteers. after one year of scit we divided patients into two groups-with and without clinical improvement. results: in allergic patients we have observed decreased levels of t reg cd4 + cd25 high compared to control group before the start for scit. during immunotherapy, the percentage of t reg cd4 + cd25 high increased after reaching the maintenance dose, however it did not reached the level of healthy volunteers. again, levels of t reg cd4 + cd25 high decreased before and during the grass pollen season, and increased after one year of scit. we compared also patient with significant improvement of clinical symptoms, and without. and we observed that the level of t regs cd4 + cd25 high decreased during pollen season in improved group, and increased in the group without clinical improvement. conclusion: in patients with grass pollen allergy, during the grass pollen season, decrease of t reg cd4 + cd25 high cells might be conresults: in the group of patients treated with sit gene expression analysis revealed significant change in ifng expression (p = 0.03) (comparison between sample a and b). comparison between samples a and c showed significantly different expression in genes: afap1l1 (p = 0.006), commd8 (p = 0.001), pik3cd (p = 0.027), and twist2 (p = 0.0003). duncan's multiple range test confirmed difference between sample a and c for commd8 (p = 0.004) and also revealed new significant difference in tbx21 in samples a and b (p = 0.035; in wilcoxon's test p = 0.08). k nearest neighbors algorithm was built based on ifng, pik3cd, commd8 expression. the results of the study indicate, that there is a significant change in the expression of a few genes during the build-up phase of sit. it may be suspected, that this change contribute to the mechanisms involved in the building tolerance to allergen. k nearest neighbors algorithm may be useful for sit efficacy prediction. 1569 | regulation of cytokine thymic stromal lymphopoietin (tslp) in modulating tgf-ß1induced interstitial inflammation and cellular fibrosis background: thymic stromal lymphopoietin (tslp) has previously been linked to allergic inflammatory diseases, tissue fibrosis and organ dysfunction. it remains unclear, however, whether tslp plays any role in the occurrence of renal fibrosis, so this study investigated that underlying mechanism. method: an in vitro fibrosis model was established by treating normal rat kidney fibroblast (nrk-49f) cells with transforming growth factor-β1 (tgf-β1), after which the levels of various fibrogenic markers (e.g., fibronectin) and downstream fibrogenic signal proteins (e.g., smad 7) were investigated. also, tslp shrna was used to silence the effects of tslp, while an elisa was conducted to evaluate the fibronectin secretions. results: the level of fibronectin in the nrk-49f cells was doseand time-dependently increased by the administration of exogenous tslp (p < 0.05). tslp also significantly increased the level of fibrosis signaling, in addition to inducing a marked decrease in the down-regulation of smad7. interestingly, the application of tslp shrna caused a dramatic reversal of the tgf-β1-induced cellular fibrosis while simultaneously leading to the suppression of fibronectin and fibrogenic signal proteins. conclusion: taken together, these observations provide insights into how extracellular matrices develop and could lead to potential therapeutic interventions for the suppression of renal inflammation and fibrosis. abstracts | 779 1571 | effects of two years treatment with the recombinant b cell epitope-based grass pollen allergy vaccine bm32 on allergen-specific b and t cell responses background: bm32 contains recombinant fusion proteins of nonallergenic peptides from ige-binding sites of the four major timothy grass pollen allergens phl p 1, 2, 5 and 6 and pres protein from the hepatitis b virus as a carrier. in a multicentre, double-blind, placebocontrolled trial, 181 grass pollen allergic subjects were treated for two years either with bm32 or placebo. here we investigated in detail the effect of immunization with bm32 on allergen-specific t and b cell responses. during the study from 35 subjects treated in the vienna centre (bm32: n = 25, placebo: n = 10) were investigated regarding proliferation using 3 h thymidine incorporation and cytokine production in response to various recombinant allergens at 9 different time points. grass pollen allergen-specific ige, igg 1 and igg 4 levels were determined by immunocap and elisa. results: a significant increase of allergen-specific igg 1 and igg 4 levels was found in the bm32-but not in the placebo group in both years (year1 > year2) after treatment. there was no difference regarding t cell proliferation in response to phl p 1 and phl p 5 after first grass pollen season between actively and placebo-treated patients whereas proliferation in particular of phl p 1-specific responses seemed to be blunted in the active group in the second year. no significant differences regarding allergen-specific th1, th2 and tolerogenic (i.e., il-10) cytokines were observed between bm32and placebo-treated patients. the findings indicate that the bm32 induces high levels of allergen-specific blocking antibodies which may reduce allergen-specific t cell proliferation but does not induce significant increases of regulatory cytokines in t cells. this study was supported by grants f4605, f4613 and dk 1248-b13 of the austrian science fund (fwf). 1 hospital universitario ramón y cajal, madrid, spain; 2 department of immunology iis-fundación jiménez díaz, uam, madrid, spain background: shiitake mushroom (sm) (lentula edodes) is an edible fungi native to east asia. it is traditionally cultivated and used in many asian countries and its consumption is increasing worldwide. direct skin exposure to sm can cause cutaneous reactions, including allergic contact dermatitis and urticaria, while its oral intake may prompt "shiitake flagellate dermatitis" (sfd), which is a distinctive itching linear erythematous eruption. sfd is usually considered a toxic reaction to lentinan, a thermolabile polysaccharide that increases interleukin-1. we report 3 cases (p1, p2, p3) of shiitake flagellate dermatitis studied in our centre. method: skin prick tests (spt) to environmental allergens-including moulds -, prick-by-prick and patch test with raw and cooked sm were carried out. total ige and specific ige to mushroom, white mushroom and environmental moulds were also determined. a raw and cooked shittake mushroom extracts were prepared. both extracts were analyzed in all the patients by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page). results: skin prick tests, prick-by-prick, patch tests and specific ige were all negative except for p1, who had positive prick-by-prick to raw shiitake mushroom. sds-page ige immunoblotting assays with the patient's sera revealed ige-reactivity with proteins ranging from 16 kda to 32 kda for p1, p2 and p3. we report 3 cases of shiitake flagellate dermatitis with demonstrated ige-sensitization. physicians should take into account that some cutaneous reactions considered as toxic might be allergic reactions. vorozhko i 1 ; sokolnikov a 1 ; sentsova t 2 ; donnikov a 3 ; ilyenko l 2 ; denisova s 2 background: allergic diseases such as asthma, rhinitis and food allergy have increased in recent decades in tropical countries. the tropics has climatic, environmental and ecological peculiarities that allow us to emit several hypotheses that could explain this phenomenon. in one of them, it is postulated that the increase of the sensitization to food, is due to the presence of lower serum levels of vitamin d, product of the adoption of a western lifestyle, with lower sun exposure, which in its turn diminishes the immunomodulatory action of this vitamin at intestinal level, favoring the sensitization against food antigens. we evaluate differences between the titers of serum antibodies against food antigens between two populations with african ancestry but different environment (rural vs urban) and investigate the influence of vitamin d levels. method: an observational, cross-sectional and descriptive study was carried out on 200 afro-descendant children living in san basilio de palenque (rural) or in the city of cartagena, bolivar (urban). the sensitization was determined by a positive skin prick test to allergen extracts, including foods, and, specific ige, iga and igg4 to egg, milk and peanut extract, as well vitamin d, were measured by elisa. antibody and vitamin d titers were correlated by spearman's test and comparisons between groups were done using the wilcoxon rank sum test. a p < 0.05 was considered significant. results: atopy was more prevalent in the urban population (24% vs 7%, p < 0.001). however, none participant tested was positive for food allergens. regarding vitamin d levels, these were found to be higher in the rural population compared to the urban group (p < 0.001). among the antibodies analyzed, only ige against peanut showed differences, which were higher in rural population (p < 0.001) as well as those of iga to peanut, which were higher in the urban population (p < 0.001). we observed only a significant correlation between peanut specific ige (rho 0.2127259, p < 0.005) and iga (rho -0.342434, p < 0.001) response and vitamin d. conclusion: in our study, we found differences between the peanut specific ige and iga response in urban and rural populations. the correlation between the levels of specific ige and iga to peanut and vitamin d, suggest that this vitamin may influence peanut sensitization in this population, besides other components like diet and genetic and environmental factors. 1580 | evaluation of inhaled allergen sensitivity in patients with food allergies younger than two years of age kulhas celik i 1 ; aldemir es 2 ; buyuktiryaki b 1 ; ginis t 1 ; toyran m 1 ; dibek misirlioglu e 1 ; kocabas cn 3 ; civelek e 1 carbohydrates and pyruvate was observed in the severe group compared to the rest of allergic groups. in addition, an increment in lactate was noticed. these metabolites were closely associated with the energy metabolism. other metabolic changes included increased levels of fatty acids such as myristate, palmitate and laureate. these fatty acids might be precursors of arachidonic acid, a key molecule in inflammation. finally, alterations in some amino acids and adenosine were found method: to evaluate these critical processes, 22 five-week old germ-free c3h/hen mice were split into two groups; 12 were intraperitoneally sensitized to the peanut allergen ara h 2 and 10 remained ns. upon reaching 8 weeks of age, mice were intragastrically challenged with purified ara h 2. mice were harvested in two groups: 30-minutes and 60-minutes post-gavage. upon harvest, the left lobe of the liver was collected and sera were removed. sera and livers were evaluated for drp-ara h 2 using an in-house quantitative sandwich enzyme-linked immunosorbent assay (elisa). a sample of the proximal small intestine was monitored for drp-ara h 2 using immunohistochemistry (ihc) and for mast cell degranulation using toluidine blue stain. results: sensitization does not have a large effect on the concentration of allergen present in the sera or liver. however, s mice allowed to digest ara h 2 for 60-minutes were more likely to display tissues positive for detection of drp-ara h 2 than ns mice at the same time point. conclusion: there is drastic biological variation among mice in their capacity to absorb and transport allergens. the elisa used in these analyses proved effective in the quantitative detection of drp-ara h 2 in both liver and sera samples, while ihc provided inconsistent results for the detection of drp-ara h 2 in tissues. however, in positive ihc samples, staining was indicative of paracellular transport across the epithelial barrier. 1583 | eczema induces a high ovalbuminspecific ige/igg1 ratio and affinity maturation during the lactation period irahara m; kido h; shinahara w inst. for enz. res., tokushima university, tokyo, japan background: recent articles have revealed that ingestion of foods induces oral tolerance and cutaneous sensitization induces food allergy. relationships with levels of immunoglobulin subclasses, affinity of allergen-specific ige, and development of food allergy have also been indicated. however, relationships with levels and affinity of specific immunoglobulins and eczema during early infancy remain poorly understood. therefore, the present study aimed to elucidate these relationships. method: this study enrolled women who visited naruto hospital (tokushima prefecture, japan) in late pregnancy and their children. blood samples and information on skin condition were taken every 2 months from neonate to 6 months old. egg white and milk allergen-specific immunoglobulin subclasses and affinity of ovalbumin (ova)-specific ige levels were measured using the densely carboxylated protein (dcp) microarray with 20 μl of serum. results: this study included 84 infants whose parents agreed to join this study. of these, 42 infants (50%) were diagnosed with eczema by 6 months old. egg white (ew) and milk-specific igg4 were detected in a few subjects at 6 months old. however, these specific ige and igg1 were detected in some subjects at that time ew-and ova-specific ige levels and ige/igg1 ratios were significantly higher in participants with eczema than in those without eczema at 6 months old. moreover, subjects with high ova-specific ige/igg1 ratios showed higher affinity ova-specific ige antibodies than subjects with low ova-specific ige/igg1 ratios. these results were not reflected in milk-specific ige levels. the milk-specific ige level differed between breast feeding and formula-fed infants, with no difference in the ige/igg1 ratio. conclusion: eczema contributed to high ew-and ova-specific ige levels and ige/igg1 ratios. high ova-specific ige/igg1 ratios involved high affinity ova-specific ige antibodies. however, the milk source during early infancy had no effect on the specific ige/igg1 ratio with eczema. these results suggest different sensitization routes provoke different results in levels and affinity of immunoglobulins. 1584 | purification and characterization of naturally occurring post-translationally cleaved ara h 6, an allergen that contributes substantially to the peanut allergome background: the 2s albumin ara h 6 is one of the most important peanut allergens. a post-translationally cleaved ara h 6 isoform has been described in the past but had not been characterized in detail, nor had its relevance for peanut allergy been investigated. method: post-translationally cleaved ara h 6 (para h 6) and intact ara h 6 (intact ara h 6) were purified from virginia type peanuts and the cleavage site was mapped using high-resolution mass spectrometry. biochemical characteristics were determined by sds-page, uv absorbance spectroscopy, far uv cd spectroscopy, and immunochemical reactivity of both forms of ara h 6 was compared by igg immunoblotting and ige-elisa using sera from individuals sensitized to peanut. reversed-phase liquid chromatography was applied to study the occurrence and abundance of para h 6 in various peanut types. results: compared to intact ara h 6, para h 6 lacks a 5-amino acid stretch, resembling amino acids 43-47 (uniprot accession number q647g9) in the non-structured loop. consequently, para h 6 consists of 2 chains; a n-terminal chain of approximately 5 kda, and a c-terminal chain of approximately 9 kda, held together by disulfide bonds. intermediate post-translationally cleaved products, in which this stretch is cleaved but not removed, are also present. the secondary structure and ige-binding of para h 6 resembles that of intact ara h 6, indicating that the loss of the non-structured loop is not critical for maintaining conformational ige-epitopes. both forms of ara h 6 were reactive with several commercially available igg antibodies. the peanut cultivars runner, virginia, valencia, and spanish contained para h 6 at equivalent levels, suggesting para h 6 is a consistent and important constituent of the peanut proteome. conclusion: a post-translationally cleaved form of ara h 6 is abundant in the main peanut market types, and has ige-binding comparable to intact ara h 6. this should be taken into account when ara h 6 is investigated in peanut-containing products. 1585 | release of major peanut allergens from their matrix at various ph and at saliva conditions; ara h 2 and ara h 6 are quickly bioaccessible background: the oral mucosa is the first immune organ that encounters allergens upon ingestion of food. peanut is often consumed in solid form, and it is not known if peanut allergens are released from the food already in the mouth. we set out to investigate the solubility of individual peanut allergens at conditions that mimic the first exposure site, i.e. the mouth. method: light roast peanut flour was suspended in buffers of various ph mimicking saliva. protein concentration was measured in supernatant, and release of major allergens ara h 1, ara h 2, ara h 3, and ara h 6 was assessed by sds-page. also, the allergen profile of un-dissolved material was assessed. results: peanut protein solubility is poor in the ph range 3-6, while at low ph (1.5) and at moderately high ph (>8), the solubility is higher. at all conditions tested, there was a substantial amount of un-dissolved protein. this indicates that the ph range of saliva, between 6.5 and 8.5 in healthy individuals, may be critical for the release of peanut protein from its matrix. in this ph range from 6.5 to 8.5, ara h 2 and ara h 6 are readily released, while ara h 1 and ara h 3 are poorly released. increasing the ph from 6.5 to 8.5 slightly increased the release of ara h 1 and ara h 3, but still the recovery was low (approximately 20% for both ara h 1 and ara h 3) compared to that of ara h 2 and ara h 6 (approximately 100% and 65%, respectively). this remarkable difference in extraction kinetics suggests that ara h 2 and ara h 6 are the first allergens an individual is exposed to upon ingestion of peanut-containing food. conclusion: based on our observations, we conclude that the peanut allergens ara h 2 and ara h 6 are quickly bio-accessible in the mouth upon ingestion of peanut. this new insight may contribute to the understanding of the extraordinary allergenicity of ara h 2 and ara h 6 compared to other peanut allergens. background: in proven cases of non-ige mediated cow's milk allergy clinical response can be partial even when treated with amino acid formulae e.g pain in infants. residual intestinal symptoms can be related to ongoing nerve hypersensitivity, changes in microbiome or motility disturbance. mast cells are thought to play crucial role in non-ige mediated food allergy. ketotifen is a first generation h1 antihistamine which has mast cell stabilising properties with pain blocking and anti tnf-a effect. hence ketotifen could have important role in symptom resolution where diet elimination has not been successful. we aim to find out effectiveness of ketotifen to unresponsive/partially responsive symptoms such as pain in non-ige mediated cow's milk allergy infants. method: children who presented to single specialist centre over 11 years had their case notes reviewed retrospectively. inclusion criteria were those children with confirmed non-ige mediated cow's milk allergy by elimination of cow's milk with improvement of symptoms and worsening of symptoms on reintroduction of dairy. where symptoms partially responded e.g pain, ketotifen was used at 0.5-1 mg once at night for 4 weeks and symptoms were reassessed. statistical analysis was performed using r v3.3.3 with significance was set at p = 0.05. results: 1031 patients were identified with 675 patients excluded due to unconfirmed non-ige mediated allergy. of the 358 case (206 males, age 3-50 months), atopic co-morbidities were found in 62% children. common symptoms were abdominal pain (51%), vomiting (47%), back arching (37%), constipation (35%), bloating (33%), food aversion (29%) and diarrhoea (26%). we compared the children who had symptom improvement on ketotifen and cow's milk elimination against children who improved on cow's milk elimination alone. significant difference of symptom improvement was found with abdominal pain; 69% using ketotifen compared to 53% who did not use results: a total of 3548 analysis for ttgiga have been performed for a total of 2965 patients during the study period (2010, 2012, 2014, 2016) . 128 patients showed at least once a positive ttgiga. among these, 11 had a negative result at first testing, and were positive at the second (n = 10) or third testing (n = 1). despite an increasing number of ttgiga requests, the number of positive results decreased. wige were rarely requested but were positive in about 45% of tested sera (table 1a and 1b). the amount of laboratory requests for ttgiga has increased, while those for wige remains stable and is rare. wheat allergy seems to be rarely investigated in our center and may deserve more attention. case report: eosinophil-associated gastrointestinal disorders (egids), including eosinophilic gastroenteritis (eog), are a inflammatory diseases, characterized by gastrointestinal symptoms and eosinophilic infiltration. patients with eoe have an increased incidence of allergy, with increased ige mediated food and inhalant sensitivities. use of either a targeted food allergen avoidance approach (based on allergy testing) or untargeted approach (based on food allergen avoidance) results in the resolution of eosinophilia in the gastrointestinal tract of 50%-70% of adult. we describe a case of a 27-year-old patient diagnosed with eosinophilic enteritis, associated to protein-losing enteropathy. the patient experienced severe diarrhea, nausea, vomiting and weight loss, that caused a severe dysproteinaemia and electrolytes abnormalities. an upper and lower endoscopy was performed, showing an ulcerative ileitis. the histological pattern was characterized by eosinophilic infiltration of ileum and duodenum>40 hpf. she presented also high levels of total ige (800 k/ui), high serum tryptase (20 μg/l, n.v. ≤9.0) and sensitization to the lipid transfer protein (ltp) of peach. the patient was prescribed to a six-food elimination diet (sfed) and underwent high doses of oral and intravenously corticosteroids, but a satisfactory therapeutic response was not achieved. we hypothesized that ige has a role in the mechanism of aeg and that blocking ige would have improved disease symptoms and reduced allergic inflammation, as measured by a decrease in intestinal tissue eosinophilia. we started off-label administration of omalizumab 300 mg/month subcutaneously, the same dosage schedule used in allergic asthma and, by other authors, in eosinophilic gastrointestinal disease after achieving informed consent by patient. except for an exacerbation of symptoms occurred 9 months after starting the therapy, when a further endoscopy, showing a gastrointestinal eosinophilic infiltration>50 hpf, was performed, a significant improvement of both gastrointestinal and cutaneous symptoms was observed during therapy, together with a normalization of laboratory parameters. after 30 months a clinical remission of disease was obtained and administration was stopped. although a histological remission during the first few months of treatment was not obtained, in a subset of aeg patients, ige plays a role in the pathophysiology of the disease and that anti-ige therapy with omalizumab may result in disease remission. 1590 | fullerene c60 reduces the allergic inflammation in food allergy mouse model background: a food allergy (fa) is an abnormal immune response to food. the signs and symptoms may range from mild to severe. they may include itchiness, swelling of the tongue, vomiting, diarrhea, hives, trouble breathing, or low blood pressure. food allergy is becoming increasingly common. fullerene c60 has the unique electronic properties making it an attractive candidate for allergic diseases therapy. the main purpose of our research was to assess therapeutic effect of fullerene c60 in a mouse model of fa. method: new efficient method for producing a water-soluble fullerene c60 has been developed. fa experimental model was induced in balb/c mice by the intragastrical (ig) ova administration after subcutaneous (sc) sensitization. fullerene c60 was administrated ig once a week, or twice a week, or daily. ova-specific antibodies were assessed by elisa. splenocytes cytokine production upon ova in vitro stimulation was detected by elisa. samples of jejunum of the small intestine were removed for histological examination immediately after the last ig allergen administration. results: it was shown that ova-specific ige and il-5 level were significant decreased in groups treated with water-soluble fullerene c60. the greatest effect was observed in mice receiving fullerene c60 daily. the ifn-gamma level was significantly higher in ig c60treated groups. the histologic analysis of jejunum of the small intestine samples showed that c60-therapy improved the histologic picture. the greatest effect was observed in mice receiving fullerene c60 daily too. conclusion: taken together, these results demonstrate that the water-soluble fullerene c60 exhibits a significant anti-inflammatory effect in a mouse model of fa, and possesses a high therapeutic potential. background: a 47-year-old male reported eight episodes of anaphylaxis after exercise. all the ingested food eight hours before each episode was analyzed. before each episode, he had always eaten chicken or turkey meat, and he tolerated these foods without exercising. in one of the episodes the patient had taken a tablet of dexketoprofen a few hours before. since several years, he referred chest tightness after eating some fish (emperor, salmon and whiff), however he tolerated others. the patient denied having eaten fish before any of the episodes of anaphylaxis. method: commercial skin prick tests (spts) and prick by prick tests (pp) with all food ingested and fishes were performed. tryptase, total serum ige (ige) and specific ige (sige) (immunocap. thermo-fisher scientific, uppsala, sweden) to the foods involved were determined. a controlled oral provocation test (opt) with dexketoprofen was performed. results: spt was positive to tuna extract (3 mm) and negative (0 mm) for the rest of fish extracts. it was also negative for chicken meat extract and other foods tested. pps were positive for raw and cooked turkey meat (3 mm), raw and cooked tuna (6 and 8 mm), raw emperor (11 mm), raw and cooked whiff (9 and 8 mm) and raw hake (18 mm). pps were negative to raw and cooked chicken meat. ige was 700ui/ml and tryptase 2.95 ng/l. sige was slightly positive to hake, cod and chicken meat. dexketoprofen opt was negative. at that moment, we recommended the patient to avoid chicken and turkey, as well as the fishes which he had symptoms with. since then, he has not suffered any new episode of anaphylaxis despite exercising daily. protein extracts from turkey meat, tuna, emperor, salmon, hake and whiff were prepared and analyzed by sds-page. conclusion: recently, triosephosphate-isomerase (28 kda) has been identified as a new chicken meat allergen. this allergen could be responsible for the cross-reactivity between bird and fish meat and the episodes of anaphylaxis after exercise in our patient. the triosephosphate-isomerase has not been implicated previously as a cross-reactive allergen involved in the fish-chicken syndrome. results: twenty-three patients were included, 61% male, median age 30 years (iqr 12.5), 48% atopic, 30% asthmatic. non-specific lipid transfer proteins (nsltp) were implicated in 70% (n = 16) and ω-5-gliadin in 30% (n = 7). eighteen (78%) patients referred anaphylaxis in the reaction with co-factor, 7(22%) urticaria/angioedema, 2 had both depending on the co-factor. all patients in which ω-5-gliadin was the allergen involved had anaphylaxis in the presence of co-factor, with tolerance to wheat without it. in patients in which nsltp was the allergen involved, 12 (75%) had anaphylaxis in the presence of co-factor. reaction with co-factor was more severe than without in 13 (81%) patients; 4 patients had no previous history of reaction and subsequently tolerated the culprit food. only 1 patient had anaphylaxis in the absence of co-factor; the remaining presented oral allergy syndrome and/or urticaria. exercise was the main co-factor, present in 22 patients. nonsteroidal anti-inflammatory drugs (nsaids) were the only co-factor in 6 patients; all of them had anaphylaxis, 4 with allergy to ω-5-gliadin, 2 to nsltps. all subsequently tolerated the nsaids involved. conclusion: nsltps and ω-5-gliadin were the most frequently involved allergens in cefa, with exercise being the most frequent co-factor. nsaids were relevant co-factors, even when ω-5-gliadin was the allergen involved. several patients subsequently tolerated culprit foods and nsaids, difficulting the diagnosis and further emphasizing the importance of a correct cofactor evaluation. molecular allergens had an important role in the diagnosis, avoiding unnecessary ofc. information about co-factors must be included in all patients with allergy to nsltps and ω5-gliadin. 1594 | allergy to wheat-dependent exerciseinduced anaphylaxis (wdeia) proteins, without? 5-gliadins as responsible ferreira a 1 ; castillo m 2 ; martins s 1 ; pineda f 2 1 unidade de imunoalergologia hospital das forças armadas., lisbon, portugal; 2 departamento de aplicaciones. diater laboratorios, madrid, spain background: the second well-characterized form of allergy to wheat proteins is wheat-dependent exercise-induced anaphylaxis (wdeia), with the ω5-gliadins (part of the gluten protein fraction) being the major group of proteins which are responsible, but other forms of food allergy have also been reported, with the proteins responsible including gluten proteins, cm proteins and non-specific lipid transfer proteins. the patient was a 38-year-old man who visited the hospital with acute urticaria just eat bread before run (30 minutes). according the components of bread. it was formed by a mixture of wheat, rye and barley. with a history (for several years) of episodes of severe urticaria after intake a mixture of cereals and/or different kinds of beer. results: prick test and specific ige with wheat, rye and barley were negative and the proteins from allergenic extract from these cereals, and also the gliadins and glutenins fractions were transferred onto a pvdf membrane to carried out a western blot technique with the patient's serum. the patient's serum recognized several proteins from wheat and millet gliadins not compatible in molecular mass with a ω5-gliadins. the association of w5 gliadin as responsible for the symptoms produced after the intake of products containing wheat and exercise is well referenced but in the case of this patient could have other proteins involved as triggers of their symptoms. suksawat y phramongkutklao hospital, bangkok, thailand background: food-dependent, exercise induced anaphylaxis (fdeia) is an anaphylactic condition that develops in patients who ingest specific food followed by exercise. a variety of foods have been described to be the cause including shellfish, wheat and vegetables. the mechanisms of fdeia is believed that exercise increases allergen absorption or decreases threshold of mast cell. the investigations such as skin prick test or specific ige for food are useful because food sensitization is demonstrated. however, a challenge test including ingestion of suspected food followed by exercise is the only method to diagnose this disease. we report a case of fdeia in a 17-year-old adolescent male. result: he presented with generalized urticaria and hypotension after eating a barbecue buffet which was one hour followed by playing taekwondo. after treatment with intramuscular adrenaline, antihistamine and systemic steroid, his condition was improved. the barbecue buffet consists of many kinds of food including shrimp, squid, salmon and pork meat which were previously tolerated. he had no past history of anaphylaxis or drug allergy. he was referred to our allergy unit for investigation. we performed skin prick test with food allergens and many kinds of fresh foods that he ate on that day and the result was positive to shrimp (6 mm. in diameter). three-day challenge protocol was set up a month after recovery and we used aspirin as a cofactor. on the first day, open challenge for 500 gram of shrimp was administered and the result was negative. on the second day, exercise challenge test based on the american thoracic society guideline was also negative. however, on the last day, he developed generalized urticaria five minutes after the same exercise challenge test which was 1 hour preceded by aspirin intake and 500 gram of shrimp ingestion. but his vital signs appeared to be stable. the patient was administered intramuscular adrenaline and antihistamine with full recovery. he was strongly advised to avoid shrimp for 4-6 hours before exercise and carry an adrenaline autoinjector. the-three day challenge protocol is a definite tool to confirm the diagnosis of fdeia. a correct diagnosis is important to avoid unnecessary restricted diet. 1596 | food dependent exercise induced anaphylaxis in peach allergic patient-case report consumption of different types of food (pancakes with cream cheese and fruit, peach, chinese dish, sandwiches-all eaten on other occasions without symptoms) and co-occurring physical exercise (dancing, shopping, walking). during diagnosis we performed spt with inhaled and food allergens (allergopharma), prick by prick tests with peach, banana, apple, pear and bread. we established the concentration of allergen specific ige (peach, wheat flour, peanuts, hazelnuts) and the level of ige specific to allergen components (immunocap isac). we performed exercise provocation test and open food challenge with peach. results: spt were negative with all tested food and inhaled allergens (inc.egg; milk; cocoa; tomato; carp; apple; banana; strawberry; rye flour; wheat flour; peanuts; hazelnut; citrus, d. farinae; d. pteronyssinus; grass; weeds; clad. herbarium; alt. tenuis; dog; cat; poplar; hazel; alder; birch; mugwort). prick by prick tests were positive with fresh peach. concentration of peach specific ige was 0.55 ku/l. in immunocap isac we found elevated levels of ige specific to ltps from different allergen sources (jug r 3 -1.3; pru p 3 -0.8; pla a 3 -0.6; tri a 14 -0.5 [isu-e]). open food challenge with a medium size peach was negative. exercise provocation test without allergen exposition was negative. exercise provocation test after eating a medium size peach concluded with severe lip and eyelids edema, followed by whizzing, dyspnea and urticarial. patient received adrenaline 0.5 mg im, steroids and antihistamines with good clinical effect. conclusion: patient was diagnosed with food dependent exercise induced anaphylaxis (fdeia) due to ltp allergy. she was advised to eat peeled fruit and vegetables, avoid cofactors of allergic diseases and carry rescue set (adrenaline, steroids and antihistamines). cases reports: we report 1case of fdeia and 3 cases of wdeia. case 1:24-year-old woman with intermittent severe allergic rhinitis and food allergies since childhood. in the last 7 years she registered weekly episodes of fdeia (urticaria, angioedema, wheeze, drop of bp) especially when during effort and after alcohol intake. prick tests were positive for grass pollen, mugworth, ragweed, dust mites, celery, soy, sesame, pistachio, mango, honey and shellfish. she followed 3 years of subcutaneous immunotherapy for grasses pollen. the fdeia episodes frequency and severity diminished during it. provocation test for celery and mango were positive, for alcohol, soy, sesame, pistachio, honey and shellfish were negative. case 2:23-year old man with a 7-year history of acute gluten induced urticaria, recently developed 2 episodes of wdeia (flushing, severe urticaria and angioedema, wheeze) when he went for gym. skin test was highly positive for wheat flour and dust mites, in vitro tests for 5 omega gliadin and wheat-specific ige were positive. food challenge for wheat was positive. he followed oral immunotherapy for wheat. the wdeia episodes were rare and mild. case 3:30-years old female with mild allergic rhinitis and controlled asthma, with wdeia (urticaria, angioedema, wheeze, drop of bp) in the last 3 years. skin tests showed positive results for wheat flour, dust mites and dog hair. omega-5 gliadin and wheat specific ige were high, but the provocation test for wheat was negative meanwhile combined wheat and effort provocation test was positive. 25-year-old female athlete, otherwise healthy, experienced three episodes of fdeia following running sessions. two reactions were preceded by intake of salad containing lettuce, tomato and sunflower seeds and the third one occurred after eating celery salad. the patient denied occurrence of any symptoms with physical exertion or food ingestion alone. physical examination and blood testing did not reveal any abnormalities. the differential diagnosis was performed. in skin prick test and specific serum ige antibodies sensitization to house dust mite, grass and mugwort were found. the spt and specific ige assay to culprit and most common food allergens were negative. the molecular diagnostic has been applied (faber, caam, rome, italy). the test scored positive for art v, blo t, der f, der p, eur m, lol p, phl p. no positive results for available food molecules including celery, tomato, sunflower seeds and lettuce were found. the detection of culprit food in fdeia is of crucial meaning as the syndrome can be life-threatening. the molecular diagnostic has been applied already in diagnosis of wheat-dependent exercise-induced anaphylaxis (wdeia) proving ω-5-gliadin sensitization in the majority of the cases. as the presented case did not reveal sensitization to culprit food in traditional allergy tests the molecular diagnostic was performed. this test did not show sensitization to culprit food either. however, not all of the molecules are available in molecular assays yet. cross-reactivity reaction to mugwort and grass has to be considered. the pathophysiological components of physical exertion has to be taken into consideration as well. this could contribute to the assessment of reasonability of molecular approach in diagnostic work-up for fdeia and to the establishment of standardized protocols for diagnosis and management of that syndrome. case report: food allergy to wheat is rare in adults, often reported in exercise-induced anaphylaxis. food-dependent exercise-induced anaphylaxis (fdeia) is a form of food allergy induced by exercise. fdeia symptoms can include urticaria/angioedema, respiratory and gastrointestinal manifestations and hypotension/shock. a 61-year-old male patient presented to the emergency department, was admitted after an episode of hives, hypotension and loss of consciousness. his consciousness was restored after treatment with epinephrine, glucocorticoids as well as fluids, and thereafter, the patient reported that the anaphylactic episode occurred when he started rapidly walking 1 hour after eating a slice of pizza. he mentioned that the offended food was tolerated always when it was not followed by a physical exercise. review of his past medical history and family one were non-contributory with respect to this episode. the allergy skin prick testing for common foods revealed a positive response only to wheat, while and other laboratory test values were within normal ranges. the patient is discharged after instructions on the use of epinephrine auto-injector. he was also advised to avoid wheat containing products up to 6 hours prior to physical exercise. our case demonstrated that fdeia can be characterized by the onset of anaphylaxis soon after physical exercise, when preceded by the ingestion of the responsible food. avoidance of the combination of the exposure to respective allergen and exercise is the most efficient precautive measure toward subsequent fdeia episodes. 1600 | residual exercise-induced allergic reactions after successful rush oral immunotherapies for milk and wheat method: we conducted roit for 55 children (median 8.7 years old) with milk allergy and 46 children (median 6.8 years old) with wheat allergy during 2010-2015. after 5-12 days of the rush phase in the hospital and a slow-increasing phase at home, patients consumed the maintenance dose (6.5 g milk protein or 5 g wheat protein). after at least three months of the maintenance phase without allergic symptoms, we conducted an exercise provocation test (ept) after eating the target food. if the ept was positive, we repeated it after a couple of years to check for remission. the presence or absence of eiars was based primarily on the results of epts but also on the clinical history in some cases. results: as of december 2017, 44 milk-and 41 wheat-allergic patients were able to continue ingesting the maintenance dose (desensitization). in these patients, 38 milk-and 38 wheat-allergic patients underwent the first ept at a median of 635 (228-2538) days after roit, and the result was positive in 19 (50.0%) and 19 (50.0%) patients, respectively. among these ept-positive patients, 10 milk-and 9 wheat-allergic patients conducted a second ept at a median of 504 (119-1120) days after the first ept. the result of the second ept was positive in 7 milk-and 7 wheat-allergic patients. in addition, clinical histories of eiars were subsequently observed in 4 milk-and 4 wheat-allergic patients after negative results on an ept. altogether, 19 (43.2%) milk-and 21 (51.2%) wheat-allergic patients still had eiars even after getting desensitization as of december conclusion: patients with persistent milk and wheat allergy often have residual eairs even after three to five years of desensitization due to the administration of successful roit. abstracts | 791 1601 | anaphylaxis caused by omega-5-gliadin initially diagnosed as idiopathic anaphylaxis: a case report tziotou m 1 ; syrigos k 2 ; syrigou e 1 ; sinaniotis a 1 1 department of allergy,, athens, greece; 2 gpp,, athens, greece case report: we report the case of a 59-year-old man who experienced two episodes of wheat dependent exercise induced anaphylaxis, initially diagnosed as idiopathic anaphylaxis. first episode: the patient woke up in the morning and drove to his resort. while driving, he ate a piece of cheese and ham pie. when he arrived, he walked some meters to the garden and started feeling pruritus and dizziness. he lost consciousness and recovered by himself. he was carried to hospital where his vital signs were normal. the patient has a history of atrial fibrillation and has been on flecainide bid and aspirin at noon for the last four years. a month after the first episode he visited an allergist. skin prick tests to aeroallergens and prick to prick tests to the ingredients of the pie were negative. tryptase levels were within normal limits and skin biopsy was negative for mastocytosis. an endocrinology workup was also negative. the patient was prescribed an epinephrine autoinjector and was asymptomatic for eight months. second episode: that morning he had a cup of milk and two slices of toast for breakfast and started working in the garden. two hours later he experienced pruritus and urticaria and fell unconscious. his wife had to administer two epinephrine autoinjectors before he regained consciousness. after the second episode it was decided to start treatment with omalizumab. two months later he experienced an episode of urticaria while working in the garden. he could not recall what he had eaten before. based on history, we thought that a cofactor might contribute to the occurrence of anaphylaxis. we performed skin prick tests with peach (ltp) and gliadin, allergens associated with food dependent exercise induced anaphylaxis in our region. the test to gliadin was positive. specific ige in serum to omega-5-gliadin was also positive, while specific iges to all ltps tested were negative. the patient was advised to avoid wheat and has been asymptomatic ever since. cases diagnosed with idiopathic anaphylaxis may actually be cases in which the culprit allergen has not been identified. detailed history and extensive workup may contribute to the successful management of these patients. written informed consent has been obtained from the patient. 1602 | wheat-dependent exercise-induced anaphylaxis (wdeia) and nsaids: clinical history is crucial conclusions :we present a case clinically compatible with wdeia with nsaids intake as augmenting factor. this case emphasizes that a carefully and thoroughly taken medical history is of crucial importance, otherwise wdeia can easily be unrecognized. as a result, non-allergic hyperreactivity to nsaids could be excluded and the diagnose of selective allergy to arylpropionic acids was made. exercise challenge test could not be performed in our case. case report: kounis syndrome (ks) has been defined as an acute coronary syndrome that manifests as unstable vasospastic or non-vasospastic angina, and even as acute myocardial infarction. it is triggered by the release of inflammatory mediators following an allergic insult. a 77-year-old woman with type ii diabetes and hypertension, and unstable angina pectoris as cardiovascular risk factors, has consumed chamomile tea. thirty minutes later, she developed generalized itching, skin rash, swelling of the face and the throat, chest tightness, dyspnea and syncope. the patient was transferred immediately to the emergency department, and sodium chloride, 50 mg prednisolone, 8 mg dexamethasone, 80 mg methylprednisolone, 5000ui heparin, 75 mg voltaren were intravenously administered along the subsequent 60 minutes under simultaneous treatment with oxygen therapy. an ekg examination is performed based on the patient disease's history, showing a 2.5-3 mm st-depression on d1-d3 leads, 1 mm on v3, and 3.5 mm on the v4-v6 ones. in addition, 2.5 mm st-elevation on the avr and 1 mm on the v1 derivation was observed, associated by a negative t-wave on the v1, v2, and avr leads. blood tests revealed a normal troponin i level (of 0.07 ng/ml). five hours later in the ekg was noticed: isolined st-segments, and negative t waves on the d3, avr, and v1 leads. ultrasound examination revealed normal heart kinetics and function. following the heart changes, the patient was administered sol. heparin 5000ui twice i.v., nebivolol 5 mg, plavix 75 mg, atorvastatin 80 mg, abstracts | 793 monocinque 20 mg, ordinary insulin 10ui s.c., glargine insulin 20ui s.c., and sol. furosemide 20 mg i.v. the patient progressed favorably, and four days after the anaphylactic episode the ekg revealed a 0.5 mm st-depression on leads v4, and v5, negative t-wave on avl lead, and normalized one on the v3 one. this case emphasizes the role of serious allergic reactions as cause of acute coronary syndrome in patients with altered coronary arteries and food intake as cause of kounis syndrome. 1605 | recall urticaria in two young patients with alpha-gal-syndrome after tick bites case report: patient a (m, age 25) had suffered from 7-8 anaphylactic reactions (hives, nausea, dyspnea and dizziness) within the past 15 months. all episodes occurred 3-5 hours after ingestion of red meat, once with alcohol as a co-factor. all episodes started with a wheal measuring about 0.5 cm in exact the same spot where he had been bitten by a tick one year before. specific ige to galactose-alpha-1,3-galactose (alpha-gal) was positive (2.55 ku/l). skin prick testing using raw pork kidney suspension and intradermal testing with gelafundin ® 4% diluted 1:10 also showed positive reactions. we performed an oral challenge with cooked pork kidney under careful monitoring being able to reproduce the recall urticaria as described above with a cumulative dose of 8 g pork kidney. we stopped the challenge and treated the patient with antihistamines and corticosteroids. patient b (m, age 22) reported on several anaphylactic reactions within the past 4 years with symptoms including abdominal pain, diarrhea, as well as dyspnea and loss of consciousness in one of the episodes. all episodes occurred several hours after ingesting food. furthermore the patient remembered a tick bite about 2 years before the first anaphylactic reaction, which repeatedly became inflamed and only healed completely over months. every episode started with pruritus and a wheal in the area of the former tick bite (in loco). specific ige to galactose-alpha-1,3-galactose was positive (4.37 ku/l) as well as the skin prick testing with cooked pork kidney and intradermal testing with gelafundin ® 4% diluted 1:10. this patient refused performance of oral challenge tests. an elimination diet of red meat for 12 months resulted in the absence of the symptoms as described. the diagnosis of alpha-gal-syndrome with recall urticaria in loco was made in both cases. this symptom may also be useful in evaluating results of oral challenge tests as well as an important clinical sign in medical history. pali-schöll i 1 ; meinlschmidt p 2 ; purschke b 2 ; hofstetter g 1 ; einhorn l 3 ; mothes-luksch n 3 ; jensen-jarolim e 3 ; jäger h 2 background: insects have gained interest as alternative nutrient source for humans and animals. however, being a "novel food" in the industrialized part of the world, several safety aspects, like allergenicity, need to be thoroughly addressed. in the present work we evaluated the cross-recognition of ige from patients allergic to crustaceans, house dust mite or stable flies, using house cricket acheta domesticus (ad), desert locust schistocerca gregaria (sg) and mealworm tenebrio molitor (tm). we further investigated changes of immune-recognition in terms of ige-binding in differently processed insect extracts. method: migratory locust locusta migratoria (lm) was subjected to different extraction methods, enzymatic hydrolysis or thermal processing, whereas tm larvae (tml) were evaluated after different centrifugation modes and ph levels. results: we revealed that ige from patients with crustacean allergy shows cross-recognition of acheta domesticus, schistocerca gregaria and stable flies. ige from house dust mite allergic individuals binds to acheta domesticus and schistocerca gregaria. importantly, the cross-reactivity to lm can be deleted by enzymatic hydrolysis with different enzymes or heat treatment (cooking, autoclaving), but not by different extraction methods. changes of ph and varying centrifugation steps are not sufficient to reduce ige-binding to tml. our results show that patients allergic to crustaceans, house dust mite or stable flies-allergic patients cross-recognize desert locust and house cricket proteins, and crustacean-allergic patients also flies proteins. furthermore, we confirm that the appropriate food processing method of insect proteins can reduce the risk of cross-reactivity for crustaceans-and house dust mite-allergic patients. the study was supported by the austrian science fund fwf (grant sfb f4606-b28 to ejj). results: the mean age at diagnosis was 10 years in children and 34 years in adults, more frequent in males (3:1). in the pediatric group, three had first-degree relatives with eoe and three had celiac disease. two children had performed milk oral immunotherapy and five adults aeroallergens subcutaneous immunotherapy. most of them were atopics with sensitization to aeroallergens (77.5% of children and 82.5% of adults) and food allergens (75% of children and 82.5% of adults), without statistically significant differences. the most frequent foods were fruits and nuts in both groups. we found significant statistical differences in fruits (35% of children abstracts | 795 and 57.5% of adults; p = 0.007) and cereals sensitization (5% of children and 47.5% of adults; p < 0.001). in the clinical presentation we observed significant statistical differences in impaction (22.5% of children and 82.5% of adults; p < 0.001), dysphagia (42.5% of children and 77.5% of adults; p < 0.001) and abdominal pain (25% of children and 7.5% of adults; p = 0.034). in the endoscopic findings children had more frequently exudates (92.5%; p < 0.001) and adults had esophageal trachealization (50%; p < 0.001). significant statistical differences were found in the treatment with topical corticosteroids (30% of children and 77.5% of adults; p < 0.001) obtaining a variable positive response. 77.5% of patients in both groups received food elimination diet, 45% with four or more foods. conclusion: eoe presents differences in the sensitization profile, clinical manifestations and endoscopic findings according to the age of presentation. the response to pharmacological treatment is variable and a high percentage of patients receive food elimination diets. it is a pathology difficult to control, therefore new non-invasive techniques would be useful in order to facilitate its management. 1610 | modulation of gut microbiota in patients with nickel allergy and ibs after diet and probiotics supplementation mb 1 ; garcía-figueroa be 2 department of allergy1 department of allergy fang l 12 united states 1431 | bcx7353 improves health-related quality of life in hereditary angioedema with c1-inhibitor deficiency bygum a 4 fang l 16 former yugoslav republic of; 6 division of clinical immunology huissoon a 2 bygum a 3 ; panovska vg 4 united states callejas fdb 1 alobid i 1 ; muñoz-cano r 1 izquierdo i 2 icahn school of medicine at mount sinai method: the case report form was developed by experienced allergists, and the web-based registry was established in cooperation with a professional medical software team. twenty-two departments from 16 hospitals took part during the first year 8%), whereas in adults, drugs (54.5%) were more common than foods (31.4%). the most common food triggers were eggs (27.1%), milk (15.8%), and walnut (7.9%) in children, and shrimps (21.1%), wheat (15.8%), and crab (7.9%) in adults. among drug triggers in adults, antibiotics (50.0%) were the most common cause followed by nsaids (16.7%), and h2-blockers (13.6%). the onset time was≤10 minutes in 48.6%. in children, home was the place of occurrence in more than half of the cases, whereas adults experienced anaphylaxis in out-of-home settings more often than children. cofactors were present in 19%. among the 289 cases registered via the emergency department of participating hospitals, epinephrine was administered in 66.8% (62.5% in adults, 69.8% in children) and the route of administration was im in 90.8%, iv in 6.3%, both im and iv in 2.1%, and subcutaneous in 0.7%. the number of epinephrine administration was single in 83% conclusion: this multicenter prospective registry would provide a better understanding of anaphylaxis, and provide visionary modalities to improve the management and prevention of anaphylaxis in future a case of kounis syndrome after chamomile tea consumption characterized by symptoms related to esophageal dysfunction and esophageal mucosal infiltration by eosinophils objective: characterize patients (pts) with eoe diagnosis and analyze the differences between pts with diagnosis at pediatric (ch, <18 years old) and adult age epicutaneous tests(epict)], serum total ige and eos, findings in upper digestive endoscopy (ude) and biopsies. the correlation between food sensitization, clinical severity (visits to er services or hospitalization due to complications of eoe, sclin) or severe histology results: 74 pts (81% male, average age 27 ± 17 years) ad 31 and 34, respectively. 96% ch and 67% ad were atopics. the most frequent symptoms of eoe were dysphagia (73%) and gastroesophageal reflux(46%) in ch; impaction(85%) and dysphagia(46%) in ad. 92% ch and 71% ad had aeroallergens sensitization. 77% ch and 69% ad had food sensitization. the most frequent positive tests were for ch: spt to milk(25%) and shellfish(19%), epict to shellfish(50%) and meat(19%); for ad: spt to milk(21%), fresh fruits and nuts both 18%), epict to shellfish(35%) and meat(13%). ude showed: 65% striation and white plaques in 50% ch shist (46%) was associated with sclin (35%), p = 0.001 in ch; but this was not observed in ad group there was no correlation between food sensitization and sclin or shist in both groups(p > 0.01). the average values of serum total ige (kua/l) were 653 in ch and 458 in ad; eos were 679 and 413, respectively in ch and ad allergy unit-fondazione policlinico universitario a. gemelli, università cattolica del sacro cuore conclusion: ltp with a fixed dose (2000 iu in 4 ml) of ready-touse shp616 led to fewer severe attacks, a higher proportion of attack-free patients, and a clinically meaningful and statistically significant reduction in cumulative attack severity and daily severity in hae patients relative to placebo. background: c1-inh-hae is a rare, potentially life-threatening disease characterized by episodes of subcutaneous and/or submucosal swelling. apex-1 was a phase 2, double-blind, placebo-controlled study to evaluate the prevention of attacks with bcx7353, a once daily oral kallikrein inhibitor, in patients with c1-inh-hae.method: patients with c1-inh-hae with a history of at least 2 hae attacks per month were randomized to receive four different doses of bcx7353 (350 mg, 250 mg, 125 mg, 62.5 mg) or placebo for 28 days. blood samples for bcx7353 concentrations and kallikrein inhibition were obtained from patients before dosing and for 24 hours post-dose on day 14. pk analyses and pk-pd modeling were done in phoenix winnonlin v 8.0 and sas v 9.3. the pk population included 16, 14, 14, and 7 subjects in the 350 mg, 250 mg, 125 mg, and 62.5 mg groups respectively.after daily dosing achieved steady state, c max was reached at a median of 3-4 hours after dosing. there was a greater than dose proportional increase in exposure (auc tau and c max ) over the 62.5-mg to 350-mg dose range, with an approximate 14-fold increase in exposure with a 5.6-fold increase in dose. at doses ≥125 mg, which showed statistically significant and clinically meaningful reductions in hae attack rates, geometric mean plasma trough concentrations (c tau ) were maintained at or above the minimum target concentration (4-fold ec 50 ) estimated to be required for adequate plasma kallikrein inhibition. percentages of study subjects at steady-state with bcx7353 plasma concentrations>4-fold ec 50 were 0%, 57%, 100% and 100% in the 62.5, 125, 250, and 350 mg dose groups, respectively. a 125-mg dose provided a mean c tau of slightly above 4.0fold ec 50 , with a corresponding reduction in hae attack rate of 69% (p < 0.001) compared with placebo. consistent with the exposure data, a dose dependent inhibition of kallikrein was observed with bcx7353 treatment over the dose range. the drug effect on kallikrein inhibition was highly correlated with exposure (r = 0.867). in patients with c1-inh-hae, bcx7353 treatment at doses ≥125 mg resulted in clinically meaningful reductions in the mean weekly hae attack rate. concentrations of bcx7353 at doses ≥125 mg were maintained at or above a c tau of 4-fold the kallikrein inhibition ec 50 in most patients, and kallikrein inhibition was highly correlated with bcx7353 plasma concentrations.background: c1-inh-hae is a rare, life-threatening disease characterized by recurrent episodes of subcutaneous and/or submucosal swelling that lead to considerable morbidity and a poor quality of life (qol). apex-1 was a phase 2, double-blind, placebo-controlled study to evaluate the prevention of attacks with bcx7353, a once daily oral kallikrein inhibitor, in patients with c1-inh-hae.method: patients with c1-inh-hae with at least 2 hae attacks per month were randomized to four different bcx7353 doses (350 mg, 250 mg, 125 mg, 62.5 mg) or placebo. subject-reported qol assessments were conducted at the start and end of treatment using the disease specific angioedema quality of life (ae-qol) questionnaire that measures 4 domains (function, fatigue, nutrition, fear/ shame) and has minimal clinically important difference (mcid) of 6 points. the changes from baseline in total and domain scores was compared between the treatment and placebo groups. modified angioedema activity score (aas) values across 4 domains (daily activities, appearance, physical discomfort, overall severity) were calculated for each attack and a total score was derived for each subject by summing scores from each attack. total scores were compared to placebo using an ancova model with adjustment for qualifying attack rate. reduction of attacks was statistically significant for all 3 top doses and there was a dose related increase in adverse events.results: in the 125 mg dose group, qol assessed by ae-qol was significantly improved after 4 weeks of treatment compared to placebo for ae-qol total score (-26.0, p < 0.001) as well as across all 4 domains (function: -28.2, p = 0.002; fatigue: -12.7, p = 0.05; fears/shame: -36.5, p < 0.001; nutrition: -23.4, p = 0.012). all other treatment groups showed a trend towards improvement. qol improved the most in the125 mg group, and 92% of subjects in the 125 mg group showed ae-qol reduction of more than 6 points. disease activity as assessed by the aas was significantly reduced in the 350 mg, 250 mg and 125 mg dose groups as compared to placebo, whereas there was no significant reduction in the 62.5 mg dose group. results: there were no marked differences in the age, sex, total ige titer, comorbidity of bronchial asthma and atopic dermatitis or the starting dose of rush oit among the groups. all patients in the low-bmfi group achieved ≥100% of the target dose, whereas 28.6% of the middle-bmfi group and 50% of the high-bmfi group failed to achieve the target dose (p < 0.05). results: a total of 473 infants were diagnosed with food allergy (ige-mediated and mixed type) at our center during the study period, 305 of these patients underwent prick test for inhalant sensitivity, and 220 (64.9% male) of these were younger than 2 years of age. conclusion: sensitivity to inhaled allergen were found in 14 of 292 (4.9%) patients with food allergy. therefore, we believe that inhalant sensitivity should be evaluated in these patients. there is also a requirement for further studies to identify the influence of inhaled antigens on the disease activity of patients with allergic conditions. method: in this study, we aimed to perform the metabolic profiling of severe profilin mediated food allergic patients looking for biomarkers that might both, predict the prognosis of the disease and understand the molecular mechanisms of inflammation underneath. other allergic patients (mild and moderate) and non-allergic were recruited in the study as comparative groups. the allergic patients class was predicted using a mathematical algorithm from non-allergic vs severe model results: plasma samples from non-allergic subjects, mild, moderate and severe allergic patients were measured using gas chromatography coupled to mass spectrometry (gc-ms). the samples were from 4 different hospitals in spain covering the areas with the highest pollen exposure. the metabolic profile was composed of 95 metabolites for each sample. results after the statistical analysis showed differences between the groups. firstly, a clear reduction of several abstracts conclusion: we found, as expected, a predominance of males with eoe diagnosis. ch were more frequently atopic and had aeroallergen and food sensitization. impaction and esophageal stenosis were more frequent in ad than ch. shist was associated with sclin only in ch. method: three children with ee, boys aged 1.5-11 years, were assessed over 6 months to 3 years. results: 2 cases developed in infancy. one had dyspepsia and low weight gain in infancy soon after feeding began. another had symptoms in infancy but not diagnosed until age 6 with dysphagia and esophageal stricture.. the third case was diagnosed at 11 years old after 2 episodes of food impaction in the esophagus beginning at age 9. all cases had allergic comorbid diseases including atopic dermatitis in all 3 and allergic rhinitis in 2. skin prick tests were positive to several food allergens (cow's milk, egg protein) in 2, to dust mite in 2 and to pollens in 1. serum total ige levels ranged from 490 to 1039 iu/ml. eosinophils in peripheral blood were elevated in all 3, reaching 10%-13%. treatment included restricted diet and topical budesonide 1-2 mg daily depending with periodic endoscopic biopsy.in all cases clinical improvement occurred by one month of treatment, with endoscopic confirmation. morphological improvement folthe study aimed to evaluate the effects of probiotic supplementation, in addition to diet, in ibs and snas patients, in terms of modulation of faecal microbiota population, reduction of gi and cutaneous symptoms, increase of patient's quality of life and modification of gut dysbiosis.method: forty patients aged between 18 and 65 years, affected by ibs, ni sensitization and ltp sensitization were enrolled to evaluate gut dysbiosis. dna extraction method (next generation sequencing) with commercial kit (microbiopassport ® ) was performed on stool samples. ibs patients were divided in two groups, according a gluten free diet prescription or a low fodmaps diet prescription for three months. similarly, (suspected) snas patients (confirmed by 5% ni sulfate in petrolatum patch test) were prescribed a low ni diet (100 μg/kg nickel content during the first four weeks and then up to 200 μg/kg up to three months). two ltp (lipid transfer protein) sensitized patients underwent a ltp free diet.gut dysbiosis was re-assessed after a fixed probiotic supplementation. a sex-age matched group of individuals without history of ibs, snas or any gastrointestinal disease was considered as control.gastrointestinal symptoms were evaluated using the visual analogue scale before and after treatment. conclusion: our preliminary findings suggest that probiotic implementation could be useful in patients with snas on a low-ni diet to increase population diversity, which could contribute to restore the intestinal homoeostatic conditions. key: cord-023157-0lqlx2rv authors: nan title: poster sessions date: 2013-04-18 journal: j diabetes doi: 10.1111/1753-0407.12032_1 sha: doc_id: 23157 cord_uid: 0lqlx2rv nan polycystic ovarian syndrome (pcos) is the most common endocrinopathy in women before menopause, the purpose of this study was to determine the effects of a diet and physical activity trial for improving pcos clinical and laboratory findings in obese women. this semi-experimental single-blind controlled trial was conducted among 50 women with pcos (25 cases and 25 controls). initially, all the patients′ information was evaluated in terms of: demographic, menstrual status, clinical characteristics of hyperandrogenemia, as well as biochemical, hormonal and abdominal ultra-sonography. the intervention plan consisted of a 12-week exercise and diet program for the experimental group. all baseline assessments were repeated in both groups within 3 months after beginning of the trial, and the results were analyzed. the comparison between the two groups revealed that in the experimental group, the interventions have been effective for the following variables: follicle stimulating hormone (p = 0.01), the luteinizing hormone (p = 0.01), total testosterone (p = 0.005), free testosterone (p = 0.004), estradiol (p = 0.03), selfglobulin band sex hormone (p = 0.001), triiodotyronine (p = 0.001), thyroxine (p = 0.001), tsh (p = 0.001), hydroxy progesterone (p = 0.03), triglycerides (p = 0.03), total cholesterol (p = 0.05), body mass index (bmi; p = 0.03), the ultrasound exam (p < 0.001), oligomenorrhea (p < 0.001), of hirsutism (p < 0.001), acne (p < 0.001) and alopecia (p = 0.01). it seems that regular exercise and dietary intervention are effective to improve pcos among obese women. a series of studies have recently demonstrated that the oxidative stress, nuclear factor-kappa b (nf-jb) activation and the subsequent coordinated inflammatory responses played an important role in the pathogenesis of urate nephropathy (un). polydatin has been suggested to have the properties of anti-oxidative, anti-inflammatory and nephroprotective effects. however, the possible protective and beneficial effects of polydatin on un are not fully elucidated. therefore, we investigated the potential beneficial effects and possible mechanisms of polydatin on un. in this study, polydatin showed inhibitory activities on xanthine oxidase to repress the level of serum uric acid in vivo and in vitro. further investigations revealed that polydatin displayed little toxic effects and significantly ameliorated the renal function in fructose-induced un mice. the nephroprotective activities of polydatin was not only due to the effects on remarkably attenuating the oxidative stress induced by uric acid, but also on markedly suppressing the oxidative stress-related inflammatory cascade, including decreasing the expressions of nf-jb p65, cox-2 and inos proteins and inhibiting the productions of tnf-a, pge 2 and il-1β. these findings elucidated that polydatin exhibited prominent nephroprotective activities and low toxic effects. drug discovery, obetherapy, evry, france many obesity related genes have been proposed as targets for the treatment of obesity. however, these obesity genes did not provide efficient drug therapy for obesity treatment. this is mainly due to the redundancy of the biochemical pathway involved in obesity and the lack of specificity of the gene targets. it is therefore a challenge to identify crucial gene(s) targets involved in energy metabolism associated with "lean or starvation phenotype". congenital enteropeptidase defficiency is an extremly rare pathology which answer to all these criteria. enteropeptidase catalyzes the conversion of inactive trypsinogen into active trypsin via the cleavage of the acidic propeptide from trypsinogen. we have generated knock out transgenic mice for enteropeptidase which shows the same phenotype like in human. these data and in vivo preclinical data using per os small molecule for long term treatment (9 weeks) will be presented. f. nasiri amiri 1 , f. ramezani tehrani 2 , m. simbar 2 1 babol medical sciences university, babol, 2 shahid beheshti university of medical sciences, tehran, iran polycystic ovary syndrome (pcos) is the most common chronic endocrine disorder with a prevalence estimated at 4-25% depending on the diagnostic criteria used. it has significant and diverse clinical consequence including reproductive, metabolic, psychological morbidity and some cancer. it is unclear how pcos symptom influences such women's experiences of their bodies. this study aimed to explain women's experiences of their health concerns when living with pcos. this research is a qualitative study. semi-structured opened interviews were conducted with 20 women aged 18-39 years who were diagnosed with pcos. interviewing the participants were continued to reach data saturation. all the interviews were recorded and transcribed. the data were analysed using content analysis. four closely intertwined themes were disclosed: physical consequences of pcos, fear of future, economic burden of desease and coming to terms with a chronic condition. our findings suggest that healthcare professionals working with pcos patients should consider providing peer support groups as a means to alleviate patients concern and to promote self management activities such as lifestyle modification. ideally, groups should be designed to meet patients support needs and expectations, and should be evaluated regularly. m. kidron 1 , e. arbit 2 1 oramed pharmaceuticals, jerusalem, israel, 2 biomedical engineering, nyu -oramed pharmaceuticals, englewood, nj, usa introduction: the major cause of hyperglycemia in t2dm is inappropriate, non-suppressible hepatic glucose production due to hepatic insulin resistance (hir)) and elevated glucagon levels. hir is a result of fat deposition in the liver which in turn begets a local inflammatory process, a common thread observed in t2dm, the metabolic syndrome and obesity which are all known risk factors for morbidity. the only antidiabetic drug that addresses specifically hir, up to now is metformin and its effects wanes over time. there is an unmet need for other drugs that target the liver specifically. insulin has a direct effect on hepatocytic receptors and thus has shown to be capable to reduce hepatic steatosis and improve glycemic control, as well as reduce hepatic insulin resistance. insulin administered orally is absorbed into the portal-hepatic vein and reaches the liver at high concentrations. this route of administration may confer physiologic advantages over systemic insulin administration and lower the risk of hypoglycemia. results: bmi, hdl and cu levels were found to be significantly higher in women compared to men (p < 0.001). _ it was established that blood copper levels, similar to hscrp, predicted ms and ms parameters, but predicted different ms parameters at different sensitivities and specifities. we obereved that there was a more significant correlation between tg level, blood pressure and cu, compared to hscrp (tables 1, 2 and 3). conclusion: based upon these results, it may be stated that cu predicts ms and ms parameters as well as hscrp does and that it is even a better predictor for ms and atherosclerosis since it is less influenced from inflammatory events than crp. short-chain fatty acids which then up regulate proglucagon (precursor to glp1) and pyy gene. these gut hormones are collectively known as "incretin" and they primarily regulate insulin level after eating. very recently, two new classes of drugs based on incretin action have been approved for lowering blood glucose levels in type 2 diabetes milliets. one is exenatide, long-acting agonist of the glp-1 receptor and other is incretin enhancer known as sitagliptin, a dpp4 inhibitor. however, glp-1 agonist reduces body weight (anti-obesity therapy) and leads to hypoglycemia. similarly, liraglutide, dpp-iv-resistant glp-1 receptor agonists is also synthesized using glp-1 sequence with prolongs half-life. in conclusion, polysaccharides might be a way to decrease the hurdle in diabetes managements and special attention should be paid to naturally derived polysaccharides. ms is often associated with inflammation caused by latent infections or sibo. we investigated whether treatments of such items may help to control ms. method: we prospectively studied 17 patients presenting with ms and three concomitant causes of inflammation [helicobacter pylori (hp = 17 cases); oral papillomavirus virus (hpv = 9) or ebv (5); sibo (h2 or ch4 breath tests: 17 cases)] who were all successfully treated for hp, hpv or ebv, and sibo (decrease of h2 or ch4 > 50% and decrease of circulating th17 cells >50% which are correlated with intestinal chronic inflammation). patients were followed-up 6 months. conclusion: treatment of latent infection and sibo benefits mainly to patients younger than 60, without methanogenic flora and with few circulating th17 cells. and increases the gastric emptying time, intestinal transit time, gastric acid secretion in the ms bark juice treated group. there was significant (p < 0.001) decrease in the t max , t 1/2 and increase in the c max of met in ms bark juice treated group as compare to diabetic group. present study suggests that the bark juice of ms shows significant effects in the treatment of gastroparesis and it improves the pharmacokinetic of met compared to diabetic group of rat. type 2 diabetes mellitus (t2dm) is a complex metabolic disorder. its prevalence is expected to increase exponentially around the world. insulin resistance, inflammation and dysregulation of adipokines play a major role in the pathogenesis of t2dm. among the huge growing adipose secretome, nicotinamide phosphoribosyl transferase (nampt) and vaspin emerged as novel interesting adipokines having insulinmimetic and -sensitizing effects, respectively. however, their role in t2dm is still controversial. accordingly, this study was designed to investigate their levels in t2dm patients compared to healthy control subjects, and to study the correlation between these two novel adipokines and the correlation between each of them with anthropometric parameters, insulin resistance, hyperglycemia, dyslipidemia, and also the inflammatory marker interleukin-6 (il-6). the levels of these two novel adipokines and other parameters were measured in non-obese and obese t2dm patients together with matched healthy non-diabetic control subjects. the nampt, vaspin and il-6 levels were measured by elisa while insulin levels by chemiluminescence technique. the nampt and vaspin levels were found to be significantly elevated in non-obese (25.9 ae 3.4 and 1.6 ae 0.2 ng/ml, respectively) and obese t2dm patients (45.4 ae 4.6 and 2.8 ae 0.4 ng/ml, respectively) compared to control subjects (9.4 ae 2.0 and 0.4 ae 0.05 ng/ml, respectively) at p < 0.01. furthermore, nampt as well as vaspin were found to be significantly correlated with one another and with various metabolic parameters. in conclusion, nampt and vaspin are potential candidates to play important role in the development and progression of t2dm. 2. to compare insulin resistance in sub-clinical and overt thyroid hypo-function. methods: one hundred eighteen patients with the diagnosis of hypothyroidism based on their clinical and thyroid function test profile were included in this cross sectional hospital based descriptive study with their informed consent. homa-ir as an index of insulin resistance was calculated for each subject from their fasting plasma glucose and serum insulin levels. autoimmunity against thyroid was evaluated by estimating anti tpo antibodies. results: homa-ir as an index of insulin resistance was comparable in overt (5.8 ae 3.24) and subclinical hypothyroidism (6.27 ae 3.87) but was above the reference range for this population. hypothyroid anti tpo positive cases has high tsh compared to negative cases in both overt hypothyroidism and subclinical hypothyroidism. conclusions: hypothyroidism induces insulin resistance but the degree of insulin resistance is not dependent on severity of thyroid hypofunction however is associated with autoimmunity against thyroid. .86 ae 0.35 in smoker and 3.95 ae 0.16 in non-smoker, insulin: 4.44 ae 0.95 and 2.0 ae 0.1 (p < 0.05), control: glucose level was 4.37 ae 0.14 in and 4.55 ae 0.16 mmole/l, insulin level: 3.1 ae 0.64 and 3.7 ae 0.65 mkiu/ ml, respectively. both growth in height and weight gain are accelerated during puberty. they are mainly affected by sex hormone, growth hormone and igf-1, and influenced by various factors either directly or indirectly. the aim of this study is to find out the association of various cytokines with obesity and early-or precocious puberty in female children. twenty-eight female children with breast budding before 9 years old, who underwent the lhrh stimulation test as well as cytokine analysis in their blood, were included in this study. the height, weight, and bmi were measured. we defined obesity when the bmi was 95 percentile or more, and puberty when the maximum lh level was 5 iu/l or more during lhrh stimulation test. adiponectin, leptin, ghrelin, il-1β, il-6, il-10, resistin, and tnfa levels in the blood were analyzed by luminex multiple bead technology (milliplex; millipore co., billerica, ma, bio-plex; bio-rad laboratories, hercules, ca). nineteen out of 28 children were categorized as having early-or precocious puberty. their mean il-6 level was lower in pubertal children than that in prepubertal state (4.48 ae 4.77 vs. 19 .56 ae 20.26, p = 0.057). the leptin and resistin levels were significantly higher in obesity group (n = 6) than in non-obesity group, while the ghrelin was significantly lower in obesity group (p < 0.05). in conclusion, the female children younger than 9 years of age in early-or precocious puberty did not show the increment of leptin or resistin comparing with the female prepubertal children, although the obesity group showed significantly higher levels of leptin and resistin. aims: arterial stiffness is independent risk factor of cardiovascular events. suggest that the statins benefit associated with improvement in arterial stiffness parameters beyond lipidslowering effects. to evaluate changes in pulse-wave shape in obese high risk patients with ah and dyslipidemia treated with rosuvastatin compared with atorvastatin. methods: eighty-two obese patients (age 61.5 ae 10 years) with ah, dyslipidemia were randomized to atorvastatin group (n = 41) or rosuvastatin (n = 41). acei and thiazide diuretics added blood pressure control. pulse-wave characteristics measured before and after 5 weeks of treatment using finger photoplethysmographic device. stiffness index (si), reflection index (ri), augmentation index (aix), systolic bp in aorta (spa), digital pulse amplitude augmentation (paa) were accessed. results: before the treatment impared si, elevated ri, aix, spa were shown. lipids and bp goals were achieved in all patients validating further analysis. decrease in si (d si, м/c à0.87 atorva and à0.89 rosuva), ri (dri, % à7.89 atorva and à7.21 rosuva) were revealed in both treatment arms (p > 0.05), whereas significant trends towards aix decrease were demonstrated only in rosuvastatin-treated patients (dai, % à1.88 atorva and à1.92 rosuva, p < 0.05) rosuvastatin group demonstrated better increasing in paa than atorvastatin group (paa (atorva): before treatment 1.58 ae 0.42 and after 5 weeks: 1.82 ae 0.51, p = 0.06 vs. paa (rosuva): before 1.68 ae 0.22 and after 5 weeks 1.95 ae 0.23, p < 0.05). conclusions: pulse-wave analysis in obese ah patients demonstrated increasing vascular stiffness. both atorvastatin and rosuvastatin treatment resulted in arterial stiffness parameters, whereas only rosuvastatin treatment was significantly associated with trends in aix and paa improvement in short-term follow-up. z. wang 1 , m. xu 2 1 methods: four thousand two hundred and twenty-six adults above 60 years of age and 3145 adults under 60 years from a cohort investigated in 2010-11 at the medical examination center of zhongnan hospital were recuited. cases of fld was identified through ultrasound imaging. the risk factors measured were bmi, and plasma concentrations of alt, ast, tc, tg, hdl, ldl and serum uric acid (sua). the probability of steatohepatitis with advanced fibrosis was calculated according to the body mass index, age, alt, and triglyceride (baat) and ast/alt ratio (aar). results: the prevalence of fld was higher in elderly (26.7%) than in non-elderly (22.8%) and similar in elderly between men and women (26.6% vs. 27.0%, p > 0.05). multiple regression analyses showed that obesity, high tc, tg, sua, low hdl, and elevated alt, aar < 1 were closely related to the elderly fld the prevalence of steatohepatitis estimated as baat index ! 3 was 2.4% in all subjects, and was higher elderly fld patients than in the non-elderly fld patients. conclusion: the prevalence of fld is higher in the elderly, and is broadly related to the same metabolic risk factors as in the nonelderly. however, female-sex is no longer protective with increasing age, and the prevalence of steatohepatitis with advanced fibrosis is estimated to be considerably higher in the elderly fld patients than in the non-elderly fld controls. vanderbilt, nashville, tn, usa bariatric surgery improves glucose tolerance and may be a viable strategy to prevent the progression from obesity-induced insulin resistance to overt diabetes; however, the recurrence of diabetes is significant following the surgery. we sought to determine whether gastric bypass surgery protected pancreatic beta cells and prevented disease progression to overt diabetes. gastric bypass (roux-en-y gastric bypass, rygb) was performed in young (6 weeks old) prediabetic blks db/db null (bks-db) mice, young (6 weeks old) new zealand obese (nzo) male mice that were fed high-fat diet (hfd) post-surgery, c57bl/6 db/db null (b6-db) mice that developed insulin resistance, and streptozotocin (stz)-induced diabetic mice. rygb resulted in sustained normoglycemia and improved glucose tolerance in pre-diabetic bks-db mice and hfd-fed nzo mice. remarkably, rygb preserved beta-cell mass and increased plasma insulin with reduced beta-cell apoptosis which was independent of weight loss and body fat reduction. rygb neither reversed hyperglycemia when performed in diabetic bks-db and nzo mice nor resulted in resolution of diabetes in stz-induced diabetic mice. the results demonstrate that gastric bypass prevents beta-cell failure if performed prior to onset of severe beta-cell damage in genetic obese mice. l. ruzic 1 , g. sporis 2 , m. prasek 3 1 sport and exercise medicine, 2 applied kinesiology, faculty of kinesiology, university of zagreb, 3 vuk vrhovac university clinic, zagreb, croatia the aim of the study was to examine the influence of strength training program on pre-and post workout glycemia. twelve able-bodied diabetic patients using insulin pump (mean age 31.4 ae 4.44, 3m/9f) were enrolled into gym programs 39/week (15 min warm-up aerobic workout, intensity at 70% hrmax plus 8 strength training exercises involving different muscle groups) for 3 months. no other interventions were introduced. we were interested in effects of the programe on pre-and post workout glycemia, so the comparisons between the first 2 weeks and the last 2 weeks of the study were performed. also the subjects were asked about hypoglycemic episodes. there was a great variability observed in pre-and post glucose concentration within and between subjects. the mean pre-workout glucose concentration in the first 2 weeks of the program was 7.98 ae 3.91 vs. 7.57 ae 3.08 mmol/l in last 2 weeks of the program and the difference was not significant (p = 0.523). nevertheless, the mean glucose decrease after workout was higher in the last 2 weeks of the program (deltaglu: 2.52 ae 0.73 vs. 3.62 ae 1.38 mmol/l; p < 0.05). no hypoglycemic episodes were reported. the 3 months strength training program did not influence large variability in glucose levels before the workout as it depends on many other factors. the only observed effect were larger pre-to post workout glucose differences. it seems that over the time, the strength training may stimulate the body to use more glucose during the workout, which might be explainable with the expected increase of lean body mass. aim: the aim of this study was to evaluate the level of c-reactive protein in gestational diabetes mellitus. materials and methods: sixty-five healthy pregnant women aged 29.75 ae 5.59 years between the 20th and 25th weeks of gestation were studied. all women referred for a 75-g oral glucose tolerance test following an abnormal result on a screening. the demographic data, waist circumference, height, and weight of the participants were recorded. fasting levels of insulin, triglycerides (tg), c-reactive protein (crp), fasting blood glucose (fbg) and hba1c were measured. results: based on oral glucose tolerance testing participants were divided into two groups: normal glucose tolerance (ngt; n = 17) and gestational diabetes mellitus (gdm; n = 48). the mean crp level was highest in gdm group (9.67 ae 5.57 mg/l), followed by ngt (2.56 ae 1.73 mg/l), (p < 0.0001). the mean fbg (4.98 ae 0.51 vs. 4.48 ae 0.58 mmol/l, p < 0.05), homa-ir (3.14 ae 1.63 vs. 1.83 ae 0.62, p < 0.05) and tg levels (1.95 ae 0.65 vs. 1.35 ae 0.58 mmol/l, p < 0.05) in the women with gdm were significantly higher than those in the ngt group. methods: in 2010, an observational, prospective study started in france on request of the health authorities (has). one thousand seven hundred and two type 2 diabetic patients treated by vildagliptin were recruited through a national representative sample of gps and diabetologists. we report the data of interim analyses after 18 months of follow-up. results: one thousand four hundred and sixty-three patients are included in this interim analysis: 60% males, aged 63 ae 11 years, with mean bmi of 30 kg/m 2 . hba1c was equal to 7.8 ae 1.2% at vildagliptin initiation, then 7.1 ae 1.0%, 7.0 ae 1.1% and 7.0 ae 0.9%, while mean weight decreased from 86 to 86, 85 and 84 kg at 6, 12 and 18 months respectively. vildagliptin, rarely prescribed when not recommended, was well tolerated: asat and/or alat were >120ui in 0.5% at initiation of vildagliptin then 0.2% at 18 months, with a slight decrease for mean asat and alat. mean gfr was 82 ml/mn at initiation of vildagliptin then 83 ml/mn at 18 months, with a stable percentage of gfr < 60 ml/mn (14%) and <30 ml/mn (0.3%). six severe hypoglycemic episodes occurred in six patients (incidence = 0.30/100 patient-years), all treated by insulin and/or sulfonylurea in addition to vildagliptin. the proportion of patients still treated by vildagliptin at 18 months was 89.3%. conclusions: over 18 months, in real-life conditions of care, vildagliptin showed a sustained effectiveness in terms of hba1c reduction, a good tolerance, very few severe hypoglycemic episodes, rare treatment discontinuations and was most often prescribed as recommended. background: hdl lipoproteins are known to play a causative role in atherosclerosis and its clinical manifestation-coronary artery disease (cad). carotid intima media thickness (imt) is considered as a marker of atherosclerosis and in prediction clinical coronary events. aim: to determine the associations between plasma lipids and subclinical atherosclerosis measured by the common carotid intimamedia thickness (imt) in cad patients. methods: hdl subclasses were separated with 3-31% pag electrophoresis, and imt was determined using high-resolution b-mode ultrasound in 92 cad patients, with normal levels of traditional lipid risk factors. results: mean value of left and right carotid artery measurement was selected as value for correlation with hdl subclass size in each patient. the mean hdl size was 9.34 ae 0.74 nm, and the mean imt in all patients was 0.89 ae 0.13 mm (0.6-1.2 mm). hdl size was not correlated with imt (r = à0.03; p > 0.05). by univariate analyses, carotid imt was the most closely related to systolic pressure (r = 0.510, p < 0.001), followed by diastolic pressure (r = 0.480, p < 0.001) and age (r = 0.25, p < 0.05). stepwise multiple linear regression analysis revealed that diastolic pressure (β = 0.322, p < 0.016), systolic pressure (β = 0.237, p < 0.067) and age (β = 0.192, p < 0.037) were independent predictors of determining carotid imt (adjusted r 2 < 0.290, p < 0.001). conclusion: these results indicate that diastolic pressure, systolic pressure and age are an important, independent determinants of carotid imt in cad patients. no other traditional risk factors imparted imt. objective: antinuclear antibodies (ana) are present in approximately 20-40% of patients with non-alcoholic steatohepatitis (nash). a recent study implied the relationship between obesity and autoimmunity. the purpose of this study was to investigate the relationship between seropositivity for ana and metabolic abnormalities including insulin resistance, obesity and hepatic steatosis in patients with nash. methods: the severity of hepatic steatosis and fibrosis was scored by the nafld activity score system. seropositivity for ana was defined as titers of 1:40 or higher by an indirect immunofluorescence method using hep-2 cells. insulin resistance and obesity were evaluated by the value of homa-ir and bmi, respectively. the diagnosis of autoimmune hepatitis (aih) was based on the simplified scoring system. results: nine (45%) of 20 patients with nash had ana. overall bmi in nash patients with ana was higher than that in those without ana. laboratory analyses revealed significantly higher mean igg level (1904 ae 440 vs. 1358 ae 236 mg/dl, p = 0.0056) and the mean value of homa-ir (7.43 ae 3.33 vs. 4.15 ae 2.79, p = 0.0348) in nash patients with ana than those in those without ana. histological examinations exhibited that nash patients with ana had higher scores in hepatic steatosis (2.44 ae 0.73, vs. 1.91 ae 0.83, p = 0.1448) and fibrosis (2.33 ae 1.00 vs. 1.64 ae 1.12, p = 0.1535) than those without ana. however, none of nash patients fulfilled the criteria for "definite" aih. conclusion: nash patients with ana had clinical characteristics of significantly higher serum igg levels and severe insulin resistance, and they tended to have more severe obesity, hepatic steatosis and fibrosis than nash patients without ana. r.f. alponti 1,2 , p.f. silveira 1 1 pharmacology, instituto butantan, 2 physiology, instituto de biociencias/universidade de sao paulo, sao paulo, brazil introduction: although irap (ec3.4.11.3) is well-known, the existence of other aminopeptidases (aps) related to energy homeostasis remains unclear. objectives: to search a diverse array of aps in high (hdm) and low (ldm) density microsomes and in plasma membrane (mf) of retroperitoneal adipocytes from healthy control (c), monosodium glutamate (msg) obese and food deprived (fd) rats with their in vitro responses to insulin (is), vasopressin (avp), angiotensin (ang)-ii and ang-iv. methods: ultracentrifugation and spectrofluorometry. results: dipeptidyl-peptidase-iv (dppiv) and aps basic (apb), neutral puromycin-sensitive (psa) and -insensitive (apm), and methionyl (metap) were found. compared with non-stimulated, these aps were unaffected by is; avp increased apb/apm (ldm) and dppiv/psa (hdm) of msg-fd and metap/psa (hdm) of c; ang-ii increased apm (fm-ldm) of c, dppiv (fm) of msg and psa (fm) of fd; ang-iv increased apb (fm) of fd, apm (hdm) of msg and dppiv (fm) of c and msg. aps were modulated by avp in hdm-ldm, by ang-ii in fm-ldm and by ang-iv in fm-hdm. compared with ldm, metap decreased in fm of c and increased in fm of msg-fd without peptide stimuli; and only diabetes-related enzyme dppiv exhibited peptide-mediated intracellular translocation, which was from ldm to hdm (stimulated by avp) in msg-fd, and from ldm and hdm to fm in msg and msg-fd (stimulated by ang-ii) and in c and msg (stimulated by ang-iv). conclusion: novel peptide-modulated apb, apm, psa, metap and dppiv are found in adipocyte, this last with altered subcellular trafficking under metabolic distress. supported by fapesp, cnpq and capes. nutritional and environmental sciences, university of shizuoka, shizuoka city, japan previous studies have suggested that (-)-epigallocatechin-3-gallate (egcg) exerts antioxidative and anti-inflammatory actions in various tissues, which might be beneficial for reducing risks of development of diabetes. however, an optimum intake level of egcg is unknown. in this study, we have examined the effect of a diet containing egcg on the expression of inflammation-related genes in various tissues including visceral adipose tissue and the muscle of non-obese type-2 diabetes animal model, goto-kakizaki (gk) rats. gk rats at 9 weeks of age were fed a control high-fat diet (45 energy% as fat) or the high-fat diet containing 0.1%, 0.2% or 0.5% egcg for 25 weeks. the mrna and protein levels of il-1β, il-18, mcp-1, cd11s, il-6, tnf-a, resistin and pai-1 were significantly reduced in the adipose tissue of rats fed a diet containing 0.1% egcg, but not in those fed diets containing 0.2% or more egcg, as compared with control. the mrna levels of tnf-a, ifn-g, il-1b, il-6 and il-18 in the muscle of rats fed a diet containing 0.1% egcg were also significantly lower than those in the control. these results suggest that there is an optimum range of intake of egcg, which may suppress the expression of genes involved in inflammation in the adipose tissue and the muscle. diabetes and hypertension are the most relevant factors leading to vascular disease and cardiovascular problems. since both pathologies are greatly increasing nowadays, there is a need to detect at early stages the occurrence of target organ damage associated with them. this is the relevance of identifying biomarkers that can detect or predict the onset of cardiovascular and renal damage associated with diabetes and hypertension. given that the role of osteoprotegerin in bone metabolism is well known, and some evidence of its putative relationship with diabetes-associated pathologies has been found, to this date there is no evidence linking this molecule with target organ damage associated with diabetes. in this study, we analyze whether osteoprotegerin may be used to detect and evaluate cardiovascular and renal pathologies associated with diabetes in an in vivo model. we used normotensive and hypertensive rats, a subset of rats of each group receiving a single streptozotocin injection in order to induce diabetes. we performed an 8-month followup, periodically collecting blood and urine samples and monitoring both blood pressure and blood flow in the lower limbs. our results showed that osteoprotegerin was associated with the presence of diabetes, suggesting that it might be used as a biomarker for the occurrence of cardiovascular damage or to detect cardiovascular risk under these circumstances. blood flow in the lower limbs decreased soon after diabetes onset, as osteoprotegerin levels increased. our data suggest the potential use of serum levels of osteoprotegerin as a biomarker for diabetes and hypertension-associated endothelial dysfunction. purpose: this study examined whether breakfast meal composition alters the incretin response and glycemic control following both the breakfast and lunch meal. methods: seven subjects with t2d completed two conditions where they consumed either a 500 kcal high protein (pro: 40% carbohydrate, 40% protein, 20% fat) or high carbohydrate (cho: 65% cho, 15% protein, 20% fat) breakfast for 6 days of acclimatization. on day 7, they underwent meal testing in which they consumed the respective breakfast followed by a lunch meal (500 kcal: 65% cho, 15% pro, 20% fat). blood samples were collected over the 8-h period and analyzed for glucagon, insulin, glucagon-like peptide-1 (glp-1) and glucose-dependent insulinotropic polypeptide (gip) concentrations. incremental area under the curve (iauc) for the 4-h post-breakfast and 4-h post-lunch period was calculated. results: the iauc for insulin, glucose and glp-1 were not significantly different between conditions or between meals. the gip response to the pro breakfast (5485 ae 1369 pg/ml*min for 4-h) was lower (p = 0.049) compared to the cho breakfast (7756 ae 759 pg/ ml*min for 4-h), with the opposite effect occurring in response to the lunch meal (pro: lunch 9226 ae 2726; cho: lunch 8174 ae 1105 pg/ ml*min for 4-h, p < 0.049). conclusion: despite no differences in glucose and insulin levels, a pro breakfast, compared to a cho breakfast, resulted in lower gip levels for the initial meal with a greater second meal effect after a lunch meal. however, a pro breakfast potentiates gip levels after a lunch meal. further research is needed to determine the physiological role of changes in circulating gip. high risk pregnancy, kasralainy university, cairo, egypt background: pregnancy tends to reset the glucose homeostasis in the direction of diabetes. about 1-2% of all pregnant women develop an abnormal glucose tolerance in pregnancy, but most often glucose tolerance returns to normal postpartum. this condition is called gestational diabetes mellitus (gdm). aims: comparative study between gestational and pregestational diabetes in relation to glycemic control as regarding fetal and neonatal outcome. methods: this study was conducted in kasralainy maternity hospital from september 2011 to march 2012 and it included 60 pregnant women complicated by dm attending outpatient clinic or inpatient. patients were classified into two groups, gestational diabetes: 30 pregnant women complicated by dm which is diagnosed for the first time during pregnancy and pregestational diabetes: 30 pregnant women who have dm that has been diagnosed prior to pregnancy. the two groups were compared according to fetal (macrosomia and intrauterine fetal death) and neonatal (respiratory distress syndrome and birth injuries) complications. all patients were 18-40 years old, singltone pregnancy, with time of termination after completed 37 weeks. results: fetal macrosomia occurred more with gdm, on the otherhand birth injuries and rds occurred more with pre-gdm. macrosomia and rds were commoner among poor glycemic control in pregnant diabetic females than birth injuries and iufd. conclusions: glycemic control started as early as possible (the best being preconceptional) is important to decrease the incidence of birth injuries, macrosomia, fetal mortality, the need for nicu admission (rds). adiponectin, an adipocyte-derived hormone, is implicated in type 2 diabetes and atherosclerosis. this study was designed to investigate whether serum adiponectin levels in coronary artery disease (cad) patients with type 2 diabetes (t2dm) are lower than in patients with cad alone and healthy controls. we measured serum adiponctin levels in 198 subjects, 138 patients with cad (72 subjects of whom had both cad and t2dm), and also 60 healthy subjects were selected as controls. all patients were subjected to anthropometric indexes assessment and biochemical measurement of serum adiponectin, interleukin six (il-6), insulin, lipid profile and glucose by standard methods. the results revealed significant differences in serum adiponectin levels between cad patients with t2dm and cad patients without t2dm (3.80 ae 1.52 vs. 5.25 ae 2.35 lg/ml, p = 0.001), between patients with cad and healthy controls (4.50 ae 2.54 vs. 7.04 ae 3.32 lg/ml, p = 0.000), and between men and women (4.62 ae 2.81 vs. 5.97 ae 3.15, p = 0.002). serum adiponectin levels were correlated significantly with insulin, total cholesterol, low density lipoprotein, body mass index, glucose, homa-ir, il-6 (r = à0.178, p = 0.013, r = à0.313, p = 0.000; r = à0.154, p = 0.016; r = à0.171, p = 0.016; r = à0.202, p = 0.006; r = à0.251, p = 0.001; r = à0.217, p = 0.042, respectively). adiponectin was correlated positively only with high density lipoprotein (r = 0.422, p = 0.000). conclusions: we conclude that low serum adiponectin levels and insulin resistance coexist in cad patients with t2dm. it is speculated that subjects who have very low levels of serum adiponectin may be at increased risk of developing both t2dm and cad. objective: to simultaneously examine the impact of self-reported and newly-identified hypertension upon subsequent type 2 diabetes (t2d). methods: two community-based adult prospective cohort studies, with the same protocols, instruments and questionnaires, were conducted during 2004-2007 and 2007-2010 in urban areas of nanjing, china. data from these two cohorts were pooled and analyzed. t2d (outcome variable) was identified using the 1999 world health organization diagnosis criteria. based on status of blood pressure (independent variable), participants were categorized into three groups: (i) people with normal blood pressure at baseline survey and during follow-up period (normal blood pressure), (ii) subjects with self-reported hypertension at baseline survey (self-reported hypertension) and (iii) those who did not report hypertension at baseline but were diagnosed having hypertension during follow-up period (newly-identified hypertension). all covariates were assessed at baseline and in the third-year follow-up survey. results: among 4550 participants, the 3-year cumulative incidence of t2d was 14.1%, 6.5% and 3.2% for participants with newly-identified hypertension, self-reported hypertension and normal blood pressure, respectively. after adjustment for potential confounders, compared to people with normal blood pressure, participants with newly-identified (or = 4.25; 95%ci = 3.04, 5.93) or self-reported (or = 1.76; 95% ci = 1.25, 2.48) hypertension were more likely to develop t2d. furthermore, subjects with newly-identified hypertension (or = 2.53; 95% ci = 1.71, 3.73) were at elevated risk of developing t2d relative to their counterparts with self-reported hypertension. the similar associations of hypertension with t2d were also found in both men and women, separately. hypertension, either self-reported or newly-identified, is an independent risk factor for developing t2d among urban chinese adults. methods: a total of 60 patients with type 2 diabetes receiving standard glucose-lowering therapy were enrolled. efficacy of the lowcalorie diet (1500 kcal/day) with inclusion of specialized nutrison advanced diason product was assessed in two similar groups in during 2 weeks: main group patients (n = 30) received diet with nutrison advanced diason in the amount of 1000 ml as the only food for the day twice a week for 2 weeks; control group patients (n = 30) received a low-calorie diet with the inclusion of a standard meal at 1000 calories twice a week. results: the average weight loss in the main group was 800 ae 70 g, in the control group -500 ae 40 g per day. according bioimpedance complex therapy with specialized product increased the content of lean mass by an average of 1.1 ae 0.4 kg and decreased fat mass by an average of 4.4 ae 0.3 kg. in main droup was a reduction of the basal level of glucose to 8.0 ae 0.7-6.0 ae 1.0 mmol/l (p = 0.01), in control groupfrom 8.0 ae 0.8 to 6.9 ae 0.9 mmol l (p = 0.01). total cholesterol level in the main group decreased from 6.8 ae 1.0 to 5.9 ae 0.7 mmol/l (p = 0.01), in the control groupfrom 6.8 ae 0.8 to 6.3 ae 0.7 mmol/l. the inclusion of the specialized product nutrison advanced diason in the standard low-calorie diet allows to raise the efficacy of dietary therapy in patients with type 2 diabetes. liver injury and regeneration involve complicated processes and are affected by various physio-pathological factors. this study was designed to investigate the mechanisms of steatosis-associated liver injury and impaired regeneration in a mouse partial hepatectomy model. male c57bl/6j and db/db mice were used as mice with normal and steatotic liver, respectively. liver regeneration and injury were evaluated chronologically after hepatectomy. initial regeneration of the steatotic liver was markedly impaired after hepatectomy. although hepatocyte proliferation was not significantly suppressed, intense liver injury with oxidative stress occurred immediately. fasl/fas expression was up-regulated in the steatotic liver, whereas the expression of anti-oxidative and anti-apoptotic molecules (catalase/mn-sod/ref-1 and bcl-2/bcl-xl/flip, respectively) and p62/sqstm1, a steatosis-associated protein, were down-regulated. interestingly pro-survival akt was not activated/phosphorylated in response to hepatectomy though it was sufficiently expressed/ phosphorylated even before hepatectomy. suppression of p62/sqstm1 increased fasl/fas-expression and reduced nrf-2-dependent are activity and anti-oxidative responses in steatotic and non-steatotic hepatocytes. exogenously added fasl induced intense cellular oxidative stress and necrosis/apoptosis in steatotic hepatocytes, only the necrosis being inhibited by pretreatment with anti-oxidants, suggesting that fasl/fas-induced oxidative stress mainly leads to necrosis. furthermore, p62/sqstm1 re-expression in the steatotic liver markedly reduced liver injury and improved tissue regeneration. in the steatotic liver, reduced expression of p62/sqstm1 induced fasl/fas expression and suppressed anti-oxidant genes through nrf-2 inactivation, which together with hypo-responsiveness of akt, caused post-hepatectomy necrotic and apoptotic liver injury in redoxdependent and -independent manners, respectively. p62/sqstm1 may be a key molecule in post-hepatectomy acute liver injury and impaired regeneration in fatty liver in mice. objective: mastication can accelerate satiety sensation and lipolysis through activation of histamine neurons; however, the data on antiobesity effects of mastication are limited. we therefore examined the effect of chewing on postprandial satiety and energy metabolism in humans. methods: satiety, energy expenditure, and fat oxidation were measured in 10 lean young women on separate occasions, before and 3 h after consumption of a solid meal with 30 chewing per bite or after swallowing the same mashed meal without chewing, in a randomized, crossover design. each test meal consisting of 2510 kj (60% of energy as carbohydrate, 25% of energy as protein, and 15% of energy as fat) was consumed between 20 and 25 min regardless of texture difference. the thermic effect was greater after the solid meal (5.4 ae 0.6%) than after the mashed meal (2.7 ae 0.6%, p < 0.001). time course of fat oxidation tended to be higher after the solid meal than after the mashed meal (p = 0.09). in addition, solid meal was more satiating than mashed meal (p = 0.005). the results suggest that mastication contributes to postprandial satiety and thermic effect of meal. chewing meal thoroughly could be a useful eating behavior for preventing weight gain. introduction: raven noted an association between insulin resistance and cardiovascular consequences and type 2 dm. mathew prescribed the method of insulin resistance designation using mathematic model homa-ir. aim: to estimate insulin resistance homa-ir in adult population according to gender, assess selected parameters in people with homa-ir ! 2.5 (i group) and homa-ir < 2.5 (ii group) and determine dependence between homa-ir and the above parameters. materials and methods: one hundred and fifty-four people medium age 52.5 years, from tarnawa city constituted the material. bp was examined. blood samples were obtained for plasma glycaemia, lipidogram and serum insulinaemia. all people were measured and weighed, bmi and homa ir were calculated, waist circumference was obtained. the cut-off point 2.5 for homa-ir was accepted. results: 24.7% of the examined population had insulin resistance without a difference between women and men. significant differences between i and ii group were observed according to tg and waist circumference. in women all examined parameters were significantly different except hdl. in men bmi, waist circumference and hdl were significantly different in i and ii group. in the female group correlations between homa ir concerned all examined parameters, in the male group-hdl and waist circumference. conclusions: there was no significant difference between women and men according to insulin resistance in the whole study group. the difference between selected parameters in the two examinated groups were more strongly in women. body mass was the main factor determining insulin resistance in the whole study group as well as in men and women. background: the immune system is altered in obesity and diabetes, through changes in adipocytes, liver, pancreatic islets, vasculature and circulating leukocytes, with increased cytokine and leukocytes activation, suggesting inflammation participation in diabetes. minocycline presents a potent anti-inflammatory activity, as evaluated in vivo and in vitro. objectives: minocycline anti-diabetic effects were assessed in alloxaninduced diabetes through biochemical parameters measurements, histological and immunohistochemistry analyses. methods: alloxan was injected to rats, blood collected 48 h later and after oral treatments (5, 10 and 30 days) for glycemia, triglycerides, cholesterol and transaminases measurements. diabetic controls and diabetic plus minocycline (25 and 50 mg/kg) or glibenclamide (5 mg/ kg) were used. furthermore, pancreas, liver and kidney were submitted to histological and immunohistochemistry (cox-2 and tnf-a) analyses. results: decreases in glycemic and triglyceride levels, at the 5th and mainly 30th days after minocycline treatments, were observed. he staining showed that minocycline partly reversed tissue alterations, and decreased expressions of cox-2 and tnf-a, as compared to untreated animals. conclusions: beneficial minocycline effects in diabetes could be due to its anti-inflammatory and antioxidant properties and, by inhibiting microglial activation, it may be an important therapeutic strategy in diabetes where inflammation plays a significant role. methodology: forty-eight hours after alloxan-induced diabetes, blood from male wistar rats was collected for biochemical measurements. then, diabetic rats untreated or treated (1 week, 1 or 3 months) with pentoxifylline (5, 25, 50 and 100 mg/kg, p.o.) or glibenclamide (2 or 5 mg/kg, p.o., alone or associated to pentoxifylline) were divided into eight groups with 10-20 animals each. after treatments, biochemical measurements were repeated. glycated hemoglobin determinations, and histological and immunohistochemistry analyses for inos were also performed. results: pentoxifylline brought hyperglycemia and triglycerides towards normality. glycated hemoglobin was improved. the use of diazoxide showed the mechanism of action of pentoxifylline partly related to atp-dependent k + channels. pentoxifylline improved histological alterations in pancreas, liver and kidney, and decreased inos cell expression. conclusions: pentoxifylline effects are probably related to its action on oxidative stress and inflammation, decreasing pro-inflammatory cytokines. thus, pentoxifylline is a potential candidate for diabetes mellitus therapy, since patients with vascular complications present beneficial effects, as shown in clinical practice. recently, a close relationship between the development of adiposity and gut-derived hormonal dysregulation has been clearly established. for instance, studies of gut-derived peptides such as pyy 3-36 , glp-1, oxyntomodulin, discovered more than 20 years ago and, later on ghrelin have significantly improved our understanding of mechanisms underlying ectopic lipid infiltration in organs and tissues. the etiology of non-alcoholic fatty liver disease (nafld) is intimately related to the capacity of hepatocytes to acquire an "adipocyte-like" phenotype. we previously reported that unacylated ghrelin (uag) is more potent than acylated ghrelin (ag) to stimulate adipogenesis. the present study intends to investigate the relevance of uag and ag as mediators of lipid accumulation in hepatocytes. hepg2 hepatocytes were treated with a control, uag or ag (1 nmol/l and 1 pmol/l) in the presence or absence of oleate (50 nmol/l) to measure lipid droplet (ld) number and size. gene expression analyses were performed for key mediators of pre-adipocyte differentiation or liver functions. in response to uag or ag treatment in the presence or absence of oleate, ld number was markedly increased in hepg2 cells. similarly, increased ld size was noted following the treatment of hepg2 cells with uag or ag. however this effect could not be detected in the presence of oleate. gene expression of ppar-c and c/ebp-a was increased while that of ppar-a was decreased in response to ag treatment. these results are first to describe mechanisms through which uag and ag could promote the development of lipid infiltration in liver. deakin university, melbourne, vic., australia the world health organization believes that type 2 diabetes mellitus is an important public health problem in the world. however some statistics showed that only 2.8% of all world population had diabetes in 2000, but the health professionals argue, this amount will reach to 4% in 2014. diabetes australia -nsw estimates that almost 1 million australians have diabetes and also around 2.1 million australian populations are at risk of this disease and unfortunately, the number of australian with diabetes will be approximately 4.5 millions in 2014. obviously, direct and indirect medical costs due to health care of diabetic's patients are very high and 2% of the australian government's health budget is spent on health care of type 2 diabetes. therefore, the health professionals have tried to provide the optimal public health programs for control and management of type 2 diabetes. many researchers and health professionals argue that control of weight by change diet habits and exercise are major keys in lifestyle programs for prevention of type 2 diabetes. in this case, the results of several studies like the finnish study, american study, the swedish malmo study and chinese study showed that the focus on diet and exercise programs are significant approaches in type 2 diabetes prevention, while some other studies claim that the change diet habits and exercise may not sufficient and some medicines for control of insulin sensitivity and energy expenditure may also be necessary. therefore, lifestyle intervention programs and pharmacologic intervention programs are the major prevention programs. objectives: metabolic responses to acute endurance exercise may be affected by time of day because the nervous and endocrine systems have circadian rhythms clearly. the purpose of this study was to investigate the influence of acute endurance exercise in the morning and evening on metabolic responses in young men. methods: ten healthy young men completed two trials in a randomized cross-over design: 1. morning (09:00-10:00) and 2. evening (17:00-18:00) trials. in the morning and evening trials, participants walked for 60 min at 60% of maximal oxygen uptake on a treadmill. pulmonary gas exchange was determined breath-by-breath by a gas analyzer. blood samples were collected to determine hormones and metabolites at preexercise, immediately and 2 h after exercise. results: plasma interleukin-6 and adrenaline concentrations were significantly higher immediately after exercise in the evening trial than in the morning trial (p < 0.01 and p < 0.05, respectively). serum growth hormone concentrations were significantly higher immediately after exercise in the evening trial than in the morning trial (p < 0.01). serum free fatty acids concentrations were significantly higher in the evening trial than in the morning trial at 2 h after exercise (p < 0.01). however, there was no significant difference in fat oxidation between the morning and evening trials. conclusion: these findings suggest that acute endurance exercise in the evening is more effective on lipolysis compared to that in the morning in young men. nutrition, school of public health, university of sao paulo, sao paulo, brazil background: the rs9939609 snp of fto (t>a) has been associated with obesity and its comorbidities. in a allele carriers, physical activity (pa) minimizes the deleterious impact on body weight, which could reduce cardiovascular risk. objectives: to investigate whether pa level and television viewing modulate the effects of a allele of fto on adiposity and markers of inflammation in individuals at high cardiometabolic risk. methods: this cross-sectional study included 158 prediabetic individuals (103 women, 54.7 ae 12.5 years, bmi 30.5 ae 5.6 kg/m²). physical activities were measured by the long-version of ipaq; individuals were genotyped and stratified according to total pa (<150 or ! 150 min/week) and television watching (<15 or ! 15 h/week). anthropometric, biochemical and inflammatory data were obtained. subgroups of individuals with or without a allele were compared by student's t test. results: among individuals who watched tv < 15 h/week, those carrying the a allele had significantly higher concentrations of total and ldl-cholesterol, apolipoprotein b (88.3 ae 27.0 vs. 96.8 ae 21.1 mg/dl) and interleukin-6 (2.37 ae 2.06 vs. 3.54 ae 3.07 pg/ ml), but did not differ regarding anthropometric measures. among those who watched tv ! 15 h/week, no difference in any clinical data was detected comparing carriers and non-carriers of a allele. regarding pa, the presence of the variant allele did not influence the metabolic profile. the presence of the variant allele at fto gene seems to favor a deleterious metabolic and inflammatory profiles particularly for individuals less exposed to sedentary activities such as watching tv. for inactive individuals, the allele presence might not affect predisposition to adiposity-induced disturbances. cardiovascular disease is the leading cause of death in brazil and in the world and its development is directly related to lifestyles and habits acquired in childhood. the objective of this study was to evaluate the anthropometric data and blood pressure levels of schoolchildren to verify that the body mass index and waist circumference maintain a relationship with blood pressure. the descriptive study was conducted with 1074 schoolchildren of both sexes in schools of cruzeiro do oeste, a small town in southern of brazil. the evaluations consisted of: measurement of weight (w) and height (e) to calculate the body mass index (bmi), waist circumference (wc) and blood pressure (bp). associations between variables were determined using the chi-square test. we observed a high percentage of overweight, wc modified and high pressure measurements. both bmi and wc showed significant association with the high pressure measurements. there is an association between increased blood pressure and increasing age. increases in bmi and central adiposity are associated with higher risks of high pressure measurements, and consequently other chronic diseases in 6-10 years old children. this study, descriptive and cross-sectional, had as objective to evaluate the relationships between anthropometric measures and lifestyle habits with the lipid profile of brazilian schoolchildren (n = 135). the variables obtained were: weight and height to calculate body mass index (bmi), waist circumference (wc), blood pressure (bp), physical activity and dietary habits, serum lipid profile and glycemia. the results were analyzed by the mann-whitney test, the chi-square test and a relative measure of effects odds ratio. the significance was set at 5% (p < 0.05). anthropometric variables showed 19.3% of schoolchildren are overweight, 59.3% showed elevated levels of total cholesterol. there was no significant association between nutritional status and lipid profile. this evidence suggests that an appropriate bmi is not indicative of the absence of changes in lipid profile components. the risk factors associated with the development of cardiovascular disease among the children evaluated were: absence of mean meals, lack of daily physical activity, physical activity <4 times a week, and the consumption of salty snacks more than four times a week. the physical activity was negatively associated with dyslipidemia, suggesting that physical activity may underlying mechanisms by which zinc and magnesium influence glucose metabolism involved oxidative stress and inflammation. this crosssectional study investigated whether intakes of zinc and magnesium are useful to indicate oxidative stress, inflammation and insulin resistance in individuals at cardiometabolic risk. two hundred and five individuals (55.2 ae 12.6 years) with pre-diabetes were evaluated regarding dietary (24-h food recalls) and clinical variables and compared according to zinc and magnesium intake tertiles by anova. multiple linear regression analysis was employed including adjustments for age, gender and bmi (model 1), and saturated fat acid intake, smoking status and physical activity (model 2). stratified according to tertiles of magnesium intake, no significant differences in anthropometric, plasma glucose, lipid variables, superoxide dismutase (sod), oxidized ldl, inflammatory markers and homa-ir were found. mean values of fat mass (35.9 ae 8.7 9 34.6 ae 9.0 9 31.8 ae 9.4%) and homa-ir (2.3 ae 1.8 9 2.1 ae 2.0 9 1.6 ae 1.8) were significantly lower in the highest tertile of zinc intake; significant trends to decrease fat mass, homa-ir and c-reactive protein were also observed (p-trend < 0.05). in linear regression models, intakes of both micronutrients were inversely associated with homa-ir but not with inflammatory markers. direct associations were found between magnesium intake and sod concentrations in adjusted models. our findings suggest that assessment of zinc and magnesium intakes may indicate oxidative stress, inflammatory status and insulin resistance in at risk individuals. our design does not allow establishing cause-effect relationship. longitudinal studies are needed to clarify if increased consumption of these micronutrients would ameliorate these pathophysiological processes. a. cezaretto, b. almeida-pititto, c.r. barros, a. siqueira-catania, s.r.g. ferreira nutrition, school of public health/university of sao paulo, sao paulo, brazil introduction: lifestyle changes remain a major challenge for reducing cardiometabolic risk. psychological disorders coexist with risk factors such as unhealthy life habits, decreasing quality of life (qol) and favoring dropouts during interventions. objectives: to compare clinical and psychological characteristics of individuals who dropped or not from 2 lifestyle interventions, traditional or interdisciplinary. methods: one hundred and eighty-three prediabetic individuals (54.7 ae 12.3 years; bmi 30.8 ae 5.8 kg/m²) were allocated to 18month interventions on diet, physical activity and stress management. traditional intervention was based on quarterly medical visits, while participants of interdisciplinary intervention also had psychoeducative group sessions. depressive symptoms were assessed by the beck depression inventory (score ! 12) and qol by the sf-36. independent t-test was used to compare dropouts and non-dropouts. results: body adiposity, blood pressure, fasting glycemia, lipids, depression and qol improved in both interventions, but, in general, improvements were greater in the interdisciplinary intervention. comparing baseline data of dropouts and non-dropouts at the 18th month, dropouts of traditional intervention had higher bmi (32.4 vs. 28.9; p = 0.024), depression score (18.2 vs. 8.7; p = 0.002) and lower qol (59.0 vs. 74.0; p = 0.013) than non-dropouts. however, no difference between individuals who dropped or not was found in the interdisciplinary intervention. conclusion: interdisciplinary approach may be more effective to improve clinical features and qol. in addition, this intervention avoids dropout of individuals with worse risk profile concerning adiposity, depression and qol when compared with those having only medical visits. interdisciplinary approach may favor the retention of a subset of individuals at higher risk, which may result in long-term cardiovascular protection. aims: consumption of high fat diet (hfd) leads to accumulation of intramuscular bioactive lipids: long-chain acyl-coa (lcacoa), diacylglycerols (dag) and ceramides (cer) which are implicated in induction of muscle insulin resistance. the aim of this study was to elucidate the role of hfd, myriocin (an inhibitor of ceramide de novo synthesis) and metformin on skeletal muscle lcacoa, dag, cer and acyl-carnitine content and their impact on proteins of insulin pathway. the experiments were performed on male wistar rats: 1-fed standard rodent chow (control), 2-fed hfd, 3-fed hfd treated with myriocin, (hfd/myr) and 4-fed hfd treated with myriocin and metformin (hfd/myr/met). muscle bioactive lipids were analyzed by lc/ms/ms and plasma glucose and insulin concentration by standard assays. content and phosphorylation of akt and glycogen synthase (gs) was measured by western blot. results: compared to control, all measured lipids were elevated in hfd group. these changes were accompanied by decreased phosphorylation of akt and gs and impaired glucose disposal. myriocin caused a decrease in cer but an increase in the other lipids content as compared to hfd. introducing metformin to hfd/myr group attenuated levels of lipid metabolites known to interfere with insulin signaling (cer, dag, lcacoa) and increased acyl-carnitine content as compared to hfd/myr. in hfd/myr and hfd/myr/met groups insulin-sensitizing effects were accompanied by enhanced akt and gs phosphorylation when compared to hfd. conclusions: simultaneous treatment with myriocin and metformin redistributes fatty acids to β-oxidation which attenuates muscular content of bioactive lipids and improves glucose tolerance by promoting akt phosphorylation. pro-inflammatory state leading to abnormal production of hormones like leptin and adiponectin. the present study was designed to explore endocrine dysfunction of adipose tissue in metabolic syndrome. the study included 170 subjects; 84 metabolic syndrome cases as defined by international diabetes federation and 86 age and sex matched controls. the blood samples were collected for estimation of serum triglycerides, hdl-c, insulin, leptin, adiponectin and fasting plasma glucose. leptin and adiponectin were estimated by elisa and insulin by electrochemiluminescence immunoassay. other biochemical parameters were estimated on clinical chemistry analyzer by standard methods. homa-ir as an index of insulin resistance was calculated from fasting plasma glucose and serum insulin levels. results: serum leptin level was significantly higher in metabolic syndrome patients (26.45 ae 6.17 ng/ml) in comparison to control group (14.76 ae 6.42 ng/ml). serum adiponectin was significantly lower in metabolic syndrome patients (6.75 ae 3.22 lg/ml) in comparison to control group (8.44 ae 3.90 lg/ml) levels. significant difference of homa-ir was between metabolic syndrome cases and controls (p < 0.05). conclusions: it is concluded that altered endocrine functions of adipocytes are associated with metabolic syndrome and leptin and adiponectin has potential to be incorporated as components of diagnostic criteria of metabolic syndrome. aim: to devise noninvasive method to diagnose nafld. methods: one hundred and eight patients were examined: biochemical blood analysis, endotoxin and nitric oxide in blood, shortchain fat acids (sfa) in faeces, biopsy of liver. patients have been divided into two groups depending on activity of hepatitis: 1. steatosis; 2. steatohepatitis, and into three groups according to the therapy: 1. statin; 2. probiotic; results: we didn′t revealed direct correlation between biochemical parameters of liver function and heaviness of morphological changes in liver. however patients with steatohepatitis had increased level of endotoxin (0.37 ae 0.01) and nitric oxide (72.25 ae 2.22) in blood. total quantity of sfa in faeces was also decreased in those patients. there was more effective decrease of lipids, endotoxin and nitric oxide in those patients, who received probiotic in combination with statin. if patient received only probiotic there was not any changes of lipids, but the level of endotoxin and nitric oxide became lower. if nafld progressed in steatohepatitis and there was high level of c-gtp and transaminases we didn′n notice such effective decrease of cholesterol. 1. the increase level of endotoxin and nitric oxide in blood is the marker of steatohepatitis. 2. application of statin in combination with probiotic is more effective in achievement of target levels of lipids and decreasing endotoxin and nitric oxide. 3. if patient has steatohepatitis, effectiveness in the hypolipidemic therapy decreases (in comparison with steatosis), because the metabolism of drugs in liver decrease. methods: one hundred and eight patients with nafld were examined: biochemical blood analysis, endotoxin and no in blood, shortchain fat acids (sfa) in faeces, biopsy of liver (index of steatosis (is) and index of histological activity (iha)). patients have been divided into two groups: 1. steatosis; 2. steatohepatitis. results: total quantity of sfa in faeces was decreased in all patients, but there wasn′t difference between i and ii groups. all patients with had increased level of endotoxin (0.37 ae 0.01) and nitric oxide (72.25 ae 2.22). there was correlation between alt and ggtp with endotoxin and no (ast/endotoxin r = 0.34; ggtp/endotoxin r = 0.88; ast/no r = 0.34; ggtp/no r = 0.88; p < 0.05). the biopsy of liver showed all signs of nafld including changes of tiny bilious ductules. we didn′t revealed direct correlation between biochemical parameters of liver function and heaviness of morphological changes in liver (appearance of morphological attributes of steatohepatitis precedes development of cytolytic and cholestatic syndrome). however there was reliable direct correlation between endotoxin and no with histological parameters of nafld (is and iha): is/endotoxin r = 0.81; iha/endotoxin r = 0.84; is/no r = 0.72; iha/no r = 0.69; p < 0.05. 1. endotoxin and no may be used in diagnosing of nafld. 2. increase of level of endotoxin and no in blood in patients with nafld is associated with progression of inflammation in liver. thereby blood analysis for endotoxin and no can be used in diagnosing of nafld progression. 3. we also can judge about effectiveness of treatment of nafld by the level of endotoxin and no in blood. of this study was to analyze incidence obesity and insulin resistance in patients with fatty liver. the study involved 130 patients with diagnosis non alcoholic fatty liver at the clinical centre pristina. there were analyzed demographic and anthropometric characteristics of the examined patients. of clinical characteristics, there were determined smoking habit, arterial pressure. routine biochemical analyses were carried out by a standard laboratory procedure. homa-ir was used to evaluate insulin resistance. results: in the study group, fatty liver was demonstrated by ultrasound in 72 (55.38%) subjects and in the control group there were 58 respondents (44.62%) without pathological findings of ultrasound and with the level of p < 0.001. the values of bmi (34.56 ae 6.05 kg/m 2 vs.; p < 0.001), waist circumference (110.13 ae 11.22 vs. 88.63 ae 10.32 cm; p < 0.001) and glucose (6.23 ae 0.95 vs. 4.82 ae 0.38 mmol/l; p < 0.00), level of insulin (16.56 vs. 7.37 mu/i; p < 0.001) and homa-ir (4.16 vs. 1.6; p < 0.001) were significantly higher. conclusions: these results confirm that obesity and insulin resistance are associated with fatty liver infiltration. background and aim: this study was designed to determine the impairment of the skin structure in experimentally-induced diabetes with injection of streptozotocin (stz). material and methods: experimental groups consisted of controls (group 1, n = 10) and diabetes groups (group 2, n = 10). dorsal skin was removed for routine histological tissue procedures. hematoxylene and eosin (he), masson's trichrome and periodic acid schiff (pas) stainings, immunohistochemical connexin 43 (cx43) and type iv collagen stainings were applied. morphometry of epidermal thickness were also determined. results: group 2 revealed decrease in epidermal thickness with disintegration of epithelium and decrease of dermal collagen fibers. stratum spinosum were morphologically abnormal for group 2. measurements of epidermal thickness revealed statistically significant decrease (p0.000). pas staining for group 2 revealed disruption of the basement membrane. epithelial scar formation, deterioration of transformation in the polyhedral cells, degradation of epidermis and decrease in pas staining for vascular structures were observed, whereas the reticular dermis and hair follicles were normal. collagen fiber density in group 2 were found to be prominently decreased in dermis with masson's trichrome staining. evident decrease in immunostaining of cx43 and type iv collagen were also shown in diabetic group in comparison to the controls. conclusion: diabetes not only induced impairment of the epidermal integrity and deterioration in the epidermis via loss of gap junctions (the most prominent cellular junctional complex), but also caused dramatically negative impact on the dermal collagen content, and integrity of the basement membrane. background and aims: obesity, a major obstacle in the improvement of human health, is associated with an increased risk of development of numerous diseases. on the other hand, plant metabolites showed various bioactivities on affecting food intake, lipase activity, energy expenditure and lipid metabolism that may have potential on preventing body weight gain. therefore, this study aims to discover a new metabolite with anti-obesity efficiency. brief methods: adipocyte differentiation was induced by dexamethasone, 3-iso-butyl-1-methylxanthine and insulin in 3t3-l1 preadipocyte. in vivo, male c57bl/6 mice were fed a normal diet (nd), high-fat diet (hfd), a lucidone-supplemented hfd in lower dosage of 250 mg/kg of diet (lsh/l) or in higher dosage of 1250 mg/ kg of diet (lsh/h) for 12 weeks. record the changes of body weight and food consumptions. physiological parameters in plasma were determined after sacrificed. epididymal and perirenal adipose tissues were collected for further histological analysis. results: lucidone at 40 mmol/l suppressed adipogenesis in 3t3-l1 cells by reducing transcription levels of adipogenic genes, including pparg, c/ebpa, lxr-a, lpl, ap2, glut4 and adiponectin. lsh/ h mice showed lowered body and liver weights, decreased food efficiency, and lowered plasma cholesterol, triglyceride, glucose, and insulin levels. dissection of adipose tissue from lucidone-fed mice showed reductions in the average fat-cell size and percentage of large adipocytes. conclusion: these results provided evidence that dietary intake of lucidone alleviates high fat diet-induced obesity in c57bl/6 mice and reveals the potential of lucidone as a nutraceutical on preventing obesity and consequent metabolic disorders under unhealthy eating habits. introduction and aim: obesity and type 2 diabetes (t2d) are accompanied by intramyocellular lipid accumulation which might lead to mitochondrial dysfunction and insulin resistance. our aim was to compare changes in peripheral insulin sensitivity and mitochondrial respiration after a diet-and subsequently a gastric bypass induced weight loss in obese patients with or without t2d. materials and methods: sixteen subjects (4m/12f; 6 with (t2dm) and 10 (ob) without t2d) reported thrice to the lab after an overnight fast: prior to weight loss (a), 2 mo later just prior to operation (b) and 4 mo after operation (c). at each visit tree tests were performed: day1: dexa scan for body composition and stationary graded bicycle test for vo 2 max. day2: hyperinsulinaemic euglycemic clamp for peripheral insulin sensitivity. prior to the clamp a vastus lateralis muscle biopsy for high resolution respirometry was obtained. respiration protocol is shown in fig. 2 . results: anthropometric, body composition and vo 2 max data are shown in fig. 1 . insulin sensitivity (gir ffm ) and maximally coupled respiration (gmso3) data are shown in fig. 2 . conclusion: in spite of a marked difference in insulin sensitivity, maximally coupled mitochondrial respiration was similar in the two groups. moreover, with marked improvements in t2dm insulin sensitivity, due to the massive weight loss, mitochondrial respiration remained unchanged. these results speak against an association of mitochondrial respiratory capacity and insulin resistance in skeletal muscle in obese and t2d patients. introduction: increased circulating levels of resistin was proposed as a possible link between obesity and insulin resistance. we investigated if increased resistin expression in adipose tissue affected age-related changes in insulin resistance of peripheral tissues, including brown adipose tissue (bat), and oxidative stress parameters in the spontaneously hypertensive rats (shr). methods: sixteen months old male shrs expressing the mouse resistin gene under control of adipose tissue specific ap2 promoter (shr-tg) and age matched non-transgenic shrs were used. results: transgenic expression of resistin was associated with impaired glucose tolerance measured during ogtt (auc 1429 ae 169 vs. 725 ae 14 mmol/l/2 h, p < 0.01); elevated serum insulin (0.34 ae 0.02 vs. 0.28 ae 0.03 nmol/l, p < 0.05) and triacylglycerols (2.00 ae 0.15 vs. 1.34 ae 0.12 mmol/l, p < 0.01) levels. shr-tg exhibited lower insulin sensitivity measured as insulin stimulated incorporation of 14 c-glucose into lipids in epididymal adipose tissue (621 ae 38 vs. 1654 ae 302 nmol gl./g/2 h, p < 0.01) and into skeletal muscle glycogen (p < 0.05). glucose oxidation in soleus muscle (p < 0.05), and bat (p < 0.05) were reduced. shr-tg displayed decreased 14 c-palmitate oxidation to co 2 in bat (37.6 ae 4.5 vs. 53.8 ae 4.9 nmol/g/2hod, p < 0.05). in adipose tissue of shr-tg rats was increased concentration of lipid peroxidation products (conjugated dienes and tbars), reduced activity of antioxidant enzymes: superoxide dismutase (1.931 ae 0.187 vs. 3.023 ae 0.307 u/ mg protein, p < 0.05) and glutathione peroxidase (118 ae 6 vs. 163 ae 4 lmol nadph/min/mg protein, p < 0.001). conclusion: chronic transgenic expression of resistin gene might contribute to insulin resistance and associated metabolic disorders suggesting possible involvement of reduced bat metabolic activity in age-induced prodibetogenic effect of resistin. examination of 230 patients with type 2 diabetes mellitus (t2dm) revealed 83 patients (36.1%) with non-alcoholic fatty liver disease (nafld). nafld met more often in the patients with obesity (54.5%) and overweight (37.5%) and only in 20.8% patients with normal body weight. all the patients had abdominal type of adipose tissue distribution regardless of body weight. in patients with t2dm and normal body weight the nafld met more often in women, and in overweight and obesity in men. in the examined patients with t2dm and nafld complaints peculiar to dyspepsia and asthenovegetative syndrome prevailed. the average level of hba1c corresponded the state of subcompensated carbohydrate exchange (6.86%). these patients had marked dyslipidemia with predominance of ii and iib types of hyperlipidemia. cytolytic, cholestatic, mesenchymal-inflammatory syndromes were marked out as well as syndrome of hepatic-cellular insufficiency. increased level of immunoreactive insulin iri (17.65 ae 2.56) mu/ml and index of homa ir (8.30 ae 1.50) mu/ml testified the presence of expressed insulin resistance in the patients with t2dm and nafld. the patients revealed the increase of free fatty acids level (2.19 ae 0.10) mmol/l/ml, that meaningfully differed from such indexes in the control group (p < 0.001) and low level of adiponektin (3.70 ae 0.70) mg/ml that confirms their role in development of nafld even for persons with normal body weight after the presence of abdominal type of adipose tissue distribution. study object: diene (dc), triene (tc), oxydien (odc), tetraen (trc) conjugates, malondialdehyde (mda), aspartate aminotransferase (ast), alanine aminotransferase (alt). the study involved 37 patients with type 2 diabetes mellitus and non-alcoholic fatty liver disease (nafld) -14 men and 23 women among them and 10 diabetes women without nafld. all subjects are representative on age and diabetes duration. correlation analysis has been conducted with the calculation of spierman rank correlation coefficient. study results: the increase of tc and odc levels has been marked in type 2 diabetes men with nafld vs. women -(46.71 ae 3.15); (113.37 ae 8.32) and (38.86 ae 2.66); (91.54 ae 5.52), respectively, p < 0.05, as well as ast and alt levels -(0.87 ae 0.1); (1.46 ae 0.2) and (0.61 ae 0.08); (0.93 ae 0.2), respectively, p < 0.05, p < 0.001. the positive correlation of ast, alt with odc and trc has been found among type 2 diabetes women with nafld (r = 0.38, r = 0.4, p < 0.05) and (r = 0.36, r = 0.37, p < 0.05) unlike women without nafld -(r = à0.002) between ast and trc and (r = 0.32) between alt and trc. ast, alt are in positive correlation with ct in men (r = 0.56, r = 0.57, p < 0.05). in type 2 diabetes men with nafld the oxidative stress displays are more clearly seen. it is expressed in increasing of tc and odc combined with ast and alt hyperactivity. trc levels are increased in women group with ast and alt increasing vs. type 2 diabetes women without nafld. the above mentioned can serve as a diagnostic marker of nafld in type 2 diabetes women. i. kara, b. bonardo, f. peiretti umr 1062, inserm, marseille, france gamma secretase and the triggered notch signalling are involved in the regulation of the differentiation/proliferation equilibrium of progenitor cells. gamma secretase consists of four subunits: presenilin 1 or 2, nicastrin, pen-2 and aph1 (stoichiometry 1:1:1:1). in humans, there are two aph1 proteins (aph1a and aph1b). in rodents there is an additional protein, aph1c issued from a duplication of the aph1b gene. it has been reported that inhibition of c-secretase improves insulin resistance and stimulates adipogenesis. these data led us to study the impact of obesity in the regulation of the c-secretase composition. expression of c-secretase subunits was measured by quantitative pcr (tissues and cells) and western blot (cells). the expression of aph1b is significantly increased in the adipose tissue of obese mice whereas the expression of the other subunits of the c-secretase is not altered. the increased expression of aph1b is not observed in muscles and liver of obese mice. the expression of aph1b is also increased in visceral adipose tissue of human obese subjects. in addition, the expression of aph1b/c increases during adipocyte differentiation of 3t3l1 cells. in conclusion, the c-secretase composition is specifically altered in hypertrophied adipocytes. this result provides the first evidence of a pathophysiological regulation of aph1b/c expression. an approach to knockdown adipocyte expression of aph1b/c is in progress. the analysis of the consequences of this manipulation on csecretase activity, notch signalling and the biology of the adipocyte will help assess the importance of the alteration of c-secretase composition during adipocyte differentiation. background and aim: certain legumes and barley kernels may favorably modify metabolic risk parameters in semi-acute studies in healthy subjects. this investigation assessed the medium-term effect of a diet combining specific legume and whole grain barley products on markers associated with the metabolic syndrome (mets) in mature women. design and methods: randomized crossover intervention in 46 women (50-72 years-old, bmi 25-33, normal fasting glycemia), comparing a diet rich in kernel-based barley products, brown beans and chickpeas (d1) with a control diet (d2) of similar macronutrient composition but lacking legumes and barley. d1 included 172 g (as eaten)/day legumes, 58 g whole grain barley kernels and 200 g barley kernel bread. both diets followed the nordic nutrition recommendations and provided similar amounts of dietary fiber (46.9 g/day, d1; 43.5 g/ day, d2), with wheat-based products as main fiber supplier in d2. each diet was consumed for 4 week under weight-maintenance conditions. blood lipids, glucose, insulin, crp, gamma-glutamyl transferase (ggt) and blood pressure were the measured outcomes. results: both diets decreased serum cholesterol fractions, but d1 had greater effect (p < 0.01) on total and ldl-cholesterol (changes from baseline: à12% and à11%, respectively) than d2. d1 also reduced ggt (à8%, p < 0.05), diastolic blood pressure (à3%, p < 0.05) and the framingham cardiovascular risk estimate (à11%, p < 0.05), while d2 had no effect. methods: group i (n = 70)women with ms were examined proinflammatory cytokines polymorphisms, group ii (n = 52)testing for hereditary and acquired forms of thrombophilia. all subjects had the fetal loss syndrome, fetoplacental insufficiency, severe preeclampsia in previous pregnancies. results: group ipolymorphisms il-1β -31t/c in 95.7%, il-6 -174 g/c-in 68.6%, tumor necrosis factor-a -308g/ain 27.1% of cases. in ii group the multigenic defects were verified in 100% of cases; the feature of multigenic defects is that the 4g/5g polymorphism of plasminogen activator inhibitor-1 (pai-1) gene was found in 92.3% of cases, the 4g/4g phenotype of the gene pai-1 was verified in 61.5% of cases. the polymorphism in the tissue-type plasminogen activator i/d gene, in the angiotensin-converting enzyme i/d gene, in the fibrinogen 455g/a gene were found in 65.4%, 48.1%, 55.8% respectively. acquired antiphospholipid antibodies were verified in 15.4% of cases. all women received antithrombotic therapy from the fertile cycle involved low molecular-weight heparin (enoxaparin sodium, daily dose 0.4-0.8 ml), vitamins b, folic acid. pregnancy was achieved in 100%. there were not recurrent fetal loss, severe pre-eclampsia, placental abruption in the study groups. live births was in all cases. conclusions: we suggest that proinflammatory and thrombophilic status is an important pathogenetic factor of recurrent fetal loss, severe pre-eclampsia and other obstetric complications in women with ms. timely antithrombotic prophylaxis may be a key of successful outcome of pregnancy. the prescription of an individualized rehabilitation program based on the crossover point of substrate utilization (cop) could be particularly relevant in patients with metabolic syndrome (ms). the aim of the study was to examine: 1. the effects of a 12 week rehabilitation program at an intensity corresponding to cop on the anthropometric characteristics of women with ms, and 2. these effects after a 2 month follow-up period without supervision. nineteen women with ms (54.8 ae 8.1 years; 89.0 ae 12.2 kg; bmi = 34.5 ae 4) performed an indirect calorimetry test to determine their cop and then participated in a supervised rehabilitation program (3 9 45 min/week at cop on cycle-ergometer over 12 weeks). afterward, they were instructed to continue to practice a physical activity without supervision over 2 months. the target exercise intensity at cop was 38.3 ae 15.3% maximal aerobic power. mass, body mass index, waist and hip circumferences, waist-hip ratio, relative fat mass and fat-free mass were significantly decreased whereas energy balance was significantly increased after the rehabilitation program. there were no significant differences at the end vs. 2 months after the rehabilitation program. two months after the rehabilitation program, only both circumferences, waist-hip ratio and energy balance were not significantly different from baseline values. an individualized rehabilitation program over 12 weeks at cop allowed to improve the anthropometric characteristics in women with ms. however, although the energy balance remains unchanged during the 2 month follow-up period, only the effects on circumferences are maintained 2 months after the rehabilitation program. objective: hypoxia contributes to adipose tissue inflammation and fibrotic remodeling in obesity. we have recently shown that longchain n-3 polyunsaturated fatty acids (pufa) ameliorate adipose tissue inflammation in obese subjects. here we investigated, whether long-chain n-3 pufa also reduce adipose tissue hypoxia and may prevent adipose tissue remodeling. patients and methods: in a randomized controlled clinical trial we treated 49 severely obese subjects (bmi > 40 kg/m 2 ) with either 3.4 g/ day highly purified long-chain n-3 pufa or control for 8 weeks. at the end of the treatment we sampled visceral and subcutaneous adipose tissue (vat and sat) during elective bariatric surgery. we quantified gene expression of hypoxia and fibrosis markers in tissue specimen by real-time quantitative rt-pcr. statistical analyses were performed by one-way anova. results: adipose tissue gene expression of hif1a correlated positively with cd40 expression, a m1 macrophage marker and expression of both genes was significantly reduced after n-3 pufa treatment in sat (p < 0.05). furthermore, hif1a expression correlated negatively with the anti-inflammatory fatty acid index in the treatment group. n-3 pufa significantly downregulated the pro-fibrotic markers tgfb, mmp3 and col6a3 in sat. conclusions: mitigation of adipose tissue inflammation by n-3 pufa is paralleled by the amelioration of hypoxia and prevention of tissue remodeling as estimated by marker gene expression. these data emphasize beneficial effects of high-dose n-3 pufa in obesity. biochemistry, university of medicine and pharmacy "carol davila", 2 faculty of medicine, university "titu maiorescu", 3 delta hospital, 4 nidnmd "n.c. paulescu," bucharest, romania background and aim: the diabetes "epidemic" nowadays appeared at the same time with the obesity "epidemic", their relationship being a causal one. weight gain cannot be conceived without a good sensitivity to insulin. all three paraoxonases have antioxidant properties. these protect vascular tissues from oxidative damage by modulating the effects of the main actors in the atherosclerotic process, namely the hdl and ldl particles and the macrophage. in this study we aimed to investigate the role of paraoxoanse2 (pon2) in the relationship between obesity and type 2 diabetes. materials and methods: ten obese patients with type 2 diabetes mellitus were compared with 12 non-diabetic obese subjects. we determined the pon2 in preadipocytes cells obtained from bariatric interventions and in differentiated adipocytes (day 4, 8, 12, 16, 20) . elisa method was used for leptin and adiponectin measurements. results: the obesity antropometric markers (waist and hip circumference, visceral fat index) were positively correlated with the value for leptin and negatively with the value for adiponectin (p < 0.001). the intracellular pon2 tented to be lower with the degree of diferentiation and these was associated with adipokines changes both in serum and cell lysates. conclusion: resuming our data, we presume that pon2 is present in pre/adipocytes and could be implicated in prevention of type 2 diabetes obesity associated complications. acknowledgements: this work was supported by a grant of the romanian national authority for scientific research, cncs-uefiscdi, project numer pn-ii-id-pce-2011-3-0429". dr. lixandru was supported by the postdoctoral program posdru/89/ 1.5/s/60746, from european social fund. 1. the effects of a 12 week individualized rehabilitation program (irp) coached at an intensity corresponding to the crossover point of substrate utilization (cop) on hqrol, peak of oxygen uptake (vo 2peak ) and power out peak (p peak ); 2. the effects on hqrol after a 2 month follow-up period without coach. nineteen women with metsyn (54.8 ae 8.1 years; 89.0 ae 12.2 kg) performed a test to exhaustion to estimate the vo 2peak and p peak , and an indirect calorimetry test to determine their cop used during a irp (3 9 45 min/week) on cycle-ergometer over 12 weeks. afterward, they were instructed to continue their physical activity without coach over 2 months. vo 2peak and p peak were increased after the irp (p < 0.001). hrqol were no different in every domain at the end and 2 months after the irp. only the question about their change of health compared to 1 year ago has improved but it was at the verge of significativity (p = 0.06). women with metsyn undergoing irp coached at cop improved vo 2peak and p peak, but showed no improvement in hrqol after a 12 week coached irp nor after the 2 month follow-up period without coach. consequently, physical fitness seems not be associated with self-perceived quality of life in women with metsyn. t.b. domagala 1 , k. kotula-horowitz 2 , r. januszek 2 , m. janczura 3 , j. zagajewski 4 , j. musial 2 micronized fenofibrate (215 mg/day) for 6 months. fifty cardiovascular risk male volunteers (mean age 40 years) with no prior myocardial infarction or stroke were enrolled as the controls. brachial fmd and homocysteine levels were measured at baseline and after 6 months. the respective groups also differed in: bmi (p < 0.0001), plasma total (p = 0.0001), hdl cholesterol (p = 0.007) and triglycerides (p = 0.0001), but not in baseline plasma homocysteine levels (11.04 ae 3.69 vs. 9.98 ae 2.08 lmol/l; p = ns). at baseline, mean brachial fmd were lower in the subjects with metabolic syndrome (6.69 ae 2.41% vs. 8.56 ae 3.12%; p < 0.05). in those subjects fenofibrate therapy significantly improved fmd (6.69 ae 2.41% vs. 9.83 ae 3.31%; p < 0.0001) and increased plasma homocysteine levels (11.04 ae 3.69 vs. 9.98 ae 2.08 lmol/l; p < 0.0001). no such differences were noted in the controls, neither before nor after 6 months. conclusions: fenofibrates improve endothelium-dependent fmd of the brachial artery, irrespective of the simultaneously induced hiperhomocysteinemia. a. picu 1 , l. petcu 1 , d. lixandru 2 , i. stoian 2 , c. cristescu 3 , e. rusu 3 , c. ionescu-tirgoviste 1 1 nidnmd "n.c. paulescu", 2 biochemistry, university of medicine and pharmacy "carol davila", 3 "titu maiorescu" university, bucharest, romania background and aims: oxidative stress leading to macrophage foam cell formation is the hallmark of the early atherosclerotic lesion. the aim of these study was to assess the relationship between clinical, biochemical and oxidative stress risk factors with obesity in newly diagnosed type 2 diabetes (n-dm). materials and methods: one hundred and forty-five patients with n-dm registered in the nidnmd ambulatory, "n.c.paulescu" and 30 healthy volunteers were included. the n-dm patients were divided into two subgroups according to the bmi (body mass index): group1overweight (bmi = 28.4-29.9 kg/m²) and group2-obese (bmi ! 30 40 kg/m²). in circulating monocytes, the ability to produce free radicals vs. their neutralizing capacity was determined by measuring nadph oxidase activity (respiratory burst; rb) and intracellular activity of pon2. we also determined the serum leptin and adiponectine by elisa, total antioxidant capacity (teac) and the concentration of non-protein thiols (shneproteic) by spectrophotometric methods. results: in diabetic patients vs. normal subjects, leptin, proinsulin and rb were increased (p < 0.001), while adiponectin and pon2 decreased (p < 0.001). the differences were accentuated in the obese group. the strongest correlation was between proinsulin and leptin (r = 0.59, p < 0.001) and both contribute for the high cardiovascular risk in type 2 diabetes. our results showed imbalance pro/antioxidant of obese diabetic patients, low pon2 activity and increased rb being influenced by the degree of obesity. introduction: cardio-protective role of human hdl-associated serum paraoxonase (pon1) is believed to be due, at least partly, to its antioxidative properties. pon1 activity is genetically determined by major polymorphism q192r. brachial flow-mediated dilation (fmd) is a non-invasive physiological measurement used to quantify endothelial dysfunction. fibrates are known to exert hypolipidemic effects, while their effect on pon1 activity remains unclear. aim: to assess pon1 activity and fmd in the subjects with metabolic syndrome on fenofibrate treatment. methods: forty male subjects (mean age 42 years) with hipertriglicerydemia, as well as metabolic syndrome, were treated with micronized fenofibrate (215 mg/day) for 6 months. pon1 activity in serum and brachial fmd were measured at baseline and after 6 months. pon1 q192r polymorphism was determined by pcr amplification and restricted digestion; serum pon1 activity was assayed spectrophotometrically. results there were 26 subjects with 192qq genotype (group a) and 14 carriers of 192r allele (group b). at baseline both groups differed in pon1 activity only (102 ae 59 vs. 190 ae 39 u/l; p < 0.0001). fenofibrate treatment reduced pon1 activity in all subjects, nonetheless significantly in those with 192qq genotype (102 ae 59 vs. 93 ae 54 u/l; p = 0.001), unlike in the 192r allele carriers (p = ns). at baseline mean brachial fmd did not differ between groups a and b. fenofibrate therapy significantly improved fmd (7.12 ae 2.83% vs. 13.30 ae 1.72%; p = 0.001) in group a subjects, unlike group b (6.66 ae 2.50% vs. 9.61 ae 2.60%; p = ns). this study was to investigate the effect of nicotine on insulin sensitivity and explore the underlying mechanisms. treatment of sprague-dawley rats with nicotine (3 mg/kg/day) for 6 weeks significantly reduced blood insulin level but had no effect on blood glucose level. both insulin tolerance test and glucose tolerance test demonstrated that nicotine treatment enhanced insulin sensitivity. pretreatment of rats with hexamethonium (20 mg/kg/day) to antagonize peripheral nicotinic receptors except for a7 nicotinic acetylcholine receptor (a7-nachr) had no effect on the insulin sensitizing effect of nicotine. however, the insulin sensitizing effect of nicotine was totally abrogated in a7-nachr knockout mice, indicating the involvement of a7-nachr. further, chronic treatment with pnu-282987 (0.53 mg/kg/day), a selective a7-nachr agonist, significantly enhanced insulin sensitivity not only in normal mice but also in amp-activated kinase-a2 knockout mice, an animal model of insulin resistance with no sign of inflammation. moreover, pnu-282987 treatment enhanced phosphorylation of signal transducer and activator of transcription 3 (stat3), a molecule involved in regulating insulin signaling, in skeletal muscle, adipose tissue and liver in normal mice. pnu-282987 treatment also improved glucose uptake in c2c12 myotubes and this effect was totally abrogated by stat3 inhibitor, s3i-201. all together, these findings indicate that nicotine enhances insulin sensitivity in animals with or without insulin resistance, at least in part via stimulation of a7-nachr-stat3 pathway independent of inflammation. our results not only contribute to the understanding of the pharmacological effects of nicotine, but also identify new therapeutic targets against insulin resistance and type 2 diabetes. calorie restriction (cr) is one of the most reproducible treatments for weight loss and slowing aging. however, how cr induces these alterations is still unclear. this study was designed to understand whether nicotinamide phosphoribosyltransferase (nampt, also known as visfatin) plays a role in the beneficial effects induced by cr using a specific chemical inhibitor of nampt (fk866). sprague-dawley rats were treated for 8 weeks in four groups: ad libitum (al), cr, al + fk866 and cr + fk866 groups. al and al + fk866 animals were allowed unlimited access to standard chow, while the cr and cr + fk866 animals were restricted to 60% of the food intake consumed by al and al + fk866 animals. fk866 (1 mg/kg/ day) was given via drinking water. we found cr upregulated nampt mrna and protein levels in rat skeletal muscle and white adipose tissue. inhibition of nampt did not affect the sirt1 upregulation by cr but suppressed the cr-induced sirt1 activity and deacetylation of foxo-1. furthermore, inhibition of nampt not only weakened the cr-induced decrease of oxidative stress (ros, superoxide o 2 à˙a nd mda levels), but also greatly abolished the crinduced improvements of anti-oxidative activity (total-sod, gsh and gsh/gssg ratio) and mitochondrial biogenesis. in addition, inhibition of nampt blocked the cr-induced insulin sensitization, akt signaling activation and enos phosphorylation. collectively, our data demonstrate that cr-induced beneficial effects in oxidative stress, mitochondrial biogenesis and insulin sensitivity require functional nampt. background: the anti-diabetic effect of camel milk (cmk) gained increasing recognition in folk medicine and recent clinical and experimental studies. however, the mechanism(s) by which cmk influence the glucose homeostasis is yet unclear. objectives: to investigate the effects of cmk on the blood glucose regulatory mechanisms in control and diabetic animals. materials and methods: experimental diabetes was induced by intrapertional injection of streptozotocin in 40 wistar rats divided into (d & d-cmk) groups. two healthy (c& c-ckm) groups served as control. camel milk (50 ml/rat/day) was administered orally to the (d-cmk) and (c-cmk) groups for 8 weeks. the (d) and (c) groups received no treatment. the changes in body weight, blood glucose, glucose tolerance, serum insulin, blood lipids, incretin hormones (glp-1 and gip), tnf-a, tgf-β1, homa-ir and atherogenic index (ai) were investigated. results: the untreated diabetic animals (d-group) exhibited significant hyperglycemia, hyperlipidemia, increased homa-ir and ai, elevated serum icretins, tnf-a and tgf-β1 levels. this was associated with weight loss and high mortality. camel milk administration to the d-cmk group inhibited the weight loss and mortality and caused significant hypoglycemia, hypolipidemia, insulin secretion, low homa-ir and ai. serum glp-1 and gip levels show significant elevation but tnf-a and tgf-β1 were reduced. conclusion: augmented insulin release and decreased insulin resistance together with enhanced incretin hormones release are anticipated to contribute to the anti-diabetic effect of cmk. besides, the peculiar composition of cmk and its anti-inflammatory properties propose it as a valuable adjuvant anti-diabetic therapy. however large-sized clinical studies are still needed. prediabetics reflect dysregulation in glucose homeostasis with obesity is known risk factor in western countries. different degree of obesity contribution toward prediabetics is observed in lean populations. aim: to investigate the role of plasma leptin as a predictor of prediabetics in lean subjects. method: plasma leptin were measured in 950 non-diabetic subjects aged 14-90 years old with bmi < 25. prediabetes (n = 100) was defined based on fasting plasma glucose and 2-h oral glucose tolerance test. plasma leptin were measured in all subjects using commercial elisa kit. binary logistic regressions were used to estimate the age and sex adjusted odds ratio of leptin and other metabolic parameters. results: plasma leptin levels were significantly and positively correlated with bmi, percent total body fat, fasting plasma insulin and blood sugar and homa-ir values. plasma leptin were significantly higher in lean prediabetics (31.04 ae 2.22) compared to euglycemic lean subjects (20.11 ae 0.62; p < 0.0001). increase in plasma leptin value by one increases the risk of prediabetics with or 1.019 (95% ci: 1.007-1.031). discussion: in a population of relatively lean and non-diabetic subjects, plasma leptin levels were associated with insulin resistance and prediabetes. furthermore, plasma leptin levels increases risk of prediabetics in non-obese subjects independent of age and sex. our data suggest that leptin as biomarker for screening individuals at high risk for prediabetes in lean population. f. al-zadjali, r. bayoumi latest update from the international diabetes federation shows that the prevalence of type 2 diabetes (t2dm) in the arabian gulf countries is among the top 10 worldwide. the rapid increase in t2dm prevalence in these countries suggests that psychological and behavioral factors, more than genetic factors, are primarily responsible for this trend. aim: to estimate heritability of plasma levels of adipocytokines in a large isolated arab pedigree. method: blood samples and anthropometric data were obtained from a large arab pedigree of 469 individuals with high level of consanguinity. plasma levels of il-6, il-1b, leptin, total adiponectin, svcam-1 were measured using commercial kits. heritability parameters were measured using measured genotype analysis. results: fout hundred and sixty-nine individuals were structured in a large pedigree which consisted of interrelated 148 nuclear families founded by 109 founders. the heritability values were adjusted using age and gender as covariates. percentage of variation of plasma adipocytokines attributed to genetic factors were as following: leptin (41%), total adiponectin (19%), il-1b (21%), il-6 (38%) and svcam-1 (38%). discussion: adipocytokines play central role in the development of t2dm. our data shows high degree of heritability of leptin, il-6 and svcam-1 indicating their production is under tight genetic control. furthermore, our heritability values are different from previously reported heritability values of these adipocytkines studied in different ethnic populations. therefore, our results suggests that arab population are distinct and further genetic association and behavioral studies should be conducted for better understanding of the nature of t2dm and its increasing trend in middle-east region. results: all treatments were well tolerated and no subjects was withdrawn from the study. compared to the baseline, the group treated with chrome picolinate only experienced a mild but significant decrease in fpg (à1.8 ae 2.9 mg/dl). compared to the baseline, the group treated with chrome picolinate-berberine experienced a mild but significant decrease in bmi (à0.1 ae 0.05 kg/m 2 ), tc (à19.1 ae 22.7 mg/dl), tg (à31.7 ae 45.3 mg/dl), non-hdl cholesterol (à18.6 ae 22.7 mg/dl), and fpg (à3.2 ae 7.2 mg/dl). compared to the baseline, the group treated with chrome picolinate-berberine-banaba experienced a significant reduction in bmi (à0.1 ae 0.1 kg/m 2 ), waist circumference (à2.4 ae 1.4 cm), sbp (à3.0 ae 4.0 mmhg), tc (à25.1 ae 15.7 mg/dl), tg (à32.7 ae 15.8 mg/dl), ldl-c (à19.5 ae 13.4 mg/dl), non-hdl cholesterol (à26.1 ae 15.9 mg/dl), fpg (à5.3 ae 2.3 mg/dl), insulin (à1.95 ae 1.47 mu/l), homa index (à0.72 ae 0.41) and hba1c (à0.13 ae 0.25%), while hdl-c increased (+1.1 ae 1.2 mg/dl). waist circumference, sbp, ldl-c, hdl-c, insulin, homa index and hba1c, also significantly improved when compared with other groups. the tested combined nutraceutical appears to significantly improve insulin-sensitivity and a large number of related parameters in subjects with ifg and metabolic syndrome. antioxidative mechanisms prevent human body from the damaging action of free radicals and reactive oxygen species. glutathione and related to it enzyme systems detoxicate h 2 o 2 and hyperoxide and from there, reduced glutathione is a potential marker of oxidative stress level. purpose: to investigete glutathione of erythrocytes in patients with prediabetes (igt and ifg) and type 2 diabetes. matherial and methods: we examined 25 patients with t2d and concomitant coronary heart disease (chd) and atherosclerosis (group 1), 20 patients with prediabetes (igt and ifg) newly diagnosed and the same co-morbidity (group 2) and 47 almost healthy person (group 3). examinations included clinical laboratory evaluation, fasting glicose, hba1c. total glutathione (gsht) and oxidized glutathione (gssg) were measered by use of glutathione reductase reaction. redox potential (e h ) of glutathione was evaluated by use of nernst equation. results: concentrations of gsht and gsh were greater in group 2 to compare with groups 1 and 3 (by 4.2 times, p = 0.012 and by 3.1 times, p = 0.005 respectively). inrease of concentration of gssg in group 1 may change extracellular redox condition. calculation of e h revealed its change to oxidized condition in group 1 to compare with group 2 (19.0 mv greater) and group 3 (15.4 mv greater). conclusion: antioxidant defense in red-cells is overactivated during prediabetes (igt and ifg) with concomitant chd and atherosclerosis while in patients with t2d was noticed depletion of compensatory mechanisms with increased gssg and e h . reduction in 2gsh/gssg suggests that intracellular antioxidant is lost; herewith cells become sensitive to oxidative stress. clozapine increases meal size and meal duration, effects similar to the pharmacological blockade or congenital deficiency of cck-1 (cck-1r) receptor. we aimed to investigate the role of cck-1r in clozapine-induced weight gain (wg) and insulin sensitivity (is) in cck-1r deficient, male otsuka long evans tokushima fatty rats (oletf). long evans tokushima otsuka (leto) rats served as healthy control. animals were orally treated with either clozapine or its vehicle over 25 days. daily food intake was measured by means of metabolic cages. the is was determined by hyperinsulinemic euglycemic glucose clamping (hegc). hypothalamic mrna expression of cck-1r and cck-2r was measured by real-time pcr, plasma insulin was determined by radioimmunoassay. clozapine failed to induce wg or increment in food intake in either oletf or leto rats. the fasting plasma insulin and blood glucose level was significantly higher in oletf than in leto rats, but clozapine failed to modify these parameters. the glucose infusion rate during the steady state of the hegc was lower in oletf than in leto rats and clozapine did not modify it. the insulin sensitivity index (isi) was lower in oletf rats than in leto and the isi was further decreased by clozapine. the metabolic clearance rate of insulin changed parallel with isi indicating the possible source of the surplus of insulin, which is responsible for the decrement in is. clozapine induced insulin resistance without hyperphagia and wg in male leto and oletf rats. changes in cck-1r and cck-2r expression were inconsistent with the changes in the isi. clinical biochemistry & immunology, peterborough and stamford nhsft, peterborough, uk the national institute for clinical evidence has published guidelines for the treatment of prediabetes. these stress diet and exercise as shown by the dpp study a decade ago. however, recently published dpp follow-up data show how difficult it is for patients to follow that advice long-term. metformin was also found to be effective and is cheap and generally well-tolerated. this study shows the long-term effectiveness of routine metformin use in patients with igt. all patients attending two rural lipid clinics from 2003 who were found to have igt after a standard 75 g ogtt were offered metformin 500 mg bd or given diet and exercise advice if they did not wish medication. metformin was preferred over diet and exercise despite being shown to be inferior in dpp because of the extra cardiovascular benefits shown in ukpds. in 2011, after a mean (range) follow-up of 3.3 (0.4-7.0) years, it was decided to review the data to see if the (off-label) treatment was effective. metformin significantly reduced t2dm threefold after 6 years (18% v 55%). further, it was possible to estimate that metformin could prevent 2/3 ever becoming diabetic (p = 0.014). the effect was due to restoring normoglycymia (fbg < 5.6 mmol/l) as the patients' weights and triglyceride levels did not change significantly. patients regularly present to lipidologists with fasting hypertriglyceridaemia. those with igt appear particularly likely to revert to normoglycaemia with metformin. routine use of this inexpensive and well-tolerated drug reduces the lifetime risk of developing t2dm by over 70%. k. salehzadeh, m. shirmohammad zadeh azarbaijan shahid madani university, tabriz, iran as a fermented drink probiotic doogh [1] can help promote the health of the society. studying crp [2] changes as an important inflammatory index that predicts coronary-heart diseases such as arteriosclerosis has received attention in medical and nutrition studies. the present study was carried out to study the effects of 10 week drinking different types of dough (probiotic and plain) on the changes of liver crp and other blood factors of male athletes with normal weight. the research sample of this study included 30 healthy male athlete students with equal bmi characteristics that were divided into two testing groups (15 each) and were homogenized and randomly divided and went through some exercises. the results of the paired t test showed that drinking plain doogh resulted in significant reduction of blood's urea (12.5%) and crp (66.87%) and significant increase of hdl (110.86%; p < 0.05). the probiotic doogh also resulted in significant reduction of crp (79.37%) and significant increase of hdl (75.67%). the comparison of the two groups showed that the only statistically significant difference was in the amount of crp and the reduction effect of probiotic doogh on crp was more compared to the plain doogh (p < 0.05). also the amount of record changes in the probiotic group (à28 s) was significantly better than the plain doogh (+2 s). background and aims: nutrition has significant effects on human health and diseases. diabetes is a chronic disease that can be affect by nutritional status. therefore, monitoring of nutritional status in these patients is one of the main components of prevention and controlling of complications. therefore, in this study the nutritional status of patients that referred to health center in songhor city was assessed. materials and methods: this descriptiveanalytical study was done on 300 patients with type 2 diabetes (56 ae 11 years old) with no insulin therapy. anthropometric mesearments and dietary intake assessment were done. ffq and three 24 recall quessionare were filled with face to face interview. n4 software were used for analysis. mean ae sd of variables were reported. background: the aetiology of type 2 diabetes and obesity may involve dysregulation of brain appetite control networks. we explored the impact of systemic insulin resistance, common to both conditions, on the effect of meal ingestion on brain responses to food cues. methods: eight insulin resistant (ir, homa2-ir 1.81 ae 0.32) and 14 insulin sensitive (is, homa2-ir 0.56 ae 0.16) non-obese non-diabetic right-handed men were studied twice, 16 min after consuming 50 ml water (fasted) or 630 kcal mixed meal (fed) in random order. brain responses upon viewing high (hc) and low (lc) calorie food images vs. non-food object (nf) and gaussian blurred (gb) images presented in a block design paradigm were measured using blood oxygenation level dependent functional magnetic resonance imaging. results: the meal reduced hunger (f 20 = 12.27, p = 0.002) and increased fullness (f 20 = 20.34, p < 0.001). meal ingestion had no specific impact on responses to lc in either group. in contrast, on viewing hc, there were decreased precuneus responses (vs. gb and nf) and superior temporal gyrus/insula responses to hc (vs. nf) in ir subjects, whilst in is subjects, meal ingestion increased responses to hc (vs. gb) in somatosensory cortex and to all food images vs. gb in putamen/insula, with a trend towards increased dorsolateral prefrontal cortex activity. conclusions: meal ingestion augmented activity in brain regions involved in sensation, interoception and inhibitory control in is subjects during food cue exposure. by diminishing activity in regions involved in imagery and interoception, insulin resistance may influence central appetite control networks to promote further eating after consuming a satiating meal. methods: we examined 76 children with obesity in the endocrinological department of university hospital (minsk) over the year 2011. the number of boys (b) 42 (55.3%), mean ae sd age 13.3 ae 2.5 years; girls (g) 34 (44.7%), mean ae sd age 13.7 ae 3 years (p = 0.6). ultrasound (u/s) of the abdominal cavity; the levels of insulin (i); total cholesterol (tc); triglycerides (tg); high-density (hdlc), low-density (ldlc), very low-density (vldlc) lipoprotein cholesterol; atherogenic coefficient (ac); standart oral glucose tolerance test (ogtt) with the calculation of homa-ir and caro indexes; the levels of blood pressure (bp) and body mass index (bmi) were held to all patients. results: sds bmi b was 6.24 ae 2.23, g 5.74 ae 2.35 (p = 0.7). bp more than 95th percentile was in 15 (35.7%) b and 17 (50%) g (p = 0.03). the signs of steatohepatosis were determined in 19 (45.2%)b, 16 (47%)g. the average levels of tc and tg were in normal limits, gender differenses weren′t noted (p = 0.8), (p = 0.1). the concentration of vldlc 0.76 ae 0.36 mmol/l (0.3-0.45), 0.73 ae 0.3 (p = 0.9). hdlc b and ldlc b didn′t exceed normal limits in boys and girls (p = 0.2), (p = 0.4). ac b 3.35 ae 0.79 (2-3), g 3.67 ae 0.92 (p = 0.3). basal and postprandial plasma glucose levels didn′t exceed normal limits regardless of gender (p = 0.6), (p = 0.1). the levels of i b 38.7 ae 5.5 mu/ml (2.1-22), g 33.3 ae 9.8 (p = 0.3). homa-ir b 8.9 ae 5 (<2.77), g 7.1 ae 2 (p = 0.5), caro 0.2 ae 0.15 (>0.33), 0.25 ae 0.13 (p = 0.8). conclusions: obesity in children was accompanied with arterial hypertension, steatohepatosis and dyslipidemia. the development of insulin resistance with maintaining the basal and postrandial normoglycemia was noted by conducting ogtt in all patients regardless of gender. objective: to evaluate the effect of short-term, moderately high-fat diets on body weight, lipid profile and serum leptin levels. the study was undertaken to create a model with an acute metabolic stress without marked obesity. design: the laboratory mice were fed either a moderately high-fat diet or control diet. body weight, energy intake, body composition, and fasting plasma leptin were compared after 3 and 8 week of dietary treatment. results: after 3 week, abdominal fat mass was 10% greater in mice fed the high-fat diet than in those fed the control diet (p < 0.05). however, plasma leptin concentrations did not change much in animals fed the high-fat diet. from 3 to 8 week, animals fed the highfat diet gained weight twice the normal diet group and consumed 28 kj/day more than controls (both p < 0.05). at 8 week, plasma leptin concentrations per unit abdominal fat mass were 20% lower in mice fed the high-fat diet (p = 0.058) and there was a significant negative association between leptin concentrations per unit abdominal fat mass and body weight (r = 0.46, p < 0.05). conclusions: feeding for 8 weeks moderately high-fat diet is associated with lower than expected circulating leptin concentrations, which correlate with a higher body weight. a high-fat diet may therefore contribute to weight gain by reducing leptin secretion in adipose tissue. the significance of these findings will be discussed. objective: to describe diabetes-related risk factors associated with lower respiratory tract infection (pneumonia) complications among children. methods: we obtained claims data on all hospital admissions to acute care hospitals for children patients with diabetes and pneumonia who were aged 1-15 years in bosnia and herzegovina and sarajevo between november 1, 1995, and november 1, 2012 . these data are checked for accuracy and validity by the pulmologist and diabetologist pediatricians that oversee data collection. in both data sets, we linked diabetic kids patient charts to allow for calculation of previous admission and rehospitalization diabetic kids rates with complications of pneumonia in children. children prescribed more than one antibiotic class contributed to determining the rates of each class. objectives: to examine the extent to which adherence with multiple concomitant healthy lifestyle traits will be associated with the avoidance of the future development of t2d. methods: five independent cohorts comprising 18,649 men and 20,052 women aged 25-74 and free of diabetes and cvd at baseline were examined in 1982, 1987, 1992, 1997 objective: to investigate the effects of raloxifene on the insulin sensitivity and lipid profile in insulin sensitive and insulin resistant postmenopausal women. study design: this placebo-controlled, double-blind, randomized study involved 64 postmenopausal women aged between 45 and 55 years. all subjects were screened with the insulin resistance homeostasis model assessment (ir-homa) and those patients in the lowest quartile (n = 16) were assigned as insulin sensitive and those in the highest quartile as insulin resistant (n = 16). patients in both groups received either raloxifene hydrochloride (60 mg/day) or a placebo, for a period of 12 weeks. insulin sensitivity, the serum lipid profile, and anthropometric measurements were established before and after therapy. results: women with the highest ir-homa scores were associated with a significantly higher weight, body mass index, waist, and waistto-hip ratio (p < 0.05). raloxifene significantly reduced the ir-homa scores from 5.76 ae 2.91 to 1.93 ae 0.96 (p = 0.02) and modified the lipid profile in insulin resistant patients when compared to the placebo group and those patients receiving raloxifene in the insulin sensitive group. conclusion: raloxifene reduced insulin resistance and modified the lipid profile in insulin resistant postmenopausal women. diseases, smoking, alcohol consumption, the use of sleeping pills and other potential confounders was also gathered. statistical analyses were done by spss ver. 19. the average age of the participants was 66.5 years old; 24% was reported current pet ownership. mean hba1c was 6.9%, bmi was 24, and systolic and diastolic blood pressure was 131 and 75 mmhg respectively. pet owners were significantly younger and slightly more overweight; and dog owners were much younger and more overweight than cat owners. in unadjusted analyses, dog owners had a lower rate of taking sleeping tablets than cat owners (p = 0.047%), and dog owners were more likely to be smokers than cat owners (p = 0.044%). however, pet ownership was not associated with hba1c, systolic or diastolic blood pressure, bmi, or the prevalence of diabetic complications. conclusions: our results suggest that pet ownership may be associated with insomnia and smoking habits. however, pet ownership is not independently associated with control of diabetes, obesity and prevalence of diabetic complications. objectives: to non-invasively assess the muscular oxygenation response using near-infrared spectroscopy (nirs) and to determine the association between increasing levels of a1c and oxygenation response in subjects with type 2 diabetes. materials and methods: forty-five subjects with uncomplicated type 2 diabetes were categorized into two groups: group i (a1c < 7) and group ii (a1c ! 7). nirs measurement of vastus lateralis was performed at rest and during a symptom-limited maximal treadmill exercise test to determine muscular oxygenation response. at rest and during peak exercise, deoxygenated hemoglobin (deoxy-hb), oxygenated hemoglobin (oxy-hb) and total hemoglobin (total-hb) were measured with characteristics of light absorptance from nirs. muscle saturation (%) was expressed as the ratio of oxy-hb to total hb volume, an index of tissue de-oxygenation. results: group i subjects (n = 26 with 15 females, 60.3 ae 5.2 years) were older than subjects in group ii (n = 19 with 13 females, aged 56.0 ae 5.9 years). the peak vo 2 was comparable between groups. significant lower muscle saturation (54.0 ae 9.4% vs. 49.3 ae 11.8%, p < 0.05) and more decline (à8.5 ae 4.0% vs. à11.8 ae 6.1%, p < 0.05) at peak exercise, was noted in group ii. a significant correlation between a1c and decline of saturation (r = à0.361, p < 0.05) was noted in this study, whereas no other significance were demonstrated in deoxy-hb or oxy-hb. conclusions: this study provided evidence of imbalance of oxygen supply to exercising muscle in asymptomatic and uncomplicated diabetic subjects with elevated a1c levels. the insufficient perfusion occurred before vasculopathy and might develop metabolic impairments even myopathy in diabetic subjects with poor glycemic control. study design and method: the lifestyle intervention was conducted as a cluster randomized trial (n = 190) in semi-urban setting in sri lanka. the intervention group (100) received a family centered lifestyle intervention package from family health workers while the control group (90) received a brief advice from a doctor. duration of the study was 6-months. results: this study has shown effectiveness in reduction in fasting blood glucose (p < 0.01), systolic blood pressure (p = 0.03), diastolic blood pressure (p = 0.01), weight (p = 0.03), added sugar (p = 0.03) and fat consumption (p < 0.01) while improving physical activity (p < 0.01) and insulin sensitivity (p < 0.01) in the intervention group when compared with the control group at follow up assessment. this lifestyle intervention sessions (4) were carried out by family health workers during their routine house visits. aim: we aimed to compare an experimental type 2 diabetic animal model generated by applying high fat diet combined with single shot of streptozotocin (stz) at 40 mg/kg bw sprague-dawley (sd) rats. methods: we generated and selected rats based on fasting glucose, oral glucose tolerance, and %hba1c. once sever type 2 diabetic rats were identified, serum proteins were subjected to 2d-dige proteomics or comparative 2d-proteomics after phosphorylation or glycation. results: seven protein targets differentially expressed in severe type 2 diabetic rats (st2d) were identified as albumin, vitamin d-binding protein precursor, and transthyretin, respectively. in terms of serum proteins extracted by affinity columns for phosphorylation and glycation, the affinity of 14 phosphorylated protein targets and 23 glycated protein targets were modified under st2d condition. results of the ingenuity pathway analysis (ipa) for those targets indicated that antigen presentation, humoral immune response, and inflammatory response are associated networks highly modified in st2d group. kidney and liver damages under those st2d rats were proposed by ipa and validated by histopathology. polymorphism of patatin-like phospholipase-3 (pnpla3) has been associated with susceptibility to non-alcoholic fatty liver disease (nafld); whereas genetic studies of nafld in asian indians are not investigated. we investigated the association of polymorphism rs738409 of pnpla3 with clinical, anthropometric and biochemical profiles in asian indians with nafld. methods: in this case-control study, 162 cases and 173 controls were recruited. abdominal ultrasound, clinical, anthropometry and biochemical profiles were determined. fasting insulin levels and value of homeostasis model assessment of insulin resistance (homa-ir) was determined. polymerase chain reaction and restriction fragment length polymorphism of pnpla3 gene was performed. the associations of this polymorphism with clinical, anthropometric and biochemical profiles were investigated. results: higher frequency of c/g and g/g genotypes of rs738409 polymorphism was obtained in cases as compared to controls (p = 0.04), as a consequence frequency of the minor allele g was significantly higher in cases (p = 0.003). the c/g+g/g genotypes was associated with significantly higher fasting insulin (p = 0.002), homa-ir (p = 0.05), alanine transaminase (p = 0.003) and aspartate transaminase (p = 0.04). using a multivariate logistic regression model after adjusting for age, sex, body mass index and fasting insulin, subjects with g/g genotype showed higher risk of nafld (or, 1.98, 95% ci: 1.43-2.73, p = 0.04). results: group 1; before diet: bg-4.3 ae 0.4 mmol/l; 3 months on diet: 6 ae 0.5 mmol/l (6.1-7.8 mmol/l) 4 months: 10.6 ae 2.4 mmol/l (8.6-12.6 mmol/l); histology: marked necrosis and destruction of bcells on 30-35% islet's surface in 32 islets from 47 (68%); in 32% not marked necrobiosis or without changes; decreasing of insulin content in b-cells: ig-1.22 ae 0.03 (intact-1.89 ae 0.04); histology: without changes; group 2: before diet: bg-4.1 ae 0.5 mmol/l; 3 months on diet + vitamin b6: -bg-5.8 ae 0.6 (5.4-6.3 mmol/l); 4 months diet + vitamin b6: bg-6.2 ae 0.5 mmol/l (5.9-6.7 mmol/l); histology: partial necro-biosis on 15-20% islet surface in 18 islets from 54 (33%); in other 67% -without changes; ig-1.61 ae 0.04 (intact-1.86 ae 0.07). conclusions: 2.5 months prolonged administration of vitamin b6 result not marked decreasing of insulin content in b-cells of animals contained on diabetogenic diet and treated by pyridoxin but not protect b-cells of part islets of necrobiosis. aim of this study: to determine the behavior of oxidative stress markers and mitocondrial dysfuncton in non-proliferative diabetic retinopathy. patients and methods: a cross-sectional study was designed with four groups: group 1: 5 healthy-volunteer subjects. group 2: 12 patients with mild-non-proliferative diabetic retinopathy (npdr). group 3: 18 patients with moderate-npdr. group 4: 22 patients with severe-npdr. serum oxidative stress markers: lipid-peroxidation (lpo measured by malondialdehyde and 4-hidroxyalkenals), nitric oxide (no metabolites measured by nitrites/nitrates), total antioxidant capacity (tac), activities of antioxidant-scavenger-enzymes in erythrocytes (gpx glutathione-peroxidase and catalase), and mitochondrial dysfunction (expressed in erythrocyte/platelet membrane fluidity and platelet hydrolytic activity of adenosine-thriphosphatase enzyme; atpase). the markers were quantified by colorimetric method. results: patients with npdr had a significant increased serum lpo and no metabolites levels compared to the group 1. the tac in patients with npdr had a significant decreased compared to the control group. a significant increased in the activities of gpx, catalase and atpase was shown in the experimental groups compared to healthy-volunteer subjects. a significant reduction was shown in membrane fluidity in patients with npdr. the results show that oxidative stress and mitochondrial dysfunction are associated to npdr and its severity. background: weight loss after rous-y gastric bypass (rygb) surgery is associated with alteration of body composition and visceral fat mobilization. we analyzed the amount of fat and fat-free mass reduction in order to identify factors that induce more favorable adiposity change. methods: morbidly obese 40 patients (age = 41 ae 11 years) underwent rygb between september 2009 and july 2001. anthropometry, dual-energy x-ray absorptiometry (dexa), computed tomography (ct), life style report, and laboratory test results were registered prior to and 1 year after rygb. results: follow-up rate was 97.5%. dexa demonstrated 27.6 ae 18.0 of %ffml (percentage of weight lost as fat-free mass) with reduction of 44% of total fat amount. ct demonstrated higher rate of visceral adipose tissue (vat) reduction rather than subcutaneous adipose tissue with 2.1 of %dv/%ds (percentage change in vat vs. sat). patients with diabetes mellitus preoperatively showed more favorable visceral fat reduction (%dv/%ds were 2.4 in dm and 1.5 in non-dm) even though they lost more fat-free mass. objective: prader-willi syndrome (pws), a genetic disorder characterized by childhood-onset obesity, is reported to have elevated levels of adiponectin. the actions of adiponectin are mediated by adiponectin receptors (adipors) which include adipor1 and adipor2. several cytokines such as adiponectin, tnf-a, and il-6, have been known to be involved in insulin sensitivity. methods: thirty pws children (median age 7.1 year, 18 boys, 12 girls) who were being receiving growth hormone (gh) therapy and 32 obese children not receiving gh therapy (median age 9.1 year, 15 boys, 17 girls) were compared. the relative expression of adiponectin, adipors, several proinflammatory cytokines including tnf-a, and il-6 measured in peripheral blood mononuclear cells (pbmcs) using real-time pcr. their correlation was analyzed by homeostasis model assessment insulin resistance index (homa-ir). the pws children showed increased expression of adipor2 (p = 0.02) and decreased expression of il-6 (p = 0.03) compared to the obese children. there was a significant positive correlation between the adipors and tnf-a (adipor1 vs. tnf-a: r = 0.66, p < 0.001 in pws, r = 0.80, p < 0.001 in control group; adipor2 vs. tnf-a: r = 0.69, p < 0.001 in obese group). the adipors in the obese group showed significant negative correlation with homa-ir (adipor1 vs. homa-ir; q = 0.41, p = 0.02, adipor2 vs. homa-ir; q = 0.46, p < 0.01). conclusion: in result, inflammatory cytokine expression was closely associated with the expression of the adipors in the pbmcs of both the children with pws and the obese group. adipor2 expression was highly expressed in the pbmcs of the children with pws. august to november 2012, homeostasis model assessment of insulin resistance (homa-r) were calculated. as a control, subjects were selected, who received oral glucose tolerance test (ogtt) between the same period showing normal glucose tolerance pattern. we used mann-whitney u-test and p-values <0.05 were considered statistically significant. results: there was no significant difference in sex, age (51.7 ae 13.6 vs. 45.3 ae 13.7), bmi (21.5 ae 2.01 vs. 21.4 ae 2.38), hba1c (5.48 ae 0.29 vs. 5.31 ae 0.37) and fasting plasma glucose level (93.0 ae 6.99 vs. 90.7 ae 7.61) between the gd group (n = 6: two males and four females) and control group (n = 6: one male and five females). the gd group showed significantly higher homa-r than control group (1.49 ae 0.35 vs. 1.01 ae 0.25, p < 0.05). discussion: this study showed that gd patients with normal thyroid function had insulin resistance independent of bmi and hba1c. conclusion: gd, even in euthyroid state, is associated with insulin resistance. introduction: it is still remain uninvestigated the correlation of prebiotics and leptin and ghrelin level in frame of inflammatory process in liver. aims and methods: the aim of our study was investigation of nifuroxazide and prebiotic therapy influence on leptin and ghrelin levels in nonalcoholic fatty liver disease patients. determination of leptin and ghrelin (elisa), insulin, il1-β, tnf-a were performed in all patients. results: n = 73 patients, 57 female, age 42 ae 7 years, bmi 32 ae 2 kg/m 2 . nafld in 97%, nash in 33% and ir in 58%. leptin was higher in women (24.0 ae 3.2 vs. 16.2 ae 2.2 ng/dl, p = 0.01), but ghrelin was similar (22.5 ae 2.1 vs. 21.1 ae 3.4 pg/ml). leptin positively correlated with bmi (r = 0.48, p = 0.01) and ghrelin correlated (r = 0.25, p = 0.05). ghrelin correlated with glycaemia (r = 0.245, p = 0.030) and correlated to homa (r = 0.52, p = 0.03). both hormones positively correlated to il1-β and tnf-a (ghrelin: r = 0.39, p = 0.01; r = 0.31, p = 0.01, respectively; leptin: r = 0.48, p = 0.04; r = 038, p = 0.02, respectively). prebiotic (npc bic) consumption significantly decreased ghrelin, insulin, il1-β, tnf-a level and had no influence on leptin level. the results of liver biopsy (histological examination) and fibromax test showed, that in patients additionally treated by prebiotics, the progress of hepatic fibrosis was significantly slowly, then in cg (fibromax test result: f1 vs. f3; p < 0.05). conclusion: our data showed that nifuroxazide and prebiotic therapy decreased inflammation activity and ghrelin level in nafld. cardiology, 2 physiology, medical university of białystok, bialystok, poland aim: ceramide (c) is considered to be an important factor reducing insulin sensitivity. the aim of the present study was to investigate the effect of reduction of the sphingolipid synthesis in the solid tissues on the level of c, and other bioactive sphingolipids, namely sphingosine-1-pohosphate (s1p) sphingosine (sp), sphinganine (sa) and sphinganine-1-phosphate (sa1p) in different blood compartments. the experiments were carried out on three groups of male wistar rats, 230-250 g of body weight fed ad libitum on a commercially available diet: (i) control, (ii)treated with myriocin (an inhibitor of sphingolipid de novo synthesis) and (iii) treated with nicotinic acid (it reduces the level of plasma free fatty acids). the level of the above mentioned bioactive sphingolipis was determined with the use of mass spectrometry. results: both treatments reduced profoundly the level of each of the examined compounds in the plasma. treatment with nicotinic acid did not affect the level of either compound in erythrocytes but reduced it to a great extend in the platelets. treatment with myriocin reduced the level of each compound, with the exception of sh, in erythrocytes. the compound reduced the level of each compound in the platelets to the level comparable to nicotinic acid. 1. the level of the examined sphingolipids in the plasma, erythrocytes and platelets depends thoroughly on their supply from solid tissues. 2. short-term reduction in the plasma free fatty acids level very efficiently reduces the level of bioactive sphingolipids in different blood compartments. background: maternal nutrition plays major role in fetal growth and development. low birth weight and impaired early postnatal growth predispose the offspring to an increased risk for future chronic diseases such as metabolic syndrome. stunting in south african children has previously been documented. the study aimed to investigate effects of maternal nutrition on fetal and early postnatal growth in the population of 78 black urban pregnant south african women, employing ffq, ultrasound and anthropometry measurements. results: based on the z-scores (at birth, 3 weeks and 6 month), babies in the current study were born lighter (à0.2) and shorter (à0.05) with larger head circumference (+0.38) in comparison with the 2010who child growth standards. the z-scores for both the weight and the lengthfor-age decreased after birth with subsequent increase at 6 months, being significant for length (à0.63, p = 0.003) after birth and for weight (+0.14, p < 0.001) at 6 months. maternal intake of polyunsaturated fat in early pregnancy and of total protein in late pregnancy were found associated with the fetal head-to-abdomen circumference (β = à0.222, p = 0.048 and β = à0.366, p < 0.001 respectively) and with the lengthfor-age z-score at 3 weeks (β = 0.320, p = 0.010 and β = 0.316, p = 0.009 respectively). early plant protein intake significantly correlated with fetal growth rate (β = 0.316, p = 0.004). conclusion: low maternal pregnancy intake of protein, namely of plant protein, and of polyunsaturated fat affected fetal linear growth, and resulted in a possible "brain sparing effect" in fetus. maternal dietary manipulation during pregnancy may therefore affect fetal and postnatal growth and thus modulate the risk of chronic disease later in life. aims and methods: to assess the efficacy of hb in cp patients in a two centre randomised, double-blind, placebo-controlled, crossover trial. one hundred and twenty-six patients with chronic pancreatitis were exposed to screening blood tests and test of elastase in stool before randomization to placebo or hb (buscopan, boehringer ingelheim) for 6 weeks. all patients followed the basic treatment scheme include dietary and physical regimen. drug effect was optimized by dose titration during weeks 1-2 starting at 30 mg daily, increasing (max 60 mg t.d.s) or decreasing as required. methods: brown beans, or white wheat bread (wwb, reference product) were provided as evening meals to 16 healthy young adults in a randomized crossover design. markers of glucose-and appetite regulation, glp-2, and markers of inflammation were measured at a following standardized breakfast, i.e. 11-14 h after the evening meals. colonic fermentation activity was estimated from measurement of plasma short chain fatty acids (scfa) and breath hydrogen (h 2 ) excretion. results: an evening meal of brown beans, in comparison with wwb, lowered blood glucose (à15%, p < 0.01)-and insulin (à16%, p < 0.05) responses, increased satiety hormones (pyy 51%, p < 0.001), suppressed hunger hormones (ghrelin: à14%, p < 0.05), and hunger sensations (à15%, p = 0.05), increased glp-2 (8.4%, p < 0.05) and suppressed inflammatory markers (il-6 à35%, and il-18 à8.3%, p < 0.05) at a subsequent standardized breakfast. breath h 2 (141%, p < 0.01) and plasma scfa (propionate 16% and isobutyrate 18%, p < 0.05) were significantly increased after brown beans. results: the present study revealed significantly lower levels of adiponectin in diabetic children compared to the controls (10.1 ae 1.57 vs. 11.23 ae 1.14 lg/ml). it also showed significantly increased carotid intima media thickness (cimt) in diabetic children compared to the control group (0.55 ae 0.08 vs. 0.43 ae 0.04 mm). there was also positive correlation between the mean cimt and age of the patient, age of onset of diabetes mellitus, hba 1 c, and bmi measurements in diabetic children. there was negative correlation between mean cimt and adiponectin level. conclusions: subclinical microvascular disease in type 1 diabetes mellitus begins early in diabetic children, which emphasize the importance of early detection and control of vascular risks in diabetic children. the study also suggests that adiponectin may prove to be useful marker of cardiovascular risks, and potential therapeutic target for risk prevention in diabetics. in 22 overweight and obese individuals (7f/11m; age 33 ae 10 years, bmi = 30 ae 5 kg/m 2 ). an intra-venous glucose tolerance test, hyperinsulinaemic-euglycaemic clamp and circulating markers relevant to age signalling were performed before and after each diet. results: the high age diet was fivefold higher in age content than the low age diet. the high age diet reduced insulin sensitivity by à1.1 mg/kg/min (95% ci, à2.0 to à0.1; p = 0.03) while the low age diet improved insulin sensitivity by +1.0 mg/kg/min (+0.1 to +1.9; p = 0.03). the overall change in insulin sensitivity was 2.0 mg/ kg/min (1.0-3.1, p = 0.001). the change insulin secretion was correlated inversely with the change in plasma age (cml) concentration (r = à0.52, p = 0.02). to investigate the level of transforming growth factor (tgfbl) and basic fibroblast growth factor (bfgf), non-specific markers of inflammation: interleukin-6 (il-6), tumor necrosis factor-a (tnf-a) in patients with coronary heart disease (chd) depending on the presence of dm2. the study involved 38 people: one group à18 patients with chd without carbohydrate metabolism disorders, two group -20 patients with dm2 and chd. blood samples were taken from the cubital vein and the aorta during coronary angiography. .03)], as in arterial and venous blood in the presence of chd and dm2 (p < 0.05). 1. availability dm in patients with chd was associated with significant increase in the level of tgfb1, il-6. these observation reflect the effect of chronic hyperglycemia on the restructuring of the connective tissue and the vascular wall. 2. tgfb1 direct correlation with lipid markers confirms the relationship of connective tissue disorders and lipid metabolism in the pathogenesis of atherosclerosis. obesity can be induced with high fat diets (hfd) and is associated with inflammation in white adipose tissue (wat) and liver. the factors that control the early metabolic responses to hfd and that trigger inflammatory gene expression are only poorly understood. a time-resolved analysis of differentially expressed genes in expanding adipose tissue of mice (12 weeks hfd feeding) identified specific clusters of lipid metabolism-related genes and inflammation-related genes with similar time expression profiles. subsequent promoter analysis of the clustered genes revealed that specific master regulators (among which fos, esr1, hnf4a, jun, ppara, pparg, nr1 h2/lxrb, nfkb, srebf1 and 2, sfpi1, smad2, sp1) orchestrate metabolic adaptations and early inflammatory responses in wat. some of these transcription factors (esr1, jun, fos, pparg, sp1) have a dual role and regulate the adjustment of lipid metabolism as well as expression of inflammatory genes such as cxcl1/kc, ccl5/rantes, complement factors, asc, granzyme a ccl5/rantes, ccl6, ccl7/mcp3. subsequent analysis of corresponding livers revealed comparable molecular responses on the level of transcription factors. more specifically, many master regulators identified in wat were also involved in the liver response to hfd as demonstrated by analysis of hepatic target gene expression in conjunction with transcription factor binding activity analysis. our findings support the view that metabolic and inflammatory processes are interlinked in wat and liver, and that responses to hfd are controlled in a similar way on the transcription factor level. distortions of the mechanisms which control metabolic homeostasis in these organs may thus also affect their inflammatory tone. aim: to investigate the effects of iptakalim, a novel sur2b/kir6.1type atp-sensitive potassium channel opener, on endothelial dysfunction induced by insulin resistance (ir) and to determine whether iptakalim improved ir associated with hypertension in fructose-fed rats (ffrs) and spontaneously hypertensive rats (shrs). methods: the levels of endothelial vasoactive mediators and enos protein expression were determined usingelisas or western blot. in both ffrs and shrs, hyperinsulinemic-euglycemic clamp was used to evaluate ir states. 1. cultured human umbilical vein endothelial cells (huvecs) incubated with the pi3-kinase inhibitor wortmannin (50 nmol/l) and insulin (100 nmol/l) induced endothelial dysfunction characterized by reduced release of no and expression of enos protein, and increased production of et-1. pretreatment with iptakalim (0.1-10 lmol/l) could potently prevent the endothelial dysfunction by increasing no production and inhibiting et-1 release. 2. in ffrs, the levels of sbp, fasting plasma glucose and insulin were elevated, whereas the glucose infusion rate (gir) and insulin sensitive index (isi) were significantly decreased, and the endothelium-dependent vascular relaxation response to ach was also impaired. these changes could be prevented by administration of iptakalim for 4 weeks. the imbalance between serum no and et-1 was also ameliorated by iptakalim. 3. in 2-4 month-old shrs (ir was established at the age of 4 months), oral administration of iptakalim for 8 weeks significantly ameliorated hypertension and increased the gir to the normal level. conclusion: iptakalim could protect against ir-induced endothelial dysfunction, and ameliorate ir associated with hypertension, via restoring the balance between no and et-1 signaling. m. ste z pie n 1 , a. ste z pie n 2 , r.n. wlazeł 3 , m. paradowski 3 , m. banach 4 , j. rysz 2 material and methods: patients (f 34, m 20) were divided according to bmi into three groups: a-obesity i 0 (n = 25); b-obesity ii 0 (n = 14) and c-obesity iii 0 (n = 15) and into other four groups: females and males and patients treated with statins or fibrates (n = 31) or untreated (n = 23). results: leptin was significantly higher in group c compared to group a and b [57.2 (31.9-94.9) vs. 34.3 (12.5-49.0) ng/ml, p < 0.01 and vs. 30.8 (26.0-50.2) ng/ml, p < 0.05 respectively]; hs-crp were higher in group c than in group a [3.6 (2.3-5.0) vs. 1.7 (1.1-2.6) mg/ l, p < 0.01]. adiponectin and leptin were higher in females [18.0 (12.9-26.7) vs. 10.9 (6.4-14.6), p < 0.0001 and 49.6 (37.1-63.1) vs. 16.9 (7.9-30.4), p < 0.0001, respectively]. resistin, hs-crp and il-6 were higher in untreated patients' group [1.04 (0.86-1.31) vs. 0.83 (0.70-1.04) ng/ ml, p < 0.05; 3.0 (1.8-5.0) vs. 1.7 (1.1-3.5) mg/l, p < 0.05 and 14.5 (10.3-19.9) vs. 8.9 (8.9-13.1) pg/ml, p < 0.01 respectively]. leptin positively correlated with hs-crp in the whole population (r = 0.318, p < 0.05), in treated patients' group (r = 0.434, p < 0.05) and with tnf-a in group c (r = 0.506, p = 0.05). conclusions: leptin may be associated with chronic inflammation in obese hypertensive patients. serum leptin and adiponectin levels are sex dependent. hypolipemic treatment has impact on chronic inflammation and resistin. lean body mass in genetically obese (ob/ob) or anorectic/cachectic subjects is severely reduced. similar outcomes of two different pathological states prompted us to wonder if leptin, adipokine well known from its control of appetite interacts with myogenesis. apparently, recombinant leptin (100 ng/ml) stimulated dna synthesis in mononuclear myoblasts together with the increase of t 202 /y 204 p-erk1/2 protein expression levels. additionally, leptin reduced cell viability and muscle fiber formation from c2c12 mouse myoblasts. detailed short-and long-term examination with the use of metabolic inhibitors revealed that both jak/stat3 and mek/mapk but not pi3-k/akt/gsk-3beta signaling pathways were stimulated by leptin, and that stat3 (y 705 p-stat3) and mek (t 202 /y 204 p-erk1/2) control these effects. in turn, insulin promoted pi3-k-dependent phosphorylation of akt (s 473 ) and gsk-3beta (s 9 ) and insulin overruled leptin-dependent inhibition of myogenic differentiation in pi3-k-dependent manner. gsk-3beta might play dual role in muscle development. insulin-induced effect on gsk-3beta (s 9 p-gsk-3beta) facilitated myotube formation. in contrast, leptin through mekdependent manner led to gsk-3beta phosphorylation (y 216 p-gsk-3beta) with resultant retardation of myoblast fusion. in summary, to some extent opposite effects of insulin and leptin on skeletal muscle development emphasize the importance of intercellular signaling between adipose tissue and skeletal muscle. insulin and leptin determine how muscle mass is gained or lost, respectively. objective: obesity is linked to both increased metabolic disturbances and increased adipose tissue macrophage infiltration. however, whether macrophage infiltration directly influences human metabolism is unclear. the aim of this study was to investigate if there are obesity-independent links between adipose tissue macrophages and metabolic disturbances. methods: expression of macrophage markers in adipose tissue was analyzed by dna microarrays in the sos sib pair study and in patients with type 2 diabetes and a bmi-matched healthy control group. results: the expression of macrophage markers in adipose tissue was increased in obesity and associated with several metabolic and anthropometric measurements. after adjustment for bmi, the expression remained associated with insulin sensitivity, serum levels of insulin, c-peptide, high density lipoprotein cholesterol (hdl-cholesterol) and triglycerides. in addition, the expression of most macrophage markers was significantly increased in patients with type 2 diabetes compared to the control group. conclusion: our study shows that infiltration of macrophages in human adipose tissue, estimated by the expression of macrophage markers, is increased in subjects with obesity and diabetes and associated with insulin sensitivity and serum lipid levels independent of bmi. this indicate that adipose tissue macrophages may contribute to the development of insulin resistance and dyslipidemia. hp gene has been demonstrated to be a major determinant of susceptibility to cvd and in the development and progression of dn. hp2 allele is defective in its ability to protect against oxidative stress cvd and dn. we decide to assess the intracellular localization of iron in the pctcells and its potential toxicity in the development and progression of dn. methods: wild type c57b1/6 mice have only an hp 1 allele. we genetically engineered a murine hp 2 allele and inderted it in the murine hp locus by homologous recombinatiobn. we induced dm, by stz for 5 days, at 10 weeks of age we assessed lysosomal membrane integrity, redox-active iron in kidney lysosomes. 1. increased iron-rich deposits in lysosomes of pct cells in hp 2-2 dm vs. hp 1-1 dm (65 ae 4% of all lysosomes) compared with hp 1-1 dm mice (41 ae 4% of all lysosomes, p < 0.05). 2. intralysosomal redox-active iron concentrations are markedly increased in hp 2-2 dm mouse kidneys lysosomes of hp 2-2 dm mice (0.56 ae 0.07 lmol/l) as compared with those from hp 1-1 dm mice (0.23 ae 0.14 lmol/l, p = 0.036). lysosomal membrane lipid peroxides are increased in hp 2-2 dm proximal tubule cell (p < 0.0001). vitamin e supplementation resulted in a 45% reduction in lysosomal redox-active iron in hp 2-2 dm mice (p < 0.04). conclusion: a novel mechanism whereby the hp genotype may predispose to renal injury in the setting of dm via increased iron deposition in the lysosomes of pct. purpose: we aimed to use other less hazardous route of insulin injection to prevent pharmacokinetic problems of sc insulin and find out response categories in diabetics and prediabetic patients. besides, fbs and hba1c are markers for treatment evaluation; we wondered whether aitt could be a marker of residual pancreatic activity regeneration or decelerating progression of type ii diabetes. methods: five hundred diabetic and 65 patients with impaired glucose tolerance were enrolled to this trial. three hours after breakfast they receive 10-50 unit equivalents of insulin into the external auditory cannel. blood glucose level was measured every 15 min for 3 h. this was repeated 6 months later in both groups; while 35 prediabetic patients received some sorts of therapy while the other 30 patients remained on their previous lifestyle. auc and best fit curve were evaluated by partial mathematical integration. results: six different prototypic curves were extrapolated from diabetic patients with the possibility of excellent theoretic explanations about etiology and multifactoriality, lifestyle change, drug(s) of choice and prognosis. parallel to this prediabetic patients showed comparable curves. the area under curve in treated prediabetics showed a small but significant reduction compared to untreated (217 vs. 226, p = 0.04). conclusions: as shown elsewhere the area under curve after an auditory insulin tolerance test is a nice marker in diabetes response evaluation and prognosis and an excellent marker for progression to diabetes and effectiveness of treatment in prediabetics (<215 compared to >235). introduction: type ii dm is generally regarded as a progressive disease with control of bs becoming more difficult with time. we wondered whether optimal therapy with "mega-treatments" that can let the pancreas rest could reverse the disease. materials and methods: thirty five patients received optimal doses of insulin through the auditory channel for 6 months. contributing factors such as obesity, anxiety, h. pylori infection and reduced physical activity were treated appropriately. at beginning and every month they were evaluated by way of auditory insulin tolerance test after 24 h of drug vacation. the general appearance of the curve, the partial integration of it and the overall area under curve were compared monthly. results: auc declined from a mean of 289-257 from first to sixth month meaning reduced insulin resistance (p < 0.03). this decline was steady during the whole 6 months and minor variations in different patients were not significant. with mathematical calculation it seems very unlikely that this decline becomes asymptotic so that on extension of the curve for 3-5 years at least 30% of the patients eventually fall into the nondiabetic range with auc under 450. (p < 0.1) although different definitions exist for dm, auditory insulin tolerance test might be functionally more illuminating. these results are in concordance with available literature that denotes early aggressive treatment might be better. we add to this notion that early aggressive treatment can possibly reverse type ii diabetes. physiology, universidad de guadalajara, guadalajara, mexico background: c-reactive protein (crp) is an acute stage protein whose serum levels become raise by infection or because an undergoing metabolism disruption. our group has found crp variable serum levels in hypertensive patients that course with obesity and dyslipidaemia. the crp polymorphism rs1205g>a has been associated to lowered crp serum levels in autoimmune diseases. the role of c-reactive protein (crp), a marker and mediator of inflammation, in the pathogenesis of metabolic syndrome and its complications such as non-alcoholic fatty liver disease (nafld) remains to be elucidate. in this study we investigated whether increased levels of human crp itself can promote increase of glucose and lipid metabolism disorders associated with metabolic syndrome. materials and methods: spontaneously hypertensive rats (shr) with transgenic expression of human crp gene under apolipoprotein e promoter (shr-tg) in age 1-year and age matched non-transgenic shr controls were used. both groups were fed a high fructose diet (60% cal fructose) for 2 weeks. parameters of insulin resistance and oxidative stress were measured by commercially available kits. results: transgenic expression of crp was associated with significant increase of serum triacylglycerols (0.84 ae 0.05 vs. 0.64 ae 0.037 mmol/l, p < 0.05) and insulin levels (p < 0.05), markedly decrease in insulin stimulated 14 c-glucose incorporation into muscle glycogen (174 ae 8 vs. 278 ae 35 nmol/g/2 h, p < 0.05), reduced serum adiponectin (2.4 ae 0.3 vs. 4.3 ae 0.6 mmol/l, p < 0.05), and microalbuminuria (p < 0.005). transgenic expression of crp was associated with increased liver triglyceride concentrations (7.62 ae 0.9 vs. 4.30 ae 0.57 lmol/g, p < 0.05), decreased liver glutathione peroxidase activity (p < 0.01) and reduced glutathione concentration (p < 0.01). liver lipidperoxidation were elevated in shr-tg rats: tbars (p < 0.01). conclusion: overexpression of human crp induced insulin resistance, oxidative stress and liver steatosis in shr rats. these finding indicate that chronically inflammation might directly contribute to the pathogenesis of metabolic syndrome and nafld. one of the most topical issues of modern pediatrics is obesity in children and adolescents, which has a tendency to doubling every three decades in almost all countries. the aim of our research was to investigate the fatty acid composition of the blood in children with obesity. patients and method: we investigated 50 children aged from 7 to 15 years, divided into two groups: group i -25 children (64% boys and 36% girls) who are overweight or obese (bmi 29.68 ae 1.21 kg/ m 2 ), group ii -25 persons (85% boys and 15% girls) with normal body weight (bmi 19.45 ae 0.05 kg/m 2 ). research methods: quantitation of omega-3 and omega-6 polyunsaturated fatty acids, determination of the total amount of fatty acids, the total content of eicosapentaenoic and docosahexaenoic acids by gas chromatography with mass selective detection and determination of the omega-3 index (the ratio of the sum of eicosapentaenoic and docosahexaenoic acids to total content of fatty acids in %). results: there was a direct correlation between the value of bmi and the increasing of omega-3 index and the omega-6 pufas in children with obesity, while for the children with normal body weight the connection of bmi with the change of the fatty acid composition of blood was not obtained. thus changes of the quantitative and qualitative fatty acid composition of blood in obesity children and adolescents had multidirectional nature and require further dynamic study. a. kopp, a. schmid, m. mü ller, a. schä ffler internal medicine i, university hospital regensburg, regensburg, germany introduction: proteins secreted by adipocytes (adipokines) play an important role in the pathophysiology of type 2 diabetes mellitus and the associated chronic and low-grade state of inflammation. it was the aim to characterize the anti-inflammatory potential of the new adipokine, c1q/tnf-related protein-3 (ctrp-3), which shows structural homologies to the pleiotropic adipokine adiponectin. in earlier studies, recombinant ctrp-3 has been shown to inhibit lps and lauric acid induced release of pro-inflammatory cytokines and chemokines dose-dependently in monocytes and adipocytes. methods: for in vivo analysis, male c57bl/6 mice were treated by intraperitoneal lps administration for 2 h. anti-inflammatory effects were tested by pre-treatment (30 min) with ctrp-3. after killing, epididymal adipose tissue was collected for cytokine mrna expression analysis (real-time rt-pcr) and blood for measurements of circulating cytokine levels (elisa). the anti-inflammatory potential previously found in vitro is also seen in lps-treated c57bl/6 mice. animals pre-treated with ctrp-3 have lower levels of inflammatory cytokines such as interleukin-6 (il-6) and macrophage inflammatory protein-2 (mip-2). furthermore, mrna expression of il-6 and mip-2 in the epididymal adipose tissue is significantly reduced by ctrp-3. conclusion: ctrp-3 acts anti-inflammatory in cells and tissues that are involved in obesity and type 2 diabetes mellitus. therefore, it might be an interesting drug target in treating obesity-related chronic inflammation. objective: the aim of the study was to assess the burden of some metabolic syndrome (ms) risk factors in polish adolescents from two less-urbanized regions as well as the relationship between abdominal obesity, dyslipidemia and hypertension incidence. the study involved 299 adolescents aged 13-18. all subjects lived in two less-urbanized regions of poland (small towns and villages in the central and north-eastern regions). the concentration in blood of triglycerides (tg) and hdl-cholesterol (hdl-c), as well as systolic blood pressure (sbp) and waist-to-height ratio (whtr) were determined. using logistics regression, an odds ratio (or) of the incidence of high whtr ( ! 90 percentiles), high tg ( ! 150 mg/dl), high spb (>90percentiles) and low hdl-c (boys: <40 mg/dl; girls: <50 mg/dl) was calculated. results: high whtr was found in 4% of adolescents, high tg in 5%, high spb in 11% and low hdl-c in 14%. adolescents with abdominal obesity (whtr ! 90 percentiles) had an or rated as high spb at 4.24 (95% ci = 1.19-15.14; p < 0.05), an or rated as high tg at 3.31 (95% ci = 0.65-16.93; p > 0.05) and an or rated as low hdl-c at 2.49 (95% ci = 0.72-8.62; p > 0.05) in comparison to adolescents with normal whtr (between 10 and 90 percentiles). adolescents from north-eastern poland had an or rated as high spb at 0.08 (95% ci = 0.02-0.26; p < 0.05) and an or rated as high tg at 0.06 (95% ci = 0.01-0.46; p < 0.05) in comparison with the central region (or = 1.00). conclusions: dyslipidemia and hypertension were the main metabolic syndrome risk factors in polish adolescents from less-urbanized regions, especially in central poland. the risk of hypertension rose over 4 times in adolescents with abdominal obesity. objective and aims: as employment has become more sedentary in nature, there is a potential for more working individuals to be at risk of developing the metabolic syndrome (mets). physical activity (pa) is recommended for prevention of such chronic conditions. this study investigates self-reported pa and presence of mets in the workplace as part of the established "prosiect sir gâr" initiative in south wales, uk. methods: two hundred and twenty-one male steel workers (sw) and 92 male local health board (lhb) employees were screened and their data analysed. anthropometric data, blood pressure and self-reported physical activity (gppaq) were all recorded alongside obtained blood samples which were subsequently analysed for high-density-lipoprotein cholesterol (hdl-c). presence of the mets was determined based on the following idf criteria: central obesity (waist circumference: ! 94 cm), reduced hdl-c levels (<1.03 mmol/l) and either systolic ( ! 130 mmhg) or diastolic ( ! 85 mmhg) hypertension. results: the proportion of males diagnosed with mets was comparable between worksites (sw: 25.8% vs. lhb: 26.1%; p = 0.96) despite the sw reporting being more physically "active" or "moderately active" than their lhb counterparts (92.8% vs. 67.3%; p < 0.001). central obesity, reduced hdl-c levels and diastolic hypertension were comparable between worksites (p > 0.05), although systolic hypertension was higher amongst lhb employees (64.1% vs. 39.8%; p < 0.001). however, more sw were found to be clinically obese (33.2% vs. 25.3%; p = 0.02). introduction: diabetes mellitus is often a silent disease and its prevalence is increasing rapidly worldwide. nonalcoholic steatohepatitis is certainly the less estimated complication of diabetes in frequency and severity. the aim of our study was to evaluate the prevalence of steatohepatitis and to identify its clinical and biological risk factors. materials and methods: the prospective transversal study was comparative between 120 type 2 diabetics (group 1) and 60 healthy people (group 2) with no chronic hepatopatic diseases, no alcoholism and no intake of hepatotoxic drugs. all have benefit from a clinical exam, biological assay and abdominal echography seeking for steatohepatitis. results: group 1 has statistically a higher frequency of android obesity, insulin resistance, steatohepatitis, high blood pressure, hypertriglyceridemia, chronic inflammation and silent myocardial ischemia in comparison with group 2. the steatohepatitis was present in 69% of patients vs. 24.6% of healthy people; p < 0.001. also, patients from group 1 with steatohepatitis (group 1a) had a body mass index (bmi), waist circumference, a percentage of fat mass, a level of triglycerides, of alanine aminotransferase (alat) higher than diabetics without steatohepatitis (group 1b), with a risk of metabolic syndrome multiplied by three. with multivariate analysis, we found that in group 1, alat and bmi were directly associated to steaohepatitis. our study notifies the frequency of steatohepatitis in diabetics. it could be prevented and treated by loss of weight and regular physical activity in order to reduce insulin resistance. objectives: vascular dysfunction and complications are the major cause of mortality in diabetic patients. arterial stiffness has been known as a useful predictor of atherosclerosis and lipoprotein level is one of major risk factors of atherosclerosis. to investigate the association of lipoprotein level with arterial stiffness, we studied the vascular characteristics of patients with diabetes. methods: fifty patients (male:female = 26:24, mean age 63.3 ae 11.3 years), with diabetes and without coronary artery disease, were enrolled and evaluated. all patients have been taking angiotensin receptor blocker or angiotensin converting enzyme inhibitor. arterial stiffness was assessed by measuring the carotid-radial pulse wave velocity (pwv). the cardiovascular risk factors, including body mass index, lipid profile, pulse pressure, c-reactive protein, flow-mediated vasodilatation (fmd) were also measured. the pwv was significantly higher in patients with metabolic syndrome than those without metabolic syndrome (1654 ae 127 vs. 1485 ae 131 cm/s, p = 0.03) in diabetic patients. multivariate analysis revealed that hdl cholesterol level, body mass index and metabolic syndrome were highly associated with pwv (p = 0.01). pulse pressure, crp, hdl cholesterol level and metabolic syndrome were significantly associated with fmd. conclusion: in diabetic patients, metabolic syndrome and hdl cholesterol level were highly associated with arterial stiffness and fmd as in non-diabetic patients. objective: our goal was to compare a weight loss diet with high caloric intake during breakfast to an isocaloric diet with high caloric intake at dinner. research design and methods: obese women (bmi 32.4 ae 1.8 kg/m 2 ) with metabolic syndrome were randomized into two isocaloric (~1400 kcal) weight loss groups, a breakfast (bf; 700 kcal breakfast, 500 kcal lunch, 200 kcal dinner) or a dinner (d) group (200 kcal breakfast, 500 kcal lunch, 700 kcal dinner) for 12 weeks. anthropometric measurements, oral glucose tolerance test (ogtt) and meal tests were performed. the bf group showed greater weight loss (à8.7 ae 1.4vs. à3.6 ae 1.5 kg) and waist circumference reduction (à8.5 ae 1.9 vs. à3.9 ae 1.4 cm) compared with the d group. although fasting glucose, insulin and ghrelin were reduced in both groups, fasting glucose, insulin and homa-ir decreased significantly to a greater extent in the bf group. mean triglyceride levels decreased by 33.6% in the bf group, but increased by 14.6% in the d group. after ogtt, the extent of reduction of aucglucose and aucinsulin was greater in the bf (à22% and à58%, respectively) compared with the d group (à15% and à30%, respectively). in response to meal challenges, the overall daily aucglucose aucinsulin, aucghrelin and mean hunger scores were significantly lower, whereas mean satiety scores were significantly higher in the bf group. conclusions: an isocaloric diet with switched caloric intake during breakfast or dinner differentially influences weight loss and metabolism. high-calorie breakfast with reduced intake at dinner might be a useful alternative for the management of obesity and metabolic syndrome. centre for biomedical sciences, cardiff metropolitan university, cardiff, uk introduction: we have reported that exercise alters markers of monocyte/macrophage polarisation in peripheral polymorphonuclear cells, decreasing the m1:m2 marker ratio. since the m2 phenotype is considered "anti-atherosclerotic", this may be beneficial in reducing cardiovascular disease risk. this study aimed to determine whether exercise specifically promotes m2 marker expression in purified monocytes, and to elucidate the molecular mechanisms involved in this process. methods: twenty-six healthy, sedentary individuals (43 ae 10 years) participated in a low-intensity exercise programme (walking, 45 min, thrice weekly for 8 weeks). peripheral monocytes were isolated using magnetic immuno-separation, and gene expression was determined by rt-pcr. serum lipids, insulin and glucose levels were measured by standard biochemical methods. the presence of serum pparc ligands was determined by gene reporter assay. results: during the 8-week exercise programme, pparc ligand generation and cox-2 gene expression were increased [1.5 ae 0.6-and 3.9 ae 2.5-fold, respectively (p < 0.05)]. similarly, pparc-dependent genes (e.g. cd36) and m2 marker (e.g. dectin-1) expression increased (2.5 ae 1.8-(p < 0.05) and 1.8 ae 0.7-fold (p < 0.1), respectively), while exercise-associated increases in pro-inflammatory m1 markers were blunted [e.g. mcp-1 0.7 ae 0.8-fold (p < 0. conclusion: low-intensity exercise may prime monocytes for differentiation into m2-polarised macrophages (possibly via pparcdependent events) and contributes to improved insulin sensitivity. this data supports exercise prescription for the prevention and management of inflammatory-linked diseases such as atherosclerosis and diabetes. quantify and determine possible differences and their correlation with plasma leptin and ghrelin levels. for this study, 40 male rats were underwent a case control study. the fecal bacteria composition was investigated by pcr-denaturing gradient gel electrophoresis and realtime qpcr. in restricted eaters, we have found a significant increase in the number of proteobacteria, bacteroides, clostridium, enteroccoco and prevotella and a significant decrease in the quantities of actinobacteria, firmicutes, bacteroidetes, b. coccoides-e. rectale group, lactobacillus and bifidobacterium with respect to unrestricted eaters. we also found a significant positive correlation between the quantity of bifidobacterium and lactobacillus and plasma leptin levels, and a significant and negative correlation among the number of clostridium, bacteroides and prevotella and plasma leptin levels in all the experimental groups. furthermore, plasma ghrelin levels were negatively correlated with the quantity of bifidobacterium, lactobacillus and b. coccoides-eubacterium rectale group and positively correlated with the number of bacteroides and prevotella. in conclusion, nutritional status and physical activity may have an impact on the gut microbiota composition affecting the diversity and similarity. the significant increase in the quantity of lactic acidproducing bacteria and butyrate-producing bacteria that would also implied an increase in their bacterial metabolites in the exercise rats without dietary restriction could be responsible of the plasma ghrelin levels decrease found in these rats which affect the food intake and the body weight. the aim of this study was to evaluate the relation between weight gain classified according to iom recommendations and macronutrient intake. methods: a cross-sectional study was conducted on 400 women hospitalized in the maternity during august-september 2010. prepregnancy weight was self-reported. gestational weight gain and weight rate gain was determined by means of the weight registered in the maternity before delivery. nutrient intake during pregnancy was evaluated with a 127-item food frequency questionnaire. results: on average, protein provided 15.79% (95%ci: 15.55-16.03), carbohydrate 53.75% (95%ci: 53.09-54.42) and fat 31.69% (95% ci: 31.11-32.28) of the total energy intake. there was a positive and consistent association between energy intake and maternal weight gain among the pregnant women (p = 0.041). the percent of energy from protein was associated with maternal weight gain rate (p = 0.022). women with a weight gain higher then media + sd had a higher intake of protein compared with those with medium (p = 0.04) and lower (p = 0.03) weight rate gain. no significant differences regarding fats and carbohydrates intake was noted. similarily, women with weight gain above the iom recommendations had a significant higher protein intake compared with those with a lower weight gain (p = 0.019). conclusion: a strong association was found only between protein intake and weight gain in the studied sample. a dietary pattern analyses would clarify the relation with weight gain during pregnancy. excessive weight gain and obesity before pregnancy was associated with large for geastational age newborns and prematurity. the aim of the study was to analyse the relation between maternal anthropometric indicators and apgar score. a cross-sectional study was conducted on 400 women hospitalized in the maternity during august-september 2010. multiple pregnancy and obstetrical pathology represented exclusion criteria. self-reported weight was used to estimate prepregnancy bmi. gestational weight gain was determined by means of the weight registered in the maternity before delivery. apgar score was determined by the neonatologist immediately after birth. results: in the sample studied, 5.4% newborn had an apgar score lower than 8, thus indicating neonatal distress. there weren't noticed significant differences according to weight gain during pregnancy. among the mothers with newborns which had an apgar score lower than 8, 18.18% had a weight gain lower, 40.91% higher and 40.91% within the iom′ s recommendations. however, excessive prepregnancy weight was associated with neonatal distress (p = 0.036): 9.37% of women with prepregnacy bmi ! 30 kg/m 2 ; 6.25% of the normal weight and 0% of the underweight gave birth children with low apgar score. conclusion: prepregnancy excesive weight was associated with neonatal distress, strenghtening the importance of achieving an ideal weight before conception. patients and methods used: six obese women (age 28.6 ae 5.4 years, bmi 39.2 ae 4.7 kg/m 2 ) had fasting metabolic measurements taken via indirect calorimetry before and after 8 weeks of a low starch diet. respiratory gases were collected for 30 min using standard procedures in order to determine resting metabolic rate (rmr), respiratory exchange ratio (rer), and macronutrient oxidation (carbohydrate and fat). a paired sample t-test was used to evaluate significant mean differences from pre-to post-diet. results: after 8 weeks on a low starch diet, subjects lost an average of 9.1 ae 1.9 kg despite the fact that this was not designed as a weight loss diet. rer decreased from 0.86 ae 0.05 to 0.76 ae 0.02 (p = 0.007) from pre-to post-diet measurements. further, carbohydrate oxidation decreased from 13.7 ae 5.3 to 5.5 ae 2.6 g/min (p = 0.009), and fat oxidation increased from 4.6 ae 1.7 to 7.5 ae 1.1 g/min (p = 0.013). changes in rmr were not significantly different. background: weight loss improves insulin resistance and hyperandrogenism in obese women with pcos but is unnecessary in lean women with pcos; however, meal timing and composition may influence glucose metabolism and hyperandrogenism. objective: to investigate the effects of two isocaloric diets with different meal timing on insulin resistance and hyperandrogenism in lean women with pcos. methods: sixty lean women with pcos were randomized to one of two 1800 kcal isocaloric diets with different meal timing: the number of young adults with ms is steadily increasing, but ms in children mainly incomplete according to the pediatricians' reports. so it was suggested that there are some flaws in the diagnostic criteria that lead to ms hypodiagnosis. hypothesis: using the clarified criteria may improve quality of ms detection in children. methods: in 208 obese adolescents the ms components were analyzed by idf and clarified criteria. additionally abdominal obesity was analyzed by bmi sd and waist to height ratio; glucose intolerance by ogtt and homa-ir; dyslipidemia by fasting tg, ldl, hdl, tc (according to percentiles by ncep for children); blood pressure by the fourth report on the diagnosis, evaluation and treatment of high blood pressure in children and adolescents. results: the total number of detected ms components by idf vs. clarified ones: "0" in 9.13 ae 3.96% by idf; "1" in 40.87 ae 6.82% vs. 6.77 ae 3.48% (p < 0.001); "2" in 21.15 ae 5.65% vs. 6.77 ae 3.48% (p < 0.001); "3" -11.06 ae 4.33% vs. 20.83 ae 5.63% (p = 0.013); "4" in 12.98 ae 4.66% vs. 34.90 ae 6.61% (p < 0.001); "5" in 4.81 ae 2.97% vs. 30.73 ae 6.39% (p < 0.001). conclusion: using clarified criteria improves the quality of ms detection in children, which corresponds to a ms global concept of identifying high cardiovascular risk group. aim: polycystic ovary syndrome (pcos) is the most common endocrinopathy among women of reproductive age. pcos is connected with ovulatory disorders, oligomenorrhea, hyperandrogenism, infertility, and an increased miscarriage rate, and is frequently associated with insulin resistance (ir). multiple factors in the follicular fluid affect the fertilization and early embryonic development, including in women with pcos. the aim of this study is to evaluate the predictive value of follicular adiponectine regarding pregnancy outcomes in pcos vs. non pcos patients who underwent in vitro fertilization. material/methods: we designed a prospective study. group 1: (n: 59) non pcos and group 2: (n: 27) pcos patients according to roterdam criteria were included the study. basal ovarian reserve parameters, endometrial thickness, follicular luid adiponectine levels, clinical and laboratory ivf outcomes, pregnancy rates were studied. results: there were statistically significant differences in used gonadotropine dose, total oocyte number, mii oocyte number in non pcos vs. pcos groups (p: 0.001). but there were no statisticaly significant differences between the groups according to follicular fluid adiponectine levels (p > 0.05). we evaluated the correlation with adiponectine and ivf outcomes as endometrial thickness, mii oocytes number, fertilization rates and pregnancy rates, but there were no statisticaly significant correlation (p > 0.05). we could not find differences of follicular fluid adiponectine levels in the pcos vs. non pcos groups. although we could not find correlation with adiponectine and pregnancy rates, further investigations with larger numbers of cases are needed to clarify this subject. hypothesis: using the clarified criteria may improve quality of cardiovascular risk (cvr) evaluation in pediatric group. methods: comparative ms components detection in obese children by idf and clarified criteria as well as prognostic value relative to the myocardial remodeling and diurnal blood pressure patterns. results: it was established that the idf criteria (>3) are highly specific (sp = 0.96), but low sensitive (se = 0.28) with deterioration negative predictive value (npv = 0.29). clarified criteria (sum score >4) correspond to higher sensitivity (se = 0.86) without loss of specificity (sp = 0.96). conclusion: using the clarified criteria improves screening sensitivity and helps determine exact cvr. obesity is a global problem associated with numerous health issues includes type2 diabetes. bariatric surgery provides sustained weight loss and partial or complete diabetes resolution. micrornas (approximately 22 nucleotides long) are post transcription regulators, which play important roles in conditions such as obesity and diabetes. the aim of this study is to investigate whether microrna profile is changed by bariatric surgery in postprandial state and if the altered microrna expression could contribute to long term post bariatric surgery benefits. both bariatric and sham-operated rats were given 5 g of food 40 min before sacrifice. total plasma and tissue rna from post-operative bariatric and sham operated rats were isolated and the microrna component was examined. principle component analysis clearly showed that bariatric surgery dramatically changed circulating microrna expression. correlation between microrna expression and metabonomics data indicated that certain plasma metabolites (for example, ketone bodies) are highly correlated. we also investigated liver microrna expression, microrna targeted mrna and metabonomics profile. mirna regulated key metabolic enzymes and receptors involved in the tca cycle, pentose phosphate pathway, gluconeogenesis and amp-activated protein kinase pathway. liver alanine, pyruvate and glucose levels were altered in bariatric compared to sham-operated rats. in summary, our data show that bariatric surgery changed both circulating and tissue microrna expression. we suggest that these alterations contribute to post bariatric surgery benefits by regulating key metabolic enzymes and receptors. the purpose of the study was to investigate serum concentrations of the asymmetric (adma) and symmetric dimethylarginine (sdma) and high sensitivity c-reactive protein (hs-crp) in hyperuricemic adolescents. patients and methods: the study group consisted of 58 hyperuricemic patients aged median 16.15 years. the control group (c) contained 27 healthy individuals with normal serum uric acid (sua) level. serum adma and sdma were measured by immunoenzymatic elisa commercial kits and were expressed in lmol/l. serum hs-crp was determined using nephelometric method (behring) and expressed in mg/l, sua was measured on the hitachi apparatus. results: hyperuricemic patients showed increased sdma (median: 0.54 q1-q3 (0.44-0.63) lmol/l vs. controls 0.51 q1-q3 (0.39-0.53) lmol/l, p < 0.01). serum adma values did not differ between two estimated groups (p > 0.05). the positive correlation was observed between adma and sua (r = 0.27, p < 0.01) and sdma with sua and hs-crp concentrations (r = 0.37, p < 0.05; r = 0.48, p < 0.01, respectively). we demonstrated that in adolescents with hyperuricemia increased serum sdma, but not adma levels were observed. no significant differences for adma and sdma between hypertensive and normotensive patients with hyperuricemia were found. the large, multicentre, prospective studies are needed to confirm if sdma might play a role in chronic inflammation in patients with hyperuricemia. objective and aim: supplementation of n-3 polyunsaturated fatty acids expresses anti-diabetic effect by enhancing insulin sensitivity and improving lipid metabolism. in this study we wanted to assess whether supplementation of n-3 can improve antioxidant status, preferably pon1 level, in the serum of type 2 diabetic (t2dm) patients. subjects and methods: twenty t2dm patients (12 females, eight males, age 53.15 ae 5.85 years, bmi 27.97 ae 1.61 kg/m 2 ) were randomized to intake 4 g marine oil (1.2 g epa and dha) during 12 weeks, after 6 weeks washout period. anthropometry, blood pressure measurements and fasting blood samples for metabolic parameters (glucose, hba1c, insulin, cholesterol, hdl-cholesterol, triglycerides), hscrp, as well as oxidative stress enzymes [pon-1, catalase (cat), superoxide dismutase (sod), glutathione peroxidase (gsh-px)] were obtained before and after treatment period. results: serum pon-1 activity response was increased by 123% from 22.07 to 49.43 (p < 0.05) as well as sod from 39.56 to 48.20 (p < 0.05) by epa and dha supplementation. it was associated with serum n-3 pufa increase by 152% (p < 0.01). there were no changes in anthropometry, glycemic control, insulin resistance index, the levels of total cholesterol, triglycerides and parameter of chronic inflammation (hscrp). only hdl-cholesterol increased after supplementation, from 1.04 to 1.25 mmol/l, with borderline significance (p = 0.05). conclusion: supplementation with 1.2 g n-3 pufa improves antioxidative status, pon1 activity and sod level, and repairs dyslipidemia in t2dm patients. aim of this study: to determine the behavior of endogenous antioxidants markers and metabolic profile in non-proliferative diabetic retinopathy patients and methods: a cross-sectional study was designed with four groups: group 1: five healthy-volunteer subjects. group 2: 12 patients with mild non-proliferative diabetic retinopathy (npdr). group 3: 18 patients with moderate npdr. group 4: 22 patients with severe npdr. serum endogenous antioxidants markers (determined by ua and b), glycemic profile (measured by fasting plasma glucose and hemoglobin a1c), lipid profile (measured by total colesterol, triglycerides, high and low density lipoproteins-cholesterol), arterial pressure, renal profile (urea and creatinine analysis). serum-markers were quantified using enzymatic-colorimetric methods. results: patients with npdr had a significant increased serum lipid profile, glycemic profile and renal profile levels compared to the control group. ua and b levels showed increased trend in retinopathy groups compared with group 1. introduction: bone tissue has been recognized as an endocrine organ. recently it′s been found that levels of osteocalcin, a protein produced by osteoblast, is associated with glucose metabolism and lipids in human being. experimental studies showed associations between adiposity and levels of osteocalcin, however in the adolescent population is not quiet elucidated. objective: to establish the association between adiposity indexes and serum levels of osteocalcin in children and adolescents. materials and methods: in this study we included 276 children and adolescents participants of the project "health worker cohort study" adiposity index were establish through bmi, waist circumference and dexa. the total concentration of osteocalcin was determined by chemiluminescence. through multiple lineal regression we evaluated the association between osteocalcin levels and the adiposity index. results: prevalence of overweight and obesity was 33.4% abdominal obesity 23.5%. it was observed a negative correlation between bmi, percentage of fat mass, waist circumference (p < 0.05). independently of the adiposity index included in the model, have obesity increases the risk of presenting low levels of osteocalcin (or: 2.09; 95% ci: 1.39-4.70, p < 0.05) and adolescents with higher percentage of fat mass have five times more risk than adolescents with less fat mass of present low levels of osteocalcin (or: 5.12; 95% ic: 1.89-13.84, p < 0.05). conclusions: adolescents with higher adiposity have more probability of have low levels of osteocalcin. objective: to evaluate the association between physical activity (pa) with presence of burnout syndrome (bs) and its three dimensions: emotional exhaustion, despersonalization and low personal accomplishment in health workers and educational sector. methods: a sample of imss workers, insp and uaem was obtained. after signing informed consent subjects answer a lifestyle questionnaire that included a pa questions and the burnout scale designed by maslach. anthropometric measurements were performed by previously standardized personnel (concordance coefficient of 0.83-0.9), with conventional stadiometer and calibrated scales previously tanita brand. we determined body mass index (bmi) based on the criteria of the center for disease control (cdc). pa was considered sufficient to perform 30 or more minutes of daily pa. the data analysis was performed in the stata statistical package version 9. results: from the analysis of 1992 health workers and educational study participants′ cohort health workers. "female sex predominated by 67%. the mean age was 39 ae 9 years. seven out of ten participants performed <30 min of pa per day. a trend of higher prevalence of bs in those performing <30 min of pa per day was showed, to perform ! 30 min of pa reduces the likelihood of developing bs in a 31% according to grunfeld criteria and 65% according to the ramı´rez's criteria. conclusions: to perform at least 30 min a day is a protective factor to avoid the presence of bs, but only three out of ten participants to perform af. aims: with research showing that more than one third of this current generation of australian teenagers likely to become obese in adulthood, the need to arm our children with the skills to maintain a healthy weight has never been so urgent. the need for feed cooking program has been developed for teenagers to improve their ability to prepare and cook healthy food, and boost their knowledge about nutrition and support them to develop healthy habits for the future. methods: need for feed is delivered using school facilities and home economics teachers, but is delivered outside of school hours in an informal and engaging format. the program, which delivers 20 h of tuition, focuses on improving participants' food preparation and cooking skills, budgeting and meal planning, basic nutrition knowledge, and attitudes and behaviours associated with healthy eating. results: evaluation demonstrates that participants improve in both skills and confidence in preparing and cooking healthy food, improve their knowledge of healthy eating guidelines and make tangible changes to their eating habits through increased consumption of fruit and vegetables, and reduced consumption of sugary soft drinks, cakes, chocolate and potato chips. conclusion: while the factors contributing to the obesity epidemic and growing rates of type 2 diabetes are complex, the importance of arming the community with skills and confidence in cooking at home is being increasingly recognised. this program has shown that teenagers can develop the necccessary skills to support healthy eating through a targeted and engaging cooking education program. postprandial hyperglycemia is an early defect of type 2 diabetes and the one of primary antidiabetic targets. the therapeutic approach for the treatment of postprandial hyperglycemia can be achieved by inhibiting a-amylase and a-glucosidase, key enzymes for starch digestion and further glucose absorption. in this study, the inhibitory activities of microalgal fucoxanthin against a-amylase and aglucosidase were determined as well as antidiabetic effect to induce differentiation in 3t3-l1 preadipocytes using oil red-o staining. fucoxanthin exhibited weak inhibitory activity against rat-intestinal aglucosidase, while strong inhibitory activity against pancreatic aamylase in a concentration-dependent manner with ic 50 values of 4.75 mmol/l and 680 lmol/l, respectively. microalgal fucoxanthin significantly increased glucose uptake in 3t3-l1 cell by 31.3% at 5 lmol/l. fucoxanthin inhibited lipid accumulation during adipocyte differentiation of 3t3-l1 cell and no cytotoxicity was recorded for preadipocytes up to 20 lmol/l. these results suggested that fucoxanthin may be useful approach for the prevention of type 2 diabetes by inhibiting carbohydrate-hydrolyzing enzymes. university of vermont, burlington, vt, usa aims: we compared the effects of ad libitum low-fat, high-fiber and low-carbohydrate diets on caloric intake, anthropometry, and cardiometabolic risk markers in adults with the metabolic syndrome. methods: twenty-three women and men (32-62 years) with the metabolic syndrome completed a randomized crossover comparison of two 4-week diets. all meals (low-fat, high-fiber: 55-60% carbohydrate, 20-25% fat, 15-20% protein, 38-48 g fiber/day; low-carb: 15-20% carbohydrate, 55-60% fat, 25-30% protein, 9-11 g fiber/day) were prepared by a research dietician and consumed ad libitum. results: caloric intake was lower on low-carb (2007 ae 383 vs. 2177 ae 398 kcal/day; p = 0.002). only low-carb reduced body fat (à1.02 ae 1.57%; p = 0.006) and tended to produce greater weight loss (à2.7 ae 1.9 kg vs. à1.7 ae 1.6 kg; p = 0.068). both diets reduced fasting insulin (à17.3% to à21.7%; p < 0.05) and non-hdl cholesterol (à5.0% to à5.6%; p < 0.05). low-fat, high-fiber reduced serum cholesterol (à6.1%), while triglycerides (à34.1%) and vldl cholesterol (à23.3%) were reduced on low-carb (all p < 0.05). plasma ffa were elevated on low-carb (+16.7%; p = 0.018). although flow-mediated dilation (fmd) was unchanged after low-fat, high-fiber (9.5 ae 1.3% vs. 10.4 ae 1.5%; p = 0.609), a trend for reduced fmd was observed after low-carb (10.1 ae 1.4% vs. 7.2 ae 1.1%; p = 0.061), producing a significant diet interaction (p = 0.012). introduction: better insulin schedules are needed to prevent hypoglycemic attacks. materials and methods: bs/time curve was plotted in a series of 500 diabetic patients with and without treatment. the best fit curve was used by partial integration and mathematical extrapolation. the first and second derivatives of these curves were plotted by the use of special software and modified matlab. points of major mathematical and lifestyle interest were marked within the curve for correlation. daily variations were also registered. based on mathematical results, 52 patients received auditory insulin and were compared with themselves and other patients under the same dose of insulin. results: although the bs/time curve and its first derivative are completely chaotic due to daily activity and unplanned meal intake the second derivative has some regularities and in regard to cutting the x axis every lambda minutes (37 min ( 51 min under 95% confidence interval. this shows that the innate glucose reducing potential of the body is exhausted after this time giving us the opportunity to treat with lower doses at these points. with this schedule patients needed less insulin and showed extremely better bs control. conclusion: preventing superimposition of innate bs reducing potency and external drugs can give rise to smoother bs control and less hypoglycemic attacks. besides, this can also be exploited in the management of prediabetic patients. introduction: type ii dm is generally regarded as a progressive disease with control of bs becoming more difficult with time. we wondered whether optimal therapy of the disease with "megatreatments" that can let the pancreas rest could reverse the disease. materials and methods: ten patients received optimal doses of insulin through the auditory channel for 3 months. they were previously on oral hypoglycemic agents and relatively in good control. their bs were measured daily for 2 months during the oral regimens before and after the auditory instillation. these two sets of measurements in addition to other subjective and objective parameters such as weight, sense of well being and hba1c were compared. results: insertion of a strict insulin regimen in the form of auditory insulin instillation between two intervals of the same oral hypoglycemic agents (sandwich effect) causes a significant improvement in glycemic control and possibly needed dose. conclusion: as fairly good controlled patients were selected there is probably no doubt that more strict control of bs can reverse type ii diabetes progression. we have postulated that rest of the pancreas in the form of auditory insulin or strict diet can restore normal pancreatic response in a later occasion. based on a mathematical model of extrapolation we suggested that strict bs control for 3-5 years with a mean bs of 80 mg/dl and possibly no attacks above 162 mg/dl can reverse type ii diabetes in at least 30% of the patients. the diabetes epidemic could be controlled with effective prevention programs. these programs require accurate identification of subjects at risk. unfortunately, current prediction models have several deficiencies and are not being used. we present results comparing a novel model using association rule mining (arm) against three traditional models applied to data from the electronic medical record (emr). we used a cohort of 53,393 adults olmsted county, mn, residents without diagnosis of diabetes who visited mayo clinic between 1999 and 2004. we collected 29 pertinent baseline characteristics available as structured data in the emr. we assessed 5-year incidence of diabetes; a total of 2349 subjects developed diabetes during the follow up period. we used c-statistic (mean ae sd) to compare risk of diabetes estimated by framingham score, san antonio index, finrisk index and our arm model. results: some variables used by traditional risk models were not available as structured data in the emr. they were imputed as the mean of the variable as measured on the population in which the index was developed. with the exception of finrisk, all indices preformed similarly. the c-statistic for framingham score, san antonio index, finrisk index and arm model was 0.75 ae 0.01, 0.76 ae 0.0.02, 0.68 ae 0.02 and 0.77 ae 0.02, respectively. conclusions: our novel risk model using arm compares favorably to traditional models and has the advantage that it does not depend on a limited number of risk factors. this model is implementable at the point-of-care or population-based care where emr is in use, assuring universal utilization and improving implementation of preventive management. this study aimed to compare the effects of two types of interventions: diet vs. diet plus sea buckthorn pulp oil intake, in childhood obesity. the results were focused on fmd and imt values and risk factors for endothelial dysfunction. two groups of matched age, sex and bmi obese children (n = 36, 8-18 years old) and 30 controls were enrolled. the measurements in the obese children were done before and after interventions: diet (low caloric, lipid, sugar intake for 2 months) or diet plus sea buckthorn pulp oil intake (800 mg/day for 2 months). ultrasounds were used for fmd and imt measurements and colorimetric and elisa methods for biochemical parameters. obese children vs. lean ones had significantly increased values for imt, leptin, apob/apoa1 ratio, fasting c peptide, c reactive protein (crp), and reduced values for fmd and adiponectin. in the obese group treated only with diet, a light reduction in weight was observed, but no modification of atherosclerotic markers or metabolic improvement. in the obese group treated with diet and sea buckthorn pulp oil, fmd and adiponectin were unchanged, while imt was significantly reduced (p < 0.03), together with the other atheroscleroric risk factors: fasting c peptide (p < 0.049), leptin (p < 0.05). in conclusion, this study shows that sea buckthorn pulp oil intake has beneficial effects by preventing atherosclerosis in obese children. aims: as the rates of type 2 diabetes continue to skyrocket across the globe, public health measures to reduce obesity appear to have limited success. while social marketing campaigns for obesity prevention have their place amongst a suite of strategies, their impact can be limited due to the gap between the campaign messages and individual's ability to identify opportunities for change in their own life. in queensland, a group of peak health agencies are working together to bridge that gap. methods: swap it, don′t stop it is an australian social marketing campaign promoting simple, everyday food and activity changes people can make to improve overall health. a multi channel health promotion and public relations strategy has been implemented to integrate the key messages of the swap it don't stop it campaign with grassroots healthy lifestyle opportunities to support individuals to make changes. this has been achieved by harnessing the collective actions of the non-government health sector to promote clear and consistent obesity prevention messages and by implementing a communications strategy that has connected community and stakeholders with tangible opportunities for healthy lifestyle changes at the local level. outcomes: through collaborative action amongst a broad network of service providers, the national campaign messages have been amplified through community events, radio, print and television stories that have highlighted examples of personal success and provided information that has inspired queenslanders to make small steps towards better health and reduce the risk of type 2 diabetes and other chronic diseases. background and aims: proposed changes to gdm diagnostic criteria are anticipated to increase diagnosis rates leading to rising demand for support during pregnancy. lack of awareness of risks associated with gdm acts as a barrier to healthy lifestyle adaptation and postpregnancy screening (1, 2) . the you2 program delivers a range of strategies to support women at diagnosis, facilitate behaviour modification (2) and re-enforce importance of post pregnancy screening. methods: newly diagnosed women and their support networks are educated about gdm, risk reducing behaviours and the health care system through a purpose built website and a suite of print resources. specific resources were developed for at risk groups including indigenous australians and women from non-english speaking backgrounds. women are encouraged to join a national register to receive post pregnancy screening reminder letters and text messages in conjunction with regular newsletters containing family-friendly information on healthy lifestyle behaviours and screening. nominated gp's are also sent screening reminders to prompt women to attend screening. results: engagement with the website and newsletters is high, screening reminders evaluated positively with focus groups and participants indicated that they intended to attend their post natal screening. conclusion: you2 delivers a multi-strategy approach that engages and supports women at diagnosis and re-enforces the importance of screening and behaviour modification during and post pregnancy. family members and peers were recognised as important sources of support and educated to encourage screening and behavioural changes. these low-cost interventions can help reduce prevalence or morbidity in women affected by gdm associated type 2 diabetes. however smoking cessation has many benefits for smokers, but the most important challenge for smoking cessation is withdrawal symptoms during the first days and months of cessation which can be an important reason for unsuccessful in the smoking cessation process. however, many methods are available to help quit smoking, but acupuncture is used for the treatment of nicotine dependency with the aim of reducing of withdrawal symptoms experience. therefore, the aim of this study is to determine the role of acupuncture in success at quit smoking in the iranian samples. methods: one hundred and thirty-two men smokers who were ready to quit smoking were participated in the study. the participants were divided to two groups by randomized sample method. one group was for acupuncture and another group was for acupuncture in shame points. the mean age of smokers was 42 ae 65 years. all participants followed for 1 year and the quit rate evaluated in 1, 6, 12 months of the study. the results show, while 22.4% in case-control and 19.2% in control group could quit smoking in the first month of study; but in 12 the month of the study 14.8% in case-control and 8.6% in control group were at quit rate. the quit rate short and long term in case group was higher than control group. therefore, physicians should play an active rate in control of smoking by ensuring and counseling and use of various pharmacology and non pharmacology methods for increase quit rate. panjab university, 2 pgimer, chandigarh, india introduction: 1,2-dimethylhydrazine (dmh)-induced colon carcinogenesis in rats is a reliable model to explore molecular mechanism involved in progression of colorectal cancer from adenoma to carcinoma sequence. objective: to study the transcriptional and translational levels of various genes involved in tumorigenesis pathway of dmh induced rat model. methods: two groups of chow-diet-fed, male sprague-dawley rats, aged 10 weeks (n = 12/group) were fed a normal diet and injected subcutaneously for two time durations of 10 and 20 weeks dmh at a dose of 30 mg/kg body weight/week or with ethylene diamine tetraacetic acid (edta)-saline. macroscopic and microscopic analyses were performed for confirmation of adenoma and carcinoma. mrna expression of nfkb and caspase-3 genes were determined by real-time pcr. immunohistochemistry was also performed for expression of above genes. results: gross examination of 10 weeks dmh treated colon showed polypoid lesions and multiple tumors were formed after 20 weeks dmh treatment. histopathological studies confirmed the colon carcinogenesis from adenoma-carcinoma sequence by type of tumor, degree of differentiation and invasion of tumors. in adenomatous and carcinomatous colonic tissues, mrna expression of nfkb was increased by 5.8 and 8.2 fold respectively, whereas expression of caspase-3 was reduced by 13.2 and 4.5 fold respectively. immunohistochemistry studies showed the increase expression of nfkb and reduced expression of caspase-3 in colonic tissues of dmh induced rat model as compared to controls. the observed data strongly implicates that dmh induced colon carcinogenesis altered the apoptotic machinery by modulating the expression of various genes involved in this pathway. objective: identify anthropometric indicators that present the greatest correlation in the diagnosis of some metabolic syndrome components in adolescents and its association with gender, age, and family history. methods: cross-sectional study with 487 adolescents between 16 and 19 years of age of a public high school in the mexico city. bmi was used to diagnose overweight and obesity according to cut-offs proposed by the international obesity task force. waist circumference (wc) and waist-to-height ratio (whr) were used as fat distribution indexes. results: 52.9% of the students were male. the prevalence of overweight and obesity were 22.9% and 4.4%. there were association between overweight-obesity and the average values of wc, whr, and blood pressure (p < 0.001). the correlation between waist circumference and bmi and waist-height were the strongest (r = 0.823 and r = 0.845, respectively). we recommend using wc and whr in clinical practice as simplified indicators to predict risk of metabolic syndrome in mexican adolescents. c. saely, a. muendlein, a. vonbank, k. geiger, p. rein, h. drexel background: the novel adipocytokine chemerin has been suggested to be linked to insulin resistance and to the metabolic syndrome (mets). its association with coronary artery disease (cad) is unclear. we hypothesized that chemerin is associated with both angiographically determined cad and with the mets. we measured serum chemerin in 498 patients undergoing coronary angiography for the evaluation of established or suspected stable cad; the mets was defined according to ncep-atpiii criteria; significant cad was diagnosed when coronary stenoses ! 50% were present. results: chemerin was higher in mets patients (n = 150) than in subjects without the mets (184 ae 77 vs. 150 ae 62 ng/ml; p < 0.001). it did not differ significantly between patients with significant cad (n = 250) and those without significant cad (p = 0.327). when both, mets and cad status were considered, chemerin was higher in mets patients both among those who had significant cad (182 ae 80 vs. 152 ae 60 ng/ml; p = 0.002) and among those who did not have significant cad (187 ae 73 vs. 148 ae 63 ng/ml; p < 0.001); it did not differ significantly between patients with significant cad and subjects without significant cad among mets patients (p = 0.248) nor among subjects without mets (p = 0.263). analysis of covariance (ancova) showed that a large waist circumference as well as elevated trigylcerides were independent predictors of elevated serum chemerin (f = 12.5; p < 0.001 and f = 8.5; p = 0.004). we conclude that chemerin is significantly associated mets but not with angiographically determined cad. the overall association of chemerin with the mets is carried by its association with visceral obesity and elevated triglycerides. objective: resistin is a novel adipocyte-secreted hormone proposed to link obesity with diabetes. the role of resistin gene polymorphism in insulin resistance and metabolic syndrome is controversial till date. the present study was attempts to investigate the relationship between resistin gene polymorphism with circulating resistin level, metabolic risk factor and insulin resistance in north indian population. methods: this is a case-control study; total 455 healthy subjects were selected for the study. out of which 225 (age 31.91 ae 6.05 years) were with metabolic risk factor and 230 age matched control (age 30.96 ae 7.01 years) were without metabolic risk factors. we estimated homeostatic model assessment (homa) index, circulatory resistin, and lipid profiles. the genotyping of resistin-c420g were carried out using pcr-rflp method digested with bbsi restriction enzyme. results: homozygous mutant genotype (cc) (cc v/s cg + gg) (p = 0.03: or = 1.61: 95% ci = 1.06-2.47) and mutant allele (g) (p = 0.01: or = 1.53: 95% ci = 1.11-2.08) of the 420c/g polymorphism was significantly less frequently observed in the control population as compared to study group. furthermore, on dividing the subjects into two groups according to the absence (resistin -1) or presence (resistin-2) of the mutant g allele, significantly high levels of resistin (p = 0.006, or = 1.90, 95%ci = 1.22-2.98) and insulin resistance (p = 0.03, or = 1.46, 95%ci = 1.22-2.98) were observed in resistin-2 group as compared to resistin-1 group. conclusion: our results suggest that the 420 c/g mutation of the resistin gene is likely to play an important role in the development of metabolic syndrome and metabolic abnormalities. king george's medical university, lucknow, 2 rims, saifai, etawah, india objective: adiponectin has been shown to be an insulin-sensitizing hormone and negatively associated with components of metabolic syndrome. present study was attempted to investigate the adiponectin polymorphism in metabolic syndrome and insulin resistance. methods: the adiponectin t45g and g276t polymorphism has been studied in 305 females with metabolic syndrome and 310 control without metabolic syndrome according to ncep atp iii criteria, 2001. circulating adiponectin and leptin levels were determined by sandwich elisa method and insulin resistance by the homeostasis model assessment (homa) index. the polymorphism of adiponectin 45 t/g and 276 g/t gene were determined by pcr-rflp method. results: significant difference were found for circulating leptin level except adiponectin level (20.21 ae 10.85 vs. 29.56 ae 13.46), and in metabolic risk factors among metabolic syndrome and without metabolic syndrome females. homozygous mutant genotype (gg) (tt vs. tg + gg) (p = 0.0169: or = 1.55: 95% ci = 1.09-2.19) and mutant allele (g) (p = 0.0080: or = 1.49: 95% ci = 1.12-1.99) of the -t45g gene and mutant allele (t) (p = 0.0278: or = 1.36: 95% ci = 1.04-1.77) of the g276t polymorphism were significantly less frequently observed in the control population as compared to study group. the results of the present study concluded that the mutation of the adiponectin 45 t/g and 276 g/t gene might play a important role in obesity associated metabolic syndrome and metabolic abnormalities except insulin resistance, glucose level and insulin levels in the north indian women due to mutation of the adiponectin gene is associated with decreased adiposity which is protective one for metabolic syndrome design: cross-sectional study of patients with good (excess body mass index lost (ebl) >60%) and poor weight loss (ebl 50%) >12 months after rygb. material and methods: sixteen patients with good weight loss and 17 patients with poor weight loss were included in the study. the patients underwent dual energy x-ray absorptiometry scan, indirect calorimetry and a 9-h multiple-meal test with measurements of glucose, insulin, total bile acids (tba), glucagon-like peptide (glp)-1, peptide yy3-36 (pyy), cholecystokinin (cck), ghrelin, neurotensin, and pancreatic polypeptide (pp) as well as assessment of early dumping and appetite. results: suppression of hunger was more pronounced in the good than the poor weight loss group in response to the multiple-meal test (p = 0.006). in addition, the good weight loss group had a larger release of glp-1 (p = 0.009) and a greater suppression of ghrelin (p = 0.037) during the test, whereas the postprandial secretion of cck was highest in the poor weight loss group (p = 0.005). pyy, neurotensin, pp and tba release did not differ. early dumping was also comparable. differences in resting energy expenditure between the groups were entirely explained by differences in body composition. conclusion: favorable meal-induced changes in hunger and gut hormone release in patients with good compared to poor weight loss support the role of gut hormones in the weight loss after rygb. background: the objective of this study is to explore changes in the vascular tone over the endothelial, neurogenic and myogenic frequency ranges during a contralateral cold pressor test by performing the wavelet analysis of skin temperature fluctuations and to compare the results obtained in healthy subjects and in patients with metabolic syndrome (ms) and type 2 diabetes. methods: thirteen adults with type 2 diabetes aged 45-65 years (average diabetes duration of 10.6 ae 1.3 years) and 14 adults with ms aged 36-62 years participated in this pilot study. the control group included practically healthy men and women aged 39-60. the lowfrequency fluctuations of skin temperature in the appropriate frequency ranges, registered during contralateral cold test, were used as a characteristic reflecting the mechanisms of vascular tone regulation. results: the response to cold pressor test in patients with type 2 diabetes and with ms differs essentially from that of healthy subjects. patients with ms show changes in the amplitude of skin temperature fluctuations similar for patients with type 2 diabetes in the endothelial range. the endothelial dysfunction occurs in the pre-clinical stage of diabetes and manifests, in particular, as a disturbance of the endothelial part of vascular tone regulation. with progression of the glucose metabolic disorders the pathological process is worsened due to violation of the neurogenic vasodilatation mechanisms. fetal metabolic programming states that early life nutrition is implicated with the risk of later disease development and both underand overnutrition during gestation might predispose individuals to develop obesity or diabetes later in life. obesity operations called "gastric bypass" operations have shown unexpected involvement of the small intestine in diabetes pathophysiology as it in most cases result in a complete resolution of the diabetes before weight loss. therefore we hypothesize that the small intestine is a subject of metabolic programming and that this programming can predispose for diabetes development. twin-pregnant ewes where fed a normal, a low or a high diet during the last 6 weeks of gestation and the twin lambs where fed either a conventional or a high fat, high carbohydrate (hchf) diet during the first 6 months of life. feeding challenge tests were performed on all lambs and some were slaughtered with collection of intestinal tissue for qpcr. the hchf diet increased the blood level of glucose, insulin and tg and increased the intestinal expression of a range of genes involved in growth, vascularization as well as digestion and absorption. the maternal low and high diet had effects on gene-expression, however the results vary between genes. these observations suggest that small intestine function has been programmed by the late-gestation low or high diet at gene expression level, whereas the physiological metabolic functions has mainly been affected by the hchf diet at such a young age. further investigations on the long-term effects of early nutrition are required. background: liposuction is considered as the treatment for the metabolic complications of obesity. the aim of this study was to evaluate the effect of abdominal liposuction on leptin and interleukin-6 (il-6) expression in adipose tissue and serum concentration. material and methods: the study included 32 consecutive patients (25 females, seven males) aged 38 ae 10 years with bmi 25 ae 3 kg/m 2 , non-diabetic, apparently healthy, who underwent liposuction in mandala beauty clinic in pozna n, poland. the patients were examined clinically and the blood was withdrawn for routine laboratory tests (hematology, glucose, lipids, coagulation). leptin (r&d) and high sensitivity il-6 (hsil-6) (abcam) were analyzed in the supernatant of adipose tissue homogenate and in serum, by means of elisa. results: the expression of leptin in adipose tissue positively correlated with white blood cells count before liposuction (rs = 0.425, p = 0.0431). it was also higher (p = 0.0397) in patients with bmi ! 25.0 (0.64; 0.40-1.199 ng/mg protein; median, interquartile range) than in subjects with bmi < 25.0 (0.38; 0.26-0.67 ng/mg protein). following liposuction, after 1 month, serum leptin levels were lowered in smokers (18.03; 10.22-22.28 ng/ml), when comparing with baseline (28.14; 17.00-34.87 ng/ml). serum level of the adipokine, both before (rs = 0.535, p = 0.0048), and after (tau kendall = 0.352, p = 0.0275) liposuction correlated with tissue expression of leptin. no differences in adipose tissue expressions and serum levels of hsil-6 were observed 1 month after liposuction. conclusion: a count of circulating leukocytes, even presented within references, affects the increasing adipose tissue expression of leptin. smoking status favorably affected the influence of liposuction on serum leptin levels. liposuction has no short-term effect on serum il-6 concentrations. r.a.l. sertié , s.a. sertié , a.r.g. proenc ßa, t. lima-salgado, a.c. oliveira, f.b. lima introduction: all adaptations acquired through physical training are reversible during inactivity. significant reductions in maximal oxygen uptake (vo 2max ) are observed within two-four weeks of detraining. conversely, the consequences of detraining on adipose tissue are poorly known. aim: to investigate the physical detraining effects on metabolism and cellularity of rat periepididymal adipose tissue. methods and results: male wistar rats, ageing 6 weeks, were divided in three groups: trained (t) for 12 weeks; detrained (d), (trained for 8 weeks and detrained for 4 weeks), and age-matched sedentary (s). training consisted in treadmill running sessions (1 h/day, 5 day/week, 50-60% of the maximal capacity). the morphometric analysis of pe tissue disclosed significant differences between the groups. the adipocyte sectional area of group d was significantly bigger than t and s (3474 ae 68.8 lm 2 vs. 1945.7 ae 45.6 lm 2 vs. 2492.4 ae 49.08 lm 2 , respectively). compared to t the cells of d animals showed 48% increased ability to perform: lipogenesis, either spontaneously or insulin stimulated and isoproterenol-stimulated lipolysis. basal lipolysis did not change. a 15% reduction in apoptosis was observed in groups t and d in relation to s. some gene expressions were changed in d vs. s: adiponectin (three-fold up) and ppar-gamma (two-fold up). pref-1 gene was three-fold higher in t vs. s. conclusions: these results suggest that adipogenesis was stimulated in this group. detraining causes significant increase in adipocyte size and lipogenic capacity. as pe fat cell apoptosis was reduced in d and t. background: chronic red wine (rw) consumption has been associated with a decreased cardiovascular disease risk, mainly attributed to an improvement in lipid profile. rw intake is also able to change gut microbiota composition. high-fat intake has recently been reported to increase metabolic endotoxemia. the gut microbiota has been proposed as the main resource of plasma lipopolysaccharides (lps) in metabolic endotoxemia. objective: to analyze the effect on lps concentrations of chronic rw consumption and acute rw intake in relation to high-fat intake in middle-aged men. design: for the chronic study 10 middle-aged male volunteers were randomized in a crossover trial and after a washout period all received rw, dealcoholized red wine (drw), or gin for 20 days. the serum lps concentration and changes in fecal microbiota were quantified before and after the treatments. for the acute study, five adult men underwent a fat overload or a fat overload together with rw, drw or gin. baseline and postprandial serum lps concentrations and postprandial chylomicron lps concentrations were measured. results: chronic rw consumption led to a significant decrease in lps concentrations compared to baseline. in addition, lps concentrations correlated negatively with bifidobacterium and prevotella levels. there were no differences in postprandial serum or chylomicron lps concentrations between acute rw, drw or gin intake together with a fatty meal. postprandial chylomicron lps concentrations correlated positively with the increase in triglyceride concentrations. conclusions: chronic rw consumption decreases lps concentrations, but it is not able to attenuate the postprandial lps increase induced by a fat overload. physical inactivity increases the risk of metabolic disease, associated with perturbations of muscle energy metabolism. the aim of this study was to assess the effect of exercise training on adiposity, physical fitness and muscle energy metabolism in obese individuals with attributes of ms. methods: sedentary individuals (m/f 16/8; age 36.7 ae 5.5 years, bmi 31.8 ae 3.6 kg/m 2 ) completed 3-months endurance or strength (n = 11/13) training program (1 h, 3-times/week). subcutaneous and visceral adiposity was measured by mri. phosphorus mr spectroscopy ( 31 p-mrs) was used to assess the energetic status of muscle in vivo by measuring phosphocreatine (pcr), atp, inorganic phosphate (pi) and maximal oxidative flux (q max ), after exercise-or magnetic-induced equilibrium perturbation (n = 11). the samples of skeletal muscle were taken by needle biopsy (vastus lateralis). cytochrome c oxidase (cox) activity of permeabilized muscle fibers was measured by oxymetry and maximal aerobic capacity (vo 2 max) by bicycle ergometry. free-living ambulatory activity was monitored by accelerometers. results: subcutaneous adiposity did not change with training. however, strength training decreased visceral adiposity (p < 0.05). training increased physical fitness (vo 2 max, n = 24, p = 0.01) and in vivo muscle energy metabolism ( 31 p-mrs: q max , pcr, atp, n = 11, p < 0.05), without a significant effect on cox activity (n = 0.1). depletion of muscle pcr was negatively associated with cox activity (p = 0.01). vo 2 max was not associated with muscle metabolism (p > 0.05). free-living activity increased during training (p < 0.05). conclusions: three months training of obese individuals was sufficient to increase both cardiorespiratory fitness and muscle metabolism, assessed by 31 p-mrs. strength training was more efficient in decreasing visceral adiposity. background: the amelioration of metabolic complications of excessive body mass by liposuction is intensively discussed, as the metabolic surgery is proposed for future diabetes therapy. we have undertaken the study on the short-term effect of liposuction on insulin resistance parameters. material and methods: we included in the study 32 consecutive patients (25 females, seven males) aged 38 ae 10 years with bmi 25 ae 3 kg/m 2 , who underwent liposuction in mandala beauty clinic in pozna n, poland. the non-diabetic, apparently healthy patients were examined clinically, blood was withdrawn for baseline routine laboratory tests (hematology, glucose, lipids, coagulation). insulin levels in serum and its content in adipose tissue were evaluated by means of elisa (diasource, sunrise tekan). insulin resistance/ sensitivity indexes: homa-ir, quicki and mcauley were calculated. one month observation included re-evaluation of glucose, lipids and insulin along with the indexes. data are presented as mean ae sd or median; interquartile range. we have observed the decrease in glucose concentrations (85 ae 8 vs. 91 ae 10 mg/dl, p = 0.0130) and insulin levels (19;16-22 vs. 21;18-25 mu/l, p = 0.0202) 1 month after liposuction, comparing to baseline. homa-ir (5.2 ae 1.6 vs. 4.3 ae 1.4, p = 0.0474) and mcauley index (5.12 ae 0.97 vs. 5.73 ae 1.04, p = 0.0448) were improved 1 month after liposuction, however no effect on quicki has been found. triglycerides concentrations 1 month after liposuction were lowered comparing to baseline (76;70-97 mg/dl vs. 108;91-155 mg/dl; p = 0.0110). in multiple regression analysis age was independent factor that affected insulin content in adipose tissue in males (b = 0.1779, p = 0.0451). conclusion: liposuction causes beneficial effect on insulin resistance parameters in short-term observation of non-diabetic patients. background: a large number of studies showed that community-based health promotion is an effective way of preventing and treating chronic diseases. however, participation rate is very low for health education programs in community health centers (chcs); that of the program is not attractive, in part, because it is not individualized. using diabetes risk assessment tool based on electronic health records (ehr) data for diabetes risk prediction could help to early identify high-risk groups and reduce the incidence of diabetes through health education and lifestyle changes to control risk factors. objectives: to conduct community research to understand the situation of community diabetes prevention and control; to initially established prediction model of diabetes risk factors based on ehr data in chcs. the project randomly selects four districts in shanghai, using a combination of quantitative research and qualitative research method. get ehr data from each chc, attempts to establish the predicted model for diabetes, and test the sensitivity and specificity of the model. outcome measures: the prediction model include age, gender, blood pressure, smoking, drinking, body mass index, family history of diabetes based on ehr data. as pr(d) ! 0.5, the sensitivity is 69.6%, specificity is 67.4% and area under roc curve (auc) is 0.7407. the empirical results will contribute to a better understanding of how the diabetes management in chc is important and provider the targeted self-management materials to improve health outcomes as well as evidences directly applicable in improving china's health policy reforms. a. schmid, a. kopp, m. bala, s. leszczak, i. ober, m. mü ller, a. schä ffler internal medicine i, university hospital regensburg, regensburg, germany introduction: the adipokine chemerin has an important role in insulin sensitivity and insulin secretion. elevated systemic chemerin concentrations correlate with obesity and insulin resistance. this study investigated chemerin serum levels of healthy volunteers undergoing an oral fat tolerance test (oftt) and their correlations with gender and markers of insulin sensitivity and inflammation. we further tested effects of the sex hormones estradiol and testosterone on chemerin secretion from adipocytes in vitro. methods: hundred overnight fasted healthy volunteers participating in the study underwent an oftt. after oral uptake of lipid suspension, venous blood was drawn at 0, 2, 4 and 6 h. subjects were characterized by anthropometric and standard laboratory parameters. chemerin levels were measured by elisa. mature murine 3t3-l1 adipocytes were stimulated with estradiol and testosterone. concentrations of secreted chemerin were measured by elisa. results: while there were no significant changes in individual chemerin concentrations resulting from the lipid load, mean chemerin levels in sera of female probands were found to be significantly higher when compared to males (116.7 ae 37.9 vs. 97.1 ae 24.7 ng/ml, p = 0.004). the correlation of chemerin with insulin and c-peptide concentrations also was gender-specific. along with these findings, chemerin secretion from mature 3t3-l1 adipocytes was shown to be affected by estradiol and testosterone treatment. conclusion: human chemerin concentrations are higher in females and there is a gender specific regulation upon stimulation with sex steroids. chemerin is not responsive to an oral lipid load. objective: to study the predictors of change in bmi with respect to time among physically active subjects. methodology: the present study was conducted among 14 physically active individuals in delhi. data was collected longitudinally with a monthly follow up of 12 months. various anthropometric and physiological measurements were taken using standard protocol. the analysis has been performed with mixed effect modeling to assess subject's variation for parameters with respect to time. r 2.10.10 software with nlme package was used. an akaike information criterion (aic) was applied to find the consistency and measure the relative goodness of fit of a statistical model. : bmi as an important factor was taken as dependent variable and using aic, gender, blood pressure and weight were independent variables or fixed and random variables for 12 months of longitudinal study. using generalized linear model (glm), bmi was dependent variable and other parameters i.e. gender, systolic blood pressure (sbp), diastolic blood pressure (dbp) and weight were independent variables. the regression coefficient obtained for gender with respect to bmi (as response variable) was à0.59. the estimated values for sbp, dbp and weight were à0.15, 0.05 and 0.05 respectively. the reduction of bmi among females was more in comparison to males. the reduction rate of sbp, dbp and weight with bmi was very low with respect to time. a.e. andreazzi 1 , s. grassiolli 2 , j.c. de oliveira 2 , r. torrezan 2 , s.t. paes 1 , m.s. silva 1 , r.m.g. garcia 3 , p.c.d.f. mathias 2 1 department of physiology, federal university of juiz de fora, juiz de fora, 2 biology, state university of maring a, maring a, 3 biology, federal university of juiz de fora, juiz de fora, brazil obesity is a worldwide epidemic and the most important factor in metabolic syndrome onset. the involvement of sympathoadrenal axis activity in obesity onset was investigated using the experimental model of treating neonatal rats with monosodium l-glutamate (msg). to access general sympathetic nervous system activity, we recorded the firing rates of sympathetic superior cervical ganglion nerves in animals. catecholamine content and secretion from isolated adrenal medulla were measured using the trihydroxyindole fluorescence method. high-performance liquid chromatography (hplc) was used to measure plasmatic adrenaline. intravenous glucose tolerance test was performed, and isolated pancreatic islets were stimulated with glucose and adrenergic agonists. msg treatment increased the epididymal and retroperitoneal fat pad mass by 60.5% and 57.2%, respectively, compared with control animals (p < 0.001). the nerve firing rate of obese rats was decreased 58.8% compared to the rate for lean rats (p < 0.05). pre-diabetic rats showed a 60% reduction in basal catecholamine secretion from the adrenal medulla and 32% lower plasma adrenaline concentration compared with the control animals (p < 0.05); whereas catecholamine secretion induced by carbachol, elevated extracellular potassium and caffeine in the isolated adrenal medulla were all increased in obese rats compared to control. both glucose intolerance and hyperinsulinaemia were observed in obese rats. adrenaline strongly inhibited glucose-induced insulin secretion in obese animals (72%, p < 0.05). these findings suggest that low sympathoadrenal activity contributes to impaired glycaemic control in pre-diabetic obese rats. objective: the aim of the present study was to evaluate the influence of physical activity patterns on cardiovascular health. the study was conducted in delhi, among adult punjabi males and females. total sample of 286 subjects was taken for the study. data was collected crosssectionally using multistage stratified sampling. according to pattern of physical activity using self administered proforma, two groups were formed, one regular physical activity group (rpa; at least 5 days/week brisk walk for 30 min, doing yoga for 45 min), and the other irregular physical activity group (ipa; <5 days a week with no consistency). various anthropometric and physiological measurements were taken using standard protocol. results: higher percentage of subjects with irregular pattern of pa was at risk of developing high bp, obesity and triglycerides. higher values of whr, whtr among them showed that group was more disposed to cardiovascular health problem. males with ipapattern had 5.51times, 3.81 times and 13.21 times more risk of increasing regional adiposity using whtr, whr and wc respectively. among females, whtr, whr and wc respectively showed 9.53 times, 3.96 times and 6.00 times higher risk of developing regional adiposity with irregular pattern of pa. the increased risk of being obese was 7.16 times more among males with irregular pattern of physical activity while among females fat percentage and obesity level using bmi category were found to be at a risk of 2.74 times and 2.78 times respectively. conclusion: subjects with regular pattern of pa showed higher percentage of normal values of various cardiovascular and obesity markers. irregular pattern of pa increased the risk of regional and general adiposity markers besides that of high bp. materials and methods: serum glycemia, insulin and c-peptide level were measured at ogl hours 0-1st-2nd in: seven healthy controls, 15 patients with ms and 13 with cushing; mean age was 29.8-31.4-29.8, bmi 26-40-35, ogl glycemia: 4.2-7.6-4.9; 5.0-7.5-4.8; 5.1-9.1-5.7 respectively. all patients had bp >145/90 mmhg and triglyceride level >2.0 mmol/l. homa assessment: (fasting insulin, pmol/l 9 fasting glucose, mmol/l):22.5. c-peptide and insulin increase above fasting level (in pmol) per 1 g of ingested glucose was calculated. results: (*p < 0.05 vs. control). homa was 20.1-38.8*-43.1* and fasting c-peptide and insulin level were 417/108, 1007/173*, 1160/196* pmol/l in control, ms and cushing respectively. to the 1st hour of ogl the corresponding data were: 1720/486, 2342/614* and 2590/ 547* and increase of c-peptide and insulin concentration per 1 g of ingested glucose was almost equal: 26/7.5-27/8.8-29/7.0 pmol/l. the degree of c-peptide increase to the 1st hour was twice less than in control and increased fasting hepatic insulin clearance lowered almost to control level (judging by c-peptide/insulin ratio). to the 2nd hour of ogl c-peptide and insulin level became similar to fasting data. conclusion: ir characteristics were similar in ms and cushing disease. insulin requirement increase on the top of ogl was equal to control. judging by c-peptide/insulin changes ir stops manifesting on the top of glycemia during ogl (probably due to normalizing hepatic insulin clearance). introduction: ir is atfr of high potential, but also one of the first signs of a ateroscl. process. condition when the body has too much insulin at in the same time too much glucose is the resistance of peripheral tissues to the action of insulin. objectives: to investigate the relationship of concentrations of insulin and glucose in the peripheral blood, but also the conc. of gh, becose normalisation of gh secretion could corrects hiperinsulinemia and insulin resistance. on the other hand reduced gh secretion often points to the high expression of somatostatin and the presence of stress, which leads to secretion of somatostatin. further increase in visceral adipose tissue and ffa relise under stress can worsen the condition of and insulin resistance. aims of study: conducted 2 therapeutic program (combination of dietary and physical activity), in which only the group edu implemented and behavioral therapy. monitoring parameters of glycemic control: fasting glucose, fasting insulin and gh and insulin sensitivity of peripheral tissues to insulin, always from in one blood sample, parallely in two different phases of therapy. we used anova-manova statistics to determine the significance of an differencies. material and methods: determining of glucose, insulin, and growth hormone. as and qucki index for determining od insulin sensitivy conclusion: ours finding indicate that the synergistic effects of dieting and increased physical activity improves glucoregulation, reduce excessive insulin secretion and restoration of gh secretion. but only behavioral therapy may lead to permanent changes in nutritional habits and lifestyle. the metabolic and endocrine dysfunctions that may occur with polycystic ovary syndrome (pcos) can be associated with future comorbidities such as diabetes, cardiovascular disease, and endometrial cancer. although a definitive link between pcos and these chronic illnesses has not been demonstrated, there is significant overlap in the clinical characteristics of these disorders. consequently, the issue of identifying and measuring potential conditions that may be associated with pcos is a priority and should be the standard of practice in its management. hiperhomocysteinemia has been shown as independent predictor of cardiovascular events in patients with atherosclerosis. the aim of our study was to determinate levels of homocysteine in woman with polycystic ovary syndrome compared with healthy woman. thirty patients (age, 23, 5 ae 5.5) with pcos and 24 (age, 25.5 ae 4.3) healthy woman were involved in the study. blood samples were collected in early follicular phase. total homocysteine was measured using fluorescent immunoassay. statistically significant differences in serum concentration of homocysteine were observed between groups. mean homocysteine level we found as (10.2 ae 2.9 vs. 7.0 ae 1.5) in pcos and normal group respectively (p < 0.05). for macedonian population we found statistically significant increased homocysteine levels in woman with pcos. although the mean homocysteine levels are within normal limits, there are significant higher mean homocysteine concentrations between these two groups. because an increased concentration of thcy has been shown as and independent risk factor for cardiovascular alterations, it is essential in this group of woman to be taken measures for early prevention. a. alkandari, n.j. gooderham, h. ashrafian surgery and cancer, imperial college london, london, uk globally 500 million people are obese and prevalence is increasing. obesity and its many co-morbidities are leading causes of mortality and morbidity and pose substantial socioeconomic burdens on health services. bariatric surgery is a form of gastrointestinal surgery that leads to sustained weight loss, a decrease in cancer risk and resolution of type 2 diabetes. micrornas are a family of small, endogenous, non-coding rnas that regulate gene expression at the posttranscriptional level. micrornas control expression of over half the human transcriptome and are involved in processes fundamental to both normal physiology and disease, including obesity, diabetes and cancer. we hypothesise that urinary micrornas are biomarkers for bariatric surgery reduction of type 2 diabetes. here, we looked at expression of micrornas involved in diabetes in urine samples prior to bariatric surgery and at 2 months and 1 year postoperatively. urinary rna was obtained using the mirvana paris kit and microrna expression was determined through quantitative pcr. we found significant increases of two to three fold postoperatively in the expression of three micrornas involved in diabetic nephropathy. these findings are consistent among patients undergoing laparoscopic roux-en-y gastric bypass and sleeve gastrectomy. our results indicate that bariatric operations can modulate post-translational effects in end-organs postoperatively and may contribute to the beneficial effects noted after these procedures. hepatic insulin resistance and the ensuing impairment of hepatic glucose metabolism is a major contributor to hyperglycemia in metabolic syndrome. various factors appear to act in parallel to elicit hepatic insulin resistance. while impaired fatty acid handling with a resulting activation of pkc isoforms is one established route, a subacute inflammation also appears to contribute and the impact of cytokines on the insulin signaling cascade is well established. by contrast, the role of small lipid mediators e.g. prostaglandins and sphingolipids, which are also affluent in inflamed tissue, is not well characterized. the current study addressed this question. in a wide array of genetic and diet-induced mouse models of the metabolic syndrome, hepatic expression of key enzymes of prostaglandin formation was induced. prostalgandin e 2 , which is released predominantly from kupffer cells, directly attenuated insulindependent hepatic glucose utilization by an erk1/2-dependent serinephosphorylation of irs and hence attenuation of insulin-dependent akt-phosphorylation. in addition, pge 2 enhanced lipid accumulation in hepatocytes by inhibiting mitochondrial fatty acid oxidation and vldl formation. furthermore, pge 2 in an autokrine feed forward loop increased the formation of oncostatin m in kupffer cells, which in turn inhibited insulin signaling in hepatocytes by inducing socs3. similar to pge 2 , sphingosine-1-phosphate, whose production was strongly enhanced by exposing hepatocytes to palmitate, attenuated insulin-stimulated glucose use and the induction of glycolytic enzymes in hepatocytes. in summary, the study provides first evidence that in addition to the established mechanism, small lipid mediators like prostaglandins and sphingolipids may impact on hepatic insulin resistance. background: the progression of childhood cardio-metabolic risk factor to adulthood suggests early origin of pathogenic pathways leading to chronic non-communicable disease as diabetes. objective: we sought to examine the distribution of insulin resistance (homa-ir) among children inhabiting delhi and clustering of cardio-metabolic risk variables among them. research design and methods: a cross sectional study was conducted among children aged 10-18 years. estimates of insulin resistance were derived on the basis of homeostatic model assessment. the 75th percentile of homa-ir for normal weight subjects with normal fasting glucose was considered as cutoff for insulin resistance. total body fat was assayed using body composition analyser (tbf-310h14a) employing bioelectric impedance technique. bmi was converted to age-and sex-standardized percentiles and subjects were classified as underweight if bmi <5th and overweight if bmi was >85th percentile. children with blood pressure >95th percentile, adjusted for age and sex were categorised as hypertensive. information on socio-demographic features and family history of diabetes was obtained through standardized questionnaire. result: independent student t-tests showed significantly higher percentage of body fat among girls (21.12%) as compared to boys (14.55%). mann-whitney u-test analysis depicts significantly higher level of fasting sugar among girls (3.96 mmol/ml) than boys (3.83 mmol/ml). out of 98 children 27 (27.6%) were insulin resistant. among them five were underweight while seven were overweight, 6/4 (sbp/dbp) were hypertensive and 14 had family history of diabetes. conclusion: high prevalence of insulin resistance, a precursor of diabetes among these children foreshadows a worrisome trend for the burden of type 2 diabetes in near future. both aims, losing weight as well as improving metabolic conditions, should be reached within the first weeks of an intervention program already. therefore, results of a short-time intervention concept should be presented. in answer to a regional tv-report dealing with "successful weight loss", 55 overweight adults contacted the freiburg institute of preventive medicine (ipm) to participate in a 6-week intervention program. after looking for exclusion criteria, 48 patients (49.9 ae 13.9 years, 34.2 ae 4.7 m/kg 2 bmi) could be included. the ipm concept consists of an initial consultation in energy balance and life style changes, the use of a soy-yoghurt-honey product (almased â , 29 per day as meal replacement), and the facility for guidance by phone. forty patients completed the program attending the exit examination. starting from a comparable bmi, the females were younger (n = 23; 46.2 ae 14.1 years) than the males (n = 17; 55.0 ae 12.1 years) and showed less associated risk factors. of significantly older age was the subgroup of patients with metabolic syndrome (ms) or type 2 diabetes mellitus (t2dm) (n = 12; 60.8 ae 5.9 years). all completers were successful in losing weight (pre-post diff. 5.5 ae 3.0 kg) and showed a comparable weight reduction after intervention: females à5.3%, males à5.6%, ms/t2dm patients à6.0%. the weight reduction was impressively accompanied by improvements of the metabolic milieu (tg, ldl-c, fbg, hba1c) particularly in the ms/ t2dm patients. the results confirm that the ipm concept is a successful way to lose weight initially and to improve the metabolic milieu within a shorttime period using a product with a high impact of bioactive compounds. objective: menopause-related changes in female body are associated with the greater risk of metabolic syndrome (ms), which includes obesity, dyslipidemia, impaired glucose tolerance, hypertension. the purpose of our study: was to reveal peculiarities of fat and lean mass distribution between postmenopausal women with abdominal obesity and with ms. design and method: the sample consisted of 43 postmenopausal 60-69 years old women (age: mean = 64.8; sd = 0.4); duration of menopause: mean = 14.5; sd = 0.9). the diagnosis of ms was considered according to idf (2005 year) criteria. lean and fat mass distrubution were measured by dual-energy x-ray absortiometry, and were compared for the cohorts with and without ms. data were analyzed using statistical package 6.0 (statsoft). background and aims: attempts to curb the ongoing epidemic of obesity and diabetes in the us and other developed countries will benefit from better understanding of the broad and upstream determinants of the population prevalence of these conditions. this study explores how individual psychosocial characteristics in addition to the social and physical community environment correlate with prevalent obesity and markers of pre-diabetes in a statewide representative sample of the population of wisconsin, usa. methods: we used cross-sectional data from the 2008 to 11 cycles of the ongoing survey of the health of wisconsin (show) a geographically diverse population-based research study of adults, age 21-74 years (n = 2479). obesity (bmi ! 30 kg/m 2 ) and hemoglobin a1c levels were the main outcomes. independent variables included individual socioeconomic status (ses), food insecurity, psychosocial status (depression, anxiety and stress), perceived discrimination and neighborhood resources, and access to health are. contextual predictors included county and census-block group socioeconomic nutrition, and built environment indicators. results: the prevalence of obesity and prediabetes was positively associated with lower ses, food insecurity, markers of depression and anxiety, perceptions about lower access to healthy food and physical activity resources in the neighborhood, and poor health care access. lower community-level ses and a poor nutritional and built environment were also associated with higher frequency of obesity and prediabetes. conclusion: our findings on the psychosocial and contextual correlates of obesity and prediabetes offer insights regarding the profile of individuals and subgroups and where tailored individual and community level interventions are most needed. objective: gastric bypass (gbp) is currently the most effective way of treating obesity. interestingly, the majority of type 2 diabetes (t2d) patients display remission of the disease after gbp. the underlying mechanisms behind this remission are not known. we used a porcine model to study how hormonal and metobolite profiles are affected by gbp. in addition we studied the impact of gbp on endocrine cell populations in the gut and pancreatic islets. methods: gbp-pigs were subjected to oral (ogtt) and intravenous (ivgtt) glucose tolerance tests before and after surgery. shamoperated, pair-fed pigs served as controls. results: during ivgtt gbp-pigs displayed lower glucose and higher insulin levels compared to controls. during ogtt, gbp-pigs displayed higher glucose and a more rapid and robust insulin response than controls. in line with this, gbp-pigs had higher beta cell mass and more extra-islet beta cells. further, during ogtt gbp-pigs displayed robustly elevated gip levels, whereas glp-1 levels were unchanged. furthermore, gbp-pigs displayed elevated density of gipproducing k-cells, but reduced density of glp-1-producing l-cells in the gut. metabolomic analyses revealed a difference in the metabolite pattern between the two groups, mainly explained by the fact that gbp provoked lower levels of free fatty acids (ffa) and higher levels of branch-chain amino acids (bcaa). conclusions: gbp in pigs provokes, 1. enhanced insulin secretion and increased beta cell mass. background: several authors have recently reported that both hiv replication and antiretroviral therapy (art) may influence adiponectin expresion which is correlated with insulin sensitivity via glucose transporter type 4 recruitment to plasma membrane. we assessed serum adiponectin patterns in a cohort of hiv-1 positive caucasian patients undergoing cart in relation to insulin resistance (ir) and hiv replication. a cross-sectional study was performed in a cohort of hiv-1infected patients attending the national institute of infectious diseases, bucharest. blood samples were tested for hiv viral load and adiponectin. insulin resistance was estimated by homoeostasis model assessment. in order to evaluate differences between groups we used mann-whitney-wilcoxon and t-tests. results: eighty patients (56.3% males) with a median age of 28 years (iqr 21 years) were included in the study. the median time from hiv diagnosis was 74.0 months and the median time on cart was 68.0 months. most patients (71.3%) had undetectable serum hiv loads. median adiponectin serum value was 10.34 lg/ml (iqr 14). most patients (68.8%) had insulin resistance. insulin resistant patients had significantly lower median levels of adiponectin (9.3 vs. 19.7 lg/ ml, p = 0.04). there were no significant differences between median adiponectin serum levels in groups with persistent and undetectable hiv replication (p = 0.57). no significant correlation was noted between insulin resistance and hiv replication. conclusions: in our cohort of young hiv-1 patients with a high prevalence of ir decreased adiponectin serum levels were associated with decreased insulin sensitivity. hiv replication may not influence in vivo adiponectin expression. objective: to examine obesity and metabolic disorders associated with vitd deficiency/insufficiency in children older people. methods: cross sectional study in 1106 community-dwelling subjects 60-98 year (766 women) residing in santiago chile. plasma levels of 25(oh)d were determined by radioimmunoassay. glucose, insulin and crpus, were measured in a fasting blood sample. blood pressure and complete anthropometry were measured. results: mean serum 25(oh)d was 64.3 ae 33.6 nmol/l (men 67.8 ae 33.0; women 62.6 ae 33.7, p < 0.01). 25(oh)d levels were under 75 nmol/l in 65.0% of men and 71.9% of women (p = 0.020). insulin resistance was present in 17.4% of all subjects and obesity in 38.5% of women and 26.1% of men (p < 0.001). significant negative crude association between 25(oh)d across bmi categories was found in the total sample (p < 0.001). crude association of vitd <50 nmol/l with obesity (p = 0.002), waist circumference (p = 0.011), insulin resistance (p < 0.001), metabolic syndrome (p = 0.004), hta (p < 0.001) and age ! 70 year (p < 0.001) was observed. after age, sex, waist circumference and season adjustment, vitd <50 nmol/l was associated with increased risk of insulin resistance, or 1.65 (ic 95% 1.18-2.32) p = 0.003. conclusion: high prevalence of vitd deficiency/insufficiency was observed in the chilean older people. vitd deficiency is associated with insulin resistance. in the future, randomized controlled trials are needed to establish a cause-effect relationship between vitd deficiency, obesity and its metabolic consequences. surrogate markers are used to estimate degree of steatosis and liver fibrosis in nafld. oxidative stress is important in the pathophysiology of nafld. the aim of this study is to find correlations between blood antioxidants and laboratory variables that are routinely determined in patients with nafld in clinical practice and used in fibrosis test. thirty-five obese children (8-16 years old) with increased liver echogenicity on ultrasounds and 30 healthy lean children were enrolled. other causes of chronic hepatitis, such as chronic viral hepatitis, were excluded. erythrocyte superoxid dismutase (sod), glutathione peroxidase (gpx) activities and plasma levels for albumin, uric acid and bilirubin were measured as antioxidants. the nafld fibrosis score was calculated by an altgoritm including: age, bmi, glycaemia, platelet number, albumin, ast/alt. pearson correlations were calculated. none of the obese children had fibrosis according to the test score. obese children with nafld had lower levels for albumin (p < 0.01), but higher levels for uric acid (p < 0.01), sod and gpx activities (p < 0.05) vs. lean children. all the measured antioxidants were related with variables included in the fibrosis test. for p < 0.05, the calculated correlations were: sod activity with alt activity (r = à0.52) and albumin (r = 0.55), gpx activity with ast/alt ratio (r = 0.56), uric acid with alt activity, age and bmi (r = 0.42). this study demonstrates strong relations between blood antioxidant defence systems with fibrosis test variables in nafld in obese children. women; diabetes prevention programs (dpp) are only available for adults at high risk of developing t2d. 1. to develop a dpp for post-gd women. 2. to assess the feasibility/acceptability of this dpp through a pilot rollout. methods: a working group was formed to develop a dpp that incorporated the needs of a mother with a young family around established lifestyle modification goals. this program was piloted with a group of nine post-gd women. the pilot had high attendance levels (70-90% for group sessions), but only 20% of participants attended all sessions (illness and travel commitments causing non-attendance). the original magda dpp was restructured to: an individual session, five group sessions and two follow-up telephone conversations. childcare arrangements were investigated, but participants elected to have their children attend sessions. participants reported the program to be acceptable and suited to their needs. the pilot determined that the program was feasible and met the needs of the target population. currently, recruitment is underway for 574 post-gd women from three hospital sites for the magda study, which uses a dpp designed for post-gd women and has a whole-of-family focus and tackles common barriers to success. objectives: assess the risk factors associated to physical inactivity (pi). a cross-sectional study was performed in 2006 in subjects aged ! 18 years, in the urban area of montes claros, brazil. the physical inactivity level was determined by the international physical activity questionnaire short version. the subjects were classified in active ( ! 150 min per week) and sedentary (<150 min per week). the univariate analysis was first performed and were included in the model when associated with p < 0.2. the poisson regression with robust variance was realized and the prevalence ratio (pr)-crude and adjusted-and 95% confidence intervals (ci) were estimated to determine the relationship between pi and risk factors like sex, age group, skin color, marital status, income, education, hypertension, dyslipidemia and overweight. the variables that remained significant when adjusted. the statistic was performed in stata. results: a sample of 528 individuals were studied (74.8% women). the prevalence of pi was of 27.6%. the variables were associated with pi were sex, the age group, marital status, education, hypertension, dyslipidemia and overweight with p < 0.05. the following variables were significantly associated with pi after adjustment for confounding variables were: sex, female (rp = 0.71; ic: 0.53-0.96) and marital status, separated/divorced/widowed (rp = 1.71; ic: 1.12-0.60). the gender and marital status were associated with pi with pr higher for these variables in this population, which shows the need for development of effective public policies, integrated to investigate the biological causes, but also the social risk factors. objective: to determine whether an interactive mhealth exercise intervention is more effective than standard of care exercise in patients with metabolic syndrome. methods: participants [n = 127, mean age 57.4 (sd 9.1) year, 74% female] reported to the laboratory at baseline (v0) and follow-up [12 (v1) and 52 (v2) weeks]. anthropometrics, heart rate (hr) and blood pressure (bp) were measured and blood drawn to examine fasting glucose (fg) and glycated hemoglobin (hba 1c ). fitness (vo 2max ) was assessed and individualized exercise programs were prescribed. the intervention group received a smartphone data portal and bluetooth tm enabled biometric tracking. differences between groups in outcomes (v1-v0) were examined using analyses of covariance, which adjusted for baseline levels of the outcome of interest. group differences from v0 to v2 were examined with two-way repeated measures anovas. results: at v1, systolic bp was reduced in both groups but significantly more in the control group (difference in mean change: à5.68; 95% ci: à10.86, à0.50, p = 0.03). there were no differences between groups at v1 for other outcomes. across the follow-up period, systolic bp, diastolic bp, resting hr, weight, body mass index, waist circumference and hba 1c were decreased, and vo 2max and target hr were increased for the entire study population (p < 0.001) with no difference in rate of change between groups. fg was significantly higher in the intervention group across the entire follow-up period (p = 0.02) and both groups had increasing levels over time (p = 0.03). conclusion: over 52 weeks, cardio-metabolic risk factors improved with both standard and mhealth supported exercise interventions. background and aims: breastfeeding improves glucose tolerance in the early postpartum period of women with prior gestational diabetes (gdm), but it is unclear whether future risk of metabolic alterations, like type 2 diabetes, is reduced. the aim of this study was to investigate the effect of lactation, 3 years after pregnancy, on glucose metabolism and beta cell function in women with prior gdm. material and methods: women with prior gdm (carpenter and coustan criteria) were evaluated with comparison of results for "lactating" [bf] vs. "non lactating women" [non bf]. breastfeeding was defined exclusive if lasting more than 4 weeks. each woman performed a 75-g ogtt to analyze glucose tolerance, insulin sensitivity/resistance and b-cell function. lipid and inflammatory profile was also studied. statistics: paired and un-paired t-test, mann-whitney and v 2 tests. methods: subjects completed three randomly ordered conditions: mie (50% vo 2 peak), hie (82% vo 2 peak), and seated rest (control). exercise energy expenditure was equated to 200-kcal. one-hour postexercise (or control), subjects received a 75-g oral glucose tolerance test (ogtt). plasma glucose and insulin concentrations were measured before and at frequent intervals after glucose ingestion. si was derived using the following models (i) oral minimal model (omm), (ii) matsuda composite index, (iii) cederholm index, and (iv) stumvoll index. exercise induced changes in insulin action were expressed relative to the control condition (exercise-control). spearman correlation coefficients and rm-anova were used to compare relative changes in insulin sensitivity. results: si calculated during the control condition was moderately correlated among the various indices (r-value range: 0.38-0.77, pvalues: 0.13-0.001). relative to control, si after mie ranged from 2% higher (cederholm) to 22% higher (omm), and after hie ranged from 5% lower (matsuda index) to 30% higher (omm excess caloric intake leads to metabolic overload and is associated with development of type 2 diabetes (t2dm). current disease management concentrates on risk factors of the disease such as blood glucose, however with limited success. we hypothesize that normalizing blood glucose levels by itself is insufficient to treat the disease and the development of complications, and that dietary interventions which diminish metabolic overload may be more efficacious in retarding the disease. we explored the efficacy and systems effects of pharmaceutical interventions vs. dietary lifestyle interventions (dli) in developing t2dm and complications. high fat diet (hfd)-fed ldlr à/à mice with already established disease phenotype, to mimic the human situation, were treated with 10 different drugs mixed into hfd or subjected to dli (switch to lowfat chow), for 7 weeks. interventions were compared to untreated reference mice kept on hfd or chow only. although most of the drugs improved hfd-induced hyperglycemia, drugs only partially affected other risk factors and also had limited effect on disease progression towards microalbuminuria, hepatosteatosis and atherosclerosis. by contrast, dli normalized t2dm risk factors, fully reversed hepatosteatosis and microalbuminuria, and attenuated atherosclerosis. the comprehensive beneficial effect of dli was reflected by normalized metabolite profiles in plasma and liver. analysis of disease pathways in liver confirmed reversion of the metabolic distortions with dli. this study demonstrates that the pathogenesis of t2dm towards complications is reversible with dli and highlights the differential effects of current pharmacotherapies and their limitation to resolve the disease. introduction: obesity may induce an oxidative stress in adipose tissue, leading to deregulated expression of inflammatory cytokines which could be an early instigator of obesity-associated diabetes and cardiovascular diseases. thus, the biological effect of natural micronutrients such as plant polyphenols that may increase the antioxidant capacity of the body is of high interest. aim: our objective was to explore the antioxidant polyphenol content of three medicinal plants (gouania mauritiana, antirhea borbonica, doratoxylon apetalum) and their impact on the viability, production of reactive oxygen species (ros) and inflammatory response of preadipocytes exposed to oxidative stress. methods: polyphenol-rich extracts from plants were analyzed for their radical-scavenging capacity by dpph method. then, their ability to modulate 3t3-l1 preadipocyte viability and protection against h 2 o 2induced oxidative stress was assessed by both mtt viability and ldh death assays, as well as by dcfh-da test evaluating intracellular ros production. finally, il-6 secretion was measured by elisa. results: all plant extracts exhibited high levels of antioxidant polyphenols which protected preadipocytes against oxidative stress by decreasing ros generation and modulating the inflammatory response. such an antioxidant activity of plant extracts could be partly mediated through their radical-scavenging capacity. we identified three medicinal plants naturally rich in antioxidant polyphenols which exerted antioxidant and antiinflammatory properties on preadipocytes exposed to oxidative stress. further studies are in progress to clarify the molecular mechanism as well as in vivo potential effects of such medicinal plants to protect against metabolic and inflammatory disorders known to play a key role in obesity-related insulin resistance. background: increased fasting plasma glucose is known to lead to diabetes, and diabetes associated complications often manifesting prior to the identification of type 2 diabetes. we aimed to determine the association of fasting plasma glucose levels with cholesterol levels and oxidative stress markers. methods: one hundred and sixty two participants attended the diabetes screening clinic, at charles sturt university, australia between february 2006 and june 2008. participants were investigated based on the american diabetes association′s diagnostic criteria of diabetes mellitus and prediabetes, i.e. fasting bgl. results: atherogenic index of plasma (aip) was elevated in the prediabetes group (0.076 ae 0.06) and continued to increase in the diabetes group (0.18 ae 0.057) compared to controls (à0.06 ae 0.04; p < 0.05). serum 8-hydroxy-2-deoxy-guanosine (8-ohdg) level was greater in the prediabetes (525.8 ae 149.7 pg/ml) compared to controls (155.7 ae 100.3 pg/ml; p < 0.05). the diabetes group (2000.7 ae 132.7 pg/ml) had the highest level of 8-ohdg. these changes paralleled by a reduction in erythrocyte reduced glutathione (gsh) from controls (70.7 ae 2 mg/100 ml, p < 0.05) to prediabetes (66.9 ae 3 mg/100 ml; p < 0.05) and the diabetes group (61.3 ae 2.6 mg/100 ml, p < 0.001). conclusion: this increase in 8-ohdg may be related to the decrease in erythrocyte gsh antioxidant capacity. a statistical significant positive correlation (pearson's r = 0.255; p < 0.001) between aip and 8-ohdg suggests that 8-ohdg may be a useful additional biomarker to determine the degree atherogenic risk in the presence of elevated lipids. rowett institute of nutrition and health, university of aberdeen, aberdeen, uk, 2 ilsi europe, brussels, belgium, 3 lund university, lund, sweden, mechanistic evidence suggests that elevated blood glucose levels contribute to the development of t2dm. adoption of a nutritional approach to manage postprandial glycaemia could deliver a cost-effective t2dm prevention and management strategy, applicable across the population. to implement a successful strategy it is essential to understand the impact of dietary modulation on the postprandial rise in blood glucose concentrations. for this reason, a systematic and comprehensive literature review was undertaken, using the highest quality data. included were the major macronutrients (carbohydrate, protein, fat), micronutrient vitamins and minerals, non-nutrient phytochemicals and additional foods including low-calorie sweeteners, vinegar and alcohol. the strongest corroboration of efficacy for improving glucose homeostasis was for insoluble and moderately fermentable cereal-based fibre and monounsaturated fatty acids as replacement of saturated fat. postprandial glucose levels were decreased by intake of viscous soluble fibre and this was considered to be predominantly by delaying absorption of coingested carbohydrates. weaker but substantial evidence demonstrated that certain phytochemical-rich foods were likely to be effective and this may be associated with the suggestion that the gut microbiota plays an important role in metabolic regulation, including provision of phytochemical and other metabolites. it is clear that dietary components have significant and clinically relevant effects on blood glucose modulation. this suggests that employing a dietary regimen to attenuate the postprandial rise in blood glucose levels along with previously identified targets (reducing excess body weight and an increase in physical activity) will benefit the health of the population and limit the increasing worldwide incidence of t2dm. knowing their metabolic control, and lipid profile, through an early intervention, we can reduce their risk factors for cardiovascular disease, and diabetes complications. the aim of our study was to determine the lipid profile of patients newly diagnosed with type 2 diabetes. patients and methods: hundred patients, selected at the outpatient policlinic nr. 3 in tirana, the capital of albania. all the patients had completed anthropometric measures, hba1c and lipid profile after a 12-h fast. the persons younger than 30 years, diabetes diagnosed prior to 6 months, or uncompleted data were excluded from the study. results: we obtained all the data for 75 patients. males 48 (64%), mean age 55.6 ae 9.32 years, mean bmi 28.7 ae 4.2 kg/m², mean hba1c 7.44 ae 2.54%. 47.6% of the patients had a total cholesterol >220 mg/dl, 16% of the patients had triglycerides >250 mg/dl, and 41.3% of them had tg <150 mg/dl, and 64% had the ldl >130 mg/ dl 83.3% of males had hdl <45 mg/dl and 100% of women had hdl <55 mg/dl respectively. conclusions: in our study the lipid profile of albanian patients was somehow different from the common profile of patients newly diagnosed with t2 diabetes. even in the previous studies we have found a lipid profile with high total cholesterol levels, and especially very low hdl levels, probably due to the sedentary lifestyle, which needs further evaluation, because the metabolic control of our patients was not very bad. background and aims: physical activity (pa) in people with type 2 diabetes helps to improve metabolic control, lipid profile, and to reduce weight. the aim of our study was to demonstrate the effect of a single session of pa to the glycaemic profile, and the effect of increasing daily physical activity on the weight and lipid profile. patients and method: fifty patients were recruited for a 8 weeks training session. every pa session lasted 30 min of fast walking. the number of daily steps was measured through a pedometer, and all the participants were encouraged to complete at least 10,000 steps/day. all the patients had completed anthropometric measures, fat body composition and lipid profile at the beginning and the end of study period. background: homocysteine is a cardiovascular risk factor for the development of vascular pathology. large population studies have been conducted demonstrating a positive association between homocysteine levels and risk of developing cardiovascular disease. paradoxically in type 2 diabetes, if renal function is within a normal range, homocysteine levels are either similar or reduced when compared to levels in the normal non-diabetic population. whether the same is true for pre-diabetes has not been previously explored. our study examines the plasma levels of homocysteine in controls, and participants with prediabetes and diabetes from a screening program in regional new south wales. methods: seven hundred and forty-four participants attended the diabetes screening clinic, at charles sturt university, australia. participants were investigated based on the american diabetes association′s diagnostic criteria of diabetes mellitus and prediabetes, i.e. fasting blood sugar levels. venous plasma homocysteine samples were measured using the fluorescence polarization immunoassay on the imx â analyzer (abbott laboratories, abbott park, il). results: median age across the three groups ranged from 61 to 64 years. the median levels for plasma homocysteine between groups were not significantly different. there was a trend for plasma homocysteine (μmol/l) levels to be higher in the prediabetic group 9.15 ae 2.7 (sd) compared to the control group with 8.4 ae 2.84 (sd) and in the diabetic group of 8.7 ae 2.7 (sd). conclusion: there is a non-significant median increase in homocysteine in the pre-diabetes group. further larger cross-sectional population studies would be able to address whether this is a chance effect and also establish statistical significance if present. clinical endocrinology, institute for endocrine pathology problems, kharkiv, ukraine introduction: active acromegaly is associated with increased morbidity attributed by systemic complications such as carbohydrate dysfunction. aims: to evaluate the effects of chronic excess of gh and igf-i on prevalence and structure of carbohydrate dysfunction in patients with active acromegaly. subjects and methods: ninety-seven patients (37 men and 60 women; aged 18-76 years) with macroadenoma of hypophysis (67somatotropinoma, 30-somatomammo-tropinoma) were under investigation. blood samples for gh, igf-1, immuno-reactive insulin (iri), glucose were taken in fasting state and on 120 min ottg. insulin sensitivity and b-cell function were estimated by homa calculator v.2.2 data are given as m ae se and coefficient of determination (r 2 ) of multiple regression analysis. results: in 40.2% of patients with acromegalia (gh -22.8 ae 3.5 ng/ ml; igf-1 -620.81 ae 301.30 ng/ml) different types of carbohydrate dysfunctions (cd) were found out: fasting hyperglycemia (10.2%); impaired glucose tolerance (17.9%), and diabetes mellitus (71.8%). 23.3% of patients have had hyperinsulinemic state. it was no sex differences in the cd structure. iri level was 22.3 ae 3.5 mu/ml, insulin sensitivity -73.2 ae 7.45%, β-cell activity -161.4 ae 10.3%. homa2_ir was positively associated with gh (r 2 = 28.5%, p = 0.001) and igf-1 (r 2 = 36.6%, p = 0.001). homa2_b% was positively associated with gh (r 2 = 17.4%, p = 0.001) and igf-1 (r 2 = 15.2%, p = 0.01), and negatively with disease duration (r 2 = 12.8%, p = 0.04). the gh hypersecretion and length of its pathologic action predetermine the stage of carbohydrate dysfunction from fasting hyperglycemia, impaired glucose tolerance to overt diabetes mellitus. a. vlassopoulos, m. lean, e. combet human nutrition, school of medicine, university of glasgow, glasgow, uk background: protein glycation is a key mechanism behind chronic diseases in both diabetic and non-diabetic individuals. about 12-18% of circulating proteins are glycated in vivo in normoglycaemic blood, but in-vitro studies have hitherto failed to demonstrate glucose-driven glycation below concentration of 30 mmol/l. methods: albumin, mercaptalbumin (40 g/l) and plasma was incubated with glucose at different concentrations (0-20 mmol/l) for 4 weeks at 37°c. to investigate the effect of oxidation on protein glycation, all protein models were used as native proteins or oxidized proteins (exposed to 10 nmol/l h 2 0 2 for 8 h prior to incubation with glucose and throughout the incubation period). fructosamine was measured at 2 and 4 weeks (nitroblue tetrazolium method). oxidised mercaptalbumin and plasma had higher fructosamine concentrations at 2 weeks at 5 mmol/l glucose compared to native controls (p < 0.05). at 10 mmol/l glucose, the same effect was observed for albumin and mercaptalbumin but not plasma. only oxidized albumin was significantly glycated at 2 weeks with 5 mmol/l glucose (compared to glucose-free control) when native was not. at 4 weeks no effect of oxidation was observed. the current study has for the first time demonstrated the importance of oxidative stress in physiological protein glycation, interacting with glucose and promoting glycation in the early stages of the process. k. elksne 1 , z. paunina 1 , a. jurka 2 , d. rezeberga 1 , p. tretjakovs 2 1 obstetrics and gynecology, 2 physiology, rigas stradins university, riga, latvia introduction: a growing number of obese women in their reproductive years increases their risk for obstetric complications, but still the exact mechanisms is not known. we created a prospective study in which clinical data were collected from antenatal visits in pregnant women. obstetric outcomes were assessed at delivery. obstetrical outcomes depending on fetal macrosomia and maternal bmi were analysed using the fisher exact test. relationships between pre-pregnacy bmi, gestational weight gain and fetal weight were analysed using spearman rank correlations. results: overweight women had lower total weight gain during pregnancy, but higher risk for obstetric complications such as fetal macrosomia, uterine dysfunction, ceasarean section and ruptures of the birth canal. fetal macrosomia positively correlated with pre-pregnancy weight, but not with gestational weight gain during pregnancy. also male gender is a risk factor for birth weight above 4000 g. it is important to increase the number of planned pregnancies, pre-pregnancy visits to doctor in order to inform women about the impact of excess weight on perinatal outcome as fetal macrosomia is associated with impaired maternal bmi. women with bmi normal before pregnancy increased their weight above recommendations during gestation. as excessive weight gain may be a risk factor for long term complications, more information about diet should be provided in antenatal visits. obesity and fetal macrosomia correlate with complications during labour, so additional attention in birth conducting should be paid. background: the obesity epidemic is widely blamed for the rise in type 2 diabetes, but new research suggests it is also linked to the increase in type 1 diabetes (t1d). the combination of obesity and t1d has a negative impact on metabolic control in patients with t1d in particular during pregnancy. aim: to evaluate the rate of overweight or obese women with t1d at pregnancy onset according to their body mass index (bmi) and its relation to metabolic control. we performed an analysis of 50 singleton pregnancies of 38 women with t1d, who consulted our diabetes outpatient clinic since 2007. results: all women were caucasians with mean age of 32 ae 6 years and average diabetes duration of 18 ae 9 years. only 45% of all women with t1d had a bmi of <25 kg/m 2 , 49% were overweight with bmi between 25.0% and 29.9% and 6% were obese. the metabolic control was similar between the group with normal bmi and bmi >25 kg/m 2 (7.5 ae 1.3% vs. 7.2 ae 1.5%). in the patients with normal bmi only 30% started the pregnancy with a hba1c <7%, which was mainly due to unplanned pregnancies. conclusion: our data revealed a high proportion of overweight women with t1d at onset of their pregnancy, confirming the worldwide trend of rising obesity rates and which calls for preventive measures in the preconception counseling. hypertension is more prevalent in hivinfected subjects than in general population, contributing to increased cardiovascular risk in hiv+ patients. moreover, hiv patients more frequently showed metabolic alteration than general population. aim of this study was to evaluate the effect of 144 weeks of administration with telmisartan 80 mg daily in hiv+ patients. we enrolled 13 hiv+ caucasian male patients treated with combined antiretroviral therapy (cart) and discovered to be na€ ıve hypertensive. systolic (sbp) and diastolic (dbp) blood pressure, viro immunological parameters and triglycerides, cholesterol, insulin resistance (homa-ir), inflammatory markers, c-reactive protein (crp), indexes of renal function and cardiovascular risk, microalbuminuria, cystatin c, were measured at baseline (t0), and after 24 (t24), 48 (t48), 72 (t72), 96 (t96), 120 (t120) and 144 (t144) weeks. treatment with telmisartan decreased sbp and dbp levels during the 144 weeks of observation. we also observed improved in totalcholesterol, triglycerides, and in total cholesterol/hdl cholesterol ratio. we also observed an microalbuminuria and cystatin c improvement at the end of study. throughout in the course of the trial our patients showed a significant improvement of the percentage of cd4+ and cd8. telmisartan doesn't interfere with the recovery of immunological parameters in this patients. telmisartan has confirmed durability and effectiveness, excellent tolerability and an high persistance with a good blood pressure control. therefore telmisartan should be the first choice in the treatment of hypertension in hiv+. methods: the subjects studied were patients diagnosed with gdm at ska˚ne university hospital, lund, sweden, 2004 -2011 . sera were analyzed for antibody positivity (gada, ia-2a and znt8a) with commercially available elisa and snps were studied with restriction fragment length polymorphism. results: combinations of two or more autoantibodies (1.5%) were less frequent than single positivity for gada (2.9%) or znt8a (2.6%), but not ia-2a (0.3%). patients that developed t1d postpartum often had combinations of autoantibodies. heterozygosity for r325w was increased (62%) in patients that developed znt8a compared to znt8a negative patients (35%). however this was not statistically significant (p = 0.06). conclusions: the previously reported prevalence of znt8a in gdm was confirmed. positivity for znt8a did not seem to be a good independent predictor for development of t1d. the tendency of increased r325w heterozygosity in znt8a positive gdm patients is a novel finding and of interest since the snp has been suggested to be of importance in both t1d and t2d. among several consequences, obesity seems to have detrimental effects on reproductive function, causing low levels of sex hormones and reduced sperm concentration. the excess of lipids on diet influences metabolism and affects testis reproductive function. the relationship between obesity, reproductive changes and metabolic syndrome is not yet fully clarified. thus, the aim of the study was evaluate the effect of high fat diet, containing soybean oil, on body composition and male reproductive system of young rats. wistar rats, at 21 days, received diet containing 7% (control diet) or 19% (high fat diet, hf) of soybean oil, until 30 and 60 days of age. food intake and body mass were monitored. at the end, body composition was evaluated by dexa and blood, liver, adipose tissue, testis and epidydimis were collected. glucose, triglycerides, cholesterol, hdl, vldl, insulin and leptin were measured. food intake, body mass gain, lean mass, total fat mass and bone mineral content did not differ between the groups at 30 and 60 days. at 30 days, glycemia and epidydimal adipose tissue mass were increased in rats fed with hf. at 60 days, glycemia, leptinemia and the mass of mesenteric adipose tissue, liver and epidydimis were high after hf ingestion, while tg was low. the excess of lipids reflected negatively on intra-abdominal adipose tissue since 30 days, accompanied by hyperglycemia. the hf ingestion maintained high glucose and leptin concentrations without insulin alteration. at the same time, it could predict some testis function change, once epidydimis mass increased in these young animals. adipose tissue is linked to cardio-vascular and metabolic complications of obesity by increased local production of adipokines.to investigate serum levels of adipokines (adiponectin and leptin) and relationship with obesity anthropometric markers and insulin resistance in overweight/obese patients with type 2 diabetes. two groups of subjects were selected: group with type 2 diabetes mellitus (n = 80) and control group (n = 50). the first group was subdivided in two subgroups, according with bmi (overweight and obese). in all individuals were assessed bmi, waist and hip circumference, visceral fat index, serum levels of adiponectin, leptin, insulin and proinsulin and homa-ir was calculated. the determined parameters were modified significantly in the diabetic patients vs. control. comparing the obese diabetic patients with the overweight, serum levels of leptin were higher on obese/overweight (p < 0.001) and levels of adiponectin were lower (p < 0.05). also, serum levels of insulin and proinsulin were higher in diabetic vs. control group maintained their statistical significance difference in the subgroups of overweight/obese (p < 0.01 and, respectively, p < 0.001). bmi was positively correlated with leptin (p < 0.001) and adiponectin negatively (p < 0.005). moreover, leptin was positively correlated with visceral fat index (p < 0.001), waist circumference (p < 0.001) and homa-ir (p < 0.01); adiponectin was negatively correlated with waist circumference (p < 0.01), visceral fat index (p < 0.05) and homa-ir (p < 0.05). in conclusion positive correlation (leptin) and negative (adiponectin) with anthropometric markers of obesity and homa-ir, demonstrating the role of adipokines in the pathogenesis of peripheral insulin resistance in patients with "diabesity." materials and methods: based on research institute of nutrition we randomly chose 12 patients with varying class obesity, who were outpatient for this disease. all patients were tested on the personal scale manifestations of anxiety (d. teylor in adapting t. nemchina). patients were asked to read a set of sentences (41 questions) about their traits. if they agreed with the statement they should answer "yes" if they did not agree -"no". the test had a ball-evaluation, in which anxiety is defined as very high, high, medium and low. objectives: the process of converting prediabetes to diabetes remains a matter of debate. the aim of the study was to assess insulin sensitivity/resistance indices and markers of oxidative stress in prediabetic persons. methods: obese non-smoking caucasians, using neither special diet nor medication, without acute and chronic disorders, were qualified for ogtt and assigned to groups: normal glucose tolerance-ngt, impaired fasting glycemia-ifg, impaired glucose tolerance-igt and newly diagnosed type 2 diabetes-t2dm, each group n = 22 subjects: 11f/11 m; age 52:46-58 (median: quartiles 1-3). plasma glucose (siemens) and insulin (biosource) were determined during ogtt (0',120'). plasma total antioxidant status-tas (randox), thiobarbituric acid-reacting substances-tbars (sigma), lipids and hscrp (siemens), as well as hba 1 c (hplc) were assayed fasting. different indices of insulin sensitivity/resistance, fasting steady-state and derived from ogtt, were calculated. results: the groups did not differ according to age, bmi, waist, blood pressure, lipids and hscrp. insulin sensitivity/resistance indices presented variety of sensitivities and specificities for prediction of dysglycaemia categories, as well as different correlations with metabolic parameters, including oxidative stress, i.e. isi 0,120 &tas (r = 0.37), isi 0,120 &tbars (r = à0.41) in the population n = 88. the comparison of ngt-ifg-igt-t2dm, showed the highest tas in ifg and the lowest in t2dm (p = 0.000), together with increasing tbars from ngt to t2dm (p = 0.001). the results of roc curves analysis pointed isi 0,120 (91% sensitivity; 100% specificity) for prediction of igt, and tas (86% sensitivity; 90% specificity) in differentiation between igt and t2dm. conclusions: insulin-sensitivity indices predict prediabetes in obese subjects near perfectly, while the development of diabetes is preceded by antioxidant insufficiency rather. m. smiraglia 1 , g. bott a 2 , e. orsi 1 1 endocrinology and diabetolgy, fondazione ca' granda irccs -ospedale maggiore policlinico di milano, 2 biomolecular sciences and biotechnology, university of milan, milan, italy t2dm is a metabolic disorder and evidences show that medical nutrition therapy improving glucose metabolism reducing the risk of complications, leads to an improvement in life quality and increase life expectancy. the aims of diabetes management are normalize blood glucose levels and weight control. obesity influences the development of t2dm and complicates its management. the study aim was to value the effect of two different nutritional treatments (nt) on the anthropometric parameters and glucose metabolism in patients with t2dm and obesity. the two nt are: the "low gi diet" that promotes the carbohydrates quality and the food choice is made on the glycemic index; the "low-carbohydrate diet" is based on the concept that glucose metabolism is influenced also by the carbohydrates amount. subjects, selected on including and excluding criteria, were randomly divided in two groups, to each of which one of the two nt was assigned. during the study participants were measured three times. all statistic differences in the values were analyzed with the use of anova test. the "low-gi group" showed a significant decrease in hba1c (à12.26%; p = 0.09). data suggest that this nt positively influences the glycemic control. the "low-carb group" showed a significant decrease in insulin level (à26.5%; p = 0.03), a greater reduction in bmi and whr and a homa-insulin resistance index reduction (à30.97%; p = 0.06). the daily reduction of carbohydrate intake influenced positively the blood glucose response and significantly reduced plasmatic insulin levels. data suggest that either nt improves glycemic control and peripheral insulin sensitivity. aims of study: the use of metformin to control glucose in pregnant women with gestational diabetes mellitus (gdm) to evaluate its safty use during the first trimester of pregnancy. design: cases report of four patients with gdm from the first trimester of pregnancy non smoking with no family history of congenital malformation disease, aged between (20 and 45) and have no liver diseases put on metformin were participated in this study and who had indicating good comliance at more than one visit over several month until delivery. result: all four patient in oure study delivered healthy babies. internal medicine, ist medical clinic, umf iuliu hatieganu cluj-napoca, 2 immunology laboratory, emergency county clinical hospital, cluj-napoca, romania introduction: the potential role of oxidative stress (os) in metabolic syndrome (mets) is rapidly evolving. reported results support the concept that increased os may play a key role in the development of atherosclerosis, hypertension and diabetes. study aim: the purpose of the present study was to analyze the impact of mets and its individual components on os and on the antioxidant status. material and methods: seventy-two hospitalized patients with a mean age 59.18 ae 5.37 years were taken under study between october 2010 and june 2011. mets was diagnosed based on aha/nhlbi/idf 2009 definition. os was assessed by urinary 8iso-prostaglandinf2a (8iso-pgf2a) (immunometric assays) and plasmatic uric acid. antioxidant status was evaluated by plasmatic gluthatione peroxidase (gpx). these data were compared to those of 100 biologically and clinically healthy subjects (mean age 59.93 ae 4.7 years). results: all biomarkers were significantly higher in mets patients as compared with healthy individuals (p < 0.05), except gpx which was significantly lower (p < 0.001). gpx and uric acid were statistically significant correlated. in multivariate analysis 8iso-pgf2a concentrations were influenced by hypertension, fasting glucose and triglycerides, uric acid levels were directly influenced by hypertension, waist circumference, fasting glucose and triglycerides. gpx levels were inversely correlated with blood pressure (all p < 0.05). only gpx was influenced by the number of mets components. objective: the purpose of this study was to compare changes in the plasma metabolome during an intravenous glucose tolerance test (ivgtt) among persons with low or high insulin sensitivity (si <2.5 or >4.5). methods: ivgtts were performed in 12 men and 12 women, 52 ae 1 years, with low si (1.45 ae 0.10) and 12 men and 13 women, 53 ae 2 years, with high si (7.82 ae 0.44). targeted electrospray ionization, tandem mass spectrometry was used to measure plasma concentrations of 15 amino acids and 45 acylcarnitines during the first phase of the ivgtt. other metabolites (glucose, insulin, etc.) were measured by conventional methods. results: fasting glucose, insulin, triglycerides, glycerol, free fatty acids, leucine/isoleucine, tyrosine, glutamate/glutamine, 3-hydroxypalmitoleylcarnitine (c16:1-oh)/dicarboxytetradecanoylcarnitine (c14:1-dc), and docosanoylcarnitine (c22) were significantly higher in the low si group, whereas ornithine was significantly lower. in response to glucose infusion, rates of disappearance of alanine, proline, valine, leucine/ isoleucine, methionine, and phenylalanine were 105%, 92%, 78%, 27%, 129%, and 79% lower, respectively, in the low si group. there were no group differences in changes in circulating concentrations of free fatty acids, glycerol, triglycerides, or acylcarnitines. conclusions: insulin resistance is associated with lower rates of disappearance of neutral, non-polar amino acids during the first phase of the ivgtt, suggesting insulin-mediated clearance of amino acids and/or suppression of protein catabolism may be impaired. cigarette smoking is worrisome in adults and also in adolescents. actually, smoking rates have grown, especially among girls, and little is known about the damages caused in these organisms still young or the consequences in adulthood. this study aim evaluates the influence of smoking in puberty and the consequences of withdrawal, body composition of female and male mice. at 35 days, mice were exposed to 3r4f cigarette (tobacco and health research institute) smoke, 8 h/day for 15 days (s, n = 24). then, half of animals were evaluated and the other half was maintained and evaluated 30 days after stop exposure (30d as). an unexposed group accompanied the events (ns, n = 24). body mass, body composition (dexa), food intake, blood, ipgtt and adipose tissue were evaluated. during exposure food intake was similar all groups, however, s groups showed low body mass gain. 30das, s groups increased body mass and food intake in females. no alterations body composition were in males, while in females, increased total body and trunk fat during exposure and lean mass 30das. fat was high epidydimal and retroperitoneal in females. in regard blood, males did not alter insulin or ipgtt during exposure whilst females had high insulin levels and glycemia at 30 min. withdrawal induced in males, high insulin and low glycemia the ipgtt and in females normal insulin and low glycemia. the set of results indicates different response cigarette smoke in young that seem to start in females and lead malefic metabolic alterations in adulthood. methods: this retrospective study included 763 children and youths (344 boys) from the children's obesity clinic. data were measured at the time for inclusion and included values of body mass index (bmi) standard deviation score (sds), blood pressure, gender, and biochemical measures, including the fasting concentrations of blood glucose, serum insulin, hba1c, and serum lipids. the bmi sds was median 2.64 (range 1.33-5.10) and the age was median 11.6 (range 2.4 -24.7) years. prediabetes was classified as a fasting blood glucose ! 5.6 and <6.9 mmol/l. the mann-whitney-wilcoxon test was used for the analyses. results: prediabetes was present in 97(47 boys) patients. these patients were older, age median 12.29 vs. 11.50 years (p = 0.015), had a higher bmi sds median 2.74 vs. 2.61 (p = 0.005), an increased fasting hba1c median 36 vs. 34 mmol/mol (p < 0.0001), an increased fasting serum insulin median 110.5 vs. 64 pmol/l (p < 0.0001), and an increased median homa-ir 5.01 vs. 2.34 (p < 0.0001), respectively, compared to the patients with a fasting plasma glucose <5.6 mmol/l. no differences were found in any of their fasting serum lipid levels or blood pressure (p > 0.05). in this large group of overweight and obese children prediabetes is prevalent. thus it seems important to identify obese subjects with prediabetes in order to prevent development of diabetes during childhood. introduction: epidemiological spreading of diabetes and developing in early age urge for finding causes and prevention before manifestation. aim of the study was testing accelerator hypothesis t. wilkin and colleges. method: the study is combined retrospective prospective. we examined history and follow up till age of 16 years 200 children who were referred to pediatrician endocrinologist because of obesity or diabetes. results: children from obese mothers and children with lower birth weight have more metabolic disturbances (insulin resistance, neonatal hypoglycemia, obesity, diabetes, precocious puberty, menstrual irregularity). subjects and methods: twenty-two obese women (bmi 35 ae 5 kg/m 2 ) were followed during di that consisted of a 28 days′ very-low-caloriediet (vlcd) and subsequent 2 months low-calorie-diet followed by a 3 months′ weight maintenance diet (wm). mrna expressions of adipokines (leptin, adiponektin, interleukines (il) -6,-8,-18, tnfa, macrophage-chemoattractant-protein (mcp-1), haptoglobin) were measured, using rt-pcr, in samples obtained from abdominal scat at baseline and at the end of vlcd and wm, respectively. results: body weight (bw) and plasma crp decreased during vlcd and at the end of wm (bw: 98.1 ae 16.5 vs. 90.7 ae 15.7 vs. 87.8 ae 15.3 kg (p < 0.001), crp: 6.3 ae 0.8 vs. 2.6 ae 0.3 vs. 2.9 ae 0.3 mg/l (p < 0.001), respectively). the decrease of plasma crp during the entire 6 months′ di correlated positively with the decreases of mrna expression of mcp-1. (r = 0.54, p < 0.001) and il-18 (r = 0.38, p < 0.05) during the entire di and with the decrease of leptin mrna during initial vlcd (r = 0.532, p < 0.01). no correlations were found for other adipokines. objective: to determine associations between change in lean body mass (lbm), fat mass and muscle strength with is following 4 month of resistance training in obese postmenopausal women. methods: thirty-four (12 with and 22 without metabolic syndrome) non-diabetic obese postmenopausal were recruited. participants completed a 4 month resistance training program (3 times/week). body composition (dxa), handgrip strength and quadriceps strength were measured. fasting glucose and insulin levels as well as ogtt derived is indices (stumvoll, matsuda) and homa were also measured. non parametric correlations were performed with spss (17.0). results: significant increases in muscle strength, is and lbm as well as decreases in fat mass were observed after the intervention. however, no correlations were observed between changes in is with changes in muscle strength, lbm or fat mass in all participants as well as in subjects with or without the metabolic syndrome. conclusion: our results suggest that the changes in muscle strength, lbm and fat mass may not be associated with changes of is in obese postmenopausal women. therefore, other potential variables seem to be implicated in the changes of is in our cohort. objective: to examine the relation between total work (tw) performed and total heart rate work performed (trimps) with changes in cardiometabolic risk factors after resistance training in obese postmenopausal women. methods: thirty seven postmenopausal women (age: 60 ae 4.87, bmi: 34.24 ae 3.53) completed a 4 month resistance training program (3 times/week). body composition (dxa), blood pressure, metabolic risk factors (insulin sensitivity, 2 h glucose, lipid profile and crp) and muscle strength were measured before and after the intervention. moreover, tw was calculated in each session by multiplying the number of repetitions, sets, weight and range of motion. thereafter, we added all training sessions. a heart rate monitor was used to assess trimps during each session. subjects were also categorized into two groups based on the top or lower 50th percentile of tw and trimps (high (n = 17) vs. low (n = 17)). results: we showed that tw negatively correlated with systolic blood pressure and 2 h glucose levels (p < 0.05). in addition, crp was negatively associated with trimps (p < 0.05). furthermore, high workers had lower levels of fat mass percentage and 2 h glucose as well as higher lean body mass content than low workers. as for the high trimps group, we observed lower levels of crp compared to the low tripms group (p < 0.05). conclusion: results indicate that the quantity of work performed during resistance training is associated with better changes in metabolic risk factors in obese postmenopausal women. research design and methods: this cross sectional study included gestational diabetes patients who were diagnosed with gdm by a 100 g ogtt and who underwent a standardized mixed meal tolerance test (mmtt). patients were divided into three groups according to the number of abnormal hyperglycemic values (group i for 2 values, group ii for 3 values, and group iii for 4 values). glycemic parameters were compared to assess glycemic control (glucose, a1c, ga) and gluco-metabolic homeostasis (homa-β, homa-ir). results: a total of 92 gdm patients were recruited for this study. subjects whit a greater number of hyperglycemic values were also more hypertensive and obese, and had decreased insulin secretory functions than those with a lower number of hyperglycemic values (lnhoma-β, 5.37 ae 0.84 vs. 5.14 ae 0.46, 4.65 ae 0.64, p < 0.001). those with higher hyperglycemic values tended to have increased insulin resistance, but this result was not significant (lnhoma-ir, 0.25 ae 0.61 vs. 0.47 ae 0.59, 0.65 ae 0.76, p = 0.062). lnhoma-β correlated negatively with fasting glucose, a1c, ga and ga/a1c. multivariable regression analysis revealed that age and ga were significant independent predictors for lnhoma-β (standardized β = à0.202, à0.423, respectively, p < 0.05) but not a1c. conclusions: korean women with dysfunctional pancreatic β-cells and increased insulin resistance are prone to gestational diabetes. ga, but not a1c, is significantly correlated with pancreatic β-cell function. conclusion: altered adipokine profile is associated with increased cvd risk in pcos patients. these markers can serve as potential therapeutic target for decreasing their cv risk. objective: to determine the prevalence and determinants of nonalcoholic fatty liver disease (nafld) in a sample of adult iranian general population. method: this was a cross-sectional study being performed in shiraz, southern iran during a 10-month period from november 2010 to september 2011 through cluster random sampling of iranian general population in shiraz region. all individuals underwent anthropometric and blood pressure measurements and thorough medical history and physical examinations. laboratory measurements included fasting blood glucose (fbs), lipid profile, complete blood count (cbc) and liver function tests. nafld was diagnosed by transabdominal ultrasonography. result: overall we included 819 subjects in this study among which there were 340 males (41.5%) and 479 females (58.5%) with the mean age of 43.1 ae 14.1 years. nafld was diagnosed in 176 (21.5%) subjects. patients with nafld were significantly older (p < 0.001), had higher proportion of male gender (p = 0.004) and had higher bmi (p < 0.001). they also had higher prevalence of hypertension (p < 0.001), high fbs (p < 0.001), high cholesterol (p = 0.026), high triglyceride (p < 0.001) and high waist circumference (p < 0.001). taking all these together, patients with nafld had significantly higher prevalence of metabolic syndrome when compared to healthy subjects (p < 0.001). conclusion: the prevalence of nafld in this group of iranian adult general population is 21.5%. nafld in iranian population is associated with male gender, old age, obesity, and features of metabolic syndrome. a. othman 1 , t. hornemann 2 1 university hospital zü rich, 2 clinical chemistry, university hospital zü rich, zü rich, switzerland 1-deoxysphingolipds (dsl) are atypical and neurotoxic sphingolipids which are formed by the enzyme serine-palmitoyltransferase (spt) due to a promiscuous use of l-alanine over its canonical substrate l-serine. pathologically elevated dsl levels were identified as a cause for the rare and inherited sensory neuropathy hsaniwhich is associated with several missense mutations in spt. significantly elevated dsl levels were also found in patients with mets or t2dm as demonstrated in various clinical studies. principal component analysis identified the dsls as important descriptors for the mets statecomparable to triglycerides and superior to conventional mets biomarkers like fasting glucose or wcf. partial correlation analysis showed an independent correlation to plasma glucose and triglycerides. strikingly, recent data from prospective clinical studies identified plasma dsls as highly significant and independent predictors for the risk to develop t2dm. elevated dsl levels were also confirmed in plasma and liver of stz rats. the dsls are therefore clinically relevant blood biomarkers for an impaired glucose homeostasis but might also be directly involved in the pathology of diabetes related sequelae. like observed in hsan1 the 1-dsl formation is significantly suppressed in response to an oral l-serine supplementation. feeding an l-serine enriched diet to stz rats resulted in a significant reduction of plasma dsls, a significant improvement of neuropathic symptoms and reduced cataract formation. our findings strongly support the value of dsls as novel and clinically relevant biomarkers in mets and t2dm but also as therapeutic targets for the treatment of the diabetic neuropathy and other sequelae. objectives: prevention of cardiovascular disease focused on the early stages of atherosclerosis, including endothelial dysfunction, should arouse attention of clinicians in dysglycaemia patients especially. the aim of the study was to assess plasma e-selectin concentrations in newly diagnosed type 2 diabetes (t2dm) and in t2dm patients treated with metformin, comparing with normoglycaemic individuals. material and methods: excess body mass non-smoking males and females, 41-73 years old, presented with neither acute nor chronic disease, were enrolled into the study. oral glucose tolerance test (ogtt) was performed to find normal glucose tolerance, group-1 (n = 20, age: median 58; interquartile range 52-63) and newly diagnosed t2dm, group-2 (n = 21, age: 57; 54-67). group-3 consisted of t2dm patients who take medication (metformin) for at least 1 year (n = 22, age: 58; 54-65) and present no retinopathy, nephropathy and neuropathy, as well as no history of coronary incident or stroke. all participants were measured plasma glucose (g-0, g-120), fasting lipids and insulin, and hba1c level. e-selectin concentration in plasma was assessed using elisa method (r&dsystems). data are shown as median and interquartile range. results: groups 1-2-3 did not differ in respect to their age and bmi. the comparison among groups, followed by post hoc analysis, revealed different (p = 0.001) e-selectin concentrations, ng/ml: group-1: 23.3 (18.5-34.5), group-2: 43.5 (36.3-51.7), group-3: 26.6 (18.7-32.7). in combined group1 + 2 (n = 41) the correlation e-selectin&g-120' was observed independently from bmi and triglycerides (multiple regression β = 0.506; r 2 = 0.35; p = 0.0025). conclusion: metformin therapy may limit early stages of atherosclerosis in t2dm patients not only works to decrease plasma glucose. aim: to evaluation clinical efficacy of candesartan and its effect on parameters of vascular elasticity, lipid profile, lipid peroxidation and antioxidative protection against a background of single-candesartan therapy in females with arterial hypertension (ah) and abdominal obesity (ao) during menopause. methods: forty-six patients were divided into two groups. group1 (25 subjects) were given candesartan and group 2 (21 subjects)enalapril. twenty-four hour blood pressure monitoring (bpm), pulse wave velocity; plasma lipids, diene conjugates (dc), malonic dialdehyde, superoxide dismutase (sod) and homocysteine, uric acid were estimated. the dynamics of all parameters was evaluated initially and in 6 weeks. results: candesartan is a more effective hypertensive drug that has a valid effect on the readings of systolic blood pressure (sbp) and diastolic blood pressure (dbp) measured in the doctor's office and on the parametrs of bpm (daily sbp and dbp, daytime and nighttime sbp and dbp, sbp and dbp variability, the rate of morning rise in sbp and dbp). we revealed a favorable effect of the medication on vascular wall elasticity, valid reduction of cholesterol, low-density lipoprotein cholesterol, triglycerides, dc, homocysteine and uric acid increase in sod in group 1. the proved antihypertensive effect of the candesartan therapy against a background of normalization of vascular wall elasticity, plasma lipids and processes of lipid peroxidation, in the presence of differently directed correlations between clinical and biochemical characteristics, shows that candesartan has pathogenetic mechanisms of correcting ah in females with ah and ao during menopause. background: high intensity interval training (hiit) may improve insulin action in skeletal muscle, but this has never been shown. objective: to study the effect of hiit on insulin mediated glucose uptake rate in skeletal muscle. methods: four healthy sedentary males [age 53 ae 4 years (mean ae se), bmi 28.4 ae 1.2 kg/m 2 ] were included. a total of eight one-legged training sessions were performed on an ergometer bicycle as 10 9 1 min high intensity exercise (workload >60% of one-legged vo 2 -peak, and heart rate >80% of maximal heart rate) with 1 min recovery between each interval. lean leg mass before and after the training period was assessed using dual-energy x-ray absorptiometry. forty hours after the last training session, a two-step isoglycemic, hyperinsulinemic clamp was performed in combination with arteriofemoral venous catheterization. blood flow was measured with doppler ultrasonography. data were expressed per kg lean leg mass, and differences were tested by t-test. results: insulin stimulated glucose clearance rates were significantly higher in trained compared with untrained legs in both steps of the clamp (figure). the lean mass of the trained legs did not significantly differ from the untrained legs (p = 0.51). conclusion: hiit increases insulin stimulated glucose uptake in skeletal muscle in the leg after a very short training period. it is a time-effective training modality which may be attractive in the treatment of insulin resistance and type 2 diabetes. (htgpos) and that this htgpos is related with the degree of insulin resistance. our aim was to characterize the adipose tissue of morbidly obese patients with mild or severe htgpos after a fat overload though the expression of a battery of genes involved in lipid metabolism. methods: we studied 57 morbidly obese patients who had mild or severe htgpos after fat overload (patent p201030776). measurements of anthropometric and biochemical variables and oxidative stress biomarkers were done. samples of visceral adipose tissue were obtained during bariatric surgery in the morbidly obese patients. the rna isolation from adipose tissues was done using rneasy lipid tissue mini kit and the gene mrna expression levels were assessed by real-time pcr using an abi prism 7000 sequence detection system. results: no significant differences were observed in biochemical variables except in triglyceride levels between the two groups of morbidly obese patients with postprandial mild or severe htgpos. the morbidity obese patients with severe htgpos had a higher oxidative stress levels. genes involved in the management of triglycerides or lipid metabolism were up-regulated in morbidly obese patients with severe htgpos. conclusion: morbidly obese patients with severe htgpos had a more active adipose tissue regarding the expression of genes involved in lipid metabolisms, these data could indicate a greater flow of lipids and a greater insulin resistance in these patients. patients and methods: three hundred and nine workers (78m/231f; aged 50 ae 13; bmi 33.7 ae 5.3 kg/m 2 ), without previous cardiovascular events, were enrolled. all subjects were evaluated for 13 biochemical analytes (fibrinogen, c-reactive protein, uric acid, creatinine, triglycerides, t-cholesterol, hdl, ldl, homocysteine, glucose, insulin, hba1c, 25 (oh) d). all parameters were routinely assayed in corelab. these parameters together with homa-ir, systolic and diastolic blood pressure, bmi, age, percent of fat and waist circumference were processed by neural networks (autocm). the autocm matrix of connections preserves non linear associations among variables, while at the same time capturing elusive connection schemes among clusters that are often overlooked by traditional cluster analyses. results: with an appropriate pre-processing able to handle each variable according to its high and low values, auto-cm showed a clear association between 25 (oh) d and metabolic status with graph links suggesting a protective role of high 25 (oh) d against increase in bmi, waist circumference and abdominal fat. these associations were not clearly visible with traditional data mining tool. the neural networks map identifies the key role of vitamin d respect to all metabolic parameters considered in our study in the development of prediabetes drawing a physiopathologic road map for obesity and type 2 diabetes. patients and methods: the study included 62 type 1 diabetic patients and 30 healthy volunteer of the same age and sex. blood sample was taken for assessment of omentin and oxldl by elisa technique. also blood sample were taken for analysis of glycosylated hemoglobin, lipid profile and urine sample was taken for assessment of albumin/creatinine ratio. twenty-four hour holter was also done. the study included 62 patients with type 1 diabetes, their mean age were 16.3 ae 1.5 years (14.0-19.0 years), and mean duration of diabetes were 9.4 ae 2.9 years (5.0-16.5 years). omentin was significantly lower, while oxldl was significantly higher than controls. omentin had a significant negative correlation with oxldl and albumin/creatinine ratio and 24 h holter (minimal hr, rms) and positive correlation with vldl. conclusion: a significant reduction of omentin and elevation of oxldl imply that they influence glucose metabolism in type 1 diabetes. omentin had a significant relation to 24 h holter may reflect its role in cardiac affection. while, albumin/creatinine ratio had a significant negative correlation with omentin and positive correlation with oxldl reflect their role in renal affection. patients and methods: the study included 62 type 1 diabetic patients and 30 healthy volunteer of the same age and sex. blood sample was taken for assessment of chemerin, vaspin, adma and oxldl by elisa technique. also blood sample were taken for analysis of glycosylated hemoglobin, lipid profile and urine sample was taken for assessment of albumin/creatinine ratio. twenty-four hour holter was also done. the study included 62 patients with type 1 diabetes, their mean age were 16.3 ae 1.5 years (14.0-19.0 years), and mean duration of diabetes were 9.4 ae 2.9 years (5.0-16.5 years). chemerin, vaspin and oxldl were significantly higher, while adma was significantly lower than controls. chemerin had a significant positive correlation with vaspin, adma and oxldl. vaspin had a significant positive correlation with waist/height ratio, sdann, sdrr and sddrr. albumin/creatinine ratio had a significant positive correlation with chemerin, adma and oxldl. conclusion: a significant reduction of adma and elevation of chemerin, vaspin and oxldl imply that they influence glucose metabolism in type 1 diabetes. vaspin had a significant relation to 24 h holter may reflect its role in cardiac affection. while, albumin/ creatinine ratio had a significant positive correlation with chemerin, adma and oxldl reflect their role in renal affection. patients and methods: the study included 62 type 1 diabetic patients and 30 healthy volunteer of the same age and sex. blood sample was taken for assessment of apelin, nitrous oxide and preptin by elisa technique. also blood sample were taken for analysis of glycosylated hemoglobin, lipid profile and albumin/creatinine ratio in urine. m mode echocardiography was also done. results: the study included 62 patients with type 1 diabetes, their mean age were 16.3 ae 1.5 years (14.0-19.0 years), and mean duration of diabetes were 9.4 ae 2.9 years (5.0-16.5 years). nitrous oxide was significantly lower, while apelin, preptin and albumin/creatinine ratio were significantly higher than controls. nitrous oxide had a significant positive correlation with lvedd, lvesd, pwt and lv mass and negative correlation with preptin and albumin/creatinine ratio. conclusion: a significant reduction of nitrous oxide and elevation of apelin and preptin and their relation to echocardiographic data imply that early assessment of these markers may unmask the initial endothelial dysfunction in type diabetic patients before overt microalbumin and renal impairment supervenes. obesity co-morbidities may appear already early in life in high-risk individuals. today we have no means to identify which obese children who are at highest risk. consequently, we have implemented a study with the objective to identify factors in obese children that could indicate early development of related co-morbidities. in this abstract we have concidered fasting glucose, 2 h ogtt, and degree of obesity as predictors. severely obese children and adolescents treated for obesity between 1996 and 2007 (n = 600), but not undergone bariatric surgery, are included. currently, follow-up measurements have been conducted in 15 subjects. study participants undergo extensive examinations during 2 days including e.g. cardio respiratory fitness, body composition, both oral and intravenous (ivfsgtt) glucose tolerance tests. average age of the first subjects is 20.4 years (17.4-22.1). follow-up time varies from 4.7 to 13.3 with an average of 6.8 years. bmissds at baseline 5.6 (4.2-7.0) correlates with bmi at follow-up (y = 6.0x + 10.9, r 2 = 0.24). no other correlations with degree of obesity at baseline could be found. fasting glucose at baseline correlates with crp 6.8 years later. however, fasting glucose was not correlated with 2 h ogtt, insulin sensitivity, diabetes, or prediabetes at follow-up. no correlations between 2 h ogtt value at baseline and later co-morbidity was found. in the first studied subjects all, but one, of the severely obese children remained obese in early adulthood. higher level of fasting glucose at baseline predicted higher crp at follow-up. analyses from more collected data will be presented. background: obesity acts as an independent cardiovascular risk factor by mechanisms that are not fully understood. elevated levels of the pro-inflammatory cytokines interleukin(il)-6, il-8 and plasminogen activator inhibitor (pai)-1 are found in obese patients. recent studies suggest that inflammation could be an adaptive response to hypoxia within the expanding adipose tissue mass. in this study we investigated the impact of hypoxia on pai-1, il-6 and il-8 regulation in human adipose tissue ex vivo and in vitro. methods: primary human preadipocytes and adipocytes were prepared from subcutaneous and visceral adipose tissue. explants, preadipocytes and adipocytes were cultured under hypoxic conditions. pai-1, il-6 and il-8 antigen were quantified by elisas, mrna levels were determined by realtimepcr. results: pai-1, il-6 and il-8 secretion was significantly increased under hypoxic conditions in subcutaneous and visceral adipose tissue explants. hypoxia significantly upregulated il-6 production in preadipocytes and adipocytes up to 5-fold and 10-fold. il-8 and pai-1 were significantly increased by hypoxia in preadipocytes and adipocytes up to 3-fold and 2-fold (il-8) and 7.5-fold and 6.5-fold (pai-1), respectively. these results were confirmed on the level of mrna expression. conclusion: our data show that hypoxia increases il-6, il-8 and pai-1 production in adipose tissue explants and in cultured human preadipocytes and adipocytes. we therefore hypothesize that hypoxia promotes the pro-inflammatory state seen in obese patients and thus could contribute to the elevated risk for cardiovascular diseases. this study sought to characterize the antioxidant properties and interaction of phenolic (free and bound) extracts from clerodendrum volubile (a leafy vegetable commonly grown and consumed in south eastern part of nigeria), with key enzymes relevant to non-insulin dependent diabetes mellitus (a-amylase and a-glucosidase) in vitro. the free phenolics of clerodendrum volubile were extracted with 80% acetone, while the bound phenolics were extracted from the alkaline and acid hydrolyzed residue with ethyl acetate; and their interaction with the enzymes were assessed. the phenolic extracts inhibited a-amylase, aglucosidase and fe 2+ -induced lipid peroxidation in pancreas (in vitro) in a dose-dependent manner. however, bound phenolics had significantly higher (p < 0.05) a-glucosidase inhibitory activities, than free phenolics while there was no significant difference (p > 0.05) in their a-amylase inhibitory activities. the stronger inhibition of a-glucosidase when compared to a-amylase in both extracts is of pharmacological relevance. the stronger action of the bound phenolic extract on a-glucosidase may explain the possible bioactivity of the phenolics at the brush border end. the phenolic profile in both extracts revealed the presence of phenolic acids and flavonoids. moreover, the inhibitory properties of phenolic rich extracts from clerodendrum volubile on a-amylase, a-glucosidase and fe 2+ -induced lipid peroxidation in pancreas could be attributed to the antioxidant properties of the extracts. from the study, clerodendrum volubile could serve as functional foods and nutraceuticals for early intervention and management of non-insulin dependent diabetes mellitus. department of kinesiology and nutrition, university of illinois at chicago, chicago, il, usa background: alternate day fasting (adf), consisting of a feed day (24-h ad libitum food intake) alternated with a fast day (75% energy restriction), is effective in reducing body weight and modulating adipose tissue physiology. however, the ability of adf in combination with endurance exercise to improve the above variables has never been tested. objective: accordingly, this study examined whether the combination of adf plus exercise produces superior changes in body weight and plasma adipokine levels, when compared to each treatment alone. methods: obese subjects (n = 64) were randomized to one of four groups for 12 weeks: 1. combination (adf + endurance exercise), 2. adf, 3. exercise, or 4. control. results: body weight was reduced (p < 0.05) by 7 ae 1, 5 ae 1, and 1 ae 1 kg, and fat mass decreased (p < 0.001) by 6 ae 1, 4 ae 1 and 1 ae 1 kg in the combination, adf and exercise group, respectively. fat free mass was retained in all groups. adiponectin and resistin values did not change in any group post-treatment. leptin levels significantly decreased (p < 0.05) by 38 ae 10, 15 ae 6 and 15 ae 6 ng/ ml in the combination, adf, and exercise group, respectively. conclusion: these findings suggest that the combination of adf plus exercise produces superior changes in body weight, body composition and leptin levels, when compared to each intervention alone. the plasma total cholesterol and triglycerides concentration were higher than other groups. adipokines results were that resistin, adiponectin, leptin, and tnf-a were significantly lower than [hf], [br] and [hc] . so it had a effect on anti-obesity. these results showed that functional rice especially giant embryonic components will probably be useful in the management of high fat diet-induced chronic disease. m.g. watve 1 , m.s. diwekar 1 , p. patil 2 1 biology, indian institute of science eduction and research, 2 physiology, bharati vidyapeeth medical college, pune, india a crucial link in the classical thinking of type 2 diabetes (t2d) is that insulin resistance (ir) is primary and hyperinsulinemia develops to compensate. high levels of insulin are associated with an insulin resistant state. there have been alternative suggestions that insulin overproduction is primary and ir develops to compensate it. it is also likely that both are secondary effects of an unknown primary cause. we critically examine the alternative possibilities in the light of theory and evidence. temporally hypoglycemia and/or hyperinsulinemia precede insulin resistance in human and animal iugr models. various gene knockouts show that primary muscle and adipose insulin resistance does not lead to hi. in insulinomas, where hyperinsulinemia is primary, insulin resistance develops almost invariably and removal of such tumors increases insulin sensitivity. if ir sets in first, pancreas must sense the ir and increase the insulin production accordingly. if raised blood glucose mediates the response, we expect a positive correlation between fasting glucose and fasting insulin in prediabetic individuals. however, such a correlation is not seen in a large set of data on normoglycemics. no other mechanisms have been postulated that can measure the level of ir and in turn regulate the insulin secretion by beta cells. on the other hand many pathways exist that can induce ir when beta cells overproduce insulin. overall there is substantially more evidence for the hyperinsulinemia-first hypothesis. this can potentially undermine our current understanding of t2d and the entire chain of processes leading to t2d needs to be re-examined. non-alcoholic fatty liver disease (nafld) is emerging as the most common liver disease in industrialized countries. the discovery of food components that would ameliorate nafld is therefore of interest. betulinic acid (ba) is a pentacyclic triterpenoid showing many pharmacological activities, but effect of ba on fatty liver is largely unknown. to explore the anti-fatty liver activity and mechanism of ba, insulin resistant hepg2 cells, primary rat hepatocytes and liver tissue of icr mice fed on hfd were utilized. oil red o staining revealed that ba significantly suppressed the excessive triglyceride accumulation in hepg2 cells and liver of mice fed on hfd. ca +2 -calmodulin dependent protein kinase kinase (camkk) and amp-activated protein kinase (ampk) were both activated by ba treatment. in contrast, protein expression of sterol regulatory element-binding protein 1 (srebp1), mammalian target of rapamycin (mtor) and s6 kinase (s6k) were all suppressed when hepatocytes were treated with ba for up to 24 h period. ba activated ampk by phosphorylation, suppressed srebp1 mrna expression and nuclear translocation and repressed srebp1 target gene expression in hepg2 cells and primary hepatocytes, leading to reduced lipogenesis and lipid accumulation. these effects were completely abolished in the presence of sto-609 (a camkk inhibitor) or compound c (an ampk inhibitor), indicating that bainduced anti-hepatic steatosis was mediated through modulation of camkk-ampk-srebp1 signaling pathway. taken together, our results suggest that ba can effectively ameliorate intracellular lipid accumulation in liver cells, and thus it may be a potential therapeutic agent for treating fatty liver disease. blood glucose concentration, insulin level, glucokinase, g6pase and pepck were significantly decrease relative to the [hf] . in addition, antioxidantresults were that cat, sod and gpx were higher than [hf] . effect of rice supplementation 2 type of giant embryonic on erythrocyte tbars and plasma tbars showed significantly decrease than other groups. these experiments suggest that components of giant embryonic help to lower the level of and blood glucose which reduces the risk of heart disease and diabetes. method: a 75 g o-gtt was performed in 289 normal-weight (n) healthy females, 562 females with overweight and 696 females with obesity (grades 1-3), with measurements of glucose and insulin. homa-insulin resistance (ir) and belfiore-insulin sensitivity (is) were caldulcated. the 90th percentile in the n group was used as cut-off. results: ir was present in 22.5% of women with normal weight and in 45.0% of women with overweight/obesity. 65.4%, 74.2% and 89.6% of women have ir in the obesity grade 1, 2 and 3, respectively. further risk factors for ir are age, hip-waist ratio, triglycerides, hdl, uric acid, pai-1 and fibrinogen (p < 0.00001). hba1c is irrelevant for the detection of ir. hormonal imbalances (hyperandrogenemia, igf1 deficiency, hypothyroidism, elevated estradiol) play a major role in obese women. postmenopausal women receiving hormone replacement therapy (hrt) develop hyperglycemia, as expected by age, but not ir. hrt improves is. discussion: ir is the common pathogenetic factor for risk components summarized as "metabolic syndrome": dyslipidemia, hypertension, hyperandrogenism and obesity. the vicious circle is intensified with the degree of ir. during the 15 to 20-year interval between the onset of ir and the manifestation of dm, significant micro-and macroangiopathies can develop. early detection of ir is therefore crucial for the prevention of dm and cvd. the costs involved for diagnosis are minimal (60-80 €) compared to the cost of treatment for dm and cvd. the effect of dietary feeding of "superjami" on the glucose metabolism and antioxidative status in mice under high fat diet conditions was investigated. the mice were randomly divided and given experimental diets for 8 weeks: normal control (nc group), high fat (hf group), and high fat supplemented with heukjinju (hf + hj group), suwon 425 (hf + sw group) and superjami (hf + sj group). at the end of the experimental period, the hf group exhibited markedly higher blood glucose level. however, diet supplementation of superjami was found to counteract the high fatinduced hyperglycemia and oxidative stress via regulation of antioxidant and hepatic glucose-regulating enzyme activities. these findings illustrate that superjami was similarly effective in improving the glucose metabolism and antioxidant defense system in high fat-fed mice and they may be beneficial as functional biomaterials in the development of therapeutic agents against high fat diet-induced hyperglycemia and oxidative stress. objective: to evaluate the effectiveness of two types of intervention: personal and group therapy in preventing or delaying diabetes. methods: two hundred and twenty-two pre diabetes adults were assigned to both interventions, which included physicians, dietitians, social workers and physical activity consultants, to modify patients' lifestyle and reduce weight. glucose, total cholesterol, ldl, hdl; tryglesirides and bmi were measured before (t1), immediately after (t2) and post 18 months (t3) of the intervention. mix linear; logistic regression and cox models were employed. results: mean age was 51.9 and 55.4 (p = 0.001) for the personal and group therapy, respectively. no significant differences in time trends for all the clinical measurements between the groups were observed. however, (i) reduction (à5.68 p < 0.0001) in glucose for the personal therapy group between t2 and t1; (ii) reduction in total cholesterol (à12.5 p = 0.001; à14.8 p < 0.0001) in personal and group therapy, respectively, between t1 and t3; (iii) significant hdl increase in both groups between t1, t2 and t3; and (iv) significant decline in ldl, triglycerides and bmi between t3 t2 and t1. 39.3% and 38.7% of the group and personal therapy, respectively (p = 0.933), developed type 2 diabetes during the study period. no differences to time to onset of the disease between the two groups. conclusion: for patients with pre diabetes, both types of intervention were effective in delaying and preventing the disease. group therapy is recommended since it requires fewer resources and can be implemented for the benefit larger population. methods: the present population-based case-control study was performed in shiraz, southern iran, over a 12-month period from december 2010 to 2011, on a randomly selected study population group consisting of inhabitants of the metropolis of shiraz in southern iran. all the patients underwent anthropometric and blood pressure measurements as well as thorough medical history and physical examinations. laboratory parameters including fasting blood glucose, lipid profiles, liver enzymes and ferritin, in addition to liver ultrasonography and cimt, were performed for all subjects. the cutoff value for the cimt was set at 0.8 mm and the measured values were correlated with other risk factors. we evaluated 290 patients with nafld and the same number of controls. subjects with nafld had a significantly higher prevalence of increased cimt (or: 1.66, p < 0.001). in patients with nafld the age of 50 years represented an appropriate cut-off value for predicting increased cimt. a systolic blood pressure (sbp) of 117 mmhg and a diastolic blood pressure (dbp) of 72 mmhg were shown to be appropriate cut-off values for predicting increased cimt. conclusion: cardiovascular risk factors such as increased intimamedia thickness (imt) occur more frequently among nafld patients when compared to healthy individuals. we recommend a careful evaluation of not only the liver, but also of the cardiovascular system in these patients, in order to prevent later morbidity related to atherosclerosis. mitochondrial capacity of oxidizing fatty acids and increased mitochondrial ros production. the peroxisomal β-oxidation, which starts the oxidation of the long-chain fatty acid and contributes with approximately 35% of all oxygen peroxide produced in the cell, was increased in ovx mice. these effects could lead to oxidative damage, a condition that was, in fact, evidenced by the reduced levels of reduced glutathione and elevated levels of tbars found in livers of ovx mice. the decreased mitochondrial capacity of oxidizing fatty acids could contribute, at least in part to development of hs in ovx mice. besides, the higher mitochondrial and peroxisomal ros generation resulted in oxidative damage in livers from ovx mice. background: elevations in high-sensitivity c-reactive protein (hs-crp) are associated with an increased risk of insulin resistance (ir). investigation of relationship between hs-crp, parameters of glucose metabolism and leptin in patients with t2dm and igt could be help to assess the role of hs-crp in development of ir. aim: of the study was investigation of relationship between hs-crp and hepatic glucose production (hgp), leptin and hba1c in women with t2dm and igt. materials and methods: forty-eight women with igm (31newly diagnosed t2d and 17 igt were observed). hs-crp and leptin were assessed in fasting states. intravenous glucose tolerance test was performed (0.75 г glucose on kg of body mass). mathematical analysis of results with definition of hgp (the h-index, mmol/l) was done with the special program (accessible in internet: www.diabet.ru/ivgtt). results: bmi was significantly correlated with hs-crp, r = 0.48, p < 0.001 and the level of hs-crp in women with bmi >30 kg/m 2 was almost four times greater compare to hs-crp in women with bmi >30 kg/m 2 [3.4 (2.5-4.5) and 11.3 (4.1-17.8) accordingly, (p < 0.05)]. between hs-crp and hgp and between hs-crp and leptin direct correlation (r = 0.40, p < 0.05) and (r = 0.45, p < 0.005, accordingly) was revealed. correlation between hs-crp and hba1c wasn't found. conclusions: association between hs-crp and leptin level, hs-crp and hgp could be demonstrate the role of hs-crp in development of insulin resistance. institute for sport and physical activity research (ispar), university of bedfordshire, bedford, uk objective: hypertriglyceridemic waist (hw) and waist-to-height ratio (whtr) are simple clinical tools that identify adults at risk of cardiometabolic disorders and cardiovascular disease. whether this applies in youth is under-researched and this study therefore investigated whether the hw phenotype and whtr are associated with cardiometabolic disorders in children and adolescents. methodology: this was a cross-sectional design study. anthropometry, biochemical variables, and cardiorespiratory fitness were assessed in 234 participants (122 girls) aged 10-19 years from bedfordshire, united kingdom. the hw phenotype was defined as a waist circumference ! 90th percentile for age and sex, and triglyceride concentrations ! 1.24 mmol/l, and a high whtr defined as >0.5. ancova and logistic regression were used in the analysis. results: in participants with the hw phenotype, clustered risk score was lower (p < 0.05), the odds of having high cardiorespiratory fitness (ml/kg/min) lower (0.045; 95% ci 0.01, 0.42), and the odds of having low hdl-cholesterol (4.41; 1.50, 12.91), impaired fasting glucose (3.37; 1.06, 10.72), and ! 1 (4.78; 1.32, 17.29) and ! 2 risk factors (7.16; 2.38, 21.54) higher than those without the phenotype. those with a high whtr had lower clustered risk (p < 0.05), higher odds of having low hdl-cholesterol (2.57; 1.11, 5.95), high diastolic blood pressure (3.21; 1.25, 8.25) , and ! 2 risk factors (5.57; 2.05, 15.17) than those with normal whtr. conclusion: the hw phenotype may be a better simple marker than whtr for identifying children and adolescents at risk for cardiometabolic disorders. treatment with ace inhibitors (acei) and at1 receptor blockers (arbs) has been shown to reduce the number of new-onset dm2, improve insulin sensitivity and reduce adipocyte size 1,2 . we investigated additional metabolic effect of renin-angiotensin system (ras) blockade with arb in comparison with acei. specifically we studied the effect of candesartan cilexetil therapy on glucose metabolism and parameters of subcutaneous adipose tissue (sat) in hypertensive subjects. antihypertensive treatment with acei was replaced by candesartan for 6 months in 18 subjects with essential hypertension. experimental procedures involved measurements of anthropometric data, blood pressure, oral glucose tolerance test, ras components and adipokines gene expression in sat obtained by biopsy. intersticial fluid from sat was collected by using microdialysis. six months after replacement acei by candesartan, the systolic blood pressure decreased by 6.95 ae 4.07 mmhg (p < 0.05), diastolic blood pressure decreased by 5.68 ae 2.35 mmhg (p < 0.05) and fasting plasma glucose decreased by 0.84 ae 0.06 mmol/l (p < 0.01). insulin sensitivity index (matsuda) tended to increase (p = 0.08). among the adipokine and ras genes studied in sat only pparc expression tended to increase (p = 0.052) after candesartan treatment. as expected, candesartan had blood pressure lowering effects comparable to those of acei. seeing that candesartan reduced fasting glycemia and strongly tended to increase pparc expression in sat, we speculate that arbs treatment might have additional positive effect on glucose metabolism compared to acei. the study is continuing and samples of serum and microdialysate are currently under analysis of ras peptide content. purpose: diastolic dysfunction in the metabolic syndrome/type 2 diabetes (d) is an epidemic without evidence-based treatment strategies. studies on dietary interventions are scarce. we tested the hypothesis, that a low-carbohydrate diet (lc) improves cardiac function in overweight-obese d more than the traditionally recommended low-fat diet (lf). methods: two groups of 16 d without cardiac disease (bmi 34 ae 6 kg/m 2 ) were studied in a parallel and partial cross-over design during a 3-week rehabilitation program with either lc or lf. the group on lf (carbohydrate 55%, fat 25%, protein 20%) had subsequent 2 weeks on lc (25%, 45%, 30%, respectively). cardiac function was assessed as myocardial velocity during systole and diastole (e′) and metabolic control before and 2 h after (pp) a standardized breakfast (400 kcal). both groups had supervised aerobic training 2 h a day. results: in the parallel groups, both diets induced similar and significant reductions of weight, hba1c and cholesterol. lc considerably improved insulin resistance, triglycerides, systolic and diastolic blood pressure and e′ (9.5 ae 1.0-10.4 ae 1.5 cm/s, p = 0.023), but lf did not whereas all these variables improved significantly after subsequent lc (e′ from 10.6 ae 1.5 to 11.5 ae 1.4 cm/s, p = 0.016). intact proinsulin was unchanged with lf but decreased with subsequent lc fasting and pp (p = 0.032 and 0.004). conclusions: these data indicate, that a lc but not lf nutrition modulates diastolic dysfunction in overweight diabetics, improves insulin resistance and may prevent or delay the onset of diabetic cardiomyopathy and the metabolic syndrome. methods: we studied 358 adolescents, males (n = 144) and females (n = 214), who were collected waist circumference, office bp, serum glucose and lipids. they underwent a 24-h abp monitoring (abpm) to record 24-hs, awake and sleep bp, and the bpv was calculated as the standard deviation bp. the ms was defined according to the national cholesterol education program, adult treatment panel iii modified for adolescents. statistical analysis: the variance ratio test (f-test) was applied to compare the bpv between adolescents with ms and those without ms. results: the ms prevalence was 11.5% (n = 41) in all, 22.9% (n = 33) in males and 3.7% (n = 8) in females (p < 0.0001). the abp values (systolic/diastolic) were the following: 116/56, 116/68 and 108/ 58 mmhg in adolescents with ms; and 105/78, 107/65 and 100/ 57 mmhg in adolescents without ms, for 24-hs, awake and sleep periods, respectively. the highest statistically significant values in systolic and diastolic abp during 24-hs and awake and systolic abp in sleep were showed by ms group. likewise, adolescents with ms showed significant higher 24-hs systolic bpv than those without ms (10.36 vs. 9.40 mmhg, p < 0.05). the ms has important effects on the abp values in adolescents. also, systolic bp variability during 24-hs is associated with ms. these findings suggest that ambulatory blood pressure monitoring is important in adolescents with ms to detect subjects in cardiovascular risk. montfort hospital research institute, university of ottawa, ottawa, on, canada organochlorine compounds (oc), are chemicals that were mostly used historically as pesticides, solvents, flame retardants, and other applications. oc have been recognized to be of environmental and potential toxicologic concern. due to their persistence and lipophilicity, these compounds will remain present in the environment for decades and accumulate in living organisms. the presence of oc is a major concern since they could act as endocrine disruptors and have recently been associated with the development of hepatic steatosis and lipotoxicity. the objective of this project is to define intra-hepatic molecular mechanisms implicated in the development of hepatic steatosis and lipotoxicity in oc exposed hepatocytes and rats. results: lipid quantification was assessed in human hepatocytes using steatosis colorimetric assay (cayman chemical company). lipid accumulation was higher in hepatocytes exposed to pcb126 [2.5 lmol/l] for 72 h compared to chloroquine (15%), a potential lipid droplet infiltration inducer, as well as our control group treated with dmso (38%). conclusion: understanding the emerging role of oc in the physiology of hepatic steatosis and lipotoxicity is of great importance as it is becoming clear that chronic lipotoxicity is strongly related to the suppression of insulin receptor signaling in the liver and activation of the apoptotic pathway. these results only represent the necessary first step on oc and their impact on the liver metabolic activity. experiments in oc-contaminated rats are presently undertaken, as well as protein quantification involved in intra-hepatic mechanisms in cells. nutrition, nutrition research institute, the university of north carolina at chapel hill, kannapolis, nc, usa insulin a precursor of t2dm, various cardiocerebrovascular disorders, non-alcoholic fatty liver, alzheimer's disease, and many other major health problems associated with excess calories. to study the mechanism of insulin resistance, we investigated the role of insulin and each category of macronutrients (glucose, fat, and amino acids) in the development of insulin resistance. our results show that glucose (hyperglycemia) does not cause insulin resistance in the absence of insulin in cultured cells or animals. dietary carbohydrate is not necessary for high fat diet (hfd) induction of insulin resistance but a small amount of it can promote insulin resistance in the maximal level in animals on hfd. our results also show that fat is necessary for the development of insulin resistance although fat can't induce insulin resistance in the absence of insulin. supplementation of amino acids such as leucine does not necessary cause insulin resistance in animals on hfd. finally, our results show that insulin can cause insulin resistance through (i) accumulation of long-chain acyl coas and cholesterol in mitochondria, (ii) inhibition of production of new mitochondria; and (iii) turnover of aged/damaged mitochondria. in summary, our results show that insulin and each category of macronutrients are necessary for the development of persistent insulin resistance. wellness institute, cleveland clinic, cleveland, oh, usa background: lifestyle factors are key in the development and progression of chronic disease. here we report outcomes for prediabetic participants in the lifestyle 180 â program, a 12-month intervention for patients with chronic diseases that integrates education in cooking, nutrition, exercise, and stress management. methods: pre-post changes in biometric, laboratory, and psychosocial variables were evaluated by paired student's t-test or wilcoxon test. results: of participants who met ada criteria for prediabetes (n = 270), most were female (68%) and obese (85%) and had hyperlipidemia (52%) or hypertension (54%). one fifth (21%) had depression. average age was 56 ae 10 years. at month 12, biometric, laboratory, and psychosocial data were available for 143 (53%), 172 (64%) and 108 (40%) participants, respectively. average bmi decreased by 7% (35.7 ae 6.4-33.2 ae 6.1) and there were decreases in waist circumference, blood pressure, resting heart rate, fasting glucose, and hba1c (p < 0.001 for all). insulin decreased from 17.5 ae 10.8-12.1 ae 7.6 μu/ml (à31.2%; p < 0.001). more diabetic medications were stopped or reduced in dose than were started or increased in dose (ratio 4.6:1). average depression (ces-d10) and perceived stress (pss-4) scores decreased by 32% and 25% respectively (p < 0.001 for both). physical, mental, and perceived health (measured by veterans rand 12 item health survey) improved from 16% to 63% (p < 0.001). conclusion: participation in a comprehensive lifestyle modification program of adults with prediabetes results in significant and clinically meaningful improvements in biometric, laboratory, and psychosocial outcomes. diabetes is frequently associated with both extracellular and intracellular magnesium (mg) depletion. epidemiologic studies found high prevalence of hypomagnesaemia in subjects with type 2 diabetes, especially with poorly controlled glycemic control. erythrocyte and serum magnesium levels were measured in 35 healthy control subjects, 35 non-diabetic normal glucose tolerant offspring of type 2 diabetic subjects and 35 non-diabetic impaired glucose tolerant offspring of type 2 diabetic subjects in addition to routine laboratory investigations, hba1c, lipid profile, fasting insulin by elisa and homa-ir. our study showed that fasting, 2 h postprandial plasma glucose, serum insulin level, hba1c, homa-ir index, serum ldl-c, serum triglycerides, serum cholesterol and serum malondialdehyde levels were higher and serum hdl-c and both serum and erythrocyte magnesium levels were lower in impaired glucose tolerant offspring of type 2 diabetes as compared to each of normal glucose tolerant group and control group, while there was no significant difference in those parameters between normal glucose tolerant and control groups. we can conclude that the magnesium depletion and the increase in malondialdehyde level in non diabetic impaired glucose tolerant offspring of type 2 dm were associated with increasing severity of insulin resistance and dyslipidemia and, this may increase the risk of development of type 2 diabetes complications in there offspring. possily use of mg suplemenaion in those subject may delay and prevent the development of type 2 diabetes. obstetrics and gynaecologist, catholic university of sacred heart, rome, italy background and aims: gdm, overweight and gestational weight gain (gwg) are important determinants for an adverse pregnancy outcome and particularly for having large for gestational age (lga) newborn. the aim of this study was to evaluate the pregnancy outcome among women affected by a gdm, belonging four different classes of pre-pregnant bmi and to investigate the specific role of the anthropometric parameters and therapeutic regimen on the foetal growth. material and methods: this was a prospective study including 183 pregnancies affected by gdm. only women with a mean pre-prandial glucose level <92 mg/dl and 2 h post-prandial values <120 mg/dl were enrolled. we considered other variables such as gwg, excessive gwg (egwg), therapeutic regimen used and maternal age to investigate their role in foetal growth. results: at the linear univariate analysis, a dependence of neonatal birth weight from pre-pregnant bmi class, egwg, gwg and gestational week at delivery was found (p < 0.001). the dependence from the pre-pregnant bmi (p = 0.001) and the egwg (p = 0.002) was confirmed even when the percentile was considered. at the multivariate analysis the birth percentile resulted associated both with the prepregnant bmi (p < 0.001) and the gwg (p = 0.003). conclusions: pre-gestational bmi, gwg and egwg are important parameters involved in fetal growth. their control during pregnancy could be an important way to improve pregnancy outcome even in women with gdm. obstetrics and gynaecologist, 2 internal medicine, catholic university of sacred heart, rome, italy background and aim: continuous subcutaneous insulin infusion (csii) may be an alternative treatment to multiple daily injections (mdi) in pregnant subjects with type 1 diabetes(¹). the aim of this study was to compare metabolic control and obstetric outcome of these pregnancies treated with csii vs. pregnancies treated with different type of insulin analogs administrated by mdi. material and methods: we studied 172 pregnancies in 150 women, three twin pregnancies, affected by type 1 diabetes. thirty-four pregnancies treated with csii and 138 treated with mdi treatment were evaluated. in the group of women treated with mdi 78 women used human analogs, 32 used insulin aspart and 28 used insulin lispro. metabolic control and obstetric outcome were compared between the groups. statistically significant differences were found in metabolic control in term of hba1c mean level for each trimester of pregnancy. conclusion: no clear advantage of csii vs. mdi treatment is shown in our study for pregnancies complicated by type 1 diabetes in term of metabolic control or obstetric outcome(²). however, it might be that some selected patients with unstable metabolic control or high hba1c levels, who become pregnant with a better metabolic profile after csii treatment, may have a better pregnancy outcome. poblations and methods: a cross-sectional study was performed on 424 children and adolescents (244 female and 180 male) between 6 and 18 years old, without cardiovascular, metabolics, oncology and immunology diseases. were excluded all subjects who took drugs with effects on glucose, insulin, lipids or weight. each subject was determined: weight, height, bmi, neck, waist, hip circumferences, adiposity, blood pressure and glucose, insulin, cholesterol, tryglicerides, c-ldl, c-hdl. was performed oral glucose tolerance test to glucose and insulin postprandial. was estimated insulin secretion and insulin sensitivity. conclusi on: neck circumference is a potential marker for to identify children and adolescents with metabolic and cardiovascular risk factors. aim: to assess the efficacy of a lifestyle modification in preventing diabetes mellitus type 2 (dm 2) among subjects with risk factors of dm 2 including impaired glucose tolerance and impaired fasting glucose (igt/ifg), obese adults and first-degree relatives of patients with diabetes. materials and methods: the study included 100 patients (32 m, 68 f) 25 -65 years old at risk factors of dm 2. all patients received recommendation on a balanced diet and physical activity. we measured fasting plasma glucose (fpg), 2-h plasma glucose concentrations. fasting serum insulin (fi) levels were detected by sensitive elisa. index homa-r were used as the index of insulin resistance. results: in 48 weeks our study 56 patients carried out this recommendations (research group) and 44 patients did not it (control group). patients of the research group demonstrated mean reduction of weight (à5.3 kg), bmi (à2.6 kg/m 2 ) and whr (à0.2) (p < 0.01) and persons of the control group had significant increase these parameters (p < 0.05). among subjects with igt, glucose levels normalized in 56% of patients from the research group and 4.5% in control group (p < 0.001). fi and homa-r in research group decreased from 11.9 ae 4.3 to 9.6 ae 4.5 lu/ml and from 3.6 ae 1.1 to 2.7 ae 1.4 accordingly (p < 0.05). in control group the specified parameters had increased significantly (p < 0.01). the risk reduction of development dm 2 among patients of the research group was 53.7% comparer the control group. conclusion: thereby, lifestyle modifications are effective in preventing dm 2 development in individuals at risk living in belarus. objectives: it is suggested that the kind and frequency of snacks as well as dairy consumption can influence body weight and composition. as the number of students are increasing rapidly in iran and regarding their unhealthy food habits, we decided to carry out this study. methods: one hundred and twelve female students who were selected from university dormitories inhabitants randomly, participated in this cross-sectional study. weight, height and waist circumference (wc) of the participants were measured. demographic and food frequency questionnaires were filled face to face. data were analyzed using spss # 16. the results showed that 14.6% of participants were under weight, 75% normal and 10.4% overweight. besides, 94.80% and 5.2% had normal and above normal wc respectively. sixty-one percent of female students had just one snack. fruits (63%) and confectionaries (61%) were the most popular snacks. mean ae sd of the dairy consumption was 1.78 ae 1.20 serving per day. there were no correlations between bmi and wc and snacking pattern or dairy intake. conclusion: it is assumed that as most of our subjects had normal weight and wc, we did not find any correlation between bmi and wc and snacking pattern or dairy intake. conducting similar studies, using both overweight and normal weight subjects is suggested. obstetrics and gynaecologist, catholic university of sacred heart, rome, italy background and aims: the management of pregnancies complicated by t1dm has changed with the introduction of new short acting insulin analogs. the aim of this study was to have a comparison of obstetric outcome among the different insulin regimens. methods: one hundred and sixty singleton pregnancies were assessed. seventy-four women were treated with human analogs, 46 with insulin aspart and 40 with insulin lispro. results: no significative differences were found in term of incidence of pre-eclampsia, iugr, large for gestational age newborn, cesarian sections, preterm delivery, apgar score <7, mean week at delivery, mean birth percentile. a significative difference was found in term of incidence of newborn infants with a birth weight ! 4000 g (23.33% in the human analogs group, 2.22% in the aspart group t and 29.03% in the lispro group; p = 0.14); a trend (p = 0.06) was observed comparing the mean birth weight among the groups (3277.30 ae 689.77 g in human analogs group, 3000.00 ae 685.20 g in aspart group and 3313.59 ae 720.07 g in lispro group). no significative differences were found in term of hba1c mean levels for each trimester of pregnancy. conclusions: the use of insulin aspart is correlate with a lower incidence of fetal macrosomia and with a lower mean birth weight. in vitro observations highlighted that lispro and aspart have a similar affinity for insulin receptor; regarding the affinity for igf-1 receptor lispro is 1.5 fold more potent than human insulin in binding igf-1 receptor. further investigations are necessary to explain the correlation between insulin affinity for igf-1 receptor and fetal growth. methods: this study was performed on 180 women diagnosed with gdm (gestational dm) 3-4 months after delivery. the jewish and bedouin women were divided into, intervention and control groups. the intervention group was instructed in healthy lifestyle habits every several months by a dietician and sports instructor. the control group was informed of the potential risks for diabetes following gdm and advised to improve lifestyle. all women had fasting insulin, glucose and lipid level tests 3-4 months post partum. height, weight, bmi, blood pressure, waist circumference were measured. food frequency and exercise questionnaires were filled. the same tests were repeated at 1 and 2 years follow up. results: the prevalence of gdm was 5.7% in jewish and 3.5% in bedouin women. lack of pregnancy care was greater among the bedouin. all the metabolic tests post gdm period improved in the intervention group in both ethnicities compared to control. there was a decrease in insulin, glucose, ldl, triglycerides and homa-ir (homeostasis model assessment) values and an increase in hdl. a significant decrease in carbohydrate consumption, calorie consumption, fat consumption and increase in protein consumption was observed and an increase in physical activity. the intervention program significantly improved the metabolic indices measured in both jewish and bedouin populations. the results underscore the need to provide lifestyle optimization guidance to women with gdm in order to reduce the risk of type 2 diabetes. introduction: fatty liver is strongly associated with type 2 diabetes. however, it is unclear whether hepatic triglyceride accumulation causes diabetes, or merely is a consequence of excess adipose tissue, that is related to insulin resistance. we aimed to investigate the association between hepatic triglyceride content (htgc) and insulin resistance, and whether this association could be explained by measures of adiposity. methods: in this cross-sectional analysis of the netherlands epidemiology of obesity (neo) study, fasting glucose and insulin concentrations were measured. abdominal fat depots were measured using mri, htgc using mrs. we performed linear regression analysis of htgc with the updated homeostasis model assessment (homa2-ir), adjusting for age, sex, ethnicity, education, smoking, alcohol consumption, visceral adipose tissue (vat) and total body fat (tbf). results: after exclusion of participants with missing data (n = 65) or known diabetes (n = 69), alchohol consumption >4 glasses/day (n = 72), 1160 participants were included with a median (iqr) age of 56 (50.61) years, mean fasting glucose: 5.6 ae 0.8 mmol/l, 52% men. per sd htgc (9.6%), the homa2-ir increased with 37% (95% ci: 22%, 54%), this attenuated to 23% (95% ci: 11%, 36%) after adjustment for vat and to 21% (95% ci: 10%, 33%) after additional adjustment for tbf. per sd vat (0.06 dm 3 ) the homa2-ir increased with 55% (95% ci: 47%, 63%), this attenuated to 43% (95% ci: 33%, 53%) after adjustment for htgc and to 27% (95% ci: 18%, 36%) after additional adjustment for tbf. the association between hepatic fat and insulin resistance was for a large part explained by vat. vat may be most important in the etiology of insulin resistance. the aim was to carry out complex estimation of mutual relations between the proinflammatory condition and basic components of the metabolic syndrome (ms) in patients with ischemic heart disease hospitalized for coronary artery bypass grafting. forty-three patients were examined by standard methods and were divided into three groups depending on presence of separate ms components: 1st group with abdominal obesity (ao), arterial hypertension (ah), type ii diabetes, dyslipidemia; 2nd group with ao, ah, hyperglycemia; 3rd group with ao, ah, dyslipidemia. indicators of chronic subclinical inflammation have been revealed before operation in all groups of patients. the strongest ones have appeared in the 2nd group of patients. hyperuricemy was more frequently registered in patients of the 3rd group (18.8%). the correlation analysis revealed authentic interrelations of uric acid level with lipid profile and inflammation markers. a strong inverse relation with triglycerides, a strong direct relation with low density lipoprotein cholesterol and inverse one with total amount of leukocytes were revealed in the 1st group. the strong direct relations with segmented neutrophils and the inverse ones with lymphocytes were registered in the 2nd and 3rd groups. the average direct correlation with crp was revealed in the 3rd group. activation of inflammatory process was noted in patients of all groups after operation, and it was already of acute nature in the 3rd group that allows attributing it to a risk group. the study of expressiveness of inflammatory reaction depending on presence of the ms components allows revealing risk groups among patients. introduction: the soft drink intake has increased worldwide and its high consumption is associated with the development of metabolic syndrome. objective: to estimate the association between the consumption of sugar-sweetened beverages (ssb) and the burden metabolic syndrome diseases (bmsd). a cross-sectional telephone survey was conducted with 3419 adults (46.03% men and 53.97% women, mean age 41.7 years) in the urban area of belo horizonte, brazil. data from the telephone-base brazilian surveillance system for chronic diseases was used (vigitel -2008/2010). burden metabolic syndrome diseases were defined as the self-reported of at least two of the following factors: diabetes, dyslipidemia, hypertension and obesity. the intake of ! 5 days/week soft drinks and artificial juices was assessed. sociodemographic, selfreported health status and lifestyle habits were also used. odds ratios (or) and 95% confidence intervals were estimated by multivariate logistic regression. results: in this sample, 24.07% of participants consumed ! 5 days/ week sugar-sweetened beverages (ssb). the prevalence of bmsd in the sample was 15.61%. the prevalence of bmsd in people who consumed ssb ( ! 5 days/week) was lower than people didn't consume the beverages (or: 0.53; 95% ci: 0.39-0.71) five or more days per week. the final model was also adjusted for physical activity, education, marital status, poor self-reported health and poor consumption of fruits and vegetables. obesity is an heterogeneous condition due to fat distribution and storage. the aim of our study was to compare the predicting role of homa index and of bmi on early vascular impairment in 65 morbidly obese subjects (bmi 44.6 ae 7 kg/m 2 ) before and after sleeve gastrectomy (sg). glycemia, insulinemia, lipids, flow mediated dilatation (fmd), carotid intima media thickness (imt) and visceral fat area (vfa) by ultrasound were performed in all subjects. patients were divided on the basis of homa-ir median values: ! 3.5 group 1, <3.5 group 2. group 1 had significantly higher values of bmi (p < 0.05), waist circumference (p < 0.0001), vfa (p < 0.0001), triglycerides (p < 0.005), glycemia (p < 0.0001), and lower hdl-c (p < 0.005) than group 2; fmd was significantly lower (p < 0.05) and imt significantly higher (p < 0.05) than in the group 2. the same population divided on bmi median values did not show any difference in lipids, imt and fmd. at stepwise regression analysis vfa was the independent predictor of reduced fmd (β à0.541, p 0.002). homa-ir (beta 0.399 p < 0.001) was the independent predictor of imt (beta 0.413, p = 0.001). in 30 patients, re-evaluated 9 months after sg, bmi, vfa and homa were significantly reduced (p < 0.0001); fmd significantly increased only in the subgroup with homa-ir pre intervention ! 3.5 (p 0.003). bmi, index of overall adiposity, seems less useful in the prediction of early atherosclerosis in morbidly obese; homa-ir, strictly related to visceral fat, is expression of metabolic impairment thus able to predict early vascular damaging. methods: swiss albino mice were given a high fat diet containing lard (h) (23.9% wt/wt), supplemented with as or f and in combination alongwith h (f + as + h) for 8 weeks. the control mice (c) were fed with normal diet. after the treatment period the physical, physiological and cellular parameters were evaluated by body weight, liver weight, biochemical estimation, western blot, pcr or ihc. results: the h mice exhibited increased body and liver weight; treatment with f or as and (f + as + h) in the diet significantly counteracted the hfd induced body and liver weight gain, hyperlipidemia; hepatic lipid profile; level of ros; hyperglycemia; hyperinsulinemia; tnf-a, il-6 level; nuclear translocation of nf-jb; lipid peroxidation. activities of antioxidant enzymes (sod, cat, gsh, frap) were up regulated significantly in f or as and (f + as + h) mice. therefore simultaneous treatment with as or f and their combination protected against hfd induced weight gain and oxidative stress. conclusion: this novel approach of combinatorial preventive medicine is validated not only with the parameters of metabolic syndromes yet it is evidenced with oxidative stress and crucial molecular targets. this study illustrates for the first time that as and f has relatively similar hypolipidemic, antioxidative, anti inflammatory actions and the as + f combination along with hfd has shown prominent preventive effects as compared to other treated groups. s. sivapraksh 1 , i. shabir 1 , n. gupta 1 , j. john 1 , a. ammini 2 1 aiims, 2 department of endocrinology, aiims, new delhi, india background: insulin resistance/hyperinsulinemia is associated with a variety of reproductive endocrine dysfunction in girls. however, there is a scarcity data regarding hypothalamic pituitary testicular axis functioning among boys with hyper insulinemia. aim: aim of this study was to assess effect of hyper insulinemia on pubertal development in boys. subjects and methods: children of subjects with type 2 diabetes mellitus (t2dm) were invited to participate in this study. children and adolescent with any chronic medical condition were excluded. detailed medical history, physical examination including anthropometry, haemogram, liver, renal functions, oral glucose tolerance test (ogtt), insulin, lh, fsh, prolactin and testosterone were done for all subjects. results: seventy-four boys, 6-18 years of age participated in this study. results are given in table 1 . thirty boys (all except 1 were overweight) had gynaecomastia. objectives: to find out the prevalence and factors associated with dyslipidemia among adults aged 18 years and above in a resettlement colony located in central delhi. a cross sectional study, that included a random sample of 200 adults, was designed. a study tool based on the world health organisation (who) stepwise approach to surveillance of noncommunicable diseases and their risk factors (steps) questionnaire was used. fasting venous blood sample was collected. criteria based on the third report of the national cholesterol education program expert panel on detection, evaluation and treatment of high blood cholesterol in adults (ncep atp iii), were used to define the cut offs for dyslipidemia. data was analysed using the statistical package for social sciences (spss) version 17. results: out of a total of 200 study subjects, 34% had raised cholesterol levels ( ! 200 mg%). thirty-eight percent had raised low density lipoprotein (ldl) levels ( ! 130 mg%), 40% had raised triglyceride levels (triglycerides ! 150 mg%) and 39% had low high density lipoprotein (hdl) levels (<40 mg%). age, hypertension, alcohol consumption and abdominal obesity were found to be associated with increased odds of dyslipidaemia, using the logistic regression model. aim: in the present study we explored whether oral consumption of the probiotic lactobacillus casei shirota bacteria in form of the yakult probiotic drink has any effects on energy metabolism in patients with impaired metabolic functions. a cross-over-study with daily intake of one portion yakult light during 12 weeks was carried out in 66 patients with impaired glucose regulation or metabolic syndrome. fasting blood samples were collected, anthropometric parameters were measured and a frequently sampled oral glucose tolerance test was performed at 3 time points (week 0, 12 and 24). effects on the human gut bacterial communities were studied via t-rflp fingerprinting of 16s rrna genes amplified from microbial dna preparations from fecal samples. our results reveal a significant increase of total cholesterol (mean increase: 7.9 mg/dl; p = 0.044), triglycerides (mean increase: 19.9 mg/dl; p = 0.004) and a significant decrease of hdl-cholesterol (mean decrease: 2.0 mg/dl; p = 0.030) after 12 weeks of daily intake of yakult. no significant influence of yakult light consumption on indices of insulin sensitivity, hba1c and ldl-cholesterol could be demonstrated. a small decrease of fasting glucose levels after yakult consumption was revealed (mean decrease: 2.9 mg/dl; p = 0.005). no consistent changes in the community profiles of the gut microbiota were observed following 12 weeks of yakult light consumption. in mexico, breakfast is one of the most important meals during the day, but changes in food consumption habits, also known as nutritional transition have been associated with a poor food quality intake. scholars, during the recess use to buy some food in internal shops of schools and this food are not generally the healthiest. the aim of this study was to evaluate the quality of the food consumed during the recces by scholars in tepic nayarit, mexico. scholars (563, about 9-12 years) from 10 different public schools were evaluated by means of food register questionnaires during 2 days, there has determined the ingestion of nutrients and nourishment healthy index (ias) using the nutrition program dial. anthropometric data were measured for weight and height, and body mass index (bmi) was calculated. the bmi showed that 7.6% of children have low weight for height, 44.6% have normal weight and 48.7% overweight/obesity. twenty percent of scholars declared that they did not take breakfast until the hour of recess. energy intake during recess was on average 25% of the daily requirement. the foods most frequently consumed were sweetened beverages (400 ml) containing 44 g of simple sugars, fried-wheat snacks (68 g/24 g fat), candies (19 g/simple sugars) and some fruit such as mango, apple or watermelon (100 g/portion) with chilli. these combinations of foods are risk factors to the development of diabetes in scholars were the prevalence of overweight/obesity is in high levels. introduction: incidence of type 1, 2 and gestational diabetes mellitus (gdm) are increasing worldwide. given that women with previous gdm have a higher risk of diabetes development later in life compared to women with a physiological pregnancy, the aims of our study were (i) to ascertain a frequency of early postpartum conversion of gdm into permanent diabetes or persistent impaired glucose tolerance and (ii) to find an eventual significant predictive factors from those routinely measured during the gdm follow up. methods: we carried out a retrospective epidemiological analysis of anamnestic, anthropometric, biochemical and clinical data of female patients (n = 1090) from faculty hospital brno with gdm diagnosis during the 2005-2011 period that underwent repeated ogtt up to 1 year after the delivery. results: any degree of impairment of glucose tolerance postpartum was detected in 11.7% subjects, of those 4.1% had manifest dm (2.8% t2dm and 1.3% t1dm). glycaemia in all three time-points of baseline ogtt, area under the curve (auc ogtt ) and baseline hba1c were significantly associated (p < 0.05, mann-whitney) with the postpartum disorder. using regression analysis predictive risk model was developed using these baseline parameters. conclusions: parameters of glucose metabolism measured during 24-28th week of pregnancy fulfilling criteria of gdm diagnosis exhibited highly statistically significant differences between women with and without persistent postpartum glucose metabolism abnormality and conferred significant predictive potential. considering generally lowcompliance of gdm women any more specific assessment of future risk stratifying gdm population could enable more effective screening of postpartum glucose metabolism disorder. institute of molecular biomedicine, comenius university medical faculty, 2 department of health, bratislava self-governing region, 3 regional public health office, bratislava, slovak republic background and aims: in the adults, microalbuminuria is considered as biomarker of present/future cardiovascular and/or renal disease. the roots of these diseases extend back into childhood/adolescence. data on prevalence of microalbuminuria, renal excretion of albumin (albumin/creatinine ratio, acr) and its relationship to obesity and blood pressure in apparently healthy adolescents are scares. methods and results: fourteen to 21-years-old apparently healthy students of secondary schools in bratislava district were enrolled (n = 2139, boys: 53%). acr was determined in a spot urine sample. in underaged subjects overweight/obesity was classified according to age-and sex-based slovak population tables from 1991. blood pressure values were recorded. acr (iqr: 0.29-0.67 mg albumin/ mmol creatinine vs. 0.25-0.51 mg albumin/mmol creatinine, p < 0.001), and the prevalence of microalbuminuria (3.6% vs. 1.2%, chi-square: p = 0.024) were higher in girls than in boys. prevalence of overweight (15.3% vs. 10.4%) and obesity (15.6% vs. 8.7%), as well as blood pressure values in pre-hypertensive (51.6% vs. 13.4%) and hypertensive (10.6% vs. 0.9%) ranges were higher in boys if compared with girls. in underweight subjects, particularly boys, acr was significantly higher if compared with the overweight/obese subjects. acr correlated inversely with the markers of peripheral and central obesity. conclusions: our data suggest the need of specific interpretation of data on acr in the adolescents, and the need of further analysis of this (in the adults risk) marker in population of adolescents with regard to other important determinants of acr, such as insulin sensitivity and other metabolic syndrome risk factors. inclusions criteria were bmi between 35 and 45 kg/m 2 , morbidities as diabetes type 2 more than 10 years in oral treatment and without control, hypertension in oral treatment too, moderate or severe liver steatoses diseases and lipid diseases. results: all patients in a both of groups (laparoscopic and robot approach) had a effective loss of weight, with mean about 25-48 kg (25-55%) after 1 year period. one case of robot approach presented a staple line bleeding at first postoperative day, without transfusion. morbidity rate after rasg was 9%, but no gastrointestinal leaks occurs. conclusions: rasg can be a safely and feasible tool to the surgical treatment of obese patients and co-morbidities, with good results and satisfactory outcomes. results: patients were nine males and six females with mean age of 50 years (range 32-58). seven patients were under insulin treatment and 8 with oral medications only. glycemia at 6 months post-surgery (132.0 ae 40.8 mg/dl; n = 8) was significantly reduced (p = 0.012) compared to time 0 (188.2 ae 68.3 mg/dl). the same occurred with hba1c at 6 months (6.2 ae 0.6%; n = 8) compared to time 0 (8.8 ae 1.8%) (p = 0.006). bmi was also reduced at 1 month (26.3 ae 2.0; n = 12) compared to time 0 (28.5 ae 1.4) and 6 months (25.1 ae 2.8; n = 10) compared to time 0 (28.6 ae 1.7) (p = 0.001). only one patient remained under insulin after surgery. the procedure was considered safe and significantly improved metabolic control of non-obese t2dm patients, although moderate weight loss was also observed. recent studies have shown that the practice of a proper diet provides health benefits. also, it has been observed that the body weight gain is associated with consumption of unbalanced diets. the objective of this study was to evaluate the quality of the diet in adult residents of tehcnological institute of tepic and establish differences for normal weight (nw) and overweight/obesity (ow/ob). forty two adults >18 years were evaluated, they were taken anthropometric data for weight, height and body mass index (bmi, kg/m 2 ) was calculated. for the diagnosis of nw and ow/ob, were taken established by world health organization (who). it was used a 3-day dietary record, for diet analysis was used dial software, which contains tables of food composition mexicans. dietary data were adjusted for the degree of discrepancy between energetic intake and energy expenditure obtained estimate. for statistical analysis was used the program rsigma babel and statistical differences were established <0.05. the caloric profile was unbalanced in 85% of the study population: a high consumption of fat in detriment of carbohydrates. the difference between groups were not significant (p > 0.05) in the caloric profile, but nor in lipidic profile. the analysis of the discrepancy intake/energy expenditure describes a probable undervaluing of the diet in both groups, but higher in adults with ow/ob compared to nw ( patients with long standing diabetes had a higher leptin, hsp70, hba1c and triglyceride than controls. serum leptin levels were significantly lower in patients with newly diagnosed diabetes. women with type 2 diabetes had a higher leptin levels compared to men, both before and after treatment. we showed a positive correlation between leptin-hsp70 in women with type 2 diabetes. the correlation was highest in women with newly diagnosed diabetes (r = 0.59) and was attenuated in women who were on treatment (r = 0.3). the significance of this correlation was only observed in women with type 2 diabetes. there was no correlation between leptin and hsp70 in men. the positive correlation between leptin and hsp is observed in chronic inflammation such as type 2 diabetes. it could be hypothesized that the observed correlation between serum hsp70 and leptin imply a higher state of chronic inflammation. materials and methods: carotid atherosclerosis (measurement of intiamedia thickness -imt) and the presence of steatosis were assesed using ultrasonography in 254 subjects (40.16% men and 59.84% women, mean age 54.75 ae 16.25 years), selected from a rural population (were excluded those with known liver disease and those with alcohol consumption >20 g/day). results: 89.19% of subjects had different degrees of steatosis: 28.38% mild, 28.83% moderate, 31.98% severe. the metabolic syndrome and all its individual traits, were significantly more frequent in nafld patients, especially in those with severe steatosis (p < 0.001). the mean value for imt was 0.8545 ae 0.10492 mm, increasing in parallel with the severity of steatosis. also, subjects with ms had significantly higher imt values than those without ms (p < 0.005). in the group without ms, imt mean was significantly higher in patients with steatosis than in those without steatosis (p = 0.032), and in patients with moderate/severe steatosis than in those with mild steatosis (p = 0.022). by multiple regression analysis, larger waist circumferince (r = 0.423), increasing alcohol consumption (r = 0.219) and the presence of steatosis (r = 0.213), were significant predicting factors for increased imt. conclusions: these data confirm the hypothesis that the presence of steatosis, independently of the ms, is associated with a significant risk for development of atherosclerosis and its detection should be an alert for the existence of an increased cardiovascular risk. the prevalence of the metabolic syndrome (ms), a cluster of central obesity, hyper/dyslipidemia, hyperglycemia, and hypertension is constantly increasing worldwide. although, the exact mechanisms underlying the development of the ms are not completely understood, modern lifestyle of physical inactivity and unhealthy nutrition, obesity, and their interaction with genetic factors are considered largely responsible. this study was carried out to identify the association between physical inactivity and burden metabolic syndrome diseases (bmsd) in an urban brazilian population. a cross-sectional telephone survey was conducted with 3419 adults (46.0% men and 54.0% women, mean age 41.7 years) in the urban area of belo horizonte, brazil. data from the telephone-base brazilian surveillance system for chronic diseases (vigitel -2008/ 2010) was used. burden metabolic syndrome diseases (bmsd) was defined as the self-reported of at least two of the following factors: diabetes, dyslipidemia, hypertension and obesity. physical activity (pa) indicators were evaluated in free time (leisure), work, home and transportation domains. sociodemographic, health status and lifestyle habits were also used. odds ratios (or) and 95% confidence intervals were estimated by multivariate logistic regression. in this sample, 13.5% of participants were physically inactive in all domains and the prevalence of bmsd in the sample was 15.6%. physical inactivity was independently associated with bmsd (or = 0.02; 95%ci: 1.03-1.80), adjusted to age, schooling, poor selfreported health and gender. in this sample, bmsd is a significant public health problem. the evidence from this study shows that physical inactivity was independently associated with proxy of metabolic syndrome. aim: to assess the prevalence of insulin resistance (ir) and glucose homeostasis alterations (gha) in overweight and obese children and the risk factors for ir and gha. method: we collected data from 145 subjects: 66 girls and 79 boys aged 2.8-17.5 years (median: 9.3). gha was measured by fasting glucose. glucose ! 90 mg/dl was treated as high normal fasting plasma glucose (hnfpg). ir was estimated by homeostasis model assessment (homa-ir), quantitative insulin sensitivity check index (quicki) and fasting glucose/insulin ratio (fgir). we adopted the following cut-off points for diagnosing ir: homa-ir ! 2.5, quicki 0.34 and fgir 7.0. results: table 1 . fasting glucose, insulin and ir-indexes. in obese children we observed a significant (p < 0.05) positive correlation between homa-ir and age (r = 0.46), bmi (r = 0.49), fasting insulin (r = 0.99), triglycerides (r = 0.29), crp (r = 0.2) and a negative correlation with birth weight (r = à0.24) and hdl the results provide evidence for an association between pd and sm in the adolescent population, which would mean that depression may influence ms in this group that is more vulnerable to pd due to hard changes typical of their life stage. the aim of this study was to evaluate the electrical activity of the pelvic floor muscles (pfm) in women with polycystic ovary syndrome (pcos) diagnostic and insulin resistance (ir). through a crosssectional study, it was recruited 42 women with pcos. the total pcos patients was divided into two groups according to the presence of ri (group a) and without ir (group b). the diagnosis of ri was made through fasting insulin. the muscle tone (mt) and maximal voluntary contraction (mvc) was evaluated by electrical activity, that it was measured by surface electromyography. among the pcos patents, 18.4% had insulin resistence. there was significant difference between the tone (p = 0.01) and cvm (p = 0.005) between groups a and b. the correlation test showed a strong negative correlation between muscle tone and fasting insulin levels in group b (r = à0.8, p = 0.01). considering the results, it seems that the presence of insulin resistance may adversely affect the electrical capacity of the pelvic floor muscles. central hospital of the army, 2 cpmc, algiers, algeria the polycystic ovary syndrom is a disorder affecting approximatively 5-10% of reproductive age women. hyperinsulinemia and insulinore´sistance are common features of a larger number of patients affected by pcos. objective: the objective of this study was to characterized the prevalence of the insulinore´sistance in a cohort of algerian women with pcos. methods: a total of 181 patients with evidence of pcos defined by the rotterdam consensus were recruted for a prospective study. all women had a standard oral glucose tolerance test with the mesure of glycemia, insulinemia and shbg. the prevalence of insulinore´sistance grows significantly with bmi (p = 0.0002) but didn't change with age (p = 0.25). the non hyperandrogenic phenotype is less insulinoresistant (33.3% vs. 68.7%). conclusion: in our population of pcos near half of them are insulinresistant and need to be treated in order to avoid metabolic features. the aim of this study was to analyze the parameters of glycemic index (ig) in foods intended for feeding patients with impaired glucose metabolism. for this purpose, a new design selected food products, characterized by a higher nutritional value, that pass attempts technological and toxicological studies and have been classified as human food. the food consisted of inulin, buckwheat, pumpkin seeds, buckwheat hulls, the addition of mulberries, kale and beans. the products were characterized by nutritional value and composition. designed food products were the nature of small snacks, designed to supplement the basic diet and enriching it with ingredients desirable from the standpoint of prevention of civilization diseases. characterized by elevated natural origin and health-related properties. the study took part of 20 healthy, non smokers volunteers between 20 and 30 years of age, with proper blood biochemical parameters. the results allow to characterize these foods as relative low glycemic index and possible to use to enrich the diet in the components with pro-health effect. for all the products the dose and single-serving size consumption was described and proposed. the possible potential changes in glucose levels after consumption were characterized. the results are the test pilot study of the project aimed to the creation of the increased food with bioactive healthy properties and are the result of the first stage of the researchclinically-nutritional studies of those products. financial supported by the ue project nr po ig 01.01.02.00-061/09. one of the fastest growing trend in food production is designing of health promoting foods, including so-called bioactive food. only food products enriched with bioactive components of natural origin might be perceived as bioactive. it is highly possible that well established technological process of bioactive foods production leads to obtain products useful in prevention and non-pharmacological treatment of metabolic diseases. the project aims to designing these kind of prohealth food, which would simultaneously be organoleptically attractive to the consumer and be an equivalent of conventional, habitually eaten products. the important aspect of bioactive foods is their antioxidant potential, which positive impact on various diseases (obesity, diabetes mellitus and hypertension) is already well known from prior literature. in the studies the antioxidant capacity of the selected bioactive foods designed in the project was assessed. the method used for assessment of antioxidant potential was orac, as the most widely applied and the most reliable one. the analysis included products from three following groups of enriched foods: fruit and vegetable juices, bakery products and confectionery, and meat products, as well as the corresponding placebo products. most of the products enriched in bioactive components was characterized by significantly higher orac values in comparison to placebo correspondents. the results support the correctness of applied production technology and proper selection of bioactive components. based on the antioxidant potential of new products, one can decided which of them should be use for further controlled clinical trial. background: obesity in adolescent tends to persist into adulthood which associated to metabolic disease thereby increasing mortality and morbidity. objective: to investigate the association between central obesity and inflammation marker, insulin resistance and dyslipidemia in adolescent with central obesity. methods: this study was case-control study, compared central obesity adolescent and normal to hscrp, homa-ir, triglyseride, ldlcholesterol, hdl-cholesterol, and sdldl-cholesterol. results: sixty two adolescent were included, 41 were central obesity and 21 were normal. we found that they were with bmi >25 kg/m 2 had hscrp level higher compared with non obesity, p-value < 0.001 (or: 10.293; 95% ci: 0.002-0.004) and homa ir, p-value < 0.021 (or: 8.711; 95% ci: 0.019-0.025). adolescent with wc ! 91 cm for boys and ! 85 cm for girls, had hscrp levels, homa-ir and hdlcholesterol higher than they were with wc < 84 cm, p-value < 0.001 (or: 11.899; 95%ci: 0.001-0.002), p < 0.022 (or: 8.208; 95%ci: 0.020-0.026) and p-value < 0.036 (or 4.397; 95%ci: 0.064-0.074), respectively. the visceral fat was associated with hscrp levels, p < 0.02 (or: 5.247; 95%ci: 0.031-0.38), and hdl cholesterol p < 0.007 (or 7.242; 95%ci: 0.008-0.012). triglyceride and sdldlcholesterol had not significanty for bmi, wc and visceral fat, however ldl-cholesterol significantly association with visceral fat, p < 0.008 (or: 6.947; 95%ci: 0.011-0.016). conclusion: central obesity adolescents showed increased inflammatory markers and insulin resistance with consequent increased the risk of metabolic diseases so that early intervention in obese adolescents should be done. aims: consumption of high fat diet (hfd) leads to accumulation of intramuscular ceramide (cer). cer is implicated in induction of muscle insulin resistance. the initial step in the de novo cer synthesis is catalyzed by serine palmitoyltransferase (spt). the aim of the present study was to elucidate the role of hfd, myriocin (an spt inhibitor) and metformin on the content of skeletal muscle cer and key proteins implicated in lipid and glucose metabolism in hfd insulin resistant rats. the experiments were performed on male wistar rats, divided into groups: cfed standard rodent chow (control); hfdfed high fat diet; hfd/myrfed hfd and treated with myriocin; hfd/myr/metfed hfd and treated with both the myriocin and metformin. muscle cer and plasma free fatty acids (ffa) were analyzed by lc/ms/ms. content of spt, carnitine palmitoyltransferase (cpt1a), fatty acid transporters (cd36, fabppm) and glucotransporter 4 (glut4) was measured by wb. results: compared to control values, plasma ffa and muscle cer content and expression of all lipid-related proteins were elevated in hfd group. myriocin decreased muscular cer but increased plasma ffa and the expression of both the spt and cpt1a. introducing metformin to hfd/myr group decreased the plasma ffa and muscular cer, lowered the expression of spt, cpt1a, cd36 and fabppm, but increased muscular glut4 expression as compared to hfd and hfd/myr group. conclusions: simultaneous treatment with myriocin and metformin decreases the plasma ffa and muscular cer and improves glucose tolerance by augmentation of muscle glut4 expression. internal medicine, new london hospital, new london, nh, usa hyperglycemia, and hyperlipidemia. understanding whether side effect profiles differ between the most commonly prescribed agents could greatly inform prescribing decisions. objectives: to quantify differences in weight gain and metabolic side effects between aripiprazole, quetiapine, and risperidone in adults and children requiring chronic antipsychotic therapy. selection criteria: randomized controlled trials that compared at least two of the three atypical antipsychotics of interest and reported change in weight. all dosing ranges were included and there were no age or diagnosis restrictions. data collection and analysis: two blinded clinicians independently completed data extraction with a piloted, standardized data collection form. study quality was assessed through the cochrane risk of bias tool. we calculated weighted mean differences (wmd) and 95% confidence intervals (ci) using random effects models on review manager 5. main results: of 95 studies identified through our search, 15 published trials involving 8923 study participants met full inclusion criteria. quetiapine resulted in more weight gain than risperidone and risperidone resulted in more weight gain than aripiprazole). no trials directly compared aripiprazole to quetiapine, but an indirect method of comparison demonstrated that quetiapine causes more weight gain than aripiprazole. similarly, quetiapine led to a greater increase in glucose levels, a greater increase in blood pressure, and a greater increase in total cholesterol than risperidone. non insulin dependent diabetes mellitus (niddm) as a most common form of diabetes is a major public health problem; there is a subgroup of niddm patients which develops the disease at an early age and shows a dominant mode of inheritance. this type is nominates maturity onset diabetes of the young (mody). the prevalence of mody is difficult to access as patients with mody genes mutations are often identified during routine screening for other purposes. mody2 was linked to mutations in glucokinase gene (gck), and account for 8-56% of mody, with the highest prevalence being found in the southern europe. the aim of this study was to examine the prevalence and nature of mutations in gck gene in iranian paients. we have screened gck mutations by single stranded conformation polymorphism (sscp) technique of polymerase chain reaction (pcr) in 12 iranian families with clinical diagnosis of mody, included 30 patients (eight males and 22 females) and their 21 family members. pcr products with abnormal mobility in denaturing gradient gel electrophoresis (dgge) were directly sequenced. we identified six novel mutations in gck gene in iranian families (corresponding to 36.6% prevalence). our findings and the last study on mody1 highlight that in addition to gck, other mody genes such as mody3 and modyx may play a significant role for diabetes characterized by monogenic autosomal dominant transmission. this is clear that the knowledge of the specific defect can be used to pre-symptomatically identify family members at risk for developing mody. background: several epidemiological studies proposed an association between helicobacter pylori (h. pylori) infection with insulin resistance (ir) and metabolic syndrome (mets). however, up to date there is no conclusive evidence regarding this association. objectives: to investigate the prevalence and correlates of h. pylori infection among lebanese adults and to evaluate its association with ir and mets. materials and methods: stored blood samples of adults participating in the national nutrition and non-communicable diseases risk factors survey conducted in lebanon were used for this study (n = 308). h. pylori-specific immunoglobulin g antibody titers were measured by elisa. data available included, in addition to anthropometric measurements, sociodemographic and lifestyle characteristics, blood pressure, biochemical indices (serum insulin, hdl, ldl, tag, glucose). a homa -ir level was used to assess insulin resistance. the international diabetes federation criteria were used to classify study participants with mets. results: the prevalence of h. pylori infection in the study sample was 52% (95% ci: 46.43-57.57%). a higher crowding index was associated with a 50% increase in the odds of infection (or: 1.52, ci: 1.04-2.37). blood pressure, waist circumference, serum hdl, ldl, tag, and glucose levels were comparable between h. pylori positive and negative subjects. the odds of ir and mets were not significantly different between the two groups. comparable to other developing countries. furthermore, our findings suggested no association of h. pylori infection with ir or mets. eradication of h. pylori infection to prevent ir or mets is not warranted. the economic burden resulting from diabetic neuropathy (dn) consumes a major portion of resources allocated for health-care services. the present study was undertaken to assess the costeffectiveness of medical intervention in patients with dn. two hundred patients with dn were purposively selected from out-patient department of birdem hospital, bangladesh. of them 100 were late in detection of dn (ldn) and 100 were detected early (edn). in ldn group, 22% had diabetic peripheral neuropathy (dpn), 17% had diabetic autonomic neuropathy (dan), 11% had diabetic proximal neuropathy (dpxn) and 9% had diabetic focal neuropathy (dfn). in edn group, 16% had dpn and 7% had dan. the average annual cost of care was us$ 26846 (direct us$ 17893 and indirect us$ 8953), with an average us$ 134 per patient. among the average annual cost ldn consumed us$ 18918 (us$ 189 per patient) and edn us$ 7928 (us$ 79 per patient). the annual medical costs increased with the increased number of complications from us$ 1320 to 2296 to 3989 and to 6520 in ldn with one, two, three and more than three complications which is increasing at a rapid rate and us$ 917 to 1556 to 1872 and to 2073 in edn respectively, increasing at a diminishing marginal rate. the regression equation showed that medical cost is significantly related to complications tested in both univariate (p < 0.0001) and multiple linear regression analyses (r 2 = 0.69; f-81.5, p < 0.0001). proper management with regular screening substantially reduces the expenditure related to care and complications. a. alizadeasl 1 , z. ojaghi-haghighi 2 , r. azarfarin 1 1 tabriz university of medical sciences, tabriz, 2 rajaei hospital, tehran, iran background: metabolic syndrome (ms), the combination of hypertension obesity, dyslipidemia, and insulin resistance, is a precursor of diabetes mellitus (dm) and highly prevalent among patients with acute myocardial infarction (ami). diabetes mellitus is associated with larger infarct size and worse outcomes after ami. this study examind the clinical presentation and hospital outcomes among nondiabetic patients with ms following ami. this investigation is prospective analytic study (cohorts type) in 500 consecutive infarct survivors who admitted to our heart centers through 2 years (2009) (2010) (2011) . patients with diabetes (n = 129) were excluded. those with ms (n = 227) included patients with three or more of the following criteria: hypertension, elevated fasting blood glucose, hypertriglyceridemia, low high-density lipoprotein, and obesity [body mass index (bmi) >or = 30]. the control group (n = 144) included patients without ms or dm. results: baseline characteristics were similar except for hypertension, bmi, and dyslipidemia, which by study desing were higher in the ms group. the nondiabetic ms group had larger left ventricular dimension (p = 0.03), left atrium dimension (p = 0.01) and higher rate of ejection fraction 40% (48% vs. 29%, p < 0.05). also nondiabetic ms patients had higher rates of in-hospital death (14.3% vs. 6.9%, p < 0.05); post infarction angina (14.5 vs. 5.1, p < 0.05) and more frequent left main coronary artery or three-vessel disease than the control group (54.2% vs. 23.8%, p < 0.05). conclusion: nondiabetic metabolic syndrome is common in patients with ami and strongly associated with poor outcome of them. background: this study assessed the prevalence of the metabolic syndrome (ms) and its impact on hospital outcomes in patients with acute myocardial infarction (ami) using both ncep atp iii and idf definitions. this investigation is propective analytic study (cohort type) in 500 consecutive ami survivors (mean age: 60.3 ae 10.09 years; 308 men and 192 women), who admitted to our heart centers through 2 years (2009) (2010) (2011) . results: q-wave myocardial infarction (mi) was present in 72.8% of patients and non-q-wave mi, in 27.2%. the ms was found in 56.6% of the patients and was significantly more common in women than in men (64.8% vs. 49.6%, p = 0.003). one component of the ms was found in 15.3% of patients; two, in 21.9%; and none, in 6.2%. 31.3% of the patients had four or five components. hypertension was the most common component of the ms (70.2%). 49.6% of ms patients had triple-vessel disease on coronary angiography in comparing with 27.7% in non-ms, ami patients (p < 0.001). the ms group had larger infarct size as determind by peak creatine kinase-mb (85.4 ae 97.5 vs. 48.1 ae 53.9, p < 001). overal inhospital complications (mechanical and electrical) were higher in patients with ms (46.8% vs. 21.6%, p = 0.01). ms is associated with a 8.9-fold increased risk of acute renal failure after mi (p = 0.0001). conclusion: ms in patients with ami is prevalent and associated with larger infarct size, more in-hospital complications, and marked increase of acute renal failure. background and aims: according to current views metabolic syndrome (ms) and obesity, especially with increased amount of visceral fat is characterized by prothrombogenic changes of hemostasis and fibrinolysis. methods: cross-sectional observational study included 165 postmenopausal women (age 48-65) with ms. coagulant system was assessed by measurement of parameters of plasma haemostasis [activated partial thromboplastin time (aptt), fibrinogen concentration], and the activities of coagulant factors vii (fvii), viii (fviii) and ix (fix) in plasma. in order to investigate the functioning of anticoagulant system the activities of antithrombin iii. also we assessed adiponectin (adp) and homocysteine (hm) concentrations in plasma. results: prothrombotic alterations were observed in 86.1% women. the aptt was decreased in 13.9%, hyperfibrinogenemia was revealed in 49.6%, the increased activity of: fviiin 26.0%, fviiiin 19.4%, fixin 21.8%, and the decreased activity of antithrombin iiiin 11.5%. the hm levels was increased in 76.4%, the adp levels was decreased in 39.3%. there were a significant positive associations between some of revealed prothrombotic alterations (fvii, fviii and fibrinogenemia) and anthropometric markers of visceral obesity and ms (weight, body mass index, and waist circumference). the adp levels were inversely correlated with the hm levels (r = à0.57, p < 0.05) and the activity of fvii (r = à0.34, p < 0.05). conclusion: results of our study demonstrate high prevalence of various prothrombogenic abnormalities in coagulation and anticoagulation systems in patients with visceral obesity and ms. plasma b-type natriuretic peptide (bnp) and blood urea nitrogen (bun) are elevated in heart failure. renal function is known to be an important factor related to bnp and bun determination. the aim of the present study was to examine whether bnp and bun are associated with left ventricular diastolic dysfunction (lvdd) in patients with type 2 diabetes (t2dm) without chronic kidney disease (ckd). the subjects in this study were 107 consecutive patients with t2dm [68 men (64%); age 65 ae 9 years old (mean ae sd); diabetic duration 10 ae 8 years; hba1c 7.9 ae 1.7%]. subjects with overt heart failure or nyha class >1, history of coronary artery disease, severe valvulopathy, chronic atrial fibrillation, and estimated glomerular filtration rate <60 ml/min/1.73 m 2 were excluded from the study. all patients underwent clinical evaluation, laboratory tests including bnp determination, and echocardiographic examination. doppler echocardiographic indices including peak early diastolic mitral annular velocity (e′) and early diastolic myocardial velocity (e) were obtained in each patient. none of the patients exhibited systolic impairment of left ventricular function (ejection fraction >50%), whereas lvdd (e/e′ >8) was detected in 91 cases (85.0%). e/e′ correlated with age (r = 0.344, p < 0.0001), sex (r = 0.204, p = 0.020), diabetic retinopathy stage (r = 0.207, p = 0.033), systolic blood pressure (r = 0.222, p = 0.026), bnp (r = 0.306, p = 0.002), and bun (r = 0.257, p = 0.007). in multiple regression analysis, age (b = 0.220, p = 0.024), sex (b = 0.191, p = 0.041), bnp (b = 0.260, p = 0.005), and bun (b = 0.210, p = 0.026), correlated independently with e/e′. bnp and bun could be useful tools to screen for preclinical ventricular diastolic dysfunction in patients with t2dm without ckd. h. kawano, y. nagayoshi, y. kinoshita objective: this study compared the effects of combination statin and fibrate therapy with either statin or fibrate monotherapy on lipid profiles in patients with impaired glucose tolerance (igt) and a high risk for cardiovascular disease. methods and patients: forty-five patients with igt and dyslipidemia (men 25, women 20, mean age 61.7 ae 2.4 years) were assigned randomly to the three treatment groups for a 6-month period. results: after 6 months of treatment, low density lipoprotein levels decreased in every group, especially the statin and statin + fibrate groups. triglyceride levels also decreased in all three groups, especially the fibrate and statin + fibrate groups. high density lipoprotein cholesterol and fasting blood glucose levels did not change in any group. the levels of remnant like cholesterol particles decreased in the fibrate and statin + fibrate groups. there was no change during the study in the levels of creatine phosphokinase, lactate dehydrogenase, or creatinine. conclusion: combination statin and fibrate therapy results in greater improvement in lipid profiles than monotherapy with either drug. no marked adverse effects were observed with combination therapy during the study. background: gout is considered a metabolic disease and ranked among the diseases connected with obesity, such as an arterial hypertension, coronary artery disease, stroke, and type 2 diabetes mellitus (who, 2000) . it has been proven that intake of a considerable quantity of meat products is predictor of acute gouty arthritis. for this reason there is great interest in studying the prevalence of gout among inhabitants of the republic of sakha (yakutia) where a lipid-protein diet prevails. this is a preliminary report on the incidence of gout requiring hospitalization in 2006-2011. objectives: patients hospitalized in the yakut city hospital with gouty arthritis were studied. methods: patients are being studied by means of a questionnaire developed by the institute of rheumatology (moscow). results: forty-four patients were registered (42 men and two women). the majority of patients (n = 35) are inhabitants of yakutsk city. median age of the subjects is 56 years, with a range of 35-76 years; four patients are over 65. secondary forms of gout and relapses of disease are common. accompanying pathology includes: ah in 22 patients, cad in seven patient, type 2 dm in four patients, glucose intolerance + obesity in one patient, metabolic syndrome + obesity in one patient, uncomplicated obesity in one patient, metabolic syndrome without obesity in one patient, chronic renal insufficiency in one patient, and cardiovascular accidents in three patients. the research proceeds. results will be used for characterization of the incidence and diagnostic features of gout in yakutia especially among young patients with accompanying metabolic abnormalities. background: the prevalence of obesity, a major public health problem, is rising in many countries including iran. non-drug dependent interventions for obesity management include physical activity, dietary restriction and acupuncture. primary study objective: we examined the effects of body electroacupuncture and low-calorie diet on plasma leptin in obese and overweight individuals with the excess or deficiency pattern (according to chinese medicine). methods/design: the case group received authentic electroacupuncture and the placebo group received sham acupuncture. both groups consumed a low-calorie diet for 6 weeks. settings: this study was conducted in nutritional clinic of ghaem hospital, mashhad, iran. participants: people (n = 86), aged between 18 and 65 years with body mass indexes (bmi) between 25 and 45 kg/m 2 were randomized into two groups. interventions: comparison of either real or shame electro acupuncture combined with a low caloric diet was investigated in this trial. primary outcome measures: plasma leptin, body fat mass (bfm), body weight and body mass index were measured before and after treatment. results: in volunteers in the case group with both the excess and the deficiency patterns, we found a significant reduction in plasma leptin (24.96%, p = 0.001) and bfm (8.29%, p = 0.001). in the placebo group, we found a less significant reduction in leptin and bfm. the difference between the two groups was significant for leptin (p = 0.03) but not for bfm (p = 0.8). conclusions: body electroacupuncture with a low-calorie diet may reduce plasma leptin concentration; through a mechanism that will require further clarification. m. darbandi 1,2 , s. darbandi 1,2 , a.a. owji 1 , p. mokarram 1 , m. ghayour mobarhan 2 effects of auricular acupressure combined with low-calorie diet on the leptin hormone level. methods: volunteers (n = 86) with body mass indexes (bmi) between 25 and 45 kg/m 2 were randomised into a case (n = 43) or a control (n = 43) group. the participants in each group received a low-calorie diet for 6 weeks. the case group was treated with auricular acupressure and the control group received a sham procedure. plasma leptin levels, body fat mass, body weight (bw) and bmi were measured before and after treatment. results: participants who received auricular acupressure showed significant reductions in their plasma leptin levels (18.57%, p < 0.01) as well as in their body fat mass (4%, p < 0.05). these changes were not observed in the control group. the reduction in leptin was significantly greater in the acupressure group than the controls. conclusion: auricular acupressure combined with a low-calorie diet significantly reduced plasma levels of leptin. however, the mechanism of this reduction is not clear. background: premature arteriosclerosis may be one of the mechanisms linking pre-diabetes mellitus (pre-dm) and cardiovascular disease. we assessed premature arteriosclerosis in pre-dm using arterial stiffness indices and analyzed the associated contributors of this process. methods: we collected clinical data of 33 patients without dm, 53 pre-dm patients, and 34 dm patients. both the compliance index (ci) and stiffness index (si) were measured to indicate large and peripheral arterial stiffness. results: patients with pre-dm and dm had lower ci (3.8 ae 2.1 vs. 5.2 ae 3.0 units; p < 0.05 and 3.6 ae 1.8 vs. 5.2 ae 3.0 units; p < 0.05, respectively) and higher si (8.0 ae 2.0 vs. 6.7 ae 1.6 m/s; p < 0.01 and 9.4 ae 2.3 vs. 6.7 ae 1.6 m/s; p < 0.001, respectively) than patients without dm. both pre-dm and dm patients had higher glucose and hemoglobin a 1c , higher homa index, higher hscrp, and a lower adiponectin level than patients without dm. using multivariate linear regression analysis, age, heart rate and homa index were independent determinants for si (whole model: r 2 = 0.47, p < 0.001), whereas male gender, hscrp, and homa index were independent determinants for ci (whole model: r 2 = 0.34, p < 0.01). conclusions: homa index was an independent determinant for arterial stiffness. increased insulin resistance may associate with increased arterial stiffness both at large and peripheral arteries in pre-dm patients. king's college london, london, uk, 2 uppsala university, 3 regional cancer centre, uppsala, sweden, 4 guy's & st thomas' nhs foundation trust, london, uk, 5 karolinska institute, stockholm, sweden background: impaired glucose metabolism has been linked with increased cancer risk, but the association between serum glucose and cancer risk remains unclear. we used repeated measurements of glucose and fructosamine to get more insight into the association between the glucose metabolism and risk of cancer. methods: we selected 11,998 persons (>20 years old) with four prospectively collected serum glucose and fructosamine measurements from the apolipoprotein mortality risk (amoris) study. multivariate cox proportional hazards regression was used to assess standardized log of overall mean glucose and fructosamine in relation to cancer risk. similar analyses were performed for tertiles of glucose and fructosamine and for different types of cancer. results: a positive trend was observed between standardized log overall mean glucose and overall cancer risk (hr = 1.08; 95% ci: 1.02-1.14). including standardized log fructosamine in the model resulted in a stronger association between glucose and cancer risk and a statistically significant protective effect of fructosamine (hr = 1.17; 95% ci: 1.08-1.26 and hr: 0.89; 95% ci: 0.82-0.96, respectively). the highest risk for cancer was among those in the highest tertile of glucose and lowest tertile of fructosamine. similar findings were observed for prostate, lung, and colorectal cancer. the contrasting effect between glucose, fructosamine, and cancer risk suggests the existence of distinct groups among those with impaired glucose metabolism, resulting in different cancer risks based on individual metabolic profiles. further studies are needed to clarify whether glucose is a proxy of other lifestyle-related or metabolic factors. patients in two groups were compared according to age, parity, prepregnancy weight, family history of diabetes mellitus, history of macrosomia in their previous pregnancies, history of hypertension, previous malformed fetus, history of iufd and abortion. based on the obtained results there was a statistically significant difference between two groups in terms of age (p < 0.05), parity (p < 0.05), prepergnancy weight (p < 0.05), family history of diabetes mellitus (p < 0.05), previous macrosomia (p < 0.05), history of hypertension (p < 0.05), previous malformed fetus (p < 0.05) and previous iufd (p < 0.05), but abortion was not significantly different. older age, parity (three or more) obesity, family history of diabetes mellitus, history of macrosomia, hypertension, malformed fetus and also previous iufd are risk factors for gestational diabetes. therefore, these women should be screened and handled for gestational diabetes in their pregnancies, and controlled for possible diabetes mellitus in the future. methods: this family based study was conducted on 529 nuclear families from among tehran lipid and glucose study with two biological parents and at least two offspring (1066 parents and 1394 offspring), aged 3-80 years. selected families had at least one person with overweight or obesity. all obesity-related variables (height, weight, hip, waist circumference (wc), body mass index (bmi), body size (bs), resting energy expenditure (ree), waist to hip ratio (whr)) were measured and calculated. the heritability estimate of continuous variables was calculated using a standard quantitative genetic variance-components model which was implemented in the solar software. results: the heritability estimates for obesity-related variables such as height, weight, hip, wc, whr, bmi, bs, and ree, after adjustment for sex, age were 16%, 31%, 21%, 32%, 27%, 43%, 51%, and 25%, respectively. the h 2 for the above mentioned variables, expect of bs, after adjustment for sex, age, and body size varied to 28%, 32%, 21%, 26%, 25%, 32%, and 47%, respectively (p < 0.05). we clearly demonstrated a significant heritability of obesity-related variables among tlgs families. the results of the present study confirmed the important impress of genetic factors on the obesity-related variables phenotype. method: pwv determined with bpuls apparatus using left external carotid and left dorsalis pedis arteries as "central" and "peripheral" points respectively. pulses picked up by infrared sensors and recorded simultaneously with single lead ecg. time difference between pulses measured. shorter time delay or faster pwv indicates decreased arterial elasticity. materials: nine hundred and fifty-seven asymptomatic filipinos studied. males-447, females-500. age 17-84 years. three hundred and twenty-seven hypertensives. classified into groups according to bmi and wc. average pwv time (adjusted for height) for each group noted. relationship of increased bmi and wc to variations in pwv time determined. results: elevated bmi does not significantly influence pwv time in: all subjects-(p < 0.1397); females-(p < 0.2372); normotensives-(p < 0.0866); and, hypertensives -(<0.1548). however, for every centimeter increase of wc there is a corresponding decrease of pwv time by: 0.000743 s (p < 0.0001) in all subjects; 0.00063 s (p < 0.0001) in females; 0.000759 s (p < 0.0001) in normotensives; and, 0.00035 s (p < 0.0001) in hypertensives. discussion: abnormal pwv is a high cvd risk factor. in four groups above, elevated bmi does not significantly influence pwv time. however, in the very same groups, increased wc significantly affects pwv time. this indicates that if we rely solely on bmi to predict cvd we will miss cases which are at high risk as shown by abnormal wc. correlation (r = 0.22). an increased risk of cvd and cancer was identified with elevated levels of ggt or crp or both markers (ggt-crp score ! 3); the greatest risk of cvd and cancer was found when ggt-crp score = 6 (hr: 1.40 (95%ci: 1.31-1.48) and 1.60 (1.47-1.76) compared to ggt-crp score = 0, respectively). conclusion: while ggt and crp have been shown to be associated with metabolic abnormalities previously, their association to the components investigated in this study (hyperglycaemia and dyslipidaemia) was limited. results did demonstrate that these markers were predictive of associated diseases, such as cancer and cvd. metabolic syndrome is the aggregation of conditions that together increases the risk of cardiovascular disease and diabetes mellitus in both normal glucose tolerance (ngt) and impaired glucose tolerance (igt) subjects. it is estimated that around 20-25% of the world′s adult population have the metabolic syndrome. over the last 20 years the prevalence of metabolic syndrome has steadily increased in all populations, and making it one of the major global public health challenge. the objective of this study is to estimate the prevalence of metabolic syndrome and cardiovascular risk factors in impaired glucose tolerance (igt) subjects. two hundred and four impaired glucose tolerance and 30 normal glucose tolerance subjects of both genders were selected for the present study according to the american diabetes association ada criteria, on the base of 2 h glucose tolerance test. anthropometric characteristics like waist circumference, bmi, systolic blood pressure, and diastolic blood pressure were measured with standard techniques. biochemical parameters like fasting blood sugar, fasting insulin, cholesterol, triglycerides, hdl-c, and ldl-c were determined by standard techniques, the homa-ir values were calculated with the help of formula. it is concluded from the present study that the prevalence of metabolic syndrome is significantly increased according to aace, atpiii definition criteria's in impaired glucose tolerance subject, the study emphasizes strongly that ms is major factor to enhance the incidence of type 2 diabetes and cardiovascular diseases in impaired glucose tolerance subjects. it is suggested that preventive measures and treatment can reduce incidence of cvd, type 2 diabetes in our population. anthropology, vidyasagar university, midnapore, india waist circumference (wc) and waist-hip ratio (whr) were reported as imperative abdominal obesity related measures having influence on many cardiovascular disease (cvd) risk factors. present study attempts to evaluate the possible association of abdomen circumference (ac) with hypertension and dyslipidaemia among the bhutias, a tribe residing the sub-himalayan state of sikkim, india. five hundred and eleven bhutias of both sexes aged ! 20 years inhabiting gangtok, the state capital and its neighbourhood were systematically selected to participate in the study. after providing informed consent data on blood pressures, blood lipids, anthropometrics and other lifestyle related variables such as perceived stress, diet, energy expenditure, socioeconomic status and so on were collected following standard methods. the results evidently indicate that the ac is the best predictor of the selected cvd risk factors. after log transformation, the effect of age on cvd risk factor variables and anthropometrics was found and regressed out through linear regression. data was pooled for sex as significant sex difference was absent for most of the variables. after adjusting for significant lifestyle related predictors, multiple logistic regression was carried out to evaluate the significant obesity related predictors of hypertension and dyslipidaemia. receiver operating characteristic (roc) curve finally showed ac as the best predictor of hypertension and dyslipidaemia. although wc and whr were found to be important abdominal obesity related predictors of cvd risk factors in many studies, present population based cross sectional study has proved the importance of considering ac as a central obesity related predictor of different cvd risk factors. the achievement of therapeutic goals is of great importance in patients with chronic diseases such as diabetes mellitus (dm), because of its impact on morbidity and mortality. objective: to explore the relationship between therapeutic adherence and metabolic control in patients with dm-2, who were seen at a hospital in the city of medellı´n (colombia). methods: cross-sectional study involving patients with the following selection criteria: age ! 18 years, diagnosis of dm-2 ! 6 months, who signed informed consent. the summary of diabetes self-care activities (sdsca) score was applied for the measurement of therapeutic adherence and duke-unc score for the assessment of perception of social support. glycosylated hemoglobin (hba1c) was processed by turbidimetric inhibition immunoassay (tinia) cobas c-501 equipment. adequate metabolic control was defined as value of hba1c 7%. version 19.0 of the spss program was used for the statistical analysis. results: seventy patients studied: 66% are female, 76% suffer from hypertension, 70% have dyslipidemias and 16% smoke. in addition, 59% are insulin dependent, 70% have inadequate glycemic control and 87% has no social support. items with the highest proportion of adherence were: medication (79%), foot care (71%) and diet (62%). whereas the lowest were: hba1c (32%) and exercise (28%). hba1c correlated significantly (p < 0.05) with adherence to diet, blood glucose monitoring, foot care, social support and sex. conclusion: therapeutic adherence was associated with metabolic control in patients with dm-2. mellitus. however, it is unclear if severity of dr is associated with lvdd, which is recognized to result in subsequent heart failure. the subjects in this study were 120 consecutive patients with type 2 diabetes mellitus (t2dm). all patients underwent clinical evaluation, laboratory tests, and echocardiographic examination. doppler echocardiographic indices including peak early diastolic mitral annular velocity (e′) and early diastolic myocardial velocity (e) were obtained in each patient. the patients were divided into three groups according to presence of dr and its severity: no diabetic retinopathy (n = 80), simple retinopathy (n = 20), and preproliferative or proliferative retinopathy (n = 20). no patients showed systolic impairment of left ventricular ejection fraction (lvef >50%), whereas impaired lv diastolic function (e/e′ >8) was detected in 104 cases (87%), furthermore lvdd (e/e′ >15) was detected in 19 cases (16%). e/e′ was correlated with age (r = 0.303, p = 0.001), sex (r = 0.208, p = 0.021), diabetic duration (r = 0.279, p = 0.002), dr stage (r = 0.241, p = 0.007), systolic blood pressure (r = 0.199, p = 0.031), and serum creatinine level (r = 0.265, p = 0.003). in multiple regression analysis, age (b = 0.322, p < 0.001) and dr stage (b = 0.266, p = 0.002) were independently correlated with e/e′. in this study, we showed that almost all subjects had asymptomatic lvdd, and severity of dr was associated with lvdd in patients with type 2 diabetes mellitus. objectives: this study explores the contribution of the sasang constitutional types as a risk factor for hypertension by examining the prevalence and risk for hypertension across different constitutional types (se, sy, te, and ty types) and investigating whether certain constitutional types can increase the risk of hypertension in an individual. design: this retrospective chart review evaluated the charts of patients who had visited the clinic for routine physical check-up. subjects: among 12,140 visitors, those aged between 21 and 80, with complete data concerning the past medical and social history, blood pressure and body size measurements, results of blood test, and confirmatory constitutional typing (excluding the ty type) were included in the final analysis group (n = 1701). results: especially, even after adjusting for the different variables, the odds ratio for hypertension in the te type was found to be 1.72 (ci 1.14-2.62) (taking the se type as the reference group), indicating that the te constitutional type can act as a risk factor for hypertension. moreover, when comparing the te type to non-te types, the odds ratio was found to be 1.37 (ci 1.06-1.78), implying a weak but valid contribution of the te constitutional type toward increasing the risk of hypertension. the results of this study show that the prevalence of hypertension differs across different constitutional types, and that the constitutional type (the te type) can act as a risk factor for hypertension independently of other possible variables. a.b. shetty 1 , p.k. roy 2 1 physical therapy, university of st mary, leavenworth, ks, 2 bon secours hospital, baltimore, md, usa introduction: the purpose of this research was to determine whether habits of individuals contributed to changes in metabolic rate. as the daily physical activity levels increase, there is an increase in the metabolic rate and caloric expenditure. it can be inferred that people who are overweight may not participate in physical activities due to social, psychological, and physical reasons. they may spend more time in less physically demanding activities during their leisure time. hypothesis: it was hypothesized that the individuals who like to read for pleasure during leisure will have higher body-mass-index. subjects: a group of 37 college aged, 18-25, females volunteered to be the subjects for the study. method: each subject's weight and height were collected to determine bmi. the leisure time activities that were included were reading, sleeping, watching television, use of computers, and sports and physical activities. a likert scale questionnaire was developed and had four levels of answers for each of the leisure time activities. a person-product moment correlation was used to determine the relationship between bmi and five leisure time activities. the results indicate that a positive correlation of 0.57 between bmi and leisure time reading habits. there is a negative correlation of à0.73 between bmi and sports and physical activities. conclusion: this research demonstrates that the bmi is significantly higher for individuals who like to read during their leisure time activities. therefore, it is also important to develop physical activities as habits during early years that may carry into adulthood. objectives: the main goal of this retrospective cohort study was to compare the incidence of over-all and site-specific cancers among israeli arabs with diabetes mellitus (dm) with that of israeli arabs without dm. methods: a retrospective cohort study was conducted in northern israel, involving all arab subscribers of clalit healthcare services (chs), which is the major hmo service in the region, serving over 70% of the concerned population. results: during a period of 10 years (1999-2008) of follow-up, 752 and 2045 incident cases of cancer were found among 13,450 people with dm and 74,484 adults free of dm, respectively. the follow-up time involved 817,506 person years. dm was associated with a standard incidence ratio (sir) of 3.27 (95%ci: 1.49-5.05) and 2.87 (95%ci: 1.25-4.50) for pancreas cancer in men and women, respectively. a significantly reduced sir was observed for esophageal, stomach and intestine cancer 0.67(95%ci: 0.36-0.99) in men. our findings support an association between dm and increased risk cancer of the pancreas in arab men and women. a significantly reduced risk of all other cancers was observed only in arab men. a.e. berezin 1 , o.a. lisovaya 2 1 methods: seventy-two mild-to-moderate arterial hypertension patients within 1-2 weeks after ischemic stroke were enrolled to the scrutiny at baseline. both vegf-1 and mmp-9 plasma levels were measured at the study entry and in 6 months after baseline by elisa. we has been assessed all new cardiovascular events including myocardial infarction (mi), unstable angina (ua), recurrence stroke (rs), tia, advance heart failure (hf) during study period. results: analysis of obtained outcomes have been shown that all cases (n = 28) of new cardiovascular events identified during first 4 weeks after start of observation are correlated well with vegf-1 plasma levels (r = à0.58; p < 0.001) measured at baseline. on the other hand, 4-weeks survival rate was 87.0% and 68.6% respectively for group subjects (p < 0.01) with top and low quartile of vegf-1 plasma level at baseline. however, lack of tightly interrelationship between cardiovascular outcomes and vegf-1 (r = 0.2; p = 0.16) in 6 months after study entry. the mean mmp-9 plasma levels were significantly higher in dead patients in comparison to survival subjects of the study end. both new events associated with rs and tia incidences independently study period are correlated well with vegf-1 (r = à0.63; p < 0.05 and r = à0.58; p < 0.02 respectively) only. we has been proposed that circulating vegf-1 might have more predicting value in comparison with mmp-9 concentration among hypertensive patients during early ischemic stroke period. introduction: clinician should change the health care system from on diseases treatment to prevention of diseases. obesity increase mortality, morbidity and psychological problems through life but beneficial effects of risk reduction by changing life style have been documented. objective: sedentary life style is the most important factor for promoting weight gain in people. it's clear that obesity occurs when calorie intake exceeding calorie output. obesity is common in families but seems environmental factors can affect body weight beside genes role. a main factor of life style change is energy balance. we recommend one simple healthy life guideline for managing of environmental factors in obese people that includes a diet and physical activity program. body weight should be reduced 7-10% during first year by following of this guideline and continued until achieving to optimal body weight. obese people are visited and monitored some parameters for program adjusting and changed each item basis on individual response. objective: to assess trend and associations between blood sugar level, blood pressure, overweight/obesity and lifestyle among tribe of northeast india experiencing transition. method: cross-sectional study was carried out among 603 tangkhul nagas, aged between 20 and 70 years. subjects were divided into five decadal age groups to assess age trend in biological and socioeconomic variables. height, weight, waist circumference, hip circumference, random blood sugar level, bp, bmi, wsr and whr were evaluated. result: the prevalence of prediabetes and diabetes were 9.15% and 2.7% respectively. diabetes was found among older age groups only. hypertension was highly prevalent with higher percentage among males (37%) than females (20.8%). mean bp was higher among males but sugar level among females, which corresponds to their higher adiposity level. prevalence of overweight/obesity in females was 27.1% while in males, it was 17.6%. sugar level and bp had positive correlation with age, bmi, wsr and whr. the prevalence of hypertension and prediabetes/diabetes were higher among centrally obese subjects. central obesity indices were stronger predictors of diabetes and hypertension than general obesity. odds ratio showed urbanization, higher socioeconomic status and sedentary lifestyle as significant risk factors for development of overweight/obesity, which in turn was risk factor for development of diabetes and hypertension. conclusions: tangkhul nagas has been experiencing socioeconomic and lifestyle change which led to the increasing prevalence of overweight/obesity and cardiometabolic health problems, contributing to the escalating global epidemic of obesity and metabolic syndrome. there was close associations between socioeconomic status, age, overweight/obesity, hypertension and blood sugar level. background and aims: trace elements play an essential role in metabolism of carbohydrates, lipids, etc. these results stipulated the present work. we studied 30 patients (eight females, 22 males, age 40-80 years) with type 2 diabetes mellitus. duration of the disease varied from 1 month to 25 years. as a control data the hair composition of practically healthy individuals (n = 67) were used. as an analytical method instrumental neutron activation analysis (inaa) was used. results: data obtained shows statistically significant decrease of cu, cr, and zn and increase of na, fe, br, and sb levels in hair in the group with a blood glucose concentration <7 mmol/l. in the group with a blood glucose concentration more than 7 mmol/l levels of cu and la were decreased and na, fe, co, br, and i levels increased. in the group with a blood glucose concentration more than 7 mmol/l glucose concentration correlates with sc (r = 0.68), cr (r = à0.55), and mn (r = +0.64). conclusion: it was found that mn level in blood increases with diabetes. this may be a manifestation of mn metabolism dysfunction in diabetes and excretory role of hair. it is also confirms the important role of some elements, especially cr, in diabetes. concentration of this element significantly decreases especially in the first year of illness. unexpected are correlations for sc. usually sc is considered as an element that has no biological role. these findings are additional reasons to study the biological role of less studied ultra trace elements. objective: investigate the prevalence of hyperglycemia in patients undergoing coronary angiography. methods: six hundered and ninety-five consecutive brazilian subjects undergoing coronary angiography were assessed for fasting plasma glucose and hba1c (hplc) levels and for previous history of diabetes mellitus (dm). we classified those without previous diagnosis in three groups according to hba1c levels (hplc): normoglycemic (n); hba1c -<5.7%; prediabetes (pd) -hba1c 5.7% à6.4%; and dm (d) -a1c ! 6.5%. cad defined by any visible lesion ! 50%. we also classified glycemic status using fasting glucose levels by ada criteria. results: patients were aged 60.3 ae 10.5 years, 55.4% male. the group had a high prevalence of cardiovascular risk factors: dyslipidemia -93%, hypertension -91%, 70% overweight/obese, metabolic syndrome (idf) in 72% of subjects. cad was detected in 69.5% of them. dm was newly diagnosed using hba1c in 11.1% of the whole sample (77 individuals) and pd in 209 (30%). in those without dm according to hba1c, 27 (3.8%) had glucose levels ! 7 mmol/l and 196 (28.3%) had glucose ! 5.6 mmol/l. in this particulary population with coronary artery disease and/or cardiovascular risk factors we observed a high prevalence of undiagnosed hyperglycemia -dm or prediabetes (32-41%) depending on the criteria used for identification/diagnosis (fasting glucose or hba1c). this finding reinforces the importance of implementing a systematic screening for hyperglycemia in this high risk population. national university of singapore, singapore, singapore, 2 university of sydney, sydney, nsw, australia objectives: to evaluate associations between metabolic syndrome (mets) components and mortality and whether these associations change over time. methods: three thousand eighty-six eligible residents aged ! 49 years were prospectively followed in the blue mountains eye study, west of sydney, australia. mets components were measured at baseline (1992 -1994) , and after 5-years and 10-years (2002-2004) . using cox proportional hazards and competing risks models with mets as a time-dependent covariate, we estimated effects of mets on all-cause and cause-specific mortality. receiver operating characteristic (roc) curve analyses were used to identify which individual or combined mets components best predicted mortality. . roc analysis suggested that glucose, body mass index (bmi) and blood pressure (bp) best predicted all-cause and chddeath, bmi and bp best predicted stroke-death and glucose and triglycerides best predicted cancer-death. national university of singapore, singapore, singapore, 2 university of sydney, sydney, nsw, australia objectives: to investigate the effect of metabolic syndrome (mets) and its components on the incidence of different sub-types of cataract (cortical, nuclear and posterior subcapsular cataract (psc)) over 10 years and whether these associations change with time. methods: a prospective cohort of elderly aged ! 49 years were followed up over a period of 10-years in the blue mountains eye study, west of sydney, australia. mets components were measured at baseline (1992-1994), 5-years (1997-1997) and 10-years (2002) (2003) (2004) . the incidence of different types of cataract was obtained from standard photographic grading at 5-and 10-year (n = 1997). using random-effect complementary log-log regression model with cataract status as interval-censored data, we estimated the effect of mets on the incidence of different types of cataract at different time-intervals. results: after taking into account the changes in mets (components) and controlling for possible confounders, mets was found to be associated with increased 5-year incidence of cortical cataract [hazard ratio (hr) 1.48, 95% confidence interval (ci) 1.05-2.09] and psc (hr 1.75, 95% ci 1.01-3.04). amongst the five components of the mets, high glucose and obesity predicted the 5-year incidence of cortical cataract and at 10-year, high glucose and low-hdl was associated with increased incidence of psc and cortical cataract, respectively. conclusions: changes in mets predicted the 5-year incidence of cortical cataract and psc. different mets components predicted the incidence of different sub-types of cataract at varying time-intervals. material and method: this study was a done on a sample of isfahan cohort study (ics) participants. subjects which met atpiii criteria, entered in our study. aobesity indices such as body mass index (bmi), waist circumference (wc), waist to hip ratio (whr) and waist to height ratio (whtr) measurements were done by trained nurses. serum lipids, fasting blood glucose, interleukin-6(il-6) and interleukin-10(il-10), adiponectin ghrelin, and crp were measured. the mann-whitney u-test was used for comparisons between the level of inflammatory markers in obese subjects with and without mets. then subjects with mets were selected and correlation between inflammation biomarkers and obesity indices correlation coefficients were obtained by pearson correlation test. results: wc and bmi were significantly higher in subjects with mets (p < 0.001). the median (iqr) of crp was significantly higher in subjects with mets (p < 0.001). the correlation between inflammatory markers and obesity indices was determined by nonparametric analysis. mets subjects had a significantly positive correlation between il6 and whtr (r = 0.367, p = 0.015). no significant relationship found between il-10, ghrelin, adiponectin and crp with, bmi, whtr and waist. significant negative correlation was observed for adiponectin and the waist (r = à0.367, p = 0.026). negative correlation between adiponectin with whr, whtr and bmi among subjects without mets (r = à0.509, r = à0.454 and à0.475 respectively). conclusion: present study indicate that the observed inflammatory concentrations cannot be explained by type of obesity. objective: the incretin effect is known to be decreased in type 2 diabetes (t2d). however, no study has directly measured the incretin effect in non-caucasian subjects. because asian patients with t2d are characterized by decreased insulin secretion, this study set out to examine the incretin effect in korean subjects with normal glucose tolerance (ngt) or t2d. research design and methods: we performed 75 g ogtts and corresponding isoglycemic intravenous glucose infusion (iigi) studies in subjects with ngt (n = 14) or t2d (n = 16). the incretin effect was calculated based on the incremental area under the curves (iaucs) of insulin, c-peptide, or insulin secretion rate (isr). the plasma levels of total glucagon-like peptide-1 (glp-1) and glucosedependent insulinotropic polypeptide (gip) were measured by elisa. results: the incretin effect was not different between the subjects with ngt and t2d (43 ae 6% vs. 47 ae 4%, p = 0.575 by insulin; 29 ae 7% vs. 38 ae 4%, p = 0.253 by c-peptide; 28 ae 7% vs. 35 ae 5%, p = 0.372 by isr, respectively). however, the gastrointestinally mediated glucose disposal (gigd) was decreased in t2d (28.5 ae 4.2% vs. 59.0 ae 4.3%, p < 0.001). the plasma levels of the total glp-1 and gip during the ogtts were comparable between the two groups. conclusions: in koreans, the secretion of glp-1 or gip during ogtts and the incretin effect were comparable between subjects with ngt and t2d, whereas the gigd was significantly decreased in patients with t2d. a.e. berezin 1 , a.a. kremzer 2 1 internal medicine, 2 clinical pharmacology, state medical university, zaporozhye, ukraine the aim of this study was to evaluate the interrelation between circulating osteoprotegerin (opg) and coronary vasculature damage in type 2 diabetes mellitus patients. methods: one hundred and twenty-six subjects with stable diabetes mellitus 2 type with previously angiographic documented asymptomatic cad were enrolled to the study. vessel-wall and plaque geometrical and compositional parameters were measured on contrast-enhanced ct angiography. the volume of intramural calcium of >320 hu in major coronary vessels was measured in 428 coronary segments with a highly standardized method. coronary artery calcification was quantified by calculating the agatston' score index and calcification mass measurement. opg plasma levels were measured with elisa. results: circulating opg level was increase in 105 patients [5201 pg/ ml [95% confidence interval (ci) = 3605-6280 pg/ml] and was normal in 21 subjects (880 pg/ml; 95% ci = 745-1140 pg/ml; p < 0.0001). the relationship between coronary artery calcium by agatston' score index and percent atheroma volume (pav) was determined by linear regression. pav and remodeling index were significantly higher in patients with elevation of opg plasma level when compared with those who have normal opg [adjusted odds ratio (or) = 4.60 (95% ci = 2.23-14.50); p = 0.006]. there was significant correlation between agatston' score index and pav [r (2) = 0.46, p = 0.008]. in conclusion, we demonstrated that opg plasma level can associate with vessel-wall thickening, percent atheroma volume, and agatston' score index value in type 2 diabetes mellitus patients with previously angiographic documented cad. along with the prevalence of diabetes continues to grow, the occurrence of microvascular chronic complications caused by diabetes has increasingly become a serious social problem. diabetic cardiomyopathy, a microvascular disease, results in chronic heart failure and causes a heavy economic burden. endoplasmic reticulum as an intracellular organelle, which occurs the stress leads to cell apoptosis. recent studies indicate that bis(maltolato)oxovanadium can alleviate the endoplasmic reticulum stress to protect cell function. in this study, stz (50 mg/kg)-induced diabetic rats were divided into two groups, the diabetic control group, and diabetes + vanadium group; the normal group without diabetes were randomly divided into two groups, normal group and the normal + vanadium group. the bis(maltolato)oxovanadium significantly improve the endoplasmic reticulum stress in stz-induced diabetic rat heart cells and reduce the cardiac damage. in this study, bis(maltolato)oxovanadium can reduce the endoplasmic reticulum stress in diabetic myocardial cells delay the development of diabetic cardiomyopathy. it can be a new way to explain the mechanism in the function of bis(maltolato)oxovanadium. between mets and type 2 diabetes families without diabetic family related. subjects and methodology: forty-three mets subjects with type 2 diabetes parental (1st group), 27 mets subjects without type 2 diabetes parental (2nd group) and 30 no mets subjects but with type 2 diabetes parental (3rd group). this study was conducted over 12 months at diabetologia department hospital. diabetes screening was achieved by oral glucose tolerance testing (ogtt). metabolic parameters were determined by spectrophotometry, insulin by radioimmunoassay. insulin sensitivity was also assessed by the homeostasis model assessment (homa) approach (glucose 9 insulin/22.5). results: in the 1st and the 2nd groups there is a significant hyperinsulinemia and to a lesser extent in the 3rd group (72%, 66% and 57%, respectively). the homa model confirms an acute insulin resistance (79%, 75% and 65% increase respectively). hypertriglyceridemia was observed only in the 2nd group. hdl dyslipidaemia has been identified in women of the 2nd group (<0.39 g/l). our study seems to confirm that type 2 diabetes genetic predisposition is not the only factor, but also environmental factors with or without diabetes family. aims: to examine, the predictors of incident chronic kidney disease (ckd) in a community-based cohort of middle east population, during a mean follow-up of 9.9 years. methods: in a sample of 3313 non-ckd iranian adults ! 20 years the estimated glomerular filtration rate (egfr) was calculated at baseline and at 3 year intervals during three consecutive phases. the egfr <60 ml/min/1.73 m 2 was defined as ckd. multivariate logistic regression analysis was used to determine the independent variables associated with incident ckd. results: the incidence density rates of ckd were 285.3 and 132.6 per 10,000 person-year, among women and men, respectively. female gender per se was associated with higher risk of ckd, compared with males. among women, age, egfr, known diabetes, being single or divorced/widowed, hypertension (marginally significant) and current smoking were independent risk factors for ckd; however the intermediate degree of education and family history of diabetes decreased the risk by 40% (p < 0.05). among male subjects, independent predictors of developing ckd included aging and hypertension (with significantly higher risk than in women, p for interaction <0.05), egfr, new diagnosed diabetes, high normal blood pressure; abdominal obesity decreased the risk of ckd about 30% which was marginally significant. in the iranian population, >2% of individuals develops ckd each year. our findings confirmed that sex-specific risk predictors should be considered in primary prevention for incident ckd. introduction: obesity is a silent killer and a forerunner of many complications if persists long. various studies with animal model have identified the role of leptin, the hormone of adipose tissue; in obesity and its associated complications like diabetes and atherosclerosis inlater stages. the exact mechanism to know how leptin influences insulin action in body and thereby leading to diabetes or post diabetic atherosclerosis is still not completely evaluated. hypercholesterolemia was only found common to all these three states. aim and objectives: the present study, therefore, evaluated the role of obesity on the expression of ldlr receptor, insulin receptor and leptin receptor. method: receptor expression was done by immunohistochemistry/ western blot. the serum level of lipids were measured by enzyme based kit method. the serum level of insulin and leptin and its soluble receptor were measured by elisa based kit. the blot for insulin expression shows no chamge with body weight; the blot for leptin receptor shows decrease expression with weight gain and blot for ldlr shows decrease expression with weight gain. the serum levels of insulin and leptin are increased with weight gain but soluble receptor for leptin did not change significantly. even the obese group showed decrease tyrosine phosphorylation of insulin receptor. background: visfatin and apelin are two new adipokines that recently gained special interest in diabetes research. however, the relationship between them has not been elucidated and their role in coronary artery disease (cad) complication of diabetes has not been adequately studied. objective: this study was conducted to study the interplay between these two novel adipokines and to study their correlation with other inflammatory and biochemical parameters in type 2 diabetic (t2d) postmenopausal women with cad. the levels of visfatin, apelin and other parameters were measured in t2d patients without cad, both nonobese and obese t2d patients with cad, together with healthy nondiabetic control subjects. visfatin and apelin were measured by enzyme-linked immunoassay (elisa). results: visfatin was found to be significantly higher in the following groups: t2d patients without cad, non-obese and obese t2d patients with cad, (26.10 ae 0.83, 24.53 ae 0.76 and 31.58 ae 1.04 ng/ml respectively) when compared to control group (12.53 ae 1.82 ng/ml) at p < 0.05. apelin was found to be significantly lower in both non-obese and obese t2d patients with cad (27.93 ae 1.07 and 1.36 ae 0.70 ng/ ml respectively) when compared to control group (34.52 ae 0.14 ng/ml) at p < 0.05. furthermore, visfatin and apelin were found to be significantly associated with each other and with other biochemical parameters in both simple and multiple regression analyses. the current study provides evidence for the novel interplay between visfatin and apelin through the inflammatory milieu characteristic of t2d and sheds light on their possible role in the pathogenesis of cad complication of t2d. aim: the aim of this study was to investigate the relationship between hscrp, il-6, and hcy levels and cardio metabolic risk factors in subjects with and without mets in a sample of tehranian population. in this cross-sectional study, 365 individuals including 134 men and 231 women, aged ! 19 years were selected randomly from among participants of tlgs. the sera of il-6, hscrp and hcy were determined using elisa method. results: of the total 365 subjects, aged mean 46.1 ae 16.1 year, mets was presented in 160 (43.8%) individuals. the levels of hscrp, hcy, and il-6 were higher in subjects with mets compared to normal group. a gradual significant increase just in the level of hscrp with increasing number of mets components was found after adjustment for sex and age. a strong linear augmentation in hscrp levels was observed as the number of mets components increase from 0 to ! 3 with median hscrp levels of 6.27, 6.59, and 7.51 (ng/ml) (p trend = 23 9 10 à5 ). also, an increase of 0.40 in hscrp levels (ci 95%: 0.17-0.63; p = 69 9 10 à5 ) was observed with an increase in each components of mets in linear regression analysis adjusted for age and sex. the best predictor for hscrp, il-6, and hcy, in subjects with mets was hip, whtr, and height, respectively, compared with whtr and wrist which were the best predictors for hscrp and hcy levels in the subjects without mets. conclusion: hip and whtr are significant predictors of the hscrp and il-6 elevation associated to mets, respectively. methods: in a nested case-control study, conducted on a group selected from participants of the tehran lipid and glucose study, those cases with new diagnosed type 2 diabetes during the 3.6 year follow-up (191 subjects) were matched to 570 controls for age, sex, body mass index andseason of entrance to study. multivariate conditional logistic regression analysis was used tocalculate the odds ratio (or) with 95% confidence interval of type 2 diabetes for vitamin dcategories, vs. the first quartile ( 11.02 ng/ml) as reference. results: the unadjusted ors of type 2 diabetes were 0.73 (0.74-1.13), 0.54 (0.34-0.85) for the second and third tertiles, respectively. after adjustment for family history of diabetes, systolicblood pressure, triglycerides to high density lipoprotein cholesterol ratio, and fasting plasmaglucose, the corresponding ors were 0.62 (0.33-1.17) and 0.43 (0.22-0.84) for second and third tertiles respectively. multivariate cubic spline modeling analysis indicated that 10 ng/ml 25hydroxyvitamin d levels was the optimal cut point for distinguishing those who were at risk ofdeveloping diabetes and those who were not. also addition of vitamin d to multivariate model, improved the net reclassification by cut-point based nri of 12%. discussion: in a prospective cohort study, we demonstrated a nonlinear independent associationof vitamin d with incident diabetes, with prominent increase in risk at a cut-off <10 ng/ml. research department-ayush projects, svyasa university, bangalore, india type 2 diabetes mellitus (t2dm), the seventh leading cause of death, is a biggest challenge for mankind. despite of fascinating advances in pharmaco-therapeutic agents, the prevalence of t2dm is growing every year. complementary and alternative medicine (cam) as classified by national centre of cam have documented various positive results of t2dm. whole ancient medical systems like ayurveda, siddha, and tcm etc use natural herbs either as a single drug therapy or in the form of formulations, depending upon principles of respective medical system. studies done on various herbs and mind body intervention like, yoga, acupuncture etc are found to be beneficial for t2dm. this resulted, large number of physicians either referring to or practicing some of the more prominent and well known forms of cam. the concepts proposed and used by these cam systems (that are kept alive by cam practitioners for thousands of years) appear very mysterious to the present day biomedical practitioners. these models of therapies seem to have evolved through several phases of internal research just like the present day drug trials that go through four phases of intensive statistical evaluation. it appears that today's scientists need to follow the footsteps of research used by the ancient seers of tcm, ayurveda, siddha, yoga or homeopathy to unravel these mysterious theories. this review talks about generating evidence not only for the efficacy of these holistic systems but also in carrying out systematic research by biomedical scientist who have the knowledge of both the western and eastern sciences. background: the epidemic of obesity, over the last two decades, in the middle and high income countries is associated with marked rise in the incidence of metabolic syndrome. objective: to measure the prevalence of metabolic syndrome (ms) and determine its association with ratio of omega-6/omega-3 fatty acids in the diet. design and methods: cross-sectional surveys were conducted in 20 urban streets in the city of moradabad, india. randomly selected subjects with ms aged 25 years and above were evaluated and graded according to omega-6/omega-3 ratio in the diet. physical examination, sphygmomanometer, questionnaire and blood tests were done. results: the overall prevalence of ms was 19.3% (n = 387) without any gender difference. the prevalence of ms, type 2 diabetes, cad and hypertension showed a higher rate, in relation to omega-6/omega-3 ratio in the diet. subgroup analysis showed that subjects eating low omega-6/omega-3 ratio (<5.0) diets had significantly lower prevalence of ms, and related components compared to higher ratio diets, among both sexes. multivariate logistic regression analysis after adjustment of age showed, that hypertriglyceridemia (odds ratio 0.90 in men, 0.76 in women) was strongly (p < 0.01) associated with ms. hypertension, hdl-c, and central obesity were weakly associated with ms in both sexes. hypercholesterolemia was weakly associated with ms only in women. conclusion: ms has become a public health problem in india. higher w-6/w-3 ratio is a major risk factor of ms and cad. it is possible that a low w-6/w-3 ratio in the diet (<5.0) may be protective against ms. objective: investigate the differences in clinical and laboratory features (including some adipocytokines) among hyperglycemic patients with or without cad. methods: five hundred and sixty-one consecutive hyperglycemic subjects undergoing coronary angiography. hyperglycemia [diabetes mellitus (dm)/pre-diabetes (pd)] previously known dm or diagnosed at the moment of recruitment by an hba1c (hplc) levels-! 5.7-6.4%-pd, ! 6.5%-dm (ada criteria). cad defined by any lesion ! 50% on angiography. results: 55.4% were men. age was 60.3 ae 10.5 (mean ae sd), cad was detected in 64.3% of individuals. patients with cad were older (61 ae 10 vs. 58 ae 11 years, p = 0.002), more often male (61% vs. 46%, p = 0.001). additionally, they showed a worse metabolic profile, with higher hba1c (7.1 ae 1.8% vs. 6.8 ae 1.4%, p = 0.01), fasting plasma glucose (135 ae 60 vs. 121 ae 41 mg/dl, p = 0.002), triglyceride [135 (101-191 mg/dl) vs. 122 (85-166 mg/dl), (p = 0.004)] and plasma visfatin/nampt levels [(5.1 (3.6-9 .8 ng/ml) vs. 4.9 (3.1-8.7 ng/ml), (p = 0.014), median/interquartile range], and lower hdl-cholesterol levels (39 ae 12 vs. 42 ae 11 mg/dl, p < 0.001), even after correction for statin use (except for hdl and visfatin/nampt). diabetic patients with cad had greater diabetes duration than diabetic individuals without cad: 6.0 (0.7-10) vs. 3.0 (2-10) years, median and interquartile range, (p = 0.038). systolic and diastolic blood pressure, homa%-s, homa-ir, total and high-molecular weight adiponectin levels were similar in both groups. conclusions: patients with hyperglycemia and cad have a worse glycemic and lipid profile and higher visfatin/nampt levels as compared to those without cad. one hundred and sixty-nine patients (p) with the mets were studied to find out the incidence of cardiovascular events, especially myocardial infarction and strokes. the mean age was 60 years. fifty-five percent were males and 45 females. the mean body mass index was 30 kg/m 2 . the mean fasting blood sugar was 165 mg/dl. ninety-seven percent were diabetic type ii and 3% diabetic type i and the lipid profile was normal. the studied population showed a higher incidence of atrial fibrillation, when compared with a comparison group of diabetics (12% vs. 5.9%) p < 0.001. no ventricular tachycardia was observed. the mets group showed a subnormal ejection fraction (49 ae 4%) when compared with a comparison group (62 ae 12%) p < 0.001. no myocardial infarctions or strokes were detected. coronary angiography was done in 60% of the (p) all were negative except one. the lower ejection fraction was explained on basis of diabetic cardiomyopathy. the genetics profile of the puertorrican, a hispanic population, is more of an european, indian and african. probably, this produces a culture less sensitive to the atherosclerotic factors, producing a less aggressive mets syndromeless myocardial infarcts or strokes. this suggest that genetics and culture is an important aspect of the expression of mets, especially in the hispanic world. objectives: controversy surrounds the annual progression from prediabetes (impaired glucose regulation) to type 2 diabetes. current uk statistics suggest a 10% progression rate. our aim was to assess subjects in our locality at high-risk of progression from pre-diabetes to type 2 diabetes within a 12 month period. this will establish whether current recommendations for follow-up of high-risk subjects successfully identify those with pre-diabetes. methods: analysis was performed on data from 127 high-risk subjects who underwent ogtt with baseline clinical and biochemical measurements. the ogtt and biochemical measures were repeated at 12 months. results: of the 127 subjects, 24 (18.9%) developed diabetes within the 12 month period, significantly higher than the estimated annual progression rate (p 0.001). as expected, baseline fasting plasma glucose was higher for subjects that progressed to diabetes (diabetes vs. no diabetes: 6.4 ae 0.5 vs. 5.7 ae 0.2 mmol/l; p 0.001), as was 2 h post-prandial plasma glucose (diabetes vs. no diabetes: 10.4 ae 2.8 vs. 6.7 ae 1.8 mmol/l; p 0.001). additionally, baseline hba1c was higher for those that developed diabetes [diabetes vs. no diabetes: 6.3 (6.0-6.6) vs. 6.0 (5.7-6.3) %; p = 0.017]. interestingly, both sbp and dbp were significantly higher at baseline in those that developed diabetes (sbp: 150 ae 22.0 vs. 140 ae 17.6 mm hg; p = 0.019) (dbp: 87 ae 10.1 vs. 80 ae 11.3 mm hg; p = 0.006). conclusions: within this cohort, progression to type 2 diabetes was almost twice the current estimated annual rate. fasting, 2 h-glucose, hba1c and blood pressure were associated with progression from prediabetes to type 2 diabetes over 12 months. methods: tissue lysates from visceral fat samples of subjects undergoing abdominal surgery (predominantly bariatric and routine non-acute for non-malignant conditions) were collected from 51 subjects categorised as l (19), o (14) or odm (18). a commercially available comet assay (cell biolabs, inc) was used to assess cellular dna damage using a single cell gel electrophoresis method. additionally, results indicate a greater percentage of dna migration from the comet "head" to its "tail" within odm samples (l vs. o vs. odm: 69.6 vs. 67 .0 vs. 71.9%; p = 0.427). in conclusion, results suggest that visceral fat from obese subjects with type 2 diabetes are subject to higher levels of oxidative burden resulting in a higher proportion of damaged dna. community medicine, faculty of medicine, kuwait university, jabriya, kuwait background: the prevalence of overweight and obesity are high in kuwait. metabolic syndrome is associated with both. it is expected to find the syndrome to higher than in other countries. objective: to assess the prevalence of ms using two different diagnostic criteria, the international diabetes federation (idf) and the national cholesterol education program-third adult treatment panel modified for age (ncep-atp iii). study design: a multi-stage random sample study. methods: the analysis of data for this study was based on a sample of 303 male kuwaiti adolescents, 10-19 years of age selected from intermediate and high schools. anthropometric measurements and biochemical tests on blood samples were carried out. the idf criterion requires waist circumference (wc) plus two of the following criteria: triglycerides (tg), high density lipoprotein (hdl), fasting blood sugar (fbs) and blood pressure (pb). the atp iii criterion requires three of the above parameters. the parameters mentioned must show increase in their values except for hdl which must show decrease in either criterion used. results: each of the two criterion revealed that the prevalence of ms was 14.8% and 19.5%, using the idf and the atp iii criteria, respectively. hdl decreased in each of the two diagnostic criteria and the other four parameters increased, satisfying the diagnostic requirements of either criterion. conclusions: significant implications may be drawn from these results, especially when it comes to being at risk of type 2 diabetes (t2d) and cardiovascular disease (cvd). m. saghebjoo 1 , j. shabanpour omali 1 , r. fathi 2 1 university of birjand, birjand, 2 mazandaran university, babolsar, iran background and aims: resistance training has been shown to be beneficial in older adults. however, very little data exist on the effects of resistance training in older diabetics. chemerin is a adipokine that has been shown to induce insulin resistance in skeletal muscles. here we investigate the role of 8 weeks of progressive resistance training (prt) on plasma chemerin levels in older men. methods: a total of 18 sedentary men with type 2 diabetes, aged between 45 and 60 years, were randomized to the 8 weeks supervised prt (n = 10) and control (n = 8) groups. chemerin, insulin, glycosylated hemoglobin (hba1c), and fasting blood glucose (fbg) were measured before and 72 h after the training period. results: plasma chemerin levels decreased significantly (p = 0.01) in the , and exhibited significant reductions in plasma insulin (p = 0.01) and fbg (p = 0.05). fbg decreased by 28.08% in prt group. conclusion: reduced plasma chemerin concentration may contribute to improved insulin sensitivity. these results recognize that increased intensity of exercise may produce greater benefits, but may not be appropriate for some individuals. some studies have demonstrated the presence of metabolic syndrome in children and adolescents, but few have investigated this syndrome in brazilian children. the aim of this study was to investigate the prevalence of metabolic syndrome in children and its association with gender and nutritional status. this transversal epidemiological study involved 385 children of both genders between 7.0 and 9.9 years old. there were collected sociodemographic, anthropometric, metabolic and hemodynamic data. the nutritional status was obtained according to the body mass index. to diagnose metabolic syndrome was used the criteria proposed by the national cholesterol education program′ s adult treatment panel iii adjusted for age. there were used two separate proposals (cook et al., 2003; ferranti et al., 2004) . the prevalence of metabolic syndrome ranged from 2.9% to 10.6%, depending on the criterion. the nutritional status showed 72.0%, 18.4% and 9.6% of children with normal weight, overweight and obesity, respectively. significant association was observed between the metabolic syndrome and nutritional status, but not in relation to gender. metabolic syndrome was present in different nutritional status of children, especially those with excess body weight, thus indicating the importance of early diagnosis and the adoption of primary prevention measures already in pediatric patients. introduction: ifg (fpg 5.6-6.9 mmol/l) is a risk-factor for developing type 2 diabetes and cardiovascular disease. we quantified changes in ifg prevalence (post-hoc analysis) from a double-blind, placebo-controlled trial investigating the effects of liraglutide 3.0 mg on maintenance of diet-induced weight loss (primary endpoint). methods: overweight/obese adults ( ! 18 years, bmi ! 30 or ! 27 kg/m 2 with comorbidities) who lost ! 5% weight after 4-12 week run-in with low-calorie diet (1200-1400 kcal/day) and exercise were randomised to once-daily subcutaneous liraglutide (n = 212) or placebo (n = 210), plus 500 kcal/day deficit diet and exercise. the full-analysis-set comprised 413 of 422 randomised individuals [age 46.2 ae 11.5 years, bmi 37.9 ae 6.2 kg/m 2 (mean ae sd)]. during run-in, participants lost 6.3 ae 1.6 kg weight and ifg prevalence decreased from 54% (224/413) to 40% (164/413). at week 56, liraglutide-treated participants lost an additional 5.7 kg from randomisation (table 1) , whereas placebo-treated participants lost no additional weight [treatment-difference à5.9 kg (95%ci à7.3; à4.4); p < 0.0001]. moreover, the proportion of participants with ifg at week 56 was lower for liraglutide (6.3%) than placebo (28.7%; odds-ratio 6.0 [3.1; 11.6]; p < 0.0001). at week 68, after 12 weeks off treatment, the liraglutide group regained~2 kg lost weight and ifg prevalence increased. mean weight loss remained greater for liraglutide vs. placebo [treatment-difference à4.0 kg (à5.8; à2.2); p < 0.0001] but ifg prevalence did not differ (odds-ratio 1.3 [0.78; 2.3]; p = 0.30). conclusion: liraglutide decreases ifg prevalence in overweight/obese individuals who have already lost weight by diet and exercise, potentially due to additional weight loss and weight-loss independent mechanisms. objective: we wanted to explore the association between polymorphisms of irs1 (rs1801278), tcf7l2 (rs7903146 and rs12255372), adrb1 (rs1801253), pparg (rs1801282), and hhex (rs5015480) genes with insulin resistance, lipid profile and atherogenic risk in mets patients from the mexican social security institute. methodology and results: four hundred and thirty-one mets patients and 547 controls were selected. the association between the snps and the atherogenic index was evaluated by multiple linear regression and multinomial logistic regression models. adrb1 c/g were associated with an increase in ldl-c levels (β = 6.56, 95% ci = 1.35, 11.77 p = 0.014), the hhex t/c variant were statistically associated with an increase in total cholesterol levels (β = 6.58, 95% ci = 0.85, 12.32 p = 0.024), and with an increase in ldl-c levels (β = 5.88, 95% ci = 0.88, 10.88 p = 0.021). the adrb1 gene showed a statistically significant association with high-risk atherogenic index, (or = 2.58, ic 95% 1.29-5.16; p = 0.007) for the arg/gly variant and for the dominant model (or = 2.66, ic 95% 1.35-5.27; p = 0.005). conclusions: the arg389gly polymorphism of the adrb1 gene may be a good biological marker to predict the risk of developing cardiovascular diseases given a high-risk atherogenic index. aims: describe the knowledge of major risk factors for type 2 diabetes held by primary health care (phc) nurses involved in the community management of diabetes. methods: random sample (26%) of phc nurses in auckland. information was collected from postal and telephone questionnaires, on education, experience, knowledge and diabetes management practice. results: responses were received from 287 phc nurses (86% response) comprising 210 practice nurses (pns), 49 district or homecare nurses (dns) and 28 specialist nurses (sns). most nurses (96%) were able to identify excess body weight as a major risk factor for type 2 diabetes. only 54% of sns and a third of pns and dns identified lack of physical activity, and fewer than 30% of sns and pns, and only 16% of dns identified hypertension as risk factors. even fewer respondents were able to identify individual lipidsalthough significantly more sns identified elevated triglycerides (14%, p = 0.02) and reduced high-density-lipoprotein cholesterol (11%, p = 0.003) as major risk factors compared with only 1-5% of pns and no dns. risk factors for diabetes-related complicationsapart from hyperglycaemia identified by 86%were not well identified, particularly smoking (17%), hypertension (31%), triglycerides (9%) and reduced high-density-lipoprotein cholesterol (8%). in general, phc nurses had a good knowledge of overweight as a risk factor for type 2 diabetes and hyperglycaemia as a risk factor for diabetes-related complications, but poor knowledge of cardiovascular risk factors, particularly smoking. yoga and life sciences, swami vivekanand yoga anosandhana samsthana (s-vyasa university), bangalore, india metabolic syndrome is clustering of different metabolic abnormalities which encompass central obesity, insulin resistance and hypertension. it is characterized by an overdrive of the sympathetic nervous system, increased oxidative stress, elevated pro-inflammatory enzymes, and impaired circadian cycle. lack of physical exercise, improper eating habits, and psychological stress are also common contributing factors to this condition. thus, life style change which includes adequate exercise, proper diet and stress management are the keys in prevention control and treatment of metabolic syndrome. yoga, one of the ancient sciences on earth, is known to bring balance at all aspects of human existence. its different components like asana (physical postures), pranayama (breathing techniques), meditation and relaxation techniques (yoga nidra) etc have been proven to influence body and the mind towards balance and equanimity. recent scientific studies on yoga have proven its safety and efficacy in the management of many metabolic disorders which includes diabetes mellitus, obesity, hypertension and atherosclerosis. yoga therapy has also caused reduction in the pro-inflammatory cytokines in such conditions. yoga reduces sympathetic tone as well as oxidative stress; it helps in reducing insulin resistance by enhancing secretion of melatonin and hepatic insulin sensitizing substance through bringing the parasympathetic dominancy. thus, we propose that yoga has an important role in prevention and treatment of metabolic syndrome as a complementary or as an alternative to conventional line of treatment. s. debnath 1 , s. addya 2 1 medical laboratory technology, women's polytechnic, 2 medical officer, government of tripura health services, agartala, india introduction: saliva is an excellent biological matrix that offers several opportunities for scientific inference of diagnostic, toxicological and in forensic importance. numerous salivary metabolites proffer great potential in clinical and epidemiological research. sample collection is non invasive and analysis require simple modifications. establishing good correlates is the need. objective: our objective was to study the association between anthropometric measures (ams) with salivary metabolites in a subsample (n = 8) of college women in suburban north east india. method: saliva and blood samples were photometrically analyzed for glucose, protein and urea. ams as bmi and waist circumference (wc) were measured in female students of menstruating age (17-21 years) following who guidelines. ams were correlated (pearson's correlation, ′r′) to salivary biochemistry. results are reported with intra assay coefficient of variation (cv < 10% all assays) in the text. ′ four day diet dairy′ was maintained over the period. results: salivary biochemistry found to be significantly correlated to ams in the studied subsample. salivary protein was negatively correlated (′r′ = à0.710) with bmi and with wc (′r′ = à0.604) whereas salivary glucose (′r′ = 0.569 with bmi and ′r′ = 0.056 with wc) and urea (′r′ = 0.612 with bmi and ′r′ = 0.676 with wc) had positive correlation. all assays performed in triplicates .we observed very good repeatability of results. medicine -cardiology, massachusetts general hospital, boston, ma, usa introduction: obesity is an independent risk factor for cardiovascular disease. supine bike stress echocardiography is suggested to be of particular utility for evaluating for coronary artery disease (cad) in obese individuals given the lack of mechanical impact with this modality. database for all recumbent bicycle stress echocardiography examinations performed between january 1, 2006 and july 31, 2009 . all tests performed to evaluate for cad were reviewed. two groups were formedpatients achieving ! 85% of maximum predicted heart rate (mphr) and those achieving <85% mphr. medical records of these patients were then reviewed. comparisons between groups were made using unpaired t-tests and correlations between patient characteristics and hr were assessed using pearson's correlation. results: four hundred and sixty tests were done to evaluate for cad. one hundred and eighty-one (39.3%) patients failed to reach 85% mphr during testing. forty-three percent of patients failing to achieve 85% mphr were obese (bmi >30) vs. 36% of all patients referred for stress testing. image quality among obese patients was not notably worse compared to leaner patients. those failing to achieve 85% mphr had a significantly higher bmi (30.2 vs. 28.7; p = 0.044) than those with adequate hr response. bmi exhibited a negative correlation with percent mphr (r = à0.14, p = 0.006). conclusion: obesity is associated with failure to achieve ! 85% mphr during supine bike stress echocardiography done to evaluate for cad. use of adjunctive pharmacologic agents should be considered when evaluating an obese patient for cad with stress echocardiography. (8):704-12), though the extent to which body weight may act as a confounder or as mediator in this relationship is uncertain. the aim of this study was to analyze whether the association between crf and mets risk is mediated by body mass index (bmi). methods: cross-sectional study including 1158 schoolchildren, 8-11 years old from the province of cuenca, spain. we measured height, weight and crf (20-m shuttle run test). a validated mets index (diabetes care 2010;33:1370-2) was estimated by summing standardized z scores of waist circumference, triglyceride-to-hdl-c ratio (tg/hdl-c), mean arterial pressure (map), and fasting insulin. to test whether the association between crf and mets index and its components was mediated by bmi, linear regressions models were estimated according to baron and kenny procedures for mediation analysis (j pers soc psychol. 1986; 51:1173-82) . results: in girls, bmi acts as a fully mediator for the relationship between crf with mets index and all its components, except for tg/ hdl-c ratio. in boys, bmi acts as a fully mediator for the relationship of crf with tg/hdl-c ratio and map; and partial mediator for the relationship between crf with mets index and the rest of components. the obesity mediates the association between crf and mets in schoolchildren. good levels of crf are associated with lower mets risk, but only when accompanied by weight reduction. pre intervention measurement was applied. no statistical differences were found for prediabetes knowledge, physical activity, eating habits, bmi, weight, waist circumference and clinical parameters (glucose, triglycerides and total cholesterol) between both groups in basal measurement. the control group received the usual care from health centers. in the study group, the intervention was developed during 6 months per patient (received usual care plus remote care model′s components). the rct will end at january 2013, and the final results will be presented. chronic conditions. this study compares the prevalence of the metabolic syndrome (mets) between genders in the workplace. methods: as part of the established "prosiect sir gâr" initiative in south wales, uk, 459 female and 313 male employees from either the local steel works or local health board were screened and their data analysed. anthropometric data, blood pressure, self-reported physical activity (gppaq) and smoking status were all recorded. in addition, blood samples were obtained and analysed for high-densitylipoprotein cholesterol (hdl-c). presence of the mets was determined based on the following idf criteria: central obesity (females waist circumference: ! 80 cm; males waist circumference: ! 94 cm), reduced hdl-c levels (females: <1.29 mmol/l; males: <1.03 mmol/l) and either systolic ( ! 130 mmhg) or diastolic ( ! 85 mmhg) hypertension. results: a higher proportion of males were diagnosed with mets than females (25.9% vs. 18.7%; p < 0.001) despite the males being more physically "active" or "moderately active" (85.3% vs. 60.8%; p < 0.001). rates of central obesity and systolic hypertension were comparable between genders (p > 0.05), however prevalence of diastolic hypertension, reduced hdl-c levels, current smokers and individuals either overweight or obese were higher in the male cohort (p < 0.05). conclusion: despite being more physically active, males were more susceptible to the mets than females, likely due to a higher percentage of smokers and either overweight or obese. conclusions: the addition of glp1 analogues to insulin therapy seemed to be superior to the enhancement of insulin therapy regarding to weight loss, decreases of hba1c, hypoglycemic episodes and requirements of insulin. it was also observed a significant improvement in insulin sensitivity and in beta cell function. background and aims: type 2 diabetes is often associated with nonalcoholic fatty liver disease (nafld). patients with nafld may be at greater risk for cvd than those without. the relationship between nafld and metabolic syndrome (ms) is very well recognized. the aim: to determine the association of nafld and cvd between type 2 diabetic patients with and without ms. materials and methods: one hundred and thirty type 2 diabetic patients (m: 71, f:59, mean age 59.32 + 11.94), were studied. all subjects were assessed for diabetes duration, the obesity degree, cv risk factors, hba1c, c reactive protein and lipid profile. nafld was assessed by patient history and ultrasound. ms was defined based on ncep-atp 3 criteria. the previous and current cvd (myocardial infarction, angina or revascularization) was assessed. we categorized four groups: ms (à) and nafld(+), ms(à) and nafld(à), ms(+) and nafld(+), ms(+) and nafld(à). results: the prevalence of cvd in type 2 diabetic patients with nafld was higher than in those without nafld (44.2% vs. 38.1%). the prevalence of chd in type 2 diabetic patients with ms was higher than in those without (40.1% vs. 35.8%). the risk of chd in patients with ms was significantly increased by the presence of nafld (53.4% vs. 32.3%). in type 2 diabetic patients with ms(+) and nafld(+) the number of components of ms, bmi, and systolic bp were positively associated with chd. conclusions: what this study suggests to us is that the presence of nafld increases the risk of cvd in type 2 diabetic patients with ms. 2007 and to determine associations between the levels of fasting blood sugar and its comorbid conditions. this is a retrospective study and uses chart review in the outpatient section of the family medicine clinic. it also uses percentages, means and its standard deviations, and chi square to detect associations. there were 302 patients with impaired fasting glucose aged between 18 and 84 years old (mean age 55.94 ae 13) and all were of filipino ethnicity. there were more females than males. the mean body mass index was 23.87 ae 4.44 kg/ m 2 and the mean fasting blood sugar (fbs) was 111.6 ae 7.23 mg/dl. there was no association between the fbs level and obesity (p > 0.5) and hypertension (p > 0.2) in this study. however, fbs >113 mg/dl was associated with the presence of family history of diabetes in this population (p < 0.01). it seemed that ifg can occur to young, nonobese patients with family history of diabetes. this study recommends that screening for ifg among filipinos should be done to all patients with a strong family history of diabetes regardless of age, gender, bmi status or presence of hypertension. h. hasan, v.l. raigangar, a.r. abdullah methods: a cross-sectional study of 80 young females (mean age 21.0 ae 2.4 years. leptin, insulin, high sensitivity c-reactive protein (hs-crp), c-peptide, ua, bg, hdl-cholesterol and triglycerides were estimated from a fasting blood sample. anthropometric parameters (wc, height and weight) and bp were measured. homa-insulin resistance was also calculated. results: mean wc of the studied population was 76.6 ae 11.7 cm, 30% (n = 24) had wc >80 cm. for the 80 subjects, all studied parameters were within normal limits except leptin which was high; 30.1 ae 15.4 ng/ml. ua showed highly significant positive correlation with wc (r = 0.55, p < 0.001), significant positive correlations with cpeptide, hs-crp and leptin with (r = 0.26, p = 0.02), (r = 0.032, p = 0.003) and (r = 0.027, p = 0.017) respectively. ua demonstrated significant negative correlation with hdl only (r = à0.24, p = 0.034). multiple linear regression revealed that wc was the only significant predictor of ua levels (b = 0.04, p = 0.00, ci 95%: 0.01-0.06). conclusion: this study stresses the importance of ua levels in cvd particularly due to its strong association with wc, the main indicator of abdominal obesity in mets that is often missed. ua may hence be considered a valuable biomarker for early prediction/detection of mets in young females. background: adipocyte-secreted cytokines are associated with inflammation and metabolic disturbances but it is unclear how their snps interfere on the response to lifestyle interventions. we assessed associations of selected snps with the changes induced by interventions. methods: this 9-month intervention on diet and physical activity included 134 brazilians at cardiometabolic risk (prediabetes or metabolic syndrome without diabetes). changes in clinical variables were analyzed according to the presence of the tnfa-308g/a, il-6-174g/c and adipoq45t/g snps; individuals with at least one variant allele were grouped and compared with the reference genotype. afterwards, individuals carrying simultaneously the 3 genotypes associated with no glycemic response were grouped and compared to the remainder sample. results: the entire sample (56.5 ae 11.6 years) had lower energy intake, higher physical activity, and improved anthropometric and metabolic variables after intervention. carriers of the tnfa variant allele but not the reference group decreased plasma glucose. il-6 and adipoq variant allele carriers had worse glucose, lipid and inflammatory responses. grouping the subset of 49 carriers of tnfa-308g + il6-174c + adipoq45g, they showed a significant increase in mean fasting glucose after intervention. this increment differ significantly from the behavior of the remainder sample (+2.8% vs. à3.8%, p = 0.025). the tnfa-308g/a but not the adipoq45t/g and il-6-174g/c may predispose a better response of glucose metabolism to a lifestyle intervention. the combination of three worst genotypes can maximize the adverse effect on glucose metabolism in at-risk brazilians. further studies are needed to direct lifestyle interventions to specific subgroups of individuals. obesity is a metabolic disorder, which is associated with an increased risk of various conditions, including sexual dysfunction. objective: to investigate anthropometric indicators of body fat in postmenopausal women influencing their quality of life. material and methods: the study was performed at the scientific and clinical study of endocrinology, uzbekistan public health ministry. a total of 230 women (mean age of 44 years) was examined. bmi, waist circumference and waist to hip ratio were obtained for anthropometric evaluation. lipid specters, glycemia and insulin were the parameters to evaluate. we have used a menopause quality of life questionnaire and female sexual function index questionnaire (fsfi). the quality of life of the obese and overweight patients was compared by age, education, marriage and matched with healthy normal weight controls. results: overweight and obesity were observed in 120 of women. values of wc >81 cm were above normal levels in 55%, 47.8%, respectively, and hdl was normal in 85.2%. homa -ir was measured in 31.3% of the women. women with overweight and obesity had worse general health-related quality of menopausal symptoms, life and psychological and sexuality scores than athletic and lean women (bmi ! 25). in fsfi it was significant differences in such as scales as desire and orgasmic disorders in women with metabolic syndrome (ms) in comparison with the controls (p < 0.05). aim: to determinate groups of high cardiovascular risk and start early hypolipidemic therapy due to genetic polymorphism of lipoproteidlipaseone of the main enzymes of lipid metabolism in metabolic syndrome and nafld patients, treating with combined therapy of statins and ursodeoxycholic acid (udca). background: health staff receive more health messages and information in working hours than others. moreover, they are supposed to be healthy models for the community. we conducted a screening of metabolic syndrome on shahid beheshti university health department staff (who are responsible for health affairs of more than 10 million people in tehran province, iran) to assess their health level. we invited all of the health staff for screening on "the world heart day". weight, height, waist circumference, systolic and diastolic blood pressure were measured and a fasting blood sample was taken for lipid and glucose level testing. results: fifty percent of the staff (85 person) participated in the screening. the mean age was 41.3 ae 7.3 years. only 36% were in normal weight range. overweight, obesity class 1 and class 2 rates were 36%, 25.8% and 2.2%, respectively. 37.1% of all and 6.3% of normal bmi cases had a high waist circumference. metabolic syndrome was identified in 8.3% of men and 10.2% of women. high blood pressure was detected in 4.5% of the cases. in 3.4% of all and 3.5% of overweight or obese cases fasting blood glucose was elevated. these rates for blood total cholesterol level were 50.6% and 60.4%, for triglyceride were 34.1% and 37.7%, and for ldl were 75.3% and 84.9%, respectively. conclusions: contrary to our expectations, a high rate of metabolic syndrome was identified in this group. it seems that interventional programs targeting nutritional habits and physical activity of the staff are needed along with routine educational programs. introduction: physiologically, brain natriuretic peptides (bnp) and lipolysis are closely linked. obesity is been identified as a major risk factor for the development of cardiovascular diseases (cvd) and has been reported to have an impact on bnp in apparently healthy subjects but also in cvd patients. thus, we speculate that bnp could play an important role in lipid metabolism and may affect the pathophysiology of obesity in cvd patients. methods: serum samples were obtained from cvd elderly patients distributed in two groups: i-non-obese and ii-obese. the plasma mature form of brain natriuretic peptide (nt-probnp) was measured by a sandwich enzyme immunoassay with spectrophotometric detection at 450 nm. conclusion: overall, these data demonstrates that obesity is an important and independent determinant of bnp expression in patients with cvd. inverse relationship between bnp and body mass index may suggest "beneficial" effects of obesity, but clearly lower levels did not confer a more favourable prognosis. the precise mechanisms linking obesity to cvd remain unsolved and may be due either to release attenuation or increases in clearance receptors. these effects should be taken into account for appropriate bnp reference values, so lower cut-points should be used for obese patients and a higher cutpoint for lean patients to increase specificity. v. mladenovic 1 , a. djukic 1 , s. djukic 1 , n. arsenijevic 2 , s. zivancevic simonovic 2 aim: the aim of this study was to investigate dynamic of changes of oxidative stress during acute myocardial infarction depending on development phases of metabolic syndrome x. method: the research included 29 patients; inclusion criteria were diagnosed metabolic syndrome and acute myocardial infarction. according to the movements of glycemia and insulinemia all patients were divided in four development phases of metabolic syndrome x. to evaluate oxidative status we determinated: lipid peroxids (malonyldialdehide), total antioxidative status, as well as oxidative stress coefficient. results: during hospitalisation in patients with acute myocardial infarction concentration of lipid peroxids increased, with maximum in the day 7th of hospitalisation, mostly in hyperinsulinemic phases of metabolic syndrome. at the 1st day of myocardial infarction total antioxidant status increased, and decreased during next 7 days. these changes are independent on the phase of metabolic sindrome x. as result of inverse dynamic changes of these parametars, during period of exam came to progressive increase of oxidative stress coefficient, particularly in patients in hyperinsulinemic phase of metabolic syndrome x. conclusion: during first 7 days after acute myocardial infarction lipid peroxids concentration progressively increased, with decrease of total antioxidant status, that results in increase of oxidative stress. these changes are most distinctive in patients with hyperinsulinemic phases of metabolic syndrome x. introduction: glp-1 (exenatide -lilly) is a good therapy for overweight or obese type 2 diabetic patients, but bid administration might not be effective over 24 h. aim: we intended to evaluate 24 h effectivness of this drug in our outpatients using continuous glucose monitoring (gcm-medtronic). methods: we asked our outpatients to undergo a continuing glucose monitoring for 4 days. twelve of them accepted [3f and 9 mol/l, median age 62 (range 39-74), median disease lenght 10 years (range 2-20)] and signed an informed consense; they had stable metabolic control with hba1c <50 mmol/mol and had been on therapy with exenatide for 8 months at least. results: glycemic profile demonstrates that no patient had experimented prolonged hypoglycemia and all subjects, expept for one, had mainteined a long period of euglycemia in range 70-160 mg/ dl during the blood glucose monitoring. conclusions: it′s our opinion that these data demonstrate exenatide can be surely used in type 2 patients to obtain a good and stable metabolic control during all 24 h inspite of the bid somministration and, moreover, the age or the disease lenght are not to be considered a contraindication to its use. several parameters of vascular function and structure have a predictive value for cardiovascular morbidity-mortality and for the presence of associated target organ damage in diabetic and hypertensive patients. superoxide dismutase (sod) is an intracellular antioxidant defense mechanism, which catalyses the dismutation of superoxide radical into h 2 o 2 and oxygen, and it is easily detectable in human plasma; oxidative stress is associated with cardiac and vascular defects leading to hypertension and atherosclerosis and with diabetic cardiomyopathy. on the other hand, osteoprotegerin (opg) is an indicator of diabetes-associated vascular pathologies as hypertension, endothelial dysfunction and cardiovascular risk. we have assessed the relationship between serum levels of sod, opg and parameters of vascular function and structure as well as cardiovascular risk in type 2 diabetic patients with and without hypertension. there are negative correlations between sod and endothelial dysfunction (evaluated by pressure wave velocity, peripheral and central augmentation index and ambulatory arterial stiffness index), pulse pressure, diastolic and systolic night/day ratio, serum opg and plasma hdl-cholesterol, as well as positive correlations between sod and plasma uric acid, liver enzymes got, gpt and ggt, triglycerides and haemoglobin. on the other hand, serum opg is correlated to endothelial dysfunction, intima media thickness, pulse pressure, systolic night/day ratio and d′agostino cardiovascular risk index. our study shows that both sod and osteoprotegerin plasma levels are indicators of cardiovascular events and target organ damage associated with diabetes and hypertension. objective: we investigated the future coronary artery disease (cad) event rate in diabetic patients with and without chest pain in a prospective cohort study performed in a korean population. we also investigated the impact of chest pain on cad risk according to the presence or absence of diabetes mellitus. research design and methods: the ansung-ansan cohort was established for a prospective large-scale community-based epidemiologic study to investigate chronic diseases in korea. the data from a baseline survey performed from 2000 to 2001 and two subsequent prospective biennial surveys were analyzed. results: among 9810 subjects (4632 men and 5178 women) without a history of cad, 0.8% and 2.2% of non-diabetic and diabetic subjects, respectively, reported newly developed cad events during 4 years of follow-up. diabetic patients had a significantly higher risk of future cad events (age-and sex-adjusted odds ratio, 1.998; 95% confidence interval, 1.277-3.125; p = 0.002). although the presence of chest pain at baseline was also significantly associated with an increased risk of cad of more than 2-fold in both non-diabetic and diabetic subjects (p < 0.01), the hazard ratio for cvd event in asymptomatic diabetic patients compared to non-diabetic subjects with chest pain was not significantly different from 1.0 (hazard ratio, 0.875; 95% confidence interval, 0.470-1.632). conclusions: diabetes and the presence of chest pain are independently and significantly associated with future cad event risk. asymptomatic subjects with diabetes have a comparable risk of cad events to non-diabetic subjects presenting chest pain. aim: the aim of the study was to select and analyze patients with diabetes mellitus and acute stemi or nstemi myocardial infarction from all patients hospitalized in cardiology department in 2010. results: more patients with nstemi underwent pci in the history than patients with stemi (n = 32, 37.7% vs. n = 8, 17.8%); p = 0.0195 and more pci and cabg (n = 10; 11.8% vs. n = 0; 0%); p = 0.0166. concentrations of cpk (nstemi vs. stemi) (u/l) (436 ae 854 vs. 1286 ae 1616) p < 0.001, ckmb (u/l) (55 ae 84 vs. 135 ae 149), p < 0.001, glucose on admission (mg/dl) (182 ae 125 vs. 217 ae 82), p < 0.0011. patients with nstemi had less critical changes in the left anterior descending artery (lad) (43.5% vs. 62.2%); p = 0.0426, less bare metal stents (bms) implanted to the lad (8.2% vs. 26.7%one stent), and (0% vs. 4.4%two or more stents), p = 0.002. management of this patients shows figure 1 . patients with stemi require more intensive treatment of more advanced concomitant metabolic disturbances. revascularization should by performed urgently because of higher death rate. during qualification for invasive diagnostic and treatment (pci and/or cabg), not only the risk of death and cardiovascular events should be taken into account, but also a history of revascularization modalities (pci and cabg). methods: a total of 150 diabetic patients both sexes aged 41.58 ae 11.33 years were studied. the levels of total cholesterol (tc), triglycerides (tg), hdl-cholesterol (hdl-c), ldl-cholesterol (ldl-c), vldl-cholesterol (vldl-c), hba 1c and renal function tests were assessed. patients were classified as normoalbuminuric (albumin excretion rate <30 mg/24 h, n = 38), microalbuminuric (albumin excretion rate 30-300 mg/24 h, n = 79) and proteinuric (albumin excretion rate >300 mg/24 h, n = 33). results: the duration of diabetes was 15.70 ae 8.81 years. the level of tc was significantly highest in proteinuric (6.90 ae 0.36 mmol/l), followed by microalbuminuric (5.38 ae 1.29 mmol/l) and followed by normoalbuminuric (3.22 ae 0.66 mmol/l), (p = 0.002, p = 0.0006, respectively). patients with proteinuria had significantly higher level of ldl-c compared to the patients with normoalbuminuria (4.14 ae 0.95 vs. 1.71 ae 0.47 mmol/l, p = 0.001). patients with microalbuminuria had significantly higher level of ldl-c compared to the patients with normoalbuminuria (3.48 ae 1.19 vs. 1.71 ae 0.47 mmol/l, p = 0.015), as well. the level of hba1c in normoalbumiuric patients was significantly lower than in microalbuminuric (7.83 ae 1.12 vs. 10.40 ae 1.59%. p = 0.004). there were no significant differences in levels of tg, hdl-c, vldl-c between patients with normoalbuminuria, microalbuminuria and proteinuria. conclusion: we showed that higher levels of ldl-cholesterol and tg were associated with microalbuminuria and proteinuria in patients with type 1 diabetes. lowering atherogenic lipids may retard nephropathy progression in these patients. s. bonakdaran 1 , b. kharaqani 2 1 endocrine research center, 2 mashhad university of medical sciences, mashhad, iran background: the relationship between elevated serum uric acid level and metabolic syndrome (ms) has been debated. we aimed to determine the prevalence of hyperuricamia and its association with ms in type 2 diabetes mellitus (dm). methods: this was a cross-sectional study in 1978 diabetic patients. hyperuricamia was defined as uric acid ! 7 and ! 5.5 mg/dl for men and women respectively. diagnosis of metabolic syndrome was based on atpiii criteria. clinical and biochemical parameters in hyperuricaemic and normouricaemic patients compared with other. results: the prevalence of hyperuricaemia and metabolic syndrome was 12.7% and 65.5% respectively. the prevalence of ms significantly increased in the highest quartile of uric acid levels compared with lowest quartile (55.9% vs. 74.4%, p < 0.001). serum uric acid had positive association with cholesterol, triglyceride, non-hdl cholesterol and a negative association with fasting blood sugar (fbs), glycosylated hemoglobin (hba1c) and hdl cholesterol. possible biochemical predictors of hyperuricamia were cholesterol, triglyceride, creatnine and fbs. conclusion: the prevalence of ms and its components increase with increasing levels of uric acid in type 2 diabetes. regular assessment of uric acid could give information for predicting of ms and prevention of atherosclerosis in type 2 diabetes. materials and methods: in 75 subjects (aged 53.0 ae 8.3) who have received recommendations on hypotensive and hypoglycemic therapy at diabetes school. psycho-correctional work has been carried out with 37 people (group 1) in order to increase treatment motivation and the work has not been carried out with 38 people (group 2). the effect has been evaluated after 3 months according to the results of 24-h blood pressure monitoring (24-h bpm). results: according to moriski-green test 65% appear to be nonadherent to treatment. as a result only in group 1 there has been recorded reduction in variability indices sbp (mm hg) at night (10.4 ae 1.8 vs. 12.0 ae 2.1; p = 0.031), daily index sbp (7.0 ae 1.3 vs. 12.0 ae 2.1%; p < 0.001) and dbp (8.7 ae 7.2 vs. 16.5 ae 6.6%; p = 0.003), rate in morning increase sbp (7.9 ae 2.1 vs. 15.4 ae 3.4 mm hg; p = 0.004) and dbp (5.9 ae 2.0 vs. 19.9 ae 4.6 mm hg; p < 0.001), which are high risk indicators of cardiocerebral catastrophe. after 3 months 19 of 37 patients have become adherent to treatment in group 1 (p = 0.002), only 2 patients of 38 in group 2 (p = 0.821) (moriski-green test). conclusion: psycho-correctional training improves adherence to treatment, which is accompanied by positive dynamics of 24-h bpm indicators. introduction: quality of life is particular decreased in elderly with diabetes mellitus (dm), even not associated with other chronic illness. the purpose of the study was to analyze the quality of life in a group of elderly diabetic patients without major complications. the study group consisted of 58 patients, males and females, aged over 65 years old [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] , diagnosed with type 2 dm. they had no severe dm complication, nor other debilitating chronic diseases. the romanian version of the sf-36 questionnaire was used to measure quality of life (qol). diabetic subjects were compared with age-and gender-matched controls from a random standard population sample of the romanian population. results: reliability of scales by coefficient alpha cronbach was >0.7 for all scales except social functioning. qol scores for study group were significantly lower compared with controls. there are no significant differences between patients following insulin therapy and patients with other therapeutic protocols. role limitations due to emotional problems correlate with disease duration and female gender. there is a significant correlation between energy/fatigue scores and female gender, hba1c, fasting plasma glucose and obesity. there are no other statistically significant correlations between sf-36 scores and analyzed variables. discussions and conclusions: patients with dm have statistically significant impairment of all aspects of qol. dm put a substantial burden on affected individuals. insulin use didn't seem to have a negative impact upon qol. glycemic control is crucial in preventing long terms complications and provides better qol for diabetic patients. the study involved 85 patients with nstemi and 91 with ua hospitalized in cardiology department in 2010. the data obtained from patients were statistically analyzed to determine the significance of the differences between the groups. results: glucose on admission was lower in patients with ua (148 ae 65 mg/dl) than with nstemi (182 ae 125 mg/dl), p = 0.0092. treatment of dm in patients with nstemi and ua shows figure 1 . patients with nstemi presented lower left ventricular ejection fraction (45 ae 12%) vs. ua (54 ae 10%); p < 0.001 and higher mortality during hospitalization. seven patients (7.1%) with nstemi died, whereas no patient died in ua group (p = 0.0099). 2. to determine whether aaa patients found to be at higher risk of osa were also at increased risk for cardiometabolic disease, compared to those at low or no risk of osa. design: the berlin questionnaire to estimate osa was administered to 318 elderly patients with or at increased risk of aaa disease along with several measures of cardiometabolic risk, including the lipid accumulation product (lap), a measure that combines triglyceride levels with central obesity (waist circumference), also known as hypertriglyceridemic waist. results: in this sample (n = 318), 184 subjects (57.3%) scored clinically positive for risk of osa on the berlin questionnaire. next, individuals were separated into groups based upon the three subclasses of the berlin questionnaire: snore (frequent loud snoring), eds (excessive daytime sleepiness), and htn-bmi (hypertension and high bmi). the combination of snore + eds subclasses, when used to differentiate subject's risk levels for osa, was highly accurate for discriminating low, moderate, and high cardiometabolic risk, based upon significant differences in bmi, insulin (lu/l), waist circumference (cm), waist/hip ratio, low density lipoprotein cholesterol (ldl-c mg/dl), and lap (anova; p < 0.05). conclusions: these results give credence to the clinical use of the snore + eds portions of the berlin questionnaire to provide simple estimates of increased risk of t2dm and cvd and/or need for further evaluation such as polysomnography or oral glucose tolerance. materials and methods: from 2007 to 2009, 2642 t2dm were cumulatively collected for study. accordingly, anthropometric and biochemical data, lifestyle measurements (lifestyle i : no smoking, no alcoholic and regular exercise; lifestyle ii : smoking and/or alcoholic and/or no exercise), total daily caloric intakes and macronutrient consumptions were recorded. the eating habits were classified by fat consumption, high fat diet (fat >35%) and low fat diet (fat 30%). the mets defined was based on the atp iii criteria. patients were obligatorily classified into six groups, mets with waist component, waist required and non-waist required; mets without waist component, with two and more than two components; non-mets with waist and without waist component. each component of mets in these six groups with different combination of lifestyle and eating habit, were presented by case number and percentage distribution. or for the clustering of each metabolic component in these six groups were analyzed. results: the case number, percentage distribution and corresponding or for the clustering of each metabolic component in these six groups were shown in table 1 and 2. the clustering of metabolic components of tg, hdl-c and bp were significantly higher in lifestyle ii, lifestyle i and high fat diet eaters respectively. objectives: early detection of metabolic syndrome is important in minimizing morbidity and motility. we conducted a study to detect wether serum leptin can be used as a biological marker to detect metabolic syndrome. methods: a group of 128 healthy pre-menopausal females, aged 25-50 years was selected from the local community, randomly, stratified according to their bmi. leptin was assessed by the elisa method and, blood pressure was measured using digital blood pressure monitor and chemical analysis for fasting blood, glucose and lipid level were conducted using colorimetric method. body weight and height, hip and waist circumferences were measured, using standard protocols. in subjects with and without metabolic syndrome mean serum leptin values were 10.15 (ae3.45) and 8.46 (ae3.66) ng/ml respectively. compared with subjects without metabolic syndrome, subjects with metabolic syndrome had a higher serum leptin level (mean difference à1.68, and 95% confidence interval à3.14 to à0.22, p = 0.024). conclusion: subjects with metabolic syndrome had a higher serum leptin level in comparison with subjects without metabolic syndrome therefore leptin can be utilized as a surrogate marker to predict metabolic syndrome. conducted a study to detect relationship between serum leptin and cardiovascular risk factors in obese and non obese individuals. methods: a group of 128 healthy pre-menopausal females, aged 25-50 years was selected from the local community, randomly, stratified according to their bmi. body weight and height were measured, using standard protocols. serum leptin was assessed by elisa and, blood pressure was measured using digital blood pressure monitor and chemical analysis for fasting blood, glucose and lipid level were conducted using colorimetric method. results: there was a statistically significant inverse correlation between serum leptin level and mean blood pressure in obese (bmi ! 30) individuals and subjects with systolic blood pressure >140 mmhg. corresponding r values were r = à0.32 and r = à0.45 (p < 0.05 for both). in subjects with bmi <30 or in normotensive subjects, there were no statistically significant correlations between serum leptin and mean blood pressure. correlations between serum leptin and total cholesterol, triglycerides, hdl, ldl, cho/hdl and fasting plasma glucose were 0.15, 0.13, 0.16, 0.07, 0.05, 0.13 respectively and not statically significant (p 0.001 for all). conclusion: due to the presence of inverse correlation between serum leptin and mean blood pressure in obese, and individuals with high systolic blood pressure, it can be concluded that serum leptin may play a different role in pathogenesis of cardiovascular diseases in such individuals. r. irzma nski, j. błaszczyk, l. pawlicki, j. kowalski the aim: the aim of our study was to assess the influence of metabolic syndrome (ms) risk factors on vascular complications in patients with ms. material and methods: the study comprised 108 patients (72 women and 36 men, avg. age 57.0 ae 8.5 years) with ms, which was diagnosed according to idf criteria 2005. results: the prevalence of micro and macrovascular complications was assessed: vascular changes in the fundus of the eye -72%, ischaemic heart disease -54.9%, egfr <90 ml/min -38.9%, diabetic foot -5.55%, cerebro-vascular accident -3.7% of patients. the negative correlation between hdl concentration, creatinine levels and level of vascular changes in the fundus of the eye was found. moreover the level of obesity and fasting glucose level had positive correlation with the intensitz of vascular changes in the fundus of the eye. objectives: to estimate malnutrition prevalence among newly hospitalized overweight/obese patients; to characterize malnutrition by body weight category; and to assess associations between bmi, duration of hospitalization and in-hospital death in malnourished patients. methods: this cross-sectional survey assessed nutrition status in all adults newly admitted to internal medicine and surgical departments at the e. wolfson medical center, holon, israel. data were recorded during the 5-week data acquisition period and screening for malnutrition risk was performed using the nrs 2002. an age-adjusted score of ! 3 on the nrs 2002 defined malnutrition. malnutrition was compared across body weight categories: underweight (bmi <18.5 kg/m 2 ), normal (bmi 18.5-24.99 kg/m 2 ), overweight (bmi 25-29.99 kg/m 2 ) and obese (bmi ! 30 kg/m 2 ). overweight/obese subjects were compared by malnutrition status. results: a total of 431 individuals were analyzed, of whom 243 were overweight/obese (bmi ! 25 kg/m 2 ). of these, 58 (23.9%) were malnourished. compared to adequately nourished overweight/obese subjects, malnourished overweight/obese patients had significantly prolonged duration of hospitalization: 11.7 ae 18.9 (median 5, 1-123 days) vs. 5.3 ae 6.7 (median 4, 0-65 days), (p = 0.001). in-hospital mortality was 6.9% among malnourished vs. 0.5% among adequately nourished overweight/obese patients, p = 0.003. malnutrition increased duration of hospitalization and in-hospital mortality risk in both overweight/obese and normal weight patients. discussion: malnutrition is a frequent finding in newly hospitalized overweight/obese adults. elevated bmi does not affect duration of hospitalization. in-hospital mortality rates are similar for normal weight and overweight/obese individuals. background: the purpose of this study was to examine differences in rmr in lean and obese subjects and to determine correlation between rmr and cardio metabolic risk factors. methods: ninety nine healthy subjects (47 normal; 52 obese and over weights) participated in this case-control study. sex and physical activity were matched. blood pressure, plasma insulin, glucose, homa-index, lipid profile, uric acid and c-reactive protein concentrations, anthropometric measurements, body composition, rmr and macronutrient intake were measured. result: fpg (p = 0.016), uric acid (p = 0.043), crp (p = 0.002), insulin (p < 0.001), homa index (p < 0.001), systolic and diastolic blood pressure (p = 0.008; p = 0.003), anthropometric measurements (except height and whr) (p < 0.001) and body composition (p < 0.001) were significantly higher in case group. there were no significant differences between both groups by the means of dietary intake. in obese and overweight group, rmr was statistically higher than control group (p = 0.001). there were no significant difference between both groups in adjusted rmr for ffm and fm (p = 0.484). with multiple linear regression, rmr was significantly associated with ffm (p < 0.001) and uric acid (p < 0.001) and negative significant correlation was observed between rmr and waist circumference (p = 0.028) and hdl (p = 0.046). the finding of a similarly specific metabolic rate in obese and lean subjects at first sight contradicts the idea that a low rmr is a main cause of obesity. one reason for the undetected association between low rmr and obesity may be that obesity-related metabolic risk factors mask the lower metabolic rate that initially contributed to weight gain. background: pain may cause some patients to avoid self-monitoring, which could impair glucose control. cooling the fingertip prior to puncture may reduce pain. objectives: to examine the efficacy of coolsense, a device designed to cool the fingertip prior to puncture for glucose self monitoring. methods: adults with type 2 diabetes treated at the e. wolfson medical center diabetes unit outpatient clinic were randomized to treatment with the coolsense device or a sham device which appeared identical to the active intervention but did not cool the fingertip. patients completed a demographic and medical history interview and were asked to rate the device for discomfort and satisfaction. results: a total of 180 patients were recruited and randomized to intervention group (coolsense vs. sham, n = 90 in each group). participants were 55 ae 5.9 years of age, had been diagnosed with diabetes 6 ae 6.4 years prior to study onset and had mean hba1c of 7.3 ae 0.4%. patients reported performing 1.3 ae 1 blood glucose checks per day. the extent to which glucose checking caused pain was rated at 1.96 ae 0.8 out of a possible five points, one being most severe. the coolsense device was a significant independent predictor of pain reduction, even after controlling for age, sex, hba1c, aspirin, neuropathy and baseline rating of pain associated with glucose check. conclusions: cooling the fingertip with the coolsense device significantly reduced pain at the puncture site compared to a sham device. it is possible that this reduction in pain will increase glucose self monitoring events in patients with diabetes. f. saad 1,2 , a. yassin 3,4 , g. doros 5 1 results: after 5 years the following changes were observed: weight (kg) decreased by 7.77 kg from 100. introduction: testosterone treatment in hypogonadal men is standard therapy, particularly in younger men with congenital forms of hypogonadism. methods: three hundred thirty-three patients (147 with primary hypogonadism including 38 patients with klinefelter's syndrome, 100 with secondary hypogonadism and 87 with late-onset ("mixed" or "metabolic") hypogonadism aged 15-72 years (mean 42 ae 15 years) received intramuscular injections of 1000 mg of testosterone undecanoate during a maximal treatment time of 15 years, overall corresponding to 1403 treatment years. hypogonadism was defined as total testosterone below 12 nmol/l and occurrence of symptoms. physiological functions of bile salts include modulation of cholesterol and triglyceride metabolism, insulin sensitivity, the intestinal endocrine response to meals and energy homeostasis. several of these functions are mediated via the membrane g-protein coupled receptor tgr5 (also called gpbar1 and gpr131) which has been shown to promote release of glucagon like peptide-1 from enteroendocrine cells and increase energy expenditure in brown adipose tissue. methods: based on comprehensive discovery platform developed at satrx several in vivo active tgr5 inhibitors were discovered. active scaffolds were identified during the high throughput screening campaign of 50,000 tgr5 biased small heterocyclic molecules library that was optimized for medchem parameters and cell permeability. confirmed selective hits were evaluated for functional activity of glp1 secretion in nci-h716 endocrine cell line. in vivo activity was confirmed in db/db mice diabetes model. active compounds were administered chronically at doses 30, 3 and 0.3 mg/kg and produced stable and statistically significant anti-diabetic effect. results: the most active compound showed efficacy comparable to 10 mg/kg doses of sitagliptin. the actual activity data and experimental details will be provided. the interaction between excess of body fat (total and abdominal) and increased cardiovascular risk is well established in all age groups [3, 9] . however, there are few studies that analyze the pattern of body fat distribution and its association with cardiovascular risk factors (crfs) in a population with more advanced age. objective: to analyze the presence of crfs according to the pattern of body fat distribution, in brazilian aged 80 years or older. methods: one hundred and thriteen subjects, randomly selected, 83.4 ae 2.9 years, of both sexes. the percentage of total and abdominal body fat, hypertension and lipid profile were used for characterization of crfs. the chi-square test was used to assess proportions of crfs and mann-whitney test was used to compare the results between distributions of adiposity. results: eutrophic subjects showed lower triglycerides (p = 0.017), total cholesterol (p = 0.001) and prevalence of hypertension (p = 0.003) and hypertriglyceridemia (p = 0.007). subjects with higher abdominal adiposity showed higher total cholesterol (p = 0.026) and prevalence of hypertriglyceridemia (p = 0.011) and hypercholesterolemia (p = 0.026) than no excess of abdominal adiposity. higher value of one outcome already reflects the higher prevalence of hypertension (p = 0.039) and, the higher values of both outcomes reflect the high values of total cholesterol (p = 0.028) and triglycerides (p = 0.001). conclusion: that obesity, whether abdominal or total, is associated, in the same way, with dyslipidemia and hypertension in the subjects aged 80 years. background/aim: there is growing consensus in the literature that inflammation plays a central role in the pathophysiology of obesity and type 2 diabetes mellitus (t2dm) and cardiovascular complications. neutrophil-to-lymphocyte ratio (nlr) provides a simple method for assessment of inflammatory status and it is a new, inexpensive marker. the aim of the present study was to investigate the predictive value of preprocedural (before the ogtt) nlr on development of prediabetes (pd) and type 2 diabetes (t2dm) in morbid obesity patients (mop). methods: 220 mop and 85 normal weight patients with normal ogtt [fasting plasma glucose (fpg) <100 mg/dl. two-hour glucose during ogtt < 140 mg/dl] were evaluated in this study. results: the mean ae sd nlr of mop were significantly higher than that of patients with normal weight healthy patients (4.23 ae 1.65 vs. 2.36 ae 0.95, p < 0.001, respectively). in receiver operating characteristics curve analysis, nlr >3.61 had 80% sensitivity and 75% specificity in predicting pd and nlr >3.84 had 61% sensitivity and 68.1% sepesifity in predicting t2dm. logistic regression analysis showed that elevated nlr (or: 3.883, 95% ci: 2.524-7.258, p < 0.001) was an independent variable for predicting t2dm in mop. conclusion: mop have higher nlr than healthy controls. high nlr is a powerful and independent predictor of pd and t2dm in mop. elevated nlr levels are usually considered as an inflammatory marker. the results of this study suggested that inflammation plays a role in the pathogenesis of pd and t2dm with mop. division of human nutrition, wageningen university, wageningen, 2 general surgery, rijnstate hospital, arnhem, 3 gastroenterology, rijnstate hospital, arnhem, the netherlands objective: endoscopic implantation of a duodenal-jejunal bypass liner (djbl), or endobarrier, is a novel bariatric technique to induce weight loss and remission of type 2 diabetes (t2d). placement of the djbl mimics the duodenal-jejunal bypass component of the roux-en-y gastric bypass (rygb) procedure. as gut hormones are known to change substantially after rygb surgery, in our study we now evaluated gut hormone responses after implantation of the djbl. methods: fourteen (eight male, six female) obese t2d subjects (bmi 33.7 ae 0.9 kg/m 2 , duration of type 2 diabetes 8.0 ae 1.4 years) were selected for implantation of a djbl. fasting plasma levels of glucose, c peptide, hba1c and gut hormones ghrelin, gip and glp-1 were analysed before and at 7 and 28 days after djbl implantation. results: plasma hba1c levels were significantly decreased after djbl implantation and a 40% reduction was found in diabetes medication usage (p < 0.05). ghrelin was found significantly elevated, with the highest induction in the first 7 days post-implant. although the gip response showed high variation between subjects, gip tended to decrease 28 days after implantation (p = 0.09). glp-1 levels showed a significant "dip" at day 7 post-implant, which correlates with the intake of solely pureed/liquid food in the first week post-implant. conclusions: implantation of a djbl results in an early substantial remission of t2d, comparable to results seen after rygb surgery. interestingly, in contrast to rygb surgery, implantation of the djbl seems to preserve normal physiological responses of gut hormones that are related to nutritional status. conflicting data exists as to the association between body mass index (bmi) and the rate of chronic kidney disease (ckd). in a cross sectional analysis of database from a screening center in israel we assessed the rate of ckd defined as estimated glomerular filtration rate (egfr) < 60 ml/min per 1.73 m 2 in relation to increasing bmi subcategories. the study population included 21,880 subjects, 32% women aged 20-80, out of whom 167 men and 45 women had ckd. subjects with a bmi of 25-29.9 (kg/m 2 ) compared to subjects with bmi <25 (kg/m 2 ), had an odds ratio (or) [95% confidence intervals (ci)] for ckd of 1.8 (1.2-2.7) and 3.4 (1.5-7.7) for men and women respectively. for subjects with a bmi of 30-35 (kg/m 2 ) the or were 2.5 (1.6-4.0) and 4.5 (1.7-11.7) for men and women respectively. for subjects with bmi >35 (kg/m 2 ) the or rose to 2.7 (1.3-5.5) and 15.4 (6.4-36.7) for men and women respectively. this association became insignificant in men after multivariate adjustment for age, hypertension and diabetes mellitus but persisted in women even after multivariate adjustment. the correlation between bmi and ckd in women was attributed to the subcategory of severely obese women with bmi above 35 (kg/m 2 ). our study suggests that for both men and women a positive correlation exists between the degree of bmi and the rate of ckd. this correlation persisted in severely obese women even after multivariate adjustment, suggesting that in women, obesity may be an independent risk factor for developing ckd. the enteroendocrine cell line pgip/neo stc-1 was used to evaluate the effects of gspe on glp-1 secretion in different nutrient conditions. a cytotoxicity detection kit (ldh) and brdu labelling and detection kit iii (roche) were used to determine cytotoxicity and cell proliferation, respectively. glp-1 levels in cell culture medium were determined using a glp-1 (active) elisa kit (millipore). cytotoxicity and proliferation cell assays (n = 3) demonstrated that the maximum non-toxic gspe treatment for these cells was 150 mg/l gspe over a period of 3 h. surprisingly, after 3 h of gspe treatment, glp-1 secretion (n = 6) was significantly inhibited. this inhibitory effect was observed at concentrations as low as 5 mg/l. furthermore, inhibition of glp-1 was also observed when stc-1 cells were co-incubated gspe and 100 lmol/l dihomo-ϒ linoleic acid. in conclusion, acute gspe incubation decreases glp-1 secretion in cultured enteroendoncrine cells. interestingly the effect occurs under either on basal or nutrient (fatty acid) stimulated conditions. c. higa 1 , f. novo 2 , m.s. donato 2 , n. rizzo 2 , g. ciambrone 2 , e. korolov 2 , p. comignani 2 1 coronay unit, 2 hospital aleman, buenos aires, argentina introduction and aim: there is no data available respect of the prognostic value of albumin:creatinin ratio (acr) in non-st-segment elevation acute coronary syndrome (nseacs). the purpose of our study was to evaluate the long term prognostic value of acr in patients with nseacs. methods: we analyzed a prospective cohort of nseacs in whom acr was determined at admission. roc curves were constructed to determine best cut off value associated with primary end-point of death or nonfatal myocardial infarction (d/ami). independent variables for d/ami were assessed by a cox regression model. : seven hundred ten patients with nseacs were analyzed. thirty percent were female and median age was 63 years. median follow up time was 24 months. best cut-off point of acr for primary end point was 20 mg/g. thirty-four percent of patients had acr ! 20 mg/g. acr correlated with higher incidence of primary end point both in diabetics and in non-diabetic patients: or 3.5 (ic 95 2.3-14), p = 0.001 and or 5.4 (ic 95% 2.4-11), p = 0.02, respectively. by multivariable cox regression analysis, acr was an independent predictor of d/ami at long-term follow up: hr 5.8 (ci 95% 2-11), log rank 2 p < 0.0001 in a model that included age, female gender, diabetes mellitus, serum creatinine, creatinine clearance, glucemia at admission a, elevated cardiac markers and st segment deviations. conclusions: acr was an independent predictor of adverse outcomes in nseacs. this simple and accesible marker should be considered for risk stratification in this high risk setting. aim of the work was to examine possible connection of methabolic syndrome with endothelium dysfunction in pre and postmenopausal women with and without ischemic heart disease (ihd). methods: hundred and eight-nine pre and postmenopausal women with ihd were examined and 83 in same age without ihd. all women were measured arterial pressure using korotkov method (according to jnc-6 recommendations). vasa regulating function of endothelium examined with ultrasound in triplex scanning using reactive hyperemia probe (method by d. celermajer). willebrant factor determined in blood plasma. results: in group of women with ihd 126 (24%) had arterial hypertension: among them 47 (24%) in pre and 79 (40%) postmenopausal. among women without ihd 23 (28%) had hypertension: 6 (7%) were pre-and 17 (21%) postmenopausal. during reactive hyperemia probe diameter of radial artery was: women with ihd 4.29 ae 0.05 mm; speed of blood flow 0.56 ae 0.05 m/s, willebrant factor 177.45 ae 10.91; women without ihd corresponding 4.55 ae 0.09 mm (p < 0.05) and 0.69 ae 0.05 m/s (p < 0.05), willebrant factor 128.15 ae 10.8 (p < 0.01) where there significant difference was noted. findings allows us to suppose, that women with ihd have higher abundance of arterial hypertension, that in connection with other components of metabolic syndrome, specify development of endothelium dysfunction, in comparison with women without ihd, which have arterial hypertension, endothelium dysfunction infrequently. objective: recently, incretin-related drugs are widely used for improving blood glucose level in clinical practices. in this study, we tried to examine the efficacy and safety of these drugs of the japanese approved dose. we compared the efficacy and safety of the human glp-1 analogue liraglutide (the approved dose 0.9 mg) with the dipeptidyl peptidase-4 (dpp-4) inhibitor sitagliptin (the approved dose 50 mg) once daily in the obese adults with type 2 diabetes, over 52 weeks. methods: japanese obese out-patients whose glycemic control was inadequately controlled with oral hypoglycemic agents received either maximum 52 weeks of treatment with 0.9 mg lilaglutide (n = 120) or 50 mg sitagliptin (n = 154) once daily within the period from march 2010 to october 2012, at chubu-rosai hospital. the primary endpoint was change in body weight. other measurements were hba1c, lipid profiles, body weight, body mass index, serum creatinine, estimated gfr, and adverse events before and at 4, 12, 24, 52 weeks after liraglutide or sitagliptin administration. results: at the japanese approved dose, the greater reduction of mean body weight was achieved with liraglutide than with sitagliptin (à6.2 kg, p < 0.001, +0.2 kg, p = n.s. vs. baseline). the degrees of lowering hba1c (both 7.6% at baseline) were à0.80% for liraglutide and à0.53% for sitagliptin (both p < 0.001 at baseline). the egfr of the sitagliptin group worsened from 74.0 to 67.8 ml/min/1.73 m 2 (p < 0.001 at baseline). the most common adverse events were gastrointestinal symptoms (24 (19%) patients on 0.9 mg liraglutide). conclusion: liraglutide provides greater body weight reduction over 52 weeks even at the japanese approved dose of 0.9 mg. objective: to examine prevalence of cardiovascular risk factors in patients with type 2 diabetes and stroke in primary care setting. methods: study was conducted at primary health center tuzla and included all patients with type 2 diabetes and stroke who were registered in one family medicine team. we also calculated absolute cardiovascular risk according to the european guidelines on cardiovascular disease prevention in clinical practice. results: prevalence of stroke in diabetic patients was 24.1%. significantly more women had stroke than men (65.1% vs. 33.9%; p = 0.001). mean age of patients was 70.58 ae 8.44 years. men were significantly older than women (72.22 ae 8.10 vs. 69.74 ae 8.60; p = 0.002). majority of participants belonged to age group >70 years (57.4%). mean duration of diabetes was 10.15 ae 5.41 years. more than half of patients (66%) had diabetes 5-10 years, 28.3% had diabetes 11-20 years, and 5.7% patients had diabetes >20 years. family history for diabetes had 43.4% patients. the most prevalent cardiovascular risk factors were hyperlipidemia (88.7%) and hypertension (86.8%). obesity was present in 49% patients, and 47.2% patients had family history for premature cardiovascular disease. unhealthy diet had 30.2% patients, 30.2% were physically inactive, and 17% diabetic patients were smokers. mean absolute cardiovascular risk was 5.49 ae 3.57%; 7.72 ae 3.80% in men, 4.34 ae 2.88% in women. men had significantly higher absolute cardiovascular risk than women (p = 0.001). conclusion: prevalence of cardiovascular risk factors in patients with type 2 diabetes and stroke were very high. it indicates more effective strategies in primary care setting to reduce risk of macrovascular and microvascular complications. the prevalence of metabolic syndrome was determined as a crosssectional study among 600 healthy saudi adults (52% males and 58% aged 35-50 years) attending national guard clinics using the definition proposed by ncep atpiii. the prevalence of metabolic syndrome was 21%. only one third of the participants had normal weight (bmi = 18.5-24.9). central obesity based on waist circumferences was noted in 21% of males and 22% of females. low hdl-c showed the highest prevalence (29%) followed by high tg (24%). about 14% of participants had impaired fasting blood glucose ( ! 110 mg/dl). only 6% had high blood pressure ( ! 130/85 mmhg). more than three quarters (77.3%) of the respondents had ! 1 component of metabolic syndrome. in conclusion, metabolic syndrome needs to be addressed as an important health problem in the gulf region. background/aim: adequate treatment and monitoring of diabetes can significantly improve quality of life and extend life expectancy in diabetic subjects. aim of this study was to examine metabolic control in patients with type 2 diabetes in primary care setting. methods: study was conducted at primary health center tuzla and included 107 randomly selected patients with type 2 diabetes, aged 40 years and over, from one family medicine team. we evaluated parameters of metabolic control in patients with type 2 diabetes according to the european guidelines on cardiovascular disease prevention in clinical practice. results: there were significantly more women than men (63.6% vs. 36.4%; p < 0.0001). mean age of patients was 68.06 ae 8.84 years. mean duration of diabetes was 10.06 ae 5.43 years. only 25 (23.4%) patients had blood glucose <6.0 mmol/l, and 39 (36.4%) patients had hba1c <7.0%. controlled blood pressure 130/80 mmhg had 47 (43.9%) patients. only 18 (16.8%) patients had total cholesterol <4.5 mmol/l, while 35 (32.7%) patients had triglycerides <1.7 mmol/l. normal body mass index (bmi) <25 kg/m 2 had 12 (11.21%) patients and recommended waist circumference 33 (30.8%) patients. mean blood glucose was 8.34 l ae 2.78 mmol/l, hba1c 7.77 ae 1.47%, systolic blood pressure 134.49 ae 17.68 mmhg, diastolic blood pressure 78.83 ae 9.75 mmhg, total cholesterol 5.76 ae 1.38 mmol/l, triglyceride 2.6 ae 2.07 mmol/l, bmi 29.96 ae 4.08 kg/m 2 , waist circumference 99.07 ae 9.97 cm. conclusion: metabolic control of type 2 diabetes in family medicine practice was inadequate which indicates more effective interventions in order to achieve appropriate metabolic control of diabetes and reduce risk of complications. aim: the aim of the study was to determine the metabolic responses to two different liquid milk protein diets in prediabetic and healthy volunteers. the diets were composed of 1. 50 g whey or casein protein, 50 g maltodextrin 19 and 10 g lactulose or 2. 50 g maltodextrin 19 and 10 g lactulose (control). procedures: fifteen prediabetic and 15 healthy volunteers consumed all liquid test diets with blood sampling over a 4 h time period. blood glucose, incretin hormones, nefas, hydroxybutyric acid and plasma amino acids were analyzed. the feeling of hunger was determined using a visual analog scale. results: in both, prediabetic as well as healthy volunteers, postprandial blood glucose levels were significantly decreased and plasma insulin levels were significantly elevated with the protein diets compared to the control diet. gip and glp-1 levels also increased after the protein-contaning test-diets. in the prediabetic volunteers, changes in glucose, insulin, amino acids, non-esterified fatty acids and hydroxybutyric acid displayed time delayed changes. a reduced feeling of hunger was reported in the prediabetic compared to the control group. conclusion: both protein components elevate insulin secretion and cause reduced blood glucose auc and these effects are more pronounced in prediabetics. however, despite differences in the amino acid composition, casein and whey protein did not reveal significantly different effects. postprandial changes in plasma metabolites in prediabetics suggest more metabolic pertubations than just impaired glucose disposition. methods: ins-1e cells or freshly isolated rat islets were incubated for 48 h in the presence of either leptin or tnf-a at lower (0.5 nmol/l or 2 ng/ml) or higher concentration (10 nmol/l or 20 ng/ml) individually or in combination. proinsulin mrna was detected by real time-pcr, insulin by radioimmunoassay kit, β cell proliferation were tested with mtt assay and β cell apoptosis was determined with annexin v-fitc/pi by fluorescent activated cell sorting. results: higher leptin (10 nmol/l) or tnf-a (20 ng/ml) significantly suppressed gsis (p < 0.05) and reduced the intracellular proinsulin mrna level and insulin content (p < 0.01) in ins-1e cells and primary islets. whereas, lower leptin (0.5 nmol/l) or tnf-a (2 ng/ml) did not show such effects. however, the inhibition effects were compromized when two factors were simultaneously administrated in either cultured ins-1e cells or pancreatic islets. similarly, higher leptin or tnf-a was able to inhibit ins-1e cell proliferation and promote cell apoptosis. again, these effects were alleviated in the presence of both factors. furthermore, western blotting showed that tnf-a inhibited leptin receptor (ob-rb) expression, decreased the phosphorylation of stat3. conclusion: tnf-a exerts antagonistic effects on leptin actions in pancreatic β cells by interfering with ob-rb/jak/stat3 signaling pathway. increased tnf-a level may contribute to the pathogenesis of hyperinsulinemia and disturbed glucose metabolism in people with obesity. aims: identifying socioeconomic factors associated with the metabolic syndrome (mets) is useful to target preventive measures. our objective was to estimate, in france, the prevalence of metabolic syndrome (mets) and to investigate the association between socioeconomic position and mets. methods: the french national nutrition and health survey (enns) cross-sectional national multistage sampling was carried out from february 2006 to march 2007. data included waist circumference and blood pressure measurements, blood sample and sociodemographic and medication information. the prevalence of mets was assessed using several definitions, including the most recent joint interim statement (jis). association with sociodemographic covariates was assessed using logistic regression models. results: among the 1856 participants 18-74 years of age, mets prevalence was found to vary from 14.6% according to the national cholesterol education program definition to 21.1% according to the jis definition, without difference between genders. after adjustment for other covariates, risk of mets increased with age in both men and women (for 1 year: ora = 1.07, p < 10 à3 and ora = 1.05, p < 10 à3 ; respectively). in women, mets risk was inversely associated with education level; women with the lowest education level facing a six fold greater risk of mets (ora = 6.3, p < 10 à3 ). in men, risk of mets was higher in men born outside of france (ora = 3.01, p = 0.003) than in french-born males. conclusions: mets prevalence is lower in france than in most industrialized countries. lifestyle modifications, targeted to migrants and persons living in low socioeconomic conditions, should contribute to further reducing mets. folate. patients were divided into two groups: group 1 (homocysteine <15 lmol/l) and group 2 (homocysteine ! 15 lmol/l). results: the mean age was 53.11 ae 8.80 years. homocysteinemia was 12.55 ae 3.47 mmol/l. a significant correlation was found between homocysteine levels and the number of criteria for ms (p = 0.009). besides, a significant decrease of hyperhomocysteinemia (from 26.7% to 3.3%, p = 0.039), systolic blood pressure (from 136.71 ae 19.82 mmhg to 126.77 ae 28.80 mmhg, p = 0.035), uric acid (from 271.18 ae 90.93 lmol/l to 250.08 ae 81 841 lmol/l; p < 0.001) and a significant improvement of hdl levels (1.06 ae 0.22 mmol/l to 1.14 ae 0.24 mmol/l, p < 0.05) were reported. a non significant improvement was observed for the other parameters. furthermore, a correlation was noted between the variation of homocysteine and hdl levels (p = 0.023). the association found between homocysteine and the severity of ms suggests a reflection on the cardiovascular risk caused by such association and on the importance of hyperhomocysteinemia screening in patients with ms. object of study and method: the 151 patients with ms: 88 of them presented with the recurrent form of af, 63 had no arrhythmia. the groups were comparable in terms of age, concomitant disorders, arterial hypertension duration, arterial pressure, and severity of chronic heart failure. patients with permanent af, hemodynamically significant heart disease, myocardial infarction with wave q in the medical history, were excluded from the study. in a review of the diagnostic efficiency parameters showed statistically significant differences in the two groups, with the evaluation of their specificity (se), sensitivity (sp) and or (odds ratio). results: patients with ms having abdominal obesity and arterial hypertension over 10 years (se 0.81; or 2.8 (95% 1.3-5.6); x 2 = 6.6; p = 0.01); homa ir index more than 2.77 (se 0.82; or 5.48 (95% 2.2-12.8); x 2 = 13.4; p < 0.00001); reduced hdl cholesterol level below 1.1 mmol/l (se 0.71; or 3.73 (95% 1.8-7.2); x 2 = 13.4; p < 0.001); left atrial dilation (se 0.81; or 3.68 (95% 1.7-7.4); x 2 = 11.4; p = 0.001); albuminuria more than 60 mg/day (sp 0.85; or 7.9 (95% 3.1-18.6); x 2 = 19.7; p < 0.001); waist circumference more than 104 cm (sp 0.71; or 3.13 (95% 1.6-6.1); x 2 = 9.86; p = 0.002) were at high risk of af. the duration of abdominal obesity and hypertension for 10 years, insulin resistance index >2.77, reduced hdl <1.1 mmol/l, an increase in albuminuria >60 mg/day, an increase in waist circumference >104 cmrisk factors for af in ms. patients and methods: fifty-three patients with ms over 60, comparable in duration af, comorbidity and treatment. patients with permanent af, hemodynamically significant heart disease, myocardial infarction with wave q in the medical history, were excluded from the study. following a successful cardioversion all patients assigned amiodarone. group 1 (n = 16) in the adjuvant therapy prescribed metformin over 1700-2000 mg/day; group 2 (n = 20) metformin <1700 mg/day, group 3 (n = 17) metformin is not assigned. the observation period is 6 months. results: in group 1 homairind decreased to 2.04 ae 1.08 (p < 0.05 compared with baseline), in the 2nd to 2.88 ae 1.2 (p > 0.05) and 3rd to 3.04 ae 1.2 (p > 0.05) (p1-3 < 0.05), and improvement of lipid profile and increased gfr (60.1 ae 15.3 (p < 0.05 compared with baseline), 51.3 ae 9.2 and 51.7 ae 8.2 ml/min, in the 1st, 2nd and 3rd groups, p = 0.04 between groups); decreased volume of the left index atria (37.05 ae 8.96 (p < 0.05 compared with baseline), 38.1 ae 10.4 and 41.9 ae 9.4 ml, in the 1st, 2nd and 3rd); reduction in waist circumference (103.3 ae 6.3 (p < 0.05 compared with baseline), 109.1 ae 8.1 and 108.3 ae 7.5 cm, in the 1st, 2nd and 3rd). patients in group 1 was maintained sr for much longer (163.4 ae 39.4 days) than in patients of the 2nd (120.9 ae 47.7) and the 3d group (124.5 ae 50.9) (p = 0.01; p1-2 <0.05; p1-3 <0.05). aims: metabolic syndrome (mets) is the serious health problem worldwide and is associated with increased risk of cardiovascular disease. mets has a significant genetic component which is estimated on 30-60%. apolipoprotein a5 (apoa5, omim acc. no 606368) gene and its variants have been associated with the plasma lipids, mainly triglycerides and its role in mets development is recently discussed. we have analyzed, if there is the association between the apoa5 gene haplotypes (based on the rs662799 and rs3135506 variants) and mets in middle european -slavic population. methods: apoa5 haplotypes based on the presence of either common or at least one minor apoa5 allele (rs662799, c-1131 and rs3135506, g56) and the presence of mets were analysed in 2559 adults (1191 males and 1368 females, 25-65 years old) selected according the monica protocol and examined at 1998/7 and 2000/2001. the presence of mets was analysed according the ncep-atp iii criteria. results: in females with at least one minor apoa5 allele, the prevalence of mets was significantly higher both at 1997/8 (38.3% vs. 32.6%, p < 0.04) and at 2000/1 (36.9% vs. 30.4%, p < 0.02). in males, association between apoa5 gene and mets was not detected neither at 1998/7 (48.1% vs. 45.1%, p = 0.29) nor at 2000/1 (45.7% vs. 42.2%, p = 0.41). a. blazquez 1 , o. garcia-sanchez 1 , y. quiros 1 , v. blanco-gozalo 2 , m.j. montero 1 , j.m. lopez-novoa 1 , c. martinez-salgado 3 , f.j. lopez-hernandez 3 1 universidad de salamanca, 2 ibsal, 3 iecscyl-ibsal-university of salamanca, salamanca, spain diabetes commonly causes a type of chronic nephropathy, namely diabetic nephropathy (dn). diagnosis of dn is presently accomplished late in the course of disease. microalbuminuria anticipates progression towards dn only in a subset of patients. another subset of diabetic, microalbuminuric patients never develop dn. accordingly, new biomarkers are necessary to more accurately identify diabetic patients progressing towards dn. ngal has also been associated to progression to dn. on the other hand, hypertension is a well-known factor of co-morbidity in diabetic nephropathy. we decided to study the capacity of ngal to detect the additive effect of hypertension on progression to diabetic nephropathy. in the present work we aimed at unraveling the origin of the increased urinary ngal. spontaneously hypertensive rats (shr) or normotensive wistar rats were rendered hyperglycemic by a single administration of streptozotocin, or not (as controls). renal function was monitored and ngal was measured in urine, plasma and tissue samples. their kidneys were perfused in situ with krebsdextran solution (containing or not exogenous ngal), and urine was collected. plasma and renal tissue ngal was also measured by western blot, and renal ngal expression was determined by rt-pcr. our results suggest that the urinary ngal is increased by the coexistence of diabetes and hypertension, but not by each of these conditions. ngal excretion results from its altered tubular handling. this subtle and primary renal alteration might be studied further as an early marker of the increased risk of chronic renal disease posed by the co-morbidity of hypertension and diabetes. the increasing prevalence of obesity is a major public health concern. more than 1.4 billion adults, 20 and older, were overweight, over 200 million men and nearly 300 million women of whom were obese in 2008. large-scale studies have demonstrated that overweight and obesity increase the risk of developing several forms of cancer, including several that are not classically viewed as hormonedependent. in both men and women, increasing bmi is significantly associated with higher death rates from cancers of the oesophagus, colon and rectum, liver, gall bladder, pancreas and kidney, as well as non-hodgkin lymphoma and multiple myeloma. the associations between obesity and particular maignancies may be affected by body fat distribution, and may result from diverse factors including diet and abnormal levels of hormones and inflammatory cytokines. recently, there is more and more sufficient evidence that excess body weight is an avoidabl e cause of excess cancers including gastrointestinal, endometrial, esophageal adenocarcinoma, colorectal, postmenopausal breast, prostate, and renal cancers. the mechanism that obesity association with cancer is remains not well understood. there are some most studied hypothesized mechanisms such as, high levels of insulin and free levels of insulin-like growth factors (igfs), sex hormones, adipocytokines, inflammatory cytokines, c-myc oncogenic transcription factor, obesity-induced hypoxia and warburg effect, and so on. in the future, the potential mechanisms and conclusions in obesity associated with increased risk for developing cancer, and the underlying cellular and molecular mechanisms will be studied. results: among 76 patients with ms (mean age: 58 years; 67% male), 65 (86%) had abdominal obesity, 56 (74%) had a1c > 5.6%, 53 (70%) had fpg > 100 mg/dl and 25 (33%) had homa > 2.6. if we consider as ir indicators: 1. presence of prediabetes criteria (fpg > 100 mg/dl and/or a1c > 5.6%); and/or 2. homa > 2.6, 60 patients (79%) met at least one marker of ir, but 36 of them (60%) had homa <2.6 [11 (31%) of them were on therapy with metformin, an insulin-sensitivity drug]. in the multivariate analysis, presence of type 2 diabetes (hr 1.2; p < 0.05) and presence of prediabetes criteria (hr 1.3; p < 0.05) were associated with homa > 2.6 discussion: although most of the patients with ms have clinical markers of ir (fpg > 100 mg/dl, a1c > 5.6%), only a third part of them have homa levels associated with ir. these observations suggest that: of spilberger's test. from 1995 to 2010 women were followed for the incidence of ah. results: high level of anxiety (hla) in studied cohort revealed in 60.4% of women. women with hla more often tried unsuccessfully to quit smoking compared to lower levels of anxiety (10.7% and 3.9%, respectively; x 2 = 11.25, p < 0.05). women with hla in twotimes less likely to follow the diet (x 2 = 20.87, p < 0.001) and assess their physical activity more passive (x 2 = 18.13, p < 0.01). relative risk (hr) of development of ah in women with hla during the first 5 years of study was in 2.38-fold higher (95.0% ci: 1.137-4.993; p < 0.05), over 10 years it was 1.85 (95.0% ci: 1.075-3.194; p < 0.05) and hr was 1.46 (95.0% ci: 1.023-2.079; p < 0.05) over 16 years of follow-up compared to those with lower anxiety levels. depending on age groups the risk of ah incidence within 10 years was highest in older group with hla aged 55-64 years (hr = 10.2; 95.0% ci: 1.244-83.609, p < 0.05). conclusions: there is high prevalence of hla in russian female population aged 25-64. during 16 years of follow-up women with hla have significantly higher risk of ah especially in older age groups. material and methods: under the third screening of the who "monica-psychosocial" (mopsy) program random representative sample of women aged 25-64 years (n = 870) were surveyed in novosibirsk. d was measured at the baseline examination by means of test "mmpi". from 1995 to 2010 women were followed for 16 years for the incidence of ah. results: the prevalence of d in women aged 25-64 years was 55.2%. women with major d (12%) significantly extended negative behavioral habits: smoking and unsuccessful attempts to give it up, low physical activity, they were less likely to follow a diet. relative risk (hr) of ah in women with d during the first 5 years of study was higher in 1.6-time compared to women who had no d (95.0% ci: 0.86-2.98; p < 0.05). with regard to age groups hr was significant in oldest age category 55-64 years (hr = 1.86; 95% ci: 0.44-7.8; p < 0.05). hr of incident ah in persons with d within 10 years was 1.74 (95.0% ci: 1.01-3.01; p < 0.05) and there were no significant differences in age groups. we did not have risk of ah over 16 years of follow-up in women with d (p > 0.05). the prevalence of d in women aged 25-64 years is more than 50%. women with d had unfavorable lifestyle and higher relative risk of ah over the first 5-10 years of the study. purpose: study the association of high level of anxiety (hla) with vntr polymorphisms d4 and dat genes; determine the relative risk (hr) of arterial hypertension in men with hla. the who " monica-psychosocial" in 1984 monica-psychosocial" in , 1988 monica-psychosocial" in , 1994 surveyed a random representative sample of men aged 25-64 years (2149 men). to assess the level of anxiety was used spielberger's test. cox proportional regression was used for hr assessment. results: the hla in an open population of men aged 25-64 years was 50.9%. since hla genotype was significantly associated 4/6 drd4 gene and genotype 9/9 gene dat. for 24 years hla maximizes the hr of hypertension in the first 5 years. conclusion: there is high prevalence of hla at male aged 25-64 in russian. hla were significantly associated with certain vntr polymorphisms of genes drd4, dat; hla increases the hr of hypertension in the first 5 years. inhibiting beta cell proliferation and promoting its apoptosis which concomitantly leads to decreased β cell mass. dermatology, faculty of medicine, cairo university, cairo, egypt background: psoriasis is a disorder with genetic and immunologic background. leptin can regulate the t-helper response. objective: our primary goal is to study the functional polymorphism (g-2548a) of the leptin (lep) gene in the genetic predisposition of psoriasis, and our secondary goal is to examine factors affecting plasma leptin levels in psoriasis, and to compare patients with and without metabolic syndrome (ms). methods: the study involved 94 psoriatic patients and 100 healthy controls. analysis of g-2548a polymorphism of the lep gene was made by the pcr and restriction fragment length polymorphism technique. the relationship between lep gene polymorphism and the clinical features of the patients was analyzed. plasma leptin levels and proportions of comorbidities in patients vs. controls were compared. results: in controls the ga, aa and gg frequencies were 50%, 30% and 20% respectively, while in patients the distribution of genotypes was 42.5%, 20.2% and 38.3% respectively, with significant difference (p = 0.014) between patients and controls. in patients with ms the gg, ga and aa frequencies were 61.5%, 23.1% and 15.4% respectively, while in patients without ms the distribution of genotypes was 29.4%, 50% and 20.6% respectively, with significant difference (p = 0.014) between both groups. plasma leptin showed a significant higher levels in the patients vs. the controls (p < 0.001), and among the different lep genotypes (p < 0.001) in the patients′ group. conclusion: lep g-2548a polymorphism could be a predictor for higher plasma leptin and increased risk of psoriasis and could be used as a marker for psoriasis-related comorbidity risk. introduction: contractile dysfunction, associated with disturbances in excitation-contraction coupling, has been widely demonstrated in diabetic heart. aims: the aim of this study was to investigate the pattern of mrna encoding cardiac muscle proteins that are involved in the process of excitation-contraction coupling in early onset type 2 diabetic goto-kakizaki (gk) rat. methods: experiments were performed in gk and wistar control rats aged 8-10 weeks. gene expression was assessed in ventricular muscle with real-time rt-pcr, shortening and intracellular ca 2+ were measured in ventricular myocytes with video edge detection and fluorescence photometry, respectively. results: expression of genes encoding some membrane pumps and exchange proteins were unaltered (atp1a1/2, atp1b1, slc8a1) whilst others were either upregulated (atp1a3) or downregulated (slc9a1) in gk ventricle compared to control. expression of genes encoding some calcium (cacna1c/1 g, cacna2d1/2d2, cacnb1/b2), sodium (scn5a) and potassium (kcna3/5, kcnj3/5/8/11/12, kchip2, kcnab1, kcnb1, kcnd1/2/3, kcne1/4, kcnq1, kcng2, kcnh2, kcnk3, kcnn2) channel proteins were unaltered whilst others were either upregulated (cacna1 h, scn1b, hcn2) or downregulated (hcn4, kcna2, kcna4, kcnj2) in gk ventricle compared to control. the amplitude of ventricular myocyte shortening and intracellular ca 2+ transients were unaltered however, the tpk shortening was prolonged and thalf decay of the ca 2+ transient was shortened in gk myocytes compared to controls. conclusions: early changes in expression of genes encoding various cardiac muscle proteins are associated with disturbances in myocyte shortening and intracellular ca 2+ transport. results: analysing level of tnf-alpha, we found that in group 1, there were significantly higher values than the control group by 6.1 times (90.4 ae 1.7 pg/ml: 14.9 ae 0.8 pg/ml, respectively), and significantly higher than patients without insulin resistance (76.4 ae 2.1 pg/ml). analyzing level of il-4, the presence of ir, level of this cytokine increases by only a factor of 2 (61.2 ae 2.9 pg/ml) relative to that of the control group (30.2 ae 1.7 pg/ml). in the second group, the level of il-4 (101.4 ae 3.3 pg/ml) was significantly higher than in group 1 and exceeded the performance of control group by 3.4 times. analyzing the ratio of the level of tnf-alpha and il-4, we have detected a significant increase in the coefficient tnf-alfa/il-4 in group 1 (1.48 ae 0.12) compared with group 2 patients (0.76 ae 0.09). that is, ir in chc patients gives a significant shift in balance of cytokines toward proinflammatory interleukins, which can be observed by increase of tnfalfa/il-4 over 1.37 (p < 0.001 by mann-whitney). conclusions: progression of ir in patients with chc is accompanied by an increase in ratio tnf-alfa/il-4 serum. beyond the value of 1.37, chc patients will an require in-depth study for carbohydrate metabolism. cardiovascular medicine, international university of health and welfare sanno hospital, tokyo, japan background: hyperglycemia has been suggested as a significant factor in coronary microangiopathy in patients with type 2 diabetes (t2dm), but whether it can be reversed through treatment of hyperglycemia is unknown. aim: to clarify whether glycemic control can improve coronary microangiopathy in t2dm. methods: subjects were 34 t2dm who underwent coronary angiography and 17 age-matched controls. myocardial segments perfused by angiographically normal coronary arteries were studied. baseline myocardial blood flow (mbf, ml/min/100 g) and mbf during dipyridamole administration (0.56 mg/kg/min) were measured using positron emission tomography (pet). myocardial flow reserve (mfr) was calculated by the ratio of mbf during dipyridamole administration to the baseline mbf. after the first pet study, patients were subdivided into an additional intensive therapy group (atg) and no-additional therapy (natg) group. second pet scan was performed 6-9 months later. results: baseline mbf was comparable among the atg (87.5 ae 24.1), natg (87.2 ae 16.9) and controls (78.2 ae 33.3). however, mbf during dipyridamole administration was significantly lower in both the atg (170 ae 52.0) and natg (192 ae 52.6) than in controls (293 ae 159, p < 0.01) as was the mfr (atg, 1.96 ae 0.52; natg, 2.27 ae 0.63; controls, 3.69 ae 1.09; p < .01 respectively). mfr was significantly improved in the atg (2.51 ae .77; p < .05), but not in the natg (2.14 ae 0.69; p = ns). there was a significant inverse relationship between percent change in mfr and percent change in glycemic control. however, no significant relationships were seen between the percent change in mfr and percent change in plasma lipid fractions. conclusion: coronary microangiopathy in t2dm can be reversed by intensive therapy for hyperglycemia. l. drimba, r. s ari, j. né meth, z. szilv assy, b. peitl background: our aim was to investigate the effect of "sui generis" hyperinsulinaemia on proarrhythmogenic electrophysiological changes and on cardiac arrhythmias. methods: euglycaemic hyperinsulinaemia was induced in chronicallyinstrumented conscious rabbits equipped with a right ventricular pacemaker electrode catheter. hyperinsulinaemia was induced by either 5 or 10 mu/kg/min insulin infusion and a variable rate of glucose infusion ensured the maintenance of euglycaemia (5.5 ae 0.5 mmol/l). the effect of hyperinsulinaemia on cardiac electrophysiological parameters and arrhythmia inducibility was studied by means of 12-lead surface ecg recording and by programmed right ventricular stimulation (prvs). the role of adrenergic activation was investigated by determination of plasma catecholamine level and by intravenous administration of beta adrenergic blocking agent, propranolol. results: both 5 and 10 mu/kg/min insulin infusion prolonged the pq and the tpeak-tend intervals and shortened the rverp, but no significant changes on other measures of ecg (hr, qt, qtc) were observed. the incidence of prvs-induced ventricular premature beats and non-sustained ventricular tachycardia was higher during euglycaemic hyperinsulinaemia than that of fasting state. we found that higher plasma level of insulin was occurred, the more inducibility of arrhythmias was seen. no change in plasma catecholamine level was observed, but the propranolol restored the prolonged tpeak-tend interval. our results indicate the "sui generis" proarrhythmic effect of hyperinsulinaemia due to reduction of the repolarization reserve in otherwise healthy rabbits. propranolol can be used safely for prevention of arrhythmia in patient with hyperinsulinaemia. euglycaemic hyperinsulinaemia is suitable method to induce acquired long qt syndrome in healthy rabbits. objective: this study investigated the prevalence of metabolic syndrome (mets) and its association with demographic, socioeconomic and behavioral factors in shift workers. a cross-sectional study was conducted on a sample of 902 shift workers of both sexes in a poultry processing plant in southern brazil. the diagnosis of mets was determined according to the recommendations from "harmonizing the metabolic syndrome". the distribution of each of the components of mets was evaluated according to the demographic, socioeconomic and behavioral characteristics of the sample. the multivariate analysis followed a theoretical framework for determining mets on shift workers. the prevalence of mets on the sample was 9.3% (ic 95% : 7.4-11.2). the most frequent altered component was waist circumference (rp 48.4 ; ic 95% 45.5-51.2). after adjustment, the prevalence of mets was positively associated with women (rp 2.16; ic 95% 1.28-3.64), workers of over 40 years of age (rp 3.90; ic 95% : 1.78-8.93) and those who reported sleeping five or less hours per day (rp 1.70; ic 95% : 1.09 -2.24). on the other hand, mets was negatively associated with higher educational level (rp 0.55; ic 0.29-1.06) and having more than three meals per day (rp 0.43 ic 95% 0.26-0.73). in addition, most of the altered components of mets were associated with sociodemographic characteristics, whereas only waist circumference and altered blood pressure were associated with behavioral characteristics. conclusion: sex, age, educational level, eating habits and duration of sleep appeared as independent risk factors for mets. arab world covers a vast geographic area, consists of 23 countries with an approxiimate population of about 358 million people. geographically, arab world is variable ranging from dry desert areas to heavily raining green land. this part of the globe is also unique for its wide cultural, social and ethnic variations. most of the countries are well-heeled with significant natural resources including oil, gas and are benefited from high income. the socio-economic progress has brought benefits in the region such as improved access to health care, education, and safe drinking water. this rapid economical change has also set the scene for the modern lifestyles activities, people are eating more and exercising less. these changes in the lifestyle cause variuos metabolic syndromes including obesity, diabetes mellitus. in spite of marvelous advancement in medical sciences, the most of the metabolic syndromes are still an incurable life-long disease and swiftly increasing in all over the world. presently, six countries including saudi arabia, bahrain, united arab emirates, kuwait, oman and egypt are among the world's highest for the prevalence of metabolic syndromes especially the diabetes mellitus. metabolic syndromes placed a great burden on the public health and clinical practice in the region. v. negrean 1 , o. mislea 1 , i. cheta 1 , t. alexescu 1 , i. chisalita 2 1 internal medicine, umf iuliu hatieganu cluj-napoca, cluj-napoca, 2 umf 'victor babes' timisoara, timisoara, romania introduction: it is known that type 2 diabetes is a major cardiovascular risk factor, but the relationship between impaired fasting glucose (ifg) and the occurrence of cardiovascular events is still undefined. objectives: to determine whether the ifg is a risk factor for cardiovascular disease. results: twenty-four percent of the patients with ifg progressed to diabetes and 8% from the control group were diagnosed with this condition. twenty-one percent of the patients with ifg and 9% from the patients in the control group were diagnosed with hbp. sixty percent from the patients with ifg and 20% of the patients in the control group were obese, 51% from the subjects with ifg and 26% from the subjects in the control group show ischemic heart disease. fifty-five from the patients with ifg and 20% from the patients in the control group declared that they practice no physical activity. there was a significant correlation between the presence of ifg and the hbp (or = 3, p < 0.05), the absence of physical activity (or = 5, p < 0.05), obesity (or = 6, p < 0.05), ischemic heart disease (or = 3, p < 0.05). conclusions: our study shows that ifg is correlated with the cardiovascular risk: patients with ifg have three times higher risk to develop hbp, six times higher risk to be obese and three times higher risk to have ischemic heart diseses. introduction: : the most common investigated factors in chronic inflammation in type 2 diabetes are increased c-reactive protein level, erythrocyte sedimentation rate. the matrix metalloproteinases are a family of proteolytic molecules which contribute to adipose tissue abnormalities. metalloproteinase 2 (mmp-2) has a significant contribution to development of complications of diabetes. aim: : the assessment of the inflammation intensity in patients with type 2 diabetes mellitus and the examination of mixed meal influence on mmp-2 plasma level. materials and methods: : twenty subjects were qualified to this study. all of them were diabetics on insulin treatment. concentrations of mmp-2 were estimated at fasting state and after mixed meal challenge (elisa). we also measured body mass, bmi, systolic and diastolic blood pressure and: esr, crp, daily average glucose, hba1c. results: : mmp-2 concentration values were higher in fasting state in comparison to in postprandial state. mmp-2 correlates with esr, crp, bmi, average daily glucose level and body weight. however, there are no significant correlations between mmp-2 and hba1c. conclusions: : there was a statistically significant positive correlation between serum metalloproteinase 2 and exponents of inflammation, such as erythrocyte sedimentation rate, c-reactive protein. after the mixed meal we observed a significant decrease of metalloproteinase 2 concentration in relation to its concentration in the fasting state. it was shown that the concentration of metalloproteinase 2 depends on short-term metabolic control of type 2 diabetes, not depending on long-term control of the disease. background: several studies suggest increased oxidative stress and reduced endothelial function in obstructive sleep apnoea syndrome (osas). we assessed the association between osas, endothelial dysfunction and oxidative stress. the effect of nasal continuous positive airway pressure (ncpap) on oxidative stress and arterial dysfunction was also evaluated. we studied 138 consecutive patients with heavy snoring. patients underwent overnight home polysomnography. ten patients with severe osas were revaluated after 6 months of ncpap therapy. oxidative stress was assessed by measuring urinary 8-iso-pgf2a and serum levels of soluble nox2-derived peptide (snox2-dp). serum levels of nitrite/nitrate (nox) were also determined. flow-mediated brachial artery dilation (fmd) was measured to asses endothelial function. results: polysomnographic indices were correlated with the metabolic score, insulin levels and central obesity indices. severe osas had higher urinary 8-iso-pgf2a (p < 0.001) and serum nox2 and lower nox. a negative association was observed between fmd and osa severity. apnea/hypopnea index was correlated with urinary 8isoprostanes (r = 0.298, p < 0.001). metabolic syndrome (t = à4.63, p < 0.001) and urinary 8-isoprostanes (t = à2.02, p < 0.05) were the only independent predictors of fmd. after 6-months of ncpap treatment, a significant decrease of serum nox2, (p < 0.005) and urinary 8-iso-pgf2a (p < 0.01) was observed, while serum nox showed only a minor increase. a statistically significant increase of fmd was observed (from 3.6% to 7.0%). conclusions: our study indicates a strong association between osas and metabolic syndrome. patients with osas and cardiometabolic comorbidities have increased oxidative stress and arterial dysfunction that are partially reversed by ncpap treatment. pendyffryn medical group, prestatyn, 2 diabetes centre, royal liverpool university hospital trust, liverpool, uk introduction: metabolic syndrome (mets) and low testosterone levels are independently associated with increased all-cause and cardiovascular mortality. low testosterone levels are associated with obesity, insulin resistance and an adverse lipid profiles in men and the metabolic syndrome and type 2 diabetes have a high prevalence of testicular hypogonadism. the relationship and interaction between these conditions and the potential affect they have on each other is not fully understood. method: this study examined the correlation between testosterone and the criteria of the metabolic syndrome in 552 subjects (age 56.7 years, ae15.4,(18-90.4) (mean, sd (range)), total-testosterone 14.6 nmol/l, ae7.3,(0.1-52), free-testosterone 0.23 nmol/l, ae0.09, (0.04 -0.63), sex hormone-binding globulin level 37.2 nmol/l, ae19.5, (7-170). correlation and significance statistical testing used the pearson correlation coefficient. results: diabetes was present in 22.6% and a further 24.1%(104/431) had the metabolic syndrome. total-testosterone, free-testosterone and shbg had significant and the strongest correlations with age (r = à0.24, à0.38.0.34 respectively, p < 0.05). shbg had the strongest negative correlations with triglycerides (r = à0.31), waist circumference (r = à0.21) and hba1c (r = à0.14), (total-testosterone, triglycerides (r = à0.19), wc (r = à0.17)), all significant p < 0.05. there were positive correlations with hdl (r = 0.29 shbg, r = 0.11 tt, p < 0.05). there were no significant correlations with blood pressure and free-testosterone had no correlation with mets criteria. conclusion: lower testosterone levels have the greatest correlation with ageing but are also associated with detrimental changes in central adiposity and dyslipidaemia. shbg rises with age, however lower levels have a stronger association with cardiovascular risk. this study suggests that cardiovascular risk and androgen abnormalities might be methods: group 1 (gr1) was composed of 30 normal subjects (age = 54.50 ae 2.03; means ae sem). group 2 (gr2) consisted of 20 non-obese type ii dm pts with hypertriglyceridaemia (type iv hlp) and ath (age = 53.19 ae 1.91). group 3 (gr3) consisted of 23 nonobese type ii dm pts with mixed hyperlipidaemia (type iib hlp) and ath (age = 56.73 ae 1.96). following have been determined in serum, in fasting state: total cholesterol (ch), hdl-cholesterol (hdl-ch), atherogenicity coefficient (hac), triglycerides (tg), lipolytic activity (la), lipoprotein fractions, prostaglandins a1 and e1, prostaglandins f2alpha (pgf). following have been determined in plasma, during standard ogtt: glucose, insulin, insulin/glucose index (igi), glucagon, c-peptide, sth, somatostatin, acth, cortisol, aldosterone, beta-endorphin. results: both gr2 and gr3 pts, compared to gr1, had higher body mass, ch, tg, hac, and lower hdl-ch, la, insulin (at ogtt hour 1), igi, sth (hour 2), basal aldosterone. gr2 pts, compared to gr1, had lower sth (hours 1 and 2). gr3 pts, compared to gr1, had higher glucagon (hour 2), somatostatin (hours 0 and 1), cortisol (hours 1 and 2), pgf, and lowerc-peptide (hour 1), sth (hours 0 and 2). conclusions: altered hormonal-metabolic patterns have been observed in non-obese type ii dm pts with ath and dyslipidaemias, including decreased sth and elevated cortisol. hyperlipidemia has been indicated as an important factor of contributing to diabetes progression. however, whether native ldl or modified ldl causes dysfunctional effect of insulin secretion in islet cells is still elusive. the present study aims to identify the mechanisms of electronegtive ldl (l5) and l1 (less electronegtive ldl) acting on the insulin secretion of pancreatic β-cells. rin-m5f cells, were cultured in the complete medium with human native l1, l5, or oxldl. the intracellular concentration of reactive oxygen species (ros) measured by use of dcfhda was significantly increased after loading l5 (50 lg/ml) or oxldl (50 lg/ml), but not effected after loading l1 (50 lg/ml). the cell viability assayed by prestoblue tm reagent was suppressed to 50% after loading l5 or oxldl; however, l1 did not inhibit the cell viability. the insulin release of rin-m5f cells was determined by elisa kit. our results demonstrated that 40% decrease of secretion ratio in the phase of high glucose-induced response after the β-cells were exposed in l5 for 48 h as comparison with in normal medium. we also found that phosphor-c-jun was activated after loading l5 or oxldl, but not after loading l1. the activation of c-jun may modulate the gene expression or the process of insulin secretion. thus we suggest that l5, not l1, is the main subparticle which induces oxidative stress and then lead to dysfunction of insulin secretion responding to high glucose stimulation in β-cells. target group for diet and physical activity interventions due to their increased risk of post-natal weight gain, type 2 diabetes and related health problems. postpartum weight gain and/or retention, particularly in the first 12 months are commonly due to lack of nutrition knowledge, poor dietary habits and physical inactivity. mothers are also integral to the shaping of attitudes and eating and activity behaviours of their children. the proposed study will evaluate whether an individualized weight management program with nutrition and physical activity advice and support enhances weight loss compared with standard care in overweight and obese women and women with a history of gdm. it is hypothesized that, compared with individuals given standard diet and physical activity advice, overweight and obese women, and women with a history of gdm prescribed exercise energy expenditure targets and using heart rate (hr) and dietary intake monitoring with electronic reminders, are better able to achieve a target weight loss (5% reduction from prepregnancy weight for overweight or obese pregravid ( ! 26.5 kg/m 2 ), for overweight women (25.1-26.5 kg/m 2 ) to achieve a weight loss that places them in the healthy-weight range, and for women who were in the healthy-weight range pregravid to return to their prepregnancy weight) through changes in eating and activity behaviours. objective: diabetes is associated with moderate cognitive deficits and neurophysiological and structural changes in the brain, a condition that may be referred to as diabetic encephalopathy. we used high-fat and sugar diet and streptozotocin induced diabetic rats to observe the changes of proteins of insulin signaling which closely correlate with learning and memory. we try to illuminate the possible mechanisms of learning and memory decreased in t2dm. methods: a total of 24 rats were randomly divided into two groups: control group (c), diabetes mellitus group (dm). after 8 weeks, morris water maze was used to perform training trial and probe trial in order to detect spatial learning and memory abilities. and we detected proteins of insulin signaling such as ir, irs-1, akt, p-creb and bcl-2 in the hippocampus of the rats by western blot and immunohistochemistry staining. results: 1. water maze experiment: compared with the c group, the escape latency increased significantly in dm from the 2nd day. in the spatial probe experiment, the first time passing hidden platform prolonged significantly and the distance swimming in the quadrant of hidden platform decreased significantly in the dm. 2. immunohistochemistry staining: compared with the c group, the positive neurons of ir, irs-1, akt, p-creb, bcl-2 increased in the dm group. 3. western blot: compared with the c group, the expression of ir, irs-1, akt, p-creb, bcl-2 increased in the dm group. conclusions: the learning and memory ablities decreased, while the expression of ir, irs-1, akt, p-creb, bcl-2 abnormal increased in t2dm model rats. the results indicate that insulin signal transduction were impaired in t2dm. objectives: niacin, a widely used lipid-modifying drug, is known to induce hyperglycemia during prolonged and high-dose treatments. however, its potential mechanism (s) whereby the islets are involved remains to be determined. we thus aim to investigate the potential role of niacin and its receptor gpr109a involved in regulating islet beta-cell function and insulin resistance. methods: hfd-induced obese mice were employed to study the in vivo effects of niacin. blood glucose/serum insulin levels, oral glucose tolerance test (ogtt)/insulin tolerance test (itt), and homeostasis model of assessment-insulin resistance (homa-ir) were performed to assess glucose homeostasis. real-time pcr, western blot and immunefluorescent assays were used to study the expression of genes of interest. cyclic adenosine monophosphate (camp) and glucosestimulated insulin secretion (gsis) from isolated islets and ins-1e beta cells were determined. knockdown of the gpr109a in ins-1e cells was also examined and compared. results: eight-week treatment with niacin increased blood glucose levels by 25% in hfd-induced obese mice while the areas under curve of ogtt and itt, and homa-ir index were consistently enhanced. in addition, niacin treatment significantly decreased gsis in isolated pancreatic islets. ex vivo and in vitro studies showed niacin decreased gsis, increased mrna expression of ucp2 and gpr109a as well as inhibited intracellular camp accumulation in ins-1e cells. in corroboration, the decrease in gsis and camp levels were abolished by the knockdown of gpr109a. our data indicate that niacin treatments leads to hyperglycemia and impaired pancreatic islet function, which is probably via the activation of islet niacin receptor gpr109a-induced pathway. a once daily glp-1 analogue, liraglutide, is emerging world-wide as a drug for the treatment of diabetes and also, potentially, obesity. this agent not only acts on glycemic control, but also exerts an effect on body weight control, because liraglutide inhibits gastric emptying, resulting in appetite reduction and lower energy intake. herein, we treated 60 type 2 diabetic obese subjects with 0.9 mg/day liraglutide and examined glycemic control and body weight changes over a 6month period. glycemic control was markedly improved (baseline hba1c 9.1 ae 0.2%, endpoint hba1c 7.6 ae 0.2%) with liraglutide. based on subgroup analysis, the good responders to liraglutide were (1) bmi <25. (2) diabetes duration <10 years. (3) postprandial cpr <3.0 ng/ml. while liraglutide-induced gastric symptoms, specifically nausea, occurred in nearly all patients at baseline, it had disappeared in about half by the end of the study. unexpectedly, the hba1c improvement was not associated with the presence of nausea. while acute body weight loss (1.0 ae 0.2 kg) was observed at 1 month, there were no significant body weight changes at the end of the study. individual body weight change was associated with the presence or absence of nausea at the end of the study. in conclusion, the effects of liraglutide on body weight loss lasted only a short period and no chronic effects were observed. thus, the hba1c lowering effect of liraglutide is not due to reduced energy intake, but rather, to recovery from defects in postprandial insulin secretion. background and aims: durability of good glycaemic control may delay development of diabetic complications. early initiation of combination treatment with oral anti-diabetic drugs (oads) having complementary mechanisms of action may increase durability of glycaemic control compared with stepwise addition of oads. dpp-4 inhibitors such as vildagliptin are good candidates for early use in combination with metformin as they are weight neutral with no additional risk of hypoglycaemia. materials and methods: about 2000 drug-na€ ıve patients with type 2 diabetes mellitus (t2dm) with hba1c between 6.5 and 7.5%, will be randomised in verify, a 5-year, multinational, double-blind, parallel group study. the study will test the hypothesis whether early combination therapy with vildagliptin/metformin will result in lower treatment failure rate or in lower rate of loss in glycaemic control over time than with metformin alone. other objectives include evaluation of rate of fasting plasma glucose progression, change in hba1c over time, time to insulin initiation, development/progression of diabetic complications, changes in weight, changes in homa-β/ir, safety and tolerability. insulin secretion rate and insulin sensitivity will be assessed in annual standard meal-test. patients will also be evaluated for early changes in the vasculature, microalbuminuria and retinal microaneurysms. results and conclusions: verify is the first study to investigate the long-term clinical benefits of early combination treatment vs. the standard-of-care metformin followed by addition of oads. verify will provide valuable data on the durability of glycaemic control, βcell function, insulin resistance, safety and tolerability and explore early changes in the vasculature of patients with t2dm. patients with metabolic syndrome are at high risk for developing atherosclerosis. recent studies have suggested glucagon-like peptide-1 (glp-1) signaling to exert anti-inflammatory effects on endothelial cells, although the precise underlying mechanism remains to be elucidated. on the other hand, pparc activation was demonstrated to inhibit the transcription of factors, such as nfjb, resulting in atherosclerosis prevention via suppression of the expressions of cytokines and adhesion molecules in endothelial cells. we investigated whether pparc activation is involved in the glp-1-associated antiinflammatory action in endothelial cells. we constructed an adenovirus expressing the ppre (+)-luc reporter gene for use with the reporter assay system. when we treated huvec cells with 0.5 nmol/l exenatide, endogenous pparc translational activity was significantly elevated by 30% as compared with control cells. the maximum pparc activity enhancing effect of exenatide was observed 12 h after the initiation of exenatide incubation and was approximately 30% of that induced by 1 lmol/l pioglitazone. when incubated with exenatide and pioglitazone simultaneously, pparc activity was additively promoted, suggesting that these two agents synergistically stimulate pparc activity. as h89, a pka inhibitor, abolished glp-1 induced pparc enhancement, the signaling downstream from glp-1 cross-talks with pparc activation. in conclusion, our results suggest that glp-1 has the potential to induce pparc activity, partially explaining the anti-inflammatory effects of glp-1 on endothelial cells. cross-talk between glp-1 signaling and pparc activation would confer major impacts on treatment of patients at high risk for cvd events. in the rat hypothalamus by adenovirus-mediated gene transfer and then examined phenotypes of the rats. dnlkb1 significantly inhibited the thr 172 phosphorylation of ampk alpha2 subunits, while wtlkb1 did not alter phosphorylation, suggesting that hypothalamic ampk is activated in basal states and negatively regulated by dnlkb1. dnlkb1-overexpressing rats exhibited body weight gain and slight insulin resistance as compared with wtlkb1-overecpressing or sham operation-rats. taking into consideration that food intakes did not differ among these rats, this effect was probably due to reduced energy expenditure. in fact, the adipose tissue in dnlkb1-overexpressing rats produced smaller amounts of pgc1alpha and ucp1, resulting in increased adipose tissue weights, as compared with wtlkb1overexpresing rats. neither hepatic fatty acid synthesis nor gluconeogenesis was significantly altered. the phenotypes observed in dnlkb1-overexpressing rats appear to be like those of ampk alpha2deficient pomc neuron mice. in conclusion, our findings demonstrated inhibition of hypothalamic lkb1 to lead to reduced energy expenditure and body weight gain, suggesting that central lkb1 is involved in bodyweight regulation probably via ampk modification. material and method: anthropometric variables and lipid related factors concentration were measured. pcr and rflp were performed. the distributions of a polymorphic site and its relationship with mentioned factors were examined. result: in normal subjects there wasn't any relationship between these three polymorphism and studied profile, but in females with mets, presence of g allele in rs2075291 significantly increase diastolic blood pressure, low density lipoprotein and apolipoprotein a1 (apo a1) level and the c allele in rs662799, increase waist circumference, triglyceride and apo a1 level. while previous studies in adults demonstrated that snps in apoa5, have primarily been associated with plasma lipoprotein levels and associated downstream consequences, such as weight gain and heart disease risk, in present study we find that there are some relation between this variation and lipid profile in female with metabolic syndrome and this relationship is sex dependent. tryglicerides mean value was 155 mg/dl and cholesterol 142 mg/dl; the mean value was slightly elevated but inside the lot of the patients the variations were wide probably due to liver inssuficiency (low values) and alcooholic etiology of underlying liver disease (higher values). serum uric acid had a mean value of 6.9 mg/dl. no large variations inside the lot. medium level of serum creatinine 1.3 mg/dl; higher values in child c cirrhosis and hcc. conclusions: most of the metabolic disturbancies in hcc are correlated with the underlying liver disease. in a few cases of small lesions but mostly in large and complicated hcc these are significant and require specific treatment. background: a significant inter-individual variability in statin treatment efficacy is likely to have a strong genetic background. gene for slco1b1 belongs to the candidates with potential to influence the statin treatment efficacy. slco1b1 codes for solute carrier organic anion transporter, which has been shown to regulate the hepatic uptake of statins and some other drugs. materials and methods: slco1b1 rs4149056 (t>c) polymorphism was successfully analysed in the group of 253 patients with dyslipidemia (treated with simvastin or atorvastatin, 10 or 20 mg per day) and 470 healthy normolipidemic controls. the polymorphism was analysed using nested pcr-rflp. lipid values (total-, ldl-and hdl-cholesterol, triglycerides) were analysed before and after 10-13 weeks of treatment. results: after treatment, as expected, there was a significant decrease both in total (7. self-care management), were applied to all patients at baseline and 3month follow-up. t-test and chi-square test were used to analyze the data. after 3 months, 55 (out of 74) and 30 (out of 48) patients remained in the study. main findings revealed, a significant difference in a1c level between the groups (p < 0.001). the self-care management score increased in both groups, but the increase was significantly higher in the intervention group (p < 0.001 methods: after the treatment decision was final, patients were assigned to either vildagliptin or other oads (sulphonylurea, thiazolidinedione, glinide, a-glucosidase inhibitor or metformin except dipeptidyl peptidase-4 inhibitor or glucagon-like peptide-1 mimetic/ analogue). demographic data and patient history, especially risk factors and macro-and microvascular complications, were collected and reported by the investigators. results: in total, 45,868 patients were enrolled in 27 countries across the world. baseline characteristics are presented in the table. hba1c was better controlled in east asia and europe than in india, latin america or middle east. patients in europe had higher bmi and longer duration of diabetes than patients in east asia and india. prevalence of risk factors such as hypertension and lipid disorders was high overall, but particularly higher in europe. macro-and microvascular complications were reported in 7.3% and 12.4% of the overall study population, respectively, and their prevalence was higher in europe. conclusions: data from edge study show that the hba1c goal of 7% as recommended by international guidelines is not achieved worldwide and metabolic control varies remarkably between regions. in addition to the high prevalence of concomitant risk factors, complications were reported already after 5 years of diabetes in a substantial proportion of patients. aim: sudomotor dysfunction due to small fiber neuropathy can be observed very early in pre-diabetes. the aim of this study was to assess the predictive power of ezscan, a non invasive, quick and simple measurement of sudomotor function to identify glucose impairment. research design and methods: the study was performed in 76 german subjects at risk of diabetes. glucose metabolism was assessed by using, oral glucose tolerance test (ogtt) at baseline and after 2 year follow-up. sudomotor function was evaluated by measuring hand and foot electrochemical sweat conductances to calculate a risk score. results: at baseline, 38 patients had normal glucose tolerance (ngt), 34 had pre-diabetes (impaired fasting glucose, ifg and/or impaired glucose tolerance, igt) and four had newly diagnosed type 2 diabetes. the auc values for fpg, 2 h-ogtt glucose, 1 h-ogtt glucose, hba 1c and ezscan score to predict pre-diabetes were 0.50, 0.65, 0.64, 0.72 and 0.76 respectively. subjects having a moderate or high ezscan score (>50) at baseline had a substantially increased risk for having ifg and/or igt at follow-up visit presented by an odds ratio of 12.0 [1.4-100.5 ], the or for having 1 h-ogtt ! 8.6 mmol/l at follow-up was 9.8 [1.0-92.8 ] and for having hba 1c ! 5.7 % was 15.7 [1.9-131.5 ] compared to subjects with low ezscan risk. conclusions: this preliminary study, which must be confirmed in a larger population, shows that ezscan measurement is associated with diabetes progression which may have implications for prevention and disease management. methods: two groups are involved to the studies -the group of 50 patients with metabolic syndrome (ms) and control group (c)-20 healthy, age matched volunteers. volunteers were expose to 3-h ogtt (according to who) and 8-h oltt (contained 80 g of fat: 50% saturated, 40% monounsaturated and 10% pufa). during both tests the blood glp-1, gip, glucose, insulin and free fatty acids (ffa) levels were assessed. results: fasting level of incretins do not statistically differ between ms vs. c participants. only ffas were elevated during whole oltt, when the glucose concentrations decreased in early postprandial period. secretion of gip was activated by ogtt as well as by oltt, however concentration of gip in oltt was higher. output of glp-1 during whole ogtt was significantly lower in patients with ms. in patients with ms amount of gip released during oltt was lower compared to control patients. conclusion: low level of incretins during oxidative stress connected with fat food intake, may not provide the protective effect for metabolically stressed pancreatic beta-cells. background: who estimates that 7.5 million deaths worldwide are due to hypertension, approximately 12.8% of all deaths. this is an important risk factor forcardiovascular disease. argane oil is an integral part of the moroccan diet. several studies showed that an argane oil supplemented diet decreased systolic and diastolic blood pressure measurments in animals and suggested that consumption of argan oil may have a beneficial effect in preventing cardiovascular disease. objective: to study the effect of a regular consumption of argane oil on hypertension in healthy postmenopausal women. methods: seventy-seven postmenopausal women (55.49 ae 6.17 years) were assigned to consume 25 ml of argane oil during 8 weeks of nutritional intervention. anthropometric (weight, height and bmi) and clinical profile (blood pressure) have been determined at 0 and 8 weeks. results: showed that systolic blood pressure was significantly reduced (125.1 ae 13.4 to 119.6 ae 15.1 mmhg) after 8 weeks (p = 0.0212). diastolic blood pressure underwent a slight decrease (81.5 ae 8.6 to 79.8 ae 8.8 mmhg) but not significantly (p = 0.2307). conclusion and perspective: these results suggest that consumption of argane oil can be relevant to prevent cardiovascular disease into postmenopausal women and help to decrease cardiovascular risk. the positive impact on blood pressure recorded by a significant decrease in sbp and hypertension is a significant result, however, the mechanisms involved in obtaining this result need to be defined more accurately, focusing mainly on the effects of certain constituents of argane oil as gamma-tocopherol on the mechanisms regulating blood pressure. the epidemic of obesity is associated with multiplication of prediabetic patients. recognition of them is essential as this state is considered to be the last chance to prevent the manifestation of diabetes. our aim was to determine metabolic alterations in healthy men with (dr: n = 14) or without (h: n = 14) first degree 2dm relatives. volunteers were adjusted according to age and bmi, insulin resistance was determined with hyperinsulinaemic-normoglycemic clamps and ßsejt function by iv glucose tolerance test. fasting glucose, insulin and ffa values were not different among the groups, but at the and of ivgtt, glucose levels were higher (h:5.8 ae 1.5, vs. dr: 7.2 ae 1.4 mmol/l, p < 0.05), injected glucose did not suppressed ffa levels (h: 0.17 ae 0.1 vs. dr:0.36 ae 0.2 mmol/l, p < 0.001) and first phase insulin secretion was decreased in dr group (h:104 ae 63 vs. dr:59 ae 33; p < 0.01). there were no differences among the groups in total body-, muscle and fat tissue glucose disposal, leptin and resistin levels, but the adiponectin levels were significantly lower in dr group (h: 5.28 ae 2.3 vs. gd:2.86 ae 1.7 mg/ml, p < 0.001) and the ffa/adiponectin ratios were higher (h: 0.10 ae 0.08 vs. gd: 0.29 ae 0.2, p < 0.01). in conclusion the impairment of insulin secretion and fatty acid metabolism are the earliest sign of diabetes risk in men with first degree 2dm relatives. the measurement of ffa/ adiponectin ratio could be a simple parameter to screen adult male relatives of diabetic patients for identification of genetic risk of diabetes. the original reaven′s definition from 1988, which has been revised in 1993, missed the abdominal obesity. the definition accepted by who in 1999 was not applied due to difficult evidence of the insulin resistance. a new definition of the ms formulated by idf and ead was published in 2005. in this case, the abdominal obesity was the necessary condition of the ms diagnosis. in order to unify the criteria, five essential parameters of the ms were put on the same level by the international medical organizations in 2009. it is not easy to define the ms. it cannot be identified as a single disease, as it consists of a complex of problems. that is why the definitions of the ms were rearranged several times during past years and the used parameters are much stricter. we can assume that in the future this process will proceed. aims: the metabolic syndrome (ms) represents an obesity-related severe health problem, and its prevalence is world-wide increasing in parallel with the growing obesity epidemiology. gwa studies have shown that many single nucleotide polymorphisms (snps) in several genes are involved in common obesity. the aim of this study was to look for associations between snps in the mc4r (rs12970134, rs477181, rs502933), sirt1 (rs3818292, rs7069102, rs730821, rs2273773, rs12413112) and fto (rs1421085, rs9939609, rs9930506, rs1121980) genes and obesity and/or ms in a southern italy population. methods: one-thousand unrelated non diabetic severely obese patients (mean bmi 46.9 kg/m 2 , mean age 32.8 years) and 100 controls (mean bmi 23.2 kg/m 2 , mean age 29.9 years) entered the study. mc4r, sirt1 and fto were genotyped by real time taqman assay. anthropometric, clinical and biochemical data were collected for all enrolled subjects. ms was diagnosed according to the american heart association criteria. results: metabolic syndrome was diagnosed in 37.2% of our patients. the four fto snps were significantly associated with the obese phenotype (0.0001 < p < 0.007). at binomial logistic regression analysis, only snp rs9930609 was significantly associated to obesity after correction for sex and age (or/95%ci: 2.5/1.4-4.4 and 3.8/1.9-7.5, for the heterozygous and the homozygous mutated genotypes, respectively) and to ms presence (or/95%ci: 2.5/1. 1-5.5) . conclusions: this study confirms that fto is a susceptible gene for obesity risk, and patients bearing the polymorphic allele in the rs9939609 snp could be at high risk of ms insurgence, possibly to be addressed toward preventive programs. results: number of patients who achieved hba1c goals according age and presence of cvd are shown in table 1 . conclusion: t2d patient of middle age reached hba1c goals in lowest percentage of cases. among different treatment groups percentage of patients reached hba1c goals were the lowest in group treated by insulin with oad. the prevalence of diseases arising mainly due to the bad lifestyle is increasing. it is necessary to find optimal tools to lower the prevalence of the metabolic syndrome (ms). there is no network of institutions that systematically cooperate in the field of education and treatment of the ms. for students and academics the possibility of establishing (18) and deepening the cooperation in this scientific field is therefore limited. the role of the project is to strengthen relations between the institutions in the form of cooperating network, which will meet the essentials principles of the primary, secondary and tertiary prevention of the metabolic syndrome. the key role of the project is to open an professional discussion and deepen the communication and professional relationship across the cooperating institutions which are realized primarily through internships of students (bachelor and doctoral degree), academic staff, roundtable discussions with experts and partners from the collaborating institutions, organizing workshops and doctoral and scientific conferences. czech society of sports medicine ( cstl) is a professional guarantor of creating a network of cooperating institutions in terms of the objectives and scientific credibility. background and aims: tissue ages accumulation is thought to be a specific marker of long-term glycaemic control, oxidative stress and cardiovascular risk. prediabetes -impaired fasting glucose (ifg) and impaired glucose tolerance (igt), are considered as risk categories for the development of both type 2 diabetes and cardiovascular disease. the aim of the present study was to assess advanced glycation end products (ages) in prediabetes and their relation to anthropometric and glycaemic control parameters. material and methods: 185 subjects (mean age 51.6 ae 11.6 years, mean bmi 30.4 ae 5.2 kg/m 2 ) were enrolled. according to glucose tolerance they were divided into two age-matched groups -91 subjects with ngt and 94 with prediabetes (ifg and igt). glucose tolerance was studied during ogtt applying 2006 who criteria. plasma glucose was measured by a hexokinase method, hba1c was assessed immuno-turbidimetricaly. tissue ages accumulation was assessed non-invasively measuring the skin fluorescence of ultraviolet light on the ventral side of the lower arm (age-reader-diagnopticstm). antropomethric measurements -weight, height, waist circumference, were performed. visceral fat area was estimated by bioimpedance method (inbody720). results: no significant difference in ages accumulation was found between the groups with prediabetes and ngt. significant positive correlation was observed between ages accumulation, age (r = 0.0001) and visceral fat area (r = 0.014). the non-invasive assessment of tissue ages accumulation probably is not a sensitive enough method for identifying subjects with prediabetes and increased cardiovascular risk. at the early stages of glucose homeostasis impairment ages accumulation appears to be related to age and visceral obesity rather than to glucose tolerance. this study is an observation study for mild diabetic males with bmi of 30 or higher with subsequent 6 month-follow-up based on single administration of exenatide and dietary and exercise intervention. method: subjects were 19 cases of male patients (age: 42.3 ae 11.1, bmi 36 ae 6.1) with apnea hypopnea index (ahi)≧30/hr and 7.5>hba1c>6.5%. based on polysomnography and measurements of ct abdominal visceral fat area and various biomarkers before and 3 and 6 months after exenatide administration, the observation study was conducted for 6 months. results: decrease in bodyweight was observed by 9.7 ae 6.8 kg on average during the 6 months with no cases to gain in bodyweight and bmi. ahi was significantly improved from 39.6 ae 8.4/h to 18.3 ae 9.6/h. improvement was observed in hba1c, 8-ohdg, pro-bnp, high molecular weight adiponectin and visceral fat areas but not in hs-crp and subcutaneous fat area. conclusion: not only diabetes but also sleep apnea syndrome was improved by exenatide administration in japanese mild diabetes patient. weight reduction may have played the primary role. effect of weight reduction provided by the pharmacological property of exenatide is useful to improve sleep apnea syndrome. currently, changes in individual calorie intake and nutrient composition during the study period have been analyzed. [1.064-8.100 ], p = 0.049 in igt group. no significant association was found between baseline fasting insulin level and progression from either ifg or igt to type 2 diabetes. conclusions: individuals with ifg or igt identified through high-risk strategies in a bulgarian population, have a rather high risk of developing diabetes within 1 year. baseline proinsulin and proinsulin: insulin ratio, known to reflect beta-cell dysfunction, appear to be independent predictors for progression to diabetes in both ifg and igt. introduction: the insulin receptor substrate 1 (irs-1) seems to be an important factor involved in the modulation of insulin signalling in adipose tissue. in this study we want to check the expression of irs-1 in visceral and subcutaneous adipose tissue, its relationship with insulin resistance and with the metabolic syndrome. material and methods: we measured irs-1 expression in visceral and subcutaneous adipose tissue from 30 morbidly obese patients. we have determined the level of insulin resistance with the homa-ir index. patients were classified into two groups based on whether or not to have metabolic syndrome (according to the idf 2005 criteria) results: irs-1 expression in subcutaneous adipose tissue is significantly higher than in visceral adipose tissue (p = 0.002). homa-ir was significantly correlated with the irs-1 expression in subcutaneous adipose tissue (r = à0.449, p = 0.041), but not in visceral adipose tissue (p = 0.198, p = 0.223). the morbidly obese patients with metabolic syndrome have significantly lower irs-1 expression levels in subcutaneous adipose tissue than those without metabolic syndrome (p = 0.012). in visceral adipose tissue, the levels of irs-1 are lower but not significant (p = 0.200) in the morbidly obese patients with metabolic syndrome. the presence of metabolic syndrome in morbidly obese patients is associated with a lower irs-1 expression level in subcutaneous adipose tissue. in the general population and in hiv+ subjects the hypertension is the major risk factor worldwide for cardiovascular morbidity and mortality. this condition has been accompanied by several complication, including dyslipidemia and impaired glucose metabolism. for all these reasons identification of hypertension is of pivotal importance in hiv infected patients. aim of this study was to evaluate the incidence of hypertension and comorbidity in male hiv+ patients. we enrolled only male patients attending two clinics of the infectious diseases in center of italy (chieti and ancona). three hundred and four patients accepted to participate at the study. viroimmunological, lipid and metabolic parameters, including triglycerides, cholesterol, hcv/hbv co-infection, tabacco use were measured at the time of enrollment. the study has shown an incidence of hypertension (esh guidelines) 36% (109/304 patients), dyslipidemia 55% (167/304), diabetes 7% (22 / 304), hcv or hbv co-infection 23% (78/304) and tobacco use 49% (150/304). patients with hypertension showed dyslipidemia in 53% of cases (58/109)and diabetes in 12% (13/109). remarkable that only 49% of patients with hypertension were treated for hypertension. in addition, the average age of the patients in the study was 46.7 ae 10.3 years. the study has shown a high incidence of hypertension in hiv+ men, even considering the young age of the patients. furthermore, hypertension is associated with a high incidence of co-mobility. hypertension is associated with evidence of under treatment, it showing a poor perception of the problem in this context. method: this study was a 4-weeks', prospective trial in subjects with metabolic syndrome. metabolic syndrome was defined as the presence of at least three out of five risk factors according to the ncep-atp iii with the asian criteria of abdominal obesity (abdominal circumference; >90 cm in men, >80 cm in women). all participants received individualized education by skilled personnel with information about tlc. blood chemistry including lipoprotein profiles and anthropometric data were collected before and after 4 weeks' tlc. result: eighty-six subjects were screened, and 67 subjects with metabolic syndrome were enrolled. body weight was not significantly changed after tlc. fasting blood glucose levels were not significantly changed (from 111.8 ae 11.7 to 110.1 ae 18.4 mg/dl, p = ns). therapeutic lifestyle change did not result in significant changes in total cholesterol (from 229.6 ae 21.1 to 226.5 ae 26.3 mg/dl, p = ns) and triglyceride (from 177.0 ae 56.6 to 168.2 ae 73.4 mg/dl, p = ns). but, 4 weeks' tlc resulted in significant reduction in ldl-cholesterol levels (from 161.1 ae 21.2 to 147.6 ae 25.1 mg/dl, p < 0.001), increase of hdl cholesterol levels (from 37.7 ae 6.8 to 41.8 ae 8.4 mg/dl, p = 0.001). conclusion: four weeks' therapeutic lifestyle change improved lipoprotein profile (especially ldl-c and hdl-c) in metabolic syndrome. our findings indicate that the importance of tlc with education should be emphasized for the control of metabolic syndrome. nutrition, hasanuddin university, school of medicine, makassar, indonesia obesity in major public health and economic problem of global significance. the prevalence of obesity in children has increased significantly, although less rapidly in indonesia. from the public health view, it is disconcerting that the prevalence of adolescent obesity has increased by nearly 40% in the past two decade. case and control study design was done in makassar, the participant is senior high school students. the study aim to identify the risk factors of adolescent obesity to premetabolic syndrome by measuring body mass index, waist circumference, lipid profile (cholesterol, trigliceride, hdl, ldl and apo b) and fasting oral glucose. data was analyzed using spss program, the relationship among variables was calculated with pearson correlation and regression test. the indicators for obesity using waist circumferences and bmi, energy intake using 24 h food recall and was analysed using wfood2. the study showed, a positive correlation between waist circumference and small density ldl, apo b, cholesterol (p < 0.007, p < 0.000, p < 0.01). student with abnormal waist circumference tend to have abnormal biochemical markers (or 0.34, 5.76, and 2.99 ) as a risk premetabolic factors conclusion: nutrition education and food balance diet should be given to adolescent obesity to prevent metabolic syndrome in later of life. l. mundbjerg 1 , g.f. thomsen 2 , r. holst 3 , c. juhl 1 1 department of endocrinology, 2 department of occupational medicine, hospital of south western denmark, esbjerg, 3 institute of regional health research, region of south denmark, odense, denmark introduction: severe obesity is associated with reduced worker productivity and chronic absence from work. gastric bypass surgery is the most effective treatment of severe obesity. the objective of this study was to measure employment status in danish gastric bypass patients before and after surgery. methods: the study is a nation-wide retrospective case-control register study. data were extracted from three sources: 4927 cases were identified in the danish national patient register according to the operational code for laparoscopic gastric bypass surgery and matched on a 1:2 basis with respect to age, gender and residence municipality with control subjects. the employment status was calculated from the danish national labour market authority's databasedatabase (dream) which includes information on all public transfer incomes. by linking the databases we achieved a valid measure of the amount of working days. results: the amount of working days was significantly lower in cases compared to controls. there was a significant decline in employment status during the observational period in both groups. this decline corresponded with the timing of the financial crisis (employment data collected over the period january 1st 2007 to december 31st 2011). there was no significant difference in the decrease of employment status between groups. conclusion: gastric bypass surgery patients are affected by the economic recession in a similar degree as the general population. thus, the patients do not seem to benefit from the operation nor do they appear particularly vulnerable in periods of recession. excessive accumulation of fatty tissue in obesity associated with fat and glucose metabolism. fish oil contains n-3 pufas epa and dha had been demonstrated in several studies had effects on the expression of genes ppar-a and srebp-1c, the pathomechanism still controversial. this study aims to determine the effect of fish oil on insulin resistance in mice obesity. clinical trials using c57bl/6j mice was conducted in animal laboratory medical faculty of hasanuddin university. the mice was giving a normal diet (nd) or high fat diet (hfd) for 12 weeks. mice was divided by four groups; normal diet (nd), high-fat diet (hfd), hfd + 3 g/100 g fish oil (hfd-fo), hfd + metformin 3 g/kg diet (hfd-met) as a positive control for 8 weeks. gene expression of ppar-a and srebp-1c from the liver were measured by rt-pcr. the study showed, hfd mice had significantly higher body weight compared to nd. hfd-fo have higher blood glucose levels than other groups. ppar-a expression in liver was lower in hfd than nd, but increased in the hfd-fo and hfd-met compared with hfd alone. srebp-1c expression decreased in hfd-fo and hfd-met, glucose metabolism of hfd_fo tended to decrease, while the hfd-met is likely to increase. we conclude, fish oil improved insulin resistance, decreased expression of srebp-1c and increased expression of ppar-a in the liver tissue through decreasing lipogenesis and increased fat oxidation in the liver. methods: total 541 subjects were divided into two groups having whr >0.85 as obese and whr <0.85 as non-obese. circulating il-6 and resistin level, fasting blood glucose, insulin and lipid profile were estimated. insulin resistance was calculated by the homeostasis model assessment (homa) index. the genotype and allele frequency of il6 -g174c gene polymorphism was determined by pcr-rflp method in 269 obese and 272 non-obese adult women from north india. results: the genotype distribution of il6 -g174c gene polymorphism was statistically significant in obese women (p = <0.001; or = 9.428; 95% ci = 4.56-19.492) compared to non-obese women. the circulating level of serum resistin was highly significant in obese women (15.11 ae 8.95 vs. 7.74 ae 4.86 , p = <0.001) compared to nonobese. significant association was found with cc + gc genotype of il6 -g174c promoter gene polymorphism in case of waist circumstance, serum triglyceride, homa index and serum resistin level (p = 0.031; or = 0.498, 95% ci = 0.262-0.953). conclusion: serum resistin is associated with the disorder of metabolism of glucose and lipid in metabolic syndrome. the relationship between this hormone with cc + gc genotype of il6 -g174c promoter gene polymorphism suggests that they may take part in the development of metabolic syndrome in north indian adult women. the comorbidities associated with excessive weight are major causes of morbidity and mortality, thus causing further reduction in quality of life. the dysfunction of excessive fat and its peculiar distribution plays a decisive role in the development of metabolic disorders. the aim of the study was to estimate the prevalence and the correlates of obesity and dysglycemia in a rural community. overweight and obesity were defined according to world health organization criteria, considering both the value of body mass index (bmi) and waist circumference (wc). dysglycemia (impaired fasting glucose -ifg and diabetes) was established based on ada recommendations. the study included 3248 people, 41.28% men and 58.71% women, with the mean age of 50.58 ae 18.26 years. more than half of them (52%) had an excessive weight: 66.66% were overweight and 33.33% were obese. the crude prevalence of overweight and obesity was 33.9% and 12.2% respectively for men, and 35% and 21.3% respectively for women. the prevalence of diabetes was 5.2% and for ifg of 3%. the prevalence of dysglycemia increased with the bmi value, with the highest frequency in the grade iii obesity group (16.7% prevalence of diabetes, ifg 8.3%). large wc was identified in 74.9% of the studied population (53.5% in men, 89.9% women). the individuals with diabetes had a significant higher mean wc (106.23 cm vs. 96.6 cm, p < 0.001). these results show that excessive weight and abdominal obesity could become a public health problem in romania, thus requiring national screening and educational programs. objective: hyperuricemia is associated with obesity; however, few studies reported the effects of surgery types on uric acid metabolism for severe obesity. the current study was aimed to explore the effects of gene and bariatric surgery on uric acid reduction and to identify the potential pathways. subjects: all participants were han chinese, aged from 20 to 50 years old. a total of 508 severely obese patients with at least body mass index (bmi) of 35 were recruited in the beginning of the study, where 164 cases received laparoscopic adjustable gastric banding (lagb) and 344 cases received laparoscopic mini-gastric bypass (lmgb). a 12-month follow-up was ensued after surgery to identify the effects of bariatric surgery and estrogen receptor-a (esr1) gene on serum uric acid reduction. results: a tagsnp (rs712221) of esr1 could influence serum uric acid reduction. bariatric surgery effect on serum uric acid reduction was greater in lmgb as compared with lagb at the 12th month of post-surgery (à2.3 ae 2.1 mg/dl vs. à1.2 ae 1.1 mg/dl, p = 0.002). obese patients carrying risk genotype (tt) on rs712221 and exhibiting better glycemic control had a greater serum uric acid reduction at the 12th month of post-surgery. synergic effect of rs712221 and lmgb exhibited the highest serum uric acid reduction at the 12th month of post-surgery (à2.7 ae 1.4 mg/dl). conclusion: for severely obese han chinese, bariatric surgery appears to reduce serum uric acid levels by mediating different factors, including esr1 gene and gender, ameliorating glycemic control, and changing dietary patterns. remained significantly lower. compared to mgc, mgr but not mgrc male offspring had higher body fat % and visceral adiposity at 6 months of age. results: the average age of patients was 56.36 years, more dominant were females with 83.08%. patients with ms characterizes increased values of bmi (32.22 kg/m²), which is statistically significantly more that in the control group, where is average value of bmi 24.67 kg/m², waist 107.33 cm in the study group, and 89.09 cm within control group (p < 0.001), blood pressure 139.4/84.9 mmhg, homa index 5.16, average value of insulinemia 18.56 (lu/ml), (within control group homa1 index was 1.60), the average value of insulimenia 7.37 (lu/ml), average values of: total cholesterol 6.47 mmol/l, hdl cholesterol 1.34 mmol/l, ldl cholesterol 3.38 mmol/l, triglycerides 2.77 mmol/l, relation between ldl/hdl 2.74. arterial hypertension was present in 93.08%, hypercholesterolemia in 46.15%, reduced hdl in 14.62%, increased ldl in 66.15%, trigliceridemia in 89.23%. conclusion: abdominal obesity is characterized by manifested insulin resistance and distinct hyperinsulinemia. arterial hypertension as one of the components of metabolic syndrome represents significant cardiovascular risk factor, increased level of triglycerides represents significant risk factor which favors atherosclerosis, in our study the presence of this parameter was 89.23%. introduction: diabetes is the most feared disease because it leads to a variety of complications including end-stage vascular disease, cardiovascular damage and retinal abnormalities. the increased risk of atherosclerotic disease in diabetic subjects may be due to enhanced foam cell formation following an increased susceptibility of low density lipoprotein to oxidative modification. cardiovascular disease (cvd) is the most prevalent complication of diabetes mellitus. methods: the aim of this study was to assess the ldl susceptibility to lipid peroxidation (ldl ox) in two study groups of elderly patients (aged 67 ae 13 years): a group of patients with cardiovascular disease and a group of patient with cardiovascular disease associated with type 2 diabetes mellitus. the ldl susceptibility to in vitro induced lipid peroxidation was evaluated following its incubation with a prooxidant system. : results obtained showed the susceptibility of ldl to in vitro oxidation was increase in diabetic group (14.58 %) compared with a cardiovascular disease group. conclusion: this study indicates that low-density lipoprotein from diabetic subjects is more susceptible to oxidation. patients with diabetes have a greatly increased relative risk of developing cardiovascular disease when compared with patients without diabetes. much of this risk is related to insulin resistance and is associated with both traditional and nontraditional cardiovascular risk factors. therefore, measurement of ldlox may be helpful for identifying high-risk patients with type 2 diabetes and cvd. janus kinase (jak) and signal transducer and activator of transcription-3 (stat3) in several cell lines. objective: we sought to determine the role of hypothalamic s1pr1 in the control of jak2/stat3 signaling and food intake in vivo. materials and methods: western blot, immunohistochemistry, gas analyzer, dissection of the hypothalamic nuclei and intrecerebroventricular (icv) infusion of s1p (50 ng), leptin (10 à6 ) and cucurbitacin (50 lmol/l) were combined to evaluate the role of s1pr1 on leptin signaling and on food intake in lean and obese wistar rats and in ob/ob mice. results: high expression of s1pr1 was found in the hypothalamus when compared to other peripheral tissues. s1pr1 is mainly expressed the arcuate nucleus of the hypothalamus, in the same neurons that possess stat3. icv infusion of s1pr1 activator, s1p, increased jak2 and stat3 phosphorylation and the energy expenditure and reduced the food intake in lean rats. in addition, s1p potentiated the effects of leptin in the reduction of food intake. conversely, the pharmacological inhibition of stat3, blocked the anorectic effect of s1p. interestingly, low expression of s1pr1 was observed in the hypothalamus of ob/ob mice and wistar rats fed on high fat diet, whereas, s1p infusion reduced the food consumption and increased leptin signaling and action in obese rats. these results indicate that hypothalamic s1pr1 has a key role in the control of leptin signaling and on food intake. aim: the adifit program is an intensive 12-week multidisciplinary treatment program which promotes weight reduction among obese. during the first 6 weeks behavior change, physical exercise, and nutritional counseling is offered three times a half a day per week, thereafter once a week half a day. the aim was to evaluate efficacy of the adifit program in weight reduction, and its effect on eating behavior, and body image. methods: patients were evaluated at baseline (t0) and after 3 month (t1). bmi was assessed. psychometric instruments such as the german versions of the eating behavior questionnaire (fev), and the body image questionnaire were used at both time points. paired-sample t-test and wilcoxon signed rank test were used for statistical analyses. results: fifty patients participated, 86% were female. bmi was significantly reduced from t 0 to t 1 (mean ae sd: 41.1 ae 6.6 vs. 39.8 ae 6.6; t = 7.68, p < 0.000). results discerned significant improvements from t 0 to t 1 in fkb scale 1 (negative attitudes towards one′s own body) (34.6 ae 6.0 vs. 32.2 ae 6.1; t = à3.68, p < 0.000), and scale 2 (restricted body dynamics) (28.2 ae 7.0 vs. 31.2 ae 6.3; t = à3.86, p < 0.000). on the other hand, fev scale 1 (cognitive restraint of eating) improved significantly from t 0 to t 1 (8 vs. 15, z = à5.72, r = à0.57, p < 0.000). fev scale 2 (disinhibition) was significantly lower at t1 (11 vs. 6, z = à5.84, r = à0.58, p < 0.000) as was fev scale 3 (hunger) (8 vs. 4; z = à4.98, r = à0.49, p < 0.000). after 12 weeks patients showed significant reduction in weight, improvements in body image and eating behavior. metabolic peptides in serum and plasma samples contain status information for diabetes. previously, we have demonstrated that intrinsic protease activity caused instability of plasma and serum peptides. other reports have described that metabolic peptides, including glp-1, gip, glucagon, and oxytomodulin, are subject to instability caused by proteolytic and other enzymatic degradation intrinsic to plasma. the variability may result in inaccurate quantitative measurements of the peptides creating challenges when interpreting pharmacokinetic and pharmacodynamic data. using both mass spectrometry and elisa based detection methods; we investigated the instability of metabolic peptides in whole blood, serum, and plasma under a variety of routine clinic conditions. first, the same subjects blood was drawn into different blood collection devices including serum and edta, citrate, heparin, and an edta tube containing enzyme inhibitors. the metabolic peptides of interest were spiked into plasma for time incubation at different temperatures. samples were quenched and processed for both maldi-tof ms and elisa analyses. quantitative analysis of each marker was used to characterize the kinetics of the peptide digestion ie stability (halflife). further a blood collection tube was developed to minimize degradation using enzymatic inhibitors specifically for the stabilization of glp-1, gip, glucagon, and oxytomodulin. the addition of specific enzyme inhibitors for stabilization of each peptide reveals more insight regarding enzymatic degradation and stabilization of the peptide biomarkers. our data clearly demonstrates the need of enzymatic inhibitors for peptide stabilization, especially in a clinical setting. (198.55 ae 70.43 mg/dl) . 105 subjects underwent a standardized meal test: 50 g of bread, a boiled egg, 100 g of apple, in the morning after 10 h fasting. venous blood glucose samples:before meal test (sample 0), 1 h (sample 1)and 2 h (sample 2) after ingestion of standardized meal. neuropathy (p = 0.007). women had higher prevalence of neuropathy (p = 0.014). people with sensory neuropathy had lower weight and bmi than those with autonomic neuropathy and those without neuropathy (p = 0.060). fasting blood glucose was higher in subjects with neuropathy (p = 0.037). value of sample 1 was increased in people with neuropathy (p = 0.063). other parameters cardiometabolic risk factors were not associated with diabetic neuropathy at onset of t2d. conclusions: diabetic neuropathy is a frequent complication at diagnosis of t2d. people with higher fasting or postprandial glycemia may associate more frequently diabetic neuropathy at onset. m. metalla 1 , m. carcani 2 , g. qirjako 3 , e. demiraj 4 1 durre regional hospital, 2 endocrinology, durre regional hospital, durres, 3 statistical, mother teresa university hospital center, tirana, 4 internist, durre regional hospital, durres, albania background: metabolic syndrome is a risk factor for cardiovascular diseases and is associated with abnormal cardiac structure and function. the aim of the study: to evaluate the left ventricular structure and function in patients with metabolic syndrome, without installation of diabetes mellitus or arterial hypertension. material and methods: it was analyzed a total of 40 consecutive adults (10-{25%} males and 30-{75%} females) with metabolic syndrome and 20 healthy (10-males and 10 females) without metabolic syndrome. metabolic syndrome was defined using the atp lll criteria. assessment is done with classic echocardiography, pulsed wave doppler and tissue doppler. results: there was difference in two groups in bmi, in waist circumference, in systolic and diastolic arterial presure in total colesterol, in level of triglicerid and hdl colesterol p < 0.001. there was no diference in age, fasting serum glucose level, lvedd, sw, pw, lvm, lvmi, lav, lvef, early trans mitral inflow (e), late trans mitral inflow (a) and in the rate e/a (p = 0.2). echokardiografic measurements by tdi in peak mitral anular velocity in early diastole ea were 9.1 ae 2.4 vs. 11.9 ae 1.7 cm/sec (p < 0.001) in the metabolic syndrome and controll grups respectively. average values of sa were significantly lower in the ms than in controll group, 3.1 ae 0.2 vs. 4.3 ae 3.0 (p < 0.001). the ratee/ea mitral was 8.4 ae 2.2 vs. 6.7 ae 1.6 (p < 0.01) in metabolic syndrome and controll group respectivly. the early identification of isolated syndrome in non diabetic, non hypertensive adults may be an indication of an aggressive preventive measure. the fasting glycaemia altered and hyperinsulinemia are strong predicting factors of type 2 dm (dm2) and sedentary habits can lead to this disease, mainly associated with obesity. objectives: verify the influence of physical activity, vo 2max and anthropometric measurements on glycaemia and insulin fasting in active military over the age of 35 of the brazilian army (ba) serving in rio de janeiro. methods: two hundred and fifty subjects (aged 42.6 ae 4.8 years), in active duty in the ba, volunteered to participate in the study. the insulin and glycemic levels were measured as well as body mass, stature and waist circumference. body fat (bf) was measured by hydrostatic weighing, when body density was obtained. vo 2max was measured by maximal cardiopulmonary treadmill exercise test (cpet) using an individualized ramp protocol. oxygen consumption and carbon dioxide production were measured using a cpx-d metabolic cart. results: the prevalence of type 2 dm found was 0.8% and afg was 14.4%. vo 2max showed an inverse correlation (p < 0.001) with insulin levels, homa-ir and fasting glycaemia and also with anthropometric measures and %bf. the bmi and %bf presented direct correlation (p < 0.001) with insulin, homa-ir and fasting glycaemia. subjects in the highest quartiles of vo 2max and in the lowest quartiles of wc and of %gc also presented lower levels of fasting glycaemia and insulin and homa-ir. the data suggest that vo 2max was an inverse and nutritional state a direct relationship between glycaemia and fasting insulin. subjects with an active lifestyle have less prevalence of alterations in glycaemia and fasting insulin levels. the increase in prevalence of cardiovascular conditions, and metabolic syndrome (ms) observed in the last decades was accompanied by increase in dietary fructose (fr) consumption (also as sucrose). the aim was to assess the prevalence of fructosemia in cardiovascular patients with or without ms and to investigate the possibility of treatment of hyperfructosemia and other components of ms with oral acarbose. material and methods: fasting serum fr concentration in 245 cardiovascular patients with metabolic syndrome (group 1) and in 69 cardiovascular patients without ms (group 2) was measured by colorimetric method with commercially available biovision set. 33 patients of the first group were treated with acarbose (glucobay, bayer) increasing the dose from 150 to 300 mg per day to normalize the glucose metabolism. fasting and post oral sucrose load (150 g) serum levels of glucose, fr, insulin, nefas and uric acid (ua) were measured at baseline and in 6 days. results: fasting serum fructose concentration varied widely and among patients in both groups, and was significantly higher in group 1 (390.0 ae 81.2 vs. 349.1 ae 67.7 lmol/l). the data after 6 days and 4 weeks of acarbose use is presented in the conclusions: dyslipidaemia is found to be the risk factor for ischemic stroke in diabetic subjects, with statistically significant differences compared to nondiabetics. hdl-cholesterol was found as a protective factor for haemorhagic stroke in nondiabetic subjects. agave tequilana weber variety azul is an economically important in mexican people because it is the sole plant allowed for tequila production but because it is a potential source of prebiotics, the inulin-type agave are nondigestible/fermentable carbohydrates which are able through the modification of the gut microbiota, the pos content of agave inulin differs from inulin extracted from chicory root. the aim was to assess the efficacy and safety of inulin type agave on lipid profile in dyslipidemic obese subjects. a clinical trial, open was carried out in 30 obese, hypertrygliceridemic and hypercholesterolemic subjects between 18 and 60 years old. all the subjects received 15 g/day of inulin in the morning, during 60 days. biochemical and metabolic profiles before and after pharmacological intervention were performed. after inulin administration, there was a significant reduction of the trygliceride concentrations (197.5 ae 72.9 and 179.1 ae 37.0 mg/dl; p = 0.042). glucose serum (83.0 ae 13.4 and 77.6 ae 12.9 mg/dl; p = 0.007), and hba1c (5.8 ae 0.4 and 5.7 ae 0.3 % p = 0.007). there was not a significant reduction of total cholesterol, low density lipoprotein and very low density lipoprotein. anthropometric parameters did not change in the group and soluble fiber intake did not produce any gastrointestinal adverse effect. the increase of fiber intake (inulin type agave) are efficacy and safety to reduced trygliceride concentrations levels in dyslipidemic obese patients. stress and strain have long been associated with the work people do. we aimed to investigate occupational stress index and influence of several different work stressors on cardiometabolic risk factors: diabetes, lipid levels in hypertensive workers in south serbia methods: we studied 817 persons (professional drivers, construction workers, production line workers and bankers): 504 composed group with hypertension (40-60 years of age, majority males), 313 were age and sex matched controls without hypertension. clinical examination was performed and blood was sampled. we analyzed work stressors by using questioners with 79 different factors and occupational stress index (osi) was calculated with permission of dr belkic. comparison was made regarding total burden and the nature stress burden (underload, high demand, strictness, extrinsic time pressure, aversive/noxious exposures, threat-avoidant vigilance/disaster potential, conflict/uncertainty) results: diabetes was highly present in bankers (46%) and glucose levels were significantly higher in this subgroup. the incidence of diabetes in construction workers was 15%. total cholesterol, ldl, triglycerides were higher and hdl lower in bankers and contraction workers (p < 0.001) and linearly correlated with osi (p < 0.001). previous myocardial infarction suffered 17.3% of bankers. total osi was significantly higher in diabetic hypertensive bankers and construction workers (p < 0.001), as well as high demand, strictness and extrinsic time pressure (p < 0.001). conclusion: hypertension appearance in working population is related to high osi, especially when other cardiometabolic risk factors added. further steps are needed to reduce the level of work stressor and provide a better quality of live in individuals. background and aim: plasma levels of adipocytokines in healthy individuals or diabetes mellitus patients have been previously reported as well as salivary levels of many adipocytokines. nevertheless, salivary levels of some adipocytokines in patients with metabolic syndrome have not been investigated. the aim of this study was to evaluate salivary and plasma levels of leptin and adiponectin in patients with metabolic syndrome. results: compared with healthy individuals, leptin levels in patients with metabolic syndrome were significantly increased, both in plasma (6.624 ae 0.774 vs. 15.123 ae 1.396 ng/ml, p < .001) and in saliva (26.053 ae 4.644 vs. 41.523 ae 5.320 pg/ml, p = .036). whereas plasma adiponectin levels were decreased significantly in patients with metabolic syndrome (31.164 ae 3.331 vs. 17.084 ae 1.802 lg/ml, p = .001), salivary adiponectin levels were inversely increased (2.003 ae 0.438 vs. 6.392 ae 1.508 ng/ml, p = .017). conclusion: this study showed that, similarly to plasma, there was an elevated change in salivary leptin levels in patients with metabolic syndrome compared to healthy individuals. however, for salivary adiponectin levels, the opposite result to plasma adiponectin levels was revealed. replacing saliva to plasma for detecting altered leptin and adiponectin should be concerned in patients with metabolic syndrome. objective: to examine dietary intake in family members with diabetes type 2 (dt2) to identify contributing factors to glycemic control and development dt2. design: descriptive and prospective study involving 192 members of 40 families who provided food intake from 3 days, 2 weeks days and 1 weekend day. inclusion criteria were proband member diagnosed with dm2 with ! 2 siblings and living parents. methods: data were collected at the participants' homes or at the university clinic. the biometric measures were glucose and hemoglobin glucose (hba1c). data was entered into nutritional data system to research, the "gold standard" for nutritional analysis. also, the data entry into spss v17 was done. : of 192, (57% women, mean age 47.82 ae 17.49; , mean blood glucose 131.33 mg/dl ae 64.17; 57-347 and mean (hba1c) 7. 43% ae 2.33; . preliminary data indicate intake total fat was higher than the recommendations 94 % of participants, while carbohydrate intake was higher in 78%. method: this was a retrospective, observational study of consecutive cardiac bypass patients that underwent surgery at sultan qaboos univesity hospital in muscat, oman, between 2009 and 2012. analyses were performed using descriptive statistics. the study included a total of 101 cardiac bypass surgery patients with an overall mean age of 60 ae 10 years and 76% (76/101) were males. mean body mass index was 27 kg/m 2 ranging from 17 to 38 kg/m 2 . twenty-seven percent of the patients were either past or current smokers while 16% (16/101) had a family history of coronary artery disease. all but 2 of the patients (98%) were on statins pre-op. the most prominent co-morbidities were hypertension (93%), angina (90%), diabetes mellitus (72%), myocardial infarction (52%), and congestive heart failure (36%). the mean average pre-op total cholesterol, low-density lipoprotein cholesterol (ldl-c), high-density lipoprotein cholesterol (hdl-c), triglycerides, apolipoprotein a1 (apo a1), and apolipoprotein b (apo b) were 4.4 ae 1.2 mmol/l, 2.6 ae 1.0 mmol/l, 0.97 ae 0.27 mmol/l, 1.9 ae 1.4 mmol/l, 1.13 ae 0.18 g/l, and 0.91 ae 0.25 g/l, respectively. eighty-one percent, 13%, and 6% of the patients had 3 or 2 or 1 diseased heart vessels, respectively. there was a total of 58%, 30%, and 10% of the patients that had on-pump, off-pump, and on-pump beating coronary artery bypass surgeries, respectively. mortality was recorded in two patients (2%) in an approximately 4-year period. conclusions: patients undergoing cardiac bypass surgery were observed with a high prevalence of cardio-metabolic risk factors. aim: to evaluate the association between osteocalcin and phenotypic characteristics of metabolic syndrome. material and methods: data of 276 children and adolescents participants of the project "health worker cohort study" was analyzed. to each participant we determined: insulin, glucose, triglycerides, hdl, waist circumference and blood pressure. glucose and insulin were evaluated by glucose-oxidase method and homa. total osteocalcin concentration was determined by chemiluminescence. body mass index (bmi) was evaluated according to cdc chart′s for children and adolescents according to age and sex. using multiple lineal regression and logistic regression we evaluated the association between osteocalcin values and components of metabolic syndrome. results: the proportion of male in the study population was 51.5%. the prevalence of overweigh and obesity was 33.4% and abdominal obesity was 26.4%. the proportion of elevated glucose, hypertriglyceridemia, low levels of hdl, hypertension and metabolic syndrome were 4.4%, 17.5%, 50.6%, 2.2% and 7.0% respectively. difference of medians according to ages groups, was observed for glucose, insulin, homa and osteocalcin (p < 0.05). the study showed a negative correlation between bmi, fat percentage, waist circumference, glucose levels and crp with ostelcalcin levels (p < 0.05). conclusions: serum osteocalcin levels were associated with some phenotypic characteristics of metabolic syndrome and measures of adiposity. background: previous studies show that hyperuricemia precede the development of the metabolic syndrome and can be a risk factor to diabetes type 2, cardiovascular disease and heart failure, independently of obesity. aim: to identify the association between hyperuricemia and cardiovascular risk factors (crf). the study population included 746 adolescents, all of them participants from the "cohort study of health workers". crf were evaluated according to idf pediatrics criteria. the cut-off points for hyperuricemia was levels of uric acid >7 mg/dl, for hyperinsulinemia >20 mu/l, insulin resistant (ir) have homa >3.16. a multivariate regression analyses was used to evaluate the association between hyperuricemia and crf. resultados: the proportion of female in the study population was 52%. mean age was 14 ae 3.7 years, prevalence of bmi >p85 was 30%, abdominal obesity (26.1%), hypertension (2.8%), hyperuricemia (10.7%), glucose ! 100 (3.9%), hypertrigliceridemia (19%), low levels of hdl (62%), hyperinsulinemia (18%), ir (26%), crp (11.3%) and ms (8.85%). proportion of hyperuricemia was six times higher in men, (19 vs. 3.1, p < 0.01). the study show association between hyperuricemia and age (or:2.2;ic 95% 1.3-3.6), hypertrigliceridemia (or:2.8;ic 95% 1.6-5.0) hyperglucemia (or:3.4;ic 95% 1.2-9.4), hyperinsulinemia (or:3.1;ic 95% 1.7-5.5), ir (or:2.7;ic 95% 1.5-4.7), abdominal obesity (or:6.0;ic 95% 3.5-10.2) and ms (or:6.2;ic 95% 3.2-11.8). conclusions: hyperuricemia is associated to crf. hyperuricemia is not considered for adolescent health diagnostic. however early diagnostic is important for avoid the presence of several crf. method: pancreas of fetuses and offspring from diabetic and nondiabetic sprague-dawley rats were obtained and processed for histological and morphometric studies and for inmunohistochemical analysis of pdx. 1, insulin and glut2. results: fetuses of diabetic mothers, showed a significant delay in the morphogenesis of the islets, with a significant reduction of the diameter, area and beta cell mass and expression of pdx1, insulin and glut2. these fetal alterations had an impact on postnatal life and offspring of diabetic rats had significantly higher glucose levels. in offspring of 20 days, the alterations persisted and the expression of insulin and glut2 was significantly lower. at 28 days the islets were mature but the size, beta cell mass and expression of insulin, glut2 and pdx.1 were still significantly lower. at 90 days the structure of the islets was normal, but the reduction in size, beta cell mass and expression of glut2 persisted, but with no significant reduction in the expression of insulin and pdx.1 conclusions: in utero hyperglycemia induced a delay on the differentiation of beta cells and morphogenesis of islets, a reduction of the beta cell mass and in the expression of insulin, glut2 and pdx.1. these alterations persisted up to adulthood causing hyperglycemia to offspring. g. yoshino 1 , t. an 2 , s. nakano 2 , k. kuboki 2 1 center for diabetes, shinsuma general hospital, kobe, 2 division of diabetes, metabolism and endocrinology, department of internal medicine, toho university, tokyo, japan background and aim: it has been a matter of debate whether the intervention is necessary even for elderly metabolic syndrome subjects. present study is therefore conducted to explore whether the elderly subjects with metabolic syndrome show increased cad risk measuring plasma small, dense ldl-cholesterol, hs-crp and plaque score of the carotid artery. methods: a total of 130 subjects including 88 male ranging from 30 to 85 years-old, were recruited for this study. they are divided into four groups according to their age (below 64 and above 65 years-old) and an association of metabolic syndrome. blood sampling was done after overnight fast. small, dense ldl-cholesterol was measured employing direct assay method. results: in the non-elderly subjects, plasma triglyceride, small, dense ldl-c, hs-crp and plaque score of carotid artery were all increased in the metabolic syndrome group compared to that of non-metabolic group, while there was no significant difference in either hs-crp or the plaque score between the two groups within elderly subjects. within the elderly metabolic syndrome group, subgroup with type 2 diabetes had higher fasting blood glucose, hs-crp, small, dense ldl-c and plaque score than the subgroup without type 2 diabetes. conclusion: metabolic syndrome is an important factor for progression of subclinical atherosclerosis in the non-elderly subjects, but it cannot be a significant determinant of subclinical atherosclerosis if the subjects are limited within elderly group without type 2 diabetes. thus, intervention for metabolic syndrome may not be obligatory for elderly metabolic syndrome subjects if they are not diabetic. methods: a retrospective cross-sectional study of randomly selected 101 patients on antihypertensive drugs at the outpatient clinic at sultan qaboos university hospital, muscat, oman. the recorded variables included age, gender, history of diabetis mellitus, weight, fasting blood glucose, creatinine level, systolic blood pressures (sbp), diastolic blood pressure (dbp), and type of medication. all parameters were collected of the last two visits from (january 2011 and december 2011) for each patient who had been labeled with persistent hypertension using the hospital information system (his). analyses were conducted using descriptive statistics. results: overall mean age of the cohort was 58 ae 11 years, 56% were male, 47% were diabetic, mean sbp 137 ae 20, dbp 76 ae 14, weight 77 ae 17, and fbg 7.4 ae 3. on visit 2, 88% were on b-blockers, 26% patients were on calcium channel blocker, 26% on angiotensinconverting enzyme inhibitor and 14% were on diuretics either as a monotherapy or in combination. target bp was attained in 23.40% patients with diabetes mellitus compared to 37.04% in non diabetics after the second visit. the mean body weight in patients with controlled hypertension was 73.6 kg compared to 78.8 kg in patients with uncontrolled hypertension. the study showed that diabetes mellitus, increased body weight and high fasting blood glucose reduces the attainment of blood pressure goal in patient on antihypertensive therapy. purpose: comparable evaluation of lipid profile and inflammatory markers between patients with stable angina (sa) and patients with sa and diabetes mellitus of type 2 (dm). material and methods: a total of 66 patients with sa (mean age 57.4 ae 7.5 years) with coronary stenosis <70% were examined. group i included 37 patients with sa and dm; group ii -29 patients with sa without dm. lipid profile parameters; inflammatory markers (hs-crp, tnf-alpha, homocysteine, interleukine 1 β, 6, 8; scd40 l, mmp-9, timp-1); endothelial dysfunction markers (endothelin-1, nitrites) were measured initially and in 1 year. results: there were high levels of hs-crp, tnf-alpha, lipoprotein (a), mmp-9, triglycerides, and endothelin-1 in both groups. the level of timp-1 reduced in both groups. patients in group 1 had significantly elevated levels of total cholesterol, ldl cholesterol, homocysteine, apo-b, apo-b/apo a-1 ratio, il-1 β. in group 1 the following positive correlations were found: between glycohemoglobin and apo-b, apo-b/apo a-1 ratio, homocysteine; il-6 and hs-crp; homocysteine and ldl cholesterol, mmp-9, duration of coronary artery disease; endothelin-1 and scd40l, tnf-alpha. reliable increasing homocysteine, tnf-alpha mmp-9, ldl cholesterol were revealed in both groups. conclusions: prospective study showed that both groups had similar blood biochemical abnormalities. however in patients with dm there was an increase in the levels of atherogenic lipid fractions as well as homocysteine, tnf-alpha and mmp-9 which may indicate a higher risk of developing coronary events even in the absence of significant coronary stenosis. objectives: il-18 is a pleotropic cytokine and is reported to be involved in various inflammatory and immune-mediated disorders. previous data in apo e-/-mice demonstrated that il-18 accelerates atherosclerosis via ifn-c and cxcl16 expression and the effect was independent of t-cells. we therefore investigated whether il-18 is involved in cholesterol efflux and plaque stability. methods: two groups of chow-diet-fed, male apolipoprotein e-/mice, aged 12 weeks (n = 6/group) were fed a normal diet and injected intraperitoneally for 30 days with either recombinant il-18 (30 ng/g/ day) or with phosphate buffer saline (pbs). mrna expression of il-18, scavenger receptor cd36, mmp-9 and lxr-a genes was determined by real-time pcr. immunohistochemistry was also performed for expression of above genes. results: il-18 administration led to a significant increase in serum cholesterol and lipoproteins except hdl-c which was decreased. in heart and aortic tissues, expression of il-18, scavenger receptor cd36 and mmp-9 genes increased 3.5, 8 and 3 fold respectively, whereas expression of lxr-a gene was reduced by 0.6 fold. atherosclerotic lesion size was quantified in the ascending aorta and the aortic arch. exogenous il-18 administration significantly increased frequency of atherosclerotic lesions and lesion area in il-18 treated mice vs. control animals (1.15 ae 0.71% vs. 5.39 ae 1.70% (p < 0.01; pbs vs. il-18 group). the observed data strongly implicates il-18 as a proatherogenic and proinflammatory molecule which not only enhances inflammation but augments cd36 and mmp-9 expression which may lead to enhanced foam cell formation and plaque instability and thereby aggravate atherosclerosis. objective: the present study was attempted to found the association between tnf-a promoter gene g-308a polymorphism with metabolic syndrome and insulin resistance. the g-308a tnf-a polymorphism has been studied in 305 subject with metabolic syndrome according to ncep atp iii criteria (age 31.91 ae 6.05; bmi 28.1 ae 5.09) and 310 healthy control without metabolic syndrome (age 30.96 ae 7.01; bmi 22.98 ae 4.36). the g-308a variant was detected by pcr amplification and nco-1 digestion. furthermore insulin resistance, serum leptin and tnf-alpha levels were also measured in both the groups. polymorphism was significantly less frequently observed in the control population as compared to study group. furthermore, on dividing the subjects into two groups according to the absence (homozygous for the wild type g allele) (tnf-1 allele) or presence of the mutant a (tnf-2) allele, significantly high levels of tnf-a (p = 0.001, or = 1.80, 95% ci = 1.25-2.60) and leptin (p = 0.003, or = 2.19, 95%ci = 1.43-3.36) were observed in tnf-2 group as compared to tnf-1 group. whereas, there was a non-significant tendency toward insulin resistance in the tnf-2 group. conclusions: our results suggest that the g-308a mutation of the tnf-a gene is likely to play an important role in the development of metabolic syndrome and metabolic abnormalities. metabolic syndrome is associated with residential-area crime rates for men and perceived crime for women: gender differences in social vulnerability to metabolic syndrome introduction: crime rates and perception of crime within neighbourhoods have been linked to residents' cardiometabolic outcomes. men and women vary in their perception of crime but share exposures to residential-area crime. it is unclear whether gender differences exist in the nature of crime-related vulnerability to metabolic syndrome. aim: to assess gender-specific associations between perceived and reported crime, and metabolic syndrome (mets) methods: cross-sectional data from a biomedical cohort study of adults randomly selected from the north-west region of adelaide, south australia, were analysed (n = 1.444) (mean age = 54.5, standard deviation = 14.2). clinically measured mets was defined using international diabetes federation criteria. perceived crime was expressed as a standardised factor score reflecting six items related to neighbourhood safety and crime. reported crime rates were obtained from police data, aggregated at the statistical local area level (n = 26) and standardised. associations were evaluated using multilevel regression models accounting for area-level clustering and covariates including area-level income and participant sociodemographic factors. results: the prevalence of mets was, for men (n = 680), 41.9% (95% ci = 38.2-45.7), and for women (n = 764), 29.1% (95%ci = 25.9-32.4). for men, mets was associated with rates of violent crime (or = 1.22, 95%ci = 1.01-1.47) and total crime (or = 1.21, 95% ci = 1.01-1.46), but not perceived crime. for women, mets was associated with perceived crime (or = 1.26, 95%ci = 1.05-1.50), but not reported crime. conclusions: crime is an adverse social exposure. mets is associated with perceived crime in women and reported crime rates in men. these differentials suggest gender-specific causal pathways by which awareness and perception of adverse social exposures relate to mets independent of socioeconomic factors. background: epidemiological studies suggest that the resting heart rate (rhr) is an independent predictor of cardiovascular and all cause mortality. however, the power of the rhr to predict cardiovascular events in patients with the metabolic syndrome (mets) is not known. methods: we prospectively investigated the relationship between rhr and cardiovascular events in 756 consecutive patients undergoing coronary angiography for the evaluation of coronary artery disease (cad) over a follow-up period of 7.1 ae 0.1 years. the mets was defined according to ncep-atpiii criteria. results: in the total study population, both all cause and cardiovascular mortality were increased with an increasing rhr (standardised adjusted hrs 1.03[1.01-1.04]; p = 0.001 and 1.15 [1.03-1.47 ]; p = 0.001, respectively). from our patients, 357 (47.2%) had the mets and 399 did not have the mets. among patients without the mets, a higher baseline rhr indicated a significantly higher risk of total mortality (h = 1.14[111-1.16], p = 0.001) and cardiovascular mortality (hr = 1.13 [1.12-1.16 ], p = 0.001) after multivariate adjustment. however, the rhr did not significantly affect total mortality (p = 0.120) or cardiovascular mortality (p = 0.244) in patients with the mets. interaction terms rhrxmets were significant for both total and cardiovascular mortality (p = 0.027 and p = 0.037, respectively), indicating that the respective risks conferred by a high rhr were significantly higher in patients without the mets than in patients with mets. conclusion: we conclude that among angiographically characterized coronary patients, the metabolic syndrome status significantly affects the association of the rhr with total and cardiovascular mortality: rhr is a strong predictor of both total and cardiovascular mortality among subjects without the mets, but not among mets patients. aim: to estimate influence of diet therapy, physiotherapeutic treatment to body mass reduction and prevention of cardiovascular and metabolic risks. patients and methods: were surveyed 62 patients with a metabolic syndrome aged of 24-52 years: 35 women (56 %) and 22 men (44 %): 8 (13 %) pts were overweight, 34 (55 %) pts had bmi 32.6 ae 0.39 kg/sq.m, 20 pts (32 %) -bmi 37.6 ae 0.38 kg/sq.m. 77 % pts had hypercholesterolemia, 48 % -glucose intolerance, 27 % -high arterial blood pressure. all patients were undergone by the assessment of nutrition status, biochemical researches. there was executed food allowance correction and added complex of physiotherapeutic treatment. control examination was carried out in 4 weeks by body structure definition by bioimpedance analysis and biochemical blood tests. results: there were noted that level of cholesterol decreased from 6.4 ae 0.22 mmol/l to 5.0 ae 0.28 mmol/l (p 0.05), level of blood glucosefrom 6.27 ae 0.34 mmol/l to 5.6 ae 0.3 mmol/l. there was registered decreasing systolic arterial pressure of 92 % of patients to 135.3 ae 3.99 mmhg (p 0.05) and diastolic at 82 % pts -to 93.3 ae 1.19 mmhg (p 0.05). there were fined reduction of a fatty component on 3.1 ae 0.61 by kg, with preservation of active cellular weight -0.5 ae 0.28 in kg (p < 0.05) in patients. conclusions: physical exercises, physiotherapeutic procedures jointly balanced food allowance promote more considerable decrease in weight of body and decrease in cardiovascular and metabolic risks. results: after adjustment for sociodemographic and lifestyle factors, the relative risk (rr) of pd comparing subjects with metabolic syndrome to those without it was 0.41 and the 95% confidence interval (95% ci) 0.22-0.74. this association was especially due to high serum triglyceride levels ( ! 1.7 vs. <1.7 mmol/l, rr 0.43, 95% ci 0.23-0.81), and high plasma glucose levels ( ! 5.6 vs. <5.6 mmol/l, rr 0.52, 95% ci 0.28-0.98). the results remained after excluding the first 10 years of the follow-up. after this exclusion and further adjustment for other components of the metabolic syndrome, the suggestive association between pd and bmi was strengthened ( ! 25 kg/m 2 vs. <25 kg/m 2 , rr 1.75, 95% ci 1.00-3.07). conclusions: high serum triglycerides and plasma glucose levels predicted low pd incidence, even after excluding the presumable preclinical disease phase. also, the suggestively increased pd risk in subjects with high bmi varied according to the follow-up time, proposing that an adequate time period should be considered to account for the preclinical disease phase in pd. pancreatic intrinsic nervous system (pins) maturation goes on postnatally and is involved in the control of pulsatility and amplitude of insulin secretion (is), both altered in obese and diabetic patients. the aim of our study was to determine the impact of obesity in infancy on pins maturation and control of is. c57bl/6j mice aged 4 weeks received a normal (nd) or a western diet (wd) for 12 weeks. 4 weeks old mice were used as initial controls (t0). after sacrifice, pancreases were placed in organ incubators for 1 h. the impact of pins upon is was studied by adding to the culture the nicotinic receptors agonist dmpp in presence or absence of l-name (inhibitor of nos) or snp (no donor). insulin was assayed in culture supernatants. pins density and phenotype were determined by ihc. pins density was less in nd compared to t 0 mice, whereas there was no difference between wd and t 0 mice. cholinergic innervation significantly increased with age in both wd and nd mice whereas nitrergic innervation increased in nd mice and decreased in wd. pins stimulation by dmpp induced a time-dependent increase in is, significantly larger in nd compared to wd mice. is profile was identical in wd and t 0 mice. addition of l-name inhibited dmppinduced is in nd mice while snp tended to reduce it. neither l-name nor snp altered is in wd mice. our study suggests that wd induces neuroplastic changes in the pins that could be involved in pancreatic dysfunctions observed during obesity. purpose: inflammation drives the progression from central obesity to insulin resistance, metabolic syndrome and hypertension. whether inflammation caused by allergic diseases such as allergic rhinitis can predispose to hypertension is controversial. therefore, we studied the association between hay fever and hypertension in the united states national health and nutrition examination survey (nhanes). we analyzed data on 1883 men and 2029 women in nhanes 2005 nhanes -2006 . we included participants aged 20 years or older who had valid data on hay fever and hypertension. results: 13.5% of the participants had a previous diagnosis of hay fever and 36.2% of them had hypertension. there were ethnic differences in the prevalence of previous hay fever diagnosis (p < 0.001) and hypertension (p < 0.001). overall, there was no significant association between previous hay fever diagnosis and hypertension. in women aged 20-39, there was an association between previous hay fever diagnosis and hypertension (or = 2.59, 95% ci = 1.26-5.30, p = 0.013). this association was not diminished after adjustment for age, race and body mass index (or = 2.74, 95% ci = 1.48-5.06, p = 0.003). after further adjustment for physical activity, alcohol consumption, smoking, liver enzymes, c-reactive protein and ige level, the association remained significant (or = 2.72, 95%ci = 1.19-6.22, p = 0.021). conclusions: in this nationally representative population-based survey, previous hay fever diagnosis is not significantly associated with hypertension in adults, except for women aged 20-39. further work is needed to confirm that this is a true association. the university of hong kong, hong kong, hong kong s.a.r. objective: beta-2 microglobulin (b2m) is the light chain of the major histocompatibility complex class 1 molecule. glycation of b2m renders it toxic. serum b2m level predicts mortality in chronic kidney disease and some malignancies such as multiple myeloma. we hypothesized that it also predicts mortality in people with diabetes. research design and methods: 897 participants of the third national health and nutrition examination survey aged 20 or above who had diabetes or were on medication for diabetes were included in the analysis. results: during a median follow-up of 11.8 years (range 0.1-18.2 years) and 9.222 person-years, 207 (16.8%) and 542 (42.4%) participants died from diabetes-related causes and all causes, respectively. tertile 3 of b2m was significantly associated with allcause (hazard ratio (hr) = 3.28, 95% ci: 1.54-7.00) and diabetesrelated mortality (hr = 4.03, 95% ci: 1.20-13.59). the association was independent of cardiometabolic risk factors, cancer, microalbuminuria and impaired glomerular filtration rate. conclusions: serum b2m level is a novel independent predictor of diabetes and all-cause mortality in people with diabetes. an elevated level is associated with a substantially increased risk of death. metabolic syndrome (ms) refers to a constellation of factors that increase risk of cardiovascular diseases (cvd) and diabetes. insulin resistance and associated abnormalities is considered to be a link between obesity and cvd. our ongoing study investigates relationship of anthropometric, metabolic and cardiovascular parameters in young patients with incipient ms (young lean subjects with essential hypertension; ht) and obese otherwise healthy subjects (ob) in comparison to healthy lean controls (c) similar for age and sex. methods: all subjects underwent the oral glucose tolerance test. the insulin sensitivity index according matsuda (isimat) and ir homa were calculated. fasting serum concentration of total cholesterol (tchol), hdl cholesterol, triglycerides (tg), and uric acid as well as fasting and post load plasma glucose and insulin concentrations were measured. results: young lean ht patients had comparable bmi but higher body fat percentage (p = 0.03), higher fasting plasma insulin (p = 0.03), slightly higher uric acid (p = 0.05), and higher t-chol (p = 0.01) than c. despite ngt, ht patients had lower isimat (p = 0.02) and higher ir homa (p = 0.03) than controls., but these parameters were comparable to the ob group. 2/3 of young lean ht patients similarly to ob otherwise healthy subjects exhibited three and or more features of ms. conclusions: young lean patients with hypertension displayed signs of insulin resistance comparable to obese subjects and metabolic abnormalities typical for metabolic syndrome. early life style interventions might prevent theto development of complete ms in these young ht and ob subjects. increased prevalence of overweight in male patients, whereas women had increased prevalence of obesity (grade ii and iii). hypertensive diabetic subjects with other metabolic risk factors are more prone to microvascular and macrovascular complications. methods: eighty-six patients were divided into two groups. the group 1 included 46 subjects (mean age 47.20 ae 9.6 years) with ah and ao (mass body index (mbi) 34.5 ae 3.9 kg/m 2 ) and the group 2 -40 subjects (mean age 45.30 ae 10.2 years) without metabolic disorders. the parameters of 24-hour bp monitoring; microalbuminuria and mdrd; total cholesterol, low-density lipoprotein cholesterol, highdensity lipoprotein cholesterol, triglyceride, diene conjugates (dc), malonic dialdehyde (md), catataze and inflammatory markershomocysteine, high-sensitivity c-reactive protein (hs-crp) and fibrinogen were estimated. results: in group 1 there was registered increase in mean 24-h and daytime systolic bp, time and square indices, in night time systolic and diastolic bp variability. in renal function factors there was registered increase mdrd. in biochemical parameters there was detected increase in total cholesterol, triglyceride level, md and dc level, fibrinogen and hs-crp level; and decrease of catalase level; high density lipoprotein cholesterol compared to group 2. besides in group 1 positive correlation between lipid and inflammatory markers against each other and with parameters of 24-h bp monitoring, microalbuminuria, mdrd and mbi was found. conclusion: we revealed relationship between blood pressure profile parameters, renal function parameters, atherogenic lipid profile parameters, inflammatory markers and mbi, that is indicative of development of cardiovascular and cardiorenal risk in patient with ah and ao. there is considerable interindividual variation in therapeutic response to metformin in women with pcos and obesity. genetic factors may play an important role in therapeutic response to metformin and tcf7l2 gene could be one of such factors. tcf7l2 marker is one of the strongest risk factors known for predicting increased likelihood of conversion from prediabetes to t2d. tcf7l2 belongs to a subfamily of tcf7-like hmg box-containing transcription factors. tcf7l2 is a component of the wnt-signaling pathway and determine the glucoseinduced insulin secretion and regulates the maturation of β-cells of the pancreas from pluripotent stem cells. in addition, this gene plays an important role in adipogenesis and differentiation of adipose tissue. the aim of our study was to evaluate the efficacy of using metformin in patients with pcos and obesity in relation to their rs7903146 polymorphism of the tcf7l2 gene. 56 patients with pcos and obesity receiving therapy with metformin were examined. comparative analysis revealed a significant increase in the frequency of genotype c/t (v 2 = 14.93; p < 0.001; or = 4.89; 95% ci = 1.19-20.13) and t/t (v 2 = 14.93; p < 0.001; or = 4.61; 95% ci = 0.54-39.49) in the group of patients with positive dynamics of treatment with metformin compared with the group without positive dynamics of treatment. thus, patients with pcos and obesity who possess the genotype c/t and t/t of the tcf7l2 gene are more likely to have positive therapeutic response to metformin. objective: to investigate the relationship between resistin gene polymorphism with its circulating level, metabolic risk factor and insulin resistance in adult women. design: total 615 healthy subjects were enrolled for the study, 305 (age 30.12 ae 6.70 years) were with metabolic syndrome and 310 were age matched control (age 29.07 ae 7.28 years) without metabolic syndrome. circulatory resistin, insulin, plasma glucose and lipid profiles fasting level were estimated along with insulin resistance. resistin-c420g promoter region polymorphism were done by rflp method digested with bbsi restriction enzyme. results: homozygous mutant genotype (cc) (cc v/s cg + gg) (p = <0.001: or = 2.22: 95% ci = 1.60-3.10) of the 420c/g resistin gene polymorphism was significantly less frequently observed in the control population. on dividing the subjects further in to two groups according to the abscence (resistin-1) and presence (resistin-2) of the mutant g allele, significantly high levels of resistin (p = 0.006, or = 1.90, 95% ci = 1.22-2.98), triglyceride (tg) (p = 0.0001), plasma glucose (p = 0.01), systolic blood pressure (sbp) (p = 0.0003), diastolic blood pressure (dbp) (p = 0.0003), whr (p = <0.001) were observed in resistin-2 group . conclusion: our results conclude that the 420 c/g mutation of the resistin gene is likely to play an important role in metabolic syndrome and metabolic abnormalities. background: in both obesity and allergy, inflammation occurs. therefore, we examined the association between body mass index (bmi), a measure of general obesity, and serum ige level, a measure of allergy. excluded from this study. using the complex sampling function of spss, the unstandardized regression coefficient, b, for the relationship between bmi and the natural logarithm of ige level was calculated. results: there was a sex difference in ige level (p < 0.001). before adjustment, ige was associated with bmi in women (b = 0.451, p = 0.006) and in the overall population (b = 0.338, p = 0.001). after adjusting for race/ethnicity, age and sex except in sex-specific analysis, the association in women (b = 0.382, p = 0.018) and the overall population (b = 0.309, p = 0.006) remained significant. after further adjustments for physical activity, alcohol consumption and smoking, the association was still significant in women (b = 0.373, p = 0.010) and the overall population (b = 0.323, p = 0.003). the association was attenuated but remained significant in women (b = 0.233, p = 0.036) and the overall population (b = 0.179, p = 0.042) after further adjustment for levels of liver enzymes and c-reactive protein. conclusions: in this nationally representative population-based survey, ige level is associated with bmi. the attenuation in the association after controlling for liver enzymes and c-reactive protein suggests that hepatic inflammation accounts for some of the association. background: janus kinase (jak) 3 is involved in cytokine receptormediated intracellular signal transduction. inhibition of jak3 protects beta-cells from cytokine toxicity and has been shown to delay the onset of diabetes in the mouse model. the influence of jak3 single nucleotide polymorphisms (snps) on diabetes risk or on diabetesrelated metabolic traits is unknown. methods: we therefore investigated the association of jak3 tagging snp rs3212780 (c>t) with metabolic phenotypes and type 2 diabetes (t2dm) in a cohort of coronary patients including 1220 non-diabetic subjects and 375 patients with t2dm, totally comprising 1595 individuals. results: among non-diabetic subjects snp rs3212780 was significantly associated with hba1c (cc: 5.8 ae 0.4, ct: 5.7 ae 0.4, tt: 5.6 ae 0.4%; p = 0.001), fasting glucose (cc: 5.4 ae 0.7, ct: 5.3 ae 0.7, tt: 5.5 ae 1.1 mmol/l; p = 0.010), and hdl-cholesterol (cc: 55 ae 17, ct: 55 ae 16, tt: 51 ae 16 mg/dl; p = 0.009), as well as with total cholesterol (cc: 212 ae 44, ct: 206 ae 46, tt: 196 ae 48 mg/dl; p = 0.002) and ldl-cholesterol (cc: 134 ae 37, ct: 131 ae 40, tt: 124 ae 42 mg/dl; p = 0.013). in patients with t2dm, the jak3 variant was significantly associated with fasting glucose (cc: 8.3 ae 2.7, ct: 8.7 ae 2.8, tt: 7.4 ae 1.9 mmol/l; p = 0.036). the association between snp rs3212780 and t2dm did not reach statistical significance (allelic odds ratio = 1.18 [0.98-1.40]; p = 0.076). we conclude that jak3 tagging snp rs3212780 is significantly associated with phenotypes conferring an increased cardiometabolic risk, at least in non-diabetic coronary patients. the association between rs3212780 and the risk of t2dm warrants further investigation. l. garrido-s anchez 1 , x. escoté 1 , l. coín-aragü ez 2 , j.c. fren andez-garcía 3 , r. el bekay 4 , j. background: munc18c is associated to glucose metabolism and could play a relevant role in the insulin resistance. however, little is known on the regulation of munc18c expression. we analyze munc18c gene expression in human visceral (vat) and subcutaneous adipose tissue (sat) and their relationship with obesity and insulin. we evaluated 70 subjects distributed in 12 non-obese lean subjects, 23 overweight subjects, 12 obese subjects and 23 nondiabetic morbidly obese patients (11 with low insulin resistance and 12 with high insulin resistance). results: the lean, overweight and obese persons had a greater munc18c expression in adipose tissue than the morbidly obese patients (p < 0.001). vat and sat munc18c correlated negatively with weight (p = 0.022, p = 0.018) and bmi (p = 0.005, p = 0.016). vat munc18c correlated negatively with glucose (p = 0.027). sat munc18c correlated negatively with insulin (p = 0.032) and homa-ir (p = 0.037), and was the main determinant of the improvement in homa-ir index at 15 days after bariatric surgery (b = à2148.4, p = 0.038). sat explants cultures show that insulin produced a significant down-regulation of munc18c expression (p = 0.048). this decrease is also obtained when explants are incubated with a liver x receptors alpha (lxra agonist, either without (p = 0.038) or with insulin (p = 0.050). however, munc18c expression is not affected when explants are incubated with insulin plus a sterol regulatory element-binding proteins-1c (srebp-1c) inhibitor (p = 0.504). conclusions: munc18c gene expression in human adipose tissue is down-regulated in obesity and is inversely associated with insulin resistance. insulin may have an effect on the munc18c expression, probably through lxra and srebp-1c. lab., faculty of medical technology, 3 endocrin, tripoli-university of medical sciences, 4 biochemstry, cdc, 5 biotechnology, research centre, tripoli, a protective factor for obesity, type-2 diabetes mellitus (t2dm), polycystic ovary (pcos), and lacunars infarction. objectives: the objective of this study was to investigate the association between this polymorphism and t2dm, gestational diabetes (gdm), and obesity. methods: genotyping was achieved by pcr-relp in 227 individuals chosen randomly from the out patient's clinics of al-jala maternity hospital of tripoli and gharian hospital, including: 63 t2dm patients, 59 gdm patients, 57 obese, 47 healthy control individuals from libyan pregnant women population (north-west region). the results revealed that this polymorphism has no association with t2dm, gdm, and obesity in comparison with the control sample. conclusion: t228a polymorphism of sorbs1 gene is not associated with the pathological conditions studied. r. parhimovich, n. konovalova, r. tishenina, n. mylov moscow regional research clinical institute named after m.f. vladimirskiy, moscow, russia area (0.744) of bmi ! 23.00 kg/m 2 (bmi23) had significant larger areas than those of wcm9080 (0.658). wci8075 and bmi23 had good sensitivity (72.73% and 76.36%). wci9080 and wci8075 had good specificity (89.73% and 77.12%) and classification (89.36% and 77.08%). the proposed cutoff values were wci8075. further cohort study is needed to confirm these values. for national reference, we recommend measuring at umbilical due to its feasibility. servicio de endocrinologia y nutricion, hospital regional universitario carlos haya, 2 ciberdem, 3 servicio de cirugía, hospital regional universitario carlos haya, m alaga, spain introduction: the fndc5 gene encodes a membrane protein which is proteolytically cleaved, glycosylated and secreted into the blood as irisin. it has been described that causes a significant increase in total body energy expenditure, reduces body weight and improves dietinduced insulin resistance in mice. however, little is known in human. the aim of this study was to analyze the irisin levels in morbidly obese subjects undergoing bariatric surgery at baseline and 6 months after surgery. material and methods: we measured serum irisin levels in 33 morbidly obese subjects undergoing roux-en-y gastric bypass and in 10 healthy controls. we have analyzed anthropometric variables and the level of insulin resistance with the homa-ir index. results: morbidly obese patients have a lower serum irisin levels that control subjects (450.9 ae 208.0 vs. 955.9 ae 861.9 ng/ml, p = 0.043). serum irisin levels were similar before and after bariatric surgery in morbidly obese subjects (450.9 ae 208.0 vs. 449.1 ae 234.1 ng/ml; p = 0.786). there were no significant differences between morbidly obese subjects according to the homa-ir levels, neither before nor after surgery. irisin levels correlated negatively before surgery with glucose (p = 0.022), triglycerides (p = 0.045), cholesterol (p = 0.046), homa-ir (p = 0.045) and waist to hip ratio (p = 0.008). in a multiple lineal regression model, irisin levels were associated with waist to hip ratio (p = 0.046) after adjusting for fasting glucose, cholesterol, triglycerides, homa-ir and age. no significant correlations were found at 6 months after surgery. conclusions: irisin levels in decreased in morbidly obese subjects and is mainly related to waist to hip ratio. results: the level of uric acid increased with age in women and had a significant difference in women of 80-89 years (r = 0.18, p < 0.05). in men, the maximal level of uric acid was in the group 60-69 years. incidence of hyperuricemia among women was 17%, in men -30%. we determined that the highest level of triglyceride, cholesterol, systolic and diastolic pressure was among women and men with hyperuricemia. the higher level of uric acid was found in patients with maximal body mass index (bmi >35). incidence of hyperuricemia among women in the i group was 10.2%, in ii -15.9%; in iii -21.2%, in iv -34.2%. conclusions: it was determined that the level of uric acid was increasing with age and the highest level of some components of metabolic syndrome (triglyceride, cholesterol, systolic and diastolic pressure) was among patients with hyperuricemia. introduction: cushing's disease (cd) is the most common reason of endogenous hypercortisolaemia. the cortisol excess leads to serious metabolic and cardiovascular complications which significantly increase the morbidity and mortality in cd. objective: to assess the prevalence of preoperative glucose homeostasis alterations in cd and their influence on the effectiveness of surgical treatment. material and methods: a prospective study that included 36 patients (30 women; six men) with cd. the prevalence of prediabetes (impaired fasting glucose, impaired glucose tolerance) and overt diabetes was assessed. the relationship was evaluated between duration of cd symptoms and the presence of glucose homeostasis alterations as well as their impact on the efficacy of surgical treatment for cd. the proportion in the study group of overt diabetes was 16.7%, whereas the prevalence of prediabetes was 33.3%. 36.1% of patients were obese (bmi ! 30) and additional 44.4% were overweight (bmi ! 25). there was confirmed the association between duration of cd symptoms and occurrence of diabetes (p < 0.01) and any type of glucose homeostasis alterations (p = 0.04). there was no relationship confirmed between the presence of glucose homeostasis alterations and efficacy of transsphenoidal surgery for cushing's disease. conclusion: a longer duration of cd symptoms was associated with greater risk of metabolic complications such as: prediabetes and secondary diabetes. the efficacy of surgical treatment for corticotroph adenoma does not depend directly on the presence of preoperative glucose homeostasis alterations. objective: the study objective was to identify factors associated with depressive symptoms in elderly with metabolic syndrome. material and methods: it was a case-control study to check sociodemographic and lifestyle influencing depressive symptoms in elderly patients with metabolic syndrome. the cases were attended in program family health strategy, classified as having metabolic syndrome and depressive symptoms and the control group consisted of individuals with metabolic syndrome who didn′t have depressive symptoms. it was used as a measure of association, odds ratio (or) with confidence interval of 95% and p-value, obtained by conditional logistic regression model. conclusions: in the population studied showed that depression was associated with individuals at higher stress levels, those who had shown the mourning, elderly older than 80 years, those with cognitive deficits and negative perception of health. the results reinforce the need to train health professionals so that they can identify and intervene in this population, to improve the quality of life for seniors. introduction: vascular endothelial growth factor (vegf) is said to play key role in pathogenesis of diabetic nephropathy by upregulating the expression of endothelial nitric oxide synthase (enos). human vegf gene, located on chromosome 6 and is highly polymorphic. insertion/deletion (i/d) polymorphism of the 18 bp fragment at 2549 position of the promoter region of vegf gene has been implicated in many diseases of angiogenic origin. aim of the study: to investigate i/d polymorphism of vegf gene in patients with type 2 diabetes mellitus and to assess their possible role in diabetic nephropathy. materials and method: fifty subjects with diabetic nephropathy, 50 subjects with diabetes mellitus without nephropathy and 50 normal controls were evaluated for i/d polymorphism of the 18 bp fragment at 2549 position of the promoter region of vegf gene by polymerase chain reaction. the frequency of vegf alleles and genotype distribution were compared in diabetic subjects, diabetic nephropathy subject and control groups. results: distribution of vegf genotype was found to be significantly (p < 0.05) different amongst diabetic nephropathy subjects, diabetic subjects and controls by fisher's exact test and chi square test. statistically significant association (p < 0.05) of d allele was also found with diabetic nephropathy. conclusion: our study shows that i/d polymorphism of the 18 bp fragment at 2549 position of the promoter region of vegf gene is associated with diabetic nephropathy. university of delhi, new delhi, india between bai, bmi and percent body fat (pbf) in an endogamous population of india. design: data was collected on 578 adults with respect to bodyweight, height, skinfolds, hip and waist circumference, and blood pressure. pearson correlations was calculated for bai and bmi with pbf. differences in correlation for baivsbmi were examined using fisher z-tests. receiver operating characteristic (roc) analysis was used to compare the predictive validity, and to determine optimal cut-off values. ors were calculated to assess the risk of having hypertension using the proposed cut-off points. results: the correlation of pbf with bmi (men: r = 0.83; women: r = 0.71) were stronger than those with bai (men; r = 0.66; women: r = 0.58). the regression model for bai explained 66.4% of the variance in pbf in men and 57.7% in women whereas the corresponding regression model for bmi explained 82.8% variance in men and 70.7% in women. in men, the sensitivity and specificity of bai to predict hypertension was higher than wc, whr and whtr but lower than bmi. in women, the sensitivity of bai was higher than bmi and wc. in men, odds of hypertension on bai were lower than other anthropometric markers while in women, it was higher for other anthropometric markers. conclusions: bai can be used as an additional marker for screening population, however its validity needs to be demonstrated on other populations too. obesity is a multifactorial syndrome characterized by a chronic state of positive energetic balance. the experimental model of treating neonatal rats with monosodium l-glutamate (msg) was used. amino acids, which also participate in the formation of proteins, are precursors of signaling molecules as local hormones and mediators; indeed, glycine can induce catecholamine secretion of adrenal medulla. catecholamines of the adrenal medulla have an important role in the regulation of the metabolism, affecting the mobilization of fats. therefore, abnormalities in catecholamines secretion can contribute for obesity development. thus, the purpose of our work was verify if glycine administration has an inhibitory effect on hypothalamic obesity development. animals were treated with tap water added to glycine (0.1 g/kg). catecholamine content and secretion from adrenal medulla were measured using the trihydroxyindole fluorescence method. msg treatment induced 65.5 % enhancement of perigonadal fat pad when compared to control animals (p < 0.001). however, glycine treatment caused a reduction of almost 8% on perigonadal pad in obese group (p < 0.05); control-glycine group presented a decrease of 14.4% in perigonadal fat pad related to control (p < 0.05). msg treatment reduced 41.3% basal catecholamine secretion (p < 0.05). obese animals that received glycine presented an increase in basal catecholamine secretion (62.3%, p < 0.001). in conclusion, we showed that glycine treatment did not inhibit obesity development, but the decrease in adiposity observed in obese and control rats treated with glycine may be consequence, at least in part, of the enhacement in basal catecholamine secretion due to stimulatory effect of the glycine. background: increased high sensitivity c-reactive protein (hscrp) and c-glutamyl transferase (ggt) have been reported to be associated with metabolic syndrome (mets) and its components. the aim of this study is to determine whether these two biomarkers independently predict new occurrences of mets in koreans. methods: on-going prospective cohort study, korea health examinee study (koex), has recruited 60.398 individuals since 2004. among them, repeated survey was done in approximately 2 years for 6154 individuals. only newly occurring mets cases during follow-up was included in this study. odds ratios (or) and 95% confidence intervals (ci) for the risk of mets were estimated using multivariate logistic regression analysis. results: three hundred seventy-six newly occurring mets cases were ascertained in the follow-up survey. two years' cumulative incidence of mets was 8.0% and was higher among men than women (16.4% vs. 5.4%). after adjustment for potential covariates, an increased risks for newly occurring mets were observed among those with higher hscrp measured at baseline examination in men (or = 2.31, 95% ci 1.52-3.50; ! 0.1 (mg/l) vs. <0.05 (mg/l)) and in women (or = 2.26, 95% ci 1.52-3.35; ! 0.1 (mg/l) vs. <0.05 (mg/l)). also higher risk was observed in those with higher ggt in women (or = 3.82, 95% ci 2.54-5.75; ! 25 (iu/l) vs. <15 (iu/l)) and in men (or = 2.17, 95% ci 1.42-3.33; ! 50 (iu/l) vs. <30 (iu/l)). conclusions: metabolic markers such as hscrp and ggt are clearly predicted new occurrence of mets. clinical usefulness of these markers needs to be more investigated in the extended follow-up study. objective: thyroid dysfunctions are supposed to be implicated in metabolic risk. we assessed the effects of hyper-, hypo-and euthyroidism on patients′ lipid metabolism and oxidative stress from a 6 month follow-up clinical pilot study. methods: a study cohort of 96 probands was grouped into hyper-(n = 9), hypo-(n = 21) and euthyroid (n = 18) patients and healthy controls (n = 48). patients received their individual medication and underwent a 6 month follow-up. routine thyroid parameters, inflammatory status, lipid metabolism and oxidative stress were analyzed in patients before and after 6 months of medication and in healthy controls. additionally, we measured the body weight, length, waist and hip circumference, bmi as well as blood pressure (bp). results: analyses of routine thyroid parameters confirmed patient's status. anthropometric baseline characteristics were comparable between groups. bp was significantly higher in all baseline patient groups compared to controls. baseline vs. follow-up analyses revealed that euthyroid follow-ups had a significant increased bmi, waist circumference and waist-to-length ratio. further, euthyroid and hypothyroid follow-ups had a significantly lower bp. observations on lipid metabolism revealed that hypothyroid and euthyroid follow-ups showed declined hdl levels and significant higher hld-ldl ratios compared to their pre-treated state. cholesterin, ldl, tg, oxldl levels and inflammatory markers were comparable in baseline and follow-ups. concerning oxidative stress we found significantly declined asymmetric dimethylarginine (adma) levels in all three follow-up groups. results: in the elderly group without metabolic syndrome, autonomy was associated with increasing age, sedentary behavior, and depressive symptomatology. in the presence of metabolic syndrome, in addition to these factors, the lowest level of education, being insufficiently active in physical activities, and have at least one chronic disease is not transmissible, also correlated with worse autonomy. the strong association between obesity and cardiovascular disease stresses the necessity of elucidation the underlying molecular mechanisms linking these pathologies. adrenergic over-activation can promote cardiac hypertrophy and progression to heart failure. our aim was to evaluate a novel sensor for camp namely epac (exchange protein directly activated by camp) and downstream signalling pathways in the development of cardiac hypertrophy and susceptibility to ischaemia/reperfusion injury in a rat model of obesity-induced insulin resistance. methods: wistar rats on a hyperphagia-inducing diet, hid (supplementing normal rat chow with sucrose and condensed milk) for 8 and 20 weeks were compared to age matched controls. isolated working hearts were subjected to 20 min global ischaemia and functional recovery was measured after 30 min reperfusion. hearts were also freeze-clamped at different time points during ischaemia and reperfusion. epac activation was determined by rap1 activation kit. nfat, p38 mapk, erk 1/2, pkb and creb were determined by western blotting using appropriate antibodies. results: the heart weight increased significantly after 20 weeks on hid along with an increase in the hypertrophic marker, nuclear nfat. the 8 weeks hid hearts recovered significantly after ischaemia compared to controls, along with a significant increase in phospho-pkb after 10 min reperfusion, compared to 20 week hid hearts. however, results on epac activation showed variable responses. type 2 diabetes mellitus (t2dm) and hypertension (htn), both illnesses share pathogenic mechanisms that predispose to micro/ macrovascular complications. it is well known the role of hyperglycemia and insulin resistance of diabetes in vascular endothelial dysfunction. several studies have shown the association of serum concentrations of osteocalcin (oc) in the metabolism of glucose. oc a marker of bone formation by osteoblasts and produced hormone that regulates energy metabolism, was found recently in arteries with atherosclerosis, suggesting their direct association with vascular disease. we analyzed the relationship between serum oc concentrations and cardiovascular risk factors. a cross-sectional analytical study was carried out in 63 patients with t2dm plus htn and 51 healthy subjects (hs). oc serum levels were measured and also statistically correlated with cardiovascular risk parameters. total osteocalcin serum levels in t2dm + htn subjects were significantly higher than those in hs (p < 0.001), whereas the uncarboxylated oc concentrations were lower in t2dm + htn subjects than in hs (p < 0.05). body mass index, waist circumference, fat percentage, fasting plasma glucose, high-density lipoprotein cholesterol, fasting serum insulin, homeostasis model assessment-insulin resistance and high sensitivity-c reactive protein were negatively correlated with uncarboxylated osteocalcin (p < 0.05). in addition, carboxylated oc was also positively correlated with systolic and diastolic blood pressure (p < 0.001) and homeostasis model assessment-insulin resistance (p < 0.05). oc serum concentrations are associated with cardiovascular risk factors in patients with t2dm + htn. the oc forms (uncarboxylated/carboxylated) might play different roles in cardiovascular and endocrine physiology and hence be of different value as cardiovascular risk markers. aim: to evaluate the prevalence of ms in a group of medical students from bucharest and their habits related to nutrition and physical activity. cross-sectional study: ms was defined according to international diabetes federation criteria. medical students from bucharest were included in the survey, based on their informed consent. they were examined clinically (weight, height, waist, blood pressure), provided a blood sample (glycaemia, lipids, triglycerides, cholesterol, high and low-density lipoproteins) and answered to block adult questionnaire for food and physical activity. results: 235 students enrolled, 70.2% females. 40.9% of subjects were free of signs of ms, 46.8%, 11.1% and 1.3% met one, two and three criteria for ms diagnosis. the highest prevalence of abnormality was found in waist (above the limit in 49.4% cases), followed by hdl (decreased in 13.2% cases). significant differences among genders were found in waist. food habits: boys were found to eat significantly more calories, fats, carbohydrates and proteins compared to girls. no differences for fibres, transfats, free sugar, fruits and vegetables. physical activity: boys consumed significantly more energy through physical activity, recreational activities and vigorous physical activity compared to girls. only in 10% of cases the energetic consume was higher than the ingestion (no difference by gender). conclusion: high prevalence of unhealthy habits related to nutrition and physical activity in a particular highly educated young group are concerning for the occurrence of ms in the future. triphala an age old commonly used powered preparation of three medicinal dried plant fruits amla (emblica officinalis), harad (terminalia bellirica) and bahera (terminalia chebula) in equal proportionsis used in indian systems of medicine. present work evaluated the anti-hyperglycemic, anti-hyperlipidemic, and antioxidant potentials of 'triphala' formulation viz., (emblica officinalis: terminalia bellirica: terminalia chebula:: 4:1:1) in subjects with igt and tiidm. the therapeutic effect of 'triphala' administration (5 gm bd) in 20 tiidm, 10 igt and 10 healthy individuals was assessed by monitoring blood glucose at 30 days intervals, hba1c, lipid profile, oxidative stress markers, and liver & kidney function markers at 90 days intervals. dna damage was assessed by comet assay, flow cytometry and hoechst nuclear stain. molecular markers were determined in the beginning and at the end of therapy. results: 'triphala' (4:1:1) administration for 1 year significantly brought down blood glucose levels with a marked improvement in lipid profile in all the groups, this was further supported by increased protein expression of ampk and adiponectin. triphala provided resistance to oxidative stress generated not only by the increasing the antioxidant enzymes activity, but also by shortening comet tail length and number of cells in g 0 phase of cell cycle. our results indicated that diabetes is strongly associated with elevated levels of ar, tnf-a, il-6 and il-10, but triphala down regulated the same, proving its anti-inflammatory potential. conclusion: these observations raise the prospects of using triphala formulation for treatment of diseases associated with oxidative stress and imbalanced cytokine production. introduction: glucoregulation disorders are a state of pre-diabetes increasingly diagnosed in the general population. its association with hypertension, increases the risk of cardiovascular morbimortality objective: identify the blood pressure profile in patients with disorders of carbohydrate. patients and methods: this monocentric study has permitted the prospective recruitment of 195 patients. all had an oral glucose tolerance test (ogtt) with a mesure of plasma glucose level immediately before and 2 h after taking 75 g of glucose. clinical examination with taking blood pressure to two arms and a cardiovascular evaluation were performed. results: the glucoregulation disorders were concerned with 90 patients (46%) with an average age of 58.6 years (38-86 years). sexratio: 1.04. ifg was found in 39 patients. ogtt revealed 15 diabetes, 51 igt. only 13 patients with glucoregulation disorders have normal blood pressure. seventy-two patients have been followed for high blood pressure and five patients are newly diagnosed. more than a half of these hypertensive patients has an igt and a third is diabetic. in the family, the high blood pressure is reported in 58%, the early cardiovascular events are dominated by myocardial infarction and stroke. in high blood pressure group, 1 patient/4 is dyslipidemic and metabolic syndrom, as defined by idf 2005, is concerned with 2 patients/3. as for as cardiovascular events, they are reported in 2 patients/5. (t2dm) is an ongoing concern and adequate treatment remains an important issue. thiazolidinediones (tzds) are a class of drugs that initially showed great promise as unique receptor-mediated oral therapy for t2dm. the tzds, rosiglitazone (rosi) and pioglitazone (pio) were widely used as hypoglycemic drugs in patients with t2dm, but a host of serious side effects, primarily cardiovascular, have limited their use. aim: in the present study we have used a systems biology approach to assess specific gene expression profiles underlying the pathological processes in the heart of pre-diabetic mice treated with rosiglitazone or pioglitazone. results: our data demonstrate that both rosi and pio efficiently decreased high fat diet-induced plasma glucose and insulin levels. analysis of the heart demonstrated that rosi, but not pio, led to an increase in atherosclerotic plaque formation and an increase in heart weight to body weight ratio. a combined transcriptomics and bioinformatics approach revealed specific regulatory pathways that may explain the adverse heart effects associated with rosiglitazone but not pioglitazone treatment. conclusion: our data provide new insights into the mechanisms underlying rosiglitazone and pioglitazone action within the cardiovascular system, including drug efficacy and cardiotoxicity. a. vlassopoulos 1 , m. lean 1 , e. combet 1 1 human nutrition, school of medicine, university of glasgow, glasgow, uk introduction: the new hba1c criteria for diagnosis of pre-diabetes have been criticised for overdiagnosis, because many more people are diagnosed than with the old ogtt criteria. it is possible that some elevation of hba1c is not driven by hyperglycaemia. aims: this study assesses associations of hba1c, commonly assumed to relate solely to glucose concentration, with, 1. smoking, a major source of reactive oxygen species (ros) and 2. fruit & vegetables consumption associated with improved redox status. methods: one-way anova, chi-squared and multivariate linear regressions, adjusted for all known confounders were used to explore associations of hba1c with self-reported smoking status and fruit & vegetables consumptions in the scottish health surveys 2003-2010, among individuals without known diabetes and hba1c <6á5%. results: compared to non-smokers (n = 2831), smokers (n = 1457) were younger, consumed less fruit & vegetables, had lower physical activity levels, lower bmi, higher hba1c and crp (p < 0á05). hba1c was higher in smokers (p < 0á001) by two sds (0á08%), and 3.5 sds higher (0á14%) in heavy smokers (>20 cigarettes/day) than nonsmokers. smokers were twice as likely to have hba1c in the 'prediabetic' range (5.7-6.4%). pre-diabetes and low grade inflammation did not affect the associations. for every extra 80 g vegetable portion consumed, hba1c was 0.25 sds (0.01%) lower (p = 0.02), but fruit consumption did not impact on hba1c, within the low range of consumptions in this population. conclusion: this study adds evidence for a neglected link between oxidative stress and protein glycation, with implications for individuals exposed to ros and for epidemiological interpretation of hba1c. v. aursulesei, i.c. roca, l. mihalache objective: to assess the relation between the traits of metabolic syndrome (ms) and the parameters with independent prognostic significance for cardiovascular risk. material and methods: hundred and forty-three patients were included according to the number of traits of ms (69.2% arterial hypertension, 29.7% abdominal obesity, 43.9% low hdl-cholesterol, 61.2% high tryglicerides, 55.6% abnormal oral glucose tolerance). the markers of cardiovascular damage were assessed: pulse wave velocity (pwv -complior method), carotidian intima media thickness (imt), left ventricular hypertrophy (index of mass -lvmi and geometric patterns), flow mediated vasodilation (fmd), microalbuminuria (absent/present), ankle-brachial index (abi). results: in our study the cardiovascular damage is present in 66.8%. pwv values increase with number of traits of ms (t-test) after adjusting for confounders/other cardiovascular risk factors (ancova). decreased abi is related with each of ms traits; the power of correlation depends on criteria association. pwv and abi are both related with postprandial hyperglicaemia and systolic blood pressure (p < 0.05), while imt is strongly related with hdlcholesterol (r = 0.43, p = 0.04). lvmi and concentric hypertrophy pattern are also related with systolic blood pressure (r = 0.29, p < 0.05), while eccentric pattern relates to waist circumference (r = 0.32, p < 0.05). we cannot establish a relation between traits of ms and fmd or microalbuminuria. conclusions: hyperglicaemia and systolic blood pressure are best related with parameters of cardiovascular risk, but the clustering of ms components may interact to synergistically affect the extent of cardiovascular damage. pwv, abi and left ventricular hypertrophy should be systematically used for defining cardiovascular risk in ms. interleukin-6 (il-6) is pleiotropic cytokine with a key impact on immunoregulation and nonimmune events. sudies have investigated the role of action/lack of action of il-6 in the pathogeneses obesity, insulin resistance, type 2 diabetes. aim of the study: to evaluate il-6 activity in hypertensive patients depend on prediabetes presence. materials and methods: 73 hypertensive patients were examined. common clinical investigations were provided. il-6 plasma levels were detected using elisa. data is represented as me (q 25 -q 75 ). median test were used, p < 0.05. results: prediabetes was observed in 36.2% of hypertensive patients in 71% insulin resistance in hypertensive patients with prediabetes vs. 54 % insulin resistance in hypertensive patients were detected. hypertensive patients with prediabetes (5.90 (5.20-7.10) %) characterized by significantly higher glycated haemoglobin levels as compared hypertensive patients (5.40 (4.70-6.97) %, p < 0.05). hypertensive patients were characterized by increased il-6 activity (18.81 (13.14-26.69) pg/ml). in hypertensive patients with prediabetes decrease il-6 activity (13.94 (11.00-16.94) pg/ml) were found. conclusion: hypertensive patients were characterized by increased iladipose tissue is now recognized as a complex organ with a crucial role in energy metabolism and in the development of obesity and metabolic syndrome. modified response and metabolism of hormones has been observed in the visceral adiposity during obesity, specifically related to cortisone. the aim of this study was to assess the response to different concentrations of cortisone in adipocyte cell line 3t3l1. the expressions of 11β-hsd1, enzyme responsible for the reduction of cortisone to cortisol, and aqp7, involved in glycerol transport, were quantified after treating differentiated cells with cortisone at doses of 0, 0.1, 1, and 10 lmol/l during 0, 5, 10, 15, 20 min, and 48 h. total rna and cdna were obtained from the samples to develop a real time pcr using mnsod as housekeeping gene. results suggest time and dose dependent response of 11β-hsd1 and aqp7; increases in the expression were observed during the first 15 min of treatment (3 and 4 fold, respectively), followed by expression decrease for both in a 5 min period (p < 0.0001). for the treatment with 1 lmol/l cortisone, both proteins expressions showed quadratic tendencies, 11β-hsd1 tendency is described by the equation y = 3.131 + 0.069x à 0.094x 2 while aqp7 tendency is described by y = 5.273 à 1.232x + 0.078x 2 . it can be concluded that long term effects of cortisone over adipocyte metabolism may be modulated by the induction or repression of proteins like aqp7 and should be explored in obese individuals. objective: to present a series of cases of metabolic syndrome patients suffering sleep disorders in which the most relevant metabolic marker is serum tryglicerides levels. it has been considered that the key meatbolic alteration underlying metabolic syndrome is a resitance to insulin action. however, there could be other metabolic abnormalities that may lead to the devleopment of the clinical feautres of this diseasea. materials and methods: patients undergoing a medical check up at medica sur hospital were included. each patient was assesed in order to determine both matabolic syndrome and a current sleep disorder. anthropometric measures were taken, body rates were calculated and blood samples were taken for laboratory tests (serum glucose, tryglicerides and cholesterol levels determination). results were analysed with the spss 20 software. results: fifteen patients meeting the world health organization metabolic syndrome were included, and who referred snoring, difficulties on getting asleep or frequent waking up while sleeping. anthropic measures, and lab results were. discussion: yet, the majority of these patients do not have serum glucose abnormalities, but mainly their weights correspond to overweight and to a phenotype of central adipose tissue distribution. the most significant metabolic alteration, in this set of patients, was hypertigliceridemia. this results suggests that there may be other metabolic pathways leading to the development of this disease. introduction: prediabetes, a condition leading to diabetes and cardiovascular diseases (cvd). in cameroon, few studies have been done about this health indicator. aims: to determine the prevalence of prediabetes and diabetes in cameroon cohort, and to compare metabolic risk factors between normoglyceamia and prediabetes individuals. patients and methods: a sample of 731 cameroonian men and women (18-65 years) participated in a health survey. statistical analysis of data compared risk factors between three subgroups: normoglyceamia (ng), prediabetes (pd) and diabetes (dt). prediabetes was defined as a glyceamia of 100-126 mg/dl. metabolic syndrome (mets) was diagnosed using the national cholesterol education program (ncep) definition. results: the distribution of ng, pd and dt prevalence in the overall sample was 70.98%, 22.37% and 6.65% respectively. the prevalence of mets in those with pd (37.4%) was significantly higher than in those with ng (13%). many significant differences between ng and pd subjects were also noticed. conclusion: in this study, approximately 30% of the population who were found to be diabetic and prediabetic is at risk of cardiovascular diseases. our findings also show that prediabetes individuals are different from those with normal glycaemia in a great number of metabolic abnormalities including a higher prevalence of mets. prevention and control measures should be set urgently. research design and methods: prospective study of the thai comprehensive cirs was further evaluated by multiple logistic regressions. in determine the ability of the thai comprehensive cirs scores in predicting each outcome, the adjusted odds ratio was calculated, and backward stepwise selection was used in the statistical modeling. results: there were also prospective correlations between the baseline thai comprehensive cirs instrument and 3-month outcomes). total score at baseline emerged as significant predictors of 3-month qol (r = 0.26-0.33, p < 0.05). every subscale of the thai comprehensive cirs was significantly related to patient satisfaction (r = 0.14-0.33), with only the neighborhood subscale as a significant predictor for adl (r = 0.18). the other outcomes (self management behavior, cost of care, and dbp) were not significant concurrent baseline predictors. method: we collected laboratory results of body mass index (bmi), blood pressure, fasting glucose, lipid profiles (total cholesterol, triglycerides, ldl-and hdl-cholesterol level) and visceral fat amount of cancer survivors who visited health promotion center at seoul national university hospital. an age and sex-matched control was selected randomly for each cancer survivor from visitors of this center. we compared blood pressure, fasting glucose, lipid profiles and visceral fat amount between cancer survivors and controls using multiple regression analysis after adjustment for bmi. results: one hundred fifty five cancer survivors and controls were included in statistical analyses. cancer survivors tended to have lower bmi compared to controls (23.05 ae 3.17 kg/m 2 vs. 23.75 ae 3.14 kg/ m 2 , p = 0.051) after adjustment for bmi, cancer survivors showed lower fasting glucose level (89.06 ae 1.24 mg/dl vs. 93.40 ae 1.24 mg/ dl, p = 0.051). however there were no significant differences of blood pressure, lipid profiles or visceral fat amount. background: both insulin deficiency and resistance are reported in patients with β-thalassemia major (btm). we assessed the ogtt and 72-h continuous glucose concentration by the continuous glucose monitoring system (cgms) and calculated homeostatic model assessment (homa), and quicki in 16 adolescents with btm on regular blood transfusions and iron-chelation therapy. results: in adolescents with btm (age: 19.75 ae 3 years), ogtt, (25%) showed impaired fasting blood (plasma) glucose concentration (bg) (>5.6 mmol/l). two-hour after the glucose load, one of them had bg = 16.2 mmol/l (diabetic) and two had igt (bg >7.8 and <11.1 mmol/l). monitoring the maximum (postprandial) bg using cgms, 4 adolescents had diabetes (25%) (bg >11.1 mmol/l) and nine had igt (56%). homa and quicki revealed levels <2.6 (1.6 ae 0.8) and >0.33 (0.36 ae 0.03), respectively, ruling out significant insulin resistance in these adolescents. neither fasting serum insulin nor c-peptide concentrations were correlated with fasting bg or ferritin levels. the average and maximum blood glucose levels during cgm were significantly correlated with the fasting bg (r = 0.68 and 0.39, respectively, with p < 0.01) and with the bg at 2-h after oral glucose intake (r = 0.87 and 0.86 respectively, with p < 0.001). ferritin concentrations were correlated with the fasting bg and the 2h blood glucose levels in the ogtt (r = 0.52, and r = 0.43, respectively, p < 0.01) as well as with the average bg recorded by cgm (r = 0.75, p < 0.01). conclusion: cgm has proven to be superior to ogtt for the diagnosis of glycemic abnormalities in adolescents with btm. background: in obese children pancreatic beta-cells may not be able to cope with insulin resistance leading to hyperglycemia and type 2 diabetes (t2dm). objectives: to assess oral glucose tolerance, 72-h continuous blood glucose concentrations (cgm) and calculate homeostatic model assessment (homa), and the quantitative insulin sensitivity check index (quicki) in 13 children and adolescents with simple obesity (bmisds = 4 ae 1.06). results: ogtt performed in 13 obese adolescents (13.47 ae 3 years) revealed three cases (23%) with ifg (>5.6 mmol/l), four cases (30%) with igt (>7.8 <11.1 mmol/l), and none with diabetes. using the cgms, ift was detected in four cases, the maximum bg (2 h or more after meal) was >7.8 and <11.1 mmol/l (igt) in nine children (69%) and >11.1 mmol/l (diabetes) in one case (7.6%). five cases had a minimum bg recorded of <2.7 mmol/l (hypoglycemia). no glycemic abnormality was detected using hba1c (5.7 ae 0.3 %). 11/13 patients had homa values >2.6 and quicki values <0.35 denoting insulin resistance. beta cell mass percent (b %) = 200 ae 94.8 % and insulin sensitivity (is) = 50.4 ae 45.5% denoting insulin resistance with hyperinsulinaemia and preserved beta cell mass. in obese children and adolescents; cgms is superior to ogtt and hba1c in detection of the glycemic abnormalities, which appears to be secondary to insulin resistance. introduction: the aim of this study is to evaluate the effect of vitamin e supplementation on glycemic control, lipid profile, inflammation markers and malondialdehyde level of type 2 diabetic patients. material and method: thirty type 2 diabetic patients were participated in this randomized single blind placebo controlled clinical trial study. fasting blood glucose, serum triglyceride, total cholesterol, ldl, and hdl-cholesterol concentration, malondialdehyde (mda), hs-crp, il-6, and insulin level were measured. each subject then given a breakfast that was contained 80 g fat. subjects' post-prandial biochemical measurement was measured as well. patients' then randomly divided in two groups. treatment group received vitamin e (400 iu/per day) and control group received placebo for 6 weeks. at the end of 6 weeks baseline procedure was repeated and fasting, and 2-h postprandial biochemical markers were measured. statistical analysis: data analyzed using mann-whitney u test to compare the mean differences between both groups. basic data expressed as mean ae standard deviation, fasting and postprandial biochemical parameters before and after intervention expressed as median. results: no significant differences were found in fasting and postprandial lipid profile, glucose, insulin and homa-ir after 6 weeks of intervention between two groups. significant decreases in fasting and postprandial serum mda levels in treatment group were recorded. no significant differences were shown in fasting and postprandial inflammatory markers except fasting il-6. the result of this study shows that short term supplementation of vitamin e is safe and effective in decrease oxidative stress in type 2 diabetic patients. objective: there is evidence that patients with normal weight and central adiposity (elevated waist circumference and waist-hip index) present a higher cardiovascular and metabolic risk. it has been proven that central adiposity is a risk factor for elevated systolic and diastolic pressure amounts, low hdl cholesterol and altered glucose while fasting. materials and methods: patients treated at the integral diagnosis and treament center of medica sur hosptial were inclued. anthropometric measurments were taken and metabolic (glucose and profile of lipids with ultrasensitive reactive c protein) parameters were analyzed. results: nine hundred and forty patients were included. women with a normal body mass index but with a waist >80 cm, had metabolic alterations on serum lipids which have a statistical significance when compared with those women with a waist circumference lesser ran 80 cm. an elevated waist circumference diameter, regardless of the bmi, increases the metabolic risk. the measurement of the circumference of the waist and the hip must take place by routine in any medical evaluation. aim of the study: to study the prevalence of prediabetes in elderly age group (60 years and above) and to study cardiovascular risk factors in prediabetic group. methodology: study was conducted on 200 patients of age 60 years and above. they were screened for prediabetes as per ada guidelines 2011. such prediabetic study group (32 cases) was investigated for cardiovascular risk factors (obesity, hypertension, microalbuminuria, crp, retinopathy, dyslipidemia) along with age matched controls. standard statistical tests were applied for analyzing data. hypertension and retinopathy with the prediabetic state. risk factors are more associated with igt as compared to ifg. conclusion: prediabetes is widely prevalent in the elderly population. it has strong association with metabolic syndrome. prevalence of igt is higher as compared to ifg. similarly, cardiovascular risk factors are widely prevalent in the prediabetic population. dyslipidemia, obesity, microalbuminuria, hypertension and retinopathy share the significant association as risk factors. early identification, of prediabetic state followed by primordial and primary prevention of cardiovascular disease should be the focus of intervention. background: despite commonly using body mass index (bmi) in diagnosing obesity, the accuracy of bmi in detecting adiposity is unknown in korean. we assessed accuracy of bmi in detecting body fat percentage (bf%) defined obesity. methods: this study is a cross-sectional design of 6017 subjects (age 20-69.9 years, men 43.6%) who conducted 2009 korean national health and nutrition examination survey (knhanes iv-3) in korea from january 2009 to december 2009. we assessed the diagnostic performance of bmi using the who reference standard for obesity of bf% ! 25% in men and ! 35% in women, which were measured by dual energy x-ray absorptiometry. we tested the correlation between bmi and bf% by sex and age groups. we defined the bmi cutoff for bf%-defined obesity using roc analysis. results: bmi-defined obesity was present in 38.7% of men and 28.1% of women. according to age and sex, a sensitivity and a specificity of bmi ( ! 25 kg/m²) showed difference. the bmi cutoff value for bf %-defined obesity is 24.2 kg/m² (sensitivity 78%, specificity 71%). difference between bmi cutoff value of korean and that of american to detecting bf% is about à1.3 kg/m². in korean, bmi showed a limited accuracy to detecting adiposity by age and sex. the difference between bmi cutoff-value of korean and that of american to detecting bf% obesity is smaller than the difference of bmi cutoff-value between the asia-pacific and who criteria. background: the healthkick intervention was developed as part of a study that aimed to address diabetes risk factors in primary schools within low-resource settings in the western cape, south africa. the intervention focused on grade 4-6 learners, their parents and the educators. aim: to ascertain the risk of developing diabetes and other noncommunicable diseases among educators. method: educators (n = 297) from 16 urban and rural schools, participated in a health check in 2011. measurements included random blood glucose and cholesterol testing, and height, weight, waist circumference and blood pressure (bp) measurements. cut-off values for bp levels in adults 18 years and older of the nhlbi were used for categorising hypertension. results: educators participating in this study were mainly females, with most of them (68%) falling in the age range between 35 and 54 years, and 17% being older than 55 years. only 23% of educators had bps in the normal range. half of those on treatment for hypertension had levels above 140/90 mmhg, while 22% with bps above these levels were not on medication. about 61% of those who said they had diabetes (n = 31) knew what treatment they were on. cholesterol levels above 5 mmol/l were seen in 45%. only 20% said they smoked cigarettes, whereas 21% were previous smokers. preliminary results show that most participants had a body mass index >25 kg/m 2 . conclusions: many of the educators who participated in the healthkick study are possibly at risk for developing cardiovascular and other related non-communicable diseases, such as diabetes. s.k. kota 1 , s. ugale 2 , n. gupta 2 , k.d. modi 1 1 endocrinology, medwin hospital, 2 laparoscopic surgery, kirolskar hospital, hyderabad, india objective: the objective of the present study was to prospectively evaluate the results of laparoscopic ileal interposition (ii) with diverted sleeve gastrectomy (dsg) for control of type 2 diabetes mellitus (t2dm) and related metabolic abnormalities. methods: all patients underwent ii +dsg. they had t2dm ! 5 years with poor glycemic control despite adequate dosage of oral hypoglycemic agents (ohas) ae insulin. the primary outcome was remission of diabetes (hba1c <6.5% without ohas/insulin) and secondary outcomes were reduction in antidiabetic agent requirement and components of metabolic syndrome. we report the preliminary postoperative follow-up data of 13.1 ae 5.3 months (range: 3-26 months). there were 32 patients (m: f = 21:11) with mean age of 48.7 ae 7.8 (range: 34-66 years), duration of diabetes of 13.1 ae 5.8 years (range: 5-30 years), and preoperative body mass index of 29.1 ae 6.9 kg/m 2 (range: 22.4-39.5 kg/m 2 ). sixteen patients (50%) had hypertension, while dyslipidemia and microalbuminuria was present in 12 patients (39%) each. twenty two patients (70.5%) had diabetes remission. fifteen/sixteen (93%) patients had remission in hypertension. all participants had weight loss ranging between 15% and 25%. postoperatively statistically significant decline was observed in the glycemic and lipid parameters, microalbuminuria at all intervals (p < 0.05). patients with postoperative duration >6 months had better improvement in terms of reduction in glycemic, lipid parameters and microalbuminuria. three patients had vitamin b12 deficiency 1 year after surgery. conclusion: ileal interposition combined with dsg addresses both foregut and hindgut theories and brings about remissions in t2dm patients. background and objective: the objective of this study was to evaluate the screening potential of various anthropometric indices, namely body mass index (bmi), waist circumference (wc), waist-to-hip ratio (whr) and waist-to-height ratio (whtr) in the early detection of dysglycemia among the omani adult population. methods: our study included 1274 omani adults (476 males and 798 females) who participated in a cross-sectional, community-based study. we defined glycemic status based on american diabetes association (ada) thresholds: <5.6 mmol/l as normal, 5.6-6.9 mmol/l as impaired fasting glucose (ifg) and ! 7.0 mmol/l as diabetes mellitus (dm). the cut-off values for the anthropometric indices namely, bmi, wc, whr and whtr were based on the standard definitions. the age adjusted analysis showed that out of the four anthropometric indices, among the males the whtr at the cut-point of 0.5 gave relatively higher value of odds ratio with or = 2.29 (95% ci: 1.95, 4.31) and among the females whr at the cut-point of 0.85 showed maximum odds ratio with or = 3.66 (95% ci: 2.56.5.25). the areas under the roc curves, at the cut-point of ifg ! 5.6 mmol/l, was more for whtr in case of males and whr in case of females. conclusion: this study shows that out of the four anthropometric indices, there are differences in the predictive values of dysglycemia among the omani males and females. in case of males, the whtr ! 0.5 appears to be a better indicator for detecting dysglycemia whereas among the females the whr ( ! 0.85) is a better indicator. introduction: increased serum ferritin and iron stores are involved in the pathogenesis of insulin-resistance. polycystic ovary syndrome (pcos) is diagnosed by oligomenorrhea and hyperandrogenism. pcos and obesity were associated with elevated serum ferritin levels. the link between obesity and altered iron metabolism was proposed. object: to evaluate the association between serum ferritin levels and insulin resistance and metabolic syndrome in obese and non-obese women. methods: retrospective study. five hundred thirty-nine women, 286 of whom had pcos and 253 of whom did not have pcos, were included in the study. results: serum ferritin correlated with menstrual cycle length, sex hormone-binding globulin, total testosterone, androstenedione, triglyceride, and total cholesterol both in obese and non-obese women. obese women (bmi >25) with high ferritin (ferritin ! 45.5 ng/ml, n = 270) levels had higher insulin resistance, impaired glucose tolerance, and liver enzymes than obese women with low ferritin levels (ferritin <45.5 ng/ml, n = 269). however, among non-obese women, insulin resistance and metabolic disturbances were not significantly different between high and low ferritin groups. women with high ferritin levels had a greater risk of pcos and hyperandrogenism than women with low ferritin levels. independent of obesity, hypertriglyceridemia was the major metabolic disturbance in women with elevated serum ferritin levels. conclusions: the pathogenesis of increased iron stores correlated with insulin resistance and metabolic syndrome among obese and nonobese premenopausal women was different. the hypertriglyceridemia in women with pcos might be associated with iron metabolism. nutrition rehabilitation, unite de dietetique, pegomas, france and yogic counseling for stress management. all subjects underwent a residential program for 6 weeks followed by therapy at home for 12 weeks. results were analyzed using paired 't' test. conclusion: in remote areas organization of screening on base of diabetes bus helps to detect t2d and early glucose metabolism disorders in more than 50% of people with increased risk. findrisk scale is useful screening tool to detect these people. however, in older age groups, percentage of screen-detected t2d decreases, which may indicate lower efficiency of findrisk questionnaire at age ! 65. background and aims: changes in the cellular oxidative status are involved in the pathogenesis of obesity-associated hepatic steatosis. the possibility of gender difference in this process was examined in an experimental model of obesity induced by a western high-fat and highcarbohydrate cafeteria diet. methods: four groups of six swiss cd1 mice (21 day old) received either cafeteria diet (male and female) or balanced diet (male and female) for 14 weeks. hepatic hydrogen peroxide (h 2 o 2 ), thiobarbituric acid reactive substances (tbars), reduced glutathione (gsh) and the activity and/or gene expression of catalase (cat), glutathione peroxidase (gpx), superoxide dismutase (sod), hypoxia inducible factor (hif-1-alfa) and nuclear factor (erythroid-derived 2)like 2 (nrf2) were measured. results: higher levels of tbars and mitochondrial h 2 o 2 were found in livers from cafeteria-fed animals of both genders, with higher levels in females. the level of gsh was lower in cafeteria-fed mice of both gender; females showed higher levels than males (cafeteria or balanced diet). cat and gpx activities were reduced in cafeteria-fed mice of both sexes; the activities in females being lower than those of males. sod activity and the gene expression of cat, gpx and sod were not significantly altered when compared by gender or dietary treatment, but the expression of hif-1-alfa and nrf2 was increased in female cafeteria-fed mice. the female animals exhibited a higher susceptibility to cellular oxidative stress in cafeteria diet-induced obesity in comparison to males. the molecular mechanisms seem to be, in part at least, posttranscriptional. acknowledgements: capes, cnpq, fundac ßa˜o arauc aria. objectives: to investigate the relationship between body fat percentage and body mass index (bmi) among young adults aged 20-25 years. methods: young adults aged 20-25 years were recruited for study when they took health examination in september 2011 in taiwan. all subjects underwent bioelectrical impedance analysis (tanita bc-418) to estimate their body fat percentage. basic demographic data, height, and weight were collected, and bmi was calculated from height and weight. overweight and obesity were identified according to bmi. the correlation between body fat percentage and bmi was analyzed using sas software 9.2. results: a total of 447 male and 447 female participants were enrolled. the mean age was 22.8 ae 0.8 years old. the mean body mass index was 22.2 ae 3.5 for men and 20.1 ae 2.6 for women. the mean body fat percentage was 18.5 ae 6.2 for men and 28.4 ae 5.3 for women. categorized by bmi, the mean body fat percentage was 10.3 ae 2.1 for men and 23.5 ae 3.2 for women in bmi <18.5 group, 16.9 ae 3.6 for men and 29.3 ae 3.7 for women in bmi 18.5-24 group, and 23.0 ae 2.7 for men and 37.0 ae 2.1 for women in bmi 24-27 group and 31.6 ae 6.4 for men and 41.3 ae 2.0 for women in bmi >27 group (p < 0.05). the prevalence of too high or obese body fat percentage among young adults aged 20-25 years in taiwan was 41.6% in male and 18.1% in female according to the who and nih recommendations. the cutoff values of healthy body fat percentage for young adults in chinese population ought to be modified. it is estimated that 6-7% of women of reproductive age have polycystic ovarian syndrome (pcos). this article summarizes the recent development and findings in the cardiometabolic abnormalities in patients with pcos. patients with pcos have the clinical features of oligomenorrhoea, hirsutism and infertility; however, they also exhibit hyperinsulinemia, obesity, hypertension, dyslipidemia, and an increased pro-thrombotic state. they have an increased risk of type 2 diabetes and impaired glucose tolerance, and sleep apnea is also found more commonly in this population. however, despite the presence of cardiovascular risk factors and increased surrogate markers of cardiovascular disease it is unclear if they have accelerated atherosclerosis. end point studies are currently lacking and the available evidence are conflicting. this article will address the current evidence for the adverse cardiovascular risk in pcos and the other factors that may be implicated. finally the therapeutic options for treatment will be discussed. conclusion: results suggest that in the fasted condition, when fatty acids levels are elevated, the gluconeogenic flux is higher in cafeteriafed rats, a finding consistent with the higher glycogen levels. the lower production of 14 co 2 indicates a deviation of the citric acid cycle intermediates into the cytosol, a change which may favour the synthesis of fatty acids and triacylglycerols. these liver metabolic disturbances probably contribute to fatty liver diseases, hyperglycemia and dyslipidemia in cafeteria diet-induced obesity. certain cancers. this study aims to evaluate the association between obesity and colorectal cancer risk and also if the association varies with the blood type, age of obesity or cancer subsites. the study was carried out on 65 hospital patients, different ages, diagnosed with colorectal cancer. we determined the blood type, cancer subsites and age of symptoms. we evaluated their family history and their lifestyle in terms of physical activity, smoking and alcohol consumption. results: out of the 65 patients, 94% had the bmi ! 30 kg/m 2 and all of them showed abdominal obesity. the age of obesity was up to 5 years to 58% of the subjects and over 10 years for 24% of them. symptoms were detected over 6 months before diagnosis for 34% of the individuals and between 4 and 6 months for 32% of them. in what respects the cancer subsites, 72% of the cases were located on the descending colon, rectum and sigma. 28% of the patients had a family history of different types of cancer (colorectal, pulmonary, gastric or breast neoplasia). sixty-five percent of them were smokers, 68% were alcohol consumers and 60% carry light or no physical activity. forty-nine percent were blood group a individuals. the study shows that obesity is a statistically significant risk factor for colorectal cancer and the association is stronger for the patients with a higher age of obesity, descending colon and rectum localisation of the neoplasia and blood group a individuals. technical university munich, munich, 2 ikfe institute, mainz, germany purpose: the angiotensin ii receptor antagonist telmisartan (t) has demonstrated bifunctional effects on the hemodynamic, vascular and metabolic features of patients with metabolic syndrome (ms). accordingly, we tested the hypothesis, that antihypertensive therapy with t vs. amlodipine (a) improves diastolic myocardial function, vascular function and metabolic characteristics in ms. methods: this randomised cross-over study investigated 19 ms patients (bmi 36 ae 6 kg/m 2 ) with mild-moderate hypertension before and after 3 months therapy with t vs. a. laboratory and ultrasound data were taken in the fasting state and 2 h after a test meal (48 g carbohydrates). cardiac function was assessed by tissue doppler as systolic (s') and diastolic myocardial velocity (e') and vascular function at the common carotid artery as elasticity modulus and pulse wave velocity. results: after 3 months with t, fasting systolic blood pressure was reduced by 10 ae 12 mmhg (p < 0.002) and postmeal by 12 ae 16 mmhg (p < 0.003) vs. a (5 ae 10 mmhg (p < 0.04) fasting and insignificant 3 ae 10 mmhg (p < 0.05 vs. t) postmeal). with t but not with a, diastolic pressure dropped by 9 ae 11 (p < 0.003) fasting and postmeal (p < 0.006). with t but not a, e' increased fasting and postprandially by 0.6 ae 1.2 (p = 0.04) and 0.8 ae 1.2 cm/s (p = 0.01) and so did s' (p < 0.001 and 0.03), whereas vascular function improved postprandially alone (p < 0.002 and p < 0.001). conclusion: in ms patients, mild to moderate hypertension and without cardiac disease, monotherapy with telmisartan improved diastolic and systolic cardiac function in particular postprandially whereas amlodipine did not. introduction: the metabolic disorders at the tunisian military pilots are more and more frequent affecting more and more young subjects. the expert doctor is in front of a big problem to know the evaluation as well as the control of the evolutionary genius of these disorders to be able to minimize their complications materials and methods: it is about a retrospective study which was interested in the files of the tunisian military pilots followed regularly in the center of expertise of aeronautical medicine since 1989. on these files, we found the various types of metabolic anomalies by referring to the age and the anthropometric characters during their appearance, as well as the evolutionary follow-up of these anomalies with or without treatment results: the metabolic disorders were found at 50 % of the military pilots. these disorders are represented in the order by dyslipide´mies, then disorders of the hepatic metabolism, the hyper urice´mie and finally the not insulin-dependent diabetes. the therapeutic care ways begins with the hygie´no-dietary rules before the passage in the medicinal treatment. the metabolic disorders make the bed of the cardiovascular diseases; their complications particularly at the military navigators are very grave. the correction of these anomalies is imperative to avoid the vascular accidents which can be responsible for a sudden incapacity during flight. comparison to wt controls, and the epididymal fat weighs significantly less in the ko (p = 0.008; normalized to body weight). the irx5 ko mice showed increased feed and water intake relative to their body weight compared to wt littermates. moreover, qpcr and western blot analysis indicates differential expression of irx5 during adipogenesis in 3t3-l1 cells. our data suggest that irx5 plays a role in the development of adipose tissue. method: forty-six obese participants (both sexes, age range from 20 to 55 years) group mean ae sd, 37.0 ae 11.6 years were selected for the study based on a bmi ! 23 kg/m 2 *. the participants were randomized as two groups. all participants were assessed for bmi and lipid profile at the beginning and end of 15 days of the intervention. twenty six participants (13 from each group) were assessed for leptin and adiponectin. the yoga group practiced yoga for 45 min two times in a day for 15 days. at the same time of the day the walking group practiced 45 min of walking for two times in day. each day participants were given non calorie restricted diet regulated as 1800 kcal/day. data recorded at the beginning and at the end of the intervention were compared by repeated measures analysis of variance using spss version 18.0, followed by post-hoc analysis. results: both groups showed a significant decrease in bmi (p < 0.001) and in total cholesterol (p < 0.05). only yoga group showed significant reduction in ldl cholesterol (p < 0.05) and significant increase in leptin (p < 0.01). the walking group alone showed a significant reduction in triglycerides (p < 0.05) and in adiponectin (p < 0.05). objective: human urotensin ii is the most potent vasoconstrictor identified to data. however, association between urotensin ii and hypertension and whether the association is independent of endothelial function has been controversial. here, we studied the association under adjustment for serum nitric oxide in a case-control study. methods: hundred and ninety-seven hypertensives and 197 age-and sex-matched normotensive controls were studied. plasma urotensin ii, serum nitric oxide and other traditional biomarkers were examined. association between urotensin ii and hypertension was evaluated by multivariate conditional logistic regression analysis. results: hypertensives had higher levels of urotensin ii [median (interquartile rang): 9.32 (7.86-11.52) ng/ml vs. 8.52 (7.07-10.41) ng/ ml] and lower nitric oxide [19.19 (2.55-38.48) lmol/l vs. 23.83 (11.97-43.40 ) lmol/l] than normotensive controls. urotensin ii positively correlated with sbp (r = 0.169, p < 0.001), dbp (r = 0.113, p = 0.024) but negatively correlated with nitric oxide (r = à0.112, p = 0.027). in multivariate regression analysis, subjects in the highest 25th percentile of urotensin ii concentration ( ! 10.91 ng/ ml) had 2.33 times the risk of hypertension than did individuals in the lowest quartile (<7.30 ng/ml) (p = 0.027). both univariate and multivariate analysis in 106 pairs of serum nitric oxide level-matched cases and controls showed that risk of hypertension significantly and positively increased with levels of urotensin ii (all p < 0.05). conclusions: urotensin ii was markedly associated with hypertension and the association was independent of endothelial function. this study suggested that urotensin ii may have an etiological role in hypertension. objective: dilated cardiomyopathy (dcm) is the third most common cause of hf. the implication of transcription factors (tfs) in molecular pathways that guide heart development and cardio-specific gene expression has recently been established. however, the role of cardiac specific tfs; myocardin and tbx20 in the failing heart is unknown. the present study was designed with the aim to determine the expression profile and regulation of these tfs in failing hearts. methods: myocardin and tbx20 mrna levels were estimated by quantitative rt-pcr (qrt-pcr) in human ventricular biopsies and pbmcs of dcm patients (n = 25) and controls (subjects with ventricular septal defect) (n = 12). copy number variations in myocardin and tbx20 were determined by qrt-pcr in dcm patients (n = 150) and control (n = 100). myocardin and tbx20 promoter methylation patterns were studied in pbmcs of dcm patients (n = 80) and controls (n = 60) by methylation specific pcr (msp). results: myocardin and tbx20 mrna levels were found to be significantly six fold (p 0.004) and seven fold (p 0.00017) increased in the failing human myocardium as compared to control samples respectively. promoter hypermethylation of myocardin was observed only in patients and none of controls were found to carry methylated alleles for myocardin. we did not observe any significant difference in promoter methylation status of tbx20. there was no significant difference observed in the copy number of both the transcription factors; myocardin and tbx20 between patients and controls. conclusion: our results suggest that epigenetically regulated expression of cardiac specific tfs; myocardin and tbx20 may contribute to pathophysiology of dcm. introduction: mir-21is among the most abundantly expressed and consistently dysregulated mirnas in heart failure and has been implicated in cardiac fibrosis. however, its role in diabetic cardiomyopathy is not known. abstracts of the 5th international congress on prediabetes and metabolic syndrome normalisation of the majority of indicators of lipid and carbohydrate metabolism. a.g. kistauri 1 , g. devidze 2 , m. jibladze 1 , a.a. kistauri 3 1 internal medicine, tbilisi state medical university, tbilisi, 2 surgery, kutaisi ortodox christian hospital, kutaisi, georgia, 3 lund university, lund, sweden aims: diabetic foot syndrome is a complex of purulent-necrotic and/ or ostheoarthropatic changes of the foot. in case of purulent-necrotic complications, lethality reaches 6-22%. objectives: optimisation of infected diabetic foot treatment. methods: eight-four patients, suffering from infected diabetic foot. following was used as a material for antibioticogram: soft tissue scraping from the bottom of the injury, purulent discharge, bone biopsy. results: degree of bacterial contamination was high: between 10 5 and 10 10 -10 12 : anaerobic flora -77.3%, aerobic flora à17.7%. bacterial flora in purulent zone (decreased-38.1% to 4.7%): staphylococcus aureus, saprophyticus, epidermidis, ps. aeruginosa, enterococcus, e. coli. the highest sensibility was shown to the following drugs (decreased-98.9 % to 40.1%): tienam, meronem, amoxiklav, vankomicin, cefepim, ceftriaxon, ciprofloxacin, likacin, klindamicin. less sensitivity was detected to the following drugs (4.7-2.3%): ampicilin, doxaciclin, cefazolin, erythromycin. based on the above, the most appropriate combinations are: ftorchinolines + i-iii generation aminoglycozides (combination i) and/or iii generation cefalosporines + lincosynamides (combination ii). using the first drug combination, infection was stopped in 42.3% and in 48.1% of cases using the second. high amputations were performed in two cases, six more than half of foot. in 59 cases process was stopped and amputation was avoided. conclusions: cause of tissue necrosis during neuropathic form of diabetic foot is infection, during neuroischemiccritical ischemy and infection. neuroischemic infected injury is characterised with much faster course than infected neuropathic ulcer. the most appropriate treatment combination of diabetic foot is fluorcholines + i-iii generation aminoglycozyed and iii generation cefalosporines + lincosomydes. background: continuous glucose-monitoring system (cgms) is a tool for assessment of glycemic excursions. glucose variability is a risk factor independent of glycosylated hemoglobin (hba1c) for diabetic complications. aim: to evaluate the prevalence and extent of glycemic excursions & unrecognized hypoglycemia in type 2 diabetic patients. setting and design: the study was carried out in 50 type 2 diabetes patients on oral agents. material and methods: patients underwent continuous glucosemonitoring by cgms for 3 days. number and duration of glycemic excursions, unrecognized hypoglycemia, correlation coefficient (%) between cgms and self-monitoring blood glucose (smbg), mean absolute difference (%mad were analyzed. results: the mean age of patients was 45.6 ae 14.3 years. the mean hba1c was 9.5 ae 2.3%. the mean number of glycemic readings was 766.0 ae 186.9 times. the correlation coefficient was 0.65 and the mad was 12.3 ae 8.0%, which were considerable. twenty three (46%) patients experienced 134 hypoglycemic events. twenty seven (54%) patients had hyperglycemic events. the hypoglycemic events were found to have significant correlation with the duration of diabetes and inverse correlation with hba1c, whereas age was significantly correlated with females diabetics (p < 0.05). conclusion: this study demonstrated that type 2 diabetic patients have a considerable number of hypoglycemic and hyperglycemia events that may be missed by smbg. internal medicine department, zagazig university, faculty of medicine, university hospital, 2 medical biochemistry, zagazig university, faculty of medicine, zagazig, egypt impaired fibrinolysis increase the risk of cvd in diabetics, it has been found also in igt associating metabolic syndrome but there is no data concerning fibrinolysis in subjects with normal gt that may convert to diabetes. therefore, the aim of work was to study the fibrinolytic activity, as measured by tpa activity and t-pai-1 antigen as markers of endothelial dysfunction in normal and igt offspring of type 2 diabetes compared to subjects without family history of diabetes. we measured fibrinolytic activity (tpa activity and tpai-1 antigen) in 150 subjects, 50 healthy volunteers and 100 offspring of type 2 (50 normal gt and 50 igt), we measured fasting plasma insulin, lipid profile and insulin resistance. we found that tpa activity was significantly reduced and tpai-1 was significantly raised in igt group as compared to normal gt. surprisingly tpa activity was also significantly reduced and tpai-1 was significantly raised in normal gt. also, we found that igt increases the (tpai-1 antigen and decrease tpa activity in igt by 6.9 fold and 8.6 respectively than normal gt offspring. we can concluded hypofibrinolysis may aggravate insulin resistance and promote progression of atherosclerosis in those offspring in the future. this may explain the increased prevalence of cardiovascular diseases in early discovered diabetics. moreover, changes of these parameters can be used as a predictor for early detection of prediabetic state even before occurrence of glucose intolerance and the proper correction of this hypofibrinolysis may delay the development of atherosclerosis in diabetic stage. introduction: type 1 diabetes in children is the most common juvenile endocrinopathy. decreased bone mass has been shown to be a common complication of type 1 diabetes. the aim of our study was to evaluate the characteristics of bone mineral density (bmd) in diabetic children and their relationship with the age of diabetes, glycemic control and the biological markers of bone remodeling. with an assay of biological markers of bone turnover were performed for each child. results: sex ratio was 0.53 in the diabetic group and 1.1 in the control group. the average age of diabetics was 10.4 years and that of controls was 9.9 years. the two groups were comparable concerning the age, sex, anthropometric parameters and pubertal stage. the only difference found between the two groups concerning calcium intake that was lower in the controls. bone mineral density did not differ significantly between the two study groups. a significant difference was found comparing averages of bone markers (osteocalcin and ctx). the seniority of diabetes was correlated to the bone mineral density only in the diabetic daughter. the glycemic control represented by hba1c was inversely correlated to the bone mineral density. conclusion: the prevention of bone damage in the diabetic child must go through adequate calcium intake, a proper sports activity and especially a good control of the disease. background and aims: the aim of the present study was to investigate all-cause mortality in relation to physical inactivity and diabetic status among patients surviving an acute myocardial infarction (ami). we hypothesised that physically inactive patients had a higher mortality risk compared to those who were physically active, regardless of diabetic status. methods: we enrolled n = 1008 patients with ami admitted to the coronary care unit of the central hospital in va¨stera˚s, sweden between november 2005 and may 2011. all-cause mortality was followed-up until may 2012. the relation between self-reported leisure time physical inactivity during the last year, diabetic status and allcause mortality was analysed univariately using kaplan-meier curves and multivariately using cox regression adjusted for the confounders bmi, prior angina pectoris, prior stroke, smoking, age, sex, education level and immigration status. p-values<0.05 were considered statistically significant. results: a total of n = 972 (96.4%) of the ami patients had valid values for diabetics status and leisure time physical inactivity. of these, n = 219 (22.5%) died during follow-up. the mortality was significantly different (log-rank test p < 0.001) between physically active and inactive patients with or without known diabetes (see figure) . notably, physically inactive patients had a higher mortality regardless of diabetic status. after adjusting for confounders, the hazard rate was 1.64 (p = 0.002) for having diabetes and 2.56 (p < 0.001) for being physically inactive. conclusions: physically inactive patients had a higher mortality risk compared to those who were physically active, regardless of diabetic status. medical pharmacology, akdeniz university, antalya, turkey aims: we investigated functional effects of glp-1(7-36), glp-1(9-36), exendin-4(1-39), exendin (9-39) and role (s) of reactive oxygen species (ros) and endothelium-derived hyperpolarizing factor (edhf) in the effects of these agents in small resistance arteries from control and diabetic rats. methods: mesenteric arterial rings were suspended in wire myograph and responses to glp-1(7-36) and its analogues were recorded in the absence and presence of ros scavengers; superoxide dismutase (sod, 100 u/ml) and catalase (cat, 1000 u/ml). role of edhf in glp-1 induced responses was investigated in kcl (30 mmol/l)-contracted rings following incubation with no synthase inhibitor l-name (10 à4 mol/l) and cyclooxygenase inhibitor indomethacin (10 à5 mol/l). results: glp-1(7-36) and glp-1(9-36), but not exendin-4(1-39) or exendin (9-39) produced concentration-dependent relaxations in mesenteric arteries from control and diabetic rats that were significantly higher in control compared to diabetic rats and in endothelium-intact compared to denuded preparations. incubation of control and diabetic rat mesenteric arteries with cat did not affect responses to glp-1(7-36) and glp-1(9-36) while, sod caused a significant increase in relaxant responses only in diabetic rats. glp-1 (7-36) and glp-1(9-36) induced relaxations were significantly and similarly blunted by l-name plus indomethacin in control and diabetic rats. we provided evidence about relaxant effect of glp-1(7-36) and glp-1(9-36) in rat resistance arteries and about the reduced vasorelaxant effect of glp-1 in diabetic rats. our findings suggested that edhf played a role in glp-1-induced relaxations and that increment in certain ros and/or reduction in sod function might play a role in reduced vazorelaxant responses to glp-1 in diabetic rats. results: significant differences for systolic blood pressure, lipids levels and kidney function between the groups are depicted in table 1 . homa-ir was elevated in both groups, but significantly higher in the diabetic group (11.8 ae 9.7 vs. 5.1 ae 3.5; p < 0.001). diastolic blood pressure, crp and total cholesterol were not significantly different between the two groups. in the longitudinal group hba1c levels decreased dramatically in the diabetes group (7.5 ae 1.4% vs. 5.8 ae 0.7%; p < 0.008), but less in the normal glucose tolerance test group (5.6 ae 0.4% vs. 5.3 ae 0.4%; p < 0.001). conclusion: diabetic patients with morbid obesity have a much higher cv risk profile compared with nondiabetic subjects despite presenting with the same bmi and should therefore be the preferred candidates for metabolic surgery since capacities are very limited. objective: morbid obesity (mo) has been shown to be associated with hypothyroid disorders in some patients. we therefore investigated the relationship between thyroid function parameters, bmi and insulin resistance in patients with morbid obesity before and after bariatric surgery. in 286 patients (82.9% women) with mo (41 ae 10 years; bmi 44.8 ae 9.4 kg/m 2 ), parameters of thyroid function (tsh, ft4, ft3) and homa-ir were determined before and 2 years after bariatric surgery (weight loss à40.3 ae 19.3 kg; p < 0.001). due to the lack of normal distribution, thyroid function parameters were logarithmized for statistical evaluation. results: of the 286 patients, 47 presented with hypothyroidism (tshrange: 3.70-21.06 lu/ml) before surgery, six of them with overt hypotheroidism (ft4-range: 0.78-0.87 ng/dl). in the euthyroid patients, tsh decreased from 2.1 ae 0.8 to 1.9 ae 1.0 lu/ml (logtsh 0.28 ae 0.19 vs. 0.21 ae 0.24; p < 0.001). in the hypothyroid patients, tsh declined from 6.5 ae 3.9 to 3.4 ae 2.4 liu/ml (logtsh 0.76 ae 0.19 vs. 0.40 ae 0.49; p < 0.001). in the latter, the decline in tsh was more pronounced than in euthyroid patients (p < 0.001). concordantly, ft4 concentrations increased by 9.1% (p = 0.001) and by 3.3% (p < 0.001) in euthyroid and hypothyroid patients. homa-ir declined from 5.7 ae 4.2 to 1.9 ae 1.4 (p < 0.001) in euthyroid patients and from 6.5 ae 4.9 to 1.7 ae 1.3 in hypothyroid patients (p < 0.001), but did not correlate with the decline in tsh. the improvement of thyroid function after bariatric surgery is not associated with the reduction in insulin resistance. therefore the improvement of thyroid function could independently contribute to the positive long-term effects of this intervention. introduction: some years ago chronic diseases were considered to be a problem of the rich and elderly population. today we know that within high-income countries, poor as well as young and middle-aged people are affected by chronic conditions. for chronic disease, there are a small number of risk factors common to many diseases. the major biological risk factors identified in the world health report 2002 are: overweight and obesity, raised blood pressure, raised blood glucose and abnormal blood lipids and its subset raised total cholesterol. objective: was to carry out an epidemiological survey on prevalence of raised fasting blood glucose in kosova. material and methods: according to the who steps methodology the fasting blood glucose in blood sample from finger was measure with accutrend plus on sample of 1000 randomly selected participants aged 15-64 years. results: according who criteria raised fasting blood glucose is if capillary glucose is more than 6.0 mmol/l or more than 110 mg/dl. in kosova, females are in higher risk for diabetes than males (females 15.5% vs. males 11.9%), total 13.7%. prevalence of raised fasting glucose was increased with age. at the age 15-24 year the prevalence of raised fasting blood glucose was 0.6%, at age 25-34 year 3.6%, at age group 35-44 year 10%, at age group 45-54 year was 21.2% and at the age group 55-64 year was 34.5%. conclusion: effective preventive interventions are needed, and health systems should prepare to detect and manage diabetes and its sequel. objectives: hypertension and hyperglycemia are features of the metabolic syndrome and diabetes. methylglyoxal (mg), a reactive glucose metabolite, is elevated in diabetic patients. we investigated whether mg induces hypertension and its molecular mechanisms. methods: male 12 week old sprague-dawley rats were treated with mg (24 mg/day by continuous infusion with a minipump) for 4 weeks. aortic rings were used for vascular contractility, and other tissues alongwith cultured vascular smooth muscle cells (vsmcs) for molecular studies. hplc, western blotting and q-pcr were used to measure mg, proteins and mrna, respectively. sirna for angiotensinogen and the receptor for advanced glycation endproducts (rage) were used to study mechanisms. results: mg treated rats developed a significant increase in blood pressure, plasma aldosterone, renin, angiotensin (includes precursor and products), norepinephrine, epinephrine and dopamine levels. the aorta showed increased contractility to the a1 agonist phenylephrine. mg level and protein and mrna for angiotensin, at1 receptor, a1d receptor and renin were significantly increased in the aorta and/or kidney of mg treated rats. treatment of cultured vsmcs with mg or high glucose (25 mmol/l) significantly increased cellular mg, and protein and mrna for nf-jb, angiotensin, at1 and a1d receptors, which were prevented by inhibition of nf-jb. silencing of mrna for rage prevented the increase in nf-kb induced by mg. silencing of mrna for angiotensinogen prevented the increase in protein for nf-jb, angiotensin, at1 and a1d receptors. conclusions: mg activates nf-jb through rage and thereby increases activity of the renin angiotensin aldosterone system to cause increased vascular contractility and hypertension. background: guidelines state obesity as an indication for diabetes screening. abdominal obesity is valuable to manifest metabolic risk as much as and may be even more than body mass index (bmi). in this study the answer to "which is a better indicator for diabetes screening; bmi or waist circumference?" has been questioned. method: three hundred people attended to the invitation of diabetes screening in istanbul medeniyet university goztepe research and training hospital outpatient clinics. their demographic characteristics, bmi, waist circumference, fasting blood glucose (fbg), diabetes history and if diagnosed drug regimen were recorded. results: a total of 300 individuals (209 women, 91 men) were enrolled. one hundred forty three of 300 (48%) individuals had a bmi ! 30. among those people 44 (31%) had fbg ! 100 and <126 mg/dl and 29 (%20) had fbg ! mg/dl (table 1) . abdominal obesity was observed in 225 individuals (173 women, 52 men). thirty 2% had known diabetes mellitus (dm). among patients with abdominal obesity and without dm diagnosis, 39% had dysglycemia (table 2) . among 143 obese individuals, 37% had a history of dm. dysglycemia rate among obese persons without a history of dm, was 25% (table 3) conclusion: abdominal obesity is a strong predictor of diabetes as obesity and its presence should be considered as an indication for screening diabetes. internal medicine and cardiology, expertise centre of aviation medicine of tunis, mhiri, tunisia type 2 diabetes is a rare disease compatible with aviation activity with restrictions and constraints of regular medical checks. this retrospective study was interested in drivers with type 2 diabetes cemeda monitored since 1991 until 2010. the aim of this work is to investigate the incidence of diabetes in the tunisian population of seafarers and show the difficulties of management of diabetic driver.59 sailors have diabetes type 2 (or # 5%) and three student pilots (two civilians and military) had type 1 diabetes resulting inability final flight. average blood glucose of diabetic patients was 1.50 g/l with a range of 1.26 and 2.7 g/l; glycosylated hemoglobin a1c is an average of 7.3% [range: 6.3-11.8 %]. the average age of diabetes is 51 years and mean bmi of 30 kg/m². a mild to moderate hypertension was found in 44% of diabetics; dyslipidemia was found in two-thirds of diabetics. the treatment of diabetic subjects: rules dietary guidelines were prescribed in only about 44% of cases. moreover, these measures were associated with treatment with oral antidiabetic (biguanide alone in 27% of cases; biguanide + pioglitazone in 5% of cases associated biguanide a sulphonylurea in approximately 17% of cases. conclusion: : the prevalence of niddm is significantly lower among sailors in the general population. the balance diabetic seafarers is the only guarantor of declining maturity of micro and macrovascular disease and strengthen the doctor′s attitude towards pn medical ethics and flight safety. methods: is a retrospective cohort study that included patients who attended a preventive evaluation unit between 2001 and 2011, with at least three visits in that period. results: a total of 105 patients were included, of whom 18 (17%) had nafld at their first visit. the basal variables were similar between groups, with male predominance in the nafld group. high blood pressure and dyslipidemia predominate in the nafld group until visits 4 and 3 respectively. the other cardiovascular risk factors were similar until visit 6. the study showed a tendency to present high blood pressure, dyslipidemia and gallstones in the nafld group in a shorter period of time than the control group. levels of aspartate aminotransferase raised in a period of 6.9 vs. 8.7 years in the control group (p = 0.08). the study was not able to demonstrate a clear difference in the presentation of cardiovascular risk factors and liver function tests in patients with nafld and although there was a higher presentation of gallstones, it requires a larger sample and a longer follow up period to have significant results. objective: to determine the relationship between metabolic syndrome (ms) and exaggerated blood pressure response to exercise (ebp) in adolescents. methods: this cross sectional study was conducted in 553 adolescents (287 males, 266 females), randomly selected of high schools, in maracaibo, venezuela. they underwent an exercise treadmill testing. the ms was defined according to the definition of the national cholesterol education program, adult treatment panel iii modified for adolescents. the bp was measured at rest and during the treadmill test (bruce protocol), and it was registered the systolic bp (sbp) in maximal exercise to define adolescents with ebp ( ! 200 mmhg). statistical analysis: chi square test was used to establish associations between ms and ebp. results: the ms prevalence was 4.2% (n = 23) in all subjects, 6.3% (n = 18) in males and 1.9% (n = 5) in females (p: 0.01). the sbp in maximal exercise values were: 185.9 ae 25 in adolescents with ms and 160.3 ae 26 in adolescents without ms (p: 0.0001). the prevalence of the ebp was 8.1 % (n = 45) in all, 17.8% (n = 8) and 3% (n = 15) in subjects with ms and without ms, respectively (p: 0.0001). the ms is closely linked to the sbp response to exercise in adolescents. those adolescents with ms are more likely to have exaggerated sbp during exercise, indicating an important information about their cardiovascular risk. background: metabolic syndrome (mets) is a cluster of risk factors that carries a great risk for atherothrombotic events leading to significant morbidity and mortality. few studies evaluated the association between mets and acute stroke. the aim of this work was to study the prevalence of mets and its effect on icu mortality in acute non embolic ischemic stroke patients. patients and methods: we studied 260 patients presented with acute ischemic stroke diagnosed by ct brain in the medical icu, zagazig university hospital for the presence of mets and its relation to age and sex as well as the number of components of mets. we studied also the relative risk of mets with its different components as well as age, sex, glasgow coma scale (gcs) and apache ii score on the mortality in those patients. results: mets was found in 53.8% of patients. it was more prevalent in males (55.7%). this prevalence increase progressively in males aged 40-60 years then decrease progressively while in females, prevalence increase progressively with age. 55.7% of patients have three components of mets compared to 44.3% with more than three components. the relative risk of mortality was found to be increased with increasing age, male gender, obesity, hyperglycemia, low hdl-c, increased serum triglycerides, lower gcs, increased map, mets per se and higher apache ii score. mets and apache ii score were good predictors of mortality. contrary to the usual approach, we could leave the future open to a bi-therapy associating glp-1 mimetics and dpp-4 inhibitors (before the supplementation in metformin and sulfamids or glitazones). case report: diabetes type 2 in balance for 20 years after being treated first with metformin only then added glitazone, to end by glp-1 mimetics at maximum doses and metformin but still not under control. it was decided to associate glp-1 mimetics and dpp4 inhibitor, ending up to a glycemia 82 and insulin 5.8 and hba1c:5.2. after stopping the treatment for 10 days we observe: fast glucose 159, postprandial 111 (on a diabetic diet), relative hypoglycemia generating a multi metabolic syndrome spiral. the increase of the fast glucose being explainable by the non-control of the glucagon produced during the night. pattern proposed: glucagon would not work on demand but time continuously. it has to be inhibited thanks to the glp1 as soon as the glycemia increases and this would be irrespective of food intake. therefore, there is no reason to use the dpp4 inhibitors only in case of resistance to endogen glp1 and let the glp1-mimetics being destroyed by the dpp4. this would lead to a therapeutic failure or usage of very high doses. we could treat upstream the sequence of energetic metabolism. first of all: glp1-mimetics or dpp4 inhibitors. secondly: glp1-mimetics and dpp4 inhibitors. those 2 steps could be part of tomorrow′s decisional algorithm of type 2 diabetes. abstracts of the 5th international congress on prediabetes and metabolic syndrome aim: to evaluate the prevalence tipe 2 diabetes mellitus (dm2) and ischemic heart disease (ihd), and diabetogenic/atherogenic factors in patients with newly diagnosed long-term gout material: seventy males 33-77-years (mean 53) with untreated tophaceous primary gout 5-36 years (mean-9) duration (without renal insufficiency) were studied. uric acid level was fasting serum glucose, insulin (control-19-123, mean 99 pmol/l), total and hdl cholesterol, triglyceride, bp and ecg monitoring 3 mmol/l, insulin 134 and 192 pmol/l. one more patient, 55 years, with ihd and triglyceride 11.6, cholesterol 6.2 mmol/l, insulin 192 pmol/l levels. thus, diabetes was in one and ihd in three patients two patients (60-70 years) with 19 and 36 years of gout and hyperinsulinemia thus, long-term gout (hyperuricemia) even with concomitant high bmi, insulin, cholesterol and triglyceride levels is not an evident risk factor for diabetes and ihd development. probably, antioxidative effects of uric acid play a role montes claros, brazil background: the leptin receptor is an important regulator of leptin activity and a potential mechanism for the obesity. the polymorphism leprgln223arg could be associated with high body fat percentage (bf%) and body mass index (bmi) objective: to investigate the association leprgln223arg with obesity indexes we obtained oral swab and anthropometric measures including waist circumference (wc), bmi and bf% for subjects aged ! 18 years. the genotypes were determined by pcr-rflp method. the statistic analyze were carriedout in stata software. results: there were no differences of age average between sex (female 44.7 ae 17.9; male 44.7 ae 17.1). the prevalence of the overweight (bmi >25 kg/m 2 ) was 35.8%(38), normal, 38.7% (82) and obesity (bmi >30 kg/m 2 ) 22.6% (48). the prevalence of high wc (>80 cm for women or >94 cm for men) was 58.6% (78) and 41.8% (33) in women and men, respectively (p = 0.01) and of the high bf% (>30% for women and >25% for men) was 54 adiposity was not associated with genotypes. the frequencies of overweight were 52.5%, 33.33% and 14.17% and of the obesity were 52.08%, 31.25% and 16.67% in the genotypes aa, ag and gg, respectively. there was not also association for high wc conclusions: these findings suggest that the leprgln223arg is not associated with high bf%, bmi or wc in this population hydrogen breath test (bth 2 ), based on lack of source for hydrogen gas in humans other than bacterial metabolism of carbohydrates, is use to detect carbohydrates malabsorption aims: to evaluate the utility of bth 2 in detect carbohydrates malabsorption in overweight-obese subjects. patients and methods: hundred and six consecutive subjects (38 overweight, 68 obese; 16 males/90 females leptin injection did not suppress ei. importantly, ts treatment reinstalled leptin sensitivity as seeing a significant decrease in ei for 24 h (à39%, p = 0.023) and increase of pstat3 level (à94%, p < 0.001) in the hypothalamus after i acknowledgements: this work was supported by a grant of the romanian national authority for scientific research, cncs-uefiscdi, project number pn-ii-id-pce-2011-3-0429". dr. lixandru, was supported by the postdoctoral program posdru/89/ 1.5/s/60746, from european social fund. this cluster-randomized controlled trial study aimed to assess the effectiveness of the interactive multi-modality technology (imm) as an intervention to increase self-management among type 2 diabetic patients in a 3-month period. the imm intervention contained email, short message system (sms), and website with four main functions (i.e., selfregulation, self-monitoring and assessment, social support, and reminder system -linked to email and sms). in this trial, four public offices in bangkok metropolis were recruited and randomly assigned into either the intervention or the control group. one hundred and twenty four thai patients who had met inclusion criteria (hemoglobin a1c or a1c >7.0%, no serious illness, and internet and mobile phone accessibility) were subsequently assigned to the intervention (n = 74) and the control (n = 48) group. patients in the intervention group received the imm intervention. those in the control group received selfmanagement knowledge via email only. outcome measures, a1c and behavioral questionnaires (diabetes quality of life, self-efficacy, and division of endocrinology and metabolism, national institute of nutrition, secundrabad, india introduction: we showed earlier that increased visceral adiposity in the offspring of magnesium deficient rats was associated with altered gene expression and increased stress.aim: to assess whether maternal magnesium deficiency modulates the gene expression of adipogenesis and insulin sensitivity in utero due to increased stress.methodology: female weanling wnin rats received for 12 weeks, an ain 93g diet (control: mgc) or the same with 61% restriction of magnesium (mgr) and mated with control males. half the pregnant mgr dams were rehabilitated from conception (mgrc) while others continued on magnesium restriction. total rna was isolated from the mgc, mgr and mgrc embryos collected on day 10 of gestation and expression of 11βhsd1, pparc2 and adiponectin was quantified by real time pcr. body composition was determined in 6 month old offspring by total body electrical conductivity (tobec).results: plasma magnesium levels in mgc and mgr dams before pregnancy; and 6 month old mgc, mgr and mgrc offspring were on expected lines. mgr embryos had significantly higher expression of pparc2 and 11βhsd1 than mgc, whereas adiponectin expression was lower. on the other hand in mgrc10th day embryos 11βhsd1 and pparc2 expression was restored to mgc, adiponectin expression introduction: khaya senegalensis is presently used for the treatment of diabetes in some west african countries.objectives: this study was conducted to investigate the anti-diabetic effects of the plant using in vitro and in vivo models.results: ethanolic extract of the root sample of the plant was subjected to solvent solvent fractionation which yielded a butanol fraction that possessed significantly higher (p < 0.05) anti-oxidative activity as well as a-glucosidase and a-amylase inhibitory activities than other (aqueous, ethyl acetate and dichloromethane) fractions. enzyme kinetic studies indicated that the butanol fraction is a non-competitive inhibitor for a-glucosidase with an inhibition binding constant (ki) of 1.30 lg/ml and a competitive inhibitor of a-amylase with a ki of 7.50 lg/ml. subsequently, the butanol fraction was subjected to in vivo studies in a type 2 diabetes model of rats. after 5 weeks of intervention, the fraction, at 300 mg/kg bw, was found to improve the feed and fluid intake, body weight gain, blood glucose, glucose tolerance ability, serum insulin concentration and β cell function of diabetic animals. phytochemical analysis of the fraction through repeated column chromatography led to the isolation of bicyclo [2.2.0] hexane-2,3,5-triol. the structure of the compound was established through detailed spectroscopic methods including 1 h nmr, 13 c nmr and 2d nmr (cosy, hsqc and hmbc) experiments.conclusion: data from the study suggests that the butanol fraction of k. senegalensis contains bioactive agents that could be exploited in the management of type 2 diabetes. objective: to determine the progression rate to impaired fasting glucose (ifg), impaired glucose tolerance (igt), and diabetes (dm2) in normal glucose tolerant (ngt) people during 8 years follow up study using who 1999 and new criteria of ifg (ifg 5,6 -fasting glucose 5.6-6.9 mmol/l).research design and methods: this is an 8 year prospective study in a randomly selected urban population aged ! 40 years living in krakow, poland. 1752 persons had ngt. based on who 1999 criteria, 564 (32.2% of invited, 209 men and 355 women, aged mean 60.7 sd = 9.2) attended the follow-up assessment.. subjects underwent a physical and biochemical examination and questionnaire examination.results: the prevalence of dm2, ifg and igt according to who 1999 criteria in examined population with baseline ngt was 4.43%, 3.37% and 9.93% respectively. the prevalence of ifg 5,6 using new criteria, was 13.48%, lowering cutoff point for ifg caused 10.11% increase in the prevalence of ifg.among people with diagnosed diabetes 56% had newly diagnosed diabetes during the control study. the prevalence of dm2 and igt/ ifg was increasing with increasing age and bmi categories (p < 0.05). the lowest obesity prevalence both baseline and after follow up was found in those who remained ngt.conclusion: in the baseline ngt population high follow-up progression rate to impaired glucose metabolism was found. the implementation of new ifg diagnostic criteria increased the prevalence of ifg by 10.1%. according to our results prevention of diabetes initiatives should focus on normal body weight preservation. results: sbp was found to be elevated in 68.29% subjects whereas dbp was elevated among 23.34% subjects. out of all the subjects, 52.83% were overweight and 35.39% obese. obese subjects were found to be more hypertensive than non-obese subjects (r = 0.38). sbp had strong correlation with age (r = 0.12), bmi (r = 0.15), whr (r = 0.10), fbs (r = 0.10), chol (r = 0.11) and ldl (r = 0.13) whereas dbp was found to be strongly correlated with same parameters and triglycerides (r = 0.08) also. no significant correlation was found with hba1c levels and hdl-cholesterol. conclusion: hypertension was found to be more prevalent in males as compared to females in this diabetic population. there was an background: the medicine faculty, chiang mai university provided health screening to detect health problem and risk group for high school students who past entrance examination in this academic year 2012. there is no official cutoff value of waist circumference (wc) measured at superior iliac crest level (wci) for overweight and obesity in thai young adolescence. we aimed to determine correlation between wci and to those measured at midpoint between lower costal margin and superior iliac crest (wcm) and to define appropriated wci and body mass index (bmi) cutoff levels for admission students in northern thailand.methods: the admission students had weight (kg), height (cm), wc, and blood pressure measurements.results: there were 6344 admission students. 59.30% of them were female. the correlation between wci and wcm was 0.86. for receiver operating characteristics analysis of having high bp (systolic ! 140 mmhg or diastolic ! 90 mmhg.) using wcm of 90 cm in male and 80 cm in female as standard (wcm9080), area under curve (0.735) of wci of 80 cm in male and 75 cm in female (wci8075) andabstracts of the 5th international congress on prediabetes and metabolic syndrome all patients were asked to follow a dietary plan for 2 weeks before bth 2 , 1 day before the test, subjects were instructed to take a low fibre diet. bth 2 was carried out both after overnight fasting (t 0 ) and during oral glucose tolerance test (ogtt). hydrogen gas is detected in exhalate, cut-off value was settled at 12 ppm.results: at t 0 bth 2 was positive in 5% of overweight and 37% of obese subjects.particularly, bth 2 was positive in 46% of subjects with ms and in 30% of those without ms.during ogtt, 50% of bth 2 results, negative at t0, became positive.conclusions: obese subjects with ms had an bth 2 altered value at t 0 suggesting a intestinal dysbiosis.on the basis of our preliminary data, bth 2 at t 0 can be helpful to diagnose gastrointestinal disorders driving the idea of intestinal dysbiosis as a possible cause in pathogenesis of obesity, thus a probiotic supplementation is to be considered as baseline therapy in overweight-obesity. department of clinical physiology and pathology of locomotor apparatus, institute of gerontology ams ukraine, kyiv, ukraine aim of research: to determine uric acid level in blood serum and incidents of hyperuricemia among women and men of different age and their relation with some components of metabolic syndrome.object of research: age of examined patients was from 20 to 89 years old: women (n = 450) and men (n = 120). they were divided in following groups: i group (bmi = 18.5-24.9), ii -(bmi = 25.0-29.9), iii -(bmi = 30.0-34.9), iv -(bmi >35). average age of examined patients was 60.4 ae 0.7 years.methods: uric acid level in blood plasma was determined by uricaseperoxidase method. aim: we studied the prevalence and prognostic impact of the metabolic syndrome (ms) in acute myocardial infarction (ami) patients (pts) with normal (ngt) or abnormal glucose tolerance (agt).material and methods: a total of 295 consecutive nondiabetic ami pts performed an oral glucose tolerance test at hospital discharge being categorized using idf/nhlbi/aha 2009 ms criteria into four groups (gr): gr. i -ngt no ms (n = 137), gr. ii -ngt with ms (n = 63), gr. iii -agt no ms (n = 54), gr. iv -agt with ms (n = 41). clinical characteristics and cv events during 2.5 years follow-up were studied.results: ms was diagnosed in 35% pts, with a higher prevalence in agt vs. ngt subjects (p < 0.005). ngtms and agtms subjects were more likely to be older (p < 0.05), women (p < 0.001), hypertensive (p < 0.001), have stroke history (p < 0.05) and hypertriglyceridemia (p < 0.05) compared to pts without ms. in hospital heart failure rates were higher in gr. ii, iv vs. gr. i, iii (p < 0.05). agtms subjects showed more often atrial fibrillation and a lower lvef% compared to ngtms (p < 0.05). cv events incidence during follow-up was similar among groups excepting significantly higher mortality in pts with ms, especially in agtms subjects (rr = 1.63 (95% ci 1.0-2.6) for ngtms; rr = 2.1 (95% ci 1.17-3.77) for agtms).conclusions: ms was present in 35% of nondiabetic ami patients and was associated with high long-term mortality; its incidence and the risk of death increased in the presence of agt. methods: a cross-sectional study was realized in government workers during his medical review, from april to september 2011. the screening included height, weight, waist circumference, blood pressure measurements, plus a blood draw sample to determine glucose, triglycerides, and total cholesterol. body mass index (bmi) was used to diagnose overweight and obesity according to cut-offs proposed by the world health organization (2007). the mets was defined according with aha/nhlbi criteria (2005) . waist circumference (wc) and waist-to-height ratio (whr) were used as fat distribution indexes. all statistical analyses were performed using the spss v19.results: in total, 84 workers aged 20-62 years, of both sexes (45% males and 55% females), were included. the overall prevalence of mets was extremely high with 54% (61% in male, and 48% in female). abdominal obesity was present in 86.7%, high triglycerides in 95.61%, high-density lipoprotein cholesterol levels in 88.9%, fasting hyperglycemia in 84.4%, and hypertension in 28.9% of the sample. 85% of the overweight or obese were diagnosed with metabolic syndrome. the prevalence of overweight and obese was extremely high too, according with gender: 82% in male, and 80% in female. mets was associated with cigarette smoking, absence of physical activity, a higher bmi and a greater proportion of obesity. the mets prevalence in this sector of mexican government workers is a very serious health problem. it is urgent to develop innovative programs that improve health situation. our rehabilitation department targets obesity and take care of 1000 patients a year, either in a outpatient (op, day hospital) or in patient (ip) programme.we focus on therapeutic education, and our approach is medical, nutritional, physical, psychological and social.the aim of our study is to compare the efficiency of the day hospital vs. in patient in the rehabilitation of obesity.two samples of 40 patients who exited our unit 6 months ago were compared.investigated criteria were: loss of weight during the programme, way of life after the programme (physical activities, diet), medical and psychological follow up.the results showed:1. a weight loss between the patient admission and the trial date of 7.2 kg for in patients vs. 7.4 kg for day hospital patients.2. the diet prescription is followed by 57.5 % of ip patients and by 85.5 % of op patients.3. the prescription of physical activity was followed by 50 % of ip patients and 67.5 % of op patients.4. a psychological follow up is done by 25 % of ip patients and 22 % of op patients.the efficiency of the therapeutical education programme is proven in either ip or op patients.the day hospital allows a smooth return to normal life after a nutrition inpatient rehabilitation. research design and methods: a total of 100 asymptomatic t2dm subjects (51 men and 49 women; mean age: 56.4 ae 7.6 years) were enrolled. clinical and laboratory parameters, including hba1c, glycoalbumin, lipid profile were evaluated and cardiovascular magnetic resonance (cmr) was performed. abnormal findings of cmr were defined as any one of the followings: 1. silent myocardial infarction.2. inducible ischemia.3. suspected cad.within 90 days after taking cmr, invasive coronary angiography (ica) was performed in selective patients.results: among 100 patients, a total of 51 patients (silent myocardial infarction (n = 3), inducible ischemia (n = 11), suspected cad (n = 37)) had at least one abnormal finding on cmr and ica was recommended. finally, 20 patients underwent ica, and 13 subjects had significant cad (a total of 60 coronary territories assess) overall, in asymptomatic t2dm patients, the positive predictive value (ppv) of sp-cmr, cmr-a, c-cmr are 100%. however, the ppv of each mri to detect signinficant stenosis are 73%, 57% and 54%, respectively. in asymptomatic t2dm patients, stress perfusion cmr showed higher ppv than cmr-a. introduction: observational data suggests that low 25-hyroxyvitamin d is associated with metabolic syndrome in diabetic and non diabetic patients. we examined the difference between components of metabolic syndrome before and after treatment with calcitriol in type 2 diabetic patients with vitamin d deficiency.method and material: a total 119 type 2 diabetic patients were selected. 43 patients had vitamin d deficiency that underwent calcitriol treatment with 0.5 lg per day for 8 weeks. in all cases, clinical parameters including weight, systolic and diastolic blood pressure and laboratory parameters including levels of fasting blood glucose, insulin, lipid profile, calcium, phosphorous, hba1c and insulin resistance were measured, before and after the treatment period. the two sets of results were then compared with one another.results: following treatment with calcitriol hba1c, total cholesterol, ldl, hdl and diastolic blood pressure decreased significantly. (p = 0.01, 0.01, 0.04, 0.001 and 0.04 respectively). but the changes in other parameters were not significant. subjects and methods: the five components of mets following the criteria of the international diabetes federation (idf) were measured in 613 men (mean age 44.8 ae 10 year) and 788 women (mean age 43.8 ae 9.3 year) participating in the pep family heart study. we determined percentage body fat using skinfold thickness. we defined high %bf >34% for females and >22% for males and elevated bmi as ! 25 kg/m². spss version 19 was used for the statistical analyses; multiple linear regression models were used. two-tailed p < 0.05 was considered significant.results: mean values of wc, blood pressure (bp), fasting plasma glucose (fpg), triglycerides (tg), hdl-cholesterol and bmi were significantly higher in men than in women who had higher %bf (28% vs. 21%). using multiple linear regression models, we found significant associations with wc, %bf and bmi. bmi had the strongest associations with tg (beta 5.4; ci 95% 4.6-6.2), hdl-c (à1.07; à1.22 to à0.01), sbp (1. objective: patients with esrd have an increased risk for cardiovascular morbidity and mortality. metabolic syndrome (metsy) has been implicated in the progression of cardiovascular disease (cvd). this cross sectional study investigated the prevalence of metsy in 210 maintenance hemodialysis (hd) patients using a joint definition for metsy.patients and methods: subjects had to meet at least three of the following five criteria for metsy: elevated waist circumference, elevated triglyceride levels, low hdl cholesterol, elevated blood pressure and elevated fasting serum glucose. demographics, medical history, anthropometric and laboratory data were collected from the medical records. serum chemistries were obtained mid-week and waist circumference was measured twice after a hd session and the mean value was calculated. the study cohort consisted of 65% male patients with the mean age of 62.4 ae 14.1 years. hypertension was the leading cause of esrd (38%) followed by diabetic nephropathy (26%). metsy was identified in 69% of the cohort, with 38%, 27% and 22% having 3, 4 and 5 risk factors, respectively. the prevalence of metsy was highest amongst patients on hd for >3 years (35%). for those patients on hd <6 months, 6-12 months and >1 to <3 years, metsy was identified in 15%, 19% and 28%, respectively. metabolic syndrome was not associated with gender and age.conclusion: metsy is highly prevalent in hemodialysis patients, which suggests an additional risk for cvd. early screening for metsy may have protective role on cvd morbidity and mortality. department of family medicine, kangwon national university hospital, chuncheon, republic of korea introduction: it was reported decreased relative muscle mass was related with insulin resistance and prediabetes in us. the aim of this study is to investigate the association of relative muscle mass with cardiovascular disease risk factor using the nationally representative sample of korean adults.methods: this is a cross-sectional study using the data of the 13 003 subjects who participated in the korean national health and nutrition examination survey (knhanes) in 2008-2010. multiple linear regression analysis for survey design was used to explore the association between relative muscle mass and the factors of metabolic syndrome and homa-ir with adjustment for confounding factors.we also conducted multiple logistic regression analysis for survey design to investigate the relationship of relative muscle mass with metabolic syndrome.results: in comparison with subjects in the first quintile of relative muscle mass, the odds ratio (95% confidence interval) for metabolic syndrome for subjects in the fifth quintile was 0.292 (0.183, 0.467) in the age <60 group and 0.155 (0.088, 0.272) in the age ! 60 group respectively after adjusting for confounding variables. relative muscle mass was inversely associated with sbp, dbp, serum tgs, fbs and homa-ir in both age <60 and age ! 60 group, showing significant liner trend.conclusions: decreased relative muscle mass was inversely associated with the prevalence of metabolic syndrome, and the factors of metabolic syndrome except hdl-c. the causal relationship is not exactly known and would be elucidated through further longitudinal study. endocrinology, moscow regional research clinical institute, moscow, russia background: there has been growth in registered prevalence of type 2 diabetes (t2d) in russia. real prevalence of t2d is much more higher. new approaches for early detection of glucose metabolism disorders are important.aim: assess effectiveness of new approach to screening of glucose metabolism disorders based on using diabetes bus in remote areas of moscow county. objective: to study the frequency of metabolic syndrome according to the atpiii criteria among elderly people attending the family practice clinic at the jordan university hospital, and to investigate the pattern of antihypertensive medications used for patients with metabolic syndrome.design: a total of 635 elderly people (298 males and 337 females) aged 60 years or more attending family practice clinic at jordan university hospital.materials and methods: elderly patients included were studied regarding the frequency of metabolic syndrome and its individual components according to the atpiii criteria. antihypertensive medications used by elderly patients with metabolic syndrome were also investigated.results: the frequency of metabolic syndrome was found to be 46.6%. hypertension was the most frequent risk factor among all patients including males and females (58% in the whole sample, 58.7% in males and 57.3% in females). all risk factors except hypertension were significantly more frequent among patient with metabolic syndrome compared to those with no metabolic syndrome. the most commonly used antihypertensive medications were the angiotensin converting enzyme inhibitors (41.5%), followed by beta blockers (34.6%), calcium channel blockers (33%), and finally angiotensin receptor blockers and thiazide diuretics (both 13.3%).conclusion: the frequency of metabolic syndrome was relatively high, which highlights the need to take some action to combat the syndrome. hypertension in particular showed the highest frequency among all risk factors. optimum control of hypertension by following the guidelines is essential in this context to better achieve control without adversely affecting the metabolic syndrome out come. introduction: metabolic syndrome is associated with a significantly increased risk of morbidity and mortality.objective: to assess incidence of the metabolic syndrome in patients of the team 1 in the family medicine centre in kalesija through the medical audit.patients and methods: we have analyzed the medical records of all the patients with team 1 with 20 years of age and over (1200 records).we have used the international diabetes federation and the american heart association (aha) criteria for the definition of metabolic syndrome diagnosis. the analysis is done separately by sex, age and body mass index (bmi). the results of this study shows that 375 (31%) of adults meet the criteria for the metabolic syndrome. of these, 245 (65%) were women and 130 (35%) men. 32% of patients in age group of 40-59 years meets the criteria for the metabolic syndrome. 12% of patients 20-39 years old, and 56% of patients ages 60 and older, meets the criteria for the metabolic syndrome. out of the total number of female patients who meet the criteria for the metabolic syndrome, 78% had increased and 22% had normal bmi. from the male patients who met the criteria for metabolic syndrome, 67% had increased, and 33% had normal bmi.conclusion: the incidence of the metabolic syndrome in family medicine is high. metabolic syndrome is prevalent and significantly increased with age and bmi. more effective interventions in primary care are needed in order to reduce cardiovascular morbidity and mortality. methods: after 15 weeks of high-fat diet, dio mice (n = 40) were divided into two groups received either intraperitoneal (ip) injection of ts (10 mg/kg, daily) or saline for 20 days. another group of mice were fed low-fat diet (lf) as control (n = 20). then both dio mice and lf mice were given intracerebroventricular (i.c.v.) injection of leptin or saline. results: ts significantly decreased final body weight gain (à210%, p < 0.001) and average ei (à24%, p < 0.001) in dio mice. ts significantly decreased pro-inflammatory markers (tnf-a, il-6, il-1β, p-ikk and p-ijba) in epididymal fat, liver and hypothalamus of dio mice. in lf mice, i.c.v. injection of leptin significantly suppressed ei compared to saline injection (à29%, p = 0.001). however, central leptin sensitivity was blunted in dio mice evidenced aim: non-alcoholic fatty liver disease (nafld), which is characterized by the accumulation of fat in the liver in the absence of alcohol intake, strongly linked to metabolic syndrome. recently, proinflammatory cytokines and oxidative stress mechanisms have been implicated in the pathogenesis of psychiatric disorders. in addition, patients with drug-free schizophrenia have significantly higher body mass index than in aged-matched healthy controls. the purpose of the study was to investigate the effects of metabolic syndrome on the apomorphine-induced stereotypy in a rat model of nafld.materials and methods: eighteen male sprague-dawley rats were included in the study. in order to develop nafld model, rats (n = 12) were provided with drinking water containing with 35 % fructose for 8 weeks, while control group (n = 6) received only tap water. after the verification of fatty liver by ultrasonography, apomorphine-induced stereotypy was investigated as described by kenneth and kenneth (1984) . then, all rats were sacrificed; homovanillic acid (hva), a dopamine metabolite, levels were measured in brain homogenates. prefrontal cortical il-2 immunoexpression was evaluated by immunohistochemistry and hepatocellular changes were determined histologically.results: histological evaluation of liver sections confirmed macrovesicular steatosis in nafld rats. moreover, the stereotyped behavior scores, brain hva levels and il-2 expression were found significantly higher in nafld group than in the control group (p < 0.05). our results suggest that metabolic syndrome and fatty liver significantly induce dopaminergic activity and stereotyped behavior in rats. the neuromodulatory effects of pro-inflammatory cytokines and imbalance between oxidative and anti-oxidative status may underlie these alterations. the classical thinking in type 2 diabetes is that the plasma glucose levels are regulated by interplay between insulin, glucagon and other peripheral mechanisms. a combination of insulin resistance and relative insulin insufficiency is considered to be causal to hyperglycemia. this traditional wisdom is now being challenged and an alternative model is proposed where the central nervous system (cns) plays a vital role in plasma glucose regulation. the role of cns in glucose regulation is well known. for example it has been shown that intracerebroventricular injection of glucose results in a decrease in plasma glucose level in rats. however whether and to what extent the cns has an active role in diabetic hyperglycemia is not known. we make mathematical models of different versions of the peripheral and central mechanisms and make differential correlational predictions which can be tested on oral glucose tolerance test ( background and aims: high calorie diets leads to fat liver and changes in the metabolic pathways. therefore the gluconeogenic capacity of the livers in a condition of high levels of exogenous fatty acids was evaluated in cafeteria-fed rats.methods: two groups of weaned wistar rats received cafeteria or balanced diet during 98 days. the fasted livers were perfused with 2 mmol/l lactate plus 0.2 mmol/l pyruvate in absence or in presence of 2 mmol/l stearate plus traces of [1-14 c]-stearate. measured parameters: glucose, ketone bodies, [ 14 c]co 2 , oxygen uptake, hepatic contents of lipids and glycogen.results: cafeteria-fed rats presented increase in: body and liver weight, fat in the tissues and hepatic glycogen. perfusion experiments revealed that in cafeteria-fed rats the gluconeogenic flux was lower than in the control, but the infusion of stearate, caused a higher stimulus on glucose production. the 14 co 2 and ketone bodies production was reduced during the active gluconeogenesis in cafeteriafed and control rats, being the former less sensitive, whereas no differences were found after stearate infusion. background: despite advances in option of treatement diabetes, optimal glycemic control is not often achieved. glucose homeostasis is dependent on a complex interplay of multiple hormones and glucagon like peptide-1 (glp-1) receptor agonists are a new class of drug for the treatment of type 2 diabetes. they are not considered as initial therapy for the majority of patient with type 2 diabetes and their clinical use, long-term benefits, risks and their role in combination with other diabetes medication are still under investigation.study: in the retrospective study in 2011 we enrolled 51 patients with poorly controlled type 2 diabetes on one or two oral agents, who received additional therapy with glp-1 receptor agonist: liraglutide or exenatide. we monitored hba1c value and body weight before and after modification of therapy.results: fifty-one patients, 26 men, were 27-78 years old (mean 58 years). their mean body weight was 116.24 kg (sd ae 24.8) and mean hba1c 8.2% (sd ae 2). after 3-6 months of combined therapy with metformin and/or sulfonylurea and glp-1 agonist their mean hba1c dropped to 7.1% (sd ae 1.4) and their body weight reduced to 107.56 kg (sd ae 33.3). in patients with type 2 diabetes and suboptimal control on one or two oral agents adding glp-1 receptor agonist was effective in improving glycemic control and body weight reduction. despite lack of evidence on their clinical use, long-term benefits and side effects these agents have the potential to change the diabetes treatment by replacing traditional secretagogues because of superior control and association with weight loss. objective: the metabolic syndrome (ms) is a particular affection that has been associated with so many factors such as hypertention. both increase cardiovascular diseases. due to the higher prevalence of hypertension in our country, this study aim to determine the frequency of ms in newly diagnosed hypertensive patients in oder to improve the follow -up of these subjets.methodology: over a period of 5 months, we conducted a crosssectional and descriptive study on patients recently diagnosed as hypertensive for <3 months. the selection of patients was done during external consultation in the cardiology unit at the university teaching hospital of yaounde. it was based on high blood pressure, anthropome´tric parameters such as weight, bmi, waist, abdominal circonference and biological values (lipid profile, blood glucose, uric acid). we identified 74 patients aged between 21 and 83 years, with a mean age of 52 years. people aged more than 39 years were mostly affected. stage 3was the prominent stage of hypertension.the prevalence of metabolic syndrome was 64.9% with a higher rate in female.the cardiovascular risk factors were obesity, alcoholism, sedentarity and hypo hdlc factors differenciating population with metabolic syndrom was obesity (p = 0.00), hyperglycemia (p = 0.00), hypo hdlc (p = 0.00) while the phenotypes most represented were hyperglycemia, abdominal obesity and hypo hdlc respectively. the metabolic syndrome is frequent in newly diagnosed hypertensive subjects; the females were most affected. metabolic syndrome should be investigated systematically in all hypetensive patients. background: although mainly inhibits osteoclastogenesis, osteoprotegerin is produced by vasculature too. serum opg (sopg) is elevated in both diabetics and patients with coronary artery disease (cad) but there are still insufficient data for its concentrations in impaired glucose tolerance (igt) subjects. aims: to determine sopg in males with igt and concomitant cad and to investigate its relationship with certain glucometabolic parameters.materials and methods: sopg was measured in 26 males with performed percutaneous coronary interventions for cad-14 with igt and 12 normoglycemic, and in 11 age-and bmi-matched healthy normoglycemic controls. glucose abnormalities were screen-detected using a standard ogtt. mean intima-media thickness (imt) of common carotid arteries was measured by b-mode ultrasonography. opg was measured by elisa.results: sopg was significantly higher in igt patients compared to controls (5.09 ae 0.7 vs. 2.87 ae 0.29 pmol/l; p = 0.01) but did not differ between igt and normoglycemic cad patiens (5.09 ae 0.7 vs. 3.79 ae 0.6 pmol/l, p = 0.2). in all participants, sopg correlated positively with imt (p 0.008; pr 0.58). similarly, in igt males with cad, sopg correlated only with imt (p 0.02; pr 0.75). there were no associations with fasting and postchallange plasma glucose, hba1c, fasting insulin, homa-ir, lipid parameters, blood pressure, bmi or waist circumference. we found higher sopg in males with igt and cad compared to the controls. opg did not correlate with glucose parameters but rather with markers of atherosclerosis. we speculate in cad patients with igt, the increased sopg might reflect the vascular damage and not glycemic status which requires further investigations. internal medicine, umf iuliu hatieganu cluj-napoca, cluj-napoca, 2 umf 'victor babes' timisoara, timisoara, romania objective: the pro12ala polymorphism in the pparg2 gene (c>g) is associated with less weight loss after treatment for obesity. this study aimed to investigate the association of rs 1801282 polymorphism with weight loss 1 year after bariatric surgery. the sample was composed of individuals with grade iii obesity undergoing roux-en y gastric bypass. anthropometric data were collected in the preoperative period and 1 year after surgery. genotyping was performed by the method of allelic discrimination in real time pcr (polymerase chain reaction) using the taqman predesigned snp genotyping assays kits (applied biosystems, foster city, ca, usa). individuals with at least one variant allele were grouped and compared with those with the reference genotype.results: 173 subjects (79.2% females, mean age 40.8 ae 10.3 years) participated in the study. genotyping showed 82% (n = 142) of individuals homozygous for the c allele (c/c) and 18% (n = 31) heterozygous (c/g). there was no homozygous mutant (g/g). individuals with the c/c and c/g genotypes respectively showed a loss of 47.7 ae 15.8 and 48 ae 18.8 kg; 34.4 ae 8 and 33.6 ae 10.3% of initial weight and 61.3 ae 15 and 59.5 ae 18.2% of excess body weight. there was no difference in weight loss between groups.conclusion: the pparg pro12ala genotype seems to have no association with weight loss 1 year after bariatric surgery. objective: evaluated determine the prevalence of ir, ms and associated factors among banking workers in southeastern brazil.methods: out of 501 males and females banking employees, 20 years old were evaluated by cross-sectional study including demographic, biochemical, anthropometric and hemodynamic. the ms was determinate by ncep and idf. the ir was determined by homa-ir, with the cut off >2.71.results: 86 (17.2% 95% ci 13.8-20.6) and 113 (22.6% 95% ci 18.8-26.3) of employees with ms, according to the ncep and idf, respectively and 52 (10.4%) with ri. the likelihood of developing the syndrome is greater in individuals with high level of education (or 2.16 (95% ci 1.11-6.13) and among those with overweight and obesity, the possibility of having ms is 12, 6 (95% ci 4.80-33.26, p = 0.000) and 43.6 (95% ci 16.05-118.88, p = 0.000) times over, respectively. persons who are overweight are at risk of 4.97 (95% ci 1.31-18.83) times more likely to have elevated homa, and among those who are obese, the risk rises to 17.87 (95% ci 40.36-73.21).conclusion: this study showed the higher number of employees who have similar characteristics of the total active working, that despite high education, also have ms and ir, and the consequent risk of developing cardiovascular disease. prediabetes is considered as a strong risk factor for type 2 diabetes (dm2). tcf7l2 is a gen involved in dm2 susceptibility related with glp-l. objective: study the relationship of rs7903146 and rs12255372 variants of the tcf7l2 gene with insulin levels, c-peptide and glp-1in normoglycemia and prediabetes subjects. we included 108 pre-diabetic and 54 normoglycemic subjects. we measure fasting glucose, lipids, insulin, c-peptide and glp-1). insulin resistance and beta-cell function were calculated with the homa model. tcf7l2 polymorphisms rs7903146c/t and rs12255372g/t were determined by pcr-rflp. for analyses two groups were compared: wild-type and carriers of two allele risk. we included a total of 162 subjects aged 44 ae 12 years. weight, bmi, systolic and diastolic blood pressure and triglycerides were significantly lower in the group of healthy subjects. glp-1 was higher in normoglycemic subjects. the frequency of rs7903146t allele was higher in pre-diabetic subjects, while the allele frequency of rs12255372t was not significantly different between the groups. in carriers of rs7903146t, serum insulin and homa-beta were significantly higher. plasma levels of glp-1 were lower in prediabetic subjects with and without the risk allele variant. in prediabetic subjects carriers of the rs12255372g/t or tt had also lower levels of serum insulin, homa-ir and homa-beta. the glp-1 concentrations were lower in both groups.conclusions: in this work the rs7903146 polymorphism tcf7l2 gene was associated with prediabetes. the frequency of the rs12255372t allele tcf7l2 gene was not different between groups. objective: determine hba1c, igfbp1, fgf21 and other metabolic markers in non-diabetic, prediabetic, type 2 diabetics (t2dm) of recent diagnosis. material and methods: a cross-sectional population based study was carried in 35-65 years old subjects classified with fasting glucose (fg) and after 2 h 75 g oral glucose load (gt), as normal with impaired fg (ifg), impaired gt (igt), with both alterations (ifg/igt) and dm2. after 3-4 weeks subjects were re-classified and registration of anthropometric data, food intake and determination of a1c, lipids, insulin, fgf21 and igfbp1.results: we found 25.1% for prediabetes and 10.1% for dm2 (n = 174). in the re-classification we classified: 35 normal, 48 ifg, 3 igt, 32 ifg/igt, 32 dm2. with anova analysis we found that hba1c in dm2 patients was different from normal, ifg, ifg/igt subjects (p < 0.002, p < 0.011 and p < 0.004 respectively). fgf21 levels was different among all groups (p < 0.05). normoglycemic subjects were better educated and had higher income than the prediabetic and dm2 subjects. dm2 subjects reported more screen time. prediabetic subjects had higher hdl-cholesterol. with multiple regression analysis we found igfbp1 positively correlated with daily caloric intake and triglycerides and negatively with bmi. the fgf21 correlated positively with insulin. the prevalence of pre-diabetes is more than twice of that of undiagnosed diabetes. we find fgf21 and insulin related with the metabolic status. igfbp1 was negatively correlation with bmi finding compatible with an association with insulin resistance. we have previously found that the expressions of several homeobox transcription factors, including irx5, were reduced in subcutaneous fat in human obesity. a knock-out (ko) mouse line was acquired for the purpose of investigating the in vivo effects of irx5 deficiency with the main perspective on how these mice would handle a high-energy dietary intake. though initially undersized, male irx5 ko mice fed a high-fat (45 %) diet approached similar weight and size as their wildtype (wt) littermates. yet, mri show that the irx5 ko mice have smaller adipose tissue depots and store less fat around organs in aim: to examine the role of mir-21 in the pathogenesis of diabetic cardiomyopathy.objectives: to study the cardiac expression of mir-21 and its effect on regulation of genes involved in akt-pkb signalling pathway in animal model of diabetic cardiomyopathy (dcm).methods: type 2 diabetes was induced in adult wistar rats by high fat diet and i.p streptozotocin injections. the animals were sacrificed at 12 weeks and development of cardiomyopathy was confirmed by heart/body weight ratio, histopathological examination, myocardial fibrotic and hypertrophic genes. cardiac tissues were examined for expression of mir-21, its target genes by real time pcr.results: myocardial expression of mir-21 was significantly increased and showed a positive correlation with myocardial mrna levels of fibrotic genes (ctgf, fgf-b & tgf-b) in diabetic rats. myocardial mrna levels of potential targets of mir-21, phosphatase and tensin homolog deleted on chromosome 10 (pten) and programmed cell death protein 4 (pdcd-4), were increased and their protein expression was decreased in dcm group and in fibroblasts exposed to high glucose (p < 0.05). increasing mir-21 levels promoted, whereas knocking down mir-21 attenuated, pten, pcdc-4 activity in cardiac fibroblasts. our results suggest that mir-21 contributes to cardiac fibrosis in dcm by modulating activity of akt/pkb pathway through pten and pcdc-4. objectives: many studies have revealed that prevalence of metabolic syndrome may be related to lifestyle components. the aim of this study was to investigate the influence of dietary patterns on prevalence of metabolic syndrome (mets). the data was based on health and nutrition survey for 534 japanese men and women aged 20-69 year. factor analysis was used to obtain dietary patterns applying to intake of food groups. the definition of mets was followed by the modified version of the criteria of idf that is applied to japanese population. values of waist circumference, hdl-cholesterol, blood pressures, and hba1c were used to identify mets. logistic regression analysis was used to examine the association between dietary patterns and prevalence of mets.results: three dietary patterns of "fruits", "rice", and "meat" were identified. in males, greater values of "fruits" pattern was associated with a significantly lower prevalence of mets after adjustment for age, physical activity level, smoking and drinking status and other confounding variables (or = 0.62, 95% ci 0.34-0.96).conclusions: fruits consumption was inversely associated with mets and this association may be explained by much intake of mandarin orange specific to this surveyed population. this finding necessitates further investigation on the mechanisms of fruits consumption on health benefits. introduction: the impaired glucose regulation shows a double risk of cardiovascular morbimortality and development of type 2 diabetes, especially in patients with high cardiovascular risk.objective: identify the carbohydrate abnormalities in unknown diabetic patients with high cardiovascular risk.patients and methods: hundred and forty-eight non-diabetic patients were involved in this single-center study, with at least three known vascular risk factors, metabolic syndrome or with known cardiovascular event over 3 months.all had a test load of 75 g oral glucose (ogtt) followed by 2 h after dosing plasma glucose, serum glycated hemoglobin (hba1c) and lipid abnormalities exploration. cardiovascular exploration also concerned all patients.results: among these patients, 64 were in primary prevention and 84 in secondary prevention. 95 cardiovascular events were reported to be dominated by myocardial infarction (35.7%) and stroke (38.09%).dysglucoregulation concerned 77 patients (52%); the sex ratio was 0.97 and the average age was 59, 73.33 unknown diabetes, 34 ifg and 42 igt were diagnosed.these glucose abnormalities were found equally in both primary and secondary prevention.in the family, diabetes was concerned with 38% and cardiovascular events mainly expressed by early myocardial infarction and stroke were observed in 40%.the vascular risk factors were more present in igt group.hypertension was found in 90%, followed by dyslipidemia in 49%; 68% met metabolic syndrom's criteria (idf 2005) .conclusion: in primary prevention context, ogtt not only detects unknown diabetics but identifies igt patients who dysplay a high risk of cardiovascular events. the aim was to study some indicators of inflammatory process and oxidative metabolism in the patients arriving for surgical treatment of pathological obesity. laboratory tests have been carried out by conventional methods in 17 patients with pathological obesity of abdominal type with a body mass index >40 kg/m 2 before and after biliopancreatic shunting operation.rise of blood glucose level up to 139.3% (p < 0.001), uric acid level up to 157.9% (p < 0.001), as well as dyslipidemia were determined in patients. the content of malondialdehyde (mda) increased 1.6 times, the general antioxidant activity (aoa) decreased 1.8 times. concentration of c-reactive protein (crp) exceeded 2.6 times the control value. the long-term results in these patients within 6-20 months have shown normalisation of level of blood glucose, uric acid, high and very low density lipoprotein cholesterol, a tendency of decrease in the general cholesterol concentration. triglycerides decreased by 17 %, still remaining at sufficiently high level. mda concentration decreased by 32.4 %, aoa increased by 38.0 %. crp level which was considerably raised before operation decreased within the next months but in 9 months its average value increased five times in relation to normal rate.the obtained results are evidence of the interrelation of inflammation process and oxidative stress in patients with pathological obesity and metabolic syndrome. efficiency of surgical treatment was shown in the insulin resistance syndrome includes well-recognized cardiovascular risk factors such as low hdl levels, hypertension, hypertriglyceridemia, which is highly correlated with small dense ldl and increased lp(a) levels, which is cardiovascular risk factor. we aimed to study dyslipidemia, especially lp(a) level in addition to carotid intima media thickness (ca-imt) as a marker of atherosclerosis in normal and impaired glucose tolerance offspring of type 2 dm. 150 subjects were included and divided into two main groups; group(i) 50 apparently healthy subjects with no family history of diabetes mellitus, as control group, group(iia) 50 non diabetic offspring of type 2 dm with normal glucose tolerance and group(iib) 50 non diabetic offspring of type 2 dm with impaired glucose tolerance. a statistically significant increase in mean ae sd of serum level of lp(a) and ca-imt in non diabetic impaired glucose tolerant offspring of type 2 diabetes as compared to non diabetic normal glucose tolerant group, significant positive correlation between lp(a) and ca-imt vs. each of ldl-ch, triglycerides and cholesterol. also, a significant positive correlation between lp(a) and ca-imt and hdl-ch. in non diabetic impaired glucose tolerant offspring of type 2 diabetes as compared to non diabetic normal glucose tolerant group. in conclusion the increase in serum level of lp(a) and increase of ca-imt in non diabetic offspring of type 2 dm were associated with increasing severity of insulin resistance and increasing the degree of atherosclerosis explaining the high prevalence of cardiovascular diseases in subjects who are genetically prone to the development of diabetes. a. kostrzewa-tarnowska, m. człapka-matyasik, m. fejfer, j. jeszka weight loss can be achieved by any means of energy restriction, but the challenge is to achieve sustainable weight loss and prevent from weight gain without increasing the risk of chronic diseases. conventional high carbohydrate diets, even when based on wholegrain foods, increase insulinemia and may compromise weight control via mechanisms related to appetite stimulation and metabolic rate. the success of low fat diets has given a push for researches on alternative dietary strategies, including high content of bioactive compounds and low glycemic index (gi) diets.the aim of this study was to determine if high content of bioactive compounds using capillary blood sampling lead to any significant difference between the gis of the products mentioned. investigated group consisted of 25 healthy people with normal weight, aged 20 ae 2 years (bmi 23.5 ae 1.5 kg/m 2 ).foods with a low gi produce a lower peak in postprandial glucose and a less overall blood glucose increase during the first 2 h after consumption compared with foods with a high gi. studies have shown that bioactive products lead to increased satiety, delayed return of hunger, and decreased food intake after ingestion of low-gi compared with high-gi foods.low-gi products may also play an extensive role in weight loss. this strategy can be associated with reducing total energy intake, modifying the macronutrient composition of the diet and lower postprandial glycaemia.research funded within the framework of the project po ig 01.01.02.00-061/09. physiology, school of medicine, national university of ireland, galway, ireland introduction: current diagnostic criteria for type 2 diabetes mellitus rely on increased blood glucose concentration following an oral glucose tolerance test (ogtt) or increased glycated haemoglobin. changes in these parameters occur long after insulin resistance manifests as plasma glucose concentration is homeostatically defended. in contrast, glucose oxidation measured by the 13 c-glucose breath test ( 13 c-gbt) is reduced as a direct consequence of insulin resistance. it is important to establish normative data and define the reliability of any diagnostic or scientific test therefore the aim of this study was to determine the reliability of the 13 c-gbt as a diagnostic tool for type 2 diabetes.methods: sixteen apparently-healthy controls {21.7(0.5) yrs, 79.6 (12.6) kg, 168.5(12.0) cm, all data mean (sd)} underwent simultaneous ogtt and 13 c-gbt on two separate occasions following a 12 h overnight fast. mixed capillary blood and breath samples were collected at baseline and every 15 min for 4 h following ingestion of a 63.35 g glucose solution (shamrock, ireland) labelled with 0.15 g 1-13 c glucose.results: mean baseline 13 co 2 was 23.8 ae 0.6 delta viennapeedeebelemnite (d-vpdb) and peak 13 co 2 was à11.9 ae 2.1 d-vpdb. fasting and 2 h mixed capillary glucose concentrations were 4.9 ae 0.5 mmol/l and 5.6 ae 0.9 mmol/l respectively. per cent dose recovered 13 c had a cronbach's alpha of 0.656, while fasting and 2 h glucose concentration were 0.682 and 0.403 respectively. we established normative data for the 13 c-gbt. the 13 c-gbt is a reliable test of whole body glucose oxidation which has the potential to be developed as a diabetes diagnostic test. introduction: outcome studies in morbidly obese patients (mo) have shown that diabetic patients have the highest benefit from bariatric surgery interventions. therefore it was of interest to study a variety of cardiovascular risk factors in a very large group of patients with mo (n = 1498), who were investigated in our center. we included 1498 patients with mo, of whom 22.9% (n = 343; mean hba1c 7.7 ae 1.6%) were diabetic. in a subset (n = 300), patients were followed up 2 years after bariatric surgery of whom 25.3% (n = 76) were diabetic. all patients without manifest type 2 diabetes underwent a 75 g oral glucose tolerance test, insulin levels were assessed, homa-insulin resistance (ir) was calculated. diabetes was diagnosed after ada criteria. apart from that demographic, cardiovascular risk-markers (blood pressure, lipids) renal and inflammation parameters were assessed. background: obex â is a dietary supplement to help lose weight. in addition, this supplement is specifically made with natural antioxidants molecularly activated to enhance their biological properties without altering their molecular structure.objectives: the purpose of this study was to evaluate the effect of obex â on anthropometric measurements and metabolic disorders in overweight and obese subjects. this was an open label pilot study conducted with 29 overweight and obese adults (bmi > 25 kg/m 2 and <45 kg/m 2 ), aged between 23 and 61, who took obex â (without changes in lifestyle), at a dose of two sachets before the two main meals of each day for 3 months. in addition to anthropometric measures and blood pressure, fasting plasma glucose, lipid profile, insulin, creatinine and uric acid were determined. insulin resistance by homa-ir and betacell function by homa-b were assessed. three indirect indexes were used to calculate insulin sensitivity.results: compared to baseline, obex â significantly reduced body weight (p = 0.012), body mass index, waist circumference, waist/hip ratio, waist/height ratio (p < 0.0001, respectively) and conicity index (p = 0.001). there was also a reduction in fasting glucose levels. compared to baseline, the use of obex â improved insulin secretion (homa-b) and hdl-c concentration (p < 0.005). no adverse effects were seen in any of the participants during the pilot study.conclusion: short-term treatment with obex â improved visceral and abdominal obesity, as well as ameliorating levels of select markers of metabolic disease risk in overweight and obese adults, indicating that further studies are warranted. methodology: a descriptive cross sectional study was conducted with 84 families. the sample was 258 family members (partner, offspring, blood family members and not family related individuals-all living in the same house). body mass index (bmi), lipid profile and glucose measured. also, the family functionality (ff) and perceived health status (phs). results: age mean was 40.02 years (sd, 20.08); 151 female (58.5%) and 107 males (41.7%). the rf were analyzed by the role in the family. seventy-five percent of the housewives were overweight, with high levels of glucose (15.8%), cholesterol (31.9%), triglyceride (38.6%) and low hdl (21.30%). husbands had overweight (85%), with high levels of glucose (16.2%), cholesterol (51.4%), and triglyceride (37.8%). the children aged between 10 and 24 years, had glucose and cholesterol normal, but were overweight (21.9%), and had high level of triglyceride (15.5%) as well as low hdl (37.5%). additionally, children and parents had only 1-2 portions of vegetables intake in 1-3 days in 1 week; similarly 61.8, had 1-2 portions of fruits or juice, but only 1-3 days by month. family functionality was reported with a mean of 69.69 (sd, 12.67)-0-100 scale and 71.72 (sd, 19.85) in phs. the family members in the ua are at high risk for developing dt2. among them are underscored obesity, high levels of lipids, low consumption of vegetables and fresh fruits. ff and phs are also risk factors in these families. this study designed to compare the effect of three dietary oils: canola, rice bran and grape seed on lipid profile and paraoxanase activity of hyperlipidermic rats. method: hyperlipidemia was induced in 40 wistar male rats by atherogenic diet. once hyperlipidemia was reached, the rats were randomly divided in four groups of 10 animals according to the treatment received. treatment groups were fed canola, rice bran and grape seed oil for 7 weeks. control rats fed regular rat chow diet which contains corn oil. baseline fasting blood lipid profile and paraxonase activity of experimental and control rats were compared at the beginning and at the end of the experiment.result: after 7-weeks of treatment, a significant decrease was found in serum triglyceride, total cholesterol and ldl-c concentration of rice bran and grape seed oil fed rats (p < 0.05), serum hdl-c concentration also increased significantly (p < 0.05). canola oil fed rats showed a significant decrease in total cholesterol, and triglyceride level and an increase in paraoxanase activity (p < 0.001), however, no significant differences were found in ldl-c and hdl-c concentration. the present study suggest that consumption grape seed, canola and rice bran oil may have beneficial effect on serum lipid profile, but in comparison of the tree dietary oils grape seed oil showed more beneficial effect in reducing hyperlipidemia. key: cord-006229-7yoilsho authors: nan title: abstracts of the 82(nd) annual meeting of the german society for experimental and clinical pharmacology and toxicology (dgpt) and the 18(th) annual meeting of the network clinical pharmacology germany (vklipha) in cooperation with the arbeitsgemeinschaft für angewandte humanpharmakologie e.v. (agah) date: 2016-02-06 journal: naunyn schmiedebergs arch pharmacol doi: 10.1007/s00210-016-1213-y sha: doc_id: 6229 cord_uid: 7yoilsho nan in vitro systems and mechanistic investigations i the demand of alternative test systems which closely mirror the in vivo situation is one of the main challenges in modern toxicity testing. the major goal is the development of in vitro systems that partly display the complexity of an organism and thus may mimick in vivo conditions. despite great efforts in the past no adequate in vitro systems are available yet. on the other hand, cell cultures from almost every organ are easily accessible and therefore may help to roughly assess the toxic potential of substances at target structures. nonetheless, the complex interactions which take place in vivo cannot be addressed in single cell cultures. in the liver, hepatocytes comprise 80% of the total liver volume while non-parenchymal cells -endothelial cells, stellate cells and kupffer cells (that is, liver resident macrophages) -contribute only 6.5% of the volume, but 40% of the total cell number (kmiec 2001) . it has been increasingly recognized that in the liver neighboring non-parenchymal cells release molecules which contribute to the inflammatory damage and even aggravate it (adams et al. 2010) . in our project a human in vitro co-culture system was established by combining a hepatic and a monocytic cell line, the latter of which can be differentiated to a macrophage-like phenotype. in this system the hepatotoxicty of substances has been analyzed, and the results were compared to single cultures and to published data from in vivo studies. using ketoconazole, an antifungal, as a known hepatotoxic substance, inflammatory markers were studied and proved to be significantly upregulated only in coculture. conversely, cultures of hepatic cells only did not display this increase in inflammatory markers. at the same time, a negative substance, caffeine, failed to show any hepatotoxic potential in the co-culture system. our results demonstrate that this novel in vitro co-culture model represents a promising tool to evaluate the hepatotoxic potential of substances. in drug research, it might help to reduce animal testing as drugs with a high dili potential can be dropped early in the development phase. question: raman spectroscopy (rs) is a highly sensitive analytical method for markerfree and non-invasive identification and characterization of cells. here, we present rs as a novel tool for gentle yet precise cell analysis in three independent experiments, focusing on monitoring cellular reactions upon treatment. we could provide evidence that rs is a suitable tool to monitor cell differentiation, analyze cell modification and study cell apoptosis after drug application. methods: in a first experiment, mesenchymal stem cells (mscs) were treated with erythropoetin (epo) for certain time points and subsequently fixed with paraformaldehyde (pfa) for raman analysis. in addition, skbr3 breast cancer cells were exposed to the anti-cancer drug herceptin (20μg/ml). cells were then fixed in pfa for rs. in a last experiment, molm-13 cells were separately cultivated in microwells and treated with thymidine for different time points prior to raman analysis. results: raman spectroscopy was able to monitor differentiation of epo treated mscs and found that around 35% of treated cells showed fibroblast like raman profiles. in case of herceptin treated skbr3 cells, rs found internal changes of the cell´s metabolism as reaction on drug application. analyzing the most prominent differences in raman spectra revealed discrimination of cells to be mainly due to changes in amid i, lipid and protein content. in the last experiment, rs was able to follow apoptosis of molm-13 cells after thymidine application and discriminate early from late apoptotic states. discussion: rs is a photonic marker for gentle yet highly specific cell analysis, which allows monitoring of single cell reaction after drug treatment. thereby, rs provides information about changes within the entire metabolome on a single cell level. raman spectra are as characteristic as a "fingerprint". rs works label-free and non-invasive and thus does not impare cell viability. this allows to gain new insights in pharmacological development and toxicological survey. acknowledgement: this project received funding from the eu 7th health program grant agreement no 279288. deutsches zentrum für herzinsuffizienz, würzburg, germany extracellular signal-regulated kinases 1 and 2 (erk1/2) are essential for the regulation of cell growth and cell survival and their kinase activity is up-regulated for example in different types of cancers and pathological cardiac hypertrophy. while inhibition of erk1/2 activity by kinase inhibitors prevents tumor growth, it can also lead to exacerbated cardiomyocyte death and impaired heart function. interestingly, we have previously identified an erk autophosphorylation at threonine 188 as a prerequisite for nuclear erk1/2 signaling and erk-mediated cardiac hypertrophy. here, we investigated an alternative strategy to interfere with erk1/2 signaling: since activation of erk1/2 triggers erk dimerization, a prerequisite for erk t188autophosphorylation, we chose the erk dimer interface as a possible target to selectively interfere with erk t188 -phosphorylation. first, we investigated the impact of monomeric erk2 on cardiac function. to address this issue, we generated mice with cardiac overexpression of monomeric erk2 ∆174-177 and performed transverse aortic constriction (tac) to induce cardiac hypertrophy. compared to wild-type mice, erk2 ∆174-177 overexpression attenuated tac-induced cardiac hypertrophy, interstitial fibrosis and mrna expression levels of collagen and brain natriuretic peptide (bnp), while cardiomyocyte survival and cardiac function were largely preserved. because of the positive effects of monomeric erk ∆174-177 in the heart, we designed a peptide to interfere with endogenous erk dimerization. cross-linking and coimmunoprecipitation experiments showed that the peptide binds to erk2 and prevents its dimerization. moreover, the peptide effectively inhibited erk t188 -phosphorylation and nuclear translocation of yfp-tagged wild-type erk2 after phenylephrine stimulation. further, adenoviral or adeno-associated virus serotype 9 (aav9)-induced overexpression of the peptide in neonatal rat cardiomyocytes (nrcm) or mouse hearts resulted in a significantly reduced hypertrophic response to phenylephrine or tac, background: g i -proteins have been proposed to be cardioprotective. it's matter of debate whether this depends on the particular g i isoform and/or on the particular conditions (e.g. cardiac "stress") only. in our study we investigated effects of a gα i2knockout on cardiac function and survival in a murine heart-failure model of cardiac β 1adrenoceptor overexpression. methods and results: β 1 -adrenoceptor overexpressing mice lacking gα i2 (β 1 -tg/gα i2 -/-) were compared to wild-type (c57bl/6) mice and littermates either overexpressing cardiac β 1 -adrenoceptors (β 1 -tg) or lacking gα i2 (gα i2 -/-). at 300 days of age mortality of mice only lacking gα i2 was higher compared to wild-type or β 1 -tg, but similar to β 1tg/gα i2 -/mice. beyond 300 days mortality of β 1 -tg/gα i2 -/mice was enhanced compared to all other genotypes (mean survival time: 363±21 days). echocardiography revealed similar cardiac function of wild-type, β 1 -tg and gα i2 -/mice, but significant impairment for β 1 -tg/gα i2 -/mice (e.g. ejection fraction 14±2% versus 40±4% in wild-type mice). significantly increased ventricle-to body-weight ratio (0.71±0.06% versus 0.48±0.02% in wild types), left-ventricular size (length 0.82±0.04 cm versus 0.66±0.03 cm in wild types) and anp and bnp expression (mrna: 2819% and 495% of wild type, respectively) clearly indicated hypertrophy. gα i3 was significantly upregulated in gα i2 -knockouts (protein compared to wild-type mice: 340±90% in gα i2 -/and 394±80% in β 1 -tg/gα i2 -/-, respectively). radioligand binding experiments confirmed cardiac overexpression of β 1adrenoceptors in β 1 -tg mice. of note, overexpression levels differed depending on the particular wild-type background. on an fvb/n background we found the overexpression level to be more than 2-fold higher (b max : 1425 ± 68 fmol/mg) than on the otherwise used c57bl/6 background. accordingly, fvb/n-based β 1 -tg mice showed a significantly impaired cardiac function at an age of 300d while c57bl/6-based β 1 -tg mice did not. conclusions: gα i2 -deficiency combined with cardiac β 1 -adrenoceptor overexpression strongly impaired survival and cardiac function. on a c57bl/6 background β 1adrenoceptor overexpression alone had not induced cardiac hypertrophy or dysfunction at day 300 while there was overt cardiomyopathy in mice additionally lacking gα i2 . we propose an enhanced effect of increased β 1 -adrenergic drive by lack of protection via gα i2 . the observed gα i3 upregulation was not sufficient to compensate for gα i2 deficiency suggesting an isoform-specific and/or a concentration-dependent mechanism. the role of gα i3 is currently addressed in a subsequent study using β 1 -tg and gα i3deficient mice. heart failure is accompanied by morphological and functional alterations (e.g. hypertrophy, decreased contractility) which are summarized by the term "cardiac remodeling". while the β-adrenergic signaling pathway is essential for short-term modulation of cardiac performance, its chronic stimulation by elevated plasma catecholamines and the subsequent activation of camp-dependent signal transduction pathways is regarded as a fundamental factor in the pathogenesis of cardiac remodeling. however, the mechanisms mediating the transition of physiological conditions of short-term to detrimental remodeling under long-term β-adrenergic stimulation are not understood in detail so far. in this context, icer, an isoform of the camp dependent transcription factor crem (camp responsive element modulator), acts as an early response gene strongly induced by beta-adrenergic stimulation via camp responsive elements (cre) in its promoter. contrary to its cre-mediated induction, icer is a strong inhibitor of cre-mediated transcription by itself. here we study the role of icer induction in the catecholamine-induced cardiac remodeling in a time dependent manner by the use of icer deficient mice (iko) and wild type (wt) controls, which were treated with isoproterenol (iso; 10 mg/kg per d) for 6 and 24 hours and 7 days. overall 7 days of iso stimulation resulted in an elevation of cardiomyocyte length in iko (in µm; 7d iso 156±1) vs. wt cardiomyocytes (7d iso 143±2). at this time point a 29 % decrease of cardiac output and a 16 % decrease of the maximal rate of rise of left ventricular pressure (dp/dt max ) in iko vs. wt animals was detectable. the maximum increase of icer mrna in wt cardiomyocytes already occurred after 6 h (75-fold), and declined after 24 h (29-fold) to 2.5 fold increase after 7 days, while icer mrna was not detectable in iko mice. this raised the hypothesis, that the early induction of icer modulates transcriptional processes after beta-adrenergicstimulation, involved in cardiac remodeling of the heart. profiling of mrna expression levels between iko vs. wt cardiomyocytes at the different time points revealed: 55 regulated genes (up-regulated: 45 %) in untreated; 103 altered genes (up 35 %) after 6h; 1437 changed genes (up 97%) after 24 h and 131 altered genes (up 19%) after 7 days of iso treatment. in summary, the absence of icer induction in myocytes resulted in an increase of cardiomyocytes length and a decrease of heart performance after 7 days of betaadrenergic stimulation. this is preceded by upregulated mrna levels of several hundred genes at 24 h, which is going along with the induction of transcriptional inhibitor icer after a few hours of beta-adrenergic stimulation. this suggested a protective role of icer by inhibiting the progression of cardiac remodeling after betaadrenergic stimulation in an early responsive manner. (supported by the dfg) the performance of the adult heart is tightly regulated by g protein-coupled receptors. adrenergic and angiotensin receptors efficiently control heart rate and contractility. muscarinic receptors, on the other hand serve as master regulators of the conduction system, which is often lost upon myocardial infarction. this function of muscarinic receptors has been well described in the adult or late embryonic heart. here we provide evidence that muscarinic receptors are crucial to constrain pacemaker cell identity. we applied subtype-specific inhibitors of muscarinic receptors to zebrafish embryos of different stages. we observed that both, early cardiac function as well as specification are specifically regulated by muscarinic m3 receptors, while m2 receptors appear to exert a heart-specific function only at later stages. continuous m3 blockage renders zebrafish with greatly altered cardiac morphology, particularly of the conduction system. furthermore, embryos with m3 inhibition display impaired ventricular function most likely due to an av-block and substantial arrhythmia in the atrium. importantly, to observe these phenotypes it was sufficient to block m3 receptors during stages of cardiac differentiation, which is long before a heart tube has formed. we corroborated our findings regarding these morphological changes using marker gene analysis. furthermore, we obtained evidence for m3 receptors preventing a transcriptional program towards the induction of pacemaker cells at the expense of av canal cells. importantly, this is not only true during heart development. a pacemaker program is also induced in adult hearts upon m3 inhibition. taken together, we postulate that muscarinic m3 receptors confine a pacemaker lineage during early steps of heart development as well as in the adult heart. our data suggests m3 receptors as potential new therapeutic targets for the regeneration of hearts with an injured sinoatrial node. systemic inhibition of mir-21 has proved effective against fibrosis of the myocardium and in other organs. mir-21 has been reported to exert detrimental effects in cardiac fibroblasts and protective roles in cardiac myocytes and other myocardial cell types. a better definition of the cell types that contribute to the beneficial effects of inhibiting mir-21 in vivo may aid the development of strategies with enhanced therapeutic efficacy. thus far, no approach to selectively manipulate micrornas in the non-myocyte population of cardiac cells in vivo has been available. in this study, we developed an icre-encoding mml virus for application in mir-21 fl/fl mice. delivery of this vector to neonates achieved targeted genetic ablation of mir-21 in non-myocyte cardiac cells. immunohistochemistry and flow cytometry confirmed that mmlv was highly selective and effective for cardiac fibroblasts and endothelial cells. in parallel, an aav9-icre vector allowed for specific and almost complete deletion of mir-21 in cardiac myocytes. when tested in a model for chronic left ventricular pressure overload, mmlv-icremediated deletion of mir-21 in cardiac fibroblasts and endothelial cells significantly reduced cardiac fibrosis and hypertrophy and improved cardiac function. the benefit of this cell-type-specific inhibition exceeded that observed upon global genetic deletion of the mir-21 gene in mice. aav9-mediated deletion of mir-21, albeit lowering cardiac hypertrophy, had no effect on fibrosis or cardiac function. taken together, neonatal delivery of engineered icre-encoding viruses enabled for the first time a differential gene targeting in non-myocyte and myocyte cells in myocardium. non-myocyte deletion of mir-21 demonstrated that mir-21 exerts its cardiac profibrotic activity directly in cardiac fibroblasts and in endothelial cells. this novel finding should encourage tailoring of antimir-21 therapy towards cellular tropism. chronic inflammatory diseases, such as psoriasis or rheumatoid arthritis, are characterized by constant leukocyte infiltration and ongoing angiogenesis in the inflamed tissue. as current anti-inflammatory pharmacotherapy is not always satisfying, there is a great demand for the discovery of new drug leads as well as novel drug targets. the synthetic carbazole alkaloid derivative c81 acts as a multikinase inhibitor. results of a thermal shift assay revealed that c81 shows by far the highest binding affinity to the bmp-2-inducible kinase (bmp2k/bike). bmp2k represents an as yet largely uncharacterized protein, which is not regulated by bmp-2 in endothelial cells. therefore, we aimed to analyze (i) the pharmacological potential of c81 and (ii) the role of bmp2k in angiogenic and inflammatory processes in the vascular endothelium. initial experiments show that only high concentrations of c81 affected the viability of human umbilical vein endothelial cells (huvecs). both c81 and the knock-down of bm2k (rnai) reduced the migratory capacity of a human microvascular endothelial cell line (hmec-1). also the proliferation of hmec-1 was reduced by c81 treatment (ic 50 : 7 µm). a tube formation assay on matrigel demonstrated that c81 significantly impaired the formation of capillary-like structures in a dose-dependent manner. interestingly, the analysis (western blot) of signaling molecules in huvecs that play a crucial role in cell proliferation (e.g. erk, akt) revealed that these pathways are not influenced, neither by c81 treatment nor by bmp2k gene silencing. in regard to inflammatory processes, c81 treatment or bmp2k silencing of huvecs decreased the adhesion of thp-1 cells, a monocytic cell line, onto the activated endothelial cells. as the interaction of leukocytes is mainly mediated by cell adhesion molecules (cams), the effect of c81 or bmp2k silencing on their expression was analyzed (flow cytometry, qpcr). while the expression of cams was strongly decreased after c81 treatment, the knock-down of bmp2k did not markedly affect their expression. furthermore, both approaches did not lead to the reduction of tnf-induced iκbα degradation (western blot) or p65 translocation into the nucleus (microscopy). our study provides first insights into the anti-inflammatory and anti-angiogenic potential of the carbazole alkaloid derivative c81 in vitro. the precise role of bmp2k in angiogenic and inflammatory endothelial processes as well as the involved pathways during bmp2k silencing and c81 treatment will be further elucidated. moreover, since the inhibition of bmp2k seems not to be responsible for all actions of c81, we will investigate the role of other kinases affected by the compound in these processes. the chemokine receptor cxcr4 is a multifunctional receptor which is activated by its natural ligand c-x-c motif chemokine 12 (cxcl12). cxcr4 seems to be part of the lipopolysaccharide sensing complex, suggesting that an intervention with cxcr4 agonists or antagonists could result in reduced tlr4 signaling. however, the role of cxcr4 and the influence of different cxcr4 ligands in acute as well as chronic inflammatory diseases are still contradictious. therefore, we aimed to characterize the systemic effects of cxcr4 activation in severe systemic inflammation and to evaluate its impact on endotoxin induced organ damages by applying a sublethal lps dose (5 mg/body weight) in mice. the plasma stable cxcl12 analog ctce-0214d was synthesized and administered subcutaneously shortly before lps treatment to ensure a delayed release and thereby a prolonged effect of the drug. 24 hours following lps administration, mice were sacrificed and blood was obtained for tnf alpha, ifn gamma and blood glucose evaluation. additionally, histopathological changes and oxidative stress in the liver and spleen were assessed and liver biotransformation capacity was determined. finally, cxcr4, cxcl12 and tlr4 expression patterns in liver, spleen and thymus tissue as well as the presence of different markers for oxidative stress and apoptosis were evaluated by means of immunohistochemistry. ctce-0214d improved the health status and distinctly reduced the lps mediated effects on tnf alpha, ifn gamma and blood glucose levels by approximately 35%, 50% or 70%, respectively. it attenuated oxidative stress in the liver and spleen tissue and enhanced liver biotransformation capacity unambiguously. ctce-0214d diminished the lps induced expression of cxcr4, cxcl12, tlr4, nf-κb, cleaved caspase-3 and gp91 phox, whereas heme oxygenase 1 expression and activity were induced above average. furthermore, tunel staining revealed anti-apoptotic effects of ctce-0214d in all organs. the cxcr4 is undoubtedly involved in inflammation. its activation was accompanied by anti-inflammatory, anti-oxidative and cytoprotective effects as ctce-0214d attenuated tlr4 signaling, induced heme oxygenase 1 activity and mitigated apoptosis. thus, the administration of cxcl12 analogs seems to be a promising treatment option to control acute systemic inflammation, especially when accompanied by a hepatic dysfunction and an excessive production of free radicals. the neurodegenerative disease friedreich ataxia (frda) is caused by a gaa triplet repeat expansion in the first intron of the frataxin gene, which results in a reduction of the corresponding mitochondrial protein. despite several cellular and animal models the exact function of frataxin is still a matter of debate, but the role of frataxin in iron sulfur cluster biosynthesis is generally accepted. however, we still don't know which primary metabolic events are caused by a frataxin deficit and until now, there is no therapeutic option available. we developed a new cellular model for frda by using the cre/loxp recombination system in mouse embryonic fibroblasts (mef). c57bl/6j mouse strains with a loxp flanked exon 4 of the frataxin gene and an tamoxifen-inducible cre-recombinase (creer t2 ) were crossed and several mef cell lines isolated. after selection by genotype and growth manner the fx-mef 2-1 (fxn -/-) and fx-mef 2-8 (fxn +/-) cell lines were finally choosed. the generation of the homozygous or heterozygous frataxin knockout was successfully tested on rna and protein level. long maintenance of the frataxin depleted fibroblasts revealed a strong growth inhibition consistent to earlier observations in other cell systems. therefore, we established a pattern of treatment over 12 days, with medium and substance changes at day 4 and 8, which allows us to get a fully functional knockout and overcome the growth inhibition problem. endpoint measurements of known metabolic phenomena from mammalian and non-mammalian models were studied at day 12 of our novel cell system. the induced total disruption of frataxin leads to a clearly reduced aconitase activity, cell division and oxygen consumption as well as an increase in ros production. in the heterozygous knockout with residual frataxin activity no such changes were observed. in addition, our pattern of treatment enables us to monitor the full and partial frataxin knockout in the course of time, to detect early and late metabolic events after frataxin disruption. therefore we analysed the mentioned parameters (with additional atp and iron content) in parallel at day 3, 5, 7 and 10 and could identify an initial event followed by secondary consequences and parameters, which seem to play only a minor role in the frda pathogenesis. on the contrary, a partial deficit of frataxin didn't result in any differences over time and suggests that there are only cellular alterations below a critical threshold. in conclusion, our new established mammalian cellular frda model mimics typical metabolic consequences of the human disease and seems to be qualified for frda research. the model shows for the first time six different metabolic events in the course of time in parallel and reveals insights into primary and secondary events of frda pathogenesis. these observations can be used to better understand the function of frataxin and can help to develop new therapeutic strategies to address the consequences of frataxin deficiency. moreover, the transfer of this cell model into 96well plates offers the possibility for a high-throughput screening of potential therapeutic substances. the disease diphtheria is caused by the diphtheria toxin (dt) which belongs to the group of single-chain ab-type bacterial protein toxins. receptor-binding of the b-domain on the target cell surface is followed by receptor-mediated endocytosis and internalization into early endosomal vesicles. endosomal acidification triggers membrane insertion and pore formation of the transmembrane (t) domain together with translocation of the (partially) unfolded catalytic (c) domain into the cytosol. herein, dta catalyzes adp-ribosylation of elongation factor 2 which leads to disruption of protein synthesis and finally causes cell death [1] . in hela cells, these events are related to cell-rounding functioning as a specific endpoint to monitor the uptake of dta into the cytosol of the host cell. as for other adp-ribosylating toxins such as c. botulinum c2 toxin, c. perfringens iota toxin and c. difficile cdt, also in case of native dt we demonstrated the involvement of several host cell factors during the translocation step of the catalytic domain across the endosomal membrane [2, 3] . in detail, we confirmed the involvement of the host cell chaperone hsp90 and the thioredoxin reductase (trxr), the latter presumably responsible for the reduction of the interchain disulfide bond between the dta and dtb moieties [4, 5, 6] . furthermore, we identified another group of protein folding helpers, the family of peptidyl-prolyl cis/trans isomerases (ppiases) including cyclophilin a (cypa), cyp40 and fk506-binding protein (fkbp)51 as required cytosolic factors for dta translocation. to characterize the role of the protein folding helpers in more detail, we investigated their interaction with purified dta in vitro by performing dot blot analysis with immobilized recombinant host cell factors, co-precipitation of cellular factors with dta from hela lysate and isothermal titration calorimetry with purified proteins therewith determining the thermodynamic parameters of the individual binding events. thereby, we detected binding of dta to hsp90, cypa, cyp40, fkbp51 and fkbp52. the data increase the knowledge of the molecular mechanisms underlying dt uptake and especially dta translocation which can be medically used to develop novel therapeutic strategies against the disease diphtheria. [1] murphy (2011) toxins 3, 294-308. [2] barth (2011) naunyn-schmied arch pharmacol 383, 237-245. [3] kaiser et al. (2012) cell. microbiol. 14, 1193-1205. [4] dmochewitz et al. (2011) cell. microbiol. 13 , 359-373. [5] ratts et al. (2003) j. cell biol. 160, 1139-1150. [6] schnell et al. (2015) novel afflictions as for example clostridium (c.) difficile associated diseases (cdad) caused by clostridium difficile are on the increase and challenging to treat. cdad most frequent occur in hospitalized patients after prolonged treatment with antibiotics. cdad includes among others diarrhea and the severe form of pseudomembranous colitis. not only the treatment of the infection, but also the treatment of the toxins has a high clinical significance. c. difficile secretes the exotoxins a (tcda) and b (tcdb), which glycosylate and thereby inactivate rho-gtpases in mammalian cells. tcda and tcdb are considered as the causative agents of cdad. in the last few years, more and more hypervirulent strains of c. difficile were described. in these hypervirulent strains, the adp-ribosyltransferase cdt was found as a third toxin in addition to tcda and tcdb. given the lack of agents effective against antibiotic-resistant bacterial strains and bacterial exotoxins, the development of novel pharmacological strategies is needed. the antimicrobial activity of naturally occurring substances is already known for a long time. one important mechanism of the innate immune system is the production of natural peptides showing antibiotic features. in recent years, it was shown that human antimicrobial peptides as important part of the native innate immune system plays a crucial role not only in inactivation of bacteria but also in inhibition of bacterial toxins (1). prompted by these result, we found that only human α-defensin-1 (hnp-1) but not human β-defensin-1 (hbd-1), both important effectors of the innate immune system, protected cultured epithelial cells from intoxication with tcda and cdt when applied prior to the toxins to the cells. moreover, α-defensin-1 prevented also the cytotoxic effects of all three c. difficile toxins tcda, tcdb and cdt combined in the medium. the combined investigation of all three toxins might be even more suitable to mimic the situation after an infection with hypervirulent c. difficile. the inhibition of the toxins was monitored by cell rounding caused by each of the toxins. currently, the molecular mechanisms underlying the inhibitory effects are still unknown and will be investigated in different cell lines. in conclusion, our results demonstrate that hnp-1 causes a loss of cytotoxicity of the c. difficile toxins and may act as novel drugs to cure c. difficile infections that contribute to cdad. neurodegenerative diseases like parkinson´s disease (pd) are accompanied by altered gene expression levels in the brain. recent studies support a role of regulatory noncoding rnas, such as micrornas (mirnas), which silence a specific set of mrnas at the post-transcriptional level. upon their aberrant expression, they are likely involved in the pathophysiology of specific neuronal loss. manipulation of neuronal gene expression is pivotal for understanding the function of proteins and the development of new therapeutic strategies. rna interference (rnai) strategies can be employed through the administration of small interfering rnas (sirna), which mediate the specific knockdown of a selected target gene. however, the main challenge is the delivery of these rnas into the neurons of interest. in this pilot study, we present a method for delivering sirnas in polymeric nanoparticles based on low molecular weight polyethylenimines (peis). their intracerebroventricular (icv) injection leads to in vivo silencing of neuronal gene expression in the brain of mice overexpressing α-synuclein (thy1-asyn mice). in a first step, pei-complexed sirna tagged with afluorescencedye were injected to track the localization and distribution after icv administration. five days later, fluorescent cells were visible throughout the brain, with the highest fluorescence intensity around the ventricles. fluorescence was also observed in large cells of the lumbar spinal cord. moreover, preliminaryresultsdemonstrate a 42.6% knockdown (p<0.05 student's t-test, n=6) of human α-synuclein (snca) in thetargetstructurestriatum upon a single icv injection of pei-complexed specific sirna compared to the control injection group (n=9). hence, our first results support the usability and efficacy of pei nanoparticle-mediated delivery of short rnas, namely sirnas, for rapidly and efficiently reducing the expression of a neuronal target gene of interest in the brain in vivo. this may allow the development of gene therapy strategies for the treatment of neurodegenerative diseases. is a propenylic alkenylbenzene found in several plants, e.g. acorus calamus. bacontaining plant materials are used to flavor foods, and are active ingredients in traditional plant medicines. thus, human exposure results primarily from the intake of bitters and teas, as well as from calamus-containing medicines and plant food supplements. although many (positive) pharmacological properties/effects of asarone isomers are described in the literature, ba was found to be carcinogenic in rodents (liver, duodenum) when given daily or in a single dosage. early experiments indicated that ba is not activated via hydroxylation and sulfonation as it is the case for known hepatocarcinogenic allylic alkenylbenzenes such as estragole or methyleugenol. because the mechanism of metabolic activation of ba in not known, we investigated the metabolism of ba in liver microsomes and human cytochrome p450 (cyp) enzymes, the mutagenicity of ba and its metabolites in the ames fluctuation assay and the dna adduct formation in primary rat hepatocytes. we found that side-chain epoxidation (leading to diols and a ketone) was by far the most dominating metabolic route of ba in liver microsomes and human cyp enzymes. ba was mutagenic in the ames test (+s9 mix), as was the synthesized ba-epoxide (-s9 mix). furthermore, we were able to synthesize and characterize a ba epoxide-derived dna adduct with deoxyguanosine. this dna adduct was formed in a concentration-dependent manner in rat hepatocytes incubated with ba. our results strongly indicate that ba is genotoxic with the side-chain epoxide being its ultimate carcinogen. morbid obesity is an independent risk factor for cardiovascular disease, type 2 diabetes mellitus and certain types of cancer. bariatric surgery -with the roux-en-y gastric bypass (rygb) being the gold standard -has become the therapeutic option of choice as a sustained weight loss and improvement of associated morbidity is achieved in the majority of patients. there is, however, a lack of evidence focusing on bariatric surgery induced sustained weight loss and its possible impact on cancer risk. we investigated the association between obesity, oxidative stress and genomic damage after roux-en-y gastric bypass surgery (rygb) or caloric restriction induced weight loss in the obese zucker rat. obese male zucker fa/fa rats were divided into three groups: sham surgery (sham), rygb and caloric restriction (cr) and were compared with lean controls (lean; zucker fa/+ rats). shams showed impaired glucose tolerance and elevated plasma insulin levels, which were less severe in rygb and cr. oxidative stress was elevated in kidney, liver and colon tissue of sham and reduced again after weight loss induced by either rygb or bwm. urine-derived oxidization products of lipids, dna and rna increased in shams and decreased after weight loss (rygb and cr). dna double strand breaks were more frequent in shams than in the weight loss groups or lean. dna damage in zucker fa/fa rats correlated with their basal plasma insulin values. obese rats showed elevated oxidative stress and genomic damage in comparison to lean rats. after body weight loss, achieved by either rygb or caloric restriction alone, oxidative stress level and genomic damage were decreased. this may indicate a reduction of the elevated cancer risk in obesity. ) mice were treated with the nocrelated compound azoxymethane (aom) followed by the administration of dextran sodium sulfate to trigger crc. tumors were quantified by non-invasive mini-endoscopy, which revealed a non-linear increase in crc formation in wt and aag -/mice. in contrast, a linear dose-dependent increase in tumor frequency was observed in mgmt -/and mgmt -/-/aag -/mice. the data was corroborated by hockey stick modeling, which yielded similar carcinogenic threshold doses for wt and aag -/mice. o 6 -meg levels and depletion of mgmt activity correlated well with the observed dose-response in crc formation. aom dose-dependently induced double strand breaks (dsbs) in colon crypts including in lgr5-positive colon stem cells, which coincided with atr-chk1-p53 signaling. intriguingly, mgmt-deficient mice displayed significantly enhanced levels of γ-h2ax, suggesting the usefulness of γ-h2ax as an early genotoxicity marker in the colorectum. this study demonstrates for the first time a non-linear dose-response for alkylation-induced carcinogenesis and reveals dna repair by mgmt, but not aag, as a key node in determining a carcinogenic threshold at low alkylation dose levels [2] . obesity is characterized as a status where the excessive accumulation of fat in adipocytes leads to local inflammation and hypoxia; both contributing to severe obesity associated co-morbidities such as cardiovascular disease and type 2 diabetes mellitus. local inflammation is mediated by macrophages, stromal vascular cells, preadipocytes, and adipocytes as well as by a number of proinflammatory cytokines and chemokines (1). in particular, the chemokines monocyte chemoattractant protein (mcp-1, ccl2), interleukin-8 (il-8/cxcl8) and stromal cell-derived factor 1 (sdf-1a/cxcl12) secreted by stromal vascular cells, preadipocytes, and adipocytes exert paracrine effects by recruiting neutrophils, monocytes/macrophages, and t-and b-cells. interestingly, deficiency in cxcl14 was shown to attenuate obesity in mice (2) . the cc chemokine ccl2 is known to stimulate ccr2 receptors, and the cxc chemokines cxcl8 and cxcl12 to activate cxcr1 and cxcr2 or cxcr4 and cxcr7, respectively. the receptor(s) activated by cxcl14 is currently unknown. a role of cxcl14 in modulating cxcr4 signaling has been proposed. we initiated our studies to determine the presence and functional significance of chemotaxin receptors in human adipocytes and their precursor cells. to this end, the mrna expression pattern of cc chemokine receptors, cxc chemokine receptors formylated peptide receptor fpr1 and the related receptor fprl1, and cc and cxc chemokines was analyzed during in vitro adipose differentiation of human simpson-golabi-behmel-syndrome (sgbs) preadipocytes, and under conditions mimicking an inflammatory response. in particular, we focused on the expression pattern of human ccr2 receptors, since previous reports indicated a role in adipogenic differentiation. however, our comprehensive analysis using different sources of adipocytes and their precursors indicated that ccr2 receptors were absent (3) . yet, the analysis revealed appreciable levels of mrna encoding ccl2, cxcl12, and cxcl14, and ccr1, cxcr2, cxcr7, fpr1, and fprl1, and cxcr4. of interest, cxcr4-and cxcr7-mrna were found to be up-regulated under the proinflammatory conditions. to analyze the responses of adipocytes and their precursors to chemokine receptor agonists, we used chemokine-mcherry fusion proteins purified from baculovirus-infected insect cells, e.g ccl2, cxcl12, cxcl14. while sgbspreadipocytes and adipocytes did not accumulate ccl2-mcherry upon stimulation, they showed a small accumulation of cxcl12-mcherry, and a strong accumulation of cxcl14-mcherry in the endosomal compartment. similar results were obtained in murine 3t3l-1 preadipocytes. using mass spectrometry analysis, we set out to identify the cxcl14-binding putative receptor protein(s) in murine 3t3l-1 preadipocytes. (1) makki, k. et al., (2013) in personalized medicine tumors are screened for several mutations in oncogenes or tumor suppressors. however, the cellular protein content not exclusively depends on the dna. we identified new rac variants generated on the mrna level in androgenindependent prostate cancer cells. all variants represent active forms of the gtpase. they are capable to suppress rhoa-induced apoptosis and additionally, mediate the synthesis of genes which are under the control of the androgen receptor. importantly, expression of the rac variants is sufficient to support tumor growth in mice. we prove the existence of the variants and verify their clinical appearance and relevance in tissue samples of a prostate cancer patient. dna analysis, however, revealed the wildtype sequence of rac. therefore, routine analysis of patient tumor tissue would miss the detection of active rac which precludes the success of therapy. the existence of active rac variants in prostate cancer tissue that promote resistance towards androgen deprivation suggest rac inhibition as an effective add on therapeutic strategy against prostate cancer. the bacterial effector protein exotoxin y (exoy) of pseudomonas aeruginosa is delivered into host cells via the bacterial type iii secretion system. once arrived in the host cell nucleotidyl cyclase activity of exoy is activated by a yet unknown cofactor and thus has a profound effect on concentrations of cyclic nucleotides: in addition to production of cyclic amp (camp) and cyclic gmp (cgmp) there is a massive synthesis of cyclic 3', and to some extent of the corresponding cytidylyl analogue ccmp 1,2 . currently, the role of cump and ccmp during the pathogenesis of p. aeruginosa infection remains unknown 3 . one of our hypotheses is that these cyclic nucleotides fulfil a role as first messengers, e.g. in the communication between individual bacteria or bacterial populations during establishment of acute or chronic infections. to test this hypothesis, the intra-and extrabacterial concentrations of cyclic nucleotides were measured via hplc-ms/ms at different time-points in liquid cultures of p. aeruginosa, either in a complete (lb medium) or a starving medium (vogel-bonner medium). additionally, we tested if supplementation of the media with extrinsic cump or ccmp had an effect on these measured concentrations. the influence of extrabacterial cyclic nucleotides on the bacterial metabolism and homeostasis was evaluated with a microarray of bacterial total cdna extracted at different time points of p. aeruginosa liquid culture with or without extrinsic cump/ ccmp. furthermore we investigated a potential function of the cyclic nucleotides in biofilm formation. cyclic ump and cyclic cmp have differential roles in bacterial metabolism and communication. for example, whereas ccmp is synthesized by p. aeruginosa when the bacteria are in a nutrient-rich environment, we could not detect bacterial cump under any tested circumstance. in our biofilm formation assays, only ccmp had a biofilmpromoting effect, but only in very high concentrations. the currently ongoing analysis of gene expression data in the presence or absence of cump may reveal a role of this cyclic nucleotide as first messenger, too. in further studies we will elucidate the signal transduction processes underlying the observed cump / ccmp effects, for example by identifying cump and ccmp binding proteins and their coupling mechanisms to intracellular signalling cascades. synapses are complex computational platforms that transmit information encoded in action potentials but also transform their functionality through synaptic plasticity. g protein-coupled receptors (gpcrs) play a major role in modulating the strength of the synapses via the second messenger camp 1 . however the spatio-temporal dynamics of the mode of action of camp underlying synaptic plasticity are still controversial. the role of this study was to investigate the dynamics of camp signaling at the drosophila neuromuscular junction, where octopamine binding to its receptors has been shown to cause camp-dependent synaptic plasticity 2 . for this purpose, we generated a transgenic drosophila expressing the camp sensor epac1-camps 3 in motor neuron. this allowed us to directly follow the octopamine-induced camp signals in real time by fluorescence resonance energy transfer (fret) in different compartments of the motor neuron (i.e. cell body, axon, boutons). we found that octopamine induces a steep camp gradient from the synaptic bouton (high camp) to the cell body (low camp), which was due by higher pde activity in the cell body. high octopamine concentrations evoked a response also in the soma. notably, these signals were independent and isolated form each other. moreover, application of octopamine by iontophoresis to single synaptic boutons induced bouton-confined camp signals. these data reveal that a motor neuron can posses multiple and largely independent camp signaling compartments, and provide new basis to explain how camp could control neurotransmission at a level of a single synapse. 1 kandel, e.r., dudai, y. & mayford, m.r. the molecular and systems biology of memory. cell 157, 163-186 (2014) . 2 koon, a.c., et al., autoregulatory and paracrine control of synaptic and behavioral plasticity by octopaminergic signaling. nat neurosci. 14 (2): p. 190-9 (2010) . 3 nikolaev vo, bünemann m, hein l, hannawacker a, lohse mj novel single chain camp sensors for receptor-induced signal propagation. j. biol. chem.279, 37215-37218 (2004) cardiovascular pharmacology 039 hyaluronic acid deposition determines engineered heart muscle characteristics and can be pharmacologically targeted to enhance function s. schlick background: engineered human myocardium (ehm) can be generated from psc derived cardiomyocytes (cms) and primary fibroblasts suspended in a collagen i hydrogel (70%:30%:0.4 mg/ml). ehm development encompasses an early consolidation phase followed by functional maturation. the presence of fibroblasts is essential for consolidation into a force-generating ehm. here we assessed the hypothesis that fibroblasts of different origin support ehm formation differentially as a function of hyaluronic acid deposition. methods and results: oscillatory rheology (1% strain, 1 hz) on cell-free and cell containing collagen i hydrogels directly after casting revealed enhanced consolidation in the presence of human foreskin fibroblasts (ffbs) compared to primary adult cardiac fibroblasts (cfbs) -change in storage modulus over time (pa/min): collagen 0.03; collagen + cms 0.03; collagen + cms + cfbs 0.09, collagen + cms + ffbs 0.2. we next generated ehm with cms and ffbs or cfbs. after 4 weeks of culture under serum-free conditions, we assessed ehm function by contraction measurements. ffb-ehms developed a significantly (p<0.01) higher force of contraction (foc) per cross sectional area (csa) than cfb-ehms (maximal foc/ csa are in mn/mm 2 : 1.8±0.1, n=29 vs. 0.3±0.1, n=20). cross sectional area (csa) of tissues was greatly increased (p<0.01) in cfb-ehms (csa in mm 2 : 1.6±0.1, n=20 vs. 0.6±0.03, n=30) and nonmyocyte content was higher in cfb-ehms (5.6±0.7, n=9 vs. 3.1±0.4, n=15; x10 5 cells/ml). histological analysis revealed that cardiomyocytes were only poorly matured in cfb-ehms compared to ffb-ehms. extending ehm functional data, principal component analysis of rnaseq data revealed distinct expression patterns for ffbs and cfbs, in which hyaluronic acid synthase 2 (has2) enzyme was significantly (p<0.01) upregulated. based on these findings, we pharmacologically intervened with has2 mediated hyaluronic acid (ha) deposition by treating cfb-ehms with hyaluronidase during all 4 weeks of culture. interestingly, ecm manipulation with low concentrations of enzyme significantly (p<0.01) reduced csa (csa in mm 2 : control 1.8±0.4, n=8; hyaluronidase of concentrations from 0.15u to 150u 0.9±0.2, n=4) with a concurrent, statistically significant (p<0.01), increase in contractile function and improved cardiomyocyte morphology on a histological level (maximal foc/csa in mn/mm 2 : control 0.2±0.05, n=8; hyaluronidase of concentrations from 0.15u to 150u 0.4±0.08, n=4). summary and conclusions: our data suggest that ehm consolidation is influenced differentially by fibroblasts of different tissue origin with hff-ehm being functionally superior to cfb-ehm. cfb-ehm could be rescued by hyaluronidase leading to reduced ha deposition. the latter demonstrates that extracellular matrix composition is centrally involved in ehm development. angiogenesis is the process of formation of new blood vessels from the pre-existing ones. vascular endothelial growth factor (vegf) is the most studied regulator of this process. by binding to its type 2 receptor (vegfr2), it has been shown to activate a variety of different signaling-pathways leading to enhanced angiogenesis. camp, on the other hand, is a versatile second messenger which regulates various endothelial functions including barrier function. it directly activates protein kinase a (pka) or the exchange protein directly activated by camp (epac) which is a guanine exchange factor (gef) for the small monomeric gtpase rap. as human umbilical vein endothelial cells (huvec) express both camp effectors (epac1 and pka), we investigated the role of camp-signaling using a spheroid based sprouting assay as an in vitro model for angiogenesis. interestingly, the activation of β-adrenergic receptors with 5 µm of isoproterenol significantly increased the cumulative sprout length. similarly, the selective activation of epac with 30 µm of the camp analog 8-pcpt-2'-o-camp (007) significantly increased the basal and the vegf-induced cumulative sprout length. in accordance, sirna-mediated depletion of epac1 in huvec decreased the basal and vegf-induced sprouting. surprisingly, 10 µm of forskolin increased basal and vegf-induced cumulative sprout length stronger than 007, indicating an additional role of pka. in accordance, 1 µm of myristoylated pki, a membrane-permeable specific pka inhibitor, significantly attenuated the forskolin-induced increase in sprouting. in all conditions tested, 50 ng/ml of vegf always showed an additive effect to the same extent on cumulative sprout length. therefore, our data indicate that the vegf-pathway is acting independently of the camp-pathway in the regulation of the sprouting angiogenesis. the β-adrenergic receptor-mediated activation of camp signaling in huvec induces angiogenic sprouting by activation of epac1 and pka. introduction: hypertension is one major risk factor for the development of chronic heart and kidney disease. mineralocorticoid receptor (mr) antagonists are a cornerstone in the therapy of heart failure and there is first evidence for a beneficial effect on the kidney as well. inflammation plays an important role in hypertensive organ injury. thus, this study was designed to evaluate and directly compare the effect of mr deletion in endothelial cells on blood pressure and cardiac vs. renal injury in a mouse model of deoxycorticosterone acetate-induced hypertension. methods and results: mice lacking the mineralocorticoid receptor in endothelial cells (mr cdh5cre ) were created using the cre/loxp system. mr cdh5cre and cre-negative littermates (mr wildtype ) underwent unilateral nephrectomy and received 1 % nacl with drinking water for 6 weeks. the mineralocorticoid deoxycorticosterone acetate (doca, 2.5 mg/d) was delivered by subcutaneous pellets. untreated mice served as controls (ctrl). ambulatory blood pressure was determined by implantable telemetry in awake mice. doca/salt treatment increased mean blood pressure in mr wildtype (141.7 ± 8.0 vs. ctrl 97.8 ± 3.2 mmhg, p<0.001) and mr cdh5cre (150.0 ± 3.0 vs. ctrl 104.1 ± 4.7 mmhg, p<0.001) without differences between genotypes. cardiac hypertrophy after doca/salt treatment was ameliorated in mr cdh5cre mice (ventricle weight 162.4 ± 4.2 mg vs. mr wildtype 189.0 ± 10.9 mg, p<0.05). doca/salt significantly increased cardiac fibrosis and the expression of fibrotic marker genes in mr wildtype but not in mr cdh5cre mice. this was accompanied by an increased expression of the vascular cellular adhesion molecule (vcam1) in mr wildtype cardiac endothelial cells. renal function was not altered by mr deletion in endothelial cells at baseline. doca/salt treatment lead to marked interstitial fibrosis in the kidneys of mr wildtype (sirius red fibrosis score: 2.4 ± 0.2 vs. ctrl 0.1 ± 0.1, p<0.001) and mr cdh5cre (2.3 ± 0.2 vs. ctrl 0.1 ± 0.1, p<0.001) mice. mrna expression of the fibrosis marker gene col3a1 (mr wildtype 4.3 ± 0.9-fold; mr cdh5cre 3.9 ± 1.1-fold vs. ctrl) was similarly increased. periodic acid-schiff staining revealed glomerular injury in both genotypes. this was associated with a marked rise in urinary albumin / creatinine ratio (mr wildtype 20.4 ± 5.7fold; mr cdh5cre 34.3 ± 12.5-fold vs. ctrl). in the kidney vcam1 mrna expression and the number of macrophages was increased by doca/salt treatment independently from endothelial mr deletion. conclusion: in conclusion, mr deletion from endothelial cells ameliorated doca/saltinduced cardiac but not renal inflammation and remodeling independently from blood pressure. these findings suggest different mechanisms for the beneficial effect of mr antagonists in hypertensive heart vs. kidney disease. platelets are relevant cells implicated in morbidity and mortality provoked by cardiovascular thrombosis. even with the actual antiplatelet therapy there is still a substantial incidence of arterial thrombosis. therefore, a better understanding of the mechanisms involved in platelet activation and aggregation is required to develop improved antiplatelet therapies. the increase in the intracellular ca 2+ concentration due to ca 2+ entry from the extracellular space is critical for platelet activation and aggregation. ca 2+ entry follows activation of plasma membrane receptors including gqcoupled receptors for adp, thromboxane a 2 (txa 2 ) or thrombin, as well as the collagen receptor glycoprotein vi (gpvi). the cellular signalling pathways downstream these receptors involve activation of phospholipase c and second messengers that are known to mediate activation of trpc channels. trpc proteins form receptor-operated cation channels, but their regulation and permeability differ depending on the cell type. it has been proposed that trpc proteins might contribute to platelet function as constituents of agonist-activated ca 2+ entry channels; however, the experimental approaches used so far and the lack of specific agonists or antagonists have not allowed to determine the individual contribution of trpc proteins for agonist-induced ca 2+ entry in platelets, aggregation and thrombosis formation. we detected the expression of trpc3 and trpc6 in human and mouse platelets. we identified that those proteins together are essential components of a system of coincidence detection in cellular ca 2+ signalling. this coincidence detection triggered by simultaneous stimulation of both thrombin and collagen receptors is required for the phosphatidylserine exposure in human and murine platelets, indicating the role of trpc3/c6 proteins for procoagulant activity. in addition, we detected the expression of s12 trpc1 transcripts in mouse platelets. therefore, we tested trpc1/c3/c6-deficient mice in an in vivo model of arterial thrombosis where they showed reduced thrombus formation. regardless of the protective effect of trpc1/c3/c6 inactivation observed in the thrombosis model, no differences were detected in tail bleeding. to evaluate the relevance of these trpc proteins in platelet aggregation we measured in vitro platelet aggregation in platelets from trpc1/c3/c6-deficient mice and we observed that the aggregation was reduced after adp (3µm) or txa 2 -analogue (1µm) stimulation, but not after collagen stimulation (10µg/ml). we are currently analyzing the in vitro aggregation and the agonist-evoked ca 2+ response in platelets from different trpc-compound and single deficient mouse lines to understand the mechanisms behind this phenotype with regard to its complementarity to actual antiplatelet therapy. background: thrombin signaling initiates inflammatory events directly and through activation of platelets. endogenous and pharmacologic inhibitors of thrombin are therefore of relevance during atheroprogression and for therapeutic intervention. the small leucine-rich proteoglycan biglycan (bgn) is such an endogenous thrombin inhibitor that acts through activation of heparin cofactor ii (hcii). here, the effect of genetic deletion of bgn on thrombin activity, inflammation and atherosclerosis was addressed. methods and results: bgn concentrations were elevated in the plasma of patients with acute coronary syndrome. in apoe -/mice, bgn was detected in the plasma as well as in the glykokalyx of capillaries. additionally, bgn expression occurred in the subendothelial matrix of arterioles as well as in atherosclerotic plaques. in line with a role of bgn in balancing thrombin activity, apoe -/-/bgn -/0 mice exhibited higher activity of circulating thrombin and increased numbers of activated platelets than did apoe -/mice. furthermore, higher concentrations of circulating cytokines in apoe -/-/bgn -/0 mice suggested a pro-inflammatory phenotype. likewise, immunohistochemistry and facs analysis of the aorta demonstrated increased macrophage content in atherosclerotic lesions of these mice. in addition, apoe -/-/bgn -/0 mice exhibited higher aortic plaque burden and larger atherosclerotic lesions at the aortic root. of note, apoe -/-/bgn -/0 mice showed progressive dilatation of the aortic arch corresponding to a decrease in collagen fibril density suggestive of an outward remodelling in the absence of bgn. no differences were evident with respect to lipid content of the aortic root plaques or circulating plasma lipids. treatment with the thrombin inhibitor argatroban reversed platelet activation and aortic macrophage accumulation in apoe -/-/bgn -/0 mice. conclusions: the present results strongly suggest a protective role of bgn during the progression of atherosclerosis by inhibiting thrombin activity and platelet activation, and ultimately macrophage-mediated plaque inflammation. the exposure to environmental or human-made xenobiotics including drugs induces the hepato-intestinal transcription of metabolizing enzymes and transporters. the time-span of induction is thought not to exceed xenobiotic exposure, in order to minimize disturbances of endobiotic metabolism. in contrast, we find cross-generational transmission of the induction of the phase i enzyme cyp2b10 (1200-fold in females, 700-fold in males) in 6-day old offspring of adult female mice exposed one week prior mating to tcpobop (3 mg/kg i.p.) , the model ligand of the xenosensing nuclear receptor car. such cross-generational effects of xenobiotics are of great clinical interest as they could have profound consequences on the health status of the offspring, including interferences with drug therapies. the multigenerational transmission of tcpobop-driven induction could be mediated by pre-uterine/pre-conceptional epigenetic changes of oocytes. alternatively, they could be brought about by direct intrauterine/post-conceptional contact with tcpobop released from long-term depots. to discriminate between these mechanisms we conducted embryo transfer experiments. both donor mothers and foster mothers were injected with tcpobop (3 mg/kg) prior to mating. the analysis of hepatic cyp2b10 expression in 6-day-old offspring is clearly consistent with a post-conceptional onset of tcpobop effects. thus, offspring of solvent-injected donor mothers transferred to tcpobopexposed foster mothers display a 700-fold induction while offspring from the reciprocal experiment show no changes. cesarean sections on day e18.5 followed by crossfostering proved transmission to be mediated predominantly via lactation (f1 hepatic cyp2b10 induction 3000-fold) and only to a minor part via intra-uterine exposure (300fold). this mechanism is consistent with the absence of induction transmission via the male germline. to analyze if tcpobop leads to functional consequences in drug metabolism of f0 and f1 generation, we conducted in vivo zoxazolamine paralysis assays taken as a functional test for cyp2b10 catalytic activity. in both tcpobop-pretreated f0 and in their f1 descendants, the induction reduced the duration of paralysis evoked by zoxazolamine by >50%. the characterization of cross-generational tcpobop-mediated effects on other processes controlled by car such as energy and bone metabolism is in progress. first tests indicate a transmission of anabolic effects on bone, as evidenced by the induction of serum osteocalcin expression by 45% in 12-weeks-old offspring. in summary, the car-mediated cyp2b10 induction by tcpobop is transmitted to the offspring mainly via lactation, resulting in lasting phenotypic consequences in drug and bone metabolism. the effects of similarly lipophilic drugs and anthropogenic environmental pollutants are currently being investigated. such compounds could affect offspring despite discontinuation of intake or exposure well ahead of pregnancy. age-related cognitive decline can eventually lead to dementia, the most common mental illness in elderly people and an immense challenge for patients, their families and caregivers. cholinesterase inhibitors constitute the most commonly used antidementia prescription medication. the standardized ginkgo biloba leaf extract egb 761 ® is approved for treating age-associated cognitive impairment and has been shown to improve the quality of life in patients suffering from mild dementia. a clinical trial with 96 alzheimer´s disease patients indicated that the combined treatment with donepezil and egb 761 ® had less side effects than donepezil alone (yancheva et al., 2009) . in an animal model of cognitive aging, we compared the effect of combined treatment with egb 761 ® or donepezil monotherapy and vehicle. we compared the effect of chronic treatment (15 days of pretreatment) with donepezil (1,5 mg/kg p. o.), egb 761 ® (100 mg/kg p. o.), or the combination of the two drugs, or vehicle in 18 -20 month old male ofa rats. learning and memory performance were assessed by morris water maze testing, motor behavior in an open field paradigm. in addition to chronic treatment, the substances were administered orally 30 minutes before testing. compared to the first day and to the control group, only the combination group showed a significant reduction in latency to reach the hidden platform on the second day of testing. moreover, from the second day of testing onwards, the donepezil, the egb 761 ® and the combination group required less time to reach the hidden platform compared to the first day. the control group did not reach the same latency reduction until day three. there were no effects on motor behavior. these results suggest a superiority of the combined treatment of donepezil with egb 761 ® compared to monotherapy. literature: yancheva, s., ihl, r., nikolova, g., panayotov, p., schlaefke, s., & hoerr, r. (2009) . ginkgo biloba extract egb 761(r), donepezil or both combined in the treatment of alzheimer's disease with neuropsychiatric features: a randomised, double-blind, exploratory trial. aging ment health, 13 (2) , [183] [184] [185] [186] [187] [188] [189] [190] institut für vegetative physiologie und pathophysiologie, göttingen, germany values are well below the plasma concentrations (3-28 µm; just et al. expert opin emerging drugs 20: 161-164, 2015) observed in patients treated with dantrolene. although not yet proven directly, oat3 may be involved in renal secretion of dantrolene and 5-oh dantrolene by mediating the first step, i.e. the uptake across the basolateral membrane of proximal tubule cells. the second step, the release of these compounds into the urine across the luminal membrane, is possibly mediated by mrp4. since oat3 was also detected in the cytoplasmic membrane of skeletal muscle cells (takeda et al. europ. j. pharmacol. 483: 133-138, 2004) , dantrolene may reach its target, the intracellular ryanodine receptor, ryr1, by influx through oat3, where it inhibits calcium efflux by ryr1 thereby preventing severe muscle contraction and malignant hyperthermia. this assumption, however, awaits a direct demonstration of dantrolene transport by oat3. in addition, we identified, besides dantrolene and 5-oh dantrolene, several further fdaapproved drugs such as tyrphostin ag 1478, ceefourin 1, glafenine, nalidixic acid, and prazosine, as inhibitors of es uptake by oat3. controls 1 with other defects and controls 2 with genetic disorders. all drugs used in the first trimester were identified from the database, and were cross-referenced against previously compiled lists of drugs with reactive intermediates and drugs with faa. drugs with reactive intermediates, with systemic absorption and with a daily dose ≥50mg were considered os-inducing drugs. when there was an association between os-inducing drugs and a group of birth defects, we further investigated two different faa exposure categories: concurrent exposure to both os-inducing drugs and faa drugs (os+/faa+) and exposure to os-inducing drugs only (os+/faa-). when the number of subjects allowed (at least five cases/controls were exposed), we examined the role of folic. odds ratios (ors) with 95% confidence intervals were adjusted for maternal smoking and alcohol use in the first trimester in controls 1 and additionally adjusted for maternal age in controls 2. results: a total of nine groups of birth defects were investigated. only nervous system defects were associated with os-inducing drugs. exposure rates were 65/464 (14.0%) for cases, 512/6033 (8.5%) for controls 1 and 130/1564 (8.3%) for controls 2 and adjusted ors (95%cis) were 1.71 (1.29-2.26) and 1.77 (1.27-2.46), respectively. this association was unchanged when we examined os+/faa+ and os+/faa-separately. the os+/faa+ category, however, had slightly higher or values than the os+/faa-(2.41 vs. 1.61 for controls 1, and 2.55 vs. 1.67 for controls 2). because of the low number of exposed subjects, we could only examine folic in relation to os+/faa-. using os-/faa-/folic+ as reference, we found the highest risk with os+/faa-/folicand a lesser magnitude with os+/faa-/folic+ (ors being 2 and 1.6 times respectively for both controls). conclusion: our study suggests an increased risk of having a child with nervous system defects in mothers who were exposed to os-inducing drugs during pregnancy, and a potential risk reduction with folic. background: inhibition of rho-gtpases with statins as well as specific inhibition of the small gtpase rac1 protects non-transformed cells from topoisomerase ii-(top2)-poisoninduced cleavable complex formation and thereof derived dna double-strand breaks. this effect rests at least partially on rac1-mediated regulation of topoisomerase ii activity. however, the link between rac1 and top2-poisoning is only poorly understood. furthermore, it is unclear whether mitochondrial or nuclear type ii topoisomerases are the most relevant target for top2-poison-induced cytotoxicity. here, we investigated the relevance of rac1-regulated actin cytoskeleton integrity as well as mitochondrial integrity in top2-poison-induced dna damage responses as well as cytotoxicity under situation of rac1 inhibition. methods: since endothelial cells are the first barrier for any kind of systemically administered chemicals and cardiomyocytes are particular sensitive to anthracyclines, endothelial cells (h5v) as well as cardiomyocytes (h9c2) were chosen as in vitro model systems for top2-poisoning. the cells were pre-treated with rac1 inhibitors, statins or actin cytoskeleton disruptors and were subsequently treated with the topoisomerase ii poisons doxorubicin or etoposide. to compare the levels of induced dna damage, γh2ax foci quantifications as well as the comet assay were employed. actin disruption was visualized by phalloidin-fitc staining. to be able to detect relevant changes in mitochondrial mass or integrity, high doses of top2-poisons had to be used in both cell lines. changes in mitochondrial homeostasis as well as integrity were detected by the jc1-assay, mitotracker assay as well as atp-assay. additionally, pcr-and gelelectrophoresis-based methods were used for detecting mitochondrial dna damages. selected components of the dna damage response machinery as well as factors of mitochondrial homeostasis were detected by western blot. results: disruption of the integrity of the actin cytoskeleton attenuated the dna damage response to a similar extent as seen by rac1 inhibition, pointing to a role of actin filaments in the dna damage response after genotoxic insults. the actin cytoskeleton seems to participate in genotoxin-induced dna damage, -repair or in the dna damage response as reflected by reduced numbers of nuclear h2ax-foci as well as the comet assay after treatment with doxorubicin. this was not related to nuclear import or export of doxorubicin. disturbance of mitochondrial homeostasis or integrity was only detectable at high doses of topoisomerase ii poisons. this was largely unaffected by pre-treatment with statins or rac1-inhibitor. top2-poison-induced raise in mitochondrial mass was slightly enhanced by the rac1-inhibitor and statins. interestingly, inhibition of rac1 counteracted doxorubicin-induced phosphorylation of the amp-kinase in endothelial cells but not in cardiomyocytes. conclusion: mitochondrial toxicity seems to play only a minor role in top2-poisoninduced cytotoxicity in h9c2 and h5v cells. the data point to a role of rac1-regulated filamentous (nuclear?) actin in the dna repair and/or dna damage response after treatment with top2-poisons. poly(adp-ribose) polymerase 1 (parp1) and the recq helicase werner syndrome protein (wrn) are important caretakers of the genome. they physically interact with each other and are both localized in the nucleus and in particular in the nucleoli. both participate in various overlapping mechanisms of dna metabolism, in particular genotoxic stress response and dna repair [1] . previously, we and others have shown in biochemical studies that enzymatic functions of wrn are regulated by parp1 as well as by non-covalent poly(adp-ribose)-wrn interaction [2] [3] [4] . furthermore, pharmacological parp inhibition as well as a genetic parp1 ablation in hela cells alters the recruitment kinetics of wrn to sites of laser-induced dna damage [5] . here we report a novel role for parp1 and poly(adp-ribosyl)ation in the regulation of wrn's subnuclear spatial distribution upon induction of oxidative stress. we could verify previous reports that wrn is transiently released from nucleoli upon induction of oxidative stress, camptothecin (cpt) treatment, and laser-induced dna damage in a time-dependent manner. while, cpt-induced translocation appears to be a parpindependent process, our results reveal that upon h 2 o 2 -induced oxidative stress, parp1 is essential for the translocation of wrn from the nucleoli to the nucleoplasm. parp1 activity only partially contributes to wrn release from nucleoli, underlining the importance of a direct wrn-parp1 interaction for subnuclear wrn redistribution. furthermore, we identified a novel par-binding motif within the wrn sequence that is located in its rqc domain, which also harbors the binding site for parp1 and is necessary for wrn's nucleolar localization under non-stress conditions. currently, we are testing corresponding wrn mutants to analyze if this region is responsible for the parp1-dependent release of wrn from nucleoli to sites of dna damage. in conclusion, we provide novel insight into the role of parp1 in wrn's spatio-temporal regulation in the nucleus during the oxidative stress response. host-cell reactivation (hcr) is an assay used to determine dna repair capacity of cells. in its canonical layout, the test utilised a virus or a plasmid with a marker gene, inactivated by uv-damage [1, 2] . among the infected or transfected host cells types, only those with functional dna repair pathway would re-activate the damaged dna, thus providing a rationale for identification of dna repair genes in the mutant screens. an obvious advantage of hcr is that repair can be measured in cells that have not been exposed to a damaging agent. however, because of multiple variables of the damage generation, transfection and interpretation of results, the assay has been hard to harmonise and develop into a widely accepted quantitative dna repair assay. over the last years, my team has developed and validated several major improvements of the mammalian hcr assay. exploiting sequence-specific nicking endonucleases and customised design of the reporter vectors, we proposed an innovative and very efficient technique for incorporation of synthetic oligonucleotides, containing single structurally defined dna base and backbone modifications, into desired gene elements [3] . this ad hoc approach allows examination of the repair in a stand-specific manner and at single nucleotide resolution. we efficiently applied hcr in its new layout for measurement of the nucleotide excision repair of various dna adducts. moreover, we demonstrated that the enhanced hcr assay can differentiate between the transcription-coupled (tc-ner) and global genome (gg-ner) subpathways of ner [4] . we further obtained new significant insights into the lesion-specific mechanisms of base excision repair of several endogenously occurring aberrant dna bases [5 -7] and plan to adapt the assay to the detection of mismatch repair and translesion dna synthesis. in addition to the applications in the dna repair field, the enhanced hcr assay provides a tool for investigation of the dynamics and transcriptional impact of the regulatory dna bases 5methylcytosine and 5-hydroxymethylcytosine as well as their derivatives (5formylcytosine and 5-carboxycytosine a coordinated and faithful dna damage response is of central importance for maintaining genomic integrity and cell survival. transcriptional activation of dna repair genes is an important regulatory mechanism contributing to the adaptation of cells to genotoxic stress and protection against genotoxin-mediated cell death. here we show that exposure to a low dose of benzo(a)pyrene 9, , the active metabolite of benzo(a)pyrene (b[a]p), which represents the most important carcinogen formed by incomplete combustion during food preparation and smoking, causes upregulation of several dna repair genes. combined induction of the nucleotide excision repair (ner) genes ddb2, xpc, xpf and xpg enhanced repair activity and protected cells against a subsequent bpde exposure. furthermore induction of the translesion polymerase polh was also involved in protection against bpde-induced apoptosis, however led to an enhanced mutation frequency in the surviving cells. activation of these dna repair pathways was also observed upon exposure to b[a]p and in vivo in buccal cells of male individuals upon smoking, indicating that this mechanism may be involved in the formation of smoking-related cancers. altogether, we could show that low-dose bpde exposure activates a complex network of transcriptional alterations, leading to protection against cell death, at the cost of increased mutation frequency, highlighting the danger of occasional smoking. poly(adp-ribosyl)ation (parylation) is an essential posttranslational modification with the biopolymer poly(adp-ribose) (par). the reaction is catalyzed by poly(adp-ribose) polymerases (parps) and plays key roles in cellular physiology and stress response by regulating physico-chemical properties of target proteins. of the 17 members of the human parp gene family, at least four have been shown to exhibit par-forming capacity. upon dna damage parp1 is catalytically activated and is thought to contribute to the bulk of the cellular par formation. parp inhibitors are currently being tested in clinical cancer treatment, in combination therapy, or as monotherapeutic agents by inducing synthetic lethality (mangerich and bürkle 2011, mangerich and bürkle 2015) . here we generated a genetic knock out of parp1 in one of the most widely used human cell systems, i.e. hela parp1 ko cells, via talen-mediated gene targeting and characterized these cells with regards to parylation metabolism and genotoxic stress response. furthermore, by reconstituting hela parp1 ko cells with a series of artificial and natural parp1 variants, we analyzed structure-function relationships of parp1 in a cellular environment without interfering with endogenously expressed wt-parp1. we confirmed that the parp1e988k mutant exhibits mono-adp-ribosylation activity and extended previous reports by demonstrating that the parp1l713f mutant is constitutively active in a cellular environment, leading to high cellular par levels even in unchallenged cells. additionally, both mutants exhibited significantly altered recruitment and dissociation kinetics at sites of laser-induced dna-damage, which can partially be attributed to non-covalent parp1-par interaction via at least one specific par binding motif located in zinc finger 2 of parp1. expression of both artificial mutants led to distinct cellular consequences, caused by the altered cellular biochemistry. while the expression of parp1l713f itself triggered apoptosis, parp1e988k expression led to a strong g2-arrest during cell cycle and sensitized cells to camptothecin treatment. interestingly, pharmacological parp inhibition with abt888 mitigated effects of the e988k mutant, suggesting distinct functions of mono-adp-ribosylation. finally, by reconstituting parp1 ko cells with a natural cancer-associated parp1 snp variant (v762a), as well as a newly identified parp1 mutant present in a patient of pediatric colorectal carcinoma (f304l-v762a), we demonstrate, that these variants exhibit altered biochemical and cellular properties, potentially supporting carcinogenesis. together, this study establishes a novel model to study parp1-dependent parylation during genotoxic stress response and reveals new insight into the structure-function relationships of artificial as well as natural parp1 variants in a cellular environment, with implications for parp research in general. mangerich, a. and a. bürkle (2011) . "how to kill tumor cells with inhibitors of poly(adpribosyl)ation." int j cancer128 (2): 251-265. mangerich, a. and a. bürkle (2015) . multitasking roles for poly (adp-ribosyl) ation in aging and longevity. parp inhibitors for cancer therapy, springer: 125-179. leukemic cells frequently overexpress the transcription factor wilms tumor 1 (wt1) and the persistence of wt1 expression after chemotherapy indicates remaining leukemic stem cells. hydroxyurea induces replicative stress by its ability to inhibit ribonucleotide reductase, an enzyme that catalyzes the synthesis of dntps from ntps. we demonstrate that the expression levels of wt1 determines the extent of dna damage and apoptosis in a panel of leukemic cells treated with hydroxyurea. accordingly, inhibiting apoptosis through chemical inhibition of caspases or by overexpression of mitochondrial anti-apoptotic bcl proteins prevents the hydroxyurea-induced depletion of wt1 and cell death. in addition, we show that an rna interference-mediated elimination of wt1 sensitizes leukemic cells to the pro-apoptotic and dna damaging effects of hydroxyurea. furthermore, such a loss of wt1 suppresses hydroxyurea-induced erythroid differentiation. pharmacological approaches that diminish wt1 also sensitize cells to hydroxyurea. these include the tyrosine kinase inhibitor (tki) imatinib or epigenetic modifiers belonging to the histone deacetylase inhibitor (hdaci) group. thus, an inhibition of wt1 is therapeutically exploitable for a targeting approach against leukemic cells undergoing replicative stress. our novel findings reveal that wt1 is a novel biological target of hydroxyurea and they suggest that wt1 has a previously unrecognized ability to prevent dna damage when replication forks halt and eventually collapse. fret (fluorescence resonance energy transfer)-based cell assays were developed to directly monitor receptor activation and receptor-stimulated camp response. mutant ß 1 ar were generated by insertion of cyan and yellow fluorescent proteins (cfp and yfp) into the third intracellular loop and the c-terminus, respectively (bornholz et al., cardiovasc res 97:472, 2013) and stably transfected to hek 293 cells (hekß 1 -fret). to monitor the camp response the epac1-based fret sensor of camp, was stably transfected alone (hekwt-e1) and together with a moderate level of native ß 1 ar to hek293 cells (hekß 1 -e1; nikolaev et al. jacc 50: 423, 2007) . fret-activity was measured with recently developed fluorescence detectors (12 channels) equipped with fast semiconductor technology, avoiding any movable optical and mechanical parts, using 438 nm for excitation and 483/540 nm for the emmission ratio. cells were cultivated in 96-format 12-well strips, incubated in physiological hepes-buffered salt solution and treated with ßar agonists of different selectivity and affinity to determine their ßar-subtype preference. catecholamines tested in hekß 1 -fret cells exhibited ec 50 -values (-log, m) which matched k d -values (-log, m) known from native heart receptor membranes (isoprenaline, iso: 6.9±0.1, adrenaline 5.7±0.1, noradrenaline 6.2±0.1). ßar-expression levels were controlled by radioligand binding with [ 3 h]-(-)-cgp12,177 resulting in different densities of ~4x10 6 and ~1.4x10 4 receptors/cell in hekß 1 -fret and hekß 1 -e1, respectively, whereas in hekwt-e1 cells only ~1000 ß 2 ar were found. surprisingly, the low level of ßar in hekwt-e1 cells allowed the measurement of the action of ß 2 -sympathomimetics (ß 2 sym), e.g. fenoterol, thereby amplifying receptor binding (pk d~6 .7) to an effective regulation of fret activity in the presence of 0.06 mm ibmx (pec 50~8 .3±0.1), nearly matching the ~100-fold amplification of iso (pk d~6 .9; pec 50~8 .9). in order to determine ß 1 ar-mediated side effects of ß 2 sym, hekß 1 -e1 cells characterized by a 14-fold higher level of ß 1 ar over ß 2 ar were assayed for fret activity. fenoterol maximally inhibited fret activity with a pec 50~8 .4 whereas the high affinity ß 2 sym salmeterol acted a partial agonist (~75% of iso maximum, pec 50~8 .6), both compounds being rather insensitive against the highly effective ß 1 ar-blockade with 1 µm cgp 20,712 a. for that reason hekß 1 -fret cells were used characterizing fenoterol as partial agonist (~25%) whereas salmeterol activated less than 10% of maximum receptor activation by iso. thus, it has to be concluded that the low level of effectively coupled wt-ß 2 ar present in hekß 1 -e1 cells precludes the exact determination of ß 1 ar-mediated side effects of ß 2 sym, and that cfp/yfp labelled receptors have to be used for the determination of the subtype specific intrinsic activity of an agonist. they account for about one third of all drug targets. their regulation from desensitization to internalization and alternative signal transduction is largely dependent on phosphorylation of intracellular serine and threonine residues of the activated receptor. even though the β 1 -adrenoceptor is of tremendous importance in a number of diseases its phosphorylation remains poorly understood. we addressed this question in a qualitative and quantitative way. by using radioactive phosphorylation assays and mass spectrometry, we were able to elucidate the phosphorylation pattern of the human β 1 -adrenoceptor in vitro. we identified ten previously unknown phosphorylation sites in the third intracellular loop and the receptor's c-terminus. labeling hek293 cells with stable heavy isotopes (silac) lead to the discovery of a stimulation-dependent regulation of several of these phosphorylation sites. furthermore, mutagenesis studies in stably transfected hek293 cells revealed the impact of phosphorylation for arrestin binding and internalization of the receptor. fluorescence resonance energy transfer experiments with β 1 -adrenoceptor variants carrying point mutations of putative phosphorylation sites identified two c-terminal phosphosites that determine arrestin recruitment. our current goal is to further investigate the functional implications of these newly identified phosphorylation sites on downstream signal transduction, with an emphasis on the map kinase pathway. a moderate increase in arrestin affinity to the β 2 -adrenergic receptor is sufficient to induce arrestin internalization. the homologous desensitization of g-protein-coupled receptors is a two-step process. initially, g-protein-coupled receptor kinases phosphorylate agonist-occupied receptors which are subsequently bound by arrestins. in many cases, the resulting receptorarrestin complex is then internalized via clathrin-coated pits. dependent on the identity of the receptor and the ligand, the complex between receptor and arrestin may exist only in the proximity of the plasma membrane or internalize into the cell interior. we constructed mutants of the β 2 -adrenergic receptor carrying three additional serine residues in various positions at the c-terminal tail. one of these mutants which carried the serine residues in close proximity to the endogenous grk phosphorylation sites (β 2 ar-sss) showed increased isoprenaline-stimulated phosphorylation and differences in arrestin-3 affinity and trafficking. the affinity of arrestin-3 to the receptor was measured by fluorescence resonance energy transfer (fret) between the receptor and arrestin-3 and by two-color fluorescence recovery after photobleaching (frap). in the fret assay, arrestin-3 dissociation from the β 2 ar-sss receptor upon agonist washout was prolonged approximately two-fold compared to the wild-type receptor. frap was performed with an n-terminally tagged receptor immobilized with an antibody against the n-terminal tag either in solution or on a micropatterned surface. in these assays, the recovery of arrestin-3 into the bleached region was prolonged between two-and fourfold for the β 2 ar-sss receptor compared to the wild-type. even though this two-to fourfold increase in affinity seemed rather modest, it resulted in the trafficking of receptorarrestin complexes to the early endosome whereas the wild-type receptor interacted only transiently with arrestin at the plasma membrane. furthermore, the increased affinity of arrestin led to more efficient internalization of the β 2 ar-sss compared to the wild-type receptor. however, recycling to the plasma membrane after agonist washout was very similar for both receptors. we conclude that even a modest change in affinity between a g-protein-coupled receptor and arrestin can lead to substantial alterations in arrestin trafficking which in turn may have effects on cellular signaling. despite recent structural research allow for better understanding of gpcr structure, the crucial aspects of the selectivity mechanism of receptor -g protein subtype coupling remain unresolved. based on the hypothesis that the affinity of the ternary complex (agonist/gpcr/g-protein) in the nucleotide-free state determines the selectivity of gpcr-g protein coupling, we set out to measure gpcr-g protein interaction in membranes of single cells. in order to quantify the affinity of gα-subunit towards gpcrs in single cell, we determined the lifetime of the receptor-g protein complex in living cells upon agonist withdrawal under conditions of gtp-depletion. therefore, we utilized förster resonance energy transfer (fret) based assays to study interactions between fluorescent muscarinic receptors and heterotrimeric g proteins in single permeabilized hek293t cells transfected with the appropriate cdnas. here we focused on muscarinic m1-, m2-, and m3-receptors and characterized the kinetics of agonist-induced binding of go/i -and gq/11-proteins to muscarinic receptors and their subsequent dissociation in the absence of nucleotides. as a measure of affinity we calculated the rate constant of g protein dissociation from the receptor after agonist withdrawal. the dissociation kinetics of go protein from m3-and m1-achrs was found to be 10-fold faster in comparison to gq. similarly, we observed a 15-fold right shift of the concentration-response curves of go proteins binding to m3-achr in comparison to gq. in order to ensure, that the affinity of the ternary complex correlates with the efficiency of g protein activation, we performed experiments on the g protein activity in intact cells expressing non-fluorescent m3-achr by using a fret-based assay. our results showed that gq activation required 10-fold lower agonist concentration compared to go activation, suggesting that indeed the stability of the ternary complex in the absence of nucleotides determines the selectivity of gpcr-g protein coupling. we further explored the subtype selectivity of m2-achr for gi family members by comparison of dissociation kinetics of gi1-, gi2, gi3-, and go-proteins from m2-achr under nucleotide depleted conditions. k off of gi1 and gi3 were found to be two-fold higher in comparison to gi2 and go proteins, indicating the higher affinity of the latter ones to m2-achr. our fret-based assay to study receptor-g-protein interactions in membranes of single cells has been proven to be a fast and reliable method to quantify the affinity of the ternary complex. the g protein subtype dependent differences in the affinity towards activated receptors correlate with the g protein coupling efficiency of this receptor. despite their tremendous pharmacological relevance and potential for the development of new drugs, our understanding of g protein-coupled receptor (gpcr) architecture and signaling mechanisms are still limited. major reasons for this are the low abundance and poor biophysical properties of gpcrs, which makes them one of the most challenging class of proteins for structural and biophysical studies. among the superfamily of gpcrs, the class b receptors comprising 15 receptors are structurally least understood because to date it has not been possible to obtain a crystal structure of this receptor class. to overcome these limitations, we have developed a method for improving functional expression and simultaneous thermo-stabilization of gpcrs by directed evolution which is based on expression of receptors in saccharomyces cerevisiae and subsequent selection of highly expressing variants by flow cytometry with fluorescent ligands. by this strategy, key residues within a receptor sequence can be rapidly identified that are responsible for improved biophysical properties without greatly affecting the pharmacological features of the receptor. we have now applied this method to the human parathyroid hormone 1 receptor, a member of the class b of gpcrs which is a major regulator of calcium homeostasis in the body and a key target for the treatment of osteoporosis. from two rounds of directed evolution in yeast we obtained several mutants of parathyroid hormone 1 receptor that exhibit strongly improved expression levels and that remain stable after solubilization in detergents. these receptor variants are ideal candidates for subsequent structural and biophysical analysis. opioid drugs exert nearly all of their clinically relevant actions through stimulation of mors (μ-opioid receptors). the molecular biology of endogenous opioid peptides and their cognate receptors has been studied extensively in vitro. for mor, signaling efficiency is tightly regulated and ultimately limited by the coordinated phosphorylation of intracellular serine and threonine residues. morphine induces a selective phosphorylation of serine 375 that is predominantly catalyzed by g protein-coupled receptor kinase 5. as a consequence, the selective morphine-induced s375 phosphorylation does not lead to a robust beta-arrestin mobilization and receptor internalization. by contrast, high-efficacy opioid agonists such as fentanyl or etonitazene not only induce phosphorylation of s375 but also drive higher order phosphorylation on the flanking residues threonine 370, threonine 376, and threonine 379 in a hierarchical phosphorylation cascade that specifically requires grk2 and grk3 isoforms. as a consequence, multisite phosphorylation induced by potent agonist promotes both betaarrestin mobilization and a robust receptor internalization. however, little is known about agonist-selective phosphorylation patterns in vivo after acute and chronic drug administration. to learn more about mor regulation in vivo we have generated a new μopioid receptor knock in mouse with an n-terminal ha-tag. using these mice, we were able to study in vivo phosphorylation of an endogenous g protein-coupled receptor using both mass spectrometry and phosphosite-specific antibodies. we were also able to address the question which of the many putative mor splice variants detected on the mrna level are indeed expressed as functional receptors in mouse brain. ion channels 061 hcn4 in thalamic relay neurons is necessary for oscillatory activity in the thalamocortical system institut für physiologie i, westfälische wilhelms-universität, münster, germany hcn channels underlie the i h current and are involved, among other functions, in the genesis of epilepsy. the significance of hcn1 and hcn2 isoforms for brain function and epilepsy has been demonstrated, however the role of hcn4, the third major neuronal hcn subunit, is not known. here we show an unexpected role of hcn4 in controlling oscillations in the thalamocortical network. hcn4 is predominantly expressed in several thalamic relay nuclei, but not in the thalamic reticular nucleus and the cerebral cortex. hcn4-deficient thalamocortical relay neurons showed a massive reduction of i h and strongly reduced intrinsic burst firing. evoked thalamic oscillations in a slice preparation were completely abolished. in vivo, brain-specific hcn4 null mutants were protected against induced spike-and-wave discharges (swd), the hallmark of absence seizures. our findings indicate that hcn4 is necessary for rhythmic intrathalamic oscillations and that the channels constitutes an important component of swd generation. ludwig-maximilians-universität, walther-straub-institut für pharmakologie und toxikologie, münchen, germany trpc4 and 5 channels are members of the classical transient receptor potential (trpc) family whose activation mechanism downstream of phospholipase c (plc) largely remained elusive until now. while trpc3/6/7 channels are directly activated by diacylglycerol (dag), trpc4 and 5 channels are commonly regarded as daginsensitive. in contrast to trpc3/6/7 channels, they contain a c-terminal pdz-binding motif allowing for binding of na + /h + exchanger regulatory factor (nherf) 1 and 2. interestingly, performing electrophysiological measurements, co-immunoprecipitations and intermolecular dynamic fret experiments, we found that dissociation of nherf proteins from the c-terminus of trpc5 confers dag-sensitivity on trpc5 channels. trpc5 channels were dag-sensitive under the following experimental conditions: inhibition of protein kinase c, amino acid exchange in the c-terminal pdz-binding motif, pip 2 depletion with and without involvement of plc, over-expression of g-protein coupled receptors, down-regulation of endogenous nherf1 and 2 proteins and overexpression of a nherf1 mutant incapable of trpc5 binding. these findings strongly argue for nherf proteins as molecular determinants for channel activation. interestingly, pip 2 depletion itself caused slight trpc5 current increases while during pip 2 depletion, the membrane permeable dag analogue oag evoked even higher trpc5 currents suggesting that pip 2 depletion induces an active and dag-sensitive channel conformation. receptor mediated pip 2 depletion also resulted in dissociation of nherf1 and 2 from the c-terminus of trpc5 thereby eliciting a dag-sensitive trpc5 channel conformation. thus, our findings suggest that dag-sensitivity of trpc5 is the result of an activation cascade starting with pip 2 depletion and subsequent dynamic dissociation of nherf1 and 2 from the c-terminus of trpc5. altogether, dagsensitivity is a unifying functional hallmark of all trpc channels. the melastatin-related transient receptor potential channel trpm3 is a heat-activated nonselective cation channel expressed in sensory neurons of dorsal root ganglia. since trpm3-deficient mice show impaired inflammatory thermal hyperalgesia, the pharmacological inhibition of trpm3 may exert antinociceptive properties. fluorometric ca 2+ assays and a compound library containing approved drugs were used to identify trpm3 inhibitors and to characterize their potency and selectivity. biophysical properties of the block were assessed using electrophysiological patch-clamp methods. microfluorometry in fura-2-loaded single cells was applied to monitor [ca 2+ ] i signals in isolated dorsal root ganglion (drg) neurons. analgesic effects were assessed applying pregnenolone sulfate (pregs)-induced chemical pain and heat stimuli at mice. in the screening approach using stably transfected hek trpm3 cells we identified the nonsteroidal anti-inflammatory drug (nsaid) diclofenac, the tetracyclic antidepressant maprotiline and the anticonvulsant primidone as highly efficient trpm3 inhibitors. the compounds exhibited half-maximal inhibitory concentrations of 0.6-6 µm. the selectivity profiles of maprotiline and primidone for trpm3 were promising with no inhibitory effects on trpm2, trpm8, trpa1, trpv1, trpc5, trpc6 and p2x7 receptor channels. primidone inhibited pregs-induced [ca 2+ ] i signals in rat drg neurones, indicating a block of native trpm3 channels. consistently, primidone attenuated nocifensive responses of mice to paw-injected pregs. furthermore, intraplantar primidone reduced nociception in healthy and hyperalgesic cfa-inflamed paws in the hot plate test. the finding that an approved drug can inhibit trpm3 at concentrations that may be therapeutically relevant and thereby can act as an analgesic, provides a method to study trpm3-related effects by acutely challenging the channel´s function. pharmacological interference with trpm3 applying an approved drug or optimised successor compounds may pave the way to better understanding of physiological functions of trpm3 in humans and may represent a novel concept for analgesic treatment. excitotoxicity, calcium deregulation, mitochondrial dysfunction and neuroinflammation contribute to progressive cell death in many neurodegenerative diseases. therefore, proteins that prevent deregulation of these pathways are considered as drug targets. potential therapeutic approaches may benefit from modulation of small-conductance calcium-activated potassium (sk) channels, since recent data supports the hypothesis that sk channel activity promotes neuronal survival against cellular stress via a dual mechanism of action: i) by controlling neuronal excitability and ii) by preventing mitochondrial dysfunction and inflammation. our previous studies showed that activation of sk channels in neurons exerted protective effects through inhibition of nmdarmediated excitotoxicity. further, we revealed recently that in a model of glutamate oxytosis, activation of sk channels attenuated mitochondrial fission, prevented the release of pro-apoptotic mitochondrial proteins, and reduced cell death. however, little is known about the function of sk channels in cell metabolism and neuroinflammatory processes in non-neuronal cells, such as microglial cells. in this study, we addressed the question whether sk channel activation affected primary mouse microglia activation upon lps and α-synuclein challenge. we found that activation of sk channels significantly reduced activation of microglia in a concentration-dependent manner, as detected by real-time xcelligence cell impedance measurements. further data on cytokine (tnf-alpha and il-6) analysis revealed that activation of sk channels attenuated α-synuclein-induced cytokine release. inhibition of glycolysis prevented microglial activation and cytokine release. although sk channel activation slightly reduced atp levels, it attenuated α-synuclein-induced no release. furthermore, glycolytic products and ampk signaling were evaluated. overall, our findings show that activation of sk channels attenuates microglial cell activation. thus, sk channels are promising therapeutic targets for neurodegenerative disorders, where neuroinflammation and cell metabolic deregulation are associated with progression of the disease. mutant of the residue pair e103/k308 can be crosslinked efficiently in both states, the closed-and open state of the p2x2r. interestingly, oxidative crosslinking of cysteine substitution mutants of each individual residue pair significantly reduced the atpinduced current amplitudes. charge reversal or swapping mutagenesis and cysteine modification by charged mts-reagents indicated the electrostatic nature of the pairwise interactions in these four residue pairs. furthermore, preliminary data from triple, tetra and penta mutant cycle analysis indicated energetic coupling between the residue pairs e84/r290, e103/k308, e167/r290 and e167/k308 and thus indicates the cooperative interaction in a larger salt bridge network. together with the markedly reduced current amplitudes following disulfide crosslinking, our data suggest that the salt bridge network serves to stabilize the closed-state conformation of the p2x2r. the comparison of the closed-state and open-state model of the rat p2x2r showed that atp promotes a marked rearrangement of the side chains of the residues r290 and k308 to enable the strong ionic coordination of the γ-phosphate oxygen of atp. in summary, our data are in line with the concept that the electrostatic interaction of r290 and k308 with atp competitively releases e84, e103 and e167 from their strong electrostatic coupling and thus initiates a destabilization of the closed-state, which favors channel opening. fig. 1 in a similarity search using sequence motifs conserved amongst various members of the trp protein family we identified three non-annotated putative membrane proteins that we initially termed tmem1, tmem2 and tmem4. expression analysis using the nanostring ncounter system, northern blotting and rt-pcr showed that murine tmem2 is expressed in various tissues including heart, brain, lung, endothelium, colon, cardiac myocytes, cardiac fibroblasts, embryonic fibroblasts, mast cells and pancreatic acinar cells. hydropathy analysis predicts that tmem2 proteins exhibit 6 to 10 plasma-membrane spanning domains, but fluorescently labeled tmem2 fusion constructs expressed in mouse embryonic fibroblasts revealed a vesicular subcellular localization pattern. in contrast to the prediction by the psort ii algorithm, tmem2-eyfp could not be identified in the plasma membrane of fibroblasts, cardiac myocytes, mast cells or pancreatic acinar cells but showed a significant colocalization with markers and fusion constructs specific for acidic compartments including lysosomes. in tmem2 -/mice, a marked elevation of amylase and lipase plasma levels was observed. we found that constitutive but not stimulated amylase secretion from tmem2-deficient acinar cells is elevated indicating a cell autonomous defect. calcium (ca ++ ) is an important signaling molecule regulating stimulated as well as constitutive secretion from pancreatic acinar cells. microfluorimetric measurements using fura-2 or indo-1 indicate higher resting ca ++ concentrations in tmem2-/-pancreatic acinar cells correlating with elevated basal enzyme secretion. in tmem2-yfp-knock-add-on mice we identified tmem2 in organelles of the apical acinar cell pole and a partial colocalisation with lamp2 proteins. furthermore, largely increased elevations in cytoplasmic ca ++ concentration were observed upon osmotic lysis of lysosomes triggered by gly-phe β-naphthylamide (gpn) or by nh 4 cl application. the role of tmem2 for ca ++ release was evaluated by stimulation with low concentration of cholecystokinin 2pm) in the absence of extracellular ca ++ using both microfluorimetric recording of cytosolic ca ++ transients as well as electrophysiological recordings of ca ++ -activated chloride currents. these measurements revealed a higher frequency of intracellular ca ++ oscillations and a larger area under the curve of ca ++ activated chloride currents upon cck-8 stimulation indicating that tmem2 inactivation leads to an enhancement of the globalization of cck-8 evoked ca2+ release from intracellular organelles. taken together, our study identifies tmem2 as a novel regulator of ca ++ release from intracellular organelles including endo-lysosomes and as a critical determinant of constitutive protein secretion in pancreatic acinar cells. while generally highlighting toxicology as a translational science that requires academic anchoring, gundert-remy and co-workers [1] have called for efforts to improve the relevance of in vitro methodologies in predicting in vivo effects. against this background, the german society of toxicology working group on alternative approaches to animal testing proposes specific quality criteria (qc) for in vitro methods and for research work using in vitro methods. these qc may serve to evaluate in vitro methods that are developed or applied in-house or that are described in work plans, peer reviewed articles, etc. for the time being, the qc focus on in vitro cell or tissue culture methods that address human health endpoints in the context of substance-related regulatory toxicity testing. nevertheless, these qc are also generally applicable to in vitro research conducted for other toxicological purposes. relevant work from, e.g., the organisation for the economic co-operation and development has been taken into account in specifying the qc that cover the following aspects:  the 3rs impact of an in vitro method in replacing, reducing (and refining) a specific animal test for a specific toxicological endpoint. this aspect also includes scientific hurdles that, in the past, had impeded the successful development of in vitro methods for the given toxicological endpoint.  scientific relevance and reliability, i.e. which fundamental requirements should an in vitro method meet to ensure that its results are relevant and reliable.  practicability and applicability, i.e. what is the expected expenditure for the in vitro method, and have relevant authorities and industrial sectors been involved in the development of the in vitro method. qc related to the scientific relevance of research work using a specific in vitro method provide a tool to justify, e.g., the suitability of the selected test system and in vitro endpoint(s) for the given purpose; the selection of test substances, positive and negative controls; the setting and control of test concentrations; and the definition of acceptance criteria to determine the relevance of test results. the proposed qc may serve as a framework to assess the relevance of in vitro methods and in vitro research work. thereby, they aim at improving in vitro predictivity of in vivo toxicological effects, which in return contributes to reducing and replacing the need for animal testing. [1] gundert-remy, u. et al. (2015) . toxicology: a discipline in need of academic anchoring -the point of view of the german society of toxicology. arch toxicol 89: 1881-93. during the past decades, considerable progress has been made in implementing 3r approaches in routine safety assessment. in spite of these achievements, animal tests still need to be conducted if legal requirements prescribe in vivo tests or if no reliable, accepted alternative method exists. efforts to foster 3r approaches and make them 'ready for use' focus on three levels: development and validation of scientific methods/strategies, regulatory acceptance of acknowledged approaches and global harmonization of standards. strong cooperation between toxicological experts from scientific bodies, national and international authorities and industry is needed to advance on all three levels for the benefit of animal welfare. 3r approaches that have already been implemented in routine safety assessment of consumer goods do not focus solely on replacement of animal testing by use of accepted alternative methods. in cases where reliable alternative approaches are not yet available, reduction in animal numbers and refinement of testing procedures can be achieved on a case-by-case basis. a tailor-made, tiered testing strategy is usually pursued that involves knowledge on specific characteristics of the test item and makes use of all available data, including details on exposure and results obtained with structural homologues. hurdles to apply alternative approaches can even occur for established methods. as legislations give different priority to alternative approaches, it remains challenging to fulfil conflicting legal requirements in different regions of the world, or even to address horizontal legislations of the same region. furthermore, successfully validated and legally implemented alternative approaches might not always provide the safety assessor with meaningful test results. with gaining experience, limitations of test systems can become evident that affect for example the applicability domain of the method, as has been the case both for some in vitro and in vivo methods. in these cases, the new information needs to be shared not only among safety assessors, but also with method developers and regulators to facilitate refinement of scientific approaches and/or amendments of regulations. leibniz-institut für arbeitsforschung (ifado), vistox, dortmund, germany two-photon microscopy facilitates imaging of biological processes in vivo. establishing this recent technique in mouse liver allowed us to record in a real-time the sequence of events during acetaminophen (apap) induced-liver damage. although apap is intensively studied and described in vivo imaging revealed so far unknown scenarios of cell death. the hepatocytes close to the central vein of a liver lobule went within hours into cell death as commonly described due to the toxic metabolite napqi. surprisingly, we observed a distinct way of cell killing at the outer border of the dead cell area which is accompanied by bile acid decompartmentalization. there, within an hour after apap administration dilatation of bile canaliculi was observed. subsequently, bile acids containing invaginations arouse from the apical side of a hepatocyte into the cytosol. these invaginations ballooned until the bile leaked into the hepatocyte volume and subsequently the plasma membrane of the affected hepatocytes lost its integrity leading to cell death. this mechanism emerged in an environment for hepatocytes where moderate napqi levels meet intracellular high bile salt concentrations of the midzonal region. in conclusion, establishing in vivo imaging in mouse liver enabled us to identify new cellular mechanisms which cannot be discovered by conventional methods. universitätsklinikum düsseldorf, institut für toxikologie, düsseldorf, germany introduction: lung inflammation and fibrosis are considered as major toxicities after thoracic cancer radiotherapy. up to now effective pharmacological interventions for normal tissue protection are largely missing. hmg-coa reductase inhibitors (statins), which are used in the clinic for lipid-lowering purpose, are reported to have multiple inhibitory effects on genotoxic stress responses. for this reason we aim to investigate the usefulness of statins to protect normal lung cells in vitro and lung tissue in vivo from damage provoked by ionizing radiation (ir). methods: according to clinically relevant anticancer radiation regimens, we used fractionated irradiation schemes (4 x 4 gy) for both in vitro as well as in vivo experiments. we analyzed the effect of lovastatin on ir-induced dna damage formation and repair, dna damage response (ddr) and cell death in non-proliferating human lung fibroblasts, epithelial as well as endothelial cells. furthermore, we established an irradiation device that is useful to selectively irradiate the right lung of mice and investigated the influence of lovastatin on lung damage following fractionated and selective irradiation of the lung in vivo (balb/c mice). results: compared to lung fibroblasts and epithelial cells, endothelial cells exhibited the highest radiosensitivity and underwent ir-induced apoptosis which was partly prevented by lovastatin. by contrast fibroblasts and epithelial cells did not undergo apoptosis upon irradiation. lovastatin did not affect initial dna damage formation in any of these cells. in all three lung cell types lovastatin enhanced the repair of dna double-strand breaks as analyzed 24 h after the last irradiation by γh2ax nuclear foci formation. depending on the cell type lovastatin affected various components of the ddr machinery in vitro. in vivo, lovastatin prevented ir-mediated increase in breathing frequency as determined two and four weeks after fractionated irradiation. moreover, statin treatment attenuated the level of residual dna damage and ir-induced apoptosis as analyzed four weeks after irradiation. these results were mimicked when eht1864, a small molecule inhibitor of the small rho-gtpase rac1, was applied in vivo, pointing to an involvement of rac1 in statin-mediated radioprotective effects. conclusion: bearing in mind that statins are well tolerated in humans, we suggest the application of statins as a promising pharmacological strategy for the prevention of irradiation-induced damage of the lung. targeted genome engineering by crispr/cas9 is an evolving tool for generating specific knockout cell lines. co-expression of crispr/cas9 allows for efficient dna cleavage and introduction of so called indel mutations (insertion/deletion point mutations) that lead to either misfolded non-functional proteins or complete knockout. we exploited this tool to generate a bid (bh3-interacting domain death agonist) knockout cell line in neuronal ht-22 cells. bid has been shown to be involved in regulated cell death pathways like oxytosis where its activation mediates mitochondrial demise, subsequent release of apoptosis inducing factor (aif) and cell death. in the cell death model of oxytosis the cystine/glutamate antiporter (x c -) is inhibited by high extracellular glutamate concentrations. following events such as increasing lipid peroxidation and ros production resemble major characteristics of another emerging cell death pathway, called ferroptosis. in this study we generated a bid crispr/cas9-knockout cell line to elucidate the role of bid as a potential link of oxytosis and ferroptosis in the ht-22 cell line. in order to investigate the potential mechanistic overlap at the level of mitochondrial death pathways, we induced oxytosis with glutamate or ferroptosis with erastin in wild-type cells and analyzed the respective effects of the well-established inhibitors ferrostatin-1 and the bid inhibitor bi-6c9 on cell death and mitochondrial paradigms. these results were then compared to the effects of glutamate or erastin in crispr/cas9-bid-knockout cells. bi-6c9 inhibited glutamate-induced morphological changes of ht-22 cells and also prevented cell death as assessed using the mtt assay and annexin v/pi staining. similar results were observed with ferrostatin-1 in the model of erastin-induced ferroptosis. subsequent facs analysis of lipid peroxidation by bodipy staining demonstrated that bi-6c9 abolishes lipid peroxide formation in the erastin model and ferrostatin-1 in the model of oxytosis. facs analysis was further employed for the detection of mitochondrial ros formation. mitosox staining revealed a significantly decreased production of mitochondrial ros by bi-6c9 and ferrostatin-1 in the respective model systems. investigating the crispr/cas9-bid-knockout ht-22 cell line revealed that bid knockout prevented cell death, lipid peroxidation and mitochondrial toxicity in both model systems of cell death, oxytosis and ferroptosis. in conclusion, the present study exposes bid as a pivotal molecular link between the previously separated cell death pathways oxytosis and ferroptosis at the level of mitochondria. parkinson's disease is a common neurodegenerative movement disorder characterized by midbrain dopaminergic neuronal loss in the substantia nigra that has been linked to alpha-synuclein toxicity. however, the molecular mechanisms underlying alphasynuclein-mediated toxicity in human dopaminergic neuronal loss are not well defined. the goal of this study was to investigate the deleterious effects of alpha synuclein in particular mitochondrial toxicity in human dopaminergic cells. therefore, we have generated neuron specific, adeno associated virus type 2 (aav2) expressing cytosolic as well as mitochondrial targeted alpha synuclein and egfp expressing viruses used as respective controls. overexpression of both, the cytosolic and the mitochondrial variants of alpha synuclein severely disrupted the dendritic network, induced loss of cellular atp, enhanced mitochondrial ros production, and was associated with activation of caspases and dopaminergic cell death in a time-dependent manner. in addition, real-time analysis of mitochondrial bioenergetics using the seahorse bioscience system following aav infection elicited a complete damage to mitochondrial respiration capacity in the dopaminergic neurons. our results suggested that mitochondrial targeted expression of alpha synuclein appeared to be more toxic than the cytosolic form of alpha synuclein. in addition, ultrastructural mitochondrial morphological analysis by transmission electron microscopy illustrated a number of deformed cristae in cells expressing the cytosolic alpha synuclein and a complete loss of cristae structure and massively swollen mitochondria following the expression of mitochondrial targeted alpha synuclein in the human dopaminergic neurons. in addition, we found that inhibition of caspases by the broad spectrum caspase inhibitor qvd significantly ameliorated alpha synuclein-induced dopaminergic neuronal death. interestingly, inhibition of caspases preserved neuronal network integrity, atp levels and mitochondrial respiration capacity in both paradigms of cytosolic and mitochondrial alpha synuclein overexpression. overall, our findings show that cytosolic as well as mitochondrial targeted expression of alpha synuclein is detrimental to human dopaminergic neurons, while inhibition of caspases amend alpha synuclein toxicity at the level of mitochondria. thus, caspase inhibitors provide promising therapeutic potential to prevent dopaminergic neuronal death in parkinson's syndromes that are associated with alpha synuclein toxicity. degradation of and adverse effects caused by tattoo and permanent make-up pigments upon sunlight exposure and laser removal have been occasionally reported in the last decades. until now, only the ban of certain azo-pigments has been addressed in the national legislation. the regulation was based on a number of studies showing the cleavage of azo-bonds by ultra violet light and laser-irradiation leading to the formation of carcinogenic aromatic amines. as a result, especially german tattoo ink manufactures switched to the use of more light-fast polycyclic pigments assuming these would be safer for this kind of application when compared to azo-pigments. to assess the potential risks of polycyclic pigments in terms of decomposition in the skin, we compared the photochemical cleavage of the widely used azo-pigment orange 13 and the polycyclic pigment copper phthalocyanine blue. main decomposition products are qualitatively and quantitatively analyzed after q-switched laser irradiation of 1 mg/ml aqueous suspensions and tattooed pig skin. irradiated specimen were extracted with ethyl acetate and analyzed with gas chromatography coupled to mass spectrometric detection (gc/ms) using liquid injection and head-space sampling techniques. we were able to confirm the cleavage of pigment orange 13 at the azo-and other weak bonds in our experimental set-up (fig.1a) . amongst other substances, the carcinogens aniline (max. conc. 1.01 ± 0.12 µg/ml) and 3,3-dichlorobenzidine (max. conc. 0.88 ± 0.18 µg/ml) are formed. despite the lack of such weak bonds, the highly stable porphyrin-like structure of copper phthalocyanine blue is as well decomposed upon laser-irradiation (fig. 1b) . here, 1,2-benzenedicarbonitrile (max. conc. 1.11 ± 0.12 µg/ml) were found as the main decomposition product in all experimental setups. concentrations of cleavage products were generally higher in aqueous suspensions compared to pig skin extracts with both pigments. additionally, the highly toxic gas hydrogen cyanide (max. conc. 35.8 ± 4.32 µg/ml) and the human carcinogen benzene (max. conc. 0.19 ± 0.06 µg/ml) were formed from both pigments, dependent on the laser wavelengths used. cyanide levels of ≥50 µg/ml evolving upon ruby laser irradiation of >1.5 mg/ml aqueous suspensions of phthalocyanine blue were proven to significantly reduce cell viability in human skin cells in vitro. reference 1 schreiver, i., hutzler, c., laux, p., berlien, h. p. & luch, a. formation of highly toxic hydrogen cyanide upon ruby laser irradiation of the tattoo pigment phthalocyanine blue. sci rep 5, 12915 (2015) . understanding the interactions between nanoscaled objects and living cells is of great importance for risk assessment, due to rising application of nanomaterials in foodrelated products. several studies show that silver nanoparticles can reach the intestinal epithelia in nanoform in a human in vitro digestion model. nevertheless, only sparse data concerning the direct quantification of cellular uptake of silver nanoparticles are available. therefore, this study was focused on a systematical quantitative comparison of the cellular uptake of differently coated silver nanoparticles of comparable size. intracellular uptake was determined quantitatively via a transwell tm -system with subsequent elemental analysis (aas) and ion beam microscopy (ibm). silver nanoparticles were coated with poly (acrylic acid) and polyvinylpyrrolidone and characterized extensively by tem, dls, saxs, zetasizer and nanosight. agpure tm as a widely used reference nanoparticle coated with tween 20 and tagat to v was also used for comparison. different intestinal cell models were applied to get closer to the complex in vivo situation: beside the widely used caco-2 model we also investigated particle uptake in a model which considered the enterocyte-covering mucus layer, as well as in a model specialized on particle uptake, the so-called m-cell model. our findings suggest that silver uptake is clearly a particle-and not an ion-related effect. the internalization of silver nanoparticles was enhanced in uptake-specialized m-cells, although no enhanced transport through the cells was observable. furthermore, the mucus did not providing a substantial additional barrier for nanoparticle internalization. rutherford backscattering spectrometry (rbs) via ibm allowed distinguishing between adsorbed an internalized material and the results were in accordance with the transwell tm -data. additionally, ibm investigations via particle-induced x-ray emission (pixe) showed intracellular association of silver with sulfur. the quantification of silver nanoparticle internalization revealed a clear particle-specific and a coating-related uptake. furthermore, a high amount of silver nanoparticles is taken up in cell models of higher complexity. thus, an underestimation of particle effects in vitro might be prevented by considering cell models with greater proximity to the in vivo situation. analyzing iron oxide nanoparticles for drug delivery -innovative investigation tools for nanotoxicology nanoparticles offer promising new possibilities for medical applications including therapy and diagnosis of various diseases. especially nanoparticle systems with magnetic cores provide a broad application spectrum as contrast agents, magnetic transporters, or heat carriers in hyperthermia treatment. for bench to bedside translation of superparamagnetic iron oxide nanoparticles (spions) for medical applications, safety issues have to be clarified. for that, reliable standards must be established on the basis of comprehensively validated physicochemical and biological characterization methods. spions consisting of maghemite and magnetite are usually of brown or black color. due to these special properties, spions and other metal oxide nanoparticles are prone to interfere with classical toxicological assays relying on optical detection of colorimetric, fluorescence or luminescence signals. particularly, nanoparticle concentration and cellular uptake are further influencing factors. consequently, for reliable analysis of nanoparticle mediated effects, alternative robust and interference-free readouts have to be established. based on long lasting experience working with spions, we suggest a combination of complementary methods to analyse nanoparticle-mediated effects: multiparameter analyses in flow cytometry deliver statistically relevant data and link uptake of nanoparticles (side scatter increase) with cellular effects in a high-content style. combination of noninvasive, label-free impedance measurements (xcelligence system) with real-time (fluorescence) microscopy enables us to monitor cellular proliferation and morphology over several days without interference by nanoparticles. additional experiments in multicellular tumor spheroids provide information about tissue infiltration and thus, more closely resemble the in vivo situation. using those complementary methods, several drug-loaded spion systems dedicated for medical applications have been successfully characterized previously. in sum, nanotoxicology is a complex and interdisciplinary challenge, where physicochemical parameters, as well as in vitro and in vivo behavior of nanoparticles have to be considered. to address these basic requirements, we are working on a stringent standardized road of characterization for iron oxide nanoparticles synthesized for medical applications. reference: lyer s, tietze r, unterweger h, zaloga j, singh r, matuszak j, poettler m, friedrich rp, duerr s, cicha i, janko c, alexiou c. nanomedical innovation: the seon concept for an improved cancer therapy with magnetic nanoparticles. nanomedicine (lond). 2015; 10 (21) acrylamide (aa) is an α,β-unsaturated compound, which is categorized as probably carcinogenic to humans [1, 2] . aa is known to arise in foods by heat treatment in the course of the maillard reaction between reducing sugars and amino acids at processing temperatures > 120 °c [3] . dietary aa exposure has mainly been estimated on the basis of dietary recall, assessing consumption of foods with known aa contents. the use of human biomarkers of aa exposure, primarily haemoglobin adducts of aa and its genotoxic metabolite, glycidamide ( ga) in red blood cells, as well as mercapturic acids excreted in the urine, is a promising alternative. such biomarkers are to be validated by exact measurement of aa uptake in duplicates of food as consumed (duplicate diet studies) [4] . we here present results of a nine-day human intervention study with 14 healthy male volunteers. aa contents were determined in duplicates of servings as consumed and kinetics of aa-associated mercapturic acids (aama and gama) monitored in total urine [5] . the study design included washout periods with an aa-minimized diet (21 -41 ng /kg bw), a low aa intake day (0.6 -0.8 µg /kg bw) as well as a high aa intake day (1.3 -1.8 µg /kg bw). after a three-day washout period an aama baseline level of 93 ± 31 nmol/d was determined. low aa intake led to an aama excretion within 24 h of 225 ± 37 nmol/d, high intake to 404 ± 78 nmol/d corresponding to an aama excretion rate of about 30% of the ingested aa dose within 24 h, whereas aama output within 72 h corresponded to 58% of the respective aa intake, the aama baseline after 3 days washout corresponds to a net exposure level of 0.2 -0.3 μg aa/kg bw/d. whether this represents a true baseline level is to be clarified in a follow-up study. in summary, this study provides important quantitative information on kinetics of urinary short-term exposure biomarkers validated by analytically verified dietary aa intake at present day food contamination levels. [1] deutsche forschungsgemeinschaft (dfg), mak-und bat-werte-liste 2013 , 2013 doi: 10.1002 iarc, iarc monographs on the evaluations of carcinogenic risks to humans 1994, 60. [3] tareke et al., j. agric. food chem. 2002, 50, 4998-5006. [4] efsa panel on contaminants in the food chain (contam), efsa journal 2015; 13(6):4104 [321 pp.] . [5] ruenz et al., arch. toxicol. 2015 doi: 10.1007 /s00204-015-1494 heinrich-heine-universität, institut für toxikologie, düsseldorf, germany objective: flavonoids are known to modulate distinct signaling pathways thereby causing different physiological effects. effects of the flavonoids baicalein and myricetin as well as several methylated derivatives were analyzed in the nematode caenorhabditis elegans and in in hct116 colon carcinoma cells and to get insights in molecular mechanisms modulated by these compounds. methods: radical-scavenging activity (teac, dcf), stress resistance (sytox, sodium arsenite), modulation of signaling pathways (nrf2/skn-1, daf16), life span. results: baicalein enhances the resistance of c. elegans against lethal thermal and sodium arsenite stress and dose-dependently prolongs the life span of the nematode (median life span: + 57%). using rna interference we were able to show that the induction of longevity and the enhanced stress-resistance were dependent on skn-1 (homolog to mammalian nrf2), but not daf-16 (homolog to mammalian foxo), another pivotal transcription factor. negletein was the only methylated derivative which was able to enhance the life span of the nematode. in hct116 cells, baicalein activates nrf2; the methylated derivatives oroxylin a and negletein showed a comparable redox-active potential in these cells, but only negletein was able to activate nrf2. the dietary flavonoid myricetin as well as the methylated derivatives laricitrin, syringetin and myricetintrimethylether strongly enhance life span of c. elegans, decreased oxidative stress (dcf) and accumulation of lipofuscin. in contrast to myricetin, the methylated compounds strongly enhanced the resistance against thermal stress. furthermore, treatment with the derivatives induced a much stronger nuclear localization of the daf-16 transcription factor. conclusion: baicalein increases stress-resistance and life span in c. elegans via skn-1 but not daf-16. experiments with methylated baicalein derivatives suggest that the redox-active potential has a minor impact on the nrf2/skn-1 activation since only distinct derivates activate this pathway. in case of myricetin, the methylation increases the stress resistance of the flavonoid. methylation seems to enhance the biofunctionality of the flavonoids. our results may be useful to understand molecular mechanisms of flavonoids and methylated derivatives used as food supplements or pharmacological extracts. the loss of progesterone during menopause is linked to common sleep complaints of the affected women. consequently, a previous study of our laboratory demonstrated sleep promoting effects of oral progesterone replacement in postmenopausal women [1] . the oral administration of progesterone, however, is compromised by individual differences in bioavailability and metabolism of the steroid. we therefore investigated the sleep-eeg effects after intranasal application of progesterone in 12 healthy postmenopausal women (50-70 yrs).in a randomized doubleblind protocol each subject received four treatments, 2 doses of intranasal progesterone (4.5mg mpp22; 9.0 mg mpp22), 10mg of zolpidem and placebo. the 4 conditions consisted of 2 experimental nights (adaptation + examination) separated by at least one week. during each examination sleep eeg was recorded from 23:00 to 07:00. simultaneously blood was collected every 20 min between 22:00 and 07:00 by long catheter for later analysis of the hormones growth hormone (gh), cortisol, melatonin and progesterone. conventional sleep-eeg was statistically evaluated by multivariate analyses of variances (manovas) with repeated measures designs after removal of two outliers, which showed a low sleep efficiency index (sei) after 4.5 and 9.0mg mpp22. univariate f-tests in the manovas pointed to the following results (significant p-values at α=0.05). sei was higher after zolpidem than after the other three treatments. after 9.0mg mpp22 sei was elevated significantly in comparison to placebo. subjects spent more time in nonrem sleep and less time in intermittent wakefulness after 9.0mg mpp22 and after zolpidem than after placebo. total sleep time was elevated and wake after sleep onset (waso) was reduced after 9.0mg mpp22 and after zolpidem. after all active treatments with mpp22 and zolpidem the time spent in sleep stage 2 was higher than after placebo. the amount of slow-wave sleep was higher after zolpidem than after placebo. in addition, the higher dose of mpp22 resulted in an increase of spindle and β frequencies combined with a decrease of δ oscillations during nonrem sleep. in comparison, administration of zolpidem resulted in strong increase of δ, spindle and high β frequencies as well as strong decrease in θ and α frequencies. nocturnal progesterone levels increased after 9.0 mg mpp22. no other changes of hormone secretion were found. our study show sleep promoting effects of 9.0 mg mpp. as expected the sleep promoting effect of zolpidem was confirmed. the spectral signature of intranasal progesterone partly resembled the well-known sleep-eeg alterations induced by gaba active compounds. progestereone levels were elevated after 9.0 mg mpp22. no other endocrine effects were observed. introduction: anticholinergic drugs or drugs with anticholinergic side effects are commonly used for the treatment of various diseases in the elderly population. elderly patients are particularly vulnerable to anticholinergic-related cognitive effects. moreover, there is a relationship between anticholinergic exposure and cognitive impairment. however, there is currently a lack of data on the anticholinergic burden in geriatric patients in germany. it was therefore the aim of this study to evaluate the anticholinergic burden in a large representative cohort of geriatric patients. materials and methods: in this retrospective cohort study, (co)-prescriptions of anticholinergic drugs as well as anti-dementia drugs were evaluated using the discharge medication of geriatric patients between january 2013 and june 2015 from the geriatrics in bavaria-database (gib-dat). anticholinergic drugs were classified according to the anticholinergic cognitive burden (acb) scale in three groups (definite anticholinergics with a score of 2 or 3 and possible anticholinergics with a score of 1). the acb scale was modified by omitting trospium and by adding the three drugs biperiden, metixen and maprotilin, which are used in germany, with a score of 3. a patient's individual score of 3 or higher is considered to be clinically relevant. in total, 130,186 geriatric patients (median age 82 years, 66.3% female, median no. of drugs 9) were evaluated. of these, 41,456 (31.8%) patients took at least one drug with anticholinergic properties. two or more anticholinergic drugs were coprescribed in 10,941 (26.4% of the patients taking anticholinergic drugs) patients. 11,241 (27.1% of the patients taking anticholinergic drugs) patients had a score of 3 or higher. the most common anticholinergic drug combinations involving two definite anticholinergic drugs were amantadine/quetiapine (58), amitriptyline/quetiapine (41) and amitriptyline/carbamazepine (35). 2,885 (7.0%) patients received anticholinergic drugs in combination with anti-dementia drugs. conclusions: one third of patients in a large geriatric population were prescribed at least one anticholinergic drug. one quarter received a co-prescription of anticholinergic drugs. caution is advised prescribing anticholinergic drugs to elderly patients especially with dementia. the antiglaucoma agents brimonidine and timolol are novel substrates of the organic cation transporters oct2 and mate1 expressed in human eye c. neul purpose: glaucoma is a leading cause of visual loss in the world population. lowering intraocular pressure by topical administration of antiglaucoma agents is still the mainstay for glaucoma treatment. 1,2 although many effective drugs exist, the major challenge is their efficient intraocular delivery, which is estimated to amount to 3 the involvement of membrane drug transporters in the intraocular delivery of the widely prescribed antiglaucoma prostanoid latanoprost has been described. 4 however, it is currently unknown whether the cationic drugs brimonidine and timolol, which are also commonly used antiglaucoma agents, are similarly transported by drug transporters and whether these transporters are expressed in human eye. brimonidine is an α 2 -adrenergic agonist, which inhibits the activity of the adenylate cyclase subsequently leading to a reduced production of aqueous humor. timolol is a β-adrenergic receptor antagonist, which blocks β-receptors on the ciliary epithelium also resulting in a reduced aqueous humor production. the aim of the present study was to determine whether brimonidine and timolol are substrates of the organic cation drug transporters oct1 (encoded by slc22a1), oct2 (slc22a2), oct3 (slc22a3) and mate1 (slc47a1). a further aim was to investigate whether these transporters are localized in different human eye substructures. experimental design: transport of brimonidine and timolol was studied using the mammalian cell line hek293 stably expressing the organic cation transporters oct1, oct2, oct3 or mate1. 5 intracellular accumulation of brimonidine and timolol was analyzed by mass spectrometry. immunohistochemistry and immunofluorescence experiments were performed to study the localization of these transporters in different substructures from glaucomatous and non-glaucomatous human eyes. results: uptake experiments revealed that brimonidine is transported by oct2 and mate1 in a time-and concentration-dependent manner, but not by oct1 or oct3. timolol is only transported by mate1, but not by the octs. as shown by immunolocalization studies, the oct2 and mate1 transporter proteins were expressed in all anterior eye substructures of non-glaucomatous and glaucomatous eyes, i.e. the cornea, the conjunctiva and the ciliary body. conclusion: our data demonstrate that oct2 and mate1 may play a role in the ocular disposition of the antiglaucoma drugs brimonidine and timolol and may contribute to interindividual variability of drug concentrations and effects. references: 1. zhang et al., nat rev drug discov. 2012 jun 15; 11(7) :541-59. 2. lavik et al., eye (lond). 2011 may; 25(5):578-86. 3. gaudana et al., pharm res. 2009 may; 26(5):1197 -216. 4. kraft et al., invest ophthalmol vis sci. 2010 51(5):2504 -11. 5. nies et al., plos one. 2011 6(7) :e22163. supported by the robert bosch foundation, stuttgart, germany. immature platelet count or immature platelet fraction as optimal predictor of antiplatelet response to thienopyridine therapy c. stratz 1 , t. nuehrenberg background: previous data suggest that reticulated platelets impact significantly on antiplatelet response to thienopyridines. it is unknown which of the parameters describing reticulated platelets is the optimal predictor of antiplatelet response to thienopyridine therapy. methods: this study is a prespecified subanalysis of the excelsiorload trial that randomized elective patients undergoing coronary stenting to loading with clopidogrel 600mg, prasugrel 30mg or prasugrel 60mg (n=300). adp-induced platelet reactivity was assessed by impedance aggregometry before loading (=intrinsic platelet reactivity) and on day 1 after loading. multiple parameters of reticulated platelets were assessed by an automated whole blood flow cytometer: immature platelet fraction (ipf, proportion of reticulated platelet of the whole platelet pool), highly immature platelet fraction (hipf), absolute immature platelet count (ipc). results: each parameter of reticulated platelets correlated significantly with adpinduced platelet reactivity: ipf (r s =0.18; p=0.002), hipf (r s =0.18; p=0.002), ipc r s =0.26; p<0.001). in a multivariable model including all three parameters, only ipc remained as significant predictor of platelet reactivity (p<0.001). after adjustment to known predictors of on-clopidogrel platelet reactivity including cytochrome p450 2c19 polymorphisms (*2 and *17), age, body mass index, diabetes, smoking and intrinsic platelet reactivity, ipc s21 was the strongest predictor of on-treatment platelet reactivity (partial η 2 =0.045; p<0.001) followed by intrinsic platelet reactivity (partial η 2 =0.034; p < 0.002). these findings prevailed when analyzing subgroups of patients on clopidogrel or on prasugrel. conclusion: immature platelet count is the strongest platelet count derived predictor of antiplatelet response to thienopyridine treatment. given its easy availability together with its even stronger association with on-treatment platelet reactivity when compared to known predictors including the cyp 2c19*2 polymorphism, immature platelet count might become the preferable predictor of antiplatelet response to thienopyridine treatment. cutaneous squamous cell carcinoma (cscc) is the second most common human cancer with continuously rising incidences worldwide. primarily caused by cumulative uvb exposure, cscc accounts for considerable costs for health care systems and poses a deadly risk especially to organ transplant recipients [1] . current chemotherapy needs to be improved, because even the topical treatment for cscc's carcinoma in situ bears limited efficacy and painful adverse effects [2] . however, animal-based approaches in preclinical development contribute to the frequent failure of investigational new drugs in clinical trials [3] . herein, we characterized a human cell-based cscc model, normal reconstructed human skin (rhs) served as control. whereas rhs exhibited low proliferation, the co-culture with cscc increased ki-67 index 23-fold in the cscc model (p£0.001). while the presence of claudin-4 and occludin were distinctly reduced, zonula occludens protein-1 was more wide-spread, and claudin-1 was heterogeneously distributed within the cscc model compared with rhs. this is in accordance to the in vivo situation [4] and likely contributes to the impaired barrier function of the cscc model, as demonstrated for 2.6-fold increased caffeine permeation. finally, the ingenol mebutate effects in the cscc model and rhs closely mimic the anti-tumor effect and the adverse reactions in patients [2] , both linked to the drug's inherent cytotoxicity. in conclusion, the thoroughly characterization of disease models fosters both advanced preclinical drug development and improved cscc treatment. funded by the german government, the berlin-brandenburg research platform bb3r with integrated graduate education was launched in 2014. the aim of this research platform, along with the associated graduate school, is to close substantial knowledge gaps in the fields of the 3rs and to find alternatives to animal experimentation within the next years. a panel of 3r experts has been set up to provide advice and assistance and to raise awareness in society for 3r-related issues. research in bb3r investigates physiological functions on different levels to establish alternative methods for preclinical drug development and basic research. the principal investigators aim at facilitating research collaborations and sustainable research activities in the region berlin-brandenburg and abroad. an integrated bb3r graduate program has been developed to offer structured training to graduate students in a specific, mandatory course program on 3r including modules on ethics and legislation. currently, 14 phd students are qualified for management positions in professional areas related to the 3rs, and three junior research groups are now ready to expand their regional research activities nationwide. furthermore, the concept of a novel lecture series for master students and undergraduates has been designed and awarded the animal welfare research award for berlin-brandenburg in teaching and education. the state government of berlin supports the research platform bb3r and will be funding an additional professorship at the fu berlin to further promote research on alternative testing. finally, co-operations with national and international partners are being built to facilitate the project-based exchange of scientists and joint research. currently, the identification and evaluation of skin sensitizers is mainly restricted to animal testing using the guinea pig maximization test, buehler test or the murine local lymph node assay. recently, an adverse outcome pathway of skin sensitization has been released by the oecd, identifying the key events leading to allergic contact dermatitis. in vitro tests address these key events and two assays are now regulatory adopted (oecd 442c and 442d). the use of the current in chemico and in vitro models is, however, limited since they do not reflect dermal penetration, complete biotransformation and cell cross-talk in an organotypic environment. in this study, we aimed to overcome these limitations by establishing reconstructed skin tissues containing langerhans cells (lcs). in vitro generated immature monocyte-derived (molcs) or mutz-3-derived cells (mutz-lcs) cultivated with keratinocytes on a dermal compartment with fibroblasts form a stratified epidermis after 14 days as indicated by the expression of epidermal differentiation markers. molcs or mutz-lcs were mainly localized in suprabasal layers of the epidermis and distributed homogeneously in accordance with native human skin. topical application of the extreme contact sensitizer 2,4-dinitrochlorobenzene (dncb) induced il-6 and il-8 secretion in skin models with lc-like cells, whereas no change was observed in control rhs lacking immune cells. increased gene expression of cd83 and pd-l1 in the dermal compartment indicated lc maturation. we confirmed the enhanced mobility from epidermal to dermal compartments for mutz-lcs and molcs in the presence of dncb. in summary, we successfully integrated immature and functional lc-like cells into reconstructed human skin. this fosters the development of animal-free test systems for advanced and potentially individualized hazard assessment of skin sensitization. computational methods for prediction of in vitro activity of new chemical structures. background: with a constant increase in the number of new chemicals synthesized every year, it becomes highly important to employ the most reliable and fast in silico screening methods to predict their safety and activity profiles. in recent years, in silico prediction methods received great attention as alternatives to animal experiments for evaluation of various toxicological endpoints, complementing the theme of replace, reduce and refine (3rs). various computational approaches have been proposed for prediction of toxicity of chemicals ranging from quantitative structure activity relationship modeling to molecular similarity based methods and machine-learning methods. within the "toxicology in the 21st century" screening initiative, a crowdsourced platform was established for development and validation of computational models to predict the interference of chemical compounds in nuclear and stress receptor pathways based on a training set containing more than 10,000 compounds tested in high-throughput screening assays. methods: here we present the results of various molecular similarity-based and machine-learning-based methods over an independent evaluation set containing 647 compounds. further, we compare the performance of these methods when applied individually and together. in retrospect we also discuss the reasons behind the superior performance of an ensemble approach, that combines a molecular similarity approach and a naïve bayes machine learning algorithm in achieving best prediction rates in comparison with other individual methods, explaining their intrinsic limitations. results and conclusions: our results suggest that, although prediction methods were optimized individually for each modeled target, an ensemble of similarity and machinelearning approaches provides promising performance indicating its broad applicability in toxicity prediction. charité -universitätsmedizin berlin, structural bioinformatics group, berlin, germany a multitude of drug candidates (approx. 20 %) fail in the late drug development due to toxicity and adverse effects [1] . immunotoxicity can be divided into four types of immune-mediated adverse effects: immunosuppression, immunostimulation, hypersensitivity and autoimmunity. current safety evaluations of drug candidates with respect to immunotoxicity are comprised of in vivo and in vitro assays. here, we present an in silico approach for predicting immunotoxic substances based on immune suppressive and not toxic compounds using the laplacian-modified naϊve baysian model as a supervised machine learning method. therefore, we examined the relations between about 51,000 compounds and 115 cancer cell lines from the national cancer institute's (nci) nci-60 growth inhibition data [2] with focus on five immune cell lines (rpmi-8226, ccrf-cem, . different fingerprints encoding the chemical structures have been evaluated for their predictive power (e. g. extended-connectivity fingerprints, substructure fingerprints) and showed good prediction rates in a retrospective analysis. acting in phase ii metabolism, sulfotransferases (sult) transform endo-and exogenous molecules such as drugs into more hydrophilic entities that are easily excreted from the human body [1] . although serving detoxification, sult-mediated transformation of molecules has also been associated with the formation of chemically reactive metabolites that could promote adverse reactions [2] . the development of a computerbased model that allows prediction of molecules susceptible to metabolism would improve drug development and drug safety [3] , and encourage the reduction of in vivo testing according to the principles of the 3rs (replacement, reduction and refinement). in our study, we developed an in silico model to predict human sult subtype 1e1 activity acting in phase ii metabolism. structure-based molecular modelling of sult activity is challenging due to the broad and overlapping substrate spectrum of sult subtypes. this low substrate specificity can be attributed to the high degree of conformational flexibility of the enzyme, particularly in the active site. therefore, molecular dynamics simulations were performed to address enzyme flexibility and the broad substrate spectrum of sult ( figure 1 ). based on a collection of selected enzyme conformations from molecular dynamics simulations and a dataset of active sult1e1 ligands, ensemble docking was utilized to generate ligand-protein complexes that served as a basis for 3d pharmacophore development. eight specific 3d pharmacophores were created that allow identification of sult1e1 substrates and inhibitors. for further refinement of the computer-based prediction, machine learning models and post-filtering steps were generated that allow classification of predicted hits. the final prediction model was used to screen the drugbank (a database comprising over 6,500 experimental and approved drugs). a major part of the predicted hits could be confirmed from literature. from the remaining hits, a representative selection of molecules was experimentally tested for sult1e1 inhibition or biotransformation. experimental results were in agreement with our computer-based models and revealed previously-unknown biotransformation by or inhibition of sult1e1 for compounds listed in the drugbank. introduction: vascularization of the dermal equivalent of full-thickness skin constructs by endothelial cells is highly desirable, because such constructs closely mimic the architecture of real skin. unfortunately, the realization of a capillary network in skin constructs is still difficult. in our study of full-thickness skin constructs, following the methodologies of küchler et al. (2011) , there were alterations in the epidermal differentiation after endothelialization of the dermal equivalent. the aim of this study was to characterize these changes on a morphological level. material and methods: non-endothelialized constructs (keratinocytes, fibroblasts) were prepared according to küchler et al. (2011) . to obtain endothelialized constructs, the dermal equivalent of the non-endothelialized constructs was enriched with endothelial cells. after two weeks of in vitro culture, the skin constructs were processed for quantitative as well as qualitative assessment by light and electron microscopy. results: both types of skin construct developed all strata, i.e., stratum basale, spinosum, granulosum, corneum of a stratified soft-cornified epidermis, although the two constructs displayed differences in every stratum: significantly more mitoses occurred in the epithelial germ layers of the endothelialized constructs (p=0.013). in addition, significantly more keratohyalin granules were counted within their stratum granulosum (p=0.010). 50% of the shapes of the spinous and the granulosum cells were irregular and these cells were separated by wide intercellular spaces. the typical epidermal lamellar bodies appeared in the endothelialized constructs more often than in the nonendothelialized ones. at the stratum granulosum -stratum corneum interface, no cohesion between the strata was present. background and novelty: during the last decade organ-on-a-chip technologies received increasing attention in the scientific community. the idea of combining different tissue types on a physiological-like system creates completely new options on how substances can be characterized without the use of animal experiments. animal models were used for the investigation of skin sensitization as a standard method until animal testing for cosmetic substances was banned in the e.u. in 2013. by combining skin models with an immunological counterpart, new data will be presented to see if the multi-organ-chip add value to the current need of alternative methods regarding skin sensitization and immunotoxicity testing. experimental approach: the multi-organ-chip platform is designed to combine different human cell and tissue types like 3d-spheroids, barrier models or biopsies in one microfluidic system. a peristaltic on-chip micropump enables circulation of medium, allowing for a constant perfusion between the compartments. first experiments were performed using a dendritic-cell-only approach in the 2-organ-chip. in subsequent cocultivation experiments ex vivo human epidermis and dendritic cells were cultivated each in one culture compartment connected by the microfluidic channels. for analysis we measured the typical activation marker cd86 and the vitality of the dendritic cells by flow cytometry. functionally different sensitizers were selected to investigate their effects in our model. finally a more complex 3d matrices including different immunological cell types to emulate in vivo-like reactions like in the human lymph node were cultivated in the 2-organ-chip. results and discussion: our data show a strong influence of pump pressure and pumping frequency on the activation of dendritic cells. hence, we established an adequate set up by cultivating the dendritic cells in cell culture inserts, preventing cell activation due to shear stress. compared to existing sensitization assays, the main advantage of the perfused 2-organ-chip sensitization assay is the presence of an epidermis equivalent, partially integrating important parameters such as metabolism and skin barrier function. we compared our data with reference cd86-values from the pbmdc (peripheral blood monocyte derived dendritic cells) skin sensitization assay. for identical substances, we observed differences in dendritic cell activation between the pbmdc assay and the 2-organ-chip perfused assay. here we present the first-time cultivation of primary derived immune cells cultivated on our microfluidic system which is a promising enhancement to integrate immunological reactions on further multi-organ combinations. due to growing social and political pressure, the interest in alternatives to animal testing has constantly increased during the past 10 years stimulating the development and validation of new in vitro test systems including reconstructed skin models. additional to toxicological studies and permeability assays, skin models are of high interest for fundamental research to elucidate basic physiological and pathophysiological processes in human skin [1, 2] . as for today, most of the in vitro skin models are grown from primary keratinocytes and fibroblasts that were either isolated from excised human skin or from juvenile foreskin following circumcision. in this project, we aimed for the generation of in vitro skin models using hair folliclederived cells. therefore, different methods to optimize cell isolation and expansion of outer root sheath (ors) cells from human hair follicles were systematically investigated. the best procedure for isolation of ors cells was the direct cell outgrowth on a cell culture insert which was co-cultured with a feeder layer of postmitotic human dermal fibroblasts. following outgrowth, the cells were either further cultivated with feeder cells in specific serum-enriched cell culture medium to obtain hair follicle-derived keratinocytes or using the same culture medium without feeder cells to obtain fibroblasts. afterwards, the generation of hair follicle-derived fibroblasts and keratinocytes was verified via the fibroblast-specific markers vimentin and desmin and the keratinocyte marker cytokeratin (ck) 14 clearly showing that vimentin and desmin are expressed in hair follicle-derived fibroblasts and in dermal fibroblasts. as expected, these cells were negative for ck14, which was abundantly expressed in hair folliclederived keratinocytes. moreover, the expression of collagen type i, iv, tgf-beta, alpha sma and il-1 alpha in hair follicle-derived fibroblasts and dermal fibroblasts showed no significant differences. ultimately, hair follicle-derived keratinocytes and fibroblasts were used to grow full-thickness skin models which were subsequently characterized with regard to epidermal differentiation, skin permeability and skin surface ph. again, no significant differences compared with skin models grown from skin-derived cells were detected showing the potential of using hair follicle-derived cells for generating in vitro skin models. [1] vávrová, k., henkes, d., strüver, k., sochorová, m., školová, b. et al. filaggrin deficiency leads to impaired lipid profile and altered acidification pathways in a 3d skin construct. the journal of investigative dermatology. 134, 746-753 (2014 bundesinstitut für risikobewertung, experimentelle toxikologie und zebet, berlin, germany background: the eu directive 2010/63 has been drawn up with the aim of ultimately replacing animal testing. wherever animal experimentation is necessary, the 3-rprinciple of russel and burch (replace, reduce, refine) has to be observed. the primary goal of the 3-r-principle is to replace animal testing with alternative methods. if no alternative method can be applied, the total number of animals is supposed be reduced. consequently, some animals are used multiple times in the course of an experiment. for example, in imaging studies, rodents are exposed to anesthesia several times in order to control the progress of a disease. however, the directive claims that "the benefit of reusing animals should be balanced against any adverse effects on their welfare, taking into account the lifetime experience of the individual animal". objective: we are looking into whether multiple exposures to anesthesia cause more stress than a single exposure. methods: the most common mouse strain c57/bl6 j and anesthetics isoflurane and the combination of ketamine/xylazine are used. with regard to recent studies, the animals are anesthetized six times for 45 minutes over a period of three weeks. all parameters observed are compared between controls, animals with a single and repeated anesthesia. the interval between the administration of the anesthesias is three to four days. when the animals are under anesthesia, their vital parameters are continuously monitored and afterwards their general condition is examined. the grimace scale is scored 30 and 150 minutes after anesthesia. besides pain, the grimace scale can also assess anxiety, stress and malaise. the display of so-called luxury behaviors like nest building and burrowing behavior serves as an indicator of wellbeing. furthermore, activity, food and water intake are monitored for 24 hours. a behavioral test battery including the free exploratory paradigm, open field, balance beam and rota rod test is performed one, seven and ten days after the last anesthesia. motor coordination and balance are assessed by the balance beam and rota rod. the open field is a test to investigate anxiety-related and exploratory behavior, the free exploratory paradigm estimates trait anxiety. moreover, corticosterone metabolites are measured in feces and fur in order to prove evidence of cumulative stress. results: the first results of our study will be presented at the 82 nd meeting of dgpt. conclusion: we are confident that the results of our study will contribute to the assessment of the severity level caused by multiple exposures to anesthesia and in this way be a benefit for the welfare of laboratory rodents. bb3r is funded by bmbf. in 1959 the 3r-principle was defined by the british scientists william russel and rex burch in their book 'the principles of human experimental techniques'. the 3r refer to replace, reduce, and refine. they set the goals to use alternative non-animal methods (replace), to reduce the number of laboratory animals (reduce) and to minimize the distress of laboratory animals and to refine their welfare (refine). the implementation of the 3r-concept is the overall goal of scientific animal welfare. article 4 of the 'directive 2010/63/eu on the protection of animals used for scientific purposes' state that the research on refinement is as important as on replacement and reduction [1] . according to the current statistics on laboratory animals, the mouse is the most commonly used animal in experiments with approximately 70 % [2] [3] . effective pain treatment is crucial not only for ethical and legal considerations but also to achieve highquality results [4] . pain in mice is only obvious if it is severe or acute pain. however, it is difficult to identify slight or moderate pain. the determination of pain levels and effective dosages of analgesics is therefore challenging. the most commonly used analgesics for postsurgical pain treatment in mice are opioids. however, the recommendations for their use show vast dosage ranges. for example, the recommended dose of buprenorphine ranges from 0.05 to 2.5 mg/kg per bodyweight [5] [6] [7] depending on the pain model used. additionally, putative pharmacogenetic strain differences have to be considered. for example, the analgesic treatment with morphine shows mouse strain specific differences in pain sensitivity [8] . the goal of the project is the refinement of the recommendations for effective dosage of opioid analgesics in laboratory animals for mouse inbred strains by incorporating strain specific differences. regarding the phenotype, we want to identify a putative inbred strain dependent pain threshold and measure the drug level in plasma and brain. additionally the metabolic capacity and mrna expression level will be investigated. genotype identification is based on a data bank analysis used for correlation with phenotypical parameters. [1] rl 2010/63/eu. [2] bmel (2014) . anzahl der für versuche und andere wissenschaftliche zwecke verwendeten wirbeltiere. [3] eu commission (2013) . seventh report on the statistics on the number of animals used for experimental and other scientific purposes in the member states of the european union. [4] carbone l (2011) pain in laboratory animals: the ethical and regulatory imperatives. plos one 6, e21578. [5] gv-solas, a.f. a.d. (2013) . empfehlung zur schmerztherapie bei versuchstieren. [6] carpenter, j.w., t.y. mashima, and d.j. rupiper (2001) . exotic animal formulary, 2nd edition, w.b. saunders co., phila. [7] flecknell, p. (1996) . laboratory animal anaesthesia, 2nd edition, academic press, san diego, ca. introduction: göttingen minipigs™ are frequently used large animal models for orofacial research, for example dental implant surgery. requests from experimental surgeons for detailed anatomical information can not be answered because there is no existing data, especially not age-related. because of unavailable data and the false choice of animal age, surgical interventions fail or lead to enormous post-operative suffering. therefore the aim of this study is the acquisition of detailed anatomical data of the mandibula and other organs and structures without sacrificing pigs for this reason. animals, materials and methods: ct scans of a 64-slice scanner were collected from 18 female minipigs, consisting of 6 animals aged 12 months (group 1, n=6) and 12 animals (group 2; n=12) examined at the age of 17 and 21 months. these minipigs were involved in experiments, approved by the regional office for health and social affairs, berlin. image analysis was performed using vitrea advanced® (vital images). more than 50 parameters concerning teeth, the mandibular body, frame and canal, coronoid process and mandibular condyle were defined and measured. for now, we focused on the development of the mandibular canal and the distance between the dorsal borders of the mandibular canal to the alveolar ridge at the most posterior mental foramen, parameters immensely important for planning interventions when testing new dental implants. results: the measurements by computed tomography showed variations of several parameters between left and right ramus mandibulae and within the different age groups. the volume of the canalis mandibulae increases in time. animals of the same age show significant differences in volume, with a range of up to 65% where the largest volume was 13,4 ml and the lowest 4,7 ml. the distance between the dorsal borders of the mandibular canal to the alveolar ridge decreases between 12 and 17 months of age. comparing 17 and 21 months old minipigs, no significant difference in distance could be observed. from the age of 17 months the position of the mandibular canal in relation to the alveolar ridge remains constant. conclusion: the decrease of the distance between the mandibular canal and the alveolar ridge between 12 and 17 months of age indicates ongoing anatomical changes of this parameter until the age of 17 months. therefore animals should be older than 17 months if included in long-term studies after orofacial experiments, like implant surgery of the mandibula. because of the described individual differences, the authors strongly suggest to support the planning of orofacial interventions by ct imaging or other radiographic techniques. background: laboratory housing conditions are standardized to a high level. under these conditions, the occurrence of stereotypic behaviour (sb) can be observed. stereotypies are commonly known as deviations from normal behaviour that are repetitive, invariant and without any obvious function or aim for the animal [1]. worldwide it is estimated that over 85 million animals perform sb, with the highest prevalence in laboratory animals and the agricultural sector. fvb/n is a typical inbred mouse-strain that shows different types of stereotyped movements. it is known that environmental enrichment decreases the incidence of sb, yet they still occur [2] . since animal's behaviour highly influences its metabolism and immune system, differences in handling, caring and keeping can lead to varying results, even with an identical experimental setup [3] . aim: of the study aim of the study is to observe different life stages of fvb/n-mice and immediately detect the development of sb. observations are linked to various behavioural tests and the characterisation of the metabolic and immunological phenotype. the results will lead to a better understanding of the mechanisms driving the development of sb and clarify its implication to animal welfare or to what extent the performance of stereotypies even reflect emotional suffering. the strain-related behaviour and sb are recorded with computer-assisted programmes. observational periods already begin with the parental generation. as possible indicators for later developing sb, data on reproductive success and maternal care are collected, such as different behavioural tests. the animals are characterized by a specific protocol for detecting the metabolic and immunological phenotype. finally, histological and molecular biological analyses follow. outlook: it is of paramount importance to take good care for the welfare of laboratory animals (3r-refinement). though, the knowledge about the ethological particularities of animals and the motivational base of animals performing sb are not enough to generally avoid stereotypies. therefore the meaning according to the character of sb has to be analysed more intensively to understand the needs of laboratory animals and evolve recommendations for optimizing the breeding and keeping such as for the assessment of possible distress in animals performing sb. objective: thermoregulation is a vital function in both humans and animals with the serotonin (5-ht) system, in particular the 5-ht 1a receptor, playing a major role. activating 5-ht 1a receptors by the 5-ht 1a receptor agonist 8-hydroxy-2-(dipropylamino)tetralin (8-oh-dpat) leads to reduced body temperature. while there is consensus that hypothermia is induced by the stimulation of postsynaptic 5-ht 1a receptors in rats and humans, the regulatory mechanisms in mice are less clear. in our group, within phenotyping a transgenic mouse line permanently overexpressing the 5-ht 1a receptor in serotonergic projection areas, bert et al. (2008, pmid: 18396339) revealed exaggerated 8-oh-dpat-provoked hypothermic response. thus, the objective of the present study was to substantiate the contribution of postsynaptic 5-ht 1a receptors to thermoregulation, more precisely to the hypothermic effect of 8-oh-dpat, in mice. methods: we used radio telemetry technique to monitor the basal body temperature and the hypothermic effect of different doses of 8-oh-dpat (0.1 mg/kg -4 mg/kg i. p.) in male transgenic mice in comparison to nmri wild-type males. additionally, we investigated whether reduction of serotonergic activity by pretreatment with the 5-ht synthesis inhibitor parachlorophenylalanine (pcpa; 100 mg/kg, i. p. on four consecutive days) would alter the effects of 8-oh-dpat on body temperature in transgenic mice postsynaptically overexpressing the 5-ht 1a receptor. results: 5-ht 1a overexpressing mice revealed lower levels of basal body temperature than wild types (transgenic mice: 36.0 °c; nmri wild-type mice: 37.4 °c). in both genotypes, systemic administration of 8-oh-dpat dose-dependently decreased body temperature, being significantly more pronounced in mutant mice (-2.8 °c compared to -1.5 °c in nmri wild types). dose response curves of 8-oh-dpat revealed an ed 50 = 0.4 mg/kg in transgenic and an ed 50 = 0.57 mg/kg in nmri wild-type mice. pcpa pretreatment did not alter the hypothermic response to 8-oh-dpat in mice. the dose-response curves indicate a higher potency of 8-oh-dpat in transgenic mice. the exaggerated hypothermic response to 8-oh-dpat in mutant mice implies that postsynaptic 5-ht 1a receptors could be involved in thermoregulatory function in mice. this assumption is further confirmed by the fact that 8-oh-dpatevoked thermal responses were not influenced by pretreatment with pcpa, most notably in transgenic mice overexpressing 5-ht 1a receptors postsynaptically. genetic variation within g protein-coupled receptors compromises the therapeutic application of drugs targeting these receptors. one of the most intensely studied variation is p.arg389gly in the human beta1-adrenoceptor (adrb1). the adrb1 carrying arginine at position 389 in helix 8 in the proximal carboxy terminus is more frequent among caucasians and is hyperfunctional. yet, the molecular basis for the differences between the beta1-adrenoceptor variants arg389-adrb1 and gly389-adrb1 is poorly understood. despite its hyperfunctionality, we found the arg389-variant of the adrb1 to be hyperphosphorylated upon continuous stimulation with norepinephrine when compared to the gly389-variant. using adrb1 sensors to monitor activation kinetics by fluorescence resonance energy transfer (fret), the arg389-adrb1 exerted faster activation speed and arrestin recruitment than the gly389-variant. both depended on phosphorylation of the receptor as shown by knockdown of g protein-coupled receptor kinases and by fret experiments using phosphorylation-deficient adrb1 mutants. point mutation of single serines and threonines in the carboxy terminus of the adrb1 finally revealed a variant-specific phosphorylation pattern that determines arrestin recruitment. taken together, these findings suggest that differences in receptor phosphorylation determine the differences in activation speed, efficacy and arrestin recruitment of adrb1 variants. opioid drugs are the most potent analgesics, which are used in the clinic; however, by activating the μ-opioid receptor (mor) they also produce several adverse side effects including constipation, antinociceptive tolerance, and physical dependence. there is substantial evidence suggesting that g protein-coupled receptor kinases (grks) and βarrestins play key roles in regulating mor signaling and responsiveness. following phosphorylation by grks, β-arrestins bind to phosphorylated mors, which prevents further interactions between the receptor and g proteins even in the continued presence of agonist resulting in diminished g protein-mediated signaling. we have previously shown that agonist-induced phosphorylation of mor occurs at a conserved 10-residue sequence, 370 trehpstant 379 , in the carboxyl-terminal cytoplasmic tail. morphine induces a selective phosphorylation of serine 375 (s375) in the middle of this sequence that is predominantly catalyzed by g protein-coupled receptor kinase 5 (grk5). by contrast, high-efficacy opioids not only induce phosphorylation of s375 but also drive higher-order phosphorylation on the flanking residues threonine 370 (t370), threonine 376 (t376), and threonine 379 (t379) in a hierarchical phosphorylation cascade that specifically requires grk2/3 isoforms. to investigate this mechanism further, we have developed novel β-galactosidase complementation assays to monitor agonist-dependent recruitment of grk2 and grk3 to activated mors. using this assay, we were able to show that activation of mor by high-efficacy agonists such as damgo results in a robust translocation of grk2/3. in contrast, activation by low-efficacy agonists such as morphine results in a much less pronounced recruitment of grk2/3 isoforms. interestingly, damgo-induced β-arrestin recruitment was strongly inhibited by sirna knock down of grk2 or grk3. conversely, the morphine-induced β-arrestin recruitment was strongly enhanced by overexpression of grk2 or grk3. mutation of s375 to alanine strongly inhibited both grk and β-arrestin recruitment. however, mutation of all 11 carboxyl-terminal serine and threonine residues of mor was required to completely abolish its interaction with arrestins and grks resulting in a complete loss of mor internalization and desensitization. heterotrimeric g proteins are located at the inner leaflet of plasma membranes and are a major primary transducer of cell signaling initiated by g protein-coupled receptors (gpcrs). based on sequence similarity, heterotrimeric g proteins can be subdivided into four main classes, i.e. gi/o, gs, gq/11, and g12/13, which interact with distinct cellular effectors to shape the final cellular response 1 . identification of new selective and cell-permeable g protein inhibitors is of great interest as these may be beneficial in complex pathologies that involve signaling of multiple gpcrs 2 . mechanistically, small molecule g protein inhibitors may, for instance, block nucleotide exchange by inhibiting gdp release (i.e. guanyl nucleotide dissociation inhibitors, gdis) or allow gdp release but block gtp entry by stabilizing the g protein in an empty pocket conformation 3 . here, we set out a new approach to classify g protein inhibitors regarding their mechanism of action in radioligand binding experiments. in particular, we investigated the influence of the specific gα q/11/14 inhibitor fr900359 4 on agonist-radioantagonist binding experiments performed with membranes of cho cells stably expressing the muscarinic m1 acetylcholine receptor (cho-m1). agonistradioantagonist competition under these conditions is biphasic because agonists bind with higher affinity in the ternary complex consisting of agonist, receptor and nucleotidefree g protein compared with a g protein-free receptor 1,5 . we show that fr900359 can be classified as a gdi as it significantly reduced high affinity binding of carbachol in cho-m1 membranes. in contrast to this, bim-46187, a pan g protein inhibitor with a cell-type-dependent preference for gq, did not influence high affinity agonist binding and thus stabilized gq in an empty pocket conformation 3 . interestingly, inhibition of high affinity agonist binding by fr900359 was incomplete in agonist-radioantagonist displacement studies and also when a radioagonist was applied. to fully prevent high affinity agonist binding by fr900359, combined uncoupling of both gi and gs proteins from m1 was required by pre-treatment with pertussis toxin and cholera toxin, respectively. these data demonstrate that not only gq, but also gi, and gs, contribute to the high affinity fraction of m1 receptors. taken together, our findings show that radioligand binding experiments are an attractive approach to classify new g protein inhibitors according to their mechanism of action. universität, würzburg, germany g protein-coupled receptors (gpcrs) are cell surface receptors which, upon a conformational change in the receptor protein induced by an extracellular stimulus, can transduce the signal onto intracellular adaptor proteins such as heterotrimeric g proteins [1] . gpcr-induced cell signaling can be rather complex as several gpcrs may activate multiple different adaptor proteins and can additionally be activated via distinct binding sites, i.e. the orthosteric transmitter binding site and other "allosteric" binding sites [2] . in the present work, we wanted to investigate the influence of an allosteric binding site on receptor activation of muscarinic acetylcholine receptors (machrs). to this end, we employed the orthosteric full agonists acetylcholine and iperoxo as well as several dualsteric compounds consisting of iperoxo linked to an allosteric phthalimide (phth) or naphthalimide (naph) moiety through alkyl chains of different length or through a diamide linker (fri). binding of the allosteric part to the receptor protein may restrict the conformational flexibility of the receptor protein and thus interfere with receptor activation [2] . therefore, application of different linker length may control the signaling outcome. here, we applied the human m1 machr which preferentially activates g proteins of the g q/11 type but can also promiscuously stimulate g s proteins. g q/11 -and g sdependent signaling pathways were analyzed by application of cho cells stably transfected with the human m1 machr in ip1 and camp accumulation assays, respectively. in comparison to the orthosteric building block iperoxo, all dualsteric compounds under investigation showed a decrease in potency for both g q -mediated and g s -mediated signaling. our findings show that the bulkier allosteric naph residue impaired both signaling pathways to a greater extent than the smaller substituent phth. particularly, the compound iper-6-naph completely lost intrinsic activity for both g q/11 and g s activation at the m1 machr. moreover, g s -mediated pathway activation is more sensitive to spatial restriction in the allosteric vestibule than g q -signaling. interestingly, longer linker length led to improved signaling for both pathways (g q and g s ) in both hybrid series. iper-7-phth seems to be an exception as it had a higher intrinsic efficacy for g s -dependent signaling than the other phth hybrids with longer linker chains. strikingly, only iper-fri-phth, which corresponds to iper-8-phth in linker length, but is able to engage increased hydrogen bonding with the receptor protein, acted as a full agonist on m1 machr for both signaling pathways under investigation. taken together, these data strongly suggest that, in comparison to g q/11 -mediated signaling, activation of the g s protein in m1 machr is more sensitive to spatial restriction in the allosteric vestibule. thus, it appears to be possible to control signaling of the m1 machr by allosteric constraint of the receptor's conformational flexibility. for more than three decades 3-(1h-imidazol-4-yl)propylguanidine (sk&f-91,486 (1) [1] ) is known as the prototypic pharmacophore of highly potent histamine h 2 -receptor (h 2 r) agonists of the guanidine class of compounds including, e.g., impromidine and arpromidine. [2] in order to get more insight into the structure-activity relationships of alkylated analogues of sk&f-91,486, we characterized 78 newly synthesized derivatives including several bivalent compounds (e.g., 2) by using different pharmacological in-vitro methods. [3] the potential h 2 r agonists were subjected to a broad screening procedure utilizing radioligand binding assays with membranes of sf9 cells [4] (hh 1,2,3,4 r). compounds were also functionally characterized in the [ 35 s]gtpgs assay (hh 2 r, sf9 cell membranes). [5] selectivity vs. hh 1,3,4 r was determined for selected derivatives also using this technique. organ bath studies (gph 1 r (ileum), gph 2 r (right atrium)) yielded functional data in a more physiological environment. the major part of the new sk&f-91,486 analogues displayed partial or full agonism via hh 2 r and gph 2 r, respectively. the most potent analogue, bivalent thiazole-type bisguanidine 2, was a partial agonist (e max = 88%) and 250-times as potent as histamine vis-à-vis the gph 2 r. attempts to antagonize the positive chronotropic effect of (partial) agonists by preincubation with cimetidine, or by adding a cimetidine bolus at the end of the concentration-response curve, respectively, were successful and furnished pa 2 values for the antagonist (5.87 -6.38) which are in accordance with literature data. however, in the functional in-vitro assay on gph 2 r, the positive chronotropic response evoked by sk&f-91,486 was surprisingly resistant to antagonism by cimetidine and other typical h 2 r antagonists (ranitidine, famotidine), although the compound so far has been unanimously classified by others as a weak partial h 2 r agonist. this behaviour is unique within the large series of sk&f-91,486 analogues studied so far under similar conditions. probably the positive chronotropic effect of the lead compound is -at least in part -the result of a second molecular interaction which has been overlooked so far. [1] parsons, m.e. et al.; agents actions 1975, 5, 464. [2] buschauer, a.; j. med. chem. 1989 , 32, 1963 -1970 [3] pockes, s.; dissertation, univ. regensburg 2015. [4] seifert, r. ; j. pharmacol. exp. ther. 2003, 305 (3) , 1104-1115. the nociceptin/orphanin fq (n/ofq) peptide (nop) receptor is the fourth most recently discovered and least characterized member of the opioid receptor family (mor, kor and dor). nop receptor is widely distributed and modulates several physiological processes by its endogenous ligand nociceptin. the nop receptor is a potential target for the development of ligands with therapeutic use in several pathophysiological states such as chronic and neuropathic pain. consequently, there is increasing interest in understanding the molecular regulation of nop receptor. recently, we generated two phosphosite-specific antibodies directed against the carboxyl-terminal residues serine 351 (s351) and threonine 362 /serine 363 (t362/s363), which enabled us to selectively detect either the s351-or the t362/s363-phosphorylated forms of the receptor. our results show that nociceptin, mcoppb, sch221510 and ro64-6198 induce a stably phosphorylation at s351 and t362/s363 followed by a profound internalization of the receptor. the nociceptin-induced s351 and t362/s363 phosphorylation can be blocked by selective nop receptor antagonists such as j113397 or sb612,111. nnc63-0532, buprenorphine and norbuprenophine failed to induce a phosphorylation at these sites. in the presence of nociceptin, s351 phosphorylation occurred at a faster rate than phosphorylation at t362/s363 indicating that s351 is the primary site of agonistdependent phosphorylation. activation of pkc by phorbol 12-myristate 13-acetate facilitated receptor phosphorylation only at s351 but not at t362/s363, indicating that s351 can also undergo heterologous phosphorylation. using nop-gfp knock in mice, we detected nop receptors in brain, spinal cord and dorsal root ganglia (drg). we were also able to demonstrate a dose-dependent nop receptor phosphorylation at t362/s363 in mouse brain in vivo using western blot and mass spectrometry. in contrast, mcoppb and sch221510 failed to induce phosphorylation in vivo. together, these data provide new insights into the molecular regulation of the nop receptor in vitro and in vivo. several findings indicate that inflammatory diseases are initiated or maintained by an imbalance of receptor baised signaling; the latter referring to the ability of distinct ligands to endue individual receptors with qualitatively different g-protein-and/or b-arrestindependent signaling (1). chemokines and their receptors regulate a wide array of leukocyte functions, including chemotaxis, adhesion, and transendothelial migration, and thus play important roles in regulating inflammation (2) . in man, two cc chemokine receptors ccr2a and ccr2b are present that only differ in their carboxyl terminal portions; the latter known to interact with multi-protein complexes made up of heterotrimeric g proteins (pertussis toxin sensitive and -insensitive) and non-g-protein components, including b-arrestin (2) . interested in differential signaling of ccr2a and ccr2b we comparatively analyzed ligand-induced g-protein-regulated signaling pathways (e.g. activation of phospholipase c isoenzymes and rhogtpase-induced serum response factor [srf] activity) and b-arrestin-regulated pathways (e.g. internalization of receptors and phosphorylation of erk1/2) in the presence of ccl2, ccl8, ccl7, and ccl13. all these chemokines have been shown to interact with human ccr2 receptors. in addition, the structural requirements of the carboxyl terminal portions involved in determining specificity in g-protein-dependent signaling was addressed by using ccr2 receptor mutants. the comparative analysis revealed that differences in ligand-induced activation of g-protein-dependent (pertussis-toxin-sensitive versus pertussis-toxin-insensitive) and/or b-arrestin-dependent signaling exist. for example, activation of ccr2b receptors by ccl2 induced both rho-dependent srf activation and receptor internalization, while ccl8-stimulation resulted in srf but little if any receptor internalization. in contrast, ccr2a-expressing cells showed ccl2dependent srf-activation but any receptor/ligand internalization. analysis of the structural requirements of ccr2 receptors for coupling to g proteins revealed that arginine 313 within the putative 'eighth helix' of the carboxyl-terminal portions of ccr2a and ccr2b is not involved in ga i -mediated induction of erk1/2 and plays a minor role in ccr2b receptor internalization, but is specifically required for the ccr2a/ and ccr2b/ga q -mediated activation of srf. serotonin 5-ht 2c receptors (5-ht 2c r) are functional engaged with gq proteins and expressed in the central nervous system (cns). 5-ht 2c r significantly regulate emotion, feeding, reward or cognition and thus might serve as promising targets for drugs against psychiatric disorders or obesity. due to the technical difficulties in isolating cells from the cns and the hitherto lack of suitable cell lines that would endogenously express 5-ht 2c r, our knowledge about this receptor subtype is rather limited. recently established hypothalamic mhypoa2-10 cells show some characteristics of appetite-regulating hypothalamic neurons of the paraventricular nucleus, where 5-ht 2c r in vivo expression has been detected. thus, we tested mhypoa2-10 cells for putative 5-ht 2c r expression by performing single cell calcium imaging. we observed that serotonin and the specific 5-ht 2c r agonist, way161,503 induced robust calcium transients, which were strongly inhibited by two unrelated 5-ht 2c r-selective antagonist (sb206,553, rs102,221). serotonin and way161,503 also activated a camp response element-dependent reporter gene construct and induced significant phosphorylation of extracellularregulated kinases-1/2 in a sb206,553 and rs102,221 sensitive manner, providing further evidence for functional 5-ht 2c r expression in mhypoa-2/10 cells. intrinsic activity of way161,503 ranged between 0.3 and 0.5 compared to serotonin in all assays, defining way161,503 as a partial 5-ht 2c r agonist. in conclusion, we provide convincing data that hypothalamic mhypoa-2/10 cells endogenously express 5-ht 2c r and thus represent the first cell line to analyse 5-ht 2c r pharmacology, signaling and regulation in its natural environment. optical and electrophysiological methods allow detection and characterization of g i/o -protein coupled receptors j. straub 1 1 walther-straub-institut für pharmakologie und toxikologie, münchen, germany g-protein mediated signaling pathways are essential components of basic cellular functions. of note, g-protein coupled receptors (gpcrs) constitute one of the major drug targets in modern medicine. however, despite their clinical importance, fundamental properties of these receptors remain unknown. in particular, regulation of the major second messenger camp by g s -and g i/o -protein coupled receptors is of special interest. the classical biochemical method to detect receptor-mediated camp level changes uses pre-labeling with 3 h-adenine and calculation of the conversion rate to 3 h-camp. although, this multi-cellular method is highly sensitive and reproducible, it lacks time resolved and spacial assessment of camp formation in single living cells. to measure g s -protein induced increases of intracellular camp levels in single living cells in a time resolved manner, the fret-based camp-sensor epac is commonly used. however, it was unknown whether this sensor might be suitable to detect g i/o -protein mediated decreases of intracellular camp levels as well. in this study, we show that fret-based camp sensors can be deployed to reliably monitor g i/o -protein mediated camp level decreases. fret experiments with adrenergic α 2a or µ 1 opioid receptors in combination with different fret-based camp sensors showed a notable reduction of intracellular camp levels upon receptor activation which could be significantly reduced by selective receptor antagonists. of note, hek293 cells had to be pre-incubated with forskolin in submaximal concentration to increase basal camp levels. our findings suggest that fret based epac sensors are suitable to detect g i/o -protein activation similar to electrophysiological whole-cell measurements with g i/o -protein coupled receptors and trpc5 or heteromeric kir3.1/kir3.2 or kir3.1/kir3.4 channels coexpressing cells. hereby, agonist stimulations caused current increases with characteristic current-voltage relationships. altogether, our findings indicate that both optical and electrophysiological approaches allow time resolved detection and characterization of g i/o -protein coupled receptor activation in single living cells. histamine can exert positive inotropic and chronotropic effects in humans via histamine h 2 -receptors. we have generated and partially characterized transgenic mice (tg) which overexpress the human histamine h 2 -receptor specifically in cardiomyocytes via the α-myosin heavy chain promoter. in these mice, but not in wild type mice (wt), histamine increased heart rate and ejection fraction (ef) measured in vivo by echocardiography under isoflurane anesthesia. to investigate some aspects of the signaling pathway in these mice, we crossed the tg mice with pp2a mice leading to double transgenic mice (h 2 xpp2a = dt). pp2a mice (j biol chem 2004; 279:40827) overexpress the catalytic subunit of protein phosphatase 2a (pp2a) in cardiac myocytes and develop a cardiac hypertrophy. at an age of about 240 days we noted reduced ef in pp2a (33.1 ± 3.5 %, n=16) compared to wt (54.4 ± 4.5 %, n=10) and tg (59.8 ± 2.9 %, n=10). interestingly, in dt the ef (43.9 ± 4.9 %, n=11) was higher than in pp2a at similar heart rates. e´/a´ was elevated in dt compared to wt. relative heart weights were unchanged between these groups. in summary, we demonstrated that pp2a is involved in h 2 -receptor signaling and we tentatively conclude that the h 2 -receptor is able to ameliorate systolic but not diastolic cardiac function of pp2a mice. serotonin (5-ht) can exert positive inotropic and chronotropic effects in humans via 5-ht 4 -receptors. we have generated transgenic mice (tg) which overexpress the human 5-ht 4 -receptor selectively in cardiomyocytes via the α-myosin heavy chain promoter. in these mice, but not in wild type mice (wt), serotonin induced increases in heart rate and ejection fraction. we treated the mice with 30 µg lps (lipopolysaccharide, i.p.; a standard model of sepsis) per g body weight or isotonic sodium chloride solution (as solvent control). echocardiography with isoflurane anesthesia was performed before and 3, 7 and 24 hours after lps treatment. lps led to a time-dependent deterioration of cardiac function in both tg and wt. the deterioration included systolic function (left ventricular ejection fraction=ef) as well as diastolic function (height of a and e waves through the mitral valve: e/a). for instance, ef amounted to 58.8 ± 20.7% seven hours after lps in wt and to 61. [4] [5] [6] [7] [8] [9] [10] [11] [12] p<0 .05 vs pre-lps value). however, 24 hours after lps, diastolic function, measured as e/a, amounted to 1.86 ± 0.43 in p<0 .05 tg vs. wt). moreover, after 24 hours a less pronounced decline in body temperature (probably due to superficial abdominal hyperemia) occurred in tg versus wt. in contrast, while all flow parameters declined after 3 and 7 hours of lps, they were not different between wt and tg. for instance, maximum flow (in mm/s) through the ascending aorta declined from 1158.3 ± 212.2 to 782.5 ± 154.3 in wt and from 1208.4 ± 235.1 to 798.8 ± 170.1 in tg (tg vs. wt, p>0.05, n=10-12; after 7 hours). we tentatively conclude: 5-ht 4 -receptors overexpression protects the heart against sepsis, putatively by interference with the intracellular biochemical pathway of lps in cardiomyocytes. histamine can exert positive inotropic and chronotropic effects in humans via histamine h 2 -receptors. we have generated transgenic mice (tg) which overexpress the human h 2 -receptor specifically in cardiomyocytes via the α-myosin heavy chain promoter. in tg, but not in wild type mice (wt), histamine (ec 50 = 34 nm) or amthamine (ec 50 = 10 nm), a more selective and potent h 2 -receptor agonist, induced positive inotropic effects (pie) and positive chronotropic effects (pce) in isolated left and right atrial preparations, respectively. in order to investigate the signal transduction for the pie in atrium, contractile studies using partially depolarized preparations were performed. therefore, left atrial preparations were equilibrated in the organ bath to 44 mm potassium chloride. thereafter, histamine (100 µm) induced a pie (31.1 ± 10.4 % of control, n=7) in tg but not in wt preparations whereas isoprenaline (10 µm) increased force in both wt and tg. the pie of histamine in potassium treated tg atrium could be blocked by cimetidine. compound 48/80, a releasing agent of endogenous histamine, increased force of contraction in tg left atria to a higher extent than in wt. furthermore, we tested whether analgetics known to release histamine were inotropically active in tg. however, morphine (10 µm) was unable to affect contractility in wt or tg, whereas ketamine and fentanyl increased left atrial contractility in both tg and wt. in summary, we demonstrated an involvement of the l-type calcium channel current in the h 2 -receptor mediated pie in tg atria. we failed to release inotropically active histamine using classical analgetics, arguing that a direct effect also in the human heart is unlikely to occur. the initial step in the homologous desensitization of g-protein-coupled receptors is their phosphorylation by one of the g-protein-coupled receptor kinases (grks). we demonstrate here measurement of the interaction of grk2, a ubiquitously expressed grk, with the μ-opioid receptor (µor) by fret and its dependence on agonist efficacy. hek293t cells transfected with yfp-tagged µor and mturquoise-tagged grk2 as well as non-fluorescent gα i1 , gβ and gγ subunits showed a robust increase in fret upon superfusion with 10 µm [d-ala 2 , n-mephe 4 , gly-ol]-enkephalin (damgo) which was reversible upon agonist washout. the partial agonist morphine (30 µm) also caused a fret increase but the amplitude of the fret signal was reduced to approximately 15-20% of that of the corresponding damgo signal. grk2 binds g-protein βγ (gβγ) subunits, and therefore we aimed to find out how cotransfection of grk2 affected the interaction of gβγ with the µor. however, we could not measure any damgo-induced fret changes between yfp-tagged µor and mturquoise-tagged gβ in the presence of non-fluorescent gα i1 and gγ. this was unexpected because we had previously successfully determined interactions between gβγ and the α 2a -adrenergic receptor or the m 3 muscarinic acetylcholine receptor. this lack of fret was not due to an inability of the tagged gβ to interact with the µor as we could measure damgo-induced fret changes between yfp-tagged gα i1 and gβγ (gβ tagged with mturquoise) in the presence of non-fluorescent µor. moreover, when we attempted to establish an effect of grk2 on the interaction between the µor and gβγ, we could pick up fret between the µor and gβγ. comparison of the on-and off-kinetics of the µor-grk2 interaction with that of the µor-gβγ interaction in the presence of grk2 revealed similar time constants both for the on-and off-kinetics (grk2: k on 0.16 s ). this suggests that, in the absence of grk2, the orientation of the two fluorophores on the µor and gβγ may be unfavorable or the interaction may be too short-lived to produce an appreciable fret signal. in the presence of grk2, however, gβγ changes its position relative to the µor in a way that allows the interaction of the grk2-gβγ complex with the µor to be detected by fret. similarly, we measured fret between gβγ and the α 2a -adrenergic receptor or the m 3 muscarinic acetylcholine receptor in the absence and presence of grk2 and compared the kinetics with the kinetics of grk2 binding and unbinding to these receptors. in both cases, we found that grk2 and gβγ in the presence of grk2 associate and dissociate from these receptors with comparable kinetics. our results suggest that ligand efficacy for µors is already apparent on the level of receptor-grk interaction. institute of pharmacology, university medical center göttingen, ag lutz, göttingen, germany introduction: the monomeric gtpase rhoa is dysregulated in heart disease and in vivo models provide evidence of rhoa signaling being involved in the progression of cardiac fibrosis and hypertrophy. how rhoa is regulated within this context on a cellular level is not defined. objective: the goal of this study was to analyze rhoa activation in adult cardiomyocytes (amcm) from normal and diseased mouse hearts in response to g protein-coupled receptor activation. this project also aimed at providing new insight into the dependence of rhoa localization and activation on the signaling organizing caveolae in neonatal as well as adult cardiomyocytes and engineered heart muscles. methods: cardiomyocytes from sham-operated c57bl/6 mice, from mice subjected to transverse aortic constriction (tac) or from neonatal rats were either directly fixed after isolation or cultured in the presence or absence of methyl-b-cyclodextrin (mβcd). for analyses of rhoa activation cells were treated with endothelin-1 (et-1) for 90 sec. cells were prepared for immunofluorescence analysis or lyzed for immunoblotting. imaging was performed using confocal microscopy. effects of mβcd were further studied in 3d engineered heart muscles (ehm) made from total neonatal rat cardiac cells. the contractile function of ehm was assessed in the organ bath and cells were studied in sections by immunofluorescence analysis. results: in amcm from sham mice active rhoa mainly localizes at the sarcolemma and is augmented in response to et-1 treatment. in tac-amcm the basal level of active rhoa is increased and surprisingly et-1 had no further effect. immunoblot analysis demonstrated that in tac-amcm rhoa expression was per se higher and the major caveolae protein caveolin-3 was reduced. to test the influence of caveolae on rhoa activation and expression, we treated nrcm with mβcd and found the expression of rhoa as well as of rhoa target genes ccn1 and ccn2 to be moderately up-regulated after 24h. in addition, an intensified longitudinal alignment of sarcomeric actin fibers was detectable, which could also be seen in mβcd-treated ehm. however, mβcd had no effect on ehm twitch tension but increased the resting tension compared to control. we further treated amcm with mβcd and found rhoa expression to be increased and its activity less sensitive to et-1 treatment. finally, we could show that the perinuclear localization of cav3 and rhoa was strongly reduced after mβcd treatment. whereas g-protein coupled receptors (gpcrs) have been long believed to signal through cyclic amp only at cell surface, our group has previously shown that gpcrs not only signal at the cell surface but can also continue doing so once internalized together with their ligands, leading to persistent camp production (1). this phenomenon, which we originally described for the thyroid stimulating hormone receptor (tshr) in thyroid cells, has been observed also for other gpcrs (2) (3) (4) . however, the intracellular compartment responsible for such persistent signaling was insufficiently characterized. the aim of this study was to follow by live-cell imaging the internalization and trafficking of tshr, tsh and effector proteins in thyroid cells. mouse primary thyroid cells were transfected with fluorescent-protein tagged tshr, g-proteins, nanobody specific for active g-proteins and/or subcellular markers by electroporation, stimulated with fluorescently labeled tsh and visualized using highly inclined thin illumination (hilo) microscopy. our results suggest that tsh is internalized in complex with its receptor and they traffic retrogradely via the trans golgi network (tgn). while we could not find any evidence of internalized tsh/tshr complexes activating g-proteins in early endosomes, we show that tsh/tshr complexes meet the intracellular pool of gαs in the tgn and activate it, as visualized in real-time by a nanobody specific for active gαs. upon acute brefeldin a-induced golgi collapse, the retrograde trafficking of tsh/tshr via tgn is hindered. bulk tsh stimulations in primary mouse thyroid cells isolated from transgenic mice expressing the camp sensor, epac1-camps, also show a significantly reduced camp production in the presence of brefeldin-a. these data provide evidence that internalized tsh/tshr complexes meet and activate g-proteins at the tgn, which might serve as the main platform of persistent camp signaling after receptor internalization. objective: sphingosine 1-phosphate (s1p) is generated by sphingosine kinase (sk)-1 and -2 and acts mainly as an extracellular ligand at five specific g protein-coupled receptors, denoted s1p 1-5 . after activation, s1p receptors regulate important processes in the progression of renal diseases, such as mesangial cell migration methods and results: here we demonstrate that dexamethasone treatment lowered s1p 1 mrna and protein expression levels in rat mesangial cells measured by taqman® and western blot analyses. this effect was abolished in the presence of the glucocorticoid receptor antagonist ru-486. in addition, in vivo studies showed that dexamethasone downregulated s1p 1 expression in glomeruli isolated from c57bl/6 mice treated with dexamethasone (10 mg/kg body weight). functionally, we identified s1p 1 as a key player mediating s1p-induced mesangial cell migration. using boyden chamber assays, we could show that dexamethasone treatment significantly lowered s1p-induced migration of mesangial cells. this effect was again reversed in the presence of ru-486. conclusion: we suggest that dexamethasone inhibits s1p-induced mesangial cell migration via downregulation of s1p 1 . overall, these results demonstrate that dexamethasone has functional important effects on sphingolipid metabolism and action in renal mesangial cells (koch et al., biol. chem. 2015; 396: 803-12) . the g protein-coupled receptor mrgd is a receptor for angiotensin-(1-7) involving g alphas , camp, and phosphokinase a rationale: angiotensin (ang)-(1-7) has cardiovascular protective effects and is the opponent of the often detrimental ang ii within the renin-angiotensin system. although it is well-accepted that the g-protein coupled receptor mas is a receptor for the heptapeptide, the lack in knowing initial signalling molecules stimulated by ang-(1-7) prevented final verification of ligand/receptor interaction as well as the identification of further hypothesized receptors for the heptapeptide. objective: the study aimed to identify a second messenger stimulated by ang-(1-7) allowing confirmation as well as discovery of the heptapeptide's receptors. we identified camp as the second messenger for ang-(1-7). the heptapeptide elevates camp concentration in primary cells such as endothelial or mesangial cells. using camp as readout in receptor-transfected hek293 cells, we provided final pharmacological proof for mas to be a receptor for ang-(1-7). more important, we identified the g-protein coupled receptor mrgd as a second receptor for ang-(1-7). consequently, the heptapeptide failed to increase camp concentration in primary mesangial cells with genetic deficiency in both mas and mrgd. furthermore, we excluded the ang ii type 2 receptor at2 as a receptor for the heptapeptide, but discovered that the at2 blocker pd123119 can also block the mas and mrgd receptors. conclusions: our results lead to an expansion and partial revision of the reninangiotensin system, by identifying a second receptor for the protective ang-(1-7) but excluding the at2 receptor, and by enforcing the revisit of such publications which concluded at2 function by using pd123119 as a specific at2 blocker. members of the g protein-coupled receptor (gpcr) superfamily are integral membrane proteins that are activated by extracellular ligands and induce cell signaling via g proteins and other adaptor proteins. rhodopsin, the prototypical gpcr that mediates vision, is activated by photons that isomerize its covalent ligand. spectroscopic analyses of the cognate agonist retinal allow a detailed description of rhodopsin dynamics at submillisecond resolution. using rhodopsin as a model, it has been demonstrated that receptor activation, i.e. a switch from the fully inactive to the fully active state, occurs within 1 ms 1 . activation kinetics of other receptors have been studied mainly using fluorescence resonance energy transfer (fret) which allows kinetic studies with high resolution in living cells 2 . in contrast to rhodopsin, activation constants of several gpcrs using agonist superfusion have been determined to range between 30-80 ms 3 . however, it is unknown if all gpcrs with diffusible ligands are really activated on a longer timescale or if ligand diffusion to the binding site is rate limiting. in this study, we intend to overcome ligand diffusion by using photodestruction of caged ligands. we monitor activation-related conformational changes of homodimeric metabotropic glutamate receptor 1 (mglur1) sensors by fret after uncaging of an inert glutamate derivative. 4-methoxy-7-nitroindolinyl-l-glutamate (mni-glutamate) is a caged derivative of glutamate that does not activate mglur1s. upon pre-incubation of hek-tsa cells expressing both cfp-and yfp-tagged mglur1 protomers with mniglutamate, a short uv laser pulse releases active l-glutamate close to the receptor binding site. we demonstrate very rapid mglur1 activation kinetics and this allows us to study the process of signal transduction of this homodimeric gpcrs opioids are still the mainstay of modern pain treatment. most of the clinically established substances primarily exert their effects via the µ-opioid receptor (mor). however, many side effects such as tolerance, constipation and respiratory depression limit their therapeutic use. the efficacy of mor agonists in the treatment of chronic pain is unsatisfactory. in general analgesic effects can be mediated by all four members of the opioid receptor family. the nociception receptor (nop) is the latest member of the opioid receptor family. there is a rapidly growing interest for the development of novel nop and combined mor/nop agonists. the aim of this developement is novel therapeutic agents with improved analgesic characteristics and less classical mor-mediated side effects. here we used buprenorphine as a clinically established opioid which exerts its effect on multiple opioid receptor subtypes. recently, nalfurafine, a potent kappa-opioid receptor (kor) agonist was granted by japanese authorities for the treatment of uremic pruritus. even though kor agonists are known to mediate dysphoria and hallucinations this has not been reported for nalfurafine. rudolf-virchow-zentrum für experimentelle biomedizin, würzburg, germany g protein-coupled receptors (gpcrs) belong to a superfamily of cell surface signaling proteins that mediate many physiological responses to hormones and neurotransmitters. they represent the prime targets for therapeutic drugs in healthcare. however, due to the limited knowledge about the pharmacology of the majority of gpcrs, only few of them are employed as therapeutic target. in our lab, the activation kinetics of the α 2aadrenergic receptor, among others receptors, has been extensively studied in single cell assays (1) (2) . the activation kinetics of the labeled α 2a -adrenergic were monitored by förster resonance energy transfer (fret). the goal of our study is to design a sensor to monitor receptor activation kinetics in high throughput screening assays. for the proof of concept, we used the α 2a -adrenergic receptor as a prototype. to optimize the fret efficiency we exchanged the previous acceptor (yfp) with the halotag technology (3) . additionally, we used halotag in combination with the nanoluc (4) to explore the possibility of using bret as a high-throughput approach to monitor receptor activation kinetics. the fret-based sensor α 2a -halo/cfp showed an increase in fret upon application of the full endogenous agonist norepinephrine with an ec50 value in accordance with the previously published data. this suggests the functionality of the fret-based α 2a -halo/cfp sensor. similar results were obtained with the α 2a -adrenergic receptor bret-based sensor. in contrast to the full agonist norepinephrine, the inverse agonist, yohimbine, decreased the ratio in both, fret as well as bret-based α 2aadrenergic receptor sensors. this suggests the sensitivity of the methods to discriminate among agonist (increased ratio) and antagonist (decreased ratio) induced receptor kinetics. our results show the feasibility of using halotag to monitor receptor activation via fret in a single cells format and halotag, in combination with nanoluc, can be used to monitor receptor activation in high-throughput format. , c. hoffmann the homologous visual arrestins) that has recently been solved by x-ray crystallography. here we investigated both the interaction with gpcrs and β-arrestin conformational changes in real time and in living cells with a series of fret-based β-arrestin2 biosensors. upon stimulation, β2-adrenergic receptors bound β-arrestin2 with a time constant τ = 1.3 ± 0.17 s, indicating that β-arrestin2 binding rapidly terminates their gprotein signaling. we observed a subsequent receptor-mediated conformational change in β-arrestin2 with a τ = 2.2 ± 0.22 s. stimulation of β2-adrenergic vs. m2 muscarinic or ffa4 receptors resulted in different patterns of conformational changes in the various β-arrestin2 sensors and also in downstream kinase signaling, revealing receptor-specificity in β-arrestin2 activation. upon agonist removal, first the interaction (delay = 1.9 ± 0.51 s) and only then the active state of β-arrestin2 (delay = 4.2 ± 0.85 s) were reversed. accordingly, β-arrestin localization at the cell membrane lasted much longer than the direct interaction with β2-adrenergic receptors. our data indicate a rapid, receptorspecific, two step binding and activation process between gpcrs and β-arrestins; they further suggest that β-arrestins remain active following dissociation from receptors, allowing them to remain at the cell surface and presumably signal independently. thus, gpcrs trigger a rapid, receptor-specific activation/deactivation cycle of β-arrestins, which permits their active signaling. patghogenic clostridium difficile produce two large glucosyltransferases, tcda and tcdb, which are the main pathogenicity factors. the cytotoxin tcdb is about 1,000 fold more potent than tcda. tcda and tcdb are a/b structure toxins exhibiting an enzymatically active (a) domain and a binding/translocation domain (b) to deliver the active glucosyltransferase domain into the cytosol of host cells. beside its glucosyltransferase activity by which the substrate proteins mainly of the family of rho gtpases are inhibited, tcdb has additional cytotoxic effects that are independent of rho glucosylation. to investigate the mechanism by which tcdb induces early cell death, we applied chimeras of tcdb from different toxinotypes where different glucosyltransferase domains were combined with different translocation domains. to this end we cloned tcdb from strain vpi10463 (historical strain), strain 1479 (serotype f, variant tcdbf), strain r20291 (hypervirulent strain, ribotype o27), and strain r9385 (hypervirulent strain with tcdbf characteristics). we were able to investigate the impact of the glucosyltransferase domains with different substrate specificity when translocated into the host cell by identical translocation domains. furthermore, we tested different translocation domains to deliver the same glucosyltransferase domain into host cells. we found that the glucosyltransferase domain of tcdbf (strain 1470) is less cytotoxic with respect to early cell death mediated by reactive oxygen species than that from reference strain vpi10463. in addition, the translocation domain also showed significant impact on cytotoxicity, probably by faster intracellular delivery of the gtd. by using glucosyltransferase deficient mutants where the highly conserved dxd-motif was changed to nxn, we were able to show that glucosylation of rho gtpases counteracts the cytotoxic effect, since the mutants were more cytotoxic than wildtype toxins. in conclusion, the cytotoxicity of tcdb mainly depends on the translocation efficiency into the host cell and on the kinetic of glucosylation of their substrate gtpases. thus, sensitivity of target cells towards cytotoxic effect also depends on receptor abundancy and intracellular status of rho gtpases, whereas the cytopathic effect, i.e. cell rounding, is predominatly determined by the substrate specificity. introduction: p63rhogef activates the g protein-coupled receptor (gpcr)-mediated induction of rhoa in different cells. however, its role in cardiac fibroblasts (cf) is not defined yet. thus we studied its localization and function in cf in 2d and 3d culture experiments. methods: neonatal rat cardiac fibroblasts (nrcf) and adult ventricular fibroblasts (amcf) from wild type mice and p63rhogef-knockout mice were adenovirally transduced for 48 to 72 h with recombinant adenoviruses or directly used. for 2d studies the cells were treated with angiotensin ii (ang ii). the location of the involved signaling components, rhoa activation and down-stream effects were studied by confocal microscopy and biochemical analyses. in addition, cf were used to prepare cf-containing engineered connective (ect) or muscle (ehm) tissues. results: we could show that p63rhogef locates at the plasma membrane, adjacent to the golgi apparatus and at the base of primary cilia. in accordance, p63rhogef regulates in response to ang ii the expression and secretion of the connective tissue growth factor (ctgf) in nrcf involving the serum response factor. in ect, p63rhogef increases the stiffness of these tissues and in ehm containing cf expressing gain-and-loss-of-function p63rhogef variants it influences the contractility. interestingly, the increase in ect stiffness was independent of p63rhogef's regulatory function of ctgf, as overexpression of ctgf in cf had no impact on ect properties arguing for a more general role of p63rhogef in auto-and paracrine signaling. moreover, our data on amcf with a genetic deletion of p63rhogef implies that p63rhogef is not only a transducer of gpcr-dependent rhoa activation as its loss led to an increase in rhoa expression accompanied by an increase in rhoa-dependent gene expression suggesting a role of p63rhogef in the feedback regulation of this signaling cascade. conclusion: in summary, our data show that p63rhogef regulates auto-and paracrine signaling in cardiac fibroblasts. the atrophic rhinitis is characterized by a drastic destruction of nasal turbinate bones in different animals. it leads to shortening and twisting of the snout and a growth retardation of young pigs. this bone degradation is induced by pasteuralla multocida toxin (pmt), a toxin produced by p. multocida serogroups a and d. this destructive effect indicates an interaction of pmt with bone cells like osteoclasts and osteoblasts. we demonstrated that pmt stimulates the differentiation of osteoclasts and inhibits the differentiation of osteoblasts in a gq-dependent mechanism. the underlying molecular mechanism of the toxin is the deamidation of an essential glutamine residue in the αsubunits of heterotrimeric g proteins, which results in the constitutive activation of the g protein. until now only the function and the pmt-dependent effects on osteoblasts and osteoclasts were studied in detail, but there is also a third important cell type in bone, the osteocytes. osteocytes are discussed as the regulator of the bone turn-over by interacting with osteoclasts and osteoblasts e.g. via secretion of several osteoclastogenic and osteoblastogenic cytokines. therefore, we studied the effects of pmt on the function of osteocytes in more detail. we utilized an osteocyte like cell line and primary osteocytes isolated from tibiae and femurs from mice. the susceptibility of primary osteocytes and the osteocyte like cell line towards pmt was demonstrated by detection of toxin-induced deamidation of g proteins. we also observed a pmt-induced secretion of different cytokines, like rankl, il-6 and tnf-α, which are known to induce osteoclastogenesis or inhibit osteoblastogenesis. furthermore, we studied the underlying signal transduction pathways and other pmtinduced effects on osteocytes, like morphological changes. in summary, we show that pmt acts on osteocytes by stimulating heterotrimeric g proteins. this might have impact on overall bone metabolism due to modulation of osteoblast and osteoclast activity. pasteurella multocida is an opportunistic pathogen often residing in the nasal pharyngeal space of animals. one virulence factor of p. multocida serogroups a and d is the protein toxin pmt (p. multocida toxin), which is the causative agent of atrophic rhinitis characterized by degradation of nasal turbinate bones in pigs and other animals. on the molecular level, pmt activates distinct members (g q/11 , g 12/13 and g i ,) of heterotrimeric g proteins leading to a modulation of bone metabolism: the toxin stimulates osteoclastogenesis but blocks osteoblastogenesis which results in bone loss. this mechanism of action of pmt might be exploited to counteract the human disease fibrodysplasia ossificans progressiva (fop), a rare and highly disabling disorder of extensive heterotopic bone growth. the underlying cause of fop is a point mutation in the activation domain of acvr1 (r206h), a bmp (bone morphogenic protein) type 1 receptor. this mutation leads to an inflated bmp-signaling and heterotopic osteoblastogenesis. here, we report that c2c12 cells, a mouse myoblast cell line often used as a fop model, are susceptible to pmt intoxication. pmt induces deamidation of g proteins in these cells. furthermore, pmt very efficiently inhibits bmp4-induced osteoblast differentiation in c2c12 cells. this has been shown by measuring alkaline phosphatase expression which is an early marker of osteoblast differentiation. additionally, the impact of pmt on acvr1 r206h induced osteoblastogenesis will be investigated and the involved cellular signaling pathways will be characterized in detail. the data indicate that activation of heterotrimeric g-proteins might be a rationale for pharmacological therapy of fop. p63rhogef is an activator of the monomeric gtpase rhoa and was shown to be expressed in the heart. in cardiac fibroblasts and smooth muscle cells, p63rhogef regulates rhoa in response to angiotensin ii and controls the actin cytoskeleton as well as protein expression and secretion. its role in cardiomyocytes, however, has not been elucidated so far. cardiomyocytes were isolated from neonatal rat hearts (nrcm), wild type mouse hearts (wt-amcm) and homozygous (ko-amcm) p63rhogef knockout mouse hearts. the cells were either directly fixed or adenovirally transduced for 48 h in culture. for activation of the g q/11 -signaling the cells were treated with endothelin-1 (et-1), angiotensin ii (ang ii) or phenylephrine (pe) for 90 s. rhoa activation was assessed by affinity binding or with a specific active-rhoa antibody. other proteins were detected by immunoblot or immunofluorescence analysis with subsequent confocal imaging. in nrcm p63rhogef is involved in the regulation of the et-1-induced rhoa activity and thus increases the expression and secretion of the rhoa target gene ctgf. in accordance, p63rhogef was found to be localized at the sarcolemma as well as in intracellular membrane compartments. the strongest co-localization was detected with the kdel-receptor 3 (kdelr3) which resides in the endoplasmatic reticulum membrane. next, we analyzed rhoa activation in wt and ko-amcm and could show that a loss of p63rhogef led to an increase in basal rhoa activity and an uncoupling from the gpcrs. interestingly, in the ko-amcm caveolin-3 the major component of caveolae, in which several gpcrs are clustered, was reduced in its expression and a shift in localization from transverse to longitudinal membrane tubules was found, arguing for a role of p63rhogef in intracellular protein transport. in accordance, golgi apparatus particles, which were demonstrated to play role in caveolae formation, were reduced in size in ko-amcm. to further address the role of p63rhogef in the transport of membrane proteins, we overexpressed p63rhogef in wt-amcm and could show that this led to an increase in the expression of the kdelr3 and its co-localization with p63rhogef in the perinuclear region and at the sarcolemma. no sarcolemmal localization of kdelr3 was found in control-transduced cells. further, p63rhogef was localized adjacent to golgi apparatus particles which were similar reduced in size as detected in the ko-amcm. finally, we expressed the dominant negative construct p63dn and detected similar changes with respect to kdelr3 localization and golgi structure suggesting that this regulatory function of p63rhogef is not dependent on its activity. conclusion: p63rhogef mediates the activation of rhoa from gpcrs coupled to g q/11 proteins. moreover, it has a function in intracellular transport and distribution of membrane proteins independent of its activity. universität bonn, pharmacology and toxicology section, bonn, germany g protein signaling is a means allowing cells to quickly respond and adapt to environmental changes. four major g protein classes (gs, gi/o, gq/11, g12/13) exist in mammals and these must suffice to convey signals from about 800 g protein-coupled receptors to the cell interior. as such, g proteins receive, interpret, and finally route the gpcr signals to diverse sets of downstream target proteins and thereby permit cells to respond to their ever changing environment. 1 understanding contribution of individual g protein classes or even isoforms to complex signaling networks in living cells requires capacity to activate or inactivate proteins with great precision and selectivity. one approach towards inactivation of g protein function is via chemical inhibition. however, "true specificity" of chemical inhibitors for their associated targets may often be debated. in this study we posit that fr900359, a cyclic depsipeptide isolated from the leaves of ardisia crenata, may clearly be designated as "truly specific" for inhibition of gq signaling. using a broad set of complementary methods based on label-free holistic cell sensing, classical endpoint assays, and bioluminescence resonance energy transferbased g protein biosensors we assign exceptional selectivity to fr for inhibition of gq/11/14 over all other mammalian isoforms ("on-target effects"). in holistic label-free recordings using hek293 cells that lack functional gq/11 alleles by crispr-cas9 genome editing, bona-fide gq stimuli were undetectable. however, reintroduction of gq into the knockout background was required and sufficient to fully restore both, agonist responses and their inhibition by fr. moreover, fr was completely ineffective in cells lacking gq/11 using phenotypic assays that examine basic cellular functions such as cell growth, viability, morphology and expression of housekeeping genes ("off-target effects") 2 . from these results we conclude that fr is of outstanding value as molecular probe to unravel contribution of gq signaling in complex biological processes in vitro, ex vivo and in vivo. just as pertussis toxin, applied world-wide by numerous laboratories to diagnose signaling of gi/o proteins, we anticipate fr to stand out at least equally for investigations into the biological relevance of gq. binary actin adp-ribosylating toxins like c. perfringens iota toxin and c. difficile transferase cdt cause depolymerisation of the cortical actin cytoskeleton, induce the formation of microtubule-based cell membrane protrusions and redirect rab-dependent intracellular traffic (schwan et al. 2009 ). here, we employed the model of toxin-induced protrusions to study the formation of cilia. we found that toxin-induced microtubule-based protrusion formation at the cell membrane depends on recruitment of septins, which are highly conserved, small gtpbinding proteins. similar to toxin-caused protrusions, septins are also recruited to the site of ciliogenesis. inhibition of septins by shrna-based knockdown inhibits ciliogenesis as well toxin-induced protrusion formation. septins are suggested to be involved in exocytotic processes, which are important for ciliogenesis and also for toxin-induced protrusion formation. accordingly, translocation of septins is accompanied by a recruitment of rab proteins and proteins of the exocytotic machinery. the data indicate that septins function as a scaffold at the base of cellular processes like cilia and toxin-induced protrusions, organizing the cross-talk between the actin cytoskeleton and microtubules to regulate the vesicle traffic-and exocytotic machinery. hypervirulent clostridium difficile strains are associated with increased morbidity and mortality. these strains produce the actin-adp-ribosylating clostridium difficile toxin cdt. cdt depolymerizes the actin cytoskeleton, causes formation of microtubule-based protrusions and increases pathogen adherence. septins are essential for cdt-induced protrusion formation. sept2, 6, and 7 accumulate at predetermined protrusion sites and form collar-like structures at the base of protrusions. the septins are a prerequisite for protrusion formation. the inhibitor forchlorfenuron or knock-down of septins inhibit protrusion formation. septins colocalize with active cdc42 and its effector borg which act as up-stream regulators of septin polymerization. microtubules interact with septin structures. precipitation and surface plasmon resonance studies revealed high-affinity binding of septins to microtubule plus end tracking protein eb1 thereby guiding incoming microtubules. the data indicate that cdt hijacks conserved regulatory principles involved in microtubule-membrane interaction, depending on septins, cdc42, borgs and restructuring of the actin cytoskeleton. the zebrafish danio rerio has become an important vertebrate model organism for a wide range of scientific questions [1] . current studies are mainly focused on development, genetics and disease for which the zebrafish is particularly well suited due to its small size, rapid development, short generation time, optical transparency of embryos and larvae as well as conservation in functional domains [1] . hitherto, nothing is known about the composition and endogenous level of different cnmps in various developmental stages and organs of danio rerio. therefore, we used the zebrafish in our study as a vertebrate model to characterize systematically the temporal-and organspecific occurrence(s) of all cnmps including cump in vivo. cyclic nucleotides were quantified by high performance liquid chromatography quadrupole tandem mass spectrometry. we observed specific cnmp patterns in developmental stages and different organs from adult zebrafish, which is in support of the hypothesis of a distinct cnmp signaling code [2] . camp, cgmp and cump were present in tissue samples of both developmental stages (embryos at 24 hours post fertilization, larvae at 5 days post fertilization) and within all harvested organs. remarkably, these three cnmps were the only ones detected in the brain. camp concentration of entrails as well as camp and cgmp concentrations in the brain were similar to those previously described in mouse tissues [3] . ccmp was detected throughout development and was present in all organs except the brain. the identity of ccmp and cump in the zebrafish was confirmed by high performance liquid chromatography quadrupole time-of-flight mass spectrometry (hplc-ms/tof). thus, we unequivocally show for the first time that cump occurs in vertebrates. furthermore, we detected cimp in several developmental stages of the zebrafish, and observed the highest concentrations in testes and heart, but we were unable to unequivocally identify cimp via hplc-ms/tof. in the zebrafish, sac is evolutionarily not conserved and absent, since a search in the ncbi gene data base revealed no entry for sac (also referred to as ac10). therefore, sac can be excluded as cump-and ccmp generator in this system and sgc remains as the only bona fide cump-and ccmp generator in the zebrafish. to test this hypothesis, the effects of no donors, sgc stimulators and sgc activators on cump levels in zebrafish should be examined in future studies. recently, induction of apoptosis in mouse lymphoma cells by ccmp-am has been described [4] . thus, it would be interesting to examine the effect of ccmp-am on zebrafish embryos in future studies as well. [1] seifert, r.: ccmp and cump: emerging second messengers, trends in biochemical sciences 40, 8-15 (2015) . ccmp, 3',5'-cump and 3',5'-cimp were ineffective. to further characterize the action of 3',5'-cgmp on hut-78 cells, we determined apoptosis (propidium iodide/annexin v staining) and proliferation (carboxyfluorescein succinimidylester staining). 3',5'-cgmp significantly increased apoptosis (ec 50 = 75 µm) and inhibited proliferation (ec 50 = 63 µm) of okt3-activated hut-78 cells. interestingly, also 2',3'-cgmp exhibited comparable effects on apoptosis and proliferation with ec 50 values of 92 µm and 75 µm, respectively, while 2',3'-camp, 2',3'-ccmp and 2',3'-cump were ineffective. this indicates that the pro-apoptotic and antiproliferative action of cgmp does not depend on the position of the phosphodiester bond. we also tested 3',5'-cgmp degradation products under the same experimental conditions and found that both 5'-gmp and guanosine increased apoptosis and inhibited proliferation with ec 50 -values between 50 and 100 µm. by contrast, adenosine did not influence cell growth and viability, suggesting that adenosine receptors are not involved in the observed effects. our results suggest that the guanosine moiety is responsible for the pro-apoptotic and antiproliferative effects of 3',5'-cgmp, 2',3'-cgmp, 5'-gmp. it has been reported earlier that guanosine is toxic to jurkat cells, another t cell lymphoma cell line [1]. 3',5'-cgmp may be hydrolyzed by an ekto-phosphodiesterase on the cell surface of hut-78 cells (e.g. enpp1), yielding 5'-gmp, which could be further degraded to guanosine by the 5'-ekto-nukleotidase cd73. a similar pathway may lead from 2',3'-cgmp to guanosine. a previous analysis of phosphodiesterases (pdes) revealed that the dual-specific pde isoforms 3a and 3b as well as the cgmp-selective pde9a also degrade the emerging second messenger cump [1, 2] . we analyzed the enzyme kinetics of pde3b-mediated cump-hydrolysis using recombinant gst-tagged pde3b and a highly sensitive and specific hplc-ms/ms method. our data show that pde3b is a low-affinity enzyme for cump with a cump k m -value of >100 µm. the pde3-selective competitive inhibitor milrinone inhibited pde3b-mediated cump degradation, suggesting that cump binds to the catalytic center. pde3b is highly expressed in adipose tissue [3, 4] . thus, we differentiated murine 3t3-l1-mbx-fibroblasts into adipocytes and analyzed differentiation-dependent alterations of pde3b expression and basal cnmp-concentrations. in both differentiated and undifferentiated 3t3-l1 cells cump and ccmp were detected in addition to the established second messengers camp and cgmp. differentiation to adipocytes reduced camp and cgmp by 66 % and 60 %, respectively, while ccmp was reduced by 78 % and cump even by 85 %. these findings suggest that cump plays a distinct role in adipocyte differentiation. the cump-hydrolyzing pde3b was upregulated ~1000-fold on mrna level after adipocyte differentiation, which may contribute to the observed reduction of basal cump concentrations. we currently investigate the potential biological role of cump in differentiation and lipolysis experiments, analyzing the effects of the membrane-permeant cumpacetoxymethyl ester cump-am. in future experiments, we will also analyze the enzyme kinetics of pde9a-mediated cump hydrolysis. pde9a is the first example of a cgmp-"specific" cump-hydrolyzing pde. background: cgmp and camp are cyclic nucleotide messengers relevant to many physiological and pathophysiological conditions. live-cell imaging with fret-based biosensors is a powerful method to study the spatiotemporal dynamics of cgmp and camp under close-to-native conditions. however, with the existing biosensors it is difficult to resolve potential membrane-associated cgmp microdomains and to monitor cgmp and camp signals in parallel in the same cell. we have generated novel versions of the "green" cfp/yfp-based cytosolic cgmp biosensor, cgi500. they comprise a "green" membrane-targeted version (mcgi500) and a "red" variant (red cgi500) that contains the fluorophores tsapphire and dimer2. methods: the sensors were expressed and characterised in primary vascular smooth muscle cells (vsmcs). intracellular cgmp was elevated in intact vsmcs by application of a nitric oxide donor or natriuretic peptides, and the sensor's sensitivity to each stimulation and its signal-to-noise ratio were determined. to test each sensor's sensitivity and specificity for cgmp versus camp, sensor-expressing cells were permeabilised with β-escin and exposed to defined concentrations of cyclic nucleotides. results: the original cgi500 sensor showed a good signal-to-noise ratio, an ec 50 value of ≈1.1 µm for cgmp, and a high selectivity for cgmp over camp (>100-fold). flincg3, a non-fret-based cgmp sensor, showed similar properties as cgi500. the new membrane-targeted mcgi500 and the new red cgi500 displayed ec 50 values of ≈0.4 µm cgmp and a high selectivity for cgmp over camp (>300-fold). in vsmcs, the red cgi500 showed a better signal-to-noise ratio than the previously described "red" cgmp sensor, red cges-de5. the "green" fret-based camp sensor, epac1-camps, showed a signal-to-noise ratio comparable to that of cgi500, an ec 50 value of ≈3 µm for camp and a selectivity for camp over cgmp of ≈6-fold. finally, imaging of cells expressing both the epac1-camps and the red cgi500 demonstrated the feasibility of combined visualisation of camp and cgmp signals in the same cell. the new cgmp biosensors should be useful for a broad spectrum of applications requiring real-time monitoring of cgmp signals. for example, mcgi500 would be useful to investigate membrane-associated cgmp compartments and red cgi500 to study the crosstalk between cgmp and camp signalling in living cells and tissues. the cgmp-system is a major regulator of blood pressure. cgmp-dependent protein kinases (cgks), located in the smooth muscle layer of vessels, enable cells to dilate and therefore cause a decrease in blood pressure (bp). to the contrary, the reninangiotensin-aldosteron-system (raas) acts as opponent and causes an increase in bp; furthermore, it influences fluid-electrolyte balance. renin, which is secreted from reninproducing cells located in the juxtaglomerular apparatus (jga), is the key regulator enzyme in this system. pharmacological inhibition of the raas, e.g. via ace-inhibitors or at1-receptor-antagonists, is a powerful tool to treat hypertension, but chronically challenges this endocrine system, resulting in an enhancement of renin expression. this is caused by an increased number of renin-expressing cells (the so-called reninrecruitment), which derive from a reversible metaplastic retransformation of extraglomerular and smooth muscle cells of afferent arterioles. next to regulation of renin-function via camp/pka, it has been shown that enos-derived no supports this recruitment via activation of sgc and subsequent generation of cgmp [1] . whether this causes an activation of cgks is not known. these enzymes exist in 3 different isoforms, cgkiα, cgkiβ und cgkii. in contrast to the β-isoform, cgkiα (as well as cgkii) is highly expressed in the jga [2] , [3] . therefore, we analyzed, whether cgkiα also plays a role regarding renin synthesis, secretion or recruitment. to characterize the function of cgkiα in jga-cells, we generated renin-cell specific cgkiα-knockout mice (ren cre-cgki fl/fl) and stimulated renin recruitment via administration of a low salt diet (0.02% na + ) and enalapril (10mg/kg/d) for 3 weeks. we analyzed blood pressure, mrna and renal protein content of renin and cgkiα, plasma renin activity and renin recruitment. furthermore, we activated the cgmp-system in these mice using bay41-8543, a sgcstimulator, and re-analyzed the above-mentioned parameters. our results indicate that cgkiα could be an additional system supporting renin recruitment but is not a crucial pre-requisite. in contrast, the basal renin concentration and activity appears to be downregulated in ren cre-cgki fl/fl-mice, thus, cgki could be an important regulator of renin synthesis. universität regensburg, pharmakologie und toxikologie, regensburg, germany jaw1/lrmp (lymphoid-restricted membrane protein) is a type 2 membrane protein, localised to the cytoplasmic face of the endoplasmic reticulum. it encodes a 539 amino acid protein with a highly conserved coiled-coil domain in the middle third of the protein and a cooh-terminal transmembrane domain [1] , [2] . jaw1 and irag have a limited homology throughout the length of the protein. the coiled-coil domain and the putative transmembrane anchor at the c-terminus of jaw1 and irag share the highest homology [3] , [4] . the coiled coil domains of irag and jaw1 are important for the interaction with ip 3 rs. as already known, irag forms a trimeric complex with cgkiβ and ip 3 r1 and gets phosphorylated by cgkiβ [5] . hence we examined if jaw1 is a new target protein of cgkiβ. the recognition site, where a substrate can be phosphorylated by cgki, is composed of the following amino acids: (k/r)(k/r)x(s/t). in the amino acid sequence of jaw1 possible phosphorylation sites can be found. our in vitro studies with jaw1 and cgki showed that jaw1 gets phosphorylated in a cgmp-dependent manner by cgkiβ. in contrast, jaw1 was not phosphorylated by cgkiα. furthermore, no stable interaction between jaw1 and cgkiβ was detected. to examine the importance of jaw1 in vivo, we generated a conditional knockout mouse. mating with a cmv cre mouse, resulted in an ubiquitous deletion of jaw1. mrna analysis and western blot analysis approved the deletion. the expression pattern revealed high expression in the thymus and weaker expression in the lung, spleen, colon, pancreas and the tongue. as already published by shindo et al., jaw1 was found in sweet, bitter, and umami taste receptor-expressing cells of mouse circumvallate, foliate, and fungiform papillae. we confirmed these results by x-gal staining and mrna analysis. therefore, we decided to analyse if jaw1 influences taste perception. two bottle preference tests did not result in significant differences between wildtype and knockout mice, indicating that taste perception is not altered by jaw1. hence, the function of jaw1 in taste receptor expressing cells has to be further examined in future studies. the cyclic purine nucleotides adenosine 3',5'-cyclic monophosphate (camp) and guanosine 3',5' cyclic monophosphate (cgmp) are well-characterized second messengers. both are generated by nucleotidyl cyclases and degraded by phosphodiesterases. several binding partners of camp and cgmp were already identified and functionally analyzed, e.g. camp-dependent protein kinase (pka) and cgmp-dependent protein kinase (pkg) as well as exchange protein activated by camp 1 and 2, hyperpolarization-activated cyclic nucleotide gated channels and phosphodiesterases. recent data indicate that the cyclic pyrimidine nucleotides cytosine 3',5'-cyclic monophosphate (ccmp) and uridine 3',5'-cyclic monophosphate (cump) also fulfill the criteria of second messengers [1, 2] . the interaction of ccmp with the regulatory subunits of pka (pkariα and pkariiα) has already been shown by using ccmpagarose [3] . additional ccmp-and cump-binding proteins such as calnexin (chaperone), myomegalin (phosphodiesterase-interacting protein) and akap9 (a-kinase anchoring protein) were identified by mass spectrometry analysis. to verify the interaction of ccmp and cump with these potential target proteins, ccmp and cump linked to biotin were used as another approach. the biotin-constructs exhibit lower steric interference than the ccmp-and cump-agarose matrices, which were previously used to confirm the binding of pkariα to ccmp and cump [4] . flag-tagged calnexin, flag-tagged myomegalin and myc-tagged yotiao (smallest splice-variant of akap9) were examined as potential ccmp and cump target proteins. hek293 cells were transiently transfected with the cdna of the respective proteins. the lysates of the protein-overexpressing cells were then incubated with ccmp-and cumpbiotin matrices and bound proteins were purified using strep-tactin® beads (iba). afterwards, the interaction of ccmp and cump with the potential binding partners was analyzed by western-blotting. a pkariα antibody was used as a positive control. analogous experiments were also performed using ccmp-and cump-agaroses. once the interaction between the cyclic pyrimidine nucleotides and the potential binding partners has been confirmed, deletion mutants will be cloned to localize the ccmp-and cump-binding area of the target proteins in further studies. axonal branching is essential for the correct formation of neuronal circuits and enables the simultaneous transmission of information throughout the body. in mice, the bifurcation of axons of sensory neurons at the dorsal root entry zone of the developing spinal cord depends on a cgmp signaling cascade that includes c-type natriuretic peptide (cnp), natriuretic peptide receptor 2 (npr2, also termed gc-b), and cgmpdependent protein kinase iα. in this study it was investigated, if a disturbance of cgmp signaling induced by manipulation of cgmp breakdown or cnp scavenging affects axon bifurcation of murine embryonic dorsal root ganglion (drg) neurons. rt-pcr screens, in situ hybridization, and fret-based cgmp imaging in living neurons revealed phosphodiesterase 2a (pde2a) as the major enzyme for degradation of cnp-induced cgmp in embryonic drg neurons. interestingly, cgmp measurements and dii labeling of pde2a knockout embryos indicated that a strongly elevated concentration of cgmp does not impair sensory axon bifurcation of drg neurons in vivo. the natriuretic peptide receptor 3 (npr3) was found to be expressed in the roof and floor plate of the spinal cord as well as in the dorsal roots of e12.5 embryos. because npr3 binds natriuretic peptides, but does not generate cgmp, it is thought to act as a natriuretic peptide clearance receptor. by scavenging cnp, npr3 could lower the activity of the cnp-npr2-cgmp signaling cascade in drg neurons. in the absence of npr3, the majority of sensory axons showed normal bifurcation, but »13 % of the axons turned only in rostral or caudal direction. this study shows (1) that pde2a is important for the degradation of cgmp in embryonic drg neurons, (2) that the bifurcation of sensory axons in the spinal cord can tolerate high levels of intracellular cgmp in the absence of pde2a, and (3) in the central nervous system, no-dependent cgmp signalling is associated with many different developmental processes and brain functions, and plays an important role in memory consolidation and cognition. to analyse cgmp signals in primary cells, a knock-in mouse was generated which stably and ubiquitously expresses a fret-based cgmp indicator (cgi500). cultured cortical and hippocampal neurons were found to respond to exogenously applied no (gsno). in these cell types, endogenous no is mainly generated by the neuronal no synthase (nnos) isoform which requires a rise in intracellular calcium for activation of the no/cgmp-signalling cascade. here, we show that ampa-type ionotropic glutamate receptors were capable to induce cgmp response in cultured cortical and hippocampal neurons. surprisingly, amparinduced cgmp signals were independent of nmdar activation, as inhibition of nmdars with the nmdar antagonist d-apv (d-2-amino-5-phosphonopentanoic acid) did not block ampar-induced cgmp response. however, cgmp accumulation depends on no synthase activation as the nos inhibitor l-nna (ng-nitro-l-arginine) completely abolished cgmp accumulation. whether ampar-induced nos activation depends on calcium influx via calcium permeable ampars, vgccs (voltage gated calcium channels) or calcium release from intracellular stores will further be investigated in detail. cyclic adenosine monophosphate (camp) is an important and ubiquitous cellular second messenger. a dogma in signaling is that camp is distributed homogenously in the cell and that its concentration changes equally upon stimulation. in contrast, a large body of evidence suggests the existence of concentration gradients (so-called microdomains) of camp. in this regard, phosphodiesterases (pdes), the only enzymes which can degrade camp, have been suspected to be responsible for maintaining those gradients. however, how pdes establish camp gradients is entirely unknown. here, we measure local camp levels in hek cells and cytosolic fractions thereof using the camp fretsensor epac1-camps fused to a phosphodiesterase (pde4a1). we demonstrate the existence of low camp concentrations in close proximity to pdes and show that this gradient is maintained by pde hydrolytic activity. further we establish that camp gradients cannot be maintained solely on the basis of pde activity as the camp turnover is very slow. we provide evidence that pdes are structurally organized in yet unspecified 'microstructures' in which camp diffusion must be considerably slowed down. taken together, we suggest that camp gradients are established by pde hydrolytic activity in cellular regions with slow diffusion of camp. our study sheds light on the organization and maintenance of signaling compartments in cells. the influence of pde hydrolytic activity on camp gradients universität würzburg, pharmakologie und toxikologie, würzburg, germany phosphodiesterases (pdes) are a family of enzymes that degrade cyclic amp (camp) and cyclic gmp (cgmp) to their respective monophosphates. although several pdes have been shown to play an important role in a wide variety of physiological and pathological processes, their complexity and function in cell signalling is only beginning to be understood. it is especially astonishing that eukaryotes express more than 100 different pde isoforms while their single function is to degrade camp, cgmp or both. in recent years, a large body of evidence has suggested that pdes (especially isoforms of the pde families 3, 4 and 5) are key players in establishing signalling compartments. these so-called microdomains are yet unspecified regions in cells where the concentrations of camp and cgmp are higher or lower than in the bulk cytosol. in a companion abstract (bock & lohse) we provide evidence that camp nanodomains, i.e. regions of low camp concentrations, exist in cells in the direct vicinity of pde4. however, the mechanisms by which pdes establish and maintain camp gradients are largely unknown. here we study if establishing camp gradients is a general role of pdes and, moreover, if the size of camp nanodomains mainly depends on pde hydrolytic activity. by fusing an ultra-fast pde2a3 (v max = 120 µmol/min/mg) to a camp fret sensor (epac1-camps) we monitor camp concentrations in direct vicinity of pde2a3. in comparison to pde4, we show that pde2a3, albeit displaying a high camp turnover, only establishes a small camp gradient in both cytosol preparations of transfected hek cells and in living cells. interestingly, this gradient can be increased by deleting the nterminal regulatory domains while maintaining fast camp turnover. biochemical mapping of the camp gradient gives an estimate of the size of nanodomains. taken together, our data suggest that establishing camp gradients does not exclusively depend on pde hydrolytic activity. arglabin is a plant-derived sesquiterpene lactone used for cancer therapy in kazakhstan and russia. signaling pathways targeted by arglabin are poorly understood. we have isolated arglabin by using high performance liquid chromatography from a methanolic extract of artemisia glabella, a plant endemic in kazakhstan. mass spectrometric analyses confirmed the chemical structure and the purity of the isolated arglabin. in j774 macrophages, arglabin strongly induced accumulation of lc3 type ii protein in the absence of inflammasome activators, and also in cells activated with lps and cholesterol crystals. in addition, arglabin induced clustering of lc3-ii at autophagosomal membranes, as evidenced from its punctuated pattern in confocal microscopic images of arglabin-treated macrophages, which is a characteristic sign for autophagy. since autophagy activation leads to increased degradation of nlrp3 and pro-interleukin (il)-1β, we further analyzed whether arglabin inhibits the nlrp3 inflammasome. arglabin reduced expression of nlrp3 and pro-il-1β, inhibited activation of caspase-1, and release of mature il-1β by lps-and cholesterol-crystal-stimulated macrophages consistent with inhibition of the nlrp3 inflammasome. intraperitoneal injection of arglabin into female apoe2.ki mice fed a high fat diet resulted in significantly decreased plasma levels of proinflammatory il-1β. moreover, arglabin markedly reduced mean lesion areas in the sinus and whole aorta in mice. thus, arglabin may represent a new promising drug to treat diseases associated with inflammasome activation, e.g. atherosclerosis. this work was supported in part by a grant from the nouvelle société francophone d'athérosclérose (nsfa). recently, we found that activation of the proteinase-activated receptor (par) 2 stimulates renin release in the isolated perfused kidney model. therefore, we determined in the current experiments the response of plasma renin concentration (prc) to acute intraperitoneal administration of the par2 activating peptide sligrl (100µg/kg), hydralazine (2 mg/kg), isoproterenol (10 mg/kg), losartan (3 mg/kg) and furosemide (40 mg/kg) in conscious wild-type (wt) and par2-deficient mice. prc was measured in plasma obtained by tail vein puncture. renal renin expression was determined by quantitative rt-pcr. renal protein expression was measured by immunohistochemistry. on a control diet (0.6% nacl), plasma renin concentration (in ng angiotensin i per ml per hour) was significantly lower in par2-deficient mice than in wild-type mice (190 ± 35 versus 380 ± 91). renin mrna expression was 50 ± 9% of wt. renin-expressing cells were located at the juxtaglomerular position and renal renin protein expression was lower in par2-deficient mice. as measured by tail-cuff method, systolic blood pressure was not different between par2 (-/-) and wt mice. administration of sligrl increased renin secretion about 6-fold (p<0.01). acute stimulation of renin release by furosemide, isoproterenol, losartan and hydralazine caused significant increases of plasma renin concentration in both par2 (-/-) and wt mice. the absolute changes (delta prc) were similar (3780 ± 770, 2230 ± 400, 2780 ± 70, 1390 ± 460 in wt, and 2320 ± 270, 2530 ± 250, 3010 ± 210, 1230 ± 470 in par2 (-/-)). in conclusion, chronic absence of par2 reduces basal renin expression and renin release. however, par2-deficiency does not alter renin release in response to typical stimuli for renin secretion. therefore, par2 does not appear to be a mandatory and specific requirement for acute regulatory responsiveness. increased myofilament ca 2+ sensitivity could be the underlying cause of diastolic dysfunction. we evaluated acute effects of epigallocatechin-3-gallate (egcg), which has been shown to decrease myofilament ca 2+ sensitivity, on cardiac myocyte contractility and force-ca 2+ relationship of skinned cardiac muscle strips in an hcm mouse model with left ventricular hypertrophy and both systolic and diastolic dysfunction. methods: the hcm mouse model used in this study carries a point mutation in the cardiac myosin-binding protein c gene at the homozygous state (mybpc3-targeted knock-in; ki). we isolated ventricular myocytes from adult ki and wt mice and analyzed sarcomere shortening and ca 2+ transients at 37 °c under 1 hz pacing using the ionoptix system in the absence or presence of egcg (1.8 µm). furthermore, force-ca 2+ relationships of skinned cardiac muscle strips of ki and wt mice were obtained ±egcg (30 µm). results: in baseline settings and absence of fura-2, ki cardiomyocytes displayed higher sarcomere shortening ( type-1 serine/threonine protein phosphatase (pp1) comprises a family of enzymes that dephosphorylate cardiac regulatory proteins, thereby modulating ca 2+ handling and contractility. all pp1 heterodimers possess a catalytic subunit, which is selectively inhibited by inhibitor 2 (i-2). it has been shown by our group that the heart-directed overexpression of a truncated, constitutively active form of i-2 resulted in an improved basal ca 2+ handling and contractility. in contrast, chronic pressure overload by transverse aortic constriction exacerbated the progression of cardiac remodeling and heart failure in transgenic mice. in the present study, we tested whether the overexpression of a full-length form of i-2, regulated by gsk3-dependent phosphorylation at thr 72 , resulted in comparable functional alterations using a model of induced heart failure. for this purpose transgenic (tg) and wild-type (wt) mice were subjected to chronic application of isoprenaline (iso, 30 mg/kg/d) via osmotic minipumps. iso-stimulated mice were compared to mice treated with 0.9% nacl (n=5). after one week of iso administration, cardiac hypertrophy was comparable in tg and wt. ca 2+ transients were measured in isolated, indo-1-loaded myocytes. the peak amplitude of [ca] i was reduced by 39% in tg nacl compared to wt nacl (p<0.05), whereas chronic iso application was associated with comparable effects in tg and wt. [ca] i decay kinetics were comparable in nacl-treated groups but hastened by 25% in tg iso compared to wt iso (p<0.05). consistently, sr ca 2+ load was diminished by 28% in tg nacl compared to wt nacl (p<0.05). chronic iso stimulation led to an unchanged sr ca 2+ content in tg and wt myocytes. biochemical analyses revealed that chronic βadrenergic stimulation was accompanied by a more than 4-fold higher phospholamban phosphorylation at ser 16 in tg (p<0.05). thus, these findings suggest that overexpression of i-2 is able to reduce the progression of heart failure by an improvement of myocyte ca 2+ handling. the ligand-activated farnesoid x receptor (fxr) is a nuclear receptor highly expressed in gastrointestinal and metabolic tissues, such as the duodenum, jejunum, ileum, colon and the liver, but also in lower amounts for instance in macrophages. the endogenous agonists for this receptor are bile acids with the primary bile acid chenodeoxycholic acid as the most active one. activation of fxr regulates the transcription of target genes relevant in bile acid homeostasis, glucose and lipid metabolism, liver protection, inflammation, and cancerogenesis. agonists for fxr have been discussed as possible therapeutic options for the treatment of obesity and the metabolic syndrome. atherosclerosis is the main pathology underlying cardiovasular diseases and often occurs side-by-side with the metabolic syndrome. cholesterol deposition and the formation of cholesterol-loaded foam cells from macrophages lead to the formation of atherosclerotic plaques. this can be prevented by stimulation of cholesterol efflux from macrophages. based on leoligin, a lignan-type secondary plant metabolite, naturally occuring in leontopodium alpinum cass., 168 derivatives were synthesized and subjected to a fxr pharmacophore-based in silico screening. testing of 56 virtual hits in a luciferase-based fxr transactivation assay yielded one compound with promising activity on fxr. moreover, the heterodimer partner of fxr, rxrα, was not activated by this leoligin derivative in a luciferase-based rxrα assay. in addition, this compound was able to increase cholesterol efflux in thp-1 macrophages without affecting cell viability. western blot experiments revealed an increase in atp-binding cassette transporter a1 (abca1) expression in human thp-1 macrophages by this leoligin derivative. the transporters abcg1 and scavenger receptor class b (sr-bi), which also play a key role in macrophage cholesterol efflux, will be investigated. moreover, the effect of the leoligin derivative on liver x receptor activation, the nuclear receptor responsible for upregulation of these transporters, has to be studied. based on this data, further characterization of the molecular mechanism underlying the described effects will provide valuable insights in a possible crosstalk between macrophage cholesterol efflux and fxr activation. background: rho-associated kinases rock1 and rock2 are serine/threonine kinases that are downstream targets of the small gtpases rhoa, rhob, and rhoc. rock1 and rock2 are known to play a pivotal role in the pathogenesis of myocardial fibrosis. however, their specific function in cardiac fibroblasts (cf) remains unclear. remodelling of the diseased heart results in the transition of fibroblasts to a myofibroblast phenotype exemplified by an increased proliferation, migration rate and synthesis of extracellular matrix (ecm) proteins. therefore, we sought to investigate whether rock protein signalling intermediates have an impact on cellular characteristics, intracellular protein expression and mechanical properties in cf and engineered tissues. methods: neonatal cardiac fibroblasts were isolated from wild type rats and downregulation of rock1 and rock2 by 75% was achieved by lentiviral transduction or transfection. wild type fibroblasts were treated with 10 μm fasudil or 3 µm h1152p for general rock inhibition and 3 µm slx-2119 for inhibition of rock2. protein expression and modification was determined by immunoblot analysis, gene expression by qpcr analysis, cf morphology and the localisation of cytoskeletal proteins by immunofluorescence analysis, cell proliferation by automated nuclei counting, cell migration on a planar surface by life cell imaging, and rigidity of engineered tissues by rheological measurement. results: our results show that both rock1 and rock2 influence cf morphology, gene expression, proliferation and migration. the knockdown and inhibition of rocks was associated with changes in cf morphology accompanied by a disorganization of higher-order actin structures including stress fibers and geodesic domes. moreover, the knockdown of rock1 and rock2 in cf increased adhesion velocity, whereas proliferation was attenuated. interestingly, downregulation of rock2, but not of rock1 led to a significantly decreased migration velocity and distance suggesting an isolated principle role for rock2 in cardiac fibroblast migratory behavior. analysis of a three dimensional engineered tissue model composed of cardiac fibroblasts (engineered connective tissue, ect) suggested that rocks are involved in the regulation and turnover of the extracellular matrix (ecm) and thus influence viscoelastic properties of engineered tissues. destructive tensile strength measurement in ect treated with rock inhibitors showed that rigidity was significantly reduced when compared to control tissues. rna sequencing of ect treated with the rock inhibitor h1152p and qpcr analysis of cf with a downregulation of rock1 and rock2 showed that both rocks are involved in the regulation of ecm proteins, such as collagens 4a2, 6a, and 8a1, biglycan, decorin, elastin and its respective degrading enzyme mmp12. conclusion: this study demonstrates that rock signalling controls myofibroblast characteristics of cf via remodelling of the cytoskeleton and the ecm. background: regulation and fine-tuning of gene transcription in cardiomyocytes (cms) is a centerpiece of cardiac development, function, and disease. in order to obtain authentic data, cell type-specific analyses are indispensable. recently, high-purity isolation protocols for cm nuclei were established [bergmann, exp cell res, 2011] and employed for detailed genetic and epigenetic studies on cardiac gene transcription [gilsbach, nat commun, 2014] . however, corresponding protein analyses, which bridge from transcriptional control to cm function are still lacking. therefore, we aimed to map the landscape of nuclear protein expression in newborn and adult mice in order to complement and extend our epigenetic studies. methods and results: cardiac nuclei were isolated from homogenized adult and p1 mouse frozen hearts by sucrose gradient centrifugation. magnetic-assisted cell sorting (macs) with pcm-1 as a nucleus-specific marker was used to enrich cm nuclei to >95%. proteins were extracted from nuclear lysate with 2% sds. quantitative protein data were obtained from silac-based liquid chromatographytandem mass spectrometry (lc-ms/ms) experiments with an ltq orbitrap xl mass spectrometer after in-gel digestion with trypsin. nuclear protein extracts from 3 murine cell lines served as silac (lys8/arg10)-labeled internal standard. finally, protein data are correlated with corresponding mrna data obtained by rna-sequencing. we identified 1041 proteins, 688 of which are annotated to the nucleus. 21%/23% (adult / p1) of nuclear proteins are annotated as dna-binding. 1.5%/1.4% belong to transcription factor complexes; 7.3%/7.5% are able to bind transcription factors. 7.9%/8.3% have chromatin modification functions; 4.3%/4.4% modify histones. nuclearenriched go terms include mrna processing and transport, transcription, nucleosome assembly and protein degradation. 71% of proteins are shared between p1 and adult nuclei. 142 proteins are exclusive to p1, 34 are only found in adult. 93 proteins are enriched (1.3-fold increase in abundance) in p1 hearts, 89 proteins in adult proteins related to heart development, gene silencing, and dna replication are more prominent in or exclusive to newborn mice. adult nuclei strongly express proteins related to regulation of actin fibers and cm function, proteins involved in protein degradation, and chaperones. although high mrna expression increases the chance of protein identification, a significant correlation between mrna and protein level could not be observed on a genome-wide scale. conclusions: we present a comprehensive and specific protein landscape of newborn and adult cm nuclei. young cm nuclei appear as a developing tissue, show the ability for proliferation, and indicate ongoing alterations in gene expression. adult cm nuclei prominently display a focus on regulation of contractile fibers and cm function, as well as chaperones and proteasomal proteins indicative of its arduous function. background: fibrosis is a hallmark of many myocardial pathologies and contributes to distorted organ architecture and function. recent studies have identified premature senescence as regulatory mechanism of tissue fibrosis. however, its relevance in the heart remains to be established. objective: to investigate the role of premature senescence in myocardial fibrosis. methods: murine models of cardiac disease and human heart biopsies were analyzed for characteristics of premature senescence and fibrosis. results: senescence markers p21 cip1/waf1 , senescence-associated ß-galactosidase (sa-ß-gal) and p16 ink4a were increased 2-, 8-and 20-fold (n=5-7; p < 0.01), respectively, in perivascular fibrotic areas after transverse aortic constriction (tac) when compared to sham-treated controls. similar results were observed with cardiomyocytespecific β1-adrenoceptor transgenic mice and human heart biopsies. senescent cells were positive for vimentin (92 ± 0.9%), platelet derived growth factor receptor α (94 ± 0.9%) and α-smooth muscle actin (71 ± 2.3%), specifying myofibroblasts as the predominant cell population undergoing premature senescence in the heart. conclusion: our data provide first evidence for an essential role of premature senescence of myofibroblasts in myocardial fibrosis. it is tempting to speculate that pharmacologic modulation of premature senescence might provide a novel therapeutic target for anti-fibrotic therapies in the heart. introduction: the endocannabinoid system is increasingly studied in cardiac research due to its role in fibrosis, inflammation and cell fate modulation. the deregulation of this system has been implicated in myocardial infarction (mi) and consequent heart failure development. a recent study suggests cannabinoid receptor 1 (cb1) inhibition to improve cardiac function and to reduce adverse remodeling after cardiac stress, but the exact underlying molecular mechanisms of these beneficial effects are still unknown. micrornas (mirnas, mirs) provide a complex layer of post-transcriptional regulation modulating key biological processes such as tissue remodelling in heart failure. the aim of the present study was to explore microrna pathways in the chronic effect of cb1 receptor inhibition after angiotensin-fibrosis induction and left ventricular remodeling. methods and results: adverse cardiac remodelling was induced in mice by chronic administration of angiotensin ii (angii, 1,5 mg/kg/day) with osmotic minipumps for 14 days. treatment with cb1 antagonist, or vehicle was performed every second day during the angii administration period. hemodynamic parameters were measured by echocardiography and cardiac pressure volume catheter and tissue samples were taken for molecular and histological analysis. after two weeks of angii infusion, left ventricular dysfunction was prevented by cb1 antagonist treatment. this was shown by significantly improvements of the myocardial performance index and end-diastolic pressure values. at the tissue level, anti-fibrotic effects of cb1 antagonist treatment was confirmed histologically and by expression analysis of pro-fibrotic genes. these beneficial effects were also observed in cb1 ko mice and in an aging mice model. the particular role of tissue fibroblast in aii-induced cardiac fibrosis was further explored. primary cardiac fibroblasts (cf) from each experimental group were isolated and analysed by next generation deep rna sequencing to identify differentially regulated micrornas. microrna-181a/b family was downregulated by in vivo angii delivery and vice versa upregulated after cb1 antagonist treatment and foxb1 (a direct target of mir-181a family) was differentially regulated, suggesting a possible mechanism of action for the benefits of cb1 receptor inhibition. conclusion: we found that in angii-induced cardiac remodelling, lv function is preserved by chronic cb1 antagonist treatment and that cardiac fibrosis is reduced with concomitant downregulation of fibrogenic genes. also, cf-enriched mir-181a/b family seems to be sensitive to cb1 antagonist treatment, thereby affecting cardiac fibrosis. the current study employs a novel concept regarding chronic cb1 inhibitor treatment and may provide important details and novel targets for anti-fibrotic approaches in heart failure. background: low homoarginine (harg) was recently identified as an emerging biomarker for stroke, myocardial infarction, and heart failure in clinical and epidemiological studies. harg competes with arginine as a substrate for nitric oxide (no) synthase and weakly inhibits arginase. both mechanisms might lead to increased no formation in vivo. the aim of this study was to investigate whether harg effects the development of atherosclerosis as a potential underlying mechanism of cardiovascular diseases. methods: harg-deficient agat-knockout (agat -/-) and wildtype (wt) mice were crossed with apolipoprotein e (apoe) deficient mice and fed with high fat diet (hfd) for three months to induce atherosclerosis. harg plasma concentrations were determined using mass spectrometry. en face preparation of aortae followed by red oil staining of atherosclerotic plaques and quantitative evaluation of plaque areas was performed for female mice. endothelial function of male mice was tested with acetylcholine (ach) and nitroglycerin (ntg) after contraction with prostaglandin f2α. background: the direct oral thrombin inhibitor dabigatran etexilate (dabigatran) is used for the prevention and treatment of venous thromboembolism. obese patients as well as patients with type 2 diabetes mellitus (t2dm) have an increased risk for thrombotic disease and show enhanced thrombin generation. besides its role in blood coagulation, thrombin is known to be involved in many pro-inflammatory processes. in obesity, adipose tissue (at) inflammation plays a crucial role in the development of insulin resistance and t2dm and contributes to atherosclerosis development. the aim of the present study was to analyse the effects of dabigatran on at inflammation in a mouse model of diet-induced obesity in the context of accelerated atherosclerosis. methods: 10-week-old female low-density lipoprotein receptor-deficient (lldr -/-) mice were fed a high-fat diet containing 5 mg/g dabigatran or respective placebo for 20 weeks. results: analysis of visceral at revealed a significant increase in adipocyte size in dabigatran-treated mice, although body weight, fat mass, glucose tolerance, and insulin resistance were unchanged between groups. this effects seemed to be directly mediated by thrombin, as treatment with another thrombin inhibitor (argatroban) also resulted in the development of adipocyte hypertrophy. accordingly, in vitro studies in 3t3-l1 cells revealed an inhibitory effect of thrombin on lipid accumulation in adipocytes. the amount of pro-inflammatory cd11c-positive macrophages (atms) in visceral adipose tissue was significantly reduced, and the secretion of pro-inflammatory il-6 from visceral at was significantly lower in dabigatran-treated animals. in vitro studies using 3t3 l1 cells and primary bone marrow-derived macrophages revealed that the changes in macrophage polarization were not directly mediated by thrombin, but indirectly by a change in the secretion profile of adipocytes. a similar reduction in proinflammatory macrophages as detected in at could also be observed in the aortic wall of dabigatran-treated mice. conclusions: the direct thrombin inhibitor dabigatran inhibits at inflammation and the accumulation of pro-inflammatory macrophages in vat but also the aortic wall of ldlr -/mice. these anti-inflammatory effects of dabigatran might contribute to the known atheroprotective effects of dabigatran. background: sarco/endoplasmic reticulum ca 2+ -atpase (serca2a) and its inhibitor phospholamban (pln) are critical determinants of cardiomyocyte calcium cycling and hence, cardiac contractility. pln exists in an equilibrium between mono-and pentamers. while monomeric pln has been implicated in direct serca2a inhibition, a functional role for the pentamers remains ambiguous. recently it has been shown that pln pentamers modulate pka-dependent phosphorylation of pln monomers in vitro. 1 using transgenic mouse models we now investigated the effects of pln pentamers on pln phosphorylation, myocyte ca 2+ cycling and contractility in cardiac myocytes. methods: phosphorylation patterns of pln were analyzed by western blot using phospho-specific antibodies as well as phosphate affinity sds-page. to assess the phosphorylation at baseline, pln knockout (pln-ko) mice expressing either wild type pln (tgpln) or the solely monomeric pln afa mutant (tgafa) transgene were deeply anesthetized, whereas pln phosphorylation by pka was induced using the betaadrenergic agonist isoproterenol. the consequences on myocyte ca 2+ kinetics were measured in isolated, fura2-loaded and electrically paced (0.5hz) cardiomyocytes as the time to 50% decay of the ca 2+ signal (t 50% ). the time to 50% baseline of sarcomere length (t 50% baseline) characterized the speed of myocyte relaxation. results: under basal conditions, we found stronger phosphorylation of pln pentamers than monomers, pointing at pentamers as the preferred pka target. pln afa monomers showed 3.3-fold stronger phosphorylation signals if pentamers were absent (p<0.0001). consistent with a higher basal phosphorylation of pln afa monomers, measurements of calcium kinetics revealed a faster decay of calcium signals in tgafa compared with tgpln cardiomyocytes (t 50% [ms]: 92±8 and 122±8, respectively, p<0.05). notably, t 50% of pln-ko myocytes was 79±5ms (p= not significant versus tgafa), indicating that the strong basal phosphorylation of monomers leads to near complete inactivation of pln in tgafa. upon stimulation of pka, pln monomer phosphorylation and calcium kinetics of tgpln and tgafa mouse myocytes were indistinguishable, because monomer phosphorylation and the speed of cytosolic ca 2+ clearance strongly increased only in tgpln. acceleration of sarcomere relaxation upon pka stimulation was also more pronounced in tgpln than in tgafa and pln-ko myocytes (increase of t 50% baseline [ms]: 32±8 in tgpln versus 7±4 in tgafa-pln and 5±7 in pln-ko, p<0.05). even high-dose isoproterenol induced phosphorylation of only about half of all protomers of pln pentamers suggesting a high capacity of pentamers to attenuate monomer phosphorylation by acting as a phosphate scavenger. conclusions: our data demonstrate that pln pentamers reduce basal phosphorylation of pln monomers in myocytes. nevertheless, pentamers allow strong phosphorylation of monomers during beta-adrenergic stimulation, thereby extending the range within which pln can modify diastolic ca 2+ kinetics and myocyte relaxation. therapeutic inhibition of micrornas is a promising field in cardiovascular research. vector-based overexpression of an inhibitor construct (e.g. microrna sponge) is one approach to achieve sustained inhibition with potential applicability in humans. yet the strength of expression achieved by the currently available gene therapy vectors (e.g. aavs) in humans remains a limiting factor, therefore inhibitory constructs with increased potency would provide an improvement of this approach and bring it closer to therapeutic application. micrornas are believed to discriminate between potential binding sites, based on additional factors provided by the endogenous untranslated regions at the 3' end of mrnas (3' utrs) and the proteins that are bound to them. aim of this project was to investigate whether selected endogenous 3' utrs can likewise increase the potency of microrna inhibitory constructs. to this end several known targets for a cardiac microrna were selected and their relative potencies of microrna inhibition was compared. to accurately assess changes in the activity of the respective micrornas we constructed dual-fluorescent reporter plasmids and established an automated fluorescent microscopy acquisition and analysis pipeline. among several tested utr constructs we found one which strongly increased the inhibitory potency of the microrna binding site in primary rat cardiac myocytes (nrcms). furthermore a similar effect was obtained when the binding site was exchanged for that of a different microrna and analyzed in the nih-3t3 fibroblast cell line. we therefore conclude that endogenous utr contexts can indeed be successfully applied to increase the potency of vector-based microrna inhibitors. the g-protein-coupled protease-activated receptor-2 (par2) regulates inflammatory responses including monocyte migration and cytokine release. par2 is activated by the coagulation factor-xa or by the tissue-factor (tf)/factor viia complex. the immunomodulatory lipid sphingosine-1-phosphate (s1p) is released from activated platelets and interlinks blood coagulation and inflammation. this study investigates the impact of s1p on the expression of par2, tf and of the anticoagulant protein thrombomodulin (tm) in human monocytes and after pma-induced differentiation into macrophage-like cells. monocytic thp1 and u937 cell lines were used as human monocyte models. primary monocytes were isolated from healthy volunteers using a magnetic bead-based monocyte isolation kit. expression of par2, tf and tm was measured by quantitative real-time pcr and western blotting. differentiation of monocytes into macrophage-like cells was induced by incubation with 50 ng/ml pma (phorbol 12-myristate 13-acetate) over 72 h. calibrated automated thrombin (cat) generation was determined in plateletrich plasma from healthy volunteers. in thp1 and u937 cells s1p induced a time-(1 to 24 h) and concentration-dependent (0.1 to 10 µm) significant upregulation of par2 mrna and total protein expression. par2 total protein was upregulated maximally (about 1.5-fold, n=7) with 1 µm s1p after 16 h incubation. comparable effects were seen in human primary monocytes. in comparison, tf mrna and protein were only marginally elevated in non-differentiated thp1 monocytes and tm was not regulated by s1p. after differentiation of cultured monocytic cells with pma into adhesive macrophage-like cells, incubation with s1p resulted in a time-(1 to 24 h) and concentration-dependent (0.1 to 10 µm) significant upregulation of tf expression within 3 to 6 h of incubation. conversely, par2 total protein expression was reduced by about 50% after 24 h s1p incubation. the expression of tm was again not affected. the generation of thrombin in platelet-rich plasma was determined using pma-differentiated thp1 cells as tf source. timedependent incubation with s1p (1 µm) in differentiated monocytes shortened the time to the onset (lag time) of thrombin generation in plasma from 8.0±1.6 to 6.5±1.4 min and elevated total the thrombin generation capacity from 814±130 to 1286±129 nm. peak thrombin formation was elevated from 66±31 to 130±30 nm/min (control versus s1p for 6h, mean±sd, n=5, respectively). these data suggest that s1p induces an enhanced expression of par2 in undifferentiated human monocytes while tf and tm are not regulated. in differentiated monocytes/macrophages, s1p does upregulate tf expression but attenuates par2 levels. since par2 is involved in regulation cell migration, s1p may stimulate a phenotypic switching from a migratory to a procoagulant phenotype during differentiation of monocytes into macrophages. the pro-inflammatory cytokine interleukin-6 (il-6) plays an important role in vascular inflammation. coagulation factors such as the activated factor-x (fxa) may regulate local inflammatory responses of the vessel wall. in this study we investigated whether fxa regulates il-6 expression and secretion in human vascular smooth muscle cells (smc) as well as in failed thrombosed vein grafts. also, we analysed its possible prothrombotic impact on monocytes. il-6 mrna expression was determined in primary human saphenous vein smc by taqman® real-time pcr. secretion to the cell culture media was measured by elisa. tissue factor (tf) expression in monocytic thp-1 cells was determined by western blot. immunostainings for il-6 and the smc marker smoothelin were performed on paraffin embedded tissue sections from failed thrombosed vein grafts and control veins. incubation of cultured human venous smc with fxa (30 nm) induced a time-dependent (3 -16 h) increase in il-6 mrna expression. maximum expression was observed within 6 h to a 7.2±3.3 fold increase (mean±sd, n=5, p<0.05). incubation with an inhibitor of p38 map kinase (sb203580, 10 µm) or pi3k (ly294002, 10 µm) significantly attenuated fxa-induced il-6 mrna expression (n=5). inhibition of p42/44 mapk, rho kinase or nf-kb had no significant effect. stimulation with fxa for 24h resulted in a markedly increased il-6 secretion into the smc culture media from 0.6±0.2 to 1.1±0.3 ng/ml (p<0.5, n=4). stimulation of thp-1 cells with il-6 (1 ng/ml) induced a time dependent (6 -24 h) increase of up to 2.1±0.6 fold (p<0.05, n=5) in tf protein expression. immunostainings of tissue sample of failed vein grafts revealed enhanced il-6 expression in smc-rich regions in vessel walls compared to non-thrombosed control veins suggesting an elevated il-6 regulation in thrombosed vein grafts in vivo. in conclusion, fxa induced il-6 expression and secretion in venous smc which may be regulated via p38 and pi3k signaling. il-6 enhanced tf expression in thp-1 monocytes and was found in smc-rich regions in failed thrombosed vein grafts. fxa-stimulated il-6 release may be involved in regulating local pro-thrombotic processes during vascular inflammation and possibly vein graft failure. rationale: the transcription factors camp-response element binding protein (creb) and camp-responsive element modulator (crem) bind to camp response elements (cres) and mediate a camp dependent gene regulation. suppression of cre mediated transcription is linked to atrial remodeling in genetic mouse models. inhibition of creb target genes is associated with atrial fibrillation (af) susceptibility in patients. creb and crem affect histone acetylation recruiting the creb-binding protein (cbp/p300). the histone acetyltransferase (hat) activity of cbp facilitates gene transcription by loosening chromatin structure. histone deacetylases (hdacs) catalyze the inverse reaction: histone deacetylation with consecutive gene silencing. mice with heart directed expression of the human cardiac isoform crem-ib∆c-x (tg) show atrial dilatation, morphological and physiological alterations in atria preceding spontaneous-onset af. the hdac inhibitor (hdac i ) valproic acid (vpa) reduced atrial weight and af incidence in tg mice. here we tested the hypothesis, that vpa attenuates the structural remodeling in tg atria by reversing changes in atrial gene regulation due to the transgene. methods and results: tg and wt mice were treated from week 5-16 with vpa (0.71% in drinking water, ad libitum) or vehicle (veh). atrial ultrastructure was studied by electron microscopy (em) (week 7 and 16). veh-treated tg atria showed a progressive dysorganisation of sarcomeres (sm) with less mitochondria and more collagen fibers between cardiomyocytes as compared to veh-treated wt atria. the fraction of sm structure in veh-treated tg atria was significantly reduced as compared to veh-treated wt atria (week 7: tg veh : 33±2%, wt veh : 47±1%; week 16: tg veh : 19±2%, wt veh : 44±1%, p<0.05). vpa led to a more organized ultrastructure and restored, at least partially, the degradation of the sm in the tg atria (tg vpa at week 7: 40±2%, tg vpa at week 16: 34±1%, p<0.05 vs. tg veh ). the structure of wt atria was not affected by vpa. we further analyzed the protein abundance profiles in the groups of all animals (wt veh , wt vpa, tg veh , tg vpa) by using lc-ms/ms. between veh-treated genotypes (tg veh vs. wt veh , p<0.05) 998 proteins were significantly changed while 854 proteins were differentially abundant between vpa-treated groups (tg vpa vs. wt vpa, p<0.05). 109 proteins were regulated by vpa in wt atria (wt vpa vs. wt veh , p<0.05), whereas vpa affected 525 proteins in tg atria (tg vpa vs. tg veh , p<0.05), out of which 43 proteins were common. 295 prominent changed proteins between veh-treated tg and wt atria were significantly regulated by vpa in tg atria in the opposite direction. a functional pathway analysis showed that pathways activated in tg atria such as cardiac fibrosis, mitochondrial dysfunction were inhibited by vpa treatment. conclusion: similar to human af, crem-tg mice present atrial dilatation, ultrastructural changes and impaired conduction and spontaneous af. while vpa had little to no effect in wt mice, valproate improved the tg phenotype by interfering with pathways involved in structural remodeling. this supports the idea that hdac inhibition by vpa antagonizes effects of crem expression in atria. in isolated mouse cardiac preparations, histamine is ineffective regarding inotropic or chronotropic effects, presumably because of lack of receptor protein expression. on the other hand, histamine can exert positive inotropic and chronotropic effects in humans via cardiac histamine h 2 -receptors. hence, we have generated transgenic mice (tg) which overexpress the human h 2 -receptor specifically in cardiomyocytes. in isolated left and right atrial preparations of these mice, we investigated the histamine metabolism on a functional level. preparations of wild type mice (wt) served as control. histamine induced positive inotropic effects (pie) and positive chronotropic effects (pce) in left and right atria of tg mice, respectively, but not in wt. interestingly, the inhibitor of histamine oxidation, aminoguanidine (1 mm), shifted the concentration response curves for the pie of histamine from ec 50 = 110 nm to 37 nm (p<0.05). furthermore, the unspecific inhibitor of mono amine oxidase, tranylcypromine (10 µm), shifted the pie of histamine from ec 50 = 70 nm to 38 nm and increased the efficacy of histamine for the pie (p<0.05). these data indicate that exogenously applied histamine is subject to degradation in the mouse heart by two different pathways namely via diaminoxidase and mono amine oxidase. drugs that inhibit theses enzymes could conceivably alter cardiac function also in the human heart. protein phosphorylation by kinases and dephosphorylation by protein phosphatases has a crucial function in cell signal cascades. it has been shown that cardiomyocyte specific overexpression of serine /threonine protein phosphatases pp1, pp2a, pp2b (calcineurin) and pp5 in mice leads to cardiac hypertrophy and alters cardiac function. to examine the function of another important protein phosphatase in the heart we established a mouse model overexpressing protein phosphatase 2cβ (pp2cβ) under control of the α-myosin heavy chain promoter. cardiac overexpression was demonstrated by western blotting. like other serine/threonine phosphatases, pp2cβ can lead to cardiac hypertrophy. in transgenic mice (tg), relative ventricular weight was increased (4.24 ± 0.14 mg/g) compared to wild type (wt) littermates (3.78 ± 0.20 mg/g; p<0.05) whereas weights of right and left atria were unchanged. therefore, relative heart weight was increased in tg (4.78 ± 0.17 mg/g) vs. wt (4.13 ± 0.22 mg/g; n=8-12; 10-11 months of age; p<0.05). left ventricular function, measured in vivo by echocardiography under isoflurane anesthesia was diminished in tg compared to wt (ejection fraction: 58.33 ± 3.16 % (tg) versus 76.94 ± 0.92 % (wt); n=12-16; 8-11 months; p<0.05 and fractional shortening: 30.84 ± 2.02 % (tg) versus 44.94 ± 0.87 % (wt); n=12-16; 8-11 months; p<0.05). the left ventricle was dilated (systolic diameter: 2.78 ± 0.21 mm (tg) versus 1.84 ± 0.06 mm (wt); n=12-16; 8-11 months; p<0.05; diastolic diameter: 3.97± 0.19 mm (tg) versus 3.33 ± 0.07 mm (wt); n=12-16; 8-11 months; p<0.05). in contrast, atrial function measured as response to β-adrenergic stimulation in isolated left and right atrial preparations was unchanged in tg vs. wt. in summary, our results indicate that pp2cβ overexpression can lead to ventricular dysfunction and hypertrophy. the underlying signal transduction pathways need to be elucidated. the insulin-like growth factor binding protein 5 (igfbp5) -a potential developmental gene is regulated upon cardiac stress m. wölfer background: cardiac remodeling is a complex biological adaptation process of the failing heart accompanied by a re-activation of embryonic gene expression, which so far has unclear pathophysiological relevance. we and others showed that insulin-like growth factor binding protein 5 (igfbp5) is expressed in the early pre-cardiac region in mouse embryos and its up-regulation impairs cardiac progenitor differentiation. igfbp5 functions as an extracellular growth factor binding protein for igf and also has igfindependent activities. the role of this factor in the context of cardiac remodeling is still unknown. the aim of this study was to investigate the relevance of igfbp5 in cardiogenesis and cardiac remodeling and its role as a potential target for ameliorating stress-induced cardiac remodeling methods and results: we investigated the expression of igfbp5 in murine cardiac tissue at different developmental stages by qpcr normalized to tpt1 (tumor protein, translationally-controlled 1). this analysis showed temporal changes of cardiac igfbp5 expression from developing to postnatal hearts, where a high expression was detected in early heart stages, which decreased during cardiac development and became low in the postnatal heart. the analysis of igfbp5 expression in different heart cells showed a very low igfbp5 in adult cardiomyocytes in contrast to a high expression in undifferentiated sca-1 positive cells. in a mouse model with cardiac specific wnt/βcatenin activation, which led to cardiac dysfunction, igfbp5 was found up-regulated (p<0.05). further we found an increased igfbp5 expression after pressure induced cardiac hypertrophy using mice with transverse aortic constriction (tac) (p<0.01). in line with this data, an in vitro model of human heart muscle hypertrophy using engineered cardiac heart muscle (ehm) showed an up-regulation of igfbp5 upon adrenergic activation via norepinephrine stimulation accompanied by a functional deterioration in comparison to untreated controls (p<0.05). all these findings were further supported by rna-sequencing analysis from human aortic stenosis patient samples, where igfbp5 expression was found increased in patients with compensatory hypertrophy and in a higher extent in patients with heart failure in comparison to non-failing heart samples. interestingly, the expression of igfbp5 in angiotensin 2 or norepinephrine stimulated neonatal murine cardiomyocytes, as well as in hearts of mice treated with angiotensin 2, showed the opposite results, namely a reduction in its expression (p<0.01; p<0.05, respectively). summary and conclusion: our results show active igfbp5 transcription in the early developing heart but a low expression in the postnatal heart. a re-activation of expression was found in the process of pathological heart remodeling in mouse and human, in vivo as well as in vitro, indicate the participation of igfbp5 in a conserved manner. we hypothesize that igfbp5 may participate in the developmental gene program becoming activated in the diseased adult heart again. the functional role and regulation of igfbp5 is under investigation. we have recently shown that perivascular adipose tissue (pvat) plays a crucial role in obesity-induced vascular dysfunction. in pvat-free aortas isolated from male c57bl/6j mice fed a high-fat diet (hfd) for 22 weeks, the endothelium-dependent nitric oxide (no)-mediated vasodilator response to acetylcholine remained normal. in contrast, a clear reduction in the vasodilator response to acetylcholine was observed in aortas from obese mice when pvat was left in place. these results suggest that the reason for vascular dysfunction in diet-induced obese mice is a pvat dysfunction rather than an endothelial dysfunction. treatment of hfd mice during the last 4 weeks with crataegus extract ws® 1442 (150 mg/kg/day) completely normalized vascular function in pvatcontaining aorta. the expression of endothelial no synthase (enos) was not changed by ws® 1442, neither in pvat nor in aorta. phosphorylation at serine 1177 is the most important positive modulation of enos activity. hfd-induced obesity was associated with a reduction in enos phosphorylation at serine 1177 in pvat, but not in aorta. ws® 1442 treatment significantly improved enos serine 1177 phosphorylation selectively in pvat but had no effect in aorta. a major upstream kinase for enos serine 1177 phosphorylation is akt. the activity of this kinase is inhibited in the pvat of hfd mice, which was largely reversed by ws® 1442 treatment. in addition, ws® 1442 treatment enhanced the mrna expression of the nad-dependent deacetylase sirtuin-1 (sirt1), known also as a longevity gene. the activity of sirt1 depends, among others, on the intracellular content of its cofactor nad. ws® 1442 treatment led to an upregulation of nicotinamide phosphoribosyltransferase (nampt), a rate-limiting enzyme in the salvage pathway of nad biosynthesis. one of the non-histone substrates of sirt1 is enos. deacetylation of enos at lysine residues 497 and 507 by sirt1 enhances the activity of the enos enzyme. currently, we are studying the effect of ws® 1442 on nad synthesis, sirt1 activity, and enos (de)acetylation. in conclusion, crataegus extract ws® 1442 reverses obesity-induced vascular dysfunction by improving pvat function. the molecular mechanisms may involve enos phosphorylation at serine 1177 and upregulation of sirt1. the raf kinase inhibitor protein (rkip) inhibits g-protein-coupled receptor kinase (grk2) and the raf-erk1/2 pathway. these two functions of rkip could counteract each other. while grk2 inhibition is cardio-protective, inhibition of the pro-survival erk1/2 axis promotes signs of heart failure in patients and experimental models. in view of this ambivalent nature, the function of rkip in vivo is not clear. furthermore, rkip could have a pathophysiological role because heart specimens from patients with heart failure showed rkip up-regulation (ref. 1). to investigate the impact of cardiac rkip upregulation in vivo, we generated transgenic mice with myocardium-specific expression of the human rkip gene (pebp1) under control of the alpha-mhc promoter. two different rkip-transgenic lines with 2.7-fold and 3.4-fold increased cardiac rkip protein level were generated (ref. 2, and jax id number 911819). we investigated the cardiac phenotype and found that tg-rkip mice developed cardiac hypertrophy with a significantly increased heart weight to body weight ratio and a decreased left ventricular ejection fraction relative to non-transgenic fvb controls, as early as 10 weeks of age. histology analysis revealed progressive atrial and ventricular enlargement of tg-rkip hearts. ecg abnormalities, a lower maximum rate of left ventricular pressure rise, and a strongly decreased left ventricular ejection fraction of 32.9±2.3 % (n=6; ±s.d.) were documented at an age of 8 months. down-regulation of the transgenic rkip by lentiviral transduction of an rkip-targeting mirna retarded the cardiac phenotype of tg-rkip mice. thus, dual-specific inhibition of the grk2 and raf-erk1/2 axis by the human rkip gene (pebp1) triggers signs of heart failure in vivo, and the documented upregulation of the cardiac rkip in heart failure patients could aggravate disease pathogenesis. these findings are in contrast to rodent rkip (pebp1), which does not seem to inhibit the raf-erk1/2 axis in vivo but instead confers grk2 inhibitionheterodimerization between the at1 receptor (at1r) for the vasopressor peptide, angiotensin ii, and the b2 receptor (b2r) for the vasodepressor peptide, bradykinin, enhances angiotensin ii-stimulated signalling in cells. in addition, at1r--b2r heterodimerization has a major pathophysiological role and contributes to the angiotensin ii hypersensitivity in women with preeclampsia hypertension. to analyse the vascular function of the at1r--b2r heterodimer in vivo, we generated a transgenic model of at1r--b2r heterodimerization (tg-b2r+) by transgenic expression of the b2r gene (bdkrb2) in the b2r-deficient tg-b2r-/-strain. fluorescence resonance energy transfer (fret) imaging was applied to analyse the interaction between different gprotein-coupled receptors in the aorta of transgenic mice. we report here that fret imaging detected the close interaction between the aortic at1r and b2r at a distance of less than 9 nm in tg-b2r+ mice whereas the at1r--b2r heterodimer was absent in tg-b2r-/-mice. in contrast, fret was not detectable between the endothelin eta receptor (etar) and the b2r in the aorta of tg-b2r+ mice, although immunofluorescence and immunohistology confirmed the aortic (co-)-localization of both, etar and b2r. the efficient at1r--b2r heterodimerization in tg-b2r+ mice was accompanied by an enhanced angiotensin ii at1r-stimulated vasopressor response relative to that of tg-b2-/-mice, which lack the at1r--b2r heterodimer. as a control, the endothelin-1-stimulated vasopressor response mediated by the etar, which did not dimerize with b2r, was not significantly different between tg-b2r+ and tg-b2r-/-mice. together these findings provide strong evidence that at1r--b2r heterodimerization occurs in vivo and enhances the angiotensin ii at1r-stimulated vasopressor response. dysfunction of the cardiac energy substrate metabolism is a characteristic feature of late-stage heart failure. the dysfunctional cardiac substrate metabolism contributes to insufficient energy generation and has limited treatment options. in search for a treatment approach, we investigated whether inhibition of g-protein-coupled receptor kinase 2 (grk2) could confer cardioprotection by targeting the dysfunctional cardiac substrate use. the impaired substrate metabolism of late-stage heart failure was reproduced in a transgenic model with myocardium-specific expression of fatty acid synthase (fasn), which is the major palmitate-synthesizing enzyme. experiments with a seahorse xf24 extracellular flux analyzer revealed that in an adult-like lipogenic milieu, fasn-transgenic cardiomyocytes reproduced the overall depressed substrate use of late-stage heart failure with a switch from fatty acid to predominant glucose utilization. the impaired substrate use was largely retarded by co-expression of a small peptide inhibitor of grk2, grkinh. the grkinh-mediated protection against cardiometabolic remodelling required an intact raf-erk1/2 axis and involved the erk1/2-dependent inactivation of the heart failure-promoting peroxisome proliferatoractivated receptor gamma (pparg) by phosphorylation of serine-273. as a consequence of erk-dependent phosphorylation of pparg on serine-273, the expression of heart failure-related pparg targets such as fatty acid synthase, resistin and adiponectin was decreased. the importance of pparg serine-273 phosphorylation was further shown in transgenic mice with myocardium-specific expression of the phosphorylation-deficient pparg serine-273a mutant, which was resistant to the cardioprotective activity of grkinh. taken together our experiments show that grk2 inhibition could target cardiometabolic remodelling by inhibition of the heart failure-promoting transcription factor pparg. the effect of sodium valproate on the action potential of atrial myocytes of crem ib∆c-x transgenic mice introduction: in mouse, cardiomyocyte directed over-expression of transcription factor crem (camp response element modulator) causes an atrial phenotype characterized by hypertrophy, reduced contractility and increased duration of the monophasic action potential (map). moreover, this animal model (crem ib∆c-x) showed spontaneous atrial fibrillation (af) episodes as early as 5 weeks of age in homozygous mice and 10-12 weeks of age in heterozygous mice (phenotype delayed towards adult stage). previous studies in heterozygous mice targeted hdac2 inhibition by sodium valproate (vpa, an anticonvulsant drug, acting also as inhibitor of hdac class i>ii). vpa treatment delayed significantly the development of atrial hypertrophy and the incidence of af episodes, without affecting cellular hypertrophy. our aim was to investigate the effect of chronic vpa treatment on the electrical activity of atrial myocytes isolated from crem ib∆c-x and wild type (wt) littermate mice. methods and results: atrial myocytes were isolated from 12 weeks old wild type mice (wt) and heterozygous crem ib∆c-x transgenic mice with enlarged atria (tg), treated for 7 weeks with vpa (0.4 mm in the drinking water) vs. water (vehicle control). action potentials (ap) were measured at room temperature using the patch-clamp technique. atrial myocytes of water treated tg mice had the ap amplitude significantly reduced by 7 mv compared to water treated wt, and, in line with previous results for the map, the tg cells depolarized with a slower slope of 90.2±8 v/s (tg: n=33 cells) vs. 109.8±5.1 v/s (wt: n=30, p=0.05). moreover, ap of atrial myocytes isolated from water treated tg mice had longer duration (apd) at 50% (tg: 11.6±1.4 ms, n=35 vs. wt: 6.9±0.5 ms, n=30, p<0.01), at 70% (in ms: 21.2±2.5 vs. 13.6±1, p<0.05) and at 90% (in ms: 46.8±4.5 vs. 34±2.5, p<0.05) repolarization. vpa treatment reduced ap amplitude in wt mice by 6 mv (n=22, p<0.05) vs. water treated wt, without altering the slope of depolarization or the apd. in vpa treated tg mice the apd was reduced (50%: 7.3±0.8 ms, 70%: 13.8±1.5 ms, 90%: 30.9±3.2 ms, n=21, p<0.05 vs. untreated tg), the amplitude was increased by 5 mv (n.s.) and the slope of depolarization was increased by 21% (p=0.16, n.s.). membrane capacitance evaluation, as an estimation of atrial myocyte size, showed that in untreated tg mice the cells were larger than in wt (tg: 104±7.2 pf, n=27 vs. wt: 55±5.4 pf, n=30, p<0.001), in line with the occurrence of cellular hypertrophy in tg atria. chronic vpa treatment did not change the cell size in either genotype (wt-vpa: 64.8±7 pf, n=17 n.s. vs. wt; tg-vpa: 88±7 pf, n=14, p<0.05 vs. wt-vpa; p<0.01 vs. wt; n.s. vs. tg). conclusions: in hypertrophied atrial myocytes of crem-ib∆c-x, ap were characterized by smaller amplitude, slower onset of depolarization and increased duration compared to wt cells. despite having no effect on atrial myocytes size, vpa treatment reduced the duration and showed a tendency to increase the amplitude and the slope of depolarization of the action potential in tg mice to values similar to wt. these data suggest that chronic treatment with vpa restored partially the electrical activity of atrial myocytes and may reverse the electrical remodeling via hdac2 inhibition. (supported by the dfg) of the transcription factor crem (camp response modulator) icer, smicer and crem-ib∆c-x are inducible by β-adrenergic stimulation and code for similar or even identical proteins. thus, these isoforms are able to repress expression of respective target genes in response to camp and might play a role in an arrhythmogenic remodeling during the development of chronic heart diseases. here we test this hypothesis in a mouse model with transgenic expression of crem-ib∆c-x (tg). these mice develop not only spontaneous onset atrial fibrillation but likewise arrhythmogenic alterations in the ventricle. patch clamp experiments revealed an increased na + -ca 2+ exchanger current (i ncx ) and decreased transient outward current (i to ) in tg ventricular cardiomyocytes (vcms) vs. wild-type controls (ctl). these alterations were associated with an increased arrhythmogenicity in tg vcms. action potentials were prolonged in tg vcms vs. ctls leading to an increased proportion of vcms displaying early afterdepolarizations. ca 2+ imaging revealed that the transduction rate of spontaneous sub-threshold ca 2+ -waves into supra-threshold transient-like ca 2+ -events which is mediated by the ncx was increased in tg vcms. at the same time the serca mediated ca 2+ transport rate (r serca ) was enhanced in tg vcms potentially limiting ca 2+ extrusion by the ncx. underlining the in-vivo relevance of our findings ventricular extrasystoles (ves) were augmented in ecgs of tg mice (ves/mouse during 10 -6 m isoproterenol challenge, tg: 3.65*, ctl: 0.4; n=20/condition). the increase in i ncx and r serca and the decrease in i to went along with an increase of ncx1, serca2a and decrease of kchip2 protein levels. however, the respective mrna levels (slc8a1, atp2a2 and kcnip2) were unaltered between groups pointing to a post-transcriptional regulation of these genes. in a mrnasequencing approach we identified the downregulation of precursor mirnas inter alia for mir-369 (fold change in tg: 0.04*) and mir-1 (fold change in tg: 0.13*) (n=10/condition). atp2a2 is a predicted target of mir-369 and mir-1 has recently been shown to regulate ncx1 and i to related potassium channel subunits. (*p<0.05 vs. ctl) our results demonstrate that transgenic expression of crem-ib∆c-x in mouse vcms leads to distinct arrhythmogenic alterations. they further indicate that the repression of micro rnas by short crem repressor isoforms may lead to the upregulation of genes in the context of an arrhythmogenic remodeling. since crem-repressors are inducible by chronic β-adrenergic stimulation our results suggest that the inhibition of credependent transcription contributes to the formation of an arrhythmogenic substrate in chronic heart disease. ( chronic overstimulation of cardiac β-adrenergic receptors (β-ar) is a major trigger for the development and maintenance of cardiac hypertrophy and heart failure. although the camp activated proteinkinase a (pka) is known as a prominent downstream effector of β-ar signaling, its functional contribution to pathological cardiac remodeling is neither well understood nor directly studied so far. to address this issue we used mice carrying a point mutation in the regulatory pka subunit riα (pkariαb), which prevents binding of camp and consequently diminished kinase activity. this dominant negative mutation was controlled by a tamoxifen (tam) inducible αmhc promotor driven cre transgene which allows a selective expression in the ventricular myocardium. the inducible and tissue specific gene expression was analyzed and confirmed by pcr, rt-pcr and immunohistochemistry. furthermore diminished phosphorylation of several pka targets verifies impaired pka activity in tam treated double transgenic animals. hypertrophic response in ventricular pka mutants was studied in genetic, pharmacological and surgical mouse models of heart disease as well. genetically induced heart failure was observed following tam treatment in mice expressing an inducible myocardial-specific cre transgene. this deleterious cardiac phenotype develops independently of the presence of the floxed transgene. 8 days after tam treatment, controls displayed elevated heart weight to bodyweight ratio (hbr) and heart weight to tibia length (htr). hbr shifted from 6.2(mg/g) in untreated control animals to 10.9(mg/g) in tam injected mice. in contrast pka inhibited mutants displayed a minor increased hbr of 7.7 (mg/g). for the pharmacological induction of cardiac hypertrophy we implanted osmotic mini pumps, delivering a combination of isoproterenol and phenylephrine. control animals showed a significantly increased hbr (8.4 mg/g) compared to saline treated animals (6.8mg/g) and pka mutants (7.1 mg/g). paradoxically, all pka inhibited animals displayed a consistent elevation in important hypertrophic markers like anp. surgical constriction of the aortic arch (transverse aortic constriction tac) led to a pressure induced hypertrophic response (hbr: 6.5 vs 7.9 mg/g) followed by a pronounced elevation in several hypertrophic factors such as anp, myh6/7 ratio and myocytes size. in contrast pka mutants displayed an irregular progression of cardiac hypertrophy presented by two groups with either an unchanged (6.9 mg/g) or a strongly elevated hbr (13.5 mg/g). however, additional hypertrophic factors including anp, myh6/7 ratio and myocyte size were significantly increased in both groups. to our knowledge this is the first report, which directly studies the role of ventricular pka activity in cardiac hypertrophy in a genetically altered mouse model. our results suggest that in an early stage of cardiac remodeling pka inhibition alleviates cardiac weight gain but provokes a detrimental shift during further progression, which implicates a protective role of ventricular pka activity in cardiac disease. acetyl-coa carboxylase catalyzes the first step in the biosynthesis of fatty acids in bacterial and eukaryotic cells, i.e. the conversion (carboxylation) of acetyl-coa into malonyl-coa. acc-generated malonyl-coa functions as a substrate for de novo lipogenesis and acts as an inhibitor of mitochondrial β-oxidation of fatty acids. because of its role in lipid metabolism this enzyme has become an interesting target in drug discovery in the field of metabolic diseases and cancer. despite this interest in acc, no attention has as yet been given to the role of acc in endothelial cells. we aimed to investigate the role of acc in two functional key aspects of angiogenesis: endothelial cell proliferation and migration. we used the acc inhibitor soraphen a, a polyketidic natural compound isolated from the myxobacterium sorangium cellulosum, as well as an rnai-based approach to inhibit the function of acc. primary human umbilical vein endothelial cells (huvecs) were used as in vitro model. first, we analyzed the action of soraphen a on cell viability. the compound did neither lower the metabolic activity of huvecs up to a concentration of 100 µm after 24 and 48 h (ctb assay) nor increase in the apoptosis rate after 24, 48, or 72 h up to 100 µm. measuring adenosine triphosphate (atp) levels revealed that 30 µm soraphen a does not alter the atp levels in huvecs after 24 h treatment. in contrast, a 48 h treatment significantly lowered the atp levels by 12 %. also gene silencing of acc1 in huvecs attenuated the atp levels by 11 %. mitochondrial membrane potential (mmp) assays showed decreased mmp levels (10 %) in soraphen a-treated cells after 24 h. interestingly, the compound inhibited the proliferation of endothelial cells with an ic 50 value of 34 µm. cell cycle analysis showed that soraphen a decreases the amount of cells in the g 0 /g 1 phase by 26 % and increases the number of cells in the g 2 /m phase by 50 %. the compound also inhibited the activation of akt (western blot analysis). in a wound healing/scratch assay, 30 µm soraphen a lowered the migration of endothelial cells by 65 %. gene silencing of acc1 in huvecs strongly decreased endothelial migration, whereas a knockdown of acc2 had no influence. furthermore, boyden chamber assays revealed that soraphen a can also lower chemotactic migration by 34 %. since actin rearrangement is necessary for migratory processes, we analyzed the factin cytoskeleton (microscopy) and found that soraphen a decreases the number of filopodia by 60 % but did not influence stress fiber formation. surprisingly, soraphen atreated cells did not exhibit significant alterations in their capacity to form tube-like structures on matrigel. in summary, we could gather first hints that inhibiting acc has an immense impact on the proliferation and migration of primary endothelial cells. the mechanistic basis of this phenomenon will be investigated in future studies by analyzing the lipid profile and the transcriptome of endothelial cells. acknowledgement: this work was supported by the german research foundation (dfg, for 1406, fu 691/9-2) . introduction: statins are among the best examined drugs with excellent efficacy and safety profiles. lowered low-density lipoprotein (ldl) cholesterol goals, new indications for treatment and new knowledge about their pleiotropic effect have promoted a considerable increase in statin use. but as statin use becomes more widespread, awareness of their adverse effects as well as the recognition of statin intolerance problems increase. statin intolerance is a significant problem in the treatment of dyslipidemia, understood as the inability to tolerate a dose of statin required to reduce individual cardiovascular risk sufficiently and could result from different statin-related side effects. muscle-related adverse events, elevation of liver enzymes, cognitive problems and new onset diabetes mellitus have all been described, especially at higher doses. although muscle symptoms are the common side effects observed, excluding other adverse events might underestimate the number of patients with true statin intolerance. these patients represent a target population for the newest lipid lowering drug category i.e. the proprotein convertase subtilisin/kexin type 9 (pcsk9) inhibitors. this work aimed to give an overview of published definitions derived from clinical studies, associations as well as major drug regulatory agencies. we discussed overlaps, differences and limitations in the current definitions. methods: literature based search included pubmed and uptodate publications in english and german language until october 2015. we performed hand searches of the references retrieved and performed an overview. results: a definition of statin intolerance of the european medicines agency (ema) or the us food and drug administration (fda) is not available. in clinical studies, different definitions are chosen and the results are not comparable. also different associations, such as the american heart association (aha), the european atherosclerosis society (eas), the canadian working group or the national lipid association (nla) are not able to agree on one common definition. statin intolerance definitions included different types of muscle symptoms, integration of ck levels and minimal requirements of statin doses. there are currently no validated questionnaires or specific laboratory parameters available. in addition, the term 'myopathy' is often considered as a synonym to statin intolerance. overall, only a few major studies have been conducted with statin intolerant patients so far using inconsistent definitions. discussion and conclusion: there is an unmet need to find a robust and clear definition of statin intolerance as overemphasizing it might hinder appropriate clinical use of this important drug class. thus, further work is required to develop a consensus definition on statin intolerance or a more focused definition regarding statin-associated muscle symptoms only. subsequently, these definitions could be implemented in patient care and their relevance being analyzed and tested in future studies. background: development of cardiac hypertrophy is characterized by reactivation of genes involved in cardiac development. wnt/β-catenin signaling is essential for embryonic cardiac development and is known to be dysregulated in pathological heart remodeling. our previous work suggested a cardiac specific protein complex regulating wnt/b-catenin/tcf transcription in the adult heart. we aim to identify and to characterize this complex in order to find potentially interesting targets for pharmacological therapy preventing maladaptive cardiac remodeling and the onset of heart failure. results: we previously demonstrated that the krüppel-like-factor 15 (klf15) is a βcatenin interaction partner and a cardiac specific nuclear inhibitor of the wnt/β-catenindependent transcription. because klf15 and β-catenin are ubiquitously expressed, we suggest the existence of cardiac specific co-factors responsible for cardiac specificity in this complex. we identified the basic leucine zipper and w2 domain containing protein 2 (bzw2), a phylogenetically conserved protein, as a β-catenin and klf15 interaction partner using yeast-two-hybrid screen. in vitro overexpression experiments and coimmunoprecipitation validated these interactions, which were also confirmed by mass spectrometry. in the developing mouse embryo bzw2 mrna expression is detectable in the heart, neuronal tissue, somites, limbs and branchial arches as shown by whole mount in situ hybridization. in the adulthood expression of bzw2 is confined to the heart, predominantly in cardiomyocytes and in cardiac progenitor cells compared to cardiac fibroblasts (*p<0.05, cm n=4, cfb n=4, cpc n=2), and skeletal muscle. bzw2 was localized in both the cytosol and in the nucleus. mutation analysis showed the importance of the n-terminus of bzw2, containing a putative bzip dna interaction domain, for the nuclear placement of the protein. bzw2 protein expression was significantly increased under cardiac wnt/β-catenin signaling activation in vivo in two mouse models (klf15 knockout (ko) mice **p<0.01 n=3, and in a cardiac specific β-catenin stabilized mouse model, **p<0.01 n=6). a mouse model with constitutively bzw2 loss of function (bzw2 ko) showed cardiac specific upregulation of β-catenin on rna level (***p<0.001, p<0.05, ctrl n=4, bzw2 ko n=5) and on protein level (**p<0.05, ctrl n=7, bzw2 ko n=9). echocardiography analysis in eight-weeks-old bzw2 ko mice showed increased left ventricle wall thickness indicating a hypertrophic phenotype at baseline. we also observed increased levels of bzw2 expression in angiotensin ii treated mice as a model for cardiac hypertrophy (*p<0.05 ctrl n=4, angii n=3) as well as in human samples derived from patients with dilated cardiomyopathy and ischemic cardiomyopathy (*p<0.05 ctrl n=3, dcm n=11, icm n=10). conclusion: these data demonstrated that bzw2 is associated with components of the canonical wnt cascade and suggest its relevance in the constitutive regulation of the wnt/β-catenin components specific in the heart. this study further contribute to the elucidation of the tuning of the wnt-off/-on states aiming to establish a proof-of-concept model for wnt-modulation as a therapeutic strategy in hypertrophic-induced heart failure. objective: sphingosine-1-phosphate (s1p) is involved in the regulation of cell growth, survival, migration and adhesion. it is formed by sphingosine kinases and degraded by phosphatases and s1p lyase [1] . mice that lack s1p lyase are characterized by the accumulation of s1p and sphingosine in their cells and tissues, and by lymphopenia, generalized inflammation, multiple organ damage, and a strongly reduced life span [2] [3] [4] . on the other hand, embryonic fibroblasts from s1p lyase-deficient mice (sgpl1 -/--mefs) are resistant to chemotherapy-induced apoptosis [5] , in part due to an upregulation of multidrug transporters of the atp-binding cassette (abc) transporter family [6] . interestingly, s1p lyase-deficient mice have elevated plasma levels of cholesterol and triglycerides, while suffering from strongly reduced body fat [7] . the aim of the present study was to analyze the link between s1p lyase deficiency and altered cholesterol homeostasis using sgpl1 background: cardiac gene expression changes during cardiac development and under pathophysiological conditions. these alterations in gene expression are regulated by several processes and the exact regulation of gene expression is essential for the proper development and function of the heart. crucial steps in transcription regulation are rna polymerase ii (pol ii) recruitment and changes in pol ii activity. pol ii activity is tightly linked with phosphorylation at serine-2 (p-ser2) of the carboxyterminal domain of pol ii. thus, the aim of the present study was to identify cardiomyocyte-specific genomewide pol ii and p-ser2-pol ii enrichments to get insight into pol ii activity and recruitment in development and disease. methods and results: to get insight into rna polymerase ii dynamics, genome-wide maps of rna polymerase ii occupancy were generated by chromatinimmunoprecipitation in cardiomyocyte nuclei purified from normal neonatal and adult mouse hearts. in addition, cardiomyocyte nuclei were obtained from adult hearts after 4 weeks of pressure overload induced by transverse aortic constriction (tac). cardiomyocyte nuclei were isolated by magnetic beads with an anti-pcm1 antibody. nuclei were used for pol ii chromatin-immunoprecipitation followed by deep sequencing (chip-seq). to test if pol ii marks correlate with nuclear mrna expression in cardiomyocyte nuclei all coding genes were ranked according to their expression level. genes expressed in cardiomyocyte nuclei (> 0.062 fpkm, gene expression rank < 12,653) showed high pol ii enrichment at promoters as well as in genomic regions. many of the gene promoters showed high levels of pol ii accumulation at the transcription start site, as compared to genic regions, which have been associated with pol ii pausing. in contrast, p-ser2-pol ii showed enrichment downstream of the transcription start site. the genomic region of troponin i type 1 (tnni1) which is expressed in cardiac muscle only during development but not in adult cardiomyocytes, was enriched for pol ii in neonatal cardiomyocytes. at the tnni1 gene, pol ii was absent in adult or pressure-overloaded cardiomyocytes. in contrast, pol ii enrichment at the alpha actin 1 (acta1) locus was only present in pressure-overloaded cardiomyocytes. these data are consistent with cellular rna-seq data showing an induction of acta1 after tac. furthermore no pol ii enrichment could be detected in the genomic region of biglycan (bgn), a matrix proteoglycan that is not expressed in cardiomyocytes. this confirms a high purity of cardiomyocyte chromatin. conclusions: this study provides, for the first time, cardiomyocyte-specific landscapes of rna polymerase ii occupancy in heart development and disease. cardiac myocyte maintenance dna methyltransferase 1 is essential for embryonic heart development but is dispensable for cardiac function and remodeling postnatally t. nührenberg background: recent studies have identified dynamic changes in dna methylation in cardiac myocytes during development, postnatal maturation and in disease. however, the enzymes involved in shaping the cardiac myocyte dna methylome are only partially known. here, we explored the role of maintenance dna methyltransferase dnmt1 in cardiac development and in remodeling after chronic left ventricular pressure overload. methods: in mice, deletion of the dnmt1 gene was accomplished by use of two different cre recombinases. crosses of homozygous dnmt1 fl/fl mice with heterozygous dnmt1 fl/+ mice expressing a cre recombinase under control of the atrial myosin light chain gene promoter (myl7-cre) resulted in embryonic deletion of dnmt1 (ko). embryos without myl7-cre served as controls (ctl). embryos were dissected and genotyped at e11.5, e12.5, e13.5 and e14.5. rna-seq and pyrosequencing of genomic dna was performed on e11.5 hearts, histology on e12.5 hearts and electron microscopic imaging on e13.5 hearts. for deletion of dnmt1 in adult mice, homozygous dnmt1 fl/fl mice expressing an inducible cre recombinase (myh6-mcm) were given tamoxifen i.p. over 4 days. homozygous dnmt1 fl/fl mice not carrying myh6-mcm as well as myh6-mcm carrying mice without dnmt1 fl alleles were also injected with tamoxifen and served as controls. cardiac phenotyping including histology, echocardiography and qpcr was carried out without (sham) or with left ventricular pressure overload induced by transverse aortic constriction (tac). results: myl7-cre mediated loss of dnmt1 resulted in progressive embryonic lethality with absence of living ko embryos after e14.5. ko embryos displayed loss of cardiomyocyte gene expression patterns, decreased promotor cpg methylation of aberrantly expressed genes and ultrastructural features of wide-spread cardiac myocyte cell death. in contrast, tamoxifen-induced ablation of dnmt1 in adult mice did not affect survival of ko mice. cardiac phenotyping of adult mice revealed no significant differences between ko and ctl mice under sham and tac conditions. conclusion: dna methyltransferase dnmt1 in embryonic cardiac myocytes is essential for proper heart development. in adult cardiomyocytes, dnmt1 is dispensable for normal cardiac function and for adaptation to chronic cardiac pressure overload. background: recent studies showed that mice with general deletion of the oxidoreductase tet3 involved in dna demethylation are embryonic lethal, the underlying cause remaining unknown. this prompted us to investigate wether embryonic lethality is caused by cardiomyocyte-specific loss of tet3. methods: female mice homozygous for tet3 flox and male mice heterozygous for tet3 flox and heterozygous for myl7-cre were mated and offspring were genotyped after weaning. mice homozygous for tet3 flox and heterozygous for myl7-cre (ko) or homozygous for tet3 flox without myl7-cre (controls) were sacrificed at 12 weeks of age and ventricles were harvested. mrna of the ventricles was isolated and expression of cardiomyocyte-specific genes was evaluated by quantitative real-time pcr. cardiomyocyte-specific genomic dna from ko and control mice was obtained from facs-sorted cardiomyocyte nuclei and bisulfite-converted for analysis of dna methylation by pyrosequencing. results: ko mice showed embryonic lethality of nearly 50 %. born ko mice developed without phenotypic abnormalities (normal heart weight/tibia length ratio) and displayed compensatory upregulation of tet1 and tet2. cardiomyocyte genomic dna of ko mice showed significantly higher methylation levels in the body of the atp2a2 gene and at the binding site of the transcription factor gata4 but not near the promotor and the binding site of the transcription factor tbx5. higher methylation levels were not accompanied by changes in atp2a2 expression. however, both myh7 and nppb were upregulated in ko mice compared to control mice. conclusions: our findings suggest that tet3 is involved in dna demethylation in cardiomyocytes. loss of tet3 expression resulted in embryonic lethality. compensatory upregulation of tet1 and tet2 isoenzymes may contribute to the incomplete penetrance of this phenotype. further studies are ongoing to investigate the functional relevance of tet3 in cardiomyocytes. to identify novel proteins secreted by the myocardium, we have previously conducted a genetic screen, which led to the identification of protease inhibitor 16 (pi16). a recent gwas analysis showed an association of a genetic variant in the pi16 genomic locus (rs1405069) with chemerin plasma levels. here we tested the hypothesis that pi16 determines chemerin plasma levels through regulation of chemerin processing. we generated mice deficient for pi16, which did not display an overt phenotype under basal conditions. plasma levels of chemerin were found significantly lower in pi16-deficient animals compared to littermate controls. to investigate whether pi16 and chemerin interact, we performed co-immunoprecipitation experiments. indeed, we found pi16 to co-precipitate with chemerin from both murine plasma and cell culture supernatants. as chemerin is proteolytically processed and activated, we next asked whether the presence of pi16 would affect the processing of pro-chemerin to its processed forms in native tissue. western blot analysis on cardiac and adipose tissue lysates that detected both the unprocessed precursor and the processed forms of chemerin showed a significant shift towards the processed forms upon genetic deletion of pi16. when we assayed for the activity of the chemerincleaving protease cathepsin k, we found recombinant pi16 to potently inhibit cathepsin k activity. taken together, we propose pi16 to act as a regulator of chemerin processing. the transient receptor potential canonical 6 (trpc6) is a second messenger-gated cation channel, which mediates depolarization and ca 2+ entry. it is known to be activated by diacylglycerol derivatives (dag, 1-oleoyl-1-acetyl-sn-glycerol oag) [1] in a pkc-independent manner and plays important roles in lung and kidney physiology. gain-of-function mutations in the trpc6 gene can cause focal segmental glomerulosclerosis (fsgs), a kidney dysfunction leading to end stage renal disease. [2] thus, the discovery of potent inhibitors of trpc6 may help to develop new therapeutic strategies. urban et al. discovered that larixol, a natural product with a labdane skeleton found in the oleoresin of the european larch (larix decidua), blocks the oag-dependent activation of trpc6. [3] larixyl acetate, another component of the resin showed an even higher potency in trpc6 inhibition (ic 50 = 0.26 µm) and a 12-fold selectivity compared to trpc3. these findings led to the idea that further modifications of the larixol lead structure may reveal even more potent inhibitors. furthermore, changes in selectivity and efficacy of such compounds may also provide a deeper insight about relevant structural elements for channel binding. as larixyl carbamate was assumed to exhibit a higher metabolic stability as larixyl acetate, this compound was already investigated in previous studies. it showed a potent and subtype-selective inhibition of trpc6. hence, the development of further carbamates was a priority objective. as an alternative to the use of different isocyanates for the introduction of a carbamate function at the c1 position of the molecule we found an elegant way via formation of an active ester with carbonyldiimidazole. this precursor allowed the design of several isosteric compounds like larixyl methylcarbamate, larixyl hydrazide and larixyl methylcarbonate, which were all able to block trpc6 with similarly low ic 50 values. the introduction of more bulky side chains appeared to diminish the bioactivity of the compounds, the stereochemistry at the c1 position, however, seems to play no important role for the inhibition of trpc6 currents. larixyl methylcarbamate lead to trpc6 inhibition with an ic 50 value of 0.15 ± 0.06 µm. compared to larixyl carbamate and larixyl methylcarbonate, which are also very potent blockers of trpc6, this compound bears the benefit of high subtype selectivity towards trpc3. even with concentrations up to 50 µm of larixyl methylcarbamate, no complete inhibition of the ca 2+ influx via trpc3 channels could be achieved. this fact distinguishes this larixol derivative as a very promising compound for further studies of trpc6 in health and disease. poisoning by organophosphorus compounds (opc) including pesticides and highly toxic nerve agents is based on irreversible inhibition of acetylcholinesterase (ache) resulting in an excess of acetylcholine causing accumulation. the subsequent overstimulation at nicotinic and muscarinic receptors finally leads to respiratory arrest due to paralysis of the respiratory muscles. therapy focuses on competitive antagonism at muscarinic acetylcholine receptors and reactivation of inhibited ache by bisquarternary pyridinium oximes. thereby, nicotinic malfunction is not directly approached. for that reason, an alternative strategy appears rational using nicotinic acetylcholine receptor (nachr) active substances to counteract the effects of accumulated acetylcholine and thus to restore the loss of function of nachrs. different bispyridinium-non-oxime-compounds (bps) have been demonstrated to be able to serve as target structures for the identification of new positive allosteric modulators of nicotinic receptors. unlike nicotinic agonists, positive allosteric modulators can reinforce the endogenous cholinergic neurotransmission despite of acetylcholine accumulation in the synaptic cleft. to this end, the following electrophysiological in vitro study investigated the effect of twelve diversely substituted bps on human α7 nachr using whole-cell patch clamping under voltage-clamping conditions (-70 mv) performed with planar electrodes in an automatic system (nanion technologies gmbh, munich). cholinergic currents of hα7 nachr that have been expressed in stably transfected cho cells were activated by the agonist nicotine. measurements of the effect of various bps concentrations in the presence of nicotine were performed to establish concentration-response relations. cholinergic inward currents were generated by human α7 nachrs in response to low nicotine concentrations. at high concentrations of the drug the currents were decayed reflecting both, desensitization of the receptors and presumably block of the open channel by high agonist concentrations. four out of twelve bps co-applicated with nicotine showed a concentration-dependent enhancement of peak agonist-evoked currents and, most pronounced, 4-tert-butyl-substitued-bp, also demonstrated a marked elongation of the evoked response. this suggests a positive allosteric effect of these compounds on the nicotinic receptor. however, at high bp concentrations in the presence of agonist, responses were decayed significantly, presumably resulting from an open channel block induced by bps. hence, further compounds have to be synthesized to identify promising candidate compounds for improvement of effective therapy against nerve agent poisoning. the transient receptor potential channels (trp) are a family of tetrameric nonselective cation channels, which are involved in a variety of physiological and pathological processes (1). among the 28 mammalian trp channels, the canonical channel 5 (trpc5) is a ca 2+ -permeable ion channel, which is predominantly expressed in the brain (2) . many aspects of trpc5 function are still elusive although behavioral experiments with trpc5-knock-out mice suggest a role in innate fear-response (3) and some studies indicate a trpc5-mediated down regulation of neurite outgrowth in nerve cells (4, 5) . to elucidate trpc5 function on a cellular level, selective and potent compounds are required to acutely control channel activity. despite extensive research, trpc5 modulators often lack selectivity or exhibit toxicity, limiting their applicability in vivo (6, 7) . thus, there is still a need for identifying novel and efficient trpc5 modulators. we therefore screened a compound library (chembionet) and identified a benzothiadiazine derivative (btd) as a novel, potent, and selective trpc5 activator. hek293 cells heterologously expressing trpc5 upon tetracycline induction (hek trpc5 ) show a btd-induced concentration-dependent activation in both ca 2+ assays (ec 50 = 0.71 µm) and in electrophysiological whole cell patch clamp recordings (ec 50 = 1.1 µm). btd elicits currents with an n-shaped i/v curve, typical for trpc5. the resulting activation is long lasting, reversible and sensitive to clemizole, a recently established trpc5 inhibitor (8) . mtt assays revealed that incubating hek trpc5 cells for 24h with btd concentrations above 1 µm results in a concentration-dependent decrease in viability and cell proliferation, indicating a ca 2+ -mediated cytotoxic effect in consequence of sustained channel activity. non-induced control cells remain unaffected by btd at concentrations up to 10 µm. ca 2+ assays showed no influence of btd on closely related trpc4 channels, as well as trpc3/6/7 at concentrations up to 10 µm. the same applied to more distantly related trpv and trpm channels. besides a homotetrameric organization, trpc5 subunits can also assemble to heteromeric channel complexes with their closest relatives trpc1 and trpc4 (9) . trpc1/5 and trpc4/5 heteromers can also be activated by btd as evident from their typical i/v curves in patch clamp experiments, suggesting a high selectivity of btd for channel complexes bearing at least one trpc5 subunit. transient receptor potential canonical channels 3/6 and 7 are controlled by membrane lipids and highly expressed in neuronal and cardiac tissues. the involvement of these channels in development and (patho)physiology of these tissues is well documented, while our understanding of structure-function relations, specifically in terms of the lipid sensing machinery, in these channel proteins is still incomplete. using a homology model of trpc3, based on the recently available structural information on trpv1, we performed structure-guided mutagenesis and identified a single residue in transmembrane domain 6 (g652), which is conserved within the canonical family of trp channels. single point mutations at position 652 in trpc3 largely eliminated lipid sensitivity. trpc3 g652a expressed in hek293 cells was found resistant not only to activation via the phospholipase c pathway but also to direct administration of diacylglycerols. on the contrary, a synthetic agonist of trpc3/6/7 channels (gsk1702934a) activated wild-type trpc3 and trpc6 channels as well as the respective lipid insensitive mutants (trpc3 g652a, trpc6 g709a ). interestingly, the synthetic activator was found to generate substantially enhanced trpc conductances in cells expressing the lipid-insensitive mutants as compared to wild-type proteins. closer inspection of sensitivity of the wt and mutant proteins to various gsk derivatives argue against a contribution of g652 to gsk recognition by trpc3. our results demonstrate the existence of two different mechanisms of trpc3/6 activation supposedly involving distinct gating movements in the channel complex. we suggest that lipid gating of trpc3/6 involves a hinge-point and/or requires a certain level of flexibility within transmembrane segment s6 provided by g652. lipids and synthetic activators of trpc3/6 may be capable of initiating markedly different structural rearrangement in these channels. objective: organophosphorus compounds (opcs), i.e. nerve agents or pesticides, are highly toxic due to their strong inhibition potency against acetylcholinesterase (ache). inhibited ache results in accumulation of acetylcholine in the synaptic cleft and thus the desensitisation of the nicotinic acetylcholine receptor (nachr) in the postsynaptic membrane is provoked. as the therapeutic efficacy of oximes is limited, e.g. poisoning by soman or tabun, the direct targeting of nachr may be an alternative therapeutic approach. studies with the non-oxime bispyridinium compound (bp) mb327 (1,1'-(propane-1,3-diyl) bis (4-tert-butylpyridinium) di(iodide)) demonstrated a therapeutic effect against soman in vitro and in vivo. consequently, studying the affinity of bps at muscle-type nachrs and functional effects are topics of interest. to identify potential candidates, homologous series of substituted and non-substituted analogues (linker c 1 -c 10 ) of mb327 were investigated by using binding and functional assays. experimental procedures: crude membranes from frozen electric organ of torpedo californica were purified by sucrose-gradient density centrifugation and used in both affinity and functional assays. in competition radio-ligand binding assays, the influence on [³h]epibatidine binding sites of torpedo muscle-type nachr was determined. functional assessments were carried out with a bilayer method to investigate the effect on the cholinergic signal induced by 100 µm carbamoylcholine. results: bispyridinium compounds bearing unsubstituted pyridinium rings and long alkyl linkers (> c 7 ) inhibited the binding of [³h]epibatidine and decreased the cholinergic signal of 100 µm carbamoylcholine in the functional assay. mb327 and several bispyridinium structure analogues (mainly c 2 -c 4 linker) exhibited no regular displacement curves at [ 3 h]epibatidine binding sites and enhanced the carbamoylcholine-induced signal. the results demonstrate that the described affinity and functional screening methods detected some structure-activity-relationships (sar). depending on linker length and substitution pattern, the investigated bispyridinium compounds seemed to interact as positive allosteric modulators. further research is necessary to verify this hypothesis. non oxime bispyridinium compounds with an effect on soman-blocked respiratory muscle function have no effect on normal muscle function the life threatening toxicity of organophosphorus compounds (op), like nerve agents or pesticides, lies in the inhibition of acetylcholinesterase (ache) which causes cholinergic crisis. the accumulated acetylcholine in neuromuscular synapses results in the desensitization of nicotinic acetylcholine receptors (nachr) and paralysis of respiratoric muscles. the 4-tert-butyl-substituted bispyridinium compound mb327 showed therapeutic efficacy in soman and tabun poisoned guinea pigs in vivo. partial restauration of neuromuscular transmission by bispyridinium compounds (bp), e.g. mb327 or mb420, could also observed in soman paralysed respiratory muscles in vitro and was partly attributed to an interaction of bp with nachrs. however, it is unknown, whether these bp might affect normal respiratory muscle function in the absence of cholinergic crisis. therefore this study investigated the effect of bp on physiological rat diaphragm muscle function. force generation of rat diaphragm hemispheres was determined after incubation with increasing bp concentrations (1-300 µm) and compare to sham treatment. the diaphragm hemispheres were stimulated every ten minutes by an indirect electrical field (20, 50, 100 hz). muscle force was analyzed as time-force integral and is expressed as percentage of the individual control values, measured at the outset of the experiment. the muscle force dropped during the experiment. the application of the bispyridinium compounds mb327 (1,1′-(propan-1,3-diyl) bis (4-tertbutylpyridinium) di(iodide)) and mb420 (1,1′-(propan-1,3-diyl) bis (2-ethylpyridinium) di(iodide)) in the tested concentration range (1-300 µm) did not change muscle force production compared to the sham treated muscle. this was equally true for low (20 hz) and high (50 and 100 hz) stimulation frequencies. this study showed that bispyridinium compounds which can partially reverse somaninduced neuromuscular block in rat diaphragms show no effect on respiratory muscle function in absence of the op-induced neuromuscular block. these results suggest that the bp tested in this study interacted with desensitised nachrs only, but do not affect physiological neuromuscular transmission. this effect needs to be investigated with further, promising bp compounds. interaction of recombinant pain-relevant atp-and proton-gated ion channels in an expression system; potentiation of the p2x3 receptor-induced current by the opening of asic3 channels g. stephan 1 , p. illes 1 1 universität leipzig, rudolf-boehm-institut für pharmakologie und toxikologie, leipzig, germany the p2x3 receptor (r) is a ligand-gated cationic channel, which is activated by extracellular atp. the acid sensing ion channel 3 (asic3) belongs to the enac/degenerin family and is gated by extracellular protons. despite their different amino acid sequences both ion channels share the same structure and pore architecture, by i.e. consisting of three identical subunits. besides, they are both located at partially overlapping subpopulations of dorsal root ganglia neurons and are implicated in acidic pain signaling. consequently, their physical interaction in the cell membrane or even the formation of heteromeric receptor channels from p2x3 and asic3 subunits has to be taken into consideration. we transfected rat (r)p2x3r and rasic3 constructs individually or together in a ratio of 1:1 into cho cells. we further used the whole cell patch clamp technique to analyze the current responses either elicited by the application of α,β-methylene-atp (α,β-meatp) or by a decrease in the extracellular ph value. the functionality of the individually transfected p2x3r-and asic3-constructs was verified by recording concentration-response curves for the agonists α,β-meatp and protons, respectively. after co-transfection of both ion channels, a ph-shift from 7.4 to 6.7 caused a rapidly desensitizing current response and a subsequent strong potentiation of the α,β-meatp-induced current. an even larger potentiation was achieved after a decrease of the ph value to 6.5. the opening of asic3 channels failed to facilitate the p2x3r current when 2-guanidine-4-methylquinazoline was used to stimulate a non-proton ligand-sensor of asic3. then, we substituted ca 2+ in the extracellular medium by ba 2+ or decreased the intrapipette concentration of egta, to modify the free intracellular ca 2+ concentration. in cells individually transfected with the receptor-channels, external ba 2+ increased the effect of α,β-meatp but decreased the effect of protons. the lowering of intrapipette egta modified p2x3r-and asic3-specific currents in a similar manner as external ba 2+ . in cells co-transfected with p2x3r/asic3, the ionic manipulations mentioned above abolished the potentiation of the α,β-meatp currents by asic3 activation. taken together, our results suggest that p2x3r and asic3 interact with each other, since the activation of asic3 had a marked impact on the p2x3r specific current response. further experiments are required to clarify the mechanism of this interaction, although it has been shown that extra-and intracellular ca 2+ and the proton sensor of asic3 appear to critically participate in this process. university of duisburg-essen, institute for anatomy, essen, germany background: the sperm acrosome reaction is an all-or-none secretion process, mainly following the conserved principles of calcium-regulated exocytosis in neurons and neurosecretory cells. however, the relationship between the formation of hundreds of fusion pores and the required mobilization of calcium from the lysosome-related acrosomal vesicle has only been partially defined. hence, the second messenger, nicotinic acid adenine dinucleotide phosphate (naadp), known to promote efflux of calcium from lysosome-like acidic compartments, was analyzed for its ability to trigger acrosome reaction in mouse sperm. in addition, the expression of two-pore channel (tpc) proteins, which are primarily localized in lysosome-related acidic organelles and which present potential molecular targets of naadp were examined in mammalian spermatozoa. methodology/ principal findings: our results show that treatment of spermatozoa with naadp resulted in a loss of the acrosomal vesicle, which shows typical properties, described for tpcs: (i) registered responses were not detectable for its chemical analogue nadp, and (ii) where blocked by the naadp antagonist trans-ned-19. in addition, (iii) two narrow bell-shaped dose-response-curves were found, with maxima either in the nanomolar or low micromolar naadp concentration range. performing immungold-electron microscopy with a tpc1 specific antibody, a co-localization with naadp-binding at the acrosomal region was detectable. moreover, quantifying loss of the acrosomal vesicle in tpc1 null sperm upon application of different naadp concentrations, responsiveness to low micromolar naadp concentrations was completely abolished. conclusions/significance: our finding that two convergent naadp-dependent pathways are operative in driving acrosomal exocytosis and that zona pellucida induced acrosomal exocytosis is prevented by trans-ned-19 support the concept that both naadp-gated cascades match local naadp concentrations with the efflux of acrosomal calcium, thereby ensuring reliable and complete fusion of the large acrosomal vesicle. since the acrosome reaction shares the same basic sequence of events typical for the conserved process of calcium regulated exocytosis, such as tethering, docking, priming and final vesicle fusion, the sperm model system may also be useful to comparatively examine whether the same convergence of naadp-dependent pathways is also operative in cellular systems with many secretory vesicles. walther-straub-institut, münchen, germany trpv4 channels are members of the vanilloid family of trp proteins. the channel is nearly ubiquitously expressed and can be found in brain, kidney, skin, heart, blood vessels as well as in the lung. pulmonary expression of trpv4 has been identified in endothelial cells (1), epithelial cells (2) and arterial smooth muscle cells (3) . most interestingly, the channel is known to be involved in the development of several lung diseases such as cough, asthma and pulmonary edema formation, due to its activation by heat, changes in osmolarity and shear stress (reviewed in 4). it is a matter of debate however if trpv4 activation in pulmonary endothelial as well as epithelial cells induces disruption of the barrier and an increased fluid leak into the alveolus as described for trpc6 (5) which is also expressed in both cell types. to analyze the potential role of trpv4 on ischemia-reperfusion-injury(iri)-induced pulmonary edema formation we utilized the isolated perfused mouse lung model. much to our surprise, we detected a significantly enlarged edema formation after 90 minutes of ischemia in trpv4-deficient mice in comparison to wild-type (wt) mice. this effect was observed by constant weight measurements as well as wet-to-dry ratio gain and was not dependent on the initial perfusion rate. most interestingly, edema formation of trpv4/trpc6 double deficient lungs was indistinguishable from wt lungs, indicating antagonizing effects of both channels, because trpc6 deficiency protected lungs from iri-induced edema (5) . moreover, we identified reduced expression levels of aquaporin 5 in trpv4-deficient lung lysates compared to wt lungs. these findings raise the intriguing possibility that trpv4 might be involved in the regulation of aquaporin expression in lung endothelial cells. endosomes and lysosomes are cell organelles involved in transport, breakdown and secretion of proteins, lipids, and other macromolecules. endolysosomal dysfunction can cause storage disorders such as mucolipidoses, sphingolipidoses, or neuronal ceroid lipofuscinoses, but is also implicated in the development of metabolic and neurodegenerative diseases, retinal and pigmentation disorders, trace metal dishomeostasis, infectious diseases, and cancer. endolysosomal ion channels and transporters are highly critical for the tight regulation of the multiple endolysosomal fusion and fission processes including endo-and exocytotic events as well as the regulation of proton and other ionic concentrations in the lumen of endolysosomal vesicles. methods to patch-clamp endolysosomal organelles are continuously improving. yet until now it has not been possible to selectively enlarge endosomal or lysosomal organelles with pharmacological tools for patch-clamp experimentation. we show here by using a combination of two small molecules that we can selectively enlarge early endosomes to a degree sufficient for patch-clamp experimentation. the ability to more selectively patch-clamp intracellular organelles will substantially improve the functional investigation of endolysosomal ion channels under physiological and pathophysiological conditions. the transient receptor potential (trp) channels are a superfamily of non-selective ion channels involved in a variety of physiological processes and in the pathgenesis of many disorders. in the kidney, trp channels have been implicated to be involved in diabetic nephropathy, focal, segmental glomerulosclerosis, polycystic kidney disease, hypomagnesemia with secondary hypercalcemia and idiopathic hypercalcuria. the melastatin-like trp channel subfamily 3 (trpm3) has been shown to be expressed in human kidney 1, 2 . using newly developed anti-trpm3 antibodies, we are able to visualize trpm3 protein in epithelial cells of proximal tubule as well as collecting ducts in the mouse kidneys. therefore, we compared renal function of male, five month old mice lacking trpm3 (trpm3 the atp-gated p2x7 receptor (p2x7r) is a non-selective cation channel widely expressed in epithelia, endothelia, and cells of hematopoietic origin. it plays a central role in cytokine release and studies in p2x7 -/animals indicate its involvement in inflammatory and neurodegenerative diseases. in addition, accumulating data suggests a functional role in neurons and its involvement in neurotransmitter release in the brain. however, despite its importance as a drug target, its precise localization and its molecular and physiological functions remain poorly understood. in particular, the location and function of p2x7r in neurons remain a matter of ongoing debate. to clarify the cellular and subcellular distribution of the p2x7r and to investigate its physiological and pathophysiological role in the brain we generated bac transgenic mouse models in which murine polymorphic variants of egfp-tagged p2x7r are overexpressed under the control of the endogenous p2x7 promoter. the egfp-tagged p2x7rs are efficiently overexpressed in the plasma membrane and can be directly visualized by green fluorescence or indirectly by anti-egfp antibodies. the obtained mouse lines show different expression levels but identical expression patterns with predominant expression in the cerebellum, hippocampus, and thalamus. using cell type-specific markers, p2x7-egfp was identified in almost all microglia and subpopulations of oligodendrocytes and astrocytes in the brain. in the spinal cord, numerous astrocytes in the white matter showed egfp immunoreactivity. so far, no egfp immunostaining was found on map2-and neun-positive cells indicating that under non-pathological conditions, the p2x7 receptor is not expressed in neurons of the cns. interestingly, a higher expression level of cd68 protein was observed in the p2x7r-overexpressing mice. these results suggests that overexpression of p2x7rs alone is sufficient to induce microglia activation, even under non-pathological conditions. since cd68 primarily localizes to lysosomes and endosomes, this further supports a role of the p2x7r in the regulation of phagocytosis. to validate these data, conditional knockout mice are generated. the current status of the project will be presented. the two-pore channels (tpcs) -tpc1 and tpc2 -are located in membranes of intracellular organelles of the endo-lysosomal system. the tpc-protein-monomer contains two homologous domains with six transmembrane α-helices each. a functional tpc probably consists of a dimer of two tpc-proteins resembling an ion channel architecture with the typical four domain organization like voltage gated na + or ca 2+ channels or such as trp channels. due to their biophysical properties, tpc1 and tpc2 are assumed to be involved in the efflux of ca 2+ from intracellular organelles and thereby contribute to fusion/fission processes of endosomes and lysosomes. thus, tpcs are supposed to be important regulators for vesicle trafficking, sorting and degradation/recycling processes. recently, it was shown that virus entry and replication of certain strains of filoviridae -such as ebola -depends on functional tpcs and that either block or genetic inactivation of tpcs reduces virus infectivity. a large family of bacterial protein toxins elicit their effects by modification of intracellular target proteins of host cells. these toxins are taken up by receptor mediated endocytosis and follow different endosomal routes to reach their final cytosolic destination. these toxins principally use two different intracellular routes: the first group uses an entry route via early or late endosomes (short-trip toxins), the second group takes a retrograde route via endosomes, golgi network and the endoplasmic reticulum (long-trip toxins) to get access to the cytosol. translocation of short-trip toxins -such as diphtheria toxin (dt), pasteurella multocida toxin (pmt) and bacillus anthracis lethal factor (pa/lf) -from early and late endosomes into the cytosol is driven by ongoing acidification. long-trip toxins -including cholera toxin (ct) -are retrogradely transported after endocytosis via the golgi apparatus to the endoplasmic reticulum (er). within the er a specific peptide-motif allows the translocation into the cytosol. due to the role of tpc1 for vesicle fusion & fission processes we investigated a potential impact of tpc1 on the uptake of bacterial toxins. first we determined the precise localization of tpc1 in intracellular compartments. to deduce its role for trafficking processes we performed co-localization and correlation studies with a whole set of established markers such as rab-gtpases and pips. second we intoxicated wild-type and tpc1 deficient cell lines with different bacterial protein toxins such as cholera (ct), diphtheria (dt) or pasteurella multocida (pmt) toxin. using cell viability and other intoxication assays we investigated the consequences of tpc1-deletion on bacterial toxin uptake, translocation and cytotoxicity. universität des saarlandes, institut für experimentelle und klinische pharmakologie und toxikologie, homburg/saar, germany trpm3 ion channels are considered to be involved in hormone release from pancreatic islets and the pituitary gland 1,2 . the trpm3 gene encodes a number of different splice variants that differ in their permeation properties and their activity in response to agonists 3, 4 . variations include the presence or absence of five stretches of 10 to 25 amino acid residues within the aminoterminus, long or short pore loops and long or truncated carboxytermini 5 . furthermore, three different aminotermini of human and mouse proteins have been described 5 , but presumably these differences are caused by the activity of different promoters. screening of a mouse pituitary gland cdna library identified 12 variants that differ in exons 8, 13, 15, 17 and 20 . however, only variants bearing the short, ca 2+ permeable pore loop were detected. 3´ rapid amplification of cdna ends (3´race) revealed that trpm3 proteins of the pituitary gland carry truncated c-termini exclusively. 5´race identified five independent regions of transcription initiation within the trpm3 gene implying the presence of five independent promotors. the different transcripts encode four trpm3 amino termini α, β, γ and δ of 1-155 amino acid residues including those described in humans (β,γ). however, the activity of these variants after stimulation with pregnenolone sulfate varied largely just as their frequency in the pituitary with shortened γ-variants being most abundant (~76 %). two-pore channels (tpcs) are a small family of ion-channels found throughout the endolysosomal system of eukaryotic cells. phylogenetically tpcs belong to the voltagegated ion channel superfamily sharing common traits with ca v / na v and trp channels. tpcs show a duplicated architecture with two homologous trans-membrane domains. each domain is build up by six membrane spanning alpha helices linked by short loops. it is very likely that tpcs form dimers maintaining the four-fold symmetry found in other members of the voltage-gated ion channel superfamily. due to their localization in the endolysosomal system tpcs are not accessible to conventional patch clamping. to investigate tpcs electrophysiological properties we use black lipid bilayer measurements. purified channels are integrated into an artificial phospholipid bilayer that separates two chambers enabling us to apply different buffers. upon activation by naadp ions flow through the channel and can thereby pass the diffusion barrier. the movement of charged molecules through tpcs results in currents which can be amplified and recorded. the controlled environment of the lipid bilayer setup allows testing of different ions as well as putative activating and inhibitory substances. one of the major drawbacks of conventional lipid bilayer setups are long preparation times between each measurement. stability of the phospholipid bilayer can pose another issue. often several membranes have to be established before a measurement can be performed making it very time consuming to achieve adequate experiment numbers. new multichannel systems resolve this issue supporting fast formation of bilayers while allowing measurement of up to 16 different bilayers at a time. here we utilize the "orbit" multichannel system with a meca16 chip by ionera to measure tpc1 channel activity. we will present data generated by the "orbit" system and a conventional bilayer setup using different channel constructs and charge carriers. cardiac action potentials are generated and propagated through the coordinated activity of multiple ion channels, including voltage-gated sodium channels (nav1) and potassium channels (like kv1, kv4). the voltage-gated na + channel nav1.5 initiates the cardiac action potential (ap), is essential for rapid depolarization, and is also known to control the ap duration in cardiomyocytes. the voltage-gated k + channel kv4.3 is responsible for the early repolarization of action potentials in human heart. similar to many membrane proteins, nav1.5 and kv4.3 have been found to be regulated by several interacting proteins. the transmembrane β subunit dipeptidyl aminopepidase-like protein (dpp) 10 is known to interact with the kv4.3 channel complex, modulating kinetics and voltage dependence. the overexpression of dpp10 in ventricular cardiomyocytes of rats revealed strong reduction of ap amplitude and significant slowing of ap upstroke velocity and ap duration, which could not be explained by the effects on cardiac kv4 channels. to study the potential influence of dpp10 on nav1.5 channels, we performed whole-cell patch-clamp analysis of transiently transfected cho-k1 cells, expressing scn5a alone or with dpp10. surprisingly, we observed significant effects of dpp10 on nav1.5 channel voltage dependence and kinetics. thus, the co-expression of dpp10 significantly shifted the half-maximal voltage of steady-state activation and steady-stateinactivation to more positive potentials compared to nav1.5 channels alone. in addition we analysed the effects of dpp10 on the kinetics nav1.5 currents. while time to current peak was not affected in cells co-expressing nav1.5 and dpp10 compared to nav1.5 alone, dpp10 slightly accelerated the inactivation. in addition, the time course of recovery from inactivation was clearly accelerated in cells expressing both nav1.5 and dpp10 compared to nav1.5 alone. in summary, we provide first evidence that dpp10 not only interacts with kv4 channels, but also influences nav1.5 channels modulating the depolarization as well as the early repolarization phase of the cardiac ap. therefore, it becomes likely that these ion channels are part of large, multi-protein complexes, and that the pore-forming subunits kv4.3 and nav1.5 behave very differently depending on the expression of its associated proteins like dpp10. cardiac fibroblasts (cf) comprise the most abundant cell type of the mammalian heart and it is known that they contribute to maladaptive cardiac remodeling processes. in response to pressure or volume overload, ischemia-reperfusion injury or myocardial infarction, cardiac fibroblasts proliferate and transdifferentiate into myofibroblasts which produce collagen and pro-hypertrophic cytokines influencing cardiomyocyte function and size. it was shown that β-adrenergic stimulation of cfs with isoproterenol leads to angiotensin ii (at-ii) production and autocrine stimulation of these cells (jaffre et al, 2009 ). activation of phoypholipase c triggered by at-ii leads to formation of inositol trisphosphate (ip 3 ) and subsequent release of calcium from intracellular stores as well as calcium entry across the cell membrane. the focus of our research is the identification of the plasmalemmal channel proteins such as trpc channels mediating this calcium entry, and whether these calcium entry pathways in cfs contribute to pathological remodeling. to date the precise role of calcium entry for these pathological processes is largely unknown. trpc channels are candidates for the analysis of calcium homeostasis in cf. recently, we showed that trpc1/c4-deficient mice are protected from maladaptive cardiac remodeling after neurohumoral stimulation or pressure overload, respectively, which can be explained by a significant reduction of a background ca 2+ -entry (bcge) pathway in cardiomyocytes; this bgce is enhanced by stimulation with agonists such as isoproterenol or angiotensin ii and it critically depends on trpc1 and trpc4 (camacho londoño et al., 2015) . nevertheless, the role of trpc1/c4 for calcium homeostasis in cfs has not been analyzed so far. we established an in vitro model that allows the analysis of calcium release and entry triggered by several (patho)physiological agonists in cultured primary adult cfs from mice. cfs were isolated using langendorff-perfusion and were cultured for maximal 6 days. our results show that there is no difference in 100 nm at-ii induced ca 2+ -release or ca 2+ -entry in trpc1/c4-deficient cf compared to wt. to evaluate whether the lack of trpc1/c4 can be compensated by other trpc channels we currently analyze cfs from trpc hepta ko mice lacking all seven trpc channel proteins concerning at-ii induced ca 2+ -release and ca 2+ -entry and we will also analyze the influence of other agonists on cfs which are known to evoke a longer lasting rise in the [ca 2+ ] i like isoproterenol, 5-ht, thrombin and endothelin-1. cardiovascular and metabolic diseases are currently the primary cause of morbidity and mortality in the western world and are spreading to the rest of the world following globalization. adipose tissue, in particular perivascular adipose tissue (pvat) is recognized as an important player in the development of these diseases. the release of relaxing factor(s) from the pvat has been a matter of interesting and highly spirited debates about its nature, the channels that govern its activities and its role in vascular dysfunction. data from our laboratory indicate that adipose-derived relaxing factor (adrf) is an important player, however the potential channels necessary for its downstream activities are still under study. our recent research primarily focuses on kv7.1 channels, which are known to be expressed in vascular smooth muscle cells. k v 7.1 voltage-gated potassium channels are expressed in vascular smooth muscle cells (vsmc) of diverse arteries, including mesenteric arteries. based on pharmacological evidence using r-l3 (k v 7.1 opener), hmr1556, chromanol-293b (k v 7.1 inhibitors), these channels have been suggested to be involved in the regulation of vascular tone. however, the specificity of these drugs in vivo is uncertain. we used kcnq1-/-mice to determine whether k v 7.1 plays a role in the regulation of arterial tone. we found that r-l3 produces similar concentration-dependent relaxations (ec 50 ~1,4 µm) of wild-type (kcnq1+/+) and kcnq1-/-arteries pre-contracted with either phenylephrine or 60 mm kcl. this relaxation was not affected by 10 µm chromanol-293b, 10 µm hmr1556 or 30 µm xe991 (pan-k v 7 blocker). the anti-contractile effects of pvat were normal in kcnq1-/-arteries. chromanol-293b and hmr1556 did not affect the anti-contractile effects of perivascular adipose tissue (pvat). isolated vsmcs from kcnq1-/-mice exhibited normal peak k v currents. the k v 7.2-5 opener retigabine caused similar relaxations in kcnq1-/-and wild-type vessels. we conclude that k v 7.1 channels are apparently not involved in the control of arterial tone by alpha 1 adrenergic vasoconstrictors and pvat. in addition, r-l3 is an inappropriate pharmacological tool for studying the function of native vascular k v 7.1 channels in mice. introduction: according to international guidelines [1] [2] [3] [4] [5] [6] [7] , both human and animal skin in vitro models have been used and validated to predict percutaneous penetration in humans. excellent correlations have been found for domestic pig as surrogate for human skin [8] [9] . material and methods: tissue the skin samples are descended from female pigs of german landrace (50 kg weight, approx. 4 month). process approved under german welfare law. after narcotization and euthanization the animals were shaved with an electric shaver, washed and dried. microbiological investigation swabs from 10 different skin area (fig. 1) were taken before next preparation step. skin areas were cut, stretched and subcutaneous fatty tissue was carefully removed. the skin was harvested at thickness of 1.000 µm by dermatome. after dermatomization, samples were taken for histological examination. skin disks of 30 mm were punched out from the frozen skin stripes and stored at -20°c. hplc and skin absorption waters corporation hplc containing 2767 sample manager, 2545 binary gradient pump, 2998 pda detector (optional: 3100 electron spray mass spectrometer); column: nucleodur® 100-5 c18 ec 50mm x 4.0 mm id. hanson microette™ vision® diffusion test system (hanson vision® autoplus™ autosampler/autofill™ collector, 6-cell drive system with vertical diffusion cell "standard". the permeation experiment was performed over a period of 48 h at 32°c. the dosage compartments of each cell were filled with approximately 300 µl of the caffeine solution (10 mg/ml). samples of 1 ml are taken after 0, 12, 24, 36 and 48 hours from receptor medium of each cell. aliquots from each vdc are analyzed by hplc in duplicate. microbiology staphylococcus spp. were found explicitly on pig skin surface. bacteria are facultative anaerobe, gram-positive bacteria that are physiologically colonizing the skin, oropharynx and the gastrointestinal tract. histology and skin thickness he staining and mechanical skin thickness determinations confirmed intact dermatomizing process of skin (fig. 2) . thickness was in the order of magnitude between 897 to 1.230 µm and intra variations were less than 10 %. caffeine skin absorption permeability coefficients and lag-phases recorded are in the same order of magnitude of previous work [10], demonstrating intact barrier properties of the membranes after 3 month storage process. until today, intra-assay variations are superior or equal to interregional and inter-animal variations. discussion: well characterized dps1000 provides ready to use research tool for locally and systemic skin investigations. ongoing experiments will cover skin structure analysis by raman spectroscopy, biophotonics and storage impact on skin barrier function. respirable biopersistent granular dusts (gbs) should fulfill the criteria of i.) a negligible solubility in physiological lung fluid that ii.) do not exhibit a specific surface chemistryrelated toxicity at volumetric non-overload conditions in lungs. in 2012, the mak commission derived a new threshold value of 0.3 mg/m 3 for gbs with a density of 1, recognizing that at this concentration a chronic inflammation and increase of the lung cancer risk will not occur. -the objectives of the project were i.) to determine an experimental dissolution value for 'low soluble' gbs using six candidate dusts; ii.) in addition, to measure the inflammatory response in lung lavage fluid and to decide on the criterion 'inert dust'; iii.) to investigate whether nanoscaled dusts could possibly fulfill the criteria to be included in the gbs class. -six micro-and nanoscaled dusts (one of them a well-characterised inert tio 2 dust (microscaled; rutile modification) were compared analysing the solubility in the lung fluid (day 3, 28 and 90) and the lung toxicity after intratracheal instillation in rats (day 3 and 28): tio 2 (rutile, micro), tio 2 (anatase, nano), eu 2 o 3 (micro-nano mixed), baso 4 (micro), zro 2 (micro) and amorphous sio 2 (nano). two doses of 0.5 and 1.5 µl per rat were administered to wistar rats; these volume doses resulted in a non-overload and moderate overload of lungs, respectively. -the differential cell count showed only slight inflammatory cell levels after treatment with tio 2 (rutile) and baso 4 (pmn < 5% after 3 days in the low dose group; < 15% in the high dose group; full recovery after 28 days). in contrast, the tio 2 (anatase) showed a stronger response (pmn > 30% after 3 and 28 days). the rare earth eu 2 o 3 (micro-nano) dust showed the strongest effect (approx. 40% pmn after 3 and 28 days) including a red-coloured lung lavage fluid. µ-zro 2 and amorphous sio 2 showed a strong acute response after 3 days, however, mostly complete recovery after 28 days. the low solubility criterion was met by the following dusts: tio 2 (both) and zro 2 . -similar volumetric lung burdens were deposited in a parallel validation experiment (14-day subacute inhalations). overall the physiological inhalation route confirmed the results obtained in the instillation study, thus suggesting the applicability of the latter as a tool for identification for gbs dusts. however, for nanoscaled dusts an individual toxicological characterization seems to be adequate. polycyclic aromatic hydrocarbons (pah) represent a complex mixture of compounds and occur in considerable amounts as contaminants in the environment and food. some pah have been demonstrated to be carcinogenic and mutagenic. benzo[a]pyrene (bp), the most known and studied member of the potent carcinogenic pah, is classified by iarc as group 1 carcinogen and is present in a wide variety of food items. however, other non-carcinogenic pah such as pyrene (pyr) and fluoranthene (fa) are also found in substantial amounts in the diet and are strongly suspicious to cause interactive effects. reporter gene assays were used for analyzing interactive effects of a ternary mixture including bp, pyr and fa in relative proportions occurring in food on the nuclear receptors aryl hydrocarbon receptor (ahr) and constitutive androstane receptor (car). the observed activations were verified at the gene expression level in the human hepatoma cell line heparg. beside the well characterized ligand bp, 25 µm of pyr and 20 µm fa also activated the ahr, even though to a much lesser extent. no significant higher activation over the level of bp alone was reached when testing different pah mixtures. however, in heparg cells the analysis of cyp1a1 gene expression as a model target gene of ahr showed synergistic effects after pah co-exposure. in addition, the activation of human car was analyzed. pyr and fa are each strong agonists, whereas bp was less potent. for the ternary pah mixture with bp a strong decrease of the induction was observed. this inhibiting effect was verified at the mrna level using the model car target gene cyp2b6 in heparg cells. in conclusion, really occurring mixtures with non-carcinogenic pah can modulate the effects of carcinogenic pah. such effects warrant to be investigated in more detail to enhance our knowledge of interaction of pah mixtures at the molecular level. cytochrome p450 enzymes and transporters are important for the turnover of pharmaceutical compounds. their expression levels and activity influence bioavailability and convey drug-drug interactions. moreover, transporters mediate barrier maintenance of several organs such as blood-brain-barrier and placenta-barrier. overexpression of export transporters in tumors can lead to multiple drug resistance. however, membrane associated proteins are difficult to quantify by conventional bioanalytical methods such as sandwich immunoassays because of their hydrophobicity. antibody -based analysis of cytochrome p450 enzymes and transporters is challenging due to their sequence homology. therefore, we developed a test system for protein quantification which combines the sensitivity of immunoprecipitation and the specificity of mass spectrometry: this method is especially convenient for hydrophobic proteins because denatured samples are analyzed on peptide level. one peptide from each protein, which can be assigned unambiguously, is identified via tandem ms and quantified by means of an isotope labeled reference. prior to ms-read-out, the peptides are enriched by antibodies which recognize a very short c-terminal epitope. these epitopes are selected in such way that they are common in peptides derived from target proteins and therefore allow the analysis of protein groups with few antibodies. the major advantage of this method is that whole cell or tissue lysates -without preparation of microsomal fractions -can be used for quantification by lc-ms. also, samples from different model organisms can be analyzed with the same assay which enhances the comparability of experiments. physiologically based toxicokinetic modeling (pbtk) is an in silico tool to predict compound kinetics based on test substance related properties and physiological parameters of the organism. pbtk is a key element for inverse dosimetry to relate effect concentrations in vitro to external, e.g. oral doses. in our investigations, we use 8 compartment models for the rat including adrenals and testes or ovaries. test substance specific properties taken for pbtk modeling are molecular weight and logp o/w as well as ivis based tissue specific partition coefficients, hepatic clearance, intestinal permeability and plasma protein binding. berkeley madonna software was applied to solve consequent differential equations. here we present the above described model for the 3 test substances bisphenol a (bpa), fenarimol (fen) and ketoconazole (keto). using the lowest effect concentrations (loecs) of bpa, fen and keto from 1) an in vitro yeast based assays with human estrogen and androgen receptor combined with a reporter gene and 2) the interaction of steroidogenesis model calculations were made to relate in vitro concentrations to oral doses in the rat. model calculations, based on in vitro loecs of 10 µm (bpa), 3 µm (fen) and 0.01 µm (keto), for concentrations in target organs resulted in estimated oral loels of 16, 4 and 0.04 mg/kg. when calculations were made for plasma levels oral loels were estimated to be 608, 77 and 16 mg/kg for bpa, fen and keto, respectively when compared to existing in vivo data with endocrine related loels of 375 mg/kg bw day for bpa (1), 50 mg/kg day for fen (2) and 6 mg/kg day for keto (3) , it can be concluded that for the exemplary test substances addressed, ivis related risk assessment approaches based on target tissues seems overpredictive, whereas plasma related loels were closer to the in vivo situation, to study the activation of the nuclear receptor pxr a gal4/uas-based pxr transactivation assay was used. the pxr-mediated induction of cyp3a4 promotor activity was investigated using a pxr-dependent cyp3a4 reporter gene assay. cyp3a4 induction was analyzed at the mrna and protein levels in hepg2 cells using qpcr and western blot. to cover the most frequently occurring pa structures (retronecine, heliotridine and otonecine type as well as monoester, open-chain diester and cyclic diester) the four pa senecionine, heliotrine, echimidine and senkirkine were selected as representative pa for initial analyses. out of the four investigated pa only echimidine activated pxr. accordingly, pxrmediated induction of cyp3a4 promotor activity could only be detected for echimidine. cyp3a4 induction by echimidine was verified at the mrna and protein level in hepg2 wildtype and hepg2 pxr-overexpressing cells. taking heinrich-heine universität düsseldorf, institut für toxikologie, düsseldorf, germany introduction: in higher concentrations, the blood pressure regulating hormone angiotensin ii (ang ii), leads to vasoconstriction, hypertension and oxidative stress by activation of the renin angiotensin system (ras). here we investigate if nadphoxidases are responsible for ras-mediated oxidative stress in kidney and heart. nadph-oxidases (7 isoforms are known, nox 1-5, duox 1 & 2) are membrane-bound enzymes that produce reactive oxygen species (ros) for example during the immune response and cell signaling. material and methods: to clarify the role of nadph-oxidases, wildtype mice and nox 1-, nox 2-and nox 4-deficient mice were equipped with osmotic minipumps, delivering ang ii in a concentration of 600 ng/kg during 28 days to stimulate high blood pressure. kidney and heart were investigated for steady-state ros levels and dna damage (dna single and double strand breaks). results: in wildtype mice, ang ii leads to hypertension, a declined renal function, formation of ros in kidney and heart and to dna single and double strand breaks in the kidney. all nox-knockout mice exhibited ang ii-mediated hypertension and albuminuria. the lack of nox 2 and nox 4 could neither protect from the formation of oxidative stress in the kindey nor from dna double strand breaks in the kidney. initial findings from the nox 1-knockout mouse do not show an increase in dna double strand breaks in the kidney. discussion: contrary to published results of nox 1-knockout mice, we observed a constant rise in blood pressure over the treatment period compared to the control. this can possibly be due to different ang ii doses. in nox 4-knockout mice we observed increased oxidative stress and increased renal dna damage already in untreated control animals, which is in line with reports suggesting a protective effect of nox4. conclusion: separate eliminations of 3 nadph-isoforms did not allow the identification of the enzyme which is responsible for ang ii-induced oxidative stress. a possible explanation is that oxidative stress is caused by more than one nox-isoform or other enzymes like xanthine oxidase or nitrogen synthase take major part in the formation of ros. of mice (rats, cats, dogs, monkey) and men -how to measure kidney biomarkers across species introduction and objectives: there is a need for reliable biomarker assays to detect organ toxicity induced by drug candidates. in the last 50 years about 60 drugs were withdrawn from the market due to liver and/or kidney damage. for the detection of druginduced organ injury, safety-tox studies in rodents, dogs, non-human primates and humans are mandatory in the drug development process. currently, several protein biomarkers for kidney, liver and cardiovascular organ toxicitity are being clinically validated by international consortia like the safer and faster evidence-based translation (safe-t) or the predictive safety consortium (pstc). we are developing mass-spectrometry-based immuoassays suitable for the detection of these markers in animal models to support these efforts method: urinary proteins are proteolytically digested to peptides using trypsin. subsequently synthetic isotope-labelled peptide standards are spiked in at known concentrations. we employ multi-specific antibodies (txp-antibodies) targeting cterminal amino acid motifs for the enrichment of peptides derived from the protein biomarkers. finally, the protein biomarkers are quantified using nanolc-parallel reaction monitoring-ms. the use of our group-specific txp-antibodies allows protein analysis of samples from different species using the same antibody. results and discussion: we established an ms-based immunoassay platform for the analysis of kidney (diki) injury protein biomarkers in urine across 5 species; human, cynomolgus, mouse, rat and dog. we analyzed the potential diki biomarkers aquaporin 2, podocin, synaptopodin, retinol-binding protein 4, clusterin and osteopontin in urine samples from toxicity studies in cynomolgus monkeys, rodents and humans. conclusion: the application of group-specific txp-antibodies and mass spectrometry allows the quantification of biomarkers in urine of all relevant model organisms. the results strongly support the validation of translational drug-induced organ injury protein biomarkers. although effective anticancer-therapeutic regimen are available, they are accompanied by severe adverse effects on normal tissue, for instance chemotherapy induced peripheral neuropathy (cipn) caused by platinum compounds. the pathophysiology of this clinically highly relevant side-effect is still unknown and neither prophylaxis nor specific treatment is available. therefore, further research elucidating the underlying molecular mechanisms of platinating anti-tumor drugs leading to cipn is required as basis for future development of preventive or therapeutic strategies. in general, platinum compounds lead to cell death mainly via dna damage induction (mostly intrastrandcrosslinks) and through interference with the redox homeostasis of cells. here, we introduce and suggest the well-known nematode model organism c. elegans to elucidate mechanisms of neurotoxicity triggered by platinating agents. so far, we determined doses for cis-and oxaliplatin, which have only moderate effects on development, reproduction and body movement (muscular read-out). however, these doses are sufficient to trigger apoptosis in c. elegans and to induce a considerable amount of 1,2-intrastrand crosslinks in dna (measured by south-western blotting). even more important they lead to strong neurotoxicity in a functional read-out (pharyngeal pumping). with regard to redox homeostasis, we determined the oxidative stress resistance showing that e.g. cisplatin sensitizes c. elegans to reactive oxygen species (ros), which could be prevented if worms were co-or pretreated with n-acetylcysteine. furthermore we determined the level of ros in living c. elegans after treatment with platinating agents and also in combination with protective compounds. using the advantages of c. elegans as a genetic model system, we will further clarify the relevance of different defense mechanisms, including dna repair (nucleotide excision repair, base excision repair), detoxification systems (antioxidative stress factors, metallothioneins) as well as drug transporters and signaling proteins. this will be achieved by using rna interference approaches that allow targeting either the whole animal or specific tissues (i.e. neurons) only. first results of this approach will be presented. finally we aim to use this setup to identify neuroprotective compounds that prevent chemotherapy induced peripheral neuropathy induced by platinating anti-tumor drugs. clostridium botulinum c3 exoenyzme (c3) exclusively adp-ribosylates rhoa, b and c leading to reorganization of the actin cytoskeleton and morphological changes. in addition to the enzyme-based inhibition of rho-gtpases, c3 promotes in an enzymeindependent manner axonal and dendritic growth in neurons. as c3 lacks the canonical binding and translocation domains of bacterial protein toxins, cell entry is currently not well understood. based on overlay assays and mass spec analyses the intermediate filament vimentin was identified as the putative membrane receptor for c3. knock down of vimentin by sirna and application of the selective vimentin disruptor acrylamide led to a significantly delayed uptake of c3. moreover, addition of extracellular vimentin to cells induced an enhanced uptake of c3. proof of principle experiments in astrocytes and neurons from vimentin knock out mice showed c3-induced morphological changes (astrocyte stellation and axon growth) to a reduced extent and a significantly delayed uptake of c3 compared to wild type cells. as vimentin knock out did not completely inhibit c3 uptake into cells, an additional uptake mechanism or additional receptor for c3 is likely. nevertheless, our data reveals that c3 employs a specific endocytosis mechanism with involvement of the intermediate filament vimentin to gain access to host cells. the primary target organ of organic hg species-mediated toxicity is the central nervous system (cns). humans are exposed to organic hg mainly in the form of methylmercury (mehg) via the consumption of contaminated fish and other seafood products. in terrestrial food sources hg is mostly found as inorganic hg. thiomersal is a further organic hg compound which is used as a preservative in medical preparations. exposure to organic hg promotes primarily neurological effects. the understanding of transfer mechanisms regarding the cns is an important precondition for an evaluation of hg species-induced neurotoxicity. thus, primary porcine in vitro models of the bloodbrain barrier and the blood-cerebrospinal fluid (csf) barrier were used to investigate effects of mehgcl, thiomersal and hgcl 2 on the barriers as well as transfer properties into and out of the cns in vitro. the results show significant transfer differences of the various incubated species as well as in the different barrier systems. whereas the bloodbrain barrier seems to account for the transfer of organic hg species from the blood side to the brain side, these species are transferred in the contrary direction by the blood-csf barrier. inorganic hgcl 2 was not transferred across both brain barriers towards the brain side but was able to leave the brain side across the blood-brain barrier. additionally, cytotoxic effects of the hg species by themselves as well as the combination of organic and inorganic hg species have been investigated in human astrocytes and human differentiated neurons. differentiated neurons were much more sensitive towards all hg species. organic species exerted stronger cytotoxic effects in both cell types as compared to hgcl 2 . interestingly, a coincubation of organic and inorganic hg species led to an increased cytotoxicity in the astrocytes. this cocytotoxic effect is currently investigated in differentiated neurons. the species-specific differences with respect to both, effects on and transfer across the blood-brain and the blood-csf barrier in vitro as well as toxic effects in brain target cells, clearly emphasizes the necessity for comparative analyses. introduction: the neural crest is a multipotent stem cell population that arises at the neural plate border during early fetal development. neural crest cells (nccs) migrate to target sites in the periphery, where they differentiate into multiple cell types, including melanocytes, cranial bones and peripheral neurons. failure of ncc migration can lead to severe disorders, such as hirschsprung's disease. aim: to test whether toxicants interfere with human ncc migration, a high-throughput migration assay was established. this test system was used to screen an 80 compound library of potential developmental toxicants. methods: nccs were derived from human embryonic stem cells. the cells were allowed to migrate for 24 h before toxicants were added to the cells. migration and viability of the cells were then measured after another 24 h by high-content image analysis and a custom-developed software package. results: the screening library was assembled by the us national toxicology program (ntp) and consisted of different substance classes, e.g. organophosphates, organochlorines, drug-like compounds, pesticides and polycyclic aromatic hydrocarbons (pahs). out of the tested potential developmental toxicants, 26 compounds reduced ncc migration at non-cytotoxic concentrations. hit-confirmation testing confirmed 23 of the compounds as concentration-dependent inhibitors of ncc migration. among the potential developmental toxicants identified here, there were several organophosphates (e.g. chlorpyriphos) and drug-like compounds as well as polybrominated diphenyl ethers (pbdes) and organochlorine pesticides (e.g. ddt and dieldrin), while none of the tested pahs inhibited ncc migration. the negative controls in the screening library, like acetylsalicylic acid, acetaminophen and saccharin, proved to be non-toxic. conclusion/outlook: the newly established test system allows screening of potential developmental toxicants in a high throughput manner for interference with human ncc migration. confirmation in other types of migration assays is ongoing, and selected compounds from amongst the screen hits are undergoing mechanistic evaluation. oxidative stress is regarded as a major trigger for neuronal dysfunction and death in the ageing brain and in multiple neurodegenerative disorders. how oxidative stress mediates neuronal death and whether the associated mechanisms are accessible for therapeutic intervention strategies is not clarified. increasing evidence suggests, however, that oxidative stress triggers molecular mechanisms of regulated necrosis that involve the activation of receptor interacting protein 1 (rip1) independently of death receptor activation. here, we show that erastin-induced ferroptosis which involves inhibition of the glutamate-cystein transporter (xc -), glutathione depletion and lethal formation of reactive oxygen species (ros) 1 , triggers mechanisms of regulated necrosis independent of tnfα-signaling. in hippocampal ht-22 cells erastin promotes activation of rip1 and subsequent rip1-rip3 necrosome formation which has been investigated as a hallmark of regulated necrosis 2 . in fact, silencing of rip1 by sirna or by the rip1 inhibitor necrostatin-1 prevents ferroptosis-induced cell death whereas the ferroptosis inhibitor ferrostatin-1 fails to protect cells against tnfα-induced classical necroptosis, a form of programmed cell death that is mediated by receptor interacting protein-1 (rip1) and rip3 kinases downstream of death receptor activation (e.g. tumor necrosis factor receptor tnfr) 2, 3 . recently, a genome-wide sirna screen linked cylindromatosis (cyld) to rip1/rip3-dependent necroptosis 4 and also in the present paradigm of ferroptosis, cyld depletion promotes neuronal survival and decreases rip1-rip3 complex formation, suggesting a role of cyld in intrinsic pathways of regulated necrosis triggered by oxidative stress. the ns5a inhibitor daclatasvir is used in combination with other antivirals such as the polymerase inhibitor sofosbuvir for treatment of chronic infection with the hepatitis c virus. daclatasvir is embryotoxic and teratogenic in rats and rabbits at exposures at or above the clinical exposure. in contrast, no teratogenic effects were observed in rat and rabbit developmental toxicity studies with ledipasvir, another ns5a inhibitor. we studied these compounds in the embryonic stem cell test (est) alone and in combination with sofosbuvir. the ns5a inhibitors were obtained from selleckchem, the main metabolite of sofosbuvir, psi-6202, was from medchem express. murine embryonic stem cells (es-d3) were obtained from atcc. they were kept in iscove's modified dulbecco's medium (imdm). substances were dissolved in dmso at a final dmso-concentration of 0.1% in the culture medium. a cytotoxicity assay as well as a differentiation assay were performed. after 10 days in culture the cells were evaluated. cytotoxicity was measured by an mtt test. differentiation into contracting myocardial cells was determined using direct phase contrast microscopy. the substances were tested at concentrations between 0.1 and 30 mg/l, which is a broad coverage of the therapeutically relevant concentrations reached in patients. at a concentration of 10 mg daclatasvir / l medium and higher the substance inhibited differentiation of cells. we observed contracting myocytes in 23, 22 and 2 wells out of 24 wells in total at concentrations of 1, 3 and 10 mg/l. at 30 mg/l no differentiation was observed. effects on cell viability were observed at 30 mg/l. unexpectedly, we found a higher potency with ledipasvir. at the low, therapeutically relevant concentration of 1 mg/l this nsa5-inibitor showed a clear impact on differentiation with 6 out of 24 wells affected and no differentiation at higher concentrations. addition of sofosbuvir or its main metabolite psi-6206 at concentrations up to 30 mg/l had no influence on the concentration effect curves established for daclatasvir or ledipasvir. this is the first indication of an embryotoxic potential of ledipasvir. the difference to the results from the routinely performed animal experiments is unknown. possibly, metabolic activity in the maternal organism is responsible for this discrepancy. dimoxystrobin is a european-registered pesticidal active ingredient. biologically it is acting as an inhibitor of the fungal respiratory chain. for the purpose of european registration a full set of toxicological studies has been conducted with dimoxystrobin, including reproduction toxicity studies (according to the most recent oecd tg 416) and developmental toxicity studies (oecd tg 414) in rats and rabbits. dimoxystrobin interferes with the iron transport in rats and mice. this leads to lower serum iron levels and anemia in rats after repeated exposure. this holds true for treated dams and offspring in reproduction toxicity studies. furthermore, offspring effects seen at the high dose of the 2-generation toxicity study were a hypochromic microcytic anemia, impaired body weight development, which only developed postnatally, and reversible cardiomegalies in some 21-days old pups. for all effects clear noaels were determined. in the 2-generation toxicity study no dose adjustment during pregnancy and lactation was performed, which resulted in considerably higher food and compound intakes in dams and offspring during these lifestages. as a result, it seemed, that pups were more severely affected by body weight effects compared to the parental generation. by performing a life-stage specific comparison of body weight and substance intakes, as well as benchmark dose calculations (bmd) for these parameters, it could be demonstrated that the point of departures (pods) and the loaels for direct dimoxystrobin-related effects were comparable for offspring and parents. the heart effects (cardiomegaly), which were reversible, occurred only after direct dimoxystrobinexposure and are considered to be secondary to the detected offspring anemia. both effects (lower body weights and offspring cardiomegalies) only occur postnatally and are not the consequence of in-utero exposure, as no respective effects at higher doses in rat prenatal toxicity studies were seen. two new mechanistic studies (1-generation toxicity study and a 3-week study in young and adult rats, additionally investigating serum iron levels and anemia) confirmed, that pups and young rats were not more sensitive than adult animals to develop anemia or decreased serum iron levels. in 2006, dimoxystrobin was classified with r63 (possible risk of harm to the unborn child) by the ecb, which was the european authority responsible for classification and labeling, before echa in helsinki was formed. the r63 (which has been translated into the ghs classification repr. 2, h361d) was based on offspring body weight and heart effects seen in the 2-generation toxicity study. based on a comprehensive re-evaluation of existing and on new data of dimoxystrobin, the conclusion can be drawn, that a classification for reproduction toxicity is scientifically not justified and should be reconsidered. perfluorooctanesulfonic acid (pfos) and perfluorooctanoic acid (pfoa) are perfluorinated substances (pfas) which are used for the fabrication of surfaces with water-and dirt-repellent properties. due to their reprotoxic properties and their persistence in the environment, the use of pfos was restricted in 2009 and a restriction program for pfoa was initiated in 2013. therefore, industry switches to pfoa and pfos substitutes, which are predominantly pfas with a shorter carbon chain length, or structure-related compounds. in contrast to pfoa and pfos only few toxicological data are available for their substitutes. aim of this study was to examine endocrine effects of the substitutes perfluorohexanesulfonic acid (pfhxs), perfluorobutanesulfonic acid (pfbs), perfluorohexanoic acid (pfhxa), perfluorobutanoic acid (pfba) and 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propionic acid (genx) in comparison to pfoa and pfos. a hek-293t cell-based dual-luciferase reporter gene assay was used to investigate the potential of these compounds to affect the activity of the human estrogen receptors herα and herβ. the reporter gene assay revealed no activation of herα or herβ by the pfas tested in this study. to investigate the potential inhibition of herα and herβ by pfas, a coincubation with the estrogen receptor agonist 17β-estradiol was performed. none of the tested pfas inhibited herα or herβ activity. however, in the case of herβ an enhancement of 17β-estradiol-stimulated activity was observed. thus, pfas do not directly activate or inhibit the human estrogen receptors but have an impact on herβ activity as they amplify the activation mediated by 17β-estradiol. further studies will be conducted to examine this synergistic effect in more detail. the xeer-reporter cell line: a novel dual-color luciferase reporter assay for simultaneous detection of estrogen and arylhydrocarbon receptor activation p. tarnow consumers are exposed to a multitude of anthropogenic and natural substances capable of activating or inhibiting ligand activated transcription factors, respectively. this in turn can lead to adverse health effects, particularly for substances acting on signalling pathways that are subject to regulatory crosstalk such as xenoestrogens and polycyclic aromatic hydrocarbons (pahs). xenoestrogens are known to activate human estrogen receptors (ers), whereas pahs or dioxins act on the arylhydrocarbon receptor (ahr). importantly, both receptor signalling pathways are interconnected by a complex crosstalk on multiple levels. this ranges from direct protein-protein interactions to competition for common co-factors. however, although this cross-talk has been long known we still lack a deeper understanding of its molecular mechanisms and physiological implications. one reason for this is a lack of tools to visualise and investigate receptor interaction in vivo. based on the breast cancer cell line t47d we thus developed a dualcolour reporter assay which allows time-resolved simultaneous monitoring of the activation of er and ahr in living cells. the assay uses two beetle luciferases emitting luminescence in the red (slr) and the green (eluc) spectrum, respectively. while eluc is expressed under the control of a 6-fold repeated xenobiotic response element (xre) slr is subject to transcription regulation by a 6-fold repeated estrogen response element (ere). both constructs were stably transfected into t47d human breast cancer cells, which endogenously express erα and ahr and are thus ideally suited for monitoring interactions with both receptors. the respective "xeer"-cell line has been successfully subjected to proof of principle studies, using prototypical er-and ahrligands as well as various phytochemicals and xenobiotics. besides e2 and tcdd ligands included various pahs, polychlorinated biphenyls, alpha-and betanaphthoflavone, cosmetic ingredients (butylparaben, benzophenone-2 and 4-mbc), bisphenol a, genistein, resveratrol, diindoylmethane as well as pharmacological antagonists of both receptors. asian women consuming soy rich food throughout life possess lower levels of 17betaestradiol (e2) in plasma (pl) than western women, whose diet is characterized by less soy consumption during early life and possible intake of soy based dietary supplements during adulthood. however, the impact of these soy exposure scenarios on estrogen (biotrans)formation and the consequence thereof in female mammary glands (mg) has not been investigated yet. thus, female august copenhagen irish rats were fed either isoflavone (if) depleted diet (idd, western exposure scenario without if supplement) or if rich diet (ird, asian exposure scenario) until the end of the study at postnatal day (pnd) 81. furthermore, rats fed idd until pnd74 were fed ird for 7 days (idd+ird, western dietary exposure scenario with if supplement). estrous was determined histologically. levels of transcripts were determined by qpcr and e2 and estrone (e1) in pl and mg were quantified by gc-ms/ms. statistical analyses of estrogens were performed by kruskal wallis and unpaired wilcoxon tests and of transcript levels by linear regression models considering the explanatory variables tissue levels of e2 and diet (idd vs ird and idd vs idd+ird). e2 levels in pl and mg did not coincide with those predicted by estrous. furthermore, median levels of e1 and ratios e2/e1 in mg and ratio of e2 levels in pl/mg were not affected by diet. in contrast, diet tended to affect e2 concentrations in pl (p=0.1211) due to an increase in the ird group (p=0.0056) whereas e2 levels in the idd+ird group only tended to be elevated (p=0.0788). in mg, ird and idd+ird increased e2 levels only weakly (p=0.0788 each). likewise, besides significant changes in transcript levels of cyp1a1 and 1a2, putatively decreasing oxidation of e2 to catechols, in the idd+ird group and (not significantly) also in the ird group, no changes in transcript levels putatively affecting e2 levels were observed. moreover, no decrease in levels of transcripts indicative for cellular (oxidative) stress (gclc, tp53, mt1a) was observed in the idd+ird group. e2 mg levels were significantly associated with an increase in transcript levels of areg and pgr, indicating activation of estrogen receptor (er). in contrast, ird was associated with a significant and idd+ird with a not significant decrease in pgr transcript levels. e2 levels but not diet were significantly associated with gata3 transcript levels, indicating tissue differentiation. furthermore, levels of transcripts involved in intercellular communication (egfr, wnt4) were significantly decreased by idd+ird and not significantly by ird and differed from that affected by e2 (increase in gdf15, hgf, igf1r, wnt5a). bmf, a marker transcript for apoptosis was increased by ird, but not affected by e2 and even decreased not significantly by idd+ird. taken together, despite an increase in e2 levels in pl, less er activation was observed after dietary exposure to if. whereas e2 and transcript levels of enzymes involved in e2 (biotrans)formation as well as er activation and cellular communication were affected similarly but to a different extend in both asian and western if exposure scenarios, differences in apoptosis were observed between ird and idd+ird groups. supported by dfg le 1329/10-1. august copenhagen irish (aci) rats with 17β-estradiol (e2)-releasing implants are an accepted model to study the etiology of breast cancer, but neither e2 (biotrans)formation in mammary gland tissues (mg) during tumorigenesis, nor the impact of isoflavones (if) shown to affect tumorigenesis in aci rats, has been investigated, yet. therefore at postnatal day (pnd) 75 and 175, placebo (-e2) or silastic implants containing 4 mg e2 were implanted in female aci rats exposed to either if depleted diet (idd) or if rich diet (ird) since conception until the end of the study at pnd 285. palpable mg tumors (pt) and 1-2 mg per animal without pt were characterized histologically and categorized into normal (-e2 group, n=12), hyperplasia and non-pt and pt with and without solid tumors (+e2 group, n=32). e2, estrone (e1), their hydroxylation products and methylation (meo-) products thereof, as well as conjugates of e1 and e2 in plasmas and mg were analyzed by gc-and uhplc-ms/ms, respectively. levels of 49 transcripts involved in (biotrans)formation of e2 and estrogen receptor (er) activation were determined by taqman®-pcr. without exogenous e2, plasma e2 as well as e1 and (borderline) e2 levels in mg were higher in ird. plasma e2 as well as e1 and e2 levels in mg were lower in the -e2 group than that in the +e2 group. e2 levels as well as e2/e1 and e2 mg/plasma ratios were elevated in pt, accompanied by a significant increase in transcript levels indicative for estrogen receptor activation (areg, pgr) and proliferation (mki67). ird increased e2/e1 ratio in pt and, although ird did not affect er activation (areg, pgr), ird increased differentiation (gata3) in normal and hyperplastic tissues and tended to decrease proliferation in hyperplastic (ccnd1) tissues. levels of e1 and 2-meo-e1 were highest in hyperplastic tissues, accompanied by an increase in transcript levels of hsd17b2 (conversion e2 to e1) and cyp1a1. transcript levels of gstm1 and gstm2 were decreased in the whole +e2 group and of gstt1 and gstt3 in hyperplastic tissues, possibly decreasing inactivation of electrophilic metabolites. accordingly, maximum transcript levels of tp53 and mt1a indicating cellular (oxidative) stress were observed in hyperplastic tissues. ird did neither affect levels of 2-meo-e1 nor cellular stress (gclc, mt1a, tp53). of note, neither 4-meo-e1, nor e1 catechols, nor e2 catechols nor methylation products of the latter were observed in any sample. furthermore, no conjugates of e1 or e2 were detected in plasmas and mammary gland tissues. thus, changes in transcript levels of conjugating enzymes induced by tumorigenesis and by ird were not related with detectable conjugate levels of e1 or e2. taken together, whereas hyperplastic tissues were characterized by maximum oxidative metabolism of e1 and cellular (oxidative) stress, pt exhibited highest e2 levels and er activation. ird increased differentiation and decreased proliferation in normal and hyperplastic tissues but increased e2/e1 ratio in pt. supported by dfg le-1329/10-1. level of 17beta-estradiol (e2) in human breast tissue is considered to affect breast cancer initiation, promotion and progression. although putatively beneficial and adverse effects of soy isoflavones (if) on the human mammary gland, in particular in western women, have been discussed extensively, the influence of if levels on estrogen formation in human mammary gland tissue has not been investigated yet. thus, glandular tissues were dissected from 37 mammoplasty specimen obtained from women (age 18-66 years old) not taking estrogen active drugs. 14 of these women had been exposed to if by their usual diet or by intake of a soy-based dietary supplement for 7 days prior to mammoplasty. information on soy consumption and lifestyle were collected by questionnaire and tissues were characterized histologically. genistein, daidzein their conjugates (n=12) and bacterial metabolites (n=7) as well as the estrogens estrone (e1)-sulfate, e1, e2 and 2-methoxy-e1 were determined by uhplcand gc-ms/ms, respectively and transcript levels of 19 enzymes involved in e2 (biotrans)formation were quantified by taqman®-pcr in glandular tissues. isoflavonoids were categorized into the if parameters aglycones (agl) and conjugates (con) of either genistein, daidzein or sum of both and were further statistically analyzed by spearman`s rank correlation analysis. a positive correlation of e2/e1 ratio with agl(+con) was observed in glandular tissues (r=0.49, p=0.002), accompanied by a significant negative correlation of e1 levels with agl (r=-0.35/p=0.032), possibly due to reduction of 17beta-hydroxysteroid dehydrogenase 2 (conversion of e2 to e1) expression as indicated by a weak negative correlation of transcript levels of 17beta-hydroxysteroid dehydrogenase 2 with agl+con (r=-0.25, p=0.080). further statistical analysis taking into account multiple variables using linear regression models will provide more insights into variables affecting e1/e2 ratio. taken together, estrogen profile in human glandular breast tissue seems to be affected by if levels. supported by dfg le-1329/10-1. allergic contact dermatitis (acd) is a widespread disease often caused by substances in consumables. the eu prohibits the testing of cosmetic ingredients in vivo. this urges the development of reliable in vitro testing strategies. activation of dendritic cells (dcs) represents a key step during sensitization as they are essential for selection and priming of allergen specific effector t cells. in an integrated omics approach we aimed to further elucidate the molecular mechanisms of dc activation using quantitative metabolomics and proteomics. monocytic thp-1 cells were used as a model system and treated with the sensitizer 2,4dinitrochlorobenzene (dncb; 5, 10 and 20 µm) and the irritant sodium dodecyl sulfate (sds; 100 µm). samples were taken after 4, 8 and 24 hours. thp-1 activation was analyzed by measuring the established activation markers cd86 and cd54 after 24 hours. a targeted lc-ms/ms approach was used to analyze 188 metabolites including amino acids and lipids. protein levels were quantified by nano-lc-maldi-ms/ms after stable isotope labeling by amino acids in cell culture (silac). data sets were examined by multivariate analyses for identification of biomarker candidates. regulated metabolites and proteins were subjected to pathway analysis. the data presented might contribute to the further development of suitable in vitro testing methods for chemical-mediated sensitization. drug induced liver injury (dili) is one of the most frequent causes of acute liver injury and a main cause for drug withdrawals. currently there are no reliable models to test the dili potential of new compounds available. kupffer cells (kc) play an important role in hepatic cell stress mediated through chemokines and release of endogenous proteins. kc activation by damaged or stressed hepatocytes can lead to activation of the nf-κb signaling pathway transmitted by reactive oxygen intermediates (roi). we have recently established a liver model composed of primary human hepatocytes (phh) and kc which enables investigation of immune reactions after induction of hepatocyte stress (kegel et al., 2015) . aim of the present study was the kinetic investigation of hepatic cell stress induction and macrophage activation after treatment with subtoxic concentrations of hepatotoxic drugs. primary human hepatocytes (phh) and kc were isolated from human liver resectates using a two-step collagenase perfusion technique. initial kc activation was characterized by the dcf assay and immunofluorescence staining. phh were incubated with different concentrations of acetaminophen (apap) and diclofenac (dic) for different time intervals. cell stress was evaluated by measurement of oxidative stress (dcfassay) and viability (xtt-assay). in order to simulate macrophage activation following hepatocyte damage, kc and macrophages derived from the monocytic cell line thp-1 were incubated with supernatants of phh treated with hepatotoxic compounds. kc and thp-1-macrophage activation were investigated by measuring intracellular formation of roi using the dcf-assay and cell activity using the xtt-assay. the characterization of kc activation revealed a donor and disease dependent kc activation resulting in kc differentiation to pro-and anti-inflammatory macrophages. therefore, kc were substituted by macrophages derived from thp-1 cells. evaluation of hepatic cell stress showed the strongest effect on thp-1-macrophages when phh were incubated with apap or dic for 4 h.treatment of kc and thp-1-macrophages with supernatants of phh challenged for 4 h with hepatotoxic compounds indicates that thp-1-derived macrophages react similar to kc when treated with phh supernatants in terms of cell activity and roi-production. in conclusion, thp-1 derived macrophages might be a suitable alternative to kc concerning macrophage activation. the evaluated kinetic window of 4 h covering hepatic stress induction and immune reaction allows to perform these measurements in a since the use of cerium dioxide nanoparticles is known to be beneficial e.g. in terms of reducing fuel consumption when added to diesel fuel it has become a frequently used nanomaterial. to compensate the concurrent lack of information on its toxicology a 90day nose-only inhalation study was initiated. by comparing the results to a combined chronic inhalation toxicity and carcinogenicity study using the same test items and experimental conditions (basf, ludwigshafen, germany) early indicators for genotoxic and carcinogenic effects should be determined. rats were exposed to 0, 0.1, 0.3, 1 and 3 mg/cm³ ceo 2 as well as 50 mg/m³ baso 4 nanoparticles (6 h/day, 5 days/week, 13 weeks). animal dissections were conducted at five time points (exposure day 1 and 28; recovery day 1, 28 and 90) aiming for endpoints mandatory according to oecd guideline 413. additionally, gene expression analyses in isolated pneumocytes type ii were performed using pathway arrays for inflammation, oxidative stress, genotoxicity, apoptosis and lung cancer. the given results intend the identification of marker genes displaying modulated expression in response to nanoparticle exposure. investigations on ceo 2 and baso 4 retention in the lung are also included in this project. in bronchoalveolar lavage fluid (balf) a time and dose-dependent increase of inflammatory cells has been detected up to the end of exposure. the amount of inflammatory cells decreased during post-exposure; however, in the high dose group a persistent inflammation up to 90 days was detected by balf and histopathology examination. based on our current results effects of ceo 2 nanoparticles on the respiratory system are suggested. its relevance in the context of long term effects such as tumor development needs to be estimated considering all investigations included in this study. the inhalt-90 project is funded by the german federal ministry of education and research (bmbf) -03x0149a. core or coating material? what dictates the uptake and translocation of nanoparticles in vitro? nanoparticles are becoming increasingly important role in consumer-related products. understanding the interactions between nanoscaled objects and living cells is therefore of great importance for risk assessment. in this context, it is generally accepted that nanoparticle size and shape are crucial parameters regarding the potential of nanoparticles to penetrate cell membranes and epithelial barriers. current research in this field additionally focuses on the particle coating material. in order to distinguish between core-and coating-related effects in nanoparticle uptake and translocation behavior, this study investigated two nanoparticles equal in size, coating and charge but different in core material. silver and iron oxide were chosen as core materials to ensure similar nanoparticles characteristics after particle synthesis. nanoparticles were coated with poly (acrylic acid) (pas) and extensively characterized by tem (transmission electron microscopy), saxs (small-angle x-ray scattering), zetasizer tm and nanosight tm . for uptake and transport studies the widely used human intestinal caco-2 model in a transwell tm -system with subsequent elemental analysis (aas) was used. for evaluation and particle visualization transmission electron microscopy (tem) and ion beam microscopy (ibm) were conducted. although similar in size, charge and coating material, the behavior of particles in caco-2 cells was quite different. the internalized amount was comparable, but pas-coated iron oxide nanoparticles were additionally transported through the cells. by contrast, pascoated silver nanoparticles remained in the cells. our findings suggest that the coating material influenced only the uptake of the nanoparticles whereas the translocation was determined by the core material. in summary, a core-dependent effect on nanoparticle translocation was revealed. both the uptake and transport of nanoparticles in and through cells should be considered when discussing nanoparticle fate and safety. nanotechnology is having a great impact not only on basic research but also on many sectors of industry opening the market for numerous new applications ranging from electronics to the health care system. besides their great innovative potential, the large variety of existing synthetic nanomaterials used in the last decade represents a major challenge for scientists and regulators in terms of measuring and assessing the potential hazard caused by the materials or the products themselves. equally, consumers often miss reliable and easy-to-understand information on nanomaterials and nanotechnology and do not know where to get such information. therefore, the international dana 2.0 expert team brings together its expertise and knowledge from different research areas dealing with all aspects of nanosafety research in order to create and provide easy-to-understand, up-to-date and quality-approved nanomaterials' knowledge base on www.nanopartikel.info. this information platform covers the 25 market-relevant nanomaterials focusing on their effects on the safety of humans and the environment.in order to manage and asses the rapidly increasing number of publications related to nanosafety issues, the dana 2.0 project developed a customised methodology «literature criteria checklist», which includes mandatory and desirable assessment criteria covering physico-chemical characterisation, sample preparation and (biological) testing parameters. this checklist facilitates the discrimination between high-and low quality publications and all positively evaluated literature is then fed into the dana knowledge base. accounting for the need to harmonise experimental practices, the dana team also developed a template for standard operation procedures (sop) to support careful scientific practice. validated protocols generated within the german bmbf-funded nanosafety research projects are presented together with results from the swiss ccmx project v.i.g.o. and available for download. another unique feature of the dana knowledge base is the integrated application-based database that provides a unique link between nanomaterials in real applications (e.g. environmental remediation or medical products) and their potential impacts/ toxicological effect(s) that can be easily accessed by the interested visitor. additionally, dana2.0 provides a list of faqs, a link platform with contact data to other information portals and the opportunity to directly pose questions to our experts via e-mail. dana 2.0 is also present on twitter, follow us @nano_info. background: particulate matter of combustion processes enhances cardio-vascular diseases and increases associated mortality rates. around 13% of total pm10 emissions are emitted by wood burners (uba 2006) . how wood combustion aerosols (particles and gasses) can affect human lung cells and how such cellular responses depend on the usage of different wood types and burners is widely unknown. methods: in an exposure chamber imitating the human respiratory tract human alveolar cells (a549) were exposed at an air-liquid-interface (ali) to gasses and particles of wood combustion aerosols. log wood of beech, birch and spruce was burnt in a conventional oven and compared to the combustion of wood pellets in a modern pellet burner. the combustion aerosols were diluted 1:40 and directly delivered to the exposure chamber. after 4h exposure the lung cells were lysed and rna was isolated. in an array based transcription analysis of the whole genome the effects of the aerosol exposures on lung cells was assessed. in parallel, physical and chemical parameters of the combustion aerosols were analyzed. results: the combustion aerosol of wood pellets contained less organic substances than the log wood aerosols, but was higher in its zinc content. genome-wide we found a higher number of regulated genes with combustion of pellets compared to combustion of log wood. the gas phase alone (filtered aerosol) showed comparable gene regulatory activity as the particle-containing total aerosol. aerosol from log wood burning induced mainly genes of the xenobiotic metabolism and cellular signaling. pellet aerosols additionally regulated apoptosis and dna repair processes. conclusions: modern pellet burners reach better combustion efficiencies than conventional log wood ovens, but their emissions seem to stress human lung cells stronger. one reason might be the higher zinc content of wood pellet aerosols. multiwalled carbon nanotubes (mwcnts) may pose as a risk similar to asbestos in causing cancer, notably mesothelioma, which is a malignant tumor originating from mesothelial cells. to identify molecular cues leading to mesothelioma development, we performed genome-wide transcriptome analysis using microarrays in primary human peritoneal mesothelial lp9 cells treated with two different tumor-inducing tailor-made mwcnts (rat model; rittinghausen et al. 2014 part fibre toxicol 11:59), or amosite asbestos at 3 µg/cm2 for 24 h. specifically, we determined how the transcriptomic changes of the highly tumorigenic mwcnt a would differ from another tumor-inducing mwcnt with albeit lesser potency (mwcnt d), long amosite asbestos as positive control, and milled mwcnt a as material control. initial analysis using bioinformatic tools, revealed 3788 significantly differentially regulated genes for mwcnt a, 1680 for mwcnt d, 145 for amosite, and 4 for milled mwcnt. further analyses with ingenuity pathway analysis comparing the two different mwcnt types and amosite, found common as well as exclusive biomarkers. interestingly, we identified many differentially regulated genes implicated in cellular senescence, a growth arrest in response to different stressors including dna damage, disrupted chromatin, and strong mitogenic signals. paradoxically, cellular senescence can represent both tumor suppression and tumor promotion mechanisms. more important, we found differential expression of genes associated with senescence-associated secretory phenotype (sasp) such as inflammatory cytokines, chemokines, proteases, and growth factors, which were manyfolds up-and down-regulated in mwcnt a, compared to mwcnt d and amosite. the mechanisms leading to mesothelioma induction by mwcnts are from far clear, but the key information emerging from the present transcriptomic data, together with our previously identified senescence markers, indicate that cellular senescence has a likely role. nanotechnology offers great advantages for the food industry despite its partly unknown risks, whose enlightenment is the main target of nanotoxicology. due to variability in terms of size, material, shape, surface texture and several endogenous influences, the toxicity of most frequently used and ingested nanomaterials is difficult to estimate. therefore, the aim of this study was the in vitro investigation of toxicological endpoints such as cell viability, dna integrity and the induction of apoptotic processes in human colon carcinoma cells (ht29). for this purpose ht29 cells were exposed for 24 hours with metal nanoparticles (gold, silver) and metal oxide nanoparticles (copper oxide, titanium dioxide, zinc oxide) in concentrations of 2-10 µg/ml. at first the cellular uptake of the nanoparticles by means of an icpms was determined. the influence of cell viability was demonstrated by the trypan blue staining and the mtt assay. the alkaline comet assay gave information about possible dna damages and the use of the repair enzyme formamidopyrimidine dna glycosylase (fpg) additionally allowed the detection of oxidized bases. the induction of programmed cell death was examined using by annexin v fitc assay. the icp-ms data showed a maximum particle content of 1.39 pg per cell for the used concentration range. the metal oxide nanoparticles resulted in a significant reduction of cell viability with a decrease up to 40 % after copper oxide and zinc oxide treatment. for metal particles, only for silver a reduced cell metabolism of about 50 % was detectable by the mtt assay. low genotoxic effects could be determined for silver nanoparticles (tail intensity about 12%; control about 6%), while for titanium dioxide the amount of oxidized bases was additionally increased (tail intensity about 20%; control about 6%) for concentrations above 8 µg/ml. induction of apoptosis was determined for silver particles (up to 24% early apoptotic and 20% late apoptotic cells) as well as for titanium dioxide and zinc oxide (10% each early apoptotic cells), whereby the most significant increase in late apoptotic cells was detected for zinc oxide (up to 90%). the results obtained in our studies indicate a clear particle-dependent influence on cell viability and apoptosis-triggering processes, depending on the used material or the concentration deployed, while only minor changes of dna integrity were detected. the evolution in the field of nanotechnology led to a variety of novel materials at the nanoscale. among them are different carbon materials like buckyballs, graphene nanoplates and carbon nanotubes (cnts). cnts are hollow carbon fibres with either one (sw) or multiple sidewalls (mw). mwcnts usually show a diameter of up to 100nm and can be several micrometres long. because of their nanoscale diameter cnt-uptake can take place directly through the plasma membrane of cells by the so called nanoneedle effect [1] . additionally cnts, like most nanomaterials, show a high surface to volume ratio and, because of their micro scale length, a potentially high loading capacity. these properties make cnts interesting for the potential use as drug delivery carriers (ddcs). mwcnts, produced via chemical vapour deposition with a diameter of 45nm and 16µm length, were used in three different forms, unmodified, acid oxidized (ox_cnt) and ground. cytotoxicity testing was performed in human umbilical vein endothelial cells (huvec). the cells were seeded in 48-well plates and exposed to doses of 1, 5, 10 and 25µg/cm² growth area of the respective cnt type for 24h. the wst-8 assay was applied for testing cell viability and the ldh cytotoxicity assay to identify potential damage to the plasma membrane and to calculate overall cytotoxicity. the results show that an increased oxidation time for the ox_cnts, in a h 2 so 4 /hno 3 mixture, leads to decreased cytotoxicity in huvec, compared to unmodified mwcnts. during the oxidation reactive oxygen groups are formed on the cnt surface [2] . these groups lead to a reduced hydrophobicity of the cnt surface which could be responsible for the decline in cytotoxicity. future investigations will include the toxicological analysis of mwcnts functionalized with polyethylene glycol (cnt-peg). the hydrophilic polymer peg will be covalently bound to the cnt surface and is expected to further reduce the cytotoxic effect. for these investigations different analytical methods will be used. among others, cell cycle analysis, the brdu assay, pathway arrays and qrt-pcr for the investigation of gene expression and cytokines will be measured. these methods will involve a co-culture model of huvec and human umbilical vein smooth muscle cells (huvsmcs) for a better approximation to the cellular in vivo situation. additionally the peg modified mwcnts will be tested for their loading capacity and efficacy with the anticancer drug doxorubicin for a potential use as an intravenous drug delivery carrier in vivo. although aluminium is one of the most common elements in the biosphere, up to now little is known about its impact on human health. aluminium and its chemical derivatives are highly abundant in food, food contact materials and consumer products what leads to an exposition via the gastrointestinal tract (gi tract), the lung and via skin contact. recently, aluminium is hypothized to cohere with cancer and neurodegenerative disorders. lately, due to an increasing attentiveness on this topic, limiting values for food additives have been tightened by the eu commission. however, cellular effects of aluminium and especially aluminium-containing nanomaterials, are in the focus of our research activities, for example in the international solnanotox project. we established an in vitro simulation system of the gi tract, where nanomaterials undergo the different physiological, chemical and proteinbiochemical conditions of saliva, gastric juice and the intestine. the artificially digested nanomaterials, as well as soluble aluminiumchloride as ionic control substance, were subjected to several analytical and biochemical methods to characterize their change of appearance and their cytotoxic effects on intestinal cellular models. we observed the fate of the nanomaterials during typical ph-values of saliva, gastric and intestinal juice with dynamic light scattering measurements and icp-ms in the single particle mode. after observable disappearance at ph 2 the particles recovered in the simulated intestinal fluid. the simulation of the gi tract, mainly the change of ph settings, may lead to a certain chemical activation of aluminium that can increase bioavailability in the intestine after oral uptake of aluminium-containing food products. in vitro assays like ctb, mtt and cellular impedance measurements showed that there were no acute cytotoxic effects measurable after a period up to 48h after incubation, comparable to undigested particles. in contrast, high amounts of aluminium ions showed additional effects on cell viability compared to non-digested aluminium ions. although toxicological potential of al ions to healthy tissue appears to be low, increased hazardous potential cannot be ruled out to pre-damaged tissue and can have a relevance for special consumer groups with for example chronical intestinal inflammation or dietary eating behavior combined with high exposure to al-containing food products. in the eu, there is a strong need for solutions to substitute halogenated flame retardant (hfr) additives, employed in the fabrication of fr thermoplastic and thermoset materials. these materials are used in diverse commercial products, applications and markets, such as electrical/electronic devices, low-voltage wires or household appliances. the phoenix project, funded by the european union 7 th framework program (grant agreement no. 310187), therefore investigates e.g. several tailor-made, nano-layered hybrid particles as alternatives to hfr additives. considering "safer-bydesign" strategies, potential fr nanomaterials (nm) were physico-chemically characterized (e.g. particle size distribution, zeta potential) and screened early in development for their (geno)toxic and pro-inflammatory potential to timely reject nm with high health hazard. as inhalation is the most important exposure route for nm, lungrelevant cells were used as in vitro screening models. to better enable detection and differentiation of the biologic effects of the most promising nm, screening was started with primary rat lung alveolar macrophages (am; cells of first contact for inhaled nm), at a high concentration of 50µg/cm 2 (24h of incubation), using membrane damage (lactate dehydrogenase release assay), direct dna-damage (alkaline comet assay), and il-8 liberation (elisa) as primary endpoints, and quartz dq12 and al 2 o 3 as particulate positive and negative controls, respectively. in this screening system, biologically inert nm could be differentiated from more active ones. thereby, mg(oh) 2 nanoplatelets (mg1; mean lateral size: 1,5-2 µm; mean thickness: 15-20 nm) represented the least, and pristine few layers graphene nanoplatelets (gr1; mean lateral size: 2 µm; mean thickness: 3 nm; graphene layers: 8 ± 0.5) the most biologically active nm. clear concentration dependencies were detected for gr1 in follow-up experiments. mg1 and gr1 were further tested in other lung-relevant cell types, i.e. mrc-5 primary human lung fibroblasts and a549 lung adenocarcinoma epithelial cells. interestingly, mrc-5 cells were less sensitive towards biological effects of gr1, compared to am, whereas a549 cells showed nearly no effect, keeping in mind that lung epithelial cells are the target cells of lung tumor development. to test the hypothesis that the observed cellspecific differences in sensitivity might in part be based on cellular uptake, cells were exposed for 24 h to 12.5 or 25 µg/cm 2 of gr1 on chamber slides. slides were finally stained with dapi and analyzed by dark field microscopy. cells indeed demonstrated differences in uptake capacity and also showed unique pattern of cellular localization of gr1, i.e. with tight perinuclear agglomeration in am, a more scattered cytoplasmic distribution in mrc-5 cells and limited uptake in a549 cells. additionally, biological activity of the diverse nm seemed also to correlate with cellular uptake, as determined by light and dark field microscopy in am and mrc-5 cells. in conclusion, an am-based screening system was able to differentiate biological activity of diverse nm, with morphology, physico-chemical characteristics, and related cellular uptake most likely to be key for nm-and cell type-specific hazard. lung carcinogenicity and putative systemic effects of low-dose life-time inhalation exposure to biopersistent nanoparticles were examined in a chronic inhalation study performed according to oecd test guideline no. 453 with several protocol extensions. female rats (100/group) were exposed to cerium dioxide (nm-212, 0.1; 0.3; 1; 3 mg/m³) for two years; a control group was exposed to clean air. after one year exposure, 42 µg/lung was found in animals exposed to 0.1 mg/m³ and 2.6 mg/lung in animals exposed to 3 mg/m³. histological examination of lungs revealed several adverse and non-adverse effects in the lung. the non-adverse effects comprised accumulation of particle-laden macrophages in alveolar/interstitial areas and in the balt, particle-laden syncytial giant cells in the balt and bronchiolo-alveolar hyperplasia (alveolar bronchiolization). the adverse effects included (mixed) alveolar/interstitial inflammatory cell infiltration, alveolar/interstitial granulomatous inflammation, interstitial fibrosis and alveolar lipoproteinosis. the incidence and severity of the effects were concentration-related. alveolar lipoproteinosis was not observed at low concentrations of 0.1 and 0.3 mg/m³ ceo 2 . neither pre-neoplastic nor neoplastic changes were observed after 12-months exposure. a no observed adverse effect concentration could not be established in this study. the comprehensive histopathological examinations of lungs and other tissues will be finalized in 2017. this project is part of the eu project nanoreg. moreover, german federal ministry for the environment, nature conservation, building and nuclear safety, german federal institute for occupational safety and health, german federal environment agency funded this project. lung carcinogenicity and putative systemic effects of low-dose life-time inhalation exposure to biopersistent nanoparticles were examined in a chronic inhalation study performed according to oecd test guideline no. 453 with several protocol extensions. female rats (100/group) were exposed to cerium dioxide (nm-212, 0.1; 0.3; 1; 3 mg/m³) and barium sulfate (nm-220; 50 mg/m³) for two years; a control group was exposed to clean air. lung burdens and burdens in extrahepatic tissues were measured at various time-point. the two year exposure period was successfully terminated and 50 animals per dose group were examined for organ burden and histopathology. the remaining animals currently are kept exposure-free for maximally 6 additional months. up to two years exposure to both nanoparticles did not lead to body weight reduction compared to control animals. the mortality rates were in an acceptable range. macroscopically evident tumors were not detected after two years. the ceo 2 lung burdens were maximally 3.5 mg/g lung tissue at the highest exposure concentration of 3 mg/m³. in comparison, highest ceo 2 burdens in organs remote to exposure were liver and spleen with maximally roughly 1 x 10 -3 g/g tissue. in brain, maximum ceo 2 levels were 7x10 -6 mg/g lung tissue. baso 4 lung burdens were comparatively low (1 mg/g) within the first 13 weeks of exposure and steeply increased to 6 mg/g lung tissue after one year. the comprehensive histopathological examinations of lungs and other tissues will be finalized in 2017. the european centre for ecotoxicology and toxicology of chemicals (ecetoc) 'nano task force' proposes decision-making framework for the grouping and testing of nanomaterials (df4nano) that consists of 3 tiers to assign nanomaterials to 4 main groups, to perform sub-grouping within the main groups and to determine and refine specific information needs. the df4nanogrouping covers all relevant aspects of a nanomaterial's life cycle and biological pathways, i.e. intrinsic material and systemdependent properties, biopersistence, uptake and biodistribution, cellular and apical toxic effects. use (including manufacture), release and route of exposure are applied as 'qualifiers' within the df4nano to determine if, e.g. nanomaterials cannot be released from a product matrix, which may justify the waiving of testing. the four main groups encompass (1) soluble nanomaterials, (2) biopersistent high aspect ratio nanomaterials, (3) passive nanomaterials, and (4) active nanomaterials. the df4nano aims to group nanomaterials by their specific mode-of-action that results in an apical toxic effect. this is eventually directed by a nanomaterial's intrinsic properties. however, since the exact correlation of intrinsic material properties and apical toxic effect is not yet established, the df4nano uses the 'functionality' of nanomaterials for grouping rather than relying on intrinsic material properties alone. such functionalities include system-dependent material properties (such as dissolution rate in biologically relevant media), bio-physical interactions, in vitro effects and release and exposure. the df4nano is a hazard and risk assessment tool that applies modern toxicology and contributes to the sustainable development of nano-technological products. it ensures that no studies are performed that do not provide crucial data and therefore saves animals and resources. the the grouping decisions of df4nano for 24 nanomaterials were validated against grouping by results of existing in vivo data and demonstrated 23 concordant grouping decisions. serum concentrations in cell culture medium influence the effect of cerium oxide nanoparticles on human lung a549 cells: cytotoxicity, proinflammation, cellular uptake, and particle properties k. burchardt the wide use of cerium oxide (ceo 2 ) nanoparticles (np), e.g. as fuel additive and for industrial and biomedical applications, evoked intense studies to understand the effects those particles might have on living organisms. contradictory results of ceo 2 nanoparticle toxicity have been published as they depend on many variables like shape, size, diluent and others. in our work we used an in vitro model of ceo 2 nanoparticles and lung carcinoma cells to investigate the role of serum content in the cell culture medium on cellular toxicity, particle uptake, proinflammation and particle characteristics. proinflammatory ceo 2 np with an average diameter of 15-30nm and different concentrations were diluted in cell culture medium with different fetal calf serum (fcs) concentrations (0.01, 0.1, 1 and 10%) and were used to expose human lung adenocarcinoma cells (a549) for up to 24 hours. at 100µg/ml ceo 2 np showed little to no toxic effecton growth arrested a549 cells at fcs concentrations of 1% or below, but cell viability was decreased to about 80% in proliferating cells in cultures with 10% fcs. the proinflammatory effect of ceo 2 np was investigated through measurement of il-8 mrna expression after 3 hours and cellular il-8 secretion after 24 hours. the qrt-pcr showed that the expression of il-8 mrna in cells treated with 100 µg/ml ceo 2 np in 10% fcs medium was three times higher than in cells treated with lower fcs concentrations. this finding correlated with the cellular il-8 secretion, which showed a stronger increase by cells treated at 10% fcs. differences in cellular uptake of ceo 2 np was determined by fluorescence-activated cell sorting (facs) after 2 and 24 hours of exposition. after 2 hours, the cells treated with ceo 2 np in 10% fcs medium showed a lower mean granularity (∆gmean) as measure for cellular particle uptake than those with less fcs in medium. after 24 hours all probes showed about the same granularity. to examine the effect the fcs concentrations on ceo 2 np characteristics in cell cultures we used dynamic light scattering (dls) and phase contrast microscopy (pcm). dls measurements revealed an increasing hydrodynamic diameter of the particles with decreasing fcs concentrations (about 1030nm (10% fcs) to 4090nm (0.01% fcs)), which was correlated by an increasing particle agglomeration shown by pcm. our results show that the fcs concentration in cell culture medium has a direct or indirect impact on the cytotoxicity, the proinflammatory effect, the facs parameter for cellular particle uptake as well as on particle properties, which should be taken into account when designing, performing and interpreting in vitro experiments to investigate the toxicity of nanoparticles. toxic and inflammatory effects of shape-engineered titanium dioxide nanoparticles in nr8383 rat alveolar macrophages. knowledge about the contrasting toxicity of nanoparticles (np) of different chemical composition has steadily increased over the past decade. however, available literature often reveals considerable differences in effects within a specific type of nanomaterial. these contrasts have been contributed to different handling and testing protocols as well as to sample-specific differences in physico-chemical properties of np that could affect their mode of interaction with cells. within the nanometrology project setnanometro, the highly controlled generation and characterisation of a large set of shape-engineered tio 2 np allows us to investigate the potential role of subtle shape-and surface structure changes on np toxicity. as inhalation represents the most relevant uptake route of np, the nr8383 rat alveolar macrophage cell line was selected for in vitro toxicological testing. since oxidative stress and inflammation are considered as key biological pathways in nanotoxicity, we evaluated the expression of the oxidative stress marker genes heme oxygenase-1 (ho-1) and g-glutamylcysteine synthetase (g-gcs) as well as the pro-inflammatory genes interleukin (il)-1β, il-6, il-18 and inducible nitric oxide synthase (inos) by qrt-pcr. protein levels of il-1β and tumour necrosis factor-α (tnf-α) were measured by elisa. cytotoxicity testing of the tio 2 np by wst-1 assay overall revealed only minimal toxicity in comparison to sio 2 np which were used as reference material. ho-1 and g-gcs mrna analyses indicated that specific tio 2 np triggered a moderate induction of oxidative stress. il-6 was only induced after sio 2 treatment, whereas il-18 was not affected by any of the tested np. in contrast, various tio 2 np caused a significant induction of il-1β mrna expression. however, no significant induction of il-1β and tnf-α protein secretion was observed for any of the tio 2 np. the results obtained from these and ongoing investigations will be linked to the physico-chemical database as being developed for all tio 2 np within the setnanometro project, with the overall aim to build and model nano-structure activity relationships (nsar) for this widely applied type of nanomaterial. acknowledgements: the setnanometro project is supported by the eu-fp7 programme. specific types of tio 2 particles were obtained by solaronix (switzerland), evonik and cristal. potential of silver and silver nanoparticles to reduce n-acetyltransferase 1 (nat1) activity j. lichter 1 , b. blömeke 1 1 trier university, department of environmental toxicology, trier, germany humans are exposed to various kinds of engineered nanoparticles including silver, which is frequently used in consumer and biomedical product due to its bactericide properties. despite their widespread usage, knowledge about influences on cellular functions is still incomplete. n-acetyltransferase 1 (nat1), an enzyme which is ubiquitously expressed in human tissues, catalyzes the transfer of an acetyl group to its substrates and although its endogenous function is not clear yet, it is well known to be involved in the n-acetylation of arylamines. in addition nat1 enzyme activity is known to modulated by non-substrates including metals and certain nanoparticles, however, the influence of silver on nat1 has not been analyzed yet. to address whether human nat1 is a target of silver nanoparticles and released irons on protein, purified nat1 was exposed to silver ions (agno 3 ) and silver nanoparticles (ag10-cooh, average size 10 nm, carboxyl functionalized), and nat1 enzyme activity was analyzing the n-acetylation of the nat1 substrate para-aminobenzoic acid (paba). therefore, purified nat1 (1 ng/µl) was co-exposed for 20 min to paba (1 mm) and agno 3 or ag10-cooh (0.01, 0.1, 1, 10 and 100 µg/ml each), resulting in a nat1:silver relation of 1:0.01-100 (w/w). both, agno 3 and ag10-cooh inhibited the n-acetylation of paba in a concentration dependent manner. using equal amounts of silver and nat1 (w/w, 1 µg/ml) enzyme activity was reduced about 98±0.2% (agno 3 ) and 82±0.6% (ag10-cooh). the lowest concentration analyzed (0.01 µg/ml) reduced nat1 activity about 24±5% (agno 3 ) and 17±5% (ag10-cooh). fifty percent activity reduction was caused by 0.11 ± 0.01 µg/ml of agno 3 and 0.21 ± 0.04 µg/ml of ag10-cooh, which is 10-fold lower compared to the published ic50 values for other metal oxide nanoparticles (3-15 µg/ml). these data indicate that both chemical silver species are able to modulate paba acetylation. further studies will be performed to clarify whether silver ions and/or silver nanoparticles could affect the specific n-acetylation of arylamines in human cells. colloidal silver has been used in medicine for centuries and nanosilver is present in many consumer-related products. however, despite of intense research in the past few years, the potential of nanosilver to induce effects different from ionic silver in vivo and in vitro, is still under debate. in this study, we compared proteomic effects of nanosilver (agpure™) and ionic silver (silver acetate) in the kidney of male rats after repeated oral delivery in a rat 28-days toxicity study. in order to avoid overt signs of toxicity, silver was dosed moderately in amounts of 60 and 6 mg/kg body weight for nanosilver and corresponding amounts of silver acetate (9.3 and 0.93 mg/kg). accordingly, no pathologic effects, including results from clinical chemistry and hematology, were reported. kidney tissue protein crude extract was separated by 2-d gel electrophoresis and differentially expressed spots were identified by maldi-ms. 374 unique proteins, showing a log 2 ratio of ≤ -0.3 for downregulation and ≥ 0.3 for upregulation were identified in all treatment groups. protein lists were analyzed with ingenuity pathway analysis (ipa). when comparing effects of particulate and ionic treatments, similar alterations were indicated for canonical pathways associated with glycolysis, gluconeogenesis and tricarboxylic acid cycle. regarding inflammatory responses, stronger effects were derived for ionic treatments. for both types of silver exposures, changes of protein expressions were linked to changes of fatty acid metabolism and nrf2-mediated stress. mitochondrial dysfunction was highlighted for both nanosilver treatments only, as well as activation of the insulin receptor. in the top-scored network of the higher dose nanosilver treatment, upregulated 14-3-3 protein zeta (ywhaz) displays a central position. ywhaz, an important regulator of cell cycle and apoptosis, interacts with the insulin receptor and is well known to be envolved in many types of cancer. overall, both forms of silver treatment revealed similar patterns of affected cellular and molecular functions in rat kidney, supporting common and overlapping mechanisms of particulate compared to ionic silver. because of the widespread application of nanomaterials and the fact that for some nanomaterials effects on different organisms where shown, nanomaterials are still in focus of interest. moreover the fate of nps is only partiallyassessed over the lifecycle of products containing nanomaterials. while general toxicological properties of nps are well described in diverse in-vitro and in-vivo experiments, the distribution of these particles during the whole and complex process of waste incineration shows big knowledge gaps. in the "nanoemission"-project the entire route from the residual material via incineration, filtering of the exhaust gas up to a possible release into the environment are considered together with the toxicological evaluation of effects on humans and the environment. in these experiments the influence of thermal waste treatment on the toxicological profile of nanoparticles, contained in the waste, will be described. after a complete characterization of the two types of baso 4 -nps from two different manufacturers by scanning electron microscopy/ energy dispersive x-ray analysis (sem/edx), measurement of the specific surface area (bet) and dynamic light scattering (dls) we investigated the impact of the pure baso 4 -nps on primary cells (normal human bronchial epithelial cells (nhbec) and peripherial lung cells (plc)). both materials show statistically significant cytotoxic effects in the resazurin-assay (decreased viability below 40 % for 1 mg/ml after 72 h). in general the effects of both nps were almost similar. additionally the effects of the baso 4-nps were compared more in detail. uptake of the baso 4 was quantified by icp-ms after 24 h and 72 h as well as the release of ba-ions into the cell culture medium after centrifugal separation. the incubation with baso 4 of plc and nhbec to 0.1 mg/ml over 24 h and 72 h leads to ~200 µg baso 4 /1 mio cells. the uptake is dose dependent but not time dependent. the impact on the secretion of inflammatory cytokines was determined by bead-based multiplex-elisa flow cytometry. tnf-α, il-8 and il-33 could be detected in nhbec and plc after np incubation. the possible induction of apoptosis was measured by flow cytometry as well. first investigations showed no induction of apoptosis for both materials. the impact of both nps on the intracellular glutathione level was measured by hplc and showed a decrease of gsh after 72 h. summing up baso 4 -nps showed toxic effects in primary human lung cell cultures after 72 h for concentrations under 1 mg/ml. c3 exoenzyme from c. botulinum is an adp-ribosyltransferase that inactivates selectively rhoa, b and c by coupling an adp-ribose moiety. rho-gtpases represent a molecular switch integrating different receptor signalling to downstream transcriptional cascades that regulate various cellular processes, such as regulation of actin cytoskeleton, cell proliferation and apoptosis. previous studies with the murine hippocampal cell line ht22 revealed a c3-mediated inhibition of cell proliferation and a prevention of serum-starved cells from apoptosis (rohrbeck et al., 2012) . former results of studies on map kinase signalling indicated c3-induced modulations of downstream signalling modules. therefore, ht22 cells treated with 500 nm c3 for 48 h were applied for screening of the activity of 48 various transcription factors followed by luciferase reporter assays. five transcription factors namely sp1, atf2, e2f-1, cbf, stat6 were identified as significantly regulated in their activity. for validation of identified transcription factors, studies on the protein level of certain target genes were performed. western blot analyses exhibited an enhanced abundance of sp1 target genes p21 and cox-2 as well as a raise in phosphorylation of c-jun. in contrast, the level of apoptosisinducing gadd153, a target gene of atf2, was decreased. our results suggest that c3 is able to modulate the activity of transcription factors whose target genes are involved in the regulation of cell proliferation and apoptosis. via covalent binding of chemical compounds to dna, adducts can be formed. as a consequence, mutations may occur, which represent stages of chemical mutagenesis and carcinogenesis. the isolation of leucocytes represents an essential field of work within dna-adductomics of blood cells, in which adducts serve as markers of exposure for biological monitoring. peripheral mononuclear blood cells (pbmc) comprising monocytes and lymphocytes can be separated from other blood cells like granulocytes, erythrocytes (and dead cells) by isopycnic density gradient centrifugation of buffy coats (bc´s). bc´s are blood concentrates rich in leucocytes and thrombocytes, prepared from whole blood samples thorough removal of plasma and erythrocytes. for the experiments only bc´s from healthy donors were used. while erythrocytes contain no dna, lymphocytes, which constitute a subcategory of leucocytes, carry genetic information. a variety of commercially available fluids with a density of 1.077g/cm 3 , based on different polymers, exists. these materials form the gradient required for the centrifugation. furthermore, there are several methods available, which differ in the ratio blood vs. fluid, duration of the different work up steps, centrifugation parameters and others. the aim of this work was to compare the effectivity of the different fluids and optimize the workflow. for isolation of pbmc the fluid was put in a tube and then covered by a thin layer of blood. after centrifugation, five layers were obtained (figure 1 ). the interphase was removed carefully, washed and the cells were counted. the yield is expressed as percentage of isolated vs. total leucocytes in the bc, which was based on the leucocyte count of the whole blood sample. as separation fluids, ficoll® paque plus (ge healthcare), histopaque® (sigma aldrich) and lsm® (ge healthcare) were tested. no significant differences between the different fluids were observed. although dilution is often recommended in literature, it was found that dilution had no effect on the yield. similarly, using different fluids (rpmi or pbs) for the washing steps did not reveal any differences. increasing centrifugation speed from 400 to 500 xg resulted in higher yields. in general, large variations in yield (46.8% ± 17.1%) among the various bc´s arose. this result is due to the different parameters such as age, storage, leucocyte and erythrocyte counts of the different bc´s used. therefore, the test conditions were optimized using the same batch of bc. the results show that the low priced separation fluids are comparable in performance tothe more expensive ones. by the direct lamination with bc (without dilution) the wastage could be minimized, the yield increased and thus the isolation made more efficient. the franz cell is a well-established model in lots of skin research fields. we adapted the diffusion cell system to additives and contaminants of consumer products which are designated for skin contacts. we were aimed to simulate real exposure as realistic as possible. to meet requirements like longevity, haptic properties and factory costs, different polymers are used as the raw material of choice and modified by a variable number of additives in the majority of commodities. besides additives with a defined function such as plasticizers, stabilizers, colorants and vulcanization accelerators, contaminants as well as decomposition products or so-called non-intentionally added substances (nias) can be part of the material, among them potentially harmful substances. in a first step, polymers of consumer products like flashlights or tools have been characterized concerning additive composition. possible breakdown products were identified by means of gc-ms/ms or pyrolysis-gc-ms. we focused on analytes of toxicological relevance including antioxidants such as n-phenyl-2-naphthylamine (neozon d), which is suspected of causing cancer, and on degradation products like cresol and its derivatives (e.g., mesitol or 2-tert-butyl-4-methylphenol). subsequently, analytes of interest were brought into direct skin contact using porcine, human and artificial skin models in the franz cell chamber assay. the analytes were either followed up layer by layer using tape stripping or examined utilizing cryo sections. for visualization purposes, analytical evaluation has been completed by use of imaging techniques like he staining or atr-ftir (attenuated total reflectance fourier transform infrared) microscopy. the latter was used with intrinsic markers for tissue specific distribution. our project provides evidence for the potential of polymer components to overcome the natural epidermal barrier and, in part, to enter the viable layers of the epidermis. during skin contact with consumer products several substances migrate out of the matrix and penetrate the skin, among them substances which are hazardous to health such as neozon d. depending on its dose, the blister agent sulfur mustard (sm) may lead to painful erythema, blistering with complicated wound healing, pulmonary edema, pulmonary bleeding and temporary blindness. sm is listed as a schedule 1 chemical in the chemical weapons convention and thus its production, stockpiling and use is prohibited. sm still represents a serious threat for civilians and military forces, especially in asymmetric, terroristic and accidental scenarios. after exposure, the highly reactive molecule alkylates nucleophilic sites in endogenous biomacromolecules forming the characteristic and stable hydroxyethylthioehtyl (hete) residue. hence, bioanalytical methods targeting theses adducts in forensic post-exposure verification analysis are of high interest. herein, we present an optimized, accurate and comparably simple method to detect adducts of sm and human serum albumin (hsa) alkylated at its cysteine 34 -residue. since albumin extraction from human plasma is a time-consuming and expensive step of an established procedure, an alternative method for direct proteolysis of human plasma was developed. plasma samples were cleaved directly with pronase resulting in the alkylated dipeptide hete-cysteine-proline (hete-cp) which is detected by micro-liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µlc-esi hr ms/ms). in order to optimize reproducibility and yield of proteolysis, kinetics were investigated for different kinds of plasma (edta-, citrate-and heparin-) as well as serum. two different mass spectrometers, a triple quadrupole system (4000 qtrap) and a hybrid quadrupole time-of-flight instrument (tt5600 + ), were compared. the latter one proved to be the more selective and sensitive system. the method was successfully applied to in vitro and in vivo samples of real cases of sm poisoning. organophosphorus compounds (op), which were originally intended to be used as pesticides to increase agricultural yields at the onset of the 20 th century, still represent a considerable threat to human health. by irreversible inhibition of acetylcholinesterase op lead to cholinergic crisis due to uncontrolled increase of acetylcholine in the synaptic cleft. finally, sudden death by respiratory failure may result if medical countermeasures are lacking. therefore exceptionally toxic compounds were designed und synthesized as chemical warfare agents (cwa), among which v-type nerve agents, i.e. vx, chinese vx and russian vx, belong to the most toxic artificial substances. recent events like the first gulf war, terrorist attacks in tokyo and the conflict in syria underline the need for ongoing and strict surveillance of cwa prohibition by the chemical weapons convention. unambiguous evidence of such substances (verification analysis) plays an important role with great political and legal impact. a variety of such bioanalytical methods have been established at the bundeswehr institute of pharmacology and toxicology in munich, which is accounted for medical chemical defence in germany. exhibiting quite short half-lives in vivo nerve agents can hardly be detected days or even weeks after exposure. accordingly, there is a great need for additional long-term biomarkers like specific protein adducts. consequently, the current work focuses on examination of adducts between nerve agents and human serum albumin (hsa) as its high abundance and stability in vivo provide relative ease of sampling. after incubation of hsa with v-type nerve agents in vitro the protein was subjected to proteolysis. subsequently resulting peptides were separated using microbore high-performance liquid chromatography (µlc) and detected on-line by modern high-resolution tandemmass spectrometry (hr ms/ms). this allowed unambiguous identification of already known phosphylated tyrosines as well as novel adducts between cysteine-proline dipeptides and the thiol-containing leaving group of v-type nerve agents. simultaneous detection of both biomarkers was realized by a new method, which was applicable even at the very low toxicologically relevant concentrations of v-type agents. therefore, this method represents a valuable and novel supplement of existing methods for verification. fatty acid esters of glycidol (glycidyl esters) are processing contaminants formed as byproducts of industrial deodorizing of plant fats or during other heating processes. following oral intake, glycidyl esters are mainly cleaved to release the reactive glycidol in the gastrointestinal tract. according to the national toxicology program (ntp), glycidol is carcinogenic, genotoxic and teratogenic in rodents. it is classified as probably carcinogenic to humans (iarc group 2a). the exposure assessment of the oral intake of glycidyl esters in humans is difficult, because the current data set for glycidyl ester contents in food is incomplete. we developed a method for the determination of the internal exposure to glycidol by mass spectrometric quantification of a hemoglobin adduct reflecting the total glycidol burden over approximately three months. a modified edman degradation was adapted for the cleavage of the valine residues from the n-termini of hemoglobin by fluoresceinisothiocyanat (fitc) (von stedingk et al. (2011 ) chem res toxicol 24, 1957 resulting in the formation of dihydroxypropyl-valine-fluorescein thiohydantoin (dhp-val-fth). the target analyte is purified with mixed-mode anion-exchange solid-phase extraction and analyzed by lc-ms/ms. a major advantage of the technique is the application to whole blood samples, which renders the time-consuming isolation of erythrocytes unnecessary. we synthesized dhp-d 7 -val-fth as an internal standard for the quantification of the glycidol adduct by lc-ms/ms multiple reaction monitoring. a limit of detection of 5 fmol per injection (5 pmol adduct/g hemoglobin) was achieved. the application of this method will possibly allow future monitoring of the internal exposure of glycidol in human studies. glycidol and 3-monochloropropane-1,2-diol (3-mcpd) are carcinogenic food contaminants, which are present in heat-processed oils and fats mainly in form of fatty acid esters. the risk assessment concerning human consumption of these substances is complicated by various reasons. for example, the data on the occurrence in food stuffs is incomplete. also, the amounts of the proximate carcinogens released from ester hydrolysis in the gastrointestinal tract in humans are not known. monitoring of the internal exposure would be an alternative strategy to support the assessment of possible health risks related to the intake of glycidol and 3-mcpd and their fatty acid esters. for short-term monitoring of the internal exposure, urinary metabolites are suitable biomarkers. we study the potential use of two different substances as descriptors of the oral intake of glycidol and 3-mcpd. the metabolite 2,3-dihydroxy mercapturic acid (dhpma) is generated following glutathione conjugation of both compounds. the second target analyte is 3-mcpd itself, which may also be formed from glycidol in the reaction with hydrochloric acid in the stomach. a method for the quantification of urinary 3-mcpd by gc-ms is currently developed. an lc-ms/ms multiple reaction monitoring technique was devised for the quantification of dhpma in urine samples with the isotope-labeled reference compound 13 c 2 -dhpma. the limit of quantification is 10 µg dhpma/l. related to creatinine, the analyte was detected to be in a relatively small concentration range in urine samples from humans. the average concentration in urine samples (n = 45) of one male volunteer collected over ten days was 154 ± 21 µg dhpma/g creatinine. a meal of a highly contaminated, commercially available frying fat (containing 1.1 mg of glycidol equivalents) did not lead to a visible increase of the urinary concentrations. the considerable background levels of dhpma in urine of humans and also in urine samples of other mammals support the hypothesis that dhpma may be also be formed from an endogenous c 3 -metabolite, as already reported by eckert et al. furfuryl alcohol is a common food contaminant formed by acid-and heat-induced dehydration from pentoses. it induced renal tubule neoplasms in male b6c3f1 mice and nasal neoplasms in male f344/n rats in a study of the national toxicology program (ntp). the neoplastic effects may originate from sulfotransferase (sult)-catalyzed conversion of furfuryl alcohol into the dna reactive and mutagenic 2-sulfoxymethylfuran. the incomplete data set of furfuryl alcohol contents in food does not allow estimating the human exposure. thus, we sought a method for the determination of the internal exposure. recently, the dna adduct of furfuryl alcohol n 2 -[(furan-2-yl)methyl]-2′deoxyguanosine was detected in specimen of human lung tissue. however, human biomonitoring of dna adducts has various disadvantages. for example, dna adducts are removed by various repair systems and human dna samples are usually not accessible in sufficient quantities. we decided to develop a biomarker for the internal exposure to furfuryl alcohol using blood proteins as dosimetric targets. bladder cancer (bc) is a smoking and occupation related disease showing a substantial genetic component. though the prognosis is generally good, a major problem is the frequent relapses affecting about half of the patients. n-acetyltransferase 2 (nat2) is well-known to modulate bc risk in persons heavily exposed to carcinogenic aromatic amines. we aim to investigate the impact of nat2 genotypes, in particular, the ultraslow genotype, on relapse-free time after first diagnosis in 772 bladder cancer cases. we used follow-ups of three case-control studies from lutherstadt wittenberg (n=207), dortmund (n=167) and neuss (n=398). nat2 was genotyped using seven characteristic polymorphisms (rs1801279, rs1041983, rs1801280, rs1799929, rs1799930, rs1208, rs1799931). haplotypes were reconstructed using phase v.2.1.1. we compared slow to rapid acetylators. additionally, we differentiated between the most frequent slow nat2*5b/*5b and *5b/other slow haplotypes as well as between the ultra-slow *6a/*6a and *6a/other slow haplotypes compared to rapid acetylators. chi-square tests used to check the frequency of relapses in ultra-slow, slow and rapid acetylators. genotype differences in relapse-free time up to 5 yr after first diagnosis of bc were analysed using cox proportional hazards models adjusted for age, gender, smoking habits, invasiveness and study group. a total of 371 (49%) patients showed a relapse within the first 5 yr after bc diagnosis. slow acetylators show a higher frequency of relapses than rapid acetylators (51% vs. 46%, p=0.154). this frequency is even higher in ultra-slow acetylators (61%, or=1.81, p=0.019) but not in slow *5b/*5b genotypes (49%, p=0.609). ultra-slow acetylators had a significantly shorter relapse free time within 5 yr after bc diagnosis than rapid acetylators (median 0.66 vs. 0.94 yr, hr=1.57, p=0.009). this trend was not that pronounced in all slow acetylators combined (0.78 yr, hr=1.19, p=0.127) nor in the subgroup of nat2*5b/*5b genotypes (0.79 yr, hr=1.20, p=0.255). the effect of ultraslow nat2 is even more pronounced in smokers (hr=1.78, p=0.003) but absent nonsmokers (hr=0.89, p=0.781). ultra-slow nat2 seems to be associated with a higher recurrence risk and a shorter relapse-free time, especially in smokers. slow nat2 in general seems to have less impact on recurrence. aalborg university, department of biotechnology, chemistry and environmental engineering, aalborg, denmark xenoestrogens with the potential for endocrine disruption like bisphenol a (bpa) may bind to the estrogen receptors (ers) and modulate expression of er target genes mimicking the natural ligand 17β-estradiol (e2). the potential for endocrine adversity is still predominantly assessed in vivo as existing in vitro tests have only limited value for an exposure-based risk assessment. thus, the development of reliable bioassays for the detection of endocrine disruptors is one of the paramount challenges faced by modern toxicology. a targeted metabolomics approach in mcf-7 cells treated with e2 or bpa revealed potential biomarkers for the estrogenic potency of the studied compounds. among them were several phosphatidylcholines and amino acids. we further addressed proline levels that were found to be strongly increased. investigations of proline levels over time showed a clear proliferation correlated concentration dependency after both e2 and bpa stimulation. furthermore, sirna knockdown experiments suggested an influence of the oncogenic transcription factor myc and the dependency of erα activation on the estrogen-mediated proline increase. our study demonstrates metabolomics as a powerful tool for biomarker identification and hypothesis generation. the results could be used further to develop bioassays for the detection of endocrine disruptive chemicals. children are considered to be more sensitive to most chemicals than the general population due to a variety of factors, including dynamic growth and developmental processes as well as physiological, metabolic and behavioral differences [1]. however, only a few data are available on the magnitude of preschool children's exposure to most chemicals present in many consumer products. several of these chemicals are linked to endocrine disrupting effects in animal studies and are suspected to have also adverse effects e.g. on development and function of the reproductive organs as well as on neurological and behavioral development in humans. among of the chemicals that have been a major focus of discussion in the last years are phthalates, dinch, parabens, bisphenol a, and triclosan due to their suspected health effects. therefore we aimed to investigate exposure levels to metabolites of different phthalates and parabens as well as to bisphenol a and triclosan in urine samples collected from preschool children in german day-care centers from north rhine-westphalia (lupe iii; 2011/12). urine specimens from children aged from 20 to 80 months from 23 different day-care centers were analyzed. in total, 253 preschool children were recruited with mean age of 54 months. our study results show that nearly all children (>95 %) of the study population had urine concentrations equal to or above the limit of quantification for five most common phthalates metabolites (mnbp, mibp, 5oh-mehp, 5oxo-mehp, 7oxo-minp), for bisphenol a and methylparaben. triclosan was detected in 16 % of the study population. in general, the median urinary concentrations of the above mentioned phthalate metabolites were about 5-50 µg/l in spot urine samples. the highest amount among the phthalate metabolites was observed for mibp with maximal values of about 1000 µg/l. median urinary concentration for methylparaben and bisphenol a were about 48 µg/l and 2 µg/l respectively. the maximum methylparaben, bisphenol a and triclosan level found were 1770 µg/l, 72.4 µg/l and 55,6 µg/l respectively. in conclusion, our study shows a widespread exposure of young children to various phthalates, parabens and bisphenol a in north rhine-westphalia, germany. a follow-up human biomonitoring study (2014/2015) has finished recruitment and is in the process of analyzing data. polycyclic aromatic hydrocarbons (pahs) represent a large group of organic compounds that are common environmental contaminants. they are formed by incomplete combustion of organic matter such as coal or crude oil and are often known to be carcinogenic, mutagenic and teratogenic. the acute toxicity of pahs is rather low, but because of their stability and lipophilic character those compounds can accumulate in the human body and cause severe chronic effects. additionally pahs may enter the food chain when preserving meat or fish by exposure to smoke. in the european union maximum levels of 2 µg/kg benzo[a]pyrene and 12 µg/kg as the sum of benzo[a]pyrene, benz[a]anthracene, benzo[b]fluoranthene and chrysene in the meat of smoked fish and smoked fishery products are set, respectively. smoked fish is often handmade in small fishery stores in schleswig-holstein, where self caught fish is prepared in smoke houses. this technique implies the danger of pahs to accumulate in smoked fishery products above allowed maximum levels. here, we report our findings of pahs in smoked fishery products bought in local convenience and fishery stores in schleswig-holstein and give a brief overview about actual contaminant levels. hplc with fluorescence detection was used to determine the quality and quantity of several toxic pahs in smoked fishery products made locally. pahs may constitute risks for human health when exposed to hazardous levels and therefore it is important to have knowledge about given contaminant levels. colorectal cancer (crc) is one of the most frequent cancers worldwide and is tightly linked to dietary habits. epidemiological studies provided evidence that the intake of red meat is associated with an increased risk to develop crc [1]. red meat contains high amounts of heme iron, which is thought to play a causal role in tumor formation. the underlying molecular mechanism, however, remains elusive and may involve increased cell proliferation and dna damage induction by heme-iron. in this study, we set out to analyze the genotoxic and cytotoxic effects of heme-iron in human colonic epithelial cell lines. we used hemin (fe iii ) as commercially available heme source, which was compared to inorganic iron chloride (fecl 3 ). first, the time-dependent internalization of hemin and fecl 3 into hct116 cells was determined using icp-ms/ms analysis. treatment of cells with inorganic iron resulted in a maximum of intracellular iron content after 1 h at all doses tested, while hemin particularly at high doses caused an iron accumulation up to 24 h. hemin catalyzed the formation of reactive oxygen species (ros) in a dose-dependent manner in caco-2 and hct116-cells as shown by flow cytometry. consistent with this finding, hemin dose-dependently induced the oxidative dna lesion 8-oxoguanine (8-oxog) as revealed by slot blot analysis and fpg-modified alkaline comet assay. using a pharmacological inhibitor of mutt homologue 1 (mth1), which protects the nucleotide pool by hydrolysis of 8-oxogtp, 8-oxog dna adduct levels in hemin-treated cells were further enhanced. in contrast, inorganic iron hardly affected the cellular ros level and only slightly increased oxidative dna damage. subsequently, a time-and dose-dependent activation of the dna damage response (ddr) by hemin was shown in hct116 and caco-2 cells using western blot analysis, which was followed by a reduction in cell viability at high doses after 72 h. finally, the cytotoxic effects of hemin and inorganic iron were tested using an ex vivo model of intestinal crypt organoids. preliminary results indicate that hemin is highly cytotoxic in organoids, whereas inorganic iron does not impair their viability. taken together, this study demonstrated that hemin induces oxidative stress and dna damage, resulting in the activation of the ddr and subsequent cytotoxicity. in contrast, inorganic iron displayed only modest effects. further in vivo studies using dna-repair deficient and proficient animals will shed light on the contribution of specific dna lesions to hemeassociated colorectal carcinogenesis. red and processed meat consumption is known to be a crucial risk factor in the development of colon cancer, which is one of the most common cancers worldwide. a diet rich in red and processed meat increases n-nitrosation within the colon leading to an increase in endogenously formed nitroso compounds. these compounds are able to alkylate dna of gastrointestinal tract cells, resulting in the formation of dna adducts such as -meg is repaired by mgmt, the potential of this enzyme to repair o 6 -cmg in cells is not well characterized. additionally, adenomas containing a k-ras gc-at transition mutation have lower mgmt levels than adenomas without this mutation. therefore, the aim of this study is to determine the role of mgmt in protecting colorectal cells from genotoxicity by repairing o 6 -cmg adducts. for this purpose, an mgmt-deficient non-transformed human colon epithelial cell line was established by stable transfection via rna interference to inhibit the expression and therefore the activity of mgmt. the transfected cell line was analyzed for complete mgmt gene silencing by activity and expression analyses and cell characterization. results confirmed that there was neither expression nor activity for mgmt in the transfected cells, and the cell characterization data showed that mgmt deficiency did not lead to differences in growth behavior or cell morphology or malignant cell transformation. cytotoxicity experiments performed in the transfected and nontransfected cell lines by treatment with dna alkylating agents suggest that the mgmtdeficient cell line is more sensitive to dna alkylating agents than the non-transfected cell line. these results were also supported by dna damage analysis via comet assay. asarones are secondary plant constituents mainly occurring in acorus calamus l. and guatteria gaumeri. the essential oil of the rhizome of acorus calamus l. is used for flavoring of food and alcoholic beverages. the concentration of β-asarone (ba) in these oils varies between 0.3% and 95%. the bark of guatteria gaumeri, which is rich in αasarone (aa), is used as cholesterol-lowering drug and to treat gallstones in traditional mexican medicine. both, aa and ba are carcinogenic in rodents and mutagenic in the ames test after metabolic activation and thus classified as genotoxic carcinogens. [1, 2] previously, the major metabolites in microsomal incubations with aa and ba were identified and synthesized in our laboratory. side-chain epoxides, the corresponding diols, side-chain alcohols and aldehydes were identified as the major metabolites of aa and ba. [3] the investigation of the mutagenic potency in the ames fluctuation test showed positive results in the salmonella strain ta 100 for aa and ba with metabolic activation and for aa-and ba-epoxide without metabolic activation. while it is known that epoxides are reactive against nucleophiles we set up the hypothesis of dna-adduct formation by epoxides to explain the mutagenic effect. this adducts are currently isolated, characterized by nmr-spectroscopy and used to quantify adducts in cells. at the same time primary rat hepatocytes were incubated with non-cytotoxic concentrations of aa and ba for 24 h. after harvest and lysis of the cells, the dna was isolated by chloroform/phenol extraction and enzymatically hydrolyzed. [4] the residual hydrolysates will be used to identify the dna-adducts formed in cells and to determine the adduct formation rate. preliminary experiments indicate that both, aa and ba also form dna-adducts in intact cells in a concentration-dependent manner. [1] göggmann, schimmer (1983) . mutagenicity testing of α-asarone and commercial calamus drugs with salmonella typhimurium. mutation research. 121, [191] [192] [193] [194] wiseman et al. fatty acid esters of 3-chloro-1, and of 2-chloro-1, are food process contaminants that are formed, e.g., during refinement of vegetable oils. after ingestion, the esters are hydrolyzed in the gut, thereby releasing free 3-mcpd and 2-mcpd. 3-mcpd has been identified by the international agency for research on cancer (iarc) to be possibly carcinogenic to humans (class 2b) and is therefore in the focus of food safety authorities. to elucidate the toxicological properties of these compounds at the molecular level (mode of action) a proteomic study was conducted in which 10 mg/kg b.w./day of either 3-mcpd or 2-mcpd were orally administered to rats over a period of 28 days. total protein was extracted from different tissues of the animals and separated via twodimensional gel electrophoresis (2de). among others, the redox sensor protein dj-1 was identified to be deregulated in liver, kidney, testis, and heart of rats treated either with 3-mcpd or 2-mcpd. up to six different isoforms of dj-1 were identified by 2de-western blot analysis, all of them having the same molecular weight but different pi values, indicating protein modifications of low molecular weight but with a strong impact on the protein charge. treatment of the animals with either 3-mcpd or 2-mcpd predominantly led to a shift of the abundance between two dj-1 isoforms in the rat tissues. this effect was more pronounced with 3-mcpd compared to 2-mcpd. mass spectrometric analysis of these two isoforms identified an oxidation of a conserved cysteine residue (cys106) of dj-1 to a cysteine sulfonic acid to be the protein modification induced by treatment of the rats with either 3-mcpd or 2-mcpd. dj-1 is discussed to participate in a number of biological processes such as proteolysis, protein folding, or redox regulation. oxidation of cys106 was shown to be crucial for the activity of dj-1, and the irreversible oxidation of cys106 to a cysteine sulfonic acid as observed in the present study was shown to result in a loss of protein function and was correlated with diseases related to oxidative stress such as parkinson's disease. thus, the potential impact of 3-mcpd and 2-mcpd on cellular oxidative stress and on associated neurodegenerative diseases has to be considered in the ongoing risk assessment of these food contaminants. pyrrolizidine alkaloids (pa) are secondary plant compounds and widely spread in plant kingdom. humans can therefore be regularly exposed via direct or indirect contamination of food, like herbal teas, honey, wheat or salad. 1,2-unsaturated pa are known for their potentially harmful effects. an acute intoxication may cause venoocclusive disease leading to hepatomegaly, ascites as well as liver hardening resulting in high mortality. on the other hand, chronic pa intoxications due to regular consumption of small amounts of pa are characterized by hepatic necrosis, fibrosis and cirrhosis. an initial whole genome transcriptome study in hepatocytes revealed that molecular pathways related to cancer development, cell cycle regulation and cell death are regulated by the four structurally different pa echimidine, heliotrine, senecionine and senkirkine in short-term exposure (24h). additionally, lipid and bile acid metabolism was affected. however, the uptake of pa by food is more likely to be continuous than singular. therefore, the aim of this study was to investigate molecular effects of longterm exposure (14d) comparatively to short-term exposure (24h) in the hepatoma cell line heparg with the four structurally different pa. in this context we analyzed selected cellular parameters like cytotoxicity and morphology. in contrast to short-term exposure, structure-and concentration-dependent cytotoxicity was found for the long-term exposure (sn>he>em/ski). furthermore, obvious morphological changes such as destructuration and perforation of the heparg cell monolayer were seen after 14d of incubation. based on these findings, a possible induction of apoptosis or necrosis by pa was examined. effects of long-term exposure to pa on gene expression were investigated for a set of genes found to be regulated in the short-term whole genome transcriptome study. the identified regulation of gene expression was confirmed for both terms, with the strongest regulation for cyp7a1 (down-regulation), a gene involved in cholesterol metabolism. therefore, the effects of pa on various parameters related to cholesterol metabolism were analyzed, showing pa effects on cholesterol levels and bile acid secretion. short-term exposure to pa did not affect cell viability. however, repeated doses of pa resulted in severe effects on hepatic cells, concerning viability and morphology. at the mrna level, short-and long-term incubation with pa seem to affect a wide range of signaling pathways. in conclusion, we show for the first time that heparg cells can serve as an in vitro model for hepatotoxicity following chronic intake of pa. shiga toxin-producing e. coli (stec) strains cause a diversity of enteric symptoms in humans, ranging from mild diarrhea to severe diseases such as the hemolytic uremic syndrome (hus). hus is a life threatening disease with microvascular endothelial damage in the gastrointestinal tract and the kidneys, which often leads to haemolytic anemia, thrombocytopenia and acute renal failure. shiga toxin plays a major role for the pathogenesis of hus but the subtilase cytotoxin, which was identified during an hus outbreak in 1998 in stec strains, might contribute as an additional potent enterotoxin. like shiga toxin, subab is an ab 5 protein toxin. the pentameric binding/transport subunit (subb) delivers the enzymatic active subunit (suba), a protease, into the endoplasmic reticulum (er) of human target cells. in the er, suba cleaves the chaperone bip/grp78, which results in cell stress and caspase 3/7dependent cell death. recently, we discovered that higher concentrations of suba (10 mg/ml) causes cytotoxic effects in human epithelial cells (hela, in the absence of subb. the cytotoxic effects were investigated in hela cells in more detail. here, suba binds in a concentration dependent manner to the cell surface and induces dramatic morphological changes as well as caspase-dependent cell death [1] . in contrast to hela and caco-2 cells, cho fibroblasts did not respond to suba. currently, further cell types including macrophages are tested for suba effects and the molecular mechanisms underlying the cytotoxic effects caused by suba and the cell type selectivity are investigated. although there are strong cytotoxic effects caused by suba on some human epithelial cells in vitro, the situation in vivo and the pathogenic relevance of the newly observed suba effect are not known. thermal treatment of fat-containing foodstuff in the presence of salt leads to formation of 3-mcpd and its esters. high amounts of 3-mcpd esters detected in food raised toxicological concern. recent studies revealed that food may also contain significant amounts of structurally related 2-mcpd and its fatty acid esters. toxicological studies indicate genotoxicity in vitro and a carcinogenic potential of 3-mcpd, pointing to kidney and testes as main target organs. 3-mcpd esters were shown to cause similar, but milder effects. few unpublished data exist for 2-mcpd, showing similar organ toxicity compared to 3-mcpd and identifying heart as additional target organ. no such data exist for 2-mcpd diesters. in consequence, further toxicological data were required in order to complete risk assessment for 3-mcpd, 2-mcpd and their esters. hence, an oral 28-days study was performed in male rats in order to fill data gaps and provide essential information for risk assessment. a proteomic analysis was included in order to compare molecular effects induced by 3-mcpd and 2-mcpd and its dipalmitic ester in rat liver, kidney, testes and heart. in order to avoid overt toxicity, moderate doses of 3-mcpd + 2-mcpd (10 mg/kg body weight), and 2-mcpd-dipalmitate (53 + 13.3 mg/kg body weight) were applied. accordingly, no pathologic effects were reported. here, we present proteomic results obtained after analysis of heart tissue. after separation of heart tissue protein crude extract by 2-d gel electrophoresis , differentially expressed spots were identified by maldi-ms. a total of 270 unique proteins deregulated at a log 2 ratio of ≤ -0.5 for downregulation and ≥ 0.5 for upregulation at p ≤ 0.05 were identified. comparing deregulated spots induced by different treatments revealed that a higher number of spots was deregulated by 2-mcpd versus 3-mcpd. dipalmitate treatment even caused more deregulation than 2-mcpd. upregulated cytochrome b-c1 complex subunit rieske (ucri) and downregulated acetyl-coa acetyltransferase (thil) were among the top deregulated proteins after 2-mcpd and 2-mcpd dipalmitate treatment. pronounced upregulation of respiratory chain protein ucri, not deregulated after 3-mcpd treatment, indicates an effect on energy metabolism. downregulation of thil, involved in ketone body metabolism, was only weakly affected after 3-mcpd treatment. protein dj-1 (park7), a multifunctional redoxsensitive chaperone and protease protecting the cell against oxidative stress, was significantly downregulated after treatment with 3-mcpd and the higher dose of the 2-mcpd diester. tropomyosin beta chain (tpm2) was commonly upregulated after 3-mcpd and 2-mcpd treatments, possibly indicating a tgf-beta induction of actin stress fibers. for rat heart, data show that similarities but also some significant differences of 2-mcpd-and 2-mcpd dipalmitate-induced proteomic changes exist compared to 3-mcpd, indicating different mechanisms of toxicity for this structural analogues. oxidation products (oxy) of cholesterol (chol) such as 7α-hydroxy-chol (7α-ho-chol), 7β-hydroxy-chol (7β-ho-chol), 7-keto-chol (7-o-chol), 5,6α-epoxy-chol (α-epoxy-chol) and 5,6β-epoxy-chol (β-epoxy-chol) are formed by autoxidation of chol and are discussed as biomarkers for inflammation and oxidative stress in human tissues to be used in the identification of risk factors for disease. however, oxy-chols are also present in processed foodstuff where 7β-ho-chol (milk) and 7-o-chol (fish, meat, and egg) tend to represent the main oxychols, whereas epoxy-chols, are generally minor constituents. thus, levels of oxychols in tissues may result from both endogenous formation as well as dietary exposure. since quantitative profiles of oxy-chols have not been determined in human adipose tissues yet, levels of oxychols and chol were quantified using gc-ms/ms (internal standards: deuterated oxychols) and gc-fid (internal standard: 5α-cholestan-3β-ol), respectively in breast adipose tissues of 48 healthy women undergoing mammoplasty. furthermore, tissue levels of 25 fatty acids in adipose tissues were determined by gc-fid (internal standard: undecanoic acid) to assess relative levels of pentadecanoic acid, docosahexaenoic acid and elaidic acid, indicative for consumption of dairy products, fish, and processed fats, respectively. all oxychols were detected in all breast adipose tissues. the most abundant oxychol was β-epoxy-chol (median: 0.36 nmol/g tissue, range: 0.06-1.55 nmol/g), followed by α-epoxy-chol > 7-o-chol > 7α-ho-chol> 7β-ho-chol (0.009 nmol/g, range: 0.002-0.11 nmol/g). tissue levels of chol (2.8 micro mol/g, range: 1.88-3.87 micro mol/g) did not correlate (spearman's rank analysis) with that of epoxy-chols and correlated even negatively with that of 7α-ho-, 7β-ho-, and 7-o-chol (r = -0.29, p=0.024-0.044) median oxychol/chol ratios ranged from 0.0119 (5, to 0.0003 (7β-ho-chol). 7-o-chol and 7-ho-chols correlated strongly with each other (r=0.81-0.91, p oxy-chol levels did not correlate with relative amounts of pentadecanoic acid and docosahexaenoic acid, whereas total oxychol, 7β-ho-chol, and β-epoxy-chol levels correlated with relative amounts of elaidic acid (r=0.30, 0.30, and 0.31, respectively, p=0.037, 0.034, 0.028, respectively) . no correlations of oxychol levels, individual or total oxychol/chol ratios with age or bmi were observed. taken together, oxychol profiles in breast adipose tissues were different from that usually present in food but could be affected by dietary habits. classification and labelling of hazardous substances and hazardous consumer products (1) has proven to be a very efficient tool for risk communication. consumer products, such as glue, varnish, or washing and cleansing products need to be classified and labelled if they contain dangerous ingredients that render the mixture hazardous. personal care products, however, need not be classified and labelled if they contain dangerous substances above the thresholds for classification. they are excluded in the clp regulation. what would happen without this exception? when i applied the criteria for classification and labelling to a selection of cosmetic product formulas in a conservative approach, most products would have to be labelled and classified, mainly due to hazardous effects to the eye and to the skin (2) . benefits of personal care products can go along with unwanted properties such as the hazards for the human health, and consumers should be informed about them (3) . risk communication for every day products like personal care products should be clear, easily and quickly understandable. according to the cosmetic regulation (4) the ingredients must be listed on the containers. it must be questioned whether the listing of the ingredients without hazard pictograms on the products could be considered as a clear, easily and quickly understandable risk communication instrument (3). the results show that it is urgent to inform consumers better about the potential dangers of personal care products, because cosmetics need to be applied even with more care than any other consumer product. it is strongly recommended to delete the exception provision in the clp regulation for personal care products. the infochemical effect describes that anthropogenic substances can interfere with the chemical communication and influence organisms so that they perceive their chemical environments differently (1,2). infochemicals play a role in life history, habitat search, food related aspects and survival which shows that disturbed communication (infodisruption) could affect population vulnerability at various decisive points (3). the classical ecotoxicological standard tests do not allow to observe the infochemical effect. systematic analyses are needed to find out more about the relevance of this new chapter in ecotoxicology for natural ecosystems. the first crucial step is to select suitable test substances. repellents (substances used to keep away target organisms, e.g. invertebrates such as midges or fleas via olfaction) enter surface waters mainly indirectly via wastewater discharges from sewage treatment plants or directly by being washed off from the skin and clothes of bathers. there are various indications that repellents which are not toxic for aquatic animals could induce effects like organismic downstream drift of non-target species (downstream dislocation of e.g. crustacean and insect larvae in streams). in a literature study, three repellents were identified to be suitable test compounds for proof of concept of the infochemical effect. deet (cas 134-62-3), icaridine (cas 119515-38-7) and ebaap (cas 52304-36-6) (4). these substances are investigated in new test designs in a subsequent experimental part of the project which are designed to detect and quantify the infochemical effect. persistent, bioaccumulative and toxic (pbt) substances or very persistent and very bioaccumulative (vpvb) substances are compounds with hazardous properties. the non-biodegradability (persistence) and high accumulation in organisms (bioaccumulation) may elicit long-term adverse effects in the environment. persistent and bioaccumulative substances concentrate in the environmental compartments (water, sediment, soil, air) and can be distributed in the food chains. ecotoxicological effects are strengthened by bioaccumulation and appear often in remote areas like marine and polar regions. in the framework of pbt assessment, contrary to the risk assessment, the substances are evaluated regardless of the emission into the environment. an evaluation of medicinal active ingredients under assessment in the german federal environment agency (uba) identified less than 10% as potential pbt candidates. due to data lacks in many cases a definite pbt classification is not possible. the poster presents results of an extensive literature research on the global occurrence of pharmaceuticals in the environment. data on measured environmental occurrences from more than 1,000 international publications have been transferred to a database, with more than 120,000 entries. according to the database, pharmaceuticals have been found in the environment of 71 countries worldwide in all five un regions. most published data are for the compartments surface water and sewage effluent; less information is available for groundwater, manure, soil, and other environmental matrices. more than 600 active pharmaceutical substances (or their metabolites and transformation products) have been detected in the environment. most findings have been published for industrialized countries. monitoring campaigns are increasingly being conducted in developing and emerging countries. these have revealed the global scale of the occurrence of pharmaceuticals in the environment. for example, diclofenac, a non-steroidal inflammatory drug, has been detected in the aquatic environment in 50 countries worldwide. a number of globally marketed pharmaceuticals have been found in both developing and industrialized countries. the available ecotoxicological information indicates that certain pharmaceuticals pose a risk to the environment at measured concentrations. options for cooperative action to address the risk of are also presented. the aim of the research presented was to support the discussion of the proposed emerging policy issue pharmaceuticals in the environment under the strategic approach to international chemicals management (saicm), which is a global initiative of united nation environment programme (unep). the european chemicals' legislation reach aims to protect man and the environment from substances of very high concern (svhc). chemicals with (very) persistent, (very) bioaccumulative and toxic properties (pbt and vpvb compounds), substances that are carcinogenic, mutagenic and toxic to reproduction (cmr compounds), as well as chemicals of comparable concern like endocrine disruptors (eds) may be subject to authorization. the identification of eds is limited as yet, because specific experimental assessments are not required under reach. evidence is currently based on a combination of few experiments, expert judgement and structural analogy with known eds. structural alerts for the identification of potential eds: predictions of properties and effects from chemical structures are based on similarities with either active or inactive substances. structural alerts for the identification of potential estrogen/androgen-ed activities are relevant parts of the structures of compounds that are known to interact with estrogen and androgen receptors as either agonists or antagonists. in addition to the backbones of the chemical structures (pharmacophore) for steric fit to the receptors, modulating factors may be small substructures for local interactions with receptor binding sites and physicochemical properties related to the strength of binding to the receptor. comparison of evidence from in silico, in vitro and in vivo assays for potential eds: identification of potential eds based on findings from mammalian long-term reproduction studies, fish life-cycle tests, receptor assays, and chemical alerts were compared and differences analysed. agreement is limited because in vivo, in vitro and in silico methods address different aspects of potential effects on endocrine systems regarding toxicological targets, modes of action and functional similarity of chemical structures. a combination of toxicological and chemical assays can provide comprehensive and complementary information to support evidence-based identification of potential eds among the chemicals released into the environment. application of structural alerts for the identification of potential eds to the einecs inventory: more than 33000 discrete organic einecs compounds are within the applicability domain of the structural alerts for potential estrogen/androgen-ed activities. among them, 3585 chemicals (ca. 11%) are indicated as potential candidates for endocrine effects based on structural alerts. due to the possibility that these chemicals may interact with estrogen/androgen receptors they should be subject to further investigations regarding their potential for endocrine effects, eventually leading to regulatory actions. within the imi (innovative medicine initiative) project "intelligence-led assessment of pharmaceuticals of the environment " (ipie;http://i-pie.org/), a more intelligent environmental testing and tools for prioritisation of legacy compounds shall be developed. regarding the evaluation of fish toxicity, screening approaches in fish embryos are pursued. while the standard fish embryo toxicity (fet) test is restricted to lethal parameters more relevant as substitutes for acute effects, additional sublethal endpoints may provide expanded applicability of the fet assay for chronic effect assessment in fish. in this respect, the analysis of the metabolome could provide additional insights into biochemical processes elicited by pharmaceutical compounds and could potentially support the extrapolation from fish embryo to chronic fish toxicity as displayed in the standard early life stage (els) test. in the context of ipie a pilot study was performed with the aim to quantify and comparatively assess changes in the metabolic signatures of fish and fish embryos induced by the reference compound amikacin, an aminoglycoside antibiotic, in order to identify metabolic patterns applicable as biomarker profiles that can be linked to apical endpoints in terms of an integrated approach. therefore, toxic effects in fathead minnow embryos and els fish were investigated following a 4 and 32 days exposure, examining conventional endpoints and additionally using a combined direct injection and lc-ms/ms assay for metabolite identification and quantification. metabolic endpoints were found to be at least as sensitive as standard apical endpoints such as growth and mortality as detected in the longer-term fish study. furthermore, multivariate data analysis (pca-x, opls-da) revealed substance induced metabolic perturbations specific for fish and fish embryos, respectively. beyond that, the statistical approach of shared and unique structure (sus) identified some metabolites from the classes of amino acids, biogenic amines and lipids to be similarly changed in both developmental stages related to amikacin treatment, representing shared biomarker candidates. in this first pilot study, the integrated metabolomics approach yielded insights into the molecular consequences of amikacin exposure and provided indications for biomarkers for common effects in fish embryos and fish. due to the different exposure levels in the fet (1 -100 mg/l) and els study (0.0003 -0.03 mg/l), more definitive conclusions regarding the predictivity of metabolic responses in fish embryos for apical endpoints in chronic fish tests are yet not possible. further studies are ongoing with a range of pharmaceutical compounds of different chemical classes which will reveal more substantial information on the applicability of this technology in the prediction of longterm effects in fish. the human cationic amino acid transporter hcat-1 (slc7a1) represents the major uptake route for arginine and other cationic amino acids (such as the essential amino acid lysine) in most mammalian cells. it thus provides these amino acids for protein synthesis as well as for essential metabolic pathways. in endothelial cells, special attention has been given to the role of hcat-1 for supplying arginine to nitric oxide synthase. in spite of its wide distribution, hcat-1 expression is highly regulated both, on the transcriptional and post-transcriptional level. however, the genetic elements involved in transcriptional regulation a largely unknown. here we studied the expressional regulation of hcat-1 in human ea.hy926 endothelial cells. starvation of these cells from cationic amino acids led to a pronounced induction of both, hcat-1 mrna and protein. the mrna induction was almost completely abolished by the transcription elongation inhibitor drb (5,6-dichloro-1-β-dribofuranosylbenzimidazole), suggesting the involvement of transcriptional regulation. we thus aimed at identifying the promoter elements in the hcat-1 gene responsible for this regulation. to our surprise and in contrast to data published by others 1 the chromosomal region up to 5 kb upstream of the first hcat-1 exon exhibited no promoter activity in either endothelial or dld-1 colon carcinoma cells that exhibit a very strong endogenous hcat-1 expression. we could however identify a promoter element within the first intron of the hcat-1 gene. transcriptional activity of this element increased upon amino acid starvation in a similar way as endogenous hcat-1 expression. our results indicate starvation-induced transcriptional regulation of hcat-1 through newly identified promoter regions distinct from those published previously. the transport of a multitude of drug molecules into the cell is mediated by multispecific organic cation transporters (octs), belonging to the solute carrier group (slc). one of these families within this group of membrane transport proteins is the slc47-family consisting of the two multidrug and toxin extrusion transporters mate-1 (slc47a1) and mate2-k (slc47a2). while mate-1 is highly expressed in several different tissues like kidney, liver, skeletal muscle, adrenal glands, testes and heart, mate2-k exclusively occurs in the apical membrane of proximal tubular epithelial cells within the kidney. both transport proteins translocate organic cations in exchange of protons into as well as out of the cell. to define the affinity of both transporters for the anti-diabetic drug metformin and to investigate their interactions with 26 different anti-neoplastic agents comparative transport experiments with the model substrate 1-methyl-4-phenylpyridinium (mpp) have been carried out. therefore stably transfected hek293-cells expressing mate-1 or mate2-k transport proteins generated by portacelltec biosciences gmbh and vector transfected hek293-cells were used. the interaction analyses were carried out by determining the uptake of [ 14 c]-metformin and the inhibition of [ university of basel, basel, switzerland drug transporters play a pivotal role in pharmacokinetics by modulating the cellular entry or efflux of compounds. one transporter facilitating the transport of bile acids, steroid hormones, and statins is the organic anion transporting polypeptide (oatp) 2b1 that is highly expressed in placenta, liver, and small intestine. especially its activity in small intestine and liver is assumed to be basis for specific drug-drug interactions. understanding mechanisms involved in pharmacokinetics is a prerequisite in drug development. to test whether there are species differences in transport activity we compared the expression and function of the human and rat orthologue. first, we determined the transport activity of the known oatp2b1 substrate estrone 3sulfate (e 1 s), and observed a significantly lower k m for mdck-hoatp2b1 compared to .00 ± 10.23 µm, *p<0.05 student´s t-test) whereas there was no difference in v max (mdck-hoatp2b1 119.4 ± 11.3 fmol/min/µg protein; mdck-roatp2b1 102.3 ± 12.8 fmol/min/µg protein). the human oatp2b1 exhibits multiple binding sites for its substrates that may explain specific drug-drug interactions [1] . to identify whether rat oatp2b1 owns the same characteristics, the cellular accumulation of 50 µm e 1 s (low affinity site) or 0.005 µm e 1 s (high affinity site) was determined in presence of specific inhibitors/substrates of oatp2b1. as observed for atorvastatin, a known inhibitor of both affinity sites, the rat transporter failed to exhibit the low affinity site. in detail, while atorvastatin reduced the accumulation of e 1 s in mdck-hoatp2b1 cells (110.0 ± 14.6 fmol/min/µg protein vs. 60.7 ± 7.5 fmol/min/µg protein, *p<0.05 student´s t-test), there was no inhibition of e 1 s accumulation in mdck-roatp2b1 cells (53.2 ± 13.2 fmol/min/µg protein vs. 49.0 ± 17.4 fmol/min/µg protein). additionally, we observed a different membrane localization of both transporters as assessed by immunofluorescent staining showing an apical localization for rat oatp2b1 while human oatp2b1 is localized at the basolateral pole of the cellular model. absolute quantification of mrna (copy number per 1000 ng of total rna) in different tissues of rat revealed a high expression of oatp2b1 in liver (159.6 ± 45.5), a moderate expression in small intestine (14.9 ± 4.0) and colon (57.0 ± 12.3), and a low expression level in kidney (2.3 ± 0.8) . the latter is in accordance with previous findings showing that oatp2b1 is abundant in rat kidney as quantified by absolute proteomics technics [2] . our data demonstrated species differences in localization and activity of the drug transporter. further studies are warranted to proof whether this knowledge will help in future drug development and which molecular cause is responsible for the observed data. background: intestinal drug absorption depends on various factors including aqueous volume, site-specific ph and intestinal motility. moreover, the expression of efflux-and uptake-transporters vary in dependence of the anatomical localization making the gut a complex barrier for drug transfer into the body. in a recent study, site-specific protein and mrna expression levels of 10 drug transporters were determined along the entire length of the human gut. interestingly, discrepancies between mrna expression and protein levels were observed. moreover, there were quantitative differences between small intestine and colon. as a consequence the question arose if this observation could be explained by small non-coding rnas acting as highly tissue-specific posttranscriptional regulators of gene expression. hence, in our current study, we aimed to investigate the impact of mirnas on site-specific transporter expression along the human intestine. methods: total rna was isolated from biopsies obtained from six disease-free organ donors. tissue samples were acquired from the duodenum, the upper and lower jejunum, the upper and lower ileum, and the transversal or descending colon. the expression of 754 mirnas was measured using rt-pcr based low density arrays. expression of all detected mirnas was correlated with transporter protein data recently determined by lc-ms/ms-based targeted proteomics. mirnas and transporter genes showing an inverse pearson's correlation between mirna and protein expression underwent an in-silico search (microcosm targets v.5, targetscan7.0) for putative mirna/mrna interaction. to investigate those interactions in more detail, reporter gene assays and site directed mutagenesis were conducted. results: out of 754 mirnas, 248 were detected in all tissue types. out of ten transporters five showed significant inverse correlations with 10 putatively targeting mirnas (e.g. pept1 and hsa-mir-27a, r= -0.498, p=0.002). reporter gene assays indicated interactions of mir-193a/b with pept1 3'-utr (p = 0.0025 and p = 0.0012) as well as of mir-27a with abcb1 3'-utr (p = 0.0006). the site-specific abundance of intestinal drug transporters is significantly affected by microrna-mediated post-transcriptional regulation under physiological conditions as exemplified for pept1 and abcb1. background: the human uptake transporter nact [gene symbol slc13a5; also known as mindy, the human orthologue of the drosophila indy (i´m not dead yet) transporter] is expressed in liver and brain. nact is important for energy metabolism and brain development and mediates the uptake of tricarboxylic acid (tca) intermediate such as citrate and succinate. reduced expression of this transporter, as studied in knock-out mice, mimics aspects of dietary restriction, promotes longevity and protects against insulin resistance and adiposity. furthermore, mutations in the human slc13a5 gene are associated with epileptic encephalopathy with seizure onset in the first days of life, possibly due to the reduced uptake of tca intermediates into neurons. to gain more insights into the role of nact in drug transport and into structure-function relationships, we studied the inhibition of nact-mediated citrate transport by various drugs and analyzed the effect of known mutations in the slc13a5 gene on nact-mediated transport. methods: drug inhibition studies were performed using hek cells stably expressing human nact with citrate as prototypic substrate. twenty-four drugs were used as potential inhibitors of nact-mediated uptake. the effects of eight mutations, three of them (nactp.g219r, -p.t227m and -p.l488p) associated with epileptic encephalopathy, were analyzed using a transient transfection approach. furthermore, the first computational-based structural model of the nact transporter was established and the impact of all mutations on substrate transport was modeled. results: inhibitions studies demonstrated that only a few drugs (three out of 24 tested) inhibited nact-mediated citrate transport at the tested drug concentration of 100 µm. from these, benzbromarone shows the strongest inhibition with an ic 50 value of 83.2 µm. furthermore, citrate transport was also slightly inhibited by pioglitazone and rosiglitazone. citrate transport of the mutated proteins nactp.g219r, -p.g219e,p.t227m, -p.l420p and -p.l488p was totally abolished and the effect of these mutations could be explained on the basis of the structural model. conclusion: inhibition studies demonstrated that simultaneously administered drugs can inhibit nact-mediated uptake of the prototypic substrate citrate. nact-mediated uptake is abolished by mutations in the slc13a5 gene associated with epileptic encephalopathy. the effect of these mutations can be explained on the basis of the first structural model of this uptake transporter. the atp-binding cassette subfamily b member 1 (abcb1) is a drug efflux pump responsible for the classic multi-drug resistance phenotype in cancer cells. increased activity of abcb1 in cancer cells contributes to protection against a wide range of chemotherapeutic agents. this dramatically decreases therapeutic options and the chance of patient survival. knowledge of the underlying mechanisms for abcb1 deregulation is a critical step for the reversal of this unfavorable condition. of note, phosphatidylinositol-4,5-bisphosphate (pip 2 ) is a known regulator of abcb1. the protein "myristoylated alanine-rich c-kinase substrate" (marcks) is known for its ability to bind and sequester the phospholipid pip 2 . in various forms of colorectal cancer the deregulation of marcks protein goes hand in hand with an increase in malignancy and therapeutic resistance. in this study, we characterized the enigmatic marcks as a modulator of abcb1 activity. we focused on a subgroup of colon cancers, where marcks resides in a hyperphosphorylated state for which the established cell line ht-29 served as a model. we employed various in-vitro methods for the measurement of abcb1 activity, in combination with imaging experiments, assays for cellular viability and classical methods of molecular biology. we combined these approaches with pharmacological inhibition of marcks phosphorylation state or rnai-mediated depletion of marcks. with these interventions we could modulate endogenous abcb1 activity and re-sensitize our cell model against chemotherapeutic agents like 5-fluorouracil which are known to be substrates of abcb1. taken together, our findings suggest a new way how a cancer cell can gain a state of therapeutic resistance. the exploitation of marcks as modulator of abcb1 might be a new approach to target resistant tumors without interfering with the natural function of abcb1 in non-malignant tissue. background: in several large clinical studies low blood concentrations of homoarginine were identified as independent risk marker for stroke, cardiovascular events and mortality. experimental data suggest an active role of homoarginine deficiency in disease development. interference with l-arginine-dependent pathways and signaling has been implicated as a possible mechanism. it was the aim of the present study to identify transport mechanisms involved in the cellular uptake and tissue distribution of homoarginine, which is poorly understood, so far. the experiments focused on cationic amino acid transporters (cats) and possible interactions with known cat substrates. methods: uptake assays were performed using [ 3 h]-labeled homoarginine as substrate and human embryonic kidney (hek293) cells stably overexpressing human cat1 [gene: slc7a1 (solute carrier family 7)], cat2a (slc7a2a) or cat2b (slc7a2b). cells transfected with an empty vector were used as control. unlabeled known catsubstrates l-arginine and asymmetric dimethylarginine (adma) were investigated as inhibitors. results: compared to the uptake into control cells, uptake of homoarginine was significantly higher in hek cells overexpressing cat1 (7-fold), cat2a (1.6-fold) or cat2b (2.2-fold) after 2.5 min using 100 µm substrate (each p<0.001). apparent k m values for cellular net uptake of homoarginine were 174.6 µm for cat1, 3640 µm for cat2a and 522.8 µm for cat2b. homoarginine uptake by the three cats could be inhibited by addition of l-arginine and adma. conclusion: the protective biomarker homoarginine is a substrate of the human cationic amino acid transporters cat1, cat2a and cat2b. compared to other cat substrates, the cat1-and cat2b-mediated homoarginine transport is characterized by relatively high affinity. the uptake of homoarginine by all three cats can be inhibited by l-arginine and adma. taken together these findings make cat-mediated transport of homoarginine a possible target for interactions and pharmacological interventions aimed at homoarginine homeostasis. this project is supported by the doktor robert pfleger-stiftung. background and aim: organic cation transporter 1 (oct1, alternative name slcc22a1) is a polyspecific membrane transporter, which has been suggested to play a role in absorption, distribution and elimination of drugs and toxins. besides endogenous compounds like thiamine (vitamin b1), known oct1 substrates are toxins like mpp + as well as drugs like metformin, o-desmethyltramadol, ranitidine, and sumatriptan. tissue distribution of oct1 has been shown to have strong inter-species differences. in rodents oct1 is strongly expressed in both the sinusoidal membrane of hepatocytes and the basolateral membrane of kidney epithelial cells. human oct1 is strongly expressed on the sinusoidal membrane of hepatocytes, but not in the kidney. furthermore, substrate specific differences have been observed between the human and rodent oct1 orthologs. the aim of this study is to characterize the mechanisms causing substrate specificity between rodent and human oct1 orthologs. methods: overexpression of oct1 orthologs in mouse, rat and human cells was performed by targeted chromosomal integration of t-rex™ 293. the cells were characterized in detail for their ability to transport the model substrates tea + , mpp + , and asp + , the drugs ranitidine, sumatriptan and fenoterol. results: mouse and rat oct1 orthologs showed similar transport activity for all the model substrates and drugs tested. however, significant differences were observed when rodent orthologs were compared to the human oct1. human oct1 showed a 5fold higher v max for the uptake of asp + and 2-fold increase of v max for sumatriptan in comparison to the rodent oct1 orthologs. conversely, human oct1 showed a 4-fold lower v max for the uptake of fenoterol compared to rodent oct1s. there was no difference between rodent and human oct1 in the uptake of ranitidine. these differences were further characterized in detail using chimeric mouse-human oct1 constructs and by comparing the effects of key point mutations in mouse and human oct1 orthologs. conclusions: these data demonstrate strong differences in the substrate specificity between rodent and human oct1 orthologs. therefore oct1 pharmacokinetic data obtained in mouse or rat models could not be directly extrapolated for use in human. furthermore, comparative functional analyses of orthologs may help reveal the mechanisms underlying polyspecificity of oct1. ranitidine is a histamine h 2 -receptor antagonist which is commonly used without prescription for the treatment of pyrosis and gastric ulcers. approximately 30% of the systemic clearance of ranitidine is via hepatic metabolism. ranitidine is known to be a substrate of the hepatic organic cation transporter oct1 [1]. oct1 is expressed on the sinusoidal membrane of human hepatocytes and is highly genetically variable. sixteen major oct1 alleles are known [2] . thereof 12 alleles confer partial or complete loss of oct1 activity. the observed loss of activity was highly substrate specific and should be analyzed substrate by substrate. in this study we analyzed the effects of genetic polymorphisms in oct1 on the uptake of ranitidine. we used hek293 cells stably transfected to overexpress wild type or variant oct1 isoforms. the variant oct1 alleles oct1*1a (met408val), oct1*1c (phe160leu), oct1*1d (pro341leu/met408val), oct1*2 (met420del), oct1*3 (arg61cys), oct1*4 (gly401ser), oct1*5 (gly465arg/met420del), oct1*6 (cys88arg/met420del), oct1*7 (ser14phe), oct1*8 (arg488met), oct1*9 (pro117leu), oct1*10 (ser189leu), oct1*11 (ile449thr), oct1*12 (ser29leu) and oct1*13 (thr245met) were analyzed. we characterized in ranitidine uptake determined k m and v max for the different polymorphic oct1 isoforms. wild type oct1 showed a time and concentration dependent uptake of ranitidine with a k m of 62.91 ± 4.32 µm and a v max of 1125.41 ± 86.12 pmol/min/mg protein. variants oct1*5, oct1*6, oct1*12 and oct1*13 completely lacked ranitidine transport activity. variants oct1*2, oct1*3, oct1*4 and oct1*10 showed v max values decreased by 64, 77, 91 and 50 %, respectively. oct1*8 variant showed an increase of v max by 25 %. there was no significant changes in ranitidine uptake by variants oct1*1a, oct1*1c, oct1*1d, oct1*7, oct1*9 and oct1*11 compared to the wild type. there were no significant differences in the k m values between the wild-type and variants. in conclusion, we confirmed ranitidine as an oct1 substrate and demonstrated that genetic polymorphisms in oct1 can strongly affect ranitidine uptake. the effects of oct1 polymorphisms on ranitidine pharmacokinetics in humans remain to be analyzed. otto-von-guericke university, institute for pharmacology and toxicology, magdeburg, germany serotonergic hallucinogens ( s hgs), such as lysergic acid diethylamide (lsd), induce profound alterations of human consciousness, which are thought to be mediated by activation of 5-ht 2a receptors. with repeated application, the mind-altering effects of most s hgs rapidly are undermined by tolerance. the only exception seems to be dimethyltryptamine (dmt), whose mind-altering effects for reasons unknown even with repeated application do not decrease. assuming that dmt differs from other s hgs in its capacity to regulate 5-ht 2a receptors, we here compare lsd and dmt with regard to processes of 5-ht 2a downregulation. in heat-exposed rats, lsd and dmt induce a marked increase in body core temperature (hyperthermia) accompanied by "defensive hypersalivation". both effects are mimicked by the 5-ht 2 selective agonist dimethoxybromoamphetamine (dob) and can be blocked by the selective 5-ht 2a antagonists ketanserin and mdl100907. after repeated application, the temperatureregulatory effects of lsd are subject to tolerance, whereas the ones of dmt are not. tolerance to lsd (as measured by dob induced [ 35 s]gtpуs binding) is paralleled by a desensitisation of frontocortical 5-ht 2 receptors; for dmt, there is no such decrease. applying techniques of immunocytochemistry, transphosphatidylation, and quantitative real-time pcr to (ha-5-ht 2a transfected) hek293 and (endogenously 5-ht 2a expressing) c6 glioma cells, respectively, we furthermore demonstrate that dmt in contrast to lsd fails to internalise 5-ht 2a receptors, fails to activate the phospholipase d (which is needed for 5-ht 2a internalisation), and fails to inhibit the synthesis of 5-ht 2a receptors. given that dmt unlike lsd turns out to be inactive as to all processes of 5-ht 2a downregulation investigated, our data suggest that the differential tolerance development noted for dmt and lsd indeed might be accounted for by differential regulation of 5-ht 2a receptors. lsd and dmt both have recently regained scientific attention as potential therapeutics in the treatment of depression and/or anxiety disorders. providing mechanistic insights into their action, thus, is of timely clinical relevance. increased gaba release in human neocortex at high intracellular sodium and low extracellular calcium -an anti-seizure mechanism? ] e , this reduction might induce an anti-seizure mechanism by augmenting gat-mediated gaba release, a mechanism absent in rats. aging is complex on the systems as well as on the molecular level. the process of aging is characterized by a progressive loss of physiological functions and accumulation of cellular damage. one hallmark of aging is an impaired protein homeostasis. the imbalance of the quality control of both de novo protein synthesis and protein degradation, therefore, is likely to contribute to the phenotype of aging. we investigated protein turnover rates with the state-of-the art techniques funcat (dieterich et al., 2010) and sunset (schmidt et al., 2009) in aging neuronal cell cultures . using these techniques we show a prominent decrease in protein synthesis and degradation that progressed gradually in aging neuronal cells cultured up to div 60. in order to rejuvenate the protein turnover in aged neuronal cells we applied the selective eukaryotic elongation factor-2 kinase inhibitor a 484954 and the polyamine spermidine and observed protein translation utilizing funcat and sunset. whereas both a 484954 and spermidine had no effect on de novo protein synthesis in juvenile neurons (div 20) , both substances increased the de novo protein synthesis to a juvenile level in aged neuronal cultures (div60). this effect is seen in neuronal somata and dendritic spines. the molecular function of spermidine as an "anti-aging agent" is not defined yet. thus, additional pharmacological interventions are used for further examination of specific molecular spermidine targets. in conclusion, the described experimental setup is used to investigate impaired protein homeostasis as one hallmark of aging. agents with a presumed "anti-aging" effect can be tested for a potential rejuvenating effect on the level of protein homeostasis. a screening approach to test tolerability of multitargeted drug combinations for antiepileptogenesis in mice a large variety of brain insults can induce the development of symptomatic epilepsies, particularly temporal lobe epilepsy. in the latent period after the initial insult multiple molecular, structural, and functional changes proceed in the brain and finally lead to spontaneous recurrent seizures. prevention of these developments, called antiepileptogenesis, in patients at risk is a major unmet clinical need. several drugs underwent clinical trials for epilepsy prevention, but none of the drugs tested was effective. similarly, most previous preclinical attempts to develop antiepileptogenic strategies failed. in the majority of studies, drugs were given as monotherapy. however, epilepsy is a complex network phenomenon, so that it is unlikely that a single drug can halt epileptogenesis. recently, multitargeted approaches were proposed ("network pharmacology") to interfere with epileptogenesis. developing novel combinations of clinically used drugs with diverse mechanisms that are potentially relevant for antiepileptogenesis is a strategy, which would allow a relatively rapid translation into the clinic. we developed an algorithm for testing such drug combinations in a screening approach, modelled after the drug development phases in humans. tolerability of four repeatedly administered drug combinations was evaluated by a behavioral test battery: a, levetiracetam and phenobarbital; b, valproate, losartan, and memantine; c, levetiracetam and topiramate; and d, levetiracetam, parecoxib, and anakinra. as in clinical trials, tolerability was separately evaluated before starting efficacy experiments to identify any adverse effects of the combinations that may critically limit the successful use in preclinical studies on antiepileptogenesis and translation of these preclinical findings to the clinic. based on previous studies, we expected that tolerability would be lower in epileptic mice than in nonepileptic mice. therefore nonepileptic mice were used as a first step, followed by epileptic mice and mice during the latent period shortly after status epilepticus. except combination b, all drug cocktails were relatively well tolerated. in contrast to our expectations, except combination c, no significant differences were determined between nonepileptic and post-status epilepticus animals. as a next step, the rationally chosen drug combinations will be evaluated for antiepileptogenic activity in mouse and rat models of symptomatic epilepsy. major depression is one of the most common mental disorders worldwide, with serious social and economic consequences. there are many different hypotheses concerning the pathophysiology of this disease. the complex brain serotonin system and particularly the serotonin 1a receptors (5-ht 1a r) apparently play a pivotal role in the development of depression. the involvement of an altered, 5-ht 1a r-mediated signalling in adult neurogenesis is also discussed. however, in this context the effects of pre-and postsynaptically located 5-ht 1a rs have not been clarified yet. mice with a permanent overexpression of postsynaptic 5-ht 1a rs (oe mice) represent a unique tool to elucidate the effects of postsynaptic 5-ht 1a rs in adult neurogenesis and depressive-like behaviour. previous studies demonstrated an increased proliferation and survival of newborn cells in the adult dentate gyrus of female oe mice in comparison to controls. in the present study, we investigate the proliferation and survival of adult born cells after chronic treatment (15 days) with the selective 5-ht 1a r agonist 8-oh-dpat (0,5 mg/kg/day) in young adult oe and wt mice. on the last three days of treatment, newly generated cells of oe and wt mice are labelled by injections with bromodeoxyuridine (brdu; 50 mg/kg/day). mice are sacrificed either one day (proliferation) or 21 days (survival) after the last injection. we hypothesise that the data we will present will confirm our previous results, with possibly more pronounced proneurogenic effects and differences in male mice. further immunohistochemical studies post-exercise and behavioural analyses are in progress to identify the relation between chronic postsynaptic 5-ht 1a r stimulation, depressive-like behaviour and hippocampusdependent learning. dystonia is a common movement disorder characterized by intermittent and prolonged muscle contractions resulting in involuntary movements and/or abnormal postures. the lack of knowledge of the pathophysiology of dystonia hampers the development of effective therapeutics. although benzodiazepines can improve dystonic symptoms, tolerance and side effects limit their use. there is evidence for striatal dysfunctions in human dystonia. gabaergic striatal interneurons (in) are important for the regulation of striatal signaling. in the dt sz mutant hamster, a model of paroxysmal dystonia, immunoreactive in were reduced at the age of maximum severity of dystonia (33 days), but not after spontaneous remission (age 90 days). as indicated by unaltered homeoboxprotein nkx 2.1 (cell density, mrna), the age-dependent deficit seems not to be related to a disturbed migration, but to a retarded maturation of in in mutant hamsters. here we further determined the maturation of striatal gabaergic neurons in the dt sz hamster compared to healthy controls. kcc2 and cavii mrna, used as markers for the gaba-switch, were unchanged in 18 and 33 day old mutant hamsters, indicating that there is no general delay in gabaergic maturation. as a retarded maturation seems to be specific for in, we used another marker for gabaergic maturation: the expression of specific gaba a receptor (gaba a r) subunits (mature striatal in express the alpha1 subunit). by stereological determination, we found a 46% decrease in alpha1 subunit expressing neurons. a lower immunoreactive intensity was restricted to the somata of dorsomedial striatal in (32%) of dt sz hamsters, indicating both a reduced density as well as a delayed maturation. these findings prompted us to examine the effects of the αlpha1 gaba a r preferring compound zolpidem in comparison with the benzodiazepine clonazepam. zolpidem (2.0 and 10.0 mg/kg i.p.) only exerted moderate antidystonic effects compared to the benzodiazepine clonazepam (0.5 and 1.0 mg/kg i.p.) in the dt sz hamster. examinations of αlpha2 gaba a r preferring compounds are ongoing. in summary our studies indicate that there is no general defect in striatal gabaergic maturation in the dt sz mutant but a specific alteration of striatal gabaergic interneurons which express αlpha1 gaba a r subunits. changes in αlpha1 gaba a r subunit expression and differences in the antidystonic efficacy of zolpidem and clonazepam indicate that further investigations on the role of gaba a r subunits could lead to new therapeutic approaches for the treatment of dystonia. 2005) . therefore, the hypothesis of the present study was that pregnenolone attenuates the inhibition of synaptic transmission elicited by cannabinoids. methods: 250 µm-thick slices containing the cerebellum and the nucleus accumbens were prepared from the brains of mice and rats. spontaneous and electrically evoked gabaergic inhibitory postsynaptic currents (sipscs and eipscs) and evoked glutamatergic excitatory postsynaptic currents (eepscs) were analyzed in superfused brain slices with patch-clamp electrophysiological techniques. results: a) the synthetic cannabinoids jwh-018 (5 x 10 -6 m) and jwh-210 (5 x 10 -6 m) inhibited the spontaneous gabaergic synaptic input (sipscs) to purkinje cells in mouse cerebellar slices. the inhibition by jwh-018 was not affected by pregnenolone (10 -7 m), the inhibition by jwh-210 was only marginally attenuated. b) the depolarization of the purkinje cells induced suppression of the gabaergic input to purkinje cells (dsi); pregnenolone (10 -7 m) did not affect this endocannabinoid-mediated form of synaptic suppression. c) in rat nucleus accumbens slices, gabaergic and glutamatergic synaptic input to medium spiny neurons was activated by electrical stimulation of axons. ∆ 9 -tetrahydrocannabinol (2 x 10 -5 m) suppressed the gabaergic and glutamatergic synaptic transmission in the nucleus accumbens. these suppressive effects of ∆ 9tetrahydrocannabinol were not changed by pregnenolone (10 -7 m). d) finally, we tested whether we can observe neurosteroid-mediated effects in our brain slice preparations. tetrahydro-deoxycorticosterone (thdoc, 10 -6 m) markedly prolonged the decay time constant (τ) of spontaneous gabaergic postsynaptic currents (sipscs), similarly as in previous experiments (ej cooper et al., j physiol 521: 437-449, 1999) . the results show that inhibition of gabaergic and glutamatergic synaptic transmission by synthetic-, endogenous,-and phyto-cannabinoids is not changed by pregnenolone. therefore, it is unlikely that interference with cannabinoid-induced inhibition of synaptic transmission is the mechanism by which pregnenolone attenuates behavioural and somatic effects of ∆ 9 -tetrahydrocannabinol in vivo. the hypothalamus is one of the key players in the regulation of the energy homeostasis. cold stress leads to an activation of neurons in the paraventricular hypothalamic nucleus (pvn) and increases thermogenesis. the thyrotropin-releasing-hormone (trh) neurons have an important function in this effect. however it is hardly understood which role the trh neurons exactly play and how they are connected to other regions of the brain. we have transduced neurons in the pvn of mice with a recombinant adeno associated virus which contains an activating "designer receptors exclusively activated by designer drugs" (dreadd) system under the control of a shortened trh promotor. two weeks after transduction the animals were injected with clozapine-n-oxide (cno). to analyse the physiological function of this neurons we performed indirect calorimetry, measured rectal temperature and thermogenesis in the brown adipose tissue (bat), analysed drinking feeding behaviour and the home cage activity. after stimulation we measured the expression of genes in bat as well plasma hormone levels of pituitary hormones. propranolol and the specific β3-antagonist sr59230a were used to analyse the relevance of the sympathetic system. to further characterise the transduced neurons and their projections we used immunohistochemistry methods. after stimulation with cno the energy expenditure and body temperature were increased. these effects were mostly driven through an activation of the brown adipose tissue (bat). in dreadd transduced trh-receptor 1 (trh-r1) knockout mice this effects were abolished. in parallel the plasma levels of tsh, the ucp1 mrna level in the bat, the home cage activity as well the food and water intake were increased. after the treatment with propranolol and sr59230a the effects on the thermogenesis were reduced, but the home cage activity was not affected. sr59230a treatment normalised the food intake and increased in parallel the plasma leptin concentrations after cno stimulation. transduced neurons project into the raphe nucleus, the medial part of the thalamus and the spinal cord. with our experiments we could provide strong evidence for a sympathetic connection of the transduced neurons in the pvn to the bat and for the involvement of thr neurons in these effects. therefore, this system is a suitable tool to investigate the metabolic relevance of trh neurons in detail. background & objective: during obesity development, tissue factor signalling contributes coagulation-independently to inflammatory and metabolic dysfunction of adipose tissue. adipogenesis involves proliferation and differentiation of preadipocytes, apoptosis and hypertrophic growth of differentiated adipocytes, angiogenesis and extracellular matrix reorganisation. the coagulant protease thrombin promotes similar processes in various cell types, through activation of protease-activated receptors par-1, par-3 and par-4. in human adipose tissue, par-1 is found in vascular stromal cells and par-4 in preadipocytes and differentiated adipocytes. thrombin stimulates mitogenic kinase signalling and induces inflammatory cytokine and angiogenic growth factor secretion in adipocytes. we have examined the contribution of thrombin receptor activation to adipogenesis processes in 3t3l1 cells. results: differentiation of 3t3l1 preadipocytes with insulin, dexamethasone and isobutylmethylxanthine increases leptin and pparg gene expression and accumulation of triglycerides and oil red o-stained lipids. par-4 is time-dependently upregulated in maturing cells while par-1 expression is detectable but not altered. in preadipocytes, thrombin (3 u/ml) activates the mitogenic kinase erk1/2, promotes cell proliferation and induces gene expression of the maturation markers leptin and pparg and the inflammatory marker tumor necrosis factor alpha (tnfa). repeated stimulation of differentiating adipocytes with thrombin suppresses induction of leptin and pparg and attenuates lipid accumulation, while expression levels of the proliferation marker ki67 and the inflammatory cytokine interleukin (il)-6 are increased compared to differentiated control cells. similar proliferative and anti-adipogenic effects are seen with the selective par4-activating peptide (gypgkf, 100 µm) and cathepsin g, a proteolytic par-4 activator released from neutrophils and mast cells. repeated exposure of maturing 3t3l1 cells to conditioned medium from degranulating mouse peritoneal mast cells (mccm) augments lipid accumulation and il-6 expression. pretreatment of mccm with a par-4 inhibitor further drives lipid accumulation, the induction of il-6 by contrast is suppressed. conclusion: par-4 activation by thrombin or inflammatory cell-derived cathepsin g appears to suppress adipogenesis, possibly by maintaining proliferative capacity and preventing the growth arrest essential for initiating matuation. increased par-4 expression in maturing adipocytes may instead support inflammatory changes, thereby promoting the onset of insulin resistance. the chemokine receptor cxcr4 antagonist amd3100 exerts deleterious effects in endotoxemia in vivo s. seemann 1 , a. lupp the chemokine receptor cxcr4 is a multifunctional receptor which is activated by its natural ligand c-x-c motif chemokine 12 (cxcl12). although a blockade of the cxcr4/cxcl12 axis revealed beneficial outcomes in chronic inflammatory diseases, its importance in acute inflammatory diseases remains contradictious and not well characterized. as cxcr4 seems to be part of the lipopolysaccharide sensing complex, cxcr4 agonists or antagonists may have a positive impact on tlr4 signaling. additionally, cxcr4 is involved in the production of pro-inflammatory cytokines, suggesting the receptor to be a promising target in terms of mitigating the cytokine storm. therefore, we aimed to investigate the impact of a cxcr4 blockade on endotoxemia by applying a sublethal lps dose (5 mg/body weight) in mice. the selective cxcr4 inhibitor amd3100 was administered intraperitoneally shortly after lps treatment to ensure an immediate effect after endotoxemia onset. 24 hours after lps administration, the clinical severity score, the body temperature and the body weight of the animals were determined. afterwards, the mice were sacrificed and serum tnf alpha as well as ifn gamma levels were measured. furthermore, the oxidative stress in the brain, liver, lung and kidney tissue was assessed. in addition, the biotransformation capacity of the liver was evaluated and finally, the expression of gp91 phox as well as of heme oxygenase 1 in the spleen and liver were determined by means of immunohistochemistry. the mice of the amd3100 plus lps treatment group displayed a significantly impaired general condition, a reduced body temperature and a decreased body weight in comparison to the control and to the lps treated animals, respectively. tnf alpha levels were significantly increased by more than 200% or 35% when compared to the control or to the lps group, respectively, whereas ifn gamma levels were elevated by about 11% in comparison to mice which had received lps only. in all investigated organs, but especially in the liver and in the kidney, co-administration of amd3100 and lps caused massive oxidative stress. furthermore, the protein contents and the activities of several cyp enzymes in the liver were significantly reduced. immunohistochemistry revealed gp91 phox to occur above average, whereas heme oxygenase 1 expression was distinctly decreased. our results indicate that a blockade of the cxcr4 in endotoxemia is disadvantageous and even worsens the disease. co-administration of amd3100 and lps impaired the health status of the animals, caused massive oxidative stress and diminished the biotransformation capacity. thus, handling acute systemic inflammation with a cxcr4 antagonist cannot be recommended, hence indicating the activation of cxcr4 to be an attractive treatment option. toll-like receptors (tlrs)recognizemicrobial pathogens and trigger inflammatory immune responses to control infections. in acne vulgaris, activation of tlr2 by propionibacterium acnes contributes to inflammation. although glucocorticoids have immunosuppressive and anti-inflammatory effects, acne can be provoked by systemic or topical treatment. enhanced tlr2 expression by glucocorticoids has been reported in undifferentiated keratinocytes, however, human skin cells of different epidermal and dermal layers have not been investigated. in this study, the modulation of tlr expression by dexamethasone was assessed in monolayer cultures of primary human keratinocytes and dermal fibroblasts, as well as the immortalized keratinocyte cell line hacat. constitutive tlr2, tlr1 and tlr6 mrna and protein expression was confirmed in basal keratinocytes, calcium-induced differentiated keratinocytes, hacat cells and fibroblasts by qpcr and western blotting. dexamethasone induced tlr2 expression in a time-dependent and concentration-dependent manner and reduced tlr1/6 expression in keratinocytes but not in hacat cells or fibroblasts. stimulation with dexamethasone in the presence of the pro-inflammatory cytokines tnfα or il-1β further increased tlr2 mrna levels. gene expression of mapk phosphatase-1 (mkp-1) was also upregulated by dexamethasone. glucocorticoid-induced tlr2 expression was negatively regulated by p38 mapk signalling under inflammatory conditions through mkp-1 induction which functions to deactivate mapks. as expected, dexamethasone inhibited the immune responses linked to tlr2 signalling as demonstrated by reduced il-8, il-1β, mmp-1 and mcp-1 levels. however, the expression of traf6, a critical cytosolic regulator of tlr-and tnf family-mediated signalling, was further upregulated by the tlr2 agonist hklm (heat-killed lysteria monocytogenes) in dexamethasonepretreated basal keratinocytes.conclusively, our results provide novel insights intothe molecular mechanismsof glucocorticoid-mediatedtlr expressionand function in human skin cells. psoriasis is a cutaneous chronic inflammatory disease characterized by increased amounts of il-1 cytokines and t helper 17 (th17) related cytokines in lesional psoriatic skin. treatment with beta-adrenoceptor antagonists is associated with induction or aggravation of psoriasis, however, the underlying mechanism is poorly understood. previously, we could demonstrate a pivotal role for langerhans cells and dermal dendritic cells in antimalarial-provoked psoriasis by maintaining a potent th17 activity. in the present study, we investigated the effect of propranolol on human monocyte-derived langerhans-like cells (molc) and dendritic cells (modc) under inflammatory conditions. in the presence of il-1β, propranolol induced the th17 priming cytokines il-23 and il-6 in a concentration-dependent manner. the increased cytokine release was not mediated by camp suggesting gpcr-independent pathways. in contrast, il-36γ and lps failed to increase il-23 release in molc and modc in the presence of propranolol but further induced secretion of il-1β. autophagy has been linked with the secretion of il-1 family cytokines that are upregulated in chronic inflammatory disorders such as psoriasis. propranolol upregulated the expression levels of the autophagy marker p62 and lc3-i to lc3-ii conversion, induced accumulation of lc3 positive vesicles, as well as expression of il-1 signalling downstream adapter molecule traf6, indicating a late-stage block in autophagy. in summary, our results suggest a prominent role of cutaneous dendritic cell subtypes in psoriasis-like skin inflammation mediated by propranolol and possibly other beta blockers. langerhans cells (lcs) represent a highly specialized subset of epidermal dendritic cells (dcs), yet not fully understood in their function of balancing skin immunity. in the present study, we investigated in vitro generated langerhans-like cells obtained from the human acute myeloid leukaemia cell line mutz-3 (mutz-lcs) to study tlr-and cytokine-dependent activation of epidermal dcs. mutz-lcs revealed high tlr2 expression and responded robustly to tlr2 engagement, confirmed by increased cd83 and cd86 expression, upregulated il-6, il-12p40 and il-23p19 mrna levels and il-8 release. tlr2 activation reduced ccr6 and elevated ccr7 mrna expression and induced migration of mutz-lcs towards ccl21. similar results were obtained by stimulation with pro-inflammatory cytokines tnf-α and il 1β whereas ligands of tlr3 and tlr4 failed to induce a fully mature phenotype. despite limited cytokine gene expression and production for tlr2-activated mutz-lcs, co culture with naive cd4+ t cells led to significantly increased ifn-γ and il-22 levels indicating th1 differentiation independent of il-12. tlr2-mediated effects were blocked by the putative tlr2/1 antagonist cu-cpt22, however, no selectivity for either tlr2/1 or tlr2/6 was observed. computer-aided docking studies confirmed non-selective binding of the tlr2 antagonist. taken together, our results indicate a critical role for tlr2 signalling in mutz-lcs considering the leukemic origin of the generated langerhans-like cells. the stagnation in the development of new antibiotics during the last decades and the concomitant high increase of resistant bacteria emphasize the urgent need for new therapeutic options. antimicrobial peptides are promising agents for the treatment of bacterial infections and recent studies indicate that pep19-2.5, a synthetic antimicrobial and lps-neutralizing peptide (salp), efficiently neutralizes pathogenicity factors of gram-negative and gram-positive bacteria and protects against sepsis. in the present study, we investigated the potential of pep19-2.5 and the structurally related compound pep19-4lf for their therapeutic application in bacterial skin infections. primary human keratinocytes responded to tlr2 (fsl-1) but not tlr4 (lps) activation by increased il-8 production, as determined by elisa. western blot analysis showed that both salps inhibited fsl-1-induced phosphorylation of nf-κb p65 and p38 mapk. furthermore, the peptides significantly reduced il-8 release and gene expression of il-1β, ccl2 (mcp-1) and hbd-2 as assessed by qpcr. no cytotoxicity (mtt test) was observed at salp concentrations below 10 µg/ml. in lps-stimulated monocyte-derived dendritic cells, the peptides blocked il-6 secretion, downregulated expression of the maturation markers cd83 and cd86, as analysed by flow cytometry, and inhibited ccr7-dependent migration capacity. similarly, monocyte-derived langerhans-like cells activated with lps and pro-inflammatory cytokines showed reduced il-6 levels and cd83/cd86 expression in the presence of salps. in addition to acute inflammation, bacterial infections often result in impaired wound healing. since re-epithelialization is a critical step in wound repair, we tested whether pep 19-2.5 affects keratinocyte migration using the scratch wound assay. the peptide markedly promoted cell migration and accelerated artificial wound closure at concentrations as low as 1 ng/ml and was equipotent to tgf-β. conclusively, our data suggest a novel therapeutic target for the treatment of patients with acute and chronic skin infections. recently, we and others have shown that the transcription factor nuclear factor erythroid 2-related factor 2 (nrf2), a major regulator of the cellular antioxidant defence system, is activated by mechanical ventilation. during ventilator-induced lung injury, nrf2 exerts a protective role by interaction with the stretch-induced growth factor amphiregulin. in the current study, we aimed to investigate the role of nrf2 in acid-induced lung injury, a model for aspiration-induced ards. methods: nrf2-deficient (nrf2 -/-) mice and wild type (wt) littermates were tracheotomised and ventilated for 30min (v t =16ml/kg, f=90min -1 , peep=2cmh 2 o, fio 2 =0.3), before 50 µl hydrochloric acid (hcl) with ph=1.5 or ph=1.3 were instilled intratracheally, controls received nacl. mice were then ventilated for further 6h under monitoring of lung mechanics and vital parameters. blood gases as well as proinflammatory mediators, neutrophil recruitment and microvascular permeability were examined to assess lung injury. results: instillation of hcl ph=1.5 induced mild lung injury, indicated by hypoxemia (po 2 /fio 2 ~300mmhg) and continuously increasing lung tissue elastance (stiffness), from which nrf2 -/mice were protected. pulmonary inflammation, characterized by liberation of cytokines, chemokines and oedema formation, was attenuated in nrf2 -/mice. in contrast, hcl ph=1.3 caused more severe lung injury (po 2 /fio 2 ~200mmhg) with a steeper incline in elastance and more severe inflammation in both wt and nrf2 -/mice. conclusion: we conclude that the presence of nrf2 augments mild acid-induced lung injury, but plays no role in more severe injury. these discrepant results will be elucidated in future investigations. uniklinik rwth aachen, institut für pharmakologie und toxikologie, aachen, germany 2 fakultät für maschinenwesen der rwth aachen, werkzeugmaschinenlabor, aachen, germany rationale: reproducibility is key to science. in recent times, the reproducibility of biomedical research has been questioned increasingly. this reproducibility crisis also affects complex animal experiments, which -if not reproducible -might also be regarded as unethical and lose public acceptance. part of the problem is frequently that the provided documentation is not sufficient for reproduction. therefore, in this study we analyzed the potential of conventional quality management tools -used as standard in machine production -as an approach to improve the documentation and ascertain the quality of complex animal experiments. methods: quality management tools were transferred to an experimental animal set up -the mouse intensive care unit (micu) -which we use for lung injury studies. the tools included visualization of the experimental set-up, transfer of the experimental procedures to an event-driven process chain (epc) and statistical process control (spc) of all crucial pulmonary and cardiovascular parameters. data from ventilator-and acidinduced lung injury studies acquired in the micu were analyzed retrospectively. results: schematic visualization of the micu resulted in a chart comprising medical components, hardware, software and generated data types. the customized epc included all important activities and the resulting events for preparation of the mouse and the workplace, the actual animal ventilation experiment and sample-taking. in addition, checklists were provided for these activities and events, to ensure standardization of every work step. lung impedance and cardiac functions from ventilator-and acid-induced lung injury models were analyzed by spc and correlated with events in the epc. the spc proved to be suitable to identify outliers, predict processes and thereby validate the lung injury models. conclusions: conventional quality management tools were successfully adapted to analyze the quality of lung injury experiments in the micu. we suggest that this new approach is suitable to standardize animal testing procedures and increase the reproducibility of animal studies. background: a dysfunctional endothelial l-arginine-nitric oxide (no) pathway is a key pathomechanism of idiopathic pulmonary arterial hypertension (ipah) that can be provoked by hypoxia in cell culture models [1] [2] [3] [4] . the small peptide apelin is involved in the maintenance of pulmonary vascular homeostasis and angiogenesis although its precise mechanism of action is still unclear [5] . asymmetric dimethylarginine (adma) is known to be an endogenous inhibitor of endothelial no synthase and is associated with several cardiovascular diseases [6] . adma is degraded by dimethylarginine dimethylaminohydrolase 1 and 2 (ddah) enzymes [7] . objective: to determine the effect of apelin on the l-arginine/no pathway in human pulmonary microvascular endothelial cells (hpmecs). methods: hpmecs were cultured under normoxic and ph-related hypoxic conditions and treated with apelin. the expression of regulators of the l-arginine/no pathway were analysed using real-time pcr. the effect of apelin on the phosphoinositide-3 kinase (pi3k)/akt signalling pathway was determined using immunoassays and specific inhibitors[lh1] . apelin and adma concentrations were measured in cell culture supernatants using an enzyme-linked immunosorbent assay and a liquid chromatography-tandem mass spectrometry assay. results: treatment with apelin resulted in a reduced expression of the apelin receptor (aplnr) on hpmecs suggesting a negative feedback mechanism. apelin directly influenced the l-arginine/no pathway by increasing the expression of ddah1 and ddah2 enzymes. thus, the concentration of adma was decreased in hpmecs supernatant following treatment with apelin. the effect of apelin could be abrogated by modulation of the pi3k/akt pathway. conclusion: apelin modulates the l-arginine/no pathway and mediates enhanced degradation of adma via an upregulated expression of ddah 1 and 2 enzymes. the pi3k/akt pathway might play a decisive role in regulation of the effect of aplein. an apelin receptor agonist could be a novel and promising therapeutic option for ipah treatment. background and purpose: there is presently no proven pharmacological therapy for the acute respiratory distress syndrome (ards). recently, we and others discovered that the heptapeptide angiotensin (ang)-(1-7) shows significant beneficial effects in preclinical models of acute lung injury (ali). here, we aimed to identify the best time window for ang-(1-7) administration to protect rats from oleic acid (oa) induced ali. experimental approach: the effects of intravenously infused ang-(1-7) were examined over four different time windows before or after induction of ali in male sprague-dawley rats. hemodynamic effects were continuously monitored, and loss of barrier function, inflammation, and lung peptidase activities were measured as experimental endpoints. key results: ang-(1-7) infusion provided best protection from experimental ali when administered by continuous infusion starting 30min after oa infusion till the end of the experiment (30-240min). both pretreatment (-60-0min before oa) and short-term therapy (30-90 min after oa) also had beneficial effects although less pronounced than the effects achieved with the optimal therapy window. starting infusion of ang-(1-7) 90min after oa (late-term infusion) achieved no protective effects on barrier function or hemodynamic alterations, but still reduced myeloperoxidase and angiotensin converting enzyme activity, respectively. conclusions and implications. our findings indicate that early initiation of therapy after ali and continuous drug delivery are most beneficial for optimal therapeutic efficiency of ang-(1-7) treatment in experimental ali, and presumably accordingly, in clinical ards. airway epithelium functions as a physicochemical barrier against dust, air pollutants and other pathogens and plays a critical role in physiological and pathological processes including modulation of the inflammatory response, innate immunity and airway remodeling such as in human asthma, copd and equine recurrent airway obstruction (rao). models of the airway epithelia are, indeed, missing for the horse; thus, we established long-term equine bronchial epithelial cell cultures using the rock inhibitor y-27632 and cell growth and differentiation was characterized. bronchial epithelial cells (ebec) from adult horses were cultured in the presence and absence of 10 µm y-27632 under conventional and air-liquid-interface (ali) culture conditions. cell proliferation and differentiation were analyzed. formation of a functional epithelial barrier was investigated by transepithelial electric resistance (teer) measurement and immunocytochemical staining of the tight-junction-protein zonula occludens-1 (zo-1). under conventional culture, y-27632 induced higher growth rate of primary ebec and increased the passage number up to 5 passages with retained epithelial cell behavior. in the presence of y-27632, ebecs under ali showed higher teer values. expression of zo-1 correlated with the increase in teer, but in y-27632-treated ebec tight-junctionformation was more rapid, indicating accelerated differentiation, as well h/e-staining and scanning electron microscopic imaging showed a higher amount of cilia and microvilli and pas-positive cells. in conclusion, the data suggest that the rock inhibitor y-27632 facilitates long-term culture of equine bronchial epithelial cells which can be used to study airway disease mechanisms and to identify pharmacological targets. leishmaniasis is a neglected disease of tropical and subtropical regions with millions of people at risk of infection with severe consequences including death. current antileishmanial drugs exhibit serious side effects and also development of resistances is rising. this disease is caused by protozoal organisms from the genus leishmania. in their insect vector they exist in the promastigote form, while in the mammalian host they survive as amastigotes inside the phagolysosomes of macrophages. this makes a specific pharmacotherapy complicated. due to the success of artemisinin in malaria therapy, it was of interest whether endoperoxides are also useful to treat leishmaniasis. in a previous study we demonstrated that ascaridole, an endoperoxide from chenopodium ambrosioides, can cure cutaneous leishmaniasis in a mouse model and exhibited ic 50 values for the viability in the low micromolar range [1] . even though in chemical model systems some basic ideas about the mechanism of activation of these endoperoxides exist, in biological systems including leishmania parasites this activation step has never been demonstrated. therefore, we set up experiments to identify primary drug intermediates formed from ascaridole by activation in leishmania tarentolae promastigotes using electron spin resonance spectroscopy in combination with spin trapping methods. ascaridole was activated in a cell-free system by fe 2+ . the radicals were trapped by 2-methyl-2-nitrosopropane (mnp). the resulting esr spectra consisted of the triplet of duplets. spectral simulations revealed coupling parameters of a n = 16.8 g, and a h = 1.8 g. these coupling constants are compatible with iso-propyl radicals as primary intermediates. in the cellular system, consisting of leishmania tarentolae promastigotes, instead of mnp the less cytotoxic 5,5-dimethyl-1pyrroline-n-oxide (dmpo) was used for spin trapping. without addition of fe 2+ a six line esr signal was observed. spectral simulations of the dmpo spin adduct revealed coupling constants of a n = 16.1 and a h = 24.6 g. according to previously published data [2] from other spin trapping experiments, this corresponds to the formation of carboncentered radicals from ascaridole by leishmania parasites. additional experiments using iron chelators and antioxidants as well as a comparison with the endoperoxide artemisinin were performed. in summary, this study for the first time demonstrated the activation of the endoperoxide ascaridole by a protozoal organism to its active intermediate as a prerequisite to understand its mechanism of action. [1] l. monzote, j. pastor, r. scull, and l. gille. antileishmanial activity of essential oil from chenopodium ambrosioides and its main components against experimental cutaneous leishmaniasis in balb/c mice. phytomedicine 21:1048-1052, 2014. nitric oxide (no), produced by the inducible nitric oxide synthase (inos) has many functions in physiological and pathophysiological pathways. after induction of inos expression by cytokines and other agents the enzyme produces high amounts of no in a ca 2+ -independent way. this high no production can have beneficial microbicidal, antiparasital, antiviral and antitumoral effects. in contrast, aberrant inos induction may have detrimental consequences and seems to be part of many diseases such asasthma, arthritis, multiple sclerosis, colitis, psoriasis, neurodegenerative diseases, tumor development, transplant rejection or septic shock. analysis of the human inos-mrna structure revealed the existence of an upstream open reading frame (µorf) and a putative internal ribosome entry site (ires) in the 5' untranslated region (5'utr) in front of the start codon of the inos coding sequence (cds). to analyze the function of the µorf and the putative ires we cloned different egfp and luciferase reporter constructs and transfected them into the human colon carcinoma cell line dld1. using a plasmid construct with the µorf fused with the egfp cds, we could show that the µorf can be translated. however, compared to the positive control plasmid less egfp was produced, which can be explained by a weak kozak sequence of the µorf. blocking the mrna cap-dependent translation by cloning a stem loop structure in front of the inos 5'utr within a luciferase reporter plasmid led to a remarkable loss of luciferase production. thus, the expression of inos seems to be cap-dependent. furthermore, transfection experiments with dld1 cells using constructs coding for a bicistronic renilla-firefly luciferase mrna showed that there is no ires in front of the inos cds. taken together, the inos expression seems to be cap-dependent and without influence of an ires, while the µorf is translatable. therefore we speculate that inos expression is only possible due to a leaky scanning mechanism depending on the weak kozak sequence of the µorf. objectives: vascular oxidative stress is considered a pathophysiologic factor promoting cardiovascular diseases such as coronary artery disease, heart failure, diabetes and hypertension. there are several sources of superoxide in vascular smooth muscle and endothelial cells but whether an impairment of the catalytic function of enos and thus generation of oxidative stress is involved in blood pressure (bp) regulation and/or the development of hypertensive disease states is unknown. methods: we generated a mutant enos in which one of the two essential cysteines required for the coordination with the central zn-ion, correct dimer formation and normal activity is replaced by alanine (c101a-enos). normal enos (enos-tg) or a novel dimer-destabilized c101a-enos described previously (antioxid redox signal. 2015 sep 20; 23(9) :711-23) were introduced in c57bl/6 in an endothelial-specific manner. mice were monitored for enos expression and localization, aortic relaxation, systolic blood pressure, levels of superoxide and several post-translational modifications indicating activity and/or increased vascular oxidative stress. some groups of mice underwent voluntary exercise training for 4 weeks or treatment with sod mimetic tempol. results: c101a-enos-tg showed significantly increased superoxide generation, protein-and enos-tyrosine-nitration, enos-s-glutathionylation, enos 1176/79 phosphorylation and amp kinase (ampkα) phosphorylation at thr172 in aorta, skeletal muscle, left ventricular myocardium and lung as compared to enos-tg and wild type (wt) controls. the localization of enos-c101a-tg was restricted to endothelium as evidenced by immunohistochemically staining for enos and an endothelial-specific marker cd31. exercise training increased phosphorylation of enos at ser 1176/79 and of ampkα at thr 172 in wt but not in c101a-enos-tg. aortic endothelium-dependent and endothelium-independent relaxations were similar in all strains. in striking contrast, c101a-enos-tg displayed normal blood pressure despite higher levels of enos, while enos-tg showed significant hypotension. tempol completely reversed the occurring protein modifications and significantly reduced bp in c101a-enos-tg but not in wt controls. conclusions: by means of a novel transgenic mouse model we demonstrated that vascular oxidative stress generated by endothelial-specific expression of a dimerdestabilized variant of enos selectively prevents bp reducing activity of vascular enos, while having no effect on aortic endothelial-dependent relaxation. these data suggest that oxidative stress in microvascular endothelium may play a role in the development of essential hypertension. the herbal medicinal product myrrhinil-intest ® consists of myrrh, chamomile flower dry extract and coffee charcoal. clinical data prove the effectiveness of this herbal preparation for inflammatory intestinal disorders. to further investigate the anti-inflammatory potential of the single components as part of a multi-target principle, an ethanolic (my) and aqueous (mya) myrrh extract, ethanolic chamomile flower extract (ka) and aqueous coffee charcoal extract (cc), were examined in an in vitro tnbs inflammation model using rat small intestinal preparations. the effect of the plant extracts on tnbs induced inflammatory damage was characterised based on tnfα-gene expression analysis, isometric contraction measurement and histological analysis. furthermore, tnfα-release from lpsstimulated thp-1 cells was determined. budesonide was used as positive control. additionally, microarray gene expression analysis was performed in lps/ifnγ stimulated native human macrophages to determine potential underlying mechanisms. the tnbs-induced overexpression of tnfα-mrna was reduced after ka (0.1 mg/ml) and mya (1 mg/ml) treatment down to 24% and 16% resp.; tnbs-induced loss of contractility and reduction of mucosal layer thickness was inhibited after ka (3 mg/ml) treatment by 26% and 25% resp.; after mya (0.1-1mg/ml) treatment by 17% and 44% resp. lps-induced tnfα release from thp-1 cells was inhibited concentrationdependently by my (ic50 = 60.65 μg/ml; 97% inhibition), ka (ic50 = 439 μg/ml; 71% inhibition) and cc (ic50 = 1886 μg/ml; 44% inhibition). furthermore, ka (200 µg/ml) and cc (500 µg/ml) inhibited the lps/ifnγ-induced expression of genes associated with chemokine signalling up to 100-fold (for cxcl13). the presented study demonstrates further evidence for anti-inflammatory properties of the herbal components which contribute to the reported clinical effectiveness. introduction: the purine nucleoside adenosine, which is involved in a variety of physiological functions, regulates immune and inflammatory responses and acts as a modulator of gut functions. although it is present at low concentrations in the extracellular space, stressful conditions, such as inflammation, can markedly increase its extracellular level up to micromolar range. by activation of different receptor subtypes adenosine is able to induce anti-inflammatory or pro-inflammatory impacts. aim: the current study examined the impact of adenosine a2a receptors (a2ar) and adenosine a2b receptors (a2br) to regulate contractility in untreated and inflamed rat colon preparations using a specific a2ar agonist (cgs 21680) and an a2br antagonist (psb-1115) on acute inflammation in rat colon preparations. further it focused on interactions of the multi-herbal drug stw 5 with a2ar as a possible mechanism of the protective effect of stw 5 in gastrointestinal disorders. methods: inflammation was induced by intraluminal instillation of 2,4,6-trinitrobenzene sulfonic acid (tnbs). contractions were measured isometrically in an organ bath set up. gene expression was determined using rt-pcr. radio ligand binding assays (competition experiments) were carried out with rat brain homogenates. morphological changes were estimated after van gieson staining. results: all four adenosine receptor subtypes were expressed in untreated colon preparations. activation of a1, a2b, and a3 receptor with specific agonists reduced the acetylcholine (ach, 10µm)-induced contractions, while activation of a2br enhanced it. after incubation with tnbs morphological damages in colonic mucosa and muscle walls were detectable followed by reduced ach-contractions. the tnbs-mediated decrease of ach-contractions as well as the morphological damages were partially normalized by co-incubation of tnbs with cgs 21680 (10µm) or with psb 1115 (100µm). the same effects with smaller intensity were found for stw 5 (512 µg/ml) in female but not in male colon preparations. these results are in accordance with ligand binding studies indicating that stw 5 interact with the a2ar. conclusion: anti-inflammatory mechanisms and cell protective actions of stw 5 are partly due to the interaction with adenosine receptors. the results give a clear-cut correlation with symptom improvements in clinical trials and thereby highlight the relevance of stw 5 as a therapeutic approach in ibs. (allescher 2006) . therefore, a multi-target approach is a promising therapeutic strategy, as is exemplified by stw 5(ottillinger et al. 2013) . stw 5 (iberogast®) is a fixed combination of nine plant extracts with iberis amara (stw 6) as one of its components. it is successfully used for treatment of functional dyspepsia and irritable bowel syndrome (ibs). to allow an overview of targets addressed by stw 5 and the role of its components in relation to the different forms and causes of functional gi diseases, an evaluation of the data, which have been gained from more than 150 pharmacological tests, is needed. all data from studies including stw 5 alone, or stw 5 and its components, were retrieved and sorted according to types of study models (human and animal systems, animal disease models, gi-preparations, cell cultures, in vitro-systems) and respective etiologic mechanisms related to fgds and then visualized in the form of 2d histograms (lorkowski et al. 2015) . results: more than 150 pharmacological tests indicated anti-oxidative activity, electrophysiological effects, ulcer protection, anti-inflammatory actions, pro-kinetic and spasmolytic effects as well as reflux and acid reduction. moreover, the analysis indicated that the components of stw 5 contribute differently to the overall effect of stw 5. altogether, the evaluation of the data shows that stw 5 is active in response to multiple etiologic factors involved in fgds, especially functional dyspepsia and irritable bowel syndrome, and to which extent the herbal extract components of the combination are relevant for the different mechanisms of action and their translation to clinical efficacy. conclusion: multi step clustering allows the transformation of complex data sets. it makes the allocation of specific actions to the different components of stw 5 manageable, so also giving support to its clinical use in patients with different symptoms. introduction: stw 5 ii has been recently developed in an effort to reduce the number of active extracts in the mother multi-component herbal preparation, stw 5 (iberogast ® , steigerwald arzneimittelwerk gmbh, darmstadt, germany) without affecting the overall therapeutic efficiency. stw 5 consists of a mixture of 9 standardized extracts: bitter candytuft (iberis amara), lemon balm (melissa officinalis), chamomile (matricaria recutita), caraway fruit (carum carvi), peppermint leaf (mentha piperita), liquorice root (glycyrrhiza glabra), angelica root (angelica archangelica), milk thistle (silybum marianum) and celandine herb (chelidonium majus), whereas stw 5 ii lacks the last 3 components. stw 5 was shown to be effective clinically to treat functional dyspepsia (1) and irritable bowel syndrome (2) and was shown experimentally to be effective to guard against the development of radiation induced intestinal mucositis (3) and in the management of ulcerative colitis (4) . the present study was initiated to show whether stw 5 ii with the reduced component extracts would also be as effective in the latter condition. this was induced in wistar rats by feeding them with 5% dextran sodium sulfate in drinking water for 1 week when lesions were observed in the colon evidenced by histological examination as well as colon shortening and reduction of colon mass index. this was associated with a rise in myeloperoxidase and a fall in reduced glutathione, glutathione peroxidase, and superoxide dismutase in colon homogenates as well as a rise in tnfα in serum. oral administration of stw 5 in doses of 2 and 5 ml/kg or stw 5 ii in a dose of 2 ml/kg for 1 week before and continued during dss feeding tended to normalize all the changes in a fashion comparable to sulfasalazine, used as a reference drug in a dose of 300 mg/kg. conclusions: the modified preparation, stw 5 ii thus proved to be as effective as stw 5, thereby reflecting its potential usefulness in ulcerative colitis possibly by virtue of its anti-inflammatory and anti-oxidant properties. (1) schmulson mj (2008) the emetic pathways include the action of neurotransmitters dopamine, serotonin and substance p in the emetic centers localized in the brainstem, area postrema and vagal nerve afferents. previous in vivo studies in beagle dogs revealed that the plant alkaloid lycorine potentially induce nausea and emesis. though antagonists of the tachykinin receptor 1 (maropitant) and serotonin receptor 3 (ondansetron) prevented lycorinemediated emesis, the molecular mechanism of nausea and vomiting remain still unknown. to study the mechanism of action of the emetic agents, we analyzed the effect of lycorine (direct activation of nk1) and channel opening (activation of 5ht3) on the intracellular calcium homeostasis (using fluorometric ca 2+ analysis) and cell proliferation rates in endogenously nk1 and 5ht3 receptor expressing cell lines as well as in cho and hek cells stably expressing the receptors. neither endogenously receptor expressing nk1 or 5ht3 cells nor receptor overexpressing cells showed calciumflux or calcium mobilization after stimulation with lycorine. furthermore, we are measuring the receptor number and subtypes using radioligand binding studies. it is planned, moreover, to obtain fluorescent labeled constructs of the nk1 receptor to gain insights into the involvement of receptor internalization which might mediate emesis. by characterizing these molecular principles of the nk1 and 5ht3 receptors, we are attempting to obtain more information in predicting drug-induced side effects such as nausea and emesis. the intestinal epithelium is completely renewed every 4-5 days. this process is driven by stem cells, which reside within specialized niches in the intestinal crypts and give rise to several differentiated cell types, including enterocytes, paneth, enteroendocrine, goblet and tuft cells. however, the molecular mechanisms that establish and maintain differentiated cell numbers and proportions remain largely unknown. here, we systematically analyzed the intestinal expression of semaphorins and plexins, which constitute a ligand-receptor system that plays central roles in cell-cell communication in various biological contexts. we identified plexin-b2 and its semaphorin ligands to be highly expressed in intestinal epithelial cells. genetic inactivation of plexin-b2 in intestinal organoids strongly reduced the number of enteroendocrine cells. our data suggest that semaphorin-plexin-b2 signaling promotes differentiation of intestinal epithelial cells towards the enteroendocrine lineage. the gastric epithelium contains several types of differentiated cells, including foveolar cells that produce mucus, parietal cells that secrete gastric acid and intrinsic factor, chief cells that synthesize pepsinogen and gastric lipase, and enteroendocrine cells that release different hormones. these differentiated cell types all originate from multipotent stem cells, yet little is known about how this differentiation process is regulated on a molecular level. the gap protein rasal1 controls the activity of small gtpases of the ras family, and its expression levels have been shown to inversely correlate with progression of stomach cancers. however, functional studies on the physiological role of rasal1 in the gastric epithelium are lacking. here, we established and characterized a mouse line with inactivation of the rasal1 gene. we observed that these mice showed increased numbers of enteroendocrine cells in the gastric mucosa. conditional inactivation of rasal1 in enteroendocrine cells, using a mouse line in which cre expression is driven by the atoh1 promoter, further corroborated that rasal1 expression in enteroendocrine cells determines enteroendocrine cell numbers. these findings identify rasal1 as a regulator of gastric epithelial cell differentiation. ). the present study investigates the impact of two faah inhibitors (arachidonoyl serotonin [aa-5ht], urb597) on a549 lung cancer cell metastasis and invasion. lc-ms analyses revealed increased levels of faah substrates (aea, 2-ag, oea, pea) in cells incubated with either faah inhibitor. in athymic nude mice faah inhibitors were shown to elicit a dosedependent antimetastatic action. in vitro, a concentration-dependent anti-invasive action of either faah inhibitor was demonstrated, accompanied with upregulation of tissue inhibitor of matrix metalloproteinases-1 (timp-1). using sirna approaches, a causal link between the timp-1-upregulating and anti-invasive action of faah inhibitors was confirmed. moreover, knockdown of faah by sirna was shown to confer decreased cancer cell invasiveness and increased timp-1 expression. inhibitor experiments point toward a decisive role of cb 2 and transient receptor potential vanilloid 1 in conferring the anti-invasive effects of faah inhibitors and faah sirna. finally, antimetastatic and anti-invasive effects were confirmed for all faah substrates. collectively, the present study provides first-time proof for a pronounced antimetastatic action of the faah inhibitors aa-5ht and urb597. as underlying mechanism of its anti-invasive properties an upregulation of timp-1 was identified. regenerative activity in tissues of mesenchymal origin depends on the migratory potential of mesenchymal stem cells (mscs). the present study focused on inhibitors of the enzyme fatty acid amide hydrolase (faah), which catalyzes the degradation of endocannabinoids (anandamide, 2-arachidonoylglycerol) and endocannabinoid-like substances (n-oleoylethanolamine, n-palmitoylethanolamine). in boyden chamber assays, the faah inhibitors, urb597 and arachidonoyl serotonin (aa-5ht), were found to increase the migration of human adipose-derived mscs. lc-ms analyses revealed increased levels of all four aforementioned faah substrates in mscs incubated with either faah inhibitor. following addition to mscs, all faah substrates mimicked the promigratory action of faah inhibitors. promigratory effects of faah inhibitors and substrates were causally linked to activation of p42/44 mitogen-activated protein kinase (mapk), as well as to cytosol-to-nucleus translocation of the transcription factor, peroxisome proliferator-activated receptor α (pparα). whereas pparα activation by faah inhibitors and substrates became reversed upon inhibition of p42/44 mapk activation, a blockade of pparα left p42/44 mapk phosphorylation unaltered. collectively, these data demonstrate faah inhibitors and substrates to cause p42/44 mapk phosphorylation, which subsequently activates pparα to confer increased migration of mscs. this novel pathway may be involved in regenerative effects of endocannabinoids whose degradation could be a target of pharmacological intervention by faah inhibitors. background: the hematopoietic disorder chronic myeloid leukemia (cml) is one of the most extensively studied neoplasms. it is caused by translocation between chromosomes 9 and 22 leading to the formation of the philadelphia chromosome and the bcr-abl fusiongene. first-line targeted therapy is still the tyrosine-kinase inhibitor imatinib (im), which led to tremendous success in treatment. however, the amount of therapeutic resistances is increasing, caused either by bcr-abl-dependent mechanisms (e.g. bcr-abl amplification/overexpression, point mutations) or bcr-ablindependent mechanisms. these might be linked to alterations in drug transporter expression or particularly, microrna-expression levels. in our previous study, we analyzed the changes of microrna expression profiles during the development of imresistances in the leukemic cell line k562. an inverse correlation of mir-212 expression and protein levels of the efflux transporter atp-binding cassette transporter g2 (abcg2) was observed in cells resistant to different im-concentrations, pointing to a relation of mir-212 to im-resistance. hence, we investigate in current studies, how the influence of mir-212 on im-sensitivity could be explained. methods: we transfected k562 cells, sensitive treatment-naïve cells and cells resistant to various im-concentrations, either with mir-mimic pre-mir-212 or inhibitory anti-mir-212, challenged them with im and analyzed effects on cell viability, activation of apoptosis and cell death using wst-1-, caspase glo 9-assay and cell counting. in addition, we analyzed changes in abcg2 expression using flow cytometry and qrt-pcr and investigated alterations in im-efflux using hplc and hoechst efflux assay. results: under im-treatment, sensitive k562 showed an effect of mir-212-inhibition using anti-mir-212. this led to a significant promotion of cell survival apparent on the level of respiratory chain function (p<0.01) and cell membrane integrity and reduced capase-9 activity (p<0.05). furthermore, these mirna-effects are dose-dependent as confirmed in concentration row-experiments. regarding transport and abcg2 expression, we found that 2 µm im-resistant k562 do not express higher amounts of abcg2, but showed higher transport rates of im or the abcg2-substrate hoechst 33342. conclusions: overall, these experiments indicate that mir-212 does not only affect abcg2-expression, but also influences cell sensitivity to im in a more direct manner. further analysis will now be performed to reveal the underlying mechanism, how cell sensitivity to im is altered and if these effects occur due to a direct regulation of abcg2. in summary, these findings could be relevant in cml-therapy, overcoming imresistances with a better understanding of mirna-and drug transporter alteration in cml. acknowledgments: we would like to thank all the authors for their contribution to this project. this work was funded by the university hospital schleswig-holstein. oxidized silicon nanoparticles and iron oxide nanoparticles for radiation therapy s. klein radiation therapy often combined with surgery and/or chemotherapy is applied to more than 50 % of patients at some point of their treatment. the cytotoxic effects of ionizing radiation occur from their ability to produce dna double-strand breaks through the formation of free radicals within cells. however, the curative potential of radiotherapy is often limited by intrinsic radio resistance of cancer cells and normal tissue toxicity. to overcome this resistance and enhance the effectiveness of ionizing radiation, radio sensitizers are used in combination with radiotherapy. in our studies we used amino functionalized, oxidized silicon nanoparticles (sinp), superparamagnetic iron oxide nanoparticles (spion) and iron doped silicon nanoparticles (fe(1%)-sinp) to increase the formation of reactive oxygen species (ros) in cells. cancer and tissue cells loaded with the various nanoparticles were irradiated with a single dose of 1-3 gy using a 120 kv x-ray tube. after irradiation, the formation of the different ros species including superoxide, hydroxyl radicals and singlet oxygen was investigated. sinps with sizes around 1 nm can easily cross the cell and nuclear membrane. the positively charged amino functionalized sinps stick in all membranes as well in those of the mitochondria. irradiation of the mitochondria may cause the depolarization of the mitochondrial membrane, which enables the release of cytochrome c and simultaneously, an inhibition of the respiratory chain, which leads to an increased generation of superoxide. amino functionalized sinps, as being embedded in the outer mitochondrial membrane, evidently enhance the depolarizing effect of the x-ray radiation on the mitochondria and therefore increase the concentration of superoxide. [1] oxidized sinps with larger sizes accumulate in the cytoplasm and generate mainly singlet oxygen after irradiation. spions enter the cells via endocytosis, whereas the uncoated spions remain in the vesicles and the citrate coated spions accumulate in the cytoplasm. cells loaded with citrate coated spions show no higher ros concentration than in media-cultured cells. but after irradiation, the ros formation increased drastically. this enhancing effect is explained with the impact of x-rays onto the surface of spions which is due to the destruction of surface structures. the freed spion surface contains easier accessible iron ions. this ions can participate in the fenton and haber-weiss chemistry and thus, catalyze the hydroxyl radical formation. [2] 1 to 5 % iron doped sinp increase the formation of hydroxyl radicals as well as the generation of singlet oxygen after irradiation. chronic pain in response to tissue damage (inflammatory pain) or nerve injury (neuropathic pain) is a major clinical health problem, affecting up to 30% of adults worldwide. currently available treatments are only partially susceptible and are accompanied with therapy limiting side effects. thus it is important to elucidate molecular mechanisms of pain signaling in detail to obtain new insights in potential future therapies. recent data indicate that hydrogen sulfide (h 2 s) contributes to the processing of chronic pain, however pro-as well as antinociceptive effects have been described so far. moreover the sources of h 2 s production in the nociceptive system are only poorly understood. here we investigated the expression of the h 2 s releasing enzyme cystathionine g-lyase (cse) in the nociceptive system and characterized its role in chronic pain signaling using cse deficient mice. paw inflammation and peripheral nerve injury led to upregulation of cse expression in dorsal root ganglia. however, conditional knockout mice lacking cse in sensory neurons as well as global cse knockout mice demonstrated normal pain behaviors in inflammatory and neuropathic pain models as compared to wt littermates. thus, our results suggest that cse is not critically involved in chronic pain signaling in mice and that sources different from cse mediate the pain relevant effects of h 2 s. this work was supported by the deutsche forschungsgemeinschaft (sfb815-a14) and in part by loewe-schwerpunkt "anwendungsorientierte arzneimittelforschung". heinrich-heine-universität, institut für toxikologie, düsseldorf, germany 2 heinrich-heine-universität, urologie, düsseldorf, germany background: cisplatin (cispt) is frequently used in the therapy of advanced stage urothelial cell carcinoma (ucc). yet, inherent and acquired drug resistance limits the clinical use of cispt. here, we comparatively investigated the response of epithelial-like (rt-112) and mesenchymal-like (j-82) uc cells following cispt treatment. methods: upon selection with equitoxic doses of cispt for months, we obtained cispt resistant variants (rt-112 r , j-82 r ). cell viability was measured using the alamar blue assay. cell cycle distribution was analysed by flow cytometry. immunocytochemistry was used to quantify the number of nuclear γh2ax and 53bp1 foci representing dna double strand breaks (dsbs), while western blot was used to unravel the role of dna damage response (ddr) to acquired cispt resistance. qrt-pcr was performed to analyse the mrna expression of genes associated with cispt resistance. j-82 and j-82 r cells were treated with different concentrations of lovastatin and selected ddr inhibitors to elucidate their influence on cell viability. results: untreated rt-112 cells showed an about 2-3-fold higher resistance to cispt than j-82 cells. both cell lines differed in the expression pattern of genes that are associated with cispt resistance. rt-112 r and j-82 r revealed a 2-3-fold increased cispt resistance as compared to the parental cells. during the selection procedure, we observed that acquired cispt resistance goes along with morphological alterations that resemble epithelial mesenchymal transition (emt). cell cycle analysis of rt-112 r cells disclosed a reduced apoptosis and enhanced g2/m arrest following cispt exposure as compared to rt-112 wild-type cells. by contrast, induction of cell death was similar in j-82 and j-82 r cells. notably, j-82 r cells showed a reduced formation of cispt-induced dsbs. correspondingly, the related ddr was diminished in j-82 r as compared to their parental cells. this was not found when ddr was comparatively analysed between rt-112 r and rt-112 cells. data obtained from qrt-pcr analysis indicate that different mechanisms contribute to acquired drug resistance of j-82 r and rt-112 r . unexpectedly, j-82 r and rt-112 r shared the upregulation of xaf-1. treatment of j-82 r cells with statins and protein kinase inhibitors revealed an enhanced sensitivity to pharmacological inhibition of chk-1 and, moreover, re-sensitization to cispt by chk-1 inhibitor. based on the data we suggest that mechanisms of acquired cispt resistance of epithelial and mesenchymal uc cell lines are different with apoptosisrelated mechanisms appear to be more relevant for epithelial-like rt-112 cells and ddr-related mechanisms dominating cispt susceptibility in mesenchymal-like j-82 cells. furthermore, our findings indicate that chk-1 might be an appropriate target to deal with acquired cispt resistance in ucc. in many patients, gastric cancer treatment with conventional cytostatic agents shows only limited clinical response. novel therapeutics, which inhibit rtk signaling by targeting c-met or her family receptors, have demonstrated some efficacy; however, primary resistance of gastric cancer cells against these inhibitors is still a major problem. in the present study we investigated the mechanism of heregulin (hrg)-promoted survival of gastric cancer cells after treatment with c-met inhbitors or sirna-mediated downregulation of c-met. we found that hrg treatment of gastric cancer cells with a c-met amplification partially rescued the cells from the antiproliferative effects of pharmacological c-met inhibition or sirna-mediated downregulation of c-met. moreover, c-met inhibition or downregulation led to an induction of her3 expression on mrna and protein level, whereas other her family receptors were unaffected. downregulation of her3 impaired the hrg-mediated rescue of cell survival upon c-met inhibition. in other tumor entities the chromatin organizer special at-rich sequence-binding protein 1 (satb1) has been described as a regulator of her family receptor expression involved in adaptive responses of tumor cells. thus, we investigated the contribution of satb1 in the upregulation of her3 after c-met inhibition. of note, c-met inhibitors as well as c-met-specific sirnas markedly induced satb1 expression in gastric cancer cells, and the downregulation of satb1 by sirnas completely prevented the induction of her3 upon c-met inhibition. in contrast, her1 or her2 expression levels were not affected by satb1-specific sirnas. the function of satb1 as transcriptional regulator is controlled by its phosphorylation status, which in turn is modulated by pkc activity. thus, we also tested the effect of pkc inhibitors on her3 expression after c-met inhibition. interestingly, the upregulation of her3 in gastric cancer cells was significantly reduced by pkc inhibitors. to summarize, satb1 and pkc are critically involved in the regulation of her3 expression in gastric cancer cells after treatment with c-met inhibitors and the oncogene her3 plays a crucial role for tumor cell survival in this context. thus, inhibition of pkc or satb1 may help to overcome resistance against c-met inhibition in this tumor entitiy. in the rising field of nanomedicine, development of new approaches in diagnosis and treatment of cancer is a challenging task. typically, a nanocarrier is synthesized and linked to functional compounds displaying either diagnostic or therapeutic effects in cancer models. recently, nanomaterials combining both diagnostic and therapeutic properties, so-called 'theranostics', became of primary interest. here we used a human albumin-polyethylene glycol (peg) copolymer (hsa) as a theranostic platform for molecular integration of the chemotherapeutic drug doxorubicin (dox) and the magnet resonance imaging (mri) contrast agent gadolinium (gd) yielding gd-hsa-dox nanoparticles. besides in vitro testing, which demonstrated cytotoxic efficacy of gd-hsa-dox, we used the chorioallantoic membrane (cam) of fertilized chick eggs as a preclinical xenotransplantation model. the cam assay, which in legal terms does not represent an animal experiment, allows testing of compounds in an in vivo setting. this model is particularly helpful to narrow the gap between in vitro and in vivo applications in rodents, because it can help to reduce number of elaborate experiments with typically nude mice, and it reduces or even avoids exposure of those animals to adverse effects and distress. treatment-resistant mda-mb-231 breast cancer cells stably transfected with luciferase were xenotransplanted onto the chorioallantoic membrane. after formation of solid breast cancer xenografts, gd-hsa-dox was injected intravenously and its antiproliferative effect was evaluated by ivis imaging of luciferase activity and by immunohistochemical analysis of the tumor xenografts for the ki-67 proliferation antigen. in comparison to conventional dox, gd-hsa-dox showed increased antiproliferative efficacy and reduced general toxicity in the cam assay. on the basis of these findings, a rodent model was established, where the mda-mb-231 breast cancer cells were orthotopically xenotransplanted into the mammary fat pads of female nmri nu/nu mice. in this model, we further investigated biocompatibility, as well as diagnostic and therapeutic properties of the engineered nanomaterial. after repeated administration of gd-hsa-dox into the tail vein of the animals, biocompatibility of gd-hsa-dox was confirmed by uncompromised liver, kidney and hematopoietic parameters. to warrant diagnostic properties, accumulation of the nanomaterial in tumor tissue is indispensable. by small animal mri of gd, kinetics of intravenously applied gd-hsa-dox in tumor tissue was monitored. an enhancement of the engineered nanomaterial in tumor tissue was detected for up to 47 h after injection indicating successful enrichment of gd-hsa-dox within the tumor tissue, which can be ascribed to the enhanced permeability and retention (epr) effect observed in the microenvironment of many solid tumor tissues. we are currently investigating the antitumor efficacy of gd-has-dox in this mouse model and preliminary data seem to indicate a dose-dependent anticancer effect. supported by the volkswagenstiftung. tubulin-binding agents are the most important anti-tumoral drugs. due to the side effects and the development of resistances, the discovery of new agents is still of importance. recently, pretubulysin (pt), a naturally occurring precursor of the myxobacterial compound tubulysin, was identified as a novel tubulin-binding compound. in the dfg research group for 1406, pt was characterized as anti-tumoral, antiangiogenic and vascular-disrupting compound. moreover, pt was also found to inhibit the formation of metastases in vivo. aim of the present study was to gain first insights into the mechanisms underlying this anti-metastatic effect by investigating the influence of pt on the interaction of endothelial and tumor cells in vitro. pt treatment of primary human endothelial cells (huvecs) strongly increased the adhesion of breast cancer cells (mda-mb-231) on huvecs, but limited their transmigration through the endothelium (transwell assay). based on this data, the gene expression of presumably involved adhesion molecules was determined by qrt-pcr: icam-1, vcam-1, e-selectin, n-cadherin, and galectin-3. moreover, the chemokine system cxcl12/cxcr4 was analyzed. it could be demonstrated that the mrna level of endothelial n-cadherin is upregulated by pt. while total protein expression of ncadherin was enhanced in pt treated huvec, its surface expression was not largely influenced by pt (western blot, flow cytometry). in line with this, blocking endothelial ncadherin by a neutralizing antibody revealed that this protein is not involved in ptevoked tumor cell adhesion. interestingly, pt strongly augmented the mrna and protein expression of cxcl12 in huvecs (qrt-pcr, western blot), whereas its endothelial secretion was not affected by pt (elisa). an autocrine action of cxcl12 could be excluded, since blocking the cxcl12 receptor cxcr4 on endothelial cells with plerixafor did not influence cancer cell adhesion. by microscopic analyses, we observed that pt treatment causes transient gaps in the huvec monolayer, where tumor cells prefer to adhere. since β1-integrins on the tumor cells could mediate interactions between cancer cells and extracellular matrix proteins in the gaps (e.g. collagen), their influence in cell adhesion and transmigration assays was examined. both the pt-evoked increase in cell adhesion and decrease in transmigration was completely abolished when β1-integrins were blocked on mdas by a neutralizing antibody. these results indicate that the anti-metastatic action of pretubulysin might be based on the trapping of tumor cells on the endothelium. whether this effect is also relevant in vivo, will be analyzed in future studies using intravital microscopy. this work was supported by the german research foundation (dfg, for 1406, fu 691/9-2). introduction: tyrosine kinase inhibitors (tkis) for the treatment of non-small cell lung cancer (nsclc) patients harboring activating mutations in the epidermal growth factor receptor have shown prominent success. nevertheless, patients treated with tkis eventually acquire resistance and relapse (1). based on an evolutionary cancer model (2) , weekly high dose-pulsed tki regimens were proposed to delay resistance. using data from nsclc bearing mice treated with erlotinib at different dosing regimens, we developed a semi-mechanistic pharmacokinetic/pharmacodynamic model for erlotinib effects on tumor killing and resistance development. methods: data was available from experiments in xenograft mice bearing nsclc tumors (pc9 and hcc827 cell lines; both erlotinib sensitive) (3). plasma concentrations from two single-dose groups, 30mg/kg and 200mg/kg, were used for pharmacokinetic modeling. relative tumor volume changes in mice randomized to five dosing regimens (15mg/kg daily, 30mg/kg daily, 200mg/kg every 2 days, 200mg/kg every 4 days, or vehicle) was the pharmacodynamic endpoint. a tumor growth inhibition model was developed by testing linear, exponential and logistic models to account for the tumor growth kinetics, as well as fitting an emax model to explain the effect of exposure on killing the sensitive tumor cells, and resistance development. analysis was performed using nonmem 7.3. results: absorption was dose dependent, and a precipitate compartment accounted for dissolution limited absorption for the 200mg/kg dose. a 1-compartment model with first order elimination kinetics described distribution and elimination. to describe tumor volume changes, a tumor was assumed to be a mixture of sensitive and resistant cells (represented by distinct compartments and ordinary differential equations). exponential kinetics best described natural growth (doubling times: 13 and 52 days, for sensitive and fully resistant cells, respectively). a tumor was found to transit through a less sensitive phase before acquiring full resistance. an e max model (less than linear) best described effect on the sensitive cells (ec 50 =0.53μm for both cell lines), and on the partially sensitive transit phase (ec 50 =1.24μm and 3.00μm, for hcc827 and pc9 cell lines, respectively), urging to provide adequate trough erlotinib concentrations for optimal effects. conclusions & future perspectives: an exposure-driven tumor growth inhibition model accounting for the kinetics of resistance development was developed. the model emphasizes the need for establishing an adequate trough erlotinib concentrations to delay disease progression. extracts of the stem bark of ficus platyphylla (fp) have been used in traditional nigerian medicine to treat psychoses, depression, epilepsy, pain and inflammation. previous studies have revealed the analgesic and anti-inflammatory effects of fp in different assays including acetic acid-induced writhing, formalin-induced nociception, and albumin-induced oedema. in this study, we assessed the effects of the standardised extract of fp on hot plate nociceptive threshold and vocalisation threshold in response to electrical stimulation of the tail root in order to confirm its acclaimed analgesic properties. we also investigated the molecular mechanisms underlying these effects, with the focus on opiate receptor binding and the key enzymes of eicosanoid biosynthesis, namely cyclooxygenase (cox) and 5-lipoxygenase (5-lo). fp (i) increased the hot plate nociceptive threshold and vocalisation threshold. the increase in hot plate nociceptive threshold was detectable over a period of 30 min whereas the increase in vocalisation threshold persisted over a period of 90 min. (ii) fp showed an affinity for µ opiate receptors but not for δ or κ opiate receptors, and (iii) fp inhibited the activities of cox-2 and 5-lo but not of cox-1. we provided evidence supporting the use of fp in nigerian folk medicine for the treatment of different types of pain, and identified opioid and non-opioid targets. it is interesting to note that the dual inhibition of cox-2 and 5-lo appears favourable in terms of both efficacy and side effect profile. despite the fact, that the enormous economic burden and individual suffering caused by gastrointestinal infections permanently persists in developing and newly industrialized countries, healthcare systems in first world countries underestimated its significance for a long time. the alarming prevalence of multidrug-resistant gram-negative bacteria, combined with a high epidemic potential of gastrointestinal pathogens, however, demonstrates the urgent need for new antibiotics and antiinfectives worldwide. 2,5 million deaths per year were actually caused by acute diarrheal infections. the most common causative agents of acute diarrheal infections, amongst others, are yersinia enterocolitica, campylobacter jejuni, salmonella spp., shigella spp., escherichia coli, vibrio cholerae, and clostridium difficile. the established treatment based on antibiotics is mostly ineffective or may even have adverse side effects and result in prolonged shedding. in either way, antibiotic treatment also eradicates at least parts of the intestinal microbiome, and thereby disrupts colonization resistance, fosters overgrowth of pathogens and prolongs shedding times. therefore, the development of future drugs should be focused on highly specific antiinfectives, which enable a direct pathogenspecific treatment. one very promising strategy is the inhibition of the biogenesis of outer membrane virulence factors. due to the fact that many decisive virulenceassociated outer membrane proteins (omps) of gram-negative enteropathogens are substrates of the periplasmic chaperone sura exclusively, we developed a new assay format to determine sura in vitro chaperone activity. previous publications by behrens et al., 2001 and buchner et al., 1998 documented an assay to determine sura in vitro chaperone activity with extremely limited sensitivity and minimal detectable concentration, which was not suitable for high throughput screening (hts). we now developed a luciferase-based screening assay. this highly sensitive and robust test system has been validated extensively and now gives reliable output with an appreciable z-factor of > 0,6. in cooperation with the hzi braunschweig (germany) and the hzi saarbrücken (germany), we were able to screen over 7000 purified compounds and over 500 extracts of myxobacteria. during the ongoing screening period, the assay generated four validated primary actives, which corresponds to a positive hit rate of 0,05 %. additionally, we developed an elaborate follow-up strategy to validate positive hits, which includes a well-established mouse infection model. we are looking forward to escalate our screening efforts and would like to use this abstract to invite all scientist who are interested in testing compound/natural extract libraries for an activity against the target structure sura. the potential atypical antipsychotic and dopamine d 2 receptor partial agonist 2bromoterguride antagonizes phencyclidine-and apomorphine-induced prepulse inhibition and novel object recognition deficits in rats e. tarland objectives: schizophrenia is a disabling mental disorder affecting more than 21 million people worldwide. available medical therapies are effective in the treatment of psychosis and other positive symptoms, however come with considerable side effects and often fail to ameliorate cognitive deficits and negative symptoms of the disorder. the dopamine d 2 receptor partial agonist 2-bromoterguride (2-bt) has recently been shown to exhibit antipsychotic effects in rats without causing adverse side effects common to antipsychotic drugs [1]. to determine its atypical character in vivo, the ability of 2-bt to antagonize the disruptive effects of phencyclidine (pcp) and apomorphine on sensory motor gating was determined in the prepulse inhibition paradigm. the effect of 2-bt on cognitive deficits was assessed in the novel object recognition (nor) test after object recognition memory deficits were induced by pcp treatment. method: 10 week old male sprague-dawley rats were injected with 2-bt (0.1 or 0.3mg/kg; i.p.) followed by pcp (1.5mg/kg; s.c.) or apomorphine (0.5mg/kg; s.c.). prepulse inhibition was measured in two sound-proof startle chambers. the attenuating effect of 2-bt (0.1 or 0.3mg/kg; i.p.) on visual learning and memory deficits following subchronic administration of pcp (5.0mg/kg; i.p. twice daily for 7 days) was assessed in the nor task consisting of a 3min acquisition trial and a 3min retention trial separated by a 1h inter-trial interval. clozapine (5.0mg/kg; i.p) or haloperidol (0.1mg/kg; i.p) were used as positive controls. results: the dopamine d 2 receptor partial agonist 2-bt (0.3mg/kg) and the typical antipsychotic haloperidol successfully antagonized apomorphine-induced ppi-deficits. interestingly 2-bt also ameliorated the pcp-induced ppi-deficits to the same extent as the atypical antipsychotic clozapine. preliminary data from the nor test indicate that 2-btreduces subchronic pcp-induced cognitive deficits in novel object recognition analogous to clozapine. the disrupting effects of pcp on ppi are mediated by non-competitive antagonism at nmda sites indirectly influencing a series of neurotransmitter systems. our results indicate that 2-bt mediates actions at multiple neurotransmitter receptors as it successfully ameliorated both the pcp-and apomorphine-induced ppi disruptions in rats, showing an atypical antipsychotic character. furthermore, our preliminary results support the potential atypical antipsychotic effect of 2-bt as it restored performance in the nor test, a test with good predictive validity. due to the previously shown properties and antipsychotic-like effects of 2-bromoterguride [1], this substance may be a promising candidate for treatment of schizophrenic patients. ongoing experiments investigate the potency of 2-bt to improve social deficits following a sub-chronic pcp regime in rats. background and objectives: cannabinoid-1 receptor signaling increases the rewarding effects of food intake and promotes the growth of adipocytes, whereas cb2 possibly opposes these pro-obesity effects by silencing the activated immune cells that are key drivers of the metabolic syndrome. pro-and anti-orexigenic cannabimimetic signaling may become unbalanced with age because of alterations of the immune and endocannabinoid system. methods: to specifically address the role of cb2 for age-associated obesity we analyzed metabolic, cardiovascular, immune and neuronal functions in 1. 2-1.8 year old cb2 -/and control mice, fed with a standard diet and assessed effects of the cb2 agonist, hu308 during high fat diet in 12-16 week old mice. results: the cb2 -/mice were obese with hypertrophy of visceral fat, immune cell polarization towards pro-inflammatory sub-populations in fat and liver and hypertension, as well as increased mortality despite normal blood glucose. they also developed stronger paw inflammation and a premature loss of transient receptor potential responsiveness in primary sensory neurons, a phenomenon typical for small fiber disease. the cb2 agonist hu308 prevented hfd-evoked hypertension, reduced hfdevoked polarization of adipose tissue macrophages towards the m1-like proinflammatory type and reduced hfd-evoked nociceptive hypersensitivity but had no effect on weight gain. conclusion: cb2 agonists may fortify cb2-mediated anti-obesity signaling without the risk of anti-cb1 mediated depression that caused the failure of rimonabant. leishmaniasis is a neglected tropical disease caused by leishmania, eukaryotic protozoal organisms, which infect humans and other mammals. this disease is transmitted by sandflies of the genus phlebotomus. due to global warming the endemic region of these vectors expands further to northwards and threatens south european countries as well. the treatment of leishmaniasis is difficult due to toxicity and resistance development for current drugs. the so far unexplored inhibition of mitochondrial functions in leishmania by natural products or even food ingredients seems to be an interesting alternative. two food ingredients, resveratrol (res) and xanthohumol (xan), were widely studied in mammalian cells but little is known about their actions on protozoal parasites. therefore, we compared the influence of res and xan on the function of leishmanial and mammalian mitochondria. anti-leishmanial activities of xenobiotics were assessed in cell culture of leishmania tarentolae promastigotes (ltp), leishmania amazonensis amastigotes (laa) and compared to peritoneal macrophages from mouse (pmm) using viability assays. furthermore, mechanistic studies regarding mitochondrial functions were conducted in ltp, mitochondrial fractions isolated from ltp and bovine heart submitochondrial particles using oxygen consumption measurements, assays of individual mitochondrial complex activities, membrane potential and superoxide radical formation by photometry, fluorimetry and electron spin resonance spectroscopy. in ltp, xan inhibited the viability more effective than res (ic 50 : xan 23 µm, res 161 µm). likewise, xan and res demonstrated anti-leishmanial activity in laa (ic 50 : xan 7 µm, res 14 µm) while had less influence on the viability of pmm (ic 50 : xan 68 µm, res > 438 µm). in contrast to res, xan strongly inhibited oxygen consumption in leishmania. further studies demonstrated that this is based on the inhibition of the mitochondrial electron transfer complex ii/iii by xan which was less pronounced with res. however, xan also demonstrated inhibitory activity on mammalian mitochondrial complex iii. in addition, xan caused no decrease of the membrane potential in leishmanial mitochondria, while res resulted in mitochondrial uncoupling. neither xan nor res increased mitochondrial superoxide release in ltp. these data show that res, a major polyphenol from red wine, and xan, an ingredient of hop-containing beer, may have selective anti-leishmanial activity. tryptophan hydroxylase (tph) is the rate-limiting enzyme in serotonin (5-ht) biosynthesis. its two existing isoforms are exclusively expressed in the periphery (tph1), or the raphe nuclei of the brainstem (tph2) and the respective 5-ht populations are distinctly separated by the blood-brain barrier, offering the possibility to pharmacologically modulate central and peripheral functions in an independent manner. peripheral 5-ht is mainly produced by tph1-expressing enterochromaffin cells of the gut and taken up into platelets and transported in the blood stream. upon platelet activation, 5-ht is rapidly released and locally induces multiple effects, such as vasoconstriction, cell proliferation or fibrosis and is furthermore involved in the regulation of e.g. vascular tone, gut motility, primary hemostasis, insulin secretion and t-cell-mediated immune response. following the classical early drug development pathway, we developed a fluorescencebased tph activity assay and performed a high-throughput screening of about 37000 small chemical compounds. we discovered a novel class of tph inhibitors, which was thoroughly validated in a variety of in vitro assay setups. combining medicinal chemistry and x-ray crystallography, we further aimed to develop these inhibitors into preclinical drug candidates. to date we were able to generate and patent a series of novel tph inhibitors with optimized affinity and an in vitro ic 50 in the low nanomolar range. this novel class of tph inhibitors could potentially be used to treat a variety of disorders with aberrant peripheral 5-ht signaling, such as gastrointestinal disorders (e.g. irritable bowel syndrome, crohn's disease, various forms of diarrhea), cardiac valve diseases, pulmonary hypertension, chronic respiratory diseases and some neuroendocrine (carcinoid) tumors. primary hepatocellular carcinoma (hcc) is the most frequent type of liver cancer. therapeutic options are rare. beside sorafenib, a tyrosinkinase inhibitor, which is only used in end stage liver cancer, the surgical intervention is the only successful clinical treatment option. hence there is an urgent need to develop new therapeutic strategies and to identify new drugs for therapy of hcc. hcc often arises in fibrotic or cirrhotic liver, which is accompanied by a change of the extracellular matrix (ecm) composition. in addition it was shown that hepatoma cells express different integrins, which interact with ecm and intracellular cell signaling, compared to hepatocytes. snake venoms have gained increased attention, as it was shown that some of their enzymes and peptides directly act on tumor cells and their multicellular arrangement or indirectly by influencing the stroma environment of the tumor. aim of the present study was to investigate the effect of snake venoms on liver cancer related cell lines as well as their specific action on the ecm-integrin axis. the effects of the snake venoms vipera palestinae (vp), calloselasma rhodostoma (cr) and echis sochureki (es) on a cellular level (mtt, ldh release), on cell-cellconnections (caco2 permeability assay) and on cell-matrix-interactions (adherence test) were investigated. cell-matrix interactions were tested with an adhesion assay using collagen i (c-i), collagen iv (c-iv), fibronectin (fn) and laminin (lm) as ecm compounds. in our in vitro models we used hepg2 as a hcc tumor cell line and the fibroblast cell line fi301 as stroma simulation. additionally caco2 cells were used, a colon carcinoma cell line representing colorectal liver metastasis. the toxicity of snake venom on liver cancer related cell lines was determined in the range of 0.01 -100 µg/ml and plotted into dose response curves. the noaels were calculated from these dose response curves: vp: 0.5 µg/ml -cr: 1 µg/ml -es: 5 µg/ml. performance of the caco2-transwell permeability assay revealed no influence of the tested venom concentrations on the integrity of the cellular arrangement. investigations for integrin inhibition revealed that the venom from vp reduced adherence on lm coated plates and the venom of ec reduced adherence on lm and fn coated plates compared to untreated cells. there was no effect on the adherence on any matrix from the venom of cr observable. co-incubation of the snake venoms of vp and es (below or near noael concentrations) with 5-fluorouracil (5fu), which is used as a chemotherapeutic agent, caused a reduction of its ic50 values. the results indicate that components of vp and ec inhibit the formation of cell-matrixinteractions possibly acting as disintegrins. the co-incubation experiments demonstrated a synergistic effect of 5fu and snake venoms. further experiments should enable the isolation of therapeutic active venom compounds, identification of disintegrins and their role in synergistic mechanisms in liver cancer therapy. modulation of the blood-brain barrier with peptidomimetics to improve drug delivery s. dithmer after decades of research, the blood-brain barrier (bbb) still remains a major problem for successful delivery to the brain for the vast majority of drugs. the main component forming the bbb is the brain microvascular endothelium. the paracellular permeation is limited by tight junctions (tjs), a multiprotein complex composed of the members of the claudin family claudin-1, -3, -5, -12. claudin-5 is known to be the key tj protein tightening the bbb. therefore, claudin-5 has been selected as target to modulate the bbb. for this reason, drug enhancer peptides (peptidomimetics) were designed to modulate transiently claudin-5 and, thereby, permeabilize the bbb. by combining biochemical protein/peptide interaction and tissue culture methods, we identified, validated and optimized peptide sequences modulating claudin-5 containing barriers. the claudin-5 targeting peptides decreased the transcellular electrical resistance and increased the permeability through mdck-ii cell monolayers stably expressing yfpclaudin-5 and immortalized brain endothelial cells (bend.3). the peptides decreased the amount of claudin-5 and zo-1 at cell-cell contacts and changed the cell morphology from spindle-shaped to more round-shaped. all tested peptides showed no signs of toxicity on cell cultures and in vivo (intravenous injection). permeability measurements in mice proved enhanced permeation of na-fluorescein (376 da) through the bbb, which was confirmed by magnet resonance imaging of contrast agents (gd-dtpa, 547 da). in summary, we identified new peptides with the potential to enhance cerebral delivery of small molecules through the bbb. treatment of cerebral diseases is limited by the capability of pharmacologically active agents to penetrate the blood-brain barrier (bbb). this paracellularly tight diffusion barrier is formed by brain capillary endothelial cells. the paraendothelial cleft is sealed by tight junctions (tjs), a multiprotein complex. cerebral tjs predominantly consist of claudin-5 (cldn5) which tightens the bbb for molecules <800 da. consequently, cldn5 is a potential target for transient and size-specific modulation of the bbb to improve cns penetration for pharmaceutically active agents. in high throughput screening using a cldn5 assay, the barrier opener 1 (bo1) was identified as a cldn5 modulator. initially, a significant removal of cldn5 from the plasma membrane was shown by confocal microscopy using epithelial and endothelial cell lines. measurement of transcellular electrical resistance and of paracellular permeability using lucifer yellow (mw 521 da) demonstrated the effect of bo1. concentration dependent treatment (50-150 µm) of cell monolayers with bo1 reduced tightness of the tjs between some hours and 24 h. applying 2-hydroxypropyl-ß-cyclodextrin as a solubilizer, opening activityof bo1 became detectable in mice. due to short stability (< 2 h) of bo1 in the bloodplasma repeated administration (1.5 mg/kg i.v.) was required to induce significantly increased permeability of the bbb for na-fluorescein(mw 376 da). the small molecule bo1 is a promising new approach for transient opening of the bbb in vivo. further modification of the stability and solubility of bo1 is necessary to optimize its applicability. the complex of tight junction (tj) proteins is located between opposing epithelial or endothelial cells. tjs restrict the paracellular permeation of ions and other solutes. tricellulin (tric) tightens tricellular tjs (ttjs) and regulates bicellular tj (btj) proteins like claudins and occludin (occl). current data suggest an important role of ttjs at the blood-brain barrier (bbb). a main pharmacological problem is modulation of the bbb to improve drug delivery to the cns. therefore, tricsi has been developed as a peptide taken from tric to open tissue barriers specifically and transiently.initially, a recombinant protein was generated based on a sequence of an extracellular loop of tric, tagged with maltose binding protein. the fusion protein caused down-regulation of tric, internalization of both tric and occl (confocal laser scanning microscopy), and a significant decrease in transcellular electrical resistance (ter) of a human epithelial colorectal adenocarcinoma cell line. then, studies with the synthetic peptide tricsi indicated its capacity of cell barrier openingafter about 16 h of incubation with concentrations varying from 100 to 150 µmaffecting the membrane localization of tricand occl. barrier opening was proven by decreasing ter, increasing permeability coefficient of lucifer yellow (457 da) and fitc-dextran (10 kda); the localization of tric elongated from ttjs towards btjs and cldn1 was weakened at btjs.physiochemical properties of tricsiexamined by circular dichroism spectroscopy suggested ß-strand structure and no helical propensity. taken together, a tric-derived peptide has been identified increasing the paracellular permeability of tissue barriers and redistributing the cellular localization of tj proteins. tricsi is a novel, promising tool to overcome cerebral barriers with the potential to improve drug delivery to the cns. further experiments are needed to better understand the role of tric in tissue barriers as well as to clarify the mode of action of tricsi. introduction: lung transplantation has become an established treatment option for a variety of end-stage lung diseases, but the long-term survival is often disappointing. the leading cause of death is generally chronic rejection which is characterized by inflammation and fibrous obliteration of the small airways, progressively leading to a reduction of the airflow. the mouse heterotopic tracheal transplantation model is widely used as an experimental model to study the development of obliterative airway disease. despite its widespread application, the heterotopic transplantation model does have a number of limitations, as for example the lack of airflow. the present study provides a description of the orthotopic tracheal transplantation mouse model, which shares more similarities with transplant situation in humans, and provides the analysis of airway obliteration via micro ct and histological evaluation. methods: a seven-ring donor trachea from balb/c mice was implanted into the recipient c57bl/6 mice. c57bl/6 mice without transplantation were used as normal controls. donor c57bl/6 mice to recipient c57bl/6 mice were served as the isograft group. 42 days after transplantation, mice were scanned using an in vivo small animal µct (skyscan 1176). tracheal tissue was harvested and fixed in formalin, embedded in paraffin, cut and stained with hematoxylin and eosin (h&e) as well as sirius-red/fast-green. results and conclusions: histologic evaluation showed luminal narrowing with subepithelial inflammatory cell infiltrates and fibrosis, as well as partially damaged and flattened epithelium. the aerated volume of the allogeneic grafts, analyzed by micro ct was significantly reduced compared to the isogenic control grafts and normal controls. non-invasive imaging via micro ct may offer an option for longitudinal monitoring of the progression of obliterative airway disease as well as response to treatment. c. elegans is a well-established model organism to study the aging process as well as effects of various substances in vivo. its lifespan is regulated by multiple signaling pathways (e.g. insulin or mtor signaling), which are well conserved up to humans. the insulin/igf-1 pathway was the first pathway shown to effect ageing in animals. mutations that decrease the activity of daf-2 (igf1r) lead to a significant increase of lifespan accompanied by a decrease of age pigment accumulation in c. elegans. the relevant effector of the insulin/igf-1 pathway is the transcription factor daf-16 (hfoxo3a). inhibition of hmg-coa reductase (enzyme of mevalonate pathway) by statins, which are frequently used as cholesterol-lowering agents in the clinic, has been shown to attenuate protein prenylation and glycosylation. notably, prenylated-, membrane-bound small gtp-binding proteins are important for the regulation of the afore mentioned age-related signaling pathways like the insulin/igf-1 pathway. recently, a cohort study showed that a decreased mortality rate in humans between age 78 -90 correlates with statin treatment, but is independent of total cholesterol levels. as c. elegans harbors the mevalonate pathway, but the branch leading to cholesterol synthesis is missing, it is a well-suited model to study cholesterol -independent effects of statins on aging-associated phenotypes and the underlying molecular mechanisms. here, we show that exposure of c. elegans to statins substantially decelerated the accumulation of age pigments. while the level of age pigments roughly doubled in control animals, there was only a slight increase in the lovastatin group. the use of atorvastatin gave comparable results indicating a more general effect of the inhibition of the hmg-coa reductase. the retarded accumulation of age pigments could be partly phenocopied using an inhibitor of the small gtpase rac1 or using rnai against the hmg-coa reductase. a reduced level of age pigments is prognostic for an elevated mean lifespan (about 20%) in c. elegans. a post reproductive treatment with lovastatin, mimicking the use of statins in patients of advanced age increased the mean lifespan in c. elegans even further. in addition, we could show a mild reduction of fertility and a developmental delay as well as a marked increase in acute thermal stress resistance mediated by lovastatin. besides the reduced accumulation of age pigments and the increased lifespan these are phenotypes which are usually observed under accumulation of daf-16 overactivity. consequently we found an increased nuclear localization of daf-16 in the presence of lovastatin and lovastatin completely failed to reduce age pigments in a daf-16-ko mutant background. rt-qpcr brought jnk-1, a known activator of daf-16, into play as a possible effector induced by statins. this is currently under investigation. in summary, statin exposure induces a longevity phenotype in c. elegans, which might be daf-16 dependent. this findings indicates that a product of the mevalonate pathway might influence the insulin/igf-1 pathway and particularly the transcription factor daf-16. the high-fat diet (hfd)-fed, streptozotocin (stz)-treated rat model is one of the experimentally-induced animal models of diabetes. this model is often used to evaluate the antidiabetic activity of several agents. according to srinivasan et al. (2005) , prolonged exposure of high-fat diet leads to insulin resistance, and the development of diabetes occurs only in insulin-resistant hfd-fed rats following low dose stz, because the hfd-fed rats are already mildly hyperglycemic due to insulin resistance (1). in hfd/stz model, the rats are fed with high-fat diet for 2-4 weeks or for a relatively long time (≥ 3 months) in order to simulate the insulin resistance and/or glucose intolerance. after induction of diabetes with multiple or single low-dose of stz (30-35 mg/kg), some of the diabetic rats receive treatment (2) . in this way, the impact of treatment can be determined by comparing the differences between groups. despite the lack of methodological information concerning the feeding time in some studies, all rats should be allowed to continue to feed on their respective diets until the end of the study. but what would happen if the hfd was switched to normal pellet diet in these diabetic rats? in our experience, the feeding of npd for 4 weeks significantly decreased fbg in diabetic rats compared to hfd-fed diabetic rats (234.40 ± 42.71 mg/dl vs. 464.00 ± 23.88 mg/dl, p < 0.05). although diet regulation could not restore normal blood glucose, such a decrease was unexpected. in addition, the body weights of the npd-fed diabetic rats were significantly lower than the body weights of the hfd-fed diabetic rats (249 ±6.00 gvs. 288.00 ±4.41 g, p < 0.05). there was no significant difference in body weight between nondiabetic control rats and diabetic rats fed npd for 4 weeks. further details can be found in table 1 . diet regulation and weight loss may prevent, control and reverse diabetes. however, at later stages of the disease, it is difficult to improve blood glucose control without medication, because the disease progresses from insulin resistance to insulin deficiency (3) . according to some diabetes researchers, the amount of residual functional betacells mass is an important issue, and another important question is whether hfd/stz rat mimics an early or late stage of type 2 diabetes (4). these preliminary findings suggest the possibility that hfd/stz rat model may simulate the characteristics of early stage more than the final stage of type 2 diabetes, and hyperglicemia in the experimental model can partially reverse with diet regulation. references: 1. srinivasan, k., viswanad, b., asrat, l., kaul, c. l., ramarao, p. (2005) . combination of high-fat diet-fed and low-dose streptozotocin-treated rat: a model for type 2 diabetes and pharmacological screening. pharmacol res 52 (4): 313-320. 2. oztürk z, gurpinar t, vural k, boyacıoglu s, korkmaz m, var a. (2015) . effects of selenium on endothelial dysfunction and metabolic profile in low dose streptozotocin induced diabetic rats fed a high fat diet. biotech histochem 90 (7): 506-515. 3. franz, m. j. (2007) . the dilemma of weight loss in diabetes. diabetes spectr 20 (3) animal models are pivotal for studies of pathogenesis and treatment of movement disorders. dystonia, characterized by sustained or intermittent muscle contractions causing twisting movements/postures, is regarded as a basal ganglia disorder. the pathophysiology is however poorly understood. in mouse models of dyt1 dystonia, which is caused by a gag deletion in tor1a that encodes for the protein torsin a, ex vivo electrophysiological studies have shown an abnormal d2 receptor mediated release of acetylcholine from striatal interneurons. in these models, which do not exhibit a dystonic phenotype, the functional relevance of the increased d2 receptor mediated acetylcholine release has not been examined yet. the aim of present study was to (1) generate more powerful tests to detect behavioural alterations in the dyt1 knock-in mouse and to (2) examine the behavioral effects of the d2 receptor agonist quinpirole. for this purpose, a sequence of cognitive, motoric and sensorimotor tests were performed in this mouse model. only the adhesive removal test that explores sensorimotor connectivity revealed significant impairments in the dyt1 knock-in mice compared to controls. to induce a more characteristic and stronger phenotype, the "rotating beam test" was developed. this motoric test measures motor coordination and balance. interestingly, dyt1 knock-in mice showed significant motor deficits in the rotating beam test. based on these results, the acute effects of quinpirole (0.25 -1 mg/kg i.p.) were tested in dyt1 knock-in and wildtype mice. subsequent to the injections, mice were tested in the open field, the rotating beam test and the adhesive removal test, respectively. in the open field test, dyt1 knock-in mice showed increased thigmotaxis at a dose of 0.5 mg/kg quinpirole. in the rotating beam test, both groups showed a dose-dependently reduced performance. in the adhesive removal test, quinpirole improved the reaction time in dyt1 mice independently of dosage, while no effects were observed in the wildtype littermates. however, in vehicle follow-up (post-drug control), this effect remained consistent in the dyt1 model, suggesting a habituation effect. in conclusion, we generated a new test, i.e., the rotating beam test which improves the detection of mild motor impairments in dyt1 knock-in mice. furthermore, the adhesive removal test revealed sensorimotor dysfunctions in this animal model. these results represent an important step for our ongoing optogenetic examinations on the role of abnormal neuronal plasticity in dyt1 dystonia and for pharmacological studies. the first data on the effects of quinpirole do not indicate a critical role of d2 dysregulated acetylcholine release, but this has to be clarified by ongoing local striatal injections of quinpirole and by pharmacological manipulations of the cholinergic system. renal fibrosis is characterized by decreased nitric oxide (no) bioavailability and pronounced transforming growth factor β (tgfβ) signalling subsequently excessive extracellular matrix (ecm) deposition. here, the effects of the soluble guanylate cyclase (sgc) stimulator bay 41-8543 after unilateral ureter obstruction (uuo) have been studied. kidney fibrosis was induced by unilateral ureter obstruction (uuo) in wild type (wt) and cgki knock-out (cgki ko) mice. starting one day after uuo, the sgc stimulator bay 41-8543 was (4mg/kg/daily) i. p. injected for seven days. biomarkers indicating remodelling processes in the kidney were analysed via mrna expression and protein expression. bay 41-8543 administration influenced the activity of the ecm degrading matrix metalloproteases (mmp2 and mmp9) and their inhibitor timp-1, the expression pattern of extracellular matrix proteins (e.g. collagen and fibronectin) of profibrotic mediators (e.g. connective tissue growth factors (ctgf) and plasminogen-activator inhibitor-1 (pai-1)) and the secretion of cytokines, e.g. il-6. thereby, bay 41-8543 increments the cgmp pool among others via modulation of endothelial no synthase (enos) expression. agents, which enhance no and cyclic guanosine monophosphate (cgmp) ameliorate the progression of fibrotic tissue. however, the molecular mechanism by which cgmp via cgki affects the development of kidney fibrosis has not fully been elucidated. accordingly, the present study investigates the functional role of sgc stimulation in regulating the fibrotic process, the signalling pathway and the underlying mechanisms involved. we hypothesize that the antifibrotic potential of bay 41-8543 might be related to the increased cgmp pool and the inhibition of the mapk and smad signalling pathway. the elucidation of the signalling allows the development of new therapeutic options. infection of mice with listeria monocytogenes (lm) results in a strong t-cell response that is critical for an efficient defense. here, we demonstrate that the adapter protein sly1 (sh3-domain protein expressed inlymphocytes 1) is essential for the generation of a fully functional t-cell response. the lack of sly1 leads to reduced survival rates of infected mice. the increased susceptibility of sly1 ko mice was caused by reduced proliferation of differentiated t cells. ex vivo analyses of isolated sly1 ko t cells displayed a dysregulation of forkhead box protein o1 (foxo1) shuttling after tcr signaling, which resulted in an increased expression of cell cycle inhibiting genes, and therefore, reduced expansion of the t-cell population. foxo1 shuttles to the cytoplasm after phosphorylation in a protein complex including 14-3-3 proteins. interestingly, we observed a similar regulation for the adapter protein sly1, where tcr stimulation results in sly1 phosphorylation and sly1 export to the cytoplasm. moreover, immunoprecipitation analyses revealed a binding of sly1 to 14-3-3 proteins. altogether, this study describes sly1 as an immunoregulatory protein, which is involved in the generation of adaptive immune responses during lm infection, and provides a model of how sly1 regulates t-cell proliferation (schäll et al., eur j immunol. 2015) . the catalytical isoforms p110γ and p110δ of phosphatidylinositol-4,5-bisphosphate 3kinase γ (pi3kγ) and pi3kδ play an important role in the pathogenesis of asthma. two key elements in allergic asthma are increased eosinophil and ige levels. whereas dual pharmacological inhibition of the catalytical subunits p110γ and p110δ reduces asthmaassociated eosinophilic lung infiltration and ameliorates disease symptoms, it has been shown that dual genetic deficiency in pi3kγ and pi3kδ in p110γ ko δ d910a mice increases serum ige and basal eosinophil counts in mucosal tissues and blood. this suggests that long-term inhibition of p110γ and p110δ might exacerbate asthma. here we analysed p110γ/δ -/mice and determined ige and eosinophil counts in a basal state and the immune response to ovalbumin (ova)-induced allergic asthma. we found that serum concentrations of ige, il-5 and eosinophil numbers in blood, spleen and bone marrow were significantly increased in p110γ/δ -/mice in comparison to single knock-out (ko) and wildtype (wt) mice. nevertheless, p110γ/δ -/mice were protected against ovainduced infiltration of eosinophils, neutrophils, b cells and t cells into the lung tissue and the bronchoalveolar space. moreover, p110γ/δ -/mice, but not single ko mice, showed a reduced bronchial hyperresponsiveness as measured with the isolated and perfused lung. we conclude that although the dual deficiency of p110γ and p110δ causes eosinophilia and ige hyperproduction, p110γ/δ -/mice are not prone to develop ovainduced allergic asthma. an increase of plasma extravasation induced by activation of constitutively expressed endothelial bradykinin type 2 receptors (b2) has been shown to contribute to the development of angioedema occurring as a sometimes life-threatening side effect of angiotensin-converting enzyme inhibitors such as enalapril (new engl j med 2015:372; 418-425) . these drugs inhibit the degradation of bradykinin and increase its vascular steady-state concentration. hence, it is reasonable to assume that bradykinin may destabilise the endothelial barrier, i.e. may increase physiologic extravasation. while the commonly used miles assay provides a useful and relatively easy tool to study the effect of permeabilizing mediators in-vivo, it does not distinguish between intravascular and interstitial evan's blue dye. likewise, extravasation can only be quantified at one particular time point per animal, usually 20-30 min. furthermore, evaluation of physiologic extravasation is not possible. in contrast, non-invasive twophoton laser microscopy may allow separating the intravascular from the interstitial compartment and thereby investigations of changes of the physiologic endothelial barrier induced by drugs or transgenes. therefore, we have evaluated this methodology for its suitability to study endothelial permeability in mice in vivo. to establish this, we used two different fluorescent dyes of different molecular weight. a 200,000 kda dextran equipped with a green fluorescent chromophore which cannot leave vascular lumen was injected intravenously to visualize small dermal blood vessels of the mouse ear located approximately 200 µm below the surface. after stabilization of the green fluorescent signal, a 10,000 kda dextran equipped with a red fluorescent chromophore which easily traverses the endothelial barrier was applied by intravenous injection. the red fluorescence permeates into the interstitium during physiologic extravasation and accumulates in the interstitial space. this process can be followed by measuring the decrease of intravascular red fluorescence over various time periods. using this methodology we have studied whether endothelial-specific overexpression of b2 changes physiologic endothelial permeability. this newly developed transgenic mouse line (b2 tg ) was established using a plasmid consisting of pbluescript ii sk+ -vector, the tie-2-promotor, the human b2 cdna, the sv40 poly-a-signal and a tie-2 intron fragment. we observed that b2tg showed a significantly stronger extravasation than their transgene negative littermates as evidenced by the more rapid extravasation of the 10,000 kda dextran at each time point (fig. 1) .we conclude that two-photon laser microscopy is suitable to study endothelial permeability non-invasively in-vivo and that this methodology allows to study the effects of drugs and transgenes on the endothelial barrier under non-inflammatory conditions. furthermore, our results suggest that endothelial-specific overexpression of b2 increases physiologic extravasation. non-allergic angioedema such as angioedema induced by angiotensin converting enzyme inhibitors (acei) develops as a consequence of increased activation of bradykinin receptor type 2 (b2). using a plasmid consisting of pbluescript ii sk+ -vector, the tie-2-promotor, the human b2 cdna, the sv40 poly-a-signal and a tie-2 intron fragment a transgenic mouse line harbouring an endothelial-specific overexpression of b2 was generated and backcrossed to c57bl/6 for more than 10 times (b2 tg ). lung mrna using primers specific for the human or the mouse b2 cdna revealed a 12.5-fold stronger expression of human b2 in b2 tg (n=6), while the expression of murine b2 mrna was unchanged and similar to transgene negative littermates (b2 n ). we have evaluated the specificity of several antibodies directed against b2 and found that a rabbit monoclonal anti b2 antibody appears to be reliable, i.e. there was just a faint staining in lung tissue of b2 -/mice. however, this antibody primarily stains rodent b2 and has only little cross-reactivity to human b2. hence, we were not able to detect a significant increase of b2 protein in tissues of b2 tg . previous experiments have shown that bradykinin induced concentration-dependent constrictions of aortic rings with a maximal effect at 1 µm of bradykinin. the contraction due to bradykinin was completely inhibited by icatibant or diclofenac indicating that it is mediated by endothelial b2 activation and dependent on cyclooxygenase activity. in striking contrast to their transgene negative littermates b2 n , we found a significant icatibant sensitive aortic dilation in b2 tg following preincubation with diclofenac which indicates functional overexpression of b2 in conductance vessels of b2 tg . to evaluate whether this applies to dermal micro vessels we used the miles assay to quantify dermal extravasation of the albumin-bound dye evans blue following intradermal injection of 30 µl of either vehicle, bradykinin, labradimil and histamine (control). increasing concentrations of bradykinin caused a significant increase of extravasation reaching 4.41±0.11 fold at 18.9 nmol bradykinin in c57bl/6 (n=6 each, p<0.0001 vs. vehicle). a similar increase was found in b2 n (4.45±0.25 fold, n=7, p<0.0001 vs. vehicle), while there was a stronger response in b2 tg (5.50±0.16 fold, n=7, p<0.0001 vs. vehicle) which was significantly different to b2 n (p<0.01) and c57bl/6 (p<0.01). in another set of experiments the specific and ace-resistant b2 agonist labradimil (1.89 nmol) was used instead of bradykinin. labradimil increased extravasation by 3.736±0.121 fold in c57bl/6 (n=6 each, p<0.0001 vs. vehicle), by 4.51±0.11 fold in br2 n (n=7 each, p<0.0001 vs. vehicle) and 4.88±0,21 fold in b2 tg (n=6, p<0.0001 vs. vehicle) which was significantly different to c57bl/6 (p<0.01) but not to b2 n (p>0.05). the effects of bradykinin and labradimil were largely blocked by 10 nmol icatibant (i.v.) in c57bl/6, b2 n and b2 tg mice (p<0.0001) and hence mediated by activation of b2. these data suggest that overexpression of b2 in b2 tg is functionally active in endothelial cells of large conductance and small dermal vessels. therefore, b2 tg represents a new animal model suitable for cardiovascular and non-allergic angioedema research. pharmacokinetic pharmacodynamic modeling of irreversible effects: the rituximab example f. keller 1 1 universitätsklinikum, innere 1, nephrologie, ulm, germany background: for pharmacokinetic-pharmacodynamic modeling usually the sigmoid emax model is used as described by the hill equation. however, treatment regimens exist where the effect is only exerted as long as the drug concentration increases whereas decreasing concentrations produce no longer an effect. examples are the pulse anti-cancer therapy such as originally proposed by the devita protocols. methods: here, the new model for irreversible drug action is derived from the time dependent change of the concentration that must be larger than the time-dependent growth of the number of target cells (tumor or bacteria). the irreversible effect can be assumed if the is no further growth of the target cells occurs. de/dt = + dc/dt -dn/dt dn/dt = 0 a solution for the above differential equation can be obtained by use of the integral exponential function iec based on the euler-mascheroni constant (gamma = 0.5772 …). this model of an irreversible effect was applied.to the example of rituximab where the initial effect on cd19+ and cd20+ b-cells completely persists for 6 month. to obtain a numerical solution, the following parameters are needed to be determined: the target concentration ctarget = 100 mcg/ml and the infusion time t = 2 hours. results: it can be shown that a plausible result for rituximab can be estimated only under the condition that a short distribution half-life is assumed of t1/2 = 2 hours (not shorter than the time t of infusion). with the terminal half-life of 460 hours no plausible solution is obtained. under these conditions two observations are made: there is a negative effect both, initially for low concentrations and after cessation of the infusion when concentrations decrease (in reality this means no effect in both cases). the irreversible effect is proportional to the target concentration. the shorter the half-life comes out relative to the infusion time (t1/2 < t) the stronger is the effect (figure) . occasionally there are specific questions occurring on the ruminant xenobiotic metabolism: 1) are the observed metabolites ruminant specific and formed directly in the rumen? 2) are ruminants able to cleave plant specific metabolites like glycosides to the respective aglycon? in the past new additional in vivo goat metabolism studies with at least one animal were performed. the aim of the project was to elucidate an alternative in-vitro method to replace the existing in vivo method in order to address robustly specific questions on xenobiotic metabolism in ruminants for registration of ppp. fresh sheep rumen fluid was incubated in-vitro >7 days by using rumen simulation technology (rusitec). the conservation of the physiological conditions were proven by measurement of ph (~ph 6.6) and redox potential (~-300 mv). the microflora composition and their viability (bacteria, protozoa and fungi) of the rumen were monitored by microscopy, incubation on agar plates and performing several viability tests (e.g. glycosidase-test, nh3 and short fatty acids). all the tests showed that the rusitec is a successful tool to maintain sheep rumen fluid for at least 7 days in -vitro. the metabolic behavior and performance of the rumen fluid was tested by e.g. incubating 14c-triazol derivative metabolites (tdm) like triazol-alanin (ta); triazol-acetic-acid (taa) and triazol-lactic-acid (tla), which are usually formed in plants after application of triazol-containing fungicides. it was shown that ta was cleaved within 72 h to 1.2.4.-triazol, while taa and tla were stable under these conditions. these data are in a good accordance with available in vivo data in cows. moreover glycosides (12c-polydatin, octyl-14c-β-d-glucopyranosid) were cleaved within 1 hour completely. all these data show, that the rumen fluid maintained its metabolic performance by using rusitec. basf identified the rusitec method, which is usually used in different areas (e.g. investigation of methane production in-vitro) as suitable and adapted this method for the purpose of investigation of ruminant xenobiotic metabolism. it was shown that rusitec is a robust method to analyze rumen xenobiotic metabolism and therefore can clearly substitute in vivo animal studies on ruminant metabolism studies beyond oecd503 and contribute significantly to animal welfare (3r: replacement). results: by using the training set, physicochemical (e.g. lipophilicity) and pharmacokinetic characteristics of mtx (e.g. v max for active tubular secretion) were slightly adjusted. using the gfr formula of morris et al. (1982) and including an empiric correction factor, mrd for the training set was 2.49 whereas bias was 2.80 µmol/l. by applying the developed pbpk model to the test set the respective values were mrd=3.92 and bias=1.43 µmol/l. for the covariates "at least one potentially interacting co-medication" and "trimethoprime/sulfamethoxazole" a significant impact on the prediction quality was found. conclusions: using the developed pbpk-model, a good prediction of the pharmacokinetics of hd mtx in severely ill children was found. by including additional factors influencing the prediction of mtx characteristics (e.g. co-medications) an improved prediction of mtx-sl might be reached. in prospective clinical trials, those more complex models should be evaluated and might be helpful to predict hd mtx pharmacokinetics and reduce unwanted side effects. hypericin is a natural polycyclic quinone found in hypericum perforatum. although hypericin reportedly has numerous pharmacological activities, only a limited number of studies have been performed on the absorption and transport characteristics of this compound, presumably, because hypericin is a highly lipophilic compound which is poorly soluble in physiological medium. recently we have shown that quercitrin and isoquercitrin, but not hyerosid, quercetin or rutin increased the uptake of hypericin in caco-2 cells. the major aim of this study was to get a detailed understanding of the exposure and fate of hypericin in the caco-2 cell system under different experimental conditions. the permeation characteristics of hypericin (5 µm) in absence or presence of hypericum extract 145, 62.5 µg/ml) were studied in the absorptive direction. following application of hypericin to the apical side of the monolayer only negligible amounts of the compound were found in the basolateral compartment. the amount of hypericin in the basolateral compartment increased concentration-dependently in the presence of the extract (from 0 to 7.5 %). the majority of hypericin was found after cell extraction (44% in absence and 76% in presence of the extract). the recovery was in the range of 90 %, and significant amounts of hypericin found after cell extraction. fluorescence microscopy and imaging analysis revealed that hypericin is mainly accumulated in the cell membrane. the precise mechanism through which hypericin might overcome the hydrophobic barrier of cell membranes remains to be elucidated. however, our experiments demonstrated that the permeation characteristics of hypericin significantly improved in presence of the extract. background: the combination of gamithromycin (gam), a novel drug with the big advantage of a once weekly administration, and rifampicin (rif) is used in the treatment of lower airway disease in foals. both are effective in the therapy of infections with rhodococcus equi, a gram-positive coccobacillus bacterium, which is known to survive and reproduce within alveolar macrophages. macrolides are combined with rif to prevent resistance developing with single agent therapy. both drug classes reach high concentrations in the lung, penetrate into phagocytes and kill intracellular pathogens. methods: a controlled, single-and multiple dose study with four-periods was conducted in 10 healthy foals (5 ♂ and 5 ♀, age: 42-63 days, body weight: 100-177 kg) which were treated once with rif alone (10 mg/kg s.i.d., p.o., a) followed by the administration of gam (6 mg/kg once weekly, i.v., b) for 3 weeks. study period 3 ("rif-gam acute", c) includes the administration of gam and rif for 7 days with an administration interruption after the first rif dose for blood sampling. for the last study period ("rif-gam chronic", d) both gam and rif were coadministered for 2 weeks. all periods were completed with blood sampling for pharmacokinetic analysis for 48 ( . rif is also accumulated in the lung, but to a much lower degree than gam (elf/c 24 h : 1.2 ± 0.5; balc/c 24 h : 2.01 ± 1.24). conclusion: pharmacokinetic data of the present study provides surprising results. in previous studies coadministration of clarithromycin and rif show a dramatic decreased plasma exposure of the macrolide, whereas the balc/c 24 h -ratio was unaffected (peters et al. 2011) . in contrast, systemic exposure of gam increase significantly in case of the combined therapy and the balc/c 24 h -ratio was nearly halved. both macrolides have in common, that they are intensively accumulated in the lung (elf << balc). at the moment there is further research required (e.g. in vitro studies) for a better understanding of the very interesting in vivo data. 1 1 institut für klinische pharmakologie, göttingen, germany background and aim: desvenlafaxine is a selective serotonin and norepinephrin reuptake inhibitor, which is approved in the usa (but not in europe) for treatment of major depressive disorder. desvenlafaxine is the major active metabolite of the antidepressant venlafaxine. desvenlafaxin is produced by o-desmethylation via cyp2d6. direct administration of desvenlafaxine should bypass the variability in venlafaxine pharmacokinetics caused by the highly polymorphic cyp2d6. however, desvenlafaxine is less lipophilic than venlafaxine and may require carrier-mediated transport to penetrate cell membranes. based on our in vitro data, desvenlafaxine is a substrate of the hepatic organic cation transporter oct1 and common genetic polymorphisms abolished desvenlafaxine cellular uptake. about 9% of caucasians are compound heterozygous carriers of loss-of-function oct1 polymorphisms. therefore, oct1 polymorphisms may cause substantial inter-individual variability in the hepatic uptake and plasma concentrations of desvenlafaxine. in this study we evaluated the influence of genetically-determined loss of oct1 function on the pharmacokinetics and pharmacodynamics of desvenlafaxine. primary aim was dependence of desvenlafaxine plasma concentrations (represented by auc as primary endpoint) on the number of active oct1 alleles. methods: 50 mg desvenlafaxine (pristiq®) was orally administered to 41 healthy subjects preselected according to their oct1 genotypes. oct1*1 allele was regarded full active, oct1*2 to *6 alleles were regarded loss of function. plasma concentrations of desvenlafaxine and its main metabolite didesmethylvenlafaxine were quantified in plasma sampled up to 60 hours after administration using lc-ms/ms. pupillographic measurements were performed as possible surrogate markers for desvenlafaxine pharmacodynamics. results out of the 41 subjects 14 carried two active, 13 one active, and 14 zero active oct1 alleles. age, height and weight were 26.9 ± 6.4 years (mean ± standard deviation), 1.75 ± 0.11 m and 70.9 ± 12.1 kg with no significant differences among the oct1 genotypes. there were strong variations in the pharmacokinetics of desvenlafaxine and its metabolite didesmethylvenlafaxine. the auc 0-infinity of desvenlafaxine varied between 52.8 and 282.2 min*mg/l and auc 0-infinity of didesmethylvenlafaxine between 3.5 and 30.7 mg*min/l. however, neither desvenlafaxine nor didesmethylvenlafaxine pharmacokinetics significantly differed among the three oct1 genotypes. concerning pharmacodynamics of desvenlafaxine, pupil diameters at maximal constriction after a standardized light exposure were on average 14% greater around the time of t max than before administration. in line with the pharmacokinetic results there were no significant differences in maximal constriction or other pupillographic parameters among the oct1 genotype groups. conclusions: our results suggest that oct1 genotype does not affect the pharmacokinetics of desvenlafaxine and therefore no dose adjustment in respect to oct1 genotype should be considered. other factors like renal transporters or polymorphic glucuronidation may explain the great variability in desvenlafaxine pharmacokinetics. background: cardiovascular disorders and medication are highly prevalent in elderly (1). due to age related changes in the body, the elderly are particularly vulnerable to side effects and adverse drug reactions. some psychotropic drugs are linked with reports of cardiac side effects. additionally, some cardiac drugs may also cause psychiatric symptoms. of these, angiotensin converting enzyme inhibitors, beta blockers, methyldopa and calcium channel antagonists can induce or exacerbate symptoms of depression (2) . the aim of this study was to provide information on the concomitant use of cardiovascular drugs among elderly patients who took psychotropic medication. methods: we conducted a single-center, retrospective study between september 2013 and december 2013 using the medical records of elderly patients (≥65 years of age) admitted to basin sitesi polyclinic, izmir ataturk research hospital, turkey. demographic characteristics of patients, diagnoses, prescription drugs were evaluated, and spss 16.0 statistical software was used for data analysis. number, percent, mean and standard deviation were used as descriptive statistics. results: a total of 541 elderly patients with psychiatric disorders were identified. one in four patients receiving psychotropic medication took at least one cardiovascular agent concomitantly (n=135). median age was 72 (min:65, max:98), 84 patients were female (62.2%). according to medical records of 135 patients, the most commonly used drugs were escitalopram, sertraline, mirtazapine, quetiapine, mianserin and risperidone. the proportion of the concomitantly use of cardiovascular drugs was higher among the patients who took more than one psychotropic drug (69.6% vs. %30.4) compared to patients taking psychotropic monotherapy. a higher percentage of women used diuretics (65.4% vs. 33.3%) and angiotensin receptor blockers (36.9% vs. 23.5%) concomitantly with psychotropic drugs when compared to men. the proportion of men using angiotensin-converting enzyme inhibitors and lipid-modifying agents was higher than women (58.8% vs. 38%, 68.6% vs. 20.2%, respectively). conclusions: the world's population is ageing rapidly. according to world health organization, over 20% of elderly suffer from a psychiatric or neurological disorder. our data showed that use of cardiovascular drugs among elderly patients with psychiatric disorders was extensive. the effects and interactions of these drugs should be discussed and carefully evaluated before starting treatment in the elderly. further studies focusing on drug use in elderly will increase the success in geriatric pharmacotherapy. since the adoption of the ich e14 guideline [1], the thorough qt (tqt) study has become a standard element of clinical drug development. however, with the iq/csrc study [2] the ability to detect qtc-prolongations of about 10 ms in a phase i setting has been demonstrated. as a consequence, regulatory agencies have begun to grant waivers for a tqt study based on negative qt findings obtained from first-in-man studies. a concentration-response model is the key tool that gives sufficient power to an analysis based on data from single or multiple ascending dose studies. this power has been investigated in subsampling studies that simulate situations comparable to those encountered in first-in-man studies [3, 4] . other topics to be addressed are the assurance of sufficient quality of the ecgs obtained, in particular in doses that cause adverse reactions, and a replacement for the active control that is part of a tqt study. if a model based statistical analysis is used for confirmatory inference, it must be specified in advance. this pre-specification includes tests to ascertain that the model assumptions are met and alternative methods to be used in case they are violated. in particular linearity and the absence of a hysteresis, i.e. a delay between the drug concentration and the observed qt effect need to be tested. this is an area of active research. in this contribution, i will share current experience from a statistical perspective, both based on real data and on simulated studies. i will also discuss critical points in the design of first-in-man studies that are intended to be used to obtain a waiver for a tqt study. human platelets express the g-protein-coupled angiotensin receptor-like-1 (apj) receptor. apj is activated by apelin, which is produced as pre-apelin and cleaved into several bioactive peptides such as apelin-12, -13 and -17. apelin and apj are expressed in a variety of tissues such as the heart, the vessel wall, several tumor types, and in platelets. to date, there is no description or a suggested function of the apelin/apj system in platelets to date. here, we investigate apj expression and function in human platelets. apelin and apj expression were determined in platelet-rich plasma from healthy donors by immunofluorescence, western blotting and flow cytometry. in a pilot study apelin and platelet apj expression were analyzed in 23 patients with nstemi, 4 stemi patients and 14 controls. here, platelet aggregation was analyzed by light transmission aggregometry (lta); platelet cd62 and apj expression by flow cytometry and circulating plasma apelin by elisa. in resting human platelets, apj receptor expression was observed predominantly in the outer cell membrane, as determined by immunofluorescence staining and flow cytometry. activation with a selective thrombin receptor-activating peptide (ap1) resulted in decreased apj protein levels determined by western blotting in platelet lysates compared to untreated controls. preincubation of platelets with different apelin isoforms for 30 sec to 5 min reduced platelet aggregation in lta studies by up to 20 % for apelin-17. this effect was inhibited by preincubation of the platelets with the enos inhibitor l-name (300 µm), suggesting the involvement of a no-dependent mechanism. in patients with myocardial infarction the expression of platelet apj was significantly reduced compared to the control group (56,84% ± 9,28 % in mi versus 100 % ± 19,35 %in controls; p = 0.029). this reduction in apj expression on platelets was accompanied by decreased plasma levels of apelin-17 in patients with mi (14.95 ± 0.6 pg/ml versus 16.98 ± 0.6 pg/ml; p = 0.035). interestingly, the decreased apj expression on platelets in mi patients significantly correlated inversely with the troponin t plasma levels (r = -0.46; p = 0.03). this may suggest an association of apj expression with lower plasma levels of troponin t and possibly tissue damage. in conclusion, our study shows for the first time the expression of apj and a possible function in human platelets. apj may act as an endogenous inhibitor of platelet aggregation in response to certain apelin isoforms, predominantly apelin-17. upon platelet activation, apj is internalized and surface expression is reduced by about 50 %. in mi patients, plasma levels of apelin-17 and platelet apj expression were reduced. this correlated inversely with troponin t levels. reduced circulating apelin-17 levels and platelet apj expression may be associated or partly account for platelet hyperactivity in mi patients. anticholinergic drug use, m 1 receptor affinity and dementia risk-a pharmacoepidemiological analysis using claims data f. thome background: dementia is characterized by cumulative cognitive decline and progressive inability for independent living. the lack of suitable therapies for terminating the progression of this disease underlines the importance of the detection of risk factors. anticholinergic drugs have been shown to enhance cognitive decline in the elderly. the classification of anticholinergic drugs according to their anticholinergic burden, however, is inconsistent. since cholinergic transmission is mainly mediated by the m 1 muscarinic acetylcholine receptor in the brain, we classified anticholinergic drugs from anticholinergic risk lists according to their affinity for the m 1 receptor subtype and calculated the risk for the onset of incident dementia. methods: our analyses are based on claims data of the public health insurance fund aok. data include information of inpatient and outpatient diagnoses and treatment from 2004 to 2011. inclusion criteria comprised the initial absence of dementia and age of 75 years or older in 2004. anticholinergic drugs were taken from three anticholinergic risk lists. the pdsp-database and literature search were used to define k i -values for the substances. hazard ratios were calculated using time-dependent cox regression including covariates like age, gender, and several comorbidities. results and conclusion: anticholinergic drug exposure increases the risk for dementia. we found that anticholinergics with small k i -values are at a higher risk than those with greater k i -values. furthermore, conventional risk factors for dementia (e.g. age, depression, stroke) could be confirmed. in conclusion, the intake of anticholinergic drugs increases the risk for incident dementia in the elderly. taking into account the m 1 receptor affinity may be a contribution in determining the anticholinergic load in view of the risk for incident dementia. safety signal detection in a large german statutory health insurance databasefirst results of a feasibility assessment f. andersohn 1,2 , s. schmiedl 3, 4 , k. janhsen 3, 5 , p. thuermann 3, 4 , j. walker background: during the last years, approaches to routinely screen health care databases based on electronic medical records or claims to identify drug safety signals were proposed. to evaluate the performance of such methods, reference sets of index drugs have been compiled consisting of (1) drugs with a known association to a certain adverse event (=positive controls) and (2) drugs without any evidence to cause this adverse event (=negative controls). the best possible signal detection method would identify a safety signal for 100% of the positive control drugs, and for none of the negative controls. ryan et al. 2013 developed drug reference sets for four adverse events of interest (acute myocardial infarction=ami, acute kidney injury =aki, acute liver injury=ali, and upper gastrointestinal bleeding=ugib) and have shown feasibility of using these reference sets in us health care databases. if the use of these us specific drug lists for evaluation of signal detection methods is also feasible within german health care databases, is unknown. aims: to evaluate if the drug reference sets developed by ryan et al. (drug saf. 2013 ;36 suppl 1:s33-47) could be used for testing signal detection methods in a large german statutory health insurance database. methods: data source was the health risk institute (hri) database, an anonymized healthcare database with longitudinal health insurance data from approximately six million germans. new users (initiators) of index drugs in 2010 to 2013 were identified and followed-up for one year from their first prescription. exposed person-time to the respective index drug was assessed to estimate for which of these drugs an increase in risk of 50% (relative risk 1.5) compared to the background incidence of the respective adverse event (ami, aki, ali or ugib) could be identified with 80% statistical power. results: from a total of 182 index drugs in the reference sets of ryan et al., 142 (78.0 %) were also available on the german drug market and were used by at least one insurant in the hri database during the study period. a total of 5,485,722 index drug initiators were included in the analysis. for a total of 16 index drugs, a relative risk of 1.5 could be detected with 80% power. the numbers of index drugs for each of the outcomes of interest were: ami (3 positive controls; 6 negative controls); aki (3 positive controls; 1 negative control); ugib (2 positive controls; 1 negative control). as the background incidence of ali was low, no positive or negative control with sufficient power was identified for this outcome. conclusions: using the set of reference drugs proposed by ryan et al., the number of drug-event pairs with 80% power to detect a relative risk of 1.5 was low, despite the magnitude of the database used. this may be attributable to differences of drug exposure in germany and the us. hence, an adaptation of the drug list to the german drug market and consumption data might be relevant for future evaluations of signal detection methods using german databases. correlation of sativex™ doses to steady state concentrations of 11-nor-9-carboxyadministration of the oromucosal spray sativex™ represents a therapy option for treatment of spasticity in patients with multiple sclerosis. sativex™ is an extract containing equal amounts of the cannabis-derived cannabinoids ∆ 9tetrahydrocannabinol (thc) and cannabidiol (cbd). in cases of cannabis abuse a long elimination half life of some thc metabolites is known. therefore, in patients receiving sativex™, the long elimination half life of these metabolites should allow a drug monitoring under conditions of steady state. due to the fact that immunologically based methods for thc determination are very common in medical chemistry, a monitoring might be simply performed even in patients under sativex™ therapy. in a preliminary observational study 11-nor-9-carboxy-∆ 9 -thc (thc-cooh) concentrations were measured with a commercial immunoassay in urine samples of 16 patients with multiple sclerosis obtaining sativex™. in addition, thc-cooh, thc, cbd as well as the hydroxy metabolites of thc and cbd were measured by gc/ms in urine and blood samples. using this analytical technique, only an excessive dosing (as compared to the declaration by the patient) can be detected. as a result of this approach, thc-cooh concentrations determined by the immunoassay were found not to correlate to the daily applicated amount of sativex™ as indicated by the patients (spearman rang order test: p > 0.05). two patients mentioned not to have taken sativex™ on the day the samples had been taken, and one patient predicated an additional cannabis abuse. in three patients the immunological thc-cooh determination was negative or nearly negative. interpretation of the data is hampered by the fact that an incorrect declaration of sativex™ applications by the patients cannot be excluded. introduction: learning analytics seeks to enhance the learning process through systematic measurement and analysis of learning related data to provide informative feedback for students and lecturers. however, which parameters have the best predictive power for academic performance remains to be elucidated. objective: to analyze the potential of different learning analytics parameters to predict exam performance in undergraduate medical education of pharmacology. methods and results: hypertext preprocessor (php) as server-side scripting language was used to develop a learning analytics platform linked to a my structured query language (mysql) database for storage and analysis of data (www.tumanalytics.de). the database consisted of 440 lecturer-authored multiple choice questions that were made available to a cohort of undergraduate medical students enrolled in a pharmacology course (winter term 2014/15) at technische universität münchen (tum). the course consisted of a 28-day teaching period, followed by a 12-day self-study period and a final written exam. students' assessment data of tumanalytics was collected during the self-study period and correlated to the individual exam results in a pseudonymized manner. a total of 224 out of 393 (57%) students participated in the study. the coefficient of multiple correlation (r) was calculated for different parameters in relation to exam results as a measure of predictive power. of different parameters investigated, the total score and the score of the first attempt in tumanalytics had the highest positive correlation with exam performance (abb. 1). no sex-specific differences were observed. summary and conclusion: in this study we systematically investigated the potential of different learning analytics parameters to predict learning outcome and exam performance. total score and score of the first attempt were identified as parameters with the highest predictive power. in conclusion, our study underscores the potential of learning analytics as valuable feedback source in undergraduate medical education of pharmacology. in educational settings, tests (e.g., written or oral exams) are usually considered devices of assessment. however, a recent and intriguing line of evidence from basic cognitive psychological research suggests that tests may not only help to assess what students know, but may also help to improve the learning and long-term retention of information. the goal of the present study was to apply such test-enhanced learning to pharmacological teaching. after the last lecture of a pharmacology class (n=194 3 rd -year medical students, n=213 4 th -year medical students: basic or clinical pharmacology, respectively), one week prior to the final exam, students were given the opportunity to voluntarily participate in online exam. because pilot work from previous semesters had revealed relatively low levels of participation in such formative exams, students were offered bonus points for (successful) participation as an incentive. the online exam consisted of 60 items (i.e., selected pieces of information from the lectures and seminars) and was provided on the e-learning platform ilias. twenty of the 60 items were presented as statements for restudy, 20 items were tested using single-choice questions, and 20 items were tested using short-answer questions. randomly a third of the students were assigned to different sets of questions. the summative final written exam for each group consisted of 30 single-choice questions, 15 questions of which had not been used before (as a standard of comparison). the remaining 15 questions of the final exam were taken from the previous online exam, but were slightly reworded to avoid ceiling effects. each of 5 of these reworded 15 questions from the final exam corresponded to restudy items, single-choice items, and short-answer items from the online exam, respectively. the main points of interest were (i) whether the re-processing (rewording and asking for transfer of knowledge) of information in the online exam affected participants' performance on the final exam, and (ii) whether any effect depended on the specific type of re-processing (restudy vs. single-choice test vs. shortanswer test). if previous findings from basic cognitive psychological research on testenhanced learning can be generalized to more applied settings and educationally more relevant materials such as pharmacological information, students' performance in the final exam should be better for questions corresponding to previously tested items than for questions corresponding to previously restudied items. moreover, if more difficult tests lead to more test-enhanced learning than less difficult tests, as is suggested by recent findings from cognitive psychological research, performance for questions corresponding to (supposedly more difficult) short-answer items should even exceed that for questions corresponding to (supposedly less difficult) single-choice items. the present findings bear direct implications for educational practice. safe and rational prescribing is one of future physicians' key skills [1] . in order to address the persisting prescribing deficiencies [2] , we set out to develop a learning tool for pharmacotherapies of the most important diseases worldwide. the format, scope, information architecture, and functionalities of the app were identified through assessment of existing apps, literature analysis, app simulation-based student surveys, and expert advice. a fully functional offline app format for smartphones was selected based on the trends in using digital technologies for educational purposes [3] and on the unreliable internet and power availability in many learning settings. a relational database based on semantic relationships was chosen to minimize information redundancy and to enable the retrieval of drug-related information in the context of mechanisms, contra-and indications, adverse drug reactions, interactions, and common prescribing situations. the usability was optimized using a simulation of the app evaluated by medical students from germany and tanzania, and by experts. a list of 67 indications was assembled beginning with disease burden data for the seven who world bank regions. each disease accounts for at least 0.3% of life years lost due to premature death or lived with ill-health or disability (daly) in at least one region. the list was further complemented according to expert recommendations. therapeutic recommendations are based on current guidelines, considering cheaper treatment alternatives provided in the who list of essential medicines. a novel dual-scale classification system lists drug mechanisms according to the affected physiological process and to the resulting therapeutic effect [4] . contraindications, adverse drug reactions, and interactions were compiled using drug monographs of the european medical agency, the us food and drug administration, and health canada. unexpectedly, we found significant differences among these sources in respect of adverse drug reactions. this necessitated the ongoing verification through surveying general practitioners and specialists in internal medicine. during the dgpt meeting we will present the results of testing of the cardiovascular section comprising 8 indications, 28 drugs representing 25 mechanisms, and up to 120 adverse drug reactions. the european certified pharmacologists (eucp) programme was lauched in july 2014 by the federation of european pharmacological societies with the intention to identify experts in the field of pharmacology whose competency profile, in addition to their personal specialised scientific expertise, covers expert knowledge in all major fields of the discipline. seventeen ephar member societies have declared their active participation in the eucp programme so far (austria, croatia, czech republic, finland, france, germany, greece, hungary, italy, the netherlands, norway, poland, portugal, serbia, slovenia, spain, turkey) . eacpt, the european association of clinical pharmacology and therapeutics, has also recently decided to participate in the eucp programme. national programmes must meet all requirements of the eucp guidelines including a clear catalogue of criteria with respect to knowledge, practical awareness and skills, as well as general rules including rules for final assessment of candidates. such programmes may be based on existing diplomas or training schemes or may consist of a set of rules how applicants may submit credentials for their expertise with respect to the eucp criteria. so far, three ephar member societies have submitted a national eucp program: austria, italy and the netherlands. the programmes differ in structure and reflect the flexibility of the eucp programme with respect to the respective national conditions. while the italian programme is based on a catalogue of criteria where applicants have to certify and document their expertise on the basis of this catalogue and the dutch program is based on a structured phd training course, the eucp scheme submitted by the austrian pharmacological society aphar is based on the legally regulated diploma medical specialist (facharzt) in pharmacology and toxicology (aphar also plans to submit separate regulations for specialists in clinical pharmacology and for nonmedically qualified pharmacologists). the aphar eucp scheme has been approved by the eucp committee in november 2015 and its regulations are available on the eucp website (www.eucp-certification.org). the differently structured programs of the italian and dutch pharmacological societies will also be available at this website, once approved by the eucp committee and may thus serve as 'case studies' for other ephar and eacpt member societies wishing to take part in the eucp programme. introduction: the outstanding importance of pharmacovigilance (pv) for the safe use of medicines has increasingly been recognised during recent years. the multidisciplinary character of pv requires know-how in topics as different as pharmacology, clinical medicine, pharmacoepidemiology, information technology, pharmaceutical manufacturing, legal aspects, public health policies, and medical traditions in different regions of the world. in this complex situation there is a growing need for pv capacity building, in particular by professional training through high quality pv courses with different focuses and different levels of detailing. against this background, the world health organization (who) and the international society of pharmacovigilance (isop) have co-operated to create a curriculum for teaching pv which can be used for a wide range of audiences and in very different settings and situations. the purpose was to provide an inventory and systematically structured overview of pv including recent developments of topics like pharmacogenomics, consumer reporting of adrs, risk management and who-led international projects, and to propose a range of tasks for practical training. we made use of several relevant already existing packages of pv topics and concepts of pv teaching from national and international institutions. we also drew from extensive printed material as well as comprehensive reviews, textbooks and guidelines developed by international organisations which are often available online. results: the curriculum includes a main component consisting of a major part for theoretical lecture-based training and a minor component with suggestions for hands-on exercises. the theoretical part has a three-level hierarchical and modular structure with evenly divided tiers. there are 15 chapters. each of them is divided into four sections and each section into four to six sub-sections. the practical part consists of twelve times three or four proposals for practical tasks which are related to the theoretical lectures. since its launch in 2014 1 it has successfully been used in several international courses. currently a pilot project is under way to explore its use for 'crowd sourcing': it is placed on the isop homepage with a programme allowing for institutions or persons experienced in pv teaching to upload any relevant presentations they may have with a link to related chapters, sections or subsections. these presentations will be offered for downloading by interested users. the curriculum provides a comprehensive coverage of almost all areas of pv. the structure and content allows almost every kind of focusing on specific issues and going into depth, while maintaining the overall context. it offers opportunities of tailoring courses specifically to the needs of different audiences and can be applied to various forms of training, such as broad, comprehensive and intensive courses, short overviews or focuses on specific narrow topics in perspective. according to the reach regulation (ec) no. 1907/2006 chemicals produced, marketed or used within the european union have to undergo a registration process, wherein the registrants have to provide information on hazard and potential risks presented by the substances. however, the standard information requirements defined in annexes vii to x of the regulation might be waived or adapted by the registrants if adequate documentation and justification according to criteria specified in annexes vii to xi are provided. to evaluate the data availability in registration dossiers of high tonnage substances (above 1000 tpa) and their compliance with the reach regulation, the federal institute for risk assessment (bfr) in cooperation with the federal environment agency (uba) developed a systematic web-based scheme. in total, 1932 dossiers were checked for selected human health and environmental endpoints such as repeated dose toxicity, genetic toxicity and ecotoxicity. a remarkable high rate, 43% to 82 % depending on the endpoint, of the evaluated dossiers included waiving or adaptations from the standard information requirements. therefore, those dossiers were not concluded, but categorised as 'complex' (springer et al., 2015) . the use of waiving and adaptations in 'complex' endpoints were part of a follow-up project. herein, it was evaluated whether the given justifications were in accordance with the criteria set out in the respective reach annexes. the results will show the frequency and pattern of waiving/adaptation approaches for the human health as well as the environmental endpoints. besides this general overview, specific problems regarding the application of the reach regulation were identified and their significance with regard to remaining data gaps will be discussed. the german commission for the investigation of health hazards of chemical compounds in the work area has re-evaluated dimethylformamide, and classified it in the carcinogen category 4. this category is for chemicals with carcinogenic potential for which a non-genotoxic mode of action is of prime importance and genotoxic effects play no or at most a minor part provided the mak and bat values are observed. under these conditions no contribution to human cancer risk is expected. dmf was identified as a substance of very high concern by european commission. the amount of dmf manufactured and/or imported into the eu is, in the range of 10000-100 000 t/y. n,n-dimethylformamide is a hepatotoxin in humans and rats. the carcinogenicity studies in both mouse and rat were conducted with test material of an acceptable purity and physical form. the critical study involved administration of dmf via inhalation, which is relevant to human exposure. there is conclusive evidence that dmf induces significant increases of hepatocellular carcinomas in rats after exposure to 800 ml/m³ and in mice in response to 200 ml/m³ and higher. several in vitro and in vivo studies have indicated that dmf is not genotoxic. the results of the long-term studies reveal that the tumors develop in the liver only after chronic toxic inflammatory and degenerative changes have developed in this organ. the commission concluded that the tumors are a result of chronic liver damage, occurring at high exposure concentrations. the available evidence therefore suggests that there is a threshold dose for the carcinogenic effects of dmf. accordingly, dmf was classified in carcinogen category 4 with a mak-value of 5 ml/m³, an exposure concentration which does not induces liver toxicity and as a consequence is not associated with an increased cancer risk. today, a large majority of people is constantly exposed to electromagnetic radiation. many studies have been performed to investigate whether this type of radiation has a potential to affect biological systems at low intensity levels. even though no complete consensus has been reached so far in this issue, most of the investigations do not indicate a harmful potential of this radiation. two questions remain open until today, i. e. long-term effects and specific effects on children. it has been demonstrated that in comparison to adults, children absorb far higher doses of mobile phone radiation in the skull, particularly in the bone marrow, where hematopoiesis takes place. these absorptions occasionally exceed the recommended safety limits. the aim of this study was to elucidate, whether cells of the hematopoietic system can be affected by different forms of mobile phone radiation. as biological systems, two cell types were investigated, hl-60 cells as an established cell line, and human hematopoietic stem cells. the radiation was modulated according to the two major technologies, gsm (900 mhz) and umts (1950 mhz). additionally, lte (2.535 mhz) modulation was applied because this technology is used worldwide already but has not been studied sufficiently. cells were exposed for a short and a long period and with different intensities ranging from 0 to 4 w/kg. studied endpoints included oxidative stress, differentiation, dna repair, cell cycle, dna damage, histone acetylation, and apoptosis. appropriate negative and positive controls were included and three independent replicate experiments were performed. exposure to radiofrequency radiation did not induce any alterations of cell functions, measured as oxidative stress and cell cycle. cell death in the form of apoptosis was not observed. primary dna damage was not induced and dna repair capacity for nucleotide excision repair was not changed. epigenetic effects (measured as histone acetylation) were not observed. finally, differentiation was not affected. the effect of treatment with various chemicals as positive controls was different in the two cell types. all in all, mobile phone radiation did not induce effects on human hematopoietic cells. in 2014 who published guidance on evaluating and expressing uncertainties in human health hazard characterisation (hc). in this approach, the outcome of hc is expressed as an interval or distribution rather than a "traditional" deterministic point estimate, such as a reference dose (rfd), thereby communicating potential uncertainties more clearly. risk management protection goals, such as the acceptable magnitude of effect (m) and incidence (i) in the population, are made explicit quantitatively along with the confidence with which they are achieved, e.g. by an rfd. specifically, the goal of this approach to hc is to estimate the "hd m i" , i.e. the "true" human dose associated with m and i (e.g. body weight decreased by ≥ 10% (m) in 5% (i) of the population). if uncertainties are expressed by providing estimates of the hd m i as confidence intervals or uncertainty distributions, both the "degree of uncertainty" (ratio of upper and lower limit of the interval/relevant distribution segment) and the "coverage" (statistical confidence) associated with a given rfd value can be characterised. alternatively, one may start from a chosen coverage and calculate the associated "probabilistic rfd". uncertainty in each hc aspect, e.g. inter-/intraspecies or time extrapolation, can be characterised by a "generic default uncertainty distribution" which has been derived from historical data, but may be replaced by a substance-or effect-specific distribution (analogous to a chemical-specific adjustment factor in the "traditional" deterministic approach), where available. the uncertainty distributions for the individual hc aspects can then be combined into an overall uncertainty interval/distribution in (1) a simple non-probabilistic way, (2) a more refined "approximate probabilistic analysis" (aproba, a free spreadsheet tool for easy implementation also by non-statisticians is available from the who website), or (3) a fully probabilistic monte carlo analysis. the hc aspects contributing most to overall uncertainty are also identified and may be prioritised for refinement in a next assessment tier. the who approach uses a tiered strategy which may start with evaluating the uncertainties in the outcome of a "traditional" deterministic hc. in this way it represents a unified framework integrating deterministic and probabilistic hc methodologies. moreover, the concept can be easily combined with exposure uncertainty assessment. risk managers may use the additional information in better weighing potential health effects against other interests. when they consider the overall uncertainty larger than desirable in view of the problem formulation, they may decide to ask for a more refined (higher tier) assessment. if all possibilities for refinement are exhausted, the new approach can also aid in the selection of new data which might need to be generated. due to a constant improvement in analytical methods an increasing number of substances are found in drinking water. the joint project toxbox aimed for the development of a reliable test battery, allowing for a rapid evaluation of single substances in water. eleven partners either from the research (nine) or the business sector (2) formed the project. the attention was focused on genotoxic, neurotoxic and endocrine effects, which are considered to be of most concern to the consumer. by the end of the project a set of guidelines is published that describes the analytical methods in detail. the project was based on a theoretical concept, called "health-related indicator value" (hriv), which was developed by the german federal environment agency (uba) for the assessment of substances with incomplete toxicological data. depending on the type of effect a hriv between 0.1 and 3.0 µg/laws derived for the substance which had to be evaluated. during the years an increasing amount of substances as well as an increase in finds was observed in drinking water. this called for the creation of an evaluation scheme that offers rapid and at the same time reliable evaluations of chemicals for which there are no data available. the concept is in accordance with tox21, which envisages the trustworthy evaluation of relevant endpoints by two or three in vitro assays. in the context of toxbox this was provided for the endpoints genotoxicity, neurotoxicity and endocrine effects. in all cases a hierarchic strategy is applied that enables a first assessment via relatively simple test assays and only when these test give a hint towards an effective more elaborate techniques are applied for a final assessment. the ames test and micronucleus assay in combination with the umu test will form the panel for genotoxicity testing. neurotoxicity will be assessed by comparing necrotic and apoptotic effects as well as the development of reactive oxygen species in human nervous cell with human liver cells. additionally neuron specific assay like the neurite outgrowth test are performed. this is complemented by measuring the activity of acetyl choline esterase activity and the development of the side line organ in zebra fish (danio rerio). the test battery for endocrine effects consists of hormone specific reporter gene s81 assays in addition to the h295r assay. when necessary a reproduction assay in the mud snail potamopyrgus antipodarum is carried out. during the project some 60 substances were evaluated. this allowed for the development of a reliable test strategy. currently the guidelines for performing the required tests are in the making. metabolomics has gained increasing interest over the last years with numerous possible applications ranging from strain optimization for industrial production over drug discovery to improved toxicity testing. however, regulatory acceptance of this promising approach is still not reached, mostly because standardization and evaluation of reproducibility are still mostly lacking. the metamap®tox database has been developed by basf over the last ten years containing toxicity and metabolome profiles of more than 750 different compounds. to ensure maximum reliability, data was gained from plasma samples of highly standardized 4 week rat studies. animal maintenance and treatment, sampling and work-up of plasma, measurement of the metabolome as well as data interpretation and storage were standardized including thorough documentation, the compliance with sops and safe data storage. data from more than 80 control groups with each 10 males and females were analyzed to assess variability. an in depth analysis of this showed a high stability and robustness of the metabolome over a period of ten years. after artificially splitting the groups of 10 control groups into groups of five animals and comparing the number of statistically significantly regulated (false positive) metabolites, the peak of the distribution curve was located to the left of the exact (gaussian) center, but tailed off to the right more than expected under the normal distribution. from this analysis we were able to calculate density distributions (relative ratio and standard deviation) for the control values of each metabolite, which can serve as a historical control displaying the range of changes which can be expected as normal. during the course of our project we have used more than ten exact repeats to show reproducibility and reliability of the metabolome analysis (kamp et al., 2012) . comparing these exact repeats at different levels of statistical significance, we noted that at a level of statistical significance of approximately p = 0.1, the best balance between matches (metabolites regulated in the same direction) and mismatches (metabolites regulated in opposite directions) was obtained. the high quality standards applied as well as the examination of control data increase the robustness of this approach, going also hand in hand with improved data quality. this significantly facilitates decision making based on the gained data. due to these improvements a new level of transparency is reached, which might allow inclusion of metabolome data in a regulatory environment. hydroxycitric acid (hca) is a fruit acid naturally occurring in fruits of the tropical plant garcinia cambogia. a number of dietary supplements intended for weight loss contain hca, which is added in form of g. cambogia extracts. the composition of these extracts is often not clearly specified. health concerns about safety of the hca-containing supplements have been raised, based on results from animal studies, which observed toxic effects on the testis and on spermatogenesis after administration of preparations containing high hca doses. in the current risk assessment, the possible health risks associated with consumption of hca-containing dietary supplements (hca doses of approximately 1000 to 3000 mg per day) were evaluated based on relevant animal and human studies with the focus on testicular toxicity as a critical endpoint. in several published animal studies, repeated (short-term or subchronic) ingestion of certain hca-preparations (g. cambogia-extract or ca 2+ -hca salt) induced testicular atrophy (i. e. atrophy of seminiferous tubules, degenerative changes of sertoli cells at histological examination) and impaired spermatogenesis (i. e. decreased sperm counts) in male rats at high doses (noael and loael of 389 and 778 mg hca/kg body weight & day, respectively). animal studies with other hca-preparations (ca 2+/ k + -hca salt) found no such effects at the highest hca-doses tested (noael: 610,8 mg hca/kg bwt & day). human intervention studies which addressed the safety of hca in healthy test persons reported no substancespecific adverse effects after ingestion of hca doses up to 3000 mg per day over the period of up to 12 weeks. however, the question of possible adverse effects of hca on the human testes was not adequately addressed in studies with human volunteers. in a single clinical study with 24 male test subjects, no significant changes in endocrinologically relevant parameters such as serum inhibin b or fsh were observed after consumption of 3000 mg of hca for 12 weeks. however, no investigations of direct parameters that might inform on potential effects on spermatogenesis, such as sperm quality and sperm count, were conducted in this study. considering the serious adverse effects on the testes observed in several animal studies as well as in view of lack of the adequate human data on the safety of the long-term use of hca-preparations, it is concluded that knowledge gaps and substantial uncertainties exist regarding the safety with respect to human health of high amounts of hca found in commercially available food supplements, particularly with regard to the human male reproductive system. a critical look on the passing-bablok-regression b. mayer 1 1 universität ulm, institut für epidemiologie und medizinische biometrie, ulm, germany background: the passing-bablok (pb)-regression is a commonly used approach to prove the equality of different analytical methods when studying quantitative laboratory data. it is based on the assumption that the measurements of two methods are linearly related. if then one method is regressed onto the other and the respective confidence intervals of the intercept and the slope include 0 and 1, respectively, it is assumed to have a proof of methods equality. however, this conclusion is problematic in respect of an essential principle of statistical hypothesis testing. methods: in this talk the general idea behind the pb-regression is discussed critically. although the method makes use of confidence intervals a decision is made, which is why it is important to discuss how the results of a statistical hypothesis test have to be interpreted. moreover, alternative statistical approaches to investigate agreement in biometrical practice are pointed out by means of a practical example and their advantages and limitations are addressed. results: all approaches applied to a sample data set led to the same conclusions. demonstrating methods equality though necessitates an a-priori definition of an appropriate equivalence margin. conclusion: the pb-regression may give useful advice when comparing two measurement methods towards equality. however, its results are statistically inconclusive, since the pb-method does not follow the principle of equivalence testing. alternative measures of agreement should be applied instead to ensure results which are not attackable and serve as a statistical proof. insulin is an important parameter both in toxicology (toxicity to the endocrine pancreas) and pharmacology (models of diabetes and metabolic syndrome). currently available elisa and ria methodologies for insulin often require up to 100 µl plasma or serum for a single measurement. in order to meet the general trend to include more relevant parameters in animal studies and restrictions through animal welfare requirements to limit the volume of interim blood draws we explored the rat/mouse insulin singleplex assay of meso scale discovery (msd) as an alternate assay consuming only 10 µl serum or plasma or less for a single measurement. the assay is a sandwich immunoassay, whereby insulin in the sample binds to the capture antibody immobilized on the working electrode surface at the bottom of each well and recruitment of the labeled detection antibody (anti-insulin labeled with electrochemiluminescent compound, msd sulfo-tag™ label) by bound analyte completes the sandwich. voltage applied to the plate electrodes then causes the label bound to the electrode surface to emit light the intensity of which is a quantitative measure of insulin. the msd insulin assay was characterized by a robust calibration and only small variations within repeated measurements. the assay presented a broad dynamic range and differences in insulin levels of normal and rats suffering from metabolic syndrome could readily be demonstrated. furthermore, the high sensitivity may even allow the use of smaller sample volumes. these features render this assay an attractive alternative for the measurement of insulin. the lack of corresponding quality control samples for internal quality control may be considered as a relative drawback. however, the cross reactivity of the assay with human insulin provides the opportunity to use qcs designed for human assays and to possibly participate in ring trials for human insulin for external quality control if needed. surfactants are main constituents of different consumer products, e.g. detergents or cosmetic cleansing products. since surfactants show an intrinsic skin irritation potential, dilutions are used in the final products to avoid adverse effects like irritant contact dermatitis from product use. in addition, mixtures of different surfactants are typically formulated, as it is a long-standing experience that those mixtures exhibit much lower acute irritation potential than expected from the mere summation of their individual irritation potential, an effect coined surfactant antagonism. only few studies were performed to gain a more fundamental understanding of the effect, and it's mechanistic basis remains unclear. however, a thorough understanding of the surfactant antagonism is not only of value for the formulation of products that are considered 'mild to the skin'. it is also important for the classification of products according to the clp regulation in cases when data of the mixtures is missing, because summation of the ingredients' irritating effects usually results in over-classification as skin irritant. due to the progress in the development of alternatives to animal testing, different in vitro methods have become available to determine skin irritating properties of substances. methods like the oecd tg 431 and 439 especially aim at deriving a classification for skin irritation/corrosion effects according to the clp regulation. however, even though these methods became the preferred test methods for skin irritation testing, to our knowledge hitherto isolated investigations on the surfactant antagonism were only performed either by human patch test studies or by non-standard in vitro assays. in this study, the irritation potential of binary mixtures of sodium dodecylsulfate (sds), linear alkylbezene sulfate (las), cocamidopropyl betaine (cabp) and alkylpolyglucosid (apg) compared to the single compounds was investigated using open source reconstructed epidermis (os-rep) models. combinations of sds or las with cabp and apg, respectively, resulted in a clear decrease of the irritation potential compared to the irritation exerted by the single surfactants, even though the total surfactant concentration was higher in the mixtures. in addition, the effect of surfactant antagonism was also observed in a mixture of cabp and apg. the reduced irritation potential of mixed surfactants came along with both reduced skin penetration of fluorescein and reduced release of ldh. since no surfactant antagonism is observed in monolayer cultures of keratinocytes that were exposed to mixtures of surfactants, it is assumed that keratinocytes in the viable parts of the reconstructed epidermis are promptly damaged by the surfactants once the model's barrier is destroyed. hence, surfactant antagonism appears to be primarily driven by the mixture's lower ability to damage the skin model's barrier. the micronucleus (mn) test is a reliable method for the detection of cytogenetic damage in proliferating cells. in recent years, substantial progress has been made on automated, thus faster and more objective scoring of mn test samples, i.e., methods based on flow cytometry. the aim of the present study was to use the adherently growing human bladder cancer cell line rt4 to carry out a comparison between traditional (fluorescence microscopy) and automated (flow cytometry) mn scoring. for this purpose, different substances which are known to be positive controls were used. rt4 cells were either continuously incubated for 72 h (approximately 1.5 cell cycles duration) with methyl methanesulfonate (mms; 50-200 µm), benzo[a]pyrene (b[a]p; 0.1-1 µm), vincristine (3-20 nm) , and colcemid (10-20 nm) or cells were irradiated with xrays (0.5-2 sv) and then cultured for 72 h. for standard mn scoring, cells were harvested, subjected to hypotonic treatment, fixed with methanol/acetic acid, placed on glass slides, stained with acridine orange and observed by fluorescence microscopy. for the flow cytometric method, harvested cells were stained in two sequential steps. intact cells were subjected to ethidium monazide bromide followed by photoactivation (75 w, 20 min) to label dead or dying cells. then, cells were lysed and stained with sytox green for a pan-dna labelling and analyzed on a flow cytometer. both, chemically-and radiation-induced treatment led to a dose-dependent induction of mn when evaluated by fluorescence microscopy. when the flow cytometry-based method was applied, clearly positive results including a dose-dependent induction of mn, however, were obtained only for 3 out of the 5 treatments (vincristine, colcemid and x-rays); whereas, treatment with mms and b[a]p led to only minor increases in relative mn frequencies (≤2-fold), even at the maximum concentrations. in summary, flow cytometry-based mn scoring has been successfully applied in rt4 cells. however, our initial results suggest that flow cytometry-based mn scoring is less sensitive than microscopic scoring when rt4 cells are used. so far, only few adherently growing cell lines have been applied to flow cytometry-based mn scoring. further substances (positive and negative controls) and possibly other adherent cell lines need to be tested to expand our knowledge on the effectiveness of automated mn scoring in vitro and compared to traditional approaches. background: the platinating agent cisplatin is commonly used in the therapy of various types of solid tumors, especially urogenital cancers. its anticancer efficacy largely depends on the formation of bivalent dna intrastrand crosslinks, which impair dna replication and transcription. these crosslinks stimulate mechanisms of the dna damage response (ddr), thereby triggering checkpoint activation, gene expression and cell death. the clinically most relevant adverse effect associated with cisplatin treatment is nephrotoxicity, which mainly results from damaged tubular cells. here, we analyzed the influence of the hmg-coa-reductase inhibitor lovastatin on the cisplatin-induced geno-and cytotoxicity in the rat renal proximal tubular epithelial cell line nrk-52e. methods: cell viability was determined by using the alamar blue assay, as well as by electrical impedance measurements via the icelligence system. alterations in cell cycle progression were assayed by flow cytometric analysis. the formation of pt-(gpg) intrastrand crosslinks was determined via southwestern blot. the amount of dna double-strand breaks (dsbs) was quantified by measuring the level of s139 phosphorylated h2ax (γh2ax) via immunocytochemistry as well as by western blot. additionally, neutral and alkaline comet assays were performed to determine the amount of dna single-and dna double-strand breaks. mechanisms of the ddr were analyzed by western blot as well as by quantitative real-time pcr. results: the data show that pretreatment of nrk-52e cells with a subtoxic dose of lovastatin reduced the cytotoxicity evoked by high doses of cisplatin by protection from cisplatin-stimulated apoptotic cell death. moreover, lovastatin had extensive inhibitory effects on cisplatin-induced ddr, as reflected on the level of p-atm, p-p53, p-chk1, p-chk2 and p-kap1. furthermore, activation of mitogen-activated kinases (mapks) was also reduced. the lovastatin-mediated mitigation of cisplatin-induced ddr was independent of the initial formation of dsbs as well as of pt-(gpg) intrastrand crosslinks. lovastatin protects nrk-52e cells from cisplatin-induced cytotoxicity by interfering with proapoptotic mechanisms of the ddr independently from initial dna damage formation. with respect to the clinic, the data indicate that lovastatin might be useful to mitigate cisplatin-induced nephrotoxicity. the influence of oxidant tert-butylhydrochinone (tbhq) on endothelial cell migration in wrn-deficient cells k. laarmann 1 , g. fritz 1 1 institut für toxikologie, düsseldorf, germany introduction: wrn is a dna helicase and possesses a 3´-5´exonuclease and atpase activity as well as a single strand annealing activity. it is involved in dna repair, by interacting with proteins of base excision repair (ber) and nucleotide excision repair (ner). defects of wrn are marked by genome instability which, in turn, is caused by defects in dna damage repair. patients with a mutation in the wrn gene show premature aging and early mortality. the latter is mainly caused by arteriosclerosis. furthermore, wrn participates in the regulation of genotoxic stress responses stimulated by reactive oxygen species (ros) and alkylating agents. the aim of this study was (i) to investigate whether endothelial cell migration and adhesion were effected by sub-toxic (ic 10 ) and moderate toxic (ic 40 ) concentrations of the oxidant tertbutylhydrochinone (tbhq) and (ii) whether wrn influences migration and adhesion in the presence or absence of tbhq. methods: endothelial-like ea.hy926 cells were treated with different concentrations of the redox cycling and thus ros producing oxidant tbhq. viability was measured by the alamar blue assay. ic 10 and ic 40 were determined after 24h permanent treatment. to investigate the influence of wrn on endothelial cell migration and adhesion, a wrn knock-down was performed in ea.hy926 cells using rna interference. to measure migration, a confluent cell monolayer was scratched using a pipet tip, 24h after permanent tbhq treatment. pictures were taken at the time points 0h, 4h and 8h after performing the scratch. the non-closed area was measured. in a second part, adhesion of the calcein-labeled colon adenocarcinoma ht-29 cell line on the ea.hy926 monolayer was investigated. wrn-deficient or non-deficient cells were treated with 100 µm and 160 µm tbhq or with tnfα. results: for ea.hy926 cells, 100 µm and 160 µm tbhq were determined as ic 10 and ic 40 , respectively. performing the migration assay, ea.hy926 cells showed 75% gap closure, whereas wrn-deficient cells showed a closure of only 35% after 8h. the gap was closed of 65% and 55% after 100 µm and 160 µm tbhq treatment. in wrndeficient cells no remarkable effect on migration was observed after 100 µm tbhq treatment, whereas the treatment with 160 µm tbhq showed a slight decrease in migration of about 10% compared to wrn-deficient cells. no effect on adhesion was observed after tnfα treatment. after 160 µm tbhq treatment a slight increase of adhesion was detected in ea.hy926 cells. the influence of moderate tbhq concentration on adhesion was reduced in the absence of wrn. conclusion: wrn influences endothelial cell migration. in contrast to wild-type ea.hy926 cells, no significant effect of tbhq was observed on migration of wrndeficient cells. furthermore, the moderate toxic concentration of tbhq showed slightly increased ht-29 adhesion to ea.hy926, which was not found in wrn-deficient cells. outlook: in forthcoming studies we analyse the effect of alkylating agents on migration and adhesion. data will be presented and discussed. the aim of the present work was to compare the sensitivity of leukemia cell lines (hl60, jurkat and tk6) and hematopoietic stem cells with regard to the response to genotoxic agents. chromosomal damage was analyzed by evaluation of the micronucleus frequency. furthermore, changes in the proliferation index and the frequencies of apoptotic and mitotic cells were assessed. several cytostatic drugs with different mechanisms of action were used as genotoxic agents. doxorubicin was used as an intercalator, radical producer and topoisomerase ii inhibitor. also, the effects of vinblastine, a mitosis-inhibiting drug and of methyl methanesulfonate, which forms dna-adducts and stalls replication forks, were analyzed. in general, a difference in sensitivity between the different substances was observed. with regard to the formation of micronuclei after treatment with doxorubicin, jurkat and tk6 cells showed similar increasing trends, whereas hl60 cells showed a much higher increase in micronucleus frequency. a clear decrease in proliferation and the frequency of mitotic cells was observed at the highest concentration (100 nm doxorubicin) investigated, and only a slight increase in the number of apoptotic cells could be shown. the biggest differences in formation of micronuclei could be detected after treatment with vinblastine. hl60 cells showed only a slight increase of micronuclei, but the effect on jurkat cells was stronger. the highest micronucleus frequency after vinblastine treatment was detectable for the tk6 cells. the results for the highest investigated concentrations (31.6 nm and 100 nm vinblastine) showed a significant reduction of the proliferation index. this effect is reflected by the increasing numbers of apoptotic cells in all cell lines. the results for methyl methanesulfonate demonstrated only a small increase in micronucleus formation for the jurkat cells, but higher values for the tk6 cells. in contrast the hl60 cells did not lead to a concentration-dependent effect with methyl methanesulfonate. these results are complemented by preliminary findings in hematopoietic stem cells at selected compound concentrations. the different results between the leukemia cell lines and the stem cells might possibly originate from the different p53 status of hl60 (null), jurkat (multiple mutations), tk6 (wild type) and hematopoietic stem cells (wild type). this difference might also cause differences in cell cycle control or repair mechanisms, and needs further investigations. hyperinsulinemia is thought to enhance cancer risk. a possible mechanism is induction of oxidative stress and dna damage by insulin, here, the effect of a combination of metformin with insulin was investigated in vitro and in vivo. the rational for this were reported antioxidative properties of metformin and the aim to gain further insights into mechanisms responsible for protecting the genome from insulin mediated oxidative stress and damage. comet assay, micronucleus frequency test and a mammalian gene mutation assay were used to evaluate the dna damage produced by insulin alone or in combination with metformin. for analysis of antioxidant activity, oxidative stress and mitochondrial disturbances, the cell-free frap assay, the superoxide-sensitive dye dihydroethidium and the mitochondrial membrane potential-sensitive dye jc-1 were applied. accumulation of p53 and pakt were analysed. as an in vivo model, hyperinsulinemic zucker diabetic fatty rats, additionally exposed to insulin during a hyperinsulinemic euglycemic clamp, were treated with metformin. in the rat kidney samples, dhe staining, p53 and pakt analysis, and quantification of the oxidized dna base 8-oxodg was performed. metformin did not show intrinsic antioxidant activity in the cell free assay, but protected cultured cells from insulin mediated oxidative stress, dna damage and mutation. treatment of the rats with metformin protected their kidneys from oxidative stress and genomic damage induced by hyperinsulinemia. metformin may protect patients from genomic damage induced by elevated insulin levels. this may support efforts to reduce the elevated cancer risk that is associated with hyperinsulinemia. the human skin is the primary barrier against environmental and chemical impacts. as such it shields us against a plethora of xenobiotics such as potentially carcinogenic polycyclic aromatic hydrocarbons (pahs). at the same time it is the second most densely populated organ, harbouring more than 1000 bacterial species and population densities of up to 10 7 cfu per cm 2 . yet little is known about this microbiome's potential to metabolise and toxify pahs such as benzo[a]pyrene (b[a]p). previous work at the bfr showed that degradation of b[a]p and other pahs is a universal feature of the skin's microbiome (sowada et al., 2014) . the corresponding metabolites only partly overlap with those known from eukaryotic metabolism and possess cytotoxic as well as genotoxic properties. excretion of these metabolites will lead to exposure times of 20-30 hours or longer for full and partial metabolisers, respectively. while in vitro studies show the corresponding substances to exert their effects synergistically, an assessment of their potential impact on human carcinogenesis is pending. one obvious mode of action would be direct genotoxicity. however, another option is interference with uv-damage repair. ultraviolet radiation (uvr) from sunlight is regarded the main causative factor for the induction of skin cancer. it induces two of the most abundant mutagenic and cytotoxic dna lesions, that is cyclobutane-pyrimidine dimers (cpds) and 6-4 photoproducts (6-4pps). these lesions are repaired primarily by nucleotide excision repair (ner), a system that is also responsible for the removal of pah-derived dna adducts. we therefore wanted to know whether and to what extent bacterial b[a]p metabolites have the capacity to interfere with ner, potentially contributing to uv-induced dna-damage. to investigate this selected genotoxic metabolites were examined for their potential to affect the dna repair capacity of skin cells (hacat). following treatment with uva/b and bacterial b[a]p-metabolites the skin's repair capacity was assessed using a modified comet-assay. ionizing radiation (ir) is a well-established model to induce dna double-strand breaks (dna-dsbs), but it also generates a broad range of other dna lesions including dna single-strand breaks as well as oxidative dna base modifications. furthermore, ir is able to modify membrane components and triggers the activation of epidermal growth factor receptor. a more specific dsb-inducer is cytolethal distending toxin (cdt), which is produced by a variety of gram-negative bacteria and harbours an intrinsic dnase-like endonuclease activity [1] . dsbs are potent cytotoxic lesions and promote genomic instability, e.g. by formation of chromosomal aberrations. a cellular mechanism to prevent genomic instability and maintain cell homeostasis could be autophagy. this process is highly regulated involving the lysosomal degradation of damaged organelles and proteins. here, we study autophagy induction following dsb generation in human colorectal cancer cells as well as in primary human colonic epithelial cells (hcec) and analyzed regulatory mechanisms. first, the autophagy-specific marker lc3b was shown to increase in a dose-and time-dependent manner after treatment with both cdt and ir as assessed by confocal immunofluorescence microscopy and western blot analysis in hct116. similar results were obtained in sw48 and hcec cells via western blot. these findings are in agreement with the enhanced formation of autophagosomes and the dose-dependent decrease of the autophagy substrate p62 as observed by flow cytometry and western blot analysis in hct116, sw48 and hcec. cdt-and irinduced autophagy rates in hct116 increased over time correlating well with the dsb induction. importantly, a dnasei-defective mutant of cdt did neither cause dsbs nor induce autophagy. additionally, the time-dependent accumulation of the lysosomal associated membrane protein 1 (lamp-1) was observed by confocal immunofluorescence microscopy. dsb-induced autophagy was blocked by chemical inhibitors. next, we showed that both ir and, to a lesser degree, cdt induce the phosphorylation of akt at ser473. pharmacological inhibition of akt in hct116 cells enhanced the cdt-and ir-induced autophagy shown by accumulation of lc3b and lamp1 after 48 h and increased autophagosome formation. upregulation of dsbinduced autophagy by akt inhibition resulted in a decreased cytotoxicity after 72 h and significantly lower apoptosis/necrosis rates after 48 h, which were determined by mts cell viability assay and annexin-v/pi staining. ongoing studies will evaluate the impact of other dna damage response pathways and the potential protective role of autophagy against genomic instability. mustard agents are potent dna alkylating agents. among them, the bi-functional agent sulfur mustard (sm) was used as a chemical warfare agent due to its vesicant properties. although the use of sm in warfare has been banned in most countries of the world, its use in terroristic attacks or asymmetrical conflicts, such as the syrian civil war, still represents a realistic and significant threat. on the other hand, especially nitrogen mustards, such as cyclophosphamide or melphalan, have been used as chemotherapeutic agents due to their cytostatic properties. thus, mustard-induced dna damage, in particular dna crosslinks, can trigger complex pathological states, as it is observed in sulfur mustard exposed victims, but on the other hand also lead to the chemotherapeutic effects of clinically-used nitrogen mustards. mass spectrometric monitoring and quantitation of mustard-induced dna adducts can help to unambiguously identify and verify sm-exposed victims and to monitor the efficiency, as well as potential side-effects of mustard-based chemotherapy. up to now, the verification of mustard-induced nucleic acid damage is mainly based on immunohistochemical methods, which have several drawbacks such as limited specificity, sensitivity, and low dynamic range of quantitation. with this project, we aim to develop a (hplc/uplc)-ms/ms-based platform for the quantitation of the most common mustard-induced dna adducts including bis(n7-guanine-ethyl) sulfide dna crosslinks. up to date, we established methods for the quantitation of the several common dna adducts induced by the mono-functional sulfur-mustard derivative 2chloroethyl ethyl sulfide ("half mustard", cees). for that reason purification protocols, chromatographic conditions and mass spectrometric settings were developed to detect n7-ethylthioethyl-2´desoxyguanosine (n7-ete-dg) and n3-ethylthioethyl-2´desoxyadenosine (n3-ete-da) and their thermal hydrolysis products n7ethylthioethyl-guanine (n7-ete-gua) and n3-ethylthioethyl-adenine (n3-ete-ade), respectively, and the sensitivity was compared to immunohistochemical methods. additional non-radioactive isotope-labelled standards are being synthesized, which will be spiked into samples to account for technical variability during sample work-up and to improve ms-based quantitation. this procedure requires minimal cellular material and therefore should be transferred to quantitation of dna adducts in human blood samples. this will allow to monitor dna adducts as biomarkers of exposure in potential smexposed victims as well as in mustard-based chemotherapy. this method also sets a basis to investigate specific mustard-induced dna repair mechanisms and their cellular consequences. the γh2ax assay vs. comet assay for genotoxicity testing universitätsmedizin mainz, institut für toxikologie, mainz, germany dna damage leads to activation of the cellular dna damage response (ddr). this signalling network results in activation of various dna repair proteins and chromatin structure modulators. a frequent manifestation of ddr is the phosphorylation of histone 2ax (gh2ax), which can be visualised as gh2ax foci by immunocytochemistry. in the present study, we tried to assess if gh2ax is a reliable biomarker for detecting the cellular response to dna damage. we selected 14 well-characterised genotoxic compounds and compared them with 10 non-genotoxic chemicals in the wellcharacterised cho cell system. we measured quantitatively γh2ax by manual and automatic scoring of γh2ax foci, and by flow cytometry counting of γh2ax positive cells. the cytotoxicity dose-response was determined by the mtt cell proliferation/viability assay. we show that a) all genotoxic agents were able to induce dose-dependently γh2ax in the cytotoxic range whereas no induction was observed after treatment with non-genotoxicants; b) manual scoring of γh2ax foci and automated scoring gave similar results, with the automated scoring being faster and more reproducible; c) data obtained by foci counting and facs analysis of γh2ax positive cells showed a significant correlation. further we compared dna damage induced by 4 selected genotoxins at the time-points using the alkaline and neutral comet assay. significant correlation with the alkaline and neutral comet assay was observed for some but not all genotoxins and, predominantly, at earlier time points. we suggest that comet assays detect mainly primary dna damage, whereas γh2ax assay detects a specific response to dna damage which can persist longer. the γh2ax foci and flow-cytometry assays allow for a rapid and reliable determination of genetic damage in mammalian cells and can be used as additional genotoxicity assays. available in vitro methods to investigate the genotoxic potential of drugs fall short of throughput, specificity and mode of action information. a set of mechanistic biomarkers for clastogenic, aneugenic or apoptotic effects may help to overcome these limitations. thus, a staining assay amenable to flow cytometric analysis is being developed by litron laboratories, rochester, ny, supported by international collaborators. the experimental design of this assay consists of 3 stages. the objective of this work is the evaluation of this assay in the laboratories of bayer pharma ag. the biomarkers covered by the assay are associated with dna damage response pathways that have potential for class discrimination (clastogen/aneugen/cytotoxicant) of in vitro genotoxicants: dna double strand breaks (γh2ax), nuclear division (phospho-h3, dna content), apoptosis (cleaved parp). based on the pilot work at litron laboratories, tk6 cells were introduced to the genetic toxicology of bayer pharma ag. cells were exposed for 4 and 24 hrs in triplicates on a 96 microwell plate to one reference clastogen (etoposide, eto), aneugen (vinblastine, vb) and cytotoxicant (carbonyl cyanide 3-chlorophenylhydrazone, cccp). after staining, the samples were analyzed with the flow cytometer bd accuri c6 (bd biosciences, heidelberg, germany). the reference substances yielded the responses expected from the pilot study at litron laboratories: vb showed distinct increases of phospho-h3 events at 4 and 24 hrs and polyploidy at 24 hrs time point. eto induced a clear increase of yh2ax with a simultaneous reduction of phospho-h3 at 4 and 24 hrs. finally, cccp caused a reduction of phospho-h3 events, increased cleaved parp events and did not influence γh2ax. moreover, benchmarking experiments under pilot work conditions were performed with high content imaging analysis. we compared yh2ax and phsopho-h3 pilot study results as well as cleaved parp with caspase 3/7. in addition, the tunel assay (click-it ® tunel alexa fluor, thermofisher) was executed to benchmark cleaved parp. the benchmarking results support the selected biomarkers of the multiplexed assay. in stage 2, additional reference compounds (three aneugens/clastogens/cytotoxicants) were investigated. so far, the chosen biomarkers of dna damage response appear useful for class discrimination and provide additional information to existing genotoxicity tests. cell-cell contacts are involved in keeping a physiological balance between proliferation, differentiation and apoptosis. far less is known about the role of cell-cell-contacts in regulating necrosis, for instance in response to oxidative stress. previous findings of our group demonstrated that, in contrast to semi-confluent proliferating cultures, confluent murine fibroblasts (nih3t3, mef) and human keratinocytes (hacat) are protected against necrosis induced by tert-butyl hydroperoxide (t-booh). comparison of confluent cells (g0/g1 = ~70 %) and semi-confluent cultures, similarly arrested in the g0/g1 phase by serum-starvation or the mek inhibitor u0126, ascertained that the resistance against t-booh is mediated by cell-cell contacts and not by cell cycle arrest. we further revealed that confluent cultures are protected against t-booh-induced dna double strand breaks as assessed by the neutral comet assay and against mitochondrial damage detected by flow cytometric analysis of dioc6 staining. to better understand the protective role of cell-cell-contacts in ros-mediated necrosis, we started characterizing the signaling cascade induced by t-booh in semi-confluent proliferating cultures. in accordance with the observed formation of dna double strand breaks in response to t-booh, we detected phosphorylation of the checkpoint kinase chk2. however, inhibition of atm, the kinase responsible for chk2 activation, did not influence t-booh-induced cell death. interestingly, first experiments gave a hint for the participation of rip1, since the chemical rip1 kinase inhibitor necrostatin-1 (nec-1) blocked cell death up to averagely 50 %, what is described as a specific marker for regulated necrosis. in line with this observation, t-booh-induced cell death could not be blocked by the pan-caspase inhibitor z-vad-fmk strongly indicating that caspase activity is not required. moreover, parp-1 and p53 are probably not involved. deeper analyses could give evidence that nec-1 did not block formation of dna double strand breaks nor mitochondrial damage indicating that the kinase blocked by nec-1, possibly rip1, acts downstream of dna double strand breaks and / or mitochondrial damage. in the end, we could identify a crucial role of ca 2+ signaling for t-booh-mediated toxicity. as the calcium chelator bapta-am was able to completely block not only cell death, but also mitochondrial damage and dna double-strand break formation, there is a strong need for further investigations of the possible interplay between regulated necrosis and calcium, regulated by cell-cell contacts among oxidative stress. the work was supported by the hoffmann-klose-stiftung, the promotionsförderung rheinland-pfalz, the johannes gutenberg-university and the university medical center of the johannes gutenberg-university. the mammalian target of rapamycin (mtor) forms two multiprotein complexes (mtorc1 and mtorc2) and influences cell growth, proliferation, survival and metabolism. constitutively activated mtor was found to be deregulated in several cancer types, which makes it an interesting target for therapeutic cancer strategies. rapamycin is able to inhibit mtor and its downstream targets and is currently studied for its anticancer properties in clinical trials. despite previous evidence, there are studies that show an adverse effect in cancer treatment causing tumour growth, evolving the question of the effectiveness of the drug in cancer treatment. therefore, we examined the transformational potential of rapamycin in a balb/c cell transformation assay (cta) as well as markers of proliferation and protein synthesis. the balb/c 3t3 cell transformation assay mimics different stages of in vivo carcinogenicity (initiation, promotion, post-promotion phase) and is a promising alternative to rodent bioassays. balb/c fibroblasts are treated for 3 days with the tumour initiator mca (3-methylcholanthrene) followed by 13 days with the promotor tpa (12-o-tetradecanoylphorbol-13-acetate). upon treatment with these chemicals cells are transformed into morphologically aberrant foci and can be visualized after six weeks by giemsa staining. it is possible to apply additional substances during the whole assay or in several phases of transformation and evaluate the colony formation. furthermore, our improved protocol allows additional westernblot or immunofluorescence analysis. the influence on cell proliferation of different concentrations of rapamycin was investigated by cell counting (living and dead) to choose a suitable concentration for the cta. performances of balb/c ctas with 10 nm rapamycin showed, contrary to expectations, an increase in cell transformation. by administration of rapamycin only in the promotion phase we could detect an increase in colony formation, whereas a treatment with rapamycin in the post-promotion phase with already established foci, seemed to reveal its therapeutic properties. to better understand the role of mtor in our cell transformation system we used another mtor inhibitor called osi-027. surprisingly, an incubation with 3 µm osi-027 led to a decrease in colony formation. we are now able to investigate the underlying mechanism with westernblot and immunofluorescence analysis and can compare regulations of downstream targets like the marker of protein synthesis p-s6. our investigations revealed different cell transformation outcomes by comparing the two known mtor inhibitors rapamycin and osi-027, which need to be further evaluated. in the ongoing project we want to detect differences between rapamycin and osi-027 by protein analysis and identify key proteins, which are involved in this opposed colony formation of the balb/c cells. these results can be helpful to better understand mtor inhibition in matters of tumour therapy. introduction: over the past 50 years, the biguanide compound metformin has been widely prescribed as an insulin sensitizer in type 2 diabetes mellitus. interestingly, recent meta-analyses of epidemiological studies have shown that metformin might be involved in risk reduction of carcinogenesis. in vitro studies have described amp-activated protein kinase (ampk)-dependent, by inhibition of the respiratory chain complex i, as well as ampk-independent actions of metformin. however, the detailed molecular mechanisms by which metformin affects cell proliferation and carcinogenesis have not been well identified up until now. method: to evaluate the protective potential of metformin, balb/c 3t3 cell transformation assays were performed. this valid toxicological method is an alternative to in vivo carcinogenic testing and mimics the different stages of cell transformation during carcinogenesis. in detail, mouse fibroblasts are treated with metformin and/or the tumour initiator 3-methylcholanthrene (mca) and the tumour promotor 12-otetradecanoylphorbol-13-acetate (tpa). in the first experiment several metformin concentrations (0.1-1 mm metformin) were applied answering the question of an effective metformin concentration. next, metformin treatment during the different phases of carcinogenesis (initiation, promotion, post-promotion phase) was done determining the most effective phase for an intervention, i.e. chemopreventive or chemotherapeutical properties of metformin. additionally, the effect of metformin on the energy metabolism of the cells was analysed using various methods like immunoblot and oxygen measurement by clark electrode. results/discussion: analysis of different metformin concentrations revealed a concentration-dependent effect of metformin. in detail, decreased colony forming potential of balb/c cells was most prominent using 1 mm metformin. this effect was not caused by growth inhibition of metformin itself since 1 mm metformin showed no growth inhibitory properties in a cellular growth pretrial. interestingly, the 2 phase cell transformation assay showed that the metformin effect is more pronounce in the postpromotion phase than in the initiation and promotion phase pointing to a chemotherapeutical potential. investigating several energy metabolism parameters, the results indicate that metformin may affect cell respiration as well as energy-dependent mechanistic markers like ampk. the presented results support rather the idea of the chemotherapeutic potential of metformin than a chemopreventive, using 1 mm metformin. the initial analysis of energy metabolism markers discovered interesting starting points for further investigations. johannes gutenberg university, institute of toxicology, 55131 mainz, germany nvp, widely used e. g. as a monomer for polyvinylpyrrolidones (pvp) with applications in food technology or cosmetics is a known hepatocarcinogen in rats after inhalative exposure to 5, 10, and 20 ppm for 2 years. nvp is tested in a battery of genotoxicity assays (e.g. ames, hprt, mouse lymphoma, uds, chromosome aberration, cell transformation, micronucleus test (mnt) in mice bone marrow) [1]) that all yielded negative results. however, nvp induces cell proliferation in liver (loaec: 0.5 ppm) after whole body exposure to vapor [2] . to confirm the absence of genotoxicity in the context of a potentially non-genotoxic mode of action, a five day whole body inhalation study to nvp vapor with concentrations of 0, 5, 10, 20 ppm was conducted in wistar rats (six animals per gender and group, ethyl methanesulfonate 200 mg/kg bw p.o. as positive control). genotoxicity was investigated by the mnt in bone marrow and the comet assay (± fpg) in liver and lung. further investigated endpoints related to possible non-hepatogenotoxic moa were: enzyme induction (erod, prod, brod), oxidative stress (gsh-, gssg-, non-protein sulfhydryl group level), and peroxisome proliferation (cyp4a, cyanide-insensitive palmitoyl-coa-oxidase). at carcinogenic inhalative doses, the results of this study proved the absence of genotoxicity in lung, liver and bone marrow as neither the tail intensity in the comet assay nor the number of micronuclei in the mnt was increased compared to the controls. however, also the non-genotoxic parameters (cyp-enzyme activity, glutathione levels, cyanide-insensitive-palmitoyl-coa-oxidase) were not affected by nvp-treatment. as potential metabolic activation cannot be excluded and may essentially contribute to the understanding of the carcinogenic mechanism, in vitro investigations in rat liver systems (subcellular fractions, hepatocytes, precision cut liver slices (pcls)) were performed additionally. up to now, 2-pyrrolidone is the only identified in vitro metabolite. as these results cannot mimic the in vivo situation of two described, ring-and vinylmajority containing unidentified metabolites [3] detailed investigations on metabolism may be a future perspective to approach the overall understanding of the carcinogenic mechanism of nvp. introduction: in ischemic conditions such as wound healing and myocardial infarction, new vessels are generated by vasculogenesis and angiogenesis. these processes are stimulated by the signalling peptide vascular endothelial growth factor which therefore has been proposed as a promising compound for the treatment of ischemic conditions. however, results of respective clinical studies have not been fully convincing yet. here, we investigated principles underlying the selforganization of newly formed vessels to functionally adequate microvascular networks indispensable for proper tissue substrate supply. intravital microscopy of the chick chorioallantoic membrane (cam), a non animal model as defined by the american national institutes of health's office for protection from research risks, was used to study peripheral expansion of existing arteriolar and venular trees by recruiting segments of the dense polygonal capillary mesh. this process we call "emerging angiogenesis". methods: white leghorn chicken eggs were put into incubators on embryonic day 0 (e0) at 37.5°c and 82% humidity. on e3, the eggs were cracked open and transferred into petri dishes. on e10, cam microcirculation was recorded using time-lapse intravital videomicroscopy at discrete time points for up to 48 hours. to improve the visibility of the capillary mesh, videorecordings were processed offline by generating coefficient of variation images of pixel grey values over time. changes of network topology during the observation time were investigated. results: in the cam, a sequence of specific events leading to extension of existing vessel trees was observed: in a capillary mesh region near terminal branches of existing vessel trees, homogeneous flow distribution is transferred to inhomogeneous flow distribution: preferred flow pathways through the mesh evolve carrying most of the blood. over time, these flow pathways exhibit diameter increase, straighten and connect the mesh to arteriolar and venular trees. in contrast, less perfused parallel mesh flow pathways and transversal mesh segments exhibit progressive decrease of flow and diameter resulting in vessel regression. as a result, hierarchical vessel tree structures are extended into the mesh region. while newly generated tree extensions are located above the mesh at the beginning, they sink to a lower level at later stages until they are finally covered by a reconstituted mesh network. the cam ex ovo model is well suited for studying emerging angiogenesis. vessel tree extension occurs via parallel processes of vessel maturation and capillary mesh segment regression. at later stages, newly formed vessel tree branches sink and the capillary mesh is reconstituted above. in the next step of our project, we will implement these phenomena in a computer simulation and use theoretical modeling to further investigate and better understand principles underlying microvascular network maturation. this will allow us to derive effective therapeutic strategies which could be tested in the cam model. chemicals are able to induce cancer in a wide range of organs. therefore, it is very important to investigate the toxic properties of chemical substances, especially their carcinogenic potential. in this context the number of animal experiments will drastically increase in the future. in order to avoid the use of expensive and time consuming animal experiments for long-term carcinogenic studies, the development of an in vitro system to test the carcinogenic potential of a high number of chemicals in a highly reproducible manner within a short period of time is imperative. by combination of the well-established balb/c cell transformation method with the soft agar colony formation assay, we developed a high-throughput in vitro system to identify effects of chemicals on cell transformation for the first time. balb/c mouse fibroblasts are treated with 3-methylcholanthrene as a tumour initiator and 12-o-tetradecanoylphorbol-13-acetate as promotor for several days, whereby foci of transformed cells are developed. after the promotion phase of the common balb/c cell transformation assay, cells are transferred into soft agar to further monitor the anchorage independent growth of transformed cells only. the established soft agar transformation assay reproduces the foci growth of previous experiments and is performed in 96-well plate format. hence, we can analyse the carcinogenic potential of several chemical substances in parallel and are also searching for alternative endpoint analysis, e.g. the usage of fluorescing cells stably expressing irfp, instead of the former time-consuming microscopic assessment. the here presented new technique is a high-throughput and low priced alternative for the evaluation of the carcinogenic potential of chemical substances in a short period of time without animal testing. the effort to develop new or refine established in vitro test systems rises due to animal welfare, scientific and/or regulatory reasons (e.g. the animal testing ban concerning the risk assessment of cosmetic product ingredients in march 2013). this progress, among others, leads to an increased performance of cell-based assays. the majority of model cell lines are routinely cultured using medium supplemented with fetal bovine serum (fbs) in amounts between 5-10%. the application of serum-substitutes will provide a reduction of the animal number needed, which corresponds to the guiding principles of the three r's (3r), described by russel and burch in 1959. in addition, chemically defined serum-substitutes have the potential to reduce the inter-experimental variability of test conditions caused by the inherent differences in chemical composition across fbs batches 1 , resulting in a refinement of in vitro testing. in this study, human tk6 cells were gradually adapted to serum-free conditions, where they show comparable growth gradients at the exponential phase. for cells under serum-free conditions a mean doubling time of 19.3 (± 1.7) h was observed while fbs supplemented cells showed a doubling time of 13.5 (± 0.8) several non-animal test methods addressing key events in the sensitization process have passed formal validation and oecd (draft) test guidelines are available. one of these methods is the direct peptide reactivity assay (dpra) assessing the ability of a chemical to bind to proteins to form a complete antigen (oecd tg 442c). the test is used to obtain a yes/no answer on whether the substance has a protein-binding potential. for a complete risk assessment, however, an estimation of a chemical's potency is also needed. in this study we examined if an assessment of potency could be achieved by 1) determining reactivity class cut offs based on published data on 199 substances for the dpra performed according to oecd 442c to predict un ghs sensitizer classes, 2) a variant of the dpra assessing reaction kinetics (time and concentration) for 12 substances or 3) an extended protocol testing several test substance concentrations for 50 reference substances and estimating the concentration of a test substance that is needed to cause a peptide depletion of 6.38% (ec6.38%). results of the first approach indicated that cut offs to differentiate the un ghs sensitizer classes 1a and 1b could indeed be defined. secondly, evaluating the reaction time based assay in which several time points between 5 min and 24 hours were assessed, it was found that not all reactions followed ideal kinetics. hence further investigations are needed to eventually derive a reaction time based prediction model. the results of the 3 rd approach (the standard protocol of the dpra was amended by testing three concentrations i.e. 1, 10, and 100 mm) indicated that potency classes could be assigned using the ec6.38% value to assess potency. in summary, using quantitative information derived from the dpra in particular using ec6.38% value may support the assessment of the skin sensitizing potency. identification of pre-and pro-haptens with non-animal test methods for skin sensitization since pro-haptens may be metabolically activated in the skin, information on xenobiotic metabolizing enzyme (xme) activities in cell lines used for testing of sensitization in vitro is of special interest. metabolic activity of e.g. n-acetyltransferase 1 (nat1) and esterase in the keratinocyte (keratinosens tm and lusens) and dendritic cell-like cells (u937 and thp-1) was previously demonstrated. aldehyde dehydrogenase (aldh) activities were found in keratinosens tm and lusens cells. activities of the investigated cytochrome p450-dependent alkylresorufin o-dealkylases, flavin-containing monooxygenase, alcohol dehydrogenase as well as udp glucuronosyl transferase activities were below detection in all investigated cell lines. a set of 27 putative pre-and pro-haptens (no obvious structural alert for peptide reactivity but positive in vivo) was routinely tested using the above mentioned cell lines as well as in the direct peptide reactivity assay (dpra). 18 of the compounds were unexpectedly positive in the dpra und further analyzed by lc/ms techniques to clarify the reaction mechanism leading to true positive results in this assay. oxidation products like dipeptide formations or the oxidation of the peptide-based sulfhydryl group led to positive results for benzo[a]pyren or 5-amino-2-methylphenol, respectively. in contrast, covalent peptide adducts were identified for 12 putative pre-haptens, indicating the dpra to be suitable for compounds requiring abiotic oxidation to get activated. for some dpra negatives, the keratinocyte and dendritic cell based assays provided true positive results. a combination of dpra, keratinosens tm and h-clat within a '2 out of 3' prediction model provided a high sensitivity of 81% for the set of the pre-/pro-haptens. the sensitivity of this combination of non-animal test methods in the '2 out of 3' prediction model in a set of 95 direct haptens was comparable (sensitivity = 87% when compared to llna skin sensitization testing is mandatory for all substances produced or marketed in volumes larger than 1 tonne per year under the european reach legislation. with reach supporting in vivo testing only "as a last resort" and the marketing ban for finished cosmetic products with ingredients tested in animals, attention has been given to developing integrated testing strategies combining in vitro, in silico and in chemico methods. key challenges are which tests to select and how to combine non-animal methods into testing strategies. this study suggests a bayesian value of information (voi) approach for developing non-animal testing strategies, which consider information gains from testing, but also expected payoffs from adopting regulatory decisions on the use of a substance, and testing costs. the 'value' of testing is defined as the expected social net benefit from decision-making on the use of chemicals with additional, but uncertain information from testing. the voi is calculated for a set of individual nonanimal methods including dpra, oecd qsar toolbox, are-nrf2 luciferase method covered by keratinosens and lusens, and hclat, seven battery combinations of these methods, and 86 two-test and 360 three-tests sequential strategies consisting of nonanimal methods. their voi is compared to the voi of the local lymph node assay (llna) as the animal test. we find that battery and sequential combinations of nonanimal methods reveal a higher voi than the llna. in particular, for small prior beliefs (i.e. a chemicals is, prior to testing, assumed to be a non-sensitiser), a battery of dpra + lusens reveals the highest voi. if there are strong beliefs that a chemical is a sensitizer, a sequential combination of the battery dpra + lusens, followed by keratinosens + hclat at the second stage and by the oecd qsar toolbox at the third stage performs best. for given specifications of expected payoffs the voi of the nonanimal strategy significantly outperformed the voi of the llna, for the entire range of prior beliefs. this underlines strong economic potential of non-animal methods for skin sensitization assessment. a chemical series to predict the proarrhythmic potential of drugs with low solubility for which no reliable purkinje fiber results could be obtained. these validation results showed that this cardiosafety in silico model can successfully be applied in r&d to predict the proarrhythmic potential of drug candidates within the model ad. introduction: the use of p-phenylenediamine (ppd) and derivatives (tab. 1) in oxidative consumer hair dye products is considered as key in hair dye allergic contact dermatitis [1] [2] [3] [4] . in recent supplement, 2-methoxymethyl-ppd (me+) shows significantly reduced sensitizing properties [5, 6] . since overcoming the skin barrier is a prerequisite for sensitization, numerous in vitro an in vivo studies on skin penetration of ppd and derivatives have been performed. the aim of the present study is the in silico prediction of the penetration of ppds, because such computations may help in understanding the processes involved in sensitization. for the first time, software dskin [7] is challenged to simulate this class of compounds. in silico results are retrospectively compared to previously published experimental data and may assist in future tailoring of in vitro experiments. material and methods: the permeabilities, lag-times and the time-dependent accumulated amounts of ppds were computed using dskin. input parameters for the latter were a concentration of 1 mg/ml (1%), finite dosing and 30 min in use incubation periods. molecular structures were optimized ab initio and the condensed fukui functions (ff) were estimated from mulliken population analyses [8] and electrostatic potentials using gamess [9] . results: initial results agree with experimental results using ppd in white petrolatum, demonstrating the applicability of dskin to ppds. the four ppds exhibit only small differences in permeabilities in silico (tab. 1). toluene-2,5-diamine shows a higher accumulated mass due to increased lipophilicity (fig. 1) . in general, the ff were very similar for all ppds and indicated that the n atoms would be the preferred targets for radical and electrophilic attack. discussion and outlook: in silico methods may be used to model the permeation of ppds despite their low molecular weight and low lipophilicity. the low amounts of ppds under in use conditions result from oxidative conditions. computed me+ permeation was not different to other ppds, therefore other properties account for the reduced sensitization potential. the very similar ff values hint at similar reaction pathways. furthermore, ppd and its derivatives are prone to n-acetylation in living skin resulting in metabolites exhibiting higher molecular weight and greater lipophilicity than the parent compounds. the effects of n-acetylation and reactions of ppd and its derivatives with histidine and cysteine residues are subject of upcoming computations. dermal absorption is an important factor in regulatory science regarding the registration of chemicals, agrochemicals and cosmetics. the issue has gained importance since it has been realized that the skin is not completely impenetrable for chemical substances. [1] the different ways to assess dermal absorption range from qsar models to complex in vivo studies including a complete toxicokinetic examination. the choice of method depends on the question that has to be answered as different systems give different results: absorption as % of applied dose in in vivo studies or permeability coefficient and lag time in infinite dose in vitro studies [1] . ideally both data would be available. since the oecd has adopted a guideline for assessing dermal penetration in vitro in 2004 the number of in vitro studies is rising continuously. depending on the chosen method results may vary in reliability and in acceptance by regulatory authorities. the skinab database [ba1] contains data for about 600 substances on dermal absorption which has been found through the echemportal [3] and extended with data from the edetox database. for selected substances with a broad spectrum of data available further analysis has now been started. 165 chemicals have been investigated in a comparable test system; from these 79 were shown to have a low dermal absorption of less than 1% and 51 compounds showed a high absorption rate of more than 50%. for the assessment of dermal exposure either the absorbed dose in percent or the flux can be measured. data analysis showed that only for 15 substances both is available: flux data from in vitro studies and absorption data from in vivo studies. this data could be used to clarify which parameter would be most useful for exposure assessment regarding dermal exposure. seven substances in the dataset were conspicuous for their range of absorption rates in different studies: less than 1% to more than 50%. an in depth analysis revealed the complex influence that different exposure parameters have on the results of dermal absorption studies. for some chemicals the influence of exposure time on increasing absorption values could be clearly demonstrated. beside other factors such as the chosen vehicle, and the (non-)occlusion of the site of exposure especially the choice species introduced a high variability; this holds even for the most common laboratory animals t. a review published by jung in 2015 [5] which comes to the conclusion that i.e. hairless species are usually not a good model to predict dermal absorption in humans. [1]who (2006) ehc 235 dermal absorption [2] scholz et al (2014) naunyn-schmiedeberg´s arch pharmacol 3 (suppl 1):s86 [3] www.echemportal.org [4] http://edetox.ncl.ac.uk [5] jung et al (2015) in-silico methods have evolved to indispensable tools in various areas of life sciences. several stages in drug development including hit identification and lead optimization, for instance, highly benefit from an accurate estimation of binding free energies associated with biological host-guest systems. as a consequence, the need for laboratory experiments including in-vivo experiments and animal testing is considerably reduced. another field profiting from free energy calculations is human as well as ecotoxicology. upon the development and risk assessment of new chemicals, transformation products arising from biotic or abiotic degradation of the parent substance have usually been neglected. however, since few years, the risk assessment of new chemicals often includes transformation products probably causing more harm than the parent substance itself. such studies as well are mostly carried out on the basis of in-vitro and in-vivo tests. moreover, many metabolites can be detected but neither enriched nor synthesized in amount sufficient for toxicological evaluations. at this stage, computational methods come into play. using classical molecular dynamics simulations in combination with an empirical linear prediction model, we have investigated several metabolites of the drugs sulfamethoxazole and carbamazepine and prioritized them according to their estimated binding affinities to potential biological target proteins. consequently, a couple of metabolites were identified that bind to one or more human cytochrome p450 variants and the bacterial enzyme dihydropteroate synthase, respectively, which are known to be sensitive to the two drugs. the investigations were carried out in the framework of the bb3r poject funded by the german government through bmbf. instituto superiore di sanità, environment and primary prevention dept., rome, italien introduction: in vitro methods have been increasingly used to characterize pharmacological and toxicological properties of substances. to address the problem of nominal versus actual concentrations, in vitro biokinetic studies were recently undertaken (truisi et al., toxicol lett 233:172-86, 2015) . we use those data as input into a physiologically based human kinetic model (pbhkm) to model the in vivo doses leading to the in vitro measured concentrations. methods: a pbhkm was used to simulate the concentration time profile of ibuprofen in the hepatic vein after oral administration. the details of the model and the physiological parameters used have been described elsewhere (abraham et al., arch. toxicology 79: 63-73, 2004) . we modelled the concentration time profile exploring the dose which would lead to a concentration at 1 hour and at 24 hour as similar as possible to the concentration measured in the supernatant of human freshly prepared cell cultures after dosing the culture with ibuprofen. we parametrized the pbhkm with the parameters which have been estimated from the in vitro kinetic studies (clearance between 3 and 15 µm 3 /sec (truisi et al., 2015) and an absorption of 100% and an absorption rate of 1/h (cristofoletti and dressman, j pharm sci 103: 3263-75, 2014). results: the data of the in vitro study with 100 µm ibuprofen could well be modelled. when assuming a clearance of 15 µm 3 /sec the dose of 1480 mg resulted in an 1 hour concentration of 66.5 µm in the hepatic vein of pbhkm equal to 133.0 nmol/well (volume of the well = 2 ml) in the in vitro study in which the measured concentration was 138.75 nmol/well. the concentration at 24 hours of 8.7 µm (equal to 17.4 nmol/well) corresponded with the in vitro concentration (16.5 nmol/well). the modelling approach was less successful with in vitro dosing of 1000 µm. the 10 fold higher dose of 14800 mg lead to nearly double the concentration at 1 hour than measured in vitro. with a dose of 8500 mg/kg an approximation was feasible resulting in 396.7 µm in the hepatic vein at 1 hour which is equal to 793.4 nmol/well whereas the measured concentration in vitro was 782.85 nmol/well. even with a clearance value as low as the 2.5 percentile (3 µm 3 /sec), the concentration at 24 hours was modelled to be lower than the in vitro measured value (in vivo model: 98.7 µm which corresponds to 197.4 nmol/well; measured in vitro concentration: 979.2 nmol/well). discussion: this is the first attempt to use kinetic data obtained in vitro to feed in in a pbhkm for reverse dosimetry finding the dose which corresponds in vivo to the in vitro situation. in the case presented here, the in vitro dose assumed to be low in vitro (100 µm) corresponds to a dose of 1480 mg (note: the highest approved daily dose is 2,400 mg). for the high in vitro dose modelling was successful only for the concentration 1 hour after dosing and a dose of 8,500 mg. conclusions: in vitro kinetic parameters, such as clearance, can successfully be used for parametrizing a pbhkm. it is of utmost importance for the relevance of in vitro finding to assure that the concentrations used in vitro can be obtained with relevant in vivo doses. in this case, the in vitro concentrations were within (low dose) and 3.5 fold above (high dose) the in vivo relevant therapeutic concentration range. introduction: a variety of drug residues have been detected in sewage plant run-offs, rivers and lakes, but also in groundwater and tap water samples. studies have yet to identify a risk for human health from these contaminants, but adverse health effects have been reported for various species, including fish and birds. it has recently been suggested that for a comprehensive risk assessment toxicologists should also consider transformation products (tps) of such water contaminants that may arise from abiotic and biotic (metabolic) reactions. with aciclovir (acv), a well-known antiviral drug, as the parent drug we tried an in-silico approach to identify tps that might be of interest due to some mutagenic or carcinogenic toxicophores. methods: from a literature and database search we picked up 12 acv-tps. predicted acute toxicities and mutagenic / carcinogenic properties for these tps were derived from an expert system analysis using the lazar portal (http://lazar.in-silico.ch/) as front end. results: two of the identified acv-tps could not be handled by lazar because of insufficient training data in one out of eight queried categories. the highest score (4 positive out of 8 possible genotoxicity categories) was assigned to 9 of the 12 tps, including acv itself. this is a rather low score when compared to other water-borne drug residues, e.g. carbamazepine. cofa, an imidazole derivative of acv seen in advanced oxidation processes, had shown antiproliferative effects in several ecotoxicologic screening assays, e.g. [1], but was unremarkable in our tests. additionally, a computer-based simulation of the respective tps interacting with human cyp isozymes did not support concerns that these tps may pose a risk for human health. conclusions: our in-silico analyses of 12 acv-tps did not provide evidence for any adverse health effects in the micromolar concentration range. further studies are needed to clarify if the biological activity of some acv-tps in ecotoxicological assays may eventually affect yet unidentified biological targets in the human body. sulfur mustard (sm) is a chemical warfare agent which was first used in world war i, but has found use in several conflicts afterwards. although sm is prohibited by geneva protocol, terroristic attacks cannot be ruled out. latest news give rise to concern that is may be in the possession of sm and is willing to deploy it. even 200 years after the initial synthesis of sm its mode of action is not fully unraveled. thus, no antidote does exist. however, chemosensing ion channels have been shown to be activated by highly toxic chemicals and might represent a specific therapeutic target. previous studies have shown that the sm-surrogate cees (mono-functional alkylating agent) is able to activate transient receptor potential ankyrin 1 (trpa1) channels that are known to affect mapk cell signaling. mapk-pathways, especially perk1/2, are known to increase protein biosynthesis through activation of transcription factors binding to the serum response element (sre). it is unknown whether alkylating agents have also impact on mapk signaling mediated through trpa1 activation. our results demonstrate that aitc resulted in phosphorylation of the mapk perk1/2 and increased protein biosynthesis of sre-regulated genes in hek293 cells overexpressing htrpa1. cees increased perk1/2 levels already after 2.5 min which could be prevented by the trpa1 blocker ap18. activation of target genes through perk1/2 signaling was also evident, but less pronounced compared to aitc. our results demonstrate that alkylating agents have impact on cell signaling through trpa1 channel activation. thus, trpa1 might represent a promising target for counteracting sm toxicity. sulfur mustard (sm) is a chemical warfare agent that provokes severe inflammation and blistering upon exposure to the skin accompanied by disturbed wound healing. the potential use of sm in terroristic assaults amplified the interest in understanding the underlying cellular and molecular pathomechanisms in order to improve therapeutical intervention. autophagy is a highly conserved catabolic pathway in eukaryotes that ensures the degradation and recycling of cellular components through the lysosomal machinery. autophagy is important for cell survival in physiological and pathological stress situations. emerging knowledge indicates that imbalanced regulation of autophagy disturbs basal cell functions including proliferation, differentiation and migration, thus contributing to the pathophysiology of various diseases. after penetration into skin cells, sm alkylates and thereby modifies nucleic acids and proteins thus forming aggregates of dysfunctional proteins destined for autophagic disposal. in our studies, we analyzed the influence of sm on protein expression (western blotting) of autophagy-related (atg) genes as well as proliferation (wst-1) of primary normal human keratinocytes (nhek) and primary normal human dermal fibroblasts (nhdf). preliminary results demonstrate that sm strongly dysregulates the biosynthesis of atg proteins that may contribute to the diminished cell migration and proliferation under these conditions. our findings suggest that sm affects autophagy in correlation with an impairment of physiological functions in keratinocytes and fibroblasts that are essentially required for normal tissue regeneration. thus, application of pharmacological modulators of autophagy might be useful in the treatment of the delayed wound healing in skin upon exposure to sm. exposure of the respiratory tract to airborne particles is a major risk to human health. due to the ubiquitous application of these particles in the field of pharmacy, industry and in daily life, there is a strong necessity to investigate the toxic properties and the underlying pathomechanisms of these inhalable substances. in addition, the eu chemicals regulation requires not only that all substances placed on the market have to undergo a toxicological characterization, including the identification of potential toxic inhalation hazards (reach), but also that animal testing shall be undertaken only as a last resort ("3rs" principle) and the promotion of the development of alternative methods. thus, the development, establishment and validation of alternative in vitrobased test systems for the assessment of pulmonary toxicity are in the focus of current research. until now, most of the available in vitro cell culture models are limited to some extent as in those studies the exposure is either done under submerged conditions, not resembling the exposure conditions in vivo, or a homogeneous particle distribution is not guaranteed. the cultex ® radial flow system (rfs) is a specially designed in vitro modular exposure system that overcomes these limitations. it enables the homogenous exposure of human lung epithelial cells at the air-liquid interface (ali), thereby mimicking the physiological conditions of the alveolae. however, further optimizations are needed for the enhancement of the cultex® methodology. aim of this study was first the optimization of the test methodology in general (i.e. focus on clean air controls of the human lung epithelial cell line a549), and second the improvement of cultivation conditions. parameters such as handling of the cultex® device (proper closing and opening operation of the cultex® rfs module; improved washing conditions and media supply), treatment of the incubator controls, adjustment of clean air pressure and flow rates, and integration of two additional filters were sequentially adjusted in order to enhance the methodical setup. our results show that the test parameters for clean air exposure of the a549 cells were successfully optimized resulting in more accurate and robust data. cultivation conditions were improved by changing from closed-wall cell culture inserts to open-wall cell culture inserts. the openwall inserts turned out to be more suitable for exposure experiments as they provided a better medium supply and preserved humidity. deductive, the change of the cell culture inserts was identified as the deciding factor for the improvement of cell morphology. hence, we have successfully optimized the cultex ® rfs methodology for clean air exposure of a549 cells. human primary hepatocytes represent the gold standard in in vitro liver research. due to their low availability and high costs, alternative liver cell models with comparable morphological and biochemical characteristics have come into focus. the human hepatocarcinoma cell line hepg2 is often used as a model for liver toxicity studies. however, under two-dimensional (2d) cultivation conditions the expression of xenobiotic-metabolizing enzymes and typical liver markers is very low. cultivation for 21 days in a three-dimensional (3d) matrigel culture system has been reported to strongly increase the metabolic competency of hepg2 cells. in our present study we extended previous studies and compared hepg2 cell cultivation in three different 3d culture systems: collagen, matrigel and alvetex culture system. cell morphology, albumin secretion, cytochrome p450 monooxygenase (cyp) enzyme activities, as well as expression of xenobiotic-metabolizing and liver-specific enzymes were analyzed after 3, 7, 14, and 21 days of cultivation. our results show that the previously reported increase of metabolic competency of hepg2 cells is not primarily the result of 3d culture but a consequence of the duration of cultivation. hepg2 cells grown for 21 days in 2d monolayer exhibit comparable biochemical characteristics, cyp activities and gene expression patterns as all 3d culture systems used in our study. however, cyp activities did not reach the level of heparg cells. in conclusion, the increase of metabolic competence of the hepatocarcinoma cell line hepg2 is not due to 3d cultivation but rather a result of prolonged cultivation time. in vitro assessment of the neurotoxic potential of arsenolipids arsenolipids are organic, lipid-soluble arsenic compounds, which occur mainly in marine organisms. major human exposure routes are fatty fish including herring or fish oil-based food supplements. about 55 different arsenolipids have been identified so far. thereby, arsenic-containing hydrocarbons (ashc) and arsenic-containing fatty acids (asfa) represent two subgroups of the arsenolipids [1]. our in vitro studies have demonstrated high cellular bioavailability and a high cytotoxic potential of ashcs in human liver and bladder cells [2] , whereas asfas were less toxic [3] . a substantial transfer across an intestinal barrier model (caco-2) indicated that ashcs are highly intestinal available. in comparison, asfas showed lower intestinal bioavailability and underwent a presystemic metabolism [4] . moreover, in drosophila melanogaster ashcs exerted late developmental toxicity and accumulated in the fruit fly's brain. these results suggest that ashcs might pass the blood-brain-barrier due to their amphiphilic structure [5] . in order to assess the neurotoxic potential we currently investigate the toxicity of several arsenolipids in differentiated, human neurons (luhmes). after 48 h incubation with ashcs or asfas, cell number (hoechst) as well as cellular dehydrogenase activity (resazurin) were measured, with the latter endpoint turning out to be more sensitive. ashcs showed substantial cytotoxic effects (ic 50 ~ 7-12.5 µm) in a concentration range comparable to that of arsenite (ic 50 ~ 7.5 µm), whereas asfas were less cytotoxic (ic 50 > 100 µm). after incubation with ashcs the cellular arsenic concentrations increased 10-20fold as compared to incubation with arsenite. further studies indicated that one possible toxic mode of action of arsenolipids could be a disruption of the cellular energy level. therefore, the mitochondrial membrane potential was investigated after incubation with the arsenic compounds in differentiated neurons. whereas arsenite did not exert an impact, ashcs reduced the mitochondrial membrane potential significantly. this might be due to interactions of the amphiphilic ashcs with mitochondrial membranes. currently we investigate the impact of the arsenolipids on neurite outgrowth as a developmental toxicity endpoint. standard treatment of poisoning by organophosphorus compounds (op; e.g. nerve agents and pesticides) consists of co-administration of atropine and an oxime-based reactivator of inhibited cholinesterases. due to lack of efficacy of clinically used oximes against various op-inhibited human acetylcholinesterase (ache) (e.g. soman) research started focusing on new therapeutic approaches. several research groups conducted in silico screenings [1, 2] in order to identify new non-oxime reactivators, presenting amodiaquine as a promising candidate for paraoxon-inhibited hache. for decades, antimalarial drugs like amodiaquine and chloroquine have been closely investigated regarding their side effects, thereby discovering interaction with cholinesterases, which could pose a new potential therapeutic benefit for inhibited cholinesterases. therefore, in this study interactions between antimalarial agents in presence or absence of ops were examined spectrophotometrically by a modified ellman assay. reversible inhibition of cholinesterases was observed with both antimalarial agents. amodiaquine had higher inhibitory potency for hache than human butyrylcholinesterase (bche), being confirmed by ic 50 values of 0.67 ± 0.02 µm for hache and 81.28 ± 0.04 µm for hbche. ic 50 values with chloroquine were 28.37 ± 0.02 µm for hache and 55.62 ± 0.02 µm for hbche, thus representing a weaker inhibition of hache than amodiaquine. furthermore, reactivation of paraoxon-(pxe), sarin-(gb), cyclosarin-(gf), and vxinhibited hache and hbche by amodiaquine and chloroquine was determined. after 60 minutes, only paraoxon-inhibited hache (50%) and cyclosarin-inhibited hbche (10%) were reactivated by 500 µm chloroquine. on the contrary, 10 µm amodiaquine reactivated all tested ops after 60 minutes in the following order: pxe > vx > gf > gb. in contrast, with hbche the highest reactivation was generated with 100 µm amodiaquine in the following order: vx > gb > gf > pxe. due to the high reversible inhibitory potency of amodiaquine, an increased concentration does not result in a higher reactivation of op-inhibited hache. in summary, our results show that amodiaquine is a reactivator of op-inhibited cholinesterases. in the future, non-oxime reactivators that are structurally-related to amodiaquine should be further investigated. [1] bhattacharjee, a.k., marek, e., le, h.t., gordon, r.k., eur. j. med. chem., 2012, 49, 229-238. [2] katz, f.s., pecic, s., tran, t.h., trakht, i., schneider, l., zhu, z., ton-that, l., luzac, m., zlatanic, v., damera, s., macdonald, j., landry, d.w., tong, l., stojanovic, m.n., chembiochem., 2015 , 16, 2205 -2215 bundesinstitut für risikobewertung, lebensmittelsicherheit, berlin, germany development of mammary gland tumors is connected to a deregulation of breast epithelial cell differentiation, a complex process which cannot be reproduced in vitro under standard cell culture conditions. however, cultivation of cells in a tissue-like environment in an in vitro three dimensional (3d) model can mimic general architecture, function and differentiation of mammary bulks. in this project, a 3d model was used consisting of the permanent breast epithelial cell lines mcf10a (er -, estrogen receptor negative) and mcf12a (er + , estrogen receptor negative) grown in matrigel tm , which mimics the complex extracellular matrix in vivo. the 3d culture of mcf10a and mcf12a cells in matrigel tm results in the formation of growth-arrested, polarized spheroids with a lumen (acini-like organoids). in order to perform a semi-quantitative estimation on the influence of substances on the differentiation of the breast cells for the identification of non-genotoxic carcinogens a scoring method was developed. this scoring method provides information about substance-induced morphological changes of the spheroids during differentiation based on the following parameters: size of the spheroids, the formation of the lumen, and the degree of polarization. furthermore, the model allows distinguishing between erdependent (mcf12a) and er-independent (mcf10a and mcf12a) effects. the 3d in vitro model is a useful tool for toxicologists to study substance effects on differentiation processes. the system will be used to examine the potential of e.g. food contaminants such as phthalates or perfluorinated substances (pfas) to disrupt the differentiation process of breast epithelial cells and will therefore serve as a valuable in vitro tool to assess their carcinogenic potential. inflammatory episodes occur erratically throughout life and are likely to play a critical role in the alteration of the individual susceptibility of a person to idiosyncratic druginduced liver injury (idili), a particular severe form of drug-induced liver injury (dili). in concordance with the inflammatory stress hypothesis, modest inflammatory stress can lower the threshold for hepatotoxicity and make an individual susceptible to develop liver injury during exposure to therapeutic doses of a drug. in order to evaluate the role of immune cells and its secreted factors during drug therapy, we established an in vitro test battery consisting of two cell culture systems in presence or absence of proinflammatory factors (lps, tnfα): (a) the monoculture of human hepatoma (hepg2) cells and (b) co-culture systems of human monocytic or macrophage-like (thp-1) and hepg2 cells. with these different test settings we aimed to identify whether the introduction of inflammatory immune cells and/or pro-inflammatory factors could increase the sensitivity of liver cells towards idili compounds. three reference substance pairs were tested, namely troglitazone -rosiglitazone, trovafloxacin -levofloxacin, and diclofenac -acetylsalicylic acid, each of them being composed of a compound that is known to induce idili and a partner compound of the same substance class that does not induce idili. first, all compounds were tested for cytotoxicity towards the single cell systems using the wst-assay. co-culture experiments with hepg2 and thp-1 monocytes or macrophages as well as co-exposure experiments with lps or tnfα were then done at about 20% cytotoxicity of the respective substance in the most sensitive cell type. subsequently the results were compared to the experiments in the monoculture of hepg2. we observed that every idili compound showed a significant increase in cytotoxicity in a minimum of one exposure combination while this effect was not observed with the corresponding non-dili partner compound. in conclusion, a combination of different culture systems and co-exposures with proinflammatory factors is needed for a valid differentiation between non-dili and idili compounds. this test battery could provide a useful tool for the prediction of inflammation-associated idiosyncratic drug-induced hepatotoxicity. furthermore, our results support the inflammatory stress hypothesis and points to an involvement of proinflammatory factors in the development of idili. extensive animal models of carcinogenicity ensure a safe usage of chemicals. to elucidate fundamental molecular mechanisms of carcinogenicity these methods are expensive, time consuming and above all too complex. in contrast, most in vitro methods are rather simple and detect only selected endpoints, like dna damage, mutations or changes in proliferation. the balb/c cell transformation assay is a validated toxicological method to identify potential tumour initiators and promotors. first, balb/c mouse fibroblasts form a monolayer culture and get contact-inhibited after reaching confluence. upon treatment with a tumour initiator (3-methylcholanthrene) and promotor (12-o-tetradecanoylphorbol-13-acetate) transformed cells do not stop proliferation and grow as morphologically aberrant foci over the monolayer of normal cells. after fixation with methanol at day 42, morphological aberrant foci can be visualized with giemsa staining. because the balb/c assay mimics different stages of the malignant cell transformation process (initiation, promotion and post-promotion phase) and detects with the colony formation a late endpoint of carcinogenicity we improved this method for mechanistic cancer research. using the example of insulin-signalling pathway we can show that (1) several substances have a different impact on the transformation process, (2) it is possible to identify for each substance the phase with the greatest effectiveness and (3) we can detect additional endpoints to elucidate the mechanistic mode of action. therefore we used several compounds (linsitinib, metformin, rapamycin, …) to manipulate the insulin-signalling pathway on different levels (insr, ampk, mtor, …) and analysed a number of characteristic endpoints of carcinogenesis. changes on protein level and signalling (westernblot, immunofluorescence, flow cytometry) or parameters of energy metabolism (oxygen consumption, glucose or atp measurement) are measurable and enable new insights into the process of cancer origin. summing up, the balb/c 3t3 assay proves to be a cheap and short-time alternative to rodent bioassays. although this method does not mimic the whole in vivo neoplastic process, it can be used to provide essential information regarding key proteins and their signalling, during the different stages of transformation. is there hope to correctly classify severe ocular irritant agrochemical formulations using in vitro methods: a proof of concept using the isolated chicken eye test, two modified bcop protocols and an epiocular™ et50 protocol while some in vitro methods addressing ocular irritancy have gained regulatory acceptance, to date the draize rabbit eye test (oecd tg 405) is the only world-wide regulatory accepted test for the determination of the full range of eye irritation potential. further although several in vitro methods for the severe eye irritation have gained regulatory acceptance, agrochemical formulations are nor explicitly included nor excluded from the applicability domain to predict severe ocular irritant formulations. systematic analyses are only available for e.g. the hen's egg test-chorioallantoic membrane (het-cam), and bovine corneal opacity and permeability (bcop, oecd tg 437) assays both showing that the used protocols do not provide sufficient sensitivity to reliably predict severe ocular irritating formulations. the purpose of this study was to evaluate whether the regulatory accepted isolated chicken eye (ice, oecd tg 438) test including corneal histopathology (as suggested for evaluation of the depth of injury), as well two modified protocols of the bcop and/or an et50 (exposure time reducing viability of treated tissue to 50%) protocol using the reconstructed cornea model epiocular™ are useful to predict severe ocular irritant agrochemical formulations. a proof of concept comprising the testing of ten to twelve agrochemical formulations with available in vivo data in each assay was conducted. in summary, based on the ice evaluation described in oecd tg 438, one of the five severe ocular irritant formulations (un ghs cat 1) was predicted correctly. using both modified protocol versions of the bcop the result for one of the four tested un ghs cat 1 formulations was just above the un ghs cat 1 classification border for using one of the modified protocols. lastly and most promising, the epiocular™ et50 predicted four of five tested un ghs formulations correctly with the fifths being close to the classification border. additional agrochemical formulations will be tested to further evaluated the epiocular™ et50 protocol to identify severe ocular irritant agrochemical formulations. drug-induced pancreatic toxicity comprises effects on the exocrine and/or the endocrine pancreas, which both can have serious clinical implications, e.g. acute pancreatitis or diabetes mellitus. adverse effects on the pancreas are occasionally observed during drug discovery and development and often prohibit further development. hence, there is a need for reliable in vitro models to early on identify the pancreas-toxic potential of drug candidates. permanent cell lines and primary cells have many shortcomings, e.g. loss of cell-to-cell and cell-to-matrix relationships or changes in cell physiology due to the isolation procedure. pancreas tissue slices are a potential alternative, circumventing most of these limitations. their preparation is rather elaborate which may explain its rare use. so far, pancreas tissue slices have predominantly been used to address physiological or pharmacological questions, although they might also serve as valuable in vitro model for toxicological applications. therefore, this work aimed to establish and characterize rat pancreas tissue slices as in vitro model for studying drug-induced pancreatic toxicity. results will be compared to the responses of the permanent endocrine (ins-1e) and exocrine (ar42j) pancreatic cell lines to evaluate a potential added value. rat pancreas tissue slices were prepared by a protocol adapted from marciniak et al. (nat protoc, 2014 . 9(12): p. 2809 . briefly, pancreas was infused and embedded with agarose. tissue sections of app. 200 µm were prepared using a vibratome and maintained in cell culture medium for up to 6 days. cell viability was determined by daily measurement of lactate dehydrogenase (ldh) in medium supernatants and by microscopic evaluation following fixation in 10 % formalin and h&e staining. functional integrity of acinar and beta cells were assessed by cell-type specific secretory responses (i.e. insulin, amylase, lipase) to physiological stimuli. moreover, the effects of the pancreas toxins streptozotocin (stz), alloxan (all), and the cholecystokinin (cck) analogue cerulein on the viability and functional integrity of tissue slices were compared to the respective responses of the cell lines. we were able to establish an optimized isolation and cultivation procedure for rat pancreas tissue slices applying minor modifications to the original protocol. cell viability declined over the cultivation period. stimulation of the cell lines with glucose or cerulein increased secretion of insulin (ins-1e cells) or amylase/lipase (ar42j cells), respectively. the pancreas slices responded to both stimuli, demonstrating functional integrity of endocrine and exocrine cells. treatment of ins-1e islet cells with the betacell toxicants all or stz only slightly affected islet cell viability, whereas treatment of ar42j acinar cells with cerulein at supraphysiological concentrations had no effect. this set of experiments is currently completed by investigating the effects of all, stz and cerulein on the viability of acinar and islet cells in pancreas slices. our preliminary data demonstrate feasibility to prepare and cultivate rat pancreas tissue slices over a period of 6 days thereby maintaining functional integrity to some extent. coculture of human monocytes with the keratinocyte cell line hacat in serumcontaining medium leads to higher sensitivity to weak contact allergens: an improvement for the loose-fit coculture-based sensitization assay (lcsa) a. sonnenburg 1 , j. the loose-fit coculture-based sensitization assay (lcsa) has proved reliable for the in vitro detection of contact sensitizers in the past. however, the use of primary human keratinocytes has some disadvantages. to facilitate high throughput screening of chemicals, we replaced primary keratinocytes from the original assay setup (setup a) by the human keratinocyte cell line hacat. these cells were cocultured with monocytederived dendritic cells in serum-free medium (setup b) or fetal calf serum (fcs)containing medium (setup c). upregulation of the dendritic cell maturation marker cd86 assessed by flow cytometry served as endpoint. we have tested four substances known as sensitizers and four non-sensitizers in both new setups as well as in the original setup with primary cells. three out of four sensitizers (2,4-dinitrochlorobenzene, 2-mercaptobenzothiazole, and coumarin) , and three out of four non-sensitizers (glycerol, monochlorobenzene, and salicylic acid) were correctly assessed under all culture conditions. the weak sensitizing potency of resorcinol was only detected by setup b with fcs supplemented medium. a false positive reaction to caprylic (octanoic) acid in all three setups confirms earlier results from our laboratory that some fatty acids are able to induce cd86 on dendritic cells in vitro. culture in fcs supplemented medium led to generation of dendritic cells showing a more pronounced upregulation of cd86 after application of substances with rather high sensitization potency compared to dendritic cells which are formed under serum-free conditions. therefore, we characterized dendritic cells from setups b and c by flow cytometric measurement of additional dendritic cell surface markers. dendritic cells from the original setup a had been characterized extensively before (schreiner et al., toxicology 2008; 249:146-1529) . dendritic cells generated in fcs supplemented medium were cd1a+/cd1c+, whereas dendritic cells from serum free culture conditions were cd1a−/cd1c− regardless whether cocultured with primary human keratinocytes or hacat. populations with cd1a+/cd1c+ dendritic cells in coculture seem to show a higher sensitivity to weak sensitizers, which proved beneficial for the identification of resorcinol. in conclusion, modification of the lcsa protocol led to an increased sensitivity of the assay. due to ethical and social reasons, in vitro assays are being developed to replace animal tests for addressing e.g. toxicological questions. for the induction of skin sensitization by chemicals, resulting in tolerance or allergic contact dermatitis after repeated exposure, prerequisites are the induction of inflammatory responses in keratinocytes supporting maturation of dendritic cells (dc), which is needed for the t cell response. although related in vitro assays consisting of one single cell type have good hazard prediction capacities, they have limitations in predicting sensitization potency. one drawback could be the lack of communication between keratinocytes and dc. with respect to the activation of keratinocytes and maturation of dc, intercellular communication between these two cells may include the release of danger molecules such as cytokines, damage-associated molecules such as atp, and metabolized chemicals. beside this, microrna (mirna), among them those that can regulate dc activation or maturation, can be differentially expressed upon stimulation but can also be transferred between cells. for skin sensitizers, we reported already that cross talk between hacat keratinocytes and thp-1 cells, as model for dc, enhanced cyp1 enzyme activity in hacat cells exposed to benzo[a]pyrene (b[a]p) and eugenol, belonging to a subgroup of chemicals (prohaptens) whose sensitizing potential depend on prior metabolic activation e.g. via cytochrome p450 (cyp) enzymes. furthermore, coculture clearly increased the upregulation of the cell surface molecule cd86 on thp-1 cells after incubation with these prohaptens and also several other skin sensitizers. in this study we further elucidate the cross talk between thp-1 cells and hacat cells by analyzing the impact of hacat cells on the expression of mirnas in thp-1 cells by microarray technology. we identified 6 differentially expressed mirnas in cocultured thp-1 cells compared to monocultured thp-1 cells irrespective of the treatment (medium, 0.2% dmso as solvent control, b[a]p). in the presence of dmso and b[a]p (after 48h) 8 additional mirnas are differentially expressed. up to now it is not clear whether the cross talk between hacat and thp-1 cells comprises the exchange of mirna between the cocultured cells or whether it influences the expression of these mirna in thp-1 cells, or both. given that one mirna has several gene targets these results illustrate that the cross talk between thp-1 and hacat cells also impacts on the mirnome. walther-straub-institut der lmu-münchen, münchen, germany transient receptor potential (trp) proteins represent a large superfamily of nonselective cation channels sensing toxic stimuli in the human body. trpa1 expresses a high number of aminoterminal ankyrin repeats and is the only member of the trpa family. channel monomers form homotetramers in the plasma membrane with six transmembrane segments (tm) and a pore forming loop between tm5 and 6. trpa1 has been extensively described in sensory nerve endings as an important cellular detector for toxic stimuli and as an oxygen sensor (reviewed in 1). although recently two reports identified trpa1 in pulmonary epithelial and endothelial cells (2, 3) , its expression in non-neuronal tissues is still a matter of debate. after isolation and identification of different murine lung cells we were able to identify murine trpa1 protein in primary endothelial cells, pneumocytes type ii (atii) and fibroblasts by using specific antibodies in a western blot analysis, but not in cells from trpa1-deficient mice. atii cells were identified by specific cell markers such as surfactant protein c and were further differentiated to ati cells characterized by their specific expression of podoplanin. quantitative trp expression patterns will now be evaluated by quantitative reverse transcription (rt)-pcr as well as utilizing nanostring ® technology in different lung cells. to characterize trpa1 on a cellular level we cultured a hek293 cell line stably expressing trpa1 (4) . allylisothiocyanate (aitc) a specific activator as well as hypoxia and hyperoxia was able to induce ca 2+ -influx in this cell line, which was blocked by the specific inhibitor a96079. in the future, we will utilize the isolated perfused lung model (5) to quantify toxin-induced edema formation in ex vivo lungs from wt and trpa1-deficient mice after exposure to potential toxic inhalation hazards (tih see 6) to challenge the hypothesis of trpa1 as an important toxin sensor in the lung. by this strategy we hope to understand trpa1 function in lung cells and to evaluate trpa1 proteins as potential pharmacological targets for a specific therapeutic intervention during toxin-induced edema formation. metabolism by the intestinal microbiota is likely to contribute essentially to the plasma metabolite profile of the mammalian host organism and it requires adequate identification of effects of the microbiome on the endogenous plasma metabolite patterns. the current investigations present insights in the mammalian-microbiome cometabolism of endogenous metabolites. antibiotics have a profound effect on the micro-organism composition of the microbiome and hence on the mammalian-microbiome co-metabolism. the consequences, however, on the functionality of the microbiome (defined as the production of metabolites absorbed by the host) and which of these changes are related to the microbiome are not well understood. to identify plasma metabolites related to microbiome changes due to antibiotic treatment, we have employed a metabolomics approach. to this purpose broadspectrum antibiotics belonging to the class of aminoglycosides (streptomycin, neomycin, gentamicin), fluoroquinolones (moxifloxacin, levofloxacin) and tetracyclines (doxycycline, tetracycline) were administered orally for 28 days to male rats including blood sampling for metabolic profiling after 7, 14 and 28 days. fluoroquinolones and tetracyclines can be absorbed from the gut whereas aminoglycosides cannot. to distinguish between metabolite changes caused by systemic toxicity of the antibiotics and microbiome related changes, the metabolites identified in the metabolome pattern were compared to a list of metabolites known to be produced by the gastro-intestinal micro-organisms. beside changes mainly concerning amino acids and carbohydrates, hippuric acid and indole-3-acetic acid were identified as key metabolites being affected by antibiotic treatment. for each class the following gut metabolites were found to be unique: indole-3-propionic acid for aminoglycosides, taurine for fluoroquinolones, 3indoxylsulfate, uracil and allantoin for tetracyclines. for each class of antibiotics specific and selective metabolome patterns could be established. the results suggest that plasma based metabolic profiling (metabolomics) could be a suitable tool to investigate the effect of antibiotics on the functionality of the microbiome and to obtain insight in the mammalian-microbiome co-metabolism of endogenous metabolites. drug-induced liver injury (dili) is still a major reason for termination of clinical trials and thus is an important concern in drug development. identification and prediction of dili in the clinic and in preclinical safety testing still relies on the classical clinical chemistry panel and histopathology with known limitations in sensitivity and specificity. in the last years bile acids (bas) have been studied as potential biomarkers to better characterize drug-induced liver injury with promising results (ellinger-ziegelbauer et al., 2011; luo, schomaker, houle, aubrecht, & colangelo, 2014; yamazaki et al., 2013) . to evaluate whether a targeted bile acid profiling via lc-ms/ms in plasma and liver tissue can improve assessment of liver injury, methapyrilene (mpy) a known hepatotoxin, or corresponding vehicle, was administered daily to male wistar rats at a low (30 mg/kg) and a high (80 mg/kg) dose. rats were sacrificed following 3, 7, or 14 consecutive daily doses, or after recovery 10 days following 14 consecutive administrations of mpy or vehicle. in addition to bile acids which were determined both in plasma and tissue, conventional preclinical safety endpoints (histopathology and clinical chemistry) assessment and gene expression profiling was performed in liver to obtain mechanistic information about potential changes in regulation of bile acid levels. conventional findings included periportal necrosis, inflammation and biliary hyperplasia, and increased liver enzyme activity and bilirubin levels during the treatment phase. the bile acid pattern showed increased levels of conjugated and unconjugated bile acids in low dose and high dose groups compared to the controls after administration of methapyrilene. furthermore, although liver enzyme activity and bilirubin levels in serum were decreased again in the recovery groups, suggesting recovering liver injury, bile acid concentrations remained elevated with no signs of recovery. analysis of transcriptomics data revealed decreased levels of mrna encoding α-methylacyl-coa racemase (amacr) 4 and 15 days after dosing, a gene responsible for bile acid synthesis. membrane transport systems for bile acids like sodium/taurocholate co-transporting polypeptide (ntcp) and organic anion transporting polypeptide 1 (oatp1) expression were down regulated as well, indicating that the increased bile acid concentrations in plasma and tissue could be attributable to reduced uptake by the hepatocyte. in summary the data suggest that targeted bile acid profiling could be used as potential biomarkers to enhance assessment of drug-induced liver injury. photorhabdus asymbiotica is an entomopathogen and emerging human pathogen causing soft tissue infections in humans. photorhabdus asymbiotica produces the bacterial protein toxin patox, which is cytotoxic for various cell lines and kills insect larvae. previous studies have established that patox harbors two enzymatic active domains, a glycosyltransferase and a deamidase domain. the glycosyltransferase domain inactivates host gtpases of the rho family by glcnacylation of a tyrosine residue in the effector binding loop, which results in the disassembly of the actin cytoskeleton. the deamidase domain deamidates a crucial glutamine residue in heterotrimeric gα i and gα q/11 proteins, which renders the g proteins constitutive active. sequence and structural homology analyses of patox revealed a third domain (patox p ) resembling peptidases of the c58 protease family. patox p contains the conserved catalytic triade (c/h/d) of papain-like cysteine proteases and shares sequence similarity with effectors from yersinia pestis (yersinia outer protein yopt) and pseudomonas syringae (avirulence protein avrpphb). transient expression of patox p in hela cells induces cell rounding and indicates a cytotoxic potential of patox p . incubation of patox p with linearized bovine serum albumin (bsa) results in cleavage products of bsa assuming proteolytic activity of patox p . mutation of the catalytic cysteine in patox p prevents cleavage of bsa and blocks cytotoxicity. we were not able to observe autocatalytic cleavage of patox constructs under various conditions. the intracellular activity of the protease domain is most likely involved in the pathogenicity of patox. vitamin d metabolism -involved in triazole fungicide toxicity? a. lehmann 1 1 background: in a 28-day rat feeding study with the azole fungicides cyproconazole (c), epoxiconazole (e), propiconazole (p), tebuconazole (t), prochloraz (pz) as well as combinations c+e and c+e+pz, a reduction of vitamin d (vitd) receptor mrna levels was reproducibly observed in adrenals for c, e and p. transcription of various enzymes related to vitd homeostasis (including cyp2r1, gc, cyp3a, ugt1a) in liver was also affected, while initial indications for modulation of renal cyp24a1 and renal and hepatic cyp27b1 could not be confirmed. a possible induction of parathormone (pth) was noted for the high dose of c, but statistical significance could not be shown. we have now performed supporting analyses for serum vitd levels, measured additional transcript levels and will provide a framework for the interpretation of the findings. methods: male wistar rats (n=5 for single substances, n=10 for combinations) were treated for 28 days at dose levels tested based on noaels from 90-day subchronic feeding studies and ranged from noael/100 to noaelx10. quantitative rt-pcr analyses were performed on organ samples obtained at sacrifice. serum vitd levels were determined using the total (25-oh) vitamin d elisa (drg instruments gmbh, marburg, germany). results: the elisa established for diagnostic analysis of human serum and plasma samples could be applied to rat serum. vitd levels in control animals (n=30) were 64.7±8.3 ng/ml (min/max: 48/78 ng/ml), i.e. in the range of values reported previously for rats. for the high dose of c (1000 ppm in food, n=5), there was a statistically nonsignificant reduction of vitd levels to 71.3±17.6% of the concurrent control (n=5). however, for 4 of 5 animals of this group, measured vitd level were below the range observed in pooled controls (n=30). an according follow-up is ongoing. qrt-pcr analysis of adrenal tissue showed deregulation of apoptosis related genes (p21 for c, e and pz; cdk1 and gadd45a for e; cdkn1c for c), which is in agreement with an involvement of vitd in the autocrine/paracrine regulation of cell proliferation. conclusion: reduction of circulating vitd levels would be plausible as a result of induction of hepatic cyp3a1/2 and ugt1a. however, this could not be confirmed by elisa as a general mechanism for all azole fungicides under investigation. only for rats fed with 1000ppm cyproconazole, there were indications for a moderate reduction of 25-oh vitamin d, which would correlate with the previously reported moderate increase in serum pth for this group. hansen's disease during pregnancy and lactation: two babies born to a mother using antileprosy drugs z. ozturk hansen's disease, also known as leprosy, during pregnancy has been rarely reported in europe and united states. early diagnosis is important, and medication can decrease the risk of those living with leprosy patients from acquiring the disease. this report presents a case of multidrug antileprosy therapy during pregnancy and lactation. a 26-year-old multiparous woman with a known case of multibacillary leprosy presented with unplanned pregnancy. her pregnancy was discovered in the 9th week, and she has been taking a multidrug therapy (dapsone 100 mg/day, rifampicin 600 mg/month, clofazimine 50 mg /day and clofazimine 300 mg/month) for the past 8 months. diagnosis of leprosy was established in her previous pregnancy. the patient was informed about the risks of drugs used in pregnancy. the treatment was continued unchanged during pregnancy. a detailed fetal ultrasonography was offered to scan the development of the fetus at about 20 weeks. in the 8th, 22nd, 28th weeks of pregnancy, prenatal sonographic examinations revealed normal fetal growth and amniotic fluid volume. at 28 weeks pregnant, she was diagnosed with gestational diabetes. diabetes did not cause any symptoms during pregnancy, and it was controlled with a reduced-calorie diet in a week. the patient delivered a healthy baby girl by vaginal birth in the 39th week of gestation without perinatal complications. the baby was also healthy (apgar 8-9,3300 g,51 cm), and its growth and development were normal during a 6-month follow-up period. the patient decided to breastfeed while taking medication. she had a previous experience with use of anti-leprosy drugs while breastfeeding, her other child was 15 months old and healthy. as well as in the first child, skin discoloration was observed in newborn due to clofazimine during lactation. after 3 months, she stopped breastfeeding, and the infant's skin changes were reversed. for pregnant women and practitioners, treatment of leprosy in pregnancy can be complicated. physical and neurological damage may be irreversible even if cured. multidrug therapy consisting dapsone, rifampicin and clofazimine is highly effective for people with leprosy and considered safe, both for the mother and the child. antileprosy drugs are excreted into human milk but there is no report of adverse effects except for skin discolouration of the infant due to clofazimine. therefore, multidrug therapy for leprosy patients should be continued unchanged during pregnancy and lactation. methods: individuals included in the analysis were participants of the berlin initiative study (bis). bis is a population-based prospective cohort study initiated in 2009 in berlin, germany, to evaluate kidney function in people ≥ 70 years. medication was assessed through personal interviews and coded using the anatomical therapeutic chemical classification system. for estimation of glomerular filtration rate (egfr) we used the ckd-epicr equation. predictor analysis was conducted via logistic regression. results: figure 1 illustrates the percentage of drug use for the three noacs and phenprocoumon, the most common vitamin k antagonist in germany, over the course of four years. table 1 shows the characteristics of patients for each oral anticoagulant group during the four-year follow-up visit (from january 2014 until april 2015). the probability of dabigatran use rose with increasing age (+12%), and the probability of phenprocoumon use rose in case of egfr < 60 ml/min/1.73m 2 (+54%) or male sex (+82%). discussion: our data show that also in the elderly noac use increased over the past years. characteristics such as age, sex or kidney function had an impact on the choice of oral anticoagulation. objective: orthostatic hypotension (oh) is an important factor in determining cardiovascular mortality especially in older age. different factors were discussed to influence oh. arterial stiffness, medication and frailty were demonstrated as modifying factors of oh. the aim of this study was to assess prevalence of and influencing factors on oh in nursing home residents (nhr) in germany. methods: systolic (sbp) and diastolic (dbp) blood pressure as well as pulse pressure (pp) and pulse wave velocity (pwv) as markers of arterial stiffness were measured in nhr aged ≥ 65 years in 12 nursing homes in berlin, germany. measurements were first performed in the sitting position and then repeated after standing up. oh was defined as a sbp decrease of > 20 mmhg and/or dbp decrease of > 10 mmhg within 3 min after standing up. hypertension was defined as the presence of diagnosis arterial hypertension, the prescription of at least one antihypertensive drug, or mean sbp values > 139 mmhg and/or mean dbp >89 mmhg. information about antihypertensive medication was received from interviews and medical records. frailty was determined by geriatric assessments, e.g. "timed up and go test" (tug) or barthel scale. results: oh testing could be performed with 96 nhr (mean age = 84.5 ± 7.3 years). in total, 15 subjects (15.6%) had oh. the mean change in sbp from sitting to standing was 19.2 ± 15 mmhg (range +8.5 to -52.5 mmhg) in patients with oh and 1.5 ±10.9 mmhg (range +44.5 to -17 mmhg) in patients without oh. mean sbp was significantly higher (143.6 ± 17.1 mmhg) in people with oh than in those without (131.5 ± 20.1mmhg). all of the nhr with oh were hypertensive compared to 89% of the nhr without oh. sex, mean age, pwv and pp was not significantly different between individuals with or without oh (p>0.05). medication data was available for 89 patients. all individuals with oh and 60 nhr without oh (80%) had antihypertensive medication. more than 2 different antihypertensive drugs were present in 11 patients with oh (78.5%) and in 43 patients without oh (57.3%). the intake of beta-blockers had no impact on oh development. geriatric assessments did not differ significantly between the oh group and the non-oh group. more than 75% of patients in both groups reached 80 points as maximum in barthel scale defining a need for assistance and tug analyses demonstrated that around 50% of patients with oh as well as patients without oh needed more than 19 sec showing a motor slowing. conclusion: we found a relatively low prevalence of oh in our very old patient cohort and the overall bp control was good. similar to earlier publications mean sbp was significantly higher in nhr with oh. all of the other investigated factors were not associated with the occurrence of oh. the small cohort size might have limited the detection of cardiovascular, epidemiological or geriatric associations. in addition, important confounding factors such as the inability to stand of some nhr and the lack of standardized fraility assessments must be addressed. impact of reticulated platelets on the initial antiplatelet response to thienopyridine loading in patients undergoing elective coronary intervention c. stratz 1 , t. nuehrenberg are known to be involved in cell metabolism pathways and therefore ccrcc is supposed to be a metabolic disease. in order to facilitate a better understanding of cancer metabolism and to support tumor classification on the metabolite level we have developed a novel analytical approach for comprehensive metabolomic profiling of small molecules and lipids in kidney tissue. the method was established and validated based on porcine tissue and, as proof of concept, applied to a small cohort of human normal and ccrcc tissue samples for molecular tissue differentiation. methods: five fresh frozen ccrcc samples and corresponding normal tissue were used for cancer-specific metabolomic profiling and were derived from patients who underwent partial or radical nephrectomy. metabolites and lipids were recovered from tissue samples by a two-step extraction protocol. tissue homogenization and extraction of polar metabolites was performed in methanol/water (aqueous extract) by a beadbeating approach. lipids were recovered by consecutive extraction of the pellet with methanol/methyl tert-butyl ether (organic extract). metabolites in aqueous extracts were separated by hydrophilic liquid interaction chromatography whereas compounds in organic extracts were separated by reversed phase chromatography prior high resolution mass spectrometry. results: reproducibility of tissue extraction and metabolite analysis was assessed by the analysis of multiple individually prepared porcine kidney samples. more than 1000 metabolic features including amino acids, nucleotides, small organic acids, phospholipids, sphingolipids, glycerolipids and fatty acids could be reproducible (cv ≤ 30 %) analyzed with the novel non-targeted metabolomics approach. the validated protocol was applied for metabolomic profiling of kidney tissue derived from ccrcc patients. based on unsupervised multivariate statistics, a clear differentiation between cancerous and normal tissue for the small metabolites profile as well as for the lipid profile could be observed. a first subset of differentially regulated metabolites responsible for tissue differentiation could be tentatively identified. conclusion: metabolomic profiling of kidney tissue extracts enables differentiation between ccrcc and normal kidney tissue samples based on the lipid and small molecule metabolomic profiles. further studies on larger and independent sample groups are necessary to confirm and validate our preliminary findings. in summary, the presented approach provides a first basis for comprehensive metabolomics studies in human kidney tissue and thus offers great potential for the metabolic characterization of ccrcc with important prognostic and therapeutic implications in the future. introduction: clomiphene (clom) citrate as mixture of trans-and cis-isomer (60:40) is the first line therapy for the treatment of infertility caused by the polycystic ovary syndrome. treatment schedule includes dose escalation from 50 mg/d clom citrate to up to 150 mg/d in case of non-ovulation. however, therapy outcome is variable and approximately 10 -30% of patients do not benefit from clom treatment. the pro-drug clom is bioactivated via 4-hydroxylation of trans-clom by the highly polymorphic cytochrome p450 (cyp) 2d6 leading to the major active metabolite trans-4hydroxyclomiphene (trans-4-oh-clom) [1] . recently, we identified a less active trans-3-oh-clom which is also formed by cyp2d6. besides the formation of the active metabolites, their plasma concentrations are influenced by their clearance e.g. via glucuronidation and sulfation. here we investigated the glucuronidation and sulfation of both hydroxyl-metabolites. methods: isoforms of udp-glucuronosyl-transferase (ugt) and sulfotransferase (sult) responsible for conjugation of oh-clom were identified using commercially available supersomes. glucuronidation and sulfation kinetics were determined in pooled human liver microsomes. conjugated clom metabolites were quantified in plasma and urine samples obtained from healthy female volunteers who received a single dose of 100 mg clom citrate. results: incubations with human liver microsomes revealed an almost 60-fold higher glucuronidation rate for trans-3-oh-clom, which is exclusively catalyzed by ugt2b7, compared to the more potent trans-4-oh-clom. for the latter a pattern of multiple ugts was identified. in contrast, the intrinsic clearance of trans-4-oh-clom to its sulfate is 16-fold higher compared to 3-oh-clom. for both metabolites a participation of sult1a1 and sult1e1 was identified. these results were in line with previous studies, which identified the same sults [2] and ugts [3] responsible for the conjugation of the structurally related trans-4-hydroxytamoxifen. in addition, in vivo data from plasma and urine samples confirmed the reverse regioselective glucuronidation and sulfation of trans-3-oh-clom and trans-4-oh-clom. overall, concentrations of clomglucuronides were significantly higher than those of sulfates. highest concentrations in plasma and urine samples were measured for trans-clom-3-o-glucuronide. conclusion: our results suggest a new metabolic route via trans-3-oh-clom which appears to be a potential inactivation pathway of clom. 1 1 institut für pharmakologie und toxikologie der bundeswehr, münchen, germany for decades the biological effect of sm has been investigated. it is well known how sm interacts and destroys cells. unfortunately, it is still unknown if and how a cell can become resistant against sm. within the here described experiments we investigated a new approach adapting cells to the presence of sm. over a time period of nearly three years the cells were cultivated in presence of sm with increasing concentrations. before starting the initial sm sensitivity was investigated. at the beginning cells were cultivated with a concentration of 0.07 µm sm (ic 10 ). today the cells are able to tolerate a concentration of 7.2 µm sm (ic 90 ), which reflects to a concentration of which 90% of the original cells would have died. to determine cellular characteristics, the resistant cells were compared with wildtype cells. the following cell characteristics were investigated: proliferation, apoptosis, clonogenicity, size of nuclei and cytoplasm, cell-cell contacts, dna adducts formation, secretome, screening of mirna expression, next generation sequencing, vital observation and scratch assay, nad(p) + /nad(p)h, h 2 o 2 , glutathione, ca 2+ -influx, mdrchannels, resistance to other alkylating agents and the reversibility of the resistance. the resistant cells demonstrate smaller nuclei and cytoplasm, less dna adducts, a higher clonogenicity as well as proliferation and less apoptosis. the secretome analysis showed an up-regulation of anti-apoptotic acting cytokines timp and ang and the proproliferative acting cytokines timp and pdgf-aa. in contrast, immunologically active cytokines were down-regulated. concerning cell-cell contacts no differences were seen. in the mirna screening 49 significant up-regulated and 20 significant down-regulated mirnas have been observed. noteworthy was the regulation of various members of 11 different families. during vital observation and in a scratch-assay the resistant cells were show to have disadvantages. the observed resistance was not unique for sm but also towards other alkylating agents and cytostatic drugs. by analyzing the reversibility cells stayed resistant over more than 35 weeks. in conclusion, many aspects investigated in this study have an influence on the sm resistance, pointing out that it is a combination of various effects that are involved to switch on resistance. more likely, there are many aspects working together. the present results are an important step in the characterization of the sm-resistant cell line and further studies may be able to directly use these as a start for target identification in antidote or prophylactic agent discovery. the arylhydrocarbon receptor (ahr) is localized in a cytosolic complex that contains several co-chaperones and associated factors. the protein is shifted into the nucleus in response to endogenous and xenobiotic ligands. however, a transient nuclear transport does also occur in the absence of any ligands, while the predominant cytoplasmic compartmentalization is maintained by parallel export. we have analyzed the interplay between this basal nucleo-cytoplasmic shuttling and ligand induced transport in hepg2 cells, using a yfp-tagged fusion protein that is capable to respond to ligands and to trigger the induction of cyp1a1 expression. basal import was assessed in cells that had been treated with leptomycin b (lmb), an inhibitor of crm1-mediated nuclear export. interestingly, the apparent ahr import rate in lmb-treated cells was comparable with nuclear import as trigged by xenobiotic (b-naphthoflavone) or endogenous (kynurenine) ligands. this observation was confirmed for endogenous ahr in hepg2 cells, since both ligands and lmb showed comparable effects on nuclear compartmentalization. however, the basal nuclear import rate in lmb-treated cells was strongly increased by ahr ligands. ligand-induced nuclear transport was therefore confirmed as an import step in receptor activation. interestingly, lmb did also accelerate nuclear import of ahr after pretreatment of cells with ahr ligands. these data suggest that nuclear export of the ahr is maintained in the presence of ligands. receptor activation might therefore comprise several rounds of shuttling, thereby involving both accelerated import and continued export of the ahr protein fraction that has not already undergone interactions with arnt or dna. we suggest that nuclear export provides an additional kinetic control of ahr activation and function. mitochondrial toxicology: rescuing mitochondria in wilson disease avoids acute liver failure h. zischka 1 1 institut für molekulare toxikologie und pharmakologie, ag zischka, neuherberg, germany in wilson disease (wd) functional loss mutations in the hepatocyte atp7b gene cause dramatic copper overload leading to acute liver failure, posing an unmet therapeutic issue. we find that the pathology of severe wd cases is mirrored in lpp (-/-) rats carrying a functional loss atp7b mutation. this is especially apparent in the hepatocyte mitochondrial compartment. a progressive copper deposition increasingly harms the lifesustaining mitochondrial membrane integrity. thus, depleting this devastating mitochondrial copper burden is a core requirement for a treatment strategy against acute liver failure in this wd animal model. preparation for the master degree program in toxicology started in 2006 as a cooperation of charité universitätsmedizin berlin with the university of potsdam and other institutions of the region. first enrollment of students was done in 2008. the program was accredited in 2011 by the central evaluation and accreditation agency. it offers a modern curriculum encompassing a wide variety of scientific aspects with an interdisciplinary character. this training program in toxicology is organized in modules and ends with the degree "master of science" (m.sc.). the goal of this program in toxicology is to teach the basis of the interactions between substances at toxic concentrations and living organisms, as well as the molecular mechanism of the adverse effects of chemicals. the understanding of the mechanism of a toxic action is an important prerequisite for the scientifically based evaluation of a hazard associated with a substance. furthermore, only with the knowledge of the mechanism of action and a deduction of structure activity relationships it is possible to predict toxic effects of new substances. this knowledge should enable students to perform a risk evaluation of chemicals or to predict the adverse effects of chemicals with the aim that human beings and the environment can be protected from harmful consequences of chemical exposure. the program allocates 30 places per year to an average of 60 applicants. most applicants have a basic training in the fields biology, chemistry, pharmacy, veterinary medicine and nutritional sciences. about 75% of the students are female. the majority of them have a bachelor's degree before starting the master program, other degrees are diploma and state examination as pharmacists or physicians. ninety percent of the students pass the final examination consisting of the master's thesis and disputation at the end of the four semesters. afterwards, most of the graduates aim to obtain a phd degree. the program is well established in the education of toxicologists in germany. respiratory injury due to chlorine developed from consumer products. still an issue in germany u. stedtler 1 , m. hermanns-clausen 1 1 uniklinikum freiburg, vergiftungs-informations-zentrale, freiburg, germany objective: in the last decades strong effords have been took to improve product safety, especially in products intended for domestic use. hypochlorite-containing cleaners may develop chlorine gas when acidified e.g. by adding an acid sanitary cleaner. usually these cleaners contain sodium hydroxide or other strong alkalines to avoid this reaction. we analysed reports to our poisons center concerning inhalation exposure to chlorine developed from hypochlorite-containing mixtures. method: retrospective search in the case database of the poisons center. human inhalative exposures to chlorine released from mixing hypochlorite as well as human inhalative hypochlorite exposure alone were analysed. frequency and symptoms were compared. results: from 2010 to 2015 in total 85 cases of human exposures to chlorine developed from mixtures of hypochlorite and acids (0.8 of 1000 cases) were registered. in 55 cases the exposure was due to mixtures of products intended for domestic use. 94 % of the exposed patients reported symptoms. only in two cases the symptoms were not considered to be caused by the inhalation accident. most frequent symptoms reported were (percent of symptomatic patients): cough (45 %), dyspnea ( 33 %), irritated upper airway (26 %), abdominal discomfort (pain, nausea, vomiting) (21 %), thoracic pain (20 %), irritated eyes (11 %), dizziness (8%), and bronchospasm (6 %). further symptoms were malaise, headache, irritated nose, sweating, muscle pain, and others. in 12 patients (14 %) the symptoms were graded as moderate severe. main symptoms in this group were dyspnoea ( 83% ), cough, and irritated airway. one third of the patients experienced bronchial obstruction. all symptomatic patients developed symptoms while exposed or shortly after exposure. there were no severe or fatal cases (especially no lung edema) and all symptoms were expected to resolve completely. because hypochlorite containing procucts sontanously release "chlorine-like" smelling gases, we additionally analysed inhalation exposures to hypochlorite solutions alone in the same period. there were 42 patients in the same period exposed to hypochlorite evaporation alone. 36 of them (86 %) had symptoms of which in 30 cases these were considered to be caused or possibly be caused by the hypochlorite. most frequent symptoms were irritated upper airway (33 %), nausea or vomiting (30 %), cough (23 %), irritated eyes (20 %). dyspneoa was less fequent than in the mixture group (10 %). all symptoms were considered mild. there was no bronchospasm or thoracic discomfort. conclusion: respiratory injuries by chlorine from hypochlorite-containing solutions still occur despite clear warning on the label. the majority of cases was due to products for domestic use. symptoms develop shortly after exposure. the γh2ax assay for genotoxic and nongenotoxic agents: comparison of h2ax phosphorylation with cell death response perturbation of mitosis through inhibition of histone acetyltransferases: the key to ochratoxin a toxicity and carcinogenicity? regulation of chromatin by histone modifications transcriptomic alterations induced by ochratoxin a in rat and human renal proximal tubular in vitro models and comparison to a rat in vivo model in vitro gene expression data supporting a dna non-reactive genotoxic mechanism for ochratoxin a fragment ion patchwork quantification for measuring site-specific acetylation degrees combinatorial patterns of histone acetylations and methylations in the human genome inroads to predict in vivo toxicology -an introduction to the etox project value of shared preclinical safety studies -the etox database acknowledgements: support of the bfr through grant 1322-530 is gratefully acknowledged. acknowledgement: supported by the robert bosch foundation, stuttgart, germany. [1] mürdter t, et al. hum mol genet, 2011, 21:1145-54 [2] nishiyama t. et al., biochemical pharmacology, 2002, 63:1817 -1830 [3] sun d. et al., drug metabolism and disposition, 2007, 35:2006 background: infections are a major problem in patients with burn diseases (bd). due to severe injuries of their total body surface area (tbsa), burn patients have altered pharmacokinetic characteristics. therefore, insufficient plasma concentrations may be achieved, when standard dosing schedules are applied for antibiotics such as piperacillin. for time-dependent antibiotics, the duration how long drug concentration exceeds the minimal inhibition concentration (mic) is crucial for their antibacterial effects. since pseudomonas spp. is the main problematic pathophysiological bacterium for bd patients. the aim of the present study was to monitor the plasma concentrations of piperacillin during piperacillin/tazobactam treatment in bd patients. patients from intensive care units (icu) served as controls. methods: 10 bd patients (5/5 m/f, 43.4±5.3y, tbsa 40 .9±5.9%) and 5 patients (74.4±3.9y) from the icu were included in this observational study. blood samples were taken within the 3 rd interval of the 8h-lasting dosing period of piperacillin/tazobactam (4/0.5g within 0.5h) at 1, 4 and 7.5h after the end of infusion. total and free piperacillin concentrations were determined in plasma using hplc-uv after deproteinisation with acetonitrile and by ultrafiltration, respectively. pharmacokinetic parameters and dosing simulations were calculated by tdmx (www.tdmx.eu). free plasma concentrations of piperacillin exceeding at least 1xmic but preferably 4xmic over the whole dosing interval were considered to be sufficient for antibiotic efficacy (mic 16 mg/l for pseudomonas spp.,www.eucast.org). results: the pharmacokinetic parameters of total piperacillin, calculated for each bd or icu patient using the concentrations at 1, 4, and 7.5h, were as follows: c max 69.6±7.9 vs.116.3±10.4 mg/l, p<0.05, half-life 1.8±0.3 vs. 2.3±0.3h, p>0.05, clearance 12.6±1.9 vs. 6.5±0.4 l/h, p<0.05, volume of distribution 27.8±2.0 vs. 20.9±1.3l, p<0.05. free concentrations (which were included in tdmx calculations) were 87±2 vs. 81±2% (p<0.05) of total concentrations. duration per day while concentrations exceeded 1xmic (15.6±1.9 vs. 22.0±1.1h, p<0.05) or 4xmic (5.4±1.3 vs. 9 .4±0.7h, p<0.05) were lower in bd than in icu patients. moreover, tdmx simulations predicted that the duration per day for 4xmic could be enhanced to 16.2±1.5h if the piperacillin amount will be increased to 4x8 g/d and the infusion duration to 3h. pharmacokinetic parameters have, however, to be determined in a pilot study with bd patients to ensure predicted values. conclusions: standard dosage regimens for piperacillin/tazobactam could result in suboptimal plasma concentrations of piperacillin in bd patients as well as in icu patients. drug monitoring and tdmx simulation of kinetic parameters may easily help to improve piperacillin treatment in bd patients. background: high dose methotrexate (hd mtx), defined as >1000mg mtx/m 2 bodysurface-area (bsa) is used in children to treat a variety of malignant diseases since the 1950s. clinicians observe relevant rates of severe unwanted side effects. identifying patients having an increased risk for toxicity due to altered mtx pharmacokinetics is urgently needed. we aim to develop and evaluate a physiology-based pharmacokinetic (pbpk) model for hd mtx in children using pk-sim® (bayer technology services gmbh, leverkusen, germany) with a special emphasize on relevant covariates. methods: in this non-interventional observational study, children receiving hd mtx intravenously at two major german pediatric oncology departments during the years 2004-2009 were included if at least one mtx serum level (mtx-sl) was determined during clinical routine. 29 patients aged 2-18 years (male = 19, female = 10) with following diagnoses were included: acute lymphoblastic leukemia, non-hodgkin lymphoma, burkitt lymphoma, brain stem glioma and glioblastoma multiforme. in total, 103 mtx treatment cycles corresponding to 300 mtx-sl were used in this study. patients were randomized into two patient sets (training set and test set). based on literature data, mtx pbpk-models were developed and slightly adapted taking into account mean relative deviation (mrd) and bias of predicted versus observed mtx-sl of the training set. the pbpk model with the lowest mrd and bias was chosen and finally evaluated using the test set. the impact of the covariates urine ph <6.5, trimethoprime/sulfamethoxazole, proton-pump-inhibitors, non-steroidal anti-inflammatory drugs and ß-lactam antibiotics on the prediction quality was assessed using the mann-whitney u test. ochratoxin a (ota) is a wide-spread food contaminant and one of the most potent renal carcinogens [1] . recent data by our group demonstrate that ota inhibits histone acetyltransferases (hats), thereby causing a global reduction of lysine acetylation of histones and non-histone proteins [2] . based on these findings and the importance of specific histone acetylation marks in regulating gene transcription [3] , we speculated that repression of gene expression as the predominant transcriptional response to ota [4, 5] may be linked to loss of histone acetylation. in this study we therefore used a novel mass spectrometry approach, which is based on chemical acetylation of unmodified lysine residues of histones using 13 c-labeled acetic anhydride and subsequent calculation of the degree of acetylation based on the measured intensities of heavy and light acetylated isotopologues [6] , to identify and quantify site-specific alterations in histone acetylation in human kidney epithelial (hk-2) cells treated with ota. our results demonstrate ota-mediated loss of acetylation at almost all important lysine residues at histones h2a, h2b, h3 and h4. we further selected acetylation at histone h3 lysine 9 (h3k9), a well-known euchromatic hallmark that is elevated at promoter regions of transcriptionally active genes [7] and which was reduced from ~ 3% in controls to < 0.1% in response to ota, to establish a link between loss of h3k9 acetylation and expression of genes consistently shown to be down-regulated in response to ota [4, 5] . using chromatin immunoprecipitation followed by quantitative real-time pcr (chip-qpcr), we observed ota-mediated loss of h3k9 acetylation at promoter regions of the selected genes (% of controls: amigo2: 45%, clasp2: 60%, ctnnd1: 54%). overall, these data provide first evidence for a mechanistic link between h3k9 hypoacetylation as a consequence of ota-mediated inhibition of hats and repression of gene expression by ota. a new paradigm to assess the proarrhythmic potential of drugs is proposed by the cipa (comprehensive in vitro pro-arrhythmia assay) initiative combining a suite of a priori in vitro assays (7 most important ion channels for cardiac activity) coupled to in silico reconstructions of cellular cardiac action potential (ap). the etox consortium has developed a multiscale simulation in silico model based on o'hara/rudy incorporating the principles of this new paradigm. the core model simulates the effects of drugs on a virtual cardiac tissue composed by different types of cardiomyocytes. the input of this model, the blockade of a set of 3 ion channels (ikr/herg, iks, ical), can be obtained experimentally or predicted using advanced 3d-qsar models. the system predicts the % change of the qt interval at different drug concentrations in order to facilitate risk assessment. this in silico model was validated using purkinje fiber assay results (input: ap prolongation and arrhythmogenic risk assessed by early after-depolarisation occurrence) from 500 in-house drug candidates. the validation showed that predictivity is highly dependent on the model's applicability domain (ad): for some chemical series the proarrhythmic potential could not be identified, for others, however, most of the positive drugs were correctly predicted with sensitivities up to 80-90% (average prediction accuracy was 70%). retraining of this model with additional internal data should help to improve the model ad and predictivity. it is important to note that ap prolongation was correctly predicted for many proarrhythmic drugs with only low (> 30 µm) in vitro herg inhibition. furthermore, the model showed high additional benefit for read-across within bayer pharma ag, investigational toxicology, berlin, germany etox [1] [2] started in 2010 and is a public-private partnership project within the european innovative medicines initiative (imi) [3] . the etox project is building a toxicology database relevant to pharmaceutical development and to elaborate innovative strategies and software tools. the overall goal is to better predict the toxicological profiles of new chemical entities in early stages of the drug development pipeline based on existing in vivo study results contributed by the participating efpia * companies in the consortium. the etox database is a relational database with a specifically designed schema to store complex and comprehensive preclinical safety data like the study design, toxicokinetics, adme data, clinical chemistry, hematology, gross necropsy, histopathological findings and general toxic effects. in addition relevant data from public sources has been included into the database. the primary focus for data collection are systemic toxicity (up to 4 week) repeated dose studies, mostly in rodent. overall more than 7000 study reports for approximately 1400 investigated compounds. in order to optimize the usage and mapping of data from different sources the development of common ontologies was a key task within the project. this timeconsuming step was necessary to make a high quality read-across analysis possible and valuable. therefore the ontobrowser [4] tool was developed to curate and harmonize the verbatim terms to standardize terms which are used within the etox database. until now more than 13 million verbatim terms were curated. additionally to the toxicology database, a web-based user interface called etoxsys was developed to allow the retrieving of toxicity information, as well as the prediction of toxic endpoints for chemical compounds. due to the complex search capabilities, the database can be queried for structural similarity, similar target classes and specific toxicological endpoints. approximately 150 prediction models based on public data are available and first models based on in vivo data are in development. the etox database therefore represents a valuable tool for early animal-free assessment of drug candidates [5] . * european federation of pharmaceutical industries and associations cell lines background: consumers are constantly exposed to chemical mixtures e. g. to multiple residues of different pesticides via the diet. this raises questions concerning potential cumulative effects, especially for substances causing toxicity by a common mode of action. since substances are tested for regulatory purposes on an individual basis at generally high dose levels, there is only limited data available on potential mixture effects especially in the low dose range. with more than 400 active substances approved for being used in pesticides and over 100000 chemicals registered under reach there are more possible combinations than one could test with classical animal experiments. the development of in vitro tools for assessment of mixture effects consequently is of tremendous importance. methods: as a first step in the development of such in vitro tools we used a group of fungicides, (tri-)azoles, as model substances in a set of different cell lines from known target tissues, basically liver (human: hepg2, heparg, rat: h4iie) and adrenal gland (human: h295r). concentrations were taken from measured tissue concentrations in vivo to ensure that used concentrations of the (tri-) azoles reflect realistic effect levels. the cell lines were exposed with the triazoles cyproconazole and epoxiconazole as well as with the azole prochloraz as individual substances and in binary or ternary combinations of these substances at three dose levels and three different time periods. the effects of the substances were subsequently analysed by transcriptomics and metabolomics. a support vector machine will be utilized to integrate the data from the different sources to gain a complete picture of affected adverse outcome pathways and mechanistic information about the applied fungicides. first results indicate combination effects of the substances also at the omics level depending on the specific endpoint and the concentration used. some of these are comparable to effects found with similar methods in a standard toxicity test, a 28-day feeding study in the rat, thus raising hope for the development of in vitro methods suitable to detect combination effects. background: plant protection and biocide products are chemical mixtures, which contain one or more active substances as well as several co-formulants (e.g. solvents, wetting agents, thickener or preservatives). nevertheless, to this day extensive toxicological testing is performed only with the individual active substances, while the plant protection products are only evaluated for acute toxicity, ie, a single dose group experiment with rats is performed as well as testing for skin-and eye-irritation. current pesticides regulation foresees testing of potential harmful mixture effects but only when adequate methods are available making the development of such methods a high priority. several published studies both in vitro and in vivo have shown fortified toxic effects of plant protection products compared to individual active substances. methods: here we present effects of plant protection products as a whole as compared to the individual active substances or co-formulants in a set of human cell lines of hepatic and renal origin (hepg2, heparg, hek293). cytoxicity has been analysed by wst-1 and nru assay as well as gene expression of several marker genes involved in xenobiotic metabolism. additionally reporter gene assays have been conducted for nuclear receptors such as ahr and car. results: while some active substances showed lower toxicity as compared to the respective products, this cannot be confirmed as a general rule for all endpoints for all of the analysed fungicides or herbicides containing active substances such as epoxiconazole, cyproconazole, azoxystrobin or glyphosate. chemical compounds may induce skin sensitization in humans, resulting in tolerance or allergic contact dermatitis after repeated exposure. mechanistically, the activation of dendritic cells is one of the prerequisites for the induction of skin sensitization. a subgroup of sensitizing chemicals, prohaptens, need metabolic activation, e.g. via cytochrome p450 (cyp) enzymes. thus, xenobiotic metabolism may crucially impact on a chemical's potential for the induction of skin sensitization by activation, but also deactivation of reactive molecules via conjugation, which determines the concentration and the chemical species available for protein haptenation and cell activation. we established a coculture model consisting of hacat keratinocytes and thp-1 as surrogate dendritic cells for the detection of sensitizing chemicals and found enhanced cyp1 enzyme activity in hacat cells exposed to benzo[a]pyrene (b[a]p) and eugenol as well as clearly increased expression of cell surface molecule cd86 on thp-1 cells after incubation with these prohaptens (hennen et al., 2011) . here, we studied the impact of intercellular cross talk on activation and conjugation capacities in more detail. treatment of thp-1 with b[a]p and eugenol in coculture with hacat cells augmented cyp1a1 and/or cyp1b1 mrna levels, while this was not found for thp-1 monoculture. augmentation of cyp1a1 mrna needed continuous presence of hacat cells. in coculture, levels of 3-oh-b[a]p as exemplary cyp-dependent metabolite were increased compared to single cultures. in contrast to this, total glutathione contents as well as n-acetyltransferase 1 enzyme activities in both cell types were not modulated in coculture, furthermore the capacity for sulfation/glucuronidation of 3-oh-b[a]p was maintained in coculture. additionally, the decrease of the total glutathione content in thp-1 cells by 2,4-dinitrochlorobenzene (dncb) was much less pronounced when exposed in coculture with hacat cells, showing that hacat cells provide additional targets for cysteine-reactive chemicals such as dncb, diminishing the total amount of chemicals available for thp-1 cells.overall, results indicate that the cross talk between keratinocytes and antigenpresenting cells enhances their capacities for metabolic activation of chemicals, while hacat cells also provide supplementary capacities for phase ii reactions. references: hennen j et al. cross talk between keratinocytes and dendritic cells: impact on the prediction of sen-sitization. toxicol sci 2011 123:501-510.toxicology -toxic pathway analysis/aop 395 background: reticulated platelets are associated with impaired antiplatelet response to thienopyridine treatment. this interaction might be caused by intrinsic properties of reticulated platelets or a decreased drug exposure due to high platelet turnover reflected by reticulated platelets as surrogate. we investigated the impact of reticulated platelets on antiplatelet response to thienopyridines and if this effect is linked to platelet turnover. methods: this study randomized elective patients to loading with clopidogrel 600mg or prasugrel 60mg (n=200). adp-induced platelet reactivity was assessed by impedance aggregometry 30 to 120 minutes and day 1after loading but before intake of the next dose of thienopyridines. immature platelet count (ipc) was assessed as marker of reticulated platelets by whole blood flow cytometry. results: platelet reactivity increased with rising tertiles of ipc (figure) . this effect was more pronounced in patients on clopidogrel as compared to patients on prasugrel. overall, ipc correlated well with on-treatment platelet reactivity at 120min (r=0.21; p<0.001). this correlation did not change over time indicating an effect independent of platelet turnover (comparison of correlations 120min/day 1: p=0.57 for clopidogrel, p=0.76 for prasugrel). conclusion: a high immature platelet count is associated with impaired response to thienopyridine loading. this effect is independent of platelet turnover indicating a relation to intrinsic properties of reticulated platelets. introduction: one of the biggest drawbacks of protein-based therapeutics with intracellular targets is their inability to enter the cytosol. targeted toxins are known to be used in drug delivery. aim of the study was to target epidermal growth factor (egf) receptor overexpressed on pancreatic carcinoma using a novel well-defined targeted toxin consisting of egf fused to the toxic plant ribosome-inactivating protein dianthin and a glycosidic triterpenoid (so1861) as efficacy enhancer. methods: the enzymatic activity of dianthin-egf was verified by an adenine release assay. the kinetics of cytotoxicity were evaluated in pancreatic adenocarcinoma bxpc-3 and miapaca-2 cells in comparison to the non-target cell line nih3t3 with an impedance-based real time cell analyzer (xcelligence) and final cytotoxicity analyses with conventional end-point mtt assays. acute toxic of dianthin-egf was studied in male balb/c mice. a xenograft solid tumor model was developed in male nude mice by injecting bxpc-3 cells into the dorsal part subcutaneously. dianthin-egf was administered at the vicinity of the tumor and so1861 by subcutaneous injection at the neck. after the tumor reached a diameter of 2 to 3 mm in size 6 treatments were given in total. tumor volumes and body weight shifts were observed twice weekly to determine the potency of dianthin-egf when given alone and in combination with so1861 in comparison to placebo. immunohistochemical detection of egf receptor was performed according to the manufacturers's advice (dako, glostrup, denmark, k1492). complete blood count analysis was done by labor 28 gmbh, berlin. results: the adenine release mediated by dianthin-egf was 47.8 pmol adenine/pmol toxin/h. the in vitro efficacy of the targeted toxin was proven by an ic50 value of approximately 1 nm for egf receptor expressing miapaca-2 and bxpc-3 cells as compared to 100 nm for non-target nih3t3 cells. real time measurement of the cytotoxicity showed a dose-dependent decrease in cell viability from 10 pm to 1 µm. toxicity studies in balb/c mice revealed 0.4 µg/mouse to be non-toxic and maximum tolerated dose (mtd) whereas 40 µg caused moribundity accompanied with white ocular discharge. efficacy studies were performed for a period of 28 days. the combination therapy showed that the average tumor volume measured by a digital vernier caliper was found to be 80% less than for placebo whereas single therapy using dianthin-egf alone caused a further increase in tumor volume which was although yet 50% less when compared to placebo. immunohistochemistry slides showed egf receptor expression in each of all untreated xenograft tumors, which further confirms the presence of egf receptor overexpression in the target bxpc-3 cell line. enlarged spleen was only observed in untreated xenografts. no significant change in various blood parameters (rbc counts, wbc counts, hgb, hct, mcv, mch and mchc) were observed on hematological analysis except for the platelet (plt) counts in comparison to healthy male nude mice. conclusion: combination therapy with so1861 proves to be a promising approach for the targeted delivery of toxins instead of single therapy administering targeted toxin alone. the strategy is specific for egf receptor overexpressing tumors such as pancreatic cancer. introduction: moringa oleifera (mo) is a popular herbal supplement used for treatment and management of diverse diseases in sub-saharan africa. its intake among individuals infected with hiv/aids has increased recently due to the purported immune boosting property. limited information, however, is available regarding its potential to cause interactions with commonly prescribed medications that are substrates of cyp3a4 and p-glycoprotein. methods: the methanol extract and four fractions of mo were tested on recombinant cyp3a4 at different concentrations with and without nadph to determine the ic 50 shift reduction. the crude methanol extract of mo was incubated with testosterone (tst) and cryopreserved hepatocytes to evaluate its influence on clearance of tst. effect of mo on the efflux transporter, p-glycoprotein was investigated by incubating the methanol extract with mdr1 -mdckii cells. virtual screening was conducted to predict physicochemical properties, bioavailability and interaction potential of phytochemical compounds unique to mo using combination of molinspiration version 2014.11 and admetsar. results: fractions (f1-f3) indicated ic 50 shift reduction ≥5 post-incubation with and without nadph. mo showed moderate interaction (auc i /auc = 2.46) with tst in cryopreserved hepatocytes. also, mo mildly inhibited the transport of digoxin (ic 50 = 35.45 µg/ml) across mdr1 -mdckii cells. niaziminin indicated 85.57% bioavailablity via the human intestinal membrane with 61% chance of inhibiting cyp3a4. βsitostenone showed strong p-gp inhibition (83.27%) with 100% absorption via the intestine. conclusions: mo has the potential to inhibit the metabolism or excretion of other medications that are eliminated by cyp3a4 or p-glycoprotein, respectively, if adequate amounts of the active constituents such as niaziminin and β-sitostenone enter the circulation. background: herb-induced liver injury (hili) has attracted attention in the past years due to an increasing number of publications reporting cases of hepatotoxicity associated with use of phytotherapeutics. here, we present data on hili from the berlin case-control surveillance study fakos. methods: fakos was initiated in 2000 to study serious toxicity of drugs including hepatotoxicity. potential cases of liver injury were ascertained in more than 180 departments of all 51 berlin hospitals from october 2002 until december 2011. through a standardised face-to-face interview and review of medical charts information on all previous intakes of drugs or herbals, on co-morbidities, and demographic data was ascertained. inclusion criteria were an elevation of alanine aminotransferase or aspartate aminotransferase threefold above the upper limit of normal or an elevation of total bilirubin higher than 2 mg/dl. excluded were patients with underlying liver disease (e.g., alcoholic fatty liver disease). drug or herbal aetiology was assessed based on the updated council for international organizations of medical sciences (cioms) scale. results: of all 198 cases of hepatotoxicity included into the fakos study, herbs were involved in ten cases (5.1%). demographic, clinical, and laboratory characteristics of these ten cases are illustrated in table 1 . among the six patients with available liver biopsy results, five patients showed signs of necrosis, either disseminated or predominantly near the central vein. portal inflammation was more common than lobular inflammation, and the infiltrates contained mostly lymphocytes, neutrophil or eosinophil granulocytes. herbal aetiology was judged two times as probable (ayurvedic herb in patient 1, pelargonium sidoides in patient 6), and eight times as possible (valeriana in patients 3, 4, 8, 9, 10 , mentha piperita in patient 5, hypericum perforatum in patient 2, eucalyptus globulus in patient 7). in nine cases other non-herbal drugs were also suspected as potentially hepatotoxic (exception: patient 6). seven cases occurred in the ambulatory setting requiring hospitalisation, three cases occurred during hospital stay. discussion: this case series provides further information on laboratory and clinical aspects of hili. it corroborates known risks for valeriana and ayurveda treatment, and suggests that further herbals rarely or never associated with liver injury before such as pelargonium sidoides, hypericum perforatum or mentha piperita could also exhibit a hepatotoxic potential. clinical routine often requires to evaluate the cause of a newly occurring adverse event. if this event is regarded to be iatrogen, further information of the association between the drugs in the current medication list and the adverse event is needed. this information should ideally reflect the true risk and allow ranking of the drugs according to this risk to identify which drug to discontinue first. we discuss the summary of product characteristics (spc), the sider side effect resource and openvigil 2 as possible sources of information. spcs are becoming more and more a vindicative charter for pharmaceutical companies that contain misleading information which is not based on evidence (ref. 1). since it relies on the spcs, sider inherits these shortcomings and flags warnings that result from confounding factors (ref. 1, fig. 1 ). furthermore, if any rates are given, they are not easily comparable since they stem from different studies. pharmacovigilance data are biased by the very nature of the data and the collection method. however, once confounders are eliminated, pharmacovigilance offers better information om how to rank the drugs than spcs/sider. we present decision-guiding information obtained by sider and by openvigil 2 for one of our patients ( fig. 2 & 3 ) and discuss how this information was used to modify the therapy. institut für naturheilkunde und klinische pharmakologie, universität ulm, ulm, germany background: differences (polymorphisms) in target genes or genes encoding drug transport proteins or drug metabolizing enzymes may be responsible, among other factors, for observed variation in patients' response to medications. pharmacogenetics aims at identification of patients at higher, genetically determined, risk of adverse drug effects or ineffective medication, to modify dosage or switch to alternative therapy. there is, however, a lack of awareness of pharmacogenetic-based clinical practise guidelines. methods: a systematic literature review was conducted which focused on published guidelines on genotype-based (germ-line genetic variants) dosage modification or selection of drugs. we serched the medline and the pharmacogenomics knowledgebase (pharmgkb) databases. prescribing information was also screened for pharmacogenetic guidance. results: the systematic review revealed recommendations for 61 drugs (table) that enable the translation of genetic test results into actionable prescribing decisions. for 20% of these drugs the respective german drug labels recommend or even require pharmacogenetic testing (table, 3rd column). although pharmacogenetic testing is recommended, the prescribing information not always provides guidance on how to adjust the drug dosage based on the pharmacogenetic test result. compared with the german or european drug labels, the fda drug labels povide more detailed information on pharmacogenetic dose modifications. conclusions: academic working groups have a front-runner role in the development of prescribing recommendations based on genetic markers. to date, drug labels rarely contain detailed guidelines how available genetic test results should be used to adjust drug dosage. because pharmacogenetics has a growing role during drug development and pre-prescription genotyping will become more widespread, it is expected that specific pharmacogenetic guidance for the treating physicians will become increasingly important. bisphenol a (bpa) is a high production volume compound mainly used as a monomer to make polymers for various applications, including food-contact applications. people are exposed to low levels of bpa because very small amounts of bpa may migrate from the food packaging into foods or beverages. however, other potential sources of exposure, such as dermal contact have also been identified (efsa, 2015) . a substance evaluation process (corap) was initiated for bpa by the european chemicals agency (echa). as part of the safety evaluation of bpa, a study was required by echa to assess absorption and metabolism of bpa following dermal exposure to human skin. an in vitro study with human skin was requested according to oecd tg 428 under consideration of the scientific committee on consumer safety (sccs) criteria for the in vitro assessment of dermal absorption. to investigate potential dermal bpa metabolism fresh human skin was used. abdominal skin was obtained fresh from surgery from 4 different donors. split-thickness human skin membranes were mounted into flow-through diffusion cells (n=4 per dose and donor) and the receptor fluid was pumped underneath the skin at a constant flow rate. the skin surface temperature was maintained at 32°c throughout the experiment and electrical resistance barrier integrity testing was performed at the start (0 h) and end of the experiment (24 h). four test preparations at final bpa concentrations of 2.4, 12, 60, and 300mg/l were investigated. the highest concentration was chosen based on the maximum solubility of bpa in water and the lowest concenration was chosen based upon the specific activity of the radiolabelled [ 14 c]-bpa that could be used for mass balance. percutaneous absorption was assessed by collecting receptor fluid (tissue culture medium (dmem), containing ethanol (ca 1%, v/v), uridine 5'-diphosphoglucuronic acid (udpga, 2 mm) and 3'-phosphoadenosine-5'-phosphosulfate (paps, 40 µm)), at multiple time points througout the experiment. at termination the skin was removed from the cells and the stratum corneum was removed with 20 successive tape strips. the exposed epidermis was separated from the dermis using a scalpel. metabolism was investigated for the highest concentration (300 mg bpa/l) only, using a hplc with in-line radiodetection and confirmed bpa-glucuronide (bpa-g) and bpa-sulfate (bpa-s) standards for comparison. no metabolism was observed in any of the epidermis samples, however some metabolism is observed in dermis and receptor fluid samples. metabolites were identified with retention consistent with bpa-g and bpa-s, and also some more polar components. the mean total absorbed dose (receptor fluid + receptor chamber wash + receptor rinse) was between 1.7 and 3.6% of the applied dose and the mean dermal delivery (epidermis + dermis + total absorbed dose) was between 16 and 20% of the applied dose, with the majority of the radioactivity associated with epidermis samples compared to dermis and receptor fluid samples. a linear dose-response relationship is observed over the whole concentration range. anastrozole is a well-known non-steroidal aromatase-inhibiting drug approved for the second-line treatment of breast cancer after surgery and for treating postmenopausal women. treatment with the only available dosage form, anastrozole film-coated tablets for oral administration, is frequently associated with concentration-dependent unwanted side effects like hot flashes, fatigue, joint pain, joint stiffness, vaginal dryness, hair loss, skin rash, nausea, diarrhea and headache. in order to minimize the local gastrointestinal as well as systemic side effects, a system for transdermal anastrozole delivery has recently been developed. in this study, we describe the first experimental in vivo application of a transdermal therapeutic system (tts) to beagle dogs and, as a necessary prerequisite for the analysis of the time course of anastrozole release and uptake, a simple, sensitive and accurate lc-ms method for quantifying anastrozole in plasma. the detection of fragment ions at m/z 225 and 237 instead of the molecule ions (m/z 294 and 306) generated from the elevated collision energy, and the use of a deuterated internal standard resulted in increased relative abundances and improved signal-to-noise ratios.the lower limit of quantification and the limit of detection were 1.4 ng/ml and 0.5 ng/ml, respectively. the developed method was successfully applied in a pharmacokinetic study of anastrozole plasma levels in beagle dogs, measuring percutaneous drug absorption from an experimental, newly designed glycerol-based patch / tts. a distinct time course was observed, with an initial linear increase over 24 hours and a plateau thereafter. this offers promising strategies for the transdermal application of anastrozole with improved pharmacokinetics. background: the monocarboxylate transporter 4 (mct4), encoded by the slc16a3 gene, mediates h + -coupled transport of lactate across the plasma membrane. for cells with high glycolytic activity lactate export is of major importance for the maintenance of the glycolytic metabolism and for the prevention of intracellular acidification. in glycolytic tumor cells, the acidic extracellular environment resulting from export of lactate and h + , furthermore promotes anti-apoptotic effects and metastasis. clear cell renal cell carcinoma (ccrcc) is the most common subtype of renal cell carcinoma (rcc) and is characterized by a metabolic shift towards enhanced aerobic glycolysis and hence, increased lactate production. mct4 and its epigenetic regulation by slc16a3 promoter methylation has previously been identified as prognostic marker for ccrcc outcome and as target for ccrcc treatment. since metastatic ccrcc is associated with poor overall survival and represents a major challenge for treatment, mct4/slc16a3 might represent a promising prognostic marker and a target for therapeutic intervention also for metastatic disease. methods: mct4 protein expression was analysed in 130 paraffin embedded tissue samples of distant metastases derived from different organs by immunohistochemical staining of tissue microarrays. protein expression was evaluated semi-quantitatively using tissue studio v.3.6 (definiens ag). dna methylation in the slc16a3 promoter, specifically at the previously identified cpg site with prognostic potential in primary ccrcc, was analysed in 82 paraffin embedded metastasis samples by maldi tof-ms. mct4 protein expression data and dna methylation at the specific cpg site in the slc16a3 promoter were correlated with clinicopathological parameters and outcome data. results: distant metastases of primary ccrcc showed high mct4 protein expression irrespective of the affected organ. the most frequently affected organs like lung or bone, with approximately 28% and 14% in our cohort respectively, showed similar expression levels as less frequent metastatic sites such as thyroid gland or spleen. accordingly, dna methylation at the identified cpg site in the slc16a3 promoter was low in metastatic tissue in all investigated organ sites. an association of low promoter dna methylation level at the previously identified prognostic cpg site in metastases with poor tumor-specific survival of the patients was observed. conclusion: from these results we hypothesize that dna methylation at specific cpg sites in the 5'-regulatory region of mct4 may not only serve as a predictor for patient outcome and as potential novel target for therapeutic intervention in primary, but also for metastatic disease. tamoxifen is used to treat pre-and postmenopausal women with estrogen-receptor (er) positive breast cancer. as a prodrug, tamoxifen undergoes extensive hepatic metabolism resulting in a complex mixture of metabolites with estrogenic and antiestrogenic effects. while endoxifen and (z) 4-hydroxytamoxifen are the most potent antiestrogenic metabolites, bisphenol and both isomers (e) and (z) of metabolite e are the most potent compounds with estrogenic properties at the er. the mixed antagonist/agonist pharmacodynamic effects of the selective estrogen receptor modulator tamoxifen at the er have been mainly attributed to tissue specific action of er coregulators, yet little is known about agonistic metabolites contributing to its estrogenic actions. the aim of the present study was to clarify whether there is a genetic component for interindividual differences in the formation and clinical effect of agonistic tamoxifen metabolites. a genome-wide association study (gwas) was conducted on steady-state agonist plasma levels in 390 postmenopausal breast cancer patients of european origin who were treated with 20 mg/day of tamoxifen for at least 6 months. plasma concentrations of estrogenic metabolites bisphenol, (e), and (z) metabolite e were quantified using a recently established lc-ms/ms method 1 . promising snps for an association between genotype and either plasma metabolite concentration or clinical outcome were confirmed for their relevance in an independent patient cohort of 313 premenopausal breast cancer patients mainly of european descent 2, 3 . twelve snps close to or above genome-wide significance (p <5e-08) were found to be associated with allele-dependent variable (e) or (z) metabolite e plasma levels, while no genomic hit was found for the tamoxifen metabolite bisphenol. here, positive intergenic or genic regions mapped to chromosomes 1, 2 and 16 for (e) metabolite e and to chromosomes 15 and 18 for (z) metabolite e. upon genotyping of the validation cohort, two genetic loci with minor allele frequencies < 5% were confirmed as putative candidates: rs662106 was associated with a 21-39% variant allele-dependent increase of (e) and (z) metabolite e isomers (p< 0.05), and rs3731872, mapping to a gene encoding zinc finger protein znf124, was associated with increased risk of reccurrence or death (hr carriers 2.6, 95% ci: 1.3 -3.4; p < 0.005). these findings suggest the existence of genetic loci that may contribute to the formation and clinical effect of estrogenic tamoxifen metabolites and therefore could explain therapeutic failure of tamoxifen and/or the occurrence of adverse events during treatment. introduction: metabolomic monitoring of endogenous biomarkers is of increasing importance for the assessment of drug safety and efficacy during clinical drug development. myrcludex b, a novel lipopetide-based entry inhibitor for the therapy of hepatitis b and d, exerts its function through inhibition of the hepatic bile acid transporter na + -taurocholate cotransporting polypeptide (ntcp). in order to assess a myrcludex binduced metabolomic response in humans, lc/ms-based monitoring of endogenous metabolites was performed in blood and urine samples from healthy individuals before and during treatment with myrcludex b. methods: plasma and urine samples were collected from healthy volunteers participating in clinical phase i trials to evaluate safety, tolerability, and pharmacokinetics of single doses of the ntcp inhibitor myrcludex b. using quadrupole time-of-flight mass spectrometry coupled to reversed-phase chromatography (lc-qtof-ms) a set of 15 known ntcp substrates (bile acids) was quantified by targeted metabolomics. protein precipitation was performed in the presence of deuterium-labeled internal standards (istds) which allowed absolute bile acid (ba) quantification in low amounts of plasma. ba profiling in urine was performed after dilution with methanol/water (1:1) in the presence of istds. both methods were validated according to fda guidance and applied to monitor the effect of myrcludex b treatment on human bile acid homeostasis. results: dynamic quantification in plasma and urine was achieved in the range from 7.8 nm to 10000 nm depending on the ba species analyzed. intraday-and interday accuracy and precision were in the 15% tolerance range for all analytes in all matrices. matrix effects were between 39-104% (plasma) and 31-95% (urine), apparent recoveries in plasma were above 97%. basal plasma ba level (mean ± sd) in fasting healthy subjects were 667 ± 574 nm (unconjugated bas), 935 ± 629 nm (glycine-conjugated bas) and 104 ± 62 nm (taurine-conjugated bas). urinary ba level (nmol/g creatinine) were 193 ± 225 nm (unconjugated bas), 89 ± 29 nm (glycine-conjugated bas) and 6 ± 2 nm (taurine-conjugated bas). myrcludex-induced ntcp inhibition resulted in significantly elevated amounts of conjugated ba species demonstrating a spillover of ntcp substrates into the systemic circulation. furthermore, higher urinary ba level were observed during treatment indicating accelerated elimination of excessive bas from the body. conclusion: lc/ms-based monitoring of endogenous biomarkers has been successfully established and applied to study the effect of myrcludex b treatment on human ba metabolism. the results obtained by our assay demonstrate that a myrcludex-induced ntcp inhibition drastically affects human ba homeostasis. this observation provides valuable insights into the drug´s mode of action and will be indispensable for the assessment of side effects and dose-finding processes during future clinical trials. further studies are required to assess a possible role of ba modification (e.g. sulfation) in the process of ba detoxification during myrcludex treatment. key: cord-006230-xta38e7j authors: nan title: deutsche gesellschaft für experimentelle und klinische pharmakologie und toxikologie e.v. date: 2012-02-22 journal: naunyn schmiedebergs arch pharmacol doi: 10.1007/s00210-012-0736-0 sha: doc_id: 6230 cord_uid: xta38e7j nan nucleoside diphosphate kinases (ndpks) are multifunctional enzymes involved in a variety of cellular processes including cancer metastasis and heart diseases. the plasma membrane content of the three major ndpk isoforms ndpk a, b and c is increased in human heart failure. we have previously shown that the ndpk b isoform regulates camp levels and cardiac contractility through a receptor-independent gprotein activation involving direct g protein β subunit phosphorylation. the precise role of ndpk c in the heart is unknown and was the object of this study. ndpk c function was assessed in neonatal (nrcm) and adult (arcm) rat cardiomyocytes with real-time pcr, immunoblotting, and quantification of camp content. heart failure was induced by chronic treatment with isoproterenol (iso, 2.4 mg/kg/d 4 days) via minipumps. chronic iso increased mrna levels of ndpk c by 9.4±1.9-fold and its protein levels by 2.1±0.11-fold. immunoprecipitation of the g protein β subunit resulted in coimmunoprecipitation of ndpk c and the stimulatory gαs subunit: iso enhanced this interaction. upon iso stimulation, ndpk c translocated from the cytosol to the plasma membrane within 3 hours in both nrcms and arcms. adenoviral overexpression of ndpk c in nrcms caused a 1.5-fold increase in basal and iso induced camp synthesis, whereas sirna mediated knockdown of endogenous ndpk c decreased camp levels by ~50%. our results establish ndpk c as a novel and critical regulator of camp synthesis and gs signaling in the heart. the up-regulation of ndpk c and the increased responsiveness to iso in failing hearts point to ndpk c as a potential counterregulatory factor in the onset of heart failure. uptake and metabolism of methylated myricetin derivatives: studies in cell culture and c. elegans ackermann d. 1, 2 , büchter c. secondary plant compounds like flavonoids that are ubiquitary present in fruits and vegetables are believed to exert health protective effects in terms of lowering the incidence of widespread diseases such as cardiovascular diseases and cancer. besides their antioxidative effects, flavonoids may also modulate cell signaling pathways and thereby performing their disease-protective actions. though this class of dietary polyphenols has become increasingly popular as dietary supplements, only little is known about their metabolic fate in vivo. therefore, we investigated the absorption and metabolism of several flavonoids such as myricetin and its methylated derivatives laricitrin, syringetin, and myricetin-3',4',5'-trimethylether in the human colon carcinoma cell line hct116 and the human hepatoma cell line hepg2 as well as the model organism caenorhabditis elegans. all flavonoids were rapidly taken up by both cell lines as shown by hplc analyses. the intracellular amount of myricetin and laricitrin did not increase with time and was only half of that of syringetin and myricetin-3',4',5'-trimethylether. interestingly, no metabolites of these flavonoids could be detected which might at least in part be due to their low intracellular amounts. absorption as well as intracellular distribution was also evidenced by using the fluorescent dye "naturstoff reagent a" nsra) . fluorescence microscopy indicated a predominant cytosolic distribution of the employed flavonoids. in the model organism caenorhabditis elegans, the flavonoids were exclusively distributed in the intestine as visualized by nsra. the antioxidative capacity of the four flavonoids (measured by using the cell free teac assay and the h 2dcf-da assay in hct116 cells) decreased with increasing methyl groups in the b-ring. myricetin-3',4',5'-trimethylether (three methyl groups) was the least effective radical scavenging flavonoid in both the cell free system and in hct116 cells compared to myricetin (no methyl groups). in conclusion, for exerting their biological effects, uptake and distribution as well as metabolism of flavonoids in certain organs such as liver and gut are important and more research in that field is warranted. munich heart alliance, münchen, germany signaling through g protein-coupled receptors is affected by receptor polymorphisms, yet the molecular basis for the functional differences of individual receptor variants is unclear. to investigate the impact of the frequent gly389arg variant of the β1-adrenergic receptor (β1ar) on receptor conformation we used β1ar-sensors capable of fluorescence resonance energy transfer (fret). these sensors retained the pharmacological and functional characteristics of the native receptors. upon stimulation of the sensors we determined the activation characteristics of the polymorphic receptors in real time and in living cells. we found the β1ar variants to behave similar upon a single stimulation with an agonist, but to differentially respond with a change of their activation kinetics during subsequent stimulations. while the arg389-β1ar did not show altered activation kinetics after prestimulation, the gly389-β1ar became slower compared to the initial stimulation suggesting that β1ars possess a memory of previous activation. we then permeabilized β1ar-sensor-expressing cells with saponin to remove soluble cytosolic factors. upon permeabilization the β1ar variants did not display receptor memory, suggesting that the β1ar memory depended on the interaction of the receptors with soluble cytosolic factors upon their initial activation including the phosphorylation of agonist-bound receptors by protein kinase a or g protein-coupled receptor kinases. our findings suggest an intrinsic, polymorphism-specific property of βars that alters activation kinetics upon continued stimulation and that might account for individual drug responses. micro-rna replacement therapy: nanoparticle-mediated in vivo delivery of mirna-145 or mirna-33a exerts antitumor effects in colon carcinoma xenograft mouse models weirauch u. 1 micro-rnas (mirnas) control the expression of various genes, and under pathological conditions several mirnas are up-or downregulated. previous in vitro studies have established a pro-apoptotic and anti-proliferative role of mir-145, which shows decreased levels in colon carcinoma. in contrast, while mir-33a is only weakly expressed in several tumors as well, its role in cancer has not been analysed so far. in this study, we demonstrate the tumor-relevance of mir-33a and identify the protooncogenic kinase pim-1 as a target of mir-33a. pim-1 harbours a highly conserved mir-33a binding site within its 3'-utr, and seed mutagenesis of this target sequence abolishes the mir-33a-mediated downregulation of pim-1. the knockdown of pim-1 by rnai or mirna transfection inhibits proliferation in leukemia and in colon carcinoma cells by decelerating cell cycle progression, thus establishing a tumor inhibitory function of mir-33a. we furthermore introduce polyethylenimines (peis) for the therapeutic application of mirnas in vivo, which is critically dependent on the development of appropriate delivery tools. peis are able to form non-covalent complexes with mirnas, leading to mirna protection after systemic application in combination with an attractive biodistribution profile and the efficient uptake in target organs/cells. therapeutic effects of pei-mediated mirna delivery were demonstrated in subcutaneous colon carcinoma xenograft mouse models. the in vivo application of mirna-145 through systemic or local injection of pei/mirna complexes resulted in efficient mirna delivery and in antitumor effects, based on the concomitant repression of erk5. likewise, tumor growth inhibition was observed upon treatment of tumor-bearing mice with pei-complexed mir-33a. this is due to the mir-33a-mediated downregulation of pim-1 expression and resembles the pim-1 knockdown through rnai / pim-1 sirnas. taken together, in tumor xenograft mouse models we establish mirna replacement therapy through the pei-complexation of mirnas as a novel therapeutic strategy and demonstrate that mir-145 and mir-33a may be promising mirnas in colon carcinoma therapy. md 288, a hybrid of chloroquine and primaquine, is a potential drug against infectious diseases such as malaria. since one moiety of the hybrid, the known antimalarial drug chloroquine, is a known intercalator, the potential of md 288 to intercalate into dna was determined. due to the ability of intercalators to cause frame shift mutations, the mutagenic potential of md 288 was also investigated. the potential of md 288 to intercalate into dna was investigated by means of fluorescence based micro plate assay using ethidium bromide (eb) and isolated calf thymus double stranded dna. as a positive control chloroquine was used. the potential of md 288 to cause gene mutations was determined using the hypoxanthine-guanine phosphoribosyltransferase (hprt) test in chinese hamster v79 lung fibroblasts (v79 cells). v79 cells were treated with 0.4 µm, 0.8 µm and 1.5 µm md 288 or the positive control, the direct mutagen 4-nitroquinoline-n-oxide (nqo, 1 µm) for 24 h. on day 6, mutants exhibiting loss of hprt function were selected with 6-thioguanine . whereas at 1 µm chloroquine, a 38% decrease in fluorescence intensity of eb (indicating dna intercalation) was observed, 10 µm md 288 were needed to observe a similar decrease (28%) in fluorescence intensity of eb. therefore, md 288 is 10fold less potent to intercalate into dna than its moiety chloroquine. the frequency of spontaneous 6-tg resistant mutants per 10 6 colony-forming cells was 9 ± 2. as expected, 1 µm nqo caused a significant increase in the mutant frequency (mf, 168 ± 9) . in contrast, mf was not significantly affected by treatment with md 288 at both noncytotoxic (0.4 µm: 5 ± 4) and cytotoxic (0.8 µm: 9 ± 7) concentrations. in conclusion, md 288 is a less potent intercalator than the known antimalarial drug chloroquine. furthermore, md 288 does not cause gene mutations in the hprt test. since current studies show that various metabolites of md 288 are formed in vitro, their mutagenic potential is currently under investigation as well. -conducting channels but depends critically on the membrane potential. trp channels form cation entry channels thereby either contributing to ca 2+ entry or depolarisation. recently, we showed that trpm4 acts as a ca 2+ -activated non-selective cation channel and critically determines the driving force for ca 2+ influx in mast cells following fcεri-stimulation (1) . in addition to trpm4 we also identified the expression of other trp transcripts in bone marrow derived mast cells (bmmc) including those encoding trpc2, trpc3, trpc5, trpc6 and trpm7. in peritoneal mast cells (pmc), rt-pcr indicated expression of trpc1, trpc4, trpc5, trpc6, trpm2, trpm4 and trpm5. to identify the functional role of those trp channel proteins for mast cell activation we analysed ca 2+ signaling using microfluorimetry in bmmcs and pmcs after stimulation with substances known to activate trpc6 channels in other cell systems such as the diacylglycerol analogue oag, the hyperforin analogue hyp-9 and flufenamic acid (ffa), but could not evoke a rise in the [ca 2+ ]i in both pmc and bmmc. sphingosine 1phosphate and lysophosphatidylcholine, which were reported to activate trpc5 channels, induced only minor rise in [ca 2+ ]i in bmmcs, respectively. here, we will present our analysis of ca 2+ signaling following stimulation of the fcεri receptor and application of secretagogues that are supposed to affect ca 2+ -dependent mast cell activation such as adenosine, endothelin-1, substance p and compound 48/80 in bmmcs and pmcs derived from mouse lines with inactivation of trpc1, trpc3, trpc4, trpc5 or trpc6 since specific antagonists are still lacking for these trp channels. the α2a-ar is the main ar in the central nervous system and it plays a crucial role in regulating norepinephrine (ne) release from nerve terminals via presynaptic feedback inhibition. it is also associated with a number of physiological effects, including hypotension, pain perception, sedation and modulation of mood. ne, once released in the synaptic space, binds to α2a-ars and induces a rearrangement of the receptors from the inactive state into an active conformation. this allows the binding and activation of the cognate gi-protein and, hence, the transduction of the transmembrane signal to the downstream effectors. generally, α2a-ar activation has been deduced from the stimulation of a receptor-mediated biological response that could be easily followed experimentally. however, most of these approaches do not employ living cells and are normally applied under equilibrium conditions that need prolonged incubation periods incompatible with the physiological temporal dynamics of ne. here, we monitored the ne-mediated α2a-ar and gi-protein activation by using a fluorescence resonance energy transfer (fret)-based approach in living cells. to examine the effects of increasing concentrations of ne on the speed and extent of α2a-ar activation with very high temporal resolution, we took advantage of the previously described α2a-ar flash/cfp sensor [1] . the results indicate that in our system the efficacy of ne in eliciting α2a-ar flash/cfp activation increases in a time-dependent way and reaches the maximum with a half-life of ~ 70 ms. the ec50 values decrease in an exponential manner and arrive at ~ 2 µm with a half-life of ~ 330 ms. next, we analyzed the ability of increasing concentrations of ne to trigger a downstream intracellular response after α2a-ar stimulation by monitoring the kinetics and amplitude of gi activation in living cells. we applied the previously well characterized gi cfp/yfp sensor [2] . the results show that both the efficacy and the potency of ne in inducing gi activation reach the steady state slower compared to receptor activation (half-life ~ 700 ms and ~ 3,000 ms respectively). in conclusion, we were able to monitor ne-mediated events occurring in the millisecond time scale and reaching the equilibrium in a time interval compatible with physiological conditions. sphingosine-1-phosphate (s1p) is an immune modulator produced by sphingosine kinase 1 (sphk1) and sphingosine kinase 2 (sphk2) and de-phosphorylated or degraded irreversibly by s1p phosphatases and a lyase, respectively. we recently showed that tlr4-induced il-12p70 is selectively counter regulated by sphk1, s1pr1 and its extracellular ligand s1p. on the other hand, spiegel et al. have demonstrated that specific, sphk1-dependent, binding of s1p to traf2 enhances the tnf-alpha signaling. therefore we were interested whether the tlr/tir and tnf-alpha-signaling pathways are interfered with each other and are modulated by s1p. in a first approach we focused our investigations on sphk1 effects on both traf2 and traf6 stimulatory signals and cytokines produced downstream. experimentally, with gm-csf expanded, bone marrow-derived dcs we first desensitized the lps-tlr4 or cpg-tlr9 signal by a defined time period of costimulation with tnf-alpha. the initial results showed a partial decrease of il-12p70 secretion in tnf-alpha-co-stimulated dcs in contrast to lps stimulation alone. this might indicate that traf2 activated via tnf-alpha interacted with the traf6 pathway to reduce il-12p70. further series with dcs derived from sphk1-deficient mice confirmed our former results that il-12p70 in contrast to other cytokines is specifically sensitive to sphk1-s1p feedback, but did not change the effects of tnf-alpha on wt dcs il-12p70 release. in comparison, cpg-tlr9-induced il-12p70 release reached only 20% of lps-induced il-12p70 levels and was less sensitive to tnf-alpha costimulation. however, sphk1-deficiency strongly augmented cpg-dependent il-12p70 production. in ongoing experiments we started to analyze the details of traf2/rip1 and traf6/tak1 activation by ubiquitination blots in wt, sphk1-and s1plyase-deficient dcs. in conclusion, we hope to unravel possible mechanisms of the observed differential effects of s1p and its enzymes on inflammation and cancer-relevant cytokines. identification of the kh type splicing regulatory protein (ksrp) as a new important mediator of the anti-inflammatory effects of resveratrol art j. 1 , besche v. 2 , bros m. 2 , li h. 1 , handler n. 3 , bauer f. 3 , erker t. 3 , behnke f. 4 , mönch b. 5 , förstermann u. 1 , dirsch v. m. 6 , werz o. 5 , kleinert h. 1 , pautz a. university of vienna department of pharmacognosy, althanstr. 14, 1090 wien, austria resveratrol, a polyphenol derived from different plants, possesses multiple pharmacological functions such as anti-oxidative, anti-diabetic, cardioprotective, anticancer, neuroprotective and anti-inflammatory properties. many of these effects have been attributed to its anti-oxidative activity but resveratrol also modulates signal transduction pathways like the p38 mapk pathway or the activity of different transcription factors like nf-κb redox-independently. moreover, the histone deacetylase sirtuin 1 (sirt1) is an important mediator of resveratrol effects. nevertheless the direct molecular target of resveratrol remains unclear. in target fishing experiments we identified the rna-binding protein ksrp as direct resveratrol binding partner. ksrp is an rna-binding protein that controls proinflammatory gene expression on the post-transcriptional level by modulation of mrna stability. moreover, it is involved in the biogenesis of mirnas. resveratrol treatment of human dld-1 cells resulted in a decreased mrna expression of a number of well known ksrp target mrnas and enhanced mirna-155 function. downregulation of ksrp expression by sirna prevented the mrna destabilizing effect of resveratrol. as the activity of ksrp is mainly regulated on the post-translational level by phosphorylation of different serine and threonine residues we analyzed whether resveratrol changes ksrp activity by altering the phosphorylation of the protein. indeed, our immunoprecipitation experiments demonstrated that resveratrol reduces the p38 mapk-mediated phosphorylation of threonine residues in the ksrp protein and thus leads to an increase of ksrp activity. interestingly, resveratrol does not block p38 mapk activation or activity. in addition we have evidence that sirt1 is not involved in the resveratrol mediated activation of ksrp. so we believe that activation of ksrp by resveratrol is the major mechanism mediating the anti-inflammatory effects of resveratrol. in vitro testing of oecd reference nanomaterials (nm-series) in rat precision cut lung slices aumann a. 1 the oecd has defined reference nanomaterials (nm) to be tested in different endpoints concerning human health and environmental safety (1) in order to evaluate if the toxicity of nanomaterials can be linked to their physico-chemical properties. for nanomaterials, inhalation presents the major exposure route of concern and can be assessed using acute inhalation toxicity studies in rodents. however, these in vivo studies are resource intensive and animal consuming. the oecd working party on nanomaterials has named several alternative methods as being of particular interest for testing of nanomaterials; among them is the precision-cut lung slices model (pcls) to estimate respiratory toxicity. we have tested all 16 nm in pcls measuring cytotoxicity, apoptosis, oxidative stress and inflammatory response of the tissues as well as observing them histological. for in vitro exposure of pcls the test material was dispersed in medium. since it is the nature of these materials to change their surface characteristics and agglomeration state in different environments, a standardized dispersion method (nanocare) using bovine serum albumin as a stabilizing agent, was used. particle size-distributions of the nanomaterial dispersions were characterized via analytical ultracentrifugation and found the nanomaterials well dispersed. silver and zinc oxide but none of the other nm showed cytotoxicity to the lung tissue in the tested concentrations. however, differences in cytokine profiles among the nm were observed and showed several correlations to the results obtained in in vivo inhalation or instillation studies. universität des saarlandes institut für molekulare zellbiologie, gebäude 61, 66421 homburg, germany tmem2 proteins show similarities in their primary sequence to motifs that are conserved amongst various members of the trp protein family. based on hydropathy analysis these proteins exhibit 6 to 10 membrane spanning domains. in contrast to trp channels there is no evidence that these proteins form ion channels in the plasma membrane following overexpression of their cdna in hek293 cells. tmem2 -/mice are viable and show no obvious signs of disease, but exhibit increased pancreatic amylase and lipase plasma levels. microfluorimetric measurements using fura-2 revealed that the elevation of the cytosolic ca 2+ concentration after stimulation with carbachol and the cholecystokinin analogue caerulein is unchanged in tmem2-deficient acinar cells. tmem2 is expressed in several cell types including pancreatic acinar cells, cardiac myocytes, cardiac fibroblasts, but their subcellular localization is still unkown. we generated several constructs encoding tmem2 fusion proteins with fluorescence protein tags by fusing eyfp, mcherry and tagrfp-t to the n-and c-terminus of the protein, respectively. based on western blot experiments and expression in hek293 cells the tmem2-eyfp construct was most suitable for further colocalisation analysis and generation of viral vectors including adenovirus and semliki forrest virus. in contrast to the prediction by the psort ii algorithm tmem2-eyfp could not yet be identified in the plasma membrane of fibroblasts, cardiac myocytes or acinar cells but showed a vesicular subcellular localization pattern. we localized tmem2-eyfp in acidic compartments and predominantly in lysosomes (pearson coefficient (pcc) 0,79 ± 0,03, n= 4 using lysotracker ® dye). analysis of subcellular localization with independent tmem2 fusion constructs and additional vesicular markers will be presented as a framework to get insights towards the cellular function of tmem2 and to reveal the mechanisms underlying increased amylase release from acinar cells of tmem2 -/mice. the small molecule bcl-2/mcl-1 inhibitor tw-37 shows single-agent cytotoxicity in neuroblastoma cell lines bachmann h. s. 1 , akdeli n. high-risk neuroblastoma (nb) remains a therapeutic challenge in paediatric oncology. pro-survival bcl-2 family proteins critically regulate apoptosis, and may represent important therapeutic targets in nb. primary nb tumours heterogeneously express mcl-1 or bcl-2, with high expression correlating to high risk phenotype. co-expression can be detected in approximately 10% and is correlated to reduced survival. recent studies with two inhibitors that predominantly target bcl-2 and other proteins, but not or to a lesser extend mcl-1, elucidated the importance of mcl-1 inhibition for cytotoxicity in nb. tw-37 is a small molecule inhibitor that showed almost equal affinity to bcl-2 and mcl-1. to explore the effect of combined bcl-2/mcl-1 inhibition on neuroblastoma cells, four cell lines (sk-n-as, imr-5, sy5y and kelly) were treated with tw-37 and changes in growth properties were determined. furthermore, nude mice with kelly (human neuroblastoma cell line) xenografts were treated with tw-37. using sirna, we investigated the functional relevance of mcl-1 and bcl-2 in kelly cells. for in vitro cell viability we observed ic50 values of 0.59 ± 0.39 µmol/l. on treatment with 1 µmol/l dose of tw-37, all neuroblastoma cell lines analyzed showed significantly reduced proliferation and increased apoptosis rates. bcl-2 as well as mcl-1 knockdown induced apoptosis in kelly cells. interestingly, tw-37 was able to reduce, but not to abrogate growth of kelly neuroblastoma xenografts in nude mice. in conclusion, combined inhibition of bcl-2 and mcl-1 using tw-37 exhibits strong single-agent antitumor activity on human neuroblastoma cells in vitro, but limited single-agent activity in vivo. therefore, inhibition of bcl-2/mcl-1 may represent an interesting therapeutic strategy, most likely in combination with conventional chemotherapy and other specific inhibitors. localization and functional characterization of membrane transporters for sulfated steroid hormones in the human testis bakhaus k. 1 , wapelhorst b. circulating sulfated steroid hormones like estrone sulfate (e1s) or dehydroepiandrosterone sulfate (dheas) are delivered to the testis via membrane uptake carriers such as the sodium-dependent organic anion transporter (soat). inside the cell these sulfated steroids can be metabolized to active steroid hormones by the catalytic activity of the steroid sulfatase (sts), which shows high enzymatic activity in the testis ("sulfatase pathway"). in addition to soat, other candidate carriers like the organic solute carrier protein 1 (oscp1) and the organic anion transporting polypeptides oatp6a1 and oatp1c1 are predominantly expressed in the human testis and demonstrate transport activity for sulfated steroids. we aimed to evaluate the cellular expression of soat and the other steroid sulfate carriers and their co-localization with the steroid sulfatase (sts) in human testis. furthermore we want to perform functional transport studies with the steroid sulfate carriers in stably transfected hek293 cells. we detected soat by rt-pcr and western blot analysis in the human testis. single cell analysis and in situ hybridization revealed pachytene primary spermatocytes to express the soat mrna. soat expression in specimens showing maturation arrest at the level of early round spermatids seems to be severely reduced or absent. sts mrna was detected by rt-pcr in testis homogenates. preliminary immunohistochemical data showed that sts may be expressed in germ cells and interstitial leydig cells. hek293 cells stably expressing the soat carrier protein showed significant transport activity for dheas. this was demonstrated by using a radiolabeled [ 3 the hepato-intestinal induction of the detoxifying enzymes cyp3a4 and cyp3a5 by the xenosensing pregnane x receptor (pxr) constitutes a key adaptive response to oral drugs and dietary xenobiotics. in contrast to cyp3a4, cyp3a5 is additionally expressed in several, mostly steroidogenic organs, which creates potential for induction-driven disturbances of the steroid homeostasis. using cell lines and mice transgenic for a cyp3a5 promoter we demonstrate that the cyp3a5 expression in these organs is noninducible and independent from pxr. instead, it is enabled by the loss of a suppressing yin yang 1 (yy1)-binding site from the cyp3a5 promoter which occurred in haplorrhine primates. this yy1 site is conserved in cyp3a4, but its inhibitory effect can be offset by pxr acting on response elements such as xrem. taken together, the loss of yy1 binding site from promoters of the cyp3a5 gene lineage during primate evolution may have enabled the utilization of cyp3a5 both in the adaptive hepato-intestinal response to xenobiotics and as a constitutively expressed gene in other organs. our results thus constitute a first description of uncoupling induction from constitutive expression for a major detoxifying enzyme. they also suggest an explanation for the considerable tissue expression differences between cyp3a5 and cyp3a4. serum albumin adducts as biomarkers for systemic bioavailability of active metabolites of various glucosinolates in animal models and humans barknowitz g. 1 , engst w. glucosinolates (gls) are natural pesticides of brassicales, which comprise many important food and feed plants. upon physical damage to the plant, the enzyme myrosinase can convert gls to reactive metabolites (e.g. isothiocyanates). the same reaction can also be catalyzed by enzymes of the intestinal microbiota. modification of sensor proteins (e.g. keap-1) by some gls metabolites leads to adaptive responses, resulting in enhanced detoxification of reactive metabolites and other protective reactions. at least in experimental models, this mechanism can be exploited for chemoprevention of carcinogenesis induced by various chemical carcinogens. however, own research revealed that certain reactive gls metabolites can covalently bind to dna in vitro and in vivo, involving possible genotoxic and carcinogenic risks. animal models are useful for studying beneficial and adverse effects of gls. however, it has to be taken into account that the toxicokinetics of gls may differ between rodent and humans and that the active metabolites are short lived and thus difficult to detect and quantify. likewise, exposure of humans to gls and their breakdown products may enormously vary depending on (i) food preferences, (ii) cultivars, growth conditions and preparation of plants consumed and (iii) variations in human xenobiotic metabolizing system and composition of intestinal microbiota. in order to estimate individual levels of systemic exposure to reactive gls metabolites, blood protein adducts may be useful. we have developed lc-ms/ms methods for quantifying serum albumin adducts formed by glucoraphanin, glucotropaeolin and neoglucobrassicin. the method involves digestion of the protein to amino acids and the usage of isotope-labelled amino acid adducts as internal standards. serum albumin adducts were detected in mouse models after feeding broccoli and pak choi respectively, as well as after administration of purified gls and breakdown products. likewise, gls adducts were detected in human blood plasma after consumption of broccoli or cress. this work was financially supported by the bundesministerium für bildung und forschung (grant 0315370d) . barlow s. harrington house, 8 harrington road, brighton, bn1 6re, great britain values for cancer endpoints for the application of the ttc approach have been derived by linear extrapolation of results from animal carcinogenicity studies to calculate "virtually safe doses" (vsds). a vsd is defined as an exposure that represents an estimated upper bound increase in risk of 1 in a million of developing cancer during a lifetime. in 1995, from consideration of a range of vsds for carcinogens, the us food and drug administration (fda) proposed and adopted a value of 0.5 micrograms/kg of diet (0.5 ppb) as a threshold of regulation to be used for substances present in food contact materials. the fda considered that if exposure to a substance in the diet was below this value, consumers would be protected "with reasonable certainty of no harm" and no toxicological data on the substance need be submitted. the value of 0.5 ppb is equivalent to 1.5 micrograms/person per day, assuming that 1500 g of food and 1500 g of fluids diet might be consumed daily. this value was subsequently incorporated into the ttc approach to be used for assessment of substances without a structural alert for genotoxicity. in 2004, kroes and colleagues further explored cancer as an endpoint and recommended a lower ttc value of 0.15 micrograms/person per day for substances with a structural alert for genotoxicity and exclusion from the ttc approach of certain groups of high potency carcinogens (with vsds below this value). in this presentation, the data underpinning these ttc values will be discussed from the perspective of their reliability for risk assessment of substances with low exposures, for which there are no toxicity data, but which may in fact be genotoxic or non-genotoxic carcinogens. stable conjugates which are recognized by the immune system. in the current investigations, the ability of chemical pre-treatment to interfere with antibody-protein binding has been investigated using ovalbumin (ova) as the model protein and naturally occurring anti-ova igg from healthy human donors. preparation of conjugates: ova (1 mg/ml) was dissolved in sodium borate buffer (0.1m, ph 9.4) . for chemical treatment various amounts (50-200 mg) of 1-fluoro-2,4dinitrobenzene (dnfb; sensitizer) or 2,4-dichloro-1-nitrobenzene(dcnb; non-sensitizer) was added and stirred for 2 hrs at room temperature. unbound compound was removed by consecutive dialysis against phosphate buffered saline (pbs) and distilled water. inhibition elisa: plates were coated with 100 µg/ml ova and blocked with 10% fcs in pbs. ova samples (native or conjugated; 0.6 -10 µg/ml) were pre-incubated with polyclonal antibodies (ab) from pooled normal human serum for 30min and then added to the plates. human anti-ova igg abs were detected by colorimetric analysis using ortho-phenylendiamine as a substrate. the concentration of soluble native ova or conjugate required to displace 50% of ab to plate-bound ova (ic50) was calculated (minimum of n=3 independent experiments). treatment of ova with the chemical sensitizer and protein reactive dnfb resulted in increased ic50 value, whereas mock treatment resulted in comparable ic50 values to native ova. treatment with dcnb showed that the presence of a chemical per se (and possible denaturation) was not sufficient to alter the ic50 value; conjugation of the compound to the protein was required. the analysis is not test chemical specific (specific ab against compound-protein conjugates are not required) and the colorimetric analysis is unaffected by absorbance of the compound itself. thus, this method may have utility for the identification of chemical sensitizers which are directly protein reactive. 2´-deoxy-camp in human cell lines: another second messenger? beckert c. 1 , hinz c. we have shown that 2´-deoxy-camp (dcamp) is synthesized by recombinant human soluble adenylyl cyclase (sac). here, we report that dcamp can be detected and quantified by liquid chromatography coupled to mass spectrometry (lc-ms) in various human cell lines. in most cells a ratio of camp : dcamp of ~10 was observed. as a remarkable exception, in hl-60 promyelocytic leukemia cells, the dcamp concentration exceeded the camp concentration more than 3-fold. a differential regulation of camp versus dcamp was determined upon replacement of the incubation medium (proliferating condition with serum / serum-free resting condition). for example, camp was dramatically reduced in hek293 cells after 24 hours under resting conditions whereas dcamp was significantly increased. in cellular subfractions of hek293 cells ac assays (mn 2+ /forskolin-or mn 2+ /bicarbonate-stimulated) with either atp or datp as substrate revealed that comparable amounts of camp and dcamp accumulated. in addition to sac, membranous acs such as ac v were capable of forming dcamp with vmax and km values for datp comparable to those for atp. we also analyzed the substrate-specificity for several human phosphodiesterases. pde3 and pde4 hydrolyzed dcamp more effectively than camp. taken together, these data point to a putative second messenger role of dcamp in human cells. we are currently investigating the regulatory role(s) of camp and dcamp in apoptosis of hek293 cells. as a novel tool for these studies, we will use the cellpermeant dcamp-acetoxymethylester which penetrates the plasma membrane and releases dcamp intracellularly. the biogenic amine histamine is recognized by target cells via four different histamine receptors subtypes (h1r -h4r), which all belong to the family of g-protein-coupled seven-transmembrane receptors. histamine plays a crucial role in allergic reactions such as rhinitis or conjunctivitis and also in allergic asthma. previously, we showed an interaction of the effects of antagonists at the h1r and h4r in a mouse model of allergic asthma. however, not much is known about the signaling pathway activated by murine h1r and h4r. in order to analyze these signaling pathways, we established a cellular model using transfected hek 293 cells which stably express recombinant mh1r or mh4r. proper expression of the receptors was verified by western blot analysis and flow cytometry. in functional assays we demonstrated that histamine stimulation results in the increase of intracellular ca 2+ concentration ([ca 2+ ]i) in cells expressing either of both, mh1r (pec50 = 8.2) or mh4r (pec50 = 6.9). as a second readout, we analyzed the modulation of forskolin-induced camp-accumulation. in mh1r-expressing cells the intracellular camp concentration was increased by stimulation with histamine, while in mh4r-expressing cells forskolin-induced camp accumulation was reduced. the histamine-induced effects in h1r-expressing cells were blocked by the h1r antagonist mepyramine ([ca 2+ ]i: pkb = 8.6) and those in the h4r-expressing cells by the h4r antagonist jnj7777120 ([ca 2+ ]i: pkb = 8.8) or by pertussis toxin, which selectively blocks receptor gi-protein coupling. jnj7777120, which behaves as a partial mh4r agonist in the steady-state gtpase assay using membranes of infected sf9 cells, was without effect on [ca 2+ ]i and forskolin-induced camp-accumulation in the mh4r-expressing cells. currently, we are investigating mitogen-activated protein (map)-kinase pathways activated by the h1r and the h4r. using a phospho-map-kinase array, histamine dependent phosphorylation of erk1/2, p38, jnk, creb, pkb (akt), and mkk 3/6 were detected in cells expressing either of both, mh1r and mh4r. in summary, the hek 293 cell lines stably expressing selective histamine receptors are very useful tools to investigate hxr signaling pathways in-vitro. enhanced fibroblast motility in the absence of the β3 regulatory subunit of voltage-activated calcium channels belkacemi a. 1 cavβ subunits of voltage-activated ca 2+ channels are required for trafficking the poreforming cavα1 subunit to the plasma membrane and modulate the kinetics of its current. mouse embryonic fibroblasts (mefs), acutely isolated cardiac fibroblasts (cfs) and nih 3t3 fibroblasts do express cavβ2 and cavβ3 subunits, but we could not detect any voltage-activated ca 2+ influx. whereas in mouse cardiomyocytes or hek 293 cells coexpressing cavβ3 and cavα1 subunits a dihydropyridin-sensitive voltage-activated ca 2+ influx was readily detectable. apparently, cavβ subunits serve functions in fibroblasts unrelated to voltage-activated ca 2+ influx. among the proteins potentially interacting with cavβ3 are the inositol 1,4,5-trisphosphate receptors (ip3rs) [1, 2] . we therefore coexpressed mouse cavβ3 and mouse ip3r type 1, 2 or 3 in cos-7 cells and found coimmunoprecipitation of ip3rs using an antibody for cavβ3 and vice versa. to study the release of ca 2+ from ip3-sensitive stores we performed fura-2 measurements on fibroblasts isolated from wild type and cavβ3-deficient mice either in the presence of thapsigargin or after stimulation of gq-coupled receptors by par-1, lpa or bradykinin. receptor-activated ca 2+ release was more pronounced in β3-deficient mefs and cfs, whereas thapsigargin-induced ca 2+ release was the same in cells from both genotypes. in addition, ip3 production measured by a radioreceptor assay was already increased in β3-deficient cells under basal conditions. fibroblasts are migrating cells and involved in various physiological and pathophysiological processes. we therefore started in vitro assays for proliferation, migration and angiogenesis as well as in vivo assays for skin wound healing. angiogenesis and proliferation were apparently not different in both genotypes but migration (measured as transwell migration and in scratch assays) and wound healing were affected in different ways. fluorescent staining of cytoskeleton and quantification of the f-actin/g-actin ratio show similar results in both genotypes, suggesting that the increased migration rates and wound repair in β3 knockout may result, in part, from the increased amount of ip3-releasable ca 2+ . [1] berggren, yang, murakami, et al., removal of ca channel β3 subunit enhances caoscillation frequency and insulin exocytosis. cell 119, (2004) 273-284. [2] müller, haupt, bildl, et al., quantitative proteomics of the cav2 channel nanoenvironments in the mammalian brain. pnas 107, (2010) 14950-14957. bender-sigel j., closs e. i. universitätsmedizin mainz institut für pharmakologie, obere zahlbacher straße 67, 55101 mainz, germany human cationic amino acid transporters (hcat) are a family of multimembrane spanning proteins that mediate the transport of cationic amino acids through the plasma membrane. our earlier results have demonstrated that activation of either protein kinase c (pkc) by pma or cdc42 by egf leads to an internalization of these transporters. in addition, in a recent collaboration with the group of alexander sorkin (university of colorado denver) we found that ubiquitination and clathrin-dependent endocytosis are necessary for the down regulation of hcat-1-mediated arginine transport by pma (vina-vilaseca et al, j biol chem 2011 286:8697) . this mechanism requires nedd4 e3 ligases, but hcats do not contain a ppxy motif to bind the ligases, suggesting that an adaptor protein takes part in this process. however, an involvement of the adaptor protein beta-arrestin in this mechanism could be excluded. using sirna against pkc alpha we now show that pkc alpha is the major isoform that induces the reduction of arginine transport in human u373 glioblastoma cells overexpressing hcat-2a-egfp. in addition, sirna-mediated knock down of cdc42 prevented the decrease of hcat-2amediated transport induced by pma. taken together pkc seems to negatively influence the constitutive cycling of cats by activation the ubiquitination machinery and clathrinmediated endocytosis. cdc42 is part of this pathway. converging of the classical mitochondria-related pathway in parkinson and nuclear dna-repair signaling? scherr a. -l. 1 parkinson disease is the second most neurological disorder worldwide. despite the fact that most cases are idiopathic and only few can be traced back to specific genes, general progression between both tracks of the disease is comparable. the variety of clinical symptoms in motor control like tremor, rigor and postural problems all originate from loss of dopaminergic neurons in the substantia nigra pars compacta of the brain. several proteins mutated in pd are involved in surveillance pathways, monitoring functionality and integrity of proteins and organelles either by the proteasome degradation machinery or by clearance of mitochondria via autophagy (mitophagy). disturbed calcium-and redox-homeostasis seems to play a major role in susceptibility to cell death signals in dopaminergic neurons, but if this is a preceding or successive event in cell death related to pd progression is not known. on the other hand, experimentally elicited parkinsonism by oxidative stress inducers like paraquat or rotenone and mptp (inhibitors of mitochondrial complex i) lead to damage in nuclear dna and activation of the dna repair protein poly(adp-ribose) polymerase 1. by consuming substantial amounts of its substrate nad + , this enzyme can drastically decrease energy levels and disturb the redox balance within a cell, also sensitizing it to stress induced cell death. inhibition of poly(adp-ribose) polymerase 1 has been proven to be beneficial to some extent in cell culture models as well as in experimental pd in mice. our research focuses on a putative crosstalk mechanism we recently discovered between the two pathways of experimentally induced cell death in culture models, i.e. mitochondrial signaling and parp1-dependent poly(adp-ribosyl)ation. both converge on two mitochondrial chaperones, mortalin and trap1. whereas mutations in mortalin have been reported recently to be responsible for some parkinson disease cases in humans, trap1 is a specific target of the kinase pink1 (pten induced putative kinase), which is often mutated in autosomal-recessive forms of the disorder. pink1 is a central regulator of the mitophagy process, tagging mitochondria with dissipated membrane potential for destruction. we could show now that both chaperones bind to short-chain poly(adp-ribose) specifically synthesized by poly(adp-ribose) polymerase 1. we will present our most recent findings about regulation of these two chaperones after application of parkinson-inducing toxins. aldrich). the effect was reversible within ten minutes when cells were re-incubated in regular cell culture medium. stimulatory effects were not due to osmolarity or cell stress due to medium exchange. analysis of different components of both media (table 1) revealed that bicarbonate stimulates accumulation of ccmp and cump besides cgmp and camp in a time-and concentration-dependent manner. bicarbonate is known to activate soluble adenylyl cyclase (sac) and particulate guanylyl cyclase g (pgc-g), regulating sugar metabolism, sperm motility and olfaction by synthesis of camp and cgmp, respectively. in order to identify a responsible cyclase for ccmp and cump generation after bicarbonate treatment, we are currently analyzing transiently and stably transfected hek293 cells overexpressing various known adenylyl and guanylyl cyclases (sac, mac1, mac3, mac9, soluble guanylyl cyclase, pgc-g, pgc-a, and pgc-d) for their pyrimidinyl and purinyl cyclase activity in vivo and their regulation by bicarbonate. in addition, cell fractions will be analyzed for the detection of specific cyclase compartments. question: long term ventricular pacing, especially at the right ventricle (rv), results in left ventricular (lv) failure. there are several lines of evidence that disturbed ca 2+ homeostasis is involved in the pathophysiology of human heart failure. in this study we examined if ventricular pacing affects the na + -and ca 2+ -channels and the expression of ca 2+ -handling proteins and investigated if there is a differential effect between right ventricular free wall (rvfw) pacing and left ventricular apex (lva) pacing. methods: after av-node ablation 14 minipigs underwent ventricular pacing at 120 beats/min (ddd mode) for one year. 7 minipigs were paced from the rvfw and 7 minipigs from the lva, respectively. 7 minipigs with normal sinus-rhythm served as control group. patch-clamp-experiments were studied to measure na + -and ca 2+ currents. western-blots were carried out to investigate the expression of the ca 2+handling proteins l-type ca 2+ -channel, serca2 and phospholamban. results: both rvfw-and lva-pacing led to significant decreased ca 2+ -currentdensities in cardiomyocytes of the lv compared to the control group. the plateau phase of the action potential was significantly shortened after ventricular pacing in relation to control minipigs. furthermore cardiomyocytes of rvfw-and lva-paced minipigs had significant lower na + -current-densities than control minipigs. the action potential amplitude was significantly decreased after rvfw-and lva-pacing whereas the diastolic potential remained unchanged. the expression of the l-type ca 2+ -channel was significantly reduced after ventricular pacing, regardless of the pacing site. in contrast rvfw-and lva-paced minipigs showed significant increased serca2-expression. the expression of phospholamban remained unchanged after rvfw-and lva-pacing compared to control minipigs. conclusion: in a chronic animal model ventricular pacing leads to remodeling of ionchannels and ca 2+ -handling-protein-expression, regardless of the pacing site. investigation on metabolic competence of dermal systems: native human skin, in vitro skin models and keratinocytes blatz v. 1 the implementation of reconstructed human skin equivalents (rhes) as an alternative method for dermal toxicity testing became very prominent in the last decades. their advantages are e. g. the human cell origin and an organ-like 3d structure. already regulatory accepted methodologies are widely in use for testing the skin corrosion (oecd 431) and irritation (oecd 439) within rhes. but there are still some questions open, one of them the metabolic competence of such dermal systems. in this context, enzyme activities of oxidizing (cyp; fmo; adh; aldh) and conjugating enzymes (nat; ugt) were investigated in subcellular fractions of in vitro systems such as keratinocytes and rhes (epidermis model epiderm tm (mattek), full-thickness skin models epiderm tm ft (mattek) and phenion ® ft (henkel ag)) and compared to those of native human skin. activities of cyp 1a, 2b and 3a isoenzymes were measured fluorometrically by oxidative desalkylation of alkoxyresorufines. fmo 1/3 activities were evaluated by hplc/fld detection of n-oxygenated product of benzydamine [1] . adh and aldh activities were investigated by photometrical detection of nadh generation during ethanol (adh) [2] or propanal (aldh) oxidation [3] . nat1 activity was followed by hplc/uv detection of acetylated p-aminobenzoic acid. ugt1 activity was quantified fluorimetrically by glucuronidation of methylumbelliferone [1] . during the course of this study the following results were observed: (loq = limit of quantification) since the metabolic competence of rhes is confirmed, these in vitro systems are estimated as suitable for further toxicity tests (e. g. genotoxicity by comet assay), where metabolic activation of substances may play a crucial role. however, for the data assessment, the determined metabolic profiles should be taken into account. we acknowledge bmbf funding this project (0315226d). signalling pathway indicates that in b104 cells the adenine receptor couples to a gqprotein followed by activation of phospholipase c pathway. these findings represent a new signalling pathway of the adenine receptor and allow the assumption that different adenine receptor subtypes exist in the rat brain. in the scope of the project lexukon ("foodborne exposure to environmental contaminants -data analysis to support and standardise exposure assessments based on nvs ii") exposure to the heavy metals cadmium (cd), lead (pb) and mercury (hg) via food consumption has been assessed for the german adult population based on the national nutrition study ii ( the updated intake assessments show that especially foods regularly consumed such as vegetables and grain contribute mainly to exposure of cd that is about 1.5 µg/kg body weight (bw) per week for average consumers over all food groups. this corresponds to 58% of the tolerable weekly intake (twi) of 2.5 µg/kg bw for cd defined by the european food safety authority (efsa) in 2009. beverages and vegetables are the food groups most relevant for exposure to pb. about 3.7 µg pb/kg bw is taken up by average consumers that is below the benchmark dose for renal toxic effects (4.41 µg/kg bw) defined by efsa for the weekly pb intake. for hg the intake amounts for all population groups examined were significantly below the toxicological reference values. for average consumers the weekly intake of hg is 0.49 µg/kg bw that is primarily taken up by eating fish and fishery products. however, individual population groups and high consumers reach and/or exceed the toxicological reference values for the daily intake amounts for cd and pb. high consumers almost reach the twi for the cd with 94%. for pb a weekly intake of 5 µg/kg bw was estimated for high consumers that exceeds the benchmark dose for renal toxic effects for the weekly pb intake. the results show that data collection should also focus on highly consumed and not only on highly contaminated foods. further, uncertainties in concentration levels should be reduced e.g. by lowering and standardizing the analytical limits. it's recommended to consider further measures in view of the reduction of contents of environmental contaminants in foods. however, other sources can also contribute to the intake of the mentioned heavy metals (e.g. smoking). major cell biological processes are regulated by rho-gtpases, actin-mediated processes in particular. amongst others, rho-gtpases are stimulated by the receptormediated activation of gα12/13 and gαq via specific rhogefs. the p63rhogef is activated by gαq and plays a major role in the acute response of vascular smooth muscle cells to angiotensin ii treatment. the aim of the present study was to establish a fret-assay between gαq-cfp and venus-p63rhogef and characterize the dynamics of p63rhogef-gαq-interaction in single living cells. the fusion of p63rhogef with venus resulted in a functional gαq-regulated p63rhogef-protein as determined by means of rho-luziferase-assays. whereas no specific fret signal was observed between the two interaction partners in the absence of receptor stimulation, a robust and rapid fret signal developed in response to stimulation of histaminergic h1and cholinergic m3-recptors. the onset of this signal after rapid application of agonist paralleled gαq activation kinetics. similar to the kinetics of gαq-protein deactivation the dissociation of p63rhogef and gαq after withdrawal of agonist was slow (tens of seconds). the specificity of the fret signal between gαq-cfp and venus-p63rhogef was verified by introducing point mutations rendering p63rhogef unable to bind to active gαq. furtehrmore we observed a robust acceleration of the dissociation of p63rhogef and gαq upon cotransfection of rgs2, suggesting a very short lifetime of the p63rhogef-gαq-complex or the ability of rgs2 to bind to p63rhogef-associated gαq. taken together, fret-based imaging of the interactions between p63rhogef and gαq revealed fast interaction kinetics closely resembling g-protein activation kinetics, both of which can be regulated by rgs2. toxicity of silver nanoparticles in intestinal cells boehmert l. 1 the rapid development of nanotechnology has been accompanied by an increased concern for the safety of nanomaterials. especially silver nanoparticles are used in many manufacturer identified consumer products including silver coated food contact materials or hydrosol silver supplements. these products lead to an intentional or unintentional oral uptake of silver nanoparticles and hence to a contact with the intestinal barrier. the human cell line caco-2 is a well established model system in studying effects on human enterocytes. although these cells are colon carcinoma cells and exhibit typical features of cancer cells when they are kept sub confluent, these cells have the capability to differentiate into polarized cells with morphological and biochemical properties of small enterocyte cells. we investigated the effects of silver nanoparticles on these colon carcinoma (proliferating) and small intestinal epithelium like (differentiated) caco-2 cells. the silver nanoparticles agpure were commercially available from rent-a-scientist gmbh. the behaviour of these silver nanoparticles in cell culture medium were characterised using asymmetric flow-fild flow fractionation (a4f), small ankle x-ray scattering (saxs) and dynamic light scattering (dls). we investigated the particle toxicity on both cell states using cell titer blue assay, xcelligence impedance measurements, annexin-v and caspase measurements, diclorofluorescein assay and antioxidant pre incubated cells. the agpure stock solution is an aqueous suspension of silver particles with a metal core radius of 7.2 nm stabilised with tween-20 und polyoxyethylenglycerol trioleate. agpure silver nanoparticles were toxic for proliferating as well as differentiated caco-2 cells in a time and concentration depending manner. the presence of foetal calf serum in the incubation medium has a minor influence on the toxicity. prior to cell death, morphological abnormal adherence characteristics and morphological changes in the cells were observed using microscopy and quantified by xcelligence impedance measurement. it is concluded that cell death is caused by an oxidative stress related mechanism rather than apoptosis. the release of sphingosine-1-phosphate from human platelets during acute coronary syndrome is attenuated by aspirin böhm a. 1 , polzin a. in addition to atherosclerosis ttp knock out mice develop more cardiovascular dysfunctions. in tail vein bleeding assays we monitored a significant difference in the bleeding times of ttp deficient mice in comparison to wildtype mice, triggered by a stronger granulopoeisis. our results leed us to the assumption that the chonic inflammation seems to be more improtant for the development of cardiovascular diseases in ra patients than the traditional risk factors. differentially expressed cardiac genes in a mouse model with heart-specific overexpression of pp2a bollmann p., makarova e. a., gergs u., neumann j. institute for pharmacology and toxicology medical faculty, magdeburger str. 4, 06112 halle (saale) , germany in transgenic (tg) mice with cardiac myocyte-specific overexpression of the catalytic subunit of protein phosphatase 2a (pp2a) reduced cardiac protein phosphorylation, cardiac hypertrophy and impaired cardiac contractility were noted compared to wild type (wt) littermates. the hearts of tg mice also suffered from ventricular dilatation and a diminished response to β-adrenergic stimulation. analyses of mrnas expressed in tg and wt hearts (n=3) using affimetrix mouse genome microarray chips resulted in several candidate genes possibly differentially regulated. in this study, we focussed on verifying the mrna data of selected genes important for stress response and signal transduction on protein level in cardiac homogenates by western blotting. hearts from wt littermates were used as control. compared to wt heat shock protein 25 (hsp25) and calcium calmodulin dependent protein kinase type ii (camkii) mrnas were upregulated in tg but only hsp25 protein was increased (p<0.05, n=7-8) but not camkii (p>0.05). protein phosphatase type 5 (pp5) and superoxide dismutase (sod) were downregulated on mrna level in tg but on protein level this could be found only for sod (p<0.05, . in contrast, pp5 protein was upregulated (p<0.05, in tg compared to wt. for comparison the regulatory a-subunit of pp2a and hsp90 were studied. both genes were unchanged on mrna level in tg: western blotting revealed the same results for the corresponding proteins. in summary, mrna expression data could only partially be confirmed on protein level elucidating the importance of western blotting studies. these data indicate that increased pp2a activity is associated with modified gene expression in tg hearts possibly affecting stress response and regulation of cell signalling. (supported by the deutsche forschungsgemeinschaft) center for regenerative therapies, technische universtität, dresden, germany cell therapy in the form of beta cell replacement to cure diabetes has been practiced for decades without become a routine clinical therapy. more widespread clinical application is hindered by the scarcity of suitable organ donors, a dramatic loss of transplanted cells within the first days post-transplant, the requirement of long term immunosuppression to maintain graft survival, and despite this, a loss of graft function from a recurrence of autoimmunity in some patients. research is currently dedicated to overcome each of these limitations. additional beta cell sources investigated include embryonic stem cell derived insulin producing cells, human insulin producing cells lines, and xenogeneic beta cells. parallel to these efforts are the development of encapsulation devices to protect these sources from immune and inflammation mediated destruction, and transplantation into new sites such as the muscle and bone marrow to infuse beta cells. additional therapy to reduce immune suppression includes the infusion of t regulatory cells to control autoimmune and alloimmune response, and cytokine and chemokine receptor directed compounds aimed at blocking early inflammation or autoimmunity. these efforts are likely to lead to an expansion of clinical activity to replace beta cells in diabetes, and to novel pharmaceutical therapies that may be more generally applicable in patients with diabetes. pdgf-bb induces the h2s producing enzyme cystathionine-γ-lyase via a rosdependent mechanism in rat renal mesangial cells boosen m. 1 , hassan m. there is increasing evidence that hydrogen sulfide (h2s) that is endogenously produced in several cell types serves as a potent gasotransmitter in a wide variety of physiological processes involving vascular homeostasis and inflammation. in the present study we investigate the expression and the regulation of the hydrogen sulfide synthesizing enzyme cystathionine γ-lyase (cse) in cultured rat renal mesangial cells. as demonstrated by qpcr and western blot experiments, mesangial cells showed a marked time-and dose-dependent upregulation of cse mrna and protein levels after treatment with platelet-derived growth factor (pdgf-bb). the cse upregulation by pdgf-bb is accompanied by a marked increase in reactive oxygen species (ros) formation. interestingly, co-administration of the ros scavenger n-acetylcysteine, the glutathione peroxidase mimetic ebselen and the nadph oxidase inhibitor diphenylen iodonium chloride (dpi) drastically reduced pdgf-induced cse expression, indicating a role for endogenously produced ros in mediating regulation of cse. as demonstrated by electrophoretic mobility shift (emsa) experiments pdgf-bb induces binding of the redox-sensitive transcription factor nf-e2-related factor 2 (nrf2) to a consensus antioxidant response element and this effect was also diminished by co-administration of antioxidants (dpi, nac, ebselen) . furthermore, lps/ifnγ-as well as pdgf-bb-induced cse upregulation was nearly completely abolished in nrf2 -/spleen macrophages and mesangial cells, respectively. as a consequence of the elevated cse levels we could demonstrate increased h2s levels and a higher cse enzyme activity in mesangial cells after stimulation with pdgf-bb by using the colorimetric methylenblue method and a cse activity assay. importantly, in a rat model of anti-thy-1-induced proliferative glomerulonephritis we observed a marked upregulation of cse protein during the course of the disease paralleled by a stabilization of nrf2 protein. from our data, we hypothesize that pdgf-bb-mediated regulation of cse via a redox-mediated activation of nrf2 may constitute a protective mechanism during glomerular inflammatory disease. rac1 knockout protects from acute hepatic damage following doxorubicin treatment bopp a., wartlick f., fritz g. heinrich-heine-universität institut für toxikologie, universitätsstr. 1, 40225 düsseldorf, germany rac1 belongs to the best characterized members of the ras-homologous (rho) family of small gtpases, which are key regulators of the actin cytoskeleton. furthermore, rac1 is part of the activation of the nadph oxidase, which produces reactive oxygen species and regulates the activity of stress kinases (e.g. sapk/jnk) and transcription factors such as nf-κb and ap1. anticancer drugs cause dna damage, which in turn stimulates the dna damage response (ddr) regulating dna repair, cell cycle progression and, in case of non-repairable dna damage, triggers apoptosis. so far, a role of rac1 in the ddr has not been reported. based on its exceptional function as a regulator of transcription and because of its recently found ability to translocate to the nucleus, we hypothesize that rac1 may be involved in the ddr. to study the in vivo function of rac1 we used an inducible cre-based knockout mouse model (rac1 flox/flox/mxcre ). mice were treated with different doses of doxorubicin for different periods of time. we monitored gh2ax foci formation as a marker of dna strand breaks, used the masson-goldner staining for the detection of collagen accumulation, analyzed phosphorylated histone 3 as a marker of mitotic events and performed a tunel assay to detect apoptotic cells. in the absence of rac1 the basal mrna expression of pro-fibrotic ctgf was decreased. collagen levels were increased and mmp1 mrna expression was reduced in the liver of rac -/animals as compared with rac1 proficient animals. in addition we found more apoptotic cells in rac1 -/mice. 96 hours after treatment with the anthracycline derivative doxorubicin the number of gh2ax foci in rac1 -/animals was reduced in comparison to rac1 +/+ animals. we also found lower level of ctgf mrna expression and reduced amount of collagen in rac1 -/mice. none of these protective effects resulting from rac1 deficiency could be detected after administration of three consecutive doxorubicin injections over a time period of 21 days. there were no significant differences in the number of gh2ax foci or collagen accumulation. the mrna expression of ctgf was even higher in rac1 -/animals. furthermore the number of mitotic events was almost two times higher in the rac1 -/mice compared to the rac1 +/+ mice. summarizing, our findings show that impaired hepatic expression of rac1 protein is hepatoprotective against acute damage following doxorubicin exposure, but does not protect against doxorubicin-induced subacute toxicity. in vitro cytotoxicity of tbhq (tert-butyl-hydroquinone) braeuning a., vetter s., schwarz m. institut für experimentelle und klinische pharmakologie und toxikologie toxikologie, wilhelmstrasse 56, 72074 tübingen, germany at high concentrations, tert.-butyl-hydroquinone (tbhq), a phenolic antioxidant frequently used as a food preservative, exerts cytotoxic effects, which are closely linked to its ability to form reactive oxygen species as a consequence of redox cycling processes. here we describe that treatment of murine 3t3 cells with tbhq in 96-well culture plates induces the death of untreated cells in neighboring wells on the same plate. the mechanisms underlying that effect were investigated. death of the seemingly untreated neighboring cells was caused by a more toxic and volatile tbhq oxidation product which was formed in a non-enzymatic process involving metal ions and oxygen. the unexpected perturbation of cytotoxicity testing by the volatile tbhq metabolite shows that not only metabolic processes, but also non-enzymatic mechanisms have to be considered as important parameters for in vitro assays. furthermore, our data show that even cells several wells distant from the site of treatment do not necessarily constitute proper "untreated" controls when cells are treated with tbhq, e.g. in assays aimed to analyze the activity of the tbhq-inducible nrf2 pathway. s14 056 angiotensin ii causes oxidative stress and dna damage in mouse kidneys via the angiotensin ii type 1 receptor brand s. 1 , amann k. 2 , schupp n. 1 1 universität würzburg institut für pharmakologie und toxikologie, versbacherstr. 9, 97078 würzburg, germany 2 universität erlangen-nürnberg institut für pathologie, krankenhausstraße 8, 91054 erlangen, germany angiotensin ii (ang ii), the reactive peptide of the renin-angiotensin-system, causes vasoconstriction and, in higher levels hypertension, which is connected with an increased cancer risk in the kidney. treatment of male c57bl/6 mice with ang ii results in the formation of superoxide radicals and dna damage in the kidney as well as in the heart. to answer the question if the dna damage is caused by hypertension or by elevated ang ii concentrations, mice were treated with different compounds: the angiotensin-converting-enzyme blocker ramipril, the ang ii receptor blocker ramipril, the ang ii receptor candesartan, the antioxidant tempol and the vasodilator hydralazine. the effect on blood pressure and renal function of ang ii-treated c57bl/6 mice was examined. treatment with ang ii led to a significant increase in blood pressure. candesartan and hydralazine led to a decrease, whereas intervention with ramipril and tempol had no effect. equal conditions could be found by examining renal function regarding the excretion of urinary albumin, which was ameliorated by candesartan and hydralazine. in addition, histopathological changes were investigated. there was significant glomerular damage and tubulointerstitial damage in ang ii-treated animals compared to control animals, which was significantly improved by candesartan and tempol. hydralazine and ramipril mitigated the observed renal damage but were less effective than candesartan. furthermore, the ang ii-induced formation of superoxide radicals in the kidney and the heart was slightly affected by all interventions. genomic damage, in the form of double strand breaks was prevented by the ang ii receptor antagonist candesartan and the antioxidant tempol. to sum up, the results from this study show that ang ii induces the elevation of markers of kidney failure and dna damage, which is prevented by substances lowering blood pressure like candesartan, showing the receptor responsibility for the induction of dna damage. actually by substances not lowering blood pressure like tempol, the oxidative stress and dna damage was ameliorated, showing the involvement of reactive oxygen species. optimization of the balb/c-3t3 cell transformation assay by coupling a drug metabolizing system brauneis m. d., steinberg p. stiftung tierärztliche hochschule hannover institut für lebensmitteltoxikologie und chemische analytik, bischofsholer damm 15, 30173 hannover, germany the analysis of the carcinogenic potential of chemicals plays an important role in toxicology. up to now the acquisition of such data requires a large amount of animal experiments. the aim of this study is to reduce the number of experimental animals being used by further optimizing the balb/c-3t3 cell transformation assay, an already well-established in vitro method. this method, which is also well suited for high throughput screening applications, allows a quantitative analysis of the aforementioned carcinogenic potential. the incubation of balb/c-3t3 cells (murine embryonic fibroblasts) with mutagenic compounds leads to a loss of contact inhibition between these cells, which results in the development of so-called foci. these foci can be distinguished by characteristic changes in cell growth behaviour, a result of the treatment with carcinogenic compounds, and their number is therefore directly related to the genotoxic potential of the latter. a major disadvantage of the "classic" balb/c-3t3 cell transformation assay is that a number of compounds initially require a metabolic transformation to gain their full genotoxic potential. hence, without prior metabolic transformation many chemicals are not detected as carcinogenic in the abovementioned test system. to overcome this drawback the balb/c-3t3 cell transformation assay has been coupled to a drug metabolizing system, in this case the so-called liver s9. in a first step the well-known genotoxic agents benzo[a]pyrene, aflatoxin b 1 and nnitrosodimethylamine were tested in this assay. all three compounds led to a concentration-dependent increase in the number of foci formed, whereby this concentration-dependent increase was observed in a non-cytotoxic concentration range. in a next step the balb/c-3t3 cell transformation assay will be coupled to further drug metabolizing systems as well as to the soft agar assay. this study is being financially supported by the stiftung set and the doerenkamp-zbinden foundation. adenylyl cyclases (acs) synthesize the second messenger camp. the family of acs consists of nine membranous and one soluble isoforms with ac5 and ac6 being the predominantly expressed isoforms in the heart. in the heart, acs integrate β-adrenergic (β-ar) signaling as the main physiological mechanism to improve cardiac performance. although ac5 and ac6 share high sequence homology, opposing effects on cardioprotection have been reported, where disruption of ac5, as well as overexpression of ac6 both exerting beneficial effects in heart failure. prospective pharmacological treatment of heart failure on the level of ac is under investigation. our study explored the impact of ac5 ko on ac-activities in the heart at a functional level. complementary, mrna expression studies of the β-ar-g-protein-ac signaling cascade were performed to detect possible compensatory alterations. hearts from 16-20 week old homozygote ac5 knockout and wild-type male littermates were examined in this study. ac activities where measured in cardiac membrane preparations from left ventricles. ac activities were assessed under β-ar and g-protein (g s) stimulation by isoproterenol, guanosine 5'-triphosphate (gtp) and 5'-o-(3thiotriphosphate) (gtpγs) as well as for direct activation by forskolin. relative mrna expressions for ac1-9, gs-, gi-a and β1-, β2-ar where measured by quantitative realtime pcr. surprisingly, assessment of basal, β-ar and g-protein-mediated ac-stimulation as well as direct activation by forskolin revealed no changes in ac activities. besides from detection of the ac5 knockout, mrna expressions analysis of ac1-9, gs-, gi-a and β1-, β2-ar did not detect any compensatory alteration. these findings suggest that proximal adrenergic signaling in the heart does not necessarily require ac5. whether physiological integration of beta adrenergic signaling in the heart is mediated by both isoforms ac5 and ac6, or can be attributed to one main isoform remains to be elucidated. melanocortin-promoted pka activation decreases ampk activity via erk-1/2 and lkb-1 in hypothalamic gt1-7 cells breit a., ellen d., gudermann t. goethestrasse 33, 80336 münchen, germany α-melanocyte stimulating hormone (α-msh)-induced activation of the melanocortin-4 receptor (mc4r) in hypothalamic neurons increases energy expenditure and inhibits food intake. intrahypothalamic injection of melanocortins decreased food intake due to the inhibition of amp-activated protein kinase (ampk) that has recently been reported to enhance food intake in rodents. until now, it is not clear if α-msh affects ampk via direct intracellular signaling cascades or if the release of paracrine factors is involved. herein, we used a murine, hypothalamic cell line (gt1-7 cells) and monitored ampk phosphorylation at thr 172 which has been suggested to increase ampk activity. we found that α-msh dephosphorylated ampk at thr 172 and consequently decreased phosphorylation of the established ampk substrate acetyl-coa-carboxylase at ser 79 . inhibitory effects of α-msh on ampk were blocked by specific inhibitors of protein kinase a (pka) or extracellular-regulated kinases-1/2 (erk-1/2), pointing to an important role of both kinases in this process. since α-msh-induced activation of erk-1/2 was blunted by pka inhibitors, we propose that erk-1/2 serves as a link between pka and ampk in gt1-7 cells. furthermore, down-regulation of liver kinase b-1 (lkb-1), but not inhibition of calcium-calmodulin-dependent kinase kinase-β or transforming growth-factor-beta-activated kinase-1 decreased basal phosphorylation of ampk and its dephosphorylation induced by α-msh. thus, we propose that α-msh inhibits ampk activity via a linear pathway including pka, erk-1/2 and lkb-1 in gt1-7 cells. given the importance of the melanocortin system in the formation of adipositas detailed knowledge about this pathway might help to develop drugs targeting obesity. autism spectrum disorder (asd) is a complex neurodevelopmental disorder with dysfunction of social interaction and communication. a hitherto unknown complexgenetic principle of origin probably underlies asd. so far, more than 100 candidate genes were identified in literature. the patients affected with the monogenic timothy syndrome show multiorgan dysfunction including lethal arrhythmias, immune deficiency, skeleton-dysplasia, syndactylia and autism. this single gene disorder serves as a model disease for asd, giving insights in a possible pathophysiology. here, a point mutation in a highly-conserved region of the pore-forming subunit of the voltage-dependent calcium channel (ca v) cav1.2 gene (cacna1c) results in incomplete inactivation of the l-type calcium currents (splawski et al., cell 2004; 119:19-31) . functionally similar biophysical effects can be induced by structural variation β1-and β2-subunits of the voltagedependent calcium channels (herzig et al., faseb j. 2007; 21:1527-38; jangsangthong et al., pflugers arch. 2010; 459:399-411) . supported by findings in a meta-analysis of linkage data of asd patients (trikalinos et al., mol psychiatry. 2006; 11:29-36) , we are investigating a function-based candidate gene hypothesis linking the β2 subunit gene (cacnb2) with asd. we performed a case control study sequencing all exons and flanking intronic regions of cacnb2 in 155 patients with asd. we found three rare missense mutations in asd patients, but not in 259 unaffected controls. all three mutations occur at highly conserved positions and might alter protein function; additionally results one amino acid substitution highly probable in a post-translational modification by phosphorylation. so far, we characterized two of these mutations and also a phosphorylation-mimicking mutant in electrophysiological studies. all variants show a decelerated and incomplete time-dependent inactivation of the co-transfected cav1.2 subunit. furthermore, two variants exhibit a significant increased slope factor of voltage-dependent steady-state inactivation. we here present mutations in the β2 subunit gene of asd patients that result in a retardation of inactivation behavior, thus phenocopying the monogenic timothy syndrome mutations of cav1.2. β2 subunit mutations may influence neuronal function or development in some asd patients. jangsangthong, w., kuzmenkina, e., khan, i.f., matthes, j., hullin, r., and herzig, s. (2010) . inactivation of l-type calcium channels is determined by the length of the n terminus of mutant beta (1) subunits. pflugers arch 459, 399-411. herzig, s., khan, i.f., grundemann, d., matthes, j., ludwig, a., michels, g., hoppe, u.c., chaudhuri, d., schwartz, a., yue, d.t., et al. (2007) . mechanism of ca(v)1.2 channel modulation by the amino terminus of cardiac beta2-subunits. faseb j 21, 1527-1538. splawski, i., timothy, k.w., sharpe, l.m., decher, n., kumar, p., bloise, r., napolitano, c., schwartz, p.j., joseph, r.m., condouris, k., et al. (2004) . ca(v)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. cell 119, 19-31. trikalinos, t.a., karvouni, a., zintzaras, e., ylisaukko-oja, t., peltonen, l., jarvela, i., and ioannidis, j.p. (2006) . a heterogeneity-based genome search meta-analysis for autism-spectrum disorders. mol psychiatry 11, [29] [30] [31] [32] [33] [34] [35] [36] background: brain serotonin (5-ht) has been implicated in the regulation of food-intake. the ingestive effects of 5-ht are mediated by a number of different receptor subtypes under which the 5-ht1a-receptor plays a central role. former in vivo studies have shown an increased intake of food, elicted by 5-ht-receptor agonists. the aim of this behavioural pharmacologic project was to determine if the hyperphagic effect is mediated by presynaptic 5-ht1a autoreceptors in the raphe nuclei or by postsynaptic 5-ht1a heteroreceptors in serotonergic terminal structures. methods: the effect of the 5-ht1a receptor agonist 8-oh-dpat (0.1, 0.5 or 1.0mg/kg) was investigated on feeding behaviour in non-food-deprived young-adult and adult nmri and transgenic l35 mice. l35 mice are characterized by an overexpression of postsynaptic 5-ht1a receptors. results: the administration of the 5-ht1a receptor agonist induced hyperphagia in all groups of mice, except for the adult transgenic mice which showed no drug effect. conclusion: the results confirm a key role of the 5-ht1a receptor in food intake. further, we make the assumption that the hyperphagic effect of 8-oh-dpat is mediated by presynaptic 5-ht1a autoreceptors in the raphe nuclei which decreases 5-ht function in the central nervous system. it can be speculated that the aberrant feeding behaviour of the adult transgenic mice refers to a possible opposite role of the postsynaptic 5-ht1a receptors. these receptors might affect the release of neuropeptides in the hypothalamus. the efflux transporter abcc2 (mrp2) expressed at different compartment barriers is important for the elimination of various endogenous and exogenous compounds. with some evidence inflammatory processes regulate abcc2 expression and cause changes of absorption, distribution and clearance of a number of xenobiotics. the investigation of the influence of interleukin (il) 1β on abcc2 mrna and protein expression in various cell lines representing specific tissues is the aim of our study. a further aim is to characterize the signaling pathways regulating abcc2 expression while inflammation. three different cell lines a) hepg2 cells (liver tissue) and b) caco2 (colon tissue) both without naïve il1β expression and c) skhep1 cells representing physiological liver tissue with naive il1β expression, were stimulated with different concentrations of il1β (range 10 pg/ml to 10 ng/ml). over a period of 48h samples were taken at defined time points. abcc2 mrna and protein expression were quantified by qrt-pcr and western blot analysis, respectively. by using small molecule kinase inhibitors for signal transduction proteins (p38 mapk, akt, erk1 and jnk) we analysed the signal transduction pathways associated with il1β-mediated transcriptional abcc2 regulation. on abcc2 mrna level an up-regulation in caco2 cells (1, 35 fold) and hepg2 cells (3, 6 fold) within the first hour after stimulation with 1 ng/ml il1β was shown. in contrast skhep1 cells demonstrated a decreased abcc2 mrna expression (0,59-0,62 fold) in comparison to unstimulated controls. the abcc2 protein expression exhibited a time and il1β dependent regulation as well. the analysis for the signal transduction showed for p38mapk a moderat time dependet down regulated phosphorylation (15%) in hepg2 cells whereas it showed no effect in caco2 cells. concluding, the expression of abcc2 is regulated moderately by il1b in a concentration and time-dependent manner. interestingly, the effects are strongly tissue-dependent concerning abcc2 expression and signal transduction pathways and show partly contradictory results. the regulation of the different signaling pathways is currently subject of ongoing investigations. introduction: despite the remarkable success of imatinib treatment of chronic myeloid leukemia (cml), therapy resistance emerged as a major clinical problem. the aim of this study was to identify micrornas, which may serve as biomarkers for therapy response or predict pathways involved in pharmacoresistance of imatinib treatment. methods: blood was collected from 21 cml-patients, ten of whom responded to imatinib therapy. after rna extraction from leukocytes, we performed a taqman low-density array screen to determine the expression of 667 micrornas. statistical analysis using the 2 -∆ct method was performed. micrornas showing a p-value<0.01 and a fold change>2 were considered to be significantly differently expressed. in addition, by using microrna target prediction databases (targetscan 1 , mirdb 2 , pictar 3 , microcosm 4 , diana microt 5 ), selected putative target genes were further functionally investigated by the david bioinformatics database 6 . results: comparing treatment-naïve responders and non-responders four micrornas were identified to be deregulated that were predicted to target 97 genes, especially transcription regulators (21%). pathway analysis showed that six of the predicted genes are relevant in cancer pathways, four of which play a role in cml (smad4, nras, rb1, raf1). when comparing patients' expression profiles before and under treatment, seven micrornas were identified to be deregulated in responders and five micrornas in nonresponders. ninety-nine targets of the latter include transcription regulators (19%), but also cellular transporters (18%, especially uptake transporters of the slc-family). most target genes are involved in mapk signalling or endocytosis pathways. conclusion: analysis of microrna expression profiles revealed four micrornas involved in imatinib-response and 12 micrornas deregulated during imatinib treatment. predicted target genes code mainly for transcription factors as well as oncogenes relevant for cml and are involved in transporter expression and endocytotic processes. dissociations in the effects of beta2-adrenergic receptor agonists on camp formation and superoxide production in human neutrophils brunskole i. 1 , buschauer a. activation of the β2-adrenergic receptor (β2ar), a classically gs-coupled receptor, in neutrophil granulocytes results in an inhibition of inflammatory responses [1] , which could be further therapeutically exploited. the aim of the present study was to evaluate the effects of various β2ar ligands on cyclic adenosine 3',5'-monophosphate accumulation (camp assay) and n-formyl-l-methionyl-l-leucyl-l-phenylalanine (fmlp)induced superoxide anion production (o2 •assay) in isolated human neutrophils, which are a physiologically relevant native test system. camp concentration in neutrophils was determined by hplc/tandem mass spectrometry, and o2 •formation was assessed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. (-)-isoproterenol, (-)-adrenaline, salbutamol and dobutamine were more potent in inhibiting fmlp-induced o2 •production than in stimulating camp accumulation. (-)-ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the camp assay, but could partially inhibit fmlp-induced o2 •production at higher concentrations. moreover, (-)-adrenaline and dobutamine were equi-efficacious in both assays whereas the efficacy of salbutamol was more than two fold higher in the o2 •assay. this suggests that salbutamol is able to stabilize a different receptor conformation than the other two ligands. thus, ligand-directed signaling via β2ar can also occur in human neutrophils. in addition, differences between the data from neutrophils and recombinant test systems [2, 3] were noticed, pointing to the problem of insufficient comparability of effects in recombinant and native test systems. the investigation of β2ar antagonists on neutrophil granulocytes is subject of ongoing work, in order to find out whether pkb values of β2ar antagonists in the camp assay and the o2 •assay are different. such differences were previously reported for β2ar antagonists in other test systems [4] . moreover, studies with protein kinase a inhibitors should give deeper insight into the signaling events in neutrophils that result in inhibition of fmlp-stimulated o2 •production and clarify how camp increase interferes with this events. agonist-selective internalization of the human 5-ht2a receptor buchborn t., kahl e., höllt v., koch t. otto-von-guericke-universität magdeburg institut für pharmakologie und toxikologie, leipziger straße 44, 39120 magdeburg, germany the serotonin 2a (5-ht2a) receptor is a g protein coupled receptor and the molecular target of lsd-like hallucinogens. downregulation of 5-ht2a receptors is an adaptive process considered relevant for the therapeutic action of diverse serotonergic antidepressants, such as ssris. since the antidepressant targeting of 5-ht2a receptors, however, is largely restricted to indirect agonists and/or antagonists, little is known about the mechanisms and implications of their regulation by direct agonists. in the present study we, therefore, investigated the capacity of various agonists to regulate the human ha-tagged 5-ht2a receptor by internalization. using immunocytochemical techniques in stably transfected hek293 cells, we show that agonists differ in their capacity to internalize the receptor. serotonin, quipazine and doi are the agonists most efficaciously internalizing the receptor, dmt and methysergide, on the other hand, hardly internalize; other agonists like psilocin, ergotamine and lsd induce low to intermediate internalization. the specificity of the agonistic effect was demonstrated by the 5-ht2a selective antagonist ketanserin, which blocked the agonist-induced internalization. in additional experiments, we show that the internalized 5-ht2a receptors colocalize with a488-labelled transferrin receptors, and that the internalization can be blocked by high molar sucrose; these results are indicative of a clathrin associated sequestration of 5-ht2a receptors in recycling endosomes. also, we demonstrate that the proteinkinase c activator pma efficaciously induces 5-ht2a internalization in the absence of an agonist, and that the doi-induced internalization can be blocked by the proteinkinase inhibitor staurosporine. we, thus, confirm previous findings that the activation of proteinkinases seems to be necessitated for the 5-ht2a internalization to occur. overall, we conclude that the internalization of the human 5-ht2a receptor is agonist-selective, and employs a proteinkinase (possibly pkc) dependent, clathrincoated endosome associated pathway. as there is recent evidence that the regulation of 5-ht2(a) receptors by agonists might have antidepressant (-like) properties, knowledge about the agonist-selective processing of 5-ht2a receptors could help to identify agonists most promising for future (pre-)clinical research. non-clinical safety assessment of homeopathic medicinal products: criteria for establishing a first safe dilution buchholzer m. -l., werner c., knoess w. bfarm bundesinstitut für arzneimittel und medizinprodukte zulassung 4, kurt-georg-kiesinger-allee 3, 53175 bonn, germany like all human medicinal products the homeopathic medicinal products for human use must demonstrate adequate safety. in general, they are regulated according to the analogue non-clinical safety principles (points to consider on non-clinical safety of homeopathic medicinal products of botanical, mineral and chemical origin, adoption by hma 2007). one particular approach is the recently introduced concept of a first safe dilution (fsd; introduction to the list of first safe dilutions, adoption by hma 2010) . this contribution summarizes the first experiences in establishing fsds of a selection of given homeopathic preparations by bfarm. for a given preparation the major toxicological concern and available data set is identified. this determines the safety assessment route: food regulation, permitted daily exposure (pde), threshold of toxicological concern (ttc) or lowest human recommended dose (lhrd/100). finally the acceptable amount/tolerable daily intake is derived and the respective fsd is calculated. for example the draft evaluation for reserpinum (ph. eur. method 4.1.1) and for atropine (ph. eur. method 3.1.1 or 4.1.1) based on lhrd leads in each case to a suggested fsd of d7 related to 10 g of preparation. furthermore, the draft evaluation for potassium iodide (ph. eur. method 3.1.1 or 4.1.1) based on food legislation emerged a proposed fsd of d6 related to 10 g of preparation. the concept of fsd combines a scientific and at the same time pragmatic approach in differentiated risk assessment of homeopathic medicinal products. impact of myricetin and its methylated derivatives laricitrin, syringetin and myricetin-3`,4`,5`-trimethylether in c. elegans büchter c. 1 , ackermann d. polyphenolic compounds ubiquitously present in herbal food are discussed to contribute to the health beneficial effects of a diet rich in vegetables and fruits. additional to a strong antioxidative activity of various flavonoids, most of these substances display a variety of other pharmacological properties. we investigated the flavonoid myricetin found in several species of berries, as well as the methylated derivatives laricitrin, syringetin and myricetin-3`,4`,5`-trimethylether. in this study caenorhabditis elegans was used as a model to explore the impact of myricetin and its methylated derivatives in vivo and to investigate molecular modes of action. myricetin (100 µm) caused an increase in mean and median adult lifespan of c. elegans. this longevity effect was associated with a decrease of the aging marker lipofuscin as well as a decrease in ros induction, measured by using the h 2dcf-da assay. however, myrictin failed to improve heat stress resistance, an attribute often associated with longevity in c. elegans. the methylated myricetin derivatives (100 µm) showed a decrease in lipofuscin accumulation and ros induction and they further improved the heat stress resistance. in order to elucidate the basis of the life prolonging action of myricetin, we investigated its influence on factors known to have important functions in stress response and the regulation of aging, namely the foxo homologue daf-16, the nad + -dependent protein deacetylase sir-2.1 and the heat-shock transcription factor hsf-1, respectively. lifespan extension by myricetin disappeared in daf-16 and sir-2.1 loss of function mutant strains, showing the effect is at least partially dependent on these signaling molecules. by using a hsf-1 loss of function mutant strain of c. elegans, it was further shown that the life prolonging effect of myricetin is independent of hsf-1. in conclusion, our results indicate that the life prolonging effect of myricetin is at least in part dependent on daf-16 and sir-2.1, probably due to a modified expression of target genes. stimulatory and inhibitory control of phospholipase c-gamma 2 bühler a., walliser c., becker l., gierschik p. universität ulm institut für pharmakologie und toxikologie, albert-einstein-allee 11, 89081 ulm, germany activation of phospholipase c-γ2 (plcγ2) upon b cell antigen receptor (bcr) stimulation has been implicated to be a critical step in the bcr-mediated calcium signaling. therefore it is important to understand the mechanisms of how the activity of plcγ2 is stimulated and inhibited. the mammalian plcs are divided into six subfamilies, designated β, γ, δ, ε, ζ, and η. within the plcγ subfamily, the two plcγ isoforms share a number of features that are distinct from those of the other plc subfamilies. the most striking difference is the insertion of additional domains between the catalytic subdomains x and y. this specific array (sa) contains a second, split pleckstrin homology (spph) domain, consisting of two halves separated by two src homology 2 (sh2) domains and one src homology 3 (sh3) domain. there is abundant evidence in the literature that plcs are autoinhibited in their basal state by structural elements within their x/y linker, pointing to a conserved role of the x/y linker in autoinhibitory regulation of plc isozymes. data from our group show that plcγ 2 is also regulated by autoinhibitory elements within its specific array (walliser et al., 2008; everett et al., 2011) . our recent data demonstrated that plcγ2 is negatively regulated by its sa. specifically, within the sh domain tandem, the c-terminal sh2 (sh2c) and the sh3 domain in combination, but not either one alone, cause the strongest autoinhibitory control of plcγ2. plcγ2 has been shown to be phosphorylated at tyrosine residues 753 and 759 upon bcr stimulation (kim et al., 2004) . both tyrosines are located in the linker between the sh2c and the sh3 domain, which we have shown to be the major elements involved in autoinhibitory regulation of plcγ2. interestingly, a novel phosphorylation site in plcγ2 was found in non-small cell lung cancer (nsclc) tissue which is located at tyrosine residue 733 (rikova et al., 2007) . in this work, we demonstrate, for the first time, the activation of plcγ2 by phosphomimetic mutations in these three positions and the functional interplay of the three tyrosine phosphorylation targets. most interestingly, mimicking phosphorylation of tyr733 is critical to fully activate the enzyme. the results not only point to a crucial role of plcγ2 in pulmonary tumorigenesis, but also prompt and stimulate the search for the protein kinase involved in phosphorylating plcγ2 at tyr733. molecular characterization of hepatotoxic effects of perfluorooctanoic acid (pfoa) buhrke t., scharmach e., lampen a. bundesinstitut für risikobewertung (bfr) lebensmittelsicherheit, max-dohrn-str. 8-10, 10589 berlin, germany perfluorooctanoic acid (pfoa) is an industrial chemical that is used for the fabrication of numerous products with oil-, dirt-and water-repellent properties. pfoa is resistant to chemical, thermal and biological degradation and has become a global contaminant of soil, water, air and food in the meantime. the toxicological data of pfoa give cause for concern as the substance was shown to damage the liver of rodents and to impair embryo development. currently, the hazard potential of pfoa for humans is controversially discussed. in this study the human liver cell line hepg2 was employed to analyse the hepatotoxic effects of pfoa on the cellular and on the molecular level. pfoa was shown to stimulate cellular proliferation at concentrations in a range between 5 µm and 25 µm. at concentrations higher than 25 µm the substance was cytotoxic to the cells (ic 50 47µm). cytotoxicity was not due to apoptotic mechanisms as no increase of caspase activity was detected up to a level of 100 µm pfoa. on the molecular level pfoa is known to act as an agonist of the peroxisome proliferator-activated receptor alpha (pparα), and the observed hepatotoxic effects in rodents are associated with pfoa-mediated pparα activation. here we show that pfoa has the capacity also to activate the human isoform of pparα. additional human nuclear receptors were tested for activation by pfoa, and pparγ as well as the pregnane x receptor (pxr) were shown to be activated at high concentrations of pfoa whereas pparδ and the liver x receptor alpha (lxrα) were insensitive to activation by pfoa. notably, we observed a significant inhibition of the activity of the hepatocyte nuclear factor 4α (hnf4α) by incubating the cells already with moderate concentrations of pfoa at a level of about 1 µm. these findings indicate that additional, pparα-independent mechanisms may contribute to the observed hepatotoxicity of pfoa. the elucidation of novel modes of action of pfoa is relevant for the ongoing risk assessment of the substance. s17 070 human breast stem cells as a toxicological model for endocrine disruptors, such as soy isoflavones stempin s. 1 , bumke scheer m. (kao et al., 1995) . two daughter cell lines were developed from m13sv1 after x-ray irradiation (m13sv1 r2) and an additional transfection with a mutated erbb2 oncogene (m13sv1 r2-n1), resulting in high and low tumorigenicity respectively and showing a change in estrogen response after growth in minimal media (wang et al., 2010) . isolated isoflavones are currently widely used in the treatment of postmenopausal symptoms of women. according to the stem cell theory of carcinogenesis, breast stem cells are the ideal target for the proposed research. however, the epidemiological data to the effect of isoflavone intake on breast cancer is contradictory. therefore, we want to develop a toxicological model using these human breast stem cell lines to test the effect of endocrine disruptors, such as soy isoflavones in a human relevant model. in the present study we analyzed the effect of the phytoestrogen genistein, the most intensively studied soy protein, on the differentiation of the 3 hbec lines. the expression of different luminal epithelial cell markers, estrogen receptors and stem cell markers was measured on the mrna level by quantitative real time pcr. the analysis of several of these markers was also performed on the protein level using western blot. additionally, a broad number of genes related to breast cancer and estrogen receptordependent signal transduction were studied using a commercial pcr-array. in parallel we are also analyzing the changes on the protein level using 2d gel electrophoresis. we want to use this panel of different markers to establish a toxicological model that can be used in the future to analyze a wide range of different endocrine disruptors. kao cy, nomata k, oakley cs, welsch cw, chang cc. (1995) bronchial asthma is a common inflammatory disease of the airways whose occurrence has increased dramatically over the past decades. histamine plays an important role in mediating the inflammatory response leading to characteristic symptoms like wheezing, coughing, chest tightness, and shortness of breath. since antagonizing the histamine h1-receptor (h1r) shows no ameliorating effects on asthmatic symptoms, h4r antagonists may be new drugs for asthma therapy. in addition to the h3r-and h4rselective antagonist thioperamide, the selective h4r antagonist 1-[(5-chloro-1h-indol-2yl)carbonyl]-4-methylperazine (jnj7777120) is used in pharmacodynamic studies. a correct interpretation of the collected data requires the detailed knowledge of the pharmacokinetics of the applied substances. for this reason, we developed a fast and robust method based on high performance liquid chromatography coupled to tandem mass spectrometry (hplc-ms/ms) which allows the simultaneous quantitation of thioperamide and jnj7777120 as well as the selective h3r antagonist 1-({4[3-(piperidin-1-yl) propoxy]phenyl}methyl)piperidine (jnj5207852) in murine plasma and lung tissue. the treatment of plasma samples based on protein precipitation performed with a mixture of methanol and 0.2 m znso4 using 30 µl of plasma. analyte extraction from lung tissue was achieved by treating 150 -200 mg of tissue with a mixture of ethanol and water followed by rigorous mixing using a fastprep-system. ten µl of the extracted samples were transferred to a synergy polar-rp 80a mercury column (10 x 2mm; 4µm) connected to a polar-rp security guard. chromatographic separation was performed via a gradient using an acetate buffer (ph 5) and methanol at a flow rate of 0.4 ml/min. the analytical run-time was 5 minutes. for plasma samples the assay was linear over a concentration range of 0.078 -40 µg/ml for jnj7777120 and jnj5207852, and 0.313 -40 µg/ml for thioperamide, respectively. in tissues, thioperamide could be quantified in a concentration range of 0.008 -2 µg/sample, jnj7777120 and jnj5207852 in a range of 0.004 -2 µg/sample. our results show that the developed hplc-ms/ms method is suitable for the quantitation of all tested histamine-receptor ligands in murine plasma and lung tissue. the functionality of the heart greatly depends on strict homeostasis and interplay of a range of signalling cascades. deregulation of either one is always harmful and eventually detrimental for life. some of the most relevant signals in the adult heart are triggered by the stimulation of g protein-coupled receptors such as adrenergic or angiotensin receptors. those in turn are modulated by a small subset of kinases, the g protein-coupled receptor kinases (grks). interestingly, grks, which for the longest time were believed to regulate only g protein-coupled receptors were shown to modulate also non-receptor-mediated signalling pathways. by now it is well documented that grk5 plays important roles in both the physiological as well pathological setting of the adult heart. in spite of the important functions of grks in the adult heart, it must be assumed that grk5, one of the two main cardiac grks, may also be involved in signal modulation in the course of heart development. deregulation of grk5-dependent pathways may very well be causal for impaired cardiac development up to congenital heart disease. in fact, grk5 is already expressed during embryogenesis and we can detect it in the developing heart. however, grk5 has not been studied yet for a potential function during embryonic development in general or heart formation in particular. we have established zebrafish as a very time and cost efficient vertebrate model to investigate the role of grk5 on cardiac signalling and development. tools for grk5 specific loss-as well as gain-of-function analyses have been developed in our lab. we revealed an unexpected role of grk5 in the development of left-right asymmetry in zebrafish. clinically, this has been associated with disorders such as heterotaxy and other syndromes linked to ciliary dysfunction. many of those disorders are known to affect proper heart development resulting for example in septum defects or in detrimental translocation of the outflow tract. precisely, depletion of the close homolog of human grk5 in zebrafish mirrors the human syndrome called heterotaxy by displaying randomized placement of inner organs, aberrant heart looping and disrupted valve formation in the heart. in addition, loss of zebrafish grk5 results in a lower heart rate as well as dilatation of the embryonic heart at later stages of development. therefore, we believe that grk5 may potentially serve as a candidate gene for congenital heart disease. identification of a hcn3 interacting protein in mouse brain cao-ehlker x., hammelmann v., zong x., fenske s., biel m. department of pharmacy, center for drug research ludwig-maximilians-universität, munich center for integrated protein science cipsm, butenandtstr. [5] [6] [7] [8] [9] [10] [11] [12] [13] 81377 münchen, germany hyperpolarization activated cyclic nucleotide-gated cation channels (hcn) pass a depolarizing current (ih) that is involved in cardiac pacemaking and the control of numerous basic functions in neuronal circuits. the four hcn channel types (hcn1-4) display specific expression pattern in brain suggesting that each channel fulfills a distinct physiological function. while hcn1, hcn2 and hcn4 channels have been studied in quite some detail there is only little information on the particular role of the hcn3 channel. as an important step towards achieving a better understanding of hcn3 function we set out in this study to identify proteins that are assembled with hcn3 in brain tissue. to this end we performed a yeast two hybrid screen with a mouse cdna library using the hcn3 c-terminal domain as bait. several proteins were obtained and confirmed for interaction with hcn3 using heterologous coexpression in hek293 cells. here, we provide an in-depth analysis of the functional interaction between hcn3 and one of the identified interacting proteins (hip3.1). we show that hip3.1 physically binds to hcn3 channels in vitro and in vivo. still several open issues remain to be clarified i.e. the precise function of tpc1 and its tissue-specific and subcellular distribution. therefore we established a mouse model with a general deletion of tpcn1 and generated a series of tpc1 antibodies. using these tools we investigate the closer molecular and vesicular environment by different biochemical approaches i.e. affinity purification from native tissue derived from wild-type and as a control from knock-out mice, density gradient based vesicle separation, fluorescence activated organelle sorting (faos), total internal reflection fluorescence (tirf) and confocal microscopy. so far, we confirmed the tpc1 knock-out model by our self-generated tpc1 antibodies. tpc1 knockout mice are viable and do not show any obvious deficits. to isolate tpc1 containing vesicles or protein complexes, tissue or cell culture derived material was prepurified by sucrose density gradient centrifugation. for a subsequent mass spectrometric analysis this preparation was taken as a source material for coimmunoprecipitation or faos respectively. in another approach the migration pattern of tpc1 containing endosomes on linear density gradients was compared with a series of endolysosomal markers i.e. different rab-, er-, golgi-and lysosomal antibodies. potentially interesting markers were then in turn analyzed for their co-localization with tpc1 by confocal microscopy/tirf. by combining these results with that from mass spectrometric analysis of faos samples we collect data to get detailed information on the precise endolysosomal distribution pattern of tpc1. foxos are involved in a wide spectrum of cellular functions, including cell proliferation, apoptosis and regulation of oxidative stress. in order to identify novel target genes of foxo transcription factors and to achieve further insight into their role in cancer cells, dna microarray analysis was performed using wild type mcf-7 breast cancer cells and mcf-7 cells overexpressing foxo. we found that several genes involved in the tnf receptor/nf-κb pathway were differentially regulated. one of the genes that was identified to be up-regulated in foxo4 overexpressing cells was a20 a negative regulator of nf-κb signaling pathway. at both mrna and protein level foxo4-dependent up-regulation of this ubiquitin modifying enzyme was confirmed. to determine whether a20 is a direct target of foxo4, a luciferase reporter containing a 1.2 kb of the a20 promoter was co-transfected with different amounts of foxo4 wild-type expression construct. foxo4 induced a dosedependent increase in a20 promoter activity, supporting the assumption that a20 is a direct transcriptional target of foxo4. overexpression of foxo4 led to decreased activity of nf-kb signaling pathway as confirmed by reporter gene and nf-kb specific elisa assays. in addition, mcf-7 cells can be sentisized to tnf-α mediated cytotoxicity which is assocciated with a dimineshed activation of nf-κb. altogether, we identified a20, an ubiquitin modifying enzyme, as a novel foxo4 target gene. our data implicate that sustained foxo4 expression may be involved in regulation of tnf receptor/nf-κb pathway and leading to reduced cell survival. trpm7 is a bi-functional protein consisting of a transient receptor potential ion channel segment linked to an α-type protein kinase domain. trpm7 is essential for motility, proliferation and cell growth. up-regulation of trpm7 function is involved in anoxic neuronal death, cardiac fibrosis and tumor cell proliferation. recently, we have demonstrated that the recombinant trpm7 channel is inhibited by the known modulators of sk1-3 channels such as antimalarial plant alkaloid quinine, cyppa, dequalinium, ns8593, ska31, ucl 1684. the most potent of these compounds, ns8593 (ic50 1.6 µm), interferes with the regulation of trpm7 by cytosolic mg 2+ . here we show that ns8593 (10 µm) fully and reversibly inhibits native trpm7-like currents in hek 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes. furthermore, we examined whether targeting of the native trpm7 currents by ns8593 would impact cellular processes known to be affected by a genetic inactivation of trpm7. we found that ns8593 (10-30 µm) suppressed motility of hek 293 cells without a detectable effect on cell viability. taken together, our findings indicate that ns8593 is a potent and reversible inhibitor of endogenous trpm7 currents and may be a good candidate drug for pharmacological targeting of trpm7. sulfur mustard (2,2'-dichlorodiethylsulfide; sm) is a highly toxic and mutagenic warfare agent classified as a weapon of mass destruction. as soon as sm was first used as a warfare agent, research aimed at the development of an effective antidote was launched. early studies with first-generation inhibitors of poly(adp-ribose) polymerases (parp) have revealed promising therapeutic potential in sm-induced skin injury, but the underlying mechanism remains elusive. the current renaissance of parp inhibitors in cancer chemotherapy has revived the discussion on their use for treatment of sm injury. thus we established a comprehensive study aiming the elucidation of the role of parp in sm pathology based on model substance 2-chloroethyl ethyl sulfide (cees), which is not classified as warfare agent. we have recently demonstrated that parp becomes rapidly activated in living human keratinocytes (hacat) after treatment with cees. the maximal parp activity was observed 10 minutes after treatment with 3 mm cees. the activation was transient and dose dependent. to our knowledge this is the first demonstration of parp activation after treatment with mustards in the context of live cells. an important question is how parp-1 becomes activated upon treatment with mustards. parp-1 is a first-line protein involved in the cellular response to dna strand breaks. however, mustards do not directly induce large numbers of such lesions. one possibility is that parp-1 is activated by dna breaks incorporated as base excision repair (ber) or nucleotide excision repair (ner) intermediates. thus, we performed knockdown experiments of ape1 and ercc1, i.e. endonucleases involved in ber and ner, respectively. the reduction of ape1 expression had no effect on parp activity. surprisingly, the knockdown of ercc1 almost completely abolished the cellular par production after cees treatment. the functional consequence of the errc1-parp cross-talk with regards to adduct removal is under investigation. however, our present data indicate that parp activity is not obligatory for the survival of cells upon cees treatment, as revealed by the lack of effect of the potent parp inhibitor abt-888. expression and activity of g protein coupled receptor kinase 2 (grk2) are elevated in several conditions of compromised heart function. although grk2 inhibition has been characterized as a promising therapeutic strategy in heart failure, a specific grk2inhibitor is not available. raf kinase inhibitor protein (rkip) inhibits raf1 but it also acts as a physiological inhibitor of grk2 upon phosphorylation by pkc at serine153. a detailed understanding of the rkip/grk2 interaction may help to identify inhibitory compounds for grk2. since phosphorylation often induces homo-oligomerization of proteins, we investigated whether this could be implicated in switching rkip from a raf1-into a grk2-inhibitor. co-immunoprecipitation assays showed that rkip self-association was substantially increased after pkc-mediated phosphorylation of rkip. rkip mutants either lacking or mimicking s153 phosphorylation confirmed that this phosphorylation is indeed a prerequisite for rkip/rkip association. cross-linking experiments with myc-tagged rkip in living cells or with purified rkip revealed that rkip phosphorylation by pkc promotes rkip dimers -not oligomers. to test whether dimerization is a critical step for the association of rkip with grk2, we generated a peptide to inhibit rkip dimerization. intriguingly, the peptide did not only prevent rkip dimerization but also attenuated rkip/grk2 association. this implicates, that dimerization of rkip is essential to bind grk2. to determine whether rkip dimers consequently inhibit grk2 activity, we established rkip mutants with high tendency to form dimers. subsequent functional analyses demonstrated that enhanced dimerization of rkip indeed translates into increased grk2 inhibition. we conclude that pkc-mediated phosphorylation of rkip is important for dimerization and that these dimers are essential for grk2 binding and inhibition. our results reveal new insights in the molecular mechanism of rkip/grk2 interaction and will help to develop specific grk2 inhibitors. expression and function of trpm3 ion channels in epithelial mdck2 cells dembla s., meiser j., philipp s. university of saarland institute for experimental and clinical pharmacology and toxicology, kirrberger str. 1, 66421 homburg, germany trpm3 proteins build ca 2+ permeable cation channels [1] activated by steroids [2] and sensitive to increased temperatures [3] . trpm3 channels are expressed in pancreatic ßcells as well as neurons of the dorsal root ganglion, where they act as mediators of insulin release [2] or as nociceptors of noxious heat, respectively [3] . however, northern blots and in situ hybridization experiments revealed that trpm3 is also expressed in epithelial cells of the choroid plexus and the ciliary body [1] as well as in the kidney. pcr analysis of different epithelial cell lines indicated that trpm3 is also expressed in madin-darby canine kidney2 (mdck) cells. quantitative analysis of trpm3 expression by qrt-pcr revealed a ~ 5 fold upregulation in mdck2 cells grown in confluency compared to well separated, proliferating cells. in contrast the level of expression of trpm7, a related ion channel described as regulator of proliferation in other cell types, remained constant. hek293 cells overexpressing trpm3 channels did not proliferate in the presence of the trpm3 agonist pregnenolone sulphate. however, as indicated by impedance analysis, the proliferation of mdck2 cells in the presence pregs was only slightly affected. when we analysed the transepithelial resistance (ter) of mdck2 epithelial cells in transwells as a measure for the formation of tight junctions, we found that the ter of cells grown in the presence of pregs was reduced. interestingly, ca 2+ imaging experiments using fura2 revealed that pregnenolone sulphate induces ca 2+ entry in well separated mdck2 cells but not in cells growing in confluency. we hypothesize that trpm3 might act as a regulator of cell proliferation and/or the formation of tight junctions in mdck2 cells. inhibition of grk2 by rkip improves cardiac contractility and structure in a transgenic mouse model of heart failure denzinger s., schmitt j. p., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. 9, 97078 würzburg, germany the raf kinase inhibitor protein (rkip) has been identified as a physiological inhibitor of g-protein coupled receptor kinase 2 (grk2). grk2 initiates g protein coupled receptor (gpcr) desensitization. since expression and activity of grk2 are upregulated in human heart failure, it has been proposed that grk2 inhibition may resensitize badrenergic receptor activity in heart failure patients. in this study, we evaluated chronic grk2 inhibition by rkip as a potential strategy to improve cardiac function in heart failure. to analyse the effect of rkip on heart failure, rkip transgenic mice were crossed with mice carrying a mutation in phospholamban (pln r9c ). pln r9c causes severe heart failure and premature death in humans and transgenic mice. cardiac function was significantly improved in the presence of rkip as shown by left ventricular catheterization and echocardiography. expression of heart failure marker genes anf and bnp was indistinguishable between wild-type mice and mice co-expressing rkip and pln r9c . in line with these findings, the life span of double transgenic mice was significantly prolonged compared to pln r9c transgenic mice. slow calcium transport into the sarcoplasmatic reticulum was characterised as cause for dilatated cardiomyopathy of pln r9c transgenic mice. since western blot analyses of rkip transgenic heart lysates showed increased phosphorylation of important regulators of cardiomyocyte relaxation, we analysed calcium transients and contractility of isolated cardiomyocytes as possible mechanism of the rkip mediated rescue. in the presence of rkip, calcium reuptake into the sarcoplasmatic reticulum was accelerated and cardiomyocyte relaxation improved. furthermore, coexpression of rkip significantly attenuated pathological cardiac remodelling. interstitual fibrosis and apoptotic cells were quantified in histological sections after sirius red-and tunel-staining. this study revealed a protective function of rkip in a genetic mouse model of human dilated cardiomyopathy by improving cardiac contractility and attenuating interstitial fibrosis and apoptosis. a detailed understanding of this rescue may help to find a new therapeutic strategy to improve cardiac contractility in heart failure. gαi-proteins comprise a group of three highly related members characterized by specific expression patterns. based on previous work of gi-mediated signaling pathways in cardiomyocytes and platelets, we checked gαi expression in mouse heart and platelets. the analysis revealed the presence of gαi2 and gαi3 with gαi2 as the predominant isoform. gene-targeted mice lacking either gαi2 or gαi3 were analyzed to unravel the physiological role of gαi-proteins in the cardiovascular system. extraordinarily prolonged bleeding times in gαi2-deficient animals were an obvious phenomenon. detailed analysis using isolated platelets gαi2-deficient mice exhibited reduced platelet activation and attenuated aggregation in response to stimulation by various agonists accompanied by reduced thrombus formation and diminished stability on a collagen-coated surface. employing in vivo injury/thrombosis models revealed abrogated thrombus formation selectively in gαi2-deficient mice. comparable results were obtained in experiments using mice with megakaryocyte/platelet-specific gαi2deficiency. to assess the pathophysiological consequences of platelet gαi2 function, we challenged these mice in experimental models of myocardial and cerebral ischemia. the results clearly show that platelet-gαi2-deficient mice were protected from both, myocardial and cerebral ischemia. in contrast, conventional gαi2-deficient mice subjected to the heart ischemia/reperfusion model exhibited a significantly increased susceptibility to ischemic injury as compared to wild type controls. in contrast, gαi3deficient mice were strongly protected from injury. thus we suggest that gαi2 and gαi3 play distinct roles in major cardiovascular disorders pointing to specific, non-redundant functions of these two highly related gαi isoforms. the cgmp signaling pathway is activated by nitric oxide (no), natriuretic peptides (anp, bnp & cnp), and cgmp-elevating drugs. it regulates several physiological functions such as smooth muscle relaxation, platelet inhibition, and cell growth and differentiation. recent studies indicate that cgmp signaling might also play a role in tumorigenesis, but the cellular and molecular mechanisms of cgmp's potential pro-and/or anti-tumor activities are not well understood. this study has examined the expression and function of components of the cgmp pathway in melanoma cells of murine and human origin. we have found that mouse b16 melanoma cells specifically express the alpha isoform of the cgmp-dependent protein kinase type i (cgkialpha) but not the beta isoform. treatment of intact cells with the membrane-permeable cgmp analog 8-br-cgmp induced the phosphorylation of cgki substrates, vasodilator stimulated phosphoprotein and phosphodiesterase 5. anp and cnp, ligands of the membrane-bound guanylyl cyclase gc-a and gc-b, respectively, did also activate the endogenous cgmp/cgki pathway. however, b16 melanoma cells did not respond to dea-no, which stimulates no-sensitive soluble guanylyl cyclases. interestingly, activation of cgmp/cgkialpha signal transduction was associated with an increase in erk1/2 and p38 phosphorylation, growth and migration of b16 melanoma cells. similar results were obtained with wm1205 human melanoma cells. in conclusion, we have identified a gc-a/gc-b/cgmp/cgkialpha pathway in melanoma cells, which stimulates tumor cell growth and migration in vitro. pharmacologic inhibition of cgmp signaling may offer a promising strategy for the treatment of melanoma. an increasing body of evidence supports important roles for voltage-gated calcium channels in idiopathic generalized epilepsies (iges), however which calcium channels participate in ige pathogenesis and how has yet to be fully understood. recently, it has been proposed that cav2.3 (r-type) and t-type calcium channels jointly contribute to oscillatory bursting in the reticular thalamus (rt) 1 , which is associated with absence epilepsy. cav3.2 is one of the two t-type calcium channels known to be expressed in the rt 2 . it has been demonstrated that ablation of either cav2.3 or cav3.2 reduces susceptibility to experimentally induced epilepsy 3;4 and in addition that both channels share several pharmacological properties [5] [6] [7] . to gain further insight into interacting mechanisms of these two channels in epilepsy, we tested cav2.3(-|-), cav3.2(-|-) and cav3.2(-|-)xcav2.3(+|-) mice side-by-side in the kainic acid model of epilepsy. we provide first in vivo data supporting a synergistic mode of action for cav2.3 and cav3.2 calcium channels in epileptogenesis. the deubiquitinase cyld regulates mechanisms of rip1/rip3-dependent necroptosis in neuronal cells diemert s. 1 , krieg s. vivo model of cerebral ischemia, we found, that cyld -/-mice exhibit significantly reduced infarction volume compared to control littermates. overall, these data reveal a role for cyld in rip1/3-dependent mechanisms of necroptosis in a model of glutamate toxicity in neuronal cells and further suggests cyld-mediated mechanisms of neuronal cell death as a potential therapeutic target after acute brain injury in vivo. cyanamide-mediated inhibition of n-acetyltransferase 1 dierolf d., bonifas j., blömeke b. university trier department of environmental toxicology, universitätsring 15, bldg. n, 54286 trier, germany cyanamide has been used for decades for medical purposes in the treatment of alcoholism and for agricultural purposes as a plant growth regulator and bud-breaking agent. its therapeutic effect is mediated by reversible inhibition of aldehyde dehydrogenase and it was reported to be metabolised in vivo mainly via coenzyme a dependent n-acetylation by n-acetyltransferases. reported to be a substrate for n-acetyltransferases, cyanamide has a different molecular structure to arylamines and hydrazines, the preferred substrates for nacetyltransferases. therefore a more detailed investigation of its interrelations with nacetyltransferases was performed. we analysed nat1 enzyme activities after incubation of thp-1 cells with cyanamide for 24h, and found that the metabolic conversion of the classic substrate para-aminobenzoic acid was significantly reduced at physiologically relevant concentrations. in detail a significant dose-and time-dependent nat1 protein inhibition was observed for 100 and 1000 µm cyanamide using over-expressed human recombinant nat1 (insect cell cytosol containing recombinant human nat1*4). however, no inhibition was found in the presence of recombinant nat2*4. as we also provide evidence that cyanamide is not metabolised via coenzyme a dependent nacetylation in vitro by human nat1 or nat2 cytosol, by thp-1 cells or by human liver cytosol, we can conclude that this inhibition is not based on substrate-dependent downregulation of nat1. further mechanistic and kinetic studies indicated that cyanamide reacts with the active site cysteine residue of nat1, leading to its rapid inhibition. since the presence of the reduction agent dithiothreitol did not reverse the results it could be that it is possibly not caused by oxidative processes. in sum these data indicate that cyanamide is able to interact with cysteine residues of human nat1, which causes its inhibition but not by a substrate-dependent mode of action. taken together our results show, that cyanamide is not n-acetylated by human nats, but might modulate nat1 dependent detoxification and activation of arylamines. dissecting the signal transduction pathway of acute hypoxic vasoconstriction (hpv) in precapillary pulmonary arterial smooth muscle cells ( low levels of oxygen in the pulmonary airways induce acute hypoxic pulmonary arterial vasoconstriction (acute hpv) redirecting blood flow to normoxic areas of the lung to assure optimal uptake of oxygen during ventilation. acute hpv lasting several minutes occurs predominantly in the precapillary region of the pulmonary vascular tree [1] . therefore, precapillary pulmonary arterial smooth muscle cells (pasmc) have been suggested as sensor as well as effector cells and trpc6 a member of the classical transient receptor potential (trpc) ion channel family was identified to be essential for the initiation of ca 2+ influx and the subsequent contraction of pasmc [2] . however, the underlying oxygen sensor and the exact signal transduction pathway(s) in pasmc have not been fully elucidated yet. by using gene-deficient mouse models as well as downregulation of potential candidate proteins by specific small interfering rnas (sirnas), we aim to dissect signaling cascades of trpc6 channel activation in acute hpv. for pasmc isolation and culture from mice we use a technique based on magnetic separation of intrapulmonary arteries originally developed in rats [3] . trpc-expression in freshly isolated and passaged pasmc cultured in low (5%) and high fetal bovine serum (25%) was analyzed. interestingly higher passage numbers resulted in a significant down-regulation of trpc1 and trpc6 the most predominantly expressed channel monomers in pasmc, while different serum concentrations resulted in no significant changes in their expression rates. sirnas were designed, transfected and successfully tested in hek293 cells and pasmc. initial results of the dissection of the signal transduction pathway activating trpc6 and inducing acute hpv in pasmc will be presented. references [1] staub, n. c. (1985) . site of hypoxic pulmonary vasoconstriction, chest 88, 240s-245s. [2] weissmann, n. et al. (2006) . classical transient receptor potential channel 6 (trpc6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange, proc. natl. acad. sci. u.s.a. 103, 19093-19098 trpv5 is a highly selective calcium channel expressed in various tissues amongst others in placenta. the channel may be involved in transcellular calcium transport in epithelial tissues thereby playing some role in calcium homeostasis of the body. in the placenta trpv5 is assumed to contribute to the maternal-fetal calcium transport. most probably trpv5 is part of a multiprotein channel complex but most of the components of this complex are unknown so far. our aim is to find interaction partners of the trpv5 protein in the placenta that might contribute to the regulation of the trpv5 protein function. therefore we expressed the intracellularly located n-and c-terminal parts of trpv5 (aa 1-330 and 571-723) as trpv5-gst (glutathione-s-transferase)-fusion proteins and used the purified recombinant proteins for pulling down proteins from human placenta cell extracts. the proteins pulled down by this approach were analysed by mass spectrometry. we identified several potential trpv5 interacting proteins which were not associated with the gst protein only. one of the proteins which was highly enriched with the n-terminal part of the trpv5 protein is calpain 6. in contrast to the classical calpains (calcium activated cystein proteases), calpain 6 is unique in that it lacks the cysteine residue in the active site. calpain 6 is mainly expressed during embryogenesis and is reported to be involved in cytoskeleton stabilisation but with unknown function in placenta. the interaction between trpv5 and calpain 6 was confirmed in reciprocal pulldown experiments and the trpv5 binding region for calpain 6 was narrowed down by using overlapping n-terminal trpv5-gst fusion proteins. after injection of trpv5 crna into xenopus laevis oocytes calcium uptake into the oocytes was measured; this uptake was largely reduced after co-injection of the calpain 6 crna. in further experiments we want to study potential regulatory effects of the trpv5 protein on the calpain 6 function in cell culture models. comparative studies on the effects of the human carcinogen inorganic arsenite and its recently identified thiolated metabolite thio-dma v on human urothelial cells ebert f., leffers l., unterberg m., schwerdtle t. universität münster institut für lebensmittelchemie, corrensstr. 45, 48149 münster, germany it has been demonstrated that chronic ingestion of 50-200 µg/day inorganic arsenic is associated with an increased risk for cancers of the skin, the lung and the bladder, but until now the underlying toxic modes of action are still under debate. in this context, in the last five years one main focus has been given to the role of human inorganic arsenic metabolism and nowadays it is generally accepted that human biomethylation contributes to inorganic arsenic induced carcinogenicity. due to further improvements in arsenic speciation techniques recently a new thiolated arsenite metabolite, the thio analogue of the well known metabolite dimethylarsinic acid (dma v ), the so called thiodimethylarsinic acid (thio-dma v ), has been discovered in human biological samples. after synthesizing and analytically characterizing this metabolite (bartel et al. 2011 , j toxicol. 2011 we investigated its toxic effects in direct comparison with ias iii in human urothelial cells. thereby cell cycle studies revealed a g2/m-and s-phase arrest as well as subg1 peak formation in case of thio-dma v . moreover, thio-dma v induced apoptosis (subg1, caspase 3 activity) at lower concentrations and earlier time points as compared to ias iii . most likely this is partly due to the higher cellular bioavailability of thio-dma v (aas/icp-ms). regarding genotoxicity, a generation of dna single strand breaks (alkaline unwinding technique) as well as an increased formation of reactive oxygen species (ros, dcfh-da-fluorescence) occurred only at high cytotoxic concentrations. however, thio-dma v strongly increased h2o2 induced ros formation at very low nanomolar concentrations, which might result in cogenotoxic effects. since our earlier studies have shown a strong inhibition of h2o2 induced poly(adp-ribosyl)ation especially by trivalent methylated arsenic metabolites, actual studies investigate the impact of thio-dma v on cellular poly(adp-ribosyl)ation, parp-1 gene expression, protein level and cellular cleavage, which might hopefully give further hints regarding the mode of action behind thio-dma v induced apoptosis. mitochondrial dysfunction in models of alzheimer´s disease eckert a. universitäre psychiatrische kliniken basel neurobiology laboratory, wilhelm klein strasse 27, 4012 basel, switzerland the histopathological characteristics of alzheimer's disease (ad) are amyloid-ß (aß) containing plaques and neurofibrillary tangles (nfts) as well as neuronal and synaptic loss. until today, the underlying mechanisms of the interplay of plaques and tangles remained unresolved. there is increasing evidence that mitochondrial dysfunction might be a possible link, as revealed by studies in several app and tau transgenic mouse models. recently, we examined mitochondrial function in a novel triple transgenic mouse model (pr5/app/ps2) -triplead mice -that combines both pathologic features of the disease in brain. using comparative, quantitative proteomics (itraq) and mass spectroscopy we found a massive deregulation of 24 proteins, of which one-third were mitochondrial proteins mainly related to complexes i and iv of the oxidative phosphorylation system (oxphos). remarkably, deregulation of complex i was related to tau, whereas deregulation of complex iv was aß dependent, both at the protein and activity levels. the triplead mice showed synergistic effects of aß and tau already at the age of 8 months, resulting in a depolarized mitochondrial membrane potential. at 12 months, the strongest defects on oxphos, synthesis of atp and reactive oxygen species were exhibited in the triplead mice, again emphasizing synergistic, ageassociated effects of aß and tau in impairing mitochondria. evidences from ad post-mortem brain as well as cellular and animal ad models indicate that aß and tau protein trigger mitochondrial dysfunction through a number of pathways, such as impairment of oxidative phosphorylation, elevation of reactive oxygen species production, alteration of mitochondrial dynamics, and interaction with mitochondrial proteins. moreover, recent reports indicate that aß may also interact directly with intracellular proteins such as the mitochondrial enzyme abad (aß binding alcohol dehydrogenase) in executing its toxic effects. mitochondrial dysfunction occurs early in ad, and aß's toxicity seems to be in part mediated by inhibition of abad. in total, a vicious cycle as well as several vicious circles within the cycle, each accelerating the other, can be drawn emphasizing the synergistic deterioration of mitochondria by tau and aß. olesoxime is a novel mitochondrial-targeted compound that is orally active and crosses the blood brain barrier. the cholesterol-oxime targets proteins of the outer mitochondrial membrane and represents a promising drug candidate for neurodegenerative diseases 1 . we evaluated olexoxime's neuroprotective effects against mitochondrial dysfunction in an animal model for alzheimer`s disease (ad). dissociated brain cells (dbc) and mitochondria were isolated from brains of c57/bj6-thy1-appsl (ad-mice) mice that were fed with 600 mg olesoxime/kg feed for 3 months. drug plasma levels reached approx. 600 ng/ml. respiration of isolated mitochondria were significantly diminished in ad-mice due to reduced complex i, i+ii and cox activities. consequently, mitochondrial membrane potential (mmp) was significantly reduced in dbc from ad-mice. olesoxime normalized respiration chain complex activities and the mpp. to further evaluate the beneficial effects of olesoxime on complex i activity, we challenged dbc with rotenone ex vivo and observed that olesoxime treatment was protective. to further clarify the mode of action, we analyzed the ability of olesoxime to prevent opening of the mitochondrial permeability transition pore (mptp) in vitro using energized brain mitochondria by measuring ca 2+ -and atractyloside (atr) induced swelling. the opening of mptp precedes apoptosis and can be induced by mitochondrial dysfunction due to calcium overload, oxidative stress, elevated phosphate concentrations or adenine nucleotide depletion. olesoxime prevented ca 2+ -as well as atr induced mitochondrial swelling. atr inhibits the adenine nucleotide translocase (ant) that requires appropriate membrane properties to mediate mitochondrial permeability transition (mpt). since cholesterol (cho) and its derivates represent potent modulators of membrane viscosity, we related the effects of cho and olesoxime on mptp opening to membrane properties. both, cho and olesoxime reduced the flexibility of membrane acyl-chains in energized mitochondria and prevented atr induced mptp opening. however, cho didn`t prevent ca 2+ -induced mptp opening, indicating a different mode of action for olesoxime. our data confirm olesoxime as drug candidate against mitochondrial dysfunction, which is considered to play a pivotal role in neurodegenerative diseases. the work was supported by the european union under the 7th framework program for rtd -project mitotarget -grant agreement health-f2-2008-223388. several inflammatory glomerular kidney diseases are accompanied with a massive production of reactive oxygen species (ros) that may attack the glomerular filtration barrier by affecting podocyte function and may contribute to apoptotic or necrotic cell death of mesangial cells. otherwise, ros also trigger fine-tuned signaling processes that may result in cell proliferation or cell migration. to define such redox-driven signaling devices, we performed a non hypothesis-driven proteomic approach, to identify homo-or heteromeric protein complexes induced by ros. to this end, protein lysates of human podocytes were treated with or without hydrogen peroxide (250 µm) for 10 min. thereafter, the cell lysates were subjected to diagonal 2d gel electrophoresis and putative redox-affected proteins were analyzed by ms/ms-analysis. by this approach, we could identify a series of proteins that form interprotein-disulfide bonds in a redoxdependent manner. one of those proteins could be characterized as the regulatory subunit of protein kinase a (r-subunit of pka), which belongs to the family of serine/threonine kinases. to evaluate whether ros is capable to activate pka also in a more physiological setting, we treated rat mesangial cells with pdgf-bb to induce ros formation and we could demonstrate that pdgf-bb induces dimerization of r-subunits in a redox-dependent manner. to demonstrate whether pdgf-bb induces pkadependent pathways, we analyzed the effects of pdgf-bb on phosphorylation of serine 157 of vasodilater stimulated protein (vasp) a classical target of pka. in fact, pdgf-bb induced vasp phosphorylation independently of intracellular camp levels. moreover, elevating camp levels via activation of adenylate cyclase with forskolin did not change the dimerization state of r-subunits. pdgf-bb-induced dimerization of the r-subunits and subsequent phosphorylation of vasp was blocked by diphenyljodonium (dpi), indicating activation of a nadph oxidase is essential for pka activity. taken together, we demonstrate a redox-dependent activation of pka by pdgf-bb and this may hint also for a probably protective role of ros in rat mesangial cells. testing the potential sensitizing capacity of chemicals is currently done by using the murine local lymph node assay (llna). animal welfare and eu cosmetics directive demands alternative methods to animal tests. the purpose of this study was to establish an in vitro assay for the prediction of skin sensitizers. based on the finding that the majority of skin sensitizers are electrophilic or have the potential to be metabolized to electrophilic substances, it is assumed that they can activate the nrf2-keap1-antioxidant response element (are) regulatory pathway. here, we report the results obtained from the lusens assay that detects electrophilic chemicals using the nrf2 pathway. the cell line lusens was derived from immortalized keratinocyte hacat cells and carries a luciferase reporter gene under the control of an are-element from the rat nadph quinone reductase nq1. the lusens assay was in house validated with a panel of 54 chemicals and cosmetic ingredients including the 22 performance standard substances of the local lymph node assay. the predictivity of this assay was compared to the predictivity of the murine llna and to human patch test data and can be considered as reliable screening approach (accuracy of 83% compared to human data). however, in order to cope with the complex multi-step mechanism of skin sensitization, an integrated approach of in vitro assays mimicking several steps was designed; thereof, the are-dependent gene activation represents one module. time-resolved fluorescence ligand-binding assays for parathyroid hormone receptors emami-nemini a. 1 ligand-binding studies represent essential tools for pharmacological research on g protein-coupled receptors. in recent years, time-resolved fluorescence gained significant relevance as readout for ligand binding studies. however, ligand-binding assays for parathyroid hormone receptors (pthrs) utilizing fluorescent parathyroid hormone (pth) were missing. therefore, we generated various fluorescent pth analogues which exhibit properties of native pth in terms of affinity, potency and internalization. for the purposes of academic and commercial research, we utilized labeled pth to set up three time-resolved fluorescence assay formats: (i) classical separation binding assay, based on time-resolved fluorescence and suitable for native receptors; (ii) homogeneous timeresolved fluorescence resonance energy transfer (htrf) based on tag-lite technology for high through-put screening; (iii) htrf based on antibodies, a synergistic approach using htrf with minimized receptor modification. this work will facilitate the development of new drugs directed to the pthr as well as fundamental research on the pthr. anandamide production in eosinophilic granulocytes is independent of il-5 and eotaxin stimulation engeli s., reinke j., zörner a., tsikas d., jordan j. medizinische hochschule hannover institut für klinische pharmakologie, carl-neuberg-straße 1, 30625 hannover, germany introduction: some animal and in vitro studies suggest that endocannabinoids exert anti-inflammatory effects. specifically, inhaled anandamide reduced the obstructive effect of leukotrien d4 in airways, and a specific cannabinoid receptor agonist significantly reduced pulmonary inflammation in guinea pigs. we have recently shown that segmental bronchial allergen challenge is associated with significant increases of anandamide concentrations in bronchoalveolar fluid of patients with asthma. the concomitant increase in eosinophilic counts, eotaxin and il-5 concentrations in bronchoalveolar fluid led us to hypothesize that anandamide is produced by eosinophilic granulocytes in response to chemotactic stimuli. peripheral eosinophilic granulocytes were isolated from whole blood by means of percoll gradient centrifugation and magnetic separation employing cd16antibodies conjugated to magnetic beads. isolated cells were counted and anandamide measurements were typically performed in whole cell lysates of 1.5x10 6 eosinophils. we stimulated eosinophils with varying concentrations of il-5, eotaxin-1 (ccl11), eotaxin-2 (ccl24), and eotaxin-3 (ccl26). to prevent anandamide degradation, a specific fatty acid amide hydrolase (faah) inhibitor (oloxa) was employed. anandamide concentrations and faah activity were determined by stable isotope dilution using lc-ms/ms protocols. results: first, we confirmed the ability of eosinophilc granulocytes to synthesize anandamide. however, cellular anandamide content could only be measured when faah was effectively blocked with oloxa, and strong faah activity was demonstrated in eosinophils. with oloxa, typical anandamide concentrations were in the range of 2-4 pm/1.5x10 6 eosinophils. neither il5 (50-500 pg/ml), nor any of the eotaxins (50 ng/ml either alone or in varying combinations) did stimulate anandamide production after 30min of incubation. our results suggest that chemotactic molecules like eotaxin and il-5 are not responsible for increased anandamide formation in eosinophils during allergen challenge. in a next step, the effects of anandamide on eosinophils and bronchial epithelial cells need to be determined. the suprisingly high faah activity in eosinophils may point to an alternative pathway facilitating prostaglandin and leukotriene synthesis by production of arachidonic acid. screening methodology for estimatation of dermal absorption in vitro fabian e., goth c., guth k., mehling a., van ravenzwaay b., landsiedel r. basf se experimentelle toxikologie, carl-bosch-strasse 38, 67056 ludwigshafen, germany dermal absorption is used in the evaluation of the effectiveness of pharmaceutical or cosmetic formulations, but often dermal absorption is a critical parameter in risk assessment of pesticides or chemicals. therefore, knowledge of dermal absorption is e.g. helpful in formulation development. skin absorption is routinely measured in vivo or in vitro following oecd tg 427 or 428. however, these tests are complex, time consuming and expensive. therefore, a method was developed to allow simple and rapid screening. the experiment uses dermatomized skin in modified franz type diffusion cells. 10 µl of test substance preparation are applied to the skin preparation. after 6h, the skin is washed and the amount of penetrated substance is quantified. the receptor fluid and the washing solutions are optimized for subsequent analyses by lc-ms. we performed dermal absorption screenings in parallel to our routine guideline studies and demonstrated a good correlation of the results of both study types: the total recovery found in the screening studies is somewhat lower than in the corresponding guideline studies but is always in an acceptable range above 80%. the efficacy of the skin washing procedure is lower than under routine conditions, most probably due to the change to an lc-ms-compatible washing solution. overall, the dermal absorption screening is an easy, fast and cost-effective screening method for the estimation of dermal absorption of a wide variety of test substances and formulations. the p53 tumor suppressor protein is frequently inactivated in human cancers by diverse mechanisms. owing to its fundamental role in the maintenance of genomic stability and cancer prevention, p53 is an attractive target in cancer therapy and several approaches were pursued to restore p53 function in tumor cells. polyamidoamine (pamam) dendrons are positively charged molecules with a systematically branched structure that interact with the negatively charged cell membrane, inducing cellular uptake via endocytosis. in the present study, biotin-labeled p53 protein was attached to a dendronized streptavidin nanocarrier to facilitate its internalization into different tumor cell lines. first, biotin-substituted pamam dendrons were conjugated with streptavidin to allow formation of the dendronized streptavidin nanocarrier. the nanocarrier displayed uptake into hela cervix carcinoma and a549 lung adenocarcinoma cells without detectable cytotoxicity. biotin-labeled p53 was then conjugated to the dendronized streptavidin, preserving its specific dna-binding in vitro. immunoblot analysis revealed efficient internalization of biotin-p53 into hela cells in the presence of dendronized streptavidin. in line with this finding, specific cellular uptake of biotin-p53 was observed by confocal microscopy, which showed a cytoplasmic and peri-nuclear localization in hela, a549 and saos osteosarcoma cells. the internalized biotin-p53 also partially co-localized with early endosomes. importantly, the delivery of biotin-p53 into p53-deficient saos cells resulted in impaired cell viability and upregulation of caspase 3/7 activity, demonstrating its biological functionality. this study intriguingly demonstrate the efficient delivery of functional biotin-p53 into different tumor cell lines using the novel streptavidin nanocarrier, which can be further modified to allow cell-type specific targeting and combined with cytotoxic drugs such as doxorubicin. identification of novel ahr target genes in rat liver oval cells faust d. 1 , vondracek j. the aryl hydrocarbon receptor (ahr) is a transcription factor involved in physiological processes, but also mediates most, if not all, toxic responses to 2,3,7,8tetrachlorodibenzo-p-dioxin (tcdd), some polycyclic aromatic hydrocarbons (pahs) and dioxin-like polychlorinated biphenyls (pcbs), such as pcb126. activation of the ahr by these ligands leads to its dimerization with arnt and transcriptional activation of several phase i and ii metabolising enzymes. while it is generally accepted that many pahs are thereby transformed to genotoxic metabolites, this classical signalling pathway so far failed to explain the tumour promoting effects of the nongenotoxic compounds tcdd and pcb126. thus, there is an urgent need to define genetic programmes orchestrated by ahr to unravel its role in physiology and toxicology. we have recently shown that treatment of rat liver oval cells with tcdd or pcb126 leads to a release from contact-inhibition involving activation of the ahr, elevation of jund protein levels and transcriptional activation of cyclin a (1, 2) . loss of contact-inhibition is one hallmark of tumour promotion. to better understand ahr-driven pathways we identified the transcriptional programme using high density microarrays in response to pcb126. already 6 h after treatment, 69 genes were found to be upregulated and 76 genes downregulated indicating that these are direct ahr-dependent target genes. david analysis revealed that these genes are involved, for instance, in drug and lipid metabolism, cancer pathways, tgf-b signalling and cell-cell communication. ten of the 69 genes were selected for confirmation by semi-quantitative rt-pcr. using the ahr inhibitor ch-223191 and sirna directed against ahr and arnt, we further demonstrated that ahr-and arnt-function is required for transcriptional activation of the selected genes. finally, we identified the transcription factor foxq1as a novel ahr target protein in rat liver oval cells. although the function of foxq1 is poorly understood, it has been shown very recently that foxq1 is overexpressed in colorectal cancer and is involved in epithelial-mesenchymal transition in breast cancer cells. its function in ahrmediated tumour promotion, however, remains to be determined. the practical relevance of histamine h1 and h3 receptors in the brain can be easily deduced since h1 receptor antagonists of the first generation have a sedative effect and an inverse h3 agonist, pitolisant (close to its introduction to the market), is active against excessive daytime sleepiness associated with narcolepsy. in this context the question arises whether also h2 and h4 receptors possess a practical relevance in the brain. to this end, we examined whether the electrically evoked 3 h-noradrenaline release in superfused human cerebral cortex slices is affected by agonists at the above receptors. the h2 agonist impromidine 10 µm failed to affect noradrenaline release in human cortex slices although it facilitated noradrenaline release in guinea-pig cortex slices; the maximum extent of facilitation was 50 %, the pec50 was 6.9 and the pa2 of the h2 antagonist ranitidine against impromidine was 7.2. with respect to h4 receptors there is some controversy in the literature whether they occur in the brain at all. however, we were able to detect h4 receptor mrna in the human and mouse cortex by the reverse transcriptase polymerase chain reaction. in cortex slices of either species, noradrenaline release was not affected by the h4 agonist 4-methylhistamine 10 -30 µm but inhibited by histamine 10 µm via h3 receptors by 28 and 55 %, respectively. in mouse cortex membranes, 4-methylhistamine 30 µm also failed to affect 35 s-gtpγs binding although r-α-methylhistamine 3 µm, acting via h3 receptors, increased it by 20 %. in conclusion, h2 receptors facilitating noradrenaline release are detectable in the isolated guinea-pig but not human cortex. despite the presence of h4 mrna in the brain, functional readouts of this receptor, i.e. modulation of noradrenaline release (humans, mice) and modulation of 35 s-gtpγs binding (mice), could not be shown. murine cx40 promoter activity is dependent on the transcription factor creb fels b., nunes f., schmitz w., müller f. u. westfälische wilhelms-universität münster institut für pharmakologie und toxikologie, domagkstraße 12, 48149 münster, germany connexin 40 (cx40) is a gap junction protein expressed in atrial myocytes and the ventricular conduction system, mediating the electrical intercellular communication in the myocardium. alterations in cx40 function were linked to the pathophysiology of atrial fibrillation and heart failure. in the heart, camp dependent gene transcription is regulated by members of the creb/crem/atf family which bind to camp responsive elements (cres). similar to the human cx40 gene promoter, the murine promoter contains one cre. cardiomyocyte-specific overexpression of a crem-isoform (crem-ib∆c-x) led to cx40 down-regulation, suggesting that creb related transcription factors are involved in cx40 gene regulation. in order to study the functional role of creb in the regulation of the cx40 promoter we have generated a luciferase based murine cx40 promoter reporter gene construct and monitored its activity in a permanent cell line upon overexpression of constitutive-active creb (cacreb) and a non-phosphorylatable dominant-negative creb (dncreb) isoform. surprisingly, overexpression of cacreb and dncreb both lead to a reduction of cx40 promoter activity (cacreb 46% ± 5% vs control 100% ± 3%; p<0.05 vs control , n=15), dncreb 45% ± 2% vs control 100% ± 3%; p<0.05 vs control, n=18). the activity of the murine connexin 40 promoter is modulated by creb. both cacreb and dncreb led to cx40 down-regulation, which could be explained by induction of inhibitory transcription factors creb/crem/atf1 transcription factor family, which in turn could suppress cx40 promoter activity. (supported by the dfg) results: fxa increased par-2 mrna, protein and cell-surface expression and augmented par-2-mediated mitogenesis. par-1 expression was not influenced. the regulatory action of fxa on par-2 was concentration-dependent and mimicked by a par-2 selective activating peptide. the thrombin inhibitor argatroban or par-1 gene silencing did not influence fxa-stimulated par-2 expression. fxa increased oxidative stress and expression of the nadph oxidase subunit nox-1 in smc. nox-1 gene silencing prevented fxa-stimulated par-2 regulation, as did ebselen and catalase. exogenous hydrogen peroxide increased par-2 expression and mitogenic activity. fxa induced nuclear translocation and par-2 dna binding of nuclear factor kb (nfkb). inhibition of nfkb prevented fxa-stimulated par-2 expression. in separate studies, fxa promoted par-2 mrna stabilisation through increased human antigen r (hur)/par-2 mrna binding and cytoplasmic shuttling. hur gene silencing abolished fxa-stimulated par-2 expression. conclusion: expression and mitogenic activity of vascular par-2, but not par-1, is upregulated by fxa. this action involves transcriptional and post-transcriptional mechanisms mediated through nox-1-containing nadph oxidase and its downstream effectors hydrogen peroxide, nfkb and the mrna stabilising protein hur. continued generation of fxa by the mural thrombus, and the autoregulatory feedback control of par-2 may maintain the inflammatory and proliferative state of the injured vessel, thereby promoting vascular remodeling. the mrna stabilising factor hur is a critical regulator of human proteaseactivated receptor 4 aim: we recently reported that functional expression of par-4 thrombin receptors is induced in human saphenous vein smc exposed to high glucose. this effect could be attributed in part to transcriptional mechanisms mediated through nfkb but the contribution of post-transcriptional effects such as mrna stabilisation is not known. this study explored the potential role of the mrna stabilising factor human antigen r (hur) in the regulation of par-4. methods: human saphenous vein smc were serum-deprived prior to study. gene expression was determined by realtime pcr, protein expression by western blotting. gene silencing utilized commercially available sirna. hur binding to par-4 mrna was determined by immunoprecipitation ("pull-down") pcr. results: high glucose (25 mm vs 5.5 mm) slowed par-4 mrna degradation in the presence of actinomycin d. par-1 mrna decay was not affected. hur binding to par-4 mrna and nucleo-cytosolic shuttling was enhanced by high glucose, total hur protein expression was not affected. hur sirna abolished the high glucose-stimulated induction of par-4 mrna. hydrogen peroxide (h 2o2) also induced cytosolic hur shuttling and increased par-4 mrna and total protein expression. the role of endogenously generated h2o2 in the regulatory effect of high glucose on par-4 expression was investigated with the nadph oxidase inhibitors apocynin/diphenyliodonium (to prevent h2o2 generation) and cell-permeant catalase (to degrade cellular h2o2). both approaches prevented the stimulatory effect of high glucose on par-4 expression. cyclic amp has been reported to suppress hur activation and in the present study, the cyclic amp stimuli forskolin and cicaprost (prostacyclin analog) suppressed basal hur shuttling and par-4 transcript stability. cicaprost also attenuated high glucose-induced hur binding to par-4 mrna and as a consequence normalised par-4 expression and inflammatory signalling in high glucosetreated cell. conclusion: the regulation of par-4 thrombin receptors in human vascular smc is critically dependent on the mrna stabilising actions of hur. through activation of hur, high glucose and other hur stimuli such as ang ii and exogenous h2o2, increase par-4 expression, while cyclic amp agonists such as prostacyclin oppose this effect. such interactions could potentially represent a fine-tuning mechanism to control par-4 expression and ultimately also the mitogenic and inflammatory actions of thrombin in the vessel wall. nucleoside diphosphate kinase b (ndpk b) is a member of a family of ubiquitously expressed enzymes required for nucleoside triphosphate synthesis. thus, they are involved in the regulation of a variety of cellular processes, e. g. g protein mediated signal transduction. however, whether ndpk b has a specific role in the regulation of angiogenic processes in endothelial cells is unknown. therefore, we studied the function of ndpk b in the vasculature in a developmental, an ischemia-induced and an in vitro model of angiogenesis. firstly, depletion of ndpk b expression was achieved by morpholino-mediated knockdown in zebrafish embryos in which the developing vasculature can be visualized by egfp expression in the endothelium. 72h post fertilization, ndpk b knockdown larvae showed a dramatic inhibition of intersegmental and dorsal longitudinal anastomatic vessel formation compared to control injected fish. this phenotype could be rescued by early re-expression of ndpk b. secondly, ischemia driven angiogenesis was studied in ndpk b-depleted mice and wildtype littermates after excision of the left femoral artery. hind limb blood flow was assessed by laser doppler perfusion imaging immediately before and after ligation (day 0) and on postoperative days 3, 7, 14, 21, 28, and 35 . a significant reduction of recovery was observed in the ndpk b depleted mice at days 3 and 7. thirdly, in vitro-sprouting angiogenesis was analyzed in human umbilical vein endothelial cell (huvec) spheroids with and without sirna-mediated ndpk b knockdown. vascular endothelial growth factor (vegf)induced sprouting was significantly attenuated by ndpk b knock down by more than 50% in comparison with control transfected huvec. we conclude from these results that ndpk b is an essential regulator of angiogenesis. the loss of ndpk b may specifically interfere with the vegf-induced migration and proliferation during endothelial sprouting. ethylene oxide in blood of ethylene-exposed volunteers ethylene (et) is a commercially important high volume industrial chemical. inhaled and endogenous et is metabolized in mammals to ethylene oxide (eo), which is carcinogenic in rats and mice. until now, no data on the oxidation of et in et-exposed humans has been published. in the present study, we investigated the formation of eo in four male adult volunteers exposed for 4 hours to constant atmospheric et concentrations of 5, 20, or 50 ppm by means of a breathing mask. during exposure, et concentrations were measured in inhaled and exhaled air by gc/fid and eo concentrations in venous blood by gc/ms. rates of et metabolism were obtained from the product of the differences in the et concentrations with the pulmonary ventilation. in each subject, linear correlations were found between the et exposure concentration and the rate of et metabolism or the eo concentration in blood. mean rate of et metabolism was 5.5 ± 1.4 nmol/h/ppm/kg body weight. steady-state concentrations of eo in blood differed by a factor of 2 between the volunteers. these inter-individual differences likely reflect the polymorphism of glutathione s-transferase theta, the main eo metabolizing enzyme in human liver. mean eo concentration in blood at steady state was 1.5 ± 0.08 nmol/l blood per ppm of et. the data will be used for validating a physiological toxicokinetic model which will describe the et related eo tissue burdens in rodents and humans. the model predictions will support risk evaluations of et. financially supported by the lower olefins sector group of cefic. in vitro effect of stw11 on human dendritic cells fink c. 1 , bonaterra g. a. extracts of echinacea (purple coneflower) are used in the prevention and therapy of infectious diseases. the medicinal product stw 11 contains the extract from purple coneflower, and in addition, extracts of monkshood, venom of honey bee and bushmaster snake in homeopathic dilutions. previous studies showed a stimulation of the cellular and humoral immune response. dendritic cells (dcs) are antigen presenting cells that act at the interface of the innate and adaptive branches of the immune system. during stages of dc differentiation, the ability to internalize antigens varies and decreases during maturation. in this study, we determined the influence of stw11 on the expression of maturation related genes (cd1a, cd83), cytokines (tnfα, il-4, il-12), chemokines (ccr7), major histocompatibility complex ii(mhc-ii) and toll-like receptors (tlr2, tlr4). in mature (mdc) and immature dc (idc) using real-time rt-pcr. peripheral blood mononuclear cells (pbmcs) were isolated from buffy coats of human volunteers by densitygradient (ficoll ® ) and seeded in 6 well plates. non-adherent cells were eliminated. to induce idc development, 50 ng/ml il-4 and 50 ng/ml granulocyte macrophagecolony stimulating factor (gm-csf) were added. at day 6, maturation was induced by addition of lipopolysaccharide (lps) at a final concentration of 1µg/ml and cultured for additional 3 days. after incubation with different concentrations (0. in idc, compared to control, we found a significantly increased expression of cd83 (2.1-2.6 fold) and tnfα (2.6-3.3-fold) genes after treatment with 0.1-5% stw11, respectively, but no effect was found on the expression of cd1a, il-4, il12, adam19, cd11c, cd40,tlr2, tlr4, mhc-ii and ccr7. in summary, these data demonstrate a stimulatory effect of stw 11 in idc and especially in mdc, concerning an increase of various genes related to maturation (cd83), immunomodulation (tnfα, cd40), adhesion (ccr7) and antigen presentation (mhc-ii) and are in accordance with the therapeutic use in infectious diseases. waterproofing sprays are widely used consumer products containing for example fluorinated polymers or silicon based compounds dissolved in alcohols or volatile petroleum distillates. there have been repeated reports on cases of severe acute respiratory disorders especially when using products that newly entered the market. it is hypothesized that impairment of the pulmonary surfactant by deposition of inhaled respirable particles of the active compound is one of the main causes of the acute lung injury. since the inhalation toxicity cannot be predicted a priori based on the physical and chemical properties of the formulation, proper test strategies are required to ensure consumer safety. we propose to combine screening tests addressing both, exposure and acute lung toxicity. the exposure potential of the spray product is characterized by determining the release fraction of the active compound in the respirable particle size range under conditions relevant for the product application. this is carried out by spraying defined quantities of the product into a control volume and measuring the concentration of health related size fractions. this procedure takes into account spray ageing, especially size reduction of the droplets due to solvent evaporation. the isolated perfused lung is used as a model for testing acute toxicity. ventilated rat lungs are exposed to aged aerosols with proper particle size of approximately 1 µm mmad generated from the liquid spray formulation. lung compliance and lung resistance are continuously monitored during exposure. dose dependent deviations from the normal values (without exposure) are used as read-out parameters. using the combined procedure, different sprays could be ranked according to their realistic exposure risk and, most importantly, sprays with known lung toxicity could be uniquely distinguished from those that have been shown to be safe. in its current stage of development the simple test method is recommended for screening of substances only. induction of oxidative damage in calf thymus dna by the fusarium mycotoxin zearalenone after metabolic activation with liver microsomes fleck s. c., pfeiffer e., metzler m. kit -institute of applied biosciences chair of food chemistry, adenauerring 20a, 76131 karlsruhe, germany zearalenone (zen) is an estrogenic mycotoxin produced by fusarium species. the adverse effects of zen and its reductive metabolite zearalenol (zel) are often compared to those of 17-beta-estradiol (e2) and estrone (e1). these endogenous estrogens are associated with an increased risk for cancer, which may be mediated by two mechanisms, i.e. (i) hormonal activity and (ii) genotoxic effects by p450-catalyzed metabolic activation to catechols (wang et al., chem res toxicol 23, 1365 . like e2 and e1, zen and zel exhibit marked estrogenicity and also undergo aromatic hydroxylation to catechol metabolites (pfeiffer et al., mol nutr food res 53, 1123 . the aim of the present study was to examine the formation of catechol metabolites of zen by liver microsomes of various species and their potential for redox cycling. catechol metabolites are frequently associated with the generation of reactive oxygen species and subsequent oxidative damage of dna, for which 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxo-dg) is a common biomarker. the propensity of the catechol metabolites of zen and zel to cause the formation of 8-oxo-dg in isolated calf thymus dna was determined using a lc-esi-ms/ms method. to this end, zen was incubated with microsomes from human, rat, mouse, bovine and porcine liver as well as with human cyp1a2, and the incubations were extracted with ethyl acetate. the extract was analyzed with lc-ms and then added to a solution of calf thymus dna in the presence of copper(ii) ions and nadph. the formation of 8-oxo-dg could be demonstrated with each extract. the levels of 8-oxo-dg correlated directly with the extent of catechol formation, which increased from steer to swine to human to mouse to rat microsomes. 15-hydroxylated zen/zel, which is the major catechol, was more reactive than 13-hydroxylated zen/zel to form 8-oxo-dg. in conclusion, our study has shown that the catechol metabolites of zen are highly reactive and give rise to oxidative dna damage in vitro. in addition, recent research from our laboratory revealed that the catechols of zen are less efficiently inactivated by catechol-o-methyl transferase than the catechols of e2 and e1. the genotoxic potential of zen may constitute another biological activity in addition to the well-known estrogenicity. supported by deutsche forschungsgemeinschaft (grant me 574/32-1) and "food and health" of kit. thrombin regulates expression of sphingosine kinase-1 (sphk-1) in human vascular smooth muscle cells -inhibition by dabigatran reduces vascular sphk-1 expression and atherosclerotic burden in vivo flößer a. 1 results: thrombin induced a time-and concentration-dependent (1-100 nmol/l) increase in sphk-1 mrna and protein expression in human saphenous vein smc, n=6-7. this was mimicked by a synthetic par-1 ligand. inhibition of sphk-1 attenuated thrombin-induced smc proliferation but not smc migration (n=5). the regulatory action of thrombin on sphk-1 expression was suppressed by sirna against the mrna stabilisiserhur. in thrombin-stimulated smc, hur binding to sphk-1 mrna and subsequent nucleo-cytosolic shuttling was enhanced. accordingly, thrombin induced sphk-1 mrna stabilisation in smc in the presence of actinomycin d. in apoe-deficient mice, long-term treatment with the direct thrombin inhibitor dabigatran significantly reduced aortic sphk-1 expression by 50% (n=5) and plaque size by 35% compared to control animals (n=10). conclusions: thrombin induces sphk-1 expression and s1p synthesis in vascular smc via the mrna stabilising protein hur. this leads to increased smc proliferation. inhibition of thrombin by dabigatran treatment in vivo attenuates progression of plaques possibly by reducing sphk-1 expression. mycotoxin contamination and cytotoxicity of grain mill products typical grain mill products from north-rhine westphalia, i.e. grains, flour, wholemeal flour and bran (from wheat, rye and spelt) as well as typical by-products (outsourced fractions) from the milling process were analysed for their mycotoxin content by lc-ms/ms. the cytotoxicity of sample extracts with known mycotoxin composition was then assessed in v79 cell cultures by means of the neutral red uptake assay, in parallel with pure reference mycotoxin mixtures. extracts from flour and wholemeal flour samples with low levels of deoxynivalenol and enniatin b (from not detectable to 0.4 µg/g don and 0.5 µg/g ennb) induced no measurable cytotoxicity. on the other hand, although mycotoxin contamination levels were also rather low in bran, these samples induced strong cytotoxicity: extracts of bran derived from rye and spelt were more cytotoxic than those of wheat bran. the cytotoxic effects of the bran samples cannot be related to their mycotoxin content as comparable concentrations of pure mycotoxins and mycotoxin combinations tested in parallel were not cytotoxic. by-products from certain stages of the milling process (sorting and waste fractions) were found to contain mycotoxins at rather high levels: enniatin b was detected in nearly all samples, and also t-2 toxin, ht-2 toxin, ergotamine, ergocornin and deoxynivalenol were present. waste sample extracts with notable mycotoxin levels (up to 6 µg/g don, 7 µg/g ennb, 16 µg/g ergotamine, 50 ng/g ht-2 toxin) exert pronounced cytotoxicity in v79 cells. the cytotoxicity of these samples was somewhat stronger than expected when compared with mixtures of reference mycotoxins tested in parallel. in summary, the tested flour and wholemeal flour extracts contained only low levels of mycotoxins and were not cytotoxic. in contrast, bran samples showed cytotoxicity which cannot be explained solely by their mycotoxin content. this unexpected observation in real samples and combination effects of mycotoxin mixtures require further studies. the ahr is a ligand-activated transcription factor that mediates the toxicity of dioxins and related compounds. upon ligand binding the ahr translocates into the nucleus and dimerizes with arnt to modulate gene expression, e.g. of cyp1a1. recently, we have shown that uvb irradiation of human keratinocytes results in activation of the ahr and associated egfr signaling leading to an enhanced expression of cyp1a1 and proinflammatory cox-2, respectively. the initial step is the uvb induced intracellular formation of the tryptophan photoproduct 6-formylindolo [3,2b] carbazole (ficz), a high affinity ahr ligand. thus, the ficz activated ahr is an important mediator of the dna damage independent part of the uvb response. our current study aims to identify further aspects of ahr mediated uvb responses. therefore, we analysed changes in protein expression, proliferation and apoptosis in ahr+/+ and ahr-/-keratinocytes (nctc 2544) by western blot, flow cytometry and brdu incorporation. uvb exposure of nctc cells led to a dose-dependent increase in apoptosis. compared to ahr+/+ cells, ahr-/-cultures exhibited an increased amount of apoptotic cells. this finding was confirmed in irradiated ahr+/+ cells, pretreated with the ahr antagonist 3'methoxy-4'-nitroflavone. moreover, the proliferation of sham as well as uvb irradiated ahr-/-cells was significantly decreased. in ahr-/-cells we found a reduced expression of checkpoint kinase 1 (chk1), an important cell cycle regulator that arrests the cell in g2/m upon dna damage. interestingly, uvb exposure led to a higher net phosphorylation of chk1 in ahr-/-cells, indicating that this pathway is responsible for the observed ahr-dependent differences in proliferation and apoptosis. further expression analyses of chk1 client proteins emphasize our hypothesis. in conclusion our study identifies the ahr as an anti-apoptotic player in uvb irradiated human nctc cells. therefore, we propose that the ahr is a suitable target to prevent uvb induced skin diseases. synthetic progestins exert divergent effects on thrombosis in a murine model of atherothrombosis background: medroxyprogesterone acetate (mpa), a synthetic progestin often used in postmenopausal hormone replacement therapy, has previously been described to be pro-thrombotic in a murine model of accelerated atherosclerosis. however, nothing is so far known about effects of progestins with receptor profiles different from mpa (i.e. agonism or antagonism of mineralocorticoid-or androgen-receptors), such as drospirenone, levonorgestrel and norethisterone acetate. methods: apo -/mice were bilaterally ovariectomized (ovx) and substituted subcutaneously with mpa, drospirenone, levonorgestrel and norethisterone acetate as well as the respective placebo pellets for 90 days on a western-type diet. subsequently, thrombosis was induced by photochemical injury to the right carotid artery using rose bengal and a green light laser. results: compared to placebo, animals substituted with mpa showed significantly shortened times to occlusion of the right carotid artery (placebo mpa: 50.0 ± 5.8 min. vs. mpa: 33.4 ± 4.2 min., n = 6 -9, p < 0.05). in contrast, drospirenone, levonorgestrel or norethisterone acetate did not alter thrombotic responses. however, at least drospirenone (placebo drospirenone: 53.7 ± 5.4 min. vs. drospirenone: 47.5 ± 4.8 min., n = 7 -8) and levonorgestrel (placebo levonorgestrel: 47.0 ± 3.2 min. vs. levonorgestrel: 41.8 ± 2.1 min., n = 6) showed a trend towards shorter times to stable occlusion. furthermore, analysis of aortic gene expression revealed that in aortas of mpa-treated mice expression of matrix-metalloproteinase 9 (mmp-9) was induced as compared to placebo-treated mice. conclusion: mpa, a progestin with glucocorticoid effects, exerts a pro-thrombotic effect that is either progesterone-or glucocorticoidreceptor-dependent while progestins with receptor profiles different from mpa do not show a significant pro-thrombotic effect. furthermore, the pro-thrombotic effect exerted by mpa may be associated with increased expression of mmp-9, a metalloproteinase being known to destabilize atherosclerotic plaques and make them more prone to rupture. rapid screening for mitochondrial toxicity in vitro using an oxygen-sensitive phosphorescent probe freyberger a. bayer healthcare bph gdd ged toxikologie -p & cp, aprather weg 18, 42096 wuppertal, germany impaired mitochondrial function has been implicated with disease, aging, and druginduced toxicities. analyzing mitochondrial respiration (mr) rates is one of the most informative ways to assess mitochondrial function as it provides information on the the bioenergetic capacity of a tissue, however, previously measurements using polarography (clark electrode) were cumbersome with only low throughput. in this work we explored luxcel's water-soluble phosphorescent oxygen-sensitive probe mitoxpress tm for the assessment of mitochondrial toxicity in freshly isolated male rat liver mitochondria (rlm) in a 96-well plate format using glutamate/malate (20 mm/0.5 mm) and succinate (25 mm) as respiratory substrates. inhibition of mitochondrial complexes i to iv, adenosine triphosphate synthetase and the adenosine diphosphate (adp) / adenosine triphosphate (atp) antiporter by rotenone, thenoyltrifluoracetone (ttfa), antimycin a, potassium cyanide, oligomycin, and atractyloside was readily detected in adp-stimulated rlm. use of the two different substrates in parallel allowed to discriminate complex i inhibition by rotenone from complex ii inhibition by ttfa, whereas downstream of these complexes inhibition by the other model inhibitors occurred independent of the substrate used. decoupling of mr from oxidative phosphorylation by carbonylcyanid-p-trifluormethoxyphenylhydrazone (fccp) was detected best in the absence of adp. compared to polarographic measurement, the use of an oxygen-sensitive probe is superior with regard to assay cycle time and sample throughput and offers new opportunities to characterize and screen for mitochondrial toxicity, but also to support studies on mitochondria-mediated modes of action of new chemical entities. the murine local lymph node assay (llna) and the guinea pig maximization test (gpmt) have been used to study the sensitization potential of a series of unsaturated compounds by kreiling et al. (2008) . we have examined the same substances in the loose-fit coculture-based sensitization assay (lcsa), developed by our working group (schreiner et al., 2007) . eight unsaturated compounds [oleic acid (oa), linoleic acid (la), linolenic acid (lna), undecylenic acid (ua), fumaric acid (fa), maleic acid (ma), squalene (sq), 1-octyn-3-ol (oc)] and succinic acid (sua) were investigated using a coculture of keratinocytes and dendritic cell-related cells (dcrc). sensitization potential was quantified by flow cytometry measuring the increase of cd86 expressed on dcrc (ec50 = half maximal effective concentration). a pronounced induction of cd86 at low concentrations was seen with la, lna and oa (ec50: 3, 5 and 5 µmol/l, respectively). ua exhibited an intermediate response (ec50: 307 µmol/l). with oc and ma, we observed effects at higher concentrations only (ec50: 1340 and 2293 µmol/l). no significant increase of cd86 was observed with fa, sua and sq. because of poor solubility, sq could not be studied adequately. induction of cd86 was generally observed at concentrations which did not cause a major impairment of cell viability. our results show a high degree of concordance with those obtained by the gpmt, except for oa. in comparison with the results of the llna, those compounds which showed a strong effect in the llna (oa, la, lna) also induced an increase of cd86 at low concentrations, whereas those with low stimulation indices in the llna induced no significant increase of cd86 (fa, sua) or only at higher concentrations (ua). we observed a discrepancy between the tests with ma and oc, causing a strong stimulation of the murine lymph nodes, while the expression of cd86 was increased at high concentrations only. we assume that ma and oc might be false-positives in the llna, because they were also negative in the gpmt. background: the first step in elimination of many cationic drugs is their uptake from the blood into hepatocytes and renal proximal tubular cells by the organic cation transporter 1 (oct1) and oct2, respectively. the pivotal role of octs in the excretion of cationic drugs raises the possibility of drug-drug interactions in which one drug reduces octmediated elimination of a second drug. although many psychoactive drugs are cationic at ph 7.4 and some of these have already been recognized as oct inhibitors, a systematic screen of this class of compounds is missing. methods: we screened a drug library of 50 most frequently prescribed psychoactive drugs (inpatient prescriptions in germany, at least 4 million ddd each) for their inhibitory interaction with oct1 and oct2. human embryonic kidney (hek) cells stably overexpressing oct1 or oct2 and the prototypical oct substrate 1-methyl-4phenylpyridinium (mpp+) were used as a test system. cells transfected with the empty vector were used as controls. results: at 20 µm, 59% and 51%, respectively, of the tested compounds significantly decreased oct1-and oct2-mediated uptake of mpp+. the most potent inhibitors (inhibition >75%) of oct1 were chlorprothixen and clomipramine, whereas olanzapine, clomipramine and doxepin were the most potent inhibitors of oct2. in contrast, neither at 20 µm nor at 200 µm carbamazepin, haloperidol, lithium, moclobemide and valproic acid did significantly inhibit mpp+ uptake into hek-oct1 or hek-oct2 cells. there was a significant correlation between the degree of oct1 and oct2 inhibition (p conclusions: our results demonstrate that inhibition of oct function by psychoactive drugs has to be considered as a potential mechanism underlying drug-drug interactions. considering estimated peak sinusoidal concentrations e.g., of chlorprothixen and clomipramine between 30 and 80 µm in humans, inhibitory interactions of these compounds with hepatic oct1 have to be taken in account. our data will help to create a chemoinformatic model to predict potential oct-dependent interactions of psychoactive drugs with the hepatic or renal elimination of coadministered drugs. this project is supported by the german federal ministry of education and research (bmbf), project grant no. 01 ex1015b. cardiac gene expression is altered during the development of hypertrophy and heart failure compared to the healthy heart. the molecular mechanisms controlling gene expression in cardiac failure are only partially known. dna methylation is one epigenetic mechanism that regulates long-term changes in gene-expression. to elucidate whether dna methylation is altered during the development and progression of chronic heart failure, genome-wide dna methylation profiles were determined in myocardial biopsies from control patients and patients with cardiac hypertrophy or failure. cardiac biopsies were obtained from patients with aortic aneurysm who served as control and did not show clinical signs of chronic heart disease (ef: 58±3 %, n=3) and from patients with aortic stenosis. the latter group was subdivided according to the ejection fraction into hypertrophic (ef: 63±7 %, n=3) and failing patients (ef: 28±1 %, n=3). after bisulfite conversion of extracted dna, the methylation status of genomic dna was quantified using the infinium® humanmethylation450 beadchip (illumina). this microarray allows analysis of more than 485,000 methylation-sites throughout the whole genome at single-base-pair resolution. these experiments identified 1280 cpg sites in hypertrophic samples and 1365 cpg sites in failing samples which were differentially methylated compared to control specimens (delta >15%; p<0.05). 523 cpg sites were significantly altered in both aortic stenosis groups compared with control hearts. from these cpgs, 496 sites were altered concordantly in hypertrophic and failing samples. analysis of regions harbouring distinct cpg densities revealed that most changes occured in shelf regions of cpg islands whereas the methylation status in the cpg islands was more stable. further analysis showed that differences in methylation were most frequent in gene body, enhancer and 3`utr regions. specifically 9 probes spanning a cpg-island at the promotor region of the muscle-specific serine kinase 1 (srpk3) showed diminished cpg-methylation in hypertrophic (-11.5±0.02%) and failing (-11±0.02%) as compared to control biopsies. remarkably, no alterations of dna-methylation were observed in loci of classic marker genes of chronic heart failure like nppa, serca, ctgf, myh6 or myh7. these results indicate that dna methylation is specifically altered in chronic heart disease but does not affect classic marker genes of chronic heart failure. gliomas are the most abundant type of primary brain tumor in the central nervous system in adults. the current standard of glioblastoma multiforme (gbm) therapy is surgery followed by radiotherapy and chemotherapy. however the morbidity and mortality of gbm remain very high and the median survival period is only 15 months even with treatment. therefore it is important to identify novel drugs to reduce gbm cell proliferation. purine-analogues (pa) are well known for their anti-proliferative effects on eukaryotic cells. in this study novel pa were synthesized and the library of substance-derivatives was tested using different gbm cell lines namely ln18, u87-mg and gl261. the effect on proliferation and viability was assessed by using brdu and resazurin assays. using these in vitro methods we were able to identify several compounds with cytotoxic and anti-proliferative effects in vitro showing ic50 values in the deeper µm range. cytotoxicity of selected compounds was further analyzed by assessment of caspase 3 and propidium iodide based cell cycle facs analysis to discriminate between apoptosis and cell cycle arrest. based on these data purine-derivatives might inhibit proliferation and induce apoptosis in glioma cells. as a result we hypothesize that these compounds could be potentially interesting for the drug-development of gbm therapy and therefore a clue for chemical modifications. further studies are required to identify the exact underlying mechanism of action of the tested purine-analogues. the biological role of adenosine receptors in brown adipose tissue gnad t. 1 brown adipose tissue (bat) is responsible for basal and inducible energy expenditure in mammals. bat contains large amounts of mitochondria and is highly vascularized. bat lipolysis and thermogenesis are stimulated by sympathetic neurons. importantly, recent findings indicate that adult humans possess metabolically active bat 1 . here, we analyzed the expression and function of adenosine receptors in bat. adenosine receptors (ador) are members of the superfamily of g protein-coupled receptors. there are four subtypes of adors in humans referred to as adora1, a2a, a2b and a3. they are widely expressed in tissues and mediate a variety of cellular functions, mostly due to their regulation of camp levels within cells. interestingly, it has been shown that adenosine can either inhibit or stimulate lipolysis in white adipocytes through adora1 or a2a, respectively 2 . however, the role of adenosine in the differentiation of brown preadipocytes to adipocytes and in bat function is not clear. to analyze the role of adors in bat, we use preadipocytes isolated from bat of newborn mice and subjected them to a differentiation protocol. 3 abundance of adora1, a2a, a2b and a3 mrna was measured using qpcr. all four receptor subtypes are present in preadipocytes with adora2b being the most abundant. adora1, adora2a and adora3 are significantly transcriptionally upregulated -albeit at varying degree -during differentiation. adora1 is upregulated 4.4 fold (+/-0.3 fold) and 11 fold (+/-0.49 fold) at day 4 and at day 7, respectively, as compared to preadipocytes (n=5). adora2a is 23 fold (+/-1.96 fold) upregulated at day 4 and 57 fold upregulated (+/-2.48 fold) at day 7, respectively (n=4). adora3 was found upregulated 3.6 fold (+/-0.46 fold) at day 7 (n=4). in contrast to this, ador2b was downregulated to 0.87 fold (+/-0.09 fold) at day 4 and to 0.69 fold (+/-0.04) day 7 compared to preadipocytes (n=5). to investigate the functional role of ador in bati cells, we analyzed lipolysis in mature cells after acute treatment with specific agonists and antagonists. we observed that adora2a activation by cgs21680 significantly increased lipolysis by 88% (+/-0.33%) compared to untreated control. moreover, adora1 antagonist psb36 increased lipolysis by 35% (+/-0.05%) (n=4). in conclusion, ador are highly regulated during brown fat cell differentiation. lipolysis of mature brown fat cells is significantly increased by adora2a agonist or adora1 antagonist, respectively. munich heart alliance, münchen, germany activation of the sympathetic nervous system and the subsequent activation of βadrenergic receptors (βars) through catecholamines represents the strongest mechanism to increase cardiac function. however, long-term activation of cardiac βars is clearly detrimental and β-blockers have been introduced as an effective treatment modality in cardiac failure. despite their central role in cardiac physiology and disease, our knowledge about the intracellular mechanism of βar stimulation is confined to a few targets and is likely incomplete. here, we report a functional proteomics approach to directly assess the entire phosphoproteome of βar-stimulated mouse hearts in vivo. to identify proteins that are phosphorylated in response to β-adrenergic stimulation in vivo, we treated mice with isoproterenol or, as a control, with propranolol. after lysis of hearts and tryptic digest, phosphopeptides were enriched by tio 2 or immobilized metal ion affinity chromatography (imac). subsequent analysis of eluated peptides by tandem mass spectrometry (ms/ms) mapped several phosphopeptides to cardiac proteins, among which known mediators of βar signaling such as phospholamban, troponin i and myosin binding protein c. we then employed multiple reaction monitoring (mrm) as a quantitative approach to assess changes of phosphorylation after βar stimulation. using this combination of ms approaches, we identified 39 peptides with pka consensus phosphosites that were more abundantly detected under βar stimulation. among those, we found myozenin-2 (myoz2) and g protein signaling modulator 1 (gpsm1, also termed ags3) as proteins previously not related to βar signaling. we validated the βar-dependence of phosphorylation at these sites in isolated cardiomyocytes by in vivo labelling or phosphoepitope-specific antibodies. current efforts aim at the functional characterization of these novel candidate mediators of βar signaling in the heart. taken together, we report the β-adrenergic phosphoproteome of the mammalian heart in vivo. we have identified several new targets of βar signaling that may represent essential factors in cardiac physiology and disease. background: drug measurement in autopsy material is normally used to investigate the cause of death. in our study it was possible to measure concentrations of drugs that were part of a regular treatment without connection to the cause of death. metamizole is used as an analgetic and spasmolytic agent. the active metabolite maa (4-methyl-aminoantipyrin) is metabolized by the liver and eliminated by the kidney. hepatic and renal dysfunction can therefore influence maa clearance. methods: maa concentrations were measured in different samples of the autopsy material (heart blood, venous blood, urine, liver, kidney and brain) using an hplc-ms/ms method. information about the dosage and time of drug application as well as information about existing renal or hepatic disorders were taken from the corresponding patient records. because of the low number of cases an explorative single-case study was necessary. results: 10 cases with oral intake of metamizole in a customary continuous dosage could be indentified. the maa distribution into body liquids and organs depended on the time between last oral intake and death. in two cases without renal or hepatic diseases maa blood levels were below 10 µg/ml. five cases with combined renal and hepatic disorders showed either increased blood levels of 40-50 µg/ml or prolonged maa elimination half-life of up to 12 hours. in one case with manifest hepatic insufficiency an maa concentration of more than 200 µg/ml was measured in venous blood. two cases with renal insufficiency alone had maa venous blood levels of less than 10 µg/ml. (pet) . pet detects the positron emission of neutron-deficient radioactive nuclides and allows their external localization in vivo. fet, a modified amino acid, is not incorporated in proteins but accumulates in glioblastomas. one pathway responsible for its accumulation is the preferential transport into the tumor cells, probably via amino acid transporters. we investigated in more detail (a) which individual, cloned amino acid transporters accept fet as substrate and (b) which transporter is responsible for the major fet transport into glioblastoma cells. studies with xenopus laevis oocytes, expressing individual human amino acid transporters, revealed that system l, y + l and b 0+ amino acid transporters recognize fet as substrate (lat1 and 2, y + lat2, and b 0+ at, respectively). in contrast, y + lat1 and atb 0,+ did not transport fet. rna expression studies using qrt/pcr revealed that lat1 is the dominant amino acid transporter in all glioblastoma cells investigated (ln229/u373/u87mg/u251/a172/t98g). a strong lat1 expression was also shown on the protein level. to find out whether lat1 is the main transporter responsible for fet accumulation, we first studied transport of the parent amino acid l-tyrosine in ln229 glioblastoma cells. [ 3 h] tyr uptake was completely na + -independent and inhibited by leu, phe and trp, but not by arg, pro or ser. sirna-mediated down-regulation of lat1 in ln229 cells led to a concomitant decrease of lat1 mrna and tyr transport (down to 3% and 20%, respectively). these results indicate that tyr is exclusively transported by lat1 in ln229 cells. we are currently performing transport studies using [ 18 f]fet to investigate whether fet transport is also exclusively mediated by lat1 in glioblastoma cells. a further question is if lat1, a sodium-independent transporter, can be responsible for the accumulation of fet observed in glioblastoma cells. if true, other amino acid derivatives that are lat1 substrates might also proof useful in cancer diagnosis. telmisartan reduces adipose tissue inflammation and biglycan accumulation in diabetogenic ldl-receptor knockout mice grandoch m., nagy n., fischer j. w. institut für pharmakologie und klinische pharmakologie, universitätsklinikum der heinrich-heine-universität düsseldorf, moorenstraße 5, 40225 düsseldorf, germany in addition to lowering blood pressure some of the angiotensin ii at1 receptor antagonists (arb) such as telmisartan have additional beneficial effects on the onset of type 2 diabetes mellitus and obesity. this was contributed mainly to peroxisome proliferator activated receptor (ppar)γ modulating activity. hyaluronan (ha), a high molecular weight polysaccharide and the small leucine rich proteoglycans, decorin and biglycan, are known to be involved in atheroprogression. mechanistically these matrix components contribute to inflammatory processes via toll-like receptor-signalling and are supposed to modulate lipid retention. the aim of this study was to elucidate the effects of telmisartan in comparison to valsartan, an arb without pparγ activity, on extracellular matrix remodelling and inflammation in atherosclerosis and the interrelationship with adipose tissue inflammation using the ldlr-/-model of accelerated atherosclerosis. male ldlr-/-mice were fed either a diabetogenic diet alone or in combination with telmisartan (10 mg/kg), valsartan (25 mg/kg) or valsartan (50 mg/kg) from 8 weeks of age for 17 weeks. all treatment groups except of the lower valsartan dose showed significant effects on reducing the aortic plaque score. the content of ha, collagen and decorin in the aortic root were not changed. however, telmisartan reduced the content of biglycan in the aortic root significantly in contrast to valsartan. in addition, a trend towards decreased mac2-positive macrophages in abdominal adipose tissue was detectable after telmisartan treatment as well as a strong reduction in the adipose tissue mrna expression of biglycan. finally, telmisartan reduced the expression of hyaluronan catabolizing enzymes potentially leading to an increase of high molecular weight ha in the adipose tissue, which is thought to be homeostatic and antiinflammatory. in summary, the results of this study underline the pronounced anti-inflammatory capacity of telmisartan on atherosclerosis and adipose tissue inflammation in comparison to valsartan and strongly suggest that biglycan might be an additional target of telmisartan not only concerning matrix composition of atherosclerotic lesions but also concerning the structure of adipose tissue and metabolic effects of the compound. human primary malignant cancer cells derived from peritoneal effusions of a patient with colorectal carcinoma, as assessed by comet assay. the primary cancer cells were more efficient in dsb repair than ht-29 cells, and their doxorubicin ic50 was four times higher. comparative protein expression levels showed that the primary cells had less rad 51 and 52 as well as less topoiiα, while ku70 and 80 levels were similar. another very interesting protein is the mrn (mre11-rad50-nbs1) complex that initializes the phosphorylation of atm and thereby starts the signalling cascade. the newly described mrn-atm pathway inhibitor mirin interrupts mrn activity by inhibiting the exonuclease activity of mre11. the toxicity of mirin in ht-29 cells was measured using a luminescence-based assay detecting the amount of atp, which is correlated with cellular viability. mirin did not show any toxic effects up to a concentration of 100 µm and incubation times of 24 hours, indicating that mirin can be used under these conditions without detrimental effects. we are currently investigating the effect of mirin on the toxicity of topoiiα inhibitors. the inhibition of dna repair may be a valuable strategy to enhance the effect of dnadamaging anticancer drugs. since tumours (even of the same entity) are not only heterogeneous, but also polyclonal, a broad selection of response modifiers of anticancer drugs would be helpful to individually enhance chemotherapeutic effectiveness. evidence has been provided that diet and environmental factors directly influence epigenetic mechanisms associated with cancer development in humans. epigenetics play an important role in the control of gene expression. epigenetic mechanisms comprise modulation in dna methylation, histone modification and non-coding rna. several polyphenols have been reported to possess histondeacetylase (hdac) inhibitory properties [1] . histone deacetylation is generally linked to transcription repression. furthermore, hdac belongs to the group of small ubiquitin-related modifier (sumo) substrate proteins. sumoylation of hdac is associated with a modulation of its biological activity [2] . little is known so far about the mechanism by which hdac sumoylation mediates inhibition of gene transcription. we addressed the question whether sumo e1 and hdac 1 expression and whether potential hdac-sumoylation will be affected by polyphenols such as chlorogenic acid, genistein and (-)epigallocatechin-3-gallate (egcg). chlorogenic acid, genistein and egcg decreased sumo e1 protein level in the human colon carcinoma cell line ht29 after 24h of incubation measured with western blot analysis. egcg exhibited the most pronounced effect at concentrations ≥ 50 µm. hdac 1 expression was also affected by these polyphenols. the direct impact of polyphenols on the hdac sumoylation is detected by co-immunoprecipitation experiments with the respective antibodies against hdac-1 and sumo e1. these experiments are still under investigation. in conclusion, chlorogenic acid, genistein and (-)-epigallocatechin-3-gallate influenced the sumo and hdac expression in vitro. in further studies the direct impact on subtract-sumoylation will be investigated. these studies contribute to a better understanding of potential chemopreventive effects of dietary polyphenols on specific epigenetic alterations may provide chemopreventive strategies for reducing cancer risk. the no/cgmp cascade is thought to be essential for penile erection. within the smooth muscle of corpus cavernosum, nitric oxide activates the no-sensitive guanylyl cyclase (no-gc) which raises the intracellular concentration of cgmp. this second messenger activates the cgmp-dependent protein kinase i (pkgi) and subsequent phosphorylation of target proteins leads to relaxation of cavernosal smooth muscle. knock out of key enzymes of the no/cgmp cascade has led to discrepant results: the deletion of pkgi in the mouse has been shown to lead to erectile dysfunction whereas mice lacking neuronal no synthase are fertile. to investigate the role of the no receptor in fertility we have generated mice lacking no-gc (gcko), a bottleneck enzyme of the no/cgmp cascade. we have shown that lack of no-gc resulted in arterial hypertension concomitant with a totally abolished no responsiveness of vascular and gastrointestinal smooth muscle. in addition, we generated a mouse line in which no-gc was specifically deleted in smooth muscle cells (sm-gcko). using these ko strains we here examined the role of no/cgmp signaling with regards to the smooth muscle relaxation of corpus cavernosum. no failed to affect corpus cavernosum from gcko in organ bath experiments: neither exogenously produced no by no donors nor endogenous no release from neurons induced by electrical field stimulation led to relaxation. similar results were observed in the corpus cavernosum of sm-gcko mice. to our surprise, the gcko animals were fertile and produced offspring albeit at a reduced rate compared to wt animals. our data show that interruption of no/cgmp signaling results in complete absence of no-induced relaxation of penile corpus cavernosum in mice and reduces the ability to produce offspring but does not abolish fertility. novel modes of invasive cell motility regulated by the formin class of actin nucleators khan j., grosse r. philipps-universität marburg, pharmakologisches institut, karl-von-frisch-str. 1, 35034 marburg, germany pathological invasive cell migration essentially reqires actin polymerization. formins are the largest group of rho-gtpase effectors involved in actin nucleation and assembly as well as microtubule dynamics. here we studied the role of formins in cytoskeletal regulation during homotypic cancer cell invasion. we identified the actin-dependent steps and structures involved for this process. using live cell analysis we characterize the distinct actin dynamics controlled by formin-like 2 and rho function. the specific involvement of this signaling module will be discussed. formin-driven nuclear actin assembly controls mal/srf activity baarlink c., wang h. polymerization of actin in the cytoplasm is tightly linked to transcriptional activation of the srf cofactor mal (also known as mrtf-a) through release of actin/mal interactions and subsequent nuclear accumulation of mal. formins directly promote assembly of actin filaments thereby efficiently regulating mal-dependent transcription for cell shape, adhesion and motility. here we show that formins assemble f-actin and promote mal activation inside the mammalian nucleus. the rho-gtpase effector mdia2 rapidly enters the nucleus in a signal-dependent fashion and an active mdia confined to the nucleus potently promotes release of g-actin from mal to specifically activate srf. live cell imaging reveals formin-mediated nuclear actin dynamics. moreover, using actin assembly assays we find that inhibition of endogenous mdia formins controls f-actin turnover in isolated nuclear extracts. thus, formin activity is dynamically compartmentalized to the mammalian nucleus to potently regulate actindependent mrtf function. in women the placenta becomes the main source of maternal estrogens during pregnancy. placental estrogen biosynthesis is located in the syncytiotrophoblast, a syncytium that builds the main part of the placental barrier and limits the transfer of substances between the fetal and maternal compartment. since the human placenta is unable to convert cholesterol into 17-oh-pregnenolone, the placenta tissue highly depends on the supply of c-19 steroids for their conversion into c-18 estrogens. in contrast to lipophilic unconjugated steroids that penetrate the cell membrane passively via diffusion, circulating sulfated steroid hormones are delivered to the placenta via carrier-mediated transport, followed by their reactivation via the catalytic activity of the steroid sulfatase (sts). dheas of maternal and fetal origin contributes about equally to the placental formation of estrone (e 1) and estradiol (e2), while 16αoh-dheas supplied by the fetus contributes to over 90% of placental estriol (e3) synthesis. soat, a member of the slc10 family with highest expression in hormone-responsive tissues such as testis, placenta, and mammary gland has been shown to transport the sulfoconjugated steroid hormones dehydroepiandrosterone sulfate (dheas), estrone sulfate (e1s), and pregnenolone sulfate (pregs) [1] . aim of this project is to investigate the role of soat for placental estrogen synthesis by means of the choriocarcinoma cell line jeg-3 as in vitro model for the human syncytiotrophoblast. therefore, we characterized a jeg-3 cell line that transformed dhea into e2 and 16αoh-dhea into e3. by qrt-pcr we found expression of sts and aromatase, both essential for estrogen synthesis in these cells. upon transient transfection of soat the carrier was located in the cell membrane of transfected jeg-3 cells. currently we investigate the transformation of dheas of these soat-jeg-3 cells by lc-ms-ms. we could demonstrate transport of 16αoh-dheas for stably transfected soat-hek293 cells. in situ hybridization and immunohistochemistry showed coloured syncytiotrophoblasts and vascular endothelial cells in late term placenta. in conclusion, soat-mediated transport of sulfated steroids could play a pivotal role for placental estrogen synthesis from sulfated steroid hormones. developing non-animal test systems for evaluation of toxicity was important in the past and will remain essential in the future. here we present a toxicity test using the chicken yolk sac area vasculosa (cav) of fertilized white leghorn chicken eggs [1, 2] and compare it to hen's egg test on chorioallantoic membrane (het-cam) [3] for polymer toxicity testing. fertilized chicken eggs were incubated and after 72 h explanted shell less into sterile petri dishes. test substances were applied on the cav and the appearance of different effects (vascular lysis, haemorrhage, aggregation of blood components, lethality) was determined by light microscopy after 1 -48 h (fig.1 ). these effects were combined to a cav test score based on the irritation score calculation used for het-cam evaluation. different polymers like poly(ethylene glycol) (peg; neutral), poly(ethylene imine) (pei; cationic) and dextran sulphate (ds; anionic), as well as guideline-conform (recommended het-cam protocol from the interagency coordination committee on the validation of alternative methods) negative (0.9 % nacl) and positive controls (1 % sodium dodecyl sulphate (sds) and 0.1 n naoh) were investigated. additionally ld 50 values for different cationic polymers have been determined. within the selected incubation times (1 -48 h) , effects such as vessel lysis and blood component aggregation could be detected. additionally to het-cam, lethality as well as recovery of the cav could be observed. differences between neutral, positively and negatively charged polymers were obtained. pei showed strong vessel lysis and aggregation of blood components whereas ds and peg showed none of these effects. lethality was found to increase from peg < ds < pei and is concentration and time dependent. the results demonstrate that differences, regarding the toxicity of the used polymers, can be shown with this test. these findings in the cav test can be well correlated with already existing data. in summary, the cav test provides same data (testing control substances) and more information (recovery and lethality) compared to het-cam and could be a suitable model for toxicity testing of polymers. risk characterisation of chemicals consists of three steps (1) hazard identification and characterisation, based on substance-specific toxicological hazard data, (2) estimates of the level of exposure toward the substance and (3) the comparison between the toxicologically safe level and the exposure level. in contrast to the classical risk assessment approach, the threshold of toxicological concern (ttc) approach is developed as a tool to assess the risk of substances without toxicity data. its application requires (1) information on human exposure, for which it is essential that exposure is fully captured and (2) knowledge of the chemical structure to assess whether the chemical is not excluded from the application of the ttc concept. instead of chemical specific no observed (adverse) effect levels (noels/noaels), the ttc approach utilises knowledge on the empirical distribution of several hundreds of noels/noaels, originally 613, based on toxicological testing in animals (munroe et al., 1996) . with the basis on noaels, the ttc concept builds on the fundamental principle of toxicology, that toxicity is a function of dose and that a dose exists, below which no adverse effects of the substance can be detected. it is assumed that exposures below this level will not result in health risks. three separate ttcs were derived (munroe et al., 1996) by classifying the chemicals into three toxicity classes using a decision tree based on a series of 33 questions related to chemical structure, and on natural occurrence in food and in the body (cramer et al., 1978) . the ttc values are derived from empirical distribution of the noels/noaels in the class taking the 95th percentiles and dividing them by the default uncertainty factor of 100. it is assumed that the probability is very low that the unknown noael of a not tested chemical will be lower than the value of the 95th percentile in the distribution of the known noels/noaels. hence, at exposures below the ttc values, the probability of adverse effects on human health is considered to be very low. introduction: kibra, mainly expressed in kidney and brain tissue, is involved in brain development and memory formation as a postsynaptic scaffold protein. in podocytes, kibra is proposed to regulate cell motility as a linker between components of the cytoskeleton and polarity protein complexes (duning et al, jasn 2008) . furthermore, kibra has been identified as key regulator of the hippo pathway, which is involved in organ size control and tumorigenesis. in the current study, we focused on the identification of kibra gene expression regulation and functional promoter characterization. methods: serial promoter deletion constructs were generated by cloning 3627 bp of the 5'flanking region of kibra into the pgl3-vector system. deletion constructs were transiently transfected into human neuroblastoma cells (sh-sy5y) and immortalized human kidney epithelial (ihke) cells. potential transcription factors (tfs) were investigated in cotransfection experiments. transcriptional start sites (tss) were determined by rapid amplification of 5'cdna ends (5'race) . tss utilization between cell lines was assessed by semiquantitative pcr. transcriptional activity (ta) of the kibra promoter p1 was separated by ~1060 bp into two distinct regions, promoter p1a and p1b. deletion constructs harbouring promoter p1b were transcriptionally active only in ihke cells. 5'race revealed two alternative tss in both cell lines upstream of the annotated tss (nm_015238). exclusively in ihke cells, two additional tss were detected in intron 1, resulting in two alternative exons. deletion constructs harbouring the putative regulatory regions (p2 and p3) of both exons were transcriptionally active only in ihke cells. overexpression of full length tcf7l2 (transcription factor 7-like 2 [t-cell specific, hmgbox]) resulted in a ~3-fold increase of promoter p1a and intron promoter p2 ta. kibra gene expression is driven by a complex alternative promoter system comprising the constitutional promoter p1 and three alternative promoters p1b, p2 and p3. the tss utilization is cell type-specific. subsequent usage of an alternative translation start site within exon 3 could result in truncated kibra protein isoforms. tcf7l2 is involved in the differential kibra gene expression regulation. resulting kibra protein isoform and their cellular function will be assessed in further studies. skin absorption in vitro based on the study of human/animal skin ex vivo or reconstructed human epidermis, respectively, is an alternative method which is accepted by the oecd. guideline tg 428 and a corresponding technical guidance document (gd 28) give technical guidance how to perform valid experiments 1, 2 . the requirements include integrity evaluation tests for the skin samples. different tests are proposed to ensure an exclusively use of undamaged skin. to decide which test suites best to our routine test strategy, we investigated the correlation between integrity test results and absorption profiles of various penetrants (logp range: -0.07 -6.2). finite dose experiments using rat and human skin were performed with 14 c-labeled testosterone, caffeine, mcpa (4-chloro-2-methylphenoxyacetic acid) and its 2-ethylhexyl-ester mcpa-2ehe. for each experiment at least three of the five following integrity tests were conducted: transepidermal electrical resistance (teer), transepidermal water loss (tewl), transepidermal tritiated water flux (³h2o), transepidermal absorption of methylene blue (blue) , transepidermal absorption and flux of a ³h-labeled internal standard (istd); ³h-testosterone or ³h-mannitol was used as istd. the applied radioactivity of the ³h-istd was selected to show no analytical interference with the 14 c-penetrants. teer, tewl and ³h2o represent pre-study, istd concurrent and blue post-study tests. calculated maximal permeability constants (kp) and absorbed doses (ad) of the penetrants were compared to the results of the integrity tests. individual linear regression analysis was used to evaluate the correlation the correlations varied over a wide range for all five methods and four penetrants. the best correlations in average were achieved with the istd. no inverse correlations were obtained for the istd, but partly for tewl, teer, ³h2o and blue. in conclusion, the istd represents best the achieved absorption profiles of the test compounds and is based on that the most suitable integrity test for our dermal absorption studies. we will further confirm its effectiveness and generate a sufficient historical database in order to include the istd in our routine test protocol. investigation of mirna expression and dna methylation in focal and non-focal brain tissue of therapy-resistant epilepsy patients haenisch s. 1 background: resistance to anticonvulsants affects one third of all epilepsy patients. limited bioavailability of the drug at the target site caused by increased expression of efflux transporters on the blood brain barrier or alterations of target genes are potential mechanisms for therapy resistance. however, these mechanisms alone cannot completely explain the observed resistance and it is likely that multifactorial alterations lead to pharmacoresistance. there is increasing evidence that expression of micrornas probably caused by dna modifications is deregulated in many neuronal diseases. we hypothesize that mirna regulation of target genes is involved in drug resistance in epilepsy. methods: hippocampal focal and cortical non-focal brain tissue samples from 13 patients diagnosed with mts (mesial temporal sclerosis) who underwent neurosurgery have been screened for mirna expression using taqman low density arrays. in silico approaches for both a hypothesis-based (efflux-transporter and target gene) as well as a hypothesis-free approach were used to identify potential phenotype-relevant target genes. using the program r (bioconductor) a mann-whitney-u test was performed to compare mirna expression between brain regions. pyrosequencing was performed to investigate methylation status 5'-upstream of dna regions encoding for selected candidate mirnas. results: out of 754 mirnas, 150 were detected in both tissue types. the expression of one mirna was 7.2 fold higher (q=0.01) and another was 3.8 fold lower (q=0.01) in the hippocampus relative to the cortex. evidence could be found that down-regulation of the latter is possibly caused by hypermethylation of 5'-flanking region of its encoding dna locus. bioinformatic analysis has identified eight genes important for neuronal regulation and signal transmission (e.g. sox11, mecp2, bsn), as well as one abc effluxtransporter, as potential targets for these differentially regulated mirnas. conclusion: differential regulation of two mirnas could contribute to an altered function of several genes resulting in an imbalance between neuronal excitation and inhibition that is independent from mechanisms presently targeted by anticonvulsants. this work was supported by a fellowship from dfg and nih grant gm61390. recently, it has been reported that human b cells express and secrete the cytotoxic protease granzyme b (grb) after the combined stimulation of the il-21-and the b cell receptors. grb produced by b cells is enzymatically active and b cells deliver grb to sensitive cancer cell lines, thereby inducing apoptosis. to date, there is little experimental evidence on the mechanisms involved in grb expression, or its function in b cell biology. as experimental transgenic murine systems should enable us insights into these issues, we assayed for grb in c57bl/6 b cells using an extensive array of physiologically relevant stimuli, but were unable to detect either grb expression or its proteolytic activity, even when antigen specific transgenic b cell receptors were cross-linked. similar results were also obtained with b cells from dba/2, cba or balb/c mice. in vivo, infection with either influenza virus or murine γ-herpesvirus induced the expected expression of grb in cytotoxic t lymphocytes, but not in b cell populations. we also investigated a possible role of grb on the humoral immune response to np-klh, but grb-deficient mice produced normal amounts of antibody with typical affinity maturation and heightened secondary response, demonstrating conclusively the redundancy of grb for antibody responses. our results highlight the complex evolutionary differences that have shaped the immune systems of mice and humans and demonstrate the need to develop novel in vivo systems to study human humoral immune responses. investigations of the cholinergic neurotransmitter system in dyt1 mice hamann m. 1 early-onset torsion dystonia is an autosomal dominant inherited movement disorder associated with the dyt1 gene defect with deletion of a glutamic acid residue in the protein torsina. despite the gene defect, the pathophysiology is poorly understood. animal models can help to understand the underlying mechanisms and thereby to develop new therapeutic strategies. sharma et al. (2005, j. neurosci. 25 [22] , 5351-5355) initially described a transgenic mouse model (dyt1 mice) with overexpression of mutant torsina. previous studies in these mice pointed to alterations in the cholinergic system. to investigate the functional relevance of these in-vitro findings, we carried out pharmacological in-vivo experiments and determined the density of striatal cholinergic interneurons as well as the expression of choline acetyltransferase in different brain regions. the acute intraperitoneal administration of the cholinomimetic drug pilocarpine (75, 100 and 125 mg/kg) as well as a long-term treatment over 21 days (100 mg/kg/d) did not induce pronounced effects in dyt1 mice compared to wildtype controls. the higher incidence of epileptic seizures in dyt1 mice compared to controls after repeated local striatal applications of pilocarpine (25 and 50 µg/0.5 µl/hemisphere) let presume an altered synaptic plasticity in dyt1 mice. the immunohistochemical investigations revealed a moderately reduced density of striatal cholinergic interneurons in the dorsomedial subregion of dyt1 mice compared to wildtype controls, while significant differences in other striatal subregions were not detected. western blot analysis did not show clear differences in the expression of choline acetyltransferase between dyt1 and wildtype control mice. these results indicate that the cholinergic system seems not to play a key role in this line of dyt1 mice. ongoing receptor autoradiographic analysis of binding to different muscarinic receptors subtypes have to further clarify the existence of possible alterations within the cholinergic system of these dyt1 mice. inhibitors direct against cell cycle-regulatory kinases are being tested in clinical trials as anti-proliferative agents. thus, the atp-competitive kinase inhibitor pd332991 which inhibits cdk4 and cdk6 is currently tested in patients with solid tumors such as glioma. we found that pd332991 suppressed il-1-induced expression of il-8 suggesting that cdk4 or cdk6 may have unknown anti-inflammatory properties. to study the effects of cdk6 on the il-1-signaling network, we established a bidirectional doxycyline-inducible system to express a constitutively active mutant of cdk6, cdk6 s178p, in asynchronized hela cells. cdk6-expressing cells were identified by gfp which was expressed from the same promoter, isolated by laser-microdissection and analysed for mrna expression using a down-scaled rt-qpcr assay. compared to the uninduced state, cdk6 s178p enhanced il-1-induced il-8 and il-6 mrna expression. moreover, shrna-mediated suppression of endogenous cdk6 confirmed a role of this kinase in regulation of maximal il-1-induced gene expression of il-8. these data also revealed that the contribution of cdk6 to inflammatory gene expression is highest in g1, when activity of endogenous cdk6 is activated by d-type cyclins. these findings were corroborated in hela cells expressing fluorescent ubiquitin-dependent cell cycle indicator (fucci) proteins. hela-fucci cells from g1, g1/s, g2 or mitotic states were isolated by laser-microdissection and analyzed by rt-qpcr for tnf-inducible gene expression. stable knockdown of cdk6 in hela fucci or inhibition by pd332991 suppressed inducible il-8 expression. microarray experiments identified many additional genes that required active cdk6 for maximal il-1-or tnf-inducible gene expression. we also found that cdk6 co-immunoprecipitated with p65 nf-κb, colocalized with p65 in the nucleus and was recruited together with the p65 subunit to the proximal il-8 promoter as assessed by chip and re-chip experiments. collectively, these results suggest an unexpected control of inflammatory gene expression through a classical cell cycle regulatory pathway. these results also imply that pharmacological targeting of cdks may have effects and side-effects on the immune system in addition to inhibition of cell cycle progression. trp channels form a heterogeneous family of calcium-permeable channels, which play major roles in physiological functions ranging from sensory reception to cellular signal transduction. members of the trpc subfamily (classic transient receptor potential channels) are downstream targets of hormone receptors. of particular interest is the biological role of trpc6 channels. they are directly activated by diacylglycerol due to phospholipase c-driven signalling pathways which are involved in smooth muscle contractility, neuronal plasticity, keratinocyte differentiation and renal function. their impact in renal function became evident from analyzing patients suffering from familial forms of focal segmental glomerolusclerosis (fsgs) which could be linked to trpc6 mutations. since the first descriptions at least 17 different pathogenic mutations have been identified in humans to cause fsgs. in order to study the underlying pathophysiological mechanisms of trpc6 mutations, we have analysed all mutated trpc6 channels known to date heterologously expressed cells. one set of mutations showed a gain-of-function phenotype which has been previously suggested to cause an increased intracellular calcium load and subsequent cell death. hence, gain of function mutations fit to the current paradigm of fsgs pathophysiology. however, another set of mutations found in the patients showed a loss-of-function phenotype. our results enable a change in the current paradigm for the role of trpc6 in renal pathophysiology and may provide a basis for our understanding of the pathophysiology of loss-of-function mutations in familial focal segmental glomerolusclerosis. karlsruher institut für technologie (kit) institut für angewandte biowissenschaften, abteilung lebensmittelchemie und toxikologie, adenauerring 20a, 76131 karlsruhe, germany risk assessment for genotoxic carcinogens is an important challenge in toxicology. even though manifold attempts have been made to substitute carcinogens and to reduce exposures, their complete elimination appears to be not possible. thus, low concentrations of known or suspected genotoxic carcinogens are present at workplaces, in the environment and in food. in order to deal with this situation and to set priorities for risk management, different concepts have been established such as the alara principle (as low as reasonably achievable) and the margin of exposure (moe), based on the ratio between concentrations being carcinogenic in experimental animals and the actual exposure of humans for example via foodstuff. while usually linear doseresponse-relationships have been used as default assumption, analytical methods are now available to assess the induction and repair of dna lesions on low exposure conditions, including environmental background exposure, and to relate the extent of exposure-induced dna lesions to endogenous dna damage. this may be an important prerequisite to establish health-based limit values for selected genotoxic carcinogens. within this workshop, different examples will be discussed and research need will be identified. dendritic cells from h4r-deficient mice lose their ability to properly stimulate t lymphocytes hartwig c., seifert r., neumann d. mhh pharmakologie, carl-neuberg str. 1, 30625 hannover, germany the incidence of allergic airway diseases is increasing throughout the world, especially in western countries. although histamine (ha) is found at high concentrations in asthmatic lungs, a role for ha in bronchial asthma is still a neglected topic in clinical research. in particular, the capacity of ha to modulate the underlying immune reaction is far from being understood. the histamine h4-receptor (h4r) is involved in acute inflammation and th2 cytokine production. consequently, we intended to analyze the role of h4r in a murine th2 lymphocyte transfer-based model of asthma. specifically the ability of h4r expressed on dendritic cells (dcs) to modulate t cell function was analyzed. ova-specific cd4 + t cells were polarized in vitro under th2-favoring conditions with ova peptide-pulsed dcs, obtained either from wild-type or h4r -/mice. analysis of the polarized t cells after in vitro restimulation revealed a marked decrease of il-4 production in t cells polarized in the presence of h4r -/-dcs compared to those polarized in the presence of wild-type dcs. thus, on dcs, the h4r is essential for proper stimulation of spleen t cells and for directing their polarization towards a th2 phenotype. the transfer of in vitro polarized t cells into recipient mice and subsequent provocation elicited an asthma-like disease. the h4r on dcs not only affects in vitro polarization of t cells, but also the in vivo function of the obtained polarized t cells. a parameter indicating allergic inflammation is the enhanced influx of inflammatory immune cells into the lung tissue, mainly driven by eosinophils, which are virtually absent in non-asthmatics. when analyzing the number of eosinophils, a dramatic difference due to the polarizing conditions of t cells occurs. in bal fluids of mice that received t cells polarized in the presence of wild-type dcs, about 40% eosinophils were detected. in contrast, the transfer of t cells polarized in the presence of h4r -/-dcs yielded only about 10-20% eosinophils in bal fluids. in summery, the h4r on dcs plays an important role for t cell polarization and consequently affects the allergic reaction during sensitization. since the lack of the h4r on dcs reduced their ability to stimulate proper th2 polarization of cd4 + t cells, we conclude that ha via the h4r significantly affects the manifestation of asthmatic inflammation. antioxidant polyphenols and their effects on nrf2 (skn-1) signalling in a cell culture system and the model organism c. elegans havermann s., wätjen w. heinrich-heine-universität düsseldorf institut für toxikologie, p.o. box 101007, 40225 düsseldorf, germany oxidative stress has been connected with a variety of diseases, (e.g. alzheimer`s and parkinson´s disease), cancer and ageing over the last years. certain polyphenols were shown to have an antioxidant capacity as well as being able to activate the protective nrf2 signalling pathway. compared to direct radical scavengers modulators have the advantage of building up a permanent defense against oxidative insults whereas scavengers do not protect any more after consumption or may even cause stress due to redox cycling. we have employed cell culture based assays (dcf, western blot, gfp reporters) to analyse the effects of polyphenols. further we tested the coumpounds in vivo in the nematode c. elegans where skn-1 is the nrf2 homologue. baicalein and caffeic acid phenethylester (cape) protected cells and the nematode from ros accumulation after application of stress (shown by dcf assay). activation of nrf2 signalling is correlated with translocation of the transcription factor into the nucleus. in both systems nrf2::gfp accumulation in the nuclei could be observed after incubation with baicalein (fluorescence microscopy). but while cape is a potent activator of nrf2 in cells, it has no effect on skn-1 localisation. further the effect on the nrf2 protein amount was investigated by western blot analysis. the expression of target genes can be investigated by differing means: while pcr methods and western blotting are standard for in vitro studies, the vast number of available gfp reporter strains offers opportunities for research using c. elegans. we have performed congruent assays in a cell culture system and the model organism c. elegans to compare antioxidative capacity and effects of polyphenols on nrf2 signalling. therefore, depending on the substance tested, c. elegans is a suitable model system to investigate effects of natural compounds in an organism. being associated with adverse health effects, the human exposure to dehp is subject to concern. quantifying the population's exposure and determining the contributions of different exposure routes is a key task of environmental health risk assessment. the study presented comprises a review of the available data on dehp levels in foods, consumer products, and house dust. extensive survey data, e.g. from the current national nutrition survey ii and the eu rapex system were processed for modeling the exposure by the oral, inhalative and dermal path of the population in germany. the study also included analytical analyses of dehp levels in selected foods and consumer goods (incl. migration rates for mouthing). probabilistic techniques allowed elucidating the exposure's variation and the relevance of different routes. mean exposure estimates for german children and adults to dehp are 36 and 26 µg/(kg d), resp. for children, food accounts for 36% of the total exposure, followed by mouthing (30%) and house dust (19%). the adult exposure is almost entirely (82%) due to food. as dietary exposure is a result from concentration and consumption, foods exhibiting high contamination e.g. butter (8%) and dressings (mayonnaise) (14 %) as well as highly consumed foods e.g. bread and bakery (16%) and vegetables (6,8 %) contributed significantly. the mean estimate of children's dehp exposure via mouthing revealed 0,9 µg/(kg d). high exposures were estimated (95th percentile) up to 10,8 µg/(kg d). on average people in germany are exposed to dehp below the current tdi of 50 µg/(kg d). however, individual exposures exceeding the tdi still cannot be excluded. current data on dehp and other plasticizers in foods are scarce, which warrants broader monitoring. our findings highly facilitate further exposure modeling focusing on dehp substitutes and risks of combined exposure. this study was funded by the federal ministry for the environment, nature conservation and nuclear safety in the frame of the environmental research plan (umweltforschungsplan, förderkennzeichen (ufoplan) 3707 61 201). on the basis of the available measurements of dehp, the exposure assessment has been focused on 37 food categories characterising a selection of the most important food groups covering all major food classes of the german population. based on an extensive literature survey, the analysis considered the available data of dehp measurements in food, as well as the official german food control data taken from the national food consumption survey. the high amount of considered data allowed the consideration of several exposure assessment tiers (deterministic and probabilistic by using monte carlo simulation). a quantitative evaluation of the uncertainties of the estimate of the 37 food categories groups has been performed by means of a sensitivity analysis by using the methodology proposed within the 2008 who ipcs guidance document of characterising and communication uncertainty in exposure analysis. qualitative uncertainty analysis (tier 1) was applied to determine the most important sources of uncertainty, i.e. concentration of dehp in all food categories. the probabilistic monte carlo simulation (tier 2) was then used to rank the cumulative probability distributions of the exposure assessments of 37 food categories on the basis of the food categories that appear to dominate. sensitivity analyses were applied to prove the impact correlation of food groups for uncertainties. by a scenario based concept, the aggregation of the 37 food groups to 10 groups has been evaluated, as well as the sensitivities by characterising particular scenarios. for this purpose, particular "meals" have been described as fixed combined scenarios and. the aggregation leads to a considerable higher exposure estimate which can be explained by the combination of high contaminated foods with others of high consumption. the evaluation confirms the considerable role of possibly high contaminated foods e.g. fats, or mayonnaise. the evaluation shows that quantitative probabilistic sensitivity analysis is a suitable and pragmatic tool for uncertainty analysis in exposure assessment. the transcription factor camp response element (cre)-binding protein (creb) plays a critical role in regulating gene expression in response to activation of the campdependent signaling pathway, which is implicated in the pathophysiology of heart failure. we observed creb knock-out cardiomyocytes to be larger than wildtype cardiomyocytes (cell area in µm 2 ; mean±sem; creb-ko 4871±258 vs. wt 3396±171; n=68/69 cells, n=4 mice, p<0.01 vs. ctr.). the nuclear factor of activated t-cells c3, nfatc3, is another transcription factor involved in the development of heart failure and also a known positive regulator of hypertrophy. hence, we investigated whether inhibition of the cre-dependent transcriptional activation has an impact on the nfatc3 signaling pathway. we first studied the effects of an overexpression of a dominant negative creb mutant (dncreb) or of nfatc3 on the activity of a nfat-dependent model promoter in a permanent cell line. overexpression of dncreb evoked an 8.0±1.5 fold increase of the nfat model promoter activity (n=36; n=6 transfections), but had no impact on kv4.2 promoter activity which is known to be regulated by nfatc3 (1.2±0.1 fold; n=18; n=3; p<0.05 vs. ctr.). nfatc3 overexpression led to a 4.5±0.7 fold increase of the nfat-dependent model promoter activity (n=12; n=2) and to an inhibition of kv4.2 promoter activity (0.8±0.1 fold; n=18; n=3; p<0.05 vs. ctr.). we conclude that creb is a negative regulator of nfat-mediated gene transcription and that activation of nfatc3 might contribute to the observed hypertrophy of creb-ko cardiomyocytes. neuroleptika der perazin-klasse sind potente modulatoren des p2x7-rezeptors -perazine-type neuroleptic drugs are potent modulators of p2x7 receptors hempel c., nörenberg w., urban n., sobottka h., schaefer m. universität leipzig rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße 16-18, 04107 leipzig, germany p2x7 receptors belong to a family of atp-gated, non-selective cation channels, which play an important role in immune cell activation, inflammatory hyperalgesia and neuropathic pain. they differ from other p2x family members by the low atp affinity, and by the ability to form or recruit dilated pores in the sustained presence of atp. owing to its involvement in many diseased states, p2x7 is a promising target for pharmacological intervention. accordingly, p2x7 blockers are currently tested in phase ii clinical trials. in an attempt to identify p2x7-modulating properties of approved drugs or natural compounds, we performed a medium-throughput screen, using an appropriate compound library (spectrum collection) and a stably transfected hek293hp2x7 cell line. with ic50 values of 1-2 µm, the tricyclic antipsychotics prochlorperazine and trifluoperazine showed a high potency and efficacy to block the atp (1 mm)-triggered increases in the intracellular ca 2+ concentration ([ca 2+ ]i) that was mediated by human p2x7 (hp2x7). the closely related phenothiazine-class neuroleptic drugs, such as chlorpromazine or triflupromazine did not have an appreciable effect on hp2x7mediated ca 2+ influx. whole-cell inward currents, measured at -60 mv, were blocked by more than 60% by 3-10 µm prochlorperazine. the inhibitory effects of perazines developed within about 200 ms, hinting to a direct mode of action by binding to the p2x7 protein. prochlorperazine added intracellularly via the patch pipette did not substitute for the extracellularly applied drug, indicating that its binding site is accessible from the extracellular side. in addition, both compounds blocked yo-pro-1 uptake when preincubated before p2x7 stimulation with 1 mm atp or when applied subsequent to the agonist. interestingly, when added to a hek293 cell line expressing the rat p2x7, perazines potentiated the atp-induced increase in [ca 2+ ]i. measurements in human monocyte-derived macrophages confirmed the ability of prochlorperazine and trifluoperazine to inhibit atp-evoked increases in [ca 2+ ]i, changes in yo-pro-1 permeability and whole cell currents. taken together, we conclude that perazine-type neuroleptics impede on p2x7 activity in a species-specific manner, presumably by binding to an extracellularly accessible binding site of recombinant or natively expressed p2x7. similarly, pre-treatment with lov also lowered dox-induced stabilisation of p53 and phosphorylation of chek1 and sapk/jnk. while lov had no influence on ir-induced initial dna damage formation in huvec and rat cardiomyoblasts (h9c2), it decreased dox-and eto-induced phosphorylation of histone h2ax, which is a surrogate marker of dna-double strand breaks. this indicates that lov specifically protects against the genotoxicity of topoisomerase type ii poisons. in an acute and subacute balb/c mouse model lov protected from ir-induced toxicity. this effect rested on inhibition of pro-inflammatory and pro-fibrotic processes as measured via quantification of mrna levels of il6, ctgf and tnfα. the same was true for dox-induced toxicity, i.e. heart and liver damage. similar to the in vitro experiments, dox-induced hepatic dna-damage was attenuated by lov treatment. overall, liver and heart toxicity were reduced by lov as mirrored by the serum levels of gldh/gpt and ctn-i, respectively. both in liver and in heart we observed collagen rich perivascular areas following dox treatment. under situation of lov-co-treatment these areas occurred more rarely and were less pronounced, pointing to a lowered level of fibrosis. pcr-array-based mrna analyses showed inhibitory effects of lov on dox-triggered expression of genes involved in oxidative stress response, drug transport, dna repair, cell cycle progression and cell death. for instance, up-regulation of p21, wee1, cjun/fos and hmox-1 following dox administration was attenuated by lov. altogether, we suggest that including lov in current cancer therapeutic regimen might widen the therapeutic window of anticancer therapeutics by lowering normal tissue damage. the p values. in addition, array data underwent cluster analysis for identification of substantial differences of gene regulation among the three different types of biopsies. results: of particular interest in our study was the expression of genes coding for metabolism and transport proteins. therefore 42 genes from the 156 significant differentially regulated genes, were selected for the qrt-pcr analysis. genes coding for abcb1 and abcg8 transport proteins showed higher expression in the jejunal tissue one year after surgery compared to the duodenal tissue (fold change 1.80 and 1.73). moreover, cyp7a1 mrna involved in metabolic processes is higher expressed in postoperative jejunum than in the jejunum tissue taken during the surgery (fold change 1.9). in conclusion roux-en-y gastric bypass operation leeds a change of mucosal gene expression profile in the jejunum during one year. there was also a significant differential gene expression between the original duodenum and jejunum one year after surgery. these results give strong evidence that jejunum not exposed to pancreatic but only to gastric fluids may change its gene regulation. background. numerous genome-wide association studies (gwas) identified polymorphisms located in transporter genes such as slc2a9, abcg2, npt1, and urat1 as predicitive for the serum levels of urate 1 . these genes encode membrane proteins expressed in the apical membrane of human kidney proximal tubule cells and are assumed to facilitate tubular exchange of urate 2, 3, 4, 5 . importantly several single nucleotide polymorphisms (snp) located in vicinity of slc2a9 have been identified as highly associated with serum urate levels. little is known about the transcriptional regulation of slc2a9. therefore, the aim of our study was to investigate which sequences in the slc2a9 gene harbour ciselements and regulate its gene expression. we also asked whether intronic snps influence gene expression at the transcriptional level. methods and results. performing dual luciferase reporter gene assays we found gene regulating modules in the slc2a9 gene. dna from human kidney samples was then genotyped for rs6449237 being part of this region. next total slc2a9 mrna-expression levels of the samples were determined using real-time quantitative rt-pcr assay. male samples with two minor alleles of snp rs6449237 showed lower slc2a9 mrna levels than samples with the wild type alleles. the effect was not seen in females. reporter gene constructs with either minor or major allele of rs6449237 were then used in luciferase assays, however showing no significant difference in activity. furthermore mrna-expression levels of other urate transporter genes were determined in kidney samples. after linear regression a positive correlation of mrna-expression of slc2a9, urat1, npt1, and oat10, respectively was observed. conclusion. our data suggest that the slc2a9 snps rs6449237 and rs74794351 might influence slc2a9 mrna-level without controlling the transcriptional activity. it needs to be elucidated whether those snps alter mrna stability. however, the mrna coexpression of slc2a9 and other urate transporter might be attributed to a common gene regulating pathway of an "transportosome" controlling urate homeostasis. sulfotransferases mediate the bioactivation of methyleugenol to a reactive sulfate ester binding to dna in vitro and in vivo herrmann k. 1 methyleugenol (me) is a secondary metabolite occurring in many herbs and spices. although me is hepatocarcinogenic in rodents, standard genotoxicity tests were negative. this may be due to the lack of critical activating enzymes responsible for the terminal bioactivation of me to a genotoxicant. me is initially hydroxylated by cytochrome p450 enzymes yielding 1´-hydroxymethyleugenol (1´-ohme). this alcohol can be further activated by sulfotransferases (sults) to an electrophilic sulfate ester that can be easily attacked by dna. the dna adducts formed could lead to mutation and further carcinogenicity observed in animals. the aim of the present study was to clarify whether individual human (h) and murine sult forms are involved in the activation of me to a genotoxicant. in order to identify critical sults, mutagenicity tests including bacteria expressing different sult forms were conducted. (±)-1´-ohme (separated into its enantiomers) served as test compound. we could show that hsult1a1, standing out due to its high expression level in many tissues, can efficiently activate both enantiomers even at low concentrations. furthermore, dna adduct formation in hsult1a1-proficient and sult-deficient bacteria was examined after incubation with 7 µm of (+)-or (-)-1´-ohme. for selective detection and quantification of me-derived 2´-deoxyadenosine (da) and 2´-deoxyguanosine (dg) adducts we developed a sensitive tandem mass spectrometry method including stable isotope dilution analysis. adduct formation was only observed in bacteria expressing hsult1a1. the concentration dependence of adduct formation in hsult1a1-proficient bacteria was examined for (+)-1´-ohme. both adducts turned out to be concentrationdependent. to check the extent and organ specificity of adduct formation in vivo we administered 54 mg/kg bw (±)-1´-ohme (i.p.) to mice carrying the hsult1a1/1a2 gene cluster. mice getting only the vehicle served as controls. animals were sacrificed and dna from eight organs was extracted. by means of tandem mass spectrometry adducts were measured and quantified. da and dg adduct formation was observed in all tissues studied, but not in untreated animals. furthermore, adduct levels were higher than in experiments using wild-type mice. altogether, we herein could show that sulfo conjugation leads to bioactivation of me to a dna-binding intermediate in vitro and in vivo. this work was financially supported by bundesinstitut für risikobewertung. objective: the soluble adenylyl cyclase (sac) activates the na + /k + -atpase in renal epithelial collecting duct cells. nuclear sac constitutes a functional complex with camp response element binding protein (creb), suggesting a more general role of sac in overall gene regulation. we determined the chromatin binding capacities of sac at cre sequences and its influence on genes, which play a role in aldosterone signalling. furthermore, we functionally characterised expression relevant promoter portions of sac and the influence of aldosterone and camp mediated signalling pathways on sac gene regulation. design and methods: in vascular endothelial cells (ea.hy926) and in human kidney cell lines (hek293t; ihke), we performed chromation immunoprecipitation (chip) assay with antibodies against sac and creb. we conducted transfection with a cre luciferase reporter vector and sac promoter constructs, following treatment with sac inhibitors and aldosterone. total rna of ea.hy926 cells, which were treated with sac inhibitors and aldosterone, was isolated and subsequently analysed by real-time pcr for expression of genes involved in aldosterone signalling. in vivo binding of sac at cre motifs was shown using cre consensus sequences in chip experiments. specific pharmacological inhibition of sac led to a significant decrease of transcriptional activity of the cre control vector in endothelial and kidney cell lines. furthermore, we were able to show the different effects of sac on the expression of downstream targets of aldosterone signalling, e.g. mineralocorticoid receptor and na + /k + -atpase alpha1 and beta1 and sac itself. regulation of sac itself is mediated by two different promoter portions, which are influenced by aldosterone and inhibition of sac and differentially accessed in kidney and endothelial cells. sac has transcriptional trans-acting properties as it interacts with cre sites and potentially influences the expression of genes, which play a role in aldosterone signalling. transcription of sac is regulated via aldosterone and camp. the location of promoter ta is cell type-and stimulation-specific. the role of the sodium-calcium exchanger (ncx1) in cardiac pacemaking herrmann s., stieber j., ludwig a. friedrich-alexander-universität erlangen-nürnberg institut für experimentelle und klinische pharmakologie und toxikologie, fahrstrasse 17, 91054 erlangen, germany the mammalian heart is driven by the sinoatrial node, the primary cardiac pacemaker. the unique feature of sinoatrial node (sn) cells is the ability to generate a spontaneous diastolic depolarization that periodically initiates action potentials which set the heart rhythm. the molecular origin of this cardiac pacemaker activity is still a matter of debate. recent findings point to a coordinated interplay between intracellular ca 2+ -cycling processes and plasma membrane-localized ion channels which determines the origin, periodicity and rate modulation of pacemaker potentials. in this study, we investigated the contribution of the cardiac sodium-calcium exchanger (ncx1) to pacemaking. ncx1 is a key sarcolemmal protein for the maintenance of calcium homeostasis in the heart. it was speculated that the membrane depolarizing current incx, whose activity is dependent on intracellular ca 2+ -fluctuations, represents a main determinant of the spontaneous diastolic depolarization. we used an inducible and sinoatrial node-specific cre-transgene to delete ncx1 in the murine pacemaker system. the successful creation of a cardiac pacemaking and conduction system specific ncx1 knockout (cpncx1ko) was demonstrated by transcript quantification as well as immunofluorescence experiments. telemetric ecg recordings of cpncx1ko displayed a distinct cardiac phenotype. mutant animals were deeply bradycardic and lost their capability of maintaining a stable heart beat as demonstrated by various ecg abnormalities like sn arrhythmia, sn pauses, av block and ventricular tachycardia. analysis of the spontaneous activity of isolated sn preparations showed a slower and arrhythmic contraction rate of the mutant tissues strips confirming that the bradycardia and arrhythmia induced by deletion of ncx1 results from a slower and arrhythmic intrinsic pacemaker activity. a battery of experiments using different heart rate lowering as well as increasing drugs revealed an altered heart rate modulation in cpncx1ko animals as compared to controls. in conclusion, these initial results establish ncx1 as a major contributor to cardiac pacemaking. a wide variety of contaminants are ingested through food, among them the procarcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (bp) which is resorbed and partially metabolized in the enterocytes of the small intestine. previous in vitro studies revealed that bp phenols are excreted as phase ii metabolites including bp glucuronides and bp sulfates. this export is mediated by the breast cancer resistance protein (bcrp/abcg2). the ultimate carcinogenic phase i bp metabolite anti-bp-7,8dihydrodiol-9,10-epoxide (bpde) can be detoxified by glutathione conjugate formation catalyzed by various glutathione s-transferases. in the present study, differentiated human intestinal caco-2 cells were used as a model for the human small intestine to investigate the detoxification of bpde and the subsequent transport of the stereoisomeric glutathione conjugates in the presence of an inhibitor (acivicin) of the glutathione-cleaving enzyme gamma-glutamyl transpeptidase (ggt) at the surface of the cells. the results indicate that the glutathione conjugates of bpde are formed and excreted mainly to the apical and to a minor extent to the basolateral side of the polarized caco-2 monolayer. to stimulate the transport rate several inducers known to enhance gene expression of xenobiotic-metabolizing enzymes as well as transport proteins were used (quercetin, oltipraz, butyrate). however, solely oltipraz substantially increased the efflux of bpde glutathione conjugates after inhibition of ggt. inhibition studies revealed that the multidrug resistance-associated proteins (mrps/abccs) are involved in the transport of the bpde glutathione conjugates. stable abcc1, abcc2 and abcc3 knockdown cell lines were generated allowing to demonstrate that abcc1 mediates the basolateral, abcc2 the apical excretion of the bpde glutathione conjugates. in conclusion, the ultimate carcinogen bpde is detoxified via glutathione conjugation and subsequently excreted by caco-2 cells in both apical and basolateral directions. .this finding is equivalent to a transport into the feces as well as blood system in the in vivo situation. signaling via irag regulates store operated calcium entry (soce) in aortic vsmc hieke b., hüttner j., schlossmann j. university of regensburg department of pharmacology and toxicology, universitätsstr. 31, 93053 regensburg, germany the mechanisms involved in the activation of store operated calcium entry (soce) through depletion of intracellular stores and their regulation are not yet fully understood. we examined the effect of inositoltriphosphate-receptor associated cgmp-kinase substrate (irag) on soce. aortic vascular smooth muscle cells (vsmc) from wild type (wt) and irag-knock-out (ko) mice were loaded with the calcium indicator fura 2-am and soce was measured as a change in the intracellular calcium concentration. in experiments with vsmc from wt mice soce was attenuated by the application of 8-br-cgmp. this effect was not observed in vsmc isolated from irag-ko mice. these differences in the strength of the soce-signal were abolished by the replacement of extracellular sodium with n-methyl-d-glucamine. the observed sodium dependence of the soce regulation via irag suggests, that an alternated sodium conductance might be responsible to some extent for the differences detected in wt and irag-ko vsmc. as a change in sodium conductance might result in a changed membrane potential we tried to track these changes with the flipr membrane potential assay kit while executing the soce protocol with and without 8-br-cgmp. no significant differences in membrane potential could be detected in the various stages of soce. in conclusion, our results indicate that irag exhibits a dual action on calcium regulation as it inhibits not only the intracellular calcium release but also the extracellular calcium influx through soce. induction of the icer promoter in vascular smooth muscle cells hildebrandt i. 1 tokyo metropolitan institute of gerontology, tokyo japan several transcription factor isoforms are encoded by the crem (camp response element modulator) gene. one prominent isoform is the inducible camp early repressor (icer), which acts as a transcriptional repressor on so called camp responsive elements (cres) in its target gene promoters. the icer mrna expression is regulated by an intronic promoter of the crem gene. in vascular smooth muscle cells (vsmcs) crem/icer is involved in the regulation of cell proliferation and apoptosis with physiological consequences in vivo. for instance crem-knockout mice, in which none of the known isoforms can be expressed, exhibit an increased neointima formation after carotid ligation as well as an increased atherosclerotic plaque formation after high fat diet on an apoe background. these observations were associated with an increased proliferation rate in isolated crem deficient vsmcs. on this background we wanted to clarify the specific role of icer isoforms in the vasculature. in first experiments we examined the inducibility of icer in primary vsmcs and smooth muscle cell lines. reporter luciferase assays showed that the activity of the icer promoter is induced at the maximum of fourteen fold after 4 hours of stimulation with forskolin (fsk) in immortalized rat vsmcs (13.7 ± 0.93; n=12 from 3 isolations). in a7r5 rat smooth muscle cells the icer promoter showed a maximum stimulation of 3.2 ± 0.16 fold after two hours of fsk stimulation (n=20 from 4 isolations). these pilot experiments showed that the icer promoter is inducible in vsmcs by camp dependent pathways. further experiments have to be carried out to elucidate the role of icer in the vascular system for example by stimulation of primary vsmcs and analysis of icer knockout mice. (supported by the dfg) overexpression of transmembrane channel-like proteins (tmcs) uncouples receptor-mediated calcium mobilisation hill k., urban n., straub i., schaefer m. universität leipzig -universitätsmedizin rudolf boehm-institut für pharmakologie und toxikologie, härtelstr. 16-18, 04107 leipzig, germany the family of transmembrane channel-like proteins (tmcs) consist of 8 members (tmc1-tmc8) all tmc genes are predicted to encode transmembrane proteins with at least six membrane-spanning helices. mutations of tmc1 cause deafness in human and mice whereas tmc6 and tmc8 (also referred to as ever 1 and 2) are linked to epidermodysplasia verruciformis (ev), a skin disorder, which is characterised by an enhanced susceptibility towards cutanous infections by human papillomaviruses. the cell biological and physiological functions of tmc proteins still remain elusive. we have overexpressed tmc6 and tmc8 in hek293 cells to get insights into their physiological function. all tmcs were located within the endoplasmic reticulum (er) after overexpression of yfp or cfp-tagged constructs. ratiometric calcium imaging revealed that after overexpression of tmc6 or tmc8, stimulation of gq-coupled receptors with carbachol and atp resulted in a greatly reduced amount of calcium release from the er. moreover, challenging tmc6-or tmc8-expressing cells with the serca pump inhibitor thapsigargin was also not followed by a release of er-based calcium within the cell. to test whether the lack of calcium release was caused by a reduced calcium content within the er, we investigated calcium dynamics within the er using an er-targeted fret-based calcium indicator (d1er cameleon). the experiments revealed that the amount of calcium within the er was reduced upon overexpression of tmc6 or tmc8. recently, it has been reported that tmc6 and tmc8 might influence intracellular zinc distribution in human keratinocytes. we could confirm the presence of tmc6 and tmc8 mrna in a human keratinocytes cell line (hacat). upon overexpression of tmc8, hacat cells revealed the same phenotype as described above for the hek293 cells with an uncoupling of the receptor-mediated calcium mobilisation due to a depletion of the er calcium store. the mechanism by which overexpression of tmc proteins causes a reduced calcium concentration within the er remains unclear. considering that the presumed topology of the tmc proteins distantly resembles those of other ion channel superfamilies such as anoctamins, one might speculate that a conductance through the tmc protein itself leads to a calcium leak from the er. terahertz radiation is defined as radiation between 0.1 thz and 10 thz. a number of applications are currently being developed using radiation in this frequency range. these applications will lead to exposure of the general public, making it very important to study potential effects on biological systems. historically, only a few studies on effects caused by terahertz radiation have been conducted because of the lack of suitable generators and detectors. during the last decade, a number of studies on effects caused by radiation with frequencies around 100 ghz have been published. the present study investigated the genotoxic potential of terahertz radiation at three different frequencies, 0.106 thz, 0.380 thz and 2.520 thz. two skin cell types were used, primary human dermal fibroblasts (hdf) and a keratinocyte cell line (hacat). the cells were irradiated applying different exposure times and different power intensities. two genotoxicity tests were applied: the comet assay quantifies dna strand breaks as well as alkali-labile sites whereas the micronucleus test quantifies chromosomal damage. all experiments were performed and evaluated under blinded conditions as three independent replicate experiments. positive (mms-treated) and negative (untreated, sham-exposed) controls were included. in the comet assay no dna damage was observed as a consequence of the exposure under all experimental conditions. the same was true for the chromosomal damage investigated with the micronucleus test. the latter finding was particularly interesting for the experiments at 0.106 thz, because this type of radiation had been reported to cause mitotic disturbances. therefore these experiments were extended, applying higher power intensities and longer exposure periods. also with these modifications, no genomic damage was observed in the form of micronucleus formation. all in all, terahertz radiation did not induce genomic damage under the applied experimental conditions. this result is in line with published findings on genotoxicity of low-frequency terahertz radiation around 0.1 thz. the question, why the reported mitotic disturbances do not lead to manifest genomic damage remains open and requires further research. introduction: tea flavonoids derived from camomile and green tea such as apigenin and epigallocatechin gallate (egcg) can inhibit intestinal neoplasia. recurrences of adenomas and cancers were reduced in patients with resected colorectal cancer by treatment with tea bioflavonoids after tumor operation [1] . to clarify the biomolecular pathway for suppression of neoplasia we investigated the anti-inflammatory effect of a nutritional supplement flavo natin® (fn) which had been used in the clinical study on tertiary tumor prevention and of egcg in a colon tumor cell line. the aim of our study was to investigate if tea flavonoids are capable to suppress the inflammatory markers produced by tumor cells after cytokine stimulation. method: we studied the cytotoxicity of fn in the colon cancer cell line t-84 by resazurin fluorescence and compared it with the placebo supplement. additionally, the t-84 cells were incubated with fn, egcg or placebo and stimulated with tnf-alpha, if-gamma and il-1-beta. after the cytokine stimulation the mrna expression of ip-10, il-8 and tnf-alpha was measured by quantitative real-time pcr (qrt-pcr). results: stimulation of t-84 cells increased the expression of ip-10 (gamma-interferon inducible protein 10), tnf-alpha and il-8. by preincubation with fn at 10 µm the mrna expression of ip-10 was strongly reduced (log2-ratio -14). the tnf-alpha mrna was also but less decreased by fn. egcg displayed an inhibition pattern similar to fn. placebo did not influence the mrna expression of the chemokines and tnf-alpha. discussion & conclusion: clinically useful dietary tea bioflavonoids inhibit the expression of inflammatory genes in a colon cancer cell line. down-regulation of inflammatory gene products could be achieved in vivo by botanicals without clinically relevant side effects. [1] h. hoensch, b. groh, l. edler, w. kirch (2008) . prospective cohort comparison of flavonoid treatment in patients with resected colorectal cancer to prevent recurrence. world j gastroenterol, 14, 2187-2193. the cxcr7 c-terminal domain mediates efficient cxcl12 uptake and degradation hoffmann f., müller w., schütz d., schulz s., stumm r. universitätsklinikum jena institut für pharmakologie und toxikologie, drackendorfer str. 1, 07747 jena, germany cxcl12-signaling mediated by the g protein-coupled cxcr4 receptor plays a key role during embryonic development and disease states including cancer and inflammation. the second cxcl12-receptor cxcr7 modulates cxcl12/cxcr4-signaling by acting as a cxcl12-scavenger. given the distinct functions of cxcr4 and cxcr7, we hypothesized that trafficking and receptor stability are differently regulated by the distinct c-terminal domains. here, we examined epitope-tagged wild type and c-terminal mutant receptors expressed in human embryonic kidney cells (hek293) with respect to trafficking, stability, 125 i-cxcl12 radioligand degradation, and g protein-coupling. we found that the 24 c-terminal residues of cxcr7 were sufficient for cxcr7 to undergo rapid spontaneous internalization. replacement of the cxcr7 c-terminal domain with that of cxcr4 (cxcr7-4tail mutant) abolished spontaneous internalization but permitted ligand-induced internalization in conjunction with c-terminal phosphorylation. conversely, replacement of the cxcr4 c-terminal domain by that of cxcr7 caused ligand-independent internalization of cxcr4. receptor-mediated 125 i-cxcl12-uptake, release of 125 i-cxcl12-degradation products, and degradation of the receptor protein itself were accelerated with receptors bearing the cxcr7 c-terminus. while the cxcr7 c-terminus was sufficient to abolish g protein coupling in the cxcr4-7tail mutant, replacement of the cxcr7 c-terminus, cxcr7 second intracellular loop or both domains with the corresponding cxcr4 domain did not generate a g protein-coupled cxcr7 chimera. taken together, we provide evidence that the cxcr7 c-terminal domain influences the ligand-uptake/degradation rate, g protein-coupling, and stability of the receptor. this suggests that heterologous regulatory pathways targeting the cxcr7-c-terminal domain may effectively control cxcr7 functions. soluble guanylyl cyclase is a key mediator of brown adipocyte differentiation hoffmann l. s. 1 brown adipose tissue (bat) uses energy to produce heat by inducible thermogenesis. recent studies show that active bat is present in adults and involved in human energy balance, suggesting that the energy consuming property of bat might be exploited to fight obesity and related diseases. the no/cgmp signaling pathway is a key player in diverse physiological processes. recently, we have shown in bat that cgmp signaling is connected with insulin signaling and abrogation of cgmp signaling leads to impaired bat differentiation and function (haas, b. et al., sci signal, 2009 ). here we investigated the role of the cgmp generating enzyme soluble guanylate cyclase (sgc) in bat differentiation in vitro. mesenchymal stem cells isolated from bat of newborn sgcβ 1 -/mice and wt littermates were differentiated in vitro into brown adipocytes in the presence or absence of cgmp. abundance of sgc isoforms was determined by qrt-pcr and western blotting. bat differentiation was assessed by redo staining of accumulated intracellular lipids, measurement of triglyceride (tg) content, determination of expression of bat marker proteins pparγ, c/ebpα, ap2 and bat marker genes ucp1, pgc1α, cidea. the α2 and β1 isoforms of sgc were highly expressed in bat whereas α1 sgc could not be detected. redo staining of wt brown adipocytes showed basal differentiation which was increased upon addition of 8-pcpt-cgmp. in contrast, staining was lower in sgcβ1 -/cells compared to wt under control conditions and increased in the presence of cgmp. tg measurement showed that sgcβ1 -/brown adipocytes contain approximately 50% less lipids than wt cells under basal conditions. addition of cgmp doubled tg content in both genotypes. similar results were observed for marker protein expression. deletion of sgc resulted in 56-40% decrease in c/ebpα, pparγ and ap2 expression compared to wt. again, cgmp roughly doubled protein expression in sgcβ1 -/and wt cells compared to control. under basal conditions, bat marker gene expression was decreased by approximately 80% in sgcβ1 -/cells compared to wt cells. this decrease was prevented by addition of cgmp. these results show that sgc deletion leads to dysfunctional bat differentiation and emphasize the central role of cgmp signaling in bat differentiation. further investigation of sgc/cgmp signaling in bat might reveal new drugable targets bringing bat-dependent pharmacological therapy to treat obesity and related disease into closer reach. comparative inhalation toxicity of carbon-nanomaterials (multi-wall carbon nanotubes, graphene and carbon black) hofmann t. 1 carbon black is a spherical carbon anomaterial whereas multi-wall carbon nanotubes (mwcnt) are cylindrical and graphene is a laminar allotrope of carbon. processing and handling as well as abrasion processes can set free inhalable cnt particles. results of rodent studies collectively show that regardless of the process by which cnts were synthesized and the types and amounts of metals they contained, cnts were capable of producing inflammation, epithelioid granulomas, fibrosis, biochemical and or toxicological changes in the lungs (lam et al. 2004 , muller et al. 2005 , ma-hock 2009 , pauluhn 2010 . graphene possess similar physical properties as cnt but may different toxicological property. we performed short-term inhalation studies in rats to compare the toxic potency of four different cnt, two graphenes and one carbon black. the materials are characterized thoroughly according to the oecd list. the four mwcnt caused morphological changes as descriped above. several biochemical and cytological parameters in the broncho-alveolar lavage fluid were strongly increased consistent with the histological findings. two mwcnt exhibited a higher toxic potency than two other mwcnts and findings caused by one graphene typ were even less severe. the graphene with lower surface area as well as low surface area carbon black did not cause any adverse effects up to 10 mg/m 3 . the short-term inhalation studies were able to descriminate different toxic potencies of carbon-based nanomaterials and is hence used for the selection of less toxic materials for further product development as well as to define and prioritize higher-tier toxicological testing of nanomaterials. 165 synthesis and analytical assessment of possible dna adducts formed after activation of the tobacco alkaloid myosmine högg c., zwickenpflug w., gudermann t. walther-straub-institut abt.: toxikologie, nußbaumstraße 26, 80336 münchen, germany myosmine represents one of the minor tobacco alkaloids and its effective uptake from smokeless tobacco or tobacco smoke, as well as by consumption of food is not understood in detail. myosmine can be activated by peroxidation and n-nitrosation yielding 4-hydroxy-1-(3-pyridyl)-1-butanone (hpb) which is well known as reactive intermediate during activation of a variety of tobacco specific n-nitrosamines (tsna). therefore, myosmine may be a potential candidate for possible mutagenic or carcinogenic risk to human health. furthermore, myosmine n-nitrosation yields the tobacco specific nitrosamine n-nitrosonornicotine (nnn), which is classified as carcinogenic to humans. considerable efforts have been undertaken, especially in organic synthesis, to verify and elucidate the significance of the hpb precursor the pyridyloxobutyl (pob) intermediate and its dna-adducts, which were analysed only in animal experiments till now. the formation of 3-pyridylmethanol was observed under myosmine peroxidation and identified as a metabolite in rat urine after application of myosmine to rats. the formation of the 3-pyridylmethanol intermediate, might provide for the reactive electrophilic picolinium ion which could interact with dna. these possible adducts might be of special interest to elucidate the role of myosmine concerning its uptake by smokers and passive-smokers in contrast to non smokers ingesting the substance by consumption of food. dna adducts may help to obtain more information about possible risk assessment of myosmine and to differentiate between the activation from the other nicotinoids and tsna. the specific dna adducts, 5-methyl-1,3-bispyridin-3-ylmethyl-1h-pyrimidin-2,4-dion and 3-(3''-picolyl)thymidine have been synthesised as reference substances. the former was prepared by addition of diisopropylazodicarboxylate (diad) to a mixture of 3-pyridylmethanol, thymine and triphenylphosphine. the reaction product was identified using nmr ( 1 h, 13 c, cosy, hmqc, hmbc). for synthesis of 3-(3''-picolyl)thymidine the hydroxyl groups of thymidine were initially acetylated using acetic acid anhydride and 4dimethylaminopyridine (dmap). the existence of this thymidine adduct was confirmed using nmr. this adduct was labelled with 5-([4,6,-dichlorotriazin-2-yl]amino)fluorescin (dtaf) to enhance the sensitivity using hplc-fluorescence chromatography and used as reference substances for analysis of dna samples from biological tissue. supported by dfg grant (ty 81/1-1). cancer and cardiovascular diseases such as atherosclerosis are the most important causes of death in western societies. common to both diseases is a deregulation of cell death, with significant contribution of inflammatory processes. enhanced oxidative stress plays a dominant role in such events as it forms a vicious cycle with inflammation and controls multiple forms of cell demise. therefore, anti-oxidative enzyme systems gained considerable interest since control of reactive oxygen species (ros) has the capacity to regulate cell death in either direction. the human enzyme family of paraoxonases consists of three members, known as pon1, pon2 and pon3. while pon1 is found predominantly in the circulation, pon2 and pon3 are intracellular enzymes with established anti-oxidative functions. it has been shown that both pon2 and pon3 are protective against atherosclerosis. underlying mechanisms of their protective and antioxidative functions however remained elusive. here we demonstrate that both enzymes locate to the endoplasmic reticulum (er) and mitochondria where they fulfill vital functions in the control of ros generation. in particular, pon2 and pon3 were shown to interact with coenzyme q10 which diminishes mitochondrial ros formation. as a consequence, these enzymes reduce execution of mitochondrial apoptosis, such as cardiolipin peroxidation, cytochrome c release and caspase activation. moreover, pon2 and pon3 reduced er stress-triggered cell death, i.e. by diminishing jnk signaling and chop expression. while these results elucidate their protective role in cardiovascular diseases, it also establishes a relevant function in survival of tumor cells. in accordance, we demonstrate that both enzymes are frequently found overexpressed in various tumors. in cancer cell culture studies, overexpression of both enzymes granted considerable resistance against chemotherapeutics. in turn, knock-down of pon2 caused spontaneous apoptosis of several cancer cell lines. finally, our analyses also revealed that pon2-knockout mice show severe alterations of the hematopoetic stem cell compartment, which implies a significant role in leukemias where these enzymes are frequently found overexpressed. together, our results propose pon2 and pon3 as new putative anti-tumor candidates and demonstrate the efficacy of interventions targeting cellular redox-balance. steigerwald arzneimittelwerk gmbh wissenschaftliche abteilung, havelstr. 5, 64295 darmstadt, germany stw 5 (iberogast ® ), a multi-component herbal drug, is successfully used in the therapy of functional dyspepsia and irritable bowel syndrome (ibs). previous studies revealed effects of stw 5 on disturbed motility and inflammatory processes. although the antiinflammatory properties of stw 5 are well examined, the contribution each of the individual herbal constituents to the anti-inflammatory effect remains unclear. therefore, we studied the effects of stw 5 and its components on inflammation-induced cell death and on the release of the pro-inflammatory cytokine tnf-α. the aim of these investigations was to analyse additive or synergistic effects of the components. the experiments were carried out on caco-2 cells after stimulation with lps (10 ng/ml) for 2 hours. cytotoxicity was evaluated using a commercially available ldh (lactate dehydrogenase)-assay. furthermore, the release of tnf-α after lps (100ng/ml) stimulation of differentiated thp-1 cells was measured using a commercially available elisa. stw 5 (62.6 -500.5 µg/ml) reduced lps (10 ng/ml)-induced cell death in a concentration-dependent manner with a maximum inhibition of 50.0 %. the herbal components in equivalent concentrations contributed to the inhibitory effect of stw 5 to different extents. the maximum inhibition differed in a wide range between the components. stw 5 (500.5 µg/ml) reduced significantly the release of tnf-α by 87 % in lps (100 ng/ml)-stimulated differentiated thp-1 cells while having no effect in untreated cells. in concentrations equivalent to stw 5 caraway, milk thistle, lemon balm and greater celandine had no effect on the lps-induced increase in tnf-α release. bitter candytuft, peppermint, chamomile, liquorice and angelica reduced the tnf-α release, though less pronounced as compared to stw 5, indicating a possible synergistic effect. our results indicate a multi-target effect of stw 5. the anti-inflammatory effect may be due to a reduction of the cytotoxic effect on intestinal mucosa cells and to an inhibition of the release of the pro-inflammatory cytokine tnf-α from immune cells. the individual herbal components seem to contribute by different mechanisms of action to the overall effect of stw 5. immune cell-induced local steroidogenesis in the lung: implications for asthmatic disease and therapeutic intervention hostettler n. 1 , brunner t. glucocorticoids are steroid hormones with potent anti-inflammatory properties. synthetic glucocorticoids are frequently used for the therapeutic treatment of inflammatory disorders, such as asthma. endogenous glucocorticoids are predominantly produced in the adrenal glands in response to emotional, physical and immunological stress. recent years, however, revealed several alternative sources of these immunoregulatory steroid hormones. thus, we have found that the intestinal epithelium is a rich source of glucocorticoids and intestinal glucocorticoids contribute to the maintenance of local immune homeostasis (1) . as the intestinal and the pulmonary epithelium have much in common, i.e. barrier functions and transport of nutrients, resp. gases, we wondered whether the lung mucosa is also capable of synthesizing immunoregulatory glucocorticoids in response to immune cell activation. the murine lung was found to expresses all enzymes required for the synthesis of corticosterone from cholesterol. while most enzymes where expressed in a constitutive manner, cyp11a1, encoding p450ssc, was strongly induced in response to immunological tress. treatment of mice with t cell-activating anti-cd3 antibody of macrophage-activating lipopolysaccharides induced strong local glucocorticoid synthesis, which was effectively blocked by the corticosterone synthesis inhibitor metyrapone, indicating that glucocorticoids measured were produced bona fide in the lung tissue. surprisingly, allergen-induced allergic airway inflammation failed to trigger local glucocorticoid synthesis despite the massive infiltration of neutrophils, eosinophils and t cells. in contrast to that in the intestinal epithelium local glucocorticoid synthesis in the lung was found to be dependent on the presence of adrenal glands as adrenalectomy abolished pulmonary steroidogenesis. in line with the notion that the lung metabolizes steroid precursors we found that ex vivo cultured lung tissue metabolized 3 hduring the last decade small rna molecules has been identified as important regulators for gene expression. these micrornas (mirna) are single-stranded transcripts, which are expressed in many cell types, where they modulate rna stability and translation and, therefore, controlling various cellular mechanism and tissue development. against this background, in the present study the mirna expression and potential target genes were studied in the human placenta as a tissue demonstrating various developmental changes in a limited period of time. taqman®array microrna cards for profiling of 365 mirnas in placentas of different gestation times revealed a significant expression of 277 mirnas by comparing placentas of early gestation (<10.week), preterm (<30.week) and term (>37.week). when comparing the analyzed groups, 106 of these mirnas expose a continuous up-(including mir-20a, mir-379) or downregulation (including mir-9, mir-200a). while comparing placentas of early gestation to term placentas, 30% (e.g. mir-9, mir-429) were up-and 70% (e.g. mir-126, mir-373) were downregulated. by focusing on the latter group with early preterm placentas the ratio is opposed. here, 60% of mirnas showed higher (e.g. mir-107) and 40% lower expression (e.g. mir-627, mir-191). with emphasize on these mirnas, a computational prediction algorithm using the mirò database predicted a potential interaction between corticotropin-releasing hormone (crh) and the mirnas mir-9 and mir-429. specific expression analyses validate an inverse expression between mirnas and target with a reduced expression of mir-9 (93%) and mir-429 (94%) in term placentas, while crh is upregulated 114fold. this interaction was verified on functional level by reporter gene assay. a significantly suppressed luciferase activity of the reporter plasmid containing the 3´-utr sequence complementary to crh was exhibit for the predicted mir-9 binding site. here, the mirna-mrna-interaction reduces the luciferase activity by ~40%, whereas the decreased luciferase activity for the predicted mir-429 binding site is not significant. the results demonstrate a gestation dependent expression of placental mirnas, which may help to explain gestational changes in gene expression and highlight the potential of mirnas as biomarker for pregnancy-related pathologies. since homo sapiens era wound treatment by early civilizations was based on the use of local flora. scilla indica knuth liliaceae (s.i) is a perennial herb with a pear shaped, tunicated bulb, bearing fibrous roots, white flowers and leaves on the stem resembling u. maritima. it is used as cardiac tonic, against inflammation, ulcers and sinus diseases. traditionally the powdered bulb is used topically for warts treatment, while roasted and crushed bulbs are applied to corns of the feet soles. the plant contains steroid glycosides (bufadienolides 1-3%), glucoscillarene a, proscillaridine a, scillarene a, scillcyanoside ,scilliglaucoside ,mucilage and alkyresorcinol derivatives exerting skin healing properties similar to wheat bran products. the study is focused on the investigation of the restoration quality of a skin dorsal incision wound in wistar rats in three groups, control (a1) and experimental (a2,a3 ) of rats weighing 350-450g local application of dichloromethane extract of s.i in vehicle olive oil preparations of 50 and 100mg were used. animals were anaesthetized ( ether pro narcosi ), trauma 2 cm long and 2mm deep was performed by lancet on depilated dorsal area skin until the muscular aponeurosis and were treated for 4 days with 60λ of each preparation. group a3 showed increased remodelling of trauma area (limited width). granulomatous tissue was more pronounced in length, width and surface in group a2. the macroscopic ulcus dimensions were better in group a3 while the microscopic view were similar in a2 and a3 and better in comparison to control a1. a tendency to trauma remodelling was observed, without statistical significance (t =0.3) in recent years, it has been suggested that nanoparticles generated from combustion processes (e.g. diesel engine exhaust particles), may contribute to the pathogenesis of neurodegenerative diseases such as alzheimer's disease (ad). the aim of our current study was to investigate the effects of subchronic exposure to diesel engine exhaust (dee) in the 5xfad mouse model, which is characterised by progressive behavioural deficits as well as amyloid plaque formation and neuron loss. ten weeks old female 5xfad mice and their nontransgenic littermates were exposed by whole body inhalation to diluted dee (~1 mg particles/m 3 ) or clean air (controls) for 3 or 13 weeks (5 days/week and 6 hour/day). subsequently, all animals were subjected to a series of behavioural tests. at ten days post-exposure, mice were sacrificed to investigate lungs and brain tissues for pathological and biochemical and molecular-biological changes. in line with the expectations, the 5xfad mice displayed typically age-dependent behavioural deficits and amyloid plaque formation in cortex and hippocampus. a significant dee exposure-related effect was observed for the string suspension test, representing a measure of motor coordination/grip strength. dee exposure was also associated with mrna expression changes of markers of inflammation and oxidative stress in specific brain regions, including the olfactory bulb. histopathology of plaqueload in cortex and hippocampus (from a limited number of animals investigated so far) did not reveal clear evidence for increased plaque formation due to the dee exposures. further research is needed to evaluate the effects of long term exposure to nanoparticles in the central nervous system. this work is supported by funds from the research committee of the medical faculty of the university of düsseldorf (9772-365), the dfg graduate school grk1033 and the rivm -centre for environmental health, bilthoven, netherlands. mono-glucosylation of (h/k/n)ras by clostridium sordellii lethal toxin (tcsl) blocks critical survival pathways, including the pi3k/akt, the ralgef/ral, or the raf/erk, and results in apoptosis. in this study, ras glucosylation is presented to result in expression of the cell death-regulating small gtpase rhob based on transcriptional activitation. rhob expression depends on k-ras inhibition, as sirna-mediated knock-down of specifically k-ras (neither h-ras nor n-ras) provokes rhob expression. rhob expression further depends on inhibition of pi3k/akt, as activation of pi3k/akt using a pi3k activator prevents rhob expression downstream of inactivated ras. newly synthesized rhob is gtp-loaded and rapidly degraded in a proteasome-and a caspase-dependent manner, providing first evidence for caspase-dependent degradation of a rho family protein. although often characterised as a pro-apoptotic protein, rhob suppresses caspase-3 activation in tcsl-treated fibroblasts. conclusions: 1. rapid and efficacious ras inactivation by tcsl turns out to be particularly useful in the characterisation of ras inactivation-induced rhob expression as an immediate-early gene response. 2. the finding on the cytoprotective activity of rhob in tcsl-treated cells re-enforces the concept that rhob exhibits cytoprotective rather than pro-apoptotic activity on cellular background of inactive ras. the activity of the rhob promoter is suppressed by rhoa (through a not yet identified pathway or ras (through the pi3k/akt pathway. ras glucosylation by tcsl results in de-suppression of the rhob promoter and rhob expression. the calcium-binding protein annexin a4 (anxa4) is involved in diverse cellular processes including e.g. vesicular transport, ion channel regulation and transcriptional regulation. upon ca2+ binding anxa4 undergoes conformational changes, which lead to oligomerization of anxa4 to homotrimers and cause an increased affinity for membrane phospholipids, which in turn provokes the translocation from the cytosol and the nucleoplasm to plasma and nuclear membranes. in order to examine the effect of anxa4 on cre-and creb-(camp response element-binding protein) dependent transcription, a series of transient transfections using a luciferase reporter gene driven by the creb target promoter of the inducible camp early repressor (icer) was carried out. when the luciferase reporter construct was co-transfected with anxa4, there was significant reduction of basal (dmso) luciferase activity ( the implantation of drug eluting stents (des) after coronary artery intervention was an important step treating coronary artery disease. indeed, cytotoxic compounds like sirolimus used on des are responsible for reduced in-stent restenosis in comparison with bare metal stents. pimecrolimus, a potent anti-inflammatory drug has also been investigated for its efficacy on des. preclinical studies in pigs revealed promising antiproliferative effects of pimecrolimus on neointima formation. however, in humans, pimecrolimus coated stents exerted adverse effects. we hypothesize that compared to the highly active sirolimus, pimecrolimus may influence additional cellular processes leading to the worse outcome. in order to identify those processes we conducted in vitro studies in human coronary artery endothelial cells (hcaec) and smooth muscle cells (hcasmc). brdu in vitro assays of hcaec treated with pimecrolimus examined an ic50 value of 6.787 µm [5.745 to 8 .017] which is much higher than ic50 values of sirolimus described in literature [0.1nm to 1nm]. genechip array analysis comparing gene expression in pimecrolimus and sirolimus treated hcaec and hcasmc showed significant induction of several genes involved in interferon signaling. in detail, the expression of ifnβ activated genes like irf9 and ifitm1 was up regulated in cells treated with pimecrolimus while no or oppositional effects were observed with sirolimus. gene regulatory effects were validated by real time pcr. incubation with ifnβ itself showed similar effects in up regulation of genes involved in interferon signaling. furthermore, we were able to demonstrate a significant increase of ifnβ secretion in hcasmc and hcaec treated with pimecrolimus. however, comparison of ifnβ and pimecrolimus on proliferation of hcaec and hcasmc revealed different cellular responses. while ifnβ significantly decreased hcasmc and increased hcaec proliferation, treatment with pimecrolimus lead to anti-proliferative effects on both cell types. in conclusion, pimecrolimus activates pathways involved in interferon signaling but exerts different pharmacological effects, compared to the endogenous compound suggesting that infβ secretion is not the major factor contributing to the difference in pimecrolimus function. identification of novel phospho acceptor sites of the mu opioid receptor regulating receptor internalization illing s., just s., doll c., schulz s. uniklinikum der fsu jena pharmakologie und toxikologie, drackendorfer straße 1, 07747 jena, germany the opioid alkaloid morphine is among the most potent clinically used analgesics. however, the clinical utility of morphine to treat chronic pain is limited by its rapid development of tolerance and dependence. the stimulation of the mu opioid receptor (mor) with damgo or morphine results in different patterns of receptor phosphorylation and trafficking. so far, the three major phosphorylation sites of the mu opioid receptor namely serine 363 (s363) threonine 370 (t370) and serine 375 (s375) have been identified using phosphosite-specific antibodies. however, mutations of these three residues to alanine (s363a/t370a/s375a) did not prevent agonist-dependent internalization. in the present study, we have constructed a series of phosphorylationdeficient mutants showing that mutation of at least six residues (s363a/t370a/s375a/t376a/t379a/t383a) was required to completely block agonistdriven endocytosis. consequently, we generated phosphosite-specific antibodies to t376 and t379 which enabled us to provide direct evidence for agonist-dependent phosphorylation of these sites. our analysis of time-and dose-dependent phosphorylation of mor revealed that s375 was the primary phosphorylation site, whereas t370, t376 and t379 were secondary sites. moreover, the partial agonist morphine induced only the phosphorylation of s375 but not of t370, t376 of t379. our results show, that mor phosphorylation occurs in a hierarchical and agonist-selective manner directly regulating receptor sequestration. assessment of mda effects from toxicity studies with regard to endocrine modulation jäger r. 1 methylene dianiline (mda; cas no. 101-77-9) is on the usepa list 2 for endocrine disruption screen testing. in a yeast androgen screening (yas) assay (in vitro assay on receptor binding or blocking) mda revealed a significant anti-androgenic activity at a concentration of 0.01 mm. to assess the biological relevance of this observation under in vivo conditions the existing data from animal toxicity studies with mda were reviewed for indications of possible endocrine modulating (em) effects (weight-of-evidence approach). in addition, literature was searched for relevant studies on mda and structural analogues using the american chemical society scifinder client-server software. structural analogues indicated several drivers for em activity, notably the diphenolic ring structure. however, in vitro receptor binding assays showed mda had no androgenic or anti-estrogenic activitiy. one report described a weak estrogenic binding at 0.2 mm mda, while another described no effect at similar concentrations and a rat estrogen receptor binding study found no effect at 0.2 mm. overall it is concluded that mda does not bind to the estrogen receptor. endocrine related effects of mda were investigated in several species and dose routes in unvalidated research-type protocols. guideline subchronic and chronic toxicity studies with rodents revealed neither adverse organ weight changes nor histopathological alterations of sex organs. the main systemic effects from the oral 13-week studies were body weight reduction and histopathological changes of the thyroid and bile duct (lowest loael: 7.5 mg/kg bw). mda was carcinogenic to rats and mice (thyroid and liver) after oral administration over 2 years. non-neoplastic effects in the thyroid (rats and mice) were observed (lowest loael for systemic toxicity: 9 mg/kg bw). mda inhibits iodide oxidation which with concomitant decreased thyroid hormone formation is known to induce thyroid tumors. in summary, in vitro screening tests revealed no consistent endocrine related effects of mda. in vivo, there was an effect on the thyroid gland, possibly by enzyme inhibition. there were no histopathological changes of gonads and accessory sex organs and no evidence of sex hormone related em. the evidence from the full dataset on mda does not indicate androgenic or estrogenic effects. overall, based on a weight of evidence assessment there are insufficient alerts for em activity to suggest further testing should be done. the gtpase arfrp1 controls the assembly of apoa-i to and the lipidation of chylomicrons in the golgi of intestinal epithelium jaschke a. 1 background: the uptake and processing of dietary lipid by the small intestine is a multi-step process that involves luminal digestion, cellular uptake of fatty acids by the mucosa, and subsequent synthesis and export of chylomicrons. the gtpase adpribosylation factor related protein 1 (arfrp1) is a member of the arf-family and controls the arf-like 1 (arl1)-mediated golgi recruitment of grip domain proteins which in turn bind several rab-gtpases. the aim of the study was to define the role of arfrp1 in intestinal nutrient absorption. methods: for the generation of intestine-specific null mutants arfrp1 flox/flox mice were crossed with mice expressing the cre recombinase under the control of the intestinespecific villin promoter (vil-cre) and arfrp1 expression was suppressed by sirna in caco2-cells. the phenotype in respect to lipid absorption and chylomicron production in the intestinal epithelium and in caco2-cells, respectively, was analyzed. results: arfrp vil-/mice were viable but showed an early postnatal growth retardation (mean body weight of arfrp1 vil-/was 43.3±5% lower than that of control mice at the age of 28 days) arfrp1 vil-/mice displayed reduced triglycerides, free fatty acids and glucose plasma levels indicating that the growth retardation is the result of a malabsorption. uptake of glucose and amino acids were unaffected by the deletion of arfrp1. in contrast, lipid uptake as elucidated by oral fat tolerance tests was impaired in arfrp1 vil-/mice. arfrp1 vil-/enterocytes as well as arfrp1 mrna depleted caco-2 cells absorbed fatty acids normally but secreted chylomicrons with a markedly reduced triglyceride content. in addition, while the release of apolipoprotein a-i (apoa-i) was dramatically decreased apoa-i accumulated in the arfrp1 vil-/epithelium and was predominantly colocalized with rab2. our results demonstrate that the growth retardation of arfrp vil-/mice is a consequence of impaired intestinal fatty acid absorption. we suggest that arfrp1 is required for the assembly of aopa-i to the chylomicrons and for the further lipidation of chylomicrons in the golgi of intestinal epithelial cells. this finally leads to an secretion of chylomicrons with a markedly reduced triglyceride content. rhoa influences adhesion and spreading of cardiac fibroblasts via complex regulation of cytoskeletal proteins jatho a. 1 the monomeric gtpase rhoa is thought to be involved in the pathology of heart diseases, however, its role in cardiac cells is not well defined. therefore we intended to analyze the effect of rhoa in cardiac fibroblasts by using a lentivirus-based knockdown approach. by doing so, we could show that the knockdown of this gtpase by about 50% resulted in a massive change in cell morphology, but displayed no effect on cell viability. the appearance of the cells in the 2d-culture changed from a fibroblast-typical stretched morphology with intracellular stress fibers to a more epithelial-like cell morphology. by morphometrical analyses we demonstrate that fibroblasts with reduced rhoa expression display an increase in cell surface by 2.2-fold and in perimeter by 1.5fold. moreover, the depletion of rhoa significantly influences the adhesion velocity, as within the first hour after cell detachment about 20% of the rhoa knockdown cells reattach, but only 5% of the respective control cells. based on these findings, we investigated the distribution and composition of different cytoskeletal proteins by immunofluorescence stainings and immunoblot analysis. we found, that the amount of b-actin is not reduced in rhoa knockdown cells, however, the distribution is markedly changed. in these cells internal star-shape bundles of actin could be found instead of the commonly appearing stress fibers in control cells. in contrast, the cortical actin fibers, mainly consisting of g-actin, were not affected. in addition, smooth muscle-actin, which is characteristic for myofibroblasts, was clearly reduced in rhoa knockdown cells compared to control cells by 33%. this reduction might be responsible for the more relaxed cell shape. in summary, the knockdown of rhoa influences cell adhesion and the morphological characteristics of cardiac fibroblasts, without obviously affecting cell proliferation and viability. using site-specific fluorescence labeling to study uptake of pasteurella multocida toxin into eucaryotic cells jehle d., aktories k., orth j. institut für experimentelle und klinische pharmakologie und toxikologie abteilung 1, albertstraße 25, 79104 freiburg, germany pasteurella multocida is an opportunistic pathogenic bacterium living in the nasal pharyngeal space of animals. p. multocida is of particular importance in the livestock management of pigs. under special conditions infection of pigs with p. multocida leads to an atrophic rhinitis, which is characterized by the atrophy of nasal turbinate bones accompanied by a shortening and twisting of the snout. the causative agent of the atrophic rhinitis was found to be the bacterial protein toxin pmt (pasteurella multocida toxin). after entering the cell the 146-kda toxin activates various signal transduction pathways by stimulating heterotrimeric g proteins of the gαq, gα13 and gαi family. the underlying mechanism of the activation of heterotrimeric g proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the g protein. the uptake of pmt into cells is not comprehensively understood. therefore, we utilized a recently described technique, called "sortagging" (popp mw et al. nat chem biol. 2007; 3: 707) , to specifically couple fluorescence tags to the n-or c-terminus of pmt. the enzyme sortase a (srta) from staphylococcus aureus attaches proteins to the bacterial cell wall. the substrates are recognized by an lpxtg motif. srta cleaves the peptide bond after the threonine and adds a glycine-containing nucleophile. we introduced these motifs into pmt to express srta-recognized toxin and coupled the toxin with fluorescence tags, respectively. fluorescently labeled pmt was used to study the uptake of the toxin into eucaryotic cells by laser scanning microscopy. munich heart alliance, münchen, germany cells within the myocardium communicate by secreted factors and this has been suggested to contribute to cardiac remodeling. to identify novel factors secreted by the myocardium, we have previously reported a genetic yeast screen which led to the identification of protease inhibitor 16 (pi16). here we report the generation of a mouse line where pi16 can be deleted globally or conditionally using the cre/loxp system. after electroporation of the pi16 floxneo targeting vector in embryonic stem cells and injection into murine blastocysts we gained a mouse line that carried the targeted modification of the pi16 allele. global pi16 deficiency (pi16 lox/lox ) per se did not lead to a cardiac phenoytpe (hw/bw (mg/g): pi16 +/+ = 5.4 ± 0.2 (n = 6), pi16 lox/lox = 5.9 ± 0.7 (n = 4), p > 0.05; fibrosis (%): pi16 +/+ = 3.3 ± 0.2 (n = 7), pi16 lox/lox = 3.9 ± 0.8 (n = 5), p > 0.05; fractional shortening (%): pi16 +/+ = 29.8 ± 0.7 (n = 7), pi16 lox/lox = 26.4 ± 2.5 (n=5), p > 0.05). in addition we carried out an immunohistochemical analysis of pi16 expression using pi16-deficient mice as negative controls. pi16 localized to cardiac fibroblasts, to the epididymis and the trachea. in the failing heart we detected accumulation of pi16 preferentially in fibrotic areas. we are currently applying cardiac stress models to gain a broader understanding of the function of pi16 and its potential as a therapeutic target molecule. enantioselective determination of r-and s-hyoscyamine in mammalian plasma and urine samples john atropine (atr) is the racemic mixture of the tropane alkaloids s-and r-hyoscyamine (hyo). s-hyo acts as a competitive acetylcholine antagonist at the muscarinic receptors (eutomer) inducing mydriasis, excitations, hallucinations, coma and ultimately death, whereas r-hyo does not (distomer). atr is used for clinical intervention of poisoning with organophosphorus (op) pesticides or nerve agents. despite well known differences in pharmacological behavior, individual pharmacokinetics of both atr enantiomers have rarely been addressed in the literature [1, 2, 3] . therefore, we initially developed a nonchiral liquid-chromatography-electrospray tandem mass spectrometric method (lc-esi ms/ms) that allows quantification of atr and additional natural and synthetic tropane alkaloids from plasma after simple deproteinization [4] . to discriminate both atr enantiomers the sample preparation step was expanded by an enzymatic pretreatment. samples were incubated either with diluted human serum (not containing atropinesterase, atre, procedure a) or with diluted rabbit serum (procedure b). rabbit serum contains atre (ec 3.1.1.10) which is suitable for stereospecific hydrolysis of shyo into tropine and tropic acid while r-hyo remains unaffected. after sample precipitation, hyoscyamines were quantified by the lc-esi ms/ms method. following procedure a the concentration of total hyo and following procedure b remaining r-hyo were determined. s-hyo was calculated by the difference between these concentrations [3] . the impact of potential matrix ingredients that may appear in samples from oppoisoned patients under atr therapy were evaluated (oximes, op agents, carbamates) [3] . the assay was applied to diverse toxicological and pharmacological samples. i) measurement of natural s-hyo in an extract of atropa belladonna leaves as well as in plasma and urine of a female patient who was poisoned after ingestion of such leaves revealed that no biotransformation to r-hyo occurred. ii) analysis of plasma obtained from an op-poisoned female patient under atr therapy revealed faster elimination of shyo when compared to r-hyo [3] . iii) in contrast, no enantioselective differences were obvious in healthy anaesthetized swine after intravenous injection of atr [5] . data indicate that the enzymatic enantioselective procedure represents a useful tool to characterize in vivo behavior of r-and s-hyo allowing to reveal individual kinetics. failing hearts are unable to adequately supply the body with blood and oxygen. common therapeutic strategies interfere with cardiac remodelling, reduce cardiac preand afterload or aim at direct improvement of cardiac contractility. cardiac contractility is mainly controlled by β1-adrenergic receptors. resensitization of b-adrenergic receptors by inhibition of g-protein coupled receptor kinase 2 (grk2) is discussed as a potential strategy to treat heart failure. we characterized raf kinase inhibitor protein (rkip) as a physiological inhibitor of grk2 and found rkip to increase contractility of neonatal cardiomyocytes. the present study evaluated the role of rkip in heart failure. we assessed its effects on cardiac function in pressure overload induced heart failure and determined the expression patterns of rkip in failing hearts of humans and mice. transverse aortic contriction (tac) was performed on 7-week-old c57/bl6 mice to induce heart failure by pressure overload of left ventricles. after three weeks of tac, echocardiography showed distinct signs of decreased cardiac function in wild-type mice. fractional shortening was reduced and left ventricular diameters were increased. histological analyses revealed increased interstitial fibrosis, caspase-and tunelassays indicated myocyte apoptosis. western blot analysis showed significant upregulation of rkip expression in failing hearts compared to non-banded control hearts. interestingly, this upregulation of rkip expression was also detected in failing human hearts and in samples of patients with aortic valve stenosis but not in healthy control samples. to assess the effects of rkip overexpression on heart failure, we analysed heart function and structure of rkip transgenic mice and wild-type mice 3 weeks after tac. while left ventricular hypertrophy was increased to similar extents in wild-type and rkip trangenic mice, rkip mice did neither develop dilatation of the left ventricle nor a decrease in fractional shortening. in contrast to wild-type mice, the expression of the heart failure markers bnp and anp was not upregulated in banded rkip transgenic mice after 3 weeks of tac. taken together, cardiac overexpression of rkip prevented left ventricular dilatation and loss of contractile function in a mouse model of pressure overload induced heart failure. therefore, increased rkip expression may be an interesting target to prevent detrimental effects from increased left ventricular pressure. the main focus of this study was the chromatographic separation. the most frequently prescribed antibiotic drugs clarithromycin, erythromycin, clindamycin, cefuroxim, doxycyclin, amoxicillin, levofloxacin, and ciprofloxacin were selected for the screening method. method: a relatively short operation time and a sufficient separation were reached by column, eluent, and gradient optimization with poplc (phase optimized liquid chromatography). in the first step columns with the five stationary phases c18 eps 2, c18 sh 2, phenyl 2, cn 2, and c30 were used to determine the retention times of the drugs in an isocratic mode. the stationary phases, the column length and the retention times were fed in the poplc software and the optimal column was calculated. this column contained different stationary phases and was compared with customary columns. results: using the optimal column a sufficient chromatographic separation of the eight antibiotic drugs was reached. that was not possible with the customary columns. with the optimal column the time of measurement was too long. using a mobile phase gradient the measuring time could be reduced. discussion: with lc-ms/ms a complete chromatographic separation of all analytes is not necessary. but when measuring many transitions in a biological matrix two problems should be excluded: ion suppression and a too small number of measurement points per peak. especially when using positive and negative ionization in the ms a good separation is mostly necessary. to determine only the eight antibiotic drugs an optimized column is not necessary, but for a screening method with more than twenty drugs the poplc system is very helpful. we have investigated the uptake mechanism of cdt and in particular the intracellular membrane translocation of cdta. our data indicate that cdt requires acidification of the endosomal lumen for translocation of cdta across endosomal membranes into the cytosol. bafilomycin a1, an inhibitor of endosomal acidification protects vero (african green monkey kidney) cells from intoxication with cdt. consistently, translocation of cdta was observed when the acidic conditions of the endosomal lumen were mimicked at the cytoplasmic membrane of intact cells. next, we tested whether host cell factors are involved in membrane translocation of the toxin. radicicol, a specific pharmacological inhibitor of the chaperone heat shock protein hsp90 as well as cyclosporine a, an inhibitor of cyclophilins delayed the intoxication of cells with cdt but not with toxins a and b [1] . this result was confirmed by analyzing the adp-ribosylation status of actin from such cells in the presence or absence of the inhibitors. in addition, we excluded that the inhibitors of hsp90 and cyclophilins have any effect on receptor binding, endocytosis or enzyme activity of cdt. the data strongly suggest that the participation of hsp90 and cyclophilin is crucial for translocation of cdta into the cytosol. comparable results were obtained for the related binary iota toxin of c. perfringens. in vitro purified immobilized hsp90 and cyclophilin a specifically bound to the enzyme components of cdt and iota toxins. in conclusion, the results imply a common hsp90/cyclophilin a-dependent translocation mechanism for the family of binary actin-adp-ribosylating toxins. our current investigations focus on the participation of fk506-binding proteins (fkbps), another group of peptidyl-prolyl cis/trans isomerases in the membrane translocation step of these toxins. [1] kaiser, e., kroll, c., ernst, k., schwan, c., popoff, m. r., fischer, g., buchner, j., aktories, k. and barth, h. (2011) . complex multicellular in vitro coculture models represent a promising tool regarding e. g. cellular interactions with nanoparticles, since they more closely mimic the cellular composition of the body. therefore, we used our developed coculture model of the alveolar-capillary barrier composed of lung epithelial cells (nci h441) on top and microvascular endothelial cells (iso-has-1) on the bottom side of a filter-membrane to study nanoparticle cytotoxicity and cellular uptake. with a coculture period of about 10 days the cells achieve a more differentiated and polarized state and develop a tight barrier, which can be measured via ter (transepithelial electrical resistance). regarding cellular uptake of fluorescently labeled amorphous silica nanoparticles (asnps, 30 nm) the coculture took up much less asnps than conventional monocultures. besides, we could not verify a specific uptake mechanism (e. g. clathrin-, caveolae-mediated endocytosis) via immunofluorescence staining of the cells. however, we detected asnps incorporated in flotillin-1 and -2 labelled vesicles. former studies concerning cytotoxicity (lactate dehydrogenase assay) of amorphous silica nanoparticles (asnps, 30 nm) revealed that our coculture behaved much more robustly compared to conventional monocultures. however, regarding inflammatory responses (e. g. sicam, il-8 increase) the coculture responded more sensitively than conventional monocultures. in a further development we added a third cell type, the alveolar macrophage (am), to our coculture. since ams embody the front-line of alveolar defense against inhaled pathogens and particles, they play a central role in regulating lung immunity. as model we applied the human acute monocytic leukemia cell line, thp-1 (prestimulated with 8 nm pma for 4d) apically to the epithelial monolayer of the coculture. our preliminary studies concerning inflammatory responses of the tripleculture (h441/iso-has-1 with thp-1) revealed a higher sensitivity of the triple-culture compared to the double coculture. the triple-culture responded with an increased il-8 release upon lps or tnf-a stimulation. in conclusion, this triple-culture model offers a promising prospect to mimic more closely realistic cell interactions with nanoparticles in the distal lung. ethanol is a component of many herbal fluid preparations [1] , since it is an excellent extraction solvent for the phytochemical components of herbal drugs and contributes to the stability of these medicines. toxicological and pharmacokinetic evaluations [2] have shown that the small amounts of ethanol applied with therapeutic doses are safe even in children. despite that these medicines have been used safely since many decades, they have occasionally been subject of discussion in the public, triggered by the increasing problem of recreational abuse of alcoholic beverages by children and young persons [3, 4] . therefore, there is a growing need of a systematic evaluation of pharmacovigilance data on these medicines. for evaluating the experience gained from the therapeutic use of these medicines, 16 pro-and retrospective studies with 10 herbal medicinal products containing ethanol at doses of 40 to 240 mg per single dose, depending on the age group, have been analyzed, covering 49 816 patients of 0-12 years of age. in these studies, altogether 15 adverse drug effects have been described, none of which was attributable to the ethanol content of the medicines. in a survey of the worldwide use of these and other 6 herbal medicinal products it was shown that during the past few years, more than 764 mio daily doses have been sold, corresponding to more than 33 mio of patients (data obtained from manufacturers; figures available partly from 1993 onwards, partly from 2003/4 onwards). from the packages sold in germany in the years between 2005 and 2009, 48.1 mio were attributable to self-medication, 10.8 mio to prescriptions reimbursed by health insurance (ims, frankfurt). as non prescription medicines are reimbursed in germany only in children, at least the latter part of the prescriptions can be attributed mainly to children. all of these medicines are registered or licensed by regulatory authorities. adverse effects are covered by the pharmacovigilance system, and no adverse effects attributable to the ethanol content have been reported. this set of data supports the conclusion drawn from the experience of a safe use over decades, i.e. that the ethanol content of herbal medicinal products does not give any causes for concern regarding their safety even in children. dedication: this contribution is dedicated to prof. dr. hilke winterhoff, institute for pharmacology and toxicology, university of münster, who died on 9 may 2010. she has initiated this work. prucalopride was introduced as a new selective 5-ht4 receptor agonist that is approved for treating obstipation. whereas one could expect -due to the fact that 5-ht4 receptors are functionally expressed in the human heart -in clinical trials prucalopride did not show cardiac effects. this is quite surprising because other 5-ht4 receptor agonists have been withdrawn from the market just for that reason. in this study we used prucalopride for in vitro studies with atrial preparations from transgenic (tg) mice with cardiac myocyte-specific overexpression of the human 5-ht4a receptor. isolated electrically driven (1 hz) left and spontaneous beating right atria of tg mice were compared with those of wild type (wt) littermates. moreover, we used isolated electrically driven (1 hz) human right atrial preparations from patients undergoing cardiac surgery. finally gr113808, a 5-ht4 receptor antagonist, was added. prucalopride exhibited a dose dependent positive inotropic effect in left atria and a positive chronotropic effect in right atria of tg mice with a logec50 of -6.8 and -7.1, respectively (p<0.05 vs. wt, n=3). in human atrial tissue prucalopride also acts as an agonist, leading to an inotropic effect. all effects could be antagonized by 10 µm gr113808. we could demonstrate that prucalopride acts as an agonist at the 5-ht4 receptor in our transgenic mice model and also in human right atrial preparations. these findings suggest that tachycardia and arrhythmias are possible side effects, which should be carefully looked for. the involvement of psychological factors, especially stress, are known to play an important role in functional gastrointestinal diseases (1, 2) probably by affecting the brain-gut axis. based on the good correlation between stress & functional dyspepsia (fd), many animal models for fd have been developed where animals are subjected to various kinds of psychological stress either during the neonatal period or in adulthood. this stress was found to induce gastric motor dysfunction resembling symptoms of fd. two models for stressinduced fd were performed in order to choose the more adequate one for studying sensitivity changes of the fundus to various mediators as well as changes in some relevant hormone levels in the blood for subsequently testing the efficacy of treatment with stw 5 (a 9 component herbal preparation of proven clinical efficacy in fd and in irritable bowel syndrome (3, 4) ). in one model, maternal separation (5) was performed on weanling rats starting from postnatal day 2 for 3 h each day for 3 weeks. rats were then allowed to mature to an adult age. the other model was that of restraint stress (rs) (6, 7) . adult animals were restrained for 90 min/ day for 1 week. the animals of both models were eventually sacrificed, the stomach fundus was isolated and its sensitivity in vitro to carbachol, potassium chloride, serotonin and adrenaline was tested. blood samples were taken to assess levels of ghrelin, corticosterone releasing factor (crf) and corticosterone. the sensitivity of fundus strips from restrained rats towards the agents tested, partly representing autonomic responsiveness, was more depressed than those from maternally separated ones. levels of ghrelin, crf and corticosterone were also more elevated in the rs model. that model was therefore chosen to test the efficacy of stw 5 in restoring the deranged parameters. a group of animals received stw 5 orally once daily starting treatment 1 week before exposing them to rs and continuing treatment for a further week during subjection to rs. stw 5 was effective in normalizing the depressed stomach fundus responses exhibited by animals subjected to rs and to normalize to a large extent the deranged blood levels of ghrelin, crf and corticosterone. the findings lend further evidence to the role of the brain-gut axis in fd and gives supportive evidence for the first time for the clinical usefulness of stw 5 in this condition. there is good evidence that oxidative damage to dna leads to down-regulation of transcription of affected genes and epigenetic gene silencing in a mechanism dependent on the 8-oxoguanine dna glycosylase (ogg1), which generates harmful repair intermediates [1, 2] . we have recently shown that the magnitude of inhibition of transcription of an egfp reporter gene by single 8-oxo-7,8-dihydro-deoxyguanosine (8-oxodg) varies between the opposing dna strands of the gene [3] . we now have addressed the question, to which extent the transcription inhibitory potential of 8-oxodg depends on its position in the gene and on the dna microsequence surrounding the modified nucleobase. to investigate the effect of position, we produced plasmid vectors containing single 8-oxodg or dg (underlined) in the second position of an agc trinucleotide. measurements of egfp expression in transfected mammalian host cells showed that a single 8-oxodg caused a strong inhibition of gene transcription. the magnitude of this effect for 8-oxodg situated in the transcribed dna strand of the gene was the same as in the non-transcribed dna strand. similarly, there was no quantitative difference between the effects of 8-oxodg present in the 5′-versus 3′-utr of the gene. the results thus indicate that inhibition of transcription by this base modification does not depend on the position in the gene. further comparisons were done between the effects of 8-oxodg nucleobases localised in the same dna strand but within different sequence contexts. gene expression analyses in the repair proficient host cells showed that the degree of inhibition of transcription caused by single 8-oxodg was dependent on the neighbouring nucleotides. among three tested sequence motifs, the minimal effect on the gene transcription was found to correlate with a significantly less efficient base excision by the purified human ogg1 protein. the results thus support the initiatory role of ogg1 in the mechanism of transcriptional repression. in addition, the finding of the effect of dna sequence on the base excision activity of ogg1 suggests that repair rates of single base modifications in genome could also be heterogeneous. allgayer j., kitsera n., epe b., khobta a. johannes gutenberg university of mainz institute of pharmacy and biochemistry, staudingerweg 5, 55128 mainz, germany interference of dna base modifications with gene transcription is an important biological consequence of genotoxic damage [1] . an efficient method for incorporation of a single 8-oxo-7,8-dihydroguanine (8-oxog) at a defined position in the egfp gene in a plasmid vector was recently developed in our lab. the method relies on the availability of adjacent sites for a sequence-specific nicking endonuclease, which allow the insertion of a synthetic oligonucleotide containing 8-oxog in a chosen position. we further showed that this single lesion inhibits transcription after excision by ogg1 [2] . in order to determine to which extend the observed effect depends on the position of the modified base in the gene, we constructed several new plasmid vectors which allow incorporation of the same dna oligonucleotide containing 8-oxog or g in different positions in different dna-strands. dna sequence cassettes were designed to contain a 5′-cattgcttcgctagcacg nucleotide sequence in different orientations, which was flanked by two unidirectional bsrdi recognition sites (5′-gcaatgnn). adapters containing the restriction sites for directional cloning into the 5′-or the 3′-utr of the plasmid-borne egfp gene were added. the produced plasmid vectors thus allow the insertion of a single 8-oxog in the same sequence context into opposing dna strands in the 5'utr and in the 3'utr. keratinocytes are the major cell type of epidermis and are responsible for the formation of an outer barrier, the statum corneum, to protect an organism against harmful environmental influences. for generation of this barrier, keratinocytes pass through a complex differentiation program that is accompanied by synthesis of lipids, like cholesterol and ceramides. finally, the differentiation of keratinocytes leads to apoptosis. another function of keratinocytes is to sense environmental factors, some of which are decoded by members of the transient receptor potential (trp) ion channel family. trpv3, for example, is predominantly expressed in keratinocytes, and decodes different chemical and physical stimuli like the terpenoid-derived ligands camphor and thymol or temperatures above 33°c. less is known about the influence of cholesterol on trpv3 signalling. we modified the cholesterol content of hek293 cells stably transfected with trpv3 and performed flipr-based calcium measurements. these experiments revealed that cholesterol enrichment robustly potentiates trpv3 by sensitizing it to lower agonist concentrations. we verified these results with whole-cell patch-clamp measurements. in contrast, trpv2, another heat-sensing channel, was not affected by cholesterol modification. since former studies showed a defective formation of epidermal barrier in trpv3 -/mice, our results imply that a cholesterol-regulated trpv3 signalling may contribute to the progression of differentiation or initiation of apoptosis of keratinocytes. ischemia-reperfusion injury causes severe problems in the early period after lung transplantation. since transient receptor potential (trp) channels are important regulators of vascular permeability and tone, we investigated the influence of a trpc6 blocker on pulmonary function after simulated transplantation, using an ex vivo model of isolated perfused and ventilated rabbit lungs. to this end, heart-lung blocks were excised and mounted in an artificial thorax chamber. negative pleural pressure ventilation was initiated, and lungs were perfused with albumin-containing tyrode-solution (100 ml min -1 ). after equilibration in a stable perfusion and ventilation mode for 30 min, lungs were flush-perfused with perfadex ® solution, followed by an ischemic storage for 4 h on ice. subsequently, ventilation and perfusion were re-initiated to simulate a transplantation situation. in the oxygenated group, pulmonary artery po2 was adjusted to 120 mmhg, in the deoxygenated group, the perfusate inflow was gassed with nitrogen to achieve a pulmonary artery po2 of 50 mmhg. both transplantation conditions were conducted in the absence or in the presence of the trpc6 blocker larixol acetate (5 µm). hemodynamic and ventilatory parameters were continuously monitored. the weight of deoxygenated lungs steadily increased during 2 h of reperfusion from 22.1 ± 1.32 g to 35.4 ± 4.23 g. this weight gain was inhibited by supplementation of the trpc6 blocker (from 21.4 ± 0.91 g to 27.4 ± 2.42 g 2 h after reperfusion). in contrast, oxygenated lungs showed no marked weight gain after reestablishment of perfusion (from 17.5 ± 1.51 g to 21.5 ± 2.29 g 2 h after reperfusion), and the trpc6 blocker had no additional effect (initial 19.5 ± 1.28 g, 2 h reperfusion 21.3 ± 1.22 g). we conclude that a trpc6-blocking compound to the lung perfusate during simulated transplantation counteracts the endothelial permeability increase and the resulting post transplant weight gain. the results indicate a role for trpc6 in the regulation of pulmonary vessel permeability and support the concept that perfusion of donor lungs with trpc6 blockers may prevent edema formation caused by ischemiareperfusion injury shortly after lung transplantation. regulation of human inducible nitric oxide synthase (inos) expression by noncoding rnas (ncrnas) kleinert h., schmitz k., koch k., hahn s., pautz a. universitätsmedizin der johannes gutenberg-universität mainz institut für pharmakologie, obere zahlbacher str. 67, 55101 mainz, germany the transcriptome analyses of human cells showed that additionally to the 30.000 protein coding sequences the human dna codes for much more (450.000 ?) non-coding rnas 1 . beside the ribosomal, and transfer rnas (rrna and trna) involved in the mechanism of translation there are also short (snrna, sorna, mirna) and long noncoding rnas (ncrnas) implicated in regulation of gene expression (splicing, translation, chromatin packaging etc.). matsui et al. described regulation of il-1β-induced inos expression by an antisense rna (as-3-utr-rna) in rat hepatocytes 2 . a promoter located on the antisense strand 3' to the last exon (exon 27) of the rat inos gene drives the expression of an as-rna complementary to the 3'-utr of the rat inos mrna. using different sense primers with homology to the 3'-utr sequences of the human inos gene for specific rt-pcr reactions (detecting only an as-rna) we were not able to detect such an as-3-utr-rna in human cells. in addition, transient transfection analyses using constructs containing a 2.7 kb fragment of the 3'-flanking genomic sequences (as used by matsui et al. in the rat system) of the human inos gene in front of a luciferase reportergene into dld-1 cells revealed no promoter activity of these sequences. korneev et al. described the expression of a 2 kb anti-inos-ncrna in different brain tumors and showed reciprocal expression to the inos mrna in human embryonic stem cells 3 . analyzing the expression of this anti-inos-ncrna in cytokine-treated dld-1 cells also showed a basal expression of this as-rna and an enhancement of the expression by cytokine-treatment. downregulation of the anti-inos-ncrna by sirnas reduced whereas overexpression enhanced cm-induced inos expression in human dld-1 cells. this indicates that this anti-inos-ncrna regulates cytokine-induced inos expression in a positive manner. rna 14, 2030 rna 14, -2037 rna 14, (2008 . using directed evolution to improve functional expression of class b g-protein coupled receptors klenk c., scott d. j., plückthun a. universität zürich institut für biochemie, winterthurerstrasse 190, 8057 zürich, switzerland the class b of g-protein coupled receptors (gpcrs) comprises 15 peptide hormonebinding receptors which regulate important endocrine and neuroendocrine functions of the human body. several of these receptors are implicated in the pathogenesis of severe diseases such as diabetes, osteoporosis, growth disorders and depression, which makes them attractive targets for drug therapy. to develop new compounds targeting these receptors a detailed understanding of the molecular structure is required which has not been succeeded to date. structural studies of proteins by x-ray crystallography or nmr spectroscopy generally require large and homogenous quantities of protein. for gpcrs this prerequisite is difficult to achieve as the vast majority of gpcrs exhibits low endogenous expression and is very unstable in solution. therefore, improved expression conditions are necessary for the efficient characterization of new gpcr structures. here we present a method to optimize class b gpcrs for improved heterologous expression and increased thermostability by means of directed evolution. libraries of class b gpcrs were obtained by random mutagenesis and were expressed in a heterologous expression system in which functional gpcr is targeted to the inner membrane of e. coli. mutants that display increased receptor expression levels and ligand binding were selected by flow cytometry using fluorescently labeled ligands. repetitive cycles of randomization and selection allow to gradually increasing the level of protein expression and stability. with this evolutionary approach key residues within the receptor sequence can be identified rapidly that are responsible for improved biophysical properties without affecting the pharmacological features of the receptor. such gpcr mutants will become a valuable tool on the way to express high quantities of stable receptor protein for subsequent structural studies. pasireotide (som230) is currently under clinical evaluation as a successor compound to octreotide for the treatment of acromegaly, cushing's disease and carcinoid tumors. whereas octreotide acts primarily via the sst2 somatostatin receptor, pasireotide was designed to exhibit octreotide-like sst2 activity combined with enhanced binding to other somatostatin receptor subtypes. somatostatin and octreotide stimulate the complete phosphorylation of at least six carboxyl-terminal serine and threonine residues namely s341, s343, t353, t354, t356 and t359, which in turn leads to a robust endocytosis of the sst2 receptor. surprisingly, pasireotide failed to phosphorylate the four threonine residues and induced only a detectable phosphorylation of s341 and s343. somatoprim and ke108 are recently developed somatostatin analogs capable of binding to four of five somatostatin receptor subtypes. here, we performed an in vitro study comparing the effects of pasireotide, somatoprim and ke108 on sst2 somatostatin receptor binding, phosphorylation, internalization and signaling. further somatostatin, octreotide, pasireotide, somatoprim and ke108 were tested for functional selectivity at sst5 receptor mutants, which possess a carboxyl-terminal sst2tail. this approach allows detection of receptor activation by phospho-specific sst2 antibodies. compared to octreotide, somatoprim activates sst2 but has a higher activity on sst5. ke108 and pasireotide are partial agonists at the sst2 receptor. pasireotide, ke108 and somatoprim show comparable effects on sst5 receptor. however, none of these new pan-somatostatin analogs behaves like natural somatostatin on the sst5 receptor. cadmium modulates ahr-associated gene expression in the rat intestine after oral exposure kluxen f. m. 1, 2 , höfer n. cadmium has been shown to mimic steroid estrogen effects in vivo and in vitro. we have recently identified cross-talk of estrogen receptor (er) and aryl hydrocarbon receptor (ahr) in the rat uterus where 17beta-estradiol (e2) and cdcl2 modulate ahrassociated genes via er after i.p. injection in a similar fashion (kluxen et al., arch toxicol doi 10 .1007/s00204-011-0787-x). however, the predominant route of exposure to cadmium in non-smokers is via diet. moreover, uterus expresses mainly the receptor subtype eralpha, whilst small intestine and colon express mainly erbeta. thus, we now investigated by real-time rt-pcr the effects of cadmium (2mg/kg b.wt cdcl2 (cd2)) or steroid estrogen (0.1 mg/kg b.wt. 17alpha-ethinylestradiol (ee2)) on ahrassociated gene expression (i.e., ahr, cyp1a1, gsta2, nqo1) in the small intestine of rats after oral exposure (3 days gavage). the animals were also co-treated with cd2 and pure anti-estrogen (2.5 mg/kg b.wt zk191703 (zk)) or ee2, to asseess whether ermediated processes are involved. we also measured cyp1a1 mrna expression in two estrogen receptor negative colon cancer cell lines (ht-29 and caco2) treated for 5 days with cdcl2 (1µm) and e2 (0.01µm). the dose-dependency of cadmium induced ahr target gene modulation was studied in a second animal experiment, with administration of cadmium in drinking water for 28 days (0.4-9 mg/kg b.wt cdcl2 equivalent to 5, 50, and 150 ppm) and ee2 (0.08 mg/kg b.wt) as steroid reference. in summary we present two major results: the metalloestrogen cadmium modulates dose-dependently the ahr-associated gene expression in the intestine after oral exposure. yet, since the cadmium induced modulation of ahr target genes was not antagonized by anti-estrogen in the small intestine in vivo and was also found to occur in er-negative colon cells in vitro, we propose that er-independent mechanisms might play a role in this effect. meg) is the most cytotoxic lesion. if not repaired by o 6 -methylguanine-dna methyltransferase (mgmt), o 6 meg/t mismatch is recognized by the mismatch repair system (mmr) that performs futile repair cycles. during this process secondary lesions (i.e. dna single-strand brakes) are formed, which block dna replication in the next replication cycle, leading to dna double-strand breaks (dsbs). these dsbs eventually signal to apoptosis and other genotoxic endpoints. here, we wished to address the question whether autophagy is part of the cellular response triggered by o 6 meg. we also assessed whether autophagy influences apoptosis induced by tmz in glioma cells. we show that tmz induces autophagy in u-87 mg and ln-229 glioma cell lines. the maximum amount of autophagy was observed several days (96 h) after tmz treatment and mgmt proficient cells did not display significant autophagy. thus, the data show that mgmt protects against tmz induced autophagy, pointing to o 6 meg as the critical lesion responsible for induction of autophagy. using colon cancer cell lines proficient and deficient in mmr, we show that mmr is required for tmz-induced autophagy. because dsbs, which emerge during the processing of o 6 meg, are repaired preferably by homologous recombination (hr) ln-229 cells stably down-regulated for hr were tested for autophagy induction. the data indicate that dsbs are involved in tmz-induced autophagy. because autophagy following tmz treatment occurs earlier than apoptosis we hypothesize that autophagy protects glioma cells against apoptosis. using an early stage autophagy inhibitor 3-methyladenin we have shown, that autophagy inhibition sensitized glioma cells to tmz-induced apoptosis. taken together our data point out that tmz induces autophagy in glioma cells and that autophagy protects glioma cells against tmz-induced apoptosis. o 6 meg is the lesion responsible for autophagy induction. furthermore, the data also shows that mmr and dsbs are involved in the induction of autophagy after tmz treatment. work was supported by bmbf (02nuk016). retinitis pigmentosa (rp) is a severe human retinal disease characterized by a progressive degeneration of rod photoreceptors and a secondary loss of cone function. in most cases, rp finally leads to legal blindness. mutations in the regulatory subunit of the rod cyclic nucleotide-gated (cng) channel (cngb1a) have been found in patients suffering from rp. we used cngb1-deficient (cngb1 -/-) mice to establish a gene replacement therapy as a potential treatment for rp by means of recombinant adenoassociated viral (raav) vectors. the packaging limitations of raav vectors required a capacity-optimized vector of the large cngb1a cdna (approx. 4 kb). therefore, we replaced regulatory elements within the expression cassette and used a short mouse rhodopsin promoter element for rod-specific expression. after injection of therapeutic raavs (serotype 8) into the subretinal space of 2-week-old cngb1 -/mice, we assessed the restoration of vision by analyzing i) protein expression and localization, ii) retinal function and morphology and iii) vision guided behavior. we found that treated cngb1 -/mice expressed full-length cngb1a and cnga1, which were previously downregulated. both proteins co-localized in rod outer segments and formed regular cng channel complexes in the treated area of the cngb1 -/retina. using electroretinography (erg) we observed a distinct rescue of rod-driven responses. moreover, cngb1 replacement significantly preserved outer segment morphology and delayed retinal degeneration. finally, treated cngb1 -/mice performed significantly better than untreated mice in a modified water maze task designed to test for rod-mediated, vision-guided behavior. in summary, this work provides a proof-of-concept for the treatment of rod channelopathyassociated retinitis pigmentosa by raav-mediated gene replacement. most endocrine disruptors interact with hormone receptors or steroid biosynthesis and metabolism, thereby modifying the physiological function of endogenous hormones. here, we present an alternative testing paradigm for detection of endocrine modes of actions that replace and reduce animal testing through refinement. receptor mediated endocrine effects were assessed using the yeast based receptor mediated transcriptional activation yes/yas assays and effects on steroid hormone biosynthesis were assessed using the human cell line h295r in the steroidogenesis assay. in our testing paradigm we propose to complement the in vitro assays with a single in vivo repeated dose study in which plasma samples are analyzed for their metabolome profile. the combination of these methods does not only contribute to refinement and reduction of animal testing, but also has significantly increased the efficient allocation of resources and allows for a sound assessment of the endocrine disruption potential of compounds. thus, this proposal constitutes a potentially attractive alternative to epa's endocrine disruptor screening program. data on 14 reference substances for which the in vitro yes/yas and steroidogenesis assays and the in vivo metabolome analysis were performed to assess their putative endocrine mode of action is presented here. the bovine corneal opacity and permeability (bcop) test has been adopted by oecd for the identification of corrosive and severe ocular irritants (ghs category 1) for single component substances and multi-component formulations. eye irritation tests (eit) using human reconstructed tissue models (such as epiocular) have been described to predict ocular non-irritants (ghs no category). thus the ultimate repaltement of the draize rabbit eye irritation test (oecd tg 405) by a combined or tiered testing strategy could be possible. the purpose of this study was to evaluate whether the bcop with additional corneal histology together with the eit could be used to predict eye irritancy of agrochemical formulations according to different classification schemes including un ghs and epa systems. we have performed the bcop (plus histology) and the eit of 50 agrochemical formulations for which in vivo eye irritation data were already available (for registration purposes). using the oecd tg guideline evaluation scheme for opacity and permeability in the bcop was not predictive for the agrochemical formulations assessed here, while corneal histology grades and the epiocular tissue viabilities were useful predictors of eye irritancy potencies and could be applied for the different classification schemes. the nanomaterials offers extraordinary opportunities. the nano-structure can change the physical and chemical properties, and often also alters the biological effects. hence the toxicity of a nanomaterial can differ from its larger-scale material; but as of today, no new quality of a general nano-specific toxic effect has been observed. therefore the established testing methods are generally suitable. it is, however, difficult to apply the nanomaterials to in vitro test systems, since it is the nature of these materials to change their surface properties and agglomeration stateand the uptake and distribution in the body may differ from their larger-scale materials. while the methods for topical effects may be used for nanomaterials without further modification, the in vitro methods for genotoxicity testing require the dispersion in culture media. the use of reproducible and well-documented dispersion protocols andthe characterization of its particle size distribution is de rigueur . [1] . for many nanomaterials published genotoxicity studies did not give a consistent picture [2] and therefore there are rather effects of individual nanomaterials than nano-genotoxicity per se. modern toxicology is based on the insight into toxic pathways. for nanomaterials a testing strategy will include testing for their primary effects (which might be only a handful: particle effects, catalyzing the formation of reactive molecules and ion release) and their uptake, distribution and clearance. the use of dermal penetration studies in vitro for the risk assessment of sunscreen nanomaterials has been demonstrated [3] . in vitro methods for specific effects (such as inflammation, pulmonary toxicity, sensitization) are currently awaiting validation (for both chemicals/molecules as well as nanomaterials). in the meantime alternative short-term in vivo studies with optimized biological readouts can deliver information on the toxic pathways as well as the biokinetics and dose-response relation of nanomaterials in the body. a short-term inhalation test for nanomaterials has already been used successfully [4] . testing strategies based on those methods engage less animals and provides more significant data than classical testing. moreover data from these methods will serve as a benchmark and a validation for the in vitro models under evaluation. nanoparticles (np) are increasingly used in various field of industry which necessitates evaluation of their safety. also in the food industry, nps have gained strong interest, for example as food additives or to improve food packing. however, the potential risks of ingested np have been rarely investigated. inhalation studies have revealed that inflammation and oxidative stress may represent unifying mechanism for the induction of adverse health effects of toxic np. in the present study, a co-incubation model of human polymorphonuclear neutrohils (pmns) and caco-2 human intestinal epithelial cells was used as a model to address potential genotoxic effect of np during intestinal inflammation. oxidative dna damage induction (measured by the fpg-modified comet assay) was induced in the caco-2 cells by activated pmn and this effect increased with increasing pmn to caco-2 cell ratio. the crucial involvement of the phagocyte nadph oxidase complex could be demonstrated using treatment of caco-2 cells with bone marrow-derived pmn from nadph oxidase deficient mice. dna damage by pmn as well as h 2o2 was increased in buthionine sulphoximine (bso) pre-treated caco-2 cells, illustrating the importance of the cellular glutathione (gsh) status in these target cells. gsh depletion in caco-2 cells could also be shown upon treatment with various types of np. our data suggests that ingested np may increase the susceptibility of the colon mucosa to genetic damage during the occurrence of intestinal inflammation. the ingestion of seafood contaminated by acute toxic doses of the marine toxin okadaic acid (oa) is responsible for diarrhetic shellfish poisoning. it is recently known that both the rat and the human hepatic cytochrome p450 monooxygenases (cyp) are able to metabolize this toxin. currently, there is a lack of data about the toxicity and mode of action of oa after xenobiotic metabolism. the aim of our study was the measurement of the toxicity and oxidative stress status in hepg2 cells incubated with oa in the absence and presence of s9 mix. pure oa, as well as oa pre-activated with liver homogenisates (s9 mix) were used to treat human hepg2 cells that have nearly undetectable levels of functional cyp but express phase ii enzymes. the experiments were performed with both human and rat s9 fraction plus cofactors of phase i enzymes. the cell viability was measured after 4 h using mtt-test and xcelligence real time cell monitoring system. furthermore, levels of intracellular reactive oxygen species (ros) were detected by 2´,7´-dichlorofluorescein diacetate and additionally by measuring the intracellular glutathione content. in the presence of both human and rat s9 mix oa showed a higher toxicity than the parental substance. oa pre-incubated in rat s9 mix was toxic at 75 nm oa. strong effects could be observed when oa was pre-activated with human s9-mix at a concentration of 50 nm oa. pure oa was non-toxic in that concentration range. we could also detect an increase of oxidative stress in hepg2 cells treated with oa in the presence of all investigated s9-mix. these results suggest that oa is activated after oxidative xenobiotic metabolism into metabolites which possess a higher cytotoxic activity and increase the amount of intracellular ros in hepg2 cells. ballast water treatment -emerging health risks werschkun b., banerji s., krätke r. bundesinstitut für risikobewertung, max-dohrn-strasse 10, 10589 berlin, germany the introduction of invasive marine species into new environments by ships' ballast water, via ships' hulls and other vectors has severe impacts on the oceans. in 2004 the international maritime organisation (imo) launched the international convention for the control and management of ships ballast water and sediments which requires ballast water to be treated in order to eliminate alien aquatic species. ballast water treatment may include mechanical, physical or chemical measures. any ballast water management system using active substances needs imo approval. therefore identification of active substances, relevant chemicals and submission of specified datasets on their physical, chemical and toxicological properties is required in order to assess the safety for the aquatic environment and for human health. the bfr is the german federal agency responsible for health risk assessment and has been involved in more than twenty assessment and approval processes so far. the majority of imo approved systems are based on oxidative principles such as chlorination and ozonation. these methods can generate disinfection by-products (dbps), which are a mixed group mostly of halogenated organic substances like trihalomethanes, haloacetic acids and haloacetonitriles. the formation of dbps is well known from the disinfection of drinking water. some dbps are regulated under drinking water directives because of their long-term toxicity but many are unregulated and have unknown toxicological properties. the formation of dbps may vary significantly depending on the treatment system as well as on environmental parameters like temperature, ph and composition of the organic matter within the aquatic environment. in sea water, sources for dbp formation besides ballast water treatment are aquaculture and the cooling systems of coastal power plants. in order to address possible health and environmental risks from dbps formed during ballast water treatment a conference on emerging risks from ballast water treatment was held at bfr in october 2011. here we summarise the main conference findings and identify areas for future research. two presentations corroborated that significant amounts of dbps can be formed in sea water and a presentation on the toxicological properties of dbps pointed out that many have genotoxic properties. accordingly, the determination of dbp species and generated concentrations under different ballast water treatment conditions was seen as a mayor task. different approaches for health and environmental risk assessments were also discussed. appropriate human exposure scenarios and methods for exposure assessment, taking into account common approaches used in risk assessment were presented during the conference. a suitable approach based on derived pec-values for exposure quantification was proposed in order to improve the procedure available for risk assessment of chemical agents used for ballast water treatment. agonist-selective signaling of µ-opioid receptors in t lymphocytes kraus j., börner c., lanciotti s., koch t., höllt v. inst. für pharmakologie und toxikologie, leipzigerstr. 44, 39120 magdeburg, germany opioids are the most potent analgesics and irreplaceable for the treatment of severe pain. in addition to their central effects, opioids modulate a great variety of immune effector cell functions, which may result in unwanted side effects during opioid treatment. the effects of most of the commonly used opioids are mediated by µ-opioid receptors, which belong to the superfamily of g protein coupled receptors. recent data support the concept that g protein coupled receptors function as dynamic entities, which may occupy multiple conformations and activate multiple signaling pathways in a ligand-dependent manner. consequently, different ligands activating the same receptor may have different cellular effects, which has been termed "agonistselective signaling". little is known about agonist-selective signaling of µ-opioid receptors in immune effector cells. in a first attempt to understand if and why such different profiles among different opioids occur we investigated effects of different opioids in human jurkat t cells. we report that the µ-opioid receptor ligands fentanyl, methadone, loperamide and betaendorphin induce internalization of a µ-opioid receptor-green fluorescent reporter construct, whereas morphine and buprenorphine did not induce internalization. the internalization was dependent on p38 mapk and phospholipase d2. in line with this, we observed marked phosphorylation of p38 mapk and activation of phospholipase d2 induced by the internalizing opioids, but no or little such activity by morphine and buprenorphine. as a physiological result, fentanyl, methadone, loperamide and betaendorphin treatment of primary human t cells and jurkat t cells resulted in a strong, up to 100 fold induction of il-4, which was dependent on p38. in contrast, morphine and buprenorphine only showed a weak, approximately one order of magnitude lower induction of il-4. by inducing il-4 opioids significantly modulate the t helper cell balance into the type 2 direction, which influences various immune responses, e. g. the antiviral, t helper cell type 1-mediated response. considering the vital necessity of opioid use in humans, it is an intriguing goal to identify analgetically feasible opioids that have little or no immunosuppressive or -modulatory effects. modulation of cgmp signals by phosphodiesterases in smooth muscle cells krawutschke c., koesling d., russwurm m. ruhr-universität bochum pharmakologie und toxikologie, universitätsstrasse 150, 44780 bochum, germany within the cardiovascular system, cgmp mediates smooth muscle relaxation and inhibition of platelet aggregation. cgmp is formed by particulate guanylyl cyclases and nitric oxide-sensitive guanylyl cyclases, that are activated by natriuretic peptides or nitric oxide (no), respectively. besides the cgmp-forming enzymes, the cgmp-degrading phosphodiesterases strongly determine amplitude and shape of cgmp signals. in vascular smooth muscle cells, three phosphodiesterases are considered to be responsible for cgmp degradation: pde5, the cgmp-specific phosphodiesterase is activated directly by cgmp binding to its gaf domains; this activation if further stabilized by cgmp-dependent protein kinase-mediated phosphorylation. pde1, the ca2+/calmodulin-stimulated pde constitutes the majority of cgmp-hydrolyzing activity in smooth muscle cells at least in the presence of high intracellular ca2+ concentrations. and lastly pde3, the cgmp-inhibited pde displays some cgmp-degrading activity, although cgmp binding to its catalytic domain is primarily thought to inhibit the campdegrading activity of pde3. cgmp signals measurable in radioimmunoassays require stimulation with cgmpincreasing vasodilator concentrations that are orders of magnitudes higher than those required for relaxation. thus, we developed fluorescent sensors for real-time measurement of cgmp signals in single cells. by using these indicators, we analyzed the contribution of different cyclic nucleotide-degrading phosphodiesterases to the modulation of cgmp signals elicited by physiologically relevant vasodilator concentrations. hyaluronan (ha) is a major component of extracellular matrices and is thought to control cellular phenotypes such as proliferation and migration. therefore, ha synthesis may play an important role in the pathophysiology of atherosclerosis. there are three major ha-synthase isoenzymes (has1-3). the has3 gene is alternatively spliced. has3 transcript variant 2 (has3v2) encodes the smallest has isoenzyme which has a different c-terminus and contains only two transmembrane domains compared to has3 transcript variant 1. the aim of the present study was to investigate whether has3v2 is expressed by vascular cells, how it is regulated and where it is localized in cells and whether it indeed synthesizes ha. has3v2 mrna expression was monitored by quantitative real-time rt-pcr. protein expression was determined by western blotting using a polyclonal antibody that was raised in rabbit. an n-terminal eyfp-has3v2 fusion protein and a ddk-tagged has3v2 construct were expressed for subcellular localization studies and co-immunoprecipitation (co-ip). endogenous has3v2 mrna was expressed in both vascular smooth muscle cells (vsmc) and endothelial cells. furthermore, western blotting revealed has3v2 protein expression in vsmc and platelets. in vsmc has3v2 mrna expression was strongly up-regulated in response to interleukin-1β (il-1β, 10 ng/ml) whereas stimulation with interleukin-10 (10 ng/ml), platelet-derived growth factor-bb (10 ng/ml), transforming growth factor β (10 ng/ml), tumor necrosis factor α (10 ng/ml) and interferon-γ (10 ng/ml) had no effect. transfection of hek cells with eyfp-has3v2 fusion protein revealed localization to the endoplasmic reticulum but not to the plasma membrane. furthermore, co-ip experiments showed that tagged has3v2 proteins were precipitated together suggesting formation of multimeric has3v2 complexes. transfection of has3v2 did not cause increased secretion of ha into the cell culture supernatant in hek cells. in conclusion, has3v2 is present in vascular cells and responds to inflammatory cytokines such as il-1ß. because of the intracellular localization and the lack of ha secretion in has3v2 transfected cells, has3v2 may serve intracellular functions apart from ha synthesis. the tubulin antagonist pretubulysin shows strong vascular-disrupting properties in vitro several epidemiological studies indicate a correlation of human exposure to ultrafine particulate air pollution caused by incomplete combustion processes and an increase in the incidence of pulmonary immune diseases like asthma. as a possible mechanism behind this pathological phenomenon, the adjuvant effect of lung inflammation induced by poorly soluble environmental particles has been hypothesised. the aim of our study was to investigate the causal link between carbon nanoparticle-induced lung inflammation and modulations of immune cell populations during processes leading to sensitization, and allergic immune responses of the airways. therefore mice were treated with ovalbumin (ova) alone or in combination with carbon nanoparticles (cnp) by pharyngeal aspiration. the induction of inflammation and the immune adjuvant activity were studied in the lungs and lung draining peribronchial lymph nodes (pbln) at the level of sensitization, and at the level of the immune response. ova-specific ige antibodies were measured in blood serum, and the development of allergic airway inflammation was studied after ova challenge. results at the level of sensitization showed that cnp-induced immediate airway inflammation had immune adjuvant activity resulting in an increase of specific cell populations in pbln and in a stimulation of asthma-specific th2 cytokines. a specific reduction of the neutrophilic lung inflammation by application of the compatible solute ectoine significantly reduced the adjuvant effects of cnp. in ova-sensitized mice, application of cnp 12 hours prior to allergen challenge, led to a significant increase in inflammatory cell infiltrate and respective cytokines in broncho-alveolar lavages. coapplication of 1 mm ectoine together with cnp reduced the particle-induced effects. our data show a link between neutrophilic lung inflammation and adjuvant effects of cnp. a specific reduction of neutrophils by the application of ectoine attenuated this np induced adjuvant effect, indicating that particle-induced lung inflammation rather than the direct interaction of nanoparticles with immune cells is the critical step in environmentally modulated pulmonary immune diseases like asthma. introduction: drug-eluting stents (des) are commonly used in the treatment of acute artery occlusion. however, even if released cytotoxic drugs reduced neointimal proliferation significantly there is still the risk of in-stent thrombosis. it is presumed that this is due to reduced reendothelialization. it has been suggested that coating the stent with biomolecules may provide a new approach to circumvent the lack of healing of the endothelial layer. one approach would be the use of biomolecular signals, such as (poly)peptides and growth factors. rgd and redv are peptide motifs, known to enhance cell attachment and spreading. aim: the aim of our study was to evaluate the efficacy of proteins, derived from elastin-like proteins (elp) and artificial modified by incorporating with the amino acid motifs rgd,redv and p15, in terms of endothelial healing on stents and other cardiovascular devices. results: in this work, we generated vectors encoding for different biopolymers consisting of various bioactive signal molecule sequences. the peptides, e.g. based on the elastinlike matrix (vpgig)2-vpgkg-(vpgig)2, were synthesized using heterologous expression. after optimizing culture conditions and extraction procedures their biological activity was assessed using human umbilical vein endothelial cells (huvec). elastin like proteins with differently incorporated bioactive signals (redv, rgd and a small p15 peptide) were linked covalently via carbodiimide coupling to poly(l-lactide) (plla) films. huvec growth was determined on these modified surfaces using the brdu assay (cell proliferation) and resazurin assay (cell viability). the chemically modified plla surfaces conferred higher cell viability after 1 h adhesion (60%) and an enhanced proliferation (63%, 1 h adhesion, 24 h cultivation) in comparison to the unmodified plla. these results indicate that the synthesized elp incorporated with amino acid motifs promote an accelerated endothelialization of the biodegradable stent material plla. discussion: in summary, we were able to generate elastin-like proteins modified by bioactive sequences. those sequences enhanced endothelial cell proliferation and adhesion. further studies are warranted to determine the activity on smooth muscle cells of these peptides. (1) the failing heart is characterized by excessive extracellular matrix production by myofibroblasts (myocfs) causing fibrosis and myocardial stiffening. myocfs represent phenotypically transformed cardiac fibroblasts (cfs) and are characterized by the expression of contractile proteins like a-smooth muscle actin (α-sma) and enhanced secretion of growth factors (e. g. ctgf). identification of intracellular enzymes that modulate this transformation process is desired to therapeutically modulate pro-fibrotic progression in heart failure. we show that pde2a, a phosphodiesterase isoform, that is able to hydrolyse cgmp and camp, is markedly upregulated in failing hearts from patients with end-stage heart failure (2-3-fold, p<0.05, n=8). notably, pde2a protein abundance is 4-fold higher in myocfs compared to cardiomyocytes from neonatal rat hearts (p<0.05, n=7). to this end we tested whether pde2a modulates the transformation of cfs isolated from neonatal rat hearts to myocfs. indeed, as assessed by immunoblotting and fluorescent microscopy (α-sma, phalloidin, dapi), adenoviral pde2a overexpression induced α-sma expression (3.2-fold p<0.05, n≥8) and to a lower extent ctgf synthesis (1.5-fold, p<0.05, n≥5). mechanistically, pde2a showed preferential subsarcolemmal localisation with diminished total cgmp levels (-56%, p<0.05, n≥5). consistently, parallel stimulation with atrial natriuretic peptide (anp), a selective activator of membrane-bound guanylyl cyclase, normalized ctgf synthesis indicating that pde2a controls cgmp in a discrete subdomain near the plasma membrane. moreover pde2a overexpression diminished the protein levels of vasodilator-stimulated phosphoprotein, a membrane cytoskeletal component (-60%, p<0.05, n≥5). these data implicate pde2a-dependent subsarcolemmal cgmp regulation in myofibroblast formation and potentially cardiac fibrosis. therefore, targeting pde2a may lead to regression of the fibrotic remodeling associated with heart failure. several anorganic nanoparticles (np) causedhigher inhalation toxicity than the corresponding coarse particles (oberdoerster et al. 2005) . we examined an organic pigment and a polymer dispersion each as nanomaterial and as the chemical identical coarse material in short-term inhalation studies in malerats. the polymer was an anionic acrylic ester copolymer containing free carboxylic groups. three different particle sizes were synthesized by varying polymerization conditions: 12, 80 or 250 nm. although polymeric acrylic ester was reported to be irritating to the respiratory tract at 3 mg/m3, all three tested polymers -including the np (12 and 80 nm) -did not cause any changes in lavage fluid and in histopathology at 10 mg/m3. the organic pigment was a poorly soluble pyrrol with an intense orange color. the np (10 to 50 nm width and 30 to 400 nm length) and the coarse pigments (70 to 200 nm width and 0.3 to 3 µm length) are both needle-like. they were tested at 1, 3, 10 and 30 mg/m3 for the np and 3, 10 and 30 mg/m3 for the coarse materials. mild and partly reversible morphological changes were observed in lung and lymph nodes at the highest concentrations, but the more pronounced effect were found in rats exposed to the coarse material. likewise there was an increase of lavage parameters in rats exposed to thecoarse material but not to the np. these data demonstrate that inhalation of finer np is not necessarily associated with higher toxicity compared to the coarse material. the results were obtained with two organic particles of rather different size and composition but are in contrast to the more severe effects seen with several anorganic np when compared to the corresponding coarse particles. within the nanocare project a standard short-term inhalation test to examine the toxicity of inhaled aerosols from nanomaterials has been developed. the inhalation toxicity of nano-andpigmentary materials was studied: baso4, zno, ceo2, al-doped ceo2, zro2, amorphous silica, surface-coated amorphous silica, titania, carbon black and three multi-wall carbon nano tubes, all with complete phys-chem-characterization as planned for the oecd sponsorship program. quartz dust tio2 and zno were tested as pigmentary materials. rats were exposed nose-only to three concentrations of one of these materials, 6 h a day for five consecutive days. positive controls were exposed to quartz dust or pigmentary zno, negative controls to clean air. a wide range of endpoints for pulmonary toxicity were evaluated immediately after the last exposure and after 3 days and 3 weeks after the last exposure. among these parameters, polymorphnuclear granulocyte count in bronchoalveolar lavage fluid is the most sensitive early parameter indicating inflammation process in lung, while histological examination reveals the type and localization of inflammation. among these substances, we identified baso4 as having the lowest toxicity. all mwcnts were most potent in producing progressive inflammation in the lung; granulomas in lung and lung associated lymph nodes were observed without indication for fibrosis. the noaecs of the 16 substances ranged between < 0.1 and >50 mg/m 3 . generally the material was only found in the lung (surface and macrophages) and in the draining lymph nodes. surface modified amorphous silica was also found in the spleen. the data demonstrate that the method is able to differentiate the toxic potential of different nanomaterials and to indicate regression or progression of the effects effects. moreover the lung burden and potential translocation to other tissues was detecable. comparing the material properties and effects of the 16 materials, no general relationship between the toxicity and either particle size, specific surface area or aerosol particle number concentration was found. hence we must not expect to find a gerneral "nanotoxicology" or a unifying dosimetry for all nanomaterials. we must rather be prepared to test individual nanomaterials for their effects. and to develop grouping concepts not only based on material properties but also on biopersistence, biokinetics and biological effects. part of this studes has been sponsored by bmbf (nanocare). endpoint-centric search for toxicological information and data to support the information retrieval for regulatory programs landsiedel r. 1 , wächter t. the eu reach regulation no 1907/2006 requires industry to ensure the safety of chemical use and manufacturing. all substances manufactured or imported in quantities above one tonne per year must be registered. information requirements for the dossiers increase with increasing tonnage or once hazards are suspected. searching for substance specific literature and the compilation of hazard data for safety assessments are highly challenging procedures. the novel web-based search engine go3r, accessible free of charge at www.go3r.org, has been created to allow quickly finding relevant hazard information and data. go3r provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information. furthermore, go3r specifically highlights information on animal testing alternatives. search results are presented automatically linked to an "intelligent table of contents" which enables the user to sort the literature listed in pubmed or the toxicology data network (toxnet) in a fast and comprehensive manner. retrieved documents are automatically organized in categories relating to the iuclid 5 chapters. hereby, the user can browse directly through the entire 21 million documents without even having to start the search with an initial query. the semantically enriched platform supports the user during query formulation, allows for bibliographic analysis, and specifically highlights information related to the replacement, reduction, and refinement of animal experiments. search results in go3r are shown in an dynamic table of contents (left) making them browsable for the contained information on animal testing alternatives and toxicologogical endpoints. towards a differentiation therapy of acute myelogenous leukemia with histamine h2-receptor agonists laue s., burhenne h., seifert r. hannover medical school institute of pharmacology, carl-neuberg-str. 1, 30625 hannover, germany acute myelogenous leukemia (aml) is a devastating malignancy characterized by a differentiation block of myeloid progenitor cells. recently, histamine dihydrochloride in combination with interleukin 2 has been approved as orphan drug for the consolidation treatment of aml (1) . it is assumed that histamine exerts its effects by activating the histamine h2-receptor (h2r) in human neutrophils, resulting in improved anti-tumor function of t killer cells by inhibiting nadph oxidase-catalyzed superoxide formation. previous studies had shown that histamine also induces myeloid differentiation (2) . considering the fact that all-trans-retinoic acids constitutes a powerful differentiation therapy of acute promyelocytic leukaemia, a specific subtype of aml (3), we initiated a study to explore the possibility that h2r-mediated myeloid differentiation provides an alternative or complementary strategy to treat leukemias associated with differentiation blocks. as model system, we used hl-60 cells. in hl-60 cells, histamine and various h2-receptor agonists induced concentrationdependent increases in camp levels. interestingly, ligands differentially increased cytosolic calcium concentration and extracellular receptor kinase (erk) pathways, indicative for ligand-specific h2r conformations. h2r activation resulted in myeloid differentiation as assessed by enhanced formyl peptide receptor-mediated increases in cytosolic calcium concentration. h2r agonists showed no signs of cytotoxicity. intriguingly, following h2r activation, the majority of the formed camp was exported into the extracellular space via multi-drug resistance protein (mrp) 4, indicating that export is a more important pathway for signal termination than cleavage of camp by phosphodiesterases. despite effective camp export, even a short-term exposure (30 minutes) of cells was sufficient to induce expression of functionally active formyl peptide receptors. these data indicate that in contrast to previously held dogma, induction of myeloid differentiation does not require continuous presence of a camp signal. from a therapeutic point of view this is very important since "spike" therapy with campincreasing substances may be sufficient to induce a therapeutic effect in aml, thereby also reducing toxic side effects. currently, we are systematically exploring the effects of h2r agonists on signal transduction pathways and differentiation in various myeloid cell types to identify highly efficacious compounds. introduction. activation of gαq/11 protein-coupled receptors of postsynaptic neurons can elicit the production of endogenous cannabinoids (endocannabinoids), which in turn inhibit transmitter release from axon terminals by activating presynaptic cb1 receptors. the aim of the present experiments was to study the mechanism of the endocannabinoid production. specifically, we wanted to clarify the role of ca 2+ release from intracellular stores in triggering endocannabinoid production. methods. patch-clamp-and ca 2+ imaging experiments were performed on purkinje cells in mouse cerebellar brain slices. glutamatergic excitatory postsynaptic currents (eepscs) were elicited by electrical stimulation of parallel fibers. the gαq/11 proteincoupled metabotropic glutamate receptor 1 (mglur1) was activated by superfusion of (rs)-3,5-dihydroxyphenylglycine (dhpg) or -more physiologically -by burst stimulation of the parallel fibers. results. both dhpg superfusion and burst stimulation of parallel fibers elicited an increase in intracellular ca 2+ concentration in the postsynaptic purkinje cells. dhpg superfusion and burst stimulation suppressed eepscs, and this suppression was abolished in the presence of the mglur1 antagonist cpccoet. the suppression of the eepscs was also sensitive to the cb1 receptor antagonist rimonabant, pointing to involvement of endocannabinoids and cb1 receptors. the suppression of the eepscs was attenuated after depletion of the endoplasmic reticulum ca 2+ stores by thapsigargin, cyclopiazonic acid and ip3. the results indicate that after activation of the gαq/11 protein-coupled metabotropic glutamate receptor 1 (mglur1) of the postsynaptic neuron ca 2+ is released from the endoplasmic reticulum. this ca 2+ release significantly contributes to the production of endocannabinoids. the endocannabinoids diffuse in the synaptic cleft retrogradely to the terminals of afferent axons and inhibit transmitter release there through presynaptic cb1 receptors. the guanine nucleotide exchange factor dock9 controls reelin dependent cdc42effects on radial migration pichler m. 1 the regulation of blood glucose levels is under tight control of a complex system including hormone and neurotransmitter signalling. many of these cellular signalling pathways are initiated by binding of the ligand to a g-protein coupled receptor (gpcr), e.g. noradrenaline inhibits insulin secretion upon binding to a gi-coupled receptor. upon gpcr activation the heterotrimeric g-protein is activated and both the α-subunit and βγdimers are released and interact with their specific target proteins. by the usage of bordetella pertussis toxin (ptx) as a common gαi inhibitor gαi-dependent signalling pathways are interrupted which leads to increased insulin secretion, and significantly improves glucose tolerance. since the gαi-isoform specific roles in the regulation of glucose homeostasis are still debated we studied the glycemic control in gαi2-deficient mice. surprisingly and in contrast to the ptx data, glucose tolerance was unchanged in the gαi2-deficient mice compared to wild type controls. however, the plasma insulin levels were significantly reduced upon glucose challenge. these findings point to disturbed islets function and improved peripheral insulin sensitivity. analysing gαi2deficient islets we show that islet size and number of nuclei are reduced. nevertheless, in vitro insulin secretion is improved at low (3 mm) and high (16 mm) glucose concentrations and can be further stimulated upon ptx-treatment. these data indicate that gαi2 proteins influence islet development and inhibit insulin secretion. in addition, these findings support our hypothesis that gαi2-deletion influences peripheral insulin sensitivity. therefore, we investigated glucose homeostasis and pakt-levels after two hours feeding ad libitum in gαi2-deficient mice. under feeding conditions no differences in plasma insulin levels were visible although blood glucose levels were significantly reduced in gαi2-targeted mice. pakt-levels of liver and skeletal muscle were unaltered, whereas akt phosphorylation in white adipose tissue was significantly increased, indicating improved glucose uptake of adipocytes. in conclusion, gαi2 is a negative regulator of both insulin secretion and peripheral insulin sensitivity and important for the maintenance of glucose homeostasis. 11689, 1992) in a radioligand binding test and to determine their functional effects with a membrane potential test using the dye r7260 (molecular probes, 0.125 mg/ml, excitation 505 nm, emission 530 nm). the affinity of compounds in radioligand binding was slightly higher in sur2b than in sur2a-type channels, but the enantiomeric ratio in sur2a channels matched that one determined for the sur2b-type indicating some conformity of the binding pockets of sur2a and sur2b-proteins. surprisingly, however, the membrane potential tests revealed that the (3r,4s)-enantiomer acted as agonist (a) whereas the (3s,4r)-enantiomer acted as antagonist (b): (3r,4s)-bms-191095 induced membrane hyperpolarisation whereas (3s,4r)-bms-191095 repolarised cells prestimulated with submaximally effective concentrations of diazoxide. concluding, bms-191095 is not selective for sur2a as compared to sur2b-type k atp channels. its enantiomers activate and block sur2-type katp channels in a stereospecific manner. thieno-thiadiazine derivatives with full agonistic activity at sur2b-type katp channels act as partial agonists at cardiac sur2a-subtypes oldenhage c., grittner d., schmidt c., lemoine h. heinrich-heine universität, inst. für lasermedizin, mol. wirkstoff-forschung, universitätsstr. 1, 40225 düsseldorf, germany new potassium channel openers (kco) of the thieno-thiadiazine(ttd)-type initially developed as agonists for the sur1-type katp channels (nielsen et al., j med chem 45: 4171, 2002) were characterized in sur2b-type katp channels as agonists and antagonists, if r contains a quaternary (methyl-cycloalkyl) and a tertiary (r = cycloalkyl) carbon, respectively (lemoine et al., this journal 375, r45, 2007) . to investigate the selectivity of ttd-derivatives for myocardial katp channels the membrane potential actions of compounds were tested in hek 293(kir6.2/sur2a)-cells and compared to hek 293(kir6.1/sur2b)-cells as a model for smooth muscle-type katp channels. membrane potential was measured by fluorescence (excitation 505 nm, emission 530 nm) using 0.125 mg/ml of the dye r7260 (molecular probes). standard-kco induced hyperpolarisation with ~10-fold smaller potency (pec50) in sur2a. ttd-compounds with ch3-cycloalkyl residues not only lost potency but also intrinsic activity for channel activation (emax) in sur2a. possibly, this loss of emax would be much greater in native heart cells with a normal channel density. in contrast, ttd-compounds with cycloalkyl residues acted as antagonists of cells pre-hyperpolarized with diazoxide with similar affinity in sur2a and sur2b-type katp channels. concluding, selectivity of kco for katp channel-subtypes cannot only be achieved by a different affinity but also by a selective stimulation of the channel of interest. small-conductance calcium activated potassium (kcnn/sk/kca2) channels maintain neuronal calcium homeostasis, shape synaptic functions and prevent excitotoxic neuronal death. so far, little is known about the function of kca2 channels in nonneuronal cells. the aim of this study was to investigate the expression of kca2 channels in microglial cells and their potential function in microglial activation and maintenance. expression of kca2 channel subtypes in microglial cells was assessed by mrna analysis, western blots and immunocytochemistry. lipopolysaccharide (lps)-induced microglial proliferation was evaluated by the xcelligence impedance-based system and mtt assays, and immunogenic activation of microglia was determined by measuring cytokines and nitric oxide (no) release into the cell culture medium. the kca2.2 and kca2.3 channel activator cyppa (25 µm) and specific inhibitory peptides (50 µm) were applied to distinguish effects mediated by the kca2 channel subtypes. all kca2 channel subtypes were detected on mrna and protein levels in resting and in lps-activated microglial cells. xcelligence real-time measurements and mtt assays demonstrated that lps (200 ng/ml) induced microglial proliferation. the kca2.2/kca2.3 channel activator cyppa reduced lps-induced microglial proliferation in a concentration-dependent manner. specific peptide inhibitors of kca2.3 channels, but not of kca2.2 channels, reversed the cyppa-effects on lps-induced microglial proliferation. cyppa alone did not alter the production of tnf-alpha or il-6, but strongly reduced the lps-dependent cytokine production. interestingly, chelation of extracellular calcium by edta induced differential cytokine kinetics by decreasing lps-dependent il-6 production while tnf-a production was not affected. moreover, using inhibitory sk3/kca2.3 channel peptides, we demonstrated that sk3/kca2.3 channels modulate lps-induced cytokine il-6 production in a calcium-dependent manner, while the tnf-a release was independent of extracellular calcium. in summary, the present study revealed that kca2.3 channel stimulation reversed microglial activation. thus, kca2.3 channels may serve as a therapeutic target for reducing microglial activity and related inflammatory responses in cns diseases. (3r,4s)-(3s,4r)intracellular amyloid beta (aß) oligomers and extracellular aß plaques are key players in the progression of sporadic alzheimer disease (ad). still, the molecular signals triggering aß production are largely unclear. we asked whether mitochondria-derived reactive oxygen species (ros) are sufficient to increase aß generation and thereby initiate a vicious cycle further impairing mitochondrial function. complex i and iii dysfunction were induced in a cell model using the respiratory inhibitors rotenone and antimycin resulting in mitochondrial dysfunction and enhanced ros levels. both treatments lead to elevated levels of aß. presence of an antioxidant rescued mitochondrial function and reduced formation of aß demonstrating that the observed effects depended on ros. conversely, cells overproducing aß showed impairment of mitochondrial function such as comprised mitochondrial respiration, strongly altered morphology, and reduced intracellular mobility of mitochondria. again, the capability of these cells to generate aß was partly reduced by an antioxidant indicating that aß formation was also ros-dependent. moreover, mice with a genetic defect in complex i, or ad mice treated with a complex i inhibitor, showed enhanced aß levels in vivo. several lines of evidence show that mitochondria-derived ros result in enhanced amyloidogenic amyloid precursor protein processing, and that aß itself leads to mitochondrial dysfunction and increased ros levels. we propose that starting from mitochondrial dysfunction a vicious cycle is triggered that contributes to the pathogenesis of sporadic ad. comparison of methods to derive health-based guidance or limit values for chemicals licht o., voss j. -u., mangelsdorf i. fraunhofer item chemikalienbewertung, nikolai-fuchs-str. 1, 30625 hannover, germany health-based guidance or limit values are derived for chemicals to compare measured or estimated exposure concentrations with these values. if the exposure is below the limit value, adverse effect for human health can be regarded as negligible, e.g. the exposure is expected to be tolerable. in germany such values have been derived since years for chemicals that can be found in soil, water and air as well as human blood and urine (biomonitoring). in a research project sponsored by the german umweltbundesamt several methods used by the agency are compared to the method laid in reach guidance document r.8 to derive a derived no effect level (dnel). the aim was to identify possibilities for standardization as well as to figure out specific elements in individual methods. in addition to extrapolation factors the public availability of guidance and specific derivations as well as procedures for consensus on the limit value were evaluated. the comparison of extrapolation factors revealed that, although they are named differently such as extrapolation, safety or assessment factors, they are used in a comparable manner. factors for interspecies and intraspecies extrapolation are presented in more detail. the standard factor for such extrapolation is 10 in most cases. in the reach guidance this factor consists of a part for allometric scaling and remaining differences. other factors are only defined in some methods, like a factor for extrapolation from loael to noael, data quality or data gaps. a factor for data quality is not laid down in the basic scheme for setting of indoor air guidance values, but is used in some of the recent derivations of limit values. also the who guidelines for drinkingwater quality use comparable factors to account for adequacy of studies or database and nature and severity of effect. a transparent and documented derivation is necessary for acceptance of the value. the derivation methods as well as the evaluation document on a specific substance are available through publications or the internet in nearly all cases. for the dnel only the numeric value is available at the echa website, but not any information on starting point and extrapolation factors. although all guide or limit values are derived in a comparable way, differences, however, exist in some details. in most cases detailed explanation is lacking when deviating from standard or default assumptions. often such deviation is based on expert judgement. hepatocellular carcinoma (hcc) is the fifth most common cancer in the world and has a poor prognosis with limited therapeutic options. up to now, no curative systemic therapy exists emphasizing the high clinical importance of new therapies for hcc. therefore, the identification and characterization of novel drugable targets is a relevant goal. cyclin-dependent kinase 5 (cdk5) is well characterized for its function in cns development and disease. recently, few reports indicate functions of cdk5 in cancer. cdk5 was shown to regulate tumor growth, and our group discovered that cdk5 regulates angiogenesis. since hcc is a highly vascularized tumor and anti-angiogenic treatment (sorafenib) has shown some therapeutic benefit, we hypothesize that cdk5 is an interesting target for hcc therapy. the aim of this study was to characterize the function of cdk5 in hcc. histology of tissue micro arrays indicates an increased expression of cdk5 in human hcc tissue in comparison to healthy liver tissue of the same patient. to investigate the function of cdk5 in hcc, we analyzed the impact of both pharmacological inhibition of cdk5 and specific downregulation of cdk5 with rna interference on hcc cells. pharmacological inhibition of cdk5 with the small molecule roscovitine (r-roscovitine, seliciclib) decreased proliferation and clonogenic survival, induced g2/m cell cycle arrest and cell death, and reduced motility of huh7 and hepg2 cells. transient downregulation (sirna) and stable knockdown (shrna) of cdk5 also reduced proliferation, clonogenic survival, migration and invasion of huh7 cells. in a subcutaneous hcc xenograft model, treatment with roscovitine reduced tumor growth and angiogenesis, indicated by decreased tumor weight and volume, and reduced vessel density. moreover, cotreatment of hcc cells with roscovitine and tumor necrosis factor related apoptosis inducing ligand (trail) resulted in an over-additive additive effect on the induction of apoptosis. this coincided with reduced phosphorylation and activity of the anti-apoptotic transcription factor stat3 at ser727 that is directly phosphorylated by cdk5, and tyr705. in line with this, the expression of the antiapoptotic protein mcl-1 is reduced by inhibition of cdk5. our results point to an important function of cdk5 in hcc and suggest cdk5 as an interesting pharmacologically druggable target for hcc therapy. delivery of mono-biotinylated rnasea into macrophages with streptavidinconjugated clostridium botulinum c3 toxin lillich m. 1 , chen x. clostridium botulinum produces the adp-ribosyltransferase c3, which modifies and thereby inactivates exclusively the small gtp binding proteins rho-a,-b and -c. recently, we discovered a specific endocytotic internalization of c3 toxin in macrophages and myeloid leukaemia cells, but not in epithelial cells [1] . thus, c3 toxin provides a tool to target cells of the monocyte/macrophage lineage, which are involved in various diseases and are of great clinical interest. we used a biochemical crosslinking approach to design a delivery system based on an enzymatic inactive c3bot mutant (c3mut) and streptavidin. the c3 portion mediates uptake of the transporter into monocytes/macrophages and streptavidin allows for binding of biotinylated cargo molecules to the transporter. in vitro, the generated c3mut-streptavidin bioconjugate showed specific and concentration dependent binding to biotinylated oligonucleotides as demonstrated by electrophoretic mobility shift assay. cell fractionation experiments indicated an uptake of the bioconjugate into the cytosol of j774a.1 macrophages. in the next step, mono-biotinylated bovine pancreatic ribonuclease a (rnasea) was used as a model cargo for the delivery of macromolecules by the bioconjugate. rnasea is a highly stable, well studied protein which catalyzes the degradation of rna. mono-biotinylated rnasea interacts in a specific and concentration dependent manner with the c3mut-streptavidin bioconjugate in vitro as analysed with dot blot technique. the c3mut-streptavidin bioconjugate efficiently mediates the internalization of biotinylated rnasea into j774a.1 macrophages as analyzed with laser scanning microscopy in fixed cells. this finding was also confirmed by live cell imaging. furthermore, cell fractionation showed a cytosolic delivery of biotinylated rnasea in the presence of c3mut-streptavidin. as expected we could not observe a cytotoxic effect of biotinylated wild-type rnasea on j774a.1 macrophages, which is attributable to the presence of ribonuclease inhibitor protein in mammalian cells. in summary, the c3mut-streptavidin bioconjugate mediates the efficient internalization of biotinylated (macro)molecules into macrophage like cells, and therefore represents a useful tool for the transduction of exogenous molecules into macrophages. in addition, cytotoxic rnasea mutants are available and will be used in further studies. organometal compounds such as cisplatin or the second generation complexes carboplatin and oxaliplatin have become more and more important as antitumor agents. nevertheless there is still an increasing demand for novel metal-based compounds. this is necessary due to severe side effects and the occurence of resistent tumour cells. in this context we investigated the cytotoxic effects of imidazole-based phosphane gold(i) complexes as potential agents for cancer treatment. initially we have used the mtt-assay to examine the toxic potential of the gold complexes in h4iie rat hepatoma cells. in this context cw60 (a diphosphane ligand with azoyl substituents r2p(ch2)2pr2, r= thiazol-2-yl) turned out to be the compound with the highest cytotoxic potential with an ic50 value of 6,5mm (24h incubation). further investigations revealed that cw60 induced an apoptotic cell death in h4iie demonstrated by the activation of caspase 3/7 (48h incubation with 10mm cw60). in addition the induction of apoptosis was confirmed by the dna ladder formation (24h incubation with 5mm cw60). in connection with the molecular mechanisms of apoptosis induction we used the comet assay to analyse the generation of dna strand breaks as well as the dcf-assay to detect the formation of reactive oxygen species. however neither dna strand breaks nor increased levels of reactive oxygen species were detected after 1h of incubation. furthermore we analysed if the compound influences intracellular signalling pathways such as the jnk pathway and the pi3k/akt but after 24h of incubation neither pakt nor jnk were influenced. the imidazole based phosphane gold (i) complex cw60 shows strong toxic effects in h4iie cells and turned out to be a promising compound as a potential agent for cancer treatment. the high and inappropriate intake of loop diuretics in hypertensive elderly reported in former studies has again been confirmed. remembering that inappropriate intake of loop diuretics can lead to exsiccosis and electrolyte loss especially in elderly, better medical education has to follow these alarming results to improve the pattern of diuretic prescription. furthermore, our results lead us to assume a high estimated number of unreported cases of torasemide use in uncomplicated arterial hypertension in elderly. this loop diuretic agent shows a longer duration of action compared with furosemide (elimination half-life: 3-4 hrs vs. 1 hr) and is effective in decreasing blood pressure in subdiuretic doses. it must be pointed out that loop diuretics are still frequently inadequately prescribed because current guidelines recommend loop diuretics only in complicated arterial hypertension. the role of cgmp/cgki signaling and trpc channels in regulation of vascular tone loga f., domes k., hofmann f., wegener j. pharmakologie und toxikologie for 923, biedersteiner str 27, 80802 münchen, germany signaling by intracellular cgmp and cgmp-dependent protein kinase i (cgki) is the major pathway in vascular smooth muscle, by which endothelial no regulates vascular tone. recent evidence suggests that trpc channels are targets of cgki in smooth muscle and mediate, at least partially, the relaxant effects of cgmp. we tested this concept by investigating the role of cgmp/cgki signaling on vascular tone and peripheral resistance using cgki-, trpc6-, and trpc3-, and trpc3/c6-double knock-out mice. we found larger contractile responses to α-adrenergic stimulation in intact aorta from cgki-, trpc6-, and trpc3/c6-double knock-out mice as compared to aorta from ctr and trpc3-knock-out mice indicating a functional link between cgki and trpc6 channels. no differences were found if the vasodilator tone, provided by the no generation in the vascular endothelium, was inhibited by l-name. likewise, no differences were observed in the increase in peripheral resistance by α-adrenergic stimulation using the hind limb perfusion system. activation of cgki by 8-br-cgmp diminished aortic tone and peripheral resistance to a similar extent in control, trpc6-, trpc3-, and trpc3/c6-double knock-out mice. no effect of 8-br-cgmp was observed in preparations from cgki -/mice. to test the co-localization of cgki and trpc channels, we performed immunocytochemistry on isolated smooth muscle and endothelial cells from aorta of ctr, trpc3-, and trpc6-knockout mice. trpc3 could be detected in both smooth muscle and endothelial cells whereas trpc6 was only detected in endothelial cells. the results suggest that absence of cgki or trpc6 impairs the vasodilator tone induced by endothelial no production but that cgki and trpc6 channels are not functionally coupled in vascular smooth muscle. we thank profs birnbaumer (nih) and freichel (homburg) for providing us with trpc6 -/-, and trpc3 -/mice and prof. flockerzi (homburg) for the antibodies against the trpc channels. whole genome microarray analysis of the effects of tcdd and pcb 153 in human hepatic cell models lohr c. 1 , neser s. after the treatment with tcdd, however, a total of 281 genes were more than 2-fold up regulated in hepg2 e.g. cytochrome p450 1a1 (cyp1a1) (32-fold) a sensitive marker for ahr activation. additional up regulated genes in hepg2 were; arylhydrocarbon receptor repressor (ahrr) 15-fold, aldehyde dehydrogenase 3 a1 (aldh3a1) 11-fold, and cytochrome p450 1b1 (cyp1b1) 10-fold. 44 genes were more than 2-fold down regulated in hepg2 cells e.g. -proprotein convertase subtilisin/kexin type 9. markedly different findings were obtained in hheps, i.e., 117 genes were up regulated, the highest up regulated gene was cyp1b1 with a 95-fold increase in gene expression, followed by cyp1a1 (41-fold) and aldh3a1 (21-fold). only a small group of genes were significantly down regulated (17 in total), e.g., solute carrier family 2 (facilitated glucose transporter). comparing both human cell types, there was an unexpected small overlap of genes being up or down regulated. interestingly, in both cell types, only 30 in common genes were up regulated, including cyp1a1, cyp1a2, cyp1b1 and aldh1a3. only platelet-derived growth factor receptor, beta polypeptide, was down-regulated in both hepg2 and hheps. in conclusion, our data indicate pronounced differences in the patterns of tcdd-regulated genes between hepg2 cells and hheps. detection of redox modified proteins in nociceptive processing lorenz j. e. 1 , kallenborn-gerhardt w. recent data indicate that redox modifications of proteins induced by reactive oxygen species (ros) contribute to sensitization of pain pathways during persistent pain. however, little is known about the targets of ros in pain processing, because the relatively unstable nature of many reversible protein oxidation states hampers the reliable detection and identification of modified proteins. here, we used the quantitative thiol trapping technique termed oxicat to identify proteins which are redox modified during nociceptive processing. we investigated spinal cords of untreated mice, after zymosan injection into a hindpaw (inflammatory pain model) and after spared nerve injury (neuropathic pain model). we identified several proteins with marked changes in their redox states after nociceptive stimulation. our results show that the oxicat method is an efficient method to detect redox modifications in proteins and that redox modifications seem to play a role in pain processing. supported by the deutsche forschungsgemeinschaft (sfb 815/a14). additive antinociceptive effects of a combination of vitamin c and vitamin e after peripheral nerve injury lu r., kallenborn-gerhardt w., geisslinger g., schmidtko a. pharmazentrum frankfurt/zafes institute of clinical pharmacology, goethe university, frankfurt am main, germany accumulating evidence indicates that increased generation of reactive oxygen species (ros) contributes to the development of exaggerated pain hypersensitivity during persistent pain. in the present study, we investigated the antinociceptive efficacy of the antioxidants vitamin c and vitamin e in mouse models of inflammatory and neuropathic pain. we show that systemic administration of a combination of vitamins c and e inhibited the early behavioral responses to formalin injection and the neuropathic pain behavior after peripheral nerve injury, but not the inflammatory pain behavior induced by complete freund's adjuvant. in contrast, vitamin c or vitamin e given alone failed to affect the nociceptive behavior in all tested models. the attenuated neuropathic pain behavior induced by the vitamin c and e combination was paralleled by a reduced p38 phosphorylation in the spinal cord and in dorsal root ganglia, and was also observed after intrathecal injection of the vitamins. moreover, the vitamin c and e combination ameliorated the allodynia induced by an intrathecally delivered ros donor. our results suggest that administration of vitamins c and e in combination may exert synergistic antinociceptive effects, and further indicate that ros essentially contribute to nociceptive processing in special pain states. -206, -213 and -214) replaced by leucine residues. both amino acids are comparable in terms of hydrophobicity, volume and the preference for forming α-helices, but only methionine is oxidizable to a sulfoxide, in contrast to leucine. in the present study we examined the protein-protein interaction (ppi) of recombinant ac 1, expressed in sf9 insect cell membranes, with cam, cam-206, -213, -214 and -215 by measuring the catalytic activity of ac 1. cam-mutants show a 3-4-fold lower potency than cam, but they are more efficacious than cam. most prominently, cam-215 was 133 % more efficacious than cam. such striking differences between cam and cam-mutants have not yet been observed for other mammalian effector proteins. as a result of the exchange of all methionine against leucine residues in cam-215, it is more hydrophobic than cam and this leads to a better ppi with ac 1. in future studies we will examine the effects of cam inhibitors, antidepressants and antipsychotics on cam/ac 1 interaction. furthermore we will analyze the effects of oxidized cam and cam-mutants on the catalytic activity of ac 1. because oxidative stress is of great importance in aging, it is important to know more about the abovenamed interaction in view to the demographic change. taken together, our data point to a unique cam/ac 1 interaction that may be selectively targeted by small molecules. in particular, enhancers of these interaction could be useful to improve memory and learning. gender differences in fat distribution and diabetes prevalence in nzo mouse lubura m., scherneck s., zucker a., schürmann a. deutsches institut für ernährungsforschung experimentelle diabetologie, arthur-scheunert-allee 114-116, 14558 potsdam, germany background: excessive fat accumulation in visceral but not subcutaneous fat depots as well as ectopic fat storage in liver, skeletal muscle and pancreas are associated with an increased risk for the development of type 2 diabetes in humans. in this study we aimed to examine the influence of early fat distribution on onset of type 2 diabetes in mice. methods: nzo mice are regarded as insulin resistant model in which only males become diabetic. we used male and female mice fed with high-fat and standard diet. we determined fat distribution by computed tomography for three times and conducted oral glucose tolerance tests on two different time points. besides we assessed body weight and blood glucose levels on weekly basis. results: contrary to previous findings, we observed that not only male nzo mice on high-fat diet develop diabetes. blood glucose levels at the 16 th week of age and total pancreatic insulin content indicated diabetes prevalence of 68% in males and 25% in females these results lead to the conclusion that high-fat diet counteracts protective action of estrogens against diabetes. inversely to the findings in humans, female mice tend to store more fat in abdominal region than males. there was no relationship between early accumulation of fat in abdominal region and onset of type 2 diabetes. however, visceral fat was associated with liver fat in males as well as in females. furthermore, at the age of ten weeks hepatic fat content correlated with blood glucose levels (r² = 0.69) indicating that the early hepatosteatosis is a predictor for hyperglycemia. however, there was no correlation between hepatic insulin sensitivity (indicated by quantitative insulin sensitivity index-quicki) and amounts of hepatic fat we conclude that early hepatosteatosis does not predict for glucose intolerance in nzo mice. in the nzo mouse, the amount of liver fat but not the early fat distribution predicts for the later onset of type 2 diabetes. further experiments are needed to examine the gender dependent differences in the diabetes prevalence of this mouse strain. with a prevalence of about 20-30% non-alcoholic fatty liver disease (nafld) represents the most common liver disorder in europe. nafld manifestation ranges from steatosis through steatohepatitis (nash) to fibrosis and cirrhosis, followed in some cases by liver failure and hepatocellular carcinoma. fatty degeneration of liver cells, increased oxidative stress with concomitant lipid peroxidation and an induction of pro-inflammatory cytokines are proposed as possible causes for developing inflammation and fibrosis, but the exact pathogenesis of the progression of nafld into nash is still unknown. thus, besides life style modifications and weight reduction interventions, no established pharmacological therapy exists so far. to gain further insights into the pathogenesis of nafld and nash and to develop new therapeutic strategies, appropriate animal models are essential. thus, in the present study three different dietary animal models for nafld were evaluated and compared to the biochemical and metabolic alterations seen with nafld and nash in man. male adult lewis rats were given standard food or one of three different diets: fatty liver diet [fld] , methionine/choline deficient diet [mcd] or methionine/choline deficient plus high fat diet [mcd+hf] . after 1, 2, 4, 6 or 12 weeks of treatment, animals were sacrificed and body and liver weights, laboratory parameters (asat, alat) as well as histopathological changes in the livers and different parameters indicating oxidative stress or representing the biotransformation capacity of the livers were analyzed. with fld and mcd+hf a normal body weight gain was observed, whereas with mcd body weight gain was strongly impaired. liver weights were mainly increased after mcd+hf. elevation of asat and alat values and hepatic steatosis were more pronounced after mcd and mcd+hf than after fld. all three diets caused an increase in the oxidative stress in liver tissue, but especially with mcd a tremendous elevation in the hepatic levels of lipid peroxidation products was seen. with regard to liver biotransformation capacity, with all three diets mainly an induction of the cytochrome p450 2e1 and 4a1 isoforms expression and activity was observed, which was most pronounced after mcd and mcd+hf. in summary, the changes induced by mcd or mcd+hf most closely resemble the alterations described in literature for nafld in man and thus should be preferred over fld in future investigations on nafld and nash. ep3 receptors for prostaglandin e2 convey stimulatory and inhibitory effects. e.g., their stimulatory effect leads to vasoconstriction in the human pulmonary artery and their inhibitory activity to reduction of neurotransmitter release from neuron endings. the aim of our study was (1) the pharmacological characterization of ep3 receptors in human pulmonary arteries and (2) the examination of the involvement of these receptors in the regulation of the neurogenic tachycardia in pithed rats. l-826266 served as the ep3 antagonist. experiments were performed in human pulmonary arterial rings isolated from patients undergoing lobectomy during resection of lung carcinoma and in pithed and vagotomised rats. the ep1/ep3 agonist sulprostone (1 nm -100 mm) concentrationdependently contracted human pulmonary artery rings (pec50 and emax; 6.89±0.12 and 106.5±5.2%, relative to the contraction induced by kcl 60 mm). the concentrationresponse curve of sulprostone was not affected by the ep1 antagonist sc 19920 (10 µm) but shifted to the right by l-826266 (10 µm) (apparent pa2 6.22). extending the exposure time to l-826266 from 0.5 to 3 h increased its antagonistic potency to 7.39 (schild plot-based pa2; concentrations 0.1, 1 and 10 µm). in pithed rats electrical stimulation (0.66 hz, 1 ms, 50 v for 5 s) of the preganglionic sympathetic nerve fibers or intravenous isoprenaline (0.15 nmol/kg) increased heart rate (hr) by 55 beats/min. sulprostone (10 -1000 nmol/kg) did not affect the isoprenaline-induced increase in hr but inhibited the neurogenic tachycardia dose-dependently, maximally by 80%. l-826266 (3 µmol/kg) diminished the inhibitory effect of sulprostone 1000 nmol/kg on the neurogenic tachycardia by 20%. in conclusion, ep3 receptors (1) located postsynaptically strongly contract human pulmonary arteries and (2) located presynaptically on sympathetic nerve fibres supplying the heart of rats strongly inhibit the neurogenic tachycardia. -5-bromo-n[3-(5voltage-gated ca 2+ channels of the central nervous system control a multitude of ca 2+ dependent processes such as neurotransmitter release, neuronal excitability, neurite outgrowth, synaptogenesis, plasticity and neuronal survival. the cav2.1 ca 2+ channelalso known as p/q-type channel -belongs to the subfamily of high voltage activated ca 2+ -channels. ca 2+ influx via cav2.1 ca 2+ channels located at presynaptic nerve terminals triggers vesicle fusion and transmitter release at brain synapses and at the neuromuscular junction. thus, cav2.1 ca 2+ channels play a crucial role in synaptic transmission. the global cav2.1 knock-out phenotype is characterized by severe ataxia, dystonia and lethality during the first postnatal weeks and is therefore an unsuitable model to analyze the importance of cav2.1 ca 2+ channels for learning and memory. therefore, we crossed a floxed cav2.1 mouse line with nex-cre transgenic mice to establish a viable, forebrain specific knock-out mouse line (fbko-mice). results from western blot analysis confirmed an efficient knock out of cav2.1 in hippocampal and cortical preparations, whereas the expression level in the cerebellum was not altered. to investigate the specific role of cav2.1 channels in hippocampus and neocortex dependent behavior, we performed tests for motor functions and sensory abilities and in particular learning and memory tasks. mice with a forebrain specific cav2.1 knock-out show significant deficits in spatial learning & reference memory and a significant reduced recognition memory as revealed by the morris water maze and an object recognition task. the fbko-mice exhibit no obvious locomotor deficits during behavioral tasks in the open field test and elevated plus maze. some fbko-mice demonstrate episodes of seizures in the morris water maze and during different rotarod tasks. to assess motor-function of fbko-mice in a stress reduced environment, we performed home cage based running-wheel motor-learning tasks. in summary, the diverse phenotypes of the forebrain specific knock-out mouse line emphasize the critical importance of cav2.1 for learning and memory. helicobacter hepaticus-infected rag2 -/mice emulate many aspects of human inflammatory bowel disease (ibd), including the development of colitis and colon cancer [erdman et al., 2009 , pnas 106: 1027 -1032 . toward the goal of elucidating mechanisms of inflammation-induced carcinogenesis and developing biomarkers of inflammation, we undertook a comprehensive analysis of macromolecular damage products during disease progression in h. hepaticus-infected rag2 -/mice. infected mice developed severe colitis and hepatitis, accompanied by infiltration of myeloperoxidase-positive neutrophils and f4/80-positive macrophages, by 10 wks postinfection (pi), progressing into colon carcinoma by 20 wks pi. qpcr array-based gene expression profiling revealed that pathophysiological changes were associated with characteristic alterations in the expression of genes related to inflammation, dna repair, and oxidative stress response. to study inflammation-related macromolecular damage, colon and liver tissues were analyzed by isotope-dilution chromatography-coupled mass spectrometry to quantify a battery of 16 different dna, rna and protein damage products thought to represent the full spectrum of inflammation-related chemistries. our data revealed a significant predominance of chlorinated dna-, rna-, and protein damage products by 20 weeks pi. in contrast, levels of damage products arising from oxidation, nitration and nitrosation changed only modestly or remained unchanged. our analyses also revealed higher levels of damage products in rna than in dna and demonstrated organ-specific differences of oxidative damage products, such as 8-oxo-dg and its oxidation products spiroiminodihydantoin and guanidinohydantoin. collectively, these results suggest that neutrophil and myeloperoxidase-induced chlorination chemistry may serve as a biomarker of ibd and may play important roles in the pathophysiology of ibd and colitis-associated cancer. characterization of a membrane protein expressed in mouse heart and brain mannebach s. 1 recently, a novel membrane protein in drosophila was shown to be localized in presynaptic vesicles. it appears to mediate a ca influx after vesicle fusion with the plasma membrane. disruption of the corresponding gene leads to endocytic defects in drosophila [1] . apparently, this protein plays a role in exo-and endocytosis and could serve as a ca channel supplying ca required for endocytosis. we have identified a protein in mouse, c90rf7, which shares 26,3% amino acid sequence identity with the drosophila protein. it covers 171 amino acid residues. using rt-pcr the full length transcripts could be identified in brain, kidney, pancreas, heart, spleen, thymus and mast cells. coexpression of c90rf7 and the ca v2.2 channel in xenopus oocytes reduced the amount of the α1b and cavβ3 subunits of the ca 2+ channel in the plasma membrane but did not affect the gating properties of the cav2.2 channel. expression of c90rf7 alone did not yield any channel activity. we therefore started to produce recombinant protein using the his-sumo-prokaryotic expression vector. the protein was efficiently expressed as his-sumo-c9orf7-fusion in e.coli (yield 2mg at 1mg/ml). we are currently preparing the c9orf7 part of the his-sumo-c9orf7fusion protein by ulp1-protease digestion followed by various chromatographic steps. the purified recombinant protein will be used to immunize rabbits to get antibodies. in parallel we generated antisera by immunizing rabbits with peptide fragments derived from the c9orf7 sequence. we could not identify any homologues of c9orf7 in the mouse genome and to analyze its function we are currently generating c9orf7 deficient mouse lines by gene targeting. we have chosen a strategy for conditionally inactivation of the gene with the option to study the cellular localization of c9orf7 by expression of the bgalactosidase gene under the control of the endogenous c9orf7 promoter. by southern blot analysis we´ve already identified 210 homologous recombinant embryonic stem cell clones out of 300 analyzed ones and we will proceed with blastocyst injection to get chimeric mice and finally mice carrying the introduced mutations in the c90rf gene. parps are involved in various biological processes such as regulation of dna repair, cell cycle progression, and cell death. consequently, several parp inhibitors are currently in clinical development as chemo-and radiosensitizers as well as monotherapeutic agents following the concept of synthetic lethality. pharmacological and toxicological studies call for an accurate analysis of parp activity in terms of a detailed knowledge of the structure of par and a reliable method for its quantification. we have developed a sensitive, precise, and accurate bioanalytical method based on liquid chromatography coupled to electrospray tandem mass spectrometry (lc/ms-ms) to characterize and quantify par with femtmol sensitivity: par is extracted from cells and hydrolysed to specific monomeric units, i.e., ribosyladenosine, which is characteristic for linear par, diribosyladenosine, which is characteristic for branching points, and adenosine, which represents the terminal part of the polymer. using this method, we are currently analyzing par levels in different cell lines and in primary human peripheral blood mononuclear cells (pbmcs) both under physiological conditions as well as upon genotoxic stress and in the presence of potent parp inhibitors. we expect that after completing method validation this assay will be useful for a wide range of applications in pharmacology and toxicology. gene mutagenic potential and metabolite profile of 17β-estradiol in cultured v79 cells expressing human cytochrome p450 1a1 martínez jaramillo d., lehmann l. university of wuerzburg, institute of pharmacy and food chemistry section of food chemistry, am hubland, 97074 wuerzburg, germany oxidative metabolism of the female sex hormone 17β-estradiol (e2) is considered to play a major role in the initiation of hormone-induced carcinogenesis. in extrahepatic tissues, e2 undergoes metabolic activation by cytochrome p450-dependent monooxygenase (cyp) isozyme 1a1 to 2-hydroxy-(2-ho) and to a lesser extent to 4-ho-e2. if not conjugated, these catecholestrogens (ce) can further oxidize to electrophilic quinones (q), which may react with dna and induce thereby mutations. conjugation of these ce in extrahepatic tissues is mainly catalyzed by catechol-omethyltransferase. in order to identify possible mutagenic metabolites (i) the induction of gene mutations by e2 was determined in male chinese hamster lung fibroblasts (v79 cells) expressing human (h) cyp1a1 and (ii) the metabolite profile of e2 in these cells was analyzed via gas chromatography/mass spectrometry after solid phase extraction of the cell suspension in the culture medium. (i) gene mutations were assessed using the hypoxanthine-guanine phosphoribosyltransferase assay. the promutagen benzo[a]pyrene (bap) served as positive control requiring metabolic activation by hcyp1a1 and dimethylsulfoxide as solvent control. v79 hcyp1a1 were treated with 100 nm e2 for 3 weeks and the resulting 6-thioguanine (6-tg) resistant mutants selected at weeks (w) 2 and 3. the frequency of spontaneous 6-tg resistant mutants per 10 6 colony-forming cells ranged from 5 ± 1 (w2) to 9 ± 4 (w3). as expected, 0.25 µm bap induced a significant increase in mutant frequency (mf, 266 ± 13, w2 and 132 ± 46, w3) . treatment with 100 nm e2 resulted in a 3-fold (17 ± 2, w2) and a 2-fold (23 ± 4, w3) increase in mf, suggesting slight mutagenic activity. in culture medium of v79 hcyp1a1 treated with 100 nm e2, 2-ho-e2, 2-methoxy-(meo)-e2, 3-o-methyl-2ho-e2 and 4-meo-e2 (suggesting intracellular formation of 4-ho-e2) were detected. while 4-meo-e2 concentration remained constant over the exposure period, the concentration of the other metabolites increased in a timedependent manner. the maximum concentration increase was reached at w3 for methylcatechols and at w2 for 2-ho-e2, correlating with the maximum increase in mf, observed after 2 weeks as well. in conclusion, e2 possessed a slight mutagenic potential after hcyp1a1-mediated activation to 2-, 4-ho-e2 and their corresponding methylcatechols. cumulative effects of three triazole fungicides in a broad dose range in vitro rieke s., kneuer c., bumke scheer m., lampen a., hirsch-ernst k., marx-stoelting p. bundesinstitut für risikobewertung chemikaliensicherheit, max-dohrn-str., 10589 berlin, germany consumers are exposed to multiple residues of different pesticides via the diet. this raises questions concerning potential cumulative effects, especially for substances causing toxicity by a common mode of action. the aim of this work was to investigate potential combination effects of the three triazole fungicides epoxiconazol, tebuconazol and flusilazol for selected parameters in a broad dose range in vitro. parameters investigated were cytotoxicity, hormone synthesis (17-β estradiol, progesterone and β-hcg), expression of a panel of androgen-or estrogen-responsive genes in the human placental choriocarcinoma cell-line jeg-3 and transactivation via estrogen receptors α and β in stably-transfected hek 293 cells. the ability to inhibit steroidogenesis was analysed by measuring the concentrations of 17β-estradiol and progesterone in cell culture supernatants of jeg-3 cells. additionally, the placental peptide hormone β-hcg was measured. while no change in β-hcg and 17β-estradiol concentrations were observed, all triazoles induced a dose-dependent decrease in progesterone concentration and a cumulative effect was observed implying dose additivity at individual doses of >1.7 µg triazole/ml. significant activation of erβ by the three triazoles, especially by flusilazol, was observed at 5 µg triazole/ml and combined exposure showed additive effects, while no significant activation of erα was observed. based on the data, our findings suggest dose-additivity of triazole pesticides with the same mode of action for selected parameters in vitro. no significant effects were observed at lower doses [1ng -1µg triazole/ml] neither for substances applied individually nor in combination. transient receptor potential channels as mediators of catecholamine release mathar i. 1 trp proteins form cation channels that are regulated through strikingly diverse mechanisms. recently, genetic association studies identified many trp genes including trpm4 as risk factors for disease states such as arrhythmias, hypertension and cardiomyopathy. the melastatin trp channels trpm4 and trpm5 have distinct properties within the trp channel family; they form non-selective cation channels activated by intracellular calcium ions and are expressed in heart, aortic endothelial cells, kidney and adrenal gland. disruption of the trpm4 gene in mice leads to increased basal blood pressure without evidence for impairment of endothelium-or smooth muscle-dependent regulation of contractility of peripheral resistance vessels, the renin angiotensin aldosterone system, basal cardiac output or body fluid homeostasis. instead, trpm4-deficient chromaffin cells exhibit increased acetylcholine-induced exocytosis of catecholamines which is associated with elevated level of epinephrine in the plasma and its metabolites in the urine. this indicates that trpm4 serves as an inhibitory regulator of exocytotic catecholamine release, at least in chromaffin cells. whether catecholamine release is also regulated by trpm4 in other cells of the sympathetic nervous system such as perivascular neurons still needs to be clarified as well as the molecular mechanism underlying how trpm4 regulates catecholamine release. besides trpm4 we recently identified transcripts encoding additional trp channels including trpc5 and trpc6 in chromaffin cells isolated by laser capture microdissection but their functional role in these cells is still unknown. measurements of the time course of the intracellular calcium concentration before and during acetylcholine stimulation (10µm) of catecholamine release as well as the analysis of the number of released vesicles in chromaffin cells relvealed no changes in trpc1/c5/c6 triple knock out mice compared to wildtype controls. although it seems that these trpc proteins are not directly involved in catecholamine release from chromaffin cells induced by acetylcholine application in our hitherto existing experiments, their contribution to the modulation of catecholamine release by agonists of gq-coupled receptors still needs to be analysed. aims: sulfonylureas (sus) are among the most widely used oral hypoglycaemic drugs that stimulate insulin secretion. in addition, sus have pleiotropic effects on other tissues. regarding the effects of sus on adipocytes conflicting findings were reported. we have now investigated the actions of glimepiride and glibenclamide (=glyburide) in primary human adipocytes. methods: primary cultured human white pre-adipocytes were differentiated in vitro according to a standard protocol. lipid accumulation was assessed by oil red o staining and determination of triglyceride content; gene expression was measured by real-time pcr and western blotting. results: we initially characterized the genes regulated during human preadipocyte differentiation by a global microarray analysis. treatment with glimepiride and glibenclamide caused a strong accumulation of lipid droplets and an increase in triglyceride content. genes involved in lipid metabolism were induced, chemokine expression was decreased. interestingly, the effects of sus were over all qualitatively and quantitatively similar to pioglitazone. in direct comparison glibenclamide was more potent than glimepiride in respect to the induction of fabp4 (ec 50 0.32 vs. 2.8 µm), an important adipocyte marker gene. su-induced differentiation was virtually completely blocked by the pparγ-antagonist t0070907 but not affected by diazoxide, indicating pparγ activation by sus. repaglinide, causing insulin liberation like sus but being structurally different, had no effect on adipocytes. conclusions: in primary human pre-adipocytes, glibenclamide and glimepiride strongly induced differentiation, apparently by activating pparγ . thus, sus but not repaglinide may be used to influence insulin resistance beyond their effect on insulin liberation. the role of at1a and at1b receptors as mechanosensors in myogenic vasoconstriction blodow s. 1 , schneider h. arterial myogenic tone denotes the intrinsic property of vascular smooth muscle cells to constrict in response to an elevated intraluminal blood pressure. this physiological reaction is more distinct in small resistance arteries than in large conduit arteries. understanding the underlying mechanisms should provide useful information for the treatment of diseases like anaphylactic shock and systemic hypertension in which this reaction is altered. whereas the underlying signaling cascade has been extensively studied, the molecular identity of the mechanosensory elements still remains elusive. recent studies at the cellular level suggest a sensory function for a subgroup of gprotein coupled receptors (gpcrs) coupling to gq/11-proteins. by determining mrnaexpression levels of selected gpcrs in consecutive pairs of resistance and conduit vessels, we could identify a subset of gq/11-coupled receptors such as angiotensin ii at1b, vasopressin v1a, endothelin eta and etb and α1a adrenoceptor significantly enriched in resistance vessels. by pharmacological blocking of those highly expressed gpcrs by different antagonists and inverse agonists, we evaluated their influence on the formation or the intensity of myogenic tone, as measured in isolated murine mesenteric arteries ex vivo. while blocking of v1a receptor and α2a and α2ab adrenoceptors showed no differences of myogenic tone, blocking of at1a and at1b receptors by losartan and candesartan, eta receptor by bq123 and α1a adrenoceptor by prazosin caused significant reductions of the vascular response. analyzing the myogenic response of at1a -/mice with and without additional blocking of at1b receptors by candesartan suggested that especially at1b receptors play a dominant role for mechanosensitivity in mice. this was further supported by investigating the myogenic response of at1b -/mice. these findings suggest that mechanosensitive gq/11-protein coupled receptors, especially at1b receptors, play a dominant role for the development of myogenic vasoconstriction. trpm3 ion channels are activated by steroidal compounds and noxious heat and are considered to be involved in insulin secretion and pain perception. the expression of the trpm3 gene generates a variety of different transcripts which arise by alternative splicing and the use of different promoters [1] . they encode a substantial variety of isoformes and so far we have identified more than 20 distinct trpm3 proteins in mouse and rat each varying in exons 1, 2, 8, 13, 15, 17 and 24 . these variants differ enormously in their biophysical properties. for example splicing within exon 24 affects the channel pore and causes significant changes of the ionic selectivity of trpm3 channels [2] , whereas splicing of 54 nucleotides encoded by exon 13 leads to dormant trpm3 proteins. however, the frequency of these different isoformes in trpm3 expressing tissues is completely unknown. to get insight into the significance of the different trpm3 isoformes we investigated the abundance of alternative trpm3 transcripts in different tissues and cell types by reverse transcription quantitative pcr (rt-qpcr). we found that the frequency of splicing within exon 13 ranges from 5 up to 20 % in different cell types and tissues. furthermore we analyzed the trpm3 transcriptome in the choroid plexus of the brain and the pituitary gland, tissues in which trpm3 transcripts are most abundant. for that purpose we sequenced more than 120 clones, each. corresponding to our rt-qpcr result, we found a significant number of transcripts lacking exon 13. in cells of the choroid plexus nearly all (124 /126 clones) carried the short ca 2+ permeable pore. furthermore, we identified seven variants spliced in exon 20 encoding truncated trpm3 proteins. however, the composition of the trpm3 transcriptome in the choroid plexus and pituitary gland differed enormously, indicating the importance of alternative splicing for trpm3 function in different tissues. the concept of "thresholds of toxicological concern" (ttc) defines tolerable dietary intakes for chemicals without toxicity data and is widely applied to chemicals present in food in low concentrations such as flavorings. based on a statistical evaluation of the results of many toxicity studies and considerations of chemical structures, the ttc concept derives a maximum daily oral intake without concern of 1800, 540 or 90 µg/person/day for non-genotoxic chemicals depending on the allocation to so-called cramer classes i, ii or iii. for substances with a structural alert for genotoxicity a ttc value of 0.15 µg/person/day might be used. recently, it has been investigated, whether the ttc values, which were derived based on mostly chronic oral dietary rodent studies would cover all relevant toxicities (neurotoxic, repeated dose, reproductive and developmental, immune effects and endocrine-related effects). several authors using different specific databases have confirmed that the ttc values derived using cramer classes are also covering immunotoxic, neurotoxic, reproductive and developmental effects. a respective decision tree is going to be presented, also considering substances or substance classes which shall be excluded from the ttc approach. there are several areas in which the ttc concept is already used, or a ttc approach is considered useful, to assess low-level human exposures, or help in prioritizing toxicological testing; as for example the assessment of plant metabolites and degradates of pesticide active substances, feed and food additives, chemicals with a low exposure profile under reach, residues, metabolites and impurities in plants, chemicals, plant protection products or pharmaceuticals. if no structural alert for genotoxicity is given or standard genotoxicity tests are negative the cramer class iii value of 90 µg/person/day, which corresponds to a dose of 1.5 µg/kg bw is considered to represent a chronic tolerable daily intake of the test substance. examples for current and future uses of the ttc concept in regulatory toxicology are presented. objective: hyaluronan (ha), synthesized by three ha-synthases (has1, -2, -3), is a prominent matrix component of atherosclerotic lesions. the aim of the present study was to identify the has isoenzyme that is associated with ha-matrix remodeling in inflammatory regions of atherosclerotic plaques. furthermore the underlying regulatory pathways were determined and functional aspects of this regulation in vascular smooth muscle cell (vsmc) were addressed. methods and results: during atherosclerosis in apoe deficient mice the peak of macrophage invasion at 14 weeks coincided with ha deposition and induction of has3 in aortic root plaques. in human symptomatic carotid artery plaques has3 was by far the most prominent has isoenzyme as determined by quantitative real time rtpcr. in vitro, in human vascular smooth muscle cell (vsmc) has3 was specifically induced via activation of nfkb by interleukin-1β (il-1β) and tumor necrosis factor alpha (tnfa) as shown by chip assay and utilization of nfkb inhibitor bay 11-7082. has3 was also upregulated in a co-culture system by activated macrophages via paracrine release of tnfa and il-1β as verified by neutralizing antibodies. in human atherosclerotic lesions nfkb positive vsmc were frequently detected in close proximity with ha and f4/80 positive macrophages as shown by immunohistochemistry. to study the effects of has3 mediated ha synthesis in human coronary vsmc, lentiviral overexpression and knockdown of human has3 were employed. overexpression of has3 resulted in increased migration and proliferation whereas knock down had the opposite effect. the effects of has3 were mediated by both pi3k signaling and mapk signaling via hyaluronan receptors cd44 and rhamm. conclusion: the present results suggest that has3-dependent ha synthesis is induced in human vsmc by inflammatory cytokines released from activated macrophages. moreover, has3-mediated ha production induced phenotypic activation of vsmc. pulmonary inflammation and airway remodeling are major features of chronic obstructive lung disease (copd). in addition, pulmonary hypertension is a common comorbidity, which is associated with a poor prognosis of the disease. recent studies in a guinea pig model of allergic asthma have shown that increased arginase activity, which converts larginine into l-ornithine and urea and competes with nitric oxide synthases for the common substrate, contributes to allergen-induced airway inflammation, hyperresponsiveness and remodeling. there is evidence that cigarette smoke and lipopolysaccharide (lps), both involved in the pathogenesis of copd, increase the expression of arginase, however, its role in the pathogenesis of copd is currently unknown. this study aimed to investigate the role of arginase in pulmonary inflammation and remodeling, using a guinea pig model of lps-induced copd. to this aim, guinea pigs were instilled intranasally with lps or saline twice weekly for 12 weeks and were pretreated by inhalation of the arginase inhibitor (2)s-amino-boronohexanoic acid (abh) or pbs. repeated lps exposure increased lung arginase activity, resulting in increased lornithine/l-arginine and l-ornithine/l-citrulline ratio's. both ratio's were reversed by abh treatment. repeated lps exposure also induced increased il-8 levels, neutrophils, goblet cells, hydroxyproline and airway collagen content in the lung, which were all abrogated by abh. moreover, repeated lps exposure increased right ventricular mass, indicative of pulmonary hypertension, which was similarly prevented by abh. in conclusion, increased arginase activity contributes to pulmonary inflammation, airway remodeling and right ventricular hypertrophy in a guinea pig model of copd, indicating that arginase inhibitors may have therapeutic potential in the treatment of this disease. (supported by msd). behavioral abnormalities in hcn3-deficient mice michalakis s., schöll-weidinger m., mader r., cao-ehlker x., fenske s., wahl-schott c., biel m. center for integrated protein science munich (cipsm) department of pharmacy -center for drug research, ludwig-maximilians-universität münchen, butenandtstr. 5-13, 81377 münchen, germany hcn3 encodes a hyperpolarization-activated and cyclic nucleotide-gated channel, which is expressed in various brain regions including thalamic, hypothalamic and habenular nuclei as well as brain stem and olfactory bulb. in this study we performed a comparative analysis of hcn3 -/and hcn3 +/+ mice using a battery of behavioral tests and telemetric biopotential measurements to evaluate a potential role of hcn3 in central nervous system function. in general, the knockout mice showed normal motor function as assessed by the rotarod and open field tests. telemetric home cage activity and core body temperature measurements confirmed a normal circadian behavior, but revealed a lower basal activity that concurred with decreased body temperature during the light phase and the light-dark transition phase. hippocampus-dependent spatial learning was normal. by contrast, hcn3 knockout mice showed more immobility than control mice on day two of the porsolt forced swimming test, which could reflect increased depressionlike behavior. however, center exploration in the open field test as well as performance in the light-dark transition and the elevated-plus maze tests was normal in hcn3 -/mice. this suggests that general anxiety was not changed in the knockout mice. in addition, hcn3 knockout mice were less active on the second day of the open field test, which supports a habituation phenotype. finally, hcn3 -/mice had higher burying scores in the marble-burying test, which is a test for certain aspects of obsessive compulsive disorder in rodents. taken together, genetic deletion of hcn3 in mice results in distinct behavioral abnormalities related to behavioral despair and expression of repetitive behaviors in response to mild stressors. mielke h. 1 , gundert-remy u. alcohol consumption when breast feeding is discussed controversially. some groups recommend breast pumping before alcohol consumption and feeding the stored milk instead of breast feeding after drinking alcohol. this study was performed to simulate the blood concentration in the breastfed baby and to assess the health impact. method: we established a physiologically based kinetic model. its parameters were calculated (partition coefficients tissue/blood ; schmitt, 2008) silva et al.,1993) . we simulated 1. the alcohol concentration in a breastfed neonate and a 3-month-old suckling infant after the nursing mother had consumed alcohol,2. the alcohol concentration in utero/fetal compartment during pregnancy assuming the identical alcohol consumption of the pregnant woman 3. the alcohol concentration during infant´s treatment of bloating by an approved herbal drug containing alcohol. results: peak maternal alcohol concentration was 0.59 ‰ after consuming 0.25 l of wine, peak concentration was 0.0033 ‰ in the newborn, 0.0038 ‰ in the 3-month-old infant and 0.38 ‰ in the utero/fetal compartment. the peak concentration after herbal drug treatment was 0.015‰ in the neonate and 0.015‰ in the 3-month-old infant, respectively. we discuss the results of the simulations and compare it with doses and published concentrations measured in experimental animals or in vitro studies. conclusions: we conclude that the recommendation "1 to 2 glasses of wine on occasion" (agence nationale d'accréditation et d'évaluation en santé, assante 2002) is in accordance with the simulation results presented here whereas stricter rules are not scientifically sound. (2002) http://www.has-sante.fr/portail/upload/docs/application/pdf/ breastfeeding_guidelines.pdf da silva et al. (1993) adenylyl cyclases (ac) mediate physiological responses in virtually all cells, where their regulation through receptors and g proteins results in the modulation of camp. in the present study we focused on the kinetics of interactions between the alpha-subunit from inhibitory g protein type 1 (gαi1) and adenylyl cyclase type v (ac5). these proteins were labeled with cfp and yfp, respectively. the dynamics of their interactions was monitored by means of high temporal resolution fret imaging in hek cells expressing unlabeled a2a-receptor and gβg subunits. to activate the signaling pathway, we applied agonist using a rapid superfusion device. application of norepinephrine resulted in the development of a fret signal, indicating interaction between gai1-cfp and yfp-ac5. after withdrawal of agonist the fret signal recovered with a remarkably slow time course compared to the deactivation kinetics of gi proteins reported previously (bünemann et al. 2003) . to further analyze the properties of the dissociation between gai1 and ac5 we measured in parallel the offset kinetics of the interaction between gai1-yfp and gβg-cfp (gi1-fret) after agonist withdrawal under comparable conditions. in addition we tested to what degree the coexpression of rgs4 accelerated the deactivation of gi proteins and the dissociation of gai1-cfp from yfp-ac5. these experiments revealed that in the absence of rgs4 the dissociation of gai1 from ac5 after agonist withdrawal takes about 3 times longer than the deactivation of gi proteins. in the presence of rgs4 this difference is even larger due to the pronounced acceleration of g protein deactivation. the dissociation of gai1 from ac5 was only marginally accelerated by rgs4. these observations lead us to hypothesize, that ac5 might trap activated g protein-subunits and thereby affect the g protein cycle by shifting the equilibrium towards activated g proteins. if this hypothesis is true, it should result in a left-shifted dose response curve compared to g protein activation dose response. in support of this hypothesis we found that the concentration response curve for gai1-ac5 interaction was several-fold leftward-shifted compared to the concentration-response curve of gi-protein activation under very similar conditions. influencing the dynamics of the g protein cycle by effectors may represent a novel and powerful mechanism for finetuning the sensitivity of receptor evoked responses in an effector-specific manner. obesity, the excessive accumulation of white adipose tissue (wat), has reached pandemic dimensions. the factors that determine fat mass are not fully understood, but adipocyte hypertrophy and adipokine secretion are thought to be important. in present study, we investigated the role of the cyclic gmp (cgmp) signaling pathway focusing on cgmp-dependent protein kinase i (pkgi) in white adipocytes. pkgi is expressed in wat, preadipocytes and differentiated adipocytes as demonstrated by real-time pcr, western blot and immunochemistry. differentiation of pkgifl/fl preadipocytes, using an optimized protocol, resulted in an enhanced lipid accumulation as evidenced by oil red o staining. deletion of pkgi in pkgifl/fl adipocytes infected with a cre lentivirus (lv-cre, pkgi0/0) exhibited reduced differentiation. analysis of the triglyceride (tg) content revealed a significant decrease of tg levels by 65% ± 1% in pkgi0/0 as compared to pkgifl/fl adipocytes. western blot analysis of white adipocytes showed a significant decrease of c/ebpalpha (19% ± 4.8%), ppargamma (66% ± 2.9%) and ap2 (37% ± 10.6%) expression in pkgi0/0 cells as compared to pkgifl/fl. treatment of 3t3-l1 cells with cgmp resulted in increased lipid accumulation and enhanced expression of fat marker genes. lentiviral overexpression of pkgi further increased differentiation. importantly, pkgi significantly induced mitochondrial biogenesis in 3t3-l1 cells. concomitant activation of pkgi in 3t3-l1 preadipocytes and treatment with the demethylating agent 5-aza-deoxycytidine significantly increased expression of uncoupling protein-1 (ucp-1) -a unique protein of brown fat cells. we found rhoa as major target of pkgi signaling with increased phosphorylation of rhoa at ser-188 in pkgi overexpressing cells. moreover, pkgi-dependent phosphorylation counteracts the effects of rhoa on insulin signaling as well as adipokine expression. taken together, pkgi is a key player in white adipocyte differentiation that regulates cell size and has an anti-inflammatory effect. pkgi decreases the secretion of proinflammatory adipokines via inhibition of rhoa signaling. in addition, activation of pkgi can establish a brown fat cell like phenotype during white adipocyte differentiation if the ucp-1 promoter is accessible. the rag gtpases, raga, ragb, ragc, and ragd form a subfamily gtpases of the ras-related superfamiliy. rag proteins are characterized by a modified ras-like gtpbinding domain and a unique c-terminal region lacking a lipid modification motif. interestingly, rag proteins have been proposed to function as heterodimeric complexes consisting of raga or ragb associated with ragc or ragd. rag gtpases have been implicated in the control of mammalian target of rapamycin (mtor) function, in particular in regulation of the nutrient-stimulated and/or hormone-regulated mtor activity. the protein kinase mtor is found as the catalytic subunit of two larger protein complexes referred to as mtor complex 1 and 2, mtorc1 and 2. under amino-acidrich conditions, activated mtorc1 promotes protein synthesis and inhibits autophagy, while under starvation autophagy inhibition is released. increasing evidence suggests that activation of rag gtpases contributes to mtorc1 function. thus, rag proteins were found to be associated with a protein complex termed ragulator, a major regulatory protein of mtorc1 function and guanine nucleotide exchange of rag gtpases within the rag-ragulator-complex were described to promote mtorc1 translocation to its functional lysosomal compartment. however, the guanine nucleotide exchange properties of rag proteins are poorly characterized, and it is currently unknown, how amino acids promote rag proteins to facilitate the formation of the active, raptor-binding state of the rag heterodimers. to characterize the guanine nucleotide exchange properties of the rag gtpases as momomers or heterodimers in more detail, recombinant rag proteins were expressed in bacteria and purified from this source to near homogeneity. first, the parameters of gdp/gtp exchange of each of these proteins were compared using the non-hydrolysable gtp analogon gtpgs. the results showed that the rag isoforms are distinct in their guanine nucleotide exchange activities. in particular, nucleotide exchange on raga and ragc, but not on ragb and ragd, was only observed at low concentrations of gdp and mgcl 2 in extraction and assay buffers, i.e. conditions favoring the gdp/gtp exchange. these findings may indicate that guanine nucleotide exchange on raga and ragc is controlled by guanine nucleotide exchange factors and suggest specific functions of the individual rag gtpases within individual rag heterodimers. in in-vitro studies on rat and canine mast cells and human mast cell leukemia cells hmc1.2 bz at micromolar concentrations inhibited mediator release which appeared to be related to an inhibition of the intracellular camp pathway. in order to identify potential targets on/in mast cells at which bz may cause an inhibitory effect on mast cell activation, the 1,4-bz flunitrazepam (flu), clonazepam (clo) and 4chlorodiazepam (4-cd) were selected because of their different affinity and selectivity to/for the gaba-a-receptor and the translocator protein (tspo): flu and clo bind with nanomolar affinity to gaba-a receptors, whereas 4-cd is a selective ligand at tspo with nanomolar affinity to tspo but only micromolar affinity to gaba-a receptors. flu also possesses nanomolar affinity to tspo, whereas clo has no or only micromolar affinity to tspo. after incubation of hmc1.2 cells with 4-cd, flu and clo for 1, 6 and 24 hours up to 712 genes were significantly differently expressed in a substance-specific and timedependent manner. comparison of the genes differently expressed at 6 hours revealed that the expression of 217 genes was regulated by both flu and clo but only 8 genes were regulated by both 4-cd and flu suggesting that flu and clo induce gene expression by acting at a target site different from that of 4-cd. the difference between the gene regulation by flu and clo on the one hand and that of 4-cd on the other hand is also reflected in pathway analysis. since it was conceivable that the beneficial effects of the 1,4-bz could be mediated by the recognition sites targeted by the 2,3-bz, i.e. the gria2-encoded ionotropic glutamate receptor ampa2, we investigated by quantitative pcr whether hmc1.2 cells express gria2, tspo, the genes encoding the subunits of the gaba-a receptor and the gaba-forming enzyme glutamic acid decarboxylase. tspo, gabra3, gabrb3, gabre and gabrd were moderately expressed. in addition, there was a week or very week expression of gabra2, gabra4, gabrb2, gabrg3 and gabrr2. expression of gria2 was not detectable. taken together, it cannot be decided yet from our data whether the inhibitory effect of benzodiazepines on mast cell activation is due to an action at tspo or at gaba-a receptors of a novel subunit composition. monien b. h., glatt h. german institute of human nutrition (dife) department of nutritional toxicology, arthur-scheunert-allee 114-116, 14558 nuthetal, germany 5-hydroxymethylfurfural (hmf) and furfuryl alcohol (ffa) are common constituents of foodstuffs in which they are formed by heat-and acid catalyzed reactions from carbohydrates. hmf and ffa have been reported to induce the formation of hepatocellular adenomas in female mice and renal tubule neoplasms in male mice, respectively. we studied whether the carcinogenic effect of these hydroxymethylsubstituted furans may originate from sulfotransferase (sult)-catalyzed formation of electrophilic esters. hmf was inactive in in vitro mutagenicity tests using standard activating systems. in contrast, it was mutagenic in v79 cells genetically engineered for expression of human sult1a1 suggesting that hmf is converted into the reactive 5sulfooxymethylfurfural (smf). following incubation of mutagenic smf with porcine liver dna in vitro, specific methylfurfural adducts were detected using liquid chromatography tandem mass spectrometry (lc-ms/ms), i.e., n 6 -((5-formylfuran-2-yl)methyl)-2'deoxyadenosine (n 6 -ffmda) and n 2 -((5-formylfuran-2-yl)methyl)-2'-deoxyguanosie (n 2 -ffmdg). these adducts were also detected in dna from v79-sult1a1 cells incubated with hmf. in order to determine sulfo conjugation of hmf in mice in vivo, we conducted pharmacokinetic measurements showing that about 500 ppm of the hmf dose was converted to smf and reached the circulation. like hmf, ffa was negative in the standard ames test and various other in vitro genotoxicity tests. we showed that ffa is mutagenic in salmonella typhimurium ta100 engineered for expression of human sult1a1. the putative mutagen 2-sulfooxymethylfuran was synthesized and incubated with porcine liver dna, in which various nucleoside adducts were found. the main adducts, -mfda were detected in dna of ffa-exposed salmonella strain ta100-sult1a1 and in dna of liver, lung and kidney of fvb/n mice that had received about 390 mg ffa/kg body weight per day via the drinking water for 28 days. in summary, both furan derivatives form mutagenic sulfate esters in vitro and in vivo. in the future, we will use genetically engineered mice to characterize the role of single murine and human sult forms in the bioactivation of the furan derivatives and the contribution to tumor induction. background: micrornas are small non-coding rnas that can negatively regulate gene expression on a post-transcriptional level and have been shown to interact with epigenetic mechanisms like dna methylation. mecp2 (methyl cpg binding protein 2) is a protein that binds methylated dna cpgs in the promoter region of genes and can thus regulate their expression. otherwise, mecp2 is known to be a target gene for several micrornas including the cluster mir-132/212 in the brain. recently, our group could show that mecp2 expression is downregulated in human heart failure suggesting that mecp2 might be involved in cardiac pathogenesis. the aim of this project is to study the upstream regulation of mecp2 by the cluster mir-132/212 in the heart during cardiac hypertrophy in-vitro and in-vivo. methods and results: to test whether hypertrophic stimuli can induce mir-132/212 expression, we treated cultured nrcms with 40 µm norepinephrine for 72 hours. this induced cardiomyocyte hypertrophy and expression of the hypertrophy marker nppa, but also of mir-132 (1.79 ± 0.14-fold of untreated cells, p<0.01) and of mir-212-3p (1.73 ± 0.26-fold of untreated cells, p<0.05) and downregulated mecp2 mrna and protein levels (0.80 ± 0.04-fold of untreated cells, p<0.05). to check whether mecp2 downregulation also occurs by direct mir-132/212 activation we increased levels of mir-132 and mir-212 in cardiac myocytes by transfecting precursor mir-132 and mir-212-3p molecules. again, we observed nrcm hypertrophy, nppa mrna upregulation and mecp2 mrna and protein downregulation (0.75 ± 0.05-fold of control, p<0.05) after mir-132 overexpression. similar results were obtained by overexpression of mir-212-3p. to test the effects of adrenoceptor activation on the mir-132/212-mecp2 axis in-vivo, wild-type mice received isoprenaline and phenylephrine via osmotic minipumps (30 mg/kg/day each). after 7 days, cardiac ventricles were analyzed. nppa gene expression (2.81 ± 0.32 -fold of control animals), mir-132 and mir-212-3p levels (3.67 ± 0.5 and 2.62 ± 0.4 -fold of control animals, p<0.01 and p<0.05, respectively) were increased while mecp2 protein levels decreased to 77% (p<0.05) conclusion: these results suggest that in-vitro and in-vivo adrenoceptor stimulation leads to the activation of mir-132/212 expression and to downregulation of mecp2 in cardiac myocytes in-vitro and in-vivo. leopold-franzens-universität, innsbruck, austria at-1 receptor antagonists block the angiotensin ii-enhancing effect on noradrenaline release from sympathetic neurons. in a cell-free assay the binding affinity of the at-1 receptor antagonists telmisartan and valsartan to the gamma peroxisome proliferatoractivated receptor (pparγ) is close to that of the pparγ selective agonists thiazolidinediones (tzds). we tested whether the tzds rosiglitazone and pioglitazone would also modify the prejunctional facilitatory effect of angiotensin ii. left ventricular slices of rats were incubated with tritiated noradrenaline, perifused and electrically stimulated. the negative logarithm of the drug concentration that caused a 30% increase of control (pec30%) was calculated. angiotensin ii caused a concentration-dependent increase of tritium overflow induced by electrical stimulation [pec30%=8.6±0.2 (mean±sem, n=18); maximum increase=110±8%]. neither rosiglitazone nor pioglitazone (0.3-3 µm) had a direct effect. the concentrationresponse to angiotensin ii in the presence of fixed concentrations of rosiglitazone was shifted to the left with increase of the maximum (pec30%=8.8±0. 2, 9. 2±0.2 and 9.3±0.3; maximum increase=118±14%, 146±13% and 148±16%, in the presence of 0.3, 1 and 3 µm of rosiglitazone, respectively, n=4-6, each). in contrast, pioglitazone in concentrations up to 3 µm had no effect on the release-enhancing effects of angiotensin ii. results show that rosiglitazone but not pioglitazone potentiates the noradrenalinerelease enhancing effect of angiotensin ii. this action might contribute to the risk for myocardial infarction from rosiglitazone use but not from pioglitazone use. deleted in liver cancer 1 (dlc1) is a tumor suppressor whose allele is lost in 50% of liver, breast, lung and 70% of colon cancers. despite its significance, the molecular mechanisms that drive cancerous transformation upon dlc1 loss remain unclear. we found that the transcriptional coactivators megakaryoblastic leukemia 1 and 2 (mkl1/2) are constitutively localized to the nucleus in hepatocellular and mammary carcinoma cells that lack dlc1. moreover, dlc1 loss and mkl1 nuclear localization correlated in primary human hepatocellular carcinoma. nuclear accumulation of mkl1 in dlc1-deficient cancer cells was accomplished by activation of the rhoa/actin signaling pathway and concomitant impairment of erk-mediated mkl1 phosphorylation. dlc1 loss led to constitutive activation of the mkl-dependent, tumor-relevant target genes ctgf, cyr61, myl9 and myh9. furthermore, we identified a novel target gene, integrin a5, with a key role in cell migration and metastasis, that exhibited a dlc1-and mkldependent regulation. depletion of mkl1/2 suppressed not only cell migration, but also cell proliferation and anchorage-independent cell growth induced by dlc1 loss. our data provide insight into the mechanism by which dlc1 loss initiates tumorigenesis. as mkl1 and 2 have a key role in this process, this pathway may provide promising pharmacological targets for cancer therapy. universität bonn, pharma-zentrum bonn pharmazeutisches institut, pharm. chemie i, an der immenburg, 53121 bonn, germany membrane receptors activated by purine and/or pyrimidine nucleotides ("p2 receptors") are widely distributed in the body and constitute novel (potential) drug targets. they are subdivided into g protein-coupled p2y receptors (p2y1, 2, 4, 6, 11, 12, 13, 14) , and homo-or heterotrimeric ligand-gated ion channel or p2x receptors (subunits: p2x1-7). we have been interested in the identification and development of potent and subtype-selective ligands -as tool compounds and potential drugs -for the various p2y and p2x receptor subtypes. our strategy involves (i) establishment of a proprietary compound library consisting of synthetic small molecules and natural products; (ii) development of screening assays suitable for medium throughput screening; (iii) careful analysis of structure-activity relationships at each target and systematic optimization of the lead structures; (iv) pharmacological evaluation of selected compounds. this approach has led to new biological tools for several targets, including p2y and p2x receptors [1] [2] [3] [4] [5] [6] . fine particles in particulate matter (pm) are effective vehicles to transport toxicants into the lung; depending on their size, smaller particles may reach the bronchiolar or alveolar space. in recent years the pm fraction pm2.5 has especially been correlated with both pulmonary and cardiovascular diseases. in order to better characterize pm emission and distribution of environmental tobacco smoke (ets) from cigarettes (reference cigarette (rc), brand cigarette (bc)) we have developed an ets emitter to simulate human smoking emission and measured pm2.5 concentration in a telephone booth (1,75 m3 volume) as an example for small indoor spaces like cars. fine particulate matter was measured using an aerosol spectrometer with 6 sec time resolution; laser scatter allowed a size resolution from 0,25 µm to 32 µm. for the pm2.5 concentration the following values were calculated: cumulative pm2.5 concentration as auc-pm2.5 (µg/m3/sec), peak pm2.5 concentration as cmax-pm2.5 (µg/m3) and average pm2.5 concentration cmean-pm (µg/m3). in closed door condition both cigarettes produced particulate auc-pm2.5 values of 59 000 ± 15 000 µg/m3/sec (rc) to 85 000 ± 31 000 µg/m3/sec (bc after myocardial infarction (mi) inflammatory cells and cardiac fibroblasts (cf) determine the remodeling response. interleukin-6 (il-6) is induced in the ischemic myocardium and is known to stimulate the differentiation of fibroblasts to myofibroblasts. hyaluronan (ha) is an extracellular matrix component synthesized by ha-synthase isoenzymes (has 1-3) and is also known to control fibroblast phenotypes. however, it is presently unknown whether il-6 participates in the remodeling of the ha-matrix or whether the ha-matrix modulates the responses to il-6. therefore, the aim of the present study was to elucidate whether il-6 regulates the expression and function of ha-matrix in cfs. cells were isolated from c57bl/6j mice and used during passage 2-3 for experiments. cfs were stimulated with il-6 or hyper-il-6 which is a fusion protein of il-6 and soluble il-6 receptor (sil-6r). after 10 and 30 min signal transducer and activator of transcription 3 (stat3) was phosphorylated in response to hyper-il-6 but not in response to il-6. rt-pcr revealed rapid upregulation of has 1 (5.94 ± 2.49 fold of unstimulated control, 1h) in response to hyper-il-6. has2 was induced to a lesser degree (2.04 ± 0.55 fold of unstimulated control, 1h) whereas has3 was not responsive (1.49 ± 0.42 fold of unstimulated control, 1h). in contrast, il-6 had no effect on transcript levels of has isoenzymes. in turn, expression of has1 and has2 in response to hyper-il-6 was inhibited by ag490, which indicates the involvement of stat signaling. interestingly, despite induction of has1 and has2 the amount of secreted ha as determined by an elisa-like assay was not affected by hyper-il-6. this may indicate that il-6 regulates the cell surface associated ha-matrix of cfs. in conclusion, the present data demonstrate that cardiac fibroblasts respond to il-6 trans-signaling (hyper-il-6) via the soluble il-6r and subsequent stat3 signaling with increased ha-synthesis. the fact that il-6 had no significant effect suggests that the expression of the non-signaling membrane-bound il-6 α-receptor (il-6r) in cultured murine cardiac fibroblasts is not sufficient to induce has 1 and -2 gene expression. therefore, il-6 trans-signaling mediated by il-6 and the circulating sil-6r might be necessary to mediate the il-6-induced has expression in vivo. mrgprd receptor endogenously expressed in dorsal root ganglia: evidence for an activation by 3-aminoisobutyric acid müller s., hoffmann k., von kügelgen i. universität bonn institut für pharmakologie und toxikologie, sigmund-freud-straße 25, 53127 bonn, germany the gpcr mrgprd (mrgd) is highly expressed in small diameter dorsal root ganglion (drg) neurons and has been implicated to play a role in nociception. the receptor was previously shown to respond to β-alanine. in the present study we searched for agonistic activity of structural analogues of β-alanine. for further characterization of the receptor we used fura-2 fluorimetry, a nfat luciferase reportergene assay and the determination of the inhibition of forskolin-induced camp production ([ 3 h]-camp affinity assay). first, we confirmed the activation of the receptor by β-alanine and gaba. in reportergene experiments we then identified 3-dlaminoisobutyric acid as an agonist, with similar potency but weaker affinity when compared to β-alanine (ec50 165 µm). fura-2 fluorimetry showed an increase in intracellular ca 2+ levels by 3-dl-aminoisobutyric acid (300 µm). moreover, 3-dlaminoisobutyric acid reduced the forskolin-induced camp production by up to 65 % (ec50 195 µm). in addition to 3-dl-aminoisobutyric acid, we identified 3-dl-aminobutyric acid as a weak agonist acting at the mrgprd. other closely related substances failed to show significant responses. next to the agonists we further characterized antagonists inhibiting the response to βalanine mediated by mrgprd. chlorpromazine shifted the concentration-response curve of β-alanine to the right with an apparent pkb of 4.9 (nfat assay), thioridazine with an apparent pkb of 5.4 (nfat assay) and 5.3 (camp assay) and rimcazole with an apparent pkb of 5.2 (nfat assay) and 5.2 (camp assay). in conclusion we show for the first time that 3-dl-aminoisobutyric acid is an agonist at the mrgprd and that the structure-activity relationship of agonists at mrgprd is very close. the sdf-1-chemokine receptor cxcr4 plays a key role during embryogenesis and regulates functions of immune and stem cells in adult life. furthermore, cxcr4 is involved in disease states including inflammation and cancer. it is well established that sdf-1-stimulated cxcr4 receptors activate gi protein-dependent signal transduction pathways and undergo c-terminal phosphorylation and internalization. because the cxcr4 c-terminal domain contains 15 serine and 3 threonine residues, it is incompletely understood which of the potential phosphorylation sites contribute to homologous and heterologous regulation of cxcr4. here, we analyzed the phosphorylation pattern of cxcr4 at 3 c-terminal sites after stimulation of the receptor with sdf-1 and after pma-induced activation of the pkc pathway as a model for heterologous receptor phosphorylation. using phospho-specific antibodies against s324/325, s338/339 and s346/347 in immunoblot analyses, we showed that the 3 sites were phosphorylated after stimulation with sdf-1 or pma. stimulation with egf or forskolin did not induce phosphorylation at these sites. sdf-1-induced phosphorylation at s324/325, s338/339 and s346/347 was reversible after wash out of the ligand. time course analyses revealed that phosphorylation occurred first at s346/347 and then at s324/325 and s338/339. taken together, these results indicate that the c-terminus of cxcr4 is phosphorylated at multiple sites by homologous and heterologous pathways and that phosphorylation at the different sites may be hierarchically organized. human milk represents the best form of nutrition for infants early in life. however, it can also contain toxic contaminants that may adversely affect infant's development. the nephrotoxin ochratoxin a (ota) is present in human milk (tab. 1 in [1] ), but information on transfer from maternal blood to milk is scarce: published data [2] indicate that levels of ota in milk are roughly one tenth (0.1) of those in blood. but, the efficiency of the ota-transfer at various stages of breastfeeding may vary since studies in animals revealed that transfer of ota is apparently time-and dose-dependent. thus, the aim of this study was to assess the ota transfer from blood to milk at different stages of breastfeeding in humans. in a small chilean cohort, 18 lactating women were asked to provide blood and milk on the same day. these samples were collected on four different occasions within the first months after delivery and analyzed using hplc with fluorescence and/or tandem mass spectrometric detection [1] . the transfer of ota from blood to milk was quantitatively assessed by measuring the milk to plasma ratio (m/p). the average ota level in blood plasma was 235 ± 129 ng/l, and no major variations were observed over time (p = 0.19). on the other hand, ota levels found in colostrum (85 ± 60 ng/l) were higher than in mature milk (p < 0.05). in line with these data, higher m/p ratios (table) were obtained with samples collected in the first six days after delivery. this study showed that the transfer of ota from blood to milk was more efficient with colostrum (m/p 0.43 ± 0.26) than with mature milk. thus, a higher exposure to ota can be expected for neonates than for infants at later stages of breastfeeding. moreover, the lactating women have lower average ota levels in plasma than non lactating women from chile [3] , indicative of milk as additional excretion route. acknowledgement: this work has been supported by a stipend from conicyt/daad to km. exposure of infants to ochratoxin a (ota) deserves particular attention since ota is nephrotoxic, and one of the most potent rodent renal carcinogen studied to date [1] . moreover, infants may be more vulnerable to the toxic effects of contaminants than adults. ota-levels in plasma of infants are indicative of an early exposure in life [2] . but blood sampling is an invasive method not readily applicable for breastfed infants. thus, the aim of this study was to implement a non invasive biomonitoring method to assess ota-exposure in this group. to assess the exposure to ota, breast milk and infants' urine specimens were collected, from two different cohorts: chile (n= 28) and turkey (n=10, only urine). analysis of the samples was performed using enzymatic hydrolysis prior to extraction and hplc-ms/ms [3] . the magnitude of infants' exposure was assessed by calculating the ota-daily intake with human milk and relating it also to urinary ota levels. calculations of the daily intake with human milk [4] showed that infants may be exposed to ota at high levels, exceeding the tolerable daily intake (tdi) of 5 ng/kg-bw/day set for adults [1] . in both cohorts, most of the urine samples tested positive for ota (chile 72%, turkey 80%). ota levels observed in urine samples from the turkish infants (range: 116 -1,361 ng/l) were 10 fold higher than levels found in chilean samples (range positive samples: 17 -320 ng/l). further analysis of phase ii metabolites in urine confirm the excretion of ota as conjugate (glucuronide) in highly exposed infants. in conclusion, ota exposure of infants early in life was documented. given that otaintake by several infants exceeded the tdi for adults, further biomonitoring in this vulnerable group is advised including also suitable biomarkers of effect. a mixture of (e)-and (z)-clomiphene citrate is the first line therapy of female infertility. however, up to 30% of patients do not respond. (e)-clomiphene is structurally closely related to another selective estrogen receptor modulator, tamoxifen which is frequently used for the treatment of hormone receptor-positive breast cancer. like tamoxifen, clomiphene is extensively metabolised by the cytochrome p450 system. using the estrogen receptor response assay (e)-4-hydroxyclomiphene and (e)-4-hydroxy-n-desethylclomiphene (ec50: 2.5 and 1.4 nm, respectively) turned out to be 100 times more active at the er compared to the parent drug isomers and de-ethylated metabolites. using recombinant expressed human cyp isoforms and inhibitory antibodies, cyp2d6 revealed to be the major isoenzyme involved in the formation of 4-hydroxlated metabolites. n-deethylation was catalysed by cyps 3a4/5, 2d6, 2c19 and 2c8. rates of 4-hydroxylation in microsomes from 30 human liver donors correlated with the number of functional cyp2d6 genes. these in vitro results were confirmed in a pharmacokinetic study with female healthy volunteers receiving a single dose of clomiphene. in carriers of two non-functional cyp2d6 genes (poor metabolizers) cmax of (e)-4-hydroxyclomiphene and 4-hydroxy-ndesethylclomiphene was 8 and 12 times lower, respectively, when compared with subjects with at least one fully functional cyp2d6 allele. in contrast, half-life of (e)-clomiphene and (e)-n-desethylclomiphene was 10 and 50-fold higher, respectively, in poor metabolizers. our data provide first evidence of a pharmacogenetic rational for the variability in the response to clomiphene treatment. among the tested compounds, compound 1 proved to be the most active derivative, showing a significant toxicity at a concentration of 2,5 µm. compounds 2 and 3 showed significant toxic effects at a concentration of 5 µm. the compound 4 showed no toxicity up to a concentration of 100 µm. all derivatives 1, 2 and 3 have a ec50 between 10 and 15 µm. we further proved the induction of apoptosis by apo-one assay (caspase 3/7 activity) and life/dead-assay (fluorescence microscopy). in conclusion, these gold complexes exhibit an example of interesting potential candidates for future anticancer pharmaceuticals due to relatively high cytotoxicity. gene regulating effects in mouse liver subsequent to treatment with selected dioxin-like compounds and pcb 153 using whole genome microarray analysis neser s. 1 , lohr c. 1 , van ede k. i. 2 , andresen k. 3 interaction with the aryl hydrocarbon receptor (ahr), with 2,3,7,8-tetrachlorodibenzo-pdioxin (tcdd) being the most potent congener amongst the ahr agonists. recent risk assessments have employed the toxic equivalency factor (tef) concept. the current eu-project systeq aims at developing, validating, and implementing human systemic tefs as indicators of toxicity for dl-compounds. at present, the best known parameter of ahr mediated effects is the induction of cytochrome p450 isoenzymes (cyps), i.e., cyp1a1, 1a2, and 1b1. one of the major objectives of the systeq project is the identification of novel quantifiable biomarkers. in a three day study, female c57bl/6 mice were treated with single doses of six dl-congeners (tcdd, pcb 118, pcb 126, and pcb 156) , and the 'non-dioxin-like' (ndl) pcb 153. quality tested (agilent ® bioanalyzer) mrna isolated from livers was analyzed using the agilent ® mouse whole genome array (4x44k) system. the quantity of genes affected (≥ 2fold) was highly heterogeneous amongst the dl-compounds. whereas tcdd-treatment upregulated 89 genes, and down-regulated 66, 4-pncdf-treatment had impact on 3208 (up), and 2396 (down). treatment with pcb 118 led to marginal numbers of 16 up and 6 down-regulated genes. with 64 (up), and 38 (down) genes shared, the most extensive overlap occurred between tcdd-and 1-pncdd-treatment. no overlap was found due to treatments with the ndl pcb 153 (21 up, 1 down) and tcdd. when comparing the effects of all dl-congeners, minor numbers of genes of 19 up, and 5 down-regulated remain, most of them being related to drug metabolism. while pcb 118 regulated only genes involved in drug metabolism, omission of pcb 118-regulated genes resulted in consistently 51 (up), and 24 (down) regulated among dl-compounds. in conclusion, our findings suggest that the pattern of gene regulation in mouse liver elicited by pcb 153 was strictly different from tcdd, while a very limited coincidence of genes was found even among dl-compounds. comparison of these 'core' genes with data from human models is required with respect to determination of novel biomarkers. introduction: proper use of antibiotics is essential with regard to effective treatment of bacterial infections. providing adequate information for patients can contribute to achieve this aim. materials and methods: data was collected from the relational database of the drug information service at dresden university of technology. the patients, who used the service, were interviewed concerning socio-demographic characteristics, reason for enquiry, number and kind of drugs taken, and diseases. possible contact paths were phone, e-mail or letter. in the present evaluation, all enquiries from the years 2009 and 2010 were analyzed descriptively focussing primarily on systemic antibiotics as reason for the enquiry. results: in the evaluated period, 4454 enquiries were registered in total. in 4.7% of those enquiries systemic antibiotics were named with a total number of 283 drugs. 50.9% of those antibiotics were found to be the direct reason for the enquiry. most common information requested by patients corresponded to adverse drug reactions (50.7%), diagnosis/treatment (35.4%), drug application (29.2%) and drug-druginteractions (19.4%). the majority of the requesting patients (61.5%) was born before 1970. a correlation between incidence of enquiries especially concerning antibiotics and quarterly statistics could not be detected. conclusion: mainly patients aged 40 years or more seem to need or search for further information about antibiotic medication. advice is required especially regarding adverse drug reactions and diagnosis or treatment. in order to this, the advisory service can help patients to lose their insecurity and to gain more confidence in handling antibiotic drugs. colon cancer is one of the most frequent cancers in the industrialized nations. epidemiological studies show a correlation between highly processed meat and the development of colorectal tumours. it is assumed that the risk of developing colorectal cancer, among various different factors, is related to the uptake of toxic substances contained in food such as heterocyclic aromatic amines that arise during the processing of fish and meat. phip is the most abundantly formed heterocyclic compound, and therefore has the biggest impact. in a previous study, we measured the absorption of phip in different intestinal segments of the rat. in the present study we focussed on the potential mechanisms by which phip is reabsorbed. the unidirectional phip transport from the mucosal to the serosal compartment (j ms) and in the opposite direction (jsm) was examined using the ussing chamber technique and 14 c-phip as a radiotracer. the proximal jejunum and distal colon of male fischer 344 rats in short-circuit current chambers was clamped, so that mucosal and serosal compartments were built. the phip flux rates were determined at defined intervals over 120 min. the experimental conditions were selected in such a way that negative net flux rates (jnet = jms-jsm) were indicative of an active secretion. both intestinal segments showed large differences. while in the jejunum jms and jsm of phip were not significantly different, there was an active secretion in the colon. in a next step the transport proteins involved in this process should be examined. introduction: human organic anion transporter 2, oat2 (slc22a7), is abundantly expressed in kidney and liver and mediates the sodium-independent uptake of clinically relevant drugs like 5-fluorouracil, paclitaxel, bumetanide, tetracycline, and zidovudine. while immunohistochemical studies have localized human oat2 to the basolateral membrane of kidney proximal tubules, its hepatic localization is currently unknown. we, therefore, firstly determined oat2 localization in human liver. because interindividual variability of oat2 expression may affect hepatobiliary drug uptake and elimination, we next systematically investigated the influence of genetic and non-genetic factors on hepatic oat2 expression. methods: an expression profile of oat2 for 20 human tissues was determined by realtime quantitative polymerase chain reaction (taqman). oat2 mrna expression was analyzed in well-characterized human liver samples from 150 caucasians that were accompanied by detailed demographic and clinical data. oat2 was localized in human liver cryosections using a commercial rabbit polyclonal antibody and hepatic oat2 protein levels were determined. resequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and genome-wide single nucleotide polymorphism microarray technology served to genotype variants in the slc22a7 gene region. results: oat2 mrna was expressed in several human tissues, including liver. moreover, a new alternatively spliced variant of oat2 was identified in human liver. hepatic expression of full-length oat2 mrna and oat2 protein varied 37-fold and 41fold, respectively. oat2 mrna and protein levels did not correlate with each other. oat2 was localized to the sinusoidal membrane of human hepatocytes. no novel variants in the 9 exons, the 5'-flanking region, or the 3'-untranslated region of the slc22a7 gene were identified. univariate analysis showed that oat2 mrna is reduced in patients diagnosed for cholestasis (p=0.0012) and is affected by genetic variants. whereas the influence of genetic variants on hepatic oat2 expression appears to be limited, cholestasis significantly contributes to the variable interindividual oat2 expression. this indicates consequences for hepatic drug elimination of and response to oat2 drug substrates such as paclitaxel or tetracycline. the life threatening toxicity of organophosphorus (op) nerve agents is caused by the inhibition of the acetylcholine esterase (ache). oximes were shown to be potent reactivators of inhibited ache, but in poisoning by some compounds, e.g. soman, they have only a small therapeutic effect. for such cases, an alternative new strategy may be the intervention at nicotinic acetylcholine receptors (nachr). previous studies with the bispyridinium non-oxime mb327 demonstrated therapeutic effects against soman in vitro and in vivo which was partly attributed to its direct interaction with nachrs [1] . we investigated the interaction of mb327 and several structure analogous at the orthosteric binding site of human α7 nachr (hα7 nachrs), a subtype which appears to be widespread in the human body, and compared the results with data obtained from torpedo-nachrs, which show a high degree of homology with human muscle-type nachrs. interaction of compounds with the orthosteric binding site of hα7 nachrs were investigated with radioligand binding experiments performed as high-throughput method [2] . membrane preparations of gh 4c1 cells stably expressed hα7 nachrs were incubated with the nachr agonist [³h] epibatidine and appropriate concentrations of the unlabelled competitors e.g. bispyridinium compounds. after incubation, bound and free [³h] epibatidine were separated by rapid vacuum filtration. ki values of the competing compounds were calculated with nonlinear regression. three bispyridinium compounds, mb442, mb456 and mb770 exhibited ki values at micromolar concentrations while three other compounds, mb327, mb583 and the pharmacological inactive mb424 (negative control) did not show any interaction with the orthosteric binding site of hα7 nachrs. with torpedo-nachrs, ki values were in similar orders of magnitude -except mb442 which indicated significant subtype selectivity. interestingly, the affinity of monomeric pyridinium derivates did not correlate with their bispyridinium structure analogues. obviously, no correlation between the affinity to the orthosteric binding site and the functional improvement of neuromuscular transmission exists, although species-related differences cannot be excluded. in this study, we analysed the cytotoxic and clastogenic effects of the anticancer drug nimustine (acnu) in cells deficient in repair proteins involved either in homologous recombination (hr) or non-homologous end-joining (nhej). we show that hr mutants are extremely sensitive to acnu as measured by the induction of apoptosis and colony formation as well as the induction of chromosomal aberrations. the nhej mutants were slightly sensitive to acnu and differed in their sensitivity, with the ku80 mutants being moderately sensitive and the dna-pkcs mutant resistant, comparable to the wild-type (wt). cell death was mostly executed via the caspase-dependent apoptotic pathway with involvement of caspase-3 and -7, and necrosis was also induced. further, we investigated the kinetics of dna double-strand break (dsb) formation that resulted from the repair of acnu-induced interstrand cross-links by means of γh2ax and 53bp1 foci analysis in wt and mutant cells. cells mutant in hr did not repair dsb and went into the apoptotic or necrotic pathway, whereas wt cells were able to repair most of the dsb. cells deficient in ku80 formed at early times after acnu treatment less γh2ax and 53bp1 foci compared to the corresponding wt, which might be due to a reduced capacity of recognising dsb. at later times after treatment, ku80 mutant cells show foci levels similar to the wt indicating restitution of h2ax phosphorylation. we also analysed whether dsb formation after acnu treatment was replication-dependent using synchronised cells. we determined the formation of γh2ax and other dsb marker in wt cells that passed through the first cell cycle after demecolcine synchronization. the level of γh2ax foci increased significantly in the s-phase and remained at a high level during g2 where a fraction of cells remained arrested. rad51, atm, mdc-1 and rpa-2 foci were also formed and shown to co-localize with γh2ax. these foci were ameliorated significantly in s-and g2-phase, which was similar to the time course of γh2ax foci formation. in western blots, we confirmed a higher phosphorylation level of atm and chk2 and less phosphorylation of chk1 in hr mutants. the data indicate that acnuinduced dna cross-links give rise to cyto-and genotoxicity via the formation of dsbs that activate the cellular dna damage response. the endocannabinoid system has been established as a mediator of numerous central and peripheral biological functions. cannabinoids have emerged as attractive alternatives or supplements to therapy with opioids for chronic pain states. however, in human the activation of cannabinoid receptors is associated with side effects. for clinical exploitation of the analgesics properties of cannabinoids, a major challenge is to devise strategies that reduce or abolish their adverse effects on cognitive, affective and motor functions without attenuating their analgesics effect. in animal studies, the anti-nociceptive efficacy of cannabinoids has been unequivocally demonstrated in several models of inflammatory and neuropathic pain. however, there are marked inconsistencies between different reports with respect to the locus of these pain-protective effects. we are working towards establishing the contribution of cb1 receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia. using cb1 globally knock-out animal as background, we induce the expression of cb1 specifically in nociceptive neurons localized in the peripheral nervous system and test the analgesic effects of cannabinoid systemical delivery in these mice. our results support the development of peripherally acting cb1 analgesic agonist with reduced central side effects. furthermore, we are utilizing proteomics approach to identify protein complexes that interact with cb1 receptor which hold promise in understanding cannabinoid signaling in health and disease. most chemoattractants, including chemokines, complement c5a, fmlp, and leukotriene b4 are signaling through heterotrimeric g proteins of the pertussis toxin (ptx)-sensitive gi family. the functional inactivation of all gαi proteins with ptx leads to a fulminant decompensation of the immune system, whereas the constitutive inactivation of a single gαi coding gene results in mild phenotypes in mice. we are mostly interested in the nonneuronally found gαi2 and gαi3 isoforms and their redundant and specific roles in immune function and infection. for this purpose cellular in vivo and ex vivo models and in vivo infection model with listeria monocytogenes are being used. macrophages were isolated from the peritoneal cavity of wild type (wt) and gαi-deficient mice 4 days after i.p. injection of 4% thioglycolate that induces peritonitis in vivo. we confirmed previous observations that in gαi2-deficient mice the migration of macrophages into the peritoneal cavity was reduced after induction of peritonitis. regarding the expression levels of gαi and gβ isoforms in the lavage samples, the predominant gαi isoform gαi2 was upregulated in gαi3-deficient macrophages. vice versa gαi3 was upregulated in gαi2-deficient macrophages. concerning gβ isoforms, both gβ1 and gβ2 were strongly reduced in the gαi2-deficient macrophages which resulted in a reduced total amount of gβ. surprisingly, the gαi3-deficient macrophages showed reduced gβ1 protein levels only which caused a change in the gβ2/ gβ1 quotient in favour of gβ2. we are currently establishing an in vivo infection model with l. monocytogenes in gαi2and gαi3-deficient mice. our previous in vitro infection studies in mice embryonic fibroblasts provided us with information about possible distinct roles of these two isoforms as far as the uptake of l. monocytogenes in the cells is concerned. challenging the immune system of gαi-deficient mice with this pathogenic organism will give us new insights into the systemic immune response in these mice upon bacterial infection. our data indicate that we may surmount the redundancy between these two isoforms and focus on their distinct and specific roles in pathogen defense. fret-based β-arrestin2 biosensors reveal conformational changes upon binding to the β2-adrenergic receptor in real time and living cells nuber s., zabel u., ziegler n., hoffmann c., lohse m. j. institut für pharmakologie und toxikologie pharmakologie, versbacherstr.9, 97078 würzburg, germany β-arrestins are multifunctional adapter proteins that regulate seven transmembranespanning receptor (7tmr) signaling and initiate also alternative signaling pathways. studies have shown that β-arrestins undergo conformational changes upon receptor stimulation, which are thought to be necessary for its downstream actions. to investigate these conformational changes in living cells we constructed fret based biosensors of β-arrestin2, in which cfp was fused to the c-terminus and the flashbinding motif (ccpgcc) was inserted to different positions within the n-or c-domain of β-arrestin2. upon β2-adrenergic receptor (β2ar) stimulation we observed a decrease of the intramolecular fret signal between cfp and flash at the n-domain (β-arrestin2flash2), indicating a conformational change moving the c-terminus and the ndomain of β-arrestin2 relative to each other. kinetic analysis revealed that this conformational change immediately follows β-arrestin2/β2ar interaction on a timescale of seconds. a β2ar mutant that was previously shown not to interact with β-arrestin2 was utilized as control and did not induce a conformational change in the β-arrestin2 molecule. our data provide evidence that β-arrestin2 changes it`s conformation upon binding to the activated β2ar in living cells. the β-arrestin2flash2 sensor could serve as universal biosensor for gpcr activation. studies on the physiological role of annexin a4 in the heart nunes f. 1 the calcium binding protein annexin a4 has been examined in the context of heart failure in the past. annexin a4 expression level was found to be elevated in ventricles of human failing heart in comparison to expression levels in non-failing ventricles. furthermore the intracellular localization pattern in atrial cardiomyocytes was found to be altered in the failing human heart (moravec and matteo, cardiovasc res 2000). in order to gain insight into the possible physiological significance of these findings we utilized an annexin a4 gene trap model (gt) in which the annexin a4 protein content was not detectable in ventricles and atria. measurements of sarcomere shortening and calcium transient kinetics in isolated ventricular cardiomyocytes revealed a prolonged calcium transient decay at stimulation frequencies of 0.5 hz, 1hz and 2 hz as well as an increased sarcomere shortening at 1 hz and 2 hz in anxa4 gene trap animals in comparison to wild type (wt) ( the effects of the β-adrenoreceptor agonist isoprenaline (iso) on the shortening of ventricular cardiomyocytes was increased in gt as compared to wt (0,2±0.013 vs. 0.17±0.012, *=p<0.05 vs. wt; n=14-18/5). western blot analyses indicated that the expression of the sarcoplasmic reticulum (sr) ca 2+ -atpase (serca2a) and the phosphorylation status of its regulator protein phospholamban (plb) did not differ between groups (n=7). however, co-immunoprecipitation experiments suggest, that anxa4 is able to interact with hax1, which acts as a repressor of serca2a (n=4). we performed force measurements in isolated and electrically stimulated left atria in response to rising isoprenaline concentrations (10 -9 m-10 -5 m). the positiv inotropic effect of isoprenaline was significantly increased in gt atria (rel. force at 10 -4 m iso [%]: wt: 433±76; gt: 699±63 *= p<0.05 vs wt; n=8-10). in conclusion, annexin a4 contributes to the regulation of cardiomyocyte contractility. the anxa4 up-regulation might therefore contribute to diminished cardiac performance in heart failure. matteo rg, moravec cs. immunolocalization of annexins iv, v and vi in thefailing and non-failing human heart. cardiovasc res. 2000 mar;45 (4) background: pregnane x receptor (pxr) is considered the most important sensor of natural and anthropogenic xenobiotics in vertebrates. in contrast, the amphibian ortholog is involved in neural development and irresponsive to xenobiotics. instead, the xenopus laevis constitutive androstane receptor (car) was recently found to possess pxr-like properties, featuring low basal activity and a pronounced ligand spectrum. thus a structural and functional characterisation of x. laevis car may provide further insights into human car basal and ligand-induced activity. methods: the time-point of origin of car genes was determined by macrosynteny analyses of car, pxr, and vdr (vitamin d receptor) gene loci, which form the nr1i subfamily of nuclear receptors. based on a 3-dimensional protein model of xenopus laevis car, docking studies with structurally diverse agonists were conducted. proteinligand-interactions as well as sequence comparisons were performed in order to select amino acids to be mutated towards human car. the organ response to car activators was determined in xenopus laevis using rna microarrays. results: car emerged together with pxr and vdr from an ancestral nr1i gene in early vertebrates via two whole-genome duplications. this was followed by losses of car from the fish lineage and of pxr from sauropsida (reptiles and birds). amino acids important for ligand binding were identified. structural features responsible for the pronounced basal activity in human constitutive androstane receptor are not present in x. laevis car. in human pxr the inter-helical loop in front of helix 3 is part of the ligandbinding pocket and supposed to be responsible for the wide substrate spectrum. in amphibian car this inter-helical loop plays no role in ligand binding. car agonists resulted in a pronounced induction of antimicrobial peptides in the ovary. conclusions: car emerged already in early vertebrates and it is conserved in land vertebrates, whereas xenosensing pxr is found only in the fishes and mammals. we provide a comprehensive modeling and mutational analysis of this first reported amphibian xenosensor. the induction of antimicrobial peptides by car activators suggests a link between xenosensing and innate immunity. the latter one may play a previously unrecognized role in the amphibian reproduction. background: retigabine belongs to a novel class of potent anticonvulsant drugs and is currently being investigated in clinical routine. the therapeutic range of retigabine serum concentration is unknown. a therapeutic drug monitoring (tdm) is used for most other anticonvulsant drugs. the aim of this study was to develop a method for the determination of retigabine in serum of patients and to compare the effect and the side effects of retigabine with the blood levels of the drug. method: a hplc method with tandem mass spectrometric detection for the sensitive determination of retigabine was developed. solid-phase extraction (spe) of 250 µl serum with oasis hlb cartridges allowed a reliable quantification down to 5 ng/ml. in order to develop an assay with high sample throughput and to obtain maximum response for the analytes we required the shortest possible retention time. to implement the determination of retigabine in a second step in the routine tdm of anticonvulsant drugs the corresponding hplc method was selected: a purospher rp18 column (55 mm x 2 mm; 4 µm, merck) and a mobile phase with a steep acetonitrile gradient. results: the great advantage of having analytes with different molecular masses and similar retention times in combination with ms/ms detection enabled us to aim at a minimum separation that might remove some salts or matrix components that can suppress or interfere with the analyses from the target components, while maintaining good sample throughput. the method was validated. the assay is precise, accurate, fast, sensitive, and selective. discussion: the developed method is suitable for therapeutic drug monitoring of retigabine. the correlation of the serum concentration and the effect of the drug and thus the necessity of tdm have to be tested. targeting inflammatory t lymphocytes with conditional chemokine receptor antagonist expression for a tissue-specific therapy of chronic inflammatory disorders ogrissek n., giegold o., pfeilschifter j., radeke h. h. uniklinikum der goethe-universität pharmazentrum / zafes, theodor-stern-kai 7, 60590 frankfurt am main, germany chemokines and their receptors are known to be involved in the pathogenesis of chronic inflammation and autoimmune diseases. several approaches tried to use chemokine receptor antagonists as therapeutics to reduce exagerrated immune response, however, due to compensation and systemic side effects clinical trials often failed. in previous experiments our group identified three promising antagonists. cxcl11(4-79) has antagonistic function for cxcr3, cxcl12(p2g2) is able to inhibit cxcr4 and the herpesvirus 8 encoded protein vmip-ii interferes with ccr1, -2 and -5 as well as with cxcr3, -4 and cx3cr1. their expression and secretion was confirmed in pichia pastoris and antagonistic function has been proven by a reduction of t cell migration. the aim of this project is to develop a cell-based therapy for chronic inflammation with a treatment that is based on the collective effect of cxcl11(4-79), cxcl12(p2g2) and vmip-ii. with targeting of stable transduced memory t cells these antagonists should be conditional expressed and secreted directly in the centre of inflammation, resulting in inhibition of further inflammatory t cell accumulation. to realize this project we first cloned constitutive lentiviral constructs containing these antagonists and optimized transduction of t cells, such as the ova-specific memory th-1 cell clone if12 with the potential to initiate antigen specific nephritis in scid mice. next we investigated expression and secretion of cxcl11(4-79), cxcl12(p2g2) and vmip-ii with pcr, western blot and elisa. at the moment we want to measure the inhibition efficiency of t cell migration in vitro with chemotaxis and flow chamber assays. construction of an inducible lentiviral vector plasmid to ensure expression of the antagonists only upon t cell activation, is also part of our current work. finally we would like to test the chemokine receptor antagonists in vivo in two relevant mouse models of type-1-diabetes and contact dermatitis. small heterodimer partner 1 (shp-1) is a member of the superfamily of nuclear receptors (nrs). in contrast to other nrs this orphan receptor lacks the dna binding domain. however, shp-1 is known to inhibit activity of several nrs by direct proteinprotein interaction. importantly several of the interacting nrs have been shown to directly regulate shp-1 expression, suggesting that shp-1 is involved in negative feedback loops of various metabolic pathways, such as cholesterol-, bile acid-and drug metabolism and glucose homeostasis. recently binding sites for nrs were identified in the promoter region of shp-1, including hnf4α, lrh1, lxr, fxr, srebp1c and pparγ. the aim of our study was to identify single nucleotide polymorphisms (snps) in the promoter region of shp-1 and to determine their impact on the transactivation of shp-1. 120 dna samples from subjects of the population based cohort study of health in pomerania were analyzed by sanger sequencing, thereby we identified four snps namely -594t>c (rs71636795), -413g>c, -423 c>t (rs78182695) and del-195ctga (rs145613139). subsequently those polymorphisms were tested for their functional consequence performing cell based reporter gene assays testing all above mentioned modulators (lrh1, lxr, fxr, srebp1c and pparγ) of shp-1 expression. only the transactivation by hnf4α was decreased in the presence of the -423 c>t polymorphism to 69% and the -413g>c polymorphism to 75%. in conclusion we described snps with impact on transactivation. it will be aim of future studies to determine the potential impact on physiological processes or disease development. autosomal recessive polycystic kidney disease (arpkd) is a rare genetic disease, afflicting about 1 in 20.000 individuals. arpkd is characterized by cystic fusiform dilatations of the renal collecting ducts leading to massive enlargement of the kidneys and ultimately loss of renal function. in addition, the patients suffer from congenital hepatic fibrosis (chf), possibly leading to portal hypertension and liver enlargement. so far, there is no cure for arpkd. therapy is focussing on controlling the disease symptoms [1] . mutations in the pkhd1 gene cause arpkd. more than 300 different mutations in this gene have been reported, all leading to the same phenotype, though there are differences regarding the severity of the disease [2] . in animal models of autosomal dominant polycystic kidney disease (adpkd) as well as arpkd elevated levels of camp were shown [1] [2] [3] . in isolated kidney cells camp stimulates cl-secretion and activates the b-raf /mek/erk pathway. these both are important factors for cyst development and disease progression [2, 3] . intracellular camp regulation is based on conversion of atp to camp by adenylyl cyclases (acs) and degradation by phosphodiesterases . referring to this, we asked the question if there are differences in the activation and expression pattern of acs in pck rats, an animal model of arpkd [4] and in sprague dawley rats. therefore, we examined membranes in a radioactive ac activity assay using various stimulatory compounds, e.g. forskolin, a direct ac activator, or hormones like glucagon and vasopressin to characterize acs. furthermore, we examined ac isoform expression on the mrna level via rt-pcr. we observed that in pck rats ac activity was decreased in general in comparison to sprague dawley rats. in future experiments we are aiming to obtain further knowledge about the influence various hormones exhibit on pck rat acs and to biochemically characterize acs. the major pathogenicity factors tcda and tcdb from clostridium difficile monoglucosylate and thereby inactivate small gtp-binding proteins of the rho subfamily after entering host cells via receptor-mediated endocytosis. although the intracellular mode of action of the toxins is well understood, far less is known about binding structure and internalization pathway of tcda and tcdb. since antibodies directed against the c-terminal located clostridial repetitive oligopeptides (crops) are able to neutralize toxin cytotoxicity the crop domain is acknowledged to mediate receptor binding. however, we recently demonstrated that crop deletion mutants of tcda (tcda 1-1874 ) and tcdb (tcdb 1-1852 ) enter host cells and exhibit full cytopathic potency though lacking the proposed receptor binding domain. we therefore refute the accepted opinion of a solely crop-mediated toxin uptake and re-evaluate the role of the crops in toxin endocytosis. tcda 1-1874 and tcdb 1-1852 induced time and concentration dependent cell rounding and rac1-glucosylation. however, depending on the cell line, truncated toxins exhibit up to 10-fold reduced potency towards host cells compared to the respective full length toxin. the observed difference in toxin potency might reflect the recognition of different receptor structures or the use of various endocytotic routes. interestingly, pre-incubation of cells with the isolated crop domain enhances binding as well as cytotoxicity of subsequent applied truncated tcda indicating that the crops primarily determine toxin uptake. in fact, competition experiments revealed that tcda and tcda 1-1874 predominantly use different receptor structures corroborating the notion of alternative internalization processes utilized by tcda. different routes for cellular uptake might enable the toxins to enter a broader repertoire of cell types leading to the observed multifarious pathogenesis of c. difficile. thus, characterization of alternative endocytotic pathways used by the c. difficile toxins might therefore be the basis to investigate the opportunity of toxin uptake inhibition as therapeutic option. in neurodegenerative diseases, such as alzheimer´s disease and parkinson´s disease, mitochondrial pathways of apoptosis are considered as major features of the underlying neuronal cell death. such mitochondrial mechanisms of apoptosis are mediated by the bh3-only protein bid, a member of the bcl-2 family that triggers mitochondrial permeabilization and the subsequent release of death-promoting proteins into the cytosol. the pivotal role of bid in apoptotic cascades of neuronal cells has been shown in our previous studies showing a neuroprotective effect of bid sirna and small molecule bid inhibitors such as bi6c9 in vitro. in vivo, however, the available bidinhibitors failed to protect brain tissue likely because the compounds were not bioavailable or did not cross the blood brain barrier. therefore, chemical modifications of bi-6c9 were generated resulting in new structures and molecules with different pharmacophors. the aim of the present study is to identify novel potent bid inhibitors available for applications in model systems of brain damage in vivo. for the first screening of 80 compounds we used a model of glutamate toxicity in immortalized mouse hippocampal neurons (ht-22 cells). in this model system, glutamate induces a decrease of intracellular glutathione levels resulting in lipoxygenase activity and enhanced formation of toxic reactive oxygen species (ros). to investigate the compounds' ability to prevent glutamate induced cell death, we first analyzed the cell viability by the mtt assay. in addition, we examined the cell survival by using real time monitoring of cell impedance (xcelligence system) to determine the neuroprotective potency of the new structures. using these assays, we identified 10 novel molecules that significantly prevented glutamate-induced toxicity in ht-22 cells. further we were able to express and to purify recombinant bid in a high amount. in the ongoing study the purified bid protein will be used for co-crystallization with the identified neuroprotective structures for further optimization of novel bid inhibitors for therapeutic applications in experimental models of neurodegenerative diseases in vivo. polymorphic enzymes, urinary bladder cancer risk and structural change in the local industry ovsiannikov d. 1 , selinski s. in the 1990s, an uncommonly high percentage of glutathione s-transferase m1 (gstm1) negative bladder cancer cases (70 %) was reported in the greater dortmund area (golka et al., 1997) . the question arose whether this uncommonly high percentage of gstm1 negative bladder cancer cases was due to environmental and occupational exposure decades ago. thus, 15 years later, another study on bladder cancer was performed in the same area after the coal, iron and steel industries had finally closed in the 1990s. in total 196 bladder cancer patients from the st.-josefs-hospital dortmund-hörde and 235 controls with benign urological diseases were investigated by a questionnaire and genotyped for gstm1, gstt1 and the n-acetyltransferase 2 (nat2) tag snp rs1495741. the frequency of the gstm1 negative genotype was 52 % in bladder cancer cases and thus much lower, compared to a previous study performed from 1992-95 in the same area (70%). nat2 genotypes were distributed equally among cases and controls (63% slow acetylators). less gstt1 negative genotypes were present in cases (17%; controls 20%). apoptosis inducing factor (aif) has been identified as a key factor in intrinsic pathways of caspase-independent neuronal death in model systems of acute brain injury and neurodegenerative diseases, such as alzheimer's disease and parkinson's disease. aif is a mitochondrial intermembrane flavoprotein with the capacity to translocate to the nucleus where it induces chromatin condensation and large-scale dna fragmentation. previous studies revealed that aif deficiency leads to protective effects in different models of neuronal death in vitro and in vivo. however, aif also plays an important physiological role for the integrity and function of the mitochondrial respiratory chain. thus, aif deficiency may significantly alter mitochondrial functions and metabolic homeostasis thereby preconditioning the cells to tolerate subsequent stress stimuli. the present study addresses this hypothesis and investigates whether neuroprotection by aif depletion was attributed to a preconditioning effect, i.e. protecting mitochondrial function and integrity. as model system we use glutamate induced oxytosis in immortalized mouse hippocampal ht-22 neurons. silencing of aif expression by sirna (20nm) protected mitochondrial morphology and integrity against glutamate induced damage. microscopy analysis of the mitochondrial morphology revealed that aif sirna prevented mitochondrial fission. furthermore, facs analysis confirmed that mitochondrial membrane potential was stable in cells with aif silencing. this protection of mitochondrial morphology and integrity by aif depletion was associated with preserved atp levels and inhibition of cell death as detected by an mtt assay. pronounced formation of lipidperoxides as another indicator of mitochondrial damage was also attenuated in cells preconditioned by aif sirna. these protective effects of aif sirna were highly similar to effects obtained with low doses of rotenone (20nm), which was applied as an inhibitor of complex i and mediated comparable preconditioning effects in the ht-22 cells. overall, these findings support the conclusion that aif depletion mediates a preconditioning effect protecting neuronal cells from a subsequent glutamate toxicity. in order to link these preconditioning effects to complex i functions, protein expression and functional analysis of complex i are being analysed to identify the molecular mechanisms of aif dependent control of neuronal life and death. -dependent inactivation and display very slow voltage-dependent inactivation. both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained ca 2+ influx through cav1.4 channels is required to couple slowly graded changes of the membrane potential with tonic glutamate release. mutations in the gene coding for cav1.4 cause severe impairment of retinal circuitry function and have been linked to congenital stationary night blindness type 2a (csnb2), aland island eye disease (aied) and cone-rod dystrophy type 3 (cordx3). the clinical phenotypes of these eye diseases vary substantially regarding the ratio of rod to cone functional impairment. the reasons for this variability are not known. to gain more insights into the pathophysiology caused by loss of cav1.4 function we analyzed the visual phenotype of cav1.4-deficient mice. to this end, we combined immunohistochemistry, electroretinography (erg) and vision-dependent behavioral testing. immunohistochemical analysis using synaptic and postsynaptic markers revealed severe synaptic defects in cav1.4-deficient mice. heterozygous cav1.4 mice showed mosaic synaptic defects most probably caused by random x-chromosomal inactivation of the healthy allele. electroretinography revealed a loss of scotopic and photopic photoreceptor function. this loss of retinal network function resulted in impaired performance of cav1.4 knockout mice in a water maze-based behavioral test of rod and cone function. in conclusion, loss of cav1.4 channels strongly impairs rod and cone retinal function and vision in mice. lsr is the host cell receptor for clostridial iota-like toxins papatheodorou p., aktories k. albert-ludwigs-universität freiburg institut für experimentelle und klinische pharmakologie und toxikologie, albertstr. 25, 79104 freiburg, germany the human enteric pathogen clostridium difficile is the most serious cause of antibioticassociated diarrhea and pseudomembranous colitis. hypervirulent strains of the pathogen, associated with more severe disease and increased death rates, produce the binary actin-adp-ribosylating toxin cdt (c. difficile transferase) in addition to the rho glucosylating toxins a and b. cdt is member of the family of clostridial iota-like toxins, including c. spiroforme toxin (cst) and the eponym c. perfringens iota toxin. the toxins induce depolymerization of the actin cytoskeleton and the formation of microtubulebased cell protrusions that increase adherence and colonization of clostridia. using a haploid genetic screen, we identify the lipolysis-stimulated lipoprotein receptor (lsr) as the target molecule for entry of cdt into host cells. in addition, we present evidence that lsr is shared as a cell entry point by all members of the iota-like toxin family. identification of the toxin receptor provides a most valuable basis for antitoxin strategies. bisphenol a (bpa) is a chemical of high interest due to its endocrine activity. controversy exists concerning the blood concentration due to normal exposures. some authors claimed to have measured concentrations in the ng/ml range which is in contrast to kinetic properties of bpa. bpa is excreted in the urine as glucuronide and sulfate metabolites. recently, data on the in vitro metabolism of bpa by recombinant udpglucuronyltransferase 2b15 enzymes (ugt2b15) revealed that ugt2b15.2 and ugt2b15.5 had markedly lower intrinsic clearance as compared to ugt2b15.1 (hanioka et al., 2011) . using the in vitro metabolism data, we scaled the kmand vmaxvalues in an established human physiologically based toxicokinetic (pbtk) model (mielke and gundert-remy, 2009, mielke et al., 2011) to the values of the variants. for oral doses at relevant exposure levels, the maximum blood concentration (cmax) for the ugt2b15.2 variant (v2) was 5 fold and those of the ugt2b15.5 variant (v5) was 7 fold higher than that of the ugt15.1 variant. with dermal exposure at a relevant exposure level, the cmax values were 1.4 (v2) and 1.6 fold (v5) of ugt15.1 variant. a combined exposure of oral and dermal exposure, an exposure scenario, which occurs in daily life, resulted in 2.4 fold (v2) and 3.2 fold (v5) higher cmax values as compared to ugt15.1 variant. the values for the area under the blood concentration time curve (auc) were for a relevant oral dose 5.7 fold (v2) and 8.6 fold (v5), for relevant dermal exposure 1.4 fold (v2) and 1.6 fold (v5), and for combined exposure 1.9 fold (v2) and 2.5 fold (v5) of ugt15.1 variant. from the results we conclude: (1) polymorphism of udpglucuronyltransferase (2b15.2 and 2b15.5) has an impact on the blood concentrations which, however, is less than 10 fold for cmax and for auc. the effect is more pronounced for oral as compared to dermal or combined exposure. (2) polymorphism of metabolism does not explain the blood/plasma concentrations in the ng/ml range measured by some authors. hanioka n, oka h, nagaoka k, ikushiro s, narimatsu s. effect of udpglucuronosyltransferase 2b15 polymorphism on bisphenol a glucuronidation. arch toxicol. 2011, 85(11) :1373-81. mielke h, gundert-remy u. bisphenol a levels in blood depend on age and exposure. toxicol lett. 2009 8; 190(1) :32-40. mielke h, partosch f, gundertthe heart responds to maladaptive pro-hypertrophic stimuli by stimulating intrinsic signals that contrast and dampen the onset and development of hypertrophy. cyclic guanosine monophosphate (cgmp) and its downstream effector cgmp kinase i (cgki) have been suggested to be an important anti-hypertrophic signaling pathway (1) . intracellular levels of cgmp can be raised by the action of nitric oxide (no) and natriuretic peptides (anf, bnf), or by inhibiting cgmp-degrading phosphodiesterases (pde). a growing body of evidence suggests that the pde5 specific inhibitor sildenafil (sil) prevents and reverses hypertrophy and chamber remodelling in the heart of mice subjected to thoracic aorta constriction (tac) by elevating cgmp levels and cgki activation (1) . in contrast, using a mouse model that lacks cgki expression in every cell type except smooth muscle cells (βres mice; see ref. 2), we recently showed that the absence of this kinase does not alter the onset of hypertrophy induced by tac or isoproterenol infusion (2) . sil is believed to increase cardiac cgmp levels, although it is unclear, if its target (pde5) is expressed in cm (2). sil may act on other pdes, such as pde1c which is abundant in cms. it is also unclear if sil effects are mediated by other cardiac cell types, in particular by cardiofibroblast. to answer these questions, we are currently investigating whether sil is able to prevent hormone induced cardiac hypertrophy in the absence of cgki in cm. preliminary results on βres mice show that even in the case of chronic angii infusion, lack of cgki in cm does not alter the induction of hypertrophic response, at least in the initial phase (7days of angii infusion at 2mg/kg/day). interestingly, βres mice showed impaired cardiac function, as indicated by decreased fractional shortening. sil was able to partially block the onset of cardiac hypertrophy in wt animals, but not in βres mice, indicating a requirement of cgki in this process. in particular, sil was able to block the transcription of pro-fibrotic genes such as tgfβ, ctgf, collageni and fibronectin. the overexpression of the somatostatin receptors sst2 and sst5 in neuroendocrine tumors provides the molecular basis for therapeutic application of the stable somatostatin analogs octreotide and pasireotide. whereas the phosphorylation of the carboxyl-terminal region of the sst2 receptor has been studied in detail, little is known about the agonist-induced regulation of the human sst5 receptor. here, we have generated phosphosite-specific antibodies for the carboxyl-terminal threonines 333 (t333) and 347 (t347), which enabled us to selectively detect either the t333-or the t347-phosphorylated form of sst5. we show that agonist-mediated phosphorylation occurs at t333, whereas t347 is constitutively phosphorylated in the absence of agonist. we further demonstrate that the pan-somatostatin analog pasireotide and the sst5-selective ligand l-817,818 but not octreotide or ke108 were able to promote a clearly detectable t333 phosphorylation. however, none of these compounds was able to stimulate t333 phosphorylation and sst5 internalization to the same extent as the natural somatostatin. agonist-induced t333 phosphorylation was dose-dependent and selectively mediated by g protein-coupled receptor kinase 2 (grk2). like that observed for the sst2 receptor, phosphorylation of sst5 occurred within seconds. however, unlike that seen for the sst2 receptor, dephosphorylation and recycling of sst5 were complete within minutes. we also identify protein phosphatase 1g (pp1g) as sst5 receptor phosphatase. together, we provide direct evidence for agonist-selective phosphorylation of carboxyl-terminal t333. in addition, we identify grk2-mediated phosphorylation and pp1g-mediated dephosphorylation of t333 as key regulators of rapid internalization and recycling of the human sst5 receptor. termination of signaling of activated g protein-coupled receptors (gpcrs) is essential for maintenance of cellular homeostasis. although the regulation of agonist-induced phosphorylation has been studied in detail for many gpcrs, the molecular mechanisms and functional consequences of receptor dephosphorylation are far from understood. recent studies have shown that phosphatase inhibitors, such as okadaic acid and calyculin a, can block the dephosphorylation of a number of gpcrs including the ß2 adrenergic receptor, d1 dopamine receptor, parathyroid hormone receptor 1, thromboxane a receptor and the vasopressin receptor 1. however, a specific phosphatase has not been identified so far. in present studies, we have examined the mechanism and function of receptor dephosphorylation using the sst2a somatostatin receptor and the µ-opioid receptor (mor) as models. within those analyses, we have identified protein phosphatase 1beta (pp1ß) as the phosphatase for the cluster of phosphorylated threonines (353ttetqrt359) within the sst2a somatostatin receptor carboxylterminus using sirna knock down screeening. those phosphorylation sites mediate ß-arrestin binding. we have also identified protein phosphatase 1gamma (pp1γ) as mor phosphatase that catalyzed t370 and s375 dephosphorylation at or near the plasma membrane within minutes after agonist removal. here, we show the different activated phosphatases with functional selective mutants. we examined tailswap mutants which specify the different phosphatase activities. therefore we produced a mor-rsst2a chimera with the rsst2a c-terminal tail and a rsst2a-mor chimera with a mor c-terminal tail. detoxification by conjugation of glutathione? formations of dna adducts of patulin activated by glutathione pfenning c., lehmann l. university wuerzburg, institute of pharmacy and food chemistry section of food chemistry, am hubland, 97074 wuerzburg, germany as a frequent contaminant in apple juice, the mycotoxin patulin (pat) has shown mutagenic potential in cultured mammalian cells at concentrations which are equivalent to those found in marketable foods. this fact is in contrast with the assumption that conjugation to the major intracellular nucleophile glutathione (gsh) leads to detoxification of the electrophile pat. although pat reacts readily with gsh, previous studies showed that co-incubation of pat with model thiols and amine compounds increased the reactivity of pat towards amines forming mixed-type adducts. thus, we hypothesise that the potential to react with dna bases after being activated by gsh might contribute to the mutagenicity of pat. adduct formation of dna bases (adenine, guanine or cytosine) with pat in the presence and absence of gsh was studied under neutral conditions. liquid chromatography coupled with electrospray ionization tandem mass spectrometry was applied for identification and structure elucidation of putative adducts. besides published as well as hitherto unknown pat-gsh adducts, several pat-dna base adducts were formed both in presence and absence of gsh. in addition, with each of the three dna bases one product exhibiting a mass to charge ratio and fragmentation pattern suggesting a mixed thiol-amine adduct was detected. based on the fragment ions of adducts formed with gsh and chemically modified derivatives, we postulate a cyclic structure of the pat-gsh-dna base adducts, resulting from the reaction of the α-amino group of the glutamic acid residue with the c7-carbonyl function of pat. the exocyclic amino group of the dna base is linked to c1 of the pat backbone by an amid bond. thus, the present study demonstrates the reactivity of pat towards dna bases and the participation of gsh in adduct formation. the postulated structures of dna adducts could be used as biomarkers for the determination of the internal exposure to pat in humans. prescribing information detailed in the summary of product characteristics (spc) forms the officially approved basis for safe prescribing of drugs. in a project funded by the german federal ministry of education and research (bmbf) we aimed to derive an internationally valid data set for safe prescribing of psychiatric drugs and therefore analyzed and compared the content of internationally available prescribing information. a team of pharmacists and clinical pharmacologists performed an in-depth comparison of the german, swiss, british and us-american spcs of 10 top prescribed psychiatric drugs. for 7 drugs (of identical pharmaceutical form) the spcs from the same manufacturer were available for all countries, whereas for three drugs spcs were only available from different companies. in these cases the most recent prescribing information from each country was included in the comparison. in 40 spcs 2220 individual data points (55.5±17.4 per individual spc) were compared. between countries the timeliness of prescribing information for an individual drug varied by a median of 18.5 (range: 6-134) months. the respective spcs covered on average 71.4±30.3% (range: 12.5-100%) of all mentioned indications and 70.1±24.4% (range 15.4-100%) of all mentioned contraindications. the warnings and precautions section of an individual spc covered on average 59.5±17.1% (range: 12.5-93.3%) of all mentioned warnings and precautions for that drug. the variation observed was only marginally improved when restricting the analysis to the 28 spcs of the 7 drugs available in all four countries from the same manufacturer. across countries, the summary of product characteristics of individual psychiatric drugs show substantial variation in crucial prescribing information. as different manufacturers are unlikely to explain much of the observed variation, these data argue for a better international cooperation and standardization of the content of summary of product characteristics. this project is supported by the german federal ministry of education and research (bmbf), project grant no. 01 ex1015b. protein kinase ck2 (former name 'casein kinase 2') is a highly conserved serine/threonine kinase, which acts as a component of regulatory networks implicated in many cellular processes but is also linked to various types of human cancer [1, 2] . elevated ck2 activity has been associated with aggressive tumor behavior and results in growth advantage, enhanced survival and dynamic adaption to stress of cancer cells [3] . ck2 is a heterotetramer consisting of two catalytic subunits (ck2α) attached to a dimer of regulatory subunits (ck2β) [4] . ck2β stabilizes ck2α against denaturation and modulates the substrate specificity of the catalytic subunit [5] . due to the relatively small and hydrophobic ck2α-ck2β interface (832 å²) [4] , low molecular weight inhibitors are able to interfere with the ck2 subunit interaction and thus affect the kinase activity [6] . such inhibitors might exhibit an increased specificity in comparison to those compounds interacting with the conserved atp binding site [3] . we have developed an elisa-based ck2α-ck2β binding assay using recombinant human ck2 subunits. different blocking reagents were analyzed to minimize nonspecific binding. the optimized binding assay was then applied to screen for inhibitors of the ck2 subunit interaction. primary hits were further characterized by determination of the parameters ic50 and ki as well as by comparing the results from the binding assay with literature-known or recently obtained crystal structures. numerous epidemiological studies have shown associations between exposure to ultra fine particles and an increase in cardiovascular diseases such as atherosclerosis, coronary heart disease and myocardial infarction. ultra-fine particles have an aerodynamic diameter of < 0.1 µm and are highly diverse with impurities of transition metals and organic compounds (e.g. polycyclic aromatic hydrocarbons). posttranslational modification (ptm) of proteins, particularly phosphorylation, is a key element in the regulation of cell function and any disturbance can lead to multiple diseases. the present study focused on the proteomic-based identification of phosphorylated proteins to understand the mechanism behind ultrafine particle exposure and cardiovascular disease development. as one of the major sources for ufp emissions are diesel exhaust, therefore to mimic the diesel particles, carbon black (cb) and benzo[a]pyrene loaded carbon black (cb+) were used in the present study. cells of the endothelial cell line ea.hy926 were exposed for 14 days to 100 ng/ml cb and cb+. phosphoprotein extraction of whole cell lysates was carried out by the method developed in our lab 1 . the obtained proteins were then separated by two-dimensional gel-electrophoresis followed by maldi-tof-ms (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) analysis of differently expressed proteins. to further validate the results invasive potential of cells were monitored by plating exposed cells for 24 hrs on top of matrigel-coated inserts. differential expressions of 20 phosphoproteins were found in cb-treated cells while an altered expression of 33 phosphoproteins was observed in cb+-treated cells. the maldi-tof analysis revealed proteins involved in the regulation of the endothelial permeability and the cellular plasticity such as vimentin, actin and transitional endoplasmic reticulum atpase. further, the invasion assay supported these results as the cb-exposed cells showed a high invasive potential as compared to control. [1] pink m. et. al. , precipitation by lanthanum ions: a straightforward approach to isolate phosphoproteins, j.protomics, 75, 2011, [375] [376] [377] [378] [379] [380] [381] [382] [383] 302 application of the ttc approach in cosmetics platzek t. bundesinstitut für risikobewertung, max-dohrn-straße 8-10, 10589 berlin, germany regulatory toxicologists in europe have been discussing the ttc approach since more than a decade, e.g. the previous scientific committee on food in 1996. since then, the concept was further developed and is now applied in the eu for the assessment of flavouring substances by efsa. two committees are discussing possible applications: the efsa scientific committee prepared an opinion exploring options for the application in food and feed, e.g. for impurities of food additives, thermal reaction products, food contact materials, contaminants etc. in addition, an eu non-food expert committee consisting of members of sccs, scher and schenir discussed the ttc concept in general as well as additional possible fields of application with the focus on cosmetics and an opinion was already published for public consultation. the proposal to apply the ttc approach also for cosmetic ingredients was introduced by a paper published in 2007 by a colipa (the european cosmetics association) supported working group. major aspects to be considered are the following: 1. applicability domain. the chemical space of the ttc dataset (> 600 compounds) has to be compared with that of cosmetic ingredients (> 10 000 compounds). route to route extrapolation. since no adequate dermal toxicity database is available both data on oral intake used in the ttc approach and on dermal exposure to cosmetic ingredients have to be transfomed to internal exposure figures. gastrointestinal and dermal bioavailability as well as route specific differences in metabolism have to be integrated. exposure. the reliability of exposure estimation is the second pillar of the ttc approach. compared to food, data on exposure to substances in cosmetics and consumer products is scarce. a pragmatic step forward is the comparison of ttcs and noael-derived safe exposure levels for cosmetic ingredients. this work was already done with substances in food contact materials and chemicals from the elincs list. for cosmetic ingredients a similar european project is ongoing. further refinement of the ttc approach is needed taking into account the up-to-date toxicological knowledge. with cosmetics specific problems may arise in praxi: according to the new eu cosmetic legislation the safety of cosmetic products available on the market has to be assessed by the manufacturer or importer and also assessors with limited toxicological experience may apply the ttc approach, e.g. by running the toxtree software. plöttner s., marczynski b., käfferlein h. u., welge p., groth h., engelhardt b., schmitz k., erkes a., brüning t. institut für prävention und arbeitsmedizin der deutschen gesetzlichen unfallversicherung -institut der ruhr-universität bochum (ipa) toxikologie, bürkle-dela-camp-platz 1, 44789 bochum, germany polycyclic aromatic hydrocarbons (pahs) comprise several hundred compounds with different carcinogenic potentials, typically occurring in complex mixtures. due to a lack of data risk estimations for pah mixtures are usually based on those for benzo[a]pyrene (b[a]p). the aim of our present study was to explore the suitability of a permanent human lung cell line as tool for future studies on genotoxicity of pah mixtures. in this pilot study we investigated the time-and concentration-dependent generation of specific anti-benzo[a]pyrene -7,8-diol-9 ,10-epoxide (anti-bpde)-dna adducts as well as cytochrome p450 (cyp) 1a1/1b1 enzyme activities after b[a]p-incubation in vitro. we used metabolically competent a549 lung carcinoma cells which display several characteristics of alveolar epithelial type ii cells. after 24 h and 48 h incubations with different b[a]p-concentrations cytotoxic effects were assessed with the neutral red assay and cyp1a1/1b1 activities using luminescent tests. the formation of specific anti-bpde-dna adducts was determined by hplc with fluorescence detection. a time-and concentration-dependent formation of anti-bpde-dna adducts was observed with maximum rates of 340.5 ± 39.1 and 599.6 ± 132.4 anti-bpde/10 8 nucleotides after incubation with 1 µm (24 h) and 3 µm b[a]p (48 h), respectively. however, the mean adduct rates decreased at higher b[a]p-concentrations. the reduction was more pronounced after 24 h than after 48 h. increased cyp1a1/1b1 activities were observed at > 0.1 -1 µm (24 h) and > 0.1 -3 µm (48 h). a clear decrease of enzyme activities was observed at higher concentrations for both incubation times. in the neutral red assay no more than 10% cytotoxicity in relation to the negative control were found after 24 h incubation with ≥ 10 µm b[a]p and after 48 h with ≥ 1 µm b[a]p. overall, incubation of a549 cells with b[a]p resulted in a time-and concentration-dependent increase of cyp-activities and anti-bpde-dna adducts. this clearly shows that a549 cells are able to generate mutagenic dna-adducts. thus, the in vitro model used in the present work appears suitable for genotoxicity studies of individual pahs and pah mixtures, and therefore may be a useful tool for research on syncarcinogenesis. the β subunit of the cav1.2 channel complex has been shown to play a key role in cav1.2 channel trafficking and channel characteristics like opening probability. furthermore, the last exon of the cacnb2 gene coding for cavβ2, exon 14, contains several potential phosphorylation sites, e.g. for protein kinase a or ca 2+ /calmodulin dependent camkii. pka-dependent phosphorylation mediates β-adrenergic stimulation of cav1.2. potential phosphorylation sites are ser478 and ser479 (in the β2 subunit in rat, perez-reyes et al. 1992 a ) and ser1928 in the c-terminus of the poreforming α1c subunit (lemke et al. 2008 b ) . in cardiomyocytes camkii regulates ca 2+ release and reuptake from and into the sr and is involved in the facilitation of the calcium channel. potential interaction sites between the cav1.2 channel complex and camkii are thr498 in the β2 subunit (in rat, grueter et al. 2006 c ) and ser1512 and ser1570 in the cterminus of the α1c subunit (blaich et al. 2010 d ) . however, the exact pathways remained widely unclear up to now. to clarify these mechanisms and to identify the relevant phosphorylation sites for pka and camkii we established a mouse line carrying a stop codon in exon 14 after aa pro501. this mutation prevented translation of the cavβ2 c-terminus containing the corresponding potential phosphorylation sites mentioned above (cavβ2stop mouse). these mice were viable, showed unaltered expression of the truncated of cavβ protein and unchanged ecg and echocardiography. electophysiological analysis of isolated cardiomyocytes showed no differences in current density, the effect of isoproterenol, the time course of inactivation and the facilitation property when compared to cells isolated from littermate controls. for further investigations we bred the cavβ2stop mice with s1928a mice (lemke et al. 2008 b ) lacking the pka phosphorylation site s1928 in the α1c subunit (s1928aβ2stop mouse) or with sf mice (blaich et al. 2010 d ) lacking the camkii phosphorylation sites s1512 and s1570 in the α1c c-terminus (sfβ2stop). both mouse lines were viable and showed unchanged echocardiography recordings compared to their control littermates and unaltered ecg for s1928aβ2stop mice. electrophysiological investigations on cardiomyocytes of s1928aβ2stop mice showed unchanged β-adrenergic stimulation with isoproterenol compared to littermate controls. these results suggest that β adrenergic regulation of the cardiac cav1.2 channel is not mediated by these phosphorylation sites. introduction. chronic atrial fibrillation (caf) is marked by increased fibrosis which contributes to the perpetuation of the disease. in addition to the role of fibrosis in structural remodeling of cardiac tissue, fibroblasts can couple with cardiomyocytes via gap junction thereby altering the electrophysiological properties of the later and potentially participating in atrial electrical dysfunction. in order to understand the importance of fibroblasts in the pathophysiology of caf, we compared the electrical properties of atrial fibroblasts isolated from patients in sinus rhythm (sr) and caf. methods. fibroblasts were isolated by outgrowth culture from right atrial biopsies and cultivated in medium containing 10% fetal calf serum. we used whole-cell patch clamp techniques to investigate ion currents and membrane potential. results. sr and caf fibroblasts showed similar capacitance (sr: 43.6 ± 4.6 pf, n=33; caf: 54.7 ± 5.1 pf, n = 17) and membrane potential (sr: -21.0 ± 4.3 mv, n = 14; caf: -27. 4 ± 4.8 mv, n = 16) . in both groups, we observed fast activating outward currents with a mean threshold at -20 mv. interestingly, current amplitude was significantly larger in sr than caf cells (sr: 23.8 ± 4.2 pa/pf, n = 15; caf: 6.1 ± 1.0, n = 6; p < 0.05). when maintained in culture for 3-5 weeks, cells from both groups developed na + currents. surprisingly, the fraction of caf cells displaying such currents was larger than the sr counterpart (caf: 38%; sr: 15%). furthermore, na + current amplitude was significantly larger in caf fibroblasts (sr: 6.1 ± 2.0 pa/pf, n = 5; caf: 17.4 ± 4.4 pa/pf, n = 6; p < 0.05). na + currents were not altered by 100 nm tetrodotoxin (ttx), but 10 µm ttx reduced current amplitude to 42% of control, suggesting that the channel involved is the cardiac ttx-resistant isoform nav1.5. conclusion. in the context of caf, fibroblasts undergo electrophysiological changes which need to be thoroughly described. understanding whether those changes contribute to the af substrate might provide new therapeutic targets for the treatment of caf. the initial recruitment of gi to the alpha2a-adrenergic receptor is affected by gprotein-coupled receptor kinases and arrestins prokopets o. s., krasel c., 35043 marburg, germany most g-protein-coupled receptors undergo homologous desensitization after agonist stimulation. in this process, agonist-activated receptors are phosphorylated by gprotein-coupled receptor kinases (grks), followed by binding of arrestins to the still agonist-occupied, phosphorylated receptors. it is assumed that arrestin competes with heterotrimeric g-proteins for the receptor molecule and thereby causes desensitization of g-protein-mediated responses. we tested this idea by investigating the effect of grks and arrestins on the recruitment of g-proteins by the alpha2a-adrenergic receptor. hek293t cells were transfected with yfp-tagged alpha2a-adrenergic receptor and the three subunits of gi1. the g(beta) subunit was cfp-tagged. upon stimulation with noradrenaline, a very rapid, robust increase in fret between the receptor and the g(beta) subunit was observed, confirming previous observations that the alpha2aadrenergic receptor recruits gi with a half-life of around 150 milliseconds. when arrestin3 and grk2 were also co-transfected, the half-life of gi recruitment was substantially delayed, increasing to around 2 seconds. there seemed to be no effect of grk2+arrestin3 cotransfection on a second stimulation; neither the kinetics nor the extent were altered compared to the first stimulation. interestingly, grk2 alone seemed to cause a similar but slightly less pronounced delay of g(beta) recruitment to the alpha2a-adrenergic receptor. in corresponding experiments, we measured the recruitment of arrestin3-cfp to yfp-tagged alpha2a-adrenergic receptors in the presence of grk2. upon stimulation with noradrenaline, we observed a robust increase in fret between the receptor and arrestin3, confirming previous observations that the alpha2a-adrenergic receptor recruits arrestin3. co-transfection of the three subunits of gi1 had no effect on the kinetics of arrestin3 binding to the alpha2a-adrenergic receptors. based on previous results with other receptors, an attenuation of gi recruiting to the alpha2a-adrenergic receptor is quite unexpected. our data suggest that the relation between receptors, g-proteins, grks and arrestins may be more complex than previously postulated. introduction: human urinary bladder expresses mrna of the three known badrenoceptor (b-ar) subtypes (b1, b2, b3) in detrusor and urothelium. we have shown previously that only b3-ar is involved in human detrusor relaxation. to investigate the urothelium-induced modulation of b-ar-mediated relaxation, we have examined systematically whether other b-ar subtypes are involved. human detrusor tissue samples were obtained from patients undergoing radical cystectomy for the treatment of bladder cancer. detrusor strips were studied with and without an intact mucosa layer. muscle strips were precontracted with 1 µm carbachol and relaxation was studied in response to the b-ar agonist ne. a-ar mediated processes were blocked with the a-ar antagonists phentolamine and prazosin. selective b-ars antagonists were used to investigate b-ar mediated relaxation. at the end of each experiment 10 µm forskolin was used to determine maximum camp-mediated relaxation. the presence of intact urothelium reduces potency but not effectivity of ne indicating involvement of urothelium-mediated processes not only during detrusor contraction but also during relaxation. ne-mediated detrusor relaxation is mediated through b3-ar in the absence of urothelium. but in intact detrusor strips b2-ars seem to have an additional inhibitory effect. affinity of the selective b3-ar antagonist l748,337 is unchanged, therefore the intact urothelium does not interact with the function of b3-ars. aldosterone causes oxidative stress and dna damage independent of blood pressure in vivo queisser n., schupp n. universität würzburg institut für toxikologie, versbacherstr. 9, 97078 würzburg, germany background: an inappropriate increase of the mineralocorticoid aldosterone (ald) can be induced by a stimulated renin-angiotensin-aldosterone system. epidemiological studies exploring the connection between hypertension and cancer found higher cancer mortality and an increased risk to develop kidney cancer in hypertensive individuals. we recently showed that ald produces oxidative stress, activates transcription factor nf-kb and is genotoxic in kidney tubule cells. objectives: this study investigated the capacity of ald to induce oxidative/nitrosative stress, dna damage, dna repair, apoptosis, cell proliferation and the activation of nf-κb in rat kidneys. methods: mineralocorticoid-dependent hypertension was induced by ald/salt in sprague dawley rats. dna damage and oxidative/nitrosative stress markers were detected immunohistochemically. results: ald/salt treatment caused increased blood pressure compared to untreated rats. tempol, an antioxidant, and hydralazine, a vasodilator acting independent of the renin-angiotensin-aldosterone system, could lower the blood pressure, while the mineralocorticoid receptor (mr) antagonist spironolactone was administered in a subtherapeutical dose not lowering the blood pressure. ald/salt treatment caused oxidative and nitrosative stress, structural dna damage, double strand breaks, dna repair and nf-κb activation. spironolactone decreased these markers significantly. tempol was also able to reduce these markers, while hydralazine had no effect. ald/salttreated kidneys showed a tendency to lower apoptosis and to increased cell proliferation compared to control rat kidneys. discussion: this study provides a first hint of blood pressure-independent effects of ald. the mr and the production of ros seem to be crucial for the damaging effects of ald. an aberrant or long-term activation of nf-κb by persistently high ald levels could support resistance to apoptosis and the survival of cells with damaged dna, and increase cell proliferation. these actions could contribute to the increased cancer incidence in hypertension by initiating carcinogenesis. grant support by the dfg is gratefully acknowledged. creb regulating transcriptional coactivator 1 (crtc1) is a transcriptional coactivator of the transcription factor creb. we have recently shown its expression in cardiomyocytes and its activation by beta-adrenergic signaling. beta-adrenergic signaling contributes to the pathogenesis of cardiac hypertrophy, leading to heart failure, as evidenced by the therapeutic success of the beta-adrenoceptor antagonists. in order to investigate if crtc1 is involved in this process, we investigated the expression of crtc1 in hypertrophied myocardium from mice and humans. methods: protein lysates from mouse and human samples were investigated for crtc1 protein expression. we distinguished between an acquired and an inherited form of cardiac hypertrophy. acquired cardiac hypertrophy is an adaptation of the heart to an increased cardiac workload and can be found in patients with an aortic valve stenosis. in mice this kind of hypertrophy can be evoked by transverse aortic constriction (tac). the inherited form of cardiac hypertrophy is caused by mutations in genes coding for proteins of the sarcomeric apparatus and is referred to hypertrophic cardiomyopathy (hcm). as a model for hcm, transgenic mice with a mutation in the mybpc3 gene, coding for the sarcomeric protein cardiac myosin-binding protein c (cmybp-c), were used. these transgenic mice were characterized by a hypertrophied heart. in case of human samples we distinguished between patients with an acquired form of hypertrophy due to an aortic valve stenosis, and patients with a hypertrophic obstructive cardiomyopathy (hocm), a special form of hcm. results: in the tac mice and in the myppc3 mutant mice the expression of crtc1 was significantly higher than in the controls (2.1-and 1.9-fold, respectively; n=3). in both forms of human hypertrophy, we found a significantly 5-fold upregulation of crtc1 protein level in comparison to human samples from non-failing myocardium or samples from patients with a dilatative or ischemic cardiomyopathy (n=7-10 for each group). conclusions: crtc1 is upregulated in cardiac hypertrophy with acquired or geneticbased origin. the evaluation of the role of crtc1 in the heart may help to elucidate the role of beta-adrenergic signaling in the development of cardiac hypertrophy. microarray gene expression profiling reveals up-regulation of the cardiac lipid metabolic process at the onset of heart failure fu x., abd alla j., quitterer u. eth zürich molekulare pharmakologie, winterthurerstrasse 190, 8057 zürich, switzerland atherosclerosis and chronic pressure overload are major cardiovascular risk factors for the development of heart failure in patients. to mimic those risk factors in experimental models we used atherosclerosis-prone apolipoprotein e (apoe)-deficient mice, and chronic pressure overload was imposed by abdominal aortic constriction (aac). cardiac function was monitored by echocardiography. severe atherosclerosis in aged apoedeficient mice or chronic pressure overload induced signs of heart failure as evidenced by a significantly reduced cardiac ejection fraction (<30%). to investigate pathomechanisms underlying the development of heart failure, microarray gene expression analysis and qt-rt-pcr were performed of failing heart tissue relative to age-matched controls. gene ontology analysis of the microarray data revealed that the onset of heart failure, in two different experimental models, was characterized by a strong up-regulation (≥2-fold) of the cardiac lipid metabolic process and lipid overload. lipid metabolism genes were involved in lipid synthesis, storage and oxidation. the major palmitate-synthesizing enzyme, fatty acid synthase, was causally related to the development of cardiac dysfunction by enhancing cardiomyocyte apoptosis. taken together the data support that the onset of experimental heart failure is characterized by a dysfunction of the cardiac lipid metabolism promoting cardiomyocyte death. at1-receptor blockers (arbs) are established for the treatment of high blood pressure and new onset of diabetes is reduced by arbs. in the past years evidence increased that body weight may also be lessened particularly in rats and mice. however, less data are available whether arbs still reduce weight, when treatment was initiated not until animals became obese by diet. prior to drug treatment, spontaneously hypertensive rats were fed for 6 months with chow but also with a cafeteria diet (cd) to develop obesity. controls received only chow (conchow). cd-fed shr were treated for 3 months with telmisartan (tel 8mg/kg/d) or amlodipine (aml, 12 mg/kg/d), whereas controls received vehicle (concd). systolic blood pressure (sbp), feeding behaviour, body weight, abdominal fat mass (by mrt) and energy expenditure (by indirect calorimetry) was monitored. leptin sensitivity was assessed by measuring energy intake and expenditure after repetitive injections (s.c.) of leptin. insulin sensitivity was functionally determined by glucose and insulin tolerance tests. due to cd feeding body weight was increased after 6 months by more than 60 g. tel normalized sbp whereas it remained >200 mmhg in concd and conchow. tel additionally reduced cd-induced increase of body weight and abdominal fat mass. food intake was diminished during the first 4 weeks, but raised beyond control levels during the last 4 weeks of treatment. the shift of the respiratory index to lower levels indicated improved energy expenditure. in response to exogenous leptin, the food intake of concd was higher compared to conchow, indicating a leptin resistance. this assumption is further supported by high triglyceride concentrations of concd. after tel, leptininduced food intake was reduced and energy expenditure was increased compared to concd, indicating that leptin sensitivity was at least partially restored. accordingly, triglycerides were reduced. compared to concd, the insulin sensitivity was improved by tel since maximal increases in plasma concentrations of glucose and insulin in response to glucose challenge were reduced, but glucose response to insulin challenge was diminished. even though reduction in blood pressure was almost similar between tel and aml, metabolic and antiobese efficacies of aml were markedly attenuated. we conclude that telmisartan reveals wide efficacies in improving all symptoms of the metabolic syndrome. the pleiotropic effects are not related to the hypotensive action of tel. a β2-adrenoceptor -camp mediated, immediate stimulation of β2-adrenoceptor gene expression in human lung fibroblasts is opposed by a delayed up-regulation of inhibitory factors racké k., lamyel f., kämpfer n., schütz i., warnken m. univ. bonn dept. pharmacol. &toxicol., 53105 bonn, germany based on their bronchodilatory effects, β2-adrenoceptor agonists constitute essential elements in the treatment of bronchial asthma and copd. however, treatment with long-acting β2-adrenoceptor agonists has been associated with possible worsening of airway hyper-reactivity, possibly because of loss of β-adrenoceptor function. therefore, the molecular regulation of β2-adrenoceptor expression was addressed here. mrc-5 human lung fibroblasts were cultured for up to 48 h in absence or presence of test substances, followed by β2-adrenoceptor mrna determination by qpcr. β2-adrenoceptor mrna decreased with a half-life of 25 min after inhibition of mrna synthesis with actinomycin d (30 µm), but increased by 333±85%, 502±52% and 640±165% (means±sem) within 1.5, 4 and 6.5 h, resp. after inhibition of protein synthesis by cycloheximide (30 µm). the β2-adrenoceptor agonists formoterol and olodaterol (1-100 nm) induced a rapid increase in β2-adrenoceptor mrna (maximally within 1 h by 100±19% and 110±19% at 10 nm, resp.). however, after 4 h exposure to 10 nm formoterol or olodaterol a reduction in β2-adrenoceptor mrna by 59±8% and 58±6%, resp., was observed. both, the stimulatory and inhibitory effects of β2adrenoceptor agonists were mimicked by forskolin (10 µm, increase by 88±14% and inhibition by 49±4%) and cholera toxin (5 ng/ml, increase by 76±12% and inhibition by 77±7%). the formoterol-induced up-regulation of β2-adrenoceptor mrna was blocked by actinomycin d, but not by cycloheximide. moreover, in presence of cycloheximide, β2adrenoceptor agonist induced inhibition was converted into a marked stimulation. in conclusion, expression of β2-adrenoceptors in human lung fibroblasts is highly regulated at transcriptional level. the observations with cycloheximide indicate that the β2-adrenoceptor gene is under strong inhibitory control of short-living, not yet identified suppressors. although both, the time-dependent up-and down-regulation of the β2adrenoceptor gene expression by β2-adrenoceptor activation appears to be mediated via adenylyl cyclase -camp signalling, only the stimulatory effect appears to be a direct action on the β2-adrenoceptor gene. et-1 appears to be involved in the pathogenesis not only of pulmonary hypertension, but also in fibrotic remodeling associated with chronic obstructive airway diseases. since human lung fibroblasts (hlf) are a source of et-1 and have been shown to be controlled by muscarinic receptors and β-adrenoceptors, a possible muscarinic and βadrenergic modulation of et-1 expression in hlf was explored. mrc-5 hlf were cultured for up to 24 h in absence or presence of test substances, followed by prepro-et-1 (ppet-1) mrna determination by qpcr. the muscarinic agonist oxotremorine (10 µm) induced an increase in ppet-1 mrna by 180%, an effect prevented by 10 nm tiotropium. the β2-adrenoceptor agonist olodaterol (up to 100 nm) caused a reduction of ppet-1 mrna expression by 45%. the effect of 10 nm olodaterol was prevented by ici 118,551 (1 µm), but not affect by cgp 20712 (3 µm) . the pka agonist 6-bnz-camp (500 µm) caused a reduction in ppet-1 mrna expression by 65%, whereas the epac agonist 8-cpt-2'-o-me-camp (100 µm) caused only a marginal inhibition by 22%. olodaterol (10 nm) strongly opposed the stimulatory effect of 10 µm oxotremorine. an increase in ppet-1 mrna expression by 185% caused by 0.3 ng/ml tgf-β was effectively opposed by 10 and 100 nm olodaterol, resulting in an inhibition comparable to that in absence of tgf-β. however, the increase in ppet-1 mrna caused by a maximally effective concentration of tgf-β (1 ng/ml, increase by 620%) was not significantly affected by 10 or 100 nm olodaterol. likewise, the pkaagonist 6-bnz-camp (500 µm) opposed the increase in ppet-1 mrna expression caused by 0.3 ng/ml tgf-β, but not that caused by 1 ng/ml tgf-β. tgf-β caused, with an ic 50 of 0.3 ng/ml, a marked down-regulation of β2-adrenoceptor mrna expression, maximally by 90% within 6 h. et-1 expression in hlf is stimulated by muscarinic receptors and inhibited by β2adrenoceptors. the effect of β2-adrenoceptors may be mediated via pka. et-1 expression in hlf is markedly up-regulated by tgf-β, but only effects of sub-maximally effective concentrations of tgf-β are opposed by the β2-adrenoceptor -pka pathway, in part because of tgf-β-induced down-regulation of β2-adrenoceptors. since et-1 can promote pro-fibrotic features in hlf, inhibition of et-1 expression could contribute to long-term beneficial effects of long-acting β2-adrenoceptor agonists such as olodaterol and long-acting muscarinic antagonists such as tiotropium. cardiac hypertrophy leads to up-regulation of dipeptidyl aminopeptidase-like proteins in human and rat radicke s., hutschenreuther a., schaefer m. universität leipzig rudolf-boehm-institut für pharmakologie und toxikologie, härtelstr. cardiac hypertrophy is a major risk factor for heart failure and associated morbidity and mortality. functional down-regulation of k + currents is a prominent feature of cells isolated from failing ventricles. a marked decrease in the transient outward potassium current ito has been shown in various models. changes in the k + channel expression differ depending on the species, and the mechanism of induction of heart failure. to study the regulation of ito channel subunits we compared the hypertrophic responses in human ventricular tissues from failing hearts with doxorubicin-induced hypertrophy in rats and in h9c2 embryonic rat cardiac cells. specifically, we quantified mrna expression of the pore-forming subunits kv4.3 and kv4.2, the cytosolic β-subunit kchip2 and the transmembrane subunits dpp6 and dpp10 using rt-pcr. treatment with doxorubicin (2 µm) induced hypertrophy and increased the mrna expression of the hypertrophy marker genes anf, bnp and beta-mhc in h9c2 cardiac myoblasts. while kv4.3 was detected in h9c2 cells and hearts from human and rat, kv4.2 mrna was only expressed in adult rats. during hypertrophy kv4.3 was downregulated in human tissue as well as in doxorubicin-treated h9c2 cells compared to the controls. in rat hearts kv4.2 expression was increased after doxorubicin treatment. interestingly, kv4.2 was also found to be up-regulated in rat heart tissues and h9c2 cells after treatment with doxorubicin. as previously shown, kchip2 mrna expression was significantly reduced in tissues of failing hearts. in contrast, kchip2 mrna was upregulated in hypertrophic rat hearts and h9c2 cells. the expression of dpp6 and dpp10 was observed only in human hearts. but mrna levels of both were significantly increased in failing tissues. dpp6 and dpp10 were not expressed in the adult rat heart or h9c2 cells, whereas in rats with doxorubicin-induced cardiac hypertrophy and in doxorubicin-treated h9c2 cells, the mrna of dpp6 and dpp10 was up-regulated. h9c2 cells showed almost identical hypertrophic responses to those observed in human ventricles and rat hearts. this finding validates h9c2 cells as a model for in vitro studies of cardiac hypertrophy. in further studies we will investigate the consequences of a knock-down of dpp6 and dpp10 in doxorubicin-induced hypertrophic h9c2 cells. in preliminary experiments specific short-hairpin rna, targeting dpp6 and dpp10, has been designed and tested in heterologous expression systems. the nonsynonymous c.521t>c germline genetic variation in the liver-specific organic anion transporter slco1b1 is associated with methotrexate pharmacokinetics in pediatric acute lymphoblastic leukemia radtke s. 1 background: methotrexate (mtx) plasma concentration is related to its clinical effect. transport proteins, such as abcc2, slco19a1, and slco1b1, have been implicated in the disposition of mtx. here we investigated whether common reduced-function variants in abcc2, slco19a1, and slco1b1 contribute to the interindividual variability in methotrexate pharmacokinetics in children with acute lymphoblastic leukemia (all). we analyzed mtx pharmacokinetics (mtx plasma concentration at the end of infusion c24h, mtx auc24-48h, and mtx clearance cl) in an unselected population of 419 children with all from the all-bfm 2000 trial (clinicaltrials.gov: nct00430118) who received 1676 courses of mtx at 5 g/m 2 as 24 h infusions. the contribution of genes (genetic component, rgc) to the interindividual variability in mtx pharmacokinetics was estimated according to the method of kalow et al. (1998) . abcc2 c.-24c>t (rs717620), slco19a1 c.80g>a (p.his27arg, rs1051266), slco1b1 c.521t>c (p.val174ala, rs4149056) and slco1b1 388a>g (p.asn130asp, rs2306283) genotypes were analyzed by taqman polymerase chain reaction. there was substantial interpatient variability in average (± sd) mtx c24h (50.95 ± 24.15 µmol/l), auc24-48h (57.44 ± 37.52 h*mg/l), and cl (390.72 ± 223.95 ml/min/m²). the rgc values of c24h, auc24-48h, and cl ranged from 0.61-0.71 suggesting that variation in mtx pharmacokinetics has a substantial genetic component. after adjustment for age and sex by multiple regression, the slco1b1 c.521t>c snp was significantly associated with c24h (p<0.001), auc24-48h (p<0.001), and cl (p=0.011) of mtx. compared with the wildtype genotype, in patients with the tc genotype c24h and auc24-48h increased by 18% (p=0.009) and 28% (p=0.003), respectively, whereas cl significantly decreased by 15% (p=0.012). pharmacokinetic variables significantly changed with increasing number of variant c.521t>c alleles (p<0.02, jonckheere-terpstra), suggesting a per allele effect consistent with a co-dominant model of association. in contrast, the abcc2 c.-24c>t, slco19a1 c.80g>a, and slco1b1 388a>g polymorphisms did not show an association with mtx pharmacokinetics. conclusions: the nonsynonymous c.521t>c polymorphism in slco1b1 contributes to the variability of mtx pharmacokinetics in this study of high-dose mtx in pediatric all. this project is supported by the johannes und frieda marohn-stiftung. the antitumorigenic mechanism of the selective cyclooxygenase-2 (cox-2) inhibitor celecoxib is still a matter of debate. using human lung cancer cell lines (a549, h358, h460), the present study investigates the contribution of cox-2 and peroxisome proliferator activated receptor γ (pparγ) to apoptosis elicited by celecoxib. celecoxib was found to cause apoptotic cell death in a concentration-dependent manner (10 -50 µm), whereas structurally-related cox-2 inhibitors (etoricoxib, rofecoxib, valdecoxib) were inactive in this respect. apoptotic cell death by celecoxib was suppressed by preincubation of tumor cells with the selective cox-2 inhibitor ns-398, the pparγ antagonist gw-9662 and by sirna targeting cox-2 or pparγ. celecoxib-induced apoptosis was paralleled by a time-and concentration-dependent upregulation of cox-2 and pparγ at both mrna and protein level. using an established cox-2 activity assay monitoring immediate conversion of exogenously added arachidonic acid to the respective prostaglandins (pgs), ns-398 was shown to suppress celecoxib-induced cox-2 activity when added prior to arachidonic acid. among the cox-2-dependent pgs analyzed, pgd 2 and its dehydration product 15-deoxy-∆ 12,14 -pgj2 were found to induce cytosol-to-nucleus translocation of pparγ as well as pparγ-dependent apoptosis. celecoxib-elicited translocation of pparγ was inhibited by preincubation of cells with ns-398 which itself did not alter celecoxib-induced total pparγ protein expression. finally, a cox-2-and pparγ-dependent proapoptotic mechanism of celecoxib was confirmed in primary tumor cells obtained from brain metastases of two lung cancer patients. together, our data demonstrate a proapoptotic mechanism of celecoxib involving initial upregulation of cox-2 and pparγ and a subsequent nuclear translocation of pparγ by cox-2-dependent pgs. uncoagulated ppp contained 40-60 nm thrombin throughout. fxa was initially measured in clots at 10 nm. this level declined over time while clot-conditioned pbs accumulated fxa. exposure of human aortic smc to clots or native unclotted ppp for 24h only marginally influenced smc apoptosis but increased mitogenesis over 15-fold. this was reduced by all 4 inhibitors. clot-stimulated induction of tnfα and interleukin-6 mrna was also attenuated by the inhibitors. denatured ppp (no protease activity) increased smc mitogenesis to a level seen in smc exposed to clot and combined hirudin + dx9065a, reflecting the well-known mitogenic actions of serum alone. conclusion: coagulation of human plasma generates nanomolar amounts of thrombin and fxa, sufficient to stimulate the proliferative and inflammatory properties of adjacent smc. our observations validate the use of purified thrombin and fxa at nanomolar concentrations for in vitro studies, and support the individual and coagulationindependent roles of these proteases in cell proliferation and inflammation. antithrombotic therapy with argatroban or rivaroxaban may limit the cellular effects of clot-derived thrombin and fxa, while normal anti-platelet therapy would not. this aspect should be considered in the clinical use of these agents, specifically in healing processes after vessel injury. rhogef17 mediates cgmp/cgk induced adherence and relaxation of vascular smooth muscle cells rauch j. 1 , stephan-schnatz k. the guanine nucleotide exchange factor rhogef17 is the only gef known so far to be directly activated by cgmp-dependent kinase. it is expressed in various types of smooth muscle cells and has been shown to play a role in the regulation of cell integrity. in a previous investigation we showed that the knockdown of rhogef17 by a shrna approach caused a loss of actin stress fibers and a subsequent change of smooth muscle cell morphology that finally resulted in cell rounding. we now provide evidence that the expression level of rhogef17 influences the re-attachment of cultured rat aortic smooth muscle cells (rasmc) to a surface after detachment. although rhogef17 depleted rasmc were still able to adhere and spread, their cell surface area remained considerably smaller than that of control cells in the first 24 hours after seeding. cell counting revealed that 6 to 12 hours after seeding the percentage of adherent cells was significantly lower in the rhogef17 knockdown group compared to the control group. these data indicate a delay in attachment. interestingly, the knockdown of rhogef17 was paralleled by a loss in rhoa and cadherine expression. as rhogef17 mediated a cgmp-induced activation of the small gtpase rhoa in rasmc, we studied the effect of a stable cgmp analogon (8-pcpt-cgmp) on the adhesion process. in accordance with previously published data, cgmp treatment accelerated the attachment of rasmc to the surface within the first 12 hours. in contrast, the adhesion of rasmc after rhogef17 knockdown was no longer stimulated by cgmp. as these data indicate that rhogef17 mediates cgmp-dependent signalling in a physiological process we wondered whether this protein might also play a role in the regulation of cgmpdependent relaxation of vascular smooth muscle cells. thus, we performed a collagenbased contraction assay with rhogef17 depleted rasmc. while the contraction in response to serum was not affected by the depletion of rhogef17, we observed a slight decrease in basal contractility. interestingly, cgmp was not able to counteract the serum-driven contraction of rhogef17 knockdown cells. there was no cgmp-induced relaxation in these cells. we conclude that rhogef17 is involved in the cell adhesion of vascular smooth muscle cells and likely promotes the expression of specific proteins. its activation is required to mediate cgmp-induced signalling in terms of vascular smooth muscle cell adherence and relaxation. human eosinophil and neutrophil granulocytes are cells of the innate immune system. they both express formyl peptide receptors (fpr) und histamine h2 receptors (h2r). activation of fpr leads to a release of reactive oxygen species (ros). h2r activation results in an increase of intracellular 3'-5'-cyclic adenosine monophosphate (camp) concentration and inhibition of fpr-mediated ros release via adenylyl cyclase activation. in this study we compared the effects of various h2r ligands on camp accumulation and formyl peptide n-formyl-l-methionyl-l-leucyl-l-phenylalanine (fmlp)induced ros release in isolated eosinophils and neutrophils. camp concentration was determined by hplc/tandem mass spectrometry, and ros release was assessed by monitoring superoxide dismutase-inhibitable reduction of ferricytochrome c. in eosinophils, histamine, amthamine and 5-methylhistamine exhibited similar potencies and efficacies with regard to camp accumulation and inhibtion of ros release. in marked contrast, in human neutrophils, we observed dissociations in potencies and efficacies of ligands at increasing camp accumulation and inhibition of ros production. our data suggest that in human eosinophils, but not neutrophils, camp mediates inhibtion of ros production. in a broader context our data provide a compelling example of the context-dependency of the pharmacological properties of g-proteincoupled-receptors. specifically, one has to be cautious when extrapolating experimental observations from one cell type to another, even when they are very closely related to each other. comonomers and monomers are used as dental restorative materials (e.g. in dental composites). unconverted compounds can be released from dental composites and can enter the body in humans. comonomers can induce various side effects in humans. this study was evaluated to qualify and to quantify eluted compounds from various dental composites. following composites were tested (producer in parentheses): els extra low shrinkage (saremco), synergy duo shade (coltène), grandio (voco), tetric evo ceram (vivadent), venus (kulzer), gradia (g.c.), and premise (kerr).polymerized composites (100 mg) were incubated in gc vials with 1 ml dest. water or 1 ml methanol, each at 37 °c for 72 hours. aliquots were taken, and eluted compounds were analyzed with the method of gas chromatography/mass spectrometry (gc-ms) and liquid chromatography/mass spectrometry (lc-ms).from all composites 18 different compounds were found. following comonomers were quantified (µg/ml; mean ± s.d.; n=4)( fig. 1 ).following range of the eluted and detected comonomers from dental composites was found (dest. water; decreasing elution): venus > gradia > synergy duo shade > tetric evo ceram premise > grandio > els extra low shrinkage. * n.d. = not detectable (below limit of detection). triphenylstibane was detected in tetric evo ceram (5 ± 2 µg/ml). reimann c., lupp a., schulz s. institut für pharmakologie und toxikologie universitätsklinikum jena, drackendorfer str. objective: cxcr4 is a plasma membrane chemokine receptor which is involved e.g. in organogenesis, hematopoesis and inflammation. in the adult organism cxcr4 is physiologically expressed on various cell types, in particular on lymphocytes. with respect to neoplastic tissues, in the current literature an over-expression of cxcr4 is described in different types of tumors, especially in breast and prostate cancer. additionally, an involvement of cxcr4 in tumor metastasis is discussed. subsequently, detection of cxcr4 expression in a given human tumor sample would provide a valuable predictive information on disease prognosis and possible therapeutic intervention. however, previous attempts to localize cxcr4 using poorly characterized mouse monoclonal or rabbit polyclonal antibodies have yielded predominant nuclear and occasional cytoplasmic staining, but did not result in the identification of cell surface receptors. thus, the aim of the present study was to reassess the cxcr4 expression in a panel of formalin-fixed and paraffin-embedded human normal and neoplastic tissue samples by means of immunohistochemistry using the well characterized novel rabbit monoclonal anti-cxcr4 antibody umb-2. methods: in comparison to negative and positive control samples (cxcr4-knockout and wild-type mouse embryos) the extent of staining in the different normal and neoplastic tissue specimens was scored from zero (no expression) to three (high expression). results: cxcr4 was found to be expressed in all neoplastic tissue entities analyzed. in many cases, the receptor was predominantly localized at the plasma membrane of the tumor cells. however, in all cxcr4-expressing tumor entities a huge interindividual variability both in the percentage of positive cells and in the intensity of staining was noted which strongly differed also between the various types of cancer. the most intense (score three) staining was found in the samples of (small cell) lung cancer, ovarian cancer and of pheochromocytoma. additionally, lymphatic organs such as lymph nodes, spleens and tonsils were cxcr4 positive, with mainly b cells displaying a distinct staining of the plasma membrane. the rabbit monoclonal antibody umb-2 may prove to be of great value in the assessment of cxcr4 expression in different human tumor entities and of the mechanisms underlying the formation of metastases, thus helping to find new targets and strategies in cancer therapy. link between β2-adrenoceptor-mediated inhibition of formyl-peptide-induced o2 β2-adrenoceptor (adrb2)-agonists are in daily use for asthma therapy. although most cases of asthma are controlled by standard medication, a subpopulation of asthmatics remains difficult to treat. the adrb2 gene contains a total of 49 polymorphisms. this variability could cause part of the ~70 % genetically-determined differences in therapy response. genetic and corresponding functional data on adrb2 can help to understand the complex disease and, in cases of severe asthma, optimize therapy with adrb2agonists for each individual. (ortega et al. 2007; chung et al. 2011) our present study connects sequence data with pharmacological data of prototypical adrb2-ligands, namely, (r)-isoproterenol, (r,r)-and (s,s)-fenoterol, (r)-and (s)salbutamol and (r,r)-formoterol. as a pathophysiologically relevant cell type, we analysed human neutrophils from peripheral blood of healthy volunteers. formyl-peptide-induced o2 .production and its inhibition by the agonists are examined in a 96-well cytochrome-c assay. characteristic pharmacological values (pic50, emax) are obtained for each individual. the data-set for each individual is supplemented by a differential blood cell analysis and an asthma-related questionnaire. most importantly, each volunteer's adrb2-sequence variant is determined by sanger-sequencing. complete determination of a 1,490 bp sequence, including the entire adrb2 exon and part of the flanking 5'-and 3'-untranslated regions, allows mapping of the most common, but also of new or rare polymorphisms. first data demonstrate the inter-day and intra-individual robustness of the functional data. in the next step, we will link sequence variants and functional differences within a population of sixty volunteers with sufficient statistical power. collectively, this study represents a straightforward approach to link functional and genetic data of a clinically relevant receptor. cyclic nucleotide phosphodiesterases (pdes) are classified into eleven families and are essential for second messenger metabolism in human cells. 1 recently, we have shown that several human pdes possess a much broader substrate-specificity than previously assumed, being capable of hydrolyzing not only the purine nucleotides cyclic adenosine 3',5'-monophosphate (camp) and cyclic guanosine 3',5'-monophosphate (cgmp), but also pyrimidine nucleotides such as cyclic uridine 3',5'-monophosphate (cump). 2 these data were obtained using a highly sensitive hplc mass spectrometric assay which is quite expensive and whose technical requirements are available only in few laboratories. in our present study we developed a fluorimetric pde activity assay using 2'-o-(n'methylanthraniloyl) (mant)-substituted cyclic purine and pyrimidine nucleotides that can be used more broadly in the scientific community. human pde3a is important for the regulation of platelet aggregation, oozyte maturation, vascular smooth muscle relaxation and contractility of cardiac myocytes. 1 moreover, this pde shows a broad substrate-specificity, hydrolyzing camp, cgmp, cump and cyclic inosine 3',5'-monophosphate (cimp). 2 using this enzyme, here, we demonstrate that various mant-substituted cyclic nucleotides are substrates of pde3a and undergo a significant change in fluorescence whilst being hydrolyzed, thus allowing a quantitative analysis of catalysis via fluorescence detection. in fact, not only native cump but also mant-cump is a substrate of pde3a. this finding is consistent with data published by hardman and sutherland 3 who described a cump-degrading pde activity in homogenates from beef and dog heart, leading to the assumption that the pde activity described there could be attributed to pde3a. as cump has furthermore been proven to be present in mammalian cells 4 , to differentially activate camp-and cgmp-dependent protein kinases 5 and to be synthesized from utp by mammalian soluble guanylyl cyclase 6 , our present study supports the hypothesis that this cyclic nucleotide could play an important role in cell metabolism. the newly established fluorescence assay with mant-cump facilitates future studies on pde3a and the assumed second messenger function of cump. rhoa is reportedly involved in stat-dependent transcription. however, the pathway connecting the gtpase and stat signaling has not been characterized. we made use of bacterial toxins, which directly activate rho gtpases to analyse this pathway. cytotoxic necrotizing factors (cnfs) are produced by pathogenic escherichia coli strains and by yersinia pseudotuberculosis. they activate small gtpases of the rho family by deamidation of a glutamine, which is crucial for gtp hydrolysis. we show that rhoa activation leads to phosphorylation and activation of stat3 and identify signal proteins involved in this pathway. rhoa-dependent stat3 stimulation requires rock and junkinase activation as well as ap1-induced protein synthesis. the secretion of one or more factor/s activate/s the jak-stat pathway in an auto/paracrine manner. we identify ccl1/i-309 as an essential cytokine, which is produced and secreted upon rhoa activation and which is able to activate stat3-dependent signaling pathways. the knowledge about the connection between rhoa and stat signaling is crucial for understanding several deseases, especially cancer. acid sphingomyelinase-deficient mice are protected from the lethal cardiovascular effects in tnf-induced septic shock reiss l. k. 1 christian-albrechts universität institut für immunologie, michealisstrasse 5, 24105 kiel, germany introduction: the cytokine tumor necrosis factor (tnf) is a mediator of septic shock. sepsis is a major cause of acute respiratory distress syndrome (ards), a heterogeneous lung disease with a mortality of about 50%. the present study was designed to investigate the effects of high systemic tnf-levels on the lung and on the systemic circulation in wildtype and acid sphingomyelinase-deficient (asm -/-) mice. the enzyme acid sphingomyelinase generates the signaling molecule ceramide that plays a critical role in edema formation and vasodilatation. material and methods: asm -/and wildtype mice were ventilated mechanically at vt=8ml/kg and f=180min -1 with fio2=0.3 and peep=2cmh2o while lung mechanics were followed. half of the mice received 50µg of murine tnf intravenously. blood pressure was stabilized by intra-arterial fluid support and body temperature was kept at 37°c to prevent lethal shock and to allow investigation of blood gases, lung histopathology, pro-inflammatory mediators and microvascular permeability 6 hours after tnf application. results: tnf induced septic shock in wildtype mice, as indicated by metabolic acidosis, high serum levels of the sepsis marker procalcitonin, decreasing blood pressure and reflex tachycardia. interestingly, asm -/mice were protected from the tnf-induced cardiovascular effects and mortality. in the present study, circulating tnf failed to cause lung injury. lung mechanics stayed stable during ventilation in all groups and also pulmonary histopathology, cytokine levels and microvascular permeability were unaffected. conclusion: circulating tnf alone is not sufficient to cause acute lung injury. we conclude that the cardiovascular effects in tnf-induced septic shock are partly mediated by acid sphingomyelinase. cyclophilin a sirna provides mitoprotection and prevents aif-dependent neuronal cell death reuther c. 1 cyclophilin a (cypa) is a peptidyl-prolyl-cis-trans isomerase which is localized in the cytosol. recent data suggested that neuronal cell death involved cytosolic cypa translocation to the nucleus, where it formed a pro-apoptotic complex with apoptosis inducing factor (aif). this cypa-aif complex induced caspase-independent chromatin condensation, dna degradation and cell death in various paradigms of apoptosis. on the basis of these data, the selective inhibition of the aif-cypa complex was proposed as a potential strategy to prevent aif-dependent cell death in neurons. therefore, the aim of this study was to determine effects of cypa silencing in a model of glutamate toxicity in immortalized hippocampal ht22 neurons. first, we addressed the interaction of aif and cypa by immunoprecipitation and their translocation to the nucleus by immunohistochemistry and confocal fluorescence microscopy. after exposure of ht-22 cells to glutamate the translocation of aif and cyp a occurred prior to cell death. cypa sirna attenuated glutamate-induced cell death as detected by the mtt-assay, impedance measurements (xcelligence system), and by facs analysis after annexin v/ propidium iodide staining. most intriguingly, cypa sirna also preserved the mitochondrial membrane potential as shown by facs analysis after tmre staining. further, confocal microscopy showed that cypa silencing prevented mitomorphology alterations and blocked the release of mitochondrial aif to the nucleus. the inhibition of the aif translocation to the nucleus was also shown by western blot analysis. in summary this study demonstrates that silencing of cypa prevents mitochondrial disruption and attenuates glutamate toxicity in vitro. thus, cypa is a promising target for mitoprotection as a basis for novel strategies of neuroprotection. up to now the question is unresolved how the ingested dose influences the absorption and metabolism of chlorogenic acids (cga) from food. so far no studies have been performed on the impact of the dose on cga absorption, circulation and excretion. recently we performed a dose-response study in a randomized, double-blinded, crossover design with five ileostomy subjects. in three trials the volunteers consumed after a two day polyphenol free diet coffee with varying cga content (high 4525 µmol; medium 2219 µmol; low 1053 µmol). the cga concentrations in plasma, ileal effluent and urine were subsequently identified and quantified by hplc-esi-ms, hplc-esi-ms/ms and hplc-dad. the results showed that the consumption of higher cga concentrations lead to a faster ileal excretion measured in the ileal effluents. this corresponded to the renal excretion of 8.0 ± 4.9% (high), 12.1 ± 6.7% (medium) and 14.6 ± 6.8% (low) of total cga and metabolites. we found that cgas with a caffeic acid moiety are predominantly sulphated and those with a ferulic acid moiety are predominantly conjugated via glucuronidation prior renal excretion. furthermore, in the ileal effluents, sulphation of both structural units dominated. in plasma samples (after enzymatic deconjugation) the auc values were determined by the major cga classes in coffee, the caffeoylquinic acids: 551.5 ± 93.8 nm*h -1 (high); 299.3 ± 79.6 nm*h -1 (medium) and 222.8 ± 91.3 nm*h -1 (low). no major differences in the metabolic pattern were observed. additionally, we were able to identify new metabolites of cga in urine and ileal fluids. we conclude that the consumption of high concentrations of cga via coffee might influence the gastrointestinal transit time and consequently affect cga bioavailability. this study was supported by the nestlé research centre (lausanne, switzerland). interaction of antagonists with the atp binding pocket at the human p2x3 ion channel helms n., riedel t., illes p. rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße 16-18, 04107 leipzig, germany the homomeric p2x3 receptor (p2x3r) is a rapidly activating and desensitizing cation channel, gated by extracellular atp. it consists of three homomeric subunits. this representative of the p2x receptor family is highly expressed on sensory afferent neurons and plays a significant role in chronic pain, bladder reflexes and taste sensation. therefore, the development of selective antagonists for p2x3 receptors and knowledge about the binding of these antagonists are of great significance for future pain therapy and therapy of urge incontinence. to simulate the shape of the rapidly desensitizing agonist-induced current responses via p2x3 receptors, we created a specific markov model to describe the binding of agonists and competitive antagonists. this model can be used to prove the competitive character of inhibition and to calculate the association and dissociation constants of the antagonists. furthermore we use this model to fit current responses at p2x3 wild type receptors and their mutants to α,βmethylene atp in the presence of different antagonists. whole-cell patch-clamp recordings were performed on hek 293 cells, heterologously expressing the human p2x3 receptor, to determine the concentration-response relationship of different antagonists. by applying increasing concentrations, differences of antagonist potency could be observed at the wild type receptor. afterwards, we chose amino acid residues for replacement by alanine, which seem to be important for agonist binding and should be so for competitive antagonist binding as well, based on our homology model, developed from the zebrafish p2x4r crystal structure and previous mutagenesis studies. we intend to identify those amino acids which are important for competitive antagonist binding by monitoring the altered antagonist potency on the mutated receptor when compared with the wild-type receptor. analysis of the p2x3 agonist binding site by double mutant cycles riedel t., wiese s., illes p. rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße 16-18, 04107 leipzig, germany purinergic p2x receptors belong to the family of ligand-gated ion channels. they are non-selective cation channels, activated by extracellular atp. one of the seven members of the p2x receptor family, the p2x3 receptor, is localized at the plasma membrane of sensory neurons and is involved in pain perception. therefore, this receptor is a possible target for new drugs in pain treatment. the development of such drugs can be supported by an exact knowledge of the receptor structure and function. there are many hints to the atp binding site, but the interaction of the p2x receptor with its agonists and antagonists remains still unknown. in this study, we investigated the effects of single alanine substitutions of amino acid residues in the supposed atp binding site of the homomeric human p2x3 receptor on the effect of nucleotide analogues. the mutant receptors were expressed in hek293 cells and the nucleotide effects were measured by means of the whole-cell patch-clamp method. modifications in the receptor binding site changed the concentration-response dependency as well as the current kinetics during fast pulsed agonist applications. based on this fact, we were able to distinguish binding from gating, conductance, and desensitisation, using a markov model that describes the complete channel behaviour by a matrix of rate constants. the results were also checked for consistency with a structural hp2x3 model that we developed from the known zebra fish p2x4 crystal structure in the closed state. voltage-dependent modulation of alpha2a adrenergic receptor signaling rinne a., birk a., bünemann m. philipps-universität marburg institut für pharmakologie und klinische pharmazie, karlvon-frisch str. 1, 35034 marburg, germany g protein-coupled receptors (gpcrs) are proteins that regulate numerous signaling pathways by activation of intracellular g proteins. gpcrs are activated by extracellular stimuli, such as light, hormones and neurotransmitters. recent evidence suggests that some gpcrs exhibit voltage-sensitivity leading to a modulation of their activity by the membrane potential (vm). we used a fret-based biosensor of the α2a adrenergic receptor to analyze receptor activation at defined membrane potentials in hek 293 cells by means of voltage-clamp recording. the biosensor was stimulated either with the partial agonist clonidine or with the full agonist norepinephrine (ne) and receptor activation was measured as decrease in the ratio of acceptor-/donor-fluorescence. receptor stimulation by ne was inhibited at depolarizing membrane potentials but enhanced by hyperpolarization. inhibition of ne activated receptors was strong at low concentrations (500 nm: 60 % inhibition) but almost absent at saturating agonist concentrations (100 µm: 9 % inhibition). both agonist-induced and hyperpolarizationinduced receptor activation exhibited a similar monoexponential time course and speed of activation was primarily dependent on agonist concentration for both activation modes. the latter indicates that depolarization lowers the apparent affinity of the ne receptor interaction and thus causes receptor deactivation by means of ne release. application of clonidine (1 µm, vm=-90 mv) resulted in a fret response that was inhibited by 40 % at +60 mv. in contrast to ne, strong receptor inhibition at +60 mv was present even at super-saturating concentrations of clonidine (100 µm), suggesting that voltage alters the equilibrium between active and inactive conformations of the receptor. voltage-dependence of the a2a adrenergic receptor also modulated downstream receptor signaling: g protein activation or the recruitment of arrestins, which we determined in fret assays that directly detect gαi protein activation or receptor-arrestin interactions, were both substantially inhibited at vm = +60 mv. therefore we conclude that negative membrane potentials promote active conformations of the a2a adrenergic receptor, increase affinity of full agonists and enhance receptor signaling. in conclusion the present data show that scc cells extrude more ha, possibly related to increased levels of has3, in comparison to keratinocytes. increased amounts of ha appear to be essential for the uvb induced tumourgenesis of sccs in mice. this effect might be related to the pro-proliferative property of high molecular weight ha. furthermore biological active ha fragments derived from ha degradation by hyaluronidases (hyal1,2) are thought to be pro-angiogenetic, anti-apoptotic and proinflammatory, thus possibly also promoting tumour growth and malignancy. estradiol induced paracrine release of egf from keratinocytes protects the dermal hyaluronan/versican matrix during photoaging röck k. 1 , meusch m. hyaluronan (ha) and versican are key components of the dermis and are responsive to uvb induced remodeling. the aim of the present study was to investigate the molecular mechanisms of estrogen (e2) mediated effects on ha-rich ecm during actinic aging. 10 weeks of uvb irradiation (3 x 1 med (80 mj/cm 2 ), weekly) of hairless skh-1 mice caused a marked decline of dermal ha, which was aggravated by ovariectomy (ovx). subcutaneous substitution of estrogen (e2) by means of controlled release pellets abolished these effects confirming the stimulatory role of e2. the increase of dermal ha correlated with induction of ha synthase has3 by e2. in addition the ha-binding proteoglycan versican was induced by uvb and further increased by e2. however in cultured skin fibroblasts e2 reduced the expression of versican and had no effect on has3. therefore, direct upregulation of has3 and versican in fiborblasts by e2 was excluded. however, e2 increased the expression of egf in uvb irradiated skin in vivo and in keratinocytes in vitro. egf in turn upregulated the expression of has3 and versican in dermal fibroblasts. furthermore the supernatants of estradiol treated keratinocytes led to the same effects in dermal fibroblasts, which could be abolished by previous treatment of the supernatant with neutralizing egf antibody or treatment of the fibroblasts with egf receptor blocker erlotinib. functionally, dermal ha and versican induction by e2 correlated positively with proliferation and negatively with accumulation of inflammatory macrophages in the dermis. collectively these data suggest that e2 treatment increases the amount of dermal ha and versican via paracrine release of egf which may be implicated in the pro-proliferative and anti-inflammatory effects of e2 during photoaging. differential pro-and eukaryotic toxicity of silver released from nanocomposite surfaces increases the therapeutic window of silver in antibacterial treatments röhl c. 1 , hrkac t. silver has been used since ancient times as antimicrobial agent. recently, silver gained new attention due to its higher effectiveness in its nanoform. this led to new developments of silver nanomaterials, e.g., for medical devices and consumer products. though, it is generally assumed that silver is less toxic for eukaryotes than for prokaryotes, concern is raised, if nanosilver at the same time might also increase mammalian cytotoxicity. in our study we examined the toxicity of silver released from nanocomposite surfaces with that of silver from agno3 solutions for adherent bacteria and mammalian cells. therefore, we established an in vitro reference system which enabled us to compare the therapeutic window between prokaryotic toxicity and eukaryotic integrity in both exposure settings. we focussed especially on the comparability of the bacterial and mammalian cell systems and the development of characterized ag/tio2 nanocomposite coatings with well-defined silver filling factors and silver surface release, which could be varied over a wide concentration range. as reference cells the e. coli sar 18 strain and human dermal fibroblasts, which are of special relevance in the context of medical devices like implants or wound dressings, were chosen. bactericidal effects were determined by direct growth visualization of the gfp-producing e. coli strain by epifluorescence microscopy. mammalian cell growth and toxicity was determined by the mtt assay, protein measurements and phase contrast microscopy. the ag/tio2 samples were prepared by sputter co-deposition from two separate magnetron sources. the silver surface concentration release was determined by xps and the silver release by icp-ms. in solution a concentration-dependent constant silver concentration could be determined between 2 and at least 72 hours at the surface. while lowest bactericidal and cytotoxic concentrations of ag + from agno3 solutions with 0.64 and 0.95 mg/cm 2 , respectively, differed only slightly, the therapeutic window increased significantly if ag + was released from the nanocomposite surface. while the toxicity on the fibroblasts was unchanged the bactericidal potency increased at least one order of magnitude. taken together, it can be concluded that local exposure factors i) can be modulated by silver nanocomposites and ii) play an important role for the differential toxicity of surface silver on bacteria and mammalian cells. charite -universitätsmedizin institut für integrative neuroanatomie, funktionelle zellbiologie, philippstr. 2, 10115 berlin, germany c3 exoenzyme (c3bot) a clostridial adp-ribosyltransferase does not possess a cellbinding/-translocation domain. nevertheless, c3 is able to efficiently enter intact cells, including neuronal cells but the mechanism of uptake is not yet understood. in the present work, binding of c3bot to the hippocampus-derived ht22 cell line was characterized by means of binding and blot overlay assays as well as mass spectrometry analysis to identify binding partners of c3bot. the binding assays established that c3bot bound in a concentration-dependent manner to ht22 cells. in the overlay assay we detected one clear band of 55 kda. to elucidate whether glycosylation is important for the c3bot-protein interaction, ht22 cells were incubated with glycosidase f resulting in a decreased binding of c3 to the 55 kda band. to explore the involvement of phosphorylation in the binding of c3 to the putative binding protein, blot was pre-treated with cip (calf intestinal phosphatase) before overlay with c3bot. pretreatment greatly reduced the c3bot-protein interaction. moreover, inhibition of dephosphorylation by vanadate before in intact cells showed an increased level of c3botprotein interaction in the following overlay. thus, interactions between c3bot and ht22 cell proteins may require phosphorylation. to further characterize the 55 kda band as binding target of c3bot, the 55 kda band was digested with trypsin and then subjected to lc-orbitrap mass spectrometry analysis. from this 55 kda single gel band 141 proteins were identified. further analysis of the identified proteins will provide a possible interaction partner of c3bot. in sum, protein overlay assays revealed that phosphorylation and glycosylation are critical for efficient c3bot-protein interaction. s76 335 solvent effects on enzyme kinetics in vitro rokitta d., pfeiffer k., gerwin h., streich c., fuhr u. uniklinik köln institut für pharmakologie, gleueler str. 24, 50931 köln, germany kinetic parameters provide essential quantitative information for characterisation of drug metabolising enzymes. such enzymes are located in an a partially queous environment, but to solve potential lipophilic substrates for in vitro measurements organic solvents are regularly needed. to preserve the enzymes from denaturation and other solvent related effects, the concentration of these solvents must be kept low. data on nature and extent of such solvent effects is sparse. in this study, we investigated the effects of methanol, ethanol, acetonitrile and dimethylsulfoxide (1% to 4%) on the assessment of k m, vmax and clint with regard to the 1-hydroxylation of midazolam via cyp3a4 and the cyp1a2 catalyzed metabolism of caffeine to paraxanthine in vitro. the presence of acetonitrile showed the highest vmax value for paraxanthine formation but the lowest values for 1-hydroxymidazolam formation. the km value for midazolam showed no systematic effects of organic solvents, while for caffeine km was up to eightfold lower for solvent free samples compared to solvent containing samples. the present example suggests that the presence of organic solvents may considerably influence enzyme kinetic parameters beyond a mere change in apparent activity. these effects are differing between enzyme-substrate systems and solvents. it remains to be determined to which extent such effects compromise in vitro -in vivo extrapolations, and which solvents are most appropriate. atrial remodeling and arrhythmia induced by the transcription factor er81 rommel c. 1 , rösner s. introduction: the transcription factor er81 (ets related 81) belongs to the large family of ets-transcription factors that are involved in developmental processes and in the pathogenesis of cancer. er81 is activated by gq-and gs-coupled receptors leading to a phosphorylation of the transcription factor by map-kinases and protein kinase a, respectively. cardiac er81 mrna expression is increased in failing human hearts. however mechanical unloading by a left ventricular assist device leads to normalization of er81 expression. thus, the aim of the present study was to investigate the cardiac function of er81 in genetically modified mouse models. we previously generated transgenic mice overexpressing er81 under control of the cardiomyocyte-specific α-myosin heavy chain gene (αmhc) promoter by pronuclear injection and established independent transgenic lines. electrocardiography (ecg) was assessed in mice at day 5 after birth (p5) and in adult mice (3 months) during isoflurane anesthesia and by ecg telemetry in awake mice, respectively. ecg analysis revealed no differences between the genotypes at day 5 after birth. however, we found a decreased heart rate, a replacement of regular p-waves by an undulating baseline and frequent supraventricular extrasystoles in adult er81 αmhc transgenic mice. next, isometric contractile force measurements on isolated left atria were carried out in organ baths. while wt left atria responded to increasing concentrations of isoprenaline, nkh477 and calcium with an increase in contractility, the maximal positive inotropic responses to these substances were severely blunted in er81 αmhc atria. we performed western blots to identify potential aberrations of calcium handling and regulatory proteins. phosphorylation of serine 16 of phospholamban (pln) was reduced in er81 αmhc mice. in addition, protein phosphatase 1 (pp1) expression was significantly increased in er81 αmhc mice, which is consistent with the increased dephosphorylation of phospholamban. furthermore, we found a decreased expression of calsequestrin and serca2a protein in er81 αmhc atria. electron microscopy revealed the significant structural remodeling of er81 αmhc atria at 3 months of age. conclusion: increased cardiac expression of the ets-transcription factor er81 leads to structural and electrical remodeling of the atria. thus, er81 may play an important role in the pathogenesis of cardiac arrhythmias in chronic heart failure. signal transduction pathway of atp and utp in neonatal rat cardiac myocytes rothkirch d., gergs u., neumann j. institute for pharmacology and toxicology medical faculty, magdeburger str. 4, 06097 halle (saale), germany extracellular atp and utp can be released from the heart during pathological conditions such as ischemia or hypoxia. in humans, atp and utp levels are increased during myocardial infarction. atp and utp can act via p2-purinoceptors which are further divided in p2x1-7 and p2y1-14-receptors. as previously shown atp and utp can induce inotropic effects in cardiac preparations of mice and man. for rat articular chondrocytes and human intestinal cells it has been demonstrated that the mapk cascade can be activated by atp and utp. therefore, the cardiac effects of atp and utp on force of contraction probably occur via the mapk pathway. to investigate the signal transduction pathway involved, we studied the effects of atp and utp on mapk phosphorylation in isolated neonatal rat cardiac myocytes using phosphorylation-specific antibodies. 100 µm atp as well as utp transiently increased phosphorylation of erk 1/2 and p38 mapk with a maximum effect at 5 to 10 minutes after application of atp and utp in neonatal cardiac myocytes (n=3 preparations each). the maximum phosphorylation of p38 increased with atp to 284% ± 68% (p<0.05) at 10 minutes and with utp up to 204% ± 21% (p<0.05) at 5 minutes. the phosphorylation with erk 1/2 mapk increased with atp to 234% ± 46.5% (p<0.05) at 5 minutes and to 337% ± 27% (p<0.05) with utp at 10 minutes of basal values, respectively. after 20 minutes, predrug values of mapk phosphorylation were reached again. in summary, we noted an atp-and utp-induced phosphorylation of erk 1/2 and p38 mapk in isolated neonatal rat cardiac myocytes. the involved receptor subtype(s) and the link between mapk phosphorylation and inotropic effect of atp and utp need to be elucidated. hameel: use of elearning in teaching pharmacology and toxicology -the halle experience rulf k. 1 , gergs u. introduction: during the past three years, our faculty has started to integrate items of elearning into the standard curriculum of a classical medical school: the "hallesches medizinisches elearning -hameel". our hypothesis was that these new elearning tools would improve the willingness of students to spend more time into learning and this would lead to an improved outcome (in multiple choice tests). methods: hence, we offered medical students (5 th or 10 th semester) additional learning environments. the courses for students (experimental pharmacology and toxicology or clinical pharmacology) were existed of a weekly lecture and in addition tutorials (problem-based-learning style, paper cases) or classical seminars. furthermore, we offered the possibility to use an online multiple choice quiz (involving 8 -12 previously used tests) and/or an online module on heart failure each week. we used the learning management system ilias software in combination with the content management system stud.ip. all students were subjected to an introductory test (to assess knowledge prior to our teaching section and allowing us to exclude a conceivable bias due to previous knowledge, involving basic items from prior teaching opportunities), a mid-term test and a final test to assess gain of knowledge. a maximum of 60 points could be obtained as a sum of both tests. results: in the means 40% of students used the new elearning tools (quizzes, heart failure module). however, there was no association between the use of self-assessment quizzes and examination results. the usage of the online quizzes increased in the periods before the exams. however, usage of the heart failure module was accompanied by significantly increased scores in exams. moreover, in a formalized evaluation system, students positively commented on our elearning efforts. conclusions: while usage of our quizzes did not improve test marks, another more sophisticated clinically oriented elearning module seemed to be improving test outcomes marginally. targeting of erk thr188 phosphorylation attenuates cardiac hypertrophy but preserves the anti-apoptotic effects of erk1/2 ruppert c., vidal m., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. 9, 97078 würzburg, germany background and aims: the extracellular regulated kinases 1 and 2 (erk1/2) play an important role in cardiac hypertrophy and cell survival. erk1/2 are phosphorylated at the so-called tey motif, which in turn activates erk1/2. hypertrophic stimuli lead to an additional autophosphorylation threonine188 (thr188). this autophosphorylation of erk1/2 stimulates activation of nuclear erk targets, which are known to induce hypertrophy. the aim of this study is to investigate whether specific targeting of erk thr188 phosphorylation affects both erk functions -erk mediated hypertrophy and cardioprotective cell survival. methods and results: for the analysis of cardiomyocyte hypertrophy in vitro, we stimulated cardiomyocytes with phenylephrine and measured the incorporation of tritiated isoleucine. cardiac hypertrophy was assessed by echocardiography before and after transverse aortic constriction (tac). for analysis of cell survival, caspase activity and dna fragmentation was determined upon hydrogen peroxide stimulation in vitro and in response to tac in vivo. to differentiate between inhibition of erk1/2 activity and prevention of erk thr188 phosphorylation, we either inhibited erk activity with pd98059 or overexpressed a mutant of erk2, which cannot be phosphorylated at thr188 phosphorylation. while inhibition of overall erk activity with pd98059 attenuated cell survival and hypertrophy in vitro, specific targeting of erk thr188 phosphorylation by overexpression of the phosphorylation deficient mutant (erk2 t188a ) attentuated phenylephrine induced hypertrophy, but preserved the anti-apoptotic effects of erk. cardiac overexpression of erk2 t188a significantly reduced tac-induced hypertrophy compared to wild-type erk2 overexpressing mice. in line with the in vitro experiments, erk thr188 inhibition only prevented hypertrophy in the tac model without promoting apoptosis. conclusions: these results show that blockade of erk thr188 phosphorylation attenuates cardiomyocyte hypertrophy but preserves anti-apoptotic effects of erk1/2. therefore, specific targeting of erk thr188 phosphorylation might be a promising strategy for the treatment of pathological hypertrophy. intracellular camp levels are determined by interplay of camp formation by adenylyl cyclases and camp degradation by phosphodiesterases (pde). eleven families of pdes are known. one of the most recently identified pdes is pde10, a pde in principle capable of hydrolysing camp as well as cgmp. pde10 contains a tandem of so called gaf domains in its n-terminal regulatory domain that mediate activation by camp. because current knowledge about the tissue distribution of pde10 was mostly based on the analysis of mrna distribution, we generated antisera against pde10 to analyze tissue distribution of the protein level. using these antibodies, we found a prominent occurrence of the enzyme in testis and in brain, where it was confined to the striatum. thus, pde10 displays a comparably restricted tissue distribution which is in contrast to that of many other pdes. low camp levels in so called medium spiny neurons of the striatum have been implicated in schizophrenia. furthermore, studies using the nonspecific pde10 inhibitor papaverine as well as specific pde10 inhibitors suggest pde10 as a target for the treatment of schizophrenia. here we set out to analyze the contribution of pde10 to camp degradation in striatum, to identify the physiological pathways pde10 is involved in and to clarify the functional impact of the proposed phosphorylation of the enzyme. identification of cgmp-dependent kinase i substrate complexes salb k., schlossmann j. universität regensburg lehrstuhl pharmakologie, universitätsstrasse 31, 93053 regensburg, germany the cgmp-dependent kinases (cgks) are components of the no/cgmp/cgk-signalling pathway and have a great physiological importance in a multitude of tissues and organs such as smooth muscles and platelets. two isoforms of the cgki and the cgkii are known. cgkiα and cgkiβ differ only in their first ~ 100 amino acids which constitute the leucine zipper and the autoinhibitory domains. the n-terminal leucine zipper domains mediate homodimerization of the kinase and the interaction with diverse substrate proteins. since cgkiα and cgkiβ express different n-termini they interact with different substrates. the cgkiβ isoform is assembled in a macrocomplex at the endoplasmic reticulum (er) with the intracellular calcium release channel inositoltrisphosphate receptor i (insp 3r-i) and the inositol-trisphosphate receptor associated cgmp kinase substrate (irag). we investigated, whether irag also interacts with the insp3r-ii and the insp3r-iii in murine platelets and tissues. additionally, we analyzed the interaction between the 52 amino acid peptide phospholamban (plb), which is also located at the er and regulates the er calcium reuptake by the sarco/endoplasmic reticulum ca 2+ -atpase (serca), and the two cgki isoforms. we performed cgmp-agarose experiments with murine wt and irag-ko platelets to examine the irag-insp3r interactions. the insp3r-ii isoform was neither bound to cgmp-agarose nor detected in the anti-irag immunoprecipitate. on the other hand, insp3r-iii from wt but not from irag-ko platelets was bound to cgmp-agarose. hence, insp3r-iii interacts directly with irag but not with cgkiβ in murine platelets. however, in colon smooth muscle lysate, insp3r-iii not only interacted with the irag protein but was also detected in the anti-cgkiα-immunoprecipitate. phospholamban from wt and irag-ko platelets was also bound to cgmp-agarose. subsequent immunoprecipitation experiments with the respective antibodies against the two cgki isoforms revealed that plb interacted both with cgkiβ and cgkiα. these results were supported by analysis of colon smooth muscle tissue from wt and irag-ko mice. in conclusion, irag interacts with insp3r-i and insp3r-iii but not with insp3r-ii in murine platelets and colon smooth muscle tissue. moreover, phospholamban is an interacting partner of both the cgkiα and the cgkiβ isoform. the human immunodeficiency virus type 1 enhancer binding protein 1 (hivep1) is regulated by proinflammatory stimuli and statins salomon a. 1, 2 , schmitz b. objective: hivep1 binds nf-ĸb and other proinflammatory consensus sequences, and is suggested to be involved in inflammatory processes. we recently identified two tagging snps, one positioned 90 kb upstream (rs169713) and another in exon 4 (rs2228220) of the hivep1 gene, to be replicatively associated with venous thrombosis in gwas and follow-up studies (ajhg, 2010; plos one, 2011) . methods: total rna isolation was performed after treatment of vascular endothelial cells (ea.hy926) with proinflammatory cytokines or statins (24h). serial hivep1 promoter deletion constructs were cloned into the pgl3-basic vector, a potential enhancer fragment, harbouring rs169713c/t, into the pgl3-promoter vector. in ea.hy926 cells and thp1 monocytes, reporter gene assays were performed by transient transfection and overexpression of transcription factors. chip and bandshift assays were performed to identify candidate transcription factors. results: in ea.hy926 cells, endogenous hivep1 expression was increased by proinflammatory cytokines tnfα and il-1β. simvastatin (1.2 and 2.4 µm) and atorvastatin (9 µm) -but not pravastatin or aspirin -both dose-dependently decreased basal and tnfα-stimulated hivep1 expression. the construct harbouring rs169713t exerted significantly higher transcriptional activity (ta) compared to rs169713c (p<0.001). for an intronic modulator, reporter gene assays demonstrated a regulatory effect on hivep1 expression in ea.hy926 and thp1 cells. cotransfection of sp1 and egr1 led to an increase in ta, while wt1 exclusively upregulated ta of constructs comprising the intronic modulator. chip and bandshift assays combined with specific antibody detection revealed binding of sp1 to the 5'-flanking region and the intronic modulator of hivep1. conclusion: increased hivep1 expression during inflammatory conditions can be repressed by simvastatin and atorvastatin, and not by pravastatin or aspirin. basal hivep1 expression is regulated by sp1 combined in a transcription factor module with egr1 and wt1 under basal and/or inflammatory conditions. the rs169713 site harbours potential activational capacity for hivep1 gene transcription and may communicate with the sp1/egr1/wt1 module. to date, the treatment of various movement disorders of the central nervous system is still insufficient. in most cases this is due to the sparse knowledge of the pathophysiology. l-dopa-induced dyskinesias (lid) represent a severe complication of long-time pharmacotherapy in parkinson's disease that deserves novel therapeutics. an increased activity of striatal projection neurons, which express kv7.2/3 channels, seems to be involved in the pathophysiology of these spontaneous involuntary dystonic and choreatic movements. previous studies demonstrated an antidyskinetic effect of the kv7.2-7.5 channel opener retigabine after acute and chronic treatment in a rat model of lid. in order to clarify if this effect was based on the modulation of kv7.2/3 channels, we examined the acute effects of the preferred kv7.2/3 channel opener ica 27243 on lid in this animal model. four weeks post 6-ohda lesioning of the left forebrain bundle, dyskinesia was induced by chronic treatment with 10 mg/kg l-dopa and 15 mg/kg benserazide for 20 days. three subtypes of dyskinesia (limb, axial and orolingual) were rated according to a score system from 0 to 4 over 180 min. for drug testing, ica 27243 (5, 10 and 15 mg/kg) was administered intraperitoneal additionally to l-dopa (or vehicle). effects of drug action in comparison to vehicle controls were detected by adding up the severity scores of each observation time. additionally, effects on parkinsonian symptoms were examined 20 min after drug administration using the block and the stepping test. ica 24273 reduced the severity of dyskinesia significantly at all doses while no negative impact on the antiparkinsonian effect of l-dopa was observed. whereas the antidyskinetic effect was restricted to the first 20 min after the application of 5 mg, it lasted up to 110 min in rats treated with 10 mg ica 27243. a higher dose of 15 mg did not further enhance the antidyskinetic effect. the results of our study suggest that the antidyskinetic effect of the k v7 channel opener retigabine was based on its action on striatal kv7.2/3 channels. in line with the results of previous studies with retigabine, this action does not seem to interfere with the antiparkinsonian effect of l-dopa. this study was supported by the micheal j. fox foundation. background: skeletal muscle toxicity is the major side effect of hmg-coa-reductase inhibitors (statins) and can be simulated in engineered skeletal muscle. statins are known to exert "pleiotropic" effects, e.g. reducing endothelial dysfunction by inducing no synthases and no production. the role of no synthases in skeletal muscle under statin treatment is largely unknown. interestingly, some skeletal muscle pathologies (e.g. duchenne muscular dystrophy) may be exacerbated by increased inos activity. here we tested whether or not statin-induced skeletal muscle toxicity would be associated with enhanced no synthesis. we generated engineered skeletal muscle (esm) from rat skeletal muscle cells, matrigel and collagen. esms displayed typical skeletal muscle properties (differentiated muscle fibres, tetanic contractions). under baseline conditions esm expressed enos most abundantly, followed by inos and nnos (n=4-5). myotoxic cerivastatin (0.01, 0.1, 1 µm for 5 days) caused a concentration-dependent decrease of contractile force (p<0,05, n=17-20) paralleled by an increase in inos transcript (mean±sem: 0.01 µm 4±0.9-fold, n=3 p<0.05; 0.1 µm 9.3±2.8-fold, n=3 p<0.05) and protein (0.01 µm 5.1±2-fold, n=4 p<0.05; 0.1 µm 7.8±0.8-fold, n=3 p<0.05). mevalonic acid fully prevented the inos increase suggesting that the induction is hmg-coa reductase-dependent. to test whether inos may contribute to the decrease in contractile force we co-treated esm with 1400w, a specific inos inhibitor. we applied 5 µm of 1400w, a concentration found to potently reduce lipopolysaccharide (lps)induced no-production in cultured myotubes. however, we did not observe a rescue effect (n=9-15). also, l-name (10 mm), an unspecific nos inhibitor, did not improve contractile function, instead we observed increased myotoxictiy (n=6-13, p<0.05). to further investigate the role of no for muscle function we treated the esms with increasing concentrations of the no-donor snp. only high concentrations of snp (10 µm) caused a reduction of contractile force. combined treatment with cerivastatin and 0.1 µm snp showed a tendency towards improved force development in esm. conclusions: statins increase inos activity in our skeletal muscle model (esm). however, this does not seem to functionally contribute to myopathy in esm. increased production of no may in fact be a protective measure. esm may help to dissect clinically relevant functional changes in statin myotoxicity. characterization of primary skin fibroblasts of patients with 3m syndrome and mutations in the cul7 gene meyer k., hieber m., engelhardt s., sarikas a. technische universität münchen institut für pharmakologie und toxikologie, biedersteiner str. 29, 80802 münchen, germany 3m syndrome is an autosomal-recessive disorder characterized by pre-and postnatal growth retardation (< -4 sd), facial dysmorphism and skeletal anomalies. the majority of patients harbor missense mutations of the cul7 (76%) or obsl1 (16%) gene, respectively. cul7 constitutes an e3 ubiquitin ligase that is involved in the regulation of the insulin-like growth factor 1 (igf-1) signaling pathway via ubiquitin mediated degradation of insulin receptor substrate 1 (irs-1). to investigate the role of cul7 mediated irs-1 degradation in the pathogenesis of 3m syndrome. primary skin fibroblasts of seven 3m syndrome patients (six with cul7 mutations, one with a obsl1 mutation) and control fibroblasts were analyzed for proliferation rate (cell counter), cell cycle profile (facs), cell morphology and cellular senescence (histochemistry), irs-1 protein concentrations and activation of the igf-1 signaling pathway (western blot). the proliferation rate of 3m patient fibroblasts was significantly increased when compared to control cells. in contrast, irs-1 protein levels and activation of the pi3k/akt and erk mapk pathway were only increased in a subset of 3m cells that carried cul7 mutations, but not in cells from a patient with the obsl1 mutation. no significant differences in cell cycle profile, cell morphology or cellular senescence were observed in 3m patient fibroblasts when compared to control cells. to determine the pathogenetic contribution of increased irs-1 levels to the observed phenotype, human imr90 fibroblasts were stably transfected with retroviral vectors encoding irs-1. despite 20-fold overexpression of irs-1 compared to empty vector controls, no significant effect of igf-1 stimulation on proliferation rate or pi3k/akt and erk mapk signaling was observed. skin fibroblasts of 3m patients with cul7 mutations displayed an increased proliferation rate and enhanced activation of the igf-1 signaling pathways. despite accumulation of irs-1 in fibroblasts from a subset of 3m patients with cul7 mutations, no pathomechanistic role for irs-1 could be demonstrated. collectively, our data indicate that a dysregulated igf-1 signaling may contribute to the pathogenesis of 3m syndrome, yet in an irs-1 independent manner. pharmacases.de -a student-centered elearning project of clinical pharmacology zollner b., berg c., gros n., muß n., oestreicher d., engelhardt s., sarikas a. technische universität münchen institut für pharmakologie und toxikologie, biedersteiner str. 29, 80802 münchen, germany pharmacases.de is a novel e-learning website of clinical pharmacology that presents clinically relevant aspects of pharmacology and toxicology in an interactive and multimedial manner. the aim of the project pharmacases.de was to develop an innovative concept for creating high quality elearning content that i) integrates and promotes the theoretical and cooperative skills of final year medical students and ii) is easily adoptable by cooperating institutes and hospitals. a peer-teaching concept was developed in which final year medical students with the elective pharmacology (pj wahlfach pharmakologie) independently researched and wrote elearning lessions ("pharmacases"). subject-specific expertise was acquired by consulting elective students of other disciplines. at present (11/2011) , this "peer network" consists of elective students of nine cooperating institutions (pathology, microbiology, radiology, cardiology, psychiatry, dermatology, neurology, ophthalmology, pediatrics) at the technische universität münchen. the average time for the generation of one elearning lession by the peer network was 10 days. to date, the website consists of 49 pharmacases that are available to all students online (http://www.pharmacases.de). the website also contains a discussion forum and evaluation form for direct feedback. on average, pharmacases.de has 1000 visitors per month with the following evaluation results: "excellent": 76%, "good": 15% and "satisfactory": 9% (n=33). the didactic concept of pharmacases.de enabled the efficient generation of high quality elearning content in a student-centered and interdisciplinary manner. the peer-teaching approach supports the collaborative skills of final year medical students and facilitates the transfer of theoretical pharmacological knowledge into clinical practice. improved glucose tolerance, less chronic adipose tissue inflammation and reduced adipose tissue mass in mice with adipocyte-specific loss of tak1 sassmann a., offermanns s., wettschureck n. max-planck-institut für herz-und lungenforschung pharmakologie, ludwigstr. 43, 61231 bad nauheim, germany tgf-β activated kinase 1 (tak1) is known to be involved in numerous inflammatory processes by linking receptors for inflammatory stimuli like lps, interleukin-1 or tnfa to ikk, p38 and jnk activation. chronic inflammation of white adipose tissue is one of the major causes for the development of insulin resistance and impaired glucose tolerance in states of obesity. to investigate the role of tak1 in white adipose tissue, we crossed the tamoxifen-inducible white adipocyte-specific cre mouse line adipoqcreer t2 with animals carrying floxed alleles of the tak1 gene. adipoqcreer ; tak1 fl/fl animals and cre negative control littermates are viable and fertile and do not show any developmental defects. after tamoxifen induction and high fat diet feeding adipocytespecific tak1 knockout mice show improved glucose tolerance and lower fasting insulin levels compared to control animals. in line with this, serum levels of the adipose tissuespecific hormone resistin are reduced in adipocyte-specific tak1 knockout mice. these findings are accompanied by a lower state of chronic inflammation of adipose tissue as indicated by a dramatic reduction of adipose tissue macrophage number and lower serum levels of tnfα and interleukin-6. stimuli like tnfα, interleukins and tgf-β released from macrophages and adipocytes are known to promote obesity-related adipose tissue inflammation. when stimulated with these substances tak1 deficient adipocytes show reduced activation of jnk and p38 which both play an important role in the development of insulin resistance. interestingly, we observe a lean phenotype in adipocyte-specific tak1 knockout mice when fed a high fat diet which reflects a reduction of white adipose tissue mass. currently we are investigating the molecular mechanisms underlying the reduced adiposity and lower state of chronic inflammation in adipose tissue. growth of small cell lung cancer (sclc) cells is regulated via the autocrine stimulation of g protein coupled receptors (gpcrs), i. e., neuropeptide and muscarinic acetyl choline (ach) receptors. the activation of gq/11 and calcium-dependent gpcr pathways results in the stimulation of erk signaling which is necessary for the mitogenic effects of neuropeptides or ach on sclc cells. in contrast, the role of calcium-independent gpcr signaling and its interplay with gq/11-regulated pathways in sclc cells are less well defined. the aim of our studies was to characterize the molecular make-up and the interaction of these pathways, and to delineate the phenotypic effects of calciumdependent and -independent signaling cascades in sclc cells. using a panel of sclc cell lines, we found that the stimulation of neuropeptide receptors led to an increase of calcium which was independent of extracellular calcium and could be prevented by depleting internal calcium stores. this calcium increase was sufficient to activate the tyrosine kinase pyk2 and subsequently the erk1/2 cascade. the role of pyk2 for the growth of sclc cells was further supported by the fact that inhibition of pyk2 using a sirna approach or a novel specific inhibitor, pf431396, exerted pronounced cytotoxic effects on sclc cells, whereas non-sclc cells were less sensitive. interestingly, the inhibition of g 12/13 signaling by sirna-mediated g(alpha)12 or g(alpha)13 knockdown also markedly reduced the growth of sclc in vitro or in subcutaneous tumor xenografts, and increased the sensitivity of sclc cells towards certain cytostatics. to further define the role of calcium-dependent signaling via pyk2 versus the role of calcium-independent signaling via g12/13, we tested the effect of pyk2 inhibition in cells with impaired g12/13 signaling. notably, pyk2 and g12/13 double inhibition led to an even increased proliferation. thus, we propose that dysbalanced g protein signaling favoring either pyk2 activation or g12/13-dependent cascades inhibits the growth of sclc cells, whereas the parallel inhibition of both pathways restores again the balance and the growth capacity in this tumor entity. dendritic cells (dcs) are essential for the initial immune response and for the defence against inhalated pulmonary toxins and carcinogens in lung. to differentiate dcs, the cell line thp-1 were used for 7 days and stimulated with various cytokines (il-4, gm-csf, tnf-a, ionomycin). the dcs were characterized by flow cytometry with different typical dendritic cell markers (for example cd11c, cd209, cd83) and by immunfluorescence compared to monocytes. the bronchial tract contains up to 800 dcs per mm² and therefore we established a triple culture model to mimic the situation in vivo. the triple culture consists out of primary human epithelial cells from small bronchi (hbec) and lung fibroblasts which are cultured under air-liquid conditions on filter membranes for 4 weeks and dcs which were added after the differentiation phase of the bronchial cells. during the cultivation time the hbec formed an epithelial layer expressing both tight and adherens junctions. they also produced mucus, formed functional cilia with a beat frequency of between 16 to 20 hz and the transepithelial resistance values were stable between 600 to 800 ω·cm². pathomechanisms of pulmonary toxicity in vivo are difficult to investigate, so the tripleculture model is the basis for investigations of the toxic effects at cellular level. lungtoxic substances such as organophosphates are usually absorbed through inhalation. organophosphates are dangerous nerve agents for the human organism. at high concentrations organophosphates damage in the coculture without dcs the cell-cell contacts of the epithelial layer. in the triple culture dcs firstly respond to inhaled organophosphates and seem to compensate effects on the other cells. in summary, it is very important to understand the pathogenic mechanisms of lung injury in relation to the role of dendritic cells in lung. they could play an essential role in therapy against damage of organophosphates in the lung. co-purification of arf gtpase-activating protein git1 and cavb3 schalkowsky p., wissenbach u., fecher-trost c., flockerzi v. universität des saarlandes institut für experimentelle und klinische pharmakologie und toxikologie, kirrbergerstraße, 66421 homburg, germany high-voltage activated ca channels are assembled from pore-forming α1 subunits and two distinct types of auxiliary subunits, cavβ1-β4 and, maybe, α2δ1-δ4. by a cavβ3-specific antibody based affinity chromatography the cavβ3 protein was highly enriched from rat brain microsomal membranes. proteins associated with cavβ3 were identified by mass spectrometry (lc-esi-ms/ms) and include α1-subunits, α2δ-subunits and β-subunits. in addition to these expected interacting proteins additional proteins were co-purified with the cavβ3 protein, including the g protein-coupled receptor kinase-interactor 1 (git1). the 770aa git1 is a ubiquitously expressed multidomain protein which may serve as a scaffold to bring together molecules to form signaling modules controlling, for example, vesicle trafficking, cytoskeletal organization and cell migration. in rat brain lysates the git1 and cavβ3 proteins were co-immunoprecipitated by the antibodies for cavβ3 and git, respectively. we cloned the git1 cdna from mouse brain and co-expressed it with the cavβ3 subunit in hek cells. like in brain lysates the git protein was retained by cavβ3 precipitated by the antibody for cavβ3 and cavβ3 was retained by the git1protein precipitated by the antibody for git1. both proteins, cavβ3 and git1 are endogenously co-expressed in mouse embryonic fibroblasts (mef). we could not observe potassium-induced voltage-activated ca influx in these acutely prepared cells. accordingly, mefs can be used as a model system to study the impact of cavβ3-git1 interaction in the absence of functional cav channels. in addition, using mefs from cavβ3-deficient mice enables us to control the impact of cavβ3 on git1 function. vice versa down-regulation of git1 by specific sirnas might allow to control the impact of git1 on cavβ3 function. as read-outs we use cell migration assays and monitor receptor-dependent and receptor-independent calcium signaling in these cells. effects of sphingosine-1-phosphate and fty720 on epidermal hyperproliferation and inflammation in an imiquimod induced mouse model of psoriasis the sphingolipid sphingosine 1-phosphate (s1p) is a mediator that modulates various physiological functions of skin cells. s1p has distinct direct effects on keratinocytes as it diminishes proliferation and induces differentiation which is a classical goal of psoriasis therapy. furthermore, s1p modulates the function of various immune cells, mainly to an anti-inflammatory direction. thus, the strategy of targeting immune cells with locally acting s1p was explored in an experimental animal model of psoriasis vulgaris, the recently established imiquimod induced psoriasis mouse model and in the mouse tail test. topical administration of imiquimod onto back and ear skin led to a distinct inflammatory response characterized by epidermal hyperproliferation, scaling and redness which was scored with a modified pasi (psoriasis area and severity index). the positive control diflorasone diacetate and s1p, but not fty720 reduced the epidermal hyperproliferation by topical administration onto ear skin, indicating a mode of action for s1p via the s1p2 receptor, which is not activated by fty720. there was also a moderate reduction of inflammatory cell influx and edema formation in ear skin by s1p treatment, which was even more pronounced by treatment with diflorasone diacetate. the pasi determined on back skin was, however, only significantly reduced by diflorasone diacetate. the discrepancy between outcome on ear and back skin remains elusive. in the mouse tail assay, the influence of s1p in stratum granulosum formation (orthokeratosis) was tested compared to the positive control calcipotriol. whereas topical administration of calcipotriol led to the expected significant increase of stratum granulosum in mouse tail epidermis, s1p lacked such an effect, indicating a different mode of action in epidermal differentiation. taken together, these results imply that topical administration of s1p might be a new option for the treatment of mild to moderate psoriasis lesions. inhalation of toxicants such as sulphur mustard (sm), an alkylating chemical warfare agent, cause pulmonary complications like respiratory failure, pulmonary edema and secondary pneumonia. in order to investigate pathomechanisms of pulmonary toxicity, an in vitro alveolar-capillary co-culture model has been established recently by our group. in this model the human lung adenocarcinoma epithelial cell line (h441) is mimicking the epithelial site of the alveoli while the human hemangiosarcoma cell line (iso-has) represents the endothelial site. acute respiratory injuries are accompanied by disruption of the alveolar-capillary barrier that can be detected by the use of biochemical markers (e.g. ldh) and electrochemical indicators (e.g. transepithelial resistance). sm-mediated pulmonary injury is characterized by the increased secretion of proinflammatory mediators (e.g. il-6). a shortcoming of this model is the missing inflammatory component in the lung. aim of the present project is the addition of macrophages to the established co-culture model to improve the model and to investigate the relevance of inflammatory processes in toxic lung injury. the effect of sm on this triple-culture model is characterized with special regard to the interaction of epithelial cells and macrophages. the human acute monocytic leukemia cell line (thp-1) was stimulated to allow differentiation into macrophages. validation of the cellular differentiation was checked by specific clusters of differentiation (e.g. cd206) using flow cytometric analysis. after successful differentiation into macrophages, these inflammatory cells were added to the co-culture model before and after exposure with sm, respectively. the cytotoxicity of sm on the triple-culture model was evaluated by xtt assays and ter measurements. furthermore, immunohistochemical staining of tight junction proteins (e.g. zo-1) and of adherens junction proteins (e.g. e-cadherin) was conducted to enhance the knowledge of the function of the intercellular junction in injured and rejuvenated regions as well as the interaction of epithelial cells and macrophages. for the contact allergen para-phenylenediamine (ppd) we showed that concentrations above 50 µm are accompanied with inhibition of nat1 activity in human keratinocytes [1] . in the following we investigated the impact of ppd on nat1 activity in antigenpresenting cells using dendritic cell-like cells, namely the monocytic thp-1 cells. measured nat1 activity of thp-1 was comparable to those found in primary keratinocytes. a 24h treatment of thp-1 cells with physiologically relevant concentrations of ppd (10-200 µm) led to a 47% reduction of nat1 activity. comparable results were found for mono-acetylated ppd (mappd) whereas di-acetylated ppd demonstrated no inhibition. time-dependent studies found a significant decrease in enzyme activity already 8h after application of ppd or mappd while nat1 mrna levels were not modified. these results are indicative for a substrate-dependent inhibition. further investigations concentrated on the restoration of nat1 activity after treatment with ppd or mappd. here we found that n-acetylation capacities were restored after 24h cultivation of the treated cells in fresh medium. independent of the enzymatic activity, certain compounds are known to oxidise the catalytic cysteine or form adducts with the nat1 protein. therefore we studied whether ppd and/or oxidised ppd including the trimer bandrowski´s base interact additionally with recombinant nat1 protein itself in the absence of acetyl-coenzyme a. we found that all compounds but mappd bind to nat1 protein after 2h. the greatest inhibition was found for oxidised ppd (up to 50%). due to the greater inhibition by oxidized ppd we propose that oxidation products interact with the protein whereas ppd itself modulates nat1 enzyme activity in a substrate-dependent mode of action. overall we demonstrated that ppd can inhibit nat1 in two different ways. the work was partially financed by federal office of public health (foph), switzerland and stiftung zur förderung begabter studierender und des wissenschaftlichen nachwuchses objective: fabry's disease is a rare progressive multisystem disorder resulting from deficiency of the lysosomal enzyme alpha-galactosidase a (gla, ec 3.2.1.22). we hypothesize that genetic gla variants, especially those in its promoter region are of pathophysiological relevance for the development and progression of fabry's disease phenotypes. this study focuses on the characterization of the gla promoter, identification of functional genetic variants and impact of transcription factor eb (tfeb), a regulator of lysosomal genes. we screened 4011 bp of the 5'-flanking region of gla in 60 patients with fabry's disease and 60 controls for genetic variants. serial promoter deletion constructs for reporter gene assays were designed and identified genetic variants were introduced by site-directed mutagenesis. constructs were transiently transfected into immortalized human kidney epithelial (ihke) cells and human vascular endothelial cells (ea.hy926) to determine transcriptional promoter activity (ta). sequencing of patients' dna revealed five genetic variants in the 5'flanking region of gla, significantly more frequent in fabry's patients compared to control group (rs2071225; rs3027580; rs3027579; rs59647857; rs3027575; all minor alleles p<0.0027). we identified two regions, a proximal one between -110 and -425 and a distal region between 1106 and -1421 with significant ta, in both cell lines. cotransfection with tfeb activated ta of both regions significantly up to 5.3-fold (p<0.001). in ihke cells, insertion of the minor t allele (rs2071225) significantly enhanced basal ta of the proximal promoter region (p=0.0006), while insertion decreased basal ta (p<0.0001) of the distal promoter portion. the combined insertion of the minor c alleles (rs3027580; rs3027579), which were in complete linkage disequilibrium, significantly increased basal ta of the distal promoter region (p=0.0037). our results indicate that three genetic variants, overrepresented in fabry's patients, are located within transcriptionally active regions, possibly altering tf binding sites and therefore, affecting gla expression. future analysis will assess the impact of gla promoter variants and gla regulation by tfeb with respect to fabry's phenotypes. multiple sclerosis (ms) and its animal counterpart experimental autoimmune encephalomyelitis (eae) have a major inflammatory component that drives and orchestrates both diseases. ceramides (cer) are known as mediators of inflammatory processes, but until now their role in ms was not elucidated. we measured the ceramide levels in the cerebrospinal fluid of ms patients and control patients using lc-ms/ms. interestingly, the c16:0-cer levels were 1.9 fold increased in ms patients. this translates into the finding that c16:0-cer levels were also significantly elevated in the lumbar spinal cord of eae mice. the raised c16:0-cer levels in the lumbar spinal cord were caused by a transiently increased expression of ceramide synthase (cers) 6 in macrophages. nitric oxide (no) and tumor necrosis factor alpha (tnf-α) secreted by interferon gamma (infγ ) induced macrophages play an essential role in the development of ms. astonishingly, rnai experiments reveal that cers6 and its product c16:0-cer are mediators of inf-γ induced no/tnf-α release in raw macrophages. moreover, treatment of eae mice with l-cycloserine prevented the increase of c16:0-cer and of inos/tnf-α expression and caused a remission of the disease. in summary, cers6 plays a critical role in the initial phase of ms, most likely by regulating the no and tnf-α synthesis. this let us speculate, that a substance designed to inhibit cers6 and therefore to limit the inflammatory effects of c16:0-cer may represent a new drug in ms therapy. role of cgmp-dependent protein kinase i for kidney fibrosis schinner e. 1 cgmp is synthesized via nitric oxide-or natriuretic peptide-stimulated guanylyl cyclases and exhibits pleiotropic regulatory functions also in the kidney. hence, the integration of cgmp signaling via cgmp-dependent protein kinases (cgk) might play a critical role for renal physiology. both isozymes were detected in arterioles, mesangium and within the cortical interstitium. in contrast to cgkiα, the β isoform was not detected in the juxtaglomerular apparatus and medullary fibroblasts. here, we focused on the function of cgki in the renal interstitium emphasizing a functional differentiation of both isoforms. the interstitium exists mainly of fibroblasts playing a prominent role in the interstitial fibrosis. accordingly, cgki could also be involved in this pathophysiological process. therefore, we studied whether cgki influences renal fibrosis which was induced by unilateral ureter obstruction (uuo). at first we analysed the role of the no/cgmp signaling by application of cgmp increasing yc1 or isdn. thereby we detected antifibrotic effects of these substances. subsequently we tested whether these effects are mediated by cgki by using mutant mice. on the one hand we examined αsm-rescue mice (expressing cgkiα only in smooth muscle under the control of the sm22 promotor with a cgki-ko background) and cgki-ko mice (expressing no cgki). on the other hand we used tgtg mice expressing more cgkiα in smooth muscle than wt mice (transgenic cgkiα under the control of the sm22 promotor). due to the steeply increased use of nanomaterials for commercial and industrial applications, toxicological assessment of their potential harmful effects is urgently needed. moreover, the continuous development of novel materials requires the implementation of hazard-predicting models to prevent potential health effects resulting from human exposure. in the present study, we studied the toxic potential of a set of nanoparticles (np) with varying physicochemical properties in human a549 lung epithelial cells, hepg2 liver epithelial cells and hk-2 proximal tubule epithelial cells. the used nanomaterials incorporated five tio 2 samples, two zno samples (i.e. uncoated and coated), two multi-walled carbon nanotube (mw-cnt) samples and a nanoparticulate ag sample. cells were treated with np at doses ranging from 0.3 to 80 µg/cm 2 for cytotoxicity and from 06 to 40 µg/cm 2 for genotoxicity. dna damage was evaluated using the alkaline comet assay while concurrent cytotoxicity was determined by the wst-1 assay. marked contrasts in cytotoxic and dna damaging properties were observed among the different materials. the overall strongest responses were observed with the uncoated zno-np sample and with ag-np, although effects were found to depend on the cell type. notably, the dna damaging effect of ag-np could at least partly be attributed to its dispersant. present results form part of a growing data set which are generated in the framework of the eu fp7 project enpra (fp7-nmp) to establish dose-response relationships and a mathematical model to predict the hazard of nanoparticles. increased spontaneous hprt mutant frequency in v79 cells expressing human cytochrome p450 1b1 schlechtweg a., esch h. , martínez jaramillo d., lehmann l. university of wuerzburg/institute of pharmacy and food chemistry section of food chemistry, am hubland, 97074 wuerzburg, germany the hypoxanthine-guanine phosphoribosyltransferase (hprt) assay in chinese hamster v79 lung fibroblasts (v79 cells) represents a widely-used mammalian test system to detect gene mutations. since v79 cells do not express any cytochrome-p450dependent monooxygenase (cyp) isozymes, usually an activating system has to be added. therefore, v79 cells expressing human (h) cyp isozymes have been commercialized. to test these v79 cells for their use in the hprt test, v79 h1a1 and h1b1 cells were characterized regarding (i) spontaneous frequency of 6-thioguanineresistant clones per 10 6 clonable cells (smf), (ii) the stability of which over 4 weeks (w), and (iii) the mutational spectrum (ms) of cdna from mutant clones. ms of cdna was determined by isolation of total rna, reverse transcription/amplification of the coding region by polymerase chain reaction and sanger sequencing of the amplification product. activity of cyp isozymes was verified by ethoxyresorufin-o-deethylase (erod) assay. (i)/(ii) whereas the smf of v79 cells (w2:13±4; w4:2±1) and v79 h1a1 (w2:17±4; w4:3±0) only varied within the range of historical controls, smf of v79 h1b1 increased continuously over time (w2: 36±6; w4: 68±13). (iii) although the smf of v79 and v79 h1a1 were similar, the mutational spectrum of v79 cells was characterized by as many transversions as transitions and deletions of exon 4 or exon 4+5, whereas the mutational spectrum of v79 h1a1 was characterized exclusively by transversions and deletion of exon 7+8. surprisingly, with 57 out of 59 cdnas derived from v79 h1b1 mutant clones, no amplification product was detected. first results indicate that there is at least one gene mutation in the untranslated region before and behind the coding region precluding amplification with the original primers. (ii)to reduce the smf of v79 h1b1, cells with wildtype hprt activity were cloned and one clone with an erod activity which did not differ significantly from the original cell population was further characterized. initially, smf of the clone varied between 0.7±0.6 and 1.2±0.0. yet its smf was unstable reaching up to 104±6. in conclusion, the mutational spectrum differed between the v79 cell lines. furthermore, h1b1 expression seemed to enhance smf in v79 cells. even though a temporary reduction of the smf by cloning was possible, smf of v79 h1b1 cells was unstable. we wanted to investigate the possible antithrombotic effects and elucidate the chemical identity of the active principles involved in inhibitory effects against adp-induced aggregation of human platelets by wild garlic, allium ursinum l. method: bioassay-guided isolation procedure was used followed by spectrometric identification of pure active compounds. for the bioassay, blood was taken from healthy human volunteers and platelet rich plasma (prp) was prepared for turbidimetric platelet aggregation tests. prp, stimulated with 20µm adp, was treated with extracts of different polarities, fractions and isolated single compounds from allium ursinum. the extracts were investigated by thin layer chromatography, hplc, mass spectroscopy, esi-ms and 1d/2d 1h/13c-nmr spectroscopic techniques. for references the adt-antagonist mes-amp was used. result: fresh allium ursinum leaves were extracted with ethanol, which was the potent form that effectively inhibited adp-induced aggregation of human platelets. this ethanolic extract was subjected to liquid-liquid partition. whilst the aqueous phase containing the moiety of cysteine sulphoxide and thiosulphinate derivatives showed only weak activity on platelet aggregation, the ethyl acetate and especially the chloroform partitions showed highest aggregation inhibiting potency. thus, in our bioassay effects of alliins/allicins could be neglected. the chloroform phase, possessing the strongest activity, was separated into 28 fractions by gradient elution open cc on silica gel. the most active fractions 11-17 were separated again yielding 10 subfractions. this afforded 1,2-di-o-α-linolenoyl-3-o-β-d-galactopyranosyl-sn-glycerol and β-sitosterol-3-o-β-dglucopyranoside, the structures of which were determined by esi-ms and 1d/2d 1h/13c-nmr spectroscopic techniques. furthermore, the diminutive amounts of volatile oil of a.ursinum leaves obtained by steam distillation according to ph.eur. could be evaluated as a third aggregation inhibiting principle. conclusions: at the first time two active, non-sulphur-containing constituents of wild garlic, namely a galactolipid and a phytosterol, could be identified exhibiting inhibitory action on adp-induced aggregation in human blood platelets. as a major constituent, the galactolipid 1,2-di-o-α-linolenoyl-3-o-β-d-galactopyranosyl-sn-glycerol, not yet found in allium spec., appears as a new, highly useful marker substance for a.ursinum drugs, or their pharmaceutical preparations. in recent years, public attention focused more and more on risk factors which may impair sperm quality and thereby human reproduction. in this context, for example pesticides, alcohol, cigarettes, and even mobile phones are discussed. a variety of parameters exists including sperm counts as well as sperm motility, which are considered to be two of the most important parameters to evaluate sperm quality in animal models with the final aim to assess human risk. in recent years computer assisted sperm analysis (casa) devices mostly replaced the formerly used manual counting and manual motility assessment. however, although casa offers multiple opportunities and can allow for an objective and more detailed evaluation, several pitfalls exist which can alter the results profoundly and consequently compromise the quality of the data and ultimately the validity of a study. the aim of the present study was to establish and validate the casa device tox ivos sperm analyzer from hamilton thorne and thereby to gain detailed knowledge about the practical advantages but also intricacies which may alter the obtained results. in this regard healthy adult male rats (10-12 weeks old) were used. ultrasonic sound resistant sperm heads were isolated from the testis and in addition, sperms were isolated from the cauda epididymis. testicular sperm head counts and sperm motility were assessed using different isolation procedures and/or instrument settings. results different instrument settings modulate both -sperm motility and testicular sperm counts. in this regard, a wide range of results including slight changes as well as false positive/negative results were obtained. in addition, the modification of the isolation procedure can lead to variable results especially for sperm motility. conclusion isolation procedures as well as instrument settings can alter the results. consequently, in an experimental setting, potential adverse effects can be confounded with methodologically mediated apparent findings exerted via inappropriate use of the device -depending on the respective conditions in the test laboratory. this study demonstrates the relevance of standardization of testing conditions adopted for computer assisted sperm analysis and the need for a robust validation prior to use in experimental settings. orai and stim proteins have been identified as central components of the highly ca 2+ selective, store-operated current in immune cells (icrac). the molecular basis of selective orai-mediated activation of the calcineurin/nfat pathway and the crosstalk with other channel and scaffold molecules of the trpc family are still incompletely understood. using patch clamp recordings complemented by fluorescence and tirf microscopy we investigated interactions between orai1 and trpc3 in plasma membrane microdomains of rbl-2h3 mast cells. orai1-mediated crac currents, activated by passive store depletion, were found significantly reduced by over-expression of trpc3. this negative impact of trpc3 on icrac was independent of channel function as the trpc3 pore dead mutant (e630k) inhibited icrac to a similar extent as wild type trpc3. importantly, despite a reduction in icrac, nfat translocation in trpc3 overexpressing rbl cells remained unchanged, or was even slightly promoted. store depletion-induced nfat translocation in rbl cells was as well unaffected by trpc3e630k but substantially reduced by trpc3 mutants with either i) eliminated fkbp12/calcineurin binding (p704q) or ii) deficiency in pkc phosphorylation (s712a). moreover, inhibition of pkc phosphorylation by (gfx109203x; 3 µm) strongly suppressed nfat signaling. we suggest trpc3 as a scaffold that links orai-mediated ca 2+ -entry to nfat/calcineurin signaling within plasma membrane microdomains. neurally-induced bronchoconstriction in human and guinea pig precision-cut lung slices schlepütz m. 1 , rieg a. d. introduction: precision-cut lung slices (pcls) are well suited to study peripheral airway responses in different species. airway tone is under close control of the autonomic nervous system and dysregulation may contribute to airway hyperresponsiveness as observed in human lung diseases such as asthma. hence, the aim of the present study was to characterize neurally induced bronchoconstriction (bc) in guinea pigs (gp) and to compare the results with those in human pcls. methods: pcls were prepared from gp or human lung tissue. nerve endings in pcls were activated by electric field stimulation (efs) or capsaicin addition. cholinergic nerve responses were proven by atropine. capsaicin was used to show excitatory nonadrenergic non-cholinergic (enanc) responses. ruthenium red or skf96365 were used to confirm transient receptor potential (trp) channel contributions upon enanc activation. results: gp and human pcls were both sensitive to efs and airways contracted to 39±26% of the initial airway area (%-iaa) and 63±21%-iaa, respectively. in frequency response curves half maximal response was found at 7.0±1.2 hz for guinea pig pcls and 8.1±0.8 hz for human pcls. efs-induced bc was inhibited by atropine in both species. capsaicin contracted gp to 18±15%-iaa. 60% of human pcls were responsive to capsaicin and airways contracted to 72±10%-iaa, respectively. in gp ruthenium red and skf96365 blocked capsaicin-as well as efs-induced bc. conclusion: gp and human pcls contain atropine sensitive cholinergic and capsaicin sensitive enanc nerve endings. since gp pcls were sensitive to trp channel inhibitors, the involvement of those channels can be characterized with respect to lung diseases. in conclusion, gp pcls resemble the human distal lung innervation and represent a useful model to study neural airway pharmacology. the erk1/2-pathway is involved in pkc-induced nox4 up-regulation schlufter f., xia n., förstermann u., li h. universitätsmedizin mainz institut für pharmakologie, obere zahlbacher straße 67, 55131 mainz, germany nadph oxidases (nox) are major producers of reactive oxygen species in the vascular wall and nox4 is the most abundant nox isoform in human endothelial cells. we have previously shown that treatment of human ea.hy 926 endothelial cells with phorbol 12myristate 13-acetate (pma) for 48 h leads to an up-regulation of nox4 expression. this effect of pma is mediated by protein kinase cα, because it is preventable by the pkc inhibitor gö 6983 and by pkcα-sirna. the present study is aimed to investigate the signal transduction cascade downstream of pkcα. pma-induced nox4 up-regulation can be attenuated by pd 98,059 (an erk1/2 inhibitor), but not by sp 600125 (a jnk inhibitor), indicating in the involvement of erk1/2. consistently, pma treatment leads to a sustained activation of erk1/2, and sirnamediated knockdown of erk1/2 markedly reduces the pma-induced nox4 up-regulation. h89, an inhibitor of the mitogen-and stress-activated protein kinases (msks) has no effect on the pma-stimulated nox4 expression, indicating that msks are not the target molecules of erk1/2 in this scenario. on the contrary, knockdown of the transcription factor elk-1 by sirna significantly reduces the pma-induced nox4 up-regulation. in conclusion, erk1/2 and elk-1 are involved in the pkcα-induced nox4 up-regulation. determination of spontaneous mutation frequencies in normal human mammary gland tissue using the random mutation capture technique schmalbach k., lehmann l. university of wuerzburg section of food chemistry, am hubland, 97074 wuerzburg, germany annually, over 57,000 women develop breast cancer in germany. the accumulation of genetic mutations in mammary gland tissue during lifetime may be reasonable for developing breast cancer. in particular mutations in tumor suppressor genes, e.g. p53, seem to play an important role in developing cancer. up to now, lack of a method sensitive enough to determine the expected very low spontaneous mutation frequency (smf) in normal mammary gland tissue precluded the investigation of the role of spontaneous mutations acquired in the p53 gene in epidemiological studies. the only test with the potential to determine low smfs was the random mutation capture (rmc) assay, a genotype selective method which detects mutants that render the mutational sequence non-cleavable by the taqi restriction enzyme after accumulation of the target sequence. therefore, the suitability of the rmc assay to determine smf in p53 gene in normal human mammary gland tissue was evaluated. thus, the rmc assay was optimized concerning (i) dna isolation, (ii) pcr conditions, and (iii) amount of mammary gland tissue. (i) genomic dna from normal human mammary gland tissue, obtained from healthy women who underwent mamma reduction surgery for cosmetic reasons, was isolated using an extended proteinase k digestion prior to chloroform extraction. (ii) the target sequence in intron 6 of p53 gene was captured by hybridization with a complementary uracil-containing dna-probe synthesized via polymerase chain reaction (pcr), followed by magnetic separation from the remaining genomic dna. the copy number of the target sequence was quantified by competitive pcr. the number of mutants was detected after cleavage of the target dna with taqi by means of pcr with a primer set flanking the restriction site. (iii) with 2 g of normal mammary gland tissue a smf of 2.2±1.4x10 -7 per base pair was determined indicating the rmc assay suitable for smf determination. in conclusion, the smf in the p53 gene in normal human mammary gland tissue was determined for the first time, enabling the future investigation of factors influencing the smf during breast cancer development. cigarette smoke-induced release of pro-inflammatory cytokines including interleukin-8 (il-8) from inflammatory as well as structural cells in the airways, including airway smooth muscle (asm) cells, may contribute to the development of chronic obstructive pulmonary disease (copd). despite the wide use of pharmacological treatment aimed at increasing intracellular levels of the endogenous suppressor cyclic amp (camp), little is known on its exact mechanism of action. we report here that next to the β2-agonist fenoterol, direct and specific activation of either exchange protein directly activated by camp (epac) or protein kinase a (pka) reduced cigarette smoke extract (cse)-induced il-8 mrna expression and protein release by human asm cells. cse-induced iκbαdegradation and p65 nuclear translocation, processes that were primarily reversed by epac activation. further, cse increased extracellular signal-regulated kinase (erk) phosphorylation, which was selectively reduced by pka activation. cse decreased epac1 expression, but did not affect epac2 and pka expression. importantly, epac1 expression was also reduced in lung tissue from copd patients. in conclusion, epac and pka decrease cse-induced il-8 release by human asm cells via inhibition of nf-κb and erk, respectively, pointing at these camp effectors as potential targets for antiinflammatory therapy in copd. however, cigarette smoke exposure may reduce antiinflammatory effects of camp elevating agents via down-regulation of epac1. polycyclic aromatic hydrocarbons (key marker substance benzo[a]pyrene (bap)) have been assumed to play a role in the development of bladder cancer. the objective of the present study was to unravel cellular and in particular cytoskeletal response to bap. to follow the sequential steps of chemical carcinogenesis the differential proteomic profile was analyzed at early and late time points. the study was carried out in a superficial human bladder cancer cell line (rt4) exposed to 0.5 µm bap, a subacute concentration based on results of proliferation (brdu) and dna damage (tunel) tests. cells of a human bladder cancer cell line (rt4) were exposed to 0.5 µm bap for 24 h (n=5), 4 wk (n=7) and 8 wk (n=3). proteins of whole cell lysate were separated by twodimensional electrophoresis (fig. 1) . differentially expressed proteins were identified by matrix-assisted laser desorption/ionization-time of flight analysis. cortactin, actin and tubulin were immunohistochemical stained. changes in migration and colony forming ability were assessed by scratch wound-healing assay and soft-agar colony formation. results: by using several databases (uniprot, reactome, panther) the identified proteins were categorized into different functional classes such as mrna processing, translation, protein metabolic process, or several areas associated with the organization of the cytoskeleton. 45 % of the differentially expressed proteins after 24 h of treatment are involved in the processing of pre-mrna (20 %) and protein metabolism (25 %). this pattern changed after 8 wk of treatment. then, 48 % of the proteins affected the cytoskeleton whereas still 26 % were categorized to protein metabolism and only 7 % to pre-mrna processing. in the immunhistochemical staining, the treated cells appeared after 8 wk of exposure larger and rounder predominantly due to the modifications of the actin cytoskeleton. merged images of actin and cortactin revealed that both proteins colocalized in invadopodiae. after 8 wk, a higher number of treated cells moved toward the centre of the wound and they formed more soft-agar colonies compared to vehicle conditions, suggesting a transformation of the cells. in conclusion, bap exposure causes in an early phase an activation of the spliceosome which can led to an epithelial-tomesenchymal transition. two coordinators of a cell-type-specific splicing program, epithelial splicing regulatory proteins 1 and 2, are currently being validate by pcr. fused master gel : representative 2-de gel of rt4 cells exposed to 0.5 µm bap for 8 wk. protein spots which were differentially expressed compared to control and identified were marked. cannabinoids stimulate mesenchymal stem cell migration via a mitogen-activated protein kinase pathway schmuhl e. 1 , ramer r. mesenchymal stem cells (mscs) are known to be involved in various regenerative processes such as cardiac, ocular, skin and bone tissue healing. however, little is known about the pharmacotherapeutical options aiming at tissue healing steps such as the mobilization and homing of mscs. here, we show that cannabidiol (cbd), a nonpsychoactive cannabinoid, stimulates the migration of human adipose-derived mscs in both boyden chamber and in vitro scratch wound assays. in boyden chambers cbd (0.01 -3 µm) was shown to promote cell migration in a time-and concentration dependent manner. this promigratory action was inhibited by am-630 (cb2 receptor antagonist) and by o-1602 (g protein-coupled receptor [gpr] 55 agonist). moreover, cbd activated the mitogen-activated protein kinase (mapk) pathway as evidenced by increased phosphorylation of extracellular signal-regulated kinase (erk) 1/2. blockade of erk activation by pd98059 prevented cbd-stimulated msc migration, whereas inhibition of p38 mapk by sb203580 was inactive in this respect. furthermore, am-630 and o-1602 were found to attenuate cbd-induced erk activation. an erk-dependent promigratory action was likewise demonstrated for the phytocannabinoid ∆ 9 tetrahydrocannabinol and for the hydrolysis-stable anandamide analogue r(+)methanandamide. we conclude that cbd promotes msc migration via receptordependent erk activation, possibly contributing to tissue healing. the duffy antigen receptor for chemokines (darc) binds promiscuously many inflammatory chemokines without showing intracellular signal transduction. it is mainly expressed on endothelial cells of postcapillary venules and on red blood cells, where it acts as a transendothelial transporter of chemokines and as a chemokine sink, respectively. surprisingly, as shown for human and mouse brain, darc is also expressed at high density in the cerebellum. however, nothing is known about the function of darc in this location. we addressed this question by subjecting c57bl/6 wildtype and darc-deficient mice to a series of behavior experiments including morris water maze-, elevated plus maze-, rotarod-and actometer tests. while the results from the water maze experiments are ambiguous, elevated plus maze trials show a strong aversion of darc -/mice to walk to the end of the open arm, which is consistent with anxiety-like behavior. moreover, darc -/mice show greatly reduced locomotor activity, which is at least partly caused by episodes of reduced mobility occurring more frequently than in the corresponding wildtype controls (elevated plus maze, actometer). finally, darc -/mice spend a significantly reduced time on the rotating rod compared to c57bl/6 wildtype controls, which may indicate an impaired cerebellar function. we conclude that darc in fact modulates brain function. surprisingly, this appears to be happening under homeostatic conditions, although darc binds for the most part to inflammatory chemokines. it remains to be elucidated, how this effect can be caused by a non-signaling chemokine receptor. it may be an indirect consequence of altered brain chemokine concentrations or of as yet unknown signaling pathways activated by darc. transporter gene expression in human head-neck squamous cell carcinoma and epigenetic regulation mechanisms schnepf r. 1 hals-nasen-ohren-klinik, kopf-und halschirurgie, friedrich-alexander-universität erlangen-nürnberg, waldstraße 1, 91054 erlangen, germany background: membrane transporters may affect the disposition and thereby treatment efficiency of anticancer drugs in human head-neck squamous cell carcinoma (hnscc). the gene expression profile of transporters in hnscc, however, is unknown and was evaluated in this study. moreover, we evaluated mechanisms by which transporters are regulated in hnscc. we focused on the role of the nuclear pregnane x receptors (pxr, nr1i2) and epigenetic mechanisms. methods and results: real-time rt-pcr revealed a significantly increased mrna expression of slco1a2 and slco1b3 and a significantly decreased expression of transporters such as slco2b1, slco2a1 and abcc3 in human hnscc tissue samples compared to adjacent normal mucosa. moreover, an association between slco2b1 mrna levels in tumor tissues and five-year survival of hnscc patients was observed (χ 2 =6.59; p=0.010; n=34). bisulfite sequencing revealed that promoter cpg islands of abcc3 and slco2a1 were not methylated and thus these genes were not epigenetically silenced in hnscc tissues. in the hnscc-derived umscc-1 and scc-15 cell lines, transcript expression of transporters (e.g., abcc3, slco2a1; p<0.001) and pxr (nr1i2; p<0.001) was markedly induced by the dna methyltransferase inhibitor decitabine. cotreatment with the prototypical pxr activator rifampicin significantly reversed decitabine-induced abcc3 and slco2a1 expression. conclusions: transporter expression profiles significantly differed between hnscc and normal mucosa and expression levels of slco2b1 may serve as a marker for prognosis. modulation of the epigenome with the anticancer drug decitabine substantially affects transporter expression in umscc-1 and scc-15 cells, suggesting epigenetic regulation mechanisms. moreover, interactions between epigenetic and nuclear receptor-mediated mechanisms in transporter regulation occur. this work was in part supported by the johannes und frieda marohn foundation. the role of hcn2 in neuropathic and inflammatory pain schnorr s. 1 , eberhardt m. the pacemaker current ih is carried by hyperpolarization-activated cyclic nucleotidegated cation channels (hcn1-4) and contributes to cellular excitability in the heart and the nervous system. hcn1 and hcn2 are the two most abundant hcn subunits in peripheral sensory neurons with hcn2 being the prevalent isoform in nociceptive small sized c-fibre dorsal root ganglion (drg) neurons. we examined the role of hcn2 for peripheral sensitization and spontaneous neuronal activity in neuropathic and inflammatory pain. we generated a conditional deletion of hcn2 by using a nociceptor specific cre-transgene driven by the nav1.8 promoter. the nociceptor-specific knockout of hcn2 in drg neurons (nosphcn2ko) was confirmed by quantitative rt-pcr and western blot. immunohistochemical staining revealed that the deletion of hcn2 was mainly restricted to small sized drg neurons. the conditional loss of hcn2 resulted in a significant reduction of ih positive small diameter drg neurons pointing to a central role of this isoform to the hcn current in nociceptive neurons. behavioral studies showed that the lack of hcn2 did not influence basal pain responses but led to a significant reduction in mechanosensation in both neuropathic and inflammatory pain models. however, thermosensation of the mutants was only decreased in neuropathic pain conditions. in wild-type animals, intraperitoneal, intraplantar and even intrathecal injection of the hcn channel blocker zd7288 nearly eliminated tactile allodynia caused by inflammation in contrast to thermal hyperalgesia which remained unaffected. in contrast, pain thresholds in nosphcn2ko mice did not significantly increase after pharmacological block of ih. additionally, experiments revealed that the inflammatory condition induced an upregulation of hcn2 protein in the spinal dorsal horn compared to saline injected mice. our results suggest that hcn2 might be a new target in the treatment of neuropathic and inflammatory pain. the proper functioning of the central, as well as the peripheral nervous systems is of outstanding importance to the survival and well-being of humans. yet, the integrity of neuronal systems is constantly challenged by a plethora of environmental and occupational toxins. some of these toxins preferentially target neural cells. these neurotoxins can exert their devastating effects by very different modes of action. neurotoxins may induce apoptosis or necrosis of neurons, or interfere with axon growth and elongation. these processes can be identified by specialized in vitro tests. furthermore, neurotoxins have been described to alter glial function which may compromise the viability of surrounding neurons. as another important mode of action, several neurotoxins act on neurotransmitter receptors, thereby altering signal propagation within neuronal networks. countless natural and synthetic substances have been characterized for their effects on neurotransmitter receptors and today can be used for detailed studies of receptor function. however, environmental toxins of anthropogenic origin and occupational toxins that both represent constant sources for human exposure are still poorly studied with respect to their effects on neurotransmitter receptors. thus, the need for a better understanding of the susceptibility of neurotransmitter systems for toxic effects exerted by these substances is of outstanding importance for the protection of human health. here, we introduce an imaging-based approach for the screening of the effects of potential and known neurotoxins on neurotransmitter receptors of intact cells in vitro. different neuronal cells were tested for their sensitivities for classical neurotransmitters using life-cell imaging experiments. in more detail, we examined the proportion of responding cells and determined the ec50 values for the most prominent neurotransmitters in cell lines widely used for in vitro neurotoxicity studies on the one hand, namely sh-sy5y and lumes cells, and primary mouse neurons on the other hand. with these data at hand, we are now able to identify and characterize the effects of neurotoxins on receptor function in chronic, as well as acute exposition paradigms. the use of an in vitro imaging-based physiological test system is at the interface between non-functional in vitro approaches and in vivo toxicity tests, thus, giving mechanistic insight into neurotoxic processes without requiring animal experiments. apomorphine acts on trpa1 channels scholze a., schaefer m., hill k. universität leipzig -universitätsmedizin rudolf boehm-institut für pharmakologie und toxikologie, härtelstr. 16-18, 04107 leipzig, germany apomorphine is a non-narcotic derivative of morphine which acts as a dopamine agonist and is clinically used to treat "off-states" in patients suffering from parkinson´s disease. adverse effects of apomorphine treatment include dopaminergic effects such as nausea, but also ulceration and pain at the injection site. we wanted to test whether an activation of trp (transient receptor potential) channels in sensory neurones contributes to the perception of pain after apomorphine injection. while the warm/heat receptors trpv1, trpv2, trpv3, and trpv4 and the cold receptor trpm8 were insensitive towards apomorphine treatment, trpa1 could concentration-dependently be modulated by apomorphine. low micromolar apomorphine concentrations potently activated heterologously expressed trpa1 channels in a stably transfected cell line (hek293-trpa1), as well as natively expressed trpa1 in cultured dorsal root ganglion neurones. on the other hand, when using higher concentrations of apomorphine, we observed inhibition of trpa1 activity. previous studies have shown that subcutaneously administered apomorphine produces a biphasic dose response relationship in rats, inducing hyperalgesia at low doses whereas high doses of the substance cause antinociception. from our studies we conclude that such in vivo effects of apomorphine are presumably mediated by activation/inhibition of trpa1 expressed in sensory neurones agonist binding to a g protein-coupled receptor (gpcr) induces a conformational change of the receptor protein, which results in the activation of receptor-associated heterotrimeric g proteins [1] . in radioligand binding studies, conducted to investigate ligand binding to specific gpcrs, receptors are usually probed with radioantagonists. as in other gpcrs [2] , agonists of the muscarinic m2 receptor exhibit biphasic kinetics and biphasic competition curves with radioantagonists, indicating a more complex situation probably caused by g protein interactions. here, we present a detailed study of the binding of agonists to muscarinic m2 receptors including the novel super-high affinity agonist iperoxo and a differential chemical knockout of g proteins. in addition to membrane homogenates living cells were employed. we demonstrate that the high affinity fraction in biphasic curves does not differ between selected full agonist and is sensitive to pertussis toxin, thus indicating that this receptor population is associated with gi proteins. however, despite promiscuous signalling properties of m2 receptors, the low affinity fraction is not associated with any other g protein, since low affinity binding is insensitive to high concentrations of guanylnucleotides and cholera toxin. moreover, high affinity agonist binding appears solely in membrane homogenates but not in experiments conducted with living cells, probably due to their high intracellular concentration of guanylnucleotides. taken together the chemical knock-out of g proteins revealed that the high affinity binding of agonists in membrane homogenates is associated with the interaction of the muscarinic m2 receptor with gi proteins. the low affinity binding cannot be related to another g protein, although the muscarinic m2 receptor exhibits promiscuous g protein signalling properties. interestingly data obtained with living cells do not reveal any high affinity binding of agonists. prolonged stress leads to a dysregulation of the hypothalamus-pituitary-adrenal (hpa)axis and may affect the sensitivity of pain perception. however, it is not yet known whether the alterations of hpa-axis and increased pain sensitivity are related. to create a long lasting stressful situation, male wistar rats were exposed to a restraint-stress for 1 h daily over a period of two weeks. the effect of stress on the hpa-axis was determined by adrenal morphology and stress hormone levels, the influence on mechanical pain sensitivity was evaluated by the randall-selitto paw pressure test. on day 15 the animals exhibited a significant mechanical hyperalgesia. they also showed increased acth and corticosterone plasma levels and an enlarged zona fasciculata of the adrenal gland, indicating a dysregulation of the hpa-axis. for testing the correlation of hpa-axis dysregulation and hyperalgesia a persistent increase in plasma corticosterone in wistar rats was generated by the administration of corticosterone via the drinking water for two weeks. these animals also showed an increased mechanical nociceptive sensitivity with an accompanied decrease of the adrenal glands and reduced acth levels. the results show that chronic stress leads to a dysfunction of the hpa-axis with an accompanied mechanical hyperalgesia which can be mimicked by oral administration of corticosterone. thus, this in-vivo test system may provide a new animal-friendly pharmacological model for stress-related pain disorders. the alternaria mycotoxins aoh and ame induce cyp1a1 and apoptosis in murine hepatoma cells dependent on the aryl hydrocarbon receptor mycotoxins are secondary metabolites of fungi including the genus alternaria (black mold). alternaria fungi are known to infest different types of foodstuffs and produce diverse toxins amongst them the mycotoxins alternariol (aoh) and alternariol methyl ether (ame) which are potential carcinogens. as planar compounds, aoh and ame are preferentially metabolized by cytochrome p450 (cyp) 1a1 and 1a2. the most prominent regulator of cyp1a1 is the dimeric transcription factor complex ahr/arnt, which is activated by planar ligands. therefore we studied the activation of ahr/arnt by aoh and ame and monitored cyp1a1 induction in murine hepatoma cells (hepa-1c1c7). indeed, aoh and ame enhanced the levels of cyp1a1 in hepa-1c1c7 cells but not in cells with inactivated ahr (hepa-1c1c12) or arnt (hepa-1c1c4). furthermore, we studied the cytotoxicity of aoh and ame. by using a fluorescence-based microscopic readout we measured effects on cell counts, apoptosis, senescence and micronuclei formation. both aoh and ame reduce the cell number and the cell nuclei show drastic morphological changes e.g. enlargement after aoh treatment or micronuclei formation. the observed effects where, except for the induction of apoptosis, independent of ahr/arnt. in summary, aoh and ame activate the ahr/arnt pathway to induce cyp1a1 expression and apoptosis. however, the predominant cytotoxic effect of aoh and ame in hepatoma cells is a profound reduction in cell numbers, which is independent of the ahr/arnt pathway. special purpose databases are the first place for researchers in the life sciences to obtain expert curated data. naturally, such resources are limited in terms of timeliness and comprehensiveness. the literature database pubmed alone lists more than 20,000,000 scientific abstracts, and 700,000 are newly added every year. the protein sequence database uniprotkb stores over 10,500,000 sequences, a hundred times more than ten years ago. turning these data into meaningful information and making it accessible to both humans and computers have become an essential part of biological discovery and biomedical research. text mining techniques have proven useful to extract the missing links in areas such as drug-target and drug-disease prediction, the extraction of mutation-phenotype associations, or the prediction of protein-protein and protein-ligand interactions. by systematically extracting information from available literature, reports or patents, text mining techniques can help to refine existing categorical knowledge stored in databases, and hence will support drug repositioning, the discovery of novel cancer biomarkers, or help to understand causes of hereditary diseases. in the area of regulatory toxicology we developed go3r, the first knowledge-based search engine for alternative methods to animal experiments. the system not only helps retrieving information on the availability of alternative methods that allows for replacing, reducing or refining animal experiments, but also provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information and data. the up-to-date taxonomicstructured "table of contents" provided by go3r allows for search in the literature listed in pubmed or the toxicology data network (toxnet) in a fast and comprehensive manner. the semantically enriched platform supports the user during the query formulation, allows for bibliographic analysis, and reveals existing relations to replacement, reduction, and refinement of animal experiments. impaired cardiac excitation-contraction-coupling in mice with complete inactivation of the crem gene schulte j. s., tekook m., schmitz w., müller f. u. westfälische wilhelms-universität institut für pharmakologie und toxikologie, domagkstraße 12, 48149 münster, germany the structurally related transcription factors camp response element binding protein (creb) and camp response element modulator (crem) mediate a regulation of gene transcription in response to camp and are expressed in the heart. mice with complete inactivation of crem display impaired cardiac contraction and relaxation, decreased expression of serca and down-regulation of β1-adrenoceptors. to elucidate the underlying functional mechanisms on the cellular level we here investigated cellular electrophysiology and ca 2+ -cycling in ventricular cardiomyocytes from crem ko mice (ko). adult ventricular cardiomyocytes were isolated from ko and wildtype (wt) mice (age 16-20 weeks) and subsequently used for experiments within 6 hours after isolation. action potentials (aps) were recorded from ventricular cardiomyocytes (perforated patch, whole cell current clamp inactivation of crem seems to have no consequences for ap duration and possibly associated ion channels but leads to impairment of ca 2+ cycling and sarcomere shortening under basal conditions well explaining the previous findings in vivo. our results show that crem is essential for a regular excitation-contraction coupling in the mouse heart. (supported by the izkf münster) new mechanistic insights in no/cgmp actions in the vasculature schulte k., koesling d., universitätsstr. 150, 44781 bochum, germany hypertension, a major risk factor for cardiovascular diseases, is associated with vascular changes resulting in increased vascular contractility und vascular peripheral resistance. a prominent factor in the development and maintenance of hypertension is the reninangiotensin-aldostrerone system. angiotensin ii (ang ii) is the main mediator of this system and a powerful vasoconstrictor. ang ii increases the intracellular ca 2+ concentration thereby activating myosin light chain (mlc) kinase, which enhances mlc phosphorylation and subsequent vascular contraction. opposite to angii-induced vascular contraction, no/cgmp pathway promotes vascular relaxation by decreasing ca 2+ concentration and lowering mlc phosphorylation. responsible for mlc dephosphorylation is the mlc phosphatase (mlcp), whose activity is regulated by different phosphorylations. phosphorylation of mlcp via rhoa-activated rho-kinase enhances phosphatase activity while phosphorylation via the cgmp-dependent protein kinase has been proposed to decrease enzymatic activity. to investigate the interplay of angii with the no/cgmp pathway, we treated wild-type and ko mice lacking the cgmp forming no receptor, no-gc1, with angiotensinii (1.44 mg / kg bw / d) for two weeks. in addition to various cardiovascular parameters, physiological changes in vascular reactivity of aortic rings of angii-treated wt and no-gc1 ko mice were assessed in organ bath experiments and correlated with biochemical parameters as the phosphorylation state of mlc, mlcp and rho-kinase activities examined by immunoblot analysis. analysis of cgmp levels revealed that angii treatment decreased cgmp in wt mice to levels comparable to those of the ko mice which were unaltered by the treatment. our study will provide further mechanistic insights in the molecular interactions between constrictor and dilator stimuli in the vasculature. nanomaterials are already used today and offer even greater use and benefits in the future. the progress of nanotechnology must be accompanied by investigations of their potential harmful effects. for airborne nanomaterials, lung toxicity is a major concern and obviously the particle size is discussed as a critical property directing adverse effects. while standard toxicological test methods are generally capable of detecting the toxic effects, the choice of relevant methods for nanomaterials is still discussed. we have investigated two genotoxic endpoints -alkaline comet assay in lung tissue and micronucleation in polychromatic erythrocytes of the bone marrow -in a combined study 72 hours after a single instillation of 18 µg gold nanoparticles (np) into the trachea of male adult wistar rats. the administration of three test materials differing only in their primary particle size (2-, 20-and 200-nm) did not lead to relevant dna damage in the mentioned tests. the measurement of clinical pathology parameters in bronchoalveolar lavage fluid (balf) and blood indicated neither relevant local reactions in the animals' lungs nor adverse systemic effects. minor histopathology findings occurred in the lung of the animals exposed to 20-nm and 200-nm sized nanomaterials. in conclusion, under the conditions of this study the different sized gold np tested were non-genotoxic and showed no systemic and local adverse effects at the given dose. platelet dense granule secretion mediates platelet-dependent enhancement of tumor cell transmigration and formation of metastases schumacher d., strilic b., wettschureck n., offermanns s. mpi für herz-und lungenforschung offermanns, ludwigstr. 43, 61231 bad nauheim, germany tumor cell metastasis to distant organs is the primary cause of mortality in cancer patients. tumor cells leave the primary tumor, intravasate, survive in the circulation and extravasate through the endothelial cell layer to grow in the target organ. it has long been known that blood platelets play an important role in tumor cell survival and dissemination, but the mechanism by which platelets promote metastasis remained unclear. given that platelets are found closely associated with tumor cells shortly after vascular arrest, we explored whether platelets can facilitate the transmigration of tumor cells through the endothelium and thereby promote extravasation of tumor cells into the organ parenchyma. the ability of various mouse and human tumor cells like lewis-lung carcinoma cells (llc1), b16f10 melanoma cells or human neuroblastoma cells (sh-sy5y) to transmigrate through an endothelial cell layer was strongly enhanced by seeding tumor cells together with mouse or human platelets onto the endothelial cell layer. this indicates that platelets facilitate tumor cell transmigration in vitro. we found that platelet granule secretion is involved in this process as supernatant from platelets incubated with tumor cells but not from resting platelets was sufficient to enhance tumor cell transmigration. additionally, no platelet-mediated increase of tumor cell transmigration was observed in dense granule secretion-defective platelets of munc13-4 deficient mice. thus, dense granule secretion is required for platelet-dependent tumor cell extravasation in vitro. while the growth and weight of primary tumors after subcutaneous injection of llc1 and b16 cells was indistinguishable between wild-type mice and animals lacking munc13-4, the number of metastases in the lung was strongly reduced in munc13-4-deficient animals. the strong decrease in formation of metastases in munc13-4 deficient mice was also observed after i.v. injection of llc1 and b16f10 cells. thus, platelet dense granule secretion plays a critical role in tumor cell metastasis by enhancing tumor cell transmigration through the endothelial cell layer. formation of dna adducts in mouse tissues by the brassica ingredient 1methoxy-3-indolylmethyl glucosinolate and its break-down product 1-methoxy-3indolylmethyl alcohol schumacher f. 1 , engst w. glucosinolates are secondary metabolites present at substantial levels in cruciferous vegetables, such as broccoli and cabbage. after injury of plant tissue they are activated by the enzyme myrosinase to form various electrophilic degradation products like isothiocyanates. we previously showed that 1-methoxy-3-indolylmethyl (1-mim) glucosinolate (or neoglucobrassicin) is a potent genotoxicant in bacterial and mammalian cells after activation by myrosinase. the induction of mutations could be correlated with the formation of dna adducts [1] . we have identified and synthesized the major dna adducts n 2 -(1-mim)-dg and n 6 -(1-mim)-da. moreover, we developed a highly sensitive uplc-esi-ms/ms method for selective mrm quantification of these adducts using stable-isotopic labeled adducts as internal standards. while the plant enzyme myrosinase is probably almost completely inactivated after cooking the vegetables, the glucosinolates reach the gut mostly intact due to their good heat and ph stability. enzymes of individual intestinal bacteria are able to cleave the glycosidic bond of the glucosinolates, which leads to the formation of reactive metabolites within the gut lumen. we were able to detect significant levels of n 2 -(1-mim)-dg and n 6 -(1-mim)-da in a dose-dependent manner in the large intestine of mice treated orally with isolated 1-mim glucosinolate. the peak levels of n 2 -(1-mim)-dg and n 6 -(1-mim)-da in the murine large intestine were reached 8 h after a single administration of 600 µmol 1-mim glucosinolate/ kg body weight. the oral application of the relatively stable metabolite 1-mim alcohol to mice led to the formation of identical dna adducts. this benzylic alcohol can be activated by sulfotransferases to an electrophilic sulfo conjugate. in contrast to the intact glucosinolate the orally administered 1-mim alcohol generated significant levels of n 2 -(1-mim)-dg and n 6 -(1-mim)-da not only in the large intestine but also in other tissues, such as the liver, of mice. [1] h. glatt, c. baasanjav-gerber, f. schumacher, b. h. monien, m. schreiner, h. frank, a. seidel, w. engst, chem.-biol. interact., 192 (2011) human pregnane x receptor genotype of the donor but not of the recipient is a risk factor for delayed graft function after renal transplantation schwab m. 1, 2 , schaeffeler e. delayed graft function (dgf) is an important immediate complication in renal transplantation significantly contributing to decreased long-term allograft survival. in addition to donor-and recipient-related risk factors altered renal excretion of xenobiotics by membrane transporters may influence dgf as well. using recipients' and donors' dna, we assessed the impact of genetic variants on dgf for the transporter proteins, pglycoprotein (abcb1) and multidrug resistance protein 2 (abcc2), and the nuclear pregnane x receptor (pxr/nr1i2), regulating the transcription of drug metabolizing enzymes and membrane transporters. in our local cohort of transplant patients (n=178) dgf occurred in 27.5 %. logistic regression analysis using four different genetic models (i.e. co-dominant, dominant, recessive and log additive) indicates that only the donor's pxr rs2276707 8055tt genotype was significantly associated with dgf (recessive model: or, 9.0; 95%ci, p=0.004 unadjusted) , even after correction for multiple testing (p=0.035 holm-adjusted). when we performed multivariate analysis including genetic and 16 clinical co-variates (i.e. age, gender, hla mismatches, panelreactive antibodies, immunosuppression using cni, t cell-depleting agents, anti-il-2 receptor antibody and steroids, cold or warm ischemia time, living vs deceased donors or graft loss) again dgf was significantly associated only with the pxr rs2276707 tt genotype of the donor (recessive model: or, 16.08; 95% ci, 2.0-129.46; p=0.0046 unadjusted) which held true after correction for multiple testing (p=0.04). for abcc2 variants only the donor rs17222723 3563t>a genotype correlated with dgf by univariate (or, 4.65; 95%ci, p=0.005 unadjusted) as well as by multivariate analysis (or, 5.5; 95%ci, p=0 .01; table 4 ) but not after correction for multiple testing (p=0.12). for variants in the abcb1 gene no significant associations with dgf were detected for both the donor's and the recipient's genotype. in summary, our findings suggest for the first time that pxr may be a risk gene for the development of dgf independently from previously identified risk factors. supported by the robert-bosch foundation, stuttgart, germany, the bmbf grant 03is2061c (berlin, germany) and by the ferdinand eisenberger grant of the german society of urology (id krs1/fe-10). formation, morphology and structural requirements for formation of microtubule protrusions by clostridium difficile toxin cdt schwan c., kruppke a. s., nölke t., aktories k. institut für experimentelle und klinische pharmakologie und toxikologie i, albertstr. 25, 79104 freiburg, germany clostridium difficile is an anaerobe, gram-positive pathogen. it causes antibioticassociated diarrhoea and pseudomembranous colitis by production of the rho gtpaseglucosylating toxins a and b. recently emerging hypervirulent clostridium difficile strains additionally produce the binary adp-ribosyltransferase toxin cdt (clostridium difficile transferase). cdt is taken up via receptor-mediated endocytosis after binding to the lipolysis stimulated lipoprotein receptor (papatheodorou et al., pnas 2011) . in the cytosol, cdt adp-ribosylates actin at arg 177, thereby actin polymerization is blocked, resulting in disruption of the f-actin network. cdt and other binary actin-adp-ribosylating toxins induce redistribution of microtubules in the cell interior and formation of long (>150 µm) microtubule-based protrusions at the surface of intestinal epithelial cells which increase bacterial adherence (schwan et al., plos pathog 2009 ). the clostridial actin-adp-ribosyltransferases influence the dynamicity of microtubules and their capture at the cell cortex by indirectly affecting different microtubule regulating proteins like clasp2 and acf7. besides the influence of cdt on microtubule regulatory proteins, the formation of protrusions depends on plasma membrane composition. depletion of cholesterol, the breakdown of sphingomyelin or inhibition of sphingolipid-synthesis reduce the formation of microtubule-based protrusions. surprisingly, most of the cdt-induced processes contain membrane-tubules derived from the endoplasmatic reticulum (er). the remodeling of the er is microtubule dependent and is mainly mediated by stim1 that usually functions as a calcium sensor in the er and activates the store operated orai1 calcium ion channels in the plasma membrane. the data suggest that toxin-induced changes of the microtubule system including alterations of the er, may affect trafficking and er-dependent signalling. bilobalide is a neuroprotective constituent of ginkgo biloba with an unknown mechanism of action. in the present study, we first used microdialysis in mice to evaluate changes in the extracellular fluid of the brain during and after stroke. microdialysis probes were implanted into the striatum of cd-1 mice, and dialysates were obtained while a monofilament was inserted for 60 min via the common carotid artery (cca) to block perfusion through the middle cerebral artery (mca). while glucose levels dropped immediately upon middle cerebral artery occlusion (mcao), glutamate concentrations in the microdialysates -as measured with a cma 600 analyzer -rose extensively during ischemia to more than 2000% of baseline level. both glucose and glutamate levels recovered rapidly when mcao was terminated after 60 min. when bilobalide (10 µm) was perfused into the striatum through the microdialysis probe during mcao, glucose levels dropped but the neurotoxic rise of glutamate was significantly attenuated and reached only 500% of baseline level (p<0.01). in the following experiments, we investigated the activity of mitochondria in ischemic brain. ischemia was induced by mcao, and ischemic as well as "healthy" tissue from the opposite hemisphere was obtained. mitochondria were isolated and mitochondrial respiration was monitored using the oroboros ® oxygraph. significant deficits of respiration were observed after ischemia. in the healthy hemisphere, the respiratory states (leak i+ii, complex i+ii+iv, oxidative phosphorylation (oxphos) and electron transport system (ets) capacity) showed a decrease of oxygen consumption to 60-70% of sham-operated mice. in the ischemic hemisphere, several values were lower at 40% of sham-operated mice (leak i+ii, complex ii+iv,oxphos and ets) whereas complex i showed a remarkably low respiratory capacity of 16% of baseline. direct addition of bilobalide (10 µm) to post-ischemic mitochondria caused a 2-fold increase of complex i activity in vitro. pretreatment of mice with bilobalide (10 mg/kg i.p.) one hour before mcao caused a significant, 3-fold improvement of complex i respiration when measured ex vivo. these data clearly indicate that bilobalide targets mitochondrial processes within the respiratory chain, preserving complex i function during ischemia. this action likely explains its neuroprotextive activity in vivo. unreacted resin monomers such as 2-hydroxyethyl methacrylate (hema) are environmental stressors released from dental composites after incomplete polymerization. the production of reactive oxygen species (ros) is a major response of cells to monomer exposure. moreover, adverse effects of monomers including delayed cell differentiation or mineralization processes, dna damage or apoptosis are associated with increased ros production. the intracellular redox homeostasis is controlled by the major non-enzymatic antioxidant glutathione (gsh), and antioxidant enzymes. here, we hypothesized that cells exposed to hema responded by a differential expression of antioxidant enzymes such as superoxide dismutase (sod-1), catalase (cat) or glutathione peroxidase (gpx1/2). raw246.7 mouse macrophages were exposed to hema (0-8mm) for 24h, and protein expression was analyzed by western blotting. to study the influence of intracellular gsh on enzyme expression, gsh synthesis was reduced by the inhibitor buthionine sulfoximine (50µm bso), or enhanced by 2-oxothiazolidine-4-carboxylate (5mm otc) and n-acetyl cysteine (10mm nac). expression of sod-1 found in untreated cultures was decreased in the presence of hema and even further reduced by bso. in contrast, nac counteracted hemainduced inhibition of sod-1 expression. cat expression was not detected in untreated cells, however, the enhanced expression of cat in cells exposed to hema indicated the decomposition of abundant levels of hydrogen peroxide. the minor influence of bso or otc showed that expression of cat was independent of gsh levels while a decrease of hema-induced cat expression in the presence of nac indicated reduced oxidative stress. gpx1/2 was expressed in untreated cultures, and its down-regulation by bso indicated that this enzyme was primarily responsible for h2o2 decomposition. the inhibitory effect of hema on gpx1/2 expression was enhanced by bso but counteracted by otc or nac. these findings indicate that h2o2 is the predominant reactive oxygen species generated in the presence of dental resin monomers like hema. abundant h2o2 production leads to the activation of cat expression and a feed-back inhibition of sod-1 expression. the hema-induced reduction of gpx1/2 expression is most likely a consequence of reduced gsh levels because of the formation of glutathione disulfide (gssg) or by gsh-hema adducts. the life-threatening effects of certain organophosphorus compounds such as soman or fenamiphos cannot be antagonized adequately by the treatment with atropine and oximes. alternative approaches are necessary. since the adequate restoration of disturbed muscle function is considered to be life-saving, a model is needed for screening of potentially therapeutic substances. an established model for the development of such new therapies is the diaphragm preparation. however, this model requires a large number of animals and experimental available time frame is limited to some hours. here, the organotypic nerve-muscle co-culture may be an appropriate alternative, because a large number of specimens with low numbers of animals and a long period of investigation over several days is possible. in the present study, the restoration of vx paralysed muscle function with obidoxime was investigated by using both models. slices of spinal cord and muscle tissue were dissected from mice embryos (e 14-15) , fixed to coverslips and incubated in roller tubes for about 2-4 weeks. spontaneous muscle activity was recorded by video microscopic techniques and was quantified offline. muscle force production in mice diaphragm preparations was elicited by indirect field stimulation technique in a 12 chamber organ bath and quantified as time-force diagrams (auc) that were expressed as relative changes of the muscle force compared to the control data. application of the nerve agent vx (0.75 µm) resulted in a strong reduction of muscle activity in the co-culture and of muscle force production in the diaphragm muscle. after obidoxime (10 µm) was added spontaneous muscle activity in the co culture recovered from 0.45 ± 0.24 hz to 1.67 ± 0.24 hz (control 1.79 + 0.22 hz) . muscle force remained stable over the next days. the vx-blocked muscle force of diaphragm was restored to 69.9 ± 21.3 % by obidoxime compared to control. muscle force production after indirect stimulation was stable for 6 hours only. our results suggest that the organotypic nerve-muscle co-cultures may be an appropriate tool for the screening of new therapeutic approaches in restoration of blocked neuromuscular transmission. moreover, the model offers an additional advantage as long-term experiments may be performed and pre-and postsynaptic effects may be assessed directly. additionally, the number of experimental animals could be reduced. the modulation of gene expression by the transcription factor crem (camp responsiveelement modulator) represents a fundamental mechanism of gene control in response to elevation of intracellular camp levels. in vascular smooth muscle cells (vsmcs) crem is involved in the regulation of cell proliferation and apoptosis supporting its relevance for vascular proliferative diseases. mice with a global inactivation of crem (cko) showed a significant increase in neointima formation after ligation of the carotid artery and an increase of atherosclerotic plaque formation after high fat diet on an apoe knockout background compared to wildtype controls (wt). on the cellular level a crem deficiency was associated with a 1.3 fold increased proliferation rate of primary vsmcs after stimulation with the platelet-derived growth factor (pdgf; n=10 from 6 isolations). microarray analysis and subsequent realtime-rt-pcr validation revealed that the alpha-type platelet-derived growth factor receptor (pdgfra) the regulator of g-protein signaling 5 (rgs5) and peptidylprolyl isomerase a (ppia) were 1.5-2.5 fold upregulated in pdgf treated cko vsmcs compared to wt vsmcs (n=6). transcripts of rgs5 (2.6 fold, ) and ppia (1.8 fold, were also upregulated in the carotid artery of cko mice in comparison to wt mice (n=10-12). we conclude that crem deficiency is associated with transcriptional changes of rgs5, pdgfra, ppia, which might explain the increased proliferation rate in cko vsmcs and the increased responsiveness to pathophysiological conditions. (supported by the izkf münster). the role of non-catalytic p101 and p87 subunits in regulating phosphoinositide 3kinase γ by gβγ and h-ras shymanets a. 1 phosphoinositide 3-kinase γ (pi3kγ) controls a plethora of cellular responses. pi3kγ, a heterodimer formed by non-catalytic p101 or p87 and catalytic p110γ subunits, is regulated by gβγ and ras. earlier we speculated that p101 binds to gβγ to translocate cytosolic pi3kγ to the plasma membrane, enabling direct activation of p110γ (brock et al., j. cell biol. 2003) . however, the p87 subunit does not function as gβγ adapter (kurig et al., pnas 2009) . since the impact of each non-catalytic subunit in regulating p110γ by gβγ and ras still remains elusive, we studied their role in detail. gβ1γ2 variants harbouring mutations in positions involved in interaction with gα subunit were purified from sf9 cells and tested for their ability to activate pi3kγ. we observed that p101, but not p87, was able to rescue the stimulatory activity of gβ1γ2 mutants incapable to activate p110γ (shymanets et al., biochem. j. doi:10 .1042/bj20111664). to further study the functional impact of the non-catalytic subunits on pi3kγ regulation, we have designed phospholipid vesicles containing similar amounts of recombinant pi3kγ variants. although p87/p110γ exhibited stronger sensitivity to gβ1γ2 than p110γ, the activity of vesicles-associated p101/p110γ was significantly higher as compared to vesicles-associated p87/p110γ or p110γ in the absence and in the presence of gβ1γ2. to study an effect of ras proteins on pi3kγ variants, recombinantly expressed h-ras was purified from sf9 cells. the posttranslational processing and lipidation of the protein was verified by mass spectrometry analysis. the impact of h-ras on regulation of p87/p110γ and p101/p110γ differed, which may explain integration of pi3kγ variants in different signalling pathways. taken together, p87 and p101 subunits implement discrete functions in respect to (i) membrane recruitment of pi3kγ and (ii) regulation of enzymatic activity by gβγ and h-ras. preparation of consolidated exposure scenarios for mixtures under reach sica m., dorn s., mostert v. dr. knoell consult gmbh, marie-curie-str. 8, 51377 leverkusen, germany under reach, formulators of mixtures need to include substance-related information into extended safety data sheets (esds), if mixtures are classified as dangerous according to the dangerous preparation directive (directive 1999/45/ec). one way to add information on substances into esds of mixtures is to generate exposure scenarios (ess) for mixtures. in order to fulfil this task, two approaches have been developed for the identification of the risk-determining substances (lead substances) in the mixtures: the critical component approach (cca) relies on dnels and pnecs for all substances, their concentrations in the mixtures as well as substance and use-specific availability parameters (echa, 2008) . in contrast, the dpd+ method is based on the legislation for classification of preparations (directive 1999/45/ec). the dpd+ method defines a lead substance for each route of human exposure and for the aquatic environment (cefic, 2010) . however, each of these methods has certain limitations. the aim of the present work is to improve the preparation of consolidated ess for a number of mixtures and provide information about their safe use. to this end, we first adopted information on risk management measures (rmms) and operational conditions (ocs) of the lead substances using the dpd+ methodology. at the same time, we considered the specific conditions of use of the mixtures (e.g. spraying, brush painting). we then conducted risk assessments by deriving dnels for the mixtures and using exposure modelling tools recommended under reach (e.g., ecetoc tra, riskofderm, art). we compared the outcome of these assessments with results obtained from the application of the dpd+ methodology. the work presents the results of application of dpd+ approach and the cca and indicates possible improvements for the risk assessment of mixtures. to check for seasonal and weather dependent influences in the prescription rate of drugs used to treat cardiovascular and respiratory diseases (atc codes c and r) a survey covering all prescriptions of a specimen german general regional health insurance (aok plus, data for saxony, largest health insurance service, approx. 50 % of all saxonian citizens are inscribed to this service) for the years 2005 to 2007 was analysed on a monthly basis. the number of prescriptions for cardiovascular drugs changed approximately +/-20 % around the mean for the different month without a clear seasonal pattern. for respiratory drugs only the systemic anticholinergics and drugs used to treat obstructive lung disease displayed a distinct seasonal pattern with a 50 -70 % above average prescription figure during spring time (february to may) and a 25 to 40 % trough in late summer/autumn (july to october). the data have to be analysed for further cofounders (e.g. influenza prevalence, environmental conditions etc.) to fully understand the fluctuations observed. the prescription rate for cardiovascular drugs and respiratory drugs seems to be influenced by multiple factors aside seasonal influences. pasteurella multocida toxin prevents osteoblast differentiation by activation of heterotrimeric g proteins siegert p., aktories k., orth j. institut für experimentelle und klinische pharmakologie und toxikologie abt. i, albertstr. 25, 79104 freiburg i. br., germany pasteurella multocida toxin (pmt) is a major virulence factor of pasteurella multocida causing pasteurellosis in man and animals and is responsible for atrophic rhinitis in pigs. the toxin modulates various signaling pathways by acting on the heterotrimeric g proteins gαq, gα12/13 and gαi. pmt activates gq to increase inositol phosphate production via phospholipase cβ and alteration of gene expression via the jak/stat pathway. the toxin also activates rhoa via gαq and gα12/13 family proteins. we showed that the underlying mechanism of the activation of heterotrimeric g proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the g protein. because pmt is the causative agent to induce progressive atrophic rhinitis in pigs, which is characterized by loss of nasal turbinate bones leading to a twisting and/or shortening of the snout, we studied the effect of pmt on bone cells. here we studied the effect of the toxin on osteoblast differentiation in st-2 cells and in primary osteoblasts from rat calvaria. st-2 cells are stromal derived cells, which can be differentiated into osteoblasts or adipocytes. the toxin inhibits the differentiation of st-2 cells into osteoblasts studied by determination of specific osteoblast markers. additionally, pmt represses the induction of transcription factors essential for osteoblast differentiation. moreover, the principal pathways activated by pmt to induce these effects were investigated. ventilator-induced lung injury (vili) is a serious problem in intensive care medicine. its mechanisms are only incompletely understood, although it is widely accepted that ventilation-induced inflammation (biotrauma) makes an important contribution. the isolated perfused mouse lung (ipl) is a valuable tool to investigate the mechanisms of vili. several studies have shown considerable differences between various mouse strains with respect to lung mechanics and inflammatory responses. therefore, we hypothesized that the pulmonary responses to mechanical ventilation differ between c57bl/6 and balb/c mice. in addition, this study introduces the novel half lung technique that allows to obtain lung tissue from the same mouse at two different time points. isolated perfused mouse lungs from c57bl/6 or balb/c were subjected to high (25cmh 2o) or low pressure (8cmh2o) ventilation for 240 minutes. after 180 minutes the left lung was removed and used for western blot analysis. the right lung was ventilated for another 60 minutes. by the end of experiment the right lung was removed and qrt-pcr performed. during the whole experiment perfusate sample were taken from the venous catheter and used for protein quantification by elisa. it was possible to remove half of the lung and to further ventilate the other half without acute changes in lung mechanics. in both strains high pressure ventilation elicited a significantly higher cytokine release than low pressure ventilation. c57bl/6 mice showed higher tnf, il-1β and amphiregulin levels after high pressure ventilation, whereas balb/c exhibited increased production of cxc chemokines (cxcl-1, cxcl-2) and il-6. kinase activities (jnk, akt, erk1/2, p38 map kinase) were increased in high pressure ventilated animals, but were strain independent. the novel half lung technique builds on the well established ipl method. it permits to separately analyze the left and the right lung at different time points during continual ventilation. this method reduces animal numbers by 50% and allows statistical within subject analysis. using this method, the present study showed that inflammatory response to mechanical ventilation differ between c57bl/6 and balb/c. these findings show that the biotrauma response in mice depends on the strain that is studied. macrophages play an important role as an integral part of the first line of immune defense. two different macrophage populations have been described. m1 macrophages produce proinflammatory cytokines and are involved in inflammatory processes. by contrast, m2 macrophages release anti-inflammatory cytokines and extracellular matrix components. they can enhance wound repair and angiogenesis, but they can also promote tumor progression. recently, industrial nanoparticles have raised concerns because of their putative toxic effects. on the other hand, specifically designed nanoparticles can be used as clinical diagnostics and as drug carriers for pharmacotherapy. thus, investigations on the interactions of engineered nanoparticles with living cells and organisms are of great importance. macrophages as phagocytosing cells scavenge nanoparticles circulating in the bloodstream. therefore, we analyzed how nanoparticles with different surface functionalization might affect functions of human macrophages. monocytes were isolated from buffy coats and differentiated to macrophages with macrophage colony-stimulating factor. carboxy-(ps-cooh) and amino-(ps-nh 2) functionalized polystyrene nanoparticles were produced by the miniemulsion polymerization process and the average particle size, the polydispersity index and their zeta potential were determined by dynamic light scattering. the macrophages were cultured in the presence or absence of different concentrations of ps-cooh and ps-nh2 nanoparticles for up to 6 days. analysis of cell viability revealed that ps-nh2 but not ps-cooh concentration-and time-dependently reduced the macrophage viability. by annexin v/propidium iodide double staining we could show that ps-nh2 trigger apoptosis in macrophages. we further polarized macrophages to either m1 or m2 using ifn-γ and lps or il-4, respectively. these macrophage populations were characterized by their expression of extracellular markers by flow cytometry and their production of cytokines by elisa. the effects of functionalized polysterene nanoparticles on the cytokine production and surface marker expression of m1 and m2 macrophages were analyzed. our data indicate that surface functionalization is a critical parameter in the nanoparticle-induced toxicity in human macrophages. this work was supported by the dfg spp1313. loos c., lunov o., syrovets t., simmet t. ulm university institute of pharmacology of natural products & clinical pharmacology, helmholtzstr. 20, 89081 ulm, germany nanoparticles are currently used for various medical applications including imaging, diagnosis and drug delivery. due to particle size and surface area, their fundamental properties differ significantly from those of corresponding bulk materials. nanoparticles circulating in the blood are mainly sequestrated by the reticuloendothelial system that consists predominantly of phagocytic macrophages. macrophages express a variety of cellular receptors for sensing and internalizing particular material like viruses, microorganisms, and foreign particulate matter including nanoparticles. therefore, a detailed understanding of the intracellular fate and processing of the nanoparticles by macrophages is indispensable for controlled biomedical applications of nanoparticles. introducing distinct surface modifications, one might control nanoparticle uptake by different cell types and thereby target specific tissues and cellular compartments. tumor cell lines are frequently used as models for primary cells to analyze the effect of nanoparticles on cells. here we show that carboxy-(ps-cooh) and aminofunctionalized (ps-nh 2) polystyrene nanoparticles of ~100 nm in diameter are internalized by human macrophages, thp-1 monocytic leukemia cells, and by pmadifferentiated thp-1 cells via different mechanisms. in buffer, macrophages and thp-1 rapidly internalize both types of nanoparticles, yet, the carboxy-functionalized particles were taken up to a higher extent. the uptake of both nanoparticles was drastically reduced in media containing serum. using pharmacological and antisense in vitro knockdown approaches, we showed that the specific interaction between cd64 receptors and the particles determines the macrophage uptake of particles by phagocytosis, whereas particle internalization by thp-1 cells occurred via dynamin iidependent endocytosis. by contrast, pma-differentiated thp-1 cells took up the particles via macropinocytosis. in line with the in vitro data, more intravenously applied ps-cooh particles accumulated in liver tissue, whereas ps-nh 2 were preferentially targeted to tumor tissue. these data show that the amount of particle internalization, the uptake mechanisms, and kinetics differ significantly among primary cells and model tumor cells, whether differentiated or not, and that they are further critically dependent on the particle opsonisation by serum proteins. this work was supported by the dfg spp1313. specifically designed and functionalized nanoparticles hold great promise for a variety of biomedical applications. to ensure their safe application, such particles require a rigorous analysis of their effects on cell functions. here we demonstrate that aminofunctionalized polystyrene nanoparticles (ps-nh2) of ~100 nm in diameter in contrast to carboxy-(ps-cooh) and nonfunctionalized (ps) particles induce an nlrp3 inflammasome activation and the subsequent release of il-1β in human macrophages. amino-functionalized ps nanoparticles induced time-dependent lysosomal destabilization followed by release of lysosomal enzymes. this resulted in mitochondrial damage and formation of reactive oxygen species. accumulation of mitochondrial reactive oxygen species was accompanied by oxidation of thioredoxin, a protein playing a central role in maintaining the cellular redox balance. upon oxidation, thioredoxin dissociated from the thioredoxin-interacting protein (txnip). liberated txnip, in turn, interacted with the nlrp3 protein resulting in a conformational change of the pyrin domain of the nlrp3 protein as predicted by molecular modeling. txnip interaction with nlrp3 led to assembly of the nlrp3 inflammasome complex, to caspase-1 activation, and release of il-1β. using an in vitro knockdown approach, we showed that ps-nh 2 induced activation exclusively of nlrp3, whereas other inflammasomes remained unaffected. treatment of macrophages with n-acetyl-l-cysteine, a scavenger of reactive oxygen species, abolished both, the caspase-1 activation and the subsequent release of il-1β caused by ps-nh2 nanoparticles. these data reveal a novel mechanism of the nlrp3 activation induced by amino-functionalized nanoparticles and provide a strategy as to how such an effect can be functionally antagonized by supplementation with a radical scavenger. this work was supported by the dfg spp1313. the semi-permeable barrier of the endothelial cell lining of the blood vessels has important synthetic and metabolic functions including transport of cells and biomolecules, regulation of vascular smooth muscle tone, and control of hemostasis. plasmin is a serine protease, which is generated from its zymogen plasminogen under physiological and pathological conditions. small amounts of plasmin are produced in the context of contact activation during inflammation. consistently, increased generation of plasmin has been reported during atherosclerosis. we have shown previously that plasmin, in addition to its role in fibrinolysis, could induce proinflammatory activation of various cells including monocytes, macrophages, and dendritic cells. therefore, we analyzed how plasmin might affect the functions of endothelial cells, which could be relevant during inflammation and atherosclerosis. using flow cytometry, western immunoblotting and fluorescent microscopy, we show that endothelial cells of different origin express the plasmin receptor complex composed of annexin a2 and s100a10. addition of plasmin to human umbilical vein endothelial cells (huvec) induced timeand concentration-dependent cytotoxic effects in the cells. in addition, within 30 min plasmin triggered a rapid and prolonged expression of free radical oxygen species (ros) in endothelial cells as analyzed by microscopy and fluorometry using the rossensitive dye carboxy-h 2dcfda. the ros production in endothelial cells was accompanied by cell detachment. fluorometric and western blot analysis of caspase 3 activation in the cells treated with plasmin showed that plasmin induced apoptotic cell death in endothelial cells, which was evident already several hours after exposure to plasmin. thus, plasmin might induce production of ros in endothelial cells, their detachment and apoptosis, events which might be relevant for the development of atherosclerosis. this work was supported by the dfg. sesquiterpene lactones (stl) comprise a large group of secondary plant metabolites that constitute the active principle of a number of traditional anti-inflammatory phytomedicines. specifically helenalin and parthenolide have recently gained considerable attention as lead compounds or putative therapeutics for the treatment of inflammation and possibly cancer. both compounds have been shown to interfere with the signal transduction through inhibition of the nuclear factor κb (nf-κb). whereas the inhibitory effects of the stl on nf-κb are undisputed, their molecular mechanism of action remains a matter of debate. surface plasmon resonance (spr) analysis allows label-free measurement of molecular interactions. yet, analysis of the interaction of immobilized recombinant proteins with small molecular ligands remains a technically challenging task. in the present study we used spr technology to investigate the molecular interaction of the stl helenalin with putative intracellular target proteins such as the nf-κb protein p65/rela, the catalytic subunits of the ikk complex, namely ikkα and ikkβ, and the intracellular antioxidant glutathione (gsh). at physiological ph 7.4, helenalin interacts with rela (k d = 4.8 µm), yet it failed to bind either ikkα or ikkβ. hence, when dna with nf-κb binding consensus sequence was immobilized on sensor chips, the binding of rela was inhibited by helenalin with an ic50 of 5.0 µm. moreover, we provided several lines of evidence that stl may modify rela on cysteine 38 by a michael-type addition. this interaction was confirmed by molecular docking that identified the best matching interaction between rela and helenalin with predicted hydrogen bonding interactions between helenalin and residues arg35, lys37, gly44 and ile118 of rela. consistent with our hypothesis that helenalin interacts with sulfhydryl groups at ph 8.0, helenalin was also able to interact with reduced, but not oxidized, glutathione with a kd of 24 µm, though no significant interaction was observed at ph 7.4. thus, we showed that the sesquiterpene lactone helenalin interacts with the nf-κb protein rela but not with ikkα or ikkβ. moreover, at physiological ph, helenalin does not interact with glutathione to any significant extent. direct interaction of helenalin with rela leading to inhibition of rela-dna binding and transactivation might present the molecular mechanisms underlying the anti-inflammatory effects of stls. although nanosized materials are quickly taken up by macrophages, our understanding of the involved processes is still rather limited. therefore, we analyzed the uptake of diagnostically used carboxydextran-coated iron oxide nanoparticles of two different sizes, superparamagnetic iron oxide nanoparticles of 60 nm (spio) and ultrasmall superparamagnetic iron oxide nanoparticles of 20 nm (uspio), by human macrophages. by pharmacological and in vitro knockdown approaches, the principal uptake mechanism of macrophages for both particles was identified as clathrin-mediated, scavenger receptor a-dependent endocytosis. further, we created a mathematical model of the nanoparticle uptake by macrophages that permitted determination of key parameters of endocytotic process, such as the uptake rate, the mean uptake time, the number of particles taken up by a cell, and the correlation between the number of internalized particles and their extracellular concentration. the model also provided information on the individual and collective wrapping time of the nanoparticles and described the relation between biophysical parameters such as cytoskeletal forces, membrane elasticity, and the uptake time. finally, we gained information on the minimal linear spacing between simultaneously acting neighboring endocytotic pits that contain single nanoparticles and govern the collective uptake process. the calculated parameters were further confirmed experimentally using spinning disc confocal microscopy. thus, the new model provides important insights into the biophysical processes involved in endocytosis of nanoparticles by human macrophages. this work was supported by the dfg spp1313. prostaglandins (pg) are hormones which are formed during inflammatory processes from arachidonic acid by cyclooxygenases and prostaglandin synthases [4] . in the subsequent metabolism, in which the five-membered ring is dehydrated, α,β-unsaturated carbonyl compounds are generated [2, 3] . these come along with mercapto groups of amino acids in a michael addition reaction associated with activation of cellular enzyme cascades [1] that potentially contribute to their possessed antiinflammatory, antineoplastic and antiviral effects [5] . however little is known so far about possible adverse health effects.we addressed the question whether selected cyclopentenone prostaglandins (cypg) exhibit potential mutagenic and genotoxic properties in the hamster lung fibroblast cell line v79. induction of dna damage was investigated by single cell gel electrophoresis assay (scge). the impact of cypg on cellular redox status was detected by total glutathione (tgsh) assay. the induction of micronuclei and apoptosis was determined by staining with 4',6-diamidino-2-phenylindole (dapi). furthermore the hypo-xanthine-guanine phosphoribosyltransferase (hprt) assay was used for mutagenicity testing. , followed by prostaglandin a2 (pga2), showed the most distinctive genotoxicity, i.e., induction of micronuclei, and apoptotic effects. furthermore, the 15dpgj2 and pga2 -induced significant decrease in the tgsh level in v79 may contribute to the observed increase in oxidative dna-damage. however, none of the tested cypg exhibited mutagenic properties in the hprt assay. in conclusion, a potential in vitro genotoxicity of cypg has been observed which may be involved in carcinogenesis associated with chronic inflammation. parabens and methylisothiazolinone are used as preservatives in personal care products. sensitization to parabens and methylisothiazolinone is relatively rare considering their wide use in cosmetics, but only few quantitative or clinical data exist. therefore, we have tested methyl-, ethyl-, propyl-, isopropyl-, butyl-, isobutyl-, pentyl-, phenyl-and benzylparaben , and methylisothiazolinone in the loose-fit coculture-based sensitization assay (lcsa) developed by our working group. the coculture of primary human keratinocytes and allogenic dendritic cell-related cells (dc-rc) in this assay emulates the in vivo situation of the human skin. sensitization potential of the test substances was determined by flow cytometric analysis of the dc-rc maturation marker cd86. determination of the concentration required to cause a half-maximal increase in cd86-expression (ec 50) allowed a quantitative evaluation. the irritative potential of the substances was assessed by 7-aad (7-amino-actinomycin d)-staining. the concentration required to devitalize 50 % of the examined cells compared to a zero control was termed ec50%. parabens exhibited weak (methyl-, ethyl-, propyl-and isopropylparaben) or strong (butyl-, isobutyl-, pentyl-and benzylparaben) sensitizing potential, phenylparaben was found to be a moderate sensitizer, with ec50-values ranging from 16.67 µmol/l (pentylparaben) to 325.36 µmol/l (methylparaben). due to a pronounced cytotoxicity (ec50% = 70.94 µmol/l), we could not estimate an ec50-value for methylisothiazolinone. sensitization potential of parabens correlated with side chain length. parabens showed no (methyl-and ethylparaben) or weak irritative potential (propyl-, isopropyl-, butyl-, isobutyl-, phenyl-and benzylparaben) , only pentylparaben was rated to be irritative. apart from phenyl-and benzylparaben, irritative potential also correlated with side chain length but did not correlate strictly with the sensitization potential. overall, we were able to demonstrate and compare the sensitizing potential of parabens in this in vitro test. it was weak for methyl-and propylparaben, the most commonly used parabens. furthermore, we showed an irritative potential for most of the perservatives. thus the lcsa is a useful in vitro test to compare the sensitizing potential of xenobiotics. phosphorylation of the neurodegeneration-related septin 4 by protein kinase dyrk1a at serine 107 affects protein stability soppa u. 1 septins are gtp-binding proteins forming heterooligomeric complexes and filaments by interactions of the 13 family members. these complexes have important functions by building scaffolds for proteins involved in cell cycle or cell polarity but their subcellular distribution as well as their regulation remain largely unclear. septin 4 (sept4) was found in neurodegeneration related protein aggregates and is associated with migration of cortical neurons. here we first describe a potential mechanism for regulation of sept4 by phosphorylation via dual specificity tyrosine-phosphorylation-regulated kinase 1a (dyrk1a). dyrk1a is overexpressed in down syndrome and supposed to be involved in neurodevelopment and neurodegeneration. by site directed mutagenesis of flag tagged mouse sept4 and overexpression in hela cells we identified serine 107 as the major phosphorylation site of dyrk1a and generated a phosphospecific antibody. transient coexpression of sept4 and dyrk1a in hela cells increased phosphorylation of serine 107 by 50% in relation to basal phosphorylation. in contrast, cotransfection of the kinase deficient dyrk1a mutants k188r and d287n did not increase serine 107 phosphorylation. moreover we could show, that inhibition of kinase activity by the dyrk1a inhibitor harmine reduced phosphorylation of exogenous sept4 at serine 107 about 25% in hela cells. furthermore, down regulation of dyrk1a by rna interference lead to decreased phosphorylated serine 107. these results indicate that endogenous dyrk1a contributes to sept4 phosphorylation in hela cells. finally we analyzed protein stability of wild type sept4 compared to the phosphorylation resistant s107a mutant in hela cells by inhibition of translation with cycloheximide. we found that in living cells the sept4 s107a mutant is more stable than wild type sept4. in summary, our results suggest phosphorylation at serine 107 by dyrk1a as a novel mechanism to regulate sept4 stability and indicate a possible link of these proteins in cellular processes. is formaldehyde a good example for a "genotoxic carcinogen" with a threshold mode of action? speit g. institut für humangenetik, universität ulm, 89069 ulm, germany formaldehyde (fa) induces toxic and genotoxic effects in directly exposed cells (site of contact). several studies in which fa was administered to rats by inhalation showed evidence of tumor induction in nasal epithelium. there is also some epidemiological evidence that fa causes nasopharyngeal cancer in humans. although fa is a known mutagen, it is still a matter of discussion whether carcinogenesis is primarily mediated via a mutagenic mode of action. there is evidence that cytotoxicity and induced proliferation are the main causes for tumor formation. however, a decisive role of mutagenesis cannot be excluded and a mutagenic mode of action has to be considered for risk estimation. the basic assumption is that mutagens have a non-threshold mode of action. a threshold mode of action for a chemical is likely when a substance with a known mutagenic potential does not induce mutations at low concentrations due to a specific type of reaction with the genetic material and / or physiological protective mechanisms. because fa is a directly acting dna-reactive substance, a threshold mode of action may only be considered because of physiological protective mechanisms. after inhalation, pre-lesion protection occurs by unspecific binding to mucus, cellular proteins and glutathione. furthermore, fa is efficiently inactivated by enzymatic pathways. if fa reaches and damages the nuclear dna, dna repair mechanisms act as efficient postlesion protection mechanisms. in vivo inhalation studies with rats indicated that fa induces primary dna damage in the nasal epithelium but increased mutation frequencies were not measured. data are now available to show the relative distribution of endogenous versus exogenous dna adducts in different locations of the nose and other organs. considering the fa concentrations present in every living cell and the background levels of endogenous dna adducts, appropriate risk assessment and the identification of practical thresholds for fa-induced genotoxicity become feasible. fraud and misconduct in clinical trials steffen c. formerly federal institute for drugs and medical devices clinical trials unit, kurt-georg-kiesinger-allee 3, 53175 bonn, germany plagiarism in scientific publications has been the subject of public ("guttenplag") and scientific debate. plagiarism violates, however, "only" the intellectual property of its authors. in clinical trials, misconduct may also endager the health of patients. the suppression or falsification of data in clinical trials may mislead patients and doctors to use worthless treatments. clinicians may be provoked to repeat these trials, thereby wasting time and money. in clinical trials, misconduct includes everything from suppression or their repeated publication, the "correction" of unwanted results to the complete invention of the data, their intentional or negligent misinterpretation leading to the publication of biased conclusions from otherwise correctly performed clinical studies. the peer review system cannot protect against fraud, as few reviewers will have the means and the time to reevaluate original data. they will have to rely on these data, the calculations and statistics that are presented to them. some kinds of fraudulent behavior, such as double publications, can be found in the review process, but this is also time-consuming and depends on a helpful librarian. other kind of fraud, as the suppression of patient data in clinical trials (the deletion of "non-responders", according to cinderella: the good ones go into the pot, the bad ones go into your crop) can only be detected by the national competent authorities performing an inspection according to good clinical practice. even if the results of such an inspection become public as in the case of ukrain (gansauge et al. 2002) , there is no institution that will further analyze and publish the misconduct or initiate the retraction of the incriminated paper. a german agency similar to the us office of scientific integrity could foster good clinical practice in germany. although it is sometimes very difficult to decide whether improper results arise from fraud or error, a bias for the source of funding is obvious. trials funded by for-profit organizations were significantly more likely to recommend the experimental drug as treatment of choice (als-nielsen et al. 2003) . medicinal products with disputed efficacy such as orally applied enzymes for systemic action, bacterial preparations for irritable bowel syndrome and food supplements are to be reviewed with special attention. further examples from the author's experience will be presented. ]i favors further kca opening, kca may establish a feed-forward regulation of ca 2+ influx. in the present study we analyzed whether kca channels of sk4 and bk type play a role in sustaining ca 2+ oscillations at g1-to s-phase transition of primary mouse tumor cells and in human breast cancer cells, aiming towards a better understanding how kca modulate tumor cell proliferation. methods: kca expression was quantified by qpcr in human breast cancer biopsies, transgenic mmtv/c-neu + mouse mammary tumors and primary tumor cells derived thereof. the identity of the tumor cells was verified by gliolan staining. proliferation of the primary mammary tumor cells in the presence or absence of bk and sk4 modulators was tested using a real time cell monitoring system. the cellular dna content as a measure for cell ploidity was determined by propidium iodide staining and flow cytometry. changes in [ca 2+ ]i oscillations and peak amplitude were determined using ca 2+ indicator fura-2am. results: sk4, but not bk, expression is detectable in human and mouse breast cancer biopsies and in primary tumor cells derived from the mmtv/c-neu + mouse model. sk4 inhibition by tram-34 dose-dependently (0,1 to 10 µm) inhibits the growth of primary mammary tumor cells probably by a g1 cell cycle arrest. ca 2+ oscillations in proliferating mmtv/c-neu + tumor cells were ablated upon pharmacologic inhibition of sk4 channels. -dependent cell cycle progression is dependent on sk4 activity. blocking sk4 disrupts a feed-forward loop that coordinates ca 2+ influx via trp or crac channels in tumor cells. the consequences of sk4 inhibition in mammary tumors in vivo will be discussed. synthesis of a triphenylphosphonium substituted derivative of 5-hydroxymethyl-5-methylpyrroline n-oxide stolze k. 1 esr combined with spin trapping is a well-known analytical approach to detect free radicals formed in various biological systems, e.g. superoxide, hydroxyl and a series of carbon-centered free radicals, which are involved in oxidative stress. our aim was to modify the established spin trap 5,5-dimethyl-pyrroline n-oxide (dmpo) with a functional side chain, which can be used further as anchor for moieties enabling the spin trap to penetrate mitochondrial membranes, such as the positively charged triphenylphosphonium substituent. several synthetic routes were tested to introduce a 4-carboxybutyltriphenylphosphonium-substituent to the spin trap 5-hydroxymethyl-5-methylpyrroline noxide (hmmpo). while the activation of the carboxy group via the corresponding chloride was not successful, the use of a mixed anhydride with acetic acid appeared to be a promising way, although the reaction is considerably slower. preliminary spin trapping experiments have been performed with model systems generating superoxide, hydroxyl-, and carbon-centered radicals. othman e. m., stopper h. universität würzburg toxikologie, versbacher str. 9, 97078 würzburg, germany type 2 diabetes mellitus (dm2) is a growing health problem affecting more than 150 million people worldwide. it is associated with severe acute and chronic complications that negatively influence both the quality of life and survival of affected individuals. epidemiological studies clearly indicate that the risk of several types of cancer (including pancreas, liver, breast, colorectal, urinary tract and female reproductive organs) is increased in diabetic patients. diabetic patients are exposed to oxidative stress which plays a pivotal role in the pathogenesis of both micro-and macro-vascular complications. this is due to a decreased antioxidant capacity and chronic exposure to increased levels of reactive oxygen species (ros). since the insulin resistance in dm2 leads to hyperinsulinemia we studied the cellular consequences of the elevated insulin level and showed that it generates superoxide anions (o 2-) and dna damage by a nadph oxidase dependent mechanism in cultured cells. in addition, we found elevated genomic damage in the lymphocytes of diabetic patients as well as oxidative stress and genomic damage in kidneys of diabetic rats. this effect of insulin may contribute to the pathogenesis and progression of dm2 complications including the elevated cancer risk. the classical transient receptor potential (trpc) channel subfamily is regarded as nonselective, calcium permeable cation channels involved in a wide range of physiological events that require calcium signaling. until now, the specific roles of trpc channels in neuronal function are still elusive. given that trpc1 is able to form receptor-operated heterotetrameric channel complexes with other trpc channel subunits, we investigated the role of trpc1 for receptor-operated calcium influx in the heterologous expression system as well as in neurons. for this electrophysiological whole-cell measurements, fluorimetric calcium measurements, mn 2+ quenching and qpcr analysis were applied. furthermore, the effect of trpc1 knock-down on neuronal migration was monitored performing scratch assays, videomicroscopy and g-actin/f-actin assays. employing these techniques, we found that recombinant trpc1 was not able to function as a homomeric channel. instead, trpc1 subunits formed functional receptor-operated heteromeric channel complexes with trpc3, 4, 5, 6, and 7. heteromers containing trpc1 subunits showed significantly decreased calcium permeation in heterologous cell systems. mutation of amino acids in the putative pore forming region of trpc1 further reduced calcium permeability. in gnrh neurons endogenously expressing trpc1, 2, 5, and 6, downregulation of trpc1 by shrna resulted in increased basal cytosolic calcium concentrations and elevated calcium permeability. trpc1 was not involved in store-operated cation influx in gnrh neurons. moreover, trpc1 suppressed the migration of gnrh neurons without affecting cell proliferation. these findings suggest a novel regulatory mechanism relying on the expression of trpc1 and the subsequent formation of heteromeric trpc channel complexes with reduced calcium permeability, thereby fine-tuning neuronal migration. the transient receptor potential melastatin-3 (trpm3) is a calcium permeable nonselective cation channel that can be activated by the neurosteroid pregnenolonesulfate (pregs) or heat. trpm3 is expressed in various tissues, including insulin-secreting βcells and a subset of sensory neurons from dorsal root (drg) and trigeminal ganglia. the ability of pregs to evoke trpm3-like currents in pancreatic β-cells and to induce insulin secretion indicated its involvement in blood glucose regulation. however, trpm3 -/mice show so far no metabolic deficits but further investigations are recommended to evaluate its function in insulin secretion. further studies showed that trpm3 is a nociceptor channel involved in sensing heat and inflammatory thermal hyperalgesia. we performed a calcium-based screening of a compound library (spectrum collection) that identified several natural compounds as trpm3 blockers. the most potent blockers were the citrus fruit flavonoids hesperetin and naringenin as well as ononetin, a chalcon from ononis spinosa. the ic50 values of the substances are in the low micromoles ranges. electrophysiological whole cell measurements as well as calcium measurements confirmed the potency of the trpm3 blockers. furthermore, we could show that these blockers are effective on endogenous trpm3 in drg neurons from mice and isolated β-cells. by drinking grapefruit juice naringenin could be consumed in concentrations that are sufficiently high enough to block trpm3 activity in vivo. in sensory neurons, trpm3 may exert similar functions as trpv1. thus, trpm3 blocker could bear a therapeutic potential for analgesic treatment. xtt-based cell viability assay was used to determine the half-maximum effect concentration (ec50) for the investigated composite components in hgf. following concentrations of substances were used to determine the induced double strand dna breaks (dsbs): 1 /25× ec50, 1 /10× ec50 , 1 /3× ec50, and 1× ec50. each experiment was performed at least four times. hgf were incubated with various concentrations of substances for a period of 6 hours. induced dna double-strand breaks (dsbs) were tested by the γh2ax focus assay, which is a direct marker for dsbs using anti γh2ax antibodies. for quantitative γh2ax analysis foci in cell nucleus were counted by eye down using a fluorescence microscope. each experiment was performed at least four times. in the xtt test following ec50 values of substances were found (mmol/l;mean +/-sem): tmp(eo)9ta a, b 0.087 ± 0.011; 1,6-hddma b, c 4.500 ± 0.700; etma a, c ; 12.000 ± 1.100; a significantly (p < 0.05) differently to 1,6-hddma, b significantly (p < 0.05) differently to etma, c significantly (p < 0.05) differently to tmp(eo)9ta. after six hours of exposure with tmp(eo)9ta at 0.00348 mm there were induced 0.55 γ-h2ax foci-formations in hgfs, at 0.00870 mm 0.67 foci, at 0.02900 mm 0.86 foci and at 0.08700 mm 0.97 foci. after exposure with 1,6-hddma at 0.180 mm there were induced 0.45 γ-h2ax foci, at 0.450 mm 0.64 foci, at 1.500 mm 0.94 foci and at 4.500 mm 1.32 foci. after exposure with etma at 0.48 mm there were induced 0.43 γ-h2ax foci, at 1.2 mm 0.50 foci, at 4.0 mm 0.61 foci and at 12 mm 0.71 foci. the negative controls dmso and medium cultures displayed 0.31 -0.34 γ-h2ax foci/cell. it was found that the induction of foci/cell were concentration-dependet for all xenobiotics in the order of: 1,6-hddma > tmp(eo)9ta > etma. these results show that dental composite components can induce dsbs in primary oral cells and therefore these substances demonstrate a genotoxic potential. effects of antioxidants on the dna-toxicity of dental (co)monomers in human gingival fibroblasts styllou p. 1 , scherthan h. unreacted (co)monomers can be released from restorative dental materials and may show biologic activity after ingestion in the human organism. in previous studies the mutagenic/carcinogenic effect of dental monomers/co-monomers (e.g. methacrylates) on the human dna was demonstrated. in this study the effects of the antioxidants vitamin c and n-acetylcysteine on the dna toxicity of the (co)monomers triethylenglycol-dimethacrylate (tegdma) and 2-hydroxyethyl methacrylate (hema) was investigated. the induction of dna double-strand breaks with (co)monomers alone and in combination with antioxidants was investigated in human gingival fibroblasts (hgf). hgf were incubated with substances without or with antioxidants for a period of 6 hours. induced dna double-strand breaks (dsbs) were tested by the γh2ax focus assay, which is a direct marker for dsbs using anti γh2ax antibodies. for quantitative analysis of the γ-h2ax test, foci were counted by the same investigator by eye down the fluorescence microscope. each experiment was performed at least four times. the halfmaximum effect concentration ec 50 (mmol/l) of triethylenglykol dimethacrylat (tegdma) and 2-hydroxyethyl methacrylat (hema) was taken from of a previous study after using xtt-based cell viability assay. tegdma induced significantly (p < 0.05) higher dsbs compared to hema (1.91 ± 0.04 vs 1.66 ± 0.02). the mean number of cells scored and the standard deviation (sd) were calculated. when cells were exposed to tegdma in combination with the antioxidant vitamin c an increase of dsbs was observed (2.02 ± 0.06), compared to tegdma alone. when cells were exposed to hema in combination with vitamin c an increase of dsbs was observed (1.89 ± 0.07), compared to hema alone. when cells were exposed to tegdma in combination with the antioxidant nacetylcysteine a decrease of dsbs was observed (1.64 ± 0.04), compared to tegdma alone. when cells were exposed to hema in combination with n-acetylcysteine a decrease of dsbs was observed (0.76 ± 0.02), compared to hema alone. these results show that dental (co)monomers can induce dsbs in primary oral cells. it also shows for the first time that the genotoxic potential may be reduced by the addition of the antioxidant n-acetylcysteine. purpose: we aimed to investigate the role of superoxide and peroxynitrite generated by genetically destabilized enos for the development of endothelial dysfunction and vascular remodelling. methods: a mutant of bovine enos in which cys 101 was replaced by ala (c101a) resulting in destabilization of enos has been generated (enos-c101a). transgenic mice carrying c101a were generated on a c57bl/6 background using the endotheliumspecific tie-2 promoter. by breeding these mice with enos knockouts (enos-ko), mice that express enos-c101a (enos-ko/enos-c101a-tg) exclusively in the endothelium were obtained. unilateral common carotid artery ligation experiments were performed in c57bl/6, enos-ko, and enos-ko/ enos-c101a-tg to study a role of destabilized enos for vascular lesion formation. results: western blot analysis confirmed the expression of enos in enos-ko/enos-c101a-tg in aorta (37.1±8.4%, n=9), skeletal muscle (45.4±5.3%, n=10) and myocardium (17.4±4.9%, n=7) and revealed an increased phosphorylation of enos on ser1176/79 (470±47%) as compared to c57bl/6 (p<0.05, n=8). endothelium-specific overexpression of destabilized enos induced a large increase in superoxide and peroxynitrite formation in the aorta and the heart of enos-ko/enos-c101a-tg (p<0.05, n=5-8), which was abolished by nos-inhibitor l-nitroarginine (l-na) suggesting enos-c101a as a source of elevated radical generation. endothelium-specific introduction of enos-c101a at ~ 35% of c57bl/6 level almost completely restored aortic endotheliumdependent relaxation. experiments with l-na, soluble guanylyl cyclase inhibitor odq, peg-catalase and no-scavenger fe(detc)2 indicated that endothelium-dependent relaxation in enos-ko/enos-c101a-tg is nos-and cgmp-dependent and nomediated. four weeks after the carotid artery ligation, neointima formation, media thickening and luminal narrowing were observed in the ligated arteries of all studied genotypes (p<0.05, n=4-7). consistent with vasoprotective roles of enos, neointima formation was accelerated in enos-ko (n=4-7, p<0.05). despite significantly higher vascular levels of nitrotyrosine and peroxynitrite, neointima formation in enos-ko/enos-c101a-tg was substantially lower then in enos-ko and tended to be similar to c57bl/6. conclusions: increased vascular superoxide and peroxynitrite formation caused by destabilization of enos does not induce endothelial dysfunction in healthy mice and has negligible effect on neointima formation. fenton reactivity as a determining parameter for the interaction of manganese oxide nanoparticles with lung epithelial cells sydlik u. 1 , bieschke c. nanoparticles consisting of manganese oxide have been suggested for several innovative technological approaches, including the use in nanomedicine and diagnostics. therefore, the interaction of such nanoparticles with human target cells is of particular interest for the success of nanomedical approaches but also with regard to unintended side effects. to address this problem, we tested different kinds of manganese nanoparticles (mnnp) in an in vitro system which we earlier evaluated for proliferative, apoptotic, and pro-inflammatory endpoints induced by carbon nanoparticles (cnp). mnnp were synthesized by hydrothermal treatment of manganese salt solutions. the particles were subsequently characterized by scanning electron microscopy and dynamic light scattering. biological and toxic effects of the generated particles were studied in comparison to carbon nanoparticles (cnp) in experiments with rat and human lung epithelial cells (rle-6tn and 16hbe14o-). cytotoxicity was determined as measures of membrane damage (lactate dehydrogenase release) and metabolic activity (water soluble tetrazolium conversion). the oxidative capacity of the particles as well as the generation of intracellular oxidative stress was monitored using dichlorofluorescein diacetate in cell free experiments and flow cytometry assays (facs), respectively. the particle-specific phosphorylation of src family kinases (sfk) and mitogen activated protein kinases erk1/2 were investigated using western blot techniques. after physico-chemical characterization, a set of three mnnp consisting of mn3o4 or mno2 with significant differences in size and shape were selected. according to the different oxidation stages of manganese, the particles showed significant differences in fenton reactivity in the cell free system. these data did not reflect the capacity of the particles to induce intracellular oxidative stress. the characteristic to trigger membranedependent signaling processes, however, was correlated to the intrinsic oxidative capacity of mnnp than to the ability to induce intracellular ros. furthermore, the metabolic activity (wst) was negatively correlated with intracellular ros, indicating a link between mitochondrial activity and ros generation. none of the particles had effects on the membrane integrity of the cells. the data demonstrate that mnnp, unlike other poorly soluble nanoparticles (e.g. cnp), mainly trigger adverse health effects through ros production via the fenton reaction. acute ozone induced airway inflammation does not effect resting human sympathetic nerve traffic tank j. 1 numerous mediators released in inflammatory and neuropathic pain states activate gprotein-coupled receptors (gpcrs) and modulate nociception via activation of gs, gi/o, g12/13, or gq/11 g proteins. each of the g protein-coupled receptor pathways is involved in nociceptive modulation and pain processing, but the relative contribution of the individual signaling pathways in vivo has not yet been worked out. the gq/11 signaling branch is of particular interest in pain research because it leads to the activation of phospholipase c, protein kinase c, and the release of calcium from intracellular stores. using a conditional gene-targeting approach we generated double-deficient mice lacking gaq and ga11 selectively in nociceptors to investigate the contribution of the entire gq/11signaling pathway in nociceptors towards the regulation of pain. we observed that mice lacking gq/11 in nociceptive neurons show normal development of the nociceptive circuitry. the nociceptor-specific loss of gq/11 results in reduced pain hypersensitivity following paw inflammation or spared nerve injury. surprisingly, our behavioral and electrophysiological experiments also indicated defects in basal mechanical sensitivity in gq/11 deficient mice, suggesting a novel function for gq/11 in tonic modulation of acute nociception. patch-clamp recordings revealed changes in voltagedependent tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels in nociceptors upon a loss of gq/11, whereas potassium currents remained unchanged. our results indicate that the functional role of the gq/11 branch of g-protein signaling in nociceptors in vivo not only spans sensitization mechanisms in pathological pain states, but is also operational in tonic modulation of basal nociception and acute pain. provocation of arrhythmic events in single primary isolated adult mouse ventricular cardiomyocytes tekook m., fehrmann e., schulte j. s., schmitz w., müller f. u. westfälische wilhelms-universität institut für pharmakologie und toxikologie, domagkstraße 12, 48149 münster, germany ap duration and ca 2+ cycling are altered in cardiomyocytes of different genetic mouse models. here, we systematically tested various protocols to study the inducibility of arrhythmic events in mouse cardiomyocytes. adult ventricular cardiomyocytes were isolated from wildtype (wt) mice by enzymatic digestion and subsequently tested to trigger arrhythmic events within 6 hours after isolation. in patch clamp experiments (perforated patch, whole cell current clamp) aps were stimulated for 1 second (5hz; 10hz; 5hz + s2-stimulus after 50-80ms) followed by a 4s rest period. the resting-membrane-potential (rmp) was observed over 30 cycles. we observed rmp-fluctuations of different length (amplitude <5 mv; % of 13 observed cardiomyocytes, mean events/cell; <1s: 100%, 17.1; <3s: 92%, 11.5; >3s: 69%, 5.8), spontaneous depolarizations (>5 mv; 92%, 5.1) and spontaneous aps (62%, 1.9). intracellular ca 2+ transients and sarcomere shortening were measured after loading cardiomyocytes with indo-1/am. after preconditioning (10 min/1 hz) cells were measured under basal and continuous isoprenaline (10 -6 m) stimulation (iso). a 4 min pacing period was followed by a 1 min interval of no pacing. pacing frequency was reduced after each cycle (1 hz, 0.5 hz, 0.25 arrhythmic events were provocable with stimulation/rest protocols both by field stimulation and direct stimulation via patch pipette. however, low stimulation frequencies seem to lead to distinct destabilization of cardiomyocytes probably due to ca 2+ overload. we conclude that the tested stimulation protocols are able to provoke arrhythmic events even in wt single adult mouse ventricular cardiomyocytes and may serve as a tool to test for the relevance of potential proarrhythmic substrates in mouse models. ruhr-universität bochum pharmakologie ma nord1, bochum, germany cgmp is a second messenger involved in many (patho-)physiological processes such as smooth muscle relaxation, platelet inhibition, and the development and plasticity of the nervous system. however, it is not fully understood how cgmp regulates these and other processes on a mechanistic level. in particular, the existence and functional relevance of global and local cgmp signaling domains is not clear. recently, highly specific genetically-encoded optical biosensors for cgmp have been developed. these cgmp indicators are either based on fluorescence resonance energy transfer (fret), with cgmp-binding domains sandwiched between fluorescent proteins with overlapping spectra, or they consist of a single fluorescent protein fused to cgmp-binding domains. with these cgmp indicators, the spatiotemporal dynamics of cgmp signals, which result from the interplay between cgmp-producing guanylyl cyclases, cgmp-binding effectors, and cgmp-degrading phosphodiesterases (pdes), can be monitored in living cells. here, we report the generation of transgenic mice expressing the fret-based cgmp indicators cgi500 and cgi6000 with apparent cgmp affinities of 500 nm and 6000 nm, respectively. one mouse line expresses cgi6000 driven by a cmv promoter in neural cells. fret experiments were performed with isolated cerebellar granule neurons, hippocampal neurons, and astrocytes. we observed nitric oxide (no)-induced cgmp transients and analyzed the capability of other agents (natriuretic peptides, glutamate) to induce cgmp responses. in another mouse line, the sm22alpha promoter directs cgi500 expression specifically to smooth muscle cells (smcs). fret experiments have been performed with smcs isolated from aorta, bladder and colon, as well as with intact vessels in the retina and cremaster muscle of transgenic animals. in primary smcs we studied responses to no, atrial and c-type natriuretic peptide (anp,cnp). in different smc types we observed differences in the overall ability to react to these stimuli and in the kinetics of the induced cgmp transients. we also studied the effects of pde inhibitors on the no-, anp-, and cnp-induced cgmp signals. importantly, we were able to detect cgmp transients upon no stimulation in intact vessels of the retina and cremaster. we conclude that the cgi transgenic mouse lines are valuable tools to visualize cgmp signals in living cells in vitro and, possibly, also in vivo in the intact animal under physiological and pathophysiological conditions. current research data dealing with pharmacotherapy of α-ama intoxication shows a particularly high variability regarding the protective effect of silibinin. the aim of this study was therefore to evaluate the influence of the frequently used clinical antidotes benzylpenicillin, silibinin and their combination in human hepatocyte culture intoxicated with α-ama. cytotoxicity and apoptosis testing were performed after two and five days of simultaneously exposure to α-ama and/ or tested antidotes. to quantify apoptosis, necrosis and cell viability, we used cell death detection elisa plus®, toxilight® bioluminescence assay and cell proliferation kit ii (xtt). furthermore, we analysed the ways of apoptosis by using immunohistochemistry (differential detection of caspase 3, 8 and 9, activated caspase 3, and aif). exposure of hepatocytes to α-ama at concentrations of 0,2 µm, 0,5 µm and 1 µm resulted in disorder of cell cultures, apoptosis and reduction in cell viability compared with unexposed hepatocytes. in hepatocyte cultures treated with benzylpenicillin at concentrations of 30 µm and 1mm, silibinin at 50 µm and 100 µm and a combination of both (30 µm benzylpenicillin and 50 µm silibinin, 1mm benzylpenicillin and 100 µm silibinin), toxilight® values in the supernatant and xtt values were not significantly different from untreated cultures. simultaneous exposure to α-ama (at all tested concentrations) and benzylpenicillin, silibinin or combination of both showed higher cell viability and lower values of necrosis compared to the cultures exposed to α-ama alone (exept 50 µm silibinin at 0,2 µm α-ama); however, in both groups dosed with benzylpenicillin the highest hepatocyte viabilitiy was observed. this protective effect was particularly revealed at high α-ama concentrations (0,5 µm and 1 µm). in conclusion, our data suggest that benzylpenicillin in monotherapy is more effective than in combination with silibinin or silibinin alone. glucocorticoids (gcs) are important hormones in the regulation of metabolic homeostasis. synthetic gcs, such as dexamethasone (dex), play a fundamental role in the treatment of inflammatory diseases. there are numerous side effects of a dex therapy, e.g. the development of hypertension. in the pathogenesis of hypertension oxidative stress is a crucial factor. glucocorticoid-induced hypertension has been shown to be associated with an imbalance between nitric oxide (no) and superoxide. however, the source of this elevated superoxide production is unknown. we hypothesize that an uncoupling of the no synthase (enos), a key mediator of vascular homeostasis, may contribute to dex-induced oxidative stress. incubation of human endothelial cells (ea.hy 926) with dexamethasone led to a decrease in enos expression at mrna and protein levels. this effect of dex was timeand concentration-dependent. since the major cause of enos uncoupling is a deficiency of its co-factor tetrahydrobiopterin (bh 4), we analyzed the amount of bh4 in ea.hy 926 by hplc. a concentration-dependent reduction of bh4 and also bh2 (dihydrobiopterin) could be demonstrated in response to treatment with dexamethasone. bh4 can be synthesized endogenously by two different pathways -the de novo pathway (from gtp with gtp cyclohydrolase i, gch1, acting as the rate-limiting enzyme) and the salvage pathway (conversion of sepiapterin to bh4 involving dihydrofolate reductase, dhfr). treatment of ea.hy 926 cells with dex decreased mrna and protein expression of both gch1 and dhfr. because bh4 is the major "coupling switch", an enos uncoupling is likely to occur in dex-treated cells. in summary, we showed that dex treatment led to a reduced availability of the important co-factor bh4 which could lead to enos uncoupling. the uncoupled enos may possibly contribute to glucocorticoid-induced vascular oxidative stress. the cellular oncoprotein c-fos is a major component of the heterodimeric transcription factor ap-1 and has been commonly found over-expressed in tumors and cancer cells of different origin. previous work showed that mouse cells lacking the immediate-early gene c-fos are hypersensitive to ultraviolet (uvc) light. here we demonstrate that in human telomerase-immortalized vh10tert foreskin fibroblasts (behaving like primary cells) and sv40-immortalized gm637 fibroblasts, uvc-triggered induction of c-fos protein is a delayed and long-lasting event. sustained up-regulation of c-fos went along with transcriptional stimulation of the nucleotide excision repair (ner) gene xpf, carrying an ap-1 binding site in the promoter. c-fos mrna was induced in a biphasic manner. an immediate c-fos mrna expression (30-90 min after exposure) was not translated into the protein, the second wave of transcription (4-24h after uvc exposure) resulted in c-fos protein expression, 18-48h post-uv. the stress-activated/mitogen-activated protein kinases (jnk, p38k and erks) were immediately induced upon uvc exposure and stayed active for at least 24h. inhibitor experiments revealed that c-fos was phosphorylated by erks and jnk. the activation of c-fos preceded re-synthesis and the induction of xpf mrna, which was observed 24-40h post-uvc, resulting in the increased expression of the xpf protein. cells over-expressing c-fos showed an accelerated induction of xpf mrna, and consequently a faster repair of cyclobutane pyrimidine dimers (cpds). sirna-mediated silencing of c-fos (transient c-fos knockdown) resulted in abrogated uvc-triggered induction of xpf, attenuated repair of cpds and increased apoptosis. finally, we observed that the removal of cpds but not of photoproducts was significantly faster when cells were pre-exposed to a low uvc dose, indicative of an adaptive response to dna damage. the work was financed by deutsche forschungsgemeinschaft (dfg ch 665/2-1). the addition of clopidogrel to aspirin reduces ischemic events in patients with acute coronary syndrome and in those undergoing percutaneous coronary intervention (pci). however, recurrent ischemic event occurrence during dual antiplatelet therapy remains a major concern. variability in the pharmacodynamic response to clopidogrel is well recognized, and patients with higher platelet reactivity while receiving clopidogrel are at increased risk of ischemic cardiovascular events. clopidogrel is an inactive prodrug requiring biotransformation to form the platelet inhibiting metabolite. interindividual differences in clopidogrel metabolism are the major source of variability in antiplatelet response. polymorphically expressed cytochrome p450 (cyp) enzymes play a critical role in the metabolism of clopidogrel. these findings gave rise to the concept of personalized antiplatelet therapy -i.e. individual platelet function testing and correction of insufficient platelet inhibition to reduce ischemic events in patients with high on-clopidogrel platelet reactivity (hcpr). gravitas was the first study to test this concept by comparing double-dose clopidogrel to standard-dose clopidogrel in patients with hcpr. gravitas failed to correct hcpr consistently in the study arm, which coupled with a low overall event rate precluded demonstrating a substantial benefit from improved platelet inhibition. the trigger-pci trial tested the effectiveness of the more potent thienopyridine prasugrel versus clopidogrel in patients with hcpr after elective pci with implantation of drug-eluting stents (des). switching from clopidogrel to prasugrel in patients with hcpr afforded effective platelet inhibition. however, given the low rate of adverse ischemic effects using contemporary des after pci in stable ischemic heart disease, the clinical utility of this strategy could not be demonstrated and the study was terminated prematurely for futility. multiple studies have shown that both heterozygotes and homozygotes for loss-offunction cyp2c19 alleles have higher rates of adverse cardiovascular events as compared with noncarriers on approved maintenance dosing of clopidogrel (75mg qd), albeit carriage of cyp2c19 loss-of-function alleles accounted for only a minor proportion of the variability in on-clopidogrel platelet reactivity. results of ongoing studies with antiplatelet treatment stratified by cyp2c19 genotyping are awaited to assess the clinical benefit of this approach. the organic cation transporter novel type 2 (octn2/slc22a5) represents a high affinity uptake system for carnitine. besides metabolic disease like severe system carnitine deficiency, genetic variants within the slc22a5 gene have been associated with inflammatory diseases like colitis ulcerosa. against this background, we characterized octn2 expression in peripheral blood cells thereby identifying its expression in all cell types. in the present work we studied octn2 expression in monocytes and thp-1 cells as an in vitro model for this cell type. in addition we examined transcriptional regulation of the carnitine transporter in lps activated thp-1 and investigated the effect of carnitine and its analog mildronate on the respective cytokine response. octn2 expression was characterized on monocytes and thp-1 cells on mrna and protein level. transporter mrna expression could be shown in both cell types by realtime pcr. however, the protein expression was analyzed by western blot, flow cytometry and immunofluorescence microscopy demonstrating octn2 specific signals as well as a localization in the plasma membrane. following thp-1 cells were activated using lps (10ng/ml) for up to 6h, thereby indicating the expected cytokine response as demonstrated by increased tnfα (24fold induction) and il-1β (37fold induction) mrna levels. in addition, octn2 expression was analyzed identifying an initial reduction of around 60% compared to untreated cells. in parallel activated thp-1 cells were coincubated with increasing concentrations of the octn2 substrate carnitine or its analog resulting in reduced cytokine release as shown by elisa for tnfα. here, the tnfα effect was diminished by 64% in the presence of 50mm carnitine. this effect does not rely on a direct neutralization of lps by carnitine since it was also present in cells only preincubated with carnitine. in the present work we could show that thp-1 cells represent a useful model to study octn2 expression and function. in addition, we demonstrate immunosuppressive effects of octn2 substrates like carnitine. further experiments will be necessary to identify the underlying mechanism of this observation. castor oil has been used for more than 3000 years for its laxative effects and also to induce labor in pregnant women. despite its wide-spread use, the mechanism of action remained unknown. the active metabolite of castor oil is ricinoleic acid which is released from castor oil by intestinal lipases. we have found that exposure of meg-01 cells to ricinoleic acid caused an increase in [ca 2+ ]i, an effect which was dose-dependent and abolished by pretreatment of cells with pertussis toxin, suggesting the involvement of a g-protein coupled receptor. to search for a putative receptor, we determined ricinoleic acid-induced [ca 2+ ]i increases in cells transfected with a sirna library directed against human gpcrs. in this way, we identified prostaglandin e2 receptors ep3 and ep4 as mediators of ricinoleic acid-induced effects. to test if ep3 and ep4 receptors mediate pharmacological effects of castor oil in vivo, we analyzed laxative effects induced by castor oil in wild-type (wt) mice, ep3-deficient (ep3 -/-) or ep4-deficient mice (ep4 -/-). while ep4 -/mice responded similarly to the wt mice, ep3 -/animals were totally insensitive to castor oil-induced laxation. moreover, mice lacking the ep3 receptor only in the smooth muscle cells did not respond to castor oil, in contrast to mice which lack ep3 receptor only in epithelial cells of the intestinal mucosa. similarly, ricinoleic acidinduced contractions of isolated ileal segments were absent in segments lacking ep3, consistent with a preferential expression of the ep3 receptor in the longitudinal muscle layer of the intestine. also, ricinoleic acid-induced contractions of isolated uteri were dependent on the expression of ep3 receptor in the myometrium. these findings identify the cellular and molecular mechanism underlying the effects of castor oil and indicate a role of the ep3 receptor as a pharmacological target to induce laxative effects. introduction: patients seek health information from various sources. they are facing the challenge to differentiate between reliable and untrustworthy sources and at the same time identify the best drug therapy for them. furthermore generalised health information confuses more than they benefit or rather unsettle. patients are not necessarily qualified to assess the evidence of statements properly. there is thus a need for providing competent drug information, which is offered by the independent drug information service at the institute of clinical pharmacology in dresden, germany. for the present descriptive evaluation we selected 5 drugs (arimidex ® , cipralex ® pentalong ® , onbrez ® and pradaxa ® ), that were affected by new referenceprice formation, generic registration, warnings or directions in 2011. in specified time frames we assessed the increase in and the cause of enquiries. deductively we draw conclusions for a perspicuous presentation of patient information. since generic registrations of the aromatase inhibitor arimidex ® enquiries on side effects of this drug were stable, but 17 additional consultations were held on generic changeovers. the antidepressant cipralex ® as well as the long-acting β-agonist onbrez ® were assigned to reference-price groups, which resulted in an 8-times (cipralex ® : 5 → 40) and 5-times (onbrez ® : 2 → 10), respectively, increase in enquiries. main aspect was to give background information on reference prices and point out therapeutic alternatives (cipralex ® : 34 of 40; onbrez ® : 10 of 10). an additional amount of 22 conversations were carried on the fictive registered drug pentalong ® after health insurance companies advised practitioners to avoid recourse by not prescribing this organic nitrate. notable insecurity was aroused by media reporting on lethal bleeding after taking pradaxa ® for anticoagulation. every tenth enquiry in the evaluation period was focussing on these instigative reports (21 of 227). patients are confronted by current changes, but often do not get enough background information from their health care providers to become acquainted with the tidings. health seekers may find eligible data from media coverage. however the individual assessment as well as the risk-benefit-relation may not be feasible for them. the drug information service for patients is a convenient helpline to reduce lack of knowledge and uncertainties and therefore support shared decision making. insulin effects on hyaluronan production -a possible link between diabetes and cancer? twarock s., fischer j. w. institut für pharmakologie und klinische pharmakologie, universitätsklinikum der heinrich-heine-universität düsseldorf, moorenstraße 5, 40225 düsseldorf, germany background: epidemiological studies have shown an elevated incidence of certain tumor entities in diabetes type 1 and type 2 patients. to reveal the underlying mechanisms we focused on the effects of increased glucose uptake in cancer cells with respect to matrix production. abundant production of hyaluronic acid (ha) in the vicinity of gastrointestinal cancer cells is a hallmark in tumor development. esophageal cancer is a rare but severe kind of gastrointestinal cancer which is differentiated in adenocarcinoma and squamous cell carcinoma (scc) of the esophagus. we studied the effects of increased glucose levels on ha production in an scc cell line (osc1). in starving and full media, elevated glucose concentrations increased the production of ha secreted to the medium in 24h as measured by an ha-binding protein linked assay (starved, 0g/l glucose: 100±4.7%; 1g/l: 185.7±44.8%; 4g/l: 362.6±58.3%; full medium 100±15.2%; 155.3±32.3%; 422.7±32.0%). surprisingly, total ha concentrations were about 2.2-2.8 fold higher under starved conditions. to investigate whether this effect might be due to insulin actions, starved cells were treated with 10 mg/l insulin for 24h. we observed a dosedependent decrease in ha production following insulin treatment (control vs insulin, 1g/l glucose: 100±6.5% vs 67.83±4.4%; 4g/l: 100±2.2% vs 71.8±6.6%). this finding might suggest that insulin directs glucose usage to the glycolytic pathway thereby diminishing ha synthesis. a premise to this assumption is the ability of osc1 cells for insulin independent glucose uptake. to verify this thesis, mrna expression levels of insulinindependent and insulin-dependent glucose transporters (glut1, glut4) were analyzed by qrt-pcr. the relative abundance was 24.32±3.49 in favor of glut1 indicating the presence of insulin-independent glucose transport. conclusion: in osc1 the absence of insulin actions caused increased ha production which might be due to diminished insulin driven glycolysis, thus leading to the use of early glucose metabolites for ha production instead of energy gain. this finding could be important in the context of diabetes type 2, where insulin actions are also diminished because of insulin resistance. since increased ha production is of critical importance for cancer growth and spread, the cellular shift in glucose usage from glucose catabolism to ha anabolism could therefore indicate a possible link between diabetes type 2 and cancer progression. schwarz m., unterberger e. universtität tübingen, institut für klinische und experimentelle pharmakologie und toxikologie abteilung toxikologie, wilhelmstraße 56, 72074 tübingen, germany chemical hepatocarcinogenesis is a multi-stage process triggered by an intitiating mutation in a gene encoding an important cell-regulatory protein. tumour initiation may be caused by genotoxic substances which directly interact with the dna, causing mutations. cells carrying permanent mutations experience clonal expansion which may be accelerated by exposure of the experimental animals to tumour promoters during the following step of tumour promotion. it has been shown that substances which constantly activate certain nuclear receptors act as tumour promoters in rodent liver, such as the model tumour promoter phenobarbital which, amongst others, activates constitutive androstane receptor (car). since these tumour promoters do not seem to directly interact with dna causing mutations they can be regarded as non-genotoxic carcinogens. however, the molecular mechanisms of non-genotoxic carcinogenesis are still widely unknown which also poses a major problem in preclinical drug-development. the aim of the marcar (biomarkers and molecular tumour classification for nongenotoxic carcinogenesis) project is to establish early biomarkers for non-genotoxic carcinogenesis by creating a comprehensive molecular profile of tumours generated by a regimen including model tumour promoters such as phenobarbital. the ultimate aim is to differentiate spontaneous liver tumours from tumours generated by non-genotoxic carcinogens. this molecular profile includes mutational analyses, immunostaining for known tumour-specific markers, phospho-proteome analyses, genome wide and promoter-specific dna methylation analyses, as well as mirna analyses. mutation analyses were carried out with mouse and rat tissue from phenobarbital promoted liver tumours to identify mutations which phenobarbital provides a growth advantage for. furthermore, real time pcr measurements show that the expression of a particular non-coding rna and mirna precursor is up-regulated in tissue isolated from phenobarbital promoted mouse liver tumours. additional in-situ-hybridisation experiments demonstrated the localisation of this transcript in ctnnb1-mutated tumours. larch-derived diterpenes are potent and selective trpc6 blockers urban n. 1 , kübler w. the transient receptor potential channel trpc6 is a poorly ca 2+ -selective cation channel that is activated by the membrane-resident second messenger diacylglycerol (dag). consistent with the major sites of trpc6 expression, its activation has been implicated in pulmonary and renal diseases, such as pulmonary hypertension, lung edema, chronic obstructive lung disease, allergic airway disease, and focal segmental glomerulosclerosis. amongst various plant extracts, conifer oils and resins are traditionally used to treat pulmonary ailments. therefore, we reasoned that they may contain constituents with a biological activity to modulate trpc6 activity. the true turpentines, oils and resins of various coniferous genera were tested with respect to a possible inhibition of dag-or receptor-induced activation of trpc6 and trpc3. indeed, turpentines and resins, but not coniphere oils blocked trpc6 and trpc3 in a concentration-dependent manner. interestingly, the larch-derived turpentine exerted a trpc6-prevalent inhibition. we identified larixol and its mono-and diacetates as the specific compounds that are contained in larch resin and give rise to a trpc6-selective block. larixol acetates displayed an ic50 towards the dag-or receptor-stimulated trpc6 activity of about 0.3-1 µm, but did not strongly inhibit a number of other trp channels, including trpv1, trpm2, trpm3, trpm8, or trpa1. selectivity for trpc6 compared to its closest relative, trpc3, was about 30-fold. unlike conipherous oils, which contain toxic pinenes, the resin constituent larixol ant its acetates exerted no significant cellular toxicity at concentrations that are required to block trpc6. electrophysiological analysis confirmed the highly potent block, which was voltageindependent and reversible. in a murine hypoxia-induced pulmonary vasoconstriction (hpv) model, larixol acetate abrogated the euler-liljestrand mechanism and, thus, mimicked the phenotype of trpc6 -/mice. we conclude that trpc6 blockers and, more specifically, larixol-related derivatives may provide novel therapeutic strategies to treat or prevent pulmonary diseases. dioxin is an environmental contaminant, believed to affect basic biological equilibria such as calcium and iron homeostasis. however, the molecular mechanisms underlying these effects are still largely unknown. this strongly hampers the estimation of the hazard to humans associated with dioxin exposure and necessitates further studies aimed at the clarification of these mechanisms. it has been suggested that nearly all biological and biochemical processes are mediated by protein complexes. the most commonly used technology for monitoring changes in the expression of complex protein mixtures is still 2d gel electrophoresis, but this method suffers from poor expression of low or moderately abundant proteins. blue native page and subcellular fractionation form an ideal partnership when it comes to enrichment and analysis of intracellular organelles and low abundant multiprotein complexes. the aim of the study is to identify and characterize multiprotein complexes by blue native page to elucidate the network of protein-protein interactions that regulate protein function after dioxin exposure. sample preparation and subcellular fractionation rt4 cells were cultured in mccoy's 5a medium. cells at confluence were harvested and fractionated into cytosolic, membrane/organelle and nuclear fraction by using the proteoextract subcellular proteome extraction kit. first dimension (bn-page) 50 mg of protein sample was mixed with 5% of coomassie blue g-250 (cbb g-250) and loaded in each lane of 4-15% polyacrylamide native gradient gels. the lanes from the first dimension were cut into individual strips and were placed into a 12% sds gel. the gels were stained with coomassie and the spots were picked up for mass spectrometry. bn/sds-page combined with ms led to the identification of proteins involved in the regulation of both calcium and iron homeostasis in dioxin-exposed cells. these results demonstrate for the first time that dioxin exposure simultaneously affects calcium and iron metabolism. since important iron and calcium requirement changes occur during the regulation of cell growth, the protein expression changes observed in our study may be associated with dioxin-dependent cell-fate decisions. the murine protease inhibitor serpina3n inhibits mechanical allodynia in a model of neuropathic pain vicuna l. 1, 2 , simonetti m. several chronic diseases are accompanied by strong, long-lasting pain. a majority of chronic pain diseases are not well understood yet and cannot be controlled by conventional analgesics or non-pharmacological approaches. therefore, there is a major need to develop novel therapeutic principles. using a genetic screen, we identified serpina3n, a serine protease inhibitor, which is homologous to human a1-antichymotrypsin, to be a determinant of low neuropathic pain. we found that serpina3n is expressed in the dorsal root ganglia (drg) and spinal cord and that it is upregulated in these tissues in mice developing neuropathic pain. importantly, we observed that spinal delivery of recombinant serpina3n inhibits mechanical allodynia in a mouse model of neuropathic pain. we identified a novel serine protease substrate for serpina3n, which is upregulated in the spinal cord in mice undergoing neuropathic pain ('enzyme e'). recombinant enzyme e delivered intrathecally to the spinal cord of mice elicited rapid and long-lasting allodynia, which was fully blocked by concomitant administration of serpina3n. our results suggest that serine protease-serpin signaling modulates spinal neuronal and glial cell networks involved in processing pain and that activity-induced spinal release of serpina3n constitutes an endogenous defence mechanism against establishing chronic pain hypersensitivity. these data have important implications for the pathophysiology of pathological pain and potentially hold therapeutic relevance. gβγ subunits are involved in β-adrenergic receptor induced cardiac hypertrophy vidal m., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. 9, 97078 würzburg, germany introduction. activated β1-adrenergic receptors and their g protein gαs induce the development of cardiac hypertrophy. however, the hypertropic effects of direct activation of downstream effectors, such as adenylyl cyclase, camp or pka, are controversely discussed. recently, a hypertrophic pathway involving a g protein βγ subunit induced phosphorylation of the mitogenic kinases erk1/2 at threonine 188 (erk thr188phosphorylation) has been described to mediate erk-induced hypertrophy. this study aims to investigate whether erk thr188 -phosphorylation is involved in cardiac hypertrophy triggered by β-adrenergic receptors. -phosphorylation was detected in hek cells overexpressing β1-receptors, murine hearts and neonatal rat cardiomyocytes after isoprenaline treatment. we performed [ 3 h]-isoleucine incorporation assays to assess cardiomyocyte hypertrophy in vitro. neonatal rat cardiomyocytes (nrcms) overexpressing wild-type erk2 showed a significant increase in [ 3 h]-isoleucine incorporation after isoprenaline treatment. in contrast, nrcms transfected with erk thr188 -phosphorylation deficient mutants (erk2 t188a and erk2 t188s ) or pretreatment with the erk inhibitor, pd98059, significantly attenuated cardiomyocyte hypertrophy. for in vivo studies, isoprenaline was given subcutaneously for 14 days to wild-type mice and transgenic mice overexpressing either wild-type erk2 t188t or erk2 t188s . echocardiography and histological analyses revealed that erk t188s mice developed less left ventricular hypertrophy than control mice. hypertrophic target proteins of erk (e.g. elk1) are located in the nucleus. western blot and confocal microscopy analyses showed that overexpressed erk2 t188a or erk2 t188s are retained in the cytosol and prevented elk1-phosphorylation after isoprenaline stimulation. co-immunopreciptation assays in hek cells and nrcms underlined the direct involvement of g protein βγ/erk interaction upon isoprenaline stimulation. in line with this finding, direct activation of adenylyl cyclase by forskolin did not lead to gβγ induced erk thr188 -phosphorylation. conclusion. taken together, gβγ-subunits participate in β1-adrenergic receptor mediated hypertrophy by enhancing erk thr188 -phosphorylation. these findings add important insight to the molecular signaling of g proteins in cardiac hypertrophy. the protein tyrosine kinase src and its role upon alpha-toxin stimulation of human platelets vogel k. 1 , burke m. introduction: alpha-toxin, a 34 kda calcium pore forming exotoxin, is a major virulence factor in the pathogenesis of staphylococcus aureus infections. alpha-toxin affects human blood cells such as platelets and induces aggregation that is accompanied by multiple changes in platelet protein tyrosine phosphorylation and dephosphorylation (1). in the present paper, we focused our interest on the protein tyrosine kinase src, the most abundant member of the src-family kinases present in platelets (2) . by the use of various inhibitors, we studied src and its role in α-toxin-induced platelet aggregation. methods: isolated human platelets from healthy volunteers were stimulated with α-toxin in the presence or absence of the src-family member inhibitors pp1, pp2 or su6654 (referred as src inhibitors). src and autophosphorylation of src were analyzed by sds-page and western blotting using specific antibodies against src and tyr-416-phospho-src from calbiochem and cell signaling, respectively (3). furthermore, calpeptin, an inhibitor of the calcium-dependent protease calpain, was used. platelet aggregation was measured by the method of born. staphylococcal α-toxin induced platelet aggregation in a concentration-dependent manner (0.18 -3.0 µg/ml of toxin). pre-incubation with 3 src inhibitors (pp1, pp2 or su6654) reduced α-toxin-induced platelet aggregation by about 50%. similar inhibitory effects have been observed by the use of calpeptin that acts as an inhibitor of the src degrading protease calpain. with respect to src itself, a-toxin induced autophosphorylation at tyr-416 followed by a fast and complete dephosphorylation within 10 min. while calpeptin modified the time course of dephosphorylation, only little effect of the src inhibitors has been seen on tyr-416 phosphorylation/dephosphorylation. the typical calpain-dependent degradation of src can be blocked by calpeptin (1µm), but also by depletion of extracellular calcium indicating that it is a calcium-dependent process. conclusion: taken together, our data demonstrate that α-toxin of staphylococcus aureus induces platelet aggregation accompanied by src degradation and autophosphorylation at tyr-416 typically observed in activated platelets. inhibition of the cellular tyrosine kinase src as well as the protease calpeptin reduces aggregation indicating an important role of src and/or other src-family members in α-toxin-induced platelet stimulation. the five subtypes of muscarinic acetylcholine receptors belong to the superfamily of gprotein coupled receptors. the even-numbered subtypes m2 and m4 prefer coupling to gi proteins, whereas the odd-numbered receptors m1, m3 and m5 prefer coupling to gq proteins. with respect to ligand binding and m2 receptor activation, the conserved epitope trp 7.35 at the beginning of tm7 displays remarkable functional features. it is located at the junction between the orthosteric and the allosteric binding site of the m2 receptor [1] . in the inactive m2 receptor, it provides subtype-independent baseline affinity for allosteric antagonists [1] . in the active receptor, m2 trp 7.35 affords binding affinity for the full agonist acetylcholine and intrinsic efficacy for the partial agonist pilocarpine [2] . to study the role of trp 7.35 for m3 receptor activation, agonist-induced formation of dmyo-inositol-monophosphate was measured in cho-cells transfected with the respective human receptor-cdna. surface receptor expression measured by radioligand binding was similar in hm3 wild-type-cells and hm3 trp 7.35→ala-cells, amounting to 0.07 and 0.11x10 6 receptors per cell, respectively. the intrinsic efficacy of acetylcholine was not influenced at m3 trp 7.35→ala relative to m3 wild-type, whereas potency was reduced about tenfold. these findings resemble those made previously in m2 and the corresponding mutant. in the case of pilocarpine, replacement of trp 7.35 by alanine in m3 did not reduce intrinsic efficacy. this finding is in contrast to m2, where the corresponding mutation induced a loss of pilocarpine's intrinsic efficacy. the potency of pilocarpine was diminished about tenfold at the m3 trp 7.35→ala mutant relative to m3 wild-type. this finding is also in contrast to m2, at which pilocarpine's potency was not sensitive to the trp 7.35→ala mutation. taken together, the diverging sensitivity of pilocarpine to the trp 7.35→ala mutation between the m3 and the m2 receptor suggests that the role of this epitope for receptor function may differ between even-and odd-numbered muscarinic acetylcholine receptors. in vivo experiments for inhalation toxicity are time and animal consuming. thus several in vitro methods aim to replace or reduce and refine the in vivo experiments. human 3dtissue models are commercially available reconstructed from different donors (normal, smokers, chronic obstructive pulmonary diseases), which show a normal human bronchiole tissue that reveals a pseudostratified epithelial structure, numerous microvilli and cilia on the apical surface of the cultures. the presence of tight junctions and mucus secretion has also been confirmed comparable to the in vivo situation. these 3d-models are cultured on a porous membrane as air-liquid interface. test substances can be applied apically, either as solution or with an aerosol-inducer. in our in house validation to test the strengths, handling and reproducibility of such 3dmodel systems as well as determining the correlation between in vivo inhalation data, we have assessed the epiairway tm model from mattek, usa. a set of 20 substances were selected with known in vivo toxicity data and mode of action. the substances were tested in the epiairway model an in parallel, in 3t3 and a549 cell lines to assess putative unspecific cytotoxic effects of the test substances. a comparison of toxicity data from the 3d-model and the in vivo data revealed, that the model is only predictive of respiratory toxicity in vivo for a subset of substances with specific modes of action. the epiairway tm model has proven to be robust, showing high reproducibility between pre-and main-tests as well as in the concurrent controls but it will need a strict definition of its applicability domain or further development of the test protocol to achieve a wider applicability. remodeling of intracellular ca 2+ handling and cyclic amp-dependent signaling in atrial myocytes from patients with chronic atrial fibrillation. voigt n. 1 background: in atrial myocytes ca 2+ entry through l-type ca 2+ channels (ica,l) triggers a larger ca 2+ release (ca 2+ transient,cat) from the sarcoplasmic reticulum activating contractile myofilaments. reduced ica,l is a hallmark of atrial remodeling in chronic atrial fibrillation (caf) and is supposed to contribute to action potential shortening and contractile dysfunction. however, the coupling efficiency between ica,l and cat and its regulation by camp-dependent signaling in caf patients are unexplored. methods: ica,l (voltage-clamp) and cat (fluo-3) were measured simultaneously in rightatrial myocytes from sinus-rhythm (ctl) or caf patients. a saturating concentration of the non-selective β-adrenoceptor (ar) agonist isoprenaline (iso, 1µm) and the nonselective phosphodiasterase (pde) inhibitor 3-isobutyl-1-methylxanthine (ibmx, 10µm) were used to increase cellular camp content. camp content was assessed with immunoassay. results: in caf amplitudes of ica,l (3.3±0.3pa/pf, n=12/6 [myocytes/patients] vs 6.2±0.8 pa/pf, n=15/10, p<0.01) and cat (182.2±26.8nm vs 307.5±30.9nm, p<0.01) were lower than in ctl myocytes, whereas diastolic [ca 2+ ]i levels were unchanged (caf, 312.3±56.7nm; ctl, 305.3±43.6nm). the coupling efficiency between ica,l and cat was similar in ctl and caf. application of iso increased ica,l amplitude to 10.4±2.0pa/pf (n=7/4) in caf and to 14.3±1.7pa/pf (n=9/7) in ctl. the corresponding cats increased to 493.0±132.3nm in caf and to 545.0±79.0nm in ctl. although the amplitudes of ica,l and cat also increased after pde inhibition with ibmx, the magnitude of these increases was smaller than the iso-induced enhancements. both iso and ibmx had no effect on diastolic [ca 2+ ]i and coupling efficiency. however, the relative iso-induced increases in ica,l (caf, +202.3±47.3% vs ctl, +98.4±16.1%, p<0.05) and cat (caf, +215.4±57.8% vs ctl, +101.9±20.3%, p=0.06) were significantly higher in caf compared to ctl myocytes and a similar tendency was found for ibmx. basal camp levels were higher in caf compared to ctl (caf, 9.9±1.5pmol/mg, n=6 vs ctl, 5.0±0.6pmol/mg, n=7, p<0.05), pointing to an increased camp-dependent signaling in caf patients. conclusions: these data point to remodeling of camp-dependent signaling in caf patients which likely contributes to the stronger relative increases of ica,l and cat amplitudes after β-ar stimulation and pde inhibition. remodeling of camp-dependent signaling might be a novel contributor to af pathophysiology. direct visualisation of g-protein-coupled receptors and heterotrimeric g-proteins using single-molecule microscopy wagner j. 1 g-protein-coupled receptors (gpcrs) form the largest family of membrane-bound receptors and mediate the effects of several extracellular stimuli. although the basic mechanisms of gpcr signalling have been extensively studied, a full characterization of the involved protein-protein interaction is still missing, largely due to technical limitations. in this study, we developed new methods for labelling gpcrs and g-protein subunits based on snap-and clip-tags and visualise them with single-molecule sensitivity. the snap-tag is a mutant of the dna repair protein o 6 -alkylguanine-dna alkyltransferase that reacts with fluorescent benzylguanine derivatives, whereas the clip-tag is reacting specifically with o 2 -benzylcytosine derivatives. these tags allow labelling proteins directly in living cells with very high specificity and low background. snap/clip-tagged receptors and g-proteins were covalently labelled with small organic fluorophores and visualised by total internal reflection fluorescence microscopy, which allows to selectively illuminate only fluorescent molecules located on or immediately underneath the cell surface. particles were automatically analysed with previously published as well as newly developed algorithms. the results indicated that both receptors and gproteins, although diffusing with high speed on the cell surface (diffusion coefficients: receptors ~ 0.05 mm 2 /s, g-proteins ~ 0.1mm 2 /s), can be visualised and correctly tracked. a variable fraction of receptors and g-proteins are immobile or show hop movements, possibly suggesting their interaction with cytoskeletal or other membranebound proteins. our data also suggest the feasibility of performing two-colour analyses with snap-and clip-tagged proteins aimed at directly visualizing transient interactions between receptors and g-proteins or among g-protein subunits. in-vitro screening systems are particularly well suited to preclinical toxicology testing at an early stage of drug development as they have the advantage of being fast and requiring only a small amount of test substance. the demands for in-vitro screening assays for systemic toxicity are multiple and include the need of organ specific cell systems, the use of optimal cell numbers, cell passages and incubation times. even minimal changes in the conditions of the test system may lead to significant changes of the biological system. therefore a reliable normalization compensating biological variability is crucial prior to any interpretation of results generated from a biological system. basf has developed an in-vitro metabolite profiling assay and a subsequently tuned normalization strategy allowing the prediction of specific organ toxicity. the in-vitro assay consist of exposing cells lines to test substances and to determine the metabolite profile using chromatography coupled to mass spectrometry systems. herein, we compare five different normalization strategies referring to their suitability in the application to in-vitro metabolite profiling data. the strategies comprise statistical approaches, approaches referring to reference values from each individual sample or samples generated in dependent batches. best results were achieved by an individual strategy using a new reference value correlating well over a large range of cell counts previously used for generating corresponding cell extracts. statistical analysis revealed the normalization based on the new reference value greatly improved the quality of the results compared to non-normalized samples as well as to all remaining strategies. generation and application of this new reference value and the corresponding normalization strategy will be presented the first time. validation will be featured on the basis of extracts of the human hepatocellular carcinoma cell line hep g2. molecular mechanisms of the inhibitory function of rhoh in phospholipase cmediated signalling walliser c., löschmann y., ziegler v., kühne e., schilling p., rasonabe z., bühler a., vatter p., gierschik p. universitätsklinikum ulm institut für pharmakologie und toxikologie, albert-einstein-allee 11, 89081 ulm, germany rho gtpases are a subfamily of ras gtpases regulating diverse signalling pathways, for example those regulating the reorganisation of the actin cytoskeleton. among them, rac2 and rhoh show an expression restricted to the hematopoietic lineage. rhoh is constitutively active, because it carries mutations in two positions (s13 and n62) known to be important for gtp hydrolysis. hence, rhoh is controlled on the level of protein expression and, possibly, by tyrosine phosphorylation. rhoh has been implicated in human malignancies, since the gene is subject to somatic hypermutation in its noncoding regions and to translocation to the gene encoding laz3/bcl6 or to other genes in human b-cell lymphomas. furthermore, rhoh is overexpressed in primary human chronic lymphocytic leukemia (cll) cells. these findings suggested that rhoh is involved in the initiation and/or progression of cll. we previously showed that rhoh acts as a potent inhibitor of both rac2-mediated phospholipase c-β 2 (plcβ2) and plcγ2 activation in intact cells. the aim of this study was to elucidate the molecular mechanisms of the inhibitory effect of rhoh on plc activity. the results showed that rhoh directly inhibited the activity of constitutively active variants of plcγ2, plcβ2, and plcδ1, but that it had little or no effect on the activity of plcγ1 and plcε. the amino acid residues s13 and n62, likely to be the cause for the gtpase-deficiency of rhoh, are not required for the inhibitory function of rhoh. furthermore, the switch-i and switch-ii regions of rhoh are not necessary for the inhibitory effect of rhoh, since rhoh mutants carrying switch-i or switch-ii regions of rac2 caused inhibitory effects on rac2-mediated plcβ2 and plcγ2 stimulation indistinguishable from wild-type. interestingly, rhoh seems to interact with regions of plcγ2 distinct from those which are necessary for rac2 interaction, as the split pleckstrin homology domain of plcγ2, which is essential for its interaction with activated rac2, is dispensable for the inhibitory effect of rhoh. in summary, our results indicate, that rhoh acts as a plc-isozyme-specific negative regulator of the activity of plcβ2 and plcγ2, both of which are specifically expressed in hematopoietic cells. these findings suggest a novel mechanism of plc isozyme regulation by rhoh. the results also suggest that rhoh plays an important role in b cell maturation, function, and leukemogenesis by modulating b-cell-receptor-mediated plcγ2 activation. effect of rac1 inhibition on doxorubicin mediated cell response wartlick f., fritz g. heinrich-heine-universität düsseldorf institut für toxikologie, universitätsstrasse 1, 40225 düsseldorf, germany background: the small gtpase rac1 is a well characterized member of the rashomologous (rho) family. rac1 is not only a key regulator of the actin cytoskeleton but also regulates the activity of nadph oxidase, stress kinases and transcription factors (e.g. nf-κb, ap1). furthermore, rac1 can translocate into the nucleus and interacts with topoisomerase type ii (topo ii). yet the general nuclear function of rac1 is still unclear. here, we address the question how rac1 influences the genotoxicity of the topo ii poisons doxorubicin and etoposide. methods: to study the function of rac1, human hepatoma cells were pretreated with the rac1-inhibitor eht 1864 before they were exposed to doxorubicin, etoposide or, for control, ionizing radiation (ir). to check the influence of rac1 inhibition on the outcome of genotoxin treatments, cell viability and cellular stress response were analyzed by the wst-assay, western blot (wb), co-immunoprecipitation experiments, facs-analysis and the alkaline comet-assay. results: as compared to the control, cells that have been pretreated with the rac1 inhibitor showed a higher viability, less phosphorylation of h2ax (s139) and a reduced dna damage formation (measured by alkaline comet-assay) after treatment with doxorubicin and etoposide but not after treatment with ir. furthermore inhibition of rac1 resulted in a reduced phosphorylation of topo iiα (s1106) and an increased interaction of topo iiα with hsp90 in doxorubicin treated cells. the data indicate that inhibition of rac1 protects human hepatoma cells against topo ii poisons due to interference with topo iiα function. the presence of drugs or other potential toxic substances in milk has enormous toxicological and nutritional consequences for consumers of dairy products. the atpbinding cassette (abc) transport protein breast cancer resistance protein (bcrp; abcg2) is expressed in alveolar epithelial cells of the mammary gland in cows, sheeps and goats. bcrp is known to play a major role in the active secretion of a variety of xenobiotics into human milk. so far there is little information about the transport activity and substrate specificity of dairy bcrp. therefore we aimed to establish a mdck cell in vitro model expressing bcrp of dairy animals. bcrp mrna was isolated from bovine, caprine and ovine mammary gland. full-length clones were generated using race (rapid amplification of cdna ends) pcr. the final full-length bovine, ovine and caprine abcg2 cdna-clone sequences were submitted to the ncbi genebank (eu570105, gq141082 and gq241418). stable transfection of bcrp in mdck cells was performed and the subcellular localization of bcrp at the apical plasma membrane was identified by confocal laser scanning microscopy. bcrp-mediated transport of the substrate hoechst 33342 was measured and the selectivity was determined by the bcrp inhibitor ko143. inhibition studies using hoechst 33342 identified various drugs including the antibiotic enrofloxacin or anthelmintic agents like oxfendazole as substrates of bovine, caprine and ovine bcrp. to further characterize bcrp carrier activity, bidirectional transport studies were performed with transwell® filter inserts that allow studying drug transport between an apical and basolateral compartment. cell monolayer integrity was checked by measuring teer values as well as by measuring the paracellular flux marker atenolol by lc/ms. bidirectional transport studies with enrofloxacin were performed to characterize the bcrp transporter activity. our results may contribute to increase the understanding of carrier associated drug transport into the milk of dairy cattle and therefore enlarge consumer protection. acrolein and acrylamide: excretion of mercapturic acids after consumption of potato chips watzek n., scherbl d., berger f., feld j., eisenbrand g., richling e. technische universität kaiserslautern fachbereich chemie; fachrichtung lebensmittelchemie & toxikologie, erwin-schrödinger-str. 52, 67663 kaiserslautern, germany acrolein (ac) and acrylamide (aa) may be formed from food constituents during heating of food. ac is supposed to be generated via heat induced formation from glycerides/glycerol, aa is known to arise during the maillard reaction from asparagine and reducing carbohydrates. ac also has also been suggested to be formed by endogenous metabolism as a side product of carbohydrate and/or amino acid turnover or by oxidative desamination of polyamines [1] . as an α,b-unsaturated aldehyde, ac forms 1,4-michael-adducts with biomolecular nucleophiles, such as sulfhydryl and amino groups. in the organism, ac and aa are preferentially conjugated to glutathione and are excreted as mercapturic acids (ma), n-acetyl-s-(3-hydroxypropyl)-cysteine , n-acetyl-s-(carboxyethyl)-cysteine (cema), (n-acetyl-s-(2-carbamoylethyl)-cysteine (aama), and (n-acetyl-s-(2-hydroxy-2-carbamoylethyl)-cysteine (gama). data on human exposure to ac and its occurrence in the diet are scarce. in general, contents in heat treated foods are considered to be in the low ppb range (µg/kg) [2] . nevertheless, in a pilot study in humans urinary 3-hpma excretion of 14 non-smokers was reported to be about three fold higher, as compared to aama [3] . in the present human intervention study we monitored the excretion of mas in five healthy volunteers (male) after ingestion of commercially available potato chips (175 g), equivalent to an uptake of 44 µg aa (absolute amount), together with an as yet unknown amount of acrolein [4] . urinary ma contents were monitored by hplc-ms/ms following solid phase clean-up of urine for up to 24 h after test meal uptake. the results demonstrated kinetics of 3-hpma and cema excretion in human urine to be clearly related to ingestion of the potato chip meal. on the basis of auc values, total excretion of 3-hpma plus cema exceeded that of aama plus gama by a factor of about four. the results confirm earlier findings on urinary mas, suggesting markedly higher human exposure to dietary ac / potential ac precursors than to aa. it is an as yet unresolved question, whether and to what extent concomitant substantial ac exposure may influence toxicology of such dietary heat-induced toxicants. [1] stevens, j.f. and maier, c.s. (2008) molecular nutrition & food research 52; 7 [2] osorio, v. m. and de lourdes cardeal, z., (2011) journal of chromatography a 1218; 3332 [3] schettgen, t., musiol, a., and kraus, t., (2008) rapid communications in mass spectrometry 22; 2629 [4] ewert, a., granvogl, m., and schieberle, p., (2011) lebensmittelchemikertag 2011 450 protective effects of increased nad + levels in human peripheral blood mononuclear cells exposed to dna damaging agents weidele k., beneke s., bürkle a. university of konstanz molecular toxicology group, department of biology, jacob-burckhardt-str.31, 78457 konstanz, germany the dna damage-activated enzyme poly(adp-ribose) polymerase 1 (parp-1) acts as a nick sensor and modifies target proteins by covalent attachment of poly(adp-ribose) [par] using nad + as substrate. the intracellular levels of par and nad + are important parameters for biological responses to genotoxic stress and influence diverse cellular functions including dna repair or maintenance of genomic stability. notably, loss of genomic stability is a hallmark of both carcinogenesis and the ageing process. here we analysed the impact of elevated nad + levels in human blood peripheral mononuclear cells (pbmc) with regard to (i) poly(adp-ribose) formation, (ii) cell death, (iii) initial dna damage and subsequent repair, as well as the influence on (iv) genomic stability under genotoxic stress. after ex vivo supplementation of pbmc with low concentration of nad + precursor nicotinic acid (na) intracellular nad + level significantly increased up to 2 fold in unstimulated [1] and 1.5 fold in mitogen-stimulated cells. after dna damage infliction, parp activity was dramatically increased in supplemented cells, necrotic cell death was reduced and dna strand break repair was significantly affected. furthermore the frequency of micronuclei decreased significantly after irradiation damage, emphasizing the fundamental role of adequate nad + levels in maintaining genomic integrity. the cyclic purine nucleotides adenosine 3':5' monophosphate (camp) and guanosine 3':5' monophosphate (cgmp) are well-examined second messengers with many proven biological functions. in a recent study, using a highly sensitive and specific mass spectrometry method, we have shown that cyclic 3':5' cytidine monophosphate (ccmp), a pyrimidine nucleotide, is naturally occurring in several mammalian cells [1] . ccmp activates both camp-and cgmp-dependent protein kinases with low potency [2] but the physiological function of ccmp is still very poorly understood. in an effort to delineate the function of ccmp, we analyzed expression of the early response gene egr1. we chose this gene because it is regulated by numerous stimuli including camp [3] . in our first study, we showed that dibutyryl-ccmp and ccmp failed to increase egr1 gene expression levels after stimulation of kb cells under various experimental conditions using real-time pcr (taqman®). we have now changed the experimental set-up using hela cells and the new ccmp analogue, ccmp-acetoxymethyl ester (ccmp-am), still focusing on gene expression of egr1. esterification of the negatively charged cyclic phosphate of ccmp allows better transport of the nucleotide across the cell membrane, thus augmenting possible intracellular effects. hela cells were stimulated in cell culture medium with extracellularly applied ccmp-am (3, 10, 33, 100 µm) over 15 to 240 min 24 h after seeding. analysis of real time pcr (taqman®) experiments, using β-actin as a housekeeping gene, showed a significant increase of egr1 expression in a time and concentration dependent manner. these effects were specific for stimulation with ccmp-am but not the control phosphate trisacetoxymethyl ester. hela cells were also cultured in serum free resting medium (mcdb 153, sigma) that induces growth arrest, one to eight hours prior to stimulation. here, even higher egr1 expression levels through ccmp-am stimulation could be seen. these results suggest that ccmp could function as a second messenger just as camp and cgmp do. studies are in progress to further examine the mechanisms of the ccmp-am effects on egr1 expression in hela cells. methyl-cpg-binding protein 2 (mecp2) recognizes methylated dna, it is involved in chromatin remodeling and it acts as a transcriptional repressor or activator. we have previously shown that expression of mecp2 is diminished in murine and human heart failure. prevention of mecp2 downregulation in transgenic mouse models aggravated cardiac hallmarks of heart failure. in patients with rett syndrome, which is caused by mutations in the mecp2 gene, mitochondrial function was found to be altered in the central nervous system. as the impact of mecp2 on mitochondrial function in the heart is unknown, the aim of the present study was to characterize the significance of mecp2 of cardiac mitochondria in mouse models with cardiac myocyte-specific expression or ablation of mecp2. in order to investigate the cardiac function of mecp2, two genetically modified mouse models were previously generated, including mice with inducible transgenic expression of mecp2 in cardiac myocytes under control of the tetracycline-system (mecp2-tg) and mice with targeted ablation of mecp2 in myocytes (mecp2 mlccre ). these mice were analyzed under basal conditions and after chronic transverse aortic constriction (tac). at baseline, cardiac-specific overexpression of mecp2 did not cause any difference in cardiac function as compared to control mice using millar catheterization. isolated interfibrillar mitochondria showed a decrease in citrate synthase activity. after chronic pressure overload, the decrease in cardiac mecp2 expression could be completely prevented by the mecp2 transgene. cardiac contractility and relaxation were significantly decreased in mecp2-tg animals. upon electron microscopical investigation, transgenic mecp2 expression was associated with a significant reduction of interfibrillar mitochondria, clustering of mitochondria in the perinuclear region and smaller mitochondrial cross sections as compared with control specimens. in contrast, cardiac myocyte-specific ablation of mecp2 caused a rightward shift in the size distribution of mitochondria as compared with mecp2-tg hearts. epigenetic processes, including the recognition of dna methylation by mecp2, may play an important role in the control of mitochondrial gene expression, structure, subcellular localization and function in the heart. thus, precise control of mecp2 expression and function is essential to prevent deterioration of metabolic function during chronic heart failure. normalisation of blood pressure does not prevent angiotensin ii-induced dna damage in kidney and heart of ren2 rats weissenberger s., hey v., lau d., schupp n. universität würzburg institut für pharmakologie und toxikologie, versbacher strass 9, 97078 würzburg, germany increased activity of the renin angiotensin system (ras) with enhanced levels of angiotensin ii (angii) leads to oxidative stress with endothelial dysfunction, hypertension and atherosclerosis. epidemiological studies revealed a higher cancer mortality and an increased kidney cancer incidence in hypertensive patients. we could show in vitro and in vivo that angii causes structural dna damage dose-dependently in kidney cells and in the kidney. elevated angii levels therefore might contribute to carcinogenesis of the kidney. in a model of high angii organ levels, the transgenic ren2 rat, carrying an additional renin gene, dna damage in the kidney was analysed in animals of 13 and 32 weeks. untreated ren2 rats exhibit increased blood pressure from the age of 8 weeks on. therefore, the line is kept on angiotensin i converting enzyme inhibitor therapy, which normalizes blood pressure and kidney function to values of control sprague dawley rats. despite this normalized blood pressure of the ren2 animals, a significant higher superoxide production could be observed in kidneys already in 13 week old animals. also a higher frequency of structural dna damage and double strand breaks could be detected in the comet assay and with an antibody against the double strand break marker γ-h2ax in kidneys. further, fittingly, an increased dna repair activity exists in kidneys of ren2 rats compared to control rats. as another organ affected by hypertension the heart of the ren2 animals was analysed for oxidative dna damage. although only a marginal increase of superoxide production could be found, also in the heart a significant higher frequency of dna double strand breaks and cells positive for dna repair activity could be observed. our data let us conclude that normalization of blood pressure in a state of activated ras is not sufficient to prevent angii-induced genotoxicity. this further implies that also patients with treated hypertension still might suffer from endorgan-damaging effects of elevated angii levels. the d541a pore mutation leads to complete inactivation of trpv6 channels in epididymis weißgerber p. 1 , kriebs u. replacement of aspartate residue 541 by alanine (d541a) in the pore of trpv6 channels in mice disrupts ca 2+ absorption by the epididymal epithelium resulting in abnormally high ca 2+ concentrations in epididymal luminal fluid and in a dramatic but incomplete loss of sperm motility and fertilisation capacity raising the possibility of residual activity of channels formed by trpv6 d541a proteins (sci signal 4, ra27, 2011) . it is known from other cation channels that introducing pore mutations even if they largely affect their conductivity and permeability can evoke considerably different phenotypes compared to the deletion of the corresponding protein. to gain insights whether the trpv6 d541a pore mutant still contributes to residual channel activity and/or channelindependent functions in vivo, we compared important fertility-parameters between trpv6 -/and trpv6 d541a/d541a mice: the fertilization rate observed in permanent matings, the in vivo fertilization rate as judged by the rate of embryos isolated from plug positive females of matings with males homozygous for either trpv6 mutation as well as the motility, in vitro fertilization capacity and viability of sperm were reduced to the same extent in both genotypes. also, no differences were observed in copulatory behavior between trpv6 -/and trpv6 d541a/d541a males. the profound reduction in ca 2+ uptake by the epididymal epithelium was identical in trpv6 -/and trpv6 d541a/d541a males. this direct comparison of these parameters indicate that deleting trpv6 does not further aggravate the phenotype observed in trpv6 d541a/d541a mice, and -in our opinion -allows the conclusion that the d541a pore mutant of the trpv6 protein leads to complete inactivation of the trpv6 channel activity or channel-independent scaffolding functions in epididymal epithelium. characterization of a naturally occurring c-terminal mutation (n996i) on herg channel function in hek293 cells sellmaier v. 1 , moretti a. long-qt-syndromes (lqts) are acquired or inherited disorders which predispose patients to cardiac arrhythmias and sudden death. in affected individuals, the electrocardiogram shows a prolongation in the qt interval, due to an unstable repolarization of the action potential. acquired forms of lqts are often the result of treatment with medications that block cardiac potassium channels, such as class iii antiarrhythmic drugs or antihistamines. inherited lqts are caused by mutations in cardiac ion channels. congenital long qt syndrome 2 (lqt2) is caused by loss-offunction mutations in the human ether-á-go-go-related gene herg (also known as kcnh2 or kv11.1). herg encodes the pore-forming α-subunit of the rapid delayed rectifier potassium current i kr, whose physiological role is to repolarize the late phase of cardiac action potentials. herg channel α-subunits exist as 2 isoforms (1a and 1b) that are identical except for structurally divergent n termini. native cardiac ikr channels are tetraheteromers containing 2 of each α-subunit types. a loss-of-function can be due to either defects in a) channel opening (gating), b) ion permeation or c) protein maturation and trafficking. we have identified a so far uncharacterized dominant missense mutation in the herg1 gene (n996i) in a patient with lqt. both herg1a and herg1b subunits were cloned from a human heart cdna library and the specific n996i mutation introduced by site directed mutagenesis. hek 293 cells were transiently transfected with equal amounts of mutated herg1a and wild type (wt) herg1b cdnas and the resulting potassium current compared to herg1a/b wt. whole-cell patch-clamp analysis showed similar current densities for wt versus mutated channels. also the voltage-dependence of activation was unchanged with a halfmaximal activation at -27 mv for wt and -29 mv for the mutated channel assembly. differences were found for the deactivation and inactivation kinetics. the deactivation was faster in the mutated channels with t fast = 62 ms and tslow = 480 ms versus tfast = 90 ms and tfast = 620 ms in wt channels, determined from the tail current at -40 mv after a 5-second pulse to + 50 mv. the half-maximal steady-state inactivation of the tail current was shifted by 20 mv to the depolarizing direction in the mutated channel compared to the wt. a defect in channel gating appears to be the most likely explanation. background: abcc2 is adjacent to p-glycoprotein the most important efflux transporter for various endogenous and exogenous compounds and is expressed at several compartment barriers. by increasing evidence it is shown that the abcc2 polymorphisms are of clinical significance. the aim of our study is to analyze the epigenetic regulation of distinct abcc2 haplotypes by the influence of micrornas. methods: abcc2 cdna clones containing five distinct haplotypes were generated by site-directed mutagenesis, cloned into pires-zsgreen expression vectors and transfected into different cell lines for further functional analysis. one modified vector set contained a short 3'utr sequence of abcc2 whereas the other contained the full length (fl) sequence. mirnas potentially interacting with abcc2 were identified form an mirna array after rifampicin stimulation of hepg2 cells. results: we could demonstrate that there is no difference in the basal protein expression level comparing the two vector types (fl 3'utr vs. mod. 3'utr) concerning the -24c/1249g/3972c (cgc) abcc2 wt in hepg2 cells. using the fl vector construct, the expression level of cac haplotype protein was increased (136,15% ± 20%). transfection assays with the mir-397, which was identified as a candidate mirna targeting abcc2 mrna, and the two different vector constructs harboring the cac or the cgc (wt) haplotype, confirmed that mir-379 was able to down regulate the abcc2 protein expression. there was no significance in downregulation for one abcc2 haplotype, respectively (modified 3'utr: cgc 22-34%; cac 20-40%, fl 3'utr: cgc 20-25%; cac 17-44% down regulation compared to mir-negative control). discussion: differences of abcc2 protein expression level are due to the epigenetic regulation of abcc2 haplotypes. to further characterize the effect of mir-379 on tcg, tgt and cgt abcc2 haplotypes, transfection assays are currently performed using cell cultures as well human primary leukocyte cultures. sex differences affect the pathophysiology and pharmacology of leukotriene biosynthesis werz o. friedrich-schiller-university jena, institute of pharmacy, philosophenweg 14, 07743 jena, germany inflammatory diseases affect more females than males. thus, women suffer more often from asthma, rheumatoide arthritis, alzheimer´s disease, and many autoimmune diseases than men. of interest, sex differences also exist in drug responses, with respect to both pharmacodynamics and pharmacokinetics. we have recently discovered a sex bias in the biosynthesis of pro-inflammatory leukotrienes (lts) due to testosterone, which may represent a molecular basis for gender differences in inflammation and asthma. interestingly, testosterone downregulates lt biosynthesis and also causes a sex bias in the efficiency of lt synthesis inhibitors, which demands for the clinical evaluation of a gender-tailored therapy with anti-lts. we found that certain inhibitors of lt biosynthesis were more efficiently in females than in males, and that androgens are responsible for these gender differences. in fact, the flap inhibitor mk886 effectively reduced ltb 4 pleural levels in female but not in male rats treated with carrageenan, and mk886 increased survival only of female mice in the lt-related disease model of paf-induced lethal shock. administration of testosterone to female mice abolished the protective effects of mk886. in view of the current active development of lt synthesis inhibitors as therapeutics in respiratory and cardiovascular diseases, our data prompt for consideration of gender issues in the development and use of such drugs, in order to optimize medical therapy both for men and women. considering the complexity of deposition and kinetics of air-borne nanomaterials in the lung, potential pulmonary toxicity of biopersistent nanomaterials should be evaluated by inhalation studies. those studies demand special equipment and large quantities of test material. intratracheal instillation appears as a simple and low substance-consuming alternative, although bolus dosing and the more central distribution of the particles in the lung are a well known trade-off. we compared the inflammatory response of the lung to amorphous silica (as) after instillation and inhalation. for inhalation the established short-term protocol for nanomaterials was employed (ma-hock et al. inhalation toxicology, 21:102, 2009 ): male wistar rats were exposed to the test items for 6 h/day on five consecutive days. the lungs were evaluated by analysis of bronchoalveolar lavage fluid (balf) and by histopathology three days and three weeks after the end of the exposure. in a parallel study, rats were intratracheally instilled and equally evaluated three days after instillation. assuming a deposition rate of 10%, the instilled dose corresponded to the aerosol concentration of 10 mg/m 3 used for inhalation. results show that inhalation and instillation of nominally equal mounts of amourphous silica elicit different results in the lung with inhalative treatment being less harmfull. this difference may be due to the bolus effect inevitable linked to instillation. instillation stuldies with amorphous silica may, therefore, be of limited value with respect to doseresponse assessment. sunscreen products containing uv filters protect consumers from the harmful effects of uv exposure. pigmentary grades of metal oxides like zno result in an opaque whiteness as a result of scattering visible light, whereasnanoparticles result in transparent products for better consumer acceptance and thus improved protection of human skin against uv-induced damage. in addition scatter uv light is most efficiently reflected at a nanosize of 60-120 nm. in the last 2 years the toxicological properties of nanozno in comparison with pigmentary zno were examined, the results of these comprehensive studies are presented. all tests were performed according to oecd guidelines, which were modified, especially in regard of substance preparation where appropriate. nanosized zno showed no acute toxicity after dermal application, in the bcop assay as well as in the epiderm assay it showed no corrosion / irritation potential. nanosized zno does not penetrate the intact as well as the sunburned skin. a dermal absorption test in rats (oecd 427) with 65 zn-labelled test item as well as penetration tests in weanling pigs after uv radiation did not show a penetration of the zno nanoparticles through the skin. genotoxicity was tested in vitro in the ames test, in the chromosomal aberration test in v79 cells, both showing negative results whereas the mouse lymphoma mutation test / l5178y/tk+/-cells was positive. in vivo no mutagenic effect was detected in two mouse micronucleus tests, on with intraperitoneally application and another after repeated inhalation. nanosized zno was tested in 5-days, 14-days and 90-days inhalation studies, in all studies the predominant effects were reversible local inflammatory changes in the nasal cavity and lungs, with a noaec of 1.5 mg/m3 in the 90-day study. in a prenatal developmental toxicity study according to oecd tg 414, with repeated inhalation exposure to female wistar rats from gestation day 6 through 19, maternal toxicity was observed by increase lung weights and inflammations in the lungs. but no substance-related effects on reproductive parameters (conception rate, corpora lutea, implantation sites, preimplantation loss, postimplantation loss, resorptions, dead fetuses) and no increase in external and soft tissue malformations and variations could be detected. the overall result of all the toxicological studies with nanosized and pigmentary zno is that the toxicological profile of both is very similar. studies were sponsored partly by cefci lri and partly by basf se. use of reach registration data for improving thresholds of toxicological concern (ttc) wieneke n., dorn s., jakupoglu c., schäfer c., sica m., wiegand c., mostert v. dr. knoell consult gmbh, marie-curie-str. 8, 51377 leverkusen, germany the threshold of toxicological concern (ttc) concept is utilised to identify human exposures that are so low that in-depth toxicological investigations are expendable. this is called "exposure-based waiving". exposure-based waiving serves to focus available resources on substances with relevant human exposure potential. important work into establishing ttc values has been published by munro et al. (1996) . the initial report used a database of 613 organic substances compiled from publicly available sources. in total, 2941 noels were collected in this fashion. the munro concept used the cramer classification to categorise substances according to their hazard potential. we broadened the ttc database by including noaels published on the echa website as per 3 november 2011, containing data for more than 3900 registrations. only nongaseous mono-constituent substances with oral noaels were included in the ttc database. organophosphates and genotoxic substances were excluded from the database as well as noaels obtained for surrogate substances. noaels for all systemic endpoints (general toxicity, developmental toxicity, fertility, neoplasia) were taken into account. where appropriate, default assessment factors of up to 6 were used to establish chronic noaels for each substance. for every eligible substance, we collected the published clp category for acute oral toxicity as a potential predictor of overall hazard potential. this gives rise to five categories of acute oral toxicity. a ttc is calculated from the 5 th percentile of noaels in each of these categories using the reach rules for establishing dnels for workers and the general population. this poster presents the preliminary results for more than 1000 substances. the results indicate that the ttc concept becomes more robust when using the very broad echa database. it also suggests that acute oral toxicity categories can be used as a predictor for the overall hazard potential of a substance. comparison of different in-vitro models for inhalation toxicology with respect to the effects of cigarette smoke total particulate matter wiese j. 1 , schumann b. b-and l-moc can be cultivated up to 70 days without loss of viability, as determined by resazurin-assay. viability of cell cultures was determined by mtt-assay after incubation with increasing doses of tpm. exposure of h322 to tpm (30 mg/l) reduced viability to 95% or 83% after 24 or 72h, respectively. in a549 viability was 67% after 72h with tpm (30 mg/l). the same dose of tpm lead to a decrease in viability to 82% (24h) or 25% (72h) in nhbec and to 74% (24h) or 28% (72h) in plc. as a marker of oxidative stress the level of intra-cellular glutathione (gsh) was determined by hplc. in both the tumor cell lines analysed gsh-level was increased by tpm (5 mg/l). in h322 the induction was 1.6 and 1.2 fold after 24 or 72h, respectively. while in a549 it was 1.3 (24h) and 1.4 fold (72h). in nhec and plc tpm (5 mg/l) did not have a significant effect on gsh-levels after 72h. in n-moc tpm (5 mg/l) also did not modulate gsh after 24h, but it diminished gsh-level by 0.5 fold upon prolonged contact (72h). in b-moc gsh concentrations also decreased to 0.6 or 0.8 fold the level of controls after 24 or 72h incubation. the results presented show that in-vitro models of varying complexity and origin within the respiratory tract clearly differ in their response to tpm, which was used a model inhalative toxicant. the tumor cell lines used seem to be better adapted to chemical stress, while the models closer to the in-vivo situation are more vulnerable. the nongenomic effects of the mineralocorticoid receptor in transgenic mouse heart winter s. 1 , schreier b. within the renin-angiotensin-aldosterone system (raas), the mineralocorticoid receptor (mr) is important for the regulation of fluid and electrolyte balance in the kidney, salivary glands, sweat glands and colon. however, survival of patients with severe heart failure is increased when mr blockage is combined with standard therapy suggesting aldosterone, the mr ligand, as a key factor in the development of cardiovascular diseases, but the mechanism is not yet fully understood. in recent years, evidence accumulated that besides its function as a hormone-activated transcription factor the mr also functions via nongenomic pathways. to investigate the function of the nongenomic effects of the mr in cardiovascular dysfunction, we generated a transgenic (tg) mouse model expressing a truncated human mr lacking the dna-binding site (hmr def ) under control of the cardiac specific α myosin heavy chain promoter (αmhc), a model for nongenomic effects of the mr in the heart. in this mouse model no enhanced mortality could be observed. body weight (bw), heart weight and relative heart weight were not different compared to wild type (wt) while left atrial weight/bw was increased by 20 % (wt 0,25 ± 0,01 mg/g vs. tg 0,30 ± 0,02 mg/g, p<0.05, n=12). compared to wt mice neither surface electrocardiographic experiments nor echocardiographic experiments revealed modified parameters for tg mice under basal (i.e. unstimulated) conditons as well as under β-adrenergic stimulation by isoproterenol (iso, 100 µl 1 mm iso intraperitoneally applied). to uncover the role of aldosterone in the development of cardiovascular diseases treatment with aldosterone and high-salt diet (1%) was performed. after 28 days cardiac function and heart dimensions were analyzed, surface electrocardiographie uncovered increased p duration (16 ± 0.5 ms vs. 14 ± 0.7 ms, p<0.05) and qtc interval (61 ± 2 ms vs. 50 ± 3 ms, p<0.05) in tg (n=7) compared to wt (n=5) animals. these findings probably indicate more sensitive conduction pathways to aldosterone in tg mice. oligomerization is important for regulation of phospholamban activity wittmann t., lohse m. j., schmitt j. p. institut für pharmakologie und toxikologie, versbacher straße 9, 97078 würzburg, germany phospholamban (pln) is a heart specific protein located in the membrane of the sarcoplasmic reticulum. it inhibits the ca 2+ -atpase serca2a, thereby decelerating cytosolic ca 2+ clearance during diastole of the cardiac cycle. upon phosphorylation the inhibitory activity of pln on myocyte ca 2+ transport is attenuated. further, it is believed that phosphorylation favors the formation of (rather inactive) pentamers and that pln pentamers itselves were an inferior substrate for phosphorylation compared to monomers. this would suggest an important role of pln oligomerization in the regulation of pln activity. to prove this hypothesis, we are investigating the patterns and kinetics of pln phosphorylation in the context of alterations in pln structure. the introduction of specific point mutations into the transmembrane region of pln yielded mutants that are purely monomeric (l37a, i40a and c41f) or favor pentamer formation (i45a, v49a). transfected hek293 cells expressing these mutants or wildtype pln were stimulated with forskolin to induce pln phosphorylation before lysis of cells and western blot analysis using antibodies directed against phosphorylated pln. surprisingly, phosphorylation was increased for both monomeric and pentameric pln after stimulation with 0.25µm forskolin for less than one minute. at increasing forskolin concentrations phosphorylation signals increased in parallel for monomers and pentamers. for measurement of phosphorylation kinetics stimulation of cells with 2.5µm forskolin was stopped at different time points. we found phosphorylation of both pln monomers and pentamers within seconds of stimulation. differences in phosphorylation patterns became more pronounced when assays were performed at low temperature (14°c). intriguingly, preliminary analyses suggest that pka dependent phosphorylation occurs first in pentamers and that phosphorylation of monomers may catch up only after pentamer phosphorylation is almost complete. our data suggest that both pln pentamers and monomers are suitable substrates for pka dependent pln phosphorylation. unlike the prevalent assumption, kinetics of pentamer phosphorylation seem to be at least as fast as that of pln monomers in transfected hek293 cells suggesting an important role of pln pentamers in the regulation of pln activity. regulation of cardiac contractility by nucleoside diphosphate kinases in zebrafish wolf n. m. 1, 2 , abu-taha i. in the heart, nucleoside diphosphate kinases (ndpks) can interact with heterotrimeric g proteins, thus regulating camp synthesis in a receptor independent manner and thereby influencing contractility in cardiomyocytes. we further investigated the interaction of ndpk isoforms with heterotrimeric g proteins in the heart in vivo and in vitro using zebrafish embryos and embryonic fibroblast from ndpk a/b double knockout mice (ndpk a/b ko mefs). in zebrafish the morpholino-mediated knockdown of ndpk a did not lead to an obvious phenotype, although the total ndpk activity was reduced. depletion of ndpk b caused a cardiac phenotype characterized by severely impaired atrial and ventricular contractility and insufficient blood flow. the depletion of ndpk b was associated with a significant decrease of protein levels of the heterotrimeric g protein subunits gβγ, gα s and gαi. the knockdown of ndpk c led to a more restricted cardiac phenotype with markedly reduced pumping function of the ventricle, while the atrium was unaffected. in accordance to the reduced cardiac pumping function, camp levels were significantly diminished in the ndpk b and ndpk c morphants. similar findings were obtained in ndpk a/b ko mefs. the absence of ndpk a and b resulted in a decrease of the plasma membrane content of gβ and gαs and a significant reduction in camp synthesis. the protein expression of the isoform ndpk c was also significantly reduced in the ndpk a/b ko mefs. the re-expression of ndpk b but not ndpk a rescued the basal camp production and the membrane content of g proteins. interestingly, the overexpression of ndpk c led to a 5-fold enhancement of the camp level and a significant increase of the membrane content of gβ and gα, and thus rescued the knockout phenotype. our data indicate, that the ndpk isoforms b and c are essential for cardiac contractility, most likely by forming a signaling complex at the plasma membrane including ndpk b, ndpk c and heterotrimeric g proteins. the isoform ndpk c, with its n-terminal hydrophobic region, might serve as a membrane anchor for the ndpk/g protein complex. induction of apoptosis via pka-dependent and pka-independent pathways by cyclic purine and pyrimidine nucleotides in mouse lymphoma cell lines wolter s. 1 camp is a second messenger that plays an important role in intracellular signal transduction of various hormones and neurotransmitters. a major function of camp in eukaryotes is the activation of camp-dependent protein kinase a (pka). pka is involved in the control of a variety of cellular processes. pka exists as an inactive tetramer of a dimeric regulatory (r2) and two catalytic (c) subunits that releases the active c-subunits upon binding of camp. stimulation of the mouse t-lymphoma cell line s49 wild-type (wt) with dibutyryl (db)-camp induces apoptosis by an intrinsic, mitochondria-dependent mechanism. apoptosis induced by db-camp occurs via a pka-dependent mechanism, since s49 kincells lacking the catalytic subunit of pka are resistant to db-camp-mediated cell death. db-camp is cleaved by esterases into the biologically active compound n 6 -mb-camp and into 2'-o-mb-camp. other cyclic nucleotides (cnmps) in addition to camp, like ccmp and cump can also function as second messengers and activate pka and cgmp-dependent protein kinase (pkg) 1 . therefore, we investigated the effects of a series of membrane-permeable analogues of camp, cgmp, ccmp and cump in s49 wt und s49 kincells on apoptosis. stimulation with db-ccmp or db-cgmp induced neither apoptosis in s49 wt nor in s49 kincells. interestingly, we observed apoptosis in s49 wt and s49 kin cells after incubation with membrane-permeable nucleotide acetoxymethyl ester (am)-analogues of cgmp, ccmp, cump and also camp. induction of apoptosis occurs via pkadependent and also pka-independent pathways. a potential role of pkg and of the exchange protein activated by camp (epac) in the induction of apoptosis is unsolved and will be explored by specific pkg-and epac-activators in this system. investigations are on the way to identify the targets, the involved signal transduction pathways and the mechanisms of pro-apoptotic actions mediated by cnmp-ams. (1) wolter s, golombek m, seifert r (2011) differential activation of camp-and cgmpdependent protein kinases by cyclic purine and pyrimidine nucleotides. biochem biophys res commun. in press apoptosis in s49 cells : induction of apoptosis in s49 wt and in s49 cells lacking the catalytic subunit of pka (s49 kin-) with a) db-cnmps and b) cnmp-ams for 72h. nebivolol reduces vascular inflammation in spontaneously hypertensive rats wu z., xia n., förstermann u., li h. institut für pharmakologie, obere zahlbacher str. 67, 55131 mainz, germany nebivolol is a third generation β1 receptor blocker with additional effects on endothelial nitric oxide production. the aim of the present study is to investigate the antiinflammatory effects of nebivolol in vivo. 48 spontaneously hypertensive rats (shr) were divided into two groups: control or nebivolol treatment group. nebivolol treatment (5 mg/kg/day for 10 days) significantly reduced the blood pressure and the heart rate in shr. the drug had no effect on coagulation. aorta from nebivolol-treated rats showed significantly improved endothelial function. nebivolol did not change the expression levels of aortic nf-kb, but significantly reduced its dna binding activity. furthermore, nebivolol decreased the expression of adhesion molecules (e.g. icam-1, vcam-1) and pro-inflammatory cytokines (e.g. il-6). in conclusion, nebivolol reduces vascular inflammation in experimental hypertension. it inhibits nf-kb activity, decreases the expression of adhesion molecules and pro-inflammatory cytokine, and improves endothelial function. characterization of the cellular activity of pde4 inhibitors using two novel pde4 reporter cell lines wunder f., quednau r., barg m., tersteegen a. bayer pharma ag lead discovery wuppertal, aprather weg 18a, 42096 wuppertal, germany cyclic nucleotide-specific phosphodiesterases (pdes) play an essential role in cellular signal transduction by regulating the intracellular levels of camp and cgmp and, therefore, are important pharmacological targets. we report here the generation and pharmacological characterization of two novel pde4 reporter cell lines. plasmid constructs encoding human pde4b1 or pde4d3 were stably co-transfected with the beta1-adrenoceptor in a parental camp reporter cell line expressing a cyclic nucleotide-gated (cng) cation channel, acting as the biosensor for intracellular camp. in this reporter cell line, camp levels can be monitored in real-time via aequorin luminescence stimulated by calcium influx through the cng channel. by using different pde4 and non-pde4 inhibitors, we could show that our novel pde4b1 and pde4d3 reporter cell lines specifically monitor pde4 inhibition with high sensitivity. pde4-selective inhibitors alone did not increase basal luminescence levels in this experimental setting. however, these inhibitors induced concentration-dependent luminescence signals in combination with the adrenoceptor agonist isoproterenol. in contrast, in a stable beta1-adrenoceptor reporter cell line with no recombinant pde4 expression, pde4 inhibitors had no effect on isoproterenol-stimulated luminescence signals. we compared the cellular activity of different pde4 inhibitors with the in vitro inhibition of full-length and truncated (catalytic domain) pde4d3 from cell lysates. two different groups of pde4 inhibitors could be identified. the first group, including the allosteric inhibitors pmnpq and d159153, showed high cellular activity and much better inhibition of full-length versus truncated pde4d3. the second inhibitor group, including classical competitive inhibitors like roflumilast, cilomilast and piclamilast, showed comparably lower cellular activity and similar inhibitory activity on full-length and truncated pde4d3. the results imply that these novel pde4 reporter cell lines are well-suited for the characterization of the cellular activity of pde4 inhibitors and may also support a better understanding of the complex pde4 pharmacology. plexin-b2 is required for kidney regeneration after acute renal failure xia j. 1 , gröne h acute renal failure is a common clinical problem with unsatisfactory therapeutic options and high mortality in humans. therefore, unraveling the mechanisms that promote kidney regeneration and repair may provide new therapeutic strategies for acute renal injury. plexin-b2 belongs to a family of transmembrane receptors which mediate the cellular effects of semaphorins. while plexins have first been described in the context of axon guidance, several recent studies have established them as key regulators of organogenesis, the immune system and cancer. we have recently found that plexin-b2 is highly expressed in the adult kidney, particularly in tubular epithelial cells which are most sensitive to acute ischemic injury. to study the role of plexin-b2 during kidney regeneration we generated mice lacking plexin-b2 specifically in tubular epithelial cells. under physiological conditions, these mice displayed normal kidney morphology and function. in contrast, following ischemia/reperfusion injury, plexin-b2 conditional knockout mice exhibited severely impaired kidney regeneration. while the renal function of control mice fully recovered within 3 weeks after injury, plexin-b2 knockout mice had strongly elevated serum creatinine and urea levels associated with increased morbidity and mortality. this was accompanied by hyperproliferation of tubular epithelial cells and obstruction of tubular lumina. we conclude that plexin-b2 is required for regeneration after acute ischemic renal injury and that pharmacological interventions activating plexin-b2 might represent a new therapeutic strategy in acute renal failure. the nadph oxidase enzyme complex consists of two membrane-bound catalytic subunits (a nox protein and p22phox) and several cytosolic regulatory components including p47phox, p67phox, p40phox and the small gtpase rac1. we have previously shown that treatment of apolipoprotein e knockout mice with resveratrol led to a downregulation of nox2 and nox4 in the heart. our recent data demonstrated that resveratrol also reduced the enzymatic activity of cardiac nadph oxidase. because activation of nadph oxidase enzyme complex is induced by translocation of the regulatory subunits, we studied whether the reduced enzymatic activity is due to an inhibition of such a translocation. indeed, resveratrol treatment prevented rac1 membrane translocation from cytosol. resveratrol is known as an activator and expression enhancer of the longevity gene sirtuin 1 (sirt1). we then wanted to find out whether the effect of resveratrol on rac1 was mediated by sirt1. sirt1 is a histone/protein deacetylase. in vitro incubation of rac1 with sirt1 led to a reduction of lysine acetylation. deacetylation of rac1 on lysine 166 could be identified by mass spectrometry analyses. the lysine 166 lies within the p67phox-binding region of rac1. consistently, in vitro incubation of rac1 with sirt1 markedly reduced its binding activity to p67phox. in conclusion, we provide evidence that rac1 is a direct target molecule of sirt1. sirt1 deacetylates rac1 on lysine 166 and thereby inhibits its interaction with p67phox. this is a novel mechanism of nadph oxidase inhibition by sirt1/resveratrol. mutational analysis of the effects of the cardioprotective drug dexrazoxane on topoisomerase ii beta in vitro yan t., deng s., frensch i., gödtel-armbrust u., wojnowski l. universitätsmedizin der johannes gutenberg-universität mainz institut für pharmakologie, obere zahlbacher straße 67, 55101 mainz, germany dexrazoxane (drz, icrf-187) is the only approved drug shown to protect against anthracycline-induced heart failure. the protection is usually ascribed to the ironchelation by drz, which is thought to reduce anthracycline-induced oxidative stress. however, similarly to anthracyclines, drz is also an inhibitor of the anthracycline target topoisomerase ii (top2). we hypothesized that the cardioprotective effects of drz are mediated by the prevention of the anthracycline-induced dna damage mediated by top2b, the dominant cardiac top2 isoform. this was investigated using top2b mutants resistant to drz, which were expressed in cells depleted of wild-type top2 isoforms. top2b-mediated double-strand dna breaks were assessed as γ-h2ax. the levesl of dsb generated by the top2b mutants in response to the anthracycline doxorubicine (dox) was indistinguishable from that mediated by a wild-type top2b. preincubation with drz depleted wild-type top2b and this was accompanied by a decrease in the dna damage following a subsequent exposure to dox. in contrast, neither top2b depletion nor the reduction of dsb by drz was seen in drz-resistant top2b mutants. furthermore, the cardially ineffective drz analog icrf-161, capable of iron chelation but not of top2 binding, affected neither the stability of top2b, nor the dox-induced dna damage mediated by this enzyme. these results indicate that drz may exert its cardioprotective effects by reducing the dna damage mediated by doxpoisoned top2b rather than by iron chelation. they also suggest a cardioprotective function of top2b, which is currently under investigation using cardiomyocyte-specific top2b mouse knockouts. aminoglycosides are important antibiotics in the treatment of life-threatening infections, especially those caused by gram-negative bacteria. their nephrotoxic and ototoxic potential is well-known, but little is known about the effects of aminoglycosides on the male reproductive system. we studied the effects of four aminoglycosides on sertolicells in vitro. rat sertoli-cells from the cell line serw3 were cultivated for 3, 6, and 9 days in dmem supplemented with three different concentrations of amikacin, streptomycin (30 mg/l, 100 mg/l, 300 mg/l), gentamicin or tobramycin (10 mg/l, 30 mg/l, 100 mg/l). we determined the expression of two junctional proteins (connexin 43, ncadherin) and one protein of the cytoskeleton (vimentin) by western blot. cells were solubilized in lysis buffer. lysates were separated by sds-page and electroblotted on a pvdf-membrane. after incubation with primary antibodies overnight and horseradish peroxidase-conjugated secondary antibody the visualization was achieved by a chemiluminescence-detection system. in addition, proteins were detected by immunohistochemistry. after three days in culture amikacin caused the most pronounced effect. at the lowest concentration tested (30 mg/l) connexin 43 and n-cadherin were reduced to 55±19% and 92±10% of the controls (n=6). no change was recognized for vimentin (102±16%). effects obtained with streptomycin were less pronounced for these these proteins (68±16%, 96±7%, and 102±13%, respectively). similar, but less pronounced effects were observed with gentamicin and tobramycin at a concentration of 10 mg/l (connexin 43: 88±15% and 81±15%; n-cadherin: 95±18% and 103±11%; vimentin: 81±12% and 102±19%) and 30 mg/l (connexin-43: 84±19% and 78±18%; n-cadherin: 98±19% and 103±13%; vimentin: 102±8% and 115±15%). after incubation for 6 and 9 days the effects occurred in the same range. the substances showed no influence on the viability of serw3 sertoli-cells up to 300 mg/l in the mtt assay. by immunohistochemistry we showed that the localisation of the proteins -connexin 43 and n-cadherin at the cell membrane and vimentin in the cytoplasm -was not influenced by the aminoglycosides. large conductance calcium-and voltage-gated potassium (bk) channels play an important role in controlling membrane potential and calcium influx, and are strongly modulated by protein kinases at multiple sites. the stress-regulated exon (strex) adds to the bk channel c terminus a cysteine-rich insert of 59 amino acids that inverts the channel regulation by protein kinase a (pka) from excitatory to inhibitory. here we investigated the mechanisms by which the strex insert influences bk channel regulation by protein kinase c (pkc). activity of bk channels without strex insert (bk-zero), transiently expressed in hek cells, was inhibited by pkc in inside-out membrane patches (~ 50% inhibition). bk channels with strex insert (bk-strex), however, were insensitive to pkc. phosphomimetic mutation of a pkc phosphorylation site (s700e) in bk-strex, resulted in a ~50% reduction of basal channel activity, whereas the s700a mutant retained normal activity. to examine whether palmitoylation, and thus association of the strex domain with the plasma membrane, prevents pkc inhibition of bk channel gating, palmitoylation was abolished by either site-directed mutagenesis (c12:13a) or by pharmacological inhibition of palmitoyl transferases with 2-bromopalmitate (2-bp). both, mutation and pretreatment with 2-bp resulted in the expression of bk-strex channels which were sensitive to pkc (~ 50% inhibition of channel activity). no inhibitory pkc effect was observed in patches of the bk-strex s700a channel mutant pretreated with 2-bp. in a clonal rat somatomammotroph pituitary cell line (gh3b6), in which pcr products without (zero) and with the 174 bp strex exon could be identified, the pkc activator pma blocked channel activity by ~25 %. this inhibition was increased to over 50% when gh3b6 cells were pretreated with 2-bp, indicating that both channel isoforms were functionally active. in summary, the present study demonstrates that palmitoylation of strex prevents bk channel regulation by pkc, which is mediated by phosphorylation of ser700, probably by steric hindrance. our results provide further evidence for a cross-talk between palmitoylation and phosphorylation as a crucial mechanism underlying the dynamic regulation of ion channels. human pleural mesothelial met-5a cells are a limited in vitro model system in determining potential asbestos-like genotoxic effects of multiwall carbon nanotubes ziemann c. 1 , reamon-büttner s. multiwall carbon nanotubes (mwcnt) are nanomaterials with important technological impact. depending on their diameter, length, and biopersistence, however, some mwcnt seem to exhibit a fiber-like cytotoxic and genotoxic potential, similar to asbestos. thus, a project funded by the german federal ministry of education and research (bmbf contract no. 03x0109a) focuses on potential adverse biological effects of different types of mwcnt to enlarge the knowledge base about toxicity determining parameters. this project comprises both in vitro (rat) and in vivo endpoints with long amosite asbestos as a positive control. as mesothelial cells are target cells for adverse effects of asbestos, in particular mesothelioma development, the human sv40transformed, non-malignant pleural mesothelial cell line met-5a was chosen as the main in vitro model in this project. in the present study part, met-5a cells were characterized concerning their usefulness as an in vitro model to study potential asbestos-like cytotoxic and genotoxic effects of different mwcnt varieties. using an mwcnt-optimized lactate dehydrogenase liberation assay and proliferation parameters derived from cell counts, concentration-dependent cytotoxicity of long amosite asbestos (2, 10, and 20 µg/cm 2 ) was demonstrated in met-5a cells. cells also showed asbestosinduced increase in dna-strand breaks and oxidative dna-damage in the hogg1modified comet assay. thus, met-5a cells were responsive to asbestos treatment. owing to asbestos potential to induce aneugenic effects and spindle fiber damage, micronucleus induction, determination of numerical chromosome aberration, and altered meta-, ana-, and telophase morphology were planned as in vitro endpoints. met-5a cells were thus initially characterized in this regard and were found to exhibit highly variable chromosome numbers with lower than 10% cells exhibiting a normal diploid chromosome set, an up to twentyfold higher spontaneous micronucleus frequency, as compared to polychromatic bone marrow erythrocytes in rodents, and a profound number of aberrant meta-, ana-and telophases with bridges, lagging chromosomes and multipolar divisions. in conclusion, met-5a cells are of only limited value as an in vitro model system to study potential asbestos-like effects of mwcnt and also biopersistent fibers. the cells are indeed responsive to asbestos, but unfortunately demonstrate marked genomic instability and thus limited significance concerning genotoxic effects. waixenicin a inhibits cell proliferation through magnesium-dependent block of trpm7 channels zierler s. 1 transient receptor potential melastatin 7 (trpm7) channels represent the major magnesium-uptake mechanism in mammalian cells and are key regulators of cell growth and proliferation. they are abundantly expressed in a variety of human carcinoma cells controlling survival, growth and migration. these characteristics are the basis for recent interest in the channel as a target for cancer therapeutics. we screened a chemical library of marine organism-derived extracts and identified waixenicin a from the soft coral sarcothelia edmondsoni as a strong inhibitor of overexpressed and native trpm7. waixenicin a activity was cytosolic and potentiated by intracellular free magnesium (mg 2+ ) concentration. mutating a mg 2+ -sensitive site on the trpm7 kinase domain reduced the potency of the compound, whereas kinase deletion enhanced its efficacy independent of mg 2+ . waixenicin a failed to inhibit the closely homologous trpm6 channel and did not significantly affect trpm2, trpm4, and crac (calcium release activated calcium) channels. therefore, waixenicin a represents the first potent and relatively specific inhibitor of trpm7 ion channels. consistent with trpm7 inhibition, the compound blocked cell proliferation in human jurkat t-cells and rat basophilic leukemia cells. based on the compound's ability to inhibit cell proliferation through mg 2+ -dependent block of trpm7, waixenicin a or structural analogs may have cancer-specific therapeutic potential, particularly since certain cancers accumulate cytosolic mg 2+ . release of metals from different sections of domestic drinking water installations zietz b. p., richter k., laß j., suchenwirth r., huppmann r. governmental institute of public health of lower saxony division of environmental medicine and environmental epidemiology, roesebeckstraße 4-6, 30449 hanover, germany different metals were used as important piping materials in the drinking water supply for a long time. due to corrosion metals can leach into the tap water. of special importance is the toxic element lead. however other heavy metals in drinking water such as copper, nickel and cadmium can also give reason for health concerns. in this study it was investigated in which amount relevant metals were released from different parts of domestic installations into the cold water. for the spatial allocation of the emission sources a sequential water sampling protocol was used after three hours of stagnation time representing the first 5 litre of the water column. after stagnation ten sample volumes were collected in series. existing facilities of domestic installations constructed with different plumbing materials were examined predominantly from residential buildings. the elements al, as, cd, cr, cu, fe, mg, mn, ni, pb, sb, se, u and zn were detected by means of icp-ms. in total 16 water pipe strands of 11 domestic installation systems were examined. they comprised 379 single water samples and 5306 single parameters. depending upon the type of plumbing different courses and concentration ranges of the elements could be measured in the tap water samples. terminal taps or installation parts were frequently responsible for a release of nickel and in several cases of cadmium. the concentration courses of the element zinc proved as a good indicator for the allocation of the emission source to a brass containing section of the installation (zinc as an alloy component of brass). one can conclude that an investigation by means of a sequential water sampling protocol and multi-element detection can be a valuable non-destructive method for drinking water-hygienic investigations of domestic installations. novel interaction partners of the murine trpc4 protein zimmermann j., beck a., flockerzi v. universität des saarlandes experimentelle und klinische pharmakologie und toxikologie, kirrberger str. 1, 66421 homburg, germany in this work novel interaction partners of the murine protein transient receptor potential canonical 4 (mtrpc4) were identified. the trpc4 protein is the major subunit of a cation channel, residing in the plasma membrane. it comprises six trans-membrane domains and cytosolic amino and carboxyl termini. two major splice variants of the trpc4 gene exist, trpc4a (974 aa) and trpc4b (890 aa), trpc4b lacks aa 781 to 864 of the trpc4a variant. both trpc4 variants are co-expressed in endothelial cells, intestinal smooth muscle and brain. to identify trpc4-interacting proteins a yeast two-hybrid system, cytotrap®, which allows identification of protein-protein interactions within the cytosol was used. a premade mouse brain cdna library was screened by the cytosolic amino and carboxyl terminal parts of mouse trpc4a (aa 1 to 324; aa 622 to 974). for the carboxyl terminal part fourteen proteins were identified. to independently prove the interaction, the fulllength cdnas of all fourteen proteins were cloned, fused to a flag-tag and coexpressed with trpc4 in hek 293 cells. co-immunoprecipitations were performed for all candidates using both the anti-flag-antibody and the antibody for trpc4. in addition, changes of cytosolic calcium were monitored and trpc4 currents were recorded in hek 293 cells expressing the candidate cdnas and stably expressing the trpc4a or trpc4b and the muscarinic receptor type 2 cdnas. the tarbp2 protein, one of the candidates shown to interact with trpc4, changed calcium influx when coexpressed with trpc4. in order to identify the domains of trpc4 responsible for its interaction with the tarbp2 protein, six trpc4-gst-fusion proteins covering the carboxyl terminal 353 aa of trpc4a were expressed in e. coli and used for pull-down experiments. by this approach two domains of trpc4 could be identified to interact with tarbp2. one of these domains is well conserved within the trpc5 protein, corresponding to the result, that trpc5 and tarbp2 effectively co-immunoprecipitate, too. the tarbp2 protein has been shown to be a component of the risc loading complex, also known as the micro-rna loading complex which is composed of dicer1, eif2c2/ago2 and tarbp2 (chendrimada et al. 2005) . by its interaction it may link trpc4 to pre-microrna processing. increased levels of angiotensin ii provoke dna damage and have influence on dna repair in mouse kidneys zimnol a., brand s., schupp n. universität würzburg institut für toxikologie, versbacherstr. 9, 97078 würzburg, germany the renin-angiotensin system (ras) plays a crucial role concerning the blood pressure, electrolyte balance and cardiovascular homeostasis. angiotensin ii (ang ii), the active hormone of the ras, in higher concentrations leads to vasoconstriction, oxidative stress and hypertension. hypertensive patients have an increased risk to develop cancer, especially kidney cancer. we have shown in vitro and in vivo, that ang ii is capable to cause an elevation of blood pressure as well as dna damage dose-dependently. to investigate whether the high blood pressure or the enhanced levels of ang ii are responsible for dna damage, male c57bl/6-mice were equipped with osmotic minipumps, delivering ang ii in a concentration of 600ng/kg · min during 28 days. additionally they were treated with ramipril, an angiotensin-converting-enzyme blocker, with the ang ii receptor antagonist candesartan, the vasodilator hydralazine, and the antioxidant tempol. dna damage was analysed with the comet assay. we measured the base excision repair (ber)-related dna repair in the kidney with a comet-based in vitro repair assay. furthermore, the distribution and expression of the ang ii-type 1 (at1) receptor in the kidney was analyzed by immunohistochemistry. treatment with ang ii led to a significant increase of blood pressure, whereas the medication with candesartan decreased the systolic blood pressure. the intervention with hydralazine lowered the blood pressure only for a short time. the other substances had no effect at all on the blood pressure. genomic damage, quantified with the comet assay, was augmented by ang ii and improved by all interventions, particularly by candesartan and tempol. beyond that, ang ii showed a tendency to reduce dna repair. treatment with candesartan, hydralazine and tempol increased the repair capacity. furthermore, ang ii tended to result in a downregulation of the at1 receptor in kidney tubule cells. candesartan and ramipril, especially were able to augment the expression of the at1 receptor, whereas hydralazine achieved the opposite. these results demonstrate that ang ii leads to dna damage in the kidney independent of blood pressure. apparently elevated levels of ang ii affect dna repair and expression of at1 receptor. to confirm these findings we are going to examine more precisely the manifestation of other enzymes, which are implicated in dna repair. regulation of hcn channel activity by cyclic cytidine 3´, 5´-monophosphate zong x., krause s., chen c. -c., gruner c., cao-ehlker x., fenske s., wahl-schott c., biel m. lmu münchen, department pharmazie, pharmakologie für naturwissenschaften center for integrated protein science munich (cipsm), butenandtstr. [5] [6] [7] [8] [9] [10] [11] [12] [13] 81377 münchen, germany hyperpolarization-activated cyclic nucleotide-gated (hcn) channels play a key role in controlling cardiac pacemaker activity and are essential for normal function of neuronal circuits. hcn channels are principally gated by voltage but are coactivated by the cyclic nucleotides camp and cgmp which directly bind to a c-terminal binding domain. recently, cyclic cmp (ccmp) was shown to be present in various cell lines and tissues at concentrations that are comparable to cellular cgmp levels. moreover, there is recent evidence that ccmp can activate camp-and cgmp-dependent protein kinases in vivo. here, we examined whether ccmp exerts effects on hcn channels. to this end, we recorded hcn channel-mediated currents (i h) in hek 293 cells that stably express hcn1, hcn2, hcn3 or hcn4, respectively. currents were measured either with a standard patch-clamp setup or by employing the planar patch-clamp technology. in hcn2 and hcn4 channels, ccmp shifted the membrane potential for half maximal activation (v0.5) to more positive values. in addition, ccmp accelerated activation while it slowed down deactivation kinetics. the ec50 for ccmp was 30 µm which is about 5 times higher than the ec50 of cgmp. cyclic cmp is a partial agonist of hcn channels since it activates only 65 % of the maximal current obtained with camp or cgmp. to identify in vivo effects of ccmp we recorded ih of murine sinoatrial node (san) cells in the presence and absence of 1 mm ccmp. like for heterologously expressed hcn channels, ccmp shifted v0.5 of ih by about +5 mv. importantly, the steepness of the diastolic depolarization of san pacemaker potentials which is mainly determined by the amplitude of ih was profoundly increased by ccmp, compared to control conditions. as a consequence of the upregulation of ih the frequency of san pacemaker potentials was increased by about 20 % in the presence of ccmp. our results suggest that ccmp is a physiological regulator of hcn channel activity. a 3d actinic keratosis like construct for assessment of innovative tumour therapeutics zoschke c. 1 the incidence of actinic keratosis (ak) has increased dramatically in the last decades and it is considered the most frequent carcinoma in situ today. especially immunosuppressed patients are at high risk to develop invasive squamous cell carcinoma (scc) [1] which asks for most efficient and well-tolerated ak therapy. yet, current measures do not fit with these demands. nucleotide analogues, recently identified by molecular modelling [2, 3] , outperformed the current standard for the therapy of actinic keratosis, 5-fluoruracil, when tested in the tumour cell line scc25, in normal human keratinocytes and fibroblasts [4] . as next step in pre-clinical drug assessment, we aimed to characterise the effect of the most selective nucleotide analogue oxbu in reconstructed human tumour skin. based on the 3d construct of scc developed by höller and co-workers [5] for start we introduced several adaptations with respect to keratinocyte, fibroblast and scc12 seeding to grow an aklike construct with scc12 cells forming nests in particular in the epidermis. in addition, first experiments with the oxbu on the ak like constructs showed promising results for an efficient treatment of actinic keratosis. efficacy was derived from immunohistology (marker for proliferation: ki-67, marker for scc: cytokeratin-10, axl, marker for invasion: mmp2, marker for apoptosis: caspase-7, nuclei were stained with dapi) as well as the effects on the secretion of cytokeratin-18 and its caspase-induced cleavage product into the culture medium following drug exposure for up to 7 days. efficacy of a 0.05% oxbu solution proved close to or even better when compared to both a 0.1% 5fluorouracil and 0.025% aphidicolin solution. the former being the gold standard of current ak therapy, the latter is a frequently used inhibitor of human polymerase alpha and delta, however, failed to be introduced into clinical use. -in fact, in monolayer cultures aphidicolin proved most toxic for normal human keratinocytes which was not true with oxbu [4] . -therefore, these 3d tumour constructs offer a new approach to pre-clinical drug assessment and may be added to other 3d models of skin diseases currently gaining increased interest as test platforms. ima910, a multi-peptide cancer vaccine for advanced colorectal cancer, induces multiple cd8+ and cd4+ t-cell responses associated with improved survival walter s. 1 , kuttruff s. ima910 is a novel peptide-based vaccine consisting of 10 hla-a*02 and 3 hla-dr binding synthetic peptides that were identified based on natural presentation on human colorectal cancer (crc) samples. ima910 was characterized in a phase i/ii trial in advanced/metastatic crc patients being at least clinically stable after 12 weeks of first-line oxaliplatin-based therapy. all patients received a single application of low-dose cyclophosphamide for immunomodulation. this was followed by repeated ima910 vaccinations in combination with low-dose gm-csf (cohort 1; n=66) or ima910 / gm-csf plus topically applied imiquimod (cohort 2; n=26). before and post vaccination, patients were analyzed by hla-multimer assay and intracellular cytokine (ics) assay for cd8 + t-cell responses and by ics assay for cd4 + tcell responses. as immune status biomarkers, 6 phenotypically defined myeloid derived suppressor cell populations (mdsc1-6) were analyzed prior to immunotherapy. tumor status of patients was monitored repeatedly by ct/mri according to recist and corresponding tumor scans were reviewed centrally. clinical assessment included disease control rate (dcr), time to progression (ttp), progression-free survival (pfs) and overall survival (os). ima910 overall was immunogenic in 73/81 (90%) evaluable patients, with 43% and 65% of patients mounting multiple cd8 + and cd4 + t-cell responses, respectively. patients that received the immunomodulator imiquimod presented with significantly more multiple cd8 + cell responses as detected by ics (p=0.016). multiple cd8 + and multiple cd4 + responses were individually associated with significantly better clinical outcome. the association was most pronounced for patients with both multiple cd8 + and multiple cd4 + responses. these patients had significantly higher dcr at 6 months (p=0.002), improved ttp (p=0.006) and improved pfs (p=0.009) than other patients. most importantly, a trend for prolonged os was also observed in these patients (p=0.088, hazard ratio 0.53). in the study population, levels of 5 different mdsc phenotypes were significantly increased as compared to age/gender matched controls. high mdsc levels were associated with fewer immune responses and for mdsc4 and mdsc5 high frequencies were associated with shorter os (p=0.007 and p=0.019, respectively). to summarize, both hla-a*02 and hla-dr restricted peptides in ima910 were immunogenic. a significantly better clinical outcome of multi-tumap responders in comparison to patients with one/no tumap response strongly indicates clinical activity of ima910. acrylamide (aa), a genotoxic carcinogen (iarc class 2a) is formed in food by thermal treatment from different precursors. after oral ingestion, aa is metabolically epoxidized in the liver by cyp450 2e1 into glycidamide (ga). ga binds to dna, forming covalent adducts, primarily at n7 of guanine (n7-ga-gua). both, aa and ga undergo conjugation to glutathione (gsh) to be excreted via urine as mercapturic acids (ma), namely nacetyl-s-(2-carbamoylethyl)-cysteine (aama), and n-acetyl-s-(2-hydroxy-2carbamoylethyl)-cysteine (gama). in a dose response study, encompassing the dosage range from human dietary exposure levels up to 10 mg/kg bw, female sprague dawley (sd) rats on a diet devoid of detectable aa content were gavaged with single doses of aa. formation of urinary mas and of n7-ga-gua dna adducts in liver, kidney and lung was measured 16 h after application, a time point where cmax of n7-ga-gua was reached. the untreated control group was found to excrete about 0.8 nmol (aama plus gama) in the urine (16 h), indicating a background of endogenous aa formation. compared to untreated control, the lowest dosage of 0.1 µg aa/kg bw neither resulted in significantly enhanced ma excretion, nor in a detectable n7-ga-gua adduct levels in any organ tested (limit of detection, lod, 0.2 adducts/10 8 nucleotides). at the tenfold higher dose (1 µg/kg bw), adducts were found in kidney (about 1 adduct/10 8 nucleotides) and lung (< 1 adduct/10 8 nucleotides), but not in liver. at 10 and 100 µg/kg bw, adducts were found in all three organs, at levels not significantly different to those found at 1 µg aa/kg bw (about 1-2 adducts/10 8 nucleotides). the results of this in vivo study and of further recent research on aa toxicology will be discussed with respect to risk assessment. exposure of rats to single doses of aa in the range of human dietary exposure (0.1-10 µg/kg bw ) leads to n7-ga-gua adduct levels in the tissues monitored obviously not exceeding the range of steady state background dna lesions associated with endogenous/exogenous exposure to various genotoxic electrophiles. thus, the question of significant impact on human background dna damage resulting from exposure to a given genotoxic carcinogen, and on potentially ensuing biological consequences may become a highly relevant issue in risk assessment. pharmaco-economic impact of price, volume and demographic development böcking w., kirch w. institut für klinische pharmakologie, medizinische fakultät der tu dresden, fiedlerstr. 27, 01307 dresden health insurance costs in germany have grown by 3% p.a. over the last ten years and amount to approx. 280 bn eur in 2009. while costs for stationary treatment as the largest cost category have been intensely analyzed over the past years, pharmaceutical expenses have been analyzed in less detail, mostly focusing on the statutory health insurance side, even though pharmaceutical expenses have grown almost twice as much as costs for ambulant treatments. therefore, the question was asked how pharmaceutical expenses in a large german private health insurance company are allocated with respect to age and indication groups, and how those have developed from 2007 to 2011. the data of a private health insurance company with more than 600.000 customers was split into price and volume effects per age group to understand if price or volume drives the cost development. additionally, the two largest indication groups are analyzed in detail. as a result, both price and volume effects drive an overall cost increase. these effects are even stronger in older age groups. this cost increase is not sustainable for the german health insurance system over a longer period of time and will even further increase due to the ageing of the german population. a novel animal replacement system for the detection of endocrine disruptive capabilities in sexual development scheider j. 1, 2 , winter p. alternatives to animal testing for prediction of local toxicity and genotoxicity have been recently established. however, currently these methods are not suitable for measuring endocrine effects in developing organs such as e.g. embryonic gonads. here we present a phenotypic anchoring of a comprehensive study on sex-specific gene expression analysis accompanied by histological analysis of endocrine disruption in chicken embryo gonads, having the potential for an animal replacing system for endocrine disruptive toxicologic and ecotoxicologic examinations of chemicals. chicken embryos were inoculated with different amounts of tributyltin (tbt) and bisphenol-a (bpa). embryos were incubated and their gonads analyzed histologically 2 d prior to hatching. from identically treated embryos right and left testes and ovaries were separated and genome-wide transcription profiles generated using supertag digital gene expression (st-dge, supersage) profiling. male and female gonadal tissues both revealed histological aberrations in response to tbt and bpa. female gonads became masculinized in response to tbt and, viceversa, bpa-treated male gonads underwent feminization whereas in female gonads clearly visible structural aberrations occurred. in both chemicals mortality increased especially in the most affected sex (tbt: females, bpa: males). the expression profiles of more than 60 million mrnas revealed massive effects of both chemicals, tbt and bpa, on important cellular signaling pathways. gene expression differences were most pronounced in the phenotypically most affected sex. our results demonstrate that endocrine disruptive chemicals exert their effects on several levels including but not restricted to known hormone-based pathways. together with an ongoing study of gene expression differences in very young life stages and different chemicals these data will form the base for a blow-by-blow analysis of sexspecific gene expression of embryonic development. the project builds on already existing and further to generate data with the aim of the development of an in vitro method for testing chemicals at chicken eggs for 1) replacement of tests on juveniles and (sub-) adult rodents, 2) stages with impossibility of sensation of pain in the individuals, 3) highly sensitive prospects of modes of action of chemicals, which 4) might show consequences in the next generations. 3 channels are critical for oscillatory burst discharges in the reticular thalamus and absence epilepsy differential distribution of three members of a gene family encoding low voltage-activated (t-type) calcium channels hippocampal seizure resistance and reduced neuronal excitotoxicity in mice lacking the cav 2.3 e/r-type voltage-gated calcium channel transcriptional upregulation of cav3.2 mediates epileptogenesis in the pilocarpine model of epilepsy structure and functional expression of a member of the low voltage-activated calcium channel family a molecular determinant of nickel inhibition in cav3.2 t-type calcium channels histidine residues in the is3-is4 loop are critical for nickel-sensitive inhibition of the cav2.3 calcium channel substrate recognition and translocation by polyspecific organic cation transporters proton pump inhibitors inhibit metformin uptake by organic cation transporters (octs) structural determinants of inhibitor interaction with the human organic cation transporter oct2 (slc22a2) functional characterization of the human organic cation transporter 2 variant p.270ala>ser extra-adrenal glucocorticoid synthesis in the intestinal epithelium: more than a drop in the ocean? local glucocorticoid production in the mouse lung is induced by immune cell stimulation biomimetic materials in tissue engineering biomaterials offer cancer research the third dimension synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering injectable self-assembling peptide nanofibers create intramyocardial microenvironments for endothelial cells directed growth of fibroblasts into three dimensional micropatterned geometries via selfassembling scaffolds novel pcl-based honeycomb scaffolds as drug delivery systems for rhbmp-2 tissue engineering spatio-temporal vegf and pdgf delivery patterns blood vessel formation and maturation presentation of rgds epitopes on self-assembled nanofibers of branched peptide amphiphiles controlling mammalian cell interactions on patterned polyelectrolyte multilayer surfaces langmuir avintegrins as receptors for tumor targeting by circulating ligands heparin binding nanostructures to promote growth of blood vessels tirrell endothelial cell adhesion to the fibronectin cs5 domain in artificial extracellular matrix proteins design and bioproduction of a recombinant multi(bio)functional elastin-like protein polymer containing cell adhesion sequences for tissue engineering purposes stimuli-responsive thin coatings using elastin-like polymers for epac as a novel effector of airway smooth muscle relaxation ) camp inhibits airway smooth muscle phenotype modulation functional roles of epac and pka in human airway smooth muscle phenotype plasticity assessment of the sensitizing and irritative potential of preservatives by loose-fit coculture-based sensitization assay (lcsa) sonnenburg a nsc-631570 (ukrain) in the palliative treatment of pancreatic cancer. results of a phase ii trial association of funding and conclusions in randomized drug trials: a reflection of treatment effect or adverse events a general method for the covalent labeling of fusion proteins with small molecules in vivo robust single-particle tracking in live-cell time-lapse sequences correlation of structural class with no-observed-effect levels: a proposal for establishing a threshold of concern trbp recruits the dicer complex to ago2 for microrna processing and gene silencing index a 009, 019, 035 011, 014 003, 044 objective: hypertension and arterial stiffness is influenced by environmental and genetic factors. high plasma sodium concentration leads to mechanical stiffening of endothelial cells resulting in endothelial dysfunction and elevated blood pressure. here we investigated whether endothelial cell stiffness of ex vivo preparations of human arteries is linked to plasma sodium concentrations and functional genetic variants of the mineralocorticoid receptor (nr3c2), rs2070951 modulating blood pressure, renin, and aldosterone levels, and rs5534, which alters a mirna binding site. design and methods: twenty patients were enrolled after a vein stripping procedure and collateral arterial blood vessels were prepared for atomic force microscopy (afm). plasma sodium concentration was routinely determined and dna for genotyping was extracted from edta blood samples. sodium levels >140 mmol/l were defined as 'high'. after application of 5 µm amiloride, a specific blocker of the endothelial sodium channel (enac) changes in endothelial cell stiffness, were defined as 'weak' (≤10%), or 'strong' (>10%). statistical analyzes were performed by anova. results: in ex vivo artery preparations of patients with high sodium levels (n=12), mechanical stiffness of endothelial cells was tend to increase (∆ amiloride) (p=0.06). both nr3c2 variants were associated with a change >10% in endothelial stiffness after amiloride treatment. the rs2070951 c allele was significantly associated with a strong amiloride response (p=0.024), while the rs5534 a allele only showed a trend towards stronger amiloride effects (p=0.06). conclusion: our findings indicate that high plasma sodium concentration results in an increased endothelial amiloride response and thus influencing mechanical stiffness, modulated by functional nr3c2 variants. our novel approach linking patients' sodium levels and genetic status to endothelial stiffness by afm will be further evaluated in larger clinical settings. protein expression changes in bap-exposed human bladder cancer cells from spliceosome activation towards redistribution of the cytoskeleton after long-term exposure to subacute concentration schmitz-spanke s., pink m., jeske e., stempelmann k., rehn s., verma n., rettenmeier a. w. universitätsklinikum essen institut für hygiene und arbeitsmedizin, hufelandstr. 55, 45122 essen, germany deregulation of the β-catenin signaling pathway plays an important role in the development of hepatocellular tumors. activating mutations in ctnnb1 (encoding β-catenin) are frequently observed in murine and human liver tumors (e.g. human hepatoblastomas). activation of β-catenin signaling induces an overexpression of several cytochrome p450 (cyp) enzymes, including cyp2e1. cytotoxicity of acetaminophen (aap) is based on its cyp2e1-catalyzed metabolism to the electrophilic compound n-acetyl-p-benzo-quinone imine, which forms covalent adducts with cellular macromolecules if depletion of glutathione occurs. treatment with aap should therefore lead to a selective damage of cyp2e1-overexpressing ctnnb1mutated hepatoma cells. mice were injected with a single dose of the liver carcinogen n-nitrosodiethylamine (den) and subsequently treated with the tumor promoter phenobarbital to select for ctnnb1-mutated tumors. administration of a single dose of aap (300 mg/kg of body weight) followed the tumor promotion protocol. two days after treatment immunohistological analysis of the livers showed about 90% necrotic tissue in the larger tumors which were positive for glutamine synthetase (gs), a marker for ctnnb1-mutated tumor cells. by contrast, gs-negative tumors remained unaffected. at later time points we observed regeneration processes with infiltration of the necrotic tissue by inflammatory cells followed by fibrotic cells. proliferation of normal hepatocytes surrounding the damaged areas could also be observed. however, repopulation of parts of the former tumor areas by remaining gs-positive tumor cells was also detected. these results suggest that treatment with aap might serve as a future therapeutic possibility to selectively poison cyp2e1-overexpressing hepatoma. release of 5,6-epoxyeicosatrienoic acid (5,6-eet) upon neuronal activity induces trpa1-dependent mechanical pain hypersensitivity sisignano m. 1 , epoxyeicosatrienoic acids (eets) are cyp-epoxygenase (cyp450) derived metabolites of arachidonic acid (aa) which act as endogenous signaling molecules in multiple biological systems. we investigated the specific contribution of 5,6-eet to transient-receptor potential-(trp)-channel activation in nociceptor neurons, and its consequence for nociceptive processing. we found that during capsaicin-induced nociception 5,6-eet-levels increased in the drg and it is released from activated sensory neurons in vitro. 5,6-eet potently induced a calcium flux [10 nm] in cultured drg-neurons which was completely abolished when trpa1 was deleted or inhibited. in spinal cord slices 5,6-eet dose-dependently enhanced the frequency, but not the amplitude of spontaneous excitatory postsynaptic currents (sepsc) in lamina ii neurons that also respond to mustard oil (aitc), indicating a presynaptic mechanism. furthermore, 5,6-eet-induced enhancement of sepsc frequency was abolished in trpa1 null mice, suggesting that 5,6-eet pre-synaptically facilitates spinal cord synaptic transmission via trpa1. finally, intrathecal injection of 5,6-eet caused mechanical hyperagesia in wild type but not trpa1 null mice. we conclude that 5,6-eet is synthesized upon acute activation of nociceptors and leads to mechanical hypersensitivity via trpa1 at central afferent terminals in the spinal cord. sisnaiske j. 1 , hardelauf h. introduction: neurite outgrowth and plasticity of neuronal networks are essential processes e.g. during brain development and learning. thus, morphological readouts of neuronal connectivity are thought to be important endpoints to assess neurotoxic effects of environmental chemicals as well as when discovering new drugs. to analyze neurite outgrowth and connectivity level rapidly and easily in vitro we developed the network formation assay (nfa) (pct/ep2010/002811). this platform requires a spatially standardized hexagonal array for culturing neuronal networks with no need to fix or stain the cells to visualize neuritic processes. methods: to demonstrate the feasibility of the nfa we performed experiments in which we disrupted mature neurite networks or inhibited generating networks of human sh-sy5y cells with different concentrations of acrylamide (acr). we also observed the counteracting effects of brain-derived neurotrophic factor (bdnf) and calpeptin in these systems. to create the hexagonal array we used a poly(dimethylsulfoxide) bilayer stencil comprising through holes for adhesion spots and interconnecting tracks. plasma stencilling a peg-coated glass substrate produces adhesive nodes for the neurons and micron-scale-tracks for guiding neurite outgrowth and connectivity. results: in both systems, the developing and mature network, we found not only a concentration dependant effect of acr and bdnf but also a time dependant effect with a limited capability of the developing system to regenerate, even in the presence of acr. the co-treatment of the cells showed that inhibition of calpains by calpeptin might reduce the effect of intracellular elevated ca2+, a known neurotoxic mechanism of acr. moreover, the neurothrophin bdnf acts via trkb receptors on pathways stimulating neurite outgrowth and thereby counteracting the adverse effect of acr. conclusion: with the nfa we provide a rapid and simple way to analyze neurite outgrowth and connection formation in real time. by spatially standardizing the array we provide assay coordinates to streamline the analysis process and bring it towards high throughput testing. furthermore preliminary data showed that modification of the surface with biomolecules allows cell adhesion of other neuronal celltypes (e.g. primary mouse neurons) and extracellular matrix proteins (e.g. laminin) stimulate neurite outgrowth via integrins. transcriptional regulation of nox4 by histone deacetylases siuda d. 1, 2 , zechner u. 3 , prawitt d. 4 nox4 is a member of the nadph oxidase family, which represents a major source of reactive oxygen species (ros) in the vascular wall. nox4-mediated ros production mainly depends on the expression levels of the enzyme. the present study is aimed to investigate the regulation mechanisms of nox4 transcription by histone deacetylase (hdac). in cultured human ea.hy 926 endothelial cells, treatment with the pan-hdac inhibitors (scriptaid, trichostatin a, tsa, and suberoylanilide hydroxamic acid, saha) leads to a drastic decrease in nox4 mrna expression. a similar down-regulation of nox4 mrna expression can be achieved with sirna-mediated knockdown of hdac3. hdac inhibition in endothelial cells is associated with enhanced histone acetylation in the human nox4 promoter region, with no significant changes in dna methylation. consistently, scriptaid-treated cells show increased chromatin accessibility in nox4 promoter. in addition, we provide evidence that c-jun plays an important role in controlling nox4 transcription. knockdown of c-jun with sirna leads to a marked downregulation of nox4 mrna expression. in response to scriptaid treatment, the binding to c-jun to the nox4 promoter region is reduced despite the open chromatin structure. in parallel, the binding of polymerase iia to the nox4 promoter is significantly inhibited as well, which may explain the reduction in nox4 transcription. in conclusion, hdac inhibition decreases nox4 transcription in human endothelial cells by preventing the binding of transcription factor(s) and polymerase(s) to the nox4 promoter. this is very likely because of a hyperacetylation-mediated steric inhibition. cyclopentenone prostaglandins induce oxidative dna-damage in hamster lung fibroblast v79 cells solecki g. m. 1 key: cord-022633-fr55uod6 authors: nan title: saem abstracts, plenary session date: 2012-04-26 journal: acad emerg med doi: 10.1111/j.1553-2712.2012.01332.x sha: doc_id: 22633 cord_uid: fr55uod6 nan objectives: we sought to determine if the ocp policy resulted in a meaningful and sustained improvement in ed throughput and output metrics. methods: a prospective pre-post experimental study was conducted using administrative data from 15 community and tertiary centers across the province. the study phases consisted of the 8 months from february to september 2010 compared against the same months in 2011. operational data for all centres were collected through the edis tracking systems used in the province. the ocp included 3 main triggers: ed bed occupancy >110%, at least 35% of ed stretchers blocked by patients awaiting inpatient bed or disposition decision, and no stretcher available for high acuity patients. when all criteria were met, selected boarded patients were moved to an inpatient unit (non-traditional care space if no bed available). the primary outcome was ed length of stay (los) for admitted patients. the ed load of boarded patients from 10-11 am was reported the editors of academic emergency medicine (aem) are honored to present these abstracts accepted for presentation at the 2012 annual meeting of the society for academic emergency medicine (saem), may 9 to 12 in chicago, illinois. these abstracts represent countless hours of labor, exciting intellectual discovery, and unending dedication by our specialty's academicians. we are grateful for their consistent enthusiasm, and are privileged to publish these brief summaries of their research. this year, saem received 1172 abstracts for consideration, and accepted 746. each abstract was independently reviewed by up to six dedicated topic experts blinded to the identity of the authors. final determinations for scientific presentation were made by the saem program scientific subcommittee co-chaired by ali s. raja, md, mba, mph and steven b. bird, md, and the saem program committee, chaired by michael l. hochberg, md. their decisions were based on the final review scores and the time and space available at the annual meeting for oral and poster presentations. there were also 125 innovation in emergency medicine education (ieme) abstracts submitted, of which 37 were accepted. the ieme subcommittee was co-chaired by joanna leuck, md and laurie thibodeau, md. we present these abstracts as they were received, with minimal proofreading and copy editing. any questions related to the content of the abstracts should be directed to the authors. presentation numbers precede the abstract titles; these match the listings for the various oral and poster sessions at the annual meeting in chicago, as well as the abstract numbers (not page numbers) shown in the key word and author indexes at the end of this supplement. all authors attested to institutional review board or animal care and use committee approval at the time of abstract submission, when relevant. abstracts marked as ''late-breakers'' are prospective research projects that were still in the process of data collection at the time of the december abstract deadline, but were deemed by the scientific subcommittee to be of exceptional interest. these projects will be completed by the time of the annual meeting; data shown here may be preliminary or interim. on behalf of the editors of aem, the membership of saem, and the leadership of our specialty, we sincerely thank our research colleagues for these contributions, and their continuing efforts to expand our knowledge base and allow us to better treat our patients. david background: two to ten percent of patients evaluated in the emergency departments (ed) present with altered mental status (ams). the prevalence of non-convulsive seizure (ncs) and other electroencephalographic (eeg) abnormalities in this population is not known. this information is needed to make recommendations regarding the routine use of emergent eeg in ams patients. objectives: to identify the prevalence of ncs and other eeg abnormalities in ed patients with ams. methods: an ongoing prospective study at two academic urban ed. inclusion: patients ‡ 13 years old with ams. exclusion: an easily correctable cause of ams (e.g. hypoglycemia, opioid overdose). a 30-minute eeg with the standard 19 electrodes was performed on each subject as soon as possible after presentation (usually within 1 hour). outcome: the rate of eeg abnormalities based on blinded review of all eegs by two boardcertified epileptologists. descriptive statistics are used to report eeg findings. frequencies are reported as percentages with 95% confidence intervals (ci), and inter-rater variability is reported with kappa. results: the interim analysis was performed on 130 consecutive patients (target sample size: 260) enrolled from may to october 2011 (median age: 61, range 13-100, 40% male). eegs for 20 patients were reported uninterpretable by at least one rater (6 by both raters). of the remaining 110, only 30 (27%, 95%ci 20-36%) were normal according to either rater (n = 15 by both). the most common abnormality was background slowing (n = 75, 68%, 95%ci 59-76%) by either rater (n = 47 by both), indicating underlying encephalopathy. ncs was diagnosed in 8 patients (7%, 95%ci, 4-14%) by at least one rater (n = 4 by both), including 6 (5%, 95%ci 2-12%) patients in non-convulsive status epilepticus (ncse). 29 patients (26%,95%ci 19-35%) had interictal epileptiform discharges read by at least one rater (n = 12 by both) indicating cortical irritability and an increased risk of spontaneous seizure. inter-rater reliability for eeg interpretations was modest (kappa: 0.53, 95%ci 0.39-0.67). objectives: to define diagnostic sbi and non-bacterial (non-sbi) biosignatures using rna microarrays in febrile infants presenting to emergency departments (eds). methods: we prospectively collected blood for rna microarray analysis in addition to routine screening tests including white blood cell (wbc) counts, urinalyses, cultures of blood, urine, and cerebrospinal fluid, and viral studies in febrile infants 60 days of age in 22 eds . we defined sbi as bacteremia, urinary tract infection (uti), or bacterial meningitis. we used class comparisons (mann-whitney p < 0.01, benjamini for mtc and 1.25 fold change filter), modular gene analysis, and k-nn algorithms to define and validate sbi and non-sbi biosignatures in a subset of samples. results: 81% (939/1162) of febrile infants were evaluated for sbi. 6.8% (64/939) had sbi (14 (1.5%) bac-teremia, 56 (6.0%) utis, and 4 (0.4%) bacterial meningitis). infants with sbis had higher mean temperatures, and higher wbc, neutrophil, and band counts. we analyzed rna biosignatures on 141 febrile infants: 35 sbis (2 meningitis, 5 bacteremia, 28 uti), 106 non-sbis (49 influenza, 29 enterovirus, 28 undefined viral infections), and 11 healthy controls. class comparisons identified 1,288 differentially expressed genes between sbis and non-sbis. modular analysis revealed overexpression of interferon related genes in non-sbis and inflammation related genes in sbis. 232 genes were differently expressed (p < 0.01) in each of the three non-sbi groups vs sbi group. unsupervised cluster analysis of these 232 genes correctly clustered 91% (128/141) of non-sbis and sbis. k-nn algorithm identified 33 discriminatory genes in training set (30 non-sbis vs 17 sbis) which classified an independent test (76 non-sbis vs 18 sbis) with 87% accuracy. four misclassified sbis had over-expression of interferon-related genes, suggesting viral-bacterial co-infections, which was confirmed in one patient. background: improving maternal, newborn, and child health (mnch) is a leading priority worldwide. however, limited frontline health care capacity is a major barrier to improving mnch in developing countries. objectives: we sought to develop, implement, and evaluate an evidence-based maternal, newborn, and child survival (mncs) package for frontline health workers (fhws). we hypothesized that fhws could be trained and equipped to manage and refer the leading mnch emergencies. methods: setting -south sudan, which suffers from some of the world's worst mnch indices. assessment/intervention -a multi-modal needs assessment was conducted to develop a best-evidence package comprised of targeted trainings, pictorial checklists, and reusable equipment and commodities ( figure 1 ). program implementation utilized a trainingof-trainers model. evalution -1) pre/post knowledge assessments, 2) pre/post objective structured clinical examinations (osces), 3) focus group discussions, and 4) closed-response questionnaires. results: between nov 2010 to oct 2011, 72 local trainers and 708 fhws were trained in 7 of the 10 states in south sudan. knowledge assessments among trainers (n = 57) improved significantly from 62.7% (sd 20.1) to 92.0% (sd 11.8) (p < 0.001). mean scores a maternal osce and a newborn osce pre-training, immediately post-training, and upon 2-3 month follow-up are shown in the table. closed-response questionnaires with 54 fhws revealed high levels of satisfaction, use, and confidence with mncs materials. participants reported an average of 3.0 referrals (range 0-20) to a higher level of care in the 2-3 months since training. furthermore, 78.3% of fhws were more likely to refer patients as a result of the training program. during seven focus group discussions with trained fhws, respondents (n = 41) reported high satisfaction with mncs trainings, commodities, and checklists, with few barriers to implementation or use. conclusion: these findings suggest mncs has led to improvements in south sudanese fhws' knowledge, skills, and referral practices with respect to appropriate management of mnch emergencies. no study has compared various lactate measurements to determine the optimal parameter to target. objectives: to compare the association of blood lactate kinetics with survival in patients with septic shock undergoing early quantitative resuscitation. methods: preplanned analysis of a multicenter edbased rct of early sepsis resuscitation targeting three physiological variables: cvp, map, and either central venous oxygen saturation or lactate clearance. inclusion criteria: suspected infection, two or more sirs criteria, and either sbp <90 mmhg after a fluid bolus or lactate >4 mmol/l. all patients had an initial lactate measured with repeat at two hours. normalization of lactate was defined a lactate decline to <2.0 mmol/l in a patient with an intial lactate ‡2.0. absolute lactate clearance (initial -delayed value), and relative ((absolute clearance)/(initial value)*100) were calculated if the initial lactate was ‡2.0. the outcome was in-hospital survival. receiver operating characteristic curves were constructed and areas under the curve (auc) were calculated. difference in proportions of survival between the two groups at different lactate cutoffs were analyzed using 95% ci and fisher exact tests. results: of 272 included patients, the median initial lactate was 3.1 mmol/l (iqr 1.7, 5.8), and the median absolute and relative lactate clearance were 1 mmol/l (iqr 0.3, 2.5) and 37% (iqr 14, 57 ). an initial lactate >2.0 mmol/l was seen in 187/272 (69%), and 68/187 (36%) patients normalized their lactate. overall sutures on trunk and extremity lacerations that present in the ed. the use of absorbable sutures in the ed setting confers several advantages: patients do not need to return for suture removal which results in a reduction in ed crowding, ed wait times, missed work or school days, and stressful procedures (suture removal) for children. objectives: the primary objective of this study is to compare the cosmetic outcome of trunk and extremity lacerations repaired using absorbable versus nonabsorbable sutures in children and adults. a secondary objective is to compare complication rates between the two groups. methods: eligible patients with lacerations were randomly allocated to have their wounds repaired with vicryl rapide (absorbable) or prolene (nonabsorbable) sutures. at a 10 day follow-up visit the wounds were evaluated for infection and dehiscence. after 3 months, patients were asked to return to have a photograph of the wound taken. two blinded plastic surgeons using a previously validated 100 mm visual analogue scale (vas) rated the cosmetic outcome of each wound. a vas score of 15 mm or greater was considered to be a clinically significant difference. results: of the 100 patients enrolled, 45 have currently completed the study including 19 in the vicryl rapide group and 26 in the prolene group. there were no significant differences in the age, race, sex, length of wound, number of sutures, or layers of repair in the two groups. the observer's mean vas for the vicryl rapide group was 55.76 mm ) and that for the prolene group was 55.9 mm (95%ci 44.77-67.03), resulting in a mean difference of 0.14 mm (95%ci-16.95 to 17.23, p = .98). there were no significant differences in the rates of infection, dehiscence, or keloid formation between the two groups. conclusion: the use of vicryl rapide instead of nonabsorbable sutures for the repair of lacerations on the trunk and extremities should be considered by emergency physicians as it is an alternative that provides a similar cosmetic outcome. objectives: to determine the relationship between infection and time from injury to closure, and the characteristics of lacerations closed before and after 12 hours of injury. methods: over an 18 month period, a prospective multi-center cohort study was conducted at a teaching hospital, trauma center and community hospital. emergency physicians completed a structured data form when treating patients with lacerations. patients were followed to determine whether they had suffered a wound infection requiring treatment and to determine a cosmetic outcome rating. we compared infection rates and clinical characteristics of lacerations with chisquare and t-tests as appropriate. results: there were 2663 patients with lacerations; 2342 had documented times from injury to closure. the mean times from injury to repair for infected and noninfected wounds were 2.4 vs. 3.0 hrs (p = 0.39) with 78% of lacerations treated within 3 hours and 4% (85) treated 12 hours after injury. there were no differences in the infection rates for lacerations closed before (2.9%, 95%ci 2.2-3.7) or after (2.1%, 95%ci 0.4-6.0) 6 hours and before (3.0%, 95% ci 2.3%-3.8%) or after (1.2%, 95% ci 0.03%-6.4%) 12 hours. the patients treated 12 hours after injury tended to be older (41 vs. 34 yrs p = 0.02) and fewer were treated with primary closure (85% vs. 96% p < 0.0001). comparing wounds 12 or more hours after injury with more recent wounds, there was no effect of location on decision to close. wounds closed after 12 hours did not differ from wounds closed before 12 hours with respect to use of prophylactic antibiotics, type of repair, length of laceration, or cosmetic outcome. conclusion: closing older lacerations, even those greater than 12 hours after injury, does not appear to be associated with any increased risk of infection or adverse outcomes. excellent irrigation and decontamination over the last 30 years may have led to this change in outcome. background: deep burns may result in significant scarring leading to aesthetic disfigurement and functional disability. tgf-b is a growth factor that plays a significant role in wound healing and scar formation. objectives: the current study was designed to test the hypothesis that a novel tgf-b antagonist would reduce scar contracture compared with its vehicle in a porcine partial thickness burn model. methods: ninety-six mid-dermal contact burns were created on the backs and flanks of four anesthetized young swine using a 150 gm aluminum bar preheated to 80°celsius for 20 seconds. the burns were randomized to treatment with topical tgf-b antagonist at one of three concentrations (0, 187, and 375 ll) in replicates of 8 in each pig. dressing changes and reapplication of the topical therapy were performed every 2 days for 2 weeks then twice weekly for an additional 2 weeks. burns were photographed and full thickness biopsies were obtained at 5, 7, 9, 14, and 28 days to determine reepithelialization and scar formation grossly and microscopically. a sample of 32 burns in each group had 80% power to detect a 10% difference in percentage scar contracture. results: a total of 32 burns were created in each of the three study groups. burns treated with the high dose tgf-b antagonist healed with less scar contracture than those treated with the low dose and control (52 ± 20%, 63 ± 15%, and 62 ± 14%; anova p = 0.02). additionally, burns treated with the higher, but not the lower dose of tgf-b antagonist healed with significantly fewer full thickness scars than controls (62.5% vs. 100% vs. 93.8% respectively; p < 0.001). there were no infections and no differences in the percentage wound reepithelialization among all study groups at any of the time points. conclusion: treatment of mid-dermal porcine contact burns with the higher dose tgf-b antagonist reduced scar contracture and rate of deep scars compared with the low dose and controls. background: diabetic ketoacidosis (dka) is a common and lethal complication of diabetes. the american diabetes association recommends treating adult patients with a bolus dose of regular insulin followed by a continuous insulin infusion. the ada also suggests a glucose correction rate of 75-100 mg/dl/hr to minimize complications. objectives: compare the effect of bolus dose insulin therapy with insulin infusion to insulin infusion alone on serum glucose, bicarbonate, and ph in the initial treatment of dka. methods: consecutive dka patients were screened in the ed between march '06 and june '10. inclusion criteria were: age >18 years, glucose >350 mg/dl, serum bicarbonate 15 or ketonemia or ketonuria. exclusion criteria were: congestive heart failure, current hemodialysis, pregnancy, or inability to consent. no patient was enrolled more than once. patients were randomized to receive either regular insulin 0.1 units/kg or the same volume of normal saline. patients, medical and research staff were blinded. baseline glucose, electrolytes, and venous blood gases were collected on arrival. bolus insulin or placebo was then administered and all enrolled patients received regular insulin at rate of 0.1 unit/kg/hr, as well as fluid and potassium repletion per the research protocol. glucose, electrolytes, and venous blood gases were drawn hourly for 4 hours. data between two groups were compared using unpaired t-test. results: 99 patients were enrolled, with 30 being excluded. 35 patients received bolus insulin; 34 received placebo. no significant differences were noted in initial glucose, ph, bicarbonate, age, or weight between the two groups. after the first hour, glucose levels in the insulin group decreased by 151 mg/dl compared to 94 mg/dl in the placebo group (p = 0.0391, 95% ci 2.7 to 102.0). changes in mean glucose levels, ph, bicarbonate level, and ag were not statistically different between the two groups for the remainder of the 4 hour study period. there was no difference in the incidence of hypoglycemia in the two groups. conclusion: administering a bolus dose of regular insulin decreased mean glucose levels more than placebo, although only for the first hour. there was no difference in the change in ph, serum bicarbonate or anion gap at any interval. this suggests that bolus dose insulin may not add significant benefit in the emergency management of dka. ihca; 3. return of spontaneous circulation (rsoc). traumatic cardiac arrests were excluded. we recorded baseline demographics, arrest event characteristics, follow-up vitals and laboratory data, and in-hospital mortality. apache ii scores were calculated at the time of rosc, and at 24 hrs, 48 hrs, and 72 hrs. we used simple descriptive statistics to describe the study population. univariate logistic regression was used to predict mortality with apache ii as a continuous predictor variable. discrimination of apache ii scores was assessed using the area under the curve (auc) of the receiver operator characteristic (roc) curve. results: a total of 229 patients were analyzed. the median age was 70 years (iqr: 56-79) and 32% were female. apache ii score was a significant predictor of mortality for both ohca and ihca at baseline and at all follow-up time points (all p < 0.01). discrimination of the score increased over time and achieved very good discrimination after 24 hrs (table, figure) . conclusion: the ability of apache ii score to predict mortality improves over time in the 72 hours following cardiac arrest. these data suggest that after 24 hours, apache ii scoring is a useful severity of illness score in all post-cardiac arrest patients. background: admission hyperglycemia has been described as a mortality risk factor for septic non-diabetics, but the known association of hyperglycemia with hyperlactatemia (a validated mortality risk factor in sepsis) has not previously been accounted for. objectives: to determine whether the association of hyperglycemia with mortality remains significant when adjusted for concurrent hyperlactatemia. methods: this was a post-hoc, nested analysis of a single-center cohort study. providers identified study subjects during their ed encounters; all data were collected from the electronic medical record. patients: nondiabetic adult ed patients with a provider-suspected infection, two or more systemic inflammatory response syndrome criteria, and concurrent lactate and glucose testing in the ed. setting: the ed of an urban teaching hospital; 2007 to 2009. analysis: to evaluate the association of hyperglycemia (glucose >200 mg/dl) with hyperlactatemia (lactate ‡ 4.0 mmol/l), a logistic regression model was created; outcome-hyperlactatemia; primary variable of interest-hyperglycemia. a second model was created to determine if concurrent hyperlactatemia affects hyperglycemia's association with mortality; outcome-28-day mortality; primary risk variablehyperglycemia with an interaction term for concurrent hyperlactatemia. both models were adjusted for demographics, comorbidities, presenting infectious syndrome, and objective evidence of renal, respiratory, hematologic, or cardiovascular dysfunction. results: 1236 ed patients were included; mean age 76 ± 19 years. 133 (9%) subjects were hyperglycemic, 182 (13%) hyperlactatemic, and 225 (16%) died within 28 days of the initial ed visit. after adjustment, hyperglycemia was significantly associated with simultaneous hyperlactatemia (or 3.9, 95%ci 2.48, 5.98). hyperglycemia with concurrent hyperlactatemia was associated with increased mortality risk (or 4.4, 95%ci 2.27, 8.59) , but hyperglycemia in the absence of simultaneous hyperlactatemia was not (or 0.86, 95%ci 0.45, 1.65) . conclusion: in this cohort of septic adult non-diabetic patients, mortality risk did not increase with hyperglycemia unless associated with simultaneous hyperlactatemia. the previously reported association of hyperglycemia with mortality in this population may be due to the association of hyperglycemia with hyperlactatemia. the background: near infrared spectroscopy (sto2) represents a measure of perfusion that provides the treating physician with an assessment of a patient's shock state and response to therapy. it has been shown to correlate with lactate and acid/base status. it is not known if using information from this monitor to guide resuscitation will result in improved patient outcomes. objectives: to compare the resuscitation of patients in shock when the sto2 monitor is or is not being used to guide resuscitation. methods: this was a prospective study of patients undergoing resuscitation in the ed for shock from any cause. during alternating 30 day periods, physicians were blinded to the data from the monitor followed by 30 days in which physicians were able to see the information from the sto2 monitor and were instructed to resuscitate patients to a target sto2 value of 75. adult patients (age>17) with a shock index (si) of >0.9 (si = heart rate/systolic blood pressure) or a blood pressure <80 mmhg systolic who underwent resuscitation were enrolled. patients had a sto 2 monitor placed on the thenar eminence of their least-injured hand. data from the sto 2 monitor were recorded continuously and noted every minute along with blood pressure, heart rate, and oxygen saturation. all treatments were recorded. patients' charts were reviewed to determine the diagnosis, icu-free days in the 28 days after enrollment, inpatient los, and 28-day mortality. data were compared using wilcoxon rank sum and chi-square tests. results: 107 patients were enrolled, 51 during blinded periods and 56 during unblinded periods. the median presenting shock index was 1.24 (range 0.5 to 4.0) for the blinded group and 1.10 (0.5-3.3) for the unblinded group (p = 0.13). the median time in department was 70 minutes (range 22-407) for the blinded and 76 minutes (range 11-275) for the unblinded groups (p = 0.99). the median hospital los was 1 day (range 0-30) for the blinded group, and 2 days (range 0-23) in the unblinded group (p = 0.63). the mean icu-free days was 22 ± 9 for the blinded group and 19 ± 11 for the unblinded group (p = 0.26). among patients where the physician indicated using the sto2 monitor data to guide patient care, the icu-free days were 21.4 ± 9 for the blinded group and 16.3 ± 12 for the blinded group (p = 0.06). background: inducing therapeutic hypothermia (th) using 4°c iv fluids in resuscitated cardiac arrest patients has been shown to be feasible and effective. limited research exists assessing the efficiency of this cooling method. objectives: the objective was to determine an efficient infusion method for keeping fluid close to 4°c upon exiting an iv. it was hypothesized that colder temperatures would be associated with both higher flow rate and insulation of the fluid bag. methods: efficiency was studied by assessing change in fluid temperature (0c) during the infusion, under three laboratory conditions. each condition was performed four times using 1 liter bags of normal saline. fluid was infused into a 1000 ml beaker through 10 gtts tubing. flow rate was controlled using a tubing clamp and in-line transducer with a flowmeter, while temperature was continuously monitored in a side port at the terminal end of the iv tubing using a digital thermometer. the three conditions included infusing chilled fluid at a rate of 40 ml/min, which is equivalent to 30 ml/kg/hr for an 80 kg patient, 105 ml/min, and 105 ml/min using a chilled and insulated pressure bag. descriptive statistics and analysis of variance was performed to assess changes in fluid temperature. results: the average fluid temperatures at time 0 were 3.40 (95% ci 3.12-3.69) (40 ml/min), 3.35 (95% ci 3.25-3.45) (105 ml/min), and 2.92 (95% ci 2.40-3.45) (105 ml/min + insulation). there was no significant difference in starting temperature between groups (p = 0.16). the average fluid temperatures after 100 ml had been infused were 10.02 (95% ci 9.30-10.74) (40 ml/min), 7.35 (95% ci 6.91-7.79) (105 ml/min), and 6.95 (95% ci 6.47-7.43) (105 ml/min + insulation). the higher flow rate groups had significantly lower temperature than the lower flow rate after 100 ml of fluid had been infused (p < 0.001). the average fluid temperatures after 1000 ml had been infused were 16.77 (95% ci 15.96-17.58) (40 ml/min), 11.40 (95% ci 11.18-11.61) (105 ml/min), and 7.75 (95% ci 7.55-7.99) (105 ml/min + insulation). there was a significant difference in temperature between all three groups after 1000 ml of fluid had been infused (p < 0.001). conclusion: in a laboratory setting, the most efficient method of infusing cold fluid appears to be a method that both keeps the bag of fluid insulated and is infused at a faster rate. fluid bolus. patients were categorized by presence of vasoplegic or tissue dysoxic shock. demographics and sequential organ failure assessment (sofa) scores were evaluated between the groups. the primary outcome was in-hospital mortality. data were analyzed using t-tests, chi-squared test, and proportion differences with 95% confidence intervals as appropriate. results: a total of 242 patients were included: 89 patients with vasoplegic shock and 153 with tissue dysoxic shock. there were no significant differences in age (61 vs. 58 years), caucasian race (53% vs. 58%), or male sex (57% vs. 52%) between the dysoxic shock and vasoplegic shock groups, respectively. the group with vasoplegic shock had a lower initial sofa score than did the group with tissue dysoxic shock (5.7 vs. 7.3 points, p = 0.0002). the primary outcome of in-hospital mortality occurred in 8/89 (9%) of patients with vasoplegic shock compared to 40/153 (26%) in the group with tissue dysoxic shock (proportion difference 17%, 95% ci 7-26%, p < 0.0001). conclusion: in this analysis of patients with septic shock, we found a significant difference in in-hospital mortality between patients with vasoplegic versus tissue dysoxic septic shock. these findings suggest a need to consider these differences when designing future studies of septic shock therapies. background: the pre-shock population, ed sepsis patients with tissue hypoperfusion (lactate of 2.0-3.9 mm), commonly deteriorates after admission and requires transfer to critical care. objectives: to determine the physiologic parameters and disease severity indices in the ed pre-shock sepsis population that predict clinical deterioration. we hypothesized that neither initial physiologic parameters nor organ function scores will be predictive. methods: design: retrospective analysis of a prospectively maintained registry of sepsis patients with lactate measurements. setting: an urban, academic medical center. participants: the pre-shock population, defined as adult ed sepsis patients with either elevated lactate (2.0-3.9 mm) or transient hypotension (any sbp <90 mmhg) receiving iv antibiotics and admitted to a medical floor. consecutive patients meeting pre-shock criteria were enrolled over a 1-year period. patients with overt shock in the ed, pregnancy, or acute trauma were excluded. outcome: primary patientcentered outcome of increased organ failure (sequential organ failure assessment [sofa] score increase >1 point, mechanical ventilation, or vasopressor utilization) within 72 hours of admission or in-hospital mortality. results: we identified 248 pre-shock patients from 2649 screened. the primary outcome was met in 54% of the cohort and 44% were transferred to the icu from a medical floor. patients meeting the outcome of increased organ failure had a greater shock index (1.02 vs 0.93, p = 0.042) and heart rate (115 vs 105, p < 0.001) with no difference in initial lactate, age, map, or exposure to hypotension (sbp <100 mmhg). there was no difference in the predisposition, infection, response, and organ dysfunction (piro) score between groups (6.4 vs 5.7, p = 0.052). outcome patients had similar initial levels of organ dysfunction but had higher sofa scores at 24, 48, and 72 hours, a higher icu transfer rate (60 vs 24%, p < 0.001), and increased icu and hospital lengths of stay. conclusion: the pre-shock sepsis population has a high incidence of clinical deterioration, progressive organ failure, and icu transfer. physiologic data in the ed were unable to differentiate the pre-shock sepsis patients who developed increased organ failure. this study supports the need for an objective organ failure assessment in the emergency department to supplement clinical decision-making. background: lipopolysaccharide (lps) has long been recognized to initiate the host inflammatory response to infection with gram negative bacteria (gnb). large clinical trials of potentially very expensive therapies continue to have the objective of reducing circulating lps. previous studies have found varying prevalence of lps in blood of patients with severe sepsis. compared with sepsis trials conducted 20 years ago, the frequency of gnb in culture specimens from emergency department (ed) patients enrolled in clinical trials of severe sepsis has decreased. objectives: test the hypothesis that prior to antibiotic administration, circulating lps can be detected in the plasma of fewer than 10% of ed patients with severe sepsis. methods: secondary analysis of a prospective edbased rct of early quantitative resuscitation for severe sepsis. blood specimens were drawn at the time severe sepsis was recognized, defined as two or more systemic inflammatory criteria and a serum lactate >4 mm or spb<90 mmhg after fluid challenge. blood was drawn in edta prior to antibiotic administration or within the first several hours, immediately centrifuged, and plasma frozen at )80°c. plasma lps was quantified using the limulus amebocyte lysate assay (lal) by a technician blinded to all clinical data. results: 180 patients were enrolled with 140 plasma samples available for testing. median age was 59 ± 17 years, 50% female, with overall mortality of 18%. forty of 140 patients (29%) had any culture specimen positive for gnb including 21 (15%) with blood cultures positive. only five specimens had detectable lps, including two with a gnb-positive culture specimen, and three were lps-positive without gnb in any culture. prevalence of detectable lps was 3.5% (ci: 1.5%-8.1%). the frequency of detectable lps in antibiotic-naive plasma is too low to serve as a useful diagnostic test or therapeutic target in ed patients with severe sepsis. the data raise the question of whether post-antibiotic plasma may have a higher frequency of detectable lps. background: egdt is known to reduce mortality in septic patients. there is no evidence to date that delineates the role of using a risk stratification tool, such as the mortality in emergency department sepsis (meds) score, to determine which subgroups of patients may have a greater benefit with egdt. objectives: our objective was to determine if our egdt protocol differentially affects mortality based on the severity of illness using meds score. methods: this study is a retrospective chart review of 243 patients, conducted at an urban tertiary care center, after implementing an egdt protocol on july 1, 2008 (figure) . this study compares in-hospital mortality, length of stay (los) in icu, and los in ed between the control group (126 patients from 1/1/07-12/31/07) and the postimplementation group (117 patients from 7/1/08-6/ 30/09), using meds score as a risk stratification tool. inclusion criteria: patients who presented to our ed with a suspected infection, and two or more sirs criteria, a map<65 mmhg, a sbp< 90 mmol/l. exclusion criteria: age<18, death on arrival to ed, dnr or dni, emergent surgical intervention, or those with an acute myocardial infarction or chf exacerbation. a two-sample t-test was used to show that the mean age and number of comorbidities was similar between the control and study groups (p = 0.27 and 0.87 respectively). mortality was compared and adjusted for meds score using logistic regression. the odds ratios and predicted probabilities of death are generated using the fitted logistic regression model. ed and icu los were compared using mood's median test. results: when controlling for illness severity using meds score, the relative risk (rr) of death with egdt is about half that of the control group (rr = 0.52, 95% ci [0.278-0.973], p=0.04). also, by applying meds score to risk stratify patients into various groups of illness severity, we found no specific groups where egdt is more efficacious at reducing the predicted probability of death (table 1) . without controlling for meds score, there is a trend in reduction of absolute mortality by 9.7% when egdt is used (control = 30.2%, study = 20.5%, p = 0.086). egdt leads to a 40.3% reduction in the median los in icu (control = 124 hours, study = 74 hours, p = 0.03), without increasing los in ed (control = 6 hours, study = 7 hours, p = 0.50). conclusion: egdt is beneficial in patients with severe sepsis or septic shock, regardless of their meds score. background: in patients experiencing acute coronary syndrome (acs), prompt diagnosis is critical in achieving the best health outcome. while ecg analysis is usually sufficient to diagnose acs in cases of st elevation, acs without st elevation is reliably diagnosed through serial testing of cardiac troponin i (ctni). pointof-care testing (poct) for ctni by venipuncture has been proven a more rapid means to diagnosis than central laboratory testing. implementing fingerstick testing for ctni in place of standard venipuncture methods would allow for faster and easier procurement of patients' ctni levels, as well as increase the likelihood of starting a rapid test for ctni in the prehospital setting, which could allow for even earlier diagnosis of acs. objectives: to determine if fingerstick blood samples yield accurate and reliable troponin measurements compared to conventional venous blood draws using the i-stat poc device. methods: this experimental study was performed in the ed of a quaternary care suburban medical center between june-august 2011. fingerstick blood samples were obtained from adult ed patients for whom standard (venipuncture) poc troponin testing was ordered. the time between fingerstick and standard draws was kept as narrow as possible. ctni assays were performed at the bedside using the i-stat 1 (abbott point of care). results: 94 samples from 87 patients were analyzed by both fingerstick and standard ed poct methods (see table) . four resulted in cartridge error. compared to ''gold standard'' ed poct, fingerstick testing has a positive predictive value of 100%, negative predictive value of 96%, sensitivity of 79%, and specificity of 100%. no significant difference in ctni level was found between the two methods, with a nonparametric intraclass correlation coefficient of 0.994 (95% ci 0.992-0.996, p-value < 0.001). conclusion: whole blood fingerstick ctni testing using the i-stat device is suitable for rapid evaluation of ctni level in prehospital and ed settings. however, results must be interpreted with caution if they are within a narrow territory of the cutoff for normal vs. elevated levels. additional testing on a larger sample would be beneficial. the practicality and clinical benefit of using fingerstick ctni testing in the ems setting must still be assessed. background: adjudication of diagnosis of acute myocardial infarction (ami) in clinical studies typically occurs at each site of subject enrollment (local) or by experts at an independent site (central). from 2000 from -2007 , the troponin (ctn) element of the diagnosis was predicated on the local laboratories, using a mix of the 99th percentile reference ctn and roc-determined cutpoints. in 2007, the universal definition of ami (ud-ami) defined it by the 99th percentile reference alone. objectives: to compare the diagnosis rates of ami as determined by local adjudication vs. central adjudication using udami criteria. methods: retrospective analysis of data from the myeloperoxidase in the diagnosis of acute coronary syndromes (acs) study (midas), an 18-center prospective study with enrollment from 12/19/06 to 9/20/07 of patients with suspected acs presenting to the ed < 8 hours after symptom onset and in whom serial ctn and objective cardiac perfusion testing was planned. adjudication of acs was done by single local principal investigators using clinical data and local ctn cutpoints from 13 different ctn assays, and applying the 2000 definition. central adjudication was done after completion of the midas primary analysis using the same data and local ctn assay, but by experts at three different institutions, using the udami and the manufacturer's 99th percentile ctn cutpoint, and not blinded to local adjudications. discrepant dignoses were resolved by consensus. local vs. central ctn cutpoints differed for six assays, with central cutpoints lower in all. statistics were by chi-square and kappa. results: excluding 11 cases deemed indeterminate by central adjudication, 1096 cases were successfully adjudicated. local adjudication resulted in 104 ami (9.5% of total) and 992 non-ami; central adjudication resulted in 134 (12.2%) ami and 962 non-ami. overall, 44 local diagnoses (4%) were either changed from non-ami to ami or ami to non-ami (p < 0.001). interrater reliability across both methods was found to be kappa = 0.79 (p < 0.001). for acs diagnosis, local adjudication identified 252 acs cases (23%) and 854 non-acs, while central adjudication identified 275 acs (25%) and 831 non-acs. overall, 61 local diagnoses (6%) were either changed from non-acs to acs or acs to non-acs (p < 0 .001). interrater reliability found kappa = 0.85 (p < 0.001). conclusion: central and local adjudication resulted in significantly different rates of ami and acs diagnosis. however, overall agreement of the two methods across these two diagnoses was acceptable. occur four times more often in cocaine users. biomarkers myeloperoxidase (mpo) and c-reactive protein (crp) have potential in the diagnosis of acs. objectives: to evaluate the utility of mpo and crp in the diagnosis of acs in patients presenting to the ed with cocaine-associated chest pain and compare the predictive value to nonusers. we hypothesized that these markers may be more sensitive for acs in nonusers given the underlying pathophysiology of enhanced plaque inflammation. methods: a secondary analysis of a cohort study of enrolled ed patients who received evaluation for acs at an urban, tertiary care hospital. structured data collection at presentation included demographics, chest pain history, lab, and ecg data. subjects included those with self-reported or lab-confirmed cocaine use and chest pain. they were matched to controls based on age, sex, and race. our main outcome was diagnosis of acs at index visit. we determined median mpo and crp values, calculated maximal auc for roc curves, and found cut-points to maximize sensitivity and specificity. data are presented with 95% ci. results: overall, 95 patients in the cocaine positivegroup and 86 patients in the nonusers group had mpo and crp levels measured. patients had a median age of 47 (iqr, (40) (41) (42) (43) (44) (45) (46) (47) (48) (49) (50) (51) (52) , 90% black or african american, and 62% male (p > 0.05 between groups). fifteen patients were diagnosed with acs: 8 patients in the cocaine group and 7 in the nonusers group. comparing cocaine users to nonusers, there was no difference in mpo (median 162 [iqr, ] v 136 ng/ml; p = 0.78) or crp (3 [1] [2] [3] [4] [5] [6] [7] [8] [9] v 5 [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] mg/l; p = 0.08). the auc for mpo was 0.65 (95% ci 0.39-0.90) v 0.54 (95% ci 0.19-0.73). the optimal cut-point to maximize sensitivity and specificity was 242 ng/ml which gave a sensitivity of 0.42 and specificity of 0.75. using this cutpoint, 57% v 29% of acs in cocaine users vs the nonusers would be identified. the auc for crp was 0.63 (95% ci 0.39-0.88) in cocaine users vs 0.73 (95% ci 0.52-0.95) in nonusers. the optimal cut point was 11.9 mg/l with a sensitivity of 0.67 and specificity of 0.79. using this cutpoint, 43% v 88% of acs in cocaine users and nonusers would have been identified. conclusion: the diagnostic accuracy of mpo and crp is not different in cocaine users than nonusers and does not appear to have sufficient discriminatory ability in either cohort. results: 18 hrs of moderate pe caused a significant decrease in rv heart function in rats treated with the solvent for bay 41-8543: peak systolic pressure (psp) decreased from 39 ± 1.5 mmhg, control to 16 ± 1.5, pe, +dp/dt decreased from 1192 ± 93 mmhg/sec to 463 ± 77, -dp/dt decreased from )576 ± 60 mmhg/sec to )251 ± 9. treatment of rats with bay 41-8543 significantly improved all three indices of rv heart function (psp 29 ± 2.6, +dp/dt 1109 ± 116, -dp/dt )426 ± 69). 5 hrs of severe pe also caused significant rv dysfunction (psp 25 ± 2, -dp/dt )356 ± 28) and treatment with bay 41-8543 produced protection of rv heart function (psp 34 ± 2, -dp/dt )535 ± 41) similar to the 18 hr moderate pe model. conclusion: experimental pe produced significant rv dysfunction, which was ameliorated by treatment of the animals with the soluble guanylate cyclase stimulator, bay 41-8543. 1 hospital of the university of pennsylvania, philadelphia, pa; 2 cooper university hospital, camden, nj background: patients who present to the ed with symptoms of potential acute coronary syndrome (acs) can be safely discharged home after a negative coronary computerized tomographic angiography (cta). however, the duration of time for which a negative coronary cta can be used to inform decision making when patients have recurrent symptoms is unknown. objectives: we examined patients who received more than one coronary cta for evaluation of acs to determine whether they had disease progression, as defined by crossing the threshold from noncritical (<50% maximal stenosis) to potentially critical disease. methods: we performed a structured comprehensive record search of all coronary ctas performed from 2005 to 2010 at a tertiary care health system. low-tointermediate risk ed patients who received two or more coronary ctas, at least one from an ed evaluation for potential acs, were identified. patients who were revascularized between scans were excluded. we collected demographic data, clinical course, time between scans, and number of ed visits between scans. record review was structured and done by trained abstractors. our main outcome was progression of coronary stenosis between scans, specifically crossing the threshold from noncritical to potentially critical disease. results: overall, 32 patients met study criteria (median age 45, interquartile range [iqr] (37.5-48); 56% female; 88% black). the median time between studies was 27.3 months (iqr, . 22 patients did not have stenosis in any vessel on either coronary cta, two studies showed increasing stenosis of <20%, and the rest showed ''improvement,'' most due to better imaging quality. no patient initially below the 50% threshold subsequently exceeded it (0%; 95% ci, 0-11.0%). patients also had varying numbers of ed visits (median number of visits 5, range 0-23), and numbers of ed visits for potentially cardiac complaints (median 1, range 0-6); 10 were re-admitted for potentially cardiac complaints (for example, chest pain or shortness of breath), and 9 received further provocative cardiac testing, all of which had negative results. conclusion: we did not find clinically significant disease progression within a 2 year time frame in patients who had a negative coronary cta, despite a high number of repeat visits. this suggests that prior negative coronary cta may be able to be used to inform decision making within this time period. 42.7-48.6) compared to non tro ct patients. there was no significant difference in image quality between tro ct images and those of dedicated ct scans in any studies performing this comparison. similarly, there was no significant difference between tro ct and other diagnostic modalities in regards to length of stay or admission rate. when compared to conventional coronary angiography as the gold standard for evaluation of cad, tro ct had the following pooled diagnostic accuracy estimates: sensitivity 0.94 conclusion: tro chest ct is comparable to dedicated pe, coronary, or ad ct in regard to image quality, length of stay, and admission rate and is highly accurate for detecting cad. the utility of tro ct depends on the relative pre-test probabilities of the conditions being assessed and its role is yet to be clearly defined. tro ct, however, involves increased radiation exposure and contrast volume and for this reason clinicians should be selective in its use. background: coronary computed tomographic angiography (ccta) has high sensitivity, specificity, accuracy, and prognostic value for coronary artery disease (cad) and acs. however, how a ccta informs subsequent use of prescription medication is unclear. objectives: to determine if detection of critical or noncritical cad on ccta is associated with initiation of aspirin and statins for patients who presented to the ed with chest pain. we hypothesized that aspirin and statins would be more likely to be prescribed to patients with noncritical disease relative to those without any cad. methods: prospective cohort study of patients who received ccta as part of evaluation of chest pain in the ed or observation unit. patients were contacted and medical records were reviewed to obtain clinical follow-up for up to the year after ccta. the main outcome was new prescription of aspirin or statin. cad severity on ccta was graded as absent, mild (1% to 49%), moderate (50% to 69%), or severe ( ‡70%) stenosis. logistic regression was used to assess the association of stenosis severity to new medication prescription; covariates were determined a priori. results: 859 patients who had ccta performed consented to participate in this study or met waiver of consent for record review only (median age, , 59% female, 71% black). median follow-up time was 333 days, iqr 70-725 days. at baseline, 13% of the total cohort was already prescribed aspirin and 8% on statin medication. two hundred seventy nine (32%) patients were found to have stenosis in at least one vessel. in patients with absent, mild, moderate, and severe cad on ccta, aspirin was initiated in 11%, 34%, 52%, and 55%; statins were initiated in 7%, 22%, 32%, and 53% of patients. after adjustment for age, race, sex, hypertension, diabetes, cholesterol, tobacco use, and admission to the hospital after ccta, higher grades of cad severity were independently associated with greater post-ccta use of aspirin (or 1.9 per grade, 95% ci 1.4-2.2, p < 0.001) and statins (or 1.9, 95% ci 1.5-2.4, p < 0.001). conclusion: greater cad severity on ccta is associated with increased medication prescription for cad. patients with noncritical disease are more likely than patients without any disease to receive aspirin and statins. future studies should examine whether these changes lead to decreased hospitalizations and improved cardiovascular health. background: hess et al. developed a clinical decision rule for patients with acute chest pain consisting of the absence of five predictors: ischemic ecg changes not known to be old, elevated initial or 6-hour troponin level, known coronary disease, ''typical'' pain, and age over 50. patients less than 40 required only a single troponin evaluation. objectives: to test the hypothesis that patients less than 40 years old without these criteria are at <1% risk for major adverse cardiovascular events (mace) including death, ami, pci, and cabg. methods: we performed a secondary analysis of several combined prospective cohort studies that enrolled ed patients who received an evaluation for acs in an urban ed from 1999 to 2009. cocaine users and stemi patients were excluded. structured data collection at presentation included demographics, pain description, history, lab, and ecg data for all studies. hospital course was followed daily. thirty-day follow up was done by telephone. our main outcome was 30-day mace using objective criteria. the secondary outcome was potential change in ed disposition due to application of the rule. descriptive statistics and 95% cis were used. results: of 9289 visits for potential acs, patients had a mean age of 52.4 ± 14.7 yrs; 68% were black and 59% female. there were 638 patients (6.9%) with 30-day cv events (93 dead, 384 ami, 298 pci). sequential removal of patients in order to meet the final rule for patients less than 40 excluded patients based upon: ischemic ecg changes not old (n = 434, 30% mace rate), elevated initial troponin level (n = 237, 60% mace), known coronary disease (n = 1622, 11% mace), ''typical'' pain (n = 3179, 3% mace), and age over 40 (n = 2690, 3.4% mace) leaving 1127 patients less than 40 with 0.8% mace [95% ci, 0.4-1.5%]. of this cohort, 70% were discharged home from the ed by the treating physician without application of this rule. adding a second negative troponin in patients 40-50 years old identified a group of 1139 patients with a 2.0% rate of mace [1.3-3 .0] and a 48% discharge rate. the hess rule appears to identify a cohort of patients at approximately 1% risk of 30-day mace, and may enhance discharge of young patients. however, even without application of this rule, the 70% of young patients at low risk are already being discharged home based upon clinical judgment. background: a clinical decision support system (cdss) incorporates evidence-based medicine into clinical practice, but this technology is underutilized in the ed. a cdss can be integrated directly into an electronic medical record (emr) to improve physician efficiency and ease of use. the christopher study investigators validated a clinical decision rule for patients with suspected pulmonary embolism (pe). the rule stratifies patients using wells' criteria to undergo either d-dimer testing or a ct angiogram (ct). the effect of this decision rule, integrated as a cdss into the emr, on ordering cts has not been studied. objectives: to assess the effect of a mandatory cdss on the ordering of d-dimers and cts for patients with suspected pe. methods: we assessed the number of cts ordered for patients with suspected pe before and after integrating a mandatory cdss in an urban community ed. physicians were educated regarding cdss use prior to implementation. the cdss advised physicians as to whether a negative d-dimer alone excluded pe or if a ct was required based on wells' criteria. the emr required physicians to complete the cdss prior to ordering the ct. however, physicians maintained the ability to order a ct regardless of the cdss recommendation. patients ‡18 years of age presenting to the ed with a chief complaint of chest pain, dyspnea, syncope, or palpitations were included in the data analysis. we compared the proportion of d-dimers and cts ordered during the 8-month periods immediately before and after implementing the cdss. all 27 physicians who worked in the ed during both time periods were included in the analysis. patients with an allergy to intravenous contrast agents, renal insufficiency, or pregnancy were excluded. results were analyzed using a chi-square test. results: a total of 11,931 patients were included in the data analysis (6054 pre-and 5877 post-implementation). cts were ordered for 215 patients (3.6%) in the pre-implementation group and 226 patients (3.8%) in the post-implementation group; p = 0.396. a d-dimer was ordered for 392 patients (6.5%) in the pre-implementation group and 382 patients (6.5%) in the post-implementation group; p = 0.958. in this single-center study, emr integration of a mandatory cdss for evaluation of pe did not significantly alter ordering patterns of cts and d-dimers. identification of patients with low-risk pulmonary emboli suitable for discharge from the emergency department mike zimmer, keith e. kocher university of michigan, ann arbor, mi background: recent data, including a large, multicenter randomized controlled trial, suggest that a low-risk cohort of patients diagnosed with pulmonary embolism (pe) exists who can be safely discharged from the ed for outpatient treatment. objectives: to determine if there is a similar cohort at our institution who have a low rate of complications from pe suitable for outpatient treatment. methods: this was a retrospective chart review at a single academic tertiary referral center with an annual ed volume of 80,000 patients. all adult ed patients who were diagnosed with pe during a 24-month period from 11/1/09 through 10/31/11 were identified. the pulmonary embolism severity index (pesi) score, a previously validated clinical decision rule to risk stratify patients with pe, was calculated. patients with high pesi (>85) were excluded. additional exclusion criteria included patients who were at high risk of complications from initiation of therapeutic anticoagulation and those patients with other clear indications for admission to the hospital. the remaining cohort of patients with low risk pe (pesi £ 85) was included in the final analysis. outcomes were measured at 14 and 90 days after pe diagnosis and included death, major bleeding, and objectively confirmed recurrent venous thromboembolism (vte). results: during the study period, 298 total patients were diagnosed with pe. there were 172 (58%) patients categorized as ''low risk'' (pesi £ 85), with 42 removed because of various pre-defined exclusion criteria. of the remaining 130 (44%) patients suitable for outpatient treatment, 5 patients (3.8%; 95% ci, 0.5% -7.2%) had one or more negative outcomes by 90 days. this included 2 (1.5%; 95% ci, 0% -3.7%) major bleeding events, 2 (1.5%; 95% ci, 0% -3.7%) recurrent vte, and 2 (1.5%; 95% ci, 0% -3.7%) deaths. none of the deaths were attributable to pe or anticoagulation. one patient suffered both a recurrent vte and died within 90 days. both patients who died within 90 days were transitioned to hospice care because of worsening metastatic burden. at 14 days, there was 1 bleeding event (0.8%; 95% ci, 0% -2.3%), no recurrent vte, and no deaths. the average hospital length of stay for these patients was 2.8 days (sd ±1.6). conclusion: over 40% of our patients diagnosed with pe in the ed may have been suitable for outpatient treatment, with 4% suffering a negative outcome within 90 days and 0.8% suffering a negative outcome within 14 days. in addition, the average hospital length of stay for these patients was 2.8 days, which may represent a potential cost savings if these patients had been managed as outpatients. our experience supports previous studies that suggest the safety of outpatient treatment of patients diagnosed with pe in the ed. given the potential savings related to a decreased need for hospitalization, these results have health policy implications and support the feasibility of creating protocols to facilitate this clinical practice change. background: chest x-rays (cxrs) are commonly obtained on ed chest pain patients presenting with suspected acute coronary syndrome (acs). a recently derived clinical decision rule (cdr) determined that patients who have no history of congestive heart failure, have never smoked, and have a normal lung examination do not require a cxr in the ed. objectives: to validate the diagnostic accuracy of the hess cxr cdr for ed chest pain patients with suspected acs. methods: this was a prospective observational study of a convenience sample of chest pain patients over 24 years old with suspected acs who presented to a single urban academic ed. the primary outcome was the ability of the cdr to identify patients with abnormalities on cxr requiring acute ed intervention. data were collected by research associates using the chart and physician interviews. abnormalities on cxr and specific interventions were predetermined, with a positive cxr defined as one with abnormality requiring ed intervention, and a negative cxr defined as either normal or abnormal but not requiring ed intervention. the final radiologist report was used as a reference standard for cxr interpretation. a second radiologist, blinded to the initial radiologist's report, reviewed the cxrs of patients meeting the cdr criteria to calculate inter-observer agreement. patients were followed up by chart review and telephone interview 30 days after presentation. results: between january and august 2011, 178 patients were enrolled, of whom 38 (21%) were excluded and 10 (5.6%) did not receive cxrs in the ed. of the 130 remaining patients, 74 (57%) met the cdr. the cdr identified all patients with a positive cxr (sensitivity = 100%, 95%ci 40-100%). the cdr identified 73 of the 126 patients with a negative cxr (specificity = 58%, 95%ci 49-67%). the positive likelihood ratio was 2.4 (95%ci 1.9-2.9). inter-observer agreement between radiologists was substantial (kappa = 0.63, 95%ci 0.41-0.85). telephone contact was made with 78% of patients and all patient charts were reviewed at 30 days. none had any adverse events related to a background: increasing the threshold to define a positive d-dimer in low-risk patients could reduce unnecessary computed tomographic pulmonary angiography (ctpa) for suspected pe. this strategy might increase rates of missed pe and missed pneumonia, the most common non-thromboembolic finding on ctpa that might not otherwise be diagnosed. objectives: measure the effect of doubling the standard d-dimer threshold for ' 'pe unlikely'' revised geneva (rgs) or wells' scores on the exclusion rate, frequency, and size of missed pe and missed pneumonia. methods: prospective enrollment at four academic us hospitals. inclusion criteria required patients to have at least one symptom or sign and one risk factor for pe, and have 64-channel ctpa completed. pretest probability data were collected in real time and the d-dimer was measured in a central laboratory. criterion standard for pe or pneumonia consisted of cpta interpretation by two independent radiologists combined with necessary treatment plan. subsegmental pe was defined as total vascular obstruction <5%. patients were followed for outcome at 30 days. proportions were compared with 95% cis. results: of 678 patients enrolled, 126 (19%) were pe+ and 93 (14%) had pneumonia. with rgs£6 and standard threshold (<500 ng/ml), d-dimer was negative in 110/678 (16%, 95% ci: 13-19%), and 4/110 were pe+ (posterior probability 3.8%, 95% ci: 1-9.3%). with rgs£6 and a threshold <1000 ng/ml, d-dimer was negative in 208/678 (31%, 27-44%) and 11/208 (5.3%, 2.8-9.3%) were pe+, but 10/11 missed pes were subsegmental, and none had concomitant dvt. the posterior probability for pneumonia among patients with rgs&#8804;6 and d-dimer<500 was 9/110 (8.2%, 4-15%) which compares favorably to the posterior probability of 12/208 (5.4%, 3-10%) observed with rgs& #8804;6 and d-dimer<1000 ng/ml. of the 200 (35%) patients who also had plain film cxr, radiologists found an infiltrate in only 58. use of wells£4 produced similar results as the rgs&#8804;6 for exclusion rate and posterior probability of both pe and pneumonia. conclusion: doubling the threshold for a positive d-dimer with a pe unlikely pretest probability can significantly reduce ctpa scanning with a slightly increased risk of missed isolated subsegmental pe, and no increase in rate of missed pneumonia. background: the limitations of developing world medical infrastructure require that patients are transferred from health clinics only when the patient care needs exceed the level of care at the clinic and the receiving hospital can provide definitive therapy. to determine what type of definitive care service was sought when patients were transferred from a general outpatient clinic operating monday through friday from 8:00 am to 3:00 pm in rural haiti to urban hospitals in port-au-prince. methods: design -prospective observational review of all patients for whom transfer to a hospital was requested or for whom a clinic ambulance was requested to an off-site location to assist with patient care. setting -weekday, daytime only clinic in titanyen, haiti. participants/subjects -consecutive series of all patients for whom transfer to another health care facility or for whom an ambulance was requested during the time period of 11/22/2010 -12/14/2010 and 3/28/2011 -5/13/2011 . results: between 11/22/2010 -12/14/2010 and 3/28/2011 -5/13/2011 patients were identified who needed to be transferred to a higher level of care. sixteen patients (43.2%) presented with medical complaints, 12 (32.4%) were trauma patients, 6 (16.2%) were surgical, and 3 (8.1%) were in the obstetric category. within these categories, 6 patients were pediatric and 4 non-trauma patients required blood transfusion. conclusion: while trauma services are often focused on in rural developing world medicine, the need for obstetric care and blood transfusion constituted six (16.2%) cases in our sample. these patients raise important public health, planning, and policy questions relating to access to prenatal care and the need to better understand transfusion medicine utilization among rural haitian patients with non-trauma related transfusion needs. the data set is limited by sample size and single location of collection. another limitation of understanding the needs is that many patients may not present to the clinic for their health care needs in certain situations if they have knowledge that the resources to provide definitive care are unavailable. background: the practice of emergency medicine in japan has been unique in that emergency physicians are mostly engaged in critical care and trauma with a multi-specialty model. for the last decade with progress in medicine, an aging population with complicated problems, and institution of postgraduate general clinical training, the us model emergency medicine with single-specialty model has been emerging throughout japan. however, the current status is unknown. objectives: the objective of this study was to investigate the current status of implementation of the us model emergency medicine at emergency medicine training institutions accredited by the japanese association for acute medicine (jaam). methods: the er committee of the jaam, the most prestigious professional organization in japanese emergency medicine, conducted the survey by sending questionnaires to 499 accredited emergency medicine training institutions. results: valid responses obtained from 299 facilities were analyzed. us model em was provided in 211 facilities (71% of 299 facilities), either in full time (24 hours a day, seven days a week; 123 facilities) or in part time (less than 24 hours a day; 88 facilities). among these 211 us model facilities, 44% have a number of beds between 251-500. the annual number of ed visits was less than 20,000 in 64%, and 37% have ambulance transfers between 2,001-4,000 per year. the number of emergency physicians was less than 5 in 60% of the facilities. postgraduate general clinical training was offered at us model ed in 199 facilities, and ninety hospitals adopted us model em after 2004, when a 2-year period of postgraduate general clinical training became mandatory for all medical graduates. sixty-four facilities provided a residency program to be a us model emergency physician, and another 9 institutions were planning to establish it. conclusion: us model em has emerged and become commonplace in japan. the background including advance in medicine, aging population, and mandatory postgraduate general clinical training system are considered to be contributing factors. erkan gunay, ersin aksay, ozge duman atilla, nilay zorbalar, savas sezik tepecik research and training hospital, izmir, turkey background: workplace safety and occupational health problems are increasing issues especially in developing countries as a result of the industrial automatisation and technologic improvements. occupational injuries are preventable but they can occasionally cause morbidity and mortality resulting in work day loss and financial problems. hand injuries are one-third of all traumatic injuries and are the most injured parts after occupational accidents. objectives: we aim to evaluate patients with occupational upper extremity injuries for demographic characteristics, injury types, and work day loss. methods: trauma patients over 15 years old admitted to our emergency department with an occupational upper extremity injury were prospectively evaluated from 15.04.2010 to 30.04.2011. patients with one or more of digit, hand, forearm, elbow, humerus, and shoulder injuries were included. exclusion criteria were multitrauma, patient refusal to participate, and insufficient data. patients were followed up from the hospital information system and by phone for work day loss and final diagnosis. results: during the study period there were 570 patients with an occupational upper extremity injury. total of 521 (91.4%) patients were included. patients were 92.1% male, 36.5% between the age 25 to 34, and mean age was calculated 32.9 ± 9.6 years. 43.8% of the patients were from the metal and machinery sector, and primary education was the highest education level for the 74.7% of the patients. most injured parts were fingers with the highest rate for index finger and thumb. crush injury was the most common injury type. 96.3% (n = 502) of the patients were discharged after treatment in the emergency department. tendon injuries, open fractures, and high degree burns were the reasons for admission to clinics. mean work day loss was 12.8 ± 27.2 days and this increases for the patients with laboratory or radiologic studies, consultant evaluation, or admission. the 15-24 age group had a significantly lower work day loss average. conclusion: evaluating occupational injury characteristics and risks is essential for identifying preventive measures and actions. with the guidance of this study preventive actions focusing on high-risk sectors and patients may be the key factor for avoiding occupational injuries and creating safer workplace environments in order to reduce financial and public health problems. background: as emergency medicine (em) gains increased recognition and interest in the international arena, a growing number of training programs for emergency health care workers have been implemented in the developing world through international partnerships. objectives: to evaluate the quality and appropriateness of an internationally implemented emergency physician training program in india. methods: physicians participating in an internationally implemented em training program in india were recruited to participate in a program evaluation. a mixed methods design was used including an online anonymous survey and semi-structured focus groups. the survey assessed the research, clinical, and didactic training provided by the program. demographics and information on past and future career paths were also collected. the focus group discussions centered around program successes and challenges. results: fifty of 59 eligible trainees (85%) participated in the survey. of the respondents, the vast majority were indian; 16% were female, and all were between the ages of 25 and 45 years (mean age 31 years). all but two trainees (96%) intend to practice em as a career. one-third listed a high-income country first for preferred practice location and half listed india first. respondents directly endorsed the program structure and content, and they demonstrated gains in self-rated knowledge and clinical confidence over their years of training. active challenges identified include: (1) insufficient quantity and inconsistent quality of indian faculty, (2) administrative barriers to academic priorities, and (3) persistent threat of brain drain if local opportunities are inadequate. conclusion: implementing an international emergency physician training program with limited existing local capacity is a challenging endeavor. overall, this evaluation supports the appropriateness and quality of this partnership model for em training. one critical challenge is achieving a robust local faculty. early negotiations are recommended to set educational priorities, which includes assuring access to em journals. attrition of graduated trainees to high-income countries due to better compensation or limited in-country opportunities continues to be a threat to long-term local capacity building. background: with an increasing frequency and intensity of manmade and natural disasters, and a corresponding surge in interest in international emergency medicine (iem) and global health (gh), the number of iem and gh fellowships is constantly growing. there are currently 34 iem and gh fellowships, each with a different curriculum. several articles have proposed the establishment of core curriculum elements for fellowship training. to the best of our knowledge, no study has examined whether iem and gh fellows are actually fulfilling these criteria. objectives: this study sought to examine whether current iem and gh fellowships are consistently meeting these core curricula. methods: an electronic survey was administered to current iem and gh fellowship directors, current fellows, and recent graduates of a total of 34 programs. survey respondents stated their amount of exposure to previously published core curriculum components: em system development, humanitarian assistance, disaster response, and public health. a pooled analysis comparing overall responses of fellows to those of program directors was performed using two-sampled t-test. results: response rates were 88% (n = 30) for program directors and 53% (n = 17) for current and recent fellows. programs varied significantly in terms of their emphasis on and exposure to six proposed core curriculum areas: em system development, em education development, humanitarian aid, public health, ems, and disaster management. only 43% of programs reported having exposure to all four core areas. as many as 67% of fellows reported knowing their curriculum only somewhat or not at all prior to starting the program. conclusion: many fellows enter iem and gh fellowships without a clear sense of what they will get from their training. as each fellowship program has different areas of curriculum emphasis, we propose not to enforce any single core curriculum. rather, we suggest the development of a mechanism to allow each fellowship program to present its curriculum in a more transparent manner. this will allow prospective applicants to have a better understanding of the various programs' curricula and areas of emphasis. background: advance warning of probable intensive care unit (icu) admissions could allow the bed placement process to start earlier, decreasing ed length of stay and relieving overcrowding conditions. however, physicians and nurses poorly predict a patient's ultimate disposition from the emergency department at triage. a computerized algorithm can use commonly collected data at triage to accurately identify those who likely will need icu admission. objectives: to evaluate an automated computer algorithm at triage to predict icu admission and 28-day in-hospital mortality. methods: retrospective cohort study at a 55,000 visit/ year level i trauma center/tertiary academic teaching hospital. all patients presenting to the ed between 12/16/2008 and 10/1/2010 were included in the study. the primary outcome measure was icu admission from the emergency department. the secondary outcome measure was 28-day all-cause in-hospital mortality. patients discharged or transferred before 28 days were considered to be alive at 28 days. triage data includes age, sex, acuity (emergency severity index), blood pressure, heart rate, pain scale, respiratory rate, oxygen saturation, temperature, and a nurse's free text assessment. a latent dirichlet allocation algorithm was used to cluster words in triage nurses' free text assessments into 500 topics. the triage assessment for each patient is then represented as a probability distribution over these 500 topics. logistic regression was then used to determine the prediction function. results: a total of 94,973 patients were included in the study. 3.8% were admitted to the icu and 1.3% died within 28 days. these patients were then randomly allocated to train (n = 75,992; 80%) and test (n = 18,981; 20%) data sets. the area under the receiver operating characteristic curve (auc) when predicting icu background: at the 2011 saem annual meeting, we presented the derivation of two hospital admission prediction models adding coded chief complaint (ccc) data from a published algorithm (thompson et al. acad emerg med 2006; 13:774-782) to demographic, ed operational, and acuity (emergency severity index (esi)) data. objectives: we hypothesized that these models would be validated when applied to a separate retrospective cohort, justifying prospective evaluation. methods: we conducted a retrospective, observational validation cohort study of all adult ed visits to a single tertiary care center (census: 49,000/yr) (4/1/09-12/31/10). we downloaded from the center's clinical tracking system demographic (age, sex, race), ed operational (time and day of arrival), esi, and chief complaint data on each visit. we applied the derived ccc hospital admission prediction models (all identified ccc categories and ccc categories with significant odds of admission from multivariable logistic regression in the derivation cohort) to the validation cohort to predict odds of admission and compared to prediction models that consisted of demographic, ed operational, and esi data, adding each category to subsequent models in a stepwise manner. model performance is reported by areaunder-the-curve (auc) data and 95%ci. signs, pain level, triage level, 72-hour return, number of past visits in the previous year, injury, and one of 122 chief complaint codes (representing 90% of all visits in the database). outputs for training included ordering of a complete blood count, basic chemistry (electrolytes, blood urea nitrogen, creatinine), cardiac enzymes, liver function panel, urinalysis, electrocardiogram, x-ray, computed tomography, or ultrasound. once trained, it was used on the nhamcs-ed 2008 database, and predictions were generated. predictions were compared with documented physician orders. outcomes included the percent of total patients who were correctly pre-ordered, sensitivity (the percent of patients who had an order that were correctly predicted), and the percent over-ordered. waiting time for correctly pre-ordered patients was highlighted, to represent a potential reduction in length of stay achieved by preordering. los for patients overordered was highlighted to see if over-ordering may cause an increase in los for those patients. unit cost of the test was also highlighted, as taken from the 2011 medicare fee schedule. physician times. however, during peak ed census times, many patients with completed tests and treatment initiated by triage await discharge by the next assigned physician. objectives: determine if a physician-led discharge disposition (dd) team can reduce the ed length of stay (los) for patients of similar acuity who are ultimately discharged compared to standard physician team assignment. methods: this prospective observational study was performed from 10/2010 to 10/2011 at an urban tertiary referral academic hospital with an annual ed volume of 87,000 visits. only emergency severity index level 3 patients were evaluated. the dd team was scheduled weekdays from 14:00 until 23:00. several ed beds were allocated to this team. the team was comprised of one attending physician and either one nurse and a tech or two nurses. comparisons were made between los for discharged patients originally triaged to the main ed side who were seen by the dd team versus the main side teams. time from triage physician to team physician, team physician to discharge decision time, and patient age were compared by unpaired t-test. differences were studied for number of patients receiving x-rays, ct scan, labs, and medications. results: dd team mean los in hours for discharged patients was shorter at 3.4 (95% ci: 3.3-3.6, n = 1451) compared to 6.4 (95% ci: 6.3-6.5, n = 4601) on the main side, p < 0.01. the mean time from triage physician to dd team physician was 1.4 hours (95% ci: 1.4-1.5, n = 1447) versus to 2.7 hours (95% ci: 2.7-2.8, n = 4568) to main side physician, p < 0.01. the dd team physician mean time to discharge decision was 1.0 hour (95% ci: 1.0-1.1, n = 1432) compared to 2.5 hours (95% ci: 2.4-2.6, n = 4590) for main side physician, p < 0.01. the dd team patients' mean age was 42.6 years (95% ci: 41.9-43.6, n = 1454) compared to main side patients' mean age of 49.1 years (95% ci: 48.5-49.6, n = 4621.) the dd team patients (n = 1454) received fewer x-rays (40% vs. 59%), ct scans (13% vs. 23%), labs (64% vs. 85%), and medications (63% vs. 68%) than main side patients (n = 4621), p < 0.01 for all compared. conclusion: the dd team complements the advanced triage process to further reduce los for patients who do not require extended ed treatment or observation. the dd team was able to work more efficiently because its patients tended to be younger and had fewer lab and imaging tests ordered by the triage physician compared to patients who were later seen on the ed main side. ed objectives: to evaluate the association between ed boarding time and the risk of developing hapu. methods: we conducted a retrospective cohort study using administrative data from an academic medical center with an adult ed with 55,000 annual patient visits. all patients admitted into the hospital through the ed 6/30/2008-2/28/2011 were included. development of hapu was determined using the standardized, national protocol for cms reporting of hapu. ed boarding time was defined as the time between an order for inpatient admission and transport of the patient out of the ed to an in-patient unit. we used a multivariate logistic regression model with development of a hapu as the outcome variable, ed boarding time as the exposure variable, and the following variables as covariates: age, sex, initial braden score, and admission to an intensive care unit (icu) from the ed. the braden score is a scale used to determine a patient's risk for developing a hapu based on known risk factors. a braden score is calculated for each hospitalized patient at the time of admission. we included braden score as a covariate in our model to determine if ed boarding time was a predictor of hapu independent of braden score. results: of 46,704 patients admitted to the hospital through the ed during the study period, 243 developed a hapu during their hospitalization. clinical characteristics are presented in the table. per hour of ed boarding time, the adjusted or of developing a hapu was 1.02 (95% ci 1.01-1.04, p = 0.007). a median of 40 patients per day were admitted through the ed, accumulating 144 hours of ed boarding time per day, with each hour of boarding time increasing the risk of developing a hapu by 2%. conclusion: in this single-center, retrospective study, longer ed boarding time was associated with increased risk of developing a hapu. queried ed and inpatient nurses and compared their opinions toward inpatient boarding. it also assessed their preferred boarding location if they were patients. objectives: this study queried ed and inpatient nurses and compared their opinions toward inpatient boarding. methods: a survey was administered to a convenience sample of ed and ward nurses. it was performed in a 631-bed academic medical center (30,000 admissions/yr) with a 68-bed ed (60,000 visits/yr). nurses were identified as ed or ward and whether they had previously worked in the ed. the nurses were asked if there were any circumstances where admitted patients should be boarded in the ed or inpatient hallways. they were also asked their preferred location if they were admitted as a patient. six clinical scenarios were then presented and their opinions on boarding queried. results: ninety nurses completed the survey; 35 (39%) were current ed nurses (ced), 40 (44%) had previously worked in the ed (ped). for the entire group 46 (52%) believed admitted patients should board in the ed. overall, 52 (58%) were opposed to inpatient boarding, with 20% of ced versus 83% of current ward (cw) nurses (p < 0.0001) and 28% of ped versus 85% of nurses never having worked in the ed (ned) opposed (p < 0.001). if admitted as patients themselves, overall 43 (54%) preferred inpatient boarding, with 82% of ced versus 33% of cw nurses (p < 0.0001) and 74% of ped versus 34% ned nurses (p = 0.0007) preferring inpatient boarding. for the six clinical scenarios, significant differences in opinion regarding inpatient boarding existed in all but two cases: a patient with stable copd but requiring oxygen and an intubated, unstable sepsis patient. conclusion: ward nurses and those who have never worked in the ed are more opposed to inpatient boarding than ed nurses and nurses who have worked previously in the ed. nurses admitted as patients seemed to prefer not being boarded where they work. ed and ward nurses seemed to agree that unstable or potentially unstable patients should remain in the ed. 8 weeks. staff satisfaction was evaluated through pre/ post-shift and study surveys; administrative data (physician initial assessment (pia), length of stay (los), patients leaving without being seen (lwbs) and against medical advice [lama] ) were collected from an electronic, real-time ed information system. data are presented as proportions and medians with interquartile ranges (iqr); bivariable analyses were performed. results: ed physicians and nurses expected the intervention to reduce the los of discharged patients only. pia decreased during the intervention period (68 vs 74 minutes; p < 0.001). no statistically/clinically significant differences were observed in the los; however, there was a significant reduction in the lwbs (4.7% to 3.5% p = 0.003) and lama (0.7% to 0.4% p = 0.028) rates. while there was a reduction of approximately 5 patients seen per physician in the affected ed area, the total number of patients seen on that unit increased by approximately 10 patients/day. overall, compared to days when there was no extra shift, 61% of emergency physicians stated their workload decreased and 73% felt their stress level at work decreased. conclusion: while this study didn't demonstrate a reduction in the overall los, it did reduce pia times and the proportion of lwbs/lama patients. while physicians saw fewer patients during the intervention study period, the overall patient volume increased and satisfaction among ed physicians was rated higher. provider-and hospital-level variation in admission rates and 72-hour return admission rates jameel abualenain 1 , william frohna 2 , robert shesser 1 , ru ding 1 , mark smith 2 , jesse m. pines 1 1 the george washington university, washington, dc; 2 washington hospital center, washington, dc background: decisions for inpatient versus outpatient management of ed patients are the most important and costliest decision made by emergency physicians, but there is little published on the variation in the decision to admit among providers or whether there is a relationship between a provider's admission rate and the proportion of their patients who return within 72 hours of the initial visit and are subsequently admitted (72h-ra). objectives: we explored the variation in provider-level admission rates and 72h-ra rates, and the relationship between the two. methods: a retrospective study using data from three eds with the same information system over varying time periods: washington hospital center (whc) (2008-10), franklin square hospital center (fshc) , and union memorial hospital (umh) . patients were excluded if left without being seen, left against medical advice, fast-track, psychiatric patients, and aged <18 years. physicians with <500 ed encounters or an admission rate <15% were excluded. logistic regression was used to assess the relationship between physician-level 72h-ra and admission rates, adjusting for patient age, sex, race, and hospital. results: 389,120 ed encounters were treated by 90 physicians. mean patient age was 50 years sd 20, 42% male, and 61% black. admission rates differed between hospitals (whc = 40%, umh = 37%, and fshc = 28%), as did the 72h-ra (whc = 0.9%, umh = 0.6%, and fshc = 0.6%). across all hospitals, there was great variation in individual physician admission rates (15.4%-50.0%). the 72h-ra rates were quite low, but demonstrated a similar magnitude of individual variation (0.3%-1.2%). physicians with the highest admission rate quintile had lower odds of 72h-ra (or 0.8 95% ci 0.7-0.9) compared to the lowest admission rate quintile, after adjusting for other factors. no intermediate admission rate quintiles (2nd, 3rd, or 4th) were significantly different from the lowest admission rate quintile with regard to 72h-ra. conclusion: there is more than three-fold variation in individual physician admission rates indicating great variation among physicians in hospital admission rates and 72h-ra. the highest admitters have the lowest 72h-ra; however, evaluating the causes and consequences of such significant variation needs further exploration, particularly in the context of health reform efforts aimed at reducing costs. background: ed scribes have become an effective means to assist emergency physicians (eps) with clinical documentation and improve physician productivity. scribes have been most often utilized in busy community eds and their utility and functional integration into an academic medical center with resident physicians is unknown. objectives: to evaluate resident perceptions of attending physician teaching and interaction after introduction of scribes at an em residency training program, measured through an online survey. residents in this study were not working with the scribes directly, but were interacting indirectly through attending physician use of scribes during ed shifts. methods: an online ten question survey was administered to 31 residents of a midwest academic emergency medicine residency program (pgy1-pgy3 program, 12 annual residents), 8 months after the introduction of scribes into the ed. scribes were introduced as emr documentation support (epic 2010, epic systems inc.) for attending eps while evaluating primary patients and supervising resident physicians. questions investigated em resident demographics and perceptions of scribes (attending physician interaction and teaching, effect on resident learning, willingness to use scribes in the future), using likert scale responses (1 minimal, 9 maximum) and a graduated percentage scale used to quantify relative values, where applicable. data were analyzed using kruskal-wallis and mann-whitney u tests. results: twenty-one of 31 em residents (68%) completed the survey (81% male; 33% pgy1, 29% pgy2, 38% pgy3). four residents had prior experience with scribes. scribes were felt to have no effect on attending eps direct resident interaction time (mean score 4.5, sd 1.2), time spent bedside teaching (4.8, sd 0.9), or quality of teaching (4.9, sd 0.8), as well as no effect on residents' overall learning process (4.6, sd 1.1). however, residents felt positive about utilizing scribes at their future occupation site (6.0, sd 2.7). no response differences were noted for prior experience, training level, or sex. conclusion: when scribes are introduced at an em residency training site, residents of all training levels perceive it as a neutral interaction, when measured in terms of perceived time with attending eps and quality of the teaching when scribes are present. the effect of introduction of an electronic medical record on resident productivity in an academic emergency department shawn london, christopher sala university of connecticut school of medicine, farmington, ct background: there are little available data which describe the effect of implementation of an electronic medical record (emr) on provider productivity in the emergency department, and no studies which, to our knowledge, address this issue pertaining to housestaff in particular. objectives: we seek to quantify the changes in provider productivity pre-and post-emr implementation to support our hypothesis that resident clinical productivity based on patients seen per hour will be negatively affected by emr implementation. methods: the academic emergency department at hartford hospital, the principle clinical site in the university of connecticut emergency medicine residency, sees over 95,000 patients on an annual basis. this environment is unique in that pre-emr, patient tracking and orders were performed electronically using the sunrise system (eclipsys corp) for over 8 years prior to conversion to the allscripts ed emr in october, 2010 for all aspects of ed care. the investigators completed a random sample of days/evening/night/weekend shift productivity to obtain monthly aggregate productivity data (patients seen per hour) by year of training. results: there was an initial 4.2% decrease of in productivity for pgy-3 residents on average from 1.44 patients per hour on average in the three blocks preceding activation of the emr to 1.38 patients seen per hour compared in the subsequent three prior blocks. pgy 3 performance returned to baseline in the subsequent three months to 1.48 patients per hour. there was no change noted in patients seen per hour of pgy-1 and pgy-2 residents. conclusion: while many physicians tend to assume that emrs pose a significant barrier to productivity in the ed, in our academic emergency department, there was no lasting change on resident productivity based on the patients seen per hour metric. the minor decrease which did occur in pgy-3 residents was transient and was not apparent 3 months after the emr was implemented. our experience suggests that decrease in the rate of patients seen per hour in the resident population should not be considered justification to delay or avoid implementation of an emr in the emergency department. emory university, atlanta, ga; 2 children's healthcare of atlanta, atlanta, ga background: variation in physician practice is widely prevalent and highlights an opportunity for quality improvement and cost containment. monitoring resources used in the management of common pediatric emergency department (ed) conditions has been suggested as an ed quality metric. objectives: to determine if providing ed physicians with severity-adjusted data on resource use and outcomes, relative to their peers, can influence practice patterns. methods: data on resource use by physicians were extracted from electronic medical records at a tertiary pediatric ed for four common conditions in mid-acuity (emergency severity index level 3): fever, head injury, respiratory illness, and gastroenteritis. condition-relevant resource use was tracked for lab tests (blood count, chemistry, crp), imaging (chest x-ray, abdominal x-ray, head ct scan, abdominal ct scan), intravenous fluids, parenteral antibiotics, and intravenous ondansetron. outcome measures included admission to hospital and ed length of stay (los); 72-hr return to ed (rr) was used as a balancing measure. scorecards were constructed using box plots to show physicians their practice patterns relative to peers (the figure shows an example of the scorecard for gatroenteritis for one physician, showing resources use rates for iv fluids and labs). blinded scorecards were distributed quarterly for five quarters using rolling-year averages. a pre/post-intervention analysis was performed with sep 1, 2010 as the intervention date. fisher's exact and wilcoxon rank sum tests were used for analysis. results: we analyzed 45,872 patient visits across two hospitals (24,834 pre-and 21,038 post-intervention), comprising 17.6% of the total ed volume during the study period. patients were seen by 100 physicians (mean 462 patients/physician). the table shows overall physician practice in the pre-and post-intervention periods. significant reduction in resource use was seen for abdominal/pelvic ct scans, head ct scan, chest x-rays, iv ondansetron, and admission to hospital. ed los decreased from 129 min to 126 min (p = 0.0003). there was no significant change in 72-hr return rate during the study period (2.2% pre-, 2.0% post-intervention). conclusion: feedback on comprehensive practice patterns including resource use and quality metrics can influence physician practice on commonly used resources in the ed. billboards, via iphone application, twitter, and text messaging. there is a paucity of data describing the accuracy of publically posted ed wait times. objectives: to examine the accuracy of publicly posted wait times of four emergency departments within one hospital system. methods: a prospective analysis of four ed-posted wait times in comparison to the wait times for actual patients. the main hospital system calculated and posted ed wait times every twenty minutes for all four system eds. a consecutive sample of all patients who arrived 24/7 over a 4-week period during july and august 2011 was included. an electronic tracking system identified patient arrival date and the actual incurred wait time. data consisted of the arrival time, actual wait time, hospital census, budgeted hospital census, and the posted ed wait time. for each ed the difference was calculated between the publicly posted ed wait time at the time of patient's arrival and the patient's actual ed wait time. the average wait times and average wait time error between the ed sites were compared using a two-tailed student's t-test. the correlation coefficient between the differences in predicted/ actual wait times was also calculated for each ed. results: there were 8890 wait times within the four eds included in the analysis. the average wait time (in minutes) at each facility was: 64.0 (±62.4) for the main ed, 22.0 (±22.1) for freestanding ed (fed) #1, 25.0 (±25.6) for fed #2, and 10.0 (±12.6) for the small community ed. the average wait time error (in minutes) for each facility was 31(±61.2) for the main ed, 13 (±23.65) for fed #1, 17 (±26.65) for fed #2, and 1 (±11.9) for the community hospital ed. the results from each ed were statistically significant for both average wait time and average wait time error (p < 0.0001). there was a positive correlation between the average wait time and average wait time error, with r-values of 0.84, 0.83, 0.58, and 0.48 for the main ed, fed #1, fed #2, and the small community hospital ed, respectively. each correlation was statistically significant; however, no correlation was found between the number of beds available (budgeted-actual census) and average wait times. conclusion: publically posted ed wait times are accurate for facilities with less than 2000 ed visits per month. they are not accurate for eds with greater than 4000 visits per month. reduction of pre-analytic laboratory errors in the emergency department using an incentive-based system benjamin katz, daniel pauze, karen moldveen albany medical center, albany, ny background: over the last decade, there has been an increased effort to reduce medical errors of all kinds. laboratory errors have a significant effect on patient care, yet they are usually avoidable. several studies suggest that up to 90% of laboratory errors occur during the pre-or post-analytic phase. in other words, errors occur during specimen collection and transport or reporting of results, rather than during laboratory analysis itself. objectives: in an effort to reduce pre-analytic laboratory errors, the ed instituted an incentive-based program for the clerical staff to recognize and prevent specimen labeling errors from reaching the patient. this study sought to demonstrate the benefit of this incentive-based program. methods: this study examined a prospective cohort of ed patients over a three year period in a tertiary care academic ed with annual census of 72,000. as part of a continuing quality improvement process, laboratory specimen labeling errors are screened by clerical staff by reconciling laboratory specimen label with laboratory requisition labels. the number of ''near-misses'' or mismatched specimens captured by each clerk was then blinded to all patient identifiers and was collated by monthly intervals. due to poor performance in 2009, an incentive program was introduced in early 2010 by which the clerk who captured the most mismatched specimens would be awarded a $50 gift card on a quarterly basis. the total number of missed laboratory errors was then recorded on a monthly basis. investigational data were analyzed using bivariate statistics. background: most studies on operational research have been focused in academic medical centers, which typically have larger volumes of patients and are located in urban metropolitan areas. as cms core measures in 2013 begin to compare emergency departments (eds) on treatment time intervals, especially length of stay (los), it is important to explore if any differences exist inherent to patient volume. objectives: the objective of this study is to look at differences in operational metrics based on annual patient census. the hypothesis is that treatment time intervals and operational metrics differ amongst these different categories. methods: the ed benchmarking alliance has collected yearly operational metrics since 2004. as of 2010, there are 499 eds providing data across the united states. eds are stratified by annual volume for comparison in the following categories: <20k, 20-40k, 40-60k, and over 80k. in this study, metrics for eds with <20k visits per year were compared to those of different volumes, averaged from 2004-2010. mean values were compared to <20k visits as a reference point for statistical difference using t-tests to compare means with a p-value < 0.05 considered significant. results: as seen in the table, a greater percentage of high acuity of patients was seen in higher volume eds than in <20k eds. the percentage of patients transferred to another hospital was higher in <20k eds. a higher percentage arrived by ems and a higher percentage were admitted in higher volume eds when compared to <20k visits. in addition, the median los for both discharged and admitted patients and percentage who left before treatment was complete (lbtc) were higher in the higher volume eds. conclusion: lower volume eds have lower acuity when compared to higher volume eds. lower volume eds have shorter median los and left before treatment complete percentages. as cms core measures require hospitals to report these metrics, it will be important to compare them based on volume and not in aggregate. does the addition of a hands-free communication device improve ed interruption times? amy ernst, steven j. weiss, jeffrey a. reitsema university of new mexico, albuquerque, nm background: ed interruptions occur frequently. recently a hands-free communication device (vocera) was added to a cell phone and a pager in our ed. objectives: the purpose of the present study was to determine whether this addition improved interruption times. our hypothesis was that the device would significantly decrease length of time of interruptions. methods: this study was a prospective cohort study of attending ed physician calls and interruptions in a level i trauma center with em residency. interruptions included phone calls, ekg interpretations, pages to resuscitation, and other miscellaneous interruptions (including nursing issues, laboratory, ems, and radiology). we studied a convenience sampling intended to include mostly evening shifts, the busiest ed times. length of time the interruption lasted was recorded. data were collected for a comparison group pre-vocera. three investigators collected data including seven different addendings' interruptions. data were collected on a form, then entered into an excel file. data collectors' agreement was determined during two additional four hour shifts to calculate a kappa statistic. spss was used for data entry and statistical analysis. descriptive statistics were used for univariate data. chi-square and mann whitney u nonparametric test were used for comparisons. results: of the total 511 interruptions, 33% were phone calls, 24% were ekgs to be read, 18% were pages to resuscitation, and 25% miscellaneous. there were no significant differences in types of interruptions pre-vs. post-vocera. pre-vocera we collected 40 hours of data with 65 interruptions with a mean 1.6 per hour. post-vocera, 180 hours of data were collected with 446 interruptions with a mean 2.5 per hour. there was a significant difference in length of time of interruptions with an average of 9 minutes pre-vocera vs. 4 minutes post-vocera (p = 0.012, diff 4.9, 95% ci 1.8-8.1). vocera calls were significantly shorter than non-vocera calls (1 vs 6 minutes, p < 0.001). comparing data collectors for type of interruption during the same 4-hour shift resulted in a kappa (agreement) of 0.73. conclusion: the addition of a hands-free communication device may improve interruptions by shortening call length. '' talk background: analyses of patient flow through the ed typically focus on metrics such as wait time, total length of stay (los), or boarding time. however, little is known about how much interaction a patient has with clinicians after being placed in a room, or what proportion of the in-room visit is also spent ''waiting,'' rather than directly interacting with care providers. objectives: the objective was to assess the proportion of time, relative to the time in a patient care area, that a patient spends actively interacting with providers during an ed visit. methods: a secondary analysis of 29 audiotaped encounters of patients with one of four diagnoses (ankle sprain, back pain, head injury, laceration) was performed. the setting was an urban, academic ed. ed visits of adult patients were recorded from the time of room placement to discharge. audiotapes were edited to remove all downtime and non-patient-provider conversations. los and door-to-doctor times were abstracted from the medical record. the proportion of time the patient spent in direct conversation with providers (''talk-time'') was calculated as the ratio of the edited audio recording time to the time spent in a patient care area (talk-time = [edited audio time/(los -door-to-doctor)]). multiple linear regression controlling for time spent in patient care area, age, and sex was performed. results: the sample was 31% male with a mean age of 37 years. median los: 133 minutes (iqr: 88-169), median door-to-doctor: 42 minutes (iqr: 29-67), median time spent in patient care area: 65 minutes (iqr: 53-106). median time spent in direct conversation with providers was 16 minutes (iqr: 12-18), corresponding to a talk-time percentage of 19.2% (iqr: 14.7-24.6%). there were no significant differences based on diagnosis. regression analysis showed that those spending a longer time in a patient care area had a lower percentage of talk time (b = )0.11, p = 0.002). conclusion: although limited by sample size, these results indicate that approximately 80% of a patients' time in a care area is spent not interacting with providers. while some of the time spent waiting is out of the providers' control (e.g. awaiting imaging studies), this significant ''downtime'' represents an opportunity for both process improvement efforts to decrease downtime as well as the development of innovative patient education efforts to make the best use of the remaining downtime. degradation of emergency department operational data quality during electronic health record implementation michael j. ward, craig froehle, christopher j. lindsell university of cincinnati, cincinnati, oh background: process improvement initiatives targeted at operational efficiency frequently use electronic timestamps to estimate task and process durations. errors in timestamps hamper the use of electronic data to improve a system and may result in inappropriate conclusions about performance. despite the fact that the number of electronic health record (ehr) implementations is expected to increase in the u.s., the magnitude of this ehr-induced error is not well established. objectives: to estimate the change in the magnitude of error in ed electronic timestamps before and after a hospital-wide ehr implementation. methods: time-and-motion observations were conducted in a suburban ed, annual census 35,000, after receiving irb approval. observation was conducted 4 weeks pre-and 4 weeks post-ehr implementation. patients were identified on entering the ed and tracked until exiting. times were recorded to the nearest second using a calibrated stopwatch, and are reported in minutes. electronic data were extracted from the patient-tracking system in use pre-implementation, and from the ehr post-implementation. for comparison of means, independent t-tests were used. chi-square and fisher's t-tests were used for proportions, as appropriate. results: there were 263 observations; 126 before and 137 after implementation. the differences between observed times and timestamps were computed and found to be normally distributed. post-implementation, mean physician seen times along with arrival to bed, bed to physician, and physician to disposition intervals occurred before observation. physician seen timestamps were frequently incorrect and did not improve postimplementation. significant discrepancies (ten minutes or greater) from observed values were identified in timestamps involving disposition decision and exit from the ed. calculating service time intervals resulted in every service interval (except arrival to bed) having at least 15% of the times with significant discrepancies. it is notable that missing values were more frequent post-ehr implementation. conclusion: ehr implementation results in reduced variability of timestamps but reduced accuracy and an increase in missing timestamps. using electronic timestamps for operational efficiency assessment should recognize the magnitude of error, and the compounding of error, when computing service times. background: procedural sedation and analgesia is used in the ed in order to efficiently and humanely perform necessary painful procedures. the opposing physiological effects of ketamine and propofol suggest the potential for synergy, and this has led to interest in their combined use, commonly termed ''ketofol'', to facilitate ed procedural sedation. objectives: to determine if a 1:1 mixture of ketamine and propofol (ketofol) for ed procedural sedation results in a 13% or more absolute reduction in adverse respiratory events compared to propofol alone. methods: participants were randomized to receive either ketofol or propofol in a double-blind fashion according to a weight-based dosing protocol. inclusion criteria were age 14 years or greater, and asa class 1-3 status. the primary outcome was the number and proportion of patients experiencing an adverse respiratory event according to pre-defined criteria (the ''quebec criteria''). secondary outcomes were sedation consistency, sedation efficacy, induction time, sedation time, procedure time, and adverse events. results: a total of 284 patients were enrolled, 142 per group. forty-three (30%) patients experienced an adverse respiratory event in the ketofol group compared to 46 (32%) in the propofol group (difference 2%; 95% ci )9% to 13%; p = 0.798). thirty-eight (27%) patients receiving ketofol and 36 (25%) receiving propofol developed hypoxia, of whom three (2%) ketofol patients and 1 (1%) propofol patient received bag-valve-mask ventilation. sixty-five (46%) patients receiving ketofol and 93 (65%) receiving propofol required repeat medication dosing or lightened to a ramsay sedation score of 4 or less during their procedure (difference 19%; 95% ci 8% to 31%; p = 0.001). procedural agitation occurred in 5 patients (3.5%) receiving ketofol compared to 15 (11%) receiving propofol (difference 7.5%, 95% ci 1% to 14%). recovery agitation requiring treatment occurred in six patients (4%, 95% ci 2.0% to 8.9%) receiving ketofol. other secondary outcomes were similar between the groups. patients and staff were highly satisfied with both agents. conclusion: ketofol for ed procedural sedation does not result in a reduced incidence of adverse respiratory events compared to propofol alone. induction time, efficacy, and sedation time were similar; however, sedation depth appeared to be more consistent with ketofol. with propofol and its safety is well established. however, in 2010 cms enacted guidelines defining propofol as deep sedation and requiring administration by a physician. common edps practice had been one physician performing both the sedation and procedure. edps has proven safe under this one-physician practice. however, the 2010 guidelines mandated separate physicians perform each. objectives: the study hypothesis was that one-physician propofol sedation complication rates are similar to two-physician. methods: before and after, observational study of patients >17 years of age consenting to edps with propofol. edps completed with one physician were compared to those completed with two (separate physicians performing the sedation and the procedure). all data were prospectively collected. the study was completed at an urban level i trauma center. standard monitoring and procedures for edps were followed with physicians blinded to the objectives of this research. the frequency and incremental dosing of medication was left to the discretion of the treating physicians. the study protocol required an ed nurse trained in data collection to be present to record vital signs and assess for any prospectively defined complications. we used chi-square tests to compare the binary outcomes and asa scores across the time periods, and two-sample t-tests to test for differences in age between the two time periods. results: during the 2-year study period we enrolled 481 patients: 252 one-physician edps sedations and 229 3 (-7 to 13) also received bag-valve-mask 3 (2) [0.7 to 6) 1 (1) [0.1 to 4] 1 (-2 to 5) two-physician. all patients meeting inclusion criteria were included in the study. total adverse event rates were 4.4% and 3.1%, respectively (p = 0.450). the most common complications were hypotension and oxygen desaturation, and they respectively showed one-physcian rates of 2.0% and 0.8% and two-physician rates of 1.8% and 0.9% (p = 0.848 and 0.923.) the unsuccessful procedure rates were 4.0% vs 3.9% (p = 0.983). conclusion: this study demonstrated no significant difference in complication rates for propofol edps completed by one physician as compared to two. background: overdose patients are often monitored using pulse oximetry, which may not detect changes in patients on high-flow oxygen. objectives: to determine whether changes in end-tidal carbon dioxide (etco 2 ) detected by capnographic monitoring are associated with clinical interventions due to respiratory depression (crd) in patients undergoing evaluation for a decreased level of consciousness after a presumed drug overdose. methods: this was a prospective, observational study of adult patients undergoing evaluation for a drug overdose in an urban county ed. all patients received supplemental oxygen. patients were continuously monitored by trained research associates. the level of consciousness was recorded using the observer's assessment of alertness/sedation scale (oaa/s). vital signs, pulse oximetry, and oaa/s were monitored and recorded every 15 minutes and at the time of occurrence of any crd. respiratory rate and etco 2 were measured at five second intervals using a capno-stream20 monitor. crd included an increase in supplemental oxygen, the use of bag-valve-mask ventilations, repositioning to improve ventilation, and physical or verbal stimulus to induce respiration, and were performed at the discretion of the treating physicians and nurses. changes from baseline in etco 2 values and waveforms among patients who did or did have a clinical intervention were compared using wilcoxon rank sum tests. results: 100 patients were enrolled in the study (age 35, range 18 to 67, 62% male, median oaas 4, range 1 to 5). suspected overdoses were due to opioids in 34, benzodiazepines in 14, an antipsychotic in 14, and others in 38. the median time of evaluation was 165 minutes (range 20 to 725). crd occurred in 47% of patients, including an increase in o 2 in 38%, repositioning in 14%, and stimulation to induce respiration in 23%. 16% had an o 2 saturation of <93% (median 88, range 73 to 92) and 8% had a loss of etco 2 waveform at some time, all of whom had a crd. the median change in etco 2 from baseline was 5 mmhg, range 1 to 30. among patients with crd it was 14 mmhg, range 10 to 30, and among patients with no crd it was 5 mmhg, range 1 to 13 (p = 0.03). conclusion: the change in etco 2 from baseline was larger in patients who required clinical interventions than in those who did not. in patients on high-flow oxygen, capnographic monitoring may be sensitive to the need for airway support. how reliable are health care providers in reporting changes in etco 2 waveform anas sawas 1 , scott youngquist 1 , troy madsen 1 , matthew ahern 1 , camille broadwater-hollifield 1 , andrew syndergaard 1 , jared phelps 2 , bryson garbett 1 , virgil davis 1 1 university of utah, salt lake city, ut; 2 midwestern university, glendale, az background: etco 2 changes have been used in procedural sedation analgesia (psa) research to evaluate subclinical respiratory depression associated with sedation regiments. objectives: to evaluate the accuracy of bedside clinician reporting of changes in etco 2 . methods: this was a prospective, randomized, singleblind study conducted in ed setting from june 2010 until the present time. this study took place at an academic adult ed of a 405-bed (21 in the ed) and a level i trauma center. subjects were randomized to receive either ketamine-propofol or propofol according to a standardized protocol. loss of etco 2 waveforms for ‡ 15 sec were recorded. following sedation, questionnaires were completed by the sedating physicians. digitally recorded etco 2 waveforms were also reviewed by an independent physician and a trained research assistant (ra). to ensure the reliability of trained research assistants, we compared their analyses with the analyses of an independent physician for the first 41 recordings. the target enrollment was 65 patients in each group (n = 130 total). statistics were calculated using sas statistical software. results: 91 patients were enrolled; 53 (58.2%) are males and 38 (41.8%) are females. mean age was 44.93 ± 17.93 years. most participants did not have major risk factors for apnea or for further complications (86.3% were asa class 1 or 2). etco 2 waveforms were reviewed by 87 (95.6%) sedating physicians and 84 (92.3%) nurses at the bedside. there were 70 (76.9%) etco 2 waveforms recordings, 42 (60.0%) were reviewed by an independent physician and 70 (100%) were reviewed by an ra. a kappa test for agreement between independent physicians and ras was conducted on 41 recordings and there were no discordant pairs (kappa = 1). compared to sedating physicians, the independent physician was more likely to report etco 2 wave losses (or 1.37, 95% ci 1.08-1.73). compared to sedating physicians, ras were more likely to report etco 2 wave losses (or 1.39, 95% ci 1.14-1.70). conclusion: compared to sedating physicians at the bedside, independent physicians and ras were more likely to note etco 2 waveform losses. an independent review of recorded etco 2 waveform changes will be more reliable for future sedation research. background: comprehensive studies evaluating current practices of ed airway management in japan are lacking. many emergency physicians in japan still experience resistance regarding rapid sequence intubation (rsi). objectives: we sought to describe the success and complication rate of rsi with non-rsi. methods: design and setting: we conducted a multicenter prospective observational study using the jean registry of eds at 11 academic and community hospitals in japan during between 2010 and 2011. data fields include ed characteristics, patient and operator demographics, method of airway management, number of attempts, and adverse events. we defined non-rsi as intubation with sedation only, neuromuscular blockade only, and without medication. participants: all patients undergoing emergency intubation in ed were eligible for inclusion. cardiac arrest encounters were excluded from the analysis. primary analysis: we described rsi with non-rsi in terms of success rate on first attempt, within three attempts, and complication rate. we present descriptive data as proportions with 95% confidence intervals (cis). we report odds ratios (or) with 95% ci via chi-square testing. results: the database recorded 2710 intubations (capture rate 98%) and 1670 met the inclusion criteria. rsi was the initial method chosen in 489 (29%) and non-rsi in 1181 (71%). use of rsi varied among institutes from 0% to 79%. success cases of rsi on first and within three attempts are 353 intubations (72%, 95%ci 68%-76%) and 474 intubations (97%, 95%ci 95%-98%), respectively. the success cases of non-rsi on first and within three attempts are 724 intubations (61%, 95%ci 58%-64%) and 1105 intubations (94%, 95%ci 92%-95%). success rates of rsi on first and within three attempts are higher than non-rsi (or 1.64, 95%ci 1.30-2.06 and or 2.14, 95% ci 1.22-3.77, respectively). we recorded 67 complications in rsi (14%) and 165 in non-rsi (14%). there is no significant difference of complication rate between rsi and non-rsi (or 0.98, 95% ci 0.72-1.32). conclusion: in this multi-center prospective study in japan, we demonstrated a high degree of variation in use of rsi for ed intubation. additionally we found that success rate of rsi on first and within three attempts were both higher than non-rsi. this study has the limitation of reporting bias and confounding by indication. (originally submitted as a ''late-breaker.'') methods: this was a prospective, randomized, singleblind study conducted in the ed setting from june 2010 until the present time. this study took place at an academic adult ed of a 405-bed (21 in the ed) and a level i trauma center. subjects were randomized to receive either ketamine-propofol or propofol according to a standardized protocol. etco 2 waveforms were digitally recorded. etco 2 changes were evaluated by the sedating physicians at the bedside. recorded waveforms were reviewed by an independent physician and a trained research assistant (ra). to ensure the reliability of trained ras, we computed a kappa test for agreement between the analysis of independent physicians and ras for the first 41 recordings. a post-hoc analysis of the association between any loss, the number of losses, and total duration of loss of etco 2 waveform and crp was performed. on review we recorded the absence or presence of loss of etco 2 and the total duration in seconds of all lost etco 2 episodes ‡15 seconds. ors were calculated using sas statistical software. results: 91 patients were enrolled; 53 (58.2%) are males and 38 are (41.8%) females. 86.3% participants were asa class 1 or 2. waveforms were reviewed by 87 (95.6%) sedating physicians. there were 70 (76.9%) waveforms recordings, 42 (60.0%) were reviewed by an independent physician and 70 (100%) were reviewed by ras, where there were no discordant pairs (kappa = 1). there were 24 (26.4%) crp events. any loss of etco 2 was associated with a non-significant or of 4.06 (95% ci 0.75-21.9) for crp. however, the duration of etco 2 loss was significantly associated with crp with an or of 1.38 (95% ci 1.08-1.76) for each 30 second interval of lost etco 2 . the number of losses was significantly associated with the outcome (or 1.48, 95% ci 1.15-1.91). conclusion: defining subclinical respiratory depression as present or absent may be less useful than quantitative measurements. this suggests that risk is cumulative over periods of loss of etco 2 , and the duration of loss may be a better marker of sedation depth and risk of complications than classification of any loss. background: ed visits present an opportunity to deliver brief interventions (bis) to reduce violence and alcohol misuse among urban adolescents at risk for future injury. previous analyses demonstrated that a brief intervention resulted in reductions in violence and alcohol consequences up to 6 months. objectives: this paper describes findings examining the efficacy of bis on peer violence and alcohol misuse at 12 months. methods: patients (14-18 yrs) at an ed reporting past year alcohol use and aggression were enrolled in the rct, which included computerized assessment, and randomization to control group or bi delivered by a computer (cbi) or therapist assisted by a computer (tbi). baseline and 12 months included violence (peer aggression, peer victimization, violence related consequences) and alcohol (alcohol misuse, binge drinking, alcohol-related consequences). results: 3338 adolescents were screened (88% participation). of those, 726 screened positive for violence and alcohol use and were randomized; 84% completed 12-month follow-up. as compared to the control group, the tbi group showed significant reductions in peer aggression (p < 0.01) and peer victimization (p < 0.05) at 12 months. bi and control groups did not differ on alcohol-related variables at 12 months. conclusion: evaluation of the saferteens intervention one year following an ed visit provides support for the efficacy of computer-assisted therapist brief intervention for reducing peer violence. violence against ed health care workers: a 9-month experience terry kowalenko 1 , donna gates 2 , gordon gillespie 2 , paul succop 2 1 university of michigan, ann arbor, mi; 2 university of cincinnati, cincinnati, oh background: health care (hc) support occupations have an injury rate nearly 10 times that of the general sector due to assaults, with doctors and nurses nearly 3 times greater. studies have shown that the ed is at greatest risk of such events compared to other hc settings. objectives: to describe the incidence of violence in ed hc workers over 9 months. specific aims were to 1) identify demographic, occupational, and perpetrator factors related to violent events; 2) identify the predictors of acute stress response in victims; and 3) identify predictors of loss of productivity after the event. methods: longitudinal, repeated methods design was used to collect monthly survey data from ed hc workers (w) at six hospitals in two states. surveys assessed the number and type of violent events, and feelings of safety and confidence. victims also completed specific violent event surveys. descriptive statistics and a repeated measure linear regression model were used. results: 213 ed hcws completed 1795 monthly surveys, and 827 violent events were reported. the average per person violent event rate per 9 months was 4.15. 601 events were physical threats (3.01 per person in 9 months). 226 events were assaults (1.13 per person in 9 months). 501 violent event surveys were completed, describing 341 physical threats and 160 assaults with 20% resulting in injuries. 63% of the physical threats and 52% of the assaults were perpetrated by men. comparing occupational groups revealed significant differences between nurses and physicians for all reported events (p = 0.0048), with the greatest difference in physical threats (p = 0.0447). nurses felt less safe than physicians (p = 0.0041). physicians felt more confident than nurses in dealing with the violent patient (p = 0.013). nurses were more likely to experience acute stress than physicians (p < 0.001). acute stress significantly reduced productivity in general (p < 0.001), with a significant negative effect on ''ability to handle/ manage workload'' (p < 0.001) and ''ability to handle/ manage cognitive demands'' (p < 0.05). conclusion: ed hcws are frequent victims of violence perpetrated by visitors and patients. this violence results in injuries, acute stress, and loss of productivity. acute stress has negative consequences on the workers' ability to perform their duties. this has serious potential consequences to the victim as well as the care they provide to their patients. a randomized controlled feasibility trial of vacant lot greening to reduce crime and increase perceptions of safety eugenia c. garvin, charles c. branas perelman school of medicine at the university of pennsylvania, philadelphia, pa background: vacant lots, often filled with trash and overgrown vegetation, have been associated with intentional injuries. a recent quasi-experimental study found a significant decrease in gun crimes around vacant lots that had been greened compared with control lots. objectives: to determine the feasibility of a randomized vacant lot greening intervention, and its effect on police-reported crime and perceptions of safety. methods: for this randomized controlled feasibility trial of vacant lot greening, we partnered with the pennsylvania horticulture society (phs) to perform the greening intervention (cleaning the lots, planting grass and trees, and building a wooden fence around the perimeter). we analyzed police crime data and interviewed people living around the study vacant lots (greened and control) about perceptions of safety before and after greening. results: a total of 5200 sq ft of randomly selected vacant lot space was successfully greened. we used a master database of 54,132 vacant lots to randomly select 50 vacant lot clusters. we viewed each cluster with the phs to determine which were appropriate to send to the city of philadelphia for greening approval. the vacant lot cluster highest on the random list to be approved by the city of philadelphia was designated the intervention site, and the next highest was designated the control site. overall, 29 participants completed baseline interviews, and 21 completed follow-up interviews after 3 months. 59% of participants were male, 97% were black or african american, and 52% had a household income less than $25,000. unadjusted difference-in-differences estimates showed a decrease in gun assaults around greened vacant lots compared to control. regression-adjusted estimates showed that people living around greened vacant lots reported feeling safer after greening compared to those who lived around control vacant lots (p < 0.01). conclusion: conducting a randomized controlled trial of vacant lot greening is feasible. greening may reduce certain gun crimes and make people feel safer. however, larger prospective trials are needed to further investigate this link. screening for violence identifies young adults at risk for return ed visits for injury abigail hankin-wei, brittany meagley, debra houry emory university, atlanta, ga background: homicide is the second leading cause of death among youth ages 15-24. prior studies, in nonhealth care settings, have shown associations between violent injury and risk factors including exposure to community violence, peer behavior, and delinquency. objectives: to assess whether self-reported exposure to violence risk factors can be used to predict future ed visits for injuries. methods: we conducted a prospective cohort study in the ed of a southeastern us level i trauma center. patients aged 15-24 presenting for any chief complaint were included unless they were critically ill, incarcerated, or could not read english. recruitment took place over six months, by a trained research assistant (ra). the ra was present in the ed for 3-5 days per week, with shifts scheduled such that they included weekends and weekdays, over the hours from 8 am-8 pm. patients were offered a $5 gift card for participation. at the time of initial contact in the ed, patients completed a written questionnaire which included validated measures of the following risk factors: a) aggression, b) perceived likelihood of violence, c) recent violent behavior, d) peer behavior, e) community exposure to violence, and f) positive future outlook. at 12 months following the initial ed visit, the participants' medical records were reviewed to identify any subsequent ed visits for injury-related complaints. data were analyzed with chi-square and logistic regression analyses. results: 332 patients were approached, of whom 300 patients consented. participants' average age was 21.1 years, with 57% female, and 86% african american. return visits for injuries were significantly associated with hostile/aggressive feelings (rr 3.7, ci 1.42, 9) , self-reported perceived likelihood of violence (rr 5.16, ci 1.93, 13.78) , recent violent behavior (rr 3.16, ci 1.01, 9.88) , and peer group violence (rr 4.4, ci 1.72, 11.25) . these findings remained significant when controlling for participant sex. conclusion: a brief survey of risk factors for violence is predictive of return visit to the ed for injury. these findings identify a potentially important tool for primary prevention of violent injuries among young adults visiting the ed for both injury and non-injury complaints. background: sepsis is a commonly encountered disease in ed, with high mortality. while several clinical prediction rules (cpr) including meds, sirs, and curb-65 exist to facilitate clinicians in early recognition of risk of mortality for sepsis, most are of suboptimal performance. objectives: to derive a novel cpr for mortality of sepsis utilizing clinically available and objective predictors in ed. methods: we retrospectively reviewed all adult septic patients who visited the ed at a tertiary hospital during the year 2010 with two sets of blood cultures ordered by physicians. basic demographics, ed vital signs, symptoms and signs, underlying illnesses, laboratory findings, microbiological results, and discharge status were collected. multivariate logistic regressions were used to obtain a novel cpr using predictors with <0.1 p-value tested in univariate analyses. the existing cprs were compared with this novel cpr using auc. results: of 8699 included patients, 7.6% died in hospital, 51% had diabetes, 49% were older than 65 years of age, 21% had malignancy, and 16% had positive blood bacterial culture tests. predisposing factors including history of malignancy, liver disease, immunosuppressed status, chronic kidney disease, congestive heart failure, and older than 65 years of age were found to be associated with mortality (all p < 0.05). patients who developed mortality tended to have lower body temperature, narrower pulse pressure, higher percentage of red cell distribution width (rdw) and bandemia, higher blood urea nitrogen (bun), ammonia, and c-reactive protein level, and longer prothrombin time and activated partial thromboplastin time (aptt) (all p < 0.05). the most parsimonious cpr incorporating history of malignancy (or 2.3, 95% ci 1.9-2.7), prolonged aptt (3.0, 2.4-3.8), presence of bandemia (1.7, 1.4-2.0 results: there was poor agreement between the physician's unstructured assessment used in clinical practice and the guidelines put forth by the aha/acc/acep task force. ed physicians were more likely to assess a patient as low risk (42%), while aha guidelines were more likely to classify patients as intermediate (50%) or high (40%) risk. however, when comparing the patient's final acs diagnosis and the relation to the risk assessment value, ed physicians proved better predictors of high-risk patients who in fact had acs, while the aha/acc/acep guidelines proved better at correctly identifying low-risk patients who did not have acs. conclusion: in the ed, physicians are far more efficient at correctly placing patients with underlying acs into a high-risk category, while established criteria may be overly conservative when applied to an acute care population. further research is indicated to look at ed physicians' risk stratification and ensuing patient care to assess for appropriate decision making and ultimate outcomes. compartative conclusion: the amuse score was more specific, but the wells score was more sensitive for acute lower limb dvt in this cohort. there is no significant advantage in using the amuse over the wells score in ed patient with suspected dvt. background: the direct cost of medical care is not accurately reflected in charges or reimbursement. the cost of boarding admitted patients in the ed has been studied in terms of opportunity costs, which are indirect. the actual direct effect on hospital expenses has not been well defined. objectives: we calculate the difference to the hospital in the cost of caring for an admitted patient in the ed and in a non-critical care in-patient unit. methods: time-directed activity-based costing (tdabc) has recently been proposed as a method of determining the actual cost of providing medical services. tdabc was used to calculate the cost per patient bed-hour both in the ed and for an in-patient unit. the costs include nursing, nursing assistants, clerks, attending and resident physicians, supervisory salaries, and equipment maintenance. boarding hours were determined from placement of admission order to transfer to in-patient unit. a convenience sample of 100 consecutive non-critical care admissions was assessed to find the degree of ed physician involvement with boarded patients. results: the overhead cost per patient bed-hour in the ed was $60.80. the equivalent cost per bed-hour inpatient was $23.39, a differential of $37.41. there were 27,618 boarding hours for medical-surgical patients in 2010, a differential of $1,033,189.38 for the year. for the short-stay unit (no residents), the cost per patient hour was $11.36 and the boarding hours were 11,804. this resulted in a differential cost of $583,389.76, a total direct cost to the hospital of $1,616,579.14. review of 100 consecutive admissions showed no orders placed by the ed physician after decision-toadmit. conclusion: concentration of resources in the ed means considerably higher cost per unit of care as compared to an in-patient unit. keeping admitted patients boarding in the ed results in expensive underutilization. this is exclusive of significant opportunity costs of lost revenue from walk-out and diverted patients. this study includes the cost of teaching attendings and residents (ed and in-patient) . in a non-teaching setting, the differential would be less and the cost of boarding would be shared by a fee-for-service ed physician group as well as the hospital. improving identification of frequent emergency department users using a regional health information background: frequent ed users consume a disproportionate amount of health care resources. interventions are being designed to identify such patients and direct them to more appropriate treatment settings. because some frequent users visit more than one ed, a health information exchange (hie) may improve the ability to identify frequent ed users across sites of care. objectives: to demonstrate the extent to which a hie can identify the marginal increase in frequent ed users beyond that which can be detected with data from a single hospital. methods: data from 6/1/10 to 5/31/11 from the new york clinical information exchange (nyclix), a hie in new york city that includes ten hospitals, were analyzed to calculate the number of frequent ed users ( ‡4 visits in 30 days) at each site and across the hie. results: there were 10,555 (1% of total patients) frequent ed users, with 7,518 (71%) of frequent users having all their visits at a single ed, while 3,037 (29%) frequent users were identified only after counting visits to multiple eds (table 1) . site-specific increases varied from 7% to 62% (sd 16.5). frequent ed users accounted for 1% of patients, but for 6% of visits, averaging 9.74 visits per year, versus 1.55 visits per year for all other patients. 28.5% of frequent users visited two or more eds during the study period, compared to 10.6% of all other patients. conclusion: frequent ed users commonly visited multiple nyclix eds during the study period. the use of a hie helped identify many additional frequent users, though the benefits were lower for hospitals not located in the relative vicinity of another nyclix hospital. measures that take a community, rather than a single institution, into account may be more reflective of the care that the patient experiences. indocyanine background: due to their complex nature and high associated morbidity, burn injuries must be handled quickly and efficiently. partial thickness burns are currently treated based upon visual judgment of burn depth by the clinician. however, such judgment is only 67% accurate and not expeditious. laser doppler imaging (ldi) is far more accurate -nearly 96% after 3 days. however, it is too cumbersome for routine clinical use. laser assisted indocyanine green angiography (laicga) has been indicated as an alternative for diagnosing the depth of burn injuries, and possesses greater utility for clinical translation. as the preferred outcome of burn healing is aesthetic, it is of interest to determine if wound contracture can be predicted early in the course of a burn by laic-ga. objectives: determine the utility of early burn analysis using laicga in the prediction of 28-day wound contracture. methods: a prospective animal experiment was performed using six anesthetized pigs, each with 20 standardized wounds. differences in burn depth were created by using a 2.5 · 2.5 cm aluminum bar at three exposure times and temperatures: 70 degrees c for 30 seconds, 80 degrees c for 20 seconds, and 80 degrees c for 30 seconds. we have shown in prior validation experiments that these burn temperatures and times create distinct burn depths. laicga scanning, using lifecell spy elite, took place at 1 hour, 24 hours, 48 hours, 72 hours, and 1 week post burn. imaging was read by a blinded investigator, and perfusion trends were compared with day 28 post-burn contraction outcomes measured using imagej software. biopsies were taken on day 28 to measure scar tissue depth. results: deep burns were characterized by a blue center indicating poor perfusion while more superficial burns were characterized by a yellow-red center indicating perfusion that was close to that of the normal uninjured adjacent skin (see figure) . a linear relationship between contraction outcome and burn perfusion could be discerned as early as 1 hour post burn, peaking in strength at 24-48 hours post-burn. burn intensity could be effectively identified at 24 hours post-burn, although there was no relationship with scar tissue depth. conclusion: pilot data indicate that laicga using lifecell spy has the ability to determine the depth of injury and predict the degree of contraction of deep dermal burns within 1-2 days of injury with greater accuracy than clinical scoring. the objectives: we hypothesize that real-time monitoring of an integrated electronic medical records system and the subsequent firing of a ''sepsis alert'' icon on the electronic ed tracking board results in improved mortality for patients who present to the ed with severe sepsis or septic shock. methods: we retrospectively reviewed our hospital's sepsis registry and included all patients diagnosed with severe sepsis or septic shock presenting to an academic community ed with an annual census of 73,000 visits and who were admitted to a medical icu or stepdown icu bed between june 2009 and october 2011. in may 2010 an algorithm was added to our integrated medical records system that identifies patients with two sirs criteria and evidence of endorgan damage or shock on lab data. when these criteria are met, a ''sepsis alert'' icon (prompt) appears next to that patient's name on the ed tracking board. the system also pages an in-house, specially trained icu nurse who can respond on a prn basis and assist in the patient's management. 18 months of intervention data are compared with 11 months of baseline data. statistical analysis was via z-test for proportions. results: for ed patients with severe sepsis, the preand post-alert mortality was 19 of 125 (15%) and 34 of 378 (9%), respectively (p = 0.084; n = 503). in the septic shock group, the pre-and post-alert mortality was 27 of 92 (29%) and 48 of 172 (28%), respectively (p = 0.977). with ed and inpatient sepsis alerts combined, the severe sepsis subgroup mortality was reduced from 17% to 9% (p = 0.013; n = 622). conclusion: real-time ed ehr screening for severe sepsis and septic shock patients did not improve mortality. a positive trend in the severe sepsis subgroup was noted, and the combined inpatient plus ed data suggests statistical significance may be reached as more patients enter the registry. limitations: retrospective study, potential increased data capture post intervention, and no ''gold standard'' to test the sepsis alert sensitivity and specificity. ) . descriptive statistics were calculated. principal component analysis was used to determine questions with continuous response formats that could be aggregated. aggregated outcomes were regressed onto predictor demographic variables using multiple linear regression. results: 80/100 physicians completed the survey. physicians had a mean of 9.8 ± 9.0 years experience in the ed. 23.8% were female. eight physicians (10%) reported never having used the tool, while 70.8% of users estimated having used it more than five times. 75% of users cited the ''p'' alert on the etb as the most common notification method. most felt the ''p'' alert did not help them identify patients with pneumonia earlier (mean = 2.5 ± 1.2), but found it moderately useful in reminding them to use the tool (3.5 ± 1.3). physicians found the tool helpful in making decisions regarding triage, diagnostic studies, and antibiotic selection for outpatients and inpatients (3.7 ± 1.0, 3.6 ± 1.1, 3.6 ± 1.1, and 4.2 ± 0.9, respectively). they did not feel it negatively affected their ability to perform other tasks (1.6 ± 0.9). using multiple linear regression, neither age, sex, years experience, nor tool use frequency significantly predicted responses to questions about triage and antibiotic selection, technical difficulties, or diagnostic ordering. conclusion: ed physicians perceived the tool to be helpful in managing patients with pneumonia without negatively affecting workflow. perceptions appear consistent across demographic variables and experience. objectives: we seek to examine whether use of the salt device can provide reliable tracheal intubation during ongoing cpr. the dynamic model tested the device with human powered cpr (manual) and with an automated chest compression device (physio control lucas 2). the hypothesis is that the predictable movement of an automated chest compression device will make tracheal intubation easier than the random movement from manual cpr. methods: the project was an experimental controlled trial and took place in the ed at a tertiary referral center in peoria, illinois. this project was an expansion arm of a similarly structured study using traditional laryngoscopy. emergency medicine residents, attending physicians, paramedics, and other acls-trained staff were eligible for participation. in randomized order, each participant attempted intubation on a mannequin using the salt device with no cpr ongoing, during cpr with a manual compression, and during cpr with an automatic chest compression. participants were timed in their attempt and success was determined after each attempt. results: there were 43 participants in the trial. the success rates in the control group and the automated cpr group were both 86% (37/43) and the success rate in the manual cpr group was 79% (34/43 objectives: our primary hypothesis was that in fasting, asymptomatic subjects, larger fluid boluses would lead to proportional aortic velocity changes. our secondary endpoints were to determine inter-and intra-subject variation in aortic velocity measurements. methods: the authors performed a prospective randomized double-blinded trial using healthy volunteers. we measured the velocity time integral (vti) and maximal velocity (vmax) with an estimated 0-20°pulsed wave doppler interrogation of the left ventricular outflow in the apical-5 cardiac window. three physicians reviewed optimal sampling gate position, doppler angle and verified the presence of an aortic closure spike. angle correction technology was not used. subjects with no history of cardiac disease or hypertension fasted for 12 hours and were then randomly assigned to receive a normal saline bolus of 2 ml/kg, 10 ml/kg or 30 ml/kg over 30 minutes. aortic velocity profiles were measured before and after each fluid bolus. results: forty-two subjects were enrolled. mean age was 33 ± 10 (range 24 to 61) and mean body mass index 24.7 ± 3.2 (range 18.7 to 32). mean volume (in ml) for groups receiving 2 ml/kg, 10 ml/kg, and 30 ml/kg were 151, 748, and 2162, respectively. mean baseline vmax (in cm/s) of the 42 subjects was 108.4 ± 12.5 (range 87 to 133). mean baseline vti (in cm) was 23.2 ± 2.8 (range 18.2 to 30.0). pre-and post-fluid mean differences for vmax were )1.7 (± 10.3) and for vti 0.7 (± 2.7). aortic velocity changes in groups receiving 2 ml/kg, 10 ml/kg, and 30 ml/kg were not statistically significant (see table) . heart rate changes were not significant. background: clinicians recognize that septic shock is a highly prevalent, high mortality disease state. evidence supports early ed resuscitation, yet care delivery is often inconsistent and incomplete. the objective of this study was to discover latent critical barriers to successful ed resuscitation of septic shock. objectives: clinicians recognize that septic shock is a highly prevalent, high mortality disease state. evidence supports early ed resuscitation, yet care delivery is often inconsistent and incomplete. the objective of this study was to discover latent critical barriers to successful ed resuscitation of septic shock. methods: we conducted five 90-minute risk-informed in-situ simulations. ed physicians and nurses working in the real clinical environment cared for a standardized patient, introduced into their existing patient workload, with signs and symptoms of septic shock. immediately after case completion clinicians participated in a 30minute debriefing session. transcripts of these sessions were analyzed using grounded theory, a method of qualitative analysis, to identify critical barrier themes. results: fifteen clinicians participated in the debriefing sessions: four attending physicians, five residents, five nurses, and one nurse practitioner. the most prevalent critical barrier themes were: anchoring bias and difficulty with cognitive framework adaptation as the patient progressed to septic shock (n = 26), difficult interactions between the ed and ancillary departments (n = 22), difficulties with physician-nurse commu-nication and teamwork (n = 18), and delays in placing the central venous catheter due to perceptions surrounding equipment availability and the desire to attend to other competing interests in the ed prior to initiation of the procedure (n = 17 and 14). each theme was represented in at least four of the five debriefing sessions. participants reported the in-situ simulations to be a realistic representation of ed sepsis care. conclusion: in-situ simulation and subsequent debriefing provides a method of identifying latent critical areas for improvement in a care process. improvement strategies for ed-based septic shock resuscitation will need to address the difficulties in shock recognition and cognitive framework adaptation, physician and nurse teamwork, and prioritization of team effort. the background: the association between blood glucose level and mortality in critically ill patients is highly debated. several studies have investigated the association between history of diabetes, blood sugar level, and mortality of septic patients; however, no consistent conclusion could be drawn so far. objectives: to investigate the association between diabetes and initial glucose level and in-hospital mortality in patients with suspected sepsis from the ed. methods: we conducted a retrospective cohort study that consisted of all adult septic patients who visited the ed at a tertiary hospital during the year 2010 with two sets of blood cultures ordered by physicians. basic demographics, ed vital signs, symptoms and signs, underlying illnesses, laboratory findings, microbiological results, and discharge status were collected. logistic regressions were used to evaluate the association between risk factors, initial blood sugar level, and history of diabetes and mortality, as well as the effect modification between initial blood sugar level and history of diabetes. results: a total of 4997 patients with available blood sugar levels were included, of whom 48% had diabetes, 46% were older than 65 years of age, and 56% were male. the mortality was 6% (95% ci 5.3-6.7%). patients with a history of diabetes tended to be older, female, and more likely to have chronic kidney disease, lower sepsis severity (meds score), and positive blood culture test results (all p < 0.05). patients with a history of diabetes tended to have lower in-hospital mortality after ed visits with sepsis, controlling for initial blood sugar level (aor 0.72, 95% ci 0.56-0.92, p = 0.01). initial normal blood sugar seemed to be beneficial compared to lower blood sugar level for in-hospital mortality, controlled history of diabetes, sex, severity of sepsis, and age (aor 0.61, 95% ci 0.44-0.84, p = 0.002). the effect modification of diabetes on blood sugar level and mortality, however, was found to be not statistically significant (p = 0.09). conclusion: normal initial blood sugar level in ed and history of diabetes might be protective for mortality of septic patients who visited the ed. further investigation is warranted to determine the mechanism for these effects. methods: this irb-approved retrospective chart review included all patients treated with therapeutic hypothermia after cardiac arrest during 2010 at an urban, academic teaching hospital. every patient undergoing therapeutic hypothermia is treated by neurocritical care specialists. patients were identified by review of neurocritical care consultation logs. clinical data were dually abstracted by trained clinical study assistants using a standardized data dictionary and case report form. medications reviewed during hypothermia were midazolam, lorazepam, propofol, fentanyl, cisatracurium, and vecuronium. results: there were 33 patients in the cohort. median age was 57 (range 28-86 years), 67% were white, 55% were male, and 49% had a history of coronary artery disease. seizures were documented by continuous eeg in 11/33 (33%), and 20/33 (61%) died during hospitalization. most, 30/33 (91%), received fentanyl, 21/33 (64%) received benzodiazepine pharmacotherapy, and 23/33 (70%) received propofol. paralytics were administered to 23/33 (68%) patients, 14/33 (42%) with cisatracurium and 9/33 (27%) with vecuronium. of note, one patient required pentobarbital for seizure management. conclusion: sedation and neuromuscular blockade are common during management of patients undergoing therapeutic hypothermia after cardiac arrest. patients in this cohort often received analgesia with fentanyl, and sedation with a benzodiazepine or propofol. given the frequent use of sedatives and paralytics in survivors of cardiac arrest undergoing hypothermia, future studies should investigate the potential effect of these drugs on prognostication and survival after cardiac arrest. background: the use of therapeutic hypothermia (th) is a burgeoning treatment modality for post-cardiac arrest patients. objectives: we performed a retrospective chart review of patients who underwent post cardiac arrest th at eight different institutions across the united states. our objective was to assess how th is currently being implemented in emergency departments and assess the feasibility of conducting more extensive th research using multi-institution retrospective data. methods: a total of 94 charts with dates from 2008-2011 were sent for review by participating institutions of the peri-resuscitation consortium. of those reviewed, eight charts were excluded for missing data. two independent reviewers performed the review and the results were subsequently compared and discrepancies resolved by a third reviewer. we assessed patient demographics, initial presenting rhythm, time until th initiation, duration of th, cooling methods and temperature reached, survival to hospital discharge, and neurological status on discharge. results: the majority of cases of th had initial cardiac rhythms of asystole or pulseless electrical activity (55.2%), followed by ventricular tachycardia or fibrillation (34.5%), and in 10.3% the inciting cardiac rhythm was unknown. time to initiation of th ranged from 0-783 minutes with a mean time of 99 min (sd 132.5). length of th ranged from 25-2171 minutes with a mean time of 1191 minutes (sd 536). average minimum temperature achieved was 32.5°c, with a range from 27.6-36.7°c (sd 1.5°c). of the charts reviewed, 29 (33.3%) of the patients survived to hospital discharge and 19 (21.8%) were discharged relatively neurologically intact. conclusion: research surrounding cardiac arrest has always been difficult given the time and location span from pre-hospital care to emergency department to intensive care unit. also, as witnessed cardiac arrest events are relatively rare with poor survival outcomes, very large sample sizes are needed to make any meaningful conclusions about th. our varied and inconsistent results show that a multi-center retrospective review is also unlikely to provide useful information. a prospective multi-center trial with a uniform th protocol is needed if we are ever to make any evidence-based conclusions on the utility of th for post-cardiac arrest patients. serum results: mean la was 2.04, sd = 1.45. mean age was 4.5 years old, sd = 5.20. a statistically significant positive correlation was found between la and pulse, respiratory rate (rr), wbc, platelets, and los, while a significant negative correlation was seen with temperature and hco 3 -. when two subjects were dropped as possible outliers with la >10, it resulted in non-significant temperature correlation, but a significant negative correlation with age and bun was revealed. patients in the higher la group were more likely to be admitted (p = 0.0001) and have longer los. of the discharged patients, there was no difference in mean la level between those who returned (n = 25, mean la of 1.88, sd = 0.88) and those who did not (n = 154, mean la of 1.88, sd = 1.35), p = 0.99. furthermore, mean la levels for those with sepsis (n = 138, mean la of 2.18, sd = 1.75) did not differ from those without sepsis (n = 147, mean la of 1.9, sd = 1.08), p = 0.11. conclusion: higher la in pediatric patients presenting to the ed with suspected infection correlated with increased pulse, rr, wbc, platelets, and decreased bun, hco 3 -, and age. la may be predictive of hospitalization, but not of 3-day return rates or pediatric sepsis screening in the ed. background: mandibular fractures are one of the most frequently seen injuries in the trauma setting. in terms of facial trauma, madibular fractures account for 40-62% of all facial bone fractures. prior studies have demonstrated that the use of a tongue blade to screen these patients to determine whether a mandibular fracture is present may be as sensitive as x-ray. one study showed the sensitivity and specificity of the test to be 95.7% and 63.5%, respectively. in the last ten years, high-resolution computed tomography (hct) has replaced panoramic tomography (pt) as the gold standard for imaging of patients with suspected mandibular fractures. this study determines if the tongue blade test (tbt) remains as sensitive a screening tool when compared to the new gold standard of ct. objectives: the purpose of the study was to determine the sensitivity and specificity of the tbt as compared to the new gold standard of radiologic imaging, hct. the question being asked: is the tbt still useful as a screening tool for patients with suspected mandibular fractures when compared to the new gold standard of hct? methods: design: prospective cohort study. setting: an urban tertiary care level i trauma center. subjects: this study took place from 8/1/10 to 8/31/11 in which any person suffering from facial trauma presented. intervention: a tbt was performed by the resident physician and confirmed by the supervising attending physician. ct facial bones were then obtained for the ultimate diagnosis. inter-rater reliability (kappa) was calculated, along with sensitivity, specificity, accuracy, ppv, npv, likelihood ratio (lr) (+), and likelihood ratio (lr) (-) based on a 2 · 2 contingency tables generated. results: over the study period 85 patients were enrolled. inter-rater reliability was kappa = 0.93 (se +0.11). the table demonstrates the outcomes of both the tbt and ct facial bones for mandibular fracture. the following parameters were then calculated based on the contingency table: sensitivity 0.97 (ci 0.81-0.99), specificity 0.72 (ci 0.58-0.83), ppv 0.67 (ci 0.52-0.78), npv 0.97 (ci 0.87-0.99), accuracy 0.81, lr(+) 3.48 ), lr (-) 0.04 (ci 0.01-0.31). conclusion: the tbt is still a useful screening tool to rule out mandibular fractures in patients with facial trauma as compared to the current gold standard of hct. background: appendicitis is the most common surgical emergency occurring in children. the diagnosis of pediatric appendicitis is often difficult and computerized tomography (ct) scanning is utilized frequently. ct, although accurate, is expensive, time-consuming, and exposes children to ionizing radiation. radiologists utilize ultrasound for the diagnosis of appendicitis, but it may be less accurate than ct, and may not incorporate emergency physician (ep) clinical impression regarding degree of risk. objectives: the current study compared ep clinical diagnosis of pediatric appendicitis pre-and post-bedside ultrasonography (bus). methods: children 3-17 years of age were enrolled if their clinical attending physician planned to obtain a consultative ultrasound, ct scan, or surgical consult specific for appendicitis. most children in the study received narcotic analgesia to facilitate bus. subjects were initially graded for likelihood of appendicitis based on research physician-obtained history and physical using a visual analogue scale (vas). immediately subsequent to initial grading, research physicians performed a bus and recorded a second vas impression of appendicitis likelihood. two outcome measures were combined as the gold standard for statistical analysis. the post-operative pathology report served as the gold standard for subjects who underwent appendectomy, while post 2-week telephone follow-up was used for subjects who did not undergo surgery. various specific ultrasound measures used for the diagnosis of appendicitis were assessed as well. results: 29/56 subjects had pathology-proven appendicitis. one subject was pathology-negative post-appendectomy. of the 26 subjects who did not undergo surgery, none had developed appendicitis at the post 2-week telephone follow-up. pre-bus sensitivity was 48% (29-68%) while post-bus sensitivity was 79% (60-92%). both pre-and post-bus specificity was 96% (81-100%). pre-bus lr+ was 13 (2-93), while post-bus lr+ was 21 (3-148). pre-and post-bus lr-were 0.5 and 0.2, respectively. bus changed the diagnosis for 20% of subjects (9-32%). background: there are very little data on the normal distance between the glenoid rim and the posterior aspect of the humeral head in normal and dislocated shoulders. while shoulder x-rays are commonly used to detect shoulder dislocations, they may be inadequate, exacerbate pain in the acquisition of some views, and lead to delay in treatment, compared to bedside ultrasound evaluation. objectives: our objective was to compare the glenoid rim to humeral head distance in normal shoulders and in anteriorly dislocated shoulders. this is the first study proposing to set normal and abnormal limits. methods: subjects were enrolled in this prospective observation study if they had a chief complaint of shoulder pain or injury, and received a shoulder ultrasound as well as a shoulder x-ray. the sonographers were undergraduate students given ten hours of training to perform the shoulder ultrasound. they were blinded to the x-ray interpretation, which was used as the gold standard. we used a posterior-lateral approach, capturing an image with the glenoid rim, the humeral head, as well as the infraspinatus muscle. two parallel lines were applied to the most posterior aspect of the humeral head and the most posterior aspect of the glenoid rim. a line perpendicular to these lines was applied, and the distance measured. in anterior dislocations, a negative measurement was used to denote the fact that the glenoid rim is now posterior to the most posterior aspect of the humeral head. descriptive analysis was applied to estimate the mean and 25th to 75th interquartile range of normal and anteriorly dislocated shoulders. results: eighty subjects were enrolled in this study. there were six shoulder dislocations, however only four were anterior dislocations. the average distance between the posterior glenoid rim and the posterior humeral head in normal shoulders was 8.7 mm, with a 25th to 75th inter-quartile range of 6.7 mm to 11.9 mm. the distance in our four cases of anterior dislocation was )11 mm, with a 25th to 75th interquartile range of )10 mm to )12 mm. conclusion: the distance between the posterior humeral head to posterior glenoid rim may be 7 mm to 12 mm in patients presenting to the ed with shoulder pain but no dislocation. in contrast, this distance in anterior dislocations was greater than )10 mm. shoulder ultrasound may be a useful adjunct to x-ray for diagnosing anterior shoulder dislocations. conclusion: in this retrospective study, the presence of rv strain on focus significantly increases the likelihood of an adverse short term event from pulmonary embolism and its combination with hypotension performs similarly to other prognostic rules. background: burns are expensive and debilitating injuries, compromising both the structural integrity and vascular supply to skin. they exhibit a substantial potential to deteriorate if left untreated. jackson defined three ''zones'' to a burn. while the innermost coagulation zone and the outermost zone of hyperemia display generally predictable healing outcomes, the zone of stasis has been shown to be salvageable via clinical intervention. it has therefore been the focus of most acute therapies for burn injuries. while laser doppler imaging (ldi) -the current gold standard for burn analysis -has been 96% effective at predicting the need for second degree burn excision, its clinical translation is problematic, and there is little information regarding its ability to analyze the salvage of the stasis zone in acute injury. laser assisted indocyanine green dye angiography (laicga) also shows potential to predict such outcomes with greater clinical utility. objectives: to test the ability of ldi and laicga to predict interspace (zone of stasis) survival in a horizontal burn comb model. methods: a prospective animal experiment was performed using four pigs. each pig had a set of six dorsal burns created using a brass ''comb'' -creating four rectangular 10 · 20 mm full thickness burns separated by 5 · 20 mm interspaces. laicga and ldi scanning took place at 1 hour, 24 hours, 48 hours, and 1 week post burn using novadaq spy and moor ldi respectively. imaging was read by a blinded investigator, and perfusion trends were compared with interspace viability and contraction. burn outcomes were read clinically, evaluated via histopathology, and interspace contraction was measured using image j software. results: laicga data showed significant predictive potential for interspace survival. it was 83.3% predictive at 24 hours post burn, 75% predictive 48 hours post burn, and 100% predictive 7 days post burn using a standardized perfusion threshold. ldi imaging failed to predict outcome or contraction trends with any degree of reliability. the pattern of perfusion also appears to be correlated with the presence of significant interspace contraction at 28 days, with an 80% adherence to a power trendline. ventions, 11 isolation, 4 testing, 4 treatment, and 1 ''other'' category intervention were identified. one intervention involving school closures was associated with a 28% decrease in pediatric ed visits for respiratory illness. conclusion: most interventions were not tested in isolation, so the effect of individual interventions was difficult to differentiate. interventions associated with statistically significant decreases in ed crowding were school closures, as well as interventions in all categories studied. further study and standardization of intervention input, process, and outcome measures may assist in identifying the most effective methods of mitigating ed crowding and improving surge capacity during an influenza or other respiratory disease outbreak. communication background: the link between extended shift lengths, sleepiness, and occupational injury or illness has been shown, in other health care populations, to be an important and preventable public health concern but heretofore has not been fully described in emergency medical services (ems objectives: to assess the effect of an ed-based computer screening and referral intervention for ipv victims and to determine what characteristics resulted in a positive change in their safety. we hypothesized that women who were experiencing severe ipv and/or were in contemplation or action stages would be more likely to endorse safety behaviors. methods: we conducted the intervention for female ipv victims at three urban eds using a computer kiosk to deliver targeted education about ipv and violence prevention as well as referrals to local resources. all adult english-speaking non-critically ill women triaged to the ed waiting room were eligible to participate. the validated universal violence prevention screening protocol was used for ipv screening. any who disclosed ipv further responded to validated questionnaires for alcohol and drug abuse, depression, and ipv severity. the women were assigned a baseline stage of change (precontemplation, contemplation, action, or maintenance) based on the urica scale for readiness to change behavior surrounding ipv. participants were contacted at 1 week and 3 months to assess a variety of pre-determined actions such as moving out, to prevent ipv during that period. statistical analysis (chi-square testing) was performed to compare participant characteristics to the stage of change and whether or not they took protective action. results: a total of 1,474 people were screened and 154 disclosed ipv and participated in the full survey. 53.3% of the ipv victims were in the precontemplative stage of change, and 40.3% were in the contemplation stage. 110 women returned at 1 week of follow-up (71.4%), and 63 (40.9%) women returned at 3 months of followup. 55.5% of those who returned at 1 week, and 73% of those who returned at 3 months took protective action against further ipv. there was no association between the various demographic characteristics and whether or not a woman took protective action. conclusion: ed-based kiosk screening and health information delivery is both a feasible and effective method of health information dissemination for women experiencing ipv. stage of change was not associated with actual ipv protective measures. objectives: we present a pilot, head-to-head comparison of x26 and x2 effectiveness in stopping a motivated person. the objective is to determine comparative injury prevention effectiveness of the newer cew. methods: four humans had metal cew probe pairs placed. each volunteer had two probe pairs placed (one pair each on the right and left of the abdomen/inguinal region). superior probes were at the costal margin, 5 inches lateral of midline. inferior probes were vertically inferior at predetermined distances of 6, 9, 12, and 16 inches apart. each volunteer was given the goal of slashing a target 10 feet away with a rubber knife during cew exposure. as a means of motivation, they believed the exposure would continue until they reached the goal (in reality, the exposure was terminated once no further progress was made). each volunteer received one exposure from a x26 and a x2 cew. the exposure order was randomized with a 2-minute rest between them. exposures were recorded on a hi-speed, hi-resolution video. videos were reviewed and scored by six physician, kinesiology, and law officer experts using standardized criteria for effectiveness including degree of upper and lower extremity, and total body incapacitation, and degree of goal achievement. reviews were descriptively compared independently for probe spread distances and between devices. results: there were 8 exposures (4 pairs) for evaluation and no discernible, descriptive reviewer differences in effectiveness between the x26 and the x2 cews when compared. background: the trend towards higher gasoline prices over the past decade in the u.s. has been associated with higher rates of bicycle use for utilitarian trips. this shift towards non-motorized transportation should be encouraged from a physical activity promotion and sustainability perspective. however, gas price induced changes in travel behavior may be associated with higher rates of bicycle-related injury. increased consideration of injury prevention will be a critical component of developing healthy communities that help safely support more active lifestyles. objectives: the purpose of this analysis was to a) describe bicycle-related injuries treated in u.s. emergency departments between 1997 and 2009 and b) investigate the association between gas prices and both the incidence and severity of adult bicycle injuries. we hypothesized that as gas prices increase, adults are more likely to shift away from driving for utilitarian travel toward more economical non-motorized modes of transportation, resulting in increased risk exposure for bicycle injuries. methods: bicycle injury data for adults (16-65 years) were obtained from the national electronic injury surveillance system (neiss) database for emergency department visits between 1997-2009. the relationship between national seasonally adjusted monthly rates of bicycle injuries, obtained by a seasonal decomposition of time series, and average national gasoline prices, reported by the energy information administration, was examined using a linear regression analysis. results: monthly rates of bicycle injuries requiring emergency care among adults increase significantly as gas prices rise (p < 0.0001, see figure) . an additional 1,149 adult injuries (95% ci 963-1,336) can be predicted to occur each month in the u.s. (>13,700 injuries annually) for each $1 rise in average gasoline price. injury severity also increases during periods of high gas prices, with a higher percentage of injuries requiring admission. conclusion: increases in adult bicycle use in response to higher gas prices are accompanied by higher rates of significant bicycle-related injuries. supporting the use of non-motorized transportation will be imperative to address public health concerns such as obesity and climate change; however, resources must also be dedicated to improve bicycle-related injury care and prevention. background: this is a secondary analysis of data collected for a randomized trial of oral steroids in emergency department (ed) musculoskeletal back pain patients. we hypothesized that higher pain scores in the ed would be associated with more days out of work. objectives: to determine the degree to which days out of work for ed back pain patients are correlated with ed pain scores. methods: design: prospective cohort. setting: suburban ed with 80,000 annual visits. participants: patients aged 18-55 years with moderately severe musculoskeletal back pain from a bending or twisting injury £ 2 days before presentation. exclusion criteria included nonmusculoskeletal etiology, direct trauma, motor deficits, and employer-initiated visits. observations: we captured initial and discharge ed visual analog pain scores (vas) on a 0-10 scale. patients were contacted approximately 5 days after discharge and queried about the days out of work. we plotted days out of work versus initial vas, discharge vas, and change in vas and calculated correlation coefficients. using the bonferroni correction because of multiple comparisons, alpha was set at 0.02. results: we analyzed 67 patients for whom complete data were available. the mean age was 40 ± 9 years and 30% were female. the average initial and discharge ed pain scales were 8.0 ± 1.5 and 5.7 ± 2.2, respectively. on follow-up, 88% of patients were back to work and 36% did not lose any days of work. for the plots of the days out of work versus the initial and discharge vas and the change in the vas, the correlation coefficients (r 2 ) were 0.03 (p = 0.17), 0.08 (p = 0.04), and 0.001 (p = 0.87), respectively. conclusion: for ed patients with musculoskeletal back pain, we found no statistically significant correlation between days out of work and ed pain scores. background: conducted electrical weapons (cews) are common law enforcement tools used to subdue and repel violent subjects and, therefore, prevent further injury or violence from occurring in certain situations. the taser x2 is a new generation of cew that has the capability of firing two cartridges in a ''semi-automatic'' mode, and has a different electrical waveform and different output characteristics than older generation technology. there have been no data presented on the human physiologic effects of this new generation cew. objectives: the objective of this study was to evaluate the human physiologic effects of this new cew. methods: this was a prospective, observational study of human subjects. an instructor shot subjects in the abdomen and upper thigh with one cartridge, and subjects received a 10-second exposure from the device. measured variables included: vital signs, continuous spirometry, pre-and post-exposure ecg, intra-exposure echocardiography, venous ph, lactate, potassium, ck, and troponin. results: ten subjects completed the study (median age 31.5, median bmi 29.4, 80% male). there were no important changes in vital signs or in potassium. the median increase in lactate during the exposure was 1.2, range 0.6 to 2.8. the median change in ph was )0.031, range )0.011 to 0.067. no subject had a clinically relevant ecg change, evidence of cardiac capture, or positive troponin up to 24 hours after exposure. the median change in creatine kinase (ck) at 24 hours was 313, range )40 to 3418. there was no evidence of impairment of breathing by spirometry. baseline median minute ventilation was 14.2, which increased to 21.6 during the exposure (p = 0.05), and remained elevated at 21.6 post-exposure (p = 0.01). conclusion: we detected a small increase in lactate and decrease in ph during the exposure, and an increase in ck 24 hours after the exposure. the physiologic effects of the x2 device appear similar to previous reports for ecd devices. use background: public bicycle sharing (bikeshare) programs are becoming increasingly common in the us and around the world. these programs make bicycles easily accessible for hourly rental to the public. there are currently 15 active bikeshare programs in cities in the us, and more than 30 programs are being developed in cities including new york and chicago. despite the importance of helmet use, bikeshare programs do not provide the opportunity to purchase or rent helmets. while the programs encourage helmet use, no helmets are provided at the rental kiosks. objectives: we sought to describe the prevalence of helmet use among adult users of bikeshare programs and users of personal bicycles in two cities with recently introduced bicycle sharing programs (boston, ma and washington, dc). methods: we performed a prospective observational study of bicyclists in boston, ma and washington, dc. trained observers collected data during various times of the day and days of the week. observers recorded the sex of the bicycle operator, type of bicycle, and helmet use. all bicycles that passed a single stationary location in any direction for a period of between 30 and 90 minutes were recorded. data are presented as frequencies of helmet use by sex, type of bicycle (bikeshare or personal), time of the week (weekday or weekend), and city. logistic regression was used to estimate the odds ratio for helmet use controlling for type of bicycle, sex, day of week, and city. results: there were 43 observation periods in two cities at 36 locations. 3,073 bicyclists were observed. there were 562 (18.2%) bicylists riding bikeshare bicycles. overall helmet use was 45.5%, although helmet use varied significantly with sex, day of use, and type of bicycle (see figure) . bikeshare users were helmeted at a lower rate compared to users of personal bicycles (19.2% vs 51.4%). logistic regression, controlling for type of bicycle, sex, day of week, and city demonstrate that bikeshare users had higher odds of riding unhelmeted (or 4.34, 95% ci 3.47-5.50). women had lower odds of riding unhelmeted (or 0.62, 0.52-0.73), while weekend riders were more likely to ride unhelmeted (or 1.32, 1.12-1.55). conclusion: use of bicycle helmets by users of public bikeshare programs is low. as these programs become more popular and prevalent, efforts to increase helmet use among users should increase. background: abusive head trauma (aht) represents one of the most severe forms of traumatic brain injury (tbi) among abused infants with 30% mortality. young adult males account for 75% of the perpetrators. most aht prevention programs are hospital-based and reach a predominantly female audience. there are no published reports of school-based aht prevention programs to date. objectives: 1. to determine whether a high schoolbased aht educational program will improve students' knowledge of aht and parenting skills. 2. to evaluate the feasibility and acceptability of a school-based aht prevention program. methods: this program was based on an inexpensive commercially available program developed by the national center on shaken baby syndrome. the program was modified to include a 60-minute interactive presentation that teaches teenagers about aht, parenting skills, and caring for inconsolable crying infants. the program was administered in three high schools in flint, michigan during spring 2011. student's knowledge was evaluated with a 17-item written test administered pre-intervention, post-intervention, and two months after program completion. program feasibility and acceptability were evaluated through interviews and surveys with flint area school social workers, parent educators, teachers, and administrators. results: in all, 342 high school students (40% male) participated. of these, 317 (92.7%) completed the pretest and post-test with 171 (50%) completing the twomonth follow-up test. the mean pre-intervention, postintervention, and two-month follow-up scores were 53%, 87%, and 90% respectively. from pre-test to posttest, mean score improved 34%, p < 0.001. this improvement was even more profound in young males, whose mean post-test score improved by 38%, p < 0.001. of the 69 participating social workers, parent educators, teachers, and administrators, 97% ranked the program as feasible and acceptable. conclusion: students participating in our program showed an improvement in knowledge of aht and parenting skills which was retained after two months. teachers, social workers, parent educators, and school administrators supported the program. this local pilot program has the potential to be implemented on a larger scale in michigan with the ultimate goal of reducing aht amongst infants. will background: fear of litigation has been shown to affect physician practice patterns, and subsequently influence patient care. the likelihood of medical malpractice litigation has previously been linked with patient and provider characteristics. one common concern is that a patient may exaggerate symptoms in order to obtain monetary payouts; however, this has never been studied. objectives: we hypothesize that patients are willing to exaggerate injuries for cash settlements and that there are predictive patient characteristics including age, sex, income, education level, and previous litigation. methods: this prospective cross-sectional study spanned june 1 to december 1, 2011 in a philadelphian urban tertiary care center. any patient medically stable enough to fill out a survey during study investigator availability was included. two closed-ended paper surveys were administered over the research period. standard descriptive statistics were utilized to report incidence of: patients who desired to file a lawsuit, patients previously having filed lawsuits, and patients willing to exaggerate the truth in a lawsuit for a cash settlement. chi-square analysis was performed to determine the relationship between patient characteristics and willingness to exaggerate injuries for a cash settlement. results: of 126 surveys, 11 were excluded due to incomplete data, leaving 115 for analysis. the mean age was 39 with a standard deviation of 16, and 40% were male. the incidence of patients who had the desire to sue at the time of treatment was 9%. the incidence of patients who had filed a lawsuit in the past was 35%. of those patients, 26% had filed multiple lawsuits. fifteen percent [95% ci 9-23%] of all patients were willing to exaggerate injuries for cash settlement. sex and income were found to be statistically significant predictors of willingness to exaggerate symptoms: 22% of females vs. 4% of males were willing to exaggerate (p = 0.01), and 20% of people with income less than $100,000/yr vs. 0% of those with income over $100,000/ yr were willing to exaggerate (p = 0.03). conclusion: patients at a philadelphian urban tertiary center admit to willingness to exaggerate symptoms for a cash settlement. willingness to exaggerate symptoms is associated with female sex and lower income. background: current data suggest that as many as 50% of patients presenting to the ed with syncope leave the hospital without a defined etiology. prior studies have suggested a prevalence of psychiatric disease as high as 26% in patients with syncope of unknown etiology. objectives: to determine whether psychiatric disease and substance abuse are associated with an increased incidence of syncope of unknown etiology. methods: prospective, observational, cohort study of consecutive ed patients ‡18 presenting with syncope was conducted between 6/03 and7/06. patients were queried in the ed and charts reviewed about a history of psychiatric disease, use of psychiatric medication, substance abuse, and duration. data were analyzed using sas with chi-square and fisher's exact tests. results: we enrolled 519 patients who presented to the ed after syncope, 159 of whom did not have an identifiable etiology for their syncopal event. 36.5% of those without an identifiable etiology were male. 166 (32%) patients had a history of or current psychiatric disease (42% male), and 55 patients (11%) had a history of or current substance abuse (60% male). among males with psychiatric disease, 39% had an unknown etiology of their syncopal event, compared to 22% of males without psychiatric disease (p = 0.009). similarly, among all males with a history of substance abuse, 45% had an unknown etiology, as compared to 24% of males without a history of substance abuse (p = 0.01). a similar trend was not identified in elderly females with psychiatric disease (p = 0.96) or substance abuse (p = 0.19). however, syncope of unknown etiology was more common among both men and women under age 65 with a history of substance abuse (47%) compared to those without a history of substance abuse (27%; p = 0.01). conclusion: our results suggest that psychiatric disease and substance abuse are associated with increased incidence of syncope of unknown etiology. patients evaluated in the ed or even hospitalized with syncope of unknown etiology may benefit from psychiatric screening and possibly detoxification referral. this is particularly true in men. (originally submitted as a ''late-breaker.'') scope background: after discharge from an emergency department (ed), pain management often challenges parents, who significantly under-treat their children's pain. rapid patient turnover and anxiety make education about home pain treatment difficult in the ed. video education standardizes information and circumvents insufficient time and literacy. objectives: to evaluate the effectiveness of a 6-minute instructional video for parents that targets common misconceptions about home pain management. methods: we conducted a randomized, double-blinded clinical trial of parents of children ages 1-18 years who presented with a painful condition, were evaluated, and discharged home in june and july 2011. parents were randomized to a pain management video or an injury prevention control video. primary outcome was the proportion of parents who gave pain medication at home. these data were recorded in a home pain diary and analyzed using a chi-square test. parents' knowledge about pain treatment was tested before, immediately following, and 2 days after intervention. mcnemar's test statistic determined odds that knowledge correlated with the intervention group. results: 100 parents were enrolled: 59 watched the pain education video, and 41 the control video. 72.9% completed follow up, providing information about home pain education use. significantly more parents provided at least one dose of pain medication to their children after watching the educational video: 96% vs. 80% (difference 16%, 95% ci 7.8%, 31.3%). the odds the parent had correct knowledge about pain treatment significantly improved immediately following the educational video for knowledge about pain scores (p = 0.04), the effect of pain on function (p < 0.01), and pain medication misconceptions (p < 0.01). these significant differences in knowledge remained 3 days after the video intervention. the educational video about home pain treatment viewed by parents significantly increased the proportion of children receiving pain medication at home and significantly improved knowledge about at-home pain management. videos are an efficient tool to provide medical advice to parents that improves outcomes for children. methods: this was a prospective, observational study of consecutive admitted cpu patients in a large-volume academic urban ed. cardiology attendings round on all patients and stress test utilization is driven by their recommendation. eligibility criteria include: age>18, aha low/intermediate risk, nondynamic ecgs, and normal initial troponin i. patients >75 and with a history of cad or co-existing active medical problem were excluded. based on prior studies and our estimated cpu census and demographic distribution, we estimated a sample size of 2,242 patients in order to detect a difference in stress utilization of 7% (2-tailed, a = 0.05, b = 0.8). we calculated a timi risk prediction score and a diamond & forrester (d&f) cad likelihood score on each patient. t-tests were used for univariate comparisons of demographics, cardiac comorbidities, and risk scores. logistic regression was used to estimate odds ratios (ors) for receiving testing based on race, controlling for insurance and either timi or d&f score. results: over 18 months, 2,451 patients were enrolled. mean age was 53 ± 12, and 54% (95% ci 52-56) were female. sixty percent (95% ci 58-62) were caucasian, 12% (95% ci 10-13) african american, and 24% (95% ci 23-26) hispanic. mean timi and d&f scores were 0.5 (95% ci 0.5-0.6) and 38% (95% ci 37-39). the overall stress testing rate was 52% (95% ci 50-54). after controlling for insurance status and timi or d&f scores, african american patients had significantly decreased odds of stress testing (or timi 0.67 (95% ci 0.52-0.88), or d&f 0.68 (95% ci 0.51-0.89)). hispanics had significantly decreased odds of stress testing in the model controlling for d&f (or d&f 0.78 (95% ci 0.63-0.98)). conclusion: this study confirms that disparities in the workup of african american patients in the cpu are similar to those found in the general ed and the outpatient setting. further investigation into the specific provider or patient level factors contributing to this bias is necessary. the outcomes for hf and copd were sae 11.6%, 7.8%; death 2.3%, 1.0%. we found univariate associations with sae for these walk test components: too ill to walk (both hf, copd p < 0.0001); highest heart rate ‡110 (hf p = 0.02, copd p = 0.10); lowest sao 2 < 88% (hf p = 0.42, copd p = 0.63); borg score ‡5 (hf p = 0.47, copd p = 0.52); walk test duration £ 1 minute (hf p = 0.07. copd p = 0.22). after adjustment for multiple clinical covariates with logistic regression analyses, we found ''walk test heart rate ‡110'' had an odds ratio of 1.9 for hf patients and ''too ill to start the walk test'' had an odds ratio of 3.5 for copd patients. conclusion: we found the 3-minute walk test to be easy to administer in the ed and that maximum heart rate and inability to start the test were highly associated with adverse events in patients with exacerbations of hf and copd, respectively. we suggest that the 3-minute walk test be routinely incorporated into the assessment of hf and copd patients in order to estimate risk of poor outcomes. the objectives: the objective of this study was to investigate differences in consent rates between patients of different demographic groups who were invited to participate in minimal-risk clinical trials conducted in an academic emergency department. methods: this descriptive study analyzed prospectively collected data of all adult patients who were identified as qualified participants in ongoing minimal risk clinical trials. these trials were selected for this review because they presented minimal factors known to be associated background: increasing rates of patient exposure to computerized tomography (ct) raise questions about appropriateness of utilization, as well as patient awareness of radiation exposure. despite rapid increases in ct utilization and published risks, there is no national standard to employ informed consent prior to radiation exposure from diagnostic ct. use of written informed consent for ct (icct) in our ed has increased patient understanding of the risks, benefits, and alternatives to ct imaging. our team has developed an adjunct video educational module (vem) to further educate ed patients about the ct procedure. objectives: to assess patient knowledge and preferences regarding diagnostic radiation before and after viewing vem. methods: the vem was based on icct currently utilized at our tertiary care ed (census 37,000 patients/ year). icct is written at an 8th grade reading level. this fall, vem/icct materials were presented to a convenience sample of patients in the ed waiting room 9 am-7 pm, monday-sunday. patients who were <18 years of age, critically ill, or with language barrier were excluded. to quantify the educational value of the vem, a six-question pretest was administered to assess baseline understanding of ct imaging. the patients then watched the vem via ipad (macintosh) and reviewed the consent form. an eight-question post-test was then completed by each subject. no phi were collected. pre-and post-test results were analyzed using mcnemar's test for individual questions and a paired t-test for the summed score (sas version 9.2). results: 100 patients consented and completed the survey. the average pre-test score for subjects was poor, 66% correct. review of vem/icct materials increased patient understanding of medical radiation as evidenced by improved post-test score to 79%. mean improvement between tests was 13% (p < 0.0001). 78% of subjects responded that they found the materials helpful, and that they would like to receive icct. conclusion: the addition of a video educational module improved patient knowledge regarding ct imaging and medical radiation as quantified by pre-and posttesting. patients in our study sample reported that they prefer to receive icct. by educating patients about the risks associated with ct imaging, we increase informed, shared decision making -an essential component of patient-centered care. does objectives: we sought to determine the relationship between patients' pain scores and their rate of consent to ed research. we hypothesized that patients with higher pain scores would be less likely to consent to ed research. methods: retrospective observational cohort study of potential research subjects in an urban academic hospital ed with an average annual census of approximately 70,000 visits. subjects were adults older than 18 years with chief complaint of chest pain within the last 12 hours, making them eligible for one of two cardiac biomarker research studies. the studies required only blood draws and did not offer compensation. two reviewers extracted data from research screening logs. patients were grouped according to pain score at triage, pain score at the time of approach, and improvement in pain score (triage score -approach score). the main outcome was consent to research. simple proportions for consent rates by pain score tertiles were calculated. two multivariate logistic regression analyses were performed with consent as outcome and age, race, sex, and triage or approach pain score as predictors. results: overall, 396 potential subjects were approached for consent. patients were 58% caucasian, 49% female, and with an average age of 57 years. six patients did not have pain scores recorded at all and 48 did not have scores documented within 2 hours of approach and were excluded from relevant analyses. overall, 80.1% of patients consented. consent rates by tertiles at triage, at time of approach, and by pain score improvement are shown in tables 1 and 2. after adjusting for age, race, and sex, neither triage (p = 0.75) nor approach (p = 0.65) pain scores predicted consent. conclusion: research enrollment is feasible even in ed patients reporting high levels of pain. patients with modest improvements in pain levels may be more likely to consent. future research should investigate which factors influence patients' decisions to participate in ed research. conclusion: in this multicenter study of children hospitalized with bronchiolitis neither specific viruses nor their viral load predicted the need for cpap or intubation, but young age, low birth weight, presence of apnea, severe retractions, and oxygen saturation <85% did. we also identified that children requiring cpap or intubation were more likely to have mothers who smoked during pregnancy and a rapid respiratory worsening. mechanistic research in these high-risk children may yield important insights for the management of severe bronchiolitis. brigham & women's hospital, boston, ma background: siblings and children who share a home with a physically abused child are thought to be at high risk for abuse. however, rates of injury in these children are unknown. disagreements between medical and child protective services professionals are common and screening is highly variable. objectives: our objective was to measure the rates of occult abusive injuries detected in contacts of abused children using a common screening protocol. methods: this was a multi-center, observational cohort study of 20 child abuse teams who shared a common screening protocol. data were collected between jan 15, 2010 and april 30, 2011 for all children <10 years undergoing evaluation for physical abuse and their contacts. for contacts of abused children, the protocol recommended physical examination for all children <5 years, skeletal survey and physical exam for children <24 months, and physical exam, skeletal survey, and neuroimaging for children <6 months old. results: among 2,825 children evaluated for abuse, 618 met criteria as ''physically abused'' and these had 477 contacts. for each screening modality, screening was completed as recommended by the protocol in approximately 75% of cases. of 134 contacts who met criteria for skeletal survey, new injuries were identified in 16 (12.0%). none of these fractures had associated findings on physical examination. physical examination identified new injuries in 6.2% of eligible contacts. neuroimaging failed to identify new injuries among 25 eligible contacts less than 6 months old. twins were at significantly increased risk of fracture relative to other nontwin contacts (or 20.1). conclusion: these results support routine skeletal survey for contacts of physically abused children <24 months old, regardless of physical examination findings. even for children where no injuries are identified, these results demonstrate that abuse is common among children who share a home with an abused child, and support including contacts in interventions (foster care, safety planning, social support) designed to protect physically abused children. methods: this was a retrospective study evaluating all children presenting to eight paediatric, universityaffiliated eds during one year in 2010-2011. in each setting, information regarding triage and disposition were prospectively registered by clerks in the ed database. anonymized data were retrieved from the ed computerized database of each participating centre. in the absence of a gold standard for triage, hospitalisation, admission to intensive care unit (icu), length of stay in the ed, and proportion of patients who left without being seen by a physician (lwbs) were used as surrogate markers of severity. the primary outcome measure was the association between triage level (from 1 to 5) and hospitalisation. the association between triage level and dichotomous outcomes was evaluated by a chi-square test, while a student's t-test was used to evaluate the association between triage level and length of stay. it was estimated that the evaluation of all children visiting these eds for a one year period would provide a minimum of 1,000 patients in each triage level and at least 10 events for outcomes having a proportion of 1% or more. results: a total of 404,841 children visited the eight eds during the study period. pooled data demonstrated hospitalisation proportions of 59%, 30%, 10%, 2%, and 0.5% for patients triaged at level 1, 2,3, 4, and 5 respectively (p < 0.001). there was also a strong association between triage levels and admission to icu (p < 0.001), the proportion of children who lwbs (p < 0.001), and length of stay (p < 0.001). background: parents frequently leave the emergency department (ed) with incomplete understanding of the diagnosis and plan, but the relationship between comprehension and post-care outcomes has not been well described. objectives: to explore the relationship between comprehension and post-discharge medication safety. methods: we completed a planned secondary analysis of a prospective observational study of the ed discharge process for children aged 2-24 months. after discharge, parents completed a structured interview to assess comprehension of the child's condition, the medical team's advice, and the risk of medication error. limited understanding was defined as a score of 3-5 from 1 (excellent) to 5 (poor). risk of medication error was defined as a plan to use over-the-counter cough/cold medication and/or an incorrect dose of acetaminophen (measured by direct observation at discharge or reported dose at follow-up call). parents identified as at risk received further instructions from their provider. the primary outcome was persistent risk of medication error assessed at phone interview 5-10 days post-discharge. a major barrier to administering analgesics to children is the perceived discomfort of intravenous access. the delivery of intranasal analgesia may be a novel solution to this problem. objectives: we investigated whether the addition of the mucosal atomizer device (mad) as an alternative for fentanyl delivery would improve overall fentanyl administration rates in pediatric patients transported by a large urban ems system. we performed a historical control trial comparing the rate of pediatric fentanyl administration 6 months before and 6 months after the introduction of the mad. study subjects were pediatric trauma patients (age <16 years) transported by a large urban ems agency. the control group was composed of patients treated in the 6 months before introduction of the mad. the experimental group included patients treated in the 6 months after the addition of the mad. two physicians reviewed each chart and determined whether the patient met predetermined criteria for the administration of pain medication. a third reviewer resolved any discrepancies. fentanyl administration rates were measured and compared between the two groups. we used two-sample t-tests and chi-square tests to analyze our data. results: 228 patients were included in the study: 137 patients in the pre-mad group and 91 in the post-mad group. there were no significant differences in the demographic and clinical characteristics of the two groups. 42 (30.4%) patients in the control arm received fentanyl. 34 (37.8%) of patients in the experimental arm received fentanyl with 36% of the patients receiving fentanyl via the intranasal route. the addition of the mad was not associated with a statistically significant increase in analgesic administration. age and mechanism of injury were statistically more predictive of analgesia administration. conclusion: while the addition of the mucosal atomizer device as an alternative delivery method for fentanyl shows a trend towards increased analgesic administration in a prehospital pediatric population, age and mechanism of injury are more predictive in who receives analgesia. further research is necessary to investigate the effect of the mad on pediatric analgesic delivery. methods: this was a prospective study evaluating php-se before (pre) and after (post) a ppp introduction and 13 months later (13-mo). php groups received either ppp review and education or ppp review alone. the ppp included a pain assessment tool. the se tool, developed and piloted by pediatric ems experts, uses a ranked ordinal scale ranging from 'certain i cannot do it' (0) to 'completely certain i can do it' (100) for 10 items: pain assessment (3 items), medication administration (4) and dosing (1) , and reassessment (2). all 10 items and an averaged composite were evaluated for three age groups (adult, child, toddler). paired sample t-tests compared post-and 13-mo scores to pre-ppp scores. results: of 264 phps who completed initial surveys, 146 phps completed 13-mo surveys. 106 (73%) received education and ppp review and 40 (27%) review only. ppp education did not affect php-se (adult p = 0.87, child p = 0.69, toddler p = 0.84). the largest se increase was in pain assessment. this increase persisted for child and toddler groups at 13 months. the immediate increase in composite se scores for all age groups persisted for the toddler group at 13 months. conclusion: increases in composite and pain assessment php-se occur for all age groups immediately after ppp introduction. the increase in pain assessment se persisted at 13 months for pediatric age groups. composite se increase persisted for the toddler age group alone. background: pediatric medications administered in the prehospital setting are given infrequently and dosage may be prone to error. calculation of dose based on known weight or with use of length-based tapes occurs even less frequently and may present a challenge in terms of proper dosing. objectives: to characterize dosing errors based on weight-based calculations in pediatric patients in two similar emergency medical service (ems) systems. methods: we studied the five most commonly administered medications given to pediatric patients weighing 36 kg or less. drugs studied were morphine, midazolam, epinephrine 1:10,000, epinephrine 1:1000, and diphenhydramine. cases from the electronic record were studied for a total of 19 months, from january 2010 to july 2011. each drug was administered via intravenous, intramuscular, or intranasal routes. drugs that were permitted to be titrated were excluded. an error was defined as greater than 25% above or below the recommended mg/kg dosage. results: out of 248,596 total patients, 13,321 were pediatric patients. 7885 had documented weights of <36 kg and 241 patients were given these medications. we excluded 72 patients for weight above the 97%ile or below the 3%ile, or if the weight documentation was missing. of the 169 patients and 187 doses, errors were noted in 53 (28%; 95% ci 22%, 35%). midazolam was the most common drug in errors (29 of 53 doses or 55%; 95% ci 40%, 68%), followed by diphenhydramine (11/53 or 21%; 95% ci 11%, 34%), epinephrine (7/53 or 13%; 95% ci 5%, 25%), and morphine sulfate (6/53 or 11%; 95% ci, 4%, 23%). underdosing was noted in 34 of 53 (64%; 95% ci 50%, 77%) of errors, while excessive dosing was noted in 19 of 53 (36%; 95% ci 23%, 50%). conclusion: weight-based dosing errors in pediatric patients are common. while the clinical consequences of drug dosing errors in these patients are unknown, a considerable amount of inaccuracy occurs. strategies beyond provision of reference materials are needed to prevent pediatric medication errors and reduce the potential for adverse outcomes. drivers background: homelessness affects up to 3.5 million people a year. the homeless present more frequently to eds, their ed visits are four times more likely to occur within 3 days of a prior ed evaluation, and they are admitted up to five times more frequently than others. we evaluated the effect of a street outreach rapid response team (sorrt) on the health care utilization of a homeless population. a nonmedical outreach staff responds to the ed and intensely case manages the patient: arranges primary care follow-up, social services, temporary housing opportunities, and drug/ alcohol rehabilitation services. objectives: we hypothesized that this program would decrease the ed visits and hospital admissions of this cohort of patients. methods: before and after study at an urban teaching hospital from june, 2010-december, 2011 in indianapolis, indiana. upon identification of homeless status, sorrt was immediately notified. eligibility for sorrt enrollment is determined by housing and urban development homeless criteria and the outreach staff attempted to enter all such identified patients into the program. the patients' health care utilization was evaluated in the 6 months prior to program entry as compared to the 6 months after enrollment by prospectively collecting data and a retrospective medical record query for any unreported visits. since the data were highly skewed, we used the nonparametric signed rank test to test for paired differences between periods. results: 22 patients met criteria but two refused participation. the 20-patient cohort had 388 total ed visits (175 pre and 213 post) with a mean of 8.8 (sd 10.1) and median of 6.5 (range 1-44) ed visits in 6 months pre-sorrt as compared to a mean of 10.7 (sd 19.5) and median of 5.0 (0-90) in 6 months post-sorrt (p = 0.815). there were 28 total inpatient admissions pre-intervention and 27 post-intervention, with a mean of 1.4 (sd 2.0) and median of 0.5 (0.7) per patient in the pre-intervention period as compared to 1.4 (sd 1.9) and 1.0 (0-6) in the post-intervention period (p = 0.654). in the pre-sorrt period 50.0% had at least one inpatient admission as compared to 55.0% post-sorrt (p = 1.00). there were no differences in icu days or overall length of stay between the two periods. conclusion: an aggressive case management program beginning immediately with homeless status recognition in the ed has not demonstrated success in decreasing utilization in our population. methods: this was a secondary analysis of a prospective randomized trial that included consenting patients discharged with outpatient antibiotics from an urban county ed with an annual census of 100,000. patients unable to receive text messages or voice-mails were excluded. health literacy was assessed using a validated health literacy assessment, the newest vital sign (nvs). patients were randomized to a discharge instruction modality: 1) standard care, typed and verbal medication and case-specific instructions; 2) standard care plus text-messaged instructions sent to the patient's cell phone; or 3) standard care plus voice-mailed instructions sent to the patient's cell. patients were called at 30 days to determine preference for instruction delivery modality. preference for discharge instruction modality was analyzed using z-tests for proportions. results: 758 patients were included (55% female, median age 30, range 5 months to 71 years); 98 were excluded. 23% had an nvs score of 0-1, 31% 2-3, and 46% 4-6. among the 51.1% of participants reached at 30 days, 26% preferred a modality other than written. there was a difference in the proportion of patients who preferred discharge instructions in written plus another modality (see table) . with the exception of written plus another modality, patient preference was similar across all nvs score groups. conclusion: in this sample of urban ed patients, more than one in four patients prefer non-traditional (text message, voice-mail) modalities of discharge instruction delivery to standard care (written) modality alone. additional research is needed to evaluate the effect of instructional modality on accessibility and patient compliance. figure) . conclusion: cumulative saps ii scoring fails to predict mortality in ohca. the risk scores assigned to age, gcs, and hco 3 independently predict mortality and combined are good mortality predictors. these findings suggest that an alternative severity of illness score should be used in post-cardiac arrest patients. future studies should determine optimal risk scores of saps ii variables in a larger cohort of ohca. objectives: to determine the extent to which cpp recovers to pre-pause levels with 20 seconds of cpr after a 10-second interruption in chest compressions for ecg rhythm analysis. methods: this was a secondary analysis of prospectively collected data from an iacuc-approved protocol. fortytwo yorkshire swine (weighing 25-30 kg) were instrumented under anesthesia. vf was electrically induced. after 12 minutes of untreated vf, cpr was initiated and a standard dose of epinephrine (sde) (0.01 mg/kg) was given. after 2.5 minutes of cpr to circulate the vasopressor, compressions were interrupted for 10 seconds to analyze the ecg rhythm. this was immediately followed by 20 seconds of cpr to restore cpp before the first rs was delivered. if the rs failed, cpr resumed and additional vasopressors (sde, and vasopressin 0.57 mg/kg) were given and the sequence repeated. the cpp was defined as aortic diastolic pressure minus right atrial diastolic pressure. the cpp values were extracted at three time points: immediately after the 2.5 minutes of cpr, following the 10-second pause, and immediately before defibrillation for the first two rs attempts in each animal. eighty-three sets of measurements were logged from 42 animals. descriptive statistics were used to analyze the data. in most cities, the proportion of patients who achieve prehospital return of spontaneous circulation (rosc) is less than 10%. the association between time of day and ohca outcomes in the prehospital setting is unknown. objectives: we sought to determine whether rates of prehospital rosc varied by time of day. we hypothesized that night ohcas would exhibit lower rates of rosc. methods: we performed a retrospective review of cardiac arrest data from a large, urban ems system. included were all ohcas occurring in individuals >18 years of age from 1/1/2008 to 12/31/2010. excluded were traumatic arrests and cases where resuscitation measures were not performed. day was defined as 7:00 am-6:59 pm, while night was 7:00 pm-6:59 am. we examined the association between time of day and paramedic-perceived prehospital rosc in unadjusted and adjusted analyses. variables included age, sex, race, presenting rhythm, aed application by a bystander or first responder, defibrillation, and bystander cpr performance. analyses were performed using chisquare tests and logistic regression. objectives: determine whether a smei helps to improve physician compliance with ihi bundle and reduce patient mortality in ed patients with s&s. methods: we conducted a pre-smei retrospective review of four months of ed patients with s&s to determine baseline pre-smei physician compliance and patient mortality. we designed and completed a smei attended by 25 of 28 ed attending physicians and 28 of 30 ed resuscitation residents. finally, we conducted a twenty-month post-smei prospective study of ongoing physician compliance and patient mortality in ed patients with s&s. results: in the four month pre-smei retrospective review, we identified 23 patients with s&s, with a 61% physician overall compliance and mortality rate of 30%. the average ed physician smei multiple-choice pre-test score was 74%, and showed a significant improvement in the post-test score of 94% (p = 0.0003). additionally, 87% of ed physicians were able to describe three new clinical pearls learned and 85% agreed that the smei would improve compliance. in the twenty months of the post-smei prospective study, we identified 144 patients with s&s, with a 75% physician overall compliance, and mortality rate of 21%. relative physician compliance improved 23% (p = 0.0001) and relative patient mortality was reduced by 32% (p < 0.0001) when comparing pre-and post-smei data. conclusion: our data suggest that a smei improves overall physician compliance with the six hour goals of the ihi bundle and reduces patient mortality in ed patients with s&s. conclusion: using a population-level, longitudinal, and multi-state analysis, the rate of return visits within 3 days is higher than previously reported, with nearly 1 in 12 returning back to the ed. we also provide the first estimation of health care costs for ed revisits. background: the ability of patients to accurately determine their level of urgency is important in planning strategies that divert away from eds. in fact, an understanding of patient self-triage abilities is needed to inform health policies targeting how and where patients access acute care services within the health care system. objectives: to determine the accuracy of a patient's self-assessment of urgency compared against triage nurses. methods: setting: ed patients are assigned a score by trained nurses according to the canadian emergency department triage and acuity scale (ctas). we present a cross-sectional survey of a random patient sample from 12 urban/regional eds conducted during the winters of 2007 and 2009. this previously validated questionnaire, based on the british healthcare commission survey, was distributed according to a modified dillman protocol. exclusion criteria consisted of: age 0-15 years, left prior to being seen/treated, died during ed visit, no contact information, presented with a privacy-sensitive case. alberta health services provided linked non-survey administrative data. results: 21,639 surveys distributed with a response rate of 46%. patients rated health problems as life-threatening (6%), possibly life-threatening (22%), urgent (30%), somewhat urgent (37%), or not urgent (5%). triage nurses assigned the same patients ctas scores of i (<1%), ii (20%), iii (45%), iv (29%) or v (5%). patients self-rated their condition as 3 or 4 points less urgent than the assigned ctas score (<1% of the time), 2 points less urgent (5%), 1 point less urgent (25%), exactly as urgent (38%), 1 point more urgent (24%), 2 points more urgent (7%), or 3 or 4 points more urgent (1%, respectively). among ctas i or ii patients, 54% described their problem as life-threatening/possibly life-threatening, 26% as urgent (risk of permanent damage), 18% as urgent (needed to be seen that day), and 2% as not urgent (wanted to be but did not need to be seen that day). conclusion: the majority of ed patients are generally able to accurately assess the acuity of their problem. encouraging patients with low-urgency conditions to self-triage to lower-acuity sources of care may relieve stress on eds. however, physicians and patients must be aware that a small minority of patients are unable to self-triage safely. when the tourniquet was released, blood spurted from the injured artery as hydrostatic pressure decayed. pressure and flow were recorded in three animals (see table) . the concept was proof-tested in a single fresh frozen human cadaver with perfusion through the femoral artery and hemorrhage from the popliteal artery. the results were qualitatively and quantitatively similar to the swine carcass model. conclusion: a perfused swine carcass can simulate exsanguinating hemorrhage for training purposes and serves as a prototype for a fresh-frozen human cadaver model. additional research and development are required before the model can be widely applied. background: in the pediatric emergency department (ped), clinicians must work together to provide safe and effective care. crisis resource management (crm) principles have been used to improve team performance in high-risk clinical settings, while simulation allows practice and feedback of these behaviors. objectives: to develop a multidisciplinary educational program in a ped using simulation-enhanced teamwork training to standardize communication and behaviors and identify latent safety threats. methods: over 6 months a workgroup of physicians and nurses with experience in team training and simulation developed an educational program for clinical staff of a tertiary ped. goals included: create a didactic curriculum to teach the principles of crm, incorporate principles of crm into simulation-enhanced team training in-situ and center-based exercises, and utilize assessment instruments to evaluate for teamwork, completion of critical actions, and presence of latent safety threats during in-situ sim resuscitations. results: during phase i, 130 clinicians, divided into teams, participated in 90-minute pre-training assessments of pals-based in-situ simulations. in phase ii, staff participated in a 6-hour curriculum reviewing key crm concepts, including team training exercises utilizing simulation and expert debriefing. in phase iii, staff participated in post-training 90 minute teamwork and clinical skills assessments in the ped. in all phases, critical action checklists (cac) were tabulated by simulation educators. in-situ simulations were recorded for later review using the assessment tools. after each simulation, educators facilitated discussion of perceptions of teamwork and identification of systems issues and latent hazards. overall, 54 in-situ simulations were conducted capturing 97% of the physicians and 84% of the nurses. cac data were collected by an observer and compared to video recordings. over 20 significant systems issues, latent hazards, and knowledge deficits were identified. all components of the program were rated highly by 90% of the staff. conclusion: a workgroup of pem, simulation, and team training experts developed a multidisciplinary team training program that used in-situ and centerbased simulation and a refined crm curriculum. unique features of this program include its multidisciplinary focus, the development of a variety of assessment tools, and use of in-situ simulation for evaluation of systems issues and latent hazards. this program was tested in a ped and findings will be used to refine care and develop a sustainment program while addressing issues identified. objectives: our hypothesis is that participants trained on high-fidelity mannequins will perform better than participants trained on low-fidelity mannequins on both the acls written exam and in performance of critical actions during megacode testing. the study was performed in the context of an acls initial provider course for new pgy1 residents at the penn medicine clinical simulation center and involved three training arms: 1) low fidelity (low-fi): torso-rhythm generator; 2) mid-fidelity (mid-fi): laerdal simmanò turned off; and 3) high-fidelity (high-fi): laerdal simmanò turned on. training in each arm of the study followed standard aha protocol. educational outcomes were evaluated by written scores on the acls written examination and expert rater reviews of acls megacode videos performed by trainees during the course. a sample of 54 subjects were randomized to one of the three training arms: low-fi (n = 18), mid-fi (n = 18), or high-fi (n = 18). results: statistical significance across the groups was determined using analysis-of-variance (anova). the three groups had similar written pre-test scores [low-fi 0.4 (0.1), mid-fi 0.5 (0.1), and high-fi 0.4 (0. 2)] and written post-test scores [low-fi 0.9 (0.1), mid-fi 0.9 (0.1), and high-fi 0.8 (0.1)]. similarly, test improvement was not significantly different. after completion of the course, high-fi subjects were more likely to report they felt comfortable in their simulator environment (p = 0.005). low-fi subjects were less likely to perceive a benefit in acls training from high-fi technology (p < 0.001). acls instructors were not rated significantly different by the subjects using the debriefing assessment for simulation in healthcareª (dash) student version except for element 6, where the high-fi group subjects reported lower scores (6.1 vs 6.6 and 6.7 in the other groups, p = 0.046). objectives: we sought to determine if stress associated with the performance of a complex procedural task can be affected by level of medical training. heart rate variability (hrv) is used as a measure of autonomic balance, and therefore an indicator of the level of stress. methods: twenty-one medical students and emergency medicine residents were enrolled. participants performed airway procedures on an airway management trainer. hrv data were collected using a continuous heart rate variability monitoring system. participant hrv was monitored at baseline, during the unassisted first attempt at endotracheal intubation, during supervised practice, and then during a simulated respiratory failure clinical scenario. standard deviation of beat to beat variability (sdnn), very low frequency (vlf), total power (tp), and low frequency (lf) was analyzed to determine the effect of practice and level of training on the level of stress. a cohen's d test was used to determine differences between study groups. results: sdnn data showed that second-year residents were less stressed during all stages than were fourthyear medical students (avg d = 1.12). vlf data showed third-year residents exhibited less sympathetic activity than did first-year residents (avg d = )0.68). the opportunity to practice resulted in less stress for all participants. tp data showed that residents had a greater degree of control over their autonomic nervous system (ans) than did medical students (avg d = 0.85). lf data showed that subjects were more engaged in the task at hand as the level of training increased indicating autonomic balance (avg d = 0.80). conclusion: our hrv data show that stress associated with the performance of a complex procedural task is reduced by increased training. hrv may provide a quantitative measure of physiologic stress during the learning process and thus serve as a marker of when a subject is adequately trained to perform a particular task. objectives: we seek to examine whether intubation during cpr can be done as efficiently as intubation without ongoing cpr. the hypothesis is that the predictable movement of an automated chest compression device will make intubation easier than the random movement from manual cpr. methods: the project was an experimental controlled trial and took place in the emergency department at a tertiary referral center in peoria, illinois. emergency medicine residents, attendings, paramedics, and other acls trained staff were eligible for participation. in randomized order, each participant attempted intubation on a mannequin with no cpr ongoing, during cpr with a human compressor, and during cpr with an automatic chest compression device (physio control lucas 2). participants could use whichever style laryngoscope they felt most comfortable with and they were timed during the three attempts. success was determined after each attempt. results: there were 43 participants in the trial. the success rate in the control group and the automated cpr group were both 88% (38/43) and the success rate in the manual cpr group was 74% (32/43). the differences in success rates were not statistically significant (p = 0.99 and p = 0.83). the automated cpr group had the fastest average time (13.6 sec; p = 0.019). the mean times for intubation with manual cpr and no cpr were not statistically different (17.1 sec, 18.1 sec; p = 0.606). conclusion: the success rate of tracheal intubation with ongoing chest compression was the same as the success rate of intubation without cpr. although intubation with automatic chest compression was faster than during other scenarios, all methods were close to the 10 second timeframe recommended by acls. based on these findings, it may not always be necessary to hold cpr to place a definitive airway; however, further studies will be needed. background: after acute myocardial infarction, vascular remodeling in the peri-infarct area is essential to provide adequate perfusion, prevent additional myocyte loss, and aid in the repair process. we have previously shown that endogenous fibroblast growth factor 2 (fgf2) is essential to the recovery of contractile function and limitation of infarct size after cardiac ischemia-reperfusion (ir) injury. the role of fgf2 in vascular remodeling in this setting is currently unknown. objectives: determine the role of endogenous fgf2 in vascular remodeling in a clinically relevant, closed-chest model of acute myocardial infarction. methods: mice with a targeted ablation of the fgf2 gene (fgf2 knockout) and wild type controls were subjected to a closed-chest model of regional cardiac ir injury. in this model, mice were subjected to 90 minutes of occlusion of the left anterior descending artery followed by reperfusion for either 1 or 7 days. immunofluorescence was performed on multiple histological sections from these hearts to visualize capillaries (endothelium, anti-cd31 antibody), larger vessels (venules and arterioles, antismooth muscle actin antibody), and nuclei (dapi). digital images were captured, and multiple images from each heart were measured for vessel density and vessel size. results: sham-treated fgf2 knockout and wild type mice show no differences in capillary or vessel density suggesting no defect in vessel formation in the absence of endogenous fgf2. when subjected to closed-chest regional cardiac ir injury, fgf2 knockout hearts had normal capillary and vessel number and size in the peri-infarct area after 1 day of reperfusion compared to wild type controls. however, after 7 days, fgf2 knockout hearts showed significantly decreased capillary and vessel number and increased vessel size compared to wild type controls (p < 0.05). conclusion: these data show the necessity of endogenous fgf2 in vascular remodeling in the peri-infarct zone in a clinically relevant animal model of acute myocardial infarction. these findings may suggest a potential role for modulation of fgf2 signaling as a therapeutic intervention to optimize vascular remodeling in the repair process after myocardial infarction. the diagnosis of aortic dissections by ed physicians is rare scott m. alter, barnet eskin, john r. allegra morristown medical center, morristown, nj background: aortic dissection is a rare event. the most common symptom of dissection is chest pain, but chest pain is a frequent emergency department (ed) chief complaint and other diseases that cause chest pain, such as acute coronary syndrome and pulmonary embolism, occur much more frequently. furthermore, 20% of dissections are without chest pain and 6% are painless. for all these reasons, diagnosing dissection can be difficult for the ed physician. we wished to quantify the magnitude of this problem in a large ed database. objectives: our goal was to determine the number of patients diagnosed by ed physicians with aortic dissections compared to total ed patients and to the total number of patients with a chest pain diagnosis. methods: design: retrospective cohort. setting: 33 suburban, urban, and rural new york and new jersey eds with annual visits between 8,000 and 75,000. participants: consecutive patients seen by ed physicians from january 1, 1996 through december 31, 2010. observations: we identified aortic dissections using icd-9 codes and chest pain diagnoses by examining all icd-9 codes used over the period of the study and selecting those with a non-traumatic chest pain diagnosis. we then calculated the number of total ed patients and chest pain patients for every aortic dissection diagnosed by emergency physicians. we determined 95% confidence intervals (cis). results: from a database of 9.5 million ed visits, we identified 782 (0.0082%) aortic dissections, or one for every 12,200 (95% ci 11,400 to 13,100) visits. the mean age of aortic dissection patients was 58 ± 19 years and 57% were female. of the total visits there were 763,000 (8%) with a chest pain diagnosis. thus there is one aortic dissection diagnosis for every 980 (95% ci 910 to 1,050) chest pain diagnoses. conclusion: the diagnosis of aortic dissections by ed physicians is rare. an ed physician seeing 3,000 to 4,000 patients a year would diagnose an aortic dissection approximately once every 3 to 4 years. an aortic dissection would be diagnosed once for approximately every 1,000 ed chest pain patients. patients were excluded if they suffered a cardiac arrest, were transferred from another hospital, or if the ccl was activated for an inpatient or from ems in the field. fp ccl activation was defined as 1) a patient for whom activation was cancelled in the ed and ruled out for mi or 2) a patient who went to catheterization but no culprit vessel was identified and mi was excluded. ecgs for fp patients were classified using standard criteria. demographic data, cardiac biomarkers, and all relevant time intervals were collected according to an on-going quality assurance protocol. results: a total of 506 ccl activations were reviewed, with 68% male, average age 57, and 59% black. there were 210 (42%) true stemis and 86 (17%) fp activations. there were no significant differences between the fp patients who did and did not have catheterization. for those fp patients who had a catheterization (13%), ''door to page'' and ''door to lab'' times were significantly longer than the stemi patients (see table) , but there was substantial overlap. there was no difference in sex or age, but fp patients were more likely to be black (p = 0.02). a total of 82 fp patients had ecgs available for review; findings included anterior elevation with convex (21%) or concave (13%) elevation, st elevation from prior anterior (10%) or inferior (11%) mi, pericarditis (16%), presumed new lbbb (15%), early repolarization (5%), and other (9%). conclusion: false ccl activation occurred in a minority of patients, most of whom had ecg findings warranting emergent catheterization. the rate of false ccl activation appears acceptable. background: atrial fibrillation (af) is the most common cardiac arrhythmia treated in the ed, leading to high rates of hospitalization and resource utilization. dedicated atrial fibrillation clinics offer the possibility of reducing the admission burden for af patients presenting to the ed. while the referral base for these af clinics is growing, it is unclear to what extent these clinics contribute to reducing the number of ed visits and hospitalizations related to af. objectives: to compare the number of ed visits and hospitalizations among discharged ed patients with a primary diagnosis of af who followed up with an af clinic and those who did not. methods: a retrospective cohort study and medical records review including three major tertiary centres in calgary, canada. a sample of 600 patients was taken representing 200 patients referred to the af clinic from the calgary zone eds and compared to 400 matched control ed patients who were referred to other providers for follow-up. the controls were matched for age and sex. inclusion criteria included patients over 18 years of age, discharged during the index visit, and seen by the af clinic between january 1, 2009 and october 25, 2010. exclusion criteria included non-residents and patients hospitalized during the index visit. the number of cardiovascular-related ed visits and hospitalizations was measured. all data are categorical, and were compared using chi-square tests. results: patients in the control and af clinic cohorts were similar for all baseline characteristics except for a higher proportion of first episode patients in the intervention arm. in the six months following the index ed visit, 55 study group patients (27.5%) visited an ed on 95 occasions, and 12 (6%) were hospitalized on 16 occasions. of the control group, 122 patients (30.5%) visited an ed on 193 occasions, and 44 (11%) were hospitalized on 55 occasions. using a chi-square test we found no significant difference in ed visits (p = 0.5063) or hospitalizations (p = 0.0664) between the control and af clinic cohorts. conclusion: based on our results, referral from the ed to an af clinic is not associated with a significant reduction in subsequent cardiovascular related ed visits and hospitalizations. due to the possibility of residual confounding, randomized trials should be performed to evaluate the efficacy of af clinics. reported an income of less than $10,000. there were no significant associations between sex, race, marital status, education level, income, insurance status, and subsequent 30-and-90 day readmission rates. hla score was not found to be significantly related to readmission rates. the mean hla score was 18.9 (sd = 7.87), equivalent to less than 6th grade literacy, meaning these patients may not be able to read prescription labels. for each unit increase in hfkt score, the odds of being readmitted within 30 days decreased by 0.219 (p < 0.001) and for 31-90 days decreased by 0.440 (p < 0.001). for each unit increase in scbs score, the odds of being readmitted within 90 days decreased by 0.949 (p = 0.038). conclusion: health care literacy in our patient population is not associated with readmission, likely related to the low literacy rate of our study population. better hf knowledge and self-care behaviors are associated with lower readmission rates. greater emphasis should be placed on patient education and self-care behaviors regarding hf as a mechanism to decrease readmission rates. comparison of door to balloon times in patients presenting directly or transferred to a regional heart center with stemi jennifer ehlers, adam v. wurstle, luis gruberg, adam j. singer stony brook university, stony brook, ny background: based on the evidence, a door-to-balloon-time (dtbt) of less than 90 minutes is recommended by the aha/acc for patients with stemi. in many regions, patients with stemi are transferred to a regional heart center for percutaneous coronary intervention (pci). objectives: we compared dtbt for patients presenting directly to a regional heart center with those for patients transferred from other regional hospitals. we hypothesized that dtbt would be significantly longer for transferred patients. methods: study design-retrospective medical record review. setting-academic ed at a regional heart center with an annual census of 80,000 that includes a catchment area of 12 hospitals up to 50 miles away. patients-patients with acute stemi identified on ed 12-lead ecg. measures-demographic and clinical data including time from triage to ecg, from ecg to activation of regional catheterization lab, and from initial triage to pci (dtbt , and door to intravascular balloon deployment (d2b). methods: the study was performed in an inner-city academic ed between 1/1/07 and 12/31/10. every patient for whom ed activation of our stemi system occurred was included. all times data from a pre-existing quality assurance database were collected prospectively. patient language was determined retrospectively by chart review. results: there were 132 patients between 1/1/07 and 12/31/10. 21 patients (16%) were deemed too sick or unable to provide history and were excluded, leaving 111 patients for analysis. 85 (77%) spoke english and 26 (23%) did not. in the non-english group, chinese was the most common language, in 22 (20%) background: syncope is a common, potentially highrisk ed presentation. hospitalization for syncope, although common, is rarely of benefit. no populationbased study has examined disparities in regional admission practices for syncope care in the ed. moreover, there are no population-based studies reporting prognostic factors for 7-and 30-day readmission of syncope. objectives: 1) to identify factors associated with admission as well as prognostic factors for 7-and 30-day readmission to these hospitals; 2) to evaluate variability in syncope admission practices across different sizes and types of hospitals. methods: design -multi-center retrospective cohort study using ed administrative data from 101 albertan eds. participants/subjects -patients >17 years of age with syncope (icd10: r55) as a primary or secondary diagnosis from 2007 to june 2011. readmission was defined as return visits to the ed or admission <7 days or 7-30 days after the index visit (including against medical advice and left without being seen during the index visit). outcomes -factors associated with hospital admission at index presentation, and readmission following ed discharge, adjusted using multivariable logistic regression. results: overall, 44521 syncope visits occurred over 4 years. increased age, increased length of stay (los), performance of cxr, transport by ground ambulance, and treatment at a low-volume hospital (non-teaching or non-large urban) were independently associated with index hospitalization. these same factors, as well as hospital admission itself, were associated with 7-day readmission. additionally, increased age, increased los, performance of a head ct, treatment at a low-volume hospital, hospital admission, and female sex were independently associated with 7-30 day readmission. arrival by ground ambulance was associated with a decreased likelihood of both 7-and 7-30 day readmission. conclusion: our data identify variations in practice as well as factors associated with hospitalization and readmission for syncope. the disparity in admission and readmission rates between centers may highlight a gap in quality of care or reflect inappropriate use of resources. further research to compare patient out-comes and quality of patient care among urban and non-urban centers is needed. background: change in dyspnea severity (ds) is a frequently used outcome measure in trials of acute heart failure (ahf). however, there is limited information concerning its validity. objectives: to assess the predictive validity of change in dyspnea severity. methods: this was a secondary analysis of a prospective observational study of a convenience sample of ahf patients presenting with dyspnea to the ed of an academic tertiary referral center with a mixed urban/ suburban catchment area. patients were enrolled weekdays, june through december 2006. patients assessed their ds using a 10-cm visual analog scale at three times: the start of ed treatment (baseline) as well as at 1 and 4 hours after starting ed treatment. the difference between baseline and 1 hour was the 1-hour ds change. the difference between baseline and 4 hours was the 4-hour ds change. two clinical outcome measures were obtained: 1) the number of days hospitalized or dead within 30 days of the index visit (30-day outcome), and 2) the number of days hospitalized or dead within 90 days of the index visit (90-day outcome). results: data on 86 patients were analyzed. the median 30-day outcome variable was 6 days with an interquartile range (iqr) of 3 to 16. the median 90-day outcome variable was 10 days (iqr 4 to 27.5). the median 1-hour ds change was 2.6 cm (iqr 0.3 to 6.7). the median 4-hour ds change was 4.9 cm (iqr 2.2 to 8.2). the 30-day and 90-day mortality rates were 9% and 13% respectively. the spearman rank correlations and 95% confidence intervals are presented in the table below. conclusion: while the point estimates for the correlations were below 0.5, the 95% ci for two of the correlations extended above 0.5. these pilot data support change in ds as a valid outcome measure for ahf when measured over 4 hours. a larger prospective study is needed to obtain a more accurate point estimate of the correlations. background: the majority of volume-quality research has focused on surgical outcomes in the inpatient setting; very few studies have examined the effect of emergency department (ed) case volume on patient outcomes. objectives: to determine whether ed case volume of acute heart failure (ahf) is associated with short-term patient outcomes. methods: we analyzed the 2008 nationwide emergency department sample (neds) and nationwide inpatient sample (nis), the largest, all-payer, ed and inpatient databases in the us. ed visits for ahf were identified with a principal diagnosis of icd-9-cm code 428.xx. eds were categorized into quartiles by ed case volume of ahf. the outcome measures were early inpatient mortality (within the first 2 days of admission), overall inpatient mortality, and hospital length of stay (los). results: there were an estimated 946,000 visits for ahf from approximately 4,700 eds in 2008; 80% were hospitalized. of these, the overall inpatient mortality rate was 3.2%, and the median hospital los was 4 days. early inpatient mortality was lower in the highest-volume eds, compared with the lowest-volume eds (0.8% vs. 2.1%; p < 0.001). similar patterns were observed for overall inpatient mortality (3.0% vs. 4.1%; p < 0.001). in a multivariable analysis adjusting for 37 patient and hospital characteristics, early inpatient mortality remained lower in patients admitted through the highest-volume eds (adjusted odds ratios [or], 0.70; 95% confidence interval [ci], 0.52-0.96), as compared with the lowest-volume eds. there was a trend towards lower overall inpatient mortality in the highest-volume eds; however, this was not statistically significant (adjusted or, 0.92; 95%ci, 0.75-1.14). by contrast, using the nis data including various sources of admissions, a higher case volume of inpatient ahf patients predicted lower overall inpatient mortality (adjusted or, 0.51; 95%ci, 0.40-0.65). the hospital los in patients admitted through the highest-volume eds was slightly longer (adjusted difference, 0.7 day; 95%ci, 0.2-1.2), compared with the lowest-volume eds. conclusion: ed patients who are hospitalized for ahf have an approximately 30% reduced early inpatient mortality if they were admitted from an ed that handles a large volume of ahf cases. the ''practice-makesperfect'' concept may hold in emergency management of ahf. emergency department disposition and charges for heart failure: regional variability alan b. storrow, cathy a. jenkins, sean p. collins, karen p. miller, candace mcnaughton, naftilan allen, benjamin s. heavrin vanderbilt university, nashville, tn background: high inpatient admission rates for ed patients with acute heart failure are felt partially responsible for the large economic burden of this most costly cardiovascular problem. objectives: we examined regional variability in ed disposition decisions and regional variability in total dollars spent on ed services for admitted patients with primary heart failure. methods: the 2007 nationwide emergency department sample (neds) was used to perform a retrospective, cohort analysis of patients with heart failure (icd-9 code of 428.x) listed as the primary ed diagnosis. demographics and disposition percentages (with se) were calculated for the overall sample and by region: northeast, south, midwest, and west. to account for the sample design and to obtain national and regional estimates, a weighted analysis was conducted. results: there were 941,754 weighted ed visits with heart failure listed as the primary diagnosis. overall, over eighty percent were admitted (see table) . fifty-two percent of these patients were female; mean age was 72.7 years (se 0.20). hospitalization rates were higher in the northeast (89.1%) and south (81.2%) than in the midwest (76.0%) and west (74.8%). total monies spent on ed services were highest in the south ($69,078,042) followed by the northeast ($18,233,807), west ($6,360,315) and midwest ($5,899,481) . conclusion: this large retrospective ed cohort suggests a very high national admission rate with significant regional variation in both disposition decisions as well as total monies spent on ed services for patients with a primary diagnosis of heart failure. examining these estimates and variations further may provide strategies to reduce the economic burden of heart failure. background: workplace violence in health care settings is a frequent occurrence. gunfire in hospitals is of particular concern. however, information regarding such workplace violence is limited. accordingly, we characterized u.s. hospital-based shootings from 2000-2010. objectives: to determine extent of hospital-based shootings in the u.s. and involvement of emergency departments. methods: using lexisnexis, google, netscape, pub-med, and sciencedirect, we searched reports for acute care hospital shooting events from january 2000 through december 2010, and those with at least one injured victim were analyzed. results: we identified 140 hospital-related shootings (86 inside the hospital, 54 on hospital grounds), in 39 states, with 216 victims, of whom 98 were perpetrators. in comparison to external shootings, shootings within the hospital have not increased over time (see figure) . perpetrators were from all age groups, including the elderly. most of the events involved a determined shooter: grudge (26%), suicide (19%), ''euthanizing'' an ill relative (15%), and prisoner escape (12%). ambient societal violence (8%) and mentally unstable patients (4%) were comparatively infrequent. the most common injured was the perpetrator (45%). hospital employees comprised only 21% of victims; physician (3%) and nurse (5%) victims were relatively infrequent. the emergency department was the most common site (29%), followed by patient rooms (20%) and the parking lot (20%). in 13% of shootings within hospitals, the weapon was a security officer's gun grabbed by the perpetrator. ''grudge'' motive was the only factor determinative of hospital staff victims (or = 4.34, 95% ci 1.85-10.17). conclusion: although hospital-based shootings are relatively rare, emergency departments are the most likely site. the unpredictable nature of this type of event represents a significant challenge to hospital security and deterrence practices, as most perpetrators proved determined, and many hospital shootings occur outside the building. impact of emergency physician board certification on patient perceptions of ed care quality albert g. sledge iv 1 , carl a. germann 1 , tania d. strout 1 , john southall 2 1 maine medical center, portland, me; 2 mercy hospital, portland, me background: the hospital value-based purchasing program mandated by the affordable care act is the latest example of how patients' perceptions of care will affect the future practice environment of all physicians. the type of training of medical providers in the emergency department (ed) is one possible factor affecting patient perceptions of care. a unique situation in a maine community ed led to the rapid transition from non-emergency medicine (em) residency trained physicians to all em residency trained and american board of emergency medicine (abem) certified providers. objectives: the purpose of this study was to evaluate the effect of the implementation of an all em-trained, abem-certified physician staff on patient perceptions of the quality of care they received in the ed. methods: we retrospectively evaluated press ganey data from surveys returned by patients receiving treatment in a single, rural ed. survey items addressed patient's perceptions of physician courtesy, time spent listening, concern for patient comfort, and informativeness. additional items evaluated overall perceptions of care and the likelihood that the respondent would recommend the ed to another. data were compared for the three years prior to and following implementation of the all trained, certified staff. we used the independent samples t-test to compare mean responses during the two time periods. bonferroni's correction was applied to adjust for multiple comparisons. results: during the study period, 3,039 patients provided surveys for analysis: 1,666 during the pre-certification phase and 1,373 during the post-certification phase. across all six survey items, mean responses increased following transition to the board-certified staff. these improvements were noted to be statistically significant in each case: courtesy p < 0.001, time listening p < 0.001, concern for comfort p < 0.001, informativeness p < 0.001, overall perception of care p < 0.001, and likelihood to recommend p < 0.001. conclusion: data from this community ed suggest that transition from a non-residency trained, abem certified staff to a fully trained and certified model has important implications for patient's perceptions of the care they receive. we observed significant improvement in rating scores provided by patients across all physicianoriented and general ed measures. background: transfer of care from the ed to the inpatient floor is a critical transition when miscommunication places patients at risk. the optimal form and content of handoff between providers has not been defined. in july 2011, ed-to-floor signout for all admissions to the medicine and cardiology floors was changed at our urban, academic, tertiary care hospital. previously, signout was via an unstructured telephone conversation between ed resident and admitting housestaff. the new signout utilizes a web-based ed patient tracking system and includes: 1) a templated description of ed course is completed by the ed resident; 2) when a bed is assigned, an automated page is sent to the admitting housestaff; 3) ed clinical information, including imaging, labs, medications, and nursing interventions (figure) is reviewed by admitting housestaff; 4) if housestaff has specific questions about ed care, a telephone conversation between the ed resident and housestaff occurs; 5) if there are no specific questions, it is indicated electronically and the patient is transferred to the floor. objectives: to describe the effects on patient safety (floor-to-icu transfer in 24 hours) and ed throughput (ed length of stay (los) and time from bed assignment to ed departure) resulting from a change to an electronic, discussion-optional handoff system. conclusion: transition to a system in which signout of admitted patients is accomplished by accepting housestaff review of ed clinical information supplemented by verbal discussion when needed resulted in no significant change in rate of floor-to-icu transfer or ed los and reduced time from bed assignment to ed departure. background: emergency physicians may be biased against patients presenting with nonspecific complaints or those requiring more extensive work-ups. this may result in patients being seen less quickly than those with more straightforward presentations, despite equal triage scores or potential for more dangerous conditions. objectives: the goal of our study was to ascertain which patients, if any, were seen more quickly in the ed based on chief complaint. methods: a retrospective report was generated from the emr for all moderate acuity (esi 3) adult patients who visited the ed from january 2005 through december 2010 at a large urban teaching hospital. the most common complaints were: abdominal pain, alcohol intoxication, back pain, chest pain, cough, dyspnea, dizziness, fall, fever, flank pain, headache, infection, pain (nonspecific), psychiatric evaluation, ''sent by md,'' vaginal bleeding, vomiting, and weakness. non-parametric independent sample tests assessed median time to be seen (ttbs) by a physician for each complaint. differences in the ttbs between genders and based on age were also calculated. chi-square testing compared percentages of patients in the ed per hour to assess for differences in the distribution of arrival times. results: we obtained data from 116,194 patients. patients with a chief complaint of weakness and dizziness waited the longest with a median time of 35 minutes and patients with flank pain waited the shortest with 24 minutes (p < 0.0001) ( figure 1 ). overall, males waited 30 minutes and females waited 32 minutes (p < 0.0001). stratifying by gender and age, younger females between the ages of 18-50 waited significantly longer times when presenting with a chief complaint of abdominal pain (p < 0.0001), chest pain (p < 0.05), or flank pain (p < 0.0001) as compared to males in the same age group ( figure 2 ). there was no difference in the distribution of arrival times for these complaints. conclusion: while the absolute time differences are not large, there is a significant bias toward seeing young male patients more quickly than women or older males despite the lower likelihood of dangerous conditions. triage systems should perhaps take age and gender better into account. patients might benefit from efforts to educate em physicians on the delays and potential quality issues associated with this bias in an attempt to move toward more egalitarian patient selection. background: detailed analysis of emergency department (ed) event data identified the time from completion of emergency physician evaluation (doc done) to the time patients leave the ed as a significant contributor to ed length of stay (los) and boarding at our institution. process flow mapping identified the time from doc done to the time inpatient beds were ordered (bo) as an interval amendable to specific process improvements. objectives: the purpose of this study was to evaluate the effect of ed holding orders for stable adult 3.6 (3.0 -4.1) 7.8 (6.9 -8.7) 15.2 (12.8 -17.6) 4.9 (2.8 -7.0) 17.3 (12.9 -21.7) 6.5 (4.5 -8.5) inpatient medicine (aim) patients on: a) the time to bo and b) ed los. methods: a prospective, observational design was used to evaluate the study questions. data regarding the time to bo and los outcomes were collected before and after implementation of the ed holding orders program. the intervention targeted stable aim patients being admitted to hospitalist, internal medicine, and family medicine services. ed holding orders were placed following the admission discussion with the accepting service and special attention was paid to proper bed type, completion of the emergent work-up and the expected immediate course of the patient's hospital stay. holding orders were of limited duration and expired 4 hours after arrival to the inpatient unit. results: during the 6-month study period, 7321 patients were eligible for the ed holding orders intervention; 6664 (91.0%) were cared for using the standard adult medicine order set and 657 (9.0%) received the intervention. the median time from doc done to bo was significantly shorter for patients in the ed holding orders group, 41 min (iqr 19, 88) vs. 95 min (iqr 53, 154) for the standard adult medicine group, p < 0.001. similarly, the median ed los was significantly shorter for those in the ed holding orders group, 413 min (iqr 331, 540) vs. 456 min (iqr 346, 581) for the standard adult medicine group, p < 0.001. no lapses in patient care were reported in the intervention group. conclusion: in this cohort of ed patients being admitted to an aim service, placing ed holding orders rather than waiting for a traditional inpatient team evaluation and set of admission orders significantly reduced the time from the completion of the ed workup to placement of a bo. as a result, ed los was also significantly shortened. while overall utilization of the intervention was low, it improved with each month. emergency department interruptions in the age of electronic health records matthew albrecht, john shabosky, jonathan de la cruz southern illinois university school of medicine, springfield, il background: interruptions of clinical care in the emergency department (ed) have been correlated with increased medical errors and decreased patient satisfaction. studies have also shown that most interruptions happen during physician documentation. with the advent of the electronic health record and computerized documentation, ed physicians now spend much of their clinical time in front of computers and are more susceptible to interruptions. voice recognition dictation adjuncts to computerized charting boast increased provider efficiency; however, little is known about how data input of computerized documentation affects physician interruptions. objectives: we present here observational interruptions data comparing two separate ed sites, one that uses computerized charting by conventional techniques and one assisted by voice recognition dictation technology. methods: a prospective observational quality initiative was conducted at two teaching hospital eds located less than 1 mile from each other. one site primarily uses conventional computerized charting while the other uses voice recognition dictation computerized charting. four trained observers followed ed physicians for 180 minutes during shifts. the tasks each ed physician performed were noted and logged in 30 second intervals. tasks listed were selected from a predetermined standardized list presented at observer training. tasks were also noted as either completed or placed in queue after a change in task occurred. a total of 4140 minutes were logged. interruptions were noted when a change in task occurred with the previous task being placed in queue. data were then compared between sites. results: ed physicians averaged 5.33 interruptions/ hour with conventional computerized charting compared to 3.47 interruptions/hour with assisted voice recognition dictation (p = 0.0165). conclusion: computerized charting assisted with voice recognition dictation significantly decreased total per hour interruptions when compared to conventional techniques. charting with voice recognition dictation has the potential to decrease interruptions in the ed allowing for more efficient workflow and improved patient care. background: using robot assistants in health care is an emerging strategy to improve efficiency and quality of care while optimizing the use of human work hours. robot prototypes capable of performing vital signs and assisting with ed triage are under development. however, ed users' attitudes toward robot assistants are not well studied. understanding of these attitudes is essential to design user-friendly robots and to prepare eds for the implementation of robot assistants. objectives: to evaluate the attitudes of ed patients and their accompanying family and friends toward the potential use of robot assistants in the ed. methods: we surveyed a convenience sample of adult ed patients and their accompanying adult family members and friends at a single, university-affiliated ed, 9/ 26/11-10/27/11. the survey consisted of eight items from the negative attitudes towards robots scale (normura et al.) modified to address robot use in the ed. response options included a 5-point likert scale. a summary score was calculated by summing the responses for all 8 items, with a potential range of 8 (completely negative attitude) to 40 (completely positive attitude). research assistants gave the written surveys to subjects during their ed visit. internal consistency was assessed using cronbach's alpha. bivariate analyses were performed to evaluate the association between the summary score and the following variables: participant type (patient or visitor), sex, race, time of day, and day of week. results: of 121 potential subjects approached, 113 (93%) completed the survey. participants were 37% patients, 63% family members or friends, 62% women, 79% white, and had a median age of 45.5 years (iqr 18-84). cronbach's alpha was 0.94. the mean summary score was 22.2 (sd = 0.87), indicating subjects were between ''occasionally'' and ''sometimes'' comfortable with the idea of ed robot assistants (see table) . men were more positive toward robot use than women (summary score: 24.6 vs 20.8; p = 0.033). no differences in the summary score were detected based on participant type, race, time of day, or day of week. conclusion: ed users reported significant apprehension about the potential use of robot assistants in the ed. future research is needed to explore how robot designs and strategies to implement ed robots can help alleviate this apprehension. background: emergency department cardioversion (edc) of recent-onset atrial fibrillation or flutter (af) patients is an increasingly common management approach to this arrhythmia. patients who qualify for edc generally have few co-morbidities and are often discharged directly from the ed. this results in a shift towards a sicker population of patients admitted to the hospital with this diagnosis. objectives: to determine whether hospital charges and length of stay (los) profiles are affected by emergency department discharge of af patients. methods: patients receiving treatment at an urban teaching community hospital with a primary diagnosis of atrial fibrillation or flutter were identified through the hospital's billing data base. information collected on each patient included date of service, patient status, length of stay, and total charges. patient status was categorized as inpatient (admitted to the hospital), observation (transferred from the ed to an inpatient bed but placed in an observation status), or ed (discharged directly from the ed). the hospital billing system automatically defaults to a length of stay of 0 for observation patients. ed patients were assigned a length of stay of 0. total hospital charges and mean los were determined for two different models: a standard model (sm) in which patients discharged from the ed were excluded from hospital statistics, and an inclusive model (im) in which discharged ed patients were included in the hospital statistics. statistical analysis was through anova. results: a total of 317 patients were evaluated for af over an 18-month period. of these, 197 (62%) were admitted, 22 (7%) were placed in observation status, and 98 (31%) were discharged from the ed. hospital charges and los in days are summarized in the table. all differences were statistically significant at (p < 0.001). conclusion: emergency department management can lead to a population of af patients discharged directly from the ed. exclusion of these patients from hospital statistics skews performance profiles effectively punishing institutions for progressive care. background: recent health care reform has placed an emphasis on the electronic health record (ehr). with the advent of the ehr it is common to see ed providers spending more time in front of computers documenting and away from patients. finding strategies to decrease provider interaction with computers and increase time with patients may lead to improved patient outcomes and satisfaction. computerized charting adjuncts, such as voice recognition software, have been marketed as ways to improve provider efficiency and patient contact. objectives: we present here observational data comparing two separate ed sites, one where computerized charting is done by conventional techniques and one that is assisted with voice recognition dictation, and their effects on physican charting and patient contact. methods: a prospective observational quality initiative was conducted at two teaching hospitals located less than 1 mile from each other. one site primarily uses conventional computerized charting while the other uses voice recognition dictation. four trained quality assistants observed ed physicians for 180 minutes during shifts. the tasks each physician performed were noted and logged in 30 second intervals. tasks listed were identified from a predetermined standardized list presented at observer training. a total of 4140 minutes were logged. time allocated to charting and that allocated to direct patient care were then compared between sites. results: ed physicians spent 28.6% of their time charting using conventional techniques vs 25.7% using voice recognition dictation (p = 0.4349). time allocated to direct patient care was found to be 22.8% with conventional charting vs 25.1% using dictation (p = 4887). in total, ed physicians using conventional charting techniques spent 668/2340 minutes charting. ed physicians using voice recognition dictation spent 333/1800 minutes dictating and an additional 129.5/1800 minutes reviewing or correcting their dictations. the use of voice recognition assisted dictation rather than conventional techniques did not significantly change the amount of time physicians spent charting or with direct patient care. although voice recognition dictation decreased initial input time of documenting data, a considerable amount of time was required to review and correct these dictations. objectives: for our primary objective, we studied whether emergency department triage temperatures detected fever adequately when compared to a rectal temperature. as secondary objectives, we examined the temperature differences when a rectal temperature was taken within an hour of non-invasive temperature, temperature site (oral, axillary, temporal), and also examined the patients that were initially afebrile but were found to be febrile by rectal temperature. methods: we performed an electronic chart review at our inner city, academic emergency department with an annual census of 110,000 patients. we identified all patients over the age of 18 who received a non-invasive triage temperature and a subsequent rectal temperature while in the ed from january 2002 through february 2011. specific data elements included many aspects of the patient's medical record (e.g. subject demographics, temperature, and source). we analyzed our data with standard descriptive statistics, t-tests for continuous variables, and pearson chi-square tests for proportions. results: a total of 27,130 patients met our inclusion criteria. the mean difference in temperatures between the initial temperature and the rectal temperature was 1.3°f, with 25.9% having higher rectal temperatures ‡2°f, and 5.0% having higher rectal temperatures ‡4°f. the mean temperature difference among the 10,313 patients who an initial noninvasive temperature and a rectal temperature within one hour was 1.4°f. the mean difference among patients that received oral, axillary, and temporal temperatures was 1.2°f, 1.8°f, and 1.2°f respectively. approximately one in five patients (18.1%) were initially afebrile and found to be febrile by rectal temperature, with an average temperature difference of 2.5°f. these patients had a higher rate of admission, and were more likely to be admitted to the intensive care unit. conclusion: there are significant differences between rectal temperatures and non-invasive triage temperatures in this emergency department cohort. in almost one in five patients, fever was missed by triage temperature. background: pediatric emergency department (ped) overcrowding has become a national crisis, and has resulted in delays in treatment, and patients leaving without being seen. increased wait times have also been associated with decreased patient satisfaction. optimizing ped throughput is one means by which to handle the increased demands for services. various strategies have been proposed to increase efficiency and reduce length of stay (los). objectives: to measure the effect of direct bedding, bedside registration, and patient pooling on ped wait times, length of stay, and patient satisfaction. methods: data were extracted from a computerized ed tracking system in an urban tertiary care ped. comparisons were made between metrics for 2010 (23,681 patients) and the 3 months following process change (6,195 patients). during 2010, patients were triaged by one or two nurses, registered, and then sent either to a 14-bed ped or a physically separate 5-bed fast-track unit, where they were seen by a physician. following process change, patients were brought directly to a bed in the 14-bed ped, triaged and registered, then seen by a physician. the fast-track unit was only utilized to accommodate patient surges. results: anticipating improved efficiencies, attending physician coverage was decreased by 9%. after instituting process changes, improvements were noted immediately. although daily patient volume increased by 3%, median time to be seen by a physician decreased by 20%. additionally, median los for discharged patients decreased by 15%, and median time until the decisionto-admit decreased by 10%. press-ganey satisfaction scores during this time increased by greater than 5 mean score points, which was reported to be a statistically significant increase. conclusion: direct bedding, bedside registration, and patient pooling were simple to implement process changes. these changes resulted in more efficient ped throughput, as evidenced by decreased times to be seen by a physician, los for discharged patients, and time until decision-to-admit. additionally, patient satisfaction scores improved, despite decreased attending physician coverage and a 30% decrease in room utilization. ) . during period 1, the ou was managed by the internal medicine department and staffed by primary care physicians and physician assistants. during periods 2 and 3, the ou was managed and staffed by em physicians. data collected included ou patient volume, length of stay (los) for discharged and admitted patients, admission rates, and 30-day readmission rates for discharged patients. cost data collected included direct, indirect, and total cost per patient encounter. data were compared using chi-square and anova analysis followed by multiple pairwise comparisons using the bonferroni method of p-value adjustment. results: see table. the ou patient volume and percent of ed volume was greater in period 3 compared to periods 1 and 2. length of stay, admission rates, 30-day readmission rates, and costs were greater in period 1 compared to periods 2 and 3. conclusion: em physicians provide more cost-effective care for patients in this large ou compared to non-em physicians, resulting in shorter los for admitted and discharged patients, greater rates of patients discharged, and less 30-day readmission rates for discharged patients. this is not affected by an increase in ou volume and shows a trend towards improvement. background: emergency department (ed) crowding continues to be a problem, and new intake models may represent part of the solution. however, little data exist on the sustainability and long-term effects of physician triage and screening on standard ed performance metrics, as most studies are short-term. objectives: we examined the hypothesis that a physician screening program (start) sustainably improves standard ed performance metrics including patient length of stay (los) and patients who left without completing assessment (lwca). we also investigated the number of patients treated and dispositioned by start without using a monitored bed and the median patient door-to-room time. methods: design and setting: this study is a retrospective before-and-after analysis of start in a level i tertiary care urban academic medical center with approximately 90,000 annual patient visits. all adult patients from december 2006 until november 2010 are included, though only a subset was seen in start. start began at our institution in december 2007. observations: our outcome measures were length of stay for ed patients, lwca rates, patients treated and dispositioned by start without using a monitored bed, and door-to-room time. statistics: simple descriptive statistics were used. p-values for los were calculated with wilcoxon test and p-value for lwca was calculated with chi-square. results: table 2 shows median length of stay for ed patients was reduced by 56 minutes/patient (p-value <0.0001) when comparing the most recent year to the year before start. patients who lwca were reduced from 4.8% to 2.9% (p-value <0.0001) during the same time period. we also found that in the first half-year of start, 18% of patients screened in the ed were treated and dispositioned without using a monitored bed and by the end of year 3, this number had grown to 29%. median door-to-room time decreased from 18.4 minutes to 9.9 minutes over the same period of time. conclusion: a start system can provide sustained improvements in ed performance metrics, including a significant reduction in ed los, lwca rate, and doorto-room time. additionally, start can decrease the need for monitored ed beds and thus increase ed capacity. . labs were obtained in 98%, ct in 37%, us in 30%, and consultation in 23%. 18% of the cohort was admitted to the hospital. the most commonly utilized source of translation was a layman (35%). a professional translator was used in 9% and translation service (language line, marty) in 30%. the examiner was fluent in the patient's language in 11%. both the patient and examiner were able to maintain basic communication in 11%. there were 47 patients in the professional/ fluent translation group and 44 patients in the lay translation group. there was no difference in ed los between groups 288 vs 304 min; p = 0.6. there was no difference in the frequency of lab tests, computerized tomography, ultrasound, consultations, or hospital admission. frequencies did not differ by sex or age. conclusion: translation method was not associated with a difference in overall ed los, ancillary test use, or specialist consultation in spanish-speaking patients presenting to the ed for abdominal pain. emergency department patients on warfarin -how often is the visit due to the medication? jim killeen, edward castillo, theodore chan, gary vilke ucsd medical center, san diego, ca background: warfarin has important therapeutic value for many patients, but has been associated with signi-ficant bleeding complications, hypersensitivity reactions, and drug-drug interactions, which can result in patients seeking care in the emergency department (ed). objectives: to determine how often ed patients on warfarin present for care as a result of the medication itself. methods: a multi-center prospective survey study in two academic eds over 6 months. patients who presented to the ed taking warfarin were identified, and ed providers were prospectively queried at the time of disposition regarding whether the visit was the result of a complication or side effect associated with warfarin. data were also collected on patient demographics, chief complaint, triage acuity, vital signs, disposition, ed evaluation time, and length of stay (los). patients identified with a warfarin-related cause for their ed visit were compared with those who were not. statistical analysis was performed using descriptive statistics. results: during the study period, 31,500 patients were cared for by ed staff, of whom 594 were identified as taking warfarin as part of their medication regimen. of these, providers identified 54.7% (325 patients) who presented with a warfarin-related complication as their primary reason for the ed visit. 56.9% (338) each 100 hours of daily boarding is associated with a drop of 1.3 raw score points in both pg metrics. these seemingly small drops in raw scores translate into major changes in rankings on press ganey national percentile scales (a difference of as much as 10 percentile points). our institution commonly has hundreds of hours of daily boarding. it is possible that patient-level measurements of boarding impact would show stronger correlation with individual satisfaction scores, as opposed to the daily aggregate measures we describe here. our research suggests that reducing the burden of boarding on eds will improve patient satisfaction. background: prolonged emergency department (ed) boarding is a key contributor to ed crowding. the effect of output interventions (moving boarders out of the ed into an intermediate area prior to admission or adding additional capacity to an observation unit) has not been well studied. objectives: we studied the effect of a combined observation-transition (ot) unit, consisting of observation beds and an interim holding area for boarding ed patients, on the length of stay (los) for admitted patients, as well as secondary outcomes such as los for discharged patients, and left without being seen rates. methods: we conducted a retrospective review (12 months pre-, 12 months post-design) of an ot unit at an urban teaching ed with 59,000 annual visits (study ed). we compared outcomes to a nearby communitybased ed with 38,000 annual visits in the same health system (control ed) where no capacity interventions were performed. the ot had 17 beds, full monitoring capacity, and was staffed 24 hours per day. the number of beds allocated to transition and observation patients fluctuated throughout the course of the intervention, based on patient demands. all analyses were conducted at the level of the ed-day. wilcoxon rank-sum and analysis of covariance tests were used for comparisons; continuous variables were summarized with medians. results: in unadjusted analyses, median daily los of admitted patients at the study ed was 31 minutes lower in the 12 months after the ot opened, 6.98 to 6.47 hours (p < 0.0001). control site daily los for admitted patients increased 26 minutes from 4.52 to 4.95 hours (p < 0.0001). results were similar after adjusting for other covariates (day of week, ed volume, and triage level). los of discharged patients at study ed decreased by 14 minutes, from 4.1 hours to 3.8 hours (p < 0.001), while the control ed saw no significant changes in discharged patient los (2.6 hours to 2.7 hours, p = 0.06). left without being seen rates did not decrease at either site. conclusion: opening an ot unit was associated with a 30-minute reduction in average daily ed los for admitted patients and discharged patients in the study ed. given the large expense of opening an ot, future studies should compare capacity-dependent (e.g., ot) vs. capacity-independent (e.g, organizational) interventions to reduce ed crowding. fran balamuth, katie hayes, cynthia mollen, monika goyal children's hospital of philadelphia, philadelphia, pa background: lower abdominal pain and genitourinary problems are common chief complaints in adolescent females presenting to emergency departments. pelvic inflammatory disease (pid) is a potentially severe complication of lower genital tract infections, which involves inflammation of the female upper genital tract secondary to ascending stis. pid has been associated with severe sequelae including infertility, ectopic pregnancy, and chronic pelvic pain. we describe the prevalence and microbial patterns of pid in a cohort of adolescent females presenting to an urban emergency department with abdominal or genitourinary complaints. objectives: to describe the prevalence and microbial patterns of pid in a cohort of adolescent patients presenting to an ed with lower abdominal or genitourinary complaints. methods: this is a secondary analysis of a prospective study of females ages 14-19 years presenting to a pediatric ed with lower abdominal or genitourinary complaints. diagnosis of pid was per 2006 cdc guidelines. patients underwent chlamydia trachomatis (ct) and neisseria gonorrhea (gc) testing via urine aptima combo 2 assay and trichomonas vaginalis (tv) testing using the vaginal osom trichomonas rapid test. descriptive statistics were performed using stata 11.0. results: the prevalence of pid in this cohort of 328 patients was 19.5% (95% ci 15.2%, 23.8%), 37.5% (95% ci 25.3%, 49.7%) of whom had positive sexually transmitted infection (sti) testing: 25% (95% ci 14.1%, 35.9%) with ct, 7.8% (95% ci 1.1, 14.6%) with gc, and 12.5% (95% ci 4.2%, 20.8%) with tv. 84.4% (95% ci 75.2, 93.5%) of patients diagnosed with pid received antibiotics consistent with cdc recommendations. patients with lower abdominal pain as their chief complaint were more likely to have pid than patients with genitourinary complaints (or 3.3, 95% ci 1.7, 6.4). conclusion: a substantial number of adolescent females presenting to the emergency department with lower abdominal pain were diagnosed with pid, with microbial patterns similar to those previously reported in largely adult, outpatient samples. furthermore, appropriate treatment for pid was observed in the majority of patients diagnosed with pid. impact background: in resource-poor settings, maternal health care facilities are often underutilized, contributing to high maternal mortality. the effect of ultrasound in these settings on patients, health care providers, and communities is poorly understood. objectives: the purpose of this study was to assess the effect of the introduction of maternal ultrasound in a population not previously exposed to this intervention. methods: an ngo-led program trained nurses at four remote clinics outside koutiala, mali, who performed 8,339 maternal ultrasound scans over three years. our researchers conducted an independent assessment of this program, which involved log book review, sonographer skill assessment, referral follow-up, semi-structured interviews of clinic staff and patients, and focus groups of community members in surrounding villages. analyses included the effect of ultrasound on clinic function, job satisfaction, community utilization of prenatal care and maternity services, alterations in clinical decision making, sonographer skill, and referral frequency. we used qrs nvivo9 to organize qualitative findings, code data, and identify emergent themes, and graphpad software (la jolla, ca) and microsoft excel to tabulate quantitative findings results: -findings that triggered changes in clinical practice were noted in 10.1% of ultrasounds, with a 3.5% referral rate to comprehensive maternity care facilities. -skill retention and job satisfaction for ultrasound providers was high. -the number of patients coming for antenatal care increased, after introduction of ultrasound, in an area where the birth rate has been decreasing. -over time, women traveled from farther distances to access ultrasound and participate in antenatal care. -very high acceptance among staff, patients and community members. -ultrasound was perceived as most useful for finding fetal position, sex, due date, and well-being. -improved confidence in diagnosis and treatment plan for all cohorts. -improved compliance with referral recommendations. -no evidence of gender selection motivation for ultrasound use. conclusion: use of maternal ultrasound in rural and resource-limited settings draws women to an initial antenatal care visit, increases referral, and improves job satisfaction among health care workers. methods: a retrospective database analysis was conducted using the electronic medical record from a single, large academic hospital. ed patients who received a billing diagnosis of ''nausea and vomiting of pregnancy'' or ''hyperemesis gravidarum'' between 1/1/10 and 12/31/10 were selected. a manual chart review was conducted with demographic and treatment variables collected. statistical significance was determined using multiple regression analysis for a primary outcome of return visit to the emergency department for nausea and vomiting of pregnancy. results: 113 patients were identified. the mean age was 27.1 years (sd±5.25), mean gravidity 2.90 (sd±1.94), and mean gestational age 8.78 weeks (sd±3.21). the average length of ed evaluation was 730 min (sd±513). of the 113 patients, 38 (33.6%) had a return ed visit for nausea and vomiting of pregnancy, 17 (15%) were admitted to the hospital, and 49 (43%) were admitted to the ed observation protocol. multiple regression analysis showed that the presence of medical co-morbidity (p = 0.039), patient gravditity (p = 0.016), gestational age (p = 0.038), and admission to the hospital (p = 0.004) had small but significant effects on the primary outcome (return visits to the emergency department). no other variables were found to be predictive of return visits to the ed including admission to the ed observation unit or factors classically thought to be associated with severe forms of nausea and vomiting in pregnancy including ketonuria, electrolyte abnormalities, or vital sign abnormalities. conclusion: nausea and vomiting in pregnancy has a high rate of return ed visits that can be predicted by young patient age, low patient gravidity, early gestational age, and the presence of other comorbidities. these patients may benefit from obstetric consultation and/or optimization of symptom management after discharge in order to prevent recurrent utilization of the ed. prevalence conclusion: there is a high prevalence of ht in adult sa victims. although our study design and data do not allow us to make any inferences regarding causation, this first report of ht ed prevalence suggests the opportunity to clarify this relationship and the potential opportunity to intervene. background: sexually transmitted infections (sti) are a significant public health problem. because of the risks associated with stis including pid, ectopic pregnancy, and infertility the cdc recommends aggressive treatment with antibiotics in any patient with a suspected sti. objectives: to determine the rates of positive gonorrhea and chlamydia (g/c) screening and rates of empiric antibiotic use among patients of an urban academic ed with >55,000 visits in boston, ma. methods: a retrospective study of all patients who had g/c cultures in the ed over 12 months. chi-square was used in data analysis. sensitivity and specificity were also calculated. results: a positive rate of 9/712 (1.2%) was seen for gonorrhea and 26/714 (3.6%) for chlamydia. females had positive rates of 2/602 (0.3%) and 17/603 (2.8%) respectively. males had higher rates of 7/110 (6.4%) (p =< 0.001) and 9/111 (8.1%) (p = 0.006). 284 patients with g/c sent received an alternative diagnosis, the most common being uti (63), ovarian pathology (35), vaginal bleeding (34), and vaginal candidiasis (33); 4 were excluded. this left 426 without definitive diagnosis. of these, 24.2% (87/360) of females were treated empirically with antibiotics for g/c, and a greater percentage of males (66%, 45/66) were treated empirically (p < 0.001). of those empirically treated, 109/132 (82.6%) had negative cultures. meanwhile 9/32 (28.1%) who ultimately had positive cultures were not treated with antibiotics during their ed stay. sensitivity of the provider to predict presence of disease based on decision to give empiric antibiotics was 71.9 (ci 53.0-85.6). specificity was 72.3 (ci 67.6-76.6). conclusion: most patients screened in our ed for g/c did not have positive cultures and 82.6% of those treated empirically were found not to have g/c. while early treatment is important to prevent complications, there are risks associated with antibiotic use such as allergic reaction, c difficile infection, and development of antibiotic resistance. our results suggest that at our institution we may be over-treating for g/c. furthermore, despite high rates of treatment, 28% of patients who ultimately had positive cultures did not receive antibiotics during their ed stay. further research into predictive factors or development of a clinical decision rule may be useful to help determine which patients are best treated empirically with antibiotics for presumed g/c. background: air travel may be associated with unmeasured neurophysiological changes in an injured brain that may affect post-concussion recovery. no study has compared the effect of commercial airtravel on concussion injuries despite rather obvious decreased oxygen tension and increased dehydration effect on acute mtbi. objectives: to determine if air travel within 4-6 hours of concussion is associated with increased recovery time in professional football and hockey players. methods: prospective cohort study of all active-roster national football league and national hockey league players during the 2010-2011 seasons. internet website review of league sties for injury identification of concussive injury and when player returned to play solely for mtbi. team schedules and flight times were also confirmed to include only players who flew immediately following game (within 4-6 hr). multiple injuries were excluded as were players who had injury around all-star break for nhl and scheduled off week in nfl. results: during the 2010-2011 nfl and nhl seasons, 122 (7.2%) and 101 (13.0%) players experienced a concussion (percent of total players), in the respective leagues. of these, 68 nfl players (57%) and 39 nhl players (39%) flew within 6 hours of the incident injury. the mean distance flown was shorter for nfl (850 miles, sd 576 vs. nhl 1060, sd 579) miles and all were in a pressurized cabin. the mean number of games missed for nfl and nhl players who traveled by air immediately after concussion was increased by 29% and 24% (respectively) than for those who did not travel by air nfl: 3.8 (sd 2.2) vs. 2.6 games (sd 1.8) and nhl: 16.2 games (sd 22.0) vs.12.4 (sd 18.6); p < 0.03. conclusion: this is an initial report of an increased rate of recovery in terms of more games missed, for professional athletes flying commercial airlines post-mtbi compared to those that do not subject their recently injured brains to pressurized airflight. the obvious changes of decreased oxygen tension with altitude equivalent of 7,500 feet, decreased humidity with increased dehydration, and duress of travel accompanying pressurized airline cabins all likely increase the concussion penumbra in acute mtbi. early air travel post concussion should be further evaluated and likely postponed 48-72 hr. until initial symptoms subside. background: previous studies have shown better in-hospital stroke time targets for those who arrive by ambulance compared to other modes of transport. however, regional studies report that less than half of stroke patients arrive by ambulance. objectives: our objectives were to describe the proportion of stroke patients who arrive by ambulance nationwide, and to examine regional differences and factors associated with the mode of transport to the emergency department (ed). methods: this is a cross-sectional study of all patients with a primary discharge diagnosis of stroke based on previously validated icd-9 codes abstracted from the national hospital ambulatory medical care survey for 2007-2009. we excluded subjects <18 years of age and those with missing data. the study related survey variables included patient demographics, community characteristics, mode of transport to the hospital, and hospital characteristics. results: 566 patients met inclusion criteria, representing 2,153,234 patient records nationally. of these, 50.4% arrived by ambulance. after adjustment for potential confounders, patients residing in the west and south had lower odds of arriving by ambulance for stroke when compared to northeast (southern region, or 0.45, 95% ci 0.26-0.76, western region, or 0.45, 95% ci 0.25-0.84, midwest region, or 0.56, 95% ci 0.31-1.01). compared to the medicare population, privately insured and self insured had lower odds of arriving by ambulance (or for private insurance 0.48, 95% ci 0.28-0.84 and or for self payers 0.36, 95% ci 0.14-0.93). age, sex, race, urban or rural location of ed, or safety net status were not independently associated with ambulance use. conclusion: patients with stroke arrive by ambulance more frequently in the northeast than in other regions of the us. identifying reasons for this regional difference may be useful in improving ambulance utilization and overall stroke care nationwide. objectives: we sought to determine whether there was a difference in type of stroke presentation based upon race. we further sought to determine whether there is an increase in hemorrhagic strokes among asian patients with limited english proficiency. methods: we performed a retrospective chart review of all stroke patients age 18 and older for 1 year of patients that were diagnosed with cerebral vascular accident (cva) or intracranial hemorrhage (ich). we collected data on patient demographics, and past medical history. we then stratified patients according to race (white, black, latino, asian, and other). we classified strokes as ischemic, intracranial hemorrhage (ich), subarachnoid hemorrhage (sah), subdural hemorrhage (sdh), and other (e.g., bleeding into metatstatic lesions). we used only the index visit. we present the data percentages, medians and interquartile ranges (iqr). we tested the association of the outcome of intracranial hemorrhage against demographic and clinical variables using chi-square and kruskal-wallis tests. we performed a logistic regression model to determine factors related to presentation with an intracranial hemorrhage (ich background: the practice of obtaining laboratory studies and routine ct scan of the brain on every child with a seizure has been called into question in the patient who is alert, interactive, and back to functional baseline. there is still no standard practice for the management of non-febrile seizure patients in the pediatric emergency department (ped). objectives: we sought to determine the proportion of patients in whom clinically significant laboratory studies and ct scans of the brain were obtained in children who presented to the ped with a first or recurrent non-febrile seizure. we hypothesize that the majority of these children do not have clinically significant laboratory or imaging studies. if clinically significant values were found, the history given would warrant further laboratory and imaging assessment despite seizure alone. methods: we performed a retrospective chart review of 93 patients with first-time or recurrent non-febrile seizures at an urban, academic ped between july 2007 to june 2011. exclusion criteria included children who presented to the ped with a fever and age less than 2 months. we looked at specific values that included a complete blood count, basic metabolic panel, and liver function tests, and if the child was on antiepileptics along with a level for a known seizure disorder, and ct scan. abnormal laboratory and ct scan findings were classified as clinically significant or not. results: the median age of our study population is 4 years with male to female ratio of 1.7. 70% of patients had a generalized tonic-clonic seizure. laboratory studies and ct scans were obtained in 87% and 35% of patients, respectively. five patients had clinically significant abnormal labs; however, one had esrd, one developed urosepsis, one had eclampsia, and two others had hyponatremia, which was secondary to diluted formula and trileptal toxicity. three children had an abnormal head ct: two had a vp shunt and one had a chromosomal abnormality with developmental delay. conclusion: the majority of the children analyzed did not have clinically significant laboratory or imaging studies in the setting of a first or recurrent non-febrile seizure. of those with clinically significant results, the patient's history suggested a possible etiology for their seizure presentation and further workup was indicated. background: in patients with a negative ct scan for suspected subarachnoid hemorrhage (sah), ct angiography (cta) has emerged as a controversial alternative diagnostic strategy in place of lumbar puncture (lp). objectives: to determine the diagnostic accuracy for sah and aneurysm of lp alone, cta alone, and lp followed by cta if the lp is positive. methods: we developed a decision and bayesian analysis to evaluate 1) lp, 2) cta, and 3) lp followed by cta if the lp is positive. data were obtained from the literature. the model considers probability of sah (15%), aneurysm (85% if sah), sensitivity and specificity of ct (92.9% and 100% overall), of lp (based on rbc and xanthochromia), and of cta, traumatic tap and its influence on sah detection. analyses considered all patients and those presenting at less than 6 hours or greater than 6 hours from symptom onset by varying the sensitivity and specificity of ct and cta. results: using the reported ranges of ct scan sensitivity and the specificity, the revised likelihood of sah following a negative ct ranged from 0.5-3.7%, and the likelihood of aneurysm ranged from 2.3-5.4%. following any of the diagnostic strategies, the likelihood of missing sah ranged from 0-0.7%. either lp strategy diagnosed 99.8% of sahs versus 83-84% with cta alone because cta only detected sah in the presence of an aneurysm. false positive sah with lp ranged from 8.5-8.8% due to traumatic taps and with cta ranged from 0.2-6.0% due to aneurysms without sah. the positive predictive value for sah ranged from 5.7-30% with lp and from 7.9-63% with cta. for patients presenting within 6 hours of symptom onset, the revised likelihood of sah following a negative ct became 0.53%, and the likelihood of aneurysm ranged from 2.3-2.7%. following any of the diagnostic strategies, the likelihood of missing sah ranged from 0.01-0.095%. either lp strategy diagnosed 99.8% of sah versus 83-84% with cta alone. false positive sah with lp was 8.8% and with cta ranged from 0.2-5.1%. the positive predictive value for sah was 5.7% with lp and from 7.9-63% with cta. cta following a positive lp diagnosed 8.5-24% of aneurysms. conclusion: lp strategies are more sensitive for detecting sah but less specific than cta because of traumatic taps, leading to lower predictive value positives for sah with lp than with cta. either diagnostic strategy results in a low likelihood of missing sah, particularly within 6 hours of symptom onset. background: recent studies support perfusion imaging as a prognostic tool in ischemic stroke, but little data exist regarding its utility in transient ischemic attack (tia). ct perfusion (ctp), which is more available and less costly to perform than mri, has not been well studied. objectives: to characterize ctp findings in tia patients, and identify imaging predictors of outcome. methods: this retrospective cohort study evaluated tia patients at a single ed over 15 months, who had ctp at initial evaluation. a neurologist blinded to ctp findings collected demographic and clinical data. ctp images were analyzed by a neuroradiologist blinded to clinical information. ctp maps were described as qualitatively normal, increased, or decreased in mean transit time (mtt), cerebral blood volume (cbv), and cerebral blood flow (cbf). quantitative analysis involved measurements of average mtt (seconds), cbv (cc/100 g) and cbf (cc/[100g x min]) in standardized regions of interest within each vascular distribution. these were compared with values in the other hemisphere for relative measures of mtt difference, cbv ratio, and cbffratio. mtt difference of ‡2 seconds, rcbv as £0.60, and rcbf as £0.48 were defined as abnormal based on prior studies. clinical outcomes including stroke, tia, or hospitalization during follow-up were determined up to one year following the index event. dichotomous variables were compared using fisher's exact test. logistic regression was used to evaluate the association of ctp abnormalities with outcome in tia patients. results: of 99 patients with validated tia, 53 had ctp done. mean age was 72 ± 12 years, 55% were women, and 64% were caucasian. mean abcd 2 score was 4.7 ± 2.1, and 69% had an abcd 2 ‡ 4. prolonged mtt was the most common abnormality (19, 36%), and 5 (9.4%) had decreased cbv in the same distribution. on quantitative analysis, 23 (43%) had a significant abnormality. four patients (7.5%) had prolonged mtt and decreased cbv in the same territory, while 17 (32%) had mismatched abnormalities. when tested in a multivariate model, no significant associations between mismatch abnormalities on ctp and new stroke, tia, or hospitalizations were observed. conclusion: ctp abnormalities are common in tia patients. although no association between these abnormalities and clinical outcomes was observed in this small study, this needs to be studied further. objectives: we hypothesized that pre-thrombolytic anti-hypertensive treatment (aht) may prolong door to treatment time (dtt). methods: secondary data analysis of consecutive tpatreated patients at 24 randomly selected michigan community hospitals in the instinct trial. dtt among stroke patients who received pre-thrombolytic aht were compared to those who did not receive pre-thrombolytic aht. we then calculated a propensity score for the probability of receiving pre-thrombolytic aht using a logistic regression model with covariates including demographics, stroke risk factors, antiplatelet or beta blocker as home medication, stroke severity (nihss), onset to door time, admission glucose, pretreatment systolic and diastolic blood pressure, ems usage, and location at time of stroke. a paired t-test was then performed to compare the dtt between the propensity-matched groups. a separate generalized estimating equations (gee) approach was also used to estimate the differences between patients receiving pre-thrombolytic aht and those who did not while accounting for within-hospital clustering. results: a total of 557 patients were included in instinct; however, onset, arrival, or treatment times were not able to be determined in 23, leaving 534 patients for this analysis. the unmatched cohort consisted of 95 stroke patients who received pre-thrombolytic aht and 439 stroke patients who did not receive aht from 2007-2010 (table) . in the unmatched cohort, patients who received pre-thrombolytic aht had a longer dtt (mean increase 9 minutes; 95% confidence interval (ci) 2-16 minutes) than patients who did not receive pre-thrombolytic aht. after propensity matching (table) , patients who received pre-thrombolytic aht had a longer dtt (mean increase 10.4 minutes, 95% ci 1.9-18.8) than patients who did not receive pre-thrombolytic aht. this effect persisted and its magnitude was not altered by accounting for clustering within hospitals. conclusion: pre-thrombolytic aht is associated with modest delays in dtt. this represents a feasible target for physician educational interventions and quality improvement initiatives. further research evaluating optimum hypertension management pre-thrombolytic treatment is warranted. post-pds, 7% had only pre-pds, and 9% had both. the most common pds included failure to treat post-treatment hypertension (131, 24%), antiplatelet agent within 24 hours of treatment (61, 11%), pre-treatment blood pressure over 185/110 (39, 7%), anticoagulant agent within 24 hours of treatment (31, 6%), and treatment outside the time window (29, 5%). symptomatic intracranial hemorrhage (sich) was observed in 7.3% of patients with pds and 6.5% of patients without any pd. in-hospital case fatality was 12% with and 10% without a pd. in the fully adjusted model, older age was significantly associated with pre-pds (table) . when post-pds were evaluated with adjustment for pre-pds, age was not associated with pds; however, pre-pds were associated with post-pds. conclusion: older age was associated with increased odds of pre-pds in michigan community hospitals. pre-pds were associated with post-pds. sich and in-hospital case fatality were not associated with pds; however, the low number of such events limited our ability to detect a difference. ct background: mri has become the gold standard for the detection of cerebral ischemia and is a component of multiple imaging enhanced clinical risk prediction rules for the short-term risk of stroke in patients with transient ischemic attack (tia). however, it is not always available in the emergency department (ed) and is often contraindicated. leukoaraiosis (la) is a radiographic term for white matter ischemic changes, and has recently been shown to be independently predictive of disabling stroke. although it is easily detected by both ct and mri, their comparative ability is unknown. objectives: we sought to determine whether leukoaraiosis, when combined with evidence of acute or old infarction as detected by ct, achieved similar sensitivity to mri in patients presenting to the ed with tia. methods: we conducted a retrospective review of consecutive patients diagnosed with tia between june 2009 and july 2011 that underwent both ct and mri as part of routine care within 1 calendar day of presentation to a single, academic ed. ct and mr images were reviewed by a single emergency physician who was blinded to the mr images at the time of ct interpretation. la was graded using the van sweiten scale (vss), a validated grading scale applicable to both ct and mri. anterior and posterior regions were graded independently from 0 to 2. results: 361 patients were diagnosed with tia during the study period. of these, 194 had both ct and mri background: helping others is often a rewarding experience but can also come with a ''cost of caring'' also known as compassion fatigue (cf). cf can be defined as the emotional and physical toll suffered by those helping others in distress. it is affected by three major components: compassion satisfaction (cs), burnout (bo), and traumatic experiences (te). previous literature has recognized an increase in bo related to work hours and stress among resident physicians. objectives: to assess the state of cf among residents with regard to differences in specialty training, hours worked, number of overnights, and demands of child care. we aim to measure associations with the three components of cf (cs, bo, and te). methods: we used the previously validated survey, proqol 5. the survey was sent to the residents after approval from the irb and the program directors. results: a total of 193 responses were received (40% of the 478 surveyed). five were excluded due to incomplete questionnaires. we found that residents who worked more hours per week had significantly higher bo levels (median 25 vs 21, p = 0.038) and higher te (22 vs 19, p = 0.048) than those working less hours. there was no difference in cs (42 vs 40, p = 0.73). eighteen percent of the residents worked a majority of the night shifts. these residents had higher levels of bo background: emergency department (ed) billing includes both facility and professional fees. an algorithm derived from the medical provider's chart generates the latter fee. many private hospitals encourage appropriate documentation by financially incentivizing providers. academic hospitals sometimes lag in this initiative, possibly resulting in less than optimal charting. past attempts to teach proper documentation using our electronic medical record (emr) were difficult in our urban, academic ed of 80 providers (approximately 25 attending physicians, 36 residents, and 20 physician assistants). objectives: we created a tutorial to teach documentation of ed charts, modified the emr to encourage appropriate documentation, and provided feedback from the coding department. this was combined with an incentive structure shared equally amongst all attendings based on increased collections. we hypothesized this instructional intervention would lead to more appropriate billing, improve chart content, decrease medical liability, and increase educational value of charting process. methods: documentation recommendations, divided into two-month phases of 2-3 proposals, were administered to all ed providers by e-mails, lectures, and reminders during sign-out rounds. charts were reviewed by coders who provided individual feedback if specific phase recommendations were not followed. our endpoints included change in total rvu, rvus/ patient, e/m level distribution, and subjective quality of chart improvement. we did not examine effects on procedure codes or facility fees. results: our base average rvu/patient in our ed from 1/1/11-6/30/11 was 2.615 with monthly variability of approximately 2%. implementation of phase one increased average rvu/patient within two weeks to 2.73 (4.4% increase from baseline, p < 0.05). the second aggregate phase implemented 8 weeks later increased average rvu/patient to 3.04 (16.4% increase from baseline, p < 0.05). conclusion: using our teaching methods, chart reviews focused on 2-3 recommendations at a time, and emr adjustments, we were able to better reflect the complexity of care that we deliver every day in our medical charts. future phases will focus on appropriate documentation for procedures, critical care, fast track, and pediatric patients, as well as examining correlations between increase in rvus with charge capture. identifying mentoring ''best practices'' for medical school faculty julie l. welch, teresita bellido, cherri d. hobgood background: mentoring has been identified as an essential component for career success and satisfaction in academic medicine. many institutions and departments struggle with providing both basic and transformative mentoring for their faculty. objectives: we sought to identify and understand the essential practices of successful mentoring programs. methods: multidisciplinary institutional stakeholders in the school of medicine including tenured professors, deans, and faculty acknowledged as successful mentors were identified and participated in focused interviews between mar-nov 2011. the major area of inquiry involved their experiences with mentoring relationships, practices, and structure within the school, department, or division. focused interview data were transcribed and grounded theory analysis was performed. additional data collected by a 2009 institutional mentoring taskforce were examined. key elements and themes were identified and organized for final review. results: results identified the mentoring practices for three categories: 1) general themes for all faculty, 2) specific practices for faculty groups: basic science researchers, clinician researchers, clinician educators, and 3) national examples. additional mentoring strategies that failed were identified. the general themes were quite universal among faculty groups. these included: clarify the best type of mentoring for the mentee, allow the mentee to choose the mentor, establish a panel of mentors with complementary skills, schedule regular meetings, establish a clear mentoring plan with expectations and goals, offer training and resources for both the mentor and mentee at institutional and departmental levels, ensure ongoing mentoring evaluation, create a mechanism to identify and reward mentoring. national practice examples offered critical recommendations to address multi-generational attitudes and faculty diversity in terms of gender, race, and culture. conclusion: mentoring strategies can be identified to serve a diverse faculty in academic medicine. interventions to improve mentoring practices should be targeted at the level of the institution, department, and individual faculty members. it is imperative to adopt results such as these to design effective mentoring programs to enhance the success of emergency medicine faculty seeking robust academic careers. background: women comprise half of the talent pool from which the specialty of emergency medicine draws future leaders, researchers, and educators and yet only 5% of full professors in us emergency medicine are female. both research and interventions are aimed at reducing the gender gap, however, it will take decades for the benefits to be realized which creates a methodological challenge in assessing system's change. current techniques to measure disparities are insensitive to systems change as they are limited to percentages and trends over time. objectives: to determine if the use of relative rate index (rri) better predicts which stage in the system women are not advancing in the academic pipeline than traditional metrics. methods: rri is a method of analysis that assesses the percent of sub-populations in each stage relative to their representation in the stage directly prior. thus, there is a better notion of the advancement given the availability to advance. rri also standardizes data for ease of interpretation. this study was conducted on the total population of academic professors in all departments at yale school of medicine during the academic year of 2010-2011. data were obtained from the yale university provost's office. results: n = 1305. there were a total of 402 full, 429 associate, and 484 assistant professors. males comprised 78%, 59%, and 54% respectively. rri for the department of emergency medicine (dem) is 0.67, 1.93, and 0.78, for full, associate, and assistant professors, respectively while the percentages were 44%, 60%, and 33% respectively. conclusion: relying solely on percentages masks improvements to the system. women are most represented at the associate professor level in dem, highlighting the importance of systems change evidence. specifically, twice as many women are promoted to associate professor rank given the number who exists as assistant professors. within 5 years, the dem should have an equal system as the numbers of associate professors have dramatically increased and will be eligible to promote to full professor. additionally, dem has a better record of retaining and promoting women than other yale departments of medicine at both associate and full professor ranks. objectives: we examine the payer mixes of community non-rehabilitation eds in metropolitan areas by region to identify the proportion of academic and nonacademic eds that could be considered safety net eds. we hypothesize that the proportion of safety net academic eds is greater than that for non-academic eds and is increasing over time. methods: this is an ecological study examining us ed visits from 2006 through 2008. data were obtained from the nationwide emergency department sample (neds). we grouped each ed visit according to the unique hospital-based ed identifier, thus creating a payer mix for each ed. we define a ''safety net ed'' as any ed where the payer mix satisfied any one of the following three conditions: 1) >30% of all ed visits are medicaid patients; 2) >30% of all ed visits are self-pay patients; or 3) >40% of all ed visits are either medicaid or self-pay patients. neds tags each ed with a hospital-based variable to delineate metropolitan/non-metropolitan locations and academic affiliation. we chose to examine a subpopulation of eds tagged as either academic metropolitan or non-academic metropolitan, because the teaching status of non-metropolitan hospitals was not provided. we then measured the proportion of eds that met safety net criteria by academic status and region. results: we examined 2,821, 2,793, and 2,844 weighted metro eds in years 2006-2008, respectively. table 1 presents safety net proportions. the proportions of academic safety net eds increased across the study period. widespread regional variability in safety net proportions existed across all years. the proportions of safety net eds were highest in the south and lowest in the northeast and midwest. table 2 describes these findings for 2008. conclusion: these data suggest that the proportion of safety-net academic eds may be greater than that of non-academic eds, is increasing over time, and is objectives: to examine the effect of ma health reform implementation on ed and hospital utilization before and after health reform, using an approach that relies on differential changes in insurance rates across different areas of the state in order to make causal inferences as to the effect of health reform on ed visits and hospitalizations. our hypothesis was that health care reform (i.e. reducing rates of uninsurance) would result in increased rates of ed use and hospitalizations. methods: we used a novel difference-in-differences approach, with geographic variation (at the zip code level) in the percentage uninsured as our method of identifying changes resulting from health reform, to determine the specific effect of massachusetts' health care reform on ed utilization and hospitalizations. using administrative data available from the massachusetts division of health care finance and policy acute hospital case mix databases, we compared a one-year period before health reform with an identical period after reform. we fit linear regression models at the area-quarter level to estimate the effect of health reform and the changing uninsurance rate (defined as self-pay only) on ed visits and hospitalizations. results: there were 2,562,330 ed visits and 777,357 hospitalizations pre-reform and 2,713,726 ed visits and 787,700 hospitalizations post-reform. the rate of uninsurance decreased from 6.2% to 3.7% in the ed group and from 1.3% to 0.6% in the hospitalization group. a reduction in the rate of the uninsured was associated with a small but statistically significant increase in ed utilization (p = 0.03) and no change in hospitalizations (p = 0.13). conclusion: we find that increasing levels of insurance coverage in massachusetts were associated with small but statistically significant increases in ed visits, but no differences in rates of hospitalizations. these results should aid in planning for anticipated changes that might result from the implementation of health reform nationally. with high levels of co-morbidity when untreated in adolescents. despite broad cdc screening recommendations, many youth do not receive testing when indicated. the pediatric emergency department (ped) is a venue with a high volume of patients potentially in need of sti testing, but assessing risk in the ped is difficult given constraints on time and privacy. we hypothesized that patients visiting a ped would find an audio-enhanced computer-assisted self-interview (acasi) program to establish sti risk easy to use, and would report a preference for the acasi over other methods of disclosing this information. objectives: to assess acceptability, ease of use, and comfort level of an acasi designed to assess adolescents' risk for stis in the ped. methods: we developed a branch-logic questionnaire and acasi system to determine whether patients aged 15-21 visiting the ped need sti testing, regardless of chief complaint. we obtained consent from participants and guardians. patients completed the acasi in private on a laptop. they read a one-page computer introduction describing study details and completed the acasi. patients rated use of the acasi upon completion using five-point likert scales. results: 2030 eligible patients visited the ped during the study period. we approached 873 (43%) and enrolled and analyzed data for 460/873 (53%). the median time to read the introduction and complete the acasi was 8.2 minutes (interquartile range 6.4-11.5 minutes). 90.7% of patients rated the acasi ''very easy'' or ''easy'' to use, 90.6% rated the wording as ''very easy'' or ''easy'' to understand, 60% rated the acasi ''very short'' or ''short'', 60.3% rated the audio as ''very helpful'' or ''helpful,'' 82.9% were ''very comfortable'' or ''comfortable'' with the system confidentiality, and 71.2% said they would prefer a computer interface over in-person interviews or written surveys for collection of this type of information. conclusion: patients rated the computer interface of the acasi as easy and comfortable to use. a median of 8.2 minutes was needed to obtain meaningful clinical information. the acasi is a promising approach to enhance the collection of sensitive information in the ped. the participants were randomized to one of three conditions, bi delivered by a computer (cbi), bi delivered by a therapist assisted by a computer (tbi), or control, and completed 3, 6, and 12 month follow-up. in addition to content on alcohol misuse and peer violence, adolescents reporting dating violence received a tailored module on dating violence. the main outcome for this analysis was frequency of moderate and severe dating victimization and aggression at the baseline assessment and 3, 6, and 12 months post ed visit. results: among eligible adolescents, 55% (n = 397) reported dating violence and were included in these analyses. compared to controls, after controlling for baseline dating victimization, participants in the cbi showed reductions in moderate dating victimization at 3 months (or 0.7; ci 0.51-0.99; p < 0.05, effect size 0.12) and 6 months (or 0.56; ci 0.38-0.83; p < 0.01, effect size 0.18); models examining interaction effects were significant for the cbi on moderate dating victimization at 3 and 6 months. significant interaction effects were found for the tbi on moderate dating victimization at 6 and 12 months and severe dating victimization at 3 months. the computer-based intervention shows promise for delivering content that decreases moderate dating victimization over 6 months. the therapist bi is promising for decreasing moderate dating victimization over 12 months and severe dating victimization over 3 months. ed-based bis delivered on a computer addressing multiple risk behaviors could have important public health effects. figure 1 . the 21-only ordinance was associated with a significant reduction of ar visits. this ordinance was also associated with reduction in underage ar visits, ui student visits, and public intoxication bookings. these data suggest that other cities should consider similar ordinances to prevent unwanted consequences of alcohol. background: prehospital providers perform tracheal intubation in the prehospital environment, and failed attempts are of concern due to the danger of hypoxia and hypotension. some question the appropriateness of intubation in this setting due to the morbidity risk associated with intubation in the field. thus it is important to gain an understanding of the factors that predict the success of prehospital intubation attempts to inform this discussion. objectives: to determine the factors that affect success rates on first attempt of paramedic intubations in a rapid sequence intubation (rsi) capable critical care transport service. methods: we conducted a multivariate logistic analysis on a prospectively collected database of airway management from an air and land critical care transport service that provides scene responses and interfacility transport in the province of ontario. background: motor vehicle collisions (mvcs) are one of the most common types of trauma for which people seek ed care. the vast majority of these patients are discharged home after evaluation. acute psychological distress after trauma causes great suffering and is a known predictor of posttraumatic stress disorder (ptsd) development. however, the incidence and predictors of psychological distress among patients discharged to home from the ed after mvcs have not been reported. objectives: to examine the incidence and predictors of acute psychological distress among individuals seen in the ed after mvcs and discharged to home. methods: we analyzed data from a prospective observational study of adults 18-64 years of age presenting to one of eight ed study sites after mvc between 02/ 2009 and 10/2011. english-speaking patients who were alert and oriented, stable, and without injuries requiring hospital admission were enrolled. patient interview included assessment of patient sociodemographic and psychological characteristics and mvc characteristics. level of psychological distress in the ed was assessed using the 13-item peritraumatic distress inventory (pdi). pdi scores >23 are associated with increased risk of ptsd and were used to define substantial psychological distress. descriptive statistics and logistic regression were performed using stata ic 11.0 (statacorp lp, college station, texas). results: 9339 mvc patients were screened, 1584 were eligible, and 949 were enrolled. 361/949 (38%) participants had substantial psychological distress. after adjusting for crash severity (severity of vehicle damage, vehicle speed), substantial patient distress was predicted by sociodemographic factors, pre-mvc depressive symptoms, and arriving to the ed on a backboard (table) . conclusion: substantial psychological distress is common among individuals discharged from the ed after mvcs and is predicted by patient characteristics separate from mvc severity. a better under standing of the frequency and predictors of substantial psychological distress is an important first step in identifying these patients and developing effective interventions to reduce severe distress in the aftermath of trauma. such interventions have the potential to reduce both immediate patient suffering and the development of persistent psychological sequelae. figure) the predictive characteristics of pets, pesi, and spesi for 30-day mortality in emperor, including auc, negative predictive value, sensitivity, and specificity were calculated. results: the 646 of 1438 patients (44.9%; 95% ci 42.3%-47.5%) classified as pets low had 30-day mortality of 0.5% (95% ci 0.1-1.5%), versus 10.2% (95% ci 8.0%-12.4%) in the pets high group, statistically similar to pesi and spesi. pets is significantly more specific for mortality than the spesi (47.0% v 37.6%; p < 0.0001), classifying far more patients as low-risk while maintaining a sensitivity of 96% (95% ci 88.3%-99.0%), not significantly different from spesi or pesi (p > 0.05). conclusion: with four variables, pets in this derivation cohort is as sensitive for 30-day mortality as the more complicated pesi and spesi, with significantly greater specificity than the spesi for mortality, placing 25% more patients in the low-risk group. external validation is necessary. nicole seleno, jody vogel, michael liao, emily hopkins, richard byyny, ernest moore, craig gravitz, jason haukoos denver health medical center, denver, co background: the sequential organ failure assessment (sofa) score, base excess, and lactate have been shown to be associated with mortality in critically ill trauma patients. the denver emergency department (ed) trauma organ failure (tof) score was recently derived and internally validated to predict multiple organ failure in trauma patients. the relationship between the denver tof score and mortality has not been assessed or compared to other conventional measures of mortality in trauma. objectives: to compare the prognostic accuracies of the denver ed tof score, ed sofa score, and ed base excess and lactate for mortality in a large heterogeneous trauma population. methods: a secondary analysis of data from the denver health trauma registry, a prospectively collected database. consecutive adult trauma patients from 2005 through 2008 were included in the study. data collected included demographics, injury characteristics, prehospital care characteristics, response to injury characteristics, ed diagnostic evaluation and interventions, and in-hospital mortality. the values of the four clinically relevant measures (denver ed tof score, ed sofa score, ed base excess, and ed lactate) were determined within four hours of patient arrival, and prognostic accuracies for in-hospital mortality for the four measures were evaluated with receiver operating characteristic (roc) curves. multiple imputation was used for missing values. results: of the 4,355 patients, the median age was 37 (iqr 26-51) years, median injury severity score was 9 (iqr 4-16), and 81% had blunt mechanisms. thirty-eight percent (1,670 patients) were admitted to the icu with a median icu length of stay of 2.5 (iqr 1-8) days, and 3% (138 patients) died. in the non-survivors, the median values for the four measures were ed sofa 5.0 (iqr 0.0-8.0); denver ed tof 4.0 (iqr 4.0-5.0); ed base excess 7.0 (iqr 8.0-19.0) meq/l; and ed lactate 6.5 (iqr 4.5-11.8) mmol/l. the areas under the roc curves for these measures are demonstrated in the figure. conclusion: the denver ed tof score more accurately predicts in-hospital mortality in trauma patients as compared to the ed sofa score, ed base excess, or ed lactate. the denver ed tof score may help identify patients early who are at risk for mortality, allowing for targeted resuscitation and secondary triage to improve outcomes in these critically ill patients. the background: both animal and human studies suggest that early initiation of therapeutic hypothermia (th) and rapid cooling improve outcomes after cardiac arrest. objectives: the objective was to determine if administration of cold iv fluids in a prehospital setting decreased time-to-target-temperature (tt) with secondary analysis of effects on mortality and neurological outcome. methods: patients resuscitated after out-of-hospital cardiac arrest (oohca) who received an in-hospital post cardiac arrest bundle including th were prospectively enrolled into a quality assurance database from november 2007 to november 2011. on april 1, 2009 a protocol for intra-arrest prehospital cooling with 4°c normal saline on patients experiencing oohca was initiated. we retrospectively compared tt for those receiving prehospital cold fluids and those not receiving cold fluids. tt was defined as 34°c measured via foley thermistor. secondary outcomes included mortality, good neurological outcome defined as cerebral performance category (cpc) score of 1 or 2 at discharge, and effects of pre-rosc cooling. results: there were 132 patients who were included in this analysis with 80 patients receiving prehospital cold iv fluids and 52 who did not. initially, 63% of patients were in vf/vt and 36% asystole/pea. patients receiving prehospital cooling did not have a significant improvement in tt (256 minutes vs 271 minutes, p = 0.64). survival to discharge and good neurologic outcome were not associated with prehospital cooling (54% vs 50%, p = 0.67) and cpc of 1 or 2 in 49% vs 44%, (p = 0.61). initiating cold fluids prior to rosc showed both a nonsignificant decrease in survival (48% vs 56%, p = 0.35) and increase in poor neurologic outcomes (42% vs 50%, p = 0.39). 77% of patients received £ 1l of cooled ivf prior to hospital arrival. patients receiving prehospital cold ivf had a longer time from arrest to hospital arrival (44 vs 34 min, p =< 0.001) in addition to a prolonged rosc to hospital time (20 vs 12 min, p = 0.005). conclusion: at our urban hospital, patients achieving rosc following oohca did not demonstrate faster tt or outcome improvement with prehospital cooling compared to cooling initiated immediately upon ed arrival. further research is needed to assess the utility of prehospital cooling. assessment background: an estimated 10% of emergency department (ed) patients 65 years of age and older have delirium, which is associated with short-and long-term risk of morbidity and mortality. early recognition could result in improved outcomes, but the reliability of delirium recognition in the continuum of emergency care is unknown. objectives: we tested whether delirium can be reliably detected during emergency care of elderly patients by measuring the agreement between prehospital providers, ed physicians, and trained research assistants using the confusion assessment method for the icu (cam-icu) to identify the presence of delirium. our hypothesis was that both ed physicians and prehospital providers would have poor ability to detect elements of delirium in an unstructured setting. methods: prehospital providers and ed physicians completed identical questionnaires regarding their clinical encounter with a convenience sample of elderly (age >65 years) patients who presented via ambulance to two urban, teaching eds over a three-month period. respondents noted the presence or absence of (1) an acute change in mental status, (2) inattention, (3) disorganized thinking, and (4) altered level of consciousness (using the richmond agitation sedation scale). these four components comprise the operational definition of delirium. a research assistant trained in the cam-icu rated each component for the same patients using a standard procedure. we calculated inter-rater reliability (kappa) between prehospital providers, ed physicians, and research assistants for each component. objectives: this study aimed to assess the association between age and ems use while controlling for potential confounders. we hypothesized that this association use would persist after controlling for confounders. methods: a cross-sectional survey study was conducted at an academic medical center's ed. an interview-based survey was administered and included questions regarding demographic and clinical characteristics, mode of ed arrival, health care use, and the perceived illness severity. age was modeled as an ordinal variable (<60, 60-79, and ‡ 80 years). bivariate analyses were used to identify potential confounders and effect measure modifiers and a multivariable logistic regression model was constructed. odds ratios were calculated as measures of effect. results: a total of 1092 subjects were enrolled and had usable data for all covariates, 465 (43%) of whom arrived via ems. the median age of the sample was 60 years and 52% were female. there was a statistically significant linear trend in the proportion of subjects who arrived via ems by age (p < 0.0001). compared to adults aged less than 60 years, the unadjusted odds ratio associating age and ems use was 1.41 (95% ci: background: we previously derived a clinical decision rule (cdr) for chest radiography (cxr) in patients with chest pain and possible acute coronary syndrome (acs) consisting of the absence of three predictors: history of congestive heart failure, history of smoking, and abnormalities on lung auscultation. objectives: to prospectively validate and refine a cdr for cxr in an independent patient population. methods: we prospectively enrolled patients over 24 years of age with a primary complaint of chest pain and possible acs from september 2009 to january 2010 at a tertiary care ed with 73,000 annual patient visits. physicians completed standardized data collection forms before ordering chest radiographs and were thus blinded to cxr findings at the time of data collection. two investigators, blinded to the predictor variables, independently classified cxrs as ''normal,'' ''abnormal not requiring intervention,'' and ''abnormal requiring intervention'' (e.g, heart failure, infiltrates) based on review of the radiology report and the medical record. analyses included descriptive statistics, inter-rater reliability assessment (kappa), and recursive partitioning. results: of 1159 visits for possible acs, mean age (sd) was 60.3 (15.6) and 51% were female. twenty-four percent had a history of acute myocardial infarction, 10% congestive heart failure, and 11% atrial fibrillation. seventy-one (6.1%, 95% ci 4.9-7.7) patients had a radiographic abnormality requiring intervention. ing the likelihood of coronary artery disease (cad) could reduce the need for stress testing or coronary imaging. acyl-coa:cholesterol acyltransferase-2 (acat2) activity has been shown in monkey and murine models to correlate with atherosclerosis. objectives: to determine if a novel cardiac biomarker consisting of plasma cholesteryl ester levels (ce) typically derived from the activity of acat2 is predictive of cad in a clinical model. methods: a single center prospective observational cohort design enrolled a convenience sample of subjects from a tertiary care center with symptoms of acute coronary syndrome undergoing coronary ct angiography or invasive angiography. plasma samples were analyzed for ce composition with mass spectrometry. the primary endpoint was any cad determined at angiography. multivariable logistic regression analyses were used to estimate the relationship between the sum of the plasma concentrations from cholesteryl palmitoleate (16:1) and cholesteryl oleate (18:1) (defined as acat2-ce) and the presence of cad. the added value of acat2-ce to the model was analyzed comparing the c-statistics and integrated discrimination improvement (idi). results: the study cohort was comprised of 113 participants enrolled over 24 months with a mean age 49 (±11.7) years, 59% with cad at angiography. the median plasma concentration of acat2-ce was 938 lm (758, 1099) in patients with cad and 824 lm (683, 998) in patients without cad (p = 0.03) (figure) . when considered with age, sex, and the number of conventional cad risk factors, acat2-ce were associated with a 6.5% increased odds of having cad per 10 lm increase in concentration. the addition of acat2-ce significantly improved the c-statistic (0.89 vs 0.95, p = 0.0035) and idi (0.15, p < 0.001) compared to the reduced model. in the subgroup of low-risk observation unit patients, the ce model had superior discrimination compared to the diamond forrester classification (idi 0.403, p < 0.001). conclusion: plasma levels of acat2-ce, considered in a clinical model, have strong potential to predict a patient's likelihood of having cad. in turn, this could reduce the need for cardiac imaging after the exclusion of mi. further study of acat2-ce as biomarkers in patients with suspected acs is needed. background: outpatient studies have demonstrated a correlation between carotid intima-media thickness (cimt) on ultrasound and coronary artery disease (cad). there are no known published studies that investigate the role of cimt in the ed using cardiac ct or percutaneous cardiac intervention (pci) as a gold standard. objectives: we hypothesized that cimt can predict cardiovascular events and serve as a noninvasive tool in the ed. methods: this was a prospective study of adult patients who presented to the ed and required evaluation for chest pain. the study location was an urban ed with a census of 120,000 annual visits and 24-hour cardiac catheterization. patients who did not have ct or pci or had carotid surgery were excluded from the study. ultrasound cimt measurements of right and left common carotid arteries were taken with a 10mhz linear transducer (zonare, mountain view, ca). anterior, medial, and posterior views of the near and far wall were obtained (12 cimt scores total). images were analyzed by carotid analyzer 5 (mailing imaging application llc, coralville, iowa). patients were classified into two groups based on the results from ct or pci. a subject was classified as having significant cad if there was over 70% occlusion or multi-vessel disease. results: ninety of 102 patients were included in the study; 55.7% were males. mean age was 56.6 ± 13 years. there were 34 (37.8%) subjects with significant cad and 56 (62.2%) with non-significant cad. the mean of all 12 cimt measurements was significantly higher in the cad group than in the non-cad group (0.60 ± 0.20 vs. 0.35 ± 0.23; p < 0.00001). a logistic regression analysis was carried out with significant cad as the event of interest and the following explanatory variables in the model: objectives: to determine the diagnostic yield of routine testing in-hospital or following ed discharge among patients presenting to an ed following syncope. methods: a prospective, observational, cohort study of consecutive ed patients ‡18 years old presenting with syncope was conducted. the four most commonly utilized tests (echocardiography, telemetry, ambulatory electrocardiography monitoring, and cardiac markers) were studied. interobserver agreement as to whether tests results determined the etiology of the syncope was measured using kappa (k) values. results: of 570 patients with syncope, 150 (26%) had echocardiography with 33 (6%) demonstrating a likely etiology of the syncopal event such as critical valvular disease or significantly depressed left ventricular function (k = 0.78). on hospitalization, 349 (61%) patients were placed on telemetry, 19 (3%) of these had worrisome dysrhythmias (k = 0.66). 317 (55%) patients had troponin levels drawn of whom 19 (3%) had positive results (k = 1); 56 (10%) patients were discharged with monitoring with significant findings in only 2 (0.4%) patients (k = 0.65). overall, 73 (8%, 95% ci 7-10%) studies were diagnostic. conclusion: although routine testing is prevalent in ed patients with syncope, the diagnostic yield is relatively low. nevertheless, some testing, particularly echocardiography, may yield critical findings in some cases. current efforts to reduce the cost of medical care by eliminating non-diagnostic medical testing and increasing emphasis on practicing evidence-based medicine argue for more discriminate testing when evaluating syncope. (originally submitted as a ''late-breaker.'') unusual fatigue was reported by 70.7% (severe 29.7%) and insomnia by 47.8% (severe 21.0%). these findings have led to risk management recommendations to consider these symptoms as predictive of acute coronary syndromes (acs) among women visiting the ed. objectives: to document the prevalence of these symptoms among all women visiting an ed. to analyze the potential effect of using these symptoms in the ed diagnostic process for acs. methods: a survey on fatigue and insomnia symptoms was administered to a convenience sample of all adult women visiting an urban academic ed (all arrival modes, acuity levels, all complaints). a sensitivity analysis was performed using published data and expert opinion for inputs. results: we approached 548 women, with 379 enrollments. see table. the top box shows prevalences of prodromal symptoms among all adult female ed patients. the bottom box shows outputs from sensitivity analysis on the diagnostic effect of initiating an acs workup for all female ed patients reporting prodromal symptoms. conclusion: prodromal symptoms of acs are highly prevalent among all adult women visiting the ed in this study. this likely limits their utility in ed settings. while screening or admitting women with prodromal symptoms in the ed would probably increase sensitivity, that increase would be accompanied by a dramatic reduction in specificity. such a reduction in specificity would translate to admitting, observing, or working up somewhere between 29% and 61% of all women visiting the ed, which is prohibitive in terms of personal costs, risks of hospitalization, and financial costs. while these symptoms may or may not have utility in other settings such as primary care, their prevalence, and the implied lack of specificity for acs suggest they will not be clinically useful in the ed. length methods: we examined a cohort of low-risk chest pain patients evaluated in an ed-based ou using prospective and retrospective ou registry data elements. cox proportional hazard modeling was performed to assess the effect of testing modality (stress testing vs. ccta) on the los in the cdu. as ccta is not available on weekends, only subjects presenting on weekdays were included. cox models were stratified on time of patient presentation to the ed, based on four hour blocks beginning at midnight. the primary independent variable was first test modality, either stress imaging (exercise echo, dobutamine echo, stress mri) or ccta. age, sex, and race were included as covariates. the proportional hazards assumption was tested using scaled schoenfield residuals, and the models were graphically examined for outliers and overly influential covariate patterns. test selection was a time varying covariate in the 8am strata, and therefore the interaction with ln (los) was included as a correction term. after correction for multiple comparisons, an alpha of 0.01 was held to be significant. results: over the study period, 841 subjects (of 1,070 in the registry) presented on non-weekend days. the median los was 18.5 hours (iqr 12.4-23.3 hours), 57% were white, and 61% were female. the table shows the number of subjects in each time strata, the number tested, and the number undergoing stress testing vs. ccta. after adjusting all models for age, race, and sex, the hazard ratio (hr) for los is as shown. only those patients presenting between 8am and noon noted a significant improvement in los with ccta use (p < 0.0001). objectives: determine the validity of a managementfocused em osce as a measure of clinical skills by determining the correlation between osce scores and faculty assessment of student performance in the ed. methods: medical students in a fourth year em clerkship were enrolled in the study. on the final day of the clerkship students participated in a five-station em osce. student performance on the osce was evaluated using a task-based evaluation system with 3-4 critical management tasks per case. task performance was evaluated using a three-point system: performed correctly/timely (2), performed incorrectly/late (1), or not performed (0). descriptive anchors were used for performance criteria. communication skills were also graded on a three-point scale. student performance in the ed was based on traditional faculty assessment using our core-competency evaluation instrument. a pearson correlation coefficient was calculated for the relationship between osce score and ed performance score. case item analysis included determination of difficulty and discrimination. the acgme also requires that trainees are evaluated on these 6ccs during their residency. trainee evaluation in the 6ccs are frequently on a subjective rating scale. one of the recognized problems with a subjective scale is the rating stringency of the rater, commonly known as the hawk-dove effect. this has been seen in standardized clinical exam scoring. recent data have shown that score variance can be related to evaluator performance with a negative correlation. higher-scoring physicians were more likely to be a stringent or hawk type rater on the same evaluation. it is unclear if this pattern also occurs in the subjective ratings that are commonly used in assessments of the 6ccs. objectives: comparison of attending physician scores on the acgme 6ccs with attending ratings of residents for a negative correlation or hawk-dove effect. methods: residents are routinely evaluated on the 6ccs with a 1-9 numerical rating scale as part of their training. the evaluation database was retrospectively reviewed. residents anonymously scored attending physicians on the 6ccs with a cross-sectional survey that utilized the same rating scale, anchors, and prompts as the resident evaluations. average scores for and by each attending were calculated and a pearson correlation calculated by core competency and overall. results: in this irb-approved study, a total of 43 attending physicians were scored on the 6ccs with 447 evaluations by residents. attendings evaluated 162 residents with a total of 1,678 evaluations completed over a 5-year period. attending mode score was 9 ranging from 2 to 9; resident scores had a mode of 8 with a range of 1 to 9. there was no correlation between the rated performance of the attendings overall or in each 6ccs and the scores they gave (p = 0.065-0.861). conclusion: hawk-dove effects can be seen in some scoring systems and has the potential to affect trainee evaluation on the acgme core competencies. however, a negative correlation to support a hawk-dove scoring pattern was not found in em resident evaluations by attending physicians. this study is limited by being a single center study and utilizing grouped data to preserve resident anonymity. background: all acgme-accredited residency programs are required to provide competency-based education and evaluation. graduating residents must demonstrate competency in six key areas. multiple studies have outlined strategies for evaluating competency, but data regarding residents' self-assessments of these competencies as they progress through training and beyond is scarce. objectives: using data from longitudinal surveys by the american board of emergency medicine, the primary objective of this study was to evaluate if resident self-assessments of performance in required competencies improve over the course of graduate medical training and in the years following. additionally, resident self-assessment of competency in academic medicine was also analyzed. methods: this is a secondary data analysis of data gathered from two rounds of the abem longitudinal study of emergency medicine residents (1996-98 and 2001-03) and three rounds of the abem longitudinal study of emergency physicians (1999, 2004, 2009 ). in both surveys, physicians were asked to rate a list of 18 items in response to the question, ''what is your current level of competence in each of the following aspects of work in em?'' the rated items were grouped according to the acgme required competencies of patient care, medical knowledge, practice-based learning and improvement, interpersonal and communication skills, and system-based practice. an additional category for academic medicine was also added. results: rankings improved in all categories during residency training. rankings in three of the six categories improved from the weak end of the scale to the strong end of the scale. there is a consistent decline in rankings one year after graduation from residency. the greatest drop is in medical knowledge. mean self-ranking in academic medicine competency is uniformly the lowest ranked category for each year. conclusion: while self-assessment is of uncertain value as an objective assessment, these increasing rankings suggest that emergency medicine residency programs are successful at improving residents' confidence in the required areas. residents do not feel as confident about academic medicine as they do about the acgme required competencies. the uniform decline in rankings the first year after residency is an area worthy of further inquiry. screening medical student rotators from outside institutions improves overall rotation performance shaneen doctor, troy madsen, susan stroud, megan l. fix university of utah, salt lake city, ut background: emergency medicine is a rapidly growing field. many student rotations are limited in their ability to accommodate all students and must limit the number of students they allow per rotation. we hypothesize that pre-screening visiting student rotators will improve overall student performance. objectives: to assess the effect of applicant screening on overall rotation grade and mean end of shift card scores. methods: we initiated a medical student screening process for all visiting students applying to our 4-week elective em rotation starting in 2008. this consisted of reviewing board scores and requiring a letter of intent. students from our home institution were not screened. all end-of-shift evaluation cards and final rotation grades (honors, high pass, pass, fail) from 2004 to 2011 were analyzed. we identified two cohorts: home students (control) and visiting students. we compared pre-intervention (2004) (2005) (2006) (2007) (2008) and postintervention (2008-2011) scores and grades. end of shift performance scores are recorded using a fivepoint scale that assesses indicators such as fund of knowledge, judgment, and follow-through to disposition. mean ranks were compared and p-values were calculated using the armitage test of trend and confirmed using t-tests. results: we identified 162 visiting students (91 pre, 81 post) and 160 home students (90 pre, 80 post). 12 (13.2%) visiting students achieved honors pre-intervention while 31 (38.3%) achieved honors post-intervention (p = 0.000093). no significant difference was seen in home student grades: 28 (31.1%) received honors pre-2008 and 17 (21.3%) received honors post-2008 conclusion: we found that implementation of a screening process for visiting medical students improved overall rotation scores and grades as compared to home students who did not receive screening. screening rotating students may improve the overall quality of applicants and thereby the residency program. background: there are many descriptions in the literature of computer-assisted instruction in medical education, but few studies that compare them to traditional teaching methods. objectives: we sought to compare the suturing skills and confidence of students receiving video preparation before a suturing workshop versus a traditional instructional lecture. methods: 88 first and second year medical students were randomized into two groups. the control group was given a lecture followed by 40 minutes of suturing time. the video group was provided with an online suturing video at home, no lecture, and given 40 minutes of suturing time during the workshop. both groups were asked to rate their confidence before and after the workshop, and their belief in the workshop's effectiveness. each student was also videotaped suturing a pig's foot after the workshop and graded on a previously validated 16-point suturing checklist. 83 videos were scored. results: there was no significant difference between the test scores of the lecture group (m = 11.21, sd = 3.17, n = 42) and the video group (m = 11.27, sd = 2.53, n = 41) using the two-sample independent ttest for equal variances (t(81) = )0.09, p = 0.93). there was a statistically significant difference in the proportion of students scoring correctly for only one point: ''curvature of needle followed'': 25/42 in the lecture group and 35/41 in the video group (chi = 6.92, df = 1, p = 0.008). students in the video group were found to be 2.45 times more likely to have a neutral or favorable feeling of suturing confidence before the workshop (p = 0.067, ci 0.94-6.4) using a proportional odds model. no association was detected between group assignment and level of suturing confidence after the workshop (p = 0.475). there was also no association detected between group assignment and opinion of the suturing workshop (p = 0.681) using a logistic regression odds model. among those students who indicated a lack of confidence before training, there was no detected association (p = 0.967) between group assignment and having an improved confidence using a logistic regression odds model. conclusion: students in the video group and students in the control group achieved similar levels of suturing skill and confidence, and equal belief in the workshop's effectiveness. this study suggests that video instruction could be a reasonable substitute for lectures in procedural education. background: accurate interpretation of the ecg in the emergency department is not only clinically important but also critical to assess medical knowledge competency. with limitations to expansion of formal didactics, educational technology offers an innovative approach to improve the quality of medical education. objectives: the aim of this study was to assess an online multimedia-based ecg training module evaluating st elevation myocardial infarction (stemi) identification among medical students. methods: a convenience sample of fifty-two medical students on their em rotations at an academic medical center with an em residency program was evaluated in a before-after fashion during a 6-month period. one cardiologist and two ed attending physicians independently validated a standardized exam of ten ecgs: four were normal ecgs, three were classic stemis, and three were subtle stemis. the gold standard for diagnosis was confirmed acute coronary thrombus during cardiac catheterization. after evaluating the 10 ecgs, students completed a pre-intervention test wherein they were asked to identify patients who required emergent cardiac catheterization based on the presence or absence of st segment elevation on ecg. students then completed an online interactive multimedia module containing 13 minutes of stemi training based on american heart association/american college of cardiology guidelines on stemi. medical students were asked to complete a post-test of the 10 ecgs after watching online multimedia. objectives: our objective was to quantify the number of pre-verbal pediatric head cts performed at our community hospital that could have been avoided by utilizing the pecarn criteria. methods: we conducted a standardized chart review of all children under the age of 2 who presented to our community hospital and received a head ct between jan 1st, 2010 and dec 31st, 2010. following recommended guidelines for conducting a chart review, we: 1) utilized four blinded chart reviewers, 2) provided specific training, 3) created a standardized data extraction tool, and 4) held periodic meetings to evaluate coding discrepancies. our primary outcome measure was the number of patients who were pecarn negative and received a head ct at our institution. our secondary outcome was to reevaluate the sensitivity and specificity of the pecarn criteria to detect citbi in our cohort. data were analyzed using descriptive statistics and 95% confidence intervals were calculated around proportions using the modified wald method. results: a total of 138 patients under the age of 2 received a head ct at our institution during the study period. 23 patients were excluded from the final analysis because their head cts were not for trauma. the prevalence of a citbi in our cohort was 2.6% (95% ci 0.6%-7.7%) ( (dti) measures disruption of axonal integrity on the basis of anisotropic diffusion properties. findings on dti may relate to the injury, as well as the severity of postconcussion syndrome (pcs) following mtbi. objectives: to examine acute anisotropic diffusion properties based on dti in youth with mtbi relative to orthopedic controls and to examine associations between white matter (wm) integrity and pcs symptoms. methods: interim analysis of a prospective casecontrol cohort involving 12 youth ages 11-16 years with mtbi and 10 orthopedic controls requiring extremity radiographs. data collected in ed included demographics, clinical information, and pcs symptoms measured by the postconcussion symptom scale. within 72 hours of injury, symptoms were re-assessed and a 61-direction, diffusion weighted, spin-echo imaging scan was performed on a 3t philips scanner. dti images were analyzed using tract-based spatial statistics. fractional anisotropy (fa), mean diffusivity (md), axial diffusivity (ad), and radial diffusivity were measured. results: there were no group demographic differences between mtbi cases and controls. presenting symptoms within the mtbi group included gcs = 15 83%, loss of consciousness 33%, amnesia 33%, post-traumatic seizure 8%, headache 83%, vomiting 33%, dizziness 42%, and confusion 42%. pcs symptoms were greater in mtbi cases than in the controls at ed visit (30.1 ± 17.0 vs. 15.5 ± 16.8, p < 0.06) and at the time of scan (19.1 ± 12.9 vs. 5.7 ± 6.5, p < 0.01). the mtbi group displayed decreased fa in cerebellum and increased md and ad in the cerebral wm relative to controls (uncorrected p < 0.05). increased fa in cerebral wm was also observed in mtbi patients but the group difference was not significant. pcs symptoms at the time of the scan were positively correlated with fa and inversely correlated with rd in extensive cerebral wm areas (p < 0.05, uncorrected). in addition, pcs symptoms in mtbi patients were also found to be inversely correlated with md, ad, and rd in cerebellum (p < 0.05). conclusion: dti detected axonal damage in youth with mtbi which correlated with pcs symptoms. dti performed acutely after injury may augment detection of injury and help prediction of those with worse outcomes. background: sports-related concussion among professional, collegiate, and more recently high school athletes has received much attention from the media and medical community. to our knowledge, there is a paucity of research in regard to sports-related concussion in younger athletes. objectives: the aim of this study was to evaluate parental knowledge of concussion in young children who participate in recreational tackle football. methods: parents/legal guardians of children aged 5-15 years enrolled in recreational tackle football were asked to complete an anonymous questionnaire based on the cdc's heads up: concussion in youth sports quiz. parents were asked about their level of agreement in regard to statements that represent definition, symptoms, and treatment of concussion. results: a total of 310 out of 369 parents voluntarily completed the questionnaire (84% response rate). parent and child demographics are listed in table 1 . ninety four percent of parents believed their child had never suffered a concussion. however, when asked to agree or disagree with statements addressing various aspects of concussion, only 13% (n = 41) could correctly identify all seven statements. most did not identify that a concussion is considered a mild traumatic brain injury and can be achieved from something other than a direct blow to the head. race, sex, and zip code had no significant association with correctly answering statements. education (0.24; p < 0.01) and number of years the child played (0.11; p < 0.05) had a small effect. fifty-three percent of parents reported someone had discussed the definition of concussion with them and 58% the symptoms of concussion. see table 2 for source of information to parents. no parent was able to classify all symptoms listed as correctly related or not related to concussion. however, identification of correct concussion definitions correlated with identification of correct symptoms (0.25; p < 0.05). conclusion: while most parents had received some education regarding concussion from a health care provider, important misconceptions remain among parents of young athletes regarding the definition, symptoms, and treatment of concussion. this study highlights the need for health care providers to increase educational efforts among parents of young athletes in regard to concussion. figure 1 ). 2/2 (100%) of patients with baseline liver dysfunction were 25(oh)d deficient and 5/6 (83%) of deaths were patients who had insufficient levels of 25(oh)d. there was an inverse association between 25(oh)d level and tnf-a (p = 0.03; figure 2 ) and il-6 (p = 0.04). background: fever is common in the emergency department (ed), and 90% of those diagnosed with severe sepsis present with fever. despite data suggesting that fever plays an important role in immunity, human data conflict on the effect of antipyretics on clinical outcomes in critically ill adults. objectives: to determine the effect of ed antipyretic administration on 28-day in-hospital mortality in patients with severe sepsis. methods: single-center, retrospective observational cohort study of 171 febrile severe sepsis patients presenting to an urban academic 90,000-visit ed between june 2005 and june 2010. all ed patients meeting the following criteria were included: age ‡ 18, temperature ‡ 38.3°c, suspected infection, and either systolic blood pressure £ 90 mmhg after a 30 ml/kg fluid bolus or lactate of ‡ 4. patients were excluded for a history of cirrhosis or acetaminophen allergy. antipyretics were defined as acetaminophen, ibuprofen, or ketorolac. results: one hundred-thirty five (78.9%) patients were treated with an antipyretic medication (89.4% acetaminophen). intubated patients were less likely to receive antipyretic therapy (51.9% vs. 84.0%, p < 0.01), but the groups were otherwise well matched. patients requiring ed intubation (n = 27) had much higher in-hospital mortality (51.9% vs. 7.6%, p < 0.01). patients given an antipyretic in the ed had lower mortality (11.9% vs. 25.0%, p < 0.05). when multivariable logistic regression was used to account for apache-ii, intubation status, and fever magnitude, antipyretic therapy was not associated with mortality (adjusted or 0.97, 0.31-3.06, p = 0.96). conclusion: although patients treated with antipyretic therapy had lower 28-day in-hospital mortality, antipyretic therapy was not independently associated with mortality in multivariable regression analysis. these findings are hypothesis-generating for future clinical trials, as the role of fever control has been largely unexplored in severe sepsis (grant ul1 rr024992, nih-ncrr). , and caval index )0.09 ± 0.14 (ci )0.14, )0.05) and all were statistically significant. the groups receiving 10 ml/kg and 30 ml/kg had statistically significant changes in caval index; however the 30 ml/kg group had no significant change in mean ivc diameter. one-way anova differences between the means of all groups were not statistically different. conclusion: overall, there were statistically significant differences in mean ivc-us measurements before and after fluid loading, but not between groups. fasting asymptomatic subjects had a wide inter-subject variation in both baseline ivc-us measurements and fluid-related changes. the wide differences within our 30 ml/kg group may limit conclusions regarding proportionality. there were significant differences in performance on ed measures by ownership (p < 0.0001) and region (p = 0.0002). scores on ed process measures were highest at for-profit hospitals (27% above average) and hospitals in the south (5% above average), and lowest at public hospitals (16% below average) and hospitals in the northeast (8% below average). conclusion: there was considerable variation in performance on the ed measures included in the vbp program by hospital ownership and region. ed directors may come under increasing pressure to improve scores in order to reduce potential financial losses under the program. our data provide early information on the types of hospitals with the greatest opportunity for improvement. methods: design/setting -an independent agency mandated by the government collected and analyzed ed patient experience data using a comprehensive, validated multidimensional instrument and a random periodic sampling methodology of all ed patients. a prospective pre-post experimental study design was employed in the eight community and tertiary care hospitals most affected by crowding. two 5.5 month study periods were evaluated (pre: 28/06-12/12/2010; post: 13/12/2010-29/ 05/2011). outcomes -the primary outcome was patient perception of wait times and crowding reported as a composite mean score (0-100) from six survey items with higher scores representing better ratings. the overall rating of care by ed patients (composite score) and other dimensions of care were collected as secondary outcomes. all outcomes were compared using chi-square and two-tailed student's t-tests. results: a total of 3774 surveys were completed in both the pre-ocp and post-ocp study periods representing a response rate of 45%. we compared in-patient mortality from ami for patients who lived in a community with either 2.5 miles or 5 miles of a closure but did not need to travel farther to the nearest ed with those who did not. we used patient-level data from the california office of statewide health and planning development (oshpd) database patient discharge data, and locations of patient residence and hospitals were geo-coded to determine any changes in distance to the nearest ed. we applied a generalized linear mixed effects model framework to estimate a patient's likelihood to die in the hospital of ami as a function of being affected by a neighborhood closure event. results background: fragmentation of care has been recognized as a problem in the us health care system. however, little is known about ed utilization after hospitalization, a potential marker of poor outpatient care coordination after discharge, particularly for common inpatient-based procedures. objectives: to determine the frequency and variability in ed visits after common inpatient procedures, how often they result in readmission, and related payments. methods: using national medicare data for 2005-2007, we examined ed visits within 30 days of hospital discharge after six common inpatient procedures: percutaneous coronary intervention, coronary artery bypass grafting (cabg), elective abdominal aortic aneurysm repair, back surgery, hip fracture repair, and colectomy. we categorized hospitals into risk-adjusted quintiles based on the frequency of ed visits after the index hospitalization. we report visits by primary diagnosis icd-9 codes and rates of readmission. we also assessed payments related to these ed visits. results: overall, the highest quintile of hospitals had 30-day ed visit rates that ranged from a low of 17.8% with an associated 7.3% readmission rate (back surgery) to a high of 27.8% with an associated 13.6% readmission rate (cabg). the most variability was more than 3-fold and found among patients undergoing colectomy in which the worst-performing hospitals saw 24.1% of their patients experienced an ed visit within 30 days while the best-performing hospitals saw 7.4%. average total payments for the 30-day window from initial discharge across all surgical cohorts varied from $18,912 for patients discharged without subsequent ed visit; $20,061for those experiencing an ed visit(s); $38,762 for those readmitted through the ed; and $33,632 for those readmitted from another source. if all patients who did not require readmission also did not incur an ed visit within the 30-day window, this would represent a potential cost savings of $125 million. conclusion: among elderly medicare recipients there was significant variability between hospitals for 30-day ed visits after six common inpatient procedures. the ed visit may be a marker of poor care coordination in the immediate discharge period. this presents an opportunity to improve post-procedure outpatient care coordination which may save costs related to preventable ed visits and subsequent readmissions. objectives: we sought to assess the effect of pharmacist medication review on ed patient care, in particular time from physician order to medication administration for the patient (order-to-med time). methods: we conducted a multi-center, before-after study in two eds (urban academic teaching hospital and suburban community hospital, combined census of 61,000) after implementation of the electronic prospective pharmacy review system (prs). the system allowed a pharmacist to review all ed medication orders electronically at the time of physician order and either approve or alter the order. we studied a 5-month time period before implementation of the system (pre-prs, 7/1/10-11/30/11) and after implementation (post-prs, 7/ 1/11-11/30/11). we collected data on all ed medication orders including dose, route, class, pharmacist review action, time of physician order, and time of medication administration. differences in order-to-medication between the pre-and post-prs study periods were compared using a results: ed metrics that were significantly associated with lbtcs varied across ed patient-volume categories (table) . for eds seeing less than 20k patients annually, the percentage of ems arrivals admitted to the hospital and ed square footage were both weakly associated with lbtcs (p = 0.09). for eds seeing at least 20k-39k patients, median ed length of stay (los), percent of patients admitted to hospital through the ed, percent of ems arrivals admitted to hospital, and percent of pediatric patients were all positively associated, while percent of patients admitted to the hospital was negatively associated with lbtcs. for eds seeing 40k-59k, median los and percent of x-rays performed were positively associated, while percent of ekgs performed was negatively associated with lbtcs. for eds seeing 60k-79k, percent of patients admitted to the hospital through the ed was negatively associated and percent of ekgs performed was positively associated with lbtcs. for eds with volume greater than 80k, none of the selected variables were associated with lbtc. conclusion: ed factors that help explain high lbtc rates differ depending on the size of an ed. interventions attempting to improve lbtc rates by modifying ed structure or process will need to consider baseline ed volume as a potential moderating influence. objectives: our study sought to compare bacterial growth of samples taken from surfaces after use of a common approved quat compound and a virtually non-toxic, commercially available solution containing elemental silver (0.02%), hydrogen peroxide (15%), and peroxyacetic acid (20%) (shp) in a working ed. we hypothesized that, based on controlled laboratory data available, shp compound would be more effective on surfaces in an active urban ed. methods: we cleaned and then sampled three types of surfaces in the ed (suture cart, wooden railing, and the floor) during midday hours one minute after application of tap water, quat, and shp and then again at 24 hours without additional cleaning. conventional environmental surface surveillance rodac media plates were used for growth assessment. images of bacterial growth were quantified at 24 and 48 hours. standard cleaning procedures by hospital staff were maintained per usual. results: shp was superior to control and quat one minute after application on all three surfaces. quat and water had 10x and 40x more bacterial growth than the surface cleaned with shp, respectively. 24 hours later, the shp area produced fewer colonies sampled from the wooden railing: 4x more bacteria for quat, and 5x for water when compared to shp. 24h cultures from the cart and floor had confluent growth and could not be quantified. conclusion: shp outperforms quat in sterilizing surfaces after one minute application. shp may be a superior agent as a non-toxic, non-corrosive, and effective agent for surfaces in the demanding ed setting. further studies should examine sporidical and virucidal properties in a similar environment. objectives: evaluate the effect on patient satisfaction of increasing waiting room times and physician evaluation times. methods: emergency department flow metrics were collected on a daily basis as well as average daily patient satisfaction scores. the data were from july 2010 through february 2011, in a 44,000 census urban hospital. the data were divided into equal intervals. the arrival to room time was divided by 15 minute intervals up to 135 minutes with the last group being greater than 136 minutes. the physician evaluation times were divided into 20 minute intervals, up to 110, the last group greater than 111 with 46 days in the group. data were analyzed using means and standard deviations, and well as anova for comparison between groups. results: the overall satisfaction score for the outpatient emergency visit was higher when the patient was in a room within 15 minutes of arrival (88.4, std deviation 5.9), analysis of variation between the groups had a p = 0.13, for the means of each interval (see table 1 ). the total satisfaction with the visit as well as satisfaction with the provider dropped when the evaluation extended over 110 minutes, but was not statistically significant on anova analysis (see table 2 for means). conclusion: once a patient's time in the waiting room extends beyond 15 minutes, you have lost a significant opportunity for patient satisfaction; once they have been in the waiting room for over 120 minutes, you are also much more likely to receive a poor score. physician evaluation time scores are much more consistent but as longer evaluation times occurred beyond total of 110 minutes we started to see a trend downward in the satisfaction score. results: in all three eds, pain medication rates (both in ed and rx) varied significantly by clinical factors including location of pain, discharge diagnosis, pain level, and acuity. we observed little to no variation in pain medication rates by patient factors such as age, sex, race, insurance, or prior ed visits. the table displays key pain management practices by site and provider. after adjusting for patient and clinical characteristics, significant differences in pain medication rates remained by provider and site (see figure) . conclusion: within this health system, the approach to pain management by both providers and sites is not standardized. investigation of the potential effect of this variability on patient outcomes is warranted. results: all measures showed significant differences, p < 0.01. average pts/h decreased post-cpoe and did not recover post transitional period, 1.92 ± 0.13 vs 1.75 ± 0.11, p < 0.05. rvu/h also decreased post-cpoe and did not recover post transitional period, 5.23 ± 0.37 vs 4.79 ± 0.32 and 4.82 ± 0.33, p < 0.05. charges/h also decreased after cpoe implementation and did not recover after system optimization. there was a sustained significant decrease in charges/h of 4.5% ± 6.5% post cpoe and 3.6% ± 6.4% post optimization, p < 0.05. sub-group analysis for each provider group was also evaluated and showed variability for different providers. conclusion: there was a significant decrease in all productivity metrics four months after the implementation of cpoe. the system did undergo optimization initiated by providers with customization for ease and speed of use. however, productivity measurements did not recover after these changes were implemented. these data show that with the implementation of a cpoe system there is a decrease in productivity that continues even after a transition period and system customization. background: procedural competency is a key component of emergency medicine residency training. residents are required to log procedures to document quantity of procedures and identify potential weaknesses in their training. as emergency medicine evolves, it is likely that the type and number of procedures change over time. also, exposure to certain rare procedures in residency is not guaranteed. objectives: we seek to delineate trends in type and volume of core em procedures over a decade of emergency medicine residents graduating from an accredited four-year training program. methods: deidentified procedure logs from 2003-2011 were analyzed to assess trends in type and quantity of procedures. procedure logs were self-reported by individual residents on a continuous basis during training onto a computer program. average numbers of procedures per resident in each graduating class were noted. statistical analysis was performed using spss and includes a simple linear regression to evaluate for significant changes in number of procedures over time and an independent samples two-tailed t-test of procedures performed before and after the required resident duty hours change. results: a total of 112 procedure logs were analyzed and the frequency of 29 different procedures was evaluated. a significant increase was seen in one procedure, the venous cutdown. significant decreases were seen in 12 procedures including key procedures such as central venous catheters, tube thoracostomy, and procedural sedation. the frequency of five high-stakes/ resuscitative procedures, including thoracotomy and cricothyroidotomy, remained steady but very low (<4 per resident over 4 years). of the remaining 11 procedures, 8 showed a trend toward decreased frequency, while only 5 increased. conclusion: over the past 9 years, em residents in our program have recorded significantly fewer opportunities to perform most procedures. certain procedures in our emergency medicine training program have remained stable but uncommon over the course of nearly a decade. to ensure competency in uncommon procedures, innovative ways to expose residents to these potentially life saving skills must be considered. these may include practice on high-fidelity simulators, increased exposure to procedures on patients during residency (possibly on off-service rotations), or practice in cadaver and animal labs. objectives: to study the effectiveness of a unique educational intervention using didactic and hands-on training in usgpiv. we hypothesized that senior medical students would improve performance and confidence with usgpiv after the simulation training. methods: fourth year medical students were enrolled in an experimental, prospective, before and after study conducted at a university medical school simulation center. baseline skills in participant's usgpiv on simulation vascular phantoms were graded by ultrasound expert faculty using standardized checklists. the primary outcome was time to cannulation, and secondary outcomes were ability to successfully cannulate, number of needle attempts, and needle-tip visualization. subjects then observed a 15-minute presentation on correct performance of usgpiv followed by a 30-minute hands-on practical session using the vascular simulators with a 1:4 to 1:6 ultrasound instructor to student ratio. an expert blinded to the participant's initial performance graded post-educational intervention usgpiv ability. pre-and post-intervention surveys were obtained to evaluate usgpiv confidence, previous experience with ultrasound, peripheral iv access, usg-piv, and satisfaction with the educational format. objectives: this study examines the grade distribution of resident evaluations when the identity of the evaluator was anonymous as compared to when the identity of the evaluator was known to the resident. we hypothesize that there will be no change in the grades assigned to residents. methods: we retrospectively reviewed all faculty evaluations of residents and grades assigned from july 1, 2008 through november 15, 2011. prior to july 1, 2010 the identity of the faculty evaluators was anonymous, while after this date, the identity of the faculty evaluators was made known to the residents. throughout this time period, residents were graded on a five-point scale. each resident evaluation included grades in the six acgme core competencies as well as in select other abilities. specific abilities evaluated varied over the dates analyzed. evaluations of residents were assigned to two groups, based on whether the evaluator was anonymous or made known to the resident. grades were compared between the two groups. results: a total of 10,760 grades were assigned in the anonymous group, with an average grade of 3.90 (95ci 3.88, 3.91). a total of 7,122 grades were assigned in the known group with an average grade of 3.77 (95ci 3.75, 3.79). specific attention was paid to assignment of unsatisfactory grades (1 or 2 on the five-point scale). the anonymous group assigned 355 grades in this category, comprising 3.3% of all grades assigned. the known group assigned 100 grades in this category, comprising 1.4% of all grades assigned. unsatisfactory grades were assigned by the anonymous group 1.9% (95ci 1.5, 2.3) more often. additionally, 5.8% (95ci 3.8, 6.8) fewer exceptional grades (4 or 5 on the five-point scale) were assigned by the anonymous group. conclusion: the average grade assigned was closer to average (3 on a five-point scale) when the identity of the evaluator was made known to the residents. additionally, fewer unsatisfactory and exceptional grades were assigned in this group. this decrease of both unsatisfactory and exceptional grades may make it more difficult for program directors to effectively identify struggling and strong residents respectively. testing to improve knowledge retention from traditional didactic presentations: a pilot study david saloum, amish aghera, brian gillett maimonides medical center, brooklyn, ny background: the acgme requires an average of at least 5 hours of planned educational experiences each week for em residents, which traditionally consists of formal lecture based instruction. however, retention by adult learners is limited when presented material in a lecture format. more effective methods such as small group sessions, simulation, and other active learning modalities are time-and resource-intensive and therefore not practical as a primary method of instruction. thus, the traditional lecture format remains heavily relied upon. efficient strategies to improve the effectiveness of lectures are needed. testing utilized as a learning tool to force immediate recall of lecture material is an example of such a strategy. objectives: to evaluate the effect of immediate postlecture short answer quizzes on em residents' retention of lecture content. methods: in this prospective randomized controlled study, em residents from a community based 3-year training program were randomized into two groups. block randomization provided a similar distribution of postgraduate year training levels and performance on both us-mle and in-training examinations between the two groups. each group received two identical 50-minute lectures on ecg interpretation and aortic disease. one group of residents completed a five-question short answer quiz immediately following each lecture (n = 13), while the other group received the lectures without subsequent quizzes (n = 16). the quizzes were not scored or reviewed with the residents. two weeks later, retention was assessed by testing both groups with a 20-question multiple choice test (mct) derived in equal part from each lecture. mean and median test results were then compared between groups. statistical significance was determined using a paired t-test of median test scores from each group. results: residents who received immediate post-lecture quizzes demonstrated significantly higher mct scores (mean = 57%, median 58%, n = 10) compared to those receiving lectures alone (mean = 48%, median = 50%, n = 15); p = 0.023. conclusion: short answer testing immediately after a traditional didactic lecture improves knowledge retention at a 2-week interval. limitations of the study are that it is a single center study and long term retention was not assessed. background: the task of educating the next generation of physicians is steadily becoming more difficult with the inherent obstacles that exist for faculty educators and the work hour restrictions that students must adhere to. the obstacles make developing curricula that not only cover important topics but also do so in a fashion that helps support and reinforce the clinical experiences very difficult. several areas of medical education are using more asynchronous techniques and self-directed online educational modules to overcome these obstacles. objectives: the aim of this study was to demonstrate that educational information pertaining to core pediatric emergency medicine topics could be as effectively disseminated to medical students via self-directed online educational modules as it could through traditional didactic lectures. methods: this was a prospective study conducted from august 1, 2010 through december 31, 2010. students participating in the emergency medicine rotation at carolinas medical center were enrolled and received education in a total of eight core concepts. the students were divided into two groups which changed on a monthly basis. group 1 was taught four concepts via self-directed online modules and four traditional didactic lectures. group 2 was taught the same core concepts, but in opposite fashion to group 1. each student was given a pre-test, post-test, and survey at the conclusion of the rotation. results: a total of 28 students participated in the study. students, regardless of which group assigned, performed similarly on the pre-test, with no statistical difference among scores. when looking at the summative total scores between online and traditional didactic lectures, there was a trend towards significance for more improvement among those taught online. the student's assessment of the online modules showed that the majority either felt neutral or preferred the online method. the majority thought the depth and length of the modules were perfect. most students thought having access to the online modules was valuable and all but one stated that they would use them again. conclusion: this study demonstrates that self-directed, online educational modules are able to convey important concepts in emergency medicine similar to traditional didactics. it is an effective learning technique that offers several advantages to both the educator and student. background: critical access hospitals (cah) provide crucial emergency care to rural populations that would otherwise be without ready access to health care. data show that many cah do not meet standard adult quality metrics. adults treated at cah often have inferior outcomes to comparable patients cared for at other community-based emergency departments (eds). similar data do not exist for pediatric patients. objectives: as part of a pilot project to improve pediatric emergency care at cah, we sought to determine whether these institutions stock the equipment and medications necessary to treat any ill or injured child who presents to the ed. methods: five north carolina cah volunteered to participate in an intensive educational program targeting pediatric emergency care. at the initial site visit to each hospital, an investigator, in conjunction with the ed nurse manager, completed a 109-item checklist of commonly required ed equipment and medications based on the 2009 acep ''guidelines for care of children in the emergency department''. the list was categorized into monitoring and respiratory equipment, vascular access supplies, fracture and trauma management devices, and specialized kits. if available, adult and pediatric sizes were listed. only hospitals stocking appropriate pediatric sizes of an item were counted as having that item. the pharmaceutical supply list included antibiotics, antidotes, antiemetics, antiepileptics, intubation and respiratory medications, iv fluids, and miscellaneous drugs not otherwise categorized. results: overall, the hospitals reported having 91% of the items listed (range 87-96%). the two greatest deficiencies were fracture devices (range 33-66%), with no hospital stocking infant-sized cervical collars, and antidotes, with no hospital stocking pralidoxime, 1/5 hospitals stocking fomepizole, and 2/5 hospitals stocking pyridoxine and methylene blue. only one of the five institutions had access to prostaglandin e. the hospitals stated cost and rarity of use as the reason for not stocking these medications. conclusion: the ability of cah to care for pediatric patients does not appear to be hampered by a lack of equipment. ready access to infrequently used, but potentially lifesaving, medications is a concern. tertiary care centers preparing to accept these patients should be aware of these potential limitations as transport decisions are made. background: while incision and drainage (i&d) alone has been the mainstay of management of uncomplicated abscesses for decades, some advocate for adjunct antibiotic use, arguing that available trials are underpowered and that antibiotics reduce treatment failures and recurrence. objectives: to investigate the role of antibiotics in addition to i&d in reducing treatment failure as compared to management with i&d alone. methods: we performed a search using medline, embase, web of knowledge, and google scholar databases (with a medical librarian) to include trials and observational studies analyzing the effect of antibiotics in human subjects with skin and soft-tissue abscesses. two investigators independently reviewed all the records. we performed three overlapping meta-analy-ses: 1. only randomized trials comparing antibiotics to placebo on improvement of the abscess during standard follow-up. 2. trials and observational studies comparing appropriate antibiotics to placebo, no antibiotics, or inappropriate antibiotics (as gauged by wound culture) on improvement during standard follow-up. 3. only trials, but broadened outcome to include recurrence or new lesions during a longer follow-up period as treatment failure. we report pooled risk ratios (rr) using a fixed-effects model for our point estimates with shore-adjusted 95% confidence intervals (ci). results: we screened 1,937 records, of which 12 studies fit inclusion criteria, 9 of which were meta-analyzed (5 trials, 4 observational studies) because they reported results that could be pooled. of the 9 studies, 5 enrolled subjects from the ed, 2 from a soft-tissue infection clinic, and 2 from a general hospital without definition of enrollment site. five studies enrolled primarily adults, 3 pediatrics, and 1 without specification of ages. after pooling results for all randomized trials only, the rr = 1.03 (95% ci: 0.97-1.08). exposure being ''appropriate'' antibiotics (using trials and observational studies) resulted in a pooled rr = 1.01 (95% ci: 0.98-1.03). when we broadened our treatment failure criteria to include recurrence or new lesions at longer lengths of follow-up (trials only), we noted a rr = 1.05 (95% ci: 0.97-1.15). conclusion: based on available literature pooled for this analysis, there is no evidence to suggest any benefit from antibiotics in addition to i&d in the treatment of skin and soft tissue abscesses. (originally submitted as a ''late-breaker.'') primary objectives: to compare wound healing and recurrence rates after primary vs. secondary closure of drained abscesses. we hypothesized the percentage of drained ed abscesses that would be completely healed at 7 days would be higher after primary closure. methods: this randomized clinical trial was undertaken in two academic emergency departments. immunocompetent adult patients with simple, localized cutaneous abscesses were randomly assigned to i & d followed by primary or secondary closure. randomization was balanced by center, with an allocation sequence based on a block size of four, generated by a computer random number generator. the primary outcome was percentage of healed wounds seven days after drainage. a sample of 50 patients had 80% power to detect an absolute difference of 40% in healing rates assuming a baseline rate of 25%. all analyses were by intention to treat. results: twenty-seven patients were allocated to primary and 29 to secondary closure, of whom 23 and 27, respectively, were followed to study completion. healing rates at seven days were similar between the primary and secondary closure groups ( we compared 100 consecutive patients each scanned on the 64 or 320 slice ccta in 2010-2011. measures and outcomes-data were prospectively collected using standardized data collection forms required prior to performing ccta. the main outcomes were cumulative radiation doses and volumes of intravenous contrast. data analysis-groups compared with t-, mann whitney u, and chi-square tests. results: the mean age of patients imaged with the 64 and 320 scanners were 49 (sd 10) vs. 51 (13) (p = 0.27). male:female ratios were also similar (57:43 vs. 51:49 respectively, p = 0.40). both mean (p < 0.001) and median (p = 0.006) effective radiation dose were significantly lower with the 320 (6.8 and 6 msv) vs. the 64-slice scanner (12.2 and 10 msv) respectively. prospective gating was successful in 100% of the 320 scans and only in 38% of the 64 scans (p < 0.001). mean iv contrast volumes were also lower for the 320 vs. the 64-slice scanner (74 ± 10 vs. 96 ± 12 ml; p < 0.001). the % non-diagnostic scans was similarly low in both scanners (3% each). there were no differences in use of beta-blockers or nitrates. conclusion: when compared with the 64-slice scanner, the 320-slice scanner reduces the effective radiation doses and iv contrast volumes in ed patients with cp undergoing ccta. need for beta-blockers and nitrates was similar and both scanners achieved excellent diagnostic image quality. background: a few studies have demonstrated that bedside ultrasound measurement of inferior vena cava to aorta (ivc-to-ao) ratio is associated with the level of dehydration in pediatric patients and a proposed cutoff of 0.8 has been suggested, below which a patient is considered dehydrated. objectives: we sought to externally validate the ability of ivc-to-ao ratio to discriminate dehydration and the proposed cutoff of 0.8 in an urban pediatric emergency department (ed). methods: this was a prospective observational study at an urban pediatric ed. we included patients aged 3 to 60 months with clinical suspicion of dehydration by the ed physician and an equal number of control patients with no clinical suspicion of dehydration. we excluded children who were hemodynamically unstable, had chronic malnutrition or failure to thrive, open abdominal wounds, or were unable to provide patient or parental consent. a validated clinical dehydration score (cds) (range 0 to 8) was used to measure initial dehydration status. an experienced sonographer blinded to the cds and not involved in the patient's care measured the ivc-to-ao ratio on the patient prior to any hydration. cds was collapsed into a binary outcome of no dehydration or any level of dehydration (1 or higher). the ability of ivc-to-ao ratio to discriminate dehydration was assessed using area under the receiver operating characteristic curve (auc) and the sensitivity and specificity of ivc-to-ao ratio was calculated for three cutoffs (0.6, 0.8, 1.0). calculation of auc was repeated after adjusting for age and sex. results: 92 patients were enrolled, 39 (42%) of whom had a cds of 1 or higher. median age was 28 (interquartile range 16-39) months, and 53 (58%) were female. the ivcto-ao ratio showed an unadjusted auc of 0.66 (95% ci 0.54-0.77) and adjusted auc of 0.67 (95% ci 0.56-0.79). for a cutoff of 0.6 sensitivity was 26% (95% ci 13%-42%) and specificity 92% (95% ci 82%-98%); for a cutoff of 0.8 sensitivity was 51% (95% ci 35%-68%) and specificity 74% (95% ci 60%-85%); for a cutoff of 1.0 sensitivity was 79% (95% ci 64%-91%) and specificity 40% (95% ci 26%-54%). conclusion: the ability of the ivc-to-ao ratio to discriminate dehydration in young pediatric ed patients was modest and the cutoff of 0.8 was neither sensitive nor specific. background: while early cardiac computed tomographic angiography (ccta) could be more effective to manage emergency department (ed) patients with acute chest pain and intermediate (>4%) risk of acute coronary syndrome (acs) than current management strategies, it also could result in increased testing, cost, and radiation exposure. objectives: the purpose of the study was to determine whether incorporation of ccta early in the ed evaluation process leads to more efficient management and earlier discharge than usual care in patients with acute chest pain at intermediate risk for acs. methods: randomized comparative effectiveness trial enrolling patients between 40-75 years of age without known cad, presenting to the ed with chest pain but without ischemic ecg changes or elevated initial troponin and require further risk stratification for decision making, at nine us sites. patients are being randomized to either ccta as the first diagnostic test or to usual care, which could include no testing or functional testing such as exercise ecg, stress spect, and stress echo following serial biomarkers. test results were provided to physicians but management in neither arm was driven by a study protocol. data on time, diagnostic testing, and cost of index hospitalization, and the following 28 days are being collected. the primary endpoint is length of hospital stay (los). the trial is powered to allow for detection of a difference in los of 10.1 hours between competing strategies with 95% power assuming that 70% of projected los values are true. secondary endpoints are cumulative radiation exposure, and cost of competing strategies. tertiary endpoints are institutional, caregiver, and patient characteristics associated with primary and secondary outcomes. rate of missed acs within 28 days is the safety endpoint. results: as of november 21st, 2011, 880 of 1000 patients have been enrolled (mean age: 54 ± 8, 46.5% female, acs rate 7.55%). the anticipated completion of the last patient visit is 02/28/12 and the database will be locked in early march 2012. we will present the results of the primary, secondary, and some tertiary endpoints for the entire cohort. conclusion: romicat ii will provide rigorous data on whether incorporation of ccta early in the ed evaluation process leads to more efficient management and triage than usual care in patients with acute chest pain at intermediate risk for acs. (originally submitted as a ''late-breaker.'') meta background: many studies have documented higher rates of advanced radiography utilization across u.s. emergency departments (eds) in recent years, with an associated decrease in diagnostic yield (positive tests / total tests). provider-to-provider variability in diagnostic yield has not been well studied, nor have the factors that may explain these differences in clinical practice. objectives: we assessed the physician-level predictors of diagnostic yield using advanced radiography to diagnose pulmonary embolus (pe) in the ed, including demographics and d-dimer ordering rates. methods: we conducted a retrospective chart review of all ed patients who had a ct chest or v/q scan ordered to rule out pe from 1/06 to 12/09 in four hospitals in the medstar health system. attending physicians were included in the study if they had ordered 50 or more scans over the study period. the result of each ct and vq scan was recorded as positive, negative, or indeterminate, and the identity of the ordering physician was also recorded. data on provider sex, residency type (em or other), and year of residency completion were collected. each provider's positive diagnostic yield was calculated, and logistic regression analysis was done to assess correlation between positive scans and provider characteristics. results: during the study period, 15,015 scans (13,571 cts and 1,443 v/qs) were ordered by 93 providers. the physicians were an average of 9.7 years from residency, 36% were female, and 98% were em-trained. diagnostic yield varied significantly among physicians (p < 0.001), and ranged from 0% to 18%. the median diagnostic yield was 5.9% (iqr 3.8%-7.8%). the use of d-dimer by provider also varied significantly from 4% to 48% (p < 0.001). the odds of a positive test were significantly lower among providers less than 10 years out of residency graduation (or 0.80, ci 0.68-0.95) after controlling for provider sex, type of residency training, d-dimer use, and total number of scans ordered. conclusion: we found significant provider variability in diagnostic yield for pe and use of d-dimer in this study population, with 25% of providers having diagnostic yield less than or equal to 3.8%. providers who were more recently graduated from residency appear to have a lower diagnostic yield, suggesting a more conservative approach in this group. background: the literature reports that anticoagulation increases the risk of mortality in patients presenting to emergency departments (ed) with head trauma (ht). it has been suggested that such patients should be treated in a protocolized fashion, including ct within 15 minutes, and anticipatory preparation of ffp before ct results are available. there are significant logistical and financial implications associated with implementation of such a protocol. objectives: our primary objective was to determine the effect of anticoagulant therapy on the risk of intracranial hemorrhage (ich) in elderly patients presenting to our urban community hospital following bunt head injury. methods: this was a retrospective chart review study of ht patients >60 years of age presenting to our ed over a 6-month period. charts reviewed were identified using our electronic medical record via chief complaints and icd-9 codes and cross referencing with written ct logs. research assistants underwent review of at least 25% of their contributing data to validate reliability. we collected information regarding use of warfarin, clopidogrel, and aspirin and ct findings of ich. using univariate logistic regression, we calculated odds ratios (or) for ich with 95% ci. results: we identified 363 elderly ht patients. the mean age of our population was 72, 34 (8.3%) admitted to using anticoagulant therapy, and 23% were on antiplatelet drugs. 14 (3.8%) of the cohort had icb, 3 patients required neurosurgical intervention, and 1 had transfusion of blood products. of the non-anticoagulated patients, 12 (3.6%) were found to have ich, half of those (6) , and mir-223) were measured using real-time quantitative pcr from serum drawn at enrollment. il-6, il-10, and tnf-a were measured using a bio-plex suspension system. baseline characteristics, il-6, il-10, tnf-a and micrornas were compared using one way anova or fisher exact test, as appropriate. correlations between mirnas and sofa scores, il-6, il-10, and tnf-a were determined using spearman's rank. a logistic regression model was constructed using in-hospital mortality as the dependent variable and mirnas as the independent variables of interest. bonferroni adjustments were made for multiple comparisons. results: of 93 patients, 24 were controls, 29 had sepsis, and 40 had septic shock. we found no difference in serum mir-146a or mir-223 between cohorts, and found no association between these micrornas and either inflammatory markers or sofa score. mir-150 demonstrated a significant correlation with sofa score (q = 0.31, p = 0.01), il-10 (q = 0.37, p = 0.001), but not il-6 or tnf-a (p = 0.046, p = 0.59). logistic regression demonstrated mir-150 to be associated with mortality, even after adjusting for sofa score (p = 0.003). conclusion: mir-146a or mir-223 failed to demonstrate any diagnostic or prognostic ability in this cohort. mir-150 was associated with inflammation, increasing severity of illness, and mortality, and may represent a novel prognostic marker for diagnosis and prognosis of sepsis. objectives: to examine the association between emergency physician recognition of sirs and sepsis and subsequent treatment of septic patients. methods: a retrospective cohort study of all-age patient medical records with positive blood cultures drawn in the emergency department from 11/2008-1/ 2009 at a level i trauma center. patient parameters were reviewed including vital signs, mental status, imaging, and laboratory data. criteria for sirs, sepsis, severe sepsis, and septic shock were applied according to established guidelines for pediatrics and adults. these data were compared to physician differential diagnosis documentation. the mann-whitney test was used to compare time to antibiotic administration and total volume of fluid resuscitation between two groups of patients: those with recognized sepsis and those with unrecognized sepsis. results: sirs criteria were present in 233/338 reviewed cases. sepsis criteria were identified in 215/338 cases and considered in the differential diagnosis in 121/215 septic patients. severe sepsis was present in 89/338 cases and septic shock was present in 42/338 cases. the sepsis 6-hour resuscitation bundle was completed in the emergency department in 16 cases of severe sepsis or septic shock. 121 patients who met sepsis criteria and were recognized by the ed physician had a median time to antibiotics of 150 minutes (iqr: 89-282) and a median ivf of 1500 ml (iqr: 500-3000). the 94 patients who met sepsis criteria but went unrecognized in the documentation had a median time to antibiotics of 225 minutes (iqr: 135-355) and median volume of fluid resuscitation of 1000 ml (iqr: . median time to antibiotics and median volume of fluid resuscitation differed significantly between recognized and unrecognized septic patients (p = 0.003 and p = 0.002, respectively). conclusion: emergency physicians correctly identify and treat infection in most cases, but frequently do not document sirs and sepsis. lack of documentation of sepsis in the differential diagnosis is associated with increased time to antibiotic delivery and a smaller total volume of fluid administration, which may explain poor sepsis bundle compliance in the emergency department. background: severe sepsis is a common clinical syndrome with substantial human and financial impact. in 1992 the first consensus definition of sepsis was published. subsequent epidemiologic estimates were collected using administrative data, but ongoing discrepancies in the definition of severe sepsis led to large differences in estimates. objectives: we seek to describe the variations in incidence and mortality of severe sepsis in the us using four methods of database abstraction. methods: using a nationally representative sample, four previously published methods (angus, martin, dombrovskiy, wang) were used to gather cases of severe sepsis over a 6-year period (2004) (2005) (2006) (2007) (2008) (2009) . in addition, the use of new icd-9 sepsis codes was compared to previous methods. our main outcome measure was annual national incidence and in-hospital mortality of severe sepsis. results: the average annual incidence varied by as much as 3.5 fold depending on method used and ranged from 894,013 (300 / 100,000 population) to 3,110,630 (1,031 / 100,000) using the methods of dombrovskiy and wang, respectively. average annual increase in the incidence of severe sepsis was similar (13.0-13.3%) across all methods. total mortality mirrored the increase in incidence over the 6-year period ( background: radiation exposure from medical imaging has been the subject of many major journal articles, as well as the topic of mainstream media. some estimate that one-third of all ct scans are not medically justified. it is important for practitioners ordering these scans to be knowledgeable of currently discussed risks. objectives: to compare the knowledge, opinions, and practice patterns of three groups of providers in regards to cts in the ed. methods: an anonymous electronic survey was sent to all residents, physician assistants, and attending physicians in emergency medicine (em), surgery, and internal medicine (im) at a single academic tertiary care referral level i trauma center with an annual ed volume of over 160,000 visits. the survey was pilot tested and validated. all data were analyzed using the pearson's chi-square test. results: there was a response rate of 32% (220/668). data from surgery respondents were excluded due to a low response rate. in comparison to im, em respondents correctly equated one abdominal ct to between 100 and 500 chest x-rays, reported receiving formal training regarding the risks of radiation from cts, believe that excessive medical imaging is associated with an increased lifetime risk of cancer, and routinely discuss the risks of ct imaging with stable patients more often (see table 1 ). particular patient factors influence whether radiation risks are discussed with patients by 60% in each specialty (see table 2 ). before ordering an abdominal ct in a stable patient, im providers routinely review the patient's medical imaging history less often than em providers surveyed. overall, 67% of respondents felt that ordering an abdominal ct in a stable ed patient is a clinical decision that should be discussed with the patient, but should not require consent. conclusion: compared with im, em practitioners report greater awareness of the risks of radiation from cts and discuss risks with patients more often. they also review patients' imaging history more often and take this, as well as patients' age, into account when ordering cts. these results indicate a need for improved education for both em and im providers in regards to the risks of radiation from ct imaging. background: in nebraska, 80% of emergency departments have annual visits less than 10,000, and the predominance are in rural settings. general practitioners working in rural emergency departments have reported low confidence in several emergency medicine skills. current staffing patterns include using midlevels as the primary provider with non-emergency medicine trained physicians as back-up. lightly-embalmed cadaver labs are used for resident's procedural training. objectives: to describe the effect of a lightlyembalmed cadaver workshop on physician assistants' (pa) reported level of confidence in selected emergency medicine procedures. methods: an emergency medicine procedure lab was offered at the nebraska association of physician assistants annual conference. each lab consisted of a 2-hour hands-on session teaching endotracheal intubation techniques, tube thoracostomy, intraosseous access, and arthrocentesis of the knee, shoulder, ankle, and wrist to pas. irb-approved surveys were distributed pre-lab and a post-lab survey was distributed after lab completion. baseline demographic experience was collected. pre-and post-lab procedural confidence was rated on a six-point likert scale (1-6) with 1 representing no confidence. the wilcoxon signed-rank test was use to calculate p values. results: 26 pas participated in the course. all completed a pre-and post-lab assessment. no pa had done any one procedure more than 5 times in their career. pre-lab modes of confidence level were £3 for each procedure. post-lab modes were >4 for each procedure except arthrocentesis of the ankle and wrist. however, post lab assessments of procedural confidence significantly improved for all procedures with p values <0.05. conclusion: midlevel providers' level of confidence improved for emergent procedures after completion of a procedure lab using lightly-embalmed cadavers. a mobile cadaver lab would be beneficial to train rural providers with minimal experience. background: use of automated external defibrillators (aed) improves survival in out-of-hospital cardiopulmonary arrest (ohca). since 2005, the american heart association has recommended that individuals one year of age or older who sustain ohca have an aed applied. little is known about how often this occurs and what factors are associated with aed use in the pediatric population. objectives: our objective was to describe aed use in the pediatric population and to assess predictors of aed use when compared to adult patients. methods: we conducted a secondary analysis of prospectively collected data from 29 u.s. cities that participate in the cardiac arrest registry to enhance survival (cares). patients were included if they had a documented resuscitation attempt from october 1, 2005 through december 31, 2009 and were ‡1 year old. patients were considered pediatric if they were less than 19 years old. aed use included application by laypersons and first responders. hierarchical multivariable logistic regression analysis was used to estimate the associations between age and aed use. results: there were 19,559 ohcas included in this analysis, of which 239 (1.2%) occurred in pediatric patients. overall aed use in the final sample was 5,517, with 1,751 (8.9%) total survivors. aeds were applied less often in pediatric patients (19.7%, 95% ci: 14.6%-24.7% vs 28.3%, 95% ci: 27.7%-29.0%). within the pediatric population, only 35.4% of patients with a shockable rhythm had an aed used. in all pediatric patients, regardless of presenting rhythm, aed use demonstrated a statistically significant increase in return of spontaneous circulation (aed used 29.8%, 95% ci: 16.2-43.4 vs aed not used 16.8%, 95% ci: 11.4-22.1, p < 0.05), although there was no significant increase in survival to hospital discharge (aed used 12.8%; aed not used 5.2%; p = 0.057). in the adjusted model, pediatric age was independently associated with failure to use an aed (or 0.61, 95% ci: 0.42-0.87) as was female sex (or 0.88, 95% ci: 0.81-0.95). patients who had a public arrest (or 1.35, 95% ci: 1.24-1.46) or one that was witnessed by a bystander (or 1.20. 95%: ci 1.11-1.29) were also predictive of aed use. conclusion: pediatric patients who experience ohca are less likely to have an aed used. continued education of first responders and the lay public to increase aed use in this population is necessary. does implementation of a therapeutic hypothermia protocol improve survival and neurologic outcomes in all comatose survivors of sudden cardiac arrest? ken will, michael nelson, abishek vedavalli, renaud gueret, john bailitz cook county (stroger), chicago, il background: the american heart association (aha) currently recommends therapeutic hypothermia (th) for out of hospital comatose survivors of sudden cardiac arrest (cssca) with an initial rhythm of ventricular fibrillation (vf). based on currently limited data, the aha further recommends that physicians consider th for cssca, from both the out and inpatient settings, with an initial non-vf rhythm. objectives: investigate whether a th protocol improves both survival and neurologic outcomes for cssca, for out and inpatients, with any initial rhythm, in comparison to outcomes previously reported in literature prior to th. methods: we conducted a prospective observational study of cssca between august 2009 and may 2011 whose care included th. the study enrolled eligible consecutive cssca survivors, from both out and inpatient settings with any initial arrest rhythm. primary endpoints included survival to hospital discharge and neurologic outcomes, stratified by sca location, and by initial arrest rhythm. results: overall, of 27 eligible patients, 11 (41%, 95% ci 22-66%) survived to discharge, 7 (26%, 95% ci 9-43%) with at least a good neurologic outcome. twelve were out and 15 were inpatients. among the 12 outpatients, 6 (50%, 95% ci 22-78%) survived to discharge, 5 (41%, 95% ci 13-69%) with at least a good neurologic outcome. among the 15 inpatients, 5 (33%, 95% ci 9-57) survived to discharge, 2 (13%, 95% ci 0-30%) with at least a good neurologic outcome. by initial rhythm, 6 patients had an initial rhythm of vf/t and 21 non-vf/t. among the 6 patients with an initial rhythm of vf/t, 4 (67%, ci 39-100%) survived to discharge, all 4 with at least a good outcome, including 3 out and 1 inpatients. among the 21 patients with an initial rhythm of non-vf/t, 7 (33%, ci 22-53%) survived to discharge, 3 (14%, ci 0-28%) with at least a good neurologic outcome, including 2 out and 1 inpatients. conclusion: our preliminary data initially suggest that local implementation of a th protocol improves survival and neurologic outcomes for cssca, for out and inpatients, with any initial rhythm, in comparison to outcomes previously reported in literature prior to th. subsequent research will include comparison to local historical controls, additional data from other regional th centers, as well as comparison of different cooling methods. protocolized background: therapeutic hypothermia (th) has been shown to improve the neurologic recovery of cardiac arrest patients who experience return of spontaneous circulation (rosc). it remains unclear as to how earlier cooling and treatment optimization influence outcomes. objectives: to evaluate the effects of a protocolized use of early sedation and paralysis on cooling optimization and clinical outcomes in survivors of cardiac arrest. methods: a 3-year (2008-2010), pre-post intervention study of patients with rosc after cardiac arrest treated with th was performed. those patients treated with a standardized order set which lacked a uniform sedation and paralytic order were included in the pre-intervention group, and those with a standardized order set which included a uniform sedation and paralytic order were included in the post-intervention group. patient demographics, initial and discharge glasgow coma scale (gcs) scores, resuscitation details, cooling time variables, severity of illness as measured by the apache ii score, discharge disposition, functional status, and days to death were collected and analyzed using student's t-tests, man-whitney u tests, and the log-rank test. results: 232 patients treated with th after rosc were included, with 107 patients in the pre-intervention group and 125 in the post-intervention group. the average time to goal temperature (33°c) was 227 minutes (pre-intervention) and 168 minutes (post-intervention) (p = 0.001). a 2-hour time target was achieved in 38.6% of the patients (post-intervention) compared to 24.5% in the pre-group (p = 0.029). twenty-eight day mortality was similar between groups (65.4% and 65.3%) though hospital length of stay (10 days pre-and 8 days post-intervention) and discharge gcs (13 preand 14-post-intervention) differed between cohorts. more post-intervention patients were discharged to home (55.8%) compared to 43.2% in the pre-intervention group. conclusion: protocolized use of sedation and paralysis improved time to goal temperature achievement. these improved th time targets were associated with improved neuroprotection, gcs recovery, and disposition outcome. standardized sedation and paralysis appears to be a useful adjunct in induced th. background: ct is increasingly used to assess children with signs and symptoms of acute appendicitis (aa) though concerns regarding long-term risk of exposure to ionizing radiation have generated interest in methods to identify children at low risk. objectives: we sought to derive a clinical decision rule (cdr) of a minimum set of commonly used signs and symptoms from prior studies to predict which children with acute abdominal pain have a low likelihood of aa and compared it to physician clinical impression (pci). methods: we prospectively analyzed 420 subjects aged 2 to 20 years in 11 u.s. emergency departments with abdominal pain plus signs and symptoms suspicious for aa within the prior 72 hours. subjects were assessed by study staff unaware of their diagnosis for 17 clinical attributes drawn from published appendicitis scoring systems and physicians responsible for physical examination estimated the probability of aa based on pci prior to their medical disposition. based on medical record entry rate, frequently used cdr attributes were evaluated using recursive partitioning and logistic regression to select the best minimum set capable of discriminating subjects with and without aa. subjects were followed to determine whether imaging was used and use was tabulated by both pci and the cdr to assess their ability to identify patients who did or did not benefit based on diagnosis. results: this cohort had a 27.3% prevalence (118/431 subjects) of aa. we derived a cdr based on the absence of two out of three of the following attributes: abdominal tenderness, pain migration, and rigidity/ guarding had a sensitivity of 89.8% (95% ci: 83.1-94.1), specificity of 47.6% (95% ci: 42.1-53.1), npv of 92.5% (95% ci: 87.4-95.7), and negative likelihood ratio of 0.21 (95% ci: 0.12-0.37). the pci set at aa <30% pre-test probability had a sensitivity of 94.1% (95% ci: 88.3-97.1), specificity of 49.4% (95% ci: 43.9-54.9), npv of 95.7% (95% ci: 91.3-97.9), and negative likelihood ratio of 0.12 (95% ci: 0.06-0.25). the methods each classified 37% of the patients as low risk for aa. our cdr identified 29.1% (43/148) of low risk subjects who received ct but being aa (-), could have been spared ct, while the pci identified 20.1% (30/149). conclusion: compared to physician clinical impression, our clinical decision rule can identify more children at low risk for appendicitis who could be managed more conservatively with careful observation and avoidance of ct. negative background: abdominal pain is the most common complaint in the ed and appendicitis is the most common indication for emergency surgery. a clinical decision rule (cdr) identifying abdominal pain patients at a low risk for appendicitis could lead to a significant reduction in ct scans and could have a significant public health impact. the alvarado score is one of the most widely applied cdrs for suspected appendicitis, and a low modified alvarado score (less than 4) is sometimes used to rule out acute appendicitis. the modified alvarado score has not been prospectively validated in ed patients with suspected appendicitis. objectives: we sought to prospectively evaluate the negative predictive value of a low modified alvarado score (mas) in ed patients with suspected appendicitis. we hypothesized that a low mas (less than 4) would have a sufficiently high npv (>95%) to rule out acute appendicitis. methods: we enrolled patients greater than or equal to 18 years old who were suspected of having appendicitis (listed as one of the top three diagnosis by the treating physician before ancillary testing) as part of a prospective cohort study in two urban academic eds from august 2009 to april 2010. elements of the mas and the final diagnosis were recorded on a standard data form for each subject. the sensitivity, specificity, negative predictive value (npv), and positive predictive value (ppv) were calculated with 95% ci for a low mas and final diagnosis of appendicitis. background: evaluating children for appendicitis is difficult and strategies have been sought to improve the precision of the diagnosis. computed tomography is now widely used but remains controversial due to the large dose of ionizing radiation and risk of subsequent radiation-induced malignancy. objectives: we sought to identify a biomarker panel for use in ruling out pediatric acute appendicitis as a means of reducing exposure to ionizing radiation. methods: we prospectively enrolled 431 subjects aged 2 to 20 years presenting in 11 u.s. emergency departments with abdominal pain and other signs and symptoms suspicious for acute appendicitis within the prior 72 hours. subjects were assessed by study staff unaware of their diagnosis for 17 clinical attributes drawn from appendicitis scoring systems and blood samples were analyzed for cbc differential and 5 candidate proteins. based on discharge diagnosis or post-surgical pathology, the cohort exhibited a 27.3% prevalence (118/431 subjects) of appendicitis. clinical attributes and biomarker values were evaluated using principal component, recursive partitioning, and logistic regression to select the combination that best discriminated between those subjects with and without disease. mathematical combination of three inflammation-related markers in a panel comprised of myeloid-related protein 8/14 complex (mrp), c-reactive protein (crp), and white blood cell count (wbc) provided optimal discrimination. results: this panel exhibited a sensitivity of 98% (95% ci, 94-100%), a specificity of 48% (95% ci, 42-53%), and a negative predictive value of 99% (95% ci, 95-100%) in this cohort. the observed performance was then verified by testing the panel against a pediatric subset drawn from an independent cohort of all ages enrolled in an earlier study. in this cohort, the panel exhibited a sensitivity of 95% (95% ci, 87-98%), a specificity of 41% (95% ci, 34-50%), and a negative predictive value of 95% (95% ci, 87-98%). conclusion: appyscore is highly predictive of the absence of acute appendicitis in these two cohorts. if these results are confirmed by a prospective evaluation currently underway, the appyscore panel may be useful to classify pediatric patients presenting to the emergency department with signs and symptoms suggestive of, or consistent with, acute appendicitis and thereby sparing many patients ionizing radiation. background: there are no current studies on the tracking of emergency department (ed) patient dispersal when a major ed closes. this study demonstrates a novel way to track where patients sought emergency care following the closure of saint vincent's catholic medical center (svcmc) in manhattan by using de-identified data from a health information exchange, the new york clinical information exchange (nyclix). nyclix matches patients who have visited multiple sites using their demographic information. on april 30, 2010, svcmc officially stopped providing emergency and outpatient services. we report the patterns in which patients from svcmc visited other sites within nyclix. objectives: we hypothesize that patients often seek emergency care based on geography when a hospital closes. methods: a retrospective pre-and post-closure analysis was performed of svcmc patients visiting other hospital sites. the pre-closure study dates were january 1, 2010-march 31, 2010. the post closure study dates were may 1, 2010-july 31, 2010. a svcmc patient was defined as a patient with any svcmc encounter prior to its closure. using de-identified aggregate count data, we calculated the average number of visits per week by svcmc patients at each site (hospital a-h). we ran a paired t-test to compare the pre-and post-closure averages by site. the following specifications were used to write the database queries: of patients who had one or more prior visits to svcmc for each day within the study return the following: a. eid: a unique and meaningless proprietary id generated within the nyclix master patient index (mpi). b. age: thru the age of 89. persons over 90 were listed as ''90 + '' c. ethnicity/race d. type of visit: emergency e. location of visit: specific nyclix site. results: nearby hospitals within 2 miles saw the highest number of increased ed visits after svcmc closed. this increase was seen until about 5 miles. hospitals >5 miles away did not see any significant changes in ed visits. see table. conclusion: when a hospital and its ed close down, patients seem to seek emergency care at the nearest hospital based on geography. other factors may include the patient's primary doctor, availabilities of outpatient specialty clinics, insurance contracts, or preference of ambulance transports. this study is limited by the inclusion of data from only the eight hospitals participating in nyclix at the time of the svcmc closure. upstream methods: data were collected on all ed ems arrivals from the metro calgary (population 1.1 million) area to its three urban adult hospitals. the study phases consisted of the 7 months from february to october 2010 (pre-ocp) compared against the same months in 2011 (post-ocp). data from the ems operational database and the regional emergency department information system (redis) database were linked. the primary analysis examined the change in ems offload delay defined as the time from ems triage arrival until patient transfer to an ed bed. a secondary analysis evaluated variability in ems offload delay between receiving eds. conclusion: implementation of a regional overcapacity protocol to reduce ed crowding was associated with an important reduction in ems offload delay, suggesting that policies that target hospital processes have bearing on ems operations. variability in offload delay improvements is likely due to site-specific issues, and the gains in efficiency correlate inversely with acuity. methods: a pre-post intervention study was conducted in the ed of an adult university teaching hospital in montreal (annual visits = 69 000). the raz unit (intervention), created to offload the acu of the main ed, started operating in january, 2011. using a split flow management strategy, patients were directed to the raz unit based on patient acuity level (ctas code 3 and certain code 2), likelihood to be discharged within 12 hours, and not requiring an ed bed for continued care. data were collected weekdays from 9:00 to 21:00 for 4 months (september -december 2008) (pre-raz) and for 1.5 months (february -march 2011) (post-raz). in the acu of the main ed, research assistants observed and recorded cubicle access time, and nurse and physician assessment times. databases were used to extract socio-demographics, ambulance arrival, triage code, chief complaint, triage and registration time, length of stay, and ed occupancy. background: telephone follow-up after discharge from the ed is useful for treatment and quality assurance purposes. ed follow-up studies frequently do not achieve high (i.e. ‡ 80%) completion rates. objectives: to determine the influence of different factors on the telephone follow-up rate of ed patients. we hypothesized that with a rigorous follow-up system we could achieve a high follow-up rate in a socioeconomically diverse study population. methods: research assistants (ras) prospectively enrolled adult ed patients discharged with a medication prescription between november 15, 2010 and september 9, 2011 from one of three eds affiliated with one health care system: (a) academic level i trauma center, (b) community teaching affiliate, and (c) community hospital. patients unable to provide informed consent, non-english speaking, or previously enrolled were excluded. ras interviewed subjects prior to ed discharge and conducted a telephone follow-up interview 1 week later. follow-up procedures were standardized (e.g. number of calls per day, times to place calls, obtaining alternative numbers) and each subject's follow-up status was monitored and updated daily through a shared, web-based data system. subjects who completed follow-up were mailed a $10 gift card. we examined the influence of patient (age, sex, race, insurance, income, marital status, usual major activity, education, literacy level, health status), clinical (acuity, discharge diagnosis, ed length of stay, site), and procedural factors (number and type of phone numbers received from subjects, offering two gift cards for difficult to reach subjects) on the odds of successful followup using multivariate logistic regression. results: of the 3,940 enrolled, 45% were white, 59% were covered by medicaid or uninsured, and 44% reported an annual household income of <$26,000. 86% completed telephone follow-up with 41% completing on the first attempt. the table displays the factors associated with successful follow-up. in addition to patient demographics and lower acuity, obtaining a cell phone or multiple phone numbers as well as offering two gift cards to a small number of subjects increased the odds of successful follow-up. conclusion: with a rigorous follow-up system and a small monetary incentive, a high telephone follow-up rate is achievable one week after an ed visit. methods: an interrupted time-series design was used to evaluate the study question. data regarding adherence with the following pneumonia core measures were collected pre-and post-implementation of the enhanced decision-support tool: blood cultures prior to antibiotic, antibiotic within 6 hours of arrival, appropriate antibiotic selection, and mean time to antibiotic administration. prescribing clinicians were educated on the use of the decision-support tool at departmental meetings and via direct feedback on their cases. results: during the 33-month study period, complete data were collected for 1185 patients diagnosed with cap: 613 in the pre-implementation phase and 572 post-implementation. the mean time to antibiotic administration decreased by approximately one minute from the pre-to post-implementation phase, a change that was not statistically significant (p = 0.824). the proportion of patients receiving blood cultures prior to antibiotics improved significantly (p < 0.001) as did the proportion of patients receiving antibiotics within 6 hours of ed arrival (p = 0.004). a significant improvement in appropriate antibiotic selection was noted with 100% of patients experiencing appropriate selection in the post-phase, p = 0.0112. use of the available support tool increased throughout the study period, v 2 = 78.13, df = 1, p < 0.0001. all improvements were maintained 15 months following the study intervention. conclusion: in this academic ed, introduction of an enhanced electronic clinical decision support tool significantly improved adherence to cms pneumonia core measures. the proportion of patients receiving blood cultures prior to antibiotics, antibiotics within 6 hours, and appropriate antibiotics all improved significantly after the introduction of an enhanced electronic clinical decision support tool. background: emergency medicine (em) residency graduates need to pass both the written qualifying exam and oral certification exam as the final benchmark to achieve board certification. the purpose of this project is to obtain information about the exam preparation habits of recent em graduates to allow current residents to make informed decisions about their individual preparation for the abem written qualifying and oral certification exams. objectives: the study sought to determine the amount of residency and individual preparation, to determine the extent of the use of various board review products, and to elicit evaluations of the various board review products used for the abem qualifying and certification exams. methods: design: an online survey instrument was used to ask respondents questions about residency preparation and individual preparation habits, as well as the types of board review products used in preparing for the em boards. participants: as greater than 95% of all em graduates are emra members, an online survey was sent to all emra members who have graduated for the past three years. observations: descriptive statistics of types of preparation, types of resources, time, and quantitative and qualitative ratings for the various board preparation products were obtained from respondents. results: a total of 520 respondents spent an average of 9.1 weeks and 15 hours per week preparing for the written qualifying exam and spent an average of 5 weeks and 7.8 hours per week preparing for the oral certification exam. in preparing for the written qualification exam, 90% used a preparation textbook with 16% using more than one textbook and 47% using a board preparation course. in preparing for the oral qualifying exam, 56% used a preparation textbook while 34% used a preparation course. sixty-seven percent of respondents reported that their residency programs had a formalized written qualifying exam preparation curriculum of which 48% was centered on the annual in-training exam. eight-five percent of residency programs had a formalized oral certification exam preparation. respondents reported spending on average $715 preparing for the qualifying exam and $509 for the certification exam. conclusion: em residents spend significant amounts of time and money and make use of a wide range of residency and commercially available resources in preparing for the abem qualifying and certification exams. background: communication and professionalism skills are essential for em residents but are not wellmeasured by selection processes. the multiple mini-interview (mmi) uses multiple, short structured contacts to measure these skills. it predicts medical school success better than the interview and application. its acceptability and utility in em residency selection is unknown. objectives: we theorized that the mmi would provide novel information and be acceptable to participants. methods: 71 interns from three programs in the first month of training completed an eight-station mmi developed to focus on em topics. pre-and post-surveys assessed reactions using five-point scales. mmi scores were compared to application data. results: em grades correlated with mmi performance (f(1.66) = 4:18, p < 0.05) with honors students having higher mmi summary scores. higher third year clerkship grades trended to higher mmi performance means, although not significantly. mmi performance did not correlate with a match desirability rating and did not predict other individual components of the application including usmle step 1 or usmle step 2. participants preferred a traditional interview (mean difference = 1.36, p < 0.0001). a mixed format was preferred over a pure mmi (mean difference = 1.1, p < 0.0001). preference for a mixed format was similar to a traditional interview. mmi performance did not significantly correlate with preference for the mmi; however, there was a trend for higher performance to associate with higher preference (r = 0.15, t(65) = 1.19, n.s.) performance was not associated with preference for a mix of interview methods (r = 0.08, t(65) = 0.63, n.s.). conclusion: while the mmi alone was viewed less favorably than a traditional interview, participants were receptive to a mixed methods interview. the mmi appears to measure skills important in successful completion of an em clerkship and thus likely em residency. future work will determine whether mmi performance correlates with clinical performance during residency. background: the annual american board of emergency medicine (abem) in-training exam is a tool to assess resident progress and knowledge. when the new york-presbyterian (nyp) em residency program started in 2003, the exam was not emphasized and resident performance was lower than expected. a course was implemented to improve residency-wide scores despite previous em literature failing to exhibit improvements with residency-sponsored in-training exam interventions. objectives: to evaluate the effect of a comprehensive, multi-faceted course on residency-wide in-training exam performance. methods: the nyp em residency program, associated with cornell and columbia medical schools, has a 4year format with 10-12 residents per year. an intensive 14-week in-training exam preparation program was instituted outside of the required weekly residency conferences. the program included lectures, pre-tests, high-yield study sheets, and remediation programs. lectures were interactive, utilizing an audience response system, and consisted of 13 core lectures (2-2.5 hours) and three review sessions. residents with previous in-training exam difficulty were counseled on designing their own study programs. the effect on intraining exam scores was measured by comparing each resident's score to the national mean for their postgraduate year (pgy). scores before and after course implementation were evaluated by repeat measures regression modeling. overall residency performance was evaluated by comparing residency average to the national average each year and by tracking abem national written examination pass rates. results: resident performance improved following course implementation. following the course's introduction, the odds of a resident beating the national mean increased by 3.9 (95% ci 1.9-7.3) and the percentage of residents exceeding the national mean for their pgy year increased by 37% (95% ci 23%-52%). following course introduction, the overall residency mean score has outperformed the national exam mean annually and the first-time abem written exam board pass rate has been 100%. conclusion: a multi-faceted in-training exam program centered around a 14-week course markedly improved overall residency performance on the in-training exam. limitations: this was a before and after evaluation as randomizing residents to receive the course was not logistically or ethically feasible. .0 years of practice. among the nonresidency trained, non-boarded em physicians, the percentage of individuals with board actions against them was significantly higher (6.9% vs. 1.9%, 95% ci for difference of 5.0% = 3.1 to 7.5%), but the incidence of actions was not significant (1.3 vs. 3.4 events/ 1000 years of practice, 95% ci for difference of 2.1/ 1000 = )3/1000 to +8/1000), but the power to detect a difference was 30%. conclusion: in this study population, em-trained physicians had significantly fewer total state medical board disciplinary actions against them than non-em trained physicians, but when adjusted for years of practice (incidence), the difference was not significantly different at the 95% confidence level. the study was limited by low power to detect a difference in incidence. objectives: we chose pain documentation as a long term project for quality improvement in our ems system. our objectives were to enhance the quality of pain assessment, to reduce patient suffering and pain through improved pain management, to improve pain assessment documentation, to improve capture of initial and repeat pain scales, and to improve the rate of pain medication. this study addressed the aim of improving pain assessment documentation. methods: this was a quasi-experiment looking at paramedic documentation of the pqrst mnemonic and pain scales. our intervention consisted of mandatory training on the importance and necessity of pain assessment and treatment. in addition to classroom training, we used rapid cycle individual feedback and public posting of pain documentation rates (with unique ids) for individual feedback. the categories of chief complaint studied were abdominal pain, blunt injury, burn, chest pain, headache, non-traumatic body pain, and penetrating injury. we compared the pain documentation rates in the 3 months prior to intervention, the 3 months of intervention, and 3 months post intervention. using repeated-measures anova, we compared rates of paramedic documentation over time. results: our ems system transported 42166 patients during the study period, of whom 15490 were for painful conditions in the defined chief complaint categories. there were 168 paramedics studied, of whom 149 had complete data. documentation increased from 1819 of 5122 painful cases (35.5%) in qtr 1 to 4625 of 5180 painful cases (89.3%) in qtr 3. the trend toward increased rates of pain documentation over the three quarters was strongly significant (p < 0.001). paramedics were significantly more likely to document pain scales and pqrst assessments over the course of the study with the highest rates of documentation compliance in the final 3-month period. conclusion: a focused intervention of education and individual feedback through classroom training, one on one training, and public posting improves paramedic documentation rates of perceived patient pain. background: emergency medical services (ems) systems are vital in the identification, assessment, and treatment of trauma, stroke, myocardial infarction, and sepsis and improving early recognition, resuscitation, and transport to adequate medical facilities. ems personnel provide similar first-line care for patients with syncope, performing critical actions such as initial assessment and treatment as well as gathering key details of the event. objectives: to characterize emergency department patients with syncope receiving initial care by ems and their role as initial providers. methods: we prospectively enrolled patients over 18 years of age who presented with syncope or near syncope to a tertiary care ed with 72,000 annual patient visits from june 2009 to june 2011. we compared patient age, sex, comorbidities, and 30-day cardiopulmonary adverse outcomes (defined as myocardial infarction, pulmonary embolism, significant cardiac arrhythmia, and major cardiovascular procedure) between ems and non-ems patients. descriptive statistics, two-sided ttests, and chi-square testing were used as appropriate. results: of the 669 patients enrolled, 254 (38.0%) arrived by ambulance. the most common complaint in patients transported by ems was fainting (50.4%) or dizziness (45.7%); syncope was reported in 28 (11.0%). compared to non-ems patients, those who arrived by ambulance were older (mean age (sd) 64.5 (18.7), vs. 60.6 (19.5) years, p = 0.012). there were no differences in the proportion of patients with hypertension (20.0% vs 32.0%, p = 0.75), coronary artery disease (8.85% vs 15.3%, p = 0.67), diabetes mellitus (6.5% vs 9.5%, p = 0.57), or congestive heart failure (3.8% vs 6.6%, p = 0.74). sixtynine (10.8%) patients experienced a cardiopulmonary event within 30 days. twenty-eight (4.4%) patients who arrived by ambulance and 41 (6.4%) non-ems patients had a subsequent cardiopulmonary adverse event (rr 1.08, 95%ci 0.68-1.69) within 30 days. the table tabulates interventions provided by ems prior to ed arrival. conclusion: ems providers care for more than one third of ed syncope patients and often perform key interventions. ems systems offer opportunities for advancing diagnosis, treatment, and risk stratification in syncope patients. background: abdominal pain is the most common reason for visiting an emergency department (ed), and abdominopelvic computed tomography (apct) use has increased dramatically over the past decade. despite this, there has been no significant change in rates of admission or diagnosis of surgical conditions. objectives: to assess whether an electronic accountability tool affects apct ordering in ed patients with abdominal or flank pain. we hypothesized that implementation of an accountability tool would decrease apct ordering in these patients. methods: before and after study design using an electronic medical record at an urban academic ed from jul-nov 2011, with the electronic accountability tool implemented in oct 2011 for any apct order. inclusion criteria: age >= 18 years, non-pregnant, and chief complaint or triage pain location of abdominal or flank pain. starting oct 17 th , 2011, resident attempts to order apct triggered an electronic accountability tool which only allowed the order to proceed if approved by the ed attending physician. the attending was prompted to enter the primary and secondary diagnoses indicating apct, agreement with need for ct and, if no agreement, who was requesting this ct (admitting or consulting physician), and their pretest probability (0-100) of the primary diagnosis. patients were placed into two groups: those who presented prior to (pre) and after (post) the deployment of the accountability tool. background: there has been a paradigm shift in the diagnostic work-up for suspected appendicitis. edbased staged protocols call for the use of ultrasound prior to ct scanning because of its lack of radiation, and the morbidity related to contrast. a barrier to implementation is the lack of 24/7 availability of ultrasound. objectives: to evaluate the impact of the implementation of ed performed appendix ultrasounds (apus) on ct utilization in the staged workup for appendicitis in the emergency department. methods: we performed a quasi-experimental, before/ after study. we compared data from the first 8 months of 2009, before the availability of ed performed apus, with the same interval in 2011 after introduction of ed apus. we excluded patients who had appendectomies for reasons other than appendicitis or had been diagnosed prior to arrival. no patient identifiers were included in the analysis and the study was approved by the hospital irb. we report the following descriptive statistics (percentages, sensitivities, and absolute utilization changes conclusion: implementation of an ed apus in the staging work up of appendicitis was associated with a significant reduction in overall ct utilization in the ed. objectives: this study aims to evaluate ed patients' knowledge of radiation exposure from ct and mri scans as well as the long-term risk of developing cancer. we hypothesize that ed patients will have a poor understanding of the risks, and will not know the difference between ct and mri. methods: design -this was a cross-sectional survey study of adult, english-speaking patients at two eds from 6/13/11-8/13/11. setting -one location was a tertiary care center with an annual ed census of 45,000 patient visits and the other was a community hospital with annual ed census of 35,000 patient visits. obser-vations -the survey consisted of six questions evaluating patients' understanding of radiation exposure from ct and mri as well as long-term consequences of radiation exposure. patients were then asked their age, sex, race, highest level of education, annual household income, and whether they considered themselves health care professionals. results: there were 500 participants in this study, 315 (of 5,589 total) from the academic center and 185 (of 4,988 total) from the community hospital during the study period. overall, only 10% (95% ci 7-12%) of participants understood the radiation risks associated with ct scanning. 60% (95% ci 56-65%) of patients believed that an abdominal ct had the same or less radiation as a chest x-ray. 25% (95% ci 21-29%) believed that there was an increased risk of developing cancer from repeated abdominal cts. only 22% (95% ci 19-26%) of patients knew that mri scans had less radiation than ct. 44% (95% ci 39-49%) either didn't know or believed that repeated mris were associated with an increased risk of developing cancer. higher educational level, household income, and identification as a health care professional all were associated with correct responses, but even within these groups, a majority gave incorrect responses. conclusion: in general, ed patients do not understand the radiation risks associated with advanced imaging modalities. we need to educate these patients so that they can make informed decisions about their own health care. background: homelessness has been associated with many poor health outcomes and frequent ed utilization. it has been shown that frequent use of the ed in any given year is not a strong predictor of subsequent use. identifying a group of patients who are chronic high users of the ed could help guide intervention. objectives: the purpose of this study is to identify if homelessness is associated with chronic ed utilization. methods: a retrospective chart review was accomplished looking at the records of the 100 most frequently seen patients in the ed for each year from 2005-2010 at a large, urban academic hospital with an annual volume of 55,000. patients' visit dates, chief complaints, dispositions, and housing status were reviewed. homelessness was defined by self-report at registration. patients were categorized according to their ed utilization with those seen >4 times in at least three of the five years of the study identified as chronic high utilizers; and those who visited the ed >20 times in at least three of the five years of the study were identified as chronic ultra-high utilizers. descriptive statistics with confidence intervals were calculated, and comparisons were made using non-parametric tests. results: during the 5-year study period, 189,371 unique patients were seen, of whom 0.7% patients were homeless. 335 patients were identified as frequent users. there were patients who presented on the top 100 utilizer lists from multiple years. 67 (20%, 95%ci 16-25) patients were identified as homeless. 148 patients were seen >4 times in at least three of the 5 years and 23 (16%, 11-22) were homeless. 12 patients were seen >20 times in at least three of the 5 years and 5 (41%, 19-68) were homeless. our facility has a 40% admission rate; however, non homeless chronic ultra-high utilizers had admission rates of 24% and homeless chronic ultra-high utilizers were admitted 14%. conclusion: chronic ultra-high utilizers of our ed are disproportionately homeless and present with lower severity of illness. these patients may prove to be a cost-effective group to house or otherwise involve with aggressive case management. the debate over homeless housing programs and case management solutions can be sharpened by better defining the groups who would most benefit and who represent the greatest potential saving for the health system. background: the prevalence of obese patients presenting to our emergency department (ed) is 38%: obese patients present in disproportionate number compared to the general population (us rate = 27%). in spite of this, there is a disconnect in patients' perceptions of weight and health: many patients underestimate their weight and report a key barrier to weight loss is patient-provider communications; such discussions have proven to be highly effective in smoking, drug, and alcohol cessation, an important initial step toward promoting wellness. information about patient provider communication is essential for designing and implementing emergency department (ed) based interventions to help increase patient awareness about weightrelated medical issues and provide counseling for weight reduction. objectives: we assessed patients' perceptions about obesity as disease and patient communication with their providers through two questions: do you believe your present weight is damaging to your health? has a doctor or other health professional every told you that you are overweight? methods: a descriptive cross-sectional study was performed in an academic tertiary care ed. a randomized sample of patients (every fifth) presenting to the ed (n = 453) was enrolled. pregnant patients, patients who were medically unstable, cognitively impaired, or who were unable or unwilling to provide informed consent were excluded. percentages of ''yes'' and ''no'' are reported for each question based on patient bmi, ethnicity, sex, and the number of comorbid conditions. regression analysis was used to determine differences in responses between subgroups. results: among overweight/obese, white/black patients, 42.5% do not feel their weight is damaging to their health and 54.7% reported they have not been told by a doctor they are overweight. of individuals who have been told by a doctor they were overweight, 23.2% still believe their present weight is not damaging to their health. of individuals who have not been told by a doctor they were overweight, 41.5% believe their present weight is damaging to their health. differences in race and age were not found. p values <0.05 for all results. conclusion: our data point toward a disconnect regarding patients' perceptions of health and weight. timely education about the burden of obesity may lead to a decrease in its overall prevalence. (originally submitted as a ''late-breaker.'') objectives: to examine the attitudes and expectations of patients admitted for inpatient care following an emergency department visit. methods: a descriptive study was done by surveying a voluntary sample of adult patients (n = 210) admitted to the hospital from the emergency department in one urban teaching hospital in the midwest. a short, ninequestion survey was developed to assess patient attitudes and expectations towards hiv testing, consent, and requirements. analyses consisted of descriptive statistics, correlations, and chi-square analyses. results: the majority of patients report that hiv testing should be a routine part of health care screening (82.4%) and that the hospital should routinely test admitted patients for hiv (78.6%). despite these overall positive attitudes towards hiv testing, the data also suggest that patients have strong attitudes towards consent requirements with 80% acknowledging that hiv testing requires special consent and 72% reporting that separate consent should be required. the data also showed a statistically significant difference in the proportion of patients who believed that hiv testing is a part of routine health care screening by race (v2 = 6.825, df = 1, p = .009). conclusion: patients attitudes and expectations towards routine hiv testing are consistent with the cdc recommendations. emergency departments are an ideal setting to initiate hiv testing and the findings suggest that patients expect hospital policies outline procedures for obtaining consent and screening all patients who are admitted to the hospital from the ed. results: the analysis revealed a ''hot spot'', a cluster of 833 counties (24.5%) with high ca rates adjacent to counties with high ca rates, located across the southeastern us (p < 0.001). within these counties, the average ca rate was 14% higher than the national average. a ''cool spot'', a cluster of 548 counties (16.1%) with low rates, was located across the midwest (p < 0.001). in this cool spot the average ca rate was 12% lower than the national average. figures 1 and 2 show us adjusted rates and spatial autocorrelation of ca deaths, respectively. conclusion: we identify geographic disparities in ca mortality and describe the cardiac arrest belt in the southeastern us. a limitation of this analysis was the use of icd-10 codes to identify cardiac arrest deaths; however, no other national data exist. an improved understanding of the drivers of this variability is essential to targeted prevention and treatment strategies, especially given the recent emphasis on development of cardiac resuscitation centers and cardiac arrest systems of care. an understanding of the relation between population density, cardiac arrest count, and cardiac arrest rate will be essential to the design of an optimized cardiac arrest system. we defined ed utilization during the past 12 months as non-users (0 visits), infrequent users (1-3 visits), frequent users (4-9 visits), and super-frequent users ( ‡10 visits). we compared demographic data, socioeconomic status, health conditions, and access to care between these ed utilization groups. results: overall, super-frequent use was reported by 0.4% of u.s. adults, frequent use by 2%, and infrequent ed use by 19%. higher ed utilization was associated with increased self-reported fair to poor health (55% for super-frequent, 48% for frequent, 22% for infrequent, 10% for non-ed users). frequent ed users were also more likely to be impoverished, with 31% of superfrequent, 25% of frequent, 13% of infrequent, and 9% of non-ed users reporting a poverty-income ratio <1. adults with higher ed utilization were more likely to report the ed as the place they usually go when sick (10% for super-frequent, 6% for frequent, 2% for infrequent, 0.5% for non-ed users). they also reported greater outpatient resource utilization, with 73% of super-frequent, 48% of frequent, 25% of infrequent, and 10% of non-ed users reporting ‡10 outpatient visits/year. frequent ed users were also more likely than non-ed users to be covered by medicaid (34% for super-frequent, 26% for frequent, 12% for infrequent, 5% for non-ed users). conclusion: frequent ed users were a vulnerable population with lower socioeconomic status, poor overall health, and high outpatient resource utilization. interventions designed to divert frequent users from the ed should also focus on chronic disease management and access to outpatient services, rather than focusing solely on limiting ed utilization. objectives: we explored factors associated with specialty provider willingness to provide urgent appointments to children insured by medicaid/chip. methods: as part of a mixed method study of child access to specialty care by insurance status, we conducted semi-structured qualitative interviews with a purposive sample of 26 specialists and 14 primary care physicians (pcps) in cook county, il. interviews were conducted from april to september 2009, until theme saturation was reached. resultant transcripts and notes were entered into atlas.ti and analyzed using an iterative coding process to identify patterns of responses in the data, ensure reliability, examine discrepancies, and achieve consensus through content analysis. results: themes that emerged indicate that pcps face considerable barriers getting publicly insured patients into specialty care and use the ed to facilitate this process. ''if i send them to the emergency room, i'm bypassing a number of problems. i'm fully aware that i'm crowding the emergency room.'' specialty physicians reported that decisions to refuse or limit the number of patients with medicaid/chip are due to economic strain or direct pressure from their institutions ''in the last budget revision, we were [told], 'you are losing money, so you need to improve your patient mix'''. in specialty practices with limited medicaid/chip appointment slots, factors associated with appointment success included: high acuity or complexity, personal request from or an informal economic relationship with the pcp, geography, and patient hardship. ''if it's a really desperate situation and they can't find anybody else, i will make an exception''. specialists also acknowledged that ''patients who can't get an appointment go to the er and then i am obligated to see them if they're in the system.'' conclusion: these exploratory findings suggest that a critical linkage exists between hospital eds and affiliated specialty clinics. as health systems restructure, there is an opportunity for eds to play a more explicit role in improving care coordination and access to specialty care. albert amini, erynne a. faucett, john m. watt, richard amini, john c. sakles, asad e. patanwala university of arizona, tucson, az background: trauma patients commonly receive etomidate and rocuronium for rapid sequence intubation (rsi) in the ed. due to the long duration of action of rocuronium and short duration of action of etomidate, these patients require prompt initiation of sedatives after rsi. this prevents the potential of patient awareness under pharmacological paralysis, which could be a terrifying experience. objectives: the purpose of this study was to evaluate the effect of the presence of a pharmacist during traumatic resuscitations in the ed on the initiation of sedatives and analgesics after rsi. we hypothesized that pharmacists would decrease the time to provision of sedation and analgesia. methods: this was an observational, retrospective cohort study conducted in a tertiary, academic ed that is a level i trauma center. consecutive adult trauma patients who received rocuronium in the ed for rsi were included during two time periods: 07/01/07 to 07/ 30/08 (pre-phase -no pharmacy services in the ed) and 07/01/09 to 06/30/11 (post-phase -pharmacy services in the ed). since the pharmacist could not respond to all traumas in the post-phase, this was further categorized based on whether the pharmacist was present or absent at the trauma resuscitation. data collected included patient demographics, baseline injury data, and medications used. the median time from rsi to initiation of sedatives and analgesics was compared between the pre-phase group (group 1), post-phase pharmacist absent group (group 2), and post-phase pharmacist present group (group 3) using the kruskal-wallis test. results: a total of 200 patients were included in the study (group 1 = 100, group 2 = 70, and group 3 = 30). median age was 35, 48.5, and 54.5 years in groups 1, 2, and 3, respectively (p = 0.005). there were no other differences between groups with regard to demographics, mechanism of injury, presence of traumatic brain injury, glasgow coma scale score, vital signs, ed length of stay, or mortality. median time between rsi and post-intubation sedative use was 13, 15, and 6 minutes in groups 1, 2 and 3, respectively (p < 0.001). median time between rsi and post-intubation analgesia use was 80, 16, and 10 minutes in groups 1, 2, and 3, respectively (p < 0.001). the presence of a pharmacist during trauma resuscitations decreases time to provision of sedation and analgesia after rsi. background: outpatient antibiotics are frequently prescribed from the ed, and limited health literacy may affect compliance with recommended treatments. objectives: among patients stratified by health literacy level, multimodality discharge instructions will improve compliance with outpatient antibiotic therapy and follow-up recommendations. methods: this was a prospective randomized trial that included consenting patients discharged with outpatient antibiotics from an urban county ed with an annual census of 100,000. patients unable to receive text messages or voicemails were excluded. health literacy was assessed using a validated health literacy assessment, the newest vital sign (nvs). patients were randomized to a discharge instruction modality: 1) usual care, typed and verbal medication and case-specific instructions; 2) usual care plus text messaged instructions sent to the patient's cell phone; or 3) usual care plus voicemailed instructions sent to the patient's cell phone. antibiotic pick-up was verified with the patient's pharmacy at 72 hours. patients were called at 30 days to determine antibiotic compliance. z-tests were used to compare 72-hour antibiotic pickup and patient-reported compliance across instructional modality and nvs score groups. results: 758 patients were included (55% female, median age 30, range 5 months to 71 years); 98 were excluded. 23% had an nvs score of 0-1, 31% 2-3, and 46% 4-6. the proportion of prescriptions filled at 72 hours varied significantly across nvs score groups; self-reported medication compliance at 30 days revealed no difference across different instructional modalities nor nvs scores (table 1) . conclusion: in this sample of urban ed patients, 72hour prescription pickup varied significantly by validated health literacy score, but not by instruction delivery modality. in this sample, patients with lower health literacy are at risk of not filling their outpatient antibiotics in a timely fashion. has been developed, validated, and utilized to study the processes of care involved in successful care transitions from inpatient to outpatient settings, but has not been utilized in the ed. objectives: we hypothesized that the ctm-3 could be successfully implemented in the ed without differential item difficulty by age, sex, education, or race; and would be associated with measures of quality of care and likelihood of following physician recommendations. methods: a descriptive study design based on exit surveys was used to measure ctm-3 scores and likelihood of following treatment recommendations. surveys were administered to a daily cross-sectional sample of all patients leaving the ed between 7a-12a by research assistants in an urban academic ed setting for 3 weeks in november 2011. we report means and standard deviations, and analysis of variance to identify differences in ctm-3 scores for those who planned and did not plan to follow ed recommendations. results: 750 surveys were completed; patients were 43 ± 19 years old, 58% black, 61% female, 56% with at least some college education, and 38% were admitted. average ctm-3 score was 87.1 ± 21.6 (range 0-100). scores were not associated with sex (p = 0.57), race (p = 0.19), or education level (p = 0.25). lower ctm scores were associated with increasing age (p = 0.03), patient perceptions that the ed team was less likely to use words that they understood, listen carefully to them, inspire their confidence and trust, or encourage them to ask questions (all p < 0.01). those who reported they were ''very likely'' to follow ed treatment had an average score of 89 ± 21, while those who were ''unlikely'' or ''very unlikely'' to follow ed treatment plans had an average score 47 ± 28 (p = 0.00). conclusion: the ctm-3 performs well in the ed and exhibited only differential item difficulty by age; there was no significant difference by race, sex, or education level. furthermore, it is highly associated with likelihood of following physician recommendations. future studies will focus on ctm-3 scores ability to discriminate between patients who did or did not experience a subsequent ed visit or rehospitalization. age and race were found to be significant predictors of the race pathway. regression of the data by race revealed blacks (or 1.9: ci 1.3-2.6; p < 0.0002), hispanics (or 3.0: ci 1.3-2.6; p = 0.0001), and asians (or 2.3: ci 1.1-4.9; p = 0.03), were more likely to enter the race cohort than were whites; however, much of this discrepancy is accounted for by age. the mean age of minority patients was 62 years, while white patients were older at 71 years (p = 0.002). conclusion: in a diverse demographic population we found that racial minorities were presenting at younger ages for chest pain and were more likely to receive cardiac testing at bedside than their white counterparts; and hence, were selected to a lower level of care (nonmonitored unit background: expanding insurance coverage is designed to improve access to primary care and reduce use of emergency services. whether expanding coverage achieves this is of paramount importance as the united states prepares for the affordable care act. objectives: we examined ed and outpatient department use after the state children's health insurance program (schip) coverage expansion, focusing on adolescents (a major target group for schip) versus young adults (not targeted). we hypothesized that coverage would increase use of outpatient services and emergency department services would decrease. methods: using the national ambulatory medical care survey and the national hospital ambulatory medical care survey, we analyzed years 1992-1996 as baseline and then compared use patterns in 1999-2009 after schip launch. primary outcomes were populationadjusted annual visits to ed versus non-emergency outpatient settings. interrupted time-series were performed on use rates to ed and outpatient departments between adolescents (11-18 years old) and young adults (19-29 years old) in the pre-schip and schip periods. outpatient-to-ed ratios were calculated and compared across time periods. results: the mean number of outpatient adolescent visits increased by 299 visits per 1000 persons (95% ci, 140-457), while there was no statistically significant increase in young adult outpatient visits across time periods. there was no statistically significant change in the mean number of adolescent ed visits across time periods, while young adult ed use increased by 48 visits per 1000 persons (95% ci, 24-73). the adolescent outpatient-to-ed ratio increased by 1.0 (95% ci, 0.49-1.6), while the young adults ratio decreased by 0.53 across time periods (95% ci, )0.90 to )0.16). conclusion: since schip, adolescent non-ed outpatient visits increased while ed visits remained unchanged. in comparison to young adults, expanding insurance coverage to adolescents improved access to health care services and suggests a shift to non-ed settings. as an observational study we are unable to control for secular trends during this time period. also as an ecological study we are unable to examine individual variation. expanding insurance through the affordable care act of 2010 will likely increase use of outpatient services but may not decrease emergency department volumes. background: cancer patients are receiving a greater proportion of their care on an outpatient basis. the effect of this change in oncology care patterns on ed utilization is poorly understood. objectives: to examine the characteristics of ed utilization by adult cancer patients. methods: between july 2007 and march 2009, all new adult cancer patients referred to a tertiary care cancer centre were recruited into a study examining psychological distress. these patients were followed prospectively until september 2011. the collected data were linked to administrative data from three tertiary care eds. variables evaluated in this study included basic we have previously shown that reducing non-value-added activities through the application of the lean process improvement methodology improves patient satisfaction, physician productivity and emergency department length of stay. objectives: in this investigation, we tested the hypothesis that non-value-added activities reduce physician job satisfaction. methods: to test this hypothesis, we conducted timemotion studies on attending emergency physicians working in an academic setting and categorized their activities into value-added (time in room with patient, time discussing cases and educating medical learners, time in room with patient and learner), necessary non-valueadded activities (charting, sign out, looking up labs), and unnecessary non-value-added activities (looking for things, looking for people, on the phone). the physicians were then surveyed using a 10-point likert scale to determine their relative satisfaction with each of the individual tasks (1 worst part of day, 10 best part of day). results: physicians spent 46% of their shift performing value-added work, 38% of their shift performing necessary non-value-added activities, and 16% of their shift performing unnecessary non-value-added activities (waste). weighted physician satisfaction (satisfaction x [percent time spent performing the activity / percent time engaged in activity category]) was highest when the physician was performing value-added work (8.75) compared to performing either necessary non-valueadded work (3.35) or waste (2.61). conclusion: the attending physicians we studied spent the majority of their time performing non-value-added activities, which were associated with lower satisfaction. application of process improvement techniques such as lean, which focus on reducing non-value-added work, may improve emergency physician job satisfaction. background: rocuronium and succinylcholine are the most commonly used paralytics for rapid sequence intubation (rsi) in the ed. after rsi, patients need sustained sedation while they are mechanically ventilated. however, the longer duration of action of rocuronium may influence subsequent sedation dosing, while the patient is therapeutically paralyzed. objectives: we hypothesized that patients who receive rocuronium would be more likely to receive lower doses of post-rsi sedation compared to patients who receive succinylcholine. methods: this was an observational, retrospective cohort study conducted in a tertiary, academic ed. consecutive adult patients, who received rsi using etomidate for induction of sedation between 07/01/09 to 06/30/10, were included. patients were then categorized based on whether they received rocuronium or succinylcholine for paralysis. the dosing of post-rsi sedative infusions was compared at 0, 30, 60, and 120 minutes after initiation between the two groups using the wilcoxon rank-sum test. results: a total of 254 patients were included in the final analysis (rocuronium = 127, succinylcholine = 127). mean age was 52 and 47 years in the rocuronium and succinylcholine groups, respectively (p = 0.04). there were no other baseline differences between groups with regard to demographics, reason for intubation, stroke, traumatic brain injury, glasgow coma scale score, pain scores, or vital signs. in the overall cohort, 90.2% (n = 229) of patients were given a sedative infusion or bolus in the ed. most patients were initiated on propofol (n = 169) or midazolam (n = 49) infusions. median propofol infusion rates at 0, 30, 60, and 120 minutes were 20, 20, 27.5, and 30 mcg/kg/min in the rocuronium group and 20, 40, 45, and 45 mcg/kg/ min in succinylcholine group, respectively. the difference was statistically significant at 30 (p < 0.001) and 60 (p = 0.003) minutes. median midazolam infusion rates at 0, 30, 60, and 120 minutes were 2, 2, 2, and 3 mg/hour in the rocuronium group and 2, 3, 4, and 4.5 mg/hour in succinylcholine group, respectively. the difference was statistically significant at 60 (p = 0.003) and 120 (p = 0.04) minutes. conclusion: patients who receive rocuronium are more likely to receive lower doses of sedative infusions post-rsi due to sustained therapeutic paralysis. this may put them at risk for being awake under paralysis. what is the impact of the implementation of an there was a difference in presenting pain (p < 0.001), stress (p < 0.001), and anxiety (p < 0.001) among patients that received an opioid in the ed. there was a difference in presenting pain (p < 0.001) for patients discharged with an opioid prescription, but not for stress (p = 0.32) or anxiety (p = 0.90). conclusion: patient-reported pain, stress, and anxiety are higher among patients who received an opiate in the ed than in those who did not, but only pain is higher among patients who received a discharge prescription for an opioid. methods: this was a prospective, randomized crossover study on the use of gvl and dl by incoming pediatric interns prior to advanced life support training. at the start of the study, the interns received a didactic session and expert modeling of the use of both devices for intubation. two scenarios were used: (1) normal intubation with a standard airway and (2) difficult intubation with tongue edema and pharyngeal swelling. interns then intubated laerdal simbaby in each scenario with both gvl and dl for a total of four randomized intubation scenarios. primary outcomes included time to successful intubation and the rate of successful intubation. the interns also rated their satisfaction with the devices using a visual analog scale (0-10) and chose their preferred device for their next intubation. results: 29 interns were included in this study. in the normal airway scenario, there were no differences in the mean time for intubation with gvl or dl (62.9 ± 24.1 vs 61.8 ± 26.2 seconds, p = ns) or the number of interns who performed successful intubation (23 vs 22, p = ns). in the difficult airway scenario, the interns took longer to intubate with gvl than dl (92.3 ± 26.6 vs 59.9 ± 22.7 seconds, p = 0.008), but there were no differences in the number of successful intubations (17 vs 19, p = ns). interns rated their satisfaction higher for gvl than dl (7.3 ± 1.8 vs 6.5 ± 1.5, p = 0.05) and gvl was chosen as the preferred device for their next intubation by a majority of the interns (19/29, 66%). conclusion: for novice clinicians, gvl does not improve the time to intubation or intubation success objectives: to determine the time to intubation, the number of attempts, and the occurrence of hypoxia, in patients intubated with a c-mac device versus those intubated using a standard laryngoscope. methods: randomized controlled trial using exception from informed consent that included patients undergoing endotracheal intubation with a standard laryngoscope at an urban level i trauma center. eligible patients were randomized to undergo intubation using the c-mac or standard laryngoscopy. standard laryngoscopy was performed using a c-mac device laryngoscope with the video output obstructed to ensure equivalent laryngoscope blades in the two groups. data were collected by a trained research assistant at the patient's bedside and video review by the investigators. the number of attempts made, the initial and lowest oxygen saturation (spo 2 ), and the total time until the intubation was successful was recorded. hypoxia was defined as an oxygen saturation <93%. data were compared with wilcoxon rank sum and chi-square tests. results: thirty-eight patients were enrolled, 20 (70% male, median age 58, range 28 to 86, median spo 2 97%, range 79 to 100) in the standard laryngoscopy group and 18 (67% male, median age 58, range 19 to 73, median spo 2 96.5%, range 78 to 100) in the c-mac group. the median number of attempts for standard laryngoscopy was 1, range 1 to 3, and for c-mac was 1, range 1 to 2 (p = 0.43). the median time to intubation for the standard laryngoscopy group was 54 seconds (range 7 to 89) and for the c-mac group was 41 seconds (range 4 to 101)(p = 0.05). hypoxia was detected in 5/20 (20%) in the standard laryngoscopy group and 1/18 (6%) in the c-mac group (p = 0.15). the median decrease in oxygen saturation during the attempt was 5.4% (range 0% to 31%) for the standard laryngoscopy group and 2.3% (range 0% to 16%) for the c-mac group. conclusion: we did not detect a difference in number of attempts, the occurrence of hypoxia, or the diagnosis of aspiration pneumonia between standard laryngoscopy and the c-mac. the time to successful intubation was shorter for patients intubated with the c-mac. the c-mac device appears to be superior to standard laryngoscopy for emergent endotracheal intubation. (originally submitted as a ''late-breaker.'') the background: aspiration pneumonia is a complication of endotracheal intubation that may be related to the difficulty of the airway procedure. objectives: to determine the association of the device used, the time to intubation, the number of attempts to intubate, and the occurrence of hypoxia with the subsequent development of aspiration pneumonia. methods: this was a prospective observational study of patients undergoing endotracheal intubation by emergency physicians at an urban level i trauma center conducted from 7/1/2010 until 11/1/2011. the device used on the initial attempt to intubate was at the discretion of the treating physician. data were collected by a trained research assistant at the patient's bedside. the device used, the number of attempts made to intubate, the lowest oxygen saturation during the attempt, and the total time until intubation was successfully accomplished were recorded. patient's medical records were reviewed for the subsequent diagnosis of aspiration pneumonia. hypoxia was defined as an oxygen saturation <93%. data were analyzed using multinomial logistic regression and odds ratios (or). results: 654 patients were enrolled; 141 (22%) subsequently developed aspiration pneumonia. 328 were intubated with a standard laryngoscope (sl), 277 using the c-mac, 26 with an intubating laryngeal mask, and 23 with nasotracheal intubation (ni) (or 0.87, 95% ci = 0.70-1.06). comparison of individual devices versus sl did not show an association by device type. the median number of attempts for patients with aspiration pneumonia was 1, range 1 to 3, and for those without was 1, range 1 to 9 (or 0.78, 95%ci = 0.43-1.38). the median time to intubation for patients who developed aspiration pneumonia was 55 seconds (range 4 to 756) and for those who did not was 54 seconds (range 4 to 721)(or 1.00, 95%ci = 0.99-1.00). hypoxia during intubation was detected in 53/141 (38%) in the aspiration pneumonia group and 175/513 (34%) in the no aspiration pneumonia group (or 1.06, 95% ci = 0.65-1.72). conclusion: there was not an association between the device used, the number of attempts, the time to intubation, or the occurrence of hypoxia during the intubation, and the subsequent occurrence of aspiration pneumonia. background: japanese census data estimate that 35 million, or nearly 29% of the overall population, will be over age 65 by the year 2020. similar trends are apparent throughout the developed world. although increased patient age affects airway management, comprehensive information in emergency airway management for the elderly is lacking. objectives: we sought to characterize emergency department (ed) airway management for the elderly in japan including success rate, and major adverse events using a large multi-center registry. methods: design and setting: we conducted a multicenter prospective observational study using the japanese emergency airway network (jean) registry of eds at 11 academic and community hospitals in japan between 2010 and 2011 inclusive. data fields included ed characteristics, patient and operator demographics, methods of airway management, number of attempts, success rate, and adverse events. participants: patient inclusion criteria were all adult patients who underwent emergent tracheal intubation in the ed. primary analysis: patients were divided to into two groups defined as follows: 18 to 64 years old and over 65 years old. we describe primary success rates and major adverse events using simple descriptive statistics. categorical data are reported as proportions and 95% confidence intervals (cis). results: the database recorded 2710 patients (capture rate 98%) and 2623 met the inclusion criteria. of 2623 patients, 1104 patients were 18 to 64 years old (62%) and 1519 were over 65 years old (38%). the older group had a significantly higher success rate at first attempt intubation (1074/1519; 70.7%, 95% ci 68.8-72.6%) compared with the younger group (710/1104; 64.3%, 95% ci 61.9-66.7%). the older group had similar major adverse event rates (112/1519; 7.4%, 95% ci 6.3-8.5%) compared with the younger group (83/1104; 7.5%, 95% ci 6.2-8.8%). (see table 1) background: the degree to which a patient's report of pain is associated with changes in blood pressure, heart rate, and respiratory rate is not known. objectives: to determine to what degree a standardized painful stimulus effects a change in systolic blood pressure (sbp), diastolic blood pressure (dbp), heart rate (hr), or respiratory rate (rr), and compare changes in vital signs between patients based on pain severity. methods: prospective observational study of healthy human volunteers. subjects had their sbp, dbp, hr, and rr measured prior to pain exposure, immediately after, and 10 minutes after. pain exposure consisted of subjects placing their hand in a bath of 0 degree water for 45 seconds. the bath was divided into two sections; the larger half was the reservoir of cooled water monitored to be 0 degrees, the other half filled from constant overflow over the divider. water drained from this section into the cooling unit and was then pumped up into the base of the reservoir through a diffusion grid. subjects completed a 100 mm visual analog scale (vas) representing their perceived pain during the exposure and graded their pain as minimal, moderate or severe. data were compared using 95% confidence intervals. results: 90 subjects were enrolled, mean pain vas 40 mm, range 0 to 77, 49 reported mild pain, 41 moderate pain, and 0 severe pain. the percent change from baseline in vital signs during the exposure and 10 minutes after are presented in the table. conclusion: there was a wide variety in reported pain among subjects exposed to a standard painful stimulus. there was a larger change in heart rate during the exposure among subjects who described a standardized painful exposure as moderate than in those who described it as severe. the small observed changes in blood pressure and respiratory rate seen during the exposure did not differ by pain report or persist after 10 minutes. background: vital signs are often used to validate intensity of pain. however, few studies have looked at the capacity of vital signs to estimate pain intensity, particularly in patients with a diagnosis that a majority of physicians would agree produce significant pain in the ed. objectives: to determine the association between pain intensity and vital signs in consecutive ed patients and in a sub-group of patients with diagnosis known to cause significant pain. methods: we performed a post-hoc analysis of prospectively acquired data in a cohort study done in an urban teaching hospital with computerized triage and nurses records. we included all consecutive ed adult patients ( ‡16 years old), who had any level of pain intensity measured during triage, from march 2008 to november 2010. the primary outcome was the mean heart rate, systolic and diastolic blood pressure for every pain intensity level from 1 to 10 on a verbal numerical scale. our secondary outcomes where the same but limited to patients with the following diagnosis: fracture, dislocation, and renal colic. we performed descriptive statistics, one-way and two-way anovas when appropriate. results: during our study period, 42,947 patients ‡16 years old where triaged with a pain intensity of at least 1/10 and 3939 had a diagnosis known to cause significant pain. 56.5% of patients were female, with a mean pain intensity of 6.8/10, mean age of 47.9 years (±19.3), and 22.3% were ‡65 years old. there was a statistically significant difference (p < 0.05) in mean heart rate, systolic and diastolic blood pressure for each level of pain intensity, ex: difference between 1/10 and 10/10 for mean heart rate was 3.9 beats per minutes, for systolic pressure was 4.0 mmhg and for diastolic 4.5 mmhg. results are similar for painful diagnosis: difference for mean heart rate was 0.3 beats per minutes, for systolic pressure was 6.5 mmhg and diastolic 8.8 mmhg. however, these differences are not clinically significant. conclusion: although our study is a post hoc analysis, pain intensity, heart rate, systolic and diastolic pressures during triage are usually reliable data and a prospective study would likely produce the same result. these vital signs cannot be used to estimate or validate pain intensity in the emergency department. 8% had a positive urine drug screen. logistic multivariate regressions analyses revealed the following factors to be significantly associated with the risk of having an abnormal head ct: association with seizure (p = 0.0072); length of time of loss of consciousness, ranging from none to 0-30 min to >30 min (p = 0.0013); alteration of consciousness (p = 0.00009); post-traumatic amnesia (p = 0.0132); alcohol intake prior to injury (p = 0,0003); and initial ed gcs (p = 0.0255). conclusion: in an emergency department cohort of patients with traumatic brain injury, symptoms including loss of or alteration in consciousness, seizure, post traumatic amnesia, and alcohol intake appear to be significantly associated with abnormal findings on head ct. these clinical findings on presentation may be useful in helping triage head injury patients in a busy emergency department, and can further define the need for urgent or emergent imaging in patients without clearly apparent injuries. background: the etiology of neurogenic shock is classically attributed to diminished peripheral vascular resistance (pvr) secondary to loss of sympathetic outflow to the peripheral vasculature. however, the sympathetic nervous system also controls other key elements of the cardiovascular system such as the heart and capacitance vessels and disruptions in their function could complicate the hemodynamic presentation. objectives: we sought to systematically examine the hemodynamic profiles of a series of trauma patients with neurogenic shock. methods: consecutive trauma patients with documented spinal cord injury complicated by clinical shock were enrolled. hemodynamic data including systolic and diastolic blood pressure, heart rate (hr), impedance-derived cardiac output, pre-ejection period (pep), left ventricular ejection time (lvet), and calculated systemic pvr were collected in the ed. data were normalized for body surface area and a validated integrated computer model of human physiology (guyton model) was used to analyze and categorize the hemodynamic profiles based on etiology of the hypotension using a systems analysis. correlation between markers of sympathetic outflow (hr, pep, lvet) and shock etiology category was examined. results: of 9 patients with traumatic neurogenic shock, the etiology of shock was decrease in pvr in 4 (45%; 95% ci 19 to 73%), loss of vascular capacitance in 3 (33%; 12 to 65%), and mixed peripheral resistance and capacitance responsible in 2 (22%; 6 to 55%). the markers of sympathetic outflow had no correlation to any of the elements in the patients' hemodynamic profiles. conclusion: neurogenic shock is often considered to have a specific well-characterized pathophysiology. results from this study suggest that neurogenic shock can have multiple mechanistic etiologies and represents a spectrum of hemodynamic profiles. this understanding is important for the treatment decisions made in the management of these patients. -year (2008-2010) , pre-post intervention study of trauma patients requiring massive blood transfusion was performed. we divided the population into two cohorts: a pre-protocol group (pre) which included trauma patients receiving mbt not aided by a protocol, and a post-protocol group (post) who underwent mbt via the mbtp. patient demographics, 24hour blood component totals, timing of blood component delivery, trauma injury severity score (iss), initial glasgow coma scale (gcs) score, trauma mechanism, and patient mortality data were collected and analyzed using fisher's exact tests, student's t-tests, and mann-whitney u tests. results: fifty-two patients were included for study. median times to delivery of first products were reduced for prbcs (4 minutes), ffp (16 minutes), and platelets (33 minutes) between the pre and post cohorts. median time to delivery of any subsequent blood product was significantly reduced (10 minutes) in the post cohort (p = 0.024). the median number of blood products delivered was increased by 5.5 units for prbcs, 4 units for ffp, 0.5 units for platelets, and 1 unit for cryoprecipitate after implementation of mbtp. the percentage of patients receiving higher blood product ratios (>3:1) was reduced between the pre and post cohorts for prbc to ffp (25% reduction) and prbc to platelet ratio groups (7 % reduction). despite improved transfusion timing and ratios, we found no significant difference in mortality (p = 0.129) between pre and post cohorts when we adjusted for injury severity. conclusion: protocolized delivery of massive blood transfusion might reduce time to product availability and delivery, though it is unclear how this affects patient mortality in all us trauma centers. background: burns are common injuries that can result in significant scarring leading to poor function and disfigurement. unlike mechanical injuries, burns often progress both in depth and size over the first few days after injury, possibly due to inflammation and oxidative stress. a major gap in the field of burns is the lack of an effective therapy that reduces burn injury progression. objectives: since mesenchymal stem cells (msc) have been shown to improve healing in several injury models, we hypothesized that species-specific msc would reduce injury progression in a rat comb burn model. methods: using a 150 gm brass comb preheated to 100 degrees celsius, we created four rectangular burns, separated by three unburned interspaces on both sides of the backs of male sprague-dawley rats (300 g). the interspaces represented the ischemic zones surround-ing the central necrotic core. left untreated, most of these interspaces become necrotic. in an attempt to reduce burn injury progression, 20 rats were randomized to tail vein injections of 1 ml rat-specific msc 10 6 cells/ml (n = 10) or normal saline (n = 10) 60 minutes after injury. tracking of the stem cells was attempted by injecting several rats with quantum dot-labeled msc. results: by four days post-injury, all of the interspaces in the control rats (54/54, 100%) became necrotic while in the experimental group, 29/48 (60%) of the interspaces became necrotic (fisher's exact test; p < 0.001). at 7 days, the percentage of the unburned interspaces that became necrotic in the msc treated group was significantly less than in the control group (80% vs. 100%, p < 0.0001). we were unable to identify any quantum dot labeled msc in the injured skin. no adverse reactions or wound infections were noted in rats injected with msc. conclusion: intravenous injection of rat msc reduced burn injury progression in a rat comb burn model. although basic demographics of bicyclists in accidents have been described, there is a paucity of data describing the street surface involved in accidents, and whether designated bicycle roadways offer protection. this lack of information limits informed attempts to change infrastructure in a way that will decrease morbidity and/or mortality of cyclists. objectives: to identify road surface types involved in pedal cyclist injuries and determine the relationship between injury severity and the use of designated bicycle roadways (dbr) versus non-designated roadways (ndr). we hypothesized that more severe injuries would happen at intersections regardless of dbr versus ndr. methods: this retrospective cohort study reviewed the trauma database from a level i trauma center in tucson, az. we identified all bicyclists in the database injured in accidents involving a motor vehicle from january 1, 2009 1, through december 31, 2009 . the patients were then linked to a local government database that documents location (latitude/longitude) and direction of travel of the cyclist. seventy-eight total incidents were identified and categorized as occurring on a dbr versus ndr and occurring at an intersection versus not at an intersection. results: only one patient who arrived at the trauma center died. fifty-one of the accidents (65%) occurred on dbrs; 63% of accidents occurring on dbrs took place in intersections. conversely, 63% of accidents on ndrs occurred outside of intersections. the odds of an injury occurring at an intersection versus not at an intersection were 2.9 times higher (95% ci: 1.0-8.5) for dbrs compared to ndrs. the odds of a trauma being severe (admitted) versus not severe (discharged home) were 2.7 times higher (95% ci: 0.9-8.7) when a collision occurred not at an intersection versus at an intersection. conclusion: contrary to our hypothesis, in this study group severe injuries were more likely outside of an intersection. however, intersections on dbrs were identified as problematic as cyclists on a dbr were more likely to be injured in an intersection. future city planning could target improved cyclist safety in intersections. background: minor thoracic injury (mti) is frequent and a significant proportion will still have moderate to severe pain at 90 days. there is a lack of risk factors to orient specific treatment at ed discharge. objectives: to determine risk factors of having pain ( ‡3/10, on a numerical intensity pain score from 0 to 10) at 90 days in a population of minor thoracic injury patients discharged from the ed. methods: a prospective multi-center cohort study was conducted in four canadian eds, from november 2006 to january 2010. all consecutive patients, 16 years and older, with mti (with or without rib fracture), a normal chest x-ray, and discharged from the ed were eligible. a standardized clinical and radiological evaluation was done at 1 and 2 weeks. standardized phone interviews were done at 30 and 90 days. pain evaluation occurred at five time points (ed visit, 1 and 2 weeks, 30 and 90 days). using a pain trajectory model (sas), we planned to identify groups with different pain evolution at 90 days. the final model was based on the importance of difference in pain evolution, confidence intervals, and number of patients in each group. to judge the adequacy of the final model, we examined whether the posteriori probabilities (i.e., a participant's probability of belonging to a certain trajectory group) averaged at least 70% for each trajectory group. then using logistic multinomial regression and the low risk group of having pain as the control group, we identified significant predictors of patients in the moderate and high risk groups having pain at 90 days. results: in our cohort of 1,057 patients, 1,025 had an evaluation at 90 days. we identified three groups at low (34%), moderate (50.6%), and high risk (15.4%) of having pain ‡3/10 at 90 days. using risk factor identified by univariate analysis, we created a model to identify patients at risk containing the following predictors: age ‡ 30 years old, women, current smoker, two or more rib fractures, complaint of dyspnea, and saturation <95% at initial visit. posteriori probabilities for low, moderate, and high risk were 76%, 74%, and 88%. conclusion: to our knowledge, this is the first study to identify potential risk factor for having pain at 90 days after minor thoracic injury. these risk factors should be validated in a prospective study to guide specific treatment plan. the use of ultrasound to evaluate traumatic optic neuropathy benjamin burt, lisa montgomery, cynthia garza meissner, sanja plavsic-kupesic, nadah zafar ttuhsc -paul l foster school of medicine, el paso, tx background: whenever head trauma occurs, there is the possibility for a patient to have an optic nerve injury. the current method to evaluate optical nerve swelling is to look for proptosis. however, by the time proptosis presents, significant damage has already occurred. therefore, there is a need to establish a method to evaluate nerve injury prior to the development of proptosis. objectives: fundamental to understanding the pathophysiology of optic nerve injury and repair is an understanding of the optic nerve's temporal response to trauma including blood flow changes and vascular reactivity. the aim of our study was to assess the dependability and reproducibility of ultrasound techniques to sequence optic nerve healing and monitor the vascular response of the ophthalmic artery following an optic nerve crush. methods: the rat's orbit was imaged prior to and following a direct injury to the optic nerve, at 72 hours and at 28 days. 3d, 2d, and color doppler techniques were used to detect blood flow and the course of the ophthalmic artery and vein, to evaluate the course and diameter of the optic nerve, and to assess the extent of optic nerve trauma and swelling. the parameters used to evaluate healing over time were pulsatility and resistance indices of the ophthalmic artery. results: we have established baseline ultrasound measurements of the optic nerve diameter, normal resistance and pulsatility indices of the ophthalmic artery, and morphological assessment of the optic nerve in a rat model. longitudinal assessment of 2d and 3d ultrasound parameters were used to evaluate vascular response of the ophthalmic artery to optic nerve crush injury. we have developed a rat model system to study traumatic optic nerve injury. the main advantages of ultrasound are low cost, non-invasiveness, lack of ionizing radiation, and the potential to perform longitudinal studies. our preliminary data indicate that 2d and 3d color doppler ultrasound may be used for the evaluation of ophthalmic artery and total orbital perfusion following trauma. once baseline ultrasound and doppler measurements are defined there is the opportunity to translate the rat model to evaluate patients with head trauma who are at risk for optic nerve swelling and to assess the usefulness of treatment interventions. background: alcoholism is a chronic disease that affects an estimated 17.6 million american adults. a common presentation to the emergency department (ed) is a trauma patient with altered sensorium who is presumed to be alcohol intoxicated by the physicians based on their olfactory sense. often ed physicians may leave patients suspected of alcohol intoxication aside until the effects wear off, potentially missing major trauma as the source of confusion or disorientation. this practice often results in delays in diagnosing acute potentially life-threatening injuries in the patients with presumed alcohol intoxication. objectives: this study will determine the accuracy of physicians' olfactory sense for diagnosing alcohol intoxication. methods: patients suspected of major trauma in the ed underwent an evaluation by the examining physician for the odor of alcohol as well as other signs of intoxication. each patient had determination of blood alcohol level. alcohol intoxication was defined as a serum ethanol level ‡80 mg/dl. data were reported as means with 95% confidence intervals (95% ci) or proportions with inter-quartile ranges (iqr 25%-75%). results: one hundred and fifty one patients (70% males) were enrolled in the study, median age 45 years (iqr 33-56). the median score for glasgow coma scale was 15. the level of training of examining physician was a median of pgy 4 (iqr pgy 3 -attending). prevalence of alcohol intoxication was 43% (95% ci: 35% to 51%). operating characteristics: physician assessment of alcohol intoxication, sensitivity 84% (95% ci: 73% to 92%), specificity 87% (95% ci: 78% to 93%), positive likelihood ratio 6.6 (95% ci: 3.8 to 11.6), negative likelihood ratio 0.18 (95% ci: 0.1 to 0.3), and accuracy 86% (95% ci: 80% to 91%). patients who were falsely suspected of being intoxicated were 7.3% (95% ci: 4% to 13%). conclusion: although the physicians had a high degree of accuracy in identifying patients with alcohol intoxication based on their olfactory sense, they still falsely overestimated intoxication in a significant number of non-intoxicated trauma patients. the background: optimal methods for education and assessment in emergency and critical care ultrasound training for residents are not known. methods of assessment often rely on surrogate endpoints which do not assess the ability of the learner to perform the imaging and integrate the imaging into diagnostic and therapeutic decisions. we designed an educational strategy that combines asynchronous learning to teach imaging skills and interpretation with a standardized assessment tool using a novel ultrasound simulator to assess the learner's ability to acquire and interpret images in the setting of a standardized patient scenario. objectives: to assess the ability of emergency medicine and surgical residents to integrate and apply information and skills acquired in an asynchronous learning environment in order to identify pathology and prioritize relevant diagnoses using an advanced cardiac ultrasound simulator. methods: 12 em r2 residents and 12 r2 surgical residents completed an online focused training program in cardiac ultrasonography (iccu elearning, https:// www.caeiccu.com/lms). this consisted of approximately 14 hours of intensive training in cardiac ultrasound. residents were then given cases with a patient scenario that lacked significant details that would suggest a specific diagnosis. the resident was then given a list of 17 possible diagnoses and asked to rank the top five diagnoses in order of most likely to least likely. each resident (blinded to the pathology displayed by the simulator) then imaged using an ultrasound simulator. after imaging, the residents were given the same list of potential diagnoses, and asked to rank them again from 1-5. results: overall, residents ranked the correct diagnosis in the top five significantly more times post-ultrasound than pre-ultrasound. additionally, the residents made the correct diagnosis significantly more times postultrasound than pre-ultrasound. similar patterns occur for congestive heart failure, pericardial effusion with tamponade, and pleural effusion. there was no significant difference pre-and post-ultrasound for pulmonary embolism and anterior infarction. conclusion: an asynchronous online learning program significantly improves the ability of emergency medicine and surgical residents to correctly prioritize the correct diagnosis after imaging with a standardized pathology imaging simulator. mark favot, jacob manteuffel, david amponsah henry ford hospital, detroit, mi background: em clerkships are often the only opportunity medical students have to spend a significant amount of time caring for patients in the ed. it is imperative that students gain exposure to as many of the various fields within em as possible during this time. if the exposure of medical students to ultrasound is left to the discretion of the supervising physicians, we feel that many students would complete an em clerkship with limited skills and knowledge in ultrasound. the majority of medical students receive no formal training in ultrasound during medical school and we believe that the em clerkship is an excellent opportunity to fill this educational gap. objectives: evaluate the usefulness and effectiveness of a focused ultrasound curriculum for medical students in an em clerkship at a large, urban, academic medical center. methods: prospective cohort study of fourth year medical students doing an em clerkship. as part of the clerkship requirements, the students have a portion of the curriculum dedicated to the fast exam and ultrasound-guided vascular access. at the end of the month they take a written test, and 1 month later they are given a survey via e-mail regarding their ultrasound experience. em residents also completed the test to serve as a comparison group. all data analysis was done using sas 9.2. scores were integers ranging between 0 and 10. descriptive statistics are given as count, mean, standard deviation, median, minimum, and maximum for each group. due to non-gaussian nature of the data and small group sizes, a wilcoxon two-sample test was used to compare the distributions of scores between the groups. results: in the table, the distribution of scores was compared between the residents (controls) and the students (subjects). the mean and median scores of the student group were higher than those of the resident group. the difference in scores between the two groups was statistically significant (p = 0.021). conclusion: our data reveal that after completing an em clerkship with time devoted to learning ultrasound for the fast exam and vascular access, fourth year medical students are able to perform better than em residents on a written test. what remains to be determined is if their skills in image acquisition and in performance of ultrasound-guided vascular access procedures also exceed those of em residents. results: there were 106 respondents (total response rate 24.71%). compared to non-em students, students pursuing em (8 students, 7.55%) were more drawn to their specialty for work hour control (p < 0.0009) and shorter residency length (p < 0.0338). em students were less likely than non-em students to be drawn to their chosen specialty for future academic opportunities (p < 0.0085). em students formed their mentorships by referral significantly more than non-em students (p < 0.0399), though there was no statistical difference in quality of existing mentorships amongst students. of the 93 students not currently and never formerly interested in em, the most common response (25.8%) for why they did not choose em was the lack of a strong mentor in the field. conclusion: the results confirmed previous findings of lifestyle factors drawing students to em. future academic opportunities were less likely to draw students to em than students pursuing other specialties. lack of mentorship in the field was the most common reason given for why students did not consider em. given the lack of direct em exposure until late in the curriculum of most medical schools, mentorship may be particularly important for em and future study should focus on this area. background: misdiagnosis is a major public health problem. dizziness leads to 10 million visits annually in the us, including 2.6 million to the emergency department (ed). despite extensive ed workups, diagnostic accuracy remains poor, with at least 35% of strokes missed in those presenting with dizziness. ed physicians need and want support, particularly in the best method for diagnosis. strong evidence now indicates the bedside oculomotor exam is the best method of differentiating central from peripheral causes of dizziness. objectives: after a vertigo day that includes instruction in head impulse testing, emergency medicine residents will feel comfortable discharging a patient with signs of vestibular neuritis and a positive head impulse test without ordering a ct scan. methods: post graduate year 1-4 emergency medicine residents participated in a four hour vertigo day. we developed a mixed cognitive and systems intervention with three components: an online game that began and ended the day, a didactic taught by dr. newman-toker, and a series of small group exercises. the small group sessions included the following: a question and answer session with the lecturer; vertigo special tests (cerebellar assessment, dix hall-pike, epley maneuver); a head impulse hands-on tutorial using a mannequin; and a video lecture on other tests useful in vertigo evaluation (nystagmus, test of skew, vestibulocular reflex, ataxia). results: thirty emergency medicine residents were studied. before and after the intervention the residents were given a survey in which one question asked ''in a patient with acute vestibular syndrome and a history and exam compatible with vestibular neuritis, i would be willing to discharge the patient without neuroimaging based on an abnormal head impulse test result that i elicited''. resident answers were based on a sevenpoint likert scale from strongly agree to strongly disagree. twenty-five residents completed both surveys. of the seven residents who changed their responses pre to post,a significant proportion (100%) changed their answer from disagree/neutral to agree after a 4hour vertigo day (mcnemar's test, p value = 0.0082). conclusion: in this single-center study, teaching headimpulse testing as part of a vertigo day increases resident comfort with discharging a patient with vestibular neuritis without a ct scan. background: previous studies have been inconsistent in determining the effect of increased ed census on resident workload and productivity. we examined resident workload and productivity after the closure of a large urban ed near our facility, which resulted in a rapid 21% increase in our census. objectives: we hypothesized that the closure of a nearby hospital closure with a resulting influx of ed patients to our facility would not change resident productivity. methods: this computer-assisted retrospective study compared new patient workups per hour and patient load before and after the closure of a large nearby hospital. specifically, new patient workups per hour and the 4 pm patient census per resident were examined for a one-year period in the calendar year prior to the closing and also for one year after the closing. we did not include the four month period surrounding the closure in order to determine the long-term overall effect. background: emergency medicine residents use simulation for training due to multiple factors including the acuity of certain situations they are faced with, and the rarity of others. current training on highfidelity mannequin simulators is often critiqued by residents over the physical exam findings present, specifically the auscultatory findings. this detracts from the realism of the training, and may also lead a resident down a different diagnostic or therapeutic pathway. wireless remote programmed stethoscopes represent a new tool for simulation education which allows any sound to be wirelessly transmitted to a stethoscope receiver. objectives: our goal was to determine if a wireless remote programmed stethoscope was a useful adjunct in simulation-based cases using a high-fidelity mannequin. our hypothesis was that this would represent a useful adjunct in simulation education of emergency medicine residents. methods: starting june 2011, pgy1-3 emergency medicine residents were assessed in two simulation-based cases using pre-determined scoring anchors. an experimental randomized crossover design was used in which each resident performed a simulation case with and without a remote programmed stethoscope on a highfidelity mannequin. scoring anchors and surveys were used to collect data with differences of means calculated. results: fourteen residents participated in the study. residents noted most realistic physical exam findings associated with the case with the adjunct in 13/14 (93%) and that their preference was for the use of the adjunct in 13/14 (93%). based off of a five-point likert scale, with 5 being the most realistic, the adjunct-associated case averaged 4.4 as compared to 3.0 without (difference of means 1.4, p = 0.00017). average scores of residents with the adjunct were 2.5/3 with the use of the adjunct and 2.3/3 without (difference of means 0.2, p = 0.076). average total times were 28:49 with the adjunct as compared to 30:02 without. conclusion: a wireless remote programmed stethoscope is a useful adjunct in simulation training of emergency medicine residents. residents noted physical exam findings to be more realistic, preferred its use, and had approached significant improvement of scores when using the adjunct. background: prior studies predict an ongoing shortage of emergency physicians to staff the nation's eds, especially in rural areas. to address this, em organizations have discussed broadening access to acgme or aoa accredited em residency programs to physicians who previously trained in another specialty and focusing on physicians already practicing in rural areas. objectives: to investigate whether em program directors (pds) from allopathic and osteopathic residency programs would be willing to accept applicants previously trained in other specialties and whether this willingness is modified by applicants' current practice in rural areas. methods: a five-question web-based survey was sent to 200 u.s. em pds asking questions about their policies on accepting residents with past training and from rural practices. questions included whether a pd would accept a resident with prior training in other specialties, how many years from this training would the applicant be still a competitive candidate and if a physician was practicing in a rural region would the likelihood of acceptance to the program be improved. different characteristics of the residency programs were recorded including length of program, years in existence, size, type, and location of program. we compared responses by program characteristics using chi-square test. results: of the 96 (48%) pds responding to date, a large majority (87%) reported they do accept applicants with previous residency training, although directors of osteopathic programs were less likely to accept these applicants (56% vs 94% for allopathic; p < 0.001). overall, 28% of pds reported no limit on the length of time from prior training to when they are accepted at an em program. 73% reported it is very or possibly realistic they would accept a candidate who had completed training and was board certified in another specialty. a majority of all respondents (61%) felt a physician practicing in a rural setting might be viewed as a more favorable candidate, even if the resident would only be in the program for 2 years after receiving training credit. directors of newer programs (<5 years of existence) were more likely to view these candidates favorably than older programs (91% vs 53%; p = 0.02). conclusion: there appear to be many em residency programs that would at least review the application and consider accepting a candidate who trained in another specialty. a qualitative assessment of emergency medicine self-reported strengths todd guth university of colorado, aurora, co background: self-reflection has been touted as a useful way to assess the acgme core competencies. objectives: the purpose of this study is to gain insight into resident physician professional development through analysis of self-perceived strengths. a secondary purpose is to discover potential topics for selfreflective narrative essays relating to the acgme core competencies. methods: design: a small qualitative study was performed to explore the self-reported strengths of emergency medicine (em) residents in a single four-year residency. participants: all 54 residents regardless of year of training were also asked to report their selfperceived strengths. observations: residents were asked: ''what do you feel are your greatest strengths as a resident? provide a quick description.'' the author and another reviewer identified themes from within each year of residency with abraham maslow's conscious competence conceptual framework in mind. occurrences of each theme were counted by the reviewers and organized according to frequency. once the top ten themes for each year of residency were identified and exemplar quotes identified, the two reviewers identified trends. inter-rater agreements were calculated. results: representing unconscious incompetency, the first trend was the reported presence of ''enthusiasm and a positive attitude'' from residents early in their training that decreases further along in training. additionally, a ''willingness and motivation to improve and learn'' was reported as a strength throughout all the years of training but most frequently reported in the first two years of residency. entering into conscious incompetence, the second trend identified was ''recognition of limitations and openness to constructive feedback'' that was mentioned frequently in the second and third years of residency. demonstrating conscious competence, the third trend identified was the increase in identification of the strengths of ''educational leadership, teamwork skills and communication, and departmental patient flow and efficiency'' in the later years of residency. conclusion: self-reported strengths has helped to identify both themes within each year of residency and trends among the years of residency that can serve as areas to explore in self-reflective narratives relating to the acgme core competencies. training. pofu can also be used to assess the acgme core competency of practice-based learning. the exact form or frequency of pofu assessment among various em residencies, however, is not currently known. objectives: we aimed to survey em residencies across the country to determine how they fulfill the pofu requirement and whether certain program structure variables were associated with different pofu systems. we hypothesized that implementation of pofu systems among em residencies would be highly variable. methods: in this irb-approved study, all program directors of acgme allopathic em residencies were invited to complete a 10-question survey on their current approaches to pofu. respondents were asked to describe their current pofu system's characteristics and rate its ease of use, effectiveness, and efficiency. data were collected using surveymonkey(tm) and reported using descriptive statistics. results: of 158 residencies surveyed, 81 (51%) submitted complete data. 77.5% were completed by program directors and over three-fourths (76.1%) of em residencies require monthly completion of pofus. the mean total pofus required per year was 78 (95% ci 58-98), with a median of 64 and a range of 2-400. almost 2/3 (63%) of residencies use an electronic pofu system. most (84%) 4-year em residencies use an electronic pofu system, compared with half (54%) of 3-year residencies (difference 30%, p = 0.025, 95% ci 5.1%-47.2%). seven commercially available electronic programs are used by 71% of the residencies, while 29% use a customized product. most respondents (88%) rated their pofu system as easy to use, but less than half (49%) felt it was an effective learning tool or an efficient one (45%). onethird (34%) would use a different pofu system if available, and almost half (44%) would be interested in using a multi-residency pofu system. conclusion: em residency programs use many different strategies to fulfill the rrc requirement for pofu. the number of required pofus and the method of documentation vary considerably. about two-thirds of respondents use an electronic pofu system. less than half feel that pofu logs are an effective or efficient learning tool. background: certification of procedural competency is requisite to graduate medical education. however, little is known regarding which platforms are best suited for competency assessment. simulators offer several advantages as an assessment modality, but evidence is lacking regarding their use in this domain. furthermore, perception of an assessment environment has important influence on the quality of learning outcomes, and procedural skill assessment is ideally conducted on a platform accepted by the learner. objectives: to ascertain if a simulator performs as well as an unembalmed cadaver with regard to residents' perception of their ability to demonstrate procedural competency during ultrasound (us) guided internal jugular vein (ij) catheterization. methods: in this cross-sectional study at an urban community hospital during july of 2011, 15 residents in their second or third year of training from a 3-year em residency program performed us guided catheterizations of the ij on both an unembalmed cadaver and a simulator manufactured by blue phantom. after the procedure, residents completed an anonymous survey ascertaining how adequately each platform permitted their demonstration of proficiency on predefined procedural steps. answers were provided on a likert scale of 1 to 10, with 1 being poor and 10 being excellent. p values < 0.10 were considered educationally significant. results: the median overall rating of the simulator (s) to serve as an assessment platform was similar to that of the cadaver (c) with scores of 8.0 and 8.3 respectively, p = 0.89. median ratings for permitting the demonstration of specific procedural steps were as follows: conclusion: senior em residents positively rate the blue phantom simulator as an assessment platform and similarly to that of a cadaver with regard to permitting their demonstration of procedural competency for us guided ij catheterization, but did prefer the cadaver to a greater degree when identifying and guiding the needle into the ij. methods: in fall 2011, wcmc and wcmc-q students taking the course completed a 20 question pre-and post-test. wcmc-q students also completed a postcourse single-station objective structured clinical examination (osce) that evaluated their ability to identify and perform eight actions critical for a first responder in an emergency situation (table 1) . results: on both campuses, mean post-test scores were significantly higher than mean pre-test scores (p £ 0.001). on the pre-test, mean wcmc student scores were significantly higher than for wcmc-q students (p = 0.02); however, no difference was found in mean post-test scores (p = 0.895). there was no association between the scores on the osce (mean = 7.01, sd = 1.00) and the post-test (p = 0.683) even after adjusting for a possible evaluators' effect (table 2) . clinical skills course was effective in enhancing student knowledge in both qatar and new york as evidenced by the significant improvement in scores from the pre-to post-tests. the course was able to bring wcmc-q student scores and presumably knowledge up to the same level as wcmc students. students performed well on the osce, suggesting that the course was able to teach them the critical actions required of a first responder. the lack of association between the post-test and osce scores suggests that student knowledge does not independently predict ability to learn and demonstrate critical actions required of a first responder. future studies will evaluate whether the course affects the students' clinical practice. assess breathing 3 assess circulation 4 call ems 5 call ems and assess abcs prior to other interventions 6 immobilize 7 localize and control bleeding 8 splint fractured extremity and skills specific to wilderness medicine by incorporating simulated medical scenarios into a day-long adventure race. this event has gained acceptance nationally in wilderness medical circles as an excellent way to appreciate the challenges of wilderness medicine, however its effectiveness as a teaching tool has not yet been verified. objectives: the objective of this study was to determine if improvement in simulated clinical and didactic performance can be demonstrated by teams participating in a typical medwar event. methods: we developed a complex clinical scenario and written exam to test the basic tenets that are reinforced through the medwar curriculum. teams were administered the test and scored on a standardized scenario immediately before and after the 2011 midwest medwar race. teams were not given feedback on their pre-race performance. scenario performance was based on the number of critical actions correctly performed in the appropriate time frame. data from the scenario and written exams were analyzed using a standard paired difference t-test. results: a total of 31 teams participated in both the pre-and post-event scenarios. the teams' pre-race scenario performance was 71.0% (sd = 17.0, n = 31) of critical actions met compared to a post-race performance of 89.7 % (sd = 11.4, n = 31). the mean improvement was 18.7% (sd = 18.7, n = 31, 95% ci 12. 1, 25. 3) with a significant paired two-tailed t-test (p £ 0.01). a total of 95 individual subjects took the written pre-and posttests. the written scores averaged pre-race 84.5% (sd = 12.5, n = 95) and post-race 88.7% (sd = 11.5, n = 95). the mean improvement was 4.2% (sd = 11.7, n = 95, ci )7.5, 15.9), with a significant paired twotailed t-test (p £ 0.01). conclusion: medwar participants demonstrated a significant improvement in both written exam scores and the management of a simulated complex wilderness medical scenario. this strongly suggests that medwar is an effective teaching platform for both wilderness medicine knowledge and skills. palliative methods: ed residents and faculty of an urban, tertiary care, level i trauma center were asked to complete an anonymous survey (6/2010-10/2011). participants ranked 22 statements on a five-point likert scale (1 = strongly disagree-5 = strongly agree). statements covered four main domains of barriers related to: 1) education/training, 2) communication, 3) ed environment; 4) personal beliefs. respondents were also asked if they would call pc consult for 15 ed clinical scenarios (based on established triggers). results: 30/45 (67%) eligible participants completed the survey (23 residents, 7 faculty), average age was 31 years, 52% (15/29) male, and 58% (15/26) caucasian. respondents identified two major barriers to ed-pc provision: lack of 24 hour availability of pc team (mean score 4.4) and lack of access to complete medical records (4.2). listed domain barriers included: communication-related issues (mean 3.3) like access to family or primary providers, ed environment (2.8) for example chaotic setting with time-constraints, education/training (2.7) related to pain/pc, and personal beliefs regarding end-of-life (2.5). all respondents agreed that they would call pc consult for a 'hospice patient in respiratory distress', and a majority (73%) would consult pc for 'massive intracranial hemorrhage, traumatic arrest, and metastatic cancer'. however, traditional in-patient triggers like frequent re-admits for organ failure issues (dementia, congestive heart failure, and obstructive pulmonary disease exacerbations) were infrequently (10%) chosen for pc consult. conclusion: to enhance pc provision in the ed setting, two main ed physician perceived barriers will likely need to be addressed: lack of access to medical records and lack of 24-7 availability of pc team. ed physicians may not use the same criteria to initiate pc consults as compared to the traditionally established inpatient pc consult trigger models. percent of charts with an mse by ait prior to resident evaluation (a measure of reduced diagnostic uncertainty and decision-making), (4) ed volume. results: there were no educationally significant differences in productivity or acuity between the pre-ait and post-ait groups. mse was recorded in the chart prior to resident evaluation in 10.9% of cases. ed volume rose by 9.0% between periods. conclusion: ait did not affect productivity or acuity of patients seen by em2s. while some volume was directed away from residents by ait (patients treated-andreleased by ait only), overall volume increased and made up the difference. this is similar to previously reported rankings that program directors gave to the same criteria. although medical students agreed with program directors on the importance of most aspects of the nrmp application areas of discordance included higher medical student ranking for extracurricular activities and a lower relative ranking for aoa status than program directors. this can have implications for medical student mentoring and advising in the future. background: emergency care of older adults requires specialized knowledge of their unique physiology, atypical presentations, and care transitions. older adults often require distinctive assessment, treatment and disposition. emergency medicine (em) residents should develop expertise and efficiency in geriatric care. older adults represent over 25% of most emergency department (ed) volumes. yet many em residencies lack curricula or assessment tools for competent geriatric care. the geriatric emergency medicine competencies (gemc) are high-impact geriatric topics developed to help residencies meet this demand. objectives: to examine the effect of a brief gemc educational intervention on em resident knowledge. methods: a validated 29-question didactic test was administered at six em residencies before and after a gemc focused lecture delivered summer and fall of 2009. scores were analyzed as individual questions and in defined topic domains using a paired student's t-test. results: a total of 301 exams were included. the testing of didactic knowledge before and after the gemc educational intervention had high internal reliability (87.9%). the intervention significantly improved scores in all domains (table 1) . graded increase in geriatric knowledge occurred by pgy year with the greatest improvement seen at the pgy 3 level (table 2) . conclusion: even a brief gemc intervention had a significant effect on em resident knowledge of critical geriatric topics. a formal gemc curriculum should be considered in training em residents for the demands of an ageing population. the overall procedure experience of this incoming class was limited. most r1s had never received formal education in time management, conflict of interest management, or safe patient trade-off. the majority lacked confidence in their acute and chronic pain management skills. these entry level residents lacked foundational skill levels in many knowledge areas and procedures important to the practice of em. ideally medical school curricular offerings should address these gaps; in the interim, residency curricula should incorporate some or all of these components essential to physician practice and patient safety. background: the american heart association and international liaison committee on resuscitation recommend patients with return of spontaneous circulation following cardiac arrest undergo post-resuscitation therapeutic hypothermia. in post-cardiac arrest patients presenting with a rhythm of vf/vt, therapeutic hypothermia has been shown to reduce neurologic sequelae and decrease overall mortality. objectives: to explore clinical practice regarding the use of therapeutic hypothermia and compare survival outcomes in post-cardiac arrest patients. a secondary outcome was to assess whether the initial presenting cardiac arrest rhythm (ventricular fibrillation/ventricular tachycardia (vf/vt) versus pulseless electrical activity (pea) or asystole) was associated with differences in outcomes. methods: a retrospective medical record review was conducted for all adult ( ‡18 years) post-cardiac arrest patients admitted to the icu of an academic tertiary care centre (annual ed census 150,000) from 2006-2007. data were extracted using a standardized data collection tool by trained research personnel. results: 200 patients were enrolled. mean (sd) age was 66 (16) and 56.5% were male. of 58 (29.0%) patients treated with hypothermia, 27 (46.6%) presented with an initial rhythm of vf/vt and 31 (53.4%) presented with pea or asystole. nine (33.3%) patients with vf/vt were treated with therapeutic hypothermia and discharged from hospital compared to 2 (6.4%) patients with pea or asystole (d 26.9%; 95% ci: 6.4%, 46.3%). of 142 patients not treated with hypothermia, 37 (26.1%) presented with vf/vt, 93 (65.5%) presented with pea or asystole, and 12 (8.4%) initial rhythms were unknown. fifteen (40.5%) patients with vf/vt, not treated with hypothermia, were discharged from hospital compared to 13 (13.9%) patients with pea or asystole (d 26.6%; 95% ci: 10.0%, 43.5%). regardless of initial presenting rhythm or initiation of therapeutic hypothermia, 37 (88.1%) discharged patients had good neurological function as assessed by the cerebral performance category (cpc score 1-2). conclusion: although recommended, post-cardiac arrest therapeutic hypothermia was not routinely used. patients with vf/vt and treated with hypothermia had better outcomes than those with pea or asystole. further research is needed to assess whether cooling patients with presenting rhtyhms of pea or asystole is warranted. racial background: chronic obstructive pulmonary disease (copd) is a major public health problem in many countries.the course of the disease is characterised by episodes, known as acute exacerbations (ae), when symptoms of cough, sputum production, and breathlessness become much worse. the standard prehospital management of patients suffering from an aecopd includes oxygen therapy, nebulised bronchodilators, and corticosteroids. high flow oxygen is used routinely in prehospital areas for breathless patients with copd. there is little high quality evidence on the benefits or potential dangers in this setting but audits have shown increased mortality, acidosis, and hypercarbia in patients with aecopd treated with high flow oxygen. objectives: to compare standard high flow oxygen treatment with titrated oxygen treatment for patients with an aecopd in the prehospital setting. methods: cluster randomized controlled parallel group trial comparing high flow oxygen treatment with titrated oxygen treatment in the prehospital setting. in an intention to treat analysis (n = 405), the risk of death was significantly lower in the titrated oxygen arm compared with the high flow oxygen arm for all patients and for the subgroup of patients with confirmed copd (n = 214). overall mortality was 9% (21 deaths) in the high flow oxygen arm compared with 4% (7 deaths) in the titrated oxygen arm; mortality in the subgroup with confirmed copd was 9% (11 deaths) in the high flow arm compared with 2% (2 deaths) in the titrated oxygen arm. titrated oxygen treatment reduced mortality compared with high flow oxygen by 58% for all patients (p = 0.02) and by 78% for the patients with confirmed chronic obstructive pulmonary disease (p = 0.04). patients with copd who received titrated oxygen according to the protocol were significantly less likely to have respiratory acidosis or hypercapnia than were patients who received high flow oxygen. conclusion: titrated oxygen treatment significantly reduced mortality, hypercapnia, and respiratory acidosis compared with high flow oxygen in aecopd. these results provide strong evidence to recommend the routine use of titrated oxygen treatment in patients with breathlessness and a history or clinical likelihood of copd in the prehospital setting. (originally submitted as a ''late-breaker.'') trial registration australian new zealand clinical trials register actrn12609000236291. background: toxic particulates and gases found in ambulance exhaust are associated with acute and chronic health risks. the presence of such materials in areas proximate to ed ambulance parking bays, where emergency services' vehicles are often left running, is potentially of significant concern to ed patients and staff. objectives: investigators aimed to determine whether the presence of ambulances correlated with ambient particulate matter concentrations and toxic gas levels at the study site ed. methods: the ambulance exhaust toxicity in healthcare-related exposure and risk [aether] program conducted a prospective observational study at an academic urban ed / level i trauma center. environmental ambient gas was sampled over a continuous five-week period from september to october 2011. two sampling locations in the public triage area (public patient dropoff area without ambulances) and three sampling locations in the ambulance triage area were randomized for 24-hour monitoring windows with a temporal resolution of 2 minutes to obtain 7 days of non-contiguous data for each location. concentrations of particulate matter less than 2.5 microns in aerodynamic size (pm2.5), oxygen, hydrogen sulfide (h 2 s), and carbon monoxide (co) as well as lower explosive limit for methane (lel) were monitored with professionally calibrated devices. ambulance traffic was recorded through offline review of 24/7 security video footage of the site's ambulance bays. results: 4,118 measurements at the public triage nurse desk space revealed pm2.5 concentrations with a mean of 21.32 ± 27.01 lg/m 3 (median 15.95 lg/m 3 ; maximum 1,152.58 lg/m 3 ). 4,867 ambulance triage nurse desk space pm2.5 concentrations recorded a mean of 60.45 ± 53.38 lg/m 3 (p < 0.0001, unpaired t test; median 43.37 lg/m 3 ; maximum 580.78 lg/m 3 ). oxygen levels remained steady throughout the study period; co, h 2 s, and lel were not detected. ambulance activity levels had the highest correlations with pm2.5 concentrations at the ambulance triage foyer (r = 0.47) and desk area (r = 0.42) where patients wait and ed staff work 8-12 hr shifts. conclusion: ed spaces proximate to ambulance parking bays had higher levels of pm2.5 than areas without ambulance traffic. concentrations of ambient particulate matter in acute care environments may pose a significant health threat to patients and staff. an ems ''pit crew'' model improves ekg and stemi recognition times in simulated prehospital chest pain patients sara y. baker 1 , salvatore silvestri 1 , christopher d. vu 1 , george a. ralls 1 , christopher l. hunter 1 , zack weagraff 2 , linda papa 1 1 orlando regional medical center, orlando, fl; 2 florida state university college of medicine, orlando, fl background: prehospital teams must minimize time to ekg acquisition and stemi recognition to reduce overall time from first medical contact to reperfusion. auto-racing ''pit crews'' model rapid task completion by pre-assigning roles to team members. objectives: we compared time-to-completion of key tasks during chest pain evaluation in ems teams with and without pre-assigned roles. we hypothesized that ems teams using the ''pit crew'' model would improve time to recognition and treatment of stemi patients. methods: a randomized, controlled trial of paramedic students was conducted over 2 months at orlando medical institute, a state-approved paramedic training center. we compared a standard ems chest pain management algorithm (control) with a pre-assigned tasks (''pit crew'') algorithm (intervention) in the evaluation of simulated chest pain patients. students were randomized into groups of three; intervention and control groups did not interact after randomization. all students reviewed basic prehospital chest pain management and either the standard or pre-assigned tasks algorithm. groups encountered three simulated patients. laerdal simmanò software was used track completion of tasks: taking vital signs, iv access, ekg acquisition and interpretation, asa administration, hospital stemi notification, and total time on scene. results: we conducted 54 simulated-patient encounters (30 control / 24 intervention encounters). mean time-to-completion of each task was compared in the control and intervention groups respectively. time to obtain vital signs was 4:18 vs. 2:21 min (p = 0.001); time to asa administration was 3:54 vs 2:00 min (p < 0.001); time to ekg acquisition was 5:39 vs 3:42 min (p < 0.001); time to ekg interpretation was 6:43 vs 4:21 min (p < 0.001); time to iv access was 5:42 vs 4:45 min (p = 0.05); time to stemi notification was 7:19 vs 4:26 min (p < 0.001); and time to scene completion was 9:02 vs 5:27 min (p < 0.001). conclusion: paramedic student teams with pre-assigned roles (the ''pit crew'' model) were faster to obtain vital signs, administer asa, acquire and interpret the ekg, stemi notification, and overall time on scene during simulated patient encounters. further study with experienced ems teams in actual patient encounters is necessary to confirm the relevance of these findings. background: use of automated external defibrillators (aed) has remained low in the u.s. understanding the effect of neighborhoods on the probability of having an aed used in the setting of a public arrest may provide important insights for future placement of aeds. objectives: to determine associations between the racial and income composition of neighborhoods (as defined by u.s. census tracts), individual arrest characteristics, and whether bystanders or first responders initiate aed use. methods: cohort study using surveillance data prospectively submitted by emergency medical services systems and hospitals from 29 u.s. sites to the cardiac arrest registry to enhance survival between october 1, 2005 and december 31, 2009 . neighborhoods were defined as high-income vs. low-income based on the median household income being above or below $50,000 and as white or black if >90% of the census tract was of one race. neighborhoods without a predominant racial composition were defined as integrated. arrests that occurred within a public location (excluding medical facilities and airports) were eligible for inclusion. hierarchical multi-level modeling, using stata v11.0, was used to determine the association between individual and census tract characteristics on whether an aed was used. results: of 2,769 eligible cases, an aed was used in 1336 arrests (48.2%) by a first responder (n = 1,127, 40.8%) or bystander (n = 209, 7.5%). patients whose arrest was witnessed (odds ratio [or] 1.26; 95% confidence interval [ci] 1.06-1.50) were more likely to have an aed used (table) . when compared to high-income white neighborhoods, arrest victims in low-income black neighborhoods were least likely to have an aed used (or 0.54; 95% ci 0.33-0.87). arrest victims in lowincome white (or 0.57; 95% ci 0.32-1.02) and lowincome integrated (or 0.70; 95% ci 0.51-0.96) were also less likely to have an aed used. conclusion: arrest victims in black and low-income neighborhoods are least likely to have an aed used by a layperson or first responder. future research is needed to better understand the reasons for low rates of aed use for cardiac arrests in these neighborhoods. the impact of an educational intervention on the pre-shock pause interval among patients experiencing an out-of-hospital cardiac arrest jonathan studnek 1 , eric hawkins 1 , steven vandeventer 2 1 carolinas medical center, charlotte, nc; 2 mecklenburg ems agency, charlotte, nc background: pre-shock pause duration has been associated with survival to hospital discharge (std) among patients experiencing out-of-hospital cardiac arrest (oohca) resuscitation. recent research has demonstrated that for every 5-second increase in this interval there is an 18% decrease in std. objectives: determine if a decrease in the pre-shock pause interval for patients experiencing oohca could be realized after implementation of an educational intervention. methods: this was a retrospective analysis of data obtained from a single als urban ems system from 1/1/2010 to 12/31/10 and 8/1/11 to 11/6/2011. in august 2011, an educational intervention was designed and delivered to approximately 150 paramedics emphasizing the importance of reducing the time off chest during cpr. specifically, the time period just prior to defibrillation was emphasized by having rescuers count every 20th compression and pre-charge the defibrillator on the 180th compression. in order to determine if this change resulted in process improvement, 12 months of data were assessed before and 3 months after the educational intervention. pre-shock pause was the outcome variable and was defined as the time period after compressions ceased until a shock was delivered. this interval was measured by a cpr feedback device connected to the defibrillator. inclusion criteria were adult patients who required at least one defibrillation and had the cpr feedback device connected during the defibrillation attempt. analysis was descriptive utilizing means and 95% ci as well as wilcoxon rank sum test to assess difference between the two time periods. results: in the pre-intervention period there were 117 patients who received 211 defibrillations compared to 30 patients receiving 71 defibrillations in the post-intervention phase. the mean duration of the pre-shock pause pre-intervention was 35 seconds (95% ci 20-50) while the post-intervention duration was 9 seconds (95% ci 7-12). the difference in pre-shock pause duration was statistically significant with p < 0.001. conclusion: these data indicate that after a simple educational intervention emphasizing decreasing time off chest prior to defibrillation the pre-shock pause duration decreased. future research must describe the sustainability of this intervention as well as the effects this process measure may have on outcomes such as survival to hospital discharge. background: the broselow tape (bt) has been used as a tool for estimating medication dosing in the emergency setting. the obesity trend has demonstrated a tendency towards insufficient pediatric weight estimations from the bt, and thus potential under-dosing of resuscitation medications. objectives: this study compared drug dosing based on the bt with dosing from a novel electronic tool (et) that accounts for provider estimation of body habitus. methods: data were obtained from a prospective convenience sample of children ages 1 to 8 years arriving to a pediatric emergency department. a clinician performed an assessment of body habitus (average/underweight, overweight, or obese), blinded to the patient's actual weight and parental weight estimate. parental estimate of weight and measured length and weight were collected. epinephrine dosing was calculated from the measured weight, the bt measurement, as well as from a smart-phone tool based on the measured length and clinician's estimate of body habitus, and a modified tool (mt) incorporating the parent estimate of habitus. the wilcoxson rank-sum test was used to compare median percent differences in dosing. results: one hundred children (mean age 3 years) were analyzed; 47% were overweight or obese. clinicians correctly identified children as overweight/obese 23% of time (ci 0.12-0.38). adding parent estimate of weight improved this to a sensitivity of 74% (ci 0.59-0.86). the median difference between the weight-based epinephrine dose and bt dose was 11%. for the et the median difference from the weight-based dose was 7% (p = 0.05 compared to the bt), and for the mt was 1.7% (p < 0.01 compared to the bt). when a clinically significant difference was defined as ±10% of the actual dose, bt was within that range 40% of the time, et was within range 56% of the time (p = 0.02), and mt was within range 64% of the time ( background: in most out-of-hospital cardiac arrest (ohca) events, a call to 9-1-1 is the first action by bystanders. accurate diagnosis of cardiac arrest by the call taker depends on the caller's verbal description. if cardiac arrest is not suspected, then no telephone cpr instructions will be given. objectives: we measured the effect of a change in the ems call taker question sequence on the accuracy of diagnosis of cardiac arrest by 9-1-1 call takers. methods: we retrospectively reviewed the cardiac arrest registry to enhance survival (cares) dataset for january 1, 2009 through june 30, 2011 from a city, population 750,000, with a longstanding telephone cpr program (apco). we included ohca cases of any age who were in arrest prior to the arrival of ems and for whom resuscitation was attempted. in early 2010, 9-1-1 call takers were taught to follow a revised telephone script that emphasized focused questions, assertive control of the caller, and provision of hands-only cpr instructions. the medical director personally explained the reasons for the changes, emphasizing the importance of assertive control of the caller and the comparative safety of chest compressions in patients not in cardiac arrest. beginning in 2010, call recordings were reviewed regularly with feedback to the call taker by the 9-1-1 center leadership. the main outcome measure was sensitivity of the 9-1-1 call taker in diagnosing cardiac arrest. bystander cpr was reported by ems crews attending the event. we compared 2009 with 2010 and 2011 using the v 2 test and odds ratios (or). results: there were 504 ohca cases in 2009, 457 cases in 2010, and 287 in the first half of 2011 (68/100,000 population). the mean age was 57 ± 21 years, and 27% of the events were witnessed. before the revision, 40% of ohca cases were identified by 9-1-1 dispatchers; and after the revised questioning sequence, 74% were identified (or 4.3, 95% ci 3.2-5.6). the false positive rate changed little (from 56/month to 72/month). the mean time to question callers was unchanged (53 vs 51 seconds). bystander cpr was performed in 37.3% of events in 2009, 39.2% in 2010, and 49.1% of events in 2011 (p < 0.001). conclusion: emphasis on scripted assessment improved sensitivity without loss of specificity in identifying ohca. with repeated feedback, it translated to an increase in victims receiving bystander cpr. in an out-of hospital cardiac arrest population confirmed by autopsy salvatore silvestri, christopher hunter, george ralls, linda papa orlando regional medical center, orlando, fl background: quantitative end-tidal carbon dioxide (etco 2 ) measurements (capnography) have consistently been shown to be more sensitive than qualitative (colorimetric) ones, and the reliability of capnography for assessing airway placement in low perfusion states has sometimes been questioned in the literature. objectives: this study examined the rate of capnographic waveform presence of an intubated out-of-hospital cardiac arrest cohort and its correlation to endotracheal tube location confirmed by autopsy. our hypothesis is that capnography is 100% accurate in determining endotracheal tube location, even in low perfusion states. methods: this cross-sectional study reviewed a detailed prehospital cardiac arrest database that regularly records information using the utstein style. in addition, the ems department quality manager routinely logs the presence of an alveolar (four-phase) capnographic waveform in this database. the study population included all cardiac arrest patients from january 1, 2009 through december 31, 2009 managed by a single ems agency in orange county, florida. patients were included if they had endotracheal intubation performed, had capnographic measurement obtained, failed to regain return of spontaneous circulation (rosc), and had an autopsy performed. the main outcome was the correlation of the presence of an alveolar waveform and the location of the ett at autopsy. results: during the study period, 921 cardiac arrests were recorded. of these, 263 had an advanced airway placed (ett or laryngeal tube airway), and no rosc. of the 263 advanced airway cases, 73 were managed with an ett. autopsies were performed on 30 of these patients and resulted in our study cohort. the location of the ett at autopsy was recorded on all 30 of these cases. capnographic waveforms were recorded in the field in all 30 of these study patients, and 100% of the tubes were located within the trachea at autopsy. the sensitivity of capnography in determining proper endotracheal tube location was 100% in this study. conclusion: in our study, the presence of a capnographic waveform was 100% reliable in confirming proper placement of endotracheal tubes placed in outof-hospital patients with poor perfusion states. results: over 60 variables were presented to the 34 ems medical directors responding (100% survey population captured). among the myriad of responses, 14 (42%) initiate cardiopulmonary resuscitation (cpr) at 30 compressions to 2 ventilations consistent with il-cor/aha guidelines. seven (21%) initiate continuous chest compressions from the start of cpr with no pause and interposed ventilations. nine (26%) begin chest compressions only during the first 2-3 minutes, with either passive oxygenation by oxygen mask (six; 18%) or no oxygen (three; 9%). airway management following non-invasive oxygenation and ventilation by primary endotracheal intubation occurs in 12 systems (35%), while six (18%) use supraglottic devices. fourteen (42%) allow paramedics to decide between endotracheal and supraglottic device placement. thirty systems (88%) utilize continuous waveform capnography. the initial approach to non-ems witnessed ventricular fibrillation is chest compression prior to first defibrillation in 30 systems (88%). eighteen systems (52%) escalate defibrillation energy settings, with four systems (12%) utilizing dual sequential defibrillation. twenty (59%) initiate therapeutic hypothermia in the field. conclusion: wide variability in ca care standards exists in america's largest urban ems systems in mid-2011, with many current practices promoting more continuity in chest compressions than specified in the 2010 ilcor/aha guidelines. endotracheal intubation, a past mainstay of ca airway management, is deemphasized in many systems. immediate defibrillation of non-ems witnessed ventricular fibrillation is uncommon. objectives: determine the out-of-hospital cardiac arrest survival in this area of puerto rico using the utstein method. methods: prospective observational cohort study of adult patients presenting with an out-of-hospital cardiac arrest to the upr hospital ed. study endpoints will be survival and neurologically intact survival at hospital discharge, 6 months, and 12 months. results: a total of 144 consecutive cardiac arrest events were analyzed for a period of 2 years. one-hundred fifteen events met criteria for primary cardiac etiology (79.86%). the average age for this group was 68.47 years. there were 45 female (39.13%) and 70 male (60.86%) participants. the average time to start cpr was 14.60 minutes. transportation to the ed was 71.3% by ems and 25.22% by private vehicle. a total of 68 events were witnessed (59.13%). the survival rate to hospital admission was 23.66%. the overall cardiac arrest survival was 9.30% and overall neurologically intact survival was 4.30%. neurologically intact survival at 6 and 12 months was 2.15%. the rate of bystander cpr in our population was 16.13% with a survival rate of 6.66%. conclusion: survival from out-of-hospital cardiac arrest in the area served by the upr hospital is low but comparable to other cities in the us as reported by the cdc cardiac arrest registry to enhance survival (cares). this low survival rate might be due to low bystander cpr rate and prolonged time to start cpr. background: hyperventilation has been directly correlated with increased mortality for out-of-hospital cpr. ems providers may hyperventilate patients at levels above national bls guidelines. real-time feedback devices, such as ventilation timers, have been shown to improve cpr ventilation rates towards bls standards. it remains unclear if the combination of a ventilation timer and pre-simulation instruction would influence overall ventilation rates and potentially reduce undesired hyperventilation. objectives: this study measured ventilation rates of standard cpr (and pre-instruction on effects of hyperventilation) compared to cpr with the use of a commercial ventilation timer (and pre-instruction on effects of hyperventilation). we propose that use of a ventilation timer, measuring and displaying to ems providers real-time ventilations delivered, will have no difference in ventilation rates when comparing these groups. methods: this prospective study placed ems providers into four groups: two controls measuring ventilation rates before (1a) and after instruction (1b) on the deleterious effects of hyperventilation, and a concurrent intervention pair with before (2a) and after instruction (2b), with the second pair measuring ventilation rates with a ventilation timer that provides immediate feedback on respirations given. ventilation rates were measured for a 60-second period after one minute of simulated cpr using mannequins. the control set without instruction (1a, n = 12) averaged 14.21 breaths (95% ci = 10.31-18.11) and with instruction (1b, n = 13) averaged 20.23 breaths (95% ci = 16.16-24.30). the intervention set without instruction (2a, n = 11) averaged 13.04 breaths (95% ci = 9.29-16.78) and with instruction (2b, n = 13) averaged 11.77 breaths (95% ci = 8.02-15.51). there was a significant improvement (p = 0.016) in ventilation rates with use of a ventilation timer (control group versus intervention group regardless of pre-instruction). there was no statistically significant difference between groups with respect to instruction alone (p = 0.223). conclusion: the use of a ventilation timer significantly reduced overall ventilation rates, providing care closer to bls guidelines. the addition of pre-simulation instruction added no significant benefit to reducing hyperventilation. background: in 2010, the american heart association (aha) recommended a compression rate of (roc) 100/ min and a depth of compressions (doc) at least 2 inches for effective cpr. as an educational tool for lay rescuers, the aha as adopted the catch phrase ''push hard, push fast''. objectives: in this irb-exempt study, we sought to determine if persons without formal cpr training could perform non-ventilated cpr as well as those who have been trained in the past or those currently certified. methods: a convenience sample of patrons of the new york state fair was asked to perform 2 minutes of hands-only cpr on a prestan pp-am-100m adult cpr manikin. these devices provide visual indicators of acceptable rate and depth of compressions. each subject was video recorded on a dell latitude 620 laptop computer with a logitech quick cam using logitech quick cam 8.4.6 for windows software. results: a total of 175 volunteers (74 male, 102 female) aged 16-68 years participated: 52 were never certified (nc) in cpr, 73 were previously certified (pc), and 50 were currently certified (cc). there was no difference in age across the groups. the cc group had a higher proportion of females (chi-square = 9.71, p < 0.008). cc volunteers sustained roc and doc for an average of 57.1 seconds as compared to an average of 18.5 seconds (pc) and 2.3 seconds (nc) respectively. (f = 27.8, p < 0.001). the cc maintained roc of closer to 100/ min (mean 111.6/min) when compared to the pc (mean 85.3/min) and nc (mean 86.0/min) groups (f = 14.7, p < 0.001). a higher proportion of volunteers of the cc group were able to perform adequate doc (chi-square = 11.2, p < 0.004), and hand placement (chisquare = 19.21, p < 0.001) when compared to the other two groups. conclusion: compared to the target roc and doc, none of the groups did well and only 14 subjects met target roc/doc. increased out-of-hospital cardiac arrest survivability due to lay rescuer intervention is only assured if cpr is effectively administered. the effect and benefit of maintaining formal cpr training and certification is clear. background: more than 300,000 out-of-hospital cardiac arrests (ohcas) occur annually in the united states (us). automated external defibrillators (aeds) are life-saving devices in public locations that can significantly improve survival. an estimated 1 million aeds have been sold in the us; however, little is known about whether locations of aeds match oh-cas. these data could help determine optimal placement of future aeds and targeted cpr/aed training to improve survival. objectives: we hypothesized that the majority (>50%) of aeds are not located in close proximity (200 feet) to the occurrence of cardiac arrests in a major metropolitan city. methods: this was a retrospective review of prospectively collected cardiac arrest data from philadelphia ems from january 1, 2008 until december 31, 2010. included were ohcas of presumed cardiac etiology in individuals 12 years of age or older. excluded were oh-cas of presumed traumatic etiology, cases where resuscitation was terminated at the scene, and those dead on arrival. aed locations in philadelphia were obtained from myheartmap, a database of installed and wallmounted aeds in pennsylvania. we used gis mapping software to visualize where ohcas occurred relative to where aeds were located and to determine the radius of ohcas to aeds. arrests within a 200, 400, and 600 foot radius of aeds were identified using the attribute location selection option in arcgis. the lengths of radii were estimated based on the average time it would take for a person to walk to and from an aed (200 feet2 minutes; 400 feet4 minutes; 600 feet6 minutes). results: we mapped 3,483 ohcas and 2,314 aeds in philadelphia county. ohcas occurred in males (55%; 1916/3483) and the mean age was 65.4 years. ventricular fibrillation occurred in 19% (662/3483). aeds were primarily located in schools/universities (30%), office buildings (22%), and residential buildings (4%). aeds were not identified within 200 feet in 93% (3,239) of ohcas, within 400 feet of 90% (3,135) of ohcas, and within 600 feet in 79% (2,752) of ohcas. the figure (large black circles) illustrates aed/ohca within 200 feet on the left and 600 feet on the right. conclusion: aeds were rarely close to the locations of ohcas, which may be a contributor to low cardiac arrest survival rates. innovative models to match aed availability with ohcas should be explored. (originally submitted as a ''late-breaker.'') potential background: early and frequent epinephrine administration is advocated by acls; however, epinephrine research has been conducted primarily with standard cpr (std). active compression-decompression cpr with an impedance threshold device (acd-cpr + itd) has become the standard of care for out of hospital cardiac arrest in our area. the hemodynamic effects of iv epinephrine under this technique are not known. objectives: to determine the hemodynamic effects of iv epinephrine in a swine model undergoing acd-cpr+itd. methods: six female swine (32 ± 1kg) were anesthetized, intubated, and mechanically ventilated. intracranial, thoracic aorta, and right atrial pressures were recorded via indwelling catheters. carotid blood flow (cbf) was recorded via doppler. etc0 2 , sp0 2 , and ekg were monitored. ventricular fibrillation was induced and went untreated for 6 minutes. three minutes each of standard cpr (std), std-cpr+itd, and acd-cpr+itd was preformed. at minute 9 of the resuscitation, 40 lg/kg of iv epinephrine was administered and acd-cpr+itd was continued for 1 minute. statistical analysis was performed with a paired t-test. results: aortic pressure and calculated cerebral and carotid perfusion pressures increased from std < std+itd < acd-cpr+itd (p £ 0.001). epinepherine administered during acd-cpr+itd signficantly increased mean aortic (29 ± 5vs42 ± 12, p = 0.01), cerebral (12 ± 5 vs 22 ± 10, p = 0.01), and coronary perfusion pressures (8 ± 7 vs 17 ± 4, p = 0.02); however, mean cbf and etco 2 decreased (respectively 29 ± 15 vs 14 ± 7.0, p = 0.03; 20 ± 7 vs 18 ± 6, p = 0.04). conclusion: the administration of epinepherine during acd-cpr+itd signficantly increased markers of macrocirculation, while significantly decreasing etco 2 , a proxy for organ perfusion. while the calculated cerebral perfusion pressures increased, the directly measured cbf decreased. this calls into question the ability of calculated perfusion pressures to accurately reflect blood flow and oxygen delivery to end organs. hypoxia background: during cardiac arrest most patients are placed on 100% oxygen with assisted ventilations. after return of spontaneous circulation (rosc), 100% oxygen is typically continued for an extended time. animal data suggest that immediate post-arrest titration of oxygen by pulse oximetry produces better neurocognitive/ histologic outcomes. recent human data suggest that arterial hyperoxia is associated with worse outcomes. objectives: to assess the relationship between hypoxia, normoxia, and hyperoxia post-arrest and outcomes in post-cardiac arrest patients treated with therapeutic hypothermia. methods: we conducted a retrospective chart review of 190 post-arrest patients admitted to an academic medical center between january, 2000 and december, 2007 who had arterial blood gases (abg) drawn after rosc. demographic variables were analyzed using anova and chi-square tests as appropriate. unadjusted logistic regression analyses were performed to assess the relationship between hypoxia (pao 2 < 60 mmhg), normoxia (60-300 mmhg), hyperoxia (>300 mmhg), and mortality. results: on first abg (190 patients), 37 (19.5%) were hypoxic, 92 (48.4%) normoxic, and 61 (32.1%) hyperoxic. the average age of the cohort was 62.8 years (no difference for hypoxic, normoxic, and hyperoxic patients). overall mortality was 70.5% (134/190). there were no significant differences between initial heart rate, systolic blood pressure, sex, race, or pre-arrest functional status. in-hospital mortality was significantly higher when the first abg demonstrated hypoxia (94.6%; 35/ 37) than for normoxia (68.5%; 63/92) or hyperoxia (59%; 36/61). in unadjusted logistic regression analysis of first pao 2 values, hyperoxia was not associated with increased mortality (or 0.7; 95% ci 0.3-1.4) but hypoxia was associated with increased mortality (or 6.1; 95% ci 1.4-27.5). conclusion: hypoxia but not hyperoxia on first abg was associated with mortality in a cohort of post-arrest patients. background: there are over 330,000 deaths due to cardiac arrest per year in the us. the aha recommends monitoring the quality of cpr primarily through the use of end tidal co 2 (etco 2 ). the level of etco 2 is significantly dependant on minute ventilation and altered by pressor and bicarbonate use. cerebral oximetry (cereox) uses near infrared spectroscopy to non-invasively measure oxygen saturation of the frontal lobes of the brain. cereox has been correlated with cerebral blood flow and jugular vein bulb saturations. objectives: the objective of this study is to compare the simultaneous measurement of etco 2 and cereox to investigate which monitoring method provides the best measure of cpr quality as defined by return of spontaneous circulation (rosc). methods: a prospective cohort of a convenient sample of patients using out-of-hospital and ed cardiac arrest from two large eds. patients were monitored simultaneously by etco 2 and cereox during cpr. patient demographics and arrest data were collected using the utstein criteria. all patients were monitored throughout the resuscitation efforts. rosc was defined as a palpable pulse and a measurable blood pressure for a minimum of thirty minutes. results: twenty two patients were enrolled with complete data sets; 27% of the subjects had rosc. average down time of rosc subjects was 12 minutes (sd ± 14.6) and 31 minutes (sd ± 17.8) for subjects without rosc. the inability to obtain a value of 30 either for etco 2 or cereox was 50% and 75% specific with an 80% and 100% npv respectively for predicting lack of rosc. obtaining a value of 30 either for etco 2 or cereox was 66% and 100% sensitive, respectively in identifying rosc. subjects with rosc had sustained values above 30 for 1.25 mins on cereox and 4.9 mins on etco 2 prior to rosc. the increase in values over a three minute period prior to rosc was 13.5 on cereox and 1.3 on etco 2 . conclusion: the inability to obtain a value of 30 on either the etco 2 or cereox strongly predicted lack of rosc. cereox provides a larger magnitude and closer temporal increase prior to rosc than etco 2 . attaining a value of 30 on cereox was more predictive of rosc than etco 2. an discrepancies due to communicating information to multiple listeners in a short amount of time. this creates a communication barrier not always apparent to practitioners. we examine the perceptions of ems and ed personnel on the transfer of care and its correlation to missing patient data. objectives: evaluate provider perception of information transfer by ems and ed personnel and compare this to an external observer's objective assessment. methods: this is a retrospective quality improvement program at an academic level i trauma center. transfers of medical and trauma patients from ems to ed personnel were attended by trained external observers, research associates (ra). ra recorded the data communicated: name, age, past medical history (pmh), allergies, medications, events, active problems, vital signs (vs), level of consciousness (loc), iv access, and treatments given. then, ems and ed staff rated their perception of transfer on a 1-10 rating scale. results: ra evaluated 448 patient transfers (268 medical and 180 trauma). transfer time did not differ, 4.05 minutes for medical (95% ci: 3.77-4.32), 3.92 minutes for trauma patients (95% ci: 3.53-4.31)(p = 0.57). missing data between the two groups also did not differ, except loc and treatment were missed more in medical transfers, while pmh was missed more in the trauma transfers. comparing the transfers with all vs present (67%, 300/448) and all vs missing (12%, 55/ 448), with all vs missing, there was no difference in perception of transfer for ems (9.6/10 vs present vs 9.4/10 vs absent) or ed staff (9.5/10 vs present, 9.4/10 vs absent). when all vital signs were missing, ra rated 69.1% of transfers as poor, whereas when all vs were present 80.8% of transfers were considered good. conclusion: ems and ed staff felt transfers of care were professional, teams were attentive, and had similar amounts of interruptions for both medical and trauma cases. their perception of transfer of care was similar even when key information was missing, although external observers rated a significant amount of transfers poorly. thus, ems and ed staffs were not able to evaluate their own performance in a transfer of care and external observers were found to be better evaluators of transfers of care. swati singh, john brown, prasanthi ramanujam ucsf, san francisco, ca background: ems transports a large number of psychiatric emergencies to emergency departments (ed) across the us. research on paramedic education related to behavioral emergencies is sparse, but based on expert opinion we know that gaps in paramedic knowledge and training exist. in our system, paramedics triage patients to medical, detoxification, and purely psychiatric destinations, so a paramedic's understanding of these emergencies directly affects the flow of patients in our eds. objectives: our objectives were to understand the gaps in current training and develop a targeted curriculum for field providers with a long term goal of appropriately recognizing and triaging subjects to the ed. methods: data were collected using a survey that was distributed during a paramedic association meeting in october 2011. subjects were excluded if they did not complete the survey. survey questions addressed demographics of paramedics, frequency of various psychiatric emergencies and their confidence in managing these emergencies. data were collated, analyzed, and presented as descriptive statistics. results: forty-nine surveys were distributed with a response rate of 82% (n = 40/49). of the respondents, 70% (n = 28) were male and 68% (n = 27) had at least five years experience. mood, thought, and cognitive disorders were the most frequently encountered presentations and 65% (n = 26) of respondents came across psychiatric emergencies multiple times a week. many respondents did not feel confident managing agitated delirium (n = 16, 40%), acute psychosis (n = 17, 43%), and intimate partner or elder abuse (n = 14, 35%). a third to a half of the respondents felt they have little or no training in chemical sedation (n = 18, 45%), verbal de-escalation (n = 14, 35%), and triaging patients (n = 21, 53%). conclusion: we identified a need for a revised curriculum on management of psychiatric emergencies. future steps will focus on development of a curriculum and change in knowledge after implementation of this curriculum. background: prehospital endotracheal intubation has long been a cornerstone of resuscitative efforts for critically ill or injured patients. paramedic airway management training will need to be modified due to the 2011 acc/aha guidelines to ensure maintenance of competency in overall management of airway emergencies. how best to modify the training of paramedics requires an understanding of current experience. objectives: the purpose of this report is to characterize the airway management expertise of experienced and non-experienced paramedics in a single ems system. methods: we retrospectively reviewed all prehospital intubations from an urban/suburban ambulance service (professional ambulance, inc.) over a five-year period (january 01, 2006 to december 31, 2010). characteristics of airway management by paramedics with 0-5 years of experience (group 1) were compared to those with greater than 5 years of experience (group 2). airway management was guided by massachusetts statewide treatment protocols governing direct laryngoscopy and all adjunctive approaches. attempts are characterized by laryngoscope blade passing the lips. difficult and failed airways were managed with extraglottic devices (egd) or needle cricothyroidotomy. we reviewed patient characteristics, intubation methods, rescue techniques, and adverse events. results: 150 patients required airway management: 120 (80%) were performed by group 1 and 30 (20%) were performed by group 2. group 1 was both faster to intubate (1.39 vs 1.83 attempts, p = 0.0035) and less likely to use a rescue device (19.1% vs 50.0%, p = 0.0009). both are equally likely to go directly to a rescue device (10% vs 10%, p = 1.0). all patients were successfully oxygenated and ventilated with either an endotracheal tube or egd. no surgical airways were performed and no patients died as a result of a failed airway. conclusion: while intubation success rates of paramedics with less than and greater than five years of experience are similar, less experienced paramedics use fewer attempts and are less likely to use a rescue device. both recognize difficult airways and go directly to rescue devices equally. this highlights difficulties faced maintaining competence. education requirements must be evaluated and redesigned to allow paramedics to maintain competence and emphasize airway management according to the latest resuscitation guidelines. how well do ems 9-1-1 protocols predict ed utilization for pediatric patients? stephanie j. fessler 1 , harold k. simon 1 , daniel a. hirsh 1 , michael colman 2 1 emory university, atlanta, ga; 2 grady health systems, atlanta, ga background: the use of emergency medical services (ems) for low-acuity pediatric problems has been well documented. however, it is unclear how accurately general ems dispatch protocols predict the subsequent ed utilization for these patients. objectives: to determine the ed resource utilization rate of pediatric patients categorized as low acuity by 9-1-1 dispatch protocols and then subsequently transferred to a children's hospital. methods: all transports for pediatric patients from the scene by a large urban general ems provider that were prioritized as low acuity by initial 9-1-1 dispatch protocols were identified. protocols were based on the national academy of medical priority dispatch system, v12. starting on jan 1, 2010, 100 consecutive cases of patients transported to three pediatric emergency departments (ped) of a large tertiary care pediatric health care system were reviewed. demographics, ped visit characteristics, resource utilization, and disposition were recorded. those patients who received meds other than po antipyretics, had labs other than a strep test, a radiology study, a procedure, or were not discharged home were categorized into the significant ed resource utilization group. results: 93% of the patients were african american and either had public insurance or self-pay (86%, 13% respectively). the median age was 11 months (4d-13yr). 54% were female. none of these low-acuity patients were upgraded by ems operators en route. upon arrival to the ped, 45% of transported patients were classified into the significant utilization group. six of the 100 total patients were admitted, including a 2 y/o requiring emergent intubation, an 8 m/o old with a broken cvl, a 6 y/o with sickle cell pain crisis, and a 2 y/o with altered mental status. the remainder of the significant resource utilization group consisted of children needing procedures, anti-emetics, narcotic pain control, labs, and xrays. conclusion: in this general ems 9-1-1 system, dispatch protocols for pediatric patients classified as low priority did poorly in predicting subsequent ed utilization with 45% requiring significant resources. further, ems operators did not recognize a critical child who needed emergent intervention. opportunity exists to refine general ems 9-1-1 protocols for children in order to more accurately define an ems priority status that better correlates with ultimate needs and resource utilization. the objectives: determine if there is an association between a patient's impression of the overall quality of care and his or her satisfaction with provided pain management. it was hypothesized that satisfaction with pain management would be significantly associated with a patient's impression of the overall quality of care. methods: this was a retrospective review of patient satisfaction survey data initially collected by an urban als ems agency from 1/1/2007 to 8/1/2010. participants were randomly selected from all patients transported proportional to their paramedic defined acuity; categorized as low, medium, or high with a goal of 100 interviews per month. the proportions of patients sampled from each acuity level were 25% low, 50% medium, and 25% high. patients were excluded if there was no telephone number recorded in the prehospital patient record or they were pronounced dead on scene. all satisfaction questions used a five-point likert scale with ratings from excellent to poor that were dichotomized for analysis as excellent or other. the outcome variable of interest was the patient's perception of the overall quality of care. the main independent variable had patients rate the staff who treated them at the scene on their helping to control or reduce their pain. demographic variables were assessed for potential confounding. results: there were 2,759 patients with complete data for the outcome and main independent variable with 45.0% male respondents and an average age of 54.1 (sd = 22.7). overall quality of care was rated excellent by 66.0% of patients while 59.1% rated their pain management as excellent. of patients who rated their pain management as excellent, 87.9% rated overall quality of care as excellent while only 34.2% of patients rated overall quality excellent if pain management was not excellent. when controlling for potential confounding variables, those patients who perceived their pain management to be excellent were 13.9 (95% ci 11.5-16.9) times more likely to rate their overall quality of care as excellent compared to those with non-excellent perceived pain management. conclusion: patients' perceptions of the overall quality of care were significantly associated with their perceptions of pain management. objectives: the purpose of this study is to determine whether ground-based paramedics could be taught and retain the skills necessary to successfully perform a cricothyrotomy. methods: this retrospective study was performed in a suburban county with a population of 160,000 and 21,000 ems calls per year. participants were groundbased paramedics in a local ems system who were taught wire-guided cricothyrotomy as part of a standardized paramedic educational update program. as part of the educational program, paramedics were taught wire-guided cricothyrotomy on a simulation model previously developed to train emergency medicine residents. after viewing an instructional video, the participants were allowed to practice using a 16step checklist. not all of these 16 steps were automatic failures. each paramedic was individually supervised performing a cricothyrotomy on the simulator until successful; a minimum of five simulations was required. retention was assessed using the same 16-step checklist during annual skills testing, after a minimum of 6 weeks to a maximum of 3 months posttraining. results: a total of 55 paramedics completed both the initial training and reassessment during the time period studied. during the initial training phase, 100% (55 of 55) of the paramedics were successful in performing all 16 steps of the wire-guided cricothyrotomy. during the retention phase 87.3% (48 of 55) retained the skills necessary to successfully perform the wire-guided cricothyrotomy. of the 16-step checklist, most steps were performed successfully by all the paramedics or missed by only 1 of the 55 paramedics. step #8, which involved removing the needle prior to advancing the airway device over the guidewire, was missed by 34.5% (19 of 55) of the participants. step #8 was not an automatic failure since most participants immediately self-corrected and completed the procedure successfully. conclusion: paramedics can be taught and can retain the skills necessary to successfully perform a wireguided cricothyrotomy on a simulator. future research is necessary to determine if paramedics can successfully transfer these skills to real patients. helicopter emergency medical services in background: netcare911 is one of the largest private providers of emergency air medical care in south africa. each hems (helicopter emergency medical service) crew is manned by a physician-paramedic team and is dispatched based on specific medical criteria, time to definitive care, and need for physician expertise. objectives: to describe the characteristics of net-care911 air medical evacuations in gauteng province and to analyze the role of physicians in patient care and effect on call times. methods: all patients transported by a netcare911 helicopter over a one year period from january -december 2008 were enrolled in the study. injury classifications, demographics, procedures, scene and flight times were collected retrospectively from run sheets. data were described by medians and interquartile intervals. results: a total of 386 patients were transported on 384 flights originating from the netcare911 gauteng helicopter base. ninety-two percent were traumarelated, with 74% resulting from motor vehicle accidents. physician expertise was listed 30% of the time as the indication for air medical response. a total of 105 advanced procedures were performed by physicians on 93 patients, including paralytic-assisted intubations, chest tube placement, and cardiac pacing. the median total call time was 46 minutes with 10 minutes spent on scene, compared with 54 and 24 minutes when advanced procedures were performed by hems (p < 0.001). conclusion: trauma accounts for an overwhelming majority of patients requiring emergency air medical transportation. advanced medical procedures were performed by physicians in nearly a quarter of the patients. there were significant differences in call times when advanced procedures were performed by hems. objectives: we sought to evaluate the level of awareness and adoption of the off-line protocol guidelines by utah ems agencies. methods: we surveyed all ems agencies in utah 18 months after protocol guideline release. medical directors, ems captains, or training coordinators completed a short phone survey regarding their knowledge of the emsc protocol guidelines, and whether their agency had adopted them. in particular, participants were asked about the pain protocol guideline and their management of pediatric pain. results: of the 186 agencies, 182 participated in the survey (98%). of those participating, 15 agencies (8%) were excluded from the analysis: 4 (2%) who only treat adults and 11 (6%) who do not participate in electronic data entry. of the remaining 171 agencies (94%), 155 (91%) were familiar with the utah emsc protocol guidelines; 116 agencies (68%) have either partially or fully adopted the protocol guidelines. 132 agencies (77%) were familiar with the pain treatment protocol guideline; 29 (17%) had adopted it; 34 (21%) planned to either partially or fully adopt the protocol. overall, 84 agencies (49%) had offline protocols allowing the administration of narcotics to children. of those, 49 (58%) had intranasal fentanyl as an available medication and delivery route. of the 84 agencies with offline protocols for pain, 77 (83%) reported familiarity with the emsc pain protocol guideline. conclusion: the creation and dissemination of statewide emsc protocol guidelines results in widespread awareness (91%) and to date 68% of agencies have adopted them. future investigation into factors associated with protocol adoption should be explored. background: intranasal (in) naloxone is safe and effective for the treatment of opioid overdose. while it has been extensively studied in the out-of-hospital environment in the hands of paramedics and lay people, we are unaware of any studies evaluating the safety and efficacy of in naloxone administration by bls providers. in recent years in naloxone has been added to the bls armamentarium; however, most services/states require an als unit be dispatched and attempt an intercept if in naloxone is administered by the bls providers. objectives: the purpose of this study is to evaluate the safety and effectiveness of bls-administered in naloxone in an urban environment. methods: retrospective cohort review as part of the ongoing qa process of all patients who had in naloxone administration by bls providers. the study was part of a special projects waiver by massachusetts oems from february 2011 through november 2011 in a busy urban tiered ems system in the metro-boston area. exclusion criteria: cardiac arrest. demographic information was collected, as well as vital signs, number of naloxone doses by bls, patient response to bls naloxone administration (clinical improvement in mental status and/or respiratory status), als intercept. descriptive statistics and confidence intervals are reported using microsoft excel and spss 17.0. results: fifty-six cases of bls-administered in naloxone were identified, and 2 were excluded as cardiac arrests. the included cases had a mean age of 38.8 years ±13.5 (range 16-82), and 74% (ci 60-85) were male. of the 54 included cases, 76% (ci 62-87) of patients responded to bls administration of naloxone. of the responders, 17% (ci 7-32) required two doses. there were 10 protocol violations representing 19% (ci 9.2-31.4) of the total administrations, however in 100% of these 10 protocol violations the patients had a positive response to the administration of in naloxone. seven of the protocol violations were patients who required a second 2 mg dose of naloxone. eleven cases did not have an als intercept; only 1 of these 11 patients did not respond to bls administration of naloxone. there were no identified adverse events. conclusion: bls providers safely and successfuly administered in naloxone achieving a response rate consistent with studies of als providers' administration of in naloxone. given the success rate of bls providers, it may be feasible for bls to manage responders without the aid of an als intercept. background: an estimated 20% of patients arriving by ambulance to the ed are in moderate to severe pain. however, the management of pain in the prehospital setting has been shown to be inadequate, and untreated pain may have negative consequences for patients. objectives: to determine if focused education on pediatric pain management and implementation of a pain management protocol improved the prehospital assessment and treatment of pain in adult patients. specifically, this study aimed to determine if documentation of pain scores and administration of morphine by ems personnel improved. methods: this was a retrospective before and after study conducted by reviewing a county-wide prehospital patient care database. the study population included all adult patients transported by ems between 01 february 2006 and 28 february 2010 with a working assessment of trauma or burn. ems patient care records were searched for documentation of pain scores and morphine administration 2 years before and 2 years after an intensive pediatric focused pain management education program and implementation of a pain management protocol. frequencies and 95% cis were determined for all patients meeting the inclusion criteria in the before and after time period and chisquare was used to compare frequencies between time periods. a secondary analysis was conducted using only subjects documented as meeting the protocol's treatment guidelines. results: 7,999 (10%) of 77,122 adult patients transported by ems during the study period met the inclusion criteria: 4,357 in the before and 3,642 in the after period. subject demographics were similar between the two periods. documentation of pain score did not change between the time periods ( background: there is a presumption that ambulance response times affect patient outcome. we sought to determine if shorter response times really make a difference in hospital outcomes. objectives: to determine if ambulance response time makes a difference in the outcomes of patients transported for two major trauma (motor vehicle crash injuries, penetrating trauma) and two major medical (difficulty breathing and chest pain complaints) emergencies. methods: this study was conducted in a metropolitan ems system serving a population total of 800,000 including urban and rural areas. cases were included if the private ems service was the first medical provider on scene, the case was priority 1, and the patient was 13 years and older. a 12-month time period was used for the data evaluation. four diagnoses were examined: motor vehicle crash injuries, penetrating trauma, difficulty breathing, and chest pain complaints. ambulance response times were assessed for each of the four different complaints. the patients' initial vital signs were assessed and the number of vital signs out of range was recorded. a sampling of all cases which went to the single major trauma center was selected for evaluation of hospital outcome. using this hospital sample, number of vital signs out of range were assessed as a surrogate marker indicating severity of hospital outcome. correlation coefficients were used to evaluate interactions between independent and outcome variables. results: of the 2164 cases we reviewed over the 12month period, we found that the ems service responded significantly faster to trauma complaints at 4.53 minutes (n = 254) than medical complaints at 5.92 minutes (n = 1910) . in the hospital sample of 587 cases, number of vital signs out of range were positively correlated with hospital days (r = 0.11), admits (r = 0.12), icu admits (r = 0.10), and deaths (r = 0.09), but not response times (r = (-)0.08). in the entire sample, there was no correlation between vital signs out of range and response times for any diagnosis (see figure) . conclusion: conclusions: based on our hospital sample which showed that number of vital signs out of range was a surrogate marker of worse hospital outcomes, we find that hospital outcomes are not related to initial response times. adverse effects following prehospital use of ketamine by paramedics eric ardeel baylor college of medicine, houston, tx background: ketamine is widely used across specialties as a dissociative agent to achieve sedation and analgesia. emergency medical services (ems) use ketamine to facilitate intubation and pain control, as well as to sedate acutely agitated patients. published studies of ems ketamine practice and effects are scarce. objectives: describe the incidence of adverse effects occurring after ketamine administration by paramedics treating under a single prehospital protocol. methods: a retrospective analysis was conducted of 98 consecutive patients receiving prehospital ketamine from paramedics in the suburban/rural ems system of montgomery county hospital district, texas between august 1, 2010 and october 25, 2011. ketamine administration indications were: need for rapid control of violent/agitated patients requiring treatment and transport; sedation and analgesia after trauma; facilitation of intubation and mechanical ventilation. ketamine administration contraindications were: equivalent ends achieved by less invasive means; hypertensive crisis; angina; signs of significantly elevated intracranial pressure; anticipated inability to support or control airway. all patients were included, regardless of indication for ketamine administration. data were abstracted from electronic patient care records and available continuous physiologic monitoring data, and analyzed for the presence of adverse effects as defined a priori in ''clinical practice guidelines for emergency department ketamine dissociative sedation: 2011 update.'' results: no patients were identified as experiencing adverse effects as defined by the referenced literature. ketamine was utilized most often for patients with the following nemsis provider's primary impression: 25 (26%) altered level of consciousness, 23 (23%) behavioral/psychiatric, 20 (20%) traumatic injury. overall, combativeness was associated with 64 (65%) patients. the mean age was 41 years (range 3-94 years) and 50 (51%) were male. the mean ketamine dose was 150 mg (range 25-500 mg) and twenty-four (24%) patients received multiple administrations. conclusion: in this patient population, our data indicate that prehospital ketamine use by ems paramedics, across all indications for administration, was safe. further study of ketamine's utility in ems is warranted. an background: rigorous evaluation of the effect of implementing nationally vetted evidence-based guidelines (ebgs) has been notoriously difficult in ems. specifically, human subjects issues and the health insurance portability and accountability act (hipaa) present major challenges to linking ems data with distal outcomes. objectives: to develop a model that addresses the human subjects and hipaa issues involved with evaluating the effect of implementing the traumatic brain injury (tbi) ebgs in a statewide ems system. methods: the excellence in prehospital injury care (epic) project is an nih-funded evaluation of the effect of implementing the ems tbi guidelines throughout arizona (ninds-1r01ns071049-01a1). to accomplish this, a partnership was developed between the arizona department of health services (adhs), the university of arizona, and more than 100 ems agencies that serve approximately 85% of the state's population. results: ebg implementation: implementation follows all routine regulatory processes for making changes in ems protocols. in arizona, the entire project must be carried out under the authority of the adhs director. evaluation: a before-after system design is used (randomization is not acceptable). hipaa: as an adhsapproved public health initiative, epic is exempt from hipaa, allowing sharing of protected health information between participating entities. for epic, the state attorney general provided official verification of hi-paa exemption, thus allowing direct linkage of ems and hospital data. irb: once epic was officially deemed a public health initiative, the university irb process was engaged. as an officially sanctioned public health project, epic was determined to not be human subjects research. this allows the project to implement and evaluate the effect of this initiative without requiring individual informed consent. conclusion: by utilizing an ems-public health-university partnership, the ethical and regulatory challenges related to evaluating implementation of new ebgs can be successfully overcome. the integration of the department of health, the attorney general, and the university irb can properly protect citizens while permitting efficient implementation and rigorous evaluation of the effect of ebgs. this novel approach may be useful as a model for evaluation of implementing ems ebgs in other states and large counties. (20.6%-58.1% by age) were transported to non-trauma centers. the most common reasons cited by ems for hospital selection were: patient preference (50.6%), closest facility (20.7%), and specialty center (15.2%). patient preference increased with age (p for trend 0.0001) and paralleled under-triage ( figure 1 ). iss ‡ 16 patients transported to non-trauma hospitals by patient request had lower unadjusted mortality (3.8%, 95%ci 1.9-5.8) than similar patients transported to trauma centers (11.8%, 95%ci 10.7-12.8) or transported for other reasons (12.6%, 95%ci 11.4-13.7) (figure 2) . under-triage appears to be influenced by patient preference and age. self-selection for transport to non-trauma centers may result in under-triaged patients with inherently better prognosis than triagepositive patients. background: only 25% of all out-of-hospital cardiac arrest (ohca) patients receive bystander cpr (cardiopulmonary resuscitation). the neighborhood in which an ohca occurs has significant influence on the likelihood of receiving bystander cpr. objectives: to utilize geographic information systems to identify ''high-risk'' neighborhoods, defined as census tracts with high incidence of ohca and low cpr prevalence. methods: design: secondary analysis of the cardiac arrest registry to enhance survival (cares) dataset for denver county, colorado. population: all consecutive adults (>18 years old) with ohca due to cardiac etiology from january 1, 2009 through december 31, 2010. data analysis: analyses were conducted in arc-gis. three spatial statistical methods were used: local morans i (lmi), getis-ord gi*(gi*), and spatial empirical bayes (seb) adjusted rates. census tracts with high incidence of ohca, as identified by all three spatial statistical methods, were then overlain with low bystander cpr census tracts, which were identified in at least two out of three statistical methods (lmi, gi*, or the lowest quartile of bystander cpr prevalence). overlapping census tracts identified with both high ohca incidence and low cpr prevalence were designated as ''highrisk''. results: a total of 728 arrests in 142 census tracts occurred during the study period, with 595 arrests included in final sample. events were excluded if they were unable to be geocoded (n = 41), outside denver county (n = 8), or occurred in a jail (n = 3), hospital/ physician's office (n = 7), or nursing home (n = 74). for high ohca incidence: lmi identified 29 census tracts, gi* identified 45 census tracts, and the seb method identified 28 census tracts. twenty-five census tracts were identified by all three methods. for low bystander cpr prevalence: lmi identified 9 census tracts, gi* identified 16 census tracts, and 101 census tracts were identified as being in the lowest quartile of cpr prevalence. twenty-four census tracts were identified by two of the three methods. two census tracts were identified as high-risk having both high ohca incidence and low cpr prevalence (figure) . high-risk census tract demographics as compared to denver county are shown in the table. conclusion: the two high-risk census tracts, comprised of minority and low-income populations, appear to be possible sites for targeted community-based cpr interventions. objectives: we sought to assess the accuracy and correlation of geographic information system (gis) derived transport time compared to actual ems transport time in ohca patients. methods: prospective, observational cohort analysis of ohca patients in vancouver, b.c., one of the sites of the resuscitation outcomes consortium (roc). a random sample from all of the ohca cases from 12/05 through 05/07 was selected for analysis from one site of the roc epistry. using gis, ems transport time was derived from reported latitude/longitude coordinates of the ohca event to the actual receiving hospital. this was calculated via the actual network distance using arcgis. this gis-derived time was then compared to the actual ems transport time (in minutes) using the wilcoxon signed rank test. scatter plot analysis of actual vs. gis times were created to evaluate the relationship between actual and calculated time. a linear regression model predicting actual ems transport time from the derived gis-time was also developed in order to examine the potential relationship between the two variables. differences in the relationship were also investigated based on time of the day to reflect varying traffic conditions. results: 641 cases were randomly selected for analysis. the median actual transport time was significantly longer than the median gis derived transport time (7.08 minutes vs. 5.50 minutes). scatter plot analysis did not reveal any significant correlation between actual and gis-based time. additionally, there was poor approximation of gis-based time and actual ems time (r 2 = 0.20) with no evidence of a significant linear relationship between the two. the poorest correlation of time was observed during the morning hours (07:00-09:00; r 2 = 0.02) while the strongest correlation was during the overnight hours (00:00-07:00; r 2 = 0.26). conclusion: gis derived time does not appear to correlate well with actual ems transport time of ohca patients. efforts should be made to accurately obtain actual ems transport times for ohca patients. objectives: we first sought to describe the incidence of ohca presenting to the ed. we then sought to determine the association between hospital characteristics and survival to hospital admission. methods: we identified patients with diagnoses of cardiac arrest or ventricular fibrillation (icd-9 427.5 or 427.41) in the 2007 nationwide emergency department sample, a nationally representative estimate of all ed admissions in the us. eds reporting ‡1 patient with ohca were included. our primary outcome was survival to hospital admission. we examined variability in hospital survival rate and also classified hospitals into high or low performers based on median survival rate. we used this dichotomous hospital level outcome to examine factors associated with survival to admission including hospital and patient demographics, ed volume, cardiac arrest volume, and cardiac catheterization availability. all unadjusted and adjusted analyses were performed using weighted statistics and logistic regressions. results: of the 966 hospitals, 949 (98.2%) were included. in total, 44,782 cases of cardiac arrest were identified, representing an estimated 203,331 cases nationally. overall ed ohca survival to hospital admission was 23.5% (iqr 0.1%, 29.4%) in adjusted analyses, increased survival to admission was seen in hospitals with teaching status (or 2.7, 95% ci 1.7-4.4, p < 0.001), annual ed visits ‡10,000 (or 3.9, 95% ci 2.5-6.1, p < 0.001), and pci capability (or 9.1, 95% ci 1.2-68.2, p = 0.032). in separate adjusted analyses including teaching status and pci capabilities, hospitals with >40 annual cardiac arrest cases (or 3.0, 95% ci 2.2-4.2, p < 0.001) were also shown to have improved survival (figure) . conclusion: ed volume, cardiac arrest volume, and pci capability were associated with improved survival to hospital admission in patients presenting to the ed after ohca. an improved understanding of the contribution of ed care to ohca survival may be useful in guiding the regionalization of cardiac arrest care. background: prior investigations have demonstrated regional differences in out-of-hospital cardiac arrest (ohca) outcomes, but none have evaluated survival variability by hospital within a single major us city. objectives: we hypothesized that 30-day survival from ohca would vary considerably among one city's receiving hospitals. methods: we performed a retrospective review of prospectively collected cardiac arrest data from a large, urban ems system. our population included all ohcas with a recorded social security number (which we used to determine 30-day survival through the social security death index) that were transported to a hospital between 1/1/2008 and 12/31/2010. we excluded traumatic arrests, pediatric arrests, and hospitals receiving less than 10 ohcas with social security numbers over the three-year study period. we examined the associa-tion between receiving hospital and 30-day survival. additional variables examined included: level i trauma center status, teaching hospital status, ohca volume, and whether post-arrest therapeutic hypothermia (th) protocols were in place in 2008. statistics were performed using chi-square tests and logistic regression. results: our study population comprised 550 arrest cases delivered to 18 unique hospitals with an overall 30-day survival of 14.4%. mean age was 69.0 (sd 16.2) years. males comprised 54.2% of the cohort; 53.3% of victims were black. thirty-day survival varied significantly among the hospitals, ranging from 4.8% to 35.0% (chi-square 32.3, p = 0.014). ohcas delivered to level i trauma centers were significantly more likely to survive (19.5% vs. 12.7%, p = 0.05), as were those delivered to hospitals known to offer post-arrest th (19.2% vs. 11.8%, p = 0.018). hospital teaching status and ohca volume were not associated with survival. conclusion: there was significant variability in ohca survival by hospital. patients were significantly more likely to survive if transported to a level i trauma center or hospital with post-arrest th protocols, suggesting a potential role for regionalization of ohca care. limiting our population to ohcas with recorded social security numbers reduced our power and may have introduced selection bias. further work will include survival data on the complete set of ohcas transported to hospitals during the three-year study period. background: traumatic brain injury is a leading cause of death and disability. previous studies suggest that prehospital intubation in patients with tbi may be associated with mortality. limited data exist comparing prehospital (ph) nasotracheal (nt), prehospital orotracheal (ot), and ed ot intubation and mortality following tbi. objectives: to estimate the associations between ph nt, ph ot, and ed ot intubation and in-hospital mortality in patients with moderate to severe tbi, with hypotheses that ph nt and ph ot intubation would be associated with increased mortality when compared to ed ot or no intubation. methods: an analysis using the denver health trauma registry, a prospectively collected database. consecutive adult trauma patients from 1995-2008 with moderate to severe tbi defined as head abbreviated injury scale (ais) scores of 2-5. structured chart abstraction by blinded physicians was used to collect demographics, injury and prehospital care characteristics, intubation status and timing, in-hospital mortality and survival time, and neurologic function at discharge. poor neurologic function was defined as cerebral performance category score of 3-5. multivariable logistic regression and survival analyses were performed, using multiple imputation for missing data. results: of the 3,517 patients, the median age was 38 (iqr 27-51) years. the median ph gcs was 14 (iqr 6-15), median injury severity score was 20 (iqr 13-29), and median head ais was 4 (iqr 3-5). ph nt occurred in 15.8%, ph ot in 9.5%, and ed ot in 17.4%, while mortality occurred in 17.5%. the 24-, 48-, and 72-hour survival analyses are outlined in the table. survival curves for ph nt, ph ot, and ed ot are demonstrated in the figure (p < 0.001) . conclusion: prehospital intubation in patients with moderate to severe tbi is associated with increased mortality. contrary to our initial hypothesis, there was also a significant association between ed intubation and mortality. these associations persisted despite survival time, and while adjusting for injury severity. background: sbdp150 is a breakdown product of the cytoskeletal protein alpha-ii-spectrin found in neurons and has been detected in severe tbi. objectives: this study examined whether early serum levels of sbdp150 could distinguish: 1) mild tbi from three control groups; 2) those with and without traumatic intracranial lesions on ct (+ct vs -ct); and 3) those having a neurosurgical intervention (+nsg vs -nsg) in mild and moderate tbi (mmtbi). methods: this prospective cohort study enrolled adult patients presenting to two level i trauma centers following mmtbi with blunt head trauma with loss of consciousness, amnesia, or disorientation and a gcs 9-15. control groups included uninjured controls and trauma controls presenting to the ed with orthopedic injuries or an mvc without tbi. mild tbi was defined as gcs 15 and moderate tbi as having a gcs <15. blood samples were obtained in all patients within 4 hours of injury and measured by elisa for sbdp150 (ng/ml). the main outcomes were: 1) the ability of sbdp150 to distinguish mild tbi from three control groups; 2) to distinguish +ct from -ct and; 3) to distinguish +nsg from -nsg. data were expressed as means with 95%ci, and performance was tested by roc curves (auc and 95%ci). results: there were 275 patients enrolled: 54 tbi patients (42 gcs 15, 12 gcs 9-14), 23 trauma controls (16 mvc controls and 7 orthopedic controls), and 198 uninjured controls. the mean age of tbi patients was 39 years (range 19-70) with 63% males. fourteen (14%) had a +ct and 9% had +nsg. mean serum sbdp150 levels were 0.764 (95%ci 0.561-0.968) in normal controls, 1.035 (0.091-2.291) in orthopedic controls, 1.209 (0.236-2.181 ) in mvc controls, 2.764 (1.700-3.827 ) in mild tbi with gcs 15, and 5.227 (0.837-9.617) in tbi with gcs 9-14 (p < 0.001). the auc for distinguishing mild tbi from both controls was 0.83 (95%ci 0.68-0.99). mean sbdp150 levels in patients with -ct versus +ct were 2.170 (1.340-3.000) and 6.797 (2.227-11.368) respectively (p < 0.001) with auc = 0.78 (95%ci 0.61-0.95). mean sbdp150 levels in patients with -nsg versus +nsg were 2.492 (1.391-3.593) and 6.867 (3.891-9.843) respectively (p < 0.001) with auc = 0.88 (95%ci 0.77-0.98). conclusion: serum sbdp150 levels were detectable in serum acutely after injury and were associated with measures of injury severity including ct lesions and neurosurgical intervention. further study is required to validate these findings before clinical application. utility of platelet background: pre-injury use of anti-platelet agents (e.g., clopidogrel and aspirin) is a risk factor for increased morbidity and mortality in patients with traumatic intracranial hemorrhage (tich). some investigators have recommended platelet transfusion to reverse the anti-platelet effects in tich. objectives: this evidence-based medicine review examines the evidence regarding the effect of platelet transfusion in emergency department (ed) patients with pre-injury anti-platelet use and tich on patientoriented outcomes. methods: the medline, embase, cochrane library, and other databases were searched. studies were selected for inclusion if they compared platelet transfusion to no platelet transfusion in the treatment of adult ed patients with pre-injury anti-platelet use and tich, and reported rates of mortality, neurocognitive function, or adverse effects as outcomes. we assessed the quality of the included studies using ''grading of recommendations assessment, development and evaluation'' (grade) criteria. categorical data are presented as percentages with 95% confidence interval (ci). relative risks (rr) are reported when clinically significant. results: five retrospective, registry-based studies were identified, which enrolled 635 patients cumulatively. based on standard criteria, three studies were of ''low'' quality evidence and two studies had ''very low'' qualities. one study reported higher in-hospital mortality in patients with platelet transfusion (ohm et al), another showed a lower mortality rate in patients receiving platelet transfusion (wong et al). three studies did not show any statistical difference in comparing mortality rates between the groups (table) . no studies reported intermediate-or long-term neurocognitive outcomes or adverse events. conclusion: five retrospective registry studies with suboptimal methodologies provide inadequate evidence to support the routine use of platelet transfusion in adult ed patients with pre-injury anti-platelet use and tich. abnormal levels of end-tidal carbon dioxide (etco 2 ) are associated with severity of injury in mild and moderate traumatic brain injury (mmtbi) linda papa 1 , artur pawlowicz 2 , carolina braga 1 , suzanne peterson 1 , salvatore silvestri 1 1 orlando regional medical center, orlando, fl; 2 university of central florida, orlando, fl background: capnography is a fast, non-invasive technique that is easily administered and accurately measures exhaled etco 2 concentration. etco 2 levels respond to changes in ventilation, perfusion, and metabolic state, all of which may be altered following tbi. objectives: this study examined the relationship between etco 2 levels and severity of tbi as measured by clinical indicators including glasgow coma scale (gcs) score, computerized tomography (ct) findings, requirement of neurosurgical intervention, and levels of a serum biomarkers of glial damage. methods: this prospective cohort study enrolled adult patients presenting to a level i trauma center following a mmtbi defined by blunt head trauma followed by loss of consciousness, amnesia, or disorientation and a gcs 9-15. etco 2 measurements were recorded from the prehospital and emergency department records and compared to indicators of tbi severity. results: of the 46 patients enrolled, 21 (46%) had a normal etco 2 level and 25 (54%) had an abnormal etco 2 level. the mean age of enrolled patients was 40 (range 19-70) and 32 (70%) were male. mechanisms of injury included motor vehicle collision in 19 (41%), motor cycle collision in 9 (20%), fall in 8 (17%), bicycle/ pedestrian struck in 8 (17%), and other in 2 (4%). eight (17%) patients had a gcs 9-12 and 38 (83%) had a gcs 13-15. of the 11 (24%) patients with intracranial lesions on ct, 10 (91%) had an abnormal etco 2 level (p = 0.006). of the 5 (11%) patients who required a neurosurgical intervention, 100% had an abnormal etco 2 level (p = 0.05). levels of a biomarker indicative of astrogliosis were significantly higher in those with abnormal etco 2 compared to those with a normal etco 2 (p = 0.026). conclusion: abnormal levels of etco 2 were significantly associated with clinical measures of brain injury severity. further research with a larger sample of mmtbi patients will be required to better understand and validate these findings. background: acetaminophen (apap) poisoning is the most frequent cause of acute hepatic failure in the us. toxicity requires bioactivation of apap to toxic metabolites, primarily via cyp2e1. children are less susceptible to apap toxicity; one current theory is that children's conjugative pathway (sulfonation) is more active. liquid apap preparations contain propylene glycol (pg), a common excipient that inhibits apap bioactivation and reduces hepatocellular injury in vitro and in rodents. cyp2e1 inhibition may decrease toxicity in children, who tend to ingest liquid apap preparations, and suggests a potential novel therapy. objectives: to compare phase i (toxic) and phase ii (conjugative) metabolism of liquid versus solid prepara-tions of apap. we hypothesize that ingestion of a liquid apap preparation results in decreased production of toxic metabolites relative to a solid preparation, likely due to the presence of pg in the liquid preparations. methods: design-pharmacokinetic cross-over study. setting-university hospital clinical research center. subjects-adults ages 18-40 taking no chronic medications. interventions-subjects were randomized to receive a 15 mg/kg dose of a commercially available solid or liquid apap preparation. after a washout period of greater than 1 week, subjects received the same dose of apap in the alternate preparation. apap, apap-glucuronide and apap-sulfate (phase 2 metabolites), apap-cysteinate and apap-mercapturate (phase 1 metabolites) were analyzed via lc/ms in plasma over 8 hours. peak concentrations and measured auc were compared using paired-sample t-tests. plasma pg levels were measured. results: fifteen subjects completed the protocol. peak concentrations and aucs of the cyp2e1 derived toxic metabolites were significantly lower following ingestion of the liquid preparation (table, figure) . the glucuronide and sulfate metabolites were not different. pg was present following ingestion of liquid but not solid preparations. conclusion: ingestion of liquid relative to solid preparations in therapeutic doses results in decreased plasma levels of toxic apap metabolites. this may be due to inhibition of cyp2e1 by pg, and may explain the decreased susceptibility in children. a less hepatotoxic formulation of apap can potentially be developed if co-formulated with a cyp2e1 inhibitor. background: pressure immobilization bandages have been shown to delay mortality for up to 8 hours after coral snake envenomation, providing an inexpensive and effective treatment when antivenin is not readily available. however, long-term efficacy has not been established. objectives: determine if pressure immobilization bandages, consisting of an ace wrap and splint, can delay morbidity and mortality from coral snake envenomation, even in the absence of antivenin therapy. methods: institutional animal care and use committee approval was obtained. this was a randomized, observational pilot study using a porcine model. ten pigs (17.3 kg to 25.6 kg) were sedated and intubated for 5 hours. pigs were injected subcutaneously in the left distal foreleg with 10 mg of lyophilized m. fulvius venom resuspended in water, to a depth of 3 mm. pigs were randomly assigned to either a control group (no compression bandage and splint) or a treatment group (compression bandage and splint) approximately 1 minute after envenomation. pigs were monitored daily for 21 days for signs of respiratory depression, decreased oxygen saturations, and paresis/paralysis. in case of respiratory depression, pigs were euthanized and time to death recorded. chi-square was used to compare rates of survival up to 21 days and a kaplan-meier survival curve constructed. results: average survival time of control animals was 412 ± 90 minutes compared to 12,642 ± 7,132 minutes for treated animals. significantly more pigs in the treatment group survived to 24 hours than in the control group (p = 0.03). two of the treatment pigs survived to the endpoint of 21 days, but showed necrosis of the distal lower extremity. conclusion: long-term survival after coral snake envenomation is possible in the absence of antivenin with the use of pressure immobilization bandages. the applied pressure of the bandage is critical to allowing survival without secondary consequences (i.e. necrosis) of envenomation. future studies should be designed to accurately monitor the pressures applied. background: patients exposed to organophosphate (op) compounds demonstrate a central apnea. the kölliker-fuse nuclei (kf) are cholinergic nuclei in the brainstem involved in central respiratory control. objectives: we hypothesize that exposure of the kf is both necessary and sufficient for op-induced central apnea. methods: anesthetized and spontaneously breathing wistar rats (n = 24) were exposed to a lethal dose of dichlorvos using three experimental models. experiment 1 (n = 8) involved systemic op poisoning using subcutaneous (sq) dichlorvos (100 mg/kg or 3x ld50). experiment 2 (n = 8) involved isolated poisoning of the kf using stereotactic microinjections of dichlorvos (625 micrograms in 50 microliters) into the kf. experiment 3 (n = 8) involved systemic op poisoning with isolated protection of the kf using sq dichlorvos (100 mg/kg) and stereotactic microinjections of organophosphatase a (opda), an enzyme that degrades dichlorvos. respiratory and cardiovascular parameters were recorded continuously. histological verification of injection site was performed using kmno4 injections. animals were followed post-poisoning for 1 hour or death. betweengroup comparisons were performed using a repeated measured anova or student's t-test where appropriate. results: animals poisoned with sq dichlorvos demonstrated respiratory depression starting 5.1 min post exposure, progressing to apnea 15.9 min post exposure. there was no difference in respiratory depression between animals with sq dichlorvos and those with dichlorvos microinjected into the kf. despite differences in amount of dichlorvos (100 mg/kg vs 1.8 mg/kg) and method of exposure (sq vs cns microinjection), 10 min following dichlorvos both groups (sq vs microinjection respectively) demonstrated a similar percent decrease in respiratory rate (51.5 vs 72.2, p = 0.14), minute ventilation ( background: patients sustaining rattlesnake envenomation often develop thrombocytopenia, the etiology of which is not clear. laboratory studies have demonstrated that venom from several species, including the mojave rattlesnake (crotalus scutulatus scutulatus), can inhibit platelet aggregation. in humans, administration of crotaline fab antivenom (av) has been shown to result in transient improvement of platelet levels; however, it is not known whether platelet aggregation also improves after av administration. objectives: to determine the effect of c. scutulatus venom on platelet aggregation in vitro in the presence and absence of crotaline fab antivenom. methods: blood was obtained from four healthy male adult volunteers not currently using aspirin, nsaids, or other platelet-inhibiting agents. c. scutulatus venom from a single snake with known type b (hemorrhagic) activity was obtained from the national natural toxins research center. measurement of platelet aggregation by an aggregometer was performed using five standard concentrations of epinephrine (a known platelet aggregator) on platelet-rich plasma over time, and a mean area under the curve (auc) was calculated. five different sample groups were measured: 1) blood alone; 2) blood + c. scutulatus venom (0.3 mg/ml); 3) blood + crotaline fab av (100 mg/ml); 4) blood + venom + av (100 mg/ ml); 5) blood + venom + av (4 mg/ml). standard errors of the mean (sem) were calculated for each group. results: antivenom administration by itself did not significantly affect platelet aggregation compared to baseline (103.8 ± 3.4%, p = 0.47). administration of venom decreased platelet aggregation (72.0 ± 8.5%, p < 0.05). concentrated av administration in the presence of venom normalized platelet aggregation (101.4 ± 6.8%) and in the presence of diluted av significantly increased aggregation (133.9 ± 9.0%); p < 0.05 for both groups when compared to the venom-only group. to control for the effects of the venom and av, each was run independently in platelet-rich plasma without epinephrine; neither was found to significantly alter platelet aggregation. conclusion: crotaline fab av improved platelet aggregation in an in vitro model of platelet dysfunction induced by venom from c. scutulatus. the mechanism of action remains unclear but may involve inhibition of venom binding to platelets or a direct action of the antivenom on platelets. background: routine use of both breathalyzers and hand sanitizers is common across emergency depart-ments. the most common hand sanitizer on the market, purell, contains 62% ethyl alcohol and a lesser amount of isopropyl alcohol. previous investigations have documented that risk is low to the health care worker who applies frequent hand sanitizers to themselves. however, it is unknown whether this alcohol mixture causes false readings on a breathalyzer machine being used to determine alcohol levels on others. objectives: to determine the effect on the measurement of breathalyzer readings in individuals who have not consumed alcohol after hand sanitizer is applied to the experimenter holding a breathalyzer machine. methods: after obtaining informed consent, a breathalyzer reading was obtained in participants who had not consumed any alcohol in the last 24 hours. three different experiments were performed with 25 different participants in each. in experiment 1, two pumps of hand sanitizer were applied to the experimenter. without allowing the sanitizer to dry, the experimenter then measured the breathalyzer reading of the participant. in experiment 2, one pump of sanitizer was applied to the experimenter. measurements of the participant were taken without allowing the sanitizer to dry. in experiment 3, one pump of sanitizer was placed on the experimenter and rubbed until dry according to the manufacturer's recommendations. readings were recorded and analyzed using paired t-tests. results: the initial breathalyzer reading for all participants was 0. after two pumps of hand sanitizer were applied without drying (experiment 1), breathalyzers ranged from 0.02 to 0.17, with a mean above the legalintoxication limit of 0.11 (t(24) = )15.3, p < 0.001). after one pump of hand sanitizer was applied without drying (experiment 2), breathalyzers ranged from 0.02 to 0.11, with a mean of 0.06 (t(24) = )14.1, p < 0.001). after one pump of hand sanitizer was applied according to manufacturer's directions (experiment 3), breathalyzers ranged from 0.0 to 0.02 with a mean of 0.01 (t(24) = )5.1, p < 0.001). conclusion: use of hand sanitizer according to the manufacturer's recommendations results in a small but significant increase in breathalyzer readings. however, the improper and overuse of common hand sanitizer elevates routine breathalyzer readings, and can mimic intoxication in individuals who have not consumed alcohol. stephanie carreiro, jared blum, francesca beaudoin, gregory jay, jason hack objectives: the primary aim of this study is to determine if pretreatment with ile affects the hemodynamic response to epinephrine in a rat model. hemodynamic response was measured by a change in heart rate (hr) and mean arterial pressure (map). we hypothesized that ile would limit the rise in map and hr that typically follow epinephrine administration. methods: twenty male sprague dawley rats (approximately 7-8 weeks of age) were sedated with isoflurane and pretreated with a 15 ml/kg bolus of ile or normal saline, followed by a 15 mcg/kg dose of epinephrine intravenously. intra-arterial blood pressure and hr were monitored continuously until both returned to baseline (biopaq). a multifactorial analysis of variance (manova) was performed to assess the difference in map and hr between the two groups. standardized t-tests were then used to compare the peak change in map, time to peak map, and time to return to baseline map in the two groups. results: overall, a significant difference was found between the two groups in map (p = 0.01) but not in hr (p = 0.34). there was a significant difference (p = 0.023) in time to peak map in the ile group (54 sec, 95% ci 44-64) versus the saline group (40 sec, 95% ci 32-48) and a significant difference (p = 0.004) in time to return to baseline map in ile group (171 sec, 95% ci 148-194) versus the saline group (130 sec, 95% ci 113-147). there was no significant difference (p = 0.28) in the peak change in map of the ile group (75.4, mmhg, 95% ci 66-85) versus the saline group (69.9 mmhg, 95% ci 64-76). conclusion: our data show that in this rat model ile pretreatment leads to a significant difference in map response to epinephrine, but no difference in hr response. ile delayed the peak effect and prolonged the duration of effect on map but did not alter the peak increase in map. this suggests that the use of ile may delay the time to peak effect of epinephrine if the drugs are administered concomitantly to the same patient. further research is needed to explore the mechanism of this interaction. rasch analysis of the agitation severity scale when used with emergency department acute psychiatry patients tania d. strout, michael r. baumann maine medical center, portland, me background: agitation is a frequently observed and problematic phenomenon in mental health patients being treated in the emergency setting. the agitation severity scale (agss), a reliable and valid instrument, was developed using classical test theory to measure agitation in acute psychiatry patients. objectives: the aim of this study was to analyze the agss according to the rasch measurement model and use the results to determine whether improvements to the instrument could be made. methods: this prospective, observational study was irb-approved. 270 adult ed patients with psychiatric chief complaints and dsm-iv-tr diagnoses were observed using the agss. the rasch rating scale model was employed to evaluate the 17 items comprising the agss using winsteps statistical software. unidimensionality, item fit, response category performance, person and item separation reliability, and hierarchical ordering of items were all examined. a principle components analysis (pca) of the rasch residuals was also performed. results: variable maps revealed that all of the agss items were used to some degree and that the items were ordered in a way that makes clinical sense. several duplicative items, indicating the same degree of agitation, were identified. item (5.19) and person (2.01) separation statistics were adequate, indicating appropriate spread of items and subjects along the agitation continuum and providing support for the instrument's reliability. keymaps indicated that the agss items are functioning as intended. analysis of fit demonstrated no extreme misfitting items. pca of the rasch residuals revealed a small amount of residual variance, but provided support for the agss as being unidimensional, measuring the single construct of agitation. the results of this rasch analysis support the agss as a psychometrically robust instrument for use with acute psychiatry patients in the emergency setting. several duplicative items were identified that may be eliminated and re-evaluated in future research; this would result in a shorter, more clinically useful scale. in addition, a gap in items for patients with lower levels of agitation was identified. generation of additional items intended to measure low levels of agitation could improve clinician's ability to differentiate between these patients. background: attempted suicide is one of the strongest clinical predictors of subsequent suicide and occurs up to 20 times more frequently than completed suicide. as a result, suicide prevention has become a central focus of mental health policy. in order to improve current treatment and intervention strategies for those presenting with suicide attempt and self-injury in the emergency department (ed), it is necessary to have a better understanding of the types of patients who present to the ed with these complaints. objectives: to describe the epidemiology of ed visits for attempted suicide and self-inflicted injury over a 16year period. methods: data were obtained from the national hospital ambulatory medical care survey (nhamcs). all visits for attempted suicide and self-inflicted injury (e950-e959) during 1993-2008 were included. trend analyses were conducted using stata's nptrend (a nonparametric test for trends that is an extension of the wilcoxon rank-sum test) and regression analyses. a two-tailed p < 0.05 was considered statistically significant. results: over the 16-year period, there were an average of 420,000 annual ed visits for attempted suicide and self-inflicted injury (1.50 [95% confidence interval (ci) 1.33-1.67] visits per 1,000 us population). the overall mean patient age was 31 years, with visits most common among ages 15-19 (3.70; 95%ci 3.11-4.30). the average annual number of ed visits for suicide attempt and self-inflicted injury more than doubled from 244,000 in 1993-1996 to 538,000 in 2005-2008. during the same timeframe, ed visits for these injuries per 1,000 us population almost doubled for males (0.84 to 1.62), females (1.04 to 1.96), whites (0.94 to 1.82), and blacks (1.14 to 2.10). no temporal differences were found for method of injury or ed disposition; there was, however, a significant decrease in visits determined by the physician to be urgent/emergent from 95% in 1993 to 70% in 2008. conclusion: ed visit volume for attempted suicide and self-inflicted injury has increased over the past two decades in all major demographic groups. awareness of these longitudinal trends may assist efforts to increase research on suicide prevention. in addition, this information may be used to inform current suicide and self-injury related ed interventions and treatment programs. benjamin l. bregman, janice c. blanchard, alyssa levin-scherz george washington university, washington, dc background: the emergency department (ed) has increasingly become a health care access point for individuals with mental health needs. recent studies have found that rates of major depression disorder (mdd) diagnosed in eds are far above the national average. we conducted a study assessing whether individuals with frequent ed visits had higher rates of mdd than those with fewer ed visits in order to help guide screening and treatment of depressed individuals encountered in the ed. objectives: this study evaluated potential risk factors associated with mdd. we hypothesized that patients who are frequent ed visitors will have higher rates of mdd. methods: this was a single center, prospective, crosssectional study. we used a convenience sample of noncritically ill, english speaking adult patients presenting with non-psychiatric complaints to an urban academic ed over 6 months in 2011. we oversampled patients presenting with ‡3 visits over the previous 364 days. subjects were surveyed about their demographic and other health and health care characteristics and were screened with the phq 9, a nine-item questionnaire that is a validated, reliable predictor of mdd. we conducted bivariate (chi-square) and multivariate analysis controlling for demographic characteristics using sta-ta v. 10.0. our principal dependent variable of interest was a positive depression screen (phq 9 score ‡10). our principal independent variable of interest was ‡3 visits over the previous 364 days. results: our response rate was 90.7% with a final sample size of 1012. of our total sample, 313 (30.9%) had three or greater visits within the prior 364 days. one hundred (32%) frequent visitors had a positive phq 9 mdd screen as compared to 142 (20.3%) of subjects with fewer than three visits (p < 0.0001). in our multivariate analysis, the odds for having three or more visits for subjects who had a positive depression screen was 1.42 (1.03, 1.97). of subjects with three or more visits with a positive depression screen, only 116 (37%) were actively being treated for mdd at the time of their visit. conclusion: our study found a high prevalence of untreated depression among frequent users of the ed. eds should consider routinely screening patients who are frequent consumers for mdd. in addition, further studies should evaluate the effect of early treatment and follow up for mdd on overall utilization of ed services. access to psychiatric care among patients with depression presenting to the emergency department janice c. blanchard, benjamin l. bregman, dana rosenfarb, qasem al jabr, eun kim george washington university, washington, dc background: literature suggests that there is a high rate of major depressive disorder (mdd) in emergency department (ed) users. however, access to outpatient mental health services is often limited due to lack of providers. as a result, many persons with mdd who are not in active treatment may be more likely to utilize the ed as compared to those who are currently undergoing outpatient treatment. objectives: our study evaluated utilization rates and demographic characteristics associated with patients with a prior diagnosis of mdd not in active treatment. we hypothesized that patients who present to the ed with untreated mdd will have more frequent ed visits. methods: this was a single center, prospective, crosssectional study. we used a convenience sample of noncritically ill, english speaking adult patients presenting with non-psychiatric complaints to an urban academic ed over 6 months in 2011. subjects were surveyed about their demographic and other health and health care characteristics and were screened with the phq 9, a nine-item questionnaire that is a validated, reliable predictor of mdd. we conducted bivariate (chi-square) and multivariate analysis controlling for demographic characteristics using stata v. 10.0. our principal dependent variable of interest was a positive depression screen (phq 9 ‡ 10). our analysis focused on the subset of patients with a prior diagnosis of mdd with a positive screen for mdd during their ed visit. results: our response rate was 90.7% with a final sample size of 1012. 243 (24.0%) patients screened positive for mdd with a phq 9 score ‡10. of the 243 patients with a positive depression screen, 55.1% reported a prior history of treatment for mdd (n = 134). of these patients, only 57.6% were currently actively receiving treatment. hispanics who screened positive for depression with a history of mdd were less likely to actively be undergoing treatment as compared to non-hispanics (22.2% versus 46.9%, p = 0.041). patients with incomes less than $20,000 were more likely to actively be receiving treatment as opposed to higher incomes (76.3% versus 42.7% p = 0.003). conclusion: patients presenting to our ed with untreated mdd are more likely to be hispanic and less likely to be low income. the emergency department may offer opportunities to provide antidepressant treatment for patients who screen positive for depression but who are not currently receiving treatment. evaluation of a two-question screening tool (phq-2) for detecting depression in emergency department patients jeffrey p. smith, benjamin bregman, janice blanchard, nasser hashim, mary pat mckay george washington university, washington, dc background: the literature suggests there is a high rate of undiagnosed depression in ed patients and that early intervention can reduce overall morbidity and health care costs. there are several well validated screening tools for depression including the nine-item patient health questionnaire (phq-9). a tool using a two-question subset, the phq-2, has been shown to be an easily administered, reasonably sensitive screening tool for depression in primary care settings. objectives: to determine the sensitivity and specificity of the phq-2 in detecting major depressive disorders (mdd) among adult ed patients presenting to an urban teaching hospital. we hypothesize that the phq-2 is a rapid, effective screening tool for depression in a general ed population. methods: cross sectional survey of a convenience sample of 1012 adult, non-critically ill, english speaking patients with medical and not psychiatric complaints presenting to the ed between 9am and 11pm weekdays. patients were screened for mdd with the phq-9. we used spss v19.0 to analyze the specificity, sensitivity, positive predictive value (ppv), negative predictive value (npv), and kappa of phq-2 scores of 2 and 3 (out of possible total score of 6) compared to a validated cut-off score of 10 or higher of 27 points on the phq-9. the two questions on the phq-2 are: ''over the last two weeks, how often have you had little interest in doing things? how often have you felt down, depressed or hopeless?'' responses are scored from 0-3 based on ''never'',''several days'', ''more than half'', ''nearly every day''. results: 1012 subjects of 1116 approached agreed to participate (90.7% response rate), and 975 (96.3%) completed the phq-9. the phq-9 identified 225 (23.1%) subjects with mdd. table 1 outlines the percent of subjects who were positive and the sensitivity, specificity, positive, and negative predictive values and kappa for each cut-off on the phq-2. conclusion: the phq-2 is a sensitive and specific screening tool for mdd in the ed setting. moreover, the phq-2 is closely correlated with the phq-9, especially if a score of 3 or greater is used. given the simplicity and ease of using a two-item questionnaire and the high rates of undiagnosed depression in the ed, including this brief, self-administered screening tool to ed patients may allow for early awareness of possible mdd and appropriate evaluation and referral. patients. however, much of this self-harm behavior is not discovered clinically and very little is known about the prevalence and predictors of current ed screening practices. attention to this issue is increasing due to the joint commission's patient safety goal 15, which focuses on identification of suicide risk in patients. objectives: to describe the prevalence and predictors of screening for self-harm and of presence of current self-harm in eds. methods: data were obtained from the nimh-funded emergency department safety assessment and followup evaluation (ed-safe). eight u.s. eds reviewed charts in real time for 35-40 hours a week between 8/ 2010 and 11/2011. all patients presenting during enrollment shifts were characterized as to whether a selfharm screening had been performed by ed clinicians. a subset of patients with a positive screening was asked about the presence of self-harm ideation, attempts, or both by trained research staff. we used multivariable logistic regression to identify predictors of screening and of current self-harm. data were clustered by site. in each model we examined day and time of presentation, age < 65 years, sex, race, and ethnicity. results: of the 92,154 patients presenting during research shift, 24,240 (26%) were screened for self-harm. screening rates varied among sites and ranged from 4% to 32%, with one outlier at 93%. of those screened, 2,471 (10%) had current self-harm. among those with selfharm approached by study personnel (n = 1,037), 916 (88%) had thoughts of self-harm (suicidal or non-suicidal), 806 (78%) had thoughts of suicide, 444 (43%) had self-harm behavior, and 316 (31%) had suicide attempt(s) over the preceding week. predictors of being screened were: age < 65 years, male sex, weekend presentation, and night shift presentation (table) . among those screened, predictors of current self-harm were: age < 65 years, white race, and night shift presentation. conclusion: screening for self-harm is uncommon in ed settings, though practices vary dramatically by site. patients presenting at night and on weekends are more likely to be screened, as are those under age 65 and males. current self-harm is more common among those presenting on night shift, those under age 65, and whites. results: there were 1328 out-of-hospital records reviewed, and hospital discharge data were available in 1120 non-cardiac arrest patients. of the 1120 patients, 1084 (96.8%) patients survived to hospital discharge and 36 (3.2%) died during hospitalization. the mean age of those transported was 54 years (sd20), 612 (55%) were male, 128 (11%) were trauma-related, and 112 (10%) were admitted to the icu. average systolic blood pressure (sbp), pulse (p), respiratory rate (rr), oxygen saturation (o 2 sat), and end-tidal carbon dioxide (etco 2 ) were sbp = 141 (sd29), p = 95 (sd25), rr = 24 (sd9), o 2 sat = 95% (sd8), and etco 2 = 34 (sd10 conclusion: of all the initial vital signs recorded in the out-of-hospital setting, etco 2 was the most predictive of mortality. these findings suggest that pre-hospital etco 2 is a useful clinical tool for determining severity of illness and appropriate triage. background: the prehospital use of continuous positive airway pressure (cpap) ventilation is a relatively new management for acute cardiogenic pulmonary edema (acpe) and there is little high quality evidence on the benefits or potential dangers in this setting. objectives: the aim of this study was to determine whether patients in severe respiratory distress treated with cpap in the prehospital setting have a lower mortality than those treated with usual care. methods: randomized, controlled trial comparing usual care versus cpap (whisperflowò) in a prehospital setting, for adults experiencing severe respiratory distress, with falling respiratory efforts, due to a presumed acpe. patients were randomised to receive either usual care, including conventional medications (nitrates, furosemide, and oxygen) plus bag-valve-mask ventilation, versus conventional medications plus cpap. the primary outcome was prehospital or in-hospital mortality. secondary outcomes were need for tracheal intubation, length of hospital stay, change in vital signs, and arterial blood gas results. we calculated relative risk with 95% cis. results: fifty patients were enrolled with mean age 79ae8 (sd 11ae9), male 56ae0%, mortality 20ae0%. the risk of death was significantly reduced in the cpap arm with mortality 34ae6% (9 deaths) in the usual care arm compared to 4ae2% (1 death) in the cpap arm (rr, 0ae12; 95% ci 0ae02 to 0ae88; p = 0ae04). patients who received cpap were significantly less likely to have respiratory acidosis (mean difference in ph 0ae09; 95% ci 0ae01 to 0ae16; p = 0ae02; n = 24) than patients receiving usual care. the length of hospital stay was significantly less in the patients who received cpap (mean difference 2ae3 days; 95% ci )0ae01 to 4ae6, p = 0ae05). conclusion: we found that cpap significantly reduced mortality, respiratory acidosis, and length of hospital stay for patients in severe respiratory distress caused by acpe. this study shows the use of cpap for acpe improves patient outcomes in the prehospital setting. (originally submitted as a ''late-breaker.'') trial reg. anzctr actrn12609000410257; funding fisher and paykal suppliers of the whisperflowò cpap device. background: because emergency service utilization continues to climb, validated methods to safely identify and triage low-acuity patients to either alternate care destinations or a complaint-appropriate level of ems response is of keen interest to ems systems and potentially payers. though the literature generally supports the medical priority dispatch system (mpds) as a tool to predict low-acuity patients by various standards, correlation with initial patient physiologic data and patient age is novel. objectives: to determine whether the six mpds priority determinants for protocol 26 (sick person) can be used to predict initial ems patient acuity assessment or severity of an aggregate physiologic score. our longterm goal is to determine whether mpds priority can be used to predict patient acuity and potentially send only a first responder to do an in-person assessment to confirm this acuity, while reserving als transport resources for higher acuity patients. methods: calls dispatched through the wichita-sedgwick county 9-1-1 center between july 20, 2009 and october 1, 2011 using mpds protocol 26 (sick person) were linked to the ems patient care record for all patients 14 and older. the six mpds priority determinants were evaluated for correlation with initial ems acuity code, initial vital signs, rapid acute physiology score (raps), or patient age. the ems acuity code scores patients from low to severe acuity, based on initial ems assessment. results: there were 9370 calls dispatched using protocol 26 for those 14 years of age and older during the period, representing approximately 13% of all ems calls. there is a significant difference in the first encounter vital signs among different mpds priority levels. based on the logistic regression model, the mpds priority code alone had a sensitivity of 68% and specificity of 55% for identifying low-acuity patients with ems acuity score as the standard. the area under the curve (auc) for roc is 0.62 for mpds priority codes alone, while addition of age increases this value to 0.69. if we use the raps score as the standard to the mpds priority code, auc is 0.528. if we include both mpds and age in the model, the auc is 0.533. conclusion: in our system, mpds priority codes on protocol 26 (sick person) alone, or with age or raps score, are not useful either as predictors of patient acuity on ems arrival or to reconfigure system response or patient destination protocols. alternate ambulance destination program c. nee-kofi mould-millman 1 , tim mcmahan 2 , michael colman 2 , leon h. haley 1 , arthur h. yancey 1 1 emory university, atlanta, ga; 2 grady ems, atlanta, ga background: low-acuity patients calling 9-1-1 are known to utilize a large proportion of ems and ed resources. the national association of ems physicians and acep jointly support ems alternate destination programs (adps) in which low-acuity patients are allocated alternative resources non-emergently. analysis of one year's adp data from our ems system revealed that only 4.5% of eligible patients were transported to alternate destinations (ambulatory clinics). reasons for this low success rate need investigation. objectives: to survey emts and discover the most frequent reasons given by them for transportation of eligible patients to eds instead of to clinics. methods: this study was conducted within a large, urban, hospital-based ems system. upon conducting an adp for 12 months, a paper-based survey was created and pre-tested. all medics with any adp-eligible patient contact were included. emts were asked about personal, patient, and system related factors contributing to ed transport during the last 3 months of the adp. qualitative data were coded, collated, and descriptively reported. results: sixty-three respondents (26 emt-intermediates and 37 emt-paramedics) completed the survey, representing 79% of eligible emts. thirty-one emts (49%) responded that they did not attempt to recruit eligible patients into the adp in the last 3 program months. of those emts, 25 (81%) attributed their motive to multiple, prior, failed recruitment attempts. the 32 emts who actively recruited adp patients were asked reasons given by patients for clinic transport refusals: 19 (60%) cited that patients reported no prior experience of care at the participating clinics, and 23 (72%) reported patients had a strong preference for care in an ed. regarding system-related factors contributing to non-clinic transport, 24 of the 32 emts (75%) reported that clinic-consenting patients were denied clinic visits, mostly because of non-availability of same-day clinic appointments. conclusion: respondents indicated that poor emt enrollment of eligible patients, lack of available clinic time slots, and patient preference for ed care were among the most frequent reasons contributing to the low success rate of the adp. this information can be used to enhance the success of this, and potentially other adp programs, through modifications to adp operations and improved patient education. the effect of a standardized offline pain treatment protocol in the prehospital setting on pediatric pain treatment brent kaziny 1 , maija holsti 1 , nanette dudley 1 , peter taillac 1 , hsin-yi weng 1 , kathleen adelgais 2 1 university of utah, school of medicine, salt lake city, ut; 2 university of colorado, school of medicine, aurora, co background: pain is often under treated in children. barriers include need for iv access, fear of delayed transport, and possible complications. protocols to treat pain in the prehospital setting improve rates of pain treatment in adults. the utah ems for children (emsc) program developed offline pediatric protocol guidelines for ems providers, including one protocol that allows intranasal analgesia delivery to children in the prehospital setting. objectives: to compare the proportion of pediatric patients receiving analgesia for orthopedic injury by prehospital providers before and after implementation of an offline pediatric pain treatment protocol. methods: we conducted a retrospective study of patients entered into the utah prehospital on-line active reporting information system (polaris, a database of statewide ems cases) both before and after initiation of the pain protocol. patients were included if they were age 3-17 years, with a gcs of 14-15, an isolated extremity injury, and were transported by an ems agency that had adopted the protocol. pain treatment was compared for 2 years before and 18 months after protocol implementation with a wash-out period of 12 months for agency training. the difference in treatment proportions between the two groups was analyzed and 95% cis were calculated. results: during the two study periods, 1155 patients met inclusion criteria. patient demographics are outlined in the table. 93/501 (18.6%) patients were treated for pain before compared to 174/654 (26.6%) patients treated after the pain protocol was implemented; a difference of 8.0% (95% ci: 3.2%-12.8%). patients were more likely to receive pain medication if they had a pain score documented (or: 1.16; 95% ci: 1.09-1.22) and if they were treated after the implementation of a pain protocol (or: 1.27; 95% ci: 1.00-1.62). factors not associated with the treatment of pain include age, sex, and mechanism of injury. conclusion: the creation and adoption of statewide emsc pediatric offline protocol guideline for pain management is associated with a significant increase in use of analgesia for pediatric patients in the prehospital setting. background: evidence-based guidelines are needed to determine the appropriate use of air medical transport, as few criteria currently used predict the need for air transport to a trauma center. we previously developed a clinical decision rule (cdr) to predict mortality in injured, helicopter-transported patients. objectives: this study is a prospective validation of the cdr in a new population. methods: a prospective, observational cohort analysis of injured patients ( ‡16 y.o.) transported by helicopter from the scene to one of two level i trauma centers. variables analyzed included patient demographics, diagnoses, and clinical outcomes (in-hospital mortality, emergent surgery w/in 24 hrs, blood transfusion w/in 24 hrs, icu admit greater than 24 hrs, combined outcome of all). prehospital variables were prospectively obtained from air medical providers at the time of transport and included past medical history, mechanism of injury, and clinical factors. descriptive statistics compared those with and without the outcomes of interest. the previous cdr (age ‡ 45, gcs £ 13, sbp < 90, flail chest) was prospectively applied to the new population to determine its accuracy and discriminatory ability. results: 416 patients were transported from october 2010-august 2011. the majority of patients were male (59%), white (79%), with an injury occurring in a rural location (60%). most injuries were blunt (95%) with a median iss of 9. overall mortality was 5%. the most common reasons for air transport were: mvc with high risk mechanism (17%), gcs £ 13 (16%), loc >5 minutes (16%), and mvc >20 mph (14%). of these, only gcs £ 13 was significantly associated with any of the clinical outcomes. when applying the cdr, the model had a sensitivity of 100% (81.2%-100%), a specificity of 51.2% (50.6%-51.6%), a npv of 100% (98.1%-100%), and a ppv of 9.9% (8.0%-9.9%) for mortality. the area under the curve for this model was 0.92, suggesting excellent discriminatory ability. conclusion: the air transport decision rule in this study performed with high sensitivity and acceptable specificity in this validation cohort. further external validation in other systems and with ground transported patients are needed in order to improve decision making for the use of helicopter transport of injured patients. background: acute non-variceal upper gastrointestinal (gi) bleeding is a common indication for hospital admission. to appropriately risk-stratify such patients, endoscopy is recommended within 24 hours. given the possibility to safely manage patients as outpatients after endoscopy, risk stratification as part of an emergency department (ed) observation unit (ou) protocol is proposed. objectives: our objective was to determine the ability of an ou upper gi bleeding protocol to identify a lowrisk population, and to expeditiously obtain endoscopy and disposition patients. we also identified rates of outcomes including changes in hemoglobin, abnormal endoscopy findings, admission, and revisits. background: acute uncomplicated pyelonephritis (pyelo) requires no imaging but a ct flank pain protocol (ctfpp) may be ordered to determine if patients with pyelo and flank pain also have an obstructing stone. the prevalence of kidney stone and the characteristics predictive of kidney stone in pyelo patients is unknown. objectives: to determine elements on presentation that predict ureteral stone, as well as prevalence of stone and interventions in patients undergoing ct for pyelo. methods: retrospective study of patients at an academic ed who received a ctfpp scan between 8/05 and 4/09. 5497 ctfpps were identified and 1899 randomly selected for review. pyelo was defined as: positive urine dip for infection and >5 wbc/hpf on formal urinalysis in addition to flank pain/cva tenderness, chills, fever, nausea, or vomiting. patients were excluded for age < 18 y.o., renal disease, pregnancy, urological anomaly, or recent trauma. clinical data (178 elements) were gathered blinded to ct findings; ct results were abstracted separately and blinded to clinical elements. ct findings of hydronephrosis and hyrdroureter (hydro) were used as a proxy for hydro that could be determined by ultrasound prior to ct. patients were categorized into three groups: ureteral stone, no significant findings, and intervention or follow-up required. classification and regression tree analysis was used to determine which variables could identify ureteral stone in this population of pyelo patients. results: out of the 1899 patients, 105 (7.0%) met criteria for pyelo; subjects had a mean age of 39 ± 15.9 and 82% (n = 87) were female. ct revealed 31 (29%, 95% ci = 0.22-0.39) symptomatic stones, and 72 (68%, 95% ci = 0.59-0.76) exams with no significant findings. two patients needed intervention/ follow-up (1%, 95% ci = 0.0052-0.0667), one for perinephric hemorrhage and the other for pancreatitis. hydro was predictive for ureteral stone with an or = 18.4 (95% ci = 6.4-52, p < 0.0001). eleven (35%) ureteral stone patients were admitted and 9 (8%) of them had procedures. of these patients, 100% had ct signs of obstruction, 8 (88%) had hydronephrosis, and 1 (11%) had hydroureter. conclusion: hydronephrosis was predictive of ureteral stone and in-house procedures. prospective study is needed to determine whether ct scan is warranted in patients with pyelonephritis but without hydronephrosis or hydroureter. curative objectives: the specific aim of this analysis was to describe characteristics of patients presenting to the emergency department (ed) at their index diagnosis, and to determine whether emergency presentation precludes treatment with curative intent. methods: we performed a retrospective cohort analysis on a prospectively maintained institutional tumor registry to identify patients diagnosed with crc from 2008-2010. emrs were reviewed to identify which patients presented to the ed with acute symptoms of crc as the initial sign of their illness. the primary outcome variable was treatment plan (curative vs. palliative). secondary outcome variables included demographics, tumor type and location. descriptive statistics were conducted for major variables. chi-squre and fisher's exact tests were used to detect the association between categorical variables. two-sample t-test was used to identify the association between continuous and categorical variables. results: between jan 1 2008 and dec 31 2010, 376 patients were identified at our institution with crc. 214 (57%) were male and 162 (43%) were female, with mean age 60.6; sd: 13.3. thirty-three patients (8.8%) initially presented to the ed, of whom 5 (15.5%) received palliation. of 339 patients who initially presented elsewhere, 69 (20.5%) received palliation. acute ed presentation with crc symptoms did not preclude treatment with curative intent (p = 0.47). patients who presented emergently were more likely to be female (64% vs male 41%; p = 0.01) and older (65 vs. 60; p = 0.02). there was no statistically significant relationship between age, sex, tumor location, or type and treatment approach. conclusion: patients with crc may present to the ed with acute symptoms, which ultimately leads to the diagnosis. emergent presentation of crc does not preclude patients from receiving therapy with curative intent. cannabinoid (or 2.93, , and white blood cell (wbc) count ‡14,000/mm 3 (or 11.35, 95% ci 3.42-37.72). conclusion: age ‡65 years is not associated with need for admission from an ed observation unit. older adults can successfully be cared for in these units. initial temperature, respiratory rate, and pulse were not predictive of admission, but extremely elevated blood pressure was predictive. other relevant predictor variables included comorbidities and elevated wbc count. advanced age should not be a disqualifying criterion for disposition to an ed observation unit. older adult fallers in the emergency department luna ragsdale, cathleen colon-emeric duke university, durham, nc background: approximately 1/3 of community-dwelling older adults experience a fall each year, and 2.2 million are treated in u.s. emergency departments (ed) annually. the ed offers a potential location for identification of high-risk individuals and initiation of fall-prevention services that may decrease both fall rates and resource utilization. objectives: the goal of this study was to: 1) validate an approach to identifying older adults presenting with falls to the ed using administrative data; and 2) characterize the older adult who falls and presents to the ed and determine the rate of repeat ed visits, both fall-related and all visits, after an index fall-related visit. methods: we identified all older adults presenting to either of the two hospitals serving durham county residents during a six month period. manual chart review was completed for all encounters with icd9 codes that may be fall-related. charts were reviewed 12 months prior and 12 months post index visit. descriptive statistics were used to describe the cohort. results: a total of 4452 older adults were evaluated in the ed during this time period; 1714 (55.7%) had an icd9 code for a potentially fall-related injury. of these, record review identified 534 (12%) with a fall from standing height or less. of the fallers, 65.9% of the patients were discharged, 31% were admitted, and 3% were admitted under observation. of those who fell, 38.2% had an ed visit within the previous year. approximately 1/3 (33.3%) of these were fall related. over half (53.4%) of the patients who fell returned to the ed within one year of their index visit. a large proportion (44.4%) of the return visits was fall-related. follow-up with a primary care provider or specialist was recommended in 46% of the patients who were discharged. overall mortality rate for fallers over the year following the index visit was 18%. conclusion: greater than fifty percent of fallers will return to the ed after an index fall, with a large proportion of the visits related to a fall. a large number of these fallers are discharged home with less than fifty percent having recommended follow-up. the ed represents an important location to identify high-risk older adults to prevent subsequent injuries and resource utilization. objectives: we studied whether falls from a standing position resulted in an increased risk for intracranial or cervical injury verses falling from a seated or lying position. methods: this is a prospective observational study of patients over the age of 65 who presented with a chief complaint of fall to a tertiary care teaching facility. patients were eligible for the study if they were over age 65, were considered to be at baseline mental status, and were not triaged to the trauma bay. at presentation, a questionnaire was filled out by the treating physician regarding mechanism and position of fall, with responses chosen from a closed list of possibilities. radiographic imaging was obtained at the discretion of the treating physician. charts of enrolled patients were subsequently reviewed to determine imaging results, repeat studies done, or recurrent visits. all patients were called in follow-up at 30 days to assess for delayed complications related to the fall. data were entered into a standardized collection sheet by trained abstractors. data were analyzed with fisher's exact test and descriptive statistics. this study was reviewed and approved by the institutional review board. results: two-hundred sixty two patients were enrolled during the study period. one-hundred ninety eight of these had fallen from standing and 64 fell from either sitting or lying positions. the mean age for patients was 84 (sd 7.9) for those who fell from standing and 84 (sd 8.4) for those who fell from sitting or lying. there were 6 patients with injuries who fell from standing: three with subdural hematomas, one with a cerebral contusion, one with an osteophyte fracture at c6, and one with an occipital condyle fracture with a chip fracture of c1. there were 2 patients with injuries who fell from a seated or lying position: one with a traumatic subarachnoid hemorrhage and one with a type ii dens fracture. the overall rate of traumatic intracranial or cervical injury in elders who fell was 3%. no patients required surgical intervention. there was no difference in rate of injury between elders who fell from standing versus those who fell from sitting or lying (p = 1). (table) . conclusion: both instruments identify the majority of patients as high-risk which will not be helpful in allocating scarce resources. neither the isar nor the trst can distinguish geriatric ed patients at high or low risk for 1or 3-month adverse outcomes. these prognostic instruments are not more accurate in dementia or lower literacy subsets. future instruments will need to incorporate different domains related to short-term adverse outcomes. background: for older adults, both inpatient and outpatient care involves not only the patient and physician, but often a family member or informal caregiver. they can assist in medical decision making and in performing the patient's activities of daily living. to date, multiple outpatient studies have examined the positive roles family members play during the physician visit. however, there is very limited information on the involvement of the caregiver in the ed and their relationship with the health outcomes of the patient. objectives: to assess whether the presence of a caregiver influences the overall satisfaction, disposition, and outpatient follow-up of elderly patients. we performed a three-step inquiry of patients over 65 years old who arrived to the upenn ed. patients and care partners were initially given a questionnaire to understand basic demographic data. at the end of the ed stay, patients were given a satisfaction survey and followed through 30 days to assess time to disposition, whether the patient was admitted or discharged, outpatient follow-up, and ed revisit rates. chi-square and t-tests were used to examine the strength of differences in the elderly patients' sociodemographics, self-rated health, receiving aid with their instrumental activities of daily living, and number of health problems by accompaniment status. multivariate regression models were constructed to examine whether the presence or absence of caregivers affected satisfaction, disposition, and follow-up. results: overall satisfaction was higher among patients who had caregivers (2.4 points), among patients who felt they were respected by their physician (3.8 points), and had lower lengths of stay (2 hours). patients with caregivers were also more likely to be discharged home (or 2.4) and to follow-up with their regular physician (or 2.1). there was no evidence to suggest caregivers affected the overall rates of revisits back to an ed. conclusion: for older adults, medical care involves not only the patient and physician, but often a family member or an informal care companion. these results demonstrate the positive influence of caregivers on the patients they accompany, and emergency physicians should define ways to engage these caregivers during their ed stay. this will also allow caregivers to participate when needed and can help to facilitate transitions across care settings. background: shared decision making has been shown to improve patient satisfaction and clinical outcomes for chronic disease management. given the presence of individual variations in the effectiveness and side effects of commonly used analgesics in older adults, shared decision making might also improve clinical outcomes in this setting. objectives: we sought to characterize shared decision making regarding the selection of an outpatient analgesic for older ed patients with acute musculoskeletal pain and to examine associations with outcomes. methods: we conducted a prospective observational study with consecutive enrollment of patients age 65 or older discharged from the ed following evaluation for moderate or severe musculoskeletal pain. two essential components of shared decision making, 1) information provided to the patient and 2) patient participation in the decision, were assessed via patient interview at one week using four-level likert scales. results: of 233 eligible patients, 110 were reached by phone and 87 completed the survey. only 25% (21/87) of patients reported receiving 'a lot' of information about the analgesic, and only 21% (18/87) reported participating 'a lot' in the selection of the analgesic. there were trends towards white patients (p = 0.06) and patients with higher educational attainment (p = 0.07) reporting more participation in the decision. after adjusting for sex, race, education, and initial pain severity, patients who reported receiving 'a lot' of information were more likely to report optimal satisfaction with the analgesic than those receiving less information (78% vs. 47%, p < 0.05). after the same adjustments, patients who reported participating 'a lot' in the decision were also more likely to report optimal satisfaction with the analgesic (82% vs. 47%, p < 0.05) and greater reductions in pain scores (mean reduction in pain 4.6 vs. 2.7, p < 0.05) at one week than those who participated less. background: quality of life (qol) measurements have become increasingly important in outcomes-based research and cost-utility analyses. dementia is a prevalent, often unrecognized, geriatric syndrome that may limit the accuracy of patient self-report in a subset of patients. the relationship between caregiver and geriatric patient qol in the emergency department (ed) is not well understood. objectives: to qualify the relationship between caregiver and geriatric patient qol ratings in ed patients with and without cognitive dysfunction. methods: this was a prospective, consecutive patient, cross-sectional study over two months at one urban academic medical center. trained research assistants screened for cognitive dysfunction using the short blessed test and evaluated health impairment using the quality of life-alzheimer's disease (qol-ad) test. when available in the ed, caregivers were asked to independently complete the qol-ad. consenting subjects were non-critically ill, english-speaking, community-dwelling adults over 65 years of age. responses were compared using wilcoxon signed ranks test to assess the relationships between patient and caregiver scores from the qol-ad stratified by normal or abnormal cognitive screening results. significance was defined by p < 0.05. results: patient qol ratings were obtained from 108 patient-caregiver pairs. patients were 51% female, 52% african-american, with a mean age of 76-years, and 58% had abnormal cognitive screening tests. compared with caregivers, cognitively normal patients had no significant qol assessment differences except for questions of energy level and overall mood. on the other hand, cognitively impaired patients differed significantly on questions of energy level and ability to perform household chores with a trend towards significant differences for living setting (p = 0.097) and financial situation (p = 0.057). in each category, the differences reflected a caregiver underestimation of quality compared with the patient's self-rating. conclusion: discrepancies between qol domains and total scores for patients with cognitive dysfunction and their caregivers highlights the importance of identifying cognitive dysfunction in ed-based outcomes research and cost-utility analyses. further research is needed to quantify the clinical importance of the patient-and caregiver-assessed quality of life. background: age is often a predictor for increased morbidity and mortality. however, it is unclear whether old age is a predictor of adverse outcome in syncope. objectives: to determine whether old age is an independent predictor of adverse outcome in patients presenting to the emergency department following a syncopal episode. methods: a prospective observational study was conducted from june 2003 to july 2006 enrolling consecutive adult ed patients (>18 years) presenting with syncope. syncope was defined as an episode of transient loss of consciousness. adverse outcome or critical intervention were defined as gastrointestinal bleeding or other hemorrhage, myocardial infarction/percutaneous coronary intervention, dysrhythmia, alteration in antidysrhythmics, pacemaker/defibrillator placement, sepsis, stroke, death, pulmonary embolus, or carotid stenosis. outcomes were identified by chart review and 30-day follow-up phone calls. results: of 575 patients who met inclusion criteria, an adverse event occurred in 24% of patients. overall, 35% of patients with risk factors had adverse outcomes compared to 1.6% of patients with no risk factors. in particular, 28/127 (22%; 95% ci 16-30%) of patients <65 with risk factors had adverse outcomes, while 85/196 (43%; 95% ci 36-50%) of the elderly with risk factors had adverse outcomes. in contrast, among young people 2/196 (1%; 95% ci 0.04-3.8%) of patients without risk factors had adverse outcomes while 2/56 (3.6%; 95% ci 0.28-13%) of patients ‡65 without risk factors had adverse outcomes. conclusion: although the elderly are at greater risk for adverse outcomes in syncope, age ‡ 65 or older alone does not appear to be a predictor of adverse outcome following a syncopal event. based on these data, it should be safe to discharge home from the ed patients with syncope, but without risk factors, regardless of age. (originally submitted as a ''late-breaker.'') antibiotics background: adherence to national guidelines for hiv and syphilis screening in eds is not routine. in our ed, hiv and syphilis screening rates among patients tested for gonorrhea and chlamydia (gc/ct) have been reported to be 45% and 30%, respectively. objectives: to determine the effect of a sexually transmitted infection (sti) laboratory order set on hiv and syphilis screening among ed patients tested for gc/ct. we hypothesized that a sti order set would increase screening rates by at least 30%. methods: a 6-month, quasi-experimental study in an urban ed comparing hiv and syphilis screening rates of gc/ct-tested patients before (control phase) and after the implementation of a sti laboratory order set (intervention phase). the order set linked blood-based rapid hiv and syphilis screening with gc/ct testing. consecutive patients completing gc/ct testing were included. the primary outcome was the absolute difference in hiv and syphilis screening rates among gc/ ct-tested patients between phases. we estimated that 550 subjects per phase were needed to provide 90% power (p-value of £0.05) to detect an absolute difference in screening rates of 10%, assuming a baseline hiv screening rate of 45%. results: the ed census was 42,461. characteristics of patients tested for gc/ct were similar between phases: the mean age was 33 years (sd = 12) and most were female (65%), black (49%), hispanic (30%), and unmarried (84% services have recommended the use of immunization programs against influenza disease within hospitals since the 1980s. the emergency department (ed) being the ''safety net'' for most non-insured people is an ideal setting to intervene and provide primary prevention from influenza. objectives: the purpose of this study is to assess whether a pharmacist-based influenza immunization program is feasible in the ed, and successful in increasing the percentage of adult patients receiving the influenza vaccine. methods: implementation of pharmacist-based immunization program was developed in coordination with ed physicians and nursing staff in 2010. the nursing staff, using an embedded electronic questionnaire within their triage activity, screened patients for eligibility for the influenza vaccine. the pharmacist using an electronic alert system within the electronic medical record identified patients who we deemed eligible and if agreed the pharmacist vaccinated the patient. patients who refused to be vaccinated were surveyed to ascertain their perception concerning immunization offered by a pharmacist in the ed. feasibility and safety data for vaccinating patient in the ed were recorded. results: 149 patients were approached and enrolled into the study. of the 149, 41% agreed to receive the influenza vaccine from a pharmacist in the ed. the median screening time was 5 minutes and median vaccination time was 3 minutes for a total of 8 minutes from screening time to vaccination time. 74% were willing to receive the influenza vaccine from a pharmacist, and 78% were willing to receive the vaccine in the ed. the main reason given for refusing to receive the influenza vaccine was ''patient does not feel at risk of getting the disease''; only 14.6% stated they were vaccinated recently. conclusion: a pharmacist-based influenza immunization program is feasible in the ed, and has the potential to successfully increase the percentage of adult patients receiving the vaccine. 1.4 ± 0.1, p < 0.05). ed visits by hiv-infected patients also had longer lengths of ed stay (317 ± 26.0 minutes vs. 222.5 ± 5.6 minutes, p < 0.05) and were more likely to be admitted (29% vs. 15%, p < 0.05), than their non-hiv infected counterparts. conclusion: although ed visits by hiv-infected individuals in the u.s. are relatively infrequent, they occur at rates higher than the general population, and consume significantly more ed resources than the general population. the background: the influence of wound age on the risk of infection in simple lacerations repaired in the emergency department (ed) has not been well studied. it has traditionally been taught that there is a ''golden period'' beyond which lacerations are at higher risk of infection and therefore should not be closed primarily. the proposed cutoff for this golden period has been highly variable (3-24 hours in surgical textbooks). objectives: to answer the following research question: are wounds closed via primary repair after the golden period at increased risk for infection? methods: we searched medline, embase, and other databases as well as bibliographies of relevant articles. we included studies that enrolled ed patients with lacerations repaired by primary closure. exclusion: 1. intentional delayed primary repair or secondary closure, 2. wounds requiring intra-operative repair, skin graft, drains, or extensive debridement, and 3. grossly contaminated or infected at presentation. we compared the outcome of wound infection in two groups of early versus delayed presentations (based on the cut-offs selected by the original articles). we used ''grading of recommendations assessment, development and evaluation'' (grade) criteria to assess the quality of the included trials. frequencies are presented as percentages with 95% confidence intervals. relative risk (rr) of infection is reported when clinically significant. results: 418 studies were identified. four trials enrolling 3724 patients in aggregate met our inclusion/exclusion criteria. two studies used a 6-hour cut-off and the other two used a 12-hour cut-off for defining delayed wounds. the overall quality of evidence was low. the infection rate in the wounds that presented with delay ranged from 1.4% to 32%. one study with the smallest sample size (morgan et al), which only enrolled lacerations to the hand and forearm, showed higher rates of infection in patients with delayed wounds (table). the infection rates in delayed wound groups in the remaining three studies were not significantly different from the early wounds. conclusion: the evidence does not support the existence of a golden period, nor does it support the role of wound age on infection rate in simple lacerations. background: although clinical studies in children have shown that temperature elevation is an independent and significant predictor of bacteremia in children, the relationship in adults is largely unknown or equivocal. objectives: review the incidence of positive blood cultures on critically ill adult septic patients presenting to an emergency department (ed) and determine the association of initial temperature with bacteremia. methods: july 2008 to july 2010 retrospective chart review on all patients admitted from the ed to an urban community hospital with sepsis and subsequently expiring within 4 days of admission. fever was defined as a temperature ‡38°c. sirs criteria were defined as: 1) temperature ‡38°c or £36°c, 2) heart rate ‡90 beats/ minute, 3) respiratory rate ‡20 or mechanical ventilation, 4) wbc ‡ 12,000/mm 3 or <4,000 or bands ‡10%. objectives: we examined the utility of limited genetic sequencing of bacterial isolates using multilocus sequence typing (mlst) to discriminate between known pathogenic blood culture isolates of s. epidermidis and isolates recovered from skin. methods: ten blood culture isolates from patients meeting the centers for disease control and prevention (cdc) criteria for clinically significant s. epidermidis bacteremia and ten isolates from the skin of healthy volunteers were studied. mlst was performed by sequencing 400 bp regions of seven genes (arc, aroe, gtr, muts, pyr, tpia, and yqil) . genetic variability at these sites was compared to an international database (www.sepidermidis.mlst.net) and each strain was then categorized into a genotype on the basis of known genetic variation. the ability of the gene sequences to correctly classify strains was quantified using the support vector machine function in the statistical package r. 1,000 bootstrap resamples were performed to generate confidence bounds around the accuracy estimates. results: between-strain variability was considerable, with yqil being most variable (6 alleles) and tpia being least (1 allele). the muts gene, responsible for dna repair in s. epidermidis, showed almost complete separation between pathogenic and commensal strains. when the seven genes were used in a joint model, they correctly predicted bacterial strain type with 90% accuracy (iqr 85, 95%). conclusion: multilocus sequence typing shows excellent early promise as a means of distinguishing contaminant versus truly pathogenic isolates of s. epidermidis from clinical samples. near-term future goals will involve developing more rapid means of sequencing and enrolling a larger cohort to verify assay performance. conference are presented by influenza scenario in table 1 and background: antiviral medications are recommended for patients with influenza who are hospitalized or at high risk for complications. however, timely diagnosis of influenza in the ed remains challenging. influenza rapid antigen tests have short turn-around times, making them potentially useful in the ed setting, but their sensitivities may be too low to assist with treatment decisions. objectives: to evaluate the test characteristics of the binaxnow influenza a&b rapid antigen test (rat) in ed patients. methods: we prospectively enrolled a systematic sample of patients of all ages presenting to two eds with acute respiratory symptoms or fever during three consecutive influenza seasons (2008) (2009) (2010) (2011) . research personnel collected nasal and throat swabs, which were combined and tested for influenza with rt-pcr using cdc-provided primers and probes. ed clinicians independently decided whether to obtain a rat during clinical care. rats were performed in the clinical laboratory using the binaxnow influenza a&b test on nasal swabs collected by ed staff. the study cohort included subjects who underwent both a research pcr and clinical rat. rat test characteristics were evaluated using pcr as the criterion standard with stratified sub-analyses for age group and influenza subtype (pandemic h1n1 (ph1n1), non-pandemic influenza a, influenza b). results: 561 subjects were enrolled; 131 subjects were pcr positive for influenza (76 ph1n1, 20 non-pandemic influenza a, and 35 influenza b). for all age groups, rat sensitivities were low and specificities were high ( hiv infection with cd4 < 200; and among nursing home residents, inability to independently perform activities of daily living. sources for bacterial cultures included blood, sputum (adults only), bronchoalveolar lavage (bal), tracheal aspirate, and pleural fluid. only sputum specimens with a bartlett score ‡1+ were considered adequate for culturing. results: among 461 children enrolled, 7 (2%) had s. aureus cultured from ‡1 specimen, including 5 with methicillin-resistant s. aureus (mrsa) and 2 with methicillin-susceptible s. aureus (mssa). specimens positive for s. aureus included 3 pleural fluid, 2 blood, 2 tracheal aspirates, and 1 bal. two children with s. aureus had evidence of co-infection: 1 influenza a, and 1 streptococcus pneumoniae. among 673 adults enrolled, 17 (3%) grew s. aureus from ‡1 specimen, including 9 with mrsa and 8 with mssa. specimens positive for s. aureus included 5 blood, 11 sputum, and 3 bal. five adults with s. aureus had evidence of co-infections: 2 coronavirus, 1 respiratory syncytial virus, 1 s. pneumoniae, and 1 pseudomonas aeruginosa. presenting clinical characteristics and outcomes of subjects with staphylococcal cap are summarized in tables 1-2. conclusion: these preliminary findings suggest s. aureus is an uncommon cause of cap. although the small number of staphylococcal cases limits conclusions that can be drawn, in our analysis staphylococcal cap appears to be associated with co-infections, pleural effusions, and severe disease. future work will focus on continued enrollment and developing clinical prediction models to aid in diagnosing staphylococcal cap in the ed. background: emergency care has been a neglected public health challenge in sub-saharan africa. the goal of global emergency care collaborative (gecc) is to develop a sustainable model for emergency care delivery in low-resource settings. gecc is developing a training program for emergency care practitioners (ecps). objectives: to analyze the first 500 patient visits at karoli lwanga ''nyakibale'' hospital ed in rural uganda to determine the knowledge and skills needed in training ecps. methods: a descriptive cross-sectional analysis of the first 500 consecutive patient visits in the ed's patient care log was reviewed by an unblinded abstractor. data on demographics, procedures, laboratory testing, bedside ultrasounds (us) performed, radiographs (xrs) ordered, and diagnoses were collated. all authors discussed uncertainties and formed a consensus. descriptive statistics were performed. results: of the first 500 patient visits, procedures were performed in 367 (73.4%) patients, including 244 (48.8%) who had ivs placed, 47 (9.4%) who received wound care, and 42 (8.4%) who received sutures. complex procedures, such as procedural sedations, lumbar punctures, orthopedic reductions, nerve blocks, and tube thoracostomies, occurred in 49 (9.8%) patients. laboratory testing, xrs, and uss were performed in 188,(37.6%), 99 (19.8%), and 45 (7%) patients, respectively. infectious diseases were diagnosed in 217 (43.4%) patients; 78 (15.6 %) with malaria and 57 (11.4%) with pneumonia. traumatic injuries were present in 140 (28%) patients; 77 (15.4%) needing wound care and 31 (6.2%) with fractures. gastrointestinal and neurological diagnoses affected 58 (11.6%) and 27 (5.4%) patients, respectively. conclusion: ecps providing emergency care in sub-saharan africa will be required to treat a wide variety of patient complaints and effectively use laboratory testing, xrs, and uss. this demands training in a broad range of clinical, diagnostic, and procedural skills, specifically in infectious disease and trauma, the two most prevalent conditions seen in this rural sub-saharan africa ed. assessment of point-of-care ultrasound in tanzania background: current chinese ems is faced with many challenges due to a lack of systematic planning, national standards in training, and standardized protocols for prehospital patient evaluation and management. objectives: to estimate the frequency with which prehospital care providers perform critical actions for selected chief complaints in a county-level ems system in hunan province, china. methods: in collaboration with xiangya hospital (xyh), central south university in hunan, china, we collected data pertaining to prehospital evaluation of patients on ems dispatches from a ''1-2-0'' call center over a 2-month period. this call center services an area of just under 5000 km 2 with a total population of 1.36 million. each ems team consists of a driver, a nurse, and a physician. this was a cross-sectional study where a single trained observer accompanied ems teams on transports of patients with a chief complaint of chest pain, dyspnea, trauma, or altered mental status. in this convenience sample, data were collected daily between 8 am and 6 pm. critical actions were pre-determined by a panel of emergency medicine faculty from xyh and the university of maryland school of medicine. simple statistical analysis was performed to determine the frequency of critical actions performed by ems providers. results: during the study period, 1170 patients were transported, 452 of whom met the inclusion criteria. 218 (48.2%) evaluations were observed directly for critical actions. the table shows the frequency of critical actions performed by chief complaint. none of the patients with chest pain received an ecg even though the equipment was available. rapid glucose was checked in only 2.1% of patients presenting with altered mental status. a lung exam was performed in 22.7% of patients with dyspnea, and the respiratory rate was measured in 9.1%. among patients transported for trauma, blood pressure, and heart rate were only measured in 1% and 4.1%, respectively. conclusion: in this observation study of prehospital patient assessments in a county-level ems system, critical actions were performed infrequently for the chief complaints of interest. performance frequencies for critical actions ranged from 0 to 22.7%, depending on the chief complaint. standardized prehospital patient care protocols should be established in china and further training is needed to optimize patient assessment. trends little is known about the comparative effectiveness of noninvasive ventilation (niv) versus invasive mechanical ventilation (imv) in chronic obstructive pulmonary disease (copd) patients with acute respiratory failure. objectives: to characterize the use of niv and imv in copd patients presenting to the emergency department (ed) with acute respiratory failure and to compare the effectiveness of niv vs. imv. methods: we analyzed the 2006-2008 nationwide emergency department sample (neds), the largest, all-payer, us ed and inpatient database. ed visits for copd with acute respiratory failure were identified with a combination of copd exacerbation and respiratory failure icd-9-cm codes. patients were divided into three treatment groups: niv use, imv use, and combined use of niv and imv. the outcome measures were inpatient mortality, hospital length of stay (los), hospital charges, and complications. propensity score analysis was performed using 42 patient and hospital characteristics and selected interaction terms. results: there were an estimated 101,000 visits annually for copd exacerbation and respiratory failure from approximately 4,700 eds. ninety-six percent were admitted to the hospital. of these, niv use increased slightly from 14% in 2006 to 16% in 2008 (p = 0.049), while imv use decreased from 28% in 2006 to 19% in 2008 (p < 0.001); the combined use remained stable (4%). inpatient mortality decreased from 10% in 2006 to 7% in 2008 (p < 0.001). niv use varied widely between hospitals, ranging from 0% to 100% with median of 11%. in a propensity score analysis, niv use (compared to imv) significantly reduced inpatient mortality (risk ratio 0.57; 95% confidence interval [ci] 0.48-0.56), shortened hospital los (difference )3 days; 95%ci )4 to )3), and reduced hospital charges 044; 855) . niv use was associated with a lower rate of iatrogenic pneumothorax compared with imv use (0.04% vs. 0.6%, p < 0.001). an instrumental analysis confirmed the benefits of niv use, with a 5% reduction in inpatient mortality in the niv-preferring hospitals. conclusion: niv use is increasing in us hospitals for copd with acute respiratory failure; however, its adoption remains low and varies widely between hospitals. niv appears to be more effective and safer than imv in the real-world setting. background: dyspnea is a common ed complaint with a broad differential diagnosis and disease-specific treatment. bronchospasm alters capnographic waveforms, but the effect of other causes of dyspnea on waveform morphology is unclear. objectives: we evaluated the utility of capnographic waveforms in distinguishing dyspnea caused by reactive airway disease (rad) from non-rad in adult ed patients. methods: this was a prospective, observational, pilot study of a convenience sample of adult patients presenting to the ed with dyspnea. waveforms, demographics, past medical history, and visit data were collected. waveforms were independently interpreted by two blinded reviewers. when the interpreters disagreed, the waveform was re-reviewed by both reviewers and an agreement was reached. treating physician diagnosis was considered the criterion standard. descriptive statistics were used to characterize the study population. diagnostic test characteristics and inter-rater reliability are given. results: fifty subjects were enrolled. median age was 52 years (range 21-82), 50% were female, 34% were caucasian. 29/50 (58%) had a history of asthma or chronic obstructive pulmonary disease. rad was diagnosed by the treating physician in 19/50 (38%) and 32/50 (64%) had received treatment for dyspnea prior to waveform acquisition. the interpreters agreed on waveform analysis in 47/50 (94%) cases (kappa = 0.88). test characteristics for presence of acute rad, including 95%ci, were: overall accuracy 70% (55.2%-81.7%), sensitivity 69% (43.5%-86.4%), specificity 71% (51.8%-85.1%), positive predictive value 59% (36.7%-78.5%), negative predictive value 79% (58.5%-91.0%), positive likelihood ratio 2.25 (1.36-3.72) , negative likelihood ratio 0.42 (0.23-0.74). conclusion: inter-rater agreement is high for capnographic waveform interpretation, and shows promise for helping to distinguish between dyspnea caused by rad and dyspnea from other causes in the ed. treatments received prior to waveform acquisition may affect agreement between waveform interpretation and physician diagnosis, affecting the observed test characteristics. asthma background: asthma and chronic obstructive pulmonary disease (copd) patients who present to the emergency department (ed) usually lack adequate ambulatory disease control. while evidence-based care in the ed is now well defined, there is limited inform-ation regarding the pharmacologic or non-pharmacologic needs of these patients at discharge. objectives: this study evaluated patients' needs with regard to the ambulatory management of their respiratory conditions after ed treatment and discharge. methods: over 6 months, 94 adult patients with acute asthma or copd, presenting to a tertiary care alberta hospital ed and discharged after being treated for exacerbations, were enrolled. using results from standardized in-person questionnaires, charts were reviewed by respiratory researchers to identify care gaps. results: overall, 58 asthmatic and 36 copd patients were enrolled. more patients with asthma required education on spacer devices (52% vs 31%). few asthma (9%) and no copd patients had written action plans; asthma patients were more likely to need adherence counseling (53% vs 36%) for preventer medications. more patients with asthma required influenza vaccination (72% vs 39%; p = 0.003); pneumococcal immunization was low (36%) in copd patients. only 22% of asthmatics reported ever being referred to an asthma education program and 19% of the copd patients reported ever being referred to pulmonary rehabilitation. at ed presentation, 28% of the asthmatics required the addition of inhaled corticosteroids (ics) and 16% required the addition of ics/long acting beta-agonist (ics/laba) combination agents. on the other hand, 36% of copd patients required the addition of long-acting anticholinergics while most (83%) were receiving preventer medications. finally, 31% of copd and 29% of asthma patients who smoked required smoking cessation counseling. conclusion: overall, we identified various care gaps for patients presenting to the ed with asthma and copd. there is an urgent need for high-quality research on interventions to reduce these gaps. methods: this is an interim, sub-analysis of an interventional, double-blinded study performed in an academic urban-based adult ed. subjects with acute exacerbation of asthma with fev1 < 50% predicted within 30 minutes following initiation of ''standard care'' (including a minimum of 5 mg nebulized albuterol, 0.5 mg nebulized ipratropium, and 50 mg corticosteroid) who consented to be in a trial were included. all treatment was administered by emergency physicians unaware of the study objectives. patients were randomly assigned to treatment with placebo or an intravenous beta agonist. all subjects had fev1 and ds obtained at baseline, 1, 2, and 3 hours after treatment. fev1 was measured using a bedside nspire spirometer, and ds was calculated using a modified borg dyspnea score. results: thirty-eight patients were included for analysis. spearman's rho test (rho) was used to measure correlations between fev1 and ds at 1, 2, and 3 hours post study entry and subsequent hospitalization. rho is negative for fev1 (higher fev1 correlates to lower rate of hospitalization) and positive for ds (higher ds correlates to higher rate of hospitalization). at each time point, ds were more highly correlated to hospitalization than were fev1 (see table) . conclusion: dyspnea score at 1, 2, and 3 hours were significantly correlated with hospital admission, whereas fev1 was not. in this set of subjects with moderate to severe asthma exacerbations, a standardized subjective tool was superior to fev1 for predicting subsequent hospitalization. methods: this is an interim, subgroup analysis of a prospective, interventional, double-blind study performed in an academic urban ed. subjects who were consented for this trial presented with acute asthma exacerbations with fev1 £ 50% predicted within 30 minutes following initiation of ''standard care'' (includes a minimum of 2.5 mg nebulized albuterol, 0.5 mg nebulized ipratropium, and 50 mg of a corticosteroid). ed physicians who were unaware of the study objectives administered all treatments. subjects were randomized in a 1:1 ratio to either placebo or investigational intravenous beta agonist arms. blood was obtained at 1 and 1.25 hours after the start of the hour long infusion. blood was centrifuged and serum stored at )80°c, and then shipped on dry ice for albuterol and lactate measurements at a central lab. the treatment lactate and d lactate were correlated with 1 hr serum albuterol concentrations and hospital admission using partial pearson correlations to adjust for ds. results: 38 subjects were enrolled to date, 20 with complete data. the mean baseline serum lactate level was 18.1 mg/dl (sd ± 8.6). this increased to 32.7 mg/ dl (sd ± 15.0) at 1.25 hrs. the mean 1 hr ds was 3.85 (sd ± 2.0). the correlations between treatment lactate, d lactate, 1 hr serum albuterol concentrations (r, s and total) and admission to hospital are shown (see table) . both treatment and d lactate were highly conrrelated with total serum albuterol, r albuterol, and s albuterol. there was no correlation between treatment lactate or d lactate and hospital admission. conclusion: lactate and d lactate concentrations correlate with albuterol concentrations in patients presenting had asthma. fifty one percent were <21 years old and 54% were female. we found a decline of 27% (95% ci: 23%-30%, p < 0.0001; r 2 = 0.73, p < 0.0001) in the overall yearly asthma visits to total ed visits from 1996 to 2010. when we analyzed sex and age groups separately, we found no statistically significant changes for females or for males <21 years old (r 2 £ 0.016, p ‡ 0.65). for females and males >21 years old, yearly asthma visits to total ed visits from 1996 to 2010 decreased 39% (95% ci: 33%-43%, p < 0.0001; r 2 = 0.90, p < 0.0001) and 20% (95% ci: 14%-26%, p < 0.0001; r 2 = 0.80, p < 0.0001), respectively. conclusion: we found an overall decrease in yearly asthma visits to total ed visits from 1996 to 2010. we speculate that this decrease is due to greater corticosteroid use despite the increasing prevalence of asthma. it is unclear why this decrease was seen in adults and not in children and why it was greater for adult females than males. objectives: our objectives were to describe the use of a unique data collection system that leveraged emr technology and to compare its data entry error rate to traditional paper data collection. methods: this is a retrospective review of data collection methods during the first 12 months of a multicenter study of ed, anti-coagulated, head injury patients. on-shift ed physicians at five centers enrolled eligible patients and prospectively completed a data form. enrolling ed physicians had the option of completing a one-page paper data form or an electronic ''dotphrase'' (dp) data form. our hospital system uses an epicòbased emr. a feature of this system is the ability to use dps to assist in medical information entry. a dp is a preset template that may be inserted into the emr when the physician types a period followed by a code phrase (in this case ''.ichstudy''). once the study dp was inserted at the bottom of the electronic ed note, it prompted enrolling physicians to answer study questions. investigators then extracted data directly from the emr. our primary outcomes of interest were the prevalence of dp data form use and rates of data entry errors. results: from 7/2009 through 8/2010, 883 patients were enrolled. dp data forms were used in 288 (32.6%; 95% ci 29.5, 35.7%) cases and paper data forms in 595 (67.4%; 95% ci 64.3, 70.5%). the prevalence of dp data form use at the respective study centers was 11%, 16%, 18%, 31%, and 85%. sixty-six (43.7 %; 95% ci 35.8, 51.6%) of 151 physicians enrolling patients used dp data entry at least once. using multivariate analysis, we found no significant association between physician age, sex, or tenure and dp use. data entry errors were more likely on paper forms (234/595, 39.3%; 95% ci 35.4, 43.3%) than dp data forms (19/288, 6.6%; 95% ci 3.7, 9.5%), difference in error rates 32.7% (95% ci 27.9, 37.6%, p < 0.001). conclusion: dp data collection is a feasible means of study data collection. dp data forms maintain all study data within the secure emr environment obviating the need to maintain and collect paper data forms. this innovation was embraced by many of our emergency physicians. we found lower data entry error rates with dp data forms compared to paper forms. background: inadequate randomization, allocation concealment, and blinding can inflate effect sizes in both human and animal studies. these methodological limitations might in part explain some of the discrepancy between promising results in animal models and non-significant results in human trials. whereas blinding is not always possible, in clinical or animal studies, true randomization with allocation concealment is always possible, and may be as important in minimizing bias. objectives: to determine the frequency with which published emergency medicine (em) animal research studies report randomization, specific randomization methods, allocation concealment, and blinding of interventions and measurements, and to estimate whether these have changed over time. methods: all em animal research publications from 1/ 2000 through 12/2009 in ann emerg med and acad emerg med were reviewed by two trained investigators for a statement regarding randomization, and specific descriptions of randomization methods, allocation concealment, blinding of intervention, and blinding of measurements, when possible. raw initial agreement was calculated and differences were settled by consensus. the first (period 1 = 2000-2004) and second (period 2 = 2005-2009) 5-year periods were compared with 95% confidence intervals. results: of 117 em animal research studies, 109 were appropriate for review because they involved intervention in at least two groups. blinding of interventions and measurements were not considered possible in 37% and 3%, respectively. significant differences between period 1 and 2 were absent, although there was a trend towards less blinding of interventions and more blinding of measurements. raw agreement was 91%. conclusion: although randomization is mentioned in the majority of studies, allocation concealment and blinding remain underutilized in em animal research. we did not compare outcomes between blinded and non-blinded, randomized and non-randomized studies, because of small sample size. this review fails to demonstrate significant improvement over time in these methodological limitations in em animal research publications. journals might consider requiring authors to explicitly describe their randomization, allocation, and blinding methods. background: cluster randomized trials (crts) are increasingly utilized to evaluate quality improvement interventions aimed at health care providers. in trials testing ed interventions, migration of eps between hospitals is an important concern, as contamination may affect both internal and external validity. objectives: we hypothesized geographically isolating emergency departments would prevent migratory contamination in a crt designed to increase ed delivery of tpa in stroke (the instinct trial). methods: instinct was a prospective, cluster-randomized, controlled trial. twenty-four michigan community hospitals were randomly selected in matched pairs for study. following selection of a single hospital, all hospitals within 15 miles were excluded from the sample pool. individual emergency physicians staffing each site were identified at baseline (2007) and 18 months later. contamination was defined at the cluster level, with substantial contamination defined a priori as >10% of eps affected. non-adherence, total crossover (contamination + non-adherence), migration distance and characteristics were determined. results: 307 emergency physicians were identified at all sites. overall, 7 (2.3%) changed study sites. one moved between control sites, leaving 6 (2.0%) total crossovers. of these, 2 (0.7%) moved from intervention to control (contamination) and 4 (1.3%) moved from control to intervention (non-adherence). contamination was observed in 2 of 24 sites, with 17% and 9% contamination of the total site ep workforce at follow-up, respectively. two of 6 crossovers occurred between hospitals within the same health system. average migration distance was 42 miles for all eps in the study and 35 miles for eps moving from intervention to control sites. conclusion: the mobile nature of emergency physicians should be considered in the design of quality improvement crts. use of a 15-mile exclusion zone in hospital selection for this crt was associated with very low levels of substantial cluster contamination (1 of 24) and total crossover. assignment of hospitals from a single health system to a single study group and/or an exclusion zone of 45 miles would have further reduced crossovers. increased reporting of contamination in cluster randomized controlled trials is encouraged to clarify thresholds and facilitate crt design. objectives: an extension of the lr, the average absolute likelihood ratio (aalr), was developed to assess the average change in the odds of disease that can be expected from a test, or series of tests, and an example of its use to diagnose wide qrs complex tachycardia (wct) is provided. methods: results from two retrospective multicenter case series were used to assess the utility of qrs duration and axis to assess for ventricular tachycardia (vt) in patients with undifferentiated regular sustained wct. serial patients with heart rate (hr) >120 beats per minute and qrs duration >120 milliseconds (msec) were included. the final tachydysrhythmia diagnosis was determined by a number of methods independent of the ecg. the aalr is defined as: aalr = 1/n total [r (n i *lr i ) (for lr > 1) + r (n k /lr k ) (for lr < 1)], where lr i and lr k are the interval lrs, and n i and n k are the number of patients with test results within the corresponding intervals. roc curves were constructed, and interval lrs and aalrs were calculated for the qrs duration and axis tests individually, and when applied together. confidence intervals were bootstrapped with 10,000 replications using the r boot package. results: 187 patients were included: 95 with supraventricular tachycardia (svt) and 92 with vt. optimal qrs intervals (msec) for distinguishing vt from svt were: qrs £ 130, 130 < qrs < 160, and qrs ‡ 160. qrs axis results were dichotomized to upward right axis (181-270 degrees) or not ()89 to 180 degrees). results are listed in the table. conclusion: application of the qrs interval and axis tests together for patients with wide qrs complex tachycardia changes the odds of ventricular tachycardia, on average, by a factor of 3.5 (95% ci 2.4 to 6.2), and this is mildly improved over the qrs duration test alone. both a strength and weakness of the aalr is its dependence on the pretest probability of disease. the aalr may be helpful for clinicians and researchers to evaluate and compare diagnostic testing approaches, particularly when strategies with serial non-independent tests are considered. consultation for adults with metastatic solid tumors at an urban, academic ed located within a tertiary care referral center. field notes were grouped into barrier categories and then quantified when possible. patient demographics for those who did and did not enroll were extracted from the medical record and quantified. patients who did not meet inclusion criteria for the study (e.g., cognitive impairment) were excluded from the analysis. results: attempts were made to enroll 42 eligible patients in the study, and 23 were successfully enrolled (55% enrollment rate). barriers to enrollment were deduced from the field notes and placed into the following categories from most to least common: patient refusal (6); diagnostic uncertainty regarding cancer stage (4); severity of symptoms preclude participation (4); patient unaware of illness or stage (3); and family refusal (2). conclusion: patients, families, and diagnostic uncertainty are barriers to enrolling ed patients with advanced illness in clinical trials. it is unclear whether these barriers are generalizable to other study sites and disease processes other than cancer. objectives: the purpose of this study was to evaluate the use of a high-fidelity mannequin bedside simulation scenario followed by a debriefing session as a tool to improve medical student knowledge of palliative care techniques. methods: third year medical students participating in a 12-week simulation curriculum during a surgery/ emergency medicine/anesthesia clerkship were eligible for the study. all students were administered a pretest to evaluate their baseline knowledge of palliative care and randomized to a control or intervention group. during week 3 or 4, students in the intervention group participated in and observed two end-of-life scenarios. following the scenarios, a faculty debriefer trained in palliative care addressed critical actions in each scenario. during week 10, all students received a posttest to evaluate for improvement in knowledge. the pre-test and post-test consisted of 12 questions addressing prognostication, symptom control, and the medicare hospice benefit. students were de-identified and pre-and post-tests were graded by a blinded scorer. results: from jan-dec 2011, 70 students were included in the study and 5 were excluded due to incomplete data. the mean score on the pre-test for the intervention group was 3.16, and for the control group was 3.45 (p = 0.90 the results indicate that educators identify the most important scenarios as protocol-based simulations. respondents also suggested that scenarios of very common emergency department presentations bear a great deal of importance. emergency medicine educators assign priority to simulations involving professionalism and communication. finally, many respondents noted that they use simulation to teach the presentation and management of rare or less frequent, but important disease processes. the identification of these scenarios would suggest that educators find simulation useful for filling in ''gaps'' in resident education. background: prescription drug misuse is a growing problem among adolescent and young adult populations. objectives: to determine factors associated with past year prescription drug misuse defined as using prescription sedatives, stimulants, or opioids to get high, taking them when they were prescribed to someone else or taking more than was prescribed among patients seeking care in an academic ed. methods: adolescents and young adults (14-20) presenting for ed care at a large, academic teaching hospital were approached to complete a computerized screening questionnaire regarding demographics, prescription drug misuse, illicit drug use, alcohol use, and violence in the past 12 months. logistic regression was used to predict past year prescription drug misuse. results: over the study time period, there were 2156 participants (86% response rate) of whom 300 (13.9%) endorsed past year prescription drug misuse. specifically, rates of past year misuse for opioids was 8.7%, sedatives was 5.4%, and stimulants was 8.0%. significant overlap exists among classes with over 40% misusing more than one class of medications. in the multivariate analysis significant predictors of past year prescription drug misuse included female gender (or conclusion: approximately one in seven adolescents or young adults seeking ed care have misused prescription drugs in the past year. while opioids are the most common drug misused, significant overlap exists among this population. given the correlation of prescription drug misuse with the use and misuse of other substances (i.e. alcohol, cough medicine, marijuana) more research is needed to further understand these relationships and inform interventions. additionally, future research should focus on understanding the differences in demographics and risk factors associated with misuse of each separate class of prescription drugs. prospective 10 objectives: this study aims to examine the association of depression with high ed utilization in patients with non-specific abdominal pain. methods: this single-center, prospective, cross-sectional study was conducted in an urban academic ed located in washington, dc as part of a larger study to evaluate the interaction between depression and frequency of ed visits and chronic pain. as part of this study, we screened patients using the phq-9, a nineitem questionnaire that is a validated, reliable predictor of major depressive disorder. we analyzed the subset of respondents with a non-specific abdominal pain diagnosis (icd-9 code of 789.xx). our principal outcome of interest was the rate of a positive depression screen in patients with non-specific abdominal pain. we analyzed the prevalence of a positive depression screen among this group and also conducted a chi-square analysis to compare high ed use among abdominal pain patients with a positive depression screen versus those without a positive depression screen. we defined high ed utilization as >3 visits in a 364-day period prior to the enrollment visit. background: numerous studies have found high rates of co-morbid mental illness and chronic pain in emergent care settings. one psychiatric diagnosis frequently associated with chronic pain is major depressive disorder (mdd). objectives: we conducted a study to characterize the relationship between mdd and chronic pain in the emergency department (ed) population. we hypothesized that patients who present to the ed with selfreported chronic pain will have higher rates of mdd. methods: this was a single-center, prospective, crosssectional study. we used a convenience sample of noncritically ill, english speaking adult patients presenting with non-psychiatric complaints to an urban academic ed over 6 months in 2011. we oversampled patients presenting with pain-related complaints (musculoskeletal pain or headache). subjects were surveyed about their demographic and other health and health care characteristics and were screened with the phq 9, a nine-item questionnaire that is a validated, reliable predictor of mdd. we conducted bivariate (chi-square) and multivariate analysis controlling for demographic characteristics (race, income, sex, age) using stata v. 10.0. our principal dependent variable of interest was a positive depression screen (phq 9 score ‡10). our principal independent variable of interest was the presence of self-reported chronic pain (greater than 3 months). results: of 77 patients enrolled, 2 did not meet all inclusion criteria. 50 had two or more assessments for comparison. their average age was 39 (range 21-59), 70% were male, and 74% were in police custody. 38% used methadone alone; 16% heroin alone; 4% oxycodone alone; and the rest used multiple opioids. the average dose of im methadone was 10.3 mg (range 5-20 mg); all but 3 patients received 10 mg. the mean cows score before receiving im methadone was 11.19 (range 3-23), compared to 4.83 (range 0-20) 30 minutes after methadone (p < 0.001; mean difference = )6.36; 95% ci = )4.57 to )8.15). the mean wss before and after methadone was )1.54 (range )1 to )2) and )0.755 (range )2 to 2), respectively (p < 0.001; 95% ci = )1.0 to )0.57). the mean physician-assessed wss was significantly lower than the patient's own assessment by 0.78 (p < 0.001). adverse events included an asthmatic patient with bronchospasm whose oxygen saturation decreased from 95% to 88% after receiving methadone, a patient whose oxygen saturation decreased from 95% to 93%, and two patients whose amss decreased from )1 to )2 (indicating moderate sedation). background: as the us population ages, the coexistence of copd and acute coronary syndrome (acs) is expected to be more frequent. very few studies have examined the effect of copd on outcomes in acs patients, and, to our knowledge, there has been no report on biomarkers that possibly mediate between copd and long-term acs patient outcomes. objectives: to determine the effect of copd on longterm outcomes in patients presenting to the emergency department (ed) with acs and to identify prognostic inflammatory biomarkers. methods: we performed a prospective cohort study enrolling acs patients from a single large tertiary center. hospitalized patients aged 18 years or older with acs were interviewed and their blood samples were obtained. seven inflammatory biomarkers were measured, including interleukin-6 (il-6), c-reactive protein (crp), tumor necrosis factor-alpha (tnf-alpha), vascular cell adhesion molecule (vcam), e-selectin, lipoprotein-a (lp-a), and monocyte chemoattractant protein-1 (mcp-1). the diagnoses of acs and copd were verified by medical record review. annual telephone follow-up was conducted to assess health status and major adverse cardiovascular events (mace) outcomes, a composite endpoint including myocardial infarction, revascularization procedure, stroke, and death. background: aortic dissection (ad) is an uncommon life-threatening condition requiring prompt diagnosis and management. thirty-eight percent of cases are missed upon initial evaluation. the cornerstone of accurate diagnosis hinges on maintaining a high index of clinical suspicion for the various patterns of presentation. quality documentation that reflects consideration for ad in the history, exam, and radiographic interpretation is essential for both securing the diagnosis and for protecting the clinician in missed cases. objectives: we sought to evaluate the quality of documentation in patients presenting to the emergency department with subsequently diagnosed acute ad. methods: irb-approved, structured, retrospective review of consecutive patients with newly diagnosed non-traumatic ad from 2004 to 2010. inclusion criteria: new ad diagnosis via ed. exclusion criteria: ad diagnosed at another facility; chronic, traumatic, or iatrogenic ad. trained/monitored abstractors used a standardized data tool to review ed and hospital medical records. descriptive statistics were calculated as appropriate. inter-rater reliability was measured. our primary performance measure was the prevalence of a composite of all three key historical elements (1. any back pain, 2. neurologic symptoms including syncope, and 3. sudden onset of pain.) in the attending emergency physician's documentation. secondary outcomes included documentation of: ad risk factors, pain quality, back pain at multiple locations, presence/absence of pulse symmetry, mediastinal widening on chest radiograph, and migratory nature of the pain. results: 65/203 met our inclusion/exclusion criteria. the mean age was 58.4 years; 65% were male, 23 (35.4%) were stanford a. 32 (60%) presented with a chief complaint of chest pain. primary outcome measure: 6/65 (9.2%; 95%ci = 3.5,19.0) documented the presence/ absence of all three key historical elements. [back pain = 42/65; 64.6% (51.8, 76.1); neuro symptoms = 39/ 65; 60% (47.1, 72.0); sudden onset = 12/65; 18.5% (9.9, 30.0).] limitations: small number of confirmed ad cases. conclusion: in our cohort, emergency physician documentation of key historical, physical exam, and radiographic clues of ad is suboptimal. although our ed miss rate is lower than that which has been reported by previous authors, there is an opportunity to improve documentation of these pivotal elements at our institution. objectives: this study assessed the opinions of iem and gh fellowship program directors, in addition to recent and current fellows regarding streamlining the application process and timeline in an attempt to implement change and improve this process for program directors and fellows alike. methods: a total of 34 current iem and gh fellowship programs were found through an internet search. an electronic survey was administered to current iem and gh fellowship directors, current fellows, and recent graduates of these 34 programs. results: response rates were 88% (n = 30) for program directors and 53% (n = 17) for current and recent fellows. the great majority of current and recent fellows (77%) and program directors (83%) support transitioning to a common application service. similarly, 88% of current and recent fellows and 83% of program directors support instituting a uniform deadline date for applications. however, only 47% of recent/current fellows and 33% of program directors would support a formalized match process like nrmp. conclusion: the majority of fellows and program directors support streamlining the application for all iem and gh fellowship programs. this could improve the application process for both fellows and program directors, and ensure the best fit for the candidates and for the fellowship programs. in order to establish effective emergency care in rural sub-saharan africa, the unique practice demographics and patient dispositions must be understood. objectives: the objectives of this study are to determine the demographics of the first 500 patients seen at nyakibale hospital's ed and assess the feasibility of treating patients in a rural district hospital ed in sub-saharan africa. methods: a descriptive cross-sectional analysis of the first 500 consecutive patient visits in the ed's patient care log was reviewed by an unblinded abstractor. data collected included age, sex, condition upon discharge, and disposition. all authors discussed uncertainties and formed a consensus. descriptive statistics were performed. results: of the first 500 patient visits, 254 (50.8%) occurred when the outpatient clinic was open. there were 275 (55%) male visits. the average age was 25.2 years (sd ± 22.2). pediatric visits accounted for 218 (43.6%) patients, and 132 (26.4%) visits were for children under five years old. only one patient expired in the ed, and 401 (80.2%) were in good condition after treatment, as subjectively defined by the ed physicians. one person was transferred to another hospital. after treatment, 180 (36%) patients were discharged home. of those admitted to an inpatient ward, 126 (25.2%) patients were admitted to medical wards, 97 (19.4%) to pediatrics, and 60 (12%) to surgical. only six (1.2 %) patients went directly to the operating theatre. conclusion: this consecutive sample of patient visits from a novel rural district hospital ed in sub-saharan africa included a broad demographic range. after treatment, most patients were judged to be in ''good condition'', and over one third of patients could be discharged after ed management. this sample suggests that it is possible to treat patients in an ed in rural sub-saharan africa, even in cases where surgical backup and transfers to higher level of care are limited or unavailable. background: communication failures in clinical handoffs have been identified as a major preventable cause of patient harm. in italy, advanced prehospital care is provided predominantly by physicians who work on ambulances in teams with either nurses or basic rescuers. the hand-offs from prehospital physicians to hospital emergency physicians (eps) is especially susceptible to error with serious consequences. there are no studies in italy evaluating the communication at this transition in patient care. studying this, however, requires a tool that measures the quality of this communication. objectives: the purpose of this study is to develop and validate a tool for the evaluation of communication during the clinical handoff from prehospital to emergency physicians in critically ill patients. methods: several previously validated tools for evaluating communication in hand-offs were identified through a literature search. these were reviewed by a focus group consisting of eps, nurses, and rescuers, who then adapted and translated the australian isbar (identification, situation, background, assessment, recommendation), the tool most relevant to local practice. the italian isbar tool consists of the following elements: patient and provider identification; patient's chief complaint; patient's past medical history, medications, and allergies; prehospital clinical assessment (primary survey, illness severity, vital signs, diagnosis); treatment initiated and anticipated treatment plan. we conducted and video-taped the hand-offs of care from the prehospital physicians to the eps in 12 pediatric critical care simulations. four physician raters were trained in the italian isbar tool and used it to independently assess communication in each simulation. to assess agreement we calculated the proportion of agreement among raters for each isbar question, fleiss' kappas for each simulation, as well as mean agreement and mean kappas with standard deviations. results: there was 100% agreement among the four physicians on 70% of the items. the mean level of agreement was 91% (sd 0.15). the overall mean kappa was 0.67 (sd 0.10). conclusion: the standardized tool resulted in good agreement by physician raters. this validated tool may be helpful in studying and improving hand-offs in the prehospital to emergency department setting. objectives: we hypothesized that residents who were provided with vps prior to hfs would perform more thoroughly and efficiently than residents who had not been exposed to the online simulation. methods: we randomized a group of 30 residents from an academic, pgy 1-4 emergency medicine program to complete an online vps case, either prior to (vps group, n = 14 residents) or after (n = 16) their hfs case. the vps group had access to the online case (which reviewed asthma management) 3 days prior to the hfs session. all residents individually participated in their regularly scheduled hfs and were blinded to the content of the case -a patient in moderate asthma exacerbation. the authors developed a dichotomous checklist consisting of 33 items recorded as done/not done along with time completed. a two sample proportion test was used to evaluate differences in the individual items completed between groups. a wilcoxon rank sum test was used to determine the differences in overall and subcategory performance between the two groups. median time to completion was analyzed using the log-rank test. results: the vps group had better overall checklist performance than the control group (p-value 0.046). in addition, the vps group was more thorough in obtaining an hpi (p-value 0.009). specific actions (related to asthma management) were performed better by the vps group: inquiring about last/prior ed visits (0.038), total number of hospitalizations in the prior year (0.029), prior intubations (0.001), and obtaining peak flow measurements (0.030). overall there was no difference in time to event completion between the two groups. conclusion: we found that when hfs is primed with educational modalities such as vps there was an improvement in performance by trainees. however, the improved completeness of the vps group may have served as a barrier to efficiency, inhibiting our ability to identify a statistical significant efficiency overall. vps may aid in priming the learners and maximize the efficiency of training using high-fidelity simulations. training using an animal model helped develop residents' skills and confidence in performing ptv. retention was found to be good at 2 months post-training. this study underscores the need for hands-on training in rare but critical procedures in emergency medicine. methods: in this cross-sectional study at an urban community hospital, 15 residents in their second or third year of training from a 3-year em residency program performed us-guided catheterizations of the ij on a simulator manufactured by blue phantom. two board-certified em physicians observed for the completion of pre-defined procedural steps using a checklist and rated the residents' overall performance of the procedure. overall performance ratings were provided on a likert scale of 1 to 10, with 1 being poor and 10 being excellent. residents were given credit for performing a procedural step if at least one rater marked its completion. agreement between raters was calculated using intraclass correlation coefficients for domain and summary scores. the same protocol was then repeated on an unembalmed cadaver using two different board-certified em physician raters. criterion validity of the residents' proficiency on the simulator was evaluated by comparing their median overall performance rating on the simulator to that on the cadaver and by comparing the proportion of residents completing each procedural step between modalities with descriptive statistics. results: em residents' overall performance rating on the simulator was 7.4 (95% ci: 6.0 to 8.8) and on the cadaver was 6.1 (95% ci: 4.7 to 7.5). the results for each procedural step are summarized in the attached figure. inter-rater agreement was high for assessments on both the simulator and cadaver with overall kappa scores of 0.89 and 0.96 respectively. background: the environment in the emergency department (ed) is chaotic. physicians must learn how to multi-task effectively and manage interruptions. noise becomes an inherent byproduct of this environment. previous studies in the surgical and anesthesiology literature examined the effect of noise levels and cognitive interruptions on resident performance during simulated procedures; however, the effect of noise distraction on resident performance during an ed procedure has not yet been studied. objectives: our aim was to prospectively determine the effects of various levels of noise distraction on the time to successful intubation of a high-fidelity simulator. methods: a total of 45 emergency medicine, emergency medicine/internal medicine, and emergency medicine/family medicine residents were studied in a background noise environments of less than 50 decibels (noise level 1), 60-70 decibels (noise level 2), and of greater than 70 decibels (noise level 3). noise levels were standardized by a dosimeter (ex tech instruments, heavy duty 600). each resident was randomized to the order in which he or she was exposed to the various noise levels and had a total of 2 minutes to complete each of the intubation attempts, which were performed in succession. time, in seconds, to successful intubation was measured in each of these scenarios with the start time defined as the time the resident picked up the storz c-mac video laryngoscope blade and the finish time defined as the time the tube passed through the vocal cords as visualized by an observer on the storz c-mac video screen. analytic methods included analysis of variance, student's t-test, and pearson's chi-square. results: no significant differences were found between time to intubation and noise level nor did the order of noise level exposure affect the time to intubation (see table) . there were no significant differences in success rate between the three noise levels (p = 0.178). a significant difference in time to intubation was found between the residents' second and third intubation attempts with decreased time to intubation for the third attempt (p = 0.001). conclusion: noise level did not have an effect on time to intubation or intubation success rate. time to intubation decreased between the second and third intubations regardless of noise level. background: growing use of the emergency department (ed) is cited as a cause of rising health care costs and a target of health care reform. eds provide approximately one quarter of all acute care outpatient visits in the us. eds are a diagnostic center and a portal for rapid inpatient admission. the changing role of eds in hospital admissions has not been described. objectives: to compare if admission through the ed has increased compared to direct hospital admission. we hypothesized that the use of the ed as the admitting portal increased for all frequently admitted conditions. methods: we analyzed the nationwide inpatient sample (nis), the largest us all-payer inpatient care database, from 1993-2006. nis contains data from approximately 8 million hospital stays each year, and is weighted to produce national estimates. we used an interactive, webbased data tool (hcupnet) to query the nis. clinical classification software (ccs) was used to group discharge diagnoses into clinically meaningful categories. we calculated the number of annual admissions and proportion admitted from the ed for the 20 most frequently admitted conditions. we excluded ccs codes that are rarely admitted through the ed (<10%) as well as obstetbackground: the optimal dose of opioids for patients in acute pain is not well defined, although 0.1 mg/kg of iv morphine is commonly recommended. patient-controlled analgesia (pca) provides an opportunity to assess the adequacy of this recommendation as use of the pca pump is a behavioral indication of insufficient analgesia. objectives: to assess the need for additional analgesia following a 0.1 mg/kg dose of iv morphine by measuring additional self-dosing via a pca pump. methods: a three-arm randomized controlled trial was performed in an urban ed with 75,000 annual adult visits. a convenience sample of ed patients ages 18 to 65 with abdominal pain of <7 days duration requiring iv opioids was enrolled between 4/2009 and 6/2010. all patients received an initial dose of 0.1 mg/kg iv morphine. patients in the pca arms could request additional doses of 1 mg or 1.5 mg iv morphine by pressing a button attached to the pump with a 6-minute lock-out period. for this analysis, data from both pca arms were combined. software on the pump recorded times when the patient pressed the button (activation) and when he/she received a dose of morphine (successful activation). results: 137 patients were enrolled in the pca arms. median baseline nrs pain score was 9. mean amount of supplementary morphine self-administered over the 2 hour study period subsequent to the loading dose was 5.7 mg and 6.7 mg for the 1 and 1.5 mg pca groups respectively. 124 patients activated the pump at least once (91%, 95% ci: 84 to 94%). figure 1 shows the frequency distribution of the number of times the pump was activated. of those who activated the pump, the median number of activations per person was 5 (iqr: 3 to 12). there were 1124 activations of the pump. 60% of activations were successful (followed by administration of morphine), while 40% were unsuccessful as they occurred during the 6-minute lock-out periods. 19% of the activations occurred in the first 30 minutes, 29% in the second 30 minutes, 25% in the third 30 minutes, and 27% in the last 30 minutes after the initial loading dose. conclusion: almost all patients requested supplementary doses of pca morphine, half of whom activated the pump five times or more over a course of 2 hours. this frequency of pca activations suggests that the commonly recommended dose of 0.1 mg/kg morphine may constitute initial oligoanalgesia in most patients. marie-pier desjardins, benoit bailey, fanny alie-cusson, serge gouin, jocelyn gravel chu sainte-justine, montreal, qc, canada background: administration of corticosteroid at triage has been suggested to decrease the time to corticosteroid administration in the ed. objectives: to compare the time between arrival and corticosteroid administration in patients treated with an asthma pathway (ap) or with standard management (sm) in a pediatric ed. methods: chart review of children aged 1 to 17 years diagnosed with asthma, bronchospasm, or reactive airways disease seen in the ed of a tertiary care pediatric hospital. for a one year period, 20% of all visits were randomly selected for review. from these, we reviewed patients who were eligible to be treated with the ap ( ‡18 months with previous history of asthma and no other pulmonary condition) and who had received at least one inhaled bronchodilator treatment. charts were evaluated by a data abstractor blinded to the study hypothesis using a standardized datasheet. various variables were evaluated such as age, respiratory rate and 0 2 saturation at triage, type of physician who saw patient first, treatment prior to visit, in ed, and at discharge, time between arrival and corticosteroid administration, and length of stay (los background: return visits comprise 3.5% of pediatric emergency department (ped) visits, at a cost of >$500 million/year nationally. these visits are typically triaged with higher acuity and admission rates and raise concern for lapses in quality of care and patient education during the first visit. objectives: the aim of this qualitative study was to describe parents' reasons for return visits to the ped. methods: we prospectively recruited a convenience sample of parents of patients under the age of 18 years who returned to the ped within 72 hours of their previous visit. we excluded patients who were instructed to return, had previously left without being seen, arrived without a parent, were wards of the state, or did not speak english. after obtaining consent, the principal investigator (ce) conducted confidential, in-person, tape-recorded interviews with parents during ped return visits. parents answered 12 open-ended questions and 9 closed-ended questions using a five-point likert scale. responses to open-ended questions were analyzed using thematic analysis techniques. the scaled responses were grouped into three categories of agree, disagree, or neutral. results: from the 49 closed-ended responses, 86% of parents agreed that their children were getting sicker, and 92% agreed that their children were not getting better. 80% agreed that they were unsure how to treat the illness, however only 41% agreed they did not feel figure 1 : frequency distribution of number of pca activations comfortable taking care of the illness. only 29% agreed that the medical condition and/or the instructions were not clearly explained in the first visit. some common themes from the open-ended questions included worsening or lack of improvement of symptoms. many parents reported having unanswered questions about the cause of the illness and hoped to find out the cause during the return visit. conclusion: most parents brought their children back to the ped because they believed the symptoms had worsened or were not improving. although a large proportion of parents believed that the medical condition was clearly explained at the first visit, many parents still had unanswered questions about the cause of their child's illness. while worsening symptoms seemed to drive most return visits, it is possible that some visits related to failure to improve might be prevented during the first ped visit through a more detailed discussion of disease prognosis and expected time to recover. pediatric background: experience indicates that it is difficult to effectively quell many parents' anxiety toward pediatric fevers, making this a common emergency department (ed) complaint. the question remains as to whether athome treatment has any effect on the course of emergency department treatment or length of stay in this population. objectives: to determine whether anti-pyretic treatment prior to arrival in the emergency department affects the evaluation or emergency department length of stay of febrile pediatric patients. methods: a convenience sample of children, ages 0-12 years, who presented to a tertiary care ed with chief complaint of fever were enrolled. parents were asked to participate in an eight-question survey. questions related to demographic information, pre-treatment of the fever, contact with primary care providers prior to ed arrival, and immunization status. upon admission or discharge, investigators recorded information regarding length of stay, laboratory tests and imaging ordered, and medications given. results: eighty-one patients were enrolled in the study. seventy-six percent of the patients were pre-treated with some form of anti-pyretic by the caregiver prior to ed arrival. there was no significant effect of pre-treatment on whether laboratory tests or medications were ordered in the ed or whether the patient was admitted or discharged. the length of ed stay was found to be significantly shorter among those who received anti-pyretics prior to arrival (184 ± 11 vs. 247 ± 36 minutes; p = 0.03). conclusion: among febrile children, those who receive anti-pyretics prior to their ed visit had statistically significant shorter length of stays. this also supports implementation of triage or nursing protocols to administer an anti-pyretic as soon as possible in the hope of decreasing ed throughput times. background: during the past two decades, the prevalence of overweight (bmi percentile >95) in children has more than doubled, reaching epidemic proportions both nationally and globally. the public health burden is enormous given the increased risk of adult obesity as well as the adverse consequences on cardiovascular, metabolic, and psychological health. despite the overwhelming prevalence, the effect of obesity on emergency care has received little attention. objectives: the goal of this study is to determine the relation of weight on reported emergency department visits in children from a nationally representative sample. methods: weight (as reported by parents) and height along with frequency of and reason for emergency department (ed) use in the last 12 months were obtained from children aged 10-17 y (n = 46,707) in the cross-sectional, telephone-administered, national survey of children's health (nsch). bmi percentiles were calculated using sex-specific bmi for age growth charts from the cdc (2000). children were categorized as: underweight (bmi percentile£5), normal weight (>5 to <85), at-risk for overweight (85 to <95), and overweight ( ‡95). prevalence of ed use was estimated and compared across bmi percentile categories using chisquare analysis and multivariable logistic regression. taylor-series expansion was used for variance estimation of the complex survey design. results: the prevalence of at least one ed use in the past 12 months increased with increasing bmi percentiles (figure 1, p < 0.001). additionally, overweight children were more likely to have more than one visit. overweight children were also less likely to report an injury, poisoning, or accident as the reason for ed visit compared to other bmi categories (47, 55, 59, 54% in overweight, at-risk, normal, and underweight respectively, p < 0.05). conclusion: as rates of childhood obesity continue to grow in the u.s., we can expect greater demands on the ed. this will likely translate into an increased emphasis on the care of chronic conditions rather than injuries and accidents in the pediatric ed setting. results: mean pediatric satisfaction score was 84.1 (sd 3.9) compared with 81.4 (3.2) for adult patients (p < 0.001); monthly sample sizes ranged from 14-74 and from 30-125 for the two populations, respectively. both populations showed an increase in satisfaction after opening of the ped-ed. for both populations there was no significant trend in patient satisfaction from the beginning of the study period to the opening of the ped-ed, but after the opening the models of the populations differed. the pediatric satisfaction model was an interrupted two-slope model, with an immediate jump of 3.5 points in november and an increase of 0.2 points per month thereafter. in contrast, adult satisfaction scores did not show a jump but increased linearly (two slope model) after 11/2011 at a rate of 0.3 per month. prior to the opening of the ped-ed, mean monthly pediatric and adult satisfaction scores were 81.5 (2.4) and 79.5 (2.8), respectively (difference 2.0 95% ci 0.1-3.8, p = 0.04). after the opening the mean scores were 86.8 (3.1) and 83.2 (2.4), respectively (difference 3.6, 95% ci 2.1-5.0, p < 0.001). conclusion: opening of a dedicated ped-ed was associated with a significant increase in patient satisfaction scores both for children and adults. patient satisfaction for children, as compared to adults, was higher before and after opening a ped-ed. the background: there are racial disparities in outcomes among injured children. in particular, black race appears to be an independent predictor of mortality. objectives: to evaluate disparities among ed visits for unintentional injuries among children ages 0-9. methods: five years of data (2004) (2005) (2006) (2007) (2008) from the national hospital ambulatory cares survey were combined. inclusion criteria were defined as unintentional injury visits (e-code 800.0 to 869.9 or 888.0 to 929.9) and age 0-9 years. visit rates per 100 population (defined by the us census) were calculated by race and age group. weighted multivariate logistic regression analysis was performed to describe associations between race and specific outcome variables and related covariates. primary statistical analyses were performed using sas version 9.1.3. results: 21,524,000 of 585,294,000 weighted ed visits met our inclusion criteria (3.7%). per 100 persons, black children had 1.5 times as many ed visits for unintentional injuries as whites (table) . there were no racial differences in the sex ratio (1.4 boy visits: 1 girl), proportion of visits by age, ed disposition, immediacy with which they needed to be seen, whether or not they were evaluated by an attending physician, metropolitan vs. rural hospital, admission length of stay, mode of transportation for ed arrival, number of procedures, diagnostic services, or ed medications. background: sudden cardiac arrests in schools are infrequent, but emotionally charged events. little data exist that describes aed use in these events. objectives: the purpose of our study was to 1) describe characteristics and outcomes of school cardiac arrests (ca), and 2) assess the feasibility of conducting bystander interviews to describe the events surrounding school ca. methods: we performed a telephone survey of bystanders to ca occurring in k-12 schools in communities participating in the cardiac arrest registry to enhance survival (cares) database. the study period was from 8/2005-12/2010 and continued in one community through 2011. utstein style data and outcomes were collected from the cares database. a structured telephone interview of a bystander or administrative personnel was conducted for each ca. a descriptive summary was used to assess for the presence of an aed, provision of bystander cpr (bcpr), and information regarding aed deployment, training, and use and perceived barriers to aed use. descriptive data are reported. results: during the study period there were 30,603 ca identified at cares communities, of which 73 were identified as educational institutions. of these, 46 (0.15%) events were at k-12 schools with 21 (45.7%) being high schools. of the 46 arrests, a minority were children (15 (32.6%) < age 19), most (32, 84.8%) were witnessed, a majority (36, 76.1%) received bcpr, and 26 (56.5%) were initially in ventricular fibrillation (vf). most arrests 28/40 (70%) occurred during the school day (7a-5p). overall, 14 (30.4%) survived to hospital discharge. interviews were completed for 29 of 46 (63.0%) k-12 events. eighteen schools had an aed on site. most schools (84.2%) with aeds reported that they had a training program and personnel identified for its use. an aed was applied in 10 of 18 patients, and of these 8 were in vf and 4 survived to hospital discharge. multiple reasons for aed non-use (n = 8) were identified. conclusion: cardiac arrests in schools are rare events; most patients are adults and received bcpr. aed use was infrequent, even when available, but resulted in excellent (4/10) survival. further work is needed to understand aed non-use. post-event interviews are feasible and provide useful information regarding cardiac arrest care. physician background: gastroenteritis is a common childhood disease accounting for 1-2 million annual pediatric emergency visits. current literature supports the use of anti-emetics reporting improved oral re-hydration, cessation of vomiting, and reduced need for iv re-hydration. however, there remains concern that using these agents may mask alternative diagnoses. objectives: to assess outcomes associated with use of a discharge action plan using ed-dispensed ondansetron at home in the treatment of gastroenteritis. methods: a prospective, controlled, observational trial of patients presenting to an urban pediatric emergency department (census 22,400) over a 12-month period for acute gastroenteritis. fifty patients received ondansetron in the ed. twenty-nine patients were enrolled in the pediatric emergency department discharge action plan (ped-dap) where ondansetron for home use was dispensed by the treating clinician. twenty-one patients were controls. control patients did not receive home ondansetron. ped-dap patients were given instructions to administer the ondansetron for ongoing symptoms any time 6 hours post ed discharge. all patients were followed by phone at 7-14 days to assess for the following: time of emesis resolution, alternative diagnoses, unscheduled visits, and adverse events. results: all 50 patients were followed by phone. 24/29 ped-dap patients received home ondansetron. 21/29 patients had resolution of emesis in the ed. 7/29 had resolution of their emesis between time of discharge and 24 hours. 1/29 of ped-dap patients reported emesis after 24 hours from ed discharge. five patients reported an unscheduled visit. all five return visits returned to the ed (1/5 returned for emesis, 4/5 for diarrhea). 17/21 controls reported resolution of symptoms within the ed. 2/21 of controls had resolution between time of discharge and 24 hours. 1/21 of the control patients had resolution with between 24 and 48 hours post discharge. 1/21 had an unscheduled appointment with the pmd at 72 hours post-discharge for ongoing fever and nausea. in follow-up there were no alternative diagnoses identified. the effect of the ped-dap on resolution of emesis between discharge and 24 hours appears to be statistically significant (p value < 0.04). conclusion: ondansetron given in schedule with a discharge action plan appears to provide a modest benefit in resolution of symptoms relative to a control population. objectives: to determine the repeatability coefficient of a 100 mm vas in children aged 8 to 17 years in different circumstances: assessments done either at 3 or 1 minute interval, when asked to recall their score or to reproduce it. methods: a prospective cohort study was conducted using a convenience sample of patients aged 8 to 17 years presenting to a pediatric ed. patients were asked to indicate, on a 100 mm paper vas, how much they liked a variety of food with four different sets of three questions: (set 1) questions at 3 minute interval with no specific instruction other than how to complete the vas and no access to previous scores, (set 2) same format as set 1 except for questions at 1 minute interval, (set 3) same as set 1 except patients were asked to remember their answers, and (set 4) same as set 1 except patients were shown their previous answers. for each set, the repeatability coefficient of the vas was determined according to the bland-altman method for measuring agreement using repeated measures: 1.96 x ö 2 x s w where s w is the within-subject standard deviation by anova. the sample size required to estimate s w to 10% of the fraction value as recommended was 96 patients if we obtained three measurements for each patient. results: a total of 100 patients aged 12.1 ± 2.4 years were enrolled. the repeatability coefficient for the questions asked at 3 minute intervals was 12 mm, and 8 mm when asked at 1 minute interval. when asked to remember their previous answers or to reproduce them, the repeatability coefficient for the questions was 7 mm and 6 mm, respectively. conclusion: the condition of the assessments (variation in intervals or patients asked to remember or to reproduce their previous answers) influence the testretest reliability of the vas. depending on circumstances, the theoretical test-retest reliability in children aged 8 to 17 years varies from 6 to 12 mm on a 100 mm paper vas. background: skull radiographs are a useful tool in the evaluation of pediatric head trauma patients. however, there is no consensus on the ideal number of views that should be obtained as part of a standard skull series in the evaluation of pediatric head trauma patients. objectives: to compare the sensitivity and specificity of a two-and four-film x-ray series in the diagnosis of skull fracture in children, when interpreted by pediatric emergency medicine physicians. methods: a prospective, crossover experimental study was performed in a tertiary care pediatric hospital. the skull radiographs of 100 children were reviewed. these were composed of the 50 most recent cases of skull fracture for which a four-film radiography series was available at the primary setting and 50 controls, matched for age. two modules, containing a random sequence of two-and four-film series of each child, were constructed in order to have all children evaluated twice (once with two films and once with four films). board-certified or -eligible pediatric emergency physicians evaluated both modules two to four weeks apart. the interpretation of the four-film series by a radiologist, or when available, the findings on ct scan, served as the gold standard. accuracy of interpretation was evaluated for each patient. the sensitivity and specificity of the two-film versus the four-film skull xray series, in the identification of fracture, were compared. this was a non-inferiority cross-over study evaluating the null hypothesis that a series with two views would have a sensitivity (specificity) that is inferior by no more than 0.055 compared to a series with four views. a total of 50 controls and 50 cases were needed to establish non-inferiority of the two-film series versus the four-film series, with a power of 80% and a significance level of 5%. results: ten pediatric emergency physicians participated in the study. for each radiological series, the proportion of accurate interpretation varied between 0.20 to 1.00. the four-film series was found to be more sensitive in the detection of skull fracture than a two-film series (difference: 0.084, 95%ci 0.030 to 0.139). however, there was no difference in the specificity (difference: 0.004, 95%ci )0.024 to 0.033). conclusion: for children sustaining a head trauma, a four-film skull radiography series is more sensitive than a two-film series, when interpreted by pediatric emergency physicians. the objectives: we developed a free online video-based instrument to identify knowledge and clinical reasoning deficits of medical students and residents for pediatric respiratory emergencies. we hypothesized that it would be a feasible and valid method of differentiating educational needs of different levels of learners. methods: this was an observational study of a free, web-based needs assessment instrument that was tested on 44 third and fourth year medical students (ms3-4) and 29 pediatric and emergency medicine residents (r1-3). the instrument uses youtube video triggers of children in respiratory distress. a series of cased-based questions then prompts learners to distinguish between upper and lower airway obstruction, classify disease severity, and manage uncomplicated croup and bronchiolitis. face validity of the instrument was established by piloting and revision among a group of experienced educators and small groups of targeted learners. final scores were compared across groups using t-tests to determine the ability of the instrument to differentiate between different levels of learners (concurrent validity). cronbach's alpha was calculated as a measure of internal consistency. results: response rates were 19% among medical students and 43% among residents. the instrument was able to differentiate between junior (ms3, ms4, and r1) and senior (r2, r3) learners for both overall mean score (61% vs.78%, p < 0.01) and mean video portion score (74 vs. 84%, p = 0.02). table 1 compares results of several management questions between junior and senior learners. cronbach's alpha for the test questions was 0.47. conclusion: this free online video-based needs assessment instrument is feasible to implement and able to identify knowledge gaps in trainees' recognition and management of pediatric respiratory emergencies. it demonstrates a significant performance difference between the junior and senior learners, preliminary evidence of concurrent validity, and identifies target groups of trainees for educational interventions. future revisions will aim to improve internal consistency. results: the survey response rate was 87% (60/69). among responding programs, 40 (67%) reside within a children's hospital (vs. general ed); 51 (85%) are designated level i pediatric trauma centers. forty-three (72%) programs accept 1-2 pem fellows per year; 53 (88%) provided at least some eus training to fellows, and 42 (70%) offer a formal eus rotation. on average this training has existed for 3 ± 1 years and the mean duration of eus rotations is 4 ± 2 weeks. twenty-eight (67%) programs with eus rotations provide fellow training in both a general ed and a pediatric ed. there were no hospital or program level factors associated with having a structured training program for pem fellows. conclusion: as of 2011, the majority of pem fellowship programs provide eus training to their fellows, with a structured rotation being offered by most of these programs. background: ed visits are an opportunity for clinicians to identify children with poor asthma control and intervene. children with asthma who use eds are more likely than other children to have poor control, not be using controller medications, and have less access to traditional sources of primary care. one significant barrier to ed-based interventions is recognizing which children have uncontrolled asthma. objectives: to determine whether the pacci, a 12item parent-administered questionnaire, can help ed clinicians better recognize patients with the most uncontrolled asthma and differentiate between intermittent and persistent asthma. methods: this was a randomized controlled trial performed at an urban pediatric ed. parents were asked to answer questions about their child's asthma including drug adherence and history of exacerbations, as well as answer demographic questions. using a convenience sample of children 1-18 years presenting with an asthma exacerbation, attending physicians in the study were asked to complete an assessment of asthma control. physicians were randomized to receive a completed pacci (intervention) or not (control group). using an intent-to-treat approach, clinicians' ability to accurately identify 1) four categories of control used by the national heart, lung, and blood institute (nhlbi) asthma guidelines, 2) intermittent vs. persistent level asthma, and 3) controlled / mildly uncontrolled vs. moderate/severely uncontrolled asthma were compared for both groups using chi-square analysis. results: between january and august 2011, 57 patients were enrolled. there were no statistically significant differences between the intervention and control groups for child's sex, age, race and parents' education. conclusion: the pacci improves ed clinicians' ability to categorize children's asthma control according to nhlbi guidelines, and the ability to determine when a child's control has been worsening. ed clinicians may use the pacci to identify those children in greatest need for intervention, to guide prescription of controller medications, and communicate with primary care providers about those children failing to meet the goals of asthma therapy. figure) . fewer than half of physicians reported the parent of a 2-year-old being discharged from their ed following an mvc-related visit would receive either child passenger safety information or referrals (table) . conclusion: emergency physician report of child passenger safety resource availability is associated with trauma center designation. even when resources are available, referrals from the ed are infrequent. efforts to increase referrals to community child passenger safety resources must extend to the community ed settings where the majority of children receive injury care. background: pediatric subspecialists are often difficult to access following ed care especially for patients living far from providers. telemedicine (tm) can potentially eliminate barriers to access related to distance, and cost. objectives: to evaluate the overall resource savings and access that a tm program brings to patients and families. methods: this study took place at a large, tertiary care regional pediatric health care system. data were collected from 1/2011-10/2011. metrics included travel distance saved (round trip between tm presenting sites and the location of the receiving sites), time savings, direct cost savings (based on $0.55/mile) and potential work and school days saved. indirect costs were calculated as travel hrs saved/encounter (based on an average speed of 55 miles/hr). demographics and services provided were included. results: 690 tm consults were completed by 13 separate pediatric subspecialty services. most patients were school aged (86% >/= 5yrs old objectives: to analyze test characteristics of the pathway and its effects on ed length of stay, imaging rates, and admission rate before versus after implementation. methods: children ages 3-18 presenting to one academic pediatric ed with suspicion for appendicitis from october 2010 -august 2011 were prospectively enrolled to a pathway using previously validated lowand high-risk scoring systems. the attending physician recorded his or her suspicion of appendicitis and then used one of two scoring systems incorporating history, physical exam, and cbc. low-risk patients were to be discharged or observed in the ed. high-risk patients were to be admitted to pediatric surgery. those meeting neither low-nor high-risk criteria were evaluated in the ed by pediatric surgery, with imaging at their discretion. chart review and telephone follow-up were conducted two weeks after the visit. charts of a random sample of patients with diagnoses of acute appendicitis or chief complaint of abdominal pain and undergoing a workup for appendicitis in the eight months before and after institution of the pathway were retrospectively reviewed by one or two trained abstractors. results: appendicitis was diagnosed in 65 of 178 patients prospectively enrolled to the pathway (37%). mean age was 9.6 years. of those with appendicitis, 63 were not low-risk (sensitivity 96.9%, specificity 48.7%). the high-risk criteria had a sensitivity of 73.8% and specificity of 77.0%. a priori attending physician assessment of low risk had a sensitivity of 100% and specificity of 49.6%. a priori assessment of high risk had a sensitivity of 58.5% and specificity of 90.2%. we reviewed 232 visits prior to the pathway and 290 after. mean ed length of stay was similar (256 minutes before versus 257 after). ct was used in 12.1% of visits before and 7.3% after (p = 0.07). use of ultrasound increased (44.8% before versus 55.9% after, p < 0.02). admission rates were not significantly different (48.3% before versus 42.7% after, p = 0.2). conclusion: the low-risk criteria had good sensitivity in ruling out appendicitis and can be used to guide physician judgment. institution of this pathway was not associated with significant changes in length of stay, utilization of ct, or admission rate in an academic pediatric ed. computer-delivered alcohol and driver safety behavior screening and intervention program initiated during an emergency department visit mary k. murphy 1 , lucia l. smith 2 , anton palma 2 , david w. lounsbury 2 , polly e. bijur 2 , paul chambers 2 1 yale university, new haven, ct; 2 albert einstein college of medicine, bronx, ny background: alcohol use is involved in 32 percent of all fatal motor vehicle crashes and recent estimates show that at least 448,000 people were injured due to distracted driving last year. patients who visit the emergency department (ed) are not routinely screened for driver safety behavior; however, large numbers of patients are treated in the ed every day creating an opportunity for screening and intervention on important public health behaviors. objectives: to evaluate patient acceptance and response to a computer-based traffic safety educational intervention during an ed visit and one month follow-up. methods: design. pre /post educational intervention. setting. large urban academic ed serving over 100,000 patients annually. participants. medically stable adult ed patients. intervention. patients completed a self-administered, computer-based program that queried patients on alcohol use and risky driving behaviors (texting, talking, and other forms of distracted driving). the computer provided patients with educational information on the dangers of these behaviors and collected data on patient satisfaction with the program. staff called patients one month post ed visit for a repeat query. results: 150 patients participated; average age 39 (21-70), 58% hispanic, 52% male. 96% of patients reported the program was easy to use and were comfortable receiving this education via computer during their ed visit. self-reported driver safety behaviors pre, post intervention (% change): driving while talking on the phone 45%,16% ()29%, p = 0.001), aggressive driving 44%,15% ()29%, p = 0.001), texting while driving 28%,9% ()19%, p = 0.001), driving while drowsy 18%,4% ()14%, p = 0.002), drinking in excess of nih safe drinking guidelines15%,%7 ()8%, p = 0.039), drinking and driving 10%,1% ()9%, p = 0.006). conclusion: we found a high prevalence of selfreported risky driving behaviors in our ed population. at 1 month follow-up, patients reported a significant decrease in these behaviors. overall patients were very satisfied receiving educational information about these behaviors via computer during their ed visit. this study indicates that a low-intensity, computer-based educational intervention during an ed visit may be a useful approach to educate patients about safe driving behaviors and promote behavior change. prevalence of depression among emergency department visitors with chronic illness janice c. blanchard, benjamin l. bregman, jeffrey smith, mohammad salimian, qasem al jabr george washington university, washington, dc background: persons with chronic illnesses have been shown to have higher rates of depression than the general population. the effect of depression on frequent emergency department (ed) use among this population has not been studied. objectives: this study evaluated the prevalence of major depressive disorder (mdd) among persons presenting with depression to the george washington university ed. we hypothesized that patients with chronic illnesses would be more likely to have mdd than those without. methods: this was a single center, prospective, crosssectional study. we used a convenience sample of noncritically ill, english-speaking adult patients presenting with non-psychiatric complaints to an urban academic ed over 6 months in 2011. subjects were screened with the phq 9, a nine-item questionnaire that is a validated, reliable predictor of mdd. we also queried respondents about demographic characteristics as well as the presence of at least one chronic disease (heart disease, hypertension, asthma, diabetes, hiv, cancer, kidney disease, or cerebrovascular disease). we evaluated the association between mdd and chronic illnesses with both bivariate analysis and multivariate logistic regression controlling for demographic characteristics (age, race, sex, income, and insurance coverage). results: our response rate was 90.7% with a final sample size of 1012. of our total sample, 525 (51.9%) had at least one of the chronic illnesses defined above. of this group, 162 (30.9%) screened positive for mdd as compared to 82 (16.6%) of the group without chronic illnesses (p < 0.0001). in multivariate analysis, persons with chronic illnesses had an odds ratio for a positive depression screen of 1.80 (1.31, 2.50) as compared to persons without illness. among the subset of persons with chronic illnesses (n = 525), 46.9% had ‡3 visits in the prior 364 days as compared to 34.4% of persons with chronic illnesses without mdd (p = 0.007). conclusion: our study found a high prevalence of untreated mdd among persons with chronic illnesses who present to the ed. depression is associated with more frequent emergency department use among this population. initial blood alcohol level aids ciwa in predicting admission for alcohol withdrawal craig hullett, douglas rappaport, mary teeple, daniel butler, arthur sanders university of arizona, tucson, az background: assessment of alcohol withdrawal symptoms is difficult in the emergency department. the clinical institute withdrawal assessment (ciwa) is commonly used, but other factors may also be important predictors of withdrawal symptom severity. objectives: the purpose of this study is to determine whether ciwa score at presentation to triage was predictive of later admission to the hospital. methods: a retrospective study of patients presenting to an acute alcohol and drug detoxification hospital was performed from july 2010 through january 2011. patients were excluded if other drug withdrawal was present in addition to alcohol. initial assessment included age, sex, vital signs, and blood alcohol level (bal) in addition to hourly ciwa score. admission is indicated for a ciwa score of 10 or higher. data were analyzed by selecting all patients not immediately admitted at initial presentation. logistic regression using wald's criteria for stepwise inclusion was used to determine the utility of the initially gathered ciwa, bal, longest sobriety, liver cirrhosis, and vital signs in predicting subsequent admission. results: there were 123 patients who fit the inclusion criteria, with 9 admitted for treatment at initial intake and another 27 admitted during the following 10 hours. logistic regression indicated that presenting bal was a strong predictor (p = 0.01) of admission for treatment after initial presentation, as was presenting ciwa (p = 0.03). thus, presenting bal provided a substantial addition above initial ciwa in predicting later admission. no other variables added significantly to the prediction of later admission. to determine the interaction between presenting bal and ciwa scores, we ran a repeated measures analysis of the first five ciwa scores (from presentation to 4 hours later), using bal split into low (bal < 0.10) and high (bal > 0.10) groups (see figure) . their interaction was significant, f (1, 93) = 11.86, p < 0.001, g 2 = 0.11. those presenting with higher initial bal had suppressed ciwa scores that rose precipitously as the alcohol cleared. those with low presenting bal showed a decline in ciwa over time conclusion: initial assessment using the common assessment tool ciwa is aided significantly by bal assessment. patients with higher presenting bal are at higher risk for progression to serious alcohol withdrawal symptom. objectives: to describe patient and visitor characteristics and perspectives on the role of visitors in the ed and determine the effect of visitors on ed and hospital outcome measures. methods: this cross-sectional study was done in an 81,000-visit urban ed, and data were attempted to be collected from all patients over a consecutive 96-hour period from august 25 to 28, 2011. trained data collectors were assigned to the ed continuously for the study period. patients assigned to a rapid care section of the ed (24%) were excluded. a visitor was defined as a person other than a health care provider (hcp) or hospital staff present in a patient's room at any time. patient perspectives on visitors were assessed in the following domains: transportation, emotional support, physical care, communication, and advocating for the patient. ed and hospital outcome measures pertaining to ed length of stay (los) and charges, hospital admission rate, hospital los and charges were obtained from patient medical records and hospital billing. data analyses included frequencies, student's t-tests for continuous variables, and chi-square tests of association for categorical variables. all tests for significance were two-sided. objectives: to examine the effect of sunday alcohol availability on ethanol-related visits and alcohol withdrawal visits to the ed. methods: study design was a retrospective beforeafter study using electronically archived hospital data at an urban, safety net hospital. all adult non-prisoner ed visits from 1/1/2005 to 12/31/2009 were analyzed. an ethanol-related ed visit was defined by icd-9 codes related to alcohol (291.x, 303.x, 305.0, 980.0 ). an alcohol withdrawal visit was defined by icd-9 codes of delirium tremens (291.0), alcohol psychosis with hallucination (291.3), and ethanol withdrawal (291.81). we generated a ratio of ethanol-related ed visits to total ed visits (ethanol/total) and ratio of alcohol withdrawal ed visits to total ed visits (withdrawal/total). a day was redefined as 8 am to 8 am. the ratios were averaged within the four seasons to account for seasonal variations. data from summer 2008 were dropped as it spanned the law change. we stratified data into sunday and non-sunday days prior to analysis to isolate the effects of the law change. we used multivariable linear regression to estimate the association of the ratio with the law change while adjusting for time and the seasons. each ratio was modeled separately. the interaction between time and the law change was assessed using p < 0.05. results: during the study there were a total of 212,189 ed visits including 12,042 (6% of total) ethanol-related visits and 5,496 (3% of total) alcohol withdrawal visits. unadjusted ratios in seasonal blocks are plotted in the figure with associated 95% ci and best fit regression line for before and after law change, respectively. after adjusting for time and season in the multivariable linear regression, we found no significant association of either ethanol/total or withdrawal/total with the law change. this remained true for both sunday and non-sunday data. all interactions assessed were not significant. conclusion: the change in colorado law to allow the sale of full-strength alcoholic beverages on sundays did not significantly affect ethanol-related or alcohol withdrawal ed visits. background: olanzapine is a second-generation antipsychotic (sga) with actions at the serotonin/histamine receptors. post-marketing reports and a case report have documented dangerous lowering of blood pressure when this antipsychotic is paired with benzodiazepines, but a recent small study found no bigger decreases in blood pressure compared to another antipsychotic like haloperidol. decreases in oxygen saturations, however, were larger when olanzapine was combined with benzodiazepines in alcohol-intoxicated patients. it is unclear whether these vital sign changes are associated with the intramuscular (im) route only. objectives: the assessment of vital signs following administration of either oral (po) or im olanzapine, either with or without benzodiazepines (benzos) and with or without concurrent alcohol intoxication. methods: this is a structured retrospective chart review of all patients who received olanzapine in an academic medical center ed from 2004-2010 who had vital signs documented both before medication administration and within four hours afterwards. vital signs were calculated as pre-dose minus lowest post-dose vital sign within 4 hours, and were analyzed in an anova with route (im/po), benzo use (+/)), and alcohol use (+/)) as factors. significance level was set to <0.05. results: there were 482 patients who received olanzapine over the study period. a total of 275 patients (225 po, 50 im) met inclusion criteria. systolic blood pressures decreased across all groups as patients reduced their agitation. neither the route of administration, concurrent use of benzos, nor the use of alcohol were associated with significant changes in systolic bp (p = ns for all comparisons; see figure 1 ). decreases in oxygen saturations, however, were significantly larger for alcoholintoxicated patients who subsequently received im olanzapine + benzos compared to other groups (route: p < 0.001; alcohol: p < 0.01; route x alcohol: p < 0.001; route x benzos x alcohol: p < 0.05; see figure 2 ). conclusion: alcohol and benzos are not associated with significant decreases in blood pressure after po olanzapine, but im olanzapine + benzos is associated with potentially significant oxygen desaturations in patients who are intoxicated. intoxicated patients may have differential effects with the use of im sgas such as olanzapine when combined with benzos, and should be studied separately in drug trials. patients with a psychiatric diagnosis rasha buhumaid, jessica riley, janice blanchard george washington university, washington, dc background: literature suggests that frequent emergency department (ed) use is common among persons with a mental health diagnosis. few studies have documented risk factors associated with increased utilization among this population. objectives: to understand demographic characteristics of frequent users of the emergency department and describe characteristics associated with their visits. it was hypothesized that frequent visitors would have a higher rate of medical comorbidities than infrequent visitors. methods: this was a retrospective study of patients presenting to an urban, academic emergency department in 2009. a cohort of all patients with a mental health-related final icd-9 coded diagnosis (axis i or axis ii) was extracted from the electronic medical record. using a standard abstraction form, a medical chart review collected information about medical comorbidities, substance abuse, race, age, sex, and insurance coverage, as well as diagnosis, disposition, and time of each visit. results: our sample consisted of 109 frequent users ( ‡4 visits in a 365 day period) and 442 infrequent users (£3 visits in a 365 day period). frequent users were more likely to be male (68% vs. 54.5% p = 0.01), black (86% vs. 59% p < 0.0001), and had a higher average number of comorbid conditions (2.0, 95%ci 1.73,2.26) as compared to infrequent users (1.0, 95%ci 0.90,1.10). a higher percentage of visits in the infrequent user group occurred during the day (49% vs. 38.3% p < 0.0001) while a higher number of visits in the frequent users occurred after midnight (24.3% vs. 16.6% p = 0.0003). visits in the frequent user group were less likely to be for a psychiatric complaint (34.3% vs. 81.2%) and less likely to result in a psychiatric admission (18.3% versus 56.7%) as compared to the infrequent user group (p < 0.0001). conclusion: our data indicate that among patients with psychiatric diagnoses, those who make frequent ed visits have a higher rate of comorbid conditions than infrequent visitors. despite their increased use of the ed, frequent visitors have a significantly lower psychiatric admission rate. many of the visits by frequent users are for non-psychiatric complaints and may reflect poor access to outpatient medical and mental health services. emergency departments should consider interventions to help address social and medical issues among mental health patients who frequently use ed services. background: the world health organization estimates that one million people die annually by suicide. in the u.s., suicide is the fourth leading cause of death between the ages of 10 and 65. many of these patients are seen in ed, while outpatient visits for depression are also high. no recent analysis has compared these groups in the recent years. objectives: to determine if there is a relationship between the incidence of suicidal and depressed patients presenting to emergency departments and the incidence of depressed patients presenting to outpatient clinics from 2002-2008. the secondary objective is to analyze trends in suicidal patients in the ed. methods: we used nhamcs (national hospital ambulatory medical care survey) and namcs (national ambulatory medical care survey), national surveys completed by the centers for disease control, which provide a sampling of emergency department and outpatient visits respectively. for both groups, we used mental-health-related icd-9-cm, e codes and reasons for visit. we compared suicidal and depressed patients who presented to the ed, to those who presented to outpatient clinics. our subgroup analyses included age, sex, race/ethnicity, method of payment, regional variation, and urban verses rural distribution. results: ed visits for depression (1.14%) and suicide attempts (0.49%) remained stable over the years, with no significant linear trend. however, office visits for depression significantly decreased from 3.14% of visits in 2002 to 2.65% of visits in 2008. non-latino whites had a higher percentage of ed visits for depression (1.25%) and suicide attempt (0.57%) (p < 0.0001), and a higher percentage of office visits for depression than all other groups. among patients age 50-69 years, ed visits for suicide attempt significantly increased from 0.12% in 2002 to 0.44% in 2008. homeless patients had a higher percent of ed visits for depression (6.5%) and suicide attempt ( background: for potentially high-risk ed patients with psychiatric complaints, efficient ed throughput is key to delivering high-quality care and minimizing time spent in an unsecured waiting room. objectives: we hypothesized that adding a physician in triage would improve ed throughput for psychiatric patients. we evaluated the relationship between the presence of an ed triage physician and waiting room (wr) time, time to first physician order, time to ed bed assignment, and time spent in an ed bed. methods: the study was conducted from 11/2009-2/ 2011 at an academic ed with 55000 annual visits and a dedicated on-site emergency psychiatric unit. we performed a pre/post retrospective observational cohort study using administrative data, including weekend visits from noon-10pm, 8 months pre and post addition of weekend triage physicians. after adjusting for patient age, sex, insurance status, emergency severity index score, mode of arrival, ed occupancy rate, wr count, boarding count, and average wr los, multiple linear regression evaluated the relationship between the presence of a triage physician and four ed throughput outcomes: time spent in the wr, time to first order, time spent in an ed bed, and the total ed los. results: 565 visits met inclusion criteria, 280 in the 8 months before and 285 in the 8 months after physicians were assigned to triage on weekends. table 1 reports demographic data; multivariate analysis results are found in table 2 . the presence of a triage physician was associated with an 8 (95% ci 0.6-15.2) minute increase in wr time and no associated change in time to first order, time spent in an ed bed, or in the overall ed los. conclusion: use of triage physicians has been reported to decrease the time patients spend in an ed bed and improve ed throughput. however, for patients with psychiatric complaints, our analysis revealed a slight increase in wr time without evident change in the time to first order, time spent in an ed bed, or total ed los. improvements in ed throughput for psychiatric patients will likely require system-level changes, such as reducing ed boarding and improving lab efficiency to speed the process of medical clearance and reduce time spent in the unsecured wr. these findings may not be generalizable to eds without a dedicated ed psychiatric unit with full-time social workers to assist with disposition. initial assessment included ciwa scoring, repeated hourly, as well as other variables (see table 1 ). treatment and admission to the inpatient hospital was indicated for a ciwa score of 10 or higher. statistical analysis was performed utilizing repeated measures general linear modeling for ciwa scores and anova for all other variables. results: there were 123 patients who fit the inclusion criteria, with 9 admitted for treatment at initial intake and another 27 admitted during the following 10 hours. the table below compares the three most prevalent ethnic populations seen at our hospital. native americans presented at a significantly younger age (p < 0.05) than the other two ethnicities. initial ciwa scores taken on admission were significantly lower in the native american group than the other two groups (p < 0.05) and at 1 hour a difference existed but failed to reach significance. repeated measures analysis indicate that ciwa scores progressed in a u-shaped curvilinear fashion (see figure 1 ) conclusion: initial assessment utilizing ciwa scores appears to be affected by ethnicity. care must be taken when assessing and making decisions on a single initial ciwa score. further research is needed in this area as our numbers are small and differences might be seen in subsequent scoring. in addition, our study consists of primarily male patients and does not include african-american patients. background: age is a risk factor for adverse outcomes in trauma, yet evidence supporting the use of specific age cut-points to identify seriously injured patients for field triage is limited. objectives: to evaluate under-triage by age, empirically examine the association between age and serious injury for field triage, and assess the potential effect of mandatory age criteria. methods: this was a retrospective cohort study of injured children and adults transported by 48 ems agencies to 105 hospitals in 6 regions of the western u.s. from 2006-2008. hospital records were probabilistically linked to ems records using trauma registries, emergency department data, and state discharge databases. serious injury was defined as an injury severity score (iss) ‡16 (the primary outcome). we assessed under-triage (triage-negative patients with iss ‡16) by age decile, different mandatory age criteria, and used multivariable logistic regression models to test the association (linear and non-linear) between age and iss ‡ 16, adjusted for important confounders. results: 260,027 injured patients were evaluated and transported by ems over the 3-year period. under-triage increased markedly for patients over 60 years, reaching 58% for those over 90 years ( figure 1 ). mandatory age triage criteria decreased under-triage, while substantially increasing over-triage: one iss ‡ 16 patient identified for every 65 additional patients triaged to major trauma centers. among patients not identified by other criteria, age had a strong non-linear association with iss ‡ 16 (p < 0.01); the probability of serious injury steadily increased after 30 years, becoming more notable after 60 years ( figure 2 ). conclusion: under-triage in trauma increases in patients over 60 years, which may be reduced with mandatory age criteria at the expense of system efficiency. among patients not identified by other criteria, serious injury steadily increased after 30 years, though there was no age at which risk abruptly increased. background: although limited resuscitation with hemoglobin-based oxygen carriers (hbocs) improves survival in several polytrauma models, including those of traumatic brain injury (tbi) with uncontrolled hemorrhage (uh) via liver injury, their use remains controversial. objectives: we examine the effect of hboc resuscitation in a swine polytrauma model with uh by aortic tear +/) tbi. we hypothesize that limited resuscitation with hboc would offer no survival benefit and would have similar effects in a model of uh via aortic tear +/) tbi. methods: anesthetized swine subjected to uh inflicted via aortic tear +/) fluid percussion tbi underwent equivalent limited resuscitation with hboc, lr, or hboc+nitroglycerin (ntg) (vasoattenuated hboc) and were observed for 6 hours. comparisons were between tbi and no-tbi groups with adjustment for resuscitation fluid type using two-way anova with interaction and tukey kramer adjustment for individual comparisons. results: there was no independent effect of tbi on survival time after adjustment for fluid type (anova, tbi term p = 0.59) and there was no interaction between tbi and resuscitation fluid type (anova interaction term p = 0.12). there was a significant independent effect of fluid type on survival time (anova p = 0.005 background: intracranial hemorrhage (ich) after a head trauma is a problem frequently encountered in the ed. an elevated inr is recognized as a risk of bleeding. however, in a patient with an inr in normal range, a level associated with a lower risk of ich is not known. objectives: the aim of this study was to identify an inr threshold that could predict a decreased risk of an ich after a head trauma in patients with a normal inr. it is hypothesized that there is a threshold at which the likelihood of bleeding decreases significantly. methods: we did a study using data from a registry of patients with mild to severe head trauma (n = 3356) evaluated in a level i trauma center in canada between march 2008 and february 2011. all the patients with a documented scan interpreted by a radiologist and a normal inr, defined as a value less then 1.6, were included. we determined the correlation between inr value binned by 0.1 and the proportion of patients with an ich. threshold was defined by consensus as an abrupt change of more than 10% in the percentage of patients with ich. univariate frequency distribution was tested with pearson's chisquare test. logistic regression analysis was then used to study the effects of inr on ich with the following confounding factors: age, sex, and intake of warfarin, clopidogrel, or aspirin. results are presented with 95% confidence intervals. results: 751 patients met the inclusion criteria. the mean age was 55.3 years ± 29.9 and 65% were men. 267 patients (35.6%) had an ich on brain scan. we found a significantly lower risk of ich at a threshold of inr less than 1.0 (p < 0.001, univariate or = 0.37, 95%ci 0.25-0.54) and a strong correlation between the risk of bleeding for every increase of the inr (r 2 = 0.8987). in fact, after adjustment for confounding variables, every 0.1 inr increase was associated with an increased risk of having an ich (or 1.50; 95% ci 1.31-1.72). conclusion: we were able to demonstrate an inr threshold under which the probability of ich was significantly lower. we also found a strong association between the risk of bleeding and the increase in inr within a normal range, suggesting that clinicians should not be falsely reassured by a normal inr. our results are limited by the fact that this is a retrospective study and a small proportion of traumatic brain injured patients in our database had no scan or inr at their ed visit. a prospective cohort study would be needed to confirm our results. background: increasingly, patients with tbi are being seen and managed in the emergency neurology setting. knowing which early signs are associated with prognosis can be helpful in directing the acute management. objectives: to determine whether any factors early in the course of head trauma are associated with shortterm outcomes including inpatient admission, in-hospital mortality, and return to the hospital within 30 days. methods: this irb-approved study is a retrospective review of patients head injury presenting to our tertiary care academic medical center during a 9-month period. the dataset was created using redcap, a data management solution hosted by our medical school's center for translational science institute. results: the median age of the cohort (n = 500) was 26, iqr = 15-48yrs, with 62% being male. 84% had a gcs of 13-15 (mild tbi), 3% 9-13 (moderate tbi), and 13% gcs < 8 (severe tbi). 39% of patients were admitted to the hospital. the median length of hospital stay was 2 days, with an iqr of 1-5 days. of those admitted, 53% had an icu stay as well. the median icu los was also 2 days, with an iqr of 1-6days. twenty nine (6%) patients died during their hospital stay. lower gcs was predictive of inpatient admission (p = 0.0003) as well as icu days (p < 0.0001). significant predictors of re-admission to the hospital within 30 days included hypotension (p = 0.002) upon initial presentation. the prehospital and ed gcs scores were not statistically significant. significant predictors of in-hospital death in a model controlling for age included bradycardia (p = 0.0042), hyperglycemia (p = 0.0040), and lower gcs (p = 0.0003). the incidence of bradycardia (hr < 60) was 4.4%. conclusion: early hypotension, hyperglycemia, and bradycardia along with lower initial gcs are associated with significantly higher likelihood of hospital admission, including icu admission, as well as intrahospital death and re-admission. background: over 23,000 people per day require treatment for ankle sprains, resulting in lost workdays and training for athletes. platelet rich plasma (prp) is an autologous concentration of platelets which, when injected into the site of injury, is thought to improve healing by promoting inflammation through growth factor and cytokine release. studies to date have shown mixed results, with few randomized or placebo-controlled trials. the lower extremity functional scale (lefs) is a previously validated objective measure of lower extremity function. objectives: is prp helpful in acute ankle sprains in the the emergency department? methods: prospective, randomized, double-blinded, placebo-controlled trial. patients with severe ankle sprains and negative x-rays were randomized to trial or placebo. severe was defined as marked swelling and ecchymosis and inability to bear weight. both groups had 50 cc of blood drawn. trial group blood was centrifuged with a magellan autologous platelet separator (arteriocyte, cleveland) to yield 3-4 cc of prp. prp along with 0.5 cc of 1% lidocaine and 0.5 cc of 0.25% bupivicaine was injected at the point of maximum tenderness by a blinded physician under ultrasound guidance. control group blood was discarded and participants were injected in a similar fashion substituting sterile 0.9% saline for prp. both groups had visual analog scale (vas) pain scores and lefs on days 0, 3, 8, and 30. all participants had a posterior splint and were made non weight bearing for 3 days after which they were reexamined, had their splint removed, and were asked to bear weight as tolerated. participants were instructed not to use nsaids during the trial. results: 1156 patients were screened and 37 were enrolled. four withdrew before prp injection was complete. eighteen were randomized to prp and 15 to placebo. see tables for results. vas and lefs are presented as means with sd in parentheses. demographics were not statistically different between groups. conclusion: in this small study, prp did not appear to offer benefit in either pain control or healing. both groups had improvement in their pain and functionality and did not differ significantly during the study period. limitations include small study size and large number of participant refusals. methods: a structured chart review of all icd-9 radius fracture coded charts spanning march 18, 2010 to july 17, 2011 was conducted. specific variable data were collected and categorized as follows: age, moi, body mass index, and fracture location. the charts were reviewed by two medical students, with 10% of the charts reviewed by both students to confirm inter-rater reliability. frequencies and inter-quartile ranges were determined. comparisons were made with fisher's exact test and multiple logistic regression. results: 187 charts met inclusion criteria. 46 charts were excluded due to one of the following reasons: no fracture or no x-ray (14), isolated ulnar fracture (19), or undocumented or penetrating moi (13). of the analyzed patients (n = 141), distal radius fractures were most common (66%), followed by proximal (32%) and midshaft (2%). chart reviewers were found to be reliable (j = 1). age and moi were significantly associated with fracture location (see table) . ages 18-54 and bike accidents were more strongly associated with proximal radius fractures (odds ratio: 12 [2-94] and 5 [2-13], respectively). conclusion: patients presenting to our inner city ed with a radius fracture are more likely to have a distal fracture. adults 18-54 and bike accidents had a significantly higher incidence of proximal fractures than other ages or mois. background: trauma centers use guidelines to determine the need for a trauma surgeon in the ed on patient arrival. a decision rule from loma linda university that includes penetrating injury and tachycardia was developed to predict which pediatric trauma patients require emergent intervention, and thus are most likely to benefit from surgical presence in the ed. objectives: our goal was to validate the loma linda rule (llr) in a heterogeneous pediatric trauma population and to compare it to the american college of surgeons' major resuscitation criteria (mrc). we hypothesized that the llr would be more sensitive than the mrc for identifying the need for emergent operative or procedural intervention. methods: we performed a secondary analysis of prospectively collected trauma registry data from two urban level i pediatric trauma centers with a combined annual census of approximately 115,000 visits. consecutive patients <15 years old with blunt or penetrating trauma from 1993 through 2010 were included. patient demographics, injury severity scores (iss), times of ed arrival and surgical intervention, and all variables of both rules were obtained. the outcome (emergent operative intervention within 1 hour of ed arrival or ed cricothyroidotomy or thoracotomy) was confirmed by trained, blinded abstractors. sensitivities, specificities, and 95% confidence intervals (cis) were calculated for both rules. results: 8,079 patients were included with a median age of 5.9 years and a median iss of 9. emergent intervention was required in 51 patients (0.6%). the llr had a sensitivity ranging from 59.4%-59.7% (95% ci: 25.9%-93.5%) and specificity ranging from 49.5%-86.5% (95% ci: 21.6%-82.1%) between both institutions. the mrc had a sensitivity ranging from 73.6%-81.6% (95% ci: 54.7%-95.1%) and specificity ranging from 69.4%-84.7% (95% ci: 54.7%-90.1%) between institutions. conclusion: emergent intervention is rare in pediatric trauma patients. the mrc was more sensitive for predicting the need for emergent intervention than the llr. neither set of criteria was sufficiently accurate to recommend their routine use for pediatric trauma patients. droperidol for sedation of acute behavioural disturbance leonie a. calver 1 , colin page 2 , michael downes 3 , betty chan 4 , geoffrey k. isbister 1 1 calvary mater newcastle and university of newcastle, newcastle, australia; 2 princess alexandra hospital, brisbane, australia; 3 calvary mater newcastle, newcastle, australia; 4 prince of wales hospital, sydney, australia background: acute behavioural disturbance (abd) is a common occurrence in the emergency department (ed) and is a risk to staff and patients. there remains little consensus on the most effective drug for sedation of violent and aggressive patients. prior to the food and drug administration's black box warning, droperidol was commonly used and was considered safe and effective. objectives: this study aimed to investigate the effectiveness of parenteral droperidol for sedation of abd. methods: as part of a prospective observational study, a standardised protocol using droperidol for the seda-acute and delayed behavioral deficits were demonstrated in this rat model of co toxicity, which parallels the neurocognitive deficit pattern observed in humans (see figure) . similar to prior studies, pathologic analysis of brain tissue demonstrated the highest percentage of necrotic cells in the cortex, pyramidal cells, and cerebellum. the collected data are summarized in the table. we have developed an animal model of severe co toxicity evidenced by behavioral deficits and neuronal necrosis. future efforts will compare neurologic outcomes in severely co poisoned rats treated with hypothermia and 100% inspired o2 versus hbo to normothermic controls treated with 100% inspired o2. increasing in popularity, attracting more than 70,000 annual participants worldwide. prior studies have consistently documented renal function impairment, but only after race completion. the incidence of renal injury during these multi-day ultramarathons is currently unknown. this is the first prospective cohort study to evaluate the incidence of acute kidney injury (aki) in runners during a multi-day ultramarathon foot race. objectives: to assess the effect of inter-stage recovery versus cumulative damage on resulting renal function during a multi-day ultramarathon. methods: demographic and biochemical data gathered via phlebotomy and analyzed by istatò (abbott, nj) were collected at the start and finish of day 1 (25 miles), 3 (75 miles), and 5 (140 miles) during racing the planet'sò150-mile, 7-day self-supported desert ultramarathons. pre-established rifle criteria using creatinine (cr) and glomerular filtration rate (gfr) defined aki as ''no injury'' (cr <1.5x normal, decrease of gfr <25%), ''risk'' (cr 1.5x normal, decrease of gfr by 25-49%), and ''injury'' (cr 2x normal, decrease of gfr by 50-75%). results: thirty racers (76% male) with a mean (+/) sd) age of 39 + /-10 years were studied during the 2008 sahara (n = 7, 23.3%), 2008 gobi (n = 10, 33%), and 2009 namibia (n = 13, 43.3%) events. the average decrease in gfr from day 1 start to day 1 finish was 28 + /-25 (p < 0.001, 95% ci 18.5-37.6); day 1 start to day 3 finish was 29.6 + /-20.1 (p < 0.001, 95% ci 18.4-40.7); and day 1 start to day 5 finish was 30.9 ± 17.5 (p < 0.001, 95% ci 20.8-41). runners categorized as risk and injury for aki after stage 1 was 44.8 % and 10%; after stage 3 was 67% and 13%, and after stage 5 was 57.1% and 7.1% conclusion: the majority of participants developed significant levels of renal impairment despite recovery intervals. given the changes in renal function, potentially harmful non-steroidal anti-inflammatory drugs should be minimized to prevent exacerbating acute kidney injury. background: more than 10% of the elderly abuse prescription drugs, and emergency medicine providers frequently struggle to identify features of opioid addiction in this population. the prescription drug use questionnaire (pduqp) is a validated, 42-item, patient-administered tool developed to help health care providers better identify problematic opioid use, or dependence, in patients who receive opioids for the treatment of chronic pain. objectives: to identify the prevalence of prescription drug misuse features in elderly ed patients. methods: this cross-sectional, observational study was conducted between 07/2011 and 08/2011 in the ed of an urban, university-affiliated community hospi-tal that serves a large geriatric population. all patients aged 65 to 89 inclusive were eligible, and were recruited on a convenience basis. exclusion criteria included known dementia, and critical illness. outcomes of interest included self-reported history of prior prescription opioid use, substance abuse history, aberrant medication-taking behaviors, and pduqp results. results: one hundred patients were approached for participation. two were excluded for inability to read english, three were receiving analgesia for metastatic cancer, 28 had never taken a prescription opioid, and seven refused to participate beyond pre-screening. sixty patients completed the study (see table 1 ). of those, 13.3% reported four or more visits within 12 months; chronic pain was reported by 56.7%; debilitating pain by 55.9%; prior pain management referral by 18.3%; and storing opioids for future use by 30%. seventeen patients reported current prescription opioid use, and were administered the pduqp (see figure) . in this population, 47.1% thought their pain was not adequately being treated; 41.2% reported having to increase the amount of pain medication they were taking over the prior 6 months; 35.3% saved up future pain medication; 11.8% had doctors refuse to give them pain medication for fear that the patient would abuse the prescription opioids; and 29.4% reported having a previous drug or alcohol problem. conclusion: screening instruments, such as the pduqp, facilitate identification of geriatric patients with features of opioid misuse. a high proportion of patients in this study save opioids for further use. interventions for safe medication disposal may decrease access to opioids and subsequent morbidity. age extremes, male sex, and several chronic health conditions were associated with increased odds of heat stroke, hospital admission, and death in the ed by a factor of 2-3. chronic hematologic disease (e.g. anemia) was associated with a 10-12 fold increase in adjusted odds of each of these outcomes. conclusion: hri imposes a substantial public health burden, and a wider range of chronic conditions confer susceptibility than previously thought. males, older adults, and patients with chronic conditions, particularly anemia, are likely to have more severe hri, be admitted, or die in the ed. background: carbon monoxide (co) poisoning is a remarkable cause of death worldwide. co, produced by the incomplete combustion of hydrocarbons, has many toxic effects on especially the heart and brain. co binds strongly to cytochrome oxidase, hemoglobin, and myoglobin causing hypoxia of organs and issues. co converts hemoglobin to carboxyhemoglobin and makes transport of oxygen through the body impossible and causes severe hypoxia. objectives: the aim of this study is to investigate the levels of s100b and neuron specific enolase (nse) measured both during admittance and at the sixth hour of hyperbaric and normobaric oxygen therapy carried out on patients with a diagnosis of co poisoning. methods: the study is designed as a prospective observational laboratory study. forty patients were enrolled in the study: 20 underwent normobaric oxygen therapy (nbot) and the other 20 underwent hyperbaric oxygen therapy (hbot). levels of s100b and nse were measured both during admittance and at the sixth hour of admittance of all patients. demographic data, clinical characteristics, and outcome measures were recorded. all data were statistically analyzed. results: in both treatment groups, mean levels of nse after therapy were significantly lower than admittance levels. although levels of nse measured before and 6 hours after treatment in hbot group were high, the difference between groups was not statistically significant (p > 0.05). in both treatment groups, mean levels of s100b after therapy were significantly lower than admittance levels; likewise nse. although levels of s100b measured before and 6 hours after treatment in hbot group were high, the difference between groups was not statistically significant (p > 0.05). additionally, while levels of s100b measured after treatment in the hbot group were lower compared to the nbot group, the difference between groups was also not statistically significant (p > 0.05). conclusion: levels of s100b and nse as evidence for brain injury elevation in case of co poisoining and decrease by therapy according to our study as well as previous studies. decrease in levels of s100b is more significant. according to our results, s100b and nse may be useful markers in case of co poisoning; however, we did not meet any data providing more value in determining hbot indications and determining levels of cohb in the management of patients with a diagnosis of co poisoining. neurons objectives: this study was conducted to determine if neurons in the dmh, and its neighbor the paraventricular hypothalamus (pvn), were likewise involved in mdma-mediated neuroendocrine responses, and if serotonin 1a receptors (5-ht1a) play a role in this regional response. methods: in both experiments, male sprague dawley rats (n = 5-12/group) were implanted with bilateral cannulas targeting specific regions of the brain, i.v. catheters for drug delivery, and i.a. catheters for blood withdrawal. experiments were conducted in raturn cages, which allow blood withdrawal and drug administration in free moving animals while recording their locomotion. in the first experiment, rats were microinjected into the dmh, the pvn, or a region between, with the gabaa agonist muscimol (80 pmol/100nl/side) or pbs (100nl) and 5 min later were injected with either mdma (7.5 mg/kg i.v.) or an equal volume of saline. blood was withdrawn prior to microinjections and 15 minutes after mdma for ria measurement of plasma acth. locomotion was recorded throughout the experiment. in a separate experiment of identical design, either the 5-ht1a antagonist way 100635 (way, 5 nmol/100 nl/side) or saline was microinjected followed by i.v. injection of mdma or saline. in both experiments, increases in acth and distance traveled were compared between groups using an anova analysis. results: when compared to controls, microinjections of muscimol into the dmh, pvn, or the area in between attenuated plasma increases in acth and locomotion evoked by mdma. when microinjected into the dmh or pvn, way had no effect on acth, but when injected into the region of the dmh it significantly increased locomotion. background: poor hand-offs between physicians when admitting patients have been shown to be a major source of medical errors. objectives: we propose that training in a standardized admissions protocol by emergency medicine (em) to internal medicine (im) residents would improve the quality of and quantity of communication of vital patient information. methods: em and im residents at a large academic center developed an evidence-based admission handover protocol termed the '7ps' (table 1) . em and im residents received '7ps' protocol training. im residents recorded prospectively how well each of the seven ps were communicated during each admission pre-and post-intervention. im residents also assessed the overall quality of the handover using a likert scale. the primary outcome was the change in the number of 'ps' conveyed by the em resident to the accepting im resident. data were collected for six weeks before and then for six weeks starting two weeks after the educational intervention. results: there were 78 observations recorded in the preintervention (control) group and 48 observations in the post-intervention group. for each of the seven 'ps' the percentage of observation where all of the information was communicated is shown in table 2 . the communication of 'ps' increased following the intervention. this rise was statistically significant for patient information and pending tests. in the control group the mean of total communicated ps was 5 and in the intervention group, the mean increased to 6 (p < 0.005). the quality of the handover communication had a mean rating of 3.9 in the control group and 4.3 in the intervention group (p < 0.05). conclusion: this educational intervention in a cohort of em and im residents improved the quality and quantity of vital information communicated during patient handovers. the intervention was statistically significant for patient information transfer and tests pending. the results are limited by study size. based on our preliminary data, an agreed-upon handover protocol with training improved the amount and quality of communication during patients' hospital admission on simple items that were likely had been taken for granted as routinely transmitted. we recruited a convenience sample of residents and students rotating in the pediatric emergency department. a two-sided form had the same seven clinical decisions on each side: whether to perform blood, urine, spinal fluid tests, imaging, iv fluids, antibiotics, or a consult. the rating choices were: definitely not, probably not, probably would, and definitely would. trainees rated each decision after seeing a patient, but before presenting to the preceptor, who, after evaluating the patient, rated the same seven decisions on the second side of the form. the preceptor also indicated the most relevant decision (mrd) for that patient. we examined the validity of the technique using hypothesis testing; we posited that residents would have a higher degree of concordance with the preceptor than would medical students. this was tested using dichotomized analyses (accuracy, kappa) and roc curves with the preceptor decision as the gold standard. results: thirty-one students completed 130 forms (median 4 forms; iqr 2,6) and 23 residents completed 206 (6; iqr 3,12). preceptors included 24 attending physicians and 3 fellows (9; iqr 4, 21). students were concordant with preceptors in 70% (k = 0.38) of mrd while residents agreed in 79.6% (p = 0.045), k = 0.59. roc analysis revealed significant differences between students and residents in the auc for the mrd (0.84 vs 0.72; p = 0.03). conclusion: this measure of trainee-preceptor concordance requires further research but may eventually allow for assessment of trainee clinical decision-making. it also has the pedagogical advantage of promoting independent trainee decision-making. background: basic life support (bls) and advanced cardiac life support (acls) are integral parts of emergency cardiac care. this training is usually reserved in most institutions for residents and faculty. the argument can be made to introduce bls and acls training earlier in the medical student curriculum to enhance acquisition of these skills. objectives: the goal of the survey was to characterize the perceptions and needs of graduating medical students in regards to bls and acls training. methods: this was a survey-based study of graduating fourth year medical students at a u.s. medical school. the students were surveyed before voluntarily participating in a student-led acls course in march of their final year. the surveys were distributed before starting the training course. both bls and acls training, comfort levels, and perceptions were assessed in the survey. results: of the 182 students in the graduating class, 152 participated in the training class with 109 (72%) completing the survey. 50% of students entered medical school without any prior training and 49% started clinics without training. 83.5% of students reported witnessing an average of 3.0 in-hospital cardiac arrests during training (range of 0-20). overall, students rated their preparedness 2.0 (sd 1.0) for adult resuscitations on a 1-5 likert scale with 1 being the unprepared. 98% and 92% of students believe that bls and acls should be included in the medical student curriculum respectively with a preference for teaching before starting clerkships. 36% of students avoided participating in resuscitations due to lack of training. of those, 95% said they would have participated had they been trained. conclusion: to our knowledge, this is one of the first studies to address the perceptions and needs for bls and acls training in u.s. medical schools. students feel that bls and acls training is needed in their curriculum and would possibly enhance perceived comfort levels and willingness to participate in resuscitations. background: professionalism is one of six core competency requirements of the acgme, yet defining and teaching its principles remains a challenge. the ''social contract'' between physician and community is clearly central to professionalism so determining the patient's understanding of the physician's role in the relationship is important. because specialization has created more narrowly focused and often quite different interactions in different medical environments, the patient concept of professionalism in different settings may vary as well. objectives: we hoped to determine if patients have different conceptions of professionalism when considering physicians in different clinical environments. methods: patients were surveyed in the waiting room of an emergency department, an outpatient internal medicine clinic, and a pre-operative/anesthesia clinic. the survey contained 18 examples of attributes, derived from the american board of internal medicine's eight characteristics of professionalism. participants were asked to rate, on a 10-point scale, the importance that a physician possess each attribute. an anova analysis was used to compare the sites for each question. results: of 604 who took the survey, 200 were in the emergency department, 202 were in the medicine clinic, and 202 were in the pre-operative clinic. females comprised 56% of the study group and the average age was 49 with a range from 18 to 94. there was a significant difference on the attribute of ''providing a portion of work for those who cannot pay;'' this was rated higher in the emergency department (p = 0.003). there was near-significance (p = 0.05) on the attribute of ''being able to make difficult decisions under pressure,'' which was rated higher in the pre-op clinic. there was no difference for any of the other questions. the top four professional attributes at each clinical site were the same -''honesty,'' ''excellence in communication and listening,'' ''taking full responsibility for mistakes,'' and ''technical competence/ skill;'' the bottom two were ''being an active leader in the community'' and ''patient concerns should come before a doctor's family commitments.'' conclusion: very few differences between clinical sites were found when surveying patient perception of the important elements of medical professionalism. this may suggests a core set of values desired by patients for physicians across specialties. emergency medicine faculty knowledge of and confidence in giving feedback on the acgme core competencies todd guth, jeff druck, jason hoppe, britney anderson university of colorado, aurora, co background: the acgme mandates that residency programs assess residents based upon six core competencies. although the core competencies have been in place for a number of years, many faculty are not familiar with the intricacies of the competencies and have difficulty giving competency-specific feedback to residents. objectives: the purpose of the study is to determine the extent to which emergency medicine (em) faculty can identify the acgme core competencies correctly and to determine faculty confidence with giving general feedback and core competency focused feedback to em residents. methods: design and participants: at a single department of em, a survey of twenty-eight faculty members, their knowledge of the acgme core competencies, and their confidence in providing feedback to residents was conducted. confidence levels in giving feedback were scored on a likert scale from 1 to 5. observations: descriptive statistics of faculty confidence in giving feedback, identification of professional areas of interest, and identification of the acgme core competencies were determined. mann-whitney u tests were used to make comparisons between groups of faculty given the small sample size of the respondents. results: there was a 100% response rate of the 28 faculty members surveyed. eight faculty members identified themselves as primarily focused on education. although those faculty members identifying themselves as focused on education scored higher than non-education focused faculty for all type of feedback (general feedback, constructive feedback, negative feedback), there was only a statistical difference in confidence levels 4.57 versus 2.65 (p < 0.002) for acgme core competency specific feedback when compared to noneducation focused faculty. while education focused faculty correctly identified all six of acgme core competencies 94% of the time, not one of the non-education focused faculty identified all six of the core competencies correctly. non-education focused faculty only correctly identified three or more competencies 25% of the time. conclusion: if residency programs are to assess residents using the six acgme core competencies, additional faculty development specific to the core competencies will be needed to train all faculty on the core competencies and on how to give core competency specific feedback to em residents. there is no clear consensus as to the most effective tool to measure resident competency in emergency ultrasound. objectives: to determine the relationship between the number of scans and scores on image recognition, image acquisition, and cognitive skills as measured by an objective structured clinical exam (osce) and written exam. secondarily, to determine whether image acquisition, image recognition, and cognitive knowledge require separate evaluation methodologies. methods: this was a prospective observational study in an urban level i ed with a 3-year acgme-accredited residency program. all residents underwent an ultrasound introductory course and a one-month ultrasound rotation during their first and second years. each resident received a written exam and osce to assess psychomotor and cognitive skills. the osce had two components: (1) recognition of 22 images, and (2) acquisition of images. a registered diagnostic medical sonographer (rdms)-certified physician observed each bedside examination. a pre-existing residency ultrasound database was used to collect data about number of scans. pearson correlation coefficients were calculated for number of scans, written exam score, image recognition, and image acquisition scores on the osce. results: twenty-nine residents were enrolled from march 2010 to february 2011 who performed an average of 247 scans (range 118-617). there was no significant correlation between number of scans and written exam scores. an analysis of the number of scans and the ocse found a moderate correlation with image acquisition (r = 0.42, p = 0.029) and image recognition (r = 0.61, p = <0.01)). pearson correlation analysis between the image acquisition score and image recognition score found that there was no correlation (r = 0.175, p = 0.383). there was a moderate correlation with image acquisition scores to written scores (r = 0.541, p = 0.025) and image recognition scores to written scores (r = 0.596, p = 0.019). conclusion: the number of scans does not correlate with written tests but has a moderate correlation with image acquisition and image recognition. this suggests that resident education should include cognitive instruction in addition to scan numbers. we conclude that multiple methods are necessary to examine resident ultrasound competency. background: although emergency physicians must often make rapid decisions that incorporate their interpretation of an ecg, there is no evidence-based description of ecg interpretation competencies for emergency medicine (em) trainees. the first step in defining these competencies is to develop a prioritized list of ecg findings relevant to em contexts. objectives: the purpose of this study was to categorize the importance of various ecg diagnoses and/or findings for the em trainee. methods: we developed an extensive list of potentially important ecg diagnoses identified through a detailed review of the cardiology and em literature. we then conducted a three-round delphi expert opinion-soliciting process where participants used a five-point likert scale to rate the importance of each diagnosis for em trainees. consensus was defined as a minimum of 75 percent agreement on any particular diagnosis at the second round or later. in the absence of consensus, stability was defined as a shift of 20 percent or less after successive rounds. results: twenty-two em experts participated in the delphi process, sixteen (72%) of whom completed the process. of those, fifteen were experts from eleven different em training programs across canada and one was a recognized expert in em electrocardiography. overall, 77 diagnoses reached consensus, 42 achieved stability, and one diagnosis achieved neither consensus nor stability. out of 120 potentially important ecg diagnoses, 53 (43%) were considered ''must know'' diagnoses, 62 (51%) ''should know'' diagnoses, and 7 (6%) ''nice to know'' diagnoses. conclusion: we have categorized ecg diagnoses within an em training context, knowledge of which may allow clinical em teachers to establish educational priorities. this categorization will also facilitate the development of an educational framework to establish em trainee competency in ecg interpretation. ''rolling refreshers background: cardiac arrest survival rates are low despite advances in cardiopulmonary resuscitation. high quality cpr has been shown to impart greater cardiac arrest survival; however, retention of basic cpr skills by health care providers has been shown to be poor. objectives: to evaluate practitioner acceptance of an in-service cpr skills refresher program, and to assess for operator response to real-time feedback during refreshers. methods: we prospectively evaluated a ''rolling refresher'' in-service program at an academic medical center. this program is a proctored cpr practice session using a mannequin and cpr-sensing defibrillator that provides real-time cpr quality feedback. subjects were basic life support-trained providers who were engaged in clinical care at the time of enrollment. subjects were asked to perform two minutes of chest compressions (ccs) using the feedback system. ccs could be terminated when the subject had completed approximately 30 seconds of compressions with <3 corrective prompts. a survey was then completed by to obtain feedback regarding the perceived efficacy of this training model. cpr quality was then evaluated using custom analysis software to determine the percent of cc adequacy in 30-second intervals. results: enrollment included 88 subjects from the emergency department and critical care units (55 nurses, 17 physicians, 16 students and allied health professionals). all participants completed a survey and 61 cpr performance data logs were obtained. positive impressions of the in-service program were registered by 81% (71/88) and 74% (65/88) reported a self-perceived improvement in skills confidence. eighty-three percent (73/88) of respondents felt comfortable performing this refresher during a clinical shift. thirtynine percent (24/61) of episodes exhibited adequate cc performance with approximately 30 seconds of cc. of the remaining 37 episodes, 71.1 ± 29.2% of cc were adequate in the first 30 seconds with 80.1 ± 28.6% of cc adequate during the last 30 second interval (p = 0.1847). of these 37 individuals, 30 improved or had no change in their cpr skills, and 7 individuals skills declined during cc performance (p = 0.007). conclusion: implementation of a bedside cpr skill refresher program is feasible and is well received by hospital staff. real time cpr feedback improved upon cpr skill performance during the in-service session. teaching emergency medicine skills: is a self-directed, independent, online curriculum the way of the future? tighe crombie, jason r. frank, stephen noseworthy, richard gerein, a. curtis lee university of ottawa, ottawa, on, canada background: procedural competence is critical to emergency medicine, but the ideal instructional method to acquire these skills is not clear. previous studies have demonstrated that online tutorials have the potential to be as effective as didactic sessions at teaching specific procedural skills. objectives: we studied whether a novel online curriculum teaching pediatric intraosseus (io) line insertion to novice learners is as effective as a traditional classroom curriculum in imparting procedural competence. methods: we conducted a randomized controlled educational trial of two methods of teaching io skills. preclinical medical students with no past io experience completed a written test and were randomized to either an online or classroom curriculum. the online group (og) were given password-protected access to a website and instructed to spend 30 minutes with the material while the didactic group (dg) attended a lecture of similar duration. participants then attended a 30-minute unsupervised manikin practice session on a separate day without any further instruction. a videotaped objective structured clinical examination (osce) and post-course written test were completed immediately following this practice session. finally, participants were crossed over into the alternate curriculum and were asked to complete a satisfaction survey that compared the two curricula. results were compared with a paired t-test for written scores and an independent t-test for osce scores. results: sixteen students completed the study. pre-course test scores of the two groups were not significantly different prior to accessing their respective curricula (mean scores of 32% for og and 34% for dg, respectively; p > 0.05). post-course written scores were also not significantly different (both with means of 76%; p > 0.05); however, for the post-treatment osce scores, the og group scored significantly higher than the dg group (mean scores of 92.6% and 88.1%; t(14) = 1.76, p < 0.05.) conclusion: this novel online curriculum was superior to a traditional didactic approach to teaching pediatric io line insertion. novice learners assigned to a selfdirected online curriculum were able to perform an emergency procedural skill to a high level of performance. em educators should consider adopting online teaching of procedural skills. background: applicants to em residency programs obtain information largely from the internet. curricular information is available from a program's website (pw) or the saem residency directory (sd). we hypothesize that there is variation between these key sources. objectives: to identify discrepancies between each pw and sd. to describe components of pgy1-3 em residency programs' curricula as advertised on the internet. methods: pgy1-3 residencies were identified through the sd. data were abstracted from individual sd and pw pages identifying pre-determined elements of interest regarding rotations in icu, pediatrics, inpatient (medicine, pediatrics, general surgery), electives, orthopedics, toxicology, and anesthesia. agreement between the sd and pw was calculated using a cohen's unweighted kappa calculation. curricula posted on pws were considered the gold standard for the programs' current curricula. results: a total of 117 pgy1-3 programs were identified through the sd and confirmed on the pw. ninetyone of 117 programs (78%) had complete curricular information on both sites. only these programs were included in the kappa analysis for sd and pw comparisons. of programs with complete listings, 66 of 91 programs (73%) had at least one discrepancy. the agreement of information between pw and sd revealed a kappa value of 0.26 (95% ci 0.19-0.33). analysis of pw revealed that pgy1-3 programs have an average of 4.15 (range, 2-9), 3.1 (range, 1-6), 1.7 (range, 0-4), and 1.0 (range, 0-4) blocks of icu, pediatrics, elective, and inpatient, respectively. common but not rrc-mandated rotations in orthopedics, toxicology, and anesthesiology are present in 77, 80, and 93 percent of programs, respectively. conclusion: publicly accessible curricular information through the sd and pw for pgy1-3 em programs only has fair agreement (using commonly accepted kappa value guides). applicants may be confused by the variability of data and draw inaccurate conclusions about program curricula. from the gravid uterus and improves cardiac output; however, this theory has never been proven. objectives: we set out to determine the difference in inferior vena cava (ivc) filling when third trimester patients were placed in supine, llt, and right lateral tilt (rlt) positions using ivc ultrasound. methods: healthy pregnant women in their third trimester presenting to the labor and delivery suite were enrolled. patients were placed in three different positions (supine, rlt, and llt) and ivc maximum (max) and minimum (min) measurements were obtained using the intercostal window in short axis approximately two centimeters below the entry of the hepatic veins. ivc collapse index (ci) was calculated for each measurement using the formula (max-min)/max. in addition, blood pressure, heart rate, and fetal heart rate were monitored. patients stayed in each position for at least 3 minutes prior to taking measurements. we compared ivc measurements using a one-way analysis of variance for repeated measures. results: twenty patients were enrolled. the average age was 25 years (sd 5.7) with a mean estimated gestational age of 39.5 weeks (sd 1.4). there were no significant differences seen in ivc filling in each of the positions (see table 1 ). in addition, there were no differences in hemodynamic parameters between positions.ten (50%) patients had the largest ivc measurement in the llt position, 7 (35%) patients in the rlt position, and 3 (15%) in the supine position. conclusion: there were no significant differences in ivc filling between patient positions. for some third trimester patients llt may not be the optimal position for ivc filling. background: although the acgme and rrc require competency assessment in ed bedside ultrasound (us), there are no standardized assessment tools for us training in em. objectives: using published us guidelines, we developed four observed structured competency evalua-tions (osce) for four common em us exams: fast, aortic, cardiac, and pelvic. inter-rater reliability was calculated for overall performance and for the individual components of each osce. methods: this prospective observational study derived four osces that evaluated overall study competency, image quality for each required view, technical factors (probe placement, orientation, angle, gain, and depth), and identification of key anatomic structures. em residents with varying levels of training completed an osce under direct observation of two em-trained us experts. each expert was blinded to the other's assessment. overall study competency and image quality of each required views were rated on a five-point scale (1poor, 2-fair, 3-adequate, 4-good, 5-excellent), with explicit definitions for each rating. each study had technical factors (correct/incorrect) and anatomic structures (identified/not identified) assessed as binary variables. data were analyzed using cohen's and weighted k, descriptive statistics, and 95% ci. results: a total of 185 us exams were observed, including 33 fast, 53 cardiac, 53 aorta, and 46 pelvic. total assessments included 185 ratings of overall study competency, 691 ratings of required view image quality, 2998 ratings of technical factors, and 2978 ratings of anatomic structures. inter-rater assessment of overall study competency showed excellent agreement, raw agreement 0.84 (0.77, 0.89), weighted k 0.87 (0.82, 0.91). ratings of required view image quality showed excellent agreement: raw agreement 0.75 (0.72, 0.79), weighted k 0.82 (0.79, 0.84). inter-rater assessment of technical factors showed substantial agreement: raw agreement 0.96 (0.95, 0.97), cohen's k 0.78 (0.74, 0.82). ratings of identification of anatomic structures showed substantial agreement: raw agreement 0.86 (0.85, 0.88), cohen's k 0.64 (0.60, 0.67). conclusion: inter-rater reliability is substantial to excellent using the derived ultrasound osces to rate em resident competency in fast, aortic, cardiac, and pelvic ultrasound. validation of this tool is ongoing. a objectives: the objective of this study was to identify which transducer orientation, longitudinal or transverse, is the best method of imaging the axillary vein with ultrasound, as defined by successful placement in the vein with one needle stick, no redirections, and no complications. methods: emergency medicine resident and attending physicians at an academic medical center were asked to cannulate the axillary vein in a torso phantom model. the participants were randomized to start with either the longitudinal or transverse approach and completed both sequentially, after viewing a teaching presentation. participants completed pre-and post-attempt questionnaires. measurements of each attempt were taken regarding time to completion, success, skin punctures, needle redirections, and complications. we compared proportions using a normal binomial approximation and continuous data using the t-distribution, as appropriate. a sample size of 57 was chosen based on the following assumptions: power, 0.8; significance, 0.05; effect size, 50% versus 75%. results: fifty-seven operators with a median experience of 85 prior ultrasounds (26 to 120 iqr) participated. first-attempt success frequency was 39/57 (0.69) for the longitudinal method and 21/57 (0.37) for the transverse method (difference 0.32, 95% ci 0.12-0.51); this difference was similar regardless of operator experience. the longitudinal method had fewer redirections (mean difference 1.8, 95% ci 0.8-2.8) and skin punctures (mean difference 0.3, 95% ci )2 to 0.18). arterial puncture occurred in 2/57 longitudinal attempts and 7/ 57 transverse attempts, with no pleural punctures in either group. among successful attempts, the time spent was 24 seconds less for longitudinal method (95% ci 3-45). though 93% of participants had more experience with the transverse method prior to the training session, 58% indicated after the session that they preferred the longitudinal method. methods: a prospective single-center study was conducted to assess the compressibility of the basilic vein with ultrasound. healthy study participants were recruited. the compressibility was assessed at baseline, and then further assessed with one proximal tourniquet, two tourniquets (one distal and one proximal), and a proximal blood pressure cuff inflated to 150 mmhg. compressibility was defined as the vessel's resistance to collapse to external pressure and rated as completely compressible, moderately compressible, or mildly compressible after mild pressure was applied with the ultrasound probe. results: one-hundred patients were recruited into the study. ninety-eight subjects were found to have a completely compressible basilic vein at baseline. when one tourniquet and two tourniquets were applied 64 and 58 participants, respectively, continued to have completely compressible veins. a fisher's exact test comparing one versus two tourniquets revealed no difference between these two techniques (p = 0.46). only two participants continued to have completely compressible veins following application of the blood pressure cuff. the compressibility of this group was found to be statistically significant by fisher's exact test compared to both tourniquet groups (p < 0.0001). furthermore, 24 participants with the blood pressure cuff applied were found to have moderately compressible veins and 72 participants were found to have mildly compressible veins. conclusion: tourniquets and blood pressure cuffs can both decrease the compressibility of peripheral veins. while there was no difference identified between using one and two tourniquets, utilization of a blood pressure cuff was significantly more effective to decrease compressibility. the findings of this study may be utilized in the emergency department when attempting to obtain peripheral venous access, specifically supporting the use of blood pressure cuffs to decrease compressibility. background: electroencephalography (eeg) is an underused test that can provide valuable information in the evaluation of emergency department (ed) patients with altered mental status (ams). in ams patients with nonconvulsive seizure (ncs), eeg is necessary to make the diagnosis and to initiate proper treatment. yet, most cases of ncs are diagnosed >24 h after ed presentation. obstacles to routine use of eeg in the ed include space limitations, absence of 24/7 availability of eeg technologists and interpreters, and the electrically hostile ed environment. a novel miniature portable wireless device (microeeg) is designed to overcome these obstacles. objectives: to examine the diagnostic utility of micro-eeg in identifying eeg abnormalities in ed patients with ams. methods: an ongoing prospective study conducted at two academic urban eds. inclusion: patients ‡13 years old with ams. exclusion: an easily correctable cause of ams (e.g. hypoglycemia, opioid overdose). three 30-minute eegs were obtained in random order from each subject beginning within one hour of presentation: 1) a standard eeg, 2) a microeeg obtained simultaneously with conventional cup electrodes using a signal splitter, and 3) a microeeg using an electrocap. outcome: operative characteristics of micro-eeg in identifying any eeg abnormality. all eegs were interpreted in a blinded fashion by two board-certified epileptologists. within each reader-patient pairing, the accuracy of eegs 2 and 3 were each assessed relative to eeg 1. sensitivity, specificity, and likelihood ratios (lr) are reported for microeeg by standard electrodes and electrocap (eegs 2 and 3). inter-rater variability for eeg interpretations is reported with kappa. results: the interim analysis was performed on 130 consecutive patients (target sample size: 260) enrolled from may to october 2011 (median age: 61, range: 13-100, 40% male). overall, 82% (95% confidence interval [ci], 76-88%) of interpretations were abnormal (based on eeg1). kappa values representing the agreement of neurologists in interpretation of eeg 1-3 were 0.54 (0.36-0.73), 0.57 (0.39-0.75), and 0.55 (0.37-0.74), respectively. conclusion: the diagnostic accuracy and concordance of microeeg are comparable to those of standard eeg but the unique ed-friendly characteristics of the device could help overcome the existing barriers for more frequent use of eeg in the ed. (originally submitted as a ''late-breaker.'') a background: patients who use an ed for acute migraine are characterized by higher migraine disability scores, lower socio-economic status, and are unlikely to have used a migraine-specific medication prior to ed presentation. objectives: to determine if a comprehensive migraine intervention, delivered just prior to ed discharge, could improve migraine impact scores one month after the ed visit. methods: this was a randomized controlled trial of a comprehensive migraine intervention versus typical care among patients who presented to an ed for management of acute migraine. at the time of discharge, for patients randomized to comprehensive care, we reinforced their diagnosis, shared a migraine education presentation from the national library of medicine, provided them with six tablets of sumatriptan 100 mg and 14 tablets of naproxen 500 mg, and if they wished, provided them with an expedited free appointment to our institution's headache clinic. patients randomized to typical care received the care their attending emergency physician felt was appropriate. the primary outcome was a between-group comparison of the hit6 score, a validated headache assessment instrument, one month after ed discharge. secondary outcomes included an assessment of satisfaction with headache care and frequency of use of migraine-specific medication within that one month period. the outcome assessor was blinded to assignment. results: over a 19 month period, 50 migraine patients were enrolled. one month follow-up was successfully obtained in 92% of patients. baseline characteristics were comparable. one month hit6 scores in the two groups were nearly identical (59 vs 56, 95%ci for difference of 3: )5, 11), as was dissatisfaction with overall headache care (17% versus 18%, 95%ci for difference of 1%: )22, 24%). not surprisingly, patients randomized to the comprehensive intervention were more likely to be using triptans or migraine-preventive therapy (43% versus 0%, 95%ci for difference of 43%: 20, 63%) one month later. conclusion: a comprehensive migraine intervention, when compared to typical care, did not improve hit6 scores one month after ed discharge. future work is needed to define a migraine intervention that is practical and useful in an ed. background: lumbar puncture (lp) is the standard of care for excluding non-traumatic subarachnoid hemorrhage (sah), and is usually performed following head ct (hct). however, in the setting of a non-diagnostic hct, lp demonstrates a low overall diagnostic yield for sah (<1% positive rate). objectives: to describe a series of ed patients diagnosed with sah by lp following a non-diagnostic hct, and, when compared to a set of matched controls, determine if clinical variables can reliably identify these ''ct-negative/lp-positive'' patients. methods: retrospective case-control chart review of ed patients in an integrated health system between the years 2000-2011 (estimated 5-6 million visits among 18 eds). patients with a final diagnosis of non-traumatic sah were screened for case inclusion, defined as an initial hct without sah by final radiologist interpretation and a lp with >5 red blood cells/mm 3 , along with either 1) xanthochromic cerebrospinal fluid, 2) angiographic evidence of cerebral aneurysm or arteriovenous malformation, or 3) head imaging showing sah within 48 hours following lp. control patients were randomly selected among ed patients diagnosed with headache following a negative sah evaluation with hct and lp. controls were matched to cases by year and presenting ed in a 3:1 ratio. stepwise logistic regression and classification and regression tree analysis (cart) were employed to identify predictive variables. inter-rater reliability (kappa) was determined by independent chart review. results: fifty-five cases were identified. all cases were hunt-hess grade 1 or 2. demographics are shown in table 1 . thirty-four cases (62%) had angiographic evidence of sah. five variables were identified that positively predicted sah following a normal hct with 98% sensitivity (95% ci, 90-100%) and 25% specificity (95% ci, 19-32%): age > 50 years, neck pain or stiffness, onset of headache with exertion, vomiting with headache, or loss of consciousness at headache onset. kappa values for selected variables ranged from 0.75-1.0 (18% sample). the c-statistic (auc) and hosmer-lemeshow test p-value for the logistic regression model are 0.87 and 0.74, respectively (table 2) . conclusion: several clinical variables can help safely limit the amount of invasive testing for sah following a non-diagnostic hct. prospective validation of this model is needed prior to practice implementation. background: post-thrombolysis intracerebral hemorrhage (ich) is associated with poor outcomes. previous investigations have attempted to determine the relationship between pre-existing anti-platelet (ap) use and the safety of intravenous thrombolysis, but have been limited by low event rates thus decreasing the precision of estimates. objectives: our objective was to determine whether pre-existing ap therapy increases the risk of ich following thrombolysis. methods: consecutive cases of ed-treated thrombolysis patients were identified using multiple methods, including active and passive surveillance. retrospective data were collected from four hospitals from 1996-2005, and 24 distinct hospitals from 2007-2010 as part of a cluster randomized trial. the same chart abstraction tool was used during both time periods and data were subjected to numerous quality control checks. hemorrhages were classified using a pre-specified methodology: ich was defined as presence of hemorrhage in radiographic interpretations of follow up imaging (primary outcome). symptomatic ich (secondary outcome) was defined as radiographic ich with associated clinical worsening. a multivariable logistic regression model was constructed to adjust for clinical factors previously identified to be related to postthrombolysis ich. as there were fewer sich events, the multivariable model was constructed similarly, except that variables divided into quartiles in the primary analysis were dichotomized at the median. results: there were 830 patients included, with 47% having documented pre-existing ap treatment. the mean age was 69 years, the cohort was 53% male, and the median nihss was 12. the unadjusted proportion of patients with any ich was 15.1% without ap and 19.3% with ap (difference 4.2%, 95% ci )1.2% to 9.6%); for sich this was 6.1% without ap and 9% with ap (difference 3.1%, 95%ci )1 to 6.7%). no significant association between pre-existing ap treatment with radiographic or symptomatic ich was observed (table) . conclusion: we did not find that ap treatment was associated with post-thrombolysis ich or sich in this cohort of community treated patients. pre-existing tobacco use, younger age, and lower severity were associated with lower odds of sich. an association between ap therapy and sich may still exist -further research with larger sample sizes is warranted in order to detect smaller effect sizes. background: post-cardiac arrest therapeutic hypothermia (th) improves survival and neurologic outcome after cardiac arrest, but the parameters required for optimal neuroprotection remain uncertain. our laboratory recently reported that 48-hour th was superior to 24-hour th in protecting hippocampal ca1 pyramidal neurons after asphyxial cardiac arrest in rats. cerebellar purkinje cells are also highly sensitive to ischemic injury caused by cardiac arrest, but the effect of th on this neuron population has not been previously studied. objectives: we examined the effect of post-cardiac arrest th onset time and duration on purkinje neuron survival in cerebella collected during our previous study. methods: adult male long evans rats were subjected to 10-minute asphyxial cardiac arrest followed by cpr. rats that achieved return of spontaneous circulation (rosc) were block randomized to normothermia (37.0 deg c) or th (33.0 deg c) initiated 0, 1, 4, or 8 hours after rosc and maintained for 24 or 48 hours (n = 21 per group). sham injured rats underwent anesthesia and instrumentation only. seven days post-cardiac arrest or sham injury, rats were euthanized and brain tissue was processed for histology. surviving purkinje cells with normal morphology were quantified in the primary fissure in nissl stained sagittal sections of the cerebellar vermis. purkinje cell density was calculated for each rat, and group means were compared by anova with bonferroni analysis. results: purkinje cell density averaged (+/) sd) 35.9 (2.4) cells/mm in sham-injured rats. neuronal survival in normothermic post-cardiac arrest rats was significantly reduced compared to sham (10.7% (5.0%)). overall, th resulted in significant neuroprotection compared to normothermia (38.9% (15.7%) of sham). purkinje cell density with 24-hour duration th was 35.0% (11.2%) of sham and 48-hour duration th was 43.3% (15.6%), both significantly improved from sham (p = 0.245 between durations). th initiated 0, 1, 4, and 8 hours post-rosc provided similar benefit: 44.6% (21.6%), 33.2% (8.1%), 36.6% (12.9%), and 41.1% (9.3%) of sham, respectively. conclusion: overall, these results indicate that postcardiac arrest th protects cerebellar purkinje cells with a broad therapeutic window. our results underscore the importance of considering multiple brain regions when optimizing the neuroprotective effect of post-cardiac arrest th. the effect of compressor-administered defibrillation on peri-shock pauses in a simulated cardiac arrest scenario joshua glick, evan leibner, thomas terndrup penn state hershey medical center, hershey, pa background: longer pauses in chest compressions during cardiac arrest are associated with a decreased probability of successful defibrillation and patient survival. having multiple personnel share the tasks of performing chest compressions and shock delivery can lead to communication complications that may prolong time spent off the chest. objectives: the purpose of this study was to determine whether compressor-administered defibrillation led to a decrease in pre-shock and peri-shock pauses as compared to bystander-administered defibrillation in a simulated in-hospital cardiac arrest scenario. we hypothesized that combining the responsibilities of shock delivery and chest-compression performance may lower no-flow periods. methods: this was a randomized, controlled study measuring pauses in chest compressions for defibrillation in a simulated cardiac arrest. medical students and ed personnel with current cpr certification were surveyed for participation between july 2011 and october 2011. participants were randomized to either a control (facilitator-administered shock) or variable (participantadministered shock) group. all participants completed one minute of chest compressions on a mannequin in a shockable rhythm prior to initiation of prompt and safe defibrillation. pauses for defibrillation were measured and compared in both study groups. results: out of 200 total enrollments, the data from 197 defibrillations were analyzed. subject-initiated defibrillation resulted in a significantly lower pre-shock handsoff time (0.57 s; 95% ci: 0.47-0.67) compared to facilitator-initiated defibrillation (1.49 s; 95% ci: 1.35-1.64). furthermore, subject-initiated defibrillation resulted in a significantly lower peri-shock hands-off time (2.77 s; 95% ci: 2.58-2.95) compared to facilitator-initiated defibrillation (4.25 s; 95% ci: 4.08-4.43). conclusion: assigning the responsibility for shock delivery to the provider performing compressions encourages continuous compressions throughout the charging period and decreases total time spent off the chest. this modification may also decrease the risk of accidental shock and improve patient survival. however, as this was a simulation-based study, clinical implementation is necessary to further evaluate these potential benefits. objectives: to determine the sensitivity and specificity of peripheral venous oxygen (po 2 ) to predict abnormal central venous oxygen saturation in septic shock patients in the ed. methods: secondary analysis of an ed-based randomized controlled trial of early sepsis resuscitation targeting three physiological variables: cvp, map, and either scvo 2 or lactate clearance. inclusion criteria: suspected infection, two or more sirs criteria, and either systolic blood pressure <90 mmhg after a fluid bolus or lactate >4 mm. peripheral venous po 2 was measured prior to enrollment as part of routine care, and scvo 2 was measured as part of the protocol. we analyzed for agreement between venous po 2 and scvo 2 using spearman's rank. sensitivity and specificity to predict an abnormal scvo 2 (<70%) were calculated for each incremental value of po 2 . results: a total of 175 were analyzed. median po 2 was 43 mmhg (iqr 32, 55). median initial scvo 2 was 79% (iqr 70, 88). thirty-nine patients (23%) had an initial scvo 2 < 70%. spearman's rank demonstrated fair correlation between initial po 2 and scvo 2 (q = 0.26). a cutoff of venous po 2 < 57 was 90% sensitive and 20% specific for detecting an initial scvo 2 < 70%. twenty-seven patients (20%) demonstrated an initial po 2 of >56. conclusion: in ed septic shock patients, venous po 2 demonstrated only fair correlation with scvo 2, though a cutoff value of 56 was sensitive for predicting an abnormal scvo 2 . twenty percent of patients demonstrated an initial value above the cutoff, potentially representing a group in whom scvo 2 measurement could be avoided. future studies aiming to decrease central line utilization could consider the use of peripheral o 2 measurements in these patients. sessions. ninety-two percent were rns, median clinical experience was 11-15 years, and 56% were from an intensive care unit. provider confidence increased significantly with a single session despite the highly experienced sample (figure 1 ). there was a trend for further increased confidence with an additional session and the increased confidence was maintained for at least 3-6 months given the normal sensitivity analysis. conclusion: high fidelity simulation significantly increases provider confidence even among experienced providers. this study was limited by its small sample size and recent changes in acls guidelines. background: recent data suggest alarming delays and deviations in major components of pediatric resuscitation during simulated scenarios by pediatric housestaff. objectives: to identify the most common errors of pediatric residents during multiple simulated pediatric resuscitation scenarios. methods: a retrospective observational study conducted in an academic tertiary care hospital. pediatric residents (pgy1 and pgy3) were videotaped performing a series of five pediatric resuscitation scenarios using a high-fidelity simulator (simbaby, laerdal): pulseless non-shockable arrest, pulseless shockable arrest, dysrhythmia, respiratory arrest, and shock. the primary outcome was the presence of significant errors prospectively defined using a validated scoring instrument designed to assess sequence, timing, and quality of specific actions during resuscitations based on the 2005 aha pals guidelines. residents' clinical performances were measured by a single video reviewer. the primary analysis was the proportion of errors for each critical task for each scenario. we estimated that the evaluation of each resident would provide a confidence interval less than 0.20 for the proportion of errors. results: twenty-four of 25 residents completed the study. across all scenarios, pulse check was delayed by more than 30 seconds in 56% (95%ci: 46%-66%). for non-shockable arrest, cpr was started more than 30 seconds after recognizing arrest in 21% (95%ci 7-42%) and inappropriate defibrillation was performed in 29% (95%ci 13-51%). for shockable arrest, participants failed to identify the rhythm in 58% (95%ci 37-78%), cpr was not performed in 25% (95%ci 10-47%), while defibrillation was delayed by more than 90 seconds in 33% (95%ci 16-51%) and not performed in one case. for shock, participants failed to ask for a dextrose check in 71% (95%ci 51-86%), and it was delayed by more than 60 seconds for all others. conclusion: the most common error across all scenarios was delay in pulse check. delays in starting cpr and inappropriate defibrillation were common errors in non-shockable arrests, while failure to identify rhythm, cpr omission, and delaying defibrillation were noted for shockable arrests. for shock, omission of rapid dextrose check was the most common error, while delaying the test when ordered was also significant. future training in pediatric resuscitation should target these errors. background: many scoring instruments have been described to measure clinical performance during resuscitation; however, the validity of these tools has yet to be proven in pediatric resuscitation. objectives: to determine the external validity of published scoring instruments to evaluate clinical performance during simulated pediatric resuscitations using pals algorithms and to determine if inter-rater reliability could be assessed. methods: this was a prospective quasi-experimental design performed in a simulation lab of a pediatric tertiary care facility. participants were residents from a single pediatric program distinct from where the instrument was originally developed. a total of 13 pgy1s and 11 pgy3s were videotaped during five simulated pediatric resuscitation scenarios. pediatric emergency physicians rated resident performances before and after a pals course using standardized scoring. each video recording was viewed and scored by two raters blinded to one another. a priori, it was determined that, for the scoring instrument to be valid, participants should improve their scores after participating in the pals course. differences in means between pre-pals and post-pals and pgy1 and pgy3 were compared using an anova test. to investigate differences in the scores of the two groups over the five scenarios, a two-factor anova was used. reliability was assessed by calculating an interclass correlation coefficient for each scenario. results: following the pals course, scores improved by 8.6% (3.8 to 13.3), 15.7% (8.6 to 22.7), 6.3% ()1.8 to 14.3), 18.2% (9.3 to 27), and 4.1% ()3.0 to 11.2) for the pulseless non-shockable arrest, pulseless shockable arrest, dysrhythmia, respiratory, and shock scenarios respectively. there were no differences in scores between pgy1s and pgy3s before and after the pals course. there was an excellent reliability for each scoring instrument with iccs varying between 0.85 and 0.98. conclusion: the scoring instrument was able to demonstrate significant improvements in scores following a pals course for pgy1 and pgy3 pediatric residents for the pulseless non-shockable arrest, pulseless shockable, and respiratory arrest scenarios only. however, it was unable to discriminate between pgy1s and pgy3s both before and after the pals course for any scenarios. the scoring instrument showed excellent inter-reliability for all scenarios. a background: medical simulation is a common and frequently studied component of emergency medicine (em) residency curricula. its utility in the context of em medical student clerkships is not well defined. objectives: the objective was to measure the effect of simulation instruction on medical students' em clerkship oral exam performance. we hypothesized that students randomized to the simulation group would score higher. we predicted that simulation instruction would promote better clinical reasoning skills and knowledge expression. methods: this was a randomized observational study conducted from 7/2009 to 5/2010. participants were fourth year medical students in their em clerkship. students were randomly assigned on their first day to one of two groups. the study group received simulation instruction in place of one of the lectures, while the control group was assigned to the standard curriculum. the standard clerkship curriculum includes lectures, case studies, procedure labs, and clinical shifts without simulation. at the end of the clerkship, all students participated in written and oral exams. graders were not blinded to group allocation. grades were assigned based on a pre-defined set of criteria. the final course composite score was computed based on clinical evaluations and the results of both written and oral exams. oral exam scores between the groups were compared using a two-sample t-test. we used the spearman rank correlation to measure the association between group assignment and the overall course grade. the study was approved by our institutional irb. results: sixty-one students participated in the study and were randomly assigned to one of two groups. twenty-nine (47.5%) were assigned to simulation and the remaining 32 (52.5%) students were assigned to the standard curriculum. students assigned to the simulation group scored 5.34% (95% ci 2.78-7.91%) higher on the oral exam than the non-simulation group. additionally, simulation was associated with a higher final course grade (p < 0.05). limitations of this pilot study include lack of blinding and interexaminer variability. conclusion: simulation training as part of an em clerkship is associated with higher oral exam scores and higher overall course grade compared to the standard curriculum. the results from this pilot study are encouraging and support a larger, more rigorous study. initial approaches to common complaints are taught using a standard curriculum of lecture and small group case-based discussion. we added a simulation exercise to the traditional altered mental status (ams) curriculum with the hypothesis that this would positively affect student knowledge, attitudes, and level of clinical confidence caring for patients with ams. methods: ams simulation sessions were conducted in june 2010 and 2011; student participation was voluntary. the simulation exercises included two ams cases using a full-body simulator and a faculty debriefing after each case. both students who did and did not participate in the simulations completed a written post-test and a survey related to confidence in their approach to ams. results: 154 students completed the post-test and survey. 65 (42%) attended the simulation session. 48 (31%) attended all three sessions. 58 (38%) participated in the lecture and small group. 15 (10%) did not attend any session. post-test scores were higher in students who attended the simulations versus those who did not: 7 (iqr, 6-8) vs. 6 (iqr, 4-7); p < 0.001. students who attended the simulations felt more confident about assessing an ams patient (58% vs. 42%; p = 0.05), articulating a differential diagnosis (66% vs. 47%; p = 0.03), and knowing initial diagnostic tests (74% vs. 53%; p = 0.01) and initial interventions (79% vs. 56%; p = 0.003) for an ams patient. students who attended the simulations were more likely to rate the overall ams curriculum as useful (94% vs. 61%; p < 0.001). conclusion: addition of a simulation session to a standard ams curriculum had a positive effect on student performance on a knowledge-based exam and increased confidence in clinical approach. the study's major limitations were that student participation in the simulation exercise was voluntary and that effect on applied skills was not measured. future research will determine whether simulation is effective for other chief complaints and if it improves actual clinical performance. background: the acgme has defined six core competencies for residents including ''professionalism'' and ''interpersonal and communication skills.'' integral to these two competencies is empathy. prior studies suggest that self-reported empathy declines during medical training; no reported study has yet integrated simulation into the evaluation of empathy in medical training. objectives: to determine if there is a relation between level of training and empathy in patient interactions as rated during simulation. methods: this is a prospective observational study at a tertiary care center comparing participants at four different levels of training: first (ms1) and third year (ms3) medical students, incoming em interns (pgy1), and em senior residents (pgy3/4). trainees participated in two simulation scenarios (ectopic pregnancy and status asthmaticus) in which they were responsible for clinical management (cm) and patient interactions (pi). this was the first simulation exposure during an established simulation curriculum for ms1, ms3, and pgy1. two independent raters reviewed videotaped simulation scenarios using checklists of critical actions for clinical management (cm: 0-11 points) and patient interactions (pi: 0-17 points). inter-rater reliability was assessed by intra-class correlation coefficients (iccs objectives: we explored attitudes and beliefs about the handoff, using qualitative methods, from a diverse group of stakeholders within the ems community. we also characterized perceptions of barriers to high-quality handoffs and identified strategies for optimizing this process. methods: we conducted seven focus groups at three separate gatherings of ems professionals (one local, two national) in 2010/2011. snowball sampling was used to recruit 48 participants with diverse professional, experiential, geographic, and demographic characteristics. focus groups, lasting 60-90 minutes, were moderated by investigators trained in qualitative methods, using an interview guide to elicit conversation. recordings of each group were transcribed. three reviewers analyzed the text in a multi-stage iterative process to code the data, describe the main categories, and identify unifying themes. results: participants included emts, paramedics, physicians, and nurses. clinical experience ranged from 4 months to 36 years. recurrent thematic domains when discussing attitudes and beliefs were: perceptions of respect and competence, professionalism, teamwork, value assigned to the process, and professional duty. modifiers of these domains were: hierarchy, skill/training level, severity/type of patient illness, and system/ regulatory factors. strategies to improving barriers to the handoff included: fostering familiarity and personal connections between ems and ed staff, encouraging two-way conversations, feedback, and direct interactions between ems providers and ed physicians, and optimizing ways for ems providers to share subjective impressions (beyond standardized data elements) with hospital-based care teams. conclusion: ems professionals assign high value to the ed handoff. variations in patient acuity, familiarity with other handoff participants, and perceptions of respect and professionalism appear to influence the perceived quality of this transition. regulatory strategies to standardize the contents of the handoff may not alone overcome barriers to this process. miology, public health) then developed an approach to assign ems records to one of 20 symptom-based illness categories (gastrointestinal illness, respiratory, etc). ems encounter records were characterized into these illness categories using a novel text analytic program. event alerts were identified across the state and local regions in illness categories using either change detection from baseline with (cusum) analysis (three standard deviations) and a novel text-proportion (tap) analysis approach (sas institute, cary, nc). results: 2.4 million ems encounter records over a 2year period were analyzed. the initial analysis focused upon gastrointestinal illness (gi) given the potential relationship of gi distress to infectious outbreaks, food contamination and intentional poisonings (ricin). after accounting for seasonality, a significant gi event was detected in feb 2010 (see red circle on graph). this event coincided with a confirmed norovirus outbreak. the use of cusum approach (yellow circle on graph) detected the alert event on jan 24, 2010. the novel tap approach on a regional basis detected the alert on dec 6, 2009. conclusion: ems has the advantage of being an early point of contact with patients and providing information on the location of insult or injury. surveillance based on ems information system data can detect emergent outbreaks of illness of interest to public health. a novel text proportion analytic technique shows promise as an early event detection method. assessing chronic stress in the emergency medical services elizabeth a. donnelly 1 , jill chonody 2 1 university of windsor, windsor, on, canada; 2 university of south australia, adelaide, australia background: attention has been paid to the effect of critical incident stress in the emergency medical services (ems); however, less attention has been given to the effect of chronic stress (e.g., conflict with administration or colleagues, risk of injury, fatigue, interference in non-work activities) in ems. a number of extant instruments assess for workplace stress; however, none address the idiosyncratic aspects of work in ems. objectives: the purpose of this study was to validate an instrument, adapted from mccreary and thompson (2006) , that assesses levels of both organizational and operational work-related chronic stress in ems personnel. methods: to validate this instrument, a cross-sectional, observational web-based survey was used. the instrument was distributed to a systematic probability sample of emts and paramedics (n = 12,000). the survey also included the perceived stress scale (cohen, 1983) to assess for convergent construct validity. results: the survey attained a 13.6% usable response rate (n = 1633); respondent characteristics were consistent across demographic characteristics with other studies of emts and paramedics. the sample was split in order to allow for exploratory and confirmatory fac-tor analyses (n = 847/n = 786). in the exploratory factor analysis, principal axis factoring with an oblique rotation revealed a two-factor, 34-item solution (kmo = 0.943, v 2 = 23344.38, df = 561, p £.001). confirmatory factor analysis suggested a more parsimonious, two-factor, 20-item solution (v 2 = 632.67, df = 168, p £ 0.001, rmsea = 0.06, cfi = 0.92, tli = 0.91, srmr = 0.04). the factors demonstrated good internal reliability (operational stress a = 0.877, organizational stress a = 0.868). both factors were significantly correlated (p £ 0.01) with the hypothesized convergent validity measure. conclusion: theory and empirical research indicate that exposure to chronic workplace stress may play an important part in the development of psychological distress, including burnout, depression, and posttraumatic stress disorder (ptsd). workplace stress and stress reactions may potentially interfere with job performance. as no extant measure assesses for chronic workplace stress in ems, the validation of this chronic stress measure enhances the tools ems leaders and researchers have in assessing the health and well-being of ems providers. effect of naltrexone background: survivors of sarin and other organophosphate poisoning can develop delayed encephalopathy that is not prevented by standard antidotal therapy with atropine and pralidoxime. a rat model of poisoning with the sarin analogue diisoprophylfluorophosphate (dfp) demonstrated impairment of spatial memory despite antidotal therapy with atropine and pralidoxime. additional antidotes are needed after acute poisonings that will prevent the development of encephalopathy. objectives: to determine the efficacy of naltrexone in preventing delayed encephalopathy after poisoning with the sarin analogue dfp in a rat model. the hypothesis is that naltrexone would improve performance on spatial memory after acute dfp poisoning. the sarin analogue dfp was used because it has similar toxicity to sarin while being less dangerous to handle. methods: a randomized controlled experiment at a university animal research laboratory of the effects of naltrexone on spatial memory after dfp poisoning was conducted. long evans rats weighing 250-275 grams were randomized to dfp group (n = 4, rats received a single intraperitoneal (ip) injection of dfp 5 mg/kg) or dfp+naltrexone group (n = 5, rats received a single ip injection of dfp (5 mg/kg) followed by naltrexone 5 mg/kg/day). after injection, rats were monitored for signs and symptoms of cholinesterase toxicity. if toxicity developed, antidotal therapy was initiated with atro-background: one of the primary goals of management of patients presenting with known or suspected acetaminophen (apap) ingestion is to identify the risk for apap-induced hepatotoxicity. current practice is to measure apap level at a minimum of 4 hours post ingestion and plot this value on the rumack-matthew nomogram. one retrospective study of apap levels drawn less than 4 hours post-ingestion found a level less than 100 mcg/ml to be sufficient to exclude toxic ingestion. objectives: the aim of this study was to prospectively determine the negative predictive value (npv) for toxicity of an apap level of less than 100 mcg/ml obtained less than 4 hours post-ingestion. methods: this was a multicenter prospective cohort study of patients presenting to one of five tertiary care hospitals that are part of the toxicology investigator's consortium (toxic). eligible patients presented to the emergency department less than 4 hours after known or suspected ingestion and had the initial apap level obtained at greater than 1 but less than 4 hours post ingestion. a second apap level was obtained at 4 hours or more post-ingestion and plotted on the rumack-matthew nomogram to determine risk of toxicity. the outcome of interest was the npv of an initial apap level less than 100 mcg/ml. a power analysis based on an alpha = 0.05 and power of 0.80 yielded the requirement of 71 subjects. results: data were collected on 171 patients over a 30month period from may 2009 to nov 2011. patients excluded from npv analysis consisted of: initial apap level greater than 100 mcg/ml (31), negligible apap level on both the initial and confirmatory apap level (31), initial apap level drawn less than one hour after ingestion (15), or an unknown time of ingestion (1). ninety-three patients met the eligibility criteria. two patients (2.2%) with an initial apap level less than 100 mcg/ml (54 mcg/ml at 90 min, 38 mcg/ml at 84 min) were determined to be at risk for toxicity based on oh s330 2012 saem annual meeting abstracts implementation of an emergency department sign-out checklist improves patient hand-offs at change of shift nicole m ma computer-assisted self-interviews improve testing for chlamydia and gonorrhea in the pediatric emergency department is the australian triage system a better indicator of psychiatric patients' needs for intervention than the ena emergency severity index triage system? patients were given an initial dose of 10 mg droperidol intramuscularly followed by an additional dose of 10 mg after 15 min if required. inclusion criteria were patients requiring physical restraint and parenteral sedation. the primary outcome was the time to sedation. secondary outcomes were the proportion of patients requiring additional sedation within the first hour, over-sedation measured as -3 on the sedation assessment tool, and respiratory compromise measured as oxygen saturation <90%. results: droperidol was administered to 424 patients and 370 of these had sedation scores documented. presentations included 56% with alcohol intoxication. dose ranged from 2.5 mg to 30 mg, median 10 mg (interquartile range conclusion: droperidol is effective for rapid sedation for abd and rarely causes over-sedation serum creatinine (scr) is widely used to predict risk; however, gfr is a better assessment of kidney function. objectives: to compare the ability of gfr and scr to predict the development of cin among ed patients receiving cects. we hypothesized that gfr would be the best available predictor of cin. methods: this was a retrospective chart review of ed patients ‡18 years old who had a chest or abdomen/pelvis cect between 06/01/11 and 07/31/11. baseline and follow-up scr levels were recorded. patients with initial scr >1.6 mg/dl were excluded, as per hospital radiology department protocol. cin was defined as a scr increase of either 25%, 0.5 mg/dl, or a gfr decrease of 25% within 72 hours of contrast exposure. gfr was calculated using the ckd epi and mdrd formulae, and analyzed in original units and categorized form (<60, ‡60) with each additional unit decrease in ckd epi, subjects were 3% more likely to develop cin (or = 1.03) (p < 0.0281). additionally, subjects with ckd epi <60 were 3.20 (or) times more likely to have cin than subjects with ckd epi ‡60 in original units, ckd epi (p < 0.0001) and mdrd (p < 0.0016) both had a significantly higher auc than scr. conclusion: age, as an independent variable, is the best predictor of cin, when compared with scr and gfr. due to a small number of cases with cin, the confidence intervals associated with the odds ratios are wide. future research should focus on patient risk stratification and establishing ed interventions to prevent cin. 694 a rat model of carbon monoxide induced neurotoxicity heather ellsworth non-traumatic subarachnoid hemorrhage diagnosed by lumbar puncture following non-diagnostic head ct: a retrospective case-control study and decision a dass score of >14 has been previously defined as an indicator of increased stress levels. multivariable logistic regression was utilized to identify demographic and work-life characteristics significantly associated with stress. results: 53.6% of individuals responded to the survey (34,340/64,032) and prevalence of stress was estimated at 5.9%. the following work-life characteristics were associated with stress: certification level, work experience, and service type. the odds of stress in paramedics was 32% higher when compared to emt-basics (or = 1.32, 95% ci = 1.23-1.42). when compared to £2 years of experience 28-2.18) were more likely to be stressed. ems professionals working in county (or = 1 ci = 1.07-1.51) and private services (or = 1 56) were more likely than those working in fire-based services to be stressed. the following demographic characteristics were associated with stress: general health and smoking status finally, former smokers (or = 1.34, 95% ci = 1.17-1.54) and current smokers (or = 1.37, 95% ci = 1.18-1.59) were more likely to be stressed than non-smokers literature suggests this is within the range of stress among nurses, and lower than physicians. while the current study was able to identify demographic and work-life characteristics associated with stress, the long-term effects are largely unknown methods: design: prospective randomized controlled trial. subjects: female sus scrofa swine weighing 45-55kg were infused with amitriptyline 0.5 mg/kg/minute until the map fell to 60% of baseline values. subjects were then randomized to experimental group (ife 7 ml/kg followed by an infusion of 0.25 ml/kg/minute) or control group (sb 2 meq/kg plus equal volume of normal saline). interventions: we measured continuous heart rate (hr), sbp, map, cardiac output (co), systemic vascular resistance (svr), and venous oxygen saturation (svo 2 ). laboratory values monitored included ph, pco 2 , bicarbonate, lactate, and amitriptyline levels. descriptive statistics including means, standard deviations, standard errors of measurement, and confidence limits were calculated. results: of 14 swine, seven each were allocated to ife and sb groups. there was no difference at baseline for each group regarding hr, sbp, map, co, svr, or svo 2 . ife and sb groups required similar mean amounts of tca to reach hypotension one ife and two sb pigs survived. conclusion: in this interim data analysis of amitriptyline-induced hypotensive swine, we found no difference in mitigating hypotension between ife and sb lipid rescue 911: a survey of poison center medical directors regarding intravenous fat emulsion therapy michael r. christian 1 , erin m. pallasch cook county hospital (stroger), chicago, il 745 reliability of non-toxic acetaminophen concentrations obtained less than 4 hours after ingestion evaluating age in the field triage of injured background: hiv screening in eds is advocated to achieve the goal of comprehensive population screening. yet, hiv testing in the ed is sometimes thwarted by a patient's condition (e.g. intoxication) or environmental factors (e.g. other care activities). whether it is possible to test these patients at a later time is unknown. objectives: we aimed to determine if ed patients who were initially unable to receive an hiv testing offer might be tested in the ed at a later time. we hypothesized that factors preventing testing are transient and that there are subsequent opportunities to repeat testing offers. methods: we reviewed medical records for patients presenting to an urban, academic ed who were approached consecutively to offer hiv testing during randomly selected periods from january 2008 to january 2009. patients for whom the initial attempted offer could not be completed were reviewed in detail with standardized abstraction forms, duplicate abstraction, and third-party discrepancy adjudication. primary outcomes included repeat hiv testing offers during that ed visit, and whether a testing offer might eventually have been possible either during the initial visit or at a later visit within 6 months. outcomes are described as proportions with confidence intervals. results: of 824 patients approached, initial testing offers could not be completed for 120 (15%). these 120 were 62% male, 52% white, and had a median age of 41 (18-64). a repeat offer of testing during the initial visit would have been possible for 99/120 (83%), and 52/99 (53%) were actually offered testing on repeat approach. of the 21 for whom a testing offer would not have been possible on the initial visit, 14 (67%) had at least one additional visit within 6 months, and 11/14 (79%) could have been offered testing on at least one visit. overall, a repeat testing offer would have been possible for 110/120 (93%, 95% ci 85-96%). conclusion: factors preventing an initial offer of hiv testing in the ed are generally transient. opportunities for repeat approach during initial or later ed encounters suggest that, given sufficient resources, the ed could succeed in comprehensively screening the population presenting for care. ed screening personnel who are initially unable to offer testing should repeat their attempt. hiv adopt an ''opt-out'' rapid hiv screening model in order to identify hiv infected patients. previous studies nationwide have shown acceptance rates for hiv screening of 20-90% in emergency departments. however, it is unknown how acceptance rates will vary in a culturally and ethnically diverse urban emergency department.objectives: to determine the characteristics of patients who accept or refuse ''opt-out'' hiv screening in an urban emergency department.methods: a self-administered, anonymous survey is administered to ed patients who are 18 to 64 years of age. the questionnaire is administered in english, russian, mandarin, and spanish. questions include demographic characteristics, hiv risk factors, perception of hiv risk, and acceptance of rapid hiv screening in the emergency department. results: to date 145 patients responded to our survey. of the 145, 102 (70.3%) did not accept an hiv test (group 1) in their current ed visit and 43 (29.7%) accepted an hiv test (group 2). the major two reasons given for opting out (i.e., group 1) was ''i do not feel that i am at risk'' (59.8%) and ''i have been tested for hiv before'' (25.5%). there was no difference between the groups in regards to sex (p = 0.737), age (p = 0.351), religious affiliation (p = 0.750), marital status (p = 0.331), language spoken at home (p = 0.211), and whether they had been hiv tested before (73.2% in group 1 and 59.4% in group 2; p = 0.123). however, there was a statistically significant difference with regards to educational level and income. more patients in group 1 (69.0%) and 46.1% in group 2 had less than a college level education (p < 0.05). similarly, more patients in group 1 (58.3%) and only 34.8% in group 2 had an annual household income of £$25,000 (p < 0.05). conclusion: in a culturally and ethnically diverse urban emergency department, patients with a lower socioeconomic status and educational level tend to opt out of hiv screening test offered in the ed. no significant difference in acceptance of ed hiv testing was found to date based on primary language spoken at home or religious affiliation background: antimicrobial resistance is a problem that affects all emergency departments. objectives: our goal was to examine all urinary pathogens and their resistance patterns from urine cultures collected in the emergency department (ed).methods: this study was performed at an urban/suburban community-teaching hospital with an annual volume of 40,000 visits. using electronic records, all cases of urine cultures received in 2009 were reviewed for data including type of bacteria, antibiotic resistance, and health care exposure (hcx). hcx was defined as no prior hospitalization within the previous six months, hospitalization within the previous three months, hospitalization within the previous six months, nursing home resident (nh), and presence of an indwelling urinary catheter (uc). an investigator abstracted all data with a second re-abstracting a random 5% for kappa statistics between 0.697 and 1.00. group background: approximately 12-20% of patients treated with epinephrine for anaphylaxis receive a second dose but the risk factors associated with repeat epinephrine use remain poorly defined. objectives: to determine whether obesity is a risk factor for requiring 2 + epinephrine doses for patients who present to the emergency department (ed) with anaphylaxis due to food allergy or stinging insect hypersensitivity. methods: we performed a retrospective chart review at four tertiary care hospitals that care for adults and children in new england between the following time periods: massachusetts general hospital (1/1/01-12/31/ 06), brigham and women's hospital (1/1/01-12/31/06), children's hospital boston (1/1/01-12/31/06), hasbro children's hospital (1/1/04-12/31/09). we reviewed the medical records of all patients presenting to the ed for food allergy or stinging insect hypersensitivity using icd9cm codes. we focused on anthropomorphic data and number of epinephrine treatments given before and during the ed visit. among children, calculated bmis were classified according to cdc growth indicators as underweight, healthy, overweight, or obese. all patients who presented on or after their 18th birthday were considered adults.background: transitions of care are ubiquitous in the emergency department (ed) and inevitably introduce the opportunity for errors. despite recommendations in the literature, few emergency medicine (em) residency programs provide formal training or standard process for patient hand-offs. checklists have been shown to be effective quality improvement measures in inpatient settings and may be a feasible method to improve ed hand-offs. objectives: to determine if the use of a sign-out checklist improves the accuracy and efficiency of resident sign-out in the ed as measured by reduced omission of key information, communication behaviors, and time to sign-out each patient. methods: a prospective study of first-and second-year em and non-em residents rotating in the ed at an urban academic medical center with an annual ed volume of 55,000. trained clinical research assistants observed resident sign-out during shift change over a two-week period and completed a 15-point binary observable behavior data collection tool to indicate whether or not key components of sign-out occurred. time to sign out each patient was recorded. we then created and implemented a computerized sign-out checklist consisting of key elements that should be addressed during transitions of care, and instructed residents to use this during hand-offs. a two-week post-intervention observation phase was conducted using the same data collection tool. proportions, means, and non-parametric comparison tests were calculated using stata. results: one hundred fifteen sign-outs were observed prior to checklist implementation and 72 after; one sign-out was excluded for incompleteness. significant improvements were seen in four of the measured signout components: inclusion of history of present illness increased by 18% (p < 0.001), likely diagnosis increased by 17% (p = 0.015), disposition status increased by 18% (p < 0.01), and patient/care team awareness of plan increased by 19% (p < 0.01). (figure 1 ) time data for 108 sign-outs pre-implementation and 72 post-implementation were available. seven sign-outs were excluded for incompleteness or spurious values. mean length of sign out was 83s (95% ci 65 to 100) and 71.7s (95% ci 52 to 92) per patient. conclusion: implementation of a checklist improved the transfer of information but did not affect the overall length of time for the sign-out. the objectives: to determine risk factors associated with adult patients presenting to the ed with cellulitis who fail initial antibiotic therapy and require a change of antibiotics or admission to hospital. methods: this was a prospective cohort study of patients ‡18 years presenting with cellulitis to one of two tertiary care eds (combined annual census 120,000). patients were excluded if they had been treated with antibiotics for the cellulitis prior to presenting to the ed, if they were admitted to hospital, or had an abscess only. trained research personnel administered a questionnaire at the initial ed visit with telephone follow-up 2 weeks later. patient characteristics were summarized using descriptive statistics and 95% confidence intervals (cis) were estimated using standard equations. backwards stepwise multivariable logistic regression models determined predictor variables independently associated with treatment failure (failed initial antibiotic therapy and required a change of antibiotics or admission to hospital). results: 598 patients were enrolled, 47 were excluded, and 53 were lost to follow-up. the mean (sd) age was 53.1 (18.4) and 56.4% were male. 497 (99.8%) patients were given antibiotics in the ed. 185 (37.2%) were given oral, 231 (46.5%) were given iv, and 81 (16.3%) patients were given both oral and iv antibiotics. 102 (20.5%) patients had a treatment failure. fever (temp >38°c) at triage (or: 4.1, 95% ci: 1.5, 10.7), leg ulcers (or: 3.1, 95% ci: 1.4, 6.6), edema or lymphedema (or: 2.5, 95% ci: 1.4, 4.5), and prior cellulitis in the same area (or: 1.8, 95% ci: 1.1, 2.9) were independently associated with treatment failure. conclusion: this analysis found four risk factors associated with treatment failure in patients presenting to the ed with cellulitis. these risk factors should be considered when initiating empiric outpatient antibiotic therapy for patients with uncomplicated cellulitis. use background: children presenting for care to a pediatric emergency department (ped) commonly require intravenous catheter (iv) placement. prior studies report that the average number of sticks to successfully place an iv in children is 2.4. successfully placing an iv requires identification of appropriate venous access targets. the veinviewer visionò (vvv) assists with iv placement by projecting a map of subcutaneous veins on the surface of the skin using near infrared light. objectives: to compare the effectiveness of the vvv versus standard approaches: sight (s) and sight plus palpation (s+p) for identifying peripheral veins for intravenous catheter placement in children treated in a ped. methods: experienced pediatric emergency nurses and physicians identified peripheral venous access targets appropriate for intravenous cannulation of a cross-sectional convenience sample of english speaking children aged 2-17 years presenting for treatment of sub-critical injury or illness whose parents provided consent. the clinicians marked the veins with different colored washable marker and counted them on the dorsum of the hand and in the antecubital fossa using the three approaches: s, s+p, and vvv. a trained research assistant photographed each site for independent counting after each marking and recorded demographics and bmi. counts were validated using independent photographic analyses. data were entered into sas 9.2 and analyzed using paired t-tests. results: 146 patients completed the study. clinicians were able to identify significantly more veins on the dorsum of the hand using vvv than s alone or s+p, 3.26 (p < 0.0001, ci 2.89-3.64) and 2.31 (p < 0.0001, ci 1.97-2.65), respectively, as well as significantly more veins in the antecubital fossa using vvv than s alone or s+p, 2.62 (p < 0.0001, ci 2.29-2.96) and 1.93 (p < 0.0001, ci 1.62-2.42), respectively. the differences in numbers of veins identified remained significant at p < 0.05 level across all ages, races, and bmis of children and across clinicians and validating independent photographic analyses. conclusion: experienced emergency nurses and physicians were able to identify significantly more venous access targets appropriate for intravenous cannulation in the dorsum of the hand and antecubital fossa of children presenting for treatment in a ped using vvv than the standard approaches of sight or sight plus palpation. an background: mental health emergencies have increased over the past two decades, and contribute to the ongoing rise in u.s. ed visit volumes. although data are limited, there is a general perception that the availability of in-person psychiatric consultation in the ed and of inpatient psychiatric beds is inadequate. objectives: to examine the availability of in-person psychiatry consultation in a heterogeneous sample of u.s. eds, and typical delays in transfer of ed patients to an inpatient psychiatric bed. methods: during 2009-2011, we mailed a survey to all ed directors in a convenience sample of nine us states (ar, co, ga, hi, ma, mn, or, vt, and wy). all sites were asked: ''are psychiatric consults available in-person to the ed?'' (yes/no), with affirmative respondents asked about the typical delay. sites also were asked about typical ed boarding time between a request for patient transfer and actual patient departure from the ed to an inpatient psychiatric bed. ed characteristics included rural/urban location, visit volume (visits/hour), admission rate, ed staffing, and the proportion of patients without insurance. data analysis used chi-square tests and multivariable logistic regression. results: surveys were collected from 495 (91%) of the 541 eds, with >80% response rate in every state. overall, only 30% responded that psychiatric consults were available in-person to the ed. in multivariable logistic regression, ed characteristics independently associated with lack of in-person psychiatric consultation were: location within specific states (eg, ar, ga), rural location, lower visit volume, and lower admission rate. among the subset of eds with psychiatric consults available, 48% reported a typical wait time of at least 1 hour. overall, 54% of eds reported that the typical time from request to actual patient transfer to an inpatient psychiatric bed was >6 hours, and 47% reported a maximum time in past year of >1 day (median 3 days, iqr 2-4). in a multivariable model, location in ma and higher visit volume were associated with greater odds of a maximum wait time of >1 day. conclusion: among 495 surveyed eds in nine states, only 30% have in-person psychiatric consultants available. moreover, approximately half of eds report boarding times of >6 h from request for transfer to actual departure to an inpatient psychiatric bed.background: many emergency departments (ed) in the united states use a five tiered triage protocol that has a limited evaluation of psychiatric patients. the australian triage scale (ats), a psychiatric triage system, has been used throughout australia and new zealand since the early 1990s. objectives: the objective of the study is to compare the current triage system, emergency nurses association (ena) esi 5-tier, to the ats for the evaluation of the psychiatric patients presenting to the ed. methods: a convenience sample of patients, 18 years of age and older, presenting with psychiatric complaints at triage were given the ena triage assessment by the triage nurse. a second triage assessment, performed by a research fellow, included all observed and reported elements using the ats protocol, a self-assessment survey and an agitation assessment using the richmond agitation sedation scale (rass). the study was performed at an inner city level i trauma center with 60,000 visits per year. the ed was a catchment facility for the police department for psychiatric patients in the area. patients were excluded if they were unstable, unable to communicate, or had a non-psychiatric complaint. results were analyzed in spss v16. the analysis of data used frequencies, descriptive and anova. results: a total of 100 patients were enrolled in the study: 72% were african american, 14% caucasian, 13% hispanic, 1% asian, and 1% indian; 63% of subjects enrolled were male. the patients' level of agitation using rass showed 59% were alert and calm, 22% were restless and anxious, 6% were agitated, and 5% combative, violent, or dangerous to self. the only significant correlation found was among the ats and several self assessment questions: ''i feel agitated on a 0 to 10 scale'' (p = 0.031) and ''i feel violent on a 0 to 10 scale'' (p = 0.001). there were no significant correlations found among the ena triage, rass scores, and throughput times. conclusion: the ats test was more sensitive to the patient declaring that he or she was agitated or felt violent. this shows that this system might be a more useful system in determining the severity of need of psychiatric patients presenting to the ed. variations background: hemoglobin-based oxygen carriers (hbocs) have been evaluated for small-volume resuscitation of hemorrhagic shock due to their oxygen carrying capability, but have found limited utility due to vasoactive side-effects from nitric oxide (no) scavenging. objectives: to define an optimal hboc dosing strategy and evaluate the effect of an added no donor, we use a prehospital swine polytrauma model to compare the effect of low-vs. moderate-volume hboc resuscitation with and without nitroglycerin (ntg) co-infusion as an no donor. we hypothesize that survival time will improve with moderate resuscitation and that an no donor will add additional benefit. methods: survival time was compared in groups (n = 7) of anesthetized swine subjected to simultaneous traumatic brain injury and uncontrolled hemorrhagic shock by aortic tear. animals received one of three different resuscitation fluids: lactated ringers (lr), hboc, or vasoattenuated hboc with ntg co-infusion. for comparison, these fluids were given in a severely limited fashion (sl) as one bolus every 30 minutes up to four total, or a moderately limited fashion (ml) as one bolus every 15 minutes up to seven total, to maintain mean arterial pressure ‡60 mmhg. comparison of resuscitation regimen and fluid type on survival time was made using two-way anova with interaction and tukey kramer adjustment for individual comparisons. results: there was a significant interaction between fluid regimen and resuscitation fluid type (anova, p = 0.011) indicating that the response to sl or ml resuscitation was fluid type-dependent. within the lr and hboc+ntg groups, survival time (mean, 95%ci) was longer for sl, 323.5 min ( injuries are common and result from many different mechanisms of injury (moi). knowing common fracture locations may help in diagnosis and treatment, especially in patients presenting with distracting injuries that may mask the pain of a radius fracture.objectives: we set out to determine the incidence of radius fracture locations among patients presenting to an urban emergency department (ed).background: carbon monoxide (co) is the leading cause of poisoning morbidity and mortality in the united states. standard treatment includes supplemental oxygen and supportive care. the utility of hyperbaric oxygen (hbo) therapy has been challenged by a recent cochrane review. hypothermia may mitigate delayed neurotoxic effects after co poisoning as it is effective in cardiac arrest patients with similar neuropathology. objectives: to develop a rat model of acute and delayed severe co toxicity as measured by behavioral deficits and cell necrosis in post-sacrifice brain tissue.methods: a total of 28 rats were used for model development; variable concentrations of co and exposure times were compared to achieve severe toxicity. for the protocol, six senescent long evans rats were exposed to 2,000 ppm of co for 20 minutes then 1,500 ppm for 160 minutes, followed by three successive dives at 30,000 ppm with an endpoint of apnea or seizure; there was a brief interlude between dives for recovery. a modified katz assessment tool was used to assess behavior at baseline and 2 hours, 1 day, and 1, 2, 3, 4, 5, and 6 weeks post-exposure. following this, the brains were transcardially fixed with formalin, and 5 lm sagittal slices were embedded in paraffin and stained with hematoxylin and eosin. a pathologist quantified the percentage of necrotic cells in the cortex, hippocampus (pyramidal cells), caudoputamen, cerebellum (purkinje cells), dentate gyrus, and thalamus of each brain to the nearest 10% from 10 randomly selected high power fields (400x background: there remains controversy about the cardiotoxic effects of droperidol, and in particular the risk of qt prolongation and torsades des pointes (tdp).objectives: this study aimed to investigate the cardiac and haemodynamic effects of high-dose parenteral droperidol for sedation of acute behavioural disturbance (abd) in the emergency department (ed). methods: a standardised intramuscular (im) protocol for the sedation of ed patients with abd was instituted as part of a prospective observational safety study in four regional and metropolitan eds. patients with abd were given an initial dose of 10 mg droperidol followed by an additional dose of 10 mg after 15 min if required. inclusion criteria were patients requiring physical restraint and parenteral sedation. the primary outcome was the proportion of patients who have a prolonged qt interval on ecg. the qt interval was plotted against the heart rate (hr) on the qt nomogram to determine if the qt was abnormal. secondary outcomes were frequency of hypotension and cardiac arrhythmias. results: ecgs were available from 273 of 424 patients with abd given droperidol. the median dose was 10 mg (iqr 10-15 mg; range: 5 to 30 mg). the median age was 33 years (rnge: 16 to 92) and 163 were males (60%). a total of four (1%) qt-hr pairs were above the ''at-risk'' line on the qt nomogram. transient hypotension occurred in 8 (3%), and no arrhythmias were detected.conclusion: droperidol appears to be safe when used for rapid sedation in the dose range of 5 to 30 mg. it rarely causes hypotension or qt prolongation. blood background: soldiers and law enforcement agents are repeatedly exposed to blast events in the course of carrying out their duties during training and combat operations. little data exist on the effect of this exposure on the physiological function of the human body. both military and law enforcement dynamic entry personnel, ''breachers'', began expressing sensitivity to the risk of injury as a result of multiple blast exposures. breachers apply explosives as a means of gaining access to barricaded or hardened structures. these specialists can be exposed to as many as a dozen lead-encased charges per day during training exercises.objectives: this observational study was performed by the breacher injury consortium to determine the effect of short-term exposure to blasts by breachers on whole blood lead levels (blls) and zinc protoporphyrin levels (zppls). methods: two 2-week basic breaching training classes were conducted by the united states marine corps' weapons training battalion dynamic entry school. each class included 14 students and up to three instructors, with six non-breaching marines serving as a control group. to evaluate for lead exposure, venous blood samples were acquired from study participants on the weekend prior and following training in the first training class, whereas the second training class had an additional level performed mid-training. blls and zppls were measured in a whole-blood sample using the furnace atomic absorption method and hematofuorimeter method, respectively. results: analysis of these blast injury data indicated students demonstrated significantly increased blls post-explosion (mean = 7 mcg/dl, sd 2.42, p < 0.001) compared to pre-training (mean = 3 mcg/dl, sd 1.60) and control subjects (mean = 3 mcg/dl, sd 2.73, p < 0.001). instructors also demonstrated significantly increased blls post explosion (mean = 6 mcg/dl, sd 1.95, p < 0.02) compared to pre-training (mean = 3 mcg/ dl, sd 1.14) and control subjects (mean = 3 mcg/dl, sd 2.73, p < 0.001). student and instructor zppls were not significantly different in post-training compared to pretraining or control groups. conclusion: the observation from this study that breachers are at risk of mild increases in blls support the need for further investigation into the role of lead following repeated blast exposure with munitions encased in lead. direct observation of the background: notification of a patient's death to family members represents a challenging and stressful task for emergency physicians. complex communication skills such as those required for breaking bad news (bbn) are conventionally taught with small-group and other interactive learning formats. we developed a de novo multi-media web-based learning (wbl) module of curriculum content for a standardized patient interaction (spi) for senior medical students during their emergency medicine rotation.objectives: we proposed that use of an asynchronous wbl module would result in students' skill acquisition for breaking bad news. methods: we tracked module utilization and performance on the spi to determine whether students accessed the materials and if they were able to demonstrate proficiency in its application. performance on the spi was assessed utilizing a bbn-specific content instrument developed from the griev_ing mnemonic as well as a previously validated instrument for assessing communication skills.results: three hundred seventy-two students were enrolled in the bbn curriculum. there was a 92% completion rate of the wbl module despite students being given the option to utilize review articles alone for preparation. students interacted with the activities within the module as evidenced by a mean number of mouse clicks of 42.1 (sd 21.6). overall spi scores were 94.5%, (sd 4.4) with content checklist scores of 92.8% (sd 5.7) and interpersonal communication scores 97.9% (sd 4.7). five students had failing content scores (<75%) on the spi and had a mean number of clicks of 30.8 (sd 28.2), which is not significantly lower than those passing (p = 0.21). students in the first year of wbl deployment completed self-confidence assessments which showed significant increases in confidence (2.86 tobackground: pelvis ultrasonography (us) is a useful bedside tool for the evaluation of women with suspected pelvic pathology. while pelvic us is often performed by the radiology department, it often lacks clinical correlation and takes more time than bedside us in the ed. this was a prospective observational study comparing the ed length of stay (los) of patients receiving ed us versus those receiving radiology us. objectives: the primary objective was to measure the difference in ed los. the secondary objectives were to 1) assess the role of pregnancy status, ob/gyn consult in the ed, and disposition, in influencing the ed los; and 2) to assess the safety of ed us by looking at patient return to the ed within 2 weeks and whether that led to an alternative diagnosis.methods: subjects were women over 13 years old presenting with a gi or gu complaint, and who received either an ed or radiology us. a t-test was used for the primary objective, and linear regression to test the secondary objective. odds ratios were performed to assess for interaction between these factors and type of ultrasound. subgroup analyses were performed if significant interaction was detected. results: forty-eight patients received an ed us and 85 patients received a radiology us. subjects receiving an ed us spent 162 minutes less in the ed (p < 0.001). in multivariate analysis, even when controlling for pregnancy status, ob/gyn consult, and disposition, patients who received an ed us had a los reduction of 108 minutes (p < 0.05). in odds ratio analysis, patients who were pregnant were 11 times more likely to have received an ed us (p < 0.05). patients who received an ob/gyn consult in the ed were five times more likely to receive a radiology us (p < 0.05). there was no association between type of us and disposition. in subgroup analyses, pregnant and non-pregnant patients who received an ed us still had a los reduction of 140 minutes (p < 0.01) and 112 minutes (p < 0.05), respectively. sample sizes were inadequate for subgroup analysis for subjects who had ob/gyn consults. in patients who did not receive an ob/gyn consult, those who received an ed us had a los reduction of 139 minutes (p < 0.001). finally, 10% of subjects returned within two weeks, but none led to an alternative diagnosis. conclusion: even when controlling for disposition, ob/gyn consultation, and pregnancy status, patients who received an ed us had a statistically and clinically significant reduction in their ed los. in addition, ed us is safe and accurate. background: although early surface cooling of burns reduces pain and depth of injury, there are concerns that cooling of large burns may result in hypothermia and worse outcomes. in contrast, controlled mild hypothermia improves outcomes after cardiac arrest and traumatic burn injury. objectives: the authors hypothesized that controlled mild hypothermia would prolong survival in a fluidresuscitated rat model of large scald burns. methods: forty sprague-dawley rats (250-300 g) were anesthetized with 40 mg/kg intramuscular ketamine and 5 mg/kg xylazine, with supplemental inhalational isoflurane as needed. a single full-thickness scald burn covering 40% of the total body surface area was created per rat using a mason-walker template placed in boiling water (100 deg c) for a period of 10 seconds. the rats were randomized to hypothermia (n = 20) and nonhypothermia (n = 20). core body temperature was continuously monitored with a rectal temperature probe. hypothermia was induced through intraperitoneal injection of cooled (4 deg c) saline. the core temperature was reduced by 2 deg c and maintained for a period of 2 hours, applying an ice or heat pack when necessary. the rats were then rewarmed back to baseline temperature. in the control group, room temperature saline was injected into the intraperitoneal cavity and core temperature was maintained using a heating pad as needed. the rats were monitored until death or for a period of 7 days, whichever was greater. the primary outcome was death. the difference in survival was determined using a kaplan-meier analysis or log rank test. results: the mean core temperatures were 32.5 deg c for the hypothermic group and 35.6 deg c for the normothermic group. the mean survival times were 124 hours for the hypothermic group (95% confidence interval [ci] = 98 to 150) and 100 hours for the normothermic group (95% ci = 68 to 132). the seven-day survival rates in the hypothermic and non-hypothermic groups were 67% and 53%. these differences were not significant, p = 0.33 for both comparisons. conclusion: induction of brief mild hypothermia increases but does not significantly prolong survival in a resuscitated rat model of large scald burns. serum objectives: we sought to determine levels of serum mtdna in ed patients with sepsis compared to controls and the association between mtdna and both inflammation and severity of illness among patients with sepsis. methods: prospective observational study of patients presenting to one of three large, urban, tertiary care eds. inclusion criteria: 1) septic shock: suspected infection, two or more systemic inflammatory response (sirs) criteria, and systolic blood pressure (sbp) <90 mmhg despite a fluid bolus; 2) sepsis: suspected infection, two or more sirs criteria, and sbp >90 mmhg; and 3) control: ed patients without suspected infection, no sirs criteria, and sbp >90 mmhg. three mtdnas (cox-iii, cytochrome b, and nadh) were measured using real-time quantitative pcr from serum drawn at enrollment. il-6 and il-10 were measured using a bio-plex suspension array system. baseline characteristics, il-6, il-10, and mtdnas were compared using one way anova or fisher exact test, as appropriate. correlations between mtdnas and il-6/il-10 were determined using spearman's rank. linear regression models were constructed using sofa score as the dependent variable, and each mtdna as the variable of interest in an independent model. a bonferroni adjustment was made for multiple comparisons.results: of 93 patients, 24 were controls, 29 had sepsis, and 40 had septic shock. we found no significant difference in any serum mtdnas among the cohorts (p = 0.14 to 0.30). all mtdnas showed a small but significant negative correlation with il-6 and il-10 (q = )0.24 to )0.35). among patients with sepsis or septic shock (n = 69), we found a small but significant negative association between mtdna and sofa score, most clearly with cytochrome b (p = 0.001). conclusion: we found no difference in serum mtdnas between patients with sepsis, septic shock, and controls. serum mtdnas were negatively associated with inflammation and severity of illness, suggesting that as opposed to trauma, serum mtdna does not significantly contribute to the pathophysiology of the sepsis syndromes. methods: we consecutively enrolled ed patients ‡18 years of age who met anaphylaxis diagnostic criteria from april 2008 to july 2011 at a tertiary center with 72,000 annual visits. we collected data on antihypertensive medications, suspected causes, signs and symptoms, ed management, and disposition. markers of severe anaphylaxis were defined as 1) intubation, 2) hospitalization (icu or floor), and 3) signs and symptoms involving ‡3 organ systems. antihypertensive medications evaluated included beta-blockers, angiotensin converting enzyme (ace) inhibitors, and calcium channel blockers (ccb). we conducted univariate and multivariate analyses to measure the association between antihypertensive medications and markers of severe anaphylaxis. because previous studies demonstrated an association between age and the suspected cause of the reaction with anaphylaxis severity, we adjusted for these known confounders in multivariate analyses. we report associations as odds ratios (ors) and corresponding 95% cis with p-values. results: among 302 patients with anaphylaxis, median age (iqr) was 44 (31-58) and 204 (67.5%) were female. eight (2.7%) patients were intubated, 57 (19%) required hospitalization, and 139 (46%) had ‡3 system involvement. forty-nine (16%) were on beta-blockers, 34 (11%) on ace inhibitors, and 22 (7.3%) on ccb. in univariate analysis, ace inhibitors were associated with intubation and ‡3 system involvement and ccb were associated with hospital admission. in multivariate analysis, after adjusting for age and suspected cause, ace inhibitors remained associated with hospital admission and beta-blockers remained associated with both hospital admission and ‡3 system involvement. conclusion: in ed patients, beta-blocker and ace inhibitor use may predict increased anaphylaxis severity independent of age and suspected cause of the anaphylactic reaction. background: advanced cardiac life support (acls) resuscitation requires rapid assessment and intervention. some skills like patient assessment, quality cpr, defibrillation, and medication administration require provider confidence to be performed quickly and correctly. it is unclear, however, whether high-fidelity simulation can improve confidence with a multidisciplinary group of providers with high levels of clinical experience. objectives: the purpose of the study was to test the hypothesis that providers undergoing high-fidelity simulation of cardiopulmonary arrest scenarios will express greater confidence. methods: this was a prospective cohort study conducted at an urban level i trauma center from january to october 2011 with a convenience sample of registered (rn) and license practical nurses, nurse practitioners, resident physicians, and physician assistants who agreed to participate in 2/4 high-fidelity simulation (laerdal 3g) sessions of cardiopulmonary arrest scenarios about 3 months apart. demographics were recorded. providers completed a validated preand post-test five-point likert scale confidence measurement tool before and after each session that ranged from not at all confident (1) to very confident (5) in recognizing signs and symptoms of, appropriately intervening in, and evaluating intervention effectiveness in cardiac and respiratory arrests. descriptive statistics, paired t-tests, and anova were used for data analysis. sensitivity testing evaluated subjects who completed their second session at 6 months rather than 3 months. results: sixty-five subjects completed consent, 39 completed one session, and 23 completed at least two background: prehospital studies have focused on the effect of health care provider gender on patient satisfaction. we know of no study that has assessed patient satisfication with patient and prehospital provider gender. some studies have shown higher patient satisfaction rates when cared for by a female health care provider.objectives: to determine the effect of ems provider gender on patient satisfaction with prehospital care. methods: a convenience sampling of all adult patients brought in to our ed, an urban level i trauma center by ambulance. a trained research associate (ra) stationed at triage conducted a survey using press ganey ems patient satisfaction questions. there were thirteen questions evaluating prehospital provider skills such as driving, courtesy, listening, medical care, and communication. each skill was assigned a point value between one and five; the higher the value the better the skill was performed. the patient's ambulance care report was copied for additional data extraction.results: a total of 225 surveys were done. average patient age was 71, and 54% were female. scores for all questions totaled 65 (mean 62.63 ± 5.1). prehospital providers pairings were: male-male (n = 141), male-female (n = 71), and female-female (n = 13). there were no statistically significant differences in scores between our pairings (mean scores for male:male 19.3, male:female 19.1, and female:female 19.2; p = 0.73). we found nonstatistical differences in satisfaction scores based on the gender of the emt in the back of the ambulance: males had a mean score of 62.7 and females had a mean score of 62.6 (p = 0.91). we examined gender concordance by comparing gender of the patient to the gender of the prehospital provider and found that male-male had a mean score of 62.8, female-female 62.2, and when the patient and prehospital provider gender did not match, 62.5 (p = 0.71). conclusion: we found no effect of gender difference on patient satisfaction with prehospital care. we also found that overall, patients are very satisfied with their prehospital care. objectives: we set out to determine the sensitivity and specificity of eps in determining the presence of recently ingested tablets or tablet fragments.methods: this was a prospective volunteer study at an academic emergency department. healthy volunteers were enrolled and kept npo for 6 hours prior to tablet ingestion. over 10 minutes subjects ingested 800 ml of water and 30 tablets. ultrasounds video clips were performed prior to any tablet ingestion, after drinking 200 ml of water, after 10 tablets, after 20 tablets, after 30 tablets, and 60 minutes after the final tablet ingestion yielding six clips per volunteer. all video clips were randomized and shown to three eps who were fellowship-trained in emergency ultrasound. eps recorded the presence or absence of tablets.results: ten volunteers underwent the pill ingestion protocol and sixty clips were collected. results for all cases and each rater are reported in the table. overall there was moderate agreement between raters (kappa = 0.42). sub-group analysis of 10, 20, or 30 pills did not show any significant improvement in sensitivity and specificity.conclusion: ultrasound has moderate specificity but poor sensitivity for identification of tablet ingestion. these results imply that point-of-care ultrasound has limited utility in diagnosing large tablet ingestion. background: intravenous fat emulsion (ife) therapy is a novel treatment that has been used to reverse the acute toxicity of some xenobiotics with varied success. us poison control centers (pcc) are recommending this therapy for clinical use, but data regarding these recommendations are lacking.objectives: to determine how us pcc have incorporated ife as a treatment strategy for poisoning. methods: a closed-format multiple-choice survey instrument was developed, piloted, revised, and then sent electronically to every medical director of an accredited us pcc using surveymonkey in march 2011; addresses were obtained from the aapcc listserv, participation was voluntary and remained anonymous; three reminder invitations were sent during the study period. data were analyzed using descriptive statistics.results: forty-five of 57 (79%) pcc medical directors completed the survey. all 45 respondents felt that ife therapy played a role in the acute overdose setting. thirty (67%) pcc have a protocol for ife therapy: 29 (97%) recommend an initial bolus of 1.5 ml/kg of a 20% lipid emulsion, 28 (93%) pcc recommend an infusion of lipids, and 27/28 pcc recommend an initial infusion rate of 0.25 ml/kg of a 20% lipid emulsion. thirty-three (73%) felt that ife had no clinically significant side effects at a bolus dose of 1.5 ml/kg (20% emulsion). forty-four directors (98%) felt that the ''lipid sink'' mechanism contributed to the clinical effects of ife therapy, but 26 (58%) felt that there was a yet undiscovered mechanism that likely contributed as well. in a scenario with cardiac arrest due to a single xenobiotic, directors stated that their center would always or often recommend ife after overdose of bupivicaine (43; 96%), verapamil (36; 80%), amitriptyline (31; 69%), or an unknown xenobiotic (12; 27%). in a scenario with significant hemodynamic instability due to a single xenobiotic, directors stated that their pcc would always or often recommend ife after overdose of bupivicaine (40; 89%), verapamil (28; 62%), amitriptyline (25; 56%), or an unknown xenobiotic (8; 18%).conclusion: ife therapy is being recommended by us pcc. protocols and dosing regimens are nearly uniform. most directors feel that ife is safe but are more likely to recommend ife in patients with cardiac arrest than in patients with severe hemodynamic compromise. further research is warranted. levels drawn at 4 hours or more (240 mcg/ml at 5 hours, 198 mcg ⁄ ml at 4 hours, respectively). npv for toxic ingestion of an initial apap level less than 100 mcg/ml was 97.8% (95% ci 92.3-99.7%).conclusion: an apap level of less than 100 mcg/ml drawn less than 4 hours after ingestion had a high npv for excluding toxic ingestion. however, the authors would not recommend reliance on levels obtained under 4 hours to exclude toxicity as the potential for up to 6.7% false negative results is considered unacceptable. background: genetic variations in the mu-opioid receptor gene (oprm1) mediate individual differences in response to pain and addiction.objectives: to study whether the common a118g (rs1799971) mu-opioid receptor single nucleotide polymorphism (snp) or the alternative splicing snp of oprm1 (rs2075572) was associated with overdose severity, we assessed allele frequencies of each including associations with clinical severity in patients presenting to the emergency department (ed) with acute drug overdose. methods: in an observational cohort study at an urban teaching hospital, we evaluated consecutive adult ed patients presenting with suspected acute drug overdose over a 12-month period for whom discarded blood samples were available for analysis. specimens were linked with clinical variables (demographics, urine toxicology screens, clinical outcomes) then de-identified prior to genetic snp analysis. in-hospital severe outcomes were defined as either respiratory arrest (ra, defined by mechanical ventilation) or cardiac arrest (ca, defined by loss of pulse). blinded taqman genotyping (applied biosystems) of the snps were performed after standard dna purification (qiagen) and whole genome amplification (qiagen repli-g). the plink 1.07 genetic association analysis program was used to verify snp data quality, test for departure from hardy-weinberg equilibrium, and test individual snps for statistical association. results: we evaluated 178 patients (37% female, mean age 41.2) who overall suffered 13 ras and 3 cas (of whom 2 died). urine toxicology was positive in 33%, of which there were positives for 32 benzodiazepines, 26 cocaine, 21 opiates, 13 methadone, and 6 barbiturates. all genotypes examined conformed to hardy-weinberg equilibrium. the 118g allele was associated with 2.5fold increased odds of ca/ra (or 2.5, p < 0.05). the rs2075572 mutant allele was not associated with ca/ ra. conclusion: these data suggest that the 118g mutant allele of the oprm1 gene is associated with worse clinical severity in patients with acute drug overdose. the findings add to the growing body of evidence linking the a118g snp with clinical outcome and raise the question as to whether the a118g snp may be a potential target for personalized medical prescribing practices with regard to behavioral/physiologic overdose vulnerability. key: cord-015021-pol2qm74 authors: nan title: third international congress on the immune consequences of trauma, shock and sepsis —mechanisms and therapeutic approaches date: 1994 journal: intensive care med doi: 10.1007/bf02258437 sha: doc_id: 15021 cord_uid: pol2qm74 nan this issue of the journal contains the abstracts for the third international congress on the immune consequences of trauma, shock and sepsis -mechanisms and therapeutic approaches. we hope that the information contained in this special issue will stimulate you to participate in the congress, to contribute to the knowledge being developed in this field and to use this information to help you in providing better care for your patients. we thank the editors and the editorial board and publishers of the journal for their interest and support in preparation of this special issue. we also, on behalf of the scientific committee, welcome you to the third international congress in munich on 2-5 march 1994. when, in the mid-1980s, we thought of having a worldwide congress, we hoped to bring together investigators to discuss this theme. the explosion of knowledge occurring around that time provided an excellent background against which the first conference in 1988 provided stateof-the-art information and consensus on factors involved in injury and sepsis. in 1991, the second congress was held at the time of another resurgence of research, study and information on injured and operated patients. it seemed then that there would be a lull in the development of new information and therapy, and that another state-of-the art conference might not be necessary until 1995 or 1996. however, the explosion in molecular biology has continued. the wonderful world of cytokines has gone from ill to il-6 to il-11, il-12 and il-13 and beyond. the vast amount of information about mediators and their importance in disease is impressive. this has all suggested a magic bullet that might be used to alter or block inflammatory responses. this has not happened, however, and the question is "why not"? our science is powerful, but our therapy is still weak. what are the issues, then, in 1994, to be dealt with at this symposium and congress? (1) proposals for new terminology. there have been a number of proposals for new terminology and new classifications of injury, sepsis, inflammation and various other problems related to human illness. the question is whether this is the way to go. will this contribute to better clinical trials, information basis and better research? the pros and cons of this development will be reviewed by those making the proposals and those questioning the need for and wisdom of this effort. (2) magic bullets: the prospect of a magic bullet to deal with inflammation in injury and infection seemed highly promising earlier. many preclinical trials and a lot of animal research suggested the possibility of a great breakthrough in clinical care. what has become, then, of all the expensive and extensive multi-institution randomized, placebo-controlled, double-blind clinical trials of agents that block mediators and endotoxin. many such studies have yielded equivocal, marginal or negative results. the reasons for this and the future of clinical research will be the subject of presentations and discussions to set the stage for further work. (3) should future clinical trials be based on new classifications of illness such as mods, sirs, apache iii, sap ii, mrm, etc., or should trials be dedicated to specific diseases -urinary tract infections, pneumonia, trauma patients, cardiac surgery and other specific problems, rather than generalized problems of sepsis, the sepsis syndrome and other classifications? in other words, should we now begin to have clinical trials on specific diseases with causes that are known and can be attacked? the causality of disease becomes an important consideration in this regard. (4) a multitude of potential therapeutic agents has been proposed on the basis of animal studies. how should we decide which of them should be brought to clinical trial? the possibilities are endless as we develop new clinical information about the mechanisms and pathogenesis of human disease. (5) information on the pathogenic mechanism of disease states and of injury continued to emerge in an explosive fashion, and in light of our gathering knowledge we can look forward to working out a cohesive system of response to injury. (6) additional information will be provided in plenary sections, many symposia and free communication sessions and posters, which will update the participants on a variety of relevant topics presented by many of the leading in-iv vestigators in these fields. topics will range from molecular mechanisms, such as signal transduction, through the explosive growth of information on the role of cytokines and pathophysiology, to practical considerations in the design of immunomodulatory therapeutic regimens. these merely touch on a few areas, from the basic to the clinical, which will be the subjects of those symposia. all this information will fit into the jigsaw of this exciting area and its stimulus to further research study. this promises to provide an exciting, educational programme with experts and participants from all over the world. we hope it will set the stage for many years to come and will increase our understanding of trauma, shock and sepsis and help us to provide better therapy for those of our patients who are affected by such problems. a. the clinical syndrome of mods versus mof will be reviewed in detail by those who have made these proposals. b. an extensive review of the design and interpretation of clinical trials in patients with shock and injury will be provided. the reasons why so many clinical studies in the recent past have been negative will be reviewed. the therapeutic strategies that are being developed for the treatment or prevention of mods or mof will be the subject of another panel discussion by experts who have been involved in and contributed to this area. a consensus conference or controversy conference will be presented about various aspects of mods or mof, including the benefits of supernormal oxygen delivery, bacterial translocation, parenteral nutrition, the immune response and other aspects. the successes and failures of completed clinical trials will be presented by those who are involved in these clinical trials, with a refreshing review of the problems related to that injury. there will be late news about studies just being completed at present or after the beginning of 1994 and where they stand. c. the mechanisms and biochemical profiles of specific organ dysfunction or failure will be reviewed. what are the definitions? what are the mechanisms? how can organ dysfunction and/or failure be defined? an extensive review of the biological mechanisms involved in production of injury by mediators will be presented. a session will be devoted to how future ongoing trials might be better designed and what can be done about the studies recently completed, many of which are negative. d. the immunological or inflammatory pathways resulting in organ injury will be reviewed in detail in presentations and a panel discussion. we look forward to welcoming you to an exciting and rewarding conference, which undoubtedly possesses the potential to become a landmark event and major reference point for any scientific discussion about the complex of host defense dysfunctions following trauma, shock and sepsis. studies over the past 25 years have established that the contact system, which forms bradykin/~, is gax important mediator in hypotensive septicemia. in addition to hradyk{nln, another product of the contact system, kailikrein, can mediate inflammation by virtue of its chemotaetic mad neutrophj/activating properties. using functional and immunochemical tech~2ques, we have demonstrated activation of the contact system in the adult respiratory distress syndrome in typhoid fever and clin/cal sepsis. we have also been able to inhibit the hypotension but not the disseminated intravaseular coagulation in a model of primate sepsis by the use of a monoclonal antibody directed agsi~st factor xii, the initiating protein of the contact system, in volunteers given e. coil endotoxin, who did not develop hypotension, we were also able to demonstrate activation of the contact system with a rise of alpha-2macrogiebulin-kalllkrein complex. we have also examined, j~ an i~tensive care situation, patients with sirs. we found that serial measuremezzts of the contact system were useful in eva~u~ting prognosis+ these studies suggest that inhibition of kalllkrein a~d l e r bradykinin actions might be useful i~ obviating many .of the features seen in sepsis and septic shock. dextran sulfate (dxs) activates the contact system and, in vivo, produces transient hypotension. in order to better define the mechanisms underlying the dxs-induced hypotension, we investigated the effects of either the plasma kallikrein inhibitor, des-pro2-iarg]5]aprotinin (bay 4620) or the b2 kinin antagonist, hoe 140 on the hypotensive response to dxs. in the first study, anesthetized miniature pigs (5 pigs/group, randomly assigned) were given one of the following treatment protocols: 1) dxs (5 mg/kg), 2-5) dxs plus bay 4620 (45, 90, 180, or 360 rag), or 6) saline. dxs alone produced a profound but transient systemic arterial hypotension with a corresponding reduction in plasma kinin-containing kininogen. circulating kinin levels, complement fragment c3adesarg and fibrin mom)mer were all increased. bay 4620 produced a dose-dependent delay or attenuation in these effects with the highest dose completely blocking dxs-induced hypotension and elevations of kinin, c3adesarg and fibrin monomer levels. thus, the effects of dxs are solely dependent on contact system activation and this activation is sensitive to bay 4620. llowev~:r, contact system activation is known to produce changes in a variety of vasoactive mediators, all of which can affect blood pressure. in a second study, two groups of pigs (3/group) were given either dxs alone (2 mg/kg) or dxs 10 minutes after a bolus injection of hoe 140 (30 #g/kg). dxs alone produced transient hypotenmon. this response was completely blocked by hoe 140 pretreatment. both groups had identical reductions in kinin-containing kininogen. we conclude that dxs-induced hypotension is produced by activation of the contact system which results in the production of bradykinin. liberation of bradykinin is both necessary and sufficient to produce all of the hemodynamic changes observed. dr. matthias siebeck, department of surgery, university of munich, klinikum lnnenstadt, nussbaumstrasse 20, d-80336 munich, germany in experimental animals exposed to i.v. injection of endotoxin accumulation of leukocytes in various organs as lungs and the liver is a prominent feature. as a part of these morphological changes damages of endothelial ceils are regularly seen. this process, which is a part of endothelial-cellular interaction, leeds to exposure of the sub-endothelial basement membran. the basement membran is known f6r its capacity to activate the contact system of plasma. during this cascade activation, coagulation factor xii is converted to the active factor xii. this activation might produce increased plasma kallikrein activities and thereby give release of the vasoactive substance bradykinin. using a porcine model we have noticed that endotoxin infusion (0,01 mg/kg) induces elevated plasma kailikrein activities within two hours after the start of the infusion. this enzyme activity remained increased during the next hours and reached value of up to 50 u/1. in patients with sepsis we also have observed elevated plasma kallikrein activities with enzyme activities up to 200 u/1. in order to further elucidate the significance of these elevated enzyme activities, we prepared human plasma kallikrein and injected it intravenously in anaesthetized pigs (1). when very small plasma kailikrein activities (0,3 u/kg bodyweight) were given intravenously a 50% decrease in arterial blood pressure was seen in the animals. in the patients with sepsis also decreases in prekallikrein values and functional plasma kallikrein inhibition are frequently seen. furthermore, degradation of high molecular weight kioinogen is found in these patients indicating formation of bradykinin. these experimental and clinical studies underline that contact activation in sepsis might results in the release of very powerful mediator substances which can be of pathophysiological importance in this disease. a number of pathological disorders as reperfusion injury, bone marrow transplantation, polytrauma and septic shock are associated with capillary leakage. as the activation of the complement system and the contact phase play a major role in these diseases we investigated whether cl-lnhibitor (c1-inh), which inactivates cl-esterase, kallikrein and clotting factors xii and xl, could abolish vascular leakage. a capillary leakage was induced in rats by the administration of interleukin-2 (5 x 106 iu/kg). the increased vascular permeability was monitored for one hour as the extravasation of fitc marked rat serum albumin from a mesenterial vessel by a video-image processing system. ci-inh (berinert®, behringwerke) given as a single i.v. bolus in concentrations of 100, 250 or 500 u/kg dose-dependently prevented the capillary leakage. carrageenaninduced inflammation in the rat leads to vascutar leakage and to edematous swelling of the paw. ci-inh in this model leads to a dose-dependent decrease in paw edema formation. finally, we investigated the effect of ci-inh (infusion (100-125 u/kg x h) on a lps-induced shock in the rat by combination therapy with the antithrombotlc agents antithrombin ill (kybernin®) or rec. hirudin (both substances from behringwerke). in this animal model mortality was 90 % in the untreated control. both antithrombotic agents decreased mortality rates by inhibiting formation of dic; a further significant improvement of survival was achieved by the treatment with ci-inh. thus+ it could be concluded that c1-inh has a beneficial effect in diseases associated with a vascular leakage. iclb and laboratory for experimental and clinical immunology, university of amsterdam, the netherlands; 2thrombosis research center, temple university, penn., usa; 3oklahoma medical research foundation,. ok. city, usa. to evaluate the contribution of the contact system to activation of other mediator systems in an experimental model of sepsis, we investigated the effect of mab c6b7 which inhibits activation of factor xli, on activation of complement and fibrinolytic cascades and activation of neutrophils in baboons suffering from a lethal sepsis. activation of the complement system was assessed by measuring circulating levels of c3b/c and c4b/c, and a significant reduction was observed in 5 animals that had received a lethal dose of e. coli together with mab c687 (treatment group), compared to 4 animals that had received a lethal dose of e. coil only (control group). activation of the fibrinolytic system as reflected by circulating plasmin-=2antiplasmin complexes and tissue plasminogen activator, and activation of neutrophils, assessed by measuring circulating elastase-=l-antitrypsin complexes, was also significantly less in the treatment group. we conclude that activation of the contact system protein factor xll during the inflammatory response to a lethal dose of e. coil in this baboon model, modulates directly or indirectly activation of the complement and fibrinolytic systems and that of neutrophils. in a prospective study, plasma levels of c3a, c3, and c5a were measured in 30 patients from an internal intensive care unit. 20 patients were clinically septic defined by the criteria of bone et al.(l) . the remaining 10 patients were critically ill but didn't fulfill the clinical criteria of sepsis. from both groups of patients blood samples were taken over a l0 days period. during the first 3 days blood samples were drawn every 8 h, on day 4-6 every 12 h and the last 4 days once daily. mean plasma concentrations of c3a within the first 32 h after clinical onset of sepsis were 1019 + 618 pg/ml, whereas non-septic-patients exhibited mean values of only 595 +_ 312 p_g/m/. c3 levels were lower for septic-patients (840 + 480 lag/ml) than for non-septic-patients (1340 _+ 770 lag/ml). the most profound difference between both groups was found, when the c3a/c3 ratio was compared (1.84 + 2.28 for septic-patients and 0.4 _+_ 0.18 for the control group). no significant differences between both patient groups were observed in c5a plasma levels (5.9 + 2.1 ng/ml in septic-patients vs. 5.6 _+ 1.5 ng/ml in control patients). in 13 of 20 cases of clinically defined sepsis causative organisms like bacteria, protozoa or fungi could be cultured from blood, bronchoalveolar lavages and/or section materials. application of the complement parameters to survivors (n=9) and non-survivors (n=l 1) within the septic-group revealed, that the c3a/c3 ratio could also be used as a prognostic parameter for clinical outcome. the possibility of rapid and easy measurement of c3a and c3 in only 20-25 minutes (2) and the significant difference of the c3ajc3 ratio between the septic and non-septic group renders this parameter a good candidate for early diagnosis of sepsis in the intensive care unit. hirudin, a single polypeptide chain composed of 65 amino acids with 6 cysteine residues (mr 7000 daitons), is the most potent and specific thrombin inhibitor, which is now available as a genetically engineered product (rec. hirudin -hbw 023, behringwerke; marburg). the aim of our study was to establish a rabbit model of tissue factor (tf) induced activation of the extrinsic pathway of coagulation and to evaluate the therapeutic efficacy of rec. hirudin. coagulation was induced in female nzw rabbits by infusion of 0.5 p.g/kgxh thromboplastin for 7 hours. development of disseminated clotting was manifested by a decrease of fibrinogen and platelets to 27.0 % and 33,0 % respectively, and by an increase of fibrin monomers from 13.2 to > 85.0 ~tg/ml. we administered rec. hirudin to rabbits in 3 different concentrations (0.5, 1.0 and 2.0 mg/kg); treatment started simultaneously with the infusion as an i.v. bolus. rec. hirudin significantly prevented the decrease of fibrinogen, platelets and the increase of fibrin monomers. this effect was dose dependent and long lasting, even 7 hours after the administration of rec. hirudin, clotting was still significantly reduced. as could be drawn from the plasma levels, rec. hirudin had been cleared from plasma at this time. in a post-treatment study we administered rec. hirudin (0.5, 1.0 and 2.0 mg/kg i.v. bolus) as late as 2 hours after the start of tf infusion. at this time there was already a prominent activation of coagulation. even in this post-treatment regimen rec. hirudin significantly prevented disseminated clotting. hence, it was concluded, that rec. hirudin by inkihiting thrombin could be effective in the prevention of coagulation disorders including disseminated intravascular clotting (dic) induced by a septic disease. research laboratories of behringwerke ag, 35001 marburg, germany $3 novel protease inhibitory activities of the second domain of urinary trypsin inhibitor (r-020) and its effect on sepns-lnduced organ injury in rat atsuo murata 1, hitoshi toda 1, ken'ichi uda 1, hidewaki nakagawa 1 , takesada mori 1, hideaki morishita 2, tom yamakawa 2, jiro hirese 2, atsushi ni~ 2, nariaki matsuura 3 1osaka university medical school, osaka, 2mochida pharmaceutical co. ltd. tokyo, 3wakayama medical schoof, wakayama, japan inhibitory-activities of the second kuntz-type inhibitor domain of human urinary trypsin inhibitor (uti) and its effect on sepsis-induced organ injury in rat were investigated by using the recombinant protein. uti is a glycoprotein with a structure in which 2 kunitz-type inhibitor domains are linked in a row. we isolated the gene encoding the second kunitz-type inhibitor domain of uti, and then constructed expression plasmids by ligating it to the e. coli phoa signal peptide gene. these plasmids expressed the second domain in e. coil strain je5505 which lacks the membrane lipoprotein. the recombinant second domain (r-020) innb[ted trypsin, plasmin, neutrophil elastase and chymotrypsin. in addition it inhibited blood coagulation factor xa and plasma kallikrein in a concentration dependent and competitive manner. the in vivo effect of the recombinant r-020 was investigated in a rat model of septic shock induced by cecal ligation and puncture. the administration of r-020 significantly improved the survival rate of the rats and attenuated the pathological changes of lung and iiver. we found out the novel protease inhibitory activities of the second domain of uti and its protective effects on sepsis-induced organ injury. macrophages are known to secrete lysosomal proteinases,mainly cathepsin b and cathepsin l, and also ~-proteinase inhibitor (pi),related to acute phase proteins.disturbances of proteinases/ proteinase inhibitors correlates with inflammatory process,leading sometimes to noncontrol "pathglogical" proteolysis (jochum et ai.,1990) . the cathepsin l-like and cathepsin b-like activity were measured in serum of 42 patients with chronic bronchitis (14 -with obstructive, 28 -with nonobstructive bronchitis),acute bronchitis (15) and 35 healthy persons.simultaneously the level of~pi was determined in the same groups.cysteine proteinases were measured with help of fluorogenic substrates,as was presented earlier (korolenko et ai.,1991) , ~pi with help of immune enzyme method. it was shown increase of cathepsin l-like and cathepsin b-like activities during aggravation of chronic bronchitis comparatively to the controls (2-4 fold) .after treatment there was a tendency to normalization of indices,but the increase was about 30-40% more than the control values.~pi level in this group was also increased (two-fold),in patients with acute bronchitis -2-4-times more comparatively to the control.it is possible to conclude that chronic bronchitis induced increased secretion both cysteine proteinases and d{pi into blood. some peculiarities of ratio were noted in patients with emphysema. endotoxins are microbial products derived from the outer cell membrane of gram negative bacteria. the active component of endotoxin is lipopolysaccharide (lps), a complex macromolecule consisting of polysaccharide covalently bound to a unique lipid, termed lipid a. now recognized to embody the endotoxic principle of lps, lipid a consists of a/31-6 diglucosamine backbone, both ester and amide linked fatty acids, some of which are acyloxyacylated, and charged constituents such as phosphate, phosphorylethanolamine and 4 amino arbinose lps, exerts its biological effects in vivo by noncytotoxic interactions with a variety of host inflammatory mediator cells, primarily the mononuclear phagocyte and the endothelial cell, although other host cells also participate. these interactions are modulated by lps-specific binding proteins found in plasma, including lps-binding protein (lbp) scd14 and perhaps other proteins as well. specific receptors for lps have been identified on mammalian cells which mediate signal transduction via multiple pathways. lps-activated host cells are stimulated to secrete or express multiple proinflammatory mediators, including tnf-a, illa, il-1/3, ifn-a, il-6, il-8, il-10, paf, pge, ltb 4 and procoagulant activity. the overproduction of these proinfiammatory mediators results in the manifestations of endotoxemia, observed experimentally as fever, hypotension, disseminated intravascular coagulation and death. modulation of activity of these mediators protects animals against lethality. similar pathways are thought to be operative in gram negative sepsis, and control studies with human volunteers support such conclusions. immunotherapeutic approaches in clinical gram negative sepsis have, to date, been less successful. in vitro experiments and studies in animal models have recently shown that several proteinaceous bacterial exotoxins can evoke cytotoxic effects that ultimately lead to cardiovascular collapse and shock. since the possible relevance of bacterial exotoxins in the pathogenesis of septic shock has received very little attention in the past, an attempt will be made here to provide a brief overview of this generaily neglected topic. protein toxins act intracellularly or they dz~nage the integrity and function of the plasma membrane. major representatives of the former group are the adenosine diphosphate (adp)-ribosylating toxins, e.g. cholera and cholera-like toxins, diphtheria toxin), and the neurotoxins. most medically relevant toxins of this category have been studied in great detail. although often responsible for severe and sometimes fatal disease, their association with septic shock is rare. in contrast, experimental evidence is accumulating for a role of membrane<lamaging toxins in the pathogenesis of inflammatory lesions. formation of transmembrane pores is probably the most widespread mechanism via which such physical membrane perturbation can occur (1,2), the present discussion will be restricted to this category of toxins. the author shall first discuss basic properties of pore-forming proteins, and consider the factors that direct their attack to specific targets. thereafter, attention will be drawn to diverse functional consequences that may follow membrane damage so that a general concept of how pore-forming toxins may contribute towards the development of shock will evolve. i. bhakdi, s. and tranum-jensen. j. 1987 gram positive bacteria produce exotoxins that, at least in part, exhibit the unusual properties of superantigens. superantigens (sag) activate v/3 selectively t cells to produce lymphokines including i1-2 and tnf/lymphotoxin. systemic application of the sag staphylococcal enterotoxin b (seb) to d-galactosamine sensitized mice causes lethal shock within hours. seb reactive v/38 + t cells accumulate within 90-120 min mrna for the ii-2, i1-4, tnfai~ and ifn-3,. parallel in time high concentrations of bloodborne i1-2 and tnf are noted. anti tnf t~//3 neutralizing mab protect mice against seb induced t cell dependent lethal shock thus indicating a key role of tnf in effecting lethal toxicity. within 12-18 h distinct levels of tolerance to seb become discernable on ligand reactive v/38 t ceils, dependent of the dose of seb used. these levels include anergy, t cell receptor downregulation, loss of cd2, cd4, cd8 accessory molecules and programmed cell death. m.freudenberg, y.yaegashi, p.nielsen, c.galanos. the sensitivity towards endotoxin (lps) is genetically determined, however it may be increased under different experimental conditions. these include treatment with hepatotoxic agents such as d-galactosamine, and with live (infection) or killed bacteria. it is well established now that d-galactosamine sensitizes to lps by increasing the susceptibility of the host to toxic activity of lps-induced tnf~. sensitization by bacteria, especially by gram-negative once is of especial interest because it implies that infecting microorganisms not only produce lps, but als0 sensitize the host to its lethal activity. sensitization to lps by bacteria proceeds in all lps-sensitive (lps n) mouse strains that have been investigated so far. it also proceeds in lps-resistant (lps d) c3h/hej mice. the absence of sensitization to lps in lps d c57bl/10sccr mice was identified as being due to their inability to produce interferon-7 (ifn?). administration of exogenous ifn? to these mice conferred sensitivity to lps. conversely, administration of anti-ifn? to lpsn; mice inhibited the development of sensitization'by bacteria. it may be concluded therefore that ifn7 is the mediator of sensitization to lps by bacterial infection. the ifn? defect of c57bl/10sccr mice concerns only the ifn7 response to bacteria, since the response to the t-cell mitogen con a and to cd3 monoclonal antibodies was not impaired. the ifn?producing cells themselves were shown to be intact. on closer investigation the impaired ifn? response was identified as the result of a defective accessory function of macrophages. macrophages of c57bl/10sccr mice are incapable of producing interferon-~ (ifn~) which we could show to be obligatory for the production of ifn? in response to bacteria. although antibiotics are required to clear bacteremia, they do not reverse evolving shock, because endotoxin free in the bloodstream or exposed on the surface of circulating bacteria continues to activate the production of inflammatory mediators, furthermore, antibiotics have been shown to induce release of endotoxin from gram-negative bacteria during the treatment of severe infection. in an in-vitro study, using live escherichia coil, we have observed the release of endotoxin upon treatment with several antibiotics like cefuroxim, imipenem, ceftazidime and aztreonam. incubation with imipenem or ceftazidime induced an early lyeis and a mild rise in endotoxin levels, whereas incubation with cefuroxime or aztreonam resulted in the formation of filaments with a late but dramatic rise in endotoxin levels. in a second study we studied the influence of different antibiotics on the tnf-= production of e.coli stimulated human monocytes. we found again great differences in the production of this important cytokine among the different antibiotics according to their capacity to induce liberation of endotoxin. these differences in the amount of endotoxin release are probably caused by penicillin-binding-proteins (pbp) specificities of the antibiotics with resultant differential morphological changes, in a rat sepsis model treatment with imipenem or ceftazidime resulted in a transient increase in il-6 levels, whereas treatment with aztreonam resulted in progressively increasing levels of il-6 and a significant increase in mortality. the increased mortality in pbp-3 specific aztreonam treated rats might have been the result of filament formation in the abdominal cavity of the septic rats. the endotoxin sequestered at local sites may account for the precipitation of gram-negative shock. finally, we've also demonstrated that in patients under treatment for septic shock endotoxin release can be related to the administration of antibiotics. carbapenem-and cephalosporin-lnduced release of lps from smooth and rough pseudomonas aeruginosa: in-vivo relevance j. jackson and h. kropp, merck research laboratories, rahway, nj b-lactam (cell-wall active) antibiotics are generally deemed the class of antibiotics most responsible for the release of free endotoxin (lps) from gram-negative bacteria due to their cell lytic actions although all antibiotic classes studied thus far induce release of endotoxin. we have recently shown in-vitro that antibiotics within the b-lactam class (i.e. imipenem , meropenem and ceftazidime , with equivalent mics) differ in their propensities to release excessive amounts of lps from both smooth-(s-) and rough-(r-) lps laboratory and clinical and antibiotic-sensitive and -resistant p. aeruginosa isolates and that lps release is independent of cell lysis. additionally, variations in the induction of endotoxin release is antibiotic concentration dependant and correlate with antibiotic penicillin-binding protein (pbp) affinities which result in morphological changes. these studies also show that lps differences measured via chromogenic lps (c-lal) assay correlate with lps lethality in lps-sensitive mice. release of lps was compared to changes in cfus, cell mass, morphology and antibiotic pbp-specificity. corresponding in-vivo studies in cd1 mice against s-lps p. aeruginosa isolates show that, despite equivalent in-vitro protection, antibiotic efficacy in mice differ as much as 100-to lo00-fold. to establish a possible in-vivo role for antibiotic-induced release of free lps we compared antibiotic efficacy results in mice challenged with virulent parent s-lps and its relatively avirulent r-lps mutant (lacking only its 0 side chain) p. aeruginosa isolates. results show the relevance of quantity and physiochemical composition (bioreactivity) of free lps to virulence and in-vivo antibiotic efficacy. in other in-vivo studies chemical agents that destroy cells (m~) which mediate lps lethality in-vivo were used to pretreat mice prior to antibiotic therapy and bacterial challenge with wild type pseudomonas. we conclude that circumstances in which antibiotic-induced filamentation occurs are also conditions that yield excessive lps release, the in-vivo effects of which are shown through the requirement of larger amounts of antibiotic to afford equivalent protection. bacterial endotoxins have been reported to play a significant role in the pathogenesis of gram negative sepsis by their interaction with, and stimulation of, host inflammatory mediator cells to produce cytokines, biologically active lipid intermediates, and procoagulant activity (tfa). although both microbe-associated and free endotoxin are capable of stimulating mediator production, studies from our laboratory suggest that free endotoxin may be the more potent stimulus for tnf and tfa. further, our studies suggest that the majority of the proinflammatory activity released from antibiotictreated e. coli coisolates with lps by two different fractionation techniques. to assess whether actions of endotoxin directly contribute to lethality in gram negative sepsis, an experimental model was developed in which mice were made hypersusceptihle to the lethal effects of endotoxin by treatment with d-galactosamine (dgaln). challenge of cf-1 dgaln-treated mice with e. coli olll:b4 resulted in an lds0 of approximately 2.5 x 104 cfu. resolution of the peritonitis and bacteremia by treatment with cell wall-active antibiotics resulted in an increase in the ldso. ceftazidime and imipenem, which differ in their mechanism of action and in their capacity to effect endotoxin release in vitro, also differed in their in vivo capacity to provide chemotherapeutic protection (3 fold vs 8 fold respectively, p< .01). parallel studies with endotoxin hyporesponsive c3h/hej mice indicate significantly greater levels of protection with imipenem (> 80 fold vs saline controls). collectively these data suggest that endotoxin may contribute directly to the pathogenesis of experimental gram negative sepsis. bacterial lipopolysaccharides (lps) are the endotoxins of gram-negative bacteria and represent their major surface antigens. lps is made up of three chemically, biologically and genetically disctinct regions, i.e, the o-chain, the core region and the lipid a moiety whereby the latter represents the endotoxic center. it is our current understanding that lps is responsible for many of the pathophysiological events observed during gramnegative infections and that one of the major mechanisms leading to shock and death is the lps-induced activation of macrophages resulting in the production and release of lipid and peptide mediators, among which tumor necrosis factor seems to be the most important. therefore, in the fight against the lethal outcome of gram-negative infections, modern strategies, in addition to antibiotic treatment, aim at i) the neutralization of tumor necrosis factor, ii) the inhibition of the production of tumor necrosis factor or iii) the neutralization of the activation potential of lps for macrophages by monoclonal, preferably human antibodies. the latter approach, to be effective against a broad spectrum of gram-negatives, must be directed against common structures of lps (lipid a and core region). the molecular basis of this approach and the controversy in this field will be discussed. passive immunotherapy has been used since 1893, when von behring described the administration of immune horse serum to treat a patient with diphteria infection. even if this therapy was sometimes successful in bacterial infections, it has been largely replaced by antibiotics. however, antibiotics have their limitations, especially in critically-ill patients. to improve outcome, adjunctive therapies such as immunotherapy with polyclonal and monoclonal antibodies particularly against endotoxin are again considered. the role of humoral immunity in host defenses against bacterial infections is weu known. for instance, tile importance of antibodies in the defense against gramnegative infections has been established clinically by studies relating the outcome of patients with gram-negative bacteremia to tilers of antibodies directed at the offending pathogens at the onset ofbacteremia (mccabe 1972; pollack 1979) . ever since we know the role of endotoxins in the pathophysiology of sepsis, antibodies against the s-and r-lps have also been detected in sepsis patients. the aim of the administration of iv/g to the sepsis patient is as follows: 1 ) enhancing of opsonization and phagocytosis(antibactericidai activity) 2) synergistic effects with [3-1actam antibiotics 3) neutralization of endotoxin, the main pathogenic mediator of gram-negative sepsis 4) modulation and/or inhibition of cytokine release the enhancement of opsonic-and phagocytic-activity especially with igg via fc and c3 receptors has been well documented. monoclonal antiendotoxin antibodies, proven in clinical studies, do not appear to neutralize endotoxin in vitro and are not reproducibly protective in animal models of sepsis. also they can not suppress endotoxin-induced tnf-~, il-6 release in mice (baumgartner 1990, corriveau and danner 1993) . in conlrast, recent studies of a polyclonal immunoglobulin preparation, containing high levels of antibodies against gram-negative bacteria and their o-antigen of lps in igg, igm and iga classes (pentaglobin®) provide evidence to neutralize endotoxin. this effect is demonstrated in vitro (berger 1990 (berger ,1993 , in animal models (stephan 1985 , berger 1993 and also in prospective, randomized, controlled clinical trials (schedel 1991 , poynton 1992 , behre 1992 . furthermore mortali b' was reduced statistically in patients with septic shock and endotoxemia by using this preparation, as has been demonstrated by sehedel. anti-core lps monoolonal antibodies: binding specificity and biological properties f.e. di padova, r. barclay, e.th. rietschel. bacterial lps and cytokines are responsible for the pathological processes of gram-sepsis and are suitable targets for therapeutic interventions. chemical characterization and structural analysis of different lps have revealed common features. the inner core region of lps shows a high degree of similarity among e. coli, salmonella and shigella. among a large number of broadly cross-reactive murine anti-core lps mab one of these igg2ak) has been selected and chimerized into a human igglk (sdz 219-800). in elisa and in immunoblots on purified lps both sdz 219-800 and wni 222-5 show a strong reactivity with all smooth lps from e. coli and salmonella. reactivity with all the known complete core structures from e. coli and salmonella (ra) is evident. reactivity with re structures or free lipid a is not observed. this mab cressreacts with all clinical e. coli isolates from blood, urine and feces and with other enterobacteriaceae. sdz 219-800 and wni 222-5 have biological activity as they inhibit the lal assay and the secretion of monokines (il-6 and tnf) by mouse and human macrophages. moreover, sdz 219-800 and wni 222-5 inhibit the release of il-6 and tnf in vivo. in vivo sdz 219-800 as well as wni 222-5 neutralize the pyrogenic activity of e. coli lps and protect mice from lethality in d-gain-sensitized mice. the possibility to use wni 222-5 as a capture antibodies in the immunolimulus assay opens the possibility to differentiate the origin of the lps in patients with endotoxemia. franco di padova, sandoz pharma ag, ch4002 basel, $chweiz $7 presentation of lps to cd14 by lps binding protein peter s. tobias, julie gegner, katrin soldau, lois kline, loren hatlen, douglas mintz, and richard j. ulevitch. the activation of myeloid cells by lipopolysaccharides (lps) has been shown to require the serum glycoprotein lps binding protein (lbp) and binding of lps to membrane bound cd14 (mcd14). other cells such as human umbilical vein endothelial cells (huvec), smooth muscle cells, and some epithelial cells, which do not express mcd14 but nevertheless respond to lps in the presence of serum, have receptors for complexes of lps with the soluble form of cd14 (scd14). these complexes of lps with scd14 are only formed efficiently in the presence of lbp. we have begun to characterise the mechanisms by which lbp enables lps to bind to cd14, either soluble or membrane bound. with the use of fluorophore and radiolabelled reagents we have developed procedures for quantitative measurement of the association of lps with lbp and of lps-lbp complexes with cd14. these results show that the delivery of lps to scd14 is catalysed by lbp, i.e., lbp is not included with the lps-scd14 complex. in contrast, on the surface of cells, lbp does not dissociate from the cells after lps binds to mcd14. the kinetics, equilibria and stoichiometry of these reactions will be discussed in the context of models for cellular activation by lps and cellular uptake of lps. supported by nltt grants gm37696, ai32021, ai15136, gm08172, and assistance from the pharmaceutical research institute of johnson and johnson. the scripps research institute, imm-12, 10666 n. torrey pines rd. la jolla, ca usa 92037. modulation of endotoxin-induced cytokine production by lps partial structures h.-d. flad, h. loppnow, t. mattern, and a.j. ulmer department of immunology and cell biology, forschungsinstitut borstel, d-23845 borstel lipid a constitutes the active moiety of endotoxin (lps) of gramnegative bacteria. it activates mononuclear phagocytes to produce cytokines, such as tnf, i1_-1, and il-6, which are the major mediators of the endotoxic effect of lps in vivo. lipid a precursor la (synthetic compound 406) does not induce cytokines, but is able to specifically antagonize lps-or lipid a-induced mediator production in human mononuclear cells, vascular endothelial cells, and smooth muscle cells. furthermore, we present evidence for the first time that t-lymphocytes proliferate in response to lps and express mrna for interleukin-2 and interferon-~ and that these responses are also antagonized by synthetic lipid a precursor la. when comparing the agonistic and antagonistic activity of lipid a and different partial structures at the functional and binding level, the number and length of the fatty acids and the number of phosphoryl groups were pound to be of crucial importance. unexpectedly, lipid a precursor la, although biologically inactive, turned out to be both the most potent antagonist and competitor in inhibiting the binding of lps. taken together, our results provide evidence for a model in which lipid a partial structures compete with lps for specific cell surface receptor(s). in this sense, biologically inactive lipid a analogues may be good candidates as therapeutic agents for the prevention of gram-negative septic shock. two mammalian lipid a-binding proteins have been identified that are believed to have important roles in mediating the host response to endotoxin: lipopolysaccharide-binding protein (lbp) and bactericidal/ permeability-increasing protein (bpi). human lbp shares a 45% amino acid sequence identity with human bpi. despite the sequence homology, the two lipid a-binding proteins have very different functional activities. lbp is an acute phase serum protein that markedly potentiates the proinfiammatory host response to gram-negative infection by a mechanism which involves binding of the lbp-lps complex to cd14 receptors on monocytes, neutrophils and endothelial cells. in contrast, bpi is a neutrophil granule protein with potent bactericidal and lps-neutralizing activities. the divergent functional properties of these two lps-bindlng proteins can be explained by the inability of bpi-lps complexes to bind to cell-surface cd14 receptors. a recombinant protein (rbpi23), corresponding to the amino terminal 23kd fragment of human bpi, has been shown to retain the potent biological activities of the hdlo protein and may represent a novel therapeutic agent for the treatment of gram-negative infections, sepsis and endotoxemia. for therapeutic effectiveness in many clinical situations, rbpi23 will have to successfully compete with relatively high serum levels of lbp (5-60 ~g/mi) for binding to endotoxin and gram-negative bacteria. to evaluate this issue, experiments were conducted to compare the relative binding affinities of rbpi23 and human recombinant lbp (rlbp) for lipid a. the binding of both proteins to iipid a was specific and saturable with apparent kd's of 2.6 nm for rbpi23 and 58 nm for rlbp. in a competition assay format rbpi23 was approximately 75-fold more potent than rlbp in inhibiting the binding of nsi-rlbp to lipid a. these results demonstrate that rbpi23 has a significantly higher affinity for endotoxin than does rlbp and may explain the potent inhibitory activity of low concentrations of rbpi23 in a variety of in vitro functional assays for lps activation of cells despite the presence of high lbp levels. for example, rbpi23 at 0.2 ~tg/mi was able to totally inhibit lps-induced tnf release from monocytes despite a 100-fold weight excess of rlbp over rbpi23. and for heparin binding. three separate domains which inhibit the lal reaction to lps and bind to heparin were identified in amino acid regions 17-45, 65-99 and 142-169 . a single synthetic peptide (85-99) was bactericidal. these results suggest that rbpi23 contains three separate functional domains which may contribute to its high affmity interaction with gram-negative bacteria and heparin. the individual activity of each domain and the cooperative interaction among domains provide the basis for developing rbpi23 analogues with increased biologic efficacy. a considerable body of experimental data has accumulated implicating tumour necrosis factor (tnf) as a principal mediator of the pathophysiological features of septic shock. these data prompted the development of clinical strategies designed to limit excess (inappropriate) tnf production. monoclonoal antibodies (mobs) were developed and a phase ii dose escalation trial in 80 patients confirmed that the mab was safe, and suggested that it was having a beneficial effect on certain parameters. preliminary results of a large phase iii study indicated that (a) the mob was safe; (b) that it was of no discernible benefit in non-shocked patients; (c) that it reduced mortality in shocked patients, especially during the first 3 days. an alternative strategy was to take advantage of the high binding affinity of soluble receptors for tnf (stnfr). stnfr-iggfc constructs were made for both the p55 and p75 receptors. both were effective in animal models of lps challenge, but when a clinical trial was done with the p75 stnfr-fc there was unexpected mortality in the treated arm. using an animal model of live e.coli sepsis, we have shown that this may have been due to the release of bound tnf from the construct. plasma enhances while bpi inhibits lps-induced cytokine production from peripheral blood mononuclear cells (pbmc). pseudomonas species produce cytokine-inducing substances which are different from lps as indicated by the fact that polymyxin b blocks only 40% of the cytokine-inducing activity of these pyrogens. we now tested the effect of plasma and bpi on the il-1[3-inducing activity of pseudomonas maltophilia -derived pyrogens (pmp). bacteria were cultured to the log phase and filtered (300kd) to obtain prop. dilutions of pmp or lps were added to pbmc alone or to pbmc in 5% plasma +/-bpi (500 ng/ml). pbmc were incubated for 24 hours at 37°c and total il-i~ was measured by ria. results: il-i[~ in ng/ml (n=7, mear~+sem, *p<0.05 vs control). 0 control 0.1_+0 + bpi 0.1+0 5% plas. 0.2_+0 + bpi 0.2_+0 pmp (ng/ml) lps (ng/ml) 60 600 3000 1 10 3.2_+1 8.0_+1 16.3_+3 1.2_+. 2 5.2+.6 0.2_+.1 3.3_+.9" 13_+3" 0.1_+0" 0.1_+0" 2.2_+. 5 5.7+1 14_+2 5_+.6* 14_+2" 0.6+. 4" 5.3+1 15-+2 0.1-+0" 0.5_+.3" cba, c57bl/6, balb/c, akr, dba, swiss mice, guinea pigs, rabbits have been used in research work. the toxicity, immunogenicity, mitogenic and immunomodulating activity of lps have been studied. the possibility of reduction of the toxic activity of lps on macroorganism by bioglycansimmunomodulators obtained from sea invertebrates anymals (crenomytilus grayanus, stromhus gigas) have been investigated too. lps has been shown to induce specific antibody response of laboratory animals. cba mice are high responsive to lps. lps stimulates humoral immune response of mice to tdependent and t-independent antigens and suppresses intensity of the delayed hypersensitivity. the small doses of lps stimulate functional activity of macrophages, the large doses of lps -decrease one and show the cytotoxic effect. the bioglycans enhance the resistance of mice to the lethal effect of lads and provide protection 59-75% of mice. one opens possibility to use of bioglicans for reduction of toxinemia in generalizated forms of pseudotuberculosis. thus, lps from y.pseudotuberculosis is immunogen and immunomodulator wich has influence on humoral and cellular factors of immunity and plays the important role in immunopathogenesis of infection. endotoxaemia is implicated in the pathophysiology of obstructive jaundice. the lirnulus lysate (lal) assay is the gold standard method for measuring endotoxin concentrations, but inherent biochemical and technical problems limit the usefulness of this assay. the endocab elisa is a novel assay which measures endogenous antibody (igg) to the inner core region of circulating endotoxins (acga). objectives we evaluated the significance of endotoxaemia in biliary obstruction using the endocab assay and subsequently the specificity of the humoral response to endotoxin compared with an exogenous antigenic challenge [tetamls toxoid (tt) ]. materials and methods in experiment i three groups of male wistar rats (250-300g) were studied [no operation (n=39) , sham operation (n=40), and bile duct ligation for 21 days (bdl)(n=50)]. plasma was collected and assayed for bilirubin, endntoxin(lal) and acga(endocab). in experiment ii 30 rats were actively immunised with tetanus toxoid ('it) and then randomised to have no op(n=8), sham op(n=8) or bdl(n=i4). blood was taken at this time (to) and 21 days later(t20 at sacrifice for acga concentrationslendocab] and igg produced to tt(ttab) [elisa] . antibody concentrations are expressed as % increase from control values.results in bdl rats, acga concentrations were significantly increased compared with controlslp<0.001, mann-whitney]. endotoxin concentrations were sporadically elevated in the jaundiced rats but the rise was not significant. in experiment [i there was no difference between the acga or ttab concentrations in the fllree groups at to, bdl rats had a significant rise in acga concentrations by t21 [p<0,001,paired t-test] and humoral response to tt was significantly impaired in bdl rats compared with control groupslp<0.05, paired ttest data plasma endotoxin was measured by means of an endotoxinspecific endospecy test after pretreatment of the plasma with a new perchloric acid method that we developed. the normal value of plasma endotoxin is less than 9.8 pglml. polymyxin b was administered at a dose of 12,500 u every 6 hours. plasma endotoxin rapidly decreased to the normal range in 40 of the 42 patients. body temperature fall significantly. apache ii scores were also significantly improved. tumor necrosis factor-o~ and interleukin 6 decreased in survivors, while in high values tended to persist in patients died. no side effects were observed in any of the patients. in conclusion, intramuscular injection of minute of polymyxin b was useful in the treatment of endotoxemia. 19-1 uchimaru, morioka 020, japan. l e v a n t g r a m n e g a t i v organisms. m e t h o d s : u n d e r general anesthesia, 12 n o r w e g i a n b r e d landrace pigs (17-30 kg) of either sex, 6 pr group, u n d e r w e n t t r a c h e o s t o m y a n d w e r e v e n t i l a t e d on a 50/50 air a n d o x y g e n m i x t u r e a i m e d at m a i n t a i n i n g a n o r m a l p h a n d a isocapnic level. ventilation w a s not readjusted d u r i n g the observation period. the anesthesia w a s k e t a m i n e 0.6 m g / k g h a n d d i a z e p a m 2.4 m g / k g h i n t r a v e n o u s l y . h e m o d y n a m i c m o n i t o r i n g of m e a n aorta, p u l m o n a r y artery, central v e n o u s a n d p u l m o n a r y capillary w e d g e pressures w a s p e r f o r m e d w i t h a 5f s w a n -g a n z catheter a n d an aorta catheter. a continous infusion of r i n g e r ' s acetate ( 1 0 m l / k g h ) w a s g i v e n intravenously. w h e n stabilised, the a n i m a l s w e r e g i v e n 2.5 x l09 cfu of e colt intraperitoneally as a bolus in 100ml saline, the a n t i b o d y g r o u p received in a d d i t i o n 5 m g / k g e5 a n t i e n d o t o x i n i n t r a v e n o u s l y over 1 h o u r via a n infusion p u m p at the start of the observation period. the a n i m a l s w e r e observed for 6 hours. results : a t 5 a n d 6 hours, the o x y g e n c o n s u m p t i o n increased by 30 % in the a n t i b o d y treated g r o u p w h e r e a s there w a s a significant fall of 30 % in the sepsis group. in the a n t i b o d y group, the arterial p h a n d the cardiac index were also significantly h i g h e r at the s a m e p o i n t s in time. there w a s no significant difference in arterial po2. in severe bacterial infections it would be beneficial to neutralize the plasma endotoxin content with complex forming compounds. the phenothiazines are able to form complexes with endoto×in and the existence of these complexes were already shown in differential speetrophotometry and animal experiments, however, the mechanism of partial neutralization was not clarified. therefore some representative phenothiazines and structurally related compounds were tested for anti-endotoxin activity. the endotoxin neutralizinb effects of several benzophenothiazines were investigated in differential speotrophotemetry, tnf induction and in the conventional limulus test. in animal experiments some beneficial effect of complex forming compounds was found. the benzophenothiazines were not able to inactivate the biological effect of endotoxin in the limulus test. the recent findings indicates that a multifocal effect can be responsible for "anti-endotoxin action in vivo". effects of tnf inducing effect of endotoxin in leukocytes and bypotensiv action in experimental animals were reduced by some phenothiazine derivatives. monophosphoril lipid a was without effect. of microbiology, albert szemt-gydrbyi medical university, odm t~r lo, h-6720 szeged~ hunbary 46 involvement of streptococcus pyogenes erythrogenic toxins in the induction oflstreptococcal toxic shock syndrome 3 heide mgller-alou~* , joseph e. alouf , die [er gerlach , ~atherine fitting., and jean-marc ca~aillon . unit6 des toxines microbiennes and "unit6 d'immuno-allergie, institut pasteur, 28, rue du docteur roux -75015 paris (france) ; institut f~r experimentelle mikrobiologie, jena (germany). superantigen erythrogenic toxin a (eta) is thought to be involved in toxic shock syndrome in humans by inducing massive release of cytokines by patient immune cells. the cytokineinducing capacity of eta w~:s £:ompa~ed to that of lps, a gram-negative bacterial cell wall component. eta elicited weak production of il-1 d and ~, tnf ~ and il-6 in purified human monocytes whereas lps stimulated the production of high amounts of these cytokines. in the presence of t cells, eta elicited the production of significant amounts of il-i~, il-i~, il-6 and il-8. however, the most preponderant cytokine was tnf~, which peaked at i0 ng/ml after stimulation with i0 ~g eta. comparable amounts of tnfd (ca 4 ng) were induced by 0.i ~g eta and 0.i ~g lp$. in contrast to lps, eta was a strong inducer of tnf~ which was produced only in marginal amounts by lps. these results suggest that the septic shock induced by gramnegative bacteria (lps) and by gram-positive bacteria {extracellular superantigens) follows different pathogenic pathways. lps-induced shock is mainly mediated by monocytes and monocyte-produced cytokines (il-i and tnf). the eta-induced shock is mediated by t-cells or depends on t cell help for the production of monocyte-liberated cytokines. production of t cell cytokines such as tnf~ and interferon 7 in addition to the other cytokines contribute very likely to the severity of the toxic-shock resulting from s. auzeus and s. pyogenes infections in humans. the present study was utidertakc~l to cvalu~tlc the effect of soluble chemically modified giucan during septic shock. carboxylnethyl-b-i,3-glucan (ram 120000) was injected twice 48 and 24 h before the shock i.v. in a dose of 50 ing/kg. shock was induced in u~?esthetizcd (sodikm~. l)mntobarbital) rats by i.v. injection of endotoxin of escherichia colli 0127 bs, 5 mg/kg. aiiofcmg pretreated ruts survived during first 24haher ¢ndotoxine, while in controi shock group the lethality was 90%. the concentration of ~col)terin in serum was significantly elevated 24 hafterthc second cmginjection (appare~tly 80 % if compare with the control rats), but didu't chartged 60 rain and 1 s0 rain after endotoxin injectjom cardiac output in cmogroup was higher a* the i20 and 180 min after endotoxine onset ( 8i % trod 72~, respectively of initial level) than in the control shock group (64 % and 52% at the same time). pretreatment of rals with soh~ble giucan w~ts associated with beneficial effects o~ the hepatic c~ergy $ia[tls after 3 h after challenge of endotoxiae: the tissue level of lactale was ahnost twice lower than in the control ruts, me~mthne the tissue atf in cmg pretreated group was higher at 40 %. twice injected macrophage stimuhttor soluble glucan can prevent the endotoxic shock, and extremely ir~creased survival rate after endotoxine injection. the national committee of surgical infections of the spanish association of surgeons have produced a computer program for the collection and analysis of information on surgical infections. the program is suitable for ibm compatible hard disk personal computers and works through the ms-dos system. the main menu is called up on the screen when the operating disk has been installed; it reads as follows: i. new record; 2. modify records; 3. erase records; 4. searches; 5. reports; 6. configure; o. ouit. if you ask fdr a new record the screen will prompt you to enter the number of case, record number, hospital, age and sex. the next screen will come up and the words "topographic diagnosis" will flash. a menu of 34 areas or organs will be displayed. then, the words "type of pathology" (inflammatory, neoplastic, traumatic and other). days of postoperative period. type of surgery (programmed and emergency). type of operation (clean, clean contaminated, contaminated and dirty). duration of surgery. this is followed by "order of operation" and the "type of anaesthesia (general, regional or local). you are then required to supply the "diagnostic code of who" (icd9) and the "procedure code of who. analytic and concurrent illnesses (total proteins, albumin, haemoglobin, haematocrit, leucocytes, red corpuscles, glucose and bilirubin). the next screen asks for "risk factors" (obesity, uraemia, neoplasia, malnutrition, urinary catheter, distant infection, artificial valve, immunosuppressive drugs, over 65 years and anergy. this is followed by a screen headed "postoperative complications". "evolution" (the questions asked are drainage, systemic antibiotics, and on each ocasion a choice of 31 antibiotics is displayed), local antiseptics, reoperation, etc. under "microhiology" is a choice of 37 organisms and the chance of identifyin 3 organisms. finally, "sepsis score". our recent work had shown that renshen-fuzi-chaihu mixture could increase the survival rate in experimented study. the purpose of this study was to determine the effect of combined administration of renshen-fuzi-chaihu mixtuer and antibitics (sa) in patients with septic shock. the result showed that, in sa group (40 cases), the total effective rate was 87,5%, in the contral group (combined administration of gentamycin and dexamethasone, 25 cases) the total effective rate was 84%. however the obviously effective rate in sa group 70% was significantly higher than in contral group 44% (p<o.os). sa group appeared to be more obvious than the contral group in raising arterial pressure, recovering consciousness and pulse, improving cold lilmbs and reducing fever (p<o.o5). it was found that sa could inhibit the pathogeneses changes of septic shock such as the decreases of superotide dismutase (sod) and glutathione peroxidase (gsh-px) in erythrocytes and the increases of plasma free hemoglobin (phb), plasma malondialdehyde (mda) and 13-glucuronidase (13-gc) occured in septic shock. sa had stronger effect than gd in inhibiting the changes mentioned above. hence, these results suggest that sa has beneficial effect in the treatment of septic shock. sa could diminish the decrease of the antioxdase activities and inhibit lipid peroxidization and stabilize cellmembrance. this may be one of the principal mechanisms of the beneficial effect of sa in the treatment of septic shock. donna l. lehman, heather a. childers, irshad h. chaudry and alfred ayala. the thymus is thought to be a privileged sight for tcell generation. here the t-lymphocytes are either selected for their potential to recognize and react competently to foreign antigens or de-selected, due to their potential to react to auto-antigens, de-selected. tcells with this latter capacity are thought to be deselected through a process referred to as programmed cell death or apoptosis. while it is known that thymic apoptosis is elevated by a variety of stressers associated with infection and inflammation, little or no information is available concerning its presence in polymicrobial sepsis. it was, therefore, the aim of this study to determine whether thymocytes exhibit increased apoptosis during sepsis. to assess this, thymocytes were harvested from c3h/hen mice at i and 24 h following cecal ligation and puncture (clp; to induce sepsis) or sham-clp (sham). thymic yields were assessed and the extent of apetosis determined by staining the cells with propidium iodide (a dna intercalating dye) for facs analysis or by examining the extracted dna electrophoretically for the endogenous endonuclease activity the results (n=6/grp) indicate that at i h post-clp there was no marked changes in any of these parameters. however, at 24 h the thymic cell yield from clp mice was significantly decreased (sham:3.6!0.2 x l06 vs clp:0.3i0.1 x 106* cells; *,p<0.05), the % of cells apoptotic by facs analysis increased from 1.7i0.1% in sham to 8.7±3.2%* in clp, and the septic mouse genomic dna exhibited dna degradation these results indicate that clp, but not simple laparotomy, induces marked thymic apetosis, which may contribute to the lymphocytic immune deficiency seen in these mice the purpose of this retrospective study was to evaluate effectiveness of irrigation to treat septic knee joints from longterm results. from 1982 to 1992 31 patients with septic knee joints have been treated. treatment in acute cases started with asp#at[on of the effusion to evaluate for cultures. without awaiting the result treatment was continued with immediate joint irrigation under arthroscopic control. three redon tubes ( ch 16 ) were introduced for continuous irrigation and for intermittent distention of the joint cavity. in case of chronic joint infection additionally all foreign and synthetic material was removed. systemic antibiotics were given. patients could be re -examined with a mean follow-up of 6 years. re-examination showed that immediate arthroscopic lavage started at the onset of septic sings had best results (less signs of osteoarthr[tis, less synovitis, less pain while loading and less range of motion loss ). 21 patients (80%) out of 26 patients with s tre_=.tsd acute se.~tic knee joint had excellent functional recovery, whereas all 5 patients with chronic septic knee joints showed poor results. additionally, in 3 of these 5 patients with chronic septic knee joint after irrigation a further procedure was necessary due to recurrent septic knee joint. the concept that bacteria can translocate across the intestinal mucosa under a variety of circumstances is well established. however, due to the inherent limitations of in vivo studies, the cellular mechanisms underlying the process of bacterial translocation (bt) across the epithelial barrier are poorly understood. this is especially true in those circumstances in which there is no merphologic evidence of mucosal injury. consequently, we have utilized an in vitro cell culture model system to investigate the cellular events involved in the translocation process. in this model system, a polarized monolayer of intestinal cells (caco-2 cell line) is grown on a 3 micron filter in a two compartment system. after tight junction integrity is established, bacteria are placed in the apical surface chamber and bt across the caco-2 monolayer is measured by culturing the basal chamber. the results of our studies utilizing the caco-2 system indicates that; l) several strains of non-pathogenic bacteria will translocate across the caco-2 monolayer in a time-dependent and dose-dependent fashion. however, the incidence and magnitude of bt are highest for those strains of bacteria (gram-negative enterics) that are most commonly cultured in vivo. 2) caco-2 cells are actively involved in the transcellular passage of bacteria across the caco-2 monolayer, since inhibition of caco-2 microtubule or microfilament function decreases the magnitude of st. 3) lectin but not integrin receptors are involved in bacterial translocation of e. col~ across the caco-2 cell monolayer. 4) caco-2 cells have the ability to ingest and kill certain strains of bacteria. the mechanisms involved in caco-2 bactericidal activity appear similar to that of pmns to the extent that both are oxidant but not nitric oxide mediated. these in vitro studies suggest that caco-2 cells can function as "nonprofessional" phagocytes and that both bacteria and enterocytes are involved in the translecation phenomenon. bacterial translocation due to gut barrier dysfunction is considered to be a major cause of septic complications and multiple organ failure following trauma, burn injury, hypoxia, shock and shock-like states. the invasion of bacteria into the circulation from the gastrointestinal tract or even from other sources, however, should not necessarily result in systemic infection or organ cohinisation, as long as the humoral and cellular defense mechanisms are not impaired. thus, a reduced res clearance capacity and hepatic clearance function of bacteria and toxins can be observed under the same conditions which enable bacterial translocation from the gut. in order to evaluate the influence of circulatory, metabolic and humorul disorders on the elimination of bacteria out of the blood circulation, a series of experiments were performed in anesthetized and ventilated rabbits which received defined numbers (1.3 x 108 colony-furming units) of escherichia coli as a bolus injection. in unaffected control animals the intravenously injected bacteria are eliminated about 99.9% within the first 15 minutes and are totaiy cleared within 90 minutes. 57% offue total cfu counted in the cultures of the organ homogenates were found in liver and spleen, whereas only very low numbers could be detected in the lungs and kidneys. systemic injury of the animals by hemorrhagic shock, endotoxinemia, hypoxia, coagulation disorder, systemic vasoconstriction by norepinephrine and other types of injury reduced the elimination rate of bacteria and changed the pattern and degree of their dissemination and tissue distribution. the bacterial clearance was delayed mostly in rabbits subjected to hypoxemic hypoxia in which about 100 cfu of viable e.eoli per ml blood could still be counted 3 hours following their injection. the number of viable bacteria was some ten powers higher in organs of hypoxic rabbits in comparison to control ardmais, and an excessive colonisation of the lungs and kidneys with 25% respectively 11% of the total cfu of the cultared organ homoganates was observed. unilateral iscbemia of the kidney prior to the intravenous injection of e.coli only moderately delayed the bacterial clearance, bowever, caused an intense eohinisation of the affected reperfused organ. in contrast to this, inhalation injury did not promote the accumulation of bacteria in the lungs. conclusion: shock, systemic and local affections reduce the clearance of bacteria from the blood circulation and enable their dissemination and accumulation in vital organs. the degree of this reduction and the organ pattern of the bacterial distribution varies with the type of injury. thus, these factors seem to be essentially involved whether or not bacterial translocation and multiple organ failure will follow the invasion of bacteria into the circulation as in the ease of gut barrier dysfunction. movement of enteric baaterla from gut to extralumeaal sites (bacterial tra~sloeation) m~y play a role ~. nraltipl~ orga~ failure. traumatic stress (shock, hffectiort, and im~lammation) and severe illness are common alateeedents. tnt~st~aal lleus is e common proximme causal factor. we have exanlined the hypothesis that bacterial ttanslecation from the gut in expe~me~tal paaerestitis is due to bacterial overgrowth as a cuas~ucnee of a deere~ae in intestinal transit. ne~rotit_.~g pancreatitls fbliowlng bile duct iigation in the opossum is associated with colonization of the pa,se ~re, aa by gut derived organisms or salmonella of biljary origin in infected animals (previously reported). to ti~rther define the mechanism of transloemdon in panereati* inflammation, pancteatitis was induced hy iigatlon of t~e lower bile duet distal to the pancreatic duets m the rat. intestinal transit, enteric bacteriology, and tromsloeatien of bacteria to mesanterie lymph nodes, blood stream and splanelmie organs wen deletmhaed at 24 (n~ 7), 4g (n ~ 10), and 96 (n~ 10) hours with the fallowing results: ly~testinai,..transfi". geometric mean of fluorescent marker -25 % at 24 hour~ with return to 51% of gut leugth (similar to ~m) at 96 hour~. bacterial overgrowt_hff -increase in total bsctetia ~ad gram negative rod.~ at 2,; hours with return to sham control at 96 hours (9 log cfu/g veraus 6 log cfu/g ia ileum). translocation to mesenteric nodes -71, 90, and 30 percent at 24, 48, and 9fi hours compared to 49b in sham controls (n=l of 26). conclusion -bacterial translocation following panetoatieobiliary duct ljgation induced panereatitls ha the rat is tighlly linked to intestinal trausit and bacterial overgrowth within the gut lumen. speclrjlation -the ilens in in/~arm~ation may be related to activation of eytokiaes and mediated by nitric oxide. preliminary evidence for this notion will be discussed. in a series of studies we investigated: a) the time course of bacterial translocation (bt), b) the kinetics of lps and cytokine appearance (tnf, il-6, soluble tnf receptor [stnf-r] ) in the circulation, and c) the role of lps and/or tnf with regard to haemorrhagic shock (hs) related pathophysiologie alterations and mortality in baboons and/or rats. method: baboons were subjected to femur fracture, soft tissue trauma (acute experiment), and hs (40 mmhg mean arterial pressure: map for 2-4 h terminated at an accumulated oxygen debt of 180-200 ml/kg followed by resuscitation). a chronic model of hs was set up by administration of zymosan activated plasma (zap) before and after hs but without trauma. hs was induced in rats by bleeding the animals to map of 30-35 mmhg 90-180 rain followed by resuscitation. results and conclusion: in rats, lps increased in the systemic circulation, plasma tnf levels were found to be significantly elevated at 90 min and were still markedly increased at 150 rain postshock. plasma tnf, il-6, and stnf-r levels increased already during the shock period in baboons, while tnf was not detectable. our data suggest that haemorrhagic shock may lead to early bt in the intestinal wall (at 30 min following resuscitation in rats subjected to hs for 90 min, similarly at 3 h after the end of oxygen debt period in baboons ) and transient access of gut-derived lps into the circulation (levels became significant at 120 min, while in baboons higher levels up to 300 pg/ml were found at the end of shock and 1 h after reinfusion, partially accompanied by bacteremia). in addition, since the treatment of rats with anti lps measures or anti tnf therapy significantly improved the 48-hour survival rate it can be assumed that endogenous lps and lps-induced mediator(s), in particular tnf, may lead to organ injury and mortality following haemorrhagic shock. alteration of the mesenteric blood flow and the consequent ischemia / reperfusion injury to the intestine appear to be one of the earliest events in the systemic inflammatory response following severe thermal injuries. the causative relationship between the subsequent disruption of the intestinal integrity and the potentiation of the translocation of indigenous bacteria and/or endotoxins to remote body organs and the systemic circulation has been deeumented in several studies. among other effects, endotoxemia solely or acting synergistically with thermal injuries has been shown to promote bacterial translocation. as these sequences continue without adequate intervention, multiple organ failure and eventually death may become inevitable. hence, the crucial need of treatment modalities with the capacity of modulating the intestinal blood flow and preventing the manifestations of these pathological incidents. although the underlying mechanisms of this process have not yet been fully elucidated, there is accumulating evidence that various interacting vasomediators are involved. the actions of these mediators can probably be modulated by either nonspecific or selective agents. among the nonspecific agents, the composition, volume and timing of the resuscitation fluids appear to play an important role in this process. nitropmsside, a nonspecific vasodilator, has been shown to prevent the decrease of the postbum mesentefic blood flow when selectively infused in the mesenteric artery. the nonspecificity of various vasodialators with their possible undesirable systemic effects emphasizes the importance of the identification and modulation of selective vasomniiators. thromboxane a2 (txa2) and angiotensin ii (a-ii) appear to be the primary mediators of the postburn vasoconstriction. both mediators were found to be elevated following thermal injuries. in a previous study, pretreatment with oky-046, a thromboxane synthetese inhibitor was found to attenuate the postbarn mesenteric vasoconstriction. in a bum/sepsis porcine model the efficacy of dup753, an a-11 receptor antagonist as a posthum treatment modality was evaluated. treatment with dup753 prevented the early posthum and the late posteodotoxin elevation in the mesenteric vascular resistance and subsequently abrogated the fall in the mesenteric blood flow. the incidence of burn & endotoxin induced bacterial translocation was significantly reduced by dup753 from 86% to 29% (p<0.05, fisher's exact test). the indigenous flora of the gastrointestinal tract exerts a potent influence on the developmental maturation of normal systemic immunologic responsiveness. moreover, both oral and intraportal administration of experimental antigen is associated with a systemic state of immunologic hyporesponsiveness, known as oral tolerance. since trauma and critical illness are associated with changes in the flora of the gut and with altered systemic immune reactivity, we have hypothesized that the interactions of an altered gut flora with immune tissues in the gut and liver play a role in the pathogenesis of the immunologic derangements of critical illness. prolonged feeding of two clinically relevant killed organisms-pseudomonas and candida-to a rat results in significant impairment of cutaneous delayed hypersensitivity (dh) reactivity. conversely, measures directed against gram negative bacterial overgrowth in experimental peritonitis result in partial restoration of impaired dh reactivity. to ascertain the role of the liver in the pathogenesis of these alterations, we studied the differential immunologic sequelae of portal versus systemic (ivc) infusion of killed bacteria. experimental infusion of live or killed gram negative organisms into the portal vein produces dh suppression in rive, and profound suppression of mitogen-driven lymphocyte proliferation in vitro; systemic administration of the same organism has no effect on these phenomena. suppression is tgf3-dependent, and mediated by a soluble factor released by fixed tissue macrophages of the lung and spleen. the suppressive influence appears to be mediated at the level of interactions between il-2 and its high affinity receptor. release of this factor can be induced by a second circulating factor present in the plasma of portally-, but not systemically-infused animals two hours after bacterial administration. these studies suggest that the release of inducible immunoregulatory factors from the liver, and possibly from the gut, may play a role in the pathogenesis of the profound derangements of systemic immune homeostasis that accompany trauma and critical illness. acad.hospital st. radboud,postbus 9101,6500 lib nijmegen introduction. the central question being tested in this study was whether liposome encapsulated dichloromethylene-diphosphonate (ci2mdp) mediated elimination of liver and splenic maorophages would influence zymosan induced systemic toxicity (st) and bacterial lranslocation (bt). materia|s and methods. 90 male swiss (icr) mice were used weighing 21-25 g elimination of liver and spleen macrophages was accomplished by intravenous injection of 200/11 suspension of ci2mdp-liposome suspension. control mice received an i.v. injection with 200 pt phosphate-buffered saline (pbs) . two days later all mice were challenged with an i.p. injection of zymosan suspended in paraffin (z) at a dosage of either 01 mg/g, 0.5 mg/g or 1.0 mg/g body weight (n=8 in each group), signs of st and bacterial translocation bt were measured 24 hours later the st was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a four point scale. bt to the mesenteric lymph nodes (mln) and systemic organs (liver, spleen, lung and blood) was tested by culturing on blood and macconkey's agar at the time of sacrifice sections of the distal ileum were taken for histology. an additional two control groups (n=6 in each group) were tested to verify that neither paraffin nor pbs or ci2mdpqiposome suspension alone induced bt or st. furthermore survival over a 12-day period was assessed in a control and macrophage-depleted group with a dose of 1d mg/g z. results. neither the paraffin nor ci2mdp-tiposome suspension induced bt or st the incidence of bacterial translocation to the mln was similar between macrophage depleted and control groups. however, macrophage depletion was associated with a significantly higher incidence of systemic spread of bacteria. data expressed as positive tissues/total tissues tested: 11/32 vs. 1/32 at 0.1 mg/g zymosan (p~o.01); 19132 vs. 10132 at 05 mg/g zymosan (p~q05); 23/32 vs. 11/32 st 1.0 mg/g zymosan (p~o.o1).the magnitude of bt however did not show any differences. although systemic bt was greater in the macrophage depleted groups, the systemic toxic response was significantly less at doses of 0.5 mg/g zymosan (3.14 -+ 0.69 vs. 7.13 -+ 3, p~o.01) and 1.0 mg/g zymosan (4.38 -+2.50 vs. 11.25 -+ 2.5, p<_.o.01) . the 12 day mortality after 1.0 mg/g zymosan was 27% in the control group and 0% in the macrophage depleted group (p=o.05). histology did not show any differences between the control and macrophage depleted groups. conclusion. the lethal and toxic effects of zymosan in this model appear to be more related to the excessive activation of macrophages than to the systemic spread of bacteria. bacterial translocation (bt) in hemorrhagic shock was studied using a porcine model. in this study three groups were randomized: one control (n=3) and two experimental (n=6 each). a portal vein catheter was placed and remained in situ 48 hrs. a femoral artery and vein catheter were in place 12 hrs. the arterial eaanula was used for monitoring mean arterial pressure (map) in all animals and to bleed the experimental animals and the venous cannula to repelfuse with ringer's lactate (rl) or autologous blood (ab). baseline samples and values were taken in this period. control animals were observed for a sham shock period of 6.5 hrs., given 2 more hours of anesthesia, and studied for further 48 hrs. 40% of the estimated blood volume was withdrawn from each experimental animal over 30 min. these animals were kept in shock for 6.5 hrs. at the end of the shock one group was perfused with rl and the other with ab. two hours later they were extubated and further studied for 48 hrs. samples from portal blood for cultures and measurement of leukotriene b4 (ltb4), prostaglandin e2 (pge2), and nitric oxide (no) metabolites were taken from all animals at intervals beyond baseline up to 48 hrs. after 48 hrs. under general anesthesia a second laparotomy was performed and samples for culture from mesenteric lymph nodes (min), liver and spleen were taken; as well as samples for histologic study with light and scanning electron microscopy were taken from jejunum, ileum, and colon. animals were then euthanized. results: significant shock was induced as indicated by map and arterial blood gases. significant bt to min was observed in all groups. no significant bt was observed in cultures from liver and spleen in any of the groups. increased values for ltb4, pge2 and no metabolites were found during the period of shock. these results suggest that the isehemic phenomenon triggers a significant inflammatory response, and that bt to min may be an epiphenomenon without apparent significant influence on the inflammatory response. objective: to study the potential effects and mechanisms of action of systemic administration of monoclonal antibodies anti-endotoxin (ha-1a) in a animal model of gut-origin sepsis. materials and methods: exoeriment a. balb/c mice were transfused with allogeneic blood (from c3hb-iej mice). five days post-transfusion the animals were gavaged with lxl09 viable escherichia coil and randomized into 3 groups (n=22/group) to receive a sham burn (sb group) or a 20% thermal injury immediately followed by the systemic administration, via a tail vein, of monoclonal antibodies (6 mg/kg) (ha-1a group) or a 20% burn injury and administration of equal volume of sterile saline (c group). all 3 groups were resuscitated by intraperitoneal injection of 0.3 ml of saline. the animal survival rate was observed for 10 days post-burn. experiment b, transfusion and burn procedures were identical to experiment a but the mice (n=8/group) were gavaged with 109 viable e.coli labeled with mmndium oxine. four hours after burn the mesenteric lymph nodes (mln), liver, lungs and blood were harvested to determine plasma endotoxin levels and the magnitude of transtocation of labeled bacteria as measured by the residual radioactivity (dpm) in the organs. circulating endotoxin levels were determined by limulus colorimetric assay. diamine 0xidae(dao) is an enzyme existing in the mucosa of small intestines, but its activity is low in the plasma. it is evoked in the blood with the intravenous injection of much heparin. it is well known that da0 declines under the chronic mueosal injury when the small intestines are cut off or the mucosae become atrophied. aim of this paper is to confirm that da0 goes up to the measureable level by the intravenous injection of a small amounts of low molecular heparin(lmh objectives: the aims of this study is to investigate the inhibitor" effect of the prolease inhibitor, urinastatin, on endotoxin induced bacterial transleeation in mice. materials and methods:lcr mice were divided in six groups as the fotlows, group i: control mice receiving intraperitoneal injections (ip) of saline 24.5 and 24 hr prior to sacrifice, group ih treated mice receiving ip of utinastatin 24.5 hr and endotoxin (5mg/kg) 24hr prior to sacrifice,group ilk sham4reated mice receiving ip of saline 24.5 hr and endotoxin (5mg/kg) 24hr prior to sacrifice,group iv: saline control mice receiving ip twice during 30 rain intervals,group v: mice received ip of utinasmtin, and 30 min later they were received ip of endotoxin (5mg/kg), group vi: mice received ip of saline,and 30 min later they received ip of endotoxin (5mg/kg). in group l,ii and ill ,the mice were killed by cervical dislocation.using stetile techniques,a middle-line incision was made and the mesenteric lymph nodes were harvested in order to culture on selective agars for quantitation of bacterial trauslocation. in group iv, v and vi, survival was recorded for 3 days. unnastatin pretreatment could rednce mortality significantly. first surgey, shiga university of medical science, seta ,ohtsu 520-21,japan increased gut permeability correlates with inflammatory response in multiple trauma aptients. pape, h.-c. dwenger, a. grotz, m. regel, g. purpose: disturbances of intestinal permeability have been advocated as a pathogenetic factor of posttranmatic multisystem organ failure (mof). a close relationship with impairment of the stationary reticulocndothelial system (res) and a systemic inflammatory response (sir) was discussed. these tactors were investigated prospectively in patients with multiple trauma showing posttraumatie complications (multiple organ failure, mof). methods: 31 polytranmatized patients were studied prospectively. according to a mof-score (goris) those with posttrattmatic complications were selected (>7 points at 2 days), others were excluded. every second day gut permeability according to the ratio of urine concentrations of lactulose and mannitol (l/m) was evaluated (enteral application). at parallel time points res clearance capacity (k-value, invasion constant, normal range 0.6 -0.8 mind) was studied after i.v. injection of 100 mbq 99rotehuman albumin. liver perfusion was calculated from these data, total serum bilirubin (/zmol/l) was documented. serum elastase (#g/l) levels were determined enzymatieally. results 86.0 148+ 239+ liver perfusion did not ehangu, bilirubin showed progressive worsening indicating mof. a positive correlation was present between l/m and k (r=0.88) and between l/m and ela (r=0.71). conclusions: there is a positive correlation between the time pattern of intestinal permeability dysfunction and res hyperactivity as well as between intestinal permeability and the systemic intlammatory response (elastase levels). the results speak in favor of an interaction between intestinal and extraintestinal inflammatory systems, which in eombiuation are likely to be responsible for post~anmafic complications. endotoxemia, il-6 release and consecutive acute phase reaction are observed as a host response to surgical trauma. as well vasodilative prostaglandins (pg) and thromboxane (tx) are released after abdominal meaenteric traction (mt). the following hypotension and acute hypoxeraja are duo to prostacyelin (pgiz) arm can be avoided by perioperative cyclooxygenase inhibition. we therefore focused on the effect of pg and tx liberated following mt on the induction of endotoxemia. methods: in a prospective, randomized double-blinded protocol 50 patients, who were scheduled for major abdominal surgery (pancreatic or infrarenal abdominal surgery), were studied. ibuprofen (400 mg i.v.) or a placebo equivalent was administered 15 minutes before skin incision. mt was applied in a uniform fashion. baseline values were obtained before induction of anesthesia. further measurements followed before the incision of the peri[onenm (tl) and 5, 30, 90, 180 min, . the plasma concentrations (,pc) of 6-keto-pgft,, txb: and-ki-12-pgf ~ (stable metabolites of pgi2, txa2 and pge~) were determined by ria. we measured endotoxin pc by limulus-amoebocyte-lysate test and il-6 levels by elisa. data are given as mean+sem (* p< 0.05 placebo vs. [ibuprofen] ). results: endotoxin plasma levels increased before incision of the peritoneum tl both in the ibuprofen pretreated and in the placebo group. peak pc were observed 180 minutes after mt. endotoxin pc were significantly higher in the ibuprufen treated group (t5 0.16+0.02e[0.32+0.06] eu/ml). il-6 pc demonstrated an increase continuously from t4 to t7 (t7 115+12 [91 + 15] ng/l) in both groups. after intentional abdominal mesenteric traction we observed a marked increase of 6-keto-pgf~,, pc up to 6h after mt in untreated patients with a peak of 2450*[60] ng/1 at tl. also txb: and kh2pge 2 pc showed a considerabe increase up to 6h after mt in the placebo group. in ibuprofen pretreated patients the pg and tx pc remained within the normal range. discussion: our data clearly indicate a significant endotoxemia and il-6 release following major surgical trauma which is not initiated either by prostaglandin or thromboxane release. moreover endotoxemia is accentuated by ibuprofen pretreatment. therefore we hypothesize that in major abdominal surgery prostacyclin release-after mt may play a crucial physiological role in maintaining splanclmic microcirculation and thus preserving gut mucosal barrier function. objectives of the study it has been shown recently that parenteral and certain euteral diets promote the translocation of gut flora to the mesenteric lymph nodes (mln) and systemic organs, a process termed bacterial translocation (bt). in chow fed rats bt usually does not occur without further promoting factors. the goals of the present study were to determine whether the provision of defined amounts of standard lab chow during iv-tpn administration wotfld redane the incidence of bt, materials und methods male spf spragnle-dawley rats were divided into 6 groups. group 1 received standard laboratory chow feeding ad lib. in group 2 a central venous catheter was placed, ligated and secured by a spring coil tether attached to a swivel allowing free movement in the housing cage and chow was fed ad lib. in group 3 50% of the calculated daily required calory intake (drci) (307/kcal/kg) was given by iv-tpn (28% glucose, 4,25% amino acids) and 50% by limited chow administration. groups 4 and 5 received 70% and 90% of the drci by i.v. tpn and 30% and 10% respectively by chow feeding. group 6 received iv-tpn only. after 7 days the rats were sacrificed and the mln, liver, spleen and cecum removed aseptically, homogenized and cultured for bt samples of distal ileum were taken for light microscopy. the group with the least amount of chow shown to be protective against bt received the amount of non-fermentable fiber of that chow regimen during iv-tpn feeding and bt was studied. 4 5,5+0,9 4 12 23,9 -2,3 16 ,7 2/12 63+11 125~1 "7,7-+0,7 6,7-+1 5 12 24~6 -3,5 67 8/12 241+~243 170+163 8_+0,7 7+1,1 6 18 25, 5 -4 88,8 16118 2061-+3224 2211+4015 8,4~0,7 8,1-+1~1 conclusions: the administration of 30% of drci by chow feeding during iv-tpn significantly reduced the incidence of bt and maintained gut barrier function. the addition of the respective amount of dietary fiber of this group did not prevent iv-tpn-indueed bt. dr. med. m naruhn., dep. of general surgery, eberhard-karls-university, hoppe-seyler-str. 3 previous experimental studies have suggested that a disturbed ecology of the enteric bacterial population might contribute to the development of bacterial translocation from the gut in acute liver failure (alf). in the present study, the effect of oral administration of lactobacillus reuteri r2lc and oat fiber on bacterial overgrowth and translocation was investigated in rats with acute liver failure induced by subtotal (90 %) liver resection. the oatmeal soup base was anaerobically inoculated with lactobacillae and fermented for 15 hours, after which the animals were fed with either fermented or unfermented oatmeal or saline daily for 6 days prior to the operation. bacterial translocation to mesenteric lymph nodes (mln) and the systemic circulation was determined, as well as the intestinal bacterial flora and enterocyte protein content. the incidence of bacterial translocstion to the systemic circulation was nit in rats subjected to sham operation and saline treatment and 17 % in animals subjected to 90 % bepatectomy and lreatment with fermented oatmeal, while 80-90 % and 34-50 %, respectively, in rats subjected to hepatectomy and treatment with either saline or unfermented oatmeal. only one rat with fermented oatmeal demonstrated bacterial growth in mln (p < 0.05 vs hepatectomy and treatment with saline or unfermented oatmeal). the enterocyte protein content significantly decreased (p < 0.01) in salinetreated animals following 90 % hepatectomy, while there was no significant difference between bepatectomized animals with oral administration of fermented or unfermented oatmeal. the number of anaerobic bacteria, gram-negative anaerobes and lactobacillus significantly decreased and the number of e.cnli increased in the distal small intestine and colon in hepatectomized animals with enteral saline or unfermented oatmeal as compared with animals subjected to sham operation or bepatectomy with fermented oatmeal. our results thus show that the occurrence of bacterial translocatiou from the gut in 90 % hepatectomy-induced alf could be prevented by enteral administration of fermented oatmeal, maybe partly due to a positive effect on the enteric bacterial ecology. 190_+20" 16+_3" 11.7" data=mean_+sd, * stats anova p<0.05 vs control. l+air and lap groups, both exposed to exogenous i.ps shnwm:t m significant increase (p<.05) in lps gut translocation compared to control and l+co2. this correlated with a significant increase in peritoneal inflammatory responses (o2-,tnf) above that of the control and l+co2 groups, while mac-1 and cr3 opsonized phagocytosis were significantly impaired. the absence of significant differences between l+air and lap groups indicates that lps rather than wound factors is the principle mediator. thus, lps plays a significant role in regulating peritoneal responses in the early post-operative period dept of surgery, rcsi, beaumont hospital, dublin 9, ireland brlke e, berger d, staneseu a, buttenschsn k, vasilescu c, seidelmann m, beger hg in 32 patients undergoing a colonoscopy, endotoxin, endotoxin neutralizing capacity (enc), thromboxane b o (stabile metabolite of tbmomboxane ~), 6-keto-prostaglansin, leueotriene c4, interleukin 6 and the incidence of bacteremia were determined before and then every five minutes during the procedure. twenty-one of 32 patients showed a significant increase of endotoxin plasma levels during colonoscopy (p=0.003), whereas only one patient had a positive blood culture with bacteria obviously derived from the gastro-intestinal tract. the enc decreased significantly five minutes after the beginning of eolonoscopy and was diminished further thereafter. the baseline values were reached after 24 hours. ~hromboxane b o levels also increased after five min. from 89 to 376 pgyml peaking at 30 min. with 1332 pg/ml. 6-keto-prostaglandin,leucotriene c 4, ii-6 and crp remained unchanged. a control group of i0 volunteers who were not subjected to endoscopy, were prepared for eolonoscopy by orthograde lavage. the blood sampling procedure remained identical. no differences were seen in all described parameters for the controls. these data show that the gut barrier can be compromised by mininml invasive procedures, at least, concerning bacterial products. living bacteria, on the contrary, do not pass the gastro-intestinal wall. endotoxin, when determined by enc, is more sensitive than the conventional limulus-amebocyte-lysate test. no acute-phase reaction was induceri by the observed endotoxin translocation. it can be speculated from the dramatically enhanced thromboxane b 2 levels, together with its hemodynamie effects, that the thromboxane release may support translocation of bacterial products. sepsis is common after hemorrhagic shock. this study aims to demonstrate that hemorrhagic shock alone can promote translocation of gut bacteria from intestinal tract to its regional nodes and subsequently to blood. one hundred twenty mice, divided into 4 groups were subjected to 30, 60 and 120 minutes of 20%, 30% and 40% of hemorrhagic shock. on the specified time, blood cultures were taken and mice were sacrificed. the intestinal tract were histologically examined for any changes which allows translocation and its regional nodes were quantitatively cultured for translocated bacteria. there was a direct relationship between duration and degree of hemorrhagic shock and incidence of translocation (p 0.05). there was a high incidence of gut bacterial translocation to the mesenteric and mesocolic nodes in all degrees of shock (p 0.05). bacterial growth in the regional intestinal nodes increased and blood cultures were positive in direct proportion to degree and duration of shock. histologic evaluation of segments of git showed submucosal congestion to allow bacteria normally contained within the gut to cause systemic infections. translocation of gut bacteria in untreated hemorrhagic shock is clearly shown in this study on animal models. in this study, guotobiotic rats with 5 known species of bacteria were subjected to total parenteral nutrition(tpn) and subsequent hemorrhagic shock. the purpose of the study was to observe the impairment of gut barrier function following tpn and hemorrhagic shock and to study the mechanism of enterogenic infection induced by tpn and shock.the results were as follows: 1.long term(8-12 days) tpn induced impairment of gut barrier function, evidenced by atrophy of intestinal mucosa, significant decrease in diamine oxidase activity of intestinal mucosa and blood, and marked microecologic imbalance of the intestinal mucosa flora with dorminant growth of aerobes and relative decrease in anaerobes. the degree of mucosal damage were proportional to the duration of tpn. 2.in tpn+shock groups, failure of gut barrier function was found. ri,~ere were further damage in the mucosa, with a large number of gramnegative organisms invading mucosa and submucosa and a significant decrease in dao activity as compared with each relative tpn groups. these changes were significantly correlated with enhanced bacterial translocation, elevation of lps and mda levels in the plasma. these findings suggested that long term standard tpn impaired the gut barrier function, precipitating posttraumatic gut barrier failure. thus infec. fion following shock might be oi'iginated from the gut and it was obviously related to the impaired gut defence resulted from antecedent tpn. the determination of plasma dao activity might provide a valuable tool for the ear. ly diagnosis of gut injut;y during tpn and after trauma. in our earlier studies we have investigated the dynamics of granuloayte infiltration of the ischemic/reperfused s~all intestine (g. illy~s, j. hamar int. j. exp. 9athol. 73. 161. 1992.) . there was a increasing infiltration of the mucosa c111m~nating at the 3d to 4th hours of reperfusion. in the present series we have studied sc~e of the conseqn/ences and the possible role of this cellular reaction. 45 ~in isehemia was followed by a 4 hour reperfusion in the anesthetized rat. arterial ~/ad mesenteric venous blood samples were collected at 5 m_in, i, 2~ 3, and 4 hours of reperfusion. elastase and lactate concentrations were determined and hamoculture was carried out from the blood samples, and tissue pieces from the heart, lung, liver and kidney were collected for histological analyses at the above mentioned times of reperfusion. all blood samples were free of cell bacteria. staphylococci appeared only occasionally at the 4th hour in the arterial blood .and at the 3d and 4th hours in the venous blood, respectively. arterial and venous elastase activities were high throughout the reperfusion, venous concentrations being higher at all times. lactate concentrations of the arterial and mesenteric venous blood samples increased during shock. ~ranuloeyte infiltration of all organs studied appeared during the 2d hour and it increased at later times of reperfusion. it is concluded that heavy infiltration of the intestinal mucosa can block bacterial translocation in most of the cases during reperfusion. granulocytes activated either by the reperfused area or by the released cytokines infiltrate other organs contributing by this way to the mesenteric shock s!rndrc~e. intestinal motility plays an important role for maintaining nutrient transport and absorption and for balancing the enteric bacterial population. disturbances of intestinal motility may be one of the earliest notable changes in intestinal function. in the present study, we aimed at determining early alterations in intestinal transit time following ischemia-reperfusion injury induced by occlusion of the superior mesenteric artery in the rat. intestinal ischemia was induced for 20 and 30 minutes by applying a microvascular clip on the superior mesenteric artery followed by reperfusion 2, 4 and 6 hours after clip removal. intestinal transit time was measured by the propulsion of a radiolabelled solution (cr51). light microscopy was performed on intestinal samples. macroscopical pathological changes were not observed. however, microscopically, mucosal epithelial oedema, degeneration or slight ulceration occurred in rats 6 hours after reperfusion in ischemia-20 rain group and 4 and 6 hours after reperfusion in the ischemia-40 rain group. delayed small intestinal transit time was seen from 2 hours and on after intestinal ischemia for both 20 and 40 rain ischemia followed by reperfusion. the distribution of radioactivity demonstrated that most radioactivity was accumulated in the first two segments following intestinal ischemia and reperfusion, significantly differing from what was seen in animals subjected to sham operation (p < 0.01). the distribution of radioactivity in segments 4 and 5 in the group with repeffusion 6 hours after intestinal iscbemia for 20 rain was significantly higher than that noted in the group with repeffusion 6 hours after intestinal ischemia for 40 min (p < 0.01). q'he results indicate that a delayed intestinal transit time may be one of the earliest pathophysiological alterations noted, associated with duration of gut ischemia, and a potential factor for the development of bacterial overgrowth, gut barrier failure and bacterial translocation, in hypovolemic conditions. bacterial infections still constitute a major cause of morbidity and mortality in patients with acute liver failure. the present study aimed at evaluating the effect of ethylhydroxyethyl cellulose (ehec) on bacterial translocation following surgically induced acute liver failure. acute liver failure was induced by subtotal hepatectomy (90 %) in the rat. water-soluble ehec was administered orally 1 and 12 hours prior to hepatectomy. the incidence of bacterial translocation from the gut to mesenteric lymph nodes (mlns) and systemic and portal circulation was evaluated and the number of isolated bacteria from these samples and from intestinal content were determined. intestinal transit time, bacterial adherence onto the intestinal surface, intestinal mucosal mass, bacterial growth and dna synthesis, bacterial surface characteristics (hydrobiology: hydrophobicity, hydrophilicity and neutrality; surface charges: positive, negative and neutral) were also determined. hepatectomized animals showed a 80-100 % translocation rate to mlns or blood 2 and 4 hours after operation, while only 0-1% of rats subjected to sham operation or animals with 90 % hepatectomy and pre-treatment with ehec (p < 0.01). bacterial overgrowth, increased bacterial adherence onto the intestinal surface as well as decreased intestinal mucosal masses were observed in animals with subtotal liver resection alone, alterations that were prevented by enteral ehec treatment. a delay in intestinal 2-hour transit time occurred in both groups with subtotal liver resection, with or without enteral ehec. ehec inhibited bacterial growth and dna synthesis, and altered bacterial surface properties following 1hour incubation with bacteria. in conclusion, the findings in the present study imply that ehec alters enterobacterial capacities for metabolism, proliferation and invasion by effects on e.g. bacterial surface characteristics. furthermore, ehec seems to possess a trophic action on the intestine, rather than exerting its effect by enhancing intestinal motility. department of surgery, lund university hospital, s-221 85 lund, sweden disturbances in intracellular calcium signalling can potentially result in impairments of cellular responses vital to the functional integrity of both immune and non-immune cells, and thus contribute to a decrease in host resistance against infection and to multiple organ system failure during sepsis. studies in our laboratory have focused on assessments of intracellular ca 2÷ regulation and ca~+-depended cellular responses in the liver, skeletal muscle and splenic tlymphocytes harvested from rats subjected to gram-negative intraabdominal sepsis. cytosolic ca 2+ concentration, [ca2*]i, and ca 2+ fluxes were measured by the use of fluorescent ca 2+ chelating dyes (fura-2 or indo-1) and 45 ca respectively. to assess sepsis-related changes in ca 2+ dependent cellular responses, we measured the acute phase protein response in the liver, the regulation of protein and sugar metabolism in the skeletal muscle, and the proliferation response in the splenic tlymphocytes. altered ca2+ i signalling with sepsis was correlated with an exaggerated inappropriate acute phase protein response (100% ¢) in the liver, and a blunted insulin mediated sugar utilization (60% 4) and increased proteolysis (50% ~) in the skeletal muscle. in t-lymphocytes, a decrease in mitogen induced elevation of [ca2+]i by 35-40% was correlated with a significant depression in their proliferative capacity. these studies clearly suggest that altered calcium signalling is correlated with disturbances in cellular responses in both immune and non-immune cells during sepsis. the altered cellular responses adversely effect the outcome of the septic injury. (supported by nih grant gm 32288). alfred ayala, ping wang and irshad h. chaudry. changes in macrophage capacity to respond to foreign pathogens are thought to be central to the developing immunosuppression associated with traumatic injury. in this respect, the suppression seen in m~ functions following hen (a common component of traumatic injury) may be mediated by the direct or indirect inhibition of their capacity to perceive external stimuli (e.g., opsonized & non-opsonized bacteria, and their cellular components, etc.} due to the breakdown of the receptormediated signal transduction system. results of a number of studies by our laboratory and others indicate that this inability to respond to external stimuli is in part due to the loss of cell surface receptors. decreases have been documented for not only la antigen, but also c3b, fc, and tnf receptors following hem in mice. furthermore, studies which have examined second messenger generation in these cells indicate that m~ derived from the peritoneum and spleen exhibit a decreased capacity to mobilize ca +2 from intracellular stores. this protein kinase dependent process of [ca+2] i mobilization appears to be linked to the inability to synthesize inositol triphosphate. of interest, the depression in ca +2 signal generation appears to be inversely related to presence of elevated levels of camp in m~ from hen mice. we have reported that m~ priming agents, such as ifn-7 (which exhibits salutary effects on m~ function following hem), appear to restore cell signal transductive capacity while reducing the levels of camp. nonetheless, the extent to which depressed receptor signal transduction in hem, is due to receptor loss~dysfunction or elevated antagonistic second messenger levels remains to be determined. conclusions: significant impairment of calcium signaling occurs at all time-points prior to and following pha stimulation in trauma patients. tcell activation failure can, in part, be explained by the inadequacy of this essential intracellular second messenger system. restoration of immunocompetence following trauma will have to address strategies to better assess and restore this vital step in the activation sequence leading to proliferation during the antigen recognition process. patrick a. bseuerle institute 01 biochemistry, albert-ludwigs-university, hermann-herder-str. 7, d-79104 freiburg, germany the active form of the transcriptional activator nf-~b is a heteredimer composed of a 50 and 65 kda polypeptide. in this form, nf-'<b can bind with high affinity to decameric sequence motifs (consensus: 5"-gggpunnf'ypycc-3") found in enhancers and promoters of many genes activated upon a wide variety of pathogenic conditions, including viral and bactedai infections, acute phase response, oxidative stress and uv and gamma irradation. clinically important target genes for nf-k:b encode inflammatory cytokines (il-1b, tnf, [l-6, il-8, gm-csf etc), cell adhesion molecules (icam-1, elam-1, vcam-1), acute phase proteins and immunoregulatory ceil surface receptors. in most cell types, nf-~:b is sequestered in the cytoplasm and, therefore, unable to initiate transcription of these genes. the cytoplasmic form of nf-~:b contains one additionai subunit, referred to as h,;:b. i~b can reversibly suppress dna binding and nuclear uptake of nf-~b by virtue of its association with p65. upon stimulation of cells, ikb is proteolytically destroyed, allowing nf-kb to enter the nucleus and activate genes upon binding to transcriptional enhancers. we have demonstrated that various antioxidative compounds inhibit the activation of nf-~b in response to many inducers. moreover, nf-~:b was shown to be activated upon treatment of cell cultures with p.m-amounts of hydrogen peroxide. these data suggested that nf-~b prinicipaliy is an oxidative stress-responsive transcnption factor. the notion is supported by numerous reports on the induction of oxidative stress by many nf-nb-inducing agents. nf-~b activition can likewise be suppressed by protease inhibitors. these drugs apparently inhibit a yet unidentified protease responsible for the inducible decay of inb. we propose to use nf-nb inhibifors as novel drugs with a very broad application under acute phases el inflammation, injury and infection. northern blot experiments revealed that expression of the recently discovered lipopolysaccharide binding protein (lbp), a 58/60 kd glycoprotein, in the liver rises substantially during the acute phase response. experiments with an in-vivo acute phase model using new-zealand-white rabbits and also in-vitro experiments employing stimulated hep-g2 cells showed that lbp transcripts could be induced 10 to 100 ford within 24 hours after induction with cytokines. by using a newly established nonradioactive nuclear run-on technique we show that induction of lbp during the acute phase is transcriptionally regulated. furthermore we screened a human genomic library and were able to clone the lbp gone, containing the promoter 1.5 kb upstream. several liver-specific and "acute-phase-typical" potential binding sites were found fn the promoter region and reporter gone assays were set up. several caat-boxes, the il-6 responsive elements hstf-hsp 70.5 and h-apf-1 rs as well as the transcription factor hnf-5 and the glucocrticoid-responsive elements oct-2-d and gcre were found, which correlates with the induction pattern of lbp mrna in hep-g2 cells by il-6, il-1 and dexamethasone. pcr-based analysis of the exon-intron pattern of the lbp gene revealed similarities with the genes of two other lps binding proteins, namely bpi and cetp. further analysis of this novel acute phase protein, that also has an important role in endotoxin recognition and immunomodulation will help in understanding the complex acute phase mechanism and potentially lead the way to new therapeutic strategies. lps activation of nucle?/r fa-ctor:~b " (nf:~bj"in cd14.transfected fi'broblasts does not appear to require tyrosine kinase activity d. t. golenbock, r. delude, r. savedra, s. vogel and m. j. fenton lipopolysaccharide (lps) is the major constituent of the outer leaflet of gram-negative bacteria. during the course of serious infection, shock may occur as a result of the interaction of lps with macrophage lps receptors. cd14, a 55 kda glycosyl phosphatidylinositol (gpi)-linked protein, functions as an lps receptor. a critical issue in lps activation concerns the mechanism by which cd14, which has no transmernbrane domain, transduces a sig,nal after lps binding. evidence exists which suggests that cd14 may signal cells after lps binding by interacting with an lps signal transducer with tyrosine kinase (tk) activity. wild-type chinese hamster ovary fibroblasts (cho) normally do not respond to lps, but can be activated following transfection with cd14 (cho/cd14). stimulation of cho/cdi4 with as little as 1 ng of lps/ml resulted in the release of 3h-arachidonic acid (3h-20:4) into the culture supernatant. in addition to 3h-20:4 release, we examined lps-imiuced activation (transl0cation) of the transcription factor nf-~b. similar to lps-induced 3h-20:4 release, these responses were observed only in cho cells lransfected with cd14 closely resembling the same response in the marine macrophage cell line raw 264.7. maximum nf-~cb expression occurred at 30 minutes. in contrast to lps-induced 3h-20:4 release, dexamethasone, cycloheximide, and actinomycin d had no effect on activation of nf-r,.b in cho/cd14, suggesting that nf-r,b activation begins early after lps binding to cd14 and does not require transcription or translation. we then examined cells for lps-induced tk activity by western blotting cellular ly~ates with anti-phcsphot-'rosin:~ anl',bodie~" although tk activity was seen in lps-stimulated raw cells and phorbol ester stimulated cho/cd14, lps-induced tk activity in cho/cd14 was not observed. although the tk inhibitor herbimycin inhibited the release of 3h-20:4 in cho/cd14 and raw cells, neither herbimycin nor genestein effectively blocked nf-r,b activation in either cell type. these results suggest that tk activity is involved in a portion of the signaling pathway that is distal to initial signal transduction. the absence of observable lps-induced phosphotyrosine residues in cho/cd14 cells as well as the failure of tk antagonists to inhibit lps-induced nf-~cb activation suggest that the proposed cd14-related lps signal transducer in cho/cd14 is not a tymsine ldnase. dimished cardiac inotropic function and response to 13-adrenergic receptor (13-ar) stimulation are frequently seen in septic patients, these cardiac defects are also seen in animal models of entoxemia. to determine the characteristics of the decreased transmembrane signal associated with the decreased beta adrenergic response we measured the force and rate of contraction of atria harvested from sprague-dawley rats exposed to endotoxin (0:2 gm/kg). to stimulate various components of the transmembrane signal cascade of 15-ar was isoproterenol (i3-ar), acetylcholine (m2ach receptor), naf(gs and gi proteins), forskolin (adenylate cyclase), and calcium (intracellular contracttile signal) were used. to eliminate the transmembrane control of calcium and test the intrinsic contractile response, hypothermia (22°c) was used the results showed alterations in the inotropic function with no significant difference in chronotropic response. the inotropic reponse for the endotoxin exposed atria and controls is shown below these results show that there is a transmembrane signal defect both for the i~-ar signal and for ca +. these data indicate that the level of the defect appears to be at the g proteins. ischemia-reperfusion of the rat intestine produces an injmry both to the intestine and to distal organs, notably the lungs and liver. prior studies have shown that 1 h of intestinal ischemia leads to a nemtrophil independent reperfusion injury of the gut. thus, rats rendered neutropenic by pl~l antiserum have been shown to express a severe local gut injury when subjected to ischemia and reperfusion. this gut injury is postulated to he mediated at least in part by the complement system. the soluble (s) cri receptor which binds to c3b and cdb was given to rats at a dose of 12 mg/kg by intravenous bolus, 15 min before reperfusion of the ischemic gut. a control group was given pbs buffer. at 4 h of rmperfmsion the animals were sacrificed. pbs treatment led to a significant activation of both classical and alternate complement pathways while scri inhibited both pathways (fall to zero of chbo ). intestinal mucosal injury score, assessed by histological grading was reduced by scri (2.49±0.22 to 1.73_+0.17,p<0.05) . intestinal neutrophil sequestration estimated by assay of myeleperoxidase was also reduced (0.05 ± 0.01 to 0.02 ± 0.01 u/g). c3 was detected in the gut hy i~unohistochemistry. remote organ injury was modified that is lung permeability (ration of concentration of radiolabelled albumin in broncho-alveolar lavage fluid to that in blood) was reduced (11.3±1.4 x i0 3 to 3.9±0.8 x 103, p<o.05). however, neutrophil sequestration by the lungs was unaffected. this remote protection is thougth to be related to prevention of ic3b deposition on imng andotheli~ which activates adherent neutrophils. these findings support the hypothesis of complement mediated local and remote injury. we postulate that complement fragments may be deposited either as a result of the ischemia induced loss of membrane bound complement regulatory proteins, such as decay accelerating factor or membrane cofactor protein, which allow complement fragments to accumulate on the endothelial cell surface. alternatively, increased expression of p-selectin on the endothelial cell surface may, by means of its complement binding domains, act as a lattice for complement deposition. indeed, p-selectin has been detected on the surface of endothelial cells ans platelet-neutrophil aggregates recovered from the intestine of these animals. ischemia (4hr) followed by reperfusion (dhr) of rat hind limbs resuits in local (muscle) injury as well as in remote (lung) injury, which is characterized by increased vascular permeability (leakage of j2sl labeled albumin) and neutrophil accumulation (tissue content of myeloperoxidase, mpo). within 60 minutes of reperfusion following ischemia, tumor necrosis factor-o~ (tnfc 0, interleukin-i (1l-i) and il-6 plasma levels increased significantly, reaching maximum levels after 2 hours ofreperfusion. polyclonal antibodies to tnfet and 1l-i provided significant protection against muscle and lung injury. muscle and lung vascular permeability decreased in anti-tnfct treated rats by 41% and 74% respectively and mpo was reduced by 72% and 83%. antml-1 yielded a protection in muscle and lung vascular permeability of 24.7°,4 and 52.4% respectively whereas muscle mpo was reduced by 46.4% and lung mpo by 25.9°,4. these results were confirmed by the use of soluble tnf~ receptor and il-i receptor antagonist (il-ira). when icam-i expression in vivo was examined using 125mabeled antmcam-i, constititive expression of icam-1 was evident only in lung but not in muscle. during reperfusion icam-1 expression increased by 98.2%. treatment with anti-tnfcx or il-ira reduced icam-i upregulation by 73.9°,4 and 74.8% respectively. frozen sections of normal rat lung revealed no evidence of e-selectin expression, whereas lung sections obtained after ischemia and reperfusion immunhistochemically demonstrated expression of e-sdectin. lungs from rats pretreated with anti-tnfct were negative for e-selectin staining. our data indicate a role for cytokines in the upregulation of adhesion molecules in ischemia/reperfusion injury. adherence of neutrophils to the coronary endothelium is associated with significant myocardial injury following 90 min of ischemia (mi) and 4.5 h of reperfusion (r). p-selectin is expressed by activated endothelial ceils and platelets within 10 min and tethers polymorphonuclear leukocytes (pmns) to the endothelium. we examined the cardioprotective effects of a monoclonal antibody (mab) designated pb1.3 to p-selectin in a feline model of mi+r. pb 1.3 (1 mg/kg) administered 80 rain post-occlusion (10 min prior to reperfusion) significantly attenuated myocardial necrosis compared to a non-blocking mab (nbp1.6) for p-selectin (15 + 3 vs 33 + 5% of area-at-risk, p<0.01). endothelial dependent relaxation to acetylcholine was also significantly preserved in ischemic-reperfused coronary arteries isolated from cats treated with pb 1.3 compared to nbp1.6 (67 + 6 vs 11 + 3, p<0.01). furthermore, pmn adherence to ischemic-reperfused coronary endothelium was significantly attenuated in pb1.3 treated cats (64 + 7 vs 136 + 12 pmns/mm z, p<0.01). also, normal coronary endothelium activated with thrombin (2 u/mt) had increased pmn adherence under conditions of shear stress, an effect which was significantly (p<0.01) blocked by pb1.3, but not by nbp1.6. furthermore, p-selectin is markedly upregulated 10-20 min post-reperfusion in coronary arterial and venous endothelinm. similar results were obtained in a rat model of mesenteric ischemia/reperfusion, including upregulation of p-selectin on mesenteric vascular endothelium. these results demonstrate that tethering of neutrophils to endethelium by p-selectin is an important early consequence of reperfusien injury, and a specific monoclonal antibody to p-selectin exerts significant endothelial preservation and cardioprotection in ischemia and reperfusion states. as recent interest has increased in the role of extracellular matrix proteins in inflammation, we examined the role and relationship of laminin and fibronectin to the processes of cell infiltration in a rat model of heart isografts. changes occurring in this system are on a nonimmunological basis and may be an effect of the initial surgical manipulation and the warm and cold ischemic times of transplantation. lewis (lew) donor hearts were transplanted into lew recipients, naive lew acted as naive controls (nc). grafts were harvested 2, 4, 8, 16, 24, 48, 72, 120 and 168h after transplantation (n=6/time point) . snap frozen sections of organs were stained for cd-4 (w3/25), cd-8 (ox8), t-lymphecytes (tl) (ox-19), macrophages (ed1, ed2), neutrophils (pmn) (mom), icam-i (iap29), laminin and fibrinogen. results were evaluated visually by counting cells/field of view (cf) for w3/25, ox8, ox19, ed1, ed2 and mom, staining for icam1, lain and fib was assessed visually using a scale (ve=0-10). as early as 2h after transplantation, fibrinogen and laminin expression increased in i, peaking 8h after transplantation (ve= 4.5; nc: ve=i.5) . at 16 and 24h in i, on both vessels and interstitial cells laminin and fibrinogen expression had declined to baseline (ve=i.5) but rose again at 48h, peaking at 72h (ve=4) and remaining elevated thereafter (ve=3). the high expression at 8h correlated well with the maximal pmn infiltration at that time (cf=6) in i returning to baseline at 24h (cf=0.5) and thereafter. the second peak at 72h correlated with the onset of macrophage and t-lymphocyte infiltration. thus, laminin and fibrinogen seem to guide leucocyte infiltration following graft ischemia during the time of reperfusion. background. reperfusion of ischemic kidneys with xenogeneic blood leads to activation of endothelial cells and circulating leucocytes with the final consequence of microvascular thrombosis. in concordant systems, the mechanisms of rejection can be studied due to cross-reactivity of mediators with anti-human monoclona~ antibodies. aim of the study. to obtain information about the kinetics of proinflammatory cytokines and production of soluble adhesion molecules in the acute phase of reperfusion, eight kidneys from rhesus monkeys were perfused ex-vivo with human blood (group b/o) for one hour in a closed system. blood levels of il-lb, il-6, tnf-a, soluble icam and e-selectin were measured in elisa-technique under steady-state conditions. results. cytokine levels rose significantly within the 60minute interval (il-lb: 6,1__+2,6 to 161,1__+98,5 pg/ml; il-6:30.24-7,7 to 274,2__+75,8 pg/ml; tnf-a: 544,2__+363,6 to 1651,0+25,7 pg/ml; p<0.05). immediately after the beginning of reperfusion, soluble icam-1 and selectin levels were abnormally high and constantly rising throughout the observation period, reaching significance at 60 minutes. discussion. high levels of proinflammatory cytokines may lead to an induction of adhesion molecules, thus upregulating the leukocyte-endothelial interaction in an complement-independent mechanism. specific pretreatment with monoclonal antibodies against icam-1, lfa-1 or other soluble mediators may be useful in downregulating hyperacute rejection in transspecies transplantation. prolonged ischemia has been associated with later dysfunction of solid organs as it may cause upregulation of adhesion molecules, production of cytokines on vascular endothelium and migration of host cells into the graft. these early events may lead to irreversible organ injury. this study evaluates the effects of global ischemia on early immunohistological changes in cardiac grafts transplanted in rats. isografted hearts (lewis->lewis) were were divided into ischemic and non-ischemic groups (n=30/group). all donor hearts were flushed immediately with cold saline. non-ischemic hearts were then transplanted within 30 rain, ischemic hearts were stored in cold ringer's solution for 3 hours before revascularization. representative grafts were removed after 12. 24, 72hrs, 10 and 100 days, and evaluated immunohistologically (cells/field of view=c/f). restitution of ventricular activity was significantly delayed in ischemic grafts (5 vs 1 rain). after 12 hrs, all ischemic grafts exhibited an extensive interstitial edema, declining slowly thereafter. at the same time, numbers of pmn peaked (3 vs 1 c/f in non-ischemic grafts), whereas edl+macrophages (9 vs 2 c/f) and tnfe expression peaked by 24hrs. by 72 hrs t-lymphocytes began to enter ischemic myocardium and icam-1 was moderately increased. after 10 days cellular infiltration had returned to baseline, and no differences were seen among both groups after 100 days. global myocardial ischemia inhibits initial graft function, and engenders a brisk inflammatory reponse, primarily pmn and macrophages, with increased mhc class ii and cytokine expression. leukocyte -endothelial interactions are the result of endothelial activation, leukocyte activation or combination of both, which are accompanied by nee-expression, upregulation or shedding of adhesion molecules (selectins, inlegrins). such interactions differ with regard to the stimulus (e.g. thrombin or histamine for p-selectin, endotoxin or tnf/il-1 for e-selectin), the time course of response (minutes versus hours) and the localisation in different organs. recently assays are available for circulating soluble fragments of the cell bound adhesion molecules e.g. se-seleetin was found to be increased in plasma concurrent with high circulating endoloxin and cytokine levels. the importance of adhesion molecules for the sepsis event is evident, while effectiveness of anti-adhesion inolecu]e therapy is controversial e.g. beneficial anti-e-selectin therapy in baboon bacleremia but deleterious effects of amti-cd18 treatment in the same model. in other species similar controversial results with anti-cd18 therapy in sepsis were reported. steven l. kunkel,theodore standiford* and robert m. stricter. the migration of leukocytes to the lung during endotoxemia is dependent upon the coordinated expression of lung vascular adhesion molecules and the subsequent production of appropriate leukocyte chemotactic proteins. in experimental animals, neutrophils accumulate within the lung soon after the administration of endotoxin, while mononuclear cell infiltration occurs in a more distal manner. a kinetic analysis of lung leukocyte levels revealed a 3-fold increase in neutrophil numbers associated with dispersed lullg tissues 2 hours after lps treatment, while macrophage levels increased by 4-fold at the 24 hour time point. thus, the recruitment of different leukocyte populations to the lungs during endotoxemia is likely directed by different mechanisms. recent studies have identified a supergene family of small inducible chemotactic cytokines (chemukines) which possesses chemotactic and activating properties for neutrophils. the prototype of this family is interleukin-8 (il-8). interestingly, a related supergene family has been identified which possesses activity for recruiting mononuclear cells. examples of this group of inflammatory chemukines are monocyte chemotactic protein-i (mcp-i) and macrophage inflammatory protein-i alpha (mip-i). in initial in viva studies we examined whether mip-i was expressed systemically or in a compartmentalized fashion post lps challenge. assessment of plasma cytokine levels revealed maximal tnf levels occurred i hour post lps administration, returning to baseline by 4 hours, while mip-i levels were maximal at 2 hours (2,5 ng/ml), with a second peak at 24 hours after lps challenge. interestingly, aqueous extracts of liver homogenates from lps treated animals demonstrated no mip-i levels, while aqueous extracts of lung revealed a 4-fold increase in mip-i levels over control lungs. immunohistochemical analysis of the lungs from 24 hour lps treated animals demonstrated the alveolar macrophage was a rich source of mip-i protein. cell-associated mip-i was also expressed by blood monocytes adherent to the pulmonary vascular endotheliun, however the expression of monocyte-mip-i was observed by 2 hours post lps administration. immunohistochemical analysis also demonstrated that mip-i antigen is associated with the extracellular matrix on the interstitial side of the endothelium. this suggests that the extracellular matrix, which is produced during inflammation, can bind mip-i and this may serve as a depot for the prolonged presence of nip-1. in additional studies we have demonstrated that the intratracheal instillation of rmui [ip-l(loong) activation of polymorphonuclear leukocytes by inflammatory stimuli may contribute to the development of multiple organ failure in septic patients. thereby pmnl are proposed to avidly adhere to vascular endothelium causing damage by the subsequent release of toxic agents. as cellular adhesion is primarily mediated by 62-integrins and lselectins, the present study compares the expression of these adhesionmolecules on pmnl in septic patients and healthy volunteers. methods: expression of 62-integrins and l-selectins on pmnl was measured in whole blood by flow cytometry using the monoclonal antibodies ib4 and dreg 200, baseline values were determined immediatley after drawing blood. in addition cells were incubated 45 min at 37°c to allow for spontaneous regulation of adhesion molecules. 55 blood specimens from 8 septic patients were obtained during the course of their illness. control values were determined in 12 healthy volunteers. results: baseline expression of 62-integrins and l-selectins was not signifcantly different in septic and in healthy subjects. in contrast, there was a significant upregulation of g2-integrins and shedding of l-selectins of pmnl in septic patients (sp) compared to healthy volunteers (hv). the local or systemic production of inflammatory cytokines, such as tumor necrosis factor alpha (tnfc~), can serve to modulate multiple aspects of neutrophil function. the ability of neutrophils to leave the circulation and migrate to areas of infection is one essential component of host defense. l-selectin, a leucocyte-associated adhesion molecule, is responsible for the initial reversible contact between neutrophils and endothelium and the subsequent roiling action of neutrophils along the vessel wall. in contrast to other adhesion molecules, l-selectin expression is rapidly down-regulated after neutrophil activation. the loss of l-seleclin may thus be a critical determinant of how neutrophils become unbound from their endothelial attachments and enabled to proceed towards an underlying extravascular area of infection. we hypothesize that the shedding of l-selectin is a strictly controlled process, occurring primarily at localized sites of inflammation, which may be modulated by tnf~, a flow cytometric method of staining neutrophhs by monoclonal antibodies in whole blood is described whereby the kinetics of l-selectin shedding may be followed in real time. the dose response and time course of in-vitro l-selectin shedding by neutrophils from normal human subjects was assayed after exposure to n-formyl-methionylleucyl-phenylalanine (fmlp) and tnfc~. either singly or in combination, our results show that l-selectin shedding can be reliably followed over time. a significant percentage of cells shed l-selectin after exposure to 2000 pg/ml tnfc~ or 1000nm fmlp (but not at 400pg/ml tnfc~ or 200nm fmlp). greater numbers of cells were able to shed their l-selectin when fmlp and tnf~x were presented in combination rather than alone. high levels of tnfc~ did not appear to alter the threshold concentration of fmlp required to induce shedding, we conclude that the extent and rapidity of l-selectin shedding may be modified by different combinations of ligands and that shedding, by vidue of the high concentrations of cytokines or chemotactic factors required, is a process localized to sites of infection or inflammation. we prospectively studied 23 patients with severe sepsis syndrome; group a : septic shock with or without adult respiratory distress syndrome lards) (n =7, bacteremia = 6); group b : sepsis syndrome without septic shock (n = 16, bacteremia = 9). serial plasma samples obtained on day 0, 1, 3, 6, 10 and 14, were assayed using elisas method (british biotechnology), normal control levels of soluble icam-1 and e-selectin, obtained from 11 healthy volunteers, were respectively 205 ± 19.4 ng/ml and 45 ± 5.7 ng/ml (mean _+ se), acute lung injury was quantified dally on a tour-point score system (murray, am rev respir dis,1989) . compared to control mean values, initial levels of groups a and b were significantly higher for icam-1 (p < 10 -4) and e-selectin (p < 10-3). comparisons of group a and [] (* = p<0.05; ** = p<0.00t) soluble icam-1 levels of group a enhanced significantly (p<0.05) during the first 24 hours, and a sustained high levels was of bad prognosis (25% of survivors at day 6). the evolution of soluble icam-1 and e-selectin levels were significantly correlated with murray's score (spearman test : p <0.001). conclusion: these results suggest that endothelial adhesion molecules are released into the plasma of patients with severe sepsis syndrome. soluble icam-1 and e-selectin are correlated with endothelial lung damage, and loam-1 seems to be a better indicator of the severity of endothelial injury. introductory remarks to anti-adhesion molecule strategies as a therapeutic modality ch wortel, repligen corporation, one kendall square, building 700, cambridge, ma 02139, usa. the development of antimicrobial therapy represented a major breakthrough in the struggle against disease. it strengthened the notion that disease could be overcome by eliminating foreign invaders threatening the host. this paradigm has proven to be very successful, the threat of many infectious diseases has significantly changed, some have even been eradicated. nevertheless, sepsis has remained a severe condition, increasing in incidence while mortality remained very high. more recently, it has become increasingly clear that besides the nature and treatment of an exogenous agent, the reaction of the host defense itself plays a pivotal role in the outcome of the event. endogenous mediators, such as tnf, il-i, il-6 and il-8, govem many of the actions of the host defense system. while the expression of these cytokines more often than not benefit the host, (over)-expression can cause severe damage. based on this hypothesis,anticytokine strategies, such as those targeted against tnf or il-1, have been evaluated for the treatment of sepsis. results of these early studies have not yet indicated success in improving the outcome of the disease. it has been difficult to define a patient population where a benefit could be reproducibly shown. furthermore, it has been documented that synergy between cytokines occurs, but detailed knowledge of the cytokine network is not yet available. it is conceivable, that neutralization of one cytokine prompts the induction of another which will evoke the intended response in the host. recent data obtained in human endotoxemic volunteer models seem to confirm this. if this turns out to be the case, neutralizing a single cytokine may not be a successful approach. cytokines in tum, induce various adhesion molecules, such as icam-1. such molecules regulate for instance the neutrophil-endothelial cell interactions, which are thought to play an important role in the pathogenesis of systemic organ injury. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries. thus, altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by inappropriate behavior of host defense cells. targeting cellular surface interactions has been added to the efforts to change the outcome of disease. modulation oftheseprocesses seems very promising, but may temporarily leave the host without effective defense mechanism. great care therefore, must be exerted when studying this powerful two-edged sword in a clinical setting. our knowledge of the role of adhesion molecules in the intlammatory response has increased rapidly due to the availability of new reagents and mice geneticly deficient in adhesion molecules. these molecules are important in interactions of leukocytes with endothelial cells, other leukocytes, platelets, and epithelial cells. when these molecules are engaged, they can also play a role in activating leukocytes and their effector functions. in the venules of the systemic circulation, adhesion often occurs through a series of sequential interactions. initial interactions are mediated by members of the selectin family to loosely associate the leukocytes with the endothelium and are followed by firm adhesion requiring members of the integrin and immunoglobulin family. later interactions with endothelium may require pecam. adhesion molecules are usually required for leukocyte emigration in response to extravascular stimuli and for neutrophil-mediated endothelial cell injury. they are critical for host response in many diseases including infections. however, when the inflammatory response results in damage to host tissues, patients may benefit from blocking the leukocyte response. anti-adhesion molecule agents are an important potential antiinflammatory therapy. the focus of anti-adhesion therapy may be at any step of the sequence. diseases where anti-adhesion molecule therapy may benefit patients include ischemia/reperfusion injury in many organs, ards and mof, and transplantation, both to protect the donor organ from ischemia/reperfusion injury and to inhibit graft vs host disease. many strategies have been considered and include: 1) blocking the ability of adhesion molecules to recognize their ligand using antibodies that have been humanized or soluble receptors linked to igg to prolong their circulating halflife, 2) blocking the ligands for adhesion molecules using soluble adhesion molecules, peptide analogues, or oligosaccharides, and 3) blocking the production of the adhesion molecule using anti-sense oligonucleotides. because the synthesis of adhesion molecules is usually regulated by cytokines, inhibiting the action of cytokines is another potential site for interrupting the adhesion process. although important issues of safety must be evaluated, the potential for modulating the inflammatory response make this an exciting area of improvement in health care delivery. claire m. doerschuk, m.d.; riley hospital for children, room 2600; 702 barnhill drive; indianapolis, in 46202 usa. modulation of neutrophil-endothelial cell adhesion with anti-cdl i/cd18 monoclonal antibodies as a therapeutic modality. ch wortel, repligen corporation, one kendall square, building 700, cambridge, ma 02139, usa. the central role of inflammatory cells in the pathogenesis of lung and systemic organ injury is well recognized. binding of neutrophils to endothelial cells and migration into the parenchyma are largely regulated by complementary adhesion molecules. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta2 or cluster differentiation (cd)18 chain covalently linked to one of three different alpha chains (cdlla, cdllb, cdilc) and exist on the cell surface as three distinct heterodimers. cdlla/cd18 is expressed on all leukocytes, whereas cd 11 b/cd 18 and cd 11 c/cd 1.8 are restricted to cells of myeloid origin. cd i 1/ cd 18 interacts with intracellular adhesion molecule-1 (icam-i), its ligand on endothelial cells. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects with anti-cd18 antibodies have been observed in a wide variety of inflammatory, immune, and isehemia-reperfusion injuries, such as arthritis, burns, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degenemrion, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. blockage of cd18, however, would affect all leukocytes, as would antibodies to cdlla/cdi8. targeting cdllb/cd18 would affect cells of the myeloid lineage only, which could prove to be beneficial. cd11b/cd18 is not only involved in transendothelial migration, but is also implicated in adherencedependent formation of reactive oxygen species. blocking cd1 lb/cd18 may therefore not only reduce the numbe r of leukocytes accumulating in the tissue, but also attenuate the oxidant stress of infiltrated neutrophils. anti-cd 11 b treatment has been used effectively to reduce tissue injury initiated by ischemia-reperfusion, complement activation and endotoxemia. altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by leukocytes, but may also temporarily leave the host without effective defense machinery. overall, animal studies suggest that it may be safe to inhibit neutrophil adhesion for a limited period of rime. these observations will have to be confirmed in carefully designed clinical trials. c, arbobydrams are ubiquizom constir~uts of cell sv.rfaees, and possess many c~xssfies ttm~ m~,e ~em ide~. canaidates for r~ognifioa mole~ule& in m~y systems whe,~ cer udhesioa ~lays a critical ro~ car~hydram l:~dtag ~otegas have been shown to b~ad tocell surfa~ earbohydzaxes ~nd pzrl~pate in cell-ceil lumtaefion& such sys,.ems include ~rti~za~io=, deveaopmeat, l~thoge~-hcet reeog--ition ~d i~zmmadon_ in particular, tb.z recent di%~ve~ of lhe selec~ and th~ impo.~a~c~ in teukccy~udo~lelium adh~ion has -~f~m av.c~on ok l~in m~ted cell adhe~on. s~vere/poten~s/cs.rbohydr~ l~ga~s hrve ~e~l ~u~ilied for ~he s~lcc~ins. the,~ c~u be broadly di,,sded la~o ~wo m'oups -sibyl l~wis x m~ mh~.~l oligo~chadd~s, ~d sf/~ ca~ohydmma, all ~:~ ~l~dns bind m siflyl l~wis x (sie$ o!igos~ccb.e.rkms, zlthou~ w~ differing avi~re~. 'we have i~¢n~ed the functional g~oups 0a s~ex ~n~ med/a~ ~he b~u ~di~g of ~h~ c~b0hydmm = e-se/sedm we have used ~hat iv.formation to sya~esize sle ~ '~mt0gs r.he, t focus on replacing slslic ~sd ~nd fuc0s¢ wi~ simpler, more stable strunt~es. a[~ou~a ~ proeer~ is ongoing, we hve been ~ucee,.~ful a~ rep~aein8 t.ke si~ic a~id. residue wi~ std.fzte. ~ce~ or la~c amd groupa we t'we ex2aninad &e ten, bunion of ezed~ hydroxyl group of the fizeose residue ~ billding of e-, l-~nd p-selees..u. we have also found m~fi~fio~ of the reducing end ~¢.cha'i~ ~z increase mtagovsst activity. the, m¢ond. group of figs,rids a.r eontzin su~a~ 0u a ea.rbohydr~t¢ support,, und seem to bi~.d to t~e sele~ti~s wi~ dlf:ferem characteristics c0.an does sle:, s=h compounds are m2ogniz~d by l-selects. md p-selectia, bur., in genera/, not e, selecti~ these dam may mdicam r.hat l-and p-s~ ¢at~ h~d via o, second ~te thaz operates lu~.ead of, or in conjunction with ~tc sle" b~ding ~iite. dam rela~&~g to ±e, se two types of ,ml~ liga~ds have beam t~ed to desig~ potential the2~peutics for i~fi~anmat0ry disease. lr:rng maimai models of acute lung lu3ury we can demo~trate that eompmmds that inhibit seleetiu birding ~ ~i~o hzve ber~ficial effects when uc~d in rive. progressive microvascular damage in the tissue adjacent to a cutaneous burn injury results in extension of burn size. the role of leukocytes in the pathogenesis of microvascular injury was investigated by inhibition of their adherence to the microvascular endothelium using monoclonal antibodies directed to leukocyte cdi 8 or its endothelial ligaud, intercellular adhesion molecule-1 (icam-1, cd54). a model of thermal injury was developed using new zealand white rabbits. two sets of three full-thickness burns separated by two 5 x 30-mm zones were produced by applying brass probes heated to 100°c to the animals' backs for 30 sec. cutaneous blood flow determinations carried out with a laser doppler blood flowmeter were obtained for 72 hours. there were five experimental groups: controls given saline alone; animals given monoelonal antibody to the cd 18 r 15.7 prior to burn injury (pre-r 15.7); animals given r 15.7 30 min after burn injury (post-r 15.7); animals given a monoclonal antibody to icam-i, r 6.5 prior to burn (pre-r 6.5); and animals given the r 6.5 30 min postburn injury (post-r 6.5). blood flow in the marginal "zone of stasis" between burn contact sites was significantly higher in the antibody-treated animals. administration of the antibodies 30 min after injury was as effective as preburn administration in preserving blood flow. at 72 hr post-burn all antibody -treated animals had blood flow in the areas at risk for progression (i.e., the zone of stasis) at or above baseline levels while the control animals had levels equal to 34.7 _+ 12% of baseline (p < 0.05 by analysis of variance and mann-whitney u test). these results indicate that leukocytes play an important role in the pathogenesis of burn wound progression, and that this progression can be attenuated by moduiating adherence to endothelial cells. a wealth of information now supports the hypothesis that inhibition of cell adhesive mechanisms will nter the course of immunologicand inflammatory processes. what remains unclear is whether inhibition of specific mechanisms wfl[ be of therapeutic benefit in any specific human disease. current data derived from animal models are not inconsistent with the hope of therapeutic benefit, but techniques for inhibition (e.g., antibodies, antisense oligonucleotides, inhibitory peptides, inhibitory carbohydrates, smaii synthetic inhibitors, etc), tissue and species differences in the relative contributions of adhesion molecules to the inflammatory process, and the cascade model of adhesive interactions are all confounding issues, making predictions of therapeutic benefit in any specific human disease process very difficult. additional concerns involve the potential roles of adhesive mechanisms in host resistance to infection. as human therapeutictdals are initiated, more exact information on the roles qf specific adhesion molecules in human disease should emerge. inhibition of leukocyte adherence to endothelial cells can represent a novel therapeutic approach to septic shock. we performed a pilot study to evaluate the safety and tolerability to cy-1787, a monoclonal antibody against human e-selectin, in patients with septic shock. septic shock was defined by clinical signs of sepsis, a documented source of infection, and fluid-resistant hypotension requiring the use of vasopressors. eleven patients entered the study, but 2 patients who died during the first 24 hours were excluded, as this was part of the protocol. cy-1787 was administered as a single intravenous bolus of 0.1 mg/kg (n=3), 0.33 mg/kg (n=3) or i mg/kg (n=3) mg/kg. the antibody was well tolerated. none of the 9 patients died during the 28 day follow-up period. organ failure was assessed for 5 organs (cns, lungs, liver, kidneys and coagulation). the mean number of organs failing, which was initially 2.7 ± 1.2, decreased to 0.2 ± 0.4 at the end of the study (p<o.o01). these results are therefore encouraging. administration of an antibody to e-selectin in patients with septic shock requires further study. the importance of cytotoxic t lymphocytes (ctl) in the host defense mechanism against viral as well as parasitic and bacterial diseases is becoming increasingly appreciated. the design of vaccines to generate cytotoxic t ceils from nonviable or subunit constructs, however, poses challenges due to the unique characteristics of antigen processing and presentation by class i major histocompatibility complex (mhc) molecules. since the final antigen recognized by ctl are short peptides capable of high affinity binding to class i mhc molecules, we have designed strategies: a) to define the structural characteristics of peptides that are required for their high affinity binding to mhc; and b) to formulate these peptides into immunogenic vaccines that are capable of generating cytotoxic t lymphocytes. the strategy and results to accomplish these two goals will be discussed. amnon altman and erich gulbins ras proteins represent essential intermediates in receptor-mediated growth and differentiation pathways. they couple signals initiated by receptor or nonreceptor protein tyrosine kinases (ptks) at the cell surface to cytoplasmic targets which coordinate signal transduciion to the nucleus. ras also participates in t lymphocyte activation initiated by the antigen-specific t cell receptor (tcr)/cd3 complex, which leads to lymphokine production and proliferation. receptor ligation causes activation of associated ptks of the src and syk families as the earliest identifiable event in this pathway. the importance of ras in t cell activation is indicated by the findings that an oncogenic ras protein activates the interleukin-2 (il-2) gene promoter; conversely, a transdominant inhibitory ras mutant inhibits this event and the activation of map kinases. the rate-limiting step in ras activation is the exchange of bound gdp for gtp, which is catalysed by guanine nucleotide exchange factors (gefs). we identified the sh2/sh3 domain-containing vav protein, which is selectively expressed in hematopoietic cells, as a ras-specific gef coupled to several hematopoietic receptors. vav can be activated by two pathways, i.e., by ptk-mediated phosphorylation or by binding of diglyceride second messengers to a cysteine-rich, protein kinase c (pkc)homologous vav domain. these pathways are independent as demonstrated by structure/function analysis and the use of pharmacological inhibitors. the two activation pathways may enable vav to integrate signals from distinct hematopoietic cell receptors, and couple them to ras. t cells express another, ubiquitous ras gef, i.e., sos, which is known to be coupled to activated tpk receptors. t cell activation leads to membrane association of a signaling complex consisting of sos and two other signaling proteins, grb-2 and shc, via direct binding of the shc sh2 domain to the tyrosine-phosphorylated chain of the tcr/cd3 complex. thus, multiple receptors, ptks and ras activators participate in signaling pathways that lead to hematopoieitc cell growth and differentiation. the interactions and function of these various signal transducers will be reviewed. phagocytes. immunity is mediated by specific t lymphocytes, cytokines produced by these specific t cells activate antimicrobial capacities in mononuclear phagocytes, thus contributing to protection. other immune mechanisms also participate in the acquisition of resistance. on the other hand, t cells are also crucial for many pathologic sequelae of intracellular bacterial infections. although ifn-y is central to macrophage activation, synergistic and antagonistic effects with other cytokines occur. the cd/3 t cells, representing the major t cell population in experimental mice, are the main mediators of antibacterial immunity; an auxiliary role for y/~ t cells appears likely. the c~//3 t cell population further segregates into cd4 t cells which recognize microbial peptides in the context of gene products of the major histocompatibility complex (mhc) and cd8 t cells which see their peptide in the context of mhc class i. the -y/~ t cells are generally double-negative. knock-out mice in which the genes for various t cell receptor chains, for mhc class i or mhc class ii molecules, for cytokines or cytokine receptors had been deleted, have provided extremely helpful tools for analyzing the role of t cell subsets and cytokines in antimicrobial immunity. using these mutant mice, the role of distinct t cell subsets and cytokines in bacterial elimination and granuloma formation was analyzed in detail. these studies underline the central role of a//3 t cells and ifn-y in antibacterial immunity and, furthermore, show a distinct function of 7/$ t cells. moreover, these studies show that the relative contribution of cd8 or cd4 t cells to antimicrobial immunity varies in response to different pathogens. apoptosis is an active form of cell death in which the cell participates in its own demise, providing the biochemical pathways that trigger and mediate the complex events of the process. although this phenomenon has been studied for many years, it is only recently that significant progress has been made towards the elucidation of the molecular mechanisms that control apoptosis. once such mechanism came as something of a surprise: in a number of eases, apoptosis can be promoted by the action of the c-myc protein, a protooncogene product that had previously been associated only with cell proliferation (and presumably, survival) . expression of c.myc in cells can cause them to enter an apoptotic pathway or proliferate, mad this "decision" depends upon additional signals, such as growth factors that inhibit apoptosis and allow the ceils to proliferate. thus, c-myc directs cells out of rest and the choice of proliferation or death depends upon the presence or absence of survival signals. other survival signals are generated by oncogene products that can cooperate with my¢, including bcl-2 and abl there are implications of this interplay between apoptotie and anti-apoptorlc signals in cell iransformation and the resistance of some tumor ceils to therapeutic agents capable of inducing apoptosis. oncogene interactions therefore control the life or death of transformed ceils but also have important roles in normal, development processes. in the immune system, developing t ceils are "screened" for potential self-reactivity by a process of negative selection, in which activation via the t cell receptor (tcr) induces apoptosis. this process can be mimicked in t cell hybridomas, and appears to depend upon the expression of the c-mye protein. evidence that this represents a requirement for the myc/max heterodimer will be presented. as above, these cells are presented with the "choice" to proliferate or die and this depends not only upon myc but also other signals. thus, expression of functional v-an kinase in these ceils can render them resistant to the activation of apoptosis via tcr ligation, surprisingly, bcl-2 does not appear to be capable of blocking this form of apoptosis, and the reasons for this will provide new insights into the development of the immune system and the process of apoptosis. although the oncogene products discussed here probably do not directly participate in the biochemical process of apoptosis, they represent molecular controls on this complex mechanism. as such, they gave us new ways to look at the life and death of a cell recent attention has focused on macrophage release of cytokines, such as il-1, il-6 and tnf as important mediators of septic shock. however, serum cytokine levels have correlated poorly with outcome of septic shock. the purpose of this study was to compare the functional status of macrophages to serum cytokine levels in early sepsis by an analysis of splenic macrophage protein synthesis during abdominal sepsis. macrophage total protein distribution and biosynthetic activity was assessed by large format 2-d page, autoradiography of 35s-labeled proteins and computer assisted densitometric analysis. sepsis was induced in sprague dawley mts by cecal ligation and puncture (clp). untreated and sham operated rats served as controls. splenic macrophages were harvested at 3 and 24 hrs. following clp. serum il-6 levels were determined at 3,12, and 24 hours by the 7 tdi bioassay. by 12 hrs. 100% of the clp mls exhibited multi-microbiaj bacteremia. control or sham operated rats did not show bacteremia. clp resulted in a significant 286% elevation (p < 0.05) in serum il-6 levels at 12 hrs. there was no difference in il-6 at 3 and 24 hrs. when compared to the control groups. 2d page studies examined splenic macrophage total protein distribution and biosynthetic activity at 3 and 24 hrs. post-clp. at 3 hrs. macrophages from clp rats showed a substantial decrease in total protein pattern (353 protein spots vs 503 protein spots) when compared to non-septic control macrophages. a decrease in the number of lower molecular weight proteins (< 40 kd) was observed. at 24 hrs. post-clp, splenic macrophages from clp rats showed a substantial increase in the synthesis of low molecular weight (< 40 kd) proteins, when compared to non-septic controls. we conclude that: l) fulminate abdominal sepsis induces an early (3 hrs.) staining with cd14 and cd16 mabs will identify two monocyte subpopulations in human blood: a major population of regular monocytes, which strongly expresses the cd14 antigen (cd14++) and a minor population with weak expression of cd14 and expression of the cd16 antigen lcd14+/ cd16+ cells). in healthy control donors(n=35) the latter cells account for 45+22 cells/mm 3 and 9%+5% of the monocytes. in sepsis patients, the cd]4+/cd16+ cells can become a major population with more than 50% of all monocytes in 3 of 18 patients and with more than 500 cells/mm 3 in 4 of 18 cases. there was no correlation of cd14+/ cd16+ cells to any clinical parameter except for cd14+/cd16+ percentage and body temperature (p= 0.013). the cd14++ monocytes showed a substanttial decrease in cd14 antigen density in 9 of 11 patients. three-color-if shows that the cd14+/ cd16+ cells in sepsis patients when compared with the cd14++ monocytes exhibit a higher level of class ii antigen and a lower level of cdiib and cd33 antigens, consistent with a more mature nature of the cd14+/cd16+ cells. levels of il-6 were increased in sepsis patients: 3 of 5 patients with high numbers of cdi4+/cd16+ cells ( 200/mm 3) had high levels of il-6 (250u/ml). these data suggest that septicemia may lead to substantial changes in blood monocyte composition and this may be related to elevated levels of cytokines such as il-6. human peritoneal macrophages were exposed to increasing doses of lipopolysaccharide (lps) or a synthetic lipid a analogue (mrl 953) and production of the cytokines il-lb, il-6, tnf-a and g-csf was assessed at the protein-and mrnalevel. cells were also stimulated with low doses of lps and mrl 953 to study their "adaptation" to a secondary challenge with high doses of lps. tnf-a production after stimulation with lps could be almost completely relieved by preincubation of cells with low doses of lps and similarly, il-6 production was suppressed. surprisingly, however, "adapted" cells were able to synthesize even larger quantities of of g-csf and il-lb when exposed to a secondary lps challenge. the changes in tnf-a, il-6 and g-csf protein synthesis could a/so be detected on the mrna level, whereas transcript levels for il-lb remained unaltered as assessed by northern blot analysis. high doses of the synthetic lipid a analogue mrl 953 were also able to "adapt" macrophages for a secondary lps challenge by downregulating tnfa-and il-6-production, while priming secretion of g-csfand il-lb as well. taken together, here we describe for the first time the discordant adaptation of human peritoneal macrophages to a secondary lps stimulus in vitro. these findings appear to bear ramifications for the in-vivo endotoxin response during inflammation and gramnegative septicemia. shock states with inadequate cellular oxygen delivery may contribute to macrophage (mo) activation or dysregulation. in this study we compared the effect of transient hypoxia and endotoxin pretreatment (lps 1) on mo mediator release with a second endotoxin stimulus (lps2). methods: in vitro cultures of marine peritoneal exudate mo were exposed to 2 hr. of hypoxic or normoxic conditions, then incubated 22 hr under identical normoxic conditions _+ 10 ng/ml of lps 1 pretreatment. during the final 24 hours all m~l were exposed to a range of lps 2 concentrations. m~i supernatants were assayed for tnf, il-1, il-6, pge2, nitric oxide (no), and superoxide release. results: lps 1 markedly inhibited mo tnf release by lps 2 but hypoxia had no effect on lps 2-triggered tnf release. hypoxia increased mo il-6 production in the absence of lps 1, but inhibited lps 1 augmentation seen under normoxic conditions. pretreatment with lps 1 increased no production from lps 2 under normoxic conditions, but bypoxiainhibited this effect. pretreatment with lps1 signifcantly augmented mo release of il-i and pge 2, but hypoxia had no significant effect (data not shown) superoxide production was increased by lps 1 under normoxic conditions, but hypoxia significantly inhibited superoxide release (not shown a. lepape, c. docile, f. arpin, m. c. gutowski, v. banssillon and j. bienvenu. i aim of the study. increased levels of tnf are inconsistently correlated with the severity of sepsis. one possible explanation is a decrease ability of ceils to produce eytokines. the objectives of this study were m compare at different levels of of clinical severity the responsiveness of cytokine producing cells. this was evaluated by the response to a stimulation by lps as well as to the inhibitory effect of ptx. the technic used is a whole blood ex-vivo model, as described by desch et al. (1) . h materials and methods. different groups of icu patients, postoperative (n=12), sepsis (n=12), septick shock (n=13), and healthy control (n=10), were studied. blood was drawn in endotoxio free heparinized robes. stimulation was achieved by addition nf75 pl oflps (10ng/ml) to 675 id of whole blood in presence of various concentrations of ptx (0, 10 "5, 10 -4, 10 "3, i0"2m). after 5 hrs incubation at 37°c, the tubes were centrifuged and supematants frozen at -80°c. tnfa and/l6 were measured by elisa (medgenix r and immunotech r respectively). monocytes were quantified on a technicon h6000. the results are expressed as median values. non parametric tests are used for testing differences between groups. hi results, tnftx and 11,6, corrected for monocytes count, are given as % of their initial value, the baseline before stimulation or inhibition being 100%, 1) stimulation by lps. the control group is much more stimulated (>21000% for il6, >8000% for tnfa). blood samples taken postoperatively and in patients with simple sepsis are significantly less stimulated (>1500% for il6, >600% for tnfa ). the lowest stimulation was observed in patients with septic shock (median = 168%), some patients being not stimulated at all. 2)effects of ptx.the inhibitory effect of ptx on tnftx production is effective in all groups at 10 -3 m (reduction to less than '¼ of the median values), and is almost complete at 10" 2 m. the septic shock group has a decreased sensitivity to ptx. il6 production exhibits a lesser reduction at 10 -3 m (~ 'a to ½ of the median values), further increased at 10 -2 m. the septic shock group is again less sensitive to ptx. iv conclusion: the reduced ability of circulating monocytes to produce cytokines during severe infections is confirmed here. ptx is able to reduce significantly tnfc~ at 10 -3 m and the inhibition is nearly complete at 10 -2 m. surprisingly, there is a lesser, but significant suppressive action of ptx on il6, not found in experiments using purified monocytes. one possible explination could be the interplay between cytokines production. (1) lymphokine research (1989) cdna sequencing constitutes a powerful method of measuring steady-state mrna levels for all genes transcribed in a given cell or tissue at a particular stage of differentiation. by comparing transcript abundance both prior to and following differentiation, individual genes can be identified whose transcription is regulated both positively and negatively. in order to examine monocyte activation, the human monocyte line thp-1 was induced with phorbol ester (48 h) and activated for 4 h with lipopolysaccharide (lps) after which polya + rna was purified. the rna from control and lps-treated cells were each used to construct a cdna library under identical conditions, and all resulting clones were selected for cdna sequence analysis. each clone sequence was evaluated by matching with both genbank and our own gene databases. very different patterns of gene expression were seen in the two libraries, the latter reflecting very high levels of known inflammatory mediators such as il-1 and tnf. a second set of libraries were made from umbilical vein endothelial cells (huvec), both with and without lps stimulation, and were analyzed in a similar fashion. the effects of lps induction on specific gene transcription in both cell types will be discussed. t. tadros, md, th wobbes, me) phd, rja goris, md phd to investigate whether the preactivation of regional macrophuges by liposomes containing muramyl tripeptide (mtp-pe) can counteract the detrimental effect of blood transfusions on both anastomotic repair and host susceptibility to infections. methods eighty lewis rats received lmg/kg of either empty or mtp-pe encapsulated liposomes, intraperitoneally (ip). twenty-four hours thereafter, the animals underwent resection and anastomosis of both ileum and colon, and received 3 ml of either saline or blood from brown norway donors,iv. the animals were killed 3 or 7 days after surgery and examined for septic complications and anastomotic repair. the average anastomotic strength, as assessed by bursting pressure (+sd), was significantly diminished in the transfused animals, as compared to the non-transfused animals (ileum;day 3; 44-+8 vs 99+7, p<0.001). transfused animals pretreated with mtp-pe encapsulated liposomes showed a significant improvement of their anastomotic bursting pressure (93+9, p<0.001 vs transfusion). pretreatment with mtp-pe encapsulated liposomes decreased significantly the incidence of anastomotic abscesses in transfused animals ( from 90% in ileum on day 3 to 20%, p<0.001). conclusions preactivation of regional macrophges by intraperitoneal administration of mtp-pe encapsulated liposomes prevents the detrimental effects of transfusions on anastomotic repair and reduces the incidence of intraabdominal sepsis. academic hospital nijmegen, dept of general surgery, pb 910 i, 6500 hb nijmegen, the netherlands. leukemia cell line, teip-1. robin s. wa, gner*, perry v. halushka "~, and james a. cook*, departments of physiology , pharmacology "l" and medicine "t, medical university of south carolina, charleston, s.c. 29425. adherence of monoeytes to endothelium and extracella/ar matrix proteins is essential for accumulation at sites of inflammation. txa2, an arachidonic acid metabolite, inhibits human monocyte chemotactic responses suggesting that txa 2 may alter monocyte adhesiveness. we selected the thp-1 cell line, a human monocytic leukemia cell line to further investigate the effect of txa 2 on adhesion. we tested the hypothesis that txa 2 alters lpsinduced adhesion of thp-1 cells and that txa 2 exerts its effect on adhesion via a camp dependent mechanism. thp-i cells were exposed to s. enteritidis endotoxin (lp.g/ml) _+ the cyelooxygenase inhibitor lndomethacin (in), the txa 2 mimetic i-bop (0.11.tm,) or txa 2 receptor antagonists bms180291 and l657925 (10~m). cells were allowed to adhere for 24 hours and adherent protein/well was determined. lps-induced a significant (p<0.05;n=7) increase in adherence of thp-1 cells (basal, 2.4+0.85gg protein/well; lps, 20.4+_6.0p.g protein/well). the amino acid glutamine is an essential compound for synthesis of purine and pyrimidine basis and therefore necessary for rna-and dna synthesis. in human plasma the concentration of glutamine is between 0.5 -0.6 mm, and is reduced in septic patients up to 80 % (0.1 -0.2 mm). monocytes play a central part in the inunune system and it was of interest, whether glutamine is involved in the modulation of cell surface markers and phagocytosis of these cells. human peripheral blood mononuclear ceils were obtained from 500 ml heparinized blood of apparently healthy donors by ficoll-paque density gradient and isolated by counterflow elutriation. the puritiy was more than 90 %. subsequently cells were cultured in phenolred-free rpmi 1640 medium with various concentrations of glutamine (0.05, 0.1, 0.2, 0.3, 0.6, 1, 2 mm) in teflon-fluorinated ethylene propylene bottles to exclude cell adhesion and possible cell activation. aider seven days culture, cell viabilty was determined by trepan blue exclusion and varied between 70 and 90 %, independent of glutamine concentrations. cell surface markers were detected by flow cytometry, noaspecifie phagoeytosis was measured with latex beads and specific phagocytosis with opsonizied e.eoli using a facscan. lower concentrations of glutamine decreased the expression of hla-dr and icam-1/cd54 on monocytes in a dose-dependent manner. the receptor for fc'/rucd64 as well as the receptors for complement cr3/cdllb and cr4/cdllc were down-regulated. cr1/cd35 which is only slightly expressed on monocytes was not influenced. furthermore, no effects on the expression of cdi4, the receptor for transferrin cd71 and fc'friii/cd16 were seen. our data indicate, that lower concentrations of glutamme influence the phenotype of monocytes. we are now interested to study whether glutmnine influences non-specific phagocytosis, or whether specific phagocytosis correlates with the decreased expression of fc'/r and complement receptors. we investigated immunologically more than 300 patients who were admitted to icu because septic syndrom during the last four years. patients were immunologically followed up 2-3 times per week until release from icu. the expression of hla-dr antigen on monocytes turned out to be the best prognostic parameter. the persistence (> 5 days) of low hla-dr expression (< 30%) predicts fatal outcome (mortality > 85%). the altered phenotype was associated with a functional deactivation of monocytes (diminished apc, ros formation, cytokine secretion). we called this phenomenon "immunoparalysis". ifn-gamma and gm-csf were able to restore the altered phenotype and function in vitro. however, addition of autologous plasma from septic patients with "immunoparalysis" to these cultures prevented the cytokine-induced restitution. the inhibitory activity could not be removed by dialysis. therefore, we started a study to prove the therapeutic efficacy of plasmapheresis. indeed, [7 of 33 patients recovered from "immunoparalysis" following repeated plasmapheres; 16 of them survived (46 %). 7 patients recovered temporarely and 9 patients did not respond (all 16 died). the survival rate in the control group of septic patients with persistent "immunoparalysis" was 4 of 38 (11%; p<0,01). in summary, plasmapheresis in association with immune monitoring may be an alternative strategy to improve survival rate in severe sepsis. taurolidine, a synthetic taurine-formaldehyde derivative has antiadherent, bactericidal and anti-lps properties functioning primarily through binding of the lipid a region of the lps molecule. the active derivative of taurolidine, taurine, modulates calcium channel activity, critical to the initiation of a number of immunostimulatory pathways. we hypothesised that taurolidine may have direct immunostimulatory activity. the aim of this study was to investigate the immune effects of taurolidine on peritoneal macrophage (pmo) function and then determine the role of taurine in this response. study 1: in vivo stimulation:cd-1 mice (n=35) were randomized to receive taurolidine (200mg/kg bw/i.p.) or saline cor~trol. peritoneai cells were harvested after 24 hours and were assessed for pm0 function [superoxide anion generation (o2-), nitric oxide (no), tumor necrosis factor (tnf), fc/cr3-mediated phagocytic function (phago) study 2: control pm0 were harvested and cultured in vitro with taurine (1.0mg/ml for 10 hrs), after which time they were assayed for 0 2-and tnf release. in vivo stimulation with taurolidine taurolidine has specific immunological effects on m0. release of the inflammatory mediators 0 2-and tnf, and fc/cr3-mediated phagocytosis were significantly increased, while release of the endothelial relaxing factor no was significantly reduced. in addition, the amino acid taurine, which is released as a byproduct of taurolidines breakdown has an immunostimulatory effect on pmo and may be the active moeity of the compound tanrolidine. in sepsis, a number of mediators which affect vasomotor tone and cardiovascular function are produced. inasmuch as sepsis causes decrease in systemic vascular resistance (svr), attention is usually focussed on vasodilators such as lactate, tumor necrosis factor, interleukin-i & 2, and nitric oxide. but injury and inflammation als0 cause production of several vasoconstrictors whose effect may not be evident in changed svr, but may significantly affect organ blood flow or function in the paracrine environment. endothelin (et) is a 21 amino acid peptide vasoconstrictor produced by ischemic or injured endothelial cells (ec's). et is also a potent constrictor for renal mesangial and coronary vessels, an endocrine regulator, and a negative cardiac inotrope. systemic et levels increase significantly in hypoperfusion and ischemia. while et is principally produced by ec's, we asked if human monocytes might also produce et and thereby regulate vasomotor tone in areas of inflammation. monocytes from healthy donors were separated on ficoll, resuspended in rpmi 1640 +5% fetal calf serum and stimulated with i0 ug/ml endotoxin (lps). et was measured by radioimmunoassay. lps-stimulated monocytes produced 87 ! 6 fm of et/106 cells (vs. unstimulated controls of <25). this calculates to 15-30% of the amount of et observed in patients with low cardiac output, sepsis or ischemia. we conclude that et is a cytokine produced by both ec's and monocytes with potent effects on numerous cells and organs in the critically ill. wuppertal 5, germany we and other authors showed that fatal outcome in septic disease is associated with a decreased capacity of peripheral blood monocytes for the in vitro production of proinflammatory cytokines, especially tnf-alpha. we found that this monocytic deactivation is completed by a persistent and marked decrease of hla-dr expression on monocytes (< 30 % hla-dr+ monocytes) and a diminished antigen presenting activity whereas the capacity to form the antiinflammatory il-1 receptor antagonist remains high. in order to evaluate the in vivo situation and to determine at which level tnfproduction/secretion is altered we assessed the tnf-alpha mrna expression in freshly isolated peripheral blood mononuclear cells (pbmnc) from septic patients. tnf-mrna was onty rarely detected by semiqaantitative polymerase chain reaction in pbmnc's from septic patients with monocyte deactivation. meanwhile, it was found in almost all pbmncs from septic patients without monocytic deactivation. we wondered, whether il-i0, which ,is known to depress monocytic proinflammatoly response and mhc class ii expression, could be one of the mediators in fatal sepsis. in fact, we found that il-10 message in pbmncs of septic patients peaked in the beginning phase of monocytic deactivation. in further investigations we found that tnf-administration can induce monocytic deactivation in a murine model/n vivo and provoke il-10 message in human pbmncs in vitro. these results support our hypothesis that an excessive delivery of proinflammatory cytokines in a first phase can induce an overwheiming inhibitory feedback, mediated by immuninhibitory mediators like il-l0, which leads to often fatal monocytic deactivation in a second phase. interferon-gamma which is known to counteract il-10 production and the effects of il-10 on monocytes restores the function and phenotype of monocytes from septic patients with monoq, te deactivation in vitro and could be a possible therapeutic agent in otherwise fatal sepsis. our laboratory previously reported that lps dependent macrophagederived tnf-a production can be enhanced by pretreatment with lps at substimulatory lps priming doses coincident with a suppression of lps dependent nitric oxide (no) production (zhang and morrison, j. exp. med 17:511, 1993) . in order to extend the characterization of these lps priming effects in mouse macrophages, we examined the capacity of substimulatory lps to modify lps dependent il-6 production. macrophages were obtained from peritoneal exudate of thioglycollate treated c3heb/fej mice and cultured in rpmi 1640 medium containing 10% fetal bovine serum. macrophages were pretreated with various subthreshold stimulatory concentrations of lps (olll:b4) for 6 hours, washed three times, and then stimulated with the effective stimulatory concentration of lps for 18 hours. the amount of il-6 in the supernatant was measured by il-6 dependent cell line (b9 and 7td1) proliferation assay. il-6 was produced by macrophages at lower threshold doses of lps than those required for tnf-o~ or no production. subthreshold doses of lps modulated il-6 production in a biphasic manner characterized by an initial suppression and then potentiation. higher doses resulted in secretion of il-6 during the initial incubation with lps and subsequent desensitization. il-6, like tnf-~ and no, is, therefore, also affected by lps pretreatment. moreover, tnf-a and il-6 shared the similar potentiational pathway, but differed by the fact that only il-6 was inhibited. (supported by r37 ai23447 and po1 a54474.) department of microbiology, molecular genetics and immunology and the cancer center, 1000 wahl east, university of kansas medical center, kansas city, ks 66160-7832. korolenko t.,urazgaliev k.,and arkhipov s. the role of macrophage (mph) stimulation in mechanism of protective effect of new immunomodulators yeast polysaccharides -heteropolysaccharide cryelan and homopolysaccharide mannan rhodexman (both produced by petersburg chem.-pharm. inst.) was studied. in vitro according to nst test incubation of murine peritoneal mphs with cryelan or rhodexman, 150 ~g/ml,30 min was followed by increase of potencial microbicidic activity of mphs. in vivo mph stimulation by immunomodulators studied included increase rate of carbon particles phagocytosis during single i.v. or i.p. mode of administration to mice 1-14 days after (peak at 2nd day for i.v. and 5th day for i.p. mode of administration of the same dose of 5 mg/100 g b.w.).the preliminary injection of cryelan (5 mg/100 g, 24 or 48 h before) to mice with acute cold stress (-10 ° c, 1 h) revealed protective effect restorating the value of depressed phagocytosis up to the normal level;the positive effect on ultrastructure of hepatocytes was noted also.there was no changes of plasma corticosterone level between group with acute cold stress and mice with cryelan + acute cold stress (several fold increase comparatively to the control mice).as was suggested, the mechanism of protection can include mph stimulation and secretion of some acute phase proteins responsible for positive effect of immunomodulators. new yeast polysaccharides cryelan and rhodexman can be used for macrophage stimulation,especially in pathological states. immunomodulators were shown to increase production and secretion of lysosomal enzymes (like zymosan). secreted enzymes,especially cysteine proteinasescathepsins b and l -involve in the process of inflammation;however, excessive release of these enzymes may lead to noncontrolled proteolysis followed by tissue degradation (assfalg-machleidt et al.,1990) .the effect of zymosan,bcg and new immunomodulator carboxymethylglucan (cmg), second fraction on secretion of lysosomal enzymes by murine peritoneal macrophages was studied. zymosan increased the secretion of n-acetyl-~-d-glucosaminidase and ~-galactosidase into the culture medium (3-4 fold); bcg possessed similar effect.cmg in the same concentrations (50 /~g/ml) increased release of these enzymes only saightly (1.6 times).it's known that zymosan-induced secretion reflects the enzyme release from formed lysosomes (warren,1989) .it was suggested that cmg activated macrophages via interaction with scavenger-receptors,followed by weak secretion of lysosomal enzymes and as a result decrease of tissue damage. in vivo zymosan induced stimulation of mononuclear system of phagocytes followed by increase of cysteine proteinases activity in liver at the 5th day. in the same time in blood n-acetyl-~-d-glucosaminidase and n-acetyl-~-d-galactosidase activity increased 2-5 fold. it was concluded that in drug design it's possible to select such immunomodulators,e.g. cmg,which can activate mononuclear system of phagocytes and do not damage tissue. endothelin-i (et-i) is produced by injured/ ischemic endothelium, mobilizes intracellular ca ++ and is a potent vasoconstrictor. it is also a ca ++ agonist for anterior pituitary or renal mesangial cells and monocytes. et-i causes monocytes to produce interleukin-l, 6, 8, prostaglandin e2, and substances which trigger neutrophil superoxide production. et-i levels increase in shock and et may play a role in activating leukocytes post shock causing reperfusion injury. but blood flow experiments suggest splanchnic circulation changes more profoundly in shock than peripheral circulation. we therefore asked if et-4 (or vic), the et which predominates in splanchnic vessels, had any effect on monocyte cytokine production. human monocytes from health~ blood donors were separated on ficoll. 0.5 x 10ucells/ ml in rpmi 1640 + 10% fcs were incubated i0 min., 6 & 24 hrs. with 10 -8 m et-i, 10 -8 m vic or i0 ug/ml of lps. supernatants were assayed by elisa. we have shown that low dose endotoxin pretreatment (lps 1 ) for 24 hrs markedly inhibits the macrophage (mo) release of tumor necrosis factor (tnf) and increases interleukin-1 (il-i) in response to a subsequent endotoxin stimulus (lps2). in this study we examined the kinetics of lps 1 inhibition of tnf and augmentation ofil-1. methods: murine peritoneal exudate mo from balbc mice were exposed in vitro to medium or 10 ng/ml of lps1 for intervals of 0 to 24 hours. culture medium was then replaced with 0, 10 or 1000 ng/ml of lps2 for 24 hrs. tnf and il-1 in mo supernatants were measured by specific bioassays. during sepsis endotoxin (lps) activates macrophages (mo) to release mediators such as tumor necrosis factor (tnf), interleukin-6 (il-6), interleukin-i (il-i) and prostaglandin e2 (pge2). we showed that preexposure to lps (lps 1) alters the response of murine m~i to subsequent lps stimulation (lps2). we hypothesized that in vitro cytokine release by lps2 in human monocytes (mo) is also be altered by preexposure to lpsi. methods: human peripheral blood mo were obtained from healthy volunteers (n=8), cultured in vitro 24 hrs, then pretreated 24 hr _+ lps 1-cultures were then stimulated with lps 2 and mediators in mo supernatant measured: tnf, il-i, and il-6 by specific bioassays, pge2 by immunoassay kit. serum cytokine levels (specific elisa kits) were compared to in vitro supernatant levels. data is expressed as % control_+sem, lps2= 1000 ng/mh the table shows that all mediators were increased, in the absence of lps 1. pretreatment with lps 1 resulted in complete inhibition of lps2-triggered tnf release. in contrast, lps 1 significantly increased mo secretion of il-1, il-6 and pge 2 (data not shown). serum cytokine levels were as follows: tnf 407_+21, il-i 241+208, and il-6 8.0-+2.5 ng/ml. these serum levels were low, showed an extremely wide variation, and did not correlate with in vitro lps2-triggered mediator production. conclusion: human monoeyte mediator production is differentially regulated by preexposure to lps 1. provocative in vitro testing of monocytes may ultimately be clinically useful to identify prior in vivo lps exposure or mo macrophages release numerous secretory products involved in host defense and inflammation. activated macrophages with cytokines produced have been implicated in tissue damage in sepsis and multiple organ dysfunction. aimed to elucidate the organ-association phenomena,this study is to compare peritoneal macrophage(pm),alveolar macrophage(am), and kupffer cells(kc) during sepsis in terms of cellular protein contents as symbol of activation by flow cytometry analysis. sepsis were produced by cecal ligatien and perforation (clp) in wistar rats weighing 210-290g.pm were obtained by peritoneal lavage,am by bronchial lavage and kc by incubating the collegenase digested liver with pronase-e. leukocytes have been implicated as a mediator of the microvascular dysfunction associated with reperfasion of ischemic tissues. a role for ieukocytes is largely based on observations that rendering animals anutropenic with anti-neutrophil serum or preventing leukocyte adhesion with monoclonal antibodies attenuates the increased fluid and protein leakage from the vaseulature that is normally observed in postischemic tissues. we have recently undertaken studies designed to determine the relationship between leukocyte-endothelial cell adhesion and albumin leakage ia rat mesenterlc venules exposed ~o ischemia-reperfusion (i/r). leukocyte adherence and emigration as well as albumin extravasafion were monitored in single postcapillary venules using iatravital fluorescence microscopy, lschemia was induced by complete occ!usion of the superior mesenteric artery and ~dl parameters were monitored at various intervals following reperfusion. the magnitude of the leukocyte adherence and emigration, and albumin leakage elicited by i/r was positively con-elated with the duration of ischemia. the albumin leakage response was also highly correlated with the number of adherent and emigrated leukocytes. monoclonal antibodies against the adhesion glycoproteins cd18, cdllb, icam-1 and l-selectin, but not p-or e-selecdn, reduced i/r-induced leukocyte adherence and emigration as well as albumin leakage. phauoidln, an f-aetin stabilizer, largely prevented the emigration (but not adherence) of leukocytes and greatly reduced, the raicrovascular protein leakage. plateletleukocyte aggregates were formed in postischemic vemdes; the number of aggregates was reduced by antibodies against p-selecdh, cdilb, cd18, and icam-1, but not e-selectin or lselectin. a significant fraction of the mast ceils surrounding the posteapillary venules degranulated in response to ischemia/repeffusion, but mast cell stabilizers did not afford protection against the albumin leakage elicited by i/r. these results indicate that reperfusloninduced albumin leakage is tightly coupled to the adherence and emigration of leukocytes in posteapillary venules. this adhesiomdependent injury response is primarily mediated by cdllb/cdi8 on activated neutrophils and icam-1 on venular endothellum, and appears to require l-selecda dependent leukocyte rolling. mast cell degranulation does not appear to conwibate to the vascular pathology associated with i/r. m.d. rod=iek, boston, ma, usa the polymorphonuclear neutrophil (pmn) has long been known to pa~tlcipats in the inflammatory rebpons~ as a phagocyte and killer of invading organisms, but little attention has been given to its potential as a participant in the in~une interaction of lymphocytes and macrophages. we and others have shown that the pmn may have i~m~/nomcdulatory effects both in vitro and in vlvo. more recently it has been proven that the pmn can make mrna for and secrete the proinflammatory oytokines illa, il-ib, tnfs, il-6 and il-8 as does the other major circulating phagocyte, the monocyte/macrophags. furthermore it has been shown to make the potentially autoregulatory oytokines gcsf and gmcsf. these functional capabilities suggest that the pmn is not an wend cell ~, but one which has a potential role in regulation cf ~he immune response and that this potential ~cle should no longer be ignored when considering the immune abnormalities existing in patients following majo~ injury or surgery. we have investigated the proinflaznmatory oytokine secretion patter~ by pmn in patients following major ~hermal or tra~matic injury and in volunteers fellowinq endotoxemia. ?ollowing major injury there is variable pmn secretion of these cytokines when stimulated in vlero. following endotoxemia in a group of human volunteers pmn showed a hypo=esponsivenesa to lps 4 hrs following endotoxin infusion followed at 24 hre by an overshoot. pretreatment with steroids modulated this overshoot phenomenon, suggesting that receptors for steroids are involved in the regulation of cytokin® secretlon by fmn. these results sugges~ that the pmn, the most numerous cell in the circulation and the first to respond to an ins~l~ may be a so~rce of the prolnflammatory cytokine cascade following injury that has been recognized as significant in the process which often leads to multiple o;gan failure, the immunosuppresslon which occurs following major thermal injury may predispose these individuals to infection and sepsis, which remain a significant cause of morbidity and mortality. included among the many immune aheratlons are the p2 integrln (cdlla, b,c/cd18) dependent activities of adhesion, chemotaxls, diapodesls, and phagocytosls. our investigations indicate that, following major thermal injuries, the expression of the [~2 integrlns, but not cd14, is significantly decreased on neutrophlls (pmns). it remains unclear if pmns from thermally injured patients respond normally to lps, the effects of treatment in vitro with lps and f-met-leu-phe (fmlp) on the expression of cdtlb was examlned on pmns from the peripheral blood of healthy volunteers and non-septic burn patients (>30~; total body surface area, >ls~ full thickness), the pmns were incubated with lps (]ng-10p.g/ml) or f'mlp (10 "1 1 to 10"6m) et 37oc for 30 mln, in 1~; human ab serum, the expression of the 132 ]ntegrins was detected using monoclonat antibodies and flow cytometry. lps and f'mlp resulted in a slight increase (3 fold) in the expression of cd11b on pmns from burned patients compared to an 8 and 14 fold increase, respectively, on pmns from healthy individuals. this inability of lps or fmlp to increase cd11 b expression was not due to the amount of lps bound to the two cell populations. because the same defect is seen after either lps or fmlp stimulation, it is speculated that the defect must be in the amount of preformed cd1 ] b or its transport to the plasma membrane. platelet-activating factor (paf) and neutrophils have been implicated in the patbophysiology of ischemia-repeffusion injury, in addition, paf stimulates neutrophi[ (pmn) oxidative metabolism in vitro. the present study examined the potential role of paf in repeffusion injury in an in viva rabbit model. eight anesthetized rabbi~s underwent retroperitoneal exposure of the infrarenal abdominal aorta after percutaneous insertion of a catheter through the jugular vein into the infrahepatic inferior vena cava. doppler flow probes were placed around the abdominal aorta and the right common femoral artery to assess flow through these vessels. an occlusive ligature was placed around the abdominal aorta (superior to the flow probe) at t = 0 and total occlusion of blood flow to the lower extremities was maintained for g0 mins., after which the ligature was released allowing for reperfusion of the ischemic lower limbs. effluent blood from the ischemic hind-limbs was collected through the ivc catheter at the times indicated below and assayed for paf by a direct radioimmunoassay. in addition, neutrophil h202 production was determined by a previously described 2'7'-dichlorofluorescein flowcytametric assay. 185 _+ 53 amean _+ s.e.m, pg/ml blood; brelative fluoresenee (% of baseline); caortic and femoral artery flow (% of baseline); *p < 0.01 vs. baseline; 1"p < 0.05 vs. baseline. a significant elevation of paf was observed in ischemic hind-limb effluent blood at 15 min. after release of the aortic ligature during the repeffusion phase, as compared to baseline levels. in addition, pmn h20 2 production was increased by 2.3-fold above baseline values by 1 hour after ligature release during the reperfusion phase. both of these elevations were transient and returned toward baseline by 2 hours post-isehemia. tatar occlusion of hind-limb flow was achieved as evidenced by the absence of aortic or femorat flow at 90 rain. post-ischemia, however after release 01 the ligature a significant reactive hyperemia was observed by 15 mln. into the rapeffusion phase. histolog[c examination of reper[used gastrocnemius muscle revealed moderate pmn infiltration into the interstitium. in conclusion, these data indicate that paf is released into the circulation during repeffusion, and is likely involved as a mediator in the observed pmn oxidative burst activity, thereby contributing to reperfusien injury. following thermal injury and infection granulocyte function ts abnormal. to elucidate the mechanism by which thermal injury and infection affect the granulocyte's ability to polymerize and depolymedze actin, we serially measured f-actin levels in granulocytes from 14 burned patients (mean age 42,1 +_ 17.7 years, mean burn size 39.8% _+ 22.2%) during the first s weeks post injury. six of the patients had infections during the course of the study, (septicemia, wound invasion and pneumonia). actin levels in granulocytes from eleven healthy volunteers (mean age 34 years) were measured repeatedly and served as controls. lysecl white blood cell preparations were brought to 370c and incubated with n-formyl-met-leu-phe (stim) or with dulbecco's phosphate unbuffered sellne (unstim). the cells were concomitantly stained and fixed with formaldehyde, lysoleclthln and fiuoresceln phafioidin. actin depolymedzation (depol) was measured by incubating stimulated cells at 0°c before the stain-fixative was added. baseline (base) f-actln levels were assessed by adding stsln-fixatlve to icecold unstimulated cells. fluorescence was estimated in a facscan and expressed as ilnesr mean channel fluorescence_+ sem (mcf). figure 1 displays granulecyle fectln levels in infected and uninfected patients as compared to controls. f-actln levels were consistently lower in control cells than in those from burned or burn-infected patients under all measured conditions. granulocytes from infected burned patients demonstrated a significant decrement in their ability to depofymerlze f.actin compared to both uninfected burned patients and controls, while there were no significant differences between infected and 1,~ uninfected patients in the baseline, unstlmuleted and stimulated conditions. those results indicate la5 that grsnulocytas from burned and bum-infected patients contain higher levels of polymerized actln than ~ ,2s control cells. in order to study tumor necrosis factor (tnf) receptor sensitivity in septic critically ill patients we investigated 20 blood samples of such people in reaction of leucocyte migration inhibition. migration of their polymorphonuclear leucocytes (pmns) was studied with stimulation with human recombinant tnf in concentration of 166.7 u/ml (recommended by manufacturer is the range of 10-200 o/ml) and without such. ten healthy blood donors formed control group. the results obtained showed diminished pmn reactivity to tnf in patients (migration inhibition was absent) oscaring with significantly increased migration ability of their pmns (107.2% of that in control group). at the same time normal pmns in control group did show migration changes upon tnf stimulation. considering all the above we come to a conclusion that externally added tnf fails to activate pmns in critically ill patients more than they are by their endogenous tnf. moreover, this tnf no longer serves a positive chemotactic factor for such pmns. these findings may suggest that in critically ill septic patients reactivity of pmns to tnf is deeply altered. tnf receptors of pmns are either exhausted as such by excessive stimulation with endogenous tnf or further transmission of their message is impossible due to "fatigue" of the cell's activation mechanisms. we express our gratitude to reanal factory of laboratory chemicals for generously providing us with a tnf com~rcial sample. ~-sanguis medical, ekaterineburg russia; s-urals med.lnst. activated neutrophils infiltrating the local site of inflammation following trauma release high amounts of destructive lysosomal enzymes into the extracellular space. cytokines were discussed to be involved in regulation of this early process. the task of this investigation was to evaluate the possible regulatory role of interleukin-6 (il-6) and its potential immunosupressive opponent, the transforming growth factor-&, in regulation of neutrophil degranulation. we analysed the concentration of the al-proteinase-inhibitor complex of the lysosomal elastase as marker for the degranulation of neutrophils as well as the levels of il-6 and tgf-61 in the plasma probes of patients undergoing multiple trauma and severe surgeries. the time courses of il-6 and elastase were found to be highly correlated, wheras the concentrations of the cytokine tgf-e~ were found to be not significantly altered in comparison to the control group. this close temporal correlationship was confirmed by investigation of fluids derived from sites of inflammation. interstingly, the inhibitory potential (~zcproteinase inhibitor, antithrombin iii) was dramatically reduced in the early inflammatory phase. to prove this in vivo findings, the effects of il-6 and tgf-i~ 1 on the degranulation of isolated human neutrophils of healthy donors was investigated in vitro. pathological high concentrations of rhll-6 up to 104u/ml (as detected in fluids derived from local inflammatory site) were found to be capable to induce a significant release of lysosomal elastase in a concentration-dependent manner, whereas the degranulation of neutrophils was uneffected by tgf-61. in conclusion, these data suggest a contribution of il-6 in regulation of neutrophil activation at sides of inflammation. the immunosuppressive cytokine tgf-i&~ seems to have no direct regulatory effect beside its described chemotactic function on neutrephils. postirradiation chan~es of adhesive properties arid supercoiled nucleoid dna structure of blood leukocytes were studied in macaca nemestrina andrats. the dynamics of membrane chan~es after nonlethal irradiation of rats demonstrated the temporary increase of the leukocyte adherence at 24 h followed by return of this parameter to normal levels at 48 h. after lethal irradiation of both animal species the increase in adhesive leukooytes fraction was detected as early as at 3 h. this hi~her index persisted until the end of experiments (5 days). the early (3-5h) temporary loosin~ of supercoiled dna structure was demonstrated in the leukocytes of nonlethally irradiated animals. this phenomenon seems to be connected with the lymphocyte fraction chan~es. this process was not dependent on altered adhesive properties of leukocyte membranes. the membrane chan~es of leukocytes preceded decondensation of supercoiled dna after lethal irradiation of animals, in this case loosin~ of supercoiled dna pro-~ressively increased at 24 h and at the later terms of postirradiation period. the systemic inflammatory response syndrome (sirs) involves many inanunological reactions of the host including acfivatinn of inflammatory mediator cascades and depression of cellular reactivity in t-lymphecytes (1). there are reports of nentrophil dysfunction in inflammatory disorders of the skin (2), are there dysfunctions concerning the unspecific host defense in sirs, as well? in this study, we examined the reactivity of neutrophil granolocytes from patients suffering from sirs. twenty-one patients (apache ii-score 21±5) with diagnosis of sirs entered the study. granulocytes were prepared as reported previously (2) . in parallel, granulocytes from 20 healthy individuals were tested. two granulocyte functians were studied in vitro: 1. migration of the ceils in a boyden chamber through a filter matrix following stimulation with 5 different receptor dependent stimuli (c5a, intefleukin-8, platelet-activating-factor, leukotrien b4, fmlp). 2. release of 13glucuronidase following stimulation with the aforementioned activators. the results demonstrate, that the release of 13-glucuronidase in patients suffering from sirs was comparable to the enzyme release of granulocytes prepared from healthy individuals. each stimulant induced release of p-glucuronidase in a characteristic dose dependent fashion. all granulocyte preparations from the healthy donors showed a positive chemotaxis response in the migration-assay. in contrast, only ten out of twenty-one patients had granulocytes migrating after stimulation. the two groups of patients displaying reactive or non-reactive granulocytes differed clinically: the nonreactive group consisted of patients with multiple organ failure (10/11) and nonsurvivors (8/11), whereas 8/10 patients in the reactive group survived. thus, the in vitro chemotaxis of granulocytes is impaired in a subgroup of patients with sirs. this defect of the non-specific host defense may contribute to poor prognosis and outcome of these patients. dermatol. 85: 194-198, 1985 klinik ffir an~isthesiologie und operative intensivmedizin der cau kiel, schwanenweg 21, 24105 kiel, germany. objectives of the study: major emphasis has been given to the analysis of interactions of antibiotics with microorganisms. effects of antibiotics on cells of primary host defense mechanisms, such as the neutrophils, are less well known. therefore, attention has been focused on clindamycin, a member of the lincoseamide family. materials and methods: the effect of clindamycin (i -i00 ~g/ml) on 8 granulocyte functions (healthy volunteers) such as random migration, chemotaxis (agarose method), ingestion (radiometric assay), superoxide (cytochrom c reduction) and hydrogen peroxide production (phenol red oxidation), lucigenin-and luminol-amplified chemiluminescence (luminometry) and degranulation (turbidometry with micrococcus lysodeicticus) were investigated in vitro. results: motility and degranulation were inhibited, ingestion of saccharomyces cerevisiae, zymosan-induced lucigenin-and luminol-amplified chemiluminescence, superoxide and hydrogen peroxide production were stimulated in a dose dependent fashion. conclusion: clindamycin has granulocyte function modulating properties. recognition of immunomodulating effects of antibiotics may have therapeutic significance, especially in patients with long-term antibiotic therapy or immune deficiencies. the intense muscle activity (ea) of rats resulted in increase of neutrophil influx in muscles during the recovery. we investigated neutrophil proteinases involvement in neutral proteinases balance of skeletal muscles by na. the rats were submited to swim with the load (8% of body mass) till exhaustion. immediately after na the neutrophil antiserum was injected i.p. to rats of experimental group. saline was injected to control animals° injections were repeated in 12 h of the recovery and cytosol proteolytic activity (ph 7.4; fitc-casein) was determined. isolated soleus muscles were incubated also in vitro and proteolytic activity of incubation media was measured. it was found that there was 2-3 -fold proteinases activity increase in cytosols of all investigated muscles (soleus, white and red portions of quadriceps) of control animals by 6 h of the recovery (the comparison was done with the sedentary rats). in 12 h cytosol proteolytic activity decreased and then increased again by 24 h of the fast. antiserum injections resulted in relible decrease of the proteolytic activities at every investigated time. when incubating m. soleus in vitro the activities of proteinases in incubation media turned out reliably less if soleus muscles were isolated from the animals to which antiserum was injected. the conclusion is that neutrophil proteinases can be involved in the balance of rat skeletal muscle neutral proteinases after ~a. a lot and new clinical problems complicating the outcome of polytrauma, burn and septic patients in surgical intensive care units, have arisen as the care improvement prolonged the patient's survival: a progressive degradation of organ and system functions often develops, usually making its first clinical appearance by ards, followed by the other organ failure (mof) and sepsis symptoms. the clinical picture is polymorphic, the end result of a complex systemic pathophysiological reaction trigg~ed off by trauma consequences (tissues disruption, hypo~xygenatiun and necrosis). nowadays there is not a preventi~ or specific therapy to lower the mortality rate (70-80%) and-'mdy-a~ early, aggressive surgical approach .-evacuating haematomas, stopping bleeding, toileting all septic, necrotic foci and restoring anatomic continuity-, seems to be of some help this complex clinical entity has not an univocal denomination yet. the proper labelling of an illness should come from the full understanding of its pathopysiology and suggest the proper treatment choice. clinical and experimental studies demonstrated that pathophysiologic mechanisms involved in the past-traumatic illness, share the same anatomo-pathological elemem: the interstitial edema, due to a generalised endothelial micro circulatory injury. this alteration, as constantly seen in polytrauma patients, develops in a few hours after trauma as a consequence of the deregulation of the homoeostatic and immune mechanisms. in fact the overproduced oxygen free radicals and r~ombinam cytokines (il1,tnf), together with the complement degradation fragments, the proteolytic enzymes and many other mediators are all strongly h~l ~ ,_he e,,j,yheha! ceils. our~osect, atim~,-bnsed on examination of autopsical specimens from 85 polytraanm patients, showed that such endothelial damage, supporting the interstitial edema, is widely and simultaneensly distributed, ensues shortly arer trauma and shows its effects in different organs at different times, only because each apparatus has different fimctienal reserves: the lung is the first organ to fail just because its ah, celocapillary membrane is one of the most delicate bodily structure, and its function is irroplace~le. we think it will be of a great help, in planning a preventive therapy, to chose a denomination focusing the physician's attention on the earl)" generalized endothelial injury and its effects, as in trauma patients it is present -even if latenflysince the first few hours. we would like to see the generalised endothelial microcircolatory injury properly highlighted when considering the best definition and the optimal nomenclature for the post-traumatic s3mdrome. the presence of interleukin (il)-8 in bronchoalveolar lavage fluid of critically ill patients correlates clinically with the development of the adult respiratory distress syndrome lards), and inhibition of il-8 in animal models can attenuate lung injury. collectively, evidence to date suggests that il-8 attracts and activates neutrophiis (pmn), which are then responsible for the capillary leak of ards. however, an alternative explanation is that il-8 is directly toxic to the endothelial cell (ec). in this study, we have hypothesized that il-8 can disrupt endothelial integrity independent of pmn. meth ods: human umbilical vein (huv) ec monolayers were cultured to confluency on collagen-coated micropore filters. to assess ec integrity, 1251.albumi n leak was quantitated by measuring the 1251 counts which crossed the monolayer, using a gamma counter. il-8 (lpg/ml) was incubated in the culture medium with 1251.albumi n for 21 hrs. the il-8 dose was not cytotoxic. to determine the involvement of protein synthesis in this process, selected monolayers were pretreated with cycloheximide (ch) prior to 11.-8 addition. statistical analysis was performed using anovmfisher plsd. we have previously shown that platelet activating factor (paf) enhances cdt8 expression and primes pmn's for subsequent 02generation. both are important steps in pmn mediated injury and are assumed to occur in concert. following major trauma non-specific pmn inflammation is activated, however, unbridled systemic pmn activity needs to be minimized. since circulating catecholamines are high early post-injury, we hypothesised that they downregu/ate cd 18 expression and pmn priming via the [3 adrenergic signal transduction pathway. methods: normal human pmns were primed with paf (10ng/ml for 5 min) or pre-treated with 10-5m of isoproterenol (i) or forskoklin (f) for 5 rain and then primed with paf. cd18 expression was measured by flow cytometry (fig.l) and 02-generation in response to 10-rm fmlp was determined as sod inhibitable reduction of cytochrome c ( fig.2 holler** and georg w. bornkamm* lymphocyte-endothelial interactions are crucial for various immune responses, including cytokine driven inflammatory processes. protein kinase c (pkc)-inhibitors on the other hand are discussed as potential cytokine antagonists. in the present study we investigated the influence of the pkc-inhibitor gf109203x on cytokine-and endotoxin induced expression of intercellular adhesion molecule 1 (icam-1) and on adhesion of lymphocytes to cytokine activated endothelial cells. we found that tumor necrosis factor alpha (tnfo0-and lipopolysaccharide (lps)-induced icam-1 expression on human endothelioma celts (eahy926) were unaffected by the pkc-inhibitor and thus appeared to be independent of pkc activation. in contrast, gf109203x significantly reduced icam-1 expression induced by interferon-y (ifn-?) and interleukin-1 (il-1). the functional relevance of these findings was evaluated in an adhesion assay using human umbilical vene endothelial cells (huvec) and peripheral blood mononuclear cells (pbmc). in fact, the ifn-? and il-1 induced adhesion of pbmc to cytokine treated huvec could be downregulated by the pkc-inhibitor, whereas tnfc~-and lps-mediated adhesion was not influenced. additionally, the il-1 driven icam-1 expression on eahy926 cells as well as the il-1 induced adhesion of pbmc to huvec was found to be tnf-dependent, since both effects could be inhibited by an anti-tnf monoclonal antibody (195f) . these in vitro data further support the idea of examining pkc-inhibitors, such as gf109203x, for their biological relevance in cytokine related dysregulations. seiffge, d., bissinger, t., laux, v., during inflammation there are some key processes, which occur in the microcirculation: the release of mediators from various cell types, the migration of inflammatory cells towards a chemotactic stimulus in the tissue, the expression of adhesion molecules on different cells, and the extravasation of plasma proteins. the aim of the present study was to elucidate the mediator induced interaction of leukocyte adhesion and plasma leakage in postcapillary venules. using an analogous video-image analysing system we have studied the effect of different mediators on leukocyte adhesion and macromolecular permeability in the mesentery of the rat. the increase in permeability was measured as changes in optical density. we found that topical administration of leneotriene b 4 (ltb4, 5x10 "6 tool/l) or intravenous injection of interleuldn-2 (il-2, 5-106 iu/kg b.w.) and lipopolysaccharide (lps, 15 mg/kg b.w.) resulted in a significant extravasation of fitc-labelled rat serum albumin (fitc-rsa) in venules but not in arterioles. we could correlate the changes in vascular permeability with a locally increased number of rolling and sticking leukocytes in venules. both effects were dose dependently inhibited by different drugs. pentoxlfylline inhibits lps-indueed fitc-rsa extravasation and leukocyte adhesion at a dose of 3 mg/kg b.w., superoxid-dismutase (sod, 10.000 iu/kg b.w.) was able to decrease the ltb 4 effect, and the immuumodulating drug leflunomide (hwa 486) exerted inhibitory effects on il-2-induced permeability at a dose of 3 mg/kg b.w.i.v. the obtained results demonstrate that lps, ltb 4 or il-2 induced extravasation of fitc-rsa is mediated by activated leukocytes and can be deminished following administration of different drugs. platelet-endothelial cell adhesion molecule-i (pecam-i), a member of the immunoglobulin superfamily, is constitutively expressed at high levels on the endothelial cell surface. in vitro data have suggested that pecam-i functions as a vascular adhesion molecule, specifically in neutrophil transmigration across the endothelium. this current work is the first demonstrating the in vivo role of pecam-1 in neutrophil migration. blocking antibodies to human pecam-1, in which the antibodies are crossreactive with rat pecam-1, were able to block the movement of neutrophils into the rat lungs after igg immune complex deposition. furthermore, when human foreskin was transplanted into mice with severe combined immunodeficiency and the site injected with tnf-alpha, anti-pecam-i blocked neutrophil emigration into the dermal interstitium. it has already been established that neutrophil recruitment is dependent upon selectin mediated rolling, followed by firm adherence that is icam-1/ integrin mediated. these data suggest, for the first time, that a third endothelial adhesion molecule (pecam-i) is involved in the coordinated recruitment of neutrophils in vivo. to test whether trauma causes generalized activation or priming of pmns, cdi8 adherence receptors were measured with iinmunomonitoring in whole blood after lps stimulation ex vivo. anesthetized (fentanyl) mongrel pigs (15-25 kg) were subjected to 40% arterial hemorrhage + soft tissue injury and after liar, resuscitated with all the shed blood + supplemental fluid. blood was collected at 24hr intervals from unanesthetized animals with indwelling catheters, pmns were counted, and lps was added (0,1,5,10 i.tg/ml) ex vivo. after 3hr incubation at 22-24°c, %cd18 (+) pmns were determined with fitc-ib4 and flow cytometry from mean channel fluorescence histograms. 49±8 # p<0.05 vs baseline * p<0.05 vs sham $p<0.05 vs no anesthesia these observations provide direct evidence for time-dependent changes in pmn priming following major injury because cd18 expression was depressed for at ]east 24hr after trauma relative to sham but by 72hr, was enhanced, relative to sham, and because fentanyl anesthesia at 72hr had a greater effect on cd18 expression in trauma vs sham. neutrophil (pmn) adhesion to vascular endothelial cells (•c) is a key element in the inflammatory response and tissue injury. inflammatory mediators such as lps (exogenous) and tnf (endogenous) can promote pmn-ec interaction which is believed to be responsible for capillary leakage and subsequent organ injury. however, the mechanism of this injury remains unclear.we hypothesised that the mechanism of tissue injury is due to ec necrosis with release of toxic products and that activated pmn are responsible. human pmn were obtained from healthy donors, separated by density gradient, and activated with lps (50ng/ml), tnf(10ng/ml), and lps/tnf(25ng/10ng/ml). cultures of the human ec tine(ecv-304) were used as surrogates of the microvasculature, were exposed to either lps, tnf, lps/tnf and pmn activated with lps, tnf, lps/tnf and incubated for 6, 12, 18, and 24hrs. ec necrosis was assessed by a 51cr release cytotoxicity assay. pmn activation was assessed by cd1 lb receptor expression and respiratory burst activity 6hr 0_+0 1.7-+ 1 0-+0 3.2_+ 1 0_+0 4.4_+ 1 0_+0 2.3_+0.9 12hr 0+0 3.24_3 0_+0 9.3_+3 0_+0 11_+3" 0+_0 12+--2.1" lghr 04-0 0.9_+2 0+_0 284-12" o:fo 25,12" 0~0 30+-9.5* 24hr 0_+0 4.14-4 0-+0 33+_3* 0_+0 30_+9* 0_+0 32_+10" data = ec % necrosis mean_+sd stats: student's t-test with significance (*) set at p<0.05 vs control. ( our previous studies have indicated that despite the increased cardiac output and maintenance of tissue perfusion, hepatoceliular dysfunction occurs during early sepsis. nonetheless, it remains unknown whether vascular endothelial cell function (i.e., the release of endothelium-derived relaxing factor/nitric oxide) is depressed under such conditions and, if so, whether endothelial cell dysfunction also occurs at the microcirculatory level. to determine this, rats were subjected to sepsis by cecal ligation and puncture (clp), following which these and corresponding shams received 3 ml/100g bw normal saline. at 5 hr after clp (hyperdynamic sepsis) or sham operation, the thoracic aorta was isolated, cut into rings, and placed in organ chambers. norepinephrine (ne, 2xi0 -7 m) was used to achieve near-maximal contraction. responses for an endothelium-dependeut vasodilator, acetylcholine (ach, via nitric oxide), were determined. in additional studies, the small gut was isolated at 5 hr post-clp. after pre-contraction of blood vessels in the isolated gut with 5xl04 m ne, vascular responses to ach (5x10 6 m) and an endotheliumindependent vasodiiator, nitroglycerine (ntg, 5xl0 -7 m), were determined. total vascular resistance (tvr, mmhg/mi/min/100g) was then calculated as pressure/ perfusinn rate. ach-induced relaxation (%, n=6/group) in the aortic rings were: ach lxl0 i~s, st-in ~ ~ significantly at 5 hr post-clp (i.e., increased *p(o 05 vs. sham; n-4 per group. tvr) in the absence of any changes in ntginduced relaxation (fig. a) . thus, the vascular endothelial cell dysfunction observed in the aorta in early sepsis also occurs at the microcirculatory level. introduction: the cytokine-mediated adherence of leulcooytes to vascular endothelium is considered as an early step in the cascade of pathologic reactions culminating in the "systemic inflammatory response syndrome" (sirs); the purpose of this study was to evaluate the influence of interleakin-1 on leukooyteendothelial cell-interactions and microoirculation in the liver after hemorrhagic shock by means of intravital microscopy. methods: in anesthetized female sprdrats co.w. 200-230g) shook was induced by fractionated withdrawl of arterial blood within 5 rain and maintained for 1 h (map at 40 mm hg, cardiac output 50 % of baseline). rats were adequately resuscitated with 60% of shed blood and twice the volume in ringer's solution additionally. following 5 h of reperfusinn (map >100 mm hg, co >120% of baseline) the microcirculation in liver lobules was examined by intravital fluorescence microscopy after labelling of leukocytes. continuous administration of il-lra (synergen, boulder, colorado, 5mg/kg/h) was started at different time points in a randomized and blinded manner. the animals in group p (n=6) received the il-lra as pretreatment beginning 5 min prior to shock induction. in the group t (n=6) the application of il-lm started at the beginning of the reperfusion period with a bolus injection of 5 mg/kg and was followed by continuons administration of 5 mg/kg/h. the control group c (n=4) received equal volumes in nac10,9%, the sham-operated group s (n=4) was not exposed to shock. results: macrohemodynamics were comparable in all shook groups. the increased percentage of permanendy adherent leukocytes after hemorrhagic shook (s: 9,1% +1,1%; c: 42,1% _+5,4%) was significantly reduced by pretreatment or treatment with il-lra (p: 16,9% -+1,9%; p<0.001, t: 28,8% -+3,6%, p<0.01, anova). temporary adhesion of leukocytes was unaffected by application of il-lra. liver microcirculation measured by volumetric blood flow in liver sinusoids and sinusoidal diameters was impaired after hemorrhagic shock in all groups and was not affected (c: 31993iam3/s +40971um3/s, p: 32768llm3/s +4445}am3/s, t: 326211ams/s -+2998 lam3/s, s: 390201am3/s -+39041am3/s). di.seu~sinn: the results demonstrate that permanent adherence of leukocytes to endothelium is in part regulated by il-1. pathological adherence could be reduced by application of illra, even given at die time of resuscitation. the effect of ll-lm on permanent adhesion is a specific event and might be caused by reduced expression of specific receptors on sinusoidal endothelial cens and leukocytes. objectives of the study. the adhesion of activated neutrophils (pmn) to endothelial ceils (ec) and the concomitant production of reactive oxygen metabolites (rom) initiates organ damage after trauma, sepsis, shock and organ reperfusion. aien of this study was to investigate the effect on adhesion and rom production of the highly water-soluble, membrane-permeable and physiological ascorbic acid (asc). materials and methods. adhesion of pmn to nylon fiber (cell count) and simultaneous rom production (chemiluminescence-cl-response) were measured up to 6 retool/1 asc as well as adhesion, rom production and ec damage (lllln-release from labeled ec) of endotoxin-activated pmn to cultered ec moanlayers. in an in vivo animal model (sheep with lung lymph fistulas) the effect of asc (1 g/kg bw bolus, followed by 0.2 g/ kg-h infusion) on the endotoxin-induced (0.5 ixg/kg bw) neutropenia (cell count), lung capillary permeability damage (lung lymph protein clearance) and rom production of neutrophils (zymosan-induced cl response) was measured. results. asc scavenged rom dose-dependently during adhesion of pmn to nylon fiber (p<0.0005 at 6 mmol/l asc), adhesion itself was unchanged. during the activated pmn/ec interaction asc scavenged rom (p<0.025 at 6 mmol/l asc) and reduced the adhesion dose-dependently (p<0.0005 at 6 mmol/l asc); ec damage was also reduced (p<0.0005 at 6 retool/1 asc). in the in rive model asc increased the endotoxin-induced blood pmn decrease (p<0.05), decreased the protein clearance (p<0.05) as well as the zymosan-induced rom production (p<0.03), indicating the asc-mediated reduction of adhesion, rom production and lung tissue damage processes. conclusions. by in vitro and in rive experiments ascorbic acid reduced the adhesion-and rom production-initiated tissue damage. therefore, i.v. administration of ascorbic acid is recommended for oxidative stress-associated states after trauma, sepsis, shock and organ reperfusion. for neut rophi l-accumulat ion and activation. we investigated the influence of or to the activation and the expression of lecam-i and cdiib,cdi8 on neutrophils and lymphocytes. methods: from blood samples (n=5) all white blood cells (wbc) and neutrophils (nc) were isolated and cultured. or were produced via the xanthine oxidase/hypoxanthine system. after 0,5, 15,30,60 and 120 minutes a giemsa-staining to determine the granulation of neutrophils (n: normal, r : reduced ) and a facs-analysis with monoclonal antibodies detecting cdiib,cdi8 and lecam-i was performed. results: under the influence of or a degranulation of neutrophils starting at 15min was observed in wbc-cultures (n/r: 0min 91/9, 15min 76/24, 30min 68/32, 60min 52/48, 120min 49/51). these data were confirmed in the dot-plots of facs-analysis. only in wbc-cultures or induced a significant increase of lecam-i expression on neutrophils up to 30min followed by a decrease to normal values at 60min. lecam-i on lymphocytes disappeared totally during the observed period. cdllb,cdl8-expression was not altered. conclusion:increased lecam-i expression on neutrophils due to or could enhance the 'rolling' of neutrophils along the endothelium which is a prerequisite for neutrophil sticking and migration. further or are able to activate neutrophils without endothelium. these changes seem to be mediated by other wbc. introduction. multiple organ failure (mof) has been hypothesized to be the result of an excessive uncontrolled autedestructive inflammatory response. since the complement system is an important mediator and initiator of the inflammatory response, interruption of this cascade could theoretically lead to an attenuation of mof. in order to test this hypothesis we evaluated the response of c5-delicient mice in a model of zymesan indt~ed mof. materials and methods. 25 c5-deficient b2d10/oid and 25 c5-sufficient b2d10/new mice were used in this study. on day 0 all mice received an intraperitoneal injection with zymosan suspended in paraffin in a dose of 1 mg/g body weight. between day 0 and 12, biological parameters (temperature, body weight and clinical condition) were measured daily and mortality was monitored. clinical condition was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a two point scale (maximum score=4). on day 12 all surviving mice were sacrificed and relative organ weights of lungs, liver, spleen and kidneys (relative organ weight= (organ weight/body weight)x100) wore calculated. earlier experiments with our model have shown a good correlation between histological organ damage and relative organ weights. statistical analysis of biological parameter was performed using the koziol curve analysis. analysis was divided in an acute phase (day 0-4) and a late phase (day 8-12). relative organ weights were analyzed using wilcoxon's test and mortality rate using fischor's exact test. results. all zymosan injected mice showed a typical triphesic illness. deterioration of the clinical condition as indicated by the symptom score and the decrease in temperature and body weight in the acute phase were all significantly lass severe in c5 deficient mice (all p<0.005). in the late phase no differences could be noticed in the courses of biological parameters. overall mortality was 2/25 (8%) in c5 deficient mice and 8/25 (32%) jn c5 sufficient mice (p=0.049), a difference mainly due to a difference in the acute phase. organ damage assessed as the relative organ weights did not show any statistical differences for any organ between both strains. conclusion. complement factor c5 appears to play an important role in the acute hyperdynamic septic response in this model but deficiency of c5 could not prevent organ damage in the late mof phase. this suggests that other factors could be more important in the development of the inflammatory response leading to mof. proinflammatory cytokines are thought to play a critical role in the pathophysiology of multiple organ failure (mof). in mice, zymosan-lnduced generalized inflammation (ztgi) leads to mof. therefore we performed a sequential study into plasma levels of, and macrophage production capacity for, four cytokines during the development of mof in the zigi model. male young-adult c57bl/6 mice received zymosan (1 mg/g body weight) intraperitoneally. groups of 14 animals were killed after 3, 6, 8, 18 and 24h and subsequently at each day until day 12. plasma was collected and peritoneal macrophages were isolated and cultured overnight with or without lipopolysaccharide (lps). interleukin 1-ct, and -6 (il-lc~,~,), and tumour necrosis factor-o~ (tnf-c 0 were measured in plasma and culture fluid by means of a ria (detection limit 0.1 ng/ml). interleukin-6 (il~) levels were assayed using the b9 hybddoma cell proliferation assay. zymosan induces a three-phase disease in mice. after an acute phase the animals recover. around day 7, they start to develop clinical signs which resemble mof. plasma tnf-~ peaked within 3h after zymosan injection and disappeared within 8h. from day 8 onwards, tnf levels started to rise again. plasma il-6 behaved almost similarly in the acute phase, but in the mof phase plasma il-6 remained low. no circulating il-16 could be detected at any time point. macrophage lps-stimulated production of il-lcq il-11~ and tnf--c~ was suppressed immediately after zymosan injection. production of il-16 and tnf-~ was normalized within 6h, while production of il-lc~ remained lower than that in macrophages from untreated control mice. only at day 6 did production of il-i~ reach control values. il-16 production was higher than control values from day 3 onwards. il6 production was similar to that of ili-il the production of tnf-ct was strongly elevated between days 1 and 6 and again during days 10 to 12. the development of mof-like symptoms during zlgi in mice is accompanied by increased plasma levels of tnf-ct without enhanced il-16 or il-6. also, the ability of macrophages to produce excessive amounts of il-16 and tnf--~, as well as the suppressed capacity to produce il-lcq could be important mechanisms in the pathophysiology of mof. when conjugated to an asialoglycoprotein, dna and oligonucleotides are specifically taken up by the hepatocytes via the asialoglyccprotein receptor which is unique to the liver. human asialoglycoprotein (~1-acid, asgp) was derivatized with low molecular weight poly(l)lysine(pll) and complexed with 5 antisense dna's (as) complementary to the 5' region of the il-6 gpl30 receptor. the antisense were 5'-agtttagggatgagg-3' (asl), 5'-atcttcatcttctgaat-3' (as2), 5'-aagtgaatgattaaaacact-3' (as3), 5'-aaacctttataggcg-3' (as4), and 5'-cgttctacaactgcaacgt-3' (as5). using hepg2, the biological effects of these antisense complexes on the high affinity il-6 receptor were evaluated by scatchard analysis, cellular proliferation, and acute phase protein expression by radioimmunoprecipitation and two dimensional gel electrophoresis. scatchard analysis demonstrated that high affinity receptor expression was inhibited by incubation of cells with asgp-pll-asi for 24 h. underivatized asl was less effective and the complex, asgp-pll-as2, had minimal effects on high affinity binding. when the cells were treated with the conjugates and stimulated with il-6 (i00 units) asgp-pll-asi alone showed a dose dependent (0.i-0.4 ~m) inhibition of 3ss fibrinogen synthesis. two dimensional gel electrophoresis showed that expression of other acute phase proteins was also blocked. these results indicate that the targeted delivery of antisense molecules via conjugates recognized by the asialoglycoprotein receptor can block the cytokine stimulated acute phase protein response in hepatocytes, this approach may be relevant to the therapeutic management of patients with severe injury and sepsis. it has been established that immune cells are able to express neuropeptide genes and to release products that were considered to be of neuroendocrine origin. we have shown that proenkephalin (penk), a neuropeptide encoding gene, is expressed in lymphoid cells in culture. to study the physiological significance of these observations we have used the model of experimental endotoxemia. in this model, a disease state is induced by bacterial lipopolysaccharide (lps), that activates the immune system, the adrenocortical axis and the nervous system. we found that the expression of penkmrna is markedly enhanced in vivo immediately after lps injection both in the adrenal glands and in the lymph nodes. in situ hybridization analysis combined with immunohisto-chemistry indicated that the induced penk expression is confined to macrephages within the lymph nodes and chromaffin cells in the adrenal medulla. furthermore, this expression in lymph nodes is modulated by ligands of the adrenergic system. our results strongly support the notion that immune derived opioids participate in the bidirectional communication between the nervous and immune systems. of neurology hadassah university hospital, jerusalem 91120, israel. objectives of the study: multiple-organ-failure is recognized as the most severe, and often lethal, complication after multiple trauma. however there is no adeqate animal model available. our goal was to develop an animal model, in which reproducable irreversible failure of parenchymal organs is achieved in the late phase after insults in the early phase (trauma). materials and methods: l0 female merino-sheep were included (mean weight: 30kg). day 0: hemorrhagic shock (mean arterial pressure (map) 50mmhg for 2 hrs.), closed femoral nailing (ao-technique), day 1-5: bolusinjection of endotoxin (et) (0,75~tg/kgbw) und zymosan-activated plasma (zap) (20ml) every 12hrs., day 6-10: observation. bronchoalveolar lavage (bal): day 1, 6, 10. the course of representative parameters of organ function was documented: cocardiac output (i/min), svr -systemic vascular resistence (dyn ~ s cm-5), pap -putm.art.pressure (mmhg), pap2 -arterial oxygen pressure (mmhg), bill -bilirubin (;xmov1), crci -creatinin clearence (ml/min) statistics: data as means+sem, *significant from baseline (wileoxon test; p<0005) results: baseline day 4 day 6 day 8 day 10 heart: co 4,96_+0,32 5,33_+0,36 5,4_+0,2 7,30_+0,77* 7,98_+0,66* svr1520_+134 1395+141 1403_+89 1119+_122" 1089+-91" lung: pap 17,0_-20,7 19,6_+1,6" 22,0+-1,2"25,8+2,0" 28,8+-1,7' pap2 103,1+1,5101,5+-5,7 97,3_+4,7 91,9+-5,6 89,8+_3,9* liver: bill 2, 94_+0, 344, 82_+1, 34' 5, 13_+0, 68'6, 13_+0, 7" 7, 19_+0, 91" kidney:crcl 86, 5+_30, 9 109, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] 4 74, 7_+10, 868, 8+14, 1 53, 1+17, 6 histologic specimens showed all signs of fulminant mof. combination of hemorrhagic shock, femoral nailing, et und zap (insults in the early phase) lead to an irreversible organ failure in the late phase. prostaglandin e (pge) levels are elevated by trauma, shock or sepsis and can profoundly affect the immune response. pge2 is produced by many cell types including fibroblasts, macrophages, monoeytes, follicular dendritic cells, and epithelial cells and is induced by il-i, bacterial lps, components of the complement cascade, tnf, il-6 and crosslinking of surface fc receptors for igg, iga and ige. our research has shown that pge inhibits b cell activation (specifically enlargement, class ii ~c and fc~ rii expression), proliferation, igm and igg3 responses, t cell proliferation, and il-2 synthesis in the mouse model. in contrast, pge greatly promotes class switching to ige,the isotype responsible for type i allergic hypersensitivity. thus, our model mirrors th~ general immunosuppression and elevated ige titers of the trauma or sepsis patient. pge increases the number of cells secreting ige and iggl, acts on surface igm positive b cells, synergizes with il-4 and lp$ to induce preswitch germline transcripts, and induces more rapid expression of mature vdj~ mp~a than in eontro~ pge intracellular signalling occurs through cyclic adenosine monophosphate (camp) levels and can be mimicked by camp-inducing agents and blocl~ed by an inhibitor of campdependent protein kinase a. pge action requires de novo protein synthesis and candidate pge-inducible regulatory proteins have been identified by 2d gel eleetrophoresis. thus, pge inhibits a number of immune mechanisms while promoting ige production. a deeper understanding of pge immune regulation may lead to more effective treatment of immune perturbations as sequelae of trauma, shock or sepsis. during infrarenai aortic surgery mesetueric traction (re.t.) results in prostacyclin (pgi:) release and consecutively in hemodynamic disturbances (decreased systemic vascular resisteace, mean arterial pressure; increased cardiac output, heartrate). these symptomes are bypassed by cyclooxygenase inhibition. hemodynamic symptoms vanish after 30-45 rain even without cyclooxygenase inhibition although pgi2 levels remain elevated. to study the endocrine vasopressor system in a prospective double blinded protocol, we investigated 26 patients undergoing major abdominal surgery as compared to 26 ibuprofen (400 rag, i.v.) pretreated (ibu) patients. the surgeon applied m.t. in a uniform fashion. we chose a general anesthesia combined with a supplemental thoracic epidural anesthesia. at the points in time 5, 15, 30, 45, 90, 180, 360 , 1440 rain after and before (to) mesentzrie traction we determined the plasma concentrations (pc) of 6-keto-pgf~o~pr~, epinephrine, norepinephrine, dopamine, renin, aldosterone, adh and cortisol. pc of 6-k-pgf~,tp~, peaked 5 minutes after m.t. (2274_+284, ibu: 102_+12, to: 60+i ng/l) and declined monotonously over 6 h (203 +_42, ibu: 84_+ 12 ng/1). catecholamine pc "s did not exceed the reference range during the observation period. reninpc peaked after 45 rain (66_+23, ibu: 18+3, to: 16-+2/~u/ml); aldosteronc also presented a maximum after 45 rain (110 + 12, ibu: 66-+ 15, to: 56 +-7 pg/ml), whereas cortisol demonstrated irrespectively of circadian rhythms a maximum 6 h after m.t. (26+_1, ibu: 24-+3, to: 10+_1 ~g/1). adh pc peaked 5 min after m.t. (71+7, ibu: 20-+6, to: 5+_1 pg/ml) and showed analogously to 6-k-pgft~j~ pc a monotone decline over the observation period. our data demonstrate a counteractive reaction to pgiz mediated vasodilation via adh secretion. the second regulative is the renin-angiotensin-aldosterone system (raas), which is activated 45 min after m.t., the aldosterone pc does not paratlel the cortisol pc, which peaked post operafionem in both groups, probably due to the end of anaesthesia. a regulative release of catecholamines could not be documented. the activation of adh and raas after mt is not a hormonal response primaryly related to surgical trauma and/or stress but a counterregulation to systemic vasoditafion induced by prostacyclin. although adh and raas support systemic circulation, angiotensin and vasopressin may compromise local organ blood flow (e.g. splancimic vascular bed). insfitut f. klin. chemic, anaesthesiologie ~, chirurgie l*, univ. ulm, 89070 elm, expression of c-fos protein in rat brain following occlusion of superior mesenterie artery. takanobu there is general agreement that neurologic abnormalities are seen in sepsis. the aim of this study is to examine what effect does the brain receive in case of sma occlusion by immunohistochemistry using antibody to c-fos, an immediate early gene, which is recently recognized as a genetic marker of activated neurons. moreover, we investigated the correlation between c-fos induction in the brain and plasma endotoxiu level. 20 rats of them received sma clipping and others wee used as control. control and treated rats at 2, 4, 6, 8 hours were perfused and fixed. the brain were sectioned at 20 pm and stained by abc method using c-fos antibody. plasma endotoxin level of 5 rats were measured at 0, 2, 4, 6, 8 hours after the treatment by chromogenic limulus method. immunohistochemical study showed scarcely no immunoreactivity in control rat brain. in treated rat brain, the significant expression of c-los was detected in specific nuclei including the habenula, some hypothalamie nuclei, amygdala, locus ceruleus and nucleus tractus solitarii. such immunoreactivities were increased in time curse, which well corresponded plasma endotoxin levels. the mean plasma endotoxin level of 0, 2,4, 6, 8 hours after the treatment were 6.48 ± 5.57, 15.0 _-4-4.37, 10.0 _+ 4.18, 14.4 ± 3.52 and 58.4 ± 28 .6 pg/ml, respectively. the results indicate that limbic and hypothalamic-brainstem systems are involved in sma occlusion, and suggest that such neuronal actival.jon may precede the elevation of plasma endotoxin icy.el. systemic vascular resistance and increased cardiac output accompanied presumingly by a increased pulmonary shunt (qs/qt). this response is induced by prostacyclin (pgi2). we examined oxygen transport after traction on the mesentery root and the transpulmonary prostacyclin levels in a prospective placebo controlled study with intravenous ibuprofen. methods: with approval of the human [nvestigadon review board we studied 52 patients in a prospective, randomized double-blinded protocol who were scheduled for major abdominal surgery. ibuprofen (400 mg i.v.) or a placebo equivalent was administered 15 minutes before skin incision. pulmonary artery thermodilution and radial artery catheters were placed after induction of anesthesia. mt was applied in a uniform fashion. baseline values preceded the incision of the peritoneum (to). fulther assessments followed 5, 15, 30, 45, . tile plasma concentrations (pc) of 6-keto-pgft, (stable metabolite of pgi2) were determined in arterial and mixed venous blood by radioimmunoassay. at all points in time we measured arterial and mixed venous blood gases. qs/qt was calculated by standard formula. data are given as median (p < 0.05 placebo vs. [ibuprofen] [117] mmhg (*p< 0.0i). these changes were accompanied by a marked increase of 6-keto-pgf~ pc up to 180 rain after mt in arterial and mixed venous blood of untreated patients with a peak of 2450*[60] ng/l tl (*p< 0.00ol). there was no difference between arterial and mixed venous pc. ibuprofen pretreated patients (n=zr) demonstrated stabile qs/qt and pao2 while 6-keto-pgf~ pc remained within the normal range. discussion: our data clearly indicate that mesenteric traction response includes a critical rise in qs/qt followed by significant decrease of paov stable oxygen transport determinants following cyclooxygenase inhibition signify an action mediated by prostacyclin. an indicative transpulmonary gradient for 6-keto-pgft~ was not detectable. a splanchnic vascular source for pgi2 release seems to be likely, but could not be proved by our current data. department of anesthesiology, cliu. chemistry * and surgery*; university clinics uim, prittwitzstral]e 43, 89075 ulm, germany it is unclear whether injuries like bums, in general, directly result in alterations of cell-mediated immunity that, in turn, promote endotoxic and bacterial translocation or, alternatively, whether these conditions allow increased bacterial invasion that, in turn, inhibits cmi. aim: to determine whether infectious challenge, as clp alone or combined with ti causes further immune abnormalities in the days following clp. study plan: on day 1, two groups of n=36 8 week old aj mice were subjected to either a 20% scold burn (ti), or were untreated (c) n=18. on day 10, 18 mice (ti+clp) and 24 mice (clp) were subjected to clp. the two other groups (ti and c) were untreated. at days 11, 12 and 13 after thermal injury splenocytes (sp) were harvested and cultured with cona for an assay of il-2 and adherent splenocytes (as) were cultured with lps for il-1, tnf, il-6 and pge2. results: either ti + clp or clp alone result in significantly decreased secretion of all cytokines tested. in the ti group almost every cytokine production determined was elevated in comparison to ti + clp and prosmcyclin (pgi2) has been implicated in the pathophysiology of septic shock. however, pgi~'s role in the inflammatory response to sepsis is not well-defined. the purpose of this study was to identify which acute septic events are mediated by pgi 2 during graded bacteremia. methods: eleven ~nesrhetized, hemodynamically monitored adult swine were infused iv with aeromonas h. (109/ml) at rates increased incrementally from 0.2 to 4.0 mi/kg/hr over 4 hours. animals were studied in two groups: septic control (sc), graded bacteremia only (n=6); pga (n=5), graded bacteremta plus anti-pgiz antibody, 50 ml/hr iv, beginning at 2 hours. mean systemic (map) and pulmonary arterial (pap) pressures and arterial po2, mmhg, cardiac index (ci), l/min/m2, oxygen delivery index (do2i) and consumption index (vozi), ml/min/m2, and oxygen extraction (er), %, )latelet aggregometry (plt), %max., plasma pg6-keto f1 alpha ; in the first instance~ peak values of lt ~4 after i~ hrs post infarction were 6 times higher than in the controls and excess leucocyte infiltration was noted at the infarction zone. in second instance two levels of lt b4 led to weak infiltration of the infarction zone by leucocytes. a. mo~e~o, in~.~p~siolo~,d~t.e~.cardiolo~,bogotsolets4, ~ev252024, ukrmne systemic lesion$of erythron in traumatic disease and possibilities of their regulation by opioid peptides. redkin y. v., fominih s. g. using clinical (121 patients) and experimental material(128 rats and 74 dogs) we revealed general regularities of erythron lesions after hard mechanical trauma of various genesis as well as some mechanisms of development of posttraumatic anemia and possibilities of its correction with preparations of opioid peptides. the condition of central and peripheral compartments of erythron was studied with unified morphologic, immunogematological, biochemical and radiological methods. it was revealed that irrespective of the experimental animal species (dogs, rats) or in clinical experiments (patients) and irrespective of the injuring factor type (skeletal trauma, craniocerebral trauma, loss of blood) in erythron can be observed one-directed unspecific reaction realized by the considerable lowering of hemoglobin concentration, erythrocytes number and hematocrit. in the initial period (1-7 days) in the system of erythron prevail processes of distraction and elimination of er~zthrocytes relatively to the general production of stimulated erythropoiesis. the primary alterating factor is the prolonged intensification of peroxydation of membrane iipids of erythrocytes with simultaneous lowering of reserves of reduced glutathione. the distraction of erythrocytes is supported by the developing phenomena of autoallergization of organism that becomes apparent by the appearance of sensitized t cells and antierythrocyte antibodies. the intensified production of erythropoietin rules to the realization of he program of fetal and terminal (reserved) erythropoiesis. failure of erythropoiesis function is supported by disturbances of the processes of the injuring of cell metabolic apparatus. using of dalargin (15 microgram per kilogram of body mass intrap'eritoneally within 5 days after the trauma) showed the precise pharmacotherapeutic effect revealed by the diminishing of anemia of experimental rats, more . fiberbronohoscopic procedures are known to produce "peep-like" effects and to increase pulmonary artery (pa) resistance [2] . peep can affect rv function by reducing preload and ejection fraction (ef) [3] . since changes of rv function during bronchoscopy in septic patients are not reported, we measured rv parameters before, during and after fiberoptic bronchoalveolar lavage (bal). method: this 34-year-old patient (apache-ii:24) developed a hyperdynanlic septic state due to staphylococcus aureus (blood culture). we inserted a "fast response" thermistor pa-catheter (baxter-edwards) to evaluate rv performance [4] . the therapeutic procedure included volume replacement, vasopressors (dopamine 5, dobutamine 3 gg/kg/min. iv) and analgosedatior/. before bronchoscopy (olympus bf-30, od=6 mm) the patient received pancmonium for muscle relaxation. ventilation was not changed during the procedure (endotracheal tube: id=8 ram, bennett 7200a, pressure controlled mode, pm~x=25 mbar, peep=8 mbar, i:e=i:i, fio2=1.0). we measured rv enddiastolic volume (edv), stroke volume (sv), ef, heart rate (hr), cardiac index (ci) and mean pa pressure (mpap gerlach h, gerlach m, clauss m, falke kj renal hypoxia and/or ischemia initiates the development of a deteriorated medullary perfusion based on fibrin deposition in the peritubular capillaries, vasoconstriction, and perivascular edema, which is followed by a swelling of the tubular epithelial ceils, intraluminal tubular obstruction, and a backleak of fluid through the injured tubules into the renal interstitium, finally leading to an acute tubular necrosis (atn) [1 ], clinically diagnosed as acute renal failure (arf). one important pathway for induction of enhanced vascular procoagulant activity and permeability is based on the synthesis and expression of macrophage-derived cytokines, which bind to specific endothelial cell surface receptors. we recently described the identification and purification .of a new 55,000 dalton polypeptide, which is synthesized and expressed by murine macrophages after stimulation with lipopolysaccharide, and exerts procoagulant activity on cultured endothelial cells [2] . in the presented study, we demonstrate that the new polypeptid is also synthesized by macrophages under hypoxic conditions. the protein binds to specific receptors, which are expressed by endothelial cells dependent on the environmental oxygen tension. animal studies were performed after approval by the local committee for animal safety; the animals were anesthetized, treated and supervised in accordance with the guidelines of this committee. in contrast to other authors, who performed long-term hypoxia experiments in awake animals, we preferred to implement the studies under anesthesia for ethical reasons, although regulatory functions for ventilation might be influenced. animal studies demonstrated that the intravenous injection of the polypeptide initiates fibrin formation in the peritubular vessels. keeping the animals under hypoxic conditions induces similar effects, which are reduced by a rabbit-antiserum against the new protein. in conclusion, the new polypeptide obviously contributes to the pathogenesis of acute renal failure by tubular necrosis during and after hypoxic events. the use of verapamil as cardioprotective agents for management of patients with acute ischemic/reperfused heart is based on the assumption that the increased intracellular ca+ level is a key factor in causing cell death. our in vitro study was designed to focus on effects of verapamil on the metabolic potential of cardiac slices after reversible ischemia in rats. the material consisted of two main groups : group a (non ischemia/reperfusion group) and group b (ischemia/reperfusion group), each is subdivided into two subgroups (a and b). each subgroup included 10 rat hearts. group aa is the control group, group ab is verapami] added group. group ba is ischemia group without verapamil. group bb is verapamil added group. ischemic cardiac slices were obtained from rats subjected to 15 min. haemorrhage to induce reversible global ischemia. both nonischemic and ischemic cardiac slices were placed in well oxygenated krebs ringer phosphate buffer containing 150 mg% glucose & 5 gm% bovine albumin and incubated in dubnoff shaking water bath for 60min at 37°c the results revealed that there was an enhancement in release of free fatty acids (ffa) (240%) and lactate (163%) and in glucose uptake (160%) in group ba as compared with group aa. these metabolic alternations produced by ischemic cardiac slices were reversed by verapamil addition (200 ml%) but in group ab verpamil did not alter the release of ffa & lactate from non-ischemic cardiac slices, whereas it inhibited glucose uptake from these slices by 67%. the improvement of the metabolic intervention of ischemic myocardium indicates that verapamil may be of importance in reducing the extent and severity of acute myocardial ischemic injury in acute haemorrhage. severe endothelial dysfunction occurs following injury to carotid arteries which is characterized by a decreased ability of these arteries to dilate when challenged with ach or a23187, but not with a direct vasodilator (nano2). this failure to relax to ach and a23187 reflects an inability of endothelium to generate edrf, but relaxation recovers gradually to control values by 2 weeks. exogenous no donors (e.g., c87-3754 or spm-5185), accelerate the recovery of the injured endothelium in rat carotid arteries. intravenous infusion of an no donor (30 p.g/day) with an implanted osmotic pump significantly accelerated the recovery of regenerated endothelium to produce edrf at 7 days. rat carotid artery rings relaxed only 17 + 5% and 15 + 5% to 10 gm ach in vehicle treated rats and in inactive no donor treated rats respectively 7 days following injury compared with 67 + 6% in no donor rats (p<0.001). relaxation to 100 gm nan02 was normal in all groups indicating that the differences in relaxation were not the result of damage to vascular smooth muscle. contraction to l-name (1 mm) was markedly reduced by injury, but was protected by no donors (p<0.01). thus, exogenous no donors enhance the ability of the endothelium to regenerate and to release edrf in response to endothelium-dependent vasodilators. this may be due to an anti-proliferative and anti-mitogenic effect of no on vascular smooth muscle cells, allowing the endothelium to regenerate without intimal thickening. no also has been shown to inhibit platelet aggregation, and to attenuate neutrophil adherence and activation. the superoxide scavenging effect of no is not the basis for these effects since hsod is inactive in preserving endothelial function in injured arteries. thus, no exerts a variety of cytoprotective effects which may be of importance in protecting against vascular injury. much evidence has now accumulated to show that the excess production of the vasodilator nitric oxide (no) in sepsis is an important contributor to the hypotension and multiorgan failure characteristic of this condition. various cytokines play an important role in this process through their ability to induce the production of one of the enzymes responsible for no synthesis, the inducible no synthase (inos). we have studied the effects of cytokines on the induction of this enzyme both in vitro using vascular smooth muscle cells, and in a murine model of gram-negative sepsis. tn smooth muscle ceils, the cytokines il-1, ifnq', and tnf-oc show strong synergy with one another in the production of inos. in order to define the molecular basis for this synergic effect, we have linked the promoter of the inos gene to a "reporter" gene, chloramphenicol acetyl transferase (cat), and transfected these constructs into vascular smooth muscle cells. assays of cat activity reflect the activity of the promoter in this system, and by generating sets of deletion mutants of the promoter sequence we have been able to define the area within the promoter which mediates the synergic effect of these cytokines. in addition to stimufatory effects on inos production, certain cytokines are able to down-regulate the production of inos in vascular smooth muscle cells, and the effects of these counterregulatory cytokines will be discussed. the interaction of these cytokine effects in the whole organism has been studied in a murine model of gramnegative sepsis. widespread induction of inos occurs in this model as assayed by enzyme activity and through use of specific antisera to inos. neutralizing antibodies to tnf-~ and tfn-y are both able to prevent death in this model, but it is only the anti-ifn-y which attenuates the induction of inos assayed in the liver. clearly there is some redundancy in the effects of cytokines on the production of inos in sepsis, and greater understanding of the most important factors in inos production is required in order to target anti-cytokine therapy most appropriately. effects of nitric oxide on hepatocyte metabolism in inflammation. j. stadler, department of surgery, tu mqnchen, frg hepatocellular nitric oxide (no) synthesis is induced by proinflammatory mediators such as tumor necrosis factor, interleukin-1 and interferon gamma or by bacterial toxins such as lipopolysaccharide. stimulation of the hepatocytes (hc) with a combination of these agents leads to an output of no in quantities which are not seen in any other celltype. it has been demonstrated by various investigators that important effects of these cytokines and bacterial toxins on hc metabolism can be attributed to the action of no. in contrast to other celltypes hc seem to be relatively resistant to suppression of basic metabolic functions such as energy metabolism by no. therefore, cell damage has not been described to a significant extent following exposure to no. however, no does inhibit total protein synthesis. the exact biochemical mechanism of this phenomenon has not been uncovered yet, but it has been demonstrated for some specific proteins that their production is inhibited at a posttransscriptional level. as in many other celltypes cgmp generation is elevated in hc by no through activation of the soluble guanylate cyclase. cyclic gmp may possibly exert a plethora of metabolic functions, but it is interesting to note that most of the cgmp seems to be transported out of the cell. some very specific effects of no on hc metabolism include the inhibition of the glyceraldehyde-3-phosphate dehydrogenase (gapdh) and the cytochrome p450 (cyp) enzymes. inhibition of gapdh activity is mediated through nitrosylation of critical domains of the enzymes by no which enhances auto-adpribosylation. this effect on gapdh activity might be responsible for the inhibition of gluconeogenesis by no, which has been described recently. finally, no-mediated inhibition of cyps may help to explain the suppression of hiotransformation processes which is a characteristic featur,'~ r ~ "~flamed liver. nitric oxide (no) is an endogenous inhibitor of polymorphonuclear leukocyte (pmn) adhesion which limits pmn-endothelial cell interactions under normal conditions. we have previously demonstrated that following ischemia, no production by the vascular endothelinm is dramatically reduced. accordingly, we investigated the effects of no-donors on pmn accumulation and tissue injury following hemorrhagic shock and ischemia. hemorrhagic shock was induced in anesthetized rats by bleeding to 35 mmhg for 3 hours followed by reperfusion. segments of superior mesenteric artery (sma) were isolated and suspended in organ baths. in rats receiving saline sma relaxation to acetylcholine (ach, 100 nm) was reduced by 80% compared to control sma segments (p<0.01) while relaxation to sodium nitrite (100 gm) was unaffected. in addition, mesenteric tissue pmn accumulation as determined by myeloperoxidase (mpo) activity was significantly elevated compared to controls (p<0.0l). interestingly, treatment with the no-donating agent, s-nitroso-n-acetylpenicillamine (snap) significantly preserved sma relaxation (p<0.01), attenuated mesenteric mpo (p<0.05) activity, and significantly improved survival compared to saline vehicle. in anesthetized, open-chest dogs we investigated the cardioprotective actions of a novel no-donor, spm-5185 (schwarz pharma), following regional myocardial ischemia (1 hour) and reperfusion (4.5 hours) . treatment with spm-5185 (500 rim) significantly reduced myocardial necrosis by 70% (p<0.01) compared to an no-deficient analog of spm-5185, spm-5267. furthermore, mpo activity within the ischemic-reperfused zone was also significantly (p<0.01) reduced following treatment with spm-5185 compared to spm-5267 (1.6 + 0.5 vs. 3.8 + 0.3 u/100 mg tissue). these data strongly suggest that no is a potent inhibitor of pmn-mediated tissue injury following hemorrhagic shock as well as in acute myocardial ischemia-reperfusion injury. overproduction of nitdc oxide (no') may contribute to sepsis-induced hypotension. during septic shock, excess no" is produced by an isoform of nitric oxide synthase (nos) which is induced by inflammatory mediators. nonselective nos inhibitors have been proposed as a new therapeutic approach to treating hypotension in septic shock. we studied the differential hemodynamic effects of n~-methyi-l-arginine (l-nma), a nos inhibitor, in normal canines versus those challenged with endotexin (lps) and compared the activity of this drug across the venous, pulmonary and systemic vascular beds. awake canines were challenged with lps (0 mg/kg, n=15:8 mg/kg, n=21 ; or 16 mg/kg, n=30) and treated with l-nma (0, 1,2, 4,10 mg/kg/hr) for 22 hours following a 0, 10, or40 mg/kg loading dose. animals were resuscitated with iv ringers solution (10 ml/kg/hr). hemodynamic data were collected at 0, 2, 6, 10, 14, and 22 hours using intravascular catheters and radionuclide heart scans and analyzed by anova. in both normal and endotoxemic animals, l-nma at all doses studied similarly increased mean arterial pressure (p=0.07), and systemic vascular resistance index (p=0.ol) and decreased cardiac index (p=0.05) and oxygen delivery index (p=0.01). in contrast, the effect of l-nma on mean pulmonary artery pressure, central venous pressure, pulmonary capillary wedge pressure, and pulmonary vascular resistance index was greater in lps-challenged canines compared to normal animals (p<0.05), but this differential effect on the venous and pulmonary circulation occurred, >6 hours after lps challenge. l-nma did not significantly increase survival rates or times at any of the doses studied (1, 2, 4, or 10 mg/kg/h) in either the low (8 mg/kg) or high dose (16 mg/kg) lps-challenge groups. a nonsignificant (p>0.2) trend toward a beneficial effect on survival ol low dose l-nma (1 mg/kg/h) in animals given the 8 mg/kg lps-cha[lenge was not enhanced by increasing the lethality of the model or by administering higher l-nma doses. at the highest l-nma dose used in this study (10 mg/kg/h), survival time decreased significantly for both the low and high dose lps-challenge animals (p<0.05). this increased mortality was not explained by changes in plasma concentrations of either lps or tnfc~. thus, l-nma did not have a greater effect on the systemic arterial circulation in endotoxemic compared to normal canines. however, in the venous and pulmonary vascular beds, the effect of l-nma increased with time after endotoxin-challenge these data suggest the induction of nos activity by endotoxin in canines may be relatively greater in venous and pulmonary vessels compared to systernic arteries. l-nma, a nonselective nos inhibitor, did not decrease mortality in endoloxemic canines and the highest dose studied was harmful. pulmonary hypertension (ph) and arterial hypoxemia are characteristic features of the adult respiratory distress syndrome (ards). reducing pulmonary vascular pressures may promote the resolution of pulmonary edema. intravenously infused vasodilators lower ph in ards, but, as a result of their general vasodilatatory effects, systemic mean arterial pressure may also decrease. furthermore, blood flow may be increased to non-ventilated or poorly ventilated lung areas resulting in a rise of intrapulmonary shunt, thus causing a further fall in pad 2. recently, short term inhalation of low concentrations of the gas nitric oxide (no), an endogenous endothelium derived relaxing factor, which is rapidly inactivated in blood by hemoglobin, was reported to decrease ph without causing systemic vasodilation in sheep [1]. similar changes have been observed in patients with severe ards during repeated short term inhalation of no (18 and 36 ppm), which rapidly and selectively decreased the mean pulmonary artery pressure (pap) and, in contrast to intravenously infused prostacyclin, induced a remarkable increase of pad2 [2] . this improvement in oxygenation was caused by a redistribution in blood flow away from intrapulmonary shunt areas to normal ventilated lung regions. continuous no inhalation (5-20 ppm) consistently lowered the pap and augmented the pao2/f.o 2 for up to 53 days. no negative side effects were observed during the whole time span examined. in particular methemoglobin levels always remained below 1.5%. following these investigations, it could be shown that these effects may also occur using concentrations in the parts per billion range [3] , which may reduce possible toxic side effects. however, in the same study it was demonstrated that the dose-response curves for pa02 and pap have different patterns. whereas pap presented a continuous dose-dependent downward tendency with an eds o of approximately 2-3 ppm, the improvement of oxygenation had a maximum at 10 ppm and, at higher doses, drifted back towards the baseline data. the ed~o was estimated at approximately 100 ppb, i.e. more than ten times lower than for the reduction of pap. in conclusion, inhalation of no by patients with severe ards may result in persistent and reproducible decreases in pap associated with an evident improvement in pad2, thus allowing reduction of the f.o 2. no inhalation should be performed using low concentrations which are less toxic, although any possible risks still have to be considered carefully. dose-response studies for the individual patients are recommended urgently. finally, controlled randomized studies are required to demonstrate that additional no inhalation is able to reduce mortality of ards. inhibition of the activity of glyceraldehyd-3-phosphate dehydrogenase (gapdh), an enzyme of the glycolysis/gluconeogenetic pathway, through adp-ribosylation is promoted by nitric oxide (no). since no is produced in the septic liver and hypoglycemia is a major problem of late sepsis, it was investigated whether no interferes with gluconeogenesis of hepatocytes. hepatocytes (hc) were isolated from sprague-dawley rats using a collagenase perfusion technique and differential centrifugation. exogenous no was applied by incubation with the no-donors s-nitrosyl-acetylpenicillamine and sodium-nitroprusside. endogenous no synthesis was induced by incubation with cytokines (tnfcq il-1, ifnj and lipopolysacchafide (lps). 24 hrs later the incubation medium was changed to a solution containing lactate, ornithine, lysine, ammoniumchloride and glucagon for optimal conditions of gluconeogenesis. after 3 more hrs glucose and nitrite levels were determined spectrophotometrically. gapdh activity was measured by the nadh-dependent conversion of 1,3-diphosphoglycerate to glyceraldehyde-3-phosphate. incubation of hc with no-donors led to a concentrationdependent inhibition of gluconeogenesis and gapdh activity. however, gapdh activity was about 5 times more sensitive to the inhibitory effect of exogenous no. incubation of hc with cytokines and lps induced nq synthesis as measured by an increase in nitrite concentrations. endogenously produced no suppressed gluconeogenesis by 49_+3%. in contrast to exogenously applied no, the effect of endogenous no synthesis was less on gapdh activity resulting in an inhibition of only 32_+12%. in conclusion, exogenous and endogenous no inhibited gluconeogenesis as well as gapdh activity. however, there was no correlation between the extent of inhibition of these two parameters of hepatocellular glucose metabolism. we have shown that inhibition of hepatocyte (hep) synthesis of nitric oxide (no) potentiates cell injury in a model of acetaminopheninduced oxidative stress and the extent of damage was paralleled by depletion of reduced glutathione (gsh) stores. to clarify the role of no in modulating the redox state of hep, we studied the effect of inhibition of cytokine-mediated no production on hep gsh stores, in a system of isolated rat hep in primary culture, no synthesis was induced (stim) by exposure to il-1, tnf, ifn, and lps for 16 hours. 20, 60, 120 and 200 ~m of n-monomethyi-l-arginine (nmma), a specific inhibitor of no synthesis, was added. cells incubated in media alone served as controls (cont). the no metabolite (no2); aspartate aminotransferase (ast), an indicator of cell injury; and gsh were assayed. (data presented as mean + sem; n=4.) gsh (nmovma orotein) ..~ (nmol/ma orotein) cont 14.1 + 0.3 32 + 4.0# stim 13.4 + 0.6 139 + 12 stim+2o tzm nmma 12.7 + 1.1 88 + 5.0# stim+60 ~m nmma 9.9_..+ 0.4* 68 + 4.5# stim+120 pm nmma 7.6 + 0.6* 57 + 3.0# stim+200 )lm nmma 5.0 + 0.5* 37 + 2.5# anova 0,0002 0.0001 (* p < 0.05 versus stim, # p < 0.01 versus stim; anova with neuman-keuls) gsh in cont+120 i~m l-nmma was equivalent to that of cont (13.3 vs.14.1). ast release was equivalent in all treatment groups. these data show that inhibition of hep synthesis of no depletes intracellular stores of reduced gsh. we conclude that hepatocyte no production modulates cellular gsh homeostasis and as a result, may be hepatoprotective in oxidative injury. nitric oxide (no) is a modulator of immune response and may be involved in the changes in immune reactivity after major trauma and operations. we investigated no-generation in rat and mice spleen cells (sc) after partial hepatectomy (ph). c57bl/6 mice and lew rats underwent a 50% and 70% ph, respectively. sc were prepared 1-6 days after ph and plated at 1 to 2 x 10ecells per well. after 20 h incubation at 37°c, no-production was measured as nitrite levels (griess reagent). normal mouse sc did not produce no, neither basal nor in response to lps or con a starting at the second day after ph, we found a substantial production of no. in rats, also sc from control animals were able to generate no; both basal and stimulated no-generation were further enhanced after ph (table, values expressed as mean --se). after shame operation, there was only a modest elevation of noproduction in rat and mouse sc. in first experiments we could demonstrate no-production also in phagocytes from a patient 3 days aider liver partial resection (1.4 nmol nitrite/106 cells) enhanced no-production in macrophages may contribute to the changes of immune reactivity after partial hepatectomy. nitric oxide (no) is recognized as an important mediator in endotoxemia and sepsis. increased synthesis of no has been demonstrated in septic humans and animals, and no inhibitors have been used in the treatment of septic shock. recent reports have, however, suggested that this form of therapy may cause serious organ damage. in the present investigation circulatory and metabolic changes in the liver were studied during treatment with the no-synthase inhibitor n-nitro-l-arginine-methyl ester (l-name) in endotoxemia. methods: juvenile pigs were randomized to one of the following treatment groups: 1) encletoxin and l-name, 2) endotoxin, 3) naci and l-name, 4) nach preliminary results from groups 1 (n=7) and 2 (n=8) are presented. catheters for pressure measurement were introduced into the aorta, hepatic and portal veins and ultrasonic transit time flow probes were placed on the hepatic artery and portal vein. a catheter was introduced into the pulmonary artery. endotoxin (1.7 gg/kg/h) was given as a continous portal infusion over the entire observation period of 8 hrs. l-name (25 mg/kg) was given as a bolus after 3 hrs. of endotoxemia. results: endotoxin transiently reduced portal vein flow (pvf) by 25%* and hepatic artery flow (hal e) by 45%*, while l-name caused a further and lasting reduction in flow (pvf 78%, haf 90%)*. transhepatic (portal-hepatic vein) vascular resistance increased to 3 times baseline value during endotoxemia while l-name caused a further marked increase in resistance to 12 times initial value. portal oxygen saturation (so2) decreased by 60%* during endotoxemia. l-name caused a reduction in portal so2 by 87%*. arterial so2 was unchanged in both groups. hepatic oxygen uptake was not changed by endotoxin, but was markedly reduced after addition of l-name. endotoxin caused a 27% reduction in cardiac output (co). the addition of l-name reduced co by a total of 71%*. *: p < 0.05. conclusion: is the present model of endotoxemia treatment with the nitric oxide synthase inhibitor l-name markedly reduced liver perfusion and portal oxygen supply. this might explain the increased liver damage reported in previous studies using no-inhibitors. the increase in transhepatic resistance found after l-name treatment will tend to cause pooling of blood in the splanchnic veins, resulting in reduced filling of the heart and thus contribute to the observed reduction in cardiac output. institute for surgical research, rikshospitalet, the national hospital, university of oslo, 0027 oslo, norway. we have investigated the role of tumour necrosis factor (tnf) and interleukin-i (il-i) in the induction of nitric oxide synthase (nos) by bacterial endotoxin (lipopolysaccharide; lps; 2 mg kg -i i.v.) in vivo. in anaesthetized rats, pretreatment with a monoclonal antibody for tnf (tnfab; 20 mg kg -i s.c., at 16 h prior to lps) or with an il-i receptor antagonist (il-ira; 16 mg/kg bolus and 2.4 mg/kg/h infusion) ameliorated the fall in mean arterial blood pressure (map) at 90-180 min after lps. for instance, endotoxaemia for 180 min resulted in a fall in map from 114-+6 (control) to 79-+4 mmhg (p<0.01; n=8). in contrast, animals pretreated with tnfab or il-ira prior to lps injection maintained significantly higher map at 180 min when compared to lps-control: 118-+3 mmeg (n=5) and 103-+7 mmhg (n=5), respectively (p<0.01). three hours of endotoxaemia significantly reduced the contractile effects of noradrenaline (na) in the thoracic aorta ex vivo. the hyporeactivity to na was partially restored by in vitro treatment of the vessels with ng-nitro-l-arginine methyl ester (l-name, 20 min, 3x10 -4 m). pretreatment of rats with tnfab or il-ira significantly (p<0.05) prevented the lps-induced hyporeactivity of rat aortic rings ex vivo. l-name did not alter or only slightly enhanced the contractions of aortic rings obtained from tnfab or il-ira treated lps-rats, respectively. at 180 min after lps there was an induction of calcium-independent nos activity in the lung (5.14-+0.57 pmol citrulline/mg/min, n=8), which was attenuated by tnfab and !l-ira by 37-+6% and 46-+6%, respectively (n=5; p<0.05). thus, the production of both tnf and il-i contributes to the induction of nos by lps in vivo. the protective effect of agents which inhibit the release or action of tnf or il-i in shock may be, in part, due to inhibition of nos induction. neal garrison, md objective: sepsis is often accompanied by organ dysfunction, in part due to impaired microvascular perfusion. recently, nitric oxide (no) has been described as an important mediator of the hemodynamic changes of sepsis, and no synthase (no-s) inhibitors have been advocated for treatment of septic shock, but their visceral microcirculatory effects are inadequately characterized. we postulated that no-s inhibition would exacerbate the impaired organ perfusion of sepsis. methods: six groups ofdecerebrate rats were studied. bacteremia was induced with 109 live e. coli, which consistently increased cardiac output 15-20% above baseline (bl). the no-s inhibitor nm-nitro-larginine methyl ester (l-name,1 mg/kg iv), prevented this increase and elevated map by 25-30%. in the first 3 groups, total hepatic blood flow (thbf, ml/min by time transit flowmetry) and microvascular perfusion (mi-ibf, ¼ bl by laser doppler flux) were measured. in the other 3 groups, in vivo videomicroscopy was used to observe renal microvascular responses (ila=interlobular artery, aff=afferent arteriole, eff=efferent arteriole; % bl for all). results: data are 60 rains after e. cob. n=5-7/group. * p<0.05 vs bl by remanova and § p<0.05 vs e. coli alone by anova. ec+l-name 11-+2 -57_+10" § -45_+4* § -89_+3* § -33+5* -20+5* § conclusions: l-name administration in controls decreased renal blood flow, indicating no contributes to basal renal tone. bacteremia decreased mtlbf but not thbf, and mi-ibf was further impaired by no-s inhibition. e. coli caused renal preglomemlar, but not postglomerular constriction and reduced flow. l-name exacerbated these e. coli-induced alterations and caused eff constriction. these data indicate that no-s inhibition exacerbates bacteremia-induced impairment of renal and hepatic blood flow, suggesting that no is an importam compensatory dilator mechanism in these organs during sepsis. irf (iron responsive factor) is the central regulatory protein of intracellular iron metabolism able to bind to responsive rna elements (ires) present atthe 5'untranslated region (utr) of ferritin mrna and 3'utr of transferrin receptor mrna. binding of irf to ires results in repression of ferritin mrna translation and increased stability of transferrin receptor mrna leading to enhancement of transferrin receptor translation. we describe here that either tetrahydrobiopterin dependent stimulation as well as cytokine (ifn-~)/lipopolysaccharidemediated induction of nitric oxide synthase activates irf, which is due to direct interaction of nitric oxide with the iron-sulphur-cluster of irf. this was shown by gene expression studies using a plasmid containing a ferritin ire and a cat indicator box which was transfected into k562 myelomonocytic cells, which were shown to have a constitutive form of nitric oxide synthase (nos). furthermore, the increased binding of 1re to irf due to irf activation of irf by nitric oxide was demonstrated by gel shift assays. irf activity was much more increased in cellular extracts from murine macrophages (j774) where a cytokine inducible form of nos has been characterized earlier as compared with irf activity in k562 cells, where nos was stimulated by increasing the availability of the essential nos cofactor 5,6,7,8-tetrahydrobiopterin. we then demonstrated that activation of irf by nitric oxide is accompanied by alterations in ferritin translation as checked by metabolic labeling and immunoprecipitation. these results suggest a reasonable mechanism for the regulation of iron disturbances under chronic inflammatory disorders, characterized by increased concentration of immune activation parameters like ifn-5or neopterin and low serum iron and hemoglobin concentrations. taken nitric oxide, no, the putative endothelial derived relaxant factor, edrf, has been shown to be a potent inhibitor ofplatelet aggregation in vitro. in vivo evidence however, is scarce. accumulation of platelets in the lungs has been shown to occur during extracorporeal circulation. the aim of the present study was to investigate the effect of inhaled no on this reaction. materials and methods: the animals were divided into two groups, each consisting of 7 pigs. platelets were selectively labelled with luln-oxine. dialysis was instituted via catheters in the femoral vessels. in group 1, no, 50 ppm, was added to the inhaled gas from the start of dialysis. in group 2 no was not given. the activity over the lungs was followed dynamically with a gamma camera. central hemodynamics was monitored via a swan -ganz catheter. results: the activity was significantly lower in group 1, from 2 minutes after start of dialysis and onwards, indicating diminished accumulation of platelets in the lungs. parallel to this the hemodynamic response in terms of increased pulmonary artery pressure and pulmonary vascular resistance was blunted in this group conclusion: inhaled no in this model seems to affect pulmonary platelet sequestration. an associated attenuation of the changes in central hemodynamics was also seen. previous studies from our laboratory have demonstrated that vascular contractility decreased in endothelium-intact blood vessel rings in early and late stages of sepsis. although endothelium removal in early sepsis restored vascular contraction, the depressed smooth muscle contractility observed in late sepsis was not restored by endothelium removal. this indicates that impairment of smooth muscleper se may be responsible for such dysfunction in late sepsis. the aim of this study, therefore, was to determine whether or not smooth muscle-derived nitric oxide (no) plays a role in producing vascular smooth muscle dysfunction during late stages of sepsis. to study this, rats (250-300g, n=4-5/group) were subjected to sepsis by cecal ligation and puncture (clp). septic and shamoperated rats then received 3 rrd/100g bw normal saline. the animals were killed at 10, 20, or 35 h post-clp (10 h post-clp=early sepsis; 20-35 h post-clp=late sepsis), and thoracic aortic rings were prepared for contraction studies using organ chambers. the complete removal of endothelial cells was tested by the absence of any significant acetylcholine-induced vascular relaxation. contractile responses to norepinephrine (ne, 10 9 to 10 -5 m) were determined in the aortic rings without intact endothelium. ng-monomethyl-l-arginine (l-nmma, 300/~m, an inhibitor of no synthase) was then added to the organ chamber and ne-induced peak contraction was determined before and after the addition of l-nmma. the peak contraction (rag/rag tissue, mean_+sem) is shown below: the results indicate that the addition of l-nmma did not significantly affect ne-lnduced peak contraction in endothelium-denuded vessel rings at 10 and 20 h after clp. in contrast, l-nmma administration produces an 18% increase (p<0.05) in peak contraction during late sepsis. therefore, the vascular smooth muscle contractile dysfunction observed at 35 h post-clp is partially due to smooth muscle-derived no over-production. thus, unlike macrophages in which inducible nitric oxide synthase (inos) is observed in early sepsis, the inos in vascular smooth muscle appears prominent only in the late stages of sepsis. in three cases of human septic shock in which ng-monomethyi-l-arginine, (l-nmma) a nitric-oxide-synthase-inhibitor was applied, we isolated three completely different types of pathogens: candida, pseudomonas aeruginose and multiresistant coagulase-negative staphylococci. this observation suggests that endotoxin alone is not the main factor triggering hypotension in septic shock by the nitric oxide pathway. in a 68-years-old woman in severe septic shock due to a candida and pseudomonas aeruginosa infection complicated by adult-respiratorydistress-syndrome conditions deteriorated despite adequate conventional therapy. in this trial, effects of l-nmma on cytokin-levels were investigated. the study-protocol was approved by the ethical committee of the department of surgery. after two boll of 200 mg of l-nmma, a continuous infusion was installed (0.05 mg/minute and kg body weight l-nmma). as expected mean arterial blood pressure rose (62 to 134 mmhg}, heart rate stayed stable (124 + 4 b/rain), systemic vascular resistance increased (225 to 354 dyne.sec/cm5), cardiac output decreased (17 to 15.2 l/rain), and cardiac index declined (9.94 to 8.63 l/min/m2}. before and after 90 minutes while the infusion of l-nmma, blood samples for immunological measurements were taken and processed together. pulmonary-shunt-volume was observed before the application of l-nmma, after one hour and after 140 matutes. neopterine increased from 16.51 to 32.55 ng/ml, tumour-necrosis-factor-a increased from 24.16 to 36.61 pg/ml and intedeukin-6 increased from 61.9 to 98.2 pg/ml. immunoglobulines a, g, and m (3.2 to 2.9, 8.1 to 7.6, 1.1 to 0.9 g/i), complement factor c-3c and c-4 (0.54 to 0.50, 0.22 to 0.23 g/i), alpha-l-antitrypsine (3.86 to 3.83 g/i), c-reactive-protein (111.1 to 105.6 rag/i), interleukin-1 (0 pg/ml) and soluble interleukin-2 (2128 to 1983 units/ml) did not change significantly. pulmonary-shuntvolume decreased from 54.1% to 35.4% within one hour and to 36.6% after 140 minutes. in septic shock blocking nitric oxide as an intervention at the end of a not ~,et ful!y understood cascade might have important influences on pulmonary-shunt-volume and inter-cell-communication. department of surgery, pharmacy* and immunology**, university hospital of zurich, r~imistrasse 100, 8091 zurich, switzerland we previously reported that hypoferremic cba mice had an increased resistance to salmonella infection, and that injection of ammonium ferric citrate (afc) to these mice led to enhanced infection (ganthier et at. 1986. microbiol.immuno130:425) . because nitric oxide (no) is involved in the antimicrobial activity of routine macmphages towards various inttacellular pathogens, we investigated the influence of iron on the bactericidal activity of cba mouse macrophages towards s.typhimurium and on the production and activity of reactive nitrogen intermediates (rni). peritoneal macrophages hum cba mice were cultured in the presence (or not) of afc 50 ,um, ifn-,/250 u/ml, lps 1 fig/m/, ngmonomethyl-l--arginine (mmla) 2ram. nitrite (no2-) content of the supematants was determined by a standard griess reaction, and h202 release was measured by the peroxidese dependant oxidation of phenol red. for intracellular killing, macrophages monolayers were infected, and, at various intervals, lysed by triton x-100, and surviving bacteria enumerated by colony counting on agar. for in vivo experiments, mice were infected ip with 0.5 ml of a suspension of 5.10 ~" s.typhimurium, strain c5, and injected with aminoguanidine (ag) 1 mg/ml in saline. our results show that the rn[ inhibitor ag strongly accelerates the mortality of infected mice, the survival rate decreasing from 60% in the control group to 20% in the treated group, 10 days after challenge. correlatively the rni inhibitor mmla induces in vitro a decrease in the rate of bacterial killing, fxom 78% to 57%, in macrophages triggered with ifn-? + lps. the cultivation of macrophages in the presence of afc leads to a decreased no 2-accumulation, 4.7 nmole/well v.s. 21 nmole/well. conversely h202 production is enhanced from 09 nmole/well up to 2,33 nmole/well. nevertheless, macrophages cultivated in the presence of afc exhibit an increased tale of intracellular killing, 77% in iron exposed macrophages v.s, 68% in control macrophages. when triggered with ifn-~, alone, macrophages have a reduced antibacterial activity (57% v.s. 68%) whereas the addition of afc to these macrophagas restores an elevated (72%) rate of killing. in conclusion, the results show that bactericidal activity of cba macrophages towards s.typhimurium depends on the production of no by these macrophages ; but they also demonstrate that no is not the only reactive species involved in the intracellular kil/ing of s.thyphimurium ; indeed afc which strongly inhibits rni production, stimulates h20 2 release by these macrophages and increase their bactericidal activity in vitro. nevertheless afc may promote bacterial growth in vivo. crssa. unit4 de microbiologie. bp 87. 38702 la tronche cedex france. henning jahr, ulrike noack, karin braun the large amounts of no produced by the inducible no synthase in rat macrophages have direct antimicrobial effects, but inhibit the activation of the lymphocyte-dependent host defense system. the aim of this study was to investigate if complement activation influences no-generation. spleen cells from lew rats were incubated at 37°in tcm-199/5% fcs, with or without additional rat serum. after 20 h, nitrite (end product from no metabolism) was measured by oriess reagent. in rat spleen cell preparations, most of the no is produced by macrophages. complement activation in vivo was carried out by i.v. injections of 240 u cobra venom factor/kg b.w. at days 0 and 2. significantly higher (p<o.o1) lps-stimulated no-generation was found in spleen cells prepared at day 4 (13.0 4-1.3 nmol nitrite/106 cells, n=6) compared to spleen cells from non-treated animals (3.6 ± 1.3 nmol, n=6) complement activation was effective also in vitro. when incubated in 25% zymosan-activated rat serum, both basal and lps-stimulated noproduction were enhanced in spleen cells (table, values increased production of nitric oxide (no) is associated with sepsis, however, the effect of no inhibition on survival during sepsis is unclear. we examined the effect of no inhibition on host's ability to eliminate bacteria and survival in mice sepsis model. sixty female balb/c mice were injected with e.coli (10qbody) into peritoneal cavity. the treated group received n-ultro-l-arginine-methyl-ester (l-name), an inhibitor of nos, one hour before bacterial challenge. thirty mice were observed for survival after e.coli challenge and the other mice were sacrificed at 4 hours. blood was obtained by cardiac puncture and peritoneal lavage fluid (plf), liver and lung were harvested. the number of peritoneal exudative cell (pec) was counted and colony forming units (cfld of bacteria in the tissues, blood, and plf were also determined. in addition, pec was cultured in vitro with or without lipopolysaecharide (lps). tnf levels in the blood, plf, and supematants of cultured pec were measured by l929 bioassay. survival rate (%) qrql/,p n 8 hr 24 hr 4 dav control (normal saline 0.1ml/body i.p.) 10 100 50 40 n10 (l-name 10mg/kg i. administration of l-name before e.coli injection increased the number of bacteria in plf and tnf level in plasma, the result suggests that nos inhibitor may depress the host's ability to kill the injected bacteria and that it may induce greater systemic tnf production. we conclude that nos inhibitor is detrimental to animals in sepsis. the hypothesis that sod prevents reperfusion injury to the liver by protecting the endogenous endothelium derived vascular relaxing factor (no) from being inactivated by oxygen free radicals should be investigated. male wistar rats were subjected to 60 min of partial liver ischemia (70%) and 30 min of consecutive reperfusion in situ. some rats were pretreated with the no synthase inhibitor nitro-l-arginin (nla: i0 mg/kg). sod, when used, was given as mn-containing preparation at a dose of 6000 u. i.v. i0 min prior to ischemia. results (sod/ nla+sod/ ni~,, respectively): bile flow (~i/g/min) : 0.93±0.11"#/ 0.52±0.08/ 0.48±0.05. alt (u/2) • 171±40~#/ 477±173/ 506±182. gldh (u/l): 5.2±1.2e#/ 10.7±4.2#/ 24.3±9.1. in absence of nla, sod enhanced postischemic bile flow and reduced leakage of alt and gldh into the plasma, while the effects of sod disappeared, when endogenous 140~synthesis was inhibited by nla. nla had no ~ffect on untreated animals submitted to the same protocol. these results may suggest, that oxygen free radicals interfe~:e with the endogenous vasodilator no and that the benefit of sod can be attribuated in large part to a protection of no during reperfusion. nitric oxide (no) reduces pulmonary vascular resistance and increases oxygenation by inhalation in ards patients [1] . animal studies on the endogeneous no production proved that no is exhaled after synthesis in the respiratory tract [2] . with approval by the hospital ethics committee, we studied healthy volunteers and ventilated patients by measurement of inand exhaled no by no/no2-chemiluminescence in segments of the upper respiratory tract. we found that no is synthesized predominantly in the nose and in the upper nasopharynx, leading to inspiratory tracheal no concentrations of 0.07-0.13 parts per million in non-smoking volunteers; data during mask ventilation per nose were as follows: non 0.089 0.0422 0.0721 0.0311,2 parts per million (ppm) no, mean, n = 10; ~ p < 0.05 (t-test) between smokers and non-smokers; 2 p < 0.05 (t-test) between in-and expiration. about 50% of no is resorbe.d by the lung, and the exhaled no -in contrast to former presumptions -is the remaining part of the autoinhaled no. intubated and ventilated patients are deprived of no autoinhalation, and the exhaled no in the trachea decreases more than 95% {o.043 ppm before intubation vs. 0.003 ppm after intubation, ~, < 0.01), whereas the nasal no concentration increases by cumulation. hence, low-dose no inhalation in ards patients might be considered as a no replacement, especially since the tracheal no concentrations we measured in volunteers are similar to those which could be demonstrated to be effective for ards in former studies [3] . the data demonstrate that no is synthesized in the nose, and inhaled and resorbed by the lung. this phenomenon of no autoinhalation, which is reduced in smokers and in ventilated patients, does possibly contribute to the regulation of the ventilation-perfusion ratio in the lung. kikuchi 1, yoshihiro inoue 1, masao yoshida 2 1 critical care and emergency center, and 2department of bacteriology, iwate medical university. nitric oxide (no) is produced by macrophages and vascular endothelial cells. it is thought to be the true form of endotheliumderived relaxing factor, and to be involved in the fall of blood pressure during septic shock. we have reported previously that endotoxin (lps), tnf-c~ and il-2 contribute to the occurrence of septic shock (circ shock 38:264; . the present study investigated the in vitro effects of lps, tnf-o~, il-2 and ifn-'i on the production of no by macrophages. peritoneal macrophages were cultured with lps, il-2, tnf-~ and ifn-¥. in culture with lps, no was produced in a dose-dependent manner, and the production was suppressed by a no production inhibitor, l-nmma. ifn-y, il-2 and tnf-c~ used alone did not promote the production of no, but helped lps to facilitate no production. a combination of il-2 and tnf-c( enhanced lps-facilitated no production to a greater extent than il-2 or tnf-c~ alone. the above results suggest that these cytokines are involved in endotoxin shock through the mechanisms that facilitate no production. 19-1 uchimaru, morioka 020. japan. t. yolk 1,2, i. ioannidis 1, w.j. kox 2 and h. de groot 1 objectives: nitric oxide (no.) is known to play an important role in neurotransmission, vasoregulation, and bactericidal as well as tumoricidal cellular defense systems. by means of no-donating agents we studied the mechanism of no-mediated cytotoxicity against rat liver microvascular endothelial cells. materials and methods: 3-morphoiinosydnonimine-n-ethylcarbamide (sin-l) and sodium nitroprusside (snp) were used as no. donators, ldh release and trypan blue exclusion were applied to indicate cell viability. results: sin-1 (5mm), which releases both no. and o2-., caused a time-dependent cytoreduction of 60% and 80% after 3 and 6 hrs, respectively. this effect was not inhibited by superoxide dismutase (sod), which removes 02--to form h202 and 02. in contrast, the addition of catalase (cat), which removes h202 to form h20 and 02, diminished the cytotoxic effect ot sin-1 to 30%, equivalent to high concentrations of snp (20mm), which solely releases no.. in addition, the amount of h202 formed by 5mm sin-1 equaled the amount ot enzymaticany produced h202 by 5mu/l glucose oxidase (god). whereas god in this concentration and snp in low concentration (5mm) alone were not toxic to endothelial cells, the combination caused an 80% reduction of viability, comparable to the effect observed with sin-1 (5mm) alone. moreover, toxicity mediated by high concentrations of snp (20mm and 50ram) was reduced by 20% under hypoxic conditions indicating synergism with no-and 02. conclusions: we conclude, that no.-induced toxicity against endothelial cells is enhanced slightly in the presence of oxygen and is not due to an interaction with 02-. however, toxicity increases dramatically in the presence of h202. this may possibly occur either by a chemical formation of more potent reactive hydroxyl radicals or by independent actions on different cellular targets. although it is becoming increasingly clear that nitric oxide (no) is associated with otxan dysfunctions in septic patients, little is known as to die kinetics of no production in these patients to clarify these kinetics, we have investegated serum arid monocyte associated no in septic patients. five patients who had undergone gastrointestinal surgeries mid were complicated postoperatively with severe sepsis served as the subjects in this study (sgroup), using twelve healthy volunteers as a control group (c group). serum no2 and no3 levels were measul*d sliectrophotometricallymonocyte cultures were done for 24 itrs with or without lps+ifn-t stimulation mid no2 and no3 levels in the supernat,'utts were also measured spectrophotometrically. furthermore,using an no selective electrode,time course of the lps+ifn-t induced no production by monocytes was studied. l) serum no2 levels in s group were slightly lfigher than dmse in c group,bnt there were no significoatt differences between the two. 2)both no2 levels in the supematmlts of monocytes cultured with lps+ifn-t mid no2, no3 levels ill lhe supernatants of monocytes cultured without lps+ifn-y were significantly higher in s group. further,the time required for the no production by enltnred monoeytes was siglfificantly shorter also in s groap. conclusions l)in severe septic patients, monocyte no productions wei~ not only primed, but also activated concomitantly, suggesting that these monocyte activations m'e strongly associated with organ dysfunctions in sepsis. 2)based on the restdts of serum no nteasuremeuts, it can be deduced that no p~'oduced by monocytes in septic patients affects tissues and org~s ioeoregiomflly. objectives of the study: nitric oxide (no) is an important messenger which accounts for a wide spectrum of physiological and pathophysiological processes, e.g. mediating vasodilation in endoloxin shock investigating the signal lransducfion pathways involved in the regulation of the inducible nitric oxide synthase (inos), we report the involvement of phosphatidylcholinespecific phospholipase c (pc-plc) and proteinkinase c (pkc). materials and methods: no-production was induced in the murine macrophage cell line j774 with lipopolysaccharide (lps) [lljg/ml] and interferon ;f (ifny) [200u/ml]. after 18 hours of incubation no-release was determined as nitrite (no2) by using a colorimetric assay based on the griess-reaetion. results: preincubation of cells with the pkc-inhibitors staurosporine (stp), chelerythrine, bisindolylmaleimide (bim) and d609, that recently has been identified as a selective inhibitor of pc-plc, diminished significantly lpsand ]fnt-induced no2-production (p<o,001). although we observed no toxic effect on cell viability, no2-production was related to protein concentrations to exclude any inhibitory effect on celt growth. celts preincubated with stp [lo0nm] released 20% less no 2 in response to lps and ifn,[ compared to control cells cultured without inhibitor. chelerythrine [30}jm] and bim [ioijm] reduced the no2-formation by 50% and 85%, respectively. interestingly, the most potent inhibitor of no-production turned out to be the xanthate d609 cells pretreated with d609 [301~g/mt] released 96% less no 2 than stimulated controls the inhibitory effect on no 2production decreased the later the inhibitore were added after stimulation; e.g. bim added 6 and 12 hours after stimulation reduced no2-production only by 50% and 12%, respectively. accordingly, inqs activity present in stimulated cell lysates supplemented with l-arginine and co-factors was not suppressed by the inhibitors tested. conclusions: our findings suggest, that the induction of inos but not its activity is a pkc and particularly pc-plc dependent process. dr. k tschaikowsky, institut for anaesthesiologie, universital erlangen--nqrnberg, krankenhausstr 12, 91054 erlangen interleukin-1 (il-1) affects nearly every cell type by increasing the expression of a variety of genes associated with the promotion of inflammatory processes. these include upregulation of cyclooxygenase and nitric acid synthases. the il-1ri transmits a signal(s) through the cytoplasmic domain which is structurally related to the fruit fly toll protein. we have recently cloned the promotor most proximal to the 5' utr of the human il-1ri gene. the genomic sequences reveal a lack of tata and caat boxes but rather a considerable (75%) gc rich region. the translation of the type i il-1r appears under a significant suppression and may limit the biological activities of il-1 by low level of expression. on the other hand, the type ii il-1r, lacking a significant cytoplasmic domain, does not transmit a signal and acts as a decoy receptor preventing the binding to the type i receptor. there are several approaches to reducing il-1 activity; inhibition of il-1 converting enzyme which cleaves pro-il-l[3, anti-il-1ri, the il-i receptor antagonist (il-1ra) and soluble (extracellular) il-1ri and il-1rii. il-1ra is structurally related to il-1 and binds at 4°c to the il-1ri with near equal affinity as that of bona fide il-i; however, il-1ra does not transduce a signal. il-1ra has been given to humans in pharmacologic levels (mean plasma levels of 30gg/ml) without evidence of agonist activity. in addition, there has been no affect on normal homeostatic parameters, suggesting that il-i does not play a role in health. in healthy humans given intravenous endotoxin, il-ira reduced endotoxin-associated neutrophilia by 50% and completely reversed endoloxin-iuduced immunosuppression, il-tra is produced naturally and is elevated in the circulation in several diseases. in a variety of diseases, plasma levels of il-1ra are in 100-fold molar excess to those of il-i [3. it is unclear whether these endogenous levels of il-1ra are sufficient to block il-1 activity in disease. a single injection of ]l-6 or ifna in humans does not induce il-1 but rather high levels of il-1ra (10ng/ml). prof.dr.l.a. aarden interleukin 6 (il6) is a multifunctionai cytokine with a central role in host defense mechanisms and consequently in inflammation. il6 is thought to be involved in the pathogenesis of various diseases. therefore, specific inhibitors could have therapeutic value. a human-mouse chimeric monoclonal antibody to il6 has been applied in a phasei clinical trial in patients with multiple myeloma and in primate models of sepsis. no toxicity was observed and the most remarkable outcome of this study was the build up in the circulation of il6-anti-il6 complexes, suggesting that plasma il6 measurements represent a gross underestimation of il6 production in the patient. until now no natural antagonists of il6 have been described. the biological activities of il6 results from binding to a lowaffinity il6-receptor (il6r). the il6/il6r complex induces disulfide-linked homodimerization of the signal transducing receptor component, gp130. studies on the structure-function relation of il6 revealed that disruption of the gpl30-interaction site on il6 gives rise to a mutant il6 that, by virtue of its intact binding to the il6r, acts as a potent antagonist on human hepatocytes and on human b cells. in vivo studies using this molecule will be initiated in the near future. the evolution of infla~ation involves a dynamic series of cellular events controlled by specific signals which are important to the initiation, maintenance, and final resolution of the inflammatory response. the various phases of inflammation are influenced via the expression and subsequent regulation of a number of key mediators which play a predominant role during each particular stage of the developing lesion. for example, the initial elicitation of blood born leukocytes to on inflamed area is a complex system that is dependent upon the expression of early response cytokineso these proximal mediators, including both interleukin-i (il-i) and tumor necroisis factor (tnf), are important in the initiation phase by activating both immune and non-immune cells and establishing a series of cytokine networks, thus, the above proximal mediators can stimulate endothelial cells, epithelial cells, smooth muscle cells, and fibroblasts to generate more distal cytokines. these latter cytokines include interleukin-8 (il-8), macrophage inflammatory protein-i (mip-i), and monocyte cbemoattractant protein-i (mcp-i). the importance of the above chemukines can be found in the role they play in leukocyte elicitation, as well as wound repair. numerous investigations have shown the diverse cellular sources of il-8 and the ability of il-8 to elicit neutrophils in vitro at pm to nm concentrations, however, recent studies have sho%~ an additional role for il-8 during the resolution phase of the inflammatory process. using corneal pocket animal models of angiogenesis/neovascularization, il-8 was found to be a potent angiogenic factor at concentrations ranging from 2-40ng/ml. sequential fluorescein angiograms demonstrated that il-8 induced neovascularization by 14 days, which regressed by 6 weeks. in further analyses, both conditioned media recovered from either inflamed human heumatoid synovial tissue macrophages or lps-stimulated peripheral blood monocytes were found to possess potent angiogenic activity, which was effectively blocked by neutralizing antibodies directed against human il-8. in addition, an il-8 antisense nligomunclentide blocked the expression of a functionally active angiogenic factor from lps treated monocytes. the above data demonstrate that il-8 has multifumctional activities during the initiation, maintenance, and resolution of a local inflammatory response. inhibits specific t-cell responses to soluble protein antigens and alloantigens. the proliferative responses of th0, thl and th2 cd4 ÷ t-cell clones and cytokine production by these t-cell subsets are equally well inhibited. the inhibitory effects of il-10 are indirect and related to inhibition of antigen-presenting capacity and accessory function of the antigen-presenting cells (apc). however, il-10 also directly inhibits t-cell proli/eration induced by cross]inked anti-cd3 or anti-cd2 mabs (e.g. in the absence of professional apc), by specifically inhibiting il-2 gene transcription and il-2 protein production. il-10 also inhibits the production of the pro-inflammatory cytokines il-l-a, ~, il-6, and tnf-a and the chemokines il-8 and mip-i-u, which are strong chemo attractants for neutrophils and macrophage, respectively. in contrast, il-10 enhances the production of the il-1 receptor antagonist, a cytokine with anti-inflammatory activity. il-l0 produced by monocytes downregulates class ii mhc expression and il-10 production by these cells in an autoregulatory fashion. collectively, these in vitro data indicate that il-10 is a powerful suppressor factor for immune-and inflammatory responses and therefore may have potential clinical utility as an anti-inflammatory agent. this notion is supported by recent studies which indicated that il-10 also prevents lethal lps-induced toxic shock in mice. five of the chamekines (hip-i~,era~tes. ip-io and il 8) also chemoattraet t lymphocytes, while others act on mast cells. we have studied the effaces of these chemokines on t cell subsets. il 8 preferentially chemoattracts uns~imulated t cells. raises ~ttracte resting as well as activated cd4 and cd8 memory t cells. ip-10 chemoattracts only preactivated (not resting) cd4 or cd8 t cells. hip-l~ preferentially chemoattracts gd8 t calla, while hip-18 more readily attracts the cd4 t call subset. the 8 chemoklnes and ip-io were also sho~rn to promote the adherence of ~he same subsets of anti-cd3 activated t cells to ili activated human umbilical vein endothelial cells (hitvec). this was not associated with a~y i~crease in the number of lymphocyte adhesion molecules, bu~ could be inhibited by neutralizing monoclonal antibodies re lfa-i and vla-4. these chemokines also rapidly increased ~ha adhesion of resting t l~phoeytes to plates coated wi~h 8 integrlns (i-cam or v-cam) or matrix proteins such as f~bronectln. this induction of adhesion began wiehln 30', peaked by 1 hr and was completed in 3 hr. this suggests that chemoklnee rapldly increase the binding affinity of adhesion prote~ns on t cells. we also recently observed ~hat mcaf/mcpi and rantes chemoattract unatimulated mast calls. furthermore, igs actlvared mast calls were chemoattraeted by mcaf, ra~tes, pf4 and mip.i~. however. ~he chemoklnee did not induce mast cells ~o release histamines. presumably, chemokines as regulators of the adhesion, migration and homing of all kinds of leukocytes participate in many types of inflammatory diseases. natural killer cell stimulatory factor or interleukin-12 (il-12) is an heterodimerie cytokine formed by two covalently linked glycosylated chains of 35kd and 40 kd. il-12 was originally purified from the supermatant fluids of ebv-transformed human b ly~phobiastoid cell lines. however, in addition to b cells, phagocytic cells, i.e. monocytes, macrophages, and neutrophils, have been identified as the major physiological producers of il-12. phagocytic cells produce il-12 in response to bacteria, bacterial products such as lps, and intracellular parasites. the cell types on which il-12 exert biological activity are t and nk cells. the major direct biological function of il-12 on these cell types are the induction of cytokine production, primarily ifn-y, the enhancement of a generation of cytotoxic cells, and a mitogenic effect on antlgen-activated lymphocytes. in part thromgh its ability to induce ifn-y production, il-12, produced in response to infections, represents an important functional link between effector cells of innate resistance, phagocytic cells and nk cells, and effector cells of adaptiye resistance, t and b lymphocytes. the ability of il-12 to induce ifn-y production from circulating. nk cells and at least a proportion of t cells determines a rapid, antigen-independet production of ifn-y during infections. ifn-y acts as a potent enhancer of phagocytic and bacteriocidal activity of phagocytic cells, participating early in infection to activate some of the inflammatory mechanisms that represent the first innate resistance against infection. this antigem-independet activation of phagocytic cells that involves, in addition to il-12, other monocyte derived cytokines such as tnf and il-l, has been shown to be important in experimental animals for the resistance against infection by microorganisms such as limteria monocytogens and toxoplasma gondii. in addition to this role, early in infection in the antigen-independet phagocytic cell activation, il-12 has a major effect in the ensuing antigenspecific admptive immune response by facilitating the generation of t helper type 1 (th-i) response and suppressing a th-2 response. the presence of il-12 during antigen stimulation in vitro using human peripheral blood lymphocytes, or both in vivo and in vitro in experimental animals, induces generation of th-i cells producing ifn-y and il-2, and inhibits the generation of th-2 cells producing il-4. il-12 induces a direct differentiative effect on th-cells, priming them for high ifn-y production. ~is priming effect is stable and maintained even when t cell clones are cultured for extended periods in the absence of il-12, however, il-12 needs to be present during the first few days after stimulation with antigen or mitogen and established th clones are resistant to the priming effect of ifn-y. unlike the generation of high ifn-y producing clones, the il-12-mediated inhibition of il-4 producing cells in probably due to selective mechanisms, which may include a selective mitegenic effect of il-12 for thl cells and an inhibitory effect of ifn-y on th2 cell proliferation. in several experimental systems, it has been shown that the enhancing effect of il-12 on th-i responses is dependent on production of if~-y and on the presence of nk cells, possibly needed for the early production of ifn-y. the importance of phagocytic cells and nk cells in determining the characteristics of the th response during antigenic stimulation indicated that the mechanisms of innate resistance have a determining influence on the subsequent antigen-specific immune response: il-12 appears to have a key role in mediating the interaction between phagocytic cells. nk cells, and t lympocytes in these proccessem. trinchieri, g. 1993, interleukin-12 and its role in the generation of t e 1 cells, imm~ol. on peripheral blood monocyte/macrophages, il-13 behaves like il-4 in a variety of assays, and shows both similar and contrasting, activties with either il-10 or ifn-y. il-13 reduces inflammatory monokine and il-10 production. it reduces the pyrogen-induced expression of tissue factor (herbert et el, 1993, febs lett. 328, 268) . it mpregulates manmose receptor and ~c class ii expression, while reducing cdi4 expression. il-13 produces morphological changes (extensive cell processes, aggregation, giant cell formation) which have previously been observed with il-4. it inhibits niv-i production (montaner et el, 1993, i. exp. mad. 178, 743) , and interferes with hiv-induced cell fusion. the activities of il-13 and il-4 differ on t-ly•ocyte and large granular lymphocyte population. in particular il-13 does not exhibit the suppressive effects of il-4 on thl-type-helper functions (ifny production) and cytotoxic functions (perforin production). data will be presented showing that il-4 and il-l3 share a common receptor on a nu~er of cell types, but not on t-lympocytes (see also zmrawski st el. 1993, embo j.7. 2663-2670), explaining both thai common and divergent activities. the levels of il-13~a in activated peripheral blood lymphocytes are greater than or equivalent to those of il-4 mp~ja and the two ml{nas are expressed by different t lympocyte population: in particular, t8 cytotoxic lymphocytes express markedly higher levels of il-13~rna. in vivo, il-13 may thus be contributing, significantly to some of the activites previously ascribed to il-4 il-10. recently, we cloned the human cdna encoding interleukin-13 (il-13). human il-13 is a non-glycosylated protein of 132 aa with a molecular mass (mr) of 10 kd and has biological activities on monocytes and b cells, il-13, like il-4, strongly enhanced the expression of class ii mhc antigens on monocytes and induced expression of cd23 (fc~rii). furthermore, il-13, like il-4 and il~10, inhibited the production of pro-inflammatory cytokines il-1, ]l-6, tnfo~ and chemokines miplc~ and tl-8 by lps activated monocytes. both il-13 and il-4 enhanced the production of il-lra by monocytes. in contrast, il-13 lacked the t cell stimulating activities of il-4. the responses of monocytes to il-13 and il-4 were not additive or synergistic, supporting the hypothesis that il-13 and il-4 receptors are complex and share a common component which is important for signal transduction, taken together, these data indicate that il-13, il~4 and il-10 have important anti-inflammatory activities on human monocytes. in addition, these results indicate that il-13 is unique in that it can modify inflammatory reactions without stimulating (as does il-4) or inhibiting (as does il-10) t cell responses. transforming growth factor beta i (tgf-fll) belongs to a family of proteins that have potent immunoregulatory properties ranging ftom effects on the growth and dfflerentiafion of primitive stem cells to the differentiated functions of immune system effector cells. tgf-i~i is synthesized as a large secretory precursor polypeptide of 390 amino acids that is inactive until processed to a disulfide linked homodimeric molecule of 1t2 amino acids each. recently it has been found that tgf-[~i can have autocrine as well as paracrine effects on the immune system indicating that immune cells can activate the inactive secreted form of tgi~-~i. in addition, tgf-i~i has differential intraceuular effects on cell surface receptor modulation, tyrosine phosphorylation and cytokine gene transcription as well as cell mediated cytotoxicity. the systemic administration of tgf-131 has proven beneficial in a variety of animal models such as septic shock, allograft rejection and experimental forms of autoimmunity including collagen induced arthritis and experimental autoimmune encephalomyelitis. moreover the increased levels of tgf-ill and other tgf-13 isoforms found in several disease states associated with immunosuppression such as different forms of malignancy, chronic degenerative diseases and aids implicate the involvement of tgt~-i~ in the pathogenesis of some diseases. hopefully, well designed clinical trials will define the potential roles of the tgf-[3 family of proteins in the treatment of diseases of man. patient development of systemic inflammatory response syndrome (sis) after severe trauma is accompanied by a wide range of immune aberrations and altered cytokine production. in particular, t lymphocyte proliferation and ifn, t production is suppressed while monocyte (mo) tnfc~, il-6, pge2 and tgf~ production is massively increased concomitant to decreased antigen presenting capacity. recently, two new cytokines, il-10 and il-12, have been characterized as critical in regulation of mo tnfa and il-6, as well as in induction of t lymphocyte proliferation and ifn 7 production. in this study, peripheral blood leukocytes (pbl) from severe trauma patients (injury severity score >35) were analyzed for their il-10 levels, their in vitro proliferation to mitogen (pha) and their response after il-12 addition. since il-10 produced either by mo or by t lymphocytes can depress m~ antigen presenting capacity, inhibit t cell ifn,/production and directly diminish t cell proliferation, it might be suggested that immunosuppressed patients' mo and/or t lymphocytes would have increased il-10 levels. increased patient il-10 production might also be resulting from the high levels of tnfa a known stimulator of il-10. conversely, since il-12 augments mo antigenpresenting capacity, thl induction and proliferation, post-trauma leukocytes might be il-12 deficient. pbl of trauma patients were compared to normals' pbl, either unstimulated or ptta induced, and their levels of il-10 found to be dramatically and significantly reduced. patients' isolated m~, either stimulated with the bacterial cell wall analogue, mdp, or unstimulated, also had depressed il-10 production concomitant to elevated tnfa production when compared to normals' mo. mechanisms for the depressed patients' mo il-10 were explored. increases in tgf[3 may have partially contributed to the patients' depressed il-10 level, but elevated pge2 had no effect. addition of il-12 to patients' pbl significantly increased their mitogen responses. these data imply that sis is characterized by disruption in the interactions between mci and t lymphocytes so that patients' m~i produce excesses of some mediators (tnfa, il-6, pge2) and a dearth of other monokines (il-12, il-io). t lymphocytes are not activated and, therefore, unable to function in both immune defense and monocyte regulation. it is known that lge receptor-mediated or ca-ionophore-induced activation of mouse bone marrow-derived mast cells (13mmc) may result in the production of different cytokines including the interleukins (il) 3, 4, 5, and 6 as well as gm-csf and tnf-a. in the present study we analyzed the effects of exogeneously applied pro-inflammatory cytokines (il-1, 1l-6, tnf-c 0 as well as various mast cell growth factors (il-3, il-4, il-9, il-10, ngf, kl (kit ligand)) on cytokine production in primary mouse bmmc using a standard activation protocol (lxl06 bmmc/ml; ll.um ionomycin; 24-48h). the actixdties of bmmc supernatants were assessed in specific biological (il-3, il-4 il-6, 1l-9) and/or elisa assays (il-5, il-9). here we show that homogeneous populations of bmmc (>97%alcian blue+/safranln-; in vitro age: 4 weeks) generated in the presence of recombinant (r) rail-3 from normal balb/c mice produced modest amounts of 1l-6 and low or undetectable levels of il-3, -5, and -9 after induction with lp.m ionomycin only. however, a dramatic increase (5-to 10-fold) of these cytokine activities was noted, when in addition to ionomycin also human (11) rll-la was provided during the induction period. this il-1 effect was dose dependent with a maximgm at 2-10 u/ml hrll-la and specific, as pre-incubation (lh) of bmmc with 20 ng/ml hrll-1 receptor antagonist abolished the action of 2 u/ml hrll-lcc similar effects were noted with hrll-lg or rurll-lb (lng/ml, respectively), but not with rhll-6 or rmtnf-~. both mrll-10 and hrll-10 substantially enhanced ionomycin-induced 1l-6 production of bmmc in the absence or presence of il-1. il-10 significantly enhanced il-6 and il-9 production while decreasing il-3 activities to abont 50-90% of control levels, when il-i0 was provided in the presence of il-l/ionomycin. a monoclonal anti-nfil-t0 antibody (ascites 1:200) abrogated the effects of mrll-10. other mast cell-active cy~okines (]1,-3, il-4, 1l-9, ngf, or kl) added to ionomycia-or 1l-1/ionomycin-treated bmmc had no major effects on cytokine production. il-1 and il-i0 did not induce significant cytokine release in the absence of ionomycin suggesting tlmt cadependent signalling was required. at doses of 10"6m, dexamethasone, corticosterone, or hydrocortisone almost completely abolished ionomycin/il-1/ll-10induced cytokine production. the inducer cocktails per se did not interfere with the cytokine bio-assays. in case of il-9 inducibility of this cytokine in bmmc was confirmed at the mrna level by northern blot analysis. hence our data show that activated mast cells are a source of il-9 previously recognized as a product of th2type lymphocytes only. moreover, our study reveals novel functional roles for i-l-i, il-10, and ghicecorticoids in the regulation of cytoldne production in mast ceils. accumulating data suggests that cytokines, peptides involved in regulation of both physiological and pathological immunological responses, predominantly are produced at the local site of antigen stimulation. a new method was used to detect cytokine-producing cells in haman tissue at the protein level. single-cell production of 17 different httman cytokines, ilia, ill [3, illra, il2, il3, il4, il5, il6, ils, ill0, gm-csf, tnfa, ifn 7 and tgf[31.3, was identified by indirect immunohistochemical staining procedures and use of carefully selected cytokine-specific mab's. frozen sections were fixed with 4 % paraformaldehyde and permeabilized by 0.1% saponin treatment, eluting cholesterol from the membranes. the intracellular presence of all cytokines except ill, illra (late) and tfg[31_3, could be demonstrated by a characteristic perinuclear configuration in producer cells. in addition, the immunoreactivity extended over a large extracellular area encompassing the producer cell. a localization of the cytokine to the golgi-organelle was established by use of two culour staining including a haman golgi complex specific mab. this staining pattern was only evident in producer cells because injection of recombinant human cytgkines into the tissue caused a membraneous and extracellular staining pattern. both the extra-and the intracellular types of staining reaction could, however, be blocked by preincubating the cytokine specific mab with pure human interleukins. oxygen radicals (or) directly induce lipid peroxidation, indirectly they trigger adhesion and activation of pmn leukocytes. we investigated whether or also lead to a release of acute-phase response cytokins such as tnf-alpha, il-i beta or il-6 in whole blood cultures to maintain the induced inflammatory reaction. methods: blood samples from healthy volunteers (n=10) were incubated at 37°c. or were produced by the xanthine oxidase (xo)/ hypoxanthine (hx) system. after 0,30,60,120,240 and 360 minutes plasma levels of tnf-alpha, il-i beta and il-6 were determined with elisa kits. results: under the influence of or tnf-alpha plasma levels increased from 0,5 pg/ml at 0min to 14pg/ml, 236pg/ml, 343pg/ml after 60, 120 and 240 min. il-ibeta (0,3pg/ml, 0,6pg/ml, 1,5pg/ml, 81pg/ml and 169pg/ml after 0,60,120,240 and 360min) and il-6 (0,2pg/ml, l,lpg/ml, 4,1pg/ml, 58pg/ml and 72,9pg/ml after 0,60,120,240 and 360min) plasma levels were increased 60min later than tnf-alpha. summary: these data suggest that or do not only play an important role in initial accumulation and activation of pmn leukocytes but also lead to a stimulation of monocytes to produce the acute phase reaction cytokins tnf-alpha, il-i beta and il-6 to maintain and strengthen the inflammatory reaction. department of general surgery, steinhsvelstr.9, 89070 ulm, germany jan k. horn md, greg a. hamon md, robert h. mulloy md, greg chen bs, rebecca chow bs, and christof birkenmaier md. transforming growth factor-i~l (tgf-131) is released from inflammatory ceils following injury and in sepsis. in vitro experiments have confirmed that low concentrations of tgf-131 (0.1-1.0 ng/ml) are chemoattractive for monocytes, whereas higher levels of tgf-131 (>5.0 ng/ml) potentiate production of the immunedepressive prostaglandin e 2. other investigators have shown that tgf-]31 can cause the appearance of cd16 (fc immunoglobulin receptor) on monocytes exposed to 10 ng/ml of tgf-[~i for 24 hours. monocytes also express on their surface a glycoprotein that binds complexes of lipopolysaceharide (lps) and lpsbinding protein (lbp). such binding is associated with generation of proinflammatory cytokines such as tumor necrosis factor alpha. we have shown that cd14 is depressed in septic patients and therefore we hypothesized that tgf-131 could account for the down-regulation of cd14 observed in these individuals. we incubated normal human monocytes with platelet-derived tgf-[31 for 2 and 24 hours at 37°c and examined ceils for cd14 and cd16 expression using flow cytometry after immunnfluoreseent staining with appropriate monoclonal antibodies. monocytes were selected on the by usual criteria for size and granularity. non-viable ceils were excluded with the use of propidium iodide. two populations of monocytes could be found afcer incubation at 37°c alone. one displaying high density of cd14 had increased fluorescence over the homogeneous expression of cd14 in cells maintained at 4°c (baseline). the other population displayed decreased cd14 expression relative to the baseline cells. tgf-i~i (10-50 ng/ml) caused a shift of ceils from the high density into the low density cd14 population. this trend was observed within 2 hours of incubation and was complete by 24 hours. we observed a net decrease in cd14 expression 0f32% for all subjects studied (p<0.05 vs controls). phorbol myristate acetate (100 ng/ml) also caused down-regulation of cd14 to a similar degree as tfg-i~i. we also confirmed that monocytes could be induced to express cd16 after incubation with tgf-131 (10 ng/ml) for 24 hours. these studies demonstrate that monocytes incubated with immunodepressive levels of regulation of cd14 by tgf-131 deplete their surface expression of cd14 while generating cd16. this down-regulation of cd14 by tgf-131 correlates with our clinical observations of lower cd14 expression on monocytes obtained from septic patients. for over 25 years, activated t lymphocytes have been considered to be the cellular source of mif. we recently isolated and cloned the murine homolog of mif after identifying the specific secretion of this protein by lpsstimulated pituitary cells in vitro and in vivo. however, further experiments showed that mif protein is detectable both in t-cell deficient (nude) and hypophyseetomized mice, suggesting that yet additional cell types may produce mif in vivo. since monocytes/macrophages are a major source of the cytokines that appear in response to lps administration, we examined the possibility that mif also is expressed in cells of the monocyte/macrophage lineage. we found that mif is expressed constitutively in the murine macrophage-line raw264.7 and in thioglycollate-elicited peritoneal macrophages. significant amounts of mif mrna (rt-pcr) and protein (western blotting) were observed in cell lysates. in raw 264.7 cells, mif secretion was induced by as little as 10 pg/ml of lps (e.coli 011 l:b4), peaked at 1 ng/ml, but was not detectable at lps concentrations >1 txg/ml. similar data were obtained with elicited macrophages, but higher lps concentrations were required, unless the cells had been preincubated with ifn 7. production of mif by lps-stimulated (l ng/ml) macrophages peaked at 12 hr. expression ofmif mrna and tnf mrna by lps-stimulated raw 264.7 macrophages was investigated by rt-pcr. as expected tnf mrna expression increased over the range of lps concentrations (1 pg/ml to 1 p_g/ml). in contrast, levels of mif mrna correlated inversely with lps concentration. by competitive pcr, mif mrna was observed to increase approximately 2-fold after lps induction (100 pg/ml). mif secretion also was induced by tnfoc (1 ng/ml) and ifn? (10 iu/ml), but not by il-113 and il-6 (up to 10 ng/ml). lps and ifn 7 had additive effects in inducing mif secretion. in separate experiments, macrophages stimulated with recombinant mouse mif (1 gg/ml) were found to secrete bioactive tnf~ (>750 pg/ml by l929 cytotoxicity). we conclude that the macrophage is an important albeit overlooked cellular source of mif in vivo. mif secretion is induced by lps, tnfc~ and ifn?. mif also stimulates macrophages to secrete tnf. taken together with previous observations that anti-mif antibody protects against lethal endotoxemia, these data implicate mif as a critical mediator of inflammation and septic shock. inflammation is characterized by an exacerbation of proinflammatory cytokine production. cytokines such as il-4, il-10, and tgf8, have been identified as anti-inflammatory mediators thanks to their ability to down regulate the production of il-1, il-6, il-8, tnfc~ by activated monocytes / macrophages. however, other cells, including polymorphonuclear cells (pmn) do contribute to the release of pro-inflammatory cytokines. we investigated the capacity of the so-called anti-inflammatory cytokines to control the release of il-8 by activated neutrophils. human pmn were purified following glucose-dextran sedimentation and ficoli-hypaque centrifugation. the cells were cultured at 37°c for 24h in the absence or presence of lipopolysaccharide (lps) or tnfa. il-8 release was measured in the supernatants using a specific elisa. among tested cytokines, il-10 was the most efficient inhibitor of il-8 production by lps-activated pmn. il-4 was also active, whereas no down regulation was noticed with tgfp~i. when tnfa was used as a triggering agent, none of the cytokine could prevent il-8 production. northern analysis are under investigation to precise the level of the il-4-and il-10-induced inhibition of il-8 production by pmn. our data illustrate that il-10 and il-4 possess the capacity to down regulate the production of il-8 by both monocytes and pmn, whereas tgfb has a more limited inhibitory activity. ciliary neurotrophic factor (cntf), a member of the il-6 superfamily, has recently been shown to promote axonal growth and neuronal healing. cntf production is also increased during neuronal and muscle damage, associated with soft tissue injury or trauma. we postulated that production of cntf may explain the loss of skeletal muscm protein that occurs in inflammation. 40 female, wistar (175-200 gm) rats received either 250 or 25 pg/kg bw s.c. injections of recombinant rat cntf for seven days, or received sham injections and were freely-fed. additional animals were pretreated with 10 mg/kg ibuprofen lp prior to 250 pg/kg bw cntf. rats treated with 250 ,ug/kg bw cntf lost 6.8_+0.4 gms bw as compared to freely-fed controls which gained 17.0_+3.1 gms (p<o.001 by anova and lsd), and ibuprofen treatment had no effect on cntf-induced weight loss (-5.8_+0.7). animals pair fed to the high dose cntf gained body weight (10.2-+3.5), although weight gain was less than seen in freely-fed controls. rats treated with 25 pg/kg bw had reduced body weight gain (+7.8+_4.4 gin). food intake was also reduced by 29% in 250 pg/kg cntf (from 23.7+_0.7 gm/day to 16.8_+0.6 gm/day; p<o.05) and was also unaffected by prior ibuprofen treatment (18.4+-0.6 gm/day). despite the loss in body weight noted in the high dose cntf animals, there was no appreciable hepatic acute phase response, since liver weight (8.6+0,1 gms vs. 9.4_+0.3 gins), total protein (54.5+-2.9 gms/l vs 44.8+-3.6 gins/l) and albumin (29.0+1.5 gms/l vs. 26.0+_1.6 gms/l) were not different between cntf and freely fed animals. we conclude that cntf causes anorexia, and increases body weight loss in excess of the associated anorexia. unlike the cachexia associated with il-1 and tnf infusions, which is associated with an acute phase response, cntf acts predominantly on food intake and carcass weight through a prostaglandin-independent mechanism and has only minimal acute phase inducing properties, n. joseph espat, md. university of florida, department of surgery, j-box 100286, gainesville, fl. 32610. of cytokines by neurotrophic factors, the neuroendocrine system and phenotiazines. cytokines, including tnf and il-1, have a pathogenetic role in various diseases related to infection and inflammation. the action and/or production of cytokines can be regulated by drugs or endogenous mechanisms that involve the cetral nervous system (cns) we found that the antipsychotic chlorpromazine (cpz) potently inhibits tnf production and protects mice against endotoxic shock. cpz also inhibits brain tnf production in mice intracerebrally injected with endotoxin, whereas cpz analogs that do not cross the blood-brain barrier inhibit only perypheral, not brain, tnf. tnf production and susceptibility to the toxicity of endotoxin, tnf and il-1 are increased in adrenalectomized mice, indicating that endogenous corticosterone (cs) controls cytokine production and toxicity, in a feedback fashion since endotoxin and cytokines increase serum cs. increased tnf production was also observed in mice where the hypothalamus-pituitary-adrenal axis was blocked by chronic administration of dexamethasone, following a two-day washout. administration of cytokines acting through the gp130 signal transd,uction protein, including il-6 and ciliary neurotrophic factor, cntf potentiated il-l-induced elevation of cs. il-6 and cntf also induced hepatic acute-phase proteins. thus il-6 and cntf might have antiinflammatory activity, by potentiating the feedback responses of the neuroendocrine axis. these data indicate how complex can be the interregulatory relationships between the brain and the immune system, how they can be used to pharmacomodulate cytokine action and how their disregulation might exacerbate il-1-and tnf-mediated pathologies. lipopolysaccharide (lps) constitutes a well established activator of monocytes/ macrophages (mizi) and murine b lymphocytes. the hydrophobic part of lps, termed lipid a, represents the endotoxic principle of lps. we now demonstrate that lps is also capable of inducing proliferation of human t cells at a dose of 10 to 1000 pg/ml. the specificity of this stimulation was analysed by the following experiments: i) the synthetic hexaacyl escherichia coiltype lipid a (compound 506) also stimulated human t cells; ii) the synthetic tetraacyl lipid a precursor (compound 406) or the 1phosphono-oxyethyl analogue pe-4, structures known to antagonize lps-induced monokine production in human mz, also inhibited lpsinduced t-cell proliferation. the t-cell proliferation expressed the following features: i) cd4 ÷-as welt as cd8 ÷ t cells proliferate in response to lps, ii) limiting dilution analyses reveal that the frequency of responding cells was between 1/500 to 1/1000 cells; iii) purified t cells alone cannot be stimulated by lps, but need the help of monocytes. this help cannot be replaced by b cells or fixed monocytes. furthermore, for activation a direct cell-to-cell contact between t cells and monocyte is neccessary; iv) t cells stimulated with lps express mrna for il-2 and ifnf, but not for il-4 or il-5 (determined by pcr). in supernatants, ifnf was detectable (elisa); v) lps-activated cd4 ÷ as well as cd8 + t cells express cd25, the high affinity il-2 receptor. our results indicate that in contrast to previous reports human t cells respond to lps with il-2 receptor expression, lymphokine production, and proliferation. the reactive cells appear to belong to the t,1 cell type, the finding that human t cells can be activated by lps is of important relevance for the understanding of the pathomechanisms of infections by gramnegative bacteria. this may lead to new strategies for the therapy of sepsis. we have recently shown that human eosinophils produce mrna for interleukin (il)-8 when stimulated with calcium ionophore (eur. j. immunol. 23 (1993), 956). we now present evidence that human eosinophils contain preformed il-8 protein although they do not express mrna for il-8 as measured by rt-pcr. il-8 protein expression was determined using specific anti-human il-8 monoclonal antibodies by western blotting, facs analysis and immunohistochemistry. moreover, preformed il-8 was re/eased into supernatant by 99% pure eosinophils after priming with gm-csf or il-5 and subsequent 25-min stimulation with paf, rantes, ionomycin or pma, however not with il-1 or il-2, as measured by elisa. this observation implies that activation of protein kinase c and/or intracellular calcium mobilization are necessary events for il-8 release in human eosinophils. the determined amounts of preformed il-8 protein was higher in patients with asthma compared to normal individuals suggesting a role for eosinonhi! derived il-8 in asthma. since the eosinopnit is the ,,r~:=z~mmant cell in the asthmatic airways, we determined il-8 concentrations in bronchoaiveolar lavage (bal. ~ids from normal !i~.~ijvtduals and asthmatic patients. indeed, il-8 concentrations in bals from both groups were similar to those seen in the supernatants released by activated eosinophils. the role for il-8 in asthma remains to be determined. based on a well established rat model named activity-induced ulceration (asu) or activity based anorexia (aba), we have developed a rat model for chronic stress. male rats were exposed to a feeding schedule in order to reduce body weight by 20-30% within 14 days. while one group of rats was kept sedentary, the other had access to a running wheel. food restricted rats allowed to exercise developed highly increased activity as compared to ad libitum fed rats in wheels (ca. 15 versus 2 kin/day). they developed elevated plasma corticosterone levels (30_+5gg/dl) at their circadian peak, increased adrenal weight and decreased thymus and spleen weight as compared to control rats and weight matched sedentary rats, establishing this paradigm as a model of chronic stress. no differences in plasma acth were seen among the various groups, suggesting increased sensitivity of the adrenal glands to acth. the effect of chronic stress accompanied by hypercorticism on baseline and endotoxin-induced cytokine levels was studied in this model. under baseline conditions,splenic il-1 a was suppressed in both food-restricted groups. this was more pronounced in rats with access to a running wheel. il-6 and tnf activity in plasma was not affected. after administration of 1001.tg/kg lipopolysaccharide (lps), tnf activity in plasma was suppressed by food restriction but there were no differences between sedentary or exercising rats. in addition, no differences were found among groups for corticosterone, spleen il-lc~ and plasma il-6 activity. we conclude, that the food restriction-induced hyperactive rat model is a useful model for studies on the effects of chronic stress. in this model, under basal conditions il-le~ in tissues may be subject to downregulation by glucocorticoids, whereas after lps-stimulation of cytokine production only tnf activity is regulated by fast acting feedback loop between cytokines and the hypothalamo-pituitary-adrenal axis (hpa-axis). this is supported by strong correlations between plasma tnf activity and corticosterone in stressed rats. cytokine antagonists modulate cytokine actions and it appears that a balance between production of cytokines and cytokine antagonists is important to control of host responses. recent observations show that during myocardial infarction there is an increase in circulating cytokines such as tumor necrosis factor (tnf) and interleukin 6 (il-6). we tested the idea that compensatory increases in cytokine antagonists likewise occur and investigated changes in plasma concentrations of tnf and interleukin i (il-1) and those of their specific antagonists interleukin-1 receptor antagonist (il-1 ra) and soluble tumor necrosis factor receptor type i (s tnf r-i). we also measured plasma concentrations of ~-melanocyte-stimulating hormone (~-msh), an endogenous neuropeptide that occurs in pituitary, brain, skin, gut and other tissues. when administered to laboratory animals, this peptide has potent antiinflammatory and antipyretic activity, perhaps by mimicking an endogenous modulatory influence on cytokine actions. samples were obtained from 20 patients with acute myocardial infarction at presentation in the coronary unit, every three hours for 24 hours, and daily thereafter until discharge. we found elevations in the plasma cytokines il-1 and tnf only in a proportion of subjects whereas cytokine antagonists c~-msh, il-lra, and stnfr were elevated in all patients, o~-msh was elevated at presentation but it decreased progressively in successive tests, reaching normal values within 24 hr whereas stnfr and 1l-lra peaked at 3-6 hr or later. there were significant correlations between plasma concentrations of cytokine antagonists and enzymes released from injured myocardium, including creatine kinase (ck), aspartate serum transferase (ast), and lactate dehydrogenase (ldh). the data show that cytokine antagonists increase in the circulation of patients with acute myocardial infarction likely to modulate prninflammatory effects of cytokines. objects of the study: interleukin-6 (il-6) is a multifunctionai cytokine known to be involved in important homeostatic processes. il-6 levels are increased in many pathological situations, and correlates with disease activity and patient outcome. to exert its effect il-6 binds to its receptor on the membrane of its target cell. this receptor is also present in a soluble form in plasma and in urine. in this study we quantitatively measured the availability of soluble il-6 receptor (sll-6r) in biological fluids in healthy volunteers. materials and methods: blood, urine, cerebrospinal fluid (csf), and synovial fluid (sf) are obtained from healthy volunteers, sil-6r and il-6 are measured using elisa's. both elisa's are tested for interference of sil-6r mid il-6. the effect of sll-6r in the il-6 bio-assay (b9) is tested. results: baseline levels are (mean + sd): 76.6 -+ 19.3 ng/ml in serum, 3.7 -+ 1.3 ng/ml in urine, 1.64 _+ 0.4 ng/ml in csf and 11.6 + 3.3 ng/ml in sf. there is no interference of sll-6r in the il-6 elisa or bio-assay and no interference of 1l-6 in the sil-6r elisa. conclusions: in all investigated biological fluids sll-6r can be found. these data provide baseline values for investigations concerning pathological situations. both elisa's described are useful tools to do these investigations. trauma-associated immune aberrations coincide with augmented release of immunoregulatory and proinflammatory cytokines. the present study examines the effect of thermal trauma on the capacity for specific (receptormediated) response of lymphoid cells to interleukin 1 (il 1) and to turnout necrosis factor ~x (tnfc0. methods. bum patients (n=24, 25->75 % total body surface area) were studied weekly up to 42 days post-injury. the limulus amoebocyte lysate (lal) test was used to measure plasma endotoxin levels. the percentage of il 1~-and tnfcz-binding t(cd 5) lymphocytes was assessed by flow cytometry analysis. levels of il 1 receptor antagonist (il lra) in patients' plasma and cultures of peripheral blood ceils (pbc) were determined by immunoassay. results. plasma endotoxin concentrations were significantly (p<0.05) increased up to 3 weeks post-bum (means 3.5+1 in non-surviving and 2.6+ 0.6 u/ml in surviving patients vs < 1u/ml in the control). within 2 weeks of bum, the percentage oft ceils expressing receptors for tnfa and il 1[~ constitutively was elevated (by 10-15 fold). in contrast, the capacity for de novo receptor expression by activated pbc was reduced. serum levels of il ira were significantly increased (range 2.5-40x10 j pg/ml vs <0.2x10 j pg/ml in the control). in all patients, high concentrations of il lm were released spontaneously in unstimulated cultures of adherent ceils (range 20-30x10 -3 pg/ml vs 4-8x10j pg/ml in the control). however, its secretion was decreased in lps-stimulated parallel preparations. conclusions. in the bum patient, susceptibility to the immunoregulatory effect of tnfcz and tl 1~ may be modulated by infection-related products. alterations in the capacity for receptor expression and secretion of 1l lra may affect il 1-regulated biological responses including specific immune reactions. while studies suggest that il-10 is an important lymphokine involved in cell-mediated immunity, little is known about this mediator's role in hem-induced immunesuppression. our aims, therefore, were to determine: i) if il-10 contributes to depressed t-cell responses seen following hem; and 2) how other agents, known to play a role in hem, effect il-10 release. to study this, c3h/hen mice were bled to and maintained at a map of 35 mmhg for 1 h and then adequately resuscitated. mice were killed 2 h post-hem to obtain splenic t-cells (nylon-wool purified). il-10's immunosuppressant role was demonstrated by the ability of monoclenal antibody (mab) to il-10 to markedly improve the t-cell proliferative response [2.5 #g the marked increase in capacity of t-cells from hem mice to produce il-10 was significantly reduced by treatment with either ibu or mabs. since ibu, tgf-~, as well as il-6 are all reported to directly/indirectly influence prostanoid synthesis, this implies that eicosanoids play a major role in inducing il-10 release by t-cells following hem which depresses t-cell function. the mechanisms underlying immunosuppression induced by thermal injury and alcohol ingestion are in part due to cytokine dysregulatinn. il-10 down-regulates production of eytokines by maerophages and may be an important regulator of the initiation of the immune response. il-10 has also been demonstrated to inhibit the production of no by macrophages. this study examined the alterations in eytokine production and effect of inhibition of no production on immunologic function in a routine thermal injury model. methods: balb/c mice (n=40) were randomized to 4 groups: saline-sham(ns-sham), alcohol-sham(etoh-sham), ns-bum, etoh-bum. animals received 20% etoh or ns daily for 14 days by gavage. a 30% full thickness bum was induced 4 hrs after the last dose of etoh or ns. animals were resuscitated, then sacrificed 4 days post bum. splenic lymphocytes were cultured for 5 days with lps, and lps with two concentrations of n-monomethyl-l-arginine, a nitric oxide inhibitor (l-nmma 2.5ug/ml, 10ug/ml). splenocyte production of il-10, interferon-gamma, il-2, pge2 were measured, and lymphocyte proliferative response examined. results: il-10 production was significantly suppressed in thermal injury. exogenous l-nmma normalized the suppression of 11.-10 in a dose-dependent manner, indicating nitric oxide may modulate il-10 and interferon-gamma production in thermal injury. il-10 production is normal in etoh-burn animals. conclusion: il-10 and interferon-gamma production is altered in this murine thermal injury model, and may contribute to this injury-induced immunosuppression. inhibition of no synthesis normalizes il-10 production and should be investigated further as an immanomodalator in thermal injury. surgery, infection and inflammation results in the production of pro-inflammatory cytokines which mediate metabolic and immunologic host responses. the aim of this study was to characterise the elaboration of cytokine release following a variety of surgical procedures. twenty one patients undergoing elective intermediate, hip, knee and major gastrointestinal surgery were studied. levels of interleukin-1 (i1-1), interleukin-6 (i1-6), the interleukin-1 receptor antagonist (i1-1ra) and the acute phase c-reactive protein (crp) were measured in bloods drawn 0, 1, 2, 4, 6, 12, 24 and 48 hours following operation. a portion of the results are shown (mean -+ sem). 135+37 1738-+843 145_+24 one and two factor anova; *p<0.0002, #p<0.0001, §p<0.0009, ¶p<0.0014, for differences between groups i1-1 was not detected at any time point. both ii-ira and i1-6 increased after surgery. maximum responses occurred following major git and hip surgery, minimal responses were seen after intermediate and knee surgery. ii-ira levels increased within two hours and remained elevated for 24 hours; the b-ira increase was a thousand fold greater than the rise in i1-6 levels. i1-6 levels increased up to 24 hours after surgery. crp levels reflected maximum ii-ira and i1-6 levels (r2=.676, p<0.0001 and r2=.282, p<0.04 respectively). high ii-1ra and i1-6 levels reflect major surgery, however the ii-ira response is more rapid and of greater magnitude. the strong i1-1ra correlation with crp may indicate that this regulatory cytokine is itself a mediator of host responses to surgery. dept. of surgery, meath/adelaide hospitals, heytesbury st., dublin 8, ireland. change of il-6 and soluble il-6 receptor levels after surgery s. hisano, k. sakamoto, s. mita, t. ishiko, m. ogawa [objectives] under surgical stress, il-6 plays a main role in producing acute phase proteins and contributes to host defense mechanism. soluble il-6 receptor (sll-6r) is considered to be agonistic to il-6, unlike other soluble type receptors of cytokines. here we measured il-6 and sll-6r levels in the serum and drain fluid from surgical field in order to investigate the changes of il-6 and sll-6r after surgery and their origins. [materials and methods] serum and drain fluid samples from 21 cases (6 of esophagectomy and 15 of gastrectomy ) were serially collected before and after surgery. il-6 and sll-6r levels were measured by elisa. [results] (1) serum il-6 : all cases reached the maximum level on pod-l, more precisely 3-6 hours after operation. (2) il-6 in the drain : maximal il-6 levels in the drain were recognized 3-6 hours after operation, at almost the same time as serum il-6. furthermore the il-6 values in the drain were much higher, about 100 times, than those in serum. (3) sll-6r in the serum : all cases reached minimum levels 3-12 hours after operation and recovered to the preoperative levels a few days later (decrease ratio : 43.9+5.6~,, range : 9-84~'). (4) sll-6r in the drain : sll-6r levels in the drain showed almost the same value and change as serum sll-6r. [conclusions] (1) il-6 is produced from the cells gathering around operative fields whereas sll-6r is considered to be produced in the cells which do not gather around the operative fields. (2) there may be a mechanism that down-regulates sll-6r in the early stage of surgery. [objectives] il-6 plays an important role in host defense in the early stage after surgery. in the present study, we examined changes in il-6 concentration after major thoracoabdominal surgery and elucidated the effect of surgical trauma and factors influencing postoperative elevation of serum il-6. [materials and methods] thirty-eight patients undergoing elective surgery of the thoracoabdomen were classified into 6 groups according to the location of the operation. bloods and drain fluids were serially obtained and samples were frozen until measured, keukocytes were simultaneously collected for northern blot analysis. concentration of il-6 was measured by elisa and il-6 mrna was detected by northern blotting after total rna was extracted by the acid guanidium phenol chloroform method. [results] (1) serum il-6 levels reached the maximum concentration on the 1st postoperative day in all patients. (2) the il-6 peak was significantly correlated with surgical trauma as defined by the operation length and the volume of blood loss during operation (r=0.554, p<0.01, r=0.427, p<0.01, respectively). (3) the peak concentration of serum il-6 in patients undergoing esophagectomy was significantly higher than in those undergoing pancreaticoduodenectomy (p<0.05), despite a similar degree of surgical trauma. (4) peak 1l-6 concentration observed in a patient who underwent esophagectomy was about 100 fold greater in the drain fluid of thorax than in the peripheral blood. (5) il-6 mrna was demonstrated in leukocytes from thoracic and abdominal exudate at 6, 24 and 48 hours after surgery. in contrast, il-6 mrna could not be detected in leukocytes from the peripheral blood. [conclusion] il-6 is mainly produced in the operative field and subsequently enter the peripheral blood to induce cytokinemia. the operation length, volume of blood loss and thoracotomy are factors influencing the concentration of cytokine in the blood. zaragoza spain age may be an important factor influencing the function of immunocompeteut cells releasing cytokines after both accidental and surgical trauma the aim of the present paper is to ascertain if patients (pts) over 60 years old show a different serum level cytokine pattern than pts under 60 after a standard surgical procedure considered as a "medium strength trauma". patients and methods: 33 pts(22 females 11 males)with gallstone disease were perspectively studied, pts were allotted in two groups: gr.a:17 pts under 60 years(mean age:49.5+-7)gr.b:16 pts over 60 years(mean age:65.5_+5). all pts underwent cholecystectomy and cholangiography. 6 pts in gr.a and 7 pts in gr. b underwent common duct exploration. 1 spbintercctomy was performed in each group. on the day of surgery (pre) and on the 1st and 7th postoperative day(leo, 7po) : percentages of cd19, cd5, cd4, cd8 and cd16 cells we measured by means of flow cytometry using moab. and levels of il-1, il-4, il-6 and tnf "in vivo" by elisa using moab. results: ere: cd16% was 5.3_+3 in gr.a and 7. 5 objectives of the study. after surgery for esophageal cancer multiple organ damage has been reported to be caused by polymorphonuclear leukocyte (pmn)-mediated injury. we measured serum granulocyte colony-stimulating factor (g-csf) and interleukin 8 (il-8) levels to determine a role of g-csf and il-8 in pmn function after surgery for esophageal cancer. materials and methods. peripheral pmn counts, peripheral pmn chemiluminescence, serum g-csf levels, and serum il-8 levels were measured before and after surgery in 12 patients with esophageal cancer (ec), and 12 patients of gastric cancer (gc). esophagectomy with thoracotomy and laparotomy were performed for patients with ec, while subtotal gastrectomy with laparotomy were performed for patients with gc. results. peripheral pmn counts (p<0.01) and peripheral pmn chemiluminescence (p<0.05) of patients with ec were significantly decreased compared to those of patients with gc at 4 and 8 hours after surgery. serum g-csf levels of patients with ec were significantly (p<0.01) increased compared to those of patients with gc at 4 and 8 hours after surgery. serum il-8 levels of patients with ec were significantly (p<0.01) increased compared to those of patients with gc at 4, 8 and 24 hours after surgery. significant inverse correlations (p<0.0l) between peripheral pmn count and serum g-csf and il-8 levels were seen at 4 hours after surgery. conclusion. these results suggest that many circulating pmns, which are excessively activated by g-csf and il-8, may adhere to the endotherial cells and then migrate into the tissues, and cause multiple organ damage after surgery for esophageal cancer. immunnogical changes in patients with severe brain trauma receive increasing attention since morbidity and mortality ere still high. interleukin-6 (il-6) was previously detected in the cerebrospinal fluid (csf) during different pathologies of the nervous system (1, 2, 3). in our study we monitored il-6 and nerve growth factor (ngf) production in the csf after human brain trauma. since astrocytes within the brain constitute one of the major cell type contributing to the inflammatory response through the release of cytokines and other factors after injury, we investigated the functional relationship of il-6 and ngf on a single cell niveau using cultured astrocytes. methods csf was obtained from patients with severe brain injury (glasgow coma score (gcs) <8 and ct abnormatities or gcs <8 over 6 hours) after implantation of intraventricular icp monitoring device for therapeutic purpose and collected over 24 hours csf and serum. il-6 and ngf were assayed by elisa. astrocytes were isolated from neonatal mouse brain as described (4) . ngf production by cultured astrocytes was measured by elisa in the presence of csf, il-6 and il-6 antibody. astrocyte migration was tested in a chemstaxis chamber. results 17 head trauma patients were included in this study (approved by the university hospital medical ethics board) and the csf was obtained through intraventricular catheters. high levels of il-6 were detected in the csf of these patients when compared to serum during the first days after brain trauma. furthermore ngf could be found inside the intracerebral compartment. csf containing high levels of il-6 could stimulate ngf production in cultured astrocytes. this effect could be [nhibited partially by il-6 antibodies, purified il-6 exposed to cultured astrocytes in vitro, stimulated the migratory activity of these cells in a dose response fashion. il-6 was found in the csf of brain injured patients, suggesting a role for this cytokine in the pathophysiology of brain injury. since astrocytes are involved in maintaining the homeostasis of the brain, we further investigated the possible role o1 il-6 on astrocyte functions, il-6 promoted ngf production in vivo and in vitro, thus contributing to neuronal cell survival and regeneration. furthermore il-6 stimulated astrocyte migration in a dose response fashion, potentially contributing to astrocytosis following brain injury and inflammation, these results show that il-6 represents a key cytokine in traumatic human brain injury with possible systemic effects, which are at preserlt under investigation. we studied a) the role of tnf and b) the therapeutic effect of a mab to tnf with regard to haemorrhagic shock (hs) related ,pathophysiologic alterations and mortality in rats. method: a prolonged hs was induced by bleeding to a blood pressure of 30-35 mmhg for 180 pin followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over 50 rain. 15 animals received a bolus dose (20 mg/kg) of tnf mab (celltech, berkshire, uk) at 15 min after resuscitation (tn3). the control group (n = 15) was treated similar to the tn3 group but received ringer's lactate (con). results: at 180 min the prolonged hs resulted in a metabolic acidosis indicated by a significant decrease of blood ph (7.16 + 0.04), hco3-(16.3 ___ 2.0 mm), and base excess (-12.5 + 1.9 ram) values with pco2 (47.9 + 7.7 mmhg) and po2 (136.2 + 20.1 mmhg) in the tn3 with no difference to the con group. immediately after resuscitation (230 min) plasma endotoxin levels were found to be increased in both groups (48.8 + 73.3 in tn3 vs 30.1 _ 25.7 pg/ml in con group) . prior to the treatment with tnf mab (230 min) there was also no difference between plasma tnf levels of the two groups (42.1 + 60.7 in tn3 vs 60 + 141.8 pg/ml in con group). treatment with the tnf mab at 65 rain post-hs improved the 48hour survival rate to 73.3 % as compared to 26.7 % in the control group. macropathologic evaluations revealed frequency of intestinal bleeding in oniy 2 animals in the tn3 vs 11 in the con group. no bleeding in the kidneys was found in the tn3 but in 4 rats in the con group. the significant increase in lung wet weight observed in non-survivors in the con (n = 11) was prevented in animals which died in the tn3 (n = 4) group ((9.3 +_ 2.4 vs 6.3 +_ 1.0 g/kg). conclusion: our data suggest that tnf formation induced by hs in rats is an important mediator for pathophysiologic alterations leading to multi organ failure and lethality. antibodies to tnf might be a useful agent in the treatment of haemorrhagic shock related disorders. 55-+8 n=ll*$ 15-+5 n=7 78_+22 n=5* * p<0.05 vs baseline :~p<0.05 no anesthesia vs anesthesia thus 1) tnf production increased 2-3 fold by 48-72 hrs following trauma in unstimulated blood, but was reduced or not changed after lps stimulation, so circulating leukocytes are probably not an important source of tnf post trauma; 2) anticd18 had no obvious effect on tnf production in unstimulated or lps stimulated blood, relative to vehicle, which suggests that the protective mechanism of anticd18 does not involve tnf suppression; 3) fentanyl anesthesia at 72 hrs following trauma unexpectedly decreased lps-evoked tnf production, which suggests that anesthesia alone can influence an inflammatory response. proinflamrnato~ cytokines have been shown to play a signific~t role in the pathogenesis of sepsis, which is a very common occurrence in born injury. tnfa is infrequently detected in the blood of burned patients, the ability to detect the shed receptors of stnfg has not been determined. serial serum mmples from burn patients were collected from the time of admission until death from septic shock. these samples were analyzed using an enzyme-linked immunosorbent assay (elisa) for stnfr, l-ira, tnf-a, and il-ib. the patients ranged in age from 26 to 72 yeas of age. the percentages of bum ranged from 40% -90%. cytokine concenlrntions vmled from patient to padent irrespective of bum size. tnfa levels were consistentiy in the range of30pgjml -150pg/ml. peaks in the tnfa values were above 90pg/ml and were also associated with a peak in the stnfr levels. these levels began at <10,000pghnl within the in,st 6 ins of injury and gradually increased with time. clinically. ti~ appearance of eytoklnes was independent of positive wound, blood, or respiratory cultures however peak values in tnfa and stnfr were ~ialed with a fluid requirnmenl levels of il-i ra were also elevated independent of clinical findings as well as extent of injury. in pl5 there is a significant corresponding peak in il-trn (>40~8/ml) at the same time as t/~:a and stnfr levels. we aimed to characterise the pattern of secretion of interleukin-1 beta 0l-ii3), intefleukin-6 (il-6) and tumour necrosis factor alpha (tnfa) in multiply injured patients and to relate these results to their clinical condition and outcome. two hourly blood samples were taken from ten patients from the time of injury until 48 hours. cytokine levels were measured using sandwich enzyme-linked immunosorbent assays (elisas). injury severity scores (iss) were calculated and haemorrhage was assessed from the blood transfusion requirement over the 48 hours. patients' ages ranged from 17 to 86 years. iss varied from 9 to 50 and transfusion requirement from 0 to 14 units. five patients died after the study period. ]1,-6 was raised in 9/10 patients (max level 12,500 pg/ml) but was unrelated to condition or outcome. 5/8 showed a rise in il-1b (max level 90 pg/ml) which was negatively correlated to iss (i=-0.789, p<0.02). tnfa was raised in 2/10 (max level 95 pg/ml). peak tnfc~ was positively correlated with iss (1=0.676, p<0.05) and haemorrhage (i=0.602 but p<0.5). il-ib and tnfa production was mutually exclusive. there was no common cytokine profile for these patients. unlike elective surgery there was no correlation between peak 11,-6 and severity of injury: tissue damage may not be the stimulus for the cytokine response to multiple injury. periods of ischemia or hypoxia produce endothelial damage in peripheral organs. tumor necrosis factor-alpha (tnf) plays a central role for regulation of endothelial physiology during septic events, taking influence on vascular permeability and coagulant activity [1] . animal experiments demonstrated a synergism between hypoxia and septic shock on letality, leading to the hypothesis that low oxygen tension leads to enhanced sensitivity of target cells for tnf [2] . radioligand binding studies with ~261odid-tnf on cultured human endothelial cells were performed after incubation in several environmental oxygen tensions (pc2) for 12 hours. data were achieved by nonlinear regression of an idealized saturation curve according to the equation: b = n " k./(1 + k,); b = totally bound tnf; k,: association constant (concentration for half-maximal binding); n: number of binding sites per cell. p_o0o (mm h¢i): _k, (nm}: n (molecules/cell): 130 -150 1.83 ± 0.15 2600 _+ 500 55 -70 1.27 ± 0.15 3600 + 500 25 -35 0,41 ± 0.13 4800 -+ 500 10-15 0.18 + 0.07 5900 -+ 300 presented are calculated values on the idealized curve + 95 % percentiles. hypoxia induces enhanced binding of tnf to specific receptors on the endothelial cell surface in a time-and dose-dependent manner by a mechanism, which is not dependent on oxygen radicals, as shown by additional protocols with radical-scavenging drugs. with respect to former findings about a correlation between growth and tnf receptor affinity [3] , these data lead to the hypothesis that enhanced tnf binding during hypoxia is due to a biochemical conversion of the receptor protein from the low affinity to the high affinity state, possibly by posttranslational phosphorylation of the binding protein by intracel)ular kinases. the proposed involvement of tnf-dependent pathways in pathogenesis of organ dysfunction and multiple organ failure after hypoxia/ischemia may provide a basis for understanding the initiation of hypoxic vascular injury, as manifested by increased permeability and prothrombotic tendency, and, thus, merits further attention. the levels of activity of circulating cytokines (ill, il-6 and tnf-alpha) which are believed to play important regulatory role in response to trauma are determined (by hioassays and respective anti-cytokine antibodies) in mice and rats subjected to scald injury ion c,10 see, °0v bsa, ld 50) and (80 c, 30 see, 20 ~ b ~^)~ , respectively. biphasic increase of cytokine activity was noted in mice: initial increase of il-i and il-6, 1-3 hr following injury and of try activity 6hr after scald, followed by elevated levels of il-i and il-6 at 12hr, with tendency of decrease of activity at later time points. increased activity of tnf was noted 24 hr following injury, in rats, initial, short-lived increase of il-i and tnf activity was detected lhr following injury, folowed by increase on days i and 3 postburn. il-6 increase peaked 3-12 hr after scalding and levels remained elevated 3-5 days following injury. similar kinetics of appearance of proinflammatory cytokines (il-i and tnf-alpha) both in lethal and ncnlethal injury concomitant with differential profile of circulating il-6 activity (early,short-lived increase and later slow decrease of activity in lethal burn injury) with late persistent high levels of activity in nonlethai injury demonstrated in the present study highlight the need for investigation the relationship of these cytokines in burn-injury induced inflammation. zikica jovicic,lnstitute for medical research, mma,crnotravska 17,11002 belgrade~yu. asadullah k (1), woiciechowsky c (2), liebenthai c (1), doecke wd (1), volk hd (1), vogel s (2), v. baehr r (1); depts. of med. immunology (1) and neurosurgery (2) , medical school (char#d), humboldt university berlin, frg in patients after polytrauma or major abdominal surgery a hyperinflammatory phase seems to be followed by the development of a phase of monocyte inactivation. the latter is charaeterised by a decrease of monocytic hla-dr expression and a shift to anti-inflammatory cytokine production. as shown, by us and others, this phenomenon indicates severe immunodepression with a high risk of infection. however, the mechanisms leading to monocyte inactivation in the above mentioned syndromes may be multiple. to elucidate the influence of a selective, sterile trauma to the central nervous system (cns) on immune reactivity the neurosurgieal patient is an interesting model. initially, 30 patients who developed a systemic inflammatory response syndrome following neurosurgery were analysed. in all of them a marked decrease of monocytic hla-dr expression was observed soon after the operation. these results suggest that neurosurgery alone can induce immunodepression and lead us to conduct a prospective study, in which we closely monitored l0 patients undergoing neurosurgery from the first preoperative day until at least day 6 after the operation. hla-dr expression was decreased hi all patients to various extent only hours after surgery. in one patient only we found a persistently reduced hla-dr expression and this was the only patient to develop sepsis syndrome. this suggests that a prolonged, postoperatively decreased hla-dr expression is predictive of infection following cns trauma. in order to assess, whether a decrease of hla-dr expression was associated with a preceding inflammatory response, local cytokine release in the cns was compared with systemic cytokine release. for this purpose, paired samples of earebrospinal fluid (csf) from a vantricle drainage and peripheral blood plasma were obtained. in the csf extremely elevated futerleakin (il)-6 levels, peaking already a few hours after the operation were found. in plasma, by eontrast, il-6( and tnf-alpha) was detectable not until days later and only if infection was present. the antiinflammatory ili-ra, on the other hand, was also present in csf but peaked after il-6 and was detectable in peripheral plasma too. we believe there is an association between the inflammatory response in the cns and the following depression of hla-dr expression on peripheral blood monocytes. our results suggest that even a sterile cns-trauma by itself may contribute to general immunodepressinn leading to septic complications. the aim of this study was to evaluate the effect of haemorrhagic shock (hs) a) on total capacity of the host, and b) the circulating blood cells to produce tnf immediately after bleeding. in vivo studies: baboons were subjected to a limited oxygen deficit (180-200 ml/kg) hypotension phase (mean arterial pressure = map of 35-40 mmhg for 2-4 hours followed by adequate resuscitation). rats subjected to hs (map of 30-35 mmhg for 90 rain followed by reinfusion of shed blood and fluid resuscitation) were challenged with endotoxin (1 ~g/kg i.v.) at the end of shock (rhs group). the control group (rco) received the same dose of endotoxin as rhs group but without prior bleeding. in vitro studies: whole blood (wb) obtained from both baboons and rats before and at the end of hs were incubated with endotoxin (100 ng/ml) for 2 hrs at 37 °c. results: at 90 min post-lps challenge we found significantly higher plasma tnf levels in rats that were subjected to hs prior to the endotoxin challenge as compared to the control group (956 _+ 585 vs 276 + 199 pg/ml) . after hs the tpc was significantly decreased in in vitro stimulated cbc of both rats (12 + 4 post-hs vs 164 + 89 ng tnf/ml pre-hs) and baboons (8 ± 16 post-hs vs 233 ± 188 pg tnf/ml pre-hs). in contrast, the il-6 productive capacity was increased in baboons cbc (not yet analysed in rats) stimulated at the end of hs (224 ± 106 pre-vs 1545 ±_ 1254 pg il-6/ml post-hs). conclusion: from our data we suggest that despite of down regulation of the cbc to produce tnf the overall tpc is enhanced at the early stage of i-is. with regard to the related literature (chaudry's group) it can be assumed that among the macrophage/monocyte populations, as the main source only the kupffer cells (kc) exhibit enhanced tnf production capacity following haemorrhage. the mechanisms of down/up regulation of cytokine response of cbc and/or kc following hs remain to be examined. d. eg~er, s. geuenich °, c. dertzlin~er °, e. schmitt*, r. mailhammer, h ehrenreich #, p. drrmer, and l. h01mer gsf-instimt fox experimentelle h~znatologie, °medizinische kliulk iii, klinikum groghadern, munich, *institut for immunologic, johannes gutenberg universit/it, malnz, and #psychiatrische k/in& der georg-aagust-universi~t, grttingen, germany. it has been shown previously (ehranreich et al., 1992, new biol. 4: 147 ) that mouse bone marrow-derived mast cells (bmmc) synthesize and secrete endothelin-1 (et-i) and express eta-type endothelin receptors (eta). so far, however, no functions of et-1/et a in bmmc have been described. in the present study we investigated the effect of exogeneously administered et-1 on the release of histamine, serotonin, and leukotriene c 4 (ltc4) by primary mouse bmmc (in vitro age: 4 weeks) caltured with different recombinant mttrine cytokines (interleukin 3 (il-3) and/or kit ligand (kl) in the presence or absence of il4) for two weeks prior to activation. et-1 (5x10 -8 to lxl0 -6 m) induced an extremely rapid (_<lmin) and dose-dependent release of histamine and serotonin (up to 20% and 16% of total mediator content, respectively, comparable to ige/ag-indueed mediator release) from bmmc cultured with il-3 and il-4. only when cultured in the additional presence of il-4 rather than in medium containing kl or kl plus il-3 alone, bmmc released histamine and serotoaln upon challenge with et-1.furthermore, et-1 stimulated ltc 4 biosynthesis up to 4-fold in bmmc cultured in the presence of il-4. in contrast, the peptide was without major effect on ltc4 biosynthesis in bmmc culturd without il-4. the release of histamine and sereotonin was not altered if bmmc were preincubated for i h with il-4 prior to activation with et-i, suggesting that a differentiation process rather than a functional priming effect had been initiated by ]l-4. et-1 (10"6m) -induced histamine and serotouln release from bmmc was inhibited by an eta-specific antagonist (cyclic id-asp-pro-d-val-leu-d-tel ) in a dose-dependent manner, with a complete inhibition observed at an antagonist concentration of 10 -8 m. crude peritoneal cells (containing 2-3% serosal mast cells) obtained from normal balb/c-mice released 87% histamine upon challenge with et-1 (10 -6 m; 20 rain). taken together, these resu/ts demonstrate that et-1 can directly function as a histamine and serotohin secretagogue and as a stimulator of ltc 4 production in mast cells. il-4 appears to be involved in the differentiation of immature mast cell prec~trsors towards an et-l-reactive functional phenotype. when inflammatory reactions are advanced, type ii phospholipase a2 (type ii pla2) is produced in high levels by various cells at the site of inflammation. cytokines such as tnf-a and il-i activate type ii pla2 and promote the production of eicosanoid, which is one of the mechanisms by which they enhance the inflammatory reaction. in the present study, the blood levels of type ii pea2, endotoxin, tnf-c~, il-6 and il-8 were determined in patients with sepsis to examine the relationship between these levels and the patients' pathological conditions. the levels of type ii pla2 and tnf-a in these patients were 218.3 ± 179.9 ng/ml and 98.5 ± 92.1 pg/ml, respectively, the two parameters being significantly correlated (r = 0.6460, p = 0.0001 ). there were also a significant correlation between the level of type ii pla2 an il-6 (r = 0.4262, p = 0.0188). there was no significant correlation between the level of type ii pla2 and il-8. these in vivo findings suggest that tnf-a may be involved in the production of type ii pla2. a. filzmaver, g. reisbach, e. schmitt °, r. mailhammer, and l. hiittner. gsf-iustitut ff~r experimentelle h~imatologie, munich, and °iustitut fttr immunelone , johannes gutenberg universit~t, mainz, germany the product of c-kit, a transmembrane tyrosin ldnase receptor, and a corresponding kit ligand (kl) are critically involved in the control of different hemopoietic cell lineages including the proliferation, maturation, migration, and function of mast cells. it has been reported previously that interleukin (il)-4 down-regulates kit receptors in human primary myeloid leukemic cells and in a human mast celt line (sinaber et al. 1991, j. immunol. 147: 4224) . transforming growth factor (tgf) gl, was also found to down-modulate c-kit mrna and kit receptor proteins in human aml blasts, a mechanism probabely contributing to the anti-proliferative effect of tgfih on kl-stimulated aml ceils in vitro (de vos et at. 1993, cancer res. 53: 3638) . in porcine endothelial cells transfected with a retroviral construct carrying the human (hu) e-kit gena, exogenous hu kl transiently down-regulated kit receptors (blume-jeusen et al. 1991, embo j 10: 4121) . in the light of these previous findings we analyzed the effects of these exogenously applied cytokines (il-4, tgfgl, kl) on kit receptor expression and kl-mediated proliferation and chemotactic migration of primary mouse bone marrow-derived mast cells (bmmc) (in vitro age: 4 weeks). our results show that in bmmc cultures initiated with recombinant (r) marine (mu) il-3 the additional presence of rmull-4 for 3 or 14 days did not change the expression of kit protein (assessed by facs analysis with the anti-mouse c-kit antibody ack-2) nor kl-mediated proliferation or chemotaxis. in contrast, il-4 syeergistically increased kl-stimulated growth of bmmc in a short-term proliferation assay (mtt-tes0. pre-incubation of bmmc with rhutgfl~ 1 (5.0, 1.0, or 0.1 ng/ml) for 24h had no effect on kit receptor expression, although tgffil at concentrations of 0.5 -5.0 ng/ml completely suppressed the proliferative action of kl on these ceils. similar anti-proliferative effects of tgfg 1 were observed in various factor-dependent mast cell lines stimulated with different mast cell growth factors (il-3, 1i,-4, il-9, and/or il-10). moreover, pre-incuhation (24h) of bmmc with tgf-gl (>500 pg/ml) significantly enhanced spontaneous undirected cell movement (chemokinesis) and synergistically increased il-3-or kl-induced chemetaxis. when bmmc were preancuhated with rmukl (200 ng/ml) for 1, 3. or 5 days, a transient down-modulation of kit receptors with a maximum effect on day 1 was demonstrated by facs analysis and correlated well with a decreased chemotactic response of these cells. in conclusion our results show that neither il-4 nor tgfi31 affect expression of kit receptors in primary murine bmmc. it is reasonable to suggest that c-kit expression is controlled in a cell type-specific manner.interestingly, tgfgl is obviously able to dissect the proliferative from the migrational signal transducted by kl in these cells. objectives of the study: antisense strategies using dna-otigonucleofides (odn) to modulate the cytokine response are presently under investigation. odn are thought to act very specifically with little or no relevant negative side effects. we now report that odn unspeeifically protect wehi 164 cells from tnf-mediated cytolysis. material and methods: wehi 164 subclone 13 ceils (5 x 105), that are highly sensitive to the cytolytic activity of tnf, were grown on 96-well culture plates in rpm11640 medium. after 24 hours, phosphorothioate(ps)and partially ps-modified-odn as well as phesphodiester-odn (20-29bp) were added (0.1, 1 and 10 pm). four hours after incubation with odn, ce(i lysis was induced by recombinant murina tnf. after 18 hours the plates were washed and stained with crystal violet cell lysis was determined by reading the absorbance (abs) at 590 nm. results: wehi 164 ceils incubated with tnf (1-8ng/ml) were completely lysed after 18 hours (0% abs). interestingly, wehi cells incubated with tnf and odn resisted complete lysis, eg cells incubated with 2.5ng/ml tnf and 11jm odn showed still 30% of the absorbance observed in control ceils without tnf (100% abs). the protective effect of odn started at 0.1pm, reached a maximum at 1,um, and diminished at 101jm. with increasing amounts of tnf the protective effect of qdn decreased and no protection was detectable at 8ng tnf per ml conclusions: dna-oligonucleotides were found to unspecifically inhibit tnf-induced cytolysis. we hypothesize, that this protective effect of qdn results from an inhibition of the binding of tnf to its receptor, or from interference of odn with the subsequent signal transduction mechanisms. as a consequence, to discriminate the specific effect of odn in biologic systems, several control odn should be used. secondly, whether dna released by degradation of tumor cells or leukocytes can significantly impair tumor-and immune-defense mechanisms merits further investigation dr. med. michael meisner, institut for anaesthesiologie der universitat erlangen-nqmberg, krankenhausstral~e 12, d-91054 erlangen. in this study we investigated the involvement of serine protease and free radical generation in the systemic release of tumor necrosis factor-alpha (tnf) and interieukin i(il-1), in the sepsis model of lipopolysaccharide (lps, 5mg/kg i.p.) induced hepatitis in galactosamine (gain, 18rag/mouse, i.p.) sensitized mice. treatment of gain-sensitized mice with lps (gain/lps) led to dramatic increase in serum cytokine (tnf and il-i) ievels and transaminase activity at 3 hr and 8 hr respectively. pretreatment of serine protease inhibitor, c~jantitrypsin (a j-at, 50mg/kg i.p.), 30 rains prior to gain/lps treatment, fully protected the animals against the hepatotoxic challenge with significantly reduced serum tnf and il-1 levels. in order to block and scavenge superoxide generation, the mice were pretreated with xanthine oxidase inhibitor, allopurinol (al, 2 x 100mg/kg i.p.) and pyran polymer-conjugated superoxide dismutase (sod, 2 x 200 unit/mouse i.v) r6spectively. pretreatment with al and sod (24 and 1 hr prior to gain/lps) prevented gain/lps hepatitis and blocked lps induced released of tnf and il-1 into serum of the mice. the protective agents like cq-at or al/sod did not protect the mice against th~ hpp~totoxi£ ch~llpn-e indllee4 b'~ th~ recombinant mmlse tnf-o' (0.3 ~/rno~e j.p.) ~d oi~lps 1~ caln-.~dlfa%aed mlce. it-l cett~aged la tnf (x/gain treated mjde was not detectable in animals pretreated with oq-at or al/sod. our study suggests that a serine protease sensitive to cq-antitrypsin is responsible in regulating tnf release, possibly by proteolytic cleavage of a tnf-precursor or membrane bound tnf. in addition our evidence suggest that the balance of extracellular protease/antiprotease activity may be regulated by free radical generation, possible superoxide anion, resulting in inactivation of the antiprotease. il-1 release may be subsequent to tnf release. objective: during sepsis one can observe a dramatically impaired production of proinflammatory cytokines like the tumor necrosis factor alpha (tnf-a), interleukin i-alpha (il-la), intedeukin i-beta (il-i&) and interferon gamma (if~) upon in vitro stimulation of circulating cells. however there is also evidence of a decreased ability to produce cytokines in other immuno-deficient states. in this study we compared the capacity to secrete proinflammatory cytokines upon in vitro stimulation of patients in severe sepsis and patients with malignant tumors. methods: heparinized blood samples of ten patients (62+ 14 years) in severe sepsis (sepsis score > 15 according to e}ebute and stoner) were drawn at onset of disease, from fifteen patients with solid growing carcinoma (59+19 years) blood was drawn at diagnosis prior to any therapy. controls were obtained from fifteen healthy volunteers. 50 pl of whole blood were incubated either with 450/4 of a standard medium or with 400 pl of a standard medium and 50 pl of phytohemagglutinin (pha) a potent mitogen. after an incubation period of 24 hours plasma concentrations of tnf-a, il-la, il-16 and if-~ were determined by elisa. comments: our results suggest that down-regulation of cytokine secretion or of cell responsiveness to non-specific mitogens during sepsis has occurred. we observe a similar phenomenon for the group of carcinoma patients vs control significant for stimulated tnf-a and stimulated if-t. sustained immunological interactions between tumorcells and cytokine producing cells could effect responsiveness of the latter, a general increased immuno-tolerant state in patients with carcinoma has to be discussed. however we found significant differences between sepsis and cancer concerning the in vitro capacity of responsable cells to produce il-la and il-i#. the dramatically decrease of the ability to produce il-i upon in vitro stimulation could be more sensitive for a septic state than stimulated tnf-a or if-3,. objective: tumor necrosis factor alpha (tnf-a) has been implicated as a central mediator of sepsis and its sequelae. increased systemic levels of this cytoklne seem to be correlated with severity of sepsis and outcome. however mechanism of action and metabolism of tnf-g are not fully understood. in most studies blood samples for tnf-a determinations are obtained either by peripheral venipuncture, a central venous catheter or by an indwelling arterial catheter. very often blood samples are taken in different manners within the same study. in this study we measured circulating tnf-a and the amount of tnf-a released upon in vitro stimulation in arterial and central venous blood. methods: heparlnized arterial and central venous blood samples of ten patients (6 males, 4 females, mean age 62+_14) with severe sepsis (sepsis score > 15, elebute and stoner} were drawn on day 1,3,6,8, and 14 of disease. blood was immediately placed on ice and processed within 1 hour. 50 pl of whole blood were incubated with 450 pl rpmi-medium supplemented with antibiotics and l-glutamlne or with 400 pl of rpmi-medium and 50 pl phytohemagglutinin (pha) a potent mitogen. after an incubation period of 24 hours samples were centrifuged and plasma was harvested and stored at -30 ° celsius before assessment of tnf-a concentration by elisa. statistical analysis was performed with the paired student-t-test. results: we found a significant difference (p < 0,05) for circulating mean arterial tnf-a concentration (237 pg/ml _+ 34 sem} and central venous tnf-a (203 pg/ml +_ 29 sem). upon in vitro stimulation there was also a significant difference (p < 0,002) between released arterial tnf-~' {746 pg/ml _+ 88 sem) and venous tnf-a (637 pg/ml +_ 76 semi. conclusions: these results are difficult to interprete but could reflect the influence of pao 2 and sao 2 on tnf a release. it could also be the result of different concentrations of tnf-o release influencing factors like for example endotoxin, interferon-f or prostaglandin. a possible pulmonary and/or a hepatic metabolism of tnf-n and tnf-a producing cells cannot be ruled out. however for better interpretations of tnf-a release in septic states it is necessary to use either arterial or venous blood samples. early inflammatory processes following trauma and/or infections were found to be associated with the secretion of high amounts of proinflammatory cytokines. besides intedeukin-t (il-1), tumor necrosis factor-a (tnf-c 0 and interleukin-8 (il-8) the multifunctional cytokine intedeukin-6 (il-6) was described to be a central regulatory element of the primary cellular and humeral defence reaction. the previously described close temporal correlation of pathologically elevated il-6-concentrations and the extracellulary release of lysosomal enzymes from activated pelymorphnuclear neutrophils suggests, that il-6 may be a potential substrate of these preteases. the serine preteases elastase (ec 3.4.21.37 ) and cathepsin g (ec 3.4.21.20) derived from the azurophilic granules were assumed to be mainly involved in unspecific proteolysis at sites of inflammation by cleavage of structural as well as soluble proteins at random sites, if the inhibitory potential is decreased. the possible proteolytic activity of elastase and cathepsin g toward the proinflammatory cytokine interleukin-6 (il-6) was investigated. the addition of purified neutrephil elastase and cathepsin g to recombinant human il-6 leads to a rapid sequential degradation in vitro. at least two intermediate products could be detected by silver staining and western blotting following protein separation under reducing conditions. the serine protease inhibitor g-anitrypsin was shown to prevent the proteolytical degradation of intedeukin-6. furthermore the loss of the biological activity of both, recombinant and natural human il-6, was demonstrated by determination of the capacity of protease-treated il-6 to stimulate hybddoma growth (7td1 bioassay). these data suggest a possible downregulation of pathologically elevated il-6 levels by proteolytic activity of extracellulary released enzymes at sites of inflammation. the aim of the study was to compare circulating levels of three cytokines -il-1, il-6, 11_-8 -between critically ill subjects who developed gram-negative sepsis and who did not. materials and methods: the patient population consisted of 39 patients admitted to an intensive cars unit, with different underlying diseases. sepsis diagnosis was given according to pre-estabilished cdteda. nineteen cases were enrolled in sepsis group, twenty in control group. serum sampling was collected in sterile tubes at study entry and every three days until study dismissal. serum concentrations of il-1, 11_-6 and il-8 were measured using commercially available test kits, based on the dual immunometric sandwich principle. results: the causative patogens of sepsis were: pseudomonas aeruginosa, acinetobacter, eseherichia co~i, serratia marceseens, proteus mirobilis and citrobacter freundl the time of observation was equal to 12 days, for a total of four tests performed (to, tl, t2, t3). i1.-1 was not detected in any samples. the serological profiles of the two cytokines 11.-6 and 11_-8 were similar; augmented levels were found at study entry and throughout the observation period, peaking at t3 and decreasing at t4. however, in patients with sepsis, il-6 and 11_-8 concentrations were significantly higher in respect to control group. conclusion: our observations shown that in icu patients increased il-6 and il-8 release may be induced by cdtical illness; however, in subjects in which sepsis occurred, il-6 and il-8 production appears more significantly elevated, suggesting a role of il-6 and 11_-8 in the pathophysiology of sepsis. the fact that ii. objective: to check whether continuous veno-venous haemofiltration (cvvh) could remove the cytokines, namely tumour necrosis factor alpha (tnfc 0 and interleukin 6 (il-6) from the circulation of critically ill patients with sepsis ad multiple organ failure (mof). setting: the intensive therapy unit of the medical school teaching hospital. patients: nine critically ill patients with sepsis and mof treated with cvvh. methods: blood samples were collected before the cvvh had been started. then, blood and ultrafiltrate samples were collected simultaneously after 4 hours and every 24 hour. tnfct and il-6 levels were measured using the bioassays with cell lines wehi-164 ci13 and 7td1, respectively. other data were recorded from the patient notes and intensive therapy unit charts. results: no measurable concentrations of tnfct were detected in either blood or ultrafiltrate samples. il-6 was found in all the patients' plasma samples and five patients' (55.5%) ultrafiltrate samples. the il-6 blood level ranged from 17.4 to 3061.7 u/ml (mean 518.2, sd 686.8). the il-6 level in positive ultrafiltrate samples ranged from 21.0 to 1150.2 u/ml (mean 252.2, sd 401.8). conclusions: our preliminary results suggest that il-6 is present in bloodstream of septic patients. we assume we could not detect tnfa in any sample because we usually started observations when septic state had developed. cvvh could extract cytokines from the circulating blood. it remains under discussion, whether that extraction may be beneficial to patients with mof. the pattern of some significant cytokines tnf, il-1 and il-6 and their pharmacomodulation were evaluated in an experimental model of polimicrobial sepsis induced in cd-1 mice by cecal ligation and puncture (clp) in order to understand their roles. this model of sepsis, which resembles the clinical situation of bowel perforation, was also compared with that induced by administration of pure endotoxin (lps). tnf was detectable in serum and tissues during the first 4h with a peak 2h after clp at a significantly lower level than after lps. il-1 was measurable in serum only after 24h, significantly increased in spleen and liver after 4 and 8h and in mesenteric lymphonodes from 8 to 24h after clp compared with shammice. il-6 was significantly increased in serum throughout the first 16h after clp. pretreatment with dexamethasone (dex), ibuprofen (ibu) and nitro-l-arginine (n-arg) significantly reduced the survival time while chlorpromazine (cpz) and tnf did not affect it. only the antibiotics and pentoxifylline (ptx) significantly increased the survival in clp. however cpz and dex protected from lps-mor~ality. in conclusion, by inhibiting tnf with dex, cpz, ptx a reduced, unchanged and increased survival time was observed and by increasing tnf with ibu and tnf administration the survival was decreased or unchanged respectively suggesting that the modulation of this cytokine does not seem to play a significant role in clp unlike lps_ moreover the negative effects of ibu and n-arg suggest an important and protective role by prostaglandins and no in clp. to gain more insigths on the contribution of tnf~, il-i~ and if 7 to lps toxicity, we explored the time-course of the cytokine production in ealb/c mice given different doses, from the lethal (= 3 ld50) to the sublethal (= 1/3 ld50) of three different lps (e.coli oiii:b4 and 026:b6; p.aeruginosa r261) endowed with different degree of toxicity cytokines were measured in serum and organs with specific elisas up to i0 h after lps administration. results demonstrate that i) circulating and organ levels of tnf~ do not reflect lps toxicity. in fact, the lethal dose of lps 026:b6 induced as much tnf~ as the sublethal dose of lps 0111:b4; furthermore, lps r261, whose cytokine inducing capability is far lower than that of lps from e.coli, induced higher tnf~ levels at the sublethal than at the lethal dose. in addition, policlonal anti tnf ab, that were able to protect mice from e.coli lps induced mortality, failed in mice treated with lps r261 2) circulating il-i~ levels are generally low and increase significantly only in muribond animals. on the contrary, in spleen and lung very high levels of il-i~ are persistent from i to 8 h post lps administration moreover, the treatment with 6 mgr of neutralizing policlonal anti il-i~ ab, did not modify survival in lps challenged mice. 3) circulating and organ levels of if 7 are proportional to the dose and degree of toxicity of all the administered lps even if lps r261 was again a less efficient cytokine inducer than lps from e.coli. csa is an immunos~ppressive drug, able to inhibit gene expression for many cytokines, including if 7. to study the effect of cytokines modulation on lps toxicity, csa was administered to mice twice at the oral dose of i00 mg/kg before the challenge with lps. mice were monitored in terms of mortality and tnf~, il-i~ and if 7 production. together with the total ablation of if7, the strong reduction of tnfu and unmodified il-i~ levels, a significant increase of lps toxicity was also observed. these results suggest the hypothesis that the numerous factors that jointly mediate lps toxic effects, can also be protective, the final outcome depending on their relative ratio rather than on the absolute amount interleukin-1 (il-1) mediates the septic shock syndrome and affects intestinal secretion in vitro. we studied the intestinal production of il-t and its effects on diarrhea during endotoxic shock. cd-1 mice were randomized to 15 mg/kg e.coli 0111:b4 lps or saline infusion (i.p. or i.v.). diarrhea invariably occurred following lps infusion. mice were sacrificed at 0, 30', lh, 2.5h, 4h, 6h, 12h, and 24h (3 mice/group/time-point). the small bowel was compressed and the intestinal contents were weighed and expressed per g sb weight. the small (sb) and large bowels (lb) were eventually frozen, weighed, and homogenized for either cytosolic protein or total rna. il-i~ (cell-associated agonist) was measured with a radioimmunoassay specific for mouse il-l~ (detection limit 100 pg/ml) and expressed as ng/g weight + sem (lowest detectable amount 1 ng/gwt). northern analysis of total rna and in sfu hybridization of paraformaldehyde-fixed frozen tissue were done with [0~-32p]-iabeled mouse il-lc~ cdna probes. only sb had il-i~ constitutively present (6.2 + 1.6 ng/gwt). lps i.p. or i.v. induced elevation of il-lc¢ in both organs in a biphasic pattern; lps i.v. induced 3-fold more il-i~ than lps i.p. following lps i.p., il-i~ in sb was 19.2 + 0.6 ng/gwt at lh, reached maximal levels at 2.5h (31.8 -+ 0.5 ng/gw-i) and returned to baseline at 24h. saline controls maintained their constitutive il-i~ levels. sb had 2fold more il-1 ¢ than lb and identical kinetics, but lb showed a clearer doseresponse. northern analysis of sb-total rna showed induction of il-i~ mrna by lps in correlation with il-lc¢ kinetics. il-i~ mrna producing cells were mononuclear cells in the lamina propda and epithelial cells at the bottom of the crypts of ueberkuhn. mucus and fluid were increased in the small bowel post-lps in correlation with intestinal il-lc~ kinetics (r2=0.9). separate mice were pretreated with saline i.p. orthe il-1 receptor antagonist (irap, 30 mg/kg bolus i.p.) and were challenged 20 rain later with 1.5 mg/kg lps i.p. or saline i.p. specific blockade of il-1 by irap decreased intestinal secretion at 4h and 6h post-lps challenge (p<_. 0.05, student's-t-test). these data indicate that local (intrinsic) intestinal il-i~ mediates sepsis-induced intestinal changes. inflammatory cytokines initiate the host response to endotoxemia, causing severe physiological and hemodynamic changes which may lead to septic shock. among the regulatory systems that play an important rote in controlling host inflammatory responses is the pituitary. it has been known for many years for example, that hypophysectomized animals are extremely sensitive to lps lethality. while investigating the possibility that protective, pituitary mediators might explain this phenomenon, we identified the cytoldne mif to be a specific secretory product produced by pituitary cells in vitro and in vivo after lps challenge. analysis of serum mif levels in control, t-cell deficient (nude), and hypophysectomized mice revealed that pituitary-derived mif contributes significantly to the rise in serum mif that occurs after lps administration. of note, pituitary mif content (0.05% of total pituitary protein) and peak serum mif levels (80-340 ng/ml) were determined to be within the range observed for other pituitary hormones that are released after pituitary stimulation. to investigate a possible beneficial role for mif in septic shock, we co-injected mice with purified, recombinant murine mif (rmif) together with lps (15 mg/kg). surprisingly, rmif markedly potentiated lps lethality compared to control mice that were injected with lps alone (85% vs. 35%, p = 0.003). to confirm these results, mice were treated with anti-rmif antibody prior to injection of a high dose of lps (17.5 mg/kg). anti-rmif antibody fully protected mice against lps lethality, increasing survival from 50% to 100% (p = 0.0004). serum levels of tnf,~, the first cytokinc that appears in the circulation after lps challenge, were reduced by 38.0 _+ 9.5% in anti-rmif-treated mice. we conclude that pituitary derived mif contributes significantly to circulating mif in the post-acute response in endotoxemia and may act in concert with other pituitary mediators to regulate both pro-and antiinflammatory effects. moreover, mif may play a critical regulatory role in the systemic host response in septic shock. our results suggest that anti-rmif antibody might be of potential therapeutic use in the treatment of septic shock. although anti-interleukin-1 (il-1) antibodies and il-1 receptor antagonist have been shown to improve survival in animal models of endotoxemia and abrogate the lethal effects of tnf, the presence of il-1 in the serum does not correlate well with outcome. we hypothesized that this may be because il-1 acts mainly in a paracrine fashion and is metabolized before it diffuses into the circulation. methods: we measured the il-i~ mrna expression with the differential reverse transcription polymerase chain reaction (rt-pcr) using g-actin as internal standard in the peritoneal macrophages and lung tissue in normal controls and mice after cecal ligation and puncture (clp). clp resembles human intra-abdominal sepsis in that it is characterized by very slight elevations of serum il-1 levels. results: il-lg mrna levels after clp are expressed as % of normal (mean+sem, n=5 in several experimental models of infection exacerbation of disease was observed, when infected animals were depleted of tuajor necrosis factor (tnf). after sublethal cecal ligation and puncture (clp) leading to peritonitis and sepsis the survival of mice also critically depends on tnf as demonstrated in earlier studies, when clp-treated mice injected with anti-tnf antibody died, whereas mice injected with a control antibody survived after clp (echtenacher et al. 1990, j. inununol. 145: 3762) . from a panel of different cell types (macrophages, neutrophils, t lymphocytes, natural killer cells, mast cells) able to produce tnf upon activation~ the mast cell is apparantly the only one capable of storing in cytoplasmic granules preformed tnf-ct which is rapidly released following challenge. in the present study-we analyzed serum tnf after lps injections as well as the outcome of clp in severely mast cell deficient mutant mice (wav v) as compared to syngeaeic wild-type littermates (+/+). we proposed that concentrations and/or kinetics of serum tnf should be different between wavv mutants and wild-type mice, if mast cell-derived tnf significantly contributes to the rise in serum tnf levels following systemic stimulation with endotoxin. although similar levels of increased tnf were detected in the sera of both genotypes after 1 and 2 hours of lps injection (100 btg/ 0.5 ml / mouse i. p.), mast ceil-deficient mice indeed showed decreased serum tnf levels 30 iron after injection amounting to only 12 to 25% of the concentrations observed in the corresponding sera of normal wildtype mice. in the clp model of septic peritonitis we found that mast celldeficient mutant mice were dramatically more sensitive to clp than syngeneic normal mice resulting in 92% mortality in w/w v versus 8% mortality in +/+ mice 2.5 days after initiation of clp. further experiments with w/w v mutants selectively reconstituted with cultured bone marrow-derived mast cells from normal syngeneic wild-type mice and the use of an antibody specifically blocking the action of tnf tn vivo should clarify a potential protective function of mast cells in this model of septic peritonitis. interleukin-10 (il-10) inhibits cytokine production, including tumor necrosis factor (tnf), by lipopolysaccharide (lps)-aetivated maerophages. we recently observed that lps injection (e.coli 055:b5, 100 gg ip) into balb/c mice induces the rapid release of circulating il-10 (17±9 u/ml at 90 min). blocking endogenous il-10 using monocional antibody (jes5-2a5, 2 mg, 2h before lps) resulted in a massive increase in tnf production (107±47 in lps+anti-il-10 treated mice vs 6±2 ng/ml in lps alone, p<0.05, n=4 to 5 mice per group) and an enhanced lps-induccd lethality (53% vs 7% in anti-il-10+lps or lps alone respectively, p=0.01, n=15 mice per group). irrelevant igg1 rat monoclonal antibody (lo-dnp) did not influence neither tnf production nor lethality associated with endotoxin shock. this led us to study the production of il-10 during human septicemia. plasma samples were obtained from 65 patients with gramnegative (gns, n=22) or gram-positive septicemia (gps, n=43) and from 20 healthy volunteers. among these patients, 17 suffered from septic shock at the time of sampling. il-10 levels were measured by elisa (detection limit: i 1 pghrd). we found that 35 patients (54%) had increased il-10 plasma levels (range 12 to 2740 pg/nd). patients with gps had il-10 levels similar to the ones observed in gns (median: 12 vs 17.5 pg/m, respectively). patients with septic shock had higher il-10 values (median: 58 pg/ml) than septicemic patients without shock (11 pg/ml, p=0.002). no il-10 was detected in plasma from healthy volunteers. we conclude that il-10 is produced daring human septicemia. our experimental data suggest that il-10 might be involved in the control of the inflammatory response induced by bacterial products. dr arnand marchant, immunology department, hopital erasme, 808 route de lennik, 1070 brussels, belgium. to provide information about the role of tnf in sepsis and mods we measured tnf and stnfr-i levels in septic patients and investigated if there is a relation between plasma concentration of these molecules and the severity of sepsis evaluated by two scores (apache 1i and sss). patients and melhods: 20 septic patients fullfilling sepsis criteria of american college of chest physician and society of critical care medicine were studied. tnf-cc and stnfr-i (60kda) were measured by enzyme immuneassays (norms1 values = 13+8 pg/ml and 1.06_+0a ng/ml respectively). results: the mean tnf and stnfr-i values for each patient (mean+sd) were 93+64 pg/ml and 11.1+5.5 ng/ml respectively. these values are approximately seven and ten times greater than those observed in normal healthy volunteers (p<0.001). mean tnf concentrations for each patient were significantly greater in non survivors (112+52 vs 64_+74 pg/ml p<0.05); stnfr-i levels also were greater in this group, but the difference was not statistically significant (12.6+5.2 vs 8.8_+5.5 ng/ml). plasma tnf and stnfr-i concentrations were significantly correlated (r = 0.65 p<0.005). mean tnf levels were significantly correlated with apache ii (r = 0.49 p<0.05) and sss (r = 0.54 p<o.01); stnfr-i levels were likewise correlated with both scores (r = 0.80 p<0.001 and r = 0.76 p<0.001 respectively). tnf and stnfqgi levels increase with the number of dysfuctional organs; in particular tnf and stnfr-i increase when hemodynamics deteriorate and kidney failure ensues. conclusions: the correlations between tnf and stnfr-i levels and sepsis severity scores suggests that tnf is involved in sepsis and may influence the occurrence of mods and finally clinical outcome. tnf and stnfr-i are in fact especially increased when mods occurs. in particular their concentrations are elevated when cardiovascular system and kidney failure ensue, suggesting that tnf can have a direct role in hemodynamic derangements caused by sepsis and that kidneys are involved in tnf and stnfr-i excretion. lif is a pleiotropic cytokine that has been implicated in the pathogenesis of sepsis in animal models. we conducted a prospective study in 33 septic patients to correlate lif plasma levels with patient's clinical and biological data (among them il-6). plasma was drawn daily from day one to .ten and lif presence was determined with a specific elisa and with the dai,a bioassay (sensitivity of 100 pg/ml and 1 ngml respectively). risk factor associated wftt~ with elevated lif plasma level was determined by an univariate analysis. a multivariate stepwise logistic regression analysis was subsequently performed with a bmdp statistical software. lif was detected with the elisa and with the bioassay in 11 and 2 out of the 33 patients, respectively. lif peak plasma levels were statistically associated with high creatmin levels, temperature and il-6. multivariate regression analysis showed that high serum creatinin level was the major independent risk factor associated with elevated lifplasma levels. this correlation was also found for il-6. peak plasma levels of both lif and il-6 correlated inversely with patients survival duration. cox's analysis for plasma levels of lif >480 pg/ml yelded a hazard ratio of [exp (1.86)=6.44]. our study indicates that lif levels were associated with clinical and biological parameters of illness severity and significantly increased (cut-off value 480 pg/mi) in patients with fatal outcome. current consensus exists about the central role of tumor necrosis factor (tnf) alpha in initiating the systemic inflammatory response syndrome (sirs). a correlation with sirs has inconsistently been found. tnf effects its pleiotropic reactions upon two distinct cellular receptors. soluble extracel]ular fragments of the human 55 kda tnf receptor (stnfri) and the 75 kda receptor (stnfrii) are detectable in the circulation. the kinetics of these endogenously produced tnf-inhibitors were measured to evaluate their role in patients with sirs. fourteen patients of an operative icu were included with the diagnossis of sirs (mean apache ii score: 20 points). serial blood samples were obtained within 12 h after diagnosis of sirs, every 6 hrs for the first 48 hrs and every 12 hrs thereafter until patients died or recovered. soluble tnfri and stnfrii were assayed by an enzymed-linked immunological binding assay. soluble tnfri and ii could be detected in all samples with a significantly higher level (p<o,o01) compared to healthy controls (normal value: tnfrh 1,5 ng/ml; tnfrii: 2,3 ng/ml). the serum concentration of tnfri (14,1 ng/ml) as well as tnfrii (18,2 ng/ml) were significantly higher in nonsurvivors (n=6) compared to survivors (8,6 and 9,5 ng/ml; n=8) at the onset of sirs and during the course of the inflammation response (p<0,05). a positive correlation exists between both receptors (r-0,57; p < 0,01 ). increasing levels and maximal values of stnfri (59 ng/ml) and i1 (82 ng/mi) were detected only in patients who died. the serum concentrations of patients who recovered did not change and are remaining at the initial level. tnf alpha bioactivity was detected in 10 out of 14 patients. no significant correlation exists between the concentration of both receptors and tnf alpha as well as the apache ii score at the onset of sirs. elevated serum concentration of stnfri and 11 are found in patients with sirs. these naturally occuring antagonists reflect the inflammatory process. the measurement of soluble tnf receptor levels may be useful in monitorina sirs and in the prediction of outcome. 11-2 is given to patients with advanced melanoma or renal carcinoma. however, this therapy is accompanied by severe side effects, including the vascular leak syndrome (vls) that closely resembles septic shock. to investigate the involvement of cytokines and phospholipase a z in the pathogenesis of the vls we collected serial plasma samples from patients during the first 24 hours after administration of il-2. in these patients we monitored temperature, blood pressure and heart rate and assessed levels of the cytokines tnf, il-6 and il-8 using immuno assays. in these plasma samples we also measured phospholipase a 2 activity using an enzyme activity assay, since this enzyme is beleived to play a major role in the generation of lipid mediators. we demonstrate that during il-2 therapy the cytokines tnf, il-6, and il-8 are produced and that the release of these cytokines is followed by an induction of phospholipase az activity in the plasma of these patients, we further discuss the role of these mediators in the development of the vascular leak syndrome observed in patients receiving il-2. objectives of the study: sepsis caused by candida albicans (ca) is no longer a clinical rarity but a common consequence of immunosuppression and surgery, and is associated with high mortality. tumor necrosis faetor-c¢ (tnf-<~), interlcukin-lg (il-lg) and interleukin-6 (il-6) are thought to play an important role in the pathogenesis of the sepsis syndrome. we therefore studied the in vitro production of tnf-<x, il-ib and il-6 by human peripheral blood mononuclear cells (pbmc) stimulated by ca compared to stimulation by bacterial lipopolysaccharide (lps). materials and methods: pbmc were isolated from venous blood of 5 healthy volunteers and cultured at a concentration of 2.5 • 10 ~ eells/ml in culture medium with a dose-range of either heat-killed ca or lps. supernatants were harvested after 24 hours. using elisa, tnf-c~, il-ib and il-6 concentrations were measured. ca was isolated from a patient with ca sepsis and cultured under sterile conditions. results: ca and lps stimulate the production of tnf-~t, il-11] and il-6 by human pbmc. we observed a dose-dependent synthesis of these three cytokines in human pbmc. tnf-c¢, il-ib and il-6 in ng/ml as mean _+ standard error. ca/ml 0.0 1. (2) we constructed a single-chain fv-fragment (sc-fv) from the variable domains of a neutralizing antibody to human tumornecrosisfactor-alpha (tnf) because sc-fv should have some advantages in vivo. the sc-fv has a similar affinity (3,8x10-9m) as the fab-fragment (2,7x10-9m) but bind the antigen with a eightfold lower avidity compared to the parental mab (4,8x10-10). the parental mab as well as its fragments can compete the binding of rhutnf to both tnf-receptors. this was shown by competitive binding to the cell line hl-60 and to immobilized rtnf-receptors. in the nutralization assay on the cell line, l 929, the sc-fv can neutralize tnf but in comparison to the mab or its fabfragment, the capacity was three-fold lower as expected from the molar ratio of the dissociation constants. the in rive-neutralizing capacity was tested in a mice receiving toxic dose of rhutnf. the sc-fv showed a 15-fold lower neutralizing capacity in comparison with the fab-fragment. this could be explained in different manners: i) partial instability of scfv at 370c, ii) shorter half-life because sc-fv but not fab-fragments are eliminated via kidney, iii) elimination of tnf-immunocomplexes may be improved by opsonization mechanisms which requires some structures of the constant region (as in fab or mab). in summary, despite the encouraging in vitro experiments, the in vivo data suggest that sc-fv are not the right strategy for neutralizing tnf in vivo. tnf production in endotoxin non-responder mice, effect of a glucocorticoid antagonist g. iazgtr jr, e. duda, j. ol~th, e. husztik, g. iazar glucocorticoids inhibit lipopolysaccharide (lps)-induced tnf production and tnf can stimulate pituitary adrenocorticotropin secretion, resulting in the release of corticosterone. earlier studies (1) reveales that the glucocorticoid antagonist ru 38486, nufepristone, sensitizes both endotoxin responder and nonresponder, c3h/hej, mice to endotoxin lethality. we have also shown that the glucocorticoid antagonist ru 38486 sensitizes endotoxin-tolerant (endotoxin-pretreated) and normal mice to the lethal effect of septic and endotoxin shock. on this basis, we set out to study the influence of ru 38486 on tnf production induced by endotoxin in endotoxin responder and non-responder mice. one and 2 hrs following lps injection, ru 38486 significantly increased the tnf concentration in the serum, liver and spleen of treated animals as compared with the control and methylprednisolonetreated groups. in tissue cultures, ru 38486 induced the tnf synthesis of myeloid cells and increased the tnf production of genetically modified hela cells, which synthesize tnf constitutively. normal and tumor cell cultures exhibited an increased sensitivity toward tnf in the presence of ru 38486. however, the same dose of lps did not induce tnf production in the serum, liver and spleen of endotoxin non-responder mice and this was not influenced by concomitant ru 38486 treatment. these studies suggest that ru 38486 aggravates lps lethality via factors other than tnf in endotoxin non-responder, c3h/hej mice. the cytokine response to a novel exnacellular mitogenic factor, mf, produced by the group a streptococci (gas), and to the streptococcal pyrogenic exotoxins (spe) a and b, was determined by in vitro stimulation of peripheral blood mononuclear cells (pbmc) obtained from healthy blood donors. the induction and kinetics of 14 different cytokines was studied at single-cell level using cytokine specific monoclonal antibodies and intracellular immunofluorescent staining. all three mitogens induced a massive ifny and tnfi3 response in 10-16% of the pbmc after 48-96 hours of cell stimulation. in contrast, il2 and tnfc~ containing cells was only detected in 1-3% of the pbmc. the induction ofil3, il4, il5, and ill0, was weak or completely absent. thus, the response indicates activation of th1 cells. however, illc~, illl3, illra and il8, were consistently found and gradually produced, peaking at 24 hems in approximately 5-8% of the pbmc. mf induced an equally extensive cytokine as well as proliferative inducing capacity, as did spea and speb, which suggests that also mf is a superantigen. a marked inter-individual variation could be noted between lymphocytes isolated from different individuals, both in the toxin induced proliferation and cytokine induction. this may be one explanation for the varying clinical severity noted in patients after infection with clonal gas strains. introduction -tumour necrosis factor (tnf) is released by mononuclear cells in response to inflammation and sepsis. it has been implicated as a possible mediator in inflammatory bowel disease (ibd) but its pathogenic role remains uncertain. this study assessed the relationship between tnfct and disease activity by measuring plasma and faecal tnf concelnmtions in an experimcntai model of ibd. methodologo' -colitis was induced in male wistar rats by intmcolonic instillation of 30rag 2,4,6-trinitrobenzenesulphunic acid in 0.5ml 50% ethanol (n=12). control animals received an isovohlmetric instillation of normal saline (n=12). faecal pellets were collected before induction of colitis (day 0) and throughout the experiment. after 8 days the colon was excised, ueighed and the colon macroscopic score (cms) assessed plasma tnf (ptnf) samples were assayed by bioassay and elisa. faecal samples were vortcxed in water and the supernatant removed for estimation of protein and faecal tnf (itnf) content (elisa). there is a significant increase in itnf on day 4 -#p=0 02 alld the total ftnf measured during the study correlatcs with the cms oll day 8 -p=0.04 there is no correlation between ftnf and ptnf. conclusions -therc is evidence of coloific tnf excretion by macroscopically normal animals and following induction of colitis there is increased faecal tnf which correlates with disease actmty. there is no significant elevation in bioactive or imnmnoreactive plasma tnf in colitie animals this data would suggest that faecal tnf is a marker of colonic inflamrnatiort and serial tnf assessment inay be useful as an indicator &severity in ibd. to study the effect of in-vivo hypoxia/reoxygenation on cytokine elaboration by routine peritoneal macrophages. materials and methods: adult male cba mice (20-25g) were primed intraperitoneauy with either lipopolysaccharide (lps) 10ug]anlmal or saline. five days later, the animals were subjected to hypoxia (8%02) for 160, followed by 20 of normoxia. harvested peritoneal macrophages were plated in-vitro, culture supernatants were collected at 20, 40, and 60 and assayed for tumor necrosis factor (tnf), prostaglandin e2 (pge2) and nitric oxide (no). control animals underwent the same procedures, but were maintained in normoxia (21%o2 disease 5, berlin, germany the precise mechanisms of reactivation of latent human cytomegalovirus (cmv) infection are still unknown. apart from the permissive effect of diminished cellular immunity there is evidence of a cytokine mediated cmv reactivation as it is known for other systemic virus infections (e.g. hiv). we found that tumor necrosis factor-alpha (tnf) -in contrast to all other cytokines tested -can stimulate the activity of the cmv-ie-enhancer/promoter region in the human monocytic cell line, hl 60. we wondered whether our in vitro results were actually reflected by the incidence of cmv reactivation in diseases which go together with high tnfalpha levels. cellular immunodeficiency as well as at least temporarily increased tnf levels are known to occur in septic disease. we tested for the presence of cmv infection in peripheral blood mononuclear cells (pbmnc) by means of 24h centrifugation culture, immunocytological detection of different cmv antigens (apaap-technique), and pcr technique (cmv-ie dna). in striking contrast to healthy controls almost all examined septic patients were positive, irrespective of the method for cmv-ddtection applied, including the less sensitive immunocytology. follow-up studies showed that cmv-antigen positive pbmnc ceased to be detectable in patients with good outcome once septicaemia had been overcome. to further back up our hypothesis, we looked for a coincidence of enhanced tnf-alpha serum levels and cmv antigenaemia (immunocytology) in some other diseases which are known to have either enhanced tnf serum levels or increased incidence of active cmv infection and found the majority of patients with b-cell chronic lymphocytic leukemia, liver cirrhosis, and common variable immunodeficiency to be positive for these two parameters. we now wondered whether, apart from cmv reactivation, tnf-alpha could also cause cellular immunodeficiency this way promoting cmv replication and spreading and eventually causing cmv disease. in summary, we were able to show that a diminished cellular immune response results from/n vitro or in vivo methylprednisolone (mps) is used to suppress both inflammatory and immune responses. il-1 receptor antagonist (il-lra) and tnf binding protein (tnfbp) are naturally occuring anti-cytokine proteins, possibly modulating inflammatory and immunological responses. to define mps modulation of cytokine and anticytokine responses, we examined effects of mps on il-lra production and tnfbp generation as well as il-i~ and tnfa production by human peripheral blood mononuclear cells (pbmc) stimulated with lipopolysaccharide (lps). pbmc were purified from the blood of healthy volunteers, then incubated with lps (10ng/ml) supplemented with different concentrations of mps (0, 500pg/ml, 50mg/ml). after 24hrs incubation, the supernatants were collected for rias of secreted cytokines. the cell lysates obtained by 3 cycles of freeze-thaw, were also collected for r ias of cell-associated cytokines. both the supernatant for secreted cytokines and the cell lysates for the cell-associated cytokines were subjected to rias for il-i~, tnfa, il-1 ra, and tnfbp-i (tnfsrp55). il-la, il-1 ra, and tnfbp were produced mainly as cell-associated forms in response to lps, whereas most tnfa was secreted into the supernatant of lps-stimulated pbmc. mps supplement resulted in reduction of il-la, il-lra, and tnfa production. il-la production was reduced up to 5.4+1.6%(50mg/ml) by mps in a dose dependent fashion, whereas about 50% decrease in tnfc, and il-lra production was observed at both high and low dose mps supplement. however, total tnfbp generation was not reduced significantly by mps supplement. furthermore, tnfbp secretion was increased 12 times more in the supernatant of lps-stimulated pbmc with 500gg/ml of mps. these data suggest that mps could effectively block tnf activity by both reducing its production and up-regulating tnfbp generation and that mps works as an anti-inflammatory drug as well as an immunosuppressant by modulating cytokine and anticytokine responses. in severe gram-negative sepsis, excess of proinflammatory cytokines is associated with increased morbidity and the mortality. we have found that immunocytes possess functional 13-adrenergic receptors (~-ar) which, when activated, down regulate the lipopolysaccharide (lps) induced gene expression and subsequent secretion of tnf-~ in vitro. in the present study, we used a lethal endotoxic model in mice to test the hypothesis that 13-ar stimulation will down regulate the gene expression thus decreasing the circulatory levels of proinftammatory cytokines and improve the survivals in severe endotoxicosis. the animals (iso group) were injected 3 times with !3-ar agohist isoproterenol (0.5 mg/kg i.m. and 0.5 mg/kg s.c.) 4, 2 and 0 hours prior to a lethal dose of lps injection (25 mg/kg i.p.). control group (ctl) received same volume of saline at the same times before the lps insult. at the appropriate time points after lps injection animals were sacrificed and venous blood was collected from the inferior vena cava. the plasma levels of tnf-cc and il-loc were determined by elisa. in the mortality study, each grou the data showed that isoproterenol significantly decreased the mortality and the circulatory levels of tnf-c~ and il-lcc (fig. 1) . these data suggest that 13-ar activation of immunocytes down regulates cytokine gene expression, decreasing circulatory eytokine levels thus reducing endotoxicosis mortality. these results provide a new pharmacological approach to reduce the excess of proinflammatory cytokines and mortality in sepsis. introduction: cytokines released from monocytic cells are thought to play an important role in the pathophysiology of sepsis. during the last few years several investigations have been performed aimed at modulating this mediator response. in the present investigation using a full blood model we have studied the effect of a standard immune globulin and an antitipopolysaccharide (lps) immune globulin preparation and a monoclonal anti-lps antibody in modulating the endotoxin induced cytokine response. methods: citrated blood from healthy human donors was incubated at 37 °c in rotating polystyrene tubes. samples were taken before addition of 1 x 103 ng/l e. col± endotoxin and after 2 hours. plasma was assayed for the cytokines minor necrosis factor alpha (tnf-~) and interleukin-6 (il-6) using elisa methods. the eodotoxin concentration was assayed by lal and end-point chromogenic substrate technique. the blood was preincubated with standard immune globuline (human igg from pooled plasma, gammagardr, baxter) or with an anti-lps immune glubuline (human anti-lps igg obtained by screening of blood donors, novo nordisk) in the concentrations of 4mg/ml or 8 mg/ml or with the monoclonal lipid a igm antibody ha-1a for ten minutes before the endotoxin was added. results: two hours after endotoxin addition markedly elevated tnf-~ and il-6 values were found.in the plasma. cytokine values at 2 hours: tnf-ec 1689 pg/ml (mean) and 1l-6 1123 pg/ml (mean). preincubalion with 8 mg/ml standard immune globulins reduced il-6 values by 49 % (mean) while there were no effects on tnf-c~ values at 2 hours. a slight reduction in il-6 values was also found with the lower concentration, 4mg/ml, but this was not statistically significant. preincubation with anti-lps immune globulins had no effect on il-6 nor on tnf-cc values. preincubation with the monoclottal lipid a igm antibody ha-1a in variable concentrations had no effect on cytokine release in this model. in this full blood in vitro model standard immune globuline reduced endotoxin induced 1l-6 release while tnf-cc values were unchanged.the anti-lps immune glubuline preparation and the monoclonal lipid a igm antibody ha-1a had no effect on the il-6 or tnf-ct release. the effect of anti-tnf treatment in severe haemorrhagic/traumatic shock was investigated in a conscious rabbit model. the jugular vein (for administration of substances and blood withdrawal) and right femoral artery (for measurement of bp) were cannulated and an aortic thermistor probe inserted into the left; femoral artery for the measurement of cardiac output (co). the following day, upto 40% of the estimated blood volume was withdrawn over 80-40 rain, to reduce bp to 40-50mmttg and co by 2/8 mad withheld for 60 rain. shed blood (60%) was then reinfused followed by lactate solution to give a total potential dreulat%ag fluid volume of 120%. zymoeen activated plasma (0,36gg/ml) was given 10 rain prior to haemorrhage and during blood r~n%eion, all ~,~ir, ak were killed at 72h and o~gane removed for histology. rabbita received either monoclonal antirabbit tnf (r6, rat igg1, 8mgkg i.e. n=7) or emine (nffi6), 60 rain prior to haemorrhage. the cardiovascular and biochemical changes during the haemorrhage and hypotensive phase (is. haemorrhagic stimulus) were similar in both groups. upon reinfusion, haematccrit and white cell count remained sigutfia~utly (p<0.05) higher in saline compared to r6-treated animals, indicating a relative haemoconcentration (i,e. reduced circulating volume) in saline animals. the lettkocytosis persisted in the saline animals upto 72h. plasma glucose, lactate and asparteto .m~-o transferase all rose similarly in both gorups but plasma ¢rea~!~+-e levels were markedly (p<0.05) higher in ealine ve r6 animals at 48 and 72h indicative of kidney damage, using a macro and micro. pathological scoring system, major organ damage was seen in the lung, liver and kidney which was significantly (p<0.05) attenuated in r6 treated animals. in the hmg and liver, this was primarily due to a reduced bleeding and pmn infiltrate and to an additional maintenance of tubular integri~:y in the kidney. hence, in tlais model anti-tnf treatment markedly reduced the biochemical and histological indices of severe ,, ~gan damage during liaemorrhage and ranerfn~ion. thermal injuries and systemic bacterial sepsis produce a wasting diathesis with anorexia and marked loss of lean tissue and body fat. endogenously produced cytokines, including interleukin-1 (il-1 ), are thought to mediate many beneficial as well as pathologic host changes that occur during burn injury and infection. to evaluate whether bluckade of il-1 in vivo would affect survival and attenuate the anorexia and cachex[a associated with a thermal injury and chronic fulminant gram negative infection, 24 sprague-dawley rats were studied. anesthetized rats were divided in 3 groups, implanted with alzet r minipumps and randomized to receive the following treatment for 14 days:i: 25 gg/kgbw/min of il-lcc; ii: 25 ~tg/kgbw/min of il-1 receptor antagonist (il-ira); iii: buffered vehicle. the animals were subjected to a 30% bsa, full thickness scald burn and the wounds then infected with 10 + cfu/ml pseudomonas aeruginosa. eight additional rats were anesthetized, but were not burned, and served as healthy controls. . il-hx reduced food intake transiently but was otherwise without effect however, il-lra significantly attenuated weight loss over the first 8 days and improved food intake between days 5 and 8. we conclude that blockade of il-1 after a thermal injury with superimposed infection does not adversly affect the progression of the disease, but does attenuate the anorexia and weight loss associated with this model. these data indicate that il-i receptor blockade may offer a means to minimize the morbidity associated with catabolic illness. tumor necrosis factor (tnf) and its downstream induced mediators have pivotal roles in the pathogenesis of sepsis and septic shock. the suppressive effect of pentoxyfillin (ptx) on tnf production may be of clinical importance. when peripheral white blood ceils were stimulated with either lps or staphylococcus aureus, pentoxyfillin inhibited tnf production. in contrast, no inhibitory effect was observed on the induction of il-6. ptx inhibited not only the tnf production of monocytes but also the tnf secretion of both granulocytes, and unseparated whole blood. the facs analysis revealed that the expression of cd14 antigen on monocytes was not influenced by pentoxyfillin. the in vitro tnf and il-6 producing capacity in septic patients were higher than in the control group, but decreased on subsequent days especially in fatal cases. administration of ptx (200 mg/day) in septic patients resulted in tnf and il-6 productions similar to those found in control donors. it also subsequently led to an improvement of the clinical status. a further improvement in apache score could be achieved by administration of pentaglobin ° (biotest)( 5ml/kg/day ). the prevention of lpsinduced in vitro tnf and il-6 production by pentaglobin ° could be demonstrated in in vitro experiments involving the use of whole blood rather than purified lymphocytes, very probably because of the presence of lps binding protein in the plasma. the level of soluble icam-1 in sera of septic patients was significantly higher (800-1200 ng/ml) than in normal individuals (50-150 ng/ml), but it decreased following the ptx and pentaglobin ° therapy. it is presumed that ptx and pentaglobin ° can antagonize eytokine production at different levels, resulting synergistic action being beneficial in the treatment of sepsis. albert szent-gy6rgyi medical university, institute of microbiology, szeged, hungary h-6720 ddm t. 10. e~i~im~/tii, septic sh0~ '. tnf alfa/pge, and somatostatln lands ji., alvarez j., gram m., llanos k., alcalde j., sanchez ja., balibr~d jl. taurine, a semi-essential sulphur-containing t:~-amian acid, promotes neutrophil phagocytic activit3' against yeasts and enhances intracellular killing of e.coli. paradoxically it protects against hypochlorous acidmediated biomembmne oxidatio~ and abrogates the pyrogenic effects of intracerebral endotoxin. it is therefore possible that taurine mediates some of these effects by modulating the cytokine cascade. aim to assess the cytekine responses in a rat model of intraperitoneal sepsis, pretreated with supplenmntary taurine, by measuring tnf and il-6 concentrations. methodology female wistar rats (wt 160-200g) (n-6 per group) were prefed with 2% tanrine, 3.33% glyciuc ( in tap water) or tap water (tw) for 15 days prior to caecal ligation aud puncture (clp). normothermia was maintained intraoperatively and the animals received 0.9% saline subcutaneously (3 mls/100g) post-procedure. animals were venosected by direct cardiac puncture at 6 or 18 hours post-operation and the blood placed in endotoxin-free tubes. centrifilgation and storage of plasma at -70°c was completed within 2 hours. tnf and il-6 were measured using wehi and b9 bioassays respectively. results (means ± sem) bioactivc tnf concentrations were not significantly different between clp or sham groups. however plasma il-6 estimation revealed a statistically significant reduction at 6 hours in tanrine-treated animals compared to glycino and tw controls ( objective: to evaluate the effects of allogeneic blood transfusion, thermal injury and bacterial garage on interteukin 4 (il-4), tumor necrosis factor alpha (tnf) production and host mortality and to study if the administration of thymopentth (thy) could affect these events. materials and methods: balb/c mice were transfused with allogeneic blood (from c3h/hej mice) and treated daily with thy (i mg/kg)(t+thy) . control animals were transfused but not treated (t). after 5 days systemic blood was harvested to determine the circulating levels of il4 and tnf. a second group was treated with thy for 5 days and then received a gavage wide lxl09 escherichia coli and a 20% burn injury (bg+thy). one hour post-bum, blood was collected to measure cytokins. control animals received the same treatment but thy (bg). a third group was transfused, treated with thy for 5 days and then received gavage and burn (tbg+thy). one hour post-burn, blood was collected to measure cytokins. control animals received the same treatment but thy (tbg). all treated and control groups had 8 mice/group. in separate groups (n=20/group), receiving the same treatments, mortality was observed for 10 days post-burn or transfusion. the production and release of oxygen radicals by phagocytic leukomytee is one of the most relevant and important functions of these substances in biology. when neutrophilic granulocytes or certain macrophages phagocytize they produce super oxide and hydrogen peroxide and form secondary products of these activated species. all of these substances are released in to the phagosome. it has been appreciated since 1977 that the amount of oxygen consumed by phagocytes in producing free radicals is prodigious. what has not been totally appreciated is the significant contribution this 02 consumption contributes to total body oxygen consumption. our understanding of increased oxygen consumption following sepsis is compounded by a paradox of increased cardiac index and a narrow av02 difference. it was mclean in a study reported in 1967 that showed an increase in cardiac index and a narrowing of the avo z difference in humans following sepsis. other studies documented similar changes in humans and it has recently been speculated that increasing o~ delivery might have a favorable impact on outcome. at least three different explanations have been put forward as to why there are possible alterations in energy metabolism during sepsis. these include decreases in oxygen supply, peripheral shunts and direct action o~ cellular mediators on o~ delivery, a provocative review by hodgkiss and nark in 1992 shewed that in experimental animals sepsis did not cause any evidence of bioenergatic failure in muscle i liver, heart or brain tissue. they further showed that the increase in serum lactate is mot necessarily due to cellular hypoxia. they conclude that cellular hypoxia and defects in energy producing metabolites are not the cause of metabolic abnormalities in sepsis. we set out on a series of experiments to try to explain the paradox in cellular metabolism and whether or not white cells might contribute to total body oxygen consumption during sepsis. the first set of experiments involved growing rat cardiac myocytes on tissue culture. pyruvate was added to the medium as substrata and physiologic levels of peroxide were also added simultaneously, carbon 14 labeled co~ production was measured as was nucleotides and degradation products from the cell. the peroxide induced a significant inhibition of pyruvate dehydrogenase. in addition, there was a loss of cellular atp and a corresponding rise in the release of inosine and adenine from the cell. we concluded from this first sst of experiments that physiologic levels of peroxide are a potent inhibitor of pyruvate dehydrogenase in isolated cardiac mitochendria as well as the cultured myocyte. we further showed that pyruvate dehydrogenase inhibition blocks the aerobic oxidation of glucose and leads te adenine nucleotide metabolism with adenosine and inosine release from the cell. other enzymes within the tca cycle did not appear to be specifically blocked by peroxide. this would explain the increase in lactate and the ability of the cell to continue to make high energy phosphate by entry of metabolites in to other entry points of the tca cycle, the second set of experimbnts was designed to estimate the magnitude of whole body reactive oxygen generation via nadph oxidase following granuloeyte activation invivo. using a guinea pig model baseline oxygen consumption was determined. ~iaphenyl iodinium (dpi) was used as a specific inhibitor of granulocyte nadph oxidase. animals were divided into two groups; those that received dpi and those that served as control, all animals were then injected with phorbcl myristate acetate (pma) intravenously. pma is a potent stimulus for granulocyte activation and subsequent reactive oxygen generation. whole body oxygen consumption measurements were then repeated after the p~la injection. in addition, arterial blood gas amalysis was performed, circulating neutrophils were collected and assayed for their ability to generate hydrogen peroxide and the r~ght lung was removed for wet and dry weight measurement. total body oxygen consumption increased by 25 percent after pma injection. arterial oxygen tension fell significantly in the control animal. neutrophil hydrogen peroxide generation rate doubled and lung water in the untreated animals increased by 25 percent. in this animal model we could show that granulocyte activation substantially increases whole body oxygen consumption to approximately 120 percent of control values. the nadph oxidase inhibitor, dpi, decreases granulocyte hydrogen peroxide generation invivo and prevents the pulmonary injury induced by pma. using this animal modal it is possible to transpose this to the human septic state. if we assume there are 5,000 neutrophils per cubic millimeter of blood, a 70kg man would have ~1.75xi0 u circulating neutrophils. when activated ixl0 ~ neutrophils generate one nanemole of hydrogen peroxide per minute. t~erefore, the circulating neutrophil pool could generate -1.75xi0 nanomoles of hydrogen peroxide per minute. this corresponds to an oxygen consumption of -4.2ml/ojmin by the circulating neutrophil pool alone. this does not take into account the production of oxygen by macrophages or the amount of oxygen consumed to produce nitric oxide. manipulation of oxygen delivery in human sepsis may or may not be beneficial. irshad h. chaudry, ping wang, and alfred ayala life threatening blood loss, due to soft tissue and bony injuries, is a frequent complication in trauma victims. although numerous organs and cells are adversely affected by hemorrhage, the focus here will be to discuss whether or not there are any differential alterations in splenic and hepatic immune functions. in particular, m~ antigen presentation (ap) and associated processes will be described since one of the important functions of the m6 is to activate resting t-cell lymphocytes by processing and presenting foreign antigens in a mhc class ii-restricted fashion. studies have shown that splenic m6 and kupffer cell (kc) ap was significantly depressed following hemorrhagic shock and remained so for at least 96 h alter resuscitation. the presentation of antigenic fragments associated with mhc class ii antigen itself was not decreased following hemorrhage, but the catabolism of antigens in antigen-presenting cells was decreased. this is likely due to a significant loss of cellular atp. although m6 ap was depressed in splenic, as well as kc, only kc showed an enhanced capacity to produce inflammatory cytokines, il-1, il-6, and tnf. this difference in response between kc and splenic md may be due to differences in the microenvironment in which these cell populations are found, as well as potential differences in the effects that hemorrhage has on the environment from which these cells are obtained. splenic m6 from hemorrhage-resuscitated mice exhibited a significantly reduced cytotoxic response, whereas kc showed an enhanced cytotuxic capacity. the increased kc cytotoxicity is probably mediated through the increased expression of cell-associated tnf and the release of reactive nitrogen. the enhanced production of reactive nitrogen may play an important role in the clearance of microbes translocated from the gut following hemorrhage. however, excessive release of reactive nitrogen by kc may have deleterious effects on the adjacent hepatncytes. such an effect could, in part, explain the reduction in active hepatocellular function, which is seen following hemorrhage-resuscitation. thus, hemorrhage induces differential effects on splenic and kc m6 populations; it decreases splenic m6 but increases kc capacity to mount a cytotoxic response. the depressed splenic md and kc ap was, however, significantly improved by posttreatment of animals with atp-mgcia, chloroquine, diltiazem or interferon-'/. these agents also decreased the susceptibility to sepsis following hemorrhage. thus, the use of atp-mgci~, dfltiazem, chloroquine or interferon-7 offers new therapeutic modalities in the treatment of immunosuppression and for decreasing the susceptibility to sepsis, which is observed following severe blood loss. the production of macrophages from bone marrow precursor cells is a dynamic and highly regulated process. the differentiation of myeloid lineage-restricted progenitor cells is initially controlled by interleukin-3, which drives the progenitors along a maturation pathway that leads to their response to specific lineagerestricted colony-stimulating factors(csfs) we have previously hypothesized that thermal injury can initiate a self perpetuating cycle of production of defective immune cells: thermal injury can initially affect circulating immune cells. then these cells, by unbalanced production of colony stimulating factors and other mediators, can cause a derangement of hematopoiesis resulting in an abnormal production profile of tnf, il-i, and il-6 and cytotoxic activity of bone marrow derived cells. the results of our studies so far have supported parts of this hypothesis. il-3 and il-3+m-csf treatments increased the production of tnf in burn 4 day macrophages and progenitor cells compared to normal cells. il-3+m-csf+il-10 increased the production of il-6 by burn progenitor cells compared to normal macrophagee. preliminary results for the ratios of tnf/~ actin mrna indicate that il-3+m-csf increased the mrna for tnf in the 4-day macrophages derived from burned animals compared to macrophages derived from normal animals. ratios of il-6/~ actin mrna indicated that il-3, il-3+m-csf, and m-csf increased the il-6 ~rna in progenitor cells from burn animals compared to progenitor cells from normal animals. these preliminary results support our hypothesis that bone marrow derived macrophages and progenitor cells from burned animals act differently compared to cells from unburned animals regarding the production of inflanunatory cytokines. more specifically, the results suggest that the different cell types are regulated in different ways by cytokines, and cells from burned animals are regulated differently than cells from unburned animals. it i,~ to ~ssume that the translneatinn is due to a ixx~h,v functioning ~astroiutestlnal surface pro|ection system, which leads to an unacceptably high load ¢ln the imrsunc dcfcncc system, partlcul~ly the local system in the gastrointestinal wall, leading tu exh~ustiou of ihi~ system. tlae g&~lrointesliual i]'ac( can be regal'deal a.s a t~2servojf of appr 200 m ~ separating 10 i~ eucayotic ceils of the hosl ttom l0 ~4 bacterial culls. in the human body the the myriad of cndngan¢nts micrix)rganistns, exogenous itl,~ttdittg micro~ganisms altd taxies m-'e sepia"areal li'otll lhe rest of the body by a simple epithclhd layer. the barrier function is heavily tlcpcnding tm =~ proleclive layer cmtsisting ie. surl~:emts, nluclts, probiotic bacteria, and ,~ub~tances with strong antioxid~lt, antipfotease al~d other i~rotective eft'ecru, the surfaet~ml htycr consists at tile besl if1 i(.) bimolecular layers, fl is thus very iltil~, the epilhelial layer is renewed every 24 hours , and tile tanuwer of the mucus is ~so fa,~t, all attdphy of die epithelial layei induced by g&sttointestinal st~'vation does also include the mu~usprndocing cells, gnhlet cells, which leaduhg lu slmnage aad luwer quality of gi mucus, altered viscoelas,jc properties and reduced protection, ]~'obioiic bactcrla keeps the ppm's low, produce nutrients for the intestinal mllcosa, eliminates tuxia~ and stimulates the local immune system, the gl surface is conslanlly under hc wv wearing by ingested bacteria and their enzymes, ingested substaucas, chefftie,'ds at~d toxins. 'll~e n~tective flora is reduced in disease, by stress and by cm'clcss antibiotic therapy. wc have made attempts tn recondition the i,,.astroiniesli!l~d. tnucosa by rcstlpply of surfactants or sttrfactanfliko glyc~lipids, gels with pseudomuein [until.oil, and probiotl¢ laetobaeilli. by supply of glycolipids we have bt:cn able m prevent and heal intlammatory and tlleerallve injuries (and io p)'cveut microbe transhteatkln) in uppe~' and lower (.31 u'acl, this c~al be fu~'ther augmentented by shnultaueos supply el' a p.~uedomucin,likc gel like pectin. ()at contain~ one hundred times more ot membrane lipid$ th~a =nay other food, much of fiber, ~spceially watcrsnlnhlc betaglucaal~, at~d has an close to ideal amino acid cornpo~ition, <')at, termented by species-specific lactobacilli, has a strol~ ability to prevent local inflammatory and ulcerative chunges,transh)cadnn and sepsis in experimeut~d attimals mid to pl~ven~ revert mof in ba,nan.~.lt seems us, that it the mucos~lnucus/probiotic bacterial filnclinns cat'= 1%' restated by mucosa reconditioning, even in severely sick iudividuals) the hx:~d iuuuuue ~y,~teme will be capable tu pr~)tec! the ix~y ag~lls~ sepsis ~nd mof. the impaired immune response associated with multiple system organ failure (msof) has been most widely studied following surgery for inflammatory disease and trauma. the majority of late deaths following trauma are due to infection and msof. not all patients with multiple organ failure have concomitant infection present, yet most have been septic at some time in their hospital course. their generalized inflammatory condition often persists whether or not organisms have been recovered. immune failure probably precedes msof with or without overt infection and indeed may perpetuate such widespread sepsis. macrophage tumor necrosis factor-alpha (tnf) production is thought to represent an important pathogenic mechanism by which this is mediated. cecal ligation and puncture (clp) is a clinically relevant murine model of intra-abdominal infection and sepsis. serum tnf and endotoxin levels, and peritoneal macrophage tnf production are only slightly elevated in this model even in endotoxin sensitive animals. mortality in this model, therefore, does not appear to be attributed to overwhelming endotoxemia and/or tnf production. it has therefore been postulated that tnf and other cytokines may act in a paracrine fashion to cause local injury. tnf messenger rna (mrna) expression was therefore measured and found to be increased in the lung and liver following clp. a different pattern of expression was seen after endotoxin administration, characterized by a more rapid rise and fall, and enhanced tnf mrna expression in the spleen as well. route of spread of infection as well as the type of stimulus may determine the organ specific cytokine response. these data support the concept of locally produced tnf as a contributing factor in tissue damage and multiple organ failure during sepsis. the estimation of histamine's role in sirs changed over the years based on increased knowledge in shock pathogenesis, evidence later found to be faulty, and the discovery of other, more fashionable mediators. over the decades, the prevailing view has been that histamine is a noxious mediator contributing to the fatal outcome of shock. the analysis of experimental data on exogenously administered, endogenously released and/or newly formed histamine in septic/endotoxic shock suggests that histamine contributes to the early cardiovascular changes via hi-receptor vasoconstriction thus excerbating (directly and indirectly) the effects of catecholamines. in the later phase of septic shock (low out-put, high resistance states), however, it can be assumed that histamine exerts strong vasodilator and positive inotropic effects via h2-receptors. these effects are considered beneficial in counteracting or attenuating the effects of sympathetic responses of catecholamines and other mediators. thus, a blockade of the early reaction with hi-antagonists and a stimulation of the h2-receptors by h2-agonists are worthwhile therapeutic approaches. shock produc~s ~ij alterations, an encrgy crisis and eventual c~ll damage, however, shock in itse|f do~ not lead freqoenfly to r~mote organ damage. in animal ~xpcrhnents and clinical expariez:ces in korea and vietnam, shock i~r s¢ was not associated with lung damage, renal failure or ards. the same is true with g.l bleeding. however, when shock is due to trauma or associated with tlssus injmy, then organ malfunction m~d damage is frequent. shock is bclleved to increase the frequency of infection, but clinical evidcnc~ for this is scant. hemonhsgio shock akme is an unusual o~orrenee dinica[ly. immune dysfolletion dons occur witl~ experhnental hypovolemic shock and in pailcnts with hypotcnsion or after receiving blood ~ansfosions. tbc most significant factor affecting reoorrcnce of resented ca,lorectal liver mclastasis was hypotensivc episodes during operation. blood transfosions depress immune function, improve transplant ~raft survival and increase ttll'rmr rec~r'tcnos ill co[eli and head and nc~k cancers. in experimental hemorrhagic shock, we found decreased response of [ymphocytes to mitogens, el:clad mixed lymphocyte reactilln and i[,-2 decreased. othe~.x hsvc found decreased macrophage and t and b ¢¢lt function, deci~ased re,ease of/l~i, increased poe v depressed macrophagc antigen presanlatiota, increased ien y, decreased il-2, 3 and 5, and shared mscrophags funclion with loss nr f and c 3b r~ccpto~s. this is inhibited by chlornqoine, ibuprofen, dihiazem, and atp. shock may activate ncutrophils contributing to ilappitlg in cspillarics, no refluw, increased adhesion, cytotoxicity and organ damage. this may be related to decreased clearance of bacteria after shock. t~lls shock, hypotcnsion and decreased microcirculatory blued flow initiate or set the stage for immune dysfmtction. they, along with tissue injury trigger inflammatory cascades. this also contributes to immunologic dysfonctitm, which is associated with increased lufoction. thus altered bh,od flow and mediator cascades are bolh involved, the question now is can wc rapidly improve microcirculatory blood flow, and dg'masa inflsrmnatory fosponss, which is also necessary for survival the effe~ of ~nterferon ~amma @n wour~ healing was ewaluated ~n a routine mo~el. ~mma ~n~erferon we8 g~ven in doses of ~37.5 to ~2,500 u~%$ synchronous with ereatio~ of a pa~splnal woumd then daily for is ~lays. ~mteet~on ~amma impaired tensile strength a% all ~oses relative to control. m0zphology suggested ~reas~ ¢oll~gen deposition and r~du~-~ neovag~ulaeit¥ ~n t~eat~4 animals, interferon ~amma was a2so ~valuated in a murine mo~el of cecal llgatlon and punneure. one hundred to 22,s00 uni~ vere ~iv.n following ~nju~ an~ than daily. interferon qamma was associated with inco:ea~ed mortalit~ and earlier death a~/a~n ~n a dose dependant manner (p< 0,05). afmlnistra~ion the proinfiammatory cytokines tumor necrosis factor-c~ (tnf-c0, interleukin-16 (il-ii3), interleukin-6 (il-6), and interleukin-8 (il-8) have been implicated in the pathogenesis of the multiple organ dysfunction syndrome (mods) following shock and trauma. these mediators which are released in high concentrations by activated macrophages, endothelial cells, and connective tissue cells cause a systemic inflammatory response syndrome (sirs), thus leading to a shock-like state and tissue destruction. recently, a number of proteins have been characterized in in vitro and in vivo studies demonstrating potent anti-inflammatory properties. these latter cytokines include interleukin-4 (il-4), interleukin-10 (il=10), transforming growth factor-6 (tgf-j3), and the newly sequenced interleukin-13 (il-i3). they are considered antiinflammatory, since in vitro studies using purified macrophage cultures or whole blood assays revealed an inhibitory effect of these antmnfiammatory cytokines on ~he synthesis and release of tnf-cl and il-u3. in addition, they reduced the severity of disease and reduce plasma levels of tnf-c~ and il-ii3 when administered to animals with infection or inflammation. furthermore, naturally occurring substances have been described that specifically neutralize the bioactivity of il-1b and tnf-a. the il-1 receptor antagonist (il-i ra) is related structurally to il-i and binds to the same cell-surface receptors, but does not stimulate the cell. in animal models ore. cob-induced shock, inflammatory bowel disease, and peritonitis, injection of il-lra significantly reduced the severity of disease. the cytotoxicity of tnf-c~ can be inhibited through two types of soluble tnf receptors (stnfr) type i (p55) and type ii (p75). these stnfrs are proteolytic cleavage products of the cell-bound tnfreceptors. when given to baboons to combat e. coil-induced shock, soluble receptm's to the tnf type [ receptor decreased the severi~ of the shock-like state. it has been well accepted that shock and severe injury result in an increased release of proinflammatory cytokines with a high incidence of sirs and mods. our studies investigating plasma levels of anti-inflammatory mediators (il-4, il-lra, stnfrs) in patients after severe injury further suggest that shock and injury lead to an activation of anti-inflammatory processes with altered plasma levels of these mediators. however, while plasma levels of il-4 and 1l-lra were significantly increased in trauma patients compared to healthy volunteers, stnfrs did not differ from control samples. thus, shock and severe injury result in different activation patterns of anti-inflammatory processes. m,l. rodrick, boston, b~usa following severe thermal injury significant suppression of the immune response has been demonstrsfed. these changes are associated with increased susceptibility not only to co,on human pathogens, but to organisms not normally pathogenic in the uncompromlsed host. early observations included prolonged graft rejection and skin test anergy in patients following burn injury. work from our laboratory and that of numerous others has shown that the~e is a profound £mmunoeuppresslon following severe thermal injury. i~mune deficiency has been identified in these patients hyz ii lack cf t nell responses to mitogens, 2) early decreases in total t and helper t cells in the peripheral bloo~ 3} lack of response to tetanus toxoid i~uniza~io~ in spite of increased non-speoifi~ i~k~unoglobulin praduction and 4) severe and prolongei deficiency of interleukin-2 (il-2) prcductlon by peripheral blood mononuclear cells (pbmo). this il-2 deficiency could be an underlyin~ cause of all the afo~ementloned immune defects by failing to activate both helper and suppressor t cell functions. mo~a recent work has focused on the molecular mechanisms of this il-2 deficiency end shown that the defect lies post-transcrlptionally since mrna for il-2 is also depressed following thermal injury. we have also investigated the potential of a defect in ii-2 receptor status on peripheral blood mononuclear uolls from patients fqllowing burn injury and in a murine model. in both oases no decrease in il-2r was found either by phenotypic markers or by funational assays. another hallmark of thermal injury is hyperao~ivatlon of proinflammatory ~ytok~ne secretion, which may play a significant role in multiple organ failure following burn injury. therapy o~ major burn injury may reasonably be aimed, therefore, at up-regulating t cell ~unction and down-regulating hyperactive secretion of proieflaam~ntory cytokines. the majority of patients dying of bums succumb to sepsis which coincides with dysfunction in the specific and non-specific host defences. generally, concepts relating to the mechanism(s) of specific immune failure in thermal trauma imply that inhibition of t (cd4+) cell responses is imposed by the injury or infection-related factors. however, the same factors elicit strong biological reactions within the lymphoid compartment. recent evidence indicates that systemic activation is inherent in the burn patient. to establish the relation of molecular and cellular events to the development of post-bum anergy we examined the state of and the capacity for t cell activation with regard to: 1) occurrence of activation-induced cell death (aicd), 2) activation-related phenotypic and functional_ diversity in the pool of peripheral cd4+ t cells. initially, (up to 7 days post-bum), peripheral blood lympliocytes from severely injured (> 35% total body surface area) patients exhibited high levels of constitutive expression of surface receptor for ]l 2 (cd 25) and spontaneous blastogenesis. the presence of activation-related t cellproducts in bum plasma was also apparent. subsequent impairment of the t cell receptor (tcr)-regulated t cell responses in vitro was accompanied by significantly increased dna fragmentation that is associated with cell death by the mode of apoptosis. using molecular markers we established that flesh peripheral blood ceils from immunosuppressed patients also contain large numbers of apoptotic cells. fluctuations in the number of viable (pi-) peripheral blood lymphocytes involved primarily cd4+/cd45ro+ (memory) subset of t ceils. the above observations suggest that thermal trauma-associated t cell anergy develops through aicd, a phenomenon commonly associated with the tolerogenic activity of bacterial superantigens. persistence of staphylococcal infections in the burn patient may support this assumption. response following trauma jane shelby, ph.d. the immune system is integrated with other physiologic systems, and is exquisitely sensitive to changes in nervous and endocrine systems changes following traumatic stress challenge. the immune, nervous and endocrine systems interact via both direct and indirect pathways which utilize neuro and endocrine hormones, neurotransmitters, neurepeptides and immune cell products. it is now known that the immune system may be affected by all of the neuroendocrine products produced during a stress response, with evidence for innervation of iymphoid organs, lymphoid cell receptors for neuroendocdne products, and leukocyte production of chemicals which are virtually identical to certain neuroendocdne peptides (acth, endorphins). trauma induced alterations in the equilibrium of various neuropeptides and neuroendocdne hormones have a significant impact on immune response potential, affecting control of proliferation, differentiation and function of immune cells. for example, the neurohormone melatonin is thought to be a natural antagonist to counteract glucocorticeid associated immunosuppression resulting from stressful challenges, such as surgery and trauma, plasma melatonin levels are known to be significantly reduced in burn patients. the administration of exogenous me[atonin improved cellular immune response following burn injury in an animal model. melatonin was also shown to have in vivo cytokine regulatory activity, increasing the potential for il-2 secretion and downregulating excessive il-6 and ifn~ in burn injured, stress susceptible mice. the regulatory interactions between the immune, nervous and endocrine systems provide mechanistic pathways for trauma associated immune dysfunction. increased knowledge of these interactions will enhance the potential for the design of novei clinical interventions to improve immune response and decrease the risk for infection in trauma and surgical patients. . animals receiving e were given a single dose daily of either 2.4 g/kg of e in a 20% solution by garage (ge), or 1.25 g/kg of sterile ive in saline. four hours following the last dose, bum animals were subjected to a 30% body surface area bum injury to their dorsum. twentyfour hours following injury, the animals were sacrificed and spleen cells were harvested for assessment of lymphocyte function. splenocytes were prepared by mincing the spleen, followed by incubation on glass petri dishes to remove adherent macrophages. non-adherent cells were then tested for proliferative response to t-cell mitogen concanavalin a (con a) and b-cell mitogen lipopolysaccharide (lps). data were analyzed by anova. results: chronic alcohol exposure and burn injury independently inhibit lymphocyte response to con a but not to lps. the combination of e plus bum injury, however, pmfouedly decreases this response to both con a and lps as outlined in the this data clearly identifies the synergistic impairment of immune function produced by ethanol and bum injury. it is furthermore apparent that ibis effect is gut mediated and that gastrointestinal exposure to alcohol is necessary to produce this effect. further studies will work to identify cellular and subcellular mechanisms to explain this effect. in experimental animal studies and investigations on human volunteers endotoxin infusion is mgulary accompanied by the release of the cytokine tumor necrosis factor a (tnf-~) determined by elisa technique. in patients with menigococcal sepsis also elevated tnf-a values have been found using a functional assay. we have studied the role of tnf-et in surgical icu patients with sepsis. using functional technique, we were not able to detect tnf-~ activities in the patient plasmas. when this cytokine, however, was determined by immunochemicai technique (el1sa) elevated tnf-e~ values where frequently oberserved. in order to further elucidate these observations, we studied shedding of tnf receptors in the patients. in these studies, we noticed that shedding of tnf receptors oecured regulary in the patients. at the time of diagnosis, soluble tnf receptor p55 and p75 were both 2-3 fold higher than values found in plasma samples obtained prior to die diagnosis of sepsis. we also observed that the sepsis patients revealed higher maximum values of p55 and p75 during the icu stay compared to values found in surgical icu patients without sepsis. these observations indicate that soluble tnf receptors are available in sufficient amounts to bind tnf-ot which is released in surgical patients developing sepsis. this mechanism may explain why functional tnf-c~ was not detected in the patients. institute for surgical research, rikshospitalet, the national hospital, university of oslo, 0027 oslo, norway. decker, d., sch6ndorf, m., bidlingrnaier, f., hirner, a., yon rfcker, a. the advantage oflaparoscopic cholecystectomy over conventional open surgical approaches in the treatment of symptomatic cholelithiasis has been shown convincingly by clinical studies. in order to facilitate comparisons of different surgical approaches, we evaluated the cell biological characteristics of tissue trauma by measuring changes in various cell surface markers on leukocytes and eytokines in plasma as a possible means to assess tissue trauma in choleeystectomy. patients recruited into our study had experienced at least one typical bifiary colic, had ultrasound-proven cholelithiasis (stages 0-ii according to me sherry), were 35-55 years old, and presented for elective choleeysteetomy. patients could choose between laparoscopic and conventional eholeeystectomy after being informed about the advantages and disadvantages of each procedure. cell surface markers on leukoeytes were determined using whole blood techniques with the help of commercially available fluorescent monocloml antibodies and flow cytometry. shed cell surface markers in plasma and cytoldnes were measured with the help of sandwich-elisa kits. blood samples were drawn 24 h before surgery, immediately before incision (after anaesthesia), 2 h and 24 h after incision. seventeen cell surface markers were examined on different cell populations and cellular subsets in 18 laparoscopic and 15 open-surgery patients. three soluble cell surface markers and six cytokines were monitored. by statistical analyses (multivariate regression analysis, student's t test, wilcoxommann-whituey's rank sum test) the six markers/cytekines that best distinguished open surgical from laparoscopic procedurea were determined. these were 1. the interleuldn-2 receptor and im soluble form (cd25/scd25); 2. the activation antigen fd-1 and its soluble form (cd30/scd30), a member of the nerve-growth-factor receptor family; 3. the cd45ro epitope which characterizes t memory ceils; 4. the trausferrin receptor cd71; 5. the soluble adhesion molecule icam-1; and 6. the cytokines interieukin-6 and interleuldn-8. on the basis of these results, a tissue trauma activation (tta) index was calculated by combining the marker/cytoldne measurements by simple multiplication. anaesthesia and pre-ineision maneuvers did not significantly change cell marker or cytokine levels in either surgical approach as compared to 24 h before surgery. 2 h after incision the tra index in open cholecystectomy showed a distinct 22-37 fold increase, whereas in laparoseopic surgery a mere 2-3 fold increase was noted. 24 h after incision, the tra-index returned to near pre-surgery levels. in conclusion, our results demonstrate that changes in cell surface markers and cytokines can help evaluate the magnitude of tissue trauma in diffei'ent surgical approaches. the relationship between lymphocyte subpopulation changes after thermal injury and the increased susceptibility of burned patients to infection is unclear. in this study, we have attempted to correlate such subpopulation changes with the presence of infection in burned patients. peripberal blood from 19 patients was monitored for lymphocyte subpopulation changes three times weekly for three weeks postburn and weekly thereafter for three additional weeks. mean bum size was 44.7% (range 24%-84%) of total body surface and mean age was 38 years. infection was diagnosed by carefully defined clinical and laboratory criteria and its presence or absence noted each time blood was drawn. samples taken when patients had wound infection, bacteremia, or pneumonia were compared with samples taken in the absence of systemic infection. whole blood samples were stained with four monoclonal antibodies, the red blood cells lysed and the leukocytes fixed and analyzed by flow cytometry. for each patient sample, the proportion of lymphocytes falling within the light scatter gates was determined as the percentage of cells negative for cd14 and most strongly positive for cd45. this percentage was used to correct each sample for the presence of debris or nonlymphocytic cells. the proportion of cd4 and cd8 positive cells was slightly greatc~ in the samples from infected patients, while the proportion of b cells (cd20+) was unchanged and nk (cd16+) cells were decreased by ahnos[ 50% compared to sampie~ li'om uuiuleclcd patients. the percentage of cells positive for cdilb (c~ integrin) decreased sharply and cd4ro (memory cells) decreased slightly in samples from infected patients while the expression of the lymphocyte homing receptor and cd29 were unchanged. cd25 (il2 receptor) and cd69 (early activation marker) were significantly increased in the samples from the infected patients while hladr was unchanged. these changes in lymphocyte phenotype correlate with the presence of infection. if they closely precede or occur during the early development of infection they may be valuable clues to the mechanism of susceptibility following thermal injury. trauma patients are subjected to an immediate massive impact on their host defense integrity due to the combined effect of tissue trauma, shock and endotoxemia. cytoldnes are playing a crucial role within the course of an impaired cell mediated immune response (cmi) resulting from a disruption of intact m%/tcell interaction. the current study was undertaken to further elucidate the mechanisms of dysfimctional cmi following major burn and mechanical trauma -via comparative analysis of mrna expression and protein release. the major regulatory levels for different cytokines were determined in mitogen, respectively lps stimulated peripheral blood mononuclear cell (pbmc) cultures of trauma patients on consecutive days (13) t, 3, 5, 7 and 10 post injury. we analyzed the cumulative data for interleukin-1 beta (il-i[3), il-2, il-8 as well as tumor necrosis factor alpha (tnf-~) and saw a considerable impairment of the protein release in the stimulated pbmc cultures until d5 post-trauma and recovery thereafter. *p < 0.05, ** p < 0.01 vs control comparing the autoradiographies of the specific cytokine mrna expression with the protein release in the supernatants, we saw a good correlation between mrna signal intensity and protein synthesis for il-8 and 11,-2, suggesting that for these cytokines the main regulatory mechanisms are located at the pre-/transcriptional level. for the other cytokines investigated one has to suppose posttranseriptional mechanisms. the analysis of our data clearly indicates a severe impairment of forward regulatory immune mechanisms following trauma. most likely the regulatory mechanisms, that are involved are greatly different among the cytokines investigated. it may be concluded, that depressed cmi responses post-trauma are partly due to an impaired pro-inflammatory cytokine production. the severity of the injury (iss) correlated with the development at multiple organ failure (mof-score; r=0.612). the levels of mediators and markers of the inflammatory response were generally higher in the more severely injured group (iss>30, n=16). i1-6, 11-8, g-csf, fpa, and c3a -levels differed significantly (p<0.005) between the iss-groups (>-< iss 30) at the time of admission, whereas on day 3 tnfa, c3a, 11-6, and ealpi showed significant differences. beyond the first week, major differences were restricted to pge 2 and c3a. the formation of two groups with respect to later multiple organ failure (mof < 3; mof > 2 n= 14) yielded similar results. leukocyte-facs analysis revealed significant differences mainly in the cd14 (monocytes), cd3/cd25 (i1-2r + t-cells), and cd29/cd4 (th 2calls) populations. summarizing our findings we were able to detect some alterations in the surface antigens of immunocompetent cells. the inflammato d response, however, seemed to be more pronounced and correlates wi~ the further clinical course. using an experimental bum model in rodents, we have demonstrated that administration of a full thickness, scald burn involving 20% or more of the total body surface area (tbsa) elicits systemic responses which are characterized by numerous alterations in t-ceu function (i.e., lymphokine production and contact hypersensitivity (ch) responses) plus an enhanced susceptibility to bacterial infection. in the present study we questioned whether the apparent systemic effects mediated by large burns would be elicited as site-specific alterations in immune function following administration of small area burn trauma (5% tbsa). following a 20% tbsa burn, ch responses to contact sensitizing antigens were found to be altered. the depression in ch responses could be induced independent of the site used for topical skin sensitization. following a 5% tbsa thermal injury, development of ch responses were affected in a site-specific manner. immunization of 5% tbsa thermally injured mice in a site near the position of the burn resulted in depressed responsiveness, whereas immunization through a contralateral site resulted in responses that displayed both the intensity and kinetics of a ch response equivalent to sham-bumed mice. similar systemic and site-limited changes in lymphokine production were observed with 20% and 5% tbsa thermal injuries, respectively. a 20% tbsa injury affected the lymphokine producing potential of all cells regardless of which lymphoid tissue the cells were isolated from. the effect of a 5% tbsa burn was significant but site-specific. thus, ceils from lymph nodes receiving drainage from thermally injured tissue were specifically affected, whereas lymphokine production by cells from lymphoid organs receiving drainage from unaffected skin was normal. it was concluded that modulation of lymphokine production and cellular immune responses may be a normal consequence of burntrauma regardless of the size of the burn. changes in immune competence can be mediated either regionally or systemically in direct proportion to the area of skin exposed to the burn injury. this work is supported by phs grant gm46899 and the office of navy research n00014-92-j-1612. division of cell biology and immunology, department of pathology, university of utah school of medicine, salt lake city, ut 84132. post spleneetomy septic sequelae may be fatal, but the mechanisms remain unclear. the objectives ef this study were to assess the mortality from concomitant splen-'etomy and ]~eritoneal bacterial challenge and to elucidate the local cetkdar responses. 50 cd-1 mice were randomised to receive laparotomy and sham splenectomy (l) or splenectomy (s) with simultaneous ca'-cal ligation and "):mcture and the survival patterns assessed. subsequently, 150 cd-1 mice were randomised into control (c), l or s groups and peritoneal cells studied at 24 hours for bacterial phagocytosis and killi:~g, superoxide (02-) and tumour necrosis factor (tnf) production and macrophage activation vsing mac-i(cd-11b) receptor in~.ensity expressed es mean channel of fluorescence (mcf). these resides indicate that sf!enectomy predisposes to nrortal~ty from bacterial sepsis ia the early pos~ operative period compared to sham operated animals. failure ~f p'.acrophages to kill bacteria in the splenectomv group '~:cured in t?~e absence of impairment of oxygen freeradical or tnf pred:~ctien. the macrovh~ge ac!ivotion marker mac-1 was significantly reduced in both l and s groups and impaired phagocytosis of bacteria oceured in both operative groups compared to controls. laparotomy a!one reduces macrophage activity in terms of surface re:eptor mac-1 expression and !ingestive capacity. splenectomy however s~gnificantiy ~mpairs r-acrophage-wediated l~,acterial killing and this qefect rttav co~tribut~ sig~ifjcav'ly to th-~ dissemination of local infection and to n':ortalit). depts of haem~ tology & surgery, beaumont hosoital, dub!in 9,eire. introduction: loss of cell membrane integrity appears to be a common pathway of injury to tissues subjected to high-voltage electrical shock. the cell membrane is the most heat labile structure in the cell, and is also the most vulnerable to externally-imposed electrical forces. skeletal muscle and nerve cells are particularly susceptible to electroporation by clinically relevant electric fields. restoration of membrane integrity is essential for cell survival in victims of electrical shock. we have studied the effect of non-ionic triblock copolymers ( poloxamer class) on the transport properties of isolated rat skeletal muscle cells following electroporation-induced membrane disruption. 1-3 mm long adult skeletal muscle fibers were isolated by enzymatic digestion from the rat flexor digitorium brevus and maintained under standard culture conditions. they were loaded with the calcein-am dye and placed in a 37,c chamber for recording by real-time video confocal microscopy. the cells were subjected to 4 msec, 150 v/era, 1 a field pulses with a low duty cycle to allow thermal relaxation. peak temperature rise was 0,2.c. the uye content of the cell was monitored in real time. experiments were carried out in calcium-free phosphate buffered saline, with 1 mm mg%. experiments were repeated with 4 mm neutral dextran ( the aim of the present paper is to ascertain if thuracotomy induces a different pattern of variations of cytokines, immunocompetent cells and antibodies from laparotomy in the early postoperative period. 60 patients (34 males 26 females,mean age: 59.5_+5) 30 with gallstone disease and 30 with non neoplastic pulmonary disease were studied. none of these patients received blood transfusion, biological response modifiers, radiotherapy or surgery for at least 6 months before being included in our study. anaesthetic procedures were similar in all patients and none were matnourished. on the day of surgery and on the 1st and 7th postoperative days (pre, lpo, 7po) percentages of cd19, cd5, cd4, cds, cdi6 were measured by means of flow cytometry using moab., and levels of ig a, lgg, igm, ige. by nephelometry cytokine levels in peripheral blood(il-1, il-2, il-4, il-6, tnf) were measured in 10 pts. of each group by means of elisa using moab. _r. esults:variations of il-2 and il-4 were not s.s.. il-6 increased but differences between groups were not statistically significant (s.s). il-i decreased on 1po and increased on 7po in both groups but were only s.s. in the th.g., and therefore, the differences between groups were s.s (p<0.05).tnf decreased in the l.g. and increased in the th.g. on the 1po, the difference was s.s(p<0.05); on 7po, tnf decreased in the l.g. and decreased in the th.g. but these variations were not s.s. cell percentages decreased an lpo and increased on 7po, except for %cd19 cell that increased on lpo and decreased on 7po ,in both groups of pts. differences were not s.s. ig a, igm decreased and ige increased in both groups (p< 0.0i), but differences between them were not s.s. in contrast, igg decreased on 1po (p<0.01) and increased on 7po in both groups, but the decrease iu the th.g. was greater than in the l.g. twenty male children,aged from six months to 3 years,admitted for elective inguinal operation were studied. the operations were performed under balanced combined anaesthesia (fentanyl,thiopemtone,vecuronium, 70% nitrous oxide in oxygen) and blood samples were collected before flunitrazepam premedication,after anaesthesia,4 and 24 hours after anaesthesia. cells from the wound were collected with cellstick sponge which was removed from the wound 4 or 24 hours after anaesthesia. the study was approved by the local ethical committee. the percentage of neutrophils was increased and that of lymphocytes was decreased in perpheral blood after the operation.the values in the wound were close to the values found in peripheral blood. the percentage of t-lymphocytes (cd3) and helper-t-cells (cd4) decreased in peripheral blood being lower in the wound than in peripheral blood after the operation. the percentage of t-eytotoxic cells (cd8) also decreased in peripheral blood and was similar to that in the wound. b-lymphocyte (cd19) percentage was increased in pe~pheral blood after the operation and was higher than in the wound. the percentage of activated t-cells (cd3+hla-dr-positive cells) in peripheral blood increased while that of natural killer cells (cd3+cd16+leu19-pos) was increased just after anaesthesia being decreased at g and 24 hours after the operation. spontaneous lymphocyte proliferative responses didn't change while phytohemagglutinin a and concavalin a induced responses were decreased in peripheral blood samples 4 hours after the operation with recovery at 24 hours.pokeweed mitogen induced lymphocyte proliferative responses were decreased at 4 hours (p<o.01) and 24 hours after the operation we previously reported that plasma ige increased after traumatic injury. in this study, we measured plasma ige ievels in 40 trauma patients on hospital admission and 3x weekly in the intensive care unit to evaluate ige kinetics. patient characteristics were: mean age 36-+ 16 years (33 m, 9 f), mean injury severity score (iss) 26+ 10, sepsis (19 patients), sepsis syndrome (9 patients), ards (4 patients), renal failure (5 patients). ige increased in 28 patients (70 %). the mean increase was 527 ng/ml (range 0 to 7362 ng/ml; 95% ci was 160 to 895 ng/ml). increase in plasma ige was significantly greater in patients with sepsis syndrome (p=0.021) and renal failure (p<0.001). although mean plasma ige was higher with ards, pneumonia, hepatic dysfunction, and shock, these differences were not significant (p>0.05). plasma ige increase was not related to severity of injury by iss score (p =0.42). the mean day to highest ige was 9.1-+6.5. the day sepsis was first observed preceded the day of highest ige by 4.2+6.6 days. there was a significant association between the day of sepsis onset and the day of highest ige (p=0.018). eight of nine patients with sepsis syndrome had > 100% increase in plasma ige from admission. one patient's ige levels were normal (20-100 ng/ml) for 24 days and then increased to 7300 ng/ml over the next 12 days, after onset of sepsis syndrome. changes in ige plasma levels may reflect the action of cytokines, such as il-4, which concurrently regulate production of ige and il-1 receptor antagonist in a response to sepsis. sepsis remains a leading cause of late mortality in trauma and hs. although hs-induced bacterial translocation is supposed to be the major cause of sepsis and mof, depression of the res increases susceptibility to infection after injury. the purposes of this study were: a) to evaluate the res in the lung, spleen and liver after hs and subsequent hypertonic saline (hsl) treatment, and b) to document the patterns of phagocytic activity in these organs during 24 hrs. adult male wistar rats (250+_23 gin) were submitted to hs (sbp 40 tort) and after t hr (shock i hr) and 24 hrs (shock 24 hrs) hsl (nac17.5%, 3.5 ml/kg) treatment, 1131 e. coli (i 131) was injected into the portal vein 10 ~tci (n_>12). twenty minutes later, the lungs, spleen and liver were harvested and scintilographic counts obtained. data is depicted as mean_%+sem * p<0.05, ~" p<0.001 and statistical analysis was performed by analysis of variance and wilcoxon tests. one hr after treatment, lung 1131 uptake was increased and liver and spleen 1131 uptake were reduced compared to sham. twenty four hrs after treatment, all organs, except lung uptake, returned to normal values. radioautographic histological analysis revealed radiolabeled particles inside phagocytic cells of all organs. we conclude that pulmonary phagocytic activity increases after 1 hr of hs hsl reatment, diminishing by 24 hrs although still above normal values. in contrast, res suppression occurs in liver and spleen after 1 hr hs hsl treatment, returning to normal values by 24 hrs. these results may explain lung complications and immunosuppression after trauma. infusion of endotoxin as well as major surgery is followed by lymphopenia in peripheral blood. the purpose of this study was to investigate to which tissues the lymphocytes are redistributed in response to endotoxaemia and major surgery. in addition changes in lymphocyte subpopulations and expression of mecii was measured. lymphocytes were isolated from peripheral blood of 30 rabbits, labelled with 111indium-tropolene and reinjected intravenously into the rabbits, i0 rabbits received an infusion of escherichia coli endotoxin 2 ~g/kg, while i0 rabbits were subjected to a major sham operation and i0 rabbits served as a control group. the redistribution of lymphocytes were imaged with af gamma camera, and calculated with an interfaces computer before, and 2, 4 and 6 hours after major surgery or infusion of endotoxin or saline. interleukin-l~ and serum cortisol were measured. in addition we followed cd2, cd3, cdlla/b, cdis, cd20, cd44, mhcii and cd4/cd8 ratio. following endotoxaemia interleukin-lf~ increased significantly, following endotoxaemia as well as major surgery serum cortisol increased significantly. following major surgery as well as endotoxaemia there was significant lomphocytepenia in peripheral blood with a decreased cd4/cd8 ratio while the cd3 positive subpopulation increased. in addition there was a decrease in the expression of mhcii on the lymphocytes peripheral blood. the radioactivity of the lymphatic tissue in and around the intestine increased to 129% of initial values following endotoxaemia and to 125% following major surgery. the results indicate that endotoxaemia as well as major surgery induces redistribution of lymphocytes from peripheral blood to lymphatic tissue. among the lymphocytes staying in peripheral blood there was a decreased expression of mhcii and a relative decrease in cd4 cells compared to cd8 positive lymphocytes. in order to analyze the effects of immune suppressive substances on expression of mrna of interleukin-2(il-2) and interleukin-2 reeeptor(il-2r), this study was carried out. twenty male rabbits with comminuted fracture were used in the study. ten ml blood were taken at 0, i, 2, 4, 6 days after injury. the sera were tested for the effects on lymphocyte blastogenesis and induction of il-2 stimulated by concanavalin a(con a): the sera from the rabbits 2 days after injury were analyzed with sds-page gel eleetrophoresis, and divided into three groups by ultrafiltration (ufpi ttk, 30kd,milipore; centricon-10,10kd,amicon), that are less than 10kd, between i0 and 30kd, and more than 30kd. each group of the substances also was tested for the expression of il-2 and il-2r by the dot blot hybridization. the results showed that: i) all sera from the rabbits after injury had significant suppression on lymphocyte proliferation and secretion of il-2 by the con a-stimulated splenocyte in mice; 2) the sera from the rabbits 2 days after injury had more profound suppression than other injured sera; 3) there was a marked band at about 10kd in sera from the rabbits 2 days after injury, but nothing at the same position in normal sera analyzed with electrophoresis; 4) the substance with molecular weight of about iokd had more obvious suppressive action on expression of mrna of il-2 and il-2r than other groups substances, of which molecular weights are more than 10kd. it is concluded that: i) the sera from the injured rabbits can reduce immune response; 2) there is kind of substance, of which molecular weight is about 10kd, it is probable the main factor involved in the pathogenesie of postinjury suppression immune; 3} the substance can depress the expression of mrna of both il-2 and il-2r. research institute of surgery daping, chongqing, 630042 p. r. china acute ethanol uptake prior to injury modulates monocyte tnfo~, production and mononuclear cell apoptosis. g. szabo, b. verma, p. mandrekar, d. catalano monocytes (mo) have been shown to contribute to immunosuppression after both major injury and alcohol consumption. we reported that acute ethanol exposure of m(3 results in decreased antigen presentation, induces tgf-13 and pge2 while inhibiting inflammatory monokine production. we also showed that post-trauma immunosuppression is mediated by hyper-elevated mo tnfc~ and il-6. consequently, here we investigated rnonokine production in trauma patients (n=10) who had elevated (>o.lmg/dl) or had no blood alcohol level (n=t0) at the time of emergency room admission. none of the patients had chronic alcohol use history. met tnfc~ production from trauma patients with prior alcohol uptake was undetectable during days 0-3 post-injury in contrast to patients without alcohol exposure. furthermore, decreased tnf~x levels were found in alcoholic patients' mci after mdp or ifny + mdp induction. however, mcl tnfc~ levels during the 4-8 days post injury period became higher in alcoholic trauma patients. furthermore, over 9 days post-injury, alcoholic trauma patients showed significantly elevated mci tnfo~ production after adherence isolation, mdp, or ifn+mdp stimulation compared to patients without alcohol. these results suggest that acute ethanol uptake prior to injury decreases tnf(x inducibility in the early post-trauma period, but these patients' mo produce hyper-elevated tnfa levels later post-injury, thereby prolonging their cytokine shock risk. tnf ng/ml 0-3 days post-injury 9 days post injury stimulus ale. pt. pt 9.3 11.9 5.6 4.0 immunosuppression might also be increased by the elevated apoptotic activity found in trauma patients' mononuclear ceils, which was even greater in alcoholic trauma patients' cells. in non-alcoholic trauma patients' preactivated mo, in vitro acute ethanol (25-150mm) exposure resulted in a significant down-regulation of tnfc~ (p<0.001) and il-6 (p<0.01) production. in contrast, in vitro ethanol exposure increased the production of inhibitory monokine, tgfi]. these results provide both in vivo and in vitro evidence for the effect of acute ethanol exposure increasing immunosuppression and cytokine shock. the 'systemic inflammatory response syndrome' (sirs) with consecutive septic multi-organ dysfunction represents the major cause of late death following major mechanical and burn trauma. systemic hyperinflammation and concurrent depression of cell mediated immune response (cmi) render the traumatized host anergic, resulting in profound susceptibility to opportunistic infection. monooytes/macrophages (mo) play a central role within the host defense system in developing and manifesting states of injury, shock and sepsis. the mechanistic scrutiny of the synthesis patterns of crucial cccytokines appears to be a helpful tool to further analyse mo behaviour in the compromised individual. the objective of this study was to further dissect the characteristics of cytokine regulation in pbmc under stressful conditions, via analysis of the expression of cd14+ receptor, the proinflammatory mediator il-6, the macrophage activating factor ifn-5,, and neopterin (npt) a metabolite of activated mo. we investigated pbmc's on consecutive days 1, 3, 5, 7 and 10 after mechanical trauma of 9 and after bum trauma of 5 patients (mean age 38~17 years; mean iss 34±2 pts). in trauma patients we saw a massive increase of pha induced neopterin synthesis compared to controls. however, when discriminating the npt levels in the supernatants for the amount of mo stimulated, the npt output of the individual cell was lower compared to mo of nontraumatized individuals. interestingly there was a contrary coarse in the cumulative protein release patterns of il-6 and ifn-7 in mechanical versus burn trauma patients. wheras in burn patients ifn-y was decreased significantly (16+5 u/ml) compared to controls (58+6 u/ml) as well as mechanical trauma (59+12 u/ml). il-6 showed a significant suppression following mechanical trauma (662+58 u/ml) vs control (1266+91 u/ml) and bum patients. the rt~,na signal intensity for beth eytokines was in concurrence with the protein release in more than 85% of the individual patients investigated. from these data we can conclude that the inadequate low npt synthesis predominantly in bum patients appears to be a sign of cellular immaturity and is probably partly due to low t-cell ifno t signals. in addition we could state that the quality of trauma is apparently responsible for the different synthesis patterns of ]l-6 and ifn-q,. it has been postulated that bacterial invasion or endotoxemia are necessary for cytokine production following burn injury. we studied the organ distribution and kinetics pattern of il-fc~ (cell-associated il-1 agonist) in eutrophic rats subjected to either 20% tbsa cutaneous scald injury (bi), muscle scald injury of equivalent 20% tbsa (mbi), sham muscle bum (resection of skin only, up to 20% tbsa) (smbi), and sham cutaneous burn (sbi), followed by saline resuscitation (15 mukg i.p.). separate rats were infused with 15 mg/kg e.coli 0111:b4 lps or saline lv. unmanipulated rats were baseline normal controls. liver, lung, spleen, ileum, thymus, kidney, skin, and plasma were harvested at various time-points within the first 24h. tissues were frozen, weighed, homogenized, the homogenates centrifuged and the supernates assayed with a radioimmunoassay specific for rat il-l(z (detection limit 150 pg/rnl). il-lc~ was expressed as ng/g weight + sem (lowest detectable amount 1.5 ng/gwt). il-lo~ was constitutively present only in the skin (102 + 16.4 ng/gwt). cutaneous burn and sham cutaneous bum induced biphasic elevations of il-lcc in the liver and lung only, with maximal levels at 2.5h (in the liver, bi = 16.5 _+ 6.2 ng/gwt, sbi = 1.7 + 0.1 ng/gwt, p _< 0.05; in the lung, bi = 10.3 + 1.3 ng/gwt, sbi = 1.9 + 0.8 ng/gwt, p -< 0.002). of note, both bi and sbi rats had detectable il-i~ in the liver at timepoint 0 already (2 min real-time). these levels increased in parallel until 30 min and became eventually different by 1 log at 1 -2.5h. all other organs as well as plasma were below detection limits. muscle burn injury and sham muscle burn (skin resection) induced similar elevations of il-10~ in the liver at lh, indistinguishable from each other and from cutaneous burn. in contrast, lps challenge induced dramatic elevation of il-t~ in all organs tested except for the kidney; the spleen was the most responsive organ to lps-induced il-lo~ production. these data indicate that thermal or mechanical injuries induce very early and organ specific production of il-1 c~ in vivo by mechanisms other than endotoxemia. injury-induced complement and platelet activation may be involved as well as the neuro-endocrine axis, which may explain the low levels of il-lo~ induction observed in all rats at the very early time-points. trauma services, massachusetts general hospital, and department of surgery, harvard medical school. fruit, st, boston, ma 02114. j. f. schmand *#, a. ayala* and i. h. chaudry* studies indicate that i.v. infusion of the colloid hes in normal animals does not adversely affect non-specific immunity. it remains unknown, however, if lies affects cell mediated, specific immune functions after trauma and hemorrhage (hem). to study this, non-heparinized c3h/hen mice underwent midline laparotomy to induce trauma and were then bled to and maintained at a bp of 40 mmi-ig for 60 rain. the animals were then resuscitated with either 4 times (x) the shed blood vohune as lactated ringer's solution (lrs) or 2x lrs + lx 6% lies. sham mice were neither hemorrhaged nor resuscitated. at 2 or 24 hours post hem serum, peritoneal (pm~) and splenic macrophages (sm~) were obtained. bioassayes were employed to assess the levels of ii-l, il-6 ( alternatively pmqb showed no differences in il-1 release between all groups at 2 and 24h, while sm~ from the lrs + hen group showed a depression at 24h. tnf production by pm~ was depressed in all groups at 2h and remained so in the lrs + hes group at 24h. sm~b showed decreased tnf release values in both hem groups at 2 and 24h. in summary, the levels of inflammatory cytokines (particularly the values of circulating il-6) after trauma/hem are positively influenced by the administration of hes. this might be due to a protective effect on pmqb and sm~, but also on other cytokine producing cells, e.g. kupffer ceils. we conclude that hes is not only a safe, but also beneficial agent in the resuscitation of patients atler trauma/bemorrhagic shock. this study investigated endotoxemia and consecutlve immune response in 39 patients with multiple trauma (median injury severity score = 20,5). blood samples.were collected shortly after injury and after 0,5, 1, 3, s and l0 days. endotoxin was measured with limulus-amebocyte lysate test and the specific antibody content (sac) against endotoxins of the classes igg, igm and lga by elisa-technique. five antigens were used: lipopolysaccaride (lps) of e.coli (ec), lipid a of e.coli (la), lps of pseudomonas aerog. (pa), lps of vibrin cholerae (vc) and cx-hemolysin of staphylococcus anreus (oth). a nephelometer indicated the total concentrations of igg, igm and iga. differences were checked with wilcoxon-test and p<0,0s was considered significant. cross-reactivity was calculated with rank correlation coefficients. results: endotoxemia peaked shortly after injury (0-3h) at 0,425 eki/ml (median), decreased thereafter to 0,04 eh/ml at day s and remained on this level. sac oflgmclass increased to all endotoxins and peaked at day 3 revealing the lfighest level to la followed by pa (= 80% of la-sac), ec (= 65% of la-sac) and vc (= 40% of la-sac). lga antibodies increased as well but only slightly and not significant (exception: sac to la was elevated significantly at day 5). igg antibodies increased similar to iga class only slightly and again only sac to la was significantly higher at day 5 and 10. however sac to (xh of all ig-classes remained continuously on the same level troughout the observation time. correlation analysis revealed strong cross-reactivity (r>0,5; p<0,01) most often between antibodies of igm-elass (73%) followed by igaclass (40%) and lgg class (2%]. conclusions: multiple trauma is associated with temporary endotoxemia. endotoxins probably translocated from the gut cause specific increase of anti endotoxin antibodies in blood of the igm-class. endotoxins cause no increase of antibodies to gramposilave bacteria. igm antibodies are most unspecific. during cardio-pulmonary bypass, as well as postoperatively, high levels of endotoxin, interleukin-6 (ii-6) and c-reactive protein (crp) were measured in 30 patients. i0 female and 20 male, ageing from 30 to 73 with a median age of 59. blood sampling was done preoperatively, immediately after induction of anaesthesia, after thoracotomy, after cannulation of the aorta and right atrium after the first half of the reperfusion phase, after closure of the thorax, 2 and 4 hours after the operation and then every morning until the 5th postoperative day. blood was drawn into heparinized tubes (i0 iu/ml) which were free of endotoxin. crp levels were determined through the use of the behring nephelometer. 11-6 levels were measured by using commercially-available elisa test. the endotoxin level was determined by a chromogenic modification of the limulus amebocyte test. the statistical analysis was done using the wilcoxon ranks test and correlation analysis. a significant increase {p 0.01) in endotoxin plasma occurred during surgery, culminating in a peak (median value of 0.795 eu/m!) during reperfusicn. plasma levels of endotoxin continued to be slightly raised till the 5th day after surgery, whereas those of interleukin-6 rose at the end of the operation and were at their highest 4 hours later (median value of 217.5 pg/ml). crp levels were also high postoperatively with a median value of 114 mg/l, and were markedly raised on day 2 (191 mg/l). a definite, statistically significant correlation between the plasma levels of endotoxin and 11-6 during the operation was establisthed (p 0.05), leading us to conclude that the endotoxin liberated during cardiac surgery acts as the main trigger in the releasing of 11-6, and thus induces the postoperative acute phase reaction. there was no evidence of a correlation between crp and endotoxin or 11-6 plasma levels. impaired immune function is well described following trauma and hemorrhagic shock (hs). prior studies have utilized peripheral blood or spleen cells to index immune function following hs. however, changes in mucosal immunity are not weii characterized in this setting. gut origin sepsis is thought to be an important cause of organ failure and death following trauma. a rodent model was utilized to allow comparison of mucosal-associated immune function vs, systemic compartments after hs. fischer rates underwent hs (map 35±5mm hg) for 60 minutes followed by resuscitation with shed blood and lr. sham animals were instrumented only. rat tears were collected at 24 and 72 hours following hs for quantitation of slga by ria. animals were sacrificed at 24 hours and spleen (spl), peripheral lymph nodes (pln), and mesenteric lymph nodes (mln) harvested for cell population analysis using flow cytometry and mitogen stimulation analysis. cell marker expression analysis revealed no changes in t or b ceil populations following hs. mitogen mucosal immune function appears relatively spared following hs. the mechanism(s) for this variability in immune function requires further investigation. we have found that transplantation of bone marrow in a hind-limb graft to syngeneic lethally irradiated recipient is followed not only by rapid repopulafion but also overpopulation of bone marrow cavities. the question arises whether this unexpected phenomenon could be the result of stimulation of stem cells by factors (cytokines) released from surgical wound at the site of anastomosis of graft with recipient. aim of the study was to investigate which tissues damaged during the procedure of limb transplantation may be a potential source of humoral factors accelerating in vivo bone marrow proliferation. methods. experiments were carried out on lew rats in 5 groups. in group i, the hind limb was transplanted orthotopically to a syngeneic recipient; in group ii, sham operation was performed; in group iii, a four-cm long cutaneous wound was made on the dorsum; in group iv, limb skin was harvested, fragmented and implanted into peritoneal cavity; in group v, bm from femur and tibia was implanted intraperitoneally. bm, lymphoid tissues and blood were sampled 10 and 30 days later for cell concentration and phenotype evaluation. results. the yield of nucleated cells from tibia was on day 10 in the control 29.3 + 3.2, in group 1 58.1 +2.0, in group ii 47.1 + 1.6, in group iii 50.8+2.0, in group iv 48.5_+2.0, in group v 37.4_+4.9x10(6). the evident increase in bmc yield in all groups continued until day 30. increase in weight and total cell count of spleen and mesenteric lymph nodes in all but group iii was also found. no differences in percentage of maturing erythroid cells, but higher of mature myeloid cells and lower of lymphocytes were observed. conclusions. trauma of skin, muscles, and bone brought about an increase in bone marrow cellularity and acceleration of maturation of myeloid lineage. transplantation of bm ceils alone did not produce this effect. transplantation of bm in limb graft is a good model for studies of natural factors reaulatin~ bm hemormesis. this study sought to determine a relationship, if any, between the degree of hypochclesterolemia upon trauma patients' admission and their subsequent outcome. all blunt and penetrating trauma patients admitted to a level i facility from 1987 through 1991, and who had serum cholesterol assayed during the first 24 hrs were retrospectively studied for development of death or significant organ dysfunction. the mantel-kaenzel chisquared test was used to determine significance of data at the p< 0.05 level. results: 2909 trauma patients were admitted during the four-year period who had serum cholesterol assays performed in the first 24 hrs. 24 patients had cholesterol levels less than 50 mg/dl; 8 of these (33.3%) died, 3 (12.5%) developed ards, 5 (20.8%) developed acute renal failure, and 8 (33.3%) developed multisystem organ dysfunction; hypocholesterolemia in these patients was not due to liver injury or massive fluid administration. the risk of death was 5 times greater and risk of multi-organ failure 3 times greater in this group than in those with a normal serum cholesterol (>if0 mg/dl; 1820 patients; p<0.05). conclusions: admission serum cholesterol level in the trauma patient serves as a powerful marker for those at risk of subsequent organ failure or death. hypocholesterolemia in this setting may result from organ hypoperfusion and humeral mediator release. lung tissue contains many immunocompetent cells. resection, therefore, is expected to activate extensively inflammatory mediators such as pmn-elastase, pmstanoids and pteridines. in a prospective clinical study we compared 35 patients (pts) undergoing either thomcotomy with or without lung tissue msectioh and tboracoscopic lung resection concerning activation of inflammatory response. material & methods: group a pts (n=8) had thoraantomy but no lung tissue injury; group b pts (n=ls) had thoracotomy and lung tissue resection due to benign diseases; group c (n=9) represents group b tissue resection but using a thomcoscopic procedure. the following parameters were determined pre-, peri-, and postoperatively: elastase and crp as indicators of activation of pmn-leukocytes and injury severity; prostacyclin (pgi 2) and thromboxane (txa~) as parameters of lung endothelial response; prostaglandin f2~ (pgf~) and pgm representing pulmonaly metabolic activity; pge a and neopterin as proof of macmphage activation. statistics were performed using analysis of variance for repeated measures. results: group b pts revealed postoperatively an increase in crp (p<0.001) indicating a higher injury severity in comparison to the thoracoscopic procedure (c). both, controls (a) and group c pts did not show pmn-activation, whereas group b demonstrated a reversible increase in elastase. surgical trauma caused in all groups a release of pgi z and txa 2 which was more pronounced in c (p<0.05) and most in b (p<0.01). similar results were found for pge~ and pgf2=. there was no activation of maerophages since neopterin did not increase. apparently, metabolic lung function was not impaired because there was no marked rise in pgm except in b (p<0.05 vs. c). discussion: our results demonstrate that lung tissue injury aggravates the mediator release induced by thoracic traum. these mediators among others are able to increase capillary pressure and hence lung edema formation. impairment of lung function, however, seems dependent on the extent of the liberation. therefore, the maximal release reactions occured in group b and c after lung tissue resection, whereas the controls showed the highest levels immediately after the incision. we conclude that thoracoscopic procedures are superior in reducing the resection trauma per se and hence might prevent severe mediamr-induced (pulmonary/systemic) sequelae. in a prospective study we investigated 45 patients using radiochemical method according to sch~dlich (s) and photometric method according to hoffmann (h). serum of severly traumatized patients was withdrawn directly after admission at our emergency room and in narrow time intervals during first 24 hours after trauma. follow up control samples were taken daily until day ten. whereas no elevated pla-ca was found during first 24 hours, a peak was regularly observed around day four. there was high correlation between pla-ca and iss (r=0.71, p<o.o001) except patients suffering from thoracic trauma, thoracic trauma {tt) provoked similar gons-score-and pla-ca-values compared to multiple trauma (mt) despite significantly lower iss: pla-ca (h) mt 32.9 + 6.4 tt 28,7 +_ 9,5 n.s. pla-ca(s) mt 11.7 +_ 2.6 tt8.3 _+ 2.6 n.s. goris mt 3.5 4-0.7 tt3.6 _+ 1.3 n.s. iss mt 35 4-3 tt21 4-3 p<0.01 we found delayed elevation of pla-ca after severe trauma, however detection of delayed pla-ca in the serum of traumatized patients cannot give reliable information about the exact role of pla in the inflammatory reaction after trauma as latest results show that pla is secreted early but bound at lipophilic sites of vascular membranes. hern~mdez m j, amiguet ja, monreal ji, vid~n jr, jim6nez f j, l6pez-vivanco g acute phase reactant proteins increment in serum of patients with traumatisms and inflammation has been previously reported. alpha-1 antitrypsin (aat), alpha-2 macroglobulin (amg) and ceruloplasmin (cp) are evaluated in the following of 33 patients, 27 males and 6 females (mean age: 43 years), with compound fractures. 57 % of them were located in tibia and fibula. intercurrent infection developed in 4 patients and internal fixation rejection in one. measurements were made by radial immunodiffusion on days 0, 3, 10, 17, 24, 31 and 38. a control group of 30 healthy volunteers was also evaluated. aat and cp showed increased values from day 3 which kept elevated untill the end of the study. amg elevated from day 24. when infection developed, aat and cp values were even higher whereas amg values decreased. fixation rejection caused aat, amg and cp increment. aat and cp increment in non complicated fractures might be due to accompanying inflamation, by means of cytokines release and aprps hepatic synthesis induction. therefore, increment is higher when infection or rejection develop. amg behaviour at early stages of evolution is secondary to hiperconsumption, considering mean life shortening after proteases ligation. amg modifications in fixation rejection remain obscure. aprps increment modulates inflammation and bony callus formation by means their antyprotease and antioxidant properties. clinically, aprps might be useful parameters in determining complications onset in fractures evolution. hemorrhagic shock (hs) is a common complication of trauma, as well as some major surgical procedures. the alteration of the immune system by hs is thought to contribute to the increased septic morbidity and mortality seen in these patients. multiple mediators are released following hs. il-1, il-6, tnf, pge 2 and tgf-b have all been implicated in these immune system changes. in vitro, pge 2 causes many of the immune system changes following hs, yet the time course of pge 2 release has not been well deliniated. the aim of this study was to determine the time course of pge 2 release by peritoneal and splenic macrophages following hs. c3h/hen mice were bled to a mean bp of 35+5 mm hg for 60 minutes. the animals were resuscitated with the shed blood and normal saline. control animals were cannalated, but blood was not withdrawn. the animals were sacrificed at 2, 12, 24, 48, and 72 hours following hs. peritoneal and splenic macrophages were harvested from each animal and cultured with lps for 24 hours. the supernatants were collected and frozen until assayed. pge 2 was assayed using an elisa (cayman chemical). results are shown below for the peritoneal macrophages: pge 2 release by peritoneal macrophages was significantly elevated at 24, 48 and 72 hours over control. it was maximal at 48 hours, but by 72 hours it had started to decline. splenic macrophage pge2 release was much more variable. (data not shown.) there was less consistency between time points and no clear trend was seen. in conclusion, pge 2 is significantly elevated at 24 hours following hs, and reaches a peak at 48 hours. pge 2 release may be responsible for the immune system changes seen in the first few days following hs. splenic and peritoneal macrophages respond differently in their release of pge 2 following hs; which may be due to differences in their milieu. there is an obvious association between the altered immunocompetence of patients following traumatic injury of various types and the increased riskto develop complications. the present investigation was undertaken in order to analyze the value of both neopterin (n) and c-reactive protein (crp) in monitoring disease course in patients with multiple trauma. we were interested to compare nconcentrations as marker of t-cell/macrophage activation with crp levels as indicator of the inflammatory acute-phase response daily serum concentrations(n and crp) were measured in 12 patients (9 male, 3 female, mean age 31.42), admitted to our icu following serious trauma (mean iss=34) and in 10 control subjects. the clinical course of each patient was evaluated with apacheii score according to knaus. mean stay in icu was 9.25 (range 5-13). 5 patients didn't develop organ failure(nof-group), 4 patients survived, developing mof (s,mof-group), 3 patients died with mof (ns, mofgroup). three patients(one s and two ns) showed signs of septicemia and septic shock. the serum samples were measured for neopterin (immu-test-ria, henning-berlin) and for crp (immunoturbidimetric method) -at the university hospital for internal medicine, innsbruck, austria. the highest crp levels were measured 48h after trauma, but they were not accompanied by significant increase in n-values. we found significantly elevated n-concentrations from the days 5-6. n and crp levels showed a parallel release peaks on the days 6-7 and the values discriminated significantly among the three outcome groups(the second crp peak is lower in comparison to the first posttrauma peak). by the ns-group we found constant and parallel increasing crp and n levels, reaching extremly high values 24-48h and preceding clinical signs of mof and sepsis. the values increased dramatically before death. the serum levels by all 10 control subjects were upper the normal limit for n and crp.. the parallel occured peak changes in n and crp concentrations showed a significant correlation with clinical status, using a disease activity score (apac h eli). our findings suggest that both-cellular and humoral mediated immune mechanisms are activated after multiple trauma and that simultaneous determination of n-crp may be of advantage as diagnostic and prognostic parameter in polytrauma-patient monitoring panel. it is apparent, that measurement of very high levels may be of value as a srong indicator in developing of mof or septic complications and thus offer the possibility for earlier therapeutic intervention. the modification of t-cell/macrophage and acute-phase responses may be potentially useful in the treatment of icu patients, resuscitated after severe trauma, and suggest that n-crp may be relevant indicator for testing experimental immunomodulating therapies. although several in vitro studies suggested the catalytic action of iron in oxygen free radical generation, few data are available concerning iron-metabolism following ischemia-reperfusion injury and hemorrhage in vivo. aim of the present studies was to evaluate iron metabolism following mild and severe hemorrhage in the presence and absence of intraperituneal hematoma. methods. male lewis rats (300 g) divided in 4 groups (observation time 120 hrs): a: mild hemorrhage by sequential blood sampling (0.9 ml each)(0, 2, 4, 6, 24, 48, 72, 96, 120 hrs). b: a with hemoperitoneum (hp)(2 ml/100g), c: hemorrhagic shock: blood withdrawal of 3 ml/100g over 30 min and resuscitation with ringer's lactate (2 x shed blood) and isogenic donor blood; d: c with hp. parameters: serum iron ([g/dl], ferrozin-method); 59fe labeling of erythrocytas (application of 1 ci 59fe i.a. into a donor rat, 5 days later bleeding of the rat and injection intraperitoneally in experimental rat; plasma transferrin ([g/ml], ria). t-tests and mann whitney. data are expressed as mean + sem. results. compared to baseline, mild and severe hemorrhage caused a significant increase in serum iron at 24 and 48 hrs, respectively (212+30 vs. 248+35; 185+_28 vs. 255+_22). hp reduced this increase (209+_17 vs. 177+21; 194+25 vs. 197+-48, p<.05). i-ip significantly increased hemoglobin between 24 to 96 hrs in mild (12.1+_.8 vs. 14.2+.7) and severe hemorrhage (10.3+.6 vs. 13.1+.6). in mild and severe hemorrhage comparable maximal resorption (59fe labeled erythrocytes in circulating blood) of intraperitoneal hematoma occured at 24 hrs (mild: 318+_71 vs. severe: 364+-114 counts/rain). hp increased survival-rate following hemorrhagic shock (no hp: 10/17; hp: 10/13). in mild hemorrhage no alterations of plasma transferrin concentrations. in shock group without hp, there was at 96 hrs a significant higher transferrin level than with hp alone (3.2+_.5 vs. 2.23 + .2). in conclusion, hemoperitoneum with hemorrhage prevents increase in plasma iron levels by increasing hemoglobin through resorption of erythrocytes and thereby suppressing iron mobilization from endogenous iron resources, e.g. liver. this suggests that intraperitoneal hematoma not necessarily promotes iron-mediated oxygen free radical production, especially since survival in these groups was improved. increased transfarrin levels in group c at the end of the experiment suggest increased iron turnover which was not observed in shock groups associated with hemoperitoneum. primary immune response to thymus-dependent (srbc) and thymus-independent (vi-polysaccharide) antigens was determined during 30 days after cct. antigenic challenge was made on day i, 5, 15 or 30. number of spleen plaque forming cells (pfc) and serum haemagglutinin (ha) titre were studied on day 5 after immunization. cct stimulated immune response to t-dependent, but not to t-independent antigen. enhanced response was noted when srbs were given in posttraumatic day i, it was higher than preceding one after injection on day 5 and reached a maximum when antigen was presented on day 15 after trauma (pfc number 2.4 times exceeded the level of immunized, but nontraumatized rats). the rise of pfc number in spleen correlated with increased ha level. thymectomy had been performed 30 days before cct completely abolished posttraumatio stimulation of immune response to t-dependent antigen. standard thoracotomy also enhanced humoral response when srbc were administered on day 15 after the operation. thymus trauma modeled by pincett squeezing of its right lobe and performed at the same time with thoracotomy fully abrogated as well as thymectomy stimulation of immune reaction. thus, different experimental chest traumas are able to increase antibody production via thymus-dependent mechanisms. endothelin is a 21 amino acid peptide produced by ischemic or injured endothelial cells. in vitro and in animal studies, et is also a potent constrictor for renal mesangial and coronary vessels, a negative cardiac inotrope and an endocrine regulator. our lab has shown that et is an agonist for monocyte production of interleukins i, 6 & 8, prostaglandin e 2 (pge2) , and substances which trigger superoxide production. systemic et levels increase in hypoperfused and septic patients, and et may be yet another important cytokine in critical illness. but while et has been shown to be a potent vasoconstrictor in healthy dogs, systemic vascular resistance does not increase in critically ill patients with high et levels. (we hypothesize that this might be due to pgez-mediated vasodilation predominating over et-mediated vasoconstriction.) we next asked what happened to systemic et levels in patients with major burns (>20%.) ten hemodynamically stable patients resuscitated by a modified parkland formula to a urine output >30 cc's per hour had et levels drawn on admission, at i, 6 12, 24, and 48 hrs. et levels were measured by radioimmunoassay. mean levels were elevated at 205±28 pg/ml at all time points versus levels in healthy controls of 39±9. in summary, systemic et levels increase significantly in patients with major burns. et may be yet another cytokine playing a significant role in the immune, inflammatory and multiorgan dysfunction observed with major burns. restoration processes in an organism after ischemic damage are realized through ~n~lammatory mechanisms~ the intensity of which is significantly defined by blood levels of neuropeptides. myocardial infarction (mi) was chosen for studyin 9 these processes since it eradicates the influence of infectious factc~rs. 32 dogs~ in whom mi underwent different forms o¢ healer, g; bhn~ed ~h~t during the acute phase of the disease there was a characteristic rise of ne!~ropeptides in the blood. these neuropeptides had nociceptive and antinociceptive effects. particularly substance p and -endorphins triggered off the development of compensatory and adaptive mechanisms and defined the intensity of inflammatory reaction at the zone of ischem~t: damage-notable fall in substance p levels after an ~nitial increase, while the ~-endorphins stayed high was an important condition for non complicated healing of mi. on the other hand high levels of substance p with low ~-endorphin concentrations lead to increased infiltration o~ neutrophils into the infarction zone and weakened the activity of synthetic processes~ thereby leading to left ventricular aneurysm. at the same time low intitial levels of substance p slowed down the development of necrotic processes which lead to delay in refunctioning of the heart and complicated the healing process. thus, regulation of the levels of neuropeptides in the blood in trauma forms a perspective method of its treatment. of laparascopic versus open choleocystectomy c. schinkel, s. zimmer, v. lange, d. fuchs, e. faist the impairment of immune function due to surgical trauma may be followed by deleterious septic sequelae. compared to open abdominal surgical procedures (lap), laparaseopic surgery (lsc) is associated with a decrease in hospital stay and in accelerated patient recover. the aim of the study was to evaluate the sensitivity of the immune sermn parameters of il-6, saa and neopterin, the percentage of cd14+ cells, the in-vitro il-2 synthesis after mitogen stimulation and lymphocyte proliferation, in order to purposefully discriminate differences in the severity of trauma. we investigated the blood of 27 patients with cholecystolithiasis undergoing either laparascopic (16) or open (i 1) cholecystectomy on consecutive perioperative days -1, 1, 2 and 3. there was no significant difference between the two groups concerning age and sex. patients with clinical signs of acute cholecystitis were excluded from the study. operation time and hospital stay were obviously longer in lap patients (67 versus 145 minutes, 5 versus 20 days) compared to the lsc group. concerning the unspecific acute phase reaction we could show no difference in the increment of senun amyoid a (saa) synthesis in the lsc group (d-i 40 + 1 lng/ml, d1 362 + 43ng/ml) versus lap group (d-1 61 + 19ng/ml, d1 457 + 45ng/ml), while in serum il-6 levels we saw a less steep increment in the lsc group (7-fold from d-1 to d 1) compared to the lap group (10-fold from d-1 to d 1). the analysis of cd14+ receptor expression and serum neopterin did not reveal any difference between the groups. lymphocyte function showed an impairment of proliferation to antigen stimulation in lap (d -1:24.000 + 3.330 cpm, d 1:17.300 + 3.000 cpm) compared to the lsc group (d -h 22.500 + 4.200 cpm, d h 26.000 + 2.900 cpm). in both groups il-2 synthesis was decreased post-operatively. our data indicate that laparascopic cholecystectomy reusults in a less distinct unspecific acute phase reaction post-trauma compared to that following lap. neopterin serum levels and cd14 receptor expression show that these parameters apparently are less useful markers to detect differences of surgical trauma severity while it appears that the impact of lap is reflected most impressively on the lymphocyte compartment. trauma alters the host resistance of organism and is accompained by appearence of excgenic and endogenic proteins in the body. to understand the molecular mechanisms of host resistans disorders in trauma, as a first step, the genetic regulatory mechanisms of immune response after antigen injection has been studed. the appearence of specific protein factors (13-19 and 25 kda), in the nucleus of rat splenic and brain cells, accordingly, was shown after immunization with sheep erythrocytes. the stimulatory effect of these factors on the il-2 mrna and il-2 production was detected. the nucleotide sequences of the human il-2 gene regulatory region bounding by the splenic nuclear proteins were determined between +39-141 b.p. the il-2 trans-factors shows the affinity to splenic and thymic lymphocytes in vitro. thus, the antigen causes the appearence of specific protein factors in the cells,which act on the gene level,stimulate il-2 production and the host resistance. these results cause the next step of experiments using the same model, but after trauma. these investigations will let us verify the hypothesis that the protein il-2 gene trans-factors may play a definite role in the decrease of the cell immune responce after trauma. confronted with the routine procedure of prophylactic treatment of candidates for surgery in a rural african hospital, we initiated studies on the fre'quency of post-surgical malaria. in tanzania 195 non-pregnant patients from rural areas were followed. of preoperative patients 10% had a parasitaemia and those maintaining it showed no increase or complaints. nine percent of patients without detectable parasitaemia before surgery came down afterwards and one-third had malaria-like complaints. spinal and general anaesthesia were equally applied in these last patients. in burkina faso we studied 120 patients of which 16% had a parasitaemia on admission and 6% had postoperative malaria. half of the surgical patients came from rural areas, whilst only 14% of those with malaria lived in the city (with much less exposure and immunity). 57% underwent major surgery and 43% minor. bloodtransfusions (4% with parasites) never evoked a parasitaemia in recipients. post-surgical malaria is thus a reality in about 10% of the adult cases, both in east and west africa. surgery evokes a cascade of factors, varying from cortison to interleukines and acute phase proteins; immune responses may temporarily be suppressed. clinical attacks of malaria in otherwise immunes could be evoked by one of these factors. though malaria can easily be cured, the differential diagnosis is difficult because of post-surgery fevers; we found that 16% was treated without justified indication. the involvement of "student-doctors" a. this study examines glucose uptake and hexose monophosphate (i~ip) shunt activity in normal human peripheral lymphocytes and polymorphonuclear leukocytes (pmn). glucose uptake was determined by measurir,g the uptake of tritiated deoxyglucose, a non-metabolized glucose analogue. adsorption of co2 derived from [i-14c] glucose was used to determine knp shunt activity. in vitro assays were carried out in hormone concentrations approximating normal and elevated trauma blood levels. (normal -cortisol 0.12 ~g/ml, glucagon 80 #g/m1, epinephrine 20 ~g/ml, insulin 18 t~u/ml; traumaeortisol 0.40 ~g/ml, glucagon 500 /*g/ml, epinephrine 420~g/ml, insulin 36 ~ij/ml. analysis of twenty subjects showed a reduction of 207° ~mp shunt activity by lymphoeytes and a ]3% reduction in glucose uptake by p~n in normal vs. trauma hontc,nes p < 0.05. lymphocyte glucose uptake was also reduced by trauma hormones p~0.05. it ha~ be.ea~ suggested thgt idiopatno pulmonary fibrous (y.pf) [s a consequence of severe alveolar epithelial injury and is associated with an nveolar irnammamry reactio~ and the presence 0f.neutr0phils. there~bre, neutr0pk~ chemoattra~ant~ are probably important in the genegs oft.he infial lesions of ipf. the obse,"wson that stimulated macrophages are or~n histologically promin~t in fibmfio [-~gs ~.nd am capable of p~oducmg a v~dery 0f flbrogenic pep'ides also a~gues for their role ~n the pathogenic prc~e~ oflpf. the observation that stimume~ maerophages ere often histologica[iy prominent in fibrotio lungs and ~re ~pable of producing a varie~, offibroge.~e peptide~ also argues for tkek role in the pathogenic process, therefore, we ha-~e tested the potentn for iater!eukln-8 (i1..-8) and mo~tocyte chemotacde pop, de 1 (x¢cp-1) to induce neutro~hil ~d mononuclear phagocyte accumuhdon in lungs of pafient~ with pulmonary .~r~idosis and i~f. brenet~o.alveolar lavabo (bal) fluids from 12 ipf and 15 sar~qidosis patient were conexntratea by reversed-phase chromatography, ~d ii.4 arid mcp-i asso.~ed by ells& ehemotaxis mad enzyme-reieasing ~ssas's on msnocyte~ and neatrophiis. elisa revealed significenfly elevated b al-eoneentrations o£mcp-i (20.1 ng]mg aibumm) in purisms with p~monary sarcoidodis artd in ipf (41.8 ng!mg) in comparises to 1.1 normal individuals (4.24 ng/mg) and to 15 patients w~th obreic bronentis (cb) (~, 16 rig/rag). similarly, chemota*dc ac~a~' for monocles (mcp-1 e.qu/va]ent) was strongly increased in sareoidosis (86.03 ngjmg) as well as ~n 12f pag,nts (54.47 ng/mg). norra.al indlvidu~s and cb patiants hzd a 7. or 3-fold lower ~cn%i~y, re~peefively. patients with ipf and sarcoidosi~ also h~l eievated il-8 ievei~ (15.5 and 26.0 rig/rag, respe~veiy; nomzls: 2.14 rig/rag; cb: 4.23 ng/mg) mad nvatropmi ohemotax~ (60.25 ~'~d 49.68 nnmg, res!z~ztiveiy; aormals: 0.25 ng,'mg; cb: 1l06 ngmg). these data suggest that increased ievels of born mcp.1 ~d il-8 may be oharacted~tie for ~arcoidosis or ipf_ it appears iikely that both ehernoattraetants ~ontribute to the influx ofmonocytes and neutrophils into the pulmonary alveoius and interstit~um in these dlsea~es. we have recently shown that the combined administration of noninjurious doses of lps and paf in the rat produce ards-like lung injury characterized by neutrophil adhesion to lung capillary venules, neutrophil accumulation in lung parenchyma, pulmonary edema, and increased protein and neutrophil count in bal fluid. this new paradigm of lung injury was associated with elevated serum tnfc~ and pretreatment with anti tnfa mab dose-dependently prevented these responses. also, the combined administration of lps and paf induced lung mrna levels of tnfe~ (5 fold vs. lps or paf alone), ll-lg (80 fold), kc (60 fold) and il-6. taken together, these data suggest that this new paradigm of lung injury is cytokinemediated and that lps/paf in vivo can functionally couple to the activation of gone expression of a multi-cytokine network system, all of which may be involved in the pathogenesis of ards. materials and methods. the sheep model included hemorrhagic shock and closed femoral nailing at day 0, 12hourly injections of e. coli endotoxin and zymosan-activated autologous plasma at clays 1-5 and further observation and measurements at days 6-10. from venous blood and bronchoalveolar lavage(bal)fluid of ten merino sheep (mean weight 30 kg) neutrophil counts (10e6 pmn/ml blood or epithelial lining fluid-elf-), the elf/ plasma ratio of albumin (r), and the zymosan-induced (stim) and non-induced (spont) chemiluminescence response (cl) of blood (10e6 cpm/250,000 pmn), and of blood-and bal-isolated pmn (10e6 cpm/25,000 pmn) were measured. for statistical calculations the wilcoxon test was used. data of the changes in polymorphonucleur leukocyte (pivinl) metabolism have been suggested to play a pivotal part in the post-traumatic systemic inflammatory response syndrome. the underlying cellular mechanisms which control this response are not yet completely understood. since the 'ca 2+ second messenger'-system has been shown to be involved in regulation of pmnl-'respiratory burst', we investigated changes in pmnl-ca z÷ regulation in relation to oxygen free radical mediated injury. methods. in 12 polytranmatized patients (mean injury severity score = 32) arterial and venous blood samples during 15 days. daily evaluation of horowitz-quotiant (po2/fio2), plasma lactate (mg/dl) and body temperature ( results. body temperature peaked at day 6 and 7 (day 0: 36+.4; day 7: 39.8+.4). plasma lactate was significantly increased at day l (36+2) and day 7 (16.2+2). hurowitz-quotient (day 0: 344+43) was low at day 4 (199+19) and day 7 to 10 (178+25)(p<.05). at day 7 a substantial rise in venous pmnl-superoxide production (day 5: 1.9+_.45, day 7: 3.3+.7, day 9: 1.72+_.8), oecured with significant increase in plasma lipid peroxidation (day 1:0.6+0.2 ; day 7: 1.3+0.2). pivin~-myeloperoxidase activity was high at day 2 (2.3+--.5) and then continuously declined (day 7: 1.3+.3). plasma antiexidant activity (glutathione pemxidase) was reduced by 34% at day 7 (day 1: 5.3+.3; day 4: 4.4+_.2; day 7: 3.5+.2). whereas basal ca 2+ concentration remained unchanged (day 4: 68+_17, day 7: 68+_15), fmlp-stimulated cytosolic ca 2+ mobilization increased at day 7 (day 4: 457+89, day 7: 11084410, day 9: 670+262). conclusion. the present study in polytraumatized patients shows, that seven days after injury the agonist-induced pmnl ca 2+ mobilization is significantly enhanced. at the same time, pmnl-oxygen free radical release and phagocytotic activity, systemic fever response and lactate concentrations were maximal. these observations were accompanied by post-tranmatic respiratory failure and in some patients by clinical signs of multiple organ failure. preliminary data from an ongoing study using hes-and dextran-infusions in these patients show attenuation of this inflammatory response. stefan rose, m.d., trauma surgery, univ. of saarland, 66421 homburg/saar 1donnelly sc, 1haslett c, 1dransfield i, 2robertson ce, 3grant is, 4carter c, 4ross ja, 5tedder tf. 1dept's of respiratory medicine, 2accident & emergency, 3intensive care, 4surgery, university of edinburgh, scotland and 5dept. tumor immunology, dana farber cancer institute, boston. the selectins are a family of adhesion molecules (l-selectin, e-selectin, pselectin), all of whom are implicated in inflammatory cell transendothelial migration. they, as a family can be proteolytieally cleaved from their parent cell and exist in a soluble form within the circulation. ards is a disease state in whic neutrophils and neutrophil transendotheliat migration have been implicated. in this study we wished to investigate whether the levels of these circulating soluble receptors from patients at-risk of ards at initial hospital presentation, correlated with subsequent ards progression. eighty-two patients were enrolled (pancreatitis (n=19), perforated bowel (n=12), and multiple trauma (n=51)), of whom 14 progressed to ards. assays for soluble l,p & e-selectin were performed on collected plasma samples via a sandwich elisa. (ns = not significant, **** = p<o.0001) we have found a highly significant inverse correlation between a low circulating soluble l-selectin (and not soluble e & p-selectin) and subsequent ards. these results further our understanding of neutrophilendothelial interactions in the early stages of ards pathogenesis and offer the promise of sl-selectin in combination with other markers of inflammatory events being used to identify individuals at particularly high risk of ards progression on initial hospital presentation. it has been suggested that polymorphonuclear leukocytes aggregate in the lung capillaries of patients developing the adult respiratory distress syndrome (ards) and account for the endothelial damage while releasing toxic metabo-iites such as o.a. free oxygen radicals. among many antioxidants which have been investigated in in vitro systems and in animal models, n-acetylcysteine (nac) has been established to be useful as a free radical scavenger in vivo. to assess the influence of n-acetylcysteine (nac) on the activation of neutrophils in patients undergoing cardiopulmonary bypass (cpb) surgery with extracorporeal circulation (ecc), we evaluated the plasma levels of elastase and myeloperoxidase (mpo) throughout the operation and up to 24 hours after surgery. four hours after surgery also a bronchoalveolar lavage was performed. a spectrophotometric method was used for the detection of plasma elastase activity and mpo levels were measured using a radioimmunoassay. we found that pretreatment with intravenous nac prevented the increase in elastase activity (632 _+ 231 pmol/i vs 1666 _+ 187; p -0.027), but not the release of neutrophil related mediators (2.20 _+ 0.56 ng/i vs 1.80 _+ 0.17; p=0.63). besides, nac had the capability to prevent the enhancement of neutrophil numbers in lavage fluid as is normally seen during cpb (1.4 + 0.8 % vs 25.0 _+ 14.3; p =0.04). although not significant, nac prevented also the increase in elastase levels in lavage fluid (19.0 + 18.5 pmol/i vs 172.7 + 101.4; p = 0.12). it is concluded that nac is able to protect against the inactivation of a 1-proteinase inhibitor, the main inhibitor of elastase activity, and that nac interferes with adhesion and migration of neutrophils. therefore nac may be useful in the prevention of tissue damage. this study is supported by inpharzam belgium. sodium nitroprnsside (snp) has previously been shown to attenuate the neutrophil induced lung injury associated with limb ischaemia and repeffusion. glyceryl trinitrate (gtn), already widely used in aortic surgery, also increases nitric oxide but has a less marked splanchnic "steal" effect. we examined the effect of gtn on lung injury, neutrophil infiltration and activation in a model of aortic occlusion (30 rain) and repeffusion (120 min). sprague dawley rats were randomized into: control (n=i 1); ischaemia repeffusion (ir) (n=12); and ir treated with gtn (2ug/kg/min) during repeffusion (n=10). myeloperoxidase activity (mpo) measured pulmonary neutrophil influx. pulmonary endothelial permeability was measured by wet to dry weight ratio (w/d), broncboalveolar lavage protein (bal) and neutrophil counts (bal pmn). neutrophil superoxide release (mean channel fluorescence mcf) was measured by flow cytometry in a further ir v gtn experiment (n=6 per group). control 573_+t02"* 992_+125 579_+54** bal pmn/mm 3 36l+-126 3"219+1087# 820_~221 *p<0.05, #p<0.02 v control/gtn;**p<0.01 v ir. students t test the significant increase in mpo activity produced by ir was attenuated by gtn. the increase in pulmonary microvascular leakage after reperfusion was also prevented by gtn. neutrophil superoxide release increased significantly from 9.4+0.76 to 13.9_+1.92 mcf after 40 minutes repeffusion (p<.01). this increase in superoxide was prevented in the gtn treated group. these results indicate that gtn inhibits neutropbil superoxide release during limb reperfusion, thus preventing pulmonary vascular leakage and neutrophil infiltration. these data suggest that the beneficial effects of gtn in aortic surgery may be due in part to a protective effect onpulmonary microvasculature. department of surgery, beaumont hospital, dublin 9, ireland. lipton adult respiratory distress syndrome (ards) is marked by increased vascular permeability of the lung to white blood ceils (wbc). indeed, influx of wbc into the lung is believed to be the central mechanism in acute lung injury of ards. evidence is developing that the neuropeptide c~-melanocyte stimulating hormone (c~-msh), a proopiomelanocortin derivative, inhibits inflammatory reactions in animai models of inflammatory responses in humans. to test the influence of a-msh on experimental ards, the peptide was administered to rats after intratraeheal infusion of endotoxin (s. typhosa, 500 #g in 0.25 ml saline). three groups (n=10 each) received: intratracheai infusion of endotoxin plus ip injections of o~-msh (100 ~tg in 0.2 ml saline) at 0, 2, and 4 hr post-endotoxin treatment; intratracheal endotoxin infusion plus saline injections; control intratracheal saline infusion plus saline injections. the bal data showed overall differences among groups (f2,29=6.2, p<.003), o~-msh treatment inhibited endotoxin-induced wbc migration into the pulmonary tree (p<.05). circulating wbc counts were markedly lower in both groups given endoroxin than in the control group (p<.01), but there was no difference in wbc count between these two endotoxin-treated groups. the results indicate that c~-msh can reduce wbc migration in experimental lung injury. in further experiments we tested the hypothesis that c~-msh, administered alone or in combination with an inhibitor of gram-negative bacterial growth, increases survival in a model of peritonitis/endotoxemia/septie shock. cecal ligation and puncture were performed under sodium pemobarbital anesthesia (75 mg/kg, i.p.) in female balb/c mice (16-20g) that were then randomly assigned (15-17 mice per group) to receive at time 0 and 3 hr later: control saline thjectioas (i.p.), injections of ~-msh (100 ~tg), gentamicin sulfate (i0 mg/kg), or both ~-msh and gentamicin. one ml of normal saline was given s.c. to each animal for fluid replacement. both ix-msh and gentamicin treatments administered singly improved survival; when given in combination survival was even greater these findings suggest that the anticytokine ot -msh molecule might be useful for treatment of systemic inflammation, perhaps particularly when used in combination with agents that inhibit bacterial growth. previous studies showed a close relationship between xanthine oxidase activation, membrane damage and postischemic cardio-respiratory failure following aortic clamping and reperfusion. aim of the present study was to evaluate alterations of polymorphonuclear leukocyte (pmnl)-metabolism and signal lransduction systems during ischemia/reperfusion induced by aortic clamping in man. methods, in 14 patients (3 female, 59+4 years) with elective reconstruction of infrarenal aortic anearysms, arterial (a) and venous (v) blood samples were obtained before clamping and declamping, and 60 min after declamping. arterial pla 2 concentrations were higher than venous (113+32 vs. 71+30). while cytosolic basal pmnl ca 2+ concenlxation was not altered at the end of ischemia (v: 52.3+8; a: 42_+6) and during reperfusion (v: 60+8; a: 69+12), fmlp-stimulated cytosolic ca 2+ mobilization showed a significant arterial increase as compared to venous (1652_+601 vs. 638+85, p < .05). these presented data indicate that the reperfusion syndrome after infrarenal aortic clamping is characterized by 1) pulmonary membrane damage possibly due to pmnl-degranulation, 2) activation of pmnl-superoxide radical production with release of activated pmnl in arterial circulation, and 3) a significant arterial increase of pmnl cytosolic ca 2+ mobilization after fmlp-stimulation parallel to pmnl-activation. in conclusion, ischemia/reperfusion in man induces pulmonary damage and activation of pmnl superoxide production possibly due to an altered pmnl-ca 2+ messenger system by inflammatory mediators (e,g. ii-8, tnf, paf). the septic lung diseases (i.e. ards) generally show distinct alveolar damage and surfactant disturbances. it is thought that alveolar macrophages are involved in specific release products like h202 and cytokines like tnf. in this pathway the type ii alveolar epithelial cell (p2 cell) is not only an important target, but as recently shown it is also capable of producing h202. the aim of this study was to investigate the influence of endotoxin on h202 release of p2 cells. we obtained p2 cells from lungs of female wistar rats and investigated the effect of lipopolysaccharid (lps) on h202 release of fresh isolated cells and on cells cultured for 2 days in a fibronectin matrix. furthermore we conditioned isolated alveolar macrophages for 16 hours with 1 p.g/ml lps and tested the conditioned medium (mcm) on cultured p2 cells. p2 cells 5 h ex vivo were _> 70 % pure, _> 95 % vital and had an basal h202 release of 64.5 _+ 1.8 nmol h202 per hour and million cells. adding 1 p.g/ml lps to the incubation medium the h202 release decreases slightly but significantly to 54.2 _+ 2.3 nmol. adding 0.3 p.g/ml phorbol myristate acetate (pma) to the basal incubation medium the h202 release increased 3-fold to 148.3_+ 26 nmol. pma induced h202 release decreased to 128.3 + 41.5 nmol after addition of 1 p.g/ml lps. after 2 culture days the p2 cells were _> 98 % pure and showed a pma inducible h202 release of 174.8 _+ 9.9 nmol addition of 1 p.g/ml lps had the inverse effect as on freshly isolated cells as it increased the h202 release up to 216.2 _+ 2.4 nmol. addition of mcm to cultured p2 cells increases pma-stimulated h202 release to 223.3 +_ 10.7 nmol. the release decreased to 191.9 _+ 2.4 nmol when an murine anti-tnf-alpha antibody was added. vitality of cultured cells was > 96 % in all experiments. the results show that lps has an direct effect on p2 cells cultured on fibronectin. we conclude that the observed additional stimulatory effects of mcm seems to depend on tnf-alpha. the induction of h202 release of p2 cells could be important for generating internal oxidative stress in p2 cells before external oxygen radicals exceed. the produced h202 did not necessarily damage p2 ceils, but it can effect surfactant metabolism, especially when extracellular h202 release of alveolar macrophages following an immune response is increasing. introduction: primary stabilization of femoral shaft fractures in patients with multiple trauma is beneficial. however, in patients with associated lung contusion we have found an increased incidence of ards, apparently associated with primary reamed femnral nailing (rfn). previous animal studies revealed, that perioperative disturbances of lung ftmetion appear to be related to the reaming procedure, ix~ssibly due to pulmonary embolizafion of bone marrow fat. in a prospective clinical analysis we compared effects of intrameduuary nailing with and withont reaming on parameters known to be related to ards-pathoganesis. in order to gain further insight into the role of endotoxin and cytokines in the pathogenesis of the adult respiratory distress syndrome (ards), we enrolled 23 patients with severe lung injury after sepsis (17) or polytrauma (6) and obtained multiple blood samples (14 days) for endotoxin, tumor necrosis factor e (tnfa), interleukin 18 (il-18) and interleukin 6 (il-6) determination. to evaluate the cytokine releasing capacity of the blood, plasma concentrations of tnfe, il-l8 and il-6 were also determined after the "in vitro" stimulation of the whole blood samples with lipopolysaccharide (lps, 0.1 ng/ml) for 2 hours at 370 c (stimulated values). the difference among stimulated cytokines levels and the basal plasma concentrations were defined as "delta values", an expression of the cytokine releasing capacity of the blood. the pao2/fiao2 quotient was used as an index of the severity of lung injury (sli). the endotoxin plasma level was significantly higher in patients with sli < 200 (0.288 ± 0.038 eu/ml, mean values ± sem) versus the patients with a sli > 200 (0.175 ± 0.023 eu/ml, p<o.01). the basal plasma concentration of ll-6 also correlates with the sli index (p<o.o01). the delta tnfe and delta il-6 values were s~gnificantly decreased in patients with a severe lung injury. compared with the survivors (n:11), the deceased patients (n=12) showed higher endotoxin level, a reduced tnfa and il-6 releasing capacity of the blood and higher basal il-6 levels. we can conclude that endotoxemia, high il-6 plasma level and a decreased capacity of the blood to release tnfa an il-6 under endotoxin stimulation associates with the severity of lung injury. [objectives] experimental and clinical findings implicate the involvement of inflammatory cytokines in the pathogenesis of the decreased oxygenation in the lung after major surgery, though the mechanism remains unclear. in the present study, we elucidated the involvement of il-8 in the pathogenesis of lung injury defined by deterioration of pulmonary oxygenation after esophagectomy. [materials and methods] seventeen patients underwent subtotal esophagectomy through a right thoracotomy. in all patients, tbe alveolar-arterial oxygen tension difference (aado2) was measured everyday and bronchoalveolar lavage fluid (balf) samples were obtained from a single segment ($4 or $5) of the right and left lung through a bronchoscope on days l, 3, and 5 days after surgery. they were then examined for cell analysis, measurement of cytokines by elisa, and measurement of neutrnphil elastase (ne) by elisa. [results] aado2 unexceptionally deteriorated postoperatively and the mean aado2 in 17 patients reached the maximum (mean ± sem; 372 _+ 23 mmhg) on the 3rd postoperative day. the mean concentrations of il-8, ne and neutrophil count in balf also increased postoperatively and reached their maximum level (2137 + 618 pg/ml, 1603 -+ 493 gg/l, and 1561 ± 477 x 103 cells/ml, respectively) on the 3rd postoperative day. these increases in balf were recognized in both the right and left lung. a highly significant correlation was observed between il-8 and ne levels in balf (r=0.82, p=0.0001). also, significant correlations between aado2 and the neutropbil count, and the concentration of ne or 1l-8 were observed. in contrast to il-8, neither il-6, il-113 nor tnfa was detected in balf. [conclusion] major surgical trauma such as esophagectomy may induce recruitment of neutrophils to the lung and cause lung injury. il-8 increased in the lung is an important mediator of neutrophil recruitment and activation in the lung. phorbol myristate acetate (pma) is commonly used to cause edema and other tissue damages in the lung. lung injuries induced by the administration of pma has been shown to be mediated mainly by neutrophils. neutrophils recruited to the lower respiratory tract may damage lung tissues by releasing reactive oxygen species, neutral proteases and lysosomal enzymes. the present study was conducted to investigate whether c~tocopherol, entrapped in dipalmitoylphosphatidylcholine liposomes and delivered directly to the lung, could counteract some of the pma-induced lung injuries. plain liposomes or c~tocopherol-containing liposomes (8 mg c~-tocopherol/kgbody wt.) were instilled into the lungs of rats 24 h prior to pma exposure (25 pg/kg, intratracheally) and treated rats were killed 3 h later. lungs of control animals exposed to pma developed increases in lung weight and lipid peroxidation as well as decreases in lung angiotensin converting enzyme (ace) and alkaline phosphatase (akp) activities. pma treatment also caused an increase in myeloperoxidase (mpo) activity in the lung, suggestive of neutrophi] infiltration. pretreatment of pma-treated rats with plain liposomes had no effect on pma-induced injuries. in contrast, pretreatment of rats with liposomal c~-tocopherol, 24 h prior to pma administration, resulted in a significant elevation of pulmonary a-tocopherol concentration, accompanied by a concomitant reduction in mpo activity and reversal of pmainduced changes in lung edema, lipid peroxidation, ace and akp activities. these results appear to demonstrate that the intratracheal administration of a liposome-associated lipophilic antioxidant, such as a-tocopherol, can significantly ameliorate the toxic effects of reactive oxygen species, released from pmastimulated pulmonary target cells and infiltrating neutrophils. jk wojcik, g palasiewicz, w roszkowski immunology department,institute of tuberculosis and lung diseases~ warszawa, poland, m~larhospital, eskilstuna, sweden. introduction:the critical point in neutrophil migration to the alveolar space in the course of ards is the attachment between giyeoproteins of neotrophil membranes(sialyl lewis x carbohydrate epitope containing u-l-fucose) and lectin domains of endothelial p and e selectins (gmp-140,elam-i). a potential beneficial therapeutic strategy may be designep to suppress neutrophil recruitment to the lungs by preventing their rolling and attachment to the pulmonary endothelial cells. objeclive:to investigate if tz-l-fueose prevent experiments pulmonary hypertension in ards as effective as metylpredniselone. methods:only healthy rabbits (n-24) weighing 5.d-5.5 kg oaselinepao>11 kpa and mean pulmonary arterial pressure (mpap)<i.5 kpa were included in this study. anaesthesia was induced with thiopental 20-25 mg/kg bw and "blind" jntubation was performed. a 5f swan ganz catheter was introduced via the left jugular vein into the pulmonary artery. pma, as was used in our experiments activates neutrophils and produces acute respiratory failure with pulmonary hypertension and pulmonary edema. eight rabbits serving as control animals received pma 25 ug/kg bw iv only. sixteen other animals were divided into two groups receiving either ~-l-fucose 100 mg iv or metylprednisolone 150 mg iv 15 min before pma admininstration. results:after pma administration notable physiological changes including marked increase in mpap(2.0-+o.~6 kpe3 ..lere found. the increase of npap was observed to be completely blocked in both groups of animals which received ~-l-fucose or metylprednisolone. conclusion:in aur rabbit model ards cz-l-fucose pretreatment prevents pulmonary hypertension after pma administration. the effect is comparable with high dose metylprednisolone. the possible explanation is that g-l-fueose molecules block iectin domains of gnp-140 or elam-i preventing the adhesion of the neutrophils to the pulmonary endotheliom. bartens c, elmadfa i, steltzer h, fischer m, druml w introduction: various micronutrients and vitamins are particulary effective in modulating immune functions and host defense. the nutritive ant±oxidants vitamin c, e and b-carotene, as well as selenium lower the burden of reactive free radicals and protect the structural integrity of cells and tissues of the immune system, as well as other systems in the body. certain cells of the immune system like polymorphonuclear leucocytes (pmn's) use free radicals as a weapon to destroy invading pathogens. these reactive molecules, when released into the surrounding area, mediate lipid peroxidation and tissue injury. there is growing evidence that pmn's are activated in vivo by complement or immune complexes in disorders such as ards. the respiratory host defense mechanisms of patients with adult respiratory distress syndrome (ards) may be defective. the aim of this study was ta evaluate the status of the free radical scavengers and their activity in ards and to investigate the respiratory burst of ards patients. methods: seven ards patients (3 sepsis, 4 trauma) with an fie 2 of 0.59+0.(36, a peep of 8.50±0.99, and a murray score of 2.83 ±0.18 were included in this study. 15 healthy adults served as controls. superoxide anion production in granulocytes was measured photometrically, and the hydrogen peroxides by fluorimetric assay. vitamin a, e, and g-carotene concentrations were assessed by hplc, and plasma vitamin c photometrically. plasma selenium was determined by dectrothermal aas. plasma lipid peroxidation pruducts, expressed as malondialdehyde (mda), were determined by thiobarbituric acid assay and hplc. results: mda-plasma (/xmol/l) 0.74~: 0.46* 0.37 ± 0.16 *= <0.01, **=p<0.001 condusion: ards patients showed significantly decreased plasma concentrations of vitamin c, e and g-carotene, as well as selenium. these nutritive ant±oxidants are consumed during increased oxidative stress. mda, a final product of lipid peroxidation, is increased. however, a difference in rates of release of hydrogen peroxides and superoxide-anion of pmn could not be detected. therefore, oxygen radical accumulation is more likely a result of excessive inspiratory oxygen concentrations (f.io2), pro-oxidant drugs, and a high settlement of pmn in the lungs, and not an increased release of free radicals of the single pmn. kd glycoprotein secreted by the alveolar type ii cells. recent study showed that sp-a exists in the sera from normal healthy adults and that the significantly elevated serum sp-a levels were observed in the patients with interstitial pneumonia and pulmonary alveolar proteinosis. these findings means there may be a mechanism that the sp-a produced in the alveoli escapes into the bloodstream. here we examined the serum sp-a levels in the patients with critical illness including sepsis and ards. ]clinical subjects & methods] a total of 99 serum samples were collected from 39 patients hospitalized in the division of critical care medicine(icu), sappom medical college hospital. serum sp-a levels were measured by an enzyme-linked immunosorbent assay using monoclonal antibodies to human sp-a. ]results] there was no significant difference between the septic (n=12) and non-septic (n=27) patients. patients with ards (n=10, 52.0+4.85ng/ml) and with respiratory disease other than ards (n=12, 49.6+-5.35ng/ml) showed significantly higher levels of serum sp-a than those with non-respiratory disease (n=17, 27.4_+4.86ng/ml). as to the ards patients, it was likely that the serum sp-a levels were getting higher in their clinical courses. however, there was no significant correlation between the serum sp-a levels and the pa%/fio 2 ratio. ]discussion] at the present time, the exact mechanism of the escape of sp-a into the bloodstream remain to be unclear. our results may suggested that there was some relation between this leakage of sp-a and the alveolar-capillary permeability because of the higher sp-a levels observed in the ards patients. however it is also likely that higher serum sp-a level represents the proliferation of alveolar type ii cells as the regeneration of damaged lung. further studies are now being done. neutrophils contain a number of enzymes within intracellular granules,potentially damagingto lung tissue.release of these enzymes into the lung tissue from the sequestred activated neutrophils is felt to play significant role in lung injury in the course of aros. using 6-hours acute animal model of aros the activity of cathepsins,beta-glucuronidase and acid phosphatase was determined in the lung tissue. results of this research were compared with neutrophil proteases activity in serum and broncho-alveolar lavage.also hemodynamic,respiratory and biochemical investigations were performed before beginning of experiment,and in a two-hours time intervals of its duration.after 6 hours was determined total and free activity of cathepsins,beta-glucuronidase and acid phosphatase in full homogenate of the lung tissue,mitochondriallysosomal fraction and the final supernatant. in the course of our experiment we observed an increase (30.i-49.6%) both total and free activity of neutrophil proteases in all fractions of the lung tissue.generally accepted current pharmacological treatment of aros only attenuated this increase but never caused reversion to the level before beginning of our investigations. the presence of aros was histologically proved.an activity of acid phosphatase in serum and the lung tissue fractions was compared with histochemical pictures of the lung. results of our research indicate that neutrophil proteases are a very important mediators in aros and they are a cause of the lung injury. in addition neutrophil-derived proteases can amplify the inflamatory process by enzymatitally activating of many other mediators and they also may to increase an oxidant induced injury by imparing of antioxidant defenses. oepartment of general surgery, sk~odowskiej 24a 15-276 ~ia~ystok, poland, tel/fax +48 85 619742. in vitro studies demonstrated that paf activates polymorplionuclear leukocyte (pmn) 5-1ipoxygenase, but the generation of leukotrienes (lts) is critically dependent on exogenous arachidunic acid (aa) disposal (f. grimminger et at., mol. pharmacol. 41:757, 1992) . we investigated the features of paf-evoked lt synthesis in a model of pulmonary microvascular leukostasis, in the presence and absence of intravascular n-3-and n-6 -prescursor fatty acid supply. human neutrophils were infused into isolated, ventilated and perfused rabbit lungs to achieve microvascular sequestration of ligand-responsive leukecytes. leukotrienes (lt) and hydroxyeicosatetra(penta)enoic acids (het(p)e) were quantified by itplc techniques. intravascular application of paf (5 um) or arachidonic acid (aa;ioum) provoked the generation of limited quantities of 4-series lts and 5-hete (total sum of 5-1ipoxygenade products rd. 7 and rd. 27 pmol/ml.; both in pmn-preloaded and non-preloaded lungs). combined administration of pat r and aa caused amplification of 5-1ipoxygenase product formation with predominance of cysteinyl-lt synthesis both in lungs without (total sum rd. 67 pmol/ml) and, much more strikingly, with pro-application of neutrophils (total sum rd. 308 pmofml). eicosapentaeooic acid (10 um) elicited exclusive generation of 5-series lts and 5-hepe (total sum rd. 82 pmol/mi). dual stimulation with paf and epa provoked amplification of epa-derived 5-1ipoxygenase product formation, again with predominance of cysteinyl-lts, in lungs without (total sum rd. 224 pmul/ml) and in particular in those with preceding microvascular pmn entrapment (total sum rd. 545 pmol/ml). we conclude that the paf-evoked 5-1ipoxygenase product formation in the neutrophil-harbouring lung capillary bed is critically dependent on intravascular precursor fatty acid supply, with epa representing the preferred substrate as compared to aa. pmn-related transcellular eicosanoid synthesis is suggested to underly the predominant generation of cysteinyl-lts. these findings may be relevant for lipid mediator profiles provoked by infusion of n-3-versus n-6enriched lipid emulsions in diseases with pmn-related inflammators events. adult respiratory distress syndrome (ards) involves damage to the alveolar epithelium and microvascular endothelium. products released from neutrophils (pmn) are thought to be among the chief causes of this damage, while the role of mononuclear cells (mnc) is uncertain. we studied patients at risk of or with acute ards. we measured the concentration of myeloperoxidase (mpo) and elastase (el) within circulating pmn as an index of pmn activation, elastin degradation products (edp) in the plasma as an index of tissue damage, as well as the cytotoxici~y of pmn and mnc to endothelial cells (ec) in vitro. cells and plasma were prepared from venous blood of healthy controls; or systemic arterial blood of patients on ventilators in intensive care but not at risk of ards; at risk of ards because of sepsis; at risk because of multiple transfusion/major trauma; or with acute ards. lung injury, multi-organ failure and apache h scores were recorded. to measure cytotoxicity, human umbilical vein ec were labelled with cr-51, incubated with either pmn or mnc for 18 hours, and the supernatant counted. the adherence of the pmn and the mnc to the ec was also measured. standard assays were used to measure mpo and el in the pmn, mad edp in the plasma. the mnc cytotoxicity did not differ between the groups. the pmn cytotoxicity was higher in the transfusion/trauma patients compared to each other group, which did not differ from each other. there was no correlation between cytotoxicity and adherence for any group. the mpo and the el concentrations were significantly decreased in the pmn from each of the patient groups, including the patient controls, compared to healthy controls. the edp were only increased in patients at risk of or with ards. thus pmn degrannlation occurred in patients not at risk of ards, but tissue damage as measured by increased edp only occurred in patients with or at risk of ards, suggesting that factors additional to pmn activation and degranulation are required for the progression to ards. there was no correlation between the parameters measured and the indices of disease activity, or outcome. these measures of pmn and mnc activation and function are not of value as prognostic indicators in patients with or at risk of ards. the liver is made up of several different cell types which subserve distinct functions in vivo. in addition to the different functions of the distinct cell types, it is now evident that even within the population of hepatocytes substantial functional heterogeneity exists. this heterogeneity occurs in a very organized fashion in the normal liver based upon the position of the hepatocyte in the acinus. although it has been proposed that the acinar heterogeneity of hepatocytes arises from migration of immature hepatocytes from the periportal region to the perivenous region where they become senescent and die, many of the heterogeneous responses can be accutely reversed by reversal of the oxygen gradient via retrograde perfusion. under normal conditions in which the acinus is exposed to unidirectional flow, the periportal hepatocytes are exposed to relatively high levels of oxygen, substrates and hormones. as these substances are extracted along the aeinus their concentration decreases leaving relatively low concentrations for the perivenous hepatocytes. in the normal liver, the heterogeneity of response is attenuated by extensive communication via gap junctions. the major liver gap junctions are made up of hexamers of connexin 32 (cx32) which forms channels between cells capable of pas.~ing any molecule smaller than 1000 d. coupling is sufficient to allow diffusion of molecules such as atp or camp from one hepatocyte into > 20 adjacent hepatocytes within 60 seconds. during stress conditions such as endotoxemia or zymozan inflammation, expression of cx32 is markedly decreased while the secondary gap junction protein cx26 is either unchanged or even increased. while cx 26 readily effects electrical coupling, molecules > 350 d pass only very slowly. this would result in restriciton of transmission of moecules the size of atp or camp. since inhibition of gap junctions also attentuates metabolic response to hormone or nerve stimulation, it is evident that modulation of hepatocyte hetereogeneity by gap junctions must be considered in determining the mechanisms of metabolic alterations during stress. already minor haemorrhage decreases portal venous blood supply to the fiver and the reduction in portal blood flow becomes more pronounced with more profound btood loss. severe hacmorrhagic hypovolemia also reduces hepatic arterial blood supply which, however, is maintained over a vide range of haemorthage. the net effect of blood loss is a reduction in liver oxygee supply and this reduction is in proportion to the vulume iossed. however, oxygen supply to the liver exceeds the demands of the normal liver and this is the ca~ stilt following reduction of 30 % of blood volume. the situation in sepsis is more complicated. po~l venous supply to the liver is redur.~i fairly early following normovolemic sepsis while hepatic arterial blood supply is maintained at le,~t initialiy, oxygen saturation might be maintained in arterial blood but may also be slightly reduced during sepsis, oxygen saturation of portal venous blood is significantly reduced during sepsis due to increased extraction of the intestines. therefore oxygea delivery to the liver during sepsis becomes sigalfkzntly reduced. at the s,~ne time and for mai.v.ly unknown reasons the need for oxygen becomes significantly increased in the ~-~ptic liver. as a consequence liver oxygen consumption becomes flow dependent and the liver is likely to suffer from ischemia during septic conditions. $94 although liver failure is well recognized in sepsis, it is generally thought to be a late complication following pulmonary and renal failure. jaundice, hypoglycemia, encephalopathy and bleeding secondary to low levels of liver-synthesizing clotting factors are, however, signs of rather severe end-stage hepatic failure. furthermore, elevated liver enzymes (sgot and sgpt) represent hepatucyte damage and not hepatocellular dysfunction. in view of this, a more sensitive indicator of hepatic function is desirable in order to detect early hepatic abnormality. in this respect, indocyanine green (icg) is a tricarbocyanine dye that possesses several properties which makes it particularly valuable inthe assessment ofhepatic function. this dye is bound m albumin and is cleared exclusively by the liver through an energydependent membrane transport process and is nontoxic at lower doses. we propose that maximal velocity (vm~,) of icg clearance is a valuable measure of active hepatocellular function, since the total concentration of functioning receptors is directly proportional to vm~. we have utilized a fiber optic catheter and an in vivo hemoreflectometar to continuously measure the administered icg in vivo and consequently determine its clearance without the need of blood sampling. using this technique, we have found that in the early stages of sepsis (i.e., 2 and 5 h following cecal ligation and puncture), the vm~ and kinetic constant (k=) of icg clearance was significantly depressed. it should be noted that at this stage of sepsis, there was no elevation in serum enzyme levels. furthermore, hepatic blood flow and cardiac output increased at the above mentioned time points. thus, the extremely early depression in active hepatocellular function in sepsis, despite the increased hepatic blood flow and cardiac output, may form the basis for cellular dysfunctions leading to multiple organ failure during sepsis. additional studies indicated that following hemorrhage, active hepatocellular function was markedly depressed. this returned to prehemorrhage levels after ringers lactate resuscitation, however, this function was not maintained and decreased significantly after fluid resuscitation. nevertheless, the depressed active hepatocelinlar function following hemorrhage was markedly improved by post-treatment of animals with either atp-mgci2, peutoxifylline or diltiazem. thus, the use of icg clearance provides an early sensitive indicator of hepatic abnormality during sepsis and following hemorrhage and this method should be used, not only experimentally, but also in the clinical arena for the early detection of hepatocellular abnormality. although multiple organ dysfunction syndrome (mods) remains a major cause of mortality and morbidity in intensive care units, very little is known about the mechanisms that precipitate its development. since an episode of inadequate tissue oxygenation is considered to be the trigger for mods, we have proposed that a primary localized injury such as ischemia/reperfusion may be sufficient to cause a change of gene expression of remote and apparently unaffected organs. such modulation of remote organ gene expression may decrease the organ's tolerance to a subsequent stress contributing to the development of mofs. to test this hypothesis, rats were subjected to hepatic regional ischemia by clamping the blood flow (hepatic artery and portal venous inflow) of the left and median liver lobes. intestinal congestion was prevented by allowing flow through the smaller right and caudate lobes. after 60 minutes of ischemia, the clamp was removed and the blood flow restored. the animals were allowed to recover for 1, 4 and 24 hours. kidneys were removed, total rna was isolated and poly(a) ÷ selected by affinity chromatography on oligo(dt) columns. message was in vitro translated using rabbit reticulocyte iysates in the presence of radioactive amino acids. the gene products (radiolabeled polypeptides) were fractionated by two dimensional gel electrophoresis, and visualized by fluorography. analyses of the two dimensional fluorograms indicate that there is a dramatic change in the electrophoretic pattern of in vitro translated products in samples corresponding to kidneys obtained after 60 minutes of hepatic ischemia and 24 hours of reperfusion with respect to kidney samples obtained after sham operation or from control rats. the latter were not subjected to any surgical manipulation. these studies suggest that the gene expression of the kidneys is specifically modified after a remote organ injury (hepatic ischemia/reperfusion). we speculate that this change of gene expression in kidneys after an indirect injury may be part of the early events leading to the development of mods. a priming event, e.g. local ischemia, in combination with a second insult, e.g. sepsis, may amplify a host's response and lead to multiple organ failure. to better understand the mechanisms involved in the pathophysiology, male fischer rats were subjected to 20 min of hepatic ischemia followed by reperfusion (rp) and injection of 0.5 mg/kg salmonella enteritidis endotoxin (et) at 30 min of rp. et injection potentiated the postischemic liver injury as indicated by histopathology and an increase of plasma alt activities from 870+122 u/l (i/rp only) to 3900+470 u/l at 4h rp. inhibition of kupffer cells (kc) with gadolinium chloride (7 mg/kg) attenuated liver injury in this model by 70%, however, monoclonal antibodies (cl26, wt3) directed against adhesion molecules (132 integrins, cd18) on neutrophils had no effect on the injury despite the substantial accumulation of neutrophils in the liver at that time (467+52 pmns/50 hpf; baseline: 13+3). isolation of kc and neutrophils from the postischemic liver indicated a 10-fold increase of the spontaneous superoxide formation only in the kc fractions [3.85+0.68 nmol o2-/h/10%elts (kc2); 6.82_+0.92 (kca) ] at 4h rp compared to control cells. in addition, stimulation with phorbol ester or opsonized zymosan revealed a substantial priming of kc for reactive oxygen formation. in contrast to the short-term experiments (4h), the antibody wt3 (4 mg/kg) attenuated liver injury by 90% at 24 h of rp and improved survival. conclusion: liver injury during the early rp phase is mediated mainly by kc generating excessive amounts of reactive oxygen while neutrophils are primarily responsible for organ damage during the later rp period. (es-06091 and gm-42957) tumor necrosis factors (tnf) are cytokines which are cytotoxic towards some tumors in vivo and certain tumor lines in vitro. moreover, these polypeptides are powerful immunomodulators and have been found to be distal mediators in several models of septic shock and septic organ failure. one of the best-characterized experimental systems is the hepatitis caused by lps or tnf in galactosamine (galn)-sensitized mice. here we describe a cell culture system, in which the direct toxicity of tnf towards mouse hepatocytes was examined. the toxicity of tnf, as determined by ldh-release or formazan-formation, was dose-and time-dependent. the threshold of toxicity was 10 ng/ml, which corresponds to serum concentrations found in mice after lpsinjection. toxicity was only observed in hepatocytes sensitized with transcriptional inhibiters such as galn, actinomycin d (actd) or cxamanitin. sensitization was neither observed with different translational inhibitors nor with various other metabolic inlaibitors or toxins. inhibitors of protein synthesis or protein processing such as cycloheximide, puromycin, tunicamycin and ricin protected actdsensitized hepatocytes from tnf-induced cytotoxicity. tnf induced apoptotic changes and dna-fragmentation in sensitized hepatocytes which is in line with the above findings that cell death is dependent on protein synthesis. thus tnf may be a trigger of programmed cell death during inflammatory organ damage. with the purpose of studying the role of complement activation in tissue injury after ischaemia and reperfusion we blocked the complement cascade in a model of rat liver isehaemia and reperfusion, either by administration of soluble human complement receptor type 1 (scri), 25 mg/kg iv after vascular occlusion (n=8) or by depleting the complement system using cobra venom factor (cvf), 0.5 mg im, 24 and 6 hours before ischaemia (n=10). non-ischaemic rats (n=8) and ischaemic non-treated rats (n=15) were used as controls. the experimental procedure consists of the temporary interruption of arterial and portal blood flow to the left lateral and medial lobes of the liver during 45 minutes, followed by reperfusion, recording the liver blood flow and haemoglobin saturation with a laser doppler flowmeter and photometer during one hour after declamping; alt levels were assayed and immunoperoxidase stainings for c3 and c9 were performed. there were statistically significant differences between the experimental ~roups and the untreated ischaemic control group in terms of post-isehaemic blood flow (p<0.001) and haemoglobin saturation (p<0.001). c3 and c9 were present in the endothelium of the ischaemic control group. no deposits of c3 or c9 were found in the cvf group. few c3 and no c9 were found in scri treated rats. these results show that the effect of reperfusion injury in the rat liver is ameliorated either by depleting complement with cvf or by regulating complement activation with scri. hepatic dysfunction, a major cause of mortality following hemorrhagic shock, has not yet been well characterized. the present study was designed to assess the effects of liver blood flow and cytokine levels on hepatic function following resuscitation from severe hemorrhagic shock in normal and cin-hotic rats. methods: aftor pentobarbltal anesthesia, 23 control and 38 cirrhotic sprague-dawley rats were subjected to severe hemorrhage to reduce their systolic blood pressure to 50 + 5 mm hg. this level of hypotension was maintained until the skeletal muscle transmembrane potential (era) depolarized by 25%.; the animals were then resuscitated with ringer's lactate solution in three times the volume of the shed blood. serial blood samples for tumor necrosis factor (tnf) determination (a modified flow-cytomeuic wehi cell bioassay) were obtained at baseline, during hemorrhage and following resuscitation. liver blood flow measurements by low dose galactose clearance (glc) and functional bepatocyte mass (fhm; defared as galactose elimination capacity [gec] from the zero order portion of the plasma disappearance curve following an intravenous galactose bolus [500 mg/kg], divided by liver weight) were measured before shock and after resuscitation. results: higher survival rates (p < .05) were observed in control as compared with cirrhotic rats. shock produced a significant reduction in gec (to < 0.01); fhm (19 < .01); and liver blood flow (p < 0.01) in normal and cirrhotic rats. decreases in gec and fi-im were greater (p < 0.05) in cirrhotic rots. tnf levels were higher (p < 0.05) in cirrhotic rats at baseline and during induction of shock. pre gap junctions provide pathways for metabolic signals between cells. in the liver, the majority of gap junctions are composed of connexin 32 (cx32) polypeptide subunits, and are regulated by gluconeogenic hormones. since sepsis and other inflammatory states alter hepatic glucoregulatory control, we have evaluated the contribution of gap junctional conductance to the metabolic dysregulation in the liver. an acute inflammation was induced in rats by injection with e. coli endotoxin (lps lmg/kg). northern blot/hybridization analysis of total rna isolated from livers after endotoxin injection show a decrease in the steady state transcript levels of cx32 to 18% of sham controls. immunostaining of liver sections using anti-cx32 revealed punctate fluorescent staining on the plasma membrane at regions of call-cell contact in saline injected animals, whereas, staining was only observed in cytoplasmic vesicles 6 hrs after animals were treated with lps, suggesting the internalization of cx32 without replacement on the cell surface. the staining was quantitated and expressed as % of pixels above threshold. at 6hr post injection 3.6% ofpixels exceeded threshold, compared to 16.2% in sham controls. functional gap junctional communication was assessed by dye coupling using lucifer yellow in an isolated perfused liver under intravital fluorescence microscopy. dye diffusion was markedly decreased 6hr after endotoxin injection. this suggests that decreased metabolic coupling after lps injection results from decreased gap junction abundance. the present data suggest that metabolic dysregulation during sepsis may arise in part from changes in intercellular communication caused by a decrease in gap junctional expression and communication. given the marked metabolic heterogeneity of hepatocytes with respect to acinar location, metabolic signaling via gap junctions most likely serves to moderate this heterogeneity, contributing to a coordinated metabolic response. altered cellular ca 2÷ regulation might be a critical step in organ dysfunction during sepsis and ischemia/reperfusion events. the aim of the present study was to evaluate hepato-ceuular ca 2÷ regulation in isehemiah'eperfusion after hemorrhage and to assess effectiveness of tnfc~-monoclonal antibody (tnfo~-moab). methods. male sprague-dawley rats (250g, n>_6/group; pentobarbital 50 mg/kg) with hemorrhage for 60 rain at 40 mm hg. reperfusion by ringer's lactate (2 x maximal bleed out/60 min) and 60% of citrated shed blood. tnfcz-moab (tn3, ceutech, 20 mg/kg in 0.9% nac1) infused during flrst 5 min of reperfusion. at baseline, end of ischemia and 60 min of reperfusion, hepatecyte isolation by liver collagenase perfusion. " 45 hepatocyte incubation (30 mg w.w./ml) with caci 2 (0.05 2+ 2+ 2+ mbq/ml) for 60 rain (ca influx [slope, 1/mini; ca uptake [nmol ca /mg protein]) w/ and w/o epinephrine (epi, 100 nm). hepatecyte resuspension in radioisotope-free medium and farther incubation (exchangeable ca 2+ (ca2+ex) [nmol ca2+/mg protein]; ca 2+ membrane flux [nmol ca2+/mg protein'min]). during incubation, aliquots (100 ~tl) were centrifuged through oil/lanthanum gradient and acivity measured by scintillation counting. statistics: anova. mean + sem. results. hepatocyte ca2+ex and membrane ca 2+ flux were significantly increased at both, the end of ischemia (14.3+.9; 0.41+.05) and reperfusion (16.4+.9; 0.50+.2), as compared to sham-operated animals (7.6+_.5; 0.09+.02)(19<.05 ). tnfc~-moab treatment significantly prevented reperfusion-induced increase of ca2+ex (10+.8) and membrane ca 2+ flux (0.17+.03)(p<.01). fast ca 2+ influx was significantly increased by epinephrine in hepatecytes from sham-operated rats (0.23+.03 vs. epi: 0.52+.1, p< .05). this hormone effect was not observed in isehemia (0.47+.05, epi: 0.6!-_.2) or reperfusion (untreated: 0.13+.06, epi: 0.07+.01; tnft~-moab: 0.26_+.04, epi: 0.33+.07). conclusion. the present study clearly demonstrated hepato-cellular ca 2+ overload in ischemia and reperfusion as a result of hemorrhagic shock. analysis of membrane ca 2+ fluxes and hormone ca 2+ mobilization suggests disturbances of membrane ca 2+ transport mechanisms, e.g. through ca2+-atpases. reperfusion-induced oxygen free radical generation which affect exchange kinetics of cellular ca 2+ buffering compartments might also be operative. prevention by tnfct-moab indicates the pivotal role of tnf as an early inflammatory mediator of hepatocellular alterations in signal transduetion mechanisms and cellular homeostasis. although the precise mechanism has not yet been elucidated, bacterial translocation and endotoxin absorption have been frequently shown after burn, and have been postulated to be one of the underlying processes of sepsis. the purpose of the current study is to define the hemodynamic response of the liver to endotoxin release in burns, in correlation to bacterial translocation. twelve female minipigs, weighing 20-28 kg, underwent a laparotomy & transition time ultrasonic flow probes were positioned on the portal vein, the common hepatic artery, and the superior mesenteric artery. 6.5 fr catheters were inserted in the superior mesenteric vein and the left hepatic vein. a jejunostomy was also performed. after five days all animals were anaesthetized and randomized to receive 40% of tbs a third degree burn. eighteen hours after burn. 100 gg/kg e. coli lps was intravenously administered over 30 rain. ali animals were studied for additional 24 hours and then sacrificed. several recent data suggest that in severe injuries, such as shock state, the gradual activation of kupffer cells and the excessive release of destructive and immunosuppresive products from macrophages may contribute to the development of "multiple organ failure". in in vivo experiments in mice, the effect of kupffer cell phagocytosis blockade on the correlation between the tissue distribution of lps, endotoxin sensitivity and lps-induced tnf production was investigated. to depress the activity of the kupffer cells, gadolinium chloride (gdc13) or carrageenan was used. th~e studies indicate the dissociation of tissue localisation of cr jllabelled endotoxin and endotoxin lethalithy. both gdc13 and carrageenan depressed kupffer cell activity, but endotoxin sensitivity was enhanced only by carragenan treatment. however, there was a close correlation between the sensitivity to lps and lps-induced tnf production as measured in the serum, since lpsinduced tnf production was enhanced only by carrageenan treatment. on the other hand, gdc13 pretreatment significantly increased tnf production in the spleen. these results support our earlier findings that gdc13-indueed kupffer cell phagocytosis blockade leads to activation of the spleen, and may explain some of the immunological effects of gdc13. inositol(l, 4, 5) triphosphate (ip3) has been proposed as a second messenger for calcium mobilization. the addition of ip3 at low concentration has been shown to cause calcium release from intracellular microsomal store in rat hepatocytes. the effects of sepsis on the ip3 binding from microsomal fraction of rat hepatocytes during sepsis were investigated. sepsis was induced by cecal ligation & puncture (clp). control rats were sham-operated. three microsomal fractions (rough, intermediate and smooth) were isolated from rat liver. study of ip3 receptor binding was performed with tridium label ip3. the results shewed that the ip3 binding was significantly depressed by 40-47% (p<0.05) during late sepsis (18 hrs after clp), but not in early sepsis (9 hrs after clp). the ip3 binding depression during late sepsis was most significant on rough and intermediate endoplasmic reticulum (p<0.05), but not on smooth subfraction. since ip3 binding plays an important role in the regulation of intracellular calcium homeostasis in hepatocytes, an impairment in the calcium release due to depressed ip3 binding on smooth and intermediate endoplasmic reticulum during late sepsis may have a pathophysiological significance in contributing to the development of altered hepatic metabolism during septic shock. septic organ failure is currently recognized as an overactivation of the nonspecific immune system by bacterial stimuli giving rise to proinflammatory mediators. little is known about the mechanisms of the resulting cellular injury. here, a synergism is described between tnf as a major mediator of septic organ injury released by macrophages and hydrogen peroxide (h202) as a representative of reactive oxygen species as formed by e.g. neutrophils. rat hepatocytes are only slightly sensitive to either agent alone. when treated with a conbination of tnf and h#09 a stronq synergistic toxicity was found, especially w~e~ tnf-treatment preceeded challenge with h~o~. we have recently described a coculture model bfzrat liver macrophaqes and hepatocytes where lps induces hepatocyte cell death partially mediated by macrophage tnf release. when h202 was also employed in fhis more complex cellular system a similar synergism was found: the ecc~ of lps was 62 consecutive patients with liver cirrhosis admitted to the department of surgery over a 1 year period from january to december 1992 were studied for their complement profiles in relation to other parameters of liver function, the aim of the study was to determine if a direct correlation existed between low complement levels and end stage liver cirrhosis. cirrhotic patients were divided into child's a, b and c categories using child's classification. complement levels (c3, c4) were measured and functional assay for complement (ch50) were performed in each of these groupings in addition to normal blood donor controls. these results show that the qualitative c3, c4 and the functional chs0 complement assays have good predictive values in assessing deteriorating liver function• in particular, the functional assay for complement (ch50) showed marked impairment in child's c patients (p<0.000001) confirming the impaired immunological status of these patients. sera from this group of patients (child's c) were titrated with pig red blood cells (rbcs) in a haemolytic assay. the results showed that there were significantly less haemolysis of pig rbcs in these patients (p=0.0177) as compared to the controls. this findings strongly support an impaired immunological status in child's c liver cirrhosis and may explain the high incidence of sepsis as a terminal event in these patients. aim:kupffer cells(kc) have an importamt play to cause hepatocellular injury in sepsis, because these cells release many kinds of substances. we reported that oxygem radicals released by kcs stimulated by lipopolysaccharide (lps) caused hepatocellular injury. aim of this study is to investigate the relationship between imtracellmlar calcium(ca) concentration of cultured rat kcs stimulated by lps and release of oxygen radicals, and effect of prostaglandin e~ (pge~) on imtracellular ca concentration. production of acute phase proteins (c-reactive protein, crp, transferrin, tf) and £erritin (f) in rat hepatocytes (hps) and its dependence on extracellular matrix components were studied. hps isolated from the liver by collagenase perfusion were cultured at ~o5 per 0.5 ml medium fi2+dmem (1:1) with 10% fetal calf serum for 2 days on uncoated or type i collagen coated plastic surface or in the presence of dextrane sulphate in the medium. hps were stimulated by conditioned medium (gm) from i~ia-p or e. coli lps preineubated human blood mononuclear cells. production of crp, tf and f by hps was detected by elisa. it was found that both cms decreased tf synthesis in hps by 15-50% (p<o.os). the level of f synthesis didn't change or only slightly decreased. the addition of cms led to the enhancement of crp synthesis more than 2.5 times. the most reproducible results were obtained when plastics had been coated with collagen. dextran sulphate markedly increased crp synthesis. thus using this model of an acute phase reaction in vitro we found the enhancement of crp synthesis and the decrease of tf synthesis. our data correspond with nublished materials on the acute phase in vivo and point to relevance of the proposed model. metabolism of hepatocytes under traumatic illness has not been sufficiently investigated. therefore we have applied absorption and fluorescent cytophotometry techniques to study the content of rna, glycogen and its fractions in hepatocytes of patients with severe mechanical trauma, both with and without endointoxication at various stages. for these purposes slides were prepared from the repeated aspiration biopsy material according to special techniques (kudryavtseva et al., 1983) . slides were stained by gallocyanin-chromalum to measure rna or underwent fluorescent pas-reaction to measure glycogen and its fractions (kudryavtseva et al., 1970 (kudryavtseva et al., ,1974 the ability of 02 metabolites derived from the xanthine-xanthine oxidase system to inhibit mitochondrial function was examined using freshly isolated rat liver mitochondria. under uncoupled conditions, mitochondria exposed to free radicals exhibited a significant decrease in 02 consumption supported by nad+-iinked substrates, but showed almost no change in 02 consumption in the presence of succinate and ascorbate. the activity of electron transfer complex i (nadh oxidase and nadh-cytochrome c oxidoreductase) and the energy-dependent reduction of nad+ by succinate were unaltered by oxidative stress. exposure to free radicals also had an uncoupling effect at all three coupling sites. the degree of mitochondrial swelling was closely correlated with the inhibition of state 3 oxidation of site i substrates and with the increase in state 4 oxidation of succinate. the immunosuppressive agent cyclosporin a (cya) completely prevented the mitochondrial damage induced by oxygen free radicals (swelling, ca2+ release, sucrose trapping, uncoupling, and selective inhibition of the mitochondrial respiration of site i substrates). the same protective effect was found when ca2+ cycling was prevented, either by chelating ca 2+ with egta or by inhibiting ca2+ re-uptake with ruthenium red. these findings suggest that the deleterious effect of free radicals on mitochondria in the present experimental system was triggered by the cya-sensitive and ca2÷-dependent membrane transition, and not by direct impairment of the mitochondrial inner membrane enzymes. correspondence should be addressed to: naoshi takeyama the aim of this study was to clarify the critical role of reduced glutathione (gsh) on the progression of lipopolysaccharide (lps)induced liver damage of mouse. the conditions of gsh-depletion and -richness were produced by intraperitoneal administration of buthionine sulfoximine (bso), a specific inhibitor of gsh synthetase, and of gsh-monoethyl ester (gsh-me), respectively. gsh-me, which was esterified the caboxyl group of the glycine residue, is readily transported into cells and is hydrolyzed intracellularly; this leads to greatly increased the cytosolic and mitochondrial levels of gsh. bso and gsh-me were administered intraperitoneally 3 mmol/kg and 10 mmollkg, respectively, and lps (2 mg/kg) given 1 h later. hepatocytes were isolated by in situ perfusion of the liver with collagenase 6 h after lps injection. the degree of hydrogen peroxide (h202) synthesis and mitochondrial membrane potential were measured by flow cytometry using fluorescence probe dichrolofluorescein diacetate and tetrametyl rhodamine ethyl ester, respectively. the degree of mitochondria membrane permeability transition (mmp) was assessed by [014]sucrose entrapment. we found that lps treatment results in a decrease in hepatic gsh level and mitochondrial membrane potential, and an increase in h202 synthesis and mmp. lps administration to bsq-pretreated mouse worsened at[ these parameters. in contrast, gsh-me pretreatment ameliorates. all together, gsh may acts an important role in cellular defence against lps-mediated liver damage, and mmp may result in the decrease in mitochondrial membrane potential. it has been shown, up to now, that disturbances of enzymatic processes in the cell organella are the basic factor of the changes taking place during endotoxin shock it has been observed that lysosomes are biochemically changed as result of the effect of lipopotysacharides of gram negative endotoxic bacteria. the purpose of the experiment was: 1.evaluation nfthe course of the ees due to the biochemic changes 2.determination of the influence of the ees on the activity of lysosomal enzymes in liver cell 3.deterrmnatian of the influence of h and d on the activity of some lysosomal enzymes in liver cell during ees material and method: examinations ware carried out on 30 dogs. the shock was evoked by i.v. administration of endotoxin e.coli. the dogs were divided into 5 groups: icontrols, i[ -dogs with untreated shock, iii-dogs in shock treated with h, ivdogs in shock treated with d, v -dogs in shock treated with h and d. the following parameters were determined: 1.activity of acid phosphatase in the venous blood serum. 2-6.total and free activity of acid phosphatase and catepsins in the full liver cell homogenate, in mitochondrial-and-lysosomal fraction, and in the superuatunt (all after 4 hours of the experiment). conclusions 1. essential increase of activity of lysosomal enzymes was observed in the ees. 2. increased activity of lysosomal hydrolases in the liver cell homogenate corresponds, as a result of the endotoxin effect, with increased enzymatic activity in the peripherial blood. extensive investigations from our laboratory of the pathophysiology of hepatic no-flow ischemia (45 min) -reperfusion injury demonstrated substantial kupffer cell activation with reactive oxygen formation during the first few hours of reperfusion as indicated by increases in plasma glutathione disulfide (gssg) levels in vivo (am. j. physiol. 260: g355, 1991) and enhanced superoxide formation by kupffer cells (kc) isolated from the postischemic livers (free radic. res. commun. 15:277, 1991) . the early kc-induced injury initiated the later, primarily neutrophilmediated reperfusion injury (faseb j. 4: 3355, 1990 ). to test whether or not similar mechanisms are operative during hepatic ischemia induced by hemorrhagic shock, male fischer rats (230-250 g) were subjected to 60 rain of hemorrhage (mean arterial pressure 40 mm hg) followed by resuscitation (rs) (reinfusion of shed blood). hepatic atp levels declined from 2.3+0.2 txmol/g to 0.24_+0.08 (60 min shock) indicating severe ischemia, and recovered to 1.04--0.2 at 90 min rs. to test for potential oxidant stress during rs, plasma gssg conc. were measured. although gssg levels increased by 120% compared to baseline levels (0.69!-_0.31 i.tm), there was no significant difference to shamoperated controls. spontaneous superoxide formation of isolated kc was 0.32+0.05 nmol o2-/min/10ecells (kc2) and 0.51+0.11 (kc3) in controls and did not change significantly after shock and 90 rain rs. addition of phorbol ester or opsonized zymosan to isolated kc did not indicate a priming of these cells. conclusion: in striking contrast to our previous findings with hepatic no-flow ischemia, there is no evidence for a significant kc activation after 60 min of hemorrhagic shock and up to 90 rain of resuscitation. objectives-this study investigates morphometric changes in kc nltrastmcture which might account for this diminution in phagacytosis. materials and methods -three groups of wistar rats were studied: control, sham-operated [sham] and bile duct ligated [edl] . after 3 weeks animals were sacrificed and livers were "perfusion fixed" with 3% glntaraldehyde and processed for transmission electron microscopy and image analysis. results -in the the bdl group kupffer cell numbers were greatly increased. biliary ductular proliferation was evident and sinusoidal spaces were compromised. eleven nuclear and cytoplasmic parameters were measured and statistically significant results are summarised in the results: ascorbic acid levels were slightly elevated after the hs and returned to basal values after 120 rain. glutathione concentrations were increased significantly after the hs and the gsh levels kept rising continuously to as much as 4-fold of basal levels at the end of 120 min. mda showed no significant variation before and after the hemorrhagic shock. plasma concentrations of ratine, teucine, isoleucine, phenylalanine, proline, threonine and serine showed significant increase over basal levels during the whole ischemic period. glutamic acid was slightly elevated at the onset of hs and remained the same for the rest of ischemic period. hydroxyproline was significantly below the basal value at the mid-point of ischemic period. alanine, glycine, histidine and aspartate did not change significantly throughout experimental time course. conclusions: (1) oxidative stress might not be severe in the systemic hemorrhagic shock for pigs since no significant variations were observed for ascorbic acid and mda (2) the increase of gsh might be that it was released from hepatocytes when the animal was under systemic ischemia. (3) celzain amino acids might be acting as transmitters in the event of ischemia. however, fm"lher investigations are needed to clarify the detailed mechanism in regard with antioxidants and amino acids. dr. fang-ku p'eng taichung veterans general hospital talchung, taiwan 407, r.o.c. jia shi yong, huang zhi oiang and men xian jun. in patients underi~ent major hepatic surgery,obstructive jaundice has been implicated with fnoreased susceptibility to sepsis and liver failure. jaundice was said to he associated with derangement of iamune function and result in expression of pr,:.inflar...aatory cytoki~es that cause hepatooellular damage. this study is ~.o investigate the effects ,.,t lip,~polysaccharide(lps)rats. jaundice were produced in healthy ~istar rats of either sex weighing 180-220 m~ by common hi l~ duet l igation(bl)l).seven days post l igation, rats were given lps(236.gg.10)0.7rag/kg intraperitoneally. at the end ,:,f l,gand 5 hours after lp$ ehanllenge, liver were removed and prepared for forzen sections immediately. i)emonstration of inf in liver parenchyna were performed by method of abu i~munohistochemistry using r~onoolonal antibody against tnf, leve of mrna for tnf ~ere measured by in-situ hybridization using biotin-labeled edna probe. results sho'~ed that tnf stained granules appeared in kup~'fer eell~ and polymorphonuclear leukocytes(pnnl,but not in hepatoeytes nor endothelial eel is. they ~'ere located within cytoplasms and peaked at 3-5 hours after lps ohal lenge. there was vocomitant tnfmrna expression but peaked earlier 1 hour post lps challenge. tnfmrna vcere detected notably in necrotic area and barely in bdl rats ~ithout lps challenge. it is therefore postulated that production of tnf by inflammatory effeetor cells-kupffer cell and phn in site, in the obstructive jaundice rats during sepsis may atribute to the hepatocellular damage. it also provide evidence for the beneficial effects of early release of biliary obstruction. in the present study, an animal model of a0c in wlstar rats was developed by tigatlng the common bite duct and injecting d.3mt solution of e. goli % b, (g×10' efm/mt) into the bite duct. the mortality rate was 40~ at 48h after aoc. at 6, 12, 24, 48h after aoc, kcs were isotated and cultured atone or cocuttured with hepatocytes to evaluate the modulation effect of kgs on hepatoeyte protein synthesis. the results showed that tactodehydrogenase leakage was increased progressively as the injured k~ function and structure developed. tnf and il-i tn the supernatant were detected with the peak values at 12h after aoc. at 48h after aoc, ~-teueine incorporation was obviousty decreased in the normal hepatocytes eocuttured with stimulated kcs compared to the uormai hepatocyte cultured group (p<8.01), but it was slgnificantiy increased in the group cocultured with normal kcs. in the a0c group, 'h-teuclne ineorporatien was decreased progressively in the injured hepatoeytes cocuttured with stimulatedkgs and it was markedly elevated when cocuttured wlthnormat kcs at 48h after a0c (p<0. og). it is concluded that the impaired hepatocyte proterin synthesis was directly mediated hy the stimulated kc function during aoc. such a mechanism possibly may be involved in the patho0ensis of hepatic dysfunction and m0f following h06. " the project supported by nsfc. in the past few years it has become evident that cytokines are implicated in various pathophysiological mechanisms. therefore measurement of cytokines and their potential inhibitors in biological fluids may be helpful in diagnosing and monitoring of diseases. however, dependent on the type of assay used investigations performed in different laboratories have often yielded conflicting results. in order to assess reliability and reproducibility of cytokine measurements two comparative studies for the determination of cytokines in biological fluids were launched by several investigators interested in this field: one national austrian ~tudy and one european study in the context of the european workshop for rheumatnlogy research. serum and synovial fluid samples were distributed by the organisers, and participants had to measure one or several of the following cytokines in all samples: il-1, il-2, 1l-6, i58, tnf-alpha, ifn-gamma, gm-csf, and the soluble receptors for il-2 and tnf; both commercially available immunoassays and home-made assays were allowed. so far, the main conclusions emerging from these preliminary studies are: (1) the same immurzoassay (elisa) yielded comparable results in different laboratories; (2) immunoassays from different manufactureres usually measured the highest concentrations in the same samples, yielded similar relative values but disparate absolute values; (3) assays for soluble receptors yielded less discrepant results; (4) some assays seem to be more suitable for measuring cytokines in biological fluids than others. the mean retrieval of exogenous recombinant human cytokines was approximately 50% for most cytokines tested. however, it must be borne in mind that natural and recombinant cytokines may behave differently in immunoassays. this raises the question as to the necessity of setting up international standards for all cytokines. possible interferences by serum components such as (lipo)proteins, complement, rheumatoid factors, etc., which may either decrease assay sensitivity or yield false positive results, must also be considered. in addition, natural cytokine binding proteins (e.g. soluble receptors) might interfere with cytokine determination in immunoassays. the problem of immunoassays versus bioassays has not yet been approached, but is without any doubt worth indepth investigation. thus, these studies are a promising first approach to deal with the difficulties of cytokine analysis in biological samples, and it is hoped that they will help to overcome some of the major methodological problems in the near future. tumor necrosis factor (tnf) is a pleiotropic cytokine which plays a key role in a number of immunologically mediated disorders like septicemia or cachexia. tnf receptors are found on the surface of a variety of cells, where they provide molecular signals for the cytokine effect. there exist two types of soluble tnf receptors (stnf-rs), tnf-r p55 and tnf-r p75. these stnf-rs can compete with the cell surface receptors and block their activity. the expression on and shedding from the cell surface of stnf-rs is enhanced by interferon gamma. increased concentrations of stnf-rs can be found in patients with infectious as well as malignant disorders. in patients undergoing immune stimulatory therapy with interleukin-2 and interferon alpha a simultaneous increase of tnf and its soluble receptors can be seen. in patients with hiv infection a strong correlation exists between the levels of stnf-rs and markers of immune activation like neopterin or beta-2-microglobulin. likewise patients with hematological disorders show elevated stnf-r levels. patients with weight loss have higher concentrations than patients with stable weight. the final value of stnf-rs within the complex network of cytokines is not yet clear. however, there exist striking parallels between infectious and malignant disorders pointing to a key role of endogenous immune activation. biochemicatly, d-erythro-neopterin derives from guanosine triphosphate. in vitro studies show that large amounts of neopterin are produced by human monocytes/macrophages stimulated with interferon-j (ifn-j). neopterin is biologically stable, and its halflife in the blood seems to solely depend on renal excretion. specimen for neopterin determination can be stored without special precautions. increased concentrations of neopterin have been described in body fluids of patients suffering from diseases which are apparently associated with an activated cellular immune response and with enhanced endogenous formation of ifn-~', e.g,, 1 ) increasing neopterin levels predict rejection episodes in allograft recipients 2) virus infections induce increased neopterin concentrations, and the degree of neopterin elevation predicts prognosis in these patients which is particularly true in hiv-1 infection 3) in autoimmune disorders such as systemic lupus erythematosus or rheumatoid arthritis neopterin levels reflect activity of the disease 4) increased neopterin concentrations in patients suffering from certain types of cancer indicate poor prognosis. significant associations between changes of neopterin and decrease of hemoglobin as well as the development of weight loss and cachexia, e.g., in cancer patients and in patients with hiv-1 infection, support the concept that endogenously formed cytokines such as ifn-~" and tumor necrosis factor-~( are involved in the pathogenesis of such symptoms. in conclusion, neopterin monitoring is a sensitive tool to detect the activation status of the cell-mediated immune system in vivo. repair and healing or perpetuation of acute inflammation is characterized by a complex network of interacting cellular and humoral defence mechanisms. of the numerous inflammatory mediators/effectors investigated hitherto, the proteolydc lysosomal enzyme pmn elastase has turned out to be highly destructive when released extracellularly thus contributing considm:ably to organ dysfunctions in severe posttraumatic and postoperative courses. besides various cytokines, elastase in complex with its main antagonist c¢ 1-proteinase inhibitor (a 1pi) is also supposed to induce the shedding of intercellular adhesion molecules from inflammatory cells (pmns, monocytes/macrophages, endothelial cells). these soluble adhesion molecules may modulate the inflammatory process in many different ways, e.g. via acting as chemotaxins, blocking neutrophil activation or competing with the membrane-bound forms in cell-cell adhesion. thus, measuring circulating or local levels of elastase-a 1pi and soluble adhesion molecules (icam, e-selectin,-pselectin, l-selectin) may be a helpful tool in judging the severity of an acute inflammatory process. in several clinical studies on surgical patients suffering from multiple trauma and/or sepsis we could demonstrate that the measurement of these parameters in consecutive plasma samples and local body fluids was highly valuable for early diagnosis and prognosis of multiple organ failure. prof. dr. m./ochum, institut fdr klinischechemie und biochemie, lmu miinchen, nufibaumstrage 20, 80336 m0nchen, germany procalaitonin (proct) a 116 aa peptide is dramatically increased in the blood of patients with various sepsis and infections. the increase begins as soon as 3 hours after the andoto:edn release and readied maximal level with a 200 to 80b-fold increase 24 h after li~ edrmrdstration. prcc, alcitorfin response was temporally different from these of tnf~ and ii-6, that reached peak levels at 2 h and 3 h respectively. at the opposite of eytokine (tnfcz, ild, j, is), proct is a very stable molecule. the half iife of proct in ~he blood is surprising if we consider the half llfe (about nine mirtute) of mature ¢alcitonin (co. we have found that it is due to the binding of proct with a protein on the n-termktal part. thin complex of about 80 000 daltons is unable to cross the kidney and the c_n9 barriers and the half life is at least elghteen hours. this long half llfe is very useful in the evaluation of a sepsis. a very sensitive and specific sandwich in'ununoassay has been developped. the results cart be delivered in two hours. at time, we know that in the healthy adults, the values are less thau 0.1ng/ml. it is also clear that proct is not increased ~n patients wlth inflammatory dleeasea without lnfectlon. for instance, proct is not signlffcat3vely increased in purpura rhumatom, arthritis, crohn's disease, rectocolitis, also in various cancer patients with inflammatory components. am other interesting finding, is the absence of increase or a very low increase in the viral infections. at the opp0site, in all the patients with bacterial sepsis or in the malaria pat/ants, progt was dramatically increased, from i00 to 10 000 fold. p~oct can be useful to the follow up of patients with sepsis. actually, we don't know the origin of this proct production, in the united states, will also be discussed. the septest portion of this study will eventually enroll over 500 patients tentatively diagnosed as septic. the results of septest will be compared with commonly accepted symptoms and criteria associated with sepsis in order to determine the clinical utility of this endotoxin assay. preclinical results of patients and normals be presented as well as data on the time course of endotoxemia and clinical outcome of selected septic patients. preparation of dna libraries clifford w. schweinfest, ph.d. a fundamental tool of the molecular biologist today is the ability to work with purified dnas representing genes of interest with respect to the investigator's field of study. originally applied almost 20 years ago to the genetic dissection of simple organisms such as viruses and bacteria, molecfllar cloning was rapidly applied to study the more complex genetics of the mammalian cell. today, virtually any study of cell physiology which depends upon specific gene expression is approachable using the tools of the molecular biologist. therefore, stress induced gene expression (such as the heat shock gene, hsp70) or the regulation of the nitric oxide synthetase gene or the induction of cytokines and growth factors as a result of trauma or injury lend themselves to investigation at the level of the gene. during these workshops, we will discuss the means by which cdnas and genes are isolated, amplified and identified. we will discuss the strategies and methods for cloning cdnas and genomic dnas, for organizing such cloned libraries, for finding specific genes and for characterizing those genes. this speaker will focus on the techniques and considerations involved in the synthesis of cdna libraries. topics include rna isolation, mrna quality, reverse transcription, choice of cloning vectors and library quality. genomic dna library synthesis will also be discussed with respect to the vector/host systems available and applications which are specific to genomic dna. finally, polymerase chain reaction (pcr) will be discussed briefly with regard to its impact on dna cloning. the identification of the genes or gene products that have a role in cellular processes is fundamental to our understanding of genetic control and methodologies to achieve this aim are currently available. all levels of the various signal transduction pathways can b~ molecularly analyzed to define the mechanism by which critical steps in each pathway are achieved. the questions that are unresolved in each area of investigation serve to direct the scientist towards analyses of gene products or gene regulation of the gene itself. thus, selection of the type of library (genomic or cdna) to be used is dependent upon the specific goals of each investigator. to facilitate resolution of this question, an overview of the uses for the different types of libraries will be presented. at least four methodologies are currently available for screening a library, which are dependent upon specific interaction between nucleic acids, nucleic acids and proteins, antibodies and antigens or between different proteins. the use and advantages of each of these methodologies will be discussed along with a description of probe preparation. sequence methodologies required to begin characterization of gene clones will be presented. hopefully, the participants in the workshop will help define areas of emphasis and allow time for directed interaction to discuss specific applications based upon their own experimental systems. alterations of physiological conditions, such as those occurring after shock and sepsis, induce changes in gene expression of cells composing the major organ systems. the challenge is to detect and characterize these changes in geae expression and relate them to the injury. the development of cloning techniques has emerged as the methodology of choice. changes in gene expression are usually characterized by variations in the concentration of cellular messages because synthesis of the final product "the polypoptide" is usually proportional to the concentration of mrna. due to the rapid regulation of gene expression the cellular concentration of messages changes very quickly. this is commonly referred to as steady-state levels, a balance between transcription and degradation. steady-state levels of mrna can be detected in a single cell (in situ hybridization) or in total rna isolated from cells or organs and separated in agarose gels (northern blots). although analysis of mrna steady-state levels are very useful, they do not provide information about the mechanisms that regulate gene expression. changes in gene expression primarily occur at the level of transcription; characterization of the rna species being synthesized at a given time is performed by the nuclear run-off technique. when there is no a priori information about the gene that may be regulated after a particular situation such as sepsis, the total pool of messages present in cells at given time can be characterized by a combination of in vitro translation of the cellular messages and fractionation of the in vitro translated products by two dimensional gel electrophoresis. such approach permits the visualization of the general pattern of gene expression, a "finger print", of the physiologic state. in summary, researchers now have access to a great variety of techniques to identify and characterize genes expressed in response to a physiological condition that may be utilized as "molecular tools" to study the organism's responses to shock and sepsis. the acute phase proteins are a set of plasma proteins with greatly increased plasma concentrations during acute inflammations and after tissue trauma, e.g. after major surgery. the proteins counteract the source of injury, facilitate clearance of infectious particles by phagocytes, assist immune responses and initiate wound healing. the corresponding genes are expressed in liver hepatocytes and regulated by the inflammatory mediators interleukin 1 and interleukin 6 (ill, il6). class1 acute phase genes are mainly controlled by ill and by combinations of ill plus il6; class2 genes mainly by ii,6. the human c-reactive protein(crp), serum amyloid a(saa) and complement c3 genes will be discussed as examples of class1 genes, and rat c~2 macroglobulin as a prototypical class2 gene. both classes of genes use different types of ¢ytokine response elements in their promoter-proximal transcription control regions; class1 genes use the transcription factor nf-il6 or c/ebp as the key factor to mediate cytokine responsiveness; class2 genes use a different, so far unidentified factor to achieve responsiveness to il6. ongoing efforts to purify this factor, called the il6-response factor (il6-rf), from rat liver nuclear protein extracts will be described. the il6-rf apparently mediates the effects of il6 in a broad range of cell types, including b lymphocytes and other hematopoietic cells. it is therefore likely to be of equal general importance for gene regulation by il6 as nf-il6. the importance of transcription factors as potential targets for novel bioactive substances and possibly therapeutic agents will be discussed with the help of several recent examples. new methods for altering the germ lines of mice provide powerful tools for understanding the roles of individual genes in normal and pathological processes, and for reproducing specific genetic mutations that result in congenital disease. three approaches will be discussed. conventional transgeulc mice are frequently constructed using artificial transgenes that express genes from a promoter other than the normal gene promoter. this paradigm is used to produce very high levels of a gene product, for example, with a viral promoter, or to attempt to regulate gene expression using an inducible promoter. alternatively, normal promoter sequences can be hooked to a gene whose expression is lethal to the cell (e.g., diptheria toxin a chain) to ablate all cells expressing that gene. another approach uses an intact gene with its normal regulatory sequences. here, an elevated level of the gene product is delivered from the correct cells in the correct tissues at the correct developmental stages or in response to natural signals. this approach has been refered to as "genetic pharmacology," and provides a precise delivery of the gene product to the target. however, to be most effective, the transgene should contain arl regulatory sequences as welt as the entire genomic segment containing gene coding regions. the introduction of conventional transgenes is limited to roughly 50 kilobases, while genes containing all regulatory sequences frequently stretch over dozens or even hundreds of kilobases. to overcome this problem, procedures have been developed recently to introduce yeast artificial chromosomes (yacs) into mouse embryonic stem (es) cells. yacs can include over 2 mb of human dna, large enough to encompass the largest known human genes. es cells are also useful for targeted gene deletions and mutations. homologous recombination can be targeted to any known gene in es cells. when these modified cells are injected into a mouse blastocyst, they can be incorporated into all tissues of the resulting mouse, including germ cells, so subsequent generations will pass the genetic modification as an inherited trait. assessment of the effects of a null allele on development can provide valuable insights into its normal role. ultimately, these technologies may be adapted to human cells -not for embryonic intervention, but to provide modified stem cells for various tissues (e.g., gut, eye, hematopoietic system) which could then be applied to somatic therapies. with major illness/injury that respirswy faihne fi'equently followed sepsis, tilney el al nse, d the term "soqueutial syste~ failure" for patients with renal failure after solmir of ruptured abdominal aortic ~aeurysms, i then reviewed our experiences after inju~ and oporstion and suggested that multiple, progsesslve or sequential systems or organ failure was a syndrome to be reckoned with in the, 70's. eiseman et al then wrote about "multiple organ failure", especially with liver failure. polk el m found that renmte orgau failure often signalled occult anita-abdominal infection. fry et al ampbasized the role of uncontrolled infection in organ failure, border et ai described metabolic alterations with mof and used the term multiplesystems organ feilmo (msof). other early conltibutots to our knowledge of mof include faist, trunkey and miller, marshall and dimic&, cassone, catlleo, meakins and marshall, (}otis et at., mater and many others. mof or msof were used eommoifly. cerra coined the terms "post-trsumatic septic sfndromc" and "hyper metabolism otgau failure complex", and border et al, used lhe ex3~ression "gut origin seplic states" to illdioate important eurrem aspects of abe gt.'nerlc problem of organ failure and death, other terms include acute organ-system failure, multiple organ injury syndrome, post-traumatic multi-system organ failure nod post-~aumatic organ system infection s~dmmc (fi~sis), i bdieve the latin "maltiple organ failure" is clean, neat and says it all, now there is a proposal for a new set of torminolugios -mods and sirs. will they be helpful in the study and esro of patients? our speakers in this session will review thcsa proposals and the evidence as we strlvo for eoasensns. it has been well demonstrated that the administration of pro-inflammatory cytokines, and especially tumor necrosis factor (tnf) can result in a picture similar to septic shock with secondary multiple organ failure. it is also clear that tissue hypoxia can lead to organ damage. we believe that it is the interaction between the two phenomenons that can lead to mof. on the one hand, the inflammatory response is amplified in the presence of hypoxia. on the other hand, the release of the mediators of sepsis can increase tbe oxygen demand ef the tissues, alter their oxygen extraction and decrease myocardial contractility, and the combination of these elements accounts for the development of septic shock. this hypothesis is supported by two lines of evidence. first, mof is usually preceded by an episode of acute circulatory failure (even though frank circulatory shock may not be evident). second, recent experimental and clinical studies indicated that immunotherapy (especially antibodies to tnf) is particularly effective in the most severe forms of sepsis, associated with circulatory shock. hence, an effective way to prevent mof may be a combination of aggressive cardiovascular management and immunotherapy. development of the multiple organ dysfunction syndrome (mods) in the critically ill follows an heterogeneous group of stimuli including infection, sterile inflammmion, extensive tissue injury, ischemia, intoxication with a variety of substances, and immune system activation. whether the resulting physiologic abnormalities reflect a common pathologic process, or are simply a non-specific manifestation of tissue injury is unknown. moreover, the lack of clearly-defined functional criteria for the characterization of the syndrome on the one hand, and of reliable biochemical markers of its evolution on the other, has made attempts to define its epidemiology and natural history difficult. using a numeric scoring system to quanfimte the clinical severity of mods, we demonstrated that, although mods is more common in patients who have significant infection at the time of icu admission, a much stronger correlation is evident between the severity of mods and the subsequent development of nosocomial icu-acquired infection. furthermore the severity of mods correlates strongly with the severity of the clinical septic response, and with the degree of immunologic compromise evident on delayed type hypersensitivity skin testing. to determine the relative importance of the initial stimulus, and the host response to that stimulus, to the subsequent emergence of mods, we undertook a matched cohort study of 109 critically ill patients admitted to the icu with infection, matched by age, sex, and apache ii score to 109 uninfected controls. organ dysfunction scores were higher in patients admitted with infection, but were also significantly associated with the expression of a septic response at the time of icu admission, independent of the presence of infection. by stepwise regression analysis, the magnitude of the septic response was the most powerful predictor of the severity of mods in both infected and uninfected patients. these data suggest a model of mods in which a stimulus (eg infection) and the host response to that stimulus (eg the septic response) result in a state of altered systemic homeostasis (manifested in this example by immunologic dysfunction). moreover they suggest that the principle determinant of the emergence of mods is the response of the host, rather than the nature of the stimulus, and are consistent with the hypothesis that a stereotypie systemic response to an heterogeneous group of injurious stimuli underlies the pathogenesis of mods. arthur e. baue, m.d, ever since the attorapt 10 build the tower ef babel, as desclibcd in the old testament of the biblc, it has been difficult to got agreement o;x common definitions or language, although mof has served well, there will always he now classifications proposed with good reasons for them, thus mods and sirs are not the final oxpressinns in the lexico~aphic sweepstakes. attcmpts to develop standard animal shock and scpsis models (hemorrhage, endotoxin, foes[ pellets, cecal ligation and puncture, e. colt hlf0sioi0 to evaluate therapy have net been suoca.ssf~. clinical syndromes of mof, sepsis, sepsis syndrome and athers have not been aeacpte.d by all. our problem is that we arc tryittg tu d¢ffino a non-emily. mof or mods or sirs is net a disease or oven a syndrome. it is a series of complications of injury, disease and intensiva care which loads to death for many patients in intensive care units, it is not a fixed entity, but an ewilving picture. we have lumped (ugethet for therapy and definition a number of diseases and problems according to clinical manifestatieos rather than the eause of ihe problem, the search for unified mechanisms has not bean successful. will we benefit oleo from a more precise classification and definition of a non-outjty, a hodgu-podge of human disorders which have charsmeristic.s and manifestations of tissue injury, inflammation and infactian? re.hi clinical trials of potential "magle ballets" suggest that we should go in a different direction--instead of lumpors, we should be splitters, to seek effective therapy, we must fucus on specific disorders such as trauma, specific infections, inflammatory disease and chronic or acute organ damage (copd -renal failure, etc.), i will say more aboet this later ha the me.ethag. thcre is cue potentially important pert of the mods proposal, and that would be do~mcntation of organ dysfunction before fsiiure occurs and before tile need for external supporl dovelope. this could lead to hatter methods of preventing organ failure. preveution is ultimately the only answer to organ failure, m mof, mods and sirs. have wo reached a consensus?--only that we arc dealing wilh a diffl~x:lt probtolr looking for solutlons. acute lung injury (ali) and its most severe form, acute respiratory distress syndrome (ards) characterized by severe hypoxemia, diffuse infiltration of both lungs, a reduction of compliance and a wedgepresure lower than 18 mm hare related to direct or indirect injury. when aliresults from direct injury (toxic agents, lung contusion, pneumonia), the lesions of epithelial barrier and the activation of lung macrophages are the first events leading to the release of inflammatory mediators wh:ch are responsible for alteration of the membranar permeability and for invasion of the alveoli by fluids, proteins and cells. an inflammatory reaction develops, with injury to endothelial cells, adhesion of polymorpbonuclear leucocytes (pmn) and the release in blood stream of intlammatory mediators dispersing the inflammatory reaction to other organs and tissues. the failure of lung function also results into hypoxia in other organs, leading to tissular ischemia, triggering an inflammatory reaction leading to organ dysfunction. frequent complications of direct lung injury are septic pneumonia with endotoxin release, phagocytes activation and cytokine production disseminating inflammation. by this way, direct lung injury can be at the origin of the multiple organ dysfunction syndrome (mods), the frequence of which having been estimated to 40% and is increased when all has occured in patients alreadypresenting an organ dysfunction (immunodepresslon, cancer...). when ali results from redirect or extrathoraclc injury (trauma, infections, hypovolemic shock, acute pancreatitis...), inflammatory mediators and micro-aggregates released in particular organs or tissues reach the lung via the blood and lymph streams, are filtered in the organ or trapped in the lung capillaries, and are responsible for a local inflammatory reactaon (endothelial cell activation, pmn trapping and adhesion, platelet aggregation, release of mediators). sepsis acts by the way of endotoxiu and sequential eytokines release (tnf, ill,...) while trauma with important blood loss acts especially by the way of hypoperfusionreperfusion phenomena leading to a disseminatedinflammatory reaction and to a possible bacterial transloeation when intestinal hypopeffnsion is present. in these conditions of indirect injury, all (or ards) is a iocal early (often the first) manifestation of a general phenomenon of altered membrane permeability and inflammatory reaction, easily and rapidly recognized by its clin~cai signs. the iung is one of the numerous organs participating to the mods, in these conditions, the mortality rate is high, reaching more than 80% when sepsis is present or when at least 3 organs are implicated in mods. by direct or redirect injury, lung is so a target organ for mods and injured lung can be the cause of mods or simply a participant to this syndrome. jorge cervts-navarro main determinants for brain dysfunction are breakdown of the blood-brainbarrier and raise of intracranial pressure (icp), in second place decrease of systemic blood pressure, disorders of blood coagulation and respiratory function. the blood-brain-barrier of cerebral blood vessels can be opened by stroke, cerebral trauma, inflammation, convulsions, acute hypertension and changes in the blood osmolarity. the consequence is brain oedema, increase of lop and decrease of the perfusion pressure. a close correlation between intraventricular pressure and the fate of patients with severe brain injuries has been established. regional cbf values of 16 to 18 ml/100g/min are reflected in isoelectric eeg, and values about t5ml/100g/min, result in loss of somatosensory evoked potentials. for ionic pump failure the threshold has been determined by values of about 10 ml/100g/min and is reflected in massive release of intracellular potassium. in stroke and in brain trauma when the oedematogenous condition is initially localized the tissue pressure variance within and between the hemispheres lead to tentorial and falciform herniations are the common cause of death even before the critical threshold of intracranial pressure is reached. the increase of the icp over the level of the perfusion pressure leads to the arrest of the cbf. if this persists {or periods exceeding 6 seconds of global brain ischemia loss of conciousness and developing seizures appear. if it exceeds 10 min irreversible injuries of the brain appear. following cerebral ischemia and after severe head injury a tendency to increased coagulation can be detected. vascular occlusion may develop either as a result of local thrombus formation or from emboli caused by circulating platelet aggregates. the formation of microthrombi is triggered by the plateletactivating factor a mediator in central nervous injury also responsible for aggravation of posttraumatic brain edema. acute hemorrhagic leucoencephalitis and brain purpura are known to arise as complications of acute viral infections and following gram-negative septicaemia. institute of neuropathology, free university of berlin, hindenburgdamm 30, 12200 berlin, germany undoubtedly, attempts to quantify as objectively as possible the degree of dysfunction of the various organs is very valuable. however, cardiovascular failure has a peculiar place in the context of sepsis. the development of acute circulatory failure (shock) is of paramount importance as a cause rather than as a consequence of organ failure. it is therefore essential to clearly separate patients with and without circulatory failure. once this has been done, attempts to list the cardiovascular system among the other systems have been unconvincing. in a number of studies, cardiovascular complications are assimilated to a low systemic vascular resistance (svr), but since svr is basically the ratio of blood pressure over cardiac output, and since hypotension is already included in the definition of shock, then a low svr is basically equivalent to a high cardiac output, which is a characteristic rather than an ominous complication of severe sepsis. development of a low cardiac output unresponsive to fluids is usually a terminal event. it is not advisable to list other problems such as severe arrhythmias, myocardial infarction or tamponade as complications of severe sepsis. host defense dysfunction following trauma, shock and sepsis e. faist, g. f. babcock * major accidental, burn and operative injury often precipitates a profound multicentric immunologic depression that is believed to predispose patients to become anergic and thus to develop towards a higb probability of infection. anergy or a lack of immunologic reactivity stands for a total elimination of skin test reactivity and recall antigen responses in the inununocompromised patient. anergy following overwhelming trauma or major septic conditions results from a massive impairment of cell mediated immunity (cmi) together with a whole body malignant inflammationiike state. we and others have demonstrated clearly that the skeration of cmi following trauma is mainly due to the disruption of intact monocyte (moo) / t-cell interaction. within this phenomenon we see a shift of the cell ratio in the compartment of peripheral blood mononuclear cells (pbmc) with a considerable increase of prostaglandin e 2 synthesizing m~ and a simultaneous decrease of functionally competent cd3+ and cd4+ lymphocytes. t-cell dysfunction in states of profound stress is characterized by impaired synthesis of two crucial lymphokines -interleukin-2 (il-2) and gamma-interferon (y-ifn). the inability to produce adequate amounts of il-2, most likely attributable to alterations in the transmission of signals from the cell membrane to the nucleus, results in incomplete proliferative t-cell responses to antigenic stimuli, while a lack of ?-ifn results in inadequate m~ antigen processing and presentation. the role of the two functionally distinct t-helper subsets, t-helper-1 (thl) secreting principally il-2 and ?-ifn and t-helper-2 (th2) acting principally in inducing antibody formation with a secretion of il-4, il-5, il-10 and tnf-[3 has still to be established within the context of major trauma. antibody formation to common protein antigens is impaired, the predominant finding is a shift from igm to igg synthesis. although excessive secretion of prninflammatory cytokines (il-u3, tnf-c~, il-6, il-8) has been considered to be deleterious for the host in states of major inflammation and infection, in-vitro and whole blood investigation of me function has clearly revealed that there is initially a marked suppression of moo from septic patients to synthesize and secrete proinflammatory cytokines to endotoxin. this probably will result in immunodeficiency of traumatized as well as septic patients, since these cytokines in low concentrations are involved in the upregulation of the essential humoral and cellular immune functions. abnormalities of pmn function noted after serious injury have been interpreted by some investigators as signs of unresponsiveness of this cell population, others have demonstrated signs of pmn byperactivation. latest findings revealed that there is a clear pmn dysfunction in circulating blood: 70% of all pmn's in this compartment show downregulated or non existent ~-integrins within 72 hours posttrauma -these cells do not phagocytose, have a reduced respiratory burst and appear to be completely degranulated. restoration of these cell functions within 7 days post-trauma is significantly correlated to survival of traumatized patients. other reports however stress increased expression of cr3+ receptors (cdll/cd18 complex) on circulating pmn's, increasing the ability of these cells to adhere to intercellular adhesion molecules, thus contributing to play a major role in inducing the pulmonary capillary leakage. our knowledge about the perturbation of host defenses associated with serious injury and sepsis is continuously increasing and represents the precondition for the design of effective therapeutic measures to prevent and counteract the resistance to invading microorganisms. dept. of surgery, klinikum grosshadern, ludwig-maximilians-university, munich, germany. * dept. of surgery, university of cincinnati, cincinnati, ohio, usa lg thijs~ ce n~ck sepsis is the most common cause of acute disseminated intravascular coagulation (dic) and coagulation disorders as well as abnormalities of fibrinolysis are common in septic patients. some clinical and experimental studies indicate that dic is a pathogenetic factor for the development of multiple organ failure. endotoxin and various mediators (tnf, il-i) enhance tissue factor and decrease expression of thrombomodulin on endothelial cells in vitro. also, tpa activity is suppressed and release of pai-i is stimulated. in such a way the endothelial surface becomes procoagulant whereas fibrinolysis is inhibited. following administration of endotoxin to normal volunteers levels of prothrombin fragments (fi+2) and thrombin-antithrombin (tat) complexes increase, indicating thrombin generation. also the fibrinolytic system is activated as evidenced by a rise of levels of tpa and plasmin-antiplasmin (pap) complexes but this is counteracted by a subsequent release of pai-i. in severely ill septic patients levels of tat complexes are increased and concentrations of the natural inhibitors of coagulation (at iii, protein c) are usually decreased. also, tpa levels snd pap complexes as well as concentrations of pai-i are elevated. the severity of many of these alterations is related to mortality. thus, both coagulation and fibrinolysis are activated in sepsis, but not in a balanced way, thereby promoting coagulation. one of the hallmsxk pathological alterations associated with sepsis is the development of microvascular thrombosis, an entity ~lmre~erizod by microvas~tlar plugbing with leukoeyies (predominately nentrophils) and fibrin deposits. preranably, ischemia remlt~g fibm ve~-'ttlac ow,.luwion contributes sisni.ficantly to end orgen darnaje and consequent dysfatlctlon. p, er~t srldies haw focused pmdominately on the role of adhesion molecules in the pathogenesis of neu~ophil sequestration during infection and s~sis. this preparation will review the mechanisms underlying intravak-'v.ler i~brin deposition and its contribution to organ injury. the normal endothelial cell sure is generally conddered 1o be anticos~am. this state is maintained by two major anticoag~lam molecules on the cell so,ace: hepax'an su,lfale and thmmbomodulin. studies performed over the past decade have do~mentod a general shii~ towards a procoa~qtlant staw during gram negative hffeodon, aft e~| mediated mainly by changes in the endothalial cell surface. antt-tf antibodies have been shown to be pro~ective during l~'ml endotexemia. in summary, ~erova~'~ler fibrin deposition, in ~rt mediated by ~rcgulation of cellular tf, is a consistemt patholo~cal flndin~ during sepsis and contributes to organ injury. strategies designed to abrogate this event may n~nimjze the magnitude of erg~ dysftm~on. n, v, christou md phd, department of surgery, megill university, montreal, quebec, canada interactions between immune calls and the endothelium vie adhesion molecules serve to modulate immune cell traffic between the blood strum and the extrmvascular space, these families of molecules acting in a cascade and closely integrated with the oytoklna network, blood coagulation and the complement system, are responsible for the homing of leukocytes to areas of inflammation and the realrculatlon of tymphocytes. there are three types of immune ceils that must exit the blood stream into the extravascular space: lymphocytes ; polymorphonuclear leukocytes; and non-lymphocyta mononuclaar cells (monooytes, basophlls, aoslncphils). b lymphocytes exit the blood stream and recircu}ate mostly via the high endothelial venule (hev) in lymphatic tissue, rarely, in chronic }nfectlon=, they may recirqulate vie hev in non-lymphoi:t tissue. t-lymphocyte recircutation on the other hand can occur via hev in lymphoid tissue, as well as, the endothalium of the skin, returning to peripheral lymph nodes via the lymphatic channels. lymphocyte recirculation is the mechanism which allows ¢ontaot between the immune repertoire and pathogens, and homing delivers these cells to the appropriate environment for activation and for effeotor function, pmns exit the blood stream through endotbelium in close proximity to the site of inflammation after appropriate endothelial membrane receptor expression. they must reach the site of pathogen tnveslon quickly in order to be effective. their exudation to the inflammatory site can be modulated by their intravascular pdmlng, which results from appropriate receptor expression. because endotoxin leak from the gut has been postulated as an important trigger mechanism for the systemic inflammatory response syndrome seen after serious injury, the immunologic and metabolic effects of bacterial endotoxin infusion into normal volunteers can be expected to shed considerable light on the validity of this hypothesis. we and ethers have used tbis model to show that endotoxin causes a temporary paralysis in the ability of circulating monocytes to prudce the proinflammatory cytokines interleukin-i (il-i) and tnf-a. endotoxin, however, causes increased production of pge2 by the same cell population. endotoxin infusion causes significant suppression of circulating t-cell numbers and a profound suppression in the ability of the circulating t-cell population to proliferate and to produce interle~in-2 (il-2) upon in vitro stimulation. after endotoxin circulating t-cells are also more sensitive to the inhibitory action of exogenous pge2. administration of cyclooxygenase inhibitors at the time of endotoxin infusion largely abrogates the effect of endetexin on t-cell proliferation and il-2 production. endotoxin infusion also causes the release of increased levels of circulating catabolic hormones including cortisol. administration of cortisol alone or cortisol along with endetowin to volunteers has allowed dissection of cortisol effects from those of endotoxin itself. cortisol infusion alone causes a diminution in the circulating t-cell population but the remaining ceils continue to produce il-2 after appropriate stimulation. cortisol infused along with endotoxin blocks the overshoot in monocyte proinflammatory cytokine production seen 24 -48 hours after endetoxin infusion. at present, one may conclude that administration of sublethal doses of bacterial endetoxin to human volusteers produces alterations in cytokime production and tlymphocyte activation which are both similar and dissimilar to those seen following serious injury. hemodynamic support is based first on intravenous fluids and second on vasoactive agents. colloids are usually preferred to crystalloids, for their more rapid hemodynamic effects and their lesser passage in the interstitial space. in the presence of hypotension, the pharmacological profile of dopamine makes it the first agent of choice. it is only when high doses of dopamine are insufficient to restore a sufficient tissue perfusion pressure that norepinephrine should be added. even when cardiac output is not decreased, a dobutamine infusion is often indicated to counteract the myocardial depression, prevent vasoconstriction, and increase oxygen delivery to the tissues. the benefit derived from the administration of "renal" doses of dopamine is doubtful, but stimulation of dopaminergic receptors may increase blood flow also to the gut. in any case, none of these catecholamines has been shown to significantly improve the oxygen extraction capabilities of the tissues. agents with antiinflammatory properties but also with some vasodilating properties, such as n-acetylcysteine, prostaglandin el or pentoxifylline represent more attractive options to increase oxygen extraction. cardiovascular support should aim not only at the restoration of a sufficient arterial pressure but also at the maintenance of an optimal oxygen delivery to the cells. it should thus be guided also by measurements of cardiac output, mixed venous oxygen saturation and oxygen extraction, and indexes of cellular hypoxia, such as blood lactate levels, gastric intramucosal ph or vanearterial pc02 gradients. supranorma[ 02 delivery contributes to the prevention of tissue hypoxia tissue hypoxia is considered to be the common final pathway in the pathogenesis of multipte systems organ failure. it may directly contribute to cellular injury and organ dysfunction as well as enhance further mediator activation and release by a positive feed back mechanism. prevention of tissue hypoxia, is therefore, one of the most important aims in the supportive therapy of critically ill patients, especially in those with sepsis and septic shock. mismatch of cellular o= supply and demand in these patients may resuit from the impairment of the cardiocircu[atory system on all levels. 1. myocardial depression with a drop in 02 delivery (do~) 2. redistribution of blood flow between the different organ systems 3. inadequate nutritive blood flow due to tissue edema and endothelial damage (gic, leucocyte swelling etc.) these pathological changes may go along with increased metabolic needs. cellular and organ 02 supply may therefore be inadequately matched with cellular 02 needs in patients with normal and even supral normal doz. hyperdynamic circulation is often present in adequately volume resusucitated patients. it is most likely that one of the bodys main compensatory mechanisms would maintain cellular o= supply in the face of increased metabolic needs and impaired oz extraction capabillities. this pathophysiology may explain the findings in several clinical investigations that patients with normal or even supranormal doz can have signs of inadequate regional tissue oxygenation. it is also consistent with the results of different studies which demonstrated that the achievement of supranormal doz levels, either spontanuously or due to adequate supportive therapy, resuits in better patient outcome. any attempt to prevent or treat tissue hypoxia in these patients, however, has to take into account the effects of therapy not only on global dgz but especially on regional and microcirculatory blood flow. r. rossaint, d. pappert, k. lewandowski, h. gedach, k. slama, k.j. falke klinik for anaesthesiologie und operative intensivmedizin, ukrv, freie universit §t berlin, germany important contributory factors to the high mortality of severe acute respiratory distress syndrome lards) are the aggressive therapies required, such as mechanical ventilation with high inspiratory oxygen concentrations and airway pressures. as specific therapies for reducing or preventing the general inflammatory reaction of the lung resulting in severe hypoxemia is unknown, today's therapy is limited to procedures which predominantly support or maintain pulmonary function, i.e. pressure limited ventilation with peep and permissive hypercapnea, avoiding or treatment of fluid overloading, and positional maneuvers. extracorporeal lung assist combined with low frequency positive pressure ventilation has been recommended, when conventional therapy fails. today, extracorporeal gas exchange may be carried out using a system internally coated with covalently bound heparin. this allows for a lower level of systemic heparinzation reducing the risk of severe hemorrhagic complications of extracorporeal respiratory support. from april 1989 to august 1993 89 patients, aged from 15 months to 60 years, were transferred to our intensive care unit (icu) for treatment of severe ards. all fulfilled at least the slow entry criteria of the u.s. national ecmo study. due to hypoxemia 13 patients required veno-venous extracorporeal membrane oxygenation (ecmo) immediately after transfer to our icu. the other 76 patients were first treated with pressure controlled mechanical ventilation, permissive hypercapnea, prone position, and dehydration, if necessary. in 23 out of these 76 patients, in which the gas exchange did not improve in the following days, veno-venous ecmo with heparin-coated systems was additionally performed. heparin was given to achieve a partial thrombin time between 40 -55 s and an activated clotting time between 120 -150 s. if surgery was performed during ecmo, heparin infusion was interrupted. no severe bleeding complications related to anticoagulation for the extracorporeal bypass could be observed, however, in some patients, after three weeks of ecmo thrombi in the drainage cannula were observed. a problem of of membrane leakage shortened the life span of the membrane lungs to a mean of about four 4 days. in the conventionally treated group 87% survived and in the additionally with ecmo treated group 58% survived, representing a 75% survival rate in patients whose risk of death is considered more than 50%. on the basis of these experiences, we conclude that the described strategy, including veno-venous ecmo with heparin-coeted systems, may improve the survival in patients suffering from severe ards. abnormally high skeletal muscle po 2 in septic patients and its normalization during recovery: evidence against tissue hypoxia but for impaired oxygen metabolism of skeletal muscle? p. boekstegers, k. werdan department of medicine i, gro6hadern university hospital, munich, germany a pathological oxygen uptake supply dependence was suggested to indicate tissue hypoxia in sepsis-a hypothesis which is discussed controversially. because the detection of reduced oxygen transport to tissue and &tissue hypoxia would have important implications for therapy, skeletal muscle oxygenation was studied in patients with sepsis. intermittent and continuous determination of skeletal muscle po 2 by polarographic needle or catheter probes provided no evidence for skeletal muscle hypoxia even in severe stage of sepsis (boekstegers et al., crit care med 1994) . in contrast, mean skeletal muscle po 2 was found to be abnormally high in patients with sepsis (48 _+ 10,4 mmhg, n=39) compared to patients with limited infection (28, 4 + 4, 7 mmhg, p<0.001, n=16) , to patients with cardiogenic shock (22,3 + 6,2 mmhg, p<0.001, n=ls) and to healthy subjects (33,3 _+ 8,1 mmhg, p<0.001, n=10). furthermore, serial measurements in patients with sepsis showed that skeletal muscle po 2 was higher (44,1 _+ 10,3 mmhg) on days with severe sepsis than on days with intermediate state (30,1 _+ 8 mmhg) and than on days without sepsis (26, 8 _+ 5, i mmhg) . thus, a decrease of abnormally high skeletal muscle po 2 was related to improvement of sepsis. this was also demonstrated by comparing the decrease of abnormally high skeletal muscle po 2 with the decrease of apache ii score or elebute score as well as interleukin-6 serum levels in a separate study (boekstegers et el., shock, in press). in summary, our data provide no evidence for skeletal muscle hypoxia in patients with sepsis but support the assumption &impaired oxygen metabolism in severe sepsis. neutropnlic emigration from the vascular space into the extracellular matrix is important for the response to tissue injury or contamination by providing a large recruitable pool of tissue-based phagocytes. the consequences of neutrophil exposure to intravaseular chemoattractants, likely relevant for il-8 and c5a in sepsis, trauma, and in burn injury, are suppression of neutrophil attachment to endothelial cells and matrix proteins. it is probable that proteoglycan-bound attractants are of considerable biologic relevance, and may result in enhancement of responses to subsequently encountered cytokines. in the presence of connective tissue proteins expressing integrin-specific binding ligands, neutrophils respond to tnf-cq gm-csf and other cytokines by producing large amounts of hydrogen peroxide. this response is likely important in the digestion of contaminated or injured tissue by facilitating activity of neutrnphil granular proteases and inactivating plasma anti-proteases. this matrix-dependent oxidative response is important in defining potential novel targets for therapeutic intervention. the response appears to involve signalling by integrin-linked tyrosine kinases. the net effect of matrix protein injury through this mechanism is enhancement of the fibrotic response which underlies much of the prolonged ventilator dependence of patients with the adult respiratory distress syndrome. this adherence dependent response has also been implicated in direct hepatocyte injury, and may represent an early component of the syndrome of multisystem organ failure. study purpose: in this patient (pat.) population with a considerable sepsis morbidity and mortality, parameters proposed for sepsis assessment (elebute sepsis score [ele], sirs) were investigated with regard to their use in the early classification of pop. pat. at risk for developing sepsis. patients and methods: in a previous observational study on 110 consecutive pat. after elective open-heart surgery, we had determined ele daily for at least 3 pop. days (in pat. with a longer stay: until icu discharge). after publication of the newly proposed sirs definition, these criteria were retrospectively calculated and compared to ele. results: on the total of n =515 patient-days, both ele and sirs showed significant (p<0.0001) correlations with parameters reflecting the general and sepsis-related severity of the pop. clinical course. compared to sirs, the ele correlations were better (icu mortality: 0.59 vs. 0.32; icu treatment days: 0,51 vs. 0.32; days on mechanical ventilation: 0,54 vs. 0.32; days with vasopressor requirement: 0.59 vs. 0.33; number of microbiological findings: 0.44 vs. 0.29), the optimal classification criteria for the mainly sepsis-related outcome gave maximal youden indices of 0.87 for ele (ele >_12 on >_2 days, accuracy: 94%) compared to 0.42 for sirs (sirs present on >3 days, accuracy: 67%). accordingly, ele roc curve areas for both overall (0.92) as well as sepsis-related prognostic evaluation (0.99) were significantly (p<0,05) larger compared to sirs (0.79 and 0.86, resp.), this higher overall accuracy of the ele criterion was primary due to a more valid assessment already on the first and second pop. day, where sirs still had a high false positive classification rate (46% and 53%, compared to 7% and 1%, resp.). conclusion: in the early postoperative course after cardiac surgery, the sirs definition displayed a high false-positive classification rate (low specificity) for subsequent sepsis-related mortality compared to better classification results obtained by the elebute sepsis score. from the departments of medicine i and of "cardiac surgery, grosshadern university hospital, marchioninistr. 15, d-81377 munich, frg. correlation between physiological and immunological parameters in critically ill septic patients. ma rogy, h oldenburg, r trousdale, s coyle, l moldawer, sf lowry a relationship between physiological parameters of severe sepsis and immunological function has not been established. in an effort to assess such a relationship we prospectively evaluated nine severely ill septic patients. physiological risk was assessed by the apache iii score 1, while one component of immunologic function was evaluated by peripheral blood mononuclear cells (pbmc) eytokine production after in vitro lps stimulation 2. four of the nine patients died. apache iii scores at 72 h were lower in survivors (s) than in non-survivors (ns), (51-+13 vs 111-+1 5 p<0.05), while apache iii scores at admission were not significant different between s and ns (82-+13 vs 95-+13). down regulation of cytokine production by pbmc upon lps stimulation was a transient event in s. while s demonstrated an 3 fold increase of tnf-a bioactivity with[r~ 72 hours, ns did not demonstrate any increase at all. a similar pattern was demonstrated for il-1[3 and il-6 immunoactivity. tnf was measured by wehi bioactivity, il-1[~ and il-6 immunoactivity were determined by elisa. the sensitivity was 35 pg/ml for tnf, 30 pg/ml for il-ll3 and 35 pg/ml for il-6, respectively. in conclusion, both physiological as well as immunological functions of severe critically ill septic patients demonstrate predictive value for ultimate survival. while patients biological status seems to be more predictable by apache iii at day 3, p<0.05, the pattern of cytokine production by pbmc upon lps stimulation over the first 72h might be a reliable predictor as well. introduction: therapy of sepsis and its sequelae depends largely on its early recognition. many studies have investigated the change of certain mediators during sepsis and their potential to predict multiple organ failure and outcome. it was the objective of this study to investigate whether the onset of sepsis can be predicted by alterations of levels of interleukin-6 (il-6), tumour-necrosis-factor (tnf), pmn-elastase and c-reactive protein (crp). materials and methods: over a one year period, 40 polytraumatized patients were prospectively studied (mean age 42y, 71% male, iss 28). serum and edta-plasma samples were taken in 12h intervalls until the patient left the icu. il-6, tnf, elastase, and crp were determined immunologically. sepsis was defined according to the criteria of 'systemic sepsis' (veterans" administration study, 1987) with at least 4 of 7 clinical signs: (1) tachycar-dia> 100/min, (2) temperature >38,9°c, (3) blood pressure < 100mmhg, (4) mechanical ventilation, (5) leukocytosis > 15.000 /ml, (6) thrombocytopenia < 100.000/ml and (7) presence of an obvious septic focus. clinical parameters, sepsis severity and serum levels were documented on a daily basis, beginning on day 2 after trauma. results: 21 of 40 patients developed a systemic sepsis (52.5%), and 5 died. all mediator levels were elevated under septic conditions. the clinical severity of sepsis correlated well with the respective levels of mediators. in patients, who developed a sepsis the following day, il-6 (510 vs. 195 ng/l; p=0.029), crp (160 vs. 121 mg/l; p=0.035) and tnf (32 vs. 15 ng/l; p=0.045) were significantly increased as compared to those patients who remained non-septic. elastase levels were considerably elevated but did not reach the level of significance. we conclude that il-6, tnf and crp appear to be sensitive markers for prediction of septic complications in polytraumatized patients. objectives of the study: the assessment of liver function in polytraumatized patients who are at risk of developing mof is too inaccurate and late by using conventional biochemical parameters. methats: the injury severity of the patients (n=26) was determined by the injury severity score (iss). lidocaine is given at a dose of 1 mg/kgbw over 2 rain. i.v. and is metabolized in the liver by a cytochrome p-450 mechanism to monoethylglycinexylidide (megx). the metabolite is measured by a fluorescence polarization immunoassay. serial determinations of the test were performed between the 1 ~t and the 11 ~ day after trauma and were compared with other liver function tests (bilimbin, gldh, alt, ast). the systemic inflammatory response syndrome (sirs) is still a challenge concerning early diagnosis, therapy and prognosis. therefore, evaluation of inflammatory and disease activity becomes more important. c-reactive protein (crp) is a well established acute phase protein in chronic inflammatory diseases. recent reports suggest an induction of crp by interteukin-6 (il-6), a cytokine involved in the mediator cascade of sirs. on the other hand, tumornecmsisfactor alpha (tnfcx) is a very early released mediator in sirs removed very rapidly from circulation. in addition, soluble tnf receptors (stnfr~5, stnfr75) are released into circulation in the acute phase response. this study examines the kinetics of five acute phase proteins (crp, il-6, tnfot, stnfr55 , stnfr75 ) in patients suffering from sirs. eighteen patients entered the study after diagnosis of sirs. blood samples were drawn every six hours during the first two days and every twelve hours thereafter. crp was measured in an routine turbimetric assay. il-6 was detected in an biological assay using the/l-6 dependent 13-cell line 1313/29. detection of tnfc~ was performed in an elisa system using a monoclonal antibody" for tnfo~. soluble tnf receptors were also measured by elisa. crp levels were elevated (>10 mg/l) in all patients and at all time points. crp values did neither differ significantly in patients with (143±33 mg/l) or without (129a:39) multiple organ failure (mof) nor in survivors (133±41) or non-survivors (147:t:44). in contrast, 1l-6 was elevated in patients wilh mof (mean 740 pg/ml, range 0-8900 pg/ml). il-6 levels correlated especially with lung dysfunction. tnf(x levels were consistently elevated in patients with mof. crp, il-6 and tnfoc did not correlate with each other. in contrast, levels for both stnfr showed a positive correlation (r=0.7). patients could be divided into two groups by values for stnfr~5 and stnfr75: the group with higher soluble tnf receptor levels showed increasing values combined with a poor prognosis. the group with lower levels of soluble tnf receptor consisted of patients surviving mof or without mof. in conclusion, crp does not monitor the course of sirs adequately. in contrast, il-6 correlates with mof and episodes of high disease activity. high stnfr levels may indicate poor prognosis. klinik f0r an/isthesiologie and operative intensivmedizin der cau kiel, schwanenweg 21, 24105 kiei, germany. ch. waydhas, md; d. nast-kolb, ivid; m. jochum, phi); l. schweiberer, mi) objective: to evaluate the irfflarranatory response after different types of orthopedic operations and compare them with the systemic effects of accidental trauma of varying severity. patients: in 135 consecutive patients with multiple injuries (iss 40.6) the inflammatory response to trauma was prospectively studied. the patients were divided into 4 groups according to their iss points. additionally, the alterations after 79 secondary operations (>24 hr) were determined (msteosynthesis of the femur (n=28), pelvic girdle (n=ll) and spine (n=8), facial reconstruction (n=13), smaller osteosynthesis (n=13) and others (n=6)). methods: specific and unspecific parameters of the inflammatory response were determined in the trauma patients every 6 h, beginning on admission of the patient to the emergency room for a period of 48 hr, and in the operative patients on the morning of the operation, at the end of the procedure and every 6 hr during the first two days. results: lactate, neutrophil elastase, heart rate, po2/fio2-ratio, and other parameters discriminated significantly between the 4 injury severity groups during the first 48 hr (kruskal-wallis-test, p<.05). the degree of postoperative changes differed significantly (kmskal-wallis-test, p<.05) between the 5 types of operations for lactate, heart rate, po2/fio2-ratio, nitrogen excretion and showed a strong discriminating tendency for neutrophil elastase and c-reactive protein. the extent of changes were highest after operations of the pelvic girdle, followed by procedures on the femur, spine, smaller bones, and the facial region. the postoperative changes after osteosynthesis of the femur or pelvis were comparable to the alterations noticed after smaller (iss 1 to 24) or moderate (iss 25 to 40) accidental trauma for neutrophil elastuse, heart rate, po2/fio2-ratio and parameters of the coagulation system. conclusions: there is a considerable inflammatory response to operative procedures that varies with the type of surgery. large operations cause changes in the body homeostasis that resemble those after multiple injuries. it remains to be established whether the inflammatory sequelae of surgical trauma are additive to the changes caused by accidental trauma. objective of the study: we retrospectively compared characteristics of elderly patients (~65 years) and yeunger patients admitted to a surgical {sicu) and a medical intensive care unit (micu). we further studied the relations between advancing age, chronic disease, sepsis, organ system failure (osf) and mortality in the elderly group. material and methods: during a 1-year period, 538 patients were consecutively admitted into the 8icu; and during a 2-year period, 487 patients were consecutively admitted to t~mich. criteria for chronic disease, sepsis, osfsi.e. cardiovascular (cf), pulmonary (pf), renal (rf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature. results: patients from the sicu and~cu were similar in age, number of osf, and length of stay. however, when compared to sicu patients, micu patients had more cf (p<o.o1), sepsis (p<o.o01), and mortality rate (27% vs. ii~, p<o.ool). in contrast, sicu patients had more hp and nf, and prior chronic disease (all, p<o.o01). of the total group of 1,025 patients, there were 406 (40%) patients 65 years of age or older. elderly and younger patients had comparable length of stay in the icj, but elderly patients had significantly more chronic disease, sepsis, and number of osf (all, p<o.o02). all osfs, except hf and nf, were more ~o~mon i~ mdeljl, eompard to you-ger patients. ~he mortality is also higher in elderly patients (29% vs. 11% in younger patients). after multiple logistic regression, only number of osf increased mortality by a factor of 2.48 (95% confidence limits, 1.97-3.12), age did not increased [odds ratio 1.05 (i.01i.i0)], while admission to the sicu reduced risk of death by a factor of 0.15 (0.08-0.30). conclusions: based on these results, we conclude that age does not have any impact on icu outcome in the elderly. the only prognostic factor is the extent of acute disease, as measured by the number of osf. hence, prevention of oaf may influence outcome in elderly patients with critical illness. the lower risk of death in sicupatients may be explained by the large proportion of postoperative elective patients with relative mild chronic disease, and the lower incidence of sepsis. department of surgery, free university hospital, de boelelaan 1117, 1081 ~v amsterdam, the netherlands. septic shock and low output syndrome h.miyakawa, t.noguchi, s.hattori, a. mizutani, m. mori and n.honda objectives: cytckines are thought to cause the acute phase reactions under several critical conditions. we had investigated the relationships between cytokines network which included il-6,1l-8,acth and cortisol,and the outcome of the patients with septic shock or low output syndrome (los). materials and methods: in the septic group and los group,nine and four patients were enrolled respectively. furthermore, the patients in the septic group were divided into two groups,survival (n=5) and non-survival group (n=4).tn these patients,ll-6,1l-8,acth and cortisol had been measured every day after diagnosis of septic shock or los.the total number of measurements were 12 points in survival septic group, l l points in non-survival septic group and 10 points in los group.statistical analysis was used student's t-test to compare the mean of each group,and the changes were reported as clinicat significant if p<0.05. results: il-8 levels in non-survival septic group were higher than in both survival septic and los group.ll-6 levels in non-survival septic group were more significant than in both survival and los group.otherwise,there were no differences with respect to acth and cortisol among three groups. conclusions: we assumed that los would make several organs dysfunction as well as septic shock. but our results showed septic shock, especially non-survival,had different cytokins network mechanism for organs failure from los. lasson ,~, g/3ransson j, jijnsson k. eady diagnosis of complications after trauma is imperative to decrease morbidity and mortality. for this purpose an easy, cheap and fast diagnostic method is needed. the aim of this study was to analyse if complications after surgical trauma of various extent could be diagnosed earlier using preoperative or daily postoperative measurements of various plasma proteins than by using climcal signs of complications. material and methods: two acute phase proteins (c-reactive protein and antichymotrypsin), lbur main plasma protease inhibitors (alpha-2-maeroglobulin, c 1estemse inhibitor, antithrombin ill and alpha-2-antiplasmin) and one collagen precursor (procollagen iii peptide) were measured preoperatively and then once dally for 7 days postoperatively. for the plasma protease inhibitors immunorcactive as well as functional levels were measured. haemoglbin, albumin and the white blood cell count were also measured. measurements were done preoperatively in 256 patients and continued postoperatively in 78 patients, 70 % of the patients were operated on for malignancy. resulls: preoperative plasma c-reactive protein levels were higher in patients developing postoperative complications. this discrimination increased when plasma was sequentially analysed postoperatively, when a remaining high level ora second increase in plasma levels depicted a complication 1-3 days earlier than clinical signs. high levels were seen both in patients developing local as well as in patients developing general complications. there was no clear correlation between plasma protease inhibitory levels and complications, neither pre-nor postoperatively. procollagen iii paptide levels were slightly (but not significantly) higher postoperatively in patients developing complications. contusions: the acute phase response, especially concerning c-reactive protein, predicts postoperative complications better than both plasma protease inhibitors or collagen precursors. preoperative plasma c-reactive protein levels indicate the risk of postoperative complications, while daily postoperative measurements more readily depicts a complication, usually preceeding the clinical signs of complication by 1-3 days. dept. of surgery m'alm6 general hospital, s-2 i4 01 malmr, sweden. mof is a major threat to the extensive burned patients and associated with a high mortality rate. the role of endotoxemia in the pathogenesis of mof in burned patients remains controversial. in this study, we examined the relationship of plasma endotoxin levels to development of mof, and outcome in patients with thermal injury. methods: a prospective cohort study of 17 patients admitted with burns covering more than 70 per cent of body surface area was undertaken. circulating endotoxin concentrations were measured by modified limulus amebocyte lysate assay in serial samples of plasma. results: 7 out of 17 burned patients developed mof according to multiple criteria. the plasma endotoxin concentrations of patients with mof were 0.512--1.127 eu/ml, significantly higher than that of 10 patients without mof (0.216-0.553 eu/ml) on 3, 14, 21 and 28 days postburn (p< 0.05--0.01). significantly more incidence of positive endotoxin test (>_ 0.120 eu/ml) was found in patients who developed mof as compared to that of non-mof during the observation period (p<0.05). as the mean endotoxin levels increased, the prevalence of mof and death also increased (see table below), persistent endotoxemia carried a poor prognosis. conclusions: the present investigation provide further evidence that endotoxemia in severely burned patients commonly occur. cimulating endotoxin has also been found to be strongly associated with development of mof and mortality following major burn injury. multiple hemostatic changes occur in sepsis mad multiple organ failure (mof). to evaluate the role of platelcts in patients with sepsis and mof, we examined changes in surface glyeoproteins on circulating platelets of t4 patients with suspected sepsis and mof. the severity of sepsis and mof was assessed by eiebute and apache 1i scoring system, respectively.using flow cytometric techniques and platelets specific monoclonal antibodies, platelet surface expression of fibrinogen receptor on gpiib-iiia, ofvon willebrand receptor gpib, and of granula glycoproteins (thrombospondin, gmp-140, and gp53) was measured. receptor density of gpiib-illa mad gpib on circulating platelets was not affected by sepsis or mof. in septic patients surface expression of activated fibrinogen receptor (libs1 expression) was significantly elevated (p<0.05) and correlated well with severity of disease (f0.597). no significant change in surface expression ofthrombospondin, gmp-140 or gp53 was noted in septic patients. in contrast, degranulation ofgraanle glycoproteins was significantly elevated in mof (!0<0.05) that correlated well with severity of mof (gmp-140, r=0.611; thrombospondin, r=0.643).we speculate, that platelets in sepsis circulate in a hyperaggregable (fibrinogen receptor activation ) but still reversible state that results in increased risk of microthrombotic events. in the course of the disease, irreversible platelet degranulation might occur and may play an important role in development of mof. abdominal sepsis is still associated with high morbidity and mortality. the present study aimed at evaluating patients with abdominal sepsis treated at our surgical intensive care unit during a 10-year period with the aim of identifying potential prognostic factors, bacteriological cultures, diagnostic procedures, treatment and outcome. during the period 1983-1992 i66 patients with abdominal sepsis were treated at the icu at our university hospital. 60 patients were women and 106 men with a mean age of 64 (17-101) years. in 74 cases, the abdominal sepsis occurred as a postoperative complication. the patients were scored according to apache ii and bacteriological cultures and the occurrence of organ failure were noted. the patients were hospitalized in median for 40 (-273) days out of which 7 (-178) in the intensive care unit. 46 out of 166 patients (28 %) died in median after 18 (1-194) days. the primary cause of mortality was multiple organ failure (38/46; 83 %). apache ii scoring could not predict a fatal outcome. abdominal bacterial cultures were dominated by bacteria of enteric origin (83 %) and in 60 % cultures grew multiple bacteria. 134 patients bad organ failure and 64 multiple organ failure. 29/166 patients (17 %) had abdominal sepsis due to diffuse peritonitis despite a morphologically intact gastrointestinal tract and the absence of localized abscess formation. mortality in this group was significantly higher as was the percentage of positive blood cultures and the occurrence of multiple organ failure. abdominal sepsis is still associated with a high mortality, predominantly caused by multiple organ failure. abdominal culture findings are dominated by bacteria of enteric origin. in about 1/5 of patients with severe abdominal sepsis a diffuse peritonitis with intact gastrointestinal tract without localized abscess formation was found. in this group the mortality was increased as well as the risk of developing multiple organ failure. during the period from january 1985 to september 1993 56 patients, mean age 24+4 years were referred to our department of resuscitologywith the diagnosis of eclampsia. all the patients were delivered by cesarian section and were mechanically ventilated for 7.3_+5.7 days. diagnosis of sepsis was confirmed in 6 cases by clinical and microbiological methods. patients were divided in two groups: lnon septic patients, 2-patients with sepsis, the control group consisted of 20 patients after cesarian section without symptoms of eclampsia or infection. we determined plasma concentrations of immunoglobulins a,g,m(a,g,m), complement factors (c3,c4), alphal-antitrypsin (aat), trausferrin (trf) and albumin (alb) using beckman (usa) analyzer,protein concentration, using kone (finland) analyzer. a(mg/dl) g(mg/dl) m(mg/dl) c3(mg/dl) c4(mg/dl) k 203+-31 971+47 205_+21 134+12 30+-3 1 154-+29" 568-+48* 142_+24" 81-+14' 17_+4" 2 102+_21'* 420-+30** 96-+21"* 67-+10"* 11_+3" in a prospective study we investigated serum of 12 severly traumatized patients withdrawn directly after admission at our hospital (tr i). follow up controls were taken daily until day ten after trauma (tr ii). two control groups were performed: serum of healthy volunteers (co, n =24) was investigated as. well as serum of patients undergoing elective herniotomy (n=12) 24 hours before (op i) and 24 hours after operation (op ii). serum bactericidal index (sbi) was determined using a hemolytic e.coli strain 08:k87:h19. 200/11 suspension with a final concentration of 250 -400 cfu were incubated with l oopl serum. after overnight incubation sbi was calculated according a special formula. results: co 74.8 _+ 6.3 opi 77.8 _+ 6.4 opii 68.2 _+ 7.7* tri 38.4 _+ 9.1"* trii 57.6 + 8.9** (*:p< 0.05; **:p<o.01) sbi represents a simple and reproducible method to detect immunobiological alterations after trauma and elective surgery. patients undergoing elective surgery showed slight but significant diminuation of sbi 24h after operation. deterioration of sbi was observed in serum of traumatized patients directly withdrawn after admission at our hospital. ten days after trauma significant improvement of sbi was found. further investigations have to be done to proof sensitivity and prospectivity of this simple but reliable test. a sepsis like syndrome (sls) occurs in i0 % of our patients following cardiac surgery. sls is characterized by a severe decrease in systemic vascular resistance requiring the use of vasopressors to maintain adequate hemodynamics in the presence of negative blood cultures. in order to determine whether preoperative factors predict the occurrence sls we retrospectively studied 62 patients with sls (group i: age 61.1 ~ 10.8 years); they were compared to 52 control patients (group 2: age 58 ± 14.3 years). treatment of sls consisted of aggressive fluid replacement and norepinephrine infusion. rydrocortisone (200 mg/24 hrs) was given on the first day. antibiotics were switched prophylactic to cover gramnegative bacteria in 48 pts. preoperatively pts in group 1 revealed a significantly lower cardiac index (2.5 ± 1 i/min/m 2) compared to group 2 (3.1 ± 1.2 i/min/m2; p < .02) higher left atrial pressure (group i: 17.8 ~ 8.1 mmhg; group 2: 13.1 ~ 6.1 mmhg; p < .005) and lower ejection fraction (group i: 57.9 z 16.3 %; group 2 65.2 ± 10.9 %; p < .03). bypass time, minimum and mean blood pressure on bypass and aortic clamp time were not different between the groups. immediately postoperatively (pop), the white blood count was elevated (group i: 15,700 ± 8,200/ui; group 2: 10,200 ~ 4,400/ui; p < .0005) and core temperature 12 hours pop was higher (group i: 38.3 7.6 °c; group 2: 37.6 ~ 8.4 °c; p < .004). serum lactate was already elevated on arrival on the icu pop (group i: 5,0 z 1.9 mmol/l, group 2: 2.2 ~ 0.9 mmol/l) but dropped within 24 hours (p < .0005). icu stay (group i: 3.7 ! 4.3; group 2: 1.6 ± 1.9 days; p < 0.001) and mortality over 3 months (group i: 19.1 %; group 2: 3.1 %; p < .005) were significant higher in group i. sls is observed more frequently in patients with preoperative cardiac congestion and results in longer icu stay and higher mortality. although intraoperative hemodynamics are similar in both groups, the white blood count and serum lactate levels suggest an intraoperative cause for sls. further investigations will focus on possible intraoperative causes of sls. the objectives of the study were to estimate the problem of stress ulcers in critically ill patients and to compare ranitidine and omeprazole to determine their efficacy in the prophylaxis of stress ulcers. 33 patients who were admitted to the intensive care units between january and july '92 with polytrauma, sepsis or those requiring ventilatory support for more than 48 hours were selected for the study. the patients were randomized into 3 groups to receive ranitidine, omeprazole or a placebo. the changes in gastric ph were monitored 4 hourly and the patients underwent an upper gi endoscopy 48 hours after the start of the study to look for stress ulcers and hemorrhage. the drugs were withdrawn one week after the start of the study. of the 33 patients selected for the study, 14 had undergone cardiac surgery, 6 had polytrauma and 13 had sepsis. the change in average pre and post drug gastric ph was +1.1 in the omeprazole group, +0.5 in the ranitidine group and -).9 in the placebo group (p < 0.05). endoscopic evidence of stress ulcers was seen in 62% of patients not on prophylaxis, 18.1% of those on omeprazole and 54.5% of those on ranitidine. while all the patients in the placebo group who had stress ulcers showed endoscopic evidence of hemorrhage, 82% of the patients with ulcers in the ranitidine group and none of the patients with ulcers in the omeprazole group showed hemorrhage (p < 0.01). in critically ill patients the incidence of stress ulceration is high. an alkaline gastric ph is protective against stress ulcers. omeprazole when used for prophylaxis against stress ulcers in critically ill patients or those on short term ventilation will reduce the incidence of formation and hemorrhage from stress ulcers. the early and accurate diagnosis of sepsis is of key importance for critically ill patients survival. many studies have shown that tumor necrosis factor ~x (tnf ~) and interleukin-6 (il-6) are mediators of the inflammatory response in sepsis. bacterial antigens interact also with antigen specific t cell receptors and the activation of t ceils takes place. activated t cells release soluble interteuldn-2 receptors (sil-2r). the aim of this study was to evaluate: the significance oftnf c~, il-6 and sll-2r in the diagnosis of bacterial sepsis and to evaluate the correlation of clinical and laboratory parameters with serum levels of tnf ~x, il-6 and sll-2r. we examined 19 patients with clinical signs of sepsis treated in the intensive care units of university medical centre ljubljana. venous blood was drawn from each patient within 24 hours after the first clinical signs of sepsis. clinical data (body temperature, blood pressure), laboratory dam (peripheral white blood cell count with differential, platelet count, c-reactive protein), and quantitative blood cultures were recorded. serum levels of tnf c~, 11,-6 and sil-2r were measured in 19 septic patients and in 20 adult healthy controls. tnf ct, il-6 and sil-2r serum levels were measured by commercially available ria and elisa (amersharn and eurogenetics) kits. the differences in serttm levels of tnf ~, il-6 and sil-2r between healthy control and patients and correlation between clinical and laboratory parameters were calculated. we found that only sll-2r serum levels were significantly (p<0.005, student t test) increased in patients in comparison with healthy controls. of all indicators of sepsis that we compared, only hypotension was significantly correlated with tnf ~x levels and leukocytosis with ]1,-6 levels. we conclude that only the increased serum sil-2r levels were predictive of bacterial sepsis within 24 hours after the first septic signs. tnf c~ and il-6 levels were not significantly increased at that time in lifts small group of patients. in order to create high precision prognostic system for patients with sepsis and septic shock we have made prospective and retrospective analis of 200 cases of sepsis. patients were included in this analis according bone's criterions of sepsis. comparing with apache-h, we have excluded such phisiologic variables as:temperature,na+ ,k+ ,}it, ph,which had a little influence on prognos of illness. we have added the other, more important variables: biilirubin, plateles,pti. we took into account: the seat arrangement, methods of respiratory support, comorbid conditions as: caneer, diabets,obersity. in general new score has 5 headings: -physiologic variables, -age, -seat arrangement, -chronic comorbid conditions, -methods of respiratory support. roc -analis have showed a greater precision of our score, compared with apache-ii score. there is significant difference between the roc-curve of apache-ii scare. the k.p.tit~v.,i.e.gurmanchuk. objectives of the study. sepsis is a severe complication of the local infection, th e fever and the systemic inflammatory response syndrome may take place in such a case as manifistations of the inflammation induced by bacteria. the systemic fever observed in infection is known to develop due to the action of certain cytoldnes. other systemic manffistation of inflammation are the production of acute phase reactants, acute phase proteins, the activation of the complement system. ivratertals and methods. we observed 35 children with light and 45 children with severe course(l-3 class of serionuess) of the sepsis. the ftmetional activity of the complement system (ch50, level of c 1-c5, factors b told d, c3a), level of c-reactive protein (crp} and tumor necrosis factor (tnf) were investigated, results. the level of crp was increased in the children with more severe class of the septic process in both group (60%-1 st and ioo%-2 nd}( with light and hard eours of disese) of patients. parameters of the complement system -the activity of cl, c4, c3 components, activity of b and d factors-were increased in the seram of children with light course of sepsis. in children with severe course of sepsis these parameters were signffieantty decreased, especially in the group of chiidrellwith higer level of crp. we found the negative correlation of the tnp-alfa concentration with the functional activity of classical pathway components (cl~3) and the positive onewith the funfional activity of the alternative pathway factors b-and d of the complement system. conclusions. thus, in children with different course of sepsis certain changes in levels and functional activity of an acute phase o[ inflammation proteins -crp and the complement system ones -&ire characteristic. the relationship between the tnf-alfa level and the activity of classical and alternative pathways proteins indicates on its role in the complement proteins genes expression. schr6der j, braun j, rennekampff ha, schr6der dw various alterations of the immune response are known in trauma, but the course will not be complicated by multiple organ dysfunction syndrome (mods) in all patients. phenotyping of cells offers a rapid system of evaluation certain aspects of the immune response. different subsets of lymphocytes and neutrophils were determined to evaluate their prognostic role in developement of mods. in 16 trauma patients with an injury severity score (iss) > 20 (mean iss = 32; mean age 41 years) lymphocyte and neutrophil phenotypes cd 3 (t-cells), cd 4 (t-helper cells), cd 8 (t-suppressor cells), ratio cd 4/cd 8, cd 11b (receptor for cr 3) and cd 16 (fcriii) were measured on day 1,2,3,5,7 and 10 post trauma. the expression of class ii histocompatibility antigen (hladr) on monocytes (hladr+ cd 14) and il 2-receptors on t-helper cells (cd 25/cd 41 were determined as well. the percentage of cells was monitored by immunofluorescence using monoclonal antibodies and three color cytometry. the percentage of hladr+ cd 14 were significantly lower an day 2,5,7 and 10 in patients who developed mods (p<0,05) compared to patients without mods and a healthy control (p<o,o01). the expression of il 2receptors on cd 4 was significantly different only on day 2. no differences could be measured in lymphocyte phenotypes cd 3, 4, 8 and cd 4/cd 8-ratio as well as in expression of cd 11b and cd 16 on neutrophils. class ii histocompatibility antigen (hladr) on monocytes offers the best ability to reflect cellular immunosuppression in trauma. this parameter allows the best differentiation of patients with and without mods. we retrospectively studied the relative importance of age, prior chronic disease, sepsis and organ system failure (osf) to determine outcome in critically ill patients with acute renal failure (arf). arf was defined as serum creatinine > 280/zmol/i, a twofold creatinine rise in prior renal insufficiency or the need of acute renal replacement therapy. definitions for prior chronic disease and other osfs -i.e. cardiovascular (cf), pulmonary (pf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature and described previously. of the 1025 consecutively admitted patients to a surgical and a medical intensive care unit during 1 -ye0r period, 136 (13%) had arf. arf mortality was 52%. ninety-eight percent had other osf. overall, cf, pf, gf, and nf was significantly more common in nonsurvivors than in survivors (all, p<o,02). although both the number of osf before and after arf was higher in nonsurvivors than in survivors (p<0.02), the number of osf before was higher compared to after arf in both groups of patients (p<0.001). furthermore, age, cf and pf before the ontset of arf, nf thereafter, and renal replacement therapy were related to mortality (all, p<0.03). however, prior chronic disease was not related to mortality and chronic renal insufficiency was inversely related to outcome (p<0.002). after multiple logistic regression, advancing age, cardiopulrnonary failure and sepsis before arf, and nf after arf remained significant. these results show the high prevalence of arf in critically ill patients and that arf rarely occurs alone. the prognosis of arf in critically ill patients is poor, especially if associated with advancing age, additional osf, particularly cardiopulmonary failure and sepsis before arf, and nf after arf. hence, prevention of cardiopulmonary and sepsis before the outset of arf may influence outcome in arf in patients with critical illness. the search cominnes for a magic bullet ta help critically ill petienta, bat recent elini ',.ml 1rials of agents that showed ixomise in animal studies raise questions about such trials. expcrlmemai uvidan~ :in anlmats and human volanteet~ for concepts, mechanisms and treatment of inju~ ur illness can be substentlal, l~rauasive and exciting but the positive effects of potential therapy suggested by such animal and ctinieal research may be difficult to prove in sick patients. efficacy of a new txeatment can be difficult to prove became elinie.al situations, hi spi~e of important biologic phenomena, are v~iable and conrplex. there is redundancy and overlap of mediators ~d problems of timing of therapy. a majt~r cause of this dilemtns is not with the trials or hypotheses, but rather the promem of the cause or the causability of the human dise~.se that is trebled. this is/hu "causa nets" or '*specific cause" as described by stehbens, when prevention or tre.atmcnt is based em the sauna nero, extinetiem of the disease is ix~ssible as with saul/ pox. only elimination of the cause wl12 cure the disease. careful animal studit~s evaluate single pcrturbmiuns for survival or other changes. an ldso preparation may be infiuancad aignificantly by a ~mall clangs which may be insignific~lt ht pstiants, most human experiments ate carried out in normal volunleers with a single porlurbation, such as a low-level, non-lethal dose of a substance. such studies are huportant in defining mechanisms attd mediators of disease, in our world of injm'ed, operated, infected and inflslned patients, there are many diseases, mof is not one of lhem. it isn't even a syndrume, it is the final pathway for a number of diseaaes~ each of which has a cause. mecormock believes thal a "multi-causal" etiology is a euphemism for ignoranc# or a synonym fo~ unknown etiology. admission iv sn icu, a high apache seer% the sepsis syndrome, mof, muds, or sirs and having predictors indicating potential mortality, are not diseases. the inmpors are getting negative results in trials of agents on eiek or septic icu patients and aru now becoming splitters, dafining subgaoups at higher risk for death. such an approach may produce positive results if the diseases are identified and the treatmeul is specific for them, a major challenge in deterraintng the efficacy of new therapies for sepsis nnd shock is identifying patients whose e#temtc inflammatory response is appropriate from those in whom the mediator| are contributing to end organ system damage end death, traditional and recently revised categorical patient descriptions such as sepsis syndrome, sepsis syndrome with shock, multiple organ system failure, or the recently proposed systemic inflammatory response syndrome (sirs) may not be sufilcient since they identify individuals at varying levels of risk for short term mortality, the efficacy of new lmmunotherapy may be directly related to the patient's severity end risk of mortality at entry to the study. we recently reported (jama 1993;270:1z33-1~41) a comprehensive risk assessment approach for patients with sepsis syndrome, a common eategarlcal description used as an entry eriterlon for many trials. by screening s database of 58,737 recent admissions to 107 hospitals in the united states and western europe, we identified 1,195 patients who met sepsis syndrome criteria but were not enrolled tn clinical trinh, we then developed a survival model end severlty assessment method to estimate their 2g.day mortality risk. k..ente physiologic abnormalities were the most important prognostic factors (82% of total chl square) influencing outcome. the specific disease resulting in ice admission and the days the patient spent in the hospital and icu before qualification were independent risks, as were age and a clinical history of cirrhosis. the predictive model was cross-validated within the original 1,195 patients with a receiver d_perator characteristics (roc) area of 0.76 and in two independent databases, the first independent database contained 295 hospitalized patients with gram-negative infection meeting criteria for sepsis syndrome (roc=0.80); in the second, the 302 placebo patients withtn the recently completed phase ill e~aluatlan of il-1ra (rocffi0.76). in all databases the risk model was able to accurately risk stratify patients throughout the range of risks that varled from a very low 1% to a very hlglt 99%. by drawing on the ready avatlabillty of large clinically aeuurate, current databases and using the power of modern statistical techniques to model the bodfs response to sepsis, we are able to produce very aeettrate pre-treatment risk estimates, these prospectively defined and continuous risk estimates reduce reliance on multiple subgroups and data dredging that can compromise a stady's integrity. they cart provide a nnlform method for patient description across clinical trials of different attempts at immmlotherapy. risk calculations can also be useful in developing entry criteria for phase ii evaluations in order to initially test efficacy on a limited number of high risk patients, fonowing sueeessinl completion of a definitive trial, the risk estimates can be used to develop specific |ndlcattons for s drug's use and can monitor the impact of both new and multiple compounds on patient outrome, when dealing with therapeutic trials for the treatment of sepsis, there are 2 ways of checking the comparability of the groups (placebo and treated groups) -to use several description items such as age, sex, preexisting disease, number and nature of organ failures, physiology score, such as saps il (1) -to use a model for risk of death either from a score (apache ii, lii, (2) saps ii) or directly (mpm ii system (3)) before using the model for risk of death in septic patients, is it mandatory to validate it (by calibration, i.e. goodness of fit, and discrin'dnation, i.e., roc curves) first on a data base using the same definition for sepsis as in the trial and secondly on the placebo group of the trial ? or is it better to use a specific model for each trial ? the general models usually do uot fit for septic patients. there are 2 methods to make them fit : either to add variables to the original score (model for sepsis using the apache iii system (4)) or to customize the model. for saps ii the customization is applied to the equation regression, using the same saps ii score. for mpm 1i each component of the model can be customized. several remarks : only one model is published to be applied at the icu entry ( mpm ii 0) before any therapy. 2 -most of the published scores are calculated from the lsl icu day. applying these scores to the let day of sepsis could have a very different significance. 3 -is the accp classification of sepsis useful at the bedside to select oatients ? ( the pathophysiology of sepsis involves a dynamic interaction between the host and the infecting microorganism(s). a multitude of biochemical and hemodynamic interactions occur which function in concert to determine the ultimate outcome of the septic episode. the complexities of the septic process preclude outcome analysis by in vitro systems or computer models, animal studies have become indispensable in evaluating new therapeutic agents in the treatment of sepsis. these studies provide valuable information on safety, pharmacokinetics, relative efficacy and dosing strategies for potential therapeutic agents in the treatment of sepsis. there are three basic types of animal models: (1) toxicity models with injection of bacterial endotoxin or exotoxins; (2) live or killed bacterial challenge models; (3) actual infection models. all models employ some form of external manipulation under controlled conditions to induce sepsis and to analyze potential therapeutic efficacy. these experimental requirements may limit the ability of animal models to accurately predict the clinical value of new agents in human sepsis. four major end points are analyzed in the various animal models: (1) hemodynamic parameters; (2) modulation of host-derived inflammatory mediators; (3) resolution of end organ injury; or (4) lethality. each animal model has certain advantages and limitations. species-specific differences in physiologic and immunologic features make it difficult to directly extrapolate the results of animal experiments into clinical medicine. while no ideal animal model exists, consistent benefit of a new immunotherapeutic agent in multiple animal systems provides the most compelling preclinical evidence of its therapeutic potential in septic patients. consensus-assisted development of a study protocol on sepsis: an important difference from previous randomized trials there have been several, and perhaps too many metaanalyses of cliuical trials on surgical infections, but their results have only rarely been incorporated into the design of new randomized trials in sepsis. metaanalysis is retrospective. it seems more important to analyse and to criticize the design of tria/s before they are ongoing. incorporation of that into development of a study protocol is the aim of the following procedure: the time-schedule of about two years should include further cell culture and animal experiments after the first successful studies showing effectiveness. exhaustive evidence for a dose-dependent cost-banefit and near complete explanations of the pathomechanism should not be awaited for development of the protocol of the clinical study. however, a discussion forum of experts should be performed in a recently published, almost perfect trial in order to see the frontiers of the present research in cell and molecular biology, clinical pharmacology, statistics and decision making. this should be followed by a metaanalysis of at least 10 study designs on sepsis with methods of formalized medical decision making (decision tree). clinical algorithms -developed by objective methods including nominal group processes -should be developed to standardize any concomitant treatment in the centers considered for a multinentre trial. the latter is usually necessary in the field of sepsis. finally, a pilot study should be performed to demonstrate not only feasability, but to learn about all possible types of interventions which modify endpoints as response variables. especially mortality is not free from such effects. the time for the individual phases of this consensus-assisted process of protocol development have to be shortened by their temporal hiterloeldug. due to the fast discovery of new chemical factors in sirs it wilt otherwise be impossible to come up with results of randomized trials which are fascinating enough to be incorporated in daily routine treatment. severity scoring systems are intended to provide a population-based estimate for the risk of an unwanted outcome resulting from some disease process. in the area of infections and the "septic" response, this has routinely been defined as death. for anti-infective trials, there has been a reasonable correlation of severity (apache ll) with outcome measured as persistent or recurrent infection, but this is not as robust as the association with mortality. it is expected that non-lethal failure would not immediately correlate with disturbed physiology: the basis for antiinfective failure depends on a variety of factors not measured in severity scoring, such as type of infecting organisms, density and susceptibility to administered antimicrobia[s, and anatomic features of infection (e.g., pancreatic). severity scoring is important in differentiating these "categorical" variables from continuous (physiologic) variables. there are, additionally, a series of problems related to the cause of death in relation to severity scoring. in those tdals in which cause of death is analyzed, high severity scores did not routinely predict death as an immediate sequelae of sepsis/septic shock. many of the predicted deaths occurred remotely of progressive background diseases. other important problems with severity scoring have to do with lead-time bias: hew long patients were ill before entry into the study. statistical techniques for condensing various categorical and continuous variables, as well as factoring in information requiring the pharmacodynamics of agents which affect outcome, are being developed to further refine deviation from predictive equations as an outcome measure. stepwise logistic regression analysis has identified the presence of shock, intm-abdominal or unknown source of infection, hospital acquired infection, age greater than 70 years, neutropenia and inappropriate antimicmbial therapy as independent variables associated with increased mortality in sepsis. maldistribution or" these variables is a concern in targe randomized clinical studies. it should be noted that only the selection of antimicrobiai therapy could be addre.~'sed and altered at the time of enrollment in a sepsis clinics] trial. inappropriate antimicrobial therapy has been noted in 41%-66% of septic patients. well designed, randomized sepsis trials do not assure the absence of imbalance between treatment and control groups with regard to inappropriate antibiotic therapy, especially with subgroup analysis. statistical manipulation to correct for any imbalance is appropriate but avoiding imbalance is ideal. selection of antibiotics in septic patients requires such considerations as clinical setting (history, physical examination and underlying disease), appropriate laboratory tests (such as gram stain), identification of source and associated bacterial flora, antibiotic susceptibility patterns for that hospital and likelihood of resistant organisms. determination of inappropriate antibiotic therapy is made retrospectively after culture results are available. in some circunmtances the organism and antibiotic sensitivities cannot be predicted, while in others it can. leibovici et al identified hospital acquired infection, antibiotic treatment before a bacteremic episode, endotracheal intubetion and thermal trauma as independent variables which predicted isolation of a multiresistant organism. the same authors developed a predictive index for resistant organisms, which when compared to prescriptions of attending physicians, improved antibiotic selection in critically ill patients. including a standard antibiotic regimen for all septic patients in a clinical trial is impractical. there is too much interhopsital variability in flora and antibiotic susceptibility. in addition, the aggressive broad spectrum coverage necessary for some patients is inappropriate for others. a protocol which guides a prescribing physician through a logical chain of reasoning might improve antibiotic selection in critically ill patients and could be included in the design of a clinical trial in sepsis. although this approach might minimize potential for maldistribution of inappropriate antimierobial therapy, it would likely create multiple problem areas. will the patient's physicians agree to the protocol choice? what is the availability of speciftc antibiotics at a participating hospital? what is the aplpiieability of study results in typical clinical situations? if there is agreement that thin is an appropriate antibiotic protocol, should it not be used in all seriously ill septic patients in the hospital? in other conditions such as ards, the success of protocol therapy is dependent upon measurable goals such as pao 2 and highest plateau pressure, while measurable goals are not as clear in choosing antibiotics. based on the above confounding issues, a complex and extensive algorithm covering many potential possibilities of error and specific antibiotics seems impractical; however, an algorithm focusing on three to four major common errors in antibiotic selection and dealing with classes of antibiotics is plausible for inclusion in sepsis clinical trials. in this session we will attempt to outline the methodology of such a future study, emphasizing the immense difficulties involved in its proper conduction including: design, selection of patients, surgical considerations, supportive treatment, the "human factor" and ~nd points. only a close cooperation between a few dedicated surgeons, obsessively studying a large number of well selected and stratified patients over years, could make such a study possible. algorithmis have frequently been mistaken for the graphic format in which they are presented. however, an algorithm is neither a flow chart nor a list of instructions; it is a step-by-step approach to solving a problem. likewise, a clinical algorithm is a step-by-step approach to solving a clinical problem. cas are deterministic, which does not mean that they are rigid. they are practical rather than mathematical algorithms, that tan be used only with clinical judgment, and must be tailored to each patient. there are a number of types of, or uses for, cas that can improve sepsis management, including: diagnostic or severity scorm cas, a management ca for managing sepsis constructed according to recently published standards for use in teaching research protocol algorithms that must be used to collect data or guide implementation of a clinical trial aimed at validating one or more dicisions in the management ca; finally, protocol-style cas drawn from the management ca and should be validated using matching outcome studies. organisational problems peculiar to multicentre trials the organisation of any randomised clinical trial is fraught with difficulties, and that of a multicentre trial particularly so. there are problems associated not only with the principles on which the investigation is based but also with the detailed organisation and analysis. are all the participants truly unbiased about the relative merits of the regimens being studied? is like being compared with like -are all the end points and the standards for measuring them clearly defined so that they cannot be misunderstood? are all eligible patients being studied, and their results reported? is truly informed consent being sought that will satisfy not only the patient's right to choose what happens to him, but also the law of the land? and, finally, how can the participants be sure that the trial will be stopped at the right time, to ensure that no treatment is given to any patient that might be harmful? these questions may seem insoluble, but until we tackle them we shall never be certain. dr.m. ev~s, scar~mu~ hospital, scarborough, nonhyo~s~ y0126ql, u.k. increasing knowledge of the pathogenesis of sepsis and septic shock has led to the development of many new therapies and technologies capable of preventing, blocking or even reversing the deleterious cycle of events. recent results of subsequent multicenter trials testing some novel therapeutic drugs, however, did not confirm the promising sometimes overwhelming effects obtained experimentally. it is postulated that this is largely due to inappropriate design and conduct of multicenter trials as currently performed. one of the shortcomings emphasized in this paper is the "treatment mix" problem. multicenter trials in sepsis are performed because no single center is able to recruit the necessary number of eligible subjects within a reasonable time. each single center (icu) has its own individual policy which cannot be standardized for the trial (treatment mix). even if we ascribe that each icu did the "best" for their patients, it is highly probable that the outcome differs between the icus. this translates to the effect of the new therapy under investigation. ]t is therefore worthwhile to consider that icus must first demonstrate a certain standard based on the patients' outcome (audit) before becoming accepted as a participating center. this prerequisite ensures comparable quality between participating centers, led to the reduction of "noise level" and increases the probability of positive study results. the riyadh-icu program based on standard mortality ratios is recommended as an audit instrument. during the last decade most clinical trials of potential therapeutic agents in systemic sepsis have failed to demonstrate benefit from the drug under study. although in many instances it is entirely possible that the agent in question does not have clinical efficacy, an equally plausible explanation for the multitude of negative results is that the studies were improperly designed and thus could not show therapeutic benefit, if it existed. problems which have been identified in these various trials include inappropriate or unrealistic definitions of response to the drug; inadequate sample size, with attendant insufficient statistical power to demonstrate a difference, if it existed; insufficient standardization of customary therapeutic maneuvers in septic patients; imprecise criteria for patient entry into the study, which creates unacceptable inhomogeneity between control and treatment groups and invites unjustified post-study subgroup analysis; the lack of a formal sequential monitoring procedure for interim data analysis, which could potentially affect patient safety; and a primary analysis of study results that excludes protocol deviants and is not according to intent-to-treat. these and other problems have impacted upon the validity of the various trials conducted to date and may well have prevented the resolution of controversies surrounding the use of certain agents in the treatment of septic patients. the complexity of bacterially-induced septic shock involves a cascade of events that evolve over time, beginning with the proliferation of offending organisms and followed by the release of toxic mediators from the bacteria. endotoxins [e.g. lipopolysaccharides) from gram-negative bacteria initiate septic shock by precipitating a systemic inflammatory response syndrome (sirs) through release of a broad spectrum of hostderived mediators. over the last century, hundreds of these endogenous mediators have been proposed to serve as pathophysiological substances in this "alphabet soup" of cytokines, eieosanoids, biogenic amines, kinins, peptides, ions and hormones. an evaluation of the literature on septic shock leaves the investigator with a sense that, although many pieces of the septic shock puzzle have been presented, no clue was provided to indicate how these pieces fit together, in an attempt to assess objectively the causal role of individual mediators, we recently completed a collective meta-analysis of 24 mediators that were individually reviewed and subjected to koch-dale analysis of causality by distinguished authorities in the field (1). in summary, our attempt analyze available evidence to support a cause-and-effect relationship for putative mediators of septic shock revealed that only eight of the 24 mediators analyzed could be strongly inferred as playing a causal role in septic shock. evidence supported the involvement of endotoxln, endorphins, complement, interleukins, tumor necrosis factor, eieosanoids, platelet activating factor and calcium. other mediators either lacked adequate supportive evidence or were not conclusively involved. posttraumatic outcome may be reduced by an amplified stress response mediated by neural and humoral stimuli. the classical hormonal catabolic response is predominantly mediated directly or indirectly by neural stimuli, while most changes in inflammatory responses such as leukocytosis, acute phase proteins and changes immunofunction are mediated by humoral mediators. clinical experience from controlled studies suggest afferent neural blockade and modification of stress response to improve organ function and postoperative outcome, and limited experimental studies also suggest neural blockade to be of beneficial value in shock states. of special value may be the improved gastro-intestinal motility after neural (visceral) blockade. no outcome studies are available from patients with major truma, multiple organ failure or sepsis, conditions where humorai mediators may be more important to modify than neurally mediated responses. future studies should investigate the clinical outcome effect of combined neural and humoral mediated blockade, as well as the potential role of an initial neural blockade in elective trauma (first hit) to improve the metabolic scene and outcome if a postoperative complication (second hit) should occur. neutrophil elastase is the predominant protease of the human neutrophil. this enzyme, and a variety of other enzymes, including neutrophil collagenase, are released by activated neutrophils in the setting of high concentrations of reactive oxygen metabolites. in addition, in this extracellular environment, the normal antiprotease defense mechanisms of the organism may have been severely inactivated oxidatively especially along capillary endothelial cells. accordingly, release of neutrophil elastase which mediates inflammation and degrades most extracellular matrix proteins, including elastin, fibronectin and many forms of collagen, may contribute to tissue injury and organ failure in a variety of diseases ranging in severity and acuity from pulmonary emphysema to the adult respiratory distress syndrome (ards). as a result, significant efforts by the pharmaceutical industry have been directed not only toward the development of human neutrophil elastase inhibitors, but also toward their characterization in a variety of animal models of neutrophil mediated diseases, and their evaluation as potential therapeutics for the treatment of a variety of human clinical conditions. data from animal models of organ injury and failure along with data collected from a series of patients at risk for ards and with ards will be presented and discussed as a rationale for inhibition of neutrophil elastase. parameters that need to be monitored to obtain success in future clinical studies will also be presented in this context. have turned out to initiate and maintain organ dysfunctions in severe posttraumatic and postoperative courses. such proteolysis-induced disorders are especially rendered possible by the concurrently arising consumption of inhibitory regulators (e.g. a vproteinase inhibitor=a 1pi, antithrombin iii=at iii) via cofnplex formation and/of proteolytic/oxidative inactivation. besides the well-known destructive potency against protein substrates, proteinases such as elastase or thrombin (active or in complex with the serpin inhibitors ~ 1pi or at iii) are also supposed to increase the inflammatory reaction by inducing cytokine release or shedding of soiuble adhesion molecules from various inflammatory cells (pmns, monocytes/macrophages, endothelial cells). those cell-derived mediators may provoke further cell activation through an autocrine/paracrine loop thus increasing significantly the proteinase burden at the inflammatory focus. therefore, this noxious circle might be interrupted by a convenient proteinase inhibitor therapy to ameliorate the inflammatory process. to verify this hypothesis, high dosis of at iii (mean plasma levels up to 140 %) were applied in first controlled randomized studies on surgical patients suffering from multiple trauma or severe sepsis. in control patients we could clearly demonstrate the sequential appearance of proteinase/serpin-complexes (fa 1pi, tat), cytokines (il-8, il-6), and soluble intercellular adh6sion molecules (icam, elan, p-selectin) in the circulation. these factors turned out to be a helpful tool for early diagnosis and prognosis of forthcoming organ dysfunctions. interestingly, m at iii-treated patients a significantly reduced release/production of inflammatory mediators became obvious concomitant with the improved clinical course in comparision to an increasing deterioration in control patients. soluble mediators of inflammation are cytokines (e.g. il-1, il-6 and tnfe) as well as breakdown products of complement proteins (e.g. c5a and terminal complement complex). therefore, attenuation of both, release and generation of these synergistically functioning mediators together with stimulation of release of antagonists including soluble forms of receptors are potentially beneficial in all kind of inflammatory diseases. intravenous immunoglobulins (ivigs) are known to have an anti-inflammatory effect in humans (nih consensus conference on wig, 1990) . the mechanism of action becomes more and more clear. in single cells stimulated by anti-cd3 mab wig was able to down-regulate production of il-2, il-10, ifn% and tnfj~ significantly. igg coated solid-phase stimulates release from monocytes of interleukin-1 receptor antagonist (il-lra) but not of il-1; this observation is very much in contrast to the effect of endotoxin in the same in vitro system. furthermore, naturally occurring anti-cytokine antibodies can be demonstrated in apparently healthy individuals and in wig prepared from plasma pools. some of these antibodies can be detected by antigen/antibody reactions in fluid phase (anti-ll-le, anti-il-6), some only by solid-phase detection systems (anti-ll-8, anti-ifn% anti-tnfc 0. infusion of wig to patients suffering from inflammatory disorders have resulted in a decrease in il-6 in circulation. during infusion wig might induce an acute but transient rise of tnfo~, il-6, and il-8. self limitiation of this process might be due to demonstrable instant release of il-1 ra and soluble tnfo~ receptors. in vitro ivig has also been shown to attenuate complement activation via the classical pathway. furthermore, acid haemolysis of erythrocytes in paroxysmal nocturnal haemoglobinuria could partially be prevented by ivig. ivig was able to attenuate forssman shock in guinea pigs. we have seen a patient with congenitally elevated turnover of alternative pathway of complement who repeatedly showed an increase of haemolytic titres after administration of ivig. thus, wigs apparently have multiple potencies including attenuation of soluble mediators of inflammation and might therefore be of benefit in trauma, shock, and sepsis. significance of sinusoidal liuing cells and of humoral factors on hepatic function following sepsis and major surgery hirata k, katsuramaki t, yamaguchi h, mukaiya m, yamashiro k. regeneration of the liver after hepatic necrosis or partial removal has been extensively studied, but uncontrolled mechanisms to irreversible hepatic failure following sepsis or major surgery of liver or with hepatic dysfunction have so far not been well documented. we suggested the importance of the intrasinusoidal aggregation between sinusoidal lining cells and intrasinusoidal running ceils in the mechanisms of hepatocyte dysfunction. thus, the purposes of this study was to investigate the changes in each sinusoidal lining cell during this process and the effect of cytokine on hepatocyte under various oxygenation. [ materials and methods ] ( 1 ) clinical study : thirty two patients with histologically proved malignant tumor underwent major hepatectomy were devided in two groups, i_e. the cirrhotic liver group and the non-cirrhotic [aver group. most of the former were the patients with hepatocellular carcinoma and most of the latter were the patients with metastatic carcinoma of colorectal cancer. five patients were complicated septic conditions with hepatic failure. following operation in each patient, a verous blood sample was taken for measurement of serum il-1, il-6, tnf and c-reactive protein concentration. and hepatic biopsies were sometimes undergone in patients with anomalous hepatic function. (2) experimental study : kupffer ceils, sinsoidal endothelial cells and hepatocytes were separated from mouse ( c3hhen ) liver. and also polymorphonuclear cells and platelets were purified from blood and peritoneal macrophages were from peritoneal cavity of mouse. co-cultures between or among these cells with iipopolysaccharide were performed. cytokine concentrations in media and hepatocytic function were compared. [ results and conclusion ] our findings in above experiments demonstrated the cleanly different concept for the mechanism of hepatocyte dysfunction following sepsis or major surgery. namely, the effect of hepatoeytie function by cytokines or superioxide were significantly affected under low oxygenation, but not significant under good oxygenation. hirata m.d.,nishi 17,hokkaido,japan $120 470 immune complexes as .mediators of sepsis and immune dyafumcffon laa k horn, chxistof birkenmaier, and greg h2mon departmen~ of surgery, san frandsco general hospital, urdversr 3, of calif(~rrda, san frandsco. immune complexes (ic) a;:e commonly found circulating in parians during infection and sepsis; and following traumm, bums, and massive l~ensfusion. clearance of 1c occurs by phago~'tmsis of esll-bou_nd or opsonized p~tlcles. monocyte activation du~jng phmgocytos/s could lead ~o release of cytokilies ieletexiot~ to the hosh to examine tb/s phenomenon, we formed insoluble ic by precipitating iow-endotoxin bovine serun~ albumin (bsa) with rabbit ant/-bsa igg. we have ~town that these ic are ingested by monocytes and concomitantly stimv/ate re~pizatory bu~t activity measuxed by assay of in%racellul~ peroxldaffon. we have shown that ic ingesffon produces signlqcant el,era,lore in the monoey~e phenotype. spedacally, cdi4 is down-regu/ated, along wl~ cd 32 and cd64. thus responsiveness to lipopolysacchmdde (128) a~xd i=m~unoglobulin fe st~mu/mtlon is reduced. in cert,+rest, the complement rote.p tot cdc42 is u~-regulamd, indicaling mcremsed re~ponalx=~ness to opsonized pat,rotes, we examined monocyte superna~n~s for production of hzmor necrosis factor alpha (tnfg) following ic stimulation and showed that ic are capable of provoking ~elaase of hrmaunl)reac~ve tni~. j[c also increase mono=yte produchon of prnstaglandin e2. in summary, ~/~ese results demonstrate lhmt ic are capable of indudng production of ¢ytokines and the imrau-nosuppressive prostaglandin pge2. ic also allez ~he surface expression of important receptors involved in actlvaffon by lp~ and phagocyiosis. thus, ic aze competent for indud/on of the mepl/e s!0otlse. by examining m0n0cyte phenotypms, it may be possible to d~mrmkne extent ohn'u~u~e complex activation and predict immune depression iu criffcally ill pa~ents. a great deal of experimental work has focused on preventing and/or treating sepsis and organ failure following injury. many of these strategies have tried to attack mediator substances released during the systemic inflammatory response following injury. sugermann has demonstrated the improvement of pulmonary function, but not eappillary leak using [buprofen in the porcine model. in a clinical trial, faist demonstrated the effectiveness of indomethacine in amelierating the post trauma of cellular amenity using in vitro parameters. morbidity and mortality, however, were not different between control and treatment groups. baue und chaudry have suggested that atp magnesium chloride may have protective effect in renal failure um kupfer cell dysfunction seen after shock. pentoxiphyllin has been found to be effective in improving tissue oxygenation and oxygen consumption following hemorrhage, and there may be promise for this drug in the ischemia reperfusion injury. monoclonal antibodies against endothxin and inflammatory mediators seemed promising at first glance; however, preliminiary clinical data of the treatment of sepsis with either ha-ia or e5 monoelonoal antibodies against bacterial cell wall have been disappointing. initial studies with il-i receptor antagonist also appeared encouraging, but no difference was seen in the most recent trial. currently studies are ongoing with monoclonal antibodies to tumor necrosis factor which may have more efficacy given the fact that it is a macrophage mediator released by endotoxin. although we seem closer to understanding and preventing the problems of sepsis and organg failure after trauma, we must continue to appreciate the complex interrelationships and delicate balances inherent in the host defense system. overzealous attempts at restoring immunity in the absence of understanding pathophysiology may be just as dangerous at post-traumatic immlmospremsion. severe acute pancreatitis was caused by different etiologic factors, but once pancreatic tissues were infected, patients show a similar pattern of aggravation and develop organ failure. remarkable elevation of serum il-6 was unexceptionally observed in patients with severe acute pancreatitis. the serum levels were more than 500 pg/ml (normal: less than 4 pg/ml) during the first one or two days after onset. there was a significant correlation between the peak serum il-6 level and the number of organs showing failure (r=0.883). tnf-a production by isolated peritoneal macrophages following lipopolysaccharide (lps) stimulation was significantly increased in cerulein-induced pancreatitis rats. serum tnf-a activity was elevated following intraperitoneal administration of lps as the septic challenge both in pancreatitis rats and in control rats, being significantly higher in the former (p<0.05). histological findings and liver function tests revealed that lps induced more severe liver damage in pancreatitis rats than in control rats. these results indicate that increased tnf-a production by peritoneal macrophages in acute pancreatitis augmented lps-induced liver injury. organ failure in severe acute pancreatitis could be caused, at least in part, by increased cytokine production. it is clear now, that in mods, organ injury is not directly due to exogenous factors, such as bacteria or toxins, but instead is largely a consequence of the host's own endogenously produced mediators. there is an extensive body of literature on the inhibition of mediator production, release and action in different cascade systems by glucocorticoids. they can serve as a host protection against overactive defense reactions if used adequately. a new understanding of the regulation of cytokine synthesis, especially tnf, may lead to an insight of the conflicting findings between the benefits of glucocorticoid therapy in animals and its current failure in recent clinical trials. glucocorticoid shares its role with other novel therapeutic approaches also shown to be successful only in experimental settings. a mete-analysis of steroids in sepsis, however, revealed a beneficial effect in the subgroup of patients with gram-negative infections. there is now a series of arguments to reevaluate the indication for steroid thera-py and/or prophylaxis in mods and sepsis. honjo i-1-1, kumamoto 860, japan in septic shock j. briegel, m. halter, h. forst, w. kellermaun, m. bittl, k. peter imrodnetlea and methods: hydrocortisone (hc) at an infusion rate similar to the maximal secretory rate of cortisol in man improves hemodynamics in human septic shock (1). we hypothesized that the hc-induced hypercortisolemia prevents a harmful overshoot of immune reactions. to test this hypothesis we prospectively investigated 12 patients admitted to the icu in refractory septic shock. after resuscitation (mechanical ventilation, fluid resuscitation, titration of vasopressors, and initiation of antimicrobial therapy) hc was started with a loading dose of 100 mg/ 30 rain, followed by a continuous infusion (10 mggh). with hemodynamic improvement (dopamine < 2 gg/kg/min) the infusion rate nfhc was tapered over a period of 3 days. phospbolipase a 2 (pla2), c-reactive protein (crp), and elastase-proteinase inhibitor complex (e-pi) were deierminated daily. results: according to our previous results hemodynamics improved during hc infusien. after 4 days dopamine requirements decreased to less than 2 gg&g/min in all patients. this corresponded to an attenuation of the systemic inflammatory response syndrome (sirs) indicated by the abrogation of fever and the decrease in heart rate and inflammatory mediators. pla 2 activity and e-pi concentrations decreased to near normal values and crp concentrations decreased as well. after cessation of hc infusion (between day 5 and 7) a reinforcement nfthe sirs was observed despite cortisol levels within the normal range. this was first indicated by e-pi, followed by fever, increases in heart rate, crp, pla2, and finally by the relapse of septic shock in four patients on days 7,8 9, and 1o. a second infusion of hc again resulted in shock reversal and in a decrease in pla2, crp, and e-pi in the patients under study. discussion: there is growing evidence that the endogenous steroid hc and proinflammatory cytokines integrate a complex immunoregulatory feec~ack circuit. the elaboration of tnf, il-16, il-6, and il-8 is attenuated by-100 rag hc prior to endotoxin challenge in humans (2, 3) . cytokines like tnf, il-16, il-6, and il-8 are potent proinflammatory signals for acute reactants (--, clip), neutrophil degranulation (--, e-pi), and pla 2 synthesis and secretion. our data provide evidence that hc at an infusion rate similar to the maximal secretory rate of cortisol in man attenuates the systemic inflammatory response in human septic shock. to evaluate the feasibility of a nigh-dose regimen of antithrombin iii (at iii) treatment in patients with multiple injuries regarding blood levels of antithrombin iii and undesired side effcts and to analyse effects of the initibitortherapy on the systemic inflammatory response. patients and methods: 20 patients with an iss nf more than 28 points who could be treated with at iii administration within 180 rain after trauma were randomized to receive either at iii or placebo. at iii was given to reach an antithrombin iii inhibitory capacity in plasma of 140% by using the following calculation: at iii to administer = (140% -at iii measured) x body weight. at iii was given every six hours for a period nf48 hours and on the 4th day. patients were followed up throughout their stay in the icu but at least 14 days. in this abstract the data of the first 10 patients are included. results: the patients' median injury severity score was 41 points with a median age of 42 years. the patients were equally distributed between verum and placebo groups with respact to age , iss, prehospital treatment, blood pressure, hemoglobin and at nmevel on admission, and time from the accident until the test solution was given. the patients of the at iii group received 18,100 units of the inhibitor on average during the first 4 days. with our regimen, the at iii levels could be kept between 125% and 140% of normal, whereas control patients an at hi concentration of 40% to 60% was observed. patients with at ni treatment required less red blood cells bur received the same amount of ffp and fluids. there were no differences with respect to platelet count, partial thromboplastin time, l~rothrombin time and prothrombim. we did not observe bleeding disorders or any other side effect with peak concetrations of up to 206% of normal. white blood count, pmn--elastase, interleukin 6 and 8 and c-reactive protein tended to be lower in the at iii group. there were no differences with respect to renal and hepatic function parameters but the duration od mechanical ventilation was shorter in the at ii1 group (8.5 vs. 15.8 days) conclusions: we conclude that our treatment protocol (a) was able to restore at iil levels to the desired range of 140%, (b) did not lead to coagulation disorders, (c) did not increase transfusion requirements, (d) did not reveal other undesired side effects, and (e) showed a tendency towards reduced posttranmatic inflammation and mechanical ventilation requirements department of trauma surgery, 45122 essen, germany antithrombin-lll (at-ill) acts as an inhibitor of thrombin, further proteases and the alternative pathway of the complement system. inhibiting thrombin, at-ill works as a functional antagonist of paf~ therefore, in state of hypevolemic-traumatic shock, continuous supranormal serum -and tissue -concentrations of at-ill may be efficient to reduce the local posttraumatic reactions resulting from complement and pmn-activation. 20 patients with severe multiple trauma (7 treatment [at-ill] --13 controls [c]) were investigated. study cdteda were age > 16 and < 65 years, injury severity (iss) > 30 points and glasgow-coma-scale > 8 points; randomization and treatment has to be started within 6 hours after trauma. permission for the clinical study was given by the local ethic committee. bradykinin (bk) and related kinins are potent inflammatory peptides which possess the ability to induce, vasodilation, increased vascular permeability and hyperalgesia. cp-0127, a novel homodimer bk antagonist has previously been shown to increase survival in rat and rabbit models of lethal endotoxin shock and is now in clinical trials for sepsis. we have now evaluated the effect of cp-0127 in other models of inflammation. male rats were precannulated with a catheter in the carotid artery. 24h later bk was injected ia and the pain score ranked from 0 (no responses) to 5 (vocalization). cp-0127 at 3.6 umoles/kg completely inhibited the pain responses for a period of 2.5 -3h. cp-0127 at 3.6 umoles/kg s.c. was also found to inhibit the increase in paw volume and hyperalgesia induced in rats over a 3-4h period by an intraplantar injection of 0.1% carrageenan. the abdominal constriction response 1o an intraperitoneal injection of kaolin was inhibited in a dose-dependent manner by cp-0127. when 50 ul of 0.5% formalin was injected into the paw of a mouse a characteristic licking response was observed which was biphasic in nature. cp-0127 significantly inhibited both the first (0-5min) and second (15-30 min) phase responses. ]n a rat burn model, where the hind paw is immersed in water at 55°c for 15 sec the increase in paw volume was significantly reduced by pretreatment with cp-0127, 3.6 umoles/kg s.c. finally cerebrai edema was induced in rats by applying cold (-78°c for 10 sec) to the dural surface following a craniectomy. cp-0127 at 3.6 umoles/kg s.c. produced a significant reduction in the amount of edema compared with sham controls 24 h later. these data suggest that bk is an important mediator of inflammation and hyperalgesia and that the bradykinin antagonist, cp-0127, may be useful in the treatment of such inflammatory, hyperalgesic disorders. partial hepatectomy in humans is associated with a considerable morbidity due to hemodynamic and metabolic derangements, which increase the risk for organ failure and mortality. we hypothesized that endotoxemia may play a pivotal role in these complications. we therefore, investigated whether peri-operative infusion of rbpi23, a recombinant protein of the human neutrophil bpi with bactericidal and endotoxin-binding capacity, could prevent postoperative derangements following partial hepatectomy. male wistar rats (230-250 g.) received a 70% liver resection (phx) or a sham operation (sh), and a continuous intravenous infusion of either 0.2 mg/kg/hr rbpi23 (phx-bpi, n=8; sh-bpi, n=7) or the (iso-electric, iso-kd) control protein thaumatin (phx-con, n=8; sh-con, n-8). various parameters were measured 4 h after the resection or sham operation. mean arterial pressure, cardiac output and heart rate were significantly decreased in phx-con rats compared with sh rats, which effects were not observed in phx rats treated with rbpi23. blood ph was significantly decreased in the phx-eon group, whereas the leucocyte count, hematocrite and il-6 levels were significantly increased compared to sham levels. in the phx-bpi group, these parameters were restored to near sham levels. in vitro experiments with rat plasma and human mononuclear cells (mncs) revealed that plasma of phx-con rats is highly capable of activating mncs, accompanied by the release of cytokines. this activation is attenuated with phx-bpi plasma. in vitro added acd14 or polymyxin b was able to reduce the activation by phx-con rat plasma to the levels of phx-bpi rats thus, these data suggest that systemic endctoxemia, possibly of gut origin, is a major cause of postoperative hemodynamic and metabolic derangements following phx and that rbpizz can prevent these changes. more recently we reported a transient appearance of both endotoxin and tnf in the circulation of rats subjected to the haemorrhagic shock (hs) already at 90 -120 rain. similar to bpi, recombinant bpi was found to bind lps and inhibit tnf formation in vitro. the aim of this study was to investigate the effects of rbpi21 (kindly provided by xoma corporation, berkeley, ca) against haemorrhage related endotoxemia and mortality in rats. method: a prolonged hs was induced by blood withdrawal to a mean arterial pressure of 30-35 mmhg for 180 rain followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over 50 rain. rbplg. 1 was administered at a total dose of 5 mg/kg i.v. (2.0 mg/kg at the16-eginning followed by two doses of 1.5 mg/kg each at end of shock and the end of resuscitation). the control group was treated similar to the bpi group but received thaumatin as a protein control preparation at the same dose as rbpi21. results: imrffe?diately after resuscitation (230 min) the detected plasma endotoxin levels in the control group (mean = 61, range = 0-120 pg/ml) were almost neutralized by rbpi21 treatment (mean = 13, range = 0-53 pg/ml) . plasma tnf levyis were not significantly influenced by rbpi treatment at the two time points 230 and 320 min of experiment (means: 65 and 51 in bpi vs 49, 65 pg/ml in the control group). the 48-hour survival rate was improved from 6/16 (37.5 %) in the control to 11/16 (69 %). conclusion: these data suggest that haemorrhagic shock may lead to bacterial translocation and/or transient endotoxemia with concomitant cytokine formation that may play an important role in the pathogenesis after shock and trauma, rbpi21 might be a useful therapeutic agent against endogenous bacterfal/endotoxin related disorders in hemorrhagic shock. morbidity and mortality after hypoxia of the vital organs had been correlated to the production of oxygen radicle which is mediated by xanthine oxidase activity, in this study we have evaluated the survival rate after allopurinol. 42 rabbits weighed 900 + 200 grams divided into two groups. group i included 22 tabbits were treated with allopurinol 80 mg/kg for seven days before induction of haemorrhage. group ii as a control included 20 rabbits. all rabbits were subjected to 25% arterial blood loss through the central ear artery for one hour then resusciatation was done by the heparinized withdrawn blood through a marginal ear vein. during the experiment blood pressure and heart rate were monitored through the central ear artery. also uric acid, lactic acid, glutathione activity were estimated. animal survival was followed for 10 days. postmortem vital organ histochemistry and histopathology examinations were done. in group i the survival after three days was 10 out of 22 while in group ii it was two out of 20. our conc|usion, allopurinol had increased the survival in aiiopurinol pretreated rabbits which may indicate the value of allopurinol premedication for patient prepared for elective bloody surgical intervention . h2 receptor antagonists are commonly used for stress ulcer prophylaxis, but their actions on the septic response are largely unknown, in an experimental model, 26 pigs were first anesthetized, then injured with 100 joules of energy to the posterior thigh, then hemorrhaged 30-40% of their blood volume. after i hr of shock, all the shed blood plus 2x the hemorrhage volume as lactated ringers was infused. following resuscitation, ranitidine (1.5 mg/kg iv twice daily) or saline placebo was begun. the treatment group was randomly assigned in a blinded fashion. after 72 hrs, a septic challenge was administered (15 bg/kg of e. coil endotoxin (lps)). serial gastroscopy, gastric ph, hemodynamics, abg's, physiologic dead space ventilation, leukocyte counts, and tumor necrosis factor (tnf) levels were recorded for 180 min. baseline values and units were cardiac index 102 _+ 9 ml/min/kg (ci), arterial po2 228 + 11 mmhg(pao2), base excess 6.5 -+ 1 meq (be), physiologic dead space fraction 23 +_ 4% (pds), and tnf 0.4 + 0.1 units/ml. baseline gastric ph was 3.9 -+ 0.4 and 4.8 _+ 0.5 in the placebo and ranitidine groups, respectively. the gastritis following hemorrhage was marginally attenuated in the ranitidine group. following lps infusion the following were obtained: ci pao2* be* gastric* pds* peak* 120 rain 60 rain 120 rain ph 120 min tnf ranitidine 167_+17 118_+15 -4.4±1. bum injury results in hypermetabolism, fever and nitrogen wasting. endotoxin (lps) has been proposed to mediate these effects, either directly or via activation of macrophages to produce cytokines such as interleukin-6 (ii-6). this study was designed to clarify the role of lps and 11-6 in the metabolic response to bum injury. twenty-five burn patients (49 -+ 17%; 16 + 15% ft bsa burn; 32 _+ 16 years old) were studied serially for three weeks post bum. patients underwent partitional calorimetry to assess metabolic rate and compartmented heat loss. nitrogen was assayed using chemiluminescence. lps and i1-6 were measured with limulus amebocyte lysate assay and elisa. patients were excluded if they suffered smoke inhalation, showed any sign of sepsis or failed to rapidly meet their nutritional needs via the enteral route. ten patients received intravenous polymixin b (5,000 u/kg/day to bind lps). these patients did not differ for the remainder. all patients were hypermetabolic and febrile in proportion to the size of their bum wound but were not endotoxemic (3.1 +_ 5.6 pg/ml; normal <40 pg/ml). i1-6 did demonstrate a significant correlation with cole temperature (tr~ = 37.6 + 0.21ogi1-6, p=0.04) and with nitrogen excretion (nou t = -5.2 4-0.091ogi1-6 + 0.14tr, p=0.01). administration of polymixin b had no effect on metabolic rate, temperature or i1-6 levels but did reduce nitrogen excretion resulting in more positive nitrogen balance (0.t grn/day vs. -0.1 gm/day, p=0.04). although bum injury does not produce an obligatory endotoxemia, i1-6 does appear to play a role in the fever and nitrogen wasting seen with such injuries. the effect ofpolymixin b on nitrogen excretion suggests that lps may play a role either locally or in the portal system. introduction: there is substantial evidence that release of inflammatory mediators by activated kupffer cells contribute to the course of a systemic inflammatory process, e.g. after shock or lrauma. besides the systemic effects of mediators such as tnf, paf or interleukines, local actions on hepatic microvasculature and hepatic inflammatory response have to be considered. our aim was to assess the role of tnf and paf by blocking their effects using anti-tnf monoclonal antibody, pentoxifylline and a paf antagonist. methnds: in anesthetized sprd-rats, hemorrhagic shock was induced by withdrawl of arterial blood within 5 rain and shock state was hold for 1 h at a map of 40 mm hg (cardiac output of 50 %). following adequate resuscitation with 60% of shed blood and twice of this volume as ringer's solntion, animals recovered to map >1130 mm hg and co >120%. hepatic microcirculation and sinusoidal leukocyte-endothelium interactions were examined by intravital epi-fluorescence microscopy at 1, 3, or 5 hours after resuscitation. in a blinded fashion, a rat-specific monoclonal anti-tnf antibody [2 mg/kg, celltech, uk) , pentoxffylline (ptx, 50 mg/kg, hoechst, d), and a paf antagonist (web 2086, boehringer, ingh., d) were given either as pretreatment or at the time of resuscitation (n=6-8 group bolla. k*., duchateau, j., hajos, gy., mbzes, t., hern~di, f. prevention of temporary/secondary immune deficiencies or reduction of their severity and/or duration as well as the reduction of the perifocal inflammatory processes belong to the rational targets of posttraumatic/pedsurgical medication. such a targeted medication can result in less frequently occurring nosocomial infections, and in reducing the duration of the intensive care and convalescence period. the results of in vitro studies performed with the amino acid sequence 32-35 of thymopoietin, i.e., with thymocartin in whole blood and peripheral mono-nuclear celi(pbnc) cultures clearly show some characteristic effects of this immunomodulator. preincubation with the tetrapeptide significantly (p<o.ol) and concentration-dependent reduced the endotoxin(lps)-induced tnfalfa production from 95,7 to about 20 pglml, but did not abolish it. the inhibition of the lps(0.1 ug/ml)-induced tnf production occured already in presence of 0,1 pg peptide/ml and peaked at the concentration of 0,01 ng/ml. higher concentrations did not increase this effect. in contrast to the other two small thymic peptides (tp-3 and tp-5), thymocartin did not enhanced the pwm-induced pge 2 production in pbmc cultures up to a concentration of 1 ng/ml, inclusive. the selection of thymocartin (tp-4) for perisurgical medication and/or for treatment of trauma-induced temporary immune depression has been strongly supported also by targeted animal experiments. thus, mortality of about 90% registered in mice infected with pseudomonas aeruginosa after bum injury could be reduced with the tetrapeptide treatment to a level which did not differ significantly from that of the infected non-burned controls. moreover, observations gathered first of all with the inflammatory model of air poach/croton oil grenuloma (selye) clearly showed, that a very definite, antiexudative effect can be achieved with this thymic peptide. as to this effect, thymocartin has been confirmed, e.g., to be equipotent with locally administered fluocinolone acetonide and/or s.c. injected prednisolone. the interim results of two explorative double-blind clinical studies (involving more than 260 patients after polytrauma and hip-fracture untill now, respectively) correspond to the expectations. the treatment significantly reduced the occurence of nosocomial infections, the days spend in intensive care and on antibiotics as well as it shortened the bed-out time and reduced the additional medication. objective of this prospective randomized study was to further delineate the mechanisms leading to these alterations and to investigate the effects of immunomodulatory intervention. patients and methods: 20 patients (pts) formed control group a. 20 group b pts received 3x50 mg indomethacin (indo) from the first (dl) to the fourth postoperative day (d5) intravenously to block prostaglandin e2induced suppression of cmi response as well as 50 mg thymopentin (tp-5) subcutaneously preop., on d2 and d4 to augment cmi reactivity. in vitro investigations preoperatively, on dl and d7 included measurements of the percentage of cd4+ t-helper (th) cells, pbytohemagglutinin (pha)induced synthesis of interleukin (il)-2 as one cytokine primarily produced by thl-cells, and pha-induced synthesis of il-6 as one cytokine primarily produced by th2-cells. delayed type hypersensitivity (dth) skin response to an antigen skin test battery and specific antibody (ab) production following tetanus vaccination preoperatively and on d7 were used as in vivo tests for thl-and th2-induced immune response respectively. results: postoperatively, group a pts showed a persistent significant reduction of cd4+ th cells, il-2/il-6 ratio, and dth skin response as compared to baseline values (bl), while ab production increased (p<0.05 vs.bl). in group b pts no change in cd4+ th cells, il-2/il-6 ratio, or dth skin response was observed (p<0.05 vs.group a), and postoperative ab production increased significantly (n.s. vs. group a). conclusions: 1.these results clearly indicate a significant suppression of th1 induced cmi response, while th2 induced response remains normal following ohs. 2.these alterations may gain clinical significance whenever a postoperative infection requires a th1 response and may explain the susceptibility to opportunistic microorganisms observed in pts after ohs. 3 the aim of this study was to find out whether the establishment of administration of thymostimulin (tp-i) in jaundiced and anergic rats would return the rats to the state of immunocompetence. material and methods. male wistar rats weighing 250-300 g were injected with haemocyanin (klh). all rats with a positive response were included in the study and we evalu ated the t cell subpopulations and il-2. they underwent laparotomy and the common duct was ligated and divided. those rats whic become jaundiced and anergic one week after the operation, were divided in two groups: control group ( objective of study. the goal of this study was to prove the necessity of thymus derived immunomodulators treatment in acute period of infantine sepsis, materials and methods. immune status was investigated in 11o infants. clinical conditions of children were defined by syndrome of multiple polyorgan]c insufficiency. the heaviness nf condition was estimated as the sum of points according to efforts needed to restore of basic living functions -respiration, hemodynamjcs, hemocoagulatlon, metabolism, functions of liver and kidneys. it was expressed in form of clinic condition classes from 1 to 4. results.it was found that in 100% of infants with 3-4 classes of heaviness immunode[iciency took place simultaneously with ii and iii stages of hemocoagulattve syndrome. amounts of t cells and t helpers were decreased more then 2 times, ratio th/ts was reduced to 1.5 or lower. concentration of lgg and igawas reduced almost a hull in comparison to control. phagocytic and metabolic activities nf neutrophils were depressed. complement system state was changed: the functional activity of the alternative pathway factors b, d and the level or c3a subcomponent were increased but the level of c2, c3,, c4 and c5 components of the classic pathway were decreased. during the development of coagnlopathy the direct correlation between chso, levels of plasminogen and antithrombine iii was found. in greater extent the function of immune system was disodered in accordance with 2 point metabolic violations and hemocoagulative disturbances considered as disseminated inmtravaseular clotting syndrome on ii or ili stages. conclusions. the rehabilitation of the immune system functions was promoted by taetivine and thymaline in doses of i-3 mcg/kg per day and 2 mg/kg per day accordingly during a week in children with 3 -4 classes el heaviness. aiter 11-14 days tactivine had advantage. when eoagulopathy took place thynaaljne promoted restoration of (1-3)-~-d-glucan (bgc) is a major cell wall compornent of pathogenic fungi and shows many immunopharmacological activities on experimental animals. lps is also derived from gram-negative bacteria and have in~ttnomodulating activities on immune systems positively or negatively. because of difficulty to cure contaminating lps from bgc preparations, it is very hard to evaluate the exact activities of bgc. in this study, we compared the immunological activities of lps ans highly purified bgc on murine system. materials and methods: c3h/hen or c3h/hej mice, 6 to 12 wkolds of both sexes were used through experiments. highly purified (1-3 )-{5-d-glucan (gurdran; bgc)was courteous gift from seikagaku kogyo co. tokyo, japan and resolved in endotoxin-free ultra-pure water. escherichia cell lps was purchased from sigma chemicals, usa. mitogenic activities of bgc or lps on splenic cells were measured by mit dye assay. in vitro activation of peritoneal exudate macrophage (pen) have moniotored by phagocytosis and intraphagocytic killing of pseudomonas aeruginosa (z~b-139. induction of cytokine productions from pem or spleen cells induced by bgc or lps were detected by cytotoxic assay. results and (bnclnsions: i ) in vitro cultures of pem with bgc have induced enhanced phagocytic and bactecidal activities in c3h/hen and c3h/hej mice, whereas lps only induced such activities in c3h/hen mice. most effective dose of bgc was 10 ug/ml. 2) bgc-dependent proliferative response of c3h/hen splenic cells was not detected under the culture condition used. 3) induction of tnf~ prodction was apparently observed in bgc-stimulated c3h/hej pd4 cultures, but not in lps stimulated one. these findings indicate that activation mechanisms of pr94 and splenic cells by bgc are different from that by lps. to investigate the bsc-induced p~4 activation, intracellular signaling pathways of p~4 by bgc-stimulation are under consideration. yamagami k, uedono y, kawakami k, kitazawa y and tanaka t according to the latest reports of use of il-2 in a burned-sepsis model, the dose was too high to adjust for human therapy. adoptive transfer of l~ymphokine-activated killer (lak) cells has been demonstrated as a means of enhancing therapy in malignant tumors. we therefore attempt to use lak therapy on burned-infected mice instead of il-2. under anesthesia, 8-weekold male c3h-hej mice were subjected to a 20% scald or sham burn and then resuscitated. sham burned mice served as controls. scald burn mice were subjected to peritonitis by intraperitoneal innoculation of 105 organisms of p. aeruginosa 24 hr after burn. burned-infected-mice were divided into two groups. one group had no further treatment, and the other group received lak cells (2x105) intraperitonelly after septic challenge. the mice were scored for survival daily. on day 7 after burn, using moabs dual-color flow cytometry, we studied lymphocyte expression in mice with use of blood and spleen. simultaneousely we studied the alteration of il-1 and il-6 in their serum using immunoassy kits. sham burned animals had no mortality. the mortality of the lak-treated mice was significantly reduced when compared with that of the burned-infected mice. the most consistent changes observed were depressions in percentages of thyl, 2 positive cells and a remarkable depression of nk cells in spleen. after lak cell treatment, those two percentages of cells significantly increased. all the data of assay of ill and [l 6 were not detected on sham burn mice serum. due to burn and septic challenge the levels of illand il 6 (n=6) were raised to 45.9_+17.4 and 3866_+1045 pg/ml, while the levels in lak treated mice were reduced to 4.23_+2.37 and 437_+110 pg/ml, respectively. the results reported here demonstrate that lak therapy is able to improve cell-mediated immunity in burned-infected mice. this is associated with a significantly improved survival rate, since ill has been demonstrated to mediate immune and inflammatory responses. also a cause effect relationship between elevated endetoxin levels and increases of [l6 has been recently established. the aim of our present study was to investigate the effect of parenteral indomethacin on the immune system of patients under major operations. our study included 20 operated patients, 10 of which received 100mg indomethacin before and 1,2 and 3 days after surgery (group a) and 10 patients received no antiinflammatory therapy (group b). we measured total t-cells helper (cd4), suppressor (cd8), nk-cells and interleukin-1 and tnf production by pha or endodoxin (lps) stimulated peripheral blood mononuclear cells at day 0 and 1, 3 and 7 days after operation. in group a we detected a significant (p<0.05) increase in cd4/cd8 ratio on day 3 while no differences were ~oted in nk-cells or cytokine production in both groups (table 1) . it seems, therefore, that parenterai indomethacin may have a benefitial effect on the immune system of patients under major operations by increasing the t-helper /t-suppressor cell ratio while having no effect on the production of cytokines by peripheral blood mononuclear cells. this study was undertaken td dete~nnine the role of prostaglandin synthesis inhibitor on survival post traulna sepsis. analysis of the effects of prostaglandin synthesis inhibitor on survival of four groups of 15 mice each were made after laparatomy and intravenous administration of live e.coli. post trauma sepsis, group a received no treatment; group b received antibiotics im; group c received prostaglandin synthesis inhibitor im and group d received both antibiotics and prostaglandin synthesis inhibitor im. survival time was longest in group treated with prostaglandin synthesis inhibitor and antibiotics post trauma sepsis. the singular use of either antibiotic or prostaglandin synthesis inhibitor did not alter the outcome on survival. mortality rate was lowest in the group treated with combined prostaglandin synthesis inhibitor and antibiotics. use of this combination showed a reduction in bacterial growth in peritoneal and blood samples, mild morphologic changes secondary to sepsis in the heart, lungs, liver, kidney and stomach and marked enhancement of mean level of activity. the study indicates that addition of prostaglandin synthesis inhibitor in treatment of trauma-sepsis has clear beneficial effects. interferon-? (ifn-y) is a potent immunostimulant which has been shown to be detrimental when administered during septic shock. blockade of this cytokine however is beneficial during the acute septic response. the aim of this study was to evaluate the cellular effects of this cytokine and its blocking monoclonal antibody (moab) in lethal sepsis following injury. 100 female cd-1 mice were randomized into two groups: septic=lps vs injury=hind limb amputation (hla vs igg. nstats= anova=p<0.05 vs lgg ifn-y was detrimental when given to control septic animals. however its blocking moab was protective (p<0.05). conversely ifn-y was proteetive in injured septic animals compared with anti-ifn-? (p<0.05). these findings demonstrate that the immune stimulant ifn-? has therapeutic potential in the immunosuppressed injured host predisposed to sepsis, while blockade of this cytokine is required for protection in the septic host with a normal immune response. dept of surgery, rcsi, beaumont hospital, dublin 9, ireland. oxidants play a role in physiology. the potential noxious oxidant biochemistry is restrained by chemically distinct antioxidants. these antioxidants, which may reside in different cellular compartments, can act synergistically. in normal physiology an oxidant/antioxidant bai&nce exists. unbalance in favour of the oxidants is called oxidative stress. oxidative stress has been implicated in many pathologies including lung diseases(e.g, doxorubicin induced cardiotoxicity), ischemia/reperfnsion damage and central nervous system trauma. transition metals such as iron are involved in the early events leading to oxidative damage in these pathologies. this has been underlined by the finding that postischemic cns pathology closely resembles that caused by a chemical oxidative insult (e.g. iron microinjection). moreover, a protective effect of oxygen-radical scavening agents or compounds that inhibit lipid peroxidation (antioxidants) in brain ischemia/trauma is apparent. some known drugs (e.g. anti-arrhythmics or histamine h2-antagonists may act as non-specific antioxidants. howe~er, recently, interesting new membrancespecific antioxidants (e.g. 21-aminosterdids) have been designed. subt&e effects on iron redox chemistry contributes to the potent antioxidant activity of these 21aminosteroids. since a few years, one area that greeted enthusimastlcally in clinical medicine is that of reoxygenatlon injury or ischemia-repeffusion, a phenomenon accepted to make a majo:" contribution to organ dysfunction in trauma, shock and sepsis through the formation o( oxygenated free radical species. however, thei detection in vivo always remains a difficult challenge. efforts have been made recently to directly establish the formation of free radicals in biological samples as complex as blood, plasma or tissue with the use of electron spin resonance (esr) spectroscopy. using spin trapping agents, the presence of reactive carbon-and oxygen-centered radicals has been demonstrated in animals blood submitted to heart, renal and intestinal ischemia-repeffusion or to liver transplantation. recently, the in vivo formation of free radicals in the cerebra; extracellnlar fluid during cerebral isehemia-repeffusion has been assessed by the spin trapping technique coupled to brain microdialysis. esr investigations have also been conducted to detect endotoxin-induced free radical generation in rat or baboon liver and oxygen free radicals (ofr), produced both at intra-and extra-cellular levels, seem to play a major role in the pathophysiology of tissue injury and organ dysfunction in sepsis, through induction of lipid pemxidation in cell membranes. oxidative damage can result both to an ofr-overpmduction or to a depletion of endogenous antioxidant defenses, which are represented by scavenger enzymes (e.g. sod), hydrosoluble and liposoluble antioxidants (e.g. ascorbate, vitamin e), and other mechanisms such as metal-ion sequestration. in animal models, oxidative damage has been proved by detection of increased levels of lipoperoxidative products, and a depletion of antioxidant defenses was found both in plasma and tissues. in the few clinical studies a similar results have been reported, and oxidative damage appears to correlate with dic, with organ dysfunction and with red blood cell deformability. in a group of critically ill septic patients we found increased levels of conjugated dienes (cd) (bioproducts of lipoperoxidation) and decreased plasma levels of alpha-tocopherol and coenzyme qlo (coqi0)(endogenous liposoluble antioxidants). a relationship between cd and uric acid (p<0.004) was found, may be related to xantine-dehydmgenase to xantine-oxidase conversion. antioxidant depletion is due both to overconsumption and to a nutritional deficiency, even if a reduced synthesis can also be present. in fact one more relationship between coqi0 and plasma cholesterol (p<0.0005) demonstrates the commun hepatic biosynthetic defect. is there any role for antioxidative therapy in sepsis? can it achives a significant improvement in the septic course, organ dysfunction, and final outcome? several antioxidant drugs, have been evaluated in experimental and clinical sepsis: scavenger enzymes, natural antioxidants, alloporinol, 21-aminosteroids or lazaroids, have been proved to reduce lipopemxidative damage, to protect organ dysfunction, and in some cases to improve survival. several questions must be addressed in evaluating safety and utility of antioxidative therapy. more specific and standardized methods must be applied to detect and quantify the lipoperoxidative damage. antioxidant drags must act selectively on ofr-production, without interference with other than that are beneficial for the organism. antioxidants must be able to achive in adequate amount the tissue or cellular compartment where their action is required. many antioxidants have also a pro-oxidative effect which can he detrimental in some conditions. the timing of administration, the dosage used and the association wih others antioxidant drugs must be defined. nutrition plays an important role as antioxidative therapy, since it supplies all compounds needed to maintain adequately levels of endogenous antioxidant or to support their synthesis. thiobarbituric acid reactive substances (tbars) concentration in serum has been determined in a large population of healthy subjects and in patients suffering acute hepatitis and chronic cases of hepatitis c, as well as several other processes. the correlation with different physiological and clinical parameters in these populations is also presented. treatment with interferon of the chronic active hepatitis c patients, 5x10 u three times a week during two months, led in those patients whose sgpt activity normalized in serum, to a concomitant decrease in serum tbars content. the possible theoretical involvement of peroxidation and antioxidants in this beneficial effect of i~terferon in hepatitis c patients is discussed. the results presented confirm the value of tbars as laboratory test in the management of disease and as a useful tool for the study of pathogenic and/or therapeutic mechanisms. 4-hydroxyalkenals are among the different substances measured by the tbars assay. these compounds show several biological effects that have been demonstrated mainly in different experimental models. we have studied the capacity of different tissues to conjugate these compounds patients surviving the initial subarachnoid hemorrhage (sah) from a ruptured in~raeranial aneurysm are prone to develop cerebra] ischemia from vasospasm which is associated with significant morbidity and mortality. among the many treatments that have been prol~osed to treat this condition, none has been shown to be satisfactorily effective. recently, a new drug, namely the 21-aminosteroid tirilazadmesylats, has been studied in a large, prospective, multicentor trial for its effectiveness to improve the outcome of patients with aneurysma! said. the study was conducted in europe and australia in 41 neurosurgical centers and included 1023 patients. all patients received presently accepted standard treatment, including nimodipine. patients were randomized to four different groups: placebo and 8 groups of tirilazad in escalating dosage (0.6 -6 mg/kg/day). the clinical outcome was compared for these 4 groups and cerebral a~giography was performed in 75 % of patients on day 7 -11 follovang sah, to study the effect 0f'the dflf~ off cerebral vasospasm. the study reached its planned endpoint 6 months ago. preliminary results ~how a significant improvement with regardto mortality in those patients receiving the highest tirilazad dose as compared to placebo and the two lower dose groups. additional beneficial effects were seen in the 6 rag/day groups with regard to the occurence of symptomatic vasospasm and good clinical outcome. although the final analysis of all data is still pending, these results suggest that tirilazad-mesylate could represent a major improvement for the treatment of patients with sail. experimentally lipidperoxidation products (lpo) are increased in acute pancreatitis (ap) due to oxygen radicals (or). the purpose of this clinical study was to determine the antioxidant status and lpo in patients suffering from ap and to evaluate the effect of antioxidant treatment with sodium selenite (se) thus improving the function of the se dependant glutathione peroxidase which is the major detoxifying system for or. materials and methods: in z0 patients sufferin s from severe ap (c-reactlve protein >120 me/l) we determined on day 1 and day 8 after admission the lpo ma!ondialdehyd (mda), conjugated dishes (cd), reduced (gsr) and oxidized (gssg) glutathione, the vitamins a,c,e and se. moreover the patients were evaluated clinically using the ranson and the apache ii score. i0 patients were randomly treated with 600 ug/day of se for 8 days. results: all patients suffered from a severe depletion of antioxidants,especially a low concentration of se (only 1/3 of normal). thereby the increase in lpo correlated with the clinical course. during se treatment lpo decreased and the levels of antioxidant vitamins improved. se had no influence on leth-slity the lenl or the chan in rs or ap ii. background: since reperfusion injury occurs when oxygen is reintroduced into ischemic tissue, the ideal timing for administration of therapeutic compounds aimed at ameliorating oxygen radical mediated injury is at the time of initial fluid resuscitation. currently used colloid or crystalloid preparations do not provide optimal, or even significant, anti-oxidant protection. systemic iron chelation affords protection against the iron catalyzed components of oxygen and lipid radical mediated tissue injury. the conjugate resulting from chemical attachment of the clinically approved iron chelator, deferoxamine (dfo, desferal ®, ciba), to hydroxyethyl starch (hes) represents a novel approach to colloid based fluid resuscitation. hes-dfo contains 85% hes and 15% chemically bound dfo. the polymer-drug conjugate has a lower molecular weight than that of hes in order to allow more rapid excretion. results: preclinical and initial clinical trials indicate that hes-dfo is well tolerated, even at high doses. in animal studies, fluid resuscitation with hes-dfo does not significantly improve central hemodynamic recovery beyond that observed with hes, but hes-dfo seems to afford better protection of microcirculation in organs at risk (lung, liver and gut), possibly by decreasing neutrophil sequestration. in a burn model, total fluid requirements are lower and oxygen utilization higher in hes-dfo treated animals compared to hes controls, suggesting decreased vascular leak and improved tissue perfusion. conclusion: hes-dfo represents a means by which potent antioxidant protection can be administered at resuscitation. iron has been suggested to play a pivotal role in oxygen flee radical mediated tissue injury. in vitro experiments indicated its critical role as a katalyst in hydroxyl free radical generation 0fenton-reaction). since iron chelator deferoxamine administered in shock alone demonstrated severe side effects, a hydroxyethylstarch (hes)daferoxamine (dfo)-conjugute was used to modulate oxygen free radical injury during the ischemia/reperfi~ion syndrome induced by hemorrhagic shock. methods. female lewis rats (190-215 g, n>6; pentobarbital anesthesia 50 mgjkg), in hemorrhagic shock ( the aim of the study was to elucidate (1) whether the generation of or would affect lung and kidneys as primary shock organs in the very early phase of sepsis and (2) whether dfo-hes could prevent this tissue damage. methods: in 67 rats sepsis was induced by cecal ligation puncture (clp) peritonitis. the animals were randomly assessed to 2 groups: one group was treated with 3 ml dfo-hes (100 mg/kg iv), the other rats received solely 3 ml of the carrier starch solution. 30, 60, 120, and 240 min after induction of sepsis respectively, the animals were sacrificed, the organs collected, and tissue contents of glutathione (gsh), malondialdehyde (mda), myeloperoxidase (mpo) and conjugated dienes (cd) determined. plasma samples were obtained for analyses of endotoxin (chromogenic lal test). blood pressure (map) was measured via a carotid artery catheter. results: clp caused sepsis with high (> 1.84 eu/ml) endotoxin levels. map in both groups decreased slightly but significantly during sepsis regardless any treatment. in the lungs mpo concentration was increased (p<0.05) in the lies group already 30 min after sepsis induction. concomitantly, tissue gsh level decreased and lipid peroxidation was pronounced as shown by elevated mda and cd levels. dfo-hes diminished tissue pmn accumulation and mpo concentration. moreover, at each time point lung mda and cd levels were lower (p<0.02). histomorphological examination showed marked micro-atelectases, destruction of the alveolar septa, and splicing of the basal membranes in the lies group. in contrast, in dfo-hes treated rats the alveoli remained well-ventiiated and only some enlarged reticular fibers without splicing were observed. almost similar results were found for the kidneys. mpo levels differed neither within nor between both groups. the slight decrease in gsh levels seen after 60 min in the dfo-hes group seems to demonstrate an oxidative stress to a lesser degree. the most impressive effect of iron chelation, however, was revealed by the lipid peroxidation products. at each time point, mda and cd levels were lower (p<0.02) compared to the hes group. light and electron microscopic examination disclosed tubulotoxic and mitochondriat damages while dfo-hes lxeatment prevented that alterations. conclusion: both the biochemical and histological results of this study reveal an early and remarkable generation of or in peritonitis-induced sepsis. thereby, these or obviously cause pulmonary and renal tissue damages, intravenous application of dfo-hes may, however, benefit by preventing early lipid peroxidation of the tissue. the proteolytic irreversible conversion of xanthine dehydrogenase (xd) to xanthine oxidase (xo) is triggered by calcium flux. the aim of our study is to clarify ~he link between intracellular ca2+ levels and xo activity determined by uric acid release, and to evaluate the efficacy of verapamil, on the generation of hydrogen peroxide associated with reperfusion by assaying lactate & pyruva~e release and the levels of cytosolic free nad /nadh ratio. experimental protocol consisted of :(a) non ischemic/reperfused experiment in which normal cardiac slices of rats were perfusated with oxygenated kreb's ringer phosphate buffer containing glucose (150mg%) and bovine albumine (5gm%) for 60min at 37°c.it composed of 3 groups, group aa (control group), and groups ab & ac (perfusate supplemented with verapamil in the dose of loo&200 mi% respectively). (b) ischemic reperfused experiment in which ischemic cardiac slices were obtained from rats subjected to 15 min ~aemorrhage.lt was also divided into two groups; group ba and bb (verapam~/ 200mi% added to perfusate}. verapamil stimulated uric acid release from normal rat cardiac slices were 267% in group ab and 767% in group ac(dose related). rates of uric acid release is enhanced by verapamil in group bb. moreover, rates of uric acid release in groups ac & bb are insignificant. in verapmil added groups (group ab, ac & bb), increase uric acid release is associated with an enhancement in pyrurate release and with increase levels of cytosolic free nad+/nadh ratio, although it is not evident ~ ischemic group (group ba).it is concluded that the conversion of xd to xo is calcium independent. eicosanoids like thromboxane a2, leukotriene b4 and leukotriene c4 are known as promoters of initial inflammatory reactions. we investigated whether oxygen radicals (or) are able to induce a release of these eicosanoids in whole blood. blood from healthy volunteers was incubated with xanthine oxidase/hypoxanthine to generate oxygen radicals. after 0,5,15,30 and 60 minutes plasma levels of thromboxane b2 (txb2), leukotriene b4 (ltb4) and leukotriene c4 (ltc4) were determined via elisa technique. another volunteer had taken 1000mg aspirin one day before taking the blood sample (no7). results: txb2 plasma levels increased from 14pg/ml at 0min to 144pg/ml, 350pg/ml, 370pg/ml and 660pg/ml at 5,15,30 and 60min (p<0,01) . ltb4 and ltc4 plasma levels showed an increase during the first few minutes (ltb4: 0min: llpg/ml,5min: 87pg/ml; ltc4: 0min: 23pg/ml, 5min: 97pg/ml (p<0,05)) followed by a decrease to normal values at 15min. in the sample no7 the cyclooxigenase-pathway was completely inhibited, the txb2 plasma-levels did not alter at all, whereas ltb4 and ltc4-plasma levels weren't affected. opallogeneic blood transfusion jane shelby, ph.d., and edward w, nelson, m.d, there have been numerous investigations dudng the last two decades examining the effect of surgery, anesthesia, blood loss and transfusion on vadous immune parameters in humans and animal models. there appears to be concurrence among several well controlled studies that transfusion of whole blood (containing leukocytes), has regulatory effects on immune ceil function which include decreased cell mediated immune response, and inhibition of il-2 secretion. these effects occur following transfusion alone and in con.cart with the distinct immune effects of surgery, trauma and anesthesla, the clinical consequences of this immune modulation by transfusion include decreased allogeneic response to transplanted organs, which has been exploited clinicelly in renal transplant patients. additionally, there is evidence for a strong association with increased risk for infection in transfused patients following surgical procedures. aiiogeneio blood transfusions have been shown to inhibit cellular anti.bacterial mechanisms, causing increased susceptibility to bacterial pathogens, in humans and in animal models. there is also concern that allog~neic transfusion may adversely affect cancer patients, resulting in decreased disease-free survival. several stategies have been proposed to minimize the adverse effects of blood transfusion. there is evidence that the risk of immune mediated infectious complications associated with transfusion may be greatly minimized wlth the use of autologous blood and leukocyte free allogeneic blood.products in surgical and trauma patients, it also appears that the inhibition of cellular immune response and il-2 productiorl following atlogeneic blood transfusion may be mediated by increased prostaglandin e2 secretion, and that immune response may be preserved in allogeneio whole blood transfused subjects receiving c3lc~oxygenase inhibitors such as ibuprofen. among these are various alterations in immune function. efforts have therefore been made to utilize alternatives to homologous transfusions. these include the use of autologous predonation, supplemental iron therapy, and recombinant human erythropoietin. although initially considered innocuous, these therapies are now recognized to have potential deliterious immune sequelae. erythropoietin, by its ability to lower serum iron levels, can impair both lymphocyte and nk cell activity. autologous donation impairs nk cell function. finally, supplemental iron therapy can stimulate bacterial growth and increase the rate of infectious complications. this talk will present a discussion of these factors as well as a weighting of their importance. r.l rutan, rn;bsn, shriners burns institute and the university of texas medical branch, galveston tx, usa the serious sequelae of homologous blood transfusions have resulted in vigorous efforts at identifying alternate therapies for correcting red blood cell (rbc) deficits. erythropoietin (epo) was hypothesized to exist in the early 20th century, however the protein was not isolaled until 1977. the human gene was identified and cloned in 1983, which permitted the production of epo through recombinant techniques. the earliest clinical trials were performed in anemic end-stage renal failure palients on hemodialysis. treated patients experienced increases in erythropoiesis with normalization of hematocrit and hemoglobin levels, cessation of lrans-fusion requirements and improvement in general wellbeing. these studies, however, identified side effects of epo treatment such as hypertension, seizures and ee deficiency. volunteer trials have established that the hypertension is not a direct pressor effect but rather the result of abnormally rapid increases in red cell mass in the face of the incompetent volume-controlling mechanisms of the end stage renal failure patient. lower doses of epo and the subsequent gradual increases in red cell mass are associated with significantly lower incidences of hypertensive complications of epo therapy. likewise, seizure activity is not the result of a direct epileptogenie effect but parallels the incidence of hyper-tensive-related sequelae during high.dose epo treatment. in cross-over designed studies, pre-existing iron deficiency has been demonstrated to decrease or negate stimulated erythropoiesis but effective-hess can be restored with appropriate fe supplementation. exogenous epo is effective whether given by iv or sq routes and dose response curves do not vary with route of administration. increases in rbc mass are directly related to the dose of epo, both in amount and frequency of administration although there is a 3-5 day time lag between the first epo dose and laboratory indications of its action (i.e. increase in the number of reticulceytes in peripheral wood). epo is currently labelled for use in the treatment of anemias associated with end-stage renal disease and aids. however, its use in the surgical population has been explored because of its unique direct dose-response, epo has been used to effectively increase the blood harvest amounls in autologous pre-donation, significantly increase hematocrils in children following thermal trauma and successfully increase red blood cell mass following essential surgical procedures in patients with religious aversion to transfusion. by blood transfusion in colorectal cancer surgery mm heiss md, ch delanoff md, r stets md, j hofinann, e faist md, kw jauch md, fw schildberg md allogeneic blood transfusions are associated with an increased risk for postoperative infections in colorectal surgery when compared with autologous blood transfusions. attribution of this effect to immunomodulation was suspected in our previous study (lancet 1993; 342:1328-33) . task of the recent investigations was to analyze which specific effector systems were affected in-vivo by this transfusion-associated modulation. for global in-viva assessment of cell-mediated immunity (cmi) multiple recall skin-reactions were applied prior and post-operative. the specific humoral immune mechanisms were investigated by applying tetanus-toxoid one day preoperatively and deterimnating the quantitative igg-response. for indication of macrophage stimulation in-vivo tnf-levels were determinated by bioassay. dth-responses were significantly suppressed (p<0.05) in patients receiving allogeneic blood (n=36) or operated without blood transfusions (n=17). dthresponses were not suppressed and tendentiously increased in patients with autologous blood transfusions (n=24). in contrast, specific igg-levels increased sigmficantly (p<0.05) in patients receiving allogeneie blood (from 1.6 + 4.4 to 9.7 _+ 18.4 ie/ml) whereas in patients receiving autologous blood a smaller increase (from 1.9 + 4.1 to 4.0 + 6.7; p=0.068) was observed. tnflevels demonstrated a similar pattern with a higher increase in patients receiving allogeneic transfusions (l 1.4 + 12.0 to 28.5 + 11.4 u/ml) compared to those patients with autologous blood (8.7 + 12.5 to 17.1 + 23.0). in conclusion these data indicate that allogeneic blood transfusions lead to a remarkable macrophage/rhs stimulation. this is corroborated by the boostered humoral igg-response which was initiated before onset of surgical trauma and blood transfusion. concerning cmi this caused a substancial suppression probably due to a stimulated secretion of immunosuppressive monokines. objective: firstly, to analyse the concentrations of the cytokines tumor necrosis factor (tnc), interleukin-1 (il-i), interleukin-6 (il-6) and coagulatioo/fibrinolysis parameters in postoperatively retrieved blood from a surgical area, secondly to characterize the correspanding cytokine patters in the patients and thirdly to study cytokine concentrations in the initial portion of drainage blood from a surgical area. materials and methods: blood retrieval was performed in a closed-loop system without anticoagulant during 4-6 hours after surgery in 10 patients undergoing arthroplasty (9 hips and 1 knee). 7kf, il-1, it-6, thrembin-antithrombin complexes (tac) and antithrombin (at) ~ere determined in shed blood. patient plasma tn v, il-i and il-6 concentrations ~ere analysed at the beginnlqg and end of the 4-6 hour blood retrieval period. in a separate study (5 hip arthroplasties) f~f, il-i and il-6 ~ere determined in the initial portion of drainage blood. cytekine analyses ~re performed usiog ipmuooassays. an omidolytic method was used for at determinaf.ion and tac was analysed by elisa. n~n-poram~tric tests was used for the statistical comparison. results: the patient plasma il-6 coocemtratiems rose from a median value of 0 to 116 pg/ml, p<o.01, during the blood ret.rieval period. in c was detectable in 4 patients, range:15-50 pg/ml. il-i was not detectable in the patients. in retrieved blood tag was >200 mg/ml in all samples (ref:<4.1 mg/ml) and at was 0. 16-0.51 units/ml (ref:o.80-1.20) . the il-6 concentrations in retrieved blood was >1000 pg/ml in all samples. tn v or il-i was not detectable. in the separate study, (n=5), characterlzing eytokine content in the initial portiere of drainage blood, in= (range: 11-277 pg/ml) and il-i (range:5-125 pg/ml) ~re present in all samples but ii-6 (range:o-25 pg/ml) was detectable in o.qly one semple. conclusion: theses findings indicate that hypereoagulability and hic~ ccrcentratioos are present in retrieved blood. the cytokine pattern in the initial portion of blood from a surgical area differed from these observed in retrieved blood and in the systemic circulation. to identify the role of both autologous and homologous blood on postoperative infections in elective cancer surgery. materials and methods: 159 patients with colo-rectal cancer submitted to curative elective surgery were prospectively studied. on hospital admission the following nutritional measurements were assessed: serum level of albumin, cholinesterase, delayed hypersensivity response , total lymphocyte count and weight loss, as were age and sex, duration of operation , operative blood loss, amount and type of blood given, pathological dukes' stage of the disease and the attending surgeon were also recorded. results : eighty-four patients (52.8%) were perioperatively transfused. thirty-six (42.8%) patients were given autologous blood , while 48 (57.2%) received homologous blood. no patients received both autologous and homologous blood. twenty eight (17.9%) patients developed postoperative infections. non transfused patients had a 9.3 % infection rate , those receiving autologous blood had a 13.9 % infection rate, whi]e in the homologous blood group the infection rate was 33.3% (p < 0.001). univariate analysis showed that infections were significantly related to operative blood loss (p<0.01), length of operation (p<0.05) blood transfusion (p<0.05) and attending surgeon (p<0.05) . multivariate analysis identified homologous blood transfusion as the only variable related to the occurrence of postoperative infections , while the other variables failed to reach statistical significance. blood transfusion (bt) remains an essential life-saving treatment for surgical patients. however, besides the beneficial short-term impacts, negative longer-term effects are observed, which include various alterations in the immune responsiveness. in surgical patients these alterations may contribute to the increased risk for infections and cancer recurrence. since relatively few data demonstrate immunologic changes occurring in other lymphoid compartments than blood after bt, we studied the effect of et on the frequency and responsiveness of immune cells in bone marrow (bm), spleen (spl) and blood (b) in a rat model. normovalemic, 2 month old rats were transfused intravenously with syngeneic heparinized venous blood (3x2ml, every other day), and 3, 7 and 14 days after the last transfusion bm cells ( leh is an experimental oxygen-carrying resuscitation fluid. since leh is cleared from the circulation primarily by the mps, its effect on the development of sepsis and the nature of its relationship with the mps remain a major concern. preliminary in vivo data from our laboratory failed to show any leh effect on the hemodynamic and hematologic responses to endotoxin lipopolysaccharide (lps) in the rat. in contrast, leh exacerbated the lps-induced tnfa production and early mortality. the exacerbation of early mortality by leh was attenuated by pretreatment with the tnfu synthesis inhibitor rolipram. ex vivo, peritoneal macrophages from rats treated with leh and lps have shown increased il-lg mrna signal as compared to lps alone. also, leh increased tnftx production by peritoneal macrophages in response to lps stimulation in vitro. additionally, recent pilot studies indicate that leh attenuates pma-induced superoxide production from rat peritoneal macrophages and that leh augments fmlp-induced migration of human monocytes. taken together, these data strongly support possible interactions of leh with the mps and therefore the nature of such interactions should be further explored. over the last decade, we have developed liposome encapsulated hemoglobin (leh) as an artificial oxygen carrying fluid, or blood substitute. our efforts have focused on studies to define the safety and efficacy of this resuscitative solutions. leh consists of distearoyl phosphatidylcholine, cholesterol, dimyristoyl phosphatidylglyeerol, and alpha tocopherol in a 10:9:0.9:0.1 mole ratio and can encapsulate hemoglobins of different origin (bovine, human, recombinant human). leh is fabricated using hydrodynamic shear to create an average particle size of 0.3 microns. leh can be lyophilized using disaccharides and stabilized in the dry state and easily reconstituted before administration. histopathology and clinical chemistries indicate that leh rapidly accumulates in tissue resident macrophages in small animals injected in the tail vein, principai1y in the liver and spleen. the consequences of accumulation in the reticuloendothelial system are manifest by transient increases in liver transaminases (ast, alt), bilirubin, and bun over 24-48 hours with no change in biliary function (ggt, ap) . clearance through the liver and spleen is observed over the course of 1-2-weeks. more recent attention has been focused on secondary consequences of leh administration especially with regard to inflammatory eytokines. leh does not elicit expression of tumor necrosis factor in vivo and in isolated macrophage cultures, but does result in a transient increase in serum il-6. we have also examined the interaction of leh with lps in vitro macrophage culture to further understand how this blood substitute may effect the immune system. we have labeled leh with technetium-99m (99mtc) to study the biodistribution of leh non-invasively in anesthetized rabbits. rabbits were infused with a 25% topload of leh (200 mg of phospholipid, 2.5 g of hemoglobin per kg of body weight) and imaged continuously with a gamma camera. at 20 hours, images were again acquired. animals were then sacrificed and tissue counts obtained, images revealed an initial rapid uptake bythe liver, 8% at 30 minutes and 15% by 2 hours. the spleen accumulated activity at a slower rate, 3% at 30 minutes and 7% at 2 hours. at 20 hours, autopsy biodistribution studies revealed that approximately 42.6% of the dose is in the blood pool, 15.4% in liver, 18.1% in spleen, 3.2% in lungs, 2.4% in muscle and 1.6% in urine, with trace levels in kidney, brain and heart (<1 °/o). in a hypovolemic model, rats were 10% or 50% exchange transfused with 99mtc-leh. in the 10% exchange model, 99mtc-leh was rapidly taken up by the liver and spleen with minimal activity in the circulation at 20 hours. with the 50% exchange, 50% of the leh was in circulation at 20 hours. the interaction of leh with platelets labeled with indium-111 was also studied. after infusion of leh, the labeled platelets rapidly moved from the circulation to the lungs and liver. over the next 30 minutes, the platelets gradually returned to circulation. this effect was not seen with iiposomes of the same lipid composition but containing no hemoglobin. non-invasive imaging is proving to be a very useful tool for the investigation of leh. the need for a safe, efficacious and commercially viable blood substitute is unequivocal. of the several strategies pursued to invent an adequate blood substitute, liposome entrapped hemoglobin (leh) has been already established as a leading possibility. major advances in liposome technology have already resulted in liposome preparations compatible with clinical use for drug delivery. recent technological advances made by the u.s. naval research laboratories resulted in the capacity to entrap hemoglobin into liposomes in a way which secludes hemoglobin from interacting freely with biological systems. the leh produced has already been tested in in vivo systems and was foun.d to be well tolerated. moreover, the leh originally produced as a solution can be transformed into a lyophilized form which can be reconstituted and delivered as a fresh solution. while important milestones in leh development for a practical blood substitute have been achieved, several issues remain to be explored. most notably, the long term consequences of leh on host defense mechanisms and, in particular, immune cell function. in addition, it is important to understand more fully the metabolic fate and repercussions of leh delivered at clinically relevant dose/schedule regimens. finally, while leh is a highly promising strategy for a blood substitute, the present formulations consist of human hemoglobin derived from human blood, to improve the safety profile, a recombinant preparation for liposome entrapment will be much desired, aa-ginine, a semi-essendai dietary amino acid, possesses several unique and potentially pharmacologic properties. argirdun is a potent secretagogue for pituitary growth hormone and prolacfin and for pancreatic insulin and glueagon; it modulates host protein metabolism by increasing nkmgen retention and enhancing wound collagen synthesis. it also is a potent t call function regulator. ait of these effects coupled with its relative lack of toxicity and safety make it an a~antive nulritionai pharmacologic agem (t). rodents fed supplemeutal arginine exhibit increased thymsc weight which is due to increased numbers of thymic lymphocytes present in the gland. thymic lymphocytes from animals fed supplemental ar~e demonstrate increased blastogenesis in response to coma. and pha (2) . peripheral blood lymphocytes from humans given supplemental arginine also have heightened mitogunic responses to mitogen or antigens (3) . in postsurgery padents supplemental arginine abrogates or diminishes the deleterious effects of trauma on lymphocyte responsiveness and restores peripheral blood lymphocyte responses much faster than observed in controls. overall host immunity is also enhanced by arginine. allograft rejection is enhanced and septic animals survive longer when given supplemental arginine (4) . tumor bearing urginine-supplemented animals have decreased tumor growth and enhanced survival (i). lastly, asgmine can induce t cell maturation and t cell mediated responses in athyrnic nude mice. arginine also has remarkable effects on host nitrogen metabolism post-injury. in increases nitrogen retention in healthy human volunteers and in surgical patients. this beneficial effect on overall nitrogen metabolism is accompanied by a unique effect on the healing wound. supp]emental arginine increases wound collagen synthesis which also translates into increased wound breaking strength (5) . arginine has no effect ou epithelialization. douglas w. wilmom, m.d. boston, ma gintamine is the most abundant amino acid in the body, but it has long been considered a nonessential amino aeid because it is synthesized in many tissues. fohov~g st,~'vation~ injury or infection, skeletal muscle pmteln inoresses its net tale of degradation and releases amino acids into the blunds~mm at an aocelerared rate. app~o)~mately one-third of the amino nitmgea is ghitamine, which is metabolized by the kidney where it parth:~pates in acid-base homeostasis, is the primly ~ for lymphocytes, mac~optmgcs and untexocyms, and contm'butcs to the synthesis of giumth~une. olmamine degrades slowly while in ~olu~ou, especially at usual room teml~mtums. because giulamine was considered nonessential, it has beer absent r'om nil intravenous and most gluts.mine should be considered a cendittona]ly essential nutrient for individuals with serious ilinesses, uspccially those confoanded by infcctinn and inflammation. over the uc~:t 5-7 years, glutamine will be incorgorated into most feeding formulas designed for patients with critical illness. 526 o]~ga-3 pufa there continues to much interest in the application of the 0mega-3 pufa in clinical nutrition. the basic principle has been that the 0mega-3 pufa will displace arachidunic acid and result in a decrease in eic0san0id production. in addition these changes in pufa will after the physical characteristics of the membrane including flujdity, receptor function and transmembrane signals. animal studies have shown that there is omega-3 incorporation with continuou~ enteral feeding both in control and endotoxic animals within 3 days. this includes the liver, spleen, circulating and alveolar marc0phages and the lung. this incorporation resuls in significant changes in the eicosan0id production including pgf 2 and ket0-pgflalpha. there is improvement in the cardio-vascular reep0nse of these animals with ~ecreamed lactic acidosis and improved cardiac contractility. as well there is improved immune function with improved t cell response to mit0gens. the ~ of a mumber of pharmacological agents blocking cicosanoid production can enhance the cell effects of 0mega-3 pufa. clinical studies using short term entsral nutrition with 0mega-3 either alone or with other enteral supplements in a number of clinical settings have shown significant 0mesa-3 incorporation and decreased eicosan0id production. these positive results must be discussed with the additional evidence that long term omega-3 supplementation decrease eic0san0id production but als0 induce a state of immune suppression that is capable of increasing transplant sunvival. these 10ng te~ inune effects may benefit clinical conditions including rheumatoid arthritis and cr0hn' disease early enteral nutrition instituted i~mediately afte~ injury will decrease the entry of bacteria into the intestinal wall and decrease the number of bacteria that translocate into the portal blood. these reductions are associated with & decreased catabolic response, decreased plasma cortisnl levels, end decreased vma excretion in the urine and prevention of mueosal atrophy. sdecific nutrients also affect the transloeation process. addition of arginlne to the diet significantly improves the ability to kill translocated organisms. however. translooetion across the gastrointestinal barrier is not affected. in contrast, glutamine diminishes the rate of translooation across the imtestinal barrier and also improves killing of the beetarla that do translooate. the omega 3 fatty acids in the form of fish oil slightly decrease the rate of translocation but more significantly increase the ability of the animal to kill translo~ated organisms, all three dietary additives, i.e. argini~e, glu=amine and fish nil. significantly improve survival, hut adding glyoine or medium chain triglyeeridem do not, combinations of srginine and glutamlns, glutamine and fish oil, and fish ell end arginine each improve survival, and to a greater degree than a combination of all three. these studies add further evidence that translocation is an important determinant of survival after injury, early feeding with immunonutrlent enriched dices will improve survival and dsarease transloeation to varying degrees, depending upon the nutrients provided. objectives: we studied effects of supplementing a commercial enteral diet, impact r (imp, sander nutr lnc), with fiber (imp/fib) or alanyl-glutamine (imp/ag, exogenous glutamine (gln) 14 gms/l) on influencing the incidence of bt to mesenteric lymph nodes (mln) in burned mice. fiber has been shown to improve gi integrity under certain stress/treatment conditions. the dipeptide ag is a water-stable source of gln, which is a specific fuel for many cells including enterocytes. traumacal (trcal), a high-protein, high-fat enteral diet (mead johnson iuc), was also studied, as well as rodent chow (harlan teklad inc), which contains very high protein & fiber. methods: anesthetized cf-1 mice aged 8-12 wks received 32% tbsa fullthickness dorsal burns & were resuscitated with 2 cc ip saline. diets were allowed ad lib; caloric intakes were comparable in all gps except fasted gp (fast 24 hrs, chow 24 hrs). at 48 hrs postburn mln were sterily removed, homogenized and plated on heart brain infusion agar; cfu/g mln tissue were determined. bt was analyzed by fishers exact test, cfu/g by anova-bonferroni. * p<0.01, ** p<0.05 compared to imp and burn-fast gps. background. infectious complications following trauma, major operation, or critical illness adversely affect hospital cost and length of stay (los). some key nutrients have been shown to possess immune enhancing properties. this multicenter trial was conducted to determine if early administration of an enteral formula supplemented with arginine, dietary nucleotides and fish oil can decrease los and infectious complications in icu patients. methods. this was a prospective, randomized, double-blind study of 326 adult icu patients who required enteral feeding for > 7 days. patients entered the study within 48 hr of the event, were stratified by age and disease, and were randomized to receive either the supplemented formula (impact®) or the conventional formula (osmolite ® hn). feedings were initiated at full strength and advanced to at least 60 ml/hr by 96 hr after event. results. both groups tolerated administration of formula well. for patients fed > 7 days, the median los was 25% shorter (p=o.ol) for the--supplemented group (21 days) compared to the conventional group (28 days). the incidence of most infectious complications was lower in the supplemented group, but this difference reached significance only for urinary tract infections (p=o.o05). the supplemented group had a significantly shorter los from onset of infectious complication until discharge for patients with pneumonia (19 vs. 27 days) and skin/soft tissue infection (19 vs. 37 days). conclusions. administration of the supplemented formula was safe and well tolerated. when fed > 7 days, it reduced the incidence of most infectious complications, and significantly reduced los. materials and methods: twenty-seven patients were randomised into 3 groups ( n= 9 each) to receive either a standard enteral formula, the same formula enriched with arginine, rna and omega 3 fatty acids (enriched group) or isonitrogen, isocaloric parenteral nutrition. early enteral nutrition was started within 12 hours following surgery (10 ml/hour). it was progressively increased reaching a full regimen on day 4. on hospital admission and on post-operative day 1 and 8, the following parameters were assessed: serum level of transferrin , albumin , prealbumin, retiool binding protein (rbp), cholinesterase. delayed hypersensitivity response, igg, igm, iga, lymphocyte subsets and monocyte phagocytosis ability were evaluated on admission and on post-operative day 1, 4, 8. the three groups were comparable for sex, age, cancer stage, type and duration of surgery, intra-operative blood loss and amount of blood transfused . in all groups a significant drop in all the nutritional and immunological parameters was observed on postoperative day 1. comparing post-operative day 1 versus day 8 a significant increase of prealbumin (p<0.02) and rbp (p<0.005) was found only in the enriched group. with respect to immunological variables an increased phagocytosis ability (p<0.001) and a significant recovery in delayed hypersensitivity response (p< 0.005) was observed only in the enriched group. conclusions : these data are suggestive for a more effective post-operative recovery of both. nutritional and immunological status in cancer patients fed with enriched enteral formula. gastrointestinal intolerance was equivalent (18% in each group) and laboratory screening confirmed that both diets were safe. when analyzing clinical outcome for all patients, there were no significant differences in septic complications (immun-aid = 41% vs vivonex ten = 35%), mean mof score (immun-aid = l.b vs vivonex ten = 3.6), or mortality (immun-aid 0% vs vivonex ten = 6%) . kowever, when analyzing the subgroup of patients with severe injury (iss or ati _> 25), patients receiving immun-aid appeared to have fewer septic complications (33% vs 50%) and their mean mof was significantly lower (1.8 _+ 0.7 vs 5.8 + 1.7, p = 0.05, student's t-test) . these preliminary data indicate that immun-aid is tolerated well when aggressively delivered immediately postinjury. the ultimate affect on clinical outcome appears ~avorable for immun-aid, but needs to be confirmed in larger patient groups. kemp?n, m., neumann, h.a., he i[michh b: as both increased, normal and reduced phagocytic capabilities of polymorphonuclear leukocytes (pmn) and monocytes in acute batterial infections have been reported, the role of phagocytes in patients with severe sepsis is less clear.we examined pmn and monocytes from 10 patients in septic shock and 17 heailhy votunteers for phagocytic function. phagocytosis was determined by flow cytometry (facscan) and was measured by the ability of pmn and monocytes to phagocytose e.coli marked with fluorescent antibodies. a septic shock was defined by the presence of a ~ource of i, nfoctiqn with a known bacteriology, distinct signs of a systemic response and defined minimum scores in 3 icu scoring systems indicating the presence of a multiple organ failure. additionally we examined how phagocytosis is influenced when a new enteral diet formulation containing substrates suggested to improve immune function or arginine, one of its major compononts, is added in vitro in defined concentrations and incubated for 10 minutes. pmn (p{o,05) and monocytes (p<o,01) of the septic patients showed a significantly enhanced phagocytosis compared tolhe phagocytes of the healthy group. the incubation with the enteral diet in vitro was followed by a higher dose-related rate of phagocytosis, especially in the healthy group, that was not statistically significant. after addition of l-argintne we atso observed an iqcrease in phagocytosis, that was lower than in the group with the complete diet. using a modern immunefluorescence-cytometric essay we founfl an improved phagocytic function in septic shosk. there are signs for an influence of nutrient substrates on phagocytosis which seems to be complex and should be further investigated. arg and gln were lower in the 49%-bcaa vs 16%-bcaa group; arg was related directly to arg dose and inversely to bcaa dose (p<o.o01 for all). clearances of bcaa increased with increasing bcaa dose (p<o.o01); arg and gln clearances were directly related to the bcaa clearances (rz=o.65, p<o.o0i). relationships between arg, gln and other aa seem to be ruled by patterns involving specific intracellular aa transport systems. regulation of arg and gln clearances through bcaa administration may be related to an increased flux of aa into synthetic processes. intestinal segments from rats immunized by infection with the nematode trichinella spiruus undergo intestinal anaphylaxis or type i hypersensitivity when challenged in vitro with parasite antigen. immunized rats exposed to t-radiation (7gy) and sustained on rat chow,fail to fully express type i hypersensitivity up to 14 days post irradiation (dpi). we hypothesized that enteral nutritional support immediately following irradiation may be effective in preserving mucosal immune responsiveness or in reducing the recovery time after radiation-induced injury. rats were infected with 3x103 t.spiralis larvae. thirty to fifty days later rats received 7gy t-radiation from a co source as total abdominal irradiation (tai). immediately following tai, rats were fed an elemental amino acid diet (eaad) and at various dpi sacrificed and tested in ussing chambers for local anaphylaxis, expressed as antigen-induced ci secretion. the normal ci secretory response to antigenic challenge which is suppressed after irradiation,was restored by 3 dpi when rats were placed on the eaad. antigen-induced c[ secretion is dependent on the interaction o~ antigen with specific ige antibodies on the surface o5 mucosal mast cells and their subsequent degranulation. mast cell-derived 5-ht,histamine and pg, together effect ci secretion.in irradiated rats on rat chow,the failure to respond to antigenic challenge appears to be associated with the destruction of mast cells. they are undetectable with standard staining procedures and mucosal histamine levels are drastically reduced. also,el secretion can be induced by direct stimulation of epithelium with cr secrctagogues and circulating ige levels are normal. despite a more rapid recovery of immune responsiveness in irradiated rats receiving eaad,mast cells were not restored. we conclude that the eaad contributes to an adaptation that supports the expression of local anaphylaxis in the absence of mast cells. total parenteral nutrition (pn) and bowel rest may influence the host response to infection. this study examined the different host response to infection in parenterally and enterally fed animals. forty-three rats were divided into pn group and total enteral nutrition (en) group. they received identically constituted isocaloric isonitxogenous diets for seven days. animals were then challenged intraperitoneauy with 3xl(y escherichia coli and survival were observed. in other experiments, they were sacrificed before and two hours after bacterial challenge. peritoneal cavity was lavaged with 10ml saline and peritoneal exudative cells (pec) were harvested and cultured in vitro for 24 hours with and without lipopolysaccharide (lps). colony f(m, ning units of bacteria (cfu-b) in the peritoncal lavaged fluid (plf) were determined and tnf in the plf, serum and pec culture supernal,ants were measured by l929 bioassay. twenty-fonr hour survival rate in en group was significantly greater than that in pn group (60% vs 0%). the data suggest that local immune responses of pn-treated animals may be depressed with consequent disturbance in the clearance of bacteria. this may lead to a greater systemic cytokine response and resulting in a poor survival after bacterial challenge in pn group. the effects of intragastfic tube feeding of diets enriched with m-6 polyunsaturated fatty acids (pufa) from safflower oil (so) or (o-3 pufa from menhaden oil (mo) on eicosanoid production, and onthe membrane phospholipid fatty acid composition were investigated in a severe acute septic shock model. the percent calories from fat maintained at 30% in the diets was adjusted to provide 10% each of saturated, monounsaturated, and pufa using olive oil and palm oil as filler fats. after feeding for 5 days, lipopolysaccharide (lps, 9mg/100g) was injected through the tail vein. the percent of animals surviving in the mo group was 67% (14/21), and did not differ significantly compared to 57% (12/21) in the so group. the plasma concentrations of tnf ct for the groups of animals fed so (1.2+3.0 ng/ml) and those fed mo (1.8+1.1) did not differ significantly. the levels of prostaglandin(pg)e2, 6-keto-pgflc~ and tumor necrosis factor-ct (tnf-c 0 were determined 90 min after lps injection. the fatty composition (relative mole%) of the liver was determined before (lps-) and 3h after the lps administration (lps+). the data (mean_+sd; n=6) are as follows. group these data indicate that a marked reduction in the plasma levels of poe2 and 6-keto-pgflct for rats fed mo diet was associated with a reduction in aa which was displaced by epa in the membrane phospholipids. further, in these rats, we observed that there was a 2.8% reduction in the percent of epa following lps challenge compared to the basal levels. however, for the group of rats maintained on so diet, the liver membrane phospholipid fatty acid composition before and after lps administration was unaltered. these finding suggest that although a reduction in aa was associated with a decrease in the production of pro inflammatory eicosanoids after 5 days of continuous mo feeding, there was no marked effect on the survival following lps challenge. animal models have been used to examine the effects of various nutrients and nutrient combinations upon the immune system. we studied the effects of standard and specialized enteral formulations on the response of mice to infectious challenge with listeria monocytogenes, influenza a or candida albicans in order to test the efficacy of specialized ingredients. cf-1 outhred female mice (12-15 g) were fed purina #5002 chow, commercially available osmolite hn ®, perative ® or impact*. mortality and tissue burden of pathogen were examined during the 21 d period following induced infection. the results indicate that there were no differences between the groups fed the liquid formulas with regards to mean survival time or % survivors in these models of infection. examination of spleens in the groups challenged with l. monocytogenes and kidneys in the groups challenged with c. albicans revealed no differences in tissue burden of pathogenic organisms. alterations in the length of pre-feeding from 0 to 8 days prior to infection did not affect these results. these data demonstrate that, contrary to previous reports, the use of specialized nutrients did not improve resistance to disease in mice challenged with l. monocytogenese, influenza a or c. albicans as compared with mice fed osmolite hn. animals fed standard chow tended to be more resistant to infectious disease than those fed the liquid formulas perhaps due to the form of diet or complexity of ingredients. the results indicate that these animal models of infectious challenge may be of limited use to distinguish effects of modified nutrient composition of enteral formulas. laboratories the beneficial effects of sesame oil (ses) have been attributed to the presence ofsesamin, a non-fat constituent which is claimed to iultibit a-5 desaturase activity. we investigated the effects of ad libidum feeding of ses enriched diet on fatty acid composition of phospholipids from plasma, liver, on eicosanoid production, and on the protective effects in mice after cecal ligation and puncture (clp) and compared with safflower oil (so) diets. olive and palm otis were added as filler fats to the so diet to maintain the ratios of saturated, and unsaturated fatty acids similar to ses diet. thus, while the total fat remained at 15wt%, the only difference between the two diets is the presence ofsesamin in ses diet. the animals were fed for 3 weeks. the incorporation of dihomo-y-linolealc acid (dgla: 20:3 o)-6) in the phospholipids from plasma and from the liver for the group of mice fed so diet ranged between 1-3% compared to the undetectable levels for those fed so diet. there were no significant differences in the incorporation of other fatty acids either in plasma or in the cell membranes between the two groups. following an intraperitoneal administration oflps (10p.g/kg body wt), the plasma levels of both pge2, and txb2, were significantly decreased (p<0.02) by nearly 50% for the group of animals maintained on ses diet compared to those maintained on so diet. these findings suggest that sesamin inhibited the release of arachidonic acid (aa) which upon accumulation is retroconverted to dgla which could reflect a modest increase in the content of dgla. failure to decrease the formation of aa could mean that the effects of sesamin on chain elongation process may be after aa is formed, and that sesamin may not inhibit a-5 desaturase activity. on the other hand, our data suggest that sesamin present in sesame oil inhibited the activity of cyclooxygenase which converts aa to its metabolites), or alternatively could block the activity of phosphalipase a 2 (pla2) (which releases aa from membrane phospholipids) as is evident from a marked decrease in eicosanoid production. the percentage of animals surviving after cecal ligation and puncture in rite group of mice fed ses diet was 65% (13/20) which was markedly higher (p<0.01) compared to only 20% (4/20) for the so group. increase in survival in the group of mice fed ses diet was associated with nearly 50% reduction in the production of tnf in response to lps i.p. challenge, for ses group compared to so fed animals. our data suggest that sesamin, present in sesame oil, decreased the production tnf~, inhibited the activity ofpla 2 and consequently offered a significant protection against clp. thus sesamin or sesame oil appear to be novel antiinflammatory agents which are present naturally in many edible products. [dept. surgery, ~e. deasoness hosp.,harvard medical school, boston, usa] stress causes alterations in the homeostasis of an organism with major changes in various organs, lifespans of cells, protein turnovers, metabolic pathways of activation or stimulation, energy mobilization, etc. punctual changes have been described in several organs and various cell types of the nervous, endocrine and immune systems. many stored or newly synthesized proteins are released to the bloodstream to be transported to target organs or to be disposed of. as stress alters protein synthesis and requirements in the targets, the bloodstream is a useful system for monitoring the relevant changes. we present here the pattern of newly synthesized or released serum proteins in c57bl/6 mice that have been stressed by immobilization. the proteins have been labelled with 3ssmethionine in vivo and the study was performed by two dimensional gel electrophoresis based on the method originally described by o'farrel. the electrophoretic run was carried out in an isodalt apparatus. a molecular dynamics laser scanner and the kepler image analysis system (lsb, rockville, usa) were used for modelling the radiofluorographic images. the radiofluorographic images of the gels from serum samples obtained from mice injected with i mci of 3ss-methionine reveal about 440 protein spots, from which a small fraction -about 20are altered in both qualitative and quantitative manner. the major changes are in the range of 25 to 60.000 d. the analysis displays a highly reproducible pattern, where clear differences between stressed and non-stressed conditions are shown. differences between patterns attributed to chronic vs. acute stress will be presented. patients with severe trauma or major surgery are known to acquire alterations in neutrophil functions which contribute to their enhanced susceptibility towards microbial infections. we analyzed neutrophil functions from fourty patients admitted for major surgery 6 days and 1 days preoperatively and on the ist and 6th postoperative days (study-days i, 2, 3, and 4) in a randomized placebo-controlled double-blind study. patients were divided into two groups, one ~eceived 6 days preoperatively an enteral nutrition enriched with omega-3 fatty acids, arginiee, and rna (impact), the other an isocaloric, not enriched control nutrition (placebo). meutrophils were isolated from the peripheral blood and stimulated with the ca-ionophor a23187 (7.3 pm). the capacity of the respective neutrophils to generate leukotrienes was analyzed by rp-hplc. neutrophils from impact group generated significantly higher amounts of ltb5 (mean values 6.6 ng/l x i~ cells) compared to the placebo group (2.6 ng) at study-day 2 [p, 0.01). in contrast, the capacity of neutrophils to produce ltb4 was not significantly different in both patients populations at study-days 2 and 3. the omega-oxidation of ltb4 to its biologically less active metabolites oh-ltb4 and cooh-ltb4 was not significantly different in both groups. however, neutrophils from group f patients generate more ltc4 at study-day 2 (p(0/5). the capacity of the patients'neutrophils to generate reactive oxygen radicals upon stimulation with fmlp, pma or the caionopbore a23187 exhibited patient dependent differences but no correlations to the repective nutritional supplementation as was measured by luminol dependent ohemiluminescense= these data indicate that an enteral nutrition enriched in omega-3 fatty acids lead to alterations in distinct parameters of neutrophil functions. clinical patient characteristics and mean caloric intake were similar between the two groups. both formula were tolerated well and the incidence of diarrhea was low. the number of infectious and wound complications as well as the apache ii score was evaluated for the two study groups. although the number of complications in the group supplemented with arginine, rna and omega-3 fatty acids was lower no significant difference in the occurance of infectious and wound complications has been observed between the two groups. however, the apache ii score indicated a significantly improved clinical outcome in patients with supplemented diet. in conclusion, early enteral nutrition with an arginine, omega-3 fatty acids and rna supplemented diet helps to recover more rapidly after surgical total parenteral nutrition (tpn} is associated with intestinal atrophy and dysfunction attributed to the absence of the non-essential amino acid glutamine in current parenteral nutrition solutions. in this animal study with 18 male wistar-rats we tested the hypothesis that glutamine-dipeptide supplementation during tpn for 10 days would restore small bowel atrophy and tpn induced dysfunction. a conventional tpn solution (250 kcai/kg bw) was compared to an isocaloric and isonitrogenous tpn (0,5 g n/animal/d) supplemented with alanin-glutamin (ala-gin) dipeptide (0,15 g n derived from ala-gin =30% gin-n). an enteral fed cqntrol group was included. mucosal thickness, villous height and architecture, absorption of water and glucose as well as dissacharidase activity of maltase and alkaline phosphatase were evaluated. total parenteral nutrition in rats causes villous atrophy, a significantly reduced absorption rate and a decreased activity of villous enzymes compared to an enteral fed control (p<o,01). addition of ala-gin to parenteral nutrition solutions prevents intestinal atrophy and restores functional alterations with a significantly increased rate of water and glucose absorption as well as a higher dissacharidase activity compared to the standard tpn group (p<0,01). there was no significant difference found between the enteral fed control and the dipeptide supplemented animals concerning intestinal structure and absorption, the enzyme activity was significantly decreased in the ala-gin supplemented group compared to the enteral fed control (p<0,01). in this rat model supplementation of parentera] nutrition solutions with ala-gin dipeptide restores tpn induced intestinal atrophy and dysfunction. boston. ~ usa injury, infection and major o1~'ations s~nulate a variety of factors whi~ initiate the body's response to th~ st~..sses. 'i"hese reactions are m~liated by a cbmage ia the neta'al hormonal, eac.ranmemt, by the elaboration of eytokines and tl~ougb the stimu/ation of other inflammatory mediators. this eatabolio setting zesulis in body protein loss, which g-taduai1y erodes both fimetianal and stm~ tissue. wiu3_b the normally nom'ished individual can withslzod "d~ response for a brief period of time, ircolonged eatabollsm l~ associated with ~,nmunosuppmssion, poor wound healing, and proioagcd convalescence. surgeons have rej2ed on intraveaous or eater~ feedings to ~everse this process. a va~ety of data now indicates that it is extremely di~edt to replete protein-containing tissue d~g the eataholie state of illness; the umutt response to hypercaloise feedings in this setting is the incxeased accretion of body fat md water. administration of gxow~ ho~moae ~cesults i.a n shift of the body's oxidative machinery to fat rather than eazbohydmte utiq~afic~; ¢oncomitaatiy, protein synthesis is stimulated. this metabolic state may have clinical utility in p~e.n.ts who need to heal large wotmds, in those who need to gain strength in order to be w~ed 5ore veatilatory support, or those patients who ~ve mulfiorgan failure and nee~ to ealaaace protein syatsesis to in.suze recovery. 235als are now in progress to dcterm~e the benefit of growth hormone in these and other disease state.s, rn the fature, the increased use of growth hormone, will occur;, this and other growth factors will serve to su~2po~ the host and shorten convalesceace following catabolic diseases. our objective was to study effects of pre-trauma growth hormone (gh) treatment on liver and skeletal muscle metabolism in a trauma model in piglets. twenty-four piglets were randomized to three treatment groups; one group was pretreated with gh 24 iu daily three days before the experiment (gh-3), another group treated with gh 24 ie at the start of the surgical procedure (gh-1) and one group served as untreated controls (con). they underwent a standardized surgical procedure to place sampling cathethers in the portal, hepatic and femoral veins and doppler flow probes around the portal vein, the hepatic and the femoral arteries. all pigs recieved a primed constant infusion of u-14c-glutamine. substrate fluxes were measured one (lh) and five (5b) hours after termination of the surgery. nitrogen excretion was significantly decreased in the gh treated animals (gh-3:30.8+4.2 mg/kg, gh-i: 37.6+_.5.8 mg/kg, con: 55.4+-3.1 mg/kg, p=0.004). in the hindieg, there was reduced net glutamine efflux due to decreased absolute glutamine release (gh-3:0.9 + 0.8gmol/min at lh and -2.4+1.4 in.tool/rain at 5h, gh-i: -3.4+1.5 i.tmol/min at lh and -1.4+1.2 pmol/min at 5h, con: -8.7+_2.8g mol/min at lh and -11.6+3.4/amol/min at 5h, p=0.005, p=0.006), whereas the absolute uptake of glutamine in hindleg was unchanged (p=0.84). glucose uptake in the hindleg was decreased (p=0.0006). net glutamine uptake in liver was attenuated (p=0.03 at 5h), whereas net glutamate release from liver was increased (p=0.005). thus, pre-trauma treatment with gh improved nitrogen economy, attenuated net glutamine loss from hindieg due to decreased absolute release, and attenuated hindleg glucose uptake. liver net glutanaine uptake was decerased and net glutamate release increased. patients with major abdominal surgery exhibit a considerable catabolic response with negative nitrogen balance and protein breakdown, which may have detrimental effects on outcome. we investigated the effects recombinant human growth hormone on substrate metabolism in parenterally fed patients. 48 metabolic healthy patients were randomized to receive either placebo or hgh in a dosage of 0,075; 0,15 or 0,3 i.u. per kg bw. substrate oxidation rate, energy expenditure as well as short life proteins were measured daily. six patients were excluded from analysis as drop outs, due to surgical complications. we could find a dose-dependend effect of growth hormone on resting energy expenditure, carbohydrate oxidation, fat and proteine oxidation. with increasing growth hormone resting energy expenditure increased from 20.9 calories per kg body weight to 26.6 calories. this was mainly due to an increased carbohydrate and fat oxidation, while proteine oxidation decreased. this was in accordance with a decreased urea.production rate and an improvement in cumulative nitrogene balance, which increased from minus 12.7 to plus 8.5 g n / 7 days. in consequence short life proteins were also increased. the only negative side effect was an increased blood glucose concentration in some of the patients, making insuline administration mandatory in 3 patients. our results are in accordance with other studies showing improvement of nitrogen balance, maintainance of lean body mass and muscular strength. if growth hormone administration may have any impact on morbidity, mortality and length of hospital stay in these patients it should be evaluated in a larger number of patients. in 30 patient after liver transplantation (oltx) the effect of recombinant human growth hormone (rhgh) on protein metabolism and hormonal changes was studied. patients and methods: the underlying disease in all patients was end stage liver cirrhosis, non of the patients included in the study was suffering from malignancies. the patients were randomly allocated to receive either 81u of rhgh bid (sc) or no alternative medication. the study period lasted 14 days. from daily 24 hrs udne collection urinary nitrogen loss and daily loss of urea were determined. body compostion analysis (bca, bioimpedance, akern-ltaly) was performed preoperativly and at the postoperative days 7 and 14 to evaluate changes in body water and body cell mass (bcm). the effect on resting energy expenditure (ree) was analysed using indirekt calorimetry wrhin the same intervalls (metabolic monitor, datex). blood levels of gh, igf1 and igf2, igfbp3 were measured daily during the study period, on day 3 a 24 hour profile of gh was.performed. samples were collected each 20 minutes. results: cumulative udnary nitrogen and urea loss were not signifcantly different for the whole study period but for the first 4 study days. bia indicated for the rhgh-group a loss of bcm to a lesser extent than for the controls (n.s.), changes in bodywater where similar for both groups. ree changes (kcal/kg bcm) were not significantly different for the two groups. blood levels of gh and igf1 differed significantly between the groups for the whole study period, but not igf2 and igfbp-3. the circadian rhythm of endogenous gh-excretion had not been altered by gh, but absolute levels of gh were increased. conclusion: during the first 4 days after oltx we could proof the protein sparing effect of gh treatment. although the differences between the two groups were not significantly different for the whole study period the results showed on all days a less catabolic situation for the rhgh treated patients. to possibly improve protein balance after major abdominal surgery we studied the effects of gh on protein metabolism after ltx. excluding malignant diseases, 29 candidates for ltx were randomized to either postoperatively receive the usual treatment (co=control group, n=14, age range 25-59 yr, bmi 23.9+5.7 kg/m 2) or for 14 days additional gh (2x8 i.u.s.c.) (gh group, n=14, 21-59 yr, 21.8+2.2 kg/m2). metabolic studies (calorimetry and lsc-leucine infusion) were performed 5 (test a) and 11 days (test b) after surgery. tracer analysis was done by gas chromatography-mass spectrometry. during the 2-week study clinical parameters did not differ significantly between groups; however, there was a tendency for increased glucose levels, insulin need, and energy expenditure at the time of test b in the gh group. leucine oxidation (x+se) during test a;b was 117+10; 111+15 (co) and 110+7; 100±12 (gh) pmol/kg ffm/h (n.s.). protein breakdown was 134±16; 131+30 (co) and 156+14; 178+30 (gh) pmol/kg ffm/h (n.s.). nonoxidative-leuine disposal was 119±9; 127+16 (co) and 139±11; 177+20 (gh) pmol/kg ffm/h (p<0.05 for the rise in the gh group). tracer data were supported by cumulative urea nitrogen excretion (days 1-4: gh 52+3 vs co 66±7 g; p<0.05). we conclude that after major abdominal surgery gh lowers nitrogen excretion by increasing total body protein synthesis. it is not known which organs contribute to this effect. in several investigations it has been shown that the protein saving effect of recombinant human growth hormone (rhgh) in the postoperative state is accompanied by raised igf-1 levels in plasma. it was concluded that the anabolic effects of rhgh were probably, at least in part, mediated by igf-1. this study was aimed at detecting the efficacy of igf-i on modulation of the protein metabolism in the catabolic state. patients and methodes: 38 patients (both sexes, age: 40-75 years, bmi 17-30 kg/m 2) with major upper gastrointestinal surgery (gastrectomy or partial gastrectomy) received 80 ug rhlgf-1/kg body weight/twice daily or placebo during 5 treatment days beginning on the first postoperative day in a double-blind, randomized study. all patients received hypocaloric and hyponitrogenous total parenteral nutrition (3g chng, o.6g aa/kg) troughout the whole study period. cumulated nitrogen balance, 3-methyl-histidin exretion in urine as well as serum-igf-1 levels have been determined. the two tailed wilcoxon test was used for statistical analysis results: first results will be presented previous studies have suggested that growth hormone (gh) potentiates various activities of leukecytes. however, it is not clear whether gh can improve survival of animals with gram-negative sepsis. we investigated the effects of gh on host response to infection in septic mice model. methods balb]c mice were randomized to 3 groups (n=10/gronp): gh-high (4+8mg/kg/day), gh-iow (0a8mg/kg/day) or control (saline). following subcutaneous treatment (every 8 hours for 6 days) with gh or saline, mice were challenged i.p. with e.coli (lxl08/body). survival rate was recorded every 2 hours. in the next experiment, gh-high and control animals were sacrificed 4 hours after bacterial challenge. peritoneal cavity was lavaged with 3ml saline and the peritoneal exudative cells (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. pec were cultured in vitro for 24 hours with lipopolymccharide (10p.g/ml in normotensive adults growth hormone (gh) rises when clonidine challenge is performed. recently evidence was shown for gh to accelerate wound healing. while gh secretion patterns are inconstant the gh-dependant insulin like growth factor (igf-1) and the insulin like growth factor binding protein (igfbp-3) reflect the integrated gh secretion over days. since we administer clonidine to patients with acute alcohol abuse we determined plasma levels of igf-1 and igfbp-3. materials and methods: 21 patients sheduled for esophagus resection were investigated once they had given written informed consent. 8 patients showed acute alcohol abuse and were treated with clonidine postoperatively• 13 patients with no history of acute alcohol abuse served as controls. besides liver enzymes igf-1 and igfbp-3 were determined. results: both groups had a comparable hepatic capacity of synthesis with decreased cholinesterase levels as to be expected after a major operation. regression analysis of igf-1 and igfbp-3 levels showed no differences between the groups. discussion: igf-1 and igfbp-3 plasma levels are not increased in normotensive patients who are treated with clonidine for prevention of postoperative alcohol withdrawl syndrom. further studies are required to discriminate whether our results are due to impaired postoperative liver function or if clonidine has not any impact on the gh-igf-axis in patients with alcohol abuse. recent studies have revealed that igf-i has several immanoregulatory roles. however, little is known about its effects on septic animals. we tested whether igf-i could increase the survival o f mice challenged intraperitoneally (i.p.) with e. coli. m~thqd$ balb/c mice received either high dose igf-i (n=10, 24mg/kg/day), low dose igf-i (n=10, 2.4mg/kg/day) or saline (11=10) every eight hours subcutaneously for six days. animals were then challenged i.p. with lx108 e. coli. survival was observed every two hours. in the other experiment, mice that received high dose igf-i or saline were sacrificed four hours after bacterial challenge. peritoneal cavity was lavaged with 3ml saline and the peritoneal exudative ceils (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. in addition, pec were cultured/n vitro for 24 hours with lipopolysaccharide (10).tg/ml). tumor necrosis factor (tnf) in the supernatants of pec culture, plf and serum were measured by l929 bioassay. results igf-i improved the survival rate at a dose of 24mg/kg/day. severe chronic neutropenia (scn) is a disorder characterized by severe neutropenia secondary to either a maturational arrest of myelopoiesis at the level of promyelocytes (kostmann-syndrome; scn) or regular cyclic fluctuations in the number of blood neutrophils with a median absolute neutrophil count (anc) of less than 500/~tl (cyclic neutropenia). patients suffering from scn have an impaired acute inflammatory response to injury and an increased susceptibility to infections, i.e. bacterial or fungal infections, resulting in chronic oral inflammation, severe pneumonia, abscess formation and bacteraemia. we have treated 36 patients (32 scn, 4 cyclic neutropenia) with r-methug-csf (filgrastim). thirty of 32 patients with scn and all 4 cyclic neutropenia patients responded to this treatment with an increase of the median anc to greater than 1000/~tl. the dose of r-methug-csf needed to achieve and maintain the response varied between 0.8 and 120 ~tg/kg/d. long-term treatment did not exhaust myelopoiesis: the anc remained stable after up to 6 years of treatment and antibodies to r-methug-csf were not detected. the increase of anc was associated with dramatic clinical responses: significant reduction of severe bacterial infections, reduction of intravenous antibiotic treatment, and reduction of hospitalizations. no severe bacterial infections occurred in any of the r-rnethug-csf responders during longterm treatment. severe adverse events, most likely due to the underlying disease, included the development of mds/leukemia in two patients, and osteopenia/osteoporosis in 12 patients. these results demonstrate the beneficial effects of r-methug-csf treatment in severe chronic neutropenia patients. the newborn is predisposed to mortality during infections due in large part to developmentally immature host defenses. recently cytokines have been identified that regulate the development of these defenses. one such cytokine is specific for neutrophils and is termed granulocyte colony stimulating factor (g-csf). in the studies to be discussed, we have attempted to impact hematopoiesis in the fetus using exogenous rhg-csf delivered through the pregnant dam. our rationale was that an enhanced myeloid system may result from such in utero treatment leading to, perhaps, enhanced protection to microbial insult. rhg-csf crossed the placenta and reached peak fetal serum concentrations 4 hours after administration. these levels were 1000-fold lower than the levels detected in the adult dams serum. white blood cell counts were elevated approximately 2-4-fold over control newborn blood. this increase was due to circulating numbers of neutrophils. the marrows from these pups were hyperplastic consisting of predominately immature and mature neutrophilic cells. no changes were seen in the thymus or livers. pups born to mothers treated with rhg-csf since day 15 of gestation (parturition in rodents occurs on approximately day 21) survived a lethal challenge of group b-13 hemolytic streptococcus. in contrast their untreated yet infected counterparts did not survive. recent clinical developments have led to trials of rhg-csf in certain neutropenic states including cyclic and chronic neutropenia. we have identified several of these patients who were pregnant during treatment with rhg-csf. biologically and anfigenically identifiable rhg-csf was detected at increased levels in the cord serum of these neonates. we will discuss our findings with these patients in light of our animal model of neonatal myeloid development. we have previously demonstrated decreased g-csf production and g-csf mrna expression from activated mononuclear cells from newboms compared to adults (cairo, pediatr res 31:574, 1992) . we have also recently observed that administration of a single dose of rhg-cse to one-day-old newborn rats induced significant neatrophilia, while administration for 7 days induced neulrophilia and increased the bone marrow (bm) neutrophil storage and progenitor pools (cairo, blood 76:1788 , 1990 cairo, pediatr res 27:612, 1990) . we embarked on a phase i trial exploring the safety, pharmacokinetics, and biological efficacy of g-csf in newborns with presumed sepsis. 42 infants <72 hrs old with a diagnosis of presumed sepsis were stratified into three groups: very preterm (26-30 wk), preterm (31-35 wk), and term (>36 wk) and randomized to receive either a placebo or 1, 5, and 10 gg/kg/qd or 5 and 10 p.g/kg/bid of rhg-csf infused by pump over 1 hour for 3 consecutive days. cbcs were obtained at 2, 6, 24, 48, 72 and 96 hrs. tibial bone marrow aspirations were performed 72 hrs after study entry and differential counts and cfu-gm pools were determined. c3bi expression was determined at 0 and 24 hrs after rhg-csf, and g-csf pharmacokinetics were performed after the first dose of rhg-csf utilizing a sandwich elisa. a significant increase in the anc was observed at 48, 72 and 96 hrs following administration of both 5 and 10 ~tg/kg/d of rhg-csf. the maximum increase in the anc occurred 72 hrs after 5 and 10 ~tg/kg/d (397 -116%) (p<0.01) and (621% -+ 200%) (p<0.001), respectively. there was a significant dose-dapendeat increase in the bm neutrophil storage pool (18 _+ 4% vs. 62 + 6%) (p<0.01) (placebo vs. 10 ~tg/kg/d). there was no significant difference in the nantrophil proliferative pool. an increase in cfu-gm and cfu-gemm was seen at all doses tested, compared to placebo (53.5 _+ 8.6 vs. 87 -+ 15) (colonies/l(p cells/plate). c3bi expression was significantly increased 24 hrs after 10 bg/kg/d of rhg-csf (233 + 81% vs. 83 +-26%) (p<0.012). peak serum g-csf levels occurred at 2 hrs and were dosedependent. the half-life of rhg-cse was 4.44 + 0.4 hrs. most importantly, there was no observed toxicity from g-csf in all patients studied. 31 of 42 patients were on ventilators prior to administration of rhg-csf and there was no increase in pulmonary toxicity. these preliminary data suggest that rhg-csf is well tolerated at all gestational ages in newborns with presumed sepsis. a multi-center phase ii/iii randomized double-blindad placebo controlled trial is required to determine the efficacy of rhg-csf in this clinical setting. we investigated the effects of recombinant canine granulocyte-colony stimulating factor (g-csf) on survival, cardiopulmonary function, serum endotoxin levels and tumor necrosis factor (tnf) levels in a canine model of lethal bacterial septic shock (clinical research.41:240, 1993) . methods: awake 2 ylo beagles had serial cardiopulmonary and laboratory studies before and for up to 10 days after intraperitoneal placement of an e. celi infected clot. nine days before and daily until 3 days after clot placement, animals received high (n=17) or low dose (n=17) g-csf or protein control (n=20) subcutaneously. results: survival in high dose g-csf animals (14/17) was significantly improved compared to low dose (10117) and controls (12120) (p<0.04 wilcoxon). high dose g-csf also improved cardiovascular function evidenced by a higher mean left ventricular ejection fraction (day 1 after clot, p<0.001) and mean arterial pressure (day 2, p<0,02) compared to low dose and controls. high dose rcg-csf increased (p<0.001) peripheral neutrophil numbers both before and after clot implantation (2 hours to 4 days) compared to low dose and controls. in addition, high dose rcg-csf produced a more rapid (p<0.0001) rise (day 2) and fall (day 4) in alveolar neutrophils determined by bronchoalveolar lavage compared to low dose and controls. lastly, high dose rcg-csf decreased serum endotoxin (2 to 8h, p<0.002) and tumor necrosis factor (tnf, 2h, p<0.02) levels compared to low dose and controls. discussion: these data suggest that therapy with g-csf sufficient to increase peripheral neutrophil numbers during peritonitis and septic shock may augment host defense and endotoxin clearance, reduce cytokine levels (tnf) and improve cardiovascular function and survival. the use of g-csf in sepsis prophylaxis in neutropenic patients is well established and has been ascribed to accelerated recovery in granulccyte counts. here, an additional sepsis-prophylactic property could be demonstrated in healthy volunteers: eleven volunteers were employed in a sinqle-btind, controlled study and were given 480 uq g-csf or saline placebo via subcutaneous injection. blood was withdrawn immediately before and 20 or 44 hours later. lps-inducible tnf, il-1, stnf-r p75 and il-lra were assessed in the supernatant of whole blood incubations stimulated with 10 ug/ml lps from salmonella abortus equi. similarly to previous animal studies, lps-inducible tnf was attenuated by about 50% 20 hrs. after treatment. the same was true of il-lb. in contrast, lps-inducible stnf-r p75 which was indetectable in blood incubations from untreated donors increased dramatically 44 hrs. after g-csf treatment. il-lra found after lps challenge was increased tenfold by g-csf treatment. it is concluded that g-csf treatment switches peripheral leukocytes to an antiinflammatery state characterized by an attenuation of il-i and tnf releasing capacity and an augmentation of the release of cytokine antagonists. this findinq minht offer a novel concept in septic shock prophylaxis. objective.the aim of the study was to investigate the effect of recombinant human g-csf (rhg-csf) on survival, bone marrow neutrophil myelopoiesis, neutrophil counts, levels of bacteria and some important sepsis mediators in a model of rat abdominal sepsis. lethal peritonitis was induced with a 4 mm coecal perforation (cp) in male wistar rats. rhg-csf was administered as 10 /.tg/kg iv every 12 h, first dose at sepsis induction. bone marrow neutrophi] progenitors were determined as blast colonies, cfu-gm and cfu-g. neutrophils and bacteria were determined in peripheral blood and peritoneal fluid. lps, tnf, endothelin 21 and lactate were measured in blood from femoral vein. mortality rates were registered with g-csf treatment starting either 1 or 7 days before or 4 hours after cp. results. mortality was reduced from 90% to about 50% with rhg-csf intervention and there was no difference between the pretreatment and treatment groups. bone marrow blast colonies were not influenced while neutrophil myelopoiesis was augmented at the stages of cfu-gm and cfu-g. neutrophils in blood and peritoneal cavity were enhanced and numbers of bacteria in the same compartments were substantially reduced. circulating lps, tnf, endothelin 21 and lactate were attenuated the first 24 hours after cp. neutrophil myelopoiesis is augmented with increased number of neutrophils in blood and peritoneal cavity, resulting in enhanced clearance of pathogens. lps, tnf, endothelin 21 and lactate are suppressed the first 24 hours during sepsis course. a. wendel, j. barsig, g. tiegs gm-csf stimulates the proliferation and differentiation of granulocytic and monocytic progenitor cells. in addition the hemopoietic cytokine activates the inflammatory response in mature leukocytes. the priming effect of gm-csf towards lipopolysaccharide (lps)-induced cytokine production in vitro has been described, but little is known about proinflammatory gm-csf effects in vivo. we detected gm-csf in plasma of lps-challenged mice with kinetics similar to tnf, reaching peak levels 2h after lps administration. gm-csf pretreatment (50 ~tg/kg i.v.) enhanced mortality in mice challenged by a sublethal dose of lps. plasma levels of tumor necrosis factor (tnf) and interleukin-6 (il-6) were significantly enhanced. a monoclonal antibody, which neutralizes gm-csf bioactivity, rendered mice less sensitive towards lethal lps-challenge. tnf-and il-6-tevels were reduced in these mice compared to control animals without antibody treatment. in addition, severalfold potentiation of lps-induced cytokine release by gm-csf was observed in vitro in murine bone marrow cell cultures. these data demonstrate the proinflammatory capacity of gm-csf and suggest that the hemopoietic cytokine plays also a role as an endogenous modulator of lps toxicity. immune dysfunction, developing in the wake of multiple trauma, overwhelming infection and other forms of critical surgical illnes% is associated with increased infections, morbidity and mortality. the mechanisms responsible for alterations in immune regulation are incompletely understood but monocyte appear to play a central role. polymorphonuclear leukocytes (pmn) are known to play a central role in the inflammatory response of the host toward invading microrganisms. reports of defects in all the aspeots of pmn function have been accumulated in recent years. the possible role of gm-csf in modifing the state of immuno suppression detected in severe intraabdominal infected pt~. inspite of surgical appropriate procedures and in reducing the expected mortality is investigated. the safety of rh-gm-csf administration in sepsis is also evaluated. a double blind randomized study is proposed. this study include 60 icu patients who do not exhibit signs of shock and/or ards, with clinical signs and symptoms of abdominal infection. immunodepressed patients-aids, chronic chemotherapy or chronic steroid administration do not partecipate to the study. patients will receive rgm-csf (l~g/kg/day) or placebo in 24 hs. continuous infusion for 15 days. safetyandefyieacy will be assessed till to day 30. the apache ii score is adopted for risk stratification of patients because it is reliable and validated, objective and composed of information that is indipendent of diagnostic criteria. patient's entry criteria is apache ii > 16 (score 16 corresponds to expected mortality rate of 38%).in this protocol the surgeons report the judgement of the efficacy of surgical procedure to remove or not the focus of infection. objectives: infections and subsequent septic responses remain the leading cause of death among surgical intensive care (sicu) patients despite tmprovetaunts in supportive care and brond-epectrum antibiotics. usually invading bacteria are efficiently cleared by neutrophil granulocytes. however, during sepsis various neatrophil dysfunctions have been demonstrated, leading to impaired host defense. granulocyte colony-stimulating factor (g-csf) induces a sustained increase in circulating neutrophils and enhances various noutrophil functions. it was the purpose of the present study, to evaluate the safety and efficacy of g-csf (filgrastim) in sicu patients at risk of sepsis. materiel a.d methods: the study was designed as an open-label phase-ll study of filgrastim. ten consecutive slcu patients, with a therapeutic interveotion score greater than 30, were included in the study. filgrastim was given by daily continuous intravenous infusion for 7 days or discharge from the sicu. apache ll-score, multiple-organ-failure (mof) score, definitions of infections, sepsis, systemic inflammatory response syndrome (sirs), and acute respiratory failure were applied daily. a response to filgrastinl th_erapy was defined as an improvement in disease severity quantified by a decrease of > 4 apache 1i score points on day 4 after onset of treatment. results: none of the 10 patients developed a sepsis or mof later on and no patient died during hospitalization. specific postoperative complications occured in one patient ~jth a leekage of the oesophagou-gastric anastomosis after oesophageus resection. at study entry the leucocytes amounted to 10.110 + 2.000/~tl (mean + sem) and reached a level of 29.280 +_ 5.810/tal at day 6 after onset offilgrastim therapy. the apache ii score initally was 14 + 1.57 (mean + sem) and as an indicator of filgrastim response a decrease of 5 points ~dthin 4 days oceured in 7 out ot 10 patients. filgrastim was well tolerated, side effects were not noted. growth of solid tumors might be modulated by the activity of inflammatory and/or immune effector cells of undefined specificity. in this study patients undergoing surgical treatment for gastric (n= 41) or colorectal (n= 47) cancers were evaluated for endogenous serum levels of granulocyte colony-stimulatingfactor (g-csf) during a pre-and postoperative time period. from the same blood specimens mononuelcar cells (mnc) were prepared. the release of ifn-%, and il-2, which are secreted by thl cells, were stimulated in vitro by pha during a cell culture period up to 48 hours. the patients were further classified for their immunreactivity by responses in dth skin testing to seven different antigens (e.g. tetanus toxoid, ppd, diphtheria toxin, trichophyton, streptococcus, candida and proteus antigens). dth testing has been repeated in each patient two remarkable results were obtained. the serum levels of endogenous g-cse showed a biphasic increase with maximum values of 900 pg/ml (preoperative < 70 pg/ml) on day 1 and day 3 to 5 after surgical treatment. similar patterns of g-csf production were found in both groups of patients with gastric or colorectal cancers. high serum levels of g-csf were significantly (p < 0,01) correlated with infectious complications in patients whh gastric cancer (n= 17/22). secondly patients could be arranged into two groups according to an anergic (n= 25) or normergi¢ (n = 63) responsiveness in dth testing. the frequency of anergi¢ responsiveness was similar in both patients with gastric (n= 11/41) or colorectal (n= 14/47) cancers. interestingly we found a significant correlation (p < 0,01) between low serum levels of g-csf and anergy during the postoperative period in both groups. stimulation of mncs from anergic patients (n= 30) within the pre-and postoperative period resulted in reduced mean values (about 50%) for ifn-ff release (preoperative means llo0 pg/nfl), if compared to patients with normergic dth (n= 63, preoperative means 1960 pg/ml). similar, but less significant results were obtained for il-2 secretion. our results confirm a correlation between infectious complications and g-csf in the postoperative period, however elevated levels were also found in some patients without any signs of infections. more interestingly there might be an association between cytokine (c~csf, ifn-% and il-2) release and dth, which is known to be mediated by activated thl calls. to recognize anergic dth as a possible higher risk in the postoperative outcome of cancer patients extended periods of observation are needed. objectives of the study effects of recombinant huraan granulocyte colony-stimulating factor(rhc-csf)a galnst severe septic infections were investigated by its single use or by its corn b{nation with cephera antibiotlcs.we examined its effects on the mortality,and circulating blood neutrophyis counts and functlons,such as phagocytic activity and h 2 02 production using the rat severe septic model. rats were subcutaneously administsrd rhc~csf(s0orl0o ~ g/k~ body wt)after on set of peritonitis brought about by cecal ]igation and one puncture withe2-gaug e needle once a day for three days.in addjtlon,cefmetazol na(cmz)(50m$/k9 bo dy wt)was injected intrarnustularly to the rats tv~ce a day for three days. cirehlatlng blood neutrophyls counts were determoned electronically with a hem ocytometer,and blood smears stained with may~runwaldm.qlemsa~taln. neutrophyls functions in vltro,such as phagocytic activity and h 20 2 producti on using the rat severe septic model was analyzvd by automated flow cytometri c single cell-analysis methods. the reortallty rate after 2 weeks was significantly decreased by administratlon of rh~-csf(p<0,01).ln addjtion,a combination therapy of rhg-csf wlte cephern ant~biotics(cmz)showed a significantly survive] advantage and the rate had b een reached 57.1%. nextly,treatn%ent wlth rhg-csf(s0 ~ $/k9 body wt)increased the nuzaber of the peripheral blood neutrophjls slgn[fieantly(p<0.01). iv~oreover,functions of neutrophlis which were phagocytic activity and h2 02 p roduction were remarkably enhanced by admlnlstratlon of rhg-cs~(50 ~ 9/ks b ody wt) (p<0.(15). these findings suggest that combination therapy of rhcrcsf with cephern antib iotlcs(cmz)is an efficient regime against severe infectlons.and the increased ne utrophils counts and enhanced neutrophiis functions were played a important ro le about the survival advantage. granulocyte macrophage colony-stimulating factor (gm-csf) is a haematopoietic growth factor active on neutrophils and macrophages. leukopenia often occurs following renal transplantation and can be associated with infection and/or the myelosuppressive effect of azathioprine. aim: we report the use of gm-csf in 17 renal allograft recipients with leukopenia. nonglycosylated recombinant gm-csf was obtained from e. coli transvected by human gm-csf gene. m~terial ~,nd methods : written informed consent was obtained from all patients. patients were suffering from toxic neutropenia (neutrophils < 500/mm 3) with medullar hypocellularity on bone marrow aspiration, or leukopenia (neutrophils < 1000/ram 3) with cytomegalovirus infection requiring ganciclovir administtation. gm-csf was given subcutaneously at a dally dose of 2 to 5 mcg/kg/day, according to renal function. results : in all cases, neutrophil counts returned to normal levels within 1 to 4 days. in most of them, spectacular correction was observed within 24 hours, with a single injection. adverse events due to gm-csf at this dose were mild and easily managed (2 cases of bone pain treated with paracetamol). one acute rejection episode was observed after correction of leukopenia. conclusion : on the basis of this study, it appears that gm-csf at a dose below 5 mcg/kg/day is an effective treatment for renal transplant recipients with leukopenia associated with cmv infection or toxic neutropenia. department of nephrology, 161, rue de s~vres, hopital necker, 75743 paris, france. changes in serum g-csf and il-6 after surgical intervention hitoshi toda 1, atsuo murata 1, hidewaki nakagawa 1 , takesada mori 1, nariaki matsuura 2 1osaka university medical school, osaka, 2wakayama medical school, wakayama, japan we measured serum immunoreactive interleukin 6 (il-6) and granulocyte colony-stimulating factor (g-csf) levels of the patients undergoing major thoraco-abdominal surgery for esophageal cancer. serum samples were collected from eight patients on the day before surgery, at the time of operation, and thereafter at suitable intervals for one week. il-6 and g-csf were measured by means of enzyme linked immunoassay. the normal range of serum ]l-6 was less than 2 pg/ml and g-csf less than 4 pg/ml. values between groups were compared with linear regression analysis. both serum g-csf and il-6 levels reached their maximal levels at the first postoperative day and decreased thereafter. the correlation between g-csf (y) and il-6 (x) was y=3.28x+6.16 (r=0.786, n=86, p<0.01 ), showing a significant correlation. in the case who suffered from aspiration pneumonia and ards at the second postoperative day, the peak level of il-6 was 50 pg/ml and g-csf 69 pg/ml respectively. the estimated value of g-csf was 171 pg/mi by the regression equation. this means the real g-cse level was less than half of the estimated value. it suggests that low responsiveness of g-csf is one of the reason of immunodeficient state after the major surgery, neutrophils from injured patients ingest and kill bacteria less efficiently as compared to those of healthy individuals, probably reflecting the suppression in respiratoly burst which occurs after severe trauma. one of the main mechanisms of killing bacteria by neutrophil granulocytes is production of oxygen radicals (respiratory burst). granulocyte colony-stimulating factor (g-csf), a 20kilodalton cytokine, leads to a sustained, dose-dependent increase in circulating neutrophils. thus, it was investigated whether filgrastim (recombinant human granulocyte colony-stimulating factor, rhg-csf) therapy fits for prophylaxis of sepsis in postoperative/posttraumatic patients, and whether, besides an expected increase in neutrophil count, filgrastim would also augment neutrophil function. material and methods: this study was designed as an open label, prospective phase ii study of filgrastim and performed in a surgical intensive care unit (sicu) (university hospital). 10 postoperative/post-traumatic patients with a therapeutic intervention scoring system (tiss) score greater than 30 were treated with filgrastim (0.5 -1 l.tg/kg/day) for prophylaxis of sepsis on 7 days or until discharge from the sicu. production of oxygen radicals can be quantified by analysis of fmlp-and zymosan-induced chemiluminescence. neutrophil oxygen radical production was tested by fmlp-and zymosan-induced chemiluminescence by the polymorphonuclear cells (pmn) of these patients in multiple blood samples over a period of up to 14 days. results: none of the patients treated with filgrastim for prophylaxis of sepsis developed sepsis. in vitro fmlp-induced (10 -7 reel/l) neutrophil oxygen radical production was significantly increased under therapy with filgrastim by a maximum of 797% +-570% (217% -1826%) compared to pretreatment values of 100%. tapering of filgrastim resulted in a reduction of fmlp-induced neutrophil oxygen radical production within 48 hours. in contrast, zymosan-induced neutrophil oxygen radical production was not affected by filgrastim treatment. conclusions: besides its quantitative effect on neutrophil counts enhanced neutrophil function, documented here as increased fmlp-induced oxygen radical production, may account for the beneficial effect of filgrastim for prophylaxis of sepsis in posttraumatic/post-operative patients. granulocyte colony stimulating factor (g-csf) and granulocytemacrophage colony stimulating factor (gm-csf) have been recently introduced in the treatment of chemotherapy-induced neutropenia. effects of these csfs on cellular immune system were evaluated in 38 neutropenic gynecological cancer patients during chemotherapy. g-csf and gm-csf were equally able to induce a rapid recovery of white cell count within one or two days. g-csf treatment resulted in a significantly higher concentration of leukocytes measured in the peripheral blood although by gm-csf a sufficient effect was achieved (p<0.05). before initiation of csf treatment urinary neopterin was similar in both groups of patients (283+/-38 and 267+/-40 lamol/mol creatinine for gm-csf and g-csf respectively expressed as mean +/-one sd). in g-csf treated patient only a marginal induction of neopterin was observed. on day 4 the mean value was about 40% above the basal level (p<0.05). on the other hand gm-csf treated patients were characterized by a pronounced increase in urinary neopterin levels. in comparison with the basal level a more than 2 fold induction was noted and the difference between g-csf and gm-csf was highly significant (p<0.01). this effect was confirmed in vitro by investigating the effects of these csfs on interferon-gamma mediated pathways in thp-1 human myelomonocytic cells. results suggest activation of immune effector cells by gm-csf which may help the organism to overcome infections. however, activated macrophages produce several growth factors which may increase malignant proliferation, and augmented neopterin production as sign of macrophage activation has also been associated with poor prognosis m several malignancies. more data are therefore necessary to clarify whether csf mediated immune activation is beneficial or deleterious for cancer patients but considering our results caution in applying csfs in oncology seems advised. from a historical perspective, the development of humoral immunity to bacterial endotoxin has assumed a prominent position in the spectrum of therapeutic approaches which have been explored for the treatment of gram negative septic shock. predicated upon the fact that rough strains of bacteria manifest lps containing exclusively conserved structural features common to lps from all gram negatives, specific antibodies were elicited which conveyed cross protective immunity in experimental models of bacteremia and endotoxemia. such studies culminated in a well-conducted, randomized, double-blind placebo-controlled clinical trial using passively administered human polyclonal antiserum to treat patients with suspected gram negative sepsis. the efficacy of treatment established in that trial spurred efforts to develop monoclonai reagents which, to date, have not been uniformly successful in reproducing those earlier studies with polyclonai antibodies. nevertheless, the numerous successes which have been documented in experimental models of endotoxemia continue to foster promise for this immunotherapeutie approach. several recent studies with human polyclonalimrnunoglobulin preparations containing antibodies reactive with lps and lipid a have yielded promising results in treatment of patients with sepsis. in addition, the recent development of an antiidiotypic monoclonal antibody which reflects an internal image of a kdo specific monoclonal antibody has provided an alternative experimental approach to generate anti-lps antibody. immunization of mice with the antiidiotype provides significant protection against subsequent lps lethality consistent with the development of circulating immunoglobulin specific for lps. thus, the use of polyclonal immunoglobulins contrives to provide an alternative and potentially cost effective method for the treatment of endotoxin shock. supported by r37 a123447 and pot ca54474. john holaday, anne fortier, shawn green, glenn swartz, john madsen, carol naey, and jan dijkstra entremed, inc.. rockville, md, 20850. at the time of diagnosis, the signs and symptoms of septic shock are an indication that the systemic inflammatory response is well underway; thus, it has been argued that the endotoxin "cat is out of the bag", and that subsequent passive immunization may be too late to achieve therapeutic benefit. our approach has been to evaluate active immunization as a prophylax~s against sepsis. mice were inoculated twice (two weeks apart) with liposomes containing dmpc[i.8], dmpg[0.2], cholesterol [1.5] , and monophosphoryl lipid a [20-200 gg/txmole phospholipid] by several routes (i.p., i.m.), and serum was collected 10-11 days after each inoculation. after a single injection, highest tilers of ab were produced in mice inoculated i.p., but mice inoculated by all routes produced anti-lipid a ab. following the second injection. ab levels were roughly equivalent in mice inoculated by all routes, regardless of lipid a concentration. mice vaccinated i.p. with liposomes containing 0, 20 or 200 gg lipid a were treated with cyclophosphamide to produce neutroperda and then challenged with e. cole in an infection model of gram negative sepsis. the lds0 for control (liposomes with no lipid a) mice was 3x10 bacteria; ld50 for mice vaccinated with 20p.g was 24x103 (8-fold increase in resistance) and with 200~tg was 48x103 bacteria (16-laid increase in resistance). mice vaccinated as before were also treated with actinomyein d to increase sensitivity to lps (salmonella minnesota) challenge in an endotoxemia model of grain negative sepsis. the ld50 for control (liposomes with no lipid a) mice was 30 ng lps; the ld50 for 20gg lipid a was 70rig lps (2-fold increase in resistance) and for 2001xg was 570ng lps (19-fold increase in resistance). mice were similarly vaccinated and challenged with an aggressive gram negative pathogen, francfsella tularensis. the ld50 of franciseua in normal mice or mice inoculated with liposomes without lipid a was 20-40 bacteria. in contrast, mice vaccinated with liposomal lipid a (200ggl survived challenges as high as 30,000 bacteria, (3 logs of protection). the impressive protective capacity of this vaccine did not correlate with ab liter in any of the sepsis models, nor did it correlate with classic nonspeeific events, such as macrophage activation. maerophages harvested from the peritoneum of mice vaccinated and protected against sequelae of gram negative infections did not spontaneously kill the bacteria in vitro, but could be activated by ifn-y for antimicrobial activity equivalent to that of macrophages from unt#eated mice. research is underway to defme the protective mechanism(s) activated by this liposomal-lipid a vaccine. intervention by monophosphoryl lipid a in septic shock jon a. rudbach, ribi immunochem research, inc., hamilton, montana, usa monophosphoryl lipid a (mla), the clinical form of which is called mpl®-immunostimulant, has been tested extensively as an intervenient material in septic shock. mla is protective when given to experimental animals prior to a live microbial challenge or challenge with lethal doses of microbial products or certain cytokines. this is shown with gram negative and gram positive bacteria, gram negative bacterial endotoxins, and gram positive bacterial exotoxins. furthermore, animals treated with a regimen of mla which results in a refractory state to a lethal dose of gram negative bacterial endotoxin concomitantly display increased resistance to a live bacterial challenge. thus, both endotoxin tolerance and nonspeciflc resistance to infection can be manifested simultaneously. also, prophylactic doses of mla do not interfere with other therapies given subsequently; an additive or a synergistic protective effect can be demonstrated with certain combinatorial treatment regimens, such as mla followed by antiendotoxin monoclonal antibodies. the preclinical studies were extended to human trials wherein the safety of agonistic doses of mla was verified. furthermore, when mla was administered to human volunteers 24 hr before challenge with a pharmacologically active dose of reference endotoxin, febrile, cardiac, tnf, il-6, and il-8 responses were all decreased significantly as compared with the responses of subjects pretreated with a control solution and challenged with endotoxin. human trials with mla are being extended into patient cohorts which have high probabilities of developing septic shock; this will expand the safety base and establish clinical efficacy for mpl®-immunostimulant. a considerable body of in vitro evidence supports the concept that the effects of lps on cells of the immune/inflammatory systems are controlled by interactions of lps with cd14. to evaluate if blocking lps-cd14 interactions has potential as a therapeutic in septic shock we have evaluated the effect of anti-cdi4 monoclonal antibody (mab) on lps-induced cytokine production and physiologic changes in an experimental model of endotoxin shock performed in cynomolgus monkeys. a novel model has been established where animals were treated with interferongamma for three days prior to infusion of highly purified lps over an eight hour period. in this model lps challenge resulted in marked release of eytokines in the blood, substantial hemodynamic changes, release of liver enzymes and alteration in lung permeability observed over a 24 hour period. to evaluate the effect of treatment with anti-cd14 mab, animals were given either nothing, an isotype control or anti-cd14 mab (5mg/kg) 30 rains, prior to the beginning of the lps infusion. evaluation of physiologic changes including mean arterial blood pressure and cardiac output, quantitative analysis of eytoldne levels including tnfct, il-113,1i,-6, il-8 and il-10, and liver enzymes during a 24 hour period revealed that treatment with anti-cd14 mab markedly attenuated all parameters of injury including decreased mean arterial blood pressure, increased cytnkine levels and the release of liver enzymes observed in animals given the isotype control mab or those not treated. administration of anti-cd14 mab to interferon-gamma treated animals not challenged with lps did not induce any detectable physiologic changes or increases in cytoldnes. these studies suggest that strategies to block lps-cd14 interactions will have utility in diseases such as septic shock or ards where lps plays a central role in initiating injury. preclinical studies with recombinant bactericidal/permeability increasing proteins (rbpi and rbpi23). p.w. "frown, dept. of preclinical science, xoma corporation, berkeley, california, usa. bactericidal/permeability increasing protein (bpi), from neutrophils, binds to and neutralizes lipopolysaccharide (lps); it also specifically kills gram-negative bacteria (gnb). these properties, which reside in the n-terminal half of the molecule, indicate potential therapeutic application in the treatment of gram-negative sepsis. the gene for human bpi has been cloned and recombinant holoprotein (rbpi) and a 23 kd n-terminal fragment (rbpi;3) have been produced in sufficient quantities for preclinical studies. both rbpi and rbpi23 bind to lipid a and neutralize the biological activities of lps derived from a variety of organisms, rbpi23 has equivalent antibacterial activity to bpi against rough gnb but is up to 30x more potent than bpi vs. serum-resistant and smooth gnb. rbpi and rbpi23 compete with lps-binding protein (lbp) for binding to lps under physiological conditions. consequently, both rbpi and rbpi23 block the cd14-dependent lpsinduced synthesis of the cytokines tnf, il-1, el-6 and il-8 in vitro. rbpi23 has also been shown to inhibit the lps-induced synthesis of reactive 02 metabolites, endothelial adhesion molecules and the procoagulant molecule tissue factor. in animals, rbpi has been reported to increase survival of endotoxin-challenged rats and mice, to inhibit the dermal schwartzman reaction in rabbits and to increase survival of neutropenic rats with pseudomonas bacteremia, rbpi23 increases survival and decreases cytokine production in endotoxin challenged mice and rats. it normalizes lps-induced changes in hemodynamic, pulmonary and/or metabolic parameters in lps-induced rats, rabbits and pigs. treatment with rbpi23 also increases survival and decreases cytokine production in bacterial challenge models in rats and mice. rbpi23 was not toxic to rats after 10 daily consecutive i.v. doses of 10 mg/kg. this combination of properties indicate that recombinant bpi may be useful in the treatment of sepsis. phase i/ii clinical trials of rbpi23 have begun. the discovery of lps binding protein (lbp) and subsequent identification of cd14 as a receptor for lps or lps-lbp complexes has resulted in a new understanding o£ how lps responsive ceils are stimulated. cd14 is found either as a glycosylphosphatidyl-inositol (gpi)-anehored membrane glycoprotein (mcd14) of myeloid cells or as a soluble serum protein (scd14) lacking the gpi-anchor. binding of lps to mcd 14 triggers cell activation while binding of lps-scd14 complexes to cells such as endothelial or epithelial cells that normally do not express mcd14 activates these cells. these pathways are shown in schematic form below. 4 ~di mcd14 plays a crucial role in presentation of lps to additional membrane components that make up a functional lps receptor. an immediate consequence of engagement of this functional receptor is protein tyrosine phosphorylation. the molecular mechanisms leading to these events will be discussed. understanding of these pathways will lead to the development of new therapeutic approaches to controlling host responses to lps. pretreatmen t posttreatment (before or after tnf peak) d) with different antibody dosages: 15 mg/kg ---0.1 mg/kg pretreatment with anti-tnfab prevented death in most model situations (except peritonitis), but also posttreatment up to 4h after sepsis induction was successful in the few studies performed. there is additional evidence that low-dose tnfab is partially effective. especially baboon anti-tnfab studies provided many insights into the pathophysiological sequences of sepsis induction, due to crossreactivity with human reagents. those events include the cytokine sequence with tnf-dependent il-i, il-6, or il-8, but also il-lra or stnf receptor release. granulocyte as well as endothelial cell activation were shown to be partly tnf related, and the procoagulatory response was influenced by anti-tnf treatment. from many animal studies the concept that tnf plays a pivotal role in sepsis is clearly evident and therefore anti-tnf therapy is a major candidate tbr clinical studies. the beneficial or harmful effects of tnf-mediated inflammatory responses depend on the clinical context. decreasing exaggerated tnf-mediated inflammatory responses may be useful in some patients with organ failure. tnfr:fc (immunex, seattle, wa) is a recombinant human protein composed of two identical extracellular p80 tnf receptors linked by the fc region of iggl. it neutralizes tnf with an affinity for tnf<z of 10-10 m and has a t1/2 of • 63h in normal humans. methods: 18 normal volunteers were randomized to receive either tnfr:fc vehicle (n=6), or low dose (ld) tnfr:fc (10 mg/m 2 iv, n=6), or high dose (hd) tnfr:fc (60 mg/m 2 iv, n=6) infused over 30 rain prior to endotoxin (e) (escherichia coil 0:113; 4 ng/kg iv) administration. plasma cytokines were measured using elisa and tnf bioactiv'dy. systemic hemodynamics were evaluated with indwelling arterial and pulmonary artery catheters and radionuclide heart scans and compared before and after fluid loading in subjects given vehicle with hd tnfr:fc. results: endotoxin-related symptoms were unchanged after ld or hd. peak temperature occurred at a later time point (4-5h) in subjects given tnfr:fc compared to vehicle (3h) (p=.004). at 3h, 5h (post fluid load ), and 8h, subjects given vehicle developed increased cardiac index and heart rate and decreased systemic vascular resistance index (all p=.0001 ). hd tnfr:fc did not alter this hyperdynamic cardiovascular response. ld and hd inhibited the e-induced teukopenta at i h post endotoxin (p<.03) and later leukocytosis (p<0.05). peak tnf bioactivity in subjects given vehicle was 154 + 44 pg/ml and <10 pg/ml in the ld or hd tnfr:fc groups (p<.001). two immunoassays for tnf were performed because the detection of receptor bound tnf varies with elisa. tnf (biosource) was decreased in ld vs vehicle or hd (p=.0001) whereas tnf (r&d systems) was increased after hd or ld vs vehicle (p<.001). decreased levels of il-8 (p=.00t), granulocyte colony stimulating factor (p=.03), and il-1 receptor antagonist (p=.001) were found after ld or hd vs vehicle. il-6 levels were lower after ld vs vehicle or hd (p=.006). il-10 levels were similar among subjects given vehicle, ld, and hd tnfr:fc. conclusions: ld and hd tnfr:fc delayed the febrile response to e but did not alter the early hyperdynamic cardiac response. this suggests that mediators other than tnf play an important role in the initial cardiovascular response to e in humans. tnfr:fc attenuated the release of some e and tnf-induced cytokines. ti~e antiinflammatory responses of tnfr:fc may be useful in some patients with disease processes resulting from excessive tnf-mediated inflammatory responses. the biosynthesis of tnf within macrophages is regulated both at the transcriptional and the translational levels. the 5' flanking region of the tnf gene contains several transcriptional control elements, including two kb enhancers similar to those of immunoglobulin genes and a "y box" similar to that of the mhc class ii promoter. these elements are critical for the enhancement of transcription noted after stimulation of macrophages with lps. despite these and other elements, transcriptional regulation alone accounts neither for the absence of tnf protein during normal homeostasis nor the profound increasein tnf production following stimulation with lps or other secretagogues. the translation of tnf mrna into protein is also tightly regulated via mechanisms involving the octameric "ttatttat" motif present in the 3'untranslated region of tnf, human inducible no synthase, and several other cytokine genes. this region not only destabilizes mrna, but also confers a translational blockade so that tnf protein is not normally synthesized despite leaky basal transcription. lps stimulation releases translational blockade and, together with enhanced trancriptional rate, accounts for significantly increased tnf secretion. opportunities for inhibition of tnf production are available at each of these two levels. agents which increase intracellular camp (pentoxif¥1ine, amrinone) inhibit accumulation of tnf mrna; in contrast, corticosteroids act primarily at the level of translation, inhibiting lps induced translational activation. understanding the regulation of tnf biosynthesis may assist in clinical strategies designed to regulate tnf secretion. the serine protease thrombin is formed by enzymatic conversion from its proenzyme form after coagulation activation and may enhance the inflammatory response in various ways. in the last step of the coagulation cascade, thrombin cleaves its substrate fibrinogen, thus forming fibrin. the fibrin clot may prevent phagocytosis of bacteria and promote local bacterial growth. thrombin can also stimulate a specific th.rombin receptor that exists on a variety of cells. for example, minute amounts of thrombin ma), stimulate platelets to develop tissue factor and boost the activation of more thrombin, thus initiating a positive feedback loop. other cellular effects of thrombin receptor stimulation include increase in intracellular calcium of platelets and monocytes, contraction of smooth muscle cells, increase in endothelial permeability, thromboxane generation by neutrophils and lymphocytes, pulmonary vasoconstriction, and enhanced cytokine production. animal experiments have been conducted with a variety of thrombin inhibitors including heparin, antithrombin ili, hiradin, and hirudin-like peptides. an alternative approach is the inhibition of the coagulation cascade at an earlier point in the cascades, or administration of the anticoagulant enzyme protein c work from our own group in a porcine model of lipopolysaccharide-(lps)-induced shock with disseminated intravascular coagulation has shown that prelreatment with recombinant desulfatohirudin (r-h) or with a preformed complex of human donor antithrombin iii with heparin or post treatment with r-h are able to block the degradation of fibrinogen and the formation of fibrin monomer. in addition, after pretreatment with r-h it was found that lps-induced lung injury was reduced. since both natural hirudin as well as r-h were well tolerated by healthy volunteers, adminislration of r-h may be a promising therapeutic approach in patients with sepsis and sirs. corticosteroids have been used in the management of various shock syndroms including sepsis. the data concerning the effects of this therapy is, however, conflicting and in large prospective studies, the use of early administration of high doses of corticosteroids have been found to give no beneficial effects in patients with sepsis. we have particularly been engaged in studies on possible effects of corticosteroids on the plasma cascade systems and on the mediator release from leukocytes. in in vitro studies, high doses of methylprednisolone were found to facilitate endotoxin induced generation of plasma kallikrein. furthermore, plasma prekallikrein and kallikrein inhibitor values decreased together with formation of kallikrein-c1 inhibitor complexes (1). in this in vitro plasma model we also found that methylpredoisolone alone in doses of 1 mg/ml induced contact activation with the formation of kallikrein-c1 inhibitor complexes. methylprednisolone was also found to have an additive effect on endotoxine induced decreases in kallikrein inhibitor functions. in the same experimental model on endotoxemia methylprednisoione was found to give an additive effect on activation of the initial part of the complement cascade (2) . activation of the terminal part of complement, however, was inhibited by the same dose of methylprednisolone. addition of methylprednisolone alone to plasma was also found to activate the initial part of complement. using a full blood model, we studied the effect of methylpredoisolone in modulating the endotoxin induced cytokine response. when 103 ng/l e. coli endotoxin was added to citrated blood from healthy human donors preinkubation with 20 gg/ml methylprednisolone significantly reduced the release of tumor necrosis factor et (79 %) and interleukin-6 (63 % ) at two hours after exposure to endotoxin. in conclusion, these studies show that corticosteroids might facilitate contact activation of plasma and reduce the function of major protease inhibitors. furthermore, we also observed that this drag in in vitro conditions facilitated activation of complement. on the other hand, corticosteroids were found to reduce endotoxin induced cytokine release from leukocytes in whole blood. these studies also showed a dose dependence of the effects of corticosteroids on endotoxin induced cellular and cascade system activation. different predisposing conditions like extensive trauma, infection or pancreatitis result in a remarkably similar inflammatory response. although thls inflammatory response primarily aims at the removal of devitalized tissues, destruction of bacteria and reestablishment of the integrity of host tissues, it may potentially become unregulated and generalized and thereby producing deleterious effects to vital organs or the body" as a whole. cytokines play an important role in the development of the systemic inflammatory response. most of their biological effects, however, are not directly mediated but exerted through other mediator systems. when the inflammatory response results in the formation of capillary leak syndrome and refractory hypotension, the unregulated activation of complement and contact systems appears to be the cause. both systems are almost exclusively regulated by c1-1nhibitor. am unbalanced degradation of the inhibitor protein was found to be associated with the onset of capillary leakage in various conditions such as bone marrow transplantation, severe burns, and septic shock. these observations suggest a relative deficiency of biologically active c3-1nhibitor in these septic shock like syndromes. therapeutic intervention studies were initiated using high doses of c1-1nhibitor concentrate. first results are promising, as in most patients the administration of the drug was followed by a quick and marked improvement of the patient's hemodynamics. whether the administration of c1-1nhibitor alone or in combination with other drugs is also sufficient to improve long-time survival has to be investigated in double-blind, placebocontrolled studies. salutary effect of human soluble complement receptor type-1 in models of adult respiratory distress syndrome g. feuerstein, m. dimartino, and *r. rabinovici, smithkline beecham pharmaceuticals, king of prussia, pa, usa, and *jefferson medical college, philadelphia, pa, usa adult respiratory distress syndrome (ards) is a severe pulmonary insufficiency syndrome associated with diverse diseases and injuries. ards is believed to be the consequence of a massive acute inflammatory reaction consisting of activated neutrophils infiltration into the lung. neutrophil activation is likely the result of multiple factors of which complement is believed to play a major role. a potent complement regulatory system in the form of the complement receptor type-1 (cr-1) exists on many cells where protection against complement injury is provided by inhibition of both the alternative and classical pathways for c3/c5 convertase formation. we have recently purified the human recombinant cr-1 in a truncated form (brl55730, tp-io, scr-1) and tested this soluble protein for protective effects in several models of ards. specifically, we have used the following rat models: 1) endotoxin-induced ards in paf primed animals; 2) 11.-2induced ards; 3) thermal injury (30% body surface)-induced ards; 4) acid aspiration-induced ards. ards-like features included edema (increase in lung wet weight and water content), neutrophil accumulation (histological inspection and myeloperoxidase assay) and increase in cells and protein in the bronchoalveolar lavage (bad. scr-1, 1-25 mg/kg, i,v., given as prophylactic (and in some cases, therapeutic) treatment significantly inhibited edema formation and leukocyte infiltration and, in some cases (e.g., endotoxin/paf or il-2), virtually completely. these comprehensive studies strongly support the therapeutic potential of scr-1 in ards due to diverse injuries. the xanthine derivative pentoxifylline (ptx) and several analogs of ptx have been evaluated for their protective effect in various animal models of septic shock. data from these in vivo studies demonstrate that (1) ptx suppresses lps-induced tnf release from activated macrophages; (2) ptx prevents tnf-induced pmn activation; (3) ptx attenuates lps-or tnfinduced acute lung injury as evidenced by prevention of increased pulmonary vascular permeability and detoriation of gas exchange; (4) ptx exerts its .protective effects even when administered following initiation of sepsis. the sum of these actions suggest that administration of ptx in sepsis may have therapeutic potential. hans hoffmann md, dept. of surgery, klinikum grosshadern, ludwig-maximilians-universit~.t, marchioninjstrasse 15, d-81377 m0nchen, germany. pentoxifylline [pof] and related xanthins are drugs of known haemorrheologieal activity and widely used clinically. recently, new pharmacological aspects of these established drugs could be demonstrated. it was found that pof selectively inhibits the formation of tnf but not il-6 most likely by causing accumulation of intraceliular camp via inhibiting phosphodiestersse activity, and, secondly, by inducing prostacyclin in different cell types. furthermore, pof is able to counteract tnf-mediated adherence of neutrophils to vascular endothelium and to inhibit fmlp-indueed respiratory burst in neutrophils. we and others have, therefore, studied its effect in different animal models. it was found that pof protected from increased pulmonary vascular permeability and sequestration of neutrophils into the lung in different animal models of acute lung injury. moreover, in pigs with induced faecal peritonitis the drug improved haemodyrmmle variables as well as pulmonary compliance and reduced the number of pulmonary neutrophila and lymphocytes. consequently, as a general outcome, pof could be found to improve survival in different models of haemorrhagie and endotoxie shock. the protective effect of pof was dose-dependent and observed even when pof was given up to 4 hours after endotoxin. in order to evaluate these pharmacological effects of pof in humans, we studied pof under controlled conditions of endotoxlnemia in volunteers. we found that pof selectively inhibits the lps-indueed endogenous formation of tnf without affecting il-6 and il-8. moreover, pof counteracts the lps-indueed initial leukocytopenia by interfering with the adherence of neutrophils in the microvasculature. meanwhile the inhibition of endogenous formation of tnf by pof could be demonstrated under different clinical conditions of acute and chronic cytokine release syndromes, {okt3 first-dose reaction, cancer-or tuberculosis-induced cachexla}. it appears worthwile and promising to investigate wether pof exhibits beneficial effects also in septic patients. objectives: to determine the specific role of paf in both lethal primate bacteremia and nonlethal human endotoxemia. materials and methods: two double-blinded placebo controlled studies were performed. first, ten baboons (14.5 +.7 kg), were randomized to a control group receiving 101° cfu/kg of intravenously administered e. co/i (n=5) and a treatment group (n =5), pretreated with .1 mg/kg of paf receptor antagonist ro24-4736 two hours prior to e. co/i infusion. in a second study, ten healthy male volunteers were randomized to a control group receiving 4 ng/kg of intravenously administered endotoxin (lps) (n=5) and a treatment group (n=5) pretreated with 10 mg of ro24-4736 eighteen hours prior to lps administration. physiologic parameters and cytokine levels were measured continuously in both studies for 24 hours. results: baboons pretreated with the paf antagonist had improved oxygenation (96 + 11 mm hg v 59 -+ 6 in controls, p < 0.05) and creatinine clearance 12 hours post e.coli (39.8 + 23.4 ml/min v 0.1 _+ 0.1 in controls, p < 0.05). similarly, volunteers pretreated with the paf antagonist experienced fewer symptoms, including rigors and myalgias at 1 hour post lps. this was in concert with a diminished peak cortisol (668 +_ 107 mmot/l v 959 +-159 mmol/l in controls, p < 0.05), epinephrine secretion (1057 + 165 nmol/l v 2029 + 431 nmol/l in controls, p < 0.05). hemodynamics and circulating tnfa levels (data not shown) were unaffected in either study by prior paf antagonism. concluslons: paf influences some components of the multi-organ failure syndrome in lethal gram negative sepsis and the counter-regulatory hormonal system in human endotoxemia through tnf-independent mechanisms. paf antagonists may serve as adjuncts to cytokine blockade in the treatment of gram negative sepsis. the mediator role of platelet-activating factor in gramnegative septic shock pierre g. braquet, david hosford and matyas koltai institut henri beaufour, le plessis robinson, france considerable evidence has been accumulated to support the concept that a paf/cytokine autogenerated feedback network is responsible for priming and amplification of inflammatory mediator release in septic shock. cytokines, such as tnf~x, il-1 and other interleukins interact with paf which then amplifies cytokine production, therefore, paf may be the principle mediator in endotoxin shock. a single factor identified as tnf is suggested to play a pivotal role in the fatal phase of septic shock. presumably excess tnf release is the result of amplification process primarily triggered by paf. one may assume that the high tnf concentration in the blood of 'non survivors' is the consequence rather than the cause of cell death. the dubious results of clinical trials with anti-tnf antibodies and il-1ra, a naturally occurring il-1 antagonist protein, have risen debate around the exceptional role of cytokines in the pathomechanism of septic shock or systemic inflammatory response syndrome (sirs) induced by gramnegative microorganisms. there are similar problems with the interpretation of the results obtained in clinical trials of monoclonal igm antibodies against e. coli endotoxin, ha-1a and e5. future studies will answer the question as to the effectivity of inefficiency of this treatment. perhaps when the plasma concentrations of lps and cytokines exceed a critical level, treatment fails to save the patients life. with regard the causal role of lps in septic shock, the putative role of bacterial porins may have to be taken into consideration. the marked release of paf induced by endotoxin leading to excess txa2 production may be responsible for the microvascular failure and death in septic shock. antagonists of paf markedly protect against the release of txa2, and may interrupt the vicious circle of amplification process. no produced by the inducible no synthase plays a pivotal role in causing vascular paralysis in lps-induced sirs. to explore the relationship of paf to no synthesis will provide better understanding of the pathomechanism of septic shock. several paf antagonists, including bn 52021, have undergone phase ii and phase iii clinical trials. the strategy in the treatment of sirs, however, remains multifactorial, since little is known about the sirs caused by microorganisms other than gram-negative bacteria. evidence has begun to accumulate which suggests that in sepsis excess production of lps and cytokines can lead to uncontrolled production of inos and that this might in part explain the severe unresponsive hypotension seen in some septic patients. a number of strategies have been explored in an attempt to limit excess no production. a favourite target has been competitive inhibition of nos by arginine analogues such as l-nmma. in animals, some investigators have shown that the haemodynamic effects of lps challenge can be attenuated by l-nmma, but there is a narrow toxic-therapeutic ratio. in a model of live e. cell sepsis, we have been unable to reproduce these findings. localisation of no production by immunohistochemistry suggests that no production is compartmentalised, and more subtle methods of regulation may be required if this approach is to have clinical valuc. anti-adhesion molecule strategies ch wortel, repligen corporation, one kendall square, building 700, cambridge, ma 02139, usa. the neutrophil has been implicated as a pivotal player in the pathogenesis of multiple organ dysfunction. an important first step in the process of neutrophilmediated organ injury involves the binding of neutrophils to endothelial ceils. this process is largely regulated by complementary adhesion molecules, some of which are present constitutively on the cell surface and others that can be upregulated in response to chemotactic and pro-inflammatory stimuli. several different adhesion molecules have been described. l-selec tin is expressed on the plasma membrane of neutrophils and is shed from the surface early in the inflammatory process. it is therefore thought to mediate the initial interactions with endothelial cells. e-selectin and p-selectin are endothelial adhesion molecules. e-selectin is not constitutively expressed but is upregulated by inflammatory mediators, particularly il-1 and tnf. p-selectin is present in the weibel-palade bodies within the endothelial cytoplasm and can be upregulated rapidly in response to thrombin, histamine, and oxidants. all selectins recognize oligosaccharides, particularly sialylated lewis x antigen. this carbohydrate moiety is expressed on many proteins, including the selectins themselves. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta2 or cluster differentiation (cd)i 8 chain covalently linked to one of three different alpha chains (cd11 a, cd1 lb, cd1 lc) and exist on the cell surface as three distinct heterodimers. cd1 l a/cdi 8 is expressed on all leukocytes, whereas cd1 l b/ cd18 and cd1 lc/cd18 are restricted to cells of myeloid origin. cd1 i/cd18 interacts with intracellular adhesion molecule-1 (icam-1), its ligand on endothelial cells. icam-1 is a member of the immunoglobulin family of adhesion molecules. it is constitutively expressed on endothelial cells and can be upregulated by cytokines. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in alarge number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries such as arthritis, bums, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degeneration, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. since modulating the function of adhesion molecules may temporarily leave the host without effective defense machinery, safety evaluations are of utmost importance in evaluating this therapeutic approach. treatment of gram-negative septic shock with an immunoglobulin preparation: a prospective, randomized clinical trial ingolf schedel, ursula dreikhausen; birgit nentwig; marion hockenschnteder, dirk rauthmann, salim balikcioglu, rolf coldewey, helmuth deicher, md endotoxin may play a major role in the pathogenesls of muitiorgan failure in the context of the toxic process in septicemia induced by gram-negative pathogens. the hypothesis which has led to a prospective clinical study consisted in the idea of inhibiting the biological effects ofendotoxin during the early phase of septic process, and thereby attampting to attain a positive effect on the clinical course of the disease. -objecave: to evaluate the effectiveness ofa polyclonal immunoglobulln (ig) preparation containing igg, lgm, and iga as an adjunctive therapy for septic shock. -desigru prospective, randomized clinical trial. patients: fifty-five patients w~th septic shock were randomly allocated to two groups according to criteria of septic shock. -intervention: one group of patients (n = 27) received a commercially available immunoglobulin preparation (containing high titers of antibodies specific for determinants to bacteria1 endotoxin) during the first 3 days after inciosion in the study. the other randomized group (n = 28) did not receive any immunogiobulin preparation. -measuremems and main resmts: during the period of <6 wks after the beginning of clinically apparent septic shock, death related to the septic process occurred in one (4%) of 27 patients who received immunoglobulln. by comparison, nine (32%) of 28 control group patients died during this period (p <. 01 ). within the first 48 hrs after onset of the clinically apparent septie process, significaptly increased activity of circulating endotoxin and simultaneously decreased specifie igg serum titers to lipid a were detected in the group of nonsurvivors. the rationale for the use of polyvalent intravenous immunogiobulins (ivig) to treat severe infections stems from in vitro and animal studies documenting that high-dose igg enhance opsonization and phagocytosis of pathogenic bacteria. moreover, recent clinical studies have shown that in septic patients the phagocytic function of circulating polymorphonuclear neutrophils (pmn) is defective; the degree of such impairment correlates to the severity of the septic state, the beneficial effect of administering high dose igo in septic patients can be two-fold: i) ivig provides large quantities of antibodies against invading pathogens; the igg bound to the pathogens can opsonize their phagocytosis; 2) the opsonization of phagocytosis by exogenously administred polyvalent igo would be of special importance in those patients who present a significant defect of their phagocytic function, when they are challanged by a large bacterial invasion. a prospective, randomized, double-blind study was carried out comparing the administration of ivig (sandoglobulin@) vs, paeebo (human albumin) in severely septic surgical patients, only patients with gepis score >_20 (meaning a mortality risk >70%) were accepted into this protocol. patients were randomized to receive 0.4 g/kg of ivig or placebo on day 0 (when they reached sepsis score>20), repeated on day +1 and +5. at the beginning of icu treatment, the two groups of patients were similar for severity of sepsis, age, concomitant disease, type of surgical procedures, antra and perioperative procedures, antibiotic administration. the results of the study indicated a significantly reduced mortality in patients with severe surgical sepsis treated with ivig as compared to placebo control patients (mortality: 38% vs, 67% respectively; p< 0,05). in conclusion, the results of our study in patients with severe surgical sepsis were the following: 1) ivig plus multimodal treatment of sepsis, including antibiotics, reduce mortality significantly', 2) the reduction of mortality seems to be due to a decreased incidence of lethal septic shock. despite substantial clinical research, the avallable data regarding the effectiveness of supplemental immunoglobulin (ig) treatment in sepsis in adult patients do not yet allow definitive conclusions. in view of the persistently high sepsis mortality there is a need to continue clinical investxqations regarding supplemental sepsis treatmen~ in general, as well as concerning ig administration in particular. we present and discuss the protocol of the ongoing ,,score-based-immuneglobulin therapy of sepsis (sbits)" study. the protocol (theoret surg 8(1993)61-83) of this multicenter, randomized, prospective and double-blind trfal relies on the results of an observational trial on i.v. igg treatment in 163 patients with sepsis and septic shock (infection 19~1991)216-227), carried out as a prerequisite for the present trial. using microcomputer-based bedside routine score monitoring, we regard quantitative measures of severity of disease and sepsis: only patients with a certain degree of both severity of disease (apache ii score 20-35) and severity of sepsis (elebute sepsis score 12-27) will be included. by observing these previously validated inclusion criteria, this trial snould iqentify a priori and include patients with potentially optimal response to therapy, consisting o~ either placebo (0.i % albumin) or polyglobin n" -12 ml (0.6g)/kg on day 0 and 6ml (0.3 g)/kg on day i. with an anticipatedpopulation size of 800 patients the study should comply with the statlstical requirements (estimated mortality: 30%, with a 33 % reduction in 28-day mortality in the treatment groupl to prove or disprove the question of igg effectiveness in sepsis in terms of improved prognosis. up to november 1993, more than 460 patients had been included; patient enrollment will be finished in 1994. previous studies have demonstrated rhll-i ra, a naturally occurring antagonist of il-1, increases survival in animal models of andotoxemia and eschehchia coli bacteremia and attenuates the decrease in mean arterial pressure resulting from challenge with both gram-negative and gram-positive bacteria. previously, in 99 patients, rhll-lra was demonstrated to increase survival in patients with sepsis syndrome and septic shock in a dose-dependent manner. methods: a randomized, double-blind, placebo-controlled, malticenter, clinical trial enrolled 893 patients at 63 academic medical centers in europe aad north america. eligible patients received either placebo (vehicle) or rhil-lra (anakinra) 1.0 or 2.0 mg/kg/hr by continuous intravenous infusion for 72 hours. the presence of organ dysfunction (i.e., ards, dic, renal, and hepatic) at study entry was determined prospectively by a clinical evaluation committee using definitions which were developed a-priori. survival time was evaluated over 28 days utilizing a linear dose-response model, assuming a log-normal distribution. results: 563 patients had one or more sepsis-induced organ dysfunction(s) at study entry. a dose-related increase in survival time was observed with rhll-lra compared to placebo in patients with ards, dic, and renal dysfunction (p --< 0.04 endotoxin infusion releases platelet-activating factor (paf), a potent phospholipid mediator which leads to an autocatalytic amplification of cytokine release. bn 52021 (ginkgolide b), a natural paf receptor antagonist, has provided significant protection against sepsis in different animal models• a randomized, placebo-controlled, double blind, multicenter trial on efficacy (mortality at d28) and tolerance of bn 52021 (2 iv infusion of 120 mg x 2/day over 4 days) in severe sepsis has enrolled 262 pts. the 28day mortality rate was 51% for the placebo group and 42% for the bn 52021 group (p = .17). the efficacy of bn 52021 was greater in pts with gram-negative sepsis: the 28-day mortality rate was 57% for the placebo group and 33% for the bn 52021 group (p = .01). bn 52021 also reduced mortality among pts with gram-negative septic shock (mortality was 65% for placebo vs 37% for bn 52021; p = .01). using statistical adjusments for pronostic factors, the relative risk of death of the bn 52021 group was 0.61 (0.34 -1.08, 95% confidence interval; p = .09). this risk corresponds to an adjusted reduction in mortality of 39% for pts receiving bn 52021. no differences in mortality rates were found between the placebo and the bn 52021 groups in the absence of gram-negative sepsis• there were no differences in adverse events between the placebo and the bn 52021 groups. bn 52021 is a safe and promising treatment for patients with severe gram-negative sepsis. a confirming study, focused on gram negative sepsis, is in progress. v~3lliam a. kanus m.d. and the rhll-lra it has been traditional within the field of infection and sepsis to think in terms of specific indications for drugs based on the type of infecting organisms, advances in antibiotic therapy now control or ltnflt the growth of bacteria. the majority of deaths are now caused by either an initial overwhelming response to infection or subsequent multiple organ system failure attributed, in part, to the effects of intrinsic biologic responses of the host. type of organism, therefore, may not be as critical as determining the exact severity of the host's severity or risk of death from infection. we also know that both the relative benefit of a new treatment across groups and its absolute benefit for an individual patient will vary with their risk in a predictable fashion. we recently iuve~iguted the relationship between one measure of host response, the acute risk of death as prospectively estimated by u comprehensive risk mode[ for 28-day mortality (jamb. 1993; 270:12,33-1241) , by its retrospective application to the results from the phase in evaluation of recombinant human intcrlenkin-1 receptor antagonist (rhll. ira). we found that there was a significant interaction between the patient's predicted risk of mortality at the time of entry to the study and the ability of rhil-lra to prolong survival time (x2 = 7.6, p [] 0.02, log.normal) for all 893 patients in the trial• survival benefit began st approximately 24% baseline risk of 28-day mortality. for the $80 patients with a predicted risk > 24%, there was a 22% reduction (p=0,00$ log normal). when we examined the variation in patients above and below the 24% risk level with hazard functions, i.e., their daily risk of death during the study period, we found that placebo patients with < 24% risk had lltile acute daffy risk during the hlltial two days follawh~g study entry and this risk was little affected by rhil-lra, in contrast, patients with > 24% risk had high daily mortality risks during the tuttlal two days that high dose rhtl-lro substantially reduced. these results are compatible with our current understanding of outcome from sepsis and the proposed mechanism of action o£ immunotherapy, the earliest deaths from sop sis are secondary to an immediate inflammatory response followed closely by deaths secondary to multiple organ system failure, later deaths (after 14 days) are not as closely related to the acute effeete of the inflammatory cascade. because of the timing and action of most proposed tmmunotherapy, they may be capable of preventing mortality primarily in these initial two phases. in this study, an independent predicted risk of mortality reflected this mortality pattern ned illustrated the potential benefit of immtmotherapy. use of a predicted risk of mortality in the design and analysis of clinical trials could improve our understanding of the clinical benefit of these new therapeutic approaches. the systemic inflammatory response syndrome (sirs) is a term recently proposed to describe patients with systemic inflammatory responses to insults such as infections (sepsis), trauma, burns, pancreatitis, and other initiating events. patients with sirs may have similar activation of inflammatory mediators and similar outcomes independent of the initiating event. these outcomes include organ dysfunction and failure, shock, and death. challenges to the successful conduct of clinical trials in sirs include the complexity of illness in these patients and the important--but limited--clinical benefits of novel compounds that may be limited to selected patient subsets. addressing these challenges will require new tools and approaches. these will include more sensitive and appropriate endpoints, and the use of methods such as baseline risk adjustment, to allow detection of drug risk interactions not captured adequately by categorical definitions, such as sepsis syndrome. on the basis of supportive preclinical and phase i safety studies, we have initiated phase ii clinical trials of a novel bradykinin antagonist, cp-0127, in four sirs subcategofies: sepsis, multiple trauma, burns, and pancreatitis. each of these studies is designed to measure the effect of cp-0127 on mortality, organ dysfunction and failure, and activation of mediators. in addition to investigating rates of organ failure using standard definitions--a new endpoint--a continuous summary measure of organ dysfunction (the acute physiology score of apache tm iii) is being used to quantify the degree of organ dysfunction and the speed and pattern of recovery of physiologic stability. in the sepsis study, another new approach--a study specific risk model based on the apache ill database--has been developed which will be used to assign a pre-treatment baseline risk to each patient enrolled. the primary outcome variable will be risk adjusted survival time to 28 days. this type of risk-adjusted analysis may allow for more efficient and powerful trials and more accurate and useful indications for use. study purpose: in post-cardiac surgical patients (pat.) at risk for sepsis, the efficacy of early i.v. immunoglobulin (ig) treatment was compared to a matching historical control (con.) population. postoperative risk assessment: using apache ii scores lap) (first postoperative [pop.] day) in a pilot study phase, we were able to differentiate between the large population (95.2%) of pop. low-risk pat. (ap< 19; mortality: 1%) and the small groups of pop. pat. at risk lap= 19-23) and high risk lap_24) with a significantly higher mortality (14% and 76%, mainly due to sepsis). subsequently, among 1341 consecutive pop. pat. we prospectively identified and treated these pat. iq treatment reqimens: first study period (n =41): (gg (psomaglobin n a, tropon biologische pr~parate, cologne, frg, day 1:8 ml/kg, day 2:4 ml/kg). second study period (n=25): iggma (pentaglobin r, biotest, dreieich, frg, 5 ml/kg on days 1 to 3). results: ig pat. and con. were comparable in demographic data, operation characteristics and baseline disease severity lap and elebute sepsis scores). in contrast to con. (risk: n=21, high-risk: n-21), the ig pat. showed a marked improvement in disease severity (fall in ap), especially in the high-risk group (igg, n=26: p<o.05; iggma, n=13: p: 0.08). in this group, ig led to higher (p<0.05) response rates, defined as an ap score decrease ->7 within four days (igg: 54%, iggma: 62%; con.: 19%), and reduction in mortality (igg: 46%, iggma: 46%; con.: 76%), statistically significant (p<0.05) for ig treatment as a whole (igg and iggma). conclusion: given the good comparability of the study groups, our results indicate, despite the non-randomized design, that early supplemental ig treatment can improve disease severity and may improve prognosis in prospectively apache ii score-identified high-risk patients after cardiac surgery. objective. elevated plasma levels of endothelin (et) have been demonstrated in both experimental and human sepsis. et has been proposed as a sepsis mediator leading to vasoconstriction with tissue hypoperfusion and organ failure. the aim of the study was to determine the effects of sepsis treatment with volume resuscitation, antibiotics and the anti-lps monoclonal antibody es® on big et and active, 21 aminoacids et (et 21) in rat abdominal sepsis. methods. lethal peritonitis was induced with a 4 mm coecal perforation (cp) in male wistar rats. plasma levels of big et and et 21 were determined with amersham tm endothelin rias 4, 8 and 12 h after sepsis induction. experimental groups: 1. cp control, 2. volume replacement (vr); 0,9% saline 10 ml/kg/h continous iv infusion started after 2 h, 3. antibiotic; imipenem 40 mg/kg iv after 2 h, 4. e5®; 10 mg/kg iv after 2 h, 5. vr + imipenem + es® after 2 h. results. high concentrations of both big et and et 21 could be demonstrated after 2 h and lasting for 12 h after cp. neither volume replacement nor imipenem did influence the elevated plasma et. e5® significantly reduced et 21 both 4, 8 and 12 h after sepsis induction, but did not reduce big et. when es® was combined with vr and imipenem, reduction of et 21 was the same as for e5® alone. these results strongly suggest that bacteria and hypovolemia per se are not decisive stimuli for et production during sepsis. e5® reduces circulating lps and tnf which is the probable mechanism of the suppressed et 21 synthesis. the unaltered big et fraction after e5® treatment indicates conversion of big et to et 21 as the site of action responsible for reduced et 21. conclusion. lethal peritonitis in the rat is followed by elevated plasma levels of big et and et 21. e5® anti-lps antibody significantly reduces plasma et 21 while volume resuscitation and antibiotics failed to do the same. es® did not reduce plasma big et. pmx treatment on severe endotoxemia with multiple organ failure was safety and effect in prognosis, and sepsis related parameters. it was certified that reduction of plasma endotoxin was effective in severe endotoxemia. a. lechleuthner,s. aymaz, g. grass, c. stosch, s. dimmeler, m. nagelschmidt, e. neugebauer. ii. dept. surgery, university of cologne, germany. introduction: the cardiovascular therapy of hypodynarnic shock states is a challenging problem. in clinical as well as experimental studies beneficial functions of a new hg-agonist bu-e-75 in congestive heart failure has been demonstrated 03aumann, 1989). therefore, we investigated the effect of bu-e-75 in hypodynamic shock in pigs. materials and methods: pigs (deutsches hausschwein, pitrain, [20] [21] [22] [23] [24] [25] were anesthesized with fentanyl/dormicum, ventilated (n20:o 2 = 2:1) and cardiovascular parameters were monitored with a complete icu-eqnipment. the hypodynamic model was established in a pilot study (4 animals) to evaluate the effective concentration of bue-75 in healthy and endotoxin (lps)-treated animals. endotoxic shock was induced by continous infusion of 10 ~g lps/kgkg/h (055:b5, fa. difco). the hypodynamic state was defined as a decrease of cardiac output by 30 % of steady state levels. a wedge pressure of 10-12 mmhg was kept constant by volume resucitation during the experiment. in a subsequent randomized controlled trial (rtc) 3 groups with 6 animals per group were studied. the groups were treated as follows: group i, lps and 0,9 % nac1; group ii, lps and bu-e-75 (100 #g/kgkg/h); group iii, famotidine (h2-blocker) pretreatment (1 mg/kgkg), lps and bu-e-75. results: the pilot study in healthy pigs revealed, that bu-e-75 had positive inotropic effects. these effects were inhibited by the h 2antagonist famotidin. bu-e-75 however had no beneficial effects in the hypodynamic phase of endotoxic shock in the rct. cardiac index (ci) and the oxygen delivery (do2) were not significantly influenced by bu-e-75 application (group i versus group ii). bu-e-75 did not ameliorate the negative inotropic effect measuring left ventricular stroke work (lvsw) in hypodynamic shock phases. on the contrary, bu-e-75 led to a further significant decrease of lvsw (p < 0,05). famotidin pretreatment did not affect the response (group iii versus group ii). conclusion: in hypodynamic shock states the h2-agonism seemed to have no beneficial effect under these experimental conditions. receptor down regulation or changes of signal transduction under septic conditions may be responsible. cellular studies may help to identify these mechanisms. objectives. antithrombin iii inactivation of proccagulant proteases is so far the only inhibitory therapeutic approach to disseminated intravascutar coagulation (dic). we therefore set out to investigate whether cll substitution reduces coagulation activation in an endotoxin induced rabbit dic model. materials and methods. 29 male rabbits chbb:hm(spf) were randomty assigned to one of the following groups. group k : naci 0.9% (control without endotoxin, n=10). group e : endotoxin 120 tjg kg "1 bolus i.v. + naci 0.9% (control with endotoxin, n=10). group c : endotoxin 120 pg kg -1 bolus i.v. + cll 400 u kg -1 bolus + 400 u kg "1 4h "~ i,v. (treatment group, n=9). all animals were anesthetized and mechanically ventilated. blood samples were drawn prior to endotoxin administration (m1) and after 120 (m2) and 240 rain. (m3). thereafter, lung and liver tissue samples were taken intravitatly in a standardized fashion for h&e microscopic fibrin quantification using a triple score (fibs). from all blood samples the prothrombin time (pt), activated partial thromboplastin time (aptt), fibrin monomers (fm), and d-dimers (dd) were measured. for statistical significance of differences between the groups anovas and the wilcoxon test (fibs) were performed. results. fibs for lung/liver were significantly different (p<0.05) between group e (lung 180, liver 159) and c (lung 89, liver 0) (group k : lung 11, liver 0). , a synthetic serine proteinase inhibitor, has an anticoagulant activity in the absence of" antithrobim iii. gabexate has been reported to be useful in the treatment of disseminated intravascular coaguiation due to neoplastic diseases. in this study, we investigated gabexate therapy for the treatment of dic due to sepsis in the postoperative critical patients. materials and methods: from july 1992 to june 1993, 15 patients in the surgical intensive care unit met the criteria of dic or pre-dic. eleven were male and four were female with the mean age of 66.7 years. all these patients suffered from some complication of operations which led to the development of sepsis. foy was administered at the rate of 2mg/kg/hr untii the coagulation profile retumed to normal or the patient died. the coagulation parameters were monitored before and on the 1st, 3rd, 5th and 7th day. results: fourteen of these fifteen patients died despite transient improvement of the coagulation parameters in five patients. these patients suffered from sepsis resulting from surgical complications which could not be well controlled. the only survival was a case of recurrent intrahepatic duct stone with biliary tract infection complicated with sepsis and dic. after choledocholithotomy and the use of foy, the patient recovered gradually. conclusion: dic is a late manifestation of sepsis in the critical surgical patients. the most important thing is to eradicate the cause of sepsis. if the underlying septic focus cannot be controlled, dic will persist despite the use of gabexate mesilate. emergency surgery, taipei veterans general hospital, taipei, taiwan. there are 2 main types of bradykinin (bk) receptor, namely bk~ and bk z. the bk 2 receptor is constitutive. the bk 1 receptor is also constitutive but in the majority of cases is inducible and involved in chronic inflammatory syndromes such as sepsis, hyperalgesia and airways hyperreactivty in animals. the mechanism(s) involved in the upregulation of the bk 1 receptor is unclear, however a variety of agents including lps, e coil and ill are particularly efficacious in vitro and in vivo. ill and bradykinin acting at their respective receptors are believed to be involved in sirs/sepsis. we have investigated the effect of antagonists at ill (antril), bk 2 (bradycor [cp-0127]),bk~ (cp-0298) and bkz/bk 1 (cp-0364) receptors on the de novo generation of bk~ receptors (reflected by hypotensive responses to a bk 1 agonist) in the lps-treated (10ug iv) rabbit. in lps treated rabbits hypotensive responses to bk~ but not bk 2 agonists increased with time and at time 210min appeared maximally induced. constant iv infusions of cp-0127 blocked bk 2 but not bk~ and cp-0298 bk~ but not bk 2 responses. cp-0364,cp-0127+cp-0298 and antril+cp-0364 blocked both bk 2 and bk~ responses. antril alone had no effect on bk 2 or bk~ responses. within 30-60 min after stopping the infusions of antagonists the responses to bk~ and bk z agonists were the same as those in nonantagonist infused rabbits. these results indicate, at least in the lps-treated rabbit, that neither bk2,bk ~ or ill receptors alone or in combination, are involved in the de novo generation of bk 1 receptors. in vitro studies demonstrated that beth bradycor and cp-0364 (but not antril) were antagonists at both bk z and bk~ receptors. if both bk z and bk 1 receptors are significantly involved in chronic inflammatory situations in man such as sirs/sepsis then the rationale for the use of compounds such as bradycor or cp-0364 is clear. infection is a major cause of or contributor for morbidity and mortality in liver transplant recipients. effectiveness of prophylactic and therapeutic protocols is important for the success of liver transplantation ( olt ). sdd is used as prophylaxis for reduction of infection caused by gram negative or fungal microorganisms. between september 1988 and july 1993 400 olt's in 368 patients were performed at our department. the actuarial 1-year patient survival is 90 %. infection prophylaxis is started with sdd and ciprofloxacin once the patient is accepted as an olt candidate. perioperatively metronidazol, tobramycin and cefotaxim, postoperatively cotrimoxazol are prescribed additionally. the table shows pneumonia, peritonitis, major wound and urinary tract infection are common nosocomial infections following severe injury. in a series of severely injured patients from the university of louisville hospital, pneumonia was the most common infection followed by peritonitis, intra-abdominal abscess formation and burn wound infection. pneumonia is actually the leading cause of death from nosocomial infection. these are defined as occurring from 48 to 72 hours after hospital admission. this definition has important implications for antibiotic therapy because the likely pathogens and their respective sensitivities are different for community acquired pneumonia. the diagnosis of nosocomial pneumonia is difficult following major injury as many patients will have pre-existing fever, leukocytosis, tachypnea, and chest x-ray changes. reliance on sputum gram stain and culture is important and best obtained by a bronchoalveolar lavage or protected specimen brush during bronchoscopy. predisposing risk factors include severe head injury, emergent intubation and shock, and such patients have been shown to benefit by early tracheostomy. staph aureus has been the most common pathogen isolated from the sputum and the remainder gram-negative organisms with pseudomonas aeruginosa, and klebsiella pneumonia predominating. bacteria recovered by site as well as by intensive care unit is published in the six month antibiogram which also includes recent antibiotic sensitivities. this aids in empiric antibiotic selection against such nosocomial organisms. in a series of severely injured patients (iss -30), mean temp. was 101.4f, leukocytosis was 16k, pan2 was 87, fin2 was .47, and peep was 5.1 at the time of diagnosis (ards excluded). there was marked reduction in class ii histocompatibility antigen (hla-dr) density on peripheral and bal monocyte/macrophages which recovered over time with resolution of pneumonia. immune suppression occurred prior to development of pneumonia, was especially localized to the infected tissue, but recovered with clinical improvement. specific immune modulation targeted to pulmonary white cells may hasten clinical recovery and minimize pulmonary dysfunction. -clinical experience j. tnllemar amphntericin b remains the drug of choice for many systemic fungal infections. its advantages include a broad spectrum of activity and intravenous administration. the major disadvantages of amphoterlcin b is its severe side-effects, especially the nephrotoxicity. to decrease the toxic side..cffccts various liposomal amphoteficin b formulations have been produced. it was found that these liposemal formulations were as effective as amphotericin b but in contrast had a low incidence of toxicity. at present there are three ~different variations of lipid formulations under assessment: amphotericin b lipid complex (ablc), amphotericin b coloidal dispersion (abcd) or true liposomes. the ablc has a ribbon like structure. it has been shown to have a reduced toxicity and an efficacy ranging from being as effective to four times less effective that conventional amphotericin b. regarding abcd the particles have a disk-like structure with a diameter of around t00 am and a thickness of 4nm. the ami-fungal efficacy is 2-4 times less than that of conventional amphotedcin b. both ablc and abcd are presently investigated in phase ii/iii studies in the us. ambiseme is currently the only commefieally available true lipesome. ambiseme is a spherical small unilamellar lipesome with a diameter less than 100 nm with a mutina ld50 of > 175 mg/kg. it has been used in dosages up to 6 mg/kg/day in compassionate based studies with good tolerability. the mycological efficacy range from a 83% response rate for invasive candida infections to 41% response rate for aspergillosis. ambisomc have been evaluated as anti-fungal prophylaxis in randomized trials in bone marrow (bmt) and liver transplant (ltx) recipients. it was well tolerated. in bmt recipients the incidence of proven fungal infections was 8% among placebo treated patients compared to 3% for the ambisome treated patients (ns). in ltx recipients ambisome prophylaxis was effective, significantly reducing the incidence of deep fungal infections from 16% to 0% ill placebo and ambisome treated patients respectively (p<0.01). prospective randomized trials comparing these various amphotericin b preparations with conventional amphotericin b is needed to determine their future place in the therapeutical arsenal. two patlentgroups ere particularly at risk to develop serious cmv disease: cmv seronegative transplant recipients of seroposltlva donors and those patlants treated for rejection with anti t-ceil preparations, we have evaluated the value of prophylactic anti-cmv immunoglobulin (cytotect", biotest pbarma gmbh, dreieich, frg) administration in high risk heart and kidney transplant recipients, in a double blind placebo controlled study 40 kidney transplant recipients, treated for biopsy proved re)action with rabbit atg, received globullntplacebo infusions. the preparatlons were given i,v, in a dose of 100 mg/kg at day 0,7,14,35,56 and 77 after the initiation of anti = rejection therapy, passive immunization completely prevented cmv related death, although it did not reduce th~ incidence of cmv isolation, viraemia or disease, this effect was mainly observed in cmv saronegativa recipients of a serop0sitive donorktdney. seroposltive recipients did not benefit from treatment and seronegatlve recipients of a seronegetlye donor were not et risk for cmv infection at e!l. in a open study the incidence of cmv infection and disease was evaluated in 143 consecutive i~eart sllograft recipients. sixty-five patients were cmv seronagatlve and they all received passive immunlzation according to the dosage schedule used in the kidney patients, but starting on the day of transplantation, this scheme resulted in median snti-cmv igg titers of 1200 elisa units during 3 months. cmv infection occurred in 21/65 ~eronegetlve and in 40/81 seropositive recipients (n,s,), in ssronegetive donor-recipients pairs the incidence was significantly lower (3/34] , the passively immunized seronegstive recipients of e seroposltlve donorheart showed comparable incidence of cmv infection f18t29) vs the seropositive recipients. primary infection more often resulted in disease than secondary infection (11121 v8 10/40), but no difference in incidence of disease (11165 vs 10/81 ) or severity in symptoms was noted between the immunoglobulln treated serone(]ative patients and the seropositiva recipients. apparently passive immunization induces anti-cmv immunity which crossly resembles naturally acquired resistance. abdulkadirov k.,chebotkevich v., moiseev s. the incidence of infection is still high in patients underwent bmt. this complication is the major cause of mortality if it is not recognized and treated promptly and properly. our data showed that from 21 patients with different types of leucemia after autologous and allogenzc bmt 19 had the episodes of fever. in the ma i ority of these episodes the bacterial etiolog$ gram negative bacflli and gram positive cocci) can be proved. on the other hand, in 62% of the fever cases we detected also viral respiratory (corona-, adeno-, rs-and other) infection. our previous investigations showed that even in healthy persons the viral infection has influence on antibacterial immunity, in the cases of model experimental reaction in volunteers we found the decrease of delayed hypersensitivity 10-14 days after intranasal inoculation of influenza virus a (h3n2-3) to bacterial (staphylococcal, streptococcal and pneumococcal) and ~iycoplasma pneumoniae antigens in the leucocyte migration inhibition test. these results showed that respiratory viruses may be the important pathogenic factor in the development of bacterial infection in posttransplanted period. we consider the constant control of latent and visual respiratory viral infection in bmt patients to be very important. ficcb the ~ter£~li of the nation~l institute of trad/~atoloqy in budapest 1.00 consecutive cases of revision hip grafting were carried out arthroplasties wlth hemoloquous bone between the years 1990 and 1993. in the same period of time 732 pri~ total hlp replacen~nts were performed under i4entieal technical conditions. the average septic rate for the 'total hip althroplasties was less than 1%. in the selected i00 cases the septic rate was 4% indicating the role of bone grafting° homografts were prepared by deep freezing~ it .is recognized that the cells of the hl~grafts become destroyed by the ium~unological, response of the host~ and the patients develop ~ti-hl~, ar~tib'o~ies. the dead ~trix, however, has a bone-inducing capacity that stimulates host osteoblasts to recolonize the *i~/trix which serves as scaffolding. the sequence of events favours the infections. for this reason, beside preventive perioperative systemic ant/biotic treatment, local ~ntibioties were also applied in the form of antibiotic-//npregnated cement. the role of age and the .immune status of the patients .is discussed.. the purpose of this study is to evaluate the rate of toxemia in patients with acute panereatitis and to find this coudition to the activation of cascade systems that are encountered in the subsequent complications of the disease. we studied a series of 45 patients with acute pancreatitis, the severeness of which was evaluated by the ranson's criteria and the apach-ii scoring system. all of them were considered to have severe acute puncreatitis. the determination of toxemia was made using the limulus test (lal test). we also determined the levels of the third (c3) and fourth (c4) complement components as weu as the coagulation factors, iibrinolysis faeters and kimns by serial measurements. the severity of the disease was serially determined by the apach-ii scoring system. it was found that complement activation ( which was also assessed using a graphically illustrated method by a aggregometer ) was followed by an increase of morbitity and mortality .we also detected that toxemia (positive lal-test) was closely correlated with complement activation and more of the ranson's criteria. a clear relation existed between the number of ranson's signs and the enmplieations' rate (1"= -0.766, p < 0.001). the documentation of toxemia and the complement activation cannot predict the kind and the severity of complications. the study of coagulation, fibrinolysis and kinms systems didn't reveal any results with statistical significance. necrotizing pancreatitis still represents a life-threatenthg disease. infectious complications dominate among the causes of death. differences in the individual immune response could possibly explain different clinical courses even in patients with comparable pancreatic morphology. to explore the inflammatory response in acute pancreatitis, the following investigation was performed. methods: peripheral-venous blood was withdrawn on admission and furthermore twice weekly in as yet 36 patients with acute pancreatitis and tested for the parameters mentioned below. in parallel, polymorphounciear granaiocytes were isolated using density gradient centrifugation and assessed for superoxide anion and hydroxyl radical producing capacity using electron spin resonance techniques. results: total leukocyte cotmt and total lymphocyte count did neither reflect the clinical course nor predict complications. this comes tree also for serum igg, igm, iga, c3, c4, crp, alpha-l-antitrypsin and neopterth as well as for plasma il-la, il-ib, il-1ra, il-2, il-2r, il-6r, tnf-ct, tnf-~r (p75) and icam-1. in contrast, pmn-elastase, il-6 and il-8 closely correlated to the clinical course. isolated pmn's in vitro capacity to produce oxygen radicals depended on the respective radical species and was slightly elevated (superoxide anions) or decreased (hydroxyl radicals), respectively. patients with a cd4+/cd8+ ratio below i were seen at risk of developing septic complications. in contrast, a percentage of monocytes of 30 % or more among total mononuclear cells indicated an uncomplicated course, in general. conclusions: the immune status of the individual patient may significantly influence the course of acute pancreatitis. the cytokine pattern in peripheral blood is very complex and most parameters are of little use for the clinician. the pmn-elastase, il-6 and il-8, however, closely correlate to the clinical course and may prove valuable for follow-up. the cd4+/cd8+ ratio was found the best predictor of septic complications, but it failed in non-septic patients. a percentage of 30 % or more of monocytes among total mononuclear ceils indicated a rather mild course. the reduced ability of the pmns to produce hydroxyl radicals may help to explain the frequent development of septic complications in severe necmtizing pancreatitis. peroxidation of membrane lipids contributes to ceil injury in pancreatitis. overwhelming release of toxic metabolites by infiltrating neutrophils is regarded a major pathogenetic factor, too. as yet little is known about the mechanisms by which oxidative stress and leukocytes damage pancreatic cells. the present study examines (i) the susceptibility of pancreatic acinar cells to attacks by oxidants and leukocytes and (2) the potential of antioxidants to prevent such damage in order to better understand the cellular mechanisms of pancreatic injury in inflammatory states. methods: freshly isolated rat pancreatic acinar ceils were exposed to a model system of oxidative stress consisting of 20 mu/ml xanthine oxidase (xod), 500 mm hypoxanthine (hx), 50 mm fec13 and 75 mm edta. in a second set of experiments, acinar cells were exposed to excess autologous neutrophils or neutrophils obtained from patients with acute pancreatitis. neutrophils were stimulated by zymosan a, pma, and il-8. cell viability was assessed by both cellular uptake of trypan blue (tb) and by release of ldh. results: the xod/hx system caused a time-dependent acinar cell injury. this injury was effectively prevented by catalase (cat) and gfutathione peroxidase (gpx). in comrast, superoxide dismutase (sod) enhanced cell injury. addition of both sod and cat abolished the damage seen with sod alone. the non-enzymatic scavengers mannitol, dmso, dmtu and the iron chelator deferoxamine were not protective and at a higher concentration even accelerated cell decline. the newly developed antioxidants of the lazaroid type effectively prevented oxidative acinar cell damage. stimulated neutrophils, both autologous and heterologous, did not damage healthy acinar cells but had even protective effects. conclusion: pancreatic acinar ceils are very susceptible to oxidative injury. a combination of catalase and sod prevented cell damage effectively. sod when given alone may rather damage than protect aelnar cells when h202 is generated in concentrations overwhelming the capacity of endogenous catalase. therapeutic approaches to pancreatic disease using antioxidants should, therefore, include combinations of protective substances. the lazaroids seem to be candidates for clinical use as antioxidants in pancreatitis. the results argue against direct toxic effects of stimulated neutrophils to pancreatic acinar cells. are ch~act~z~ by the presence of a polymicrobial flora, the pmtotyi~ cffthese inf~ons is secend~,y bacterial pedtonitlw, whereby a pathololoeal process in the ~trointesfimd tract r~ful~ in tim disrup~on ofi~ inteffrlty and ¢ollseqtlent sptl]nge of inte~.i,o~.l gontents into the peritoneal c~iry. the ensuing infection invariably contains a mixtm~ of gt~m negative enteric bacilli, gram positive b~eria and anaerobe& experimental and clinical =t~ies have de~ed the eantrlbution of each of th¢~ components to ti~ ovemu virulence of these in~ons, gram negative enteri~ such as f.veher~chla coil ere endowed with a virulent l~l~x~lyse~haride ptill~ly t~sponsible for lethality, by contrast, bacteroldes sl~cles, which rarely c~se death, prornot~ abscess fonllation, a uniqm~ capsul~ polyseccluu'ide, particularly on b.j~ogiljs slrai~, oontributes to tjtis erect, several mecltanims have bccn pml~ed whereby or~ microorganism mi~t interact with its microbial ~net to augment the overall virulence of a r~xed im~edan. these include: l) provision of nutrients by one apexes which stimulates the growth of its ~opathoge& 2) inhibition of host deletes by one of the migroorganisms so that the other microbes might persist and exert their virulence, 3) the trant~ of vim.©n~e traits between ~renr~a.,dsms and 4) the ~.mizatian d the mi~oe~vironmental con~tion$ by one d the baetez'isl pa#, so that the other might persist. exampl~ for each of these m~banisms imv~ been provided by experimental ttudies i~stigating e.co!l-b.p~flls synergistic in~ra~ons. byproducts ofg.coli metabolim l~¢ovide essential short ebath fatty acids £~ optimal b,frosili~ ga'owth. fm-ther, oxygen ¢ons~tmption by kcelt lowers oxygen tension end redox potantial to levels eomlucive to b#a#lts gro~h. coawr~ely, b,~agtlis rolea~s proteases and fatty acids wl~¢h impair pl'tsgocy~¢ ~lt rmctlon tnd permit f-..¢oli proliferation and expression of its intrinsic virulent. in summaxy, interactions among the separate microbial cemponents of mixed infections heighten the overall virttienee of these lafectiot~, this knowledge provides ~r rationale for targetting of antibiotic therapy against the knowa eantributors of these synergistic pro~¢sses, intraabdominal abscess formation and the macrophage william g. cheadle, m.d., department of surgery, university of louisville school of medicine, louisville, ky 40292 inflammation of the peritoneal cavity following bacterial contamination has been classified into primary, secondary and tertiary, the last two relating to bacteria originating from the gastrointestinal lumen. the natural history of such infection is either resolution without clinical sequelae, which is uncommon, abscess formation, or generalized peritonitis, which occurs as a result of failure of peritoneal host defenses. early clearance of microorganisms by peritoneal fluid circulation and filtration througti subdiaphragmatic lymphatics into the thoracic duct and systemic circulation occurs as well. simultaneously peritoneal macrophages and the omentum approach the area of inflammation and lead to neutrophil influx and abscess formation adjacent to the affected viscus. we have found a shift in peritoneal macrophage function from antigen presentation to proinflarnmatory cytokine production that occurs early after experimental peritonitis produced by cecal ligation and puncture. this is also reflected by reduced class ii histocompatibility antigen expression on peripheral blood mononuclear cells and peritoneal macrophages. this is accempauied by an influx of both neutrophils and macrophages into the peritoneum and subsequent abscess formation. interestingly, there is little serum endotoxin or tnf seen in this model despite tnf mrna expression in peritoneal macrophages. we believe this model is more clinically relevant than other models of endotoxemia or bacteremia in which different patterns of cytokine expression are seen. newer agents aimed at reduction of systemic manifestations of sepsis originating from intra-abdominal infection such as monoclonal antibodies against cytokines or il-1 receptor antagonists may need to be directed against remote organ macrophage populations while preserving peritoneal macrophage function. inflammation is a complex process involving microcirculatory changes, extravasation of fluid and a cellular influx in the affected body area. in our communication, we will only consider the regulation of the cellular infiltrate which plays a major role in the defense of the peritoneum against microbial invasion. until recently, it was thought that the influx of leukocytes in the abdomen was induced by bacterial products, local humeral factors and secretions of resident macrophages. there is now increasing evidence that this view is too simplistic. many other cell types present in the abdominal cavity or composing the peritoneal membrane (mast-cells, mesothelial cells, fibroblasts) are able to release or secrete vasoactive or chemotactic substances such as histamine, prostagtandines, or cytokines. they are most likely to play a role in the regulation of intraperitoneal inflammatory reactions. the emigration of leukocytes towards the abdominal cavity is also modulated by a previous contact with gram negative bacteria. in the rat, this intriguing phenomenon is long lasting, cannot be transferred by serum and seems independent from t lymphocytes. the clinical relevance of these various regulating mechanisms has still to be determined. kinnaert paul, h6pital erasme, route de lennik 808, 1070 bruxelles belgium generalized response in secondary peritonitis the clinical course of an intraabdominal infection may depend on a variety of variables including the capacity of host defense mechanisms and the degree of the inflammatory response. if local defense mechanisms fail to restrict the inflammation to the abdominal cavity a generalized inflammatory reponse will result. in a first stage generalized signs of a local inflammation become detectable whereas the second stage comprises the overwhelming systemic inflammatory response. the extent of this systemic response determines the outcome. sometimes it may appear to be unrelated to the severity of the intraperitoneal findings. the activation of plasma systems and cellular elements leads to a fast release of cytokines, inflammatory mediators and other substances. these parameters precisely reflect the degree of the generalized response. inflammation of the peritoneum causes significant morbidity. objektives: to test the hypothesis that peritoneal mesothelial cells play a role in regulating inflammatory responses within the peritoneal cavity, we examined neutrophil-chemotactic activity (interleukin 8) and monocyte-chemotactic cytokine (mcp) release by sytokine-etimulated mesothelial cells. confluent human peritoneal mesothelial cells were exposed to varying concentrations of phorbolmyristate-acetate (pma) and the cytokines tumorneerosis factor a (tnf a) and interleukin i~ (il-i~). the supernatant was examined for il-8 by elisa and for mcp by investigating the ehemotactic activity for isolated human monocytes. mesothelial cells express low levels of il 8 and monocyte chemotactic activity when cultured. these activies were significantly increased (2-fold) after stimulation with either tnf a or il-i~. additionally macrophage inflammatory protein was detected. these observations provide a probably important mechanism whereby peritoneal mesothelial cells respond to imflammatory stimuli released during peritonitis and how leucocyte recruitment by liberation of chemotactic cytokines is regulated. the perioperative course of lps, tnfa and il-6 in 19 patients with bacteriologic proven abdominal infection (intraabdominal abscess 10, diffuse peritonitis 4, pancreatic necrosis 2, pancreatic abscess 3) was followed prospectively and evaluated for possible correlation with septic state and organ function. methods: patients were studied in a 6 to 10 hours period during their first surgical intervention because of intraabdominal infection. all were monitored for their cardiovascular, respiratory, hepatic and renal function. plasma samples for lps. tnfa and il-6 determination were drawn preoperatively, intraoperatively, and until 2h postoperatively in regular intervals (min 7/pat), results: preoperative apache ii was 12 in median (rain 4, max 25). 10 patients fulfilled the criteria of sirs. 4 of them were in septic shock.there was a significant correlation between preoperative tnfa and apache ii (p=0,000 i, spearman coefficient). preoperative cardiovascular (systol. rr<90 mmhg) and respiratory (pao2<75mm hg) dysfunction were associated with significantly elevated tnfa (cardial: p=0,000 i, wilcoxon; pulmonal: p=0,0003) and il-6 (cardial: p=0,2; pulmonal: p=0.0001) overall, lps, tnfa and il-6 values varied considerably during the observation period. however, tnfa was markedly higher in patients with sirs and septic shock (group a: n= i 0, mean 182 pg/ml) than in those who did not fulfill these criteria (group b; n=9, mean 24 pg/ml; p=0,00 i, wilcoxon). il-6 was significantly higher in group a (mean 2169 pg/ml) than in group b (mean 191 pg/ml; p=0,0o i wilcoxon). conclusion: perioperative tnfa and il-6 were shown to correlate significantly with preoperative organ function, apache ii and the severity of sepsis. these results could help to define patients that might benefit from further therapeutic strategies, e.g. antibody administration. department of surgery, university vienna, akh wien, wahringer gurtel 18-20, 1090 wien. aim of the study: the purpose of this pilot study was to establish and to prove a standardized reproducible animal model of intraperitoneal sepsis induced by e.coli-endotoxinaemia in lew.lw-rats in order to investigate early immunoserological responses to find a mediator based evaluating system of peritonitis sepsis. materials and methods: in 30 lew. lw-rats, diffuse peritonitis was induced by intraperitoneal injection of a mixture of e.coli (khu +) and autogenous haemoglobin solution. in the control animal group (n= 12) an intraperitoneally injection of physiological saline solution was done. blood samples were obtained by heart puncture after 6 hours. stastistieal calculations were performed on a personal computer with the spss programm vers. 4.01 (correlation with pearson's r, mann-whitney-u-test, descriptives statistics, discriminant analysis). results: in contrast to the sham treated rats, the peritonitis animals showed significant differences in the concentrations of endotoxin, interferon-gamma (wn-y), the pteridin derivate biopterin and serum pla2-activities [endotoxin range from 0.064 eu/i, sd=0.19 to 102.38 eu/1, sd-145.4 (p < 0 0001), ifn-¥ levels, range from 147.9 pg/ml, sd-141.6, to 4591 pg/ml, sd=6541 (p < 0.0001), circulating pla2-activities range from 116.2, sd=45.4 to 185.4 u/1, sd=105.4 (p < 0.01) and biopterin range from 54.4 nmol/l sd=17.2 to 127.8 nmol/l, sd=76.8 (p < 0.001)]. for the peritonitis group we found strong correlations between the degree of endotoxinaemia to elevated levels of ifn-'~ (rp = 0.72, p < 0.0001) and bioptefin synthesis (rv= 0.82, p < 0.0001). the increase of ifn-t levels was correlated to the regulatory synthesis of biopterin (r = 0 73 p < . .. p • , . 0.0001) and to the pla2-actwtues (rp = 0.50, p < 0.005). the biopterin synthes~s correlates slightly with the pla2-actn,ities (rp= :0.38; p < 0.05). using the para, meters of endotoxin, ifn-y levels, biopterin and the pla~ -activities only, the statistical procedure of the linear discriminant analysis makes it possible, to distinguish between non-septic animals and septic animals correctly at a rate of 87 %. anaerobes were found in 53.4%, anaerobes were isolated in 22.8%. there were aerobic and anaerobic associations in 12.1% and microflora was not found in 11.6% of the cases. express method of anaerobes discovering let to receive information on 1 -3 days early than in generally accepted nethods. intraaotal transfusion of oxygenate blood and laser irradiation of blood reduces the duration of anaerobic sow, disminishes intoxication and accelerate the patients recovery. patients with abdominal sepsis are subject to long periods of hospitalization and high associated morbidity and mortality rates. this category of patients is thus consuming extensive facilities and costs. as the age-related outcome of abdominal sepsis is not fully known, the aim of the present study was to investigate abdominal sepsis in the elderly. out of 166 patients with abdominal sepsis treated at the surgical intensive care unit during a 10-year period, 72 (43 %) had an age of 70 years or more. 28 were women and 44 were men, a sex distribution not differing with patients younger than 70 years. the patients were scored according to apache ii and septic severity score (sss) upon arrival to the intensive care unit. bacterial cultures, the occurrence of organ failure, hospitalization and outcome was noted. in median two operations were performed for both "younger and elderly" patients. the median time of hospitalization in the elderly was 29 (-183) days including in median 10 days in the icu. figures in patients less than 70 years of age were comparable (34 (-293) days out of which in median 10 days in the icu). apache ii and sss-scores did not significantly differ (14.2 vs 13 and 23.3 vs 26.5, respectively), between the groups. neither did the incidence of organ failure differ (57/73 vs 77/94). however, the incidence of multiple organ failure was significantly lower in elderly patients (21/72 vs 42/94 (p < 0.001)). the mortality rate, however, did not differ between the groups (21/72 vs 26/94). in conclusion, severe abdominal sepsis in the elderly was not associated with an increase in mortality, incidence of organ failure or hospital stay. with the help of light transmissional scanning electron microscopy morphology of erythrosytes of peripheric blood was studied in patients with different stages of diffuse peritonitis before and after intravascu!ar irradiation of blood with heliun-neon laser. peritoneal morphology was investigated in patients who died from peritonitis, it was established that in all phases of peritonitis occured stomatocytoric and echinocytoric transformation of erythrocytes which progressed simultaneously with increase of intoxication. it combined with strongly pronounced vessels variability of microcirculatory peritoneal bed which displaied by erythrocytes aggregation, stasis and microtrombogenesis. in intravascular laser irradiation of blood number of erythrocytes which underwent to stomatocytoric and echinooytorie transformation was lower than in patients without laser irradiation. it indicated that the intravascular irradiation of blood with helium-neon laser can prevent development of severe alterations of rheological property of blood and consequently variability of microcirlatory peritoneal bed in patients with diffuse peritonitis. abdominal sepsis is still associated with high morbidity and mortality rates, frequenfly caused by multiple organ failure. it has been reported that changes in capillary permeability play a role in the pathogenesis of multiple organ failure. the present study aimed at evaluating the influence of intraabdominal sepsis induced by cekal ligation and puncture on capillary permeability in multiple organs and tissues. 96 adult male sprague-dawley rats were subjected to laparotomy with separation of the cekum (sham operation) or induction of intraabdominal sepsis by cekal ligation and puneatre (n--48 in each group). at 3, 6, 12, 24, 48 and 72 hours (n=6/timepoint), the animals were evaluated concerning mortality and capillary permeability as determined by the passage of :25i-labelled albumin from capillaries to the peritoneum, the proximal and distal small intestine, cekum, colon, spleen, kidneys, lungs. the mortality rate in rats with intraabdominal sepsis was 33 % both at 48 and 72 hours. capillary permeability in the peritoneum, cekum, colon and kidneys significantly increased from 6 hours and on in rats with intraabdominal sepsis. in septic animals, capillary permeability in the lungs and spleen increased from 12 hours and on and in the proximal and distal small intestine from 24 hours and on. different types of alterations in capillary permeability seem to appear: 1) a temporary short increase e.g. in the proximal small intestine and spleen; 2) a temporary longer increase e.g. in the colon and kidneys; 3) a persisting increase e.g. in the peritoneum, cekum, distal small intestine and lungs. we conclude that experimentally induced intraabdominal sepsis induces early alterations in capillary permeability in multiple organs and tissues. such changes may contribute to explain the development of sepsis-induced multiple organ failure. despite a number of significant advances in the care of burn and non-burn traumatic injury, infection and sepsis remain major causes of morbidity and mortality. the severe immunosuppresslon often seen in patients with severe trauma or large burns may predispose these patients to life threatening infections. included among the many immune alterations are changes in the functional capabilities of neutrophlls (pmns). we have examined the expression of the p 2 integrins (cd1 l a, b,c/cd18), and the fc'?r (cd16, cd32, and cd64), as well as several functional parameters, on pmns from thermal and non-thermal traumatic injury, pmns were obtained from patients sustaining severe trauma (initial apache ii score >10) or thermal injury (>30~ total body surface area, 15% full thickness), and healthy controls. the expression of cd11 b and c and to a lesser degree cdi 1 a was significantly reduced on pmns. the expression of cd16 and cd32 but not cd64 was also significantly reduced. pmns displaying this reduction in receptor expression have a significantly reduced ability to phagocytose bacteria and undergo the oxidative metabolic burst response. thermal and traumatic injury result in global reduction in the expression of 132 integrins and for which may lead to decreased functional capabilities, these abnormalities may in turn account at least in part for the increased rate of infection in these patlems, institute, dept. of surgery, 2~1 8ethesda ave, cincinnalt, oh, usa, 45267-0s5b, antibiotic-phagocytic cell interactions: their effect on endotoxin release. c g c-emmet1, dep[baeteriolog.z, univer_sitv of glasgow, scotlan~_d increasingly it is recognised that pathogenic bacteria are capable of surviving intracellularly within phagocytic cells in addition to their capacity to produce disease whilst in the extracellular milieu. as well as providing protection from certain antibiotics which fail to penetrate the phagocyte, such intraceltular bacteria may be transported from the initial site of infection to a distant more vulnerable body site wherein they may proliferate. it is also known that some antibiotics are capable of becoming concentrated within phagocytic cells mid displaying bioactivity therein. such bioactivity might be responsible for the release of endotoxia #orn gram-negative bacteria which when liberated from the celt could ~gger the cytokine cascade. anfib,.'otic-induced damage to the ultrastructure of bacteria can also occur when the target bacteria are exposed to low (sub-mic) concentrations of certain drugs. such bacteria may present quite altered surface components m host-defense cells as well as releasing biologically active ceil wall components such as endotoxin. the nature of these interactions at the cellular level as well as the consequences for the host will be discussed. new jersey medical school: umd, newark, nj 07107 a technique of physiologic state classification has been developed based on the m~itlvariable analysis of patient derived data sets of seventeen physiologic variables. these multivariable data sets obtained from critically ill patients requiring intensive care, were aormallsed by the mean and the standard deviation of recoverin~ trauma patients who were not critically ill, the resulting normalized seventeen variable sets were then clustered. seven independent data groupings were developed. the normal stress response hyperdynamic state seen post-trauma and in compensated sepsis (a stets)/ metabolic insufficiency seen in septic decompsnsation (b stste}; early (c,) and late (e2) respiratory insufficiency associated with ards; cardlogenlc dscompensation (n state); post-trauma hyvolemla without shock (r stats). the stats closest to a new patient's values allows patient classifi0atlon with regard to his previous physiologic state. classifying observations f~om patients who lived or died who fell into these physiologic states enables a probability of death (p death) to be obtalned. utilizing this criteria for the staging of severity in 44 recent trauma patients the physiologic states accurately and significantly predicted the likelihood that the patient had an increased circulating level of the eytoklnes tnf and il-1. the probability of death (p death) as well as the cytoklne levels appear to be a function of the physiologic b state with the highest levels being seen in the b state of metabolic insufficiency and the c~ state of oombined respiratory and metabolic insqffioienoy characteristic of septlc ards. the increase in the magnltude of metabolic abnormalities associated with the transition from non-sepsls to septic a, septic b, or septic c z states was associated with an increasing probability of death (p denth)(mean a state =.28, mean b state = .57, mean ~ state = .61). the accuraay of this estimate was prospectively analyzed in this group of 44 m~itlple patients of whom 72% had sepsis and 36% had ssptlo ards. the 22 survivors had a mean p death of 0.39 and the 22 deaths had a mean p death of 0.63. the severity of post-trauma sepsis can be quantified by probability analysis and stra~ifie~ by physiologic state. serologic tests have not been extensively tes'~ed in surgical patients but seem to be of limited value. we use nystatin as the main form of chemoprophyhxis. patients "~'ith signs of infection who do not rapidly improve with antibacterial therapy are candidates for anti-funsal therapy, amphoteradn b remains the first llne of therapy although combination therapy '~'ith flueonazole is use;l with increasing freque~;c)', the recovery of c~dida from an antra-abdominal site represents a challenging problem, anti~ngal therapy in such patients depends on the underlying disease, the nature of the infected material and overall patient risk. role of neural stimuli and pain principles and practice of anesthesiology effect of combined prednisolone, epidural analgesia and indomethacin on the systemic response after colonic surgery arginine: biochemistry, physiology and therapeutic irnplications immunosfimulatory effects of arginine in normal and injured rats arginine stimulates lymphocyte immune response in heahhy humans rote of arginine in trauma, sepsis and immunity arginine enhances wound healing in humans if labrecque t, gv campion t, and the rhll-lra phase i//sepsis syndrome study group the cleveland clinic foundation a murine-anti-human tnf-monoclonal antibody known as cb6 was the first anti-tnf mab which was studied in a phase ii multinational trial in the treatment of patients with severe sepsis.this was an open-label, dose-escalation trial consisting of 80 patients who were enrolled into one of four treatment groups: (1) 0.1 mg/kg of anti-tnf mab, (2) 1.0 mg/kg, (3) 10 mg/kg or (4) 1.0 mg/kg at study entry and the second dose 24 hours later. the small sample size in each group (n=20) precludes detailed statistical inference in this study. nonetheless, a considerable amount of useful information was obtained from this investigation. irst, this study demonstrated the clinical feasibility of specific anticytoldne therapy in septic patients. second, the measurement systemic levels of tnf proved to be an elusive target; interleukin-6 may prove to be a more useful indicator of cytokine activation. third, immunologic reactions including tnf: anti-tnf mab immune complexes and human anti-routine antibodies were frequently found in these patients. despite their apparent lack of overt toxicity in this study, these immunologic reactions may complicate this form of anticytokine therapy. additionally, the potential benefits of anti-tnf mab therapy occur within the first 48 hours of therapeutic administration in these septic patients. infecting organisms differ in their potential to induce tnf in vitro and these differences correlate with circulating tnf levels observed in septic patients. rapid methods to define those patients most likely to respond to anticytokine therapy are needed to determine the ultimate therapeutic potential of these agents in clinical medicine. wherry, j., abraham e., wunderink r., silverman h., perl t., nasraway s., levy h., bone r., wenzel r., balk r., allred r., pennington j. and the tnfa mab sepsis study group.tnfa mab (bay x1351) is a murine monoclonal antibody raised against human tumor necrosis factor. tnf~ mab has been shown to reduce morbidity and mortality in animal models of septic shock and has been safely administered to septic and non septic patients.to evaluate the efficacy and safety of tnf~ mab in patients with sepsis syndrome, a prospective, multicentered, double-blind, placebo-controlled trial was conducted in 31 hospitals in north america. patients were prospectively stratified into shock or nonshock groups and then randomized to receive a single intravenous infusion either of 15 mg/kg tnf~ mab, 7.5 mg/kg tnf~ mab or placebo (0.25% human albumin).patients received standard aggressive medical/surgical care during the 28 day post dosing period.the three treatment arms were well balanced with respect to demographics, apache ii score and other parameters. for all infused sepsis syndrome patients, those who received tnf~ mab had slightly reduced 28 day all cause mortality compared to placebo. among shock patients there was a more pronounced trend towards efficacy at day 28 post dosing with lower mortality rates in both active treatment arms. among nonshock patients tn~ mab did not appear beneficial. the initial clinical experience with a chimeric anti-tnf monoclonal antibody, ca2, was undertaken in septic patients. the objectives of the study were to determine the safety, pharmacokinetics and effects on cytokine levels of ca2. as a single infusion or in combination with ha-1a in septic patients. the study was conducted with the intent to progress to an efficacy trial based on the information collected.the trial was conducted in three stages. stage 1 was an open label trial in which 4 groups of 5 patients each with the clinical diagnosis of sepsis received ascending doses of ca2 (0.1, 1, 5, 10 mg/kg). stage 2 was a randomized, double blind study in which patients received a single dose of ha-1a (100 mg) and placebo or one of 3 doses of ca2 (1,5,10 mg/kg). stage 3 was a randomized, double blind study in which patients received a single dose of placebo or one of 3 doses of ca2 (0.1, 1, 10 mg/kg). in addition to usual laboratory tests, the following assays were performed: chimeric anti-tnf concentration, anti-chimeric antibody, endotoxin, tnf, il-1, and il-6 levels.a total of 141 patients were enrolled from 13 clinical sites (20 in stage 1, 60 in stage 2 and 61 in stage 3). primary analyses were performed on patients in stage 1 and 3. there were 65 patients who received ca2 exclusively and 16 patients received placebo. administration of ca2 was well tolerated at doses up to 10 mg/kg. no patient discontinued treatment due to adverse events. human anti-chimeric antibody responses were positive in 61% (30/49) of evaluated patients. mean cma × and auc increased proportionally with increasing doses of ca2. the mean half-life was -70 hrs (58-96 hrs). a dose related decrease in tnf concentration was observed 1 hr post infusion of ca2. tnf is considered to be one of the central endogenous mediators for the inili'ation of the pathophysiological changes in patients with sepsis and septic shock. high tnf levels were demonstrated to correlate with patient outcome. blocking or neutralising tnf with specific antibodies was effective in preventing death in some animal modets of sepsis. in a placebo controlled prospective randomized study we tested the mur~ne derived antibody mak 195f. it is a f(ab')2 fragment. the fragment rather the complete antibody was selected in order to reduce the potential immunogenicity and to facilitate tissue penetration. 122 patients with severe sepsis or septic shdck were enrolied in the study, three different doses of mak 195f or placebo were administered (0,1; 0,3 and i mg/kg) over a perid of 72 hours in random order. the patients were evaluated for side effects, hemodynamics, organ dysfunction, cytokines (il 6, il 8 and tnf), and outcome. at this time only an interim analysis of 60 patients is available i indicating that mak 195f in all 3 dosage groups resulted in a decrease in il 6. this contrasted to a further in crease of il 6 in the placebo patients. no serious side effects have been reported so far. a more detailed analysis on all 122 patients in the study will be presented and discussed.$152 s154 staubach,k.h., otto, v., kooistra,a,, rosenfeid,j.a., bruch, h.p., univ. lfibeek, germany once endotoxinemia occurs in sepsis a vieieus cycle with translocation of et can be established. increasing the clearance capacity for et would therapeutically be the ulimate aim. we developed a new et on-line adsorption (ad) system in whole blood by means of polymyxin b (pb) coupled eovalently to a matrix (acrylic particles) via a 13 atom-chain spacer. the detoxification capacity was 150 ug[et/ml column material. the biocompatbility resulted in 90~ platelet recovery. the column contained 7 ml of admaterial and was sterilized by high steam autoclave, anticoagulation was achieved by heparine 1.000 iu/h in the inflowline after bolus injection of 5.000 iu. hp was performed on 8 pigs at a rate of 50 ml/min by means of a roller-pump until the animals succumbed (h). 8 animals served as controls (c). serum et levels rose from 3.40 pg/ml to 74,9 pg/ml after 5 hours in the c and from 2.77 pg/ml only to 28 pg/ml in the h group after 7 hours whieh was highly significant. survival time could be extrended from 216 to 313 min. results are listed in the following l. blinzler, p. zaar, m. leier, r. b(2rger, d. heuser clinic of anaesthesiology , city hospital nuremberg, germany sepsis and multiple organ failure (mof) are still related with poor prognosis inspire of pharmacological and technical progress. impressed by revealing reports about blood purification the continuous veno-venous hemofiltration (cvvh) was used as supporting treatment beside the critical cam basic therapy of mof. from 1989 to 1991 78 consecutive patients were treated by cwh. mof was caused by hemolrhagic-traumatic noxa in 21°,4 and by septic-toxic event in 79%. all patients required mechanical ventilation (fio2>0, 5) . 86°4 showed hyperdynamic shock. 85% had renal and 53% hepatic failure. medium appache ii score amounted to 29,8 points. cvvh was performed in postdilution mode with a polyamide membrane (fh 66) and high volume exchange (50 l/die). anticoagulation was done with heparin. hemofiltration in mof was installed, when critical cam basic therapy including adequate respiratory and hemodynamic management, pamnteral nutrition, antibiotic treatment, etc., failed to stabilize organ functions. during consequent application of cvvh most of these patients showed improvement of their clinical course. pulmonary stabilization was seen in 71%, hemodynamic in 68% and renal in 66% of the cases. 42% of the patients survived and were discharged from hospital. 10 of 45 non-survivors (58%) died because of fatal mof within 24h after admission to icu. patients with early application of cvvh in mof showed a better survival rate.mediators of mof, i.e. products of the complement cascade measured in blood and nitrafiltrate by elisa, were partially removed by cvvh. the testing ultrafiltrate by hplc demonstrated decreasing spikes ofpolypeptides during hemofiltration. mof seems to be generated by cascade-activation of immune competent cells and plasmatic mediators (e.g. bmdykinin, eicosanoides, cytokines, anaphylatoxins, etc.). therapeutic approaches aim to inactivate or eliminate single substances. cwh with high-flux membranes in combination with high-volume exchange allows elimination of many mediators with different molecular weight and therefore may contribute to improve the prognosis of mof. other significant advantages of this teqalnique like adequate nutrition, optimized fluid balance and control of body temperature should not be negicctod. introductioni pseudomonas (p) aeruginosa has to be considered an important pathogen of nosocomial pneumonia and septic organ failure. the lung seems to be the predominant target organ for the pore-forming p. aeruginosa cytotoxin, thus inducing microvascular injury. with respect to therapeutical consequences, the potential protective effects of paf-antagonist (web 2086), cyelooxygenase inhibitor (diclofenac) and specific and unspecific antibodies on cytotoxin-induced pulmonary vascular reaction and mediator release were studied in the isolated perfused rabbit lung. methods: cytotoxin (6p_g/ml) was administered into the perfusion fluid in all groups, either in the absence of inhibitors (n=6), or after pretreatment with web 2086 (5xl0-gm, n=6), or diclofenac (10#g/ml, n-6). furthermore, the application of specific antitoxin (mg/ml, n=6) was tested in comparison with the unspecific immunoglobulins (venimmun®, behring, 7.5 mg/ml) (n=6) and the combination of immunogiobulins, web 2086 and diclofenac (n=6). six experiments without toxin served as controls. the arterial pressure mad the weight gain as an indicator of edema formation were continuously monitored during the three hour peffusion phase. arachidonic-ucid metabolites, as well as lactate dehydrogenase (ldh) and k + concentrations were determined at 30 rain intervals. results: cytotoxin caused a gradual increase in pulmonary arterial pressure, reaching a maximum value of 2.5 times higher than the control, starting after 1 min and a delayed onset of edema formation resulting in a mean weight gain of 20 g after 120 min. this was paralleled by a significant increase in prostacyclin generation and a continuous release of k + and ldh. thromboxane synthesis exceeded about 10 times that of controls in the toxin treated lungs. pretreatment with web 2086 or diclofenac significantly attenuated the pressure response and edema formation evoked by cytotoxin. the addition of the unspecific immunognbulin preparation alone induced a transient pressure increase within the first minutes, but mean values remained below those of the cytotoxin group in the continuing observation period. mmost complete inhibition of the pressure reaction, the edema formation and the metabolic alterations was achieved mainly by the combination of immunoglobulin, web 2086 and diclofenac and to lesser extend by the specific toxin antibody. conclusion: the current results point towards the crucial role of paf and aa-metabolites as mediators of cytotoxin induced microvascular injury. the systemic or local application of cytotoxin antibodies or even unspecific immunoglobolins in combination with paf-antagonist and diclofenac appears to be a promising therapeutic approach in the case of infection with cytotoxin-preducing strains. cytokines have long been shown to be of particular importance in the metabolic derangements occurring in lps-induced shock. recent studies strongly imply the involvement of platelet aggregating factor (paf) in the pathogenesis of gram-negative bacterial sepsis. an autocatalytic feedback network has been postulated to exist between paf and tumor necrosis factor (tnf), a key cytokine involved in septic metabolic cascade, leading to an uncontrolled amplification of inflammatory mediator release. we have previously shown that st 899 (4-n,n,n trimethylammonium-(r)-3-isovaleroyloxy-butanoic acid z-3-(5-chlorphtalidiliden) ethyl ester bromide) was quite effective in inhibiting the "in vitro" binding of 3h-paf (ki=7.5x10 -8 m) to rabbit platelets. the present study shows that pretreatment of c57bl/6 mice with st 899, administered by different routes, dose-dependently and significantly reduces the lethality induced by endotoxin (e.coli 055:b5 injected at 30 mg/kg intraperitoneally). very interestingly, st 899 administered at the same doses as above (i.e. 1.25, 2.5, and 5 mg/kg body weight) results to be significantly effective in reducing the endotoxin-induced release of serum tnf. the reported dual activity of st 899 (i.e. paf antagonism and decreased circulating tnf levels) may turn out to be greatly beneficial, in combination with current therapies, in the treatment of diseases that involve overproduction of tnf and paf such as septic shock. introduction: recently, we reported that prophylactic whole body hyperthermia (42.5°c) induces heat shock protein ('asp) 72 and increases smvival 2-4 fold in a mouse endotoxin model (am. j. physiol. in press). other investigators reported that prophylactic pharmacologic induction of hsp-72 by sodium arsenite improves survival in a rat sepsis model (abstract a95 am. rev. resp. dis. vol. 147, 1993) . the effects of heat are complex and in addition to formation of lisp-72 include release of cytokines, changes in cellular ph etc. thus, the protective mechanisms of heat may differ from those due to pharmacologically induced . the purpose of this study was to compare the protection of heat vs the protection of pharmacologically induced hsp-72 in a mouse endotoxin model to determine if different protective mechanisms were likely to be involved.. i%'lethods: both sodium arsenite (10 mg/kg) and ethanol (400 ~1 of 39% ethanol) caused marked induction of hsp-72 in lung, gut, kidney, and liver, which was comparable to heat-induced hsp-72. female nd4 mice weighing 22-25 gms were pretreated with arsenite or alcohol 8 hours prior to challenge with escherichia coli endotoxin (-ld 80) and survival was compared to control mice. results: survival at 24 hrs. for arsenite treated and alcohol treated mice was 96% and 88% respectively and was statistically different from the 40% survival for control mice. (p<0.01) (n=25 mice per group). however, at 7 days post endotoxin, there were no differences in survival in the 3 groups, i.e., ~ 4% survival for all 3 groups. in contrast, the protective effect of hyperthermia remains present at 7 days, i.e., ~ 70% survival vs 20% survival control. conclusion: the protective effect of heat is probably due to other factors such as the effect of hyperthermia to release il-lc~ and is not due solely to hsp-72 formation. it was the aim of the study to examine whether bacteria play a causative role in the pathogenesis of anastomotic insufficiency following gastrectomy in man.the study was carried out in form of a prospective, randemised, double-blind, multicenter trial. primary endpoints were the rate of anastomotic insufficiencies, infectious-and uncomplicated postoperative courses. all pat. received a periop, i.v. prophylaxis with cefotaxim. identical numbered vial either contained placebo or polymyxin b, tobramycin, vancomycin and amphotericin b . the vials were administered 4x per day from the day be ~ fore the operation until the 7th postop, day. insufficiencies were detected by gastrographin swallow and recorded by x-ray on day 7 postop.. evaluation was carried out on an "intention to treat'basis. statistical analysis was done with the pearson's chi square and fisher's exact tests~ results: interim analysis was carried out in 3/93 after 137 pat. had been recruited. along with a significant reduction of s.aureus and enterobacteria there was a reduction in the rate of anastomotic insufficiency of the esophago-jejunostomy from 10.6 % in the placebo-group to 3.5 % in the treatment group. the difference was not yet significant. the rate of nosocomial infections (e.g. respiratory tract infection and uti) were significantly reduced from 33.3 % in the placebo-group to 16.4 % in the treatment-group (p ~ 0.0281;fisher's exact test). in march 1994 final results with more than 200 patients will be presented for the first time. (= po2 <80 mm hg, b s-creatinin >2 mg%). respiratory insufficiency was the most frequent systemic complication followed by sepsis and respiratory insufficiency. etiology of pancreatitis and initial serum increase of pancreatic enzymes predicted neither complications nor outcome. only 2 of 14 deaths occurred during the 1st week, all other deaths occurred late (after 4-12 weeks), generally as the consequence of septic complications and multi-organ failure. high levels of crp were correlated with a compliacted course and a fatal outcome. although same cytokines (e.g. 11--6) were found increased in severe disease, the predictive value of these markers was not better than the combination of ctinical scores (ranson, imrie, apache ii) with gt or crp. conclusions: intensive care medicine can often control the inital shock situation in severe pancreatitis. thus. only 15% of deaths today occur eady in the course of the disease, whereas this percentage varied between 40-70% just 10 years ago. nowadays, most deaths are caused by late septic complications and multi-organ failure. ranson-and ct-scores as well as serum crp predict a course with systemic complications; they are less helpful for prediction of sepsis and late mortality. it is doubtful whether measurements of cytokines will help to better predict the late outcome. as yet, only careful and continuous monitoring of patients (e.g. by apache scores) may help to early identify those who develop septic complications and multi-organ failure. the classic description of severe acute pancreatitis has hinged upon the release of large volumes of activated enzymes into the peritoneal cavity and thertce the lymphatics and blood stream. these activated enzymes escape from the pancreas due to disruption of cells with associated ischaemia and occasional infarction of tissue. for 20 to 25 years it has been postulated that the bocly's defence system to activated pancreatic enzymes required supplementation iu the form of anti-protease support either in the vascular space or in the peritoneal cavity. all controlled studies have shown that this is either impracftcal or unnecessary.hore recently release of a large number of cytokines from monocytes, macrophages and neutrophils have been considered to be harmful to the body and various agent~ which oppose the action of tnf alpha, paf and similar cytokines are being examined in experimental anim~is and certain clinical trials, it has clearly been shown that higher levels of cytokines are released in the patients with objectively graded severe acute pancreatitis than in those with milder disease. we now seem to be moving into an exciting phase of potentially beneficial therapy in acute pancreatitis which has had no specific effective therapy through studies utilising aprotinin, gabexate mesilate and fresh frozen plasma. inflammation cascades may play a role in the pathogenesis of acute pancreatitis. to evaluate the status of the cellular immune system we examined serum concentrations of immune activation markers in 24 patients with acute pancreatitis (14 males, 10 females; median age: 56 years, range: 32-81 years). concentrations of neopterin, serum soluble tumor necrosis factor receptor (stnf-r) and serum soluble intercellular adhesion molecule type 1 (slcam-1) were determined using immunoassays (henning, bender, t cell sciences). 13/14 had increased concentrations of stnf-r compared to the 75th percentile obtained in healthy controls (>4.2 ng/ml), and 9/24 patients had increased neopterin (> 8.7 nmol/i), 9/24 presented with elevated slcam-1 (>440 u/i). all patients with increased neopterin also had increased stnf-r, 4 patients had concentrations of all three markers outside the normal range. there existed a significant correlation between neopterin and stnf-r (rs = 0.728, p < 0.001 ). weak associations between age and stnf-r (rs=0.435, p=o.05) or neopterin (rs=0.456, p = 0.044) were also found. our results demonstrate activation of the cell-mediated immune system taking place in a sub-group of patients with acute pancreatitis. the finding of increased neopterin and stnf-r levels implies that activated monocytes/macrophages are involved in the pathogenesis of the disease. further data are necessary to evaluate potential associations between changes of marker concent-rations and the course of the disease. pancreatic injury after heart surgery was reported as soon as 1970 ( 1,2) and characterized by increased serum or urine amylase levels (in about 33% of patients) in the 5 fi~t postoperafi.'ve days. this pancreatic injury, which sometimes led to acute pancreatitis, was atreaay at~buted to inappropriate perfusion of this organ. in the 198ffs, 3 studies were published dealing with pancreatic suffering alter heart surgery, in large series of patients, concluding ~n~at panc~a~c injury (with a low incidence of pancreatifis) is more common than previously recognized and is a potential source of complication after camliac surgery (3, 4, 5) . in a recent study (6), evidence of pancreatic cellular injury was found in 80 out of 300 patients undergoing cardiac surgery, with 22 out of these 80 patients presenting abdominal signs or symptoms and 3 developing severe pancreafitis. this injury was associated w~th preoperative renal insufficiency, valve surgery, ~..stoperalive hytxxension, calcium administered periopuratively and length of bypass. we studied 300 patients submitted to cardiopulmunary bypass (cpb) for heart surgery and used the measurement of un:~sin, pancreatic iso-amylase and lipase in plasma for biochemical characterization of pancreatic cellular injury. blood samples were obtained before surgery, directly aller surgery (return to inte~ve care unit), 8 hours alter surgery and in the folfowing days alter surgery (days 1, 2, 3, 4 and 8). computed tomography scan of pancreas was performed in patients presenting hi~ levels of amylase on day 1. we measured abnormal levels of trypsin and pancteatic iso-amylase in 30% of patients and observed 2 simultaneous releases of these 2 enzymes, the fi,'st one in the 24 hours after surgery and the second more intense from day 4 and pa~icularly on day 8 after smgery. this second release was concomitant with abnormal levels of llpase. these biochemical observations were accompanied by radiological and clinical signs of pancreatic injury in about 2% of our patients : pancrealic abnormalities were revealed by scan in 7 patients and acute pancreatitis in i patient. more pronounced pancreatic suffering was observed in patients undergoing valve replacement than in patients undergoing coronam-anrtic bypass grafm~g. analysis of trypsin and pare're, tic 1so-amylase are sw.cific of pancreatic cellular injury and their simultaneous ir~rease in plasma alter cpb in our padents confirms the presence of an exocrine pancreatic injury. the presence of a simultaneous peak of lipase mcaezse~ the specificity of overt pancreatic injtu 7 diagnosis. the precise cause of th/s injury could he related to hypoperfnsion leading to ischemic injury of foe splancbnic area, pancreas being largely sensible to hypoperfnsion (7). this hypoperfosion could he responsible for the ftmt release of pancrealac enzymes observed in our patients and would contribute to the deterioration of other organs leading to an inflammatory reaction developing in the following days and responsible for the second release of pancreatic enzymes observed in our patients. patients with necrotizing pancreatitis show a heigh rate of pulmonary, renal and septic complications, whereas the course in acute interstitial pancreatitis is generally very mild. we have prospectively analysed the value of endotoxin, interleukin-6(il-6) and transferrin in compare with c-reactive protein(crp) for the early assessment of the severity of acute pancreatitis. patients aud methods: the values of endotoxin(measured by limulus-lysate-test), ii-6(elisa), transferrin and crp (nephelometry) were analysed daily along the first i0 days of hospitalisation by 28 patients with acute pancreatitis admitted to our hospital from 4/1991 to 4/1993 . it was judged whether the patients have either interstitial (aip) (n=9) or necrotizing (anp) (n=lg) pancreatitis. 5 patients with anp have died during the course of pancreatitis (mortality=17.5%). results: 1-severity o~ pancreatitis: signifcant differences (p<o.05, mann-whitney test) could be calculated between aip and anp for endotoxin, il-6, transferrin and crp during the i0 days of monitoring. 2-mortality: except of il-6 on days 2, 3, 4 and 5 after ad-mission there were no significant differences between the values of analysed parameters in patients who survived and those values in patients who died. 3-organ failure: the patients with pulmonary, renal or multiorgan failure have significantly heigher values of endotoxin, il-6 and crp along the i0 days of monitoring in compare with those patients without an organ failure. conclusions: endotoxin, il-6 and transferrin, an important endotoxin carrier, are promising parameters to differentiate between the severe and mild form of pancreatitis comparable with crp. this parameters may be helpfull in the early clinical assessment of patients with acute panereatitis. the determination of cytokines may elucidate the pathophysiologic mechanisms that produce early systemic complications in acute interstitial (i) or necrotizing (n) pancreatitis (ap). the appearence of respiratory insufficiency (ri) is used to appraise the early systemic complications and is compared to the results of cytokine blood levels. in a prospective clinical trial from 10/92 -8/93, 23 patients with ap were recruited and blood samples taken for cytokine determination with commercial elisa-kits. the differentiation between i (n=12) and n (n=l 1) ap was made through ct-scan in all and laparotomy for drainage and necrosectomy in 13 patients. the etiology of ap was gallstones in 9, alcohol abuse in 7, hyperlipidemia in 2 and other or unknown causes in 5 patients.five of 11 patients with nap died early (n=l) or of late septic complications. none died of iap.the peak of cytnkine level in the first three days of hospitalisation was used for calculation. ri was diagnosed in the first week of hospitalisation, if oxygenmask or iutubation for at least 3 days was nescessary to treat arterial hypoxemia. for statistical analysis u-test of wilcoxon, mann and whitney was applicated.the il-6 concentration in blood reached up to 2600pg/ml in the first days depending on the severity of ap and dropped to almost zero in the next days, independently of the clinical course. the differentiation of i-versus nap, using a cutoff-line of 600 pg/ml was correct in 20 patients (87%, sensitivity (se): 82%= specificity (sp): 91%, (z:0,001). high levels of il-6 over 500 pg/ml correlated well with the prognosis ifri in the first week (accuracy: 87%, se: 80%, sp: 100%, c~: 0,001). the blood levels of il-8 reached a maximum of 1381 pg/ml in the first days, depending on the severity of ap, and showed a correlation to the clinical course in the following days. the peak of 1l,-8 blood levels indicated correctly the severity of ap in 18 out of 23 patients using a cut offtevel of 200 pglml (accuracy: 78%, se: 82%, sp: 75%, ~: 0,01). there was no correlation between cytokine blood levels and mortality, also there seemed to be no correlation between the levels of il-6 and il-8. in the bloodsamples of five patients with i-or nap no c~-tnf was detectable.the blood levels of il-6, and to a lesser extent of [l-8, can predict the severity and early systemic complications of ap in men. the excessive rise of cytokines can be explained by the stimulation of immunological cells ( macrophages, lymphocytes and endothelial cells) in the course of ap. objectives: in an opossum model, ligation of the sphincter of oddi or separate ligation of the bile and the pancreatic duct together leads to severe haemorrhagic pancreatffis while single ligation of the pancreatic duct causes only an exocdne atrophy. since bo has been discussed to be a contdbutor t¢, res dysfunction, this may depdve the organism of a potent defensive mechanism thus promoting the course of pancreatitis. the present study wa,,; carded out tc develop a new method for measuring dynamic liver res func.. tion and to test the hypothesis that bo causes a res dysfunction. material & methods: 18 opossums were randomly placed in three groups. all received a central venous line. after midline laparotomy, a specially designe0 tourniquet was placed around the common hepatic duct above its junction with the pancreatic duct (group a, n=6), around the pancreatic duct (group b, n=6) or around both ducts (group c, n=6). after one week, the tourniquets were closed. using a scintillation camera, the liver uptake of nanocoll, a 99mtcalbumin colloid with a diameter of 25 nm, was measured before, 2,5 and 6 days after the obstruction. the curves were analysed by a computer and two parameters (r, s) were calculated using natural logarithmic regression. as a common measure for res function, the corrected granulopexic index (a) wa,,; determined, after day 6, the pancreas of each opossum was searched for necrosis by morphometdc methods. results: all animals survived till day 6. the opossums in groups a & 8 had a macro-and microscopic normal pancreas, while those in group c showed a severe hemorrhagic pancreatitis. the granuinpexic index showed a significan~ change to the worse starting at day 5 in group a & b (p<o,05) and at day 2 it~ group c (p<0,ol). at day 6, res function in group c was significantly worse than that in group a & b (p<0,01). the regression parameter r showed no significant change in groups a & b, but a highly significant decrease in group c starting at day 2 (p<0.005), thus showing a dysfunction of the hepatic res. conclusions: obstruction of the hepatic or pancreatic duct alone does no{ result in immediate dysfunction of the hepatic res, while obstruction of beth ducts does. because only ligation of both ducts is followed by a severe hereof.. rhagic pancreatitis, res dysfunction could be an important factor in the pathogenesis of pancreatitis and resulting organ failure.logarithmic regressinrl has proven to be a valuable tool to analyse dynamic hepatic res function. (b6) in lewis rats weighing 220-250g. there were five groups: group a (n=14) were untreated controls; group b (n=7) were given isotonic saline intraperitoneally and observed for 48 hours; group c (n=7) 2 ml x 108 e. coli intraperitoneally and observed for 48 hours; group d (n=7) were given 4 ml x 108 e. coli and observed for 48 hours; group e (n=7) 4 ml x i08 e. coli and observed for 24 hours, the rats were than killed, the spleens were removed and heparinised blood taken for immunological tests. total t-ceils, helper t-ceils, suppressor t-cells, monocytes, and the interleukin 2 receptor were determined by monoclonal antibodies, indirect immunfluorescence, and flow cytometry (facs analyser 11). statistical analysis was by the t test on rank transformed data.in group b (isotonic saline) the total t-ceils increased in the blood (p=0,003) and spleen (p=0,0001 )com pared with the untreated rats (group a), in the groups with peritonitis (c, d, and e) we found highly significant rises in suppressor t-cells in the blood and spleen (both p<0,0001 ) compared with the non-infected control groups a and b. this increase was significantly higher in group d than in groups c and e. as well as other alterations, we found a uniform increase in the number of t suppressor ceils in our model of acute peritonitis that was both time and dose dependent, department of surgery, university vienna, akh wien, w~hringer gurtel 18-20, 1090 wien. d~'na~'aics of som~ l~mnunologic indices in children with abdo;~tinal shock v.z.i~ioskalenko, s.f.vesiol$,t.v.p~bina serum (iga,:~i,g, circulating imaune co!~plexes, co:apleaentary serum activity, antimesothelialvisceral and parietal antibodies) and cellular (i-l~q~hocytes, ~s/th,b-lyaphoeytes,i nlaunoleukosis to aesothelial allergens; spontaneous blast cell transformation to phytohemaaglutinin and .nesoth<-~lial ~,togen; phagooftic neutrophil activity) im:~une indices v.'ere studied in 60 children operated v:ith symi~to'as of abdo~ainal shock. ,'~!-~ of them had appendicnlar(26-1ocalized jo-diffuse, 9-~eneralized) and 5-hemoperitoni-~is (dull abdominal trau;na with injayy of 'the spleen-4, the spleen and liver-q), flo patients tjere operated on for co~iplete iieus (~-intussnsc.sption, 5-com:lissural ileus). 2he follo~'~ing serum factors: cireula:;ing im~aune complexes and ~).esothelial antibodies are the aost i~portant ~ar~ers of the abdominal shock course. :yith the progress of the process the indices increase irresponsive of t.l~e cha±.aeter of pathology. in case of ileus regress and hemoperitonotis an abrupt drop in ~he titer of anti~aesothelial antibodies is noted. in bacterial peritonitis a high ~iter of anti:aesothelial parietal antibodies in great dilutions ("+++","++++"-q ; 720) persisted even in the period of clinical recovery. cellular factors cham'acterize dfnaaics of the abdominal shock coulees less specifically llast cell transfor~aation and phagocyue neut~ophil activit 2 can inderectly characterize transition of shock f~orn reversible into irreversible phases. the past so years have brought a number of major advances in burn care. the initial advance was the discovery that burn victims required large volumes of resuscitation fluid in the initial 24 hours to maintain hsmodynamic stability, this was followed hy the dsvelopmsnt of topical antimicrohlal agents to prevent end treat infections. next, was the reporting that early excision of burn wounds, with autografting of the excised wounds was possible.finally, was the identification of the importance of aggrsssive nutritional support for the hypermstabolic burn patient.these advances have markedly increaesd survival rates for given burn mimes, s~ch that burns which would previously have been considered to be aniformly fetal, are now readily survivable.thsre remain two unsolved problems in burn care, which are limiting survivability cf burn patients.the first ie ths treatment of inhalation injury. ths sscond is how to obtain coverage of ths maaaivsly burned patient, who has limited skin graft donor sites available.this ssssion will focus on the new treatments being developed to obtain permanent coverage of burn wounds.these include the use of various tcplcal growth £aotore which can speed the rate of reepithelialization of wounds, both the beneficial and dstrimental effects of using cultured karatinooyte preparations, to obtain in excess of one squats meter of a pseudoskin from a single fifteen square centimeter biopsy of unburned skin, will be discussed, finally, the used for artificlal dermal preparations will be discussed. integumentary wound healing consists of simultaneous epithelializalion and contraction. not long ago, it was thought that healing proceeds at an optimum fixed rate and could only be delayed. the recent explosion in molecular biology and recombinant gene techniques have produced information on the biological activity of compounds previously believed to be biologically inert. many current attempts to modulate the rate of wound healing center on the topical application of various growth factors produced by integumentary cells or agents designed to modulate growth factor expression or response. epidermal growth factor (egf), plateiet derived growth factor (pdgf), transforming growth factor (tgf) and fibroblast growth factor (fgf) have been demonstrated, both in vitro and in vivo, to stimulate the proliferation and diferentiation of their designated cells types. egf and pdgf have been clearly demonstraled to increase the rate of wound closure in partial and full thickness wounds. pdgf also has demonstrated efficacy in the closure of recalcitrant pressure sores, whereas tgf increases the tensile strength of incisiona[ wounds and fgf stimulates angiogenesis. it appears that epidermal keratinocyte migration during wound healing depends on integrin-mediated interactions with compounds and ceils extracellular matrix. additionally, the activities of extracellular glycoproteins such as fibronectin, fibrinogen, laminin and thrombospondin are coordinated by multiple integrins with specific distributions and binding affinities. currently, agents which incorporate specific protein sequences essential for the interaction of the glycoproteins with the cells of the extracellular matrix are being investigated. it is has been clearly established that the rate and nature of wound healing can be influenced by the application of various agents and that sufficient quantities of these agents can be manufactured. the future challenge is to develop techniques to now the objective evaluation of the clinical efficacy of these various agents and to employ the appropriate agents in a cost-effective manner. transplantation; and 4) the immune status/manipulation of the host.multiple interactive variables will determine the effect of employing allogeneic cells and tissue in wound coverage design and a batter understanding of the dynamics of the wound-graft microenvironment will facilitate the dove opment of clinicauy beneficial graft composites. cadaver allograft skin (cas) is the "gold standard" for wound coverage, but has limitations including varied availability & quality, immunogenicity leading to rejection, & potential for disease transmission. our development of a temporary living skin replacement (tlsr) addresses these issues; the tlsr consists of highly screened human neonatal fibroblasts (hf) cultured in biobrane a, a bilayer dressing consisting of nylon mesh laminated to a thin silicone membrane which controls water-vapor transmission. studies in our lab indicated that "fresw ~ tlsr grafts reproducibly close full-thickness wounds in mice & perform better than bb controls. we studied wound adherence & structural/molecular properties of fresh & cryopreserved (cryo) tlsr. methods: tlsrs were prepared by seeding 4000/co 2 hf in bb nylon mesh in dmem. hf quickly attached to the nylon mesh & were allowed to proliferate 3-6 wks. fibroblast mitochondrial activity was assayed by mtt assay. matrix proteins were identified by immunostaining, mrnas for specific growth factors were identified by reverse-transcription polymerase chain reaction amplification, & quantitated by gel scans. bb structure was studied by scanning electron microscopy & stretching measurements pro/post cryo. 80 grafts were cryo'd in 10% dmso, quick-thawed & sutured onto 2x2 cm excised full-thickness dorsal wounds on athymic mice. 40 "fresh" grafts were similarly placed. controls grafts were cas and aeellular bb. adherence was quantitated by a device which peeled grafts from wounds at intervals following placement, using a serve motor and a strain gauge. results: postcryo, storage and subsequent thawing tile tlsrs maintained >60% cell viability by the mtt assay, indicating continued mitochondrial activity, and bb structure & stretchability were maintained. multiple matrix proteins secreted and deposited in the bb nylon mesh (types l/iii collagen, decorin, fibroneetin) were identified by specific immunostaining. growth factor mrnas in the tlsrs (afgf, bfgf, kgf, tgf~,p~,) were present in 10-10,000x higher levels in fresh/cryo tlsrs than in adult hcs. grafts adhered to wounds on mice through 40 days of followup. histologic exams on days 6-40 showed excellent vascular ingrowth and minimal inflammation. adherence of tlsrs to wounds was >cas adherence. burn wound coverage in the massively burned patient remains a difficult problem. although cultured keratinocytes have been utilized for burn wound coverage, their impact on the patient with burns greater than 80% total body surface area has not been spectacular, with poor graft take and unstable epithelium.current investigations have been directed toward dermal replacement beneath either very thin split-thickness autografts (stag) or utilizing cultured keratinocytes. current products include: collagen dermal replacement with thin stag (burke, et al). collagen dermal replacement with cultured keratinocytes and fibroblasts (boyce, et ai). allograft dermis with cultured keratinocytes (cnno, et al). allograft dermis with thin stag (life cell). polyglactin acid mesh and neonatal human fibroblasts with thin stag (hansbrnngh, et al).investigations regarding culture media, use of growth factors, topical nutrients and antibiotics, and melanocytes for pigmentation as well as safety and efficacy are needed before any of the current products become viable options for coverage of the massively burned patient. the~ is a growing world-wide problem with the ujc of cadaver tissues and ocgans bae, au~ of the tren~m~s~km of dilemma such a; cmutzfeldt.jukob disease and iiiv as we1] as ready availability of urdform lis~ue~. on dec~mt~r 14, 1993, the fda assumed control of as1 tissue bar~s in the uldtod st=tea in an attempt to bflng ~s difficult problem of dise~s~ transmission under ¢onlrol. in europe, ~om¢ of the governments are consldofll~ a c~mplcte bat) on the use of cadaverlc fissu~s such as ddn, 'this |ncroam in regulation of cadavefle ~s,quct will incmar¢ the difficulty of obtain~g and dlslflbulmg them. however, thc nc~ for these tissues contlnue~ m incrcaso, we will discuss ~'l¢ solulion to this important pmbl~n: tissue engineering. tlssu~ engineering is an in~rdisdpllnary field that applies pdnclplc~ of angin~edng and die life sclcnce~ reward the development of ~olok~¢al sub~dtute,~ ih= mslom, maintain, or improve tissue function, "13ssuc ongln~cdng can provide ~ho nccassary tlssuoa for wound repair ~d ibe assuranoe fl'~t the lissuos are d.ls¢~¢ free. in addition, a ds~uo-cng~ne~n~l wound covering will bo u~lvemally acceptable and evntlublc as "off g~o shell", consis~t products, them are several approaches to restating thls function in a large wound, 'l'nosc i~elud~ tmmcdiete long term coverage, short t=nn coverage, uandtl~el coverage and compost= dssu¢ coverage, "flssuo onglncrcd wound coverings that meet those vaflous ne,.cds will he r~vlowod.cllni~:sl and experimental d~la in venous ulcer, dlabctl¢ ulcers, prossur~ ulcers and bum wounds wgj be mvlcw~, a~ welt as new approacl~s u~ csrtilag¢, bone, liver and bone marrow it~suos. c oomplon, k nadirs, w press, g wetland, j fallen iv, shrtners burns institute and massachusetts general hospital, boston, ma~schusetts, usa the clinical "take" rate o? cultured epithelial autografts (cea) has been observed to increase with transplantation to allodermls, but the reasons for the improved clinical performance have not yet been defined. the aim of this study was to determine the biological impact of normal human dermis on cea differentiation and maturation, biopsies of cea transplanted to engrafted and de-opldermlzed human homograft dermis have been compared to nopsles of cea transplanted to granulation tissue in tullthickness burn wound beds on the same patient, each patient serving as hls or her own control. paired test and control biopstes from six patients have acquired from as early as one week postgrafting to as late as 3 years postgrafting (one patient) and analyzed histopathologlcally, ultrastructurally and immunoh[stochemloally, results demonstrate more rapid normalization of differentiation markers (e,g., involucfln, fllaggrln, cytokeratln profiles) in the cea transplanted to allodermls compared to their corresponding controls by in all patients, the proliferation rate within the basal layer ot the epidermis as determined by ki-67 (proliferation-associated antigen) is seen to norh~altze more quickly in the cea transplanted to allodermls in every case, persistence of allodermal matrix can be dooumented in all patients by elastic tlssue-trichrome stain, allowing visualization of the dermal elastin network. the popu;atlon densities ot intraepldarmal langerhans cells are conslstently and signlflcantly higher in cea transplanted to ,allodermls, possibly reflectlng an immunologlcal reaction to the underlying allogenlc tissue. overall, these preliminary results indicate that transplantation to a normal human dermal matrix accelerates the maturation of cea-deflved epidermis, wound closure continues to be a major problem in patients who have sustained a major thermal injury, cultured epidermal autografts (cea) have been utilized extensively since 1984 when galllco et el reported theh'use in two brothers with greater than 90% total body surface area burn. unfortunately, cea take rate varies widely and the resultant skin coverage is often fragile and the cosmetic results are less than optimal however the overall take rate and durability of the coverase can be markedly improved by using nn allodermls base as the recipient bed. a review of cea applications performed by physicians using cultured outologens epithelium obtained from blusurfaoe teclmology, inc. shows a marked discrepancy in the results obtained utilizing different methods of wound bed preparation. tgf-b is an important modulator coordinating complex physiological events associated with growth and development. it is assumed that tgf-b is also involved in the well-coordinated process of cutaneous wound healing by regulating proliferation, differentiation, chemotaxis and matrix deposition. the purpose of our study was to analyze the spatial and temporal pattern of tgf-b expression during granulation tissue formation in patients with accidanutl surgical trauma (monotraumata mid polytraumata) and bum wounds. after debridement (day 0), the full thickness wounds were covered with epigard, a synthetic dressing until day 10. after this time the granulated wounds were closed by transplantation of mesh graft. biopsies of the wound center were taken from 30 patients at the beginning of surgical treatment (day 0) and after 3, 6, and 10 days. cryosections were stained with antibodies against tgf-fi s using the apaap technique and -for standard histology -with hematoxylin-eosin. for identification of the cell type expressing tgf-6, double staining immunofluorescence experiments were conducted using antibodies specific for monocytes/macrophages, polymorphoanclear neutropkils and fibroblasts. the results showed a characteristic pattern of tgf-t~ distribution during wound development. tgf-fi appearence was mainly cell-associated znd the absolute and relative number of cells that were positive increased with lime. infiltrating cells and developing blood vessels were most prominently stained; epithelial and t-cells showed no immuno-reactivity. a delay of emergence for tgf-b during the time course could be seen in one patient group. this might reflect various regulation patterns depending on the type and severity of injury.(1) pharmatec gmbh, frankfurt (2) institut fiir immonologie and serologic, heidelberg ( immune cells extravasating specifically in skin recognize and eliminate the invading antigens (bacteria, viruses, etc.) either in situ or transport them to regional lymph nodes. they also participate in the process of skin wound healing. cells which traffic through the skin can be harvested from efferent lymph drained from a given area of skin. the type of migrating cells changes after trauma, heating and infection. we have developed a method for collection of human afferent lymph in lower limbs. the method allows obtaining immune cells from normal and injured skin and their characterization. aim of the study was to characterize skin immune cells in situ and in skin lymph with use of immunohistological methods (staining, facs). results. group 1, cells migrating through skin: 75+4% t lymphocytes (cd3), 6+3% langerhans and dendritic cells (cdla, hla dr, s100), 41+9% cd4, 18+6% cd8, no b cells (cd22, 23), 74% cd45r0 (memory cells), 6+3% il2r. approximately 90% cells possessed cdlla and 18 antigens. cd1 lc was expressed only on large cells. the frequency of all phenotypes was different from the blood populations. group 2, cells in skin: langerhans cells were found only in epidermis, cd3,4 and 8, cd45r0, rb, ila/18 cells around venules, cd68 (macrophages) uniformly dispersed, no il2r and b cells. hla dr positive were endothelial and some dispersed mononuclear cells. group 3, one, three and thirty days after surgical wound (simple varicous vein extirpation): high density of epidermal langerhans cells, hla dr positive keratinocytes and all endothelial ceils, few il2r cells, perivenular infiltrates of cd68, 45r0 but less cd3 cells, high density of cdlla/18 cells. classic staining of isolated and in situ located ccl!s with mgg or he did not allow to follow kinetics of changes. conclusions. this study presents the first in the literature quantitative data of immune cell traffic through normal and injured human skin. in the controlled release of biological response modifiers for soft tissue regeneration. alan s. rudolph, helmut speilberg, mariam monshipouri, and florence rollwagen, and barry j. spargo. we have employed lipid microstructures as controlled release vehicles for the delivery of growth factors in wound repair. traditional liposomes as well as novel lipid based microcylinders have been examined for their in vitro kinetics of the release of transforming growth factor beta (tgf-b). in vitro reiease has been examined by setting up models with examine the physical release of iodinated tgf-b as well as a cell based bioassay (based on the ht3 bioassay). the hollow lipid microcylinders (50 microns in length and i micron in diameter) show an initial burst (5-10 ng) followed be zero order kinetics which result in the release of approximately i ng tgf/day. this release behavior can be modified by temperature based on the phase behavior of the lipid bilayer which comprises the microcylinder.we have also examined the cellular response to lipid microcylinders applied in vivo. the lipid microcylinders are mixed in agarose and implanted as a composite hydrogel block under the flank of a mouse. the blocks are removed 1, 3, and 5 days following implant and the cells analyzed by facs sorter analysis. the observed pattern of ceil recruitment to the blocks mimics that seen in a local inflammatory response. cell surface phenotype studies included the determination of cd3 and cd4, mac-l, and ig bearing cells. we have also begun to examine the change in cell surface phenotype and kinetics of recruitment following the inclusion of tgf-beta in the lipid microcylinders.center for biomolecular science and engineering, code 6900, naval research laboratory, washington, dc. 20375-5348. expression pattern of heat shock proteins in acute, good healing and chronic human wound tissue. abstract: wound healing is a complex biologic process that is well characterized at the histological level, but its molecular regulation is poorly understood. after clot formation, inflammatory cells are rapidly drawn into the wound, followed by migration of fibroblasts and epithelial cells that divide and repopulate the wound area. during the last decade peptide growth factors and cytokine are thought to play a key role in initiating and sustaining the phase of tissue repair. these factors which are released from different cells appear to initiate the cascade of events that lead to healing. different studys described the rapid activation of a family of proteins,named heat shock proteins (hsp) in differnt tissue that were exposed to various forms of stress (heat, toxic agents, mechanical). in this context hsp's have the ability to regulate protein folding and assembly, to transport proteins across cytoplasm and membranes, to disrupt protein complexes, to stabilize, degrade and regulate the synthesis of proteins and to take part in dna replication and repair. we now attempted to find out if hsp-gene activation is also involved in injury and wound healing, which likewise resemble a stress situation for cells. therefore we collected tissue samples during operation and single biopsies from chronic wounds (decubitus for example) and granulation tissue. after rna preparation from these samples we used rna-pcr and nothern analysis to study the expression of objectives of the study chronic, non-healing cutaneous tflcers are a challenging clinical and socioeconomic problem. several animal studies have shown that cytukines (e.g. egf, pdgf, fgf, tgfb) accelerate the healing process and tissue repair in general. results from first clinical trials indicate a promising value of cytokines in the treatment of chronic non-healing diabetic and venous ulcers. recent reports in the literature indicate that the biological activity of the solution of platlet derived wound healing formula (pdwt~) released from c~-granules (mainly pdgf & tgfi~) is greater than the activity of the recombiant single factors like e.g. pdgf-bb (robson, lancet 1992) . the aim of our study was to determine whether a correlation exits between the concentration of tgfi~ & pdgf and the time course of wound healing. materials and methods pdwhf was prepared from 200ml of auto]ogous patient blood and diluted with a special buffer to a final concentration of 30 ng/ml g-thromboglobulin. the concentrations of pdgf and tgfg were determined by elisa-tests developed in our laboratory. 22 patients with chronic non-healing ulcers have been evaluated alter treatment by topical application of pdwhf. pdfg and tgff~ concentrations of the topical solution were measured and two patient groups formed for analysis the time course of wound healing was regularly and meticulously documented and evaluated by photography and casting. the time from initiation of treatment instil o wound volume reduction to 50 go of the origional size (t50%) was noted• results: healing of extensive burn wounds can be accelerated by grafting cultured autologous or allogeneic keratinocytes. the stimulation of granulation tissue formation and reepithelialization is presumably based on growth factors and cytokines released by keratinocytes. we wanted to prove this hypothesis by investigating the bfgf expression during wound development, bfgf is mainly described as an angiogenic protein with mitogenic activity on various mesodermal and ectodermal cell types pointing to its stimulating potential in wound heating. in the present study we compared the pattern of human bfgf m-rna expression and the localization of bfgf protein during the first days of wound healing. biopsies were taken from 5 juvenile human bum patients, immediately after wound debridemerit mad on day 8 after transplantation of cultured allografts. biopsies were snap frozen and cryosected. the pattern of bfgf expression was assessed by in situ hybridization of the bfgf m-rna with a digoxigenin-labelled antisense-rna and the parallel detection of the mature protein with an anfi-bfgf monoclonal antibody. our study revealed typical patterns of bfgf-m-rna-expression and intense bfgfprotein deposition during granulation tissue formation and reepithelialjzation of healing bum wounds. 'it, is known that major thermal injuries cause early impairment of wound healing followed by decreased influx of granuiocytes st. the site of injury. the role of granuiocytes in the process of wound healing is not ~"~ "" elucidated, it is now assumed that they are not merely phagocytic cells but active participants in ~n~*'1~.,.,a+~o~: processes secreting_ a number of various cvt-;kines, in order to investigate the effect of there is accumulating evidence that neuropeptides could be involved in the pathogenesis of several inflammatory reactions. vasocactive intestinal polypeptide (vip) and substance p (sp) have been detected by immunohistochemistry in normal as well as inflammed skin mostly in perivascular and periglandular location. both vip and sp are involved in vasodilatation, mast cell degranulation and irnmunomodulation.we determined the influence of sp and vip on the proliferation of lymphocytes in patients with psoriasis and healthy individuals. peripheral blood t-lymphocytes of 6 psoriatics and 6 healthy controls were isolated by density gradient centrifugation and passage over nylon wool. cell enrichment was controlled by facs analysis, lx105 t-lymphocytes were then incubated alone or in coculture with 2x104 irradiated autologous lymphocytes in culture medium containing 10 -7 mol/i sp or vip. cell proliferation was measured semiquanfitatively by tdr uptake in a betacounter. significance was tested by the wilcoxon signed-rank test.our results show that sp and vip exert only an effect on unstirnulated t-cells. in healthy individuals but not in patients with psoriasis sp increases significantly proliferation of t-cells. vip, however stimulates significantly the blastogenesis of t-lymphocytes only in psoriatics.our results confirm the psychoneuroimmunologic component in inflammatory reactions and vip and sp could be partially implicated in their pathogenetic mechanisms. moreover psoriatic lymphocytes show an altered reaction to sp and vip. this might be due to a preexisting (genetic?) or more likely to an epiphenomenal receptor defect. the adhesive interactions between endothelial cells and circulating ~enkocytes in shock and innammatory vondltions is mediated by several distinct families of ce11-surface determinants. of particular importance are the leukocyte integrins cdib / cdlla-c. in this study monoclonal antibodies to two of the u chains (cdlla & cdiib) and the common [~ chain (cdib) have been used to investigate leukocyte-dependent and leukocyte-independent plasma leakage in tee skin of rabbite. plasma leakage was measured as the local accumulation of t2si-hsa over a 30 rain period, the chemotac~c peptide imlp (0.1. 30ng) and bradykinin were used to induce cell.dependent and cell-5ndependent leakage respectively, the antibodies used were 6.5e (cdis), nri85 (cdlla) and antibody 198 (cdllb). ]ntradermal in~ections of bradyklnin and ~dlp both caused a dose dependent increase in plasma extravasatien (15.~. ffi 2.1p.l to 54.4 z b.bttl and 16.4 ,-1.8~ to 59.8 z 6.71d respectively. 6.5e (0.0072-2.4mf,/k~ iv) caused a dose dependent inhibition of imlp-induced but not bradyldnin.inducecl plasma exudation. at 0.24mk/kg, the plasma leakage was completely inhibited, antibody nr185 produced similar results, treatment with antibody 198 did not cause inhibition o£ plasma leakage due to either tnedi~tor. in vitro, the irmnune system ex~nination in persons with bone, chest and abdominal traumatic injury (i group . 22 patients without infectious coz~lications and 2 group -53 patients with wound infections development) was carried out. to restore found immunity disorders and host defense to infection 23 patients of the 2 group were treated with thymalin-the biologically active peptides prepared from bovine thymus. the examination on t~e i-3 days after injury revealed a considerable decrease of lymphocytes, ed3",$d4 ~ and cd8 cells amo~it in the blood, cd4 /cd8 ratio and indexes of let~ocyte migration inhibition test in both groups of patients. the imm~lity disorders recovered to norm on the 7-10 days in pateents of+the i group. but stable ~eple$ion of cd3 and cd4 cells amount, lower cd4 /cd8 ratio and indexes of leukocyte migration inhibition test in patients of the 2 group were observed~ besides that, these persons showed higher cd19cells amount and ig level in the blood. after thymalin therapy valid ii~rovement of inun~e status was discovered. also good clinical effect of immunotherapy and best wo~id healing observed in 80% of cases. these results allow us to propose that the thymus involution and the reduction of cell-mediated immunity responsiveness with disturbances of immu_uoregulatio~ on the level of restriction of activated cd4 tho cells play the most important role in the pathogenesis of wound infections development in persons with traumatic injury.dept. of immunology, military-nedical academy, lebedeva str. 6, 194175, st.petersburg, russia a severe impairment of neutrophil (pmn) function often occurs following severe thermal or non-thermal traumatic injury. our laboratory has previously reported that following severe burn or non-burn traumatic injury the expression of the p2 integrlns (cd11a,b,c/cd18) and the fw receptors (cd16, and cd32) were significantly decreased on pmns, in this study, the effects of gm and g-csf on the expression of the f~ r and the ~2 integrln family on pmns were examined, pmns were obtained from severe trauma (initial apache ii score ;z 10) or thermal injury (>30~; total body surface area, >15~ full thickness) and incubated /n v/tro with gm or g-csf. the j32 integrins or fcyr were detected with monoclonal antibodies and flow cytometry. gm end g-csf induced a sllght increase in the percentage of pmns expressing cd1 lb, cd16, and cd18 while gm bur not c-csf induced an increase in the percentage expressing cdi 1 a, cd1 lc, and cd32, gm-csf and to a lesser extent g-csf induced an increase in the density (1,5 fold) of the ~2 integrlns on pmns from normal, burn, and trauma patients, these data suggest that cytoklne modulation with csfs could have a role clinically in certain situations. institute, dept. of surgery, 231 bethesda ave, cincinnati, oh, usa, 45267-0558. funl~al infections after solid organ transplantatlon(sot) lewis flint, md and ed,~-afd e. etheredge, me) dept. of surgery tullrte univ. school of medicine new orleans. louisiana infections contribute to increased gra5 loss and mortaliw following sot. pr~isposing facton include diabetes, hepatitis, leukopenia, cc.¢xistem infection, and intense, especially triple drug, immunosuppression. funga] infections occur ~s isolated conditions in 6% and in association with bacterial infection(l 1%), viral infection(3*/.), and combined infections(it%), candida sp. is the most common fungus recovered but aspecgillus, coccidiodies, cryptococcus, histoplasma, mueor~ ghizopus, tinea, and toruiop~is s?. also are pathogens. clinical syndromes vary among orga.aizms or may be variable with a single p~tthogen, for ~ample, with aggressive immunosuppression, candlda my be localized esophagitis or cystitis or systemically iavaslve with an associated high mortality. aspergilius presents ~ a diffuse pneumonia while cryptococcus causes pulmonary and centrad nervons sy'stem infection, clinical examination, ct scanning and aggressive sampling for c'ultures a.s wall as serologic tests contribute to diagnosis. empiric the~py is ind',cated where there is a high level of suspicion. preventlon of ca.adlda izfection is ~ci~itated by early remov-a.1 of central }ants, ca~hetess and stents as well as by the use of oral nystatin. amphotericin ]~ remains the drug of choice for treatment of in.save fungd infection, surgical resection of infectious loci in the lung and brain is indicated in selected patients. the main problems of diagnosis in lower respirator-), tract infection are the differentation of infection from colonization or contamination, and the isolation of a reliable and true pathogen. expectorated sputum may be unreliable in pneumonia, because of contamination by oropharyngeal flora. although blood cultures may be negative, they provide a precise diagnosis and should be obtained in all pneumonias. other more invasive procedures are transtracheal needle aspiration, fibrobronchoscopic techniques including protected specimen brush and bronchoalveolar lavage with quantitative culturing and cytological analysis, transthoracic needle aspiration, thoracoscopy -guided biopsy and open lung biopsy. recently m. e1-ebiary, a. torres et al, 1993 reported quantitative cultures of endotracheal aspirates for the diagnosis of ventilator-associated pneumonia offering reliable results in these patients and should be further investigated. any invasive procedure in a severely ill patient should be carefully directed weighing the risks as well as the benefits, whilst taking the underlying diseases and expected survival into consideration. -current therapeutic approach is based mainly on monotherapy with broad spectrum antibiotics. combination therapy is apparently indicated only in p. aeruginosa infections and severe s. aureus pneumonia. graft infection can lead to fulminant graft failure or rapid progressive cirrhosis. for prevention of graft infection immunoprophylaxis, i,e. administration of human polyclonal anti hbs hypedmmunoglobutin (hig), starting in the anhepatic phase during operation, has proved to be at least partially succesful when performed on a long term basis.from a total of 415 olt in adult patients 100 olt were performed for hbsag positive liver disease (cirrhosis n=86, fulminant liver failure n=8, retransplantation n=6) in 94 pat. all pat. received 10.000 u hig in the anhepatic phase and 2.000 u/per day for the first week. a small group of 9 pat. received hig only for i week (short term immunoprophylaxis), in all other pat. hig is administered on a long term basis to keep anti hbs serum levels above 100 uii or until graft infection occurs (long term immunoprophylaxis);one-year survival rates are 100% in pat. who were transplanted for fulminant hepatitis, 96% in pat. with cirrhosis and long term prophylaxis, and 78% ir~ pat. with short term prophylaxis. all fatalities were related to hbv graft infection. the total rate of graft infection was 100% under short term prophylaxis and was independent from preoperative hbv dna status, under long term prophylaxis graft infection occurad in 28% in pat, negative for hbv dna. in hbv dna positive pat. infection rate was 56%, the total rate of reinfection for all pat. with long term prophylaxis was 36%the results of liver transplantation in hbsag positive pat. are comparable to other indications, graft infection with hepatitis b virus ist the major risk factor for these patients. under long term therapy with hig the rate of graft infection can be significantly reduced. the crucial cellular element for mods-mof: monocyi'f_./m acrophaoe ronald v. meier, m,d., f.a,c,s. the severely :injured or crldcally ill surgical patient is at high risk for immune dysfunction. a major consequence of this immune dysfunction is multiple organ dysfunction and failure leading to death, the underlying etiology is now recognized to be an uncontrolled, unfocused, disseminated activation of the host normally protective inflammatory. ,, cascades.. the resultant "mahgnant' systemic" inflan'a'natlon produces d~ffuso multiple organ bystander injury !eading to progressive organ dysfunction and failure. systemic malignant inflammation involves diffuse actlvatton of all components of the humoral and cellular inflammatory host response. of these various components, the macropha~e is the crucial central cellular element. the tissue fixed macrophage is ideally located diffusely throughout the various organs injured to orchestrate the inflammatory process. the macrophage is long-lived and highly metabolic, the macrophage regulates both the extent and the dissemination of the inflammatory processes. the macrophage is an exu'emely active c¢11 capable of producing and releasing not only directly eytotoxlc agents, s irnil~, to the neutrophil, including oxidants and numerous proteases out also the multitude of other cytokines and initiators of the interacting inflammatory cascades. the macrophage is the central source for ehemotactic agents (il-8, ltb4, c5a) for neutrophils and other inflammatory cells, production of vasoaetive arachidonie acid metabolites (tx, pgi2, poe, lt's), complement components (c3a, csa), thrombotic agents (pca, tx), metabolic and physiologic modulators (il,1, il-6 or tnf), and immunosuppressivc agents (poe2, il-10). these products of the macrophage are highly effective in enhancing and augmenting the inflammatory response. disseminated activation otthe macrophage is critical to the induction of the long-term diffuse activation of inflammation necessary to induce multiple organ injury and failure. our ability to elucidate the molecular mechanisms that control the macrophage will lead to our ability to conu'ol the maerophage response and prevent mods-mof.flarborview medical center, 325-9th ave za-16, seattle, wa 98104 usa key: cord-005814-ak5pq312 authors: nan title: 8th european congress of intensive care medicine athens greece, october 18–22, 1995 abstracts date: 1995 journal: intensive care med doi: 10.1007/bf02426401 sha: doc_id: 5814 cord_uid: ak5pq312 nan objectives: evaluate the levels of tnf, il-6 and pai-i in different moments of the ards and the possible relationships among them. methods: 23 septic patients with ards were studied. also significant differences for: tnf, pai-i and il-6 in septic patients and both evaluations of ards with control gropup; pai-1 between septics and 2nd evaluation in ards, and between the ist and 2nd evaluation in ards; il-6 between septics and both evaluations in ards; and il-~ in both evaluations in ards patients in relation to mortality. conclusions: i) elevations of tnf, pai-i and il-6, with clinical signs, are suggestive of infection; 2) the persistent and progressive elevation of pai-i with any clinical criteria may suggest evolution to ards; 3) due to its own kynetics, il-6 takes part later in the acute phase, its levels being related to the magnitude of the injury in the tissues. objectives: the influence of long-term volume therapy with different solutions on plasma levels of circulating adhesion molecules was studied. methods: according to a randomized sequence, 30 patients with sepsis secondary to major surgery exclusively received either hydroxyethylstarch solution (10% hes, mean molecular weight (mw) 200,000 daltons, degree of substitution (ds) 0.5) or human albumin 20% (ha) for volume therapy for 5 days. plasma levels of circulating (soluble) adhesion molecules (endothelial leukocyte adhesion melecule-1 [selam -i] , intercellular adhesion molecule-1 [sicam -i] , vascular cell adhesion molecule-1 [svcam -i] , and p-selectin ) were serially measured on the day of admission to the intensive care unit (='baseline ' value) and during the next 5 days. results: selam-i, sicam-i, and svcam-i plasma levels were markedly higher than normal at baseline in both groups. in the hes-patients, selam-j decreased to normal range, whereas it further increased in the ha-group (from 89• to 106• during the study period, sicam-i and svcam-i plasma levels remained unchanged in the hes-patients, but further increased in the ha-group (from 626• to 1,329• sgmp-140 increased significatly only in the ha-group (483• to 683• only pao2/fio 2 was significantly correlated to plasma levels of adhesion molecules. conclusions: sepsis is associated with markedly elevated plasma levels of adhesion molecules indicating endothelial activation or damage. by long-term volume therapy with hes, these levels remained unchanged or even decreased, whereas volume therapy with human albumin did not have any beneficial effects on soluble adhesion. central venous catheters are frequently used in the care of the critically ill patient. the incidence of catheter related sepsis varies in the literature. we investigated the occurrence of contamination and sepsis compared to results of the epic study as part of quality assesment in our intensive care unit. from january until august 1994 all removed central venous catheters were examined for microbiological culture. the patients who showed signs of sepsis were also registered. the results of the contaminated catheters and septic patients were compared with results from the epic study. during the 8 month period ,2059 patients were hospitalized on our intensive care unit. 230 central venous catheters were examined for microbiological culture. 118 specimens appeared to be possitive (51%). 13 patients showed clinical signs of sepsis. the incidence of sepsis due to contaminated central venous catheters was 13/118 (11%). the incidence of sepsis due to the presence of all central venous lines was 13/230 (6%). the microorganisms responsible for the sepsis syndrom were : stapylococcus aureus (n=5), escherichia colt (n=7), others (n=6). in the epic study the percentage for sepsis on the icu was 17.6% for the netherlands and 17.8% for europe. despite a high number of positive culture from removed intravascular lines, a low percentage of sepsis was seen compared to results of the epic study. we recommend routine bacteriological culture of all removed central venous lines and recommend to look at colonization and sepsis due to intravascular lines as a measure of quality control in the intensive care unit. objectives: prognostic assessment of simplified acute physiology score (saps) in granulocytopenie patients with septic shock (ss). methods: the medical records of 59 admissions to an intensive care unit (icu) of granuloeytopenic patients with ss are reviewed. fiftytwo patients had haematological malignancies. seven patients had aplastie anaemia. patients were categorised as survivors (discharged from icl 0 and non-survivors (died in the icu). saps index was calculated for patients daily during their stay in icu. all patients were severe granulocytopenic (total white cell count less than 0,5 ]09]1). results: five patients (8,5%) were discharged from icu. fifty-four patients died in icu. non-survivors had saps on admission higher than survivors ( 20.9+4.6 and 16.5+3.0, respectively, p<0,01, mann-whitney u test). no patient with a saps greater than 20 survived. mortality among the 27 patients with saps from 9 to 20 was 81,5%o. the evolution of ss was rapid. the mean stay in icu among non-survivors was only 56 hours. an analysis of the saps index on admission of non-survivors showed an inverse correlation with the duration of their stay in icu (r=-0,52, p=0.001). all survivors recovered from granulocytopenia. they had normal white cell counts at the time of discharge from icu. there was inverse correlation in survivors between saps and white cell counts, when these parameters were evaluated daily. however, the saps index alone cannot be considered to be on individual predictor factor of mortality. patients who had failure of the malignancy to respond to chemotherapy and who had persistent granuloeytopenia died in icu despite saps index on admission and recovery from ss. conclusion: saps index greater than 20, failure of the malignancy to respond to chemotherapy and persistent leueopenia all point to a poor outcome of granulocytopenie patients with ss. introduction: antipyretics sometimes are used for fever control in febrile neutropenic patients with hematological malignancies(hm). we observed a dramatic fall of blood pressure(bp) and development of septic shock(ss) in some of the patients who received antipyretics. aim: to clarify can antipyretics provoke ss in neutropenic patients with infection. methods: retrospective review of medicat records of 52 neutropenic(wbc <0,5 109/1)patients with hm, admitted to the intensive care unit for ss, was performed. there was selected group of 8 patients receiving antipyretics shortly before a fall of bp. results: there was a definite causal relationship between receiving antipyretics and fall of bp in 4 from 8 patients. all patients had fever due to infection and had normal level of bp before receiving antipyretics. hypotension developed within 40 minutes up to 1,5 hours after administration of antipyretics. three patients received 0,5 g of metamisol and one 0,5 g ofparacetamol per os. in all cases we observed dramatic diaphoresis and the temperature fall to subnormal level (35.4+0.4~ accompanied'by hypotension. but in 8-12 hours the fever was coming back without blood pressure elevation. the fluid replacement was controlled by central venous or wedge pressures. there were required 1200+350 ml colloid and cristalloid solutions for volume loading. in spite of fluid administration the hypotension persisted and all patients required inotropic therapy. only one patient survived and is alive now. conclusion: it seems to us that our data offer to state that antipyretics administration can initiate ss in febrile neutropeuic patients with infection. objectives: to assess the agreement between cardiac output (co) measured by odm t and by 3 other methods used in icu patients. methods: we prospectively studied 12 adu t patients requiring hemodynamic monitoring with a pulmonary artery catheter. an esophageal doppler monitor provided measurements of co (odm), stroke volume and flow time (ft) used as an indirect evaluation of patient's volume status. patient hemodynamic status was evaluated by a modified fast response pulmonary artery catheter (baxter health care corporation, santa ana, ca), allowing co measurements by thermodilution 0"d) and an evaluation of right ventricular ejection fraction and end diastolic volume (rvef and rv-edv). in the last six patients co was measured by transthoracic echocardiography (echo) and oxygen consumption was measured by a deltatrack ii metabolic monitor (datex) allowing co calculation according to the fick formula (fick). the agreement between methods measuring co and their reproducibility, were evaluated by bland and altman analysis. results: agreement between co measurements is expressed as bias (d) and 95% limits of agreement (l of a = d_+2sd .15 td-fick -2.36 -8.06 to 3.34 fick-echo 0.60 -5.92 to 7.12 there was no correlation between ft and rv-edv. conclusions: although co measurements by odmil had the best reproducibility, the limits of agreement between the four methods tested were unacceptable for clinical purposes. further investigation is required in order to improve the accuracy of co measurement in the icu. phd, a. paltzev, v.bajbikov, b.dobryakov d.sc., a.ostanin phd, o.leplifia phd, h.chernykh phd munieip. hosp. n l, n 12; inst. of clin. immunol., novosibirsk, russia objectivies: efficiency of native cytokines used in the treatment of patients with severe surgical infections has been studied. methods: for two years 120 patients were treated with cytokine mixture (ssp) obtained by arterio-venous perfusion of swine spleen and contained the following cytokines: il-1, il-2, il-3, tnfa, ifny, gm-csf. results: ssp intravenous infusions were shown to accompany with mortality decrease from 23.4% to 12.5% in patients with abscessed pneumonia and lung abscesses and from 50% to 13% if disease course was complicated with sepsis. in patients with purulent peritonitis and sepsis efficiency of ssp was decreased due to endotoxieosis. thus, we used adoptive immunotherapy with mnc activated in vitro with ssp or recombinant il-2. intravenous infusions of such cells resulted in transformation of a pathologic process from destructive into productive one. moreover, clinical manifestations of sepsis were controlled in 81% and mortality was decreased from 46% to 19%. conclusions: the use of eytokines themselves as well as cytokine-treated lymphoeytes permits to control the disease and leads to the mortnlity decrease owing to stimulation of host defence mechanisms. background: although red blood cell transfusions (rbct) are used to increase oxygen availability in septic patients, several lines of evidence suggest that rbct may actually worsen tissue hypoxia. thus, rbct may negatively influence outcome of septic patients. objectives: to determine the association of 1) rbct ; 2) number of units transfused; and 3) mean age of the units transfused on the first day of transfusion with mortality of critically ill septic patients. methods: we prospectively identified patients who met strict criteria for sepsis syndrome (ss) seen in the icu of st. paul's hospital from 1992 to 1994 and excluded patients who died in the first 5 days after the onset of sepsis. we recorded clinical characteristics, multiple system organ failure score, and apache ii at onset of sepsis. then, we retrospectively recorded the total number and age of rbc units transfused during the first 5 days after onset of sepsis. overall 30-day mortality was 22%. results: the main results are shown in the table. the mortality of patients who received rbct was nearly double the mortality of those who did not receive rbct even after adjusting for severity of illness using apache ii. objectives: gastric mucosal acidosis is frequently observed in patients with sepsis. the aim of this study was to determine whether volume infusion using pentaspan| decreases abnormal gastric mucosal pco2 (pico2) in patients who have sepsis syndrome (ss) who have already been resuscitated using clinical endpoints. methods: we prospectively identified 5 patients who met strict criteria for ss, had a pulmonary artery catheter and a gastric tonometer in place, and pico2 > 50 mmhg. pentaspan| (500 ml) was infused in 30 rain. measurements of hemodynamics, hemoglobin, arterial lactate, blood gas analysis, and pico2 were performed before and repeated 30 miff and 2 hr after pentaspun| infusion. we calculated the pico2 -arterial pco'2 difference (pico2-paco2) and phi (using henderson-hasselbach equation). anova was used to assess statistical significance. results: all patients werereceiving adrenergie drugs. map was 73 :1:13 mmhg and lactate 1.2:1:0.6 mmol/l. pentaspan| increased ci by 22% (p<0.05) but did not change pico2 ( and increase m oxygen o* wery were simimny achieved in both groups. nevertheless, epinephrine was associated with a lactic acidosis and increased laetate/pyruvatemia ratio (l/p) that evoke a dysoxia rather than a metabolic effect. an higher gastric mucosal pco2 in the ep group compared to nor-rob suggests the hypothesis of an anaerobic production of co2 in favor of a splanchnic hypoxia. in both group, arterial ketone body ratio that reflects hepatic mitochondrial redox state, compared to a control group without shock was decreased but increased between 12 and 24 hours after restoration of arterial pressure. the association norepinephrine-dobutamine seems to be better for splanehnic circulation than epinephrine and should be used for dopamine resistant septic shock. moreover, the increase in arterial pressure with nor-dob improved gastric mueosal ph and hepatic mitochondrial redox state and argue to reconsider arterial pressure as a significant goal for resuscitation in septic shock. conclusion: significantly higher malondialdehyde and ghitathione levels and glutathione-peroxidase activity in group ns at the end of icu stay were related to mortality these findings indicate an increased generation of free oxygen radicals together with increased anfioxidant activity in this group and sapport the employment of antioxidant interventions in critically ill patients. oblecfives: to determine the role of nitric oxide (no) in the mechanism of septic shock induced by isolated limb perfuslen with recombinant tnfcr methods: we have measured tnfr~ and metebo~ites of no in 5 patients with signs ot septic shock following treatment with isolated limb perfusion for nonresectable soft tissue tumors and melanomas of a limb. perfuslen was carried out with melphalan (burroughs wellcome) and recombinant tnfcr (boehringer). tnfc~ was determined by specific radiometric assay (medgenix diagnostics), nitrate and nitrite were measured with a modification of the guess reaction ~. results: results are shown in the table. conclusions: during isolated limb pedusion with recombinant tnf~ very high levels of tnfcr were measured in arterial blood in 5 patients. they all showed signs of severe sepsis syndrome with shock from vasodilafion, probably due to leak of recombinant tnft~ from the peduslen circuit to the systemic circulation. tnfc~-induced vasodilation was not accompanied by a rise in serum no-metsbolites. our findings do not confirm the widely accepted theory, mainly based on animal experiments, that genera• of no is the key pathogenefic mechanism in septic vasodilafion 2, nor that tnfrt invariably induces forreafion of no. the precise mechanism of shock in these patients remains to be elucidated. references: 1. moshage h, kok b, huizenga jr, jansen plm nitrite and nitrate determinaiions in plasma: a critical evaluation. clin chem 1995: 41/6. 2. moncada s, higgs a. the l-argioine-nitrio oxide pathway. n engl j med 1993; 329: 2002 -2012 ec is a commonly used for prolonged, stable animal anesthesia. noting that the hypotension after iv lps was attenuated by ec, we hypothesized ec also protects against lps toxicity. sprague-dawley rats received ip saline (s), thiobutabarbita180 mg/kg (tb), or varied doses of ec, followed 2 hours later by bolus 30 mg/kg iv lps. 7-day survival is shown below: group: s tb ec(0.1gmikgi ec(0.sgm/kg) ec(i.2gm/kg) alive (n) t 0 0 3 9 ~ total (n) 10 s s 7 10 "signiflcant;y different from all other groups, p<0.0s 5/5 rats given lps followed 2 hours later by ec (1.2 gm/kg) also died. additional rats were treated with s (n=10) or 1 2 gm/kg ec (n=10) followed by 30 mg/kg lps, then sacrificed at 4 hours. blood glucose (bg, mg/dl),.hematocrit (hct), leukocyte count (wsc/mm~ platelet count (pltxl0~/mm3), bicarbonate (hco, mg/dl), gross bowel hemorrhage (bh, 0-4 scale) and lung myeioperoxidase activity (mpo, ~vmirvgm wet lung) are shown below ( we conclude that ec reduces the lethality and multiple organ toxit;~ty of lps. its diverse effects suggest asite of activity upstream from the cytokine cascade. these results are important for studies of lps which may use ec anesthesia and may have potential in the therapy of septic shock. [zo = 0 hz impedance (z; {dyn.sec.cm "5 }); zl = first harmonic z; zc = characteristic z; z1 ph. = t'trst harmonic phase angle {radians}; f, #, * at least p < 0.05 between fio2 0.4 and 0.12, fio2 0.4 and fio2 0. 4&no -0.46_+0.1 -0.26_+0.1 # -0.24+0.1 m -0.85+0.1 * -0.85+0.1 * -0.74+0.1 * -0.88_+0.1 * in hyperoxia, compared to dogs at the same q, minipigs had a higher ppa (26+1 rnmhg versus 16+1 mmhg; p < 0.01). hypoxia increased (ppa-ppao) at all levels of q by an average of 13 mmi-ig in minipigs and 2 mmhg in dogs. inhaled no inhibited hypoxia-induced (ppao-ppa)/q changes in both species. conclusions: we conclude 1 ~ that the minipig is an animal model of elevated pulmonary vascular resistance and impedance, and 2 ~ that hypoxia-induced alterations in pvz spectrum are due to changes of resistance in small arteries. objectives: 1) to determine the toxicity of ng-monomethyi-larginine (nma) administered by intravenous bolus to patients with refractory septic shock. 2) to investigate the biologic activity of nitric oxide synthase inhibitors in septic shock. methods: from august 1993 to january 1995, thirteen patients with vasopressor refractory septic shock received nma intravenously in escalating doses from 1 to 40 mg/kg. results: no hepatic, renal, gastrointestinal, or hematologic toxicity was observed at doses of nma as high as 40 mg/kg. significant biological activity was observed at all dose levels consisting of increased blood pressure (systolic blood pressure from 70.9 mm hg + 3.4 to 109.0 _+ 4.3 s.e.m., p=0.0004, systemic vascular resistance (430 + 57 to766 + 93 dyne.sec/ cm s, p=.002), and a decrease in vasopressor requirements. the magnitude and duration of these effect were dose dependent. decreased cardiac output (8.1 _+ 0.8 to 7.0 _+ 0.9 i/min p=.003) and increased pulmonary artery pressure (35.6 _+ 2.1 to 43.5 _+ 2.0 mm hg; p=.004) were also observed. no significant effects on heart rate, pulmonary capillary wedge pressure, or central venous pressure were observed. four of 13 patients survived for more than 28 days, 4 patients died of cancer complications (all 5 patients had maintained blood pressure for 24 h on nma) and 4 patients died of complication attributable to septic shock (mods, ards, dic, refractory hypotension), and 1 patient was unevaluable. conclusions: no adverse clinical effects have been observed in patients receiving bolus doses of nma as high as 40 mg/kg. the increased pulmonary artery pressures observed in septic shock patients is further augmented by nma and may limit the dose which can be administered by intravenous bolus. other schedules of drug dosing may attenuate this effect. glucose-insulin-potassium (gik) solutions have been shown to improve cardiac contractility and increase oxygen availability in experimental and clinical settings of septic shock. several mechanisms have been proposed to explain these effects including a direct improvemeut of the energy balance by glucose, a direct influence of insulin on cardiac performance or an increase in intravascular volume due to the hyperosmolarity of the solution. to explore the role of hyperosmolapity, we compared the effects of gik to those of a isoosmolar hypertonic saliue solutiou in endotoxin shock in dogs. methods : the study included 18 mongrel dogs (25• pentobarbitalanesthetized aud mechanically ventilated with air. thirty minutes after the intravenotls administration of 3 mg/kg of e. coli endotoxin, the dogs were randomized to receive a 2ml/kg infusion in 30 rain of a hypertonic (2895 mosm]l) solution iucludiug either a mixture of glucose 50 % with 750 u insulin and 2000 meq kcl/l (glk-group 1) or hydroxyethyl starch 7.5 % in naci 8.4 % (hes-group 2). in each dog, a 0.9 % saline infi~sion was continued to maintain the puhnonary arlery occluded pressure at baseline level. hemodynamic, blood gas aualysis and laboratory data were collecled at baseline and 30 miu, 60 rain, 120 rain, and 240 nunutes later.. results : eudotoxin administration was followed by a fall in mean arterial pressure (map) aud cardiac index (ci) and a rise in blood lactate levels. resuscitation with either gik or hes hypertoaic solutions resulted in similm increases in map, ci, oxygen delivery and left ventricular stroke index (table 1) . we conclude that during resuscitation from endotoxic shock the use of gik solutions is not superior to hypertouic hes solutions. the higher blood lactate levels observed in the dogs receiving gik can be attributed to the glucose metabolism. 1, 75 for group 2, 105 for group 3) were drawn and immediately analysed at 37~ using the abl500 radiometer for po2, pco2 and ph, and the osm3 radiometer for hbo2%, hbco% and methb%. psost (i.e. the ps0 at ph=7.40, pco2=40 mmhg and temperature at 37 ~ c) was calculated automatically by the instruments on mixed venous blood, as was the ps0"in vivo" (i.e. the ps0 at the patient's value of ph, pcoz and temperature), using siggaard-andersen's algorithm. the data were compared by the one-way anova test and by the t-test for paired and unpaired samples. results: the mean resulting values (in mmhg) with the statistical differences are shown in table i. in addition, the time series analysis shows the mean ps~st values as statistically below the psin vivo" in the septic patients while the opposite is shown for the cardiac patients. no differences in the time analysis are demonstrated for the second group. a possible clinical significance may be drawn from these different behaviours. objectives:toxemia degree and humoral immunity condition have been studied in 36 patients aged from 19 to 72 with progressive course of sepsis and polyorganic insufficience. methods: such toxemia and humoral immunity findings as lencositlcindex of toxication (lii), level of oligopeptides of the middle molecular mass registered at the wave length of 25nm(mmi) & 280nm (mm2), distribution index (id), immunoglobulins a,m,g, concentration of circulating immunocomplexes (cici & cic2) and also some clinical and biochemical findings on the 1,3,5 day after the operation serve as criteria for treatment effect. results: it was founded that in intensive therapy and detoxication, level of lii is successively decreased from 12.6~1.4 to 2.6+.5 on the 5-th day after the operation. true decrease of the level mm2 from .704~.09 to .402+.08 un & optimal density and increase of distribution index from .96 to 1.09 are argued. conclusions: in studlng the dynamics of the immunoglobulin's spectrum and the true increase of immunoglobulin g level from 8.4+.6g/i to i0.8+.7g/i on the 5-th day after the operation simultaneously with the decrease of cic2 from 806.9~60 to 624.7~54.8(p .05) were founded. some stages of the investigation true increase of lymphocytes from 9.0+2.6% to 15.0+1.8% was noted and it appeared to be a favourable prognosis finding for disease outcome. high correlation dependence between bacillus-and segmentonuclear neutrophils and immunoglobullns g & m (r=.5-.7 in p<.05) was discovered and it also showed positive dynamics of the course of the disease. a 34 year old male patient was admitted to the icu with severe paraquat poisoning. treatment consisted of gastic lavage and oral administration of fullers earth. because of very high plasma levels hemodialysis together with charcoal hemoperfusion was started within one hour after admission. this treatment was further continued by continuous veno-venous hemofiltration in order to remove the circulating paraquat and also circulating cytokines. nevertheless patient s condition worsened necessitating artificial. ventilation and hemodynamic support. patient died 24 hours after admission of acute multiple organ failure due to paraquat poisoning. serum levels of paraquat were determined by colorimetric method (table) . levels of interleukin 6 (il 6) and 8 (il 8), tumor necrosis factor (tnf-alpha), interleukin i receptor antagonist (il 1 ra) were determined both in plasma and ultrafiltrate ( q~!ectives : evaluate in critically ill patients the effects of tow-dose dopamine on gastric mucosal blood flow (gmbf) using laser-doppler flowmetry, a continuous non invasive method of assessing microcirculation. methods : 6 patients requiring both mechanical ventilation and pulmonary artery catheterization for multiple trauma (n=3), ards (n=2) and pancreatitis (n=l) were included. in each patient, the laser-doppler (ld) probe was inserted through a naso-gastric tube. the ld signal is proportional to the number of red blood cells moving in the measuring volume and the mean velocity of these cells. when the ld signal was satisfactory, an aspiration was created into a catheter which was fixed in parallel to the ld probe, to maintain the tip of the probe against the gastric wall at the site of measurement. data (systemic hemodynamic parameters and gmbf) were obtained at the end of a 30 rain resting period (baseline), then 30 min after dopamine (2 mcg/kg/min) infusion, and finally 30 rain after the end of dopamine infusion (recovery gmbf 185 _+ 23 (perfusion units) gmbf 0 ~a% vs baseline) * p < 0.05 vs "baseline" and "recovery". conclusions : 1) despite a slight increase in co (+13%), the dramatical increase in gmbf (+87%) with dopamine, strongly suggests a selective vasodilator effect of low-dose dopamine on gasaic mucosal perfusion. 2) laser-doppler flowmetry appears a promising method to assess gastric microcircalation in critically ill patients. increasing evidence suggests that the activation of inos is the final common pathway for vasodilation in human sepsis associated with endotoxic shock. activation of the cellular immune system induces the excessive release of the pteridines neopterin (n) and 7,8-dihydroneopterin (nh2) by human macrophages/monocytes. besides the well established diagnostic value of pteridines in several inflammatory diseases, it is speculated that these substances per se exhibit biochemical functions. thus we hypothesize that pteridines can modulate inos gene expression in vascular smooth muscle cells (vsmc) in vilro. cdtured rat aortic vsmc from female wistar kyoto rats were incubated with n (20 pm), nh2 (20 ilm), lipopolysaccharide (lps, 5 ~g/ml), and interferone-~/(ifn-~/, 100 u/ml) for 9 h, respectively, inos gene expression was measured by competitive reverse transcription polymerase chain reaction. the results are summarized in the table. the present study demonstxates a neopterin induced increase in inos mrna expression at the transcriptional level in vsmc. while coincuhation of cells with n + lps resulted in an additive effect on inos gene expression, n + ifn-7 seem to have a more than additive effect nh2 did not alter inos mrna synthesis, but it suppresses the lps as well as the ifn-yinduced augmentation of inos gene expression. we speculate that this pteridine-mediated modulation of inos gene expression is involved in the regulation of the vascular tone in endotoxic septic shock. the relationship of sepsis and coagulation abnormalities is well known, mainly in severe sepsis and septic shock. still farther, the extreme expression of hemostasis abnormalities (disseminated intravascular coagulation) in sepsis, has been extensively described. we studied the changes in several coagulation and fibrinolysis markers in septic patients, trying to correlate them with the evolution of the sepsis phenomenon, with an emphasis in its early stages, where therapeutic intervention might be more drastic. in 64 patients, 30 with sepsis, 22 with severe sepsis and 12 with septic shock, as well as in 14 healthy volunteers (control group) we measured : platelet (ptl), coagulation markers [fxii, fvii, fviii, fvw, fibrinogen (fibr) we conclude that all parts of the coagulation system are gradually changed during the evolution of sepsis phenomenon , even in the earliest stage of sepsis. the expression of an inducible nitric oxide (no) synthase (inos) plays a major role in the pathophysiology of septic shock (ss). inhibition of inos could therefore be of therapeutic value. however, such an inhibition has been shown to be detrimental, increasing tissue anoxia (and end-organ damage), possibly through the simultaneous blockade of constitutive nos (cnos). thus, selective inhibition of inos might be more suitable. we evaluated the effects of l-canavanine (can), a more potent inhibitor of inos than cnos, in an animal model of ss. method: in 30 anesthetized rats, catheters were placed in the femoral vein and artery. 23 rats were given an iv bolus of lipopolysaccharide (lps, 5 mg/kg), at baseline (to). after 1 h (t1), rats received at random an infusion of either can (20 mg/kg/h; can group, n=l 1) or an equivalent volume of 0.9% naci (2cc/kg/h; nac1 group, n=12), giyen over 4 h (t1-t5). a third group (sham group, n=7) received 0.9% nac1 in place of lps, and then was treated like the nac1 group. mean blood pressure (mbp), blood lactate and nitrates (no3) were measured each h. glucose, creatinine and asat were also measured in 18 rats (n=6 in each group). the can 98_+9* 304+52"t 3.2+1.1"~ 3.6+_1.4"t 138+46" 48+10" *p<0.05 can vs naci ?p<0.05 vs sham can suppressed the hypotension, reduced the hypoglycemia and hyperlactatemia, and attenuated the biological signs of renal and hepatic dysfunction induced by endotoxemia. these effects were associated with a lesser elevation of blood no3, confirming a partial inhibition of inos. conclusion: l-canavanine attenuates the hemodynamic and metabolic consequences of endotoxemia in the rat. these effects may be related to a partial inhibition of inos. they contrast with the deleterious effects described with non selective inhibitors of nos. l-canavanine could become a new tool for the treatment of septic shock. rocalc1tonin :marker of sepsis, ii~flammaiiur% t~ boifi .cheval*~ jf.timsit*, m.assicot**, b.misset*,/.carlet*, c.bohuon** saint joseph heap, paris**biochemistry institut g roussy, villejuif, ce bi~)l~i~ttectives_: high serum levels of procalcitoaln (proct) have been shown to be ~ss-ocinted with bacterial infection. however, few data exist about the ability of proct to differenciate septic shock and shock from other origin in which an activation of intlmmamtory mediators has been also demonstrated. methods: thirteen patients with bacterial septic shock (ss), 11 patients with non septic shock (nss), 14 patients with bacterial infection without shock (1nf) and 8 icu patients without shock and without infection (control) were compared for proct levels at dayl, 2, 3, 7, 14 . patients were classified blindly and independently fi'om proct results. twelve patients were excluded because any classification was impossible due to mixed pathology. proct was measured with ebemoluminescenee (brahms diagnostica-berlin). results: dayl, proct levels are significantly different between the four groups. dayl proct levels are correlated with saps (p=0.0002), infection (3.7+_3 vs 61_+25,p=0.0007), shock (13_+10 vs 60+.-27,p=0 .002), death at day28 (12_+7 vs 96_+44,p=0.003). when shock and infection are introduced in multifactor &nov& only infection remains correlated with day 1 proct levels (19=0.003) in patients with shock, dayl proct levels are correlated with saps, infection and death at day28, but not with arterial lactate levels (p=0.37), white blood calls (p=0.2) or fever (p=0.1). proct levels remain higher i~i septic shock patients at day 1, 2 and 3( figure) . i c0edpsion: procalcitonin levels in the first three days of shock are differen[" between septic and non septic shock patients. in patients with diseases known to induce acute an inflammatory process, procaldtonin seems to be a marker o~ infection. obiectives-to evaluate the effect of endotoxic shock on the distribution of blood flow between the mucosal and the muscular layer of the intestinal wall. methods: in 10 fasted pigs, mean aortic pressure (map, mm hg), cardiac output (co, ml/min-kg),superior mesenteric artery flow (q sma, ml/min.kg), and phi, where measured before (control) and after i.v. endotoxin (10 gg/kg). the blood flow to the mucosal and the muscular layer was measured in 3 regions (proximal jejunum (pj), mid-small intestine (mi) and terminal ileum (ti)) by colored microspheres, using 8 adjacent samples in each region. the muscular layer was separated from the mucosa by blunt dissection, and the flow determined independently in each layer. results: endotoxin with fluid resuscitation induced the expected decrease in map (95.8_+3.0 vs 53.9-+2.4, p<0.01), and phi (7.29!-_0.02 vs 7.13_+0.01, p<0.01), with a constant co (133_+4 vs 144_+8, p=0.28) and qst, aa (21.5_+0.8 vs 22.8_+0.9, p=0.09). the results of regional pertusion are presented in the table. (flow in ml/rain 9 100g of tissue; mean _+ sem ; * p<0.001 vs control by two-way anova) conclusions-these data indicate that the mucosal flow increased during septic shock. they suggest that a decrease in phi may be due to hypoper~usion of the muscular layer or to metabolic alterations within the mucosa, despite a 50% increase in flow. acute increase in wbc count (from a mean of lo.oo01mm a to 42 o00/mm~), between the 3rd and the 7th day of therapy. there was a decline of the wbc count to an average of about 25.0001mm a after decreasing the daily dose of the medication to 200 mcg there was no increase in tile absolute number of the eosinophils during the whole course of the medication. there was a slight decrease in the c3 complement between 0.26 to 0.29 g/i. normal values 0.5 to 0.9 g/i there was no change in c4 values. conclusions : an early increase in wbc count was observed (3rd day) without subsequent increase in the number of immature types from bone marrow, probably due to the mobilization of wbc from the periphery and this increase was dose dependent. there was a slight decrease in c3 fraction of complement, probably due to the consumption of this fraction in the process of opsonization. no adverse effects of the medication were observed, during the treatment with the above dose. these data sugest that cm csf may be a useful complement to tile main antimlcrobial treat,nent ~ of septic [cu patients. objectives: as part of a large multicentric, placebo-controlled, randomized clinical trial investigating the effects of interleukin-1 receptor antagonist (ii-lra) in the treatment of severe sepsis and septic shock, this substudy evaluated in dem.il the acute hemodynamic effects of ii-lra in patients who were invasively monitored. methods: in a total of 71 evaluable patients in whom vasoactive support was little altered, hemodynamic measurements were performed at baseline (twice), and i hour, 2 h, 3 h, 4 h, 8 h, and 12 h after the administration of 1 mg/kg (n=20) or 2 mg/kg (n=22) of i1-1 ra or the corresponding placebo (n =29). 58/71 patients (82 %) were treated with adrenergie agents and 66/71 (93 %) with mechanical ventilation. data were analyzed by a kruskal-wallis test. results: during the study, there was no significant difference with time or between groups in arterial pressure, cardiac filling pressures, cardiac index or left ventricular stroke work (figure). burmester, "~ man8 and h. djonlagic medical university (internal medicine, "cardiology, *'microbiology) and "**southern city hospital, lfibeck, germany obiectives: evaluation of the incidence of bacteremia and sepsis in patients with nontyphoidal salmonella (s.) infections, specification of risk factors, need of icu treatment, clinical course, and mortality in the group of the patients who developed septic complications. methods: data of all patients with microbiologically proven s. infections hospitalized in the medical university of lobeck and in the southern city hospital of l0beck from 1992 to 1994. results: within the observation period s. was isolated from the stool cultures of 748 patients. in 13 patients (g m, 4 f, median age 52 yrs) s. could be detected in blood cultures (9 s. enteritidis, 4 s. typhimurium). in addition, in 10 of these patients s. was also isolated from other specimens (urine, liquor, and tissue fluids derived from abscess punctures). in all 13 patients with positive blood cultures the clinical course of s, infection was complicated: ? patients developed mof (acute renal failure, ards, hemodynamic instability, dic) and required icu treatment for at least 4 up to 62 days, 4 of the 13 patients died. the predisposing disorders in the patients with s. bacteremia were (n=): aids (3), immunosuppressive drugs (2), chronic alcoholism (2), malignancies (2), none (4). septic complications in patients with nontyphoidal s, infections are relatively rare (in this study < 2 % of all hospitalized patients with microbiologically proven salmonellosis) but severe (mortality of approx. 30 %). patients at risk for a complicated clinical course are predominantly those with predisposing disorders but occasionally also patients without evidence for an underlying disease. age (yr) 65 + 9 58 + 14 death (n) 22 18 duration of shock (h) 27 + 37 54 + 44 noradrenaline (rag/h) 5,9 _+ 6 2 + 5 temperature (~ 38,2 + 2 38,1 + 1 pvr (dynxsecxcm -5) 1195 + 408 572 + 418 co (ljmin) 4,2 _+ 1,6 9,9 + 3,6 lactate (mmol/l) 7 + 5,9 9,2 + 9 interleukin-6 (pg/ml) 829 _+ 798 1331 + 888 interleukin-1 (pg/ml) 9,3 _+ 9,4 9,8 + 7,3 tnf-alpha (pg/ml) 23,5 + 31,7 166 + 209 neopterin (nmol/l) 43,2 + 43,7 218 + 193 crp (rag/l) 131 _+ 97 233 +_ 138 pro-ct (ng/ml) 22,6 + 50,5 77,9 + 160 there was no positive correlation between serum lactate levels, degree of shock, hypoxemia and pro-ct positivity. pts with septic shock of bacterial origin entirely developed hyperprocalcitoninemia, whereas pts with cardiogenic shock, who expired within 24 h did not. however, in late cardiogenic shock (>24h) all pts developed fever of unknown origin and consecutive hyperprocalcitoninemia. these data suggest bacterial inflammation and/or mucosal translocation of bacterial products in pts with prolonged cardiogenic shock. the use of a loading dose of quinine (16.7 mg/kg base in 4 h) is recommended in previously untreated patients (pts) with sfm, particularly in multi-drug resistance areas. this protocol is difficult to validate, since the viability of microorganisms is not assessed routinely in parasitology laboratories. objectives: to examine the evolution of parasite viability during the early phase of therapy of sfm. methods: from 02/1993 to 12/94, pts with sfm (who 1990) treated with iv quinine for less than 6 h were included prospectively. blood samples were collected at o, 6, 12, 18, 24, 36 and 48 h viability was assessed by culturing parasitized red blood cells in the presence of 3h-hypoxanthine, and radioactivity was determined at 42 h by scintillation counting. viability was expressed as the percentage of radioactivity compared to the initial sample. plasma quinine was determined by liquid chromatography. tile ratio plasma quinine (pmol/1)xlo00/icso for quinine (nmo]/]) was called the parasiticida/ index. results:5 pts were included, 42• saps1 18.6-+4.9. the initial parasitemia was 2t.4+7.2%. complications of malaria were coma (4 pts), shock (3 pts), renal failure (2 pts) and acute lung injury (2 pts). all strains were sensitive to quinine (icso 174--67 nmol/1). in 2 pts who were not given a loading dose, parasite viability increased by 63 and 157%, with concomitantly low quinine levels (22 and 19 #mow] at 12 h); 1 pt died. in 3 pts that received a loading dose (serum quinine at 12 h = 33.1--2.0 ~mol/]) a marked decrease of parasite viability (by 73+_10% at 12 h) was shown. viability was inversely correlated with plasma quinine (r=.677, p-.o11) and parasiticidal index (r=.678, p-.o1). conclusions: even with fully sensitive strains, the use of a loading dose of quinine seems warranted in severe falciparum malaria in order to reach rapidly adequate plasma quinine ]evels, necessary to inhibit significantly parasite viability. l nkka, e ruokonell j takala. critical care research program, department of intensive care, kuopio univ hospital, finland objective: to determine the incidence of positive blood cultures, their microbial subgroups and to evaluate the outcome of icu patients with different bacleremias. material and methods: we analysed all positive blood cultures in 3077 consecutive admission to a university hospital icu in 1992 -93 and the icu and hospital survival of the bacteremia patients. during these years 73 patients had 176 positive blood cultures that were considered as clinically relevant, excluding colonizations or contanfinations. results: patients with positive blood cultures had an icu survival of 65.8 % (vs. 92,7 % in all icu patients) and six month survival of 50.7 % (vs. 85.8 % in all icu patients). the most common bacteria were enterobacteriaceae (27,3 %), staphylococcus aureus ( 18,8 %) , coagulase negative staphylococci ( 14.2 %), pseudomonas ( 14.8 %) and slieptococci (9.1%). obiectives: to evaluate prognostic factors and mortality in consecutive patients (pts) with hiv infection and septic shock. methods: from 03-1991 to 12-1993 , records of consecutivepts with septic shock (crit care med 1992, 20: 864-74) admitted to the icu were reviewed retrospectively. results: among 76 pts with septic shock admitted during the study period, 28 had hiv infection-26 of whom had aids-(gr. i) and 46 were hiv-negative (gr. ill. ten gr. ii pts (21%) were irnmunosuppressed because of neoplastic or immune dlsease. mechanica] ventilation was required in 89% gr. i and 83% gr. ii pts in 8 gr . i pts (29%) a multivariate analysis demonstrated that hiv infection and sap5 i were independently predictive of death in pts with septic shock. ~onclusions: evidence of increased mortality, number of organ failures and higher severity scores (saps i does not take into account immunosuppression) is demonstrated in hi v-positive pts, infection with hiv appears to be an independent prognostic factor in pts with septic shock. the frequency of opportunistic infections (often responsible for delayed diagnosis and treatment) may contribute to the poor prognosis in this population. obiectives: to determine interleukin (il)-i 0 levels in plasma of patients with sepsis and septic shock. to analyze the relationship between plasma il-10 and the proinflammatory mediators, tumor necrosis factor-aifa (tnf) and il-6, the underlying severity of the disease and the evolution of patients with sepsis. methods: we studied 94 critically ill patients (63 men, 31 women; 18-86 years old) in three diferents groups. group i: 23 patients without evidence of infection, group i1:34 patients with sepsis and 37 with septic shock (group iii). we measured plasma il-lo, tnf and il-6 levels in the first 12 hours of diagnosis. severity of illness was estimated with the acute physiology and chronic health evaluation (apache ii) scoring sytem. results: plasma levels of il-10 were higher in group iii (median, 51 pg/ml; range, 5-6000 pg/ml) than in group ii (median, 10 pg/ml; range, 2-970 pg/ml; p <.001) and group i (median, 5 pg/ml; range, 2-133 pg/ml; p <.001). median il-10 concentrations did not differ among patients who survived (median 7 pg/ml; range, 2-6000 pg/ml) and those who died during the overall follow-up period (28 days) (median, 15; range, 5-5400 pg/ml); but patients who died in short-term (< 24 hours) with catecholamine-refractory hypotension showed the highest concentrations of il-io (median, 1200 pg/ml; range, 51-5400 pg/ml). in patients with bacteriemia (34%), levels of il-10 were higher (median, 51 pg/ml; range, 2-6000 pg/ml) than in those with negative blood culture (median, 8,5 pg/ml; range 2-5.400 pg/ml; p< .001). there was a good correlation between plasma il-io concentration and levels of tnf (r= .59; p < .001) and il-6 (r= .60; p < .001). the correlation between levels of il-10 and the apache ii score was significant only in the septic shock group (r= 0.48; p <.005). conclusions: in septic shock, il-io and proinflammatory citokines are released in high concentrations. the significant correlation observed in patients with septic shock between il-10 levels and apache ii, short-term death and bacteriemia can possibly be explained by the massive inflammatory response in septic shock with fulminant course. intensive care department -calmette hospital -59037 lille -france. in septic shock, inadequate splanchnic blood flow may play a prominent role in the pathogenesis of multiple organ failure. measurement of gastric phi has been propose to evaluate tissue oxygenation in splanchnic organs. objectives: to compare gastric phi values with hepatic icg clearance, an index of liver blood flow and function ; to determine if one of these two methods could be proposed to assess the entire splanctmic peffusion in septic shock. methods : 6 patients (age : 65• years ; saps ii : 46• were prospectively investigated (septic shock : bone criteria). following parameters were collected during 12 hours : systemic hemodynamic parameters (swan ganz catheter 93a434h -ref1 computer -baxter lab.), calculated systemic oxygen transport (do2), oxygen consumption (vo2) by indirect calorimetry (deltatrac datex lab.), gastric intramucosal pco 2 (pco2ss) and phi (trip -ngs catheter -tonometrics lab.) and plasma disappearance rate of icg (pdr dye) (femoral artery fiberoptic/thermistor catheter 2024, cold z021 computer -pulsian medizintechnik, germany). correlations were performed using a linear regression. elevated in all days with the highest value in second and third days of treatment. nonsurvivors had higher values of these parameters than survivors but differences did not reach statistical significance. another trend of changes were observed in selectin p (gmp-140) concentration. in all patients concentrations measured were elevated but in survivors after not significant decrease this parameter in second day another one had simmilar values. in patients who died we noted significant decrease in third day (p < 0.05) whereafter prominent increase, significant after seventh day, in comparison to third day value and value in survivors group. icam-1 concentrations in all patients reached high levels and in nonsurvivors after four day of treatment significant increase in comparison to survivors we found. conclusions: multiple trauma complicated with sepsis induce rapid elevation of concentrations of il-6, il-8 and increased expressior of adhession molecules (selectin e, p, icam-1) measure of icam-1 and selectin p concentration determine lung injury severity and prognosis as to health and life. (clp) .pathophysiology of cip is unclear, but changes in regional bloodflow may be a ~ignificant factor. nerve blood flow (nbf)is reduced in rat models of hemorrhagic shock (g),but no information is available in sepsis. we studied the comparative effect of acute endotoxemic shock {etx)& h on perfusion of rat sciatic nerve. methods: 20 male sprague-dawley rats were anesthetized with pentobarbital (ip), instrumented with a tracheostomy, carotid arterial & venous catheters and mechanically ventilated (fi02=0.5). the left sciatic nerve was surgically exposed. monitored variables included: a) mean arterial pressure (map,mmhg) ,b) nbf (ml/1o0 g/min) by laser doppler flow meter,c) nerve internal arterial diameter (id ~ m) by video image shearing and splitting method. after stable baseline measurements were obtained, acute hypotension was induced by randomly assigning the rats to etx (0.25 b6, difco) in saline at 1 mg/kg or h. both interventions produced 50% reduction in map within 3 min., which recovered to baseline values spontaneously in etx group, & by reinfusion of heparinized withdrawn blood in m. data were analyzed by linear regression, two-way repeated measures analysis of variance followed by bonferroni-t method. experimental stages were:(1)baseline, (2) mid-point of map reduction; (3) nadir of hypotension, (4)midpoint of map recovery, & (5) after stable recovery of map. both etx & h induced shock result in similar reduction in nbf consistent with lack of autoregulation in peripheral nerve vessels independent of etiology. since cip is primarily associated with sepsis, it is not likely that acute reduction in nbf alone causes cip. direct & indirect neurotoxic effects of mediators of sepsis need to be evaluated. .':_.~::::o4o:oc4., objectives : evaluate the relationship between il-10, a cytokine which inhibits tnf, production and protects mice from endotoxin toxicity, and the other proinflammatory cylokines, tnf~, il 6 and ils in severe sepsis and septic shock. methods : twenty-eight icu patients (19 m, 9 f, mean age 54 + 17 y) were studied as soon as they developped a severe sepsis (n = 16) or a septic shock episode (n= 12) as defined by a conference consensus in 1992 (1). tnf~, il 6, il s and il-10 plasma levels were measured by immuno-radiometrie assays from medgenix (fleurus, belgium). lc mean and range. results : the comparisons between cytokine levels in severe sepsis versus septic shock were made using the logarithm of the value in order to normalize the distribution of data, and student test. il-10 plasma levels were higher in patients with septic shock than in patients in severe sepsis. there was a significant correlation (p < 0.05) between il-10 and tnf a (r= 0.6), il-10 and il~ (r = 0.73) and il-10 and il s (r = 0.65) as well as between il-10 and apache n score (r= 0.52). patients who died (n = 13) had il-10 levels higher than patients who survived but this difference was not statistically significant (114 pg/ml vs 34.5 pg/ml; p>0.05). conclusions : during severe sepsis and sepsis shock, il-10 seems at least to follow the same evolution (increase in plasmatic level) with the severity of sepsis as the other cytokines. reference : (1) crit care med 1992;20:864-74. objectives: to evaluate the effects of steroids on hemodynamics and mortality in septic patients with konwn levels of cortisol concentration. methods: retrospectively we analyzed data ofpatients with documented septic shock who received steroids after assessment of adrenal function. in all patients hemodynamic parameters as well as the necessary vasoactive medication were assessed, before and 24 hours after corticosteroid medication. immediately before administration of corticosteroids adrenal function was evaluated with cortisol levels before and after synthetic corticotropin (0.250 mg). finally we studied mortality. we defined a positive respons on corticosteroids as an elevation of map of at least 30 mmhg and/or a decrease in the necessary vasoactive medication of at least 50% within 24 hours. adrenal insufficiency was defined as a cortisol level after stimulation of less than 500 nmol/l. results: 15 of 23 patients were found to respond to steroid medication, 8 did not. mean cortisol levels before and after corticotropin were 534 • 366 and 737 • 396 nmol/l in the responder group (rg) and 583 • and 907 • 301 nmol/l in the non responder group (nrg). in the rg 9 out of 15 (60%) were found to have an adrenal insufficiency, in the nrg 3 out of 8 (37%). in the rg 2-weeks mortality was 6.7% (l out of 15), the overall mortality 33% (5 out of 15). mortality in the nrg was 62% (5 out of 8) (p <0.01) and 75% (6 out of 8) (p <0.005) respectively. conclusions: in patients in septic shock there is a beneficial effect of steroids in case of adrenal insufficiency, but also in a subgroup with normal adrenal f{unction. obiectives: intercellular adhesion is a critical step in the accumulation of leukocytes. postischemic cardiac lymph has the capacity to stimulate icam-i. in the coronary microcirculation neutrophils can be trapped and in many cases obstruct capillaries, previously we found that troponin t (s-tnt) a marker for myocardial iechemia, was increased in septic patients. the aim of the study was to follow slcam-1 and s-tnt levels continuously starting at the beginning of sepsis. methods: 19 patients were ingluded in this institutionally approved study after relatives had given their informed consent. all patients were included within 24 hrs following the beginning of sepsis. blood was drawn every 4 hrs in the first ;~4 hrs, after 48 hrs, followed once per day for 7 days. s-tnt, icam-1, elam (elisa's, boehringer mannheim inc, r&d systems ltd.) arterial and venous blood gases were determined, an ecg and a complete hemedynamir measurement including cardiac output were obtained. all patients received adequate volume and catecholamine therapy (norepinephrine, dopamine, dobutamine; median (range) 0.6 (0.0-1.66), 3.12 (2.4-12), 6.29 (0.0-15.3) pg/kg/min, respectively). statistical analysis: wileoxon signed rank-sum test. .45 (0.06-7.6) 0.0003 13 patients had s-tnt levels >0.2pg/l. 11 of these died, whereas only 2 of 6 patients died with s-tnt values <0.2 pg/l (p=0.0296). all patients that died had elevated sjcam-1 levels (232 ilg/l:cut-off ) whereas in the survivor group only 50% had elevated icam-1 levels (p=0,043). conclusions: increased slcam-1 and s-tnt levels were found during early sepsis in the majority of patients, a high sicam-1 and s-tnt value was associated with a higher mortality. the research of the noninvasive haemodynamic monitoring accelerated recently all over the world. the aim of our study was to test whether the changes of the haemodynamk parameters measured by impedance cardiography (icg) were corresponded to clinical changes in septic patients. investigations were performed on 20 critically ill postoperative septic patients (their multiple organ failure score was 8-9/with icg monitor. in 9 cases the investigation~ were performed in septic shock. the measured parameters were: heart rate (hr), mean arterial pressure (map), cardiac output (co), peripherial resistance (svr),preejection period (pep), and ventricular ejection time (vet). these parameters were measured during 3-72 hours in every 30 minutes, depending on the patients cl~tnical condition. results: at the septic patients the hr and the co ]~reased. in septic shock the co was significantly higher the svr lower than in the septic group. in the hr there was no difference between the two groups. in septic shock noradrenalin influenced more effectively the measured parameters than dobutamin. conclusion: the trend of the measured icg parameters correlated with the clinical changes of septic patient's state. the noninvasive haemodynamic monitoring by impedance cardiography helps the planning and leading the adequate intensive therapy of these critically ill septic patients. to evaluate the development of sirs, sepsis and septic shock in hospitalized patients with fever, a prospective study was performed on 300 patients using previously defined criteria. methods: 300 normotensive patients with fever (temperature >38.0 ~ axillary), admitted to the department of internal medicine were evaluated for the existence of sirs during the first three days of the study and sepsis at inclusion. during a follow-up period of 7 days the patients were daily evaluated for the development of sepsis or septic shock. results: most patients (69%) had or developed sirs within the first three days, 16 patients (5%) did not. sepsis was present in 25% at inclusion. in patients with sirs, 76% did not progress to sepsis or septic shock, 24% progressed to sepsis (mean interval 2.55 • 1.97 days), and 1 patient (<1%) directly progressed from sirs to septic shock. in patients with sepsis, 17% progressed to septic shock (mean interval 2.08 • 1.56 days). sepsis was preceded by sirs in 40%. septic shock was preceded by sepsis in 92% and by sirs in 8%. conclusions: 94% of patients with fever in an internal medicine department develop sirs, or sepsis. furthermore, progression from sirs to sepsis or septic shock is poorly predicted by fever or sirs. nevertheless, all patients with septic shock were preceded bysirs or sepsis. taken together, this may indicate a severity hierarchy of the syndromes. however, fever, sirs and sepsis are relatively poor indicators of development of septic shock. this supports further research on additional predictors of septic shock. b. m.manuylov, v.b.skobelsky (moscow) in recent years sodium hypochlorite (sh) has been successfully used to eliminate pyo-septic complications. moreover, the mechanism of the sh effect on the immune system has not been sufficiently studied. the aim of the present investigation was to study the mechanism of sh effect in inflammatory pulmonary diseases. 20 patients with double pneumonia were subjected to the evaluation. sh in the concentration of 600 mg/l in the volume of 400-800 m1/24 hours was administered by drop infusion into the central vein. to evaluate one of the defence systems the leukocytes activity by the chemoluminescence technique was studied. in all the patients baseline secondary immunodeficiency which was indicated by the decrease in the luminescence level was established. even 1 hour after the sh administration the leukocytes activation exp-ressed by the enhancement of their chemoluminescence 0.5-5 times was observed. this supports the available findings that accumulation and liberation of the oxygen active forms (ol'oh, '02, h202) are accompanied by the increased phagocytosis, i,e. the signs of "the oxydation explosion" testify to the favourable sh effect on the course of inflammation processes. the use of sh permitted to decrease the percentage of lethality in double pneumonia by 15% in the intensive care unit over the year. at the same time, excessive activation of free radical oxygen may be a damaging factor. therefore, precise individual control over the choice of concentration, dosage and the preparation administration rate is required. prospective, double-blind, placebo-controlled, trial of atiii substitution in sepsis r. a. balk objective: pilot study to evaluate the efficacy and safety of atiii substimtion therapy in patients with sepsis. efficacy assessed using change in mortality or organ failure/dysfunction. adult patients meeting a definition of sepsis and cared for in a tertiary care academic medical center in chicago were identified and prospectively randomied to receive either atiii (kybernin p) or placebo in a double-blind treatment protocol. all other therapy and patient management were under the direction of the patient's attending physician. all patient's were followed for 28 days and the organ dysfunction/failure were scored using published scoring systems (jordan et al crit. care med. 1987 , goris et al arch. surg. 1985 , kuaus et al ann. surg. 1985 colldusions:wha~ we met the shomaeker objectiv% the mortality and the pro~os[s were i~ttc*. those criteria were obtained with file tradititmal t~ctor likr doht~mme, hut c.~vh ~,as ca1 in~aertam measure. they ac~s smxergically in the optimizatic~l of the fell vmtrictdar work index, tad fimdameatally cavh seox~s to have an impo.aat role in the better respiratory ev-altmtioa, leaving yet the possibility to coltrol the flui& r althou~l eomproved it's not aec~pt~xl file importmlce h* the diminution, of the sepsis modiat~lrs llke fnt and il-6 with h~wmotiltrafi(al, stopphlg the evolution to nmltiorganic failure mid de~easethe mortality. with ours clhlicals results, we could saythat cavii in multiol~atlie disfut~oa septic patieats, se~r~ to be an c4xilna] supoa or troatmeat maesure. of anaesthesia and intensive therapy, medical university of prcs, p~csf hungary. objectives: since some biological effects of bacterial endotoxin require an interaction between the lps molecule and a serum factor(s), we hypothesized that lps-induced no production and cgmp accumulation in vascular smooth muscle cells (vsmc), a mechanism ~thought to underlie cardiovascular collapse associated with septic shock, is modulated by serum factor(s). methods: cultured vsmc from rat aorta were challenged with e. coli lps for 4-6 hours either in the presence or absence of fetal calf serum (fbs), and no production was monitored by radioimmunoassay determination of cgmp content of hci extracts. results: in the absence of serum, 1o00 ng/ml lps was required to increase cgmp levels, whereas the presence of 10 % fbs shifted the lps concentration curve i00 times to the left. similarly to fbs, human serum also potentiated lps-induced cgmp accumulation. in contrast to lps, serum had no effect on cgmp accumulation elicited by sodium nitroprusside, a no releasing agent, suggesting that the sensitivity of vsmc to generate cgmp in response to exogenous no is not modulated by serum. heat inactivation (>80 ~ 30 min) but not removal of small molecules (<10,000 d) from the serum by dialysis, reduced the potentiation of cgmp accumulation by serum. time course studied indicated that serum is required within the first 120 min of lps exposure to increase cgmp levels. to investigate whether the effect of serum is specific for lps, we treated the cells with increasing concentration of interleukin 1-~ (il-i). 10% fbs shifted the il-iinduced cgmp responses five times to the left. conclusions: our study suggests that lower concentrations of e. cell lps and il-i require a heat labile macromolecule in the serum in order to elicit no production. this factor is present in the human serum and it may play a potentially important role during no synthesis induction in vsmc. objective: to evaluate the factors of acquisition and the outcome of methicillin resistant staphylococcus aureus (mrsa) bacteremia in an intensive care unit (icu). methods: all patients in which bacterermia due to staphylococcus aureus developed >72 hours following admission to our icu, during a 10 year period ( january 83 through january 94) were reviewed. 30 patients (pts) were included, mean age 68,1y (sd 13,1), saps 2 35,9 (sd 11,1), mac cabe (1 and 2) 53%, mortality directly due to sepsis 30%. 16 pts had mrsa bacteremia and 14 methicillin susceptible staph. aureus (mssa) . both groups were compared using the chi square (with correction of yates), fisher's exact, student's t or wilcoxon test. results: there was no statistically significant difference between mrssa and mssa regarding at age (71,8+ 4,8 vs 63,9+ 1,8) , saps2 (33,6+6,6 vs 38,2+14,1), use of vancomycin (94% vs 71%), mechanical ventilation (94% vs 100%), number of days (d) before the drawing of the first positive blood culture (median 20 d, range 7-150 d vs median 30 d, range 7-120 d). more mrsa than mssa pts had previous use of nonsteroidal anti-inflammatory drugs (nsaid) (25 % vs 0% p<0,001), central venous catheter infection due to staph.aureus (62,5% vs 14% p<0,01), but previous use of antibiotics was not significantly different (37,5% vs 21%). the outcome of the bacteremic pts was not statistically different: saps 2 at the first day of bacteremia (33,6+_.7,2 vs 40,7+14,5), severe sepsis and septic shock (31% vs 28%), persistence of the bacteremia (43% vs 78%), mortality directly due to bacteremia (25% vs 45%). conclusion: previous use of nsaid, infection of venous central catheter are more frequently associated with mrsa bacteremia. thus, similar to others studies (hershow infect control hosp epidemio11992; 13:587-593) , these results do not indicate that mrsa is associated with increased virulence. objectives: to closer definition of mosf formation mechanismes in nosocomial sepsis (ns) the complex clinicobiochemical, microbiological, immunological, functional exaroination of 62 cases with ns had been done. methods: examination of cellular and humoral immunity, nonspecific immunologic reactivity, systemic and hepatic circulation, microbiological examination of blood,electro-and echocardiography, sonography and computer tomography of chest and abdomen organs were obligatory. autopsy findings of 5 dead cases had been analized. results: in 45 cases (72,6%) opportunistic pathogen microscopic flora ( staphylococcus anreus,staphylococcus epidermidis, staphylococcus saprophyticus) had been found out in blood inoculations. in 36 cases (58%) side by side with destructive process in lungs the bacterial endo-and myocarditis with blood circulation failure had been determined.in 21 cases (34%) simultanious lesion of three organs (heart,lungs,liver) had been found. morphologic examinations of 5 dead cases (8%) internal revealed involvement of them in mosf-syndrome.hyperplasia of adenohypophysis;sclerosis of adrenal glands cortical layer;perivascular brain oedema,paralysis of brain capillaries and plasmorrhagia, cerebral thrombosis and cerebral abscess,necrobiosis of epithelium tubules of the kidney,pletora of hepar, fatty and granular degeneration of hepatocytes had been found.atrophy of white pulp and hyperplasia of red pulp, supress of lymphoid tissue, plethora and formation of infarctious had been found in spleen. mentioned changes in spleen were indispensable in ns. conclusion: in ns spleen can not secure it functions to support and appropriate detoxication potencial of organism,elimination of microbes,toxines,antoallergenes. insolvency of immunological link of antimicrobic defence is the starting mechanism of mosf developmentin ns. %neviere, jl. chagnon, b. vallet, d. mathieu, n lebleu, f. wattel ] ept of intensive care, hop calmette, lille, france ~everal studies have described tiypoperfusion of intestine during sepsis. 4owever, it is unknow whether the mesenteric blood flow is associated with nucosal hypoperfusion. additionally, the effects of resuscitation on the ntestinal microcirculation remain controversial. 3bjectives : to describe the effects of endotoxin in a porcine model during ~hock and resuscitation. ~ethods : ten pigs (30 kg) were anesthetized and instrumented for "neasurement of cardiovascular variables. gastric and gut oxygenation vere assessed by intra-mucosal ph and microvascular laser doppler lowmetry. after baseline data collection, a 30 minute intravenous infusion )f escherichia colt (serotype 34h4113, sigma, st. louis, mo) was begun ~t a rate of 150 pg/kg. an infusion of either saline at 1.7 ml/kg/min (group ; n=5) or saline and dobutamine at a rate of 5 pg/kg/min (group ii; n=5) vas begun 30 mn after the end of the endotoxin infusion. tesults : to td0 1120 t180 ~ fl0w fluid ioadin,q alone sfyras d, k perreas, e douzinas, k spanou, m pitaridis and c roussos critical care dpt, evangelismos hosp., athens univ, school of medicine. obiectives: much controversy exists concerning the beneficial effects of cvvh on sepsis. we studied the effects of cvvh application on septic patients with reference to the following parameters: i) survival rate ii) cytokines' removal and iii) timing of cwh onset. methods: 20 patients with sepsis (criteria according to accp/sccm, 1992) underwent cvvh as soon as they developed renal failure or dysfunction (urinary output<250 ml/8h, cr>2.5 mg/dl and bun>60 mgd'dl ). specimens were collected: blood samples before cvvh and therafter both blood and ultrafiltrate (uf) samples on 24, 48 and 72 hours. cytokines tnfa, i1-1 and ii-6 were measured by the immunoassay method in all specimens (uf and plasma -p) and sieving coefficient ([uf]/[p]) and 24 h solute mass transfer of tnf and i1-6 were calculated (v24 h x [uf] ). the apache ii score before cvvh onset, the duration of icu stay and the timing of cwh application related to the sepsis onset in days (ta) were recorded.with respect the mortality two groups were formed, i.e. group a (survivors) and group b (non-survivors) . the morbidity period in days of those septic patients who died in the past year and were not subjected to cwh (group c) was compared to that of group b. results: group a included 8 pts and group b 12 pts with mean+sd age (65 _+19 vs 64_+9, ns) and apache scores(24_+2 vs 24-+2.2, ns). the mean ta-+ sd was 3.6+2 vs 10-+6, p<0.05. the mean_+se morbidity period of group b vs group c was 20_+4 vs 8_+0.8 p<0.05 . the mean values of cytokines are presented in the following figures. the sieving coefficient for tnf was 0.2 and for i1-6 was 0.25. the solute mass tranfer was 6-fold the actual plasma content at a given time. . o conclusions: i) early application of cvvh seems to favourably affect the outcome of septic patients, ii) cytokine plasma levels do not decrease although cytokine removal is substantial, iii) it seems that cwh application in sepsis of any stage helps to buy time for further treatment. the most commonly monitored variables in shock stages idclude : arterial pressure, heart rate, central venous pressure, pulmonary artery wedge pressure and cardiac index. with vigorous therapy it is possible to bring these values back into the normal range in both survivors and nonsurvivors. therapeutic goal in septic shock stages is to maximize the values of cardiac index, 02 delivery (do2) and 02 consumption (c02). objectives: the main purpose of this article is to determine the relationship betwee~ 02 delivery an 02 consumption as a sign of hypoxia. fifteen patitents with septic shock were treated with intention to maximize the value of ci,d02 and v02. we compared the levels of these parameters between the survivors and nonsurvivors and found no significant differences after 24 hours. high levels of do2 and v02 may not guarantee against tissue hypoxia in early stage of septic shock. zjar~iic, dj janjic, lj. gvozdenovic, a.komareevic. t.petrovic, &marjanovic, institute of surgery, novi sad, yugoslavia objectives: evaluation and mutual comparison of clinical signs, laboratory data and microbiological monitoring in the patients with burn sepsis. method: retrospective analysis of the recorded data of all burn patients treated in our department between january 1989 and december 1994. specially attentions were given to data considering wound infection, positive haemocultures, positive urinocultures and characteristics of septic state. results: out of 372 patient there were 324 (87,09~) adults and 48 (12,9(3~) children. almost two thirds of the patients (238 -63,97~) were males. the predominantly cause (68,759~) of children's burns was scalding b~y hot liquids and flame burns ~63,97~) in adult patients. the most frequdntly species isolated from surface swat~ were pseudomonas aeruginosa (64"1796 in adult patients) and staphyloccocus epidermidis (78,57% in children). in only five patients (1,349~ 1 the haenmcultures were positive -pseudomonas aeruginosa was isolated in three and staphyloccocus aureus in two patients. urine infection was diagnosed in 6,72% of all patients. the treatment protocol included use of imipenem and polyvalent pseudomonas vaccine again~ pseudomonas aeruginosa and vancomycin and aminoglycosides against staphylococcus aureus. total mortality rate in this group of burned patients was 6,98~, but the mortality rate caused of sepsis was low (i 34%) . conclusions: early detection of any signs of wound infection and symptoms of septic state is a foundation for prevention and treatment of burn sepsis. the burn sepsis could be reliable detected by continuously monitoring the patient's status and by systematic microbacteriological monitoring of the burned patients. hyperdynamic vasoplegic septic shock p.f. laterre, p. goffette, j. roeseler, j.p, fauville, a. poncelet, p. lonneux, m.s. l~eynaert. dept. of intensive care, st. luc univ. hospital, brussels, belgium. splanchnic ischemia is described as a common feature of septic shock and could determine the development of msof. therapy such as noradrenaline (na) aiming at improving blood pressure is expected to worsen splanchnic ischemia by its vasoconstrictive effect and subsequent reduction in intestinal blood flow. ob[ective: evaluate the effect of na on splanchnic blood flow. material and method : in a patient admitted for variceal bleeding, ards and sepsis with positive blood culture, a fiberoptie catheter was positionned in the portal vein after recanalisation of its portosystemic stent shunt. blood pressure (bp-mmhg) , ci, svr, do 2 (vigilance ~ baxter), v02 (indirect colorimetry), arterial, mixed venous and portal vein blood gases, phi were determined before (to) and during (t1) na infusion (0, 1 to 0, 19 hcg/kg/min.) . changes in splanchnic flow were assessed by changes in portal oxygen saturation (sp02) and arterio-portal oxygen saturation gradient (sao, -spoe laterre, ,lp. pedgrim, th. dugernier, v. delrue, ph. hantson, p. mahieu, m.s. reynaert. dept. of intensive care, st. luc univ. hospital, brussels, belgium. aim of the study : prospective determination of plasma levels of in patients with ss and their correlation with the type of microorganism and outcome. material and methods : in 19 patients (pts) with ss and severe sepsis, plasma levels of tnfti, ill-b, il6 and il8 were determined every 8 hours for 3 days and on day 7 after fulfilling the criteria of ss and severe sepsis. results : in 9 pts, sepsis was caused by a gram (-) microorganism, in 6 pts by a gram (+) and in 4 pts no microorganism was identified. there were 12 survivors (63%) (s) and 7 non-survivors (37%) (ns) . cytokines profiles and levels were not different between gram (+) and gram (-) sepsis. ill-b levels were seldom elevated whatever the group studied. tnfot and il-6 were significantly higher in ns than in s ( objective: to evaluate the effects on the nitric oxide synthase inhibitor l-n~ hcl (546c88) on myocardial performance in human septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map<70mmhg) or the requirement for a noradrenaline (na) infusion >_ 0.i ]tg/kg/min with a map _< 90mmhg. cardiovascular support was limited to na _+ dobutamine (db), 546c88 was administered for up to 8 h at a fixed dose-rate of either 1, 2.5, 5, 10 or 20 mg/kg/h iv. during 546c88 infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above 70 mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = 0); at i h from the start of treatment (t = 1); and at the end of treatment (t = 8) with 546c88. conclusions: 546c88 can restore systemic vascular tone in patients with septic shock enabling na therapy to be reduced and/or removed. the ci tends to fall whilst lv performance is sustained over time. 546c88 is a novel vasoacfive agent for the treatment of septic shock, which is undergoing further clinical evaluation. laterre, f. thys, e. danse, j.p. pelgrim, e. florence, z roeseler, m.s. r eynaert. dept, of intensive care, st. luc univ, hospital, brussels, belgium. therapy aiming at improving blood pressure and cardiac index in septic shock (ss) might have deleterious effects on regional blood flow. objectives : compare the influence of volume loading (vl), dobutamine (dobu) and noradrenaline (na) on sushepatic oxygen saturation (shoe) and svoe-sho, gradient in treated ss. material and methods : in patients with ss, ci (thermodilution) , doe, svo,. sho,, svoe-sho e gradient and lactate (l) were determined before (to) and after (t1); vl, dobu and na. results: in 5 patients with treated ss, 16 tests were performed (vl n=8; dobu n=4; na n=4 method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map<70 mmhg) or the requirement for a noradrenaline (na) infusion ~> 0.1 ~g/kg/min with a map _< 90mmhg. cardiovascular support was limited to na + dobutamine (db), 546c88 was administered for up to 8 h at a fixed dose-rate of either i, 2.5, 5, 10 or 20 mg/kg/h iv. during 546c88 infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above 70 mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = 0); at 1 h from the start of treatment (t = 1); and at the end of treatment (t -8) with 546c88. conclusions: 546c88 is a novel vasoactive agent that can sustain map in patients with septic shock, enabling na support to he reduced and/or removed. there is a tendency for the ci to fall during treatment, which may be reflex in response to the increase in systemic vascular tone. 546c88 is a promising new therapy for septic shock, which will now be evaluated in a randomised, placebo-controlled safety and efficacy study. k. guntupalli objective: to evaluate the acute effects of the nitric oxide synthase inhibitor l-n~ hc1 (546c88) on selected indices of organ function in patients with septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < 70 mmhg) or the requirement for a noradrenaline (na) infusion --> 0.1 [xg/kg/ min with a map _< 90 mmirlg. cardiovascular support was limited to na + dobutamine. 546c88 was given for up to 8 h at a fixed dose-rate of either 1, 2.5, 5,10 or 20 mg/kg/h iv. during 546c88 infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above 70 mmhg and the cardiac index (ci) as clinically appropriate. indices of organ function were assessed at baseline (t = 0); at the end of treatment (t = 8); and 12 h after treatment (t = 20) with 546c88. results. -median values (* assessment made at 8 h or when 546c88 discontinued). conclusions: there was no appareut dose-dependent adverse effect on these indices of organ function either during or after exposure to 546c88. the plmelet count tended to fall whilst creadnine appeared to increase over time in all dose cohorts. this novel and promising therapy for septic shock will now be evaluated in a randomised, placebo-controlled safety and efficacy sludy. pharmacokinetics of 546c88 in patients with septic shock preliminary results z. hussein, b. jordan, c. fook-sheung, k. guntupalli objective: to evaluate the pharmacokinetics of the nitric oxide synthase inhibitor l-n~ hc1 (546cg8) given by continuous infusion for 8 h in patients with septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < 70 mmhg) or the requirement for a noradrenaline (na) infusion --> 0.1 ~tg/kg/min with a map _< 90 mmhg. cardiovascular support was limited to na • dobutamine. 546c88 was administered for up to 8 h at a fixed dose-rate of either 1, 2.5, 5,10 or 20 mg/kg/h iv. plasma was collected from each patient over a 24 h period and analysed for 546c88. pharmacokinetic parameters were derived from plasma concentration-time profiles using non-compartmental pharmacokinetic analysis. results: the (cm~ -maximum plasma concentration; auc -area under curve; cl -plasma clearance; v,, s -steady state volume of distribution; t'/2 -plasma elimination halflife). conclusion: the pharmacokinetics of 546c88 in patients with septic shock are dose-independent at infusion rates up to 2.5 mg/kg/h. at higher rates, clearance of 546c88 decreases without any marked change in volume of distribution. 546c88 metabolism may be partially saturable at dose-rates above 2.5 mg/kg/h. obiectives: investigate the effect of the no synthase inhibitor, l-nt-methylarginine hc1 (546c88) on the haemodynamics and survival rate in a conscious mouse model of endotoxin shock. methods: female cd-1 mice (25-35 g) were instrumented under gaseous anaesthesia (isofluorane, 2%) and connected to a swivel tether system for continuous monitoring of blood pressure and drug administration. results: after 24 h recovery, endotoxin administration (e. col• 026:b6, 6-12.5 mgkg -1 i.v.) elevated the plasma concentration of nitrite/nitrate (nox) and caused a progressive fall in mean arterial pressure (map) from 101 + 5 to 59 + 4 mmhg (n=5, p<0.05) at 12 h, with a survival rate at 24 h, 48 h and 72 h of 80%, 40% and 20% respectively. 546c88 administered as a 24 h continuous infusion (3 mgkg-th -t i.v., n=5), 4 h after endotoxin, inhibited the elevation of plasma nox and attenuated the fall in map from 105 + 2 to 70 + 3 mmhg (n=5) at 12 h, with an improved survival rate at 24 h, 48 h and 72 h of 100%, 100% and 60% respectively. conclusions: this study suggests that overproduction of no is involved in the hypotension and mortality characteristic of septic shock. inhibition of no synthase using 546c88 represents a novel and promising treatment for septic shock. cultures of e.coli (19,5%) and candida(8, 3%) were olso received from autopsy material of children;p.aeruginosa,unspored anaerobes,proteus sp.,s.aureus,b.pneumonia were found in the few cases. in adults the spectrum of bacterioflora was mo~ re limited speaking about the number of species and cultures. in generalized forms of bacterial pyo-septic pathology a wider specific spectrum of causative agents was revealed usua fly with associations. e.coli and k.pneumonia played the leading role in children as well as in adults. in general,k.pneumonia (23,7%cultures) and common e.coli(18,6%)prevailed according to the date of microbiological investigations of authopsy material in pyo-septfc pathology in 1994. objectives: .in spite of all clinical exertion sepsis is still the reason for high clinica! lethality. this study is characterizing the group of patients which survived a septi~ shock. methods: during a period of 12 months all surgical patients on icu were registrated prospectively, more than 270 parameters for each of them were documented'daily in a paradox file. results (see table 1): 20 of 286 patients fulfilled the criterion of a septic shock (r. bone, 1991 ) , 11 of them died at the 2 lth day, while the surviving group of patients stayed almost 51 days at icu. obiectives: to compare the effects of 6 and 10% pentastarch solutions to a human albumin solution on oxygen delivery (do2) in septic patients. methods: this stud}, included 41 septic patients with fever (t > 38~ tachycardia flqr > 90/rain), tachypnea (rr > 20/min) or mechanical ventilation, leukocytosis (wbc>12000/mm 3) or leukopcnla (wbc<400()/mm 3) and a clinical source of infection, who required a fluid challenge. in each patient the pulmonary arterial occlusion pressure (paop) was < 12 mmhg. patients were randomized to receive 400 ml of 4% albunun (n:i4), hydroxyethyl starch (hes -mw200/d.s. 0.5) 6% (n:14) or t0% (n=i3); 33 patients were also treated with adrenergic agents. results cardiac index (c1) increased significantly only in 10% lies (table) hemoglobin (hb) decreased significantly at 40 min in the same group. there was not significant change in oxygen delivery ( do2 ). baseline ci alb 3.7::1.3 (l'min/m 2) hes 6% 3.9=0.9 hes 10% 3. polyneuropathy of the critically ill (pci ) is a well recognized complication, acquired in the course of severe illness. we undertook a prospective study, to estimate the severity, extension and time of onset of pci in a selected group of 25 patient with established septic shock ( bone's criteria ). all patients received inotropic circulatory support and were mechanically ventilated. none received relaxants or aminoglycosides. pci was diagnose<by clinical investigation and by emg, and repeated weekly ( axonal degeneration ). after 3 days ( day 1 = onset of septic shock ) in 68% of the patients pci was demonstrable. after 14 days 80% of the patients had pci. there was a strong correlation between pci and other mof and between pci and encephalopathy ( eeg ). we conclude that i. pci is a common complication after septic shock. 2. pci develops early in the course of illness ( 85% after 5 days ). 3. pci can be considered as a part of mof, suggesting a common pathogenesis. objectives: we addressed two questions: 1) can serial measurements of vapco2 and avph also reflect the onset of tissue hypoxia during endotoxemia? 2) are whole body changes in vapco2 and avph associated with parallel changes in regional circulation? methods: in 12 anesthetized, mechanically ventilated dogs exhaled gases were sampled for determination of oxygen consumption (vo2). a catheter was positioned into the pericardial cavity to induce cardiac tamponade. ultrasonic flow probes were placed around superior mesenteric, left renal and left femoral arteries, and the corresponding veins were cannulated. group l served as control (n=6), group 2 (n--6) received 2 mg/kg of endotoxin. thirty min later, tamponade was induced to gradually reduce do2. results: systemic do2crit was higher (12.1_+2.2 vs 7.9+2.6 ml/kg.min, p <0.05) and critical oxygen extraction ratio (o2ererit) was lower (45.1+_9.7 vs 74.1+_9.1%, p<0.05) in the endotoxie than in the control group. meslenteric and femoral do2crit were higher in the endotoxic than in the control group (8.2_+2.5 vs 4.1_+0.5 mlll00 g tissue.min and 8.3_+2.3 vs 4.6_+0.9 ml/min, respectively, both p<0.05). regional o2ercrit were lower in we endotoxic than in the control group (mesenteric: 37.1_+15.4 vs 71.i_+7.4 %, renal: 30.7+ 24.6 vs 53.9-+28.7 % and femorah 48.1-+9.2 vs 75.3_+6.9 %, all p<0.05). vapco2 and avph followed a similar pattern in both groups, increasing slightly before do2crit was reached, but dramatically wh,en do2 fell below do2crit. in the presence like in the absence of endotoxin, systemic and regional do2crit calculated from vo2, from vapco2, or from avph were similar; do2crit was also significantly higher in the mesenteric and the femoral beds of the endotoxic than the control dogs. systemic and regional do2crit could be calculated from the blood lactate levels only in the control group. conclusions: during an acute reduction in blood flow i) vapco2 and avph can reflect hypoxic threshold in the presence like in the absence of endotoxemia. 2) alterations in organ oxygen extraction capabilities induced by endotoxin can be reflected by regional changes in vapco2 and avph. objectives: enhanced nitric oxide (no) release has been incriminated in the vasodilation and myocardial depression characterizing septic shock, but the administration of no blockers has yielded equivocal results. the present study explored the systemic and regional effects of a no donor linsidomine (sin-i) during endotoxic shock. methods: 14 anesthetized dogs received endotoxin (2 mg/kg iv), followed 30 min later by a saline infusion to keep cardiac filling pressures constant. oxygen uptake (vo2) was derived from the expired gas analysis. regional blood flow was measured by an ultrasonic flowmeter (transonic). results: the control endotoxie group (n=7) maintained low arterial pressure and systemic vascular resistance. the 7 dogs receiving a continuous infusion of 1,2,4 ~ug/kg.min of sin-1 starting 30 min following initial fluid resuscitation (each dose for 1 h), had a higher cardiac index (0.23-+0.03 vs 0.16_+0.08 l/min.kg, p<0.05) and lower systemic and pulmonary vascular resistance than the control group (1006-+170 vs 1460-+445 and 114-+38 vs 244-+84 dyne.sec/cm5, respectively, both p<0.05). arterial pressure and pulmonary artery pressure were not influenced. sin-1 significantly increased the mesenteric blood flow from 63-+17 to 148+49 % of baseline levels and renal blood flow from 67-+25 to 96-+24 % (both p<0.05) but did not influence femoral blood flow. the relative blood flow was increased in the mesenteric bed (at all doses), and reduced in the femoral bed (at highest dose). systemic oxygen delivery, vo2, oxygen extraction and blood lactate levels had similar course in the two groups. conclusions: in fluid-resuscitated endotoxic shock dogs, the no donor sin-1 increased cardiac index without deleterious effects on arterial pressure, and increased splanchnic blood fl0w. objectives: sepsis syndrome is associated with the release of a variety of inflammatory mediators, such as interleukins, tumor necrosis factor and platelet-activating factor (paf). administration of paf, a naturally occuring phospolipid, to laboratory animals has been demonstrated to resullt in pathological changes that closely resemble those found in sepsis. in addition, paf antagonists of various origins have been demonstrated to attenuate cardiovascular collaps and to protect against lethality in endotoxemia. in the present study the efficacy and safety of the paf-antagonist tcv-30g was investigated in sepsis syndrome patients. in addition, the putative inflammatory parameters tumor necrosis factor (tnf), interleukin il) -6 il-8, and soluble (s) e-selectin were measured in both tcv-309 and placebo treated patients. metho.~a randomized, double-biind, multi-center study was undertaken to compare the safety and efficacy of intravenously administered tcv 309 versus placebo (takeda chemical industries ltd., osaka, japan). tcv-309 was used as add-on to conventional therapy in the treatment of patients with severe sepsis and septic shock (1.0 mg/kg body weight intravenously over 2 hrs, twice a day for one week). day 28 mortality was registered, as well as vital signs, laboratory parameters, hemodynamic parameters, apache ii score and mof score. plasma samples for the determination of inflammatory parameters were collected twice a day for one week. the study was approved by each institutional review board and written informed consent was obtained for each patient. results: a total of 29 patients were included in the study. one patient had to be excluded as a result of protocol violation of the remaining 28 patients 12 patients were randomised to receive tcv-309 and 16 to be treated with placebo. there were no significant differences between the treatment group with respect to age. gender, and apache i~ score on admission. however, admission levels of il-6 and il-8, considered to reflect severity of disease, tended to be higher in tcv treated patients: 24.59. vs 2.37 ng/ml for il-6 (p.06) and 0.34 vs 0.04 ng/ml for il-8 (p.07) in tcv-309 and placebo treated patients, respectively. a remarkable difference in survival, however not significant, was observed at day 7 i.e. the end of tcv treatment (mortality: 2 out of 12 in the tcv group and 7 out of 16 patients in the placebo group). the inc;dence ui ~-,~verse events, 'n both treatment groups were comparable. plasma il-6 and plasma il-8 levels tended to decrease more rapidly in tcv treated patients compared to controls (p=.17 and p=.09, for il-6 and /l-8 respectively) conclusions: tcv-309 is safe as add-on treatment in sepsis syndrome patients. the data presented indicate that tcv-309 may enhance survival of sepsis syndrome which will be assessed in a phase ill study. objective: oxidation of lipids by free oxygen radicals playes an important role in sepsis. an important source of oxygen radicals is the respiratory burst of phagocytic leukocytes, especially~ polymorphonuclear leukocytes. clinically the hallmark of sepsis is the capillary leak, which is mediated in part by oxyradicals. the adult respiratory distress syndrom (ands} represents the most studied capillary leak phenomenon in sepsis. methods: in ii patients (df,~m, aged 20-81y.) with verified septicemia had been included in this study. beginning from day 1 oxidisable lipidautoantibodies (club), ~opterin and crp were determined in the patients serum and compared against the apache ii score during the days 1-7. 4 patients, 2f and 2m died @u= ring their stay at the icu, 3 of them in less than 48h~urs: after admission. at the begin surviving patients showed olab values between 75-119%, non-survivors between 68-104%, com= pared to neopterin values of 38-75 m~ol/l in survivors and 20-250 ~mol/l in non-survivors. apache ii score didn t show any difference. during the following days olab sbowed an in= crease in survivors, while in non-survivors olab stayed equal or showed a decrease. no difference between both groups was found in neopterin, crp and apache ii score. as result we conclude, tbat lipidperoxidation playes an important role during septicemia. olab as a marker of this process are predictive for the outcome of patients. obiectives : to evaluate the influence of cardiopulmonary bypass ongastrointestinal transit. meth~xls; gastrocoecal transit time (gtt) in 10 consecutive children on day 1 after uncomplicated open heart surgery was compared to gtt in 10 children after closed heart thoracic surgery and to healthy controls. gtt was measured by hydrogen breath testing, using lactulose (o,4g/kg in a 10% aequous solution) as nonabsorbable marker. exspiratory hydrogen content was measured using a electrochemical system (stimotron). patients in both groups had continuous infusion of morphine and midazolam and were comparable in respect to catecholamine dose. children with a history of toddler's diarrhoea and children with elevated central venous pressure were excluded. results: gastrocoecal transit was delayed in all patients after cpb, no transit was achieved within 480 minutes in 7/10. after closed heart surgery gtt was delayed 138(+-80) min vs. 56(+-20) compared to controls, but transit was achieved in all aptients within 285 rain. mean dopamine dose was 2,7mcg/kgmin after cpb and 4,8meg after closed heart surgery.. conclusions: after uncomplicated cpb gastmcoecal transit is extremely delayed. the degree of delay is not explained neither by the use of analgetic and sedative drugs neither by the use of catecholamines. factors directly related to cpb (e.g. low flow perfusion, hypothe,mia) may be responsible. these findings are of limited clinical relevance in ancomplicated cases. in critically ill patients delayed enteral feeding may contribute to infectious complications. studies of therapentic measures to accelerate gastrointestinal transit therefore are warranted. neonatal ecmo -czech experience. nekvasil r., penkova z, vasek l., plier p., bobula a novotna e., pavlicek v., hnilicka m. nicu and ecmo center brno and dept. pediatric surge13', children's hospital brno, czech republic. introduction. in spite of the fact that neonatal ecmo has been gradually considered a standard method of solving uncontrollable respiratol t failure in newborns in most countries of the western europe, the introduction of this sophisticated method in countries of the former communist block meets a lot of problem. material methods. in the course of two years, only 16 newborns, b.w. 2000 -4300 g, were reffered to neonatal ecmo because of uncontrollable respiratow failure. at the admission the average values of oxygenation index were 51 +12, and aado2 79,6 -+7,6 kpa. results. 4 newborns (25%) died shortly alter" admission or during transport without possibity to further is 5 newborns (31,3%) were treated using high-frequency ventilation (hfo or hfjv), one for conspicuous airway resistance paradoxically with low-frequency ippb. all ventilated newborns survived. ecmo was applied in 6 newborns (37,9%) and 3 of them survived the mortality rate of group mentioned was 44%. conclusion. the fact that in the czech republic (10 mil. population with natality rate of about 100.000 newborns/year) only small part of patients was reffered to neonatal ecmo has a number of causes. the most important are: unacquaintance of prognostic and indication criteria at forwarding workplaces and the aversion of neonatologist to all new.therapeutic interventions. last but not least, a negative role is also played by current change of health service systems when, under the influence of health-service insurance companies a newborn in serious condition given only a standard intensive care in primary and secondary nicu is literally "the golden calf", and therefore these workplaces either do not send him or send him too iate ~o an ecmo center. stndy aim: 8urfactant, which is being applied into the lungs for treatment of respiratory distress syndrome, can reach the circulatory blood system by absorption. it refinances local fimctiun of macrophages and lymphocytes and increases the accumulation of nentrophils into the hmgs this eunld influence the local acute phase reactiml in the hmgs after surfactant application. "ilia aim of the stud), was to investigate the influence of oatural and artificial surfactaat onto the acute phase reaction and the blood elottiog system. material :and methods: a) measurement of tnf-a, 11-6, ltb 4 and thromboxane b 2release in whole blood: each of 1.5 ml hepariaised blood of healthy adult volunteers was iucubated lbr 2 hears with eillier ill ug/ml lps, 1 mg/ml b&,ine smfactant (alveulhct/themae), 1 rag/nil artificial surfactant (exosurf/wellcome), 10 og lps + i mg/ml bovine surfactant or i0 ug lps + i mghal artificial sorfactant, tleparinised bhx-,d without additions was used for controls. '111e concentration of e)lokines and eicosanoids ".van measured semiquantilatively by eijsa or eia (dimmva/hamburg) after ceatrifagalion, b) measurement of platelet aggregation: each of 1.5 ml haparinised hltxxl of healthy adult vohmteers was inculmled lbr 2 hours with eilher 10 aghnl i,ps, i mg/ml bovine surfactant (alveafact/thomae), i mg/ml artificial snrlhctant (exosurf/wellcotne), it} ug lps + i mghnl bovine snrfactant or 10 ug lps + i mg/ml artificial surfaetant, tlepminised blood withont additions was nsed for controls. platelet aggregation in whole blood was measured by impedance in the aggregometer (g~nstaedt). c) measurement of the dotting activity: citrated bleed of healthy adult w~lunlccrs was mixed with difl'ereul couccalrations of imvine or artificial snrfactant fad then prolhrombiu tiaras and imrlial thrmnlx~plaslia filues were ineasnrcd. for measurement of the prokoagulatioo acfivily the reagent in the qnick test was replaced by surfaclant. results: dependmlt on the dose both surfactants stimulated the release of 11-6 but not or tnf-a io wlmle blood. "illere was no suppression of the lps-indaeed eytokine-release. both surfaclants did not directly influence the extrinsic blood coagulation system, while both activated the inniasie activity dependant on the dose. platelat aggregatiun was slightly stimulated by bovine snrfactunt and strongly inhibited by by artificial surthctant. in comhinafion with 1.1~ both factors showed no difft.'renca to lps ahme.'fhe reltu.tse of eiconasnid~." was stimolated more by artificial surfactant then by bovine surfilctant with regard to the cyehmxygelmse (thromboxsne b2) and the lipooxygenase (lencotrieae b4) pathways. conclusion: we cooclude: 1) i:lovine as well as artificial surfaelant stimulate the cellular inmnn]e response. 2) bovine as well as artificial surfnctant activate the intrinsic ctmgnlation cascade. 3) bovine surlilctant stimulates platelet aggregation, artificial sar-fijctant inhibits platelet aggregation.. introduction: although the use of modem noninvasive methods like measurement of tope2, tcpco 2 or san 2 leeds to an carly infonnation about pathologic alterations of the gas exdmage of ventilated patients, these methods cannot replace invasive bloodgas analysis. a new noninvasive method to monitor the capillary gas exchange is the reflection spectrophotometry. to record reflection spectra with a ldgh sensitivity on the surface of tissue we eonstnmted a special mierolightgnide photospectronmter (empito). this spectrometer enables the investigation of local helerogenities of llbo 2 and 1 lb content, capillary flow rate and oxygen uptake rate. aim of the study: 1 ) to compare the sousitivity and specifity ofthe empilo with other non-invasive method~ of monitoring the gas exchange in neonatal intensive care patients. 2) to investigate the possibility of obtainiug infonnations about the local 02nptake and the venous as well as arterial 02 satnration. patients and methods:we investigaled 16 non-selected patients of oar pediatric intensive care unit. for coulinnons monitoring we conslracted a special sappert to fix file end nf the microlightguide+ during the investigation tope2, tcpco 2 and san 2 were measured by standard methods. "fo investigate the local tisslm oxygen uptake we recorded 1000 spectres within 2 minutes by moving the lightgnide over an described area of the skin of the patient. 'lhe reselting i(gx) l ibo 2 vahms were sorted by a software package. on the assumption that the low values correspond to the tibo 2 value of the veaons ends of the capillaries and the high values to those of the arterial ends we 2) it is possible to gel infonnations about the local o2-uptake and arterial 02 saturation ia the peripheral tissue by photoslg'ctroscopy. 3) in combination with noninvasive pl i-measorement it seems possible to reduce withdrawal of blood for invasivn bhmd gas analysis. objectives:improve ventilation,tissue oxygenation and acid base balance among extremly low birth weightand premature infants. methods: fourteen cases were s~udied,their gestational age ranged from(27-32)ws.continnous positive air way pressure was applied to six cases at peep level from (3-6)cm h2o through nasal pronge,(group i),the other 8 cases were managed as routine,(group ii).blood gases, tcpo2,tcco2,resp.rate,depth and pattern were monitored for assessment of tissue oxygenation and ventilation, results: our rasults showed that early application of cpap improve ventilation among (83.3%)of cases,while (16.7%)of cases need imv.the cases of group ii need imv among (75%)of the studied cases during the second or the third day of life. conclusions:-this preliminary study showed the import-............ ance of early application of cpap before the onset of respiratory distress syndrom to improve spontaneous ve ventilation,tissue oxygenation and to gecrease the risk of intubation. comparative assessment of pediatric intensive care; a national multicenfre siudy. reinoud j.b.j. gemkc, gouke j. bonsel 2, the dutch picasso (pediatric intensive care assessment of outcome) study group. twilbelmina children's hospital and utrecht university, utrecht 2department of clinical epidemiology, academic medical center, : amsterdam, the netherlands the performance of all (10) pediatric intensive care units (icus) in the netherlands (6 tertairy and 4 non-tertiary) was analyzed in an open prospective muiticenter study. effectiveness of care was defined as the ratio of observed to prism-score derived expected mortality 0aenee, standardized mortality rate). efficiency was determined by 2 objective criteria (mortality risk > 1% or administration of at least 1 icu-dependent therapy)'. 1063 consecutive admissions aged < 18 years old were included. overall, observed and expected mortality were in good agreement (p > 0.5). between hospitals, crude mortality showed wide variations (mean 7.1%, range 1-10%). however, in each center, observed and expected mortality were similar (mean ratio 0.99, range 0.8-1.5). in tertiary care centres, severity of illness corrected mortality in high-risk patients was less than in non-tertiary care centres; paradoxically, in low-risk patients the opposite was found. probably the large proportion of low-risk tertiary care patients suffering from severe, incurable chronic disease, explains the higher mortality in this group. this indicates that simultaneous assessment of circumstances of dying and of long term morbidity in similar future studies is imperative. the average proportion of efficient icu days was 72%, however large variations between units were found (range: 22-95 %). in conclusion differences in mortality rates among pediatric icus were explained by differences in severity of illness. high efficiency rates in combination with adequate effectiveness, found in several centres suggest that admission and discharge decisions might be improved by a better selection of high risk patients requiring icu-dependent therapies, especially in less efficient centres. objectives: previously published studies showed that serum lactate levels correlated with outcome of severe ill adult, 'we hypothesized that critically ill newborns are often incurred hypopeffusion manifested by elevated lactate levels. these initial blood lactate levels should be related to nicu outcome. design: prospective study with ethical comfnittee approval. setting: the 14-bed neonatal intensive care unit of a university hospital material and method: a total of 209 consecutive outbem newborns admitted to nlod from 01,10.1991 to 31.,19.1992 were enrolled to the study. babies who died or were discharged from the unit within 48 hours of treatment were excluded from the study, mean birth weight was 2040g (+/-820r), mean gestatational age was 35 weeks (+/-3.5 wks), mean age at the admission was 50 h (+/-110hi. multiple (~_2j organ system failure occurred jn 38.3% of babies at the admission./~tertal lactates were measure/at the admission, among 22 -26 hour and 46 -50 hour of n[c'lj therapy. outcome was defined as a mortality and length of nicu stay. results" survival rate was 68.4%, mean length of nicu stay for survivors was 17.8 days (+/-15.1 day). we found high lactate levels at the admission in 82.8% babies (~9.2% with levels above 5.0 retool/i). the mean arterial lactate concentrations for nonsurvivors were signiftcahtly higher than for survivors durin~ consecutive da~ as follows: objectives: the purpose of our research was to analyze the frequency of bronchial asthma (b.a.) exacerbations in pregnant women and health status of infants. methods: the research was based on the epidemiological investigation and prolonged observation of 23 pregnant women with b.a. during the gestation period. remission of b.a. before the pregnancy in excess of 5 years was recorded in 9 patients (39.1%), 6 patients (26.1%) reported a 2-3 year remission and 8 patients (34.8%) had a remission lasting less than 12 months before they became pregnant. results: seven patients (30.4%) developed medium attacks in the second half of pregnancy, four patients (17.4%) experienced light attacks of b.a. asthma attacks were most frequently caused by acute respiratory diseases and stress factors. in two cases with grave manifestation of b.a., the pregnancy ended in abortion within the first 16-18 weeks due to the frequent and heavy choking attacks. to fight b.a. attacks, five patients used 132adrenomimetics (salbutamol, becotid) in sprays, six women were administered theophyllinum and salbutamol in the form of tablets during 1-2 weeks. a significant portion of pregnant women with b.a. (78%) exhibited frequent complications during pregnancy (toxemia, late gestosis, threat of miscarriage). our findings prove that babies born from women with b.a. of domestic and pollen origin had a low body weight (2800-2500 gr), functional immaturity and chronic antenatal and intranatal hypoxia twice as often as the infants born from healthy women without allergic background. conclusions: preventive treatment of women with b.a. prior to pregnancy is required to maintain a stable remission of the disease, which is a key to having healthy children delivered by mothers suffering from b.a. introduction. intracerebral hemorrhage (ich) is a common event in human prematudty, affecting about 25% of newborns weighing below 1500 g who are born before 32 weeks of gestation, however, little is known about the pathogenesis of ich with exception of the prematurity of the brain itself, (birth) trauma, and asphyxia. the postischemic production of oxygen free radicals (ofr) dudng reoxygenation as a cause of brain damage has been demonstrated in 9 animal research. since almost all preventive antioxidant activity of plasma is associated with ceruloplasmin and transferdn we investigated the association of such iron-oxidizing resp. iron-binding proteins and ich. we could demonstrate significantly reduced levels of both, iron-oxidizing and iron-binding proteins, in premature asphyxiated newboms pdor to development of ich. an increase of suparoxide after hypoxia in the presence of iron ions facilitates the formation ofthe highly reactive hydroxyl radicals. our data support the theory that ich may be caused by ofr, which can damage any sensitive tissue including growing endothelial cells. the estimation of transferrin-saturation and measurement of ceruleplesmin levels might help to identify an infant at dsk before the onset of ich. with the new medos | hia-vad | cardiac assist system the missing tool in the armamentarium of cardiac surgeons is available in two pediatric sizes: i0-ml and 25-ml pump volume. the right sided pumps are 10 % smaller for biventricular use. between february 1994 and may 1995 we implanted this assist system in 6 children. the indications and demographics are indicated in the following table (left ventricular assist device-lvad, right vad-rvad univentricular vad-uvad, post cardiotomy cardiac failure-pcf, dilated cardiomyopathy-cmr bland white garland syndrome-bwg, tetralogy of fallot-tof, hypoplastic left heart syndrome-hlhs). objectives: evaluate tile effeci'of inhaled nitric oxide (no) as puhnona] t vasodilating agent ill tile posloperalivc period after correclion of congenital heart defects in 3 infant. patient n.l: 4 kg, 7 lnonlhs, down syndrome undenvcnl rep~fir of atrioventricular septal defect (avsd). after surgery the puhnonary arlcry pressure (pap) slowly rose to tile syslemic dcspilc tnaximal eonvcnlional fllerapy (fentanyl 4 mcg/kg/h, hypocapnia of 27 mmhg and metabolic alcalinization). no was delivered into tile inspiratory branch of!be breathing circuit at 10 ppm, and the gas aoalyser for no and no2 (polylron dmger) were situated at the espiratory branch, a rapid dccrcasc of pap io i/3 of systemic was obtained with a dramalic improvement. no was continued at 5 ppm for six days and the baby was exlnbated if! days after surgery and discharged from the icu 5 days after. patient n.2 : 4.5 kg, 6 monlhs, onderwen! repair of avsd. the day after surgery the systemic oxygen salnralion was 76% wilh a pap at 75% of systemic. two hours of c01wenlional therapy failed 1o improve ihc patient and no administration was slarled at 10 ppm. so2 dramatically incrcased to 95%, but the pap dropped only to 50% of syslemic. nevertheless ihe clinical conditions improved and the no administration could be reduced at 5 ppm in the following 6 days. she was extubaled 8 days after surgery and discharged from the icu 20 days after. patient n.3:12 kg, 3 3'ears. underwen| hearl tral~splantalion for congenital heart disease with moderate hypoplasia of pulmonary arlcrics. at the end of cardiopulmonary bypass the transpnlnlonary al~erio-venoas gradient yeas higher than 7 mnfflg and we speculaled !hat w'ls due to a degree of puhnonary vasocostrictiont. the nsnal dose of no was otilised, however no significant modilicalion of pulmonary pressure or systemic oxygen saluralion was noled, and after 1 h no was discontinned. tile palienl was carried io the icu with maximal inotropic support, extubated after 4 d;b's and disclmrged from the icu after 15 days. in all patient no major adverse effect relaled to no admilfistration ",','as holed. conclusion: in our experience no ms a pulmonary vasodilaling agent is effective and easily adjustable to tile palienls requiemenls, however its use remains limited ill those palienl ill whoin tile alnonll! of fixed inlllllojliify vascular resistance is predominanl. we report the use of ecmo support in two unusual cases of severe tracheal disruption in which it had become impossible to achieve adequate ventilation. case 1: severe tracheal laceration due to aspiration of a share forelan bodv: a previously healthy 13 month old toddler was referred for ecmo following aspiration of a porcelain foreign body (with razor sharp edges) which had become embedded in the right mainstem bronchus with massive extrusion of air. this was removed on veno-arteda[ ecmo support, as the patient was unventilatable prior to bronchoscopy due to ongoing airieak. ecmg was continued after bronchoscopy to permit airway healing without the presence of an endotracheal tube. unfortunately, an extensive pulmonary haemorrhage on day 4 of ecmo necessited re-exploration of the airway. this revealed a posterior tracheal tear from the cricoid to the middle of the right lower lobe. following repair the patient was left on ecmo support together with high frequency oscillation ventilation (hfov), the latter being used to minimise potential aideak and maximise alveoli recruitment. ecmo was weaned after 17 days (420 hours) -the patient was extubated 7 weeks later. case 2: tracheal wound dehiscence due to seosls -tracheal transelant on ecmo: a 4 month old infant with a c[inically significant congenital long segment tracheal stenosis and left pulmonary artery sling underwent resection of the stenosis, followed by primary reanastomosis. this was complicated, 5 days later, by severe mediastinitis and complete dehiscence of the anastomosis. an autologous pericardial patch was used to repair this, however, the tracheal wound again dehisced 4 days later making mechanical ventilation impossible. in view of ongoing sepsis and a severely disrupted trachea ecmo was the only possible form of support. following resolution of the local sepsis (4 days) a definitive procedure in the form of a tracheal homograft (transplant) was undertaken on ecmo. the patient was managed on ecmo and hfov for a further 3 days, the hfov being used to optimize rapid lung inflation. unfortunately this patient died 9 months after weaning from ecmo due to complete disintegration of the homograft, which was not deemed reparable. conclusions: 1) ecmo can be used in the acute management of oxygenation when there is major airway disruption making mechanical ventilation impossible. 2) hfov was a useful adjunct in aiding recruitment of lung volume on ecmo in these two patients. backoreund: persistent pulmonary hypertension of the newborn (pphn) consists of a heterogenous group of diseases ranging from transient reversibte pulmonary hypertension to fixed primary malformations of the lung (primary pulmonary dyspfasia-ppd). inhaled nitric oxide (ino), a selective pulmonary vasodilator, has been proposed as a treatment for severe pphn. obiective and methods: ino was administered to 23 near term neonates with severe persistent pphn, oxygenation index > 25 and echocardiogrephic evidence of pulmonary hypertension, in order to further determine the clinical role of ino in the treatment of pphn. the response to ino was also analysed retrospectively to examine whether this could be of diagnostic value in differentiating at an early stage patients with reversible from fixed causes of pphn results: twenty one of the 23 patients studied responded to the initial trial of 1no (20ppm x 20 minutes), as defined by a greater than 20 percent improvement in pad2 as well as a fall in the el to < 40. these 21 patients were continued on ino therapy, with 3 patterns of response emerging: pattern 1 babies (n=8) continued to show a sustained response to ino and were successfully weaned from it within 5 days -all survived. pattern 2 babies (n=9) failed to sustain their response to ino over 24 hours, as definded by a rise in the el > 40. six survived, five with ecmo. pattern 3 babies (n=3) had a sustained dependence on ino for 3 -6 weeks. all three died and lung histology revealed severe primary pulmonary dysplasia (ppd). patients with ppd (pattern 3) not only required ino for longer periods of time than did the sustained responders (pattern 1), but also required significantly higher doses of ino we report on the air transport of 32 paediatric intensive care patients. these transports fall into three categories: 1) retrieval of critically ill neonates and paediatdc patients referred for either ecmo or inhaled nitric oxide (ino) (n = 12). one patient was transferred on ind. mean transfer time 2.2 hours (se +0.6hrs). 2) long distance international transport using chartered aircraft (n = 11). the indications for these transfers included both urgent retrievals for cardiac surgery and semi-elective transfer of stable patients back to their referring unit following treatment in tertiary centres. mean transfer time 4.4 hours (se +0.4hrs) 3) long distance international transport using commercial aircraft (n = 9). indications for transfer were either semi-elective retrieval for tertiary treatment or the return of stable chronically ventilated patients to their referring hospitals. mean transfer time 14 hours (se _+1 .fhrs, longest 24 hrs). the transport team consisted of a paediatric intensive care doctor of at least registrar grade and a registered sick chidrens nurse with intensive care experience. the administrative components of the transfer (ambulances, airlines, customs) were managed in collaboration with companies specializing in air ambulance transfers. outcome: all the patients were safely transported to their destination without mortality or morbidity. complications durino transfer ir~lv~; 1) patient complications -semielective endotracheal tube change and central access needed in the only patient brought to the commercial aircraft by the referring hospital (all others retrieved directly from referral hospital), seizure in patient with known encephalopathy, severe cyanotic spells in patient with fallots tetralogy who was retrieved for urgent surgery for this indication 2) mechanical compfications -ventilator failure, incubator battery failure, oxygen regulator failure -all occurred with equipment sent from referral hospital, this was unfamiliar and unchecked by our transport team -it was not the decision of the transfer team to use this equipment on this single occassion. 3) administrative complications -confiscation of incubator battery by airport security police, excessive delay by custom officials (2 hours) in the airport. the incidence of such problems were felt to be low and unpredictable. in conclusion: mechanically ventilated paediatric patients can be safely transported on both chartered and commercial airlines. these transports are best accomplished by trained intensive care medical and nursing staff with the backing of an air ambulance organization competent in arranging the necessary administrative details. it is essential to use your own equipment and to retrieve the patient _directly from the referrin(] hospital to minimise ootential complications. our experience with anaesthesia for paediatric electromyography _w_._pla_ti_k_a_n_o_v, r.eousseff, k.pavlova, d.marinova dpts. of anaesthesiology and int. care and clinika] neurophysiology, med. university, pleven, bulgaria ~)_b_j#~ti_v~. to t~st a " heavv sedation " regimen of anaest-es~a for the purpose of paediatric electromyography d#s~gil~ non-randomized,non-blinded human trial in the seting of an uriiversity hospetal. _m_a_t_eri_a_is_a_nd_ m_e_th_od_s_.110 children,asa i-if,median age 6 years,range 9-13 who undervent eleetrcmyography required anaesthesia. they recieved low-dose ketamine + i~iazepam or midazolam via musculary route( 25 children,age 0 -3 yrs,ketamine 2,5 mg/kg, diazepam 3-6 mg total dose ) or per os ( 85 children,ketamine 5-7 mg/kg,diazepam 0,3 mg/kg or midazclam 0,4 -0,5 mg/kg ) _resu_l_t_s. 20 -25 minutes after medication a state of heavy sedation with weak spontaneos and stimuli-provoked movements was achieved in all children, that lasted 30 -60 minutes and allowed adequate needle emg and nerve conduction investigation. 11 children recieved additional 0,6 -1,0 vol.% halothane during the placement of the needle. non -invasive blood pressure , breath and heart sounds and hb sad2 by pulse oxymetry were monitored.none of the older children disclosed memories of pain when asked after they regained adequate verbal contact.no complicationes were observed. antenatal maternal steroids reduce the risk of periventricular-intraventricular hemorrhage in very premature neonates treated with natural surfactants. i.apostolidou, c.papagaroufalis, g.touloumi, m.xanthou, n.kalpoyannis a' and b" neonatal icu "ag. sophia" children" s hosp. athens, greece. dept of hygiene and epidemiology, athens university, greece. obiectives: the aim of the study was to evaluate the association of periventricular-intraventricular hemorrhage (p-ivh) in surfactanl treated premature neonates with pre-and postnatal variables. methods: the population of the study was 88 neonates admitted during the years 1990 to 1992, with gestational age _< 32 weeks and severe respiratory distress syndrome (rds) (mechanical ventilation and arterialalveolar oxygen tension ratio (ajapo2) <0.22), who received rescue therapy of at least two doses of natural surfactants (alveofact or curosurf) and examined with ultrasound and/or autopsy for the presence of p-ivh (papile's classification). the examined factors in each neonate were the following: gestational age, birth weight, sex, multiple pregnancy, antenatal maternal steroids (complete and incomplete course of betamethasone), a/apo 2 before the administration of the 1st dose of surfeclant, delivery, apgar score at 5min, type of surfactant, pneumothorax and patent ductus arteriosus. the statistical methods used were x 2 and one-way analyses of variance followed by logistic regression medels, results: the incidence ot p-ivh was 31.8%. three factors were found to have an independent relation to p-ivh (final logistic regression model): gestalional age, a/apo 2 before surfactant administration, and antenatal administration of maternal steroids (complete and incomplete courses). for every 2 weeks of lower gestational age the neonates had an almost doubled associated risk of p-ivh (or: 1.92, 95% c1:1.14, 3.22). for every 0.02 on average decrease of a/apo 2 before surfactant administration the risk of p-ivh in the neonates was 1.27 times higher (95% ci: 1.02, 1.58). the neonates whose mothers received antenatally steroids had only one tenth of the risk of p-ivh of the neonates whose mothers had not (or: 0.10, 95% ci: 0.01, 0.82). conclusions: our results suggest that the antenatal administration of maternal steroids, even less than 24 hours before delivery, reduce the risk of pqvh in very premature neonates treated with natural surfactants, whereas the small gestational age and the lung immaturity still remain the main risk factors tor the development of p-ivh. we analysed retrospectively the management of 103 (51 boys, 52 girls) accidental ingestions of foreign bodies in children (mean age : 2.8 years, range : 7 months-10 years). no child had ingested more than 1 foreign object. the majority of the ingested foreign bodies were : coins (n : 44), toy parts (n : 11 ), jewellery (n : 3), batteries (n : 16), "sharp" materials such as needles and pins (n : 21), "large" amounts of food (n : 8). impaction of food occurs more frequently in children after oesophageal reconstruction in cases of oesophageal atresia. although according to literature "coca-cola" is reported to be effective, this was not seen in our experience. 28/103 patients had minor transient symptoms at the moment of ingestion, such as retrosternal pain. only 4 children experienced severe manifestations (cyanosis, dysphagia). in these children, endoscopy revealed oesophageal and gastric erosions. children were seen at the emergency ward within a few hours after the accident ( mean : 3 hours, range 20 min. -28 hours). chest and/or abdominal x-ray was performed as first-line investigation ( 93/103 objects were radio-opaque), and revealed an (unexpected) oeeophageal impaction in 6 children. in 87/103 the foreign body was in the stomach. batteries, sharp objects and objects trapped in the oesophagus were removed, either by endoscopy or by magnet-extraction whenever possible. the outcome of the patients was excellent. no complications were observed. extraction is recommended in symptomatic patients, and whenever the foreign body is trapped in the oesophagus, or if the foreign object is "sharp" or a battery. objectives: two strategies were used for management of malignant diphtheria in children aged from 0.5 to 13 years. methods: protocol n1 consisted of intravenous administration of diphtheria antitoxic serum, prednisolone (2 mg/kg bw/day), plasmapheresis and supportive care. protocol n2 included the use of antitoxic serum against the background of high-dose dexasone (2-3 mg/kg bw/day), hemocarioperfusion and a preventive use (before the clinical manifestation of myocardial damage) of inotropic medications, inhibitors of angiotensin-converting enzyme and pentoxyphylline. each of protocols included the monitoring of serum toxin (diphtherin) levels. results: the group of patients treated according to the protocol n1 consisted of 17 children with malignant diphtheria, 11 of them with severe malignant diphtheria (grade 2 and 3). all patients exhibited the circulation of toxin during at least three days after the start of treatment. all 11 patients with severe grade of disease demonstrated heavy cardiovascular disturbances associated with malignant diphtheria. of the 11 children in the group died seven. the children of the second group were treated according to the protocol n2. out of total of 22 patients of this group. 11 patients had severe malignant diphtheria. in all children a significant reduction in serum toxin level was revealed after hemocarboperfusion. in all but one case the satisfactory control of cardiovascular function on was achieved. of 22 children admitted to the trial 21 survived, one child with malignant diphtheria of grade 3 and congenital filbroelastosys of the left ventriculum died. the severity of neurological complications was similar in each of groups. conclusions: the use of hemocarboperfusion, high-dose dexasone and early prevention of heart failure as a adjunct to the standart treatment has been shown to be of benefit in the management of malignant diphtheria. t. schaible, i. reiss, j. m611er, l. gortner med. university of lqbeck, children's hospital, kahlhorststr. 31-35, 23538 l~beck, germany surfactant therapy seems a promising approach for the treatment of the biochemical and biophysical abnormalities of the pulmonary surfactant system in severe ards. patients and methods: over a 18 months period 10 non-neonatal pediatric ards patients (age 1-38 months) in a "pre-ecmo"-situation (oi 40 over 4 h) were treated with bovine surfactant (alveofact| the underlying conditions-of ards were pneumonia (5), sepsis (2), immunosuppression (1), near drowning (1), neurogenous ards (1). a total of 20-120 mg/kg b.w. was applied in several fractions. before surfactant therapy, we first tried different ventilation (best peep-finding, inversed i/e-ratio, hfo-ventilation) while monitoring the pulmonary mechanics. for hemodynamic stabilisation both norepinephrine and epoprostenol were used to optimize pulmonary perfusion for max. 4 hrs. if there was no improvement of the oi by at least 10, further treatment with surfactant was initiated. in addition to surfactant all patients received a treatment with dexamethasone of 1 mg/kg in 2 doses. patients with no benefit (oi remained unchanged or increased within the max. 2-4 hrs) were taken on ecmo. results: nine patients improved within 4 hours after surfactant therapy: the oi decreased from a level of 41 (mean, range 22-100) before our treatment to a level of 16 (mean, range 6-60) thereafter. in 6 patients we were able to continue the positive effects of our treatment and they could be weaned of the respirator within 3-10 days. the other 3 patients got worse despite respiratory improvement, they suffered of multiorgan failure of more than 3 organ systems. the last patient did not benefit from surfactant, he had to be put on ecmo, but died because of a complication (hemopericard)after 10 days. the autopsy of the ecmo-patient showed a pulmonary fibrosis, but the other 3 death were not due to pulmonary failure. conclusion: a different sequential ards treatment integrating surfactant therapy can reduce the number of patients requiring ecmo. but ecmo as a therapeutic tool should be available in centers involved in ards treatment. l.blindl, t.p.le, h.weinzheimer, centre for paediatrics, university of bonn, germany selective reduction of elevated pulmonary vascular resistance by inhaled prostacycliu (pgi) has been reported in adults with acute lung injury, neonates with persistent pulmonary hypertension and in one infant with idiopathic pulmonary hypertension. we report on the effect of aerosolized prostacyclin in two children with secondary pulmonary hypertension. patient 1: in a boy with down's syndrome an avsd had been surgically corrected at 11 month of age. at 5,6 yr of age a catheter examination revealed a pulmonary vascular resistance of 70 % of systemic vascular resistance in room air and at an fin2 of 1.0. prostacyclin (0.5 mcg/ml) was administered with a jet nebulizer at an fin2 of 0.21. pvr declined to 0.4 systemic vascular resistance and returned to baseline after stopping pgi-inhalation. subsequent intravenous infusion (5 ng/kg rain) had to be stopped after 5 minutes because of systemic arterial hypotension. patient 2: a 8 month old male infant with bronchopulmonary dysplasia developed suprasystemic right ventricular pressure inspire of therapy with oxygen and nifedipin. while he was spontaneously breathing 60 % oxygen via face mask pao2 was 37 mmhg, arterial ph was 7.35. systolic arterial pressure was 85 mmhg, a rv-ra gradient of 100 mmhg was measured by cw-doppler. while fio2 was maintained aerosolized prostacyclin was administered over 30 minutes. rv-ra gradient was 70 mmhg, systemic blood pressure 75 mmhg, pao2 58 mmhg. two hours later nitric oxide (20 ppm) was inhaled at an fio2 of (3,6. rv-ra gradient declined from 100 to 65 mmhg, systemic systolic blood pressure remained stable at 105 mlnhg. discussion: sporadic experience shows that aerosolized prostacyclin selectively reduces elevated pulmonary vascular resistance in some patients. in patient 2 the poor response to inhaled pgi1 compared to inhaled nitric oxide may be explained by the fact that the action of pgi is not independent from endothelial function, limiting it's effect in severe vascular disease. during the last two years (1993-94), 51 infants weighing less than 1000gr. admitted to our referral unit. thirty four of them (67%) survived, (28% of infants weighing 500-700g and 78% of infants weighing 701-1000gr survived) for the years 1986-87-88 the survival of these infants was 53% and for the years 1976-77-78, 14% (p<0.01). we analyzed the perinatal and neonatal factors influencing the outcome of these infants. the comparison among neonatal survivors (1) to neonatal deaths (2) shows: gestational age: 27.6 w (1) to 26.4 w (2) (s). birth weight: 923.5g (1) to 724.7 (2) (s). apgar score: 7,90 (1) to 7.56 (2) (ns). presentation and mode of delivery: breech presentation is associated with higher incidence of neonatal deaths. i.v.h. (at the age of 8 weeks): no one of the survival infants had evidence of i.v.h. respiratory problems: intubation, at the admittance of the infants 32.3",,(1) to 95% (2) (s) use of surfactant: 70% (1) to 95% (2). bpd observed in 62% of the babies and only one was dependent on oxygen at home. antenatal betamethasone was given in 20% of the mothers. in conclusion: 1) a great improvement in the survival rate observed in these infants the last 20 years in our unit. 2) factors with positive effect are increasing gestational age and birth weight, the absence of i.v.h. and the use of surfactant. the breech presentation and the severe respiratory problems increase the incidence of death. animal experiments demonstrated, that brain temperature determines the amount of neuronal damage caused by hypoxia and that mild hypothermia may have a protective effect. until now there is no method described and evaluated to measure brain temperature in neonatal intensive care units. we non-invasively measured brain temperature analogues, nasopharyngeal (tnasoph) and zero-heat-flux temperature (zht) at the temple whereby under zero heat flux surface temperature represents deep head and thus brain temperature. the aim of our study was to investigate the practicability of the method, the relationship of the two brain temperature analogues to rectal temperature (trect) and their dependence on insulation, thermal environment, body activity and time course. we investigated 19 healthy preterms less then 2 weeks postnatal age (gestational age 315 +_ 2.1 wks; x + sd, weight 1653 +_ 370 g) in an incubator. tnasoph was measured by a thermistor within a feeding tube, advanced to the nasopharynx, zht temple by a thermistor and a heat flux transducers both covered by an insulating pad, and trect thermal environment was characterised by operant temperature (tair .0.4 + twall 0.6). body activity was video taped. measurements were performed during the following interventions: i/ insulation increased by turning the temple with sensors onto the mattress (15rain). ii) insulation increased by a cap (30 min), iii) 30 min after its removal, iiii) increased operant temperature by 1.6 + 0.5~ (60min). results: seven children with ea had a gasless abdomen, the endoscopic procedure excluded (6) or diagnosticated an upper pouch fistula (1). in patients who suspected "h" fistula (2) broncoscopy has strong advocated method to make diagnosis and established cervical approach. from july 1992 14 newborns with ea and lower pouch tef received a selective transtracheal incannulation. we were not able to proceed just in 1 case with congenital subglottie stenosis. in these patients we provided gastric drainage by radiopaque and flexible 3-4 french catheter. the knowledge of the precise anatomic position of tef consent to adjust the tip of the endotracheal tube in order to achieve best ventilation. the presence of the catheter through the fistula helps the surgeon to identify, it quickly. no complications were correlated to the procedure and no babies had early pneumonia. alimentary continuity was achieved in all patients (30 primary anastomosis, 2 resections of tef, 6 oesophagocoloplasty and 1 died with gastrooesofagostomy). the late mortality 7.7% (3) was only directly related to the severity of associated malformations. conclusion: the advantages of this technical approach are unquestionable for the anaesthesiologist and the surgeon. in our experienc e the procedure improves perioperative management of babies and appears to be safe. relation between cytokines, prethrombotic markers and endotelial injury markers in children with septic shock objectives: to establish the relationship between cytokines (tnf, il-1, il-6) prethrombotic markers (d.d., pcam) and endothelial injury markers (tm, uwf) in pediatric patients with sepsis and bacteriemia without shock, and patients with septic shock. design and methods: prospective study, 18 children (9 months-16 years) were admitted in our picu in 1994 with the following diagnosis: bacteriemia (4) sepsis (4) and septic shock (10) according to jacob's r f criteria. measurements: il-1, il-6, tnf, tm, vnf, d.d. pcam and routine laboratory data on admision, 12, 24, 48 hours and on discharge. the prism (pediatric risk of mortality score) was also recorded. results and conclusions: two patients in the septic shock group died. significant differences were found between non-shock and septic shock patients in relation to tm, dd, pcam, il-2, il-6 and tne high levels of tnf and il-6 are closely associated with the severity of septic shock with purpura in children. low levels of pcam on admission were associated with severe shock. who underwent open hea~nt surgery, hypervotaemia with or without oliguria was the most frequent reason to start pd (77%). in 10 patients pd lasted less then one week and there were no complications; in 3 patients it lasted 13 -29 days (one child had a peritonitis). instillation of dialysis fluid into the peritoneal cavity was associated with a significant increase in central venous pressure. there were no significant changes in cardiac output or arterial oxygeu saturation. in all patients pd dhnjnished fluid overload or improved the metabolic status. 5 patients (38%) survived the postoperative course and all had complete reintegration of renal function. conclusion: pd is a useful method to treat the fluid overload and acute renal failure in paediatric patients following open heart surgery with file effects of little importance on the cardiovascular fimction. obieetives: with the marketing of computerised systems for lung function testing in newborns, there has been an increasing interest in clinical approaches. percentile curves of pulmonary parameters permit an appropriate and clinically useful interpretation. however, the manual evaluation of the results using different curves is an impractical technique. therefoi'e a computer programme was developed. methods: the percentiles (5%, 10%, 50~ 90%, 95%) of the most important pulmonary parameters were determined non-parametrically in 6 weight-classes. for the calculation we have taken results of our own as well as other laboratories using a meta-analysis of reference studies. in all, individual data of 300-600 healthy newborns ageing between 1-28 days were collated. using these percentiles, for every parameter in relation to the body-weight the cumulative distribution was calculated approximately using piecewise linear and exponential functions. as shown in the figure the results of computing are represented numerically as well as graphically and can be included in the patient report. conelusions: clinic~d experiences with the programme have shown that representation of all measured parameters on standardised 100% scales allows an easy interpretation at first sight and improves the detection of pathologic patterns in the parameters. ")supported by bmft, fp "risikoneugeborene" prism (pediatric risk of mortality) score is a well known, already validated scoring system that quantifies severity of illness based on 14 routinely clinical and laboratory variables measuring physiological instability. once computed the score by summing up the weights corresponding to the most abnormal value recorded during the first 24 hours, the overall risk of mortality can be predicted by using the coefficients estimated by a logistic regression where prism score is the main independent variable. (pollack mm et al, -pediatric risk of mortality (prism) score. crit. care med. 1988; 16:1110 -1116 . to assess the applicability and validity of prism in the italian setting we launched out a prospective data collection in a sample of 33 pediatric icus. measures of calibration (goodness of fit statistics) and discrimination (receiver operating characteristics and area under the roc curve) are planned to be adopted in the cohort of patients recruited during 1 year period. as the validation study started on july 94, data collection is still on going and validation analyses will be carried out on july 95. up to now 23 centers recruited 1116 cases. at present, characteristics of the sample recruited are the following: most of the patients were male (62%); the mean age is 3 years with 30% of patiens having less than 30 days; more than half were medical cases (59%) admitted from emergency room or from hospital floor (51%); 52% cases were admitted with an organ failure while 48% to be intensively monitored. icu-mortality was l 1%. the paper will present final results of calibration and discrimination analyses that will be carried out in the whole sample and across subgroups known to differ in terms of clinical relevance and prognosis. if calibration and discrimination assessment will produce not satisfactoty findings, a customization of the current coefficients will be made allowing a formal comparision of previous and new parameters. jf riera-faneao, m wells, j lipman. baragwanath intensive care unit, university of the witwatarsrand, south africa. [background the prism score is designed to assess the likelihood of death in ipaediatdc icu patients, using only acute physiological disturbances, age and [operative status to predict mortality. there is no evaluation of chronic health status, [including malnutrition. this may significantly affect its ability to accurately predict outcome in a population where malnutdtion is common. aim to determine the influence of nutritional insufficiency, as indicated by a low weight-for-age on outcome prediction by prism. patients & methods we analysed prism, weight and demographic data co ected prospectively from 1528 consecutive paediatdc icu admissions over a 6 year pedod. a proportional weight (pwt) was calculated as a percentage from the 50th centile of the who weight-for-age growth charts. the pwt was compared for survivors and nonsurvivors, and mortality compared for pwt categodes 0nho wellcome classification). multivariate statistical techniques were used to identity associations with non-survival and to develop a modified logistic regression equation including a measure of i nutdtional status. receiver operating characteristic (roc) analysis was performed including and excluding patients with low pwt for the odginal and modified equations. results non-survivors had a lower weight than survivors (6.8kg and 8.3kg medians p = 0 63) a lower pwt (85% and 90% medians p = 0.6"0. the incidence of malnutdtion , in our icu population was 33%. the mortality of manoudshed patients was' significantly increased (p = 0.001), with a good correlation with the degree of malnutrition. the accuracy of prism was significantly improved when malnourished patients were excluded from the analysis (roc value increased from 0.72 to 0.79). ! logistic regression and discriminant analysis identified a significant association between prism, pwt and outcome; age and operative status were not significantly related to mortality. the use of a modified equation including the raw prism score, pwt category and age can significantly improve the discriminatory power (az dm/elopmental sample 0.82, az validation sample 0.81). the modified formula is: legit = -2.864 +0.134*prism score -0.006*age + 0.463*weight category, where the probability of mortality is exp(iog/t)/1 + exp(iogio. discussion although we can improve the prediction of mortality by a modified or recelibrated formula, this still does not compare with the reference prism population. the need for validation of the score itself, in the association with outcome of the acute physiological variables themselves, is thus apparent. we conclude that while the odginal prism formula can be improved significantly, a modification of the basic variables in this and other third wodd populations may be essential. a high incidence of malnutrition is an independent risk factor of mortality, and an important cause of the poor discriminatory performance of prism. in order to improve the accuracy of prism, nutritional status should be taken into account. objectives: to assess the value of inhaled no to differentiate between pulmonary vascular constriction or fixed anatomical obstruction. methods: we assessed the response to 40 ppm inhaled no in 12 patients(9 m, 3 f, median age 4.5 months, range 1day to 17years) with signs of increased pulmonary vascular resistance, there were 5 pre and 7 postoperative patients. patients were divided into responders(+) or non-responders(-). a positive response was defined as a 20% reduction in pulmonary arterial pressure and pulmonary vascular resistance(pvr) or in the presence of a left to right shunt, a fall in pvr accompanied by increasing pulmonary blood flow. left atrioventricular valve atresia + 12 mustard pat: pulmonary atresia vsd: ventricular septal defect asd: atrial septal defect pda: patent ductus arteriosus tapvc: total anomalous pulmonary venous connection the responders(7/12) were characterised by left to right shunts or pulmonary venous hypertension(4/7). patient#11 was weaned from ecmo with inhaled no. patient#2, without congenital heart disease, underwent a lung biopsy which confirmed reversible pulmonary vascular changes. patient#1 had a pulmonary hypertensive crisis which responded to no. all non-responders(5/12) had evidence of anatomic obstruction to pulmonary blood flow (#7,10,12)or a low pvr(#8) on subsequent cardiac catheterisation. in patient #3, lung biopsy confirmed severe obliterative vascular disease. conclusions: inhaled no appears to be an effective pulmonary vasodilator. a failed response may be evidence of either irreversible pulmonary vascular disease or a residual anatomical obstruction which may be surgically remediable in the postoperative cardiac patient. therefore, inhalation of no may be a useful diagnostic test to differentiate between fixed anatomical obstruction and reversible vasoconstriction. results: during these 18 years, the incidence of sdra was 1.3% of the total of admissions. the most common etiology was meningococcic septic shock. since 1989, there is a decrease of its incidence. (from 24% to 11%) and an increase of pneumonia and immtmodeficiencies. mean age of our patients was 2,7 years (61% males, 39% females), total mortality by sdra was 59% and there is an increase up to 72% since 1989 mean time of stay of the dead was 4,3 days and 12,4 days those who survived. although during the late years we offer in the picu a better attendance quality to the patients with sdra and the mean stay is longer, both for those who die and for those who survive, mortality of patients with sdra have increased. the incidence of sdra secondary to the septic shock of a meningococcic etiology have decreased. on the contrary, the sdra secondary to infections by opportunistic germs in patients with congenital inmmunodeficiencies or acquired immuodeficiencies have a tendency to increase. in our series, this change of aetiology is the responsible for the increase in mortality. hospital infantil unlversitario "virgen de1 roclo". sevilla. espalqa aims:to assess the incidence, etiology, clinical course, sequelae and mortality of the patients admitted to a paedfiatic intensive care unit with the diagnosis of severe traumatism. material and method: 60 cases of severe traumatism in children admitted to our icu in the period from january 1990 to june 1990 were reviewed. age of patient ranged from 4 months to 9 years, 65% were males. in our series, 53% of cases suffered traumatism due to a traffic collision and 38% had a fall from a considerable height. only in one case was traumatism due to violence to the child. we assessed the first assistance received in 76% of cases: where was it performed, interval of time since the accident, and steps taken. these data were also studied in relation to the latter evolution. results: 75% of our patients suffered cranioencephalic traumadsm (ct); in 53 % it was an isolated picture and in 22 % of cases was associated to other lesions. there was participation of thoracic and/or abdominal organs in 16 % of cases. 10% of cases presented important maxillofacial involvement. only one case presented serious cervical medullar lesion. mortality in our series was 3.3%. in 8.3% important sequelae remained. all of these patients presented tepas on admission equal or lower than 4. 16% of those with traumatises had slight sequelae. 71.6% of the total evolve towards healing. a polytraumatized child is a patient that benefits considerably of it admission in a paedriatic !cu. the rapidity in receiving first aid and its quality are essential to avoid sequelae and to make mortality decrease. after unilateral lungtransplantation 20% of the patients develop a lung failure with decrease of perfusion and increase of pulmonary blood pressure in the transplantated lung. the improvement of perfusion is an importent task in the postoperative period. case report: a 14 year old girl with idiopathic pulmonary fibrosis received a left sided single lung transplantation. during the early postoperative period occured a higtter demand of oxygen and an increasment of the pulmonary vascular resistence in the left lung. the pulmonary ventilation and perfusion scintigraphy indicated in comparison with the right lung a reduced perfusion of only 30% in spite of a ventilation of 70% of the transplanted lung. to improve the perfusion of the transplant we administrated per inhalation prostacyclin in a maximal dose of 20 ng/kg/min. the arterial blood pressure decreased but the perfusion continued nearly at the same level. during the following administration of 10 ppm no in the respiratory air we achieved a significant reduction of the respiration pressure f~m 40 to 32 nun h20 and of the pulmonary arterial pressure. the perfusion in the transplanted lung increased to 70ca/of the total pulmonary perfusion. after 3 days of administration with no we were able to withdraw the axtifical respiration without any following complications. conclusions: the perfusion of transplanted lungs is a major proble_r~ in the postoperative period. this case demonstrated the advantage of no towards the inhalativ application of prostacyclin. no showed a significant improvement of perfusion in the transplanted lung of a 14 year old girl. results: a total of 11 children with ards were treated with bovine surfactant (alveofact| 9 cases were evalable. the median age was 1.3 years (range 2 weeks to 4,9 years). in six cases ards was associated with pneumonia, in two cases with lung hemorrhage; in one case isolated ards followed hemihepatectomy. the first surfactant application was performed with a median latency of 22 clays (range 3-68 days) after first symptoms of ards witha median doseof 84 mg/ kg (range 42-133mg/kg). in 9 patients 28 doses of surfactant were applied. during the hour before therapy, the median pao2 / fio2 -ratio was 70-65. within 30 min. after application of exogenous surfactant the pao2 / fio2 -ratio increased to 90 with successive decrease over a period of 8 hours to 75. accordingly, an increase in pao2 and oxygen saturation and (less significant) a decrease in ventilation parameters could be observed. analysis of broncho-alveolar lavage before surfactant application in children receiving repeated doses revealed in most examined cases either clear surfactant deficiency or pathological function. 2 of 9 treated patients survived (3 of the 11, respectively). 13 of the 28 surfactant doses were applied in the 2 surviving patients.conclusions: the application of exogenous surfactant in children with ards caused a significant increase in oxygenation, which declined over a period of 8-12 hours. the effect often could repeatedly reproduced, in one case after 11 applications. the increase in oxygenation often allowed the reduction of fio2 and/or the inspiratory pressure. no side effects were observed after exogenous surfactant application.in many cases the application of surfactant wag too late after first symptoms of disease (median latency 22 days). ards mostly due to pneumonia seemed to respond to surfactant therapy less well or not at all. permanent junctional reciprocating tachycardia (pjrt) is the most common incesant supraventricular tachycardia (svt) in children. it is usually drug resistant and its onset in early life has been associated with dilated eardiomyopathy. we report our clinical experience with 3 patients detected antenatally and another diagnosed at 2 months of age. method.diagnosis: negative p waves were detected in leads ii,iii and f, p'r > rp" and there was not warm-up at tachycardia onset.clinical records, ekg,x-rays, echo and holter were reviewed. ep studies were undertaken only with therapeutic purposes. results. in a 10 year period 13 patients under 14 y of age fullfilled diagnostic criteria; 3 were detected prenatally (25-34 weeks) and one was diagnosed at age 2 mo. the 3 fetuses had intermitent svt during gestation. all 3 of them had pjrt in the first month of life at rates between 180 and 240 bpm. they were admitted to the icu but did not develop signs of heart failure. they were controlled with digoxine (d); d and quinidine; d and propafenone in 2 to 20 days. one was in sinus rhytm until age 4y; he then showed persistent pjrt over 70% of the day on repeated holters and underwent successful radiofrecuency catheter ablation (rfca).the other two patients showed initially a lowering of tachycardia rate followed by sinus rhytm for over 90% of the day (follow-up 2ran and 4 y). the 2 mo. old infant was admitted to the icu in severe cardiac failure. echocardiogram showed marked systolic dysfunction (shortening fraction 15%) treatment with digoxine, amiodarone and propafenone were unsuccessful despite lowering heart rate to 185; rfca was performed at 3 m. of age with restoration of sinus rhytm and rapid recovery of contractility. all patients were given atp at admission with transient (15 to 35 see) recovery of sinus rhytm. ff,s162 clinical course of pjrt is variable. atp is useful only as a diagnostic tool. initial treatment with digoxine + amiodarone or propafenone is adviced. rfca is a very useful therapeutic modality and can also be performed in young infants twelve patients (5%) died. these were 4 meningitis, 2 head injury, 2 sub-arachnoid bleeds, 1 status epileptieus, 1 leukaemie, 1 drowning, and 1 multiple trauma. calculated from the a 2 admission day p edialric risk of mortality score (prism), the probability of death (p) ranged from 0-100%. of the 12 deaths, i 1 were predicted by prism analysis except for the leukaemie patient (p i%) who died from haematological complications following chemotherapy. two children predicted to die (p 43% & 73%) survived. the median length of stay was 2 days (range 1-34 days). 98 patlents(50%) received ventilatn~ support and 10 patienta(4%) were transferred to specialist units (5 neurosciences, 3 liver, 1 cardiac, 1 bums). this data supports the view that many paediatric patients are being adequately treated in a dgh icu. meningitis and other neurological illness caused the majority of deaths and respiratory problems caused most admissions. most deaths (9 of 12) occurred within a few hours of admission. ectopic junctional tachycardia (ejt) is one of the most dangerous arrhythmias in the postoperative setting of congenital heart defects since it does not respond to antiarrhythmics or defibrilation. the object of this presentation is to report on two patients who presented f_jt in the early postoperative period and developed intense congestive heart failure which could be controlled after treatment with moderate topical hypothermia. two patients, 8m and 2y, diagnosed of atdoventficular septal defect and tetralogy of fallot developed intense heart failure in the early postoperative period. taehyeardia rate was 215 and 225 bpm. medical drug therapy included weaning from vasoactive drugs, iv digitalization and iv amiodarone treatment. there was not response. they were both surfaced cooled by placing plastic bags filled with cold water over the patient's chest and abdomen. temperature was monitored to obtain a central temperature of 34~ there was a gradual decrease in heart rate in the following hours (130-150bpm) paralel to the degree of surface cooling and clinical course estabilized.both recovered normal sinus rhytm in 48 to 72 hours. there were not significant arrhytmias after the procedure and postop, was uneventful. conclusions. moderate hypothermia is a very useful manuever for the treatment of drug resistant ejt. since it lacks side effects of other antiarrthymics we beleave it should be the treatment of choice for the treatment of ejt in the postoperative patient. present understanding of the pathogenesis of sepsis, based on the theory of systemic inflammatory reaction, has risen new interest in the more invasive methods of treatment, like plasmapheresis, leucapheresis and exchange transfusion (et). obiectives: evaluate the effect of et in the treatment of neonatal sepsis. material and methods: from september 1 to december 31, 1994 a prospective study was carried out, where the severest cases of bacteriologically proven neonatal sepsis (n=9) were treated with et. in total 15 newborns were treated for culture positive sepsis in the intensive care unit during this study period. diagnosis of sepsis was based on the clinical criteria of suspected neonatal sepsis, used by mc harris et al., laboratory data and positive blood culture. newborns with severe congenital malformations were excluded. et was carried out with fresh (less than 24 hours old) adsol-conserved erythrocytes, from which buffy coat had been removed, and same donors plasma, using a slow continuous two-site technique. the mean volume of et was 164.3 ml/kg. the effect of et was assessed as a change in the score for acute neonatal physiology (snap), general treatment results were compared with a historical control group of 26 newborns, treated for culture-positive sepsis in the same icu during the first eight months in 1994. students ttest and chi-square test were used in statistical analysis of the data. results: with the use of el a significant decrease in mortality was achieved: 1 death of 15 cases during the study period, compared to 9 deaths among the 26 controls (p<0.05). no baby, receiving et, died. the incidence of severe complications did not differ in the two groups. the snap-score showed quick improvement by the first post-transfusion day (p<o.05) and the effect persisted during the five following days. conclusions: we conclude, that the use of et in the treatment of critical cases may help to lower the mortality of neonatal sepsis. it provides a quick improvement in the clinical condition of septic neonates. the granulocyte-colony stimulating factor (g-csf) is largely employed in granulocytopenic patients. experimental studies have' demonstrated the effectiveness and safety of the therapeutic use, nevertheless the human employ is reported almost exclusively in neoplastic patients submitted to chemotherapy and in patients with fungal or pseudomona~ spp infections. the authors report the data concerning three non granulocytopenic children: in the first, o girl 7 years old, affected by pseudomomm aerug/n~a pyeionephritis, the antibiotic therapy was not tolerated in consequence of renal and hepatic failure. the second patient, an infant 6 months old, has been treated with salbactam-ampiciilin, ceftriaxone and chioramphenicol because of haemepldlw iafluem~ meningitis; a late neurological aggravation required the antibiotic therapy recommencement the last, a 17 years old insulin dependent diabetic boy, was affected by cared/do spp systemic infection; the antifungnl therapy w~ not practicable in consequence of renal and hepatic involvement in all the g-csf was administred subcutaneously at the'dose of $ u for 7 days. a significant increase of granulocytes was ever noted already after the second administration until 24 hours after the last dose. clinical signs, fever and biochemical values are promptly influenced positively and all patients are restored to health. no side affects were noted. the granulocyte colony stimulating factor, at indicated doses, can prove of great help in critical patients, in who the antibiotic or antifangal therapies can result toxye or cannot be tolerated. the ~reptococcas p~, among the gram pmltlve~ represents the main causative organism of bacterial meningitis in children, with the majority of deaths occurring within hours of admission, the third generation cepohlosporim were indicated as antibiotic of first choice, and some literature reports have demonstrated no significant dlffereuces in dinical course of patients treated with ce~rlaxoae and of those treated with ampieibin and chlornmphenicol. the authors report the ease of on infant, male, 18 months old, submitted to a neurosurgical interventiol after 2 days the infant became febrile and lethargic and a bacterial meningitis by ~trepmcecctz ~ was diagnosed, in spite of he was given the eoftriaxone therapy.. an amelioration was found only when a systemic treatment with vaucomyein was added. after 2 days an intrathecal administration of vancomyein 40 m~day was started: the norbmlization of the spinal fluid profile was uoted two days later and the infant receve~d progressively. the follow-up after eight months doem't evidentiate motosensorini sequelae-the individuation' of ~cscc~ pmmmsm/ae strains heta-loctam antibiotics resistant requires alternative therapeutic regimens: the k~terity quead vitam and quoad valetudinem of these infections justifies an intrathecal therapy, especially failing o prompt answer to the systemic treatment objectives: to evaluate the incidence of the burnout (bo) of the staff in a picu in a university hospital. the bo is a syndrome charactedzed by many factors like emotional stress, lack of personality and reduced work ability. this syndrome represents a kind of abnormal response in the working habitat, above all, where psychological and personal characters are mostly involved. the symptoms of bo are represented by demoralisation, demotivation, incapability, boredom, isolation up to organic disturbances, like migraine, insomnia, dizziness and gastrointestinal disturbances. the major incidence of this syndrome was found in the hospital staff, especially among the nurses, working in an atmospher of high emotional dsk, just like in an intensive care unit. methods: from january '95 till april '95, all the staff of the picu of the a. gemelli hospital of rome, underwent a test to evaluate the bo. the test used was taken from the book "bumout: from tedium to personal growth", by pines and aronson. twenty seven people were studied (19 females, 8 males)i out of which 7 doctors, 7 residents and 13 nurses. it was considered sign!ficant as mild bo, a score between 3-4 and as severe, the score of bo >. 4. results: 10 subjects (37%) resulted positive for bo, out of which 8 were females (80%) and 2 were males (20%). the subjects with mild bo were 7/10:1 was a doctor, 3 residents and 3 nurses. the subjects with severe bo were 3/10, out of which 1 resident and 2 nurses. conclusion: the results obtained show that bo is a condition well represented in the staff of our picu. the category most at dsk seem to be the nurses (5 subjects), as well as residents (4 subjects), as in literature, which shows a major incidence of the syndrome in younger subjects and having a limited partecipation of functional decision. the results obtained obliged us to start a programme of serial controls so that the subjects most exposed can have a necessary psychological support to react adequately to this condition. the term systemic inflammatory response syndrome (sirs) was adopted by the consensus conference to denote a type of systemic response to severe infection or otherinsults in critically ill patients. when sirs occurs from infection it is called sepsis. sepsis occurs more frequently in persons with perexisting illness or severe trauma. there has been tremendous advances in prophylaxis, diagnosis, and treatment of sepsis. a comprehensive model of the disease progression from sirs to mods should be developed giving priority to severity of illness scoring system and other predictive methods. some recommendations for future clinical trials include: trials should not start with humans. before proceeding to human trials, animal studies should indicate an acceptable risk/benefit ratio. appropriate patient populations must be defined and treatment protocols should be standardized. full and rapid reporting of all results should be mandatory and a central repository of published and unpublished study results could be helpful. accrual at each center should be of sufficient size, and should include the number of patients accrued, mortality rates, and patient characteristics. pivotal trial should be preceded by sufficient pilot or phase ii studies. correct drug dosage and usage should be delineated in pilot studies. large, multicenter, trials should be used to enhance the unversality of trial results. analyses should be planned a priori. definitions for the target population should be explicit, reproducible, and include illness severity scores. outcomes should be relevant reproducible and include both measures of benefit and harm. mods and its reversal should be considered as an endpoint. quality of life should also be considered as an endpoint. the estimators of overall treatment effects should be controlled for base-line prognostic factors and subgroup anaiysis should only be used for hypothesis generation and not to modify the conclusoin of the trial. economic analysis should be included as part of clinical design. evaluatin of source control should be a critical component of any study. standardized clinical mediator assays should be pursued. placebo patients in clinical trials should be studied for a better understanding of the pathogenesis and epidemiology of sirs, evidence based medicine should be used to evaluate the validity of clinical. introduction: use of inhaled nitric oxide (no) as a modulator for optimizing ventilation-perfusion or lowering pulmonary artery pressure is becoming increasingly common. no is a free radical but little toxicological research has been published. clearance of nebulized 99mtc-dtpa is known to be, a sensitive indicator for early function impaimaent of the alveolocapillary barrier. we investigated whether exposure to no increased clearance of 9~tc-dtpa from the lung. methods: three groups of 5 white sealand rabbits (bw 3.5 kg) were anesthetized, tracheotomized and paralyzed. 2 groups were ventilated for six hours at pressure regulated volume control, set to deliver 10 ml/kg with a frequency of 30/rain, i/e ratio = 1:2 and peep = 3 cm hzo using a modified servo 300 ventilator (siemens, solna, sweden) with computerized no delivery system. gas mixture per group was either 0/25 or 20/25 [no (ppm) / fioz]. after six hours of ventilation in these 2 groups and immediately after anesthesia in group 3 (control), 99~tc-dtpa was nebulized into the inspiratory line of the breathing circuit and administered as a fine aerosol. gamma counting was measured for 20 minutes, monoexponential curves were fitted to the data and the clearance half-time (t 89 was calculated. the t~/2 mean • sd of the different groups were: t~a (mean 4-sd) h"e,i witl~ arf 1:14 di.ff:erent kinds, aged .q-ore 2 mon't.hes to [4 gears o11:1 (bodi weight .~rom 4.,5 to 45 kg), is presen .... "ed ( i,,~u::trl:e i:ibstraclive d:lse~se...14~ 2.ards'-8; :~,;,,arf o~ ::entral genes:i s .-3,0,~ :inc lud ing men ingeenceph 1 it :is-3~ reye ' s ~yrtdro~e-..#~,bri~:ln pes~.re~nimatior~ disease.."5). int:lrl~]. pa-. "iiulle'i,~s ariel regymes o+ l;mv,l;i"t"v were cle'l'.ermllled by ba'i~ier was. about 4. tuber,, dopamin tiara-:. t.io; was ~.".,,'.r:~r~led. cmv,cppv d~.!"~tion raniled -~rom f to 25 dayns.,~ <6.-:in 351, 6"t2-irl lo;and>12 davs'-in 6'l~atierr~{s i'i"ai3s:ltiol~ o4; patterers to imv, simv modee was per.r:)rmed, ~herl pif:' decrease.d to 16-17 ml~ar, fi02 ~ecreased to 0,4. lind less with 5a02=901/,,. i:lesq.lts:{ in pat:i.ents e{ group :l, who were tre,~d.ed w&th 2f'f'v, teoph :i. :1. l:i.r~ (is-24.mg/kg/day), g lucecdr t icostei~oids (2 .... :~;mg/kg/day), when r exceeded in 2,2-.];,4 times normal va i tea the e aqes/,'!:l"oln 2~j,, ite :i.~;::.!;, 8 ~ml"lrj), it was possible 't'(' ce 'e~ e aad]t:..~rom !1.20 2'.' 26i', 8 to !..';51,0-106, 1 mml-lg in ~}.. :~.[~ houi,!; ~d'l(:i to ru:}l",g'd!~l:i.2e i::h,:~e,'~c['el';i.stil obieetives : this chapter will describe what is knovca of the psychlogical responses of infant and children to hospiuiisation and attendant procedures. the factors which may modify these responses will he discussed and important considemtiorts will be outlined for optimal anaesthetic management and postoperative period of infants and children which will minimised the rise of emotional upset. methods : in this paper the autors will discttssed the probl 9 of: 1. health children (asa i, ii) facing single uncomplicated surgical elective procedures 2. various abnormal situations including neurotic children, children facing repeted operations, chronically ill, buaaes and tsaumatically impired ones 3. unfortunate young patient facing and often expoclting fatal outcome from le "ul'ukaemia, tumors, cystic fibroses or otheq" disease. : management of each child must vary greatly, ifi general the phases of emotional conditioning include home and preadmissiun preparation, admitiun preoperated and operative care and postoperative period. the authors would be happy if the child passes all stages without any trauma which could be prolonged in the future life. introduction ino is used to selectively reduce pulmonary vascular resistan(~e. we applied ino in the postoperative intensive care of patients with pulmonary hypertension and the risk of right ventricular failure after surgical correction of a congenital cardiac defect. methods 2-50 ppm no were added to the ventilatory gas mixture using a specially designed equipment (messer-griesheim, germany/austria). indications for application included pulmonary artery pressure >50% systemic pressure, critically depressed right, ventricular function or an oxygenation index >10. assessment of n oefficiacy consisted of on-off-on measurements according to the clinical stability of the patient including hemodynamic parameters, pulmonary gas exchange, continuous monitoring of ventitatory function and transesophageal echocardiography of the right heart. results in 29 situations (19 patients, age 3 days-71,1 years), ino was applied 0-628 h postoperatively. oxygenation was improved in 13 situations from 114_+51 to 171+53 mmhg pc2; pulmonary pressure was reduced in 17 situations from 70-*22% to 34_+17% of systemic pressure. in 7 situations, no reduction of pulmonary pressure was present, but measurement of cardiac output or echocardiographic analysis indicated an improvement of right ventricular function (right ventricular stroke volume +39-*12%, cardiac output +20-*11%). in 8 situations (immediately postoperativ with suprasystemic pulmonary artery pressures [n=4], multi-organ-failure [n=4]), no response to ino could be determined. conclusions for a special group of patients, the selective reduction of pulmonary vascular resistance by ino has become an important part of postoperative therapy. using this selective afterload reduction, postoperatively depressed right ventricular function can be improved. this effect of ino seems to be the most important one in the postoperative period. thus, ino appears justified to be appfleo when impaired right ventdcular function could be improved even when pulmonary artery pressure is not raised or remains unchanged. obiectives : premature infant are exposed to danger of apaea due to anaesthesia during their tirst months of life. it is yet unknown whether prematurity is corelated to any other kind of reslgratory disorder due to anaesthesia within the tirst year of life. methods : we theretbre researched retrospectively for respiratory disorders in all infants under 12 months of life belonging to asa group 1. they all had been anaesthetised in 1985.95 in our clinic for the following surgical reasons: ingvinal haemia, umbilical haemia, hydrocelae testis and phymosis. results : in 2350 cases we tbund: lafingospasm during induction in anaesthesia (0,5%), bronchospasm during induction in anaesthesia (0,22%), impaired intubation (0,1~ postanaesthetic laringospasm (0,1%), supposed aspiration (0,04%),postanaesthetic inspiratory stridor (0,05%), postinductional inngoedema (0,03%), death after 4 months in consequative of infection pneumonie (0,12%), none of these disorders was correlated the prematurity, 3 infants suffered of post anaesthetic apnea, 2 of them had premature medical history. concludions : prematurity does not enhance the risk of respiratory disorders due to anaesthesia within the first year of life, except the danger of postanaesthetic almea needs spetial cosideration. it could be demonstrated that aepgi 2 lowers pulmonary vascular resistance and indirectly improves cardiac function. this effect seemed to be selective, and was comparable to ino in the doses we have examined. therefore, aepgi 2 could represent a clinically useful alternate to inc. however, further research is necessary to work up the benefits of either therapeutic strategy. objectives: heat and moisture exchange filtem (hme) are used as artificial noses for intubated patients to prevent tracheo-bronchial or pulmonary damage resulting from dry and cold inspired gases. furthermore they are used for the prevention of bacterial contamination of the anesthetic apparatus by the patient's exspired air. so they are considered as a time-and money-saving device in anesthesia. filters are mounted directly on the tracheal tube, where they collect a large fraction of the heat and moisture of the exspired air, adding this to the subsequent inspired breath. the effective performance depends on the water-and bacteria-retention capacity of the filter. this study evaluates the efficiency of four different filters under clinical conditions. methods: four different types of filters ( dar hygrobac, gibeck humidvent, medisize hygrevent and pall bb 100 ) were investigated dudng mechanical ventilation over a pedod of 24 hours. 20 minipigs with hemorrhagic shock were intubated and ventilated for 5 days in an animal intensive care unit (icu). after 24 hours of mechanical ventilation the filter was randomly replaced maintaining the individual ventilatory conditions. the weight of the filter was determined before use and after removal after 24 hours. the airway pressure was monitored online to record changes during use. tracheal secretions and both sides of the filter were microbiolologically tested to see whether bacteria of the animal's respiratory system could be found on the patient's side of the filter or if they even would have penetrated the barrier. results and discussion: over a pedod of 24 hours 3 of 4 types of filters showed an increase in weight of 10 + 6% and airway pressure. bactedal celonisation 0ccured in nearly all fillers (93 of 100) on the patient's side, whereas only three of four types of filters showed identical bacterial colonisation on both sides. the only filter that did not show bacterial penetration, increase in weight or airway pressure was the pall-hme, a condensation humidifier without hygroscopic salts for moisture retention. with respect to our data one should use a condensation humidifier if airway conditions should remain stable dudng mechanical ventilation and desinfection of the anesthetic apparatus should be avoided after each patient. aim: to assess the clinical uses of, and experiences with, the hayek oscillator. this is a non-invasive device capable ef delivering not only continuous negative pressure (cnp) but also external oscillatory ventilation around a negative baseline (eov-nb) using an external cuirass. this type of ventilation avoids the need for intubation and intermittent positive pressure ventilation (ippv) and facilitates weaning in ventilator dependent patients. patients and methods: 21 patients in respiratory failure, age range 3 weeks to 15 years in a total of 29 patient episodes were treated using either cnp or eov-nb mode. duration of treatment varied from 4 hours to 8 days. indications for use ef the device were: 1) to facilitate weaning from ippv 2) prevent reintubation of patients following unsuccessful extubation, and 3) avoid intubation and ippv altogether using the hayek oscillator as the on[y means of respiratory support. results: there was an increase in pao2:fio2 ratio after cnp and eov-nb (p <0.0001, and p=0.01 respectively, wilcoxon signed rank test). patients who were in respiratory failure with hypercapnia showed a statistically significant reduction in paco2 both with eov-nb and cnp (p=0.02 and p=0.01 respectively) but the magnitude of change was individually greater in the patients who were treated with eov-nb. all patients, however, showed a fall in respiratory rate (p<0.0001) after the application of the cuirass in cnp mode. there was no physiological deterioration related to the application of external extrathoracic negative pressure in either cnp or eov-nb modes. conclusion: the improvement in pao2:fio2, the fall in paco2 and respiratory rate were indicators of an improvement in ventilation. the proposed mechanisms include improvement in frc, recruitment of additional alveolar units, and improvement in secretion clearance resulting in reduction in the work of breathing. meek to ~ month of the lifo,the bemodyuanicfacls were defined uitb the help of tetropolar reography method!. the excretion of !he catbocholauines fcfi] mith the urine gas detertend by taylor ll,laoorsy ~ iacg/dayl. hsaltl in the hypercuagulation stage of bic we deflorteeed the acliuutiun of the tbrubio and plasiin syaet~ mitb the increase of the inhihitnrs, in this case we registered in full uahe dot this process coabined uitb the dayl~ excreliou with lho urine epinopbr ne e], nor~pinopbr no tel and dophanine io], lbat shod the inlensificatiou of the s~nthosis prnoe-s~es and the release of ea in blood fron hissue deport the actffat on of the svnpathadrenui systen ]sfisl assisted to furl the b?perd~nanical rosins of the eircuidion and increase the ,icrocirculatinn, the klinicai sings of the insufissieutly of the circulalion have not defined,that has been associated the conpensatury character uf the ehan~es of ~ and heludy~enic status, t~e uun~u|p-lion ceugulupatby bus been donoustraled in the hypocougulatien stage ~bat man xauifosted b 7 the exhaust of lhe confulalion nod oessel-platel heuostasis, the consuxptton of cnnpononts tbronbln ,plnstin, kallek~eiu-kinln s~slots and the forniration eat in fell canoe clot uas accoqaued bs docrea,e of fl,nfl,o, the products of the xotabolisx of c~ and the activation of xonoaninoxydasu. the decrease of the extoll'on g and the exhaust deport co indicahd about t!e ]ou fund/anal reserve of ~fl~. it was one of the lain reason of ~bo heiod~uanic disbroed iheat insnfissient]~] and the uicrncireulaflion lintestinal codeme with the low effectife periferal flow] and nul[iplay organ failure,the distrued deport of sos mitb throubocytupenin no; be one of the nechanisn the dislrood of uessej-plalol heioshasis, the correlation bolueeo changes of boiostosis c~ and circulation ore reguired aduinistration nedidns, thai reslore the love s of c~ in the blood, prevent uulliplay organ failure and hetorrnge in children with sepsis, ~b~ectives: multi-measured correlative analysis of the most number of non-invasive indices of the cardiorespiratory system function was made to determine the structure of their interrelation and the ways of their adequate and effective correction. hethods: spiremetry, capno~raphy, oxygenography, indirect fick method at recurrent respiration, plethysmography, integral rheography -in all 52 indices were used. the received data were processed on a computer by a standard package of statistical bmdp programs. results: 70 women with ~h-gestosis (i group) and 48 somatically healthy pregnant women (ii group) were studied. cluster analysis has shown that the rate of the mean correlation connection between ventilation indices was 94% in the ist group and 90% in the iind group; gaseous metabolism -91% and 86%, respectively; central hemodynamics was 87~ in both groups. conclusion: cluster interpretation allowed to suggest that an increase of the rate of the mean correlation connection between the indices was characteristic of effective adaptation as the system was multi-component and well-regulated. on the contrary, the increase of the rate of strong correlation connection between the indices reveals the rigidity of the system and the tensity of adaptation mschaniams, i.e. the proximity to decompensation. it follows from this that in cases of eph-gestgsis, the reliability of regulating ventilation and gaseous metabolism decreases. seve/e hypoxemia in non intubated patients represents a major contraindicafion to fiberoptic bronehoscopy (fob) and bronehoalveolar levage (bal), but these procedures are often required for a correct diagnosis of the causative agent of pneumonia. aim of this investigation was to veaify the safety and efficacy of bronehoseopic procedures during pressure support ventilation administered through facial mask (fm-psv). five intensive care patients, all immunoeompromised, (3 males and 2 females; mean age 41.6• were enrolled in the study. all patients presented criteria for pneumonia with pao2/fio2 ratio ~ 100 and were responders to fm-psv. fob and bal were performed afte~ topical anesthesia with fm-psv ( ps = 16 em h20; peep = 5 emh20; trigger = -lemh20) continuously admires" tered ( 10' before fob fio2 = .7; during fob, fio2 =1 and for 90' alter fob, fio2 = 0.7). pao2/fio2 ratio as well as 02 saturation (sat) did not show signifteative changes during the procodure (fig.l) . no complication was observed and hemodynamic conditions were stable for all patients. cmv, pnenmoeystiis (2), legionella and mycobaetermm tuberculosis were identified from bal allowmg a prompt and targeted therapy. we concluded that mask psv can represent an excellea~ technique to pexform fob and bal in severely hypoxemic patients without deterioration of gas exchanges and avoiding endotraoheal intubation. intensive care unit, hospital general of albacete, albacet~ spain. objective: to analyze the current incidence and epidemiology of total parenteral nutrition (tpn) among critically ill patients placed on mechanical ventilation. design: prospective observational study. setting: medical intensive care unit in a tertiary hospital. patients: a total of 113 consecutive l'ritically ill patients with non-coronary related disease needing mechanical ventilation admitted in our icu during a 12 months period. measurements: data of sex, age, diagnosis, and outcome were recorded. severity of illness and therapeutic effort in the first 24 hours were measured using acute physiology score and chronic health evaluation (apache ii) and therapeutic intervention scoring system (ties). r~ults: 113 mechanically ventilated patients, 76 male and 37 female, were studied. only ten patients needed tpn and their main diagnoses were: five cases of multiple organ failure secondary to pneumonia (2), ards (2) and septic shock (1); two eases of acute panereatitis; and one mesenteric throngmsis, one status epilepticas, and one ,prolonged cholinergic crisis b~ suicidal organophnsphate insecticide subcutaneous injection. no statistically significant differences between both tpn and non-tpn groups were found: objectives: evaluate the efficacy of prone position in ards and determine its importance in the therapeutic algorithm. methods: 43 consecutive patients with severe ards (murray-score > 2,5; pao2/ fit 2 < 160 mmhg; 29 male, 14 female, mean age 62 years) were conventionally ventilated (pcv, peep 6-16 mbar, i:e=i:i, ppeak < 30 mbar). if after 24 hours pulmonary function did not improve patients were placed in prone position. change from prone to supine position was done every 12 hours. beside ultimate survival, parameters investigated were aado2, pao2/fio2, and venous admixture (qs/qt). results: during the first 12 hours in prone position 39 of 43 patients showed a significant decrease in qs/qt (25.3% vs. 17.8%) and aado 2 (235 vs. 187 mmhg), and an increase in pao2/fio2 (151 vs. 201 mmttg). changes were most pronounced in patients with high qs/qt, and in patients with an onset of ards less than 48 hours before first application of prone position. after an average of 6 position changes (2 to 16) 28 of 43 patients could be weaned from the ventilator. 22 patient could leave tile hospital. i11 the later course letality was primarily determined by additional organ failures and by the severity of the underlying disease. negative side effects were minor, including slight cardio-vascular depression and increase in p~co2, and never posed a limitation to continuation of prone position. especially in patients with septic shock skin lesions in exposed areas could not always be prevented, prone position could easily be combined with all ventilation modes and with all intensive care interventions. also immediately after major surgery and in patients with open packing prone position was possible. conclusions: in this investigation prone position proved to be an efficient and safe method in the treatment of severe ards. patients with a pronounced ventilation/ perfusion mismatch and patients in the early stages of ards appear to profit most from prone position. though the immediate effect on oxygenation is striking, still more the 40% of all patients die from multi organ failure and underlying diseases. a proposed therapeutic algorithm for ards is as follows: if under conservative ventilation (pcv, peep < 20 mbar, ppeak < 30 mbar) pulmonary function does not improve within 12 -24 hours prone position should be applied. when after 2 -3 position changes no lasting effect can be achieved further ventilation modes (e.g. pc-irv, aprv, no, etc.) should be used in addition to prone position. standard intensive care principles, such as fluid restriction and optimization of circulation, apply also to patients in prone position. objectives: nitric oxide reacts with superoxide to form peroxynitrite, an extremely reactive and toxic species. we quantified the presence nitrotyrosine, the stable product of the interaction ' of peroxynitrite with tyrosine residues in the lungs of pediatric patients that died with respiratory distress syndrome (rds). methods: paraffin embedded lung sections, obtained at autopsy, were incubated with a polyclonal antibody raised against nitretyrosine, followed by a secondary fluorescent antibody. alveolar structure-associated fluorescence was quantified using existing methods. results: tissue sections from patients who died with rds exhibited significant specific immunostaining which was uniformly distributed across the blood-gas barrier. in contrast only background levels of fluorescence were seen in the lungs of patients who died from non-pulmonary causes. intense staining was also seen in the lungs of rats that breathed 100% 02 for 60 h, a condition known to result in rds-type illness; no immunostaining was observed in air-breathing rats. conclusions: significant levels of peroxynitrite may be formed in the lungs of patients with acute lung injury. peroxynitrite may be contributing to the pathology of rds by damaging key components of the alveolar epithelium including the pulmonary surfactant system. mechanical ventilation time was prolonged 16,g • 10 days in patients with ardsvs 1,7 _+ l,4 days in control . mean staylcuwas lg _+ 10,g days in the ards group vs 4,9 • 2,7 days in control group postoperative mortality rate was 53% in ards patients vs 5,7% in those without respiratory failure. 1-ards incidence in liver transplantation is low (11,2% in our sene) but it causes high mortality (53%) page, gas ventilation of the perfluorocarbon-f'dled lung, supports gas exchange and circulation in small animals (<15kg) with lung disease. we hypothesized that large animals could be supported by page without adverse effects on bemodynamics. we first elucidated the determinants of gas exchange in normal sheep, and applied them to a model of adult respkatory distress syndrome (ards). methods: using the ventilator settings determined to be optimal in our pilot study (fio2 of 0.6, peep of 5 cm h20, imv of 6 bpm, it of 50%, and tv of 16 ml/kg), sheep weighing 58.9 ~ 8.3) kg had lung injury induced by instilling 2 ml/kg of 0.05n hc1 into the trachea. ten minutes after injury, sheep with pao2<100 ton" were randomized to continue gas ventilation (control, n=9) or to institute page (n=9). page was instituted by instilling 1.6 l of unoxygenated pefflubron into the trachea and resuming gas ventilation at the previous settings. abg's were drawn at baseline, 10 minutes after injury, 30 minutes after injury, and then every 30 minutes for 4 hours. objectives: inhaled nitric oxide (no) can improve oxygenation and decrease mean pulmonary artery pressure (papm) in hypoxemic patients with ards. in severe hypoxemic copd patients, it is not known whether inhaled no can exert a similar effect on hemodynamics and gas exchange. therefore, we investigated die response of inhaled no in hypoxemic copd patients and the results compared with those obtained in a group of ards patients. methods: ten copd patients (age 71_+2y;fev~ 0.98_+0.12l) and 11 ards patients (age 57_+5; lis 2.8_+0.1) mechanically ventilated were studied. hemodynamic parameters were measured using a swan ganz catheter. arterial and mixed venous blood gas determinations, sao2, svo2, hb and methb were measured (abl 500,osm3). mean intratracheal concentrations of no and no2 were continuously monitored using a chemiluminescence analyzer (nox 2000) . during the study the ventilatory pattern and fioz were kept constant. the protocol was for ards group: basalt, no loppm, basal~; copd group: basalz, no lo ppm, no 20 ppm, no 30 ppm and basal2 . after a steady state of 20 rain hemodynamic and gas exchange measurements were performed. a positive noresponse was defined as a 20% increment in pao 2. results: papm was similar in both groups and decreased significantly after no (ards, basal 33.6_+9.7 mmhg, no 29.7 +6.7 mmhg, p <0.01) (copd, basal 27.8_+6.3 mmhg, no-10 24.4_+5.3 nrmhg, p<0.01). all other hemodynamic variables remained unchanged after no. basal oxygenation was higher in copd group (paojfio 2 189_+53 mmhg) vs ards group (paojfio 2 100_+40 mmhg)(p<0.01). after no-10, pao2 increased (69_+20 mmhg to 97_+40 mmhg, p<0.01) and qs/qt decreased (37+11% to 31_+10%, p<0.01) only in ards group. in both groups, significant correlations between basal papm and inhaled no-induced decrease in papm were found. inhaled no-induced increase in pao2/fio2 was not correlated with basal paoflfio2. no responders were 8/11 (73 %) in ards group and 2/10 (20%) in copd group (p<0.05). conclusions. in hypoxemic ards and copd patients, inhaled no decreased mean pulmonary artery pressure. however, oxygenation only ameliorated in ards group because die number of responders to inhaled no were higher in ards group and this effect seems not to be related to the basal hypoxemia. these results might be explained by the v/q abnormalities present in copd patients. grant fis 95/1390. objectives: it has been recently reported that expired con slope as a function of time is modulated by total respiratory system resistance (rrs) in critically ill patients (chest 1994; 105:219-223) . in this study, we analyze the relative contribution of disease (dis), endotracheal tube resistance (rtube), airway resistance (rmin), additional resistance (~rrs), autopeep (peepi) and dylmmic/static elastance (ed/es) to the co2 elimination in different clinical conditions. methods: we have studied 37 adult patients (8 controls, 11 acute respiratory failure, 9 severe ards and 9 copd) mechalfically ventilated (servo 300 and 900c, siemens) without peep. we recorded tracheal pressure, airflow and capnograms. signals were analogic to digital converted for posterior data analysis. objectives: alveolar ejection volume (van) can be defined as the fraction of tidal volume (vt) with minimal dead space (vd) contamination. according to the classical paradigm: limvd_~ [vco2/vt] =facoz, vco2 vs vt relationship tends asyntotically to a constant slope when approaches end-tidal volume. we have defined van as the volume that defines this relationship until a limit of 5% variation. methods: six subjects with normal respiratory mechanics were studied during anesthesia for minor surgery. two subjects, otherwise normals but having high values of total resistance and dynamic compliance, were also studied. capnograms were recorded in steady-state at 3 levels of vt (0.3, 0.5 and 0.8 l) and four levels of peep (0, 5, 10 and 15 cmh20 objectives: patients with ards presented lung abnormalities which originate an increase in airway resistance (rmin), in additional resistance (~rrs) and in static elastance (ers). application of peep further increases ~rrs. capnographic indexes reflect lung ventilation]per fusion inhomogeneities. in these conditions, the effects of peep on lung mechanics could be better understood by simultaneous measurement of capnographic indexes. methods: we studied 3 groups of subjects. n: 8 normal subjects scheduled for minor surgery; arf: 9 critically ill patients with mild acute respiratory failure; ards: 8 patients with early ards (< 72 h). we recorded tracheal pressure, airflow and capnograms. signals were analogic to digital converted for posterior data analysis. respiratory system mechanics was assessed by constant end-inspiratory and end-expiratory occlusions technique. at equal tidal volmne (0.5l) a peep level of 0,5,10 and 15 cmh20 was applied in all patients. we calculated ers (cmh20/l), rmin, c~rrs (cmh20/l/s) and autopeep. capnographic indexes were alveolar ejection volume (vae)/vt ratio and expired co2 slope beyond vae (sipco2 in contrast to synthetic surfactant natural suffactants (alveofact|174 are able to inhibit pmn-activation. after incubation of activated neutrophils with surfactant, l-selectin expression is decreased. these effects depends on which preparation is used. we conclude, that natural surfactant (aveofact| can perhaps influence early recruitment (,,rolling") of pmn in patients with respiratory failure like ards. with ards hormann cb, baum m, putensen c, knapp r, lingnau w, putz g . clinic for anesthesia and general lntensiv care medicine, university of lnnsbruck, anichstrabe 35, 6020 innsbruck objectives: in thoracic ct scans of patients with severe ards atelectasis and pleural effusion can be found in the dependent lung regions. by rotating these patients from left lateral position to right lateral position a redistribution of the ct densities, a recruitment of atelectasis and therefore an improvement of gasexchange is possible within a few days (1, 2). the objective of this study was to find out the mechanism of alveolar recruitment during lateral positioning by ct scanning in left and right lateral position. methodes: after approvel by the local institutional reviewboard we investigated 7 ventilated patients with severe ards (entry criterias: murray score > 2,5) in the ct scann of the university hospital. after a stabilisation period of 30 minutes in supine position a thoracic ct scan slice 1 cm above diaphragm was taken. then two different positions of the patients were studied in a randomized order: a) 60 degree of left lateral position, b) 60 degree of right lateral position. each lateral position was held for 20 minutes. at the end of each of these periods a thoracic ct scan slice 1 cm above diaphragm was taken. quantitative analysis of ct scan data was based on the frequency distribution of the ct numbers. to quantify the alveolar recruitment during lateral positioning by means of ct scan we defined 3 compartments within the lungs: a) normaly inflated lung, b) poorly inflated lung, c) noninflated lung ( = atelectases) (3). results: independant of the side of lateral positioning (l) in the non-dependent upper lung a significant increase of the normaly inflated compartment (s: 45%; l: 65%) as well as a significant decrease of the noninflated compartment (s: 34%, l: 12%) was observed in comparison to supine position (s). in the dependant lower lung the normaly inflated compartment decreased significantly (s: 45%, l: 26%) whereas the noninflated compartment increased significantly (s: 34%, l: 51%). throughout the whole studyperiode we did not observe any significant change regarding gasexchange and hemodynamic parameters. conclusions: in lateral position the non-dependent upper lung is decompressed. therefore a significant recruitment of atelectases is observed in the upper lung within 20 minutes. on the other hand the dependent lung is compressed by the weight of the upper lung and the mediastinum. a great amount of the alveoli of the dependant lung collapse in this short time intervall. therefore the net effect of recruitment of one positioning maneuver is very small. when positioning patients one should be aware, that the patient is kept in each lateral position long enough to clean up the atelectases in the non-dependant lung and short enough to compress less lung tissue in the dependant lung. objective: to analyze effects of low-dose no inhalation ia patients with severe aeut~ respiratory distress syndrome (ards) over five days. methods: we prospectively studied 10 patients (9 men, 1 woman) with severe ards admitted to our icu between may 1994 and may 1995 who required no inhalation with a dose of 5 ppm for at least 5 days. entry criteria for no injaalafioa were murray score >i 2.5 aud pat/fie 2 < 125 nun hg with peep >~ 8 em i~o for at least 24 hours. all patients were sedated, intubated and mechanicauy vantil~ed with volume assist-control ventilation, and had indwelling arterial catheters (pulmonary artery, and radial or femoral artery) to measure cardiac output (by thermodilufion) and relevant intravaseular pressures, and to calculate derived parameters. no was administered between y piece of the ventilator and endotraeheal tube and flow was adjusted to obtain 5 ppm no in the inhaled gas. the no, no 2 and no x concentrations were continuously measured at the distal end of the endouacheal tube by the chemiluminiscence method (nox 4000, see-seres, france). metahemoglobinemia levels were mesured daily. no inhalation was manteined if paojfio ~ improved at least 20 % and was stopped when the change in pao2/fio ~ was below 20% or when the patient presented a paojf02 > 150 mm hg a~er 30 minutes without no inhalation. every day we made an on-off test to determine if no inhalation improved pao2/fio ~. statistics: analysis of vmiance. data: mean + standard deviation. results: the mean age was 60.1 +_ 10.2 years and mean lung injury score was 3.3 • 0.2. mortality was 60 % (6/10), metahemoglobinemia 1.1 • 0.2 %, and no2 concentrations zero. paojf~o 2 always improved significantly al~er 5 ppm no inhalation (see :~ conclusions: reintubation in salf-extubated patients strongly depends on the type of meehamcal venfilatory support: the probability of needing a reintabation ff ese occurs during fult vontilatory support is higher than ff ese occurs during weaning. these data suggest that some patients may remain under weaning from mechanical ventilation for unnecessarily prolonged periods of time. objective: the aim of this study was to evaluate the acute effects on gas exehonge and hemodynamics due to positional changes from supine (sp) to prone (pp) in patients with severe acute respiratory distress syndrome (ards). methods: nine intubated, sedated, paralyzed and mechanically ventilated patients with severe ards were prospectively studied. all had a murray score > 2.5, and a pao2/f~o 2 < 100 with peep ~8 cm h20 for at least 24 h. all patients had indwelling arterial catheters in the pulmonary artery as well as in the radial or femoral artery in order to measure cardiac output (by thermodilution) mad relevont pressures, and to withdraw blood samples. arterial blood gases and hemodynamie parameters were measured first in sp, and then in pp after 60 minutes of stabilization. vontilatoly parameters remaing unchanged during all the study. statistical analysis was done by the non parametric wdeoxon test. data are expressed as mean ~= sd. results: there were 6 men and 3 women with a mean age of 54.2 years (21-71) and mortality was 55 % (5/9). main results are shown below: objective: to describe and compare a new method for obtaining p-v loops (p-vcv) by using a two-way collins valve (twv) with thosu obtained by the supersyringe method (p-vss). methodology: we prospectively studied 14 patients who had an aeute lung injury and were intubated, sedated and paralyzed, and mechanieany ventilated. we performed the p-vev loops and p-vss loops in random order, and the static inflation pressure was limited to 35 emh20 with both methods. pressure (p) was measured at the airway opening by means of a differential p transducer, and volume was obtained from flow (measured with a pneumotacograph) integration. the p-vse method has already been described (h~trf a,et al.bepr 1975; 11:709-28) . the p-vev method consists in the following: the inlet of a twv is connected to the ventilator's y-piece, and both outlets are couneeted to the endotraeheal tube by means of an additional y-piece; one of this outlets has a one-way rudolph valve in order to allow inspiration but not expiration during the inflation maneuver. changing the twv tap position allows basal ventilation or progressiveinflation of the respiratory system. this maneuver is as follows: during an end-expiratory occlusion, the ventilatory settings are adjusted to deliver a 100 ml v r with a respiratory rate of 20/min and i/e ratio 1:4; at the same time the twv tap is ehonged in order to divert flow through the one-way valve. inflation then begins alter releasing the expiratory oonlusion. pressure and flow signals were digitized and acquired by a computer for subsequent data analysis. we analyzed the following parameters: inflation compllonee ( objective: to analyze the variables which eventually may differentiate ards patients who do and do not respond to low doses of inhaled no. we prospectively studied 10 patients (9 men, 1 woman) with severe ards admitted to our icu between may 1994 and may 1995 who were treated with no (5 ppm). the onta'y criteria for no inhalation were murray score >/2.5 and paojfo z < 125 mm fig and peep >/8 cm i~o for at least 24 hours. all patients were sedated, intubated and mechanically ventilated with volume assist-control ventilation. tidal volume was between 6 and 10 ml&g, with constant inspiratory flow, respiratory rate was 15-25/rain, and i/e ratio between 1:2 to 1:3. all patients had indwelling arterial catheters (pulmonary artery, and radial or femoral artery) in order to measure cardiac output (by thermodiintion) and relevant intravascular pressures, and to calculate derived parameters. no was administered between y piece of the ventilator and ondotracheal tube, and flow was adjusted to obi~a 5 ppm no in the inhaled gas. the no, no 2 and no x concentrations were continuously measured at the distal end of the endotracheal tube by the chemilumiinscenee method (nox 4000, see-seres, france). metahemogtobinemia levels were measured daily. we considered a response to no inhalation when an improvement in paoz/fo 2 above 20 % was observed after the inhalation of 5 ppm no (group r) . when the cha~age in paojfi0 z was below 20 % it was considered a lack of response (group non-r small airways functional abnormalities have been recognized as a common feature of lung pathology. however peripheral airways contribute relatively little (~ 10%) resistance to flow and there disturbances can not be adequately estimated by conventional measurements of respiratory mechanics. the purpose of the study was to evaluate the relationship between raw and small airways conductance following weaning from ventilator methods. 37 patients (age:24-62 years; 22 males) with no serious complications al~er mitral or multiple valves replacements and with more than 15 hrs on mechanical ventilation have been enrolled in this study. the modified flow interrupter technique (ptg "gould" with fleish head #2; differential pressure transducer pm-131-tc "statham" w amplifier "kistler 7251") and flow-volume recording of forced expiration (fleish head #4) have been applied before surgery and following operation on mechanical ventilation (my), after extubation (t:xtijb), on 2 (2 nay) and 3 (3 day) days. airways specific conductance (sg aw) has been calculated as a mean of 7-10 consequent measurements in each patient at each stage. the sac was estimated by max expiratory flow at 50 and 25% of vc on 3-4 f-v curves (mef .~0, mef 25) all the data were statistically analyzed with t-test introduction : noninvasive ventilation (niv) reduces the need for endotracheal intubation, the length of stay in icu and the mortality rate in acute exacerbation of copd. however, some patients failed to be ventilated with niv. .objectives...; to further delineate patients who failed to be ventilated with niv and to obtain predicted factors of failure. patients : a cohort of 51 patients (72 • 10 years) presenting with acute exacerbation of copd (fevi: 610 • 396 ml, paco2:62 • 17, ph: 7.33 • 0.08) and nonmvasively ventilated (pressure support through a full-face mask) between april 1990 and may 1994 twenty-seven (53%) were successfully ventilated with niv (discharged alive without the need for endotracheal intubation) while 24 (47%) failed, requiring endotracheal intubation. .methods : patients successfully ventilated and those who failed were compared according to 35 respiratory and nonrespiratory variables univariate analysis (wilcoxon rank-sum test and fisher-exact test) was performed to select variables included in a multivariate analysis by stepwise logistic regression. results : underlying disease assessed by the simplified acute physiologic score (15 • 3 vs 11 • 3, p = 0.0003), creatinine serum concentration (122 • 45 vs 86 • 25 gm/l, p = 0.005), blood urea nitrogen (bun : 12 • 6 vs 8 9 3 mm/l, p = 0.009), age (75 • 9 vs 69 • 10, p = 0.01) were higher and encephalopathy (71 vs 30%, p = 0.005) more frequent in patients who failed. multivariate analysis showed that encephalopathic patients (or (odd ratio) = 4, p = 0.001) older than 65 years (or = 4, p = 0.04) and presenting with bun >_ 10 mmyl (or = 3, p = 0.01) failed to be ventilated with niv. variables related to the respiratory" status (i.e. paco2, pao2, fev1) were unable to predict tile failure of niv. conclusion : copd patients older than 65 years, presenting with acute exacerbation, encephalopathy and bun > 10 ram/l, should be carefully monitored because of high probability of failure with niv. methods:from february to december 1994 we studied 30 pa_ timnts,25 males and 5 females(mean age 68+/-5);18 of the se had emphysema,lo chronic bronchitis,2 dilatative car diomyopatia,with tracheostomy and emphysema.mean pac02 at admission in icu was 95+/-8mmhg,while when weaningbegan, 60+/-5.mean autopeep was 8 cmh20(4-12).all patients were ventilated in crpv as long as four hours to calculate st8 tic and dynamic cmpliance and autopeep.then the ventila tion was continued with psv+cpap(peep 7cmh20 objectives: analysis of the incidence of neurogenic pulmonary edema (npe) in a population of headtrauma patients with acute respiratory failure (arf). npe can occur after a central nervous system insult. differential diagnosis: cardiogenic pulmonary edema and other forms of non eardiogenic pulmonary edema. true incidence and pathophysiohigy remain poorly defined, however the role of catecholamines seems undeniable. early onset npe (within 12 h after trauma) is characterised by hypoxemia, transient pulmonary hypertension and bilateral central fluffy infiltrates on chestx-ray. characteristics of cardiogenic edema or pneumonia are absent. late onset npe, (beyond 12 hours after trauma), is more insidious. the clinical and radiographic picture has to clear within 24 to 48 hours. (1) methods: all headtrauma patients admitted from january 1 to december 31, 1993 in a nearotrauma icu setting were retrospectively analyzed for arf with as sole criterinm a pao2-fio2 ratio < 250. results: 151 neurotrauma patients were admitted during 1993.94 patients (63%) presented with severe head injury (gcs<8), 42 patients (27.8%) with moderate (gcs 8-12) and 15 patients (9.9%) with minor head injury (gcs 12-15). overall mortulity was 19.2% early (within 12 h. after trauma) and delayed onset respiratory incidents were distinguished, counting for 29 (19.2%), respectively 27 patients (17.8%), 7 patients (4.6%) had early and late respiratory complications. early respiratory insufficiency was caused in 9 patients (25.0%) by aspiration, in 11 patients (30.1%) by lung contusion, in 1 patient (2.7%) by fat embolism and in 15 patients (41%) by npe. in the late onset group 31 patients (91.2%) presented with pneumonia, 1 (3.0%) with fat embolism and 2 (5.8%) with npe. the npe group, 17 patients, presented as follows: 15 patients (88.2%) developed early npe, and 2 (11.8%) delayed onset npe. 9 patients (53%) died within the first days after admission, showing high mortality. gcs was less than 8 in 16 patients (94.1%), indicating severity of head injuries. conclusions: high incidence of arf with various etiology (41,7~ was found in this population. in about 10% of all admitted hcadtrauma patients (26,9% of arf) npe was causing attetial hypoxemia. occurrence of npe seems to be related to the severity of the brain injury and thus to outcome. these data call for extreme vigilance in respect of the insidious occurrence of npe. were included if recovering from respiratory failure and if in the opinion of the primary physician were ready for extubation. patients were excluded if undergoing compassionate withdrawal of support or had tracheostomies. the attending physicians were blinded to the measurements. included patients were placed on pressure support (ps) of 0 em h20 with demand-flow continuous positive airway pressure (cpap) 5 cm h20. after a minimum of 30 minutes on the above sehiogs: gastric intramucosai pc'o2, abg, and a p0.1 were measured. the padents were then disconnected from the ventilator for a period of one minute and the patients" respiratory rate and minute ventilation were measured using a wrights respirometer to calculate the frequency to tidal volume ratio (f/vt). patients were then extubated. extubafion failure was defined as the inability to maintain spontaneous ventilation for 24 hours for any reason. results: twenty patients met criteria and were studied over one month period in october 1994. six of the twenty patients (30%) failed weaning. the mean and standard deviation is outlined in failure 7.01+/-0.10 6.8+/-3.9 74.3+/-43.3 87.5+/-43.6 comparison between roc areas shows phi and p0.1 to each show a statistically significant difference from an area of 0.5 (p<o.05). the area at which the test has no discriminative value. this is not the case for either the integrative index or the f/vt ratio. the differences between areas for each curve is not statistically significant. the area's for each roc curve is shown in table #'2. results. 9 of the 21 newborns referred to us for ecmo-therapy developed barotrauma, 6 of the 9 required ecmo and one of the three other patients who did not recieve ecmo-therapy died. to determine the correlation between the risk factor barotrauma and the severity of the pulmonary situation, we determined the oxygenation index of each patient referred to us for ecmo-therapy . the table demonstrates 36, d-35392 giessen, frg. inhalation of nitric oxide (no) and aerosulizatiun of prostaglandin (pg) i: have been suggested to achieve selective pulmonary vasodilation and improvement of arterial oxygenation in patients with acute respiratory distress syndrome (ards). we directly compared these two modes of transbronchial vasodilator therapy in 16 ards patients mechanically ventilated for 24 -96 h (mean murray lung injury score [1] 2.75 + 0.05). patients were randomized to receive either first no and then pgi2 (n=8), or vice versa (n=8), with an intermediate baseline period. each drug was titrated individually to find the lowest effective dose for maximum improvement of arterial oxygenation. gas exchange variables, including data from the multiple inert gas elimination technique (miget), and hemodymmaics under application of no/pgi2 were compared to pro-and past-challenge values. no (mean dose 17.8 + 2.7 ppm) increased the mean oxygenation index (pao2/fio2) from 115 + 12 to 144 + 15 mmi-ig (p < 0.01) and reduced the shunt-flow from 33.1 + 3.6 to 26.6 5:4.5 % (p < 0.05). aernselized pgi2 (mean dose 7.5 + 2.5" ng/kg rain) augmented pao2/fio2 from 114 + 12 to 135 + 12 mmhg (p < 0.01), and decreased shunt from 33.5 + 3.8 to 26.0 + 3.9 % (p < 0.05). the miget analysis showed predominant redistribution of blood-flow from shunt areas to regions with normal ventilation-perfusion ratios in response to both agents. in 10 patients, both no and pgi2 caused an increase in pao2/fio2 by at least 10 rnmhg. two further patients displayed a corresponding improvement of arterial oxygenation in response to either no or pgiz. the mean pulmonary artery pressure decreased from 34.8 5:2.2 to 33.0 5:1.8 mmhg in response to no, and from 35.0 5:2.2 to 31.9 5:1.7 mmhg (p < 0.05) in response to pgi2. cardiac output, systemic arterial pressure, and right and left heart filling pressures were not affected by either agent. we r that individually titrated doses of inhaled no and aerosolized pgi~ effect selective pulmonary vasodilation and redistribute blood-flow from shunt-areas to weu-ventilated regions with nearly identical efficacy profiles. obieetives: the use of extraeorporeal bypass systems for oxygenation and co2-removal is performed in patients with acute respiratory distress syndrome (ards) to reduce mortality and to improve restitution of pulmonary functions. high-dose heparin -commonly used in such patients to prevent thrombotic complications -might cause incurable bleadings as a major risk. this lead to the development of heparinized bypass systems; results of animal and first clinical studies proposed that such systems might run with low-dose heparin or even without any systemic anticoagulation. methods: since 1989, 47 patients with severe ards were treated with extracorporeal lung assist (ela) using heparin-coated systems (type maxima, medtronic, carmeda coating) in our icu. they received a moderate systemic heparinization. standard clotting assays (act, aptt, tt, ptt, at-ill, fbg, fsp) as well as special tests (tat, r~-tg, pf4, pal-l, d-dimers, protein c, cl-inhibitor) were performed. results: covalent heparin-eoating of the ela-systems combined with lowdose heparinization could not prevent major changes in coagulation: 1) in mean, platelets dropped from 305/nl to 76/nl within 60 h after onset of ela. 2) tat levels were already elevated before ela start (55 ug/]) and demonstrated an additional peak 2 h after onset of ela (up to 240 ug/[). 3) in parallel, there was a transient peak of pai-i levels (from 5 to 75 au/ml} 2 h after onset of ela. 4) in mean, fibrinogen levels dropped from 470 to 310 mg/100ml within 24 h after onset of ela. 5) in mean, cl-inhibitor levels dropped significantly from 132% to 115% after onset of ela and reached the initial levels within 48 h. 6) after membrane change, there were significant changes for c1-inhibitor, fibrin-d-dimers and tt. 7) global clotting tests such as aptt, ptt and tt were within normal range. conclusions: patients with severe ards develop a prethrombotic state already before they get connected to the ela bypass system. after onset of ela, they experience additional involvement of procoagulant, anticoagulant, fibrinolytic, and complement systems. in parallel to laboratory findings, clinical data suggest that the use of heparin-coated systems with accompanying systemic low-dose heparinization does not avoid thrombembolic and hemorrhagic complications in tong-term ela patients. thus, at the present, higher dosed individually adjusted heparin treatment has to be recommended. thereby, standard clotting monitoring is not sufficient. objective: poor muscle performance contributes to prolonged dependence on mechanical ventilation. longitudinal data on muscle function in icu patients is scarce. we evaluated changes in inspiratory and peripheral muscle performance during prolonged intensive care in patients with acute respiratory failure. methods: 22 patients requiring intensive care for more than one week were studied. maximum inspiratory pressure (pimax) was measured twice a week for a maximum of three weeks. handgrip power (dynamometer) and subjective fatigue (keldet score) were obtained in survivors only. one measurement was done on the date of extubation. pimax pressure was obtained by occluding the airway for 20 seconds or at least five inspiratory breath attempts. obiectives -measurement of peep-induced changes in lung volume helps to assess respiratory mechanics in acute lung injury (ali). we evaluated the accuracy and stability of a new respiratory inductive plethysmograph (rip), in the measurement of peep induced changes in end-expiratory lung volume (eelv) and tidal volume (vt) against volume reference, pneumotachograph (pnt), methods -two hours periods of basal ventilation and stepwise increases and reductions of peep (5-10-15-10-5 cm h20) were recorded in patients with all. in addition, the new rip device (respitrace plus tm) was compared to an older rip (respigraph tm, nims, fl, usa) to determine its thermal stability using cold and warm devices and a ventilated lung model. results -the difference between the new rip and pnt in the measurement of vt was -30 _+ 3 mi (x _+ se) or a -5.2 _+ 0.6 % error. increasing peep from 5 to 15 cm h20 induced a 511 + 93 ml change in eelv. a difference of 18 + 12 ml (3.5 % of max. eelv change) (ns) was found between rip and pnt eelv readings. during controlled ventilation with constant settings the mean change in eelv was 187 _+ 69 ml/120 min (fig.i) . validation of calibration showed a mean error of 1 -+ 1.78 % after 120 rain. the new rip had a higher drift when cold (cold 28 + 10 ml/30 min vs warm 3 _+ 1 ml/30 min), whereas the old rip was thermally stable (cold -3 + 3 ml/30 rain vs warm i + 1 ml/30 min). trauma icu, hospital traumatologia y rehabilitacidn. vall d'rebron. barcelona. spain. recent data suggest that the proportion of patients with relevant discrepancies between two jugular bulb (jb) oxygen saturation (sjo 2) values is higher than suspected. objectives: determine the incidence, the significance and the pro9nosis value of those differences,if they exist,in severe head injured-patients. material and methods: 35 severe head-injured patients, coma glasgow scale (gcs) 8, with diffuse brain injury according to marshall criteria in the first ct scan, icp recorded, with an interval from accident-double jb monitoring < than 12 hours, were studied. data from bilateral sjo 2 were obtained simultaneously, every 6 hours. discrepancies were considered significant, when differences in sjo 2 were > 10%. no chan9es in treatment protocol (hyperventilation, man• etc) were carried out due to this study. results: 30 men and 5 women were studied, aged 32• yrs. at arrival at hospital, gcs were < 5 in 20 and ) 5 in to. the incidence of high icp() 20 mmhg) were 7sz at the entry. the mean therapy index level required to control lop was 4~l all patients required vasopressor therapy to maintain upp over ds mmhg. in 20 patients a s.s f swan-ganz fiberoptic catheter was used to obtain a continuous recording of sjo 2. in the others 15, sj02 were intermittently controhed.the mean time of monitoring were d.8• days. ten patients died within this period. a total of 1.240 blood samples were analized. at arrival, sjo 2 discrepancies were found in 22 patients, b2%. at 48 hours, the incidence were lower, 18/35, 51.4%. at 4th day, were h/29, 38z and at day 7, when the catheters were retired, ii[25, 44z showed discrepancies. the ct showed new injuries in g4z of patients with differences > 10~ in sd02 values throughout treatment period. none of those were considered for neurosurgical treatment. no correlation was found between iop and sjo 2 values and sjo 2 differences. conclusions: the incidence of discrepancies between sjo 2 was higher than expected in severe head-injured patients. these situation could reflect disturbances between 02 demands. when differences are known, and those lend to change, the ct scan, nearly always, will show new injuries. platelet-activating factor (paf) is an inflamatory mediator implicated in the pathogenesis of bronchial asthma and acute respiratory distress syndrome (ards). its inhalation in healthy subjects produces transient bronchoconstriction and mild ventilation-perfusion mismatch, together with peripheral leukopenia as a result of intrapulmonary neutrophil (pmn) sequestration. likewise our group has shown in healthy subjects and asthmatic patients that aaibutamol (s) inhibits both pulmonary and systemic effects of paf, suggesting that s may inhibit paf-induced venoconstriction in pulmonary microoirculation. the aim of the present study was to investigate if s inhalation decreases pmn by lung sequestration induced by paf. we studied 8 healthy, non-atop• nonsmoking subjects (6m/2f, 24+4 yr), which were pre-treated with s (300,ug) or placebo, with a randomized, double-blind, crossover, design, before paf (24,ug) inhalation. we measured the respiratory system resistance (rrs) by forced oscillation, arterial btood gases and both total white cell and pmn count every 4 min over a 30 min. period. simultaneously, we recorded continuously the lung dynamics of inm-neutrophil and tc99m-erythrocytes activity, with a gammacamara. after placebo, paf inhalation decreased white cells (from 5410 2 1125 to 33022934x109/l), and pmn(from 29752693to 1222 _+ 767 x109/l), and increased aapo 2(from 2.1 _+9.5 to 14.7+ 12.2 mmhg, p<o.05) and rrs(from 3.0 2 0.6 to 3.7 2 0.7 cmh20.s.i q ) (p < 0.05 each). the fall in total white cell count correlated with the intrapulmonary pmn retention (r =0.83, p <0.05). by contrast, after s, paf did not induce any change in white ceil count (from 596321766 to 6147+1721 x109/i), pmn count (from 354321628 to 2953 2 1793 x109/i), aapo 2 (from 7.3 2 10 to 5.6 + 8.6 mmhg), and rrs (from 2.9 2 0.5 to 2.9 2 0.7 cmh20.s.i 1). compared with placebo, after s there was a lower ~lln-pmn lung activity (2.020.4 vs 1.320.7 cts/mci/pixel, p<o.01 ), lower intrapulmonary pmn retention (15.627.7 vs 11.1 24.8 %, p=o,09), and a faster washout (0.1420.08 vs 0.1720.007 s ~, p=0,4). our results indicate that s inhibits paf-induced intrapulmonary pmn sequestration, being consistent with the hypothesis that s may offset the venoconstriction of pulmonary microcirculadon caused by this mediator. supported inpiratory muscle fatigue with failure of force generation as def'med by a tensiontime index (tti)>0.15-0.20 has been shown to occur in normal volunteers and in stable copd patients with a specific imposed breathing pattern. its role, however, in hypercapnic respiratory failure is less certain. we studied 10 failed weaning trials in 5 copd patients in which breathing pattern, tension-time index (tti) of inspimtory muscles, dynamic peepi, dynamic lung elastance, lung resistance, and arterial paco2 and ph were measured at the beginning and end of a t-piece weaning trial. in addition, the change in esophageal pressure during a mueller maneuver (apes max) was measured. a weaning trail has been prospectively defined to have failed if one of the following criteria was met: a rise in pco2 >20mmhg from baseline accompanied by a fall in ph<7.35; a respiratory frequency (f) >30/min; excessive accessory inspiratory muscle recruitment; and a marked increase in dyspnea. values are expressed as mean • se. weaning failure was characterized by a more rapid, shallow breathing pattern, worsened mechanics, hypercapnia and respiratory acidemia despite an unchanged tri and pes max. we conclude that in this setting hypercapnic respiratory failure is not a consequence of inspiratory muscle fatigue. rather the adopted breathing strategy and resultant hypercapnia may represent an adaptation to forestall the onset of muscle fatigue. concerning the investigated elf-par~eters, no stadstically signhqcant differences were detected between the pgi2 and the control group. histopathologlcal changes occured in both groups and consisted in rare focal flaaaning 0f tracheal epithelium with loss of cilia and slight inflammatory cell infiltration, as well as slight swelling of alveolar typo4 pneumoeytes. sections of generation 5, 10 and 15 from bronchial tree were free of pathological changes. conclusion: alter 8h inhalation of p~ji2 no signs of respiratory-lract tissue damage caused by the aerosol could be detected. the minor pathological findings in the trachea are most likely due to mechanical irritation by bronchoscopy, changes of the alveolar epithelium are known for long-term mechanical ventilation 3. objectives: the aim of this study was to evaluate of efficiacy of ganglion stetlate blockade in patients with respiratory failure. methods: two groups of patients were investigated: group i (n = 15) trauma patients with acute lung injury (ali), group if (n = 15) patients with asthmatic status. in all cases continuous mandatory ventilation (cmv) was used with bennett 7200 ae. in both groups bilateral ganglion stellate blockade with antero-lateral approach was performed, using 0.375 % marcain. the following parameters were analysed: pao2, sao2, paco~, pip and c~t~t. results: in trauma patients with aij after bilateral ganglion stellate blockade short -lived and slight improvement of pao 2 and sao2, decrease of pacoz and pir and increase of static compliance of respiratory system were found. in second group bilateral ganglion stellate blockade interrupted the asthmatic status and significant statistical improvement of parameters of oxygenation, ventilation and respiratory system mechanics were observed. conclusions: we suggest that the bilateral ganglion stellate blockade is a very useful method in treatment of patients with obstructive respiratory insufficiency. the aim of the study was to analyse whether there exists serum and urine electrolyte disorder in patients(pts.) with acute respiratory insufficiency(ari). the study included t8 pts. with ari (pao2:8,24@1,49 kpa. paco2: 5,01i-0,77kpa, ph:7 42~:0,59, hco3: 26,3:~8,10 mmol/1, sao2 : 90,4~-7,42%) who were hospitally treated due to pneumonia(9 pts.),emboly of the pulmonary artery(3 pts.) and severe attack of bronchial asthma (6 pts). among tham there were 12(66,7%) males and 6(33,3%) females, average age 51,5~:16,1 years, otherwise previously healthy. electrolyte concentracions were measured at the onset of the disease in serum and urine collected during 24 hours (sodium-na,potassium-k, chlorine-c1, calcium-ca,magnesium-mgand phosphorus-p). the measured serum and urine electrolyte concentrations were compared with respective referent values (rv). by serum electrolyte analysis, the following average velues were obtained: na:l 4o,94 the object of our investigation was a group of 21 pts with massive pneumonias, 14 males (66.6%), 7 females (33.3%),mean age 55 yrs.thirteen (62%) of them were smokers,8(38%) nonsmokers. only 1 pt (4.7%) had pre-existing chronic respiratory disease, and 20 (95.2%) were admitted for the first lime,with no previous respiratory anamnesis. diagnose was based on anamnestic data of productive cough in 15 pts(71.4%),physicaly ~onchial breathing in 19 i~s (90.4%),white cell count onder 10 x 109 /l in 18 pts(85.7%). radiographicly, bilateral massive homogeneous shadows were found in 7 pts (33.3%), onilateral in 12 pts(57.1%),pleural effusion in 2 pts (9.52%). abnormal renal function was found in 14 pts (66.6%). sputum culture was positive in 8 pts (38%): slr.pneumoniae, str.pyogenes, pse'udomonas aerug, in 4, 2, 2 cases respectively. all patients had remarcable hypoxernia (pao2 range from 4,75 to 8,1 kpa) without hypercalmea. all patients needed oxygenotherapy together with antibiotics and other .symptomatic therapy. nineteen pts had anaelioration of general condition and normalization of blood gas analyses, while 2 pts with the lowest hypoxcmia died.in conclusion, massive pneumonias are frequently followed by respiratory insufficiency which is one of the markers of pneumonia severity. as existing hypoxemia complicates the course of the disease,prolonges the recovery, makes therapy more complexe and may be cause of death , frequent blood gas measurement is recomanded. we studied the effects of bosentan (bos), an eta and etb receptor antagonist, to examine if endogenous et mediates pulmonary hypertension in anesthetized and ventilated dogs with acute lung injury due to oleic acid (oa). the gradient between pulmonary artery pressure (ppa) and occluded ppa (ppao), and gas exchange (evaluated by arterial blood gases and sf6 intrapulmonary shunt) were measured at controlled flow. in 8 dogs (treatment), data were collected at baseline, during long injury (obtained 90 rain after intravenous administration of oa 0.06 ml/kg), and again after bos (10 mg/kg intravenously). in 5 dogs (pretreatment), data were obtained at baseline, after bos and then after oa. in treated dogs, oa increased (ppa-ppao, mmhg, table, means + sem, * p < 0.05 vs base) and deteriorated gas exchange. after oa, bos did not affect pulmonary vascular tone nor gas exchange. in pretreated dogs, bos had no effect on baseline pulmonary vascular tone but prevented the increase in (ppa-ppao) after oa. the deterioration in gas exchange after oa was not influenced by bos pretreatment. objectives: the alveolar 02 tension is measured by the application of the alveolar air equation in which the arterial pco 2 is used or by the simplified form of this equation in which the respiratory exchange ratio is taken at the value of 0.8. the purpose of this study was to estimate the effective alveolar 02 tension (pao2eff) during spontaneous breathing with a new bedside technique which is simple non-invasive in 14 normal subjects and 27 patients with chronic bronchitis-emphysema. we also compared these values with the ideal alveolar po 2 (pao2(i)), measured from the alveolar air equation in which paco 2 was substituted by the effective alveolar pco 2 (paco2eff) and with the alveolar po 2 measured from the simplified alveolar air equation (pa02). this study is complemantary to previous work for the estimation of paco2eff. methods: the subjects breathed quietly through the equipment assembly (mouthpiece monitoring ring, fleisch transducer head) connected to a pneumotachograph and a fast response 02 and co 2 analyzer. the method is a computerised calculation of the effective alveolar po2quite similar to that of paco2eff, obtained from the simultaneously recorded at the mouth expiratory flow, 02 and co 2 concentration versus time curves. results: the results showed a mean difference (pao2eff-pa02(i)) of -0.061 kpa in normal subjects and -0,711 in patients. the mean of the difference (pao2eff-paq 2) and (pad2(i]-pao z) was much greater than 0.281 in all subjects. the limits of agreement for the difference (paozeff-pa02(i))were -0.691 to 0.568 kpa in normal subjects and -2.040 to 0.596 in patients, while those for the differences (pao2eff-pad 2) and (pao2(i)-pad 2) were very large ( > -1.5 to > 1.7) in all subjects. conclusions: the effective alveolar po 2 is very close to the ideal one in normal subjects, tn patients pao2eff may excessively deviate from pa02(i) due to the observed significant difference between the alveolar/tidal volume ratio for o 2 and that for co 2. the alveolar po 2 measured from the simplified alveolar air equation (pao 2) differed substantially from pao2eff and pad2(i) in all subjects. the essential role of glucoprotein hormone erythropoietin is to control red cell production. hypoxemia, reduced blood 02-carrying capacity and increased affinity of hemoglobin for 02 are the primary stimuli for erythropoietin production. both anemia and hypoxemia induce rapidly erythropoietin secretion. kidney erythropoietin rna levels correlate inversely with hematocrit and directly with plasma erythropoietin level. similarly, hypoxemia increases kidney erythropoietin rna and plasma erythropoietin. the effect of hyperoxemia (pa02>lo0 mmhg) on erythropoietin secretion isn't very well understood. the purpose of this study was first to evaluate the erythropoietin secretion in patients with acute respiratory failure and second to determine the effect of hyperoxemia on erythropoietin secretion in patients with and without anemia. sixteen patients with acute or acute on chronic respiratory failure needed mechanical ventilation were included in this study. these patient were divided in two groups. the patient who developed anemia were included in group i and the patients without anemia in group i1. erythropoietin was estimated in venous blood in three stages. the first sample was taken during hypoxemia, the second during hyperoxemia and third during normoxemia. all the patients had high erythropoietin level during the hypoxemia period (mean value 98• mu/ml). during hyperoxemia etythropoietin levels were reduced in both groups ( mean value 21.6+15.2 mu/ml in group i, 36.8• mu/ml in group ii). in normoxemia stage, erythropoietin increased again in anemic patients, and decreased more in the patients of group i1. we conclude that hyperroxemia inhibit erythropoietin secretion in spite of anemia and tow arterial oxygen content. hyperoxemia may be a factor of the insisted anemia in with oxygen treated icu patients. the purpose of this study was to determine the relationship between clinical features of acute lung injury (all) and parameters like total proteins, total and individual phospholipids, the presence of paf, and acetylhydrolase activity in bal of mechanically ventillated patients. acetylhydrolase catalyses the cleavage of acetyl-group from the second position of the glycerylether backbone of paf, leading to its inactivation. mechanically ventillated patients were divided to three groups. group i includes patients without all; group ii, comprisespatients with moderate degree all, (1.0<g-i1<2.5); and group iii comprises 5 patients with severe ards (all score > 2.5). broncoalveolar lavage (bal) was obtained after infusion of normal saline at 37~ to intubated patients and cooled immediately. cells were removed after mild centrifugation (350 x g, 30 min, 4oc). aliquots from the supernatant were used for total protein, phospholipid and paf analysis and determination. acetylhydrolase activity was assessed after incubation of bal with 3h-paf labelled on the acetyl group. released label was measured by liquid scintillation counter in the supernatant after trichloroacetic acid precipitation of the non-reacted substrate. kinetic characteristics of the enzymes were also studied. total phospholipids appear reduced in bal of patients with all, while total proteins increase. these factors appear to correlate with the severity of all. paf was not present in bal samples pretreatad with equal volume of 20% acetic acid to denaturate acetylhydrolase. detection limit for paf under our experimental conditions: 60 pg paf/ml bal. instead, acetylhydrolase activity was detected in amounts increasing with the total protein content. background: intubated patients without lung injury or impaired breathing control normally display an inspiratory peak flow of below 1l/s. the aim of our study was to investigate the inspiratory peak flow generated by patients with acute respiratory insufficiency (ari). we had to take into account that both an inspiratory pressure support (ips) and the resistance of the endotracheal tube considerably influence the flow pattern generated by the patient. patients and methods: to investigate the non-influenced flow pattern we developed a new ventilatory mode which automatically compensates for the flow-dependent resistance of the endotracheal tube (automatic tube compensation, atc). furthermore, the mode maintains a constant tracheal pressure in inspiration and expiratio n . consequently, the measured flow pattern exactly corresponds to the flow pattern generated by the patient except that the ventilator modified for this mode (evita, driiger liibeck, germany) was not able to deliver a gas flow of more than 2l]s. we have investigated 10 patients with ari arising from different reasons. results: the inspiratory peak flow measured in the atc-mode was 1.7l/s _+0.3l/s. the maximal deliverable flow of 2l/s was obtained in 3 of 10 patients. the figure shows the flow pattern under atc and ips in [~s] oi:) one of these patients. conclusions: patients with ari display a highly increased inspiratory peak flow. ventilators used for spontaneous breathing should therefore be able to deliver a gas flow of more than 2l/s. an overproduction of no and reactive oxygen species (ros) has been demonstratred in septic shock. ros and nitric oxide (.no) are free radicals which are known to react together leading to peroxynitrite anions that can decompose to form nitrogen dioxide (no2) and hydroxyl radical (oh~ thus, no has been reported to have a dual effect on lipid peroxidation (prooxydant via the peroxinitrite or antioxidant via the chelation of ros). in the present study we have investigated in different models the in vitro and in vivo action of no on lipid peroxidation. copper-induced ldl oxidation was used as an in vitro model of lipid peroxidation. ldl (100 ~g apob/ml) was incubated with cu 2+ (2,5 ~tm) in presence or absence of no donor (sodium nitroprussiate or glutathione-no) from 10 to 500 ~m. oxidation of ldl was monitored continuously with conjugated diene formation (234 nm) and 4 hydroxy nonenal accumulation (hne). exogenous no prevents in a dose dependent maner the progress of copperinduced oxidation. ischaemia-reperfusion injury (i/r), characterized by an overproduction of ros, is used as an in vivo model. anaesthetized rats were submitted to 1 hour renal isehaemia following by 2 hours of reperfusion. sham operated rats (sop) were used as control. lipid peroxidation was evaluated by measuring the hne accumulated in rat kidneys in presence or absence of l-arginine or d-arginine infusion. l-arginine, but not darginine, enhances hne accumulation in i/r but not in sop (<0.05 nmol/g tissue in sop versus 0.6 nmol/g tissue in i/r), showing that in this experimental conditions, no produced from l-arginine, enhances the toxicity of ros. this study shows that the pro-or antioxydant effects of no are different in vivo and in vitro and could be driven by environemental conditions such as ph, relative concentration of no and ros, ferryl species...these conditions are impaired in circulatory shock. methods:" the diagnostic and therapeutic approach was standardized so that data collected over a 10-year period were comparable. a progressive deterioration of clinical conditions and/or pulmonary gas exchanges was considered as indication for my. variables potentially predicting the need for hv were derived from clinical and arterial gas data, extrapulmonary diseases, use of drugs, chest x-ray and ecg abnormalities. results: rv, performed with external and/or internal ventilators, was necessary in 130 patients (22%). at the hospital admission, pac02 was higher and ph was lower in patients requiring rv ( pneumomediastinum, pneumothorax, ateleetasis and myocardial infarction are rarely seen in bronchial asthma. these complications occur as a result of the severe asthma.the aim of our retrospective study was to analyse the complications seen in acute asthma attacks. during the years 1990 through 1994, 244 patients were admitted to hospital in acute asthma episode. there were 11 (4,5%) pts with complications; mean age of 27 yrs; 6 females (54%). clinical history, ecg and chest radiogr~hs were analysed. the mean duration of bronchial asthma was 14 yrs (range from 2 months to 17 yrs), all patients were atopics. there were four ex-smokem and one smoker. the worsening of asthma symptoms begun two days before the admission (range from 1 to 7 days). on ecg all patients had tschycardia. rightward shift of the qrs axis and st-t changes indicative of right ventrieutur strain were found in three pts. these were the transient fmdings that improved after curing the acute asthma attack. non-q myocardial infarction oeeured in one patlent and resulted from the hypoxaemia of asthma. hyperinfl~ion was the usual finding on the chest radiograpk pneumomediastinum and subcutaneous emphysema were apparent in five pts and required no additional treatment unilateral pneumothoraccs were present in two pts and needed eontimous intrapleural drainage; one of these patienst died in eardiorespiratory insufficiency. ateleetasis of right upper lobe was present in one patient. it oceured due to inspissated secretions and needed no additional treatment all these patients, except one who died, improved on lreaanent with oxygcr~ steroids, beta-two agonists, theophylline and antibiotics. in conclusion, complications occur in acute asthma episodes as a result of the severe asthma mediastir,*l emphysema and atelectasis are not serious complications. pneumothorax and myocardial infarction are very serious life-treatening complications and always have to i:m considered in taati~ts with sev~ asthma. acute bronchial asthmatic episodes represent one of the most common respiratory mnergendes, its maximmum expression "status asthmatiens" is one entity of low incidence, still it is a risk to the physical integrity of the patient. during 1993 a total of 52 patients with diagnosis of status asthmabcas were hospitalized. out of these palients six had a near-fatsl asthma and they were subjected to a complex examination. near-fatal asthma was defined as either respiratory arrest or acute asttuua with paco2 greater than 6,7 kpa and/or an altered state of consciousness. mean age was 56,2-d:16,2 yrs, four male and two female sex. at presentation two patients suffered from coma, others were confused. they exh'bited severe dystmoes, diffieul~ speaking, used accessory muscles of respiration, increased whee~tg while two cases had silent chest on auscultation. cyanosis indicated a very severe asthma attack in all six patients. mean respiratory rate was 28~4/min and puts rate 118.d: 12 bts/imn. arterial blood gases revealed a pao2 of 6,95~1,33 kpa, paco2 of 7,87• kpa and ph of 7,274-+-0,132. area-careful evaluation they received conventional therapy (immediately continuous oxygen, impelled nebulization with high doses of betatwo agonists and ipmtropium bromide, intmvanous st~oids and theophylline). in two eases signs and symptoms of deteriorating airflow and respiratory muscle fatigue determined the need for mechanical ventilation. out of six near-fatal attacks aggressive lrealanent was suscessfull in four patients and fatal in two eases. one patient admittcxl in coma died in severe hypoxae~a upon one hour and one mechanicaly ventilated died from cardiac arrhythmia. life-threatening attacks in asthmatics in our group developed gradual worsening despite neatment which r symptoms in most other patients. one patient had "brittle asthma", other long-standing acute episodes ireated with systemic steroids. conclusions: idantitiechon of fatality prone subjects may lead to fttrther muetion of seveze episodes. respiratory affest and coma upon admission, severe dyspnoca with silent chest on ausouhation, oyanusis and use of accessory muscles of respiration constitute the basic cfinieal picture. hypoxasmia must be immediately eon'ected.the patients and physicians should be able to assess the severity of asthma, a major factor in near-fatal and fatal asthma attacks. objectives :our purpose was to asses if the evolution of patients with a adult respiratory distress syndrome (ards) ,shows any relation to the pulmonary or systemic origin of the disease and whether or not there were differences in the frequency of the syndrome in both groups. methods : randomized prospective study in multidisciplinary icu. one hundred and sixteen patients with a high risk developing ards were distributed into two groups. one was named systemic origin group(so) and the other pulmonary origth group (po).ai1 patients only showed one cause (pulmonary or systemic) with potential risk of ards.the patient's hemodynamic and respiratory status was evaluated every 6 hours the first day and every 12 hours the second and third day. at the end of 72 hours the patients were diagnosed as ards or non-ards. measurements and main results : of the total 116 patients, 57 were finally included in the so group and 59 in the po group.patients in so group and po group had comparable ages (p<.01).peep in both groups was comparable (=.06) at the mmnent of admission to the study. there were no statistically significant differences for cardiac index and systemic vascular resistances. the pulmonary vascular resistances (pvr) showed significant differences at 48 h.(p<.05) and 72 h. (p<.03).the oxygen comsumption (vo) in patients of the so group showed statistically significant differences at 48 h. (p<.05) with respect to initial values.fifteen cases of ards (26.3%) in the so group and twenty five cases (42.3%) in the po group were identified. the time of onset of ards was 35_+ 14 hours in the so group and 11 + 4 b hours in the po group.the final outcome was very similar th both groups : mortality of 36% in the so group versus 37% in the pc group. conclusions : the pathogenesis of ards depends on whether the lesion is originated at or outside the lung. the po group showed a sborter thne of onset of ards, a faster and more severe increase of pulmonary shunt and a higher percentage of patients developing ards compared with patients of the so group.the so group showed a higher and faster increase in puhnonary resitances tbat po group and a decrease th oxygen comsumption earlier and more severe than in the po group. these data thus seem to show that there could be two mechanisms involved in the genesis of ards depending on the cause. the fact that the ards genesis is shorter in the cases of pulmonary etiology with faster impairment of pulmonary shunt, and a slower increase in pulmonary resistances in this pulmonary group, would indicate that the underlying mechanisms responsible for the hypoxemia are different to those which thitiate the increase in pulmonary resistances. finally, the exclusive inapairinent of oxygen consumption, which appears earlier than the onset of ards in the systemic origth group, could show the generalized character of the process in this group. perfusion of prostacyclin (pgi2) to treat pulmonary hypertension in adult respiratory distress syndrome (ards) worse pulmonary gas exchange due to a marked impairement of ventilation/perfusion mismatch. recently has been shown that if prostacyclin is given by aerosol instead of intravenous the net effect is an improvement of arterial oxigenation due to a redistribution of blood flow to well ventilated areas. objectives: to asses the effects of inhaled proatacyclin on pulmonary haemodynamics and gas exchange in patients with severe ards. methods : two patients with severe ards (murray score >3) recived inhaled pgi 2 at 15-20 ng.kg.min "1 using an ultrasonic nebulizer. haemodynamic measurements, arterial and mixed venous blood gas analysis were performed before and after 30 rain of pgi inhalation. results: short-terro p~i 2 inhalation improved pulmonary g-~ e-'~hange in both patients. arterial oxygen partial pressure (pao2) increased from 101 to 166 mmhg in patient 1 and from 87 to 108 in patient 2, the ratio pao to the fraction of inspired oxygen increased from 1262 to 207 (patient 1) and from 124 to 154 (patient 2). venous admixture decreased from 36% to 29% and from 34% to 27% in patient 1 and 2 respectively. mean pulmonary artery pressure decreased slightly from 25 to 23 mmhg in patient 1 and from 41 to 37 mmhg in patient 2. no effects on systemic haemodynamics were observed in any patient. conclusions: pgi 2 inhalation improves gas exchange and produces selective pulmonary vaaodilation, thus can be an alternative therapy for the treatment of pulmonary hypertension and hypexemia in patients with severe respiratory falllure. methods: we treated 67 ards-patients (age 41 yr (16-75) mean, range) during 1991-94. the lowest pao2/fio2-ratio was 74 (29-140), the worst murray score 3.0 (2.3-4.0), icu-stay 41 (1-121) days and hospital mortality 40%. the costs of intensive care were calculated according to intensivity of patient care as assessed by tiss-scoring (therapeutic intervention scoring system). the more intensive the care, the higher are the costs. costs per year of life saved (=life-year" in us $) were compaired by other medical treatments (1-4). it is assumed that the mean expected length of remaining life in ards-survivors after intensive care is 25 years. treatment life-year ($) ' bone marrow transplantation (acute leukemia) 65 000 lowering cholesterol using iovastatin 51 000 treating hypertension using nifedipine 32 900 heart transplantation 28 000 intensive care of ards-patients 3300 conclusions: intensive care of patients with severe ards is highly more cost-effective as compared with many other routinely used medical treatment strategies, the usually good recovery and the reasonable quality of life in survivors justifies investments to care of these patients (5). there is a close correlation between these two methods of measuring evlw. however there is an underestimation of 38.5 % in this kind of pulmonary edema ( oleie acid induced ) with the double dilution method. although the size of the sample is small, in normal lungs there appear not to be this underestimation. the effect of peep on evlw has been studied with contradictory results, probably as a consequence oft differences in methods of measuring evlw, variations in the type and severity of lung injury, and different timings of peep application. objective= 1) to analyse the effect of different levels of peep (0, 10 and 20omh20) on evlw during hpe; 2) to establish whether increases in intrathoracic pressure due to high peep levels can obstruct lymphatic drainage. material and methodet hpe was provoked in 3 groups of dogs by inflating a foley catheter in left auricular to a pressure of 24-26 r~uhg. peep levels of 0, i0 or 20 m~hg were applied. resultst objective: to assess the effect on extravascular lung water (evlw) of the application of peep and the reduction of vt in an oleic acid pulmonary edema model in pigs, using three ventila~ary strategies. material and methods: twelve adolescent pigs (weighing over 30 kg) were randomly divided in three gmups immediately alter infusing via a central vein 0.1 ml/kg of oleic acid to produce a permeability pulmonary edema. the ventilatory parameters for each group were as follows: group i (n=4) : vt: 10-15 ml/kg; zeep. group 2:(n=4) : vt: 10-15 ml/kg; peep: 10 cm h20. group 3:(n=4) : vt: 5-10 ml/kg; peep: 10 emil20. (resulting in permissive hypereapnla) after a four-hour period of ventilation the animals were killed and the lungs excised to calculate gravimetrically the extravascular lung water using a standardized procedure ( hemoglobin content method ). ill evlw (ml/kg) group obiective: in the postoperative period, maintenance of adeguate arterial oxygen tension is a major problem in morbidly obese patients probably because of a large reduction in functional residual capacity (frc). the aim of this study was to evaluate the effects of peep on respiratory mechamcs and gas exchange in this kind of patients. methods: in nine postoperative mechanically ventilated morbidly obese patients (bmi>40 kg/m 2) we partitioned the total respiratory system mechanics into its lung (1) and chest wall (w) components using the airway occlusion technique associated with the esophageal balloon, during constant flow inflation (jap 1989; 67: 2556) . at three different levels of peep (0, 5, 10 cmh20 ) we measured: compliance (cst), airway (rim) and "additional" (dr) resistance, frc and gas exchange. obiectives. to describe the use of prone position in our icu we analyzed the clinical records of all patients admitted in 1993-94, selecting adult patients with arf defined as: intubation and pao2/fio2<250 mmhg plus an fio2>0.5 or peep>5 cm i120. results. 146 patients met the arf criteria: 40 of them (27.4%) underwent prone positioning (p+). prone position use began in the early phase of arf (3.5• days from the beginning, range 1-32, median 2).25 out of 40 p+ pts were treated with controlled ventilation (cppv or pcv), while 14 were on assisted ventilation (simv+ps) and 1 on spontaneous breathing (cpap). only 2 pts were awake when turned prone, while 11 pts required adjuncts of sedation to tolerate the change of position. the duration of prone positioning was variable (average lenght 4.7• h, range 0.5-12 h). only minor side effects were observed (eyelids and facial edema, chest and facial pressure bruises). we consider responders (r+) those patients presenting at least 12.5 mmhg increase in pao2/fio2:35/40 patients (87.5 %.) were responders when first pruned. the pao2/fio 2 changes induced by prone position are reported in the figure. pao2/fio 2 increased when patients were pruned (*p<0.001) and remained higher than baseline values when returning supine(*p<0.001). paco 2 remained unchanged. prone positioning was used at least twice in 21/40 ( conclusions. this retrospective analysis confirms that prone positioning improves oxtgenation in the majorib' of arf patients. altough we have no available criteria to discriminate in advance r+ from r-pts, we now routinely consider the use of prone position in the treatment of severe arf. palo a, otivei m*, galbusera c, veronesi r, sala gallini g, zanierato m, iotti g, braschi a.servizio anest. e rianim. i, *laboratorio biotecnologie e tecnologie biomediche irccs s. matteo, pavia, italy inhaled no can improve arterial oxygenation and reduce pulmonary hypertension in ards patients; little information is, however, available about the dose-response curves. methods seven ards patients (lis 2.7+.5) submitted to mechanical ventilation randomly received 8 inhaled no doses in increasing or decreasing sequence: 0.5, 1, 5, 10, 20, 50 and 100 ppm. reference measurements were obtained before and after the entire period of no inhalation. hemodynamic parameters and blood gases were measured after 25 min in each condition. cmv was administered under sedation and paralysis, with constant ventilation, peep (lol-_2 cmh20) and fit2 (.56+.14). the changes in vt and fit2 due to the no (1000 ppm in n2) injection in the ventilator external circuit were compensated for. results .34 the dose of 0.5 ppm, ineffective on papm, significantly improved oxygenation. the increase of pat2 and the decrease of q'va/q' and papm were nearly maximal at 5-10 ppm. no deterioration of arterial oxygenation was observed at no doses as high as 100 ppm. co2 exchange was not influenced by no inhalation. systemic hemodynamic variables did not change throughout the study. these results suggest that a concentration around 10 ppm is adequate for obtaining maximum effects on hypoxemia and pulmonary hypertension in patients with ards. low-dose inhaled nitric oxide (no) induces redistribution of pulmonary perfusion in patients with severe ards and causes improvement of oxygenation [1] . however, addition of exogenous lowdose no in the inspiratory gas mixture might be only a replacement of missing atmospheric no (2-130 ppb) in hospital central-supplied medical air. [2] we have realised nitric oxide measurements in ten healthy volunteers, (4 smokers and 6 non-smokers) breathing with a mouthpiece and occluded nostrils through a ventilator circuit, with separation of inhaled and exhaled gases by a valve. no concentration was measured with a double-chamber chemiluminometer (environnement sa, france) and with charcoal/silicate purified compressed air. there was no nitric oxide detectable in the inspirat0ry limb of the ventilator. unfiltered central supply medical air contained :20 -50 ppb of no and 10 -30 ppb of no2, whereas central supplied oxygen was no/no 2 free. samples were taken after equilibration periods of 5 minutes, with increasing fit2 levels of 0.21, 0.50 and 1.0 for subsequent 5 minutes periods; paired values were recorded every 30 s. the mean no value was 4.57 ppb (sd 2.51) and n o significant differences were found for different fit2 levels both in smokers and non-smokers. these data suggest that the no concentration of pulmonary origin in the exhaled air of' healthy volunteers is probably lower than that reported by other authors [2] and that, previously reported, differences between smokers and non-smokers are not always striking [3] . we suggest the use of activated charcoal/silicate filters for clinical trials in order to achieve standard conditions. [ objective: to compare efficacy and safety of two doses of salbutamol. methods: sixteen adults who had severe acute a~hma were randomly assigned to receive either 10rag (n=9) or 5rag (n=7) of nebulized sulbutamol. both groups were similar with respect to age, duration of a~hma, duration of attack before arrival at the hospital and severity of a~hma according to baseline measurements (table) . evaluation was performed 30, 60, and 120 rain after the start of nebulization. results: compared with 10mg regimen, 5mg regimen resulted in the same improvement in peak-flow and fischl index (figure). the changes in heart rate, respiratory rate and pace2 did not differ significantly between both groups. the incidence of side effects, which included tremor, palpitations, cardiac arrythmlas and other symptoms, was not sj~ificanfly different in the two populations. conclusion:the results of this study suggest that nebulization of 10ng of salbutamol is not more effective than 5rag in the initial treatment of acute severe asthma in adult patients. the prognostic factors of neutropenic patients admitted to the icu remain poorly known. the aim of this study was to determine the respective weight of underlying malignancy and organ system failures on the outcome of these patients. patients and methods: the charts of 107 neutropenic patients (wbc < 1000/mm 3 and/or pmn < 500/ram3), admitted to the icu between 1986 and 1990, were retrospectively reviewed. the characteristics of the neoplastic disease (h~emopathy or solid tumor, tumoral evolution, duration of cancer disease and of neutropenia), the mac cabe's score, the organ system (respiratory, hemodynamic, renal, neurologic, hepatic) failures and the severity scores (saps, saps ii ,osf) were registred within the 1 st day in the icu. when discharged from the icu, the patients were classified as alive or dead. results: fifty-seven patients (53.3%) had a h~ematologic malignancy, and 50 (46.7%) a solid tumor. fifty-nine of the 107 patients died (55.1%); the mortality rate did not differ between both groups (61.4 and 48% respectively, p = 0.16). with univariate analysis, none of the tumoral features is linked to the prognosis; only the respiratory (p < 10 -4) and cardiovascular (p < 10 -3) failures, and the number of organ system failures (p < 10 -4) are associated to the risk of death. the saps (p < 10 -3) and saps ii scores (p < 10 -4) were higher in patients who died. with multivariate analysis (logistic regression), only the respiratory failure is correlated to the risk of death (p = 10-4); neither the features of the underlying malignancy (p > 0.8), nor the duration of neutropenia before admission in icu (p = 0.83), nor the severity scores figs ii: p = 0.068) are linked to the outcome. conclusions: the tumoral characteristics do not modify the prognosis after admission to the icu. they should not influence the decision to admit or refuse a cancer patient in the icu. respiratory failure at icu admission has the predominent weight on the risk of death in the icu. patients with respiratory acidosis due to asthma occasionally require levels of mechanical ventilation that place them at risk for barotrauma. a few case reports have described the use of an extra-corporeal membrane oxygenator(ecmo) circuit as an alternative means of co 9 removal. generally, this has been used for short periods of time (<24h) without serious complications and with low blood flows through the extra-corporeal circuit. we report a case of refractory asthma who could not tolerate even small-volume breaths from a mechanical ventilator due to severe bilateral airleak. ecmo therapy was initiated at the referring hospital prior to helicoptor transport. high blood flows were used (70% of the patient's cardiac output), sufficient to achieve both co 2 removal and oxygenation. satisfactory gas-exchanged was accomplished (pco2=50-60 mmhg) with nearly total lung rest for a prolonged period (60h). however, the long ecmo duration was associated with two severe complica-ti0ns:1) bilateral hemothoraces due to anticoagu!ation in the extra-corporeal circuit, and 2) prolonged weakness as a result of neuromuscular blockade for six days. the patient was discharged from the hospital in good condition. we present the respiratory and hemodynamic features of this case aw well as the potential complications of ecmo therapy in asthma. objectives: parameters derived from tidal expiratory flow ~e) and volume (vt) can be used to detect airflow obstruction in copd patients who might be unable to perform forced spirometry (e.g., icu). however, indices such as ave/v t and at/re are highly variable (thorax, 1981: 36; 135) . methods: we investigated whether the standardized for v m effective time (teff~) of a tidal breath, which is derived by asimple mathematical procedure (teff,= j'vdt/vt2), is a more reproducible and sensitive detector of airways obstruction, we studied nine normal subjects (5 male, 31 -+5yr) and 13 copd patients (4 male, 61 -+10yr) in the seated position, with a noseclip on. they breathed quietly, through a pneumotashograph to measure flow (v). volume was obtained by numerical integration of thellow signal. each subject had an initial 10-15 min trial run, in order to become accustomed to the apparatus and procedure. when regular breathing had been achieved, all breaths over a5 min time interval were recorded. the mean value of six consecutive breaths (ers criteria) for each subject was used for analysis under the condition that within session variation of tidal volume (vt) was <10%. lung function tests were: in normals (mean-sd), fevl%pred = 100• fevl/fvc%=81-+3% , and in copd patients, fev~%pred=53__.20 and fevi/fvc%=51 --.12%. results: values are shown as mean-..+-sd in the following a su~ve~ os literature sources p~oves that t~aditlona], i.e. medicinal medication and physiothe~apeutic methods os t~eatment often p~ove to be insufficientl~ effective both currently and in the ~emote future. the goal of this study was to investigate the efficacy os t~eatment of b~onchial asti~ma patients by means os speleo-and artificial sp~ay therapy. speleotherapy t~eatment was conducted in the conditions os mic~oclimate os salt mine in solotvino hospital. a~tis sp~ay the-~apy was conducted by means os a self-made device. ou~ method is based on the p~inci-~ le os using the majo~ facto~ of speleo-he~apy -highly dispe~sed sp~ay 0s sodium chloride. the obtained ~esults ~e~e analyzed in five g~adations. at the end os the speleothe~apy improvement and considerable improvement was observed in 75,0~ os patients; inconsiderable improvement -in 15,9~ os patients. having evaluated the e~s os t~eatment using a~tis sp~ay therapy the indices a~e 68,7h and 20,5~ ~espectively. remote ~esults of t~eatment a~e an important index os t~eatment, the ~esult os ~hich ~e~e studied by means 0s a ~uestionnaive-method. patients ~ho had been t~eated by speleothe~apy mo~e f~eguently ~e-po~ted a ~elapse in disease 3ust afte~ the course o~ t~eatment (29,3h). ho~eve~, in a ]ate~ phase the ~emission ~ould last ]on-~e~ (s 6 months in 84,5~ os patients, till one yea~ in 69~9~). in 12,5~ os patients who passed the co~se os a~tificial sp~ay therapy a ~elapse was ~egiste~ed immediately as the co~se os t~eatment. then thei~ condition stabilized ~hile in 73,5~ os patients a period os ~emission lasted s ha]s a yea~. 42,9~ of patients dida't ~epo~t a ~elapse of the disease du~in~ one yea~. evangelismos hospital, critical care department, athens, greece method#: 19 mechanically ventilated patients (5 copd, 6 ards, 8 other pulmonary diseases) were studied in two phases: 1) during the acute phase of respiratory failure; 2) during recovery 5-73 days later. we measured mip and monitored the pattern of breathing while the patients were breathing spontaneously through the respirator (pressure support mode with 3-8 cmh20) until either the point they were unable to sustain spontaneous breathing (sb) any longer (phase 1) or for two hours when they could sustain sb indefinitely (phase 2). subsequently the patients were sedated, paralyzed and mechanically ventilated. then we simulated the pattern of sb at the end of the sb trial by manipulating the variables of the ventilator and assessed respiratory mechanics b y the end-inspiratory and end-expiratory occlusion technique. 1. during recovery, a combination of reduced inspiratory load and increased venfilatory capability makes a patient previously unable to sustain sb to breathe spontaneously. 2. inspiratory load is reduced during recovery, mainly because both intrinsic peep and breathing frequency are diminished. obiectives: although elevated concentrations of a few cytokines have been shown to be present in the bronchoalveolar lavage (bal) fluid (balf) of patients with the adult (acute) respiratory distress syndrome (ards), the pethogenesis of ards is largely unknown. leukemia inhibitory factor (lif), a growth factor recently recognised as a polyfunctional cytokine integrated in cytokine networks was measured in unconcentrated balf of patients from different patient groups. methods: lif was measured in balf by means of a specific and sensitive elisa (detection limit 10 pg/ml)in balf (lavage of 3 x 50 ml in the right middle lobe). results: lif was not detected in the balf of 13 healthy control patients and in only one (34 pg/ml) out of 26 patients at risk for ards (after cadiopulmonary bypass surgery) who underwent bal 4 h after the end of the extracorporeal circulation. high and detectable levels were found in the unconcentrated balf of 10 out of 12 patients with full-blown ards (196 + 80, mean + sem, range 10-985 pg/ml). there was a good correlation between the level of lif in the balf and a number of markers of inflammation: neutrophils/ml (r:0.70, p= 0.01), albumin ( r:0.75, p=0.008) and protein level (r:0.74, p=0.006). conclusions:the biological role of lif in these balfs is not readily explained by its currently known actions and it is unkwon whether lif contributes to or is a response to local tissue damage. our results indicate that this cytokine with lots of interesting _functions is a pert of the inflammatory cytokine cascade in ards. background and obiective : we recently demonstrated that cisapride -a new prokinetic drug -enhanced enteral feeding in a heter0genoas group of ventilated icu patients by significantly accelerating their gastric clearance (crit care meal, 1995 ; 23 : 481-485) . it remains unknown, however, whether certain subgroups of patients might benefit more from adding cisapfide to their enteral nutrition regimen than others. patients with chronic obstructive pulmonary disease (copd) might represent such a subgroup since their illness and its specific treatment put them at risk for gastric emptying disorders. design and setting : prospective, consecutive sample study in an adult medical intensive care unit in a university hospital. patients : 10 mechanically ventilated and hemodynamically stable copd patients. interventions : gastric emptying was evaluated by bedside scintigraphy and expressed as the time at which 50% of a tcg~-labelled test meal was eliminated from the stomach (t 1/2). baseline data (do) were recorded after enteral nutrition reached 1500 to 2000 ml daily. scintigraphic measurements were repeated 4 days after cisapride (10 ml orally, q.i.d) had been added to this regimen (d4). patients were considered cisapride responders when gastric clearance improved by more than 50% from baseline. results : normal values for the test meal and for scintigraphic acquisitions obtained in the supine position were found to be 31 + 15 min. in healthy volunteers (crit care med, 1995 ; 23 : 481-485) . five patients responded to cisapride (t 1/2 : 81 + 31 rain vs. 26 + 10 min at do and d4, respectively) and five did not (t 1/2 : 36 + 18 min vs. 33 _+ 11 rain at do and d4, respectively). in contrast with non-responders, all five responders had clinically significant maldigestion at baseline (excessive (> 150 ml) gastric residues, vomiting (> 3 times/day and abdominal distension) which disappeared in 4 of them after the administration of cisapride. conclusion : copd patients who tolerate enteral nutrition well have basal gastric emptying times which are comparable with those of healthy volunteers and are not influenced by cisapride. however, cisapride treatment provides both scintigraphic and clinical improvement in those copd patients who exhibit clinically obvious gastric emptying disorders. cernv v., dostal p., zivny p., zabka l. dept. of anesth. and critical care, charles university, faculty hospital, i-irade~ kralove 500 36, czech republic objective: the aim of the study was to evaluate the effect of early entera nutrition started within 24 hours of injury on the incidence of multiple orgar failure (mof) in trauma patients requiring vantilatory support. methods: after institutional approval 25 patients were enrolled in the study enteral feeding was begun within 24 hours of injury in 14 trauma patients (en group) admitted to icu. nasuenteric tube was placed as soon as possible after admission into the distal duodenum under endoscopy. additional parenteral nutrition was used to meet patients energy and protein requirements. the control group (pn) consisted of 11 patients fed during this period paretuerally. severity score apache ii, trauma score, cumulative balance of nitrogen (g), incidence of mof (three and more organs) and length of ventilatury support (days) were calculated. values are expressed as mean + sd. results: tab introduction : parenteral nutrition (pn) is an important aspect in the optimal treatment of patients on gastroenterology or intensive care. the aim of this bi-center study in 38 patients has been to assess tolerence and efficacy of a new protein-lipid mixture for pn from a simple preparation. patients and m~hods : patients were selected in two hospitals (tenon and saint-lazare, paris) and were divided into two groups : group a (gastroenterology~ 1 l short bowel syndrome) and group b (intensive care, 27 surgical patients). all patients likely to require pig for a period of 10 days (group a) or 7 days (group b) were studied. the pn regimens administered were the following : combination with 50 g of mct/lct fat emulsion end 9,6 g of nitrogen, in 1 liter end glucose requirements were met by imfizsion of l liter of glucose 20-30 % via a "y " connection. lipid thus provided 3040 % of the non introgen calories. total daily calorie intake was 1540 to ] 940 kced. this study monitored, before and at the end of infusions, the sennn albumin (alb), preaiburtun (prealb), triglycendes (tg), cholesterol (cs), and the serum ammotransferases (sgot and sgpt) end alkaline phosphatase (alp) activities. statistical significances were calculated using the wilcoxon-tost. introduction: many 1cu patients present a catabolic illness in response to inflammation and infection, characterized by a rapid loss in skeletal-muscle mass despite optimal nutritional support. growth hormone (gh) is responsible for a rise of lipolysis, enhancing the energetic balance, and of protein synthesis. recombinant human gh (rhgh) is nowaday available for clinical use, but its cost is very high. therefore, rhgh should only be prescribed to icu patients when its efficacy can reasonably be anticipated (ie. when the patients are catabolic or stressed, but in order to avoid overprescription for unstressed patients and for those who are overly catabolic). hence, we, as others, recently demonstrated that rhgh had no favorable effect in highly stressed icu patients. objective: to detect on a clinical basis, low (ls), mild (ms) and severe stress (ss) states in icu patients and validate this clinical judgement by objective metabolic mesurements, in order to select early those icu patients potentially able to benefit from rhgh therapy. methods: 36 consecutive icu patients were prospectively stratified as ls, ms and ss by two experienced icu senior consultants (temperature; agitation; heart rate; arterial blood pressure; presence of an infection; respiratory rate; exogenous catecholamines). anabolic (insulin, igf-1, gh) and catabolic (cortisol, ghicagon) hormones, and nitrogen balance were determined for each patient within 8 hours after admission in the icu. metabolic and clinical data were then compared. the clinical stress states determined by icu physicians correlate with an objective metabolic assessment. therefore, the patients who will more likely benefit from adjuvant rhgh therapy can be detected simply and early. a prospective study on rhgh therapy in ms icu patients is in progress. berger mm md 1, chiolero r md 1, pannatier a phd 2, berger l 2, cayeux c 1, voirol p 2, hurni m md 3. 1 surgical icu, 2 pharmacy, and 3 cardiac surgery, chu vaudois, ch-iotl lausanne, switzerland objective. nutrition of the compromised cardiac surgical patient is challenging. numerous factors influence the gastrointestinal (gi) absorption function, among which gut perfusion, which depends largely on the systemic hemodynamic status. patients in hemodynamic failure are prone to organ failure, and may benefit from an early jejunal feeding. the study was designed to assess the absorption function after cardiac surgery in patients with adequate and altered hemodynamic status, using paracetamol as tracer of gi absorption. methods. after cardiac surgery, 24 patients, aged 63_+8 years (mean_+sd) were assigned to 2 groups (anaesthesia: fentanyl 20 gg/kg + midazolam): group 1 (n=10): reference group, with normal hemodynamic status, easy recovery. group 2 ('n=14): patients in low output syndrome, cardiac index < 2.5 i/m2 on day 1 (d1) after surgery, requiring prolonged intensive care, mechanical ventilation + nutritional support. paracetamol 1 g, was given intragastrically on d1 + d3: plasma levels measured (h.p.l.c), at administration (to), t30-60-90-120-180-240 and 480 rain. hemodynamic status assessed with pulmonary artery catheter. 5 healthy subjects served as controls. results. compared to healthy controls, absorption was strongly reduced on d1 in all patients (no difference between groups). on d3, peak paracetamol level was significantly lower in group 2 (low cardiac output): in group 2 the area under the curve on d1 and d3 were similar. there was a large inter-patient variability, reflecting the hemodynamic status. conclusion. gi absorption was decreased on d1 in all patients, and reverted to normal between d2 and d3 in case of normal cardiac function, but not in case of low output syndrome. the decrease on d1 can be attributed to fentanyl, known to slow down the gi transit. in patients with cardiac failure, correction of altered absorption was correlated with the hemodynamic status, suggesting that gi absorption is dependent on adequate splanchnic perfusion. the aim of the work was to define specific significance and evaluate efficiency of enteral component of infusion therapy in the intensive care of gastroenterotogic patients of surgical profile with pyo-septic complecations. there were used the methods of radial diagnostics and polyelectrography; the laboratory control on oxygen-transporting function, volumetric and hemodynamic state, changes in metabolic, hormonal and immunologic status was conducted. from january, [992 till november, 1994 there was carried out the randomized study of 155 patients with general purulent peritonitis; among them 70 persons constituted the control group and 85 -the main one. in the main g~oup the intestinal lavage, enterosorption, enteral introduction of nutrient solutions with gradual turn to enteral nutrition by equalized mixture "ovolaet" were started from the first hours after operation. the data obtained allowed to define the specifity of the program of artificial medical nutrition in the group of examined patients, based on necessity of individual selection of media for enteral introduction depending on the stages of intestinal insufficiency syndrome. it was shown that inclusion of enteral component into the program of infusion therapy during early periods stabilized circulation in the regime of moderate hyperdynamia, considerably decreases the deficiency of circulating blood volume, normalizes the values of oxygen transport, consumption an}d extraction, provides the optimal level of mycardial adaptive possibilities without tension of its compensatory functions and pulmonary circulation overload. due to combined application of parenteral and enteral nutrition the metabolic processes are shifted towards anabolism. this is supported by decrease to normal values in the contents of blood aggresive hormones (acth,hydrocortisone) and increase in somatotrophic hormone. the complete parenteral-andenteral nutrition influences positively on restoration of cellular and tumoral immunity, activates the factors of organism nonspecific protection and recovery from immunodepression, prevents the development of immunodeficiency. impact tm vs control. s atkinson, n maynard, r grover, e sieffert, r mason, m smithies, d bihari departments of surgery and intensive care, guy's hospital, london, u.k objectives: comparison of the effect of an immunonutrient enteral feed versus a control on the outcome of a mixed intensive care unit (icu) population. methods: admissions to this multidisciplinary adu)t icu thought likely to stay more than three days and with tube access to the gi tract ~r randomised to receive either impact tm, a feed with supplemental arginine, dietary nucleotides and omega-3 fatty acids, or an isocaloric and isonitrogenous control feed. study end points included mortality and icu stay. approval was obtained from the hospital ethics committee. rosults: 390 patients were entered into the trial. the two groups were well matched for age, sex, and admission apache ii with an overall mean admission risk of death of 30.8 (std. dev. -+ 22.9). on an intention to treat basis, there was a no significant difference in icu mortality, icu stay or standardised mortality ratio (s.m.r.) between the two groups (see table) . similarly, there were no differences after stratification for patients receiving 5 or more litres of feed. conclusion: there is no evidence of an effect of impact@, an enteral immunonutrient feed, on pre-determined end-points (icu mortality, icu stay or standardised mortality ratio) in a mixed intensive care unit population over that of an isocaloric, isonitrogenous control feed. objeeflves: evaluate changes of blood laatate levels according to patient medical status after cvvhd initj,~ion using dialysate solution containing lactate. method: review of medioal records of 20 consecutive patients ~eated by cvvhd (dialysate solution hmnosol lg2, hospal,uk, lactate concentration 40 retool/l). date obtained 1 hr before and 4 -6 hrs at~er cvvhd initiation were analysed. results: all data are presented as mean + sem. in one patient, pre end post filter lactate levds were measured during standard cvvhd setting (blood flow 100ml/mlu, dialysate solution flow i 1/hr), and approximate daily lactate flux into the patient was calculated to be as high as 920 mmol/d. lactate leveh measured after cvvhd initiation increased significenfly compared to baseline levels (3.80+0.67 axtd 2.88+0.68,respectively; p<0.01,paired t-test). when patiente with increased basal lactete (~-7) were compared to paliente with normal basal values (n=13), no difference in laotete increase was fmmd (p=0.22, manova). patiente with severe liver dysfunction (2 points in mop scomlg, n=9) had higher basal laotate levels than patiente with normal or slightly abnormal liver teste (0 or 1 point in mof scoring, n=ll), rite values being 4.04 + 1.17 and 1.84 + 0.23, respectively (p<0.05, student t-test). increase in blood lactate did not differ between these two groups after cvvhd was stetted (p=0.86, manova). in 11 pafiente with invasive hemedynamio mo~, no oorrelation batween changes in lactate levels and eitlm" changes in oxygen ddivery (t2=o.ol; p--o.81) or oxygen consumption (reversed fie, k) (r2-q).o7;p--0.66) were found after cvvhd initiation. conclusion: blood lactate increases on cvvhd with dialysate soh~on rich in lactate. this increase is predominantly caused by influx of lactate into the blood via the filter end does not seem to depend on the liver fimotion and/or oxygen metabolism changes. objectives: the study was designed in order to determine the effect on plasmatic proteins, of two types of aminoacids solutions of parenteral nutrition (pn) adapted to stress, having different concentration of branched chain aminoacids (bcaa), when applying to politraumatized critical patients. methods: a prospective study was performed using a randomized double blind design of 20 polytraumafized patients, split in two groups of ten patients each, with mean ages of 35 _+ 17 an 45 -+ 20 years. due to their condition, all patients required p.n. for at least 9 days. both groups were subjected to isocalorie and isonitrogenous solutions (45 ci/kg/ day and 0.24 g of nitrogen/ks/day), varying only in the concentration of bcaa; solution a having a 10 % concentration and solution b 23 %. blood samples determinations during days 0, 3, 6, 9 after the beginning of treatment with p.n. were total proteins., albumin, trandferrine, protein binding retinol; prealbumine and fibronectine. the anova test (one and two way) was used to compare the values between the two groups. results: the administration of solution a, showed statistically significant increases in the determinations of the values of protein binding retino] (p < 0.05) and prealbumin (p < 0.05). no significant increases were observed in the values of total protein, albumin, transferrine and fibronectin. solution b produced statistically significant increases only in the values of total proteins (p < 0.05). the remaining proteins did not changed from their control values during the whole period of pn administration. comparing both groups, no statistically significant differences were observed related to the type of diet. nevertheless, differences were found in total proteins, albumin, protein binding retinoi, fibronectin (p<0.05) and prealbumin (p < 0.005) in relation to the time course of pn therapy. only the albumin values showed significant differences (p < 0.01) when considering the interaction of both the type of diet and the time course of pn. conclusions: 1. solutions of pn adapted to stress, can maintain the control values of slow turnover proteins and improve the values of rapid turnover proteins. 2. no significant differences on plasma proteins were found between the two solutions having 10 % or 23 % concentration of branched chain aminoaeids. &determination of rapid turnover proteins does not seems useful for discriminating different solutions of bcaa during pn. obiectives; the hormonal changes in the post-traumatic situation often leads to an elevated blood glucose and a negative nitrogen balance. to reduce the elevated glucose production by aminoacids the apprication of xylitol may be an alternative energy source. in a double-blind randomized study we investigated the effects of a xylitol/glucose solution (group a: aminoacids 50 g/i; glucose/xylito180 g/40 g/l) on metabolism and particularly on pancreatic and liver enzymes compared to a glucose based nutrition solution regimen (group b: aminoacids 50 g/i; glucose 120 g/i). methods: the clinical trial was carried out after the approval by the local ethical committee on 31 patients with severe brain injury. there was no difference in body mass index bmi (group a: 25.9 +/-2.7 kg/m 2 and group b: 25.1 +/-2.4 kg/m=), age, and sex. daily individual energy expenditure was measured by indirect calorimetry (deltetrac "~). nutrition was started 24 -48 hours after trauma or surgery with carbohydrates and aminoacids. fat was added 24 h after nutrition had started. to analyze the effects on pancreatic and liver enzymes we investigated the following parameters for 4 days: blood gtucose, serum lipase, serum amylase, asat, alat, ~gt, ap, and serum cholinesterase (che). results: due to the daily indirect calorimetric measurements energy requirements were satisfied. there was no difference in blood glucose concentration and cumulative nitrogen balance between the two groups. neither were there any significant changes in asat, alat, ap, and che for 4 days in both groups. serum tipase steadily rose to 202 lull in group a and 320.2 lull in group b, respectively. conclusions: there was no measurable influence of either nutrition solution on liver enzymes. the xylitol/glucose nutrition regimen does not have any advantage over the glucose based nutrition solution concerning blood glucose level or nitrogen balance. the elevation of serum lipase to a 2-fold level in either group needs further investigation on trauma patients. the effects of fat emulsions in lung function, particularly in lungdamaged patients, have been attributed to alterations in pulmonary vascular tone caused by eicosanoid production modificatione. as the eicosanoid production may depend on the fatty acid profiles of the intravenous fat emulsion, haemodynamic, pulmonary gas exchange and plasma levels of prostanoids were investigated in acute respiratory distress syndrome (ards) patients, during different intravenous lipid emulsions (providing different prostanoid precursors). we studied in a randomized double-blind design 3 groups (n=7 each) with ards. group i (lct) received a fat emulsion with long chain triglycerids (lct-20%), group ii (mct) an emulsion containing a mixture of medium and long chain triglycerids (mct/lct 50/50-20%) and group iii placebo (control), during 12 h (2 mg/kg/min each). we measured before, at the end of 12 h infusion, and 12 h after the end of the infusion: lipaemia, arterial and venous blood gases, pulmonary and systemic haemodynamics, and plasmatic levels (arterial and in mixed venous sample) of eicosanoids (txb=, 6-keto pgf~,, and ltb4). at the end of the fat emulsion, groups (i and il) to 1,10• to 0,51 • mmol/i), the paoz/fio z remained unchanged in the three groups; no changes in intrapulmonary shunt (qs/qt) were shown; neither in the mean pulmonary artery pressure. in contrast, only in the lct group: cardiac output and oxygen consumption increased significantly (12.5% and 19%) (p<0.05). eicosanoids were increased at baseline compared to reference values (p<0,05). a decrease (p<o,05l in the arterio-venous difference of the vasodilatador prostanoid (6-keto pgf~=) was shown in lct group. our results indicate that fat emulsions in ards patient do not influence pulmonary gas exchange, provided that the perfusion rate were keept at 2 mg/kg/min. the present study was designed to evaluate the metabolic changes of a carbohydrate-based diet versus a fat-based diet on oxidation rate of substrates and energy muscle metabolism in patients with an acute exacerbation of chronic obstructive lung disease. patients were mechanically ventilated with a volume-cycled respirator. after an overnight fast, patients were randomnly treated with a continuous enteral feeding over a nasogastrie tube with a carbohydrate/fat ratio of 2/1 in group i (n=6) , and a carbohydrate/fat ratio of 1/2 in group i] (n = 6) during 8 consecutive days. indirect calorimetry was set at baseline and 24 hours later the enteral feeding was started. muscular quadriceps biopsy was performed on baseline and on day 8 of enteral feeding. urine was collected during 24 hours and was used to measure 24h urea nitrogen production. the caloric intake was set according to the measured resting energy expenditure. respiratory gas exchange rate were measured using a computerized open-circuit indirect calorimeter. quadficeps femoral muscle samples were obtained by muscular biopsy after local anesthesia. analyses of glycogen, glucose 6-phosphate, lactate, phosphocreatine and adenosine triphosphate (atp) and enzymes (glycogen synthase and glycogen phosphorylase) were determined. after 24 h of nutrition patients in group i showed an increase in carbohydrate oxidation (from 17.8% to 55.4%, p=0.06), and in group ii a decrease in protein oxidation (from 27% to 21%, p=0.03). after 8 days of enteral feeding a tendency to increase in glycogen concentration (group i, p=0 .06; and group ii,p=0.1) was observed. while patients in group i showed a tendency (p=0.06) to decrease glycogen phosphorylase, patients in group ii maintained these enzymatic levels. the increased in glycogen was correlated in group li with degradation enzyme (p =0.01 ). in group i, the pao2/fio 2 ratio increased after 8 days of treatment (from 235.9 to 284.5; p=0.03), but no changes in days of mechanically ventilation, length of stay in icu, or mortality rate was evidenced between groups. it is concluded that the oxidation rate of substrates used for energy production varies according to the caloric sources administered. with nutritional therapy muscle glycogen concentration increases in both diets, with different enzymatic influence. more studies are warranted to further assess the clinical implications of the different pathophysiologic behavior of the two diets. under certain experimental and human conditions (radiation and/or thermal injury) the gi mucosa is unable to perform this protective function and could play an important role in the pathophysiology of the syndrome of multiple organ failure (smof). the objective of the present multicenter study was to know the gi mucosal permeability in critically ill patients and the relationship with their outcome. the method used to assess the integrity of the gi mucosal barrier measured mucosal permeability to various hydrophilic compounds. specifically, we measured the urinary excretion ratio (uer) of two sugars (lactulose and mannitol) that had been previously administered through a nasogastric tube. urinary excretion rate was measured in 10 healthy humans (control) and in 30 patients admitted to the intensive care unit (icu) at days 1, 3 and 5. the apache ii of the patients studied was 15_+4. there was an increase of uer in the critically ill patients compared with controls (o.26_+0.1 vs 0.036_+0.01) (p<0.001). in contrast, no changes in uer between days 1, 3 and 5 were observed (0.26 -+ 0. . significant changes were also established in the different groups: major and minor surgery, general and epidural anaesthesia , transfused and non transfused patients except for iron and ferritin plasma levels which didn't change significantly in patients who had received blood. a positive significant correlation was established between crp and ferritin (r=0.89) for patients under general anaesthesia, but not on epidural, regardless of the type of surgery. conclusions: iron and transferrin plasma levels fell acutely in surgical patients while ferritin and crp rose. transfusion implied an acute iron load.the established correlation between cpr and ferritin in patients on general anaesthesia suggests that ferritin behave as an acute phase reactant in that setting, while epidural anaesthesia may ameliorate the inflammatory response. objectives: to analize the effect of gastrointestinal complicacions (gic) related to the enteral nutrition (en) in the real volume of diet administered to icu adult patients. a prospective multicenter study was designed (comgine study) in wich en application, gic definition and his management were defined by collaborative consensus.patients treated with en in one month pedod were included. in each case, daily volume of diet prescribed (pv) and administered (av) was recorded and the volume ratio calculated (vr=pvxl00/av). patients were divided for days of en according to the presence (group 1) or absence (group 2) of gic (abdominal distension, increase of gastdc residuals,vomits or regurgitation, diarrhea,constipation and bronchoaspiration). differences between groups were analized by anova and mann-whitney u test with p<0.05 for statistical significance. (vr%) 53 61 58 89 67 63 65 72 62 60 64 59 67 7461 group2 (vr%) 85 90 90 94 94 93 92 90 92 91 92 94 90 92 tolerance to the enteral nutrition (en) has been related to the severity of illness in icu patients. the aim of this study was to evaluate this relationship. methods. a prospective, multicenter, study was performed (comgine study) in wich en application and related gastrointestinal complications (gic) were defined by collaborative consensus. adult icu patients treated with en in one month pedod were included. gic were classified as: 1) abdominal distension (abd), 2) increase of gastdc residuals (igr), 3) vomits (vom), 4) diet regurgitation (reg), 5) diarrhea (dia) and 6) constipation (con). patients were divided in two groups: group a: patients with gic dudng their evolution. group b: patients without gic. differences between groups were analized for apache ii admission score or evolutive variables ( carbon dioxide production is a major determent of work of breathing. increase in co2 production during nutritional support may precipitate ventilatory failure and difficulty in weaning from mechanical ventilation. in the present study, co2 output was calculated while passive mechanically ventilated patient were fed with different amount of calories and different proportion of protein, carbohydrate and fat. four clinical stable and mechanically ventilated patients were studied. carbon dioxide was calculated while patients were paralyzed, sedated and mechanically ventilated. six nutritional regimens were used: a) no calories, b) t00gr glucose/24h, c) calories equal to rest energy expenditure in special formula with low carbohydrates and high fat (pulmocare) d) calories equal to rest energy expenditure in standard formula (nutdson) e) calories double to rest energy expenditure in standard formula (nutrison) and f) calories equal to rest energy expenditure parenterel (tpn). carbon dioxide output was low while no calories and/or only 100 gr/24h glucose was provided, the mean values of vco2 output were 134_+_25 and 140!-_26 ml/min respectively. there was no difference in vco2 output between with low carbohydrates and high fat regimen and standard regimen, mean values were 16t .5-+27 and 163_+26 respectively.the high calories regimen and tpn resulted in higher vco2 output, mean values 184_+25 and 211• respectively. we conclude that the special with low carbohydrates and high fat regimen did not reduce vco2 output, in the contrary, high calories intake as well as tpn increased vco2 output under tested conditions. klouche k., cristol j.p., vela c., canaud b., b6raud j.j. m6tabolic intensive care unit and nephrology department. lapeyronie hospital. 34059 montpeuier france. predictive factors for rhabdomyolysis (rh) -induced acute renal failure (arf) were studied in a retrospective study from january 92 to january 95. 33 patients (pts) (male:23, female:lo; mean age: 57+4 y.o.) were admitted with rh defined as cpk>1000 iu/1. etiologies were: traumatic and ischaemic 24, infectious 4, toxic 4, excess activity 1. factors studied were: simplified acute physiologic score (saps: 10.3+1.1), organ systemic failure (osf: 1.02_-!-0.2), diagnosis delay (d: 33+_5h), clinical parameters (sepsis, dehydration), blood chemistry data (cpk, bun, creatinine, potassium, phosphorus, calcium, proteins, hematocrit) and urinary ph. severity of rh was estimated by ward score determined according to phosphorus, albumin, potassium, cpk, dehydration and sepsis. urea appearance rate (uar) and creatinine index (ci*) were determined over a 24 hours period. arf was observed in 25 pts. in non-arf and arf groups respectively, saps (5.5_+0.5 vs 11.8+1.3), deshydratation (0 vs 11), sepsis (0 vs 12), phosphorus (1.03+0.16 vs 2.21-+0.21), calcium (2.1+0.07 vs 1.8_+0.07), ward score (4_+0.65 vs 11.8+0.8) were significantly different. however, no significance was observed in uar (310-+89 vs 210-+35) and ci (26_+7 vs 34_+3). 16 patients required hemodialysis (hd) (85:2 sessions) and 9 remained dialysis free. only osf (1.1_+0.1 vs 1.9-+0.23), ward score (9.2_-/-0.95 vs 13.25_+0.92) and ci (29+_3 vs 41-+3) appeared significantly higher in pts requiring hd. 5 pts died from associated disease. all patients suffering from arf recovered a normal renal function. we confwmed that an elevated ward score (over 7) is a good predictive index of arf. in addition we found that ci is a severity factor for arf requiring hd. thus, patients suffering for rh with elevated ward score and ci, have a fair chance of dialysis and should be treated more intensively. * ci (expressed in mg/kg) = (car + feces creatinine) / weight. where car: creatinine appearance rate; feces cr~t..= mean plasmatic creatinine x 0.043. tr~er k., cetin t.e., tugtekin i., georgieff m., ensinger h. universit~tsklinik flir an~sthesiologie, 89070 uim, germany introduction: endogenous as well as exogenous adrenergic agonists have a profound effect on carbohydrate metabolism in human critical illness. in this study the effects of noradrenaline (nor) and dobutamine (dob) on carbohydrate metabolism during a 4 hr infusion were investigated. methods: after approval by the local ethic committee 14 healthy volunteers were studied. hepatic glucose production (hgp [mg/kg/min]), using 6,6-d2glucose as stable isotope tracer, as well as plasma concentrations of glucose (glc [mmol/i]) and lactate (lac [mmol/i]) were measured prior and during infusion of nor (0.14 pg/kg/min) and dob (6 pg/kg/min). blood samples were drawn before and during the agonist infusion. results: no major changes in insulin and gtucagon plasma concentrations could be found during the study period. ::i:::: :iiiii~ 8~ i ::i: ~:: : :: i:ii. mean-+sd are shown. # p<0.01, anova for repeated measurments. conclusions: the effect of nor on hgp and glc were smaller as compared to adrenaline (i) with a similar time course. in contrast to the effects of adrenaline and nor, dob had a different effect on carbohydrate metabolism: a decrease in hcp and glc, which is uncommon for a /3-adrenoceptor agonist. since hgp is an energy consuming process that might deteriorate hepatic oxygen balance in critical illness, the differential effects of adrenergic agonists may be of importance and need further clarification. the nutritional insufficiency often accompanies post-operative hypercaloric states, inanition, serious infections and weakening chronic illnesses. that is why the early nutritional support, sufficient and appropriate for each individual base, is a fundamental component of intensive care unit as an indispensable factor for recovery. per this reason, our unit, developed a software for the implementation and nutritional control of t~e assisted patients. this software is incorporated is an expert system called ~i~su, designed and developed by the computational division of our unit. this system arrives to inferred diagnoses such as : respiratory, hepatic, renal(with and without dialysis) dysfunctions, pancreatitis, ards, decrease of consciousness, diabetes. according to these data objectives: to compare the effect of short term enteral feeding versus parenteral nutrition, when a isonitrogenous and isocaloric feeding solution is administered by either mute. methods: in a prospective controlled clinical trial 30 patients were studied; all exhibited moderate degree of malnutrition, normal liver and kidneys, and a functi6ning gastrointestinal tract. the patients were randomized to receive a free amino acid and small peptide diet (15 patients) or an isonitrogenous isocaloric parenteral support (tpn) (15 patients) (total energy: 2880 kcal, nitrogen: 14.5 g, carbohydrates: 380 g, fat: 112 g, n/non protein calories: 1/175) at least for 10 days. results: there were no significant changes in anthropometric parameters within either group. nitrogen equilibrium was aqhieved by day 3 in the tpn group and by day 5 in the enteral group (66.6% of the enterally fed patients and 80% of the tpn patients maintained in positive balance the day 10 of the study). there were no significant changes in serum albumin within either group. serum level of transferrin reached a significant increase in both groups (p=0.003). thyroxine-binding prealbnmin rose significantly in both groups as well (p=0.019 and 0.004 respectively). statistically significant rises in lymphocyte counts (p=0.003 and 0.001 respectively), in levels of c 3 (p=0.009 and 0.01)1 respectively), iga (p=0.002), igg (p=0.004 and 0.003 respectively) and igm (p=0.004) occurred in either treatment group. there was a high incidence of negative skin tests at the start of the study in the enteral group (73.3%) and the tpn group (60%). by the end of the study the incidence of negative responsiveness was 40.0% and 26.6% respectively. despite maintenance of similar glucose levels in both groups, tpn led to significantly higher serum insulin levels. the serum insulin increased almost linearly over the study period and eventually prevented fat mobilization and lipolysis, so that free fatty acid levels had fallen significantly. a significant elevation of the liver enzymes over the study period occurred in 73.3% of the tpn group, but not in the enterany fed patients. conclusions: the present findings provide no evidence that enteral diets containing free amino acids and small peptides, as their nitrogen sources, are in any way inferior to isonitrogenous isoealoric regimes parenterally given. aim: the aim of this study is to describe and explore the expectations of the functions of the critical care nurse to enable the formulation of guidelines for the scope of practice for the critical care nurse with a south african context, methods: phase i was to determine the expectations of the critical care nurse, the nursing service managers and the doctors with regard to the functions of the critical care nurse. a focus group interview was held with a group of experts in the field of critical care. the results were used to compile a questionnaire. this questionnaire was sent to the critical care nurses, the nursing service managers and the doctors in south africa for completion. from these results the functions of the critical care nurse were determined. phase ii was to formulate guidelines for the scope of practice for the critical care nurse within a south african context. through usage of the date (phase i) the scope of practice was formulated. guidelines were formulated for the practise, education and research regarding the limitations of the professional-ethical authoration and the implementation of the scope of practice for the critical care nurse. objectives : high output gastric aspirates arc occasionally observed during fasting in critically ill paticnts, preventing any attempt of feeding via the enteral route. although these patients are often said to suffer from "gastroparesia", the motor correlates of this condition arc lurgcly unknown. in this stud?', wc recorded the gastrointestinal motility of critically ill patients with abundant (>250 ml/24 hours) fasting gastric aspirates. methods : antral (4 sites separated each other from 1.5 cm), duodenal (1 site) and jejunal (1 site) contractions were recorded simultaneously by ~eans of a multihimen tube assembly positioned trader fluoroscopic control (perfused catheter technique). tracings from prolonged recordings were obtained on a multichannel recorder (7758a recorder, hewlett-packard) then anal)7,ed visually, with a special attention for the following abnormalities which are characteristic of intcstinal pseudoobstmctiou: l) absence or aberrant propagation of the migrating motor complex (mmc), 2) presence of bursts (> 2min) of nonpropagated phasic pressure and 3) presence of sustained (>30 min) uncnardinate pressure activity. 11 patients with a volume of gastric aspirates of 731 • 506 (sd) [median 5001 ml/24 hrs were investigated for 538 4-271 [median 4551 minutes. results : only one patient had no detectable motor abnormality. mmcs were either absent (n=4) or migrated abnormally (retrograde propagation : n=4; retrograde and stationnary : n=2) in 10 pts. bursts of nonpropagated phasic pressure activity were present in the duodenum in 9 pts and sustained uncoordinate pressure activity was found in 2 pts. additional abnormalities included episodes of prominent pyloric activity. (n=l) and sustained antral pressure activity (n=2}. conclusion : critically ill patients with large volume of gastric aspirates have manometric evidence of intestinal pseudoobstruction. prokinetic therapy in these patients should thus focus not only on enhancing gastric motility, but also on restoring a normal propagative contractile activity in the intestine. this prospective, open-label, randomized placebo-controlled study included 20 patients with hypokalemia in whom rapid potassium replacement (20 meq kci in 1 h) was performed: 14 patients received mg sulfate (6 g in 3 hours) and 6 patients received a corresponding saline infusion. measurements were made at time 0, +1, +3 and +6 hours results: k levels increased more in mg treated patients than in the patients who received saline infusion at time 1 and 3 h (p < 0.05 -students-newman-keuls). (table 1 ). introduction. dual lumen uaso-gastrojcjunal tubes are a major ads'ance in nutritional therapy of mechanically ventilated critically ill patients since the3" authorizc jejunal feeding with concurrent gastric decompression, there,, reducing the risk for aspiration. unfortunately, placcmem of these tubes in the jejunum regularly dictates to resort to endoscopy in order to facilitate pyloric intubation. recently, the remarkable gastrokinetic properties of the well known macrolide antibiotic er}lhromycin have been demonstrated in gastroparetic critically ill patients 1. aim. in the presem stu~,, we evaluated the feasibility of placing dual lumen naso-gastrojcjunal feeding tubes at the bedside without endoscopy, using edthromycin to help iranspy'loric migration of the tube under fluoroscopic control. methnd each patient admitted in our icu during a 2 months period and requiring artificial ventilation and enteral nutrition for a period of at least 3 days was included in the study.. after inserting the tube (stayput| sandoz, usa) in the gastric anmnn, e.rythromycin (200 rag) was aduunistored intravenously, to help fluoroscopic positioning of the tube into the jejunum. the total duration of the procedure (from nasal intabatiun to jejunal placement), as well as the duration of ftuoroscopy were recorded in each patient. results. 15 patients (male/female : 13/2: mean age : 56.9 + 22.2 years; mean apacbell score : 23.t • 7.0) wore enrolled into the study.the procedure was performed within the 2 dab,s following institution of mechanical ventilation. jejunal access was obtained in all 15 patients without resort to enduscopy in 10,81 • 7.31 min.(total duration of the procedure). mean duration of fluoroscopy was 3.54 + 2.97 rain. conclusion. we conclude that placement of dual lmnen naso-gastrojejunal tubes can be obtained in mechanically ventilated critically ill patients without resort to endoscopy., provided that e rythromycin is used as gastrokinetic agent to help pyloric intubation. the following ad and dis parameters were considered in all patients: -mid arm circumference, triceps skinfold thickness, serum transferrin, albumine and lymphoeites and urinary creatinine/height index. patients whose results were bellow 80% of normal values in 3 or more of the 6 above criteria were considered undernourished (und).statistical analysis was performed using %2 analysis.statistical significance was established at p<o.o5. results: a total of 129 patients(47 female, 82 male) -with a mortality rate (~) of 31,8% -were studied, aged 56-+8 years and a median ]enght of stay of 22 days. 1 -nourished (nou) at ad and at dis -22; ~ 0%; 2 -nou at ad and und at dis -17; ) 41,2% ; 3 -und at ad and und at dis -73; ~ 45.2%; 4 -und at ad and nou at dis -17; ~ 5,9%. we observed a p< 0.05 when we compared groups 1 and 2 (p=0.0012), and groups 1 and 3 (p=0.00026). variation in nutritional status and longht of stay: 1-nou at ad and nou at dis = > median lenght of stay 18 days; 2 und at ad and und at dis = > median lengbt of stay 22days; nutritional status and age at admission: 1-age > = 60 years : nou (13) , und (53) 2-age < 60 years: nou (26), und (37) nutritional status and age at discharge: 3-age > = 60 years : nou (12) , und (54) 4-age < 60 years: nou (36), und (27) we observed a p<o.05 when we compared groups 1 and 2 (p=o.01) and groups 3 and 4 (p =0.004) conclusion: such study permits to conclude that most of our patients (69.8%), staying in icu for more than 6 days, were und, namely the elder. we observed that this kind ef patient had a longer median length of stay in icu, associated with higher mortality rate. in conclusion, the nutritional management is an essential element in suportive care of critical)y ill patients. ohiectives: sirs is associated with hypercataholisnl and resistance to nutritional support, despite raised circu&ting levels of growth hormone and insulin. serum igf-1 levels however are low; thercti~re rhigf-i administration might produce nsefnl anabolic effects. the pharmacokinetics of exogenously administration rhlge-i in such patients are likely to differ from that seen in the less seriously ill due to changes in circulating blood volume, increased capillary permeability, poor nutritional stares, and alterations in binding proteins. the ol2jective of this study was to determine the clearance, apparent vohnne (if distribution (vd), time to peak concentration (tmax) and elimination half-like (tia) of a single subctkaneous il~ieetion of rhlgf-i in patients with sirs on the intensive care unit (icu). methods: 9 icu patients with sirs were entered into the study, which had been approved by the ethics comjnittee. in each patient 6 baseline hlood samples for circulating 1gf-i were obtained over a 24 hour period, prior to il!iection of 40 p.g/kg of rhlgf-1 subcutaneously. a further l4 samples were taken fi'om each subject over the next 48 hours. circulating ige-i was measured in serum at each time point by radioimmtme assay, and the area under the curve tbllowing administration of rhlgf-i was derived tbr individual patients fixm~ baseline a~!justed igf-i levels. clearance values were caietdated by dividing the administered dose of rh[ge-[ by the total area under the etnve. vd and tv2 were calculated by regression analysis of the terminal part nf the log sertnn concentration time profiles. results: results are expressed as median (range). age 35(29-73) years, apache ii score 18(12-34), and length of icu stay 26(10-92) days. baseiine circulating igf-1 levels were low 28( < 20-166) ng/ml reaching a peak of only 84( < 20-246) ng/ml. 2 of the 9 patients had basal levels predmnhaantly below the inwel-limit of detection of the assay, and showed no appreciable rise in serum igf-[ levels tbllowing rhigf-i injection. parmacokinetic variables could not therefi)re be determined in these two patients. following rhlgf-i injection in the remaining 7 patients tmax was at 3(2-6) hours, clearance was 89(31-452)0 ml/min, vd was 0.29(0.12-0.73) l/kg, and tia 9.2(4.2-30.1) honrs. conchlsions: there was marked patient wu-iability in response to rhlgf-i administration. vd was similar to that seen in poshq~erative maior surgery patients (0.29 vs 0.33 l/kg), but clearance was greater (89 vs 25 ml/min), tmax earlier (3 vs 5 hours) and t~a shorter (9.2 vs i0.8 homts). if riaigp-i is to be administered to dtically ill patients the dose should he nlodified m the light of these findings. baekgronnd: acute bacterial endoearditis continues to be a condition with high morbidity and mortality. the majority of patients are treated with high dose antibiotics, but a patient group requires surgical treatment. methods and results: from january 1991 to march 1994, 65 patients underwent surgery for acute bacterial endocarditis: 36 had native valve endocarditis and 29 had prosthetic valve endocarditis. there were 48 men and 17 women. streptococcus viridans was the predominant infecting agent in native ~alve endocarditis and staphylococcus epidermidis in prosthetic valve endocarditis. progressive congestive failure, uncontrolled sepsis and peripheral emboli were the most common indication for surgery. although the mitral valve is more commonly involved in acute bacterial endocarditis, the aortic valve most often requires surgical inlervetion. the postoperative bleeding in the no-aprotinin group was 1902+1450cc and in the aprotinin was 850:t:80 (p < 0,0001). the mortality in icu was 10,7%, the staphyloccocical endoearditis mortality was 18,2% and the no-staphyloccocical endocarditis mortality was 2,6% (p<0,05). conclusions: the early surgery is indicated if endocarditis has no response with medical treatment. the mortality of staphyloceocical endoearditis involving left valves was higher than caused by other infecting agents. aprotinin treatment improved prosta#andin metabolism and preserved pletelet function during open heart surgery and could be useful in this type of interventions. urgent surgery had a high mortality (24%). selective digestive decontamination (sdd) has increasingly become the subject of critical discussion sine~ the development of multiresistant organisms has become an issue. moreover, a selection of gram-positive pathogens must also be taken into account when using the sdd regimen, which is primarily directed at the gramnegative bacterial specumn, with the methicillin-resistant staphylococcus aureus strains (mrsa) being of particular importance. the objective of our study was to determine the extent to which colonization with mrsa strains is promoted by sdd. method: 65 patients (ventilation period > 5 days) were randomized and allocated to the sdd group (n=32) or the control group (n=33). in their general intensive care theraw, there were no differences between the groups. the sdd regimen consisted of the four times daily administration of 50 rag polymi~ 80 mg tobramycin and 500 mg amphotericin b in the nesc, mnoth and stomach. systemic prophylactic ~dmini~/rution of antibiotics was not part of the sdd regimen. smears were taken from the nose and the rectum twice wceldy and from the pharynx and trachea once wceldy, and tested for mrsa. further samples were taken as clinically reqnircr results: 625 smears were examined in the sdd group. mrsa strains were detected in 66 samples (10.5%) from 7 patients, and in 5 patients they were detected for a period of up to 4 weeks. the positive smears were districted as follows: tracheal 11/117 (9.4%), nasal 28/199 (14.0%), pharyngeal 15/111 (13.5%) and rectal 121198 (6.1%). severe mrsa-induced infections were observed in 2 patients (infection rate 28.6% of the colonized sdd patients). 560 smears were examined in the control group. ivlrsa swains were r in 15 samples (2.6%) from 6 patients, but only repeatedly over a period of up to 10 days in 3 patients. the po~tive snmars were distributed as follows: traclmal 1/114 (0.8%), nasal 8/174 (4.6%), pharyngeal 4/98 (4.0%) and rectal 2/174 (1.1%). there were no mrsa infections in the control group. conclusion: the data collected support the view that the use of sdd promotes a selection and persistence of mrsa strains. longer-term colonization with mrsa and sovere systemic inf~ons were only found in the sdd group. although the clinical and epidemiological impact of resistance develol~ng when sdd is applied ~maine unclear, this question should be given close scrutiny. tazobactam/piperacillin (taz/p1p) is a new broad spectrum antibiotic, in which the acylaminopenicillin piperaeillin is protected by the betatactamase inhibitor tazobactam from hydrolization by bacterial enzymes. taz/pip has shown to possess a high antibacterial activity against almost all clinically relevant bacteria and is a registered drug in germany. obiectives: purpose of this investigation was to evaluate, whether faz/pip 4.5g is suited for efficient antibacterial monotherapy of severe infections and what influence dosage frequency reveals on clinical efficacy. methods: 2151 hospitalized patients have been documented in this multicenter trial during a 2 year period. as this investigation should reflect the usual clinical treatment, the only criteria for enrolment were the typical signs of infection as e.g. temperature > 38~ leucocytosis or an isolated pathogen. exclusion criteria did not exist and the patients were treated in accordance to the severeness of infection, underlying diseases, risk factors etc. with taz/pip 4.5g t.i.d, or b.i.d. results: patients suffered in most cases from infections of the lower respiratory tract (n=926), followed by intraabdominal (n=765) and skin and soft tissue infections (n=460). 61% of the 926 lrtis wvre nosocomial acquired and in 75% the treatment was conducted as monotherapy. in 53% the lrti was treated with taz/pip b.i.d, and in 45% t.i.d. pseudomonas spp. (n=138) and staph..aureus (n=134) were the most isolated pathogens pretrcatment. the clinical response rates (cured/improved) after treatment with taz/pip 4.5g b.i.d, and t.i.d, were 89% and 81% respectively. results for intraabdominal-and skin and soft tissue infections will be presented. conclusions: in hospitalized patients with severe infections successful treatment with taz/pip in monotherapy is possible. in this population a reduction of the dosage frequency to 4.5g b.i.d, revealed equivalent clinical response rates. objectives. retrospective evaluation of 9 cases of severe generalized tetanus (sgt), treated in our icu the last 7 years. we review 9 cases of sgt (6m, 3f), mean age 66.7 years. in 5 eases the entry site of c.tetanus was a skin laceration, in 1 case it proved to be the external genitalia, while in the rest no portal of entry could be determined. in the first 6 cases incubation period was short (3-11days) and so was the period of onset (1-6 days). all patients needed mechanical ventilation (range 15-58 days), initally through an orotracheal tube,and later through a tracheostomy, performed 6• days after admission. clinical manifestations of sgt included muscle rigidity and i generalized spasms, persisting for up to 6 weeks in the most severe cases. significant autonomic nervous system dysfunction was present in 3 cases occurring 5-12 days after the admission and following the time course of generalized spasm. besides general supportive measures, specific treatment included passive +active immunization, penicillin g, magnesium sulphate and sedation in a variety of regimens. neuromuscular blockade was required in 5 cases. nosocomial infections occurred in 7 eases, with sepsis and mof in one. average stay in the icu was 18-62 days. one patient died with severe septic complications and one was discharged with severe 9 disability due to anoxaemie ancephalopathy, after a cardiac arrest on admission. ~ disinfectant in suspension test, without presence of organic load, 12 disinfectants showed efficacy on lm. in the carrier test, in the presence of organic load, 6 out of 14 examined disinfectants did not exposed efficacy on lm. the results of examinations clearly showed that evaluation of disinfectant's efficacy partly depend on the used test method. antun basi6, intensive care unit, kb firule split spin~ideva 1! jugoslavia bacteremia and sepsis are frequent complications encouuntered in severe icu patients.microorganism identification with hemoculture presents the basis for adequate and successful antibiotic treatment.in many patients damage and vulnerability of the peripheral veins presents an obstacle for obtaining the blood culture from the central venous (cv) catheter sample could be also used. material and methods blood cultures were perfomed in lo4 patients on blood samples simultaneously obtained from the peripheral vein and cv catheter three times in a 24-hour period.criteria for the suspected bacteremia were body temperature above 38 c and leucocytosis above ioooo leucocytes/dl. the site for venipuncture and the cv catheter stopcock port were cleansed with povidon iodine.after the initial 5 ml of blood were discarded,lo ml were used for the blood culture.standard laboratory technique for blood cultures was used. results and discussion in 76 (73%) patients hemocultures was negative at both sites,whereas in the remaining 28 (27%) they were positive.for twentyone (26~176 of the positive patients the same results were obtained at both sites (peripheral vein and cv catheter),whereas in 7 (6.7%) patients the blood culture were positive only for the cv catheter samples.the cv catheters were in place for less than 4 days in 81 patients and for more than 7 days in 23 patients.from 7 patients with positive blood culture from the cv catheter,one patient had the catheter for three days,whereas the other 6 had the catheter from 6-1o days. we neither found significant differences in hemodynamic dates : objectives: 1, to count and evaluate bacteria isolated from endotracheal (et) suctiori samples (with and without saline). 2. to establish the exogenous source(s) of pathogens isolated from carer's hands and the equipment involved in sampling in order to reduce the incidence of contamination and infection. method~: this prospective study included 20 consecutive ventilated patients (15 male and 5 female, 56_ + 16 yr; apache ii score 19-+7) over a period of 3 months. et aspirated samples with and without saline were taken daily from day 0 of intubation until pathogen~ were presented in counts of _> 105 per ml. at the same time, samples from both carer's hands were taken before and after et suction and a swab from the ventilator tube. results: the overall length of intubation varied between 3 to 65 days. bacterial transfer between staff and patients was noted in 80% of patients until day 5 of intubation. there was no significant correlation between severity score and appearance of colonization. the incidence of pneumonia in studied patients was 45% with an overall mortality rate of 30%. acinetobacter anitratas (no 15), staphylococcus aureus (no.15), klebsiella pna~moniae (no.9) and pscudomonas aeruginosa (no.4) isolates predominated in all our specimens. we noticed increased resistance to most antibiotics with the exception of imipenem for gram (-) bacteria and vancornycin for gram (+) bacteria. conclusions: i. tracheobronchial colonization appears directly in the maiority of intubated patients. 2. there is a close relationship between the microflora of personnel, patients and equipment. 3. bacteria transfer was noted both to and from patients. 4. strict hand disinfection policy remains an important measure for the proper care of mechanically ventilated patients to reduce respiratory infections. nnseeomial pneumonia is the most common nnsocomiai infection in the icu-settiag, reported in up to 20% of patients admitted to the icu following surgery. it is associated with significant mortality that ranges from 30~ to 70%. enteric gram-negative bacilli have been implicated in 75% to 85% of ventilntor-associated pneumonias and pseudomonas aeruginosa accounts for 27% to 40% of these pneumonias. importantly, epidemics of/3-1actamnse-pruducing enterobacter spp or klebsiella spp that are resistant to extended spectrum cephalosporins or penicillins, pose serious obstacles to effective antibiotic choices. carbapenems provide in ~tro activity against a wide range of enterobacteriaceaeand other gramnegative aerobic bacteria, except steaotrophomonns maltophilia. in vitro meropcnem is more active against pseudomonas spp than imipanem (especially p. aeruginosa and p. cepacia), imipenem and meropenem are effective against more than 95% of strains responsible for nnsocomial infections. all major pathogens associated with lrti are usually covered by the carbapenems, exceptions are pathogens involved in so-called atypical pneuomouia like mycoplasma, chlamydia and legionella. carbapenems are highly stable in the presence of most chromsomal and plasmid-mediated blactumases and usually offer a postantibiotie effect lasting for three hours against most of the enterubacteriaceae. reeent studies comparing imipenem/cilastatin with other ~-lactams and fluoroquinolones in severe lrti in icu patients resulted in favourable clinical cure rates and good tolerance, but development of resistance in p. aeruginosa and 5;. aureus during treatment were of some concern. meropenem offers the advantage of greater stability against enzymatic degradation, so no concomitant administration of an enzyme inhibitor is necessary, and meropenem appears to be associated with a lower risk of seizures, particularly when used at high doses. results from studies with meropenem in lrti, especially in critically ill patients with acute exacerbations of chronic bronchitis, demonstrated excellent cure rates and better gastrointestinal tolerance of this new carbapenem. both earbapenems are effective candidates for use as empiric monotherapy in nosucominl infections of critically ill patients. qbl~ctives a favourable effect of iv immunoglobulins in septic surgical patients has been reported, but not sufficiently validated. we conducted this study on trauma patients to: i) investigate the effect of ivig on septic complications and il) quantify this effect by means of serum bactericidai activity (sba) assessment and iii) to explore the effect of temperature increase (from 37 to 40 ~ c) on the sba methods: twenty trauma patierits matched on admission for age, sex, inju~ severity score and glasgow coma scale, were allocated to receive either wig (ivig group; i0 patients) or equal volumes of human albumin 20% (control group; 10 patients). wig (sandoglobulin) was administered in a total dose of 1 g/kg divided in a four time regimen on days 1, 2, 3 and 6 post-admission. three blood collections were performe& before the first dose (day 0) and 24 hours after the third and the fourth dose (days 4 and 7 respectively). complement, lgg fractions, the sba at 37 ~ and at 40 o c and clinical parameters were recorded. results-similar lgg and igg] serum levels were found in groups ivig and control on day 0 (743+_130 vs 898• ns and 394+103 vs 472+101, ns), whereas they were significantly higher (p<0 05) in the 1v1g group on days 4 (1700_+_274 vs 799+197, p<0 05) and 7 (1740_+227 vs 864+i64, p<0.05). the various complement-fractions increased in both groups without inter-group differences the mean (• sbas (37 ~ c) at 30 rain in ivig group vs control group were: -53_+32 vs -56• ns for day 0, 9_+46 vs -54_+46 p<005 for day 4 and 7_+34 vs -54+47 p<005 for day 7. the mean (+sd) sbas (40 ~ c) at 30 rain presented a significant improvement over those of 37 ~ c but for the control group remained negative a~d were respectively as following: -~8• vs -26+33, ns for day 0, 22+_39 vs -29_+35, p<0.05 for day 4 and 24_+31 vs -27_+36, p<0.05 for day 7. the increase of temperature induced a 3-fold improvement of sba in iv1g group and 2-fold ofcontrol-~oup positive blood cultures, and the product of the infectious episodes number multiplied by days of occurence, were significantly lower (p<0 05) in the ivig group than in the control (2 vs 7, and 440 vs 1900, respectively). conclusions: our study shows a significantly favourable effect of ivig administration on septic complications and on sba of trauma patients. the increase of temperature results in a significant improvement of sba of patients that received ivig, which theoretically means a farther prevention of infection in the febrile state. pharmaceutical microbiology, university of bonn, meckanheimer aune 168, d-53115 bonn, germany infectious diseases in intensive care patients are common in comparison to patients on other wards and out-patients. the main difference is that intensive care patients are much more sensitive even to less virulent bacteria. thus, the spectrum of infecting organisms is different. strains often regarded as pathogens with low virulence cause serious infections in these patients. strains such as serratia, however, have intrinsic resistance to most commonly used agents such as 3rd generation eephalosporins. furthermore, the common pathogens like staphylococci, psoudomonas aeruginosu, enterocneei and gram-negative bacteria, enterobacteriaeceae as well as the non-fermenters are less sensitive if isolated from intensive care patients. it is difficult to generalize on intensive care units as different patient groups are in different icus aud there are great changes from one hospital to another and from one country to another. if we take s. aurens strains from one study from 1990 the'overall resistance in intensive care units towards oftoxacin was 22 %, whereas in other hospital wards the percentage of resistance was 5.3 %, in out-patients, however, only 2.$ %. the same trend was true for entercnecus faecnlis, coagulase-negntive staphylococci, and other bacteria as well as other drugs. one most striking difference was found with klebsialla pneumoniae and gantamycin resistance, which was $ times higher in intensive care units as compared with outpatients, whereas in the same species no difference was to be seen with the resistance towards carbapenems. however, differences between countries seem to be even more striking, as example gantamycin resistance and staph. anrens is given. the extreme difference is more than 60 fold. thus, it is evident that there is a general trend towards higher resistance in intensive care units, but no generalizatiouis possible. therefore, surveillance studies in intensive care units are needed and the antibiotic policy has to be adapted to the specific needs of the unit. in the icu setting the most potent antimicrobial agents are required to address problem organisms including those resistant to penicillins, cephalosporins and aminoglycosides. carbapanems would appear to present a useful option in this setting. objectives of this study was the evaluation of systemic candid•177 in postoperative cardiac surgery patients (pts) with prolonged icu stay. methods: out of 2617 postoperative adults pts of mean age 61.1+8.9 years old, with a mean icu stay of 1.6_+0.6 days, following an open heart surgery from july 1993 to april 1995, 54 pts (2%) remained in icu for more than 10 days because of severe perioperative complications. patients were included in the protocol if they had clinical signs of infection or sepsis, and fungi isolated in blood culture or in culture from at least three different sites. the patients who developed systemic candidiasis received iv fluconazole (800 mg/day) (10 patients) or amphotericin-b for at least four weeks, and then they were closely monitored. results: out of 54 postoperative pts with prolonged jcu stay, 11 pts (20.3%) developed systemic candid•177 usually after the 20th postoperative day. they were 8 males and 3 females of mean age 64+_7.4 years old. this group of pts had prolonged bypass and aortic cross-clamp time compared to control group (119 min vs 84, and 64 vs 49 min). all these pts received inotropes per• (mean value=2.3). during their icu stay, 9 pts developed sepsis of bacterial origin, while the other two severe infection, and received antibiotic regimens for prolonged period. the patients were submitted to mechanical ventilation for a median period of 50 days. the median icu and hospital stay was 58 and 60 days respectively. all pts have been improved and finally negative cultures were obtained. conclusions: 1. a significant percentage of patients who remained in the postoperative icu for more than 10 days developed systemic candidiasis. 2. all patients who developed systemic candidiasis had received antibiotics because of sepsis or severe infection, for prolonged period. 3. fluconazole seems to be a very good alternative to amphotericin-b. 4. fluconazole is a safe antifungal agent with few side effects. botulism is the most severe and an odd food poisoning. although it is more commonly related to preserved meat derivatives, preserved fish and vegetables are also responsible for a number of cases. obiectives: to evaluate four familiar outbreaks of botulism . methods: we study the patients that were admitted in our hospital because of botulism from may 1982 to february 1995. results: the thirteen pacients involved had a previous history of home preserved beans ingestion. after a 24-hours incubation period, gastrointestinal symptoms (abdominal pain, vomits, constipation) appeared and lead them to hospital consultation in the 4th to 7th day after ingestion. two patients died (acute respiratory failure before admission), seven were admitted in icu, two in ward and two of them were discharged from emergency room. clinical symptoms and the previous history of the ingestion established the diagnosis, that was emg confirmed. in all cases, symptoms were consistent with b-toxin botulism. b-toxin was isolated in serum and food proceeding from the third outbreak, and the serum was negative in the other ones. neurological symptoms were predominant: midriasis (100%), dry mouth (100 %), dysfagia (100 %), asthenia (55 %), palpebral ptosis (55 %), accomodation paralisis (66%) and urinary retention (55%). muscle weakness lead to acute respiratory failure in three patients (one of them required mechanical ventilation). four patiens developed infections (respiratory, urinary and phlebitis). both died patients and one another presented severe hypertension. all admitted patients were treated with polivalent anti-toxin. the two patients who underwent a more severe muscle weakness received also guanidine hydrochloride, with no answer in one case and provoquing a cholinergic crisis in the other one. icu length of stay was 10 days. at hospital discharge, patients continued symptomatic, mainly with dry mouth, disfagia and impaired vision. conclusions: although botulism is a serious illness, the pronostic seems favorable if treatment and support measures are avaible. usually neurological symptoms we predominant and at discharge some of them could still persist. the arrow "hands-off" (aho) thermodilution catheter (tc) is completely shielded during balloon testing, preparation, and the insertion procedure. in order to assess the value of the aho thermodilution catheter in the prevention of systemic infections associated with pulmonary artery catheterization (siapa), we conducted a randomized prospective study over an 18-month period. methods : the patients (pts) were randomly assigned to two groups : group i for a standard tc customarily used in the department, versus group 2 for the aho thermodilution catheter. the diagnosis of siapa was determined on the basis of a positive culture of tc and bacteremia with the same organism, with out any other nearby focus, in association with regression or disappearance of the clinical signs of infection after removal of the thermodilution catheter. results ( objectives: the mortality rate (mr) of tb requiring mechanical ventilation (mv) is high (70-100%). the aim of the study was to evaluate mr, associated factors, and prognostic significance of mv and hemodynamic disorders from tb in icu in 35 patients with tb. methods: clinical parameters on admission, and complications in icu were related by univariate analysis to icu, hospital, and 6 month outcome. 18 patients required mv; 10 were immunocompromised (ic) including 8 hiv. tb was pleuropulmonary in 24, disseminated in 9 and meningeal in 2. results: mr was 31% in icu, 34% in hospital and 47% at 6 month. 12/16 (75%) <0.001 mortality was associated with a high saps score, initial shock, mv and nosocomial septicemia. the mr dramatically increased when ards occurred during illness, despite the lack of correlation between mr and initial po2/fio2 ratio or initial murray score. the site of infection did not influence the mr. surprisingly, the mean therapy delay was shorter for non survivors. mr was not related to ic status, nor hivstatus, but was only related to previous steroid therapy. conclusion: mr of tb requiring icu is high (47% at 6 month). need for mv increased mortality (72% vs 18%). general severity and respiratory dysfunction seem to be major prognostic factors in icu rather than tb per se or than therapy delay. in spite of the improvement in the prognosis of pneumococcal meningitis (pm) with third generation cephalosporins (tgc), this infection still presents a great mortality which could be increased with the appearance of antibiotic resistant streptococcus pneumoniae. objectives: to asses intensive care mortality and morbidity of pm and to define patients (pts) at risk of complicated evolution. patients and methods: a retrospective evaluation of pm cases (all diagnosed by csf culture) admitted in our icu from january 1985 tit march 1995. in all pts we analized: demographic data, underlying disease, apache ii score, clinical symtomps, treatment, complications and outcome. statistical analysis was done using bmdp sofware package. results:a total 0f42 pts were studied, 26 males; mean age 55,8 _+ 16 (16-81); apache ii score 16,6 + 7,9; glasgow coma scale (gcs) at admission 12,5 _+ 1,7; 17 (40%) pts suffer from cronic pathology; 5 (12%) pts diabetes mellitus (dm), 4 (9,5 %) pts had had a previous cranial traumatism. in 22 cases the source of infection was otic and also in 22 (52 %) episodes of pm there were bacteriemia. in 21 out of 26 (80%) pts that ct was performed no radiologic abnormalities were shown, 3 of them presented cerebral oedema and 1 pts a cerebral abscess. twenty-eight percent presented seixures, 14% hemiparesia, 46,3% respiratory failure, 17,5% shock, i5% renal failure, 5,1% multiple organ failure (mof). as for treatment refers 5,5% pts recieved only penicillin, 69,4% pts only tcg, 11,1% pts tcg followed by penicillin and 8,3% pts tcg+vancomycin. seventy-five percelat of pts recieved corticosteroids and 25,6% vasoaetive drugs. the mean icu stay was 7,5 5:6 days (1-28). twelve (28,5 %) pts died, two of them presented pm relapse (resistant streptococcus pneumoniae) and another two pts developed neurological sequelae. factors associated statistically with bad prognosis were dm, the use of vasoactive drugs, shock, mof, the apache ii score at admission, the gcs at the 48 and 72 hours from admission in the icu but not the gcs at admission. didn't resulted statistiealy signifcative age, previous eronie pathology, seizures, baeteriemia, renal failure and coagulation disorders. conclusions: mortality was high and associated to apache ii score at admission, to gcs at 48 and 72 hours after admission, shock, vasoaetive drugs and mof. objectives:the aim of the study was to analyse some of significant immunologycai changes in surgical patients,requiring intensive health care,and to determinate the possibility for evaluation,dynamical examination and importance of immunologycal problems for treatment. methodes:the study concerns a number of 30 patients with expanded surgical intervention or serious postoperative complications.the results has been carried out with fiowcytometryc analyses of lymphocytic suhpopulations and routins methods for investigation of humeral immunity.the"panel" for evaluation of 2(} immunologycal parameters has been offered:t-calls total/cd3+/;t-helper/cd4+/;t-supressor/cd8+/ th/ts ratio;b-cells/cd19+/;naturai kilier/nk/cells;skin test for cellular immune function;phagocytic and oxidative activity;serum levels of immunogiobulins-g ,a,m;protease inhibitors;c-reactive protein.all patients have been studied during suffering and after surgical procedures dynamicaly. results:there have been estimated significant changes in immunologycal parameters especially:decrease of t-cells: cd3+mean=37.62%/14.3%-47.9%/and cd4+mean=22.11%/9% -28.8%/;inverted th/ts ratio ,mean=o.72/0.37-0,90/;reduced or negative skin teste;reduced phagocytic and oxidative activity before septic complications. conclusions:dynamical examination of immunologycal parameters shows,that the prolonged t-total,t-helper lymphocytopenia with functional deficience of ceils-mediated immunity correlates with the stage of clinical condition of the patients and has prognostic importance.it's clear,that immunologycal monitoring gives a possibility for immunecorrection. patients (pts) with the human tmunodeficiency virus (hiv) infection have a decreased immune response and are particularly susceptible to infectious endocarditis (ie). the aim of our study was to analyze the prevalence of ie, its clinical and therapeutic implications in a hiv population 9 we prospectively studied 245 pts, 9.4% (23/245-group ie+) with ie during the clinical course of this disease. we analyzed the following parameters: age, gender, race, type of hiv, cdc classification, number of t4 and t8 type cell population and its ratio, therapeutic with azt, type and number of opportunist infections (inf, mycobacteriosis (mb), neoplasm's (nee) 9 the echocardiographic parameters were lv internal diastolic and systolic diameters, lv percentage of fractional shortening, interventricular and posterior wall thickness, the degree of valvular regurgitations and the presence of pericardial effusion. el was located at the mv in 2.7%, tv in 6.0%, av in 2% and pv in 0.9~ and was multiple in 2.0%. hiv el+ pts had larger lv diameters and more frequent significant valvular regurgitations (39% tr, pe 33%, mortality 32%). these two groups differed significantly in the following clinical parameters: the typical symptoms were watery diarrhea, high fever, tachycardia,luekocytopenia and oligouria within 7th postoperative days. the patients with mrsa enterocolitis had positive mrsa culture from the many materials except feces.mesa strains frequently had coagulase type 2,enterotoxin a and toxic shock syndrome toxin-1 .eight of 1 6 patients had postoperative organ failure.most of the mrsa strains in japan were similar in coagulase type to our hospital and our department.all of mesa strains were susceptible to vancomycin and arbekacin,tbough most of them showed resistant to many other antibiotics.we have employed guidelines for therapies such as oral or enteral administration of vancomycin and correction of the hemodynamics for dehydration and circulatory failure due to diarrhea from 1992.futhermore we have placed colonized or infected patients in private room,worn gown and mask,and carefully washed our hands from 1992. these countermeasures for prevention of nosocomial infections after 1992 significantly reduced the incidence of mrsa enterocolitis. conclusions:earlier diagnosis and treatment, and distric prophylactic measureres against mrsa infections are very important. -cdo ivda leptespiresls affects all the organs with widespread hemorrhage that is more prominent in skin, mucosa, skeletat muscles, liver and kidneys. lung involvement is usually mild and less common. suli, it is very uncommon acute respiratory failure to be the pr6sontirlg symptom. a case with leptosplrosl..,s which was presenting with acute respiratory failure is described. a 36 year-old man admitted to icu becauso of fever, myaigla, aevere c~, hemopty~s. his blood gases showed: pao2:46mmhg with fio2:1.0, pco2:27 mmhg, ph:7.4, hco3:20mecl chest x-ray film demonstrated diffuse bilateral alveolar pattern occupying beth lung4/4). trarmamlnase, bllllrubln, ~ and esr were elevated, wbc was 8.7001mm8, platelet: 40.0001ram3, hematesrlt:30%, hemoglobin: .sgrldl=. there was no clinical or ecttlographlc evidence of left heart failure.patient fulfilled the criteria for diagnosis ards he was found to have an ~lutinatlon tlter for leptoq~lral antigens(indirect he~lutlnatlon atomy, ilia} very high (1/800, negative<ill00) and iglm antibodies also very high (elisa, a=1.492, negative<0.150) strongly suggesting leptospirosls. treatment with tetracycline and non-tnvaslve mechanical ventilation by nasal mask were started. pre~jre support mode and cpap was usod. the following days the clinical picture,the oxygenation and the chest x-ray of the patients were improved, and patient dl~herged from the icu. the control of leptosplremlc phase as well as his good cooperation during nipsv contributed to rapid recovery and excellent outcome in hiv infections, pneumonias (pnm), mainly due to pne~ mocystis carinii (pc) have an outstanding place in pu ! monary complications. the aim of this work was to compare two group> of patients admitted with pnm in our icu during the same period (1990-94): group a, 19 patients hiv+, and group b, 152 patients hiv-. apache ii was identical in the 2 groups (p=ns). group a required more often mechanical ventilation (p=0,o07), had a higher p(a-a)o2 (p=0,004) and metabolic acidosis was more frequent (p=0,001). regarding laboratorial parameters group a had a lower no. of linfocytes (p=0,02), a higher ldh (p=0,04) and a more marked hypoalbuminemia (p=o,03). mortality was higer in group a (52,6%) than in group b (29,6%), (p=0,04). analysing the a group patients, we found no significant differences between alive and deceased patients, with exception for albuminemia, which was lower in the deceased patients (p=0,02). in conclusion, the hiv+ patient's pnm have a more agres sive behavior when compared with community acquired hiv-patient's pnm. the prognosis was not influenced by the apache ii. perhaps other parameters such as p(a-a)o2, metabolic acidosis, linfocytes, ldh and albumin shoud be more evaluated as possible predictive indices. some prognostic factors, usually accepted as predictive in the analysis of hiv+ patients do not seem to be worth in the late stages of aids, mainly when they reqquire intensive care. intensive care unit, onassis cardiac surgery center, athens, greece. objectives of this study was the comparison of two different antibiotic regimens as prophylaxis in cardiac surgery patients. methods: in a prospective randomised comparative study, two different forms of antibiotic regimens were investigated : a single dose of cefuroxime (zinacef, 3 gr) (group a) given during the induction of anaesthesia, versus a four days combination of amoxiculine (amoxil, 2 gr tid) plus netilmicin (netromycin, 150 mg bid) (group b). a total of 926 patients (pts) (767 males and 159 females, of mean age 60.6+8.7 years old) were included in the study over a period of one year; 424 in group a and 502 in the group b. patients were checked for the occurrence of infection during the first postoperative month. results: the total rate of infection in cardiac surgery pts was 5.8%; 5.4% in group a and 6.1% in group b (p=ns). 34 pts (4.7%) developed infection following cabg, 17 pts (7.9%) following valve replacement and 6 pts (17.6%) after other cardiac surgery. they were 43 males (5.6%) and 11 females (6.9%). endocarditis has occurred 0.4 % in group a and 0.2 % in group b. severe wound infection was recorded in 0.4% in group a and in 0.8% in group b. one case of sepsis (0.2%) in group a and in group b (0.2%). respiratory infection occurred in 11 pts of group a (2.6%) and in 11 pts of group b (2.2%). two cases of urinary tract infection was in group a and one in group b. catheterrelated infection was occurred in 5 (1.1%) in group a and 6 (1.1%) pts in group b. 3 pts (0.6%) had fever of unclear aetiology in group b. conclusions: there was no statistically significant difference regarding the rate of infection in both groups. a single dose administration of cefuroxime is accordingly just as effective as a four days regimen of amoxicilline plus netiimicin. legionella pneumophila is a common bacteria of the environment, and it is an agent responsible for severe community acquired pneumonia (cap). we analyzed the 8 patients with lpp admitted in our icu during the last 8 years (1986) (1987) (1988) (1989) (1990) (1991) (1992) (1993) (1994) . they represented 4.6% of cap. seven patients were males and 1 female, with mean age 46.7+12.1 years. tiss was 24.1+10.9 and apache ii 21.0+5.2. all, but 1 patient, were under mechanical yen tilation (mv) during a mean period of 11.7• (min-l, max-44) days. two pneumonias occurred beyond the season, while 4 patients had an epidemiological history. only 1 patient had no risk factor. in all the others tobacco smoking and alcohol abuse was quite frequent. diagnosis was based on serologic test and culture or direct fluorescent antibody staining of bronchial secretions. seven patients had a multisystemic disease with hepatic dysfunction in 5, renal failure in 4 (due to rhabdomy~ lysis in 3). one patient had a prosthetic valve endocarditis and another developped ards. nosocomial septicaemie occurred in 3 patients. mortality rate was 50%. deceased patients had initially higher apache ii, (a-a) 02, and lower natriemia. comparing lpp with the other cap (n=84), both submitted to mv, mortality rate was similar (57,1% versus 54.7%). in conclusion lpp can occur all over the year. there was a high incidence of severe complications and outcome was similar to the other cap when requiring mv. prospective specimen brash (psb) with culture > 10 cfu 10 3 cfu/ml. broncho-alv~lat lavage (bal) ~= 104 c'fu/rnl or positive blood culture. 10 were excluded for rapture of treatment ; 63 were analysed (shift with oral antibiotic8 : 3 ; prohibited antibiotics associations : 5 ; resistant germ : 2). clinical data : age 60,6 • 18,7 ; saps 12 • 2,86 ; mac cabe i : 76,2% -ii : 22,2 % -iii : 1,6. 63,5% of the patients were intubated and under mechanical ventilation. the pneumoaiae were : primitive in 35 (55,6%), copd 9 (14,3%), aspiration pneumonia 19 (30,2%). 75 germs were isolated (psb 67, bal 1, blood culture 7) : s. pneumoniac 28 (37,3%), h. influeazae 14 (18,7%), sttep~:occns 10 (13,3%), saar6ns 10 (13,3%), enterobaetdrindr 5 (6,7%), mosexella catarrhalis 2 (2,7%), othem 6. 71/75 (94,7%) were sensitive to freatment. the ltentment was 100 mg/kg/d of ampiclllin and 50 mg/kg/d of sulbactam in continuous iv adminisu'ation during at least 10 days. clinical eff~ienev : success 46 (73%), failures 17 (27%) with superinfeetion 7, worsening or relapse 3, dead 5, side effects 2. there was no difference between etiologies : primiti~;e~ 74,3%, copd 77,8%, aspiration pneamoniae 68,4%. the bacteriological effieieacy was evaluated only for 41 patients with eradication 30 (73,2%), eradication but super~ection 6 (14,6%) : with pseadomoaas a&ogiuosa 2, eater~ac~ 3 ; beeteriological failure 5 (12,2%). in conclusion, the aasor ampicillin -sulbactam is effective for the i~eatment of severe acquired community pneumonise. objectives : to assess the efficacy of chlorhexidine (cl) gel or suspension applied in the nose and in the op for the prevention of the tmcheobronchial colonization. methods : thirty-seven patients expected to be intubated for > 48h were randomized to received topical application oga cl suspension (2%) qshrs, a cl gel (1%) q6hrs or a placebo. in addition all vpts received a nasal and a op spray (2%) of either cl or placebo administrated according to the same schedule. semi-quantitative cultures of the anterior nares, the oropharynx (op) and the trachea were obtained on admission and once a day until extubation (just before the next application). the results were assessed according to the following criteria: success = no acquisition of gnb in the trachea ; failure = acquisition of gnb in the trachea. acquisition was defined by a follow-up culture positive for a gnb not present in the trachea on admission. results : success failure nosocomialpneumonia overall morality clsusp. placebo clgel placebo n=8 n=10 n=9 n=10 5/8 6/10 7/9* 3/10 3/8 4/10 2/9* 7/10 1/8 2/10 3/9 4/10 0/8 1/10 2/9 2/10 i *p = 0,03byfisher'sexacttest conclusions : these results suggest that topical cl gel administered q6hrs may prevent tracheal colonization by gnb. f. daumal*, m. daumal**, c. plot**, v. vurmmen ~ e.colpurt**, b. manonry** * hygiene hospitali&e, ** service de r6enmmtion, * service des admissiens-urgeuces centre hospitalier g-6ndral -02 321 saint-quentin -france obiectives: evaluate the nosocemial risk due to peripheral venous inserted short catheters, and the quality of care. patients-methods: the intensive tare unit (i.c.u.) is a 9 beds unit. the prospective study includes all the patients comn~ in from 01/01/1993 to 30/03/1995. the recruitemont uses an evaluation schedule of local clinical signs. the nurses aimed to create this evaluation data which includes the place of entry site, the duration of catheterization and the cause ot withdrawal. only patients staying longer than 2 days in the i.c.u. are accounted for. the diagnosis of uosoenmial infection is assured by the physician taking care of the patient and by the hospital epidemiologist on the next signs: evident pus at the catheter entry site, positive culture of the strain, with or without the same pathogen in the blood sla'uam,the patient having no other distant source of infection. analyses were performed on epi/nfo. results: the occurrence of 3 nosoeomjal inthrtions: i abcess and 2 bacteremia during the first part of the study lent the medical staff to modify the protocol of insertion end survey of the device. so we analysed 2 different periods: period 1( 1/01/93 to 31/10/93 ) and period 2 (01/11/93 to 30/03/95 ) for all 1 .e peripheral catheters inserted in the i.c.u. period 1 44,9 % 46,2 % en infection due to peripheral venous device is a daily threat. the severity of some clinical situations requiring admission in icu proves it. the motivation of nurses for rigid adherence to established protocol, the daily survey of the entry site, the withdrawal of the peripheral catheter every 72 hours aimed to reduce significantly the local signs of inflammation end infection of peripheral catheters inserted inside the i.c.u. objectives: to investigate the use of a new metabolic monitoring device for different ips levels by comparing oxygen consumption (vo2) to measurements of the mechanical work of breathing (web) and p0.1. methods: the study was approved by the institutiotml ethics committee. eight patients were investigated during weaning after prolonged mechanical ventilation (6-75 days) for various diagnoses when the clinical physician judged the patient to be ready fur weainag. ips was setto 15, 10, 5, 0 mbar far 20 rain periods each. all patients had a peep between 5-8 mbar.. respiratory frequency (f), tidal volume (tv), minute ventilation (ve) were read from the ventilator display (7200ae, puritan bennett, carlsbad, usa). flow and airway pressure were measured at the endotracheal tube site. esophageal pressure was measured using an esophageal balloon catheter (fa. ruesch, frg). web was determined as the area subtended by the pleural-pressure-vohime curve. p0.1 was determined by using standard occlusion technique and graphical analysis of the airway pressure tracing. vo2 and vco2 were measured using the pb 7250 metabolic monitor (puritan bennett, carlsbad, usa) connected to the pb 7200ae ventilator. all data are given as mean• deviation for each ips level. comparison between the different ips levels was performed using anova for repeated measurements. significance was considered at p<0.05, compared to ips 0 mbar. results: the values for breathing pattern, web, p0.1, vo2 and vco2 are given in the table for the different ips levels; significance is indicated by ~. objectives: fluidized beds are often used in the management of critically ill mechanically ventilated patients. critically ill patients are increasingly colonized with resistent pathogens [ie: p. aeruginosa, methicillinresistent s. aureus (mrsa), extended spectrum i~-iactamase producing enterobacteriaceae ] that can ultimately cause nosocomial infection. methods: we prospectively monitored bacterial colonization of mechanically ventilated patients and of the fluidized bed (clinitron) inwhich they were treated. multiple samples for quantitative bacterial cultures were taken from oropharynx, trachea, feces and bedsores. samples of ceramic beads from the bed were also taken both during and after patient stay (after bed operation in the absence of patient). re,~ults: 13 episodes in 12 consecutive patients (mean age: 57.6 years) were analyzed. all had bedsores and/or urinary catheters and fecal incontinence, 7 patients had nosocomial pneumonia, 6 had urinary tract infection [2 with extended spectrum imactamase producing k/ebsie//a pneumoniae (ki~lse)], one had positive blood cultures with mrsa, and one patient had a ki~lse found in high concentrations (103 -10 s cfu/ml) in 2 occasions in feces. patients were heavily colonized: the , samples from ceramic beads showed no growth or became sterile without any sterilisation procedure (even in one case of presence of kf~lse) during the patient stay. conclusions: fluidized beds do not put patients at high risk of acquiring nosocomin pathogens, and cross-contamination between patients seems unlikely, even when multiple resistent organisms were initially present. the recommandation from some manufacturers to undergo extensive sterilization of fluidized beds after use does not seem warranted, at least with the bed used in this study. ant. koutsoukou, a, tahmitzi, p. kithreotis, m. koutonlidou, k. stavrakaki, kainis e, g. vlahogiorgos and e. eliopoulos icu-centre for respiratory failure -chest diseases hospital of athens. the cost-effectiveness issue is becoming vital in modern medicine and may lead to moral dilemmas since sometimes certain groups of patients may not have access to highly specialised modalifies. objective: our study compared the mean daily cost for antimicrobial medication in copd patients treated in icu versus all other patients in the context of relevant epidemiological, prognostic and outcome data. methods: age, sex apache ii score, length of icu stay (los) and in -icu fatality were retrieved from the files of all icu admissions over 1994. mean daily cost for antimicrobial therapy per patient (dcat) was estimated. these variables were statistically compared between copd and non-copd patients. significance was assumed at p<0.05 results: 140 of the total 178 admissions were fully evaluable. 38 of them (27%) were copd patients. data (m---sd) results for statistical test are given in table i . copd patients were significantly older spent more time in the icu and presented with significantly higher apache ii scores. outcome and dcat were comparable in the two groups. objectives: the use of heat and moisture exchangers (hmes) during long term mechanical ventilation (mv) is increasing. in icu patients, they are routinely changed every day, according to the recommendations of the manufacturers, but the clinical basis for such a daily practice is lacking. we therefore prospectively assessed whether changing hmes (dar hygrobac, spa, mirandola, italy) every 48h only would affect their clinical and bacteriological efficiency. methods: two consecutive groups of patients requiring mv for >48h were compared: group1= hme replaced every day, n= 61 episodes of mv in 61 patients; group 2 = hme changed every 48h, n=68 episodes in 64 patients. tubings were not changed in the same patient during the whole length of ventilatory support. diagnosis of nosocomial pneumonia (np) was based on a positive quantitative culture (~103 cfu/ml) of a protected specimen brush in patients with clinical signs of pneumonia. quantitative cultures of pharynx, trachea and y-cannector were performed every 48h. results: the groups were similar in terms of age, indication for and overall duration of mv (10+_8.6 vs 10+_9days, p=0.9), and severity of illness (saps: 16---4.9 vs 16.4+_5.5, p=0.6). the maximal values for peak airway pressure were identical in both groups (33.4-+7.8 vs 33.7• cmh20, p=0.9). obstruction of the tracheal tube was observed in only one instance in a group 1 patient who had tracheal bleeding. circuit colonization was very rare, and of low grade in both groups. the level of patient colonization and the type of organisms were identical in both groups. more importantly, the incidence of np was the same (6/61 vs 8/68, p=0.7), as was duration of mv before the occurence of pneumonia (9• vs 10.5+_4.7, p=0.6) and overall mortality rate (17161 vs 17168, p=0.7). conclusions: the clinical efficiency of this hme does not seem altered after 2 days of use. indeed, replacing this hme every 48h only neither affect circuit and patient bacterial colonization nor the incidence of np. therefore, substantial savings could be obtained changing hmes every other day only. obiectives: to evaluate the usefulness of different paraclinical investigations for the diagnosis and prognosis of acute viral encephalitis in icu patients. methods: we reviewed 13 patients (pts) admitted to our icu from july 1989 to december 1993 with the diagnosis of acute viral encephalitis. all were in coma and were initially treated as presumed herpes simplex virus (hsv) encephalitis. the causative agents were: hsv (2 cases), herpes zoster varicellae (1), measle (1), rabies (1), unidentified (8). eleven pts survived and three presented neurologic sequelae. twelve pts were investigated by mri, and eleven also by spect and multi-modality eps. including brainstem auditory eps (baeps). these investigations were obtained as soon as possible following admission and were repeated during icu stay when possible. the clinical outcome was noted. results: six pts (6/12) had an abnormal mri. among them, 2 pts made a complete recovery, in comparison with 5/6 pts with a normal mri. in one hsv infected patient, mri remained normal despite clinical deterioration and bad outcome. when repeated, mri became abnormal in 3 cases (with poor outcome in one) and was improved in one. spect was found abnormal in 10/11 pts (among them, 4 pts had thus a normal mr/). the correlation regarding the topography of brain lesions was poor between mri and spect. the findings of spect could not be correlated with a poor outcome. the baeps confmned in 56% of the pts the clinical diagnosis of brainstem involvement. changes in visual and somatosensory eps were mild in all the pts and were not helpful for the prognosis. eps were otherwise interesting for the follow-up of the coma in these sedated and ventilated pts. conclusions: the value of mri and eps for the diagnosis of acute viral encephalitis is of limited interest. spect seems to show early modifications, even in pts with a normal mri, but this test is poorly specific and does not correlate with mri changes when present. concerning the prognosis, larger studies should probably confmn that a normal mri could usually result in a good outcome. this serie illustrates also that hsv encephalitis could be demonstrated only in a small number of cases and that the prognosis of non hsv encephalitis is not easily assessed. objectives: to study the influence of gram (-) bacterial lung infections on liver function i~ mv icu pts. pts and methods: we studied 102 pts, 68 # (66,7%), 34 (33,3%). hean age:48,3• years (16-82). mean stay in icu:13,3• days (8-75). they were divided in 2 groups: a(44 pts) who did not suffer from pneumonia and b (58 pts) who developed a gram(-) bacterial pneumonia. both groups were consisted of pts with same age, sex and disease distribution and same systemic failures. we measured sgot, sgpt, total bilirubin(tb), direct bilirubin (db), alk.phosphatase (al.ph.), v-gt and albumin (alb.) 3 times: on days o, 4 and 7 of the pneumonia for group b and respectively for g~oup a. conclusions: 1) in elderly intubated pts of an icu, kp is isolated more frequently than in icu pts<65 years (p<o,01). 2) the frequency of isolation of kp in icu pts during 1985-92 was 25-30%, but now (1992-95) kp becomes more and more rare (12-15%) and is isolated especially in elderly pts. 3) its sensibility to antibiotics remains always the same. 4) the prognosis of np in our study was independent from age (p<o,1), sex, presence of bacteremia (p<0,1) and type of invading microorganism. gram neg bacteria and renal failure (rf) from aminoglycoside toxicity are common and serious complications in critically ill patients. the aim of this prospective, pilot study was to compare the safety and adequancy of the endotrecheal (et) vs the intravenous (iv) administration of aminoglycosides. were measured in the plasma and the bronchial secretions at 1 and 4 hours after the iv (bolus) or the et (nebulized) administration of 80 mg of gm in seven criticcally ill patients (3 with established renal failure). safety was defined as peak and trough plasma gm levels _< than 8 and 2 ijg/ml respectively and adequancy as gm levels in bronchial secretions _> 0,5 ijg/ml. results: gentamicin was administered by the et and iv routes in 18 and 7 separate sessions respectively. a total of 107 samples were assayed, 69 in bronchial secretions (bs) and 38 in serum. the et route resulted in higher gm levels in the bronchial secretions compared to the iv route (3,26 + 2,86 vs 2,1 _+ 2,1 pg/ml respectively, p = ns ). adequate bronchial gm levels were achieved in 100% of patients after et administration, compared to 66% after iv aaministretion. the blood levels of gm were significahtly lower after the et vs the iv route (1,56 + 1,95 vs 5,56 • 1,96 pg/ml respectively, p _< 0.01). the et administration resulted in toxic bronchia~ gm levels in 47% of the specimens. 66% of these samples were from patients with renal failure, however toxic blood levels were reached in only 12% of these. gentamicin seems to be a safe and adequate alternative route of treatment for the lrti. however, in patients with renal failure the et administration of the aminoglycosides should also be modified and continuously monitored. in order to evaluate the pathogenic role of anaerobes in nosocomial pneumonia (np), we investigated the systemic humoral response in patients who developed a np with anaerobic bacteria, especially prevotella species. methods: blood samples from 4 groups of patients were tested. group i: 13 patients with a np in which prevotella spp. was isolated from protected specimen brush (psb), group ih a control group of 30 patients with a np without anaerobic bacteria, group ill: a control group of 27 patients with dental stumps but without pulmonary infection, group iv: a control group of 30 healthy voluntary people with prevotella spp. isolated from the dental plaque. an elisa was used to evaluate the total antibodies level against a mixture of four prevotella strains and a western-blot method was done to identify the antigenic proteins. results: data are expressed as means .+ sd. the antibody levels in patients of group i (63• was statistically higher (p=o.o05) than in the 3 control groups (respectively: 29+25, 32_+25, 31_+23). using western-blot method, the intensity of the response was roughly superposable to levels obtained by elisa and the profiles were different according to the prevotella species. the occurence of a np with anaerobic bacteria (prevotella species) isolated from psb leads to an antibody response which seems specific of the prevotella species isolated. fever is common in the intensive care unit, but is not always related to an infection. we sought to define the epidemiology of febrile patients in a general medical/surgical icu. methods: we prospectively analysed the source of fever (t >38.2 ~ c) in all adult patients admitted for >-48 hours in the icu during a two month period. these patients were studied for 14 consecutive days. and werc classified in 3 groups according to the evidence of infection (center for disease control criteria) after complete evaluation: documented infection: cdc criteria + isolation of pathogen (d); possible infectron: cdc criteria without isolation of pathogen (p); unlikely infection: patients who did nol meet the cdc criteria (u). results: of a total of 208 patients studied, 74 dec'eloped fever (35 6%). including (after complete evaluation) 39 d, 15 p and 20 u palients. both the highest temperature in tile first day of fever and the maximal temperature were higher in d than in u (38.7•176 versus 38.5•176 and 39.2-~0.9~ versus 38.64-0.4, respectively p= 0.05 and p= 0.003). most common sources of infection in d were the lungs in 25 patients (64%) and urina .ry tract in 4 (10%). 14 of these patients had positive blood cultures (36%). the overall mortality was 27% (23% in d, 40% in p and 25% in u. differences ns). antibiotics were given in 100% of d, 73% of p and 15% of u (3 patients). in p there was a non significant lower mortality." in patients who received antibiotics (3/11 (27%) versus 3/4 (75%) patients, respectively). conclusions: in febrile icu patients both the highest first day" temperaturc and maximal temperature are significantly higher in infected than in non infected patients, but the differences are too small to be useful clinicall). mortality rate is not significantly influenced either by the presence of an infection or by the administration of antibiotics, obiective: retrospective study to determine the influence of candida infection on icu outcome. methods: 126 patieet with a stay of more than 7 days in inteaasive care were screened for candida infection. 70 patients were treated with antifungal therapy due to either an increased antigen titre of -> 1:8 or clinical evidence of candida colonization. serological candida-antigens (ramco, pastorex) and antibody titres (hemagglutination, lgg-, igm-elisa) were examined routinely. seroconversion was defined as a threefold increase of antibody titre or a titre of 1:640 or higher. results: the median length of stay was 37 (ranging from 8 to 132) days, the mean apache ii score on admission was 18 (+_ 5.8 sd) points. of 126 patients 31 patients died (24.6%). in the group treated with antifungnls (71 patients) 19 patients died (26.7 %). although of the 126 patients only 51 (40.4 %) developed a candida infection as defined above the mortality in the group that showed signs of infection was significantly higher (37.2% vs. 14.6%, p < 0.05 [chi-square-test]). in 34 patients an antigen concentration-> 1:16 was measured. seroconversion was found in 41 patients. the most common fungus was candida albicans (66.4 %). furtberm0re, candida glabrata was found in 21.1%. most of the patients were treated with 2 x 200 mg fluconazole (66 patients). in 38 patients therapy was changed to amphotericin b/flucytosine. in 5 patients therapy was started with amphotericine b and flucytosine. in 40 patients a threefold decrease of candida antigen titre was found. 27 patients showed a decrease of candida antibody titre. conclusions: meticulous screening for eandida infection seems to be necessary since the number of patients with fatal outcome is significantly higher in the group with signs of fungal infections and thus requires immediate antifungal treatment. objective: early diagnosis of patients with ventilator-associated pneumonia (vap), and subsequent identification of causative microorganism, and selection of the appropriate therapy are critical important points that affect morbidity and mortality. the results of the quantitative bacterial cultures are not available for at least 24 hours, while a two hours period, since the specimen are obtained is enough to know the gram stain results. the aim of this study is to determine the usefulness of gram stain in specimens obtained by bronchoaiveelar lavage (bal), through the bronchoscope. material and methods: we studied 47 patients (36 males and 11 females, age 49 + 23) with suspected ventilator-associated pneumonia. the bal gram stain was considered positive when the specimen after a centrifugation at 1500 rpm for 15 min revealed: i) more than 20 leukocytes per optic field, ii) squamous epithelial cell less than 1 percent and iii) one or more microorganisms per optic field on 1000 magnification. all patients had been receiving antibiotics, with no change during the last 3 days, prior to bronchoscopy. results: 8 patients had vap and 39 patients did not. in 5 cases the bal specimens (quantitative bacterial cultures) established the diagnosis of vap in the remaining three patients the vap diagnosis was established by other procedures (blood or pleural fluid culture, clinical outcome, autopsy). apache fl score in patients with vap was 15,7 -+ 5,5, while in patients without vap was 17,9 + 6,4. there was a significantly higher incidence of vap in patients who had i) coma (gcs <8) and ii) been receiving neuromuscular blockade (p<0.05) . the sensitivity of the gram stain for vap diagnosis was 75%, the specificity 89,5%, the positive predictive value 60%, and the negative predictive value 94,6%. conclusion: our data indicate that the gram stain of bal specimens is useful for the early diagnosis of vap and the subsequent administration of the appropriate treatment. the role of anaerobes in mechanically ventilated patients with pneumonia (mvp) have been poorly investigated aim of the study : analyse the prevalence of anaerobic isolation in mvp. methods : between october 1992 and february 1995 all suspected mvp were investigated using protected specimen brush (psb) technique. brushes were rapidly transported in shaedler broth to laboratory. a special care was tooken for anaerobic isolation. results : among the 153 psb performed for suspected mvp (132 nosocomial and 21 community-acquired pneumonia), 81 yielded at least one micro-organism (positive psb : 53%). 63 of positive psb demonstrated only aerobic bacteria and 18 (23%) yielded with anaerobes. in 14 out 18 patients, anaerobes were associated with aerobic bacteria. anaerobes were mostly isolated in nosocomial pneumonia (17/76 positive psb). 27 strains of anaerobes were isolated. prevotella species represent 19 out these 27 strains (70%) the most frequent anaerobic species were prevotella oralis (6) p. intermedia (5) and p. buccae (4). comments:using adequate methods, anaerobic bacteria are frequently isolated in mvp. it could be off importance to take in account anaerobes in the choice of empirical antibiotic therapy in mvp. objectives: the majority of patients with multiple trauma are considered immunocompromised. the aim of this study was to identify risk factors of pneumonia in mechanically ventilated patients with multiple trauma or after surgery. methods: in this prospective study we studied 64 multi-trauma patients (mean age 58 + 19 years, apache ii 16.5 + 6), admitted to a general intensive care unit (icu). all patients were intubated and mechanically ventilated. we were considered that a patient had ventilator associated pneumonia (vap) when the specimens of bronchoalveolar lavage (bal) or protected specimen brush (psi?,), ebb'ned through the bronchoscope, had one or more microorganisms in concentrations greater than 105 and 103 cfu/ml respectively. all patients had been receiving antibiotics, with no change during the last 3 days, prior to bronchoscopy. results: 14 patients had vap, and 50 patients didn't. in the bivariate analysis, the glasgow coma scale (gcs)<8 (x2=4.15, p<0.05), the administration of neuromuscular blockade (x2=7.9, p<0.05), the duration of mechanical ventilation to be greater than 5 days (x2=5.5, p<0.05), the flail chest (x2=4.1, p<0.05), the parenteral nutrition (x2=5.6, p<0.05), the ards (x2=3.9, p<0.05), the abbreviated injury scale (ais) of more than 4 for thorax (:,:2=5.9, p<0.05), the pneumothorax (x2=5.1, p<0.05) were statistically significant related to development of vap. in multivariate regression analysis, using the stepwise technique, three of the seventeen studied factors showed to have an indepantent association with the development of vap:the administration of neuromuscular blockade (f: 4.8, p<0.001), flail chest (f:3.0, p=0.003), and gcs (< 8) (f:2.1, p= 0.039). conclusions: in patients admitted to icu for multiple trauma or major surgery, the administration of neuromuscular blockade, the flail chest, and the gcs (<8), in the population under study, were the indepedent risk factors for vap. mof is a sereous complication of differem states: infection, sterile inflamation, extensive fissure injure, intoxication, ets. there is close correlation between extension of mof and death, developement of nasocomial infection. immunologic disfunction. in order to prgnose probability of risk of mof development among the patients with sepsis and septic shock, we achived an eqation, allowing to recive a coeficient, closely connected with this probabiliti. we have used retrospective analisis of 160 cases of sepsis. diagnosis of sepsis was based according to bone's criterions of sepsis. mof was assessed as disfunction of 2 or more systems according to bone's classification of mof. having used correlation analisis we have estimated factors which have had high correlation coeficient with the probability of development of mof. there were: apache-ii score points, evidenceof septic shock, endocrinopathy. with the help of multyple regression analisis we acheved next equation: y= 0,2042 + 0,0243x~ +0,2931x 2 +0,1504x3 , were x i-apache-ii score points, x2-evidence of septic shock, x3-endocrinopathy. the explanatory power of this quation was evidenced by roc of 0.88, se (v 0 -0.033 introduction: the presence of liver dysfunction in the process of multiple organ failure is associated with an adverse outcome, particularly when it becomes progressive to liver failure. disturbances of liver function may occur early and their detection may be of significant importance for the further development of organ failure. routinely used liver function tests appear to be inconsistent indicators of hepatic damage. in this study, we used p_lasma disappearance rate (pdr) of indocyanin-green dye (icg) as an early estimate of liver function. methods: we serially evaluated pdr and routine liver function tests (serum bilirubin, sgot, sgpt), as well as acute phase and non-acute phase proteins (crp, transferrin) in 26 patients during the first week after trauma or the onset of sepsis. patients: group 1: (n = 11) multiple trauma iss > 30, group 2: (n = 15): abdominal sepsis, acute necrotizing pancreatitis (anp) grade iii. patients were selected on the basis of clin4cal estimates that these patients would require continued icu observation. pdr was determined by means of a fiberoptic catheter and a computerized system (cold z-021, pulsion), which permits repeated bedside measurements. the initial values of pdr, serum bilirubin and transaminases were not significantly different in trauma, sepsis and anp. in trauma patients pdr improved during the first week. in patients with sepsis and anp pdr remained low and worsened with time. the decrease in pdr preceeded an increase in biochemical liver function tests in these patients. 1+4.4 110&-_11 7(4-9) discussion: routinely available blood tests of liver function are usually altered several days after injury. however, they are generally non-specific indicators and they are influenced by extrahepatic factors. pdr seems to be useful to evaluate impaired liver function early after the onset of sepsis and trauma. objectives: to study frequency of organ system failure (osf) and it's influence on outcome in granulocytopenic patients with hematological malignancies and septic shock(ss). materials and method: retrospective review of medical records of 52 granulocytopenie(wbc<0,5xl09) patients with hematological malignancies and ss, who were admitted to the intensive care unit (icu). frequency of osf before and after ss was analysed. the patisnts were categorised on survival and non-survival. results: signs of osf were observed in 28.8% of patients before ss and in all patients after ss. only 3 patients presented with hypotension refractory to inotropic therapy. nevertheless there was a significant increase of frequency of acute respiratory failure (arf), acute renal failure (arenf) and liver injury (li) after ss occurred(showed on the figure). only 5 frequency of organ failure before and after objectives: statusmetria allows to define the effective level of oxygen status and accordance to it means of carbon dioxide and elec-trolyte8 in critical care. the conception of syndrome int~ive care (sic) is exhausted itself and invariable outcomes of sic of multiergan system failure (mosf) confirms that. therefore, an alternative to sic should be advanced. methods: efficlenoy of treatment has been asscsaed in 257 patients with mosf using value of metabolic rate and ability of an organism to cover it by oxygen and substrate supply. oxygen pulse (op) and index of efficacy of oxygen transport (ieto2) was monitored. ~lt~.lntenaive care is considered to be homeostasis-securing therapy (hst) if energostructure deficit is eliminated and necessary for recovery regeneration rate is .restored. op in patients with mosf was 0.8 mt-m "2, and le,~ and ie'i~ w~ 2.4 units in sic. we managed to maintain op of 1.0-1.7 ml.m "2 and ieto2 of 1.9-2.6 units in hst. 41 patients from 135 with mosf survived in sic and 96 patients from 122 survived in hst. efficiency of hst appeared to be two times as much as efficiency of sic. cr of homeostasia-se-'uring therapy is advancing. the conception provides restoration of regeneration rate due to effective then in sic elimination of en=gostructure deficit. the conception may be a basis of new technology for treatment of mosf. helen f goode phd, nigel r webster phd. anaesthesia & intensive care, university of aberdeen, ab9 2zd, uk. objectives: xanthine dehydmgenase is converted under conditions of ischemia, reperfusion and endothelial damage to xanthine oxidase, with superoxide anion as a co-product of its catalytic activity. multiorgan dysfunction syndrome is associated with splanchnic vasoconstriction resulting in significant and prolonged gut ischaemia. aggressive volume resuscitation with prompt restoration of blood flow results in reperfusion of the tissue and is likely to cause xanthine oxidase-mediated release of oxygen-derived radicals. this study investigates xanthine oxidase activation and oxygen-derived free radical-mediated damage in such patients. methods: fourteen consecutive patients on itu who met established criteria for septic shock and secondary organ dysfunction were studied. serum xanthine oxidase activity was measured using oxidation of a chromagen in a dual enzyme system and plasma malondialdehyde was measured using a specific spectrephctometdc assay. apache ii scores, blood pressure, svr, cardiac output and 28 day survival were also recorded. biochemical data were compared with results from 20 healthy subjects. results: xanthine oxidase activity was 6.30 + 1.59 units/i in patients (mean :t: sem) and 0.74 + 0.12 units/i in controls (p<o.01, mann whitney u test), and was highest in the 6 patients who survived (p<0.05). malondialdehyde concentrations were elevated (0.81 • 0.11 prnol/i compared with 0.29 • 0.05 pmol/i in controls, p<0.001). xanthine oxidase activity did not relate to apache score or any of the cardiovascular parameters recorded, and was not influenced by the degree of organ dysfunction. conclusions: patients with sepsis and secondary organ dysfunction have xanthine oxidase activation and evidence of free radical damage. the finding that activity was highest in those patients who survived suggests that reperfusion was incomplete in patients who died, with decreased tissue xanthine oxidase ~tash out' into the circulation. influence objective : evaluation of the feasibility of measuring the intragastrie partial pr~sure of carbon dioxide (pco2) using a chemilumin~nt optode and fibre~ptics originally developed for intrav~cular blood gas monitorlng(p7) methods the pco 2 electrode w~ calibrated i~-vitro according to the m~ufaeturers instructions the sensor w~ then stripped of its outer c~ing and threaded down the pile tube of a g~ c enema e (gt) so ha he ens ng portion sat well within the balloon. the modified gt was inserted n~og~trically after induction of anesthesia in ten patients undergoing c~diopuimonary byp~s throughout results: according to the datas of integral rheography 70% of patients were revealed to have hypodynamic type of blood circulation (cardiac output was decreased on 35-40% and general peripheral resistance increased on 80-100%). it was effective to use complamin (xantinoli nicotinas) in the complex therapy of su~h patients. the dosage was 10-15 mg/kg during 4-6 days. as a result of such therapy was also the oliguric stage shortening (ac n 1464083).in 9,6% of the cases unsatisfactory datas of central hemodynamic,combined with high (more than 15 cm h20) venous pressure,lung oedema.then nanipruss (0,5-1,5mkg/kg/min) was succesfutly used in complex with routine therapy.in the patients with low cardiac output and low general peripheral resistance mesaton (0,25-0,5 mg/kg) and dopamine (5-10 mkg/kg/min) were effective.change for the worse in the hemodynamics datas, inspire of therapy during 5-7 days,was prognostically unfavourable. conclusions:we consider early diagnostic of hemodynamic disorders and adequate correction of them is one of the most important factors, determined the outcome and prognosis in arf patients. obiective: to evaluate the results of renal replacement therapy (rrt) in surgical icu patients with acute renal failure (arf). arf in icu patients is associated with high mortality rates. we investigated the relation between mortality and organ failure in 4 different categories of surgical icu patients receiving renal replacement therapy (rrt) for arf. methods: the records of 76 surgical icu patients with arf requiring rri-(haemodialysis or cavhd) were examined. five different patient categories were defined; 1 .ruptured aneurysm of the abdominal aorta (raaa) n =20 2.elective vascular surgery (evs) n =20 3.abdominal sepsis n -17 4.trauma n = 8 5.others ( e.g. malignancy, obstetric emergencies) n-11 seven organ systems (cns,circulatory,respiratory,hepatic,renal,digestive,haematologic) were scored for possible failure using the mof score. results: mortality was as follows: all patients 72% , group 1 75% , group 2 70% , group 3 88% , group 4 38 % , group 5 75 % . mortality increased with the number of failing organ systems; mortality for all patients with > 4 failing organsysterns was 80% the only exception being the subgroup of trauma patients where mortality under these circumstances was 5o% conclusions: mortality in surgical icu patients receiving rrt for arf is high. no significant difference in mortality is found between raaa and evs. mortality increases with the number of failing organ systems. the subgroup trauma patients shows a lower mortality compared to the group as a whole, even with > 4 failing organ systems. to look for the most accurate scoring system to measure the severity of the complications occuring in the early phase ( first 30 day) of kidney transplantation and to asses their prognostic value. methods: in our retrospective study we applied the apache li and the goris scoring system for the kidney recipients who developed multiple organ failure (mof) as a consequence of their pulmonary and. cardiovascular complications following kidney transplantation. we evaluated the recipients the distribution of the women and men ( 60% ~ 40 % ) was the same as in the kidney recipients. applying the apache ii system most of the patients had their score between 10 and 19, and the function of 2,3 or 4 organs were affected at the time of the onset of mof. the apache ii system gave adequeate information about the disturbance of the function of other organs beside the kidney failure even at the time of the transplantation. the scores and the number of the affected organs correlated with the condition of the patients in the goris scoring system but not as sensitively as in the apache ii scoring system. conclusions: both the goris and the apache ii scoring system can be applied to measure the severity of the multiple organ failure occuring during the early phase of kidney transplantation. however the apache ii system is more suitable to follow not only the stateof the patients at the time of the admission but also the changes occuring in their condition during the complication. v.v.erofeev, v.v.ivleva scientific research institute for general reanimatulogy russian amsci, moscow, russia objectives: the analysis of ssc and results of their treatment in patients following critical states showed the necessity of developing a combined antibacterial therapy. methods: according to the protocol 97 patients (18-60 years old) with combined trauma and massive hemorrhagy following vast aml traumatic operations were examined. microflora's composition and resistence to up-to-date antibiotics was studied using the anaiyser iems reader by "labsisteme"(finland). general clinical, bacteriological, immunological indices, as weil as the duration of the treatment and recovering rate served as criteria of the combined antibacterial therapy effectiveness. results: it was proved expedient to administer antibiotics in staphylococcus infection in the following combinations: riphampizin with fluoroquinolones; i-ii degeneration, cephalosporins with aminoglycosides; cephalosporins with fluoroquinolones. in case of singling out the exciters of the euterobacteriaceae family, including the pseudomonas aereginosa, -fluoroquinolones combined with modern amynoglycosides; fluuroquinolones with ureidopenicillines; ureidopenicillines with amynoglycosides; amynoglycosides with the ii-iii generation cephalosporins; cephalosporins with fluoroquinolones. in severe ssc caused by combined infection (including anaerobes) clindamicin with modern amynoglycosides was prescribed. conclusion: the combined antibacterial therapy allows: 1) to increase the effect on microbic agents and the efficacy of treatment in combined infections; 2) to lessen the possibility of the exciters'resistence to antibiotics; 3) to prevent the development of superinfection: 4) to decrease the doses of medicine and its toxic effect. objectives: two methods of blood volume measurement in a group of critically ill patients were compared to investigate the practical possibilities of a new easy to use method based on carbon monoxide (co) uptake. methods: all patients had multi-organ failure and haemodynamic monitoring with a swan-ganz catheter. mean apache ii score was 19 (10-25). when indicated, 9 patients had blood volume measurements simultaneously based on the techniques of, i) dilution of 5~cr labelled red cells, and ii) inhalation of carbon monoxide gas with measurement of the rise of carboxyhaemoglobin produced. the co was administered via a newly designed, ventilator driven, fully closed circle system ensuring co retention and co2 removal with automatic addition of oxygen to m}ttch patient uptake. a portable computer performed all necessary calculations. results: volumes obtained by co uptake were compared with the "gold standard" radiolabelling method. mean blood volume determined by the co method was 6310ml (4710-7959ml) compared with 4690ml(3755-5778ml) with slcr labelled red cells (r=0.9). regression analysis produced an intercept at 769ml. the slope of the regression line was 0.62 (0.33-0.9, 95 % confidence limits). discussion: the co method produces volumes in excess of the radiolabelling method. there appears to be a systematic error, and one possible explanation is co binding to substances other than haemoglobin. conclusion: the co method is easier to use than radiolabelling and of the lower cost, since cohb measurement only is required. aceuraey is sufficient for clinical use and our preliminary findings suggest this system will meet the requirements. objectives: this study was conducted to determine the role of nitric oxide (no) in the pathophysiologic alterations and multiple organ damage, and the possible effects of " " " (l-n -monomethyl-l-arglnlne nmma) on hemodynamics and mortality in rats caused by a prolonged hypovolemic insult. methods: a prolonged hemorrhagic shock (30-35 mmhg for 180 rain) was induced in anesthetized rats followed by adequate resuscitation. l-nmma was administered intravenously at doses of 2.0 mg/kg or 20.0 mg/kg at the end of resuscitation. results: infusion of 2.0 mg/kg l-nmma diminished the fall in mean arterial pressure, significantly increased the cardiac index (ci) and stroke volume (sv), together with remarkable protection from multiple organ damage compared to the controls. the 48 h survival rate was significantly improved from 26.7% in the control group to 68.8% in the treatment group (p<0.05). in contrast, the high dose of 20.0 mg/kg l-nmma resulted in a strong blood pressure response but a marked reduction in ci and sv concomitant with an increased total peripheral resistance index within the observation period, and caused severe damage to various organs at 2 h after treatment. in addition, marked elevation in both endotoxin and tnf levels were observed in animals subjected to shock insult. conclusions: these results suggest that no induced by hemorrhagic shock in rats is an important mediator for pathophysiologic alterations associating with cardiovascular abnormalities, multiple organ dysfunction, and even lethality. thus, regulation of no generation and use of no inhibitors might provide new aspects in the treatment of hemorrhage related disorders, and the use of l-nmma would be either deleterious or salutary in a dose dependent manner. (hebert, chest-1993) . the purpose of this study was to assess the risk factors for hepatic dysfunction in mosf. methods: 733 patients have been hospitalized in our icu from january 1992 to may 1. 994, 198 (27%) with mosf. among mosf pati~ts, 57 (29%) have had hepatic dysfunction defined according to hebert (bilirubin ~ 60 ttmop1, chest 1993). thirty six of these 57 patients acquired hepatic dysfunction after admission in the icu. these 36 patients were compared with 36 mosf patients without hepatic dysfunction selected blindly. chrorfic diseases, severity scores, eanse of admission, clinico-biologieal and hemodyunrrfic parameters, use of vesopressors, use of hepaiotoxic drugs, use of nutritional support and mortality were compared for hepatic failare and non hepatic failure groups.twenty nine patients had postmortem hepatic histologic examination, results: univaciate analysis: only parameters with p _< 0.05 are pre~nted. including these paramet~'rs in a multivariate analysis, anly c~hosis and vascular surgery remain independent risk factors for hepatic dysfunction. in particular, pao2/fio2, arterial lactate, do2 were not different between the two groups, some de~'ee of histological abnormalities was found in all liver samples, despite a normal bilirubin level in 15 % of the cases conclusions: in our patients, conu'ary to previous studies, hypoxic and hemody~anfic parameters were not independent risk factors for hepatic dysfantion. this might be due to the inadequacy of the usual biologic definition of hepatic dysfunction as well as to the poor sensitivity of general hamodynamic parameters. critical states of various origin are complicated with the mldtiorgan farm (moi~ oceuzr~ce. due to their and functional features the lungs become the primmy damage target in various critical.states. ard8 that occurs in such states is associated with pulmonary edema development because of capillary permeability increase mediated by humeral and cenular responses to 0amag/~5 factors exposure. r nmst be emphasized that mediators and effecto~rs of this respo~e affect not only puknonary capillaries, but other organs capiu~es as wellenhancing their permeability. orsans edema is a conmm~ finding at the autopsy of patients died from mof.clinical and radiolosial findings allow to have a diagnosis of pulmonmy edema before ~mi!ar lesions in other organs occm. additionally, there are some techniques that permit quantitative assessment of pulmonary edema flv.id (evlw) volume. in conclusion, we suggest that evlw changes in .dyn~rmcs in patients with mof are considered as a critical state severity measure which reflects indirectly the edema in other organs. objectives: we compared three different dialysis membranes to find out whether or not there were differences between their clearance characteristics on substances such as inuline, creatinine, urea, and phosphate to be eliminated in acute renal failure (arf). moreover, if a loss of clearance did occur we were interested in whether this was due to heparinization and a high production of the thrombine-anti-thrombine-complex (tat). methods: we carried out a randomized controlled study on 13 consecutive critically ill patients presenting with arf, most of them in association with multi-organ failure, to be treated by continuous pump-driven arterio-venous renal replacement therapy on continuous low-dose heparinization. three different types of high-flux filter membranes (f 60 tm [fresenius] , ct 190 tm [baxter] , and filtra116 tm [hospal]) were assessed. each filter was changed intentionally after a 24 hours" use. together the data of 54 filters were evaluated, each at three different times (immediately after its onset [0 hi, after 8 h, and after 24 h). the clearances of creatinine, urea, phosphate, and inuline were measured. results: there were some significant differences in clearance characteristics of inuline, creatinine, urea and phosphate between the filters (p<0,05) showing the f 60 tm membrane excelling filtra116mand ct 190 tm the more. the loss of inuline clearance (3 mi/min/m 2) after 24 h, however, was insignificant for all 3 filter types. a continuous low-dose heparinization scheme was applied without any relevant prolongation of the aptt. even lower losses were noted for the clearances of creatinine, urea, and phosphate. we found the tat-producfion increased after 8 h (p<0,05), but it did not rise any further. conclusions: as we could demonstrate in our study the clearance data of different types of filter membranes applied during continuous renal replacement therapy do show significant differences. on the other side, no relevant loss of clearance occurs during a 24 hours" period indicating a high efficiency over time. to consider commercial aspects as well it shows that inexpensive conventional filter membranes can successfully be applied even for a longer renal replacement period, if needed. a retrospective study was performed on 100 patients with acute renal failure (arf). we analysed survival in continuous (cd) and intermittent dialysis (hi)). mean age of the patients was 60 years (y), 57 patients (57 %0) were <65 y, 43 patients (43%) were >= 65 y. the incidence of dialysed arf in our mixed intensive care departement is 3%/admission/y. statistics: fischer's exact test, mann-whitney-u test. efioloev: the contribution sepsis, cardiac failure and aminnglycosidcs was respectively 70%, 44 % and 35 %. treatment: cavh (cd) or cvvh (cd) was used in 40 patients (40%), hemedialysis (hd) was used in 60 patients (60 %). data: mean apache 2 scores were the same for cd and hd (27 for both groups), patients treated with continuous dialysis techniques had significantly (p<o,05) more need for ventilation (100 % vs 70 %), elevated bilirubin (82 % vs 57 %) and a higher vasopresser need (89 % vs 46 %@ than patients treated with hemedialysis. there was a trend towards more sepsis (83 % vs 62 %; p=0.06) mad coaguiation disorders (50 % vs 27 %; p=0.07) in cd. taere were sign/ficantly more younger patients (<65 y) treated with cd (70 % vs 30 %, p<0.os). mean apache 2 score was significantly lower in patiants<65 y than as patienta>=65 y (26 vs 30; p<0.05). patients<65 y had significantly (i}<0.05) more coagulation disorders (53 % vs 17 %) and elevated bilirabin (81% vs 52 %). there was no significant difference in vasopressur need and ventihatio~ between age groups. outcome:. hi) had a better sr compared to cd (43 % vs 15~ p<0.05). patiants>=65 y had a comparable sr vs patients<65 y (3") */e vs 28 %; p----a.s.). tha global survival rate (sr) was 32 % (32 patients). conclusions : diaiysed arf has a well known lowsurvival rate (32 %): hc~raedialysed patients had a better survival rate than patients treated with continuous dialysis. this can be explained by the fact that the latter were in a worse condition considering organ failure (more vantilatian, elevated bflirubin and need for vasepressurs), apache 2 score couldn't illustrate that. patient~65 y with arf have the same survival rate as patients<65 y: although patients >=-65 y have a higher apache 2 score they have less organ faille. the avacbe 2 score is not a good oredictor of survival in p with organ failure. departments of surgery and intensive care, guy's hospital, london, u.g-obiectives: a randomised controlled trial of a management protocol utilising the regular measurement of gastric intramucosal ph (phim) to control the administration of dopexamine. methods: patients admitted to a multidisciplinary teaching hospital intensive care unit (icu) undergoing insertion of a pulmonary artery catheter were managed according to a resuscitation protocol. randomisation was to either the protocol alone or to insertion of a nasogastric tonometer and subsequent management guided by phim. phim < 7.32 initiated volume and inotrope resuscitation and, if unsuccessful in elevating phim, dopexamine was commenced. approval was obtained from the hospital ethics committee. results: 94 patients were considered for analysis and the two groups were well matched for age and sex. overall, there was a high hospital mortality of 64.9%. there was no difference in icu or hospital mortality between the two groups (see table) . objectives: to compare cardiac output (co) measurements between continuous termodilution (cco) by thermal wire on pulmonary artery catheter (cco/svo2 vigilance. baxter critical care), and co measurement using a trans-esophageal doppler (dco) ultrasound system (odm ii, abbott laboratories), in the immediate postoperative period of cardiac surgery. methods: 15 patients undergoing myocardial revascularization were monitored with cco by a swan-ganz catheter and an intra-esophageal dco probe, after induction of anesthesia. exclusion criteria were: aortic valve disfunction, previous valvular surgery esophageal disease, absense of sinus cardiac rhythm, and need of ventricular or intraaortic assistance. hemodynamic parameters, co by both cco and dco, svo2. sao2, diuresis, pha, and hemoglobin were repeatedly registered during the first 6 hours after surgery, as the patients were kept under sedation and mechanical ventilation. results were compared using the method described by bland and altman. results: 176 measurements of co were obtained, ranging 2. objectives: a decreased tissue oxygen delivery is responsible for a higher morbi-mortality rate among surgical patients; this diminished oxygen delivery/consumption rate (dojvo2) may origin the lactic acidosis observed in the gastrointestinal tract, reported in patients undergoing hypothermic cardiopulmonary extra corporeal surgery, and can be registered by tonometry as result of the gastric mucose ph. the purpose of this study is to evaluate the reliability of the intramucosal ph (phi) measurement by a nasogastric catheter as indicator of the do2/vo > its co> relation to other parameters of do2/vo 2 disturbance, and with postoperative complications and clinical course. methods: 20 patients (16 male, 4 female) undergoing cardiac surgical procedures were included (16 myocardiai revascularizations, 3 valvular substitutions, 1 constrictive pericarditis). mean age was 63 + 12 years, mean weight 70 _+ 10 kg. a nasogastric probe (trie tonometrics) was placed after anesthesia induction; phi values were registered in the postoperative period (90', 120', 240", 360' and 18 h after surgery end). the corresponding hemodynamic parameters, venous oxygen saturation (svo2), diuresis and arterial ph (pha) were also recorded. results: phi values ranged 7.20 to 7.65 (mean 7.40 (0.8); the mean values of clinical evolution were: extubation time, 20 _+ 12 hr.; discharge from postoperative care unit, 88 4-50 hr.; and hospital total postoperative time, 10 _+2.2 days. complications registered were: 2 perioperative acute myocardial infarctions, 2 cases of respiratory insufficiency, 1 occlusion of coronary bypass, an 1 ease of hyperamilasemia. all patients with severe complications needing specific treatment showed either a low phi value, or a considerable descent in comparison with the initial register. statistic correlation between low phi and presence of complications was found; the low significance (p > 0.05) degree may be due to the low population size. conclusions: phi measurement in cardiac surgery patients is a non invasive, uncomplicated method for prediction of doz/vo 2 disturbances, thus reflecting risk of increased major complications, and may precede changes in other usual indicators (svo2, pha, cardiac output, ...). work-in-progress with a greater population size may offer more significant results. references: (1) gutidrrez g: lancet 1992; 339:195-202. (2) landow i: acta anaesthesiol scand 1994; 38: 629-639. the haemoglobin-level (hb) is besides the arterial oxygen saturation and the cardiac index one of the relevant parameters of oxygen supply to the tissue. in contrast to otherwise healthy patients, there is no agreement on tile so-called transfusion-trigger in critically ill patients. in i?ont of this background the question arises, whether and to what extent blood transfusion in critically ill patients improves oxygen supply io tile tissue. this study was performed in 34 critically ill/septic patients in the postoperative period alier an inlcclive/scptie revision operation of the hip or knee joint. on cardiac/seplic reasons monitoring consisted beside other measures of a pulmonary arlery catheter and of an indwelling arterial line li~r measurering/calculating standard haem~dynamic as well as systentic oxygen parameters. the indication for blood transfusion was given by hb together with the cliuical slatus of thc patienl (asa-scorc and multiple organ dysfunction (moi))). statistical analysis w~ks performed by mann-whitney-u-test. by fisher's exact-test and by wii.coxon-test: statistical significance was set with p<0.05. according tu the pretransfusion value of hb and of lactate (lac) palicnts ;,,'ere divided into groups as follows: a: hb<8 and b: >sg/dl: i: 1ac<2.8 and ii: >2.smm. in either group blood transfusion results in zt significant increase in hb (a: 7.5_+0.4 to 9.4+0.8 g/dl; b: 9.(~0.8 tt, 10.5+0.09 g/dl; i: 8.0-+1.0 to 10.2-+0.6 jdl; i1:8.4-+0.9 to 10.1+0.7 g/dl). wlailc, however, haemodynamic parameters do not difl)r significantly from each other before and alter blood transfusion, oxygen delivery (do, -ml/min x m-') increases significantly hi either group studied (a: 467-+86 to 581-+158; b: 521+125 to 577+137; 1:512 -+1 13 to 599-+141; i1:516-+123 to 632-+214), in contrast oxygen consumption (vo~ -ml/min x m e) does not change significantly in either group (a: i68-+148 to 158-+38; b: 180-+162 to 175-+52; i: 171-+38 tu 179-+35; 11: 199-+60 to 219+_71); oxygen exlraction ratio decreases. this study in critically ill/septic patients demonstrates, that in this group of patients studied blood transfusion at a base-line-value of >7.5-+0.4 g/dl expectedly rises do~, however, it does not improve vo=; even not in septic patients with elevated lac-values. paclitaxel in a new anticancer agent, extract from the bark of the yew tree (taxus brevifolia), employed against breast and ovarian cancers resistant to chemotherapy. it promotes the polymerization of tubuline, and disrupts the normal microtubule dynamics. hematologic toxicity, hypersensitivity reactions (bronchospasm, urticaria and hypotension), and peripheral neuropathy are the main reported toxic effects. cardiac side effects are rare: atrioventricular blocks of higher degree are reported in 0.1% of patients; congestive cardiotoxicity was discussed only in one trial in patients treated with paclitaxel and doxorubicin. we describe the history of a 48-years-old worn an with a breast cancer, diagnosed in 1989, initial staging t3nim0, treated with mastectomy, axillary lymphadenectomy, andchemotherapy with a cumulative dose of anthracyclines of 678 mg/m2 until august 1994. the patient complained of dyspnea and severe hypotension immediately after an intravenous infusion of 100 mg paclitaxel, given over 1 hour for the treatment of bilateral, malignant pleural effusion. at echocardiography die left ventricular ejection fraction was reduced to 20%. she died 20 days later because of a severe cardiac low output with hepatic and renal failure; an impressive hepatic cytolysis was observed. the post mortem examination confirmed the dilatation of the cardiac cavities, especially of the right ventricle, bilateral pleural fluid, and ascites. the histology was suggestive for a cardiomyopathy secondary to anthracyclines. the electron microscopy revealed a deposition of an unusual pathological pigment in the myocytes; subsarcolemmal deposition or membranous were absent. we hypothesize that paclitaxel was the cause of a major hypersensitivity reaction with shock and severe hepatic cytolysis, worsening the myocardial damage induced by anthracyclines. the possibility that a low doge of paclitaxel could directly increase anthracyclines cardiotoxicity -as decribed in the medical literature -will be discussed. objectives: activated endothelial cells release soluble intercellular adhesion molecule-1 (sicam-1), vascular cell adhesion molecule-1 (svcam-1), and e-selectin (selam-1). sicam-1, svcam-1, selam-1, and inflammatory cytokines were determined. methods: sicam-1, svcam-1, and selam-1 were determined by elisa. tnf-a, il-6, and il-8 were also measured by elisa. endotoxin was measured by an endotoxin-specific endospecy test after pretreatment of new pea method. results: the sicam-1 and s vcam-i levels were significantly higher in the septic multiple organ failure (mof) and sepsis groups than in the non-septic mof group. the selam-1 level was slightly higher in the septic mof group than in the sepsis withut mof group and non-septic mof group. the increases of soluble adhesion molecules were not in agreement with changes of plasma endotoxin level. levels of soluble adhesion molecules were correlated with the levels of plasma tnf-a and il-8, but the level of il-6. discussion and conclusion: the slcam-1 and svcam-1 levels in septic patients closely reflected the severity of the pathophysiological conditon. it was possible that the release of sluble adhesion molecules were not stimulated by plasma endotoxin, but endotoxin in the local infectious region. tnf-c~ and il-8 also were suggested to be involved in the release of these soluble adhesion molecules. obiectives: cardiopulmonary bypass (cpb) surgery is associated with a systemic inflammatory response attributable to the release of various inflammatory mediators and the activation of complement or coagulofibrinolytic system. in addition, adhesion molecules, such as icam-1, elam-1, and vcam-1, appear to be of central importance in the inflammatory process following cpb surgery. we previously reported the effects of a synthetic protease inhibitor, fut-175, reduced release of inflammatory cytokines (tnf, il-lg, il-6), activation of complement (c3a, c4a) or coagulofibrinolytic system (tat, pic, fpa) and protected platelet function (gpib, gpiib/llla) following cpb surgery. methods: in this study, we analyzed fut-175 on soluble adhesion molecules following cpb surgery. 20 patients undergoing cpb surgery were divided into two groups, group a consisted of 10 patients who received 1omg of fut-175 in priming solution, followed by a continuous infusion at 2mg/kg/hr during cpb in addition to initial heparin dose of 3mg/kg. group b, a control group, included 10 patients who were injected with heparin only. the plasma slcam-1, selam-1, and svcam-1 concentration was measured by elisa. results: every soluble adhesion molecules decreased during cpb in both groups, and rose after cpb. selam-1 and slcam-1 reached their peaks on 3 hours after cpb and on pod 1 respectively in both groups, but they remained lower in group a (selam-i: 32.1+11.5 vs. 38.6• ng/ml, p<0.05, slcam-i: 327• vs. 483• ng/ml, p<0.05), svcam-1, in both groups, remained lower than preoperative levels, but did much lower in group a. conclusions: fut-175 reduced adhesion molecules and suggested to be the effect on postoperative organ dysfunction. in the last few :,'ears the conditions of treatment in continuous hemofiltration/hemodiafiltration were discussed controversially. a significant removal of tnf-alpha and il-i could be demonstrated in cvvhd. the aim of our study was to investigate the elimination of tnf-alpha, 1l-2, il-6, il-8, s-cd-14 and ifn-gamma in cvvh by measurement in plasma and hemofiltrate of 10 critically ill patients with an acute renal failure. the patients of our study were treated with a continuous veno-venous-hemofiltration (polysulfone-filter, blood flow: 100-130 ml/h, filtration rate 500 ml/h). the samples, hemofiltrate and plasma, were taken one hour after the start of treatment. the patients suffered from septic shock (4), the so called hepatorenal s~aldrome (3) and a severe pancreatitis (3). the cytokine concentrations were measured with elisa-method. in contrast to elevated concentrations in plasma for tnf-alpha (5 cases), scd 14 (9 cases), il-2 (l case) and il-6 (4 cases), hemofiltrates contained no activities. only il-8 was removed in significant amounts with even higher levels in hemofiltrate than in plasma. this phenomenon was described so far for tnf-alpha and il-1 and may be due to the absence of metabolic properties (possibily enz~natic) in hemofiltrate. it can be shown, that tnfalpha, il-2, il-6 could not be eliminated in cvvh with a filtration rate to 500 ml/h. in contrast to findings of other investigators with a higher filtration rate (> 1000 ml/h), we found no significant concentrations of tnf-alpha and il 6 in hemofiltrate. we conclude, that for a significant removal of important cytokines higher filtration rates (>1000 ml/h) are necessary. objectives: multiple organ dysfunction syndrome including liver and renal impairment is a fatal complication in patients with the diagnosis of sever sepsis. this study focused to the effects of removing toxic substances from inflamnatory tissue by hemodiafiltration. ~4ethods: eleven patients were admitted to the icu in emergency center and met the criteria of systemic inflammatory response syndrome in association with infection. all patients developed liver and renal dysfunction and were treated by hemodiafiltration with high flux membranes (fb-u:nipro). the hemodiafiltration were performed 19 times using nafamostat mesilate as an anticoagulant in 5 hours with 12 l of substitution fluid (hf-b:fuso). the serdm levels of endotoxin, cytokines, endothelin-i (et-]), human neutrophil elastase ~1-proteinase inhibitor complex (hne-pi), fibronectin (fn), lactate, and amino acids were measured before and after the hemodiafiltration. the hemodiafiltration would be effective to renal dysfunction by reducing endothelin and beneficial to tissue metabolism represented in fisher's ratio, but might be harmful to respiratory function by activating neutropila in patients of severe sepsss. background : intermittent hd may be poorly tolerated in the early phase of arf in hemodynamically unstable patients (pts). this technic may fail to achieve steady state urea low levels in hypercatabolic pts. method : nt = 25 consecutive pts treated with hd; n 2 = 25 consecutive pts treated with cvvhf. hemodynamic unstability is defined by arterial hypotension and requirement of inotropie support despite adequate filling. rate of change in urea (u), ereatinin (cr), k + , ph were computed from a linear regression .analysis of data vs time in each treatment group during the 5 first days of application of the two technics (anova). dally worst values were recorded. results : hd-group : apach% score = 22 _+ 7; mean number of organ system failure (osf) = 1.5 -+ 1; mean blood pressure (mbp) = 75 • 22 mmhg (first day of application of hd). cvvhf-group : apachen score : 25 + 6; osf = 3 -+ 1; mbp = 57 + 20 mmhg (first day of application of cwhf discussion : during the 5 first days of application of hd/cvvhf, u and cr decreased much more rapidly in the cwhf-group. k* and ph were maintained within normal range in the two groups. initial mbp which was much lower in the cwhf-group significantly improved during the application of cvvhf while mbp remained unchanged in the hd-group. conclusion : despite higher severity of disease in cvvhf group (apachen score, osf, lower initial mbp), we obtained a better performanco with cvvhf regarding the decrease of u and cr and the improvement of mbp. in relation to the different and continuous renal replacement techniques, the continuous venovenous one is the alternative method to continuous arteriovenous for critical patients with acute renal failure (arf). we present you our experience with cvvh in patients with mof. in our intensive care unit (icu) 20 patients with mof were treated with cvvh in the period between january in 1992 to march in 1995. the mean (• age of our patient population was 52,1• years, being 65% male and 35% female the whole patient population was with mof iust at the moment the technique was accomplished; 75% was in mechanical ventilation, 90% needed vasopressor support and 65% required both of them (mechanical ventilation and vasopressor support) apache ii score mean of the patient population was 17,84~:6,97 (range 5-28) and ati of them were with arf oligoanudc. technique: cvvh was accomplished using a single-d~al iumen catheter, ptaced in either a temoral or subclavian vein by the stand ard seld{nger technique. pol{sultone hemofitiers were also used, and the extracerporeal circuit used standard arterial-venous blcod tubing. blood flow and hence oltrafiltration pressure, within the circuit was generated by a roller blood pump. the modulus has a roller pump, a pressure transducer connected in an arterious and venous line, such as an air-transducer which is adapted to a drip-chamber in the return way. the replacement used was a peritoneal dialysis solution. medicine 1, st. george's hospital medical school, london. england. hepatic sinusoidal endothelium shows a major inflammatory response in porcine sepsis that can be attenuated by the administration of dopexamine hydrochloride. dopexamine is a beta 2 and dopaminergic receptor agonist. the specific beta 2 adrenoceptor antagonist ici 118551 has been shown to reduce the protective effects of dopexamine. we investigated the effect of this antagonist on hepatic ultrastructure in porcine sepsis. six pigs (25-30kg) divided into 2 groups were anaesthetised and intubated. cardiac output and portal blood flow were measured using standard techniques. the 2 groups were; placebo, (peritonitis induced); blocker, (peritonitis induced and 200 pg/kg ici 118551 bolus infused then given hourly). caecal content was aspirated and peritonitis induced. colloid was infused to maintain pawp at 10-12mm hg for eight hours the animals culled, hepatic tissue removed and prepared for electron microscopy. in the placebo group hepatic endothelium was swollen and the sinusoids occluded by wbc. but in the ici 118551 blocker group, much of the sinusoidal endothelium was absent and there where large extra sinusoidal spaces among the hepatocytes. an assessment of the two groups showed worse hepatic architecture in the blocker group. the b2 antagonist blocked any protective effect of endogenous beta 2 adrenoceptor agonist (adrenaline) on hepatic endothelium in porcine sepsis. george's hospital medical school, london. england. dopexamine hydr0chloride, a beta 2 and dopaminergic receptor agonist reduces hepatic damage in porcine sepsis. we tested dopexamine's effect on cerebral oedema. the beta 2 adrenoceptor antagonist ici 118551 was infused to block any protective effect of dopexamine. nine anaesthetised pigs (25-30kg) were randomised into 3 groups; placebo, (peritonitis induced); dopexamine, (peritonitis induced and 5 ~tg/kgdar of dopexamine infused); blocker, (as in dopexamine group but in addition 200 pg/kg ici 118527 bolus given then infused at that rate hourly). caecal peritoneum was induced and colloid infused to maintain pawp at 10-12mmhg for eight hours when the animals were culled, cerebral tissue removed, prepared for electron microscopy and digitisation. digitisation of the area of oedema surrounding the blood vessel and expressed as a percentage of the micrograph. 30.5_+4.1, dopexamine 13.5+2.9", blocker 31.5+3.7. data expressed as mean + sd. significance p<0.05. * dopexamine compared to placebo and blocker. in the dopexamine group the area of tissue oedema was significantly lower than either the placebo or blocker groups. there were no significant differences between the placebo or blocker groups. the 62 antagonist completely blocked the protective effect of the drug on cerebral oedema in porcine sepsis. beta 2 adrenoceptor stimulation is protective of cerebral oedema in porcine sepsis. objectives: the hemodynamie~ of hepatic circulation during multiple organ failure (mof) have not been suffleienly studied. we investigated liver hemodynamics in two subgroups of patients with mof, those with either liver or lungs as the main organ of involvement. methods: three groups of patients were created: i) mof-hepatic involvement (mof-hi) (7 patients) with bilirubin >3.5 mg/dl and lung injury score <1.8, it) mof-ards (9 patients) with respective values <2.0 and >2, iii) 5 patients with head injury with respective values <2 and <1, served as group control. all patients were in haemodynamieally stable state with an oxygen delivery index >300 ml/min/m2 prior to measurements. two swan-ganz catheters 'were inserted, one in the hepatic veins and one in pulmonary artery and the following measurements were determined: the hepatic vein free pressure (hvfp), the hepatic vein wedge pressure (hvwp), cvp, paop and co. the gradient of hvwp-hvfp represents liver perfusion pressures. by injecting contrast media at dose of iml/lokg with the balloon inflated to achieve sinusoidai image, the hepatic blood flow (hbf) was concluded by the time in seconds of media removal after balloon deflation. results: the co, cwp and cvp were comparable to all three groups. namely, for mof-hi, mof-ards and control groups the mean (+sd) value of co was 7.2_+0.8 vs 6.9_+0.3 (ns) and 6.3_+0.6 respectively, of the paop was 8.7+_1.6 vs 10+:3 (ns) and 8.2+3.1 respectively and of the cvp was 12.+.2.3 vs 11.6+2.3 (ns) and 5.8 respectively. in contrast the two mof groups were different after the cut-offinclusion criteria ie the mean (+sd) value for bilirubin was 6.8+9.5 vs 1.20+0.7 (13<0.05) and 0.8_+0.2 respectively and lung injury score was 1. objectives: oxygen delivery (do2) and oxygen consumption (vo2) are increasingly monitored parameters in the icu. there still remain controversies about an oxygen supply dependency in critical illness particularly with respect to vo 2 determination by either indirect calorimetry (vo2m) or tick calculation (vo2c). the purpose of this study was to investigate the changes in vo2m and vo2c following do 2 increase. methods: the relatives of 24 critically ill patients (mean age 63 years, mean apache ii 24, mean mof-score 9) gave their written informed consent to participate in this institutionally approved, prospective study. do 2 was increased by fluid loading (hydroxyethylstarch 10%: mean volmne 750 ml, mean duration of infusion 80 min) and catecholamine support (dobutamine: mean dose 14,3 ~g/kg/min). changes in vo2m and v02c were recorded sinmltaneously before, during and following interventions. calorimetry was obtained with the metabolic monitor 7250 integrated in the ventilator 7200 (puritan bennett, carlsbad, ca adaptive endocrine response of organism to septic shock consisting in activation of the production of adrenal hormons, renin -angiotensin -aldosterone system (raas) and other hormonal systems has an influence over microvascular changes in these states and for development of multiple organ failure (mof). in 25 patients with peritonitis of different origins (18 nonsurvivors and 7 survivors) were followed the changes in cortisol level and raas by radioimmunological methods and many variables for evaluation of respiratory, renal, hepatic function, coagulation etc. as a signs of mof. it was observed significant increase of the level of cortisol (1099 +_ 4,83 nmol/ i), aldosterone (0,895 • 0,687 nmol/i). by factorial statistical analysis we found significantly high correlations between hormonal changes and respiratory function (for example r=-0,539, p < 0,02 between cortisol and pao2; r = 0,817, p < 0,001 between cortisol and d (a-v) 02; olso renin -cao 2 r=-0,824, p < 0,001, renin d ~,vl o2 r = 0,626, p < 0,001). such significant correlations was found and for raas with respiratory, renal function, byproducts of arachidonic acid thromboxan b 2 and p6fla, soluble fibrine degradation products etc. these correlations between the degree of endocrine changes and multiple organ failure in patients with septic shock produced by peritonitis suggest that their effects upon peripheral vascular resistance and constriction of the splanchnic, splenic, renal and other organ vasculatures are not always with physiologic expediency and there are perhaps the possibilities of therapeutic influence. intredu~on : dopexamlne has previously been shown to control hyperkalaemia ia patients with acdto renal failure (arf), however effects on the subsequent course of art are undomunente~ ob_iectlv~ : to evaluate clinical progress in patients with acute renal failure (arf) in an intensive care unit (icu) with regard to biochemical control, need for -and time to -dialysis, and outcome in patients receiving dopexamine. m~ods : 14 consecutive patients meeting standard criteria for diagnosis of arf were included in the study. full cardiovas~dar, biechemical and intervention/outcome details were recorded. dopex.~min~ was infilsed at a dose of 2 pg/kg/min in conjunction with a regimen of inotropir support and blood volume optimization. resn]~ : following the intzoduetion of dopc',~mine ilrinr vohlmes increased slightly over the next 24 hrs fzom 516 + 140 ml/24 hrs to 817 + 229 ml/24 hrs (ns). data expres,uxl as mean + sem. three patients (21%) became polyuric with urine output >100 ml/hr within 3 days and did not need dialysis. in the remaining patients the time to dialysis (to correct acid-base deficits or volume overload) was 5.09 + 0.84 days. serum potassium levels were well controlled. day 5 or immediate pre-dialysis levels were 4.48 + 0.19 mmol/l compared with pre-lreatment 4.67 + 0.2 mmol/l overall mortality in this series was 4/14 (28%). duration of acute dialysis in survivors with renal recovery was 16.8 +_ 1.82 days. 2 patients (14%) progressed into chronic renal failure and needed continuing renal replacement therapy. no adverse cardiovascular altects were seen at this low dopoxami~ dose although its competitive inhibition to adrenergic reuptake mechanisms meant that doses of pressor agents could often be reduced. : dopcx:~minr nsed in conjunction with inotropic support and blood volume oplimitntion, can safely postpone, or even avoid, the necessity for acute haemodialysis in icu patients. no evidence of tachyphylaxis to the effect on serum potassium levels was seen over the duration of the study. hen'era m., suarez g., dagn d., varela a., ramos j., garoia jm, aragdm c, jurado l, medina a. icu. hospital regional. malaga. spain. objective: to evaluate the haemodinamic tolerance to the veno-venous continuous hemefiltration (vvchf) system in patients with systemic inflammatory response sindrome (sirs), and the possible beneficial effect of this technique on the haemodinamics in these patients. material: 13 patient admitted to the icu, with diagnosis of sirs and monitored with a pulmonary artery catheter at the beginning of wchf. we performed a complete haemodinamic study to all these patients (cardiac output, vascular resistanoss, ph and co2 in arterial and mixed venous blood samples, saturation of pulmonary mixed venous blood, do2 and vo2 calculations and temperature) and determined the respiratory mechanics (compliance and pao2 /fie2 relatinship) before starting the procedure, after 10 minutes operating with the ultraflltrate branch closed (without filtered fluid production), afler 30 and 60 minutes of zero fluid balance bemofiltration and after 120 minutes of filtration with negative balanos adjusted to the patients conditions. for the statistical analisis we have performed the anova test over the mentioned variables. results: we have not detected statisticaly significant differences of the analyzed variables before the beginning after operating the pun'@ for 10 minutes without filtered fluid production and after 60 minutes of zero fluid balance hf. only temperature shows a meaningful decrease in time. objectives: among many organs, playing the important role in pathogenesis of multiple organ failure, the particular place is taken by the intestine. ~ethods: the study was carried out in 5 dogs !~n"~h pi was modelled by severe operative trauma (ot). the dcm was estimated by the indices values of work time (wt), contraction frequency (cf), mean amplitude of contractions (~ac) and motility index (mi) measured by method of tensography. "sl", created on the basis of sorbit and sodium lactate (1800 mosm/l), was injected in the dose of 10.o ml/ kg into v. cephalica antebrachii after 24 hrs of ot. the results of the present study are the evidence of "sl" stimulative action on dcm and are experimental ground for "sl" using in complex therapy of pi in clinic. with splanchnic venous blood pc02 p.f. laterre p. goffette, j.p. fauville, a. poncelet, p. loneux, m.s. reynaert. intensive care unit, st. luc univ. hospital, brussels, belgium. determination of gastric intramucosal ph (phi) by gastric tonometry using the henderson-hasselback equation is expected to allow the detection of splanchnic ischemia in critically ill patients. because of bicarbonate concentration and acidbase balance influences on the calculation of phi, it has been proposed to use arterio-gastric pco,_ gradient [p(gast-a)co,] to assess splanchnic perfusion. htpothesis : pcoz in the gastric mucosa is in equilibrium with intraluminal co z and with co, in the blood leaving the stomach (mesenteric and portal blood). objective: mesure pco; and ph in portal vein blood and compare its value with pco 2 and phi obtained simultaneously by gastric tonometry. material and method : in a patient (55 y.), a fiberoptic catheter (baxter r) was positionned in the portal vein after transhepatic stent shunt repermeabilisation. hemodynamic parameters, do, (vigilance n baxter), gastric co 2 and phi (tonometrics baxter) and portal blood gas were determined at regular intervals. results : 19 sets of data were obtained and are expressed in mean + sd. gastric pco z was 46,5+18 compared to 40,4+3.5 mmhg for portal pco 2. phi was 7.32+._0,17 vs 7.34+._o,04 for portal ph. no correlation was found for these 2 parameters. p (gast-a) c02 was 6.4+15 mm hg vs 2+1.6 mm hg for p (portal-a) coz (no correlation). there was a good correlation between do e and p (portal-a) co z (r = 0,61) [figure] but no correlation with p (gast-a) c02. obiectives: desaturation is a common finding during haemodialysis (hd). pulmonary oedema might be one cause for impaired gas exchange (1). the aim of this study was to quantitate the amount of extravascular lung water (evlw) and gasexchange in chronic renal failure patients during and after a regular hemodialysis session. methods: 10 chronic renal failure patients without symptoms or diagnosis of cardiac or respiratory disease were studied at the start (i), at the end (ii) and two hours after (iii) a regular bicarbonate hemodialysis session. the double-indicator 2 dilution method, with indocyanine green and the stable isotope h20 as tracers, was used to measure evlw (2). arterial bloodgases and endtidal co2 were registered. evlw data was compared to a group of 18 renal healthy patients (0). dcp n evlw, ml -pao2, mmhg h~o +, nmol/l control group 0 18 243 -2--61 l 10 332 _+ 91"* 13 -+ 3 38 _+4 crfgroup ii 10 269 -+ 84~ 11 +-2ns 34-+2"(" iii 10 283 +-78t 11 _+3ns 34-+2t ** p < 0.01 dcp i from dcp 0, t p < 0.01 dcp li or i1 from dcp i, :~ p < 0.001 dcp ii from dcp i the evlw at the start of dialysis was larger in the crf group than in the control group. the evlw decreased significantly to a level not different from the control group in response to the reduction in weight after hd. pao~ was normal at the start of hd and showed a nun-signficant reduction after hd. paco2 (5.3+0.6 kpa) and etco2 (5.2+0.8 kpa) were unchanged while h3 o+ decreased and bicarbonate increased significantly. conclusions: the elevated level of evlw at the start of hd did not impair gasexchange. the decrease in evlw did not inhibit the decrease in pao2. the reduction in h30 + followed by a fall in alveolar vantilation is the most plausible cause for the decrease in pao2 in bicarbonate dialysis. 1. prezant lung 1990; 168: 1-14.2. wallin j appl physio11994; 76: 1868-75. a. dona~ d. battis& l col~ r danieli, d. achill~ l viglienz;~ c. giov-anaini, p. piaropao~ oblectives: to verify if intraoperative modifications of mtramucosal gastric ph (phi) below the normal lowest value 7.32, can be predictive for important complications, as perforation, sepsis, mof or death. methocls: we have considered 20 patients who andenvent major abdominal surgery. all patients received the same drugs in pre-anaesthasia, the same type of anaesthesia (balanced anaesthesia) and the same treatment with h2-bloekers. after the induction of anaesthesia a gastric tonometer was positioned and a catheter was positioned in the radial artery. during the operation, every 30 minutes, the following parameters were measured at the same time: phi, arterial ph (pha), blood lactate, mean arterial pressure. in follow up we considered death and complications happened during the hospital stay, in relation to intraoperative phi falls below 7.32. results: among the 20 patients, 9 had a drop of phi below 7.32 during surgery. in three of them this fall was a single episode and happened within the first hour after the begiluting of the operation. after that phi rose to nomml values until the end of the operation these patients had a normal post-operative period, without complications, the other 6 patients had a fall of phi during the demolitive manoeuvres. two paticots of them died. the first had a lowest phi=7.25 and the second 6.97. the first one ~zs operated on for hepatic istiecitoma, suffered a complete del'dseenco of the surgical wound on the 20th day after operation and died on the 25th day, the second one was operated on for a hepatic carcinoma had an intraoperative haemorrhage and died ~vo hours after the end of the operation. the other 4 patients with a fall of phi had a lowest phi=7.24. 7.18. 7.26. 7.28 respectively.the first patient,operated onfor sigmoid carcinoma, underwent on a second operation for a transmural necrosis of the colic segment on the 25th day; the second one, operated for carcinoma of the right colon, had a cardiac ischelnia on the 5th pest-operative day and a dehiscence of the surgical wound on the 8th day: the third one, operated on for a sigmoid carcinoma, had melena in 41h post~ operative da b, and finally the fonrth patient, operated on for carcinoma of the tight colon, suffered a fistula of the surgical enteral anastomosis.all these 4 patients were discharged alive from the hospital. the other 11 patients, who had not reductions of phi ditring the operation, had a normal pest-operative period, without complications. conclusion: phi was able to predict the arising of some complications, probably due to intraoperative ischemic events. we can say that gastric tenometry, for its low invasivi.ty, can be included among the intraoperative monitoring in patients that tmdenvent on major abdominal surgery. (ttd),t"ea~rrerj.~ of 3 hours duraticn. all l:atients nm.'-~ms_(~lly va~2ated in eantrol wcde ard_ la':'ad a a,~m--ganz catheter, with optic fibers for contirums mmsuremmt of svo mic studies were performed, c~e before the hegir~ of hd, c~e 15 rain after the ~, ~ne at the middle, ~ne 15 rain before lhe erd ard one 30 rain after the erd of hd. paired t test ~as used far slatistical eval~ti~n. results: daring i~d there was a significant'reductton (p<o.~) of: c~tr~ venc~s eres~.re (0ve)emm 9 to 7.5 ~ ~, 2) ~arn'~w capil~ ~f~e pressure (i<~p) fr~n 13 to 11 ~mg hg, 3) i~09 from 33 to 30.5 mmg hg. ~here was also a less pmnameed bat also-slatistics/ly significant r~ucticn (p<o.05) of: i) s~o~ from 67 to patients undergoing surgical reconstruction of the aorta due to anenrysms of its abdominal part may develop transient and sometimes sustained episodes of dysoxia despite the conventional appeoreneas of being adequately resuscitated. indirect masurement of gastric mussel ph (phi) is being widely evaluated as a minimally invasive and sensitive means of assessing the adequaey of tissue oxygenation in these circumstances. the duration end degree of gastric intramucosal acidosis are related to the risk developing injured gastrointestinal tract end its putative consequences, i.e. translceation, cytokine release, organ dysfunction end failure, sepsis end death. measurement of phi is obtained indirectly by measuring pc02 in the lumen of the stomach with a silicone balloon tonometer (tonometrics, inc., worcester, ma, usa) and the bicarbonate concentration in arlz,,rial blood, end substituting these two values in the henderson-hasselbalch equation. objectives: to evaluate whether perioperative phi changes are predictive of complications end outcome end how they compare to standard haemodyuamic and oxygen trensport parameters. methods: 16 patients (15 males and 1 female) urtderwent surgical replacement of abdominal aortic anonrysm with the aortic simple or bifurcated grail. 14 patients were operated on eleetively, 1 had an emergency surgery for raptured enentysm. haemodynamic, arterial ph and bicarbonate concentration measurements were taken before and after induction of anaesthesia, after cross-clamping and declamping of the aorta end at admission to itu. phi calculations ware done at the sime time except before induction, as tonometers were inserted after patient had been asleep. we used pulmonary utilizing a computer billing survey as well as hospital service transfer data from a 67 month period (1/89 -7/94), all obstetric patients that were transferred to the med/surg icu were identified, as well as their diagnoses and procedures. twenty-eight obstetric patients were transferred to the med/surg icu, compared to 12,489 deliveries occurring during that time period for a .22% rate. the following procedural indications for icu transfer occured: 4 insertions of endotracheal tubes, 2 arterial catheterizations, 1 temporary tracheostumy, and 1 venous catheter. the following medical diagnoses were found: 5 hypertensive/eclamptic episodes, 2 hypotensive episodes, 1 pulmonary embolism, 1 mitral stenosis, and 1 coagulation deficiency. there were four episodes of respiratory failure (14%). the historical rates of respiratory failure from the medical literature seen at a university hospital ore higher than those at our community hospital despite comparable icu transfer rates for critically ill ob patients. objectives: to evaluate the usefulness of several isss used in order to predict risk of death (rd). design: prospective data collection for 7 months in a multidisciplinary 18-bed icu. methods: data from 270 consecutive patients admitted to the icu from 1/10/1994 to 1/5/1995 were studied. all the necessary informations were stored in a computer using special software for every day computation of three isss (saps, saps ii, apache ii) and of the rd predicted by saps ii, mpm0, mpm24 and odin. data from 2453 days of hospitalisation were used for comparative evaluation of isss and rd, while sensitivity and specificity in death prediction (cut off point of rd = 90%) were evaluated from data collected during the day of admission (saps ii, rpm0, odin) or after 24 hrs (mpm24). agreement between rd predictions was evaluated by bland and altman analysis. results:our results were the following: objectives: a) to create and validate software specially designed for the collection of demographic data and estimation of illness severity and predicted rd and b) to provide data necessary for the evaluation of the effectiveness and the efficiency of our icu. design: prospective data collection in a multidisciplinary 18-bed icu. methods: prospective data on specific physiological variables, selected demographic characteristics, comorbidity and the reason for icu admission were recorded every day for 270 consecutive patients admitted to the icu from 1/10/1994 to 1/5/1995. data were entered into a portable computer using specially designed software allowing the calculation of usual illness severity scoring systems (isss) and the corresponding predicted rd. results: we studied 176 men and 94 women, with a mean age of 55 years. we conclude that, although observed mortality was higher than predicted rd, this difference was not statistically significant. the fact that the sd of predicted rd was too large, limits the usefulness of isss predictions for a comparative evaluation of different icus. objectives :the importance of objective evaluation of patients prognosis at their admission to an emergency department is clear. the aim of this study was to appreciate the prognosis of patients with the simplified acute physiology score ii (saps ii), and correlate this score to the prognosis and orientations of patients. methods : the score which is a reduced form of saps ii was calculated from 6 clinical variables and 6 laboratory items for 200 patients. sensibili}y (se), specificity (sp) and accuracy (ac) were calculated to evaluate the performances of saps i1. thresholds were calculated with the youden's test (se+sp-1). results : 23 patients were admitted in the intensive care unit (icu)and 177 patients in medical units; 23 patients died. the saps ii was higher in patients which died than the survivors (31,0+11,4 vs. 1,9+9,3, p<0,0001 ; respectively), the best threshold was 24, se was de 86%, sp was 92%, the ac was 920/0. thus for patients admitted in icu than in medical unit (27,0:1:13,5 vs. 12,5+9,9 , p<0,0001; respectively); the best threshold was 21, se was 69%, the sp was 78%, the ac was 77%. conclusions : these preliminary results suggest that saps ii can be used to determine the short term prognosis and the destination of patients seen in an emergency department. design: data was collected on all admissions over a six month period. all patients had a screening hiv elisa test. confirmatory ifa tests, western blot and flow cytometry were performed on hiv positive patients. the institutional ethics committee considered the major clinical impact of the study sufficient cause to waive the patient's right to informed consent, icu staff and patients were blinded to hiv results. following discharge patients were given the option of being advised of their hiv status. setting: the study was conducted in a 16 bedded surgical icu at a tertiary care teaching hospital. patients, participants: all patients admitted during the period in question were included. interventions: "all patients were treated according to standard icu protocols. there were no interventions. results: most patients were admitted following trauma. there were no patients with aids, 52 hiv positive and 350 hiv negative patients. with respect to the latter two groups, the mean age and sex distribution was similar, there was a significant difference in organ failure (71% versus 49%, p < 0.05) and septic shock (39% versus 15%, p , 0:05) but no difference in icu/hosp[tal mortality or duration of icu/hospital stay. flow cytometry changes in hiv positive patients were consistent with the quiescent phase of hiv infection. conclusion: while morbidity is higher in hiv positive patients, there is no difference in mortality. hiv status should, therefore, not be an exclusion criterion for icu admission in this patient population. 1, n= 112) and january-march 1994 (period 2, n= 127) were studied. interventions: a resident micu team led by a trained intensivist took over the medical care from the primary physicians when the patients were admitted to the micu. the intensivist also vetted micu admissions and decided on micu discharge. in addition, there was a resident respiratory therapist to attend to vendlatory care during office hours. after office hours, the care of the micu was delegated to the on-call team on a rotational basis among the medical departments. results: the patients in the two groups were similar with respect to age, sex, race, source of admission and apache 11 scores. the icu and hospital mortalities decreased from 33.8% to 27.6% (p=ns) and 38.2% and 34.2% (p=ns) respectively in period 2. the mean icu and hospital stay was also decreased from 4.6 __+ 7.1 to 3.6 + 4.0 days (p=ns) and 19.3 __+ 357 to 17.2 • 22.2 days (p=ns) respectively. the improved micu length of stay for survivors in period 2 was statistically significant (4.2 • 4.2 days vs 2.8 • 2.8 days = 0.015). there was no significant differences in the vse of peritoneal dialysis (5.4% vs 6.3%) and mechanical ventilation (55.4% vs 49.6%). however, utilisation of intra-arterial lines and pulmonary artery catheters increased from 0% in both to 23.6% and 5.5% respectively in period 2. conclusion: the duration of critical illness was reduced in period 2. hence, we believe that the closed |cu which was staffed by an intensivist and had the services of a respiratory therapist was beneficial for the care of the critically ill. the intensivist, was also more likely to utilise invasive monitoring. in the absence of liver transplantation, intensive care for patients with acute hepatic decompensation arising from alcoholic liver disease (ald) is indicated only for those who are likely to benefit from conventional means of organ support. we have attempted to elucidate potential risk factors and the efficacy of organ support in relation both to hospital outcome and icu costs so as to allow the earlier identification of relatively futile intensive care in this group of patients. methods: over a period of one year, 59 ald patients (37 males; 22 females; median age 46 years, median apache ii score 19; hospital mortality 68%)with severe complications (30 [50%] with bleeding oesophageal varices, 16 [27%] with hepatorenel failure, 5 [9%] with respiratory failure, 3 [5%] with septic shock, and 5 [9%] others) were studied in our liver icu. organ system failure, daily therapeutic interventions and icu costs were assessed using a patient management system (riyadh intensive care program). results: whilst survivors had a significantly lower admission apache ii score compared with non survivors (medians 12 and 20, p<0.0003), 2 or more organ systems in failure in the first 24 hours of icu admission was associated with a 91% (21123) mortality. mortality was also related to child's grading and the numbers of organs supported. of the 29 patients who received more than one form of organ support only one survived. organs supported mortality a (n=2) 0% none 3/13; 23% b (n=6) 33.3% one 8/17; 47% c (n=51) 74.5% two or more 28/29; 97% the icu costs for the 59 patients were only s 440 but s 687 (75%, 216 patient days) was utilised by the 40 patients who died, giving an effective cost per survivor (ecps) of s 496. although the cost of treating the 23 patients with 2 or more organ systems in failure on the day of admission was s 510 (36% of the budget) the ecps was s 755. conclusion patients with ald who have 2 or more organ systems in failure early in their icu course have a poor prognosis and in the absence of liver transplantation, traditional strategies of organ support are ineffective. this begs the question whether such costly care is appropriate as although the ecps in this sub group is comparable with other patient groups with multiple organ failure, their outcome is considerably worse. objectives: to evaluate the economic impact to the healthcare sector of using dopexamine hydrochloride infusions to deliberately increase perioperative oxygen delivery in high-risk patients. methods: effectiveness data were obtained from a controlled clinical trial in which 107 surgical patients were randomly assigned to either a control group (n = 549 to receive best standard optimal fluid resuscitation perioperatively, or a protocol group (n = 53) to receive, in addition, an infusion of dopexamine hydrochloride to increase the perioperative oxygen delivery index to greater than 600 ml/min per square metre. resources consumed by patients during treatment were recorded prospectively and costs of resources at 1993/94 prices were obtained retrospectively. cost-effectiveness ratios were estimated by dividing the total cost for each treatment group by the total number of patients in each group and by the clinical outcome of mortality 28 days postoperatively. results: in the protocol group, there was a 75 % reduction in mortality 28 days postoperatively (p = 0.015), a significant reduction in the number of complications (p = 0.008) and a reduction in length of stay in the icu and surgical ward. the average cost per protocol patient to achieve a 94.3 % survival rate 28 days postoperatively was s 9,894, resulting in an average cost of s 10,488 per surviving protocol patient. the average cost of a control patient was s 11,331 to achieve a 77.8 % survival rate 28 days postoperatively, resulting in an average cost of s 14,568 per surviving control patient. the use of dopexamine to deliberately increase oxygen delivery perioperatively generated a cost saving to the nhs of s 1,436 per patient together with a 75 % reduction in mortality at 28 days postoperatively. hence, the cost per surviving protocol patient was s 4079.8 less than the cost of a surviving control patient. conclusion: the additional cost of dopexamine in protocol patients was offset by a lower incidence of complications and a shorter stay in the icu and on the surgical ward. hence, increasing oxygen delivery perioperatively in high-risk surgical patients saves both money and lives. objective: although king's college criteria are widely used, indication and timing for liver transplantation in acute liver failure is still far from certain. long-latency sensory evoked potentials -a proven measure of metabolic brain dysfunction (grimm et al. lancet 1988; 2: 1392-94; madl et al. lancet 1993; 341: 855-58 ) -are markedly affected in patients with acute liver failure. serial sensory evoked potential recording should provide very useful information for the management of patients with acute liver failure. methods: 90 recordings of standard bilateral sensory evoked potentials were performed in 25 patients with acute liver failure. findings were focused on cortical n70 peak -a stable negative deflection occuring 70 • 9 ms after median nerve stimulation in healthy subjects, which can be recorded by skull electrodes over the sensory cortex. results: nine patients recovered spontaneously, 8 patients were referred to emergency liver transplantation, and 8 patients died. in all 9 patients who recovered spontaneously, n70 peak was detectable between 74 and 162 ms. all 8 patients who were referred to liver transplantation and 7 of 8 patients who died, developed a loss of a prior detectable n70 peak during the course of acute liver failure. objective: to determine high risk arid low risk patient groups in a medical intensive care unit regarding short term and long term outcome. methods: data on all admissions of the year 1993 were collected prospectively. according to their mode of admission patients were classified as admissions by the physician staffed ambulance service (as), admissions through the emergency department (ed)i referrals from non-intensive care wards (ni), and secondary referrals from other hospitals (oth). outcome was assessed in terms of in-unit mortality, in-hospital mortality, and long time mortality. patient groups were compared using the \2-test. life table analysis were per{ormed by kaplan-meier method comparing patient groups by log-rank test. the software spss for windows 6.0.1 was used for statistical calculations. results: in-unit mortality: 184/1853 patients (9.9%) --oth 20.0% > as 10.6%> ni 9.1% > ed 6.8%; p = 0.01. in-hospital mortality: 365/1853 patients (19.7%) --oth 30.9% > ni 20.4% > as 19.6% > ed 16.2 %; p = 0,05. mean survival time in days after discharge: as 620 < ni 682 < oth 708 < ed 777; p = 0.66. conclusions: despite an excess in-unit mortality of secondary referrals from other hospitals the iongtime course of this special patient group is not different to others. solsuam, j, marrugat*, g, mirs, j, nolla, a, vazqu~z-sanchez, l alvamz, ~ioio s xndioina i~siw. ir~itate l(~icipal da l~sti~isn l~di~*, ~ospits dal objective: to study the influence of modifiable variables (complications derived from therapeutic activities) on the prognosis of ~atients admitted to the icu indapemently on thn severity of illnsss. patients am methods: between january 1990 asd ]lay 1993 data from 1,425 patients over 14 years of aqe who retained in the icu for mare than 24 hours ~ere pr~pectively regiatered. a cohort st~ly with follo~-~ nf patients durin~ ~eir stey in the hospital was deni~.el in all patients, reasons for a~issien, principal diagnosis sad severity of illn~s moasared by the saps scare vare recorded. fastens affecting patients' outcome that my be proventsd or modified included technical :omplisafioss, heapital-acqnired infections and in~pro~riate therapeutic decisions. a logistic regression model was used to assess the relative risk (l~} for in-heapital mortality adjusted for each variable. results: ic~ mortality ~s 17.2% and in-hospitul mortality 22.7%. patients who died showed a higher spas score then survivors (15,3 ~ i0,i). after adjusting hy severity of illness, co~;licetices that statistically increased the risk of in-hospital death were septic shock secomery to hoapitul-acqdired infection (1~ 7.18; 95% el, 1.9 to 27.1), pmo~othor~x related to mocasnical ventilation (@ 6.28; 95% cl, 1.7 to 22.3) and delay in the insertion of a fln~-quidod catheter (ii~ 5.49; 95% ic, i.i to 26.9). col~lusien: registration of complicaticas derived from therapeutic activities is a valuable tool far quality central in the icu. g, ~i~5, j.l mle~ma, j, ~amqat*, j..~lla, a, vazquez-saltemz, f, alvamz , servioia de nndicina l~siu. i~stitutu ~icipal de ln~sti~acidn ~4i:a*, hospital dsl objective: to dstsr~ine the incidence of self-extebatien and its effect on ~ortality. patients and ]~etheds: betveen january 1990 and april 1994, all i~tiente in whom selfextubatien w~s registered were inclnded in a prospective study. patients were divided into @nee who needed r~intabatinn within 24 hoers and those who did not. in all patients, dsmoqraphie and ciinical data were recorded as well as icii mortality, in-hoapital mrtality and severity of illness according to saps score. 0eta were analyzed usi~ the cbj-square test for cathgorical verinbls, the analysis of varianc~ (anva) for aontinuc~ ~ria~les and a leqi3tic regression anal~is to estimate the relative risk (iiii) for mortality as result of celt-nxtt~ation after adjusting for severity of illness. results: a total of 815 intnmtsd patients amre stndied. self-extu~atien occurred in 54 (6.6%) patients and 25.6% required reintuhot~pn. when a co,arise was made between patients who did not required reint@atinn and patien~.s who did, statistically significant differences in eqe (52.1 v_s 60.4 years, p = 0.~02), ~verity of illness (11.4 ~ 13.1 spas score, p = 0.02), dia~isstia category (4s.6% v_s 66.7% of patients with res~iratury conditiono, p =0,002} and mean length of stay (10,9 ~ 20,7 days~ p = 0.05) were fo~m, a~ter ad~sti~ for severity, patients with self-ext@atinn who did not reqnired reintalatien showed a 0.4 iir for mortality (95% ci, 0.i to 0.9) as co~arod with patients in when self-ext@ation did mot occur. conclnsien: self-~extamtice that does not require reint@ation is associated with a isamr in-hospital natality probably dt~ to a prolonged period of weaming. patients' admissions to ices am often delayed doe to the shortage of beds available. @ile amaltieq icu admission, these patients are treated in observation nits of @e emergency services which bare ,either tile structure nor the trained ~reomenl that are available in leb~. objective: to daterdno the effect on the patient's proqusis of a delay in tile admission to the icu when criteria for icij admission are fulfilled. ~terials and methods: between jme am l?ece~ber 1993 all patients who fulfilled criteria to be almittod to the ic0 who for waste~r reason retained in tile observation unit for more than 24 hours were included in a prospective stedy. in all patients, des~raphic end clinical dabs amre recorded as well as severity of illness aencrdi~j to saps score. a cesucontrol dasi~ was eend with a total ss~ln of 1,425 patients who suffered no delay is admission to icii over a period of 3 years. data wen analyzed using the chl.-squ~re test (to aeons the association hetwenn in-patienty mortality end categorical vari~lns) and a maltipln logistic reqression model to sstimta odds ratio for) for in-hospital mortality as result of delay in icy admission as compared with early ad~issi| after adjusting for severity of illness end use of assisted mchenical ventilation. ~9&ults: a total of 50 patients remained in the observation nit for more than 24 hours with a del w in igd admission of 55.8 _+ 25.1 hoers. assisted mechanical ventilation was requited in 22% of patients and only monitericatien in 46%. itsse patients were cspared with 112 ntients from the tet~l sample ratchod by age, sp~ score and rennoss of admission. in-hospital mortality for cases warn 16% as compared with 17.5% for controls (p = s). after adjamtilg fen spas, age and mobamioal ventihtien, no statistically significant differences between both ~renpa were foam, altho~b there was a tendency towards a higher mortality amen@ patients with delay in icu admission (or = 0.779; 95% ci, 0,2 to 2,5). conclnnien: ~se findings suggest that prognosis of critically-ill patients is no worse as a result of admission to the loll being deln~d for 24 borers. all data appropriate for the calculation of the apache ii score (aps) together wi'th other specific cardiac details relevant to these .patients were collected daily, verified and enter~ into a computer database. results: 150 patients were studied. six patients died and five of thee underwent cardiac surgery. the mean aps was 9 for survivors and t6 for non-survivors (p < 0.001). the mortality ratio was 1.1 and the major markers of mortality were apache ![ score, presence of chronic ill health, mean duration of ventiiation, mean length of icu stay and need for emergen~ surgery. sixteen percent (233) of icu bed days were occupied by 4% of patients (non-sarvivors) which resulted in cancellation of 60 cardiac sot#cat sessions in 6 momhs. conclusions: this study concludes that apache 1t could be used as an audit tool in a cardiac surgical icu and demonstrates the severe compromis~don of cardiac surgical throughput by a few non-survivors, organ to determine the number of organ failure free days (offd) in a cohort of survivors and non-survivors with sepsis syndrome followed over a 30 day period. 2) to determine sample size requirements for clinical trials utilizing a increase in the number of organ failure free days as the primary outcome as opposed to mortality. methods: beginning december 1990 through to april 1992, patients who met inclusion criteria of the "cardiopulmonary effects of ibuprofen in sepsis syndrome" and who did not have hiv/aids. brain death or moribund state were prospectively identified. presence or absence of failure of 7 organ systems (pulmonary, cvs, renal, hepatic, gi, hematologic, & cns) was recorded daily until death or until 30 days. a score of one was assigned to each organ system free of organ failure in patients still alive, ie, maximum daily off score=7, maximum 30 day off scorn=210, sample size estimations were performed for variable detectable differences in off scores (delta). alpha was set at 0.05 (two-sided), with n/group = 2[(z a +z b ) o conclusions: a clinically relevant increase in off days may be detected with as small a sample size as 30 to 50 patients per group. this represents a significantly smaller sample size than needed to detect a change in mortality from 40% to 30% (25% relative risk reduction) where the n/group=356. scoring patients in this manner prevents a lethal inte~entien from providing an improved organ failure score. in addition, an intervention that prolongs survival must also provide greater organ failure free days in order to be counted by this scoring method. survival as an outcome provides no information about the quality of that survival. off days provides a measurement of burden of illness. interventions which lessens this burden may be just as valuable as those that decrease mortality by providing a measure of the quality of survival and by decreasing costs of care. they may also prove to be an accurate surrogate marker of mortality. the advantage of this approach is that the event rote is much higher and sample size requirements are subsequently smaller. this would mean that clinical trials can be completed faster and at lower cost. outcomes such as mortality could then be assessed at a later date utilizing recta-analysis. we suggest that the use of off days is a valid outcome measure that may be utilized in clihieal trials of sepsis syndrome. the icu is perceived by many as being a stressful environment for both patients and staff. stress has been defined in three ways: a stimulus producing a particular response; the physiological and psychological response to a stimulus; an interaction butwom an individual and their environment. stress is currently thought to be a dynamic system of stimulus and. response which takes into account the individual's perception of the stimulus and their ability to respond effectively. stress may, therefore, be positive and allow personal development but an individual unable to respond effectively to a stimulus will experience negative effects or strain. critical illness is an intense stimulus to which the body needs to respond effectively. physiological responses are vital and most of intensive care involves supporting these. alternatively, blocking them, for instance with atom(date, increases mortality. psyehological responses are also vital but often poorly appreciated because of communication problems. many of the problems patients experience in an icu are evidence of psychological strain. this can be exhibited in various ways, for instance, anxiety, depression, passivity and confusion. dealing with critically ill patients is perceived as stressful. we recently studied occupational stress in our icu. most aspects of intensive care were not generally perceived as stressful indicating a self-selectien of icu staff. the most stressful aspects of icu work for nursing staff were the structure of the organization and career opportunities. medical and nursing staff had different stressors and different coping strategies. support for occupational stress, therefore, should focus on the individual and concentrate on information and communication. atmosphere, and especially at intensive care units, we face up to daily decision making. in most cases these are taken on the basis of personal opinion and the processing of a very limited amount of information. rising need to optimize the results of medical attendance becomes necessary to set structured system of d@cision making in which ethical basis have a sp@dial significance in view of next considerations: -we live into a pluralist society in which the importance of values is different. -most persons consider health as the first value only in the event of illness. -medical resources available are limited, whereas medical, attendance demand from population increases in a way many people consider it unlimited. in consequence, it becomes necessary to set up priorities in patients treatment. ehtical basis that rule decision making are essentially these ones: i. beneficence: to provide the patient that is being treated the highest profit. 2. non maleficence: it is our first duty to avoid hurting or damaging the patient."primum non nocere" 3. autonomy: in every particular medical attendance, the patient has ability to decide by himself. 4. justice: as equity: to provide the same treatment for those who have the same pathology, ignoring another factors such as age, sex or race. severe application of these principles can cause difficulty, which resolution requires a systematization of decision making. (1-84) . the lenght of stay between survivors and non survivors didn "t show statistical significance (p =0.51 ). the mean aiii score when considering all admissions was 59,9 (8-153) . the initial score between survivors and non survivors showed ststistical difference (48.6 vs 92.5) respectively (p < 0.0001). univariate logistic regresion analysis demostrated a 90% increment in death probability for every 10 points augmentation in the aiii score with a sensitlbity of 94.9% and specificity of 62.7%, the roc curve showed that the best cut off point for death prediction was 75 points with a sensitivity of 75.6% and specificity of 79.9%. if a patient is classified as high risk (> 75) the bayesian analysis showed a 52.8 probability of death and for one class(fed as low risk (<75) a death probability < 10%. conclusions: the first day aiii score in this population showed to be a good discriminator between survivors and non survivors, and the risk of death augments as the aiii does. in this population an aiii score > 75 points is asociated with a greater risk of death. using the aiii score in conjuntion with the clinical judgement will help clinicians reducing uncertainty in the every day decision making and better predict outcome, the results from this study should been taken with caution because the data were obtained from a small sample. objective: the quality of life has been considered a "uniquely personal perception" resulting from a mixture of health related factors and social circumstances [t. m. gill, jama 1994, 272: 619] . the aim of this study was to evaluate two measures of pqol in intensive care unit (icu) admitted patients. patients and methods: during icu stay and six-months after hospital discharge, 160 co-operative icu admitted patients were directly interviewed about their pqol. we administered ftrstly the uniscale (pqolu) [sage et al crit. care med. 1986, 14: 777-782] and then a 5 step verbal scale (pqolv): best, good, fair, poor, worst. of the 160 studied patients, at the first interview, 116 were able to use both scales, but 44 (27.5%) understood only the verbal one. at the second interview, 8 patients were not able to answer, 113 used both scales and 39 only pqolv. statistical analysis was performed using wilcoxon signed ranks, spearman rank correlation, student's t and chi square tests. results: of all cardiac surgery pts, 42 pts (1.6%) died in icu. they were 33 males (1.5%) and 9 females (1.6%). their mean age was 66 (+7) years and mean ef was 0.38 (+0.1). nineteen pts (45%) had low (<0.35) preoperative ef. mortality was 0.9% in the coronary artery bypass grafting (cabg) group (n=2014) and 2.8% in the valve replacement (vr) group (n=359). in the cabg +vr group, mortality was 8.4% (n=95), and 3.3% in the remaining pts (n=149). cardiogenic shock was the sole cause of death in 24 pts (57%), septic shock in 6 pts, whereas sepsis in combination with ards in 4 pts, sepsis and stroke in two pts. in addition, 6 pts died from cerebrovascular accidents, one from ards and one from pulmonary embolism. the pts who died in the icu had a significantly longer bypass and aortic cross clamp time and received more blood transfusions (p<0.001) than a matched control group that survived to icu discharge. the duration of mechanical ventilation and length of icu stay were greater in the pts who died in the icu than in the control group. conclusions: 1. although cardiogenic shock is the main cause of death (57%)in cardiac surgery pts, sepsis and cerebrovascular accident are relatively frequent causes. 2. patients who died in the icu had longer bypass and aortic cross clamp time and received more transfusions, compared with the control group. 3. although renal or hepatic failure contributed to death in some pts, they were not the primary cause of death in any patient. objectives: evaluate the acute and follow-up outcome of 27 patients (pts) treated with primary ptca (without prior thrombolysis) in acute myocardial infarction (ami) after 12 and up to 24 hours after onset of typical thoracic pain ("late" primary-ptca). methods and patients characteristics: from 12/89 to 4/95 364 consecutive pts with ami were treated by primary ptca in the wuppertal heart center 9 27 pts (7,4%) were admitted to our hospital > 12 hours and < 24 hours after symptom onset with ongoing chest pain and typical ecg-changes.mean age was 62 years (49-78). 23 pts were male, four female. 37% had an anterior wall myocardial infarction, 63% suffered an inferior/postero-lateral wall myocardial infarction.two pts were in cardiogenic shock at admission. singlevessel-disease was documented in 70.4%, multi-vessel-disease in 29.6%. average time of onset of pain to recanalisation was 929 min (720-1440). angiography revealed timi-flow 0 in 85.2% of the pts, timi-flow i in 11.1%, timi-flow ii in 3.7%. average follow-up (fu) period was 12 months (4-28 months). timi iii lv-ef ~ 30-day major late re-late flow p.i.* aeute/fu mortality bleeds infarction mortality 92.6% 58%/63% 7.4% 7.4% 3.7% 0% early mortality occured in the two pts, who were in cardiogenic shock at admission 9 no pt required emergency coronary artery bypass grafting.restenosis >50% was seen in 37% of the pts. conclusions: "late" primary ptca achieves a favourable high recanalisation rate of about 90% (timi ill-flow) in our study group. additionally, there seems to be a trend for lv-ef improvement in follow-up. early high mortality is influenced by the patients admitted in cardiogenic shock. there might be a trend for increased major bleeding complications. objective: to assess the validity of saps ii (new simplified acute physiology score), comparing it with the previous version, (saps), in a sample of patients recruited by giviti, a network of 128 icu's representative of the italian icu system 9 methods: measures of calibration (goodness-of-fit statistics) and discrimination (receiver operating characteristics curve and area under the curve) were adopted in the whole sample and across subgroups differing in relevant prognostic characteristics. of the 3004 patients recruited during one month period, a total of 1813 patients were included in this study. for the purpose of the comparison of the two scores, patients with less than 18 years, or having cardiac surgery or staying in the icu less than 24 hours were excluded. vital status at icu discharge in the whole sample and at hospital discharge in half cases wher adopted as outcome measure. re$01~: saps ii fits the data equally well compared to the older version (goodness-of-fit p=0.29 and 0 9 in the new and old versions, respectively) but its performance is somewhat better in terms of capability to distinguish patients who live from patients who die (areas under the curve 0.81 and 0.73, respectively). furthermore, saps ii is better in terms of uniformity of fit across relevant subgroups, although substantial over prediction of mortality was observed in trauma patients and in patients admitted without organ failure to be intensively monitored. saps ii performed very wet] also in the subsample where hospital mortality was the dependent variable.satisfactory measures of calibration (goodness-of-fit p--0.47) and discrimination (receiver operating characteristics area=0.80) were observed. c0nr saps ii, a multipurpose scoring system developed in an international study, retains its validity in this independent sample of patients recruited in a large network of italian icus. although it has shown a good performance when adopted to predict icu and hospital mortality in the entire sample, further investigations are warranted. the observed over prediction of mortality in a few subgroups indeed call for a through assessment of the impact of confounders and biases on model performance when saps ii is adopted in samples that do not reflect the "average" icu patient. objectives: 1) assess the effectiveness in a group of intensive care units by means of a quality performance index (qpi); 2) assess the efficiency by means of a resource use index (rui); 3) evaluate the performance of individual icus with respect to both indices (clinical and economical) while controlling for severity of illness. 1270 critical from 17 ucis in catalonia patients alearic islands have been included in the study. inhospital mortality and weighted hospital lenght-of-stay (los) have been considered the outcome variables. severity of illness has been measured with the mpm ii at admission. in each icu, expected mortality has been obtained adding the probabilities of dying for its patients. expected los has been estimated adjusting a second order polynomial to the severity of illness. performance indices have been obtained by dividing the observed by the expected outcomes. re~ult~: the overall qpi was 1.15 and it ranged from 0.58 to 2.05 in the 17 icus. the overall rui was 1 and it ranged l~ont 0.61 to 1.51. there was not a trade-offpattern between clinical performance and resource use. objectives: teaching hospitals often provide [cu care across a variety of specialized services. overall, this approach appears to result in the best risk adjusted survival rates, but at the highest cost (critical care medicine 1993;21:1432-42): recently, there has been increasing focus on markers of overall hospital performance. however, in large teaching institutions, such markers may fail to detect intra-institntional variation at a large tertiary care medical center. methods: first intensive care unit (icu) day, acute physiology and chronic health evaluation iii (apache iii) and active therapeutic intervention scoring system (tiss) data were collected on 1621 random admissions to 8 specialty icus with 90 beds (range 8-14) between february i and december 3 l, 1994. post-operative solid organ transplant recipients were excluded. units included 2 general medical, 2 general surgical, and trauma, neurosurgery, cardio-thoracic surgery, and coronary care units. data were analyzed for risk adjusted outcomes: icu and hospital mortality and length ef stay (los); risk of requiring active 1cu treatment; and icu readmissinn using apache iii risk prediction models. results: the study icus cared for a diverse group of patients. mean apache iii scores ranged from 36.9-55.5; predicted risk of hospital death ranged from 8.5-21.1%. standardized mortality ratios ranged from 0.40 to 1.24 with 4 icus performing significantly better and 1 performing worse than predicted (p<0,05). los ratios and icu readmission rates ranged from 0.95 to 1.09 (ns) and 2.1 to 13.2% respectively. patients predicted at low risk of requiring active icu treatment ranged from 6,6 to 45.8% conclusions: there was wide variation in the mean level of patient severity between icus. after controlling for this severity, outcomes also varied widely. no clear pattern of overall institutional performance was evident. these data suggest that efforts to assess performance, improve quality, and maximize efficiency must be focused within individual units. programmatic evaluation of outcome allows for focused review of the processes of care contributing to good outcome (best practices) and where to focus ongoing quality improvement and cost reduction activities. background and method : we compared icu mortality in different age groups presenting with the same severity of disease. we assessed severity of illness by the physiological day 1 -apache~ (physio-aa) score (thus excluding the age related points). for each of the following physio-a n score intervals (0-5; 6-10; 11-15; 16-20; > 20) , we compared tcu mortality within 6 age intervals (< 40; 41-50; 51-60; 61-70; 71-80; > 80 years -10, 11-15, 16-20) . in these groups mortality may be twice higher in the > 60 years patients than in the _< 60 years. mortality does not vary with age in low (physio a n = 0-5) and high (physio a n = > 20) risk groups. in the low risk group, mortality is low in all the age intervals because of the begninity of illness. in the high risk group, extreme severity of disease probably blunts the impact of age and leads to high mortality rates in all age intervals. introduction: to access the actual social/clinical outcome of the patients who undenvent intensive care therapy oct) is rather difficult, quality of lilr is not easih.' defined and ohserver subjectivity is a prime factor in the evaluation. mortality ratio after discharge must be established and its causes understood. obieetives: the propose of this stud)-is to look into the mortality ratio that occurred on a series of patients that undorwent ict at our unit from of the ~iew point of severity of the original illness and the diagnostic groups. material and methods: during the period of one )-ear (1994), 216 patients were treated at the unit, 45 of them died, and 16 ~ere not matched in our series because os incumpletc records. thirteen patients died in hospital after their reference to other departments, twelve patients were lost after discharge. thus. at the end. only 142 patients were evaluated on the fu. the, were classified into the follov4ng three groups: acute medical, elective surge d 9 and acute and emergency postoperative. the patients were seen at 3, 6 and 12 months after discharge. the, were evaluated in accordance to their abili~, to being self supported in their daily life and capecity to fully return and hold to their pre~4ous jobs. apache 11 scores were evaluated for each of the three groups and correlated to the icu dead, hospital dead, and mortality after hospital discharge, spss package was used for statistical analysis. remlts/conclasions: data shows that 19/142 patients died after discharge from the hospital, of ~itch nine died in the first three months. seventy-eight per cent of the patients were fully self supported in their daily life and 20% showed some kind of handicap. fosty-nine per cent of the patients wore on retirement either due to age or some form of chronic disease, when admilled to our unit. thirty-two peg cent had not been able to return to work, because the" were incapacitated on discharge. only 7% had return to their fully jobs but the period of the stu~, is not enough for all of them to be fully physically recovered. preliminmy statistical analysis shows us significant differences among groups. the aim of the present study is to compare the prognostic performance of five general severity indices ou coronary patienta and to find out if a proper ntatistical hundling of these indices could provide better results in these patients. methods: saps ii, mpm ii (mpm ii0 i mpmp ii24), apach ii end gaprik were evaluated o~ 456 patients with acute myocardial infurction admitted to 17 intensive care units from catulunye. calibration and discrimination were calculated for each index. calibration was calculated by th bosmer-lemeshow test. discrimination was evaluated by the area under the relative operating characteristic (roc)curve. if a model did not show a good performance it was customized using multiple logistic regression. finally, tworeduced models were developed, one fro~ the mpm series (mpm ii24cor) and one from the group apache-saps (sapsiicor).their performances were again evaluated. results: discrimination was high enough for all models. neverthelees, oelibration of apache ii, saps ii and mpm was not satisfactory. thus,mpm ii24, saps ii and gaprik were customized for coronary patients using the logits of both models, and obtaining good calibrations. mpm ii24, and apache-saps were adapted and reduced to 5 (mpm ii24cor) end to 4 variables (sapsiicor), respectively . both models showed better oalibrutions end discriminations than the original models. conolusion| models developed for multidisciplinary patients show a good discrimination when applied on aoronar i patients, but some needed customization in order to improve calibration. the number of variables of the principal model can be reduced (even to 5 or 4 variables) without loosing prognostic accuracy. objective: to compare the ability of two methods to predict outcome for intensive care patients. methods: we included 343 consecutive intensive therapy unit (itu) admissions with an itu stay>24 hrs in a 18 month prospective study (exclusion criteria: burn injury and age <16 yrs). data were couectsd applying the criteria described by the developers [1, 2] . the definition of coma (mpm24ii) was modified and the best assessment within 24 in's, rather than the admission score, was used. statistical analysis included classification tables and receiver operaung characteristics (roc) curves to assess discriminative power, and lemeshaw-hosmer statistics and calibration curves to test accuracy of prediction. results~ average abe was 58 yrs (ranse:16-92) with a male:female ratio of 1.6:1. the actual hospital mortality was 26.8%, mean predicted death rates were 22.8% (mpmz4ii) and 15.2% (ap hi). non-survivors had siguitlcanfly higher predicted risks than survivors applying both methods (p<0.000l, t-test). the total correct classification rates (tccr) for apache iii were bett~r for all decision criteria applied (tccr, decision criterion 50%: apache ]/i 77.1%, mpm24ii 71.4%). the area under the roc curve was 0.75 (ap iii) and 0.66 (mpm24ii) confirming the better discrimination of apache ill. accuracy of risk prediction was similar for both models (ap nl ~2-59, mpm24b ;(2-52, lemeslmw-hosmer). showing some fluctuation, calibration curves lay close to the ideal line for predicted risks -<50% with increasing deviation for higher risk groups (s. figure) . apache iii underestimated the risks of hospital death for almost all risk groups (curve above diagonal), whereas considerable overestimation for predicted risks >40%0 ceenred with mpm~ii. objective: to assess the goodness-of-fit of the apache iii model for british itu patients. methods: we prospectively studied a cohort of 715 adult patients consecutively admitted to a medical-surgical itu over a period of 18 months. patients with burn injury, age < 16 yrs and itu stay < 4 hrs were excluded. using a eomputerlsed database, we routinely recorded 24 hrs apache ill scores. predicted risks of hospital death were computed by critical audit ltd, london. accuracy of risk prediefion was assessed by hosmer-lemeshaw chi square (;(2) statistics and calibration curves [1 ]. discrimination was tested employing classification tables and receiver operating characteristics curves (roc). restths: the mean age of the 453 male and 262 female patients was 59 yrs (range: 16-92 yrs). of these patients, 64% were medical admissions, 17% were admired after emergency and 21% after elective surgery. the observed hospital mortality was 25.4%, the overall mean predicted death rate was 16.8%. mean predicted risks were siguifieanfiy greater for nonsurvivors (38.0 %o) than for survivors (9.6%, p<0.00l, t-test). apache iii showed good calibration (z2-~59, lemeshaw-hosmer). however, the calibration curve lay above the diagonal for almost all risk groups reflecting the tendency to underestimate actual mortality (s. figure) . the best total correct classification rate (tccr) was 89.3% (decision criterion: 50%). the area under the roc curve was 87.6% confirming the good discriminative ability of the model. objectives: the aim of this study is to point out the discrepancies between needs and actual treatment of less severely ili patients admitted in italian intensive cam units (icus) requiring only intensive monitoring, and verify the substantial likelihood of data comparing those collected from a national short term study with a regional long ternl use. ~: less severely ill patients ("observed patients") were only monitored; they did not require intubation, even if for a short period (less than 24 houm) or major cardioeiranlatory supports, and were neurologically normal. epidemiologieal national data were obtained from giviti group (gruppo italiano valutazione interventi in terapia intensiva); this cohort study, collected 5092 patients, in two months in summer in 1992 all over italy. regional data were echieved in a three years entlection (1990-i992) in lombardia' icus from archidia group (arehivio diagnostieo), including 10065 patients. mortality, severity score, diagnostic category and some typical intensive procedures were analysed and compared in both studies. patients' disgunstie categories were defined as surgical, medical and trauma, according to the main diagnosis and the presence/absence of surgical procedures. rr162 observed patients account for 23.2% and 22% of all icu's patients respectively in national and regional data. very tow mortality rate was found in national data (2.3%) and extremely low mortality in regional data (0.6%). in both studies mortality, s.a.p.s. and length of stay were much lowor in "observed patients" than in general icu's population (mortality: 25.7% and 22.3%; 8.a.p.s. score: 10.6 and 13; iength of stay: %7 and 9). homologous distribution of patients in the two studies was noted for what concern their diagnostic category, aside from a slight prevalence of tranmatised patients in the giviti study. in the two groups the surgical patients were respectively 47% vs. 57%, medical patients were 34% vs. 31% and traumatised were 19% vs. 13%. 92% of "observed patients" in national study and 93% in the regional did not received any intensive procedure. only a minority of these patients availed haemodynamie eonu'ol with swan-ganz or renal haemofiltration. conclusions: these results underline that about one fourth patients admitted in italian icus benefit an oversized slructure i, relation to the real needs of their pathology. in hot more than 90% did non received any advanced treatment and mortality and s.a.p.s. score were substantially lower respect to general population. the results obtained from these two studies are similar, suggesting an uniform distribution of the case mix in italy, even if a different recruitment period and a different gengraphieal distribution were used. some discrepancies in the two studies were found in the diagnostic categories moreover regarding the tranmatised patients (19% vs. 13%); this can be explained from the seasonal (summer) characteristic of the national study. mutuality, yet very low, is different in the two groups, but these data do not allow any definite explanation. finally these epidemiologieal survey suggest need of further studies settling more strict criteria of admission in icu. this study aims to evaluate patients outcome, quality of care and effectivity of therapy in our intensive care unit. the main goal was to indentify factors that the most influence that outcome. during 1994. the authors collected data of patients outcome and predictor variables. overall mortality rate was 39,3%. the most common causes of death were infection. the diagnosis of sistemic inflammatory response syndrome (sirs) and multiple organ dysfunction syndrome (muds) significantly correlate with death (90%). average length of stay was 6.6 days ~. 55% patients died in the first ten hosiptal days and only 18% after 30 days. age was directly correlated with death 50% of dead were older then sixty years. an analysis of physiological variables showed that serum levels of gl~cose (55%) and natrium (71%) were in optimal physiological values. serum proteins (72%) and haemoglobin (50%) levels were inversely related to death. multivariate showed that alveolo-arterio difference in 02 content was the most informative of all mortality predictors (mean value 22,4 mmhg in 90% patients io>mrnhg). factor that most influence the patients outcome was infection (sepsis) and muds. use of predictive indicators of outcome in critically ill patients may help to assess treatment regimens and to compare patient groups. acute physiology and chronic health evaluation (apache if) score (crit. care had. 1985; 13: 818-29) and the sepsis score of elebute and stoner (br. h surg. 1983; 70: 29-31) have been used, objectives: to compare sepsis score and apache ii score in predicting outcome of critically ill patients. methods: overall survival during the past 8 years for patients in our icu was calculated = 62% (prior probability). the outcome of 230 patients who were admitted to our icu for > 72 hours was observed. apache ii score on admission, patient predicted risk of death (apache ii risk) and the sepsis score on the first day of antibiotic course were prospectively recorded. discriminant function analysis of the scores in relation to outcome was performed. results: apache ii and sepsis scores in the survivors were significantly lower than in those who died (21.6 i 7.2 v~s 25.6 • 6.5 and 10.9 • 5 v's 15.2 • 5.9 respectively p < 0.001). correct prediction of outcome by each score is shown in discussion and conclusions: although both scores have been previously evaluated in predicting outcome of icu patients, studies of the sepsis score were conducted in small numbers of patients or involved additional measurements not routinely available. this study demonstrates that the sepsis score alone or in combination with apache ii score is more effective than apache ii score in predicting outcome. objective to test the hypothesis that resuscitation titrated against gastric intramucosal ph (phi) improves survival in critically ill patients as suggested by gutierrez et al~. method emergency admissions to the intensive care unit were randomized into control and intervention groups. in the control group phi was measured at 0, 12 and 24 h while in the intervention group phi measurements were made 4 hourly for 24 h. both groups were managed according to the same guidelines to achieve the following targets: mean arterial pressure >70 mmhg, systolic arterial pressure >90 mmhg, urine output >0.5/ml/kg, haemoglobin >8 g/dl, blood glucose < 12 mmol/1, arterial oxygen saturation >94% and correction of uncompensated respiratory acidosis. if the phi was < 7.35 after achieving these targets, or after maximal therapy to achieve the targets, patients in the intervention group were given fluid to ensure an adequate cardiac preload and then dobutamine at 5 then 10 mcg/kg/h, titrated against phi. this additional therapy was continued until 24 h after entry into the study. in each year patients were subdivided in two series with random selection, so that the 1st series contained abeat 2/3 and the 2nd 1/3 of the patients. the 1st series of all the years constituted the devdoping data set and the 2nd series the validation data set. with data of the 1st series (642 patients), we created the predictive model, using stepwise logistic regression (bmdp, usa). each patient has been evaluated in die 1st, 5th, 10th and 15th day, calculating for each lime the apache ii score (for a total of 1444 records), independent variables were, besides time and apache ii of the time ( michaloudia g,, melissaki a., alexias g., gogafi c., kolotoura a., krimpeni g., pamouktaoglou f, filias n. objectives: to determine the medical staff's attitude towards various ethical issues methods : between january 1994 and february 1995,185 anonymous questionnaires were sent to 20 intensive care units, all over greece. results : 107 questionnaires (57,7%) were replied and returned back. of them 58,9% were answered by male and 41,1% by female. the doctors replied in the following rate :81,2% aged up to 34,80% aged between 3544 and 94,4% aged over 45. 36 questions were answered and were divided into 3 main topics, as following: 1. admission criteria: limited bed availability was the main cause for refusing admission in 54,5% of icu's. 54,5% evaluated each case's viability and only 10,3% used some prognostic score system. 21,5% of icu's accepted all cases and a significant percentage (64%) gave in to pressure coming from their colleagues (72,7% female and 58,7% male). 2. informing the patient/relatives: only 6,5% was willing to tell the whole truth, while 39,2% had given selective information.. in the case of iatrogenic incident, 58,9% withheld it, because either they feared legal implications (34,6%), or lost of trust (46,7%). doctors are asking consent from the patient and/or his family, in order to include him/her in research protocols, in a rate of 82,3%, while only 55,1% found informed consent necessary for the proposed treatment procedure. 3. withdrawal of therapy/dnr orders/organ donation: 80,4% were willing to withdraw complex treatment in patients with short life expectancy, except of administi'ating intravenous fluids, feeding and analgesics. in 34,6% such a decis~n was unanimous, while the percentage of those carrying it out was 69,1% (72,2% female, 63,9% male). in case of brain stem death 87,8% (67,3% female, 85,7% male) withdrew any life support. 67,3% would like therapy withdrawal to be legally established, while only 12,1% would perform euthanasia, if there was substantial legal cover. for these cases, relatives' consent was considered to be necessary from a percentage of only 11,2%. 83,2% considered organ donation to be a necessary proposal, while 10,3% refused to ask the patients' relatives for an organ donation, either because they didn't have the psychological strength for it (3,7%), or because they doubted the procedures' objectivity (4,7%). note: in greece, icu beds are less than 1% from the total number of hospital beds available. only a percentage of 35-50% of these admissions comes from the same hospital, with a potentially direct evaluation. usually an icu doctor has to be informed through the telephone. finally, employment conditions in greece are such that any changes of the medical and nursing staffare limited. conclusions: the mathematical model we found has been validated also in the second series and the discrimination capability increases with time. using this model we can evaluate the probability of survive at every, time. its application at different times permits a better evaluation of haemodinamically instable patient trend. introduction: the feasibility to assess pulmonary capillary pressure (pcap) offers the opportunity to determine the longitudinal distribution of pulmonary vascular resistance (pvr). the purpose of this study was to measure pcap and to calculate pvr to determine whether relevant shifts in the distribution of pvr could be expected after routine cardiac surgery. methods: the study population consisted of 25 consecutively admitted patients after cardiac surgery. surgical procedures included coronary artery bypass graft (cabg) (n=14) and mitral valve replacement (mvr) (n=t 1). pcap was estimated by analysis of the pressure decay tracing after pulmonary artery occlusion. after estimation of pcap precapillary (ra) and postcapillary resistance (rv) was calculated. a complete set of hemodynamic variables was obtained at 1 hour and at 6 hours after operation. results: there were no significant hemodynamic changes during the first 6 hours after surgery. the mvr group maintained pulmonary hypertension and higher levels of pcap. ra/rv, reflecting the longitudinal distribution of resistances, remained unchanged. however, rv predominated ra during the postoperative period in both groups. objectives: evaluation of the influence of long-term continuous i.v. administration of the ace-inhibitor enalaprilat on regulators of circulatory homeostasis. methods: t9 trauma and 26 sepsis patients randomly received either 0.25 mg/h (group i, n=15) or 0.5 mg/h (group 2, n=15) of enalaprilat i.v. or saline solution (control, n=15) as placebo for 5 days. plasma levels of endothelin-1 (et), atrial natriuretic peptide (anp), renin, vasopressin, angiotensin-ii, and catecholamines were measured before injection of enalaprilat (='baseline' values) and during the next 5 days. results: except for et, plasma levels of all vasoactive substances exceeded normal range at baseline. angiotensin-ii significantly decreased during enalaprilat infusion (0.25mg/h: from 53.1• to 22.1• pg/ml; 0.50mg/h: 62.1• to 17.9• whereas it remained significantly elevated in the untreated control patients. vasopressin increased only in the control group (p<0.01) and decreased after 0.50mg/h of enalaprilat. et remained almostunchanged in group 2, whereas et increased significantly in the control patients (from 4.9• to 10.t• on the 5th day). catecholamine plasma levels (epinephrine, norepinephrine) markedly increased in the control group (p<0.01), but they did not change significantly throughout the study period in both enalaprilat groups. conclusions: continuous i.v. administration of the angiotensin-converting enzyme inhibitor enalaprilat beneficially influenced systemic and local vasoactive regulators of the circulation, which are normally increased in the critically ill. thus patients at risk of (micro-) circulatory abnormalities may profit from enalaprilat infusion. objectives: to determine the time taken for hemodynamic and gas exchange variables to a reach stady-state after a change from supine to trendelenburg position (trp). methods: we prospectively studied 8 adult patients with severe sepsis or septic shock requiring hemodynamic monitoring. usual cardiorespiratory parameters were measured at baseline, 15 min after the patient was placed in a trp and again 15 min after the return to a supine position. a fiberoptic pulmonary artery catheter (svo~ oximetrix, abbott) allowing continuous svo 2 monitoring wa~used. during the protocol we also continuously measured sao~ by pulse oximetry and vco~ and vo 2 by monitoring partial concentration of o2and co 2 ir~ inspiratory and expiratory gases (deltatrac metabolic monitor, datex). therefore, we were able to monitor cardiac output variations by dividing vo~ with arteriovenous difference according to the fick equation (co-fick). results: no significant difference in hemodynamic status was observed 15 min after the patients were placed in trp. despite the fact that no significant change was observed in co and vo~ estimated by thermodilution, co-fick had a tendency to dedrease continuously in trp and then to return to its initial value when patients regained supine position. respiratory gas analysis showed a small but persistent continuous increase in vco 2 without a similar trend in vo 2 values. conclusions: we conclude that no significant hemodynamic effect was detected in our patients after 15 min in trp. evaluation of vo 2 from respiratory gases analysis after a change in body's position should be interpreted with caution, since the patient may not yet have reached a stady-state after 15 rain. since vo 2 did not change, vco~ increase was probably due to position related changes in-pulmonary gas exchange and not to a change in patient's metabolic status. objectives: to determine whether changes in svo 2 and/or other hemodynamic parameters during weaning trials could be used to predict successful weaning. methods: we prospectively studied 10 adult patients with a history or clinical evidence of cardiovascular dysfunction, who were unable to tolerate spontaneous breathing (sb) for 3 hours. for all these patients right heart catheterisation was considered necessary in order to detect hemodynamic alterations during weaning. a fiberoptic pulmonary artery catheter (svo 20ximetrix, abbott) allowing continuous svo 2 monitoring was 5sod. hemodynamic status was evaluated ~t baseline and after one hour of spontaneous breathing through a t-piece. patients were assigned to one of two groups depending on whether they tolerated sb for 3 hours. data were analysed by analysis of variance and unpaired student's t-test we also used multiple linear regression analysis to determine which hemodynamic variables were correlated with the magnitude of svo~ change and multiple discriminant analysis to determine if asy of the above variables were associated with toleration of sb for 3 hours and/or successful weaning (s-w). (j physiol 1995; 78." 696-701) . we tested the hypothesis that the ventilatory stimulation by dead space (vd) loading and 3% co2 inhalation is accompanied by a proportionate cardiovascular change. methods: six healthy subjects, mean age, 25 year, performed three incremental exercise tests in a randomized order: 1) inspiring air without vd (air control, ac); 2) inspiring air with vd of 920 ml (avd); 3) inspiring 3% co2; 21% oxygen, balance nitrogen. the ventilatory responses were examined at matched heart rate (hr) equivalent to 90% peak hr. results: ventilation (vi) was significantly greater (p<0.0001) during the avd and co2 tests than during the ac test at the same work rates. end-tidal co2 (petco2) and estimated arterial co2 (paco2) were significantly greater (p<0.01) at 150 w and 200 w. oxygen saturation was significantly lower (p<0.05) during the avd test than during the ac and 3% co2 exerdse. at matched hrequivalent to 90% peak hr, vi was significantly greater (p<0.01) during the avd and 3% co2 tests than during the ac exerdse (123 l, 121 l, and 91/). conclusion: we conclude that the increase in xri and petco2 due to vd loading and 3% co2 inhalation is not associated with an acceleration in hr. sup.ported by mrc (canada). objeetlve: the production of large amounts of oxygen radicals from the onset of ~en may be responsible, st least in part, for peroxidative damage to myocardial tissue. the aim of this study was to evaluate the time dependence of plasma tbars in patients with am] receiving thrombolytie therapy (tt). patients and m~hods: filiy eight patients admitted in icu (46 men and 12 women; mean age 50.6 4-16.02 years) rec~ving systemic tt for possible am] were ~died. all patients received recorabinant haman tissue-type plasminogen activator (r-tpa). the mean time fi'om the onset of symptoms and the be~nning of tt was 3.01 4-2.13 hours. peripheral veao~s blood samples were obtained fi'om each patient before and serially after tt (0, 3, 6 and 9 hours). tbars levels woe determined by using a spectrophotometrie technique. rq~r fusion was identified by the timing of ereatine phosphate kkmse (cpk) peak (<15 hours). table i list the variation of plasma eoneenlrations of tbars (mean 4-sd) in groups (a,b, and c) as a function of time from the beginning of tr. co,arisen oftbe 0 time cuncentzatiens reveal a difference p<o.01 between group ami (a and b) versus group c. the between-groups (a-c) comparison showed significant differences (p<~).05) at 3,6 and 9 hours fi-om de begin~g of'it. comparison ofthe 3,6 and 9 hours eonocnlratiens did not reveafl any difference becween n~ (group b) and angor (group c). condmien: we have observed a maintenance of high level of tbars in reperfused patients enly. these results suggest that the increase of tbars levels may be due to phospholipid derangament of reperfused myocytes. objective: to detemline if tbars levels may be an early index reflecting peroxidative damage in ami patients. patients and methods: by using a spectrophotometric teclmiqtm, based on a modificatien oftbe buege and aust method, tbars levels ware determined in 51 healthy subjects (group i) , 43 patients with several risk factors of ischaemic cardiopathy (group if) and 99 icu patients with possible aim (87 were confirmed ami (group ma) and 12 were angor (group iiib)). peripheral blood was obtained at the time of the icu admission in the group i11. electrecardiographic data and eehocardiographic wall motion were used to identify ami location (anterior, inferoposterinr and indeterminate). statistical analysis was made with a standard statistical package (rsigma, horus hardware). , h 4, s, , h statistically significative increase of ' ix~e/ea.ee x~* tbars levels (p< 0.001) only in pstients with con/tuned ami (group ilia) . no differences were found between the rest of the groups. the figure shows the relationship between the tbars values and the time from the onset of the aim's symptoms. conclusion: we have found that in am/patients the tbars levels can be used as an early index reflecting peroxidative damage occurring to ischemie tissues. perioperative risk in patients with ischemic heart disease. the protective role of diltiazem. lg. hatziantoniou, p. tassiopoulos, c. fakiolas, ! g. foussas~ m. lycourinas perioperatlve morbidity and mortality are mainly of cardiac origin, particularly among elderly patients (pts) ~ith ischemic heart disease. we performed a randomized trial, in order to investigate the possible protective activity of diltiazem (d). methods: 58 pts (aged zo• with a history of coronary insufficiency were subjected to major urological operations. they were randomized to two groups: group i (29 pts) -p:ithout d and group ii (29 pts) -d ~as administered 0.15 mg/kg iv bolus 12 hours prior to anesthesia and 0.15 mg/kg/h upon the end of operation and for at least 12 hours post-operatively. iv was substituted by oval administration of 2x120 mg starting the next day and for the rest of hospital stay. all pts underwent daily clinical, electrocardiographic and laboratory (ck-mb) control. results: are shown in the following interactions between the heart-lung unit and a cardiac assist device are the crucial issues for successful outcome of patients under mechanical circulatory support. several factors have been indicated and we can diffrentiate between anatomical and hemodynamic interactions. especially right heart failure and pulmonary hypertension have been proven to limit left ventricutar assist device (lvad) performance. in order to study the effects of right heart failure (rhf) and pulmonary hypertension (pht) on mechanical circulatory support, we designed an experimental study in dogs and combined in six groups left heart failure (lhf) and rhf and pht, respectively. in the control group we induced lhf with subsequent occlusions of lad and cx followed by reperfusion: in the next group we supported with an lvad during 90 minutes starting 5 minutes after cx occlusion (group if). in the next group we added right ventricular ischemia by occluding the rca simultaneously to the cx (group iii). these both groups we repeated only with pre-existing acute pht induced by the injection of 1o0 fm glass beads (groups vi and v). a last group was similar to group v (lhf and rhf and pht), but instead of a lvad, we used bvad support. in i and v no animal survived the second perfusion period (4 hours). in ii and iii (no pht), all dogs survived in stable conditions. in iv (lvf, pht), 60 % survived, the remainder died of incomplete recovery. in vi with bvad, only 40 % survived. in iii and v, during occlusion of the rca, the right ventricle stopped contracting and right atrial pressure and mean pulmonary pressure equalized. this lead to a significant drop in inflow on the left heart side, which still provided sufficient lvad performance in case of absent pht (iii) and lead to death due to "low lvad output" syndrome in v. this "low lvad output" snydrome could be partly overcome by bvad support. we conclude that heart-lung interactions play a major role in lvad performance. by optimizing the perioperative management, they all can be overcome, except the occurrence of alveolar leakage syndrome. obiectives : we propose a new approach of transmural pulmtmary artery occlusion pressure (paoptm), in presence ef peep, from the folluwing hypothesis : if respiratory swings of paop are compared to those of alveolar pressure (apalv = plateau pressure -total peep), an index of transmission of airway pressures to pulmonary vessels is obtained (i t = apaop/apalv). the value of the product of it by peep can be substracted from end-expiratery paop (paopee) to obtain a calculated paoptm (paoptrnc). thus paoftmc = paopee -(apaop/apalv x peep). methods : we tested this hypothesis in 67 patients by comparing paopee, paoptmc, and paopnadir obtained within the first 3 seconds after disconnection from the ventilator, value of reference of paoptm in absence of intrinsic peep (peepi). at zeep, peepi was absent in 38 patients (group a) but present in 29 others (group b). patients were studied under the peep level chosen by the attending physician-(gr a : 11 +_ 2 ; gr b : 11 _+ 3 cmh20 ). results : l. gr a : paopee (12 _+ 6 mmhg) differed (p < 10 -3) from paopnadir (8 • 6 mmhg) and paoptmc (8 _+ 6 mrahg). gr b : paopee, paopnadir, paoptmc differed between themselves (14 "2-6, 12 + 6, 10 + 6 mmhg respectively). 2. good correlatkm (r (i.99 ) and agreement between paopnadir and paoptm were found in gr a, while there was no agreement in gr b (bland and altman analysis). 3. lung compliance (v t/bpah,) correlated well with i t in both groups (r -0.92), suggesting that our hypt)thesis was strtmg even in patients with peepi. conclusions : paoptm under peep ventilation can be obtained, even in patients with peepi, from parameters easily measured at the bedside. introduction: cardiopulmonary bypass (cpb) may cause ischemia in several organ systems like the heart, brain and kidneys. reactive oxygen species (ros) are formed by subsequent reperfusion and stimulation of neutrophils by cpb. ros are harmful to biological systems e.g. cellular membranes, enzymes~ dna. many natural antioxidant systems protect against the effect of ros. in patients undergoing cabg we evaluated the production of ros by total plasma antioxidant status (tas) and lipid peroxidation measured by lipofuscin flip). methods: tas (randox, uk) and concentrations ofllp (tsuchida et al.) were measured in 16 consecutive patients undergoing cabg. all the patients had a normal serum creatinine clearance (>50 ml/min). serum samples were obtained a) before operation, b) after removal of the aortic crossclamp, c) at admission to the icu, d) 4 hours after operation, e) 22 hours after operation. results: tas was significantly decreased after removal of the aortic crosselamp ( b, c and d lower than a), followed by a subsequent significant increase of lip ( c and d higher than b). the levels of tas and lip returned to baseline 22 hours after operation. methods: 10 patients with preoperative lvef<40% undergoing coronary artery bypass grafting were studied. after surgery, a 3f femoral artery catheter was inserted and connoted to a fiberoptic monitoring system (cold z-02t; pulsion medizintechnik, germany); this allows, with a double-indicator dilution technique, the calculation of cardiac index (ci,l/min/m2), intrathoracic bood volume (itbv,ml/m2), pulmonary blood volume (pbv,ml/m2) and extravascular lung water (evlw,ml/kg). with a 7f pulmonary artery catheter, wedge (w,nunhg) and central venous pressure (cvp,mmhg) were measured, while 02 extraction ratio (o2exr,%) and oxygen delivery (do2,ml/min/m2) was calculed. peak inspiratory pressure (pawp,cmh20) and mean airway pressure (mawp,cmh20) were measured with a varflex flow transducer (bicore,sensormedics,us). the patients were studied after 60 minutes (to) of volume controlled standard ratio ventilation (vc), and after 60 minutes (ti) of stabilisation period of pcirv (67% inspiratory time, 0 % pause). vt,ve and total peep were held constant in every mode of ventilation. 1+_0.4" *'p < 0,05 versus to conclusions: these data show that pcirv 2:1 is a safe ventilatory support also in cardiac patients with impaired ventricular function, and monitoring of itbv is more reliable to measure and optimise circulatory volume status, than w and cvp. c.ledeki-,g.rldisis,s.karotzai,c.micheilidis,m.agioutantb, g.beltapaulos. objeolivee:to evaluate the influence of lvswl on the well known correlation of sr and svo2. paw eight patients (12 melee end 16 females) were included in this study regerdlen of the icu ~h"niseion couse. all paints were ,'~theta~ with e fiboroptir pulmonary artery catheter connected with an oxymetfir (r)~ so2/co abbot computer.for any pulmonary artery catheter insertion, two pain= of sr and svo2 were obtained, one dudng inserlion and one during taking the catheter out. for any pair obtained, we eleo collected the deta concemig with the pedient's hemodynamir and oxygenation end we calculated the lvswi. were significantly (p<o.05) higher in group i. conclusion: breathing room air, the less hypoxemic patients had a rather satidactory do2 and tissue oxygenation (pro2) because they had properly adapted their ci. the absence of this mechanism (right heart impairement ?) in the hypoxemic patients, in spite of higher blood ne and e, was responsible for the poor do2 and pro2. objectives: acute massive pulmonary embolism (pe) increases pulmonary artery pressure (pap) and dght ventdcular aftedoad, which may lead to dght ventdcular failure. inhaled nitdc oxide (no) selectively dilates pulmonary vessels. thus, inhaled no may be of potential therapeutic value in the management of the acute phase of pe. methods: following institutional approval, ten piglets (body weight 17+2 kg) were anaesthetised (midazolam/piritramide) and ventilated using a modified servo ventilator (siemens elema, sweden; fio 2 0.3). the following variables were obtained: systolic pap (spap), mean pap (mpap), diastolic pap (dpap), mean artedal pressure (map), central venous pressure (cvp), pulmonary capillary wedge pressure (pcwp), cardiac output (co), and artedal (pao2) and mixed venous (pvo2) oxygen saturation. pulmonary vascular resistance (pvr) was calculated. to induce pe, 300 pm microspheres (sephadex g50 coarse, pharmacia biotech, freiburg, germany) were injected in an amount sufficient to increase mpap to 45 mmhg initially. 45 rain after embolisation when haemodynamics were in a steady state (control 1), inhaled no was administered. further measurements were taken 5 min after 40 ppm no (no 40), 5 min after 80 ppm no (no 80), and 10 min after discontinuation of no administration (control 2). the administration of inhaled no resulted in a significant decrease in spap, mpap, and pvr. the cessation of no inhalation led to a complete return to pretreatment control values (table) . co, map, cvp, pcwp, pao?, and pv~ remained unchan no administration. objectivss:evsluate the yield of recombinant tissue plasminogen activater(rt-pa, actiiyse ~ 9oehringer ingelheim) as a thrombolytic agent ie pulmonary embolism ie .kr~auma patients. methods: in 1994 five patients with diagnosed pulmonary embolism(blood gases, chest x-ray, echocardiography, perlesion lung scans) were given 100rag of actij.yse as ~ 2hrs infusion, followed by heparin~single 5000j. ihjaction snd iefusion ie doses 300-400j./kg/day).the effectiveness of rt-pg thrembo].ytic sctier~ was svaluated im 3.24. hrs(blood gases) amd on the third dayafter t~eat-,ment(~chocardiography and lung scan). results: three hrs after actilyse was given we observed significant clinical imprevemeet,confirmed by elevation of pao 2 and sao 2 in dlood.echocardiegr~m on the third day showed regression of pulmonary hypertension and lung scans showed 45% improvement in total lung perfusien. except slight bleeding from the site of injection nc~ more thromboiytic complicatioos were observed. conclusiens: actilyse used in five hrauma patients with pulmonary embol.ism was a very effsctive and safe thrombolytic agerlt, inducing rapid and evident clinical improvement. intermittent positive pressure ventilation (1ppv) by increasing pleural pressure, decreases the pressure gradients for systemic venous return and left ventricular (lv) ejection. we have previously shown that when inspiration is synchronized with systole using synchfjv in patients with severe cardiomyopathy, that cardiac output (co) was increased relative to both ippv and non-synchronized hfjv (1). however, it is not known if similar effects could be realized in ventilatordependent patients with lesser degrees of lv dysfunction and fluid overload. we thus compared synci-ifjv to ippv in stable patients following coronary artery bypas grafting. methods: 13 normothermic who were stable (< 20 % variance/h.) were studied following admission to the sicu. heart rate (hr), mean arterial (pa), pulmonary artery occlusion pressure(ppao), co by continuous thermodilutiun (vigilance, baxter, usa), mixed venous saturation (svoz) and arterial blood gases (abg) were measured after 30 rain. of each step. ippv was simv adjusted for a rate, tidal volume and fit2 to insure both normal abg and a peak airway pressure < 35 cm h,o. syncfifjv was delivered by a hfjv ventilator (acutronic) during systole with ventilator parameters adjusted to optimize abg. protocol: patients received three ippv runs (ippvi.3,5) with synchfjv runs interposed (hfjvz4). statistics: anova for repeat measures and paired t-test were used to compareruns and demonstrate variability. results:no significant difference in any of the measured hemodynamic variables were seen among the ippv and synchfjv runs (table) . post hoc separation of patients by preoperative ejection fraction (ef; ef > 40% ; n=10 and < 40%; n=4) did not alter these results. back~ound: in man, vascular endothelium-bound ace is expressed in concentrations greater than 50x that in serum and is believed to be the site of synthesis of circulating angioteusin il it is unclear whether ace inlubitors interact similarly with ace in different vascular beds. coronary vessels possess all the components of the renin-angiotensin system, including ace which may be involved in normalcardiac homeostasis, as well as in the pathogenesis of various cardiomyopathies. obiecfive: to develop a method for assaying the interaction of ace inkibitors with coronary endothelium-bunnd ace in man, methods: ace a~aty was meas~ed in five patients undergoing cabg surgery, from the transeuronary hydrolysis of the synthetic ace substrate 3h-bpap. trace mnou~ of ~fi-bpap (4gci) were injec~d as a bolus in the root of the aorta and simultaneously blood was withdrawn from a coronary sinus catheter into a syringe containing protease inhibitors which prevented the convession of umeaet~ ai-i-bpap by blood ace. the sample was later centrifuged to separate cells from plasma and the radioactivities due to formed product (~rl-bphe) and total sh were astimated in a [b-counter. two additional such determinations of ace activity were perform~ the second in the presence of 1.5pg/kg e (coinjected with ~-i-bpap) and the third ten minutes after e. results: all subjects were hemodynamically stable throughout the course of the there were no noticeable hemodynamic effects of e. control transcorunary metabolism of~-bpap averaged g0-a:3%, in agreement with previously reported data. in the presence of e, % metabolism of ~-bpap was reduced to 21• reflecting a 85• inhibition of normal ace activity. ten minutes after e, ~ri-bfap metabolism had partially recovered to 52:l:10%, representing a 50-a:15% inhibition of control ace activity. from this data, the dissociation constant of e for coronary ace in vivo was estimated as 6.8x10 "4 sec "l. conclusions: we have demonstrated the feasibility of repeated, reproducible measures of coronary endothelium-bound ace activity and of its inhibition by e. this procedure is safe and can be used to study the role of ace in normal cardiac function and in card pathologies. objectives. primary pulmonary hypertension (pph) is a progressive fatal disease of unlmown origin, with median life expectancy of less than three years after diagnosis. the responsiveness of pulmonary hypertension to a variety of vasodilator agents led to the speculation that, concomitant with vascular renmdelling processes, persistent vasoconstriction is an important feature of the disease. long term use of ca-channel blockers and intravenous pgiz may improve mortality in certain populations of pph patients, but both of these treatments lack selectivity for tire lung vasculature. the aim of this study was to test the efficacy of aerosolised prostacyclin and its stable analogue, [loprost for selective pulmonary vasodilatation in pph. methods: in three patients with pph, we compared aerosolisation of prostaglandin iz (pgi2) and iloprost to a battery of vasodilatory agents (diltiazem, nifedipin, inhaled nitric oxide, intravenous pgiz). results: nebulisation of pgi2 and iloprost tumed out to be most favourable for achieving effective and selective pulmonary vasodilatation. pulmonary vascular resistance decreased from 1664 + 75 to 1054 -+ 93dyn*s*cm 4 (p<0.001) and pulmonary artery pressure from 63.3 + 3.1 to 528 + 3.4 mmhg (p < 0.05), cardiac output increased from 2.66 + 0.11 to 3.57 _+ 0.16 i/rain (p < 0.001), mixed venous oxygen saturation from 49.6 _+ 2.2 to 63.3 + 2.8 % (p < 0.001) and arterial oxygen saturation from 87.9 + 2.6 to 93.6 _+ 2.2 % (mean _+ sem of 7 trials in 3 patients). 5-month iloprost nebulisation in one patient (100 gg/day in six aerosol doses) demonstrated sustained efficacy of the vasodilator r~men. conclusion: aerosolation of pgi2 or its stable analogue may offer as new strategy for selective pulmonary vasodilatation in pph. endothelial adhesion molecules may play an important role in the pathogenesis of myocardial cell damage, and may contribute to the progression of heart failure. we measured the plasma soluble intercellular adhesion molecule-1 (sicam-1), vascular cell adhesion molecule-1 (svcam-1), and e-selecfin (selam-1) levels in 27 patients with acute myocardial infarction admitted within 6 hours after onset. peripheral venous plasma-samples were collected at the time of admission, 12, 24, 36, 48, and 72 hours after onset. plasma soluble adhesion molecule concentrations were determined by elisa. patients were divided into 3 groups as follows: group 1; killip's class (k) 1 and 2 without thrombolytie therapy, group 2; k 1 and 2 with thrombolytic therapy and group 3; k 3 and 4. both plasma sicam-1 and svcam-1 concentrations in group 2 and 3 were elevated rapidly and significantly and maintained at a high level during the first 3 days. plasma selam-1 level did not change in any of the groups. these results suggest that the adhesion molecules icam-1 and vcam-1 may play a role in the pathogenesis of myocardial reperfusion injury and may indicate its severity in myocardial infarction. objectives: nitric oxide (no) is known to exert cytotoxic and negative inotropic effects on cardiomyocytes. no synthase activity has been reported to be increased in infarcted area in animal model of myocardial infarction. these findings suggest that no may be an important regulator for myocardial damage and cardiac function after myocardial infarction. we measured plasma no27no3-(nox) levels and estimated serial changes in acute phase of myocardial infarction. methods: subjects were 15 patients admitted within 3 hours after onset. venous blood samples were collected at 3-hour intervals on the first day, 6-bour intervals on the 2nd day and 12-hour intervals on the 3rd day and 4 th days after onset. plasma nox concentrations were determined by griess method. results: the time course of the plasma nox levels (mea~+sem) displayed a tendency to gradually increase and to make a biphasic pattern with two peaks about 24 hours and 2-3 days after onset (basal level; 32.8_+4.9, first peak; 42.0!-_7.0, second peak;47.0+7.6 ram/l). plasma nox concentration was not influenced by the thrombolytic therapy, and nox values at the time of 60 hours after onset were significantly correlated with maximal plasma creatine kinase level (r=0.83, p<0.01). the levels of plasma nox in the early stage of myocardial infarction (from admission to the 4th day after onset) did not correlate significantly with the hemodynamic parameters (left ventricular ejection fraction, pulmonary capillary wedge pressure). conclusion: the early and late increase in no production after myocardial infarction may be implicated in the deterioration of myocardial contractility and induction of myocardial damage in the early phase of myocardial infarction. range 36-85) fullfilling the high risk criteria of shoemaker (colectomy 13, gastrectomy 10, pancreaticoduodenectomy 4, others 6). patients were admitted to the icu preoperatively. arterial and pulmonary artery catheters were inserted and hemodynamics and oxygen transport were measured at admission and after stabilization to predetermined physiological end points. patients were considered stable when ci >2.5 l/min/m 2, pcwp >10 mmhg, hb >100 g/l, sat2 >.94. objectives: evaluate the acute effects of 0,08 mg ipratropium bromide and 0,2 mg fenoterol (ibf) inhaled dose on pulmonary function in 17 nonsmocers (nb:m) and 14 smocers (s) with sever (new york heart association class ii-iii), stabile congestive heart failure(chf) and 12 healthy subjects. methods: pulmonary function tests were performed < 3h postprandial. the tests consisted el arterial blood gas aspiration followed by routine spirometry and pletismography, and single-breath gas analysis. after performance of these maneuvers, the patients was administred 4 puffs-ipratropium bromide (0,08rag) and fenoterol (0,2 rag). for 0,5 h, spirometry was repeated. results: in resting, pulmonary abnormalities observer in the s group were more severe then abnormalities observere in the nsm group. after treatment with ibf the improvement in pulmonary function was even more marked in patients who had smoked. the mean changes by forced expiratory volume in 1 second(eevt) was 8,1% (p<0,00t) improvement and 3,9% (p<0,ob), forced expiratory flow betwen 25% and 75% of the forced vital capacity (fef25.75) was 30,8% (p<0,001) and 23,6% (p<0,001) and maxamal voluntary ventilation (mw) was 21,7% (p<0,05) and 16,2% (p<o,05) improvement. the mean changes in normal subjects were mild (fev 1 increased by 3,4%,fef 25-75 by 14,5% and mvv by 6,3%). we observed no adverse heamodinamie conseevence and no arrthytmogenicity. conclusion." in patients with chf the airway response to ibf is highly significant. further study is needed to determine whether therapy with ibf will load to improvement quality of life in patients with chf with dearly documented venti/atory defects. compared with unilateral ima grafts, usage of bilateral ima grafts in cabg patients causes greater thoracic trauma and further reduces blood supply to the sternum and intercostal muscles. these effects may be associated with increased postoperative respiratory complications obiectives: to study the effects of bilateral ima grafts on postoperative respiratory morbidity in cabg patients. methods: two groups of patients undergoing cabg were prospectively studied: group 1 (n=13) received both imas as grafts and group 2 (n=12) received only the left ima. the two groups were matched for age, smoking habits, total number of grafts and preoperative ejection fraction. pao2/fio2 ratio was measured in all patients in the icu, at 30,120,240 minutes on mechanical ventilation (mv), at 30, 120, 240 minutes after extubation (ex) and on postoperative day (pod) 5. in addition, radiografic scores of atelectasis and pleural effusion were assessed postoperatively. results: group 1 had significantly longer bypass (101_+23 vs 70+15. p<0,001) and aortic cross clamp time (45+-19 vs 28+7, p=0,009). pao2/fi02 ratio was significantly higher in group 1 only at 30 minutes after extubation (table) . there was no difference in the incidence or severity of atelectasis or pleural effusion between the two groups. the duration of mv was longer in group 2 (13+_3 vs 9_+2, p=0,004), but the length of icu stay and hospitalization were similar. there was one case of pneumothorax in group 2 and one case of pulmonary infection in each group. after extubation 30 m[n 120 min 240min 30min* 120rain 240min pod 5 1' 342_+87 302_+92 369+87 278-+67 2664-79 294-+102 376_+76 2 319_+105 324_+89 320_+76 223-+52 257_+94 249_+82 400-+43 mean_+sd, *p=o, o03 conclusions: compared with unilateral ima grafts, the usage of bilateral ima grafts in cabg patients is not associated with increased postoperative respiratory morbidity. the diagnosis of rv ischaemia is difficult in the critical care setting. the purpose of this study was to determine the ability of rv endomyocardial and interstitial ph electrodes to detect rv ischaemia in an animal model of rv infarction produced by fight coronary artery ligation. we hypothesized that changes in transmural and interstitial ph would accompany the induced tissue hypoperfusion. rv interstitial (phint) and transmural endomyocardial ph (phendo) were measured in 10 adult anaesthetized pigs before and serially after coronary ligation. phendo and phint fell progressively and significantly following coronary occlusion. this was accompanied by ischaemic ekg changes. the absolute and relative rates of change were greater for phendo compared to phint, particularly after 4 minutes of ischaemia, ph was unchanged in control experiments when the electrode was placed within the fight atrial or ventricular chambers, as well as when coronary ligation was not performed. these data suggest that rv myocardial ph measurements reflect coronary ligation induced rv ischaemia, ph recorded from an electrode placed against the rv endomyocardial wall via a central vein was as useful as an interstitial measurement that required a thoracotomy. this newly described technique may be helpful in clinical settings where differentiation between ischaemic and nonisehaemic causes of rv dysfunction is important. centre. depts of cardiac surgery and anesthesiology, university of bari, italy. lung injury secondary to cardiopulmonary bypass (cpb) is well recognized. nevertheless, mechanical respiratory changes after cpb have been poorly investigated. aim of the study was to evaluate the effects of cpb on respiratory mechanics. elastance of the lung (est l), chest wall (est cw), and total respiratory system (est rs) were measured in 8 patients without previous pulmonary disease undergoing elective cardiac surgery. there were 6 males and 2 females; no pleurotomy was done. cpb was carried out with membrane oxygenator. mean cpb and cross clamping times were 69_.27 and 39• min. flow (pneumothacograph), airway opening and esophageal pressures (pressure transducers and esophageal balloon) were measured on patients sedated and paralysed. measurements were obtained before sternotomy, at the end of cpb after chest closure, four and seven hours later. est rs, est l and est cw, were measured by occluding the airways and the end of a tidal breath. before end cpb-4 h after 7 h after stemotomy closed chest cpb cpb estrs (cmh20/l) 17.7+4.9 19.94-5.6" 22.5_+5.8* 22.64-4.3* est cw " 6.7_+4.1 6.3• 6.61+4.09 6.5-+4.1 est l " 10.9--4.3 13.54-5.4" 15.9-_4.9" 16.1_+5.1" x + sd. * p < 0.05: anova vs "before sternotomy". our data show that cpb induces increase in est l, whereas est cw remains unchanged despite sternotomy. impairment in est l after cpb evolves within the first 4 hours and stabilizes 7 hours later. during the clinical course of human imunodeficiency virus infection (hiv), patients (lots) can develop congestive and dilated cardiomyopathy syndrome (dcm). the aim of our study was to analyze the prevalence of dcm among an hiv population, its clinical and echocardiographic changes under a long term follow-up and therapy with an ace-inhibitor agent (captopril). we prospectively studied 220 pts, 23% (50/220) developed dcm during the follow-up study. among this group 26% (13/50 pts) were in class i and ii and 64% (37/50 pts) in class iii and iv of the nyha classification. we divided this population in a acei+ (22 pts) and acei-(28 pts), according to the administration of captopril. we analyzed the following parameters: age, gender, race, ivda, type of hiv, cdc classification, number of t4 and t8 type cell population and its t4/t8 ratio, therapeutic with acei, type and number of opportunist infections, mycobacteriosis and neoplasm's. we analyzed several echocardiographic parameters, which included the initial (i), final (f) and variation (a) of the lv internal diastolic (lvdd/mm) and systolic (lvsdimm) diameters, lv % of fractional shortening (lv%fs/%), ivs and posterior wall thickness and lv mass (lvm/g). the two groups differed significantly in the following parameters: left ventricular functional indices of patients (pts) with iscjhemic dilated cardiomyopathy (idcm) have a critical value in terms of clinical prognosis of this disease. three-dimensional echocardiography (3-de) is a recently developed method that gives a better evaluation of lv morphology and function. the purpose of our prospective study was to assess the clinical applicability of this new 3-de method in a population with ischemic dcm diagnosis and calculate its lv indices of global and regional function. we studied a group of 16 idcm pts, mean age 58_+11 yrs, 70% male gender, using a combined 3-de tee approach. all pts were in class ill/iv of the nyha classification. first, ekg gated images were acquired through a mechanical pullback of the tee probe, and each set of data was transferred to a conventional ibm-pc system using a dedicated 3-de software. computer processing and analysis of the tee images led to a 3-de reconstruction matrix and off-line calculation of several lv global and regional indices. in all idcm lots the lv cavity was clearly reconstructed and end-systole and diastole in several orthogonat projections, out in multiple planes and its function quantitated using 3-de derived indices. mean values of 3-de lvesv=149-+20 ml, lvedv=197_+32 ml, lvstv=48_+25 ml and lvef=24-+5% were obtained. for all these 3-de parameters, good correlations were observed with 2-de lv measurements obtained through biplane tee analysis of the lv cavity (r>.70; p<.01) as well as regional analysis of sequential 3-de cut planes. conclusion: in our group of patients with the diagnosis of ischemic dilated cardiomyopathy, this new 3-de method could be applied. our results show that this method allows a better assessment of the lv morphology and spatial geometry, with the calculation of global and regional indices with critical clinical and prognostic value in this particular cardiovascular pathology. simultaneous left atrial (la) and left ventricle (lv) inflow analysis assessed by pulsed doppler tee illustrate the loading conditions and reflect the hemodynamics of the left heart. we performed a prospective tee pulsed doppler study with recordings of the transmitral lv filling and pulmonary venous (pv) flow drainage in a group of patients with dilated cardiomyopathy (dcm). a group of 23 dcm patients, mean age 57_+11 yrs, 74% male were studied. this population was divided according to tee severe lv dysfunction (group slvd+ 62% pts; group slvd-38% pts) in each pt we measured the peak velocities (vel/m/sec) and time velocity integrals (vti/m) of the transmitral early (e) and late (a) filing waves, the vel and vti of the pv systolic (s), diastolic (d) and atrial contraction (c) reversal flows. 3-de tee evaluation of the lved, lves, lvst volumes and lvef were obtained. we calculated other parameters, such as e/a, s/d and a/c ratios and the sum of c+a vel, that refelect la systolic function and lv compliance. 19+5 305-_8 0.02 simultaneous and quantitative analytical approach of the pulmonary venous and transmitral flows and ventricular volumes improve the non invasive assessment and understanding of left ventricular diastolic function and cardiac performance in dilated cardiomyopathy patients. objectives : to assess the hemodynamic effects of fluid loading (fl) in acute circulatory failure (acf) due to acute massive pulmonary embolism. methods : hemodynamic measurements (fast-response thermistor pulmonary artery catheter) were performed at baseline (baseline) and after a rapid fluid loading with 250 (fl250) and 500 (fl 500) ml of dextl'an 40 (rhemacrodex| in 12 patients free of previous cardiopulmonary disease (66 • 3 yrs) with acf (ci < 2.5 l/rain/m2) due to angiographicalty proven mpe (miller score > 21) . results : are expressed as mean _+ sem and compared by anova. a significant negative correlation (r = 0.83) was observed between baseline rvedv[ and the effects of fl on ci. such correlation was not observed between baseline rap and the fl induced increased in ci. conclusion : fusibmificantly increases ci in acf due to mpe. however, the simultaneous decrease of arterial 02 content due to hemodilution, limits the benefits expected from improved ci on peripheral oxygenation. obiective: to examine the hemodynamic effects of external positive endexpiratory pressure (peep) on right ventricular (rv) function in acute respiratory failure (arf) patients. methods: incremental levels of peep (0-5-10-15 cmh20) were applied and rv hemodynamics were studied by a swan-ganz catheter with a fast response thermistor for right ventrieular ejection fraction (rvef) measurement in 20 mechanically ventilated arf patients (lis = 2.6 ~-0.45 sd). according to the response to peep 15, two groups of patients were defined: group a (9 pts.) with unchanged or increased rv end diastolic volume index (rvedvi) and group b (h pts) with decreased rvedvi. results: in the whole sample cardiac index (ci) and stroke index (sj) decreased at all levels of peep, while rvedvi , rv end systolic volume index (rvesvi) and rvef remained anchange d. at zeep the hemodynamic parameters of the two groups did not differ. in group a, ci decreased at peep5, rvef decreased at peep10 (~0.8%)~ rvesvi increased only at peep15 (+21.5%) and rvedv[ reded unchanged. in group b, ci and rvedvi started to decrease at peep5, 'rvesvi decreased only at peep15 (-21.4%), anf rvef was unchanged. individual behaviors of the hemodynamic parameters at the 4 levels& peep were studied. rvedvi and ci were significantly correlated in 10 out of:l 1 patients in group b, and in no patient of group a. on the contrary, mpap and rvesvi were significantly correlated in 5 out of 9 patients in group a, and in no patient of group b. the slope of the relationship between rvedvi and rv stroke work index (rvswi) expresses rv myocardial performance. this relationship was significant (no change in rv contractitity)in 8 patients of group b and in 2 patients of group a. in some patients of group a, increments of peep shifted the rvswi/rvedvi ratio rightward inthe plot (rv function decrease). conclusions: in arf patients peep causes more often a preload decrease with unclmnged rv conctraetility. on the contrary, the finding of increased rv volumes during the application of peep is related to a decrease in rv myocardial performance. thus, these data suggest that application of peep might be considered as a stress test to assess rv function. right introduction: after heart transplant (ht), the right ventricle can be subject to an acute pressure overload, especially in cases where there is a preexisting severe pulmonary hypertension. this provokes right ventricular failure and, occasionally, circulatory collapse in intensive care unit. desire the advances that have been made in systems for preserving the donor heart and in post-surgical management, we have failed in our attempts to totally avoid this problem. the right ventricular function, although it usually remains within tolerable limits in these patients during the post surgery period, represents a factor which limits the results achievable in clinical transplant programmes. objectives: to determine the maximum tolerance of the right ventricle (mxtrv) when faced with acute pressure overload. to study the function of both ventricles of the healthy heart (donor) when faced with different degrees of pulmonary hypertension. to detect possible interactions between the ventricles in the absence of the pericardium to approximate the experimental model to the clinical model of ht. materials and methods: the pulmonary artery is progressively constrained in an experimental model until biventricniar failure is detected. this experiment is performed in two diffferent situations: with and without pericardial integrity. results: when pericardial integrity is maintained the mxtrv faced with a pressure overload is 73.2 + 8.56 nun hg. when this pressure is exceeded there is a circulatory collapse with a sharp fall in the cardiac output and in the aortic pressure. however, when pericardectomy is performed (model similar to ht), only 52 • 6.71 nun hg is tolerated (p < 0.01). conclusions: with the pericardium open, as in heart transplant, the maximum pressure that the right ventricle can support is significantly less than with the pericardium closed. the pericardium has a positive effect in protecting the systolic ventricular interaction. it is, therefore, advisable to close the pericardium after heart transplant. jb prrez-bernal, a ordrfiez, a. heroandez, jm borrego, map camacho, c cruz, mac s~nchez, j monterrubio, c garcia, e. gonz~lez. hospital uulversitario " virgen del rocio ". sevilla. espaiqa. introduction: nowadays cardiomyoplasty isused incases of cardiac insufficiency as an alternative to cardiac transplant. after surgery the patients show a noteable improvement with the aid of this "biological circulatory assistance". some researchers suspect that the improvement could also be due to the formation of new blood vessels from the muscle that wraps the heart, nourishing the ischemic myocardium. objectives: our cardiovascular research group has proposed as an objective, the detection of any possible myocardial neovascularization through the muscle used for cardiomyoplasty. in the case that there are new blood vessels to the diseased myocardium through the wide dorsal muscle in which it is wrapped and which aids it mechanically, it would be possible to confirm the worldng hypothesis that cardiomyoplasty not only improves the cardiocirculatory funcfinn mechanically but also by facilitating a better blood flow to the ischemic myocardium. materials and methods: the cardiomyoplasty technique is described using an experimental model of myocardial ischemia. the vascular cast is achieved by injecting methacrylate simulataneously into both the coronary tree and the wide dorsal muscle, in five experiments the connections between the coronary vascular system and the vascular structure of the wide dorsal muscle are demonstrated, conclusions: we have demonstrated that cardiomyoplasty, as well as improving ventricular function, favours the revascularization of the myocardium. cardiomyoplasty could be indicated for cases of ischemic cardiopathy in patients in whom it is not possible to perform direct revacularization using conventional methods. a the therapeutic cardiological manouevres necessary in cases of ischeima reperfusion have increased considerably: fibrinolysis, transluminal angioplasty, coronary revascnlarization surgery and cardiac transplant. the appearance of a specific pathology ht acute reperfusion has been related to free oxygen radicals (for) generated by oxidative damage. objectives: to evaluate the appearance of for during a conti-olled process of ischemia-reperfusion in an experimental biological model and compare it with that in clinical cases. materials and methods: transitory cardiac ischemia was performed in five rabbits by reversible surgical ligation of the descending anterior coronary artery. after 15 minutes coronary reperfusion was performed. blood samples were taken in the basal situation, at the end of ischemia and at 5, 10 and 15 minutes after the start of reperfusion. malondialdehyde (mda) was measured to evaluate the degree of lipid peroxidation (oxidative damage to the membrane). in ten patients undergoing conventional cardiac surgery the production of for was measured after aortic clamping. results: we observed that after 5 minutes of reperfusion there was a highly significant increase (p < 0.001) in the mda values (mean =2.00 /zmols/l). these returned to basal levels after 10 and 15 minutes of reperfusion. conclusions: an "explosion" of oxygen free radicals was detected very quicldy, just a few minutes after post-ischemia reperfusion. thus, if antioxidant agents are to be used to reduce the toxic effects of the for, these will ordy have a therapeutic effect if they are administered in the early phases of reperfusion. introduction: aortic connterpulsation is a ventricular assistance widely used in intensive care units in patients with cardiogenic shock as a provisional ventricular assistance. paraaortic or external aortic counterpnlsation is been investigated as a definitive veutricular assistance in those cases of terminal congestive heart failure and when heart transplantation is counterindicated. aims: to assess the haemodynamic effects of an aortomyoplasty in a biological model of congestive heart failure. material and method: as specimens, we used 10 "large white" pigs. mean weight was 22 kg. after the administration of conventional anaesthesia, dissection of the ladssimns dorsi muscle was performed on the samples at the laboratory of experimental surgery of our hospital. then we performed a thoracotomy at the level of the fourth intercostal space to reach the thoracic aorta. the aorta is dissecated 7 centimetres from the exit of the subclavia and it is wrapped by the dissecated muscle. a cardiomyostimulator is provided in order to allow the synchronization between the diastole and the muscle contraction. the model of heart failure was provoked using verapamil plus propanolol i.v.. results: a significant increase of the aortic diastolic pressures and a significant decrease of the left ventricle telediastolic pressures were observed. this improvement in the parameters (dpti/tti) implies an increase of the coronary perfusion in a model of heart failure. conclusions: using the external aortic counterpulsation, the aortomyoplasty improves the coronary perfnsion and the heart efficiency in patients with heart failure in whom no conventional therapeutic action is possible. the permanent character of the paraaortic counterpulsation is it main advantage. the appearance of specific pathologies as a resuk of myocardial reperfasion has been related to the oxidative damage secondary to the release of oxygen derived free radicals (ofr). during the myocardial ischemia induced during heart surgery with extraeorporeal circulation, severalsubproducts of the oxygen are produced that shall cause toxic effects after the reperfusion which could be counteracted by the physiological antioxidant systems and/or provided by the medication. aims: to asses the ofr during heart surgery. to check whether an antioxidant treatment administered in the preoperative period make decrease the levels of ofr before and after the myocardial reperfusion and to verify whether its administration have any beneficial effect on the intra and extraoperative management. material and method: the study comprehends 20 patients studied as two groups of 10 individuals each (a and b). all patients underwent conventional heart surgery of valvniar substitmion or myocardial revaseularization. group a patients were administered 400 rag/8 hours of vitamin e (tocopherol acetate) 72 hours prior to the intervention as antioxidant treatment. group b patient were not administered vitamin e. we assessed the quantity of malondialdehido (mda) to assess the degree of lipidic peroxidation or oxidative damage of the membrane during the myocardial ischemia and 15 nm after the reperfusion. conclusion: patients who underwent heart surgery and were treated with tecopherol acetate in the preoperative period presented levels of rlo significantly lower than those who were not administered the drug, both during the intraoperative period and after myocardial reperfusion. we detected in these patients a need for antiarrhythmicals and pharmacoiogical support with catecholaminas, although not significant, both in the introaperative period and the immediate postoperative period. recommendations for the treatment of pulmonary embolism (pe) in the presence of right atrial thrombus (at) are conflicting. because of a significantly higher mortality rate due to fulminam or recurrent pe, there is a necessity to treat patients (pts) with mobile type a thrombi compared to pts with adherent type b thrombi. therapeutic strategies include anticoagulation, thrombolysis (t) or surgical thrombembolectomy. combination thrombolysis (cot), predominantly used for the treatment of acute myocardial infarction proved to prevent reocclusion of the infarct related artery at a comparable rate of hemorrhagia. benefit has been related to the alteration of hemostatic proteins by non-fibrinspecific thrombolytic s. administration of cot in pe has been performed sporadically. in the present case, a 55-year old male with no history of prior cardiovascular disease developed acute dyspnea which was related to pe in the presence of deep vein thrombosis of the left femoral vein. therapeutic anticoagulation was installed for a couple of days until there were several bouts of deterioration. biplane transesophageal echocardiography (tee) was performed and revealed a large, wormlike, hypermobile thrombus within the right atrium. computer tomography (ct) of the chest detected a saddle embolus in the bifurcation of the pulmonary tmnk almost occluding the entire left pulmonary artery (pa) and parts of the right pat consisted of 100 mg frontloaded rt-pa and the subsequent continuous administration of urokinase in a dosis of 200.000 u/hr for 24 hrs followed by therapeutic anticoagulation. symptoms, blood gases and ecg improved steadily during infusion, no adverse effects, i.e. minor or major hemorragia were registered. follow-up ct promptly after termination of t showed almost complete resolution of the saddle embelus, whereas tee showed complete dissolution of the at. ' finally, the patient was switched to oral anticoagulants and had an uneventful clinical course until he was discharged. conclusion: in the present case, cot was effective for the treatment of a complicated pe without any adverse effect. introduction: nowadays we can assist hearts with problems of insufficiency by techniques other than transplant. many researchers believe that the best way of assisting insufficient heart muscle is with another muscle from the patient. this technique of ventficular assistance is known as cardiomyoplasty. we describe the surgical technique of cardiomyoplasty using a biological model. the transformed skeletal muscle is transferred to the thoracic cavity where it wraps the heart and assists it. the choice and preparation of this muscle is currently under investigation. our group has focussed on the development of protocols for electrical stimulation to transform a skeletal muscle into a muscle which resists fatigue and which is functionally similar to the myocardium. we detect the optimum time at which this muscle has been transformed, by studying the transmembrane action potentials using intracellular electrodes. when the action potential of the trained muscle behaves like cardiac muscle we consider it ready for cardiomyoplasty. conclusions: cardiomyoplasty is an alternative surgical technique to cardiac transplant, which has a great future in the treatment of patients with advanced cardiac insufficiency. we describe methodology which, by intracellular techniques, allows selection of the optimum moment of transformation of a skeletal muscle trained to perform,like cardiac muscle, without suffering fatigue. purulent pericarditis is a rare disease. its treatment associate systemic antibiotics and drainage of the pericardium. we report a ease of purulent constrictive pericarditis in which intraperieardial fibrinolysis was use. a 38 years old patient admitted in our icu for a constrictive pericarditis as a complication of a purulent pericarditis diagnosed seventeen days before. he had also an aehalasia and the o'esogastric endoscopy had found an oesophageal neoplasm. a fistula was not seen, indeed pericardial of flora was the same that oropharyngeal. hemodynamie and echographic study had confirmed a constrictive pericarditis. because of the poor state of the patient an intraperieardial fibrinolysis was prescribed (250.000 ui of streptokinase on days 23, 25, 27, 29). fluid drainage was improved and cardiac output was also improved (day 23 : 2.53 1.min "i, day 27 : 3.58 l.min'l). no change ofhemostasis was noted. a pericardeetomy and an oesophagectomy were performed after 37 days of evolution. eighteen months latter the patient was still alive. intraperieardial fibrinolysis seems an interesting therapeutic way if rapidly prescribed in the purulent pericarditis course. the decrease in the systolic pressure following a mechanical breath, termed ddown (delta down), has been shown to be a sensitive indicator of preload (1,2) . however, the clinical use of this method necessitates the introduction of a short apnea. we have therefore developed a respiratory systolic variation test (rsvt) which obviates the need for apnea. the test is based on the delivery of 4 successive breaths of increasing magnitude (5, 10, 15, and 20 ml/kg). a line of best fit is drawn between the 4 minimal systolic values (one after each breath) and the downslope calculated as the decrease in blond pressure for each increase in airway pressure ( mmhg / cmh20). in 14 mechanically ventilated patients the rsvt was performed during controlled mechanical ventilation under sedation. the test was repeated after the administration of 7 ml/kg of plasma expander. the initial mean downslope of the rsvt was -.40 + .40 mmhg/cmh20. following volume loading the downslope decreased to -.23 + .44 (ns). at the same time, cardiac output (co) increased by .96 + 1.2 l/min (p<.02), end-diastolic area (determined by tee) increased from 18.5 + 6.9 to 20.3 + 7.1 cm2 (ns), and paop increased from 12 + 8 to 17 + 9 mmhg ( p < .001). the preinfusion downslope value of the rsvt correlated significantly with the increase in the co (r = .81) and the eda (r = .70). methods: an expert system has been constructed running on a multimedia computer with the two objectives in mind, viz training of inexperienced staff, and protocol guidance with treatment regimes for all staff. the system is based on experience gained from two previous systems, the one for dealing with acid-base and electrolyte problems in icu patients; the second for stabilisation of patients with heart rate and blood pressure abnormalities. the training section takes the form of a stage-by-stage account of the insertion of the pac and displays of correct waveforms, coupled with indications of possible incorrect placements, and guidance when failing to achieve the perfect positioning. the treatment protocol section extends an existing protocol for correcting abnormalities in heart-rate and blood-pressure, and now takes account of all the indices as measured by the pac. the system will suggest treatment to correct such things as abnormal wedge pressures concomitant with parameter values throughout the rest of the cardiovascular system. the type of patient eg post-operative cardiothoracic or i. c. u. trauma, will be taken into account when recognising abnormal parameter values and when prescribing treatment. results: a working system which will be improved by the finetuning being carried out. the results and lessons learnt will be presented at the conference. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < 70 mmhg) or the requirement for a noradrenaline (na) infusion ~ 0.1 9g/kg/ rain with a map --< 90 mmhg. cardiovascular support was limited to na + dobutamine (db). 546c88 was given for up to 8 h at a fixed dose-rate of either 1, 2.5, 5, 10 or 20 mg/kg/h iv. during 546c88 infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above 70 mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = 0); at i h from the start of treatment (t -1); and at the end of treatment (t -8) with 546c88. conclusions: 546c88 does not appear to increase mpap or worsen pulmonary gas exchange in patients with septic shock, when given by infusion for up to 8 h. 546c88 is a novel vasoactive agent for the treatment of septic shock which will now he evaluated in a randomised, placebo-controlled safety and efficacy study. objectives : to compare cardiac output (q) data obtained for thermal indicators in pulmonary artery (qtpa) and aorta (qtao) and for the stable isotope 2hzo in aorta (q2v~2o) with indocyanine green (icg) in aorta (qicg) as reference. methods : an indicator solution of ice cold h20 (9.4 ml), 2h20 (0.6 ml) and icg (10 mg) was injected as bolus via the injection port of a swan-ganz catheter. qlco and qzmo was measured using a dual optical system (penn lab instruments, philadephia, pa, usa). qtpa and qtao was measured using a in contrast to the recoveries of thermal indicator in pa and 2h20 in aorta the :~covery of thermal indicator in aorta was significantly increased in group ii (n= 18 boluses) over group i (n=18 boluses) (1.3<-0.3 vs. 1.0+-0.1, p=0.04). conclusions: the "overrecovery" of thermal indicator in aorta is in agreement with " biscks deconvolution study (i) and results in erroneous values for q. the most pausible explanation is the distortion of the thermal curve caused by the slow response time of the thermal detection instrument as shown by ganz (2) objectives: to compare data obtained with the double indicator dilution method using indocyanine green (icg) and the stable isotope 2h20 for the estimation of extravascular lung water (evlw2hzo) to gravimetriu lungwater data (evlwg~). methods: an indicator solution oflcg (10 rag) and 2h20 (0.6 ml) was injected as bolus via the injection port of a swan-ganz catheter. dilution curves for icg and zh20 was registered in aorta with a dual optical system (penn lab instruments, philadephia, pa, usa). cardiac output and mean tranist time was measured for both tracers (qico, tlco, q2n2o, t202o) (1). data analysis: evlwg~av was reference for evlwzhzo calculated as q2hzo times the difference in mean transit time between t2nzo and rico (atm2n). as reference for atzn2o evlwg~,v was divided by q~cg to obtain atg~,. a reference distribution volume for 2h20 was calculated as the sum of central blood volume and evlwg=v. 54 boluses were administrated in a group (i) of 6 anaesthetized pulmonary healthy sheep while q was altered. another 18 boluses were administrated in a group (ii) of 6 anaesthetized sheep with stable oleic acid induced pulmonary oedema. evlwg~v measurement was performed postmortem. 2 results: for 72 boluses h20 parameters were not significantly different from their respective reference parameter: at2vao 5.3+_3.5 s vs. atg~, 5.4+3.2 s, evlwzh2o 332-+195 ml vs. evlwg~,~ 338+169 ml. in group i the ratio between 2hzo parameters and respective reference parameters (n=54) were independent of qlco from 1.4 to 7.1 l/min. obiectives: to assess the thermo dye method using indocyanine green (icg) and thermal indicator for the estimation of lung water (evlwt). methods: ice cold indicator solution of icg (10 mg) in water (10 ml the aim of the study was to assess left and right ventricular function in the early postoperative period after orthotopic heart transplantation to elaborate therapeutic approaches of heart function abnormalities correction. mathefial and methods. haemodynamic monitoring data of twenty one patients ( 19 men, 2 women ) age from 18 to 56 were studied. cardiac output, pulmonary artery, right atrium and pulmonary wedged pressure were measured with swan-gans catheter. central haemodynamic indices were calculated with the help of computer-based monitoring system. relations of ventricular stroke work index to it's end-diastolic pressure were used for ventficular function assessment. results. in most cases right ventricular disfunction was the main problem. isolated fight ventficular failure with high pulmonary vascular resistance (pvr) was observed in 24% (5pts), without high pvr-in 43% opts) and with left ventricular failure-in 33% (7pts). one of the most important reasons for fight ventricular failure was the time of heart ischemia more than 90min, which is of great importance in the ease of distance harvesting. the most effective treatment for cardiac failure was combination of dobutamine with i8oprotherenol, atrial pacing and vasodilatators in case of right ventfieular disfunction. all cases with isolated right ventricular failure were treated sucsessfully. biventricular heart failure was a sighn of bad prognosis and the reason of death in 2 cases. conclusion. right ventfieular disfunetion is the main problem during transplanted heart adaptation in the early postoperative period. optimal therapeutic management of cardiac disfunction includes infusion of dobutamine in combination with isoprotherenol, atrial pacing and vasodilatators. cardiology-department of clinical centre-kragujevac institution for occupational health "zastava"-kragujevac, sr yugoslavia the aim of the investigate is analisis five years survives patients with a.i.m.in dependence of locality and risk-factors. we ana~sed-39~-pat~e~ts (273 males and 118 woman), average 59,8 years. for statistic evaluation we used life-table slstem in oder to estimate prognostic determinants. patients with respkatory muscle paralysis may benefit from respiratory assistance by abdomino-diaphragmatie pneumatic belt. we used a non invasive technique, m-mode sonography, to assess the effect of this device on diaphragmatic excursion. we measured the amplitude of right diaphragm motion in seven patients with duehenne muscular dysl~ophy in supine position with various thoracic posture (0 ~ 45 ~ 75~ without and during pneumatic belt respiratory assistance. without respiratory assistance, the thoracic posture had no significant consequence on the amplitude of diapttragm motion, either in quiet or deep breathing. the pneumatic belt increased the diaphragm motion amplitude from 7.1 +__ 3.6 mm to 17.71 +_ 5.5 ram (p = 0.009) at 45 ~ tilt angle, and from 8.4 + 3.8 mm to 19.3 + 5.8 mm (p = 0.009) at 75" tilt angle. the tidal volume increased from 211 + 78 to 373 + 99 rut a145* tilt angle, and from 229 + 78 to 447 + 143 ml at 75* tilt angle (p = 0.009). two patients could not bear the horizontal position (0' tilt). in the five other patients, the pneumatic belt increased but not significantly the amplitude of diaphragm motion (9.2 + 4.9 mm to 15.5 + 7.3 ram). after an overnight respiratory assistance, pao2 increased from 66.4 +_. 8.7 to 73 + 10.1 mmhg (17 = 0.015), sao2 increased from 91.1 + 2.5 % to 91.9 +_. 3 % (p = 0.015), and paco2 decreased from 52 + 6.4 to 46.4 +_. 4 mmhg (p = 0.015) according to the ventilatory pattern result, m-mode sonography allows to measure non invasively the improvement of diaphragm kinetics obtained by pneumatic belt respiratory assistance, and may be helpful for its adjustment. objective: to study the effect of flow triggering (flow sensitivity 1 and 5 l/min) vs pressure triggering (-lcmh20) on inspiratory effort during pressure support ventilation (psv) and assited/controlled mode (a/c) in 8 stable copd patients non-invasively ventilated with a full face mask. methods: the patients were studied during randomized 15 min. runs using a bird 8400 st ventilator at zero peep (zeep). trigger values for pressure (-lcmh20) and flow (1 l/rain) were the lowest allowed by this ventilator. the transdiaphragmatic pressure time product per breath (ptpdi), dynamic intrinsic peep (peepi,dyn), maximal airway pressure drop during inspiration (apaw) andl ventilatory variables (ti,te,ttot,rr,vt and minute ventilation) were measured. results: no major problems due to airleaks or to auto-triggeriffg phenomena were observed in the patients, so that all of them were able to perform all the protocol runs. minute ventilation and respiratory pattern were not different using the two triggering systems. the ptpdi was significantly higher during both psv (10.6+6.8 cmh:0 x sec) and a/c (10.1+2.5) with pressure triggering, as respect to psv (8.9+7.5, p<0.02) and a/c (5.8+4.4, p<0.001) with flow triggering (1 l!m). no differences were observed between 1 and 5 l/min flow triggers. apaw was also significantly larger during pressure triggering; peepi,dyn was reduced during flow triggering being 5.5+1.5 cmh20 (psv flow trigger) vs 8.1+1.5 (psv pressure trigger) and 4.4+_1.3 (a/c flow trigger) vs'f~5+l (atc pressure trigger). conclusions: in stable copd patients non-invasively ventilated, flow triggering reduces the respiratory effort during both psv and aic mode as compared to pressure triggering. this may be partly due to a decrease in peepi,dyn using a flow-by system. objective. cardiac output is higher during alternating ventilation (av) (i.e. differential ventilation of the lungs with a phase shift of half a ventilatory cycle) than during synchronous ventilation (sv) of both lungs 1 . we verified the hypothesis that the higher cardiac output depended on a lower central venous pressure and intrathoracic pressure, due to a lower mean lung volume, which we attributed to part of the expansion of the inflated lung at the expense of the expiring, opposite lung 2. we studied this interaction between the lungs during one-sided inflation, which we called cross-talk. method. in 6 anaesthetized and paralyzed piglets we applied short periods (30 s) of one-sided ventilation (10 breaths per rain, bpm), while the other lung was open to the ambient air. the air flow into the non-ventilated lung during expiration of the ventilated lung was integrated to volume. we studied 1-to-r and r-to-i cross-talk at ventilatory rates of 10, 15 and 20 bpm. the amount of cross-talk was the volume displacement in the non-ventilated lung. results. during 10 bpm the r-to-i crosstalk was 23 _+ 4.7 % (mean +__ sd) of the tidal volume to the right lung and the 1-to-r crosstalk 31 _ 6.3 % of the left tidal volume. both values increased at 20 bpm to 30 _ 4.1% (p < 0.05) and 39 _ 7.7 % (p < 0.01) respectively. the values at 15 bpm were in between., conclusion. we concluded that the lower mean lung volume and lower thoracic expansion during av compared to sv depends on partial expansion of the inflated lung into the non-inflated lung, resulting in a lower mean intrathoracic pressure as the main reason for the higher cardiac output during av. obiective: natural surfactant given for rds in premature infants leads to a rapid improvement in oxygenation, but lung compliance did not improve in most studies. however, acute effects on lung mechanics during and immediately after surfactant administration have not been studied before. methods: a total of 13 administrations of bovine surfactant in recommended doses was given via a small catheter into the distal endotracheal tube either as a bolus (n = 8) or as a slow infusion (n = 5) in 10 infants with established rds. static compliance (c), resistance (r) and time constant (tc = cxr) of the lung were measured every 3 minutes with a lung function cart (sensormedics 2600) without interrupting ventilation. 3 infants receiving synthetic surfactant were studied as controls. results: after surfactant as a bolus or during infusion c first decreased but then increased, whereas r increased immediately with great fluctuations but did not return to baseline. this pattern was more pronounced in infusion than in bolus administration. change of c and r varied greatly in the individual case, maximum c was > 400 %, maximum r > 700 % of baseline value. retreatment was followed by an increase in r in all 3 patients, but c increased only in the one who was responder. patients receiving synthetic surfactant had no change of c or r and were non-responders. ob~i31ctives= acute lung injury (ali} sometimes induces severe hypoxernla which may be refractory to conventional modes of mechanical ventilation (mv). the elm of this study was to observe some cardio-pulmonary effects of an alternative method of ventilatory management of severe ali. five patients with severe ali (murray scores >3) requiring mv were studied. protocol inclusion was considered when a control-mode of mv (with a pzo~=l.0 and a peep level <15 cme=o} was not able to get either a p.ojf=o= ratio >55 or a s.o= >85%. patients were sedated, paralyzed, and a ventilator (serve 900c) was used for pressuz'e-control ventilation (pcv). fio= was maintained at 1.0 and peep removed. continuous gas flow (250• ml/kg] was humidified and jet delivered through a tube (7 ram id, 18 ml capacity, 0.09 ml/cm h=o compllancel ended in a nozzle (0.8 mm is) attached to the endotracheal tube connector. a thermodilution flcw-dlrected catheter was inserted in pulmonary artery. following variables were recorded 15 minutes before and after protocol started: tidal volume (vt), minute ventilation (vz), intratracheal pressures (p~w), wedge pulmonary artery pressure (wp), central venous pressure (cvp), mean arterial pressure (map), cardiac index (ci), arterial and mixed venous oxyhemoglobin saturation (sao=, svoa) , oxygen delivery (do~) , oxygen consumption (vo2) , intrapulmonary shunting (q./qt) , and oxygen extraction ratio (ero). this observation suggests that hfpv could allow to ventilate at lower fin2 and improve blood oxygenation during the acute phase after inhalation injury reducing toxicity risk related to high fin2. further studies are necessary to confima these results and evaluate the possible implications on mortality alter smoke inhalation and for other icu pts. objectives: to design a system for volume controlled high frequency ventilation (hfv) and to estimate the dependence of the tidal volume (vt) on frequency (f) in normocapnic ventilation in rats at frequencies 2 -25 hz. methods: a new system for volume controlled hfv was devised consisting of the generator of the constant flow during inspirium and the constant pressure during expirium. the ventilator allows ventilation at frequencies 2 -25 hz with the relative inspiratory time (ti) 0.2 -0.8. the airway pressure was measured at the proximal port of tracheostomic cannula , at the same site inspiratory and expiratory flow was measured using modified lilly-type of pressure-differential flow sensor. non-linearity of flow sensor was compensated on line by derived equation based on calibration at static and dynamic conditions. flow and pressure data were evaluated on line using original software. value of the positive end expiratory pressure (peep) was serve-regulated by analogous feed-back. in animal experiments white wistar rats (400-430 g) narcotized with ketamine/xylazine with cannulated carotid and femoral arteries were kept at the rectal temperature 37~ the arterial pressure was monitored. after traeheotomy the metal cannula (2mm [.d.) was inserted, animals were curarized and ventilated at the following condition: peep = 0.1 kpa, ti = 0.5. the dead space of ventilator including canula was 0.45 ml. the initial frequency was 2hz and 10 rain after each change of the ventitatory regimen the blood gases analysis was performed. the frequency was changed according to the following schedule : 2 hz-->4 hz-->8 hz-->4 hz-->16 hz-->4 hz--~25 hz-->4 hz. vt for each frequency was regulated to maintain normocapnie ventilation with arterial pco2 = 40 + 2 mm hg. the arterial po2 was always above 70 mm hg. results: for normocapnie ventilation in rats the following tidal volumes vt [ ml/kg] were found : vt1 = 5.91 --+ 0.30 ml/kg for ft = 2 hz, vt 2 = 4.31 +0.12 mukg for fz =4 hz, vt3 =3.30 +_0.27 ml/kg forf3 = 8 hz, vm4=2.61+0.08ml/kg forf4=16hz andvmt= 2.18 + 0.13 mukg for fs = 25 hz (presented as mean values _+ s.d., n = 6 ). the regression analysis using the mean values resulted in the equation for normocapnic vt in rats in our experiments : vtn = 37. 5 * f-e.30 . conclusions: the described system allowing ventilation in a wide frequency range 2 -25 hz with accurate measurements of airway pressures and vt might be useful for optimisation of artificial ventilation in new-barns with different lung pathologies. supported by grants iga mz cr nr 1448-3 and gacr nr 305. s122 intensive care unit. university. hospital of south manchester, uk. methods: measurements were conducted on 6 ventilated patients (puritan bennett 7200ac with metabolic monitor pb 7250 set to measure end tidal co2). all measurements were repeated with the patient stabilised at 5cm. 10cm and 15cm peep. inclusion criteria were: 1) haemedynamic stab(l( .ty for 1 hr; 2) pulmonad" anon" flotation catheter in situ: 3) volume control ventilation with plateau of 0.5s: 4) fio2~ > 11.6 to maintain pao~. > 10 kpa with 5em peep: 5) qs/ot > 20%; 6) pao2/fio2 ratio <150. measured variab!es included: r162 minute volume: plateau ainvay pressure: applied and intrinsic peep: fractional end tidal co2; arterial and mixed venous blood gases and hacmod).ttamic variables. results: statistical analysis was performed using repeated measures anova. significant decreases in cardiac index (ch p<0.01), compliance (p<t).05) and o,'gcn deliver, index (do2, p<0.02) occurred with increasing peep (table 1) . no other variable showed any significant change. methods : all consecutive patients orally intubated in the micu between january to april 1995 were studied. etts were positioned according to the reference marks mentioned above (measurements from upper incisors). the position of the ett tip in relation to the carina was measured on the chest radiograph done with the patient's head in neutral position. results : a total of 59 men and 46 women were studied (n= 105). the mean distance of ett tip to carina was 4.1 cm. using the reference markings, 29 cases (27.6%) had the ett tip < 3 cm from the carina and 5 cases (4.8%) > 7 cm. one case resulted in an endobronchial intubation. the mean height of all patients were 167 cm (153-187) for males and 155 cm (140-170) for females. of the patients with ett tip < 3 cm from carina, the mean height was 163 cm and 151 cm respectively. ~2onclusion : adopting the above quoted reference marks did not result in ideal positioning of the ett in a significant proportion of cases (32.4%). we postulate that [s because our asian population is generally shorter than those in previous studies. objectives: to measure the changes of pulmonary mechanics before and after tracheostomy in patients with prolonged mechanical ventilation and to determine factors that predict the outcome of liberation from mechanical ventilation. design: prospective. setting: respiratory intensive care unit (ricu) in a tertiary hospital. patients: twenty patients with chronic lung disease requiring long-term mechanical ventilation. tracheostomy is indicated for further care. intervention: tracheostomy. measurements and results: pulmonary mechanics including respiratory rate (rr), tidal volume (vt), peak inspiratory pressure (pip), intrinsic positive end ex~ piratory pressure (peepi), lung compliance (cld), mean airway resistance (rawm), work of breathing (wob), pressure time product (ptp) by bicore cp-100 pulmonary monitor were recorded 24 hours before and after tracheotomy. ventilator setting parameters remained the same during surgical intervention and were also recorded for comparison. generally, the mechanics including pir wob, raw~x and ptp showed improvment after tracheostomy. but only pip was significantly reduced (pre 33.4 _+ 11.8 to post 28.6 _+ 9.2, p < 0.05). changes of wobp showed significant correlation with pre-operation rr, minute volume (mv), wobp, and peep(. changes of raw m were also significantly correlated with pre-operation peep, vt, and raw m. the patients were divided into two groups according to their outcome after two week follow-up. group 1 included eight patients who were completely weaned from ventilator; group 2 included twelve patients who still remained ventilator-dependent or were mortality. there was no difference in age, duration of mechanical ventilation, pro, post or changes of several lung mechanics between the groups of patients. pre-tracheostomy peep i and cld showed significant difference between these two groups (1.1 _+ 1.6 vs 2.7 + 1.4 in peepi; 47.3 _+ 36.9 vs 28.8 _+ 16.5 in cld, p < 0.05). pre-tracheostomy ventilator setting in mode of assist/control also showed significant higher percentage in group 2 (3%5 % in group 1 vs 66.6 % in group 2). conclusion: in prolonged mechanical ventilation patients with chronic lung disease, tracheostomy will significantly improve pip and slightly reduce wobp, raw m and ptr patients who used pressure support mode before tracheostomy had better underlying lung conditions (lower lung compliance and auto-peep) will have better chance to wean from mechanical ventilation. forty-eight infants with congenital diaphragmatic hernia presenting within the first 6 hours of life, who underwent surgical rapair,were analysed prospectively in order to produce a reliable inde x of severity of disease that would reliably predict eventual outcome. there were 25 survivors and 23 deaths in this series (mortality 48%).using arterialpco 2 values measured 2 hours after surgical repairand correlating them with an index of mechanical ventilation,we have been able to clearly define two groups of diaphragmatic hernia based on their response to hyperventilation. the first group, with co 2 retention and severe preductal shunting,was unresponsive to hyperventilation with high rates and pressures the mortality was 90%. the second group responded well to hyperventilation and demonstrated reversable ductal shunting only. survival in this group was 97%. arterial co 2 accurately reflects the degree of lung development in this disease and separates those patients with severe pulmonary hypoplasia where the outcome is invariably fatal, from those with a well developed contralateral lung where there is excellent potential for survival. respiratory failure unit, dpt medicine, univ. thessaloniki, thessaloniki, greece the variability of arterial blood gases (po2, pc02) and the ph (abg) was examined in 20 stable icu patients, few hours before a successful weaning from the ventilator. all patients were lightly sedated and the ventgatory conti~ons were pressure support (ps) for 9 and ps plus intermitted mantatory ventilation in ii. [n each patient, 6 speciments of abg were measured at 10 min intervals during a 1-11 study period. at the same time with abg the arterial blood pressure (bp), the heart rate (cf), the tidal volume (tv) and the respiratory rate (n r were measured. for all the patients, the mean coefficient of variation (c) was 3.45 percent for po2, 3.53 percent for pco2 and 2.27 percent for hco3. the average sd for ph was 0.008, the corresponding c for systolic bp, diastolic bp, cf, tv, rf were 4.35, 5.21, 3.68, 2.51, 4.18 percent. we conclude that the spontaneous variability of arterial blood gases in icu patients is not substantial ~hen they have stable the heamodynamic and the ventilatory parameters. deptx?fa'aaesthesioiogy and reanimation, rhe sechenov medical academy, moscow, russia objective: ~he prevention and treatment of hypoxia in the critical patiems. methods: i~fusions of perphtoran -a blood substitute with gas-transporting fimclion based on perphtorhydrocarbon -in 496 patients with acute hypovolemia, microcirculatory distnrbance~ tissue gas exchange and metabolism; pulmonary iavage in 104; iongterm extrapulmonary oxigenation with tleoroearboa oxygenator in combination whb ~trafiltra!ion, hemosorption and hemodialysis -in 73 patients. results: pe~htoran increases blood volume, co,sv, decreases svr, improves capillary blood flow, increases the blood oxygen capacity, tissue oxygen tension, 102 del, vo2 by improving the rheologic properties of blood and plasma, normalizes 02 ext., prevents and eliminates fat embolisation and ards. decreases the need for blood transfusions and infusions of plasma expanders by 1.3-1.4 limes. alveolar venti!ation-perfusion ratio remains unchanged with its increased effective utilization. there was no surfactant destruction during lavage. extrapulmonary oxygenation of small volumes of venous blood eliminates venous destruction and then arterial hypoxia and increases pulmonary oxygenation. the use of lluorocarbon cxygenators during hemosorption and hcmodialysis provides the atraumatic and iongterm oxygenation of arterial blood and increases elimination of co2 which prevents the development of hypoxic complications. conclusions: perphtoran and fluorocarb~n oxygenators are effective in the correction of hypoxia in the criticat patients. objeqtives: to determine if there are differences in oxygen consumption (vo2) during weaning from mechanical ventilation (during total ventilatory support and spontaneous ventilation with cpap), and to compare different predictive parameters of weaning in predicting success of weaning. methods; prospective study in 20 critically ill patients treated with mechanical ventilation for at least 48h, who fulfilled at least 3 of 4 standard weaning criteria (vt>5ml/kg; respiratory frecuency (f) <35; pimax > 20 cm h20; pao2/fio2 > 150). baseline measurements: t, vt, p0.1, pimax, f/vt, p0.1*(f/vt), p0.1/pimax. study protocol: measurement of vo2, vco 2 (medgraphics), vt, f, ve, and arterial blood gases during total ventilatory support (cmv), and after 30 and 120 minutes of spontaneous ventilation with cpap 5 cm h20. the weaning trial was stopped, failure to wean diagnosed, and mv resumed it a patient presented significant tachypnea, tachycardia, bradycardia, cardiac rythm disturbances, hypertension, hypotension, hypoxemia or hypercapnia. results: four patients did not complete the weaning trial, 16 were extubatad, and 2 of them had to be reintubated before 48h, being considered also weaning failures. during cmv, vo2/kg was 4.07 + 0.2 ml/kg/min, and 5.09 _+ 0.4 mlo-2/kg/min after 30' on cpap 5 cm h20 (p < 0,01 ). 14 of 15 patients (93%) with 4 standard criteria were extubated, while only 2 of 5 (40%) with 3 criteria (p<0,01). next objectives: compare the extent and distribution of lung injury in dogs preinjured with oleic acid (oa) and ventilated with high tpp and adequate peep in the prone and supine position. methods: lung injury was induced with oa (0.06-0.09 ml/kg) in anesthetized, paralyzed, and intubated dogs (n=10) during volume controlled ventilation: rate=12/min, peep=5 cmh20, ti/ttot=0.3, fio2=0.6, vt=15 ml/kg. animals were rotated during the oa infusion and the following 90 minute stabilization period to assure uniform injury. in the supine position, peep was set 1-2 cmh20 above the lower inflection point (as determined by the pressure-volume curve), and vt was set to obtain a tpp of 35 cmh20: animals were ventilated in either the prone (n=5) or supine (n=5) position for four hours. pulmonary artery occlusion pressure was maintained constant (4-6 mmhg) with saline infusion. at the end of the protocol the lungs were removed and divided by template into dependent (d) and nondependent (nd) sections for wet weight/dry weight (v~n/dw) and grading of nstologic lung injury (hli; scale 0-3). oseillatron | is a pneumatic device that generates high frequency, oscillation by means of a reciprocating system in the form of a membrane. it generates sinusoidai wave form at 2(10 to 10(10 cycles/rain. the system does not deliver gas but must be adapted to the proximal respiratory, circuit of a conventional ventilator, resulting in ci-ifo. it was developed to enhance intrapnlmona~ diffusion during mechanical ventilation and to mobilise endebronchial secretions. methods. we measured arterial blood gases and haemedynamics during a first period of conventional ventilation (cppv) followed by. two 30 rain periods of chfo (sequences : 6(10 and 901) c/rain : group l, n = 1 l: 900 and 600 c/rain : group 2, n = 8). measurements were made at the end of each period. cardiac output was measured using thermedilution method: flu2 and peep were kept unchanged throughout the study. intrinsic peep was also evaluated by, means of an occlusive valve. results. pa02 is not significantly modified during chfo at 600 or 900 c/rain. paco2 is slightly decreased at 600 c/rain (p = 0.(16). however, intrinsic peep remains unchanged. there is no sequential effect (gr. l vs gr. 2). there is no more effect of chfo for patieets who are at a flu2 higher than 0.50 (n = 9). no changes in haemodynurmcs are observed except a slight increase in central venous pressure (cvp) during ci-ifo (p < 0.ol). obiectives: to examine the effects of inspiratory muscles unloading on neuromuscular output at controlled levels of chemical stimuli. methods: the ventilatory response to co 2 was examined in ten normal subjects using rebreathing method. ventilation ~) and respiratory muscle pressure output (pmus) at the same end-tidal partial pressure of co 2 (petco~) were compared with and without combined flow and volumeproportional pressure assist in two protocols (a and b). protocol a (n = 10): two levels of assist were studied; flow assist (fa) of 2 cmh20/i/sec and volume assist (va) of 2 cmh20/i (assist 1), and fa of 2 cmh20/i/sec and va of 4 cmh20/i (assist 2). all conditions were applied randomly. v~, tidal volume (vt) and breathing frequency (f) were measured breath by breath and plotted as a function of petco~. protocol b: in 5 subjects, in addition to above measurements, esophageal (pes) and gastric (pg) pressures were measured and the time courses of transdiaphragmatic pressure (pdi) and pmus were calculated. one level of assist (assist 2) was studied in this protocol. results: in both protocols inspiratory muscle unloading did not change the f response to c%. compared to control, with assist v t response was displaced upwards; at petco2 of 55 mmhg v t was increased significantly by 0.4+0.1 i and 0.7+0.2 i in protocol a with assist 1 end 2, respectively, and by 0.5_+0.1 i in protocol b with assist 2 (p<0.05). ~/~ responses showed similar changes as vtresponses. in both protocols the slope of v~ response (s did not change significantly with unloading. at low petco~ (50 mmhg), pdi and pmus waveforms did not differ with and without assist. with unloading, at high petco2 (59 mmhg), pdi and pmus at the end of neural inspiration decreased by 18.8-+8.3% and 11.7+15.7%, respectively, from control values. neither change was significant (p>0.05). by theoretical analysis we estimated the expected changes in vt and ~/~ when the levels of assist used in both protocols were applied in the absence of : any change in neural output response to co z. the predicted response was similar to that observed, indicating that the small difference in pdi and pmus between control and unloading runs was due to intrinsic properties of respiratory muscles end respiratory system. conclusions: these results suggest that when chemical stimulus is controlled, respiratory motor output is not downregulated with unloading. the determinants of the response of the respiratory output to inspiratory flow rates (v~) were examined in awake normal subjects. subjects were connected to a volume-cycle ventilator in the assist/control mode and v~ was increased in steps from 30 to 90 i/min and then back to 30 i/min. v~ pattern was square, and all breaths were subject-triggered. in six subjects the effects of breathing route (nasal or mouth) and temperature and volume of inspired gas (protocol a) and in 8 subjects the effects of airway anesthesia (upper and lower airways, protocol b) on the response of respiratory output to varying v~ were studied. in protocol b, in order to calculate muscle pressure during inspiration (pmus), respiratory system mechanics were measured using the interrupter method at end-inspiration. independent of conditions studied breathing frequency increased . significantly and end-tidal concentration of c% decreased as v~ increased. the response was graded and reversible and not affected by breathing route, temperature and volume of inspired gas and airway anesthesia. with and without airway anesthesia (protocol 8) neural inspiratory and expiratory time and neural duty cycle, estimated from pmus waveform, decreased significantly as v~ increased. at all conditions studied the rate of change in airway pressure prior to triggering the ventilator tended to increase as v~ increased. the changes in timing and drive were nearly complete within the first two breaths after transition with no evidence of adaptation during a given ~/~ period. we conclude that v~ exerts an excitatory effect on respiratory output which is independent of breathing route, temperature and volume of inspirate and airway anesthesia. the response most likely is neu~'al in origin, mediated through receptors not accessible to anesthesia such as those located in chest wall or below the airway mucosa. it has been shown, in mechanically ventilated awake normal humans, that increasing inspiratory flow rate (~/~) exerts an excitatory effect on respiratory output. it is not known if this effect persists during sleep. to test this seven normal adults were studied during wakefulness and nrem sleep. subjects were connected through a nose-mask to a volume-cycled ventilator in the assist/control mode and ~/t was increased in steps (3-4 breaths each) from 30 to 70 i/min and then back to 30 i/min. v~ pattern was square, and all breaths were subject-triggered. forty-one trials during nrem sleep and 10 during wakefulness were analyzed. both during sleep and wakefulness minute ventilation increased and total breath duration (ttot) decreased significantly in a graded and reversible manner as ~' increased. these changes were complete in the first breath after v{ transition. the response was significantly less during sleep than during wakefulness (p<0.05); at 30 i/min ttot, expressed as % of that at 70 i/rain, was 110.2+_1.3% during sleep and 127.8+_3.9% during wakefulness. during wakefulness, at 30 i/min, the rate of change in airway pressure prior to triggering the ventilator, an index of respiratory drive, was 60% of that at 70 i/min (p<0.05). the corresponding value during sleep, was 86% (p>0.05). in four sleeping subjects the increase in v~ was sustained for 1.5-2 min. there was no evidence for adaptation of the response; tro t, averaged over the last three breaths, did not differ from that obtained when vj was sustained for only 3-4 breaths. we conclude that 1) vt exerts an excitatory effect on respiratory output, mediated by a reflex neural mechanism and 2) the gain of this reflex is attenuated by sleep. chest radiographs is a common complementary technique for patients in critical care units, with a low cost and easily available. however, it has certain well-known limits in diagnosis, the most important derived from the low quality of some pictures. in this paper we make a general review of some new technical approaches developed for improving the quality of the images, and so incrensing the diagnostic value of conventional radiology. we begin deaeng with the correct positioning of the patient, trough the filtering techniques, the synchronization of radiology and ventilation, and we make reference to the new computerized systems for digital image processing. conclusions: the portable radiographic system is a device that probably with maintain for many years in critical care units as a basic non-invasive diagnostic tool. but we need an increase in the efficiency of it, applying means as simple as a correct positioning of the patient, or the use of fitlers or synchronizers. thus we should improve the general standards of portable radiography. "are circular circuits safe? quantifying undelivered tidal volume in pediatrics patients". objectives: to evaluate the overall influence of internal compliance of circular circuits on delivered tidad volume (vt). methods: we studied prospectively 14 asa i pediatrics patients (2 to 10 yr. old) scheduled for elective general surgery. mechanical ventilation was supplied by an ohmeda excel 210 (circular circuit). the internal compliance of the circuit (cc)-anesthesia machine plus external circuit-was determined by the supersyringe method: corrugated dar tubes of 10 mm. id and 1.5 m. long (children < 30 kg), and a corrugated dar set of 15 mm. id and 1.5 m. long (children > 30 kg) were respectively used for ccl an cc2 values of 9.3 and 9.5 ml/cm h20. a vtof 10 mlg/kg and respiratory frequency was adjusted for an end-tidal co2 (etpco2) between 30 35 mmhg. tidal volumes (measured by spirometry) and airway pressure (paw) data were recorded every ten minutes. volumes and thorax-lung compliances were calculated as follows: (vt delivered = vtadjusted-vol compressible, being vol. compressible = co x ppeak (aw). apparent compliance (ca) = vt adjusted/pplateau(aw), and true compliance (ct) = =vt delivered/pplatean(aw)). comparative statistics were separately designed between calculated compliance data and tidal volumes on a paired sample ~test basis. results: calculated values for volumes and thorax-lung compliances were: conclusions: due to the elevated internal compliance of the circular circuit there is a remarkable dilference between adjusted and delivered vt: mean undelivered vt was 38.8 % and reached as high as 64.1%. teere is also a significative error in calculating true thorax-lung compliance: its overestimation can be as high as 69.6 %. circular circuits are considered safe and cost-saving for anesthetical practice. nevertheless we conclude that anesthetists should bearin mind vt losses when using circular circuits, due to compressible volume. tracheal stenosis is one of the most serious complications of patients submitted to prolonged endotracheal intubation, in which the decrease in inner diameter of upper airway makes it very difficult to achieve a correct ventilation. objectives: compare the results of applying high frequency jet ventilation (hfjv) to some of these patients with conventional controlled ventilation (cmv). methods: we used a prototype of high frequency jet ventilator (santiago-2) developed in our university, and we developed a tracheal tube in wich we modified the distal tip (conic tip). we applied this system to two patients which were initially ventilated in the operating room with usuai controlled mecanical ventilation (cmv) following the standards of our department, and then intubated with the special endotracheal tube and ventilated with hfjv. results: we could verify a proper ventilation of both patients with cmv and hfjv. during hfjv, the airway pressures were lower than those recorded during cmv. a lower airway pressure prevents lesions due to high pressures. conclusions: hfjv is a good method of ventilation for patients with significative stenosis of the trachea, not only during surgical procedures, but also during ventilation for long periods in critically 111 patients. the ventilatory setting is pressure support mode. the pressure level and fit2 were kept constant during h/d. arterial blood gas, wbc count, and mean bp was checked according to the schedule: 0'(immediately before h/d), 15', 30', 60', 120', 180', 240'. respiratory drive (represented by poa), tidal volume(ti) and minute ventilation(ve) were continuously recorded by pulmonary mechanics monitor (bicore cp-100). the mean value of the breaths 5 minutes before blood sampling were used to represent the ventilatory status of that period. anova test is used for comparison between groups. for poa, hierarchical cluster method is applied to divide the cases into two groups of similar change. conclusions: our data suggest that pl is very useful, non invasive and low-expensive emergenc e support for arf, expecially in the elderly with severe chronic pulmonary disease and relative controindications to eti. pl seems to be an effective alternative when it is not immediatly possible to perform etl. the multiple inert gas elimination technique (miget) can be used to assess the effects of any given mode of mechanical ventilation on the pulmonary and systemic factors determining arterial po2 and pco> however, a potential problem in mechanically ventilated patients is that the 10 l mixing box (mb-10l) placed in series in the expiratory side of the circuit of the ventilator to sample mixed expired gas may provoke substantial discrepancies between the tidal votume set in the ventilator and the effective tidal volume delivered to the patient, due to the increase in the compression volume (vc) of the circuit. the effects of the mb-10l on the v c were compared with those produced by a new 1 l mixing box (mb-1 l) specifically designed to produce adequate gas mixing and to prevent loss of the two most soluble gases (ether and acetone) used in the miget. at any given peak cycling pressure (p~ak, cm h~o), the v c (ml) provoked by the mb-10l was substantially higher (vc= 7.4*ppeak) than that provoked by the new mb-1l (vc= 1.4*ppeak). at a ppeak = 50 cm h20 ~ the v c were 377 ml (mb-10l) and 67 m{ (mb-1l), respectively (p< 0.001). in a group of 6 subjects (4m/2f, 57_+6 years), for each of six the gases used in the miget, the regression line between the mixed expired partial pressures simultaneously obtained from mb-1 l and mb-10l fell on the identity line. it is concluded that the new mb-1l allows adequate assessment of the effect of different modalities of mechanical ventilatory support on pulmonary gas exchange, with less potential for gas compression and thus hypoventilation. objectives evaluate the influence of different pressure support ventilation (psv) levels on cardiovascular and respiratory funcion in icu polytrauma patients. metbed&we studied 15 polytrauma icu patients , who were in weaning process , after long term mechanical ventilation for acute respiratory failure . mean age 52 (37-71) yrs . they all were connected to servo ventilators siemens 900c , and all were in stable condition , without sedation , inotropes or diuretics. the hemodynamic studies were done with continuous svo2, swan ganz catheter (oximetrix, abbott). they all were in spontanuous mode (spent) with 5 cm h20 cpap for at least one hour. we turned them to psv with 0 inspiratory assistance (psv 0 cm h20) and after 60 rain we applied psv 10 cm h20, and after 60 min psv 20 cm h20 . hemodynamlo and respiratory measurements were done before and after the application of insiratory assistance. the results were statistically analyzed with anova. resets . respiratory variables . no significant changes in minute volume (ve). tidal volume (vt) and mean airway pressure (mpaw) increased statistically significant (p< 0.001 ) . respiratory rate (rr) decreased significantly (p<0.01) . blood gase showed no difference . cardiovascular variables. cardiac output (co) decreased ns , heart rate (hr) had no change , central venous pressure (cvp) , mean pulmonary artery pressure (mpap) , pulmonary capillary wedge pressure (pcwp) , increased ns , oxygen delivery (do2) decreased ns, oxygen consumption (vo2) decreased ns. conclusions. psv is a very useful respiratory mode helping patients to be weaned from long term mechanical ventilation . it has beneficial effects on respiratory function and oxygen consumption without affecting seriously the hemodynamic parameters, possibly due to a decrease of the work of breathing. a. michalopoulos, a. anthi, k. rellos, j. kriaras, s. geroulanos intensive care unit, onassis cardiac center, athens. objectives of this study was to examine the effect of different levels of peep on postoperative svo2 and pvo2 values in a group of patients, following open heart surgery. methods: upon transfer to icu, 67 patients (54 males and 13 females) of mean age 63_-+6 years, were randomly assigned to receive 0 (n=22), 5 (n=24), or 10 cm of peep (n=21). there were no statistically significant differences in demographic data or preoperative respiratory status among the three groups. all patients were ventilated on the assist control mode with a tidal volume of 10 ml/kg. the fraction of inspired oxygen (fio2) was adjusted to keep a pao2 around 100 mmhg. mixed venous po2 and svo2 were measured at 30 min, 4 and 8 hours after application of mechanical ventilation in the icu, just before extubation (be), half hour after extubation (ae), and at 4 hours post-extubation. differences at each study time were analysed by anova. results: mean svo2 and pvo2 values among the three groups, for all study intervals, are presented in the table. conclusion: we found no differences (p=ns) in tissue oxygenation (expressed by svo2 and pvo2) among the three groups, at any study interval, in the early postoperative course of patients following open heart surgery. intrinsic peep (peepi), and high elastance and resistance increase inspiratory work load in copd. cpap reduces work of breathing by counterbalancing peepi. pav provides flow (fa) and volume (va) assistance proportionally to patient resistance and elastance and inspiratory effort. we studied the effects of partitioned support (cpap-fa-va) on breathing pattern and inspiratory effort in five copd patients on pav compared to spontaneous ventilation (sv) and full support (fs: cpap+fa+va). flow, volume, minute ventilation (ve) respiratory rate (rr), inspiratory swing in esophageal pressure (apes), and its integral per breath (pti/b) and per minute (pti/m) were measured. objectives: to evaluate airway pressure fluctuation (apf) during spontaneous breathing in a high compliance cpap system. methods: the cpap system consisted of two 7l weighted balloons in a wedge shaped holder. ventilating gas flowed from one balloon through a low resistance one way valve into a tracheal tube (ett) provided with a pycor co 2 sensor to monitor rebreathing. the ett was connected to a piston drive mechanical lung. expired gas flowed through a low resistance valve into a second weighted balloon, from where it was exhausted through a peep valve connected in parallel with the second weighted balloon. we evaluated system performance at v r from 70 to 500ml, at rr from 10 to 120 bpm, while closely monitoring cpap airway pressure swings. at v v of 400 and 500ml the rr was limited to 60 bpm. for comparison we explored aps of a one 16l balloon cpap system, the cpap mode of the puritan bennett 7200, and siemens 300 ventilators, when connected to a healthy adult volunteer breathing through an ett. results: the compliance (cpl.) of one 7l balloon system was linear over a range from 1.0 to 3.3l, with a cpl. of 4.0 l/em h20.the cpl. of the 16 l balloon (0.5 l/em h20) was linear between a volume of 13 and 14.5 l. apf of the weighted balloon system was under 1 em h20 at all v r (except at a v r of 500ml aps was 1.5em h20), while the apf in the 16l balloon was up to 3 em h20. apf witli human volunteers with the two commercially available ventilators in the cpap mode was about 7 cm h20; while under identical conditions apf in the 16l balloon system was 1.5 emhzo; and in the two 7l balloon system was below lcm h20. conelusions: cpap using the two balloon system exhibits lower airway pressure fluctuations than a single balloon system; and is substantially lower than found in the two commercially available ventilators when used in the cpap mode. objective: to perform independent lung ventilation (ilv) with individual tidal volume (vt) set at a value generating a plateau airway pressure (pplat) < 25 crnh~o and to evaluate the usefulness of the continuous monitoring of endtidal co2 (etco2) as a guide to titrate individual lung vt during ilv and for the weaning from ilv. methods: in seven patients, ilv was performed with ttvo ventilators set with the same fio: and respiratory rate. each lung was ventilated with a vt that developed a pplat < 25 cmh~o. this setting led to a lower vt on pathological lung (pl). vt was increased in pl following etco~ and paco2-etco2 variations. ilv was discontinuated when etco~., vt and statical compliance (cst) were similar in both lungs. results: one hour after starting ilv (ti), pl mean vt was significantly lower than in normal lungs (nl) (224 + 46 ml vs 377 + 766 ml, p<0 001) two individual behaviours were observed on tl in pl: four patients presented low etco: (range 18 -31 mmhg)and normal pacoz (range 38 -42 mmhg), while three patients had normal etco2 (range 35 -45 mmhg) with high pac02 (range 44 -61 mmhg). one hour before stopping ilv (t2), vt, etc02 and paco2 were the same in each lung. the pao2/fio: ratio improved in all patients from the beginning ofllv cst of pl was 526 + 30 % of the normal lungs' cst on ti and improved to 97.6 + 27 % ofnl's cst on t2 (p<0.005 vs conclusions: setting vt of pl to a value not overcoming a pplat threshold does not impair oxygenation and is helpful in avoiding barotraumatism. measurements of differential etco2 and of the differential paco2-etco2 gradient can be used to titrate vt allocation during ilv and as a guide for the weaning from ilv. total respiratory resistance in mechanically ventilated patients exceeds values obtained in normal subjects, due to the added and highly flow dependent resistance of the endotracheal tube (rett). this can adversely effect the efficacy of pressure regulated modes of assisted ventilation, such as pressure support (psv) and proportional assist ventilation (pav). recent work demonstrates that the influence of rett during psv can be overcome by using tracheal (ptr) rather than airway opening (pao) pressure to regulate the pressure applied (intensive care med 20:$41, 1994) . the purpose of this study was to see if this approach would also be effective during pav. flow, volume, pao, ptr, and transdiaphragmatic pressure (pdi) were measured in 5 intubated patients in which either pao or ptt were used to regulate the pressure applied during pav where volume assistance was varied from 20 to 80% of respiratory elastance. representative results (mean + se) are shown below. compared to spontaneous breathing (pav 0%), pav increased tidal volume (vt) while reducing respiratory rate (rr) so that minute ventilation ('~e) also rose. this was associated with a reduction in inspiratory effort, as reflected by a decrease in the pressure-time integral ( [ p) of pes and pdi both per minute and per liter ~re. the effects on breathing pattern were similar for pao and ptr regulated pav. in contrast, the reduction in inspiratory effort was always greater for ptr regulated pav. in conclusion, the volume assistance provided by pav is more effective when ptr rather than pao is used to regulate the pressure applied. pav methods: retrospective data analysis of 596 adult patients with normal pulmonary function before operation and uneventful course following coronary artery bypass graft surgery over an 18 month period. we compared assist/controlled mandatory ventilation (s-cmv, 123 patients), synchronized intermittent mandatory ventilation with inspiratory pressure support (s-imv/psv, 431 patients) and biphasic positive airway pressure ventilation (bipap, 42 patients). results: patients ventilated with bipap had a significantly shorter mean duration of intubation (10.1 h, p< 0.05) than patients treated with s-imv/-psv (14.7 h) and s-cmv (13.2 hi. with s-cmv 39.9% of the patients required single or multiple doses of midazolam but only 13.5% in the s-imv-/psv group and 9.5% in the btpap group. the mean total amount of midazolam of these patients was significantly higher in the s-cmv group (8.8 mg) than in the s-imv/psv group (6.6 mg, p<0.05) and in the bipap group (4.3 mg, p<0.05). the consumption of pethidine and piritramide did not differ between s-cmv and s-imv/psv but was significantly lower during bipap (p<0.05). after extubation the paco2 patients was highest in the s-cmv group. conclusion: ventilatory support with bipap reduces the consumption of analgesics and sedatives and duration of intubation. unrestricted spontaneous breathing as well as fully ventilatory support allow adequate adaptation to the patients requirements. bipap seems to be an alternative to s-cmv and sqmv/psv ventilation not only in patients with severe ards but also in short term ventilated patients. _objectitives: after end-inspiratory airway occlusion we examined the ensuing gradual decrease in tracheal pressure (ptr) with the following equations proposed by bates et al. and hildebrandt: pv = p'v e'~cccl~2 +pst, rs (bates) [1] where p'tr is tracheal pressure immediately after occlusion, to= is occlusion time, "r 2 is viscoelastic time constant of respiratory system, and p t is static elastic recoil pressure of respiratory system. p~(t) = h 1 -h 2 log t (hildebrandt) [2] where h~ and h 2 are parameters depending on lung volume, and initial time is 1 s for analytical reasons. materials & methods: we studied 8 healthy patients intubated, anestethized with propofol, paralyzed with vecuronium, and mechanically ventilated with constant flow (0.5 i/s) at zeep for minor surgery. pressure was measured in the trachea. flow was measured with a pneumotachograph and volume was obtained by numerical integration. the rapid occlusions were produced by an external valve. the signals were sampled at a frequency of 200 hz and processed on a pc. the influence of the cardiac artifacts during the occlusion time (4 s) was reduced by a software low-pass filter kaiser finite duration impulse response of elevated order. results: the mean (+ sd) coefficient of correlation using eq. 1 was 0,912 -+ 0.168, and using eq. 2 was 0.884 + 0.045. the values ofz~ (eq. 1), however, decreased with increasing the tidal volume (vt) according to the following equation: "~2 = 1.52 -0.65 v t, similary, the values of h~ and h 2 increased with increasing v t according to the following functions: h~ = 4.4 + 13 v i and h 2 = 1.15 + 1.88 v t. conclusions: the behaviour of "% of eq. 1 suggests that the linear viscoelastic model is not sufficient to further describe the mechanical properties of the respiratory system over the vt range (6-14 ml/kg) in ventilated patients. infect this model predicts that "c 2 is constant and independent of tidal volume. on the other hand the plastoelastic model is not sufficient to further describe the mechanical properties of the respiratory system. in fact "r 2 obtained by fitting an exponential for data of eq. 2, is determined by the time of endinspiratory airway occlusion. obiectives: according to the viscoelastic model, the viscoelastic pressure of the respiratory system pv=rs during lung inflation with constant flow e~ is t/ r 1 2 1 wh t lsms ira tlmeand r given by:pv~c.~ = 2d~( -'e-~ )[ ] ere " ' p" tory " 2 and "r 2 are resistance and time constant of viscoelastic unit. in the past, the viscoaletic constants were determinated by performing a series of occlusions at different lung volumes, or a sedes of occlusions at a fixed lung volume achieved with various inflation flows. in the present study we have developed a new method for determining "c 2 and r 2 which requires a single constant flow inflation. our method is based on determination of pv~r, during a single breath constant flow inflation, and of z 2 during the ensuing end-inspiratory airway occiusion. dudng the occlusion the tracheal pressure p~, declines according the following function: ptr = p'lr e " too= " z2 + e~t.r= [2] where p'~r is tracheal pressure immediately after occlusion, toc c is occlusion time, p,i.rs is static elastic recoil pressure of respiratory system, and ~ is viscoelastic time constant. we first determinated "~2 by analyzing the time-course of ptr according to eq 2 and next determining r 2 according to eq. 1, using the expedmental values of p,i=~, ~ and ti, as well as "~2 obtained with eq. 2. materials & methods: we studied 8 healthy patients intubated, anestethized with propofol, paralyzed with vecurenium, and mechanically ventilated with constant flow (0.5 i/s) at zeep for minor surgery. pres-sure was measured in the trachea. flow was measured with a pneumniachograph and volume was obtained by numerical integration. the rapid occlusions were produced by an external valve. the signals were sampled at a fi'equency of 200 hz and processed on a pc. the influence of the cardiac artifacts dudng the occlusion time (4 s) was reduced by a software low-pass filter kaiser finite duration impulse response of elevated order. results: the mean coefficient of correlation with eq. 2 was 0.912. with v t of 7 ml/kg, the mean values (+ sd) of ':2 and r 2 of the 8 subjects amounted to 1.128 • 0.100 s and 3.990 • 0.890 cmh20 i "~ s. with the traditional multi breath method the corresponding values were 0.711 + 0.257 s and 4.445 _+ 1.474 cmh20 i "1 s, respectively. with the t-test the difference between new and traditional "~2 was statistically significant, between new and traditional r2 was not significant. conclusions: with the single breath method it is possible to compute ':2 and r 2. the mean values of r 2 with v t of 7 nd/kg, however, was slighuy different than those obtained with the traditional multi breath method. the application of modem principles of respiratory care and mechanical ventilation in icus has resulted in increased survival of critically ill individuals with neuromuscular, skeletal and irrevers~le pulmonary diseases. in these chronically ill individunts mechanical ventilation, long term 02 therapy (ltot) and continuous home care is considered a chronic life supporltng technique that can not be withdrawn after their discharge from an icu. the aim of this study was to present the results of a rehabilitation programme and home care that runs in our ward. twenw three patients were referred to our clinic f~om icus during 1993-94. a specific rehabilitation programme designed according to individual's needs was performed. patients that benefitted from this programme were grouped into the following disorders. 1) post tb respiratow failure 6(26%) 2) neuromuscular diseases, 3(21%) 3} undiagnosed sas 3{13%) 4) cope) 9(39%) (3 patients had a overlap syndrom). the programme consists of : 1) assessment and mechanical support ff needed of the respiratonj system with non invasive methods (nasal or via tracheostomy). 2) group and individual respiratory therapy 3) mobilization 4) nutritional support 5) educational classes for the members of the family. three from the patients passed away (during the year), 11 are under nippv during night with or without 02 supply, 13 pts recieve ltot. conclusion: the development of a programme for chronically ill individuals in especially designed wards in hospitals and the overall care at home is considered necessary at least in hospitals with icus. a rehabilitation programme and home care permits the fast but safe discharge of these patients from units of acute medicine that the cost of treatment is high and besides permits beds that are invaluable. we considered that the rehabilitation prod'amine and home care in our ward is the first performed in greek chronically ill pts and even though there is no special administxative support we think that the results are quite saltsfactory. objective: we postulated that the product of the respiratory frequency (f) and the ratio of inspiratory pressure (ip) to maximal inspiratory pressure (mip) would predict the weaning outcome in deeompensated copd patients better than either variable alone or other indices previously proposed. methods: in 28 decompensated copd patients with difficult weaning, we measured, daily, respiratory mechanics data both during mechanical ventilation and after ten minutes of spontaneous breathing. then we calculated weaning indices reported in literature and some new integrated indices. according to the results of the discriminant analysis, we considered the integrative index crop (acronym of compliance, rate, oxygenation and pressure), the rapid shallow breathing index f/vt, the load/capacity ratio ip/mip, and the following new index: f x ip/mip. we used receiver-operatingcharacteristic (roc) analysis by calculating the area under the curve considered as the overall probability of correct classification. results: main results are reported in the following objective: to evaluate the reliability of some indices of endurance in predicting the weaning outcome of decompensated copd patients. methods: in 28 decompensated copd patients with difficult weaning from mechanical ventilation (mv) we measured, daily, blood gas analysis, ventilatory and airway pressure pattern during mv, breathing pattern (frequency (f) and tidal, volume (v~)), inspiratory pressure (ip), and maximal ip (mip) during spontaneous breathing (sb). thereafter we calculated the following weaning indices: crop (compliance * mip * (pao2/pao2) / f), flvt, ip/mip. data obtained the day at which the patient was considered ready for a trial of sb on clinical grounds but weaning failed (wf) and those obtained the day of the successful weaning (ws) were compared statistically through the wilcoxon rank-sum pair analysis. in order to quantify the predictive accuracy for each index with respect to successful weaning we calculated sensitivity, specificity, and diagnostic accuracy according with the standard formulas. methods : five patients (64 + 6 yrs) suffering from ards (lung injury score > 2.5) for 48 hours or less entered into the study. irv (volume controlled, decelerating flow, 20 % inspiratory pause, lie = 2/1) was compared to conventional ventilation (cv) (volume controlled, constant flow, no inspiratory pause, iie= 1/2). these two modes were applied for 6 hours in a randomized order, with the same levels of total peep (peept = peep + peepi), tidal volume (8.0 • 0.7 ml/kg), respiratory rate (20 • 0"bpm) mad fit2 (63 • 2 %). measurements (respiratory mechanics, hemodynamics, arterial and mixed venous blood gases) were performed after 1, 2, 4 and 6 hours of application of each mode. rvsuils : are expressed as mean + sem and compared by anova. backeround and methods: periodic breathing (pb) is characterized by repetitive cyclic variation in minute ventilation. pb is considewxl to be provoked by an instability in the respiratory control. inintubated, spontaneously breathing patients conventional modes of pressure support ventilation, i.e., triggered inspiratory pressure support 0ps), do not allow patients to breathe with theirinherent breathing pattern. therefore, pb, if existing, will appear mainiy after extubation. since our new mode of pressure support ventilation" automatic tube compensation" (atc) continuonsly corrects for the flow-dependent tube resistance during insnmdon and expiration ("electronic" extubatim), it pemaits patients to maintain their own inherent breathing pattern. then, ff necessary, tracheal pressure can be additionally supported by volume-proportioead and/or by flow-proportional pressure support (proportional assist ventilation, pav). (~as~: we report the case of a 70-year-old male patient who was intubated due to acute respiratory insufficiency after acute myocardial infarction with left ventricular dysfunction. during ips of 10 mbar the patient showed a regular breathing pattem which became periodic during atc. in addition, proportional assist ventilation of 10 mbar/l increased periodic breathing in such a way that the typical cheyne-stokes breathing pattem occurred (see figure) . baqkground: the hering-breuer reflex (hbr) is characterized by an inhibition of inspiration during lung inflation. this response has been recognized as an important vagally mediated mechanism for regulating the rate and depth of respiration in newborn mammals. in adult man the hbr is considered to be active only at lung volumes well above functional residual capacity, i.e., at tidal volumes above 1000 ml. assessment of the hbr requires specialized methods such as single breath or multiple occlusion technique. methods; in the presence of desynchronization between ventilator and patient, which frequently occurs during triggered inspiratory pressure support ventilation (ips)(see figure) , prolongation of the interval between inspiratory efforts (indicated by negative deflection of the esophageal pressure) due to lung inflation exposes an active hbr. we examined the occurrence of hbr in intubated critically ill patients. strength of hbr was assessed by the formula: prolongation [%] = ((inspiratory interval of interest -preceding inspiratory interval)/preceding inspiratory interval) * 1(30. rr162 18 of 50 patients examined showed moderate to severe desynchronization. in 17 of these 18 patients a (re)activation of the hbr was found. the strength of hbr amounted to 134 + 51%. there was a significant correlation between tidal volume and strength of hbr. in contrast to previous reports, an active hbr was shown during lung inflation well below 1000 ml. b pck~round: triggered inspiratory pressure support ventilation (ips) is commonly used to support inspiration in intubated spontaneously breathing patients. despite its usefulness ips shows some disadvantages which can be deleterious in crificauy ill patients: -additional work of breathing to be performed by the patient due to the flow-dependent tube resistance -desynchronization between patient and ventilator due to inherent triggering failures of the ips mode suppression of the patient's inherent breathing pattern -inability to predict successful extubation in difficult-to-wean patients methods: based on the known flow-dependent tube resistance our new mode "automatic tube compensation" (atc) compensates for the pressure drop across the endotracheal tube ("electronic" extubation). then, if necessary, tracheal pressure can be supported by volume-proportional pressure support (vpps) and/or by flow-proportional pressure support (fpps). results: hitherto, we have examined 20 patients after open-heart surgery and 50 patients with acute respiratory insufficiency (ari) or ards using atc with/without vpps/fpps. preliminary results suggest that the new mode avoids additional work of breathing due to accurate compensation of the pressure drop across the endotracheal tube during in-/expiration prevents desynchronization between patient and ventilator allows patients to breathe with their inherent breathing pattern accurately predicts the outcome of extubation even in difficult-to-wean patients due to "electronic" extubation conclusions: the new mode atc with/without vpps/fpps allows to support ventilation in a more physiologic manner and overcomes the disadvantages of conventional modes of pressure support in intubated patients. backgound: cheyne-stokes respiration (cs) is characterized by regula]; recurring periods of hyperpnea and apnea. in normal subjects, cs may occur after hyperventilation, after arrival in high altitude, or during sleep. it has also been observed in patients with prolonged circulation time due to congestive heart failure, as well as in some neurological patients. there is no report about the influence of sedative drugs on periodic breathing (pb) and cs. methods: in intubated patients conventional modes of pressure support do not allow patients to breathe with their inherent breathing pattem. therefore, periodic breathing and cs are rarely seen. since our new mode of pressure support ventilation "automatic tube compensation" (atc) continuously corrects for the flow-dependent tube resistance during inspiration and expiration ("electronic" extubation) it permits patients to maintain their own inherent breathing pattem even if pathological, e.g., periodic. results: using this new mode of pressure support ventilation, periodic breathing was unmasked in 13 of 37 intubated patients, 6 of which showed cs. in 4 of these 6 patients the occurrence of cs was linked to impaired left ventricular function with increased circulation time. normal left ventricular and neurologic function was found in the remaining 2 patients. in 1 of these 2 patients cs disappeared after intravenous administration of the benzo-diazepine antagonist flumazenil (figure). consequently, in this patient cs was induced by benzodiazepine sedation. objecti',~s: in contrast to conventional rhodes for pressure supported spontaneous breathing, our newly developed ventilatow mode ,,automatic tube compensation" (atc) completely compensates for the flow-depandant pressure drop tlpm-r across endotracheal ttlbe (ett). in the atc mode, the ventilator supplies a flow v' in order to maintain a constant tracheal pressure p~,,~. to this end, pk,,= has to be oontinuousiy determined. since continued measurement of p,,~ by introducing a catheter via the ett is not reliable, we opted for its continuous calculation socordng to the following equation: p~ = p,,, -aperr, pw being the continuously measured airway pressure. this also requires the continual measurement .of flow v' to calculata apm-r using the non-fineer approximation: aport = kvv' + k2.w. the constant tube coefficients k~ and k2 are mathematically determined by mesns of a least-squares-fit procadum based on laboratory investigations. tracheal secretions, however, reduca the omss-saction of the ett. consequently, ~ values of ki end k2 are changed rendering the p~,ch calculations inaccurate. therefore, k1 and ~ have to be pedodcally updated to ensure an a~urete monitoring of pn,~ and a complete tube compensation under atc at any time. background: one of the first steps in weaning patients from controlled mechanical ventilation is to stop muscle relaxation and to reduce sedation. it can take several hours, however, until the patient is able to trigger the ventilator and to breathe spontaneously. during this period, many patients display a sudden increase in peak airway pressure of up to 30%. patients and methods: to investigate the reason for this potentially dangerous effect, we continuously measured lung and chest wall mechanics in post-operatively ventilated patients. lung mechanics (airway resistance and lung compliance) was measured using the esophageal balloon technique as described in [1] . chest wall mechanics (tissue resistance and chest wall compliance) was calculated from lung mechanics and total respiratory system mechanics as described in [2] . results: we found a decrease of chest wall compliance (cw) to be the main reason for episodes of sudden airway pressure increase while lung compliance (cl) remained unchanged. the decrease of c w can be intergil cano a, san pedro jm ~, sandar d, herntndez .1, carrizosa f, , herrero a. emergency and intensive care department, hospital of jerez, spain objective: 1) to determine the incidence of hypoteasion (h) associated with emergency intabatian of mechanical ventilation, and 2) to establish its relauonship with respiratory mechanics (rm) and arterial blood gases. mechanical ventilation performed in the emergency room, in a prospective eans~eative manner, were evaluated. data collected included patient demographics, diagnoses, blood pressure and arterial blood gas levels before and at~er intabatian, and p_m, including calculated pulmonary end-inspiratory volume above functional residual capacity (veic) and calculated dynamic hypetinflatien (dhc). all patients received midazolen and awaanrinm to facilitate tracheal intubatien and rm measurement. hypotension was defined as a decrease in systolic pressure higher than 40 mmhg or an absolute decrease in systolic blood pressure below to 90 mhg within 1 hour of intabatian. 14 patients were excluded because met at least one of the following exclusion criteria: preexisting shock or h (8), cardiac arrest (5) .1 there weren't any association between peepi or other airway pressures (paw) and h, but calculated pulmonary volitmes had tendency to be larger in patients with h (p < 0.1). high paco 2 before lrasheal intubatian (87.4 4-9 mmhg) with a quickly decrease alter starting mechanical ventilation was a usual finding (p < 0.01) in patients who developed h. paw. 3) thexe was a good relatienship between h and high arterial paco 2 before traqueal intahatian and its fast "washing" with mechanical ventilation. 4) because cao patients had the highest incidence of h, controned mechanicel hypoventilatien driven by paco 2 changes and pulmonary volumes monitoring instead paw, should be attempted in these patients to avoid this cemplication after tracheal intubatiert. introduction: the endotracheal tube (ett) and demand valve devices cause an added work of breathing (wobadd), which is the work necessary to overcome the resistive load of the ett and the breathing circuit (1). application of ips has been shown to partly compensate this added work (1). since tbe amount of wobadd is flow dependent, a fixed ips is not adequate to completly compensate the wobadd (2). therefore, atc has been developed as a new form of assisted spontaneous breathing (3), which provides a flow-dependent pressure support. thereby, it theoretically should compensate all the wobadd due to the tube. the purpose of this study was to evaluate the reduction of wobadd with ips and atc for different ett. methods: a mechanical lung model (ls 4000, dr*alger, liibeck, frg) was used to generate a constant spontaneous breathing pattern. the ls 4000 was connected to an artificial trachea (at, 10 cm long, 22 mm id). the at was intubated with three different tubes of 7.0, 8.0, 9.0 mm id and connected to an evita 2 ventilator modified to provide atc as an option (dfager, liibeck, frg). flow and airway pressure were measured between the y-piece and the ett for four different modes of ventilation: cpap, ips of 5 and 10 cm i420 and atc all with a peep of 10 cm h20. the tracheal pressure (ptrach) was measured in the at. total wobadd was calculated as the area subtended by the ptrach-volume curve below peep. results: the results for total wobadd in nd/1 are shown in the figure for the three different ett: breath/mln, s=success, f=failur% *~p<.05, **-p<01, ns = non significant, f versus s neveltheless, in 5/26 patients, invasive ventilation was necessary in mean 12.6_+12 hours after beginning of fmpsv. there was no significant difference between the two groups (success, failure) in following parameters : sex, age, previous histoly, medical treatment, saps1 & 2, clinical signs (rr, spo2, heart rate, blood pressure, glasgow score...), radiological and echocardiographic findings and standard biological parameters. only two parameters were related with failure : 1.a low value of pac02 on admission until the patients were intubated. 2. an increased level of cpk in relation with an acute myocardial infarction (4/5 cases in the failure group, vs 3/21 cases in the success group, x~(with continuity correction) : p<.05). conclusion : fmpsv is a noninvasive, safe, rapidly effective method of treatment in acpe, which may avoid tracheal intubation. further studies are necessary to precise if association of arf and low paco2 (<35mmhg) and/er acute myocardial infarction represents an indication of immediate invasive ventilation. introduction: since the added work of breathing (wobadd) imposed by the endotracheal tube (ets and the breathing circuit is regarded as an important contribution to the total work of breathing, considerable effort has been tmdettaken to compensate for this added work. ips has been fotmd to decrease the wobadd imposed by different ventilators (1, 2). because of the flow dependent pressure drop across the etf the tracheal pressure (ptr) should be measured to estimate the total imposed wobadd (wobtut) (3, 4). the aim of this study was to assess the circuit imposed work (wobcirc) and wobtot (including ett) for different demand valve ventilators during cpap and/ps. methods: a mechanical lung model (ls 4000, driiger, lfibeck, frg) generated a constant spontaneuus breathing pattern. the ls 4000 was connected to an artificial trachea (at), intubated with an 8.0 nun et]', end connected to one of four ventilators (servo 900c and servo 300, siemens,-elema, sweden; evita2, driiges, liibeck, frg; pb 7200ae, puritan bennett, carlsbad, usa). three different modes of ventilator settings were tested (cpap, ips 5 and 10 mbar; trigger set at maximal sensitivity, peep always 10 mbar). flow and airway pressure (paw) were measured between the y-piece and the etr; tracheal pressure (ptr) was measured in the at. wobtot was calculated as the area under the ptr-volume curve below peep, wobcirc was calculated as the area under the paw-volume curve below peep. results: in the foti g., patroniti n., cereda m., sparacino me., giacemini m., pesenti a. inst.of anesth.and intensive care-univ.of milan -sgh monza i aim of the study was to assess cpl,rs measurement obtained by the airway occlusion method during psv. we therefore studied 31 paralyzed cppv ventilated ali patients (lung injury score =2.25• that were weaned to psv. we performed end inspiratory and end expiratory airway occlusions using the hold function of the ventilator (siemens serve 900c), first during cppv and then within the 24th psv hour. airway pressure and flow signals were recorded (cpi00 bicore) for subsequent analysis. an airway pressure plateau was defined as a 0 flow tracing in which airway pressure was stable for at least 0.25 sec. end inspiratory (pel,rsi) and end expiratory (pel,rse) recoil pressures were then measured as the mean airway pressure during plateaus. cpl,rs was computed as tv/ (pel,rsi-pel,rse i) cpl,rs can be adequately estimated during psv using the airway occlusion method; 2) during psv inspiratory plateaus are longer than the expiratory ones; 3) the length of plateaus is negatively affected by the respiratory drive. foti g., de marchi l., *tagliabue m., gilardi p., giacomini m., sparacino me., pesenti a. inst.of anesth.and intensive care,-univ.of milan *dept.of radiology-sgh monza i we retrospectively compared ct scan and gas exchange findings between a group of patients successfully weaned from vcv to psv (group s = ii patients) and a group who failed the weaning (group f = 6 patients). we selected 17 ali patients (lis=2.5• in vcv mode who had available a chest ct scan performed within 4 days from the weaning trial. a psv trial was began as soon as the patient reached hemodynamic stability and a pao2 >80 mmhg, irrespective of fie2 (peep <15 cmh20). maximum psv level was < (pel,rs-peep) measured during vcv, where pel,rs was the respiratory system elastic recoil pressure at end inspiration. psv ventilation was considered successful if a respiratory rate <40 bpm, an increase in fie2 lower than 0.2 compared to vcv, a pace2 increase <20% of vcv value and hemodynamic stability were maintained during the next 48 hours of psv. if any of these conditions was not met the trial was declared a failure. interdisciplinary critical care unit, regional hospital lugano-ch *surgical critical care unit, university hospital, geneva-ch objective: to assess the degree of correlation of cardiac output measured by thoracic electrical bioimpedance and thermodilution in mechanically ventilated patients with different levels of positive end-expiratory pressure (peep). methods: prospective study with 10 ventilated patients, 7 after head injury and 3 with postoperative sepsis, with normal cardiac output: simultaneous determination of cardiac output by thermodilution and thoracic electrical bioimpedance performed with different levels of peep (0-5-15 cm h20). results: cardiac output measured by thermodilution during sequential increment of peep did not vary: 7.3 + 2.5 for peep 0, 7.4 + 2.7 for peep 5 and 6.9 + 1.7 l/rain for peep 15. simultaneously the bioimpedance device recorded a significant increase in cardiac output from 4.4 + 1.3 for peep 0 to 6.0 + 1.9 l/mi for peep 15. (p < 0,05). conclusion: cardiac output measured by bioimpedance cannot replace the invasive thermodilution methods of cardiac measurement output during mechanical ventilation with peep. we also isolated a subset (h) of 12 patients who had been hypercapnic (paco2>50mmhg) for at least 3 days (range 3 to 60 days) before the end of cv. the psv trial was started as soon as pao2 was > 80 mmhg, irrespective of fie2 and with peep < 15 cmh20 and the psv level had to be < (pplateau-peep) as measured during cv. pace2, pha, base excess (be) were collected before discontinuation of cv and on the ist day of psv: 05) . 2) weaning is more difficult in pts with head injury(p<o,05) and iss>30 (p<o,ol), in elderly(p<o,05) and in pts who need fi02>0,6 (p<o,05) and peep>io cm h20 (p<o,05). 3) pts with iss>30 need longer duration of mv (p<o,ol). 4) the mortality rate is higher in pts with iss>30 (p<o,o01), with head injury (p<o,ol) and in elderly (p<o,05). 5) paradoxally, compliance was found to be higher in pts>60 years than in pts<60 years (p<o,05). s. crotti, p.pelosi, d.mascheroni, m.cerisara, a.lissoni, l.gattinoni. istituto di anestesia e rianimazione -irccs, milano, italia. obiectives. we investigated by ct scan the effects of extrinsic peep (peepe) on lung inflation, tidal volume distribution and regional compliance in sedated, paralyzed chronic obstructive pulmonary disease (copd) patients with dynamic hyperinflation (peep• methods. two ct section (end expiration, end inspiration) were obtained at lung base level in 4 patients (individual analysis for each of the eight lungs) at 4 peepe levels: peepe = 0 cmh20 (zeep) and peepe amounting to 50%, 100% and 150% of the peep• at zeep (9.5 • 4 cmh20). tidal volume was kept constant (8 • 1.4 ml/kg). each lung section was divided into 3 zones equally spaced along the vertical axis: upper (the most ventral), middle and lower (the most dorsal) zone. for each zone we computed: gas volume at end expiration (gase) and at end inspiration (gas• tidal volume (dgas=gasi-gase) and compliance (cpl=dgas/dp;.dp=plateau pressureactual peep• we also computed the lung inflation as the entire ct section gas volume at end expiration (total gase). results. at each peep level both gase and gas• were higher in the upper than in the lower zone ( we cone!ude that in copd patients with dynamic hyperinflation only the application of peepe higher than peepi produces a further increase of lung inflation, decreasing cpl of the upper zone (the more expanded one) probably by stretching phenomena, and causing dgas redistribution from the non dependent to the dependent lung zones. i. ravagnan, p. vicardi, f. valenza, d. tubiolo. p. pelosi l. gattinoni. 1st . anestesia e rianimazione, universita' di milano, ospedale maggiore -irccs, italy objectives: to investigate the effects of peep and ps on respiratory. pattern and inspiratory effort during ps ventilation in patients with acute lung injury or acute exacerbation of chronic lung disease. methods: 4 pts with acute (a) (paco2= 39• mmhg, pao2/pao2= 0.4• and 4 pts with chronic (c)(paco2= 51• mmhg, pao2/pao2= 0.3:50.1) lung disease were studied in ps ventilation during the weaning phase. measuring airway pressure, esophageal pressure and gas flow we computed respiratory rate (ril bpm), tidal volume (tv, ml) and pressure time product (ptp, cmh20*s/min), which is an index of oxygen muscle consumption. measurements were collected, after 15 rain of stabilization, at 5 and 15 cmh20 ps and at two different peep levels (0 and 8 cndd20 conclusions: during psv: 1) breathing pattern and ptp are different between a and c; 2) in both groups peep does not modify breathing pattern; 3) peep reduces ptp in c but not in a; 4) ps modifies breathing pattern and reduces ptp in c but not in a. to test the performance of a new heat and moisture exchanger (hme): twistair, baxter. this is a hydrophobic-hydrophilic hme without hygroscopic salts. method: we evaluated the hme performance with a previously described method (i); it consists of a "lung simulator" connected to a mechanical ventilator and a flow divider, with a dry and a wet thermal probe inserted into inspiratory and expiratory limbs. on average ps level changes were associate( with opposed changes in both p0.1 and rr. changes in p0.1 were remarkable, homogeneous and highly significant (p<.002), while changes in rr were less pronounced, inhomogeneous and less significant (p<.02). it seems therefore that p0.1 is a better index than rr for estimating a change in load for the respiratory muscles. recently, the lsf method has been described as an interesitng alternative to conventional tecniques used to measure total respiratory mechanics, providing, over these latter, advantages such as no need for hold maneuvers and no need for particular flow patterns. data on the reliability of the lsf method, however, are still few. methods. 9 sets of measurements were obtained in each of 7 ards patients, paralyzed and ventilated in cmv with constant inspiratory flow and an end-inspiratory pause equal to 15% of ttot. the lsf method provided data for total compliance (ctotlsf) and total resistance (rtotlsf) by multiple linear regression analysis of airway pressure, flow and volume change, applied over the entire breath. the lsf measurements were automatic and continuous, and each value used for analysis was the average of 160 consecutive cycles. conventional measurements of dynamic.compliance (cdyn), quasistatic compliance (cqs), minimum inspiratory resistance (rmin) and maximum inspiratory resistance (rmax) were obtained by the constant flow, end-inspiratory occlusion method, each value being the average of 3 measuremts. ctotlsf was compared with cdyn and cqs, while rtotlsf was compared with rmin and rmax. results. ctotlsf showed a high correlation both with cdyn (cdyn=.87.ctotlsf+l.14, r2=.982), and with cqs (cqs =.94.ctotlsf +1.19, r2=.984). the average difference between ctotlsf and cqs, yet, was much lower (+.36+1.79 ml/cmh20) than the one between ctotlsf and cdyn (+2.43+2.40 ml/cmh20). rtotlsf correlated much better with rmax (rmax=.88*rtotlso+.98, r2=.914) than with rmin (rmin =.58.rtotlsf +. 1.71, r2=.804). the average difference between rtotlsf and rmin was +4.48+3.48 cmh20/1/s, while the one between rtotlsf and rmax was +.81+2.03, confirming the better agreement between rtotlsf and rrnax. conclusion.the agreement between rtotlsf and rmax suggests that rtotlsf takes into account both the airway and the tissue components of resistance, unlike rmin which only expresses airway resistance. the agreement between ctotlsf and cqs indicates that ctotlsf expresses the pure elastic components of the respiratory system. in the paralyzed patient, the expiratory time constant (rce) of the unit represented by total respiratory system and ventilator can be calculated from the slope of the expiratory flow-volume curve. recently it has been suggested that an estimate of this slope is provided by the ve/v'epeak ratio, ve being the exaled tidal volume and v'epeak the peak expiratory flow.the reliability of this method is still little known. methods. 9 sets of measurements were obtained in 7 mechanically ventilated ards patients during paralysis and cmv. measurements of ventilator expiratory resistance (rext) and of total respiratory mechanics were performed in order to calculate these 3 reference measurements for ve/v'epeak: rcdyn=(rmin+rext)ocdyn, rcstat=(rmax+rext)ocqs, rclsf=(rlsf+rext)~ cdyn (dynamic compliance), cqs (quasistatic compliance), rmin (minimum inspiratory resistance), rmax (maximum inspiratory resistance) were obtained by the constant flow, end-inspiratory occlusion method. clsf and rlsf respectively corresponded to measurements of total respiratory system compliance and resistance by the least squares fitting method. ve/v'epeak showed a high correlation with all 3 reference measurements, rcdyn (ve/v'epeak= 1.07orcdyn-.18, r2=.939), rcstat (ve/v'epeak = .97 9 rcstat -. 12, r2= .929), and especially rclsf (see fig the static pressure volume curve of the respiratory system (p/vst) provides valuable information, but the extensive data analysis prevents its use in clinical routine. dynamic p/vcurves are simpler to record. the objective of this study was to develop an automated method to obtain quasi-static p/v-curves (p/vqs) during low flow inflation as well as to measure resistance. preliminary tests were done in normal subjects and subjects with varying degree of lung disease. methods: a computer/ventilator interface (cvl) was constructed for control of a servo ventilator 900c during an automatic sequence: a) a normal breath, b) a prolonged expiration at zero peep, c) a low flow inflation of a predetermined volume, d) analysis of ventilator pressure and flow. pressure was corrected for tube resistance. airway resistance was calculated from a) and used to derive p/vqs from c). as reference, p/vst was obtained by the occlusion method. results: in non-obstructive diseases the method performed well at volume and pressure controlled ventilation. static and quasistatic compliance were virtually identical. p/vqs clearly reflected the lower inflection point and, when present, the upper inflection point (uip). in some patients uip was more evident in p/vqs, especially at an increased inflation flow rate. in obstructive diseases, the method for subtraction of resistive pressure was inadequate. conclusions: the system for automated computer controlled studies of lung mechanics based on a standard ventilator provides comprehensive information. differences between p/vst and p/vqs imply that the latter reflects also other factors than p/vst that contribute to impedance at hyperdistension. the clinical significance of the two curves remains to be shown. obiective: to evaluate the effect of combined high frequency jet ventilation (chfjv) in patients with severe respiratory insufficiency (sri). methods: in a retrospective study of the period 1985-1995 we analyzed patients suffering from sri who were refractory to conventional mechanical ventilation and were treated with combined high-frequency jet ventilation (chfjv). refractory to conventional mechanical ventilation was defined as a pao2/fio 2 < 100, despite maximal ventilator settings: peep > 8 cm hz0 , fit 2 > 0.6. a total of 43 patients matched these criteria, 27 males and 16 females with a median age of 44 (17-72) years. seventeen suffered from severe trauma. chfjv was started following a median period of 3 (1-22) days of conventional mechanical ventilation. prior to chfjv ventilation parameters expressed as median were the following: fit 2 0.8, pao2/fio 2 78, peep 12 cm h20 peak airway pressure (pap) 48 cm h20. chfjv consisted of high frequency jet ventilation with a frequency of 100 to 300 breaths/minute, driving pressure of 1.8 to 3.5 arm, and inspiration time of 20 to 30 percent, superimposed on the whole cycle of conventional mechanical ventilation with a frequency of l0 to 20 breaths/minute and tidal volumes of 100 to 400 ml. results: following two days of chfjv 31 of 43 patients showed an improvement of ventilatory parameters; peep could be reduced to < 8 cm h20 in 14 patients, the pap was decreased with > 5 cm h:o in 20 patients, fio 2 could be reduced to < 0.6 in 27 patients and finally the median pao2/fio 2 ratio changed from 78 to 133. during chfjv 23 patients died, 4 of respiratory failure and 19 due to multiple organ failure, 6 died within two days of chfjv. the median duration of chfjv in survivors and nonsurvivors was 6 days in both groups. conclusions: our data show that with chfjv in the majority of patients with sri who are refractory to conventional mechanical ventilatior" the ventilatory parameters can be improved. backeround and obiectives: although ventilation with peep above the inflection point (pinf) has been shown to reduce lung injury by recruiting previously closed alveolar regions, it carries the risk of hyperinflating the lungs. in the present study we set out to develop a new strategy to recruit the lung during ventilation with small vt, while maintaining peep levels as low as possible. we hypothesized that if the lung was recruited with a sustained inflation (si) to total lung capacity, recruitment would be maintained as long as the peep level was higher than the critical closing pressure of the lung, as observed on the deflation limb of the pv curve (ajrccm 1995; 151(4) :a432). the purpose of this study was to examine the hypothesis that a strategy using si and a peep<pinf would induce less lung injury during small tidal ventilation than ventilation at the same low peep level without using a recmitment strategy in a model of respiratory distress syndrome. methods: we used a randomized protocol with 4 groups to ventilate 40 nonperfused, lavaged rat lungs with small vt (5 to 6 ml/kg) for 2 hours and studied the effect on compliance and lung injury. group 1: peep>ping group 2: peep<pin f with an si, inflation to 30 on h20 over 30 sec, to recruit volume; group 3: peep<pinf without si; group 4: control group, lungs were inflated at peep<pin f, but not ventilated. results: in group 2 and group 4 static compliance did not change after ventilation (table) . in group 3 static compliance fell significantly. group 1 showed significant changes in the pv curve, which were less severe than in group 3. results from morphological examination are pending but preliminary results showed less lung injury in group 2, compared to group 3. conclusions: small tidal ventilation following a recruitment manoeuvre in a model of respiratory distress syndrome allows ventilation on the deflation limb of the pv curve at a peep below pinf. this strategy (1) minimizes lung injury as well as, or better than using peep above pint" and (2) ensures a lower peep to minimize the detrimental consequences of high lung volume ventilation. i~peep>pin~ 2 _objectives-this report is presenting the results of the clinical study for using eeg examination as a method of the evaluation of patients ability for weaning. methods: the study inclljqles 42 eeg examinations with fourier spectral analysis' of 37 patients ~vith respiratory insufficiency and prolonged control mechanical ventilation (cmv). all patients have had a-rhythm of eeg before weaning. we have followed respiratory rate, tidal volume, respiratory pa{tern, end-tidal co2 and blood gases during weaning. results: 13 patients had invariable eeg activity or short 13-waves period (till one hour). the weaning of this patients was fast arid sucsessful. other 24 patients have had a decreasing of a-activity, an appearence of 13-waves for an hour and more, a short episodes of a-and e-activity. after that this patients had gas exchange and respiratory disorders with regression of the weaning right up to cmv. conclusion: eeg could be used as a method of the evaluation of patients ability for weaning from cmv. some eeg signs shows the overstrain of compensatory systems before the change to the worse of gas exchange and respiratory pattern. s. elatrous, p. aslanian, d. touchard, d. corsi, h. lorino, l. brochard. medical intensive care unit, inserm u 296, hopital henri mender, cr~teil, france. in vitro comparison of flow triggering (ft) systems demonstrated advantages compared to pressure triggering (pt) systems for some ventilators (puritan bennett 7 200) but not others (siemens serve 300). we studied the two types of systems in two groups of 8 patients mechanically assisted with pressure support ventilation (15 + 6 cmh20). in the first group (pb 7 200) the effort of breathing, assessed by the esophageal pressure time index, was significantly lower with the ft than with the pt (139 + 40 cmh20.s/min -1 vs 158 + 32, p< 0.05). by contrast no significant difference appeared in the second group (serve 300), as predicted by the bench study despite marked interindividual differences (134 + 55 cmh20.s/min -1 vs 160 + 61, p = 0.1). we conclude that 1) rigorously performed bench studies can predict in vivo effects, 2) mild advantages can be found for the new triggering systems on some ventilators. objectives: pressore-volume curves (pv) of the respiratory system is of interest for the determination static compliance (cs0, lower (lip) and upper (uip) inflection points which indicate zones of airway recruitment and overdistension. this study aimed to compare an "automated low flow inflation" method (alfi) to the reference occlusion (oc) method. the ability of the former method to identify cst, lip and uip was tested in icu patients. me,otis: 16 (8 arf and 8 ards) sedated paralysed patients were studied using a serve 900c ventilator linked to a computer which automatically forced the ventilator to insufflate at a low constant flow a velum up to 1500-2000 ml or a maximum paw of 50 cm h20 (alfi). the quasistatic elastic pressure (pel,qs0 was obtained by subtraction of the resistive pressure of tubing and patient and related to volume for calculation of compliance cqst. for oc tidal volumes (v0 from 50 up to 1500-2000 ml were followed by a 3 s post-inspiratury pause for determination of static pal (pel,st) in relation to volume. compliance was defined from the linear part of the p/v curves. lip and uip were defined from the consistent deviation of p/v data from extrapolated the linear part. ~,~111i~: in ards, mean cst was 27.9 + 3.5 and cqst 29.7 + 3.9 ml/cm h20 (us), lipst 5.2 + 5.0 and lipqst 7.0 + 4.6 cm h20 (us), uipst 23.1 + 10.8 and uipqst 26.0 + 5~4 cm h20 (us). nosocomial pneumonias (np) are frequent and often unsuspected during ards (bell, !983). in the present study, we evaluated prospectively the onset of np during severe ards (group b of the european study). patients and methods: the charts of 15 patients with severe ards have been prospectively recorded. a plugged telescopic catheter (ptc) specimen has been systematically performed every 48 hours, for quantitative bacteriological analysis. the diagnosis of np was defined by a number > 103 colony forming units / ml. results: for the 15 patients studied, the mean saps score (+ sd) was 16+_2, the initial pao2/fio2 ratio was 100-&-_35, the duration of mechanical ventilation (mv) was 19+9 days. the mean delay before the onset of the first np was 8.6+5.6 days (5-12), and the mean pao2/fio2 ratio was 110+-28. respiratory symptoms (purulent aspirates, new pulmonary infiltrates, or gazometric changes) were present in 80% of the patients studied. alteration of gas exchange was present in 8 of the 15 patients (7 np) . a new pulmonary infiltrate was present in only 1 np (10%). an increase of fever was noted in 6 patients, an increase of leukocytosis > 20% in 8 patients, an increase of volume and purulence of sputum in 3 of the 10 patients with np. the degree ofgazometric worsening (pao2/fio2 before np minus pao2/fio2 during np) during the first episode of np was 44+17 mmhg. excluding the bacteriological criteria of np, the number of criterias of np present was 1 in 1/10 patients, 2 (5/10), 3 (2/10) or 4 (2/10). two patients only had a pulmonary colonization (ptc: < 1102 cfu / ml) before the first episode of np. the incidence of np is high (53%) during severe ards. the first episode occurs in average:at the 9 th day, and is the cause of a severe hypoxemia (pao2/fio2 110) . the onset of a np may contribute to the high mortality rate observed in our patients (93%). each worsening of hypoxemia during severe ards should induce to suspect a np. respiratory system during mechanical ventilation. the me~hod quantifies the dissipative energy consumption of the respiratory system in terms of energy loss aek, inefficiency ~k~ and respiratory dissipative resistance rk~ over a given partition of the tidal volume. the method can be applied in intensive care units with no interference to ventilatory support. it allows for monitoring the combined effects of inhomogeneities, non-linearities and visco-elastic effects, that are subject to change in the respiratory system. the method is studied on pigs~ in the presence of a log-dose response curve of methacholine (mch) induced disease. in healthy pigs~ we find a mean value of energy loss, ae, of 0.27 • j/l, a mean value of inefflency, ~ of 0.25 ~=0.05 and a mean value of resistance, 7~, of 4.40 • cm h20 s/1. the respiratory resistance, rk, shows a variation over the partition of tidal volume with armax ----3.90 • 0.66 cm h20 s/l. during methacholine provocation~ ae rises more than five-fold up to 1.48 • j/l~ doubles to 0.54 • and t~ increases to a maximum of 22 • cm h20 s/l, with armax : 15.1 • 7.0 cm h20 s/1. the variation in rk becomes more pronounced with higher doses of methacholine. methods: 10 ards patients were prospectively studied. initially they were ventilated in the amv (assist mechanical ventilation) mode with the settings prescribed by their primary physician. after stabilization, ventilatory gas exchange and hemodynamic variables were determined. patients were then ventilated in the mrv (mandatory rate ventilation) mode with 20 breaths as the target rate. in mrv the target rate is set and the ventilator autoregulates the pressure support level delivered ~o achieve this rate. after stabilization, the measurements done on amv were repeated. finally, patients were sedated and paralyzed and ventilated in cmv (control mechanical ventilation) with the ventilatory variables they had during mrv. measurements done in amv and mrv were repeated and respiratory mechanics were assessed with the constant flow end inspiratory occlusion method. results: two groups were recognized based on their response to mrv. tn group 1 patients responded to mrv by decreasing their v and increasing the t/t t ratio. ve, vo 2, and aado 2 decreased while paco 2 increased and tda vo ume and co remained unchanged. on the contrary, in group 2 v, vr and ve increased; ppeak and trr t remained unchanged, paco~ decreased while vo 2 and aado 2 increased with constant co, the pressure support level needed to achieve the target rate was much lower in group 1 than in group 2 (19,8-+1.3 vs 29.4_+2.0). obiectives : in the newly developed mode of ventilatory support ,,automatic tube compensation" (atc) the ventilator compensates for the flow-dependent pressure drop across the endetracheat tube (ett) thus allowing ,,e]ectronic extubation". the aim of the study is to investigate whether healthy subjects perceive atc in inspiration (atc-in) and in expiration (atc-in-ex) and whether atc provides an increase in subjective comfort compared with the conventional assisted spontaneous breathing mode (asb). methods : healthy volunteers (no preceding lung disease, non-smokers, male, 20-40 years)breathed spontaneously through an uncut ett of 7.5 mm id via a mouthpiece. the ett was connected with a prototype ventilator evita 2 modified by the manufacturer (drfiger, lebeck) for atc. flow and airway pressure were measured at the outer end of the ett. three ventilatory modes, (1) asb (10 mbarover 5 mbar peep), (2) atcin, (3) atc-in-ex were selected in random order. immediately following the transition from one mode to another the volunteers answered by hand sign how they perceived the new mode compared with the preceding mode: ,,better" (+1), ,,equal" (0) or ,,worse" (-1). inspiration and expiration were investigated separately by presenting 120 mode transitions (in total; including ,,placebo" transitions). results : the difference between atc and conventional asb is perceived in inspiration and in expiration. atc is positively judged; asb is nega ively judged. the diagrams show mean values _+ sd of five volunteers investigated up to now. the new mode atc is perceived as an increase in subjective comfort. our explanation is that atc preserves the natural breathing pattern better than conventional asb. objectives: to determine the role of cerebral vasoconstriction in the delayed hypoperfusion phase in comatose patients after cardiac arrest. to correlate the results with indices of cerebral oxygenation and the levels of several vasoactive hormones in the jugular bulb. methods: in comatose patients after cardiac arrest we measured the pulsatility index (pi) of the medial cerebral artery by transcranial doppler sonography. the pi is a reliable indicator of cerebral vascular resistance. we also sampled blood from the jugular bulb and measured cerebral oxygen extraction ratio and jugular bulb levels of endothelin, nitrate and cgmp. the first measurement was done within 4 hours after cardiac arrest and repeated 3, 6, 9, 12, 18 and 24 hours later. results: we studied 10 patients, 6 females, mean age 64,1+13,7 years. the pi decreased s!gnificantly between th~ first and the last measurement from 1.86 _+ 1.02 to 1.05 + 0.22 (p = 0.03). cerebral oxygen extraction ratio decreased also from 0.39+ 0.13 to 0.24 + 0.11 (.p = 0.015). endothelin levels were high, but didn't change during the studied period. nitrate levels varied in a wide range, but didn't change significantly. however, cgmp levels increased significantly from very low levels in the first measurement to very high levels 24 hours later, rasp. 2.95 pmol/ml (median; 25th 2.48-75th 5.43) and 7.5 pmol/ml (median; 25th 6.2-75th 14.00) (p = 0.02). eighteen and 24 hours after the first measurement we found a strong correlation between pi and cerebral oxygen extraction ratio ( r = 0.64, p = 0.05 and r = 0.76, p = 0.01). we.also found 12 hours after the first measurement a significant correlation between pi and cgmp levels ( r = 0.69, p = 0.03). we found no correlation between pi and endothelin or nitrate levels. conclusion.~; our results show a high cerebral vascular resistance in the first few hours after cardiac arrest, gradually decreasing during the next 24 hours. this is accompanied by an initially high cerebral oxygen extraction ratio and low cgmp levels, suggesting that the cerebral vascular resistance is induced by active vasoconstriction because of insufficient cgmp levels, leading to a decrease in cerebral blood flow and a compensatory ~ncrease in cerebral oxygen extraction. objectives: sudden cardiac arrest is a major cause of mortality in western countries accounting for over half of all cardiovascular deaths. in most cases the mechanism of death is prolonged cardio-circulatory arrest due to ver:tricular fibrillation (vf) preceding final asystole. recurrent syncopes due to idiopathic vf with good neurological prognosis have been reported in patients with and without cardiac etiology (1,2). in the past measurements of cerebral hemodynamics have been repeatedly done in humans during cpr, but until today no studies of cerebral blood flow velocity (cbfv) have been reported during controlled cardiac arrest in humans not under-going cpr. it was the purpose of our study to evaluate the acute hemodynamic effects of untreated vf on cbfv. methods: after approval by the local university ethics comittee, five male patients aged 34-48 years without evidence of cerebral disease were investigated during vf while undergoing implantation of a pacer cardioverter defibrillator system (model 7219d; medtronic| a standard anaesthetic regimen was used (propofol, fentanyl). after implantation of the automated cardiac defibrillator vf was induced by electrical countershock to test effective sensing, pacing, and defibrillation. to measure cerebral blood flow velocities (cbfvmca) the doppler probe was placed above the zygomatic arch between the lateral margin of the orbit and the ear and directed towards the m1 segment of the middle cerebral artery (mca). results: a total of 12 phases of vf were investigated. duration of vf ranged from 6 to 26 seconds, with cbfvmc a (mean_+sd, cm sec -1) flow pattern changing from pulsatile to laminar flow immediately after onset of vf. conclusions: the underlying mechanism of the laminar cerebral blood flow observed during vf in our patients is uncertain, but it may provide insight into the prognosis of patients with idiopathic vf. theoretically, the laminar cerebral blood flow observed in our pulseless patients may provide a substantial amount of cerebral perfusion even during clinical cardiocirculatory arrest objective: to investigate whether the intensive care nursing staff can inflate more accurately a specific air volume with the laerdal resuscitation bag when they receive feedback after each inflation about the delivered volume compared to no feedback. method: 42 icu nurses were asked to inflate a testlung model 10 times with a specific air volume (600 ml, 800,ml or 1000 ml) under three different conditions (normal, decreased compliance and increased resistance) without and with feedback. we measured the mean absolute difference from the specific airvolume after each ten inflations. results: the largest absolute difference was found when icu nurses inflated 600 ml (250 ml). the mean inflated volume for this group was 843 ml. when the icu nurses had to inflate 800 ml the mean absolute volume difference was 181 ml with a mean inflated volume of 913 ml. inflating 1000 ml produced an absolute volume difference of 131 ml with an mean inflated volume of 1042 ml. the absolute volume difference decreased when the compliance of the testlung was decreased and even more when the resistance of the used endotracheal tube was increased. when the icu nursing staff received volume feedback after each inflation the mean absolute volume difference was reduced between the 42 ml and 66 ml for all specific air volumes. 42% of the last 5 inflations with feedback were significantly smaller than 50 ml from the specific air volume (p < 0.05). conclusion: the majority of nurses overinflated the specific air volumes. the largest over inflation occurred when 600 ml and the smallest when inflating 1000 ml. when nurses were provided with volume feedback the performed significantly better. we concluded that icu nurses are not able to inflate a specific air volume with the laerdal resuscitation bag without receiving volume feedback. feedback is desirable in order to reduce the volume trauma. objectives: a pro_found impairment in systolic and diastolic myocardial function following successful cardiopulmonary resuscitation (cpr) has been demonstrated by using langerdorff method in rats. in the present study we have investigated post resuscitation myocardial dysfunction in a porcine model of cpr. methods: ventricular fibrillation (vf) was electrically induced by alternating current applied to the ep{cardium of the right ventricle in 11 domestic pigs. following 4 rain of untreated vf, precordial compression and mechanical ventilation was initiated and maintained for 8 min. electrical defibrillation was then attempted and 6 of 11 animals were successfully resuscitated. results: following successful cardiac resuscitation, stroke volume index (svi) decreased from prearrest value of 1.13 ml/kg to 0.74 ml/kg (p<0.05), and left ventricular stroke work index (lvswi) from 1.57 to 0.77 mmhg,ml/kg (p<0.05). both svi and lvswi remained depressed for another 3 hours. these decreases were associated with increases in heart rate from 145 bpm to 185 bpm (p<0.05). no significant changes from baseline in mean arterial pressure, mean pulmonary pressure, right atrial pressure and pulmonary artery wedge pressure were observed. prehospital resuscitation efforts c. k6ppel. g. fahron, h. lufft, a. kruger, c. th(jrk, f. bertschat, f. martens dept, of nephrology add medical intensive care, virchow-klinikum, humboldt-universit~t, d-13353 bedin, germany obiective: the success rate of prehospital resuscitation in patients with cardiocirculatory arrest in an emergency medical system (ems) may reach 30 -40% depending on the time of calling the ems, the distance to cover by the emergency ambulance and the training of the emergency physician and his staff. in the berlin ems, which is associated with the berlin fire brigade, the time between alarm and arrival at the scene ranges from 2 -31 min, mean 8 min. resuscftation is based on the advanced cardiac life support (acls) according to the guidelines of the american heart association. if resuscitation efforts fail to restore circulation, they are terminated after 30 -60 min, depending on duration of cardiocirculatory arrest, pre-existing disease, age, absence of an even transient response to cpr. however, there is a lack of practical criteria for termination of cpr in individual decision making. patients: we report 5 cases of prehospital cpr with primary asystolia terminated after 45 -60 rain of frustraneous cpr efforts including highdose epinephrine and dopamine. results: after termination of cpr, the ecg monitor remained connected and showed permanent asystolia in all patients while the emergency physician completed his records. spontaneous resumption of respiration and circulation was observed in these patients after 2 -5 min and cpr efforts were immediately resumed, nevertheless, 3 of the patients died at the scene, while 2 could be hospitalized with stable circulation. one of them died 3 hours after admission to the icu, the other survived for 3 weeks in a vegetative state. spontaneous resumption of circulation and respiration is most likely due to the development of extreme hypercapnia and acidosis, which -at least in some patients -seems to be a stronger stimulant of the circulatory and respiratory brainstem centers than cpr with high-dose catecholamines, conclusion: because of the legal and ethical implications of this rare phenomenon, emergency physicians should continue ecg monitoring for at least 5 rain. after termination of cpr efforts. pulmonary artery catheterezation is used for patient's monitoring [1]. we reported our results on such monitoring in 1969 [f.coaobbeb,r.fe6enb~-kap~monorm~,1969,n7,p.28-39] .however not all of the received criteria assessments meet demands that are necessary for early diagnosis of critical states. here we report the data on po2,pco2 (mm rg),so2,ph levels in femoral [af) and pulmonary (ap) arteries blood, as well as on summary gas pressure (sgp) calculated from pe=(po2+pco2) in mm hg in ap blood. these data were derived from:i)86 subjects free of cardiovascular pathology according to catheterization data during their spontaneous air breathing (n group in ap blood appears to be a measure of adequacy ratio between pc2 and sgp in ap blood during air breathing; partly its characteristics and variations ranges are presented earlier [2j. in control group it is equal to 1,91• mm hg. tests on sgp neither exclude nor substitute conventional (pc2 and pco2) tests, but rather include them as a part choosing only additive characteristic -pressure. they appear to be a part of general system of human metabolism regulation by pressure (arterial,venous,intracardiac, tissue,liquor,onco-osmotic,etc ietraabdeminal pressure produces perturbations of cardiac, pulmonary, and renal physiology. this most often occurs fonowing eeliotomy for peritonitis or intestinal obstruction; bowel edema and distention prevent wound closure without unacceptable compromise of blood pressure or pulmonary compliance. a variety of temporizing measures have been reported for managing wounds that cannot be closed: 1) using towel clips to reapproximate skin only, 2)i sewing silastic, marlex or other prosthetic grafts to the fascia to "enlarge" the peritoneal cavity, 3) using loosely tied retention sutures for partial closure, 4) simply packing the wound without attempts at c~osure. these techniques either traumatize the abdominal wall (complicating definitive closure), expose the bowel to damage, or allow excessive loss of fluid and heat. since 1989 we have evolved a suturelees technique which permits the abdomen to be partially closed in a quick, safe, sterile, sealed, atraumatic fashion -while providin! decompression of unphysiologic intraabdominal pressure. methods: whenever possible omentum is interposed between bowel and the open incision. viscera are covered by a layer of sterile, non-reactive plastic, placed deep to the fascia and extending we~t beneath the edges. sump tubes are placed above the plastic and covered in turn by two layers of an adhesive plastic drape which sticks to the skin and seals the wound in all directions, the patients remain intubated and paralyzed. results: we have used this technique in a total of 27 patients, four of whom suffered from compartment syndrome. all of the latter were males and ranged in age from 19 to 51. all four showed immediate physiologic improvement. all four incisions were eventually closed without complication. one compartment syndrome patient died 4t days later of multiple organ failure. there were no complications related to the closure technique in any of the 27 patients. conclusions; 1. selected patients with abdominal compartment syndrome will benefit from decompression using this temporary sutureless technique. the technique a) is quick, safe, sterile, sealed, and atraumatic, b) minimizes loss of fluid and heat, c) facilitates eventual definitive abdomina| closure. although m. brunner m. mitllncr objectives: to determine incidence and predisposing factors for cardiac arrest occurring during the first 24 hours after open heart surgery. methods: the study included patients who, following open heart surgery, had adequate cardiac function and in whom cardiac arrest was not anticipated. all data were prospectively recorded and analyzed. results: from 12/1993 through 3/1995, 2140 pts underwent open heart surgery at our hospital. of th~se, 23 pts (1%) (age 65_+9 yrs) had a cardiac arrest during the first 24 hours after transfer to icu. they were operated on for coronary artery bypass grafting (cabg) (17 pts), valve replacement (vr) (3 pts), cabg and vr (2 pts) and aortic aneurysm (1 pt). the preoperative ejection fraction was 44_+12% whereas bypass and aortic cross-clamp time were 127+70 and 72+42 rain, respectively. prior to arrest, they had a cardiac index of 2.23_+ 0.5 l/min/m 2 and were receiving 1.3+1 inotropes. arrythmias leading to cardiac arrest were ventricular tachycardia/fibrilation (10pts) and bradyarrythmia (9 pts). closed-chest cpr was initially performed on all pts and was followed by open-chest cpr in 12 pts. eighteen pts (78%) survived to icu discharge. causes of arrest included perioperative myocardial infarct (t2 pts, 52%), tamponade (3 pts, 13%), rupture of the proximal vein gra& anastomosis (1 pt, 4%), graft occlusion (2 pts, 9%); no cause was found in 5 pts (21%). conclusions: postoperative cardiac arrest in stable cardiac surgery pts is relatively infrequent (-1% incidence) and is associated with a high survival rate following successful cpr. perioperative myocardial infarct is the most common predisposing factor. group ~deptof anaesthesia and intensive care, semmelweis univ. medical school, 2 buda military hospital intensive care unit, budapest background: when a cardiac arrest occurs in-hospital, the outcome can be improved by a higher quality of basic life support provided by the witnessing health care workers until the code team arrives. this basic life ~pport (bls) should include the best available method for airway management as well. since not all medical staff are ready for carrying out endatracheal intnbation, we investigated the effieacy of the use of different airway management methods during bls. methods: we have investigated the efficacy of airway management of 25 doctors and 25 nurses from different hospital wards: internal medicine, department of surgery, trauma, urology and gynaecolagy. comparing the bag-valve-mask, laryngeal mask and the endotracheal intubafion, we have measured the following parameters: time needs for correct application (sec.), number of incorrect applications (out of ten trial), efficacy of artificial ventilation provided by the device. we used a computerised als trainer manikin for the evaluation of the performance. total performance score was created after the measurement between 0-10. after the first screening we held a 2 x 2 hours training. 8 doctors and 8 nurses were trained for the endotracheal intubation (group it1, 1t2) , 8 doctors and 9 nurses were trained to use the laryngeal mask (group lm1, lm2) . all respondent were trained to use the bag-valve-mask device. 1 day, 1 month and 3 month after the training we have carried out retention study using the same method. results: we have found that the efficacy of the artificial ventilation using the above mentioned devices were poor before the training. the average after-training performance scores of the groups are presented in the table below. (bls) should be initiated by the witnessing health care professional. the cpr study introduced a multi level code system, which means bls included sophisticated airway management, early defibrillation and early epinephrine administration provided before the code team arrives. our previous studies confirmed a poor level of cpr performance and a high demand for cpr training among health care professionals. method: we established a cpr training course centre, where doctors and nurses are being trained for in-huspital basic and advanced life support. 3 x 6 hours of training were held. after the theoretical introduction a step-by-step training method ws used for trainees to be familiar with all sequences of basic and advanced life support. then we synthetised all separated sequences. afterwards, a r01e play of rescue groups was taken in simulated situations. we also trained the multi level alarm system fur the in-hospital resuscitations. after the training all respondents had to sit for examination. the quality of performance was scored and compared to our previous results. semi-structured interviews were carried out before and aider the training among all respondents to collect information about the course. results: we have found a remarkably high interest among doctors and nurses in our cpr training courses. it was very important to use proper equipment for the training: audio-visual training facilities, computerised als trainer manikin, manual and automatic defibrillator units. the evaluation of the examination held immediately a~er the training course showed a significant higher quality of performance than before the training. the self.-eonfidence of the trainees for initiating and carrying out resuscitation had increased. their overall feeling about the course was positive and 100% responded the course "very useful". 73.6% of doctors and 79.4% of nurses claimed fur regular training facilities with als trainers, conclusion: the cpr training for health care werkers is mandatory including the training of sophisticated airway management and use of elad~l~ills~tt~r wlaa ~en ~r a~ti~atir ~nel r rm~a'*h*nr m~thnd for training will improve the efficacy, the satisfaction of trainees, therefore their compliance for further co-operation will also increase. s 144 objectives: the effect of reinfusion in emergency surgery and gynecology. methods: we had an experience of autologous blood transfusion in 22 patients whom was produce t an emergency surgical or gynecological interventions in occasion with break tubal pregnancies (45.5 %), penetrating abdominal wounds with injuries of mesenterial vessels (22.8 %), injuries of the liver (9.1%), blunt abdominal trauma with lien ruption (22.8 %). in 27.3 % patients had the previous somatic pathology. blood loss volume was 1500-4500 ml, & the reihfuside blood volume was 500-2000 ml, consisting 30-70 % of blood loss. it was needn't to fransuse donor blood in 18.2 % in further but 300-2500 ml of contanined erythrocytes were frasfused for supporting of hb concentration on the 80 g/l (8 g/dl) rate at the other patients with isovolemie hemodiluttion. results: the arterial blood pressure fast stabilisation on the perfusion level had noted after reinfusion, excluding the case, when the volume of reinfused blood had conisted just 40 % of blood loss at the patient with massive blood loss. complications have noted in two cases. one patient with slash wound, injury of arteria gastrica dextra and total blood loss of 4500 ml, has an episode of asystoly, dic (disseminated intravascular coagulation) syndrome, acute renal failure, and acute pancreatitis that we haven't connected to reinfusion. all the complications were successfully corrected and at thirty first day patient with subcapsular wound of the lien that has happened 14 days before complicated with external rupture of the capsull & massive intraabdominal bleeding, has the hemolytical shock, dic syndrome, acute renal failure developed after reinfusion. he was died. all another have no complications. posthemorrhagic anemia had corrected rapidly than in case when hemorrange corrected exclusively by donor blood. conclusions: we consider that simplicity, accessibility, high effectiveness, quite well further results of blood reinfusion, except the case of blood reinfusing that was for time-expired out of blood vessels (more than 10 days in our case) will promote to the wide spreading of this method, especially in emergency surgery, in massive injuries, & in disarters, all the cases of insufficiently of time for selection of lot of donor blood. objectives: study of a reaction of the oardioreepiratory system of pregnant women to i/v microperfusion of clophelinum which is known to eliminate hemodynsmic and endocrine nociceptive reactions and can be used for treating hypertensive syndrome in pregnancy and labor. methods: the following non-invasive methods were used: capnography, spirometry, oxygenography, indirect fick principle based on the circle breathing, plethysmography and integral rheography~ 52 functional indices of cardiorespiratory function were evaluated. results: 74 pregnant women with ~h-gestosis were examined before and after i/v infusion of i00 ml of 0.0001% clophelin solution, 0.005 mg/kg/hour. before the treatment intensification of carbohydrate metabolism, hyperventilation with moderate hypooapnia and complete respiratory compensation of metabolic acidosis~ increased alveolar ventilation, decreased alveolar volume, predomination of perfusion over ventilation, hypokinetio type of circulation with dominated load by peripheral vascular resistance to the blood flow was observed in this group of patients. microperfusion of clophelin imp~-oved the ventilation/perfusion ratio, ventilatory and gaseous exchange efficiency, resulted in a decrease of congestion in the pulmonary circulation, possibly owing to a decrease of peripheral vascular resistance by 17%, of the heart rate by io.5%, of the oardial output index by 9.5%. conclusionm: the resulted type of circulation with a decreased load on the heart both by resistance and volume allowed to improve the cardioreepiratory system function in pregnant patients. objectives: the injury severity score is a measure of severity of anatomic injuries. iss is a sum of squares of the highest degrees of the abbreviated injury scale (ais) for each of three most severity injured regions. the purpose of the study is to establish correlation between the iss values and mortality rate in older, polytraumatized patients. methods and results: iss was determined for 214 patients. the mean iss value was 27.65 + 17.36 while the median value was 21. minor injuries were present in 90 (42 %) patients with iss less than 21, while 124 (58 %) patients with iss more than 22 had severe injuries. increased mortality of the older patients was noted in the range 21-30. all patients older than 50 died while 20 % of patients below 50 yrs of age survived, indicationg correlation between iss and mortality rate in polytraumatized patients above 50 yrs of age. conclusions: this mode of evaluating severity of injuries may help in triage, determining appropriate level of care and as an indicator of future outcome of polytraumatized patients. objectives : tissue hypoxia is a non exclusive cause of hyperlactatemia. other serious medical situations induce hyperlactatemia. therefore, lactatemia could be a non specific indicator of severity in patients admitted in emergency unit. the aims of this study were to examine the correlations between lactatemia with the short term survival course prognosis and the unit of hospitalisation; intensive care unit (icu) or medicine unit, in patients admitted in our emergency department. methods -lactatemia was measured as soon as the admittance, in arterial blood sample of patients which needed arterial blond gas. sixty-one patients were included during 4 months. to assess the statistical performances of lactatemia, sensitivity (se), specificity (sp) and accuracy (ac) were calculated for the threshold determined by the youden's test (se+sp-1). results : fifteen patients were admitted in icu and 46 in a medical unit. fifteen patients died. a group of 35 patients had a lactatemia up to 2 mmol.l" 1. in this group of patients, 3 had acidocetosis, 3 had asthma, 3 had cerebral vascular ischemia, 3 had neoplasia, 2 had cardiogenic shock, 1 was epileptic, 8 had congestive heart failure, 6 had acute respiratory failure, 2 had septicaemia, 2 had hyperosmolar status finally 3 had medicinal intoxication. lactatemia was significantly higher in non survivor than survivor ( 5.5• vs. 2.3+1.0, p<o.oool, respectively), the cut point of lactatemia was 3.7 mmoll" 1 se was 60%, sp was 91% and the accuracy was 83%. on the other hand lactatemia was higher in patients admitted in icu than in medical unit (4.5+3.6 vs. 2.0• p<o.01, respectively). conclusions : this preliminary results suggest that lactatemia is a good indicator of the short term survival course in patients admitted in an emergency unit. furthermore, hyperlaotemia is present in several pathology seen in emergency. objective: to determine the pattern of injury, total peripheral white cell, neutrophil, and lymphocyte responses, and the outcome of trauma patients who are leucopaenic on admission to the icu. design: prospective, descriptive study of consecutive admissions over4 months. setting: a 16-bed surgical intensive care in a teaching hospital. patients: thirty consecutive adults admitted to the icu following trauma. leucopaenia was defined as a total peripheral white cell count of < 4 x 109, neulropaenia < 2 x 109, and lymphopaenia < 1.5 x 10 ~ cells per litre. measurement and results: the total peripheral white cell count was documented daily and the neurtophil and lymphocyte counts and differential percentages on days 0, 5, and 10. the incidence of leucopaenia was significanfiy higher in patients with gunshot wounds (p < 0.05) and hollow visceral intra-abdominal injury (p < 0.001). eight (27%) patients died. no significant differences were found in the initial mean total white cell, neutrophil, or lymphocyte counts nor in the differential percentages between survivors and non-survivors. the total peripheral white cell count increased significantly in survivors compared to those who died (p < 0.001) and significant differences were found in absolute neutrophil counts (p < 0.02) and differential percentages (p < 0.01) on days 5 and 10. no significant differences were found in lymphocyte counts or differential percentages. conclusions: there is a signficant association between leucopaenia, neutrepaenia, and intra-abdominal hollow visceral injury and peritoneal contamination. survival is related significantly to resolution of these white cell deficiencies. these findings suggest a possible role of granulocyte colony stimulating factor in the trauma patient with intra-abdominal infection and persistent neutropaenia. amelioration of organization emergency care team in order to improve caring for urgent cases. objectives: emergency care team (ect), as integral part of health, respectively as activity of primary health protection care all acute difficult conditions which vitaly danger patients and distressed. the aim of our investigations was to indicate on the possibilities of amelioration of organization ect in order to improve caring for urgent cases. methods: in our investigations we used epidemiologicai and statistical metods all informations and matherials from terrain ects. results: the number of traumatized in traffic accidents etc. have a epidemic character. it is well known that 58.1% traumatized died in first two hours. ect isn't enough qualified to care a traumatized on the place of accident and during transport. because that medical personnel must have an education with solid qualities. conclusions: 1. besides a physician specialized (vii --+ vii2 -~ viii gradus) in urgent medicine, it's necessary to introduce two (2) medical technicians instead of one as is existing now (1 ~ 2). 2.the medical technicians can drive cars and one of them always, drives the ambulance car ("b" category). 3.the technicians have been specially educated for urgent cases (iv ~ v ~ vi). 4. such a new team structure should be more capable in professional sense. 5.we can see an importance of team-work in ects. 6. such teams should also be more economic in comparison with existing one. 7. we think, that on the general medical studies need to include a new discipline-urgent medicine! objectives: to detoxicate an organism since 1983 we have used biological sorbents: isolated from liver and spleen of human or animal cells or tissue fragments. methods: cellular dialysis (164 dialyses) in patients with an acute hepatic insufficiency poisoning by hepatotropic poisons: cc14, dichloethane, mushrooms -were conducted using "artificial kidney" apparatus, by means of arteriovenous shunt, formed at forearm with disposable dialyzers. hepatocytes suspension was poured into dializing circuit of dialyzer at 5-10 mg of cells per i kg of patients weight. dialysis was performed during 1-2 hrs. 168 patients with purulent-septic states were treated as for hemoperfusion through prepared sorptional column: 40.0 ml of hemosorbent and 30.0 ml of fragmented spleen or hepatocytes suspension with the help of roller pump with the rate of 80-120 ml/min, during 90-120 min. average volume of hemoperfusion made 3.51. conclusions: in all cases we used both native and cryopreserved biomaterial. analysis of the dynamics of clinical, biochemical, scintigraphic and us-investigastions allows to conclude that application of cellular-sorptional method of detoxication enable to prolong life and reduce lethal outcome in some severe category of patients with endotoxicosis. after infusion of diazepam (0.2mg/kg),thiopentone (4mg/ kg) and vecuronium (lmg/kg),patient was intubated and mechanical ventilation was adjusted to mantain normoca= pnia. ct scan showed left parieto-occipital hypodensity with brain swelling. after admission in neurological icu,dexamethasone (24mg/ kg/die),mannitol (0.smg/kg) and diphenylydantoin (20mg/ kg/die) were administred. two hours later neurological status of the patien~ was normal and she was extubated. the risks of stereotactie radiosnkgety are related to: hemorrhgge during the latency interval;transient radia= tion effects;permanent radiatio~einduced complications; radiation induced neoplasia. no epilepsy is reported in pts with avm and no seizure before radiosurgery. in c0nclusion, epilepsy can be a posmib~e delayed eompli c~t~n, ~fe~r rgdi0surgery for cerebral avm. serum potassium deficiency is a frequently reported finding in the time course of acute myocardial infarction (ami). if this is also correct for patients with ami undergoing primary percutaneous transluminal coronary angioplasty (ptca) is unknown.for that reason we analysed in 20 patients (14 m.,6 f.,61 + 10 y.) serum potassium concentrations on ad ,mission and directly after ptca.the reference intervall for potassium in our clinical laboratory is 3,5 -5,0 mmol/l. although more than 90 % of the patients with ami undergoing ptca showed normal potassium serum concentrations on admission a measurable decrease of potassium could be found in 65 % of patients (13120) after ptca.therefore for the majority of patients with ami potassium supplementation prior to ptca can be justified to avoid electrolyte disturbances as a possible trigger factor for cardiac arrhythmias in the setting of ami and acute reperfusion due to primary ptca. objectives: diagnostic procedures are very important in the management of abdominal injures in trauma. the aim of this paper is to present our experience with peritoneal lavage as diagnostic procedure in abdominal injury. methods: we analyzed patients to whom peritoneal lavage was applied in the surgical emergency from 1st of january 1991 till 31st of december 1994. results; in this four years period there were 250 abdominal injures. there were 110 blunt injures. among them in 43 patients peritoneal lavage was applied. results were positive in 28 patients and negative in 11 patients. false positive finding were in 2 patients and false negative in two patients. in patients with positive lavage there were more than 90,000/mm3 red blood cells, more than 1,000/mm3 white blood cells and the level of amylase was up to 8 u/i. the gall, urine and stool were positive as well. in patients with negative lavage there were less than 40,000/mm3 red blood cells, less than 500/mm3 white blood ceils and amylase were negative. conclusion: peritoneal lavage is useful and important diagnostic procedure in the management of abdominal injures in trauma. our experience with urgent surgical management of the acute gastric and duodenal bleeding s.se6en md, z.milo~evi6 md, *s.srdi6 md, k.sar6ev md. clinic for abdominal and endocrine surgery, institute for surgery; *clinic for cardiology, institute for cardiovascular diseases; school for medicine novi sad, university of novi sad, yugoslavia objectives: acute gastric and duodenal bleeding are very common complication of gastric and duodenal ulcer. the aim of this paper is to present efficiency of our surgical management of these acute conditions. methods: in this paper results of surgical management of patients with acute gastric and duodenal bleeding, treated in our clinic from the 1st of january 1991 till 31st of december 1994, are presented. results: among 92 operated patients there were 31 females (33.70%) and 61 males (66.30%). there were 52 patients (56.53%) with gastric bleeding and 40 patients (43.47%) with duodenal bleeding. bleeding from gastric ulcer were treated with the following methods: excision of ulcer with suture in 23 patients (44.24%), ulcer suture in 12 patients (23.07%), billroth i in 9 patients (17.31%) and billroth ii in 8 patients (15.38%). heinecke-mikulicz-weinberger pyloroplastica was the most frequently used in the treatment of the duodenal ulcer bleeding. bilateral truncal vagotomy was done in 34 patients (85%), duodenotomy with ulcer suture was done in three patients (7.5%) and three patients underwent billroth ii operation. we had three complicationsduodenal dehiscence in pyloroplasty. there were no lethal complications. conclusions: based on our own experience we conclude that acute bleeding from gastric and duodenal ulcer can be safely managed with urgent and modern operative techniques. dept. of anaesthesiology. emergency center of serbia, belgrade, srj 0b0ectives and methods: cardiac contusion was evaluated by serial determination of cpk-mb fraction and co using non-invasive doppler technique in 60 patients with blunt trauma of the chest. results: miooardial contusion was diagnosed in 34(57%) patients, while in 26 (43%) it appeared unaffected. cpk-mb of 3% and more in comparison to total cpkwas 6.4+4,2% in 34 patients, and 1,9+0,6% in 26 pts (p<0.01).c0 of 5.8+1.17 l/min in patients with cardiac contusion, measured 16 hrs after admission at the latest, was significantly lower than in group of 26 patients with co of 4.8+0.86 l/min (p<0.01). conclusions: co values were significantly lower in patients with cardiac contusion and correlated with pathological values of cpk-mb fraction. objectives: extracorporal plasmophoresis (pph) was implemented by non-stop method in 28 patients with obstructive jaundice. methods: changes of serotonin (s) and histamine (n) in arterial (ab) and venous (vb) blood were investigated before and after one-time session of pph. s and h in blood serum were determined with fluoremetric method. results: before pph the average s contents in ab and vb did not differ. after one-time pph session s in the blood outflowing lungs was 23 % lower than in the blood inflowing. 24 hours after extracorporal detoxication implemented the indices kept the same level. similar dynamics after pph was observed in h. before the session h contens was equal both in the blood inflowing and in the blood outflowing lunge. after pph h contents in ab was 18 % lower compared to the vb and did not change for 24 hours. conclusions: dynamics of changes of biogene amines shows that under the influence of pph there takes place not only mechanical removal of their surpluses together with plasm, but there sharply grows inactivating role of lungs towards ba substances, which is confirmed by reduction of their concentration in the blood outflowing lungs. this effect is probably connected with the "deplasmation" of the lung cellular elements. simultaneously lungs' cells are freed not only from plasm but from toxic adsorbents on their surface. as a result the lung cells activate absorption of the surplus number of amines from the vessel channel. it is not excluded that s inactivation is due to growing activity of monoaminooxidase resulting in deminishing of intoxication. acute pulmonary embolism: thrombolytic therapy or pulmonary embolectomy? i.lodiena md, phd, s.shevchenko md, pphd., medical university, kharkov, ukraine objectives: pulmonary embolism (pe) remains a challenging problem for diagnosis and management for the emergency physician. the aim of this work is to identify the best treatment for massive pulmonary thromboembolism. methods: during recent 5 years 150 patients with symptomatic acute serious pe were reffered to our observations. selective pulmonary arteriography confirmed this diagnosis in all cases. 4 (2.7 %) underwent urgent surgery, of them 2 (1.33 %) with cardiogenic shock and 2 (1.33 %) with massive pulmonary embolism. 146 patients (97.3 %) underwent thrombolytic therapy. results: in the patients who received thrombolytic therapy successful results were achieved in 133 cases (91.1%), in 13 (8.9 %) patients subsequent pulmonary embolectomy was performed. mortality rate was 2 (1.36 %) in the patients who underwent pulmonary embolectomy after unsuccessful thrombolytic therapy and 4 (100 %) in the patients with cardiogenic shock and massive pe. lower extremity deep venous thrombosis caused pe in 89 % patients. pe mortality rate is repoted to be 4 %, but all patients with cardiogenic shock and massive pe died. this very high mortality rate related to the clinical status of the patients whose condition became worse despite intensive treatment. conclusions: the results of this study show that in most cases thrombolytic therapy is an effective rapid method of treating pe. however, surgical treatment is preferable when the patients reveal adverse effects to drug therapy, when medical therapy is unsuccessful or when the patients are seriously ill with reccurent cardiac arrest. high-quality pulmonary angiography remains the most accurate and reliable means of diagnosing pe. this prospective study was conducted to investigate the serum thyroid hormone levels in traumatized critical ill patients. serum thyroid stimulating hormene(tsh),total thyroxine(tr4),free thyroxine(ft4),total tri-iodothyronine(tr3),free tri-iodothyronine (ff3) levels were measured within 24 hr.of intensive care unit admission(first day) in 17 multiple tratmmtized male patients(injury severity score higher than 17). in fifth day the same hormone levels were repeated. tsh,et4,ft4,tr3 and ft3 levels were determined by radioimmueoassay(ria). the levels of 'i~sh,xt4,fr4,et3 and ft3 in survivors and nonsurvivors were compared with first and fifth days (table) . in conclusion, we are convinced that there was a high correlation between low ser~n thyroid hormone levels and mortality, serum thyroid hormone levels are prognostic value in traumatized critical ill patients. objectives: glucagon improves myokardium contractility, intensifies kidney blood flow, stops bronchospasm (1), these were the reasons of investigating its effect on hemodynamics and metabolism in hypovolemic shock expermentally and in clinic. methods : in experiments on white rats with traumatic shock glucagon (novo nordisk) was injected intraperitoneally in dose 20 rocg/kg. patients with hypovolemic shock (trauma, hemorrage) were injected glucagon intravenously in dose 1 mg on the background of infusion therapy. results : glucagon hightened recovery indexes of glucose in rats and patients with shock, whereas in groups without glucagon hyperglycemia developed. rats with glucagon injection had double times urea content than the norm ( 10,5+1,5 mmol& norm -4,9+0,3 mmol/i ), whereas rats without glucagon had urea content 30,3 +1,8 mmol/1 ( p < 0,001 ). osmolarity of blood plasma in rats having glucagon injections was 301,5 + 2,9 mosm/kg h20, without the drug -317,0 + 3,5 mosm/kg h20 ( p < 0,01), ldh activity -27,7 + 2.9 and 51,0 + 9,6 ( p < 0,01) mcmol cec-1 accordingly. lethality in the group of rats without glucagon was 70% /n=50/, with glucagon -0 (n = 70 ). in patients with hypovolemic shock in 15 minutes after glucagon administration stroke volume increased to 71% (sv), miocardium contractility -to 70% (mc), peripheral resistance of vessels ( vr ) decreased to 10%. in 1-1,5 hrs sv increased to 100%, mc-to 126%, pvr -decreased to 116%. conclusion : the study provides the evidence that glucagon has an anti-shock effect, improves hemodynamics and metabolism indexes in cases of hypovolemic shock. reference : picazo j. glucagon in acute medicine : pharmacological, clinical and therapeutic implications (norwell, mass:kluwer publishing,1993 ), 172 p. n, vasina (t) ,n.sebbota hph (2). dept; of acute endotoxicosis, n.v. s~lifosovslay scientific research institute of ermergency ltealth care, boscow, lk'ussia (i}. institute for problems of cryobiology and cryouledicine of the national academy of sciences of the f/maine, kl'k3r-key, [7maine (2), objectives: the investigation of isolated hepatocytes using's efficacy in patients with acute hepatic fai lln-e. methods: native and crioconsrved allo-and xeno-!mmatocztes fixed on semi;ermeable ~mbr~s were used. results: the artificial supporting system with isolated hepatocttes as metabolic ~its was. ap-plied in 85 patients. during tl~ treatmen~ with this cytotherapz an improvement of clinic state and biochemical rates were observed even inthe first 8 we~s. for instance, the kncreasing of glutamin acid's level in blood up to normal value and decreasing of ammontes -concentration from i9~26 mmol/l down to 89#15 mol/l were discovered. ~ae diminution of ence,%halo;athy was noted too. general lethality was' equal 37 z, there of 2i z concerned acute hepatic failure (/k~). conclusions: the using of sum~rting hepatic system is able to cope the ~nenomenon of ~i~. objectives: fat embolism (ee) is an important and insufficiently studied problem of the intensive care medicine. fe is clinically diagnosed in 25% and morphologically in 80-90% of the victims with severe multiple skeletal injury that is accompanied by the shock. methods: during the last 5 years 27 patients with the cerebropulmonary form of fe were treated in the icu. the prognosis of the probability of fe development was done using the computer system "cite-prognosis" in which the triss-method, dynamic and statistical prognosis of the injury outcome~ and the values of the most severe injury in points were realized. the treatment consisted of the surgical stabilization of the fractures with the authors' design of the rod apparatus in the acute period, long-term mechanical ventilation with peep via tracheostomy, complex fluid therapy using perfluorochemical emulsions (pe), and electrochemically active solution of na hypochlorite (snh) via the central vein. results: the treatment of 27 patients with fe gave a good result that was manifested by the complete regression of psychoneurologic and respiratory disorders. conclusions~taking into account the prognostic data~ it is necessary to provide early complex of intensive care to the polytraumatized patients. it includes the surgical stabilization of fractures, fluid therapy using pe and snh~ early long-term mechanical ventilation. dr. alexandr e. poladko, station of medical aid. kiev ukraine. objectives: the reason of investigation is to prove the necessity of beginning of the intravenous infusion of nitroglycerin before the hospitalisation. methods: from january i to june 30 1994 protocols of treatment myocardial infarction patients in kiev medical aid station. results: the results of treatment are better if infusion began early or prolonged during hospitalisation. conclusions: there were 30 patients with acute myocardial infarction treated before hospital by the cardiological groups. 100 patients were treated with the early infusion of nitroglycerin and 200 without. the results of treatment were: in the group with infusion: mortality in the first hours 5 % the full disappear of pain 90 % in the group without infusions mortality in the first hours 8 % the full disappear of pain 82 % that's why our opinion is that infusion of n. is necessary in the treatment of the patients with myocardial infarction from the first minutes of illness. dr. gennady d. kyrzhner, station of medical aid, kiev, ukraine. object: looking for safe medication which is good for the treatment of arterial hypertension and is simple for use. we inspected the effect of prazosin in the conditions of the cail during the year. method: we used 1 mg prazosin tablets sub lingua and measured blood pressure 4 ones during 2 hours. the parameters were registrated in protocol. result." one hundred patients took part in investigation. the middle age of patients was 69. the reason of hypertension was not taken into consideration. in two hours after beginning of the treatment we caught the reliable lowering of blood pressure in 90 % of patients. the general condition of patients became better in 10-15 min. it was only one accident of complication during the treatment -the orthostatic hypoter~sion, conclusion: prazosin hydrochloride in the dose 1 mg sub lingua is safe and simple for use in the urgent situations. objectives: hypomagnesemia is frequently observed in severely burned patients during the early period after injury. increased urinary excretion is insufficient to explain the magnitude of the magnesium (mg) depletion. the possibility of exudative cutaneous mg losses has been proposed, but not yet demonstrated. the study aimed at measuring the mg content of the cutaneous exudates and urine during the first week after major burns. methods: 16 patients, aged 34_+9 years (mean _+ sd), and burnt 37_+11% of body surface, were studied from day 1 (d1) to d20. serum samples were collected at do, serum and urine from d1 to d7, and on dio, d15, d20. cutaneous exudates were extracted from the textiles surrounding the patients, collected over 24h periods from d1 to d7, using bidistilled acidified water. mg supplements (8.2 to 123 mmol/24h) were prescribed from d1 to dio according to the severity of hypomg (serum mg < 0.7 mmol/i). results: mg serum levels decreased below reference ranges in 12 patients between d1 and d4, and normalised thereafter during supplementation. urinary mg excretion was increased in 11 patients, mean excretion being 4.8_+1.3 mmol/24h until d20. mean daily mg cutaneous losses, were 16 mmol/24h, i.e. 112 mmo; in 7 days. large inter-& intra-patient variability: the daily exudative losses ranged from 0 to 83 mmol/24h. conclusions: the mean daily mg cutaneous losses after burns were larger than the urinary losses, and equivalent to the recommended intakes for mg; the addition of the exudative and urinary losses largely explained the increased mg requirements after burns. complex immunologic alterations occur following thermal injury. the activation and consumption of the complement and dotting systems are suggested to play an important role in the development of the capillary leak syndrome, tn burned patients, a generalized inflammatory reaction as well as the impairment of the host defense are considered to be the major reasons for complications, such as sepsis and multiple organ failure. in a pig model we investigated a possible protective effect of cl-inhibitor (berinert, behring). before conducting the animal experiments the human cl-inhibitor concentrate was analyzed for its inhibitory capacity on purified pig cls and its pharmacoldnetic behaviour in pigs (t1/2,id50). pigs received anesthesia and analgesia and arterial, venous and pulmonary (swan ganz) katheters were placed into the carotis and jugular veins. the pigs were scalded for 25 seconds with 75~ hot water to achieve a 30% tbs partial-thickness burn. blood samples were taken prior and after surgery as well as 15, 45 minutes, 4, 8, 12 and every further 12 hours after thermal trauma. two control groups (each n=6) included animals which were either scalded or not scalded and treated only by resuscitation of ringers lactat solution. besides various parameters blood samples were analyzed for complement. the pigs (n=4) received c 1-inhibitor concentrate at an initial dose of 100 u/kg body weight either immediately or 12 hours after thermal trauma (n-2), followed by three further applications (of reduced amounts) every 12 hours. the clinical outcome as indicated by vital parameters and as well as demonstrated by reduced edema and inflammatory tissue destruction suggested a protective effect of cl-inhibitor. complement analysis revealed a faster recovery of the alternative and classical pathway activities in cl-inhibitor treated pigs as compared to the control group but only if the regulator was given immediately after thermal trauma. from these results a protective function of c 1 -inhibitor on thermal trauma can be assumed. objectives: the aim of study was to characterise the pattern of secretion of interleukin-6 (il-6) and tumor necrosis factor alpha (tnf alpha) in multiply injured or not injured critically ill patients and to relate these results to their outcome. methods: blood samples of 12 multiply injured ( 238 (73) 148 (46) 154 (57) il-6 (nis) 156 (81) 88 (54) 65 (39) il-6 (nin) 117 (68) 150 (76) 157 (04) conclusions: this data suggest tnf alpha is a better prognostic marker in patients with multiple injury, than in critically ill patients without injury. il-6 is not significantly higher in nonsurvivors of both groups. sepsis is associated with an increased production of nitric oxide (no), as reflected by a rise in plasma levels of nitrites (no2) and nitrates (no3). severe bum injury is associated with similar symptoms of inflammation like hypotension, high cardiac output low vascular resistance and increased vascular permeability so that an increased no production may be involved. the present study included 16 patients admitted to the intensive care unit of the burn center of brussels (age 35:1-18 years; burned surfaee area 374-19 %), and 6 control (non-septic) patients hospitalized in the neurological rehabilitation unit of the erasme hospital (age 64 4-18 years). the patients in both groups were fed enterally with 30 to 40 kcal/kg/day of a standard solution. in the burn group, daring the study period, 6 patients were mechanically ventilated, 4 required vasopressor (dopamine) therapy, and 5 received antibiotics; 13 patients were discharged alive from the intensive care unit, and no patient died during the study period. plasma was sampled for no2/no3 determinations (griess reaction) daily from day 1 to day 5 (n=ll) or to discharge (day 3, n=2; day 4, n=3) in the burn group, and once the control group. in the burn group, no2/no3 levels were elevated at day 1 (70_+ 12 #moles/i), reached a maximal value on day 2 (874-26 p.moles/l) and progressively decreased to day 5 (fig). these values were higher than in the control group (5.6:t:7.6 #moles/l, all differences p<0.01). however, no correlation was found between the plasma no2/no3 levels and the age of the patient or the total burned surface area; burned patients who became infected tended to have higher plasma no2/no3 levels than those who did not (177 + 131 vs 83 4-48 #moles/i, ns). the present study indicates that burn 120 noa/noa (llmol**/l) injury is associated with increasedloo] [__ ] ~ no2/no3 plasma levels, which are ao so consistent with an enhanced no pro-4o duction, ao obiectives:urapidil has been recommanded for therapy of hypotension in neurosurgical patients, because it was found not to increase intracranial pressure in patients with compromised intracranial dynamics (1). in contrast several authors reported an increase of icp after urapidil administration in patients with head injury (2). our study was designed to determin the influence of urapidil on mean lumbar cerebrospinal fluid pressure (csfp) in awake humans with uncompromised intracranial compliance. methods: after approval by the local university ethics comittee, six volunteers (asa 1, 4 male, 2 female) were studied in the lateral decubitusposition with the vertebral column positioned horizontally. a 25 gauge needle was inserted into the lumbar subarachnoid space at level l3/l4 and connected to a trancducer (mx860| medex inc.) for csfp measurements. the volunteers were advised to keep respiratory rate and etco2 constant. after a resting period of 10 minutes basline measurements were performed including csfp, cvp, map, hr, etco2 (cardiocap 2| datex). then the volunteers were given urapidil 0.2mg/kg over a period of 1 minute. 5 minutes later measurements were repeated. results: statistics: wilcoxon signed rank test, p<0.05 significant; values: mean_+sd, si = mmhg. 10-2_1" 74_+5* 64_+5* -3_+1" 35_+2 csfp: cerebrospinal fluid pressure, cpp: mean cerebral perfusion pressure. conclusions: during normoventilation urapidil leads to a decrease in map parallel by an increase in csfp and therefore in icp, most likley mediated by an autoregulatory dilatation of cerebral vessels. if this cerebrovascular response is suppressed by hyperventilation, no increase in icp is found despite a uredipil induced drop in map (2). after longterm hyperventilation cerebralbloodflow is normalized. this might be the reason why the drop in bloodpressure following urapidil administration again led to an increase in icp ( background and objectives: the intestine is one of the most sensitive tissues to ischemia-reperfusion injury. reperfusion of the intestine is often associated with increase in mucosal permeability and ulceration. d(-)-iactate is produced by indigenous bacteria found in the gastrointestinal tract and mammals do not possess the enzyme systems to rapidly metabolize it. the present study was designed to determine the kinetics of plasma d(-)-iactate alteration in rats paused by acute intestinal ischemia-reperfusion injury. methods: following the anesthesia, the superior mesentcric artery (sma) was exposed and it was occluded by a micro-bulldog clamp applied at its origin from the aorta. after 75 min of occlusion, the arterial clamp was removed and the supply area of the sma was allowed to be reperfused ( 6 h). plasma d(-)lactate levels were measured by an enzymatic spectrophotometric assay. the endotoxin content of plasma sample was assayed by the limulus amebocyte lysate test with a kinetic modification of the test kit procedure. results: intestinal ischemia for 75 min resulted in a significant increase in d(-)-lactate levels within the portal vein as compared to sham-operated animals. plasma d(-)-lactate levels had a tendency to further increase after reperfusion up to 6 h. it was showed that significant elevation of endotoxin concentrations in portal vein was alread~r detected at the end of 75-min ischemia, reaching peak at 0.5 h post-reperfusion and decreasing gradually throughout the study period. at the end of ischemia, marked mncosai damaged such as edema, hemorrhage and necrosis was observed. there was evidence of injury to ti, e muscuiaris propria together with diffuse mucosal damage and destruction following reperfusion, particular at 6 h postreperfusion. in addition, penetration of bacteria was commonly found in the intestinal wall at various time points following ischemia/reperfusion injury. conclusions: these data suggest that acute intestinal ischemia is associated with permeability change of mucosal barrier resulting in increased plasma d(-)qactate, which is also remarkably influenced by subsequent reperfusion. plasma d(-)-lactate may be a useful marker of intestinal injury following both ischemia and reperfusion insult. objective: to assess the clinical usefulness of two methods of intracranial pressatre (icp) monitoring: intraparonquimatous recording (icpc) and epidural recording (icpe), versus intraventricular icp (icpv) as a gold standard. we prospectively studied 13 patients with severe head injury (glasgow coma score < 8) admitted to our icu between fobmaly 1990 and december 1993. icpv was monitored in all pativrats by means of a multipzffolated catheter connected to a water coinnm. six of 13 patients were additionally icpc monitored by a fiber optic transducer (comma 420r~), and seven of 13 patients were icpe monitored by a couplvd fluid system (plastimedr). icpc was placed homolatetal to the focal, lesion m two cases and contralateral in three; icpe was placed homolateral to the focal lesiou in two cases and contralateral in three. all three systems were calibrated at the ear level. stali~cs: correlation coefficient and lineal regression were done between icpe and icpv, and between icpe and icpv with the hourly p~rs of values obtained dttting assistontial period. results: of the six icpc -icpv studied pa~onts, we observed a correlation coef~cieut r = 0.84 (p < 0.001) with a regres~on line y = 0.75 + 0.93x. four of ~hx lcpc -icpv patients had r > 0.85 when correlaliou eoet~dent was obtained indixddually. of the seven icpe -]cpv studied patients, we observed a cortelafiau ooeffioiont r = 0.47 (p < 0.001) with a regression line y = 7.2 + 0.43x. corralalmu eoetfieiont was inwer than 0.5 in all seven patients. corrdation eoelfieients for levals of icpv > 20 man hg, > 25 mm hg and > 30 tuna hg with icpe showed r = 0.89, r = 0.91 and r = 0.97 respectively; and with icpe r = 0.25, r = 0.13 and r = 0.09. the obtained values did not change during the study. conclusdns: in our study icpe was considered a good type of icp monitoring. /cpe signiticantly infravalorates icp values. we observed a good correlatinn between icpc and icpv values in patients with high inttacramal presanre. objective: midazolam is a benzodiazepine agonist widely used for sedation in emergency medicine. few studies in animals and humans point to a direct analgesic effect of midazolam probably mediated by spinal antinociceptive receptors and/or peripheral benzodiazepine receptors (1,2). in our experience in the berlin emergency medical system (unpublished results) with anecdotal cases of extreme chest pain due to binge drinking but no evidence of acute myocardial infarction or extreme abdominal pain due to peritonitis, acute intermittent porphyria, peutz-jeghers syndrome or testicular torsion, we found that small doses of midazolam (2 -5 mg i.v.) were much more effective in relieving pain than repeated administration of high doses of buprenorphine or morphine, which may be associated with a considerable respiratory depressant effect. the dose of midazolam required for pain relief in these patients is non-narcotic and allowed further communication on the character and localization of' the residual pain, which might be very important for the further diagnostic procedure. patients: ten patients with abdominal pain due to acute gastrointestinal bleeding, suspected pancreatitis, suspected acute porphyria, and chest pain with no evidence of acute myocardial infarction received first-line midazolam i.v. at an initial dose of 1 mg and were asked how it affected the intensity and character of pain. results: at the chosen dose of midazolam (2-8 mg), all patients were responsive to detailed questioning on basic orientation, the character, intensity and localization of the pain, and medical history. none of the patients required an additional opiate. all patients stated that the pain was tolerable after midazolam alone. conclusion: our preliminary clinical observations suggest that low-dose midazolam might be an alternative to opiates in extreme pain of presumably visceral odgin. objectives: it is known that severe head injury in elderly patients is associated with higher mortality than in younger patients. it remains however to be clarified whether the preinjury pathology which is frequent among these patients, affects the outcome. methods: in an attempt to investigate this hypothesis, 79 patients aged over 60 years suffering from head injury, with glasgow coma scale (gcs) of 8 or less, were studied retrospectively. twenty-six patients (32.9%) had preinjury pathology i.e. diabetes mellitus, arterial hypertension, heart failure, alcoholism, parkinson's disease etc. (group a) and fifty-three (67.1%) did not (group b). the following data were recorded: mortality in the i.c.u., duration of hospitalisation, incidence of infective complications and neurologic status at discharge. results: groups were comparable in terms of mean gcs (6.57 vs. 6.56) and median age (67.5 vs. 67). the incidence of brain pathology in the two groups was the following: epidural haematoma 7.69% vs. 11.32%, acute subdural! haematoma 30.7% vs. 30.19%, intracerebral haematoma 19.23% vs. 5.66%, subarachnoid haemorrhage 38.46% vs. 39.62%, diffuse haemorrhage 11.54% vs. 13.21%, contusion 26.92% vs. 49.06% and non-visible pathology (normal ct) 3.85% vs. 1.89%. unilateral pupilary dilatation was found to be 15.38% in group a and 18,87% in group b. the mortality during hospitalisation in the i.c.u. was almost the same: 50% iu group a and 47.2% in group b patients. however, group a patients had significantly more infective complications, required longer hospitalisation and had lower gcs at discharge. conclusions: the results show that the existence of preinjury pathology does not seem to affect the short-term outcome of elderly patients with severe head injury. it has however an impact on morbidity and perhaps long-term survival of these patients. the assessment of clinical development in intensive care patients with severe head injury still remains a problem. to optimize the monitoring of intracraniel prassure (icp) we rautlr~dly implant an eplduml measuring device in our hospital. the aim of this study was to prove the correlation of the icp-values with ct findings and clinical development. during a 12 month period (1993 -9r the icp was monitored in 23 p~,tients (14 male, 9 female) with severe head injury by an eplclural measuring device (epldyn~/$plegelberg| the mean age was 36.9 years (4 -83). the glasgow coma scale at admission was 6.9 (3 -15). in all cases the device was placed wfihln the first 10 hours after admission. the tcp was compared with physical examination, radioidglcal or intraoperatlve findings and cunlca! outcome. the average time of measuring was 7. 2 days (1 -19) . the traatment depended on the !cp values recorded. rising icp-valuea ~ed to radlologlcal c0ntra!s by ct-scan. in 1 case an intracranlai hemorrhage was detected and drained. the overall survival rate was 78.3 %. 113 showed a complete resolutl0n, in other 33.3 % psychological residuals like decreased mentatlon, in 17.4 % sensomotorlc residuals like cerebral nerve dysfunction and aphasia, and 11.1% of the injured remained in a comatous status. in 87 % of our cases the measured values correlated with clinical course and management. in 2 cases (8.6 %) we observed a displacement of the icp-pevice. there was no icp induced infecllon. istituto di anestesiologia e rianimazione, universit& ,,la sapienza", rome, italy * istituto superiore di sanit& -servizio di epidemiologia e biostatistica, rome, italy objectives: acute renal failure (arf) can be a severe complication of trauma. the current incidence of post-traumatic arf is associated with high mortality 1. identification of risk factors and prevention of this complication could improve the outcome of trauma patients. methods: one hundred fifty three consecutive trauma patients (age 37.6 _+ 19.6, injury severity score 28.3 +10.9) admitted to icu were studied. incidence of arf was 31.4 % (48/153). arf was defined as persisteat plasma creatinine >2mg/dl with or without oligoanuria 2. arf was defined as early when occurring within the first 96 hours (earf) and late when the onset was after the first four days (larf). results: earf occurred in 31 patients while larf developed in 17 patients. age, iss, and incidence of rhabdomyolysis and acute respiratory failure were not different in the two groups. an higher incidence of multiple organ failure (mof) and sepsis (76.6% for both) were observed in larf group, when compared to earf (25% and 23% respectively). abdominal trauma was more frequent in earf group (32% vs 18%). the gs for earf and larf were respectively 8_+4.4 and 9_+4.15 while in the group who not developed arf (narf) the gs was 10.5• conclusions: gs score difference seems suggestive and can be that an abnormal cerebral activity (hipofisary hormones?) may play a crucial role on onset of arf in these patients. moreover the frequency of acute respiratory failure in the group of arf was higher (91.7 versus 64.5) than narf group. the early ipoxia in the early phase of trauma, then, may be another crucial point for development organ failure. these are preliminary data. a more exact statistical analysis must be perform to have definitive conclusions. to compare the active compression-decompression cardiopulmonary resuscitation (acd-cpr) with the standard cardiopulmonary resuscitation (s-cpr) in out of hospital cardiac arrest patients. is a controlled, randomized study. two groups of patients with cardiac arrest out of the hospitalwere formed. group i, (acd-cpr) and group ii (s-cpr). for the acd-cpr groupweusedthecardiopumpdeviceofambulnternational. asfortherest, the erc (1992) algorithms for acls were followed. the utstein style (for out of hospitat cardiac errest) was used for listing and evaluating all cases of the study. the cpr was contucted by the crew and the doctors of our mobile intensive care units (micu). we studied 146 consequitive patients (75 in group i) and (71 in .group ii). demographics pre-cpr characteristics (e.g. ecg form of cardiac arrest) and procedures (eg bystanders or second tiers crew cpr, defibrillation, drugs) were quite similar for both groups. the mean arrival time of micu was 9min. in group i we recorded r.o.s.c. (return of spontaneous circulation) 17,5%, death 73%, continuation of cpr efforts 9,5%. while in group ii, 21%, 69%, and 9,9% respectively (recorded percentage until the admission to the hospital). no significant difference was found in anyofthe short term outcome parameters. no complications related to the acd-cpr technique, were noted. not any significant difference between the two methods was proven (from this small evaluated sample). the results of previous clinical studies are controversial (i) . more sophisticated studies proved the superiority, in a certain number of parameters (e.g pressures, flow, etc) of the new technique although there are many difficulties for establishing clinical results. in the pre-hospital setting that is related to many parameters (speed of the intervention, effectiveness of bystanders cpr, education ofparamedics, etc.)the evaluation is even harder. the superiority ofthe acd-cpr can be proven when it is performed in almost 0 times increased number of studied patients as w~ll as improvement of the technique could lead us to more established results. objectives; infectious morbidity is the major cause of mortality after burn injury, and is due to multiple factors. trace elements (te), which are involved in both humeral and cellular immunity, exhibit severely altered status after burns. te supplementation has been shown to be associated with increased leukocyte counts and shortened hospital stay. the trial aimed at studying the immune responses in severely burnt patients receiving normal te supplies or early large supplements. methods: 12 patients, aged 40_+16 yrs (mean_+sd), with burns covering 49+18 % of body surface were studied from day 1 (d1) to d30 post-injury, were randomised in 2 groups (g): g1-control receiving recommended te supplies + placebo; g2 -receiving in addition large supplements of cu, se and zn from d1 to d8. enteral nutrition was started within 12 hours of injury in all patients. immunological parameters: peripheral leukocyte counts, proliferation of mononuclear cells to mitogens, cell surface molecule expression, and neutrophil chemotaxis at d10 and d20. infectious episodes and micro-organisms were monitored until d30. results: the patients' characteristics were similar g1 & g2. the total leukocyte counts were higher in g2 between d10 and d20, due to increased neutrophils (significant from d13 to d15). total cd3+ and cdlg+ cells did not differ, whereas cd14+ (monocytes) were significantly increased at d20. proliferation to mitogens was significantly depressed in all patients. chimiotactism was not altered. the number of infectious episodes was significantly decreased in g2 with a mean of 2.0_+ 0.9 infections during the first 30 days versus 3.3_+ 0.8 in the control group (p < 0.03). conclusions: the large te supplements for 8 days was associated with a significant decrease of the number of infectious episodes. supplementation was associated with increases in total leukocyte, monoeyte and neutrophit numbers. further studies are required to determine the precise mechanism underlying the improved immune defences. objectives: evaluate the efficiency of local adsorption (la) with the use of carbon adsorbents in case of severe burns in expertment and clinic. methods: experimental studies on la were performed on a model of 20% body surface area iiib-iv burn in 335 rats. a burn eschar was excised on the 3rd day after burn, the wounds were dressed with the gauze bandages (control) or with adsorptive dressings (la), dressings were regularly changed. clinical investigations were carried out in the course treatment of 78 patients with severe thermal and radiation ilia-iv burn. in the dynamics of bum disease some indices of proteometabolism and intoyacation criteria were evaluated. results: the experiments have demonstrated that the application of la after early excision of a burn eschar exerts a pronounced normalizing effect on a protein electrophoregram and the activity of proteases and their inhibitors in burned tissues preserving vitality. thus, by the 14th day after burn infliction the activity of cathepsin d in injm'ed muscles is 6 times lower under an adsorptive dressing than under a gauze bandage (control) (p<0,05), the activity of trypsin-like proteases is 1.5 -3.4 times lower and the antitryptie activity does not differ significantly from the normal level. the cytotoxicity of extracts of burned tissues after the adsorptive dressing application fn vivo and adsorption in vitro is 25-35% and 7-20%, respectively, of the toxicity of control extracts. a similar normalizing effect of la is ok~rved for an intact muscular tissue and blood serum. the dectron-spin-resonance studies have demonstrated that la allows to normalize antitoxic activity of liver and functional activity of kidneys. the application of la in the treatment of patients with severe burns have been shown to localize a region of irreversible tissue changes, accelerate rejection of a burn eschar, attenuate an endogenous intoxication level and, as a result, shorten the time for grafting of a burn wound and accelerate wound heating. conclusions: proceeding from the obtained results, we can consider la as an effective method of localization of a region of irreversible tissue changes as well as of correction of local and general metabolism failures and overcoming burn autointoxication during burn disease. c de deyne, t vandekerckhove*, j. decruyenaere, b. vaganee, v vandewalle*, f colardyn depts of intensive care and neurosurgery*-university hospital gent-belgium. jugular bulb oximetry is the first bedside available cerebral monitoring technique providing an estimation of the adequacy of cerebral perfusion. its routine use in all patients suffering from severe head injury admitted to our ic unit enabled an extensive analysis of all very early cerebral perfusion data in order to evaluate the incidence of abnormal sjo~ data (and their possible causes) in this very eady period after traumatic insult and to search for possible implications as to the emergency management. these very early data were defined as the first 6 hours icu data and icu admission had to occur within 12h of traumatic insult. over the last 2 years, 150 pts with severe head injury (gcs<8) were monitored by jugular bulb oximetry, starting immediately after their arrival at the icu (mean of 4.8h after trauma, range between 2-9h). in a total of 85 pts (=56.6%), jugular bulb desaturatiens (<55%) were noticed during this early 6h period. in 24 pts (=16%), jugular bulb saturations higher than 75% were observed, whereas 41 pts (=27.4%) revealed no abnormal sjo 2 data (55-75%) during these first 6h. concerning the periods with too low jugular bulb saturations (n:85), we found the following correlation ; in 49 pts (=57.6%) cerebral perfusion pressure (cpp) was below 70 mmng, in 36 pts (=42.3%) paco~ was below 30 mmhg and finally in 6 pts (=7%) we found primary intracranial hypertension. for the high jugular saturations (n:24) we found a primary intracraniaf hypertension in f0 pts (=41%), and a pace 2 level above 40 mmhg in 6 pts (=25%). in all patients we could restore jugular bulb saturation within normal range (55-75%) with the correct!on of the presumed causative factor. we can conclude that ultra early jugular bulb saturation data revealed a high incidence of abnormal values, with a predominance of jugular bulb desaturations, confirming once again the high incidence of disturbed and too low cerebral perfusion within the first hours after severe head injury. these jugular bulb desaturations were especially correlated to systemic causes, as a too low cpp (caused in the vast majority by primary map insufficiency, and not by intracranial hypertension) and hyperventilation were the 2 major causes of the desaturation periods. as jugular bulb desaturatione are known to be significantly correlated to a worse neurological outcome after severe head injury, one might improve outcome by an emergency management avoiding these possible causes of jugular desaturation. therefore, extreme attention should be paid to the maintenance of an adequate mean arterial blood pressure (above 90 mmhg?) even duhng the few time spent at the emergency department. one should be as attentive to the maintenance of normoventilation during this very early period of admission and hyperventilation without any knowledge of icp or sjo2 should be abandonned. recently, indomethacine has been proposed for the treatment of therapy refractory intracranial hypertension in pts suffedng from severe head injury (1). indomethacine, a cyclo-oxygenase inhibitor, gives rise to a significant fall in cerebral blood flow by inducing cerebral vasoconstriction. therefore, its use could result in a drastic lowering of the intraeranial pressure (;cp) in pts suffering from intracranial hypertension secondary to cerebral hyperaemia and in whom the use of other cerebral vasoconstrictive drugs (barbiturates or hyperventilation) appears insufficient to control icp. for the last 18 months, we included the use of indomethacine in our therapeutic flow chart for severe head injury management. pts revealing intracranial hypertension (icp>20 mmhg) and cerebral hyperaemia (sjo~>75%) and in whom icp was not efficiently controlled by the combined use of hyperventilation and barbiturates were given indomethacine in a trial to control icp. a total of 98 head injured pts received treatment for intracranial hypertension over the last 18 months. six of them met the criteria set for the administration of indomethacine. in 2 pts, no decrease in icp or in sjo 2 was observed and both pts died due to therapy refractory intracranial hypertension. in the other 4 pts, a significant fall in icp and in sjo 2 was observed shortly after indomethacine administration. in 2 pts we observed a catastrophic fall of sjo= even below 55%, indicating an extreme cerebral vasoconstriction with the possible risk of inducing cerebral ischaemia. in one of the 4 pts, icp remained under control without further administration of indomethadne, but he died 3 days later in multiple organ failure. the other 3 pts, needed multiple indomethacine administrations (for 1 pt even during 4 consecutive days) to finally control icp. in all 3 pts, icp was finally controlled, but only 1 pt survived. both other pts died from systemic causes (multiple organ failure in 1 pt, massive gut infarction in the other tat, possibly due to the systemic vasoconsttictive effects of the indomethacine administration). in conclusion, indornethacine might have a role in the treatment of intraoranial hypertension, especially when caused by cerebral hyperaemia. we observed however a poor final outcome and a threatening high incidence of systemic events (multiple organ failure, gut infarction) in those pts receiving indomethacine for icp control. therefore, indomethacine in the treatment of intracranial hypertension should be reevaluated in controlled study settings, before its routine use can be considered. untill recently, intracranial hypertension (ich) in pts suffering from severe head injury was managed in a staircase approach, with csf drainage as first therapeutic step, mannitol as second step, hyperventilation as third step, and finally, barbiturates as the last rescue step for therapy refractory ich. this staircase approach for the treatment of tch was only guided by the intracraniat pressure, and not by other parameters such as e.g. the actual state of cerebral perfusion of the concerned pt. jugular bulb oximetry provides us with the first, bedside and continuous available, estimation of cerebral perfueion. its implementation in a rigourous flow chart, based on as well icp-as jugular bulb oximetry-data might result in an altered strategy for ich management. we adopted a '~ugular bulb saturation (sjo~)-guided approach" for ich management in 86 consecutive pts, suffering from severe head injury (gcs<8). we maintained csf drainage as first therapeutic step, but the decision for the second step was guided by sjo2 information. pts revealing ich and sjo=values above 75%, were treated with hyperventilation, and did not receive mannitol. if ich persisted, barbiturates were added as a third step. on the other hand, pts with ich and sjo= vales less than 75%, received mannitol administration as second step. hyperventilation and/or barbiturates were only added if ich persisted and if no cerebral hypoperfusion was discerned (sjo=>55%). our objectives were to prospectively analyze this new therapeuticstrategy, as compared to the formerly used staircase approach of ich. we managed 86 pts with ich, with an overall mortality of 13.7% due to therapy refractory ich. all pts received standard primary care with head elevation, full sedation and normovenfilation. fer 16 pts, csf drainage alone was sufficient to control ice of the remaining 70 pts, 38 pts received mannitol and 32 pts were hyperventilated as second approach. in the third line, 14 pts were managed with barbiturates, 12 with mannitol and 10 pts with hyperventilation. finally, barbiturates were used as the final rescue in 14 pts. these results reveal a less frequent use of mannitol as only 50 pts received mannitol, compared to the 70 pts that would have received mannitol using the former staircase approach. hyperventilalien was used much earlier in the treatment course, as 32 lots were already hyperventilated in the second line approach, were this was formerly exclusively reserved for the third line approach. finally, also barbiturates were used much eadier (14 pts received barbiturates as third approach). we may therefore conclude to a important change in the management of ich, induced by a sjo2-guided flowchart. however, future studies will have to elucidate if this new strategy for the intensive care management of severe head injury will also result in an improved outcome. obsectives: in a first series of experimental brain injury we investigated the course of brain po2, icp and cerebral blood flow after traumatic brain injury (tbi), whilst accordingly there are very few data available and the mechanisms leading to secondary brain damage are poorly understood. methods: in 6 piglets (14 days old, 3,3-5 kg) of either sex we produced a moderate brain injury (1,5 arm., 20 msec.) using a lateral fluid percussion {fp) device. complete measurements were made before and 5 min. after brain trauma and after 3, 5 and 24 hours including blood gases, cardiac output (htermodilution), heart rate, eeg, laser doppler flow probe (ldf} and icp values (camino), brain temp., po 2 by a clake type oxygen electrode (licox) and coloured microspheres for regional blood flow. results: immediately after the trauma a typical "cushing"response to the icp peak up to 130 mm hg being highly significant (before mean i0 mm hg, range 4-12 mm hg) could be observed: mean arterial blood pressure rose from appr. 85 mm hg to ii0 mm hg for 3-5 min. in two animals this was followed by an ischemic period lasting 15 min. accordingly icp values gradually returned to starting measures within 3 hours; in the ischemic animals they remained at a level of about 30 mm hg.-no secondary increase of icp could be observed, once icp dropped to starting values within 24 hours. cerebral blood flow (ldf) fell from mean values being i00 before trauma to appr. zero and recovered to around 50. brain po 2 started at mean values of 20 mm hg (range 15-30 mm hg) and fell to around zero depending upon the severity of the ischemic reaction. on average values of 15 mm hg were reached over the time course. conclusions: with our fp trauma model we can reproduce the well known "cushing"-response after brain injury; secondary icp elevations cannot be achieved, although local edema is observed. direct brain po 2 measurement seems to be a very sensitive variable for detection of cerebral ischemia and anticipates eventually following icp elevations by far. pulmonary aspiration s,traoaras. v. sgountzos, p. agouridakis, m eforakopoulou, e. ioannidou. intensive care unit (tcu) of "kat" hospital, athens, greece ob!e=ives: the reported mortality rate after pulmonary aspiration is variable in several series. the purpose of this study was to find out the influence of preexisting disease or situation on morbidity and mortality of intensive care unit (icu) patients with pulmonary aspiration. methods: patients who were treated in icu and had pulmonary aspiration, were studied, entrance's criteria in the study, all of them obliged, were: 1) suction of gastric contents from trachea during intubation, 2) presense of a predisposing factor, e.g. coma. 8) recent hypoxaemia or new infiltrates in xray. preexisting disease was recorded and correlated with complications and outcome. patients with glasgow coma scale 3, because of cerebral injury, and patients who died within 3 days from cause other than aspiration, were excluded from the study. method of statistical analysis: chi-square test, results: one hundred forty five patients were studied. the trauma patients were 96 and the non trauma patients 49. from the trauma patients, 77 had cerebral injury and 19 were polytreumatized without cerebral damage. from the non trauma patients, 13 had malignant neoplasms, 14 neurological diseases in terminal stage, 7 old age, 10 drug overdose, and 5 several diseases. eighty seven from 96 trauma patients (91%) and 45 from 49 non trauma patients (92%) manifested several complications (pneumonia, ards, etc), so there was no statistical difference in complications' frequency between the 2 groups (p>0,1). the severity of complications was also proportional in the 2 groups. eighteen deaths were recorded in the trauma patients (mortality 19%). only 7 deaths correlated directly or indirectly with the aspiration (7%). in non trauma patients, 32 deaths were recorded (71%). twelve deaths were recorded in 13 patients with neoplasms, 12 deaths in 14 patients with neurological diseases, 6 deaths in 7 aged patients, 1 death in 10 drug overdose patients, and 1 death in 5 patients with several diseases, the mortality difference in trauma and non trauma patients was statistically significant (p< 0,001). in patients with drug overdose the mortality was significantly lower from the other non trauma patients and the difference was statistically significant (p<0,001). conclusion: the preexisting disease or situation plays a major role in the outcome of the patients with pulmonary aspiration. the mortality of patients with aspiration seems to be caused by severe preexisting situations rather, that lead to death, than from the pulmonary aspiration per se, which may be a final happening in a predetermined course. obiectives; the purpose of this study was to compare fluconazole and amfotericin-b in the treatment of fungal infections in severe trauma patients. methods: thirty five severe trauma patients who were treated in intensive care unit (icu), were studied prospectively. they all developed fungal infections, prooved with blood positive cultures and at least one of the following: fever, positive urine or bronchial secretions cultures, infiltrates in xrays. the patients were separated randomly in 2 groups. the patients of group a (15 patients) received fluconazole 200 rag/day for 15 days. and the patients of group 8 (20 patients) amfotericin-b 50 rag/day for also 15 days. compaiison's criteria were the clinical responce to treatment (fever etc), the fungal elimination (blood and other cultures), the relapses of the disease, the side effects of drug, and the outcome of the patients. as method of statistical analysis was used the chi-square test. results: nine patients from 15 of the group a (60%), and 18 from 20 of the group b (90%), presented remission of fever (patients of group b had better clinical responce than patients of group a, and the difference was statistically significant, p<0,05). all the patients before treatment had positive for fungi blood cultures. after 10 days of treatment, 3 patients of group a and none of group b had positive cultures. eight patients (from 13 who had positive cultures of bronchial secretions before treatment) of group a. and 5 (from 17) of group 8. had positive cuttures of bronchial secretions after 10 days of treatment, so positive bronchial secretions were fewer in group b than in group a, but this difference wasn't statistically significant, (p<0,1 and p>0,05): ten patients (from 12) of group a and 7 patients (from 16) of group b had positive urine cultures, after 10 days of treatment (positive urine cultures were fewer in group b than in group a and this difference was statistically significant. (p<0,05). two patients of group a and none of group b had a relapse of fungal disease. in group a, no side effects were obsepced, while in group b were observed only minor side effects (small increase of serum creatinine in 2 patients, chills and fever during infusion in 3 patients, and hypokalemia in 12 patients). three patients of group a and 1 patient of group b died, because of sepsis. conclusion: amfotericin-b (even i~ short regimen of 15 days), is superior to fluconazole in the clinical and laboratory responce and also in the relapse of fungal disease, fluconazole is superior to amfotericin-b as it has no side effects. ob!ectives: flail chest after thoracic trauma is a serious injury. it is controversial if flail chest by itself orthe concomitant intrathoracic injuries e.g. pulmonary contusion, is the cause of the reported significant morbidity and mortality. in this study we searched the influence of concomitant thoracic injuries in the course and outcome of patients with flail chest. methods: eighty five patients with flail chest after isolated chest injuries were studied, for the purpose of analysis, we separated the patients into 4 groups, patients with isolated flail chest were included in group a, patients with flail chest and hemo-pneumothorax in group b, patients with flail chest and pulmonary contusion in group c, and patients with flail chest and hemo-pneumothorax and pulmonary contusion in group d. complications from the chest, duration of mechanical ventilation and mortality were compared in the 4 groups. statistical comparison of results belween groups was made using chi-square and t-studend tests. results: the patients were 85. all patients received mechanical ventilation, twenty eight patients were ihcluded in group a, 19 in group b, 20 in group c. and 18 in group d. seventy three patients manifested complications from the chest, especially pulmonary infections. there was no statistical difference among the 4 groups as to number of complications ( twenty four patients had chest complications in group a, 16in group b, 17 in group c, and 16 in group d. p>0,1}. the duration of mechanical ventilation was not statistically different among the 4 groups (the mean duration was 15,9 days in group a, 16,8 in group b, 16,5 in group c, and 17,5 in group d, p>0,1). there was also no statistical difference in mortality among the groups (six patients died in group a. 4 in group b, 4 in group c, and 5 in group d, p>0,1 ). conclusion: flail chest by itself is a serious thoracic damage with many complications, regardless of the presense of other thoracic injuries, which don't contribute to greater morbidity and mortality. the present study investigated the correlation between blood lactate mortality and organ failure in 129 trauma patients admitting between december 1, 1992 and july 31,1993 in the icu. road traffic accidents were the most common cause of trauma in this studded population. brain damage was the main cause of mortality .nevertheless, 29 of patients died from sepsis and multiple organ failure without significant brain damage and these deaths were potentially preventable. respiratory failure was the most common complication and was developed in 44 (44%) of survivors and in 26 (86%) of non survivors .we noted low fncidence 5 of renal failure may be do to the early and aggressive ittv'asive hemodynamic monitoring and cardiopulmonary support. as part of our routine case protocol serial blood lactate levels were measured in each patient at least 3 times a day until the valses returned within the normal range or until death. we analysed the blood lactate levels on admission, the highest value and the number of days until the first normal value (<l.smeq/i). initial lactate and highest lactate levels were significantly higher in patients with than without organ failure.(3.4 vs 2.4, 4.1 vs 2.8meq/l, p<0.01)the duration of hyperlactemia also was higher in the patients with organ complications. (2.2 vs 1.0 days, p<0.01). both initial lactate and highest lactate levels were higher in the non survivors than in the survivors.(4.0 vs 2.8,4.6 vs 3.4meq/l, p<0.01) the present study demonstrated that not only the degree but also the duration of hyperlactemia can be related to the development of posttraumatic complications. serial blood lactate measurements are reliable indicators of morbidity and mortality following trauma. s155 current evidence suggest that peep only affects intracrenisl pressure (zcp)when it interferes with systemic arterial pressure, although the effects of peep over cerebral oxygenation remains unknown. objective: determine the effects of peep over icp and cerebral metabolism measured by sj02, kjdo 2 and ceo 2 in severe head injured patients. meterial ~ methgds: patients with glasgow coma scale l 8 at arrival, with difusse brain injury in the first gt, according to marshall criteria,lop and 8jo 2 monitored, under analgesia and sedation, normoventilated with zeep, without mannitol treatment in the previous 4 hours, normal x ray chest and within the first 48 hours from injury, were considered candidates. peep value where increased fro~ zero to 15 cmh20, in three different steps, each one with 20 min of duration. all date were analized on line, pmax p'p ..... p.._, from the ventilator, systemlc haemodynem]c data, cerebra perfuslon pressure (cpp), icp and sjo 2, in the case of hemodynamic deterioration, during the study, ppc 6ommhg, extra-volume were administered. if persistence, dopamine were begun or increased dosage. if no good response were obtained, patients were retired from the study. results. 40 patients were candidates, 15 of them were exc;uded due to haemodynamic instability, k total of 25 completed all steps and were e~amined, 22 men and 3 women, age 32!7.9 yrs. at hospite; arrival, glasgow were < 5 in 18 patients, and >5 in the rest 7. 15 patients 20 mmhg at the beginning. zeep ob/ectives. critically ill patients are transpoded to an intensive care unit(icu), under conditions, which have not been systematically evaluated. therefore, we set suite investigate transportation and admission condition of these patients to our department. methods. we studied 36 patients(16 females), aged (mean-..+-sd) 56.2_ 17.3yrs, which were consecutively (from august 1994 to march 1995) admitted to the icu, through the greek national emergency transporta~on service. apache ii severity score upon admission was 17.4-+6.8 (range 4-31). the following data were evaluated: 1) number of medical departments, where health care was provided until final admission to the icu, 2) ambulance transportation conditions, 3) catheters and tubes inserted before admission, 4) vital signs upon admission 5) information provided by referring physician (scored on a 1 to 5 scale: history, electrocardiogram, chest x-ray, laboratory data, drug therapy already administered), 6) comparison of the state of the patient described by referring physicians, to the actual state u pen admission. resu/ts. one to four medical departments had provided health care before the palient was admitted the icu (1:22.2%, 2:47.2%, 3:27.7%, 4:4%). thirty/36 (83.4%) patients were escorted by a physician. twenty-six/36 (72.2%) were transported on oxyge n, fio2(mean__.sd): 46-+3%, pao2: 78.6-+35.2mmhg. five of the remaining 10, for whom no oxygen was provided, had pao2: 46.2-+7mmhg. twelve/36 (33.3%) were intubated and ventilated during transportation. thirtyfour/36 had a peripheral venous line, 5/36 had an arterial line, 13/36 had a nasogastdc tube, 20/36 had a urinary catheter. eleven/36 were sedated and 2/36 were paralysed. three/36 were on inotropes. vital signs upon admission were: arterial blood pressure, systolic 100.6-+44mmhg, diastolic 57-+23mmhg, heart rate 104-+22 bpm, temperature 36.3 -+2cc. patient information score was 217--. 1.7. the actual state upon admission was found substantially different, as compared to the description of the referring physician, in 28/36(77.7%) patients. conclusions. we conclude that several aspects of the greek national emergency transportation service to an icu should be reevaluated and further improved, i. e. ventilatory support, adequacy of information provided and accuracy of prior description of the patient's state. a new perspective must be applied for critically ill patients transportation since 78.8% of the patients were evaluated and treated in more than one, medical departments, mostly primary care, before they were finally admitted to our icu. dclhb is a human derived hemoglobin molecule that has been cross-linked to stabilize and permit heat pasteurization to remove residual proteins and inactivate viruses. dclhb is mixed with a lactated electrolyte solution to yield a total hemoglobin concentration of log/dl objective: to present an overview of four recently completed clinical safety studies of dclhb in the u.s. and europe, and to discuss the properties, actions and potential indications for dclhb. method: patient populations in the four studies included males and females ranging in age from 18 to 84 years. dosing ranged from 25mglkg to 300mg/kg. the controlled randomized safety studies were conducted in chronic renal failure patients, surgical patients undergoing total hip replacement or abdominal aorta repair and in hemorrhagic hypovolemic shock patients. these very diverse patient populations allowed safety evaluation of the product in patients who were generally elderly, often hypertensive with some degree of cardiovascular disease, and receiving medications for treatment of other conditions. results: over 150 patients received dclhb in the four:studies. no product related sarious adverse events occurred during the clinical trials. conclusion: results from phase itll safety studies of dclhb in patients undergoing chronic renal dialysis, abdominal aorta repair, or total hip replacement and in patients in hemorrhagic hypovolemic shock, indicate that the product was well tolerated in these distinct populations. although these studies were designed to evaluate safety, the data suggest clinical benefit. follow-up efficacy trials are indicated. prehospital emergency services represent the extension of emergency care into the community and constitutes the manpower, communications, transportations and facilities used to provide care for patients outside hospital. one of the main points of the system is how to decide the hospitalization of patients and what kind of facilities to provide : emergency medical service, fire brigade, locat general praclitionner or ambulance officers. objectives : to realize guidelines for using the prehospital emergency medical service in case of patient'calls outside hospital. methods : from 1st june 1994 to 14 july 1994, all the calls for emergency care were analysed using a questionnaire of 114 items (origin of the call, responses to the questions of an emergency practitionner, kind of emergency service provided and the issue of the patient). after taking account of the appropriatness of the decision, statistical method used was a logistic regression. results : 996 calls were analysed. the criteria, for prehospital emergency medical service using, given by the logistic regression were as following : existence of a call for emergency, thoracic pain, dyspnea, seizures, cyanosis, drug intoxication, fall of the patient, fracture, age, the state of consciousness and the neurologic reactivity. the minimal and maximal predictive values of the model given by the logistic regression are respectively 2% and 100%. the performance of the model is 88 %. conclusion : it seems possible to help medical decision of emergency medicine by using only some easy criteria and a predictive model. (italy) objective: to evaluate the incidence of blunt carotideal injury (bci) in patients admitted to our icu after head injury. methods: we reviewed the medical records of all patients diagnosed to have a bci. at admission, the severity of trauma was assessed either with glasgow coma scale (gcs) and with ct scan. bci was demostrated by doppler ultrasography (us) and by angiography (ang). results:since may 1991 to april 1995, 4 patients were admitted to our icu with bci (2m,2f, age 29+ 1 3). a history of direct trauma was present in 2 patients. admission gcs was 15 in all patients, and was associated with hemiparesis in 3 of them; the last became paretic 48 hours thereafter. two patients had concomitant injuries (a homoiateral clavicular and a controlateral zygomatic fracture, respectively). the initial ct scan was negative in every patient, and showed signs of ischemia after a variable timespan (2-4 days) after the onset of the symptoms. the bci was diagnosed with us and ang, which demonstrated a thrombosis of the internal carotid artery (ic). in two patients, an intimai dissection was also present. three patients were treated with heparin associated with antiaggregating agents and were discharged alive. the last patient was referred to our icu after the development of a massive hemispheric infarction, and died three days after the admission. at necropsy, the ic thrombosis was associated to an extensive homolateral extra and intracranial venous thrombosis. conclusions:the presence of focal neurological signs despite a negative ct scan should address the diagnosis toward a bci, thus implementing the diagnostic workup with us and/or ang. tab i: distribution of l~tients (%) in the 3 groups the outcome were monitorett results were sabmitted to statistical analysis using a continence table 3x2 in z2 test. res.cl~s: of 43 patients 34 were submitted to thrombolysts and 3 died. the higher incidence of bracb, ar~lhmias (ii degree gg p t39e 1 and 2 av block. 11i degree av block. avsb 6.141 <ii.05 sinus arrest) that requited the insertion of avsc 11.121 <cl005 temporar~ pace-maker was recorded in group a b vs c 0.004 ns in 64% of the cas6~. the rapid recognition of life tab li:;~ test. threatening conditions suggests the monitoring of v4r-lead r b' in the course of inferior ami. the authors report their own expirience in application of ringer lact.(rl)and 7,5%nacl/6%dextran 70. methods:in 40 polytraumatized patients with developed hae morrhagic shock,injures of the chest(qg)and abdomen(q2) prevalid.replacement of the volume loss 5egan in institutions of general medicine,frequently by rl and 69dextran 70 or haemaccel.after receiving necessary medical aid,polytraumatized patients wer transported to mma by helicopter,continuing replacing of circulation volume by the same solution. results:average quantities of the solution 8dministered ~o the injured wer~2375-2550ml.a group of the inoured which,after the admission to mma was resuscitated by adml nistration of the 7,5%nacl/6%dextran 7@(groupii)(4-sml/kg observed to the final surgical management,had statistically higher map(increase of 58% in relation to admission), cvp(average increase of 8,25cmh~o),diuresis(averagely 35omi after 30min.),better gas ~nalyses and acid-base status in relation to the group which was administered only rl(groupi)(increase of the map of q8%,average increase c~ of cvp 2,5cmh20 and average diuresis 20oml after 3omin). total quantity of the administered solutions was significantly smaller in the groupii(2800ml:4925ml).the only ob served complication was hyperhatr~f~a::and~moderat~hyper osmolerity of plasma which disappeared within 32hours. the quantity of replaced blodd was considerably smaller in the groupii(qooofnl:20ooml).in the group i interstitial pulmonary oedema was observed in two patients(qo%),while: in the group ii no complication was observed.coagulation i disorder and hypotension were not recorded because of the fast infusion of hypertonic/hyperoncotic solution in the groupil as the infusion of 7,5%nacl/6%dextran 70 lasted 5 minutes. w.scherzer(l~,k.lechner(1),r.simon(2). ll)dept.anaesthesiology,trauma hospital,vienna-meidlin 9 !(2)neurotraum.rehabilitation center,vienna-meidling both austrian workers'compensation board,vienna,austria what we usually call"unconcious" means only that the patient is not able to interact in any kind of way.experiences in intensive care medicine (robinson,1975) ,in general anaesthesia (bennet,1985) and with evoked potentials show,that unconcious patients are not automatically unhble to process and recall information.this implicates a challenge to start the efforts to restore the traumatica-!ly desintegrated brain function as early as possible in the acute phase ia (zieger,1992) .we present a method to ~repare the patient for the rehabilitation goals of the postacute phase ib,the phase of stabilisation ii and of rehabilitation iii within one concept. to achieve sensory stimulation in phase la we use: attempt at some kind of dialogue by speaking to and tou-ching the patient,to make him experience his body limits, ~acio-oral therapy by ice application,tast and smell pro4 vocations and trials of feeding,early adaption to circadian rhythm and guiding the patient in every-day-life-ac-tivities e.g.:in the brushing of teeth,washing etc. to achieve motor stimulation and orientation in terms of ~-ace,time and gravity in phase la we use: avoidance of perceptual impairment as produced by air-:sack-beds or habituation to supine position,using method~ according to affolter(1980) ,basal stimulation (bienstein, 1991) , bobath-therapy(1966) and facilitation of a physiological moving pattern(pnf)according to knott(1968) . conclusions: by integrating all these measures in the ~herapy and nursing even in the acute phase la one prei ares the comatose patient for multisensory stimulation nd motor training and all other rehabilitation activities in the following phases of therapy. s157 objectives." measurement of cardiac output (co) and stroke volume (sv) requires insertion of a central venous catheter and is associated with a considerable risk for complications. non-invasive methods for determination of hemodynamic parameters have been introduced recently. one of these methods is the aortic modelflow program, which provides co and sv continously using a three-element windkessel model. the aim of the study was to evaluate the accuracy of the aortic modelflow program in critically ill patients. methods: 21 patients (mean age: 69) admitted to the emergency department after cardiopuimonary resuscitation (n=ll) and for myocardial infaction (n=5), acute congestive heart failure (n=4) and anaphylactic shock (n=l) were included to the study protocol. after stabilization all patients received a swan-ganz catheter (baxter~; edwards swan-g-anz) via a central vein. co and sv were measured by the use of a cardiac output catheter and injection of 10rrd ice-cold glucose 5% non-invasive cardiac output measurement was done by using the continuous cardiac output software package modelflow (tno; portapres| results: overall, 159 paired measurements were used for the evaluation of the accuracy of non-invasive obstained sv and co. mean values, standard deviation and range of co and sv evaluated by invasive and non-invasive methods are summarized in the aortic modelflow provides reliable hemodynamic data in critically ill patients. it may be a useful alternative due to its non-invasive approach and continous measurement. objectives: to determine the feasability and accuracy of a rapid urine screening test (triage tm, merck-diagnostika) in an emergency department. methods: prospective analysis of the triage tm testkit (tt) in patients admitted because of suspected drug abuse during an observational period of 6 months. the tt is a eompetitve immunoassay which gives qualitative results within i0 minutes for methadone, benzodiazepines, cocaine, amphetamine, opiates, barbiturates and cannabis. urine samples were analysed with the tt and compared to the results of a enzyme-lihked tmmuno-assay (el.a) in our labratory (gold standard). results: during the study 25 patients were enrolled, in 19 of these patients substance abuse was proven by tt and verified by eia. we recorded no false positive or false negative results for benzodiazepines, barbiturates, opiates, cocaine and cannabis. two results were false positive and one false negative for methadone; one result was false negative for amphetamines. the triage-testldt had an overall sensitivity of 0.94 and specificity of 0.94. conclusions: the triage tm testkit seems to be a a useful rapid test device in addition to quantitative tests for drugs of abuse in an emergency department. objectives: the purpose of this study was to evaluate the influence of several factors of the renin-angiotensin-aldosterone system on blood pressure response in patients with hypertensive crises. methods: patients with systolic blood pressure >210 rorohg and diastolic blood pressure > 110 nunllg were included into the study. prior to treatment blood samples for determination of plasma renin activity (pra), angiotensin converting enzyme (ace), angiotensin ii (ang ii) and aldosterone (aldo) were collected. all patients received 5 rog enalaprilat intravenously. success of treatroent was defined as a reduction of systolic blood pressure below 180 mmi-ig and diastolic blood pressure below 95 mmi-ig within 75 minutes after start of treatment. results: 35 patients were included in our study, 20 (57%) patients responded successfully to treatment. mean arterial pressure decreased in responders by 36.5 mmhg and in non-respenders by 12.7 mmhg (p<0.001). responders and non-respenders differed signii'icantly concerning pra (p=0.001), ace (p=0.003) and ang ii (p=0.04). 0.003 0.04 the extent of blood pressure reduction correlated positively with the pretreatment pra and ang ii concentrations (correlation coefficient for pra: r=0.43; ang ii: r=0.66). conclusion: our data confirm that in patients with hypertensive crises blood pressure response to ace inhibition is mainly determined by circulatory pra, ace and ang ii. as the extent of blood pressure reduction correlates with pra, ace-inhibitors in patients with suspected high renin status cannot be recommended, as excessive blood pressure reduction, which carries a considerable risk for further organ damage, may occur. f. staikowsky, n. grillon, f.pevirieri, c.jedrecy, c. zanker, f. michard, a. haft medical emergency department. hospital bichat, paris epidemiology of acute intentional self medications-poisoning (smp) in france is especially known by data of poison control centei,s and intensive care units (icu). the purpose of this study is pro~,ided characteristics of this problem in a med for adults. method: july 1992 to june 1993, files of patients consulting to the ed for smp have been retrospectively analyzed. results: 727 patients, 482 women and 245 men, 33.3 + 12 years old (range 15-92) have been admitted for 804 episodes of smp (4% of all consultations) whose 77 relapses during the period of study. psychiatric disorders, drug addiction or hiv patients was found for respectively 42.6%, 9.1% and 2,9% of patients. the interval of time between the ingestion and emergency consultation was noted for 43% of smp (332 + 532 min, ranges 15-4320). the involved products name was known in totality in 89% of cases with an average number by episode of 1.7 + 1 drugs (ranges 1-8). the most often, 1 (52%) or 2 (21%) different products were interfered. the nonbarbiturate psychotropic drugs accounted for 76.7% of the products (benzodiazepines 67%, antidepressants 9.5%, neuroleptics 8%, carbamates 5.8%, imidazopyridines 5.1%, cyclqpyrrol0nes 2.7%). analgesics and nonsteroidal antiinflammatories represented 6.8% of all drugs, anticonvulsants 3.4%, cardiovascular drugs 2%, antiinfective agents 1.9%, drugs against cough 0.86%, muscle relaxants 0.86% and antihistamines h1 0.5%. the benzodiaz6pines were present in 531 episodes, alone in 316 episodes. in 36.5% of cases, there was a simultaneous intoxication with alcohol. the processing consisted of gastric lavage in 32.5% of cases, activated charcoal in 16.7% of cases, flumazenil in 16.9% of cases, naloxone and acetylcysteine in 3.4% of cases; orotracheal intubation was performed in 12 patients. admission in hospital was effective for 280 patients, in medical ward (n = 156), psychiatry (n = 63) or icu (n = 62); no fatal case was recorded. conelusion: smp to ed are often benign. the benzodiaz6pines are the most often incriminated but the new anxiolytics and hypnotics (imidazopyridines and cyclopyrrolones) take a growing place. the latsion burn center of athens. its planning constructive and functional refinements j. ioannovich, a. petalas-vourekus, d~ serbetis, h. carsin a 18 bed burns unit is under construction following a donation to the general hospital of athens. the plan of the unit, covering a surface of approximately 3.500 m2 is based on the principle of three identical 6 bed satelites which may function totally independent from each other. in the center of the unit the common facilities are installed, like operation theatres, storage rooms etc. this new modification in the plan of a burn unit is presented in this paper. the advantages from the fucntional, administrative and medical point of view are discussed. tiffs anisotropic conduodon could favour the ocenrence of a circular movement of the impulse that leads to tachyeardias by reentry. purposes of this work were to study, with the help of epicardial mapping, the influence of a trieyclie antidepressant, clomipramine (c), on the conduction velocity longitudinal (vl) and transverse (vt) to myocardial fiber orientation and on anisotropy (a = ratio vl/vt), and their modificutions by the sodium bicarbonate (13). method: a plaque of 64 electrodes, positioned on the left anterior ventricular wall of 9 anesthetized dogs, allowed to deliver, thanks to central electrodes, programmed electrical stimulations inducing vcuttienlar complexes, and to collect them. each entailed unipolar dectrogram was processed by a computer system that drew the isochrones and a map of activation allowing the calculation of v. the c was infused (0.5 mg/kg/min iv) during 75 rain; at t60, dogs received the b until the retuni of qrs to its initial value fro). a lengthening of qrs of at least 30% of its value at to was demanded before the administration of b. results: 1 dog was excluded because of an.~nsufficient prolongation of qrs before the administration of b. all values (map : mean arterial pressure, i-ir : heart rate, qrs andqt intervals, v) differed significatively (13<0.05) compared to values control fro)except qrs at t65. the b (7 + 6 ml/kg; ranges 2.8 and 20.5 ml/kg) modified no studied dements outside of the ( }rs. to ti5 t30 t45 t60 t65 t75 a 2,1 + 0,6 2,1 + 0,5 2,1 + 0,4 2,1 + 0,4 2,1 + 0,7 2,1 + 0,7 2,1 +-0,~ conclusion : the c slowed v l and v t without modify the anisotropy. the b did not modify the v of~conduction while the qrs prolongation was corrected. the c acts as a class i antiarrythmie drug on the inward sodium current during the phase 0 of action potential; the gap junctions have shown to be important in the conduction and an action on the gap junctions such as a modulation of the junctional resistivity, can not be rule out. is the doctor a heroe ? p. t.schies~.he, t. bauer, m. seyr dept. of anaesthesiology and intensive care, aokh krems, austria objectives: helicopter emergency services (hes) are getting popular more and more. the results concerning outcome are encouraging. however, some recent accidents with dead or badly wounded hescrew-members have shown the relatively high risk for the crews. therefore we were interested to eval0ate the motivation of physicians to participate in a hes. this survey was designed to investigate current concerns about safety and motivation of doctors on emergency call. methods: a questionnaire was sent to 205 doctors of the austrian emergency system. the survey consisted of multiple choice questions and subjective scoring tables from 1 (--full agreement) to 5 (=disagreement). overall, 64"/. of the active emergency physicians participated in the survey. results: 61.1% of the doctors assume the system is basically safe, experienced doctors tended to have less trust in safety. only 13% would not hesitate to go into action by dark. 14.8 % stdctly refuse night flights to accidents outdoors. although defibrillations are assumed to be safe dudng flight, only 29% would do it. 52.8% of the doctors would rather stop flying. the most common reasons for 9,uitting were wish of family and fear of an accident. 47.2% conclusioq: short transportation times help to avoid trauma related stress, pain and shock-induced organ complications. therefore the physiologic and economic advantages of hes are undebatable. however, the survey data indicate a considerable concern about safety of the medical personal in a hes. 14 crash landings within less than 10 years with 15 deadcases and 17 badly wounded crew members in a small country like austda make desire for safe flying conditions understandable. obiectives: to evaluate the clinical usefulness of trachlight. methods: trachlight is a new device facilitating endotracheal intubation. a stylet with a lightprobe is inserted into the endotracheal tube. intubation is guided by the light glowing through the neck tissues, thus rendering direct laryngoscopy unnecessary. intubation using trachlight was studied in 37 patients (age 21-68 years). the indication for intubation was elective surgery in 21 patients (asa i-ii) and emergency intubation in 16 patients. in the elective patients, anaesthesia was induced with thiopentone supplemented with fentanyl, and intubation was facilitated with vecuronium. the cause for intubation in the 16 emergency patients was dyspnea in 8, cardiac arrest in 2, trauma in,2 and unconsciousness due to drug overdose or seizures in 4 patients. intubation was facilitated with medication in 12 patients. results: of the elective 21 patients, 19 (91%) were successfully intubated. six patients (29 %) needed two attempts before successful intubation. the duration of intubation exceeded 30 seconds in 8 patients (38 %). of the emergency patients, 14 (88%) were successfully intubated. six patients (38%) needed two attempts, and the duration of intubation was more than 30 seconds in 9 patients (56 %). in 54 % of all 37 patients, intubation was assessed as easy. no or insufficient glow, prolonging intubation or necessitating two attempts, was noted in 11 patients (30 %). oesophageal intubation occurred in 2 patients. conclusions: trachlight may be a valuable adjunct for intubation in varoius settings provided that adequate training is provided. a learning curve was found to exist. objectives: to compare enoxaparin and standard heparin in cavhd and calculate the value of laboratory controls in the treaanent. patients and methods: twenty patients needing dialysis for acute renal failure participated in the study. the main exclusion criteria were massive bleeding or a thrombocyte level < 50 x e9/i. in each treatment the same type (av-400, fresenius ag, germany) of a polysulfone capillary haemofilter was used. the study scheme consisted of two consecutive four-day cavhd treatments, one course for each type of heparin. the order of heparin administration was counterbalanced between patients. the standard heparin was given as a continuous infusion aiming at an activated coagulation time between 200 and 250 s. the initial enoxaparin dose was 80 rag every 8:th hour intravenously, but was modified by any signs of coagulation in the dialysis blood lines or bleeding complications. results: the dialysis treatment was adequate in both treatment modes, with mean blood urea levels 24.3 and 25.2 mmol/l respectively (ns). the bleeding complications were moderate and similar in both treatment modes. the mean life-span of haemofilter using enoxaparin as an anticoagulant was some longer than using heparin (35.7 +31.6 h versus 22.3+26 h, ns). the mean aptt-levcl during heparin treatment was 79s and during enoxaparin treatment 54s (ref. 24 -34s). the mean daily dose of heparin was 422 nag, that of enoxaparin lg7mg. the mean anti-xa activities were 0.40 u/mi and 0.47 u/mi, respectively, reflecting a better bioavallability of enoxaparin. conclusions: both anticoagniation modes were equally effective and well tolerated. the amount of enoxaparin needed for a proper anticoagulation was, however, less than half of that of standard heparin. the changes in aptt level were too slight to make its use possible in controliing the dose of enoxaparin. the use of enoxaparin seems to be rather safe in cavhd even without laboratory controls. the adv~ucea in the management of computerized data of an intensive care unit have been petalled to the clinical advauces and the increasing sophistication of methods of diagnosis fop the clinical application an therapy. this has led our unit to design and develop a computational system called timbu which is used to help physicians assist patients. among its various uses, this system has a software for the hemodynsmic control of a critic patient. this program was carried out to get as fast as possible the hemodynamic data of the patients in an intensive care unit. as an example, we can mention that when we load 17 data obtained through direct measurement from the monitors and the lab, the program calculates 18 parameters that guide, intelligently, to the diagnosis and therapeutic behaviour of the hemodynamic problem through screen messages. the validation of this program in the unit of intensive care has demonstrated that its use allows a more efficient handling of the patient with serious hemodynamics and respiratory disorders. ohieetlve: traema is a heterogeneotm 'disease' that ecatr~ a~"o~s all age ~oupe with v~ying degrees of severity. this imerogeneity has made the di~e, trmma, diflkaflt to r the ehn of this stady wa~ to assr the fitaen of saps in ibis popeleties. methode: in order to compute the ~ probability, a model derived from logistic regression w~ developed. meam'e8 of calibration (goodaess-of-fit stetislj.r and di~'riminafion (roc ou~e) were adopted in developmm~ and validetlon set randomly taken from a database of 10065 pts eeeseemivety admitted in icu (arohidia). ~ witho= salm, p~ yom~ am is yam, with los ~horter thma 24 hotam wore exr fa'om thi~ mmly~ir thi~ model v~s then evahmed on the ~per ~mbgro~ (i.e., trmma pts). if'it did t~t fit the data well ~, new model wm developed rer the logit only on trm=~apm. reims: data were availabte for 8059 pts during aperiod of three .y~m , treama pts were 1156 04.3%), teats of calibration iadioaled probability model did mot provide m adequate refle~on of the mortality ezperieace in pm with ireutae, being the observed mortality lower flma the expected (figm'o). a aew model was then variable. this oastomized model fit~ the de~t of trmara pts very well (g 2=-8a7 p>0.25; roc = 0,94 ). the di:lferencea between the two modele were evident. conclusion: this ltudy shows that mortality in iramna pts is over wcfe~d when ~se~ed by menm of saps. however the r mode! meets high standmcd in terms of calibration mid dil~'iminat'~o~ ']"he advaatage of ~imd models meaas the colleotion of the ~ set of variables for all pm admitted in icu e~einat the ase of diasma specific ~oring syatex~. ("sl"): effects on cardiovascular and hemostasis systems (cvs, hss) a.oborin~ph, ~.~yndiuk~ph, b.kondratsky ~pt. of'""su~gery and transfusiology, research institute of hematology, lvov, ukraine objectives: great interest has been shown recently in the use of hoss for the initial resuscitation of hypovolemic shock. methods: the study was carried out in 6 dogs 1-~h hs was induced by jet momentary hemorrhage (h) from a. femoralls (the bloodloss volume made 29.8+1.9 ml/kg). the treatment was begun after 7.u+o.2 hrs of h. "sl", created on the basis of-sorblt and natrium lactate (1800 mosm/l) was injected into v. femofalls at the dose of io.0 ml/kg. results: it is established that before treatmen-~rterial blood and central venous pressures (abp, cvp) diminished to 30.0 mm hg and -0.6+0.2 cm h20 (p4.o01), while heart rate (hr)-increased to 190.0+9.6 per min (p<.o01). by this the indices of ~latelet counts (pic) and plasma fibrinogen (pf) lowered by 42.2% (p<.i) and 6.4% (p~.05), while fibrin degradation products (fdp) enlarged by 215.6% (p~ .001). after 30-40 min of treatment termination abp and cvp increased to 98.3+6.1 mmhg and 4.1+o.2 cm h20 (p<.o01), and ~[r diminished to t80.0+6.3 per min (p>.5). at the same time the indtces of pic and pf enlarged by 36.4% and 2.6% (p>.i), while fdp diminished by 8.2% (p>.i). one of 6 dogs survived. life duration of the other 5 dogs was 3.9+1.2 hrs. conclusions: the obtained data are ~he evidence of normalizing influence of "sl" on cvs and hss, and allow to recommend it as a mean of initial resuscitation of hs in clinic. oblectives: we prospectively studied 64 icu patients with severe head injury (hi), which cerebral lesions monitorized with sjo2 through opljcal fiber and the cerebral flux with tcd. methods: since january 1990 until june 1994, we collected 152 ht admitted to the icu, and 64 of them monitorized with optical fiber in the right jugular bulb and tcd. all patients needed mechanical ventilation related to gcs <__ 8, with ct in admission (classifing lesions according to marshall and al.) . we related the final results to the evolution of sjo 2 and tcd, with other monitorizing methods like gcs, ct and icp. ~sults: conclusions: in patients with gcs _< 8, sjo2 is useful to evaluate the evolution towards vegetative state, still more in cases with ct type ii in admission and higher apache ill. elevation of icp implies an evolutive nsk to brain death and data of tcd is a good indicator of brain death, the complete monitorization of these patients can improve the therapeutic control of this neurologic problem, , (16m,6f) , (m. age: 39+4 years), divided in two groups (a and b) under specific criteria(tremor and/or fever during admission in i.c.u., or not). the injury severity score was >25 in all studied patients. tbe group a (9 m, 41") had no tremor and/or fever on admisskm, while em group b (tin, 211 the above criteria were ix)sitive. bhx~d samplings were taken 2-9 hours after accident and 10-25 rain. after admisskm in i.c.u. micro-eli~ method was used for measuring cytokinc-levcls. statistic analysis was performed by studcnt-t test. as control group, 25 healthy people were examined. _resu!_ts-il-lct, il-ii~, il-2 and tnf-tt levels were similar to control group levels in both groups a and b. i!,-6 and g-csf levels were found increased in both groups (p<0jxjl), while il-6 levels were statistically significant comparing to group a. in con_tin_skin, during immediate post4raumatic period,proinflamatory cylokines il-i~, il-i~ and tnf.-ct, produced in an earlier stage than 11,.6, cannot be detected,whereas 11.-6 was increased significantly, especially in group b. g-csf was fimnd in increawal levels in both gr(mps, without statistically significant difference between gnmps a and i|. objectives-l~valantc proteolitic activity, disorders in" eariy, period after combined trauma and p(~.ssibilit, i' of their correction by injection of proteo[ysis inhibitors contrycal and s-fto~:nracil in combination with driving an isotonic snlu~ion of sodlum chloride and polig[ucine. methods: biochemicai studies of proteolitic activity in dogs with limited deep burn and acute bloodloss, . result:s: in case of deep 5% burn, cornplicated by bloodshed the of blood grows at 6-7 times. it; is the restdt of the pancreas glandischemi demage, caused by the centralised circulation of blood and intensifies the deviations of haemodiaamics and albumin exchange. the degree of endogene intoxication by mean mofecular peptides which are the products of albumin decay reses to 30%, and 77% in 3 hours. in 3 hours after the trauma the-process is accompanied b3! 59,6% lower inhibitory activity of blood, where as at the peak of the trauma it was 14,5~ higher. that proves the nnfavuurahle process of the shock in case a combined trauma. conclusion: the vein injection of 'proteolysis inhihitotz cnntrycal and 5-fforuraei[ in cumbination with driving an isotonic solution of sodium chloride and p.dligh]cine to refill lhe loss of blood helps to lower at 2 times the profeolitic activity of blood. but it still remains above the initial level. the degree of endogene intoxication lowers at 2 times; [15emodinamics aml albumin exchange stahilised. objectives: nimodipine, a known calcium antagonist, has been shown to dispose a beneficial effect on patients with subarachnoid hemorrhage, but its efficacy on traumatic or spontaneous intracerebral hematoma has not been justified. therefore, we studied the effect of nimodipine on the histopathological changes following an experimental intracerebral haematoma in rabbits. methods: twenty-three new zealand albin rabbits of both sexes, weighing 2-3,5 kgr and at age of 4-6 months were anesthetized and a small burr hold in the left parietal aerea was carried out under aseptic conditions. the dura was opened and 0.1 ml (this volume assuring a normal incranial pressure after kaufman 1985) of autologous blood was injected into a depth of 3 mm via a needle of 0.36 mm bore. the wound was closed and the animals were left to recover. nimodipine, of 2,1 mg/kgr of by weight per day was given via a nasogastric tube to fifteen animals for a period of time of fifteen days (group b). six rabbits were given water and served as control (group a). both groups of animals weie sacrified on the fifteenth day, their brains were removed and immersed into 10% formalin solution. tissue sections of 5 ~ were embedded into paraphin and stained with haematoxyline and eosin, mason and gfap stain for gliac cells. results: two animals died after the surgical procedure, because they developed large intracerebral bematoma. no animal developed neurological deficit except one of group a which manifested a right side hemiparesis. the results of the bistopathological changes are the following: i) the mean -+ sd diameter of the lesions in the group a was 260 --. 26 ~t while that of group b was 76 + 12 ~t (p<0,01) ii) secondary ischaemic neural tissue changes, characterized by the extravasatlon of red cells, the presence of haemosiderin-containing macrophages and signs of low grade inflammation zpredominated in the specimens of group a and were totaly absent from those of group b. iii) a ring of gliac hyperplasia and a low grade local fibrosis was found, encircling the lesions in the specimens of group a in contrast to those of group b. conclusions: nimodipine when administered in rabbits following the development of a non increasing the icp experimental intracerebral haematoma, prevents the extention and the severity of the lesion. objectives: to study the efficacy and side effects of adding intramuscular clonidine (clophelinum) to analgesic regimen in early management of patients with serious burn injury. methods: 20 pts with 20-40% bsa second to third degree flame burns (respiratory tact injury excluded) 19 to 61 yrs of age were randomised to study (n=10) and control (n=10) groups. burn shock was treated with hypertonic saline -bicarbonate solutions (250 mmol/l na +) 2ml/kg/%bsa for the first 24 hours and 1 ml/kg/%bsa for second day. analgesia in control group for the first 48 hours was provided by regular 6 hourly intramuscular administration of 20 mg of morphine sulphate and 500 mg of analgesic -antipyretic analgin with 10 mg of diphenhydramine (dimedrol). from the 3rd day regular administration of morphine was finished. in the study group 100 ixg of clonidine was added 8-hourly for 72 hours and dose of morphine halved. vas, verbal rating scale for sedation (vrs, 1 -5), sleeping time, spo2, hr, bp, diuresis, vomiting and other complications were comparatively evaluated during patients' stay in icu. results: addition of 300 ~g of intramuscular clonidine daily allowed to achieve better analgesia and sedation with halved consumption of morphine. mean vrs in study group for the first 3 days was 3.1 -3.3 vs 1.3 -1.7 in control group with twice longer sleeping time. there was significantly less tachycardia in study group; dynamics of bp for the first 24 hours did not differ considerably; later, there, was tendency for hypotension in study group without adverse effects on diuresis or other indices of tissue perfusion. because of high incidence of chronic ethanol abuse among study population 7 pts of control group suffered from psychomotor agitation or delirium, probably as a sign of alcohol withdrawal syndrome (aws). this made regular evaluation of vas impossible. in the study group only 1 pt showed sign of aws. mean vas score was in 2.4 -2.9 range for first 3 postburn days. 7 pts appeared excessively drowsy due to clonidine, but it had no adverse effect on their overall clinical course. mean spo 2 values in study group were in 95 -98 % range, among controls 90 -95 %; vomiting was absent in. cionidine group vs 4 cases among controls conclusions: clonidine could be a valuable addition to analgesic -sedative regimen in burns, especially for prevention of aws and deserves further study in this regard. hemodialysis -hemoflltration modifications and/or intratracheal gas insuflation have been recently used for blood gas exchange in several models of respiratory failure. objectives: evaluate the combination of cavh-m and igi for respiratory support in experimental acute lung injury. methods: five mongrel dogs (22-+4kgr) were mechanically ventilated inroom air, paralysed, heparinized, connected with a cavh-m system (diafilter-30 polysulphone membrane) and remained stable for one hour (pao~= 98.8• peco2=34-+8mmhg, ph=7.37-+0.07, bp=137-+12mmhg and pap=15-+2mmhg). all was induced two hours after oleic acid infusion (0.07ml/kgr) into the pulmonary artery (poo~=46.6_+6 -p<0.001, paco~-50.2_+10 -p<0.05, ph=7.10-+0.10 -p<0.01, bp=158-+25 -p=ns, and pap=29_+5 -p<0.01). fio 2 70% for the next 30 minutes did not significantly altered the b3ood gas abnormalities. afterwards, pure oxygen applied simultaneously a) through the inlet of the filtrate's compartment of the hemofilter (2l/min) while filtrate and gas were removed from the outlet port (bypass flow 220 ml/min) b) through a thin intratracheal catheter positioned 2cm above the carina (4l/min). the fio 2 given through the ventilator readjusted to 21%. results replacement fluids/filtrate during the next four hours were not exceed 0.7l/hour, whilst the blood gases and pressures were improved as follow: cavh-inlet:pao.=88. objective. to compare the changes in humoral immunity in trauma patients following massive transfusion of autologous and homologous blood. methods. we studied 3 randomised clinical groups of patients each containing 24 patients with trauma and operation of large arterial vessels. the amount of autologous or homologous blood transfused to the patients was exceeding 1 500 ml, while the patients in the control group did not recieve blood or blood products. results. we recorded most pronounced and characteristic changes on the 1-st and on the 7-th day in the group of patients recieving homologous blood transfusion, i.e. decreased amount of igg,iga,igm,c5 and c4 fractions of the complement system, haptoglobin and significant and sustained rise of circulating immune complexes up to the end of the study period. in the control group of patients the decrease was weaker and lasted only during the 1-st post-operative day; the dynamics of the circulating immune complexes level were almost the same as in the first group of patients. in the group of patients recieving autologous blood transfusion, the parameter values did not change significantly from preexisting levels after the 1-st day, while on the 7-th and on the 30-th day showed a tendency towards aslight rise. conclusions. autologous blood has a favourable effect upon humoral immunity and should be the transfusion medium of choice in cases where autologous blood reinfusion is technically possible. ivan petkov, m.d., rumen farashev, m.d. and dimitar terziiski, m. d. medicine, military medical academy, 3 g. sofiiski str.,1606 sofia, bulgaria objective. the amount of blood lost during trauma and operation could hardly be forseen and donor blood supplies are not always available in sufficient amounts. rare blood group types and/or unexpected haemorrhage pose a great challenge to the transfusion therapy and the methods of intraoperative autologous blood transfusion. methods. we report a case of a 18-year old male patient with extremely massive intraabdominal haemorrhage ( 7 300 m( blood loss ) during an abdominal aorta reconstruction following a traumatic injury of the abdominal aorta. we achieved a successful reinfusion of 6 600 ml of autologous blood using an original autotransfusion system developed by us ( pat. no 95311/ 11.10.1991) . results and conclusions. the autotogous blood in the case reported here was the only and the most suitable transfusion medium for the rapid intraoperative compensation of the acute haemorrhage and the favourable outcome of the patient. the post-operative period was smooth and no significant disorders in the clinical course as well as in the laboratory tests ( morphological,biochemical,coagulation and immunological) were recorded. there were no complications during the postoperative period despite the fact that the amount of blood reinfused to the patient was slightly exceeding his own volume of circulating blood. objective. the haemoglobin concentration and the perfusion pressure value could not be the only criteria for the early signs of tissue and organ dysfunction. because of this, we employed the extensive monitoring of oxygen transport during severe trauma in order to. achieve dynamic evaluation of physiologic compensatory mechanisms and to assess the efficacy of intensive care management. methods. we conducted a prospective controlled trial on the blood oxygenation, oxygen transport and tissue perfusion during the first 3 days after the trauma in 20 patients with polytrauma. we used a swan -ganz pulmonary artery catheter (beckton -dickinson, u.s.a.), deseret 1000 cardiac output computer (medical inc., u.s.a.) and hewlett -packard monitor (hewlett -packard, germany) to measure and calculate all the parameter values. the severity of the injury was assessed using the apache ii score system. all the patients had scores over 18. results. the results show a significant decrease in the arterial blood oxygen content and in the arterio-venous difference, as well as an increase in alveolo-arterial oxygen difference and in the transpulmonary right-to-left shunt. the tissue oxygen supply and the tissue oxygen consumption reveal a tendency towards a decrease below the physiologic minimum of adeqate values. the erythrocyte current velocity and the ratio between oxygen transport and erythrocyte current velocity also decrease inspite of the optimal blood rheology. conclusions. the dynamics in the parameters values are most pronounced between the 2-nd and the 18-th hr after trauma, which predisposes patients to the risk of developing stable hypoxemia and characterizes this period as the most critical for tissue metabolism and organ dysfunction. posttraumatic changes in immune mechanisms in lung compartment in trauma were analyzed in ao and da inbred strains of rats which differ in their immunological reactivity: the former being low responder and lat-~er hiperresponsive. methods: the levels of tnf-alpha activity in the 24 supernatants of cultured lung lobes and dynamics of cells migration from tissue explants in 6h lung cultures were assessed in ao and da rats subject ted to severe burn trauma. results: increased levels of tnf activity (160+3 pg/ml compared to 50+4.9 pg/ml in control) were found od day 3 following trauma in lung sups of ao rats while no changes in the levels of activity of this cytokine were found in lung-sups od da rats more pronounced extent and dynamics of cell emigration were noted in da rats, while almost unchanged in ao rats sharp rise in pmn percentages 3h following trauma (60-70% compared to rare pmns in control), followed by increase in lymphocyte numbers at later time points among lung cell emigrants was detected in ao rats. slower but persistent increase (25%, 3h following trauma and 60% and 50% on days 1 and 3 after trauma infliction, respectively) in pmn numbers among da lung cell emigrants was detected, which appeared to be activated, as judged by their nbt reduction capacity. increased percentages of peripheral blood pmns and increased state of leukocyte aggregation/adhesion were detected in both strains, but different levels of plasma tnf: increased levels in ao rats on days 1 and 3 following trauma, and initially but persistently high levels of plasma tnf alpha in da rats (4-5 fold higher compared to initial levels in ao rats). conclusions:different patterns of local (lung) and systemic changes in cell numbers and cytokine levels implicate differential posttraumatic migratory capacity of pmns vs. lymphocytes in lungs in ao and da rats. early diagnosis of acute intestinal ischemia by color doppler sonography e. danse, b.van beers, p.goffette, f.hammer,aav.dardenne, f.thys, p-f.laterre, m,s. reynaert, .lpringot dept of radiology (profb.maldague) and dept of intensive care ( prof m,s.reynaert), st.luc univ.hospital, brussels, belgium ob emergeny medical squad service is the most important segment in the process of saving the people, in the cases of mass accidents, like industrial accidents caused by the: explosion, fire, chemical poisoning, traffic accident, elemental catastrophes and the war. because of that, each emergency medical squad service needs to have in its motor-pool vehicle for the mass accidents/ for provoding at least 100 people, wounded as well as the people became ill/. objectives: presentation of such special vehicle, produced by "zastava-kamioni" and it's medical-technical equipment. methods: descriptive and comparative analysis of the medical and technical characteristics, based on the actual norms/din, 75080, iso 9001, yus.../ results: on the base of doctrinaired requirements of the emergency medical squad in the case of mass accidents, our researches resulted in the following medical and technical characteristics -the vehicles for mass accidents are gvw/with a payload off cca 5-8t, with the fixed, closed body, type: universal van, -technical equipment aggregates, stretches, anti-fire device, equipment for pitching the tent and for maintaing technical conditions of the work -medical equipment: linen bags with complete sets of bandage material, means for the reanimation and immobilization, for the infusion, medical instruments and remedies as well as the tent for lodging at least 50 wounded and sik people. in federal republic yugoslavia, it was proposed 24 such vehicles for the emergency medical squad needs. conclusion: we suggest to introduce this vehicle in the production range of the ambulance vehicles for saving, especially in the circles where can occur serious accidents. introduction : carbon monoxide (co) poisoning commonly generates central nervous system abnormalities though an important cardiac morbidity and mortality must be considered. long-term exposure to co with cohb levels < 30% may be more dangerous than short-term levels of 45-50%. we report a case of an adolescent who after prolonged exposure to co developed a severe reversible cardiac dysfunction with low levels of bloed cohe c a.ase history : a 15 year old boy was found comatose at home. his mother in the neighbouring bathroom died severn hours earlier of what was later proven to be a co intoxication. on arrival the gcs was 8/15 and the patient was breathing spontaneously. a postictal status with eventual postanoxic encephalopathy was suspected. a coh'b level of 10% was objectivated. the cardiorespiratory situation quickly deteriorated requiring mechanical ventilation. chest x-ray showed diffuse bilateral patchy infiltrates. ecg revealed signs of ischemia. severe left ventricular dysfunction was evidenced by pulmonary artery catheterisation and echecardiography and later by isotopic angiography (lvef 20%). treatment was intensified with inotropic support, intta-aortic balloon counterpulsation and oxygen therapy. the clinical course was further complicated by a crush syndrome and renal failure. the patient's condition gradually improved and he fully recovered without any residual lesions (lwf 80 %) conclusion : even after prolonged exposure cohb levels can be misleadingly low. high tissue levels of accumulated co can be associated with coma and fulminant cardiorespiratory failure requiring advanced life support facilities. introduction : both neuroleptics (nlp) and tricyclic antidepressive agents (tca) can induce arrhythmias, prolongation of the qt segment and the pr interval and hypotension. we report a case illustrating that combined overdose of these agents increases the toxicity of each compound and the risk for adverse cardiac events. .c, gse history : a 44 year old male ingested 2500 mg doxepin (sinequanr), a tca and 3500 mg prothipendyl (dominalr), a potent nlp in an attempted suicide. upon arrival in the emergency department the patient was unconscious (gcs 6/15), breathing superficially, and presenting signs of recent vomiting. physical examination revealed a taehycardia of 140 b.p.m., an arterial blood pressure of 90/70 mmh4g. ecg showed a brood qrs complex tachycardia. a chest x-ray revealed the presence of an aspiration pneumonia. laboratory investigation demonstrated increased levels of crcatine phosphokinase, lactate dehydrogenase and aspartate transaminase ; hyperglycemia and leucocytosis were present. the plasma concentrations of doxepin and prothipendyl were respectively 410 gg/l (toxic level 5130 #g/l) and 3900 i.tg/l (no reference). treatment consisted of mechanical ventilation, gaslric lavage and administration of activated charcoal and iv fluids and antibiotics. a hemodynamically well tolerated veatricular tachycardia developed 1 1/2 h later. nahco3 (250 meq/24 h) was administrated inducing an ectopic atrial tachycardia with a normal qrs complex and prolonged qt. 8 h after admission a normal sinus rhythm was present; the prolongation of the qt segment persisted for 2 days. the patient fully recovered. conclusion : the treatment with nahco~, alkalizing the blood and thus increasing the protein binding of the tricyclic antidepressant molecule, can readily correct the potentially life-threatening cardiac arrhythmias and therefore should be part of the routine treatment of combined tca-nlp overdose. ob/ectives: the development of diabetes insipidus (di) in patients with brain injury is a known negative prognostic sign. the aim of this study was to investigate whether this is also a reliable early prognostic sign of brain death. methods: this is a retrospective study of 85 patients treated" during a two year period (1-3-1992 to 1-3-1995) in our i.c.u who meeted the following criteria: (1) coma score _< 8 gcs within the first 24 hours, (2) positive brain ct scan on admission classified according to marshall's diagnostic classification (classes 1-6), (3) normal renal function during the entire icu stay. for the definition of di were used the usual di criteria plus hypematriaemia (serum na" >_ 155 meq/l). survival was defined up to the 30th postadmission day. conclusions: according to the findings of this study, the development of diabetes insipidus in brain injured patients seems to be a highly specific index for brain death (positive predictive value = 0.95). however, further prospective studies are needed for the definitive evaluation of these findings in such patients. emergency care in italy, despite all efforts, is still lacking a nationwide organized prehospital care system and, until today, there are only different regional solutions. the majority of these realities imply rather simple ambulance first-aid services without attending emergency physicians and without resuscitation equipment. the emergency medical service (ems) system in falconara m., italy, was implemented in august 1994 by a collaboration between the school of anesthesiology and intensive care of the university of ancona and the, already existing, volunteer rescuer organisation "yellow cross". according to the guidelines pubblished in 1992 [1] the pre-existing equipment of the volunteers was completed with type a ambulances and 1 special equiped motorcar (patient monitor, defibrillator) for ambulance indipendent physician transpur[. a special data collecting schedule was created to memorise every emergency intervention in a computerised data-base. the intraining members of the school of anesthesiology and intensive care provide 24 hour ready intervention. in this report the authors describe their experience concerning primary firstaid medical interventions. for a preliminary evaluation we considered, retrospectively, 300 consecutive emergency interventions in the time period from novembre 1, 1994 to april 30, 1995. the emergency physicians treated 131 male (44 %) and 169 female (56%) patients, 15 patients died before hospital admission and 75 patients (25%) were treated at home by the ambulance indipendent physician and did not need any further medical treatment. in the same time period 1 year earlier (november 1993 to april 1994) without attending physician the volunteer rescuers transferred all 257 first-aid interventions to near-by hospitals. we conclude that the presence of an attending, iudipendently motorised physician in emergency interventions is essential for the establishment of precise priorities and may be helpful to reduce hospital admissions by ambulance intervention, though reducing primary" health care costs. we have developed the method of liquor filtration which allows to purify the cerebrospinal liquor from blood and its decay products in the subarachnoid bloodstroke. the hemipermeable dialysis membrane was used as a filter, which lets only in water, electrolytes and substances with small molecular weight. the liquor filtration was used for the treatment of 19 patients with the subarachnoid bloodstrokes of different etiology. the perfusion of liquor was performed at the rate 3 ml/min in the recirculatory mode. its duration was 180 -240 min depending on the bloodstroke intensity. the filtration makes possible the most completely purifying of the hemorragic liquor, the reducing of the content of blood ceils and its decay products 80 -300 times as less. the monitoring of the patient's state during the perfusion didn't revealed the departure from the norm of the main vital part. the liquor filtration technique compares favo-~ rsbly with the routine method of cleaning by the absence of toxical effect of heterogenous solutions on the central nervous system. the filtrstion of the cerebrospinal liquor in the subarachnoid bloodstroke sllows to provide the the early cleaning of liqour, the regression of meningeal syndrome and to improve the patient's state of health. e3tabli~mczr 3bd ~ of rei~idnal medical first-aid zhoulittoing, ed., tan zi, m.d. dept. of sargery, the first teaching t[ospitat, 29 yejin-l)a-l)ao, wuhan 430080 fltlna objectives: the medical first-aid is the most important task of the public hc atth department. in general, single hospital model couldn't fatty, effective ly rescue mony severe patients who need mergant treatment in the scene. bub establishing the medical first-aid network, the severe patients can be given the most timely und the most scientific emergent treatment. so that, the suc cessfut rate of the saving wilt be greatly increased. methods..; our hospital is a general big hospital. through developing and cons tructlng for more than ten years, the medical first-aid network distributed art over the area under our jurisdiction has been set up. it consists of thr ee units: the medical first-aid unib center comartd and mnagment unit, co m~nlcation and tiaison unit. the principle of the network operation is with oat having to 90 far to mergoncy, specialized emergency and the best merge acy. results: the results of the network operation were notable. cmpari~ the to tat successful rate of the saving (91.6~), the successful rate of saving tra ma (93.~), the suscessfut rate of saving shock (98.~) and the successful rate of cardioputmonary resuscitation (52.4~) daring the three years after t he network operated with these before (86.6~), (91]. 4~), (92. ~) and (4ft. 5~), the successful rates after operating were remrk~iy higher ( p<i).o5 ). conctusions~ the above results showed estabiishmnt and mena0emont of region at medical first-aid network had very si~nificont action during mergentty s avin9 the severe patients. moreover, the regi~at medical first-aid network atso take an important part in prevention and health protection of the mass. objectives: se related mortality is due to the occurrence of both rv and lv dysfunction evoking cardiogenic shock and pulmonary edema (f. abroug, intensive care med 1995; s. nouira. chest 1995) . prospective evaluation of scorpion antivenon (sav), the only available specific treatment, is lacking. the aim of this study is to assess the effects of sav on the basis of a case-control study. methods:among 600 consecutive victims of se presenting to the emergency department, 135 were managed using sav (group sav+). these patients were matched to 135 envenomated patients who did not receive sav (group savchest injuries are the cause of death in 25% of trauma fatalities and a major contributing factor in an additional 50%. pneumothorax is a major complication of chest injuries and can be initially overlooked bringing to additional morbidity and mortality especially in patients who require mechanical ventilation. the real incidence of pneumothorax in severe blunt chest trauma hasn't yet been established, as many pneumothorax can be missed on the initial chest x-ray (cxr) and computed tomography (ct) has not been routinely used. to evaluate the incidence of pneumothorax in patients with severe blunt chest trauma. methods: a prospective study. from january 1 1993 to december 31 1994, all trauma patients with one of the followings conditions: fractures of four or more ribs, signs of lung contusion on the initial cxr or presenting a severe hypoxemia on admission (pao2/fio2 < 200) in the absence of pre-existing pathologies, were evaluated by initial cxr and subsequently submitted routinely to a ct. results: 52 severe trauma patients were considered. 8 of them, presenting a severe hypoxemia on admission, had no evidence of major chest trauma (ais<3). hypoxemia was the consequence of severe trauma in other districts; these patients were therefore excluded. 44 patients with severe thoracic trauma (ais>=3) were admitted into the study. the mean iss was 36.2 (16-75). thirty-six patients required artificial ventilation for at least 24 hours during the icu slay. three of them, who had a tension pneumothorax, were submitted to an emergency thoracic decompression on the field by the emergency helicopter team. in 7 cases pneumothorax was diagnosed an the initial cxr 14 more patients had a pnx which was identified only on the ct. in 4 cases a large pnx with lung collapse was missed on the cxr. in our group of severe blunt trauma patients, 54% (24/44) presented a pnx that required the insertion of a thoracic drainage. only one third (7/21) of the pneumothorax could be recognised on the initial cxr, while other 3 were decompressed before performing the cxr. as many as 58% of the cases of clinically significant pnx were missed on the cxr, and a ct performed soon after admission allowed an early diagnosis bringing to changes in the treatment. (as the patients were mechanically ventilated a chest tube was inserted in all these cases). in 4 cases, the initial cxr overlooked a huge tended pnx which was the cause of hemodynamie instability. conclusion: in patients with severe blunt chest trauma even large pnx can be missed on the initial cxr. moreover due to the non compliant compressible lung, a 20% pneumothorax which can be recegnised only on a ct, can bring to high intrapleural pressure altering eardiopulmonary function. n. andoeli6, 0.~osid, m.zesevid, m.risovid, d.stepi6, d.djokid b~rga~yc~qterclinicalcaqterafserbia, belgrade cb~ctives:~lis study ~ the use of ~rq]ofol earbired with k~t~ine (aq a~sjgh~ic s@~qt widn inirjrsic armlgesic pro~mities) or with fsqtmtyl,with psrtial azgmsis an hgenxlyn-a~ic ~ durirg ~ ~ re:~ver~ f~m ~ in hxh ~ of ~ti~. ~: yali~mial and ~bod:30 a~it p~tie~ts a~ i-ii were included in ibis shxly. patients were rsrd]nly dieided in two ~ns. all d~tie~ts ~me given 5-5 prcpofol bolus doses (o,5 ~gkg) for ird~iqn of ~. ~ia ~s m~sjn~ with an infusion 6 ~ ~ropafol. as sdflitianal were given fan-i~l (o,2 n]g) ~tely before ~ anj trad~e~ irfojoation followad by feasted bolus of o,i mg in ~ro4o l.patients in gr~4o 2 received i~ (an initial bolus dose of 35 rg slowly intcavax~ 8rd 25 mg as infusion over ~0 rain) .infusions of pro~fol or imcpofol with kg~mine ~ stopfsj 10-15 rain ]:~o~ extuhation.arterial blood ~ (sistolic arterial blood preassu-re~zap,mean ~rterial blood pr~,d~lic arterial preassure-[zp a~ h~art rate-~) ~ m~ before induction of a~ io, 30 snd 60 rain aftem ~ intutation. results: arterial blood preasstre ~s decreases duri~ irn~ction of sn~wd~sia in hy~ ~n~s,tnt mare in th~ ~ who r~eived fsqtanyl.~ere w~s statisticslly sifnific~ntly difemerme dmir~ m~ of an~ia. arterial blood r~easatre and heart rate were stable in the t-..e~min 9-~a4~. all th~,fl-e keta'nire grcqo hsd e~rly :~e~y time. ctrmlusi~s: ~e ombiretion of protxfol wilh keta/ne for irduorion a~d ~ of sn~sd~esis w~s yell accept~ by p~tierfcs anj coald he ~ as an alterrstive ~o ccnva~icrsl a~es -d~sia. objectives : assess the relation between cytokine or endotoxin release and indices of splanchnic malperfasion after hemorragic shock in multiple trauma patients. ]~r study was approved by the local ethical committee. trauma patients admitted to the emergency room who met the entrance criteria of more than 1 hour map < 60 mmhg or use of vasoactive agents or blood lactates > 5 mmol/1 were selected for study. a nasogastric tonometer (tonometrics, inc, plastimed, france) and a swan ganz catheter were placed on admission. phi, lactates, hemodynamics, plasma cytokine and endotoxin concentrations were measured on admission and at 3.6, 12, 24, 48 hrs. an immunoradiometric assay was used to determine plasma concentrations of il6 (n<0.03ng/ml) and tnfc~ (n<5pg/ml). plasma endotoxin concentrations were measured using a chromogenic limulus assay (n<0.1eu/ml)(1 endotoxine unit= 100pg). results : 9 severe multiple trauma patients (age = 42_+18 yrs, iss = 40-!-_15, saps = 19+'~, mean-+sd) were studied. they received 15+6 packed red cells during the first 24h. mean duration of collapsus before inclusion was 5.1_+2.9 hrs. death occm'red in ~tients. ~ pglml, *: ng/ml, etox : endotoxin(eu/ml), lact: lactate (retool/l) a significant correlation between initial il6 level and saps was observed. in the early post-injury period phi, sao2, svo2, vo2 were significantly associated with ;il6 release (p<0.05 at ho, h3, h6). later a significant correlation existed between lactates and ii6 (h6, h24). a peak of tnf was detected at 24 and 48 hrs. it was associated with low phi and low arterial ph of the early post-injury period (p<0.05 iat ho, h3, h6 ,h12, h24) and with high lactate levels of later period (_>h12). only the late release of endotoxins (i{48) was correlated significantly with initial !oxygea-delivered parameters. iconclusion : there was a marked increase in il6 in the early phase of trauma . i16 and tnf release after major trauma iwith hemorragic shock is associated with splanchnic malperfusion, as assess by the ivery low values of phi. lactates seem to be a later indice. toxic effects are a well-known complication of an overdosage of prescription theophylline. what is less known is that over-the-counter (otc) asthma medications contain theophylline, and that in some cases this might cause toxic effects. a case seen by us involved toxic effects from theophylline in an otc medication and to date is the only published case in the english literaturet the rationale for this study was to delineate the otc products containing theophylline from whatever data sources available. hyperthermia frequently occurs in intensive care treated patients and intentional application of whole body hyperthermia together with chemotherapy is a therapeutical access to treatment of malignant disorders. anaesthetic support is required in either condition. due to the marked decrease in systemic vascular resistance seen in hyperthermia an additional vasodilatory effect of the anaesthetic is unwanted. the vascular effects of anaesthetics in hypertherm organisms is not known in detail. therefore, we performed an experimental study to detect the effects of inhalational anaesthetics in whole body hyperthermia. in 30 sprague-dawley-rats katheters were inserted into trachea, jugular vein, and carotid artery. for continuous monitoring of cardiac output a flow probe was placed around the aortic arch. the rats were mechanically ventilated with different concentrations of inhalational agents in oxygen. we compared the effects of enflurane, isoflurane, and halothane in stepwise increased body temperature by submerging in a temperature controlled water bath. results: isoflurane lowers arterial pressure more than halothane or enflurane. the inhalational anaesthetics lower the cardiac output similarily and independently of temperature. isoflurane decreases systemic vascular resistance independently of core temperature and the decreasing effect of halothane on the resistance is completely abolished in hyperthermia. conclusions: the influence of hyperthermia on the systemic vascular resistance is dangerous. this allows no additional effect of the anaesthetic management. in spite of the vasodilating effect of inhalational agents in normotherm subjects, this effect is abolished in hypertherms using halothane. the condition of management of analgosedation in hyperthermia is different from normothermia. objectives: to evaluate a bedside computer processed cerebral function monitor for assessment of brain wave activity when clinical/visual clues are not present. methods: ten icu patients undergoing neuromuscular blockade monitored with the aspect 1000 brain wave monitor from january 1 to june 1, 1995. results: time to onset and depth of sedation were readily apparent to icu physicians not specifically trained in eeg reading. objectives: to determine whether non-depolarising neuromuscular blockade reduces oxygen consumption (vo2) in sedated, apnoeic patients. methods: haemedynamic. metabolic and oxygen transport variables were determined in 5 sedated, apnoeic patients with severe acute lung injury. all patients were ventilated using a puritan-bennett 7200ae ventilator with integrated 7250 metabolic monitor. inclusion criteria were; 1) stable cardiorespirator s" status; 2) systemic and pulmonary artery catheters already in situ; 3) inspired oxygen < 80%. patients were sedated with midazolam or propofol to abolish response to verbal stimuli, and sufficient morphine or alfentanil to abolish all spontaneous respiratory efforts. following baseline measurements, neuromuscular blockade was induced with intravenous vecuronium, 150 ug/kg, followed by an infusion of 80 ug/kg/h to maintain the train-of-four ratio at 0. a further four sets of measured and calculated variables were obtained at 20 min intervals. results: statistical analysis was by repeated measures anova. there were no significant changes in any variable over time. the changes in calculated oxygen consumption (vo2fick) , and measured oxygen consumption (vo2gas), and in energy expenditure (ee), are shown in the table. objetive: to study the effects on coronary hemodyrtamics and myocardiai metabolism of administering propofol during postoperation sedation of patients with normal coronary circulation and good ventricular function undergoing cardiac surgery. patients and methods: 18 patients (12 women and 6 men) undergoing aortic and/or mi~-a/ valvular cardiac surgery were selected, with an ejection fraction greater than 0.5 and normal coronary circulation. for postoperation sedation propofol was administered in 0.5 mg/kg i.v. bolus, followed by a 2.2 mg/kgth perfusion. all data were registered before administering propofol and after 20 minutes, the patients being hemodynamically stable and a rectal temperature of 34 _+ 0.5 -~ systemic and pulmonary hemodynamics, and global, as well as regional myocardial blood flow, and metabofic variables were measured. results: the patients studied were about 56 years old, and the average period of aortic cross-clamp was 77.50 min. the adminstering of propofol caused a decrease in the coronary blood flow (-9%), great curonary vein flow (-23%), myocardial oxygen consumption (-14%), regional myocardial oxygen constanption (-11%), myocardial oxygen extraction (-6%), regional myocardial ooxygen extraction (-10%), while coronary vascular resistances and global coronary vascular resistances did not change. oxygen saturation increased in the coronary sinus (+16%) as well as in the great cardiac vein (+32%). in no patient were significant changes suggestive of myocardial ischemia objectified. there was also found a decrease in systolic (-23%), diastolic (-20%) and mean (-25%) arterial pressure, systemic vascular resistance (-20%), and cardiac output (-8%). conclusions: in accordance with the clinical conditions of this study, the administering of propofol is not likely to cause changes in coronary autoregulation, oxygenation and myocardial metabolism. obietive: analyse the effects of 0.4% "end tidal" isoflurane (sedative dosage) on the metabolism and coronary hemodynamics during the postoperation period of patients undergoing cardiac surgery. patients and methods: 16 patients (12 women and 4 men) undergoing aortic and/or mitral valvular cardiac surgery, with an ejection fraction greater than 0.5 and normal coronary anatomy, were selected. after the surgical operation, 0.4 "end tidal" isoflurane was administered for postoperadon sedation. the determination of variables to be studied was carried out before and 20 minutes after administering isoflurane, die patients being hemodynamically stable and a rectal temperature of 34 _+ 0.5 -+c. systemic and pulmonary hemodynamics, and global, as well as regional myocardial blood flow, and metabolic variables were measured. results: the average age of the patients studied was 57 83 -+ 8.87 years. during surgical operation the period of aortic cross-clamp was 78.56 _+ 32.09 rain. the administering of isoflurane was followed by a statistically significant drop in coronary perfusion pressure (-26%), coronary vascular resistance (-29%), regional coronary vascular resistance (-29 %), regional myocardial oxygen consumption (-7%), regional myocardial oxygen extraction (-6%) and accompanied by a significant rise in oxygen saturation in the coronary sinus (+16%) and in the great cardiac vein (+32%). myocardial oxygen consumption, myocardial exu'action of lactate and regional myocardial lactate extraction did not change. in no patient were enzyme or electrocardiograph changes objectified. systolic (-23%), diastolic (-25 %), mean (-25 % ) arterial pressure, and systemic vascular resistances (-28%) decreased, while cardiac output did not. discussion: the administering of 0.4% "end ddal" isoflurane, in the clinical conditions of this study, produced a decrease in systemic arterial pressure due to a reduction of systemic vascular resistance without deteriorate cardiac output. at coronary circulation level, has and effect on coronary autoregulation but had no effect on oxygenation and myocardial metabolism. the idea of tiva implies the realisation of major anesthesia components (los of consciousness, neurovegetative inhibition, analgesia, myorelaxatiou, providing the adequate gas-exchange) through i.v. introduction of drugs exclasively. aim: providing for the main tiva components with minimal side effects of the drugs used, taking into consideration the patients characteristics and the surgery specific character. methods: 78 anaesthesias have been conducted in patients aged 15 75 years (28 females, 50 males), undergoing planned and urgent operations with the pathology of lower, extremities, perinaeum, small pelvis, hypogastrium and with reserved spontaneus respiration against a background of 100 % 02 insnffladon through mask. operations lasted from 0.5-1.5 h. anaesthesia adequacy was assested by constant monitoring: "cardiocap" (nibr hr, rr, sao2, t), through glykhaemia level and mimicry reactions. standart premedicatioo of m-cholinolytics (0.01 mg/kg) and h2-blockers (0.3 mg/kg) on the operational table was sumplemented by administration of 0.5-1.0 mg/kg of lidocaine, 1.50.0 mkg/kg of clonidine, 0.5-1.0 mg/kg of pentamidine by the tachifilaxia method. the premedication adequacy was assessed through haemodynamics characteristics. sedation: 0.05-0.1 mg/kg of droperidoi, 0.l-0.15 mglkg of diazepam and analgesia: 2-3 mkg/kg of phentanyl, 1.0--1.5 mg/kg of ketamine were introduced fractionally according to indications. infusion rate of ringer-lactat solution was 5-15 ml/kg/h and depended on the intraoperational blood loss volume and on the patients preoperational condition. the duration of postoperative analgesia was registered. results: clinical assessment of analgesia according to this techniques allowed to decrease the anaigetics dosage to the subauaesthetic levels. smooth stabilisation of haemodynamics (bp) at proper age norms in patients with the initial hypertension by the 30-th min. of anaesthesia as well as the absence of its increase in response to the additional introduction of anaesthetic have been achieved. (hr) had no abrupt changes and remained in the range of 70-80 per rain. adequate external breathing: decrease (rr) by 2-3 per rain., with sao2 increase from 9446 % to 98-100 %. hypoventilation was avoided by respirate ventilator. according to unauthentic data the glykhaemia level had been lowered by 10-t5 % to the end of the operation with the initial moderate hyperglykhaemia of up to 10 mmol/l the cutaneous covering grew warm and got pink colouring. no mimicry reactions. in the postoperative period patients were in the superficial sleep state (3-48) and analgesia lasted 6-8 b. there were no complications due to anaesthesia. conclusion: combined using of bz, opiates, neuroleptics potentiate the i.v. anaesthetics effects allowing lowering of each tiva component dosage and, as a consequence avoiding their negative influence on respiratory and heart vascular systems. complex application of adrenergetics (therapeutic doses of cionidine and pentamini with using of taehfilaxy effects) permitted to provide for analgetic and neurovegetative components of general anaesthesia under subanacsthetic doses of tiva main components, and manifestation of hyperdynamic reactions of haemodynamics decreased while using of lidocaine -the economicai activity of heart-vascular system. good level of muscle relaxation was achieved allowing for widening of surgical intervention extent without respirator ventilators and inhalation anaesthetics application. anaesthesia is easily controlled due to fractional introduction of drugs with quick recovery of cns functions after anaesthesia. postanaesthetic analgesia is increased while concurrent opiates doses are decreased. absence of marced haemodynamic, endocrine and metabolic reactions during the operation and after it resulted in shortening the period of patients staying in hospital. a 64 yo white man was admitted to hospital for dyspnea and a productive cough. he had cabg in past, but no recent cardiac ischemia. physical exam: decreased breath sounds over right lung. chest xray: consolidation of right lung. admission medications included diltiazem, furosemide (both were continued) and trazodone (which was discontinued). admission ecg: sinus rhythm, qt 0.44/qtc 0.49 sec, with st and t wave abnormalities similar to prior tracings. he required intubation and mechanical ventilation for progressive hypoventilation and hypoxemia. between icu days 8 and 16 he received haloperidol, 10-44 mg/d (cumulative dose 209 rag) for agitation and delirium. icu day 11: qt 0.46/qtc 0.57 sec. icu day 12: for better control of delirium, trazodone " 50 mg q hs was added. icu day 15: he developed frequent nonsustained ventdcular ectopy. icu day 16: qt 0.70/qtc 0.74 sec, pha 7.48, paco2 50 mm hg, pao2 72 mm hg, k 4.9 meq/l, mg 2.0 meq/l. later in icu day 16 the patient had 3 brief episodes of torsades de pointes, each responding to precordial thump, and finally rhythm stabilized with i.v. lidocaine and magnesium. haloperidor and trazodone were discontinued. ecg was unchanged and myocardial infarction was ruled out. next day, icu day 17: qt 0.42/qtc 0.53 sec. torsades de pointes, a form of ventricular tachycardia characterized by a twisting qrs axis, is commonly associated with qt prolongation. haloperidol is used frequently in icu for control of agitation and delirium, with reported doses up to 1000 mg/day. over past decade, 9 cases of torsades de pointes with prolonged qt related to haloperidol have been reported. trazodone may also prolong qt and cause ventricular arrhythmias, especially in patients with pre-existing cardiac disease. in this patient, trazodone likely exacerbated qt prolongation from halopeddol leading to torsades de pointes. critical care physicians must be aware of this interaction. it is imperative to follow the qt interval for patients receiving halopeddol, especially when another drug also known to prolong qt is added. one must consider discontinuing the drug when qt/qtc becomes prolonged. objectives: analgesics and intravenous anesthetic drugs are routinely used in critically fll patients, who often suffer from a secondary impairment of the immune system. previous in vitro studies have demonstrated inhibitory effects of these drugs on polymorpho nuclear cells (pmn). the potentially important role of endothelial cells (ec), however, was not investigated, since suitable test systems were not available until recently. therefore a physiologically more relevant in vitro migration assay through cultured human endothelial cell monolayers (ecm) we established. using this assay system, the comparative effects of fenlanyl, sufentanil, propofol and the known pmn inhibitor thiopontal were tested. methods: human umbilical vein endothelial cells (huvec) were isolated and cultured on microporous membranes (cyclopererm) until an ecm was grown. pmn from male and female volunteers were separated by standard procedures. ecm and pmn were preincubated with clinically relevant concentratious of thiopental (104 m), propofol (4p_g/ml), the solvent of propoful (intralipid), fentanyl (30ng/ml) and sufentanil (sng/ml). after preincubatiun (ecm 30 minutes, pmn 15 minutes) with the reslx~tive drug, leukocyte migration towards the chemoatfractant fmlp (1o 7 m) was measured in a two chamber 24 well system for 3 hours. the migration rate of untreated (untr.) and treated (treat.) pmn through untreated and treated ecm were determined. as a control untreated pmn and untreated ecm were used. results are given as means from 5 independent duplicate determinations and expressed as a percentage of control (table) . statistical analysis was done with student's t-test. results: clinical concentrations of fentanyl, sufentanil and prupofol showed similar inhibitor~ effects as the known pivin inhibitor thit e 1 ). 77% conclusions: for the first time we could show that analgesics and anesthetics exert their inhibitory effects not only on pmn, but mainly on the interaction of pmn with endothelial cells. moreover, we could shmv a significant suppressive effect of the opinids fentanyl and sufentanil on both ec and pmn. the known inhibitory effect of thiopental obtained in ec-free test systems were also confirmed in our physiologically more relevant assay system. objectives: to investigate when and how sedation is used in a consecutive cohort of patients admitted in a large sample of italian intensive care units (icus), gathered in a network named giviti, representative of the italian icus system. methods; the study called for a recruitment period of one month, from january 10 to february 8, 1994, data collection included age and other demographic variables, acute diagnostic broad profiles, severity of illness scores, treatments, lenght of stay and vital status at icu discharge. as concerned sedation, each patient was observed until discharge or for a maximum period of seven days. information on all the drugs used for analgesia/sedation, the route and modalities of administration, the timing, dosages and purpose of the administration have been recorded. results: the study involved the cooperation of 138 icus, 128 of which enrolled at least one case. the total sample included 2932 patients. overall, 60.7% of patients analyzed (t780/2932) received at least one prescription of sedative during their stay. globally, at least one sedative drug was prescribed to these 1780 patients in 5014 days in icu. although over 38 drugs were reported to be used, 10 pharmacological principles accounted alone for 89% of all prescriptions. opioids were actually used in 33% of prescriptions; propofol in 24% and benzodiazepine in 18.3%. as regards the way of administration, intravenous administration was applied in 74% of cases and, followed by intramuscular in 17.3%. moreover, non-steroidal anti-inflammatory drugs (nsald) were used in 19% of patients and neuromuscular blockade agents (nmba) in 23%. detailed analysis on certain subgroups (surgical, trauma, ventilated patients etc.) have been also carried out in order to describe the practice of sedation in these peculiar subgroups. findings will be widely discussed during the presentation. conclusions: these results should be interpreted keeping in mind how peculiar is the intensive care setting compared to many other less complex settings of hospital care. in conclusion we thought it was important to present the data currently available in the most neutral form, to start moving in a direction which will enable us -by means of more specific and detailed studies, and with the cooperation and involvement of all those participating in the project -to shed light on one of the many aspects of medical practice in the field of intensive care which deserve closer attention. introduction: the aged run perilously high risks in cardiac surgery: among others, of haemodynamic fluctuations, respiratory depresskm and organ failure. response to anaesthetics is a crucial determinant for post<)perative complications, none the less being reintubation due to mechanical ventilation difficulties which increase morbidity, mortality and intensive cdre unit (icu) stay. objective: we wanted to assess our a,aesthesia window (selection, and a view of the induction -extubation period) for predicting safe and swift awaking, thus: icu dismissal for the aged. methods: in 1994, 162 selected patients (pts) (>70y, 62f) followed a regular elective cardiac surgery protocol (propofol given at precisely designated time intervals). upon 1cu arrival, they were subjected to an admission protocol. our predictive criteria for early extubation at 8h included: a) alertness and ready response to commands; b) adequate gag reflex and sufficient protection for respirak)ry tract; c) pao2 >75 mmhg with flu 2 <0.4; d) stable ph>7.35 with spontaneous respiration; d) stable haemodynamics without dysrhythmias; e) adequate perfusion and diuresis (>1.(i ml/kg/h); f) mediastinal bfeeding<100ml/h for at least 2h; g) normothermia (core temp>36~ and no shivering). subsequent reintubation was for: 1) rr>35/min; 2) spontancx)us ventilation for 30 rain with paco2>50 mmhg; 3) pao2<50 mmhg with fio2>0.4; 4) ph>7.45; 5) heart rate>]20 bm; and/or 6) non mental alertness; and 7) other medical disorders, after which adequate weaning therapy was necessary. then, successful weaning after 24h was considered: 1) spontaneous breathing without any forrn of mechanical assistance; 2) stability in haemodynamics; and 3) elimination of fever threat. results: 122 pts (75%) were extubated at 8h without complication; 29 other pts (18%) at 8h but had to be reintubated because they were hypoxic and began weaning therapy; finally, they were all re-extubated by 48h. only 11 pts (7%) proved problematic. conclusion: a,aesthesia wimhlw options (selectkm, extubation, reintubation and weaning) predicted quick (times propofol administration) and safe (rigid criteria) extubation (75%=8h and 18%=24h), exempting pts with developed post-operative complications (7%=extubation<72h) unrelated to al~aesthesia window or icu protocol. dismissal and recovery then became an abbreviated question of time. fifisetll p, domeneg~i ~, sforzini i., veronesi i~, maconi a.g. *, breg~ massone p.p 30h [] ic+pca request conclusions:using e~aprenorphine, a synthetic,long-acting, ago-antagemist opinid drug as analgesic, in the major surgery we obtained the best clinic results with association of conttheus infusion of haft dose drug with bohts of pca in the first 15-20 hours and just pca in the secmad day after surgery when the patient is less sleepy. in this way we dent have a great sav~g of suppled drug but the major well-belng of patient without ~erious side-effects and quick mobilization; the dosage used don't compromise a good awake of patient: all patients are sleepy but ready for answer, no allueinatian, bradipnea but not less than 10 b/m without ipoxia. also the patient proffered this kind of truit meut than the traditional at demand. the ward staff feel it useful] and rehabl~ the negative feed-back technology of the electronic infuser system makes possible to use it safe in the ward with high drug's concentration too. the infusion rate of low dose of drug assure a continuative analgesic covering ~n the first postoperative periad; the pca mode involves the patient him-self in the managemenl of therapy and enables him to choose the best way to confront the dll~icuity of postoperative period without call medical stall using pca-device we have had no probicm~ no accident. analgesia during extracorporeal shook wave lithot ripsy a .levit, b.grinbezg regional hospital, ekaterinbu~g, russia 0b~ectives: our task was to compare ~he analgetic effect of norphin and tramel. methods: study was made of two groups of uro-li~patients aged 25-61. group a (23 patients) received baprenorphine hydrochloride (norphin) at dosages of #.52• mg/kg. group b (30 patients) received tramadel hydrochloride (t~aasl) st dosages of 1.50z0.38 mg/kg. before the procedure diazepam was administrated i.v. (0.24!0.03 mg/kg). blood saturation (spoz), hemodynamics incides (bp, hr,sv,co,sap,svr) were examined and the patients' subjective assessments of snsesthesis quality were analyzed. the hospital ethics committee approved the investigation. results: when using norphin hr increased by 17.7% on the onset of the procedure while sap and sv decreased by 8.%% and 9.6%, respectively (p<0.05). however, there were no reliable co chsnges. spoz ~educed by @.2% (p<0.05) and remained lower than the initial one after the procedure was oyez. when administrating tramsl 50 min. after ste~ting the procedure sap and svr increased by ~1.2% and 7.3% respectively. sv and co decreased insignificantly. nine patients in group b saffeting some dlscomfo~t needed additional tm~msl in~ection. in the course of the whole p~oced~e spo, was constant and was highez than that in ~he case of nozphin (p<o.05). conclusion: respiratory suppression by no~phin can limit its use in case of lithotzip@y while the advantages of tremsl a~e: lack of dyspnoe and good contact with the patti-b_ the role of uncommon agents of antinociception, placed epidurally, in the control of pain transmission in the spinal cord is well documented. somatostatin as an inhibitory agent is found in the dorsal horn of the gray matter of spinal cord. the effects of somatostatin is similar to the opiates if injected into the epidural space (ref. i, 2, 3, 4) . the aim of the present study is to present the clinical effect of above mentioned palliative therapy in the case of cancer and non cancer pain. informed consent had been obtained from every patient before treatment was initiated under the legalisation of the local ethics committee. using the permanent epidural catheter and continuous infusion pump, we administer somatostatin in the dose of 5 up to 20 microgram per hour for one up to three days. the patients paln feeling was evaluated using a vas (visual analogue scale). we found that the opiate resistant pain level decreased by 50%. the rest pain could be controlled by pereral opiate analgetics or by combination of somatostatin and an opiate plus a local anaesthetic agent epidurally. theoretically, it seems that epidural administration of somatostatin and local anaesthetic agent is useful in the control of acute pancreatic pain and systemic effect of somatostatin is beneficial in the treatment of acute pancreatitis. few studies have been performed in the critically ill (ci) and because of the complex pharmacodynamic and pharmacokinetic changes that occur, it is difficult to predict accurately drug responses and interactions in individual patients. midazolam (m) is a watersoluble benzodiazepine with a rapid onset and short duration of action in normal individuals; however its metabolism is decreased in the ci. critical care physicians every day experience suggest that among the ci there is a different pattern of response to m; here it is hypothized that such a response could be due to different metabolic behaviours of ci. m (i.v.infusion rate 1-7 rag/h) and antipyrine (1000rag p.o.) were infused to ci with (10 patients -group 1) and without (10 patientsgroup 2) endotracheal intubation. blood samples were collected at fixed times during and after m infusion. total urine nitrogen and antipyrine elimination half-life demonstrated different metabolic characteristics in group 1 chypermetabolizer") as compared to group 2 chypometabolizer"). results follow. the results of our study are preliminary and more kinetic data in critically ill patients are needed to statistically confirm our approach of "hypometabolizer" and "hypermetabolizer". objectives: some clinieai and experimental studies suggest that propofol decreases myocardial contractility in coronary artery bypass grafting (cabg) patients. the intrinsic negative inotropic action of the drug may be independent from changes of preload and afterload, whereas the myocardial depression induced by propofol may depend on changes in ca ++ ious availability. aim of this study was to verify the hemodynamic effects of propofoi in a group of cabg patients with uneompromised contractile function and to minimize myocardial depression, during induction of anesthesia, choosing to associate calcium chloride (cac12) in an other group and to define its role in producing this effect. methods: fourty patients, randomly divided in two groups (20 pts a and 20 pts b), undergone elective cabg, were enrolled in this study. anesthesia was induced by senior anesthetist in both groups by means of fentanyl 7 mg• kg < in 60", pancuronium 0.1 mg x kg -1 and propofol 2 mg • kg -1 in 60". a blind investigator administered via another venous way at same speed (60") saline in patients of group a and 10 mg x kg -1 cac12 in patients of group b. hemodynamic data (heart rate hr, mean arterial pressure map, mean pulmonary artery pressure pap, pulmonary capillary wedge pressure pcwp, central venous pressure cvp, cardiac index ci, stroke volume index svi, systemic and pulmonary vascular resistances indexed svri and pvri) were obtained at baseline (to), 2' after anesthesia induction (t1) and 2" after tracheal intubation (t2). results: hr decreased moderately in both groups (p = ns). map decreased as well, more markeatly in group a at ti and t2. this trend was statistical significant (p < 0.05) in both groups. pap, pcwp and cvp unchanged following induction in any of two groups. ci decreased in group a (p < 0.05) dramatically dropping at t2, while in group b it showed a negligible decrease at t1 (p = ns) and value similar to baseline at t2. svri and pvri did not exhibit statistically significant changes. conclusions: the use of propofol as a starter of anesthesia in cabg patients allows to reduce total dose of fentanyi, dramatically reducing post-operative intubation time. propofol-fentanyl association prevented the hyperdynamic response to stress (i. e. laryngoscopy, intubation) but severely depressed ci and svi. cac12 simoultaneous administration effectively minimized the negative inotropic effect of propofol. moreover no unwanted side-effects due to cac12 were observed. diseoordinated labor activity (dla) is one of most serious in obstetric pathology.medication sleep effect to a woman in birth is of limitation of pathologic impulsation from the central nervous system, but it does not influence processes resulting in and supporting dla on the organ level. constant discoordinated uterine contractions during medication sleep (ms) imerease disturbances of uterine blood flow, organ hepoxemia connected with hyperergia of vascular wall and myometrium to catecholamines, that reduces efficiency of anesthesiologic protection. we applied hexoprenalin in complex with ms to 20 primaparas. efficiency control was being accomlished through hysterograms, grade scale of analgetic effect where hemodynamics reaction to pain, significance of emotional reactions and their vegetative manifestations before and during ms were registered, and also through hydrocortisone level in plasma before and during ms. the examined women's average duration of ms was 2 hours more than in control and made 4 hours, though their average labor duration and in control was the same and made 12 hours. 98 % of the examined women experienced independant labor, as for the control group -only 68 %. supplementary methods of anesthesia were required in 73 % in control,that increased medicamentous depression of the fetus, but for the examined group -only in 16 %. the examinees's newborns experienced favourable terminations, in control 3 newborns were in state of light rate asphixia. analgetic effect was complete in control just in i5 %, and of examinees -in 82 %. on the women's hysterogrnms before ms discoordinated contractions were registered on the background of increased tonus. during ms in control similar uterine contractions were constantly registered, but for the examinees they were not marked at all or had the proper character. the examinees' hydrocortisone level was reduced for 42 % and in control only for 24 %. the study has allowed to make a conclusion that hexoprenalin applications in complex with ms in case of dla make anesthesiologic care safer and more effective, promote simultaneous removal of patholgic processes in the central nervous system and uterus, which lead to dla development. objectives: the aim of report is to present a clinical study results for the evaluation of anesthesia for a patients in unconscious states by eeg examination: methods: the study was conducted on 17 patients after a severe abdominal operations on stomach and intestine and on one patient after traumaticat exarticulation of upper extremity. all patients have different unconscious state levels from somnolence to coma ]. also thay hav~ had respiratory insufficiency and control mandatory ventilation have been used. we were used intrave,~ous injections of morphine hydrochloride from initial dose of 20 mg. than we were increased the dose to 80 mg i.v. by drops with an accompanying eeg monitoring and fourier spectral analysis. initially all patients have had 13-rhythm with slow 0and a-activity. an c~-activity index have been lower than 5%. results: after anesthesia the o-and a-activity have disappeared. the or-activity index have increased to 35%. also we have noted decreasing of the unconscious state levels to a possibility of a verbal contacts (13-14 points gcs). conclusion: the quality of anesthesia could be evaluated by eeg monitoring. a regaining consciousness afte~ the injections of high doses of morphine, probably, may be a result of an opiate deficiency (full or partial). anesthesia with the following drugs: clonidine, l-arginine, l-n-arginine-methyl-ester (l-name), and their combinations. tail-flick latency (tfl) was determined at time zero and 15 min after each treatment. clonidine (i-4 ~g/mouse) prolonged tfl in a dose-dependent manner. at a clonidine concentration of 4 gg/mouse, tfl increased 2.37-fold compared to time zero. l-arginine increased tfl at a high concentration (i00 gg/mouse). l-name suppressed tfl 1.2-fold compared to time zero at a concentration of 50 gg/mouse. conclusion: we have found that no is involved in clonidine spinal analgesia. studies are currently being undertaken to assess the effect of combination treatment of the above drugs on clonidine supraspinal analgesia. objectives: hemodynamic changes ussualy accompany laryngoscopy and tracheal intubation.the aim of this prospective study vas tu present the effectivness sufentanil in preventing reflex circulatory respone of laryngoscopy and tracheal intubation and provides stable hemodynamio condition for induction of balanced anaesthesia. methods: 30 adult asa i-ii patients sheduled for elective surgery.they were allocated randomly and divided into two groups:fifteen patients received single bolus of placebo prior to laryngoscopy.and tracheal intubation. anaesthesia induction was then performed with thiopental (smg/kg) and atracurium (0,5 mg/kg), followed by neurolept anaesthesia. mean arterial preassure (map) and heart rate (hr) were measured before, during and 5 min after intubation. althought ecg was recorded at the same time intervals. results: the groups were compared with regard to the changes in arterial blood pressure,heart rate and electrocardiogram. mean arterial preassure and heart rate were incresed significantly during and after laryngoscopy and tracheal intubation in control group but remained unohange the baselin values in the group who received sufentanil. control group ecg suggested more changes than sufentanil group. conclusions: the results indicated that 1,2 mg/kg sufentanil for anaesthesia induction reducing the pressor response to laryngoscopy and tracheal intubation. obiective: the aim of this experimental study was to evaluate morphologically the influence of anesthesia in liver as well as in isolated hepatocytes (hc). methods: a total of 16 female swine (20-22 kg) were used as liver donors and were randomly assigned to one of two groups regarding anesthesia protocol: group a (n=8): induction with intravenous sodium thiopental in a dose of 5 mg/kg, group b (n=8): propophol in a dose of 2 mg/kg for induction'and 10 mg/kg/h for maintainance of general anesthesia. both groups were under the same preanesthetic regimen (diazepam, ketalar and atropin) and received standarised doses of fentanyl and pancuronium during anesthesia. in beth groups total hepatectomy was performed and the liver was perfused with 4-5 it of cold (4oc) university of wisconsin solution for a period of 5 hours before hepatocyte isolation. liver tissue samples were fixed in formalin for the morphological study and stained with hematoxylin-eosin and pas. for hc isolation collagenase solution (type v, sigma, c-9263,1.3mg/ml) was infused via portal vein and the liver was incubated at 37oc for 45 rain. cells were filtered and then washed in cold hanks' solution. isolated hc were fixed and prepared for staining or simply cryopreserved (-20oc). results: histology was completed in 8/16 experiments (4 in group a and 4 in group b). mononuclear infiltration (halothane type injury) was found in all specimens (8/8). focal zonal necrosis and acidophilic bodies were found in specimens from group a (2/4 and 1/4 respectively) while portal inflammation with piece meal necrosis was found in one specimen from group b (1/4). smears of hepatocytes -beth formalin fixed and cryopreserved at -20oc -were stained with hematoxylin-eosin and showed morphologically intact hepatocytes. conclusion: pre[imineq~' study of our material reveals mote frequent livet injury in the thiopental group, but this finding has yet to be established by increasing the number of observations. objectives: in this paper we present our experience and point out the importance of sedation of those patients who suffered severe head injuries/contusic cerebri with signs of decelebration/and who were put on ventilatory machine to obtain better mechanical ventilation in neurosurgical intensive care unit of emergency center in belgrade. methods: 256 patients were treated from jan. 1994 till dec. 1994 results: in 157 patients we successed to reduce agitation and decelebration. conclusions: the major demand of sedation in neurosurgical patients are that the patient should be calm, comfortable and painless. references we compared three analogous groups of 20 patients in each, with orthopedic operations on lower extremities. in each group we applied different kind of analgesia: first group rece-ned local anesthetic (4 ml 0.5~ bupivacaine) when analgesia was first demanded; second group received 2 ml fentanyl via peridurai catheter when analgesia was first demanded, and third group received 2 m] fentanyi intravenously on demand for analgesia. we used visual analog scale (vas) for estimation of pain intensity and success of applied therapy. statistical data analysis was performed using student's t-test. tha best vas had group in which local anesthetic was applied periduraly. second was the group with periduraly applied fentanyl, and third was the group with intravenously applied fentanyl the longest duration of the effect of analgesia ~/as in the second group. there were no adverse effects and complications, apart from mild sedation in the third group. for orthopedic operations on lower extremities, application of peridural catheter for anesthesia and successful postoperative analgesia with local anesthetic, or opioid analgesics is acceptable. background and obiective : tympanic temperature measurement (ttm) is a relatively new procedure which provides accurate estimates of core temperature in stable postoperative patients and in patients admitted to the emergency ward. however, experience with this technique in hemodynamically unstable adult icu patients is limited. design : prospective study with patients serving as their own control. se:ttijlg : adult medico-surgical icu in a tertiary care hospital. patients : 51 consecutively enrolled patients with thermodilution catheters in place and without contraindieation for rectal temperature measurement and without hypothermia (core temperature <35~ interventions : tympanic temperature, measured by a commercialized tympanic thermometer (genius 3000 a first temp) was compared with pulmonary artery (pat) and rectal temperature (rt). within 10 minutes, core temperature was assessed in each patient by the same trained individual using the three techniques in random order. measurements and main results : mean bias (ix~ and its variability (sdr of method comparison pairs were calculated using the methods comparison analysis as described by bland and alunan (lancet, 1986 ; i : 307-310 objective: to measure the prevalence of pressure sores on the intensive care unit and the effect of risk calculation on pressure score prevalence and pressure score severity. method:in 1993 during a period of 5 months a series of prevalence studies was carried out on the sicu to investigate how many patient days with pressure sores were taken care for by the icu nursing staff, what kind of preventive interventions were done and what the patient risk was on developing a pressure score. results: on average there was a prevalence of pressure sores on the buttocks of 18.5% on the short stay unit and 42.8% on the long stay unit; preventive interventions increased when the pressure score was visible for the nurse and the majority of patients had a high risk or extra high risk for developing pressure sores according the pressure score risk calculator. as a result of these findings the nursing management decided in 1994 that nurses had to complete daily on every patient the pressure score risk assessment in order to be able to take adequate preventive interventions. when the results of the two studies were compared, the most important results were that: 1) there is no indication of a reduction in patient days with pressure sores (short stay unit: 14%, long stay unit: 55%); 2) there is a reduction in severity of pressure sores (grade iv decreased from 2.4 % to 0.7 %) because adequate preventive measurements are initiated by icu nurses in an earlier stage. possible explanations for the increase of patient days with pressure sores on the long stay unit are that the average pressure score risk score was increased from 17.8 to 19.8 and the mean frequency of nursing patients on alternate sides decreased from 1.4 to 0.6 times each day. conclusion: daily assessment of the individual patient risk on developing pressure sores does not decrease development but reduces the severity of the developed pressure sores. method: the conventional woundcare method of patients with an open abdomen is to change frequently the saline soaked ribbon gauze pads (4-6 times each day). this method is labour intensive for the itu nursing staff and rather uncomfortable for the patient. problems that often occur are: insufficient hygiene, frequent nursing interventions, a progressive risk for deterioration of the skin and underlying tissues, difficult to measure abdominal output and in case of bowel fistulas enteral feeding is hardly possible. in using the new method, stomahesive is applied on the skin surrounding the wound. a large size of surgical film dressing, opsite | is applied over the abdomen. two or more foley ~ catheters ch 22 are inserted through an opening in the surgical filmdressing. the application of several lateral openings in the catheters is advised. the drains are held firmly in place between two layers of sterile opsite | "flexigrid ~'' 6x7 with the so called bookend method and are connected to a low vacuum suction system (1-2 kph) results: we studied the frequency of dressings changes on 12 patients in the itu. in total they had 363 days with an open abdomen. during this period there were 63 dressing changes. this means one dressing change every 6 days. further advantages of the new method are; more hygiene in patient care, no maceration and irritation of the skin, nursing on alternate sides is possible, output can be measured, in case of bowl fistula, enteral feeding is possible and an abdominal lavage is possible on the sicu by opening the opsite | conclusion: this new method of woundcare management is more patient friendly, prevents several nursing complications and decreases the work load for the nursing staff. objective: to investigate the interaction between the ventilator and the closed suction system related to possible induced negative pressure by this sytem. method: we studied in a testlung model what the effect was of the different ventilators (evita: dr~iger; servo 900 c: siemens), ventilation modes 0ppv, simv) and ventilator settings (tv, trigger level, frequency, peep level) on the induced negative pressure by the closed suction system during suctioning. results: when using the evita the magnitude of the negative pressure increased when the ventilation frequency and tv decreased.. the largest negative pressure (-76 cm h20) was produced with a tv of 600 ml and a frequency of 6 /min in the ippv mode with trigger level on -3 cm h20 and a peep level of + 10 cm h20. the servo 900 c showed a complete different pattern. the largest negative pressures were measured when no trigger level was set (27 cm h20), the induced negative pressure was not depended on the tv or ventilation frequency but more depending on the ventilator mode. the simv mode produced the smallest negative pressures ( 1-4 cm h20 ) and the ippv mode the largest (16-27 cm h20 ). conclusion: the effectiveness of the closed suction system is very much depending on the kind of ventilator, ventilator mode and ventilator setting that is used. if the interaction with the used ventilator is not known by the icu nurse this piece of equipment can cause damage to the patient by inducing large negative pressure in the comprimised lung and there fore creating the possibility of alveolar collaps and atelectasis or oedema. introduction : ethical problems are daily and disturbing preoccupations for the icu staff. objectives : improving ethical management in icu by creation of an intelprofessional group of ethical reflexion ( iger ). methods : existing was evaluated by a preliminary formulated series of questions sended to the whole staff (q1, n = 43, 13 items) after two training sessions (laws, deontology, professional values, culture, norms of quality) followed by 38 persons (88 %) and directed by an external chairman, satisfaction and needs of the staff were evaluated by a second series of questions (q 2, n = 38, 12 items). at the issue, iger was created. results : q1 : 30 responders (70%). law's misinformation : 33 to 70 %, disagreement for limitation of intensive cares : 40 %, hope to improve collective decisions and creation of iger : 88% q 2 : 29 responders (76%) : satisfied by the training sessions : 65%, non satisfied : 31%, want to continue the project : 68 %, refuse : 10 %. iger was constitute by physicians, nurses, secretary, dietetic (n = 15). the first working theme was {< therapeutic's withholding and withdrawing decisions in icu ~>. four subgroups of iger's members (having access to an ethical library) worked independautly and submitted their reflexions in a tdmestrial plenary session of iger in the presence of an external chairman, allowing a synthesis. at the issue a report was writted to be used as a reference for bedside and individual decisions. conclusions : constitution of iger seems to improve ethical management in icu. the first result of iger is that it is now possible to began collectively a reflexion concerning therapeutic's withholding and withdrawing in icu. the work is going on and further subjects will be studied. objectives: 1) to compare the value of heat-moisture exchangers with bacterial filters (hmef) and without bacterial filters (hme) in the prevention of colonization of ventilator tubing and ventilator-associated respiratory infections. 2) to asses the temperature and relative humidity of inspired all using both types of heat-moisture exchangers. methods: 48 mechanically ventilated patients were randomized, to either hmef or hme. endotraeheal aspirates, pharyngeal swabs and samples from tubing were collected for bacterial cultures on the 1st, 2nd day mechanically ventilation and weekly thereafter. temperature and relative humidity were measured in 23 patients (13 hmef and 10 hme) 3 h and 24 h after placing the hme or the hmef. results: both groups were comparable as regards age, mechanical ventilation period, severity score (saps ii), leukocyte count, and number of patients with prior antibiotic treatment. from the hmef group, 10 (42%) ventilator tubing yielded microorganisms in, at least, one sample as compared to 7 (29%) of the hme group; p=ns. the incidence of respiratory infection was similar in both groups (25% vs 17%, p:ns, for hmef and hme respectively). among the 16 bacterial species isolated from ventilator tubing in the hmef group, 7 (44%) were not isolated from pharyngeal swabs. a similar ratio was shown in the hme group (6/15, 40%). both heat-moisture exchangers were efficacious in keeping a good relative humidity of inspired air (97% • vs 96% • 3.%; p=ns, for hmef and hme respectively). relative humidity was significantly higher after 3h of mechanical ventilation in the hme group as compared to hme group (28.5% • vs 26.5% • 2%; p=0.03). conclusions: both types of heat-moisture exchangers have the same effect on the prevention of colonization of ventilator tubing. similar relative humidities are achieved when using either type of heat-moisture exchanger. results: tumor and nontumer enhrgements of the thyroidea were present in 85~ of the operated, surgicel adrenal disease in io!, hyperplssle or persthyroid gland tumor in 2~ end endocrine pancreatic tumors in 3%. in the intensive oere unit, these patients wore screened by noninwsive monitoring in 85~ of cases: and invasive monitoring was applied in 15% of ceses.the basic noninvesive methods included: electrocardiogram with standard end precerdial leeds, percutaneous eutomotlc measurement of systolic, diastolic and mean arterial pressure, measurement of hourly diuresis and body temperature, frequency, hearing capacity and rhythm of one s own breathbng bs well as pulse oxymetry. a special plece in monitoring and control of vital parameters in postoperative period belonged to the nurse, thoroughly trained for enelysis end interpretation of the observed parameters which would be discussed in the paper. it has been believed that the leader sits at the pinnacle of power. over the years, this has proven to produce frustruation and anguish instead of the expected results. leaders have not been able to produce the changes they know are essential to their organization's survival with this command-and-control paradigm. through literature reviews and evaluating leadership styles, one can clearly see the most effective form is that of empowering people to a new level of performance -not ordering it. changing the leadership paradigm to a manner/style that has been shown to be effective and one of people empowerment shifts the focus to personal responsibility for performance. removing obstae}es~ stimulating self-directed actions, and determining focus and direction are just a few elements used to create the successful environment of empowerment. with increasing pressure in the health care arena, it becomes critical that a leader's job is to get the people to be responsible for their own performance. developing ownership, creating an environment where people want to be responsible, being a mentor or coach, and learning faster while encouraging others to do so demonstrates the commitment to effective leadership. this presentation will illustrate the critical components that are achieved when every person in the institution is empowered to perform at a level that is directed toward positive, effective results. herrera m. (md) . icu. hospital regional. malaga. spain. the systems of veno-vanous continuous haemofiltration (wchf) have a high cost and a limited life span. in an attempt of lengthening their mean life it has been proposed to accomplish programmed washes of the ~-stems. this practice supposes an increase in nursing workload. in order to evaluate the real efficiency of this practice we have accomplished this study. material: prospective randomized study of all the filters of vvchf used during the last year in our icu. we have determined two groups of filters, in the first (group a) we accomplished washed in a programmed way, and in the other (group b) only when the alarms of the system suggested a clotting of the filter. for the statistical analysis we used the kaplan-meier test for survival analysis. results: we have studied a total of 24 patient submitted to wchf during the last year. we used a total of 32 filters with this results. objectives. sounding out the nurses about the need to inform patients" relatives and the rigth kind of such information, like a preliminary approach to an information cuality assessment, methods: we inquired all the nurses of the intensive care unit of an regional hospital by an semiestructurated questionary which included personal data: age, sex, contractual relation, professional experience.., and opinion data: do you think to inform relatives is a nurse task?. which of the next informafions do you think is more important?, please, write others topics about information you think are relevant. we process the data on epi-info estatistical program and use x 2 test to compare the results. results" from 80 nurses of staff 5 refused to flu the quetionary, and 8 were not available. of the 67 remaining, 71%were v~men and 29% men. the mean age were 31.51% had an svable contract and 499( eventual, the mean professional experience were of 10 years and 44% worked in the unit since more than 6 years. the 88% answered that offer information to relatives is part of the nurse activities. we did not find differences with nurses who answered negatively comparing by sex, age, contractual relation or proffesional experience. the three information topics found out like more important were: 1) to inform about patient mood. 2) to inform about happenings from the last visit. 3) to inform about dressing instrument required by the patient, nurses who answered negatively think that to inform is a doctors task or that nurses are not competent. conclusion~ intensive care unit teams (nurses, doctors and auxiliar personnel) should get accord on who and how to inform relatives, we consider the nurses' role on information as unquestionable. objective: investigate the respiratory and cardiovascular response after discontinuing oxygen therapy durir~ intr~/]o~pital transport. desiqn: fifty-one patients (29 male and 22 female, aged 69+2,5 and 73,912,4 years respectively, ~+sym) being on 02 therapy were studied prospectively in two consecutive intrahospital transports. oxygen therapy was continued in the first transport while the second one was performed as usually, i,e, without 02. during transport each patient was monitored by pulse oxymeter and holter whereas arterlal blood gases were tested just before a~xl aft~-trar~portation. results: compared to daseline, pa02 and sa02 were signif~canthy decreased in the case of oxygen discontinuation (p<0,00i). paco2 was significantly inur~ds~i only in the subgroup of patients with obstructive lun[ disease (p<0,01) . heart rate increased in all phases of the transport when 02 administratlon was discontinued. blood pressure remained stable in either case. the percentage of supraventricu!ar extrasysto!es, ectopic v~r[hicui~r contractions and st-s6~ment depression was progressively increasing and became very high at the end of transport in the case of 02 therapy discontinuation. other arrhythmias did not change significantly. conclusion: discontinuation of oxygen therapy during intrahospital transport causes severe drop of pao2 and sa02, increases the heart rate and contributes to the appearance of arrhythmias which were not present before. methods:for evaluation of the functional state of brain the complex of methods was used,whieh included electro encephalngraphy ( brain mapping ), rheoencephalography, tetrapolar transtorax rheography. for the estimation of humoral status the level of histamine and serotonine, products of free-radical oxidation,enzimatic markers of ishemic damage of brain and of endogenous intoxication was investigated. results:92 patients with encephalopathies after resuscitation were observed.asystolia was as a result of:shock, trauma, asphyxia,poisonings,appiication of drugs, eclamp sia,injury of the heart,diseases of fhe cardiac vessels. all patients with postasystolic syndrome entranced in comafose condition.in the 1 group (reconvalescents) the depth of coma by glasgo~ pittsburg"s scale was 23,3+-1,78. the duration of coma was from 30 rain. to 48 hour,average 11,9+-4,sh.ln the 2 group (the deads) the depth of come was 13,8+-0,86.the artificial lung ventilation was used in all patients:in the 1 group 2,64+-0,92 days,in the 2~ 6,1 +-1,1 days.apallish syndrome developed in 5 cases,in 5 patients diagnozed <<brain death~. the 4th degree encephalopathy (coma) was found to be associated with disorganized eeg-pattern with predomi nant alpha-( 1 group) and slow (2 group) activity. the relative power of delta-and theta-ranges was about 77-87 per cent.the patients with apallich syndrome demonstrated greatly elevated theta-range power, that of beta -range was essentially decreased. the degree of disturbances of circulation in the both groups was different.cardiac index (ci) and stroke index (si) in the 1 group decreased on 19,5% and 44,3z, in the 2 -on 45,9% and (;3,3%.general peripheral resistance (gpr) increased in the 1 group on 40,5%,in the 2 -on (;9,4%.in the 1 group brain blood flow was increased, in the 2 -decreased on 37,6n.in the 1 group there was the normotonic type of reg-curve,in the 2 -atonic or hypotonic type (without reaction on pharmacologic influ ence).disturbances of permeability of cellular membranes (enhanced free-radical oxidation with an increase in di enic conjugates on 107 and 121n) and vascular ones (an increase of serotonin concentration on 90 and 110%, hi stamine content on 116 and 180%) resulted in hyperfermentia (an increase of concentration of creaune phosphoki nase in the 1 group on 59%,in the 2 -69%.the level of middle molecules (mm) increased in the 1 group on 79,2%, in the 2 -on 120,8%,of polyamines (p) (spermidin,spermin,putrescin).coefficient of correlation between mm and p was 0,891. immediate correction of neurocyte metabolism is impossible due to marked brain oedemaswelling,severe dis ordes of cerebral circulation, disturbances of membrane permeability.thus,the following treatment algorythm might be advisable: 1. protective inhibition of brain and reduction of its energy requirements. 2. recovery of cell and vascular membrane fuoctions.3.recovery of blood circulatiom4.oxygenation of nervous tissue and drainage of brain disintegration products.in 22 patientshbo carried out.5.active methods of detoxication (homo-and enterosorption).6.correction of cerebral metabolism.7. dehydrative therapy must be very cautious. conclusion: the activation of energetic metabolism in neurocytes must be carred out only under neurophysiolngical control and after definite preparation. (67). the clinical estimation of acute cerebral failure carried out by olasgo-pittsburg"s classification of coma. we studied such groups of patients: poisoning co (36), asystolia (8), eclampsia (5), acute renal failure (5), control group (13). methods: electroeneephalngraphy (brain mapping) was fulfilled by means of encephalograph <,medicor ~ in 8 monopolar channels. the estimation of eeg patterns by zhirmunskaya method (1984) was carried out. there were analyzed and changes of eegb by results of rapid transformation furie after rhythmical photostimulation(phts) 2,6,10,20 hz. we studied also the figures of absolute and relative power in such diapason : delta (1-4 hz), theta (5-7 hz), alpha (8-12 hz), betal(13-25hz), beta2(25 hz and more). results: all eeo changes were divided into following types: 1 -organized (3,4 groups); iii -asynehronic (8 group); iv -disorganized with prevalence of alpha-waves (12,13,14 groups); v -disorganized with prevalence of delta-and theta activity (16,17,18;19,20 groups) akkording to our model of hormonal-metabolic disbalance in pregnancy , one of the main reason of destosis is hypoergos , reesults in endotoxemia and neuro-endokrine disregulation. so, stady of central nervous system function give us the information about adaptation processes. the aim of our work is to study the degree of neurologikal deficit in destosis when olifen and lipin were used. there were studied 20 pregnants with different severe forms of gestosis. the degree of neurologikal deficit was valued by datas of neurologikal status . eeg with ,~brain mappinng~, , electrikal resistance of the skin. the komplex inteensive therapy with applikation of olifen and lipin allowed to improve the patient,s condition , decrease the degree of neurolodikal deficit and mortality . kostenko v.phd,kabanko t.,phd,galalu s.md,beliavtseva n. ,shramenko k. phd intensive care department,donetsk medical university, donetsk, ukraine plasmapheresis (pph),as other extracorporal methods of detoxicatin,has a great importance in contemporary medicine. the role of pph is special,because of its simplicity, effectiveness, atraumatic.we incalcated pph in obstetrical clinic. there are rough changes in agregate condition of blood in more of pregnants.these changes lead to disturbances of central,organ and peripheral hemodynamic.the therapeutic effect of pph is due to influence on agregate condition of blood. we considered that application of pph is very effective in pregnants with:bronchial asthma, severe forms of diabet, psorias, gestosis, allergic diseases. we used the membrane pph in pregnants: apparatus-,,hemos-pf>,, plasmofllter pmf-800,with effective area-800 cm,the volume of extracorporal contour-60 ml.such pph has no the ~ agressive effect,,, as in cases of application another extracorporal methods. this method was incalcated in our practice recently, so results will be reported in further publications. (2). post-operative cerebral neoplasm (1), post-operative subdural hematoma (1). icp was monitored via a catheter inserted in the lateral ventricle and values were continuously digitally recorded by means of a bedside computer data acquisition system (maclab). the fiberoptic tracheobroucosenpe, which guided the procedure, was passed between the nasotracheal tube and the trachea in order to avoid hypoventilalion. the patients had stable baseline hemodynaimcs. propofol infusion and fentanyl boli were administered to mantain stable mean arterial pressure values. peak (mean(sd)) icp duping the 30 minutes pre-ciaglia procedure (baseline values) were compared with values during ciaglia procedure, and the 30 minutes p0st-ciaglia procedure. data were compared with repeated measures anova. results: ciaglia procedure duration was (mean(sd)) 30 (14) objectives: transient global amnesia (tga) is a syndrome caracterized by impairment of short-term memory, inability to form new memories, retrograde amnesia and repetitive queries, without other neurological signs and symptoms. the pathophysiology of tga is unknown; thromboembolic, epileptic, migrainous and metabolic mechanisms have been suggested. to address some of these issues, we undertook a study of 25 cases of tga in whom we examined clinical, laboratory data, electroencephalogram, ct of the head, ultrasonography ecodoppler. methods: 25 patients were included in this study: 9 men and 16 women. the mean age was 64 years. all cases underwent a standard clinical examination, electrocardiogram, routinary humoral tests and x-ray, electroencephalogram (eeg), ct scan of the head, ultrasonography ecodoppler. results': the mean duration of amnesia was 5 h. 32 m. +/-7 h. 10 m. hypertension was found in 19 patients (76 %), ischemic heart disease in 4 patients (16 %), hypercholesterolemia in 10 patients (40 %), hypertrigliceridemia in 3 patients (12 %), smoking in 2 patients (8 %), atrial fibrillation in 1 patient (4 %), history of epilepsy in 1 patient (4 %), migraine history was not recorded. ct scans of the head showed multiple small deep infarcts in 4 patients (16 %), a single hypodense lesion in 4 patients (16 %). in 11 patients electroencephalogram was normal (44 %), in 8 patients there were widespread nonspecific electrical changes (32 %), in 6 patients there were focal nonspecific eeg abnormalities (24 %). conclusion: in our study tga was more common in women (64 %). we showed a prevalence of hypertension, hypercholesterolemia and cerebral infarcts compared to normal controls. we have demonstrated a higher incidence of nonspecific electrical changes in tga of lower length, while ischemic lesions in ct of the head were more frequent in tga of greater length. these data seem to be in agreement with the hypothesis that tga is a heterogeneous clinical syndrome, consisting of pure, epileptic, and ischemic types. however we did not find any correlation useful in discriminating pure from associated tga forms. from our study it is tempting to speculate that pure tga is a rare event, underlying still unknown mechanisms wich differ from ischemic, epileptic, migraineous causes. objectives: aneurysmal subarachnoid haemorrhage (sah) is special condition increasing intracranial pressure (icp) in various ways. at the other hand cerebral vasospasm and related delayed ischaemic deficit (did) could answer for the poor outcome. triple h therapy seems today a basic option to prevent did, but it may increase the icp worsening the altered intracranial pressure condition and thereby the cerebral perfusion pressure (cpp). is there any way to individualise the triple h therapy when it is necessary? methods: between sept. 94 march 95 thirty-seven patients with intracranial aneurysms were operated on within 48 hours following sah. five patients were in hunt-hess iv at admission. all patients received triple h therapy in a preventive fashion following surgery and were monitored by daily transcranial doppler ultrasonography (tcd). icp and cpp was measured in twenty-four cases. twenty-two of them received lumbar liquor drainage (lld) and nineteen were administered induced hypertension. the other group was treated by basic triple h therapy. results: in group with monitored icp the outcome was twenty-one excellent, one poor, two died (one of them died from extracranial decease). in the other group four had excellent, six moderate, two poor outcome, and one died. conclusion: according to our recent observation the patients can be divided into two groups of therapy. in group i, the patients with elevated tcd values and either low or high icp reacted to lld. we are concerned that haemodilution and slight hypervolaemia should dominate in the triple h therapy. in group ii patients having high icp with tcd and/or symptomatic vasospasm should be managed by the induced hypertensionhypervolaemia dominated therapy focusing on cpp (icp) and focal neurological signs. air emboli were detected in lo% (n=12) of natients undergoing coronary srtery bypass craftin~ (cabg). central nervous system ~ysfunction occured in 23~$ of the nstients with air embnli and in none of those ~ithhout air embo!i. hvtothermia is the classic form of oro-tect~on used dur~nc ~"~" " ~ ~ ca~.,~modu] :r, on~_,_.7 bj/oass. the surf~eon sho,;,ed thorough!~: evecnnte air from the heart, but the onesthesio!o[[ist can signifieamt!y influence the outcome by emt!oyin2~ methods to detect and treat air emboli. the changes in head rate are primarily due to alterations of autonomic tone. the heart rate variability (hrv), that express the degree of heart rate fluctuation around the mean heart rate, reflects somehow the condition of central nervous system. hrv may be measured by a number of techniques. short-term time-domain variables of hrv are reflect generally the vegal activity. in this study the changes in hrv variables of patients with brain damage, and in addition the changes in hrv measurements in comparison with the clinical evolution were evaluated. eight patient with brain damage and six normal individuals as control group were studied. a elecrocardiographer with availability of computation the sequence of beat-to-beat intervals for one minute was used. the following variables of hrv were measured: 1) standard deviation (sd) of beat to beat r-r interval differences that reflects the respiratory control, 2)the maximum/minimum (max/rain) interval that reflect variability related to baroreflex and thermoregulation and 3) the coel~cient of variation (cv), the results are shown in the in the patients with brain death and in vegetate state there were virtually no hrv. increased hrv pattern was found with clinical improvement, the changes of hrv precede of the changes of gcs, we conclude that time-domain hrv could reflects the degree of brain damage, it is good prognostic index of the brain damage and may change earlier than the gcs. objectives: cerebral co 2 vasoreactivity is an important determinant of cerebral blood flow (cbf) and has been shown to be of prognostic value in head trauma (acta anaesthesiol. scand. 1991; 35:113-122) . we wondered whether co 2 vasoreactivity could be selectively altered in one hemisphere in comatose patients. methods: 6 patients (5m/1f, age 32-65yrs, glasgow 4-8) in coma due an acute brain lesion (trauma, hemorrhage, or infection) were studied. cbf was measured bilaterally using jugular thermodilution at paco 2 25, 30, 35, and 40 mmhg by increasing pico 2 with mechanical ventilation kept constant. normal co 2 vasoreactivity was defined as an increase in cbf of at least i ml/min.100 g per mmhg paco 2. results: 2 patients had normal co 2 vasoreactivity bilaterally, 2 patients had altered co 2 vasoreactivity at both sides, and 2 patients had a normal response at one side (left or right) with an altered response on the other side (dght or left). for the 6 patients left cbf was in mean !7 ml/min.100g lower than right cbf (figure methods: following institutional approval 4 piglets (body weight 25:tl .5) were anaesthetized by 2% fluothane. a catheter was placed in the right femoral artery for blood pressure monitoring and a fiberoptic catheter (oxymetncs-3 abbott) was advanced via the right internal jugular vein to the jugular bulb for sjo 2 determinations. another catheter with a balloon on the tip was advanced in the right atrium via the right femoral vein. a mean arterial pressure (bp) at 25 mmhg was achieved by appropriate balloon inflation for 10 rain and two groups were cleated: i) the hypoxemic group by respirator disconnection (*) and it) the hyperoxemic group by fio2=l on respirator (o). samples were obtained at 0 time (1), 10' min at hypoperfusion (2) arid at reperfijsion at 1' (3), 3' (4) and 10' (5). pao2, pjo 2 and oxidative brain stress evaluation was performed from jugular bulb blood. the latter included: i) no synthase (nos) and xanthine oxidase (xo) activities by a method based on the oxidation of scopoletin detected fluorometrically, it) no levels estimated as onoo-by luminol enhanced chemiluminescence in the presence of 500~tm hydrogen peroxide (h202). resul'~s: the mean pao 2 was 34 mmt-ig for group i and methods: we retrospectively reviewed all 411 upper gi-endoscopies, performed in the period january 1992-july 1994 in 301 patients (199 men and 102 women) admitted at the 4 icu's of our hospital. results: it concerned 129 surgical, 103 medical, 50 eardiological and 19 neurological patients with a mean age of 57.9 yrs (range: 14-91). in 86%, the endoscopy was performed at the icu and in 14% at the endoscopy department. in 56% of the cases, the endoscopy was primarily diagnostic, of which 70% was performed for localization of upper gi blood loss. in 44 % the endoscopy was primarily thempentic, of which 89 % was performed for placement of a duodenal feeding canula. location of the upper gi bleeding was: variees (31%), duodenal ulcer (20%), oesophagitis (13%), gastric ulcer (11%), others (13%) and none (10%). as coincidental findings were noted: cesophagitis (37%), gastritis (16%), gastric deer (14%), duodenal ulcer (9%), duodenitis (8%), oesophageal ulcer (7%) and others (8%). conclusions: there were marked differences in indications and findings of endoscopy at the different icu's. these differences reflect an admission bias and differences in populations and treatment preferences. compared with cardiological and neurological icu's, substantially more endoscopies were performed at surgical and medical icu's. in a considerable number of cases, no source of upper gi blood loss could be found endoscopicaiiy. when upper gi blood loss was the icu admission diagnosis, the main cause was needing varices, which could be controlled endoscopically in the vast majority of cases. when upper gi blood loss was ndt the icu admission diagnosis, peigie ulcer and oesophagifis were the main causes of bleeding. because of the considerable number of coincidental almom~adities found at endoscopy, there is still room for debate whether antacid medication and/or motility stimulating agents should be given prophylactically at icu's. many studies have shown that blood lactate levels in survivors and nonsmvivors of traumatic and septic shock are significantly different. the degree of multiple organ failure is related to the duration of lactic acidosis (1). the aim of this study was to evaluate blood lactate level as a prognostic marker of high risk postoperative patients who may benefit from invasive hemodynamic monitoring and aggressive fluids administration and early inotropic support based on oxygen transport parameters. methods: 32 patients undergoing elective long term vascular and abdominal surgery (asa i-bi) were studied. blood lactate levels were measured after icu admission. in the case of blood lactate level above 2 mmoltl, measurement was repeated every 4 hours for 12 hours or until normaiisation (blood lactate level less than 2 mmol/1). type of surgery, length of surgery, amount of fluids delivered intraoperatively and postoperatively, hemoglobin levels, hemodynamic variables, diuresis, postoperative complications, length of icu stay and clinical outcome were recorded. because no attempts were made to randomisr therapy or change our standard therapy protocol institutional approval was not required. rebuts: the frequency of postoperative complications was 12,5 % and mortafity was 5,5 % in a group of patients with blood lactate level less than 2,5 mmol/l (n = 18). frequency of complications (62,5 %) was significantly increased in a group of patients with blood lactate levels 2,5-4 mmol/l (n = 8), mortality was 12,5 %. mortality (60 %) and frequency of complications (80 %) were significantly increased in a group of patients with blood lactate levels above 4 mmol/l (n = 5). conclusion: blood lactate levels can serve as early marker of high risk postoperalivr patients and may predict increased risk of postoperative complications mad ~e death. objective.~: investigated practicability and clinical value of the routine measurement of hepatic venous oxygen saturation (shvo2) after major liver surgery, as shvo 2 is considered an indirect parameter for splanchthc and hepatic blood flow. methods: 30 consecutive patients were included in this study after liver resections for primary or secondary liver tumors. 5 patients suffered from liver cirrhosis (childs a). immediately after post-operative admission on the icu a pa-catheter ,was inserted under fluoroscopy via the right jugular internal vein into the hepatic vein contralateral to the resection area. hepatic venous and arterial blood samples were drawn every two hours. shvo 2 was correlated to the clinical course, macro hemedynamics, abgs aug other established lab parameters. results: in 26 out of 30 attempts the catheter could be placed correctly. in four cases after right hemihepatectomy the left hepatic vein could not be intubated due to a dorso-lateral tilting of the left liver. this is also reflected in a significantly longer time of fluoroscopy for catheterization of the left hepatic vein (12.9 _+ %5 rain vs. 3.7 + 2.5 rain; p < 0.001). the procedure requires a total of between 45 and 75 minutes. relevant clinical complications were not observed except for short term supraventricular arrhythmias during passage of the catheter through the right atrium. hemodynamics and pulmonary function could be considered normal in all individuals at time of measurement. shvo 2 showed a span from 27.4% to 90.0% with a mean of 67.0% -+ 10.8%. the following statistically significant findings could be obtained: (a) patients with liver cirrhosis showed a significantly lower shvq than patients without (53.4% • 5.3% vs. 68.7% • 10.1%; p < 0.001). (b) a negative correlation between shvo 2 immediately after operation and the duration of intraoperative hepatic vascular occlusion could be observed (r = -0.58; p < 0.05). this correlation could also be seen for the first 12 post-operative hours (r = -0.42; p < 0.01). (c) a negative correlation between shvo2 and the difference between arterial and hepatic venous lactate levels was found (r = -0.39; p < 0.02). conclusions: the routine measurement of shvo 2 appears to be a promising extension of post-operative monitoring after major liver surgery. it is a safe method easily feasible on any major surgical icu though relatively time consuming. a further validation of this method is necessary in larger studies. therapeutic recommendations on the basis of shvo 2 findings cannot be given yet. methods: in 5 cases after major liver resection, in which abnormally low readings of shvo 2 suggested an impaired hepatic blood flow, pgi 2 was applied at a dose rate of 5 ng/kg/min. as shvo 2 can be considered an indirect parameter for hepatic blood flow, the effect of pgi 2 infusion on shvo 2 was measured. moreover, the changes of macro hemodynamics and pulmonary function were monitored. results: before the application of pgi z mean shvo 2 for all 5 patients .was 54.1% (47-9 -47-3). in three cases without major structural alteration of the remaining liver tissue the continuous intravenous administration of pgi 2 lead to a sustained increase of shvo z to an average of 67.1% (65.6 -69,1 ). the postoperative course in these three cases was uneventful. in two cases with compensated liver cirrhosis after hepatitis c no change in shvoz under pgi 2 infusion could be observed. both patients died 32 and 45 days respectively after operation in protracted liver failure. side effects of pgi 2 included a slight decrease of systemic and pulmonary vascular resistances. consequently map decreased by up to 10% as did intrapuimonary right-left shunt increase. in none of the observed patients did these side effects posed a limitation of continuous application of pgi z. conclusions: in patients without structural alteration of the liver the systemic application of prostacyclin at a dose rate of 5 ng/kg/min could significantly increase an abnormally low hepatic venous oxygen saturation after major liver resections, tn two cases of severe liver cirrhosis a similar increase could not be observed. after first clinical investigations and with the results of recent studies in animal further controlled clinical studies of prostacyclin in the postoperative management after liver surgery appear justified. any delay in gastric emptying can promote micro-aspiration and give rise to ventilator associated nosoarnnial pneumonia. h2-receptor antagonists have been suspected of promoting pneumonia by changing the gastric ph. in a few tri',ds on humans ranitidine was noted to delay gastric emptying. the aim of this prospective, randomised, blinded study was to evaluate in a ventilated icu population if there was a difference between cimetidine (c) and ranitidine (r) on the gastric filling index (gfi conclusion: in this population there was no difference in gfi between c and r; however the age and creatinine were significantly different and could have favoured the c group. also the very long t/2 could have hidden smaller differences between c and r as has been described in volunteers. between april 22, 1990 and april 19, 1993 , 102 patients with severe acute pancreatitis were admitted to 16 participating hospitals. patients were entered into the study if severe acute pancreatitis was indicated, on admission, by multiple laboratory criteria (imrie score >_ 3) and/or computed tomography criteria (balthazar grade d or e). patients were randomly assigned to receive standard treatment (control group) or standard treatment plus selective decontamination (norfloxacin, colistin, amphotericin; selective decontamination group). all patients received furl supportive treatment, and surveillance cultures were taken in both groups. results: fifty patients were assigned to the selective decontamination group and 52 were assigned to the control group. there were 18 deaths in the control group (35%), compared with 11 deaths (22%) in the selective decontamination group. (adjusted for imrie score and balthazar grade: p = 0.048). this difference was mainly caused by a reduction of late mortality (> 2 weeks) due to significant reduction of gram-negative panreatic infection (p = 0.003). the average number of laparotomies per patient was reduced in patients treated with selective decontamination (p < 0.05). failure of selective decontamination to prevent secondary gram-negative pancreatic infection with subsequent death was seen in only three patients (6%) and transient gramnegative pancreatic infection was seen in one (2%). in both groups of patients, all gram-negative aerobic pancreatic infection was preceded by colonization of the digestive tract by the same bacteria. reduction of gram-negative colonization of the digestive tract, preventing subsequent pancreatic infection by means of selective decontamination, significantly reduces morbidity and mortality in patients with severe acute necrotizing pancreatitis. ieco by sodium hypochlorite (nacio) infusion is considered to be a model of microsomal oxidation in liver on cytochrome p-450. active c10 provides oxidation of toxic metabolic products in the blood and exfused during plasmapheresis plasma, and also hydrophobic to hydrofilic transformation of substanses. sterile nacio in necessery concentrations was obtained by electrolysis of saline (0,85-0,9% naci solution) in electrochemical set e~io-4 (russin,moscow). methods: 1. the nacio in concentration 600 ragfl (400-800 ml/24h ) was administred into central veins in patients with extensive peritonitis and endotoxicosis 2-3/t. erytrocytes resistance to nacio, circulating blood volume glycemia and hemostasis were initially estimated. 2. after plasmapheresis exfused toxic plasma was mixed with nacio conccantration of i000 mg/t in 10:1 ratio in sterile "hemacons".the effectiveness of plasma detoxication and possibility of its reinfusion were evaluated by determination of albumin effective concentration (eca 35 g/l), the concanlration of medium molecular oligopeptides (mm 0,2) and other biochemical tests (bilimbin, creatinine, carbomide and so on). results: 1. the intravenous administration of nac10 excels detoxicative effect of hemosortion by 17-20% provides effictive presentation of protein components and blood cells and improves the transport function of albumin by 37%. 2. the return of exfused plasma after its purification ieco was 70-80%. only the remaning 20-30% of deficient plasma were compensated by fresh cryoplasma and albumin solutions. ischemic hepatitis (ih) is a severe complication in critically ill patients. acute circulatory failure of multiple etiology can lead to splachnic hypoperfusion and cause acute and reversible anoxic damage. over a period of 26 mos 12 pts, 8 m and 4 f, mean age 64+6.6 yrs developed liver disease compatible with ih. eight pts had a documented hypotensive episode (six pts with septic shock and two hypovolemic shock), while cardiogenic pulmonary edema in the absence of hypotension was responsible for ih in the remaining four pts. all the pts had a rapid striking elevation of ast, &lt and ldh with equally rapid resolution of these parameters to near normal wimin 9 days (mean 6.25). the mean peak level of ast, alt and ldh was 4340 iu/l (range 2105 to 7500), 3453 iu/l (range 1685 to 5150) and 2868 iu/l (range 1440 to 6960) respectively. serum total bilirubin levels rose transiently with a moan t:eak level of 1.95 mg/dl (range 1.1 to 2.7), while altered coagulation paran-,ete's (pt> 1.5 times normal) was observed in four pts and clinically significant coagulopathy with fibrin degradation products occurred in one pt (8.3%). renal impairment (cr>2.0 mg/dl) was manifest in all pts; six pts developed non-oliguric renal failure (50%) while two pts required hemodialysis. ten lots required vasoconstrictor inotropes [dobutamine (range 3-10pg/kg/min) and dopamine (range 7-25 pg/kg/min), while replacement of circulatory blood volume was performed in two pts with hypovolemic shock. eight lots expired (66.6%), but none died as a direct result of hepatic damage. the mortality rate was higher among pts with concurrent renal failure (75%). it is concluded that: 1) ih is not uncommon complication in the icu with the prognosis depending on the underlying disease. 2) clinically significant coagulopathy is uncommon complication of ih. 3) titration of inotropes is required to obtain optimal cardiac output support and subsequently liver blood flow. it is difficult to ascertain the perfusion of free flaps such as jejunal loops after surgery. objectives: to assess ischaemia as evidenced by intramural ph of jejunal free flaps used for reconstructive surgery following total pharyngolaryngectomy. methods: the sigmoid ph tonometer ( tonometrics inc.,usa ) was used to monitor intramural ph of the jejunal free microvascular flaps ( phig ) in 15 patients who underwent total pharyngolaryngectomy. a standard general anaesthetic was given and all patients were admitted to the icu for controlled ventilation and monitoring. all had similar postoperative care. phig was measured pre, post-revascularization of the flap and on icu admission, 4, 12 and 24 hours postrevascularization. objectives: to classificate the wide spectrum of itc of anp into distinct pathophysiological patterns according to presentation and course. patients (pts) and methods: 52 pts, 34 ~(65,4%), 18 (34,6%) were admitted in the icu because of anp and acute respiratory failure(arf), ilean age:54,3• years. hean stay in icu:29,2• days. 38 pts were operated, 15 of them twice. hean value of ranson's scale:4,4• (2-7). we analyzed hemodynamic measurements,arterial blood gases(abg), x-ray findings(xrf), ct-scans and operative records. results: 5 patterns of pleuropulmonary complications were identified: a)early hypoxia without xrf -33 pts. b)early ards with typical xrf -5 pts(1 died), c)early arf with xrf(atelectasis,infiltrates)-15 pts(9 died). d)late ards with typical xrf-32 pts(31 died), e)pleural effusions in various combinations with the above patterns -38 pts. overall mortality rate: 41/52 = 78,8%. conclusions: l)frequent x-rays and abg are important for the classification of itc of anp. 2)even though patterns of classification in anp are not clearly distinguishable,they facilitate an anticipatory management. 3)deterioration of abg and xrf indicates that preventive measures for arf must be intensified and agressive surgical therapy is required. 4)delay of surgical therapy is related to worse prognosis(p<o,05) and prolonged hospitalisation time(p<o,ol). in the contrary, the early timing of the operation before infection and extrapancreatic necrosis is essential for a better prognosis (p<o,05). objectives: the development of cholestasis during intensive care treatment is allied with an inferior prognosis. this study investigates the sensivity, specification and also the positive and negative forecast of patients survival -exemplary for bilirubine. methods: during a period of 12 months all surgical patients on icu were registrated prospectively (n = 1572). for documentation of disease development a standard foldcrform based on a computer-data-system was used. the results of this kind of documentation showed aapprax. 5,2 m!ll. of single patient informatioas. results: 1300 of 1572 patients didn't suffer from liver disease or have any operation on liver or bile tract. 1201 of them have had a normal scale of bilimbine in the postoperative period, a higher scale of bilirabine was regisu'atcd by 99 patients. at a ,,cut-off" of 4rag% (total-bilirubine) the sensitivity was 0,7, specivity 0,6, positive predicive level 0,8, negative predictive level 0,5 for survival criteria of a patient. the max. reached level of bilirubine, also the daily increase turned out almost the same results as they were found for other parameters nf cholcstasis like ap or gamma-gt. objective: the aim of this experimental protocol was to evaluate the morphological and functional parameters of isolated pig liver grafts, perfused in an extracorporeal liver support system. methods: the system consists of the graft liver, a membrane oxygenator (oxygenation surface 1.2 cm z, 02 flow=3 itlmin), a heater, a centrifuge pump and a fluid reservoir. twenty pig livers, mean weight 790 gr (min 610, max 870 gr) were obtained and after a mean cold ischemia time of 35 min were perfused fo} a mean of 7.5 h (min 4.5, max 9 h). perfusion solution consisted of r/l and hemacell. inflow to the graft liver was performed through the portal vein at a mean pressure of 16 (12-25) mmhg at 38~ while outflow was secured through the suprahepatic inferior vena cava. during perfusion the following parameters were evaluated through pre-and posthepatic hourly (to-t8) sampling: po2, 4" + +4" pco2, ph, hco3-, na , k , ca , osmolality, glucose, lactate, ast, alt, alp, u bilirubin and coagulation factors i, v and viii. biopsies were obtained periodicallty. b_p_~: results were as follows: mean ph fell from 7.45 at t o to 7.168 at t 8 while mean output po 2 fell from 527 at t o to 10 at t 8. mean output ast values increased from 361 at t o to >2500 at t 8 while mean output alp values increased from 3.66 at t o to 197 at t 8. mean output k + values increased from 3.93 at t o to >8 at t 8. histology revealed lesions of ischemic necrosis, more prominent after t 6. conclusion: results show that the isolated liver graft presents satisfactory function and morphology at least for a five hour perfusion period in the described extracorporeal circuit. correction of ph contributed to an increase in bile flow. between 1982 and 1993 the practice of transplantation has changed drasticaily in switzerland -besides kidneys also hearts, heart and lung, lung, iiver and pancreas transplantation has started in several centers. major information efforts have been made, organ exchange rules were set up and a national coordination center was initiated. the aim of this retrospective single center study was to assess the influence of transplantation on organ donation. in the past eleven years 205 organs were donated from 458 potential donors i139 single, 66 multi organ donations) analysis of refusal was evaluated categorized into medical and/or familiar reasons. the number of potential donors increased from 28 (1982) ,to 61 (1992) with a concomitant drastic reduction of donations from 64% in 1982 to 26% in 1992; amounting to a net unchanged number of donations over the last 10 years (1982 = 18; 1992 = 17) . the import and export of donor organs was balanced since the introduction of the national coordination center. in contrast multi organ donation increased from 0% in 1986 to 90% in 1993 despite of the more stringeant selection criteria, in conc]usion the introduction of a full range of transplantation procedures at several new university programs and the increase of multi organ donation has not had the forecasted impact on organ donation despite a sustained informative and promotional campaign, objective: monitoring hepatic venous oxygen saturation (svho2) provides online information about hepatic-splanchnic oxygen supply-demand ratio [1]. previously, x~ reported hepatic venous catheterization in patients undergoing orthotopic liver traru~lantation (olt) [2] . in the present study, we assessed the effects of nitroglycerin (ng), a vasudilator that affects the venous capacitance vessels more than arterial vessels and prostaeyclin (pgi2, flolan r~, wellcome, uk), an arterial and splanchnic vasodilator on hemodynamies and hepatic venous oxygen saturation (svho2) in human liver transplantation. methods: with institutional approval and informed consent, 14 consecutive patients, mean age 50-2-_10 years, were studied following olt. postoperatively, fiberoptic pulmonary artery catheter was inserted into the right hepatic vein. timed infusions of ng at a rate of 0.1 gg/kg/min and pgi2 at 5 ng/kg/min were initiated for a 45 rain period. each sequence was followed by baseline therapy for 45 rain. results are expressed as mean=tsd. statistical analysis was performed using friedman's-two-way-anova-test, significance was accepted at p<0,05. results: ng at 0.1 gg/kg/min induced a decrease of mean arterial pressure (map) (84_49 [baseline] vs. 75+9 mmhg) and pulmonary artery wedge pressure (pcwp) (8j:2 [baseline] vs. 65:1 mmhg). cardiac index (ci) (5-41 vs. 4+1 l/rain/m2), oxygen delivery index (do2i) (655-+108 vs. 618+123 mgnfin) and svho2 (74_~12 vs. 69-l-_19%) were decreased (p<0.05). pgi2 at 5 ng/kg/min induced a reduction in map (73• nm~. _g) and pcwp (6+1 mmhg). ci (6_+1 l/rain/m2), do2i (7555:135 ml/min) and svhoz (81+6%) were increased (!o<0.05). 9 vasedilatation induced by ng decreased systemic oxygen supply and impaired splanclmie oxygenation. 9 pgi2 increased systemic oxygen delivery in parallel with svho2, suggesting a corresponding improvement of hepatic-splanchnic okygenation. 9 thus, if vasedilator therapy is indicated in th6 15orient receiving liver grafting, pgi2 appears to be advantageous. however, due to its platelct aggregation inhibiting properties, the usefulness and safety of pgi2 in olt patients has still to be determined. objectives: to analyze the effect of steroid treatment given to donor on the early function of transplanted kidney. methods: from january, 1994 until now 56 donors were involved into this prospective study. every other donor was treated with 30 mg/kg solu-medrol one hour before organ retrieval. according to the steroid treatment of the donor the recipients were divided into two groups: group 1 -steroid pretreatment goup (y~=35), and group 2 -control group (n=37). the donors and the recipients were treated using the same kidney transplantation protocol onl~r the adults, and the first cadaver kidney transplanted patients were involved into the study. the daily routine parameters were analyzed pre-and intraoperafive, and on the 0-5th, 14th and 30th postoperative days. results: we could not show any clinically important differences between the two groups in respect of donor parameters. preoperative, the patients in group 2 had slightly lower ereatinin level (819 -+ 244 g.,non vs.923 -+ 254gmol/1) which persisted into the early postoperative phase. the values of the other examined pre-and intmoperativc parameters were almost the same. during the first 5 postoperative days the patients in group i needed less diuretics (furosemide and renal dose of dopamine) and their sodium excretion was closer to the physiological range than in group 2. the other parameters did not differ significantly. the less furosemide need in group ! pe~isted to the end of the first month. conclusions: according to our data the steroid treatment of the donors improves the early function of the transplanted kidney in some respects. to prove the real benefit of the donor steroid treatment needs more data and further analysis. objectives: severe infections may compromize the outcome of liver transplantation..determination of new parameters may increase the knowledge of pathophysiologic mechanisms and may lead to changes in postoperative therapeutic management of patients at risk. methods: between august 1993 and september 1994, 81 patients with 85 transplants were monitored for cytokines and extracellular matrix pammeters on a daily basis. serious infections (n=10) included microbiologic evidence and more than 2 secondary organ failures. patients with cholangitis (n=ll) or uneventful postoperative course (n=37) referred as control groups. results: 1-year patient survival was 88.9% (72/81): 5 patients died due to serious infections, while 4 died for other reasons. mean bilimbin, stnf-rii-, ifn-7-, il-4-, il-8-, il-10-, laminin-and neopterin levels were significantly elevated in patients with serious infections compared with patients experiencing mild cholangitis or with an uneventful postoperative course. a further increase of all parameters was observed in patients who subsequently died; tnf-ri/: 28310_+788 pg/ml vs 20452• pg/ml; ifn-7:466_+57 pg/ml vs 4.4-+1.8 pg/ml; il-4:214-+35 pg/ml vs 148-+29 pg/ml; il-8:667-+48 pg/ml vs 251_+26 pg/ml; il-10:149_+52 pg/ml vs 52• pg/ml; laminin: 3010-+312 ng/ml vs 1263-+ 117 ng/ml; neopterin: 247_+37 nmol/1 vs 96_+19 nmolb for non surviving vs-surviving patients. a significant decrease of sialic acid yeas observed in patients with serious infections; and a further decrease occurred in patients who subsequently died: 455-+31 mg/l vs 685• mg/1. conclusions: the increase or decrease of various cytokines and extracellular matrix parameters may be indicative for severity of infectiolx routine monitoring of these parameters may improve current diagnostic tools and poss~ly lead to changes in therapeutic management of patients at ~k. objectives: evaluation of the cytokine network after liver transplantation may give some insight in pathophysiologic mechanisms of rejection and may lead to detection of patients at high risk. methods: 81 patients with 85 transplants were monitored for various cytokines on a daily basis between august 1993 and september 1994. rejection was assessed by histology in combination with clinical signs of rejection and laboratory investigations. results: during the first postoperative month, 28 patients (34.6%) developed rejection; 14 patients were successfully treated with methylprednisolone (steroid-sensible rejection), while further 14 patients required additional treatment with fk506 or okt3 (steroid-resistant rejection). 4 patients subsequently developed chronic rejection. mean levels of various cytokines and extracellular matrix parameters including tnf-rii, ifn-7, il-ib, il-2r, il-4, il-6, il-8, hyaluronic acid and neopterin were significantly higher in patients with steroid-resistant than in patients with steroid-sensible rejection. a further increase of some parameters was observed in patients who subsequently developed chronic rejection; bilirubin: 18.2-+4.1 mg/dl vs 11.2-+1.7 rag/all; tnf-rii: 23374-+798 pg/ml vs 18246_+679 pg/ml; il-8:1024+-192 pg/ml vs 275-+67 pg/ml; neopterin 148_+37 nmol/1 vs 49-+21 nmol/1; hyaluronic acid: 290_+63 ~tg/l vs 223_+28 ~tg/l for patients with chronic versus patients with acute steroid-resistant ~ejection. sialic acid levels decreased in patients with acute steroidresistant rejection; and a further decrease was observed in patients who tieveloped chronic rejection: 437_+34 mg/l vs 671_+55 mg/1. ~onclusions: various cytokines and extraeeuular matrix parameters were indicative of severity of rejction. the extensive increase of bilirubin, tnf-ii, il-8, hyaluronic acid and neopterin may indicate subsequent chronic ection. monitoring of these parameters may, therefore, lead to changes in immunologic management after liver transplantation. background : combined kidney and pancreatic transplantation is being performed with increasing frequency in patients with diabetes mellitus and renal failure, as it offers more chances of success and better results than kidney transplantation alone. mycotic arterial aneurysm constitutes a devastating complication following pancreatic transplantation. all cases of mycotic arterial aneurysms have been however reported with exocrine pancreatic drainage into the gastrointestinal tract. intervention : we describe a series of 8 consecutive whole kidney-pancreas transplantation performed at the university of geneva hospitals (1500 beds) between december 1992 and may 1994. exocrine pancreatic drainage into the bladder (epdb) was performed to improve early detection of rejection episodes. epdb was hypothesized to reduce the risk of contamination from the gastrointestinal tract and the subsequent possible occurrence of potentially fatal infectious complication. in all patients the dual transplantation was performed through a median incision according to the procedure described by nghiem. results : two out of the 8 patients who received kidney-pancreatic transplant developed arterial mycotic aneurysms 15 and 35 days following surgery. aneurysms developed at the site of the arterial anastomosis used to rearterialize the homograft. both patients had peritonitis caused by candida albicans requiring surgical drainage and intravenous antifungal therapy. rupture with hemorragic shock occured in both patients leading to graft removal in one patient, and three episodes of lffetreateniug hemorragic shock followed by graft failure and removal 32 days after transplantation in the other. conclusion : arterial mycotic aneurysm constitutes an early, lifetreatening complication of kidney-pancreatic transplantation; it mandates graft removal. although exocrine pancreatic drainage into the bladder consitutes a definitive advantage for caller diagnosis of graft rejection, it does not eliminate the risk for retrograde colonization and subsequent severe infection in our experience. s. bocharov, i. teterina, regional clinical hospital, irkutsk, russia acute profound loss of blood can result from the very different injuries and hepato-pancreato-duodenai operations enter such a rank. ill-timed and inadeguate correction of operation hemorrage is one of the reasons for postoperation complications, including polyorganic insufficiency. the pathogenesis seems to be very complex. in early stages of bleeding the liquid enters the vessel bed, followed by hypoproteinosis and hematocrit fall. however, as decompensation develops, the fluid leaves the vessel system in the result of increasing postcapillary resistance and lowering col-ioidnooncotic blood pressure (cop). the resulting hypovolemia causes primarily acute disturbance of central hemodynamics and then of microcirculations and transcapillary exchange. central hemodynamic failure after acute loss of blood manifests itself through cardiac output lowering and capillary blood flow deceleration. taking into consideration, that 35 % is critical value for cpv loss and for cev it is 65 %, we consider arising the level of cop to the immediate task. cop raising allows to normalize transcapillary exchange, which we assess through cop and mcp (mean capilary pressure) gradient. the next task is to make up for globular volume till homeostasis providing level. considerable attention is given to catabolism inhibition and maximum possible enegry provision. control over high proteolitic activity of blood and callicreinkinin system activity implies direct proteases inhibitors. reologic, membrane stabilizing, antihypoxanthine and anticoagulant therapies are obligatory. virehow clinic, dept. of surgery, humboldt university berlin, germany regarding a high mortality up to 85 % of fulminant hepatic failure orthotopic liver transplantation seems to be the only promising therapeutic approach in many cases. this study shows experiences from a transplantation center. between june 1991 and april 1995 39 patients suffering fulminant hepatic failure were admitted to our surgical intensive care unit all patients showed severe liver dysfunction with grade ii to iv encephalopathy. after a period of diagnostics and conservative treatment ranging from few hours to 10 days (mean 2.4 days) we reported 22 of these patients as possible organ recipients to eurotransplant. all of these 22 patients were transplanted within 48 hours, 16 (73 %) of them even within 24 hours. the principal aetiologies were hepatitis b (7), hepatitis c (1), nanb hepatitis (5), mushroom poisoning (amanita phalloides 1). after transplantation 2 patients suffered from initial-non-function and underwent re-transplantation. the one-year-survival rate was 82 %, 5 patients died within 3 months after transplantation due to various reasons. 17 patients were not referred for liver transplantation. 10 of them never met transplantation criteria, improved by conventional therapy and could finally be discharged from hospital. the known reasons for liver failure in this group were mushroom poisoning (4), paracetamol intoxication (4) and fulminant hepatitis a (1). 7 patients suffering from fulminant hepatitis (6) or intoxication (1) were excluded from emergency liver transplantation for various contraindications. 6 of these 7 patients (86 %) died despite conventional intensive care. we don't know if some of the patients in the transplantation group would have survived without transplantation, because whenever we decided on transplantation we could perform the operation within 48 hours. but 9 the good survival rate in the transplantation group (82 %) 9 the 100 % recovery rate in the group, where there was no transplant-indication in our opinion 9 and the fatal outcome (86 % mortality) in patients with contraindications are an encouraging proof of a successful therapeutic strategy in acute liver failure. these results are based on a close cooperation between experienced transplant surgeons, hepatologists and intensive care doctors, using sophisticated laboratory and imaging techniques in a specialized center. introduction: during brain death patients suffer from multiple endocrinologic disturbances. one of the most important are those related with thyroidal axis. it is well described the euthyroid sick syndrome whose more frequent pattern consist of decreased triiodothyronine (t3), increased reverse t3 (rt3) with normal levels of tetraiodothyronine (2"4) and tsh, this lacking in "1"3 levels lead to a change from aerobic to anaerobic metabolism which results in tissular damage. objective: 1.to study thyroidal pattern in brain death patients potential organ donors. 2.to avoid organ impairment by administration of t3. 3.to study the hemodynamic and hormonal changes after the administration of t3 in these patients. material and methods:population: 22 brain death patients of any etiology potential organ donors admitted to the intensive care unit. patients were classified in hemodynamically stable (group 1) and unstable (group 2). group 2 received a bolus of 0.25p.gr/kg. and a perfusion at a dose of 2-3.5 p.gr]h of t3. hormonal assays: total t3 (tt3), total 2"4 (tt4), tsh. fxee t3 (ft3), free 1"4 (ft4) and rt3 were determine at the moment of clinical brain death (0 hrs) and in group two these assays were repeted at hours 4, 8 and 12. results: 22 patients (17 male) with a mean age of 33 years (range 17 to 71yrs.) were studied. the clinical brain death was confirm later with other explorations (eeg, doppler). there were 15 patients in group 1 (68,1%) and 7 patients in group 2 (31,8%). hormonal pattern: at the moment of brain death tt3 was normal in 21 cases (95,4%) and decreased in i (4,6%); tt4 was normal in 9 patients (40,9%) and decreased in 13 (59,1%); ft3 was normal in 3 cases (i3,6%), decreased in 19 (86,4%); fl'4 was normal in 19 patients (86,4%) , decreased in 3 (13,6%) .rt3 was normal in 17 cases (77,2%) and increased in 5 cases (22,8%). there were no statistically significant differences in hormonal pattern between the two groups. only t3 levels at hours 0, 4 and 8 were significant in group 2. in the 19 cases with ft3 decreased, the tt3 was normal in 18 (84%) and decreased in 1 (16%), tt4 was decreased in 11 (57,8%) and normal in 8 (42,1%), tsh was decreased in 1i (57,8%), normal in 7 (36,8%) and increased in i(5,2%) and ft4 decreased in 3 (15,7%) and normal in 16 (84,2%) and rt3 was normal in 14 (73,68%) and increased in 5 (26,3%). there were no statistically significant differences in cardiac index, vascular resistances and pulmonary shunt before and after the administration ef t3. conclusions: 1. the hormonal pattern most often find in brain death patients was: normal tt3, decreased tt4, normal tsh, decreased ft3, normal fr4 and normal rt3. 2 . there were discrepancies in the values of ft3 and tt3 3. there were no statistically significant differences in hemodynamic and pulmonary parameters. objectives: magnetic resonance angiographie (mra), a non-invasive procedure, provides flow-related information additionly to the anatomy of the vascular system. measurement of signal intensity and edge detection of vessel structures permits to calculate blood flow velocity and vascular diameters. we examined whether cerebral hemodynamic changes by altering the arterial pressure of carbon dioxid (pace2) could be detected by mra. methods: following institutional approval and informed consent, 10 mechanically ventilated patients without elevated intracraltial pressure underwent mra with defined periods of hyper-, hypo-and normoventilation (pace2: 30, 50, 40 mmhg; arterial blood gas probes; avl). mra was performed with a 1.5 tesla magnetom (vision, siemens). two different mra techniques were used: a conventional time-of-flight-3d-angiography (tr: 39 ms; te: 7 ms; fl: 20 deg; slab: 56 mm) for vessel diameter detection and a flash-2d-gradient-echo-sequence (tr: 28 ms; te: 5 ms; fl: 30 dog) for measurements of blood flow velocity. an axial view parallel to the ac-pc-iine (anteriorposterior-commissur-line) was used for repeated imaging of identical regions of interest 0toi) of the proximal part of the internal carotid (ica) and middle cerebral artery (mca) as well as of peripheral branches of the mca and the posterior cerebral artery (pca). results: changes of pace2 correlated with changing signal intensities, whereby under hyperventilation a decrease of 23,7% (p 0.01) and under hypoventilation an increase of 28.4% (p 0.01) was observed compared with normoventilation. blood pressures were stable throughout the whole study period, pace2 dependent changes in vessel diameters were more pronounced in peripheral branches of mca and pca. a change from normo-to hyperventilation produced a decrease in proximal vessel diameter of -3.5% (p _< 0.01) and in peripheral diameter of -22.2% (p _< 0,001). a change from normo-to hypoventilation produced an increase in proximal diameter of +6.1% (p < 0.05) and of +21.3% (p -< 0.001) in peripheral diameter. conclusions: pace2 related changes of cerebral vessel diameter can be easily detected by mra without injecting a contrast agent. the results confirm that co2-reactivity is more pronounced in peripheral cerebral vessels, which are subjected to greater changes in diameter than major basal arteries. hyperventilation leads to a decrease and hypoventilation to an increase in signal intensity thus reflecting the corresponding changes in blood flow velocity, intensive care unit (icu) of "kat" hospital, athens, greece, ob!ective$; the value of bronchoscopy in pulmonary atelectasis of icu patients is under question the presence of an air bronchogram sign in xrays, which is considered as evidence of central bronchus patency, is referred in several studies as a negative criterion for bronchoscopy, whereas its absence as a positive one. it is also referred that air bronchogram sign correlates with delayed resolution of atelectasis, probably because of obstruction of many periferal airways (not central). the purpose of this prospective study was the evaluation of the air bronchogram sign on frontal chest film as a negative criterion for bronchoscopy and as criterion of delayed resolution of atetectasis, methods: icu patients with atelectasis were studied prospectively. they underwent bronchoscopy, bronchoscopic findings, presense of air bronchogram sign, and outcome of atelectasis were recorded, correlations were made, between: 1) bronchoscopic potency of airways and air bronchogram sign 2} resolution time of atelectasis and broncoscopic potency of airways. 3) resolution time'of atelectasis and air bronchogram sign, methods of statistical analysis were the t-student test and the chi square test, results:the patients were 23, men 19 women 4, seventeen patients had atelectasis of whole lung, 3 of upper lobe, and 3 of lower lobe. ten patients had atelectasis in right and 13 in left lung. eight from 28 patients had air bronchogram sign in x-ray, there was no statistical correlation between air bronchogram sign and bronchoscopic potency of airways [6 from 8 patients with air bronchogram sign (75%) and 11 from 15 without air bronchogram sign (73%), had bronchoscopic potency of airways, p>0.1], resolution time of atelectasis didn't correlate statistically with bronchoscopic potency of airways (mean resolution time in patients with bronchoscopic potency 2,29 days and in bronchoscopically closed bronchi 2,33 days, p>0,1). there was also not a statistical correlation between resolution time of atelectasis and air bronchogram sign (mean resolution time in patients with air bronchogram sign 2,25 days, and without air bronchogram sign 2,33 days. p>0). conclusion~i; the presense of an air bronchogram sign in x-ray of icu patients with atelectasis, does not coexist obligatorily with bronchoscopic patency of airways and cannot be used as a negative criterion for bronchoscopy, neither as a criterion of delayed resolution of atelectasis. th. wertgen chest sonography (cs) is routinely used in our department to examine icu patients with clinical symptoms of pulmonary embolism, pneumonia, pleural effusion or unclear chest pain. we perform cs with a sector transducer (4.0 mhz) and a linear transducer (7.0 mhz) using acuson 128xp/10 c. the sonographic signs of pulmonary embolism and infarction are most well demarcated, mainly wedge shaped and triangular pleural based lesions, more roughly structured, observed with a hyperechoic reflex in the center corresponding to the bronchitic (fig. 1) . pneumonia is characterized by homogenously hypoechoic, wedge shaped parenchymal lesions, containing air or fluid bronchograms; they move with respiration (fig. 2) . pleural effusions are spaces of various echogenicities, from anechoic to homogeneously echogenic, which may contain floating strands or complex septa, located between visceral and parietal pleuras (fig. 3) . from march 1994 to april 1995 we did 55 examinations by cs in 34 icu patients (20 male, 14 female; age from 29-87). patients examinations pulmonary embolism 10 16 pneumonia 7 16 pleural effusion 13 19 us-guided thoracic punctions were performed in 7 patients. in two patients we found pneumonia or pleural effusion caused by a lung carcinoma. another two patients showed a normal cs (diagnosis: inflammation of the gall bladder, inflammation of the myocardium). conclusion: cs is a very useful method for icu patients with chest diseases. it takes less time and is less expensive than ctand sometimes of a higher diagnostic value than x-ray. last but not least cs is invaluable for the icu patient, because the examination is done save and quickly at bed side and the results of cs are very helpful in diagnoses and treatment. results : inter-observer reliability was evaluated as an 83 % concordance. results of the tee classification were : class 0 : n = 21 (34 %) ; class 00 : n = 13 (21%) ; class 1: n = 7 (12 %) ; class 2 : n = 8 (13 %) class 3 : n = 12 (20 %). therapeutic implications of tee in class 3 patients were : cardiac surgery in 5 patients (two cases of acute mitral regurgitation, two valvular abscesses and one hematoma compressing the left atrium), discontinuation of peep in one ventilated patient with an atrial septal defect, weaning of mechanical ventilation in one patient with an atrial septal defect, prescription of antimicrobial therapy in 8 patients with endocarditis and prescription of anticoagulant therapy in 2 patients with left atrial thrombus. the only noteworthy complication was a case of spontaneously resolving supraventrieular tachycardia. conclusion : tee is safe and well tolerated, and is useful in the management of icu patients with shock, unexplained and severe hypoxemia or suspected endecarditis. the aim of this study was to determine whether ultrasound guidance can help interns to improve the results of jugular vein access in icu. methods : in a prospective and randomized study, we compared, in 79 patients admitted to the icu, an ultrasound-guided method (ultrasound group : 37 patients) with an external landmark guided technique (control group : 42 patients). all jugular vein accesses were performed by young interns with an experience of < 5 procedures. results : internal jugular cannulatian vein was aci~ieved in all patients in the ultrasound group and in 10 patients (24 p.cent) in the control group (p < 0.01). average access time was longer in the control group (235 • 408 sec. vs 95 • 174 see. ; p = 0.06) and puncture of the carotid artery occurred in 5 patients in each group (p = 0.83). 32 patients (86 p.cent) in the ultrasound group and 23 patients (55 p.cent) ia the control group (p < 0.05) were cannulated in 3 rain. or less. the cannula was therefore unabie to be inserted within 3 minutes in 19 patients in the control group, with failure of eannulation in 10 of these patients (53 p.cent). failure was due to thrombosis (n = 1), small calibre of the internal jugular vein (< 4 ram) (n = 5), abnormal vascular relations (n = 3) or cervical irridation (n = 1). among the 10 primary failures of cannulation, an internal jugular vein catheter was able to be inserted in 4 cases by an experienced physician on the side initially selected and with ultrasound guidance in 2 cases. the catheter was inserted into the contralateral internal jugular vein under ultrasound guidance in the remaining 4 cases. jugular cannulation was obtained at the first attempt in 26 p.cent in the control group and 43 p.cent in the ultrasound group. conclusion : ultrasound guidance improved the success rate of jugular vein cannulation by inexperienced operators in icu patients. when the internal jugular vein has not been successfully eannulated within 3 minutes by the external landmark guided technique, the authors recommend the use of the ultrasound guidance. in the majority of cases right atrial or ventricular thrombi represent pulmonary emboli in transit. these may be fatal in patients (pts) treated conservatively with anticoagulation only. in literature the incidence of right heart thrombi in pts with proven pulmonary embolism (pe) is said to be in the range of 3-4%. extremely mobile, long, worm-shaped masses in the right heart cavities carry an especially high early thrombus-related mortality rate which ranges from 40-50%. current therapeutic strategies favour fibrinolytic therapy with consecutive anticoagulation. we report five cases (4 male, i female, 55-74 years) of right heart and pulmonary thromboembolism. in these pts diagnosis and regression of thromboemboli following systemic intravenous lysis therapy with recombinant tissue-type plasminogen activator (rt-pa) was documented by transesophageal echocardiography (tee). a submassive pe occured in 3 pts, a massive pe in 2 pts. one patient (pt) had a cardiac arrest. in all 5 cases tee clearly identified the extensive thrombns formation in the right-sided cavities of the heart and in the central pulmonary artery in 2 cases. all pts were treated with 100 mg rt-pa, 3 pts in a front-loaded regimen over 90 minutes, 1 pt over 120 minutes, and, due to the life threatening situation, in one case a bolus injection as ultima ratio was performed with no intracerebral bleeding complication. regression of thromboembolic masses after fibrinolytic therapy was demonstrated by transthoracic and transesophageal echocardingraphy after 1 to 15 hours. all pts survived and were put on coumadine, 1 pt developed an intracerebral bleeding with persistent hemiplegia. conclusions: the use of thrombolytic therapy is highly efficacious for the therapy of pts with pe and concomitant right or ventricular thrombus formation. transthoracic and especially transesophageal echocardiography are powerful bed-side diagnostic tools for the immediate diagnosis and follow-up of successful treatment in this life-threatening condition. although widely used, catheterisation of the femoral vein in the groin using "landmark" technique is frequently complicated by accidental arterial puncture. suboptimal hygiene and patient discomfort are also associated with this technique. with regard to these last two factors cannulation of the femoral vein 10-20 cm below the inguinal ligament would seem an attractive alternative. as "landmark" technique is not possible for the cannulation of the femoral vein in this part of the thigh, ultrasound was used to locate the vessel and the results of this technique were evaluated. methods: a portable compact ultrasound device (site rite,dymax corp.) featuring a 7.5 mhz transducer (ultrasound depth 4-5 cm) fitted with a needle guide and a 6 cm screen was used by residents with no previous experience in ultrasound guided cannulation. patients consisted of a surgical icu population. results: in 46 patients 55 catheters were introduced.in 6 cases more than one (2-4) attempt was made and in 3 patients the procedure was unsuccesfull due to the fact that the vessel was situated out of reach of the ultrasound (vessel depth > 4-5 cm), during the 55 procedures one accidental arterial punction was registered. the catheters remained in situ for a mean of 9 days (range 1-22) and were used for volume suppletion, medication, parenteral nutrition and haemodialysis.co-ionisation rates compared to those of subclavian catheters in our icu. in the first 20 patients 3 cases of asymptomatic thrombosis of the femoral vein were seer on ct-scans performed for other indications, in the following 26 patients duplex scanning performed after removal of the catheter yielded another 3 cases of asymptomatic femoral vein thrombosis. conclusions: ultrasound guided femoral vein catheterisation 10-20 cm below the inguinal ligament is a safe and simple technique that can easily be performed by residents without prior experience. the incidence and impact of thrombo-embolic complications associated with this technique are still subject to further investigation. objectives: to estimate the cost of antibiotherapy (ab-cost) in a multidisciplinary 8-bed greek icu and to correlate ab-cost with total cost of drugs and consumables and with patient's outcome, severity of illness and type of admission. methods: prospective data from 137 consecutive patients admitted to the icu from 1/10/1994 to 30/3/1995 were studied. a tick chart was designed to record all drugs, materials and consumables regularly used for icu patients, but did not include low price drugs and consumables, which are provided from hospital's pharmacy as stock and were included in a fixed icu cost calculated for a 12 month period. the chart also contained demographic details and data necessary for the calculation of several illness severity scoring systems. obiectives: over 3 years evaluate the necessary efforts and expenses to implement a cis in the routine of a 16-bed stcu. methods: in june 1992 a commercially available, unix-based cis was installed on a 16-bed surgical icu. the goal was a paperless documentation at the bedside. after more than 2 years clinical experience two aspects were investigated: what effort is necessary to install and support a cis, and what is the benefit for patients and personnel on the icu? results: the installation and support of a full-fledged cis requires a considerable effort: (a) the conceptual framework for the cis has to be defined. this includes the definition of documentation standards, as well as nursing and therapeutic standards, which is the essential basis for the configuration of any cis. (b) configuring a cis, i.e. "fine-tuning" it to the user's specific needs, is always a laborious task. moreover, constant maintenance is necessary. these tasks require the following personnel: experienced health care professionals for defining the conceptual framework, 1-3 trained health care professionals for configuration, 1 system administrator. on a single icu (12-20 beds) these are not considered full-time jobs. (c) training is best done employing the "train-the-trainers" approach. (d) beside the necessary amount of man power and money to install and purchase a cis, administrative and mis support is needed, especially when interfaces to the hospital and laboratory information systems have to be set up. in general, a cis needs the commitment of all people involved. without a really professional approach with a longterm goal any major cis can turn into an unnecessary but inevitable night mare. after 3 years clinical use and a thorough implementation of a cis on a major sicu it can be said that full-fledged cis offers an opportunity to dramatically improve the working environment on an icu. moreover, it adds to patient safety, quality of care and cost efficiency in one of the most advanced and expensive areas of medicine. conclusion: a major investment in man power and money is necessary to install and maintain a full-fledged cis. a sincere professional commitment to the goals of a cis is necessary. in exchange, a well configured and well maintained cis dramatically improves the quality of therapy and care on the icu. even return of investment and financial profitability of a cis seem feasible todayl from the clinical perspective it appears that the users themselves are the central determinant whether a cis makes a dream come tree or turns into a night mare. objectives: to establish a relationship between the activities of the staff and the occurrence of auditory alarms on the i. c.u. ard to evaluate confusion between auditory alarms. methods: laboratory based studies which investigated aspects of confusion between alarms in current use on the i. c. u. the observational studies were conducted over an 18 month period and examined the frequency and duration of alarms together with the concurrent activites being undertaken by staff on the unit. the laboratory based studies showed that there were enduring confusions between the alarms on various items of medical equipment, for example a ventilator alarm and an e. c. g. monitor alarm. the results of the observation studies demonstrated that alarms are activated when specific activities are being undertaken by staff. sounds could be used in future recommendations for alarms on medical equipment. suggestions are also discussed for improving and rationalising auditory warnings in the i. c. u. obiectives: we investigated inferior petrosal sinus (ips), the lowest affluent to jugular bulb (jb), as a possible source of contamination of samples in jb for monitoring oxyhemogiobin saturation (sjbo2). pulling back the catheter the oxyhemoglobin saturation usually rises indicating extracerebral contamination (jakobs en met al: j cereb blood flow metab 1989;9:717). methods: the study was carried out on patients undergoing ips sampling to differentiate cushing disease from ectopic acth syndrome and to lateralize any resulting pituitary lesion. we studied the value of oxyhemogiobkn saturation high in jb (sjbo2), at ips (sipso2) and at mid jugular vein (5th cervical vertebra) (smj 02) bilaterally. results: we found significant differences between right sjbo 2 and both right sipso 2 (p= 0.007) and right smjo 2 ( p= 0,017) and between left sjbo 2 and both left sipso 2 (p= 0.01) and left smjo 2 (p= 0.017) we did not fred any difference bilaterally. objectives: we studied various methods of receiving and editing of clinical datas in critically ill patients (different ethiology). 163 patients were investigated in regional intensive care center. methods : the following datas were studied : anamnesis, status praesens objectivus ( organs and systems ) ,. clinical and biochemical markers of critical condition , datas of eeg ,rheography . the medical information complex contained : 8channel electroencephalograph, 4-channel roencephalograph, ad-converter (16 analog inputs, 12 bit resolution, 60 k hz), ibm 486 dx2, software includes set of routines for spectral eeg analysis, eeg-mapping, correlative analysis, and brain bloodstream reg-monitoring (written in turbo pascal 4.0), expert programs for estimation objective and humoral patient status (written in clipper 5.0) and statistics. there were used following programme-language instruments : borland c++ 3.0, nantucket clipper 5.01, ca-clipper tools ii. as the methods of statistical processing of dates were used: t-students criterion , fisher criterion, methods of correlation analisis, calculation of the regression levels, dispersion analysis, results : there was created the optimal structure of hard and sofware complex of search steady objective regularity in dynamic of critically ill patients condition. conclusion : the created system allowed to value effectiveness of intensive care and give us new opportunities in study pathogenesis of systems disorders in critical condition . over a five year period a patient data management system has been installed which allows individualised patient data to be accurately collected. using this data a costing system has been developed which ascribes costs thus: 1. direct costs -drugs, fluids, consumables, interventions. these are ascribed to individual patients, according to data collected from the pdms. 2. indirect costs -energy, depreciation, admm costs, maintenance etc. these are summed for the year and ascribed as an overhead per patient day. n.b staffcusts contain art element of both cost types the aim is to make as many costs as possibie 'direct', hence 'activity costs' have been calculated winch comprise staff time, drugs and consumables -these are direct costs. these costs of patient care are then searnlessly integrated into the financial and budget management of the icu environment. it was found that by calculating costs in this manner 50% of the total cost of icu are captured within the 'direct' element, and so are able to be ascribed to individual patients. this is much more accurate than simply dividing the total costs of ~cu by the number of patient days. temporal costs (variations during patient stay) and cross sectional costs (cost differences between admitting specialities) were also noted with interest. results of the initial analysis of data captured by the system will be presented. little is known about the resource costs (not simply cash costs) of icu. even less is known about individual patient costs, with previous estimates of these costs varying widely. however, if cost effectiveness studies are to be undertaken accurate calculation of individual, group and total icu cost is an essential, prerequisite, which, via this system of costing, is now achievable. information about intensive care of cancer patients is limited in the literature, despite the increasing use of such facilities in oncology over the two last decades. in order to determine if and how critical care facilities can be used specifically for these patients, we performed a world-wide inquiry in anticancer centers selecting the hospitals by using the international directory of cancer institutes and organizations. we mailed a questionnaire to 146 centers and we received 84 responses (57.5 %). there was at least one uncological (i.e. with > 50 % of cancer patients) icu in 59 (% % an 18-year old woman with graves disease presents with sore throat, vomiting, diarrhea, sinus tachycardia at 155/minute and a temperature of 40~ several weeks before, treatment with propylthiouraeil had been stopped (rash and fever) and replaced by methimazole and ledide prior to a minor surgery. however, both drugs were discontinued by the patient two weeks before admission. shortly after arrival in hospital, patient's condition progressed to respiratory failure (upper airway edema), delirium and shock requiring icu admission, intubation and resuscitation with fluids and vasopressors. white blood count was 1300/mm ~ with 0 neutrophils. patient's hemodynamic data showed initial hyperdynamic profile followed by low output state with decreased sv02 (59%) (n 70-80%) and cardiac index (2,37) (n 2,5-3). echocardiogram confirmed cardiac chambers dilation as previously described in thyroid storm. lithium carbonate, corticosteroids, antibiotics and beta-blocker perfusion were given. plasmapheresis was started. free t& (n=9,2-21pmo/l) went from 143,6 to 16,6 after the first two pheresis. after a remarkable clinical recovery, sub-total thyroideetomy was done i0 days after admission. in life-threatening thyroid storm, plasmapheresis is a very effective therapy when anti-thyroid drugs are counterindicated. purpose: to compare the reliability of prognostic indexes in crhically iu patients admitted in an intesive care unit (icu) who had acute renal failure (arfi and were treated with different dialytic techniques. material and methods: 1087 patients were included in a prospective study from june 92 to november 94. 220 patients presented arf defined by creatinin serum leve(s greater than 150 pmol/l and previous normal levels. patients were divided in three groups. group i (control) : 156 patients with arf who did not receive substitutive techniques. group ih 21 patients under intermittent hemodialysis (hd) or peritoneal dialysis (pd). group ii1:43 patients under continuous hemodiafiltrstion (hf). the statistical analysis was chi-square test and analysis of variance. results: the table shows the results we obtained, we did not find any significant difference betwen the two groups of patients undergoing dialysis. d(fferences were observed only between group i and the other groups as shown below. we did not find any significant association between the theoretical mortality predicted and the observed mortality according to saps in the three groups. due to exposure to a wide variety of unpleasant stimuli, for example, tracheal suctioning, venipuneture and physiotherapy, most pataents admitted to the icu will require some form of sedation. this review will describe the suggested properties of an ideal sedative agent for use in the icu and review the current limitations of some of the available agents from this perspactive. methods used to quantify the level of sedation, such as the ramsay score, glasgow coma score, newcastle sedation score and visual analogue scores, and their deficiencies will be examined. consideration will be given to defining the optimal level of sedation and the circumstances under which sedation might be varied over the icu course will be discussed. preliminary results from an ongoing study examining the role of light versus heavy sedation and ischaemia in a cardiac surgical icu population will be presented. the pharmacceconomics of icu sedation will be briefly addressed. finally, the role that sedation may play in increasing morbidity, pastieuiarly nosocomial pneumonia, in the icu will be discussed. objectives : therapy cost(tc) in icu patients is a substantial component of total hospital care cost. estimation of tc during this year, partitioning to various groups of drugs used and attempt to minimise it, were considered practically useful. methods : in collaboration with the hospital pharmacy we were able to have a complete report of au drugs used for icu patients (including enteral and parenteral nutrition). mean apache ii severity score upon admission was 19.7 and mean length of tcu stay was 6.7 days. price per drug unit and cost per group of drugs were also available drugs were divided into two groups: antibiotics (1) cardiovascular drugs (2), gastrointestinal system drugs (3), enteral and parenteral nutrition (4), respiratory system drugs (5), sedative, analgesics and paralysing agents (6), parenteral solutions with electrolytes, vitamins and trace elements (7), anti-inflammatory agents (8), protein substitutes and immunomodulation agents (9), anticoagulative agents (10). antibiotics were further subdivided into those "freely" prescribed (a) and those whose prescription and administration requires filling of a relevant form (b). results : !) tc for icu patients/day was 30.530 drs ($122). total tc/patient was 295.195 drs ($1.108.7). ii) partitioning total tc per group of drugs reveals : (1) 43%, (2) 2.7%, (3) 2.7%, (4) 9.2%, (5) 0.3%, (6) 8.6%, (7) 9.3%, (8) 1.3%, (9) 15.8%, (10) 2.5%. t11) concerning antibiotics which consist the major cost component, group a and group b contributed by 29.1% and 13.9% to the total icu tc respectively. group b were administered to 13.9% of all icu patients. conclusions : i) for the above studied patient population antibiotics consist almost half of total tc followed by protein substitutes and immunomodulation agents. ii) if tc control could be attempted in the icu, prescription of beth groups must be reviewed. appropriate treatment should be prescribed and readily provided to any patient. clinical significance of routine protein substitution, currently controversial, should be re-evaluated. new antibiotics (third & fourth generation cephalosporins, quinolones, carbaponems) should be prescribed on the basis of strict diagnostic procedures using modern technology available. rationalisetion of antibiotic therapy will lead to cost control, redistribution of icu expenses and substantial contribution to infection policy in our country. objectives: i -to investigate the clinic efficiency of the monitoring of the rso2 cerebral, in relationship to the stroke prevention, in patient undergoing carotid surgery. 2-to determinate the variations of the rso2 during the different surgical and anesthetic procedures in these patients methods: ten patients undergoing carotid endarterectomy. precise neurological exploration previously to the surgery and in the immediate postoperative period. angiography evaluation to the extend of carotid artery disease. invasive blood pressure, ecg, pulse-oximetry ( pso2 ) and rso2 were collected previousty to the induction of anesthesia. the premedication was administered intravenously -midazolam (50 mcgr/kg) and fentanyl (i rncgr/kg) -. thiopental (4 mg/kg),fentanyl (3 mcgr/kg) and atracnrium (0,5 mg/kg) have been used for induction of anesthesia. co2te is monitoring al~er the orotraqueal intubation ! the anesthetic maintenance is accomplished with lsofluorane (0,5 -1,5%) and bolus of atracurium and fentanyh the surgical procedure is standard (without arterial shunt during the carotid cross-clamping). we register each 5 minutes: blood pressure, cardiac frequency, pso2, co2te and rso2. the rso2 cerebral variate in relation with: the anesthetic induction, blood ~ressure, co2te, cross-ulampping carotid and with the modifications of the head position. the maximum decrease of rso2 cerebral was in relation with the :ross-clampping carotid ( minimal value: 52 ). no patient had neurologic complications and postoperative stroke after carotid endarterectomy were not observed. objectives: there are more than 8000 anesthesia in chelyabinsk emergency hospital every year. to 80% patients of it emergency anesthesia is applied. more than 900 patients have ishemie heart disease (ihd), hypertansion (hp) and previos miocardial infarction (pmi). more than 5% of all patients are old patients (op). the resalts deep noninvasive bioimpedance monitoring (nbm) in surgical patients have been studied by us. methods: our nbm system "kentavr" includes 21 parameters of cardiac and vessels function. it is realised by monitors in operation theatres and computer network. moreover we are able to examine surgery patients before anesthesia and perioperatively by using special computers system for cardiovascular reflex control by fast fourie transform (fft) of 12 parameters simultaneously. results: 187 pathients extremly needed peryoperative monitoring of hemodinamics. from these 187 patients more 40% had stroke volume (sv) less than 30 ml, 18n -co less than 2.1/mim/m2, 25% -ejection fraction (ef) less than 65n and 32% -puls bioimpedans microvessels (pbm) less than 10 morn. 100 patient had intensive care in special department. 42 out of 187 died. comparing with survived with these patients before operation hr was larger, sv, co,ef, pbm and puls bioimpedance aortha was smaller. much more of these patients were with ihd, pmi, hd, op. even with survived patients these parameters decreased the towards the end of operation. surgery patients had different variability of 12 basic hemodinamical parameters with common tendency to increase power amplitude in low frequency by fft. conclusions: using of bioimpedanee noninvasive parameters allows to have criteria for corrections (infusies, vasodilatators, inotrops and others) and then us the final goal, to have more sucssesful surgery. with survived patients was perioperatively and postoperatively care more intensive. obiectives: the aim of the study was to compare the phi with the hemodynamically derived tissue oxygenation indexes as: oxygen delivery (do2), oxygen consumption (vo2), cardiac index (el), and arteriovenous difference in oxygen [(a-v)do2]. methods: 18 patients (15 males and 3 females) with major trauma or major abdominal surgery were studied. on admission, a nasogastric tube allowing phi measurement was introduced and a pulmonary artery catheter was inserted for optimal hemodynamic management. each phi measurement was accompanied with a complete hemodynamic study comprising systemic and pulmonary artery pressures, blood gases, and cardiac output measurements with the thermodilution method. derived parameters vo2, do2, ci, (a-v)do2 were measured according to the standard formula. hemodynamic parameters were opt• as soon as possible with fluids, inotrepes, and vasopressors according to repetitive hemodynamic measurements. all patients were under mechanical ventilation. after hemodynamic stabilisation phi and hemodynamic measurements were repeated every eight hours, during a 24-hour study period. a total number of 52 measurements were obtained and compared. statistics: results are presented as means + sd, correlations were performed between phi and the hemodynamically derived oxygenation parameters. a p<0.05 value was considered as significant. results: mean values were phi=7.19+0.1, do2=984+313, vo2=181+71, c.1= 3.7+ 1.2, (a-v)do2 = 4.47+1.2. no correlation was found between phi and do2, phi and vo2, phi and c.i, phi and (a-v)do2. on the contrary in 14 patients phi remained below 7.30 for more than 24 hours despite adequate hemodynamically derived tissue oxygenation parameters. mortality in this group of patients was very high (85%). conclusion: no correlation was found between phi and the hemodynamically derived tissue oxygenation parameters our data suggest that phi is a better oxygenation indicator than the hemodynamically derived tissue oxygenation parameters, because it is closely related to the patient's outcome. objectives: the pathogenesis of septic shock and multiorgan failure is believed to be related to tissue hypoxia of the gastrointestinal tract. therefore new monitoring techniques, preferably organ specific, are required to establish the adequacy of tissue oxygenation. peep is used to reduce pulmonary shunt volume and improve blood oxygenation, but is accused to impair splanchnic perfusion. we studied mucosal oxygenation and perfusion on the capillary level in the stomach and the duodenum. methods: we used the erlangen microlightguide spectrophotometer (empho ll) together with a specifically designed fibre probe (bodenseewerk ger~tetechnik, 0berlingen) in combination with a standard gastroscope. measurements were performed on 9 ventilated, traumatized patients (ages 17 -75 years), with no evidence of shock or severe infection, after informed consent was obtained from the relatives. all patients were hemodynamically stable without inotropic support. an area of 9 cm 2 was analysed in the gastric corpus, the antrum and in the duodenum. in three patients we simultaneously measured the muc0sal blood flow using a laser doppler flowmeter ( objectives: to investigate the influence of hb-o 2 affinity in the monitoring of svo~ during improvement of cardiac index (ci) in cardiogenic shock. design: to state whether changes in svo: were associated in changes in actual pso (p~0) and standard p~0 (ps0st) 22 consecutive measurements of artero-venous bga, before an.d after therapy-induced changes in ci, were evaluated in 11 patients (mean age 73-*7 y) suffering from cardiogenie shock, all under mechanical ventilation in psv modality. methods: together the hemodynamic measures, m~xed venous samples were analysed at 37 ~ c using the abl500 radiometer for po2, pco: and ph, and the osm3 radiometer for hbo2%, hbco% and methb%. psost (i.e. the p~0 at ph=7.40, pco:=40mmhg and temperature at 37 ~ c) was calculated automatically by the instruments on mixed venous blood as was the ps0"in vivo" (i.e. the pso at the patient's value of ph, pco2 and temperature), using siggaard-andersen's computerizated algorithm. mean time between paired measurements was 6.1 -* 1.2 houm. the data were compared by anova test for linear regression and t-test for paired samples. results: a dose linear relationship was found between svo2 and oxygen extraction ratio (oer), r=0.94,p=0.00000. the improvement of ci (1.41 -* 0.47 to 2.55 + 0.5 l/min/m 2, p<0.0000001) induced a significant increase in svo~ (0.495 -* 0.131 to 0.636 • 0.060 %, p<.0001). a significant decrease in p50 (32.5 • 6.7 to 27.9 • 2.5 mmhg, p<0.05) without any significant change in p~0st (29.1 • 2.2 to 28.7 • 2.3 mmhg, p=ns) was also found. these data show that either oer or the shift to the left of the oxygen dissociation curve account for increase in svo2 occurring with restoration of systemic blood flow. the program is intended to help the intensive care unit interne providing him with a practical tool when making decisions concerning patients in a critical condition. in his daily practice in intensive care unit, in this case the interne of the unit, uses this program for each patient as follows: on the first stage of data collection he should complete the following modules: (1)personal data (2)patient's pathology (3) laboratory and~ monitor lug data (4)drugs prescribed or toxic elements ingested. in this way, the system allows optionally the consult with a computerized data base about the drugs prescribed, standardized parameters and techinques performed by the central laboratory. (5)reference to an antibiotics guide regarding becterian sensitivety in our unit, whitch ee checked every six month (6) access to de questionnaired apache ii to load up new data. (7) statistcs about patient's admission and discharge. results: once all data collection is finished the system performs the followin duties: (1)detailed drugs interactions, including toxic elements (2)diagnosis starting from the clinical, laboratory and monitoring data. in some cases, it also establishes therapeutic strategies, e.g. a coagulopathy (3) give the l~narmacological incompatibilities between the drugs p~escribed and %he diagnosis established, and (4)perform dosage adjustments based upon the personal and pathological data. objeatve: to assess the power of diseri~,~ion ofa multiperpose severity score (sai~) when applied to subgroups ofpatieals (pta) according to their lemg~ of ~ay (los) in icu. design: in order to compute the saps probability, a model derived fi~m legible regression was developed. meaumree of calibration (goodmem..of.fit statistics) end discrimination (roc cm've and relative area under the cm've) were adopted in develotammtul asd validation set. the whole databue was ~ati~ed in five gronps reeked on los as follows: los = 2 days, los = 3-4 days, los = 5-7 da~, los = 8-14 days, los >15 day~. area under the carve (auc) was ud~ninted for each 8ro~. s~ing: 25 imlimlcus. patents: of 10~65 pts comec~ively admired ~ a period of three yeet~ (1990) (1991) (1992) , a total of 8059 was i~leded in this study. pts without saps, p~ yolmger them 18 yearn, p~ with los shorter ~ 24 hom'~ were excluded from this maly~is. iaterventinns: nose mema'onm~ end result: the logistic model developed gave good remits in terns of calibration md discrimin~on, both in developmental set (do.s g2: 9.24, p > 0.25; auc = 0.79i-0.01) and in validation ~t (g.o.g g2: 8.95, p > 0.50 ; auc = 0.78..+0.01). auc of each grottp showed a loss in di~zimination (i.e., prediaton) closely related with los, being 0.90i-0.01 in pts with los = 2 days el 0.59~.02 ia tm with los > 15 da~ (figure). following the present guidelines of integral management, in order to achieve optimization of sanitary resources and better use of facilities, we feel that the setting up of objetives is a key factor in the continuous process of improvement of quality care. postsurgical intensive care services maintain an interdepent relationship with other hospital services. within the general plan of the hospital it's of the utmost importance to delegate autonomy to the various depertments and service units in determining and achieving objetives. it's also necessary to establish mechanism for coordination of the activities in order to assure the succes of the program. the objetives cannot be improvised, they must be carried out in a specific manner in the following stages: 1.-analysis of the present situation (starting point). where are we?. defining objetives and making explicit the activities and methods to achieve them is to anticipate the future; it is of the utmost importance to comunicate said plans to all whom affect by encouraging them to attain the desired results. in the present paper we intend to show the guidelines to follow in carrying out a course of objetives. introduction:we presents results related to the quality of life (qol)of critical patients, from paeec project data. material and methods: the paeec project is a multicentre study define the type of patients cared for in spanish icus, and the therapeutic activity provided. ninety-five icus from spain are taking part. this study analyzes the qol of critical patients prior to their icu admission.for the evaluation of qol a questionnaire designed by our team for critical patients was used, with 15 items grouped in 3 sub-scales: physiological functions (4 items); functional capacity (8 items) and subjective aspects (3 items). qol is classified in 4 levels: normality (0 points); slight deterioration (1-4 points);moderate deterioration (5-9 points); significant deterioration (>i0 points). the we present results related to therapeutic activity in critical patients and their age, from the paeec project. material and methods: the paeec project is a multicentre study to define the type of patients in spanish icus, and the therapeutic activity provided. ninetyfive icus from spain are participating. this study analyzes therapeutic activity in the first 24 hours as evaluated by tiss, and related factors. results: the sample was 9,291 patients, sge 57.91~17.46 years. severity by apache ii system was 15.59• points. the tiss score was 19.87• points, distributed as follows: i (<i0 points): 14%; ii (i0 -19 points): 44%; iii (20 -39 points): 36%; iv (>39 points):5%.there is a positive correlation between the level of therapeutic activity and severity by apache ii (r = 0.46, p < 0.001), and a very weak but negative correlation between tiss and age (r = -0.048, p < 0.001), so that an increase in age corresponds to a lower level of therapeutic activity.patients the multivariate analysis of the relationship between tiss and age took into account: severity, existence of previous history, need for mechanical ventilation, size of hospital, diagnosis and mortality. it indicated that there continued to be a relationship between therapeutic activity and age, so that as age increased, therapeutic activity diminished. conclusions: therapeutic activity performed on critical patients is less in the oldest patients, in whom excessively aggressive procedures are limited. a relational data base management system in the icu. c. kotsavassiloglou*, d.matamis, g. dadoudis, j. kioumis, d. riggos. icu dep., g. papanicolaou gen. hosp., exohl, thessaloniki, and * a' neurological clinic of aristotelian university, thessaloniki, greece. objectives: the introduction of the information technology in the i. c. u seems to be unavoidable because of the large amount of produced data and the need for their systematic analysis. such an information system should be a) easy to use, b) friendly to the user, c) powerful and d) modular. on that basis, we created a patient data management system (pdms) according to the expectations of the medical staff of an eighteen bed multidisciplinary icu. methods: we selected paradox for windows v4.5 for the implementation of a relational data base because this program meets the above mentioned criteria. informations regarding the patients include a) demographic data, b) previous medical history, c)diseases upon admission, d)complications during hospitalization and e) outcome data. the diseases' registration consists of 421 items classified in 15 categories upon the principal system affected. specific informations about the need and duration of mechanical ventilation, nutrition, renal replacement, right heart catheterization and icp monitoring are also available. an extension was added concerning icu infections and related informations about antibiotic-resistant pathogens. all icu pathogens can be matched to their resistance or sensitivity and cost of antibiotics. the program can perform queries and various statistical analyses based on complex criteria. new modules can be added later according to the future needs and remarks of the users. results: the program was well accepted by the medical staff and 300 patients were registered as a test. the first analysis of the data related a) observed mortality versus the apache ii predicted mortality, b) mortality according to the age, gender, pathology aud duration of icu stay and c) pathology upon admission and icu related complications. conclusions: the long term use of this pdms can be an efficacious research tool. it can be used in retrospective or prospective studies by addition of necessary modules. the first data analysis revealed the iack of an international diseases' classification system. the development of a worldwide common classification system is essential for the compatibility of the data analysis among various icus. this will allow the realization of multicenter trials on a large scale. s. nanas= n. sphiris, a. precates, a. lymberis, m. pirounaki, and ch. roussos dept. of critical care, university of athens, athens, greece the complexity of the cases submitted to an icu, the variety of underline disease, tbe severity, as well as the large number of substances administered to each patient constitute obvious the need of support with an easy available dss. this system will assure the safety of the administered treatment will help to adjust the dose according to the situation of each patient and it will screen for possible interaction and incompatibilities between the administered drugs. the goal of the present effort is the design and development of a software system acting as a decision support tool to physicians of icu. the application is organised around a relation database management system (rdbms) that consist of: a) all available substances (1.850), b) all generic names of medications available in our country for each substance, c) incompatibilities (2.300 cases) and d) interactions with other substances (50.000 cases). the following figure shows the structure of the rdbms. y ta~ortato~ [ c~rs using the stored parameters for each patient the dose and the rate of administration of selected substances will be possible to calculate. the continuous monitoring of the treatment for each patient supports the medical staff to make the necessary changes of the prescriptions. the application is currently developing in wireless pen based computer systems which place patients at the centre of "islands of information" located throughout icu. in conclusion this dss is a powerful and useful tool for icu staff because it provides without additionai work to the routine of daily practice, the currently available information for each order concerning drug interaction and incompatibilities as well as treatment monitoring is to obsea~ among 113 critically ill pfdieats, stdjdivided following the diagn~s at the adn~ssio~ the diffmeax:es in the ~ and oxyplx~efic l~mmems bawe~ strvwors [s] and non sumvors ins] and to test the pc~'bih'ty to have soar survival criteria, as earliest as tx~able. method~ :we made a ~ study on 113 consexa~e ~ilically ill paliffas, subdivided in 3 series following the diastases at the admission: medical pafiea~ (29 s and 23 ns), surgical patients (19 s and 22 ns), a~d poliwauntas ( 14 s and 6 ns). follow up was done at 20 d,.ays from the admission in ice. all the patienls were ramitored with a ~ c~eter and 18 laeno:lymmi. "c and o .x.xyphorefic txuamaers va:~e couected at 7 fin~es (t): at fiae ~draission (t0), at 121x~ars from t0 (t1), at 24 (f2), 48 (y3), 72 (t4), % (t5) and 120 horus from t0 cf6). in~,h ~ies, for ~y ~ a all the lin'~ n~an and sandaid d~viation was ~ tx~h for s and for ns. th~ betw~ s and ns tl~ roeaas of ~h porarneter ~e ccmpared tt~ng t-lest and p < 0.05 w~ considered ska~ significant in each series in the t wheae the mast significative diffemx:as ~goeamd bet~en s and ns, we made a txedictive criterion, asamting as predictive indices for stnvival the i:r values, higher or lower than flae treans of the ~rar~ers of au flae patients, axx)rdhlg to those ones t~iatistically diff~'e~ betw~m s and ns. fhmlly xse co:weatxt onaong the 3 series the nrametees of the st~rs with the analysis of variance, to daserve the lxjsable differealt irea~ of sty1 hflices, following the diagn~s of admission: :nedkal, angical patient or poll~tam results: we c~ld not find ~ predictive criterion for politraonaas, perhaps ixx:ause of the few ntanber of l~fients. for high ri~ saw~cal patieras the following criterion at t2 has a sensitivi .ly of 100~ ,and a ~ecificity of 27.8%: sv1>32.89 nffmin/n~, map>92 mmhg, pmap<27 nmalqg cvp<i0 nunhg will4 nmah& lvswi>34 g m/m 2, sxo2>67~ do21>515 mlhnin/m2, o2er<31%. for lx~dical l~tienls at t5 the following criteric~a has a ser~tivi.ty of 100% and a ~zificity of 36.8~ cvp<7.5 mn~g, sao2>97%, s,g)2>74~ vo2i<133 ml/nfin/m 2, o2er<25%, shunt<19% survlvops' data of the 3 series ~ signitic~atly differenl~ both for the t~mody~nic a~ for fl~e ox3rphomfic lxlmn~s; moreover we ~ that the vatt~ of hemodynamic mad ox.~ho~tic indices were higher in politrautms. conclus'ions: acx~ording to the fftffe~mt patho!o~es, the ~ rnelabo~c needs are diffeten~ so that it is juslified to mash ~ the~alceutic goals, following the type oflmthology. 2hen~ we foru~d for high ~k mrgical pmka~ and for medical patier~s assme, ff mllslied, a good prognosis while, if n0[ ntljsfled~ the plinsliclioil ofdl~tth is no[ g(ioct finally, ab~ high iis~ supgical palieaats, according to what other atmhors say, txatws sh0~'n~ers ' therapeutic goalsvvould seem inadeqt~te, bec~jse they need a gear physiologic and themtx~ic elth~ in rdation to the rretabolic needs. figure 1) . thus, the smaller european nations had a greater participation than ~e larger ones, with the exception of norway. a similar result was evidenced for contributions to intensive care medicine (figure 2 ). these findings can be explained by different submission policies and language banners. however, there was no significant correlation with the gross national product of each country. conclusion: we conclude that the smaller european countries generally contribute more to international intensive care journals than the larger ones. objectives: to evaluate the agreement between a new and three old methods measuring ctp and to assess their reproducibility. methods: we studied 20 patients ventilated with a siemens 900c respirator. we measured ctp by dividing the tidal volume with the increase in airway pressure (paw), either with the respirator setting used (ca) or with a fixed setting (cf). by modifing the inspiratory time (ti) without changing inspiratory flow, we were able to deliver two series of 10 inflations (100, 200,... 1000 ml) before and after curarisation of the patient. the same volumes were also inflated in paralysed patients with a super syringe. at the end of each inflation a plateau of 3 sec was performed and paw was recorded. the above three sets of pressure-volume (pv) points were used to reconstruct the corresponding pv-curves ((31, c2, c3 the new method for ctp measurement without a super-syringe had the best reproducibility in paralysed patients and gave similar results without curarisation in the majority of them. however, agreement between the methods tested was unacceptable for clinical purposes. further investigation is required in order to improve the accuracy of ctp measurement in icu patients. m kunert, r.sorgenicht, l.scheuble, k.emmerich, h.g01ker med.clinic b (dept.of cardiology) i heart center of wuppertal/university witten-herdecke,germany objective to determine the accuracy of activated partial thromboplastin time (apl-l) and activated clotting time (act) studies when samples are drawn through heparinized central venous catheters (cvc). methods a total sample of 80 paired act/p't-/" values was analysed in 40 patients (28 m.,12 f.,66 + 12 y.) for monitoring heparin therapy.all patients had a cvc (certofix trio,braun,frg) in the internal jugular vein receiving a continous infusion of 20.000 u heparin via the central catheter.act (hr-act, hemotec,usa) and ap'i-f (neothromtin, behring,frg) samples were drawn from the cvc using the double syringe technique (removing and discarding 5 ml blood before drawing the sample). these blood samples were compared to act/ap'cf blood samples obtained by venipuncture (v.fem.) at the same time, act values were analysed directly in the intensive care unit (icu),api-i samples were measured in the hospital laboratory within 30 minutes. results ac-i -~ pi-f~ cact/~pi7 r = 0,62) cvc samples 132 88 +34 +22 . v.femoralis samples 1"28 84 +29 +24 p-value n.s. n.s. conclusion there is no difference in heparin anticoagulation studies drawn from heparinized central venous catheters compared to those obtained by femoral venipuncture,withdrawing 5 ml blood prior to obtaining the blood specimen is a safe way for eliminating heparin contamination.not only the aptt test but also the act test is a useful method for heparin anticoagulation assessment in the icu. objectives: evaluation of the delicate balance between filter-coagulation and patient-hemorrhage using heparin as anticoagulant in continuous renal replacement procedures. methods: from january 1991 through august 1994, we studied filter surviva[ and hemorrhagic complications during 240 filter periods in 78 critically d[ patients, treated with continuous arterio-venous hemo(dia)filtration, with special emphasis on the heparin dose, concurrent use of coumarins, systemic activated partial thromboplastin tirne(aptr), platelet count, mean arterial bloodpressure and the type of filter used. results: 141 filters (59%) were disconnected because of coagulation. mean survival of multiflow an69 filters was twofold shorter compared to survival of fh66 gambm filters. a total of 48 hemorrhagic complications occurred of which three patients died at aptt values of respectively 39, 48 and 56 seconds. after adjustment for mean arterial bloodpressure, platelet count and the type of the filter, the risk for filter-coagulation decreased 25% (relative risk 0.76, 95%c1 0.68-0.85) for each ten seconds increase in aptt. the risk for patient-hemorrhage increased 50% (relative risk 1.50, 95%ci 1.38-1.64) at an aptt-increase of ten seconds. the occurrence of filter-coagulation and patienthemorrhage was not correlated with the administered dose of heparin. concurrent use of cournarines had a positive effect on filter-survival, without increasing the overall incidence rate of patient-hemorrhage. conclusions: the systemic apt]" is a good predictor of the risk for filtercoagulation and patient-hemorrhage. heparine therapy seems optimal at an aptt between 35 and 45 seconds, although one should realize that fatal hemorrhagic complications still can occur. objectives: the alterations in vascular tone which are primarily regulated by adreno-sympathetic tone(ast) are compensatory responses in hemorrhagic patients. this study was designed to evaluate the correlation between vascular tone and ast in patients with hemorrhage, methods: the vascular tone was expressed by volume elastic modulus (ev) that is defined as; ev = ap/(av/v) (ap; the arterial pulse pressure, av/v; the volume change ratio). ev was measured using a non-invasive transmittance infrared photoelectric plethysmography (tipp) and a volume oscillometric sphygmomanometer . we prospectively studied 6 patients with hemorrhage. the initial ev measurement was performed on arrival and repeated for a 48hours duration. as a parameters of ast, serum concentrations of adrenalin (ad), noradrenalin (nor), plasma renin activity(pra) were measured simultaneously. we analyzed the correlation of ev and conventional parameters to ast by multivariate statistical analysis. results: ev values at transmural pressure 40mmhg on admission and 48hours later were respectively 864.2 + 249.5mmhg, 270.0 +_ 92.0 mmhg (mean + sd). systolic pressure(pas) and serum hormones on arrival and 48hours later were respectively, pas; 96.5_+20.4, 152+18.7mmhg, ad; 1.21_+1.02, 0.07_+0.04 ng/ml, nor; 1.60_+1.48, 0.65+0.39 ng/ml, pra; 26.6_+37.8, 2.5_+2.9ng/ml/hr. the ev values correlated significantly with ad (r=0.47, p=0.006, n=33), nor (r=0.47, p=0.005, n=33), pra (r=0.38, p=0.032, n=33). by multivariate statistical analysis, ev correlated more significantly with ad and nor and pra (p=0.00079) than the conventional parameters such as pas, heart rate and pulse pressure. conclusions: the alterations of ev correlates closely with ast. the compensatory mechanism in hemorrhagic patients can be detected noninvasively by ev monitoring. obiectives and method: autologous oxygenator blood was processed at the end of cardiopulmonary bypass (cpb) by either hemofiltration (hf 60, 1,2 m 2, fresenius) or by cell washing with a onntinous autologous transfusion system (cats, fresenius). prospectively the blood of 10 patients for each group was processed and then retransfused intravenously to the patient. besides, volume and time requirements, standard hematologic chemistry, coagulation and complement activation were measured. results (mean values for oxygenator blood at the end of cpb, and results of concentrate after processing by filtration or washing): both processing techniques show excellent hemoconcentration of the diluted cpb blood with a good transfusion effect for the patient. filtration retains all plasma proteins and large molecular weight plasma bound waste products. in contrast, cell washing with cats significantly depletes plasma proteins and waste products. the newely developped cats machine gives eonsisinnt laboratory result in a fully automatic continuous processing mode. in conclusion, both filtration and washing are effective for processing cpb blood. filtra tion yields a highly concentrated whole blood, whereas cats washing produces a high quality autologous erythrocyte concentrate. soluble fibrin has during the last years gained interest as a marker for the activation of the coagulation in connection with various clinical conditions, e.g. disseminated intravascular coagulation, deep venous thrombosis and myocardial infarction. elevated levels of soluble fibrin in plasma can be detected by the chromogenic assay coaset fibrin monomer, relying on the ability of fibrin to enhance the tpa-catalyzed conversion of plasminogen to ,plasmin. using this test, it has been shown that the level of soluble fibrin can be correlated to severeness of illness in critically ill intensive care unit patients. a revision of the coaset fibrin monomer kit has now been made and the new product, coatest soluble fibrin, is considerably more convenient to handle and gives higher resolution at low fibrin levels. the test is performed by the addition of a buffer dilution of the plasma sample to a microstrip well containing the colyophilized mixture of tpa, plasminogen and the plasmin specific cbromogenic substrate s-2403. the reaction is allowed to proceed at,. room temperature for 15 minutes before discontinuation. the absorbance at 405 nm, measured in a microplate reader, is proportional to the content of soluble fibrin in the sample. the assay is carefully standardized and calibration curves are provided in the kit. the convenient and rapid assay procedure makes the coatest soluble fibrin test well suited for single test analysis in acute situations. objectives : blood coagulation abnormalities have been reported in the systemic blood of patients with cerebral lesions. the physiopathology of such events is not yet completely understood. we compare the coagulation profile of blood from the right jugular bulb with systemic blood of patients with head injury. methods: we studied 4 patients, who were admitted to our neurosurgical intensive care unit between january and march 1995 with head injury and no other associated pathology (age 20-60 yrs), a glasgow coma score <= g, no abnormality in baseline coagulation profile and no history of coagulopaties. the patients did not undergo angiography. a one-way 16 gauge certofix catheter was inserted through the right internal jugular vein up to the jugular bulb. an identical catheter was inserted through a subclavian vein. blood was sampled from either catheter (a=atrial; j=jugular) 6-10 hours after trauma (t 1) and t 2 hours later (t2 the inddence dpontolx'rative thmmhi~e and haumord~gic complieatiom were assessed in padents treated with indobefen, heparin calcine caeca), low mollecolar weight heparin (lmwh) (f.nosheparin) and undergoing hemodiludun, blood predeposhing, intra mad postoperative blood saving. ]'he indolmfon tempota~.norks platelet aggregation through ,,elective inhibition of the cyclatygenasis and thus atacbldonicadd(1).tbe n'mimum effect occurs after 3 hours from the fast administration and is still present after 24 hours. ~-979 patients, mean age 62---11 yrs., weight 68---10 kg were studied. 321 (32.8%) were male and 658 (67.2%) female. 858 onderwent 713 hip prosthesis (7 previously plate and screw removal) 145 hip revim'un (10 stem, 33 cop and 102 stem + cop), 121 tutal knee prosthesis, in the 1st anaesthesidogy depl from 14-1992 to 30-6-1994. as for antithromboembolic ptephylam, apart from hemodihitiun 668 pts were with treated indobufen 0ndo), 199 with heparin ealdum caeca) and 112 with low mo!lecular weight hepam (lwr,91). as the slightest clinical and/or imtmmental suspidon of deep vein thrombosis (dv'i') or polmonary umbolism(pe), a phlebogram or sdndgram were respectively carried out. -the inddence of homologom transhisiom was significandy lower (p=0.000l) in the padeats treated with indobufen (4.256) compared 7.'ith heca (14.5%). the con~gency table shows statistical signifleance for the use of heca in patients with vein deficiency in the lower limbs, past dvr and/or pe, coronary heart disease (cdh'), while there is no correlation for renal, cardiac or liver defidency, obesity, systemic hypertemion, atrhythmy, diabetes, chronic bronchitis and rheumatoid arthritis. by comparing the postoperative cumplications with the risk factors, there ks a highly significant correlation (p=0.000l) between cdh and thrombotic and humord~agic complieatiom (pe, death, he~atoma, die use of hum_ologous blood). thee data show that hep~in, preferred in patients with c'dh, roost likely for leagal-tuedical reasons, did not have the de~'ed effect. conclusions -the stastisfical aar~ais shows ~nifieanfly different efflea~ (pro0.0001) between the therapies (see table) : it can be seen that in patients undergoing autotramfusiun and hemedihidon, indobufen produo~ a lower incidence of haemotrhagic complieatiens compared to heca and lmwh and is more effective in the prevention d ~c complications at clinical e~idence. the duration of i~toperadve hospital stay is signi~cantlylonger for patients transfused with homologous red ceils and treated with hec,7.a (13.7-+1.4 days) and lmwh (13.5+-1a days) compared with indo(ll.8_+1a days). one of the main causes for postoperative complications in major orthopaedic surgery is postopemtive bleeding with local effects in the operation site (hematomata, pain and delayed mobilization) and/or systemic and subsequent cardiodrculamry repercussions that are sometimes severe. the aim of this study is to assess the possibility to apply a new system of monitoring, control and saving postopemtive blood loss from the drainage. the bt797 recovery dideco (marandola, modena-italy) ~ used since it is the only apparatus capable of doing this. the apparatus consists of a pressure transducer, adjustable from -100 a +50 mmhg, which activates a peristaltic pump connected m drainage robes. the bt797 recovery display shows hourly bleeding in the first 8 hours, total bleeding, time passed since the start of monito~g and subsequent salvage and the aspimtioo pressure on the drainage robes; the latter is inserted at -10 mmhg and then modified according to bleeding/minute, g bt797 recovery also has an alarm that sounds automatically if.' blood loss is more than 300 ml/hour; air is in the circuit; the batteries are running low. materials and methods: 191 pts were studied (53 m and 138 ~), aged 63.5-+11.lyears, basal hemoglobin 13.1-+ 13(range 7.8-16.6)g/all, treated from 1st january, 1992 to mst december, 1994 in the 1st service of anesthesia and intensive care unit of our hospital. the patients underwent the following surgical treatment: total hip revision (132pts), cup revision (~ipts), stem revision (13 pts), total knee revision (2 pts). the average dumtion of the operations was 173-+58 min. intranpemtive monitoring and blood salvage was applied to all patients. genera! anesthesia was used on 57 pts. and integrated (epidural analgesia + light general) on the remaining t34. anttthromboembolic prophylaxis consisted of external pressure bandage, isovolemic hemodilution with iodobufen in 131(68.6%)pts., calalc heparin in 35(18.3%)pts., low molecular weight heparin in 25(13.1%)pts.; 1 pt did not give a predepoalt of blood, 4 gave 1 unit, 45 pts 2 units, 110 pts 2 units, 31 pts 4 units. the data obtained was statistically analysed using contingency tables and anova. results: average intmop salvage was 420-+345 ml, average postop salvage was 420-+265 mi the average intra+postop 909+-460 ml. average postop loss was 677-+359 ml. the global incidence of postop complications was: h~natomata 5.2%, dvt 1.1%, pulmonary thromboembolism 1,196, myocardiac ischemia 0.5%, acute myocardic infarction 0.5%, respiratory deflciecy 2.6%, arrhythmia 2%, cystitis 0.5% there were nn complications in 86.4% of pts. postop bleeding over 300 ml in under 60 minutes (with bleeding alarm activation) occurred in 30 pts (15.7%). this sta~tically correlates only with the type of operation performed (more frequently in total hip revision p=0.034) and with a significant decrease (p~0.003) in the pruthrombic activity detected about 8 hours after the operation. this bleeding, also made the alarm sound, calling the attention of staff who could act accordingly, by making the drainage pressure positive and incre~sthg the tension of the external pressure bandage. conclusions postop monitoring, control and blood loss salvage combined with predepoalting and intmop salvage has enabled allogenic transfusions in 16% of cases to be avoided in operations with high postop blood loss like hip or knee revision. the usefulness of the system can be seen by the fact that in the 30 patients with so much bleeding to set off the alarm, there was no significant difference in the incidence of allotransfusions and complications. references 1)borghi b., bassi a., de simone n., laguardia am., fonnaro g. an injury of the brain may result in various disorders of hemostasis caused by the release of • into the circulation through a damaged blood-brain bar tier. disseminated intravascular coagulation(dic) is one of these disorders. it is a freguent but relatively rare ly diagnosed complication of subaraohnoidal haemorrhage. the aim of this study was to evaluate some parameters of both blood coagulation and fibrynolisis in patients with sah.in addition one wanted to find out wh~ther potential changes correlated with the pa• condition in the acute phase of sah and whether they influenced the course of this disease. 60 patients with sah were studied. in 17 of them sah was due to closed eraniocerebral injury and in the rema ining 43 resulted from vascular malformation. the following parameters were evaluated:the prothrombine time,the activated partial thromboplastin time, the thrombine time,level of factor v,fibrinogen degrada tion products and fibrin monomers. the results let us show the presence of oic in 3 patients with closed craniocerebral injury and in 14 with vas. cular malformation despite the lack of clinical symptoms the tests in 5 posttraumatic patients and in 6 patients from second group showed incomplete dic.on admission patients with such changes in measured parameters were in poor condition.the course of the disease and the effe cts of treatment were also worse in these patients. the results showed ihal in patients with sah complex disorders of both coagulation and fibrynolisis occur, and they depend on clinical condition of the patient. they also influence the course of the disease. methods : charts of all patients admitted with d.i.c. over a ten year period (85-94) were reviewed. diagnosis of dic was based on the association of fibrinogen < 2 g/1 -platelets <150 109/1 -fpd > 40 ~tg/ml in the 24 hours of the admission. results : 40 patients -mean age 29+6 y -saps 8+_5 -gestanional age 35_+5weeks -the two first conditions associated with d.i.c. were placental abruption (35 %) and preeclampsia or eclampsia (22,5 %). bleeding episode was present in 22 pts (55 %) and surgical treatment has always been necessary. 26 pts (65 %) were given packed red ceils (12+10 u) and fresh frozen plasma (9+8 u). 6 patients were given platelets packs. heparin was never administered. 6 pts required mechanical ventilation and two patients hemodialysis. all the 40 patients survived. correction of prothrombin time (p.t.) and fibrinogen (f) was quick (p.t. at t24 h 69~2 % -f at t36 h 2,60+1,5 g/i). but platelets count remained low (plat. at t48 h 80 +42109/1) -no difference was observed in patients who received platelets. conclusion : prognosis of critically ill o.p. is good. blood loss is the main complication. correction of hypovolemia and anemia with concomitant surgical treatment are essential. the administration of coagulation factors or platelets is still under discussion. objectives: to evaluate the effects of antithrombin iii i at-iii) and a protease inhibitor, gabexate mesilate foy), on the coagulation and fibrinolysis in disseminated intravascular coagulation (dic). methods: after the approval of our institution and consent from patient's family, 40 patients with a dic score (1988, japan) more than 5 points (dic or having a risk for dic) entered this study. they were randomly divided into two groups, foy (i-2 mg/kg/h for 7 days or more) treated group and no foy group, each of 20 patients. platelet count (plt), fibrinogen (fen), at-iii fibrin degradation product (fdp), d-dimer (do), fibrin monomer (fm), thrombin-antithrombin complex (tat), plasmin-plasmin inhibitor complex (pic), and prothrombin time ratio (ptr) were measured before the start of treatment (at admission) and i, 3, 5 and 7 days after the admission. at-iii at 1500 units for 3 days was administered if the at-iii at admission was less than 70%. finally the patients were divided into four groups: group a, foy (+) and the at-iii ~ 70%; group b, foy (+) and the at-iii < 70%" group c, foy (-) and the at-iii 70%; group d, foy (~) anffthe at-iii <70%, each of 7 patients, to match the patients for backsrounds. all parameters, dic score and survival rate in a month following treatment were compared among the four groups. results: the at-iii and plt from day 3 to 7 were significantly higher in groups a and c than in groups b and d. the fdp, dd, tat, and pic after treatment decreased significantly from the baselines in groups a and c but not in groups b and d. the fgn and fm were not significantly different among the four groups. the ptr decreased in groups c and d but increased in group b. the dic score decreased significantly in groups a and c than in groups b and d. survival rates were 57%, 43%, 71% and 57% in groups a, b, c and d, respectively, although not significantly different. conclusions: in patients with dic or a risk for dic, foy had no expected effects but at-iii had suppressive effects on the coagulation and fibrinolysis mechanisms. a prognostic factor ? carbon monoxyde intoxication is a classical complication of inhalation injury. carbon monoxyda is also physiologically produced during the heme metabolism: heme is conversed to bi]irubin by the hemeoxygenase which is an intracellular stress protein. 20 icu patients (pts) were studied prospectively for apache ii score and carboxyhemnglobin (hbco) arterial level to assess if hbco level could be correlated with the severity of the pts. objective: to evaluate a new technique of non-surgical tracheotomy. patients: 55 adults, mean age 54 years and 11 children, mean age 18 months (2 me.-5 yrs). method: through a needle inserted in the trachea, a guide wire is retmgradely pushed out of the mouth and attached to a special device formed by a flexible plastic cone with pointed metal tip joined to an armoured tracheal cannula. this device is then pulled back through the oral cavity, larynx and trachea, and outwards across the neck wall by applying traction on the wire with one hand and counterpressure on the neck wall with the fingers of the operator's other hand. when the cone and 2/3 of the eannula have emerged, the cannula is cut off from the cone, straightened perpendicular to the skin, rotated and advanced caudally to its final position. results: endoscopic control facilitates and improves the safety of all manoeuvres. the pointed cone easily pierces the tissues, and the cannula is extracted without difficulty since it has the same outer diameter as the cone. tissue adherence around the cannula is absolute thus preventing local inflammation. the time in apnea required for dilation and cannula placement does not exceed 60 see., and it is well tolerated because within safety limits in patients hyperventilated with oxygen. only one case of bleeding occured in a patient on dialysis with severe coagulopathy. autoptic findings in subjects who died due to progression of primary disease showed a very regular stoma with an almost complete lack of hematic and flogistie infiltration in recent tracheotomies. .conclusions: translaryngeal tracheotomy (tlt), by virtue of its greater inherent safety and lower tissue trauma than percutaneous techniques, can also be carded out in infants and children, a severe test bench for any tracbeotomy technique. further specific indications are recently stemotomized patients, since tlt is associated with a low rate of infection, and short term tracheotomies after laryngeal surgery, to prevent obstructive complications. references: fantoni a., translaryngeal tracheotomy, apice, ed. gullo, trieste, 1993, 460 . background: inhalation of no has been shown to reverse hypoxic pulmonary vasoconstriction 1, to reduce pulmonary pressure in pulmonary hypertension of different origin and to improve gas exchange. in putmoflary embolism, pulmonary hypertension is caused by mechanical vascutar obstruction and by reactive vasoconstriction. the effects of inhaled no in putmonary embofism has been partiatly studied' the purpose of this study was to investigate and determine the effects of no inhalation on pulmonary hemodinamica and gas exchange in a hypoxic canine model of pulmonary embolism. methods: two groups of adult mongrel dogs were studied: group 1 (control} 5 dogs and group 2 (no inhaled) 6 dogs. both groups were anestesized with tiopental, mechanically normoventilated with an hypoxjc mixture of 02 and n~ (f[q2 0,16) and instrumented (swang-ganz catheter, femoral artery catheter) pulmonary embolism (pe) was induced by fisher's method s. no inhalation (80 ppm) in group 2 was started 15 rain. pdor to pe and kept constant throughout the experiment. no inhaled concentration was analyzecf by chemiluminiscence technique. pulmonary artery pressure (pap), central venous pressure and sistemic arterial pressure were continuosly recorded. cardiac output, artedat po~ (pan2) and mixed venous po~ were measured in both groups under hypo)dr conditions, before pe and 5, 15, 30 and 45 rain. after pe. pulmonary vascular resistance (pvr) and gas exchange (pao21fio:~ ratio), were calculate using standard formulas. data were process and analyzed with non pararnetdc test, and reported as mean -so and statistical significance was considered if p < 0,05. : no produced an increase in arterial oxigenation (pao2/fio~ ratio) and reduced pap before pe induction in group 2. after pe we found no significant difference with .respect to the time eour.se of pap, pvr and gas exchange between beth groups throughout the experiment. probably, the severe mechanical obstruction produced in pulmonary embolism masked the small effects of no inhaled. obiectives: blood volume measurement would be useful in critically ill patient management if it were easy to perform. this is not the ease and current methods are based on radiolabelled red cell dilution. inhalation and uptake of a known mass of carbon monoxide (co) gas and measurement of earboxyhaemoglobin increase can give results accurate enough for clinical use. this requires a rebreathing system providing oxygenation and carbon dioxide removal, yet complete retention of all carbon monoxide administer&l, and so most authors hand ventilate with a bag and waters soda-lime canister, adding oxygen as necessary. we aim to popularise this method by; i)design of an automatic co administration system driven by the itu ventilator and ii)writing of software for a portable computer to perform all necessary calculations 9 method: we show the computer is use estimating the co dose required and later estimating the blood volume. we also show the new gas administration system. this is a fully closed circle attached to a "bag in bottle", driven by the ventilator. the novel feature is the mechanism by winch driving gas (set to 100% 02) spills automatically into the circle, balancing o5 uptake by the patient, yet allowing no co loss. conclusions: this equipment is easy to use, reduces human error and allows optimum ventilator settings to remain. the operator merely administers the volume of co determined by the computer and takes blood on two occasions. carboxyhaemoglobin measurement is easy to perform, thus there is a cost saving also. with our modifications use of this technique may potentially become more widespread, the video demonstrates the method in use in our itu. -10 (25%) underwent conventional surgical therapeutics. 9" (90%) with resection of tracheal stenosis with end-to-end anastomosis(rts). i (10%) with broncoscopic dilatation. one patient died and the others still have stable patency(sp) without continued treatment. -29 (72,5%) have received endoscopic laser ablation with or without calibration tubes. 17 of them (58,6%) are receiving continued endotracheal treatment until now. 12 (41,4%) have sp wihout continued treatment. -i (2,5%) endoscopic laser therapeutic case turned to rts and is having sp. conclusion: conventional surgical aproach has been progressively replaced in our hospital by endoscopic laser ablation and silicone calibration tubes. this study suggests that these technics are effective and could be the elective treatment for iatrogenic stenosis. obiectives: hemorrhagic disorders due to thrombocytopenia and thrombocyiopathia remain one of the most serious complications during long-term extracorporeal membrane oxygenation (ecmo) in patients with severe acute respiratory distress ~drome (ards). in the presented study, nitric oxide (no), kwown as a potent endogenous platelet antiadhesive, disaggregating and antiaggregating compound, was evaluated for its possible antagonistic effect on platelet trapping when added to the gas compartment of membrane oxygenators (mo). meti~ods: two parallel separated extracorporeal circuits, consisting of heparin bonded hollow fiber oxygenators (minimax, medtronic, carmeda eioactive surface), tubing systems, low pressure reservoirs, and roller pumps were prepared. for each measurement, a pair of circuits was simultaneously filled blood from the same volunteer. low-heparinized fresh warm blood was obtained from four healthy volunteers, who had no drugs for at least two weeks. the gas inlets of both oxygenators received dry gas (21% oxxygen, 5 % carbon dioxide, 84 % nitrogen); gaseous no (20ppm) was added to the gas of one of the oxygenators (no-mo), whereas the other one (mo) was used as control. after 270 minutes no gas was switched off, so that the no-mo received no more no, and no was added to the gas inlet of the membrane, which had no no before_ to assure iutracircnit volume stability, drawn blood for measurements was replaced with saline, and platelet counts were corrected for dilution by hemoglobin values. the mean of four platelet counts (coulter counter) of each timepoint (start, 30, 90, 150, 210, 270, 330, 390 , and 450 minutes) was used for statistical analysis (paired sample t-test). results: in the no-mo platelets remained at 96 + 3,2 % (percentage of baseline value, mean -+ sd) until 270 min. in contrast, platelets of the mo continuously decreased after start and were significantly lower after 150 minutes ( 96,4 9 +3,5% vs90_+3,1%(p<0.05);210min. 95,9-+4,5%vs84,5_+2,2%(p< 0.05); 270 min. 82,7 _+ 2,5 % ( p < 0.05). after switching of no gas to the mo, further decrease of plateleta was stopped and platelets remained at 81,4 +_ 4,5 % until termination of circulation. platelets of the former no-mo decreased slightly after cessation of no gas to 92,6 _+ 5,4 %. conclusions: these data indicate that gaseous no significantly attenuates platelet trapping in hollow fiber oxygenators, when added to the gas compartment. this might be a new therapeutical approach for membrane oxygenator induced thrombocytopenia during long-term ecmd. objectives: nitric oxide (no) plays a pivotal role in regulation of vascular hemostasis. several studies elucidated the antiadhesive, antiaggregating, and disaggregating properties of endothelially synthesized no to platelets. additionally, agonist-induced no production in platelets by the l-arginine-no pathway was found as a negative feedback mechanism after platelet activation. although noplatelet interactions were intensively studied by several investigators, no data exist, about changes in platelet surface molecule expression in no-modulated platelets measured by flow cytometry using monoclonal antibodies (moabs). methods: p-selectin (alpha-granule-membrane protein, gmp-140, cd62p) and glycoproteiu 53 (gp53, lysosomal protein, cd63) are expressed only after platelet activation and degranulation. activation was quantified in thrombin (0.4 u/ml) and adp (0.1 ram) stimulated platelet rich plasma samples (prp). blood was obtained from healthy volunteers (n=7), who had no drugs for at least 14 days. for evahiation of no-modulated activation, the spontaneously noreleasing compound sin-i (0.1 mm) (3-morpholino-syndonimin-hydrochlorid) was added in parallel prepared samples prior to the addition of agonist. platelet surface molecule expression was evaluated with moabs directed against cd41a (gpilbliia, fibrinogen-receptor, phycoerythrin(pe)-conjugated), cd62p (fitcconjugated), and cd63 (fitc). only cd41a-positive signals were gated in sideangled light scatter, and assayed for activation marker expression (defined as percent of gated population). results: basal p-selectin expression was 1.6 + 0.7 %, and increased to 75.2 _+ 12.2 % after thrembin-activation, and to 26.7 + 5.3 % in adp-stimulated samples. addition of sin-1 attenuated p-selectin expression to 34.0 -4-193 % in thrombin (p<.001, two-tailed paired t-test), and 5.2 + 2.2 % (p<.001) in adpactivated platelets. basal gp53 expression was 1.7 _+ 0.5 % and increased to 63 .0 + 6.4 % in thrombin, and to 7.7 _+ 3.4 % in adp-stimulated samples. with sin-l, gp53 expression decreased to 346 _+ 10.4 % (p<.001) in thrombin, and 3.0 5:1.4 (p .001) in adp-stimulated samples. conclusions: these data implicate, that no leads to a significantly reduced activation of surface molecule expression in thrombin and adp-stimulated platelets. in addition, flow cytometry might be a useful tool for studying modulation of platelet activation by no or no-releasing compounds. introduction: acute cadmium poisoning is very rare. on initial presentation may mimic metal-fume fever, but acute inhalation cadmium toxicity may produce fatal chemical pneumonitis. case report: we present a case of acute fatal respiratory failure secondary to cadmium-fume irthalation. a 53 year old patient was trasferred from another hospital with acute respiratory failure presumably due to pneumonia. the last days before he had had commom cold symptoms. he had been cutting with a welder during one hour without any respiratory protective measure. three hours after exposure he developed progressive dispnea and was admitted to hospital. with presumtive diagnosis of respiratory infection, antibiotics were begun, however be failed to improve. all microbiological studies were negative. chest x-ray showed bilateral diffuse infiltrates. on seventh day he needed intubation and mechanical ventilation and on 10th he was admitted to our icu. antibiotics were stopped and new microbiological studies were performed including brochoalveolar lavage and virologic studies. all results were negative. he developed progressive hipoxemia and hipercapmia and finally, multiorganic disfunction syndrome. he died 19 days after exposure. the metal he had been working with was a 10% cadmium alleation. blood cadmilam concentration 15 days after exposure was 0.34 mcg cd/g cr, and urine cadmium concentration was 16.9 mcg/l. on postmortem examination, tissue cadmium concentrations were: blood 175 ng/ml, liver 823 ng/g, kidney 3571 ng/g and lung 1143 ng/g. these values confirm that cadmium was the cause of the fatal respiratory illness in this patient. conclusion: this case evidences the considerable hazard of acute poisoning after inhalation of eadmium-fume and stresses the need of appropiated safety measures against metal-fume poisoning. aim : lactic acidosis is considered the hallmark of cyanide poisonirig. however, the relationship between plasma lactate and blood cyanide levels has not been determined. the aim of this study was to determine the significance of plasma lactate concentration (plc) during the course of cyanide poisonings. methods : the patients were included according to the clinical suspicion of pure cyanide poisoning at the time of presentation. fire victims were excluded. serial blood samples were collected before and after intravenous hydroxocobalamin (hoco). blood cyanide concentration (bcc) was measured colorimetrically. plc was measured enzymatically. results : 8 patients were studied. on admission, plc ranged from 4.8 to 53 mmol/l, and bcc from 12.7 to 256 gmol/l. mean systolic blood pressure was 80 • 56 mm hg, mean arterial ph 7.34 • 0.14, mean anion gap was 29.7 + 7.7 mmol/l and mean pao 2 32.2 • 27.0 kpa. three patients died. before antidotal treatment, there was a significant correlation between plc and arterial ph (p = 0.008), anion gap (p = 0.008) and bcc (p = 0.016) but not with heart rate, pao2, paco 2 and blood glucose, or blood pressure. during the whole course of the poisoning, a plc _> 7 retool/1 was a sensitive and specific indicator of a blood cyanide concentration > 40 ~tmol/1. sustained catecholamine administration reduces the correlation coefficient. conclusion : baseline measurement of plc allows assessment of severity of acute cyanide poisoning. thereafter, plc may be used to assess the adequacy of antidotal treatment, more especially in patients not requiring sustained infusion of catecholamines. aim: the aim of this case report was [o study the correlation between the plasma lactate levels and several clinical, biological, and toxicological parameters serially measured during the course of a cyanide poisoning treated with a high dose of hydroxocobalamin. a 63-year-old male ingested potassium cyanide leading to cardiac arrest. cpr was performed prior to hospital arrival where the patient received 10 g hydroxocobalamin. sbp rapidly returned to normal allowing withdrawal of epinephrine. the patient remained comatose and died from brain injury 12 days after the ingestion. methods plasma lactate and blood cyanide levels were measured serially. blood cyanide levels were measured using a colorimetric method.~ plasma lactate levels were measured using an enzymatic method. for correlation spearman rank correlation test was used. results. initial plasma lactate and blood cyanide levels were 53 mmol/l and 256 gmol/l, respectively. there was no overall correlation between sbp and either blood cyanide or plasma lactate levels. similarly, there was no overall correlation between arterialvenous oxygen saturation difference with either blood cyanide or plasma lactate levels. in contrast there was a strong correlation between blood cyanide and plasma lactate levels (r=0.976, p<0.0001). the time-course of the blood cyanide concentrations was described by a mono-exponentiai decay (r2=0.968) with a blood half-life of 1.14 h. similarly, the time-course of plasma lactate levels was described by a mono-exponential decay (r2=0.986) with a blood half-life of 3.94 h. discussion. in this case of acute human poisoning, sbp was a much poorer indicator of continuing cyanide effect both before and after antidotal treatment, than was lactate production. this suggests a potential clinical role for following serial plasma lactate levels as a marker of the evolution of cyanide toxicity. aim : cyanide (cn) poisoning in fire victims is frequent and rapidly fatal. in a prospective study we tried to assess the clinical tolerance of a high dose of hydroxocobalamin (hoco) administered at the scene of the fire in fire victims suspected of cn poisoning. methods : inclusion criteria : soot in mouth or sputum ~ any degree of neurological impairment. exclusion criteria : children, pregnant women, burns of total surface body area > 20 %, multiple trauma. protocol desigrl following examination and the collection of a blood sample in dry heparin, a 5 g dose of hoco (10 g in case of cardiovascular collapse) was administered intravenously over 15 min. the systolic blood pressure was monitored before and after the administration of hoco, and one hour later. results : there were 28 females and 22 males. the mean blood cn concentration was 83 • 73 pmol/1. the mean blood carbon monoxide was 3.2• 2.1 mmol/1. nineteen fire victims eventually died. among the non-cn-intoxicated patients (blood cn < 40 ~mol/1), there was no significant change in arterial blood pressure. in the 33 cn-intoxicated patients (blood cn > 40 gmol/1) a significant increase in blood pressure was observed both immediately (p < 0.001) and 1 hour later (p < 0.001) after the admistration of hoco. no allergic reactions were observed. conclusions : in fire victims with cyanide poisoning, the administration of a high dose of hydroxocobalamin was associated with an improvement in systolic blood pressure. hydroxocobalamin is well tolerated in fire victims without cn poisoning. objectives: tricyclic antidepressant (tca) overdose can lead to serious complications including cardiac arrhythmias [ 1] . because of the known risk of early deterioration and the implication for management, emergent evaluation is essential. we determined the diagnostic usefulness of the electrocardiogram (ecg) in tca poisoning. methods: retrospective study of all patients with tca intoxication (pos. ,toxicology screening in urine and/or pos. history) in a 800-beduniversity hospital from 1989 through 1994. the severity was graded with mild= no symptoms or agitation; medium= disorientation, somnolence, tachycardia, or convulsions; and sever~ coma, significant arrhythmias or death. we analysed the first ecg after admission with a special emphasis on qrs-and qtc-intervals and the terminal 40ms frontal plane qrs-vector (tqrs), which, was reported to lie typically between +130 and 270* 798+310 915+453 5609• the best correlation with severity grade was found with qrs-and qtc-duration (p=0.0001), the tca-dose (p=0.0003) and hf (p= 0.027); tqrs did not correlate. 2 patients died (5.7%). conclusion: qrs-and qtc-prolongation in the admission ecg, and the reported dose of ingested drugs are useful predictors for severity of poisoning due to tricyclic antidepressants. we did not find additional benefit in determining the terminal 40ms frontal plane qrs-vector. objectives: since treatment of amphetamine poisoning is usually symptomatic and often associated with a fatal outcome, a search for specific drugs to help the amphetamine-intoxicated victim is sorely needed. methods: we report a case of a suicidal ingestion of large amounts of the amphetamine-derivative 3,4-methylenedioxy-ethamphetamine (mdea) and heroin (diacetylmorphine) and present the hypothesis that the two drugs produce opposing clinical effects. results: a 25 year old caucasian male was admitted to the emergency ward because of acute-onset confusion. at presentation, he was agitated and showed increased muscular rigidity. he had taken 40 tablets of "eve" (mdea, approx. 4 g) and 12 g of "smack" (heroin) by oral route approximately 2 h before admission. because of rapidly progressive tachypnea and exhaustion, the patient was intubated and ventilated. the serum concentration of "eve" on admission was 1400 ng/ml (lethal range 950-2000 ng/ml). trace amounts of cocaine and substantial amounts of heroin (115 ngtml; mean value in heroin-related deaths: 190 ng/ml) were also found in the serum. the patient was successfully weaned from the ventilator by day 4 and recovered without persistent neurobehavioral disturbance. despite high serum levels of both drugs, the patient did not present with the classic signs and symptoms normally seen during intoxication with these drugs. amphetamines in general, and mdea in particular, have opposite clinical effects to heroin or diacetylmorphine. none of these were however present in the case presented despite the high ingested doses and the serum levels in the lethal range. conclusions: the fascinating fact that, apart from the respiratory depression, none of the clinical signs reported after massive overdose with these two drugs were present, might be attributed to the opposite pharmacological effects of mdea and heroin. we believe that the patient unwittingly saved his own life by the oral coingestion of both mdea and heroin. our clinical data raise an interesting point about the pharmacological treatment of acute poisoning with amphetaminederivatives. introduction: the acute attack of aip still carries a significant risk of mortality of around 10 %. a succesful outcome depends on early diagnosis, removal of pricipitating factors and provision of intensive supportive therapy. objectives: twenty one patients (20 females, 1 male) with documented aip were seen over a 10-year period in the university hospital. 1 patient was in clinical remission and 20 were with the acute attack of aip, among them 4 with respiratory paralysis were required artificial lung ventilation and 4 -assistant ventilation with peee pathologic treatment during the attack was normosany, adenil, androgenes, glueosa, riboxin parenteral and enteral nutrition via nasogastric tube. symtomatic treatment -pethidine, propranoton, antibiotics, bronchoscopia. methods: intermittent phasmapheresis was performed on 15 patients. the following measurements were peformed: level of porphobilinogen (pbg) in the wire and delta-aminolevulinic acid in the blood. hematological and routine chemical evaluations, hepatic, hemodynamic and respiratory function. results: after plasmapheresis the median pbg excretion (normal range 1-2 mkg per/1 kgr creatinine) fill from 188 mkg on admission 140.8 mkg, then on 3-5 day raise to 193 mkg and then during treatment with normosong and prasmapheresis lowest level was 32.9 mgk. fatalities occured in two females during attacks with proforma cerebral involvement and 13 patients attained clinical remission. conclusion: after therapy with plasmapheresis normosong we found that there was consistently reduce the urinary excretion of pbg and shortening the duration of the acute attack. objectives: pigs has been reported to present with a higher pulmonary arterial pressure (ppa) and stronger pulmonary vascular reactivity than many other species, including man. aim of the present study was to compare pulmonary vascular impedance (pvz) before and after embolisation in weight-matched adult dogs and minipigs. methods: we investigated pvz spectra in 6 anaesthetized and ventilated (fio2 0.4) minipigs and 6 dogs. after baseline measurements the animals were embolised with autologous blood clots to reach a ppa above 35 mmhg. results: flow (03 and ppa matched pvz data (mean-+sem) are shown in the table. [zo = 0 hz impedance (z; {dyn.sec_em-5}); zl = first harmonic z; zc = characteristic z; z1 phase = first harmonic phase a@e {radians}; fmin = frequency of pvz the first m{n~mam; *, f p at least < 0.05 between dog and minipig, and before v~. after embolisation respectively]. before case report: a 53-yr-o]d woman affected by legs recurrent thmmbophlebitis, was admired in medmine department for tach.~pnea, chest pain, tachycardia and cyanosis. before starting two-dimensional transesophageal echocardiography (tee) to confirm the suspicion of pulmonary embolism, she suddenly had ventricular fibrillation. resuscitation and defibrillation were readily performed. when sinus rhythm was reinstituted she was in superficial coma with preserved corneal and light reflexes: right hemiplegia, poor perfusion and h~posphygrma of the left arm. tee showed dilation of rigth ventricle (rv), incomplete occlusion of pulmonary arter~ (pal at it~ hifurcation, severe tigth-to-left shunt through a patent foramen ovate, paradoxical embolism with incomplete occlusion of left subclavian artery mechanically ventilated with vt=800 ml, rr=12/mm, fio2=l, the patient had ph=7.28, pao2=57 mmhg and paco2=45.1 systemic bp was 130/80 mmhg and hr=80 b/min with low dose epinephrine (0.12 g/kg/min) a thrombolytic infusion (rtpa: 100mg/2h) through a peripheral vein was started tee imaging and clinical status 3 hours later were unmodified. a new rtpa infusion was performed through the pulmonary hole of a swan-ganz catheter with the tip close to the embolus. one hour later pa pressure decreased from 46/30 mmhg to 36/25 mmhg, etco2 increased from 26 to 30 mmhg and sao2 improved from 89% to 96% three days later the parietal, spontaneously breathing and with normalized tee scans of rv and pa, was transferred to rehabilitation service to perform physical therapy. conclusions: massive pulmonary embolism in a patient with patent foremen ovale, paradoxical embolism and refractory hypoxaemia was unaffected by systemic rtpa infusion, while intrapulmonary rtpa administration dramatically improved gas-exchange, hemodinamics and the general conditions of the patient. the presence of a large rigth-to-left _atrial shunt and the rapid rtpa metabolism could likely explain the effectiveness of its intrapulmonary administration in front of failure of systemic thrombolysis. introduction. cardiogenic shock during massive pulmonary embolism (blpe) is due to an acute increase of right ventricle (rv) afterload and possibly rv ischemia causing a failure of rv pump function. the rec~;mmended therapeutic strategies are: xoiume augmentation ~n ~rder m }ncrease rv pre-h~ad, adrenergic drugs to increase t'ontractillly and maybe coronary perfusion, fibrinolytic drugs to delermine clot lysis. there have been several reports of noradrenaline (na) as a useful drug in this setting for its sluing ~z, but also 1~, properties. case report.an obese 75 },ears old woman was transferred to our icu for tetanus. she was given the usual antibiotic and immunoglobuline therapy. l'wo thoracic epidural catheters were put in place at different levels and replenished with marcaine qid. a continous infusion of sedation (diazepam § was started together with mechanical ventilation. curarization ~,as given occasionally. fraxiparine 0.3/die was used for prophylaxis of thrombotic disease, on day 8th at 11.00 a.m. she started to be hypoxic (sa02 90%), tach3,tardic l1 l(i b/rain.), her blood pressure(rp) dropped frum norma~ values to 6r mm/hg, the central venous pressure (cvp) raised [rom lb to 27 mm/hg and the end tidal co2 was 7mm/hg lower than one hour before. the physical examination of the chest revealed a clear bilateral ventilation and the chest x-ray was normal apart from an elevation of the :tiaphragm as compared to the previous. an e.c.g. showed sinus tachycardia, right bundle branch block and a possible inferior necrosis (which was already present on admission). a trans-thoracic echozardiography was performed which showed "an acute overload of the right centricle wilh remarkable dilatation. tricuspidal regurgitation ++. paradoxical movement of septum. small left ventricle with normal wall kinetics". the cardiac enzymes were later shown to be normal. an acute massive pulmonary embolization was assumed m be present.. a bolus of streptokinase 750 x i(i 3 u. was given fonowed by a continous infusion . two liters of colloids were also given in a sh~rt time, two hours later the patient was still deeply hypotensive, hypoxemic and anurir(bp 54/32 mm/hg, cvs 23 mm/hg, spo2 90%) despite a cominnus infusion of dobutamine 20 fag/kg/min and adrenaline 0.5 ~tg/kg/min. at this stage a bolus of aoradrenaline 20 ,g was given followed by a cnntinous infusion of 0.05 !*g/kg/min. an immediate improvement of the hemodynamics was noticed and one hour later the bp was 149/77 mmhg, the cvp 24 mm/hg, the sao2 100% and a brisk diuresis started. the hemodynamics kept stable and weaning from vasoactive drugs was achieved within two days. one month iater the patient was discharged home in good conditions.. con c i u sio n.ne administration may help to restore rv coronary flow and ;~ump function during mpe. aeute putmonary t~omboembo~sm [ffe) cou16 be mamfeslated with either respiratory or cardiovascular syndromes or both. the arm of the study was to establish leading respn'atory symptoms, frequency and form of the roendganographic (rig) changes as well as blood gas disturbance degree in acute pte with dommam respiratory disease appearance. the study includes retrospeotive analysis of i 14 pte patients (pts), 63 males (average age 47,7 yrs) and .q females (average age 53,2 yrs). they were admitted at university, olinie" with suspection ofpleuropnlmonary disease, including pte. final diagnosis of pte was based o~ evident risk factors in 94,7% of the eases (deep venous thrombosis, surgery, trauma, imobilisation, malignancy ere), acceptable clinical, rtg, sdntigraphic and laboratory findings, as well as deep veins examination by dopple~-sonographie and radioisotopic -~enogmphy. respiratory symptoms appeared in all cases: sudden pleural pain (79%), dyspnea (64%), hemoptysis (49%), cough (39%) with association of two or more symptoms in 93%. chest xrays findings were abnormal in 92% with diaphragmal elevation (74,2~ lung opaeilies (69,5%), atelectasis (48,5%), plemal effusion (35,2%), main pulmonary brancah asimetry (22,8~ oligemia (19%), heart shadow changes (10,4%) and pulmonary arteries "cut off' (6,6%). the association of two or more abnormalities was found in 92,1% while normal chest x-rot was found in 8~ of the cases. hypoxemia with pao2<10,4 kpa was found in 64,4% followed with hypocapnia and respiratory alealosis in 34,6% in 27,8% of the gas exchage analysis were within normal limits. among cardiovascular symptoms short syn~cpa appeared in i0,5%, ecg changes-st q3t3 type in "~1,6%. results show high frequency of positive ~g findings in pte pts that is opposite to oppinion that chest x-ray in acute fie is the most ofran normal. leading symptoms are pleural pain and dyspnea, while hemoptysis were found in a half of the study group. blood gas changes were present in two thirds of the cases. kakkar, in his classic work ,clearly demonstrated the efficiency of low doses of heparin in prevention of deep vein thrombosis (lancet 2:669,1971) .after this first study the application of heparin prophylaxis became more and more diffused until to be considered a routine in many surgical departement.actually application of blood saving technique induces postoperative hemodilution effect. in that condition prophylaxis routinely applied seems a nonsense and can be at risk for postoperative hemorrhage. methods: to analize this problem we compared 100 patients arrived in our intensive care unit (i.c.u.) in.1980: (group a) with 100 arrived in 1994: (group b) .every patient was operated for major abdominal surgery.in each one we considered the hemoglobin (hb) value,hematocrit(hct), and coagulation pattern (c.p.) at the arrive in i.c.u. and 24 hours later. the patients was also divided in those receiving heparin prophylaxis (i) from not treated patients (ii) results:the application of blood saving technique clearly appears from the hb and hct level wich have a mean value of 11,4 +/-1,8 (hb) and 34 +/-2 (hct) in group a while in group b mean value are 9,7 4-/-1,2 (hb) and 29 +/-2(hct).patients of group a (ii) are the only one where a pathologycal c.p. with statistical significance has been demonstrated.in this goup we got four cases of evidence of venous thrombosis and one of pulmonary embolism.in patients of group b(i) we encontered the incidence of two cases of severe hemorrhage despite the absence of statistical significance in c.p.modifications. oxygen desaturation during broncho-alveolar lavage: role of oxygen saturation monitoring in prevention of acute respiratory insufficiency g. galluccio, b. valeri, s.batzella, m. di lazzaro*, servizio di endoscopia toracica, ospedale forlanini, rome, italy * servizio die anestesia a rianimazione, osp. forlanini the broncho-alveolar iavage is a diagnostic procedure employed in interstitial diseases of the lung. it requests the introduction through the working channel of a fiberoptic bronchoscope, after occlusion of a segmentary bronchus, of aliquots of saline solution at 37 c, subsequently gently reaspired, in order to remove cells and proteins from elf (endoalveolar lining fluid), which is related to interstitial medium. bronchoalveolar lavage induces deep effects on pulmonary function: -lowering of the alveolar surface of exchange; -shunt effect, depending on the perfusion of non-ventilated districts; -increased pulmonary arterial pressure, due to hypoxic vasoconstriction; -decrease of lung compliance. in this report the authors present the result of oxygen saturation monitoring in a group of patients with interstitial lung disease, who underwent diagnostic broncho-alveolar lavage. in most patients with severe interstitial involvement, the lavage performed without supplement of oxygen induced a severe fall in the oxygen saturation during the late phase of the procedure. if supplementary oxygen was delivered during bronchoscopy, since its beginning, only slight modifications of the curve were detected. in patients without thickening of interstitium, in whom the lavage was performed in order to obtain material for bacterial or cytologic examination, no modification of oxygen saturation was observed in standard procedure. as conclusion the authors strongly reccomend monitoring oxygen saturation in patients with radiologic evidence of interstitial involvement also in patients with no evidence of dyspnoea. g. galluccio, b.valeri, s.batzella, m. di lazzaro*, servizio di endoscopia toracica, ospedale forlanini, rome, italy * servizio die anestesia a rianimazione, osp. forlanini the treatment of choice in patients with alveolar proteinosis consists of pulmonary lavage. this procedure requests the introduction, through the working channel of a fiberoptic bronchoscope, segment by segment, of aliquots of saline solution at 37 c, subsequently gently reaspired, in order to remove the proteins deposited in the alveolar spaces. the method is very similar to that used in bronchoalveolar iavage, a diagnostic procedure used to obtain cells and substances from elf (endoalveolar lining fluid), which is related to interstitial medium. as known, bronchoalveolar lavage induces oxygen desaturation, because of shunt effect. understandably, one lung lavage has remarkably more deep effects on pulmonary function than bronchoalveolar lavage, for the amount of fluid introduced, the length of the procedure and the conditions of controlaterai lung. in this report the authors present the result of oxygen saturation monitoring in a patient who underwent pulmonary lavage for alveolar proteinosis. in the lavage performed without supplement of oxygen a severe fall in the oxygen saturation was observed during the late phase of the procedure. if supplementary oxygen was delivered during bronchoscopy, since its beginning, only slight modifications of the curve were detected. as conclusion the authors strongly reccomend the subministration of supplementary oxygen in pulmonary lavages, also in patients with excellent respiratory conditions. a. b. dublisky prof., m. r. isaakjan ass., v. a. zasukha, s. m. vinichuk prof., v. p. tserty ass. prof., chair of anaesthesiology, resuccitation and medicine of catastrophes, neurology of ukrainian state medical university, kiev, ukraine. objectives: detection of plasmophoresis's influence of results in treatment of ishemic insult. methods: we ve investigate 25 patients with ishemic insult, treated with reverse plasmopheresis in complex treatment. after primary infusive therapy we took 400 ml of patients' blood and separated it within 15 min with rotation frequensy of 2000/rain. after separation of erythrocytes from plasma, the latter has been returned to patients. we made 3-4 procedures during 3-4 days. hemoglobin, hematokrit, time of blood coagulation were determinated. the brain blood flow in internal carotid arteries, regional volum brain blood flow and total brain biood flow were evaluated with tetrapotar chest rheography and tetrapolar rheoencephalography. obtained date were comparised with control group after traditional treatment. results: it was found that after reverse plasmopheresis the hemoglobin and hematokrit levels decreased significantly in studied patients' plasma (from 140 + 3.2 g/l to 120 _+ 2.3 g/1 and from 44 + 2.1% to 35 _+ 1.8 % respectively). the time of blood coagulation by lee-white has increased by 2-2.5 times (up to 10-12 rain). the level of brain blood flow has been increased significantly after reverse plasmopheresis in comparison with control group. the following tests of brain blood flow have been increased: a) the total volume brain blood flow from 480.7 + 34.6 ml/min to 625.4 _+ 35.4 ml/min (p < 0.05); b) the regional brain blood flow from 52.2 _+ 2.8 ml/min to 87.1 + 6.2 ml/min (p < 0.01); c) the brain blood flow in internal carotid arteries from 166.1 _+ 12.2 ml/min to 206.3 + 14.6 ml/min (p < 0.05). conclusions: the use of reverse plasmopheresis in complex treatment of patients with ishemic insult aiiows to improve rheological blood patterns, helps to increase volume brain blood flow. it results in quicer reparation of neurological functions. objectives: a prospective evaluation of the efficacy of continuous infusion of verapamil in reducing the incidence of postoperative atrial fibrillation after pulmonary surgery. methods: a total of 199 consecutive patients, 100 on verapamil, 99 on placebo was included after lobectomy or pneumouectomy. a loading bolus of verapamil (10 mg over 2 minutes) was followed by a rapid loading infusion (0.375 mg/min) for 30 minutes and finally a maintenance infusion (0.125 rag/rain) for 72 hours. results: a mean plasma level of verapamil of 150 ng/ml was obtained only after more than 24 hours. atrial fibrillation occurred in five out of 78 patients who tolerated the verapamil infusion, and in 15 out of 99 patients on placebo (p = 0.08). verapamil infusion was not tolerated in 22 patients because of hypotension or a heart rate of less than 50/min, within 6 hours of the start of the therapy. when atrial fibrillation occurred, the ventricular response, mean _+ sd, was not significantly slower during verapamil infusion (132 + 22) compared to placebo (147 + 20). conclusions: because of its frequent side effects and the only modest efficacy verapamil should not be considered for prophylactic therapy of atrial fibrillation after pulmonary surgery, and is probably not a good first choice for slowing the heart rate in case of rapid ventricular response once atrial fibrillation has occurred in these patients. results: study of haemostasis in these patients has showed deep disturbances of blood coagulation. fibrogen level has reduced to 0.62 + 0.03 g/l, fibrinogen and/or fibrine degradation products concentration have enhanced to 0.40 _+ 0.04 g/l, monofibrin soluble complex concentration to 0.08-+ 0.04 g/l, blood plasmin level was enhanced to 34.0 + 0.2 mmol/1, plasminogen proactivator level was also enhanced to 153.0 + 0.60 ram, plateletes aggregation has decreased to 52 %. after plasmopheresis aggregation was decreased in 1.6 times. it has been connected with decrease of fibrin and/or fibrinogen degradation products level and level plasmin in 1.7 times, and plasminogtnt activator level in 4.6 times. at the same time we have observed increase in total antifibrinalitic activity of blood in 1.3 times. activity of activators plasmine and plasminogene proactivators has decreased in 1.2 times and in the same time activity of activation inhibitors and antiplasmines has increased in 7 times. conclusions: plasmapheresis leads to considerable improvement of a general condition and reduction of the haemorrhagic syndrom's sings (controlling of gastrointestinal haemorrage, reduction of intensity of subcutaneons haematoma). evaluation of continuous cardiac output (cc0) monitoring based on thermodilution technique in 35 critically ill patients. methods: cardiac output (co) was monitored continuously using a modified pulmonary artery (pa) catheter, on which a heating filament is located and by which energy is transmitted to the circulating blood. a microprocessor calculated co by a new algorithm. standard bolus thermodilution technique (10ml of ice-cold saline solution) was used to compare cc0 with intermittent bolus cardiac output (ic0) measurements. the following subgroups were prospectively studied: i. heart rate (hr) >120 beats/min, 2. cardiac output >10 i/min 3. cardiac output <4.5 i/min, 4. rectal temperature >39.0~ and 5. pa catheter was inserted for more than 4 days. results: a total of 404 pairs of ic0 and cc0 measurements were obtained from the 35 patients. bias (ico measurement minus cc0 measurement) of all measurements were 0.03• i/min and the 95% confidence limits (mean difference• were -1.01/1.06 i/min. also in the subgroups, cc0 measurement agreed closely with ico measurement (c0 >10 i/min: bias=0.16• i/min; co <4.5 i/min: bias=-0.17• i/mln). elevated temperature and prolonged lay-days of the pa catheter did influence agreement of cc0 measurement with ic0 measurement neither (>39~ bias=0.09• i/min). conclusions: monitoring of cc0 using a modified pulmonary artery catheter with a heated filament has proven to be accurate and precise also in the critically ill when compared with "standard" intermittent bolus thermodilution technique. this method enhances our armamentarium for more intensive monitoring of these patients under various circumstances. background: the number of patients who need coronary artery surgery was) grows every year. most of these surgical operations are with extrar eircuiation (ecc). since january 1994, this surgery is made without ecc in selected patients in our hospital. this technique is exceptional in spain. this type of surgery has proved useful in patients requiring revascularization of the left anterior descending, eireunflex or right coronary artery (not for grafting the pos~tefio~r descending branch}. blethods and results: since 1994, 30 patients aged 54 to 77 years (mean 66 years) underwent cas without ecc. the mortality in programmed surgery was 0%. no patient was reexplored for hemorrhage. the mean values of some clinics parameters v~ere: a) blood requeriments: 2 units per patient, b) need of mechanical ~entilation: i3,6 hours, c) postoperative bleeding: 900 cc, d) days at icui 2,5. we used the student % t test or fisber~s exact test to compare these results with the mean values of surgery with ecc: a) blood requeriments 4 per patient (p<0,0001), b) need of mechanical ventilation: 29 hours (p<0,0001), c) postoperative bleeding: 1300 cc (p<0,004), d) days at icu: 4 (p<0,001), e) programmed surgery mortality: 7% (p<0,05). conclusion: our limited experience shows that this surgery is an alternative in the treatment of coronary disease, especially for aged patients with associated pathology and in jehova's witness. the need of mechanical ventilation, days at icu, blood requeriments and morbi-mortality were fewer than surgery with ecc. to study the hemodynamic and antiarrhythmic influence of ace-inhibitor enalapril in acute myocardial infarction (mi). methods: holter ecg monitoring, heart rate variability analysis, echocardiography (3 and l0 days after beginning of the treatment), stress-echocardiography and stress ecg (8-10-th day after the onset of mi). enalapril was included into the treatment of 42 pts with mi (study group), with normal or increased blood pressure, from the 1-st day of the disease. the data were compared with 30 pts treated without enalapril (control group). results: silent ischemia during stress-test was registered in 6 pts of the study group and 8 of control group, the arrhythmia episodes during stress test -in 5 and 8 pts and episodes of silent nocturnal isehemia -in 7 and 12 pts correspondingly. enalapril importantly attenuated the hypertensi~re re~aetioh %0 stress test. in 10 pts of the study group the number of perifocal hypokinesis zones decreased; in the control group it didn't change. the quantity of ventricular extrasystoles in the patients of the study group decreased by 25%; the heart rate variability indices improved as well; in the control group the character of ventrieulir arrhythmias, heart rate and its va]~i~bili%y didn't change significantly. conclusions: the inclusion of enalapril into the treatment of mi is a useful t0ol to improve hemodynamie parameters and decrease the incidence of ventricular arrhythmias. objectives: to study left ventricular (lv) systolic function in the patients with acute myocardial infarction (ami) before and after peroral captopril test. methods: the original echocardiographic parameter of lv contractility, "coefficient of effective systolic function" (cesf), was proposed in the study. cesf is calculated from lv stroke volume (sv), obtained from doppler aortic flow in lv outflow tract and lv end-diastolic diameter (edd): cesf =sv/edd. the study included 60 patients with ami, who had local lv dyskinesia and global lv systolic dysfunction (ef<45%). besides cesf, the ejection fraction was calculated before and after administration of 25 mg eaptopril (on the fifth day of ami) by methods of bullet and simpson. results: the dynamics of these parameters, as well as heart rate (hr) and mean blood pressure (bp), is shown in the tabte. before cal~topril ef (bullet) 32.12 • 2.51 ef (simpson) 35 . introduction: the cold system is a monitoring system for measurement of right (copa) and left (coart) ventricular cardiac output, cardiac function index (cfi), fight ventricular ejection fraction crvef), fight ventricular cnddiastolic volume (rvedv), intrathoracic blood volume (!tbv), global enddiastolic volume (gedv), lung water (etv) and excretory liver function (pdr). patients and methods: 41 pts have been monitored by the cold system. above mentioned parameters are measured by thermal dye dilution and a fiheroptic femoral artery catheter. copa, rvef and rvedv measurements additionally were compared to measurements by the baxter explorer. :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ;;;k;;;;i cov (%) explorer ! 6 13 ! 1 [ gedv, itbv and pdr showed a significant decrease dufing the first 12-24h after the operation, cfi and rvef si~canfly improved after 48k wheras etv showed a i~ in the early postoperative phase and fell to normal ranges at 48h. comparison of cold/explorer m~ements sb0wed good correlations. discussion: concerning m0~toring of ri,ght ventric~ar function cold and explorer can he seen as equal. rvef gives an ar report about the performance of the right ventricle without use o f echocardiography. measuring itbv and gedv ~ improve ~gement and con~ol of th.e volume status, monitoring etv helps preventing lung edema. pdr shows good corre|ati0n to liver blood chemistry and is bedside avai|ab|e. thus the cold system offers additional parameters for comprehensive m~nitofing of pts. ~e~ ~c surgery. obiectives: to evaluate the influence of an a!'~ered cardiac function on the cardiovascular response to the increase in oxygen demand induced by an increase in core temperature. methods: this preliminary study included 12 adult critica!ly ill patients monitored by arterial and pulmonary artery catheters in whom thermodilution cardiac index {ci) and arteria! and mixed-vef)ous blood gases measurements could be obtained before and after an acute change in core temperature of at least 0.5~ (max 60 rain apartl the patients were separated in two groups according to their cardiac function: 4 patients had an impaired cardiac function as defined by a history of cardiac disease and an ejection fraction below 45% and 8 patients had normal cardiac function. results: individual data are shown in the figure. in contrast to the control group (continuous line) in which c! increased without changes in oxygen extraction (02er), the q2er in patients with impaired cardiac function (dottled line) increased without changes in ci. conclusions: the increase in oxygen demand associated with changes in temperature is met by an increase in c! in patients with unaltered cardiac function and in an increase in o2er in patients with altered cardiac function. temperature should be taken into account in the assessment of the adequacy of cardiac output in patients with impaired cardiac function. objectives: to define the hemedynamic and metabolic response to physical therapy(pt) in relation to the type/level of sedation and the cardiac status in icu patients. methods: we studied 34 mechanically ventilated icu patients (64• years) in stable hemodynamic status (no change in vasoactive treatment for at least 2 hours), separated in 4 groups: group 1 = deep sedation, cardiac dysfunction required dobutamine (n=5)r group 2= deep sedation (barbiturates), unaltered cardiac function (h=lo), group 3= moderate sedation, altered cardiac function (h=7) and group 4= moderate sedation, unaltered cardiac function (n=12). complete hemodynamic data, arterial and mixed venous blood gases, respiratory gas analysis (metabolic cart ccm, medgraphics) were obtained at baseline (2x) and twice (q.10 min) during leg mobilization. data were analyzed by anova. calcium channel blockers were used in complex preoperative preparation of 59 hypertensive surgical patients. patients were allotted to 3 groups based on their hemodynamic profile: hypokinetic: ejection fraction (ef)<0.6, 29 patients; eukinetic (ef>0.5),i6 patients and hyperkinetic (ef>0.6),i4 patients. the most noticable change in hemodynamics was in the hypokinetic group: ef and cardiac output (co) were significantly decreased (p<0.001) while systolic arterial pressure (sap) (p<0.05) and peripheral resistance (pr) (p<0.01) were elevated. the results showed that in hypokinetic patients on nifedipine ef (p<0.00t) stroke volume (sv) (p<0.0l) and co (p<0.001) were increased while pr(p<0.0t), sap(p<0.001) and diastolic arterial pressure(p<0.05) were decreased. eukinetic type patients also showed an increase in ef,albiet to a lesser extent,than in the hypokinetic group. increased sv and co(p<0.01) were observed in eukinetic patients though this was to a lesser extent than in the hyperkinetic group. in the hyperkinetic group of patients nifedipine had no effect on the aforementioned parameters except for a decrease in sap(p<0.0 i). nifedipine increased ef in all hypokinetic patients. comparative results show that isoptin was less effective than nifedipine in decreasing peripl~eral vascular resistance and had a depressive effect on the myocardium. it can be concluded that the action of calcium channel blockers normalizing the circulation in the hypertensive surgical patient depends on: the condition of myocardium, the patients hemodynamic profile and their pharmacological properties. they were most effective in the hypokinetic group. zalo/nthinos e., daniil z. zakynthinos s., armaganidis a., kotanidou a., nikolaou ch..,roussos ch. critical care department, university of.athens, evangelismos hospital, athens, greece. introduction : surgical is the optimal treatrnent for ioculated effusions and the preferable procedure when multiple bands are seen in the pericardial sac by echo. patients : 6 palients, 4 post cardiac surgery, 2 uremic (3men, 3 women) with large pericardial effusion and clinical or echocardiographic findings of tamponade or both. these particular patients displayed numerous linear echo-dense bands and s~'ands crossing the pericardial space (in one of them a ioculated effusion compressed the left ventricule). one had aptt increased, four were mechanically ventilated. technklue : a 8fr polyurethane catheter with end and multiple side holes over 18 ga needle was echo-guided to the ideal site (fluid abundant and closest to the transducer). the catheter was attached to a close system with a heimlich valve for continuous drainage (pneumothorax kit). subcostal entry was selected in one patient and chest wall in five. the patient's position was changed every hour at least. (we believe that the small changes in the position of the catheter and the mechanical breaking of the bands in relation with the movement of the heart assist the pericardial fluid to remove). results : in all cases only a small quantity of fluid was withdrawn in the first minutes(30-70ml) with some clinical and echo-findings improvement. the fluid was bloody or serosanuginous with high protein content (ht=15% ,protein 5,1gr/dl) in all cases. in first 24 hours the mean volume of fluid removed was 550ml (350to 720ml). in that period echo showed no residual fluid. the catheter remained within the pericardium 1 to 3 days .. no complications are mentioned. conclusion : cardiac tamponade due to hemorrhagic high protein pericardial effusion in uremic and postcardiac surgery patients,, as it is revealed by echo dense bands, can be faced by 2-d echo guided perieardiocentesis. a 8-fr polyurethane catheter with multiple side holes, attached to a heimlich valve was effective to evacuate the pericardial fluid. no catheter was occluded though heparin infusions were not used. multiple changes of the patient's position may be fundamental. this 2-d echo guided pericardiocentesis performed in in~nsive care unit seems to be useful , safe and quick technique. determining the best inotropic drug represents a very serious problems. the use of more selective and potential inotropic and vasodilatative drugs does not always lead to improvement of hemodynamic parameters in patients with low cardiac output syndrome. this paper presents patients with acbp who need an inotropie support after extracorporeal circulation in first 24 hours. the patients were divided into dobutamin et dopamine groups. the heart rate (hr). mean sistemic arterial pressure [map), central venous pressure (cvp). and termodilution cardiac index (ci) were measured. the measurements were without using inotropic drugs, and then using them after 5 rain, 30 min, and finally with one hour rate, within first 24 hours. the statistical analysis shows that both drugs lead to an increase in hr in the first hour of the application. the final effect of dobutamine is no change in hr, whereas the effect of dopanime is very significant increase in hr. thus. an absence of taehyeardie response selects the dobutamine as a better choice. backeround: pulmonary vascular eadothelium possesses major metabolic functions, which when altered contribute to the development of serious pathologies such as ards. one such function is the conversion of angiotensin i to angiotensin ii, catalyzed by angiotensin converting enzyme (ace), located on the luminal surface of the endothelial cells. ace activity has been extensively studied in animals in vivo, by means of indicator-dilution techniques, providing: i) under toxic conditions, an early index of lung injury, and it) under normal conditions, estimations of dynamically perfused capillary surface area (pcsa). objectives: to validate the use of these techniques in matt: i) for pulmonary endothelial function assessment, and it) for pcsa estimation. methods: ace activity was estimated in ten adult haman volunteers, with no pulmonary medical history and normal pulmonary artery pressures, undergoing cardiac catheterization for coronary artery disease assessment. single-pass traspulmonary hydrolysis of the specific ace substrate 3hbenzoyl-phe-ala-pro (bpap; 30p.ci) was measured by means of indicatordilution techniques, and expressed as %metabolism (%m) and v=-hi(1-m). bpap was injected as a bolus i) into a main pulmonary artery, and it) inside the right atrium, to assess ace activity in one and both lungs. we also calculated a,~,/i~, an index of pcsa. pulmonary plasma flow (fv) was determined by thermodilution. fp in one lung was estimated as 0.5xf v. results: similar values of %m (69.6+3.8 vs 68.9• and v (1.29• vs 1.25• were observed in both and one lung respectively. a~k~ decreased from 4185• ml/min (both ltmgs) to 2122:~175 (one lung). conclusions: i) pulmonary endothelial ace activity and thus pulmonary endothelial function may be assessed in humans by means of indicator-dilution techniques, it) our data denote homogeneous pulmonary capillary ace coneentratious and capillary transit times in both haman lungs, iii) the 50% reduction of a=~/k~ in one lung suggests that this procedure can be used to quantify pcsa in man. (supported by the fonds de la recherche en saute du quebec and the national health system of greece). objective: verify whether antioxidant activity is higher in reperfused than in no-reflow myocardium after i.v. thrombolysis for acute myocardial infarction (ami). methods: 37 patients with ami were included. blood for estimation of catalase (cat), glutathione peroxidase (gpx) and mn-superoxide dismutase (sod) was drawn before initiation of i. the mechanism of myocardial cell defence against free radicals is probably identical in both reperfusion and no-reflow phenomena. therefore, antioxidants cannot be used as reperfusion markers. objectives_ to evaluate the precipitating factors of hypothermic phrenic nerve injury following cabg with lima. methods: fifty two consecutive patients (8 females), with a mean age of 59+8 (mean +sd) years were studied. during the ischemic arrest time topical hypothermia was obtained in al~ patients wffh ice slush and no cardiac insulation pad was used. all patients received a lima graft, with or whithout additional vein grafts. supramaximai, bilateral phrenic nerve stimulation was performed percutaneously preoperatively and whithin 24 hours postoperatively. square wave stimuli of 0.1 msec duration were applied at the posterior border of the sternomastoid muscle. the compound muscle action potential of the diaphragm was recorded, using surface electrodes on the anterior chest wall. the time interval from the application of stimulus to the onset of diaphragmatic activity, phrenic nerve conduction time (pnct), was measured. values exceeding 9.75 msec were considered as abnormal. besults: preoperatively, all patients had normal (mean+sd) pnct, 7.69• msec for the left nerve and 7.98• mseo for the right nerve. on the first postoperative day, right pnct was normal in atl patients (7.93• msec) , whereas left pnct was normal in 45 patients (7.86• msec) and abnormal in 7 patients (incidence 13.5%). in 6 patients the left phrenic nerve was inexcitable and in 1 patient left pnct was prolonged (10.50 msec). comparing patients with normal and abnormal pnct there was no difference in age, gender, number of grafts used, aortic cross-clamp and bypass time. however, patients with abnormal pnct had a lower preoperative ejection fraction (45• vs 53• p=0.03). moreover, in all of them lima was dissected from its origin ligating all upper arterial branches, which provide the blood supply to the left phrenic nerve, whereas in those with normal pnct the small vessels originating from the upper 2 to 3 cm of lima were preserved (p=0.001). conclusiojel~ a hypoperfused left phrenic nerve seems to be more susceptible to hypothermic injury during cabg with a lima conduit. objectives: to test if necessary interventions on systemic vascular resistance (svr) along with preset pump flew (q) during cpb could adversely affect autoregulatory response and cause vo 2 shifts. methods: we studied 26 males (554-7 yrs) who underwent cpb for cardiac surgery. at o oesophageal temperature 27-28 c we set pump flow at 2.1 i.m~2.min -1. when map was higher than 85 mmhg we calculated vo 2 by using fick equation. then we infused sodium nitropruaside (sn) to control map at 55-65 mmhg for 10 min and we calculated vq 2. without changing the sn infusion rate we set q at 2.5 i.m'2.min "1. ten min later we measured vo 2. we took vo 2 changes into consideration if greater than 15%. statistical analysis using students-t-test for paired data and analysis of variance was used as appropriate. results: depending on the biphasic vo 2 response to sn infusion during low and high q we classified pts in four groups (table). i. vo 2 increases with sn and increases further during high q unmasking hypoperfusion and supply dependency. ii. vo 2 increases with sn but the addition of high q results in systemic shunt. iii. vo 2 increase during high q proves that vasodilatation can turn flow insufficient. iv. vo 2 does not change with any intervention. the small number of pts and the wide standard deviation did not allow any statistical significance. conclusions: cpb is an interesting model for the behavior of microcirculation. intervention on svr and q can improve or impair effective regional oxygen delivery, resulting in either better perfusion or systemic shunt. vo 2 monitoring seems necessary during cpb. preoperative cardiovascular optimization (opt) to ci > 2.8 l/min/m 2, 8 _< paop < 18 mm hg,and svri __< 30 mmhg/ll/min/m 2 decreases cardiac events (events) and mortality (mort) in peripheral vascular surgery patients (pvs). objectives: to determine if opt to the same endpeints decreases events in patients undergoing abdominal aortic aneurysm repair (aaar) and to study the r predictive value in pvs patients. methods: 44 aaar patients and 41 pvs patients were admitted to the s cu monitored with e pa and arterial catheters and treated to achieve opt. patients underwent surgery independent of success of opt data included demograph cs, incremental risk factors, laboratory and hemodynamic data pre, intra, a~nd postoperatively events, and mort. events included arrhythmias requiring treatment or prolonging the sicu stay > 24 hours, a st depression > !mm or t wave inversion, an acute mr defined by a new q wave > 0.03 sec or cpk-mb > 5%. results are presented as means _4-. sd. opt was achieved in 32 of 41 (78%) and in 36 of 44 (82%) in the pvs and aaar group, respectively. events did nat differ between groups 9 of 41 (21,9%) and 12 of 44 (27,2%) in the pvs and aaar group, respectively (p>o.05). mort was 0 of 41 (0%) and 1 of 44 (2.2%) in the pvs and aaar group, respectively (p > 0.05), while there was no difference in endpoints of opt between patients with and with.out events in the aaar group, there was a significant difference in ci between patients with and without events in the pvs group. of note, 8 of 9 (89%) patients who developed events in the pvs group had a ci < 2.8 in contrast to 4 of 12 (33%)in the aaar group. the positive and negative predictive value were 89% and 97% in the pvs and 50% and 75% in the aaar group. conciusione: f. the endpoints of opt used for pvs patients cannot be ~sed to reduce events in aaar patients; 2. pvs patients who have net achieved opt are at extraordinary risk of perioperative events; 3. preoperative card ovascu ar opt in aaar patients makes no difference in cardiac related events, background : comparison of the right and left filling pressures (cvp/pcwp ratio) is considered as a useful diagnostic clue : the normal ratio is _< 0.6; ratio >_ 0.8 may suggest right ventricul~ infarction while equalization of the cvp and pewp is a classic sign of tamponade (1). however after cardiac surgery, many conditions (diastolic dysfunction, pulmonary hypertension, positive pressure ventilation) are susceptible to modify the '*normal" cvp/pcwp ratio. material and method : we determined cvp/pewp ratio in 100 consecutive patients (pts) after uncomplicated cardiac surgery (78 coronary artery bypass grafts; 22 valvular replacements) measurements were made before and after tracheal axtubation. results :cardiac index : 3.2 _+ 0.7 1/minlm~; laotate: 144 + 68 rag/i; cvp range : 4-17 rnmhg; pewp range : 2-16 mmhg. mean cvp/pcwp ratio before extubation is 0.94 (95 % confidence imerval : 0.86-1.02) and after extubation, 0.93 (95 % confidence interval : 0.83 -.1.03), (ns, paired t-test). in 25 % of the pts, cvp was higher than pewp. there are no correlation between the cvp/pcwp ratio and c! before (r = -0.04) and after extubation (r = -0.09) nor between the cvp/pcwp ratio and mean pulmonary arterial pressure (mpap), before (r = 0.08) and after extubation (r = -0.13), discussion : cardiac performance is adequate according to ci and lactate. however the cvp/pcwp ratio is markedly higher than the "normal" (_< 0.6) ratio. this difference is not related to mechanical ventilation because the ratio is similar before and after extubation, nor to pulmonary hypetaension because of absence of any correlation with mpap, post-cpb diastolic dysfunction of the right ventricle could be an alternative explanation. in this group of pts, increased cvp/pewp is not associated with any impairment of cardiac performance (absence of correlation with ci), conclusions : cvp/pcwp ratio as high as 1 within a large range of cvp (4-17 mmhg) and pcwp (2-16 mmhg) may still be considered as normal after cardiac surgery. this emphasizes the limitations of the hemodynamic monitoring after cardiac surgery (in comparison with echographic technics). careful analysis of the morphology of the cvp and right ventricular pressure curves (x descent, y descent, dip-plateau) is mandatory rather than relying on the quantitative assessment alone. reference : (1) ntensive care.-university hospital -m~laga (spaink introduction. fibrinolitic treatment (ft) permits the treatment of acute myocardial infarction (ami) addressing the etiology, thereby eading to mproved ventncular function and a marked reduction m mortality. the main clinical oroblem is the reduced time of application. delay in hospitalization, which can be from 50 to 120 minutes, is potentially the most avoidable delay. method. to reduce delays in hospitalization, the following was carried out in two chases. 1 audit: analysis of the time lapse from onset of symptoms to start of ft. showed that during "(he period 1 june to 31 december 1994, 79 patients with chest paros were treated within a 0eriod varying from 30 minutes to 6 hours from onset of symtoms. ages ranged from 33 to 86 (average 64,4), oelng 49 males and 30 females. they were glved initial ecgs to determine st mcreases suggesting ami. median t~me for this orocedure was l0 m.. 204 potentia ami patients were then admitted to the coronary unit, 103 [)atients, under age 75 with no contraindications received ft the median time apse from admission to corona-y care and administration of ft was 15 minutes (12. 17), -he total median delay was 58 minutes ~40-i h.45min,~ delays n start of this procedure are grouped as follows: extra-hosdita delays (from onset of symtoms to arrival at hospital) diagnostic delays (from hospital arrival to ecg). treatment delays (from diagnosis to ft). 2 objectives: protocol of procedure to implement a fast-track method. a protoco was drawn up with the object of reducing diagnostic delays to 8-i 0 minutes and treatment delays to less than i 5 minutes results. following rmplementatlon of this protocol in january 1995, 30 fts were glven, with an over all average delay of 24 minutes. this fast-track method did not reveal any inappropnate ft or any increase m complications, conclusions: detailed study of the various times taken for diagnosis ane treatment of ami patients, showed up weaknesses in the system and improvements througn the protocol based on performence orocedures which led to a 59% reduction in the start of ft background: the importance of the early use of thrombo!ytic agents in acute myocardial infarction (ami) is based in the better remaining ventrictjlar function and smaller mortality rate because of the greater reperfusion and sma!ler infarction size, therefore, it is very impodant to apply this treatment to the maximum number of patients without thrombolytic contraindicati0n, and within the minimun period of time. the "thrombolytic fast track" implementation allows to optimize the time to administrate thrombelytic agents avoiding multiple delays~ methodology: we anal!ze the application of thromboly0c agents to 73 patients with suspect of ami from the begin!ng of september 1994 until the end of february 1995. in this time there are two different periods, during the first 4 months thrombolytic agent were admin!strated at intensive care unit (icu), and during the second period we carried out a protocol of quick detection and thrombolysis therapy in susceptible patients at the emergency room in order to reduce the time to treatment. ma!n results are shown in the faffewins de ay h=hours m=minutes the implementation of the fast track does not need supplementary personal or equipment but a protocelized approach and training of the personal involved the main problem detected was the usual attendance overload of the emergency department that makes difficult to follow many structurated actions. conclusions: pratocqlized changes in the management of ami can significantly reduce the detay in the administration ef thrombolytic agents. it is not necessary to eomplet the procedure iq the emergency department, as the use of bolus schedules allows to begin the treatment in this area and to transfer the patient to icu afterwards. elective cardiac surgery. b calvet, f ryckwaert, p trinh duc, p colson. anesthesia -reanimation, hopital arnaud de villeneuve, montpellier, france. obhectives: the study was aimed at analysing the incidence of renal dysfunction following cardiac surgery and its prognosis (acute renal failure, post-operative morbidity and mortality). methods: two hundred and thirty seven patients (aged from 28 to 90) were consecutively operated on for elective cardiac surgery and retrospectively included in the study. patients with preoperative infections and operated on in emergency were excluded. each patient had preoperative invasive cardiac investigation with angiography and calculated ejection fraction (ef). anaesthesia, cardiopulmonary bypass (cpb) and cardiac arrest management were similar in all patients. general body temperature was reduced to 28 -30 ~ c. renal dysfunction was defined as a 20 % increase from baseline of serum creatinine. demographic data, asa, treatments, pre-operative creaunine level, cpb and clamping (axc) times, intra and postoperative use of inotrope, serum lactate level before surgery, at the end of cpb, at the time of admission in intensive care unit (icu) and on post operative day one and apache score were compared in patients with or without renal dysfunction using anova test for repeated mesures and x2 when appropriate. data are expressed as mean +__sd. p value less than 0.5 was considered statistically significant. results: thirtytwo patients (13,5 %) suffered from renal dysfunction. age, serum lactate level at the end of cpb, at admission in icu, at pod1 and apache level at admission in icu, intra-operative use of inotropes were statistically different in patients with or without renal dysfunction (p<0,05). mortality rate was statistically different in patients with or without renal dysfunction(~,2,5 % and 0 %, respectively, p=0,001). incidence of acute renal failure following renal dysfunction was 6,2 % (2 patients required hemodialysis). conclusions: although our cdteria for defining renal dysfunction were very sensitive, the incidence of renal dysfunction following elective cardiac surgery was lower than communly accepted in the litterature (1). however renal dysfunction appeared significantly associated with a poor prognosis. reference: 1-settergren g, ohqvist g current opinion in anaesthesiology 1994, 7:59-64 r 66; 159, 200 tzelepis, g. 112, 127, 142 late complications were observed in 14% of cannulations: local infection in 2 (i,7%), catheter displacement by the patient in 4 cases (3,5%), catheter displacement during nursing care in 5 (4,4%) and malfunction in 5 cases (4,4%). conclusions: central venous catheterizations are followed by immediate and late complications in almost the same percentage acute poisoning with amphetamines (mdea) and heroin: antagonistic effects between the two drugs methods: after institutional approval and informed consent, 33 selected patients (65_+9 years) undergoing peripheral vascular surgery (n=17) or carotid endarterectomy (n=16) were investigated. patients included had either documented cad (n=18) or two or more (n=15) dsk factors (age >65 years, smoking, diabetes meltitus, hypertension, hypercholesterolaemia >240 mg/dl). 12-lead ecg recordings were carded out preoperatively, on ardval in the postanaesthetic care unit, and 20 h, 48 h, 72 h, and 84 h postoperatively. ecg recordings were analysed by an independent blinded cardiologist for signs of pmi (new st segment depression >0.2 mv and/or new t inversion). in addition results: 22 of the patients investigated developed ecg-documented pmi, 86% occurdng in the immediate postoperative phase. troponin i levels >1.6 ng/ml were found in 19 of these 22 patients thus, comparing a cardiac troponin i cut-off level of 1 ng/ml with intermittent 12-lead ecg recordings, we found a sensitivity of 86% and a specificity of 91% methods: demographic, clinical and ecg data were analyzed. 53.8% of patients were male; 46.2% female. cad was the most common underlying cardiac disease (85.7%) and 58.4% underwent open heart surgery. 69% received proeainamide for supraventricular and 31% for ven~cular arrhythmias. 27% received a loading dose. maintenance was provided by iv route in 36.8% and by po in 63.2% (40.8%sr end 22.4% ir). 40.4% of patients were obese right ventricular function following cardiopulmonary bypass: is important the mode of myocardial protection we underwent this study in order to examine its safety and usefulness in pts with trustable coronary conditions (unstable angina ua the mean age for group a was 54 • 16 years, for group b 64 • 11 years, and for group c 59 • 13 years. a history of previous myocardial infarction was present in 6 pts of group a, in 3 of group b and in 54 of group c. three pts in group a, 5 in group b and 15 in group c had previous coronary artery bypass grafting. the median time between the onset of symptoms and a was 5 days (2 -19) for group a we used a continuous fixed intravenous a infusion at a dose of 140 the sn was 100% in groups a and b, 98% in c, and sp 100% for group a, (fixed defects included) and 50% for groups b and c. there was no difference of side effects among groups: chest pain (i pt -group a, 2 pts -group b, and 8 pts -group c), transient hypotension (1 pt -group c), headache (5 pts, group c), dyspnea (1 pt -group a), while st depression was seen in 2 pts of group b and in 2 pts in group c. the rate of a infusion was decreased to 70 7/kgr/min in one group b pt due to development of chest pain s228 five year follow up of humoral immunity in paced patients athens polyclinic hospital, department of cardiology athens, greece author index a abiad ch 133 bertschat, e 141 betbes6 75 blanch, l1 177 del nogal saez 93 e1-meneza 220 nolla, j. 98 nolla-salas 24 pilz~ u puig de la bellacasa e 38 scarpa, n. 77 217 van de wetering objectives: only 50% of patients suffering from acute guillain-barr@ syndrome (gbs) respond promptly to established therapies like plasma exchange or intravenous immunoglobulines. in contrast to serum, cerebrospinal fluid (csf) of gbs and ctdp patients contains enriched portions of antiexcitatory factors(i) and cytokines (2) able to induce pronounced conduction block (3). to reduce or remove such pathologic factors we introduced a technique with direct access to the subarachnoid space. methods: with informed consent we lumbally inserted 18 g catheters in 24 gbs-and 22 cidp -patients under sterile conditions. some of them had not responded very well to established therapies. 30-40 ml of csf were withdrawn and retransfused by a bidirectional pump (flofors) after passing newly developed filters (pall). daily filtrations with several cycles were performed (200 -300 ml) over one week. results: the 24 gbs patients improved after 19 days (median) for one grade (according to the gbs-scale from the gbs study group) . the ventilator dependent patients were weaned after 16 days (median). patients not at all treated before (16/24) responded better than patients that had been pretreated (8/24) with plasmaexchange or intravenous immunoglobulines. 18/22 cidp patients drew benefit from treatment, 10 stabilized iongterm. conclusions: csf-filtration is a relatively save and well tolerated additional procedure. the costs are considerably lower (1/3) than those for plasmaexchange or intravenous immunoglobulines. references:(1)wsrz aet al: csf and serum from patients with inflammatory polyradiculopathy have opposite effects on sodium channels. muscle nerve (1995) . (2) clinical observations were made in 24 patients admitted to the clinic. they were in coma associated with acute alcohol intoxication.standard evaluations (ecg-monitoring, electrocardiography, neuromonitoring, studies of acid-alkali condition, biochemical and toxicologic investigation of blood and urine) prior to and following the treatment conducted were undertaken in all the patients.to correct irreversible impairement of functions twofold laser blood irradiation by means of alok-01 apparatus, the exposure within 20 minutes, was carried out.the data obtained confirm more rapid coma withdrawal of the patients, reconstruction of the heart and central nervous system electrophysiologic indeces, reliable reduction in complications compared with the control group. objective: to know the actual incidence of the critical illness polyneuropathy(cip). setting: fourteen intensive/critical care unit beds, in 550 bed university hospital, covering 345.000 inhabitants (majority rural area). the icu patients are medical, surgical and coronary, excluded the neurotrauma and neurosurgical. design: a conseculive and prospective study. all the patients admitted during three months, from january lth to march 31th 1993, were eligible (patients with admittance diagnosis of polyneuropathy were excluded ). methods: patients with apache ii score >10, at the admission and six days after admissions were included into the study protocol. diagnosis of sepsis, mof, and all the drugs administered days before were recorded. a complete neurological exam, by a neurologist, in absence of ssdatives and muscles reliant (7th, 25~ and 60th days after icu admittance) was made. we evaluated the nerve and muscles function with and electromyography study in all patients, at same days. in some paeents with cip we performed a nerve biopsy. results: from 285 patients ( apache ii score: 12.82) admitted in the icu, 16 (5.6%) enter the study protocol. seven (2,45%) had an axonal polyneuropathy(cip), three very severe. only four of the patients with cip had pathologic clinical exam. apache ii score: cip vs non-cip was 22.6 vs 16.6. the incidence of cip by diagnosis (cip/diagnosis) was: sepsis, 5/9 and mof, 6/11. conclusions: 1. -we think that it is necessary to define the "critically ill" for some score, before designing a study to know the incidence of this syndrome. 2. -we think that the incidence of the cip is lower that the latest papers say. objectives:acute pancreatitis(ap)is becoming a more important problem among the elderly as the population ages. the increasing presence of gallstone disease,as well as the use of certain drugs,may also contribute to the occurrence of pancreatitis. methods:all patients(>60 years)admitted to our medical department over an eight year period were included.pancreatitis was confirmed by biochemical tests and imaging techniques.scores were developed using ranson's criteria and a multiple organ system failure(mosf)index . overall, 103 patients were evaluated; 21(23%)had pancreatitis of unknown etiology . results:(1)patients with pancreatitis of ~nlqnown etiology were sicker and had greater morbidity(48% vs 22%),mortality(24% vs 8%),and longer hospital stays than p~tierf~ with pancreatitis of known cause.(2)the best predicto~of severity and outcome was the mosf index and not ranson's criteria;the higher the score,the greater the associated disease,the worse the outcome.(3)curlously,no difference existed in associated medical conditions between patierts withknown and ur6~own causes of pancreatitis. conclusions:greater organ dysfunction exists in patients with pancreatitis of unknown etiology, even though age and associated medical conditions do not differ . the application of the total enteral nutrition in the burns disease has minimized the complication rate and consequently increased the survival rate of children and adults. time of initiation, composition, duration and way of administration are very important in obtaining the optimum beneficial effect from the treatment and diminishing the complication rate and side effects. the above features will be discussed in view of our experience in 240 cases. ta buckle?,, ra freebalm, c gomersall g joynt, r young. tg short. department of anaesthesia and intensive cm+e, prince of wales hospital. the chinese university of hong kong, shatin, hong kong introduction: gastric mucosal ph (phi) monitoring has been proposed as a relatively noninvasive index of the adequacy of aerobic metabolism in the gut. to examine the accuracy of gastric intramucosal pit measurements as a function of time and as a function of the catheter itself to determine whether the measurement error between catheters is clinically acceptable. patients with a gastric tonometer (trip tm, tonometrics, worcester. ma) insitu for >3 days were studied. following informed consent two new tonometers were inserted equidistantly & correct position was confirmed radiographically. measurements of intramucosal gastric ph were then performed over a 36 hr period. eight -ten measurements were made in each of ten critically ill patients.percent differences between the two new catheters were 1.6% ie at ph 7.3 _+0.12 (95% limits) and between old & new catheters were 2.2%, ie ph 7j3 _+0.16 (95% limits). conclusions: the results suggest that the function of the tonometer deteriorates over time and that the absolute values of phi m~ not ~ufficiently accurate. however as a trend monitor phi may be useful in the clinical setting. despite a continuous decline both in li'equency and severity of gastro-intestinal stress-lesion/-bleeding (gisb) due to both improvement in preclinical support and in intensive care medicine, patients with cerebral lesion are still considered at high risk for developing gis8. therefore the question arises, whether m> specific (}lsb-prophylaxis besides general and neurological intensive care, specific pharlnaeothcrapy or even the combination of two specific drugs reveals any protective efli~ct on frequency and severity of gisb.this pntspcclive randomized study has been perfornted in 173 patients snfrering t'rttna head-injury/cerebral lesion and with a glasgow-coma-scale on admission (gcs:,)of <9. according to randomization the patients have been grouped as tbllows: h analgesia/sedation (n=37); ih analgesiajsedation plus pirenzepine 60 mg/day (n=54); .[ih anatgcsia/sedalkm plus sncraltate 6 x [ g/day (n=47); iv: analgesidsedatkm plus pirenzcpine 60 mghlay plus sucralfate 6 x 1 e/day (n=35). slalislical analysis has been performed by chl:*tt~sl. rank correlatinn and unpaired t-test; statistical significance has been set with p <0.05. 28/173 patients (16.2 %) developed gisb. although the mean gcs~-value (x -+ sd) did not reach significance between patients with and without gisb (5.61 + 1.65 vs 6.12 -+ 1.65). a significant inverse correlation between gcs:, and the incidence of gtsb (rs~ = 0.89) has been shown. the frequency of gisb among the groups is as follows: h 18.9 %; lh 18.5 %; llh 17.0 %; iv: 8.6 % (ch1 -~ =1.94; not signilicant). no gisb-induced blood translusion or mortality, respectively, could be demonstrated. survival rate between the groups did not differ significantly (chi-" =5.86; p=0.1186) and reached an overall-value of 75.1%.drug-specific glsb-prophylaxis -administered either as monotherapy (pirenzepine, sueralfate) or in combination of these two specific-drugs -reveals no additional significant influence on the incidence of gisb in patients with cerebral lesion compared to no specific prophylaxis besides the general trauma-/disease-specific intensive care measures. critical care dpt, evangelismos hospital, athens university scho~" of medicine objectives: the correlation of longterm presence of nasogastric tube (ngt) to gastroesophageal reflux (ger) is still in question. in case of positive correlation, peg should represent an alternative to tube feeding in patients unable to be fed orally. therefore, we investigated: i) the correlation between ng and ger and ii) the effect of peg on ger. methods: a 24-h esophageal ph-metry was performed in 40 patients in recumbent position at 30 ~ who had a ngt for more than 10 days and were on sucralfate for gastric mucosal protection. the tip of the ph-probe was lied 5 cm over the esophagogasttie junction, confirmed by x-rays. patients who presented a percentage of ger-total (i.e. with a ph less or more than 4) (ger-t) more than 3%, underwent ~t peg. the presence of a creseent-notch on the esophagogastric junction persisting on inspiration and the grade os endoseopic and histologic esophagitis (scale=0-3) was noted. two ph-metrles repeated on 48 h and on 7 days post-peg were compared to the pre-peg one, with the followin~ parameters taken in consideration: i) % ger-t, ii) number of ger-total per hour (no/h ger-t) and iii) the duration that ph was less than 4 (tph<4). in case ot ger persistence at the ph-metry on ?th day post-peg (group ii) another endoscopy was performed, while patients with reduced ger (group i) were considered as esophagifis-free.results: 23 out of 40 patients presented a ger-t>3%. eleven out of 23 group i group (n=6) 1i ( objectives: the aim of the present study was to compare the performance of a specially modified version of a photo-and magnetoacoustic (pa/ma) gas analyzer (br~)el & kjaer, denmark) with a conventional quadrupole mass spectrometer (ms) (innovision, denmark) in inert gas rebreathing (rb) tests such as determination of functional residual capacity (frc), pulmonary capillary blood flow (pcbf) and lung tissue volume (vtc). methods : from simultaneous readings of inert gas concentrations with the ms and the pa/ma analyzer during rb experiments a comparison was made of the pcbf, vtc and frc values. the rb tests were performed during rest and exercise (0,50 and 100 w) in ten healthy subjects. results: the differences (mean +/-sd) between simultaneous estimates of rebreathing parameters were the following (pa/ma -ms) for pooled data, pcbf: 0.18 +/-0.38 i/min, vtc: -33 +/-108 ml and frc: 0.028 +/-0.048 liters. conclusions: smell but significant differences were found between the estimates of pcbf, vtc and frc using the ms and pa/ma, respectively. reference: p. clemensen, p. christensen, p. norsk, and j. gr~nlund. a modified photo-and magnetoacoustic multigas analyzer aplied in gas exchange measurements. j appl physiol 1994; 76: 2832-2839. objectives: because transcranial doppler (tcd) has been proposed to explore cerebral co 2 vasoreactivity in brain injury (stroke 1992;23:962-6), we compared this technique with the kety-schmidt reference method to assess cerebral vasoreactivity in comatose patients. methods: 17 mechanically ventilated patients (age 30-81 yrs, glasgow 3-10) in coma due to acute brain injury were investigated during stepwise changes in paco 2 (25, 30, 35, and 40 mmhg) by increasing inspired pco 2. middle cerebral artery velocity (vm) was measured by tcd. after insertion of a catheter in the ipsilateral jugular bulb, cerebral blood flow (cbf) was determined by the kety-schmidt method, using the inhalation of 10% n20 through the inspiratory line of the ventilator. for each patient a cerebral co~ vasoreactivity index was calculated as the slope of linear relationship between vm or cbf and paco2. objectives: after cardiac surgery the fluid shill, between interstitial and intravasal space may be marked. this is due either to the intraoperative volume loading by the extracorporeal circulation or the increased postoperative diuresis. therefore, infusion of a large amount &fluids is necessary during the first postoperative hours. it still remains unclear which of the substances at disposal is the best for this purpose. aim of the present study was to compare the different fluids with special regard to postoperative bleeding and rheological behaviour. methods: 93 patients undergoing cabg-surgery were investigated and randomizedly distributed to three different groups of postoperative volume replacement to stabilize the mean arterial pressure at 80 mm hg. 1. ringer's solution, 2. 3.5% gelatine solution, 3. 6% hydroxyaethylstarch (mean m.w. 70.000). we evaluated the following parameters within intervals of 30 min: arterial and central venous pressure, heart rate, postoperative bleeding, urinary output, volume replacement. results: there was no statistically significant difference between the groups with regard to urinary output and bleeding. in spite of larger amounts of fluids necessary in the ringer treated group patients of this group showed symptoms of hypovolemia. hematocrit was increased in the ringer patients. this was statistically significant. introduction: pulmonary wedge pressure (pcwp) and central venous pressure (cvp) are frequently used as parameters for cardiac preload, although it is known that both are poorly correlated to the cardiac index (ci). it has been claimed that intrathoracic blood volume (itbv) measured with the thermal dye dilution method reflects cardiac preload better than pcwp and cvp. we studied the correlation between itbv and ci in a mixed population of critically ill patients. methods: in 17 consecutive patients (6 sepsis/sirs, 2 acute heart failure, 3 ards, 6 transjugular intrahepatic portosystemic shunt) monitored with a pulmonary artery catheter, itbv was measured on regular intervals using the pulsion cold z-021 system (pulsion, munich, germany). ci, pcwp, and cvp were recorded simultaneously. results: a total of 1ol measurements was made. pcwp and cvp did not correlate to ci, nor did apcwp or acvp correlate to aci. itbv was correlated to ci in a non-linear fashion (f -139, df = 99, p < 0.001, (figure) ). aitbv was correlated to ac1 in a linear fashion (r = 0.76, f = 134, df = 99, p < 0.o01). a rapid and efficient circulatory support system may save a patient in cardiogenic shock. left heart bypass with percutaneous and transseptal placement of the aspiration canuia simplifies the circuit and avoids the need for an oxygenator. we assessed this preclinical set-up in 5 anaesthetized pigs using a centrifugal pump with a 14 f arterial catheter and a 16 f left atrial aspiration line. animals were supported for two hours at a mean flow of 3.1 liter (3'680 rpm), a mean hematocrit of 29% and low heparinisetion (act double baseline). hemodynamic and laboratory samples were taken at baseline (a), 10 minutes (b), one hour ( pulmonary hypertension (ph) usually involves obliteration and loss of functional pulmonary microvasculature. the microvaseular endothelium normally acts as a major metabolic organ, converting angiotensin i to angiotensin ii via the angiotensin-converting ectoenzyme (ace). it is unknown whether the loss of functional vasculature and altered pulmonary blood flow seen in ph will affect lung ace metabolic activity. we therefore estimated pulmonary vascular ace activity in 9 patients with ph of various causes: 2 primary; 1 post atrial septal defect closure (asd); 2 chronic thromboembolic (te); 1 anorexigen; 1 iv drugs; 2 collagen disease. single-pass transpulmonary hydrolysis of the specific ace substrate 3h-benzoyl-pbe-ala-pro (bpap) was measured and expressed as % metabolism (%me0. we also calculated an index of peffused functional capillary surface area (amax/km). all patients with ph had an abnormality of %met or amax/km, or both. as compared to 9 control humans (mean %met = 71.4% _+ 11.3% s.d.), the mean %met in ph patients was 54.3% _+ 14%. the %met in ph patients correlated inversely with cardiac output (r=0.74), possibly reflecting more complete bpap hydrolysis with longer pulmonary transit times. amax/km was markedly decreased in ph (1663 + 536 ml/min) as compared to controls (4225 _+ 1018 ml]min), consistent with a significant loss of functional capillary surface area. patients with collagen disease, asd and anorexigen-induced ph had the most marked abnormalities. in conclusion, patients with pulmonary hypertension have decreased pulmonary endothelial angiotensin converting enzyme activity, likely due to a loss of functional or perfused pulmonary microvaseulature. supported by the funds de la recherche en same du quebec and the national health system of greece. objective: to investigate adrenocortical function in patients with ruptured aneurysm of the abdominal aorta (raaa). studies investigating adrenocortical insufficiency in critically ill patients report an incidence ranging from 20% to less than 1%. this may in part be explained by difference in methods used (single cortisol measurement vs short acth stimulation test) and populations studied (heterogenous groups of patients with great individual variation in underlying disease as well as duration and severity of illness). methods: we investigated the adrenocortical function in 32 patients with (raaa).a short acth stimulation test (synacthen test; 250 ug 1-24 acth iv) was performed at 0800 hrs within 24 hrs of admission. plasma cortisol was measured before (cort basal) and after stimulation (cort stim). a plasma cortisol level > 0.55 umol\l before or after stimulation was considered normal, severity of illness was assessed using apache ii. results: of the 32 patients investigated 6 died and 26 survived. mean cort basal in nonsurvivors was significantly (p<0.o04) higher than in survivors; 1.03 (range 0.72-1.29) vs 0.69 (range 0.24-1,14). this difference between nonsurvivors and survivors was also present for cort stim but lacked significance; 1.30 (range 0.96-2.25) vs 1.00 (range 0.57-1.53). while 8 patients showed a cort basal < 0.55, no cort stim <0.55 was found. there was no significant difference in mean age or apache ii score between survivors and nonsurvivors; 70 vs75 and 19 vs 21. conclusions: single plasma cortisol levels were inadequate to assess the adrenocortical function in the patients studied, judged by a short acth stimulation test, our investigation in patients with raaa showed no adrenocortical insufficiency. mortality in raaa is associated with elevated plasma cortisol levels. obiectives: mortality in acute myocardial infarction (ami) prinicipally depends on hemedynamic impairment. thus, patients (pts) with elevated pulmonary wedge pressure (pwp) present high in-hospital mortality. however, the complete right heart catheterization is laborious, so the central venous pressure (cvp) alone is frequently used to assess the severity of ami. the accuracy of cvp in estimating pts with ami was tested in this retrospective study. methods: 131 pts. aged 68+14 years, admitted to our ccu from 1992 to 1994 with their first ami, were inctuded in this study. all had undergone right heart catheterization because of overt or suspected heart failure. swan-ganz catheters (7f, 85cm, abbott, il, usa) had been used, every treatment had been temporarily interrupted l h before the calheferization. based on ecg findings the pts were retrospectively divided into 3 groups. in group a we included 54 pts with anterior ami, in group b, 30 pts with inferior ami, and in group c, 47 pts with inferior and right ventricular ami. the initial values of cvp and pwp were considered for the linear regression of the pwp variable on cvp and p<0.05 was accepted as statistically significant.results: in g~oup a, the cvp and pwp vaiues were 8+3 mmhg and 14_+7 mmhg respectively. despite the signifanf correlation (p<0.01) between the two variables, it was not possible fo predict the exact value of pwp based on cvp value, 19 pts (35%) presented cvp>8 mrnhg and 16 of these (84%) had pwp_>15mmhg. in group 3, the cvp was 11_+8 mmhg and the pwp, 13_+6 mmhg. significant correlation (p<0.05) between the two variables also existed, however it was impossible to predict the pwp value. 6 pts (20%) had cvp>8 mmhg but only 2 of these (33%) had pwp>15 mmhg, similar was the relation between cvp and pwp in group c (p<0.01). cvp averaged 12+6 mmhg, and pwp, 17_+8 mmhg. 33 pts (70%) had cvp>8 mmhg and 25 from these (76%) presented pwp>15 mmhg,conclusions: a single measurement of cvp in ami does not ensure an accurate assessment of pwp. because every pt with ami needs optimal values of pwp in order to prevent pulmonary congestion or manifestations of low preload, the significance of complete right heart catheterization becomes apparent. in patients (pts) with advanced hf the need and the prognosis for heart transplantation (ht) can be predicted from vo= max. indirect measure of functional capacity with the six-minute walk test can also predict smvival in moderate hf. to predict vos max from indirect astinmtions of functional capadty such as 6-1~q~/, pulmonary and heart function tests, and to assess the prediddve value of the above parameters in hf pts survival. we evaluated 35 pts (age 48+12 yeats nyha class: 12 ii, 15 hi, 8 iv) with hf for pit. they underwent a pmgmmive exercise test on cycle ergometer for vo2 max determination, a 6-mw, a right heart catheterization and a spirometry and dlco estimation. introduction: brain death causes myocardial impairment by mechanisms that are not well understood yet. the aim of this work was to assess the echocardiographic features found in these patients from the clinical onset of brain death to somatic death, methods: seven brain dead patients were studied (patients" relatives refused to allow them to be used as donors). mean age was 23.5 (18-32) years old. four of the patients were female, none of the patients had any history of cardiac disease. transthoracic echocardiogram (echo) and electrocardiogram (ecg) were obtained at the onset of clinical brain death and were repeated every 24 hours until somatic death. we we detected severe diffuse hypokinesia (ef<50%) in 2 patients and mild hypokinesia in 3 others (ef 50-60%). systolic function was strictly normal in only 2 patients. corrected qt interval (qtc) in ecg was 54.6_+5.5 msec (normal range 38-43 msec) just before somatic death (b). conclusion: in patients with brain death we observed a significant increase of left ventricular mass due mainly to ivs "hypertrophy" without any important change in the dimensions of the left ventricle. to our knowledge, this finding has never been reported before and its importantance in heart transplantations may be of particular interest. predict right ventricular outcome. l. jacquet, r. dion, p. noirhomme. m. van dijck. m. goenen cardiothoracic intensive care unit, st-luc univ. hospital(ucl) we have registred: heart rate (hr), blood pressure (bp), pulmonary artery pressures (pap), central venous pressure (cvp), pulmonary capillary wedge pressure (pcwp), pulmonary and systemic vascular resistances (pvr, svr), right ventricle end-diastolic end end-systolic volume (redv, resv), right ejection fraction (ref), right sistolyc ventricular work (rsvw) and cardiac output (co) using a thermodilution thechnique and a microprocessor (model ref-1; baxter-edwards laboratory); duration of cpb and aortic clamping, and the requirements of haemodynamic support after cpb.results: in the c group an increase post-cpb of the fc (62 + 13 95.8 + 18.4, p < 0.01) was produced without significantly changes in the redv, resv, ref, rsvw neither co. in the w group, hr increased from 58.5 + 8.8 to 79.9 + 8.6 (p < 0.01); redv was reduced from 227.7 -+ 76 to 139.14 _+ 36.4 (p < 0.05); resv was reduced from 142 • 68.7 to 82 + 35.3 (p < 0.05). there were not changes in the other haemodynamyc parameters. there was a trend (no significantly) to an increase of ref in the w group (40.07 + 9.7|• 4.7) compared with the c"group (39 • 10.9($ 38.3 • 8.5) post-cpb. the need for haemodynamic support was similar in both groups.conclusions: the warm, continuous, anterograde-retrogade myocardial protection has obtained a decrease of preload, hr, and a trend to an increase in the ref, making an improvement in the right ventricular global performance when is compared with the classic form of cold myocardial protection. objective: to evaluate the effect of dobutamine on gastric mucosal ph (phi) after coronaly artery bypass surgery. design: prospective study in a university hospital intensive care unit (icu). subjects: 20 elective cardiac surgery patients. interventions: dobutamine was infused at 4 ug/kg/min for 3 hours immediately after admission to the icu. hemodynamics were measured every 30 minute periods until 3 hours and again 2 hours after stopping dobutamine. results: there were no significant differences in mean gastric phi between the groups but mean phi decreased in both groups during the study period. oxygen delivery and consumption both increased during dobutamine infusion but decreased to the control group level after stopping the dobutamine infusion. lactate levels did not change. baseline 90 objectives: the aim of the study was to evaluate the usefulness of a low dobutamine dose in conjunction with intraaortic balloon pumping and mechanical ventilation in cardiogenic shock. we studied 21 patients 58.9-+ t4.4 years of age suffered of post infarction cardiogenic shock characterized by a systolic arterial pressure< 80mmhg, urine output< 20 ml/h and mental confusion or purpueral signs of low output, non responded to dobutamine infusion up to 9 pg/kg/min. all patients underwent mechanical assistance by the intra-aortic balloon pump (iabp). five patients were additionally placed on mechanical ventilation due to blood gases disturbances. the end points in our study were: reversion of cardiogenic shock, improvement of patients survival or both on the 15th post infarction day and 6 months later. results: three patients refused iabp treatment and 0/3 survived on the 15th day. on the 15th day 3/13 supported by the iabp and 5/5 that underwent mechanical ventilation plus iabp were alive (p <0.01). on the 6th month 2/13 supported by the iabp and 5/5 that underwent mechanical ventilation plus iabp were alive (p<0.01). conclusions: in conclusion, the combined use of mechanical ventilation and iabp assistance in severe cardiogenic shock might improve survival. obiectives: the study was aimed at analysing predictive factors of swan ganz pulmonary catheter (pc) requiremen t during elective cardiac surgery according to the need of sustained inotropic support after surgery. methods: three hundred patients (aged from 27 to 85; 89 females and 211 males)were consecutively operated on for elective coronary artery bypass surgery (cabg, n=179), valvular replacement (vr, n=98), combination of both (vr-cabg, n=15), or others (n=6) and retrospectively included in the study. each patient had preoperative invasive cardiac investigation with calculated ejection fraction (ee). anaesthesia, cardiopulmonary bypass (cpb) and cardiac arrest managements were similar in all patients. pc requirement was estimated from the need of either dobutamine, adrenaline, dopamine or enoximone use during the first 48 hours after cardiac surgery. demographic data, asa and nyha classifications, preoperative ef and treatments, type of surgery, cpb and aortic cross clamping (axc) times, and postoperative incidence of complications were compared in patients with or without inotropic support using either student's t test or x2 with continuity correction when appropriate. results: seventy4hree patients (24.5%) required inotropic support after surgery. axc .and cpb times, mean stay in icu were significantly longer in patients with inotropie support (p<0.001). type of surgery, preoperative ef, and nyha classification are the first 3 significant factors related to inotropic support (p<0.005). most patients operated on for double-vr or vr=cabg required inotropic support (57 and 60%, respectively). postoperative mortality was higher in patients receiving inotropic support (8,2% vs 0,9% 'overall mortality, p=0.003). conclusions: since pc insertion is most.often justified because inotropes are required, these results suggest that elective rather than routine systemic pc insertion could be helped by considering several but selected preoperative factors. background: cardiovascular depression due to anaesthesia, old age and major gastrointestinal surgery is becoming an increasingly frequent challenge .to the anaesthesia-surgory team. deliberate preoperative manipulation of haemodynamics and oxygen transport parametres towards prede~t~mined optimal values may prove to be effective "in reducing 12 morbidity ~nd mortality in high risk surgical patients,. a new concept of using conlimaous perioperative measurement of cardiac'output to obtain and maintain supranormal oxygen delivery (do2i) is presented. methods: continuous measurement of cardiac output is a relatively new form of on-line monitoring, in which trains of impulses are emitted from a thermal filament mounted on a pulmonary artery catheter. computer software recognizes patterns generated by minute changes in blood temperature and ealoalates cardiac output every 30-60 seconds. cardiac 3 output and mixed venous blood oxygen saturation are displayed graphically on line. in tins tm study cardiac output was measured continuously by vigilance cardiac outpu t compl/ter (baxter). preoperative haemodynamic optimization was performed with the goal of increa-2 1 sing do2i to at least 600 ml/min/m accordfing to shoemaker's algorithm . this was.done by infusing colloids (albumin or hydroxy ethyl starch (haes-steril| until the desired do21 was reached. infusion was stopped if cardiac output ceased to increase with infusion, if there were signs of pulmonary oedema or if wedge pressure reached 18 mmhg. vasoactive or inotropic drugs were infused if the desired do21 was not reached by infusion alone. anaesthetic technique included continuous thoracic epidural and isoflourane anaesthesia. expected mol:bidity and mortality rates were calculated by the "possum" score aasing preoperative clinical and paradinical estimates of organ function as well as surgery characteristics 4. materials: 15 asa group ill-iv patients with a mean age of 75 years (range 60-92) and a mean weight of 67 kg (range 36-93)) scheduled for major abdominal surgery were included. results: 2 patients were excluded because do2i could not be raised at all. mean do2i was increased from 488 ml/min/m 2 (range 384-610) to 688 ml/min/m 2 (range 490-967). mean volume of preoperativdy infused colloid was 954 ml (range 0-2000). during surgery 1230 ml (range 5002300) of colloid was infused. mean length of surgery was 150 minutes (range 60-300). mean blood loss was 920 ml (range 04800). expected mortality and morbidity rates ("possum") were 56% and 92%, respectively, whereas patient follow up upon discharge or at death revealed mortality and morbidity rates of 8 % and 54%, respectively. conclusion: based on experience from the present study, continuous measurement of cardiac output has proved to be a valuable tool for perioperative optimization of do21 in asa group ili and iv patients during major surgery. however further studies including a greater number of patients are necessary to confirm the promising preliminary findings. we studied the hemodyn~c effects of three different combinations of positiv inotropic .agents, vasodilators, diuretics and av-filtration (av) in 16 patients (pts) with severe left heart faille (left veutrieul0x filling pressure (lvfp) >25 mmhg) due to acute myocardial infarction. hemodynamic measurements (intravascular pressures (lvfp), thermodilution (cardiac index (ci)) were made before (control) and after each therapy. in 11 furosemide (f) + d0butamin (d) + nitroglycerin (ni) reduced lvfp and a small increase of ci occurred. in 6 of these pts :(group a) nitroprusside (hip) instead of ni increased ci significantly, in the other 5 pts adding of amrinone (a) resulted in a pronounced increase of ci. group c (n=5): the combination of ni and av reduced lvfp but did not increase ci which was achieved by av+d+ni. in order to optimize the treatment of acute heart failure a combination of inotropic agents, vasodilators, diuretics and av-filtration should he used guided by hemodynamic monitoring. arias jr, miragaya d, sandard, san pedro dm ~, herndndez d, valenzuela 12 . objectives: to evaluate the variation in nomdrenaline (na) plasma concentrations in patients with acute myocardial infarction (am1) after thrombolytic therapy with noniltvasive reperfusion criteria (clinical, electrocardiographic and enzymatic), in relation to infarct size and location.methods: 42 consecutive patiens with ami, from october 1, 1994 to february 28, 1995, admitted within 6 hours alter onset of symptoms, undergone successfull systemic thrombolysis. 21 of them were anterior (group a) and 21 inferior (group b) . noradrenaline plasma levels at 0 (na1), 60 (na2) and 240 (na3) minutes after admission were compared with ck-peak plasma levels by linear regression. differences were tested for significance by student-t-test for paired and unpaired values. na plasma concentration was measured by high-presssure liquid chromatography. p< ns 0.01 ns means 4-sem (normal limit for our laboratory: na < 37/0 pg/ml; ck < 170 u/i ) conclusions: 1. the na plasma levels at admission (nai) are more increased in anterior than inferior amis, probably in relation to infarct size. 2. the decrease in na is more evidence in amis with anterior location. 3. this decrease is probably due to the major efficacy of thrombolytic therapy in amis with anterior location. arias jd, miragaya (group b) , probably due to certain degree of t~cg'rfueion. 3. there is not significant variation in na in conventional treated ami (group c). v.suchanov, a.levit, p.trofimov, icu, regional hospital, ekaterinburg, russiaobjectives: our task was to improve the technique of preservation of platelet rich plasma. methods: 38 patients scheduled for multiple cardiac valve replacement in 1994 were divided into two groups: group i (10 patients) -without pp; group ii (28 patients) -pp was performed preoperatively. the first pp was made ten days and the second -3 days before the operation. prp was preserved by cryoconservation. our technique of cryoconservation is distinguished by the speed of freezing (17-18~ and absence of dmso. this made it possible to preserve 90 % functionally active platelets during 20 days. the prp was transfused back after heparin neutralization. the hospital ethics committee approved the investigation.results: the blood loss through the 1st p. o. d. was significantly greatest in the group i (725 _+ 97 ml) and all the patients required transfusion of the donor blood (480 + 112 ml) whereas the blood loss in group ii was 489 +_ 75 ml and olny 12 patients required the donor blood. the number of platelets on the 1st p.o.d, was 107 _+ 12. 109/l (group i) and 153 + 19. 109/l (group ii), p < 0.05.conclusions: our technique of prp cryoconservation makes it possible to avoid the crystallization phase during freezing of prr thus the infusion of prp may improve hemostasis after open heart surgery and limit the use of the donor blood. in-hospital outcome of women suffering an ami is generally considered worse than that of men, but it is still debated whether female sex is per sea negative prognostic factor or is merely associated with other negative determinants of prognosis. the purpose of the present study is to evaluate the independence of the association between female sex and mortality (in the 567 patients of the swiss centers) and in the 36381 patients randomized in the isis-3 trail mortality rate in women was 14.8% (1421/9600) compared to 9.1% (2417/26480) in men; in switzerland: in-hospital mortality for women was 15.5% (20/129), for men 7.1% (31/438).the table shows the results of isis-3 in terms of odds ratios and their 95% confidence intervals either after unadjusted analysis or after adjustment for age, known to be the major confounding variable when prognosis of women after myocardial infarction is considered, and for all the available clinical and epidemiological characteristics collected at trial entry: these observations suggest that there is a small but independent effect of female sex on short-term mortality after acute myocardial infarction. (27) and bubble (13) oxygenators a, ere used. anaesthesia was balanced and pts were extubated 12 to 18 hrs after cpb. pts were monitored with swan-ganz catheters (sgc) for 24 hrs after cpb. at that time qs/qt was calculate( according to )be standard shunt equation. after the sgc had been removed, an estimated shunt was calculated. measurements of qs/qt were performed: before induction of anaesthesia (1), after induction of anaesthesia (i[), 5 mins after cpb (iii) 2 (iv) and 6 (v) hrs afiter cpb, 30 rains after extubation (vi), 24 hrs after cpb (v[1) and on the 2nd, 3rd, 5th, 8th and 13tb postoperative day (pd) (viii, 1 x, x, xi, xi1, respectively). analysis of data was performed by two-way analysis of variance, p < 0.05 being regard as significant.results: the figure shows the values for qs/qt expressed as means + sd. there was a significant increase in qs/qt above b~setine throughoul the whole investigated period except on the 13th pd. qs/qt reached maximum at 30 rains after extubation (vi). objectives: many stndies have shown advantages of membrane oxygenalors over 9ubbie type oxygenators. the aim of this study was to evaluate the influence of 3x3'genator type on pulmonary shunt (as/at) after coronary surgery. methods: 40 patients (pts) gave their informed consent to the study which was approved by the university ttuman research committee. pts were divided into two groups: a1 (n = 27) with a membrane o~genator and a2 (n = 13) with a bubble oxygenalor used during cardiopulmonary bypass (cpb). ths were monitored with swan-ganz catheters (sgc) for 24 hrs after cpb. at that tfme os/ot was calculated according to the standard shunt equation. alter the sgc had been removed, an estimated shunt was calculated..measurements of os/qt were performed: betore induction of anaesthesia (i), 30 mins after extubation (11), 24 hrs alter cpb (111) and on the 2nd, 3rd, 5th, 8th and 13th postoperative day (iv, v, vi, vii> viii, respectively). analysis of data was performed by one-way analysis of variance, p < 0.05 being regarded as significant.results: the figure shows the values for qs/qt expressed as means _+ sd. os/qt was significantly greater at 30 rains after extubation (ii) in a2 group. the difl'ereuce between the two groups was no more significant from 24 hrs after cpb (iii) to the end of the investigated period. ! i * p < a.0s betw~n ~o~ conclusions: membrane ox3'genation during cpb is accomplished by reduction in blood cellular destruction and less alteration in blood. the results of our study show the influence of oxygenator type on value of qs/ot only after extubation (12 to 18 hrs after cpb). the difference in qs/qt disappeared 24 his after cpb and since that time the oxygenator type had no influence on qs/qt. it may be of particular importance in patients with severe forms of cardiopulmonary disease who are at risk of higher postoperative morbidity and mortality. objectives: hypomagnesemia has been reported with a variable prevalence (20 to 61% ) in icu patients. magnesium deficiency can induce a number of climcal symptoms (primarily cardiovascular and neuropsychiatric) but can also be clinically silent (10-65% are asymptomadc), methods: we measured whole blood ionized magnesium (lmg++) in 74 patients on admission to the icu, using a nova 8 electrolyte analyzer (nova biomedical), containing an img++ electrode. blood was collected in syringes with dry heparin (radiometer qs 50 ). normal range of img++ was found between 0.45-0.55 mmot/l (healthy volunteers). results: for the entire population, we found a 61% prevalence (45/74) of hypomagnesemia (figure 1) . among the surgical patients, the prevalence was highest after cardiac surgery (85%) and after thoracic surgery (80%) and was lowest after neurosurgery (8%). hypomagnesemia was also common in patients after liver transplantation (lvtx) or with hepatic failure (100% for both groups). conclusion: our findings confirm that hypomagnesemia is common in acutely ill patients, especially in those after cardiothoracic surgery or those with liver disease. nevertheless. it is difficult to define the associated factors with sufficient specificity, so that measurements of img++ are warranted to diagnose hypomagnesemia. hepariu influences platelet function and may lead to thrombocytopenia called heparin-associated thrombocytopenia (hat) regardless of the dose and route of administration. additinnal venous and/or arterial thrombosis may lead to life-threatening complications. the incidence of so-calied heparin-associated thrombocytopenia and thrombosis (hatt) ranges between i-5%. hatt is confirmed by a heparin induced platelet activation assay (hipa). results: from 11/93 to 11/94 1146 consecutive patients of our icu were reviewed retrospectively. all patients were treated with heparim the incidence of hatt was 1% (12). in all cases diagnosis was proven by a positive hipa. 2/12 patients died. in 3/12 hatt could be confirmed before severe thromboembolic complications occured. 4/12 patients developed a deep vein thrombosis (dvt), 2/12 dvt and pulmonary embolism (pe), 2/12 dvt, pe and arterial thrombosis (at) and 1/12 a dvt, pe~ at and a sinus thrombosis. conclusion: the incidence of hatt in a r series of 1146 pts. is 1%. presence of thrombocytopenia and thrombosis of the great 'vessels is associated with a significant mortality (2/12). computed tom0graphy (ct) and transthoracic/transesophageal echocardiography (tte/tee) are important tools in diagnosing and monitoring the extent of cenlrai venous and arterial thrombosis. a. cabral md, m. shahla md c. meneses-oliveira md and jl vincenl md.phd. department of intensive care. erasme university hospital, brussels, belgium objective: to determine extreme hemodynanuc patterns in cardiogenic shock. although ~.~xdiogenic shock is characterized by a low cardiac index (ci), high systemic w~,scular resistance index (svri), and high cardiac filling pressures, some patients may develop art atypical pattern. we reviewed the hemodyuamic pattern of 73 patients with cardiogenic shock, as defined by an initial ct below 2.5 l/rain/m: in the presence of myocardial dysfimction attributed to ischemic heart disease (n=26), heart failure (n=7), valvulopathy (n=2) or recent cardiac surgery (n=38). after exclusion of 10 patients with concurrently suspected/documented infection, this study included 63 patients, of whom 23 (36.5%) survived. treatment of shock included dopamine (n=43), dobutamine (n=56), norepinephrine (n=18) and epinephrine (n=23). 39 patients with arterial hypertension (ah) and initially law plasnla renin activity (pra) had been studied. in all patient changes of arterial pressure (ap) after single administration of enap was studied. nypotensive reaction wiht deereasin e of average ap about 20-25 mm hg ayter single drug administration observed only in 4 patients. ezap monotherapy accomplished during one week with 20 mg daily dose. hypotensive effect observed in 5 patients including ones which were susceptible to single enap administration. after that first stage of therapy all patints began to combinate enap with hypothyazid in dose of 25 mg per day~ after week of treatment such drugs combination lead to veritable ap lowering in 3 addition patients. in the remaining resistant to such drug combination patients was add corinfar in daily dose of 40 mg. this new drug combination permits to lower ap in 23 patients. subsequent discontinuation of enap administration to such patients aid not connected with increasing of again.therefore the most of the patients with ah and law pra(78,7%)did not susceptible to enap therapy and enap and hypothyazid combination. on the contrary-combination of corinfar with hipothyazid was effective in 59% patients with ah and low pra. methods: in 35 patients with cardiogenic shock due to ischemic heart disease (n=26), heart failure (n=7) and valvulopathy (n=2), hemod31aamic data including measures of intravascular pressures, cardiac output and mixed venous gases were collected at regular times intervals, at least 3 times a da?. all measurements were obtamed in a relative steady state and in the absence of severe anemia or hypoxemia. treatment of shock included dobutamine (n=30), dopamine (n=24), norepinephrine (n=i2) and epinephrine (n=7 objective: based on our previous studies of the function of isolated liver grafts, this experimental protocol aims at developing a novel extracorporeal liver support circuit, with an incorporated pig liver. methods:the graft liver was obtained from pigs weighing 15-20 kg. under general anesthesia the aqimals underwent total hepatectomy,following cannulation of the portal vein, the infrarenal aorta and the infrahapatic vena cava and peffusion wit h 4 it of heparinised r/l solution at 4~ the circuit consisted of the graft liver connected to a fluid reservoir and a centrifuge pump. ten healthy pigs weighing 30-35 kgr were connected to the circuit as follows: the rt carotid artery was connected to the portal vein of the graft and the rt jugular vein was connected to the fluid reservoir, through the centrifuge pump. the fluid reservoir collected the outflow from the graft's suprahepatic inferior vena cava. the cystic duct of the graft was ligated and the bile.duct cannulated for bile collection and measurement. bridges were adapted to the circuit to bypass the graft liver when necessary, in cases of by pass blood perfusing the graft was oxygenated through a bubble oxygenator. mean total priming volume of the circuit was 600 ml. temperature was maintained at 38~ and portal vein pressure at 16 (12-20) mmhg. the flow was 0.5-0.7 ml/gr of graft liver mass per minute. observation period was 8 hours (t8). results: results of the hemadynamic and metabolic monitoring of the recipients [map (t0=124mmhg , t8=118mmhg), hr (t0=177, t8=201), rap (t0=11mmhg , t8=19mmhg), pap (t0=26mmhg, t8=31mmhg), pcwp (t0=14mmhg, t8=15~mhg), svr (t0=1940dyn'sec/cm '5, t8=2190dyn'seclcm~ pvr (t0=206dyn.sec/cm o, t8= 354 dyn.sec/cm ,'~), co (t0=4.631t/min, t8=3.61t/min), do 2 (t0=662ml/min, t8=261.6 ml/min), vo 2 (t0=118ml/min, t8=111ml/min), o2er (t0=17.8%, t8=42.5% ), ph (to= 7.48, t8=7.39 ), po 2 (t0=292mmhg, t8=371mmhg), pco 2 (t0=28mmhg, t8=30 mmhg), pvo 2 (t0=47mmhg, t8=36mmhg), svo 2 (t0=84%, t8=66%), be, na, k, ca ++, lactate, osmolality, ast, alt, pt, aptt, revealed hemodynamic and metabolic stability of the animal. 02 consumption, co 2 production and tissue oxygenation of the graft were also studied. conclusion; the described circuit proved to be safe and well tolerated by healthy animals but its value for temporary liver support is currently being estimated, in a surgically induced experimental fulminant hepatic failure modal. introduction: prosthetic materials like silikone, dacron, teflon e.tc. produce auto immune responses and may even trigger clinical syndromes like scleroderma, sjogren, sle el.c. in our study we followed the evolution of humorial immunity parametrs for up to five years in a cohort of paced pts with implanted metallic and silicone materials. method: 24 paced pts (mean age 55+-13 yrs) without clinical or laboratory findings of malignancy or immune disorders were included. we measured the immunoglobulins, the complement, the auto antibodies and the proteins involved in inflammatory reactions every 6 months. the initial and final mean values are shown in the obiectives: hsp, a systemic leucocytoclastic vasculitis and anaphylactoid purpura can be accompanied by abdominal pain and life-threatening intestinal bleeding. recently we could disclose, that these patients develop severe fxiii-deficiency and immense haemorrhagic oedema of the intestinal wall. by the following case report we will demonstrate and discuss the importance of fxiiideficiency for pathogenesis, therapy and outcome in hsp. case report: a 41 year old man developed typical skin manifestations of hsp following an episode of severe (biliary ?) pancreatitis and percutaneous draining of a pancreatic pseudocyst. two days later he had a paralytic "ileus with immense hemorrhagic wall-oedema and massive dilatation of the small bowel. he got fever up to 39.5 ~ and developed severe gastrointestinal haemorrhage (blood transfusions necessary). the coagulation data disclosed a severe fxhi-deficiency (activity 34%), whereas quickvalues, platelet count and atiii-level were found to be within the normal range. elastase was markedly elevated. substitution of fxiii to normal levels leeds to the cessation of bleeding symptoms and abdominal pain, later resulting in a restitutio ad integrum. conclusions: hsp with intestinal involvement is a life-threatening vasculitis, in which careful and frequent examinations of the coagulation system, especially of fxiii are necessary. detailed analysis of the coagulation data suggest, that the severe fxiiideficiency is due to a specific degradation by proteolytic enzymes (like elastase) as well as consumption within the immense haemorrhagic oedema of the intestinal wall. knowing these facts, even most severe cases of hsp with intestinal involvement can be successfully treated by substitution of fxih. a 49-year-old woman presented a 3 year history of occasional self-limited episodes of weakness, generalized edema and o!!~aria. the immunologic testing showed no~nnai levels of complements, clq inhibitor, and serum chemistry values, between or during a attack, she was not treated. she was a~mitted to the hospital with symptoms including nausea, vomiting, weakness and ol!guria. on examination, the patient presented facial and g~neralized edema. the systolic blood pressure was 60 mm hg, pulse 140 beats/mir~ute, hematocrit 0.59, seln~n protein 46 9/i, and se~um albumin 23 q/l. an leg-kappa pa[apfotein was demostrated (7.82 g/l) and urine was neaative for puotein. c~'stalloid and colloid don't increased the blaod pressure but resulted in anasarca, with a total of ii lit[as of in~ravenous fluids. therapy wink flozen plasma, 1.000 units of clq inhibitor, cortlcosteroids, annihistwnines and antifibrinolytic agents was uns~iccessfull. the a~minist~ation of dopamine, norepineph~ne and epinephrine was inefective. the patient died at the 48 bores, only a few cases have been reported, all had igg paraprotein, the pathophysio!o~] is urd~no~n% but is possible that the paraprotein may be zesponsib!e for the increased capillary pe~leabilityo despite efforts to res~scinate the patients during an acute attack, the syndrome is often fatal. the variable course of systemic uapiliary leak syndrome and the unpredictability and self-limited nature of attacks cloud assessment of therapeutic inte~-vention. the purpose of the present work is to provide some information about the nursing care and results from our experience in continous arteriovenus hemofiltration (cavh).cavh is an extracorporeal technique, especially applicable in the critically ill patients, for disturbances, and for the control of azotemia.we used this method in 30 critically ill patients 16 men and 14 women ages from 32-74 who had sepsis -arf 10 congestive heart failure 8 postoperative multiple organ failure 8 and polytrauma 4.this method was applied to these patients from 24 to 168 hours. 20 % of the patients recovered completely their kidney function, 50 % improved their kidney function and 30 % died.we concluded therefore that this method was very effective for the critically ill patients to whom it was applied, but it requires excellent and continuous nursing care; under the above mentioned circumstances the method works effectivelly. an animal model with rats undergoing a dialysis procedure was designed to test the hypothesis that recovery from ischemic acute renal failure (airf) may be affected by the type of membrane used in hemodialysis. male sprague dawley rats were allocated to 2 groups: in group i, (n=48) airf was inducted by bilateral renal artery clamping for 60 rain. group h (n=48) rats underwent a sham procedure. in each group, rats were dialyzed twice (4th and 8th day) with either a cuprophan (cupro), a hemophan (hemo) or a pan (an69) minidialyscr or stayed nondialyzed (no hi)). renal function was monitored daily by measuring urea and creatinine values and by two single shot inulin clearances on the days following dialysis. additionally hemolytical activity of complement was determined. inulin clearance on day 5 was reduced significantly but there was no difference in the degree of decrement in glomular filtration rate (gfr) between dialyzed and undialyzed rats, nor between the dialyzed animals with different membranes (gfr: no hi): 0.78_+0.54; cupro: 0.84_+0.9; hemo: 0.82_+0.28; an69: 0.77_+0.31). the evaluation of renal function by day nine revealed significant recovery for all airf-groups compared to day 5 (p<0.001), irrespective of wether they underwent dialysis or not, or the type of dialysis membrane. complement activation could be detected in all dialyzed groups but no statistical differences between the animal groups dialyzed with different membranes were noticed. our findings refute the hypothesis that in airf exposure to complement-activating cellulosic membranes impairs the recovery of renal function in rats. changes patients: 150 patients who underwent first cadaver kidney transplantation in our unit between january and december in 1994 were involved. the recipients were divided into 3 groups: group i." non functioning graft (n=27); group ii: delayed graft function (n=59), group ili: good graft function (n=64). the grouping criteria were: a/haemodialysis in the fii~t 5 postoperative days, b/diuresis in the i st postoperative day, c,' scram crcatininc difference between the 1st postoperative day and the preoperative level. all of the parameters were involved into the exarainatio, which we measllre in our every, day practice. results: the preoperative haematocrit level differed significantly between group i. (0.36) and croup ii. and iii. (0.31 and 0.30, p< 0.05). intmo!0emtive significant differences were found between the different groups in systolic blood pressure (group i. 110 hgrmn, group ii. 140 hgnnn, group iii. 165 hgmm, p<0.05), mean arterial pressure (group i. 66 hgmm, vs. group ii. 83 hgnun p<0.05, vs. group iii. 116 hgmm p<0.001), and pulse-amplitude and rate-pressure product too. the second warm ishaemic time in group iii. was significantly shorter than in the other two groups (group iii. 39 inin. vs. group ii. 43 rain. p< 0.05, vs. group i. 49 rain. p< 0.00!). the rejection rate was higher in the first 5 days in the patients with non-functioning grafts (group i. 53% and group ii. 24% vs. group iii. 12 %) . the other examined parameters have not differed significantly. conclusion: according to our results the success of the kidney transplantation is mnitifactorial. the most important factors of this relationship are: the perioperative fluid-balance, the maintenance of adequate perfusion blood pressure during the operation, good surgical technique and immunological problems. key: cord-022940-atbjwpo5 authors: nan title: poster sessions date: 2016-09-07 journal: febs j doi: 10.1111/febs.13808 sha: doc_id: 22940 cord_uid: atbjwpo5 nan ** each poster has been given a unique number beginning with the letter p; the next part relates to the session in which the poster will be presented. moreover, klf5 is also acting on cellular processes such as cell migration, apoptosis, inflammation, angiogenesis and differentiation. previous studies showed a novel role for klf5 as a regulator of proliferation and differentiation in skeletal muscle stem cells. detecting klf5 at the protein level harbored technical obstacles. commercially available antibodies exhibited low affinity, low specificity and failed to recognize post-translationally modified forms that are directly relevant to the function. thus, these obstacles prevent further functional protein studies such as western blots, protein co-immunoprecipitation and chromatin immunoprecipitation (chip) assays. therefore, we used crispr/cas system to establish a stable cell line which carry v5 epitope tag into the n-terminal of klf5 gene. insertion into the target side of klf5 gene via crispr-cas9 system provided an opportunity to overwhelm the above mentioned obstacles. v5 epitope tag would not interfere with the function of the klf5 and also enable us to recognize endogenous klf5 via anti-v5 antibody in the mouse myoblast cell lines (c2c12). we confirmed the targeted insertion into the exon 1 of the klf5 gene both at the dna and protein levels. the conformational dynamics of structural domains plays an important role in functioning of many proteins. the reca proteins from e. coli are known to be the central catalyst of homologous recombination and repair in bacteria. it forms a helical filament on ssdna capable to bind homologous dsdna and catalysis of the exchange of the complementary strand. significant mobility if its c-terminal domain has been observed experimentally by cryo-electron microscopy. however its potential significance for reca protein activities still remains unclear. in this work we investigated this question by construction of a mutant reca protein with artificial disulfide bridge between central and c-terminal domains. the wild type protein has no disulfide bonds, and therefore its native mobility can be restored in vitro, by addition of b-mercaptoethanol. our data suggest that the s-s bridge decreases both the rate of atp hydrolysis in vitro and the e. coli resistance to uv in vivo. thus, our experimental results indicate that the flexibility of the c-terminal domain significantly affects recombination activity of reca protein in vivo and in vitro. hydroxiurea (hu) is an inhibitor of ribonucleotide reductasethe enzyme that catalyzes the process of free dntps synthesis in living cells. treating cells with hu causes diminishment of the nucleotide pool. as a result, single-stranded dna regions are generated, which leads to s-phase checkpoint activation. the progression of replication forks is blocked and the completion of dna replication is prevented. this results in s-phase cell arrest. nevertheless, our results demonstrate that after prolonged hu treatment, the saccharomyces cerevisiae cells seam to escape the arrest and continue the progression of their cell cycle. we show that when cells re-enter the cell cycle, mrc1, but not ctf4 is detached from chromatin. our data also shows that meanwhile, rad53 checkpoint activity is diminished in order to allow s-phase checkpoint escape and completion of the cell cycle. moreover, cells not only continue the cell cycle, but steadily surmount in the presence of hu. all this data indicates that cells have made the decision to compromise s-phase checkpoint and to adapt to the novel environmental conditions in order to survive. as both mrc1 and ctf4 are known to be responsible for polymerase and helicase harmonization during replicative arrest, our data indicates that mrc1 has a more specific role in the process of adaptation. our data demonstrates that mrc1 is a leading protein to regulate the stability of s-phase checkpoint arrested replication forks. zinc finger domain is the most common dna binding domain in metazoa. almost 100 drosophila proteins with c2h2 zinc fingers also have zinc finger associated domain (zad). several proteins with zad (zw5, pita and zipic) were found to interact with cp190 and act as insulator proteins. for some of the zad-containing proteins (for example, weekle and grauzone) it was shown that their zad domains can form dimers with each other. the ability of these proteins to dimerize appears to be especially important in the light of the model suggesting that dna-binding insulator proteins can support genome looping and organization of chromatin structure via interaction with each other. in this work we aimed to understand the role that zadmediated protein-protein interactions play in maintenance of dna loops, focusing on proteins: zw5, pita, zipic and cg6808. first, we performed co-precipitation and yeast two hybrid assays to confirm dimerization of isolated zads in vitro. we observed that only zads from the same protein can specifically interact with each other (homodimerization) and they are unable to interact with zads from different proteins (heterodimerization). we confirmed homo-but not heterodimerization of zads in vivo with coimmunoprecipitation experiments in s2 cells. furthermore, we found that zad proteins can support longdistance interactions in transgenic constructs in flies. using model system with cg6808 protein, we demonstrated that zad is essential for these interactions. proteins without zad cannot maintain loop formation. finally, analysis of chip-seq experiments for zw5, pita, zipic and cg6808 revealed that binding sites of zad proteins often overlap with regions of inter-chromosomal contacts known from hi-c experiments. we conclude that zad-containing proteins can support longdistance genomic interactions and dimerization of zads is necessary for these interactions. this study was supported by the russian science foundation (project №14-24-00166). over the years a large body of clinical knowledge has accumulated on pharmacological effects of drugs on thyroid function. antipsychotics are administered over long periods in humans; therefore their possible adverse side effects should be taken into consideration. the aim of this study is to evaluate the effects of haloperidol and clozapine on plasma t3 and t4 concentrations in adult male wistar rats. fifty rats aged between 14 and 15 weeks (270 ae 30 g) were divided into five groups (n = 10 in each group), and drugs were administered each day intraperitoneally (ip) for 28 days. the first group was a sham group. the other four groups were considered as low and high treatment doses of the drugs. after a one-week habituation period, animals was administered haloperidol (0.05 mg/kg, n = 10 and 2 mg/kg, n = 10) and clozapine (0.5 mg/kg, n = 10 and 20 mg/kg, n = 10). the rats were anesthetized with ether, and bloods were collected by direct cardiac puncture 24 hours after the last injection. the t3 and t4 plasma concentration levels were analyzed with chemiluminescent immunoassay. statistical analysis was performed with ibm spss v20.0. kruskal-wallis and bonferroni tests were used. t4 plasma concentration levels significantly differ between sham (median=8.25 mg/kg) and haloperidol (2 mg/kg) (median=7. 00 mg/kg), haloperidol (0.05 mg/kg) (me-dian=7.70 mg/kg) and clozapine (20 mg/kg) (median=8.32 mg/ kg), haloperidol (2 mg/kg) (median= 7.00 mg/kg) and clozapine (20 mg/kg) (median= 8.32 mg/kg) groups (p < 0.05). however, no significant differences between the groups regarding to t3 plasma levels were observed. in conclusion, haloperidol and clozapine increased the t4 plasma concentrations, but didn't have any significant effect on t3 plasma concentrations. p-02.09.1-003 isolation of lipase producing strains of bacillus obtained from olive wastewater and screening for substrate specificity in this work, wastewater samples of an olive factory from yusufeli (artvin, turkey) were collected carefully. after a centrifugation period of samples, supernatants were applied to a 0.45 lm filter and incubated on lb agar medium for 24 hours. based on differencies of colony morphologies, 13 isolates were selected and purified for identification. 16s lipase activity assay was carried out by rhodamine b. all of the 13 strains exhibited lipase activity. for determining the substrat specificity of isolates, 5 different substrates were used; 4nitrophenyl-butyrate, 4-nitrophenyl-caprylate, 4-nitrophenyl-laurate, 4-nitrophenyl-myristate, 4-nitrophenyl-palmitate. results were measured spectrophotometrically at 405 nm. all of the strains hydrolyzed 4-nitrophenyl-butirat, while there was no activity with 4-nitrophenyl-palmitate. bacillus sp. l3 was the most efficient strain that hydrolyzed all of the substrates. the gene encoding for lipase of bacillus sp. l3 will be cloned and expressed for more analyses and industrial applications. p-02.09.1-004 some quantitative aspects of hair follicle layers differentiation e. vsevolodov 1,2 , v. golichenkov 3 , a. mussayeva 1,2 , i. latypov 2 1 llc "kazcytogen", almaty, kazakhstan, 2 "institute of general genetics and cytology" sc mes, almaty, kazakhstan, 3 lomonosov's moscow state university, moscow, russia in the course of stable hair growth the differentiation of hair bulb cambium cells to several layers with dissimilar cytochemistry and morphology takes place. this means the activation of different genes in the cells of different layers. depending upon the hair diameter some layers may be absent (medulla in the thin hairs). the hair diameters of the carpet sheep breeds vary widely even within the same square mm of the skin. we compared the different layers thicknesses proportions for the follicles with varying hair diameters. the follicle layers were measured on 155 microphotos of transverse histological sections of the follicles made under the standard magnification. all follicles belonged to the same skin biopsy. the measurements were made at the levels just below the fissure separating the hair and inner root sheath appeared. the empirical regressions of the layers thicknesses and of ratios of different layers against hair diameters were counted. the computer model was made on the basis of these regressions which allowed to obtain the absolute parameters of the layers as well as ratios of these parameters for every chosen hair diameter. using this model we found an essential trend in changing the proportions in relative layers dimensions as we choose the follicles with more and more thick hair. when we change the follicles with 30 mcm hair diameter for those with the hair diameter 100 mcm the ratio of hair medulla diameter to hair diameter increases from 0.07 to 0.76. the ratio of hair diameter to the diameter of inner root sheath increased from 0.70 to 0.84. it means that the thicker is the hair the higher proportion of cells produced by cambium are spent to build innermost layers (medulla layer within the hair or hair within the complexinner root sheath + hair). these data may throw some light on positional information mechanism of layers differentiation. lignin is a heterogeneous polymer that constitutes 30% of woody plant cell walls. microorganisms that degrade lignin are fungi, actinomycetes and to a lesser extent, bacteria. in case of industrial applications, the use of fungi is not feasible due to the structural hindrance caused by fungal filaments, requirement of particular culture conditions such as humidity, aeration which are not compatible with industrial processing environments. bacteria are worthy of being studied for their ligninolytic potential due to their immense environmental adaptability. environmental concerns and increasingly stringent emissions standards have led the pulp and paper industry to devise ways to decrease the level of chlorinated lignin residues in its effluents through both production process changes and improved treatment technologies. bleaching with the enzymes is the most promising because the enzymes may be very efficient, and can be used under industrial conditions. the main objective of this study was to investigate the adequency of klebsiella pnuemonia gst (glutathione-s-transferase) pretreatment for bleaching of calabrian pine kraft pulp. for this purpose the following conditions were investigated: enzyme loadings from 3 to 10 u/g pulp basis and the consistecy of the pulp was between 3 and 10%. enzyme at the desired concentration was added to the pulp and the mixture was incubated at 40°c for 2 hours. after the enzymatic pretreatment to determine the optimum conditions the kappa number of all reactions were analyzed according to tappi standarts. as a result of this study we determine the optimum conditons as 5% pulp consistecy, 8u/g enzyme for pulp treatment. after the enzymatic treatment carried out under optimum conditions we are planning to submit a short bleaching sequence and analyze for physical properties such as viscosity and brightness. owing to this bleacing sequence we are going to able to compare the enzymatic and chemical treatments of pulp in bleaching prosess. p-02.09.1-006 biochemical characterization of lipase from bacillus subtilis strain a10 from olive waste water f. ay sal, m. kac ßagan, s. c ß anakc ßi, a. o. beld€ uz karadeniz technical university, trabzon, turkey lipases (triacylglycerol acyl hydrolases, ec 3.1.1.3) are regarded as mild and environment-friendly biocatalysts for triacylglycerols hydrolysis. in addition to this hydrolytic reaction, they also catalyze reverse reactions of esterification, transesterification, and interesterification in non-aqueous environments. substrate, stereo-, regio-and enantio-specificities, and chiral selectivity are certain unique attributes of lipases that make them industrially attractive. these properties are often exploited in the manufacturing of detergent formulations, synthesis of fine chemicals, useful esters and peptides, food processing, paper manufacturing, degreasing of leather as well as in bioremediation. in this study, lipase from bacillus subtilis strain a10 is partially purified and characterized. bacillus subtilis strain a10 is isolated from olive factory from soke (aydin, turkey) and identified with 16s rrna analysis. lipase activity is screened on petri supplemented with rhodamine b. bacteria was grown in lb medium supplemented with 1% olive oil (vol/vol) for 48 hour at 37°c. after incubation, cells were harvested by centrifugation at 11,000 rpm for 5 minutes, resuspended in 50 mm tris-hcl (ph 8.0) buffer, followed by sonication with sartorius labsonic m to release intracellular proteins. q-sepharose is used as ionexchange column chromatography for lipase purification. effects of temperature on activity and stability were determined spectrophotometrically using p-nitrophenyl laurate as the substrate. effects of ph on activity and stability were also determined. the effects of various metal ions and other reagents on the hydrolytic activity were assayed at 37°c. the enzyme was active and stable in the broad ph range of 5.0-10.0 and temperature range of 24-50°c. bacillus subtilis strain a10 have high lipolytic activity. after cloning this enzyme to an expression vector and detailed characterization, this may suggests its usefulness in industrial applications. p-02.09.1-007 investigation of pin1 as a nuclear factor one binding partner s. saritas, a. e. yilmaz, a. kumbasar department of molecular biology and genetics, istanbul technical university, istanbul, turkey the nuclear factor one (nfi) proteins are important regulators of gene expression in the developing embryo and in adult stem cell niches. this transcription factor family has four members: nfia, nfib, nfic, and nfix. nfi proteins bind a consensus sequence on gene regulatory regions as homo or heterodimers. each member of nfi family has a highly conserved n-terminal dna binding and dimerization domain and a diverse proline rich c-terminal transcriptional activation/repression domain. as knockouts of nfi genes display distinct developmental phenotypes, we hypothesized that specificity of nfi protein function may arise from their interactions with binding partners. a yeast-two hybrid screen identified protein interacting with never in mitosis a1 (pin1) as a potential nfib interactor. pin1 is a ubiquitously expressed protein that specifically recognizes and binds to a phospho-serine or a phospho-threonine followed by a proline (ps/pt-p motif), and catalyzes isomerization of peptidyl-prolyl bonds. interestingly, both n-terminal and c-terminal domains of four nfi isoforms contain several conserved putative ps/pt-p motifs and some of these are reportedly phosphorylated. we looked for nfi pin1 interactions in vitro by gst-pulldown and co-immunoprecipitation assays. while gst-pin1 fusion protein interacts with all of four nfi isoforms, it binds nfib most strongly, nfia and nfic moderately, nfix most weakly. moreover, deletion of the cterminal domain leads to loss of nfi affinity for pin1 implicating this domain in nfi-pin1 interactions. co-immunoprecipitation assays where we co-expressed various epitope tagged nfi and pin1 proteins in hek 293t cells showed that pin1 precipitates nfib, as well as other nfi isoforms and nfib can, in turn, precipitate pin1. we are currently carrying out site-directed mutagenesis on nfib to identify the specific residues that pin1 recognizes. we will further explore if this interaction regulates nfi function during embryonic development. that pre-adapt migrating fish to the life in seawater. among others, smoltification induces intense growth of fish that enter the ocean at a size where risk of predation is significantly reduced. skeletal muscle growth depends on a tightly controlled balance between protein synthesis and degradation. protein synthesis driven by hormone regulation is well studied in smoltified atlantic salmon; while less is known on protein degradation occurring via a number of pathways including cytosolic ubiquitin-proteasome system and calcium dependent calpains. the aim of this study was to compare calpain and proteasome enzymatic activities in the skeletal muscles of s. salar parr, pre-smolts and smolts. calpain and proteasome activities were determined by casein or suc-llvy-amc hydrolysis in the skeletal muscles of s. salar from indera river (kola peninsula, russia). our results demonstrated the significant differences in studied protease activity levels between parr and smolts. calpain and proteasome activities in s. salar smolt muscles showed a significant drop compared with that of parr. the negative correlation between proteases activity levels in the muscle tissue and overall fish growth rate was shown. so, our data indicated life stage specificity in skeletal muscle protein degradation capacity in migrating fish. we suppose that intense muscle growth in s. salar pre-smolts is supported by various mechanisms including accelerated muscle protein accretion through the reduction of protease activities. obtained results enhance our knowledge in the mechanisms of atlantic salmon smoltification. the work was supported by the russian scientific foundation, project no. 14-24-00102. p-02.09. [1] [2] [3] [4] [5] [6] [7] [8] [9] the sociodemographic characteristics of the pregnant women who double and triple prenatal screening test h. d€ ulger, s. yabanci€ un meram medical faculty, n.e.university, konya, turkey double and triple prenatal screening tests which are applicable during first and second trimesters of pregnancy predict existent abnormalities at early stage. the aim of this study is to investigate the relationship between positive results of double and triple tests, further confirmatory tests during prenatal phase, postnatal status of babies and maternal age. in this study, double and triple test results of pregnant women who were admitted to meram faculty of medicine during 2009-2013 period were scanned from archive and test results indicating risk were detected. from these results, those which were above cut-off values for down syndrome, trisomy 18, open spina bifida were determined. a questionnaire was carried out with voluntary participants by reaching to these individuals. positive-negative result ratio of all double and triple test results and sociodemographic features such as age, occupation, presence of consanguineous marriage were investigated. all data from archive and answers from survey questions were assessed statistically. participants of the study were 18 to 46 years old and their average age was 29.89 ae 6.56. 219 ofthem (69.30%) were under 35 years of age whereas 97 of them (30.70%) were above 35 years of age. number of pregnancies were scaling between 1 to 13 with an average of 2.56 ae 1.56. 207 of 316 mothers (65.50%) were not undergone amniocentesis, whereas 6 babies with chromosomal abnormalities were detected among 109 mothers who were undergone amniocentesis. in conclusion, there may be regional, sociological and such that reasons for those who were not undergone amniocentesis despite positive double and triple test results. 6 (5.50%) chromosomal abnormalities were detected among pregnancies with increased risk assessment with positive double and triple results. p-02.09. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] the effects of oil on the growth and development of amphibians l. sutuyeva, t. shalakhmetova, a. ondassynova al-farabi kazakh national university, almaty, kazakhstan currently, the pollution of ecosystems by oil and oil products is increasing everywhere. the oil gets into water and ground during oil production, transportation and accidents. as a result, terrestrial animals and hydrobionts are exposed to oil contamination. thus, populations of animals decline. it can be assumed that the most sensitive to the effects of pollutants are animals in early stages of development. amphibians have established themselves as the most convenient bioindicator species. since lake frog (rana ridibunda) and green toad (bufo viridis) are the bioindicator species in kazakhstan, the study of the effects of oil on their larvae was carried out. we used water-soluble fraction of the oil from zhanazhol field (aktobe region) in our test. the larvae of control group were kept in pure water, and larvae of test groupsin aquariums with 0.05, 0.5 and 1% concentrations of the oil fractions. the concentrations were chosen in accordance with the level of pollution of kazakhstan's water bodies with oil. mortality of larvae, morphometric parameters and morphogenesis were studied. it was found that high mortality of larvae is the most visible reaction when exposed to oil. this indicator rose noticeably depending on the doses (0.05, 0.5% and 1%) in both species with percentages 16%, 51% and 78% in r. ridibunda and 22%, 61% and 83% in b. viridis, respectively, while in the control group it was about 10%. furthermore, delayed larval development was detected. thus, the larvae from the control and 0.05% oil group reached gosner stage (gs) 36, tadpoles from 0.5% and 1% groups were at gs-32 and gs-29, respectively, by the 30th day of life. moreover, behavioral abnormalities (sluggish movements) and decreased sensitivity to mechanical stress (touch) were observed under the influence of high concentrations of oil fractions. thus, oil in low concentrations alters the growth and development of tadpoles of anurans, and causes their increased mortality in high concentrations. p-02.09.1-011 effect of catechin loaded plga nanoparticles on glioma cell line histone h1t is a linker histone which binds to dna and contribute in chromatin condensation as well as regulation of specific genes through spermatogenesis. replacement of this histone h1 subtype and hyperacetylation of histone h4 tail, facilitate the replacement of histones with sperm chromatin condensing proteins of tnps and prms. ethical approval and informed patient consent was gained from 12 infertile men referred to royan institute. testicular biopsies were collected from patients through assisted reproductive techniques (art) procedure. based on pathological results samples were classified into the following three subgroups: obstructive azoospermia (as positive control), complete maturation arrest and sertoli cell only syndrome (negative control). chromatin of tissues evaluated for presence/absence of histone h1t protein in regulatory regions of tnps and prms genes using chip-real time pcr. results showed lower incorporation of h1t protein on regulatory regions of tnps and prms genes in two spermatogenic failure group versus positive control. in this study, it can be concluded that the decreased levels of h1t histone variant in testis tissues and failure in chromatin condensation have significant association with male infertility. p-02.09.1-013 serum dickkopf-1 levels in obese children and adolescents that obesity is detrimental to bone health despite potential positive effects of mechanical loading conferred by increased body mass on bones. the wnt/b-catenin pathway is essential for normal osteogenesis. serum dickkopf-1 (dkk-1) is one of the most important inhibitors of the wnt//b-catenin pathway. the aim of this study was to investigate the serum dkk-1 levels in obese and non-obese children and adolescents. materials and methods: the study included 30 obese children and adolescents (14 males and 16 females) aged from 7 to 17 years and 30 healthy normal-weight controls (13 males and 17 females) aged from 6 to 17 years. serum dkk-1 levels were measured by elisa method using commercially available kit. results: body mass index of the obese children was significantly higher than that of non-obese children (p = 0.000). however, there was no significant difference between dkk-1 levels of the groups. (these results are preliminary and the study is continuing). discussion and conclusion: our result showed that serum dkk-1 levels were not changed in obese children and adolescents. p-02.09.1-014 transcriptional regulation of cdo by nuclear factor one proteins b. kutay, c. lektemur, v. g€ uler, a. kumbasar department of molecular biology and genetics, istanbul technical university, istanbul, turkey nuclear factor one (nfi) transcription factors play important roles in regulation of central nervous system development. three of the four members of nfi family, nfia, nfib, and nfix are expressed in neural progenitors, as well as neurons and glia in the embryo. inactivation of these genes in mice show that they function in development of neocortex and hippocampus in the forebrain, cerebellum, spinal cord and precerebellar nuclei of the hindbrain, regulating neurogenesis, gliogenesis, as well as neuronal migration, axonal outgrowth and guidance. all three neural specific nfis are expressed in precerebellar neuroprogenitors, however, only deletion of nfib leads to a delay in development of precerebellar neurons. investigation of misregulated genes in nfib à/à precerebellar neuroprogenitors identified cell adhesion associated, oncogene regulated (cdo) as a potential downstream target of nfib. interestingly, this gene has been reported to be upregulated in nfia à/à hippocampus as well. cdo, a cell surface glycoprotein of the ig superfamily, has been found to regulate neurogenesis in vivo, is highly expressed in the developing brain and can induce neural differentiation by promoting heterodimerization of basic helix loop helix transcription factors with e proteins. bioinformatic analysis of the 5 kb human cdo promoter region identified five nfi binding sites: one cluster in the first 1 kb region, another in the 3.5 kb upstream region. electrophoretic mobility shift and supershift assays showed that nfib binds to all five sites. furthermore, nfib, along with the other neural nfis, inhibits the proximal cdo promoter driven luciferase activity by up to 85% in hek293t cells. preliminary data indicate that nfis bind to sites in both clusters in human neural stem cells (hnscs) suggesting that these sites are functional in vivo. we are currently investigating this possibility through nfi overexpression and silencing experiments that will examine regulation of cdo in hnscs. differentiation. the aim of this study is to investigate bdnf and drd2/ankk1 gene variants in eos development. in this study, 111 eos patients and 138 healthy controls were used. genomic dna extraction was performed from peripheral blood leukocytes. drd2/ankk1 taq1a (rs1800497) and bdnf val66met (rs6265) polymorphisms were determined by real-time polymerase chain reaction (rt-pcr). positive and negative syndrome scale (panss) was used to determine eos severity. for drd2/ankk1 rs1800497 polymorphism, there was a significant difference in the genotype frequencies between patients and controls for the co-dominant model (p = 0.05, or = 1.723; 95% ci: 0.996-2.98). however, no significant relationship was observed in the genotype frequencies of bdnf val66met polymorphism between eos patients and controls (p = 0.489). these results indicate that, drd2/ankk1 rs1800497 genotypes may affect eos development. however, bdnf val66met polymorphism may not be associated with eos. lack of association of bdnf val66met polymorphism may be due to limited number of patients. our findings need to be confirmed by further studies. various dyes used in the textile industry are discharged in large quantities to the receiving environment in the manufacturing process. this is the beginning of a process that is difficult to compensate for environmental and human health. therefore, contaminated areas should be cleaned. in addition, technologies with high polluting potential should be integrated with biological approach and thereby the impurities consisting of dyes should be reduced. in this experiment; burdirect black meta konz (c.i. direct black 38) was intended to decolorization using laccase. firstly, enzymatic decolorization of the dye was determined using spectrophotometry. the wavelengths of maximum absorption of burdirect black meta konz (c.i. direct black 38) was determined between 200 and 800 nm. then, optimization studies have been done. for optimization studies; dye concentration, laccase activity, ph, buffer concentration, temperature, mediator effect, mediator concentration and time parameters were determined. lastly, in optimal conditions, atr-ftir and gc-ms analyzes of ensuring decolorization of dye were analyzed. decolorization of burdirect black meta konz (c.i. direct black 38) was performed successfully and the absence of any metobolite in the decolorization medium has been provided by atr-ftir and gc-ms analyzes. assessing in terms of application, it can be easily applied by provided the reaction conditions in textile factories. laccase is a tool of decolorization of dyes in environmental friendly process. thus for the development of spermatids into mature sperm able to fertilize the oocyte.one of the causes of male infertility is in fact impaired sperm fertilization capacity due to sperm chromatin abnormalities and aberrant protamine replacement.recent research has focused on protamine biology,including protamine gene and protein structure,mechanisms of protamine expression regulation and involvement of the protamines in male fertility.various studies reported abnormal expressions of protamine (prm) genes in sperm of infertile men.the aim of the study is to investigate the gene expression of prm1, prm2 and their relationship with defective spermatogenesis. materials and methods: this study has been performed on 50 infertile and 3 fertile turkish men.total rna was extracted from the sperm pellet using trizol reagent.after rna extraction and cdna synthesis,real-time quantitative polymerase chain reaction (rt-qpcr) was used to determine the expression of prm1 and prm2. results: distinct levels of spermatozoal prm1 and prm2 mrna were found in infertile patients compared to fertile control groups.we found that the mrna levels of prm1 was reduced in 23 (%47), and the mrna levels of prm2 was reduced in 33 (%64) out of 50 infertile patients.in the current study,we found statistical significant association between the prm1 expression and infertility (p < 0.05).although prm2 gene expression was decreased in most of infertile patients compared to fertile control groups,the differences between the groups were statistically insignificant (p > 0.05). discussion: the results of the study suggested that, the protamine expressions which were associated with spermatogenesis may be important in infertility treatment. further studies are required in a large series of different populations to clarify the role both prm1 and prm2 themselves and their mrna expression on male fertility. the study was conducted to characterize the processes of muscle growth in atlantic salmon (salmo salar l.) of different ages inhabited rivers indera and varzuga (kola peninsula, russia) in summer and autumn. the expression levels of genes myosin heavy chain myhc, myostatin (mstn), and myogenic regulatory factors myf5, myogenin) in white muscle were studied in salmon parr of age groups 0+, 1+, 2+ in june and october. the changes in expression levels of mrfs, myhc and mstn indicating the extent of hyperplasia, hypertrophy, and restriction of muscle growth at different ages of parr were revealed. the pattern of age-related changes differed between seasons. especially, the expression of genes myod, myogenin and myhc peaked in yearling parr (1+) in summer, that indicated the high rate of hyperplastic and hypertrophic muscle growth in yearlings (1+). at the same time, the mstn expression level, the negative regulator of muscle growth, was highest in parr at age 1+. possibly, it is the necessary regulation mechanism to attenuate hyperplasia and hypertrophy and control muscle growth. in autumn, the expression level of myhc and myogenin were higher in salmon of age 0+ and 1+ then in 2+, indicating the higher intensity of hypertrophy in parr at both first ages in comparison to 2+. there was no differences in expression level of myod, myf5 and mstn between age groups in autumn. moreover, the expression levels of genes studied were lower in autumn than in summer. thus, it indicated the decrease of muscle protein synthesis and muscle growth rate in autumn. these findings expand knowledge on age-and season-related features of muscle development in young atlantic salmon in their natural habitat. the study was supported by the grant of the russian science foundation no. 14-24-00102. p-02.09.1-020 lmp2 and lmp7 gene polymorphisms in the southeastern anatolia population of turkey d. mihc ßioglu 1 , f. ozbas gerceker 2 1 sanko university, gaziantep, turkey, 2 gaziantep € universitesi, gaziantep, turkey introduction: the low molecular weight polypeptide 2(lmp2) and low molecular weight polypeptide 7(lmp7) genes are located in the class ii region of the major histocompatability complex (mhc) locus on chromosome 6. these genes encode peptides forming the large components of the proteosome complex which degrades short-lived cytoplasmic proteins. due to the significant role of lmp products antigen presentation, these genes can be accepted as strong candidates of susceptibility factors for different diseases. population genetic studies can also contribute to understanding of the possible role of lmp gene polymorphisms. the aim of this study was to determine the allele and genotype frequencies of the lmp2 and lmp7 gene polymorphisms in southeastern anatolia population and to compare these with the frequencies in other populations previously reported. material and methods: a total of 110 healthy and unrelated individuals participated in this study. polymorphism analyses were done by polymerase chain reaction (pcr)-restriction fragment length polymorphism (rflp) method and allele/ genotype frequencies of lmp2 and lmp7 genes were determined. results: a deviation from the hardy-weinberg equilibrium (v2 = 17.97,p < 0.05) was found for the genotype distribution of lmp2 gene polymorphism, while the lmp7 genotypes found to be distributed (v2 = 0.43,p > 0.05). discussion: available allele frequency data for different populations were used to calculate genetic distances and to construct a neighbor-joining tree. among the included populations, nahuas (mexico) population was found to have the lowest genetic distance from the southeastern anatolia-turkey population. conclusion: it can be concluded that, more studies using different types of genetic markers are needed to clarify the filogenetic relationships of southeastern anatolia population with other populations and also the number of population studies on lmp2 and lmp7 genes should be increased to understand their effects as a genetic marker. p-02.09.1-021 investigation of in vitro antioxidant activity of quercetin loaded plga nanoparticles pharmacological effects. but its usage is restricted because of low aqueous solubility, poor bioavailability, poor permeability and instability in physiological medium. these problems can be overcome with encapsulation of quercetin into nanocarriers such as biodegradable plga based nanoparticles. polymeric nanoparticles which have 1-1000 nm particle size and providing controlled released of biological active agent are prepared by using biodegradable and biocompatible polymers. in this study, encapsulation of quercetin molecules into plga nanoparticles was carried with using the single emulsion (w/o) solvent evaporation method. size measurements of the obtained nanoparticles were performed by zetasizer and their size were found 882. 1; 189.25; 436 .9 nm respectively. the morphological features were examined by sem images. antioxidant activities of q5, q7 ve q10 nanoformulations have been investigated by dpph and no (nitric oxide) methods. it is thought that the nanoparticular formulations that is developed in this study can be useful model for the other antioxidant molecules and will provide a significant contribution to the food and pharmaceutical industry. "this research has been supported by yıldız technical university scientific research projects coordination department. project number: 2014-07-04-gep03". p-02.09. the effect of environmental enrichment on spatial memory and certain nmdars, and 5ht2a expressions in rat pups introduction: the aim of the study was to investigate the effect of environmental enrichment exposed during whole childhood on spatial learning and memory and certain nmdars, and 5ht2a in the hippocampi of pups. materials and methods: four-weeks old, male, weaning rats were randomised into 2 groups as enviromental enrichment (ee, n = 12) and standard cage control (scc,n = 12) groups. eeg housed in an enriched environment and sccg were kept in standard cages for 8 weeks. following the experiment the rats were trained and tested in the morris water maze (mwm) , open field test (oft) and forced swim test (fst) in order to assess the neurobehavioural effects of ee. nr2a, nr2b, 5ht2a protein levels were analyzed by western blotting from hippocampi of rats. results: the positive effect of ee was seen at the learning phase in the mwm as 'latency to locate the hidden platform' between groups thoughout the 4 training days showed that eeg located the hidden platform significantly earlier than sccg on days 2, 3 (p = 0.006, p < 0.0001). also eeg significantly spent lower time in the outer zone of the maze on days 2, 3 which was the sign of low anxiety level (p = 0.011, p = 0.049). the parameters of oft which indicated increased locomotion, exploration and low anxiety were significantly higher in eeg (p < 0.05), in fst comparison of groups showed no difference (p > 0.05). the levels of nr2b and 5 ht2a were significantly increased as compared to sccg as well (p < 0.0001, p = 0.003). discussion & conclusion: these findings showed that exposure to ee throughout the whole childhood causes several neurobehavioural effects like increased exploration and low anxiety. these effects may lead to improvement in speed of learning. increase in the nr2b and 5ht2a concentrations which are the receptors that are related to learning and memory in the hippocampi accompanied these changes which may be basis of the neurobehavioural improvements or may provide contribution to positive neurobehavioural effects. p-02.09.1-023 effects of monosodium glutamate exposure during prepubertal term on several biochemical parameters in rats h. i. b€ uy€ ukbayram, d. kumbul doguc ß, i. ilhan, a. y. ismail s€ uleyman demirel university, isparta, turkey monosodium glutamate, which is commonly used in processed foods as flavor enhancer, is considered 'generally recognised as safe' by fda; however many studies have revealed the negative effects of msg.we aimed to evaluate the effects of msg in childhood on several serum parameters. sixty-six rats, (4 weeks old) were divided into 3 groups as control (cg, n = 22; 11 + 11, male+female) , experiment 1 (msg-low dose, e1g, n = 22; 11 + 11, male+female) and experiment 2 (msg-high dose, e2g, n = 22; 11 + 11) groups. msg was administered at 25 mg/kg/d to e1g, 2.5 g/kg/d to e2g for 6 weeks by oral gavage. the rats were sacrified and blood samples were collected from aorta. the blood samples were centrifuged, the serum samples were separated and glucose, alt, total protein, albumin, creatinine, cholesterole and triglyceride levels were analysed by beckmann au 5800 autoanalyser. level of total protein was significantly increased in e1g and e2g groups when compared to cg (p < 0.05). level of alb€ umine was also increased in both egs but significant difference was seen in e2g as compared to cg. creatinine levels were significantly increased in egs when compared to cg (p < 0.05). although the glucose levels in both egs were increased, the increase in e2g was statistically significant (p < 0.05). the alt levels of in egs were also increased but the significant increase was seen in e2g (p < 0.05). the effect of msg seem to be dose dependent and especially effect on carbonhydrate metabolism. increasing doses caused increase in glucose level, and tendency to glucose intolerance. increasing doses of msg also caused increase in creatinine and urea. another apparent effect of msg was detected on alt activity. in conclusion the negative effect of msg on glucose level, liver and kidney functions depends on daily dose intake. consumption of msg seem to be inevitable it has to be restrained in children otherwise early metabolic problems may be future problems for these children. (700 mg/kg) + tartrazine (750 mg/kg) + brilliant blue fcf (600 mg/kg) + ponceau 4r (70 mg/kg) + azorubine (400 mg/kg) + indigotine (500 mg/kg) + erythrosine (10 mg/kg). artificial food color mixture were administered to g 2 and g 3 and drinking water was applied simultaneously to g 1 by oral gavage per day for 3 weeks. after application all rats were sacrificed, the total oxidant (tos)/antioxidant (tas) capacity were analyzed in rats' brain, liver, kidney homogenate and serum with rel tos-tas diagnostics assay kit.the statistical analysis was carried out by using kruskal wallis test. tas and tos levels in liver homogenate were not found significantly different between all groups (p > 0.05). in serum and kidney and brain homogenate, tas levels were not significantly different between all groups. tos levels in g 3 were higher than g 1 and g 2 in serum and kidney and brain homogenate (p < 0.05). exposure to synthetic food colors may increase oxidative stres in vitale organs such as brain, kidney in female rats. these alterations differ according to organ and dose. parallel with increasing trends on healthy eating habits, consumption of prebiotics and probiotic microorganisms have been popular due to their benefits on human health. functional dairy foods such as probiotic yoghurt and cheese are the most common foods including probiotic microorganisms. due to some considerations such as standardization and quality in bulk production, starter cultures are used in industrialised fermentative food production to start fermentation. starter culture basically refers the microorganisms which induce and maintain fermentation of the fermentative foods and starter cultures including probiotic microorganisms are called as probiotic starter cultures. in this study, probiotic cheese starter cultures as a microbial community were investigated using computational systems biology tools. a metabolic network model of probiotic cheese starter culture was reconstructed using microbial community network modeling approach. literature-based genome-scale metabolic models of commonly used lactic acid bacteria were used for the microbial community metabolic model. the microbial community metabolic model simulated metabolic interactions of the microorganisms in the probiotic starter culture. metabolic flux values computed by the metabolic network model also predicted the metabolic pattern of the glycolysis (conversion of lactose), lipolysis (conversion of fat) and especially amino acid catabolism which are associated cheese flavor metabolism. simulations obtained by metabolic network-based analysis of cheese starter cultures can also be used for other fields like genetic engineering, upstream processing of the functional cheese production. p-03.01.1-002 er quality control protein network in cf to modulate f508del-cftr rescued phase ii xenobiotic metabolizing enzymes convert parent compounds into more hydrophilic metabolite by catalyzing conjugation reactions including glutathione and amino acid conjugation, glucuronidation, sulfation and acetylation. this study was aimed to describe the best cell line model for studying phase ii xenobiotic metabolizing nqo1 and gst-pi enzymes. for this purpose, mrna and protein expression of nqo1 and gst-pi enzymes were analyzed in ht29 and sw620 (colon); hepg2 and huh7 (liver); pnt1a and pc3 (prostate) cell lines by qrt-pcr and western blotting techniques, respectively. protein expression analysis revealed that nqo1 protein was expressed in all cell lines and relative protein expression is highest in the hepg2 (100%) and pnt1a (97%) while huh7 (31.9%) showed relatively low expression of nqo1. in addition, nqo1 mrna expression was relatively high in ht29 (1.68 fold) and pnt1a (1.92 fold) when compared with liver cell line hepg2 (1.00 fold). gst-pi protein expression was found very high in huh7 (100%) while there was no expression in hepg2. gst-pi mrna expression was relatively higher in pnt1a (3.56 fold) and ht29 (2.47 fold) when compared with huh7 (1.00 fold). according to these results, choosing the best cell line as model depends on the purpose of the research. for studying metabolism of a chemical by nqo1 and gst-pi or effect of a chemical on translational regulation of these enzymes, it is better to consider protein expression of the cell lines for choosing best model. however, if the aim is to study effect of a chemical on transcriptional regulation of these enzymes, it is better to choose a cell line that expressing highest mrna of gene of interest. in conclusion, considering both mrna and protein expression levels together, the best model cell lines for studying phase ii xenobiotic metabolizing nqo1 and gst-pi are ht29 and huh7, respectively. p-03.01. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] the studies on the pancreatic cells' surface glycoconjugates profiles in rats fed with high fat with lectin labelling methods by flouresans microscopy y. mater, s. beyhan ozdas gebze technical university, kocaeli, turkey in this study, the backbone of the cellular adhesion-recognition mechanism, located in the cell membrane. the study material selected pancreas tissue, has a privileged structures. the pancreas is one of the main organs to aid in digestion. the pancreas functions as an exocrine gland and role in digestion. in addition, the pancreas also functions as an endocrine gland, secreting several hormones into the blood that control the blood levels of glucose and other nutrients. due to the pancreas have been selected for this unique feature. thus, different types of cells in the same sample will be able to study the structure of the surface glycoconjugates. generally researches about determination of carbohydrates in the cell, glycoproteins or/and glycolipids are cut with enzymes. next step, the oligosaccharide mixture obtained, than establishing the complete structure of oligosaccharides and polysaccharides requires determination of branching positions, the sequence in each branch, the configuration of each monosaccharide unit, and the positions of the glycosidic links. this is a more complex problem than protein and nucleic acid analysis. these processes are indispensable for the understanding of the chemical structure of the sugar. whereas in cells using labeled lectins specific sugars, it is possible to accurately determine. in this study, was used triticum vulgaris (wga) labeled with fluorescein (fitc). thus, the cells located on the cell surface and neu5ac (sialic acid) for wga sugar residues were investigated. according to preliminary results of this study, wga labeled with fitc is specifically binding of these sugars. when this study is completed, the differences of sugar on the surface of different type of cells in the pancreas can be distinguished in micrographs. thus, in the cells of the pancreas, the sugar units involved in adhesion-recognition can be possible to determine specifically. large scale gene networks could be topologically analyzed in order to obtain possible global system-level structure cancer gene co-expression networks can have lower connectivity as compared to normal samples. using colorectal tissue gene expression datasets, we observed that tumor specific networks are less connected than normal networks. functional enrichment analysis suggested that cell cycle genes and methylation-associated cell adhesion genes can specifically play a role in the connectivity loss of carcinoma samples. literature confirmation provided a gene network including significant genes playing roles in the intersections between cell cycle, cell adhesion, and cell skeleton dynamics. this network can provide novel insight to our understanding of the molecular mechanisms of colorectal cancer. p-03.01.1-012 tf-mirna circuits specific to epithelial cancers y. oztemur, a. aydos, b. gur dedeoglu ankara university biotechnology institute, ankara, turkey cancer is the most common cause of death in the world but there are still a lot of uncertainties about the exact mechanism taking roles in regulation of it. cancers can be classified according to cell type; in which they start. carcinomas are the most prevalent types of cancer and start in epithelial tissues. they are also named as epithelial cancers (ecs) and make up about 85 out of every 100 cancers. over the past few years, many studies are concentrated on mirnas, which have emerged as important regulators of gene expression like transcription factors (tfs). tfs are regulators at transcriptional level while mirnas are post-transcriptional regulatory key-elements. otherwise the transcription of mrnas and mirnas are known to be regulated by tfs and tfs are the targets of mirnas. therefore, it is crucial to characterize the relation of tfs, mirnas and their targets by building circuits in diseases such as in ecs. for this study, mirna and mrna expression studies including epithelial tumors and normal samples searched in geo and array express microarray databases. 4 mrna studies and 4 mirna study, which were designed for 4 different ecs (breast, lung, ovary and colorectal) were selected to be analyzed. differentially expressed (de) mrnas and mirnas between epithelial tumors and normal samples were extracted (p ≤ 0.05, 2 fold change). among de genes, transcription factors and mirnas were identified and listed for epithelial tumor vs. normal comparison. circuit analysis resulted with remarkable circuit, which was common for all the types of ecs that includes klf4 transcription factor and hsa-mir-145. in the literature hsa-mir-145 and klf4 are known as important regulators in different types of cancer, which indicated that the motifs involving tfs and mirnas might be useful for understanding the regulation of ecs. as a conclusion finding out new and common circuits may aid us in predicting new or alternative diagnostic and/or prognostic biomarkers for ecs. mesenchymal stem cells (mscs) are multipotent stromal cells that can differentiate into a variety of cell types which are used in cell therapy. although they are the most attractive cell type for cell therapy studies, primary mscs lose their differentiation potential with increasing time in culture and passage so they are of limited use. due to this disadvantage, msc lines are more suitable for in vitro researches owing to their immortality. in this study we compared primary bone marrow-derived msc (bm-msc) with bone marrow derived msc line (rcb2153) in terms of cell characteristics and gene expression profiles to determine the functional differences among mscs types. firstly, mscs were identified by using cd29, cd70, cd90 and cd105 as positive markers and cd34 as a negative marker. gene expression profilling was investigated using affymetrix hg-u133-plus2 arrays. the significant go biological process terms and kegg pathway enrichment analyses of the identified degs were performed using david (p < 0.001, fold change≥2). the analysis showed similar pathway clustering in both cell types. the resulting quantitave transcriptome of 754 genes were identified that differentially expressed in msc line versus primer mscs (538 upregulated and 216 down-regulated). functional classification of changed genes was mainly clustered in cell cycle, cell death and mismatch repair. kegg pathway analysis revealed that the genes were significantly enriched in pathways including "cell cycle, dna replication and focal adhesion" pathways. in conclusion, our results indicate that msc lines can be used instead of primary mscs. these quatitative results provide an important basis to adapt cell lines to more closely resemble physiological conditions as oppossed to animal experimentation. this could help to minimize the use of animals in research. p-03.01. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] association between loss of 18q21, gain of 20q13.33 and progression in sporadic colorectal cancer n. belder 1 , b. savas 2 , m. a. kuzu 2 , i. pak 3 , h. s€ umer c ß elebi 1 , a. ensari 2 , h. € ozdag 1 1 biotechnology institute, ankara university, ankara, turkey, 2 school of medicine, ankara university, ankara, turkey, 3 ankara oncology training and research hospital, ankara, turkey colorectal cancer (crc) is one of the most diagnosed cancer and the third leading cause of cancer deaths throughout the world. identifying of copy number variation (cnv) profiles between early and late stage cancers can be useful to understand the progression and aggressiveness of cancer. the main goal of this study was to construct a comprehensive insight of association between cnv and sporadic crc stages in order to identify novel candidate targets which may contribute to tumor progression. affymetrix 6.0 genechip snp arrays were used for characterization of cnvs in tumor and matched normal formalin-fixed, paraffin-embedded (ffpe) tissues from 5 stage i, 17 stage ii and 25 stage iii samples. paired cnv analyses were performed using partek genomic suite 6.6 and genomic segmentation algorithm was performed using a minimum of 10 markers per segment, a signal-to-noise ratio of 0.3 and the cut-off value for the gain and loss was set of 2 ae 0.3. the adjusted p-value ≤0.05 were considered to be significant. whole genome cnv analysis revealed that amplification of 20q13.33 with 4 genes was found the most frequent (76.5%) in stage ii tumors. the most frequent (36%) amplifications were 13q12.2 and 7p22.2 in stage iii tumors. while deletion of chromosome 18q21.2 in stage iii with a frequency of 36% was found the most frequent loss, deletion of 18q21.1 was seen the most frequent (64.7%) in stage ii tumors. two tumor suppressor genes smad2 and smad4 which are found in these deletion regions were common genes between stage ii and stage iii. our results showed that gain of 20q13.33 might have a significant role in the progression of cancer. loss of 18q21 comprising two tumor suppressor genes is also another important finding. 18q21 loss can be a significant prognostic value in colorectal cancer even though validation of target genes requires additional study and larger sample size. this work was supported by tubi-tak project no:109s477. p-03.01.1-016 meta-analysis based mirna signature discriminates cervical cancer from normal samples a. yucel polat, y. oztemur, a. aydos, b . gur dedeoglu biotechnology institute, ankara university, ankara, turkey gynaecological cancers are common problems in female health. squamous cell carcinoma (scc) is a type of these malignancies. this tumor type is derived from pre-cancerous lesions, which is called cervical intraepithelial neoplasia (cin). cin is classified as cin1, cin2 and cin3 according to their dysplasia grade in the cervical tissues. mirnas are small non-coding rnas that were shown to have important roles in the development and progression of various cancers. the aim of this study is identifying mir-nas, which are playing a part in progression of cervical lesions by a ranking based meta-analysis approach. two mrna and three mirna expression studies, which include normal, cin1, cin3 and scc samples were selected from arrayexpress and gene expression omnibus (geo) databases. three mirna studies were combined with anova dependent ranking based meta-analysis program which was developed in our laboratory to find out a mirna signature that can discriminate cin1, cin3 and scc samples from normal samples. the top five mirnas with the highest ranks in meta-list were selected for further analysis. predicted targets of these mirnas were identified by mirdb target prediction tool. additionally two mrna datasets were selected for mirna-target validation studies. common genes, which were obtained from meta-mirna targets and differentially expressed genes between normal and cin1, cin3 and scc groups from two independent studies, were identified and they were subjected to pathway enrichment analysis. pathway enrichment analysis that was performed with 338 common genes showed that these targets were significantly enriched (p < 0.05) in especially cell proliferation, cell survival and cell cycle pathways, which are the key players of cancer development and progression. the meta-analysis results together with validation analysis of their targets may point out the potential roles of mirnas as biomarkers for the diagnosis and the treatment of cervical cancer. p-03.01. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] the hypoglycaemic and regenerative activity of thymbra spicata in alloxanized-diabetic rats thymbra spicata (labiatae), a carvacrol and thymol containing plant, is one of the medicinal herbs used by diabetic individuals to reduce blood glucose in turkey. we investigated the hypoglycaemic and anti-lipemic effects of the aqueous extract prepared from dried leaves and flowers of this plant in alloxanized-diabetic rat model. rats were divided as: diabetic control (group 1), dia-betic+glibenclamide (group 2), diabetic+plant extract (group 3), untreated control (group 4) and control+plant extract (group 5) groups (n = 6 for each group). serum glucose, lipid levels and body weight changes were mesasured and pancreas and liver histology of the rats were examined. each rat in all groups were administered the plant extract (30 mg), and the reference drug glibenclamide (2 mg/kg) by gastric gavage every day for 8 weeks. in group 1, blood glucose, serum alt, ast, triglyceride, cholesterol and ldl cholesterol levels increased while body weights decreased. in group 2, serum glucose, alt, ast, triglyseride and hba1c levels decreased compared to group 1 while cholesterol and ldl levels were high. in group 3, serum alt, ast, trigliserit, cholesterol, ldl levels decreased significantly but serum glucose and hba1c were higher compared to group 4. body weights increased except group 1 and hdl levels were not altered. histologically degenerative changes observed on pancreas of group 1 were decreased in groups 2 and 3. there was no difference on liver histology of the groups. in conclusion, thymbra spicata showed a protective and regenerative effect on diabetic pancreas. the hypo-lipidemic effect of the plant extract was also more effective than glibenclamide possibly due to the flavonoids, saponins and triterpenoids contents in the extract. its hypoglycaemic and protective activity should be tested for different doses and extract preparations and for longer periods. our study suggests that thymbra spicata is an excellent candidate for future studies on diabetes mellitus. with three different transcriptome data sets from the public gene expression omnibus database: time dependent data of dphop mutant, dargr mutant and wild type strain. the dynamic data spanned both primary and secondary phases of the metabolism. statistical results of transcriptome data were used for reporter metabolite analysis and reporter pathway analysis, which identify the metabolites (or pathways) with a significant coordinated transcriptional change in response to gene deletion perturbation in phosphate and nitrogen metabolisms. further, the production of actinorhodin, a pharmaceutically important compound, was modeled in the two deletion strains by calculating the metabolic fluxes subject to transcriptional level constraints on enzyme-coding genes. the metabolic switch from primary to secondary metabolism was highlighted in terms of the activity of pathways and fluxes as a result of the computational analyses in this work, leading to a better understanding of the role of phosphate and nitrogen metabolisms in increasing production levels. introduction: as a member of legume family licorice (glycyrrhiza glabra l.) has been widely used by human kind for many years as food constituent. especially by folks in rural sites licorice consumed widely. beside food constituent licorice has been used for medical purposes as well. licorice found effective with scientific datas on peptic skin infections, ulcers, inflammation, eczema, alzheimer disease, liver disease, and cancers. it also has been used as natural sweetener and food additive for preparing candies, chewing gum and beverage since ancient times. like all other medicine it has not been free of adverse event or toxicological effects. material and methods: alcoholic extracts of plant obtained by maceration process. for in vitro examination of anti-oxidant profile of licorice dpph free radical scavenging, abts cation radical scavenging and cupric ion reducing antioxidant capacity assay applied. application of extract made by oral route to rats for a week. anti-oxidant profile has been evaluated by myeloperoxidase (mpo), arylesterase (ares), total oxidative stress (tos) and total antioxidant status (tas) of serum levels. determination of toxicological effects alt, ast, ldh and alp values studied. histological investigation applied on liver and kidney tissues. results and discussion: results compared with control and standarts. antioxidant potential of licorice has been observed by in-vitro assays. serum mpo and ares values also compared with in-vitro results and correlation between them has evaluated. toxicological investigations made after evaluation of ast, alt, ldh and alp values. conclusion: in vitro assays has showed that licorice has potential anti-oxidant effect. investigation revealed that a mild toxic effect of licorice by biochemical tests. toxicological profile compared with control group and alt, ast values found slightly decreased and a mild elevation has been seen in ldh and alp values. for further and detailed investigation is needed. p-03.01.1-020 on the applications of a metabolic network model of mesenchymal stem cells h. fouladiha, s. a. marashi, m. a. shokrgozar, m. farokhi mesenchymal stem cells (mscs) have several applications in tissue engineering and regenerative medicine. mscs can be very useful in stem cell therapy, because they can be isolated bone marrow or adipose of an adult. these cells have also been used as gene or protein carriers. therefore, maintaining them in a desire metabolic state has been the subject of several studies. here, we have used a genome scale metabolic network model of bone marrow derived mscs for exploring the metabolism of these cells. then, we try to validate the computational results by experimenal tests. we analyzed metabolic fluxes in order to increase stem cell proliferation using the metabolic model. consequently, the experimental results were in consistency with computational results. in the present work, the applicability of the metabolic model was successfully approved. therefore, this metabolic model can be useful in biomedical researches of stem cells. p-03.01.1-021 qtl analysis for body weight and fatness in bxd recombinant inbred mouse strains a. dogan 1 , c. neuschl 2 , r. alberts 3 , g. a. brockmann 2 1 school of medicine, istanbul kemerburgaz university, istanbul, turkey, 2 department for crop and animal sciences, humboldt-universit€ at zu berlin, berlin, germany, 3 helmholtz-zentrum f€ ur infektionsforschung, braunschweig, germany genetic variation in body weight and composition is under the influence of many genes and have different genetic architectures. in the present study, the genetic factors contributing to body weight and fatness were examined under energy rich feeding conditions. growth traits, lean and fat weight, fat mass gain were analyzed to map qtls in a set of bxd ri strains. genome-wide analyses were revealed several genomic loci that control body weight and associated bodily changes in a sex and age-specific manner. the genetic data provided evidence for significant qtls on chromosome (chr) 4, 5, 14, and 16. most likely candidate genes within or near the regions with the highest significance levels were identified. the genes 1700048f04rik, gbe1, a830060n17, and four genes cenpc1, stap1, uba6, gnrhr for example, are suggested as most likely positional candidates accounting for the qtl effects on chr 5 for fat mass, on chr 16 for fat mass gain and on chr 16 for lean weight, and chr 16 for body weight, respectively. our results showed that body composition and fatness are highly complex that many genetic factors regulating and suggested candidate genes, which may help for studies of human fatness. related to serotonergic and gaba systems in response to hormonal changes. the nutrients involved in neurotransmitter synthesis may be the cause of relationship between diet and premenstrual syndrome. therefore, the aim of this study was to investigate the effect of various nutrients and premenstrual syndrome. this study was conducted to 29 healthy women aged 20-37 years. participants were asked to fill in premenstrual assessment form. dietary intakes (three days in each phases) were recorded during premenstrual, menstrual and postmenstrual phases. energy, protein, amino acids, iron, calcium, and magnesium intakes were estimated. statistical analyses were performed using the spss software. friedman tests were conducted and differences were considered to be statistically significant for p-values lower than 0.05. 60.9% of the participants reported premenstrual symptoms and premenstrual symptoms related nutrient intake were increased in these women. it was determined that energy (p = 0.03) and protein (p = 0.001) intakes were higher in the premenstrual phase. during premenstrual phase; tyrosine (p = 0.002), isoleucine (p = 0.001), leucine (p = 0.001), lysine (p = 0.008), methionine (p = 0.008), cysteine (p = 0.008), tryptophan (p = 0.000), and glutamic acid (p = 0.005) intakes were higher than other phases. likewise, iron intake was higher on premenstrual phase (p = 0.054). on the other hand, intake of other potential premenstrual syndrome related nutrients like fat, cholesterol, calcium, magnesium, and vitamin b6 were not significantly different within the menstrual phases. amino acids including tyrosine, tryptophan, glutamine, and vitamin b6 are involved in neurotransmitter synthesis and might be related to premenstrual symptoms. consequently, elevated intakes of dietary protein and some amino acids during premenstrual phase may be related to premenstrual syndrome symptoms. until now far uv cd spectra of only two potexviruses were published. the papaya mosaic virus (papmv) spectrum, measured by leclerc and co-authors contained no obvious anomalies and was similar to the spectrum of isolated papmv coat protein (cp). but measured 30 years earlier by homer and goodmanfar uv cd spectrum of potato virus x (pvx) itself had anomalous character and differed strongly from the spectrum of isolated pvx cp. in the present work we measured far uv cd spectra for two more members of potexvirusgenus: alternanthera mosaic virus (altmv) and potato aucuba mosaic virus (pamv) and their free cps. the altmv virion and altmv cp spectra were similar to each other and to the spectra of papmv and its cp. the pamv spectrum resembled the pvx spectrum in anomalously low ellipticity of the negative band at 208 nm, but in contrast to pvx, did not have additional peak at 228 nm. homologous modeling showed that cp of the three viruses is very similar in the core structure, and the observed difference may be explained by differences in disordered parts of proteins. possible reasons of potexvirus structural variability are discussed and it is suggested that the intravirus potexvirus cps may assume different conformations in different virions of the same preparation or even along the length of one virus particle. this work was supported by the russian science foundation (grant 14-24-00007). the antimicrobial potential of different phenolics was tested on pectobacterium in search of possible mode of action. in this respect, biofilm formation, exoenzyme activity, gene expression and virulence on its natural host (potato, cabbage, calla lily) were performed. also computational approach to show interaction between phenolic compounds and target protein was carried out using docking tools. the virulence determinants of pectobacterium were significantly impaired, at compound concentrations that did not affect bacterial cell growth. these observations suggested a mechanism which specifically interferes with bacterial virulence. since, these virulence determinants in pectobacterium are controlled by quorum sensing (qs), we focused on the effect of phenolics on the qs system in pectobacteria. the study revealed an inhibiting effect of the tested compounds on the expression level of central qs system and controlled genes, using qrt-pcr. also, there was a prominent reduction in the level of qs signal molecules n-acyl-homoserine lactone (ahl) accumulation. in addition infection capability was also practically blocked, which was completely recovered by application of exogenous-ahl. these results were supported by a potential interaction of plant phenolics with qs targets, as shown by molecular docking tool. collectively, results suggest the potential interference of phenolic compounds with qs central components (expi/expr proteins). moreover, it holds potential for future development of control measures against pectobacterium, and possibly other pathogens with similar mode of virulence. saccharomyces cerevisiae has been a key experimental organism for the study of infectious diseases, including double-stranded rna (dsrna) viruses. the l-a dsrna virus family of s. cerevisiae is widely distributed in nature. several versions of l-a virus are described and new ones continue to be discovered. some s. cerevisiae strains along with l-a dsrna possess smaller dsrnas, called m satellites. these dsrnas encode a sole secretable protein, known as k1, k2, k28 and k-lus toxin. l-a genome encodes the gag major structural protein and gag-pol fusion protein, formed by ribosomal frameshifting. gag-pol has transcriptase and replicase activities are necessary for maintenance of both l-a and m satellite dsrnas. so far, it's not known whether certain l-a virus has evolved to maintain a distinct type of satellite dsrna or this phenomenon lacks inherent specificity. we developed universal strategy to obtain full length l-a and m dsrna genomes from s. cerevisiae. complete viral dsrna genomes can now be cloned, as evidenced by l-a-28 dsrna, analyzed and sequenced directly from any yeast strain by means of enzymatic manipulations on total or fractioned rna content. we have identified previously undescribed l-a variant from different yeast strains specifically associated with certain type of m satellites. moreover, we identified for the first time full 5 0 -utr and 3 0 -utr sequences of m2 satellite. highly conserved sequence regions along with variable fragments were discovered at protein level, revealing clear trend to form clusters among different l-a gag-pol proteins. the obtained data suggest that each l-a virus variant can specifically maintain a distinct type of satellite dsrna. p-04.01.1-004 physic-chemical characterization of plga adjuvants for immunization per os t. chudina, d. kolybo palladin institute of biochemisry of the national academy of sciences of ukraine (nasu), kyiv, ukraine antibodies against diphtheria toxin play the most important role in the immunity against corynebacterium diphtheriae. all current diphtheria vaccines have parenteral route of administration. undoubtedly, oral administration of antigens would be the most patient-friendly way of immunization. however, the efficacy of free antigens oral administration is limited by their degradation in the gastrointestinal tract and poor absorption by m-cells. biodegradable and biocompatible polymers, like poly (d,l lactide-co-glycolide) (plga), are widely used for the design of mucosal immunizing agents. importantly, that the way of particle preparation plays an important role in plga biodegradation and antigen release. the aim of this work was to characterize the main physic-chemical properties of two types of plga particles: with immobilized antigen (plga 1) and with encapsulated antigen (plga 2) . we have prepared two types of plga particles containing egfp-sbb proteins (non-toxic recombinant fragment b of dt fused with egfp). the antigen loading efficiency of particles was determined based on the ratio of protein concentration in solution before and after loading and shown better results for plga 2 particles (plga 1 -72.05%, plga 2 -90.02%). the flow cytometry results demonstrated that 99% of plga1 particles conjugated with egfp-sbb, and only 92.2% of plga 2 particles conjugated with protein.the particle sizes had the slight difference by the results of two different techniques (ntanumber based, the software tracks individual particles; dls -scattering intensity weighted), however demonstrate similar patterns. dls data showed that the mean plga 1 particles size was 203.3 nm and plga 2 -211.6 nm. nta data also showed that mean plga 1 particles size a little smaller than plga 2 (183.8 nm and 192.8 nm respectively) . demonstrated differences in the properties of synthesized particles may have an influence on the immunogenicity of the used for oral immunization antigen. p-04.01.1-005 a suitable system for studying the functionality of a plasmodial protein in mammalian cell lines cho-mt58, a mutant cell line was proved to be an appropriate tool for investigating intracellular function of cct. in this cell line, the endogenous cct activity decreases dramatically at 40°c, blocking membrane synthesis and ultimately leading to apoptosis. we have studied the rescuing potential of pfcct in cho-mt58 cells with the isogenic cho-k1 cells as a control. cells after transient transfection were incubated at 40°c and then analysed by facs using the fluorescence of egfp fused to pfcct. the proportion of cells undergoing apoptosis was determined by propidium-iodide staining. we have demonstrated for the first time that heterologously expressed pfcct is able to complement endogenous cct activity in mammalian cells. thus, a suitable system has been established for functional investigation of structural elements of pfcct. in order to reveal the role of different protein sequences in enzymatic function, we redesigned the structural gene of pfcct obtaining a modular system where different domains are easy to be removed or exchanged. here we designed a series of different truncation and deletion constructs to reveal the role of plasmodium specific sequences. in parallel, heterologous expression experiments of different constructs in the mutant cho-mt58 and the wild type control cell lines are performed to validate the reported model system. p-04.01.1-006 host-pathogen interactions: is there a relationship between tlr 4 polymorphisms and tuberculosis in a group of turkish patients? introduction: tuberculosis (tb) is a global health problem and according to world health organization (who) each year more than 2 million individuals die from tb and each year20,000 cases of tb are notified in turkey. malatya is the third largest city in east anatolian region of turkey and tb incidence rate is higher (28.5/100,000) comparing to the general population of the country. for this reason it is important to determine the factors that lead to tb in this population. disease agent can stay in the latent phase for long periods of time after infecting the individuals. while some infected individuals show the symptoms some others never do and even 90% of these never develop clinical disease. various mechanisms take place during the host response to infectious agents. toll-like receptor (tlr) genes are shown to be candidate genes in these responses. materials and methods: in this study 49 tb patients and 50 healthy controls were included. tlr 4 genotyping for rs4986790, rs4986791 was performed by using a commercial taqman snp genotyping assay kit. data were summarized by count and percent. hardy-weinberg equilibrium was tested by chi-square distribution with 1 df. differences between groups due to allelic and genotypic distributions were analyzed by pearson's exact or fisher's exact tests. in all comparisons significance level was considered to be 0.05. results: the single nucleotide polymorphisms (snps) which were subject of this study haven't been screened in turkish population earlier. no significant association was found between tb and the snps we screened in our group of patients. discussion and conclusion: unlike other populations results we couldn't find a significant association between the disease and the genotypes of our patients. the study should be performed in bigger populations in order to confirm the results. p-04.01.1-007 lytic action of bacteriophages as a tool for the obtaining of images p. boltovets 1 , r. radutny 2 , t. shevchenko 3 1 institute of semiconductor physics nas of ukraine, kyiv, ukraine, 2 scientific and technical center of advanced technologies nas of ukraine, kyiv, ukraine, 3 taras shevchenko national univercity of kyiv, kyiv, ukraine obtaining of images by different types of bacteria now became a very special branch of skill at the interface between science and art. however the authors did not found any scientific article, where bacterial lawn was used as the background and the image was formed by the lytic action of the virus (bacteriophage). whereas the mentioned approach could be used not only with artistic aims but for the practical use. the aim of this work was to demonstrate a possibility to obtain the image on the bacterial lawn by the lytic action of the bacteriophage. the bacterial lawn was obtained by the standard metod using the 1.5% agar with the nutrient medium and the 0.7% agar containing escherichia coli culture. stencils with the preparation of the bacteriophage t4 were applied. samples were incubated during the twenty-four hours at +37°c. after that stencils were removed and the samples were stained by coomassie blue r-250 or fuchsine (with further fixation by the 7% acetic acid). several approaches to obtain the image by the lytic action of the virus were applied. first of all stencils made from printing paper and filter paper were compared. it was demonstrated, that the use of filter paper stensil allows to obtain more accurate and controllable images, than the use of the printing paper stensil. in the next series of the experiment the possibility of the reversed stencil use (where the image is formed not by the lytic zone but by the zone of bacterial growth) was demonstrated. also the possibility of the partial staining of the obtained image was explored. it gives an opportunity to obtain polychrome images using available colorants. summarizing the above it should be noted, that it was the first time when the graphical image was obtained by the lytic action of the virus on bacteria. this approach could be used not only for the artistic aims but as well for the practical use, for example, for the restriction of the action of microorganisms in out-of-theway places. burgdorferi the identification and characterization of possible antigens is essential for the improvement of current laboratory diagnostics for lyme disease and vaccine development. in this study, several recombinant b. burgdorferi outer surface proteins have been obtained and their antigenic properties have been evaluated in an effort to characterize novel immunodominant antigens. because b. afzelii and b. garinii are the most prevalent species in latvian ticks, proteins with conserved domains were included in this study. a panel of serum samples of lyme disease patients with early and disseminate disease stage was used. the controls were matched by age and sex to the patients and represented the same geographic area. the results show that proteins of several b. burgdorferi gene families have properties with respect to their candidacy as a subunit assay for a novel lyme disease immunodiagnostic. especially, the difference in their size in a range on the western blot assay may provide good discrimination between protein bands. however, they have potential for diagnosis if used in combination with other antigens but not as a "stand alone" test. in conclusions, this study showed the existing challenges in serological testing of early lyme disease. the conservation of the sequence of antigen between species of b. burgdorferi complex is essential for the most successful serodiagnostic marker candidate. the presence of homologous proteins in treponema species could lead to the cross reactivity in syphilis patients, and should be carefully evaluated. antimicrobial resistance is one of the greatest challenges in modern medicine. there is a pressing need for better understanding of the specific mechanisms that contribute to resistance to optimize existing therapies. in 2013 in georgia extended-spectrum beta-lactamase (esbl)-producing e. coli strain was isolated from the post-surgical sample obtained from gallbladder of the patients with chronic calculous cholecystitis which belongs to the sequence type 23 (st23) complexes with ctx-m 55 gene. is this strain characterized by other differences on a proteome level? are antibiotics against which the strain is resistant inducing the changes in bacterial proteome? the present work was aimed (i) to study the differences on a proteome level (i) between e. coli 92-1917/13-g and attc e. coli-reference strain and (ii) to compare the proteomes of 1917 strain at two conditions: with and without antibiotics. 1917 strain was grown in the presence of three antibiotics: rocephin (ceftriaxone), fortum (ceftazydym) and claforan (cefotaxime sodium) together. proteomic expression was analyzed using two-dimensional gel electrophoresis and mass spectrometry. significant differences were found for several proteins, including putative abc trnsporter arginine protein 2, cystine-binding periplasmic protein, fkbp-type peptidyl-prolyl cis-trans isomerase, outer membrane protein a, d-galactose binding periplasmic protein and some others. the importance of these differences for anti-microbial resistance will be discussed. p-04.01.1-010 molecular characterization of resistance and virulence features in staphylococcus aureus clinical strains isolated from cutanaeus lesions in patients with drug adverse reactions i. lupu 1 , i. gheorghe 2, 3 , m. popa 2, 3 , a. ion 3 , m. mihai 1 , v. lazar 2, 3 , m. c. chifiriuc 2, 3 1 carol davila" university of medicine and pharmacy, bucharest, romania, 2 research institute of the university of bucharest-icub, bucharest, romania, 3 faculty of biology, university of bucharest, bucharest, romania patients treated with epidermal growth factor inhibitors often experience cutaneous adverse reactions. however, the infectious complications of these toxic effects and the contribution of specific pathogens, such as the community emergent methicillin resistant staphylococcus aureus strains. the present study was aimed to identify the types of sccmec and virulence genes profile in clinical s. aureus isolated from cutaneous lesions of different severity degrees in patients with dermatologic toxic effects. this study was conducted on a total of 42 s. aureus clinical strains isolated in 2016 from acneiform reactions pustulae and periungual lesions in patients with drug cutaneous adverse reactions. multiplex pcr was performed on genomic dna from isolates in order to identify the sccmeccentral elements and the virulence genes: bbp (bone bound sialoprotein), ebps (elastinbinding protein), fnbb, fnba (fibronectin-binding proteins), fib, clfa, clfb (clumping factors a and b), cna (collagen-binding protein), luk-pv (panton-valentine leucocidin), hlg (haemolysin), tst (toxic shock toxin). the mrsa phenotype was genetically confirmed by the presence of meci gene in case of 19.04%, meca in 14.28%, sscmec type ivd element in 11.90%, ccrb2 in 7.17% and sccmec types i, iii, iv in 4.76% of the studied s. aureus strains. regarding the virulence genes encountered in s. aureus strains, the most frequent was clfa (90.47% of the isolates), followed by clfb (88.09%), fib (35.71%), hlg (16.66%) and bbp (14.28%). these results confirm the high prevalence of mec i and sscmec type iv elements, usually encountered in communityacquired mrsa strains, in cutaneous isolates from patients with dermatologic toxic effects. more data on the virulence and genetic background of these local strains are needed to appropriately assess the risk of such infections and avoid the inappropriate administration of beta-lactams. p-04.01.1-011 analysis of toxicogenic properties of staphylococcus aureus strains isolated from cows with subclinical form of mastitis in the central area of russian federation. pore-forming toxins and enterotoxins), which are present in s. aureus strains isolated from clinically healthy cows. staphylococcus strains were isolated from cow's milk. disk diffusion method was used to determine the sensitivity to antibiotics. pcr analysis was used for detection of meca, mecc genes and genes of toxins. investigated strains were resistant to oxacillin (14%), vancomycin (8%) . it was found that all strains, which contain meca and mecc genes, showed resistant to more than 5 antibiotics. it was determined that among the investigated strains 13% contained meca, 13% -mecc, 9% contained both meca and mecc. some strains contained genes of panton-valentine leukocidin (pvl) or alpha-hemolysin and several strains contained both types of genes. enterotoxin a (sea) gene was detected in 26.7% of cases, sed -5%, seg -10%, sei -35%. genes of staphylococcal toxins b, c, e, h were not found. the presence of phenol soluble modulin biosynthesis genes was determined: genes of alpha peptide synthesis were found in 93% of strains, beta peptide toxin genes in 73%, delta toxin gene in 100%. it was determined, that clinically healthy animals are carriers of s. aureus strains that cause mastitis. high level of antibiotic resistance was found in strains containing meca and mecc genes. the major part of the strains carried genes of phenol soluble modulin biosynthesis. the role of phenol soluble modulins as well as of pvl and alpha-hemolyzin in the development of mastitis is not completely clear. we conclude that pore-forming toxins have dominant role in the latent form of mastitis. p-04.01.1-012 impact of lactoferrin on the hydrophobicity and adherence to the inert substratum of staphylococcus aureus strains isolated from patients with cutaneous drug reaction skin healing is a complex biological process that requires the involvement of different cell types and humoral effectors. one of the main factors are aggravating and delaying the healing process is represented by the supra-infection with pathogenic or opportunistic microorganisms that grow in specialized consortia embedded in a self-produced extracellular polymeric matrix, called biofilms, which are extremely resistant to any antimicrobials and host immune response. lactoferrin (lf) is an ironbinding glycoprotein which promotes skin healing by enhancing the initial inflammatory phase, but also by inducing an overabundant immune response. the aim of this study was to investigate the influence of lf, one of the main components of innate, humoral anti-infectious immunity on some microbial features, involved in the first steps of the infectious process, such as hydrophobicity and adherence of staphylococcus aureus strains isolated from maculo-pustular lesions in patients with adverse reactions to epidermal growth factor inhibitors. for hydrophobicity measurement the bacterial suspensions were grown in the presence or absence of lf, and then, the "microbial adherence to hydrocarbons test" (math) was performed. the capacity to develop biofilms on inert substrata and the influence of lf on this feature was spectrophotometrically quantified using an adapted microtiter method, after crystal violet staining. our results showed that lf decreased the hydrophobicity and limited the biofilm development of all 42 s. aureus tested strains, in a dose and time dependent manner. the decreasing effect on the microbial hydrophobicity was accompanied by a lowering effect on the adhesion of microbial strains to the inert substratum. in conclusion these observations indicate that lf exhibits a wound pro-healing effect, by limiting the microbial colonization and biofilm formation and thus, the occurrence of infectious complications of skin lesion. p-04.01. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] host-specificity determinants of bacteriophage vb_ecom_fv3 considered vehicles of s.aureus intoxication in humans throughout the world. the objective of the present study was to assess the presence of enterotoxigenic and methicillin-resistant s. aureus in water buffalo milk and dairy products. a total of 100 water buffalo milk and 100 dairy products (50 water buffalo cream and 50 water buffalo cheese) were collected from different dairy farms, smallholders and local bazaars in samsun, turkey. all samples were analyzed using the standard procedure en iso 6888-1 and isolates were confirmed for the presence of the 16s rrna and nuc gene by polymerase chain reaction. s. aureus was identified in 30 of 100 water buffalo milk (30%), 9 of 50 water buffalo cream (18%), and 17 of 50 water buffalo cheese (34%). a total of 99 isolates were confirmed as s. aureus by pcr. genotypic methicillin resistance was evaluated using pcr for the meca gene. out of 99 isolates, 9 (9%) were found to be methicillin resistant (meca gene positive) by pcr. the enterotoxigenic s. aureus was identified in 12 out of 99 (12%) isolates by the mpcr technique. five isolates produced staphylococcal enterotoxins sea (5/12; 41.6%), two isolates produced sec (2/12; 16 .6%), one isolate produced (1/12; 8 .3%) sed, one isolate produced (1/12; 8.3%) see and three isolates produced sec+sed (3/12; 25%) . none of samples were positive for seb. in conclusion, the presence of enterotoxigenic and methicillin-resistant s. aureus in milk and dairy products is of significant for public health concern and also these enterotoxin genes sea and sed are predominant toxins that can cause staphylococcus intoxication in humans. this study was funded by ondokuz mayıs university, samsun, turkey, scientific research project programs (project no: pyo. vet -1904.12 .004) and this article was part of a phd thesis. p-04.01.1-015 identification and biochemical characterization of an immune modulating protein from helicobacter pylori b. kaplan t€ urk€ oz faculty of engineering, department of food engineering, ege university, izmir, turkey helicobacter pylori is able to achieve persistent infection with minimal immune response. the first line of defence during h.pylori infection is through gastric epithelial cells which present toll like receptors (tlr). a family of bacterial proteins which share homology with the toll/il-1 receptor (tir) domain were identified. the structure of btpa from brucella showed that bacterial tir proteins (btp) mimick human tir domain proteins and act on myd88 signaling pathways to suppress tlr signaling. h.pylori might also produce a similar protein. a putative h. pylori tir protein was found based on sequence homology and the corresponding gene; hp1437; was cloned in fusion with an n terminal cleavable 6his-tag. the recombinant protein, 6his-1437 was purified using nickel affinity chromatography. 1437 was subjected to limited proteolysis and the bands were analyzed by peptide mass fingerprinting (pmf). oligomerization of 1437 was investigated by in vitro pull-down and size-exclusion chromatography. 1437, a 239 amino acid protein, has a predicted c terminal tir domain similar to other btps and sequence alignments verified the presence of tir domain signature regions. recombinant 6his-1437 was produced with a yield of 10 mg/l culture. a structurally stable 25 kda fragment was obtained from limited proteolysis which contained the tir domain as verified by pmf. in vitro pull down assays showed 1437 interacts with itself forming dimers as shown by size-exclusion chromatography. tir domain proteins function by interacting with themselves and other tir domains. our results showed that 1437 also form dimers, supporting that it is a btp. current research is focused on solving the structure of 1437 and investigating its interaction with myd88. 1437 might play a direct role in reduced immune response against h.pylori by binding to myd88 analogous to other btps. further characterization of 1437 will provide the first solid evidence of presence of a tir domain protein in h.pylori. p-04.01. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] lipopolysaccharides with different lipid a acylation status from vibrio cholerae and campylobacter jejuni contribute differently to il6 production by bone marrow-derived macrophages k. korneev 1,2 , e. sviriaeva 1, 2 lipid a is a biologically active part of lipopolysaccharide (lps) from gram-negative bacteria that is responsible for the activation of the innate immunity through interaction with toll-like receptor 4 (tlr4) and subsequent production of proinflammatory cytokines. bacteria frequently transform their lipid a so that its recognition by tlr4 is not sufficient for induction of effective antibacterial immune response. we compared biological activity of various lps from pathogenic bacteria vibrio cholerae and campylobacter jejuni. we purified r-form lps for each strain by hydrophobic chromatography. the biological activity of lps preparations was evaluated by their ability to activate production of proinflammatory cytokine il6 by bone marrow-derived macrophages from c57bl/6 mice, using tlr4-deficient macrophages to control for specificity of tlr4 signaling. lps from e. coli and inactive lps from f. tularensis were used as positive and negative controls. lps from v. cholerae demonstrated biological activity similar to that of lps from e. coli, consistent with the presence of highly acylated lipid a in both strains. however, the former was a slightly weaker activator than the latter, because lipid a from v. cholerae had on average shorter acyl chains. lipid a from c. jejuni had on average longer acyl groups than in e. coli, while degree of acylation was lower, and as a result its lipid a displayed significantly lower biological activity. our study demonstrates importance of functional groups of lipid a in the ability of lps to activate production of il6 by macrophages. in line with our previous reports, we confirmed a direct correlation between biological activity of various lps species with their lipid a acylation status: the biological activity increases with increase in the length and in the number of the acyl chains. excess proinflammatory cytokine production through tlr4 activation can cause sepsis, while inefficient activation may result in the failure to clear bacteria. clostridium perfringens phospholipase c (cpplc) is the most toxic extracellular enzyme produced by this bacterium and it is an essential virulence factor in the pathogenesis of gas gangrene. cpplc may lead to cell lysis at concentrations that causes extensive degradation of plasma membrane phospholipids. however, at sublytic concentrations it induces cytotoxicity without causing evident membrane damage. the results of this work demonstrated that the cytotoxic effect of cpplc requires its internalization and the activation of the mek-erk pathway. cpplc internalizartion occurs through a dynamin-dependent mechanism and in a time progressive process: first, cpplc colocalizes with caveolin both at the plasma membrane and in vesicles, and later it colocalizes with early and late endosomes and lysosomes. the results also showed that cpplc requires endocytosis in order to activate mek-erk, because treatment with the dynamin inhibitor, dynasore, prevents cpplc endocytosis, erk 1/2 activation and cytotoxcity. cholesterol sequestration as well as inhibition of actin polymerization also prevents cpplc internalization and cytotoxocity, involving endocytosis in the signaling events required for cpplc cytotoxic effect. once internalized, cpplc induces reactive oxygen species production through the activation of pkc, mek/erk and nfjb dependent pathways. inhibition of either of these signaling pathways prevents cpplc's cytotoxic effect. in addition, it was demonstrated that nfjb inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of cpplc in mice. these data provide new insights about the mode of action of this bacterial phospholipase c, previously considered to act only locally on cell membrane. understanding the role of these signaling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridial myonecrosis. p-04.01.1-020 apoptosis induced by clostridium perfringens phospholipase c is mediated by reactive oxygen species m. flores-d ıaz 1 , l. monturiol-gross 1 , m. j. pineda padilla 1 , c. araya-castillo 1 , a. alape-gir on 1 bacterial phospholipases are lipolytic esterases surface associated or secreted by a wide variety of bacterial pathogens. clostridium perfringens, the most broadly distributed pathogen in nature, secretes a prototype phospholipase c (plc), also called a-toxin, which plays a key role in the pathogenesis of gas gangrene. this toxin causes death to cultured cells and extensive myonecrosis when injected intramuscularly in experimental animals. the results of the present study showed that c. perfringens plc (3-5 ng/ml) induces morphological and biochemical changes characteristic of apoptosis in cultured cells, as determined by scanning electron microscopy. nuclei condensation and fragmentation were observed by fluorescence microscopy and a typical ladder fragmentation pattern of genomic dna was detected by dna in agarose gels. cell death was prevented by the caspases inhibitors z-devd-fmk and z-vad-fmk. c. perfringens plc induces oxidative stress in cultured cells as determined by fluorescence microscopy and flow cytometry using the membrane permeable probe dcfda. different antioxidants including the gluthation precursor nac, several iron chelators and the free radical scavengers tiron and edaravone prevent cell death induced by c. perfringens plc in cultured cells or in mice challenged intramuscularly with 1.2 lg of that toxin. thus, this work provides compelling evidence that superoxide, hydrogen peroxide, and the hydroxyl radical are involved in the cytotoxic and myotoxic effects of c. perfringens plc. furthermore, the data demonstrated that edaravone, a clinically used hydroxyl radical trap, reduced the myonecrosis and the mortality caused by c. perfringens in a murine model of gas gangrene, induced by intramuscular bacterial injection of 10 8 bacteria. this knowledge provides new insights for the development of novel therapies to reduce tissue damage during clostridial myonecrosis. lectins are ubiquitous proteins able to recognize mono-and oligosaccharides with high specificity and low affinity. lectins do not have any catalytic activity, unlike enzymes, and they are not products of the immune system in contrast to antibodies. lectins play a crucial role in cell interactions on molecular level showing their importance in various physiological and pathophysiological processes as well as both mutualistic and parasitic interactions between microorganism and hosts. photorhabdus luminescens is a gram-negative bacterium from the family enterobacteriaceae. the bacteria have a complex life cycle that involves mutualistic and pathogenic interaction with two different invertebrate hosts. it is highly pathogenic towards insect larvae. in addition, p. luminescens lives in the intestine of infective juveniles of nematode heterorhabditis bacteriophora, together forming an effective entomopathogenic complex. we have identified several soluble lectins produced by p.luminescens. in this study, we focus on 4 proteins from p. luminescens, which show a high sequence homology with each other. a wide range of methods was used for structural and functional studies of photorhabdus lectins, e.g. surface plasmon resonance, isothermal titration calorimetry, analytical ultracentrifugation and x-ray crystallography. all lectins from p.luminescens recognize l-fucose and d-mannose. despite being closely related, they differ in fine binding specificities. to determine their biological function, knock-out mutants of p. luminescens are being prepared to study its interaction with axenic nematodes and insect larvae. breast cancer is the major disease of women in developed countries occuring predominantly after the age of 65. triple negative breast cancer (tnbc) is a typical subtype of epithelial breast cancer which lacks estrogen receptor (er), progesterone receptor (pr) and human epidermal growth factor receptor 2 (her2) all together. although various researches have been focused on characterizing tnbc and enlightening different molecular markers with the aim of improving the overall outcome, currently the sole affective therapy action for tnbc is chemotherapy. thus chemoresistance is the main clinical challange and accounts for 90% of failures in terms of treating the disease. multidrug resistance (mdr) is defined as simultaneous resistance towards the drugs which do or do not demonstrate structural resemblance and have different effects on their molecular targets. p-glycoprotein (p-gp) is a membrane protein coded by abcb1 (mdr-1) gene. p-gp is an atp-dependent pump which pumps a wide range of drugs out of the cells including chemotherapeutic agents such as doxorubicin (dox) and pactilaxel. in the present study, tnbc cell line mda-mb-231 was treated with increasing doses of dox, cell viability was examined with srb assay and development of mdr was investigated through mdr assay and rt-pcr. results demonstrated that cell viabiliy decreased significantly with the treatment of higher doses. mdr was shown to be increased when cells were treated with 50, 200 and 800 nm of the drug respectively along with 15 lm of p-gp inhibitor verapamil. rt-pcr results were obtained to be consistent with mdr assay results and indicated increased mdr-1 gene expression with the treatment of dox. especially after 400 nm of dox treatment, mdr-1 was overexpressed to be 59 fold when compared to control. in conclusion, it was demonstrated that mda-mb-231 cells have shown to display elevated resistance to higher doses of dox. p-05.01.1-005 targeting dna damage response pathway in cancer cells under heat stress and the mechanical effect of ultrasound y. furusawa 1 , t. kondo 2 1 toyama prefectural university, imizu-shi, japan, 2 university of toyama, toyama, japan ultrasound (us) has been widely utilized for diagnosis and therapy in many medical fields. the biophysical modes of us are divided into three classes, thermal, cavitation and non-thermal non-cavitation effects. in clinical use for cancer therapy, the thermal effect was utilized for hyperthermia therapy with focusing us on cancer to rise the temperature from 41°c to 44°c, or further which could induce thermal ablation of cancers. cavitation leads to a variety of mechanical stress such as shear stress, shock wave, high pressure, and chemical stress such as free radical formation, both of which have been inferred to act simultaneously on all biological materials. it has been indicated that us induces cell killing, cell lysis, loss of viability, and loss of clonogenicity. recently, we found that heat stress as well as us without thermal effect induce not only dna single-strand breaks but also dna double-strand breaks, a most cytotoxic region of dna, in chromatin dna detected by both gammah2ax staining and neutral comet assay. in response to the stresses which induce dna damage, the dna damage sensor protein kinase, ataxia telangiectasia mutated (atm), atm and rad3 related (atr), and dna-dependent protein kinase (dna-pk) become activated form to initiate signal transduction pathways activating cell-cycle checkpoints, dna repair, and apoptosis. the molecules consisting of dna damage response pathway were expected as therapeutic targets because defects in the response to dna damage agents can be lethal. this work was designed to explore the possible therapeutic targets of the molecules in dna damage response pathways for future us-aided therapy. finally, several kinases (e.g., checkpoint kinase) on dna damage response pathway seems to be the targets for hyperthermia and us therapy. (ural branch) , ekaterinburg, russia, 2 shemyakin and ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia based on the recently synthesized (s)-(2-aminopurin-6-yl) amino acids (gly, ala, val, phe, pro), we obtained a series of novel modified nucleosides using the transglycosylation reaction. for the first time, it has been demonstrated that the corresponding nucleobases are good substrates for the genetically engineered recombinant e. coli purine nucleoside phosphorylase (conversion to nucleosides reached 90-98%). nucleosides, such as ribosides, 2-deoxyribosides, and arabinosides were obtained in high yields (70-88%). it has been found that yield in the transglycosylation reaction does not depend on the structure of the amino acid fragment. the nucleosides synthesized are considered as potential inhibitors of intracellular adenosine deaminase (ad), the increasing activity of which is observed in hepatitis, cirrhosis, hemochromatosis, obstructive jaundice, prostate and bladder cancer, hemolytic anemia, rheumatic and typhoid fever, gout, and cooley's anemia. cytotoxicity of the synthesized nucleosides was tested in the jurkat (model of human t-lymphoblastic leukemia) and el-4 (model of mice t-lymphoblastic leukemia) cell lines. the compounds studied did not exhibit cytotoxic activity compared to the activity of the known antitumor agent nelarabin. the work was financially supported by the russian science foundation (grant 14-13-01077). p-05.01.1-007 dna binding, dna cleavage, antimicrobial activities, antimutagenic and anticancer studies of a schiff base and its complexes n. yildirim 1 , n. demir 2 , m. yildiz 3 1 health services vocational school, c ß anakkale onsekiz mart university, c ß anakkale, turkey, 2 department of biology, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, 3 department of chemistry, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey schiff bases are considered as favored and the most widely used ligands, due to their metal complexes having variety of applications as antibacterial and anticancer agents. the rational design and synthesis of new schiff bases and their metal complexes have been drawing great interest because of their diverse biological and pharmaceutical activities. so, exploring and designing novel molecules that have biological activities and capable of interacting with nucleic acids has a great significance for disease defence and to discover new dna-targeted anticancer drugs for chemotherapy. in this study, we report the synthesis and characterization of a novel schiff base and its ni(ii) and cu(ii) complexes. the minimal inhibitory concentration (mic) of the compounds was screened in vitro against bacteria and yeast cultures using broth micro dilution test. dna binding and dna cleavage activity of the compounds were investigated by uv-vis spectroscopy and agarose gel electrophoresis. antimutagenic activity of compounds were tested in the absence of microsomal enzymes (s9-). also, cytotoxicity of the compounds against hepg2 cell lines was assayed by the mtt (3-(4,5-dimethylthyazolyl-2)-2,5-diphenyltetrazolium bromide) method. consequently, uv-vis spectroscopy studies indicated that the compounds interact with calf thymus dna (ct-dna) via intercalative binding mode. dna cleavage activity studies showed that the cu(ii) complex can effectively cleave pbr322 plasmid dna. compounds inhibited the base pair mutation with high inhibition rate in the absence of s9. also, schiff base complex had cytotoxic activity towards hepg2 cell line, that it was found to be more potent than the control cisplatin. p-05.01.1-008 single particle electron tomography of rnap elongation complex, stalled at position +24 genome in vivo is constantly exposed to the damaging effects of the environment. single-strand breaks (ssbs) are the most frequently occurring dna lesions. accumulation of unrepaired ssbs can interfere with the cells metabolism and increase genomic instability. in vivo, ssbs are repaired in specific pathway, but, in eukaryotic nuclei, dna is organized in chromatin that could affect the accessibility of lesions to sensor proteins. breaks in a template strand induce arrest of rna polymerase ii (polii) in vitro and in vivo and can be revealed in a transcription-dependent manner. our recent biochemical studies identified two key intermediates formed during transcription through a nucleosome by rnap that are nearly homogeneous, active and stable by biochemical criteria (complexes stalled after entering 24 or 42 bp into the nucleosome; ec+24 or ec+41, respectively). hear we produced two complexes, both stalled in the +24 position, one without break in the dna, and the other with introduced ssb at position +12 of a non-template dna strand. complexes were purified using affinity chromatography and applied to a carbon-coated, glow-discharged em grid. tomographic studies were performed at ae70°in a jeol microscope at 200 kv accelerated voltage. images were recorded using a gatan ccd camera. image analysis was performed using the imod software. the resulting structure of the ec+24 complex with no break in dna consist of two domains, connected by a single dna string. the complex with a break introduced into the dna has a more compact appearance and its two domains were connected by two dna strings, thus forming an intranucleosomal dna loop. our data suggest that ssbs in a non-template strand can induce the formation of stable non-productive transcription intermediate. the inhibitory effect of ssbs onto transcription may suggest a possible mechanism for their recognition in vivo with a transcription-dependent pathway. this work has been supported by the rsf grant #14-24-00031. colorectal cancer (crc) is one of the leading causes of cancerrelated deaths in the developed countries. according to 2014 who report new incidence rate of crc in turkey is 6.5% among other cancer types. owing to difficulty of the low allele frequency variations detection, genetic association profiles of crc have not been entirely identified. low allele frequency variations mlh1 à93g>a (rs1800734) promotor substitution, mlh1 415g>c (rs28930073) exonic substitution, mthfr c677t (rs1801133) and apc 1307 t>a (rs1801155) were investigated in this study. these 4 snps "rs1800734, rs28930073, rs1801133, rs180115" are located on 1p36.3, 5q21, 3p22 respectively. colonoscopic investigations were performed on both cancer and control group. the 4 snps were genotyped using kompetitive allele specific pcr technology in 1014 cases and 805 healthy controls. statistical analysis was carried out with cochran-armitage chi-square test. in this study these 3 of the 4 snps in mlh1, mthfr genes were examined for the first time in turkish sporadic crc cases. statistical analysis showed no significant association within our turkish sporadic crc population. percentage of mlh1 à93aa genotype in group aged ≥ 65 was found to be 5.7% in cancer versus 2% in control group. moreover apc 1307a, mlh1 415c alleles were detected only 1 and 3 allele respectively. previously, apc 1307a allele was determined in 53.3% of a turkish cohort. however in the present study apc 1307a allele was detected on 1 allele only. studies showed mlh1 à93 promoter variation as a risk factor for microsatellite instabile crc but for the current study this data is not available. in spite of literature mthfr c677t and mlh1 415g>c snps were not found to be associated with sporadic crc in turkish population. this research demonstrates that importance of population based studies in multifactorial disease. p-05.01.1-010 excision of damaged bases from transcription intermediates by fpg/nei superfamily dna glycosylases k. makasheva, d. zharkov sb ras institute of chemical biology and fundamental medicine, novosibirsk, russia oxidative lesions are abundant due to constant presence of reactive oxygen species in living cells. repair of oxidative base lesions is initiated by dna glycosylases. for example, bacterial fpg and nei dna glycosylases excise oxidized purines and pyrimidines, respectively, from dna. their human homologs, neil1 and neil2, have been reported to show preference towards oxidized lesions in dna bubbles. from these observations, it had been hypothesized that neil proteins may be involved in the repair of lesions in dna bubbles generated during transcription. however, it is not presently clear how neils would behave on bubbles more closely resembling transcription intermediates (e. g., containing the rna strand), and bacterial homologs fpg and nei had never been investigated with bubble substrates. we have studied excision of either 8-oxoguanine (8-oxog) or 5,6-dihydrouracil (dhu) by e. coli fpg and nei and human neil1 and neil2 from single-strand oligonucleotides, perfect duplexes, bubbles with different number of unpaired bases (6 to 30), d-loops with dna or rna and from complexes with rna polymerase. fpg, neil1 and neil2 efficiently excised dhu located inside a bubble. fpg and neil1 was generally more active than neil2 in excision of 8-oxog from ssdna and bubbles. nei, on the other hand, was active only on dhu located in dsdna (either perfect duplex or dna/dna d-loop). fpg and neil1 also have shown activity in d-loops with rna. the presence of an additional unpaired 5 0 -tail of the third strand of d-loops didn't affect the glycosylases activity. the activity of fpg was observed in pre-assembled transcriptional complexes with e. coli rna polymerase and depended on the position of the lesion in the transcription bubble, possibly reflecting local accessibility of the lesion within the elongation complex. this work was supported by rsf (14-24-00093). nucleotide excision repair (ner) is a multistep process that eliminates a wide range of lesions in dna, including uv photoproducts and base modifications by many carcinogenic and chemotherapeutic agents. one of the advanced approaches to ner process investigation is based on reproducing the repair reaction by mixing protein extracts from mammalian cells with model linear dnas, bearing lesions. long linear dnas (137 bp) containing efficiently recognized and processed by ner system lesions (fluoro-azidobenzoyl photoactive lesion fab-dc, nonnucleoside lesions nflu and nant) in both strands have been synthesized. we have demonstrated that dnas containing closely positioned lesions in the both strands represent difficult-to-repair (fab-dc/nflu(+4), fab-dc/nflu(à3)) or unrepairable (nflu/nflu (+4), nflu/nflu(à3), nant/nflu(+4), nant/nflu(à3)) structures. besides, it has been shown that model dnas bearing 2 bulky lesions in opposite positions (fab-dc/nflu(0), nflu/nflu(0)) represent unrepairable structure as well. the model substrates with increasing distance between lesions in the duplex demonstrated the full recovery of substrate properties in ner process (fab-dc/nflu(+8), fab-dc/nflu(à10), fab-dc/nflu(à21), nflu/nflu (+8), and nant/nflu(+8)), whereas the level of specific excision from nflu/nflu(à10), nflu/nflu(à21) and nant/nflu(à10), nant/nflu(à21) was approximately 50% of the nflu/dg or nant/dg dna respectively. it has been shown that modified dna-duplex (54 bp) with fab-dc has decreased structurally dependent affinity for xpc-hr23b compared to duplexes containing lesions in both strands being analyzed (fab-dc/dg, fab-dc/nflu(+4), fab-dc/nflu (à3), fab-dc/nflu(+8), fab-dc/nflu(à10), fab-dc/nflu(-21)) and increased compared to umdna. the data provide an argument that the ner system of higher eukaryotes recognizes and eliminates injured dna fragments on a multi-criteria basis. it is well known that dna plays crucial role in the biological system because of including all the genetic information for cellular function. therefore, the interaction of molecules with dna has gained interest in the medicinal chemistry to explore new anticancer agent. photodynamic therapy which is alternative cancer treatment method depends on free radicals and singlet oxygen to destroy tumor tissue via necrosis and apoptosis. phthalocyanines (pcs) are used for photodynamic therapy because of their absorption of high wavelength light ability and they have high triplet quantum state yields and long lifetimes in triplet states. also they do not have any toxic effect without light. in this study the novel synthesized 4[2-(2morpholin-4-ylethoxy) ethoxy]phthalonitrile substitued zinc(ii), manganese(ii) and copper(ii) phthalocyanines were used. the potential properties of phthalocyanine compounds for photodynamic therapy were purposed to reveal by the preliminary work. for this aim, the mode of dna binding, photocleavage and topoisomerase i inhibition of these compounds were investigated. 4[2-(2-morpholin-4-ylethoxy) ethoxy]phthalonitrile substitued zinc(ii), manganese(ii) and copper(ii) phthalocyanine compounds have been synthesized. the interaction of novel pcs compounds with calf thymus (ct) dna was investigated by using uv-vis spectroscopy, thermal denaturation studies and viscosity measurements. additionally, dna photocleavage and topoisomerase i inhibition studies were performed to pbr322 dna by using agarose gel electrophoresis. the interaction studies indicated that pcs compounds powerfully bound via an intercalation mechanism with ct-dna. these compounds showed efficiently dna photocleavage under irradiation at 650 nm. the all of pcs inhibited topoisomerase i in a dose-dependent manner. all the experimental studies showed that pc compounds might be used agents for photodynamic therapy. p-05.01.1-014 target search by base excision repair dna glycosylases e. dyatlova, g. mechetin, d. zharkov institute of chemical biology and fundamental medicine, novosibirsk, russia the problem of rapid target search in dna is faced by transcription factors, restriction endonucleases, dna repair enzymes and other sequence-or structure-specific dna-binding proteins. theoretically, the fastest target search in dna can be achieved by combining one-dimensional diffusion along the dna contour (processive search) and three-dimensional diffusion (distributive search). the balance between these search modes depends on many factors affecting dna-protein interactions, such as the presence of mono-and divalent cations, competing proteins, crowding effect, etc. presently, the mechanisms of target search are understood only for a handful of enzymes. we have recently developed an assay to study target search by dna repair enzymes, based on cleavage of oligonucleotide substrate containing two targets. thus, the distance between the targets can be precisely controlled, and any modification can be introduced into dna. subsequently, the probability of correlated cleavage (p cc ) is estimated, reflecting the efficiency of enzyme transfer between the specific sites. in this work, we have investigated five repair enzymes: e. coli endonuclease viii (nei), its human homologs neil1 and neil2, and uracil-dna-glycosylases (ung) from e. coli and vaccinia virus. as expected, p cc of all enzymes depended on the ionic strength of the solution and the presence of mg 2+ . ung from vaccinia virus was the most sensitive to these factors, raising questions about its proficiency as a suggested processivity factor of viral dna polymerase. nei, neil1 and neil2 showed a peak of p cc at low but non-zero ionic strength indicating that nonpolar interactions contribute to binding of these proteins to nonspecific dna. this conclusion was also supported by analyzing amino acid conservation in the catalytic core of nei. introduction of bulky fluorescent group between two specific sites greatly reduced the ability of glycosylases to slide along dna. this work was supported by rsf (14-24-00093). p-05.01.1-015 does causes 1800 mhz magnetic field application kras and p53 mutations in colon?: occurences histopatologically and microbiologically changes in colon determination of kirsten rat sarcoma (kras) and p53 gene mutations in colon. materials and methods: in this study, three groups were prepared as control,sham and electromagnetic field (emf) group.1800 mhz radiofrequency (rf) radiation was produced by using an electromagnetic energy generator.the emf group rats were exposed to electromagnetic field for 12 weeks as 45 minutes per day.at the end of experiments, rats were sacrificed under ethyl ether anesthesia and the rat colons were dissected.fecal speciments were collected.fecal dna (for detection of fusobacterium and bacteroides) and colonic dna (for detection of kras and p53 mutations) were isolated.rt-pcr tchnique was used for detection of bacterias and mutations. results: no any differences was observed histopathologically between control and sham groups.erosions and partial losses were observed at mucosal epitelium in the emf group.the corrupted gland structure, the mucosal edema and the inflammatory cell infiltration were observed.the amout of collagen was increased and fibrosis was detected in emf group.goblet cell number decreased statistically significant when compared to control and sham groups (p < 0.05).the amount of fusobacterium increased significantly in emf group compared to controls.the difference was not detected between groups in the amount of bacteroides.all the samples analysed for kras and tp53 mutations in the colon tissue were found to be wild type.no significant difference was observed between the control group and the emf applied group. discussion and conclusion: in conclusions,for 12 weeks 45 minute/day exposure to 1800 mhz emf caused histopatological damage in rat colon.the amount of fusobacterium is increased.emf exposure did not caused to kras and p53 mutations in colon tissue. p-05.01.1-016 synthesis, antimicrobial activity, genotoxicity, dna binding and dna cleavage studies of new glycine methyl ester derivative schiff base there has been an increasing focus on the binding study of small molecules to dna during the last decades, since dna is an important genetic substance in organisms. therefore, the current growing interest in small molecules that are capable of binding and cleaving dna is related to their utility in the design and development of synthetic restriction enzymes, new drugs, dna agents, and also to their ability to probe the structure of dna itself. in recent years, schiff bases have found increased application in pharmaceutical research, organic synthesis, and bio-processes. schiff bases are considered as favored and the most widely used ligands, due to their metal complexes having variety of applications as antibacterial and anticancer agents. in this study, we report the synthesis and characterization of a novel glycine methyl ester derivative schiff base. the minimal inhibitory concentration (mic) of the compound was screened in vitro against bacteria and yeast cultures using broth micro dilution tests. antimutagenic activity of compound was tested in the absence of metabolic activation. also, dna binding and dna cleavage were investigated of compound by uv-vis spectroscopy and agarose gel electrophoresis respectively. consequently, this compound differs significantly in its activity against tested microorganisms. this difference may be attributed to the fact that the cell wall in gram-positive bacteria is a single layer, whereas the gram-negative bacteria cell wall is a multilayered structure, and the yeast cell wall is quite complex. the compound inhibited the base pair mutation in the absence of s9 with high inhibition rate. uv-vis spectroscopy studies of the interactions between the compound and calf thymus dna (ct-dna) showed that the compound interacts with dna via intercalative binding. to date a large number of the sequences in the human genome (g4 motifs) with the potential to form a spatial structure, gquadruplexes is known. g4 motifs were found in the promoter regions of most of the known oncogenes. recent experimental studies have shown that genome instability directly related to the non-canonical dna structures, including g-quadruplexes. in this work we study the distribution of somatic snvs within the g4 motifs in tumor samples with the aim to identify involvement of the motifs in the process of mutagenesis in pancreatic cancer. using the access kindly provided by the international icgc consortium to the database, we analyzed 68 samples of pancreatic ductal adenocarcinoma and 27 samples of pancreatic endocrine neoplasms. we considered only the promoter regions as the richest with g-quadruplex motifs. we found that quadruplex sequences have the ability to focus somatic snvs. this could be explained by the errors of polymerase during replication through secondary dna structures. furthermore, the snvs occur much more often in loops of g4 motifs than in g blocks, without changing the motive. in addition, t>g(a>c) and t>c(a>g) substitutions occur significantly more likely in loops which in turn stabilize the g-quadruplex structure. the cancer-related mutations tend to increasing the length of g blocks. the conservation of g4 motifs may indicate an important functional significance of g-quadruplex structures in human genome. supported by project no. 16-14-10396 of the russian science foundation. background: multiple myeloma (mm) is a rare, leading to bone destruction and marrow failure, largely incurable malignant disease of plasma cells. anemia (mostly normocytic normochromic) is seen in most patients. mean platelet volume (mpv) is a laboratory marker of platelet function and activity, the most accurate measure of platelet size. the aim of this study was to investigate the mean platelet volume (mpv) values in this disease. materials and methods: whole blood samples were collected from 60 healthy controls and 105 patients with mm. the mean age for controls and patients were 54.5 ae 8.5 and 57.4 ae 8.1 years, respectively. mpv levels were calculated with cancer is a chronic disease in the world which is the second leading cause of death, after cardiovascular diseases. benzimidazoles have been known to act as antiproliferative or anticancer agents in chemotherapeutic drug research area. in this regard we aimed to investigate the cytotoxic and apoptotic properties of novel benzimidazole derivatives bearing pyridyl/pyrimidinyl piperazine moiety against a549 lung adenocarcinoma cells. a549 lung adenocarcinoma cell lines were used in the studies. the cytotoxic activities of the tested compounds were determined by mtt assay. detection of apoptosis was performed using annexin v-fitc apoptosis detection kit bd, pharmingen according to the manufacturer's instruction. all measurements were performed on a facs-calibur cytometer. the ic 50 values of the compounds were determined for a549 cell line. compounds 4, 7 and 12 which were including 4-chlorophenyl, 4-nitrophenyl on pyridine ring; 4-fluorophenyl on pyrimidine moiety, had significant cytotoxic activity with ic 50 values lower than 55.33 ae 23.40 lg/ml. compound 12 showed the highest cytotoxic activity with a ic 50 value of 16.67 ae 2.24 lg/ml, whereas cisplatin ic 50 values were 14.0 ae 2.0 lg/ml lg/ml against a549 cells. cytotoxic activity of compound 4 and 7 with a ic 50 value were 55.3 ae 23.4 and 21.7 ae 8.4 lg/ml, respectively. also, compound 12 showed the highest population of early apoptotic cells (39.4%) of the tested compounds which was 5.88-fold higher than for cisplatin. compound 7 produced a comparable population of apoptotic cells with a percentage of 9.5%, respectively according to cisplatin's percentage of 6.2%. it was determined that synthesized compounds 4, 7 and 12 had considerable anticancer activity against a549 cell lines compared to cisplatin. compound 12 including 4-florophenyl on pyrimidine ring was the most cytotoxic compound against the a549 cell line. our study results demonstrated that compound 7, 12 also induced apopototic pathway on a549 cells. p-05.01. in vitro/in vivo antimitotic activity and structure-activity relationships of new glaziovianin a isoflavone series glaziovianin a (gva), isolated from the leaves of the astelia glazioviana, demonstrated cytotoxicity, disrupting microtubule structure and dynamics of hl-60 cells. the aim of the present work was to devise a concise synthetic route toward gva and its derivatives in order to expand structure-activity relationship studies and to investigate their anti-mitotic effect. a concise six-step protocol for the synthesis of gva and its alkoxyphenyl derivatives 9 starting with readily available plant metabolites from dill and parsley seeds was developed. the sea urchin embryo tests confirmed that gva directly affects tubulin/ microtubule dynamics and structure. the b-ring substitution pattern of gva derivatives exhibited strong effects on activity. according to the assay results, the anti-mitotic activity decreased in the following order: gva > myristicin ≥ 3,4,5-trimethoxyphenyl = 4-methoxyphenyl > dillapiol > 3-methoxyphenyl> 3,4dimethoxyphenyl > 2,3,4,5-tetramethoxyphenyl derivatives. a methylenedioxy moiety was essential for the activity of compounds substituted with four b-ring alkoxy groups. the mts assay of the limited panel of cancer cell lines shows that gva displayed the highest inhibitory activity, with ic50 values ranging from 0.27 (a375 cells) to 2.2 lm (mda-mb-231 cells). compounds, containing 3,4,5-trimethoxy and apiol-derived b-rings, respectively, were less active. other isoflavones did not affect cancer cell growth up to 10 lm. anti-proliferative effects of isoflavones 9 observed in both the sea urchin embryo model and human cancer cell lines correlated well. importantly, none of the synthesized isoflavones 9 demonstrated cytotoxicity in human pbmcs, up to 10 lm. in summary, gva and its analogues were synthesized via a scalable six-step reaction sequence. the gva and its analogues containing 3,4,5-trimethoxy and apiol-derived b-rings were found to be promising anti-mitotic microtubule destabilizing agents with low toxicity against human pbmcs. bag-1 is a multifunctional protein which has interactions with a number of cellular proteins; nuclear hormone receptors, bcl-2, hsp70/hsc70 family, growth hormone receptors, raf-1, ubiquitin machinery and dna to regulate cell survival. for this reason, bag-1 is a critical molecular player in the regulation of cell survival signaling and apoptosis mechanism. elevated expression levels of bag-1 are associated with progression of cancer. in the treatment of breast cancer, silencing tools as a promising combined therapy strategies in the presence of classical chemotherapeutics gain importance to investigate interaction networks of cell death and survival signaling pathways. therefore, we aim to understand potential role of bag-1 silencing in the treatment of breast cancer cells with apoptotic agents; cisplatin or paclitaxel. our results showed that, silencing of bag-1 enhanced cisplatin or paclitaxel-induced apoptosis in mcf-7 cells by down-regulating antiapoptotic and upregulating proapoptotic bcl-2 family proteins, changes on cell cycle, upregulation on subg1 phase, activating caspases and cleavage of parp. in addition, knockdown of antiapoptotic bag-1 has a suppressive role in pi3k and akt signaling pathway in mcf-7 breast cancer cells through inhibition of akt phosphorylation and downregulation on pi3k. investigation targets of akt pathway showed that mtor cell survival pathway also affected through bag-1 silencing. bag-1 silencing inhibited mtor signaling via downregulating both rictor and raptor proteins which are the members of rapamycininsensitive mtorc2 and rapamycin-sensitive mtorc1 complexes, respectively. knockdown strategies of bag-1 is important to enlighten the network interactions of bag-1 and clarify its interaction partners in the cells. therefore utilization of bag-1 targeted strategies might further increase therapeutic efficiency of drugs through inhibiting cell survival machinery in the treatment of metastatic breast cancer. p-05.01.1-024 biological activity evaluation of new 3,5,6trisubstituted triazine derivatives bearing different heterocyclic rings against lung cancer cell lines l. yurttas, g. akalin c ß iftc ßi, h. e. temel, b. demir anadolu university, eskisehir, turkey cancer is one of the major death causing disease worldwide. among the various cell types occurs on different organs, lung cancer is one leading cause of cancer death accounting for approximately 26% of all female and 28% of all male cancer deaths in 2013. the resistance development, cytotoxicity and inadequacy are the main encountered problems by the treatment with existing chemotherapeutic agents. therefore, there is continuous need to discover new active and non-toxic molecules. 1-[4-(5,6-bis(4-substituted phenyl)-1,2,4-triazin-3-yl)piperazin-1-yl]-2-[benzimidazole/benzoxazole/benzothiazole-2-yl)thio]ethanone (1-9) derivatives were synthesized with a four-step synthetic procedure using toluil, anisil and 4-chlorobenzil as starting materials. the anticancer activity of the compounds was evaluated using the methods mtt (3-(4,5-dimethylthiazol-2-yl )-2,5-diphenyltetrazolium bromide), brdu (bromodeoxyuridine) assays and flow cytometric analysis against lung cancer cell lines. the lipoxygenase enzyme inhibition activity of the compounds were also investigated using the method described by baylac and racine. compounds was found to have (inhibition concentration) ic 50 values between 135-500 lg/ml. the early and late apoptotic cell percentage was determined as 6.6 for compound 8 by flow cytometric analysis. the lox inhibition activity was found 48.35 ae 3.08 for compound 1. compound 8 bearing 8-chlorobenzil and benzoxazole moieties was found as the most active compound when we evaluate anticancer potential of all compounds. the lox enzyme inhibition was indicated for the compound 1 including methyl substituent on phenyl rings. the dna synthesis inhibition of the compounds has been still studied at the concentrations ic 50 /2, ic 50 and ic 50 x2. p-05.01.1-025 single amino acid substitutions and deletions modulate the drp-lyase activity of human dna polymerase iota n. miropolskaya, i. petushkov, a. kulbachinskiy, a. makarova institute of molecular genetics, moscow, russia dna polymerase iota (pol ι) is a y-family dna polymerase that possesses an unusual combination of properties. due to the special organization of the active site pol ι has a very low accuracy of dna synthesis but possesses an ability to bypass a variety of dna lesions. in addition to the dna polymerization activity, human pol ι also possesses an intrinsic 5 0 -deoxyribose phosphate (drp)-lyase activity. removal of the drp group is a pivotal step in base excision repair (ber) in vivo. although pol b plays a key role in the drp group cleavage and dna synthesis during ber, pol ι was shown to complement the in vitro single-nucleotide ber deficiency of pol b null cell extracts and was suggested to be involved in ber under oxidative stress. the drp-lyase active site in pol ι is still not known. to address the mechanism of the drp-lyase activity of pol ι we obtained a series of pol ι mutant variants including point mutations of conserved lysine residues and deletions in different locations. we purified human pol ι variants from yeast saccharomyces cerevisiae and tested the effect of mutations on the cleavage of an internal 5 0 -drp group in oligonucleotide dna substrates in the presence or absence for me 2+ ions. the experiments revealed several point amino acids substitutions that significantly affected the drp-lyase activity of pol ι, thus suggesting a possible location of the drp-lyase active site. furthermore, we showed that deletions in the n-terminus of pol ι and metal ions modulate its drp-lyase activity, which may play an important role in the regulation of pol ι activities in vivo. this work was supported by russian foundation for basic research grants 15-04-08398-a and 15-34-70002-mol-a-mos and by the russian academy of sciences presidium program in molecular and cellular biology. rosmarinus officinalis, commonly known as rosemary, is an aromatic plant belongs to lamiaceae family. from past to now, rosemary have been used as a traditional medicine to cure for various illnesses such as diabetes, rheumatism and cancer. recent studies have shown that rosemary is effective for various cancer types. in this study we aimed to investigate the effect of rosemary in glioblastoma cells (gbm) by comparison with etoposide and the effect of rosemary by concurrent application with the etoposide. gbm cells (u87 mg) were seeded into the 24 well plates and cultured with dmem supplemented with 10% fetal bovine serum. rosmarinus officinalis tea was prepared just as traditional usage and filter sterilized. at the second day of the culture rosemary in 1/75 (v/v) dilution ratio was given to first group, 40 lm etoposide was given to second group, 1/75 (v/v) diluted rosemary and 40 lm etoposide together were given to third group. after one day incubation cell viability was measured by neutral red assay. it was observed that rosemary reduced the viability of gbm cells by nearly %38, etoposide reduced the viability by nearly % 49 and rosemary with the etoposide reduced the viability by nearly %50. the results showed that rosemary was able to reduce the viability of gbm cells but hadn't got an increasing or inhibiting potential over the etoposide's cytotoxic effect. from our previous studies we know that rosemary increases the proliferation of mouse embryonic fibroblasts. it is considered that rosemary might have a protection potential from dna damages and when rosemary is used with etoposide during the cancer treatment, it might reduce the side effects on healthy cells. in conclusion rosemary promises hope for developing new cancer treatment strategies and reducing the side effects of chemotherapeutics. for further studies it is aimed to examine the effects of rosemary with other chemotherapeutics and if rosemary has got a protection potential from the genotoxic stress. morpholine moiety has been found to be an excellent pharmacophore in medicinal chemistry and a number of molecules possessing morpholine skeleton are the clinically approved drugs. in this present study, we aimed to investigate the possible underlying apoptotic mechanism for the cytotoxicity of new morpholine dithiocarbamate derivatives bearing 1-(2-aryl-2-oxoethyl)-2-substituted benzimidazole moiety on c6 glioma. c6 glioma cell lines were used in the studies. the cytotoxic activities of the tested compounds were determined by cell proliferation analysis using standard (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (mtt) assay. detection of apoptosis was performed using annexin v-fitc apoptosis detection kit bd, pharmingen according to the manufacturer's instruction. all measurements were performed on a facs-calibur cytometer. the ic50 values of the compounds were determined for c6 cell line. compounds 1, 2, 8, 9, and 10 , which were including hydrogen, 4-methyl, 3-methoxy, 3-chloro and 3-floro substituents on phenyl acetyl moiety, had significant cytotoxic activity with ic 50 values lower than 55 lg/ml. compound 10 showed the highest cytotoxic activity with a ic 50 value of 28 lg/ml, whereas cisplatin ic 50 values were 15 lg/ml against c6 cells. cytotoxic activity of compound 1, 2, 8, 9 and 10 with a ic 50 value were 42, 55, 50 and 30 lg/ml, respectively. compound 2, 9 and 10 showed the highest population of early apoptotic cells as 11.5, 10.1, and 10.3% respectively compared to cisplatin (6.2%). also, compounds caused dna synthesis inhibition depend on their ic50 values by brdu assay. conclusions: it was concluded that synthesized compounds had considerable anticancer activity against c6 cell lines. however, compound 2, 9 and 10 including 4-methyl, 3-chloro and 3-floro substituents were the most active compounds against the c6 cell line. also our study results showed that compound 2, 9, 10 induced apoptosis in c6 glioma cells. rutin is a glycosided flavonoid and known to have antioxidant and anti-inflammatory properties.trail induces the apoptosis of tumor cells and has no significant toxic effect on normal cells. although trail is a promising anticancer agent, trail resistance is a major barrier to effective cancer therapy. this study was conducted to examine the utility of the combined use of rutin and trail in prostate cancer cells. pc-3 and du145 prostate cancer cells were treated with rutin (1-1000um) and/or trail (20 ng/ml), cell viability and migration were examined. cell viability was determined by trypan blue exclusion and mtt assay. cell migration was determined by wound healing assay. furthermore, lactate dehydrogenases (ldh) levels of medium were determined as biochemical markers of cell viability. pc-3 and du-145 prostate cancer cells were treated with rutin for 24 and 48 hours incubation and ic 50 doses for 48 hours incubation were determined 250um and 1000um respectively. treatment with rutin, pc-3 cells is more sensitive than du145 cells. rutin and rutin plus trail inhibit prostate cancer cell growth in a dose-dependent manner. treatment with trail has no effect at inhibiting growth of pc-3 and du145 prostate cancer cells. the combination of rutin and trail elicit a synergistic antitumor effect on pc-3 and du145 prostate cancer cells. there is a significant increased in rutin and rutin+trail treatments group of ldh activities with respect to control and trail group. conclusion: present data show that rutin efficiently enhanced trail effects in prostate cancer cells. combined treatment with rutin and trail is more effective than the individual treatments of trail at inhibiting growth of prostate cancer cells. p-05.01.1-030 determination of antigenotoxic, proliferative and cytotoxic properties of ellagic acid since ancient time, people use plant for traditional treatment. plants or fruits are produced different type of secondary metabolites. particularly phenolic phytochemicals from plants play an important role in the prevention and treatment of radical damage by inactivating the reactive oxygen compounds due to their antioxidant properties. however, the structure and the activities of many herbal products are not fully elucidated yet and there are several studies about the toxicity of herbal antioxidants and their possible risks to human health. ellagic acid, phenolic compounds, is an important substance. ellagic acid is a naturally occurring plant phenol found in numerous fruits, including blackberries, raspberries, strawberries, cranberries, walnuts, pecans, pomegranates and wolfberries. different researchers give some information about the biological activities of ellagic acid. in this study, we aimed to determine the cytotoxic, proliferative and antigenotoxic effects of ellagic acid, which is phenolic compounds found in natural products. cytotoxic effects of ellagic acid on huvec is investigated by lactate dehydrogenase (ldh) and cell proliferation (wst-1) methods; and antigenotoxic effects against ccl 4 on human lymphocytes is investigated by single cell gel electrophoresis (comet) methods. the rusults showed that high concentration (100 and 200 lm) of ellagic acid has cytotoxic and mutagenic effects, but showed antiproliferative effects. on the contrary, low concentrations (4, 8, 12.5 lm) of ellagic acid has anticytotoxic and antimutagenic effects. as a conclusion, low concentrations of ellagic acid might be use treatment of some disease. but high concentrations of ellagic acid constitute a risk factors for people. keywords: cytotoxicity, antiproliferation, wst-1, ldh, rtca-sp the constitutive nuclear factor kappa b (nf-kb) activation is widely found in diverse types of hematologic malignancies such as acute myeloid leukemia (aml) and chronic myeloid leukemia (cml) as well as solid tumors. inhibition of nf-kb signaling via proteasome inhibitors such as bortezomib can induce apoptosis in myeloid leukemia cell lines. however it is not clear whether the cytotoxic effects of bortezomib on myeloid leukemia cell lines is due to direct inhibition of nf-kb or another pathway, such as dna damage. in this study, cml cell line k562 and aml cell line hl-60 were treated with bortezomib (bor) , etoposide (eto) and camptothecin (cpt) alone or in dual combination with these drugs, following by measuring the effects on cell viability, apoptosis and signal pathways. the effect on cell viability was determined using the mtt assay. the data were used in combination index and isobologram analysis. the expression levels of apoptototic genes (bcl2, bax and caspase 3), the related dna damage genes (atm and atr) and the involved genes in nf-kb signaling (rela and p50) were determined by real time rt-pcr. we showed that combinations of bor with topoisomerase inhibitors (cpt and eto) exhibited synergistic cytotoxic effect in k562 cell line but not in hl-60 cell line. the combination treatment increased apoptosis and dna damage response. dnadamage-sensing kinases were detected in k562 and hl-60 cells following treatment with bor as similar as topoisomerase inhibitors. bor increased the mrna levels of atm and atr dramatically, which indicated active dna damage in the myeloid cell lines. furthermore, bor induced apoptotic cell death by decreasing bcl2 and increasing bax and caspase 3 levels. these effects of bor were observed to correlated with increasing the p65 expression levels. this study on the mechanism of action of bor indicates that this compound affects several pathways involved in the control of cell cycle progression, apoptosis and dna damage. p-05.01.1-032 analysis of molecular cytogenetic alterations in gastric and colon carcinoma by array-based comparative genomic hybridization (array cgh) introduction: genomic dna regions are frequently lost or gained during tumor progression. we aimed to evaluate tumor samples of patients with gastric cancer and colorectal carcinoma to show these genetic alterations by array-based comparative genomic hybridization (array cgh) method. materials and methods: dna isolation was performed from the tumor samples obtained from sixteen patients with primary gastric adenocarcinoma and twelve patients with colon adenocarcinoma. then, agarose gel electrophoresis was performed in those dna samples. following electrophoresis of dna, array cgh procedure was performed to four patients with gastric adenocarcinoma and three patients with colon adenocarcinoma who had dna breaks with 100-200 kb. results: after array-cgh study, many common genetic changes in gastric and colon cancer genome were determined. in gastric cancer dna samples, common losses were detected in chromosome 1p34. 1, 1p13.3, 1q22, 3q29, 5p13.2, 6q24.1, 7q11.23, 7q22.1, 8q 12.1, 9p12, 11q13.1, 12 q24.31, 14q32.31, 16q22.1, 17q21.2, 19q13.3, and 20q11 .23, and also common gains were detected in chromosome 3p26.1, 6q27, 8q12.1, 15q11.2 and xq25. in colon cancer dna samples, common losses were detected in chromosome 1p34. 1, 3q29, 7p22.2, 7p11.21, 9q34.11, 12q24.31, 17q21.2, 17q25.1, 19p13.3, 19p13.33, 20q11.21, and 20q11.23 , and also common gains were detected.in chromosome 14q32. 33, xp22.33, xp22.11, xp13.3, xp11.1 and xq25 . both in gastric and colon cancer dna samples, common losses were detected in chromosome 12q24. 31, 17q21.2, and 19p13.3 , and common gains were detected in xq25. discussion and conclusion: we think that these common changes, generally in dna loss areas harboring tumor suppressor genes and dna gain areas harboring oncogenes, may important in gastrointestinal tumorigenesis. the dna of every cell is under a constant attack by various mutagenic factors which damage the dna and can cause cell cycle arrest and even cell death. accumulation of dna damage is the basis for cancer development and one of the reasons for aging of the organisms. in order to preserve the integrity of its dna cells have evolved an impressive array of dna repair pathways, which are precisely coordinated with the progression of the cell cycle. one of the first events at the site of dna damage is poly(adp-ribose) polymerase 1 (parp1) recruitment which is a sensor for single strand breaks in dna. parp1 catalyzes the synthesis of poly(adp-ribose) or par which is needed for the recruitment of many other dna repair proteins by means of par-binding domains. we used high speed confocal spinning-disk microscopy of living cells to obtain precise kinetics of recruitment of par-dependent proteins to the sites of laser induced dna damage. our results show that the investigated par-dependent proteins are recruited to dna damage sites in the matter of seconds, they reach peak intensities for 20 to 30 seconds after damage infliction and start dissociating. the recruitment of the proteins is entirely dependent on par because addition of parp inhibitor abbrogated their recruitment. the use of spinning-disk microscopy of living cells allowed us to obtain the kinetics of recruitment of the studied proteins to the sites of dna damage. the results are consistent with the fact that parp1 and par-dependent proteins are quickly recruited to damage sites and generation of par is essential for other dna repair protein recruitment. the precise kinetic curves may serve as a basis for investigating how they will change or if they will change at all when cells are put in different conditions or treated with various chemical substances affecting dna metabolism and repair. introduction: chronic myeloid leukemia (cml) is a myeloproliferative disease associated with reciprocal translocation between chromosomes 9 and 22. bcr-abl fusion gene which exhibits constitutively active tyrosine kinase activity has a main role in cml. the tyrosine kinase inhibitor imatinib is used as a first line treatment in cml patients, but imatinib resistance leads to failure in therapy. the application of imatinib in combination with other anticancer agents may be a strategy to increase the antileukemic effect of imatinib. in this study, we have investigated the antiproliferative effect two novel agents: a benzamide derivative xt5 and a benzoxazole derivative xt2b in combination with imatinib. these molecules were investigated in imatinib-sensitive (k562s) and imatinib-resistant (k562r) cml cell lines. materials and methods: antiproliferative and apoptotic effects were assessed by mtt assays and flow-cytometry, respectively. we also evaluated the effects of these compounds on the expression of apoptosis-related genes bax, bcl-2, bad, bim, bcl-xl and mcl1 by real-time quantitative pcr. results: treatment of k562 cells with xt5 increased the expression levels of the pro-apoptotic genes bax, bad and bim in both sensitive and resistant cells. however, xt2b was not found to have similar effects on k562r and k562s cells. combined application of xt5 increased cell death in the mtt assay. mtt assay demonstrated that ic50 for xt5 treated cells in k562r with imatinib (ic50 = 3.5) is lower than k562r without imatinib (ic50 = 8.5). discussion and conclusion: our results showed that combining xt5 with imatinib has more antiproliferative and apoptotic effect on a cml cell line. as a result combination of xt5 with imatinib can be an alternative approach to overcome imatinib resistance. introduction: the mmr(mismatche repair) system recognizes base-base mismatches and insertion or deletion loops in doublestranded dna, and it degrades the error-containing region of the newly synthesized strand, allowing the polymerase to correctly resynthesize the second strand according to the template sequence. the human mmr system includes the mlh1 and msh2. alteration in expression or a defect in mlh1 or msh2 can cause resistance to anti-cancer drugs used in chemotherapy. the attempt of the mmr system to detect drug induced dna damage, triggers the activation of apoptosis, a mechanism which may enhance the cytotoxicity of chemotherapy. loss of the mmr system would make the neoplastic cell less able to initiate apoptosis. inability to initiate apoptosis could be a mechanism of resistance to drugs. chronic myeloid leukemia (cml) is a clonal disease originating from aberrations in hematopoietic stem cell. imatinib, a tyrosine kinase inhibitor has significantly improved clinical outcome for cml patients. however, patients develop resistance when the disease progresses to the blast phase (bp) and there are several mechanisms involved in imatinib resistance. in this study we investigated the role of mmr system in imatinib resistance. materials and methods: k562s (sensitive) and k562r (resistance) were grown in rpmi-1640. k562r cells were maintained in rpmi-1640 medium supplemented with 5 lm imatinib rna isolation, cdna synthesis, rt-pcr was performed respectively. results: the results demonstrated that expression of mlh1 in k562r cells is dramatically lower than equal amount of imatinib treated k562s cells, whereas msh2 expression level did not change in both cell lines. conclusion: it can be suggested that alteration and down-regulation of mlh1 genes leads to imatinib resistance. p-05.01.1-037 characterization of interaction between rad51 inhibitor dids and human serum albumin d. velic, s. henry, c. charlier, m. popova, p. weigel, j. masson, i. nabiev, f. fleury cnrs/university of nantes, nantes, france 4 0 -diisothiocyanostilbene-2,2 0 -disulfonic acid (dids) has been largely used during the last 30 years for its inhibitory effect on anion transporters and channels. more recently, ishida and colleagues have described a possible mechanism by which dids inhibits rad51-mediated homologous pairing and strand exchange, key processes in dna repair by homologous recombination. thus, dids could act as a potential revertant of radioand chemo-resistance in cancer cells, which is the major cause of failure during therapeutic protocols. new drugs targeting rad51 protein have since been developed with potential use for medical applications. in this context, we attempted to determine the behaviour of dids towards blood and plasma proteins such as serum albumins. firstly, we analysed the effects of several environmental factors such as solvent polarity, which may affect the stability of the molecule. secondly, we analysed the spectroscopic properties of dids in the presence of human or bovine serum albumin proteins. uv-visible absorption, circular dichroism, fluorescence spectroscopy and isothermal calorimetry were used. here we show for the first time that dids can interact with both serum albumins. we have also determined the characteristics of these interactions. the comparison of several dids derivatives led us to identify the essential chemical moiety of this compound involved in the interaction. moreover, by using site competition approaches we show that the main binding site for this molecule is in subdomain ib of the protein. these findings show that the binding of dids to serum albumin proteins may change the equilibrium between the free and bound dids forms, thereby affecting its bioavailability and efficiency against the rad51 recombinase protein. p-05.01.1-038 mechanism of tap73 beta-mdm2 autoregulation p73 is a transcription factor which is the member of a p53 family. it regulates many cellular processes, such as apoptosis, cell cycle, and senescence. in contrast to p53, p73 is rarely mutated in tumors and elevated p73 expression is observed in many types of cancers including hepatocellular carcinoma, neuroblastoma, and lung. defining regulatory mechanisms which control p73 protein abundance and activity will be crucial for the development of new therapeutic strategies for cancers. mdm2 is known as the key player in regulation stability and activity of p53. in addition, p53 induces mdm2 transcriptional activity, and caspase-2, 3 activations which cleave mdm2 n-terminal at asp 367. cleaved form of mdm2 binds p53 and promotes its stabilization. mdm2 suggested as a candidate to modulate p73 activity and stability too. however, an interaction between p73 and mdm2 has not defined well. in this study, we aimed to analyze the role of mdm2 in p73 stability. to define this relationship, firstly, we overexpressed the tap73beta isoform using trex system in hep3b. tap73 beta and mdm2 protein levels were determined by western blot. to examine whether mdm2 mediate tap73 beta protein degradation by the proteasomes, cells were treated with proteasome inhibitor, mg132 for 4 hours prior to analysis. previous studies showed that p53-induced caspase-2 and caspase-3 activation cleaves mdm2. considering this, we firstly examined caspase-3 activation by western blot in hep3b tap73 beta cells. then we analyzed expression of cleaved mdm2 and tap73 beta levels following caspase inhibitor, z-vad-fmk treatment. as a conclusion, tap73 beta-induced full-length mdm-2 expression. furthermore, tap73 beta enhanced cleavage of mdm2 via increased caspase-3 activation. in addition, inhibition of caspase-3 activation caused a decrease in cleaved-mdm2 levels in parallel with tap73 beta expression repression. our results suggested positive regulation between mdm2-tap73 beta. hepatocellular carcinoma (hcc) is one of the most common type of liver cancer and third leading cause of cancer related deaths in worldwide. discovery of new targets is important in survival of hcc patients. p73 is a transcription factor which is the member of p53 family. it has two promoters; while p1 promoter expresses apoptotic ta isoforms, p2 promoter expresses anti-apoptotic dn isoforms. in addition, alternative splicing in c terminal creates many isoforms of ta and dn p73. it has been shown that both tap73 and dnp73 isoforms are expressed in hcc patient tissue and cell lines. the ratio between tap73 and dnp73 affects the apoptotic response, drug response and prognosis. accordingly, identification of the role of p73 and its targets are important in discovery of new treatment strategies in hcc. to understand the role of p73 isoforms in hcc, firstly we performed mtt assays following dna-damaging drugs and multikinase inhibitor, sorafenib treatment to categorize hcc cell lines as resistant or sensitive. after that, we analyzed the expression levels of tap73 isoforms via western blot in all hcc cell lines. then we overexpressed the tap73beta isoform using trex system in hep3b and snu449 cells. these two clones were analyzed for dna damaging drug response by mtt, cell cycle and apoptosis by flow cytometry, and tumor formation by in vitro and in vivo experiments. in scope of our study; 1. only tap73 alpha isoform is expressed in a few hcc cell lines. 2. there is no correlation between basal expression of p73 isoforms and drug responses in hcc cell lines. 3. there is no change in expression of p73 isoforms after treatment of drugs. 4. we showed that the ectopic expression of tap73beta in hep3b arrested the cell cycle in g1/ s and decreased the colony formation. therefore, the capacity of tumor formation of the cells dramatically decreased in scid mice. as a result, we revealed that tap73 beta play role in tumor formation, cell cycle arrest, dna damage responses in hcc. p-05.01.1-040 biochemical characterization of exonuclease iii-family ap endonuclease point mutants reveals role of conserved amino acid residues in the nir-specific enzymes a. mursalimov, z. koshenov, t. yeleussizov, m. redrejo-rodriguez, a. ishchenko, b. t. matkarimov, m. saparbaev national laboratory astana, astana, kazakhstan oxidative dna damage caused by reactive oxygen species is believed to be a major type of endogenous cellular damage. oxidatively damaged dna bases are substrates for two overlapping repair pathways: dna glycosylase-initiated base excision (ber) and apurinic/apyrimidinic (ap) endonuclease-initiated nucleotide incision repair (nir). in the ber pathway, an ap endonuclease cleaves dna at ap sites and 3 0 -blocking moieties generated by dna glycosylases, whereas in the nir pathway, the same ap endonuclease incises dna 5 0 to a number of oxidized bases. majority of characterized ap endonucleases possess classic ber activities and about half of them are able to catalyze nir activity. at present, the molecular basis of dna substrate specificities of various ap endonucleases remains unclear. here, we examined amino-acid sequence requirement of the nir activity of human major ap endonuclease 1 (ape1). amino acid sequence alignment of various ap endonucleases including e coli exonuclease iii (xth), human ape1 and archaeal mth212 revealed conserved amino acid residues in the nir-specific ap endonucleases ape1, mth212 and exoa that are absent in xth. based on these data, we constructed four ape1 point mutants y128h, n174q, g231s and t268d and examined their dna substrate specificities. results obtained from biochemical characterization of ape1 mutants are discussed in the light of the evolutionary conserved dna repair functions of ap endonucleases and whether these functions can be mutationally separated from. since its discovery some 40 years ago, cisplatin has evolved for its efficacy in one of the most used drugs in treatment of various cancer types. huge effort was invested in understanding the action of cisplatin and development of more potent drugs. they target mainly neighboring purine bases of nuclear dna forming covalent intra-or inter-strand cross-links that affect inhibition of replication and transcription, cell cycle arrest, and attempted repair of the damaged nucleotides. if such damage cannot be removed the cell dies. we have studied the details of the binding site of the short oligonucleotide modified by a platinum compound using complementary solution techniques used in modern structural biology, including raman spectroscopy with dft calculations aided interpretation of the obtained vibrational spectra. moreover, the calculated structure of the dna duplex was verified using saxs (small angle x-ray scattering) curve. in our contribution, we will present an nmr structure of a dna cross-linked with a cisplatin derivative containing a cyclohexane ring. at this atomic level resolution, structural features probably influencing cytostatic effects are described and compared with previously published structures. common structural features of previously determined structures are: a significant roll (25-60°) of the guanine bases involved in the cross-link, bending and unwinding of the double helix at the site of cross-link and orientation towards the major groove. also, the platinum-guanine plane angle varies between 19 and 54°. although the experimental structures were often used as the starting models for molecular dynamics (md) simulations, results of these md still leave many questions unresolved. the results of this research have been acquired within ceitec 2020 (lq1601) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. p-05.01.1-043 ercc2/xpd polymorphisms and colorectal cancer risk: a case control study in a north eastern iranian population j. mehrzad islamic azad university, neyshabur, iran excision repair cross-complimentary group 2 (ercc2) is one of the important dna repair genes.ercc2 codon 751 and 312 polymorphisms has been shown to modulate cancer risk. we therefore assessed the relationship between the ercc2 polymorphisms and the susceptibility to colorectal cancer in a case-control study. there were 105 lung cancer cases and matched healthy controls in this study. information concerning demographic and risk factors was obtained, each person donated 2 ml blood for biomarker testing. ercc2 genotypes were determined by t-arms-pcr method. all of the statistical analyses were performed with spss (v 20.0). there was significant difference between the frequencies of ercc2 polymorphism in cancer cases and controls (p < 0.05). the frequencies of ercc2 751 gln allele were 6.2% in controls and 13.8% in cancer cases. the individuals with lys/gln+gln/gln combined genotype were at an increased risk for lung cancer as compared with those carrying the lys/lys genotype (adjusted or=2.80, 95%=ci 1.21à6.48). the above findings indicate that the genetic polymorphism in the ercc2 codon 751 is associated with the risk of colorectal cancer in an iranian population (neyshabur citizenship). peptide pore blockers are potent tools to study structure and function of potassium voltage-gated channels (kv). kcsa-kv1.x chimeras, in which a ligand-binding site of eukaryotic kv-channel is inserted into bacterial kcsa channel, mimic properly the pore domain of kv-channels. a fluorescence-based approach to study the binding of peptide blockers with kcsa-kv1.1-kcsa-kv1.3 chimeras was developed by us. this approach rested on high-level expression of kcsa-kv1.x chimeras in e.coli inner membrane, binding of fluorescently-labeled toxin at the surface of the spheroplast and analysis of competitive binding of studied ligands by laser scanning confocal microscopy (lscm). here we report on a new analytical system for search and study of kv1.6-channel blockers that combines bl21 (de3) cells expressing kcsa-kv1.6 and rhodamine-labelled agitoxin 2 (rh-agtx2) as a fluorescent probe. by tuning cultivation conditions, the high-level of membrane expression of kcsa-kv1.6 was achieved. it was found that lowering both the growth temperature and the concentration of inducer resulted in significant increase in membrane-embedded kcsa-kv1.6. for system validation, wellknown kv1 channel blockers were studied by the method of competitive binding, and equilibrium dissociation constants were estimated for agtx2, osk1, and kaliotoxin. a new system was applied to study molecular determinants of peptide-kv1.6 channel binding using a number of agtx2 mutants constructed by us, whose affinities to kcsakv1.6 were measured. a new bioengineering fluorescent system is a robust and sensitive assay for assessing the binding activity of kv1.6 channel blockers. it can be used to study interaction interfaces of toxinchannel complexes, to search for novel peptide blockers and to develop new potent and selective kv1.6-blockers for scientific and medical purposes. the work was supported by the grant 14-14-00239 from russian science foundation. asparagus racemosus root extracts (ar) have been exhibited to show a wide range of pharmacological benefits. in this study, liposomes of ar were developed and assessed their physicochemical properties and anti-inflammatory activity in monocytic leukemia cell line (thp-1). liposomes containing ratios of ar to lipid and phosphatidylcholine to cholesterol ratio were synthesized by thin-film hydration (tf), reverse-phase evaporation (rev), and polyol dilution (pd). the in vitro anti-inflammatory activity was assessed in terms of inhibition of tumor necrosis factor alpha (tnf-a) in lipopolysaccharide activated thp-1 by elisa. the size of ar liposomes prepared by tf were larger, whereas those prepared by rev and pd were smaller. ar to lipid ratio was shown to have no influence on particle size, whereas zeta potential enhanced with increasing ar to lipid ratio. ar liposomes with lipid ratio of 1:5 achieved the highest value of entrapment efficiency and were at the highest with polyol dilution method. ar was found to have no toxic effects on thp-1 cells. the anti-inflammatory activities of ar and ar liposomes in terms of tnf-a in thp-1 cells were was exhibited to possess the highest values of around 52% at ar concentration of 1 lg/ml and % tnf-a inhibition tended to decline with the increasing amount of ar. this result may be attributed to the increased amount of liposomal particles being uptaken into the cells as a result of the increasing ar concentrations. it can be suggested that ar liposomes could be an alternative choice of topical/transdermal drug delivery for anti-inflammatory activity. p-mis-002 inhibition of ire1 signaling enzyme increases the expression of tumor suppressor genes and modifies their hypoxic regulation in u87 glioma cells d. tsymbal, o. minchenko palladin institute of biochemistry of the national academy of sciences of ukraine (nasu), kyiv, ukraine gliomas constitute one of the most aggressive groups of malignant neoplasms with poor survival prognosis and scarce therapeutic options. plentiful studies have proven the connection between endoplasmic reticulum stress and malignant growth. we have studied the effect of inhibition of ire1 (inositol requiring enzyme 1), which is a central mediator of endoplasmic reticulum stress and controls cell proliferation and tumor growth, on hypoxic regulation of the expression of different proliferation related genes in u87 glioma cells. it was shown that inhibition of ire1 leads to up-regulation of the expression of krt18, cd24, mest, cenpu, myl9, ing1, ing2, mybl1, and mybl2 genes at the mrna level in u87 glioma cells, with more profound changes for mest, mybl1, and cd24 genes. hypoxia leads to up-regulation of the expression of cd24, ing1, and ing2 genes and to down-regulationof krt18 gene in glioma cells. at the same time, inhibition of ire1 modifies the effect of hypoxia on the expression of all studied genes: suppresses effect of hypoxia on ing1 gene, eliminates hypoxic regulation of krt18, cd24, and ing2 genes in glioma cells. the present study demonstrates that inhibition of ire1 enhances the expression of all studied genes and modifies the hypoxic regulation of these gene expressions in gene specific manner and thus possibly contributes to slower glioma cell proliferation, but several aspects of this regulation remain to be further clarified. amplification and clonig of dna polymerase 1 (pol1) of thermus scotoductus k1 isolated from an armenian goethermal spring a. saghatelyan, h. panosyan, a. trchounian, n. birkeland yerevan state university, yerevan, armenia the most important enzyme ''mined'' from thermophilic microorganisms is dna polymerase, which widely used in molecular biological studies. although dna polymerase produced by thermus aquaticus (taq polymerase) was launched into the market long back, isolation of more processive, reliable and stable dna polymerases from other species is a demand. the purpose of this work was to amplify and clone the pol1 gene of t. scotoductus strain k1 recently isolated from an armenian geothermal spring. the draft genome sequence of strain k1 was deposited under accession number ljjr00000000.1. genomic dna was isolated using genelute bacterial genomic dna kit. primers for the pol1 gene were designed manually. the gene was amplified using pfu polymerase, and amplicons (~2.5 kb) were ligated into the pet-21b(+) vector (novagen) and transformed into chemically competent top10 escherichia coli. inserts were sequenced with t7prom and t7term primers, which showed that the gene sequence was correct and in the right reading frame and could be expressed in mesophilic e.coli. dna polymerases patented form different species of thermus are mostly comparable, suggesting that only limited natural variations in taq-like dna polymerase may be discovered. the pol1 gene from k1 shares 99% and 83% similarity with pol1 of t. scotoductus sa-01 (2.0 kb) and t. aquaticus, respectively. although the difference is not huge at sequence level, possible functional differences (e.g. stability, proofreading activity, resistance to different pcr inhibitors etc.) may occur. therefore, it is important to express and purify dna polymerase from strain k1 for further investigations. peptide ligands of the immunoglobulin g fc region identified by screening phage libraries and site-directed mutagenesis n. kruljec, p. molek, t. bratkovic young researcher, ljubljana, slovenia affinity chromatography based on immunoglobulin (ig)-binding proteins, such as staphylococcal protein a and streptococcal protein g, typically represents the initial step in therapeutic antibody purification process. however, this approach suffers from high cost, poor ligand stability and the requirement for relatively harsh elution conditions that can negatively impact activity and immunogenicity of antibodies. compared to protein ligands, peptides represent an interesting alternative due to higher stability and less expensive production. furthermore, the expected lower affinity for immunoglobulins should allow for elution under milder conditions. the aim of our research was to identify short peptide ligands for the fc region of human iggs. we have screened three commercially available phage display libraries of random cyclic and linear peptides for binding to human fc region in solution using an optimized biopanning approach. five non-homologous linear peptides were shown to specifically interact with the fc portion of immunoglobulins as verified by a set of phage elisa assays. individual phage-displayed peptides were able to recognize specific subclasses of igg. the highest-affinity peptide (12l-19fc), which competed for fc binding with protein a, was subjected to mutagenesis studies. we displayed on phage several variants of 12l-19fc with individual amino acid residues exchanged for alanine as well fragments of the parent peptide of different lengths and evaluated binding to fc with phage elisa to identify the minimal binding motif. binding characteristics of the minimized peptide were further analyzed using spr biosensor. the details will be disclosed at the meeting. diverse effects of ganoderma lucidum in combination with tamoxifen citrate and doxorubicin in mcf-7 breast cancer cells ganoderma lucidum, an edible medicinal fungus, has been known with its anti-metastatic, anti-carcinogenic bioactivities and widely used in asian countries in complementary and alternative medicine. however, there is no information regarding its combined usage with tamoxifen and doxorubicin in breast cancer treatment. we investigated the interactions between ganoderma lucidum and tamoxifen or doxorubicin in mcf-7 human estrogen receptor positive breast cancer cell line. anti-proliferative properties of six extracts were assessed by wst-8 method. the most effective extract in inhibition of mcf-7 cell viability was then evaluated in terms of its anti-metastatic activity by boyden chamber assay. apoptosis and cell cycle assays were performed by flow cytometry. ganoderma lucidum ether extract (g.ether) was the most effective extract on inhibition of cell viability among others with ic (50) values of 100 lg/ml and 12.82 lg/ml at 48 h. and 72 h. respectively. we found that g.ether is capable of inducing apoptosis and changing cell cycle dynamics. however, incubation with g.ether did not affect mcf-7 cell motility significantly. we then assessed the interactions between g.ether and tamoxifen or doxorubicin in mcf-7 cells. the interactions between g.ether and cancer therapeutics were examined by combination index analysis and macsynergy ii software. interestingly, g.ether increased the anti-proliferative effect of tamoxifen although exhibited strong antagonism with doxorubicin in mcf-7 cell line. testing the best matrix/analyte combination for maldi tof mass spectrometric detection of steroid hormones, amino acids, vitamins and carbohydrates in spite of numerous advantages, there are serious drawbacks of the application of matrix assisted laser desorption/ionization time-of-flight mass spectrometry (maldi tof ms) for smallmolecule analyses (below 500 da) and quantification. the main problem is the background interference from commonly used maldi matrix materials. the aim of this work is to evaluate maldi tof mass spectra of physiologically relevant small molecules: steroid hormones, vitamins, amino acids and carbohydrates, acquired with several organic, traditional matrices. small volume, 0.5 ll, of each sample solution (testosterone, progesterone, estradiol, l-cysteine, l-alanine, dl-methionine, glutathione, d-(+)-glucose, d-(+)-maltose, vitamin a, vitamin e) was mixed on the sample plate with the same volume of organic matrix solutions (dhb, thap, chca, 9-aa). for each molecule/matrix pair, we determined quantitative and qualitative parameters of ms analysis. to calculate within day and day-today variation we used excel tools (anova tests). in addition, homogeneity of the sample/matrix distribution on the target was also calculated and expressed as the coefficient of variation of a series of measurements. our results show selectivity of the detection of individual molecules related with the matrix applied. the statistical analysis of certain molecule/matrix pairs gave within and day-to-day variations less than 15%. additionally, homogeneity of the sample/ matrix mixture distribution on the target plate was with some matrices, also less than 15%. some of the used matrices have a great potential for the analysis of small molecules with good analytical parameters, with low variations and high homogeneity of samples on the maldi target plate. these results hold potential for quantification of metabolically-significant small molecules and are very promising for future applications of maldi tof ms analyses. stress causes different expression of mitochondrial biogenesis markers in rat steroid-producing cells of adrenal gland and testes i. starovlah, s. radovic, t. kostic, s. andric faculty of science univeristy of novi sad, novi sad, serbia functional mitochondria of steroid producing cells of adrenal cortex and leydig cells of testes are essential for steroid hormones biosynthesis and regulation. the aim of this study was to determine transcriptional profile of mitochondrial biogenesis markers in adrenal cortex and leydig cells by applying in vivo and in vitro studies. immobilization stress (imo), was performed for 2 hours daily for one (1ximo), two (2ximo) or ten (10ximo) consecutive days. in in vitro studies, primary cultures of purified leydig cells from undisturbed rats were stimulated with stress hormone adrenaline, propranolol (nonselective b-adrs-blocker) and prazosin (the selective a1-adrs antagonist). rq-pcr results showed that the transcription of the main regulator of mitochondrial biogenesis, ppargc1a and ppargc1b, significantly decreased in adrenal cortex of 10ximo rats. oppositely, the significant increase of the same transcript was registered in leydig cells from the same rats. in parallel, transcription of ucp1, the mediator of regulated proton leak, decreased in adrenal cortex, but increased in leydig cells of the same group of rats. incubation of leydig cells with adrenaline, increased transcription of the main markers of mitochondrial biogenesis (ppargc1a, ppargc1b, nrf1 and nrf2a). nonselective b-adrsblocker attenuated this effect. the selective a1-adrs antagonist did not change adrenaline-induced stimulation of ppargc1a, ppargc1b, nrf1 and nrf2a transcription in leydig cells, indicating that the most of the effects are probably mediated by b-adrenergic receptors, not by a1-adrs of leydig cells. in summary, the results suggest that reduction of transcription of mitochondrial biogenesis markers could be a possible mechanism that protects body from excessive glucocorticoid production from adrenal glands in stress conditions, while at the same time stimulation of mitochondrial biogenesis markers transcription in leydig cells could serve as mechanism to preserve testosterone production. p-mis-008 generation of new mitochondria is possible protection mechanism of basal steroidogenesis in leydig cells s. radovic, i. gak, t. kostic, s. andric faculty of science, university of novi sad, novi sad, serbia mitochondria are the most important component of stress response in all cells and for steroid-hormones-producing cells they are the starting point for steroid biosynthesis. here we investigated the parameters of mitochondrial biogenesis in these cells from rats exposed to the psychophysical stress by immobilization (imo). imo stress was applied for 2 hours daily for one (1ximo), two (2ximo) or ten (10ximo) days.hormone levels were measured employing eia, elisa kit or ria. mitochondrial membrane potential (δwm) was measured by tmre fluorescence, mitochondrial mass was detected by quantitative analysis of mitotracker-green fluorescence as well as relative intensity of fluorescence, since number of mitochondria and mitochondrial architecture were defined using transmission electron microscopy. relative gene expression and proteins analyses were performed by rq-pcr and western blot. there was positive correlation between δw m of leydig cells and androgens production of leydig cells. both of them were reduced in all stressed rats but partially recovered in 10ximo group. the mitochondrial mass in leydig cells from 10ximo group was increased. transmission electron microscopy analyses showed that acute and two times repeated stress altered architecture of mitochondrial cristae, while 10ximo increased number of mitochondria and recovered mitochondrial architecture. there was significant increase in the expression of the all markers of mitochondrial biogenesis in leydig cells from 10ximo rats compared with other groups. accordingly, stress-triggered mitochondrial biogenesis represents an adaptive mechanism and does not only correlate with but also is an essential for testosterone production, being both events depend on the same regulators. supporting the evidence that stress, a constant factor in life of humans, induces mitochondrial biogenesis in leydig cells, our results indicate this mechanism probably protects the basal steroid production in stress conditions. targeting survival pathways in leukemic cells through synergism of metformin and thymoquinone u. glamoclija, m. suljagic international university of sarajevo, sarajevo, bosnia and herzegovina generation of resistance to current treatment options is common problem in the therapy of many hematological malignancies. combined therapies utilizing compounds with low toxicity that act synergistically, are proposed to overcome this problem. metformin and thymoquinone (tq) are two molecules which have proven safety profile and represent potential candidates for treatment of hematological malignancies. there are more than 150 clinical trials, at different stages, exploring metformin anticancer activity. metformin activates amp activated protein kinase (ampk) leading to inhibition of the mammalian target of rapamycin (mtor) and induction of apoptosis in different cancers. however, human leukemic cells with increased basal protein kinase b (akt) phosphorylation were shown to be resistant to metformin-induced apoptosis. it was found that activity of metformin can be enhanced by combination with akt and/or nuclear factor 'kappa-lightchain-enhancer' of activated b-cells (nf-jb) inhibitors. tq is phytochemical compound that has shown inhibitory capacity on both of these targets. wst-1 assay was used to evaluate the effects of metformin and tq in dhl4 (b cell lymphoma) and k562 (chronic myelogenous leukemia) cell lines. compusyn software was used in order to calculate the combination index (ci). the ci value indicates whether two drugs have synergistic (ci<1), additive (ci=1) or antagonistic effects (ci>1). we have shown that separately, metformin and tq, exhibit dose dependent inhibition of dhl4 and k562 cells. in combinatorial study with fixed constant ratio and simultaneous drug exposure, in dhl4 and k562 cell lines, ci values were 0.68 and 0.66, respectively. to our knowledge, this is the first report showing synergistic effects of metformin and tq in lymphoma and chronic myelogenous leukemia derived cell lines. these promising data are currently being investigated in order to obtain the insight into their molecular mechanisms. for the last decade many methods of calculating and analysing the physical characteristics of dna has been developed. these methods allow to estimate distributions of free energy, propensity to bend, stress-induced duplex destabilization (sidd), electrostatic potential (ep) etc. and most of them have been used for prediction of genomic regulatory site positions. the main idea of such approach is that proteins recognize genome regulatory sites by these physical and chemical properties, so the physical characteristics are used to predict the location of regulatory sites. most of the characteristics mentioned above describe properties of dna at equilibrium or steady state, but we propose to use characteristics of internal dna dynamics. in this work we used the coarse-grained model of dna, developed recently, to simulate dynamics of the dna open states. with this model we were able to calculate trajectories of the open states moving along the molecule and their dynamical characteristics, such as: open state activation energy, size, half decay time and sound velocity in dna. we use distribution of four dynamical characteristics around transcription start site of experimentally found e.coli promoters taken from regulon db to organise them in stable clusters. clusterization was made with ward method and consensus clustering technique was applied to clusterization results for analysis of its consistency. the same procedure was applied to equilibrium dna characteristics for comparison. distribution of go functions among clusters was also analysed. stable promoter clusters obtained with different physical properties share some similarity. it was not surprise that clusters obtained with dynamical characteristics of dna more similar to sidd clusters then to ep clusters. the data highlights the possible role of dna dynamical properties in transcription initiation and its applicability to promoter identification together with other physical and textual properties of dna. chromium complex with 5-hydroxyflavone acts on metabolic pathways the development of novel therapeutic strategies for obesity treatment are urgently required as obesity is currently the main leading cause in type ii diabetes and insulin resistance. among natural compounds, flavonoids have recently gained interest due to their positive role in maintaining blood glucose levels and insulin secretion. their association with trace elements, wellknown for their capacity in increasing the efficiency of insulin, might potentiate flavonoids biological effects. in this context, the aim of our study was to investigate the in vitro changes in energetic metabolism related genes expression profile in the presence of a chromium complex with 5-hydroxyflavone. dna microarray technology was used for a large scale screening of differentially expressed genes in human adipose stem cells (hascs) after 3 weeks of adipogenic induction in the presence of the chromium complex with 5-hydroxyflavone. moreover, perilipin expression was assessed by flowcytometry. the chromium complex with primuletin negatively regulates the expression of key genes involved in adipogenesis and also modulates the expression of the genes associated with triglyceride synthesis and subsequent fat storage in mature adipocytes. consequently, the chromium complex with 5-hydroxyflavone can be further employed in studies on animal models to investigate the possible improvement of metabolic disorders. deinococcus radiodurans is a highly radioresistant and stress-resistant bacterium. despite extensive studies, the mechanisms of transcription regulation that contribute to the stress-resistance are still poorly understood. d. radiodurans encodes multipe stress-related proteins including three members of the gre-family of transcription factors: grea, gfh1 and gfh2. while grea is a universal bacterial factor that stimulates rna cleavage by rna polymerase (rnap), the functions of lineage-specific gfh proteins remain unknown. we cloned, expressed and purified d. radiodurans rnap and gfh factors and their mutant variants and analyzed their properties using various in vitro transcription approaches. we tested gfh effects on rnap activity in promoter, elongation and termination complexes assembled on natural and synthetic dna templates under different conditions. we found that the gfh factors strongly enhance site-specific pausing and intrinsic transcription termination by d. radiodurans rnap but do not act on active transcription complexes and do not compete with the grea factor. uniquely, the pause-stimulatory activity of gfh is greatly enhanced by manganese ions, which are accumulated in d. radiodurans cells under stress conditions, and is modulated by the secondary rna structure. we revealed functionally important regions in the gfh factors and the rnap active site involved in transcriptional pausing. we propose that gfh factors inhibit rna extension in paused complexes through binding within the secondary rnap channel, coordinating metal ions in the rnap active site and stabilizing an inactive enzyme conformation. this may serve as a sensitive mechanism to regulate transcription under stress conditions and coordinate it with dna repair and replication. our data suggest that gre and gfh proteins target different structural states of the transcription elongation complex and reveal functional diversity of the factors that bind within the secondary channel of rnap. from planktonic to biofilm state of growth, flagella formation is turned off, and the production of fimbriae and extracellular polysaccharides is activated. bola protein is widespread in nature and has been associated with several cellular processes. using high-troughput techniques we showed that bola protein is a new bacterial transcription factor, which regulates the switch between motile and sessile lifestyle. it negatively modulates flagellar biosynthesis and swimming capacity in escherichia coli. moreover, bola overexpression favors biofilm development, involving fimbriae-like adhesins and curli production. our recent results show that bola action in these pathways is related with cdi-gmp a relevant intracellular signaling molecule involved in biofilm formation. we demonstrate that bola contributes to a fine-tuned expression of different diguanylate cyclases and phosphodiesterases and c-di-gmp has a negative influence in the bola mrna transcription. herein we propose that bola is a key player in motile/adhesive transcriptional switch, contributing to a fine-tuned regulation of these important pathways. background: deep venous thrombosis (dvt) is an important health problem worldwide. its pathophysiology is multicausal and involves environmental, genetic and acquired factors. factor v leiden (fvl), prothrombin g20210a (pt g20210a), and methylenetetrahydrofolate reductase (mthfr) gene mutations are to predispose to venous thrombosis. the aim of this study was to compare the frequency of fvl, pt g20210a and mthfr polymorphisms between patients with dvt and healthy controls. methods: this study was conducted at the bozok university hospital. total 220 participants were included in this study, 126 patients with dvt and 94 healthy blood donors. in order to identify fvl, pt g20210a, mthfr c677t and mthfr a1298c, the polymerase chain reaction (pcr) method was utilized combined with the amplification refractory mutation system. results: in 126 patients fvl was present in 54 (42.8%) patients while in controls fvl was present in only 18 (19.1%). frequency of fvl was significantly higher in cases as compared to controls (p < 0.05). pt g20210a mutation was present in 13 patients (10.3%) and in 5 healthy participants (5.3%). mthfr c677t and mthfr a1298c polymorphisms were almost equally distributed among patients and healthy participants. however, the concomitant presence of fvl and double heterozygous polymorphisms of mthfr c677t/a1298c was found in 11 patients (8.7%) and in 2 healthy controls (2.1%), showing significant association with deep venous thrombosis. conclusion: in this study, the frequencies of fvl and pt g20210a polymorphisms were found significantly higher in patients with dvt than those in healthy participants. thus, fvl and pt g20210a polymorphisms have a contributory role on the development of dvt in contrast, mthfr c677t and mthfr a1298c genotypes were not associated with a predisposition to development of dvt. but, a combination of double heterozygous polymorphisms of mthfr c677t/a1298c with fvl may be associated with increased risk of dvt. p-mis-016 self-assembling micellar clusters comprising drugs, nanoparticles and fluorescent compounds for bilogical applications when designing drug carriers, the drug-carrier ratio is an important consideration, because the use of wrong drug-carriers relation can result in toxicity as a consequence of poor metabolism and elimination of the carriers. solubility problem of various substances also plays an important role in many aspects of fundamental science and practical field. specifically, it is an important parameter as well as bioavailability, which determines the required concentration of drug in the body needed to achieve a pharmacological response. among the variety of solubilization methods micellar solubilization is widely used as an alternative to the dissolution of poorly soluble drugs. here, we show a specific approach based on sequential selfassembly of nonionoc detergent micelles (t 9 100, tx114) followed by enacpsulation of various nanoparticles (noble metals, magnet etc.), drugs, fluorescent compounds leads to the formation of stable micellar nano-amd microcomplexes. we propose 3 ways of micellar clusterisation. in the first one micelles are modified by semi-hydrophobic chelator followed by addition of metal ion to make cross-linking. the second way is similar to the first one and suggests application of the metal complex with incresed denticity instead of naked metal ion, and the third one involves micelles clusterisation by semi-hydrophobyc metal complex directly. therefore, one can stabilize micellar network by means of 'interactions on interface': semi-hydrophobyc metal complexes are embedded inside micelle due to hydrophobyc interactions. hydrophobic fluorescent compounds-loaded micellar complexes demonstrates better optical response in aqueous media without crystallization. such obtaining clusters are also very flexible and can be modified by nanoparticles to obtain various nanocomposites, such as fluoromagnetic clusters. this work was supported by russian foundation of basic research grants no. 15-43-03214 r_center_a (2015 no. 15-43-03214 r_center_a ( -2016 no. 15-43-03214 r_center_a ( ) and 15-33-20002 mol_a_ved (2015 no. 15-43-03214 r_center_a ( -2016 . lamellipodia and membrane blebs utilize different signalling pathways to induce directional movement of walker carcinosarcoma wc 256 cells in a physiological electric field clear if those reactions are mediated by similar mechanisms. to establish that, we performed proteomic analysis and subsequent investigation of the role of differential signalling pathways in electrotaxis of cells representing various strategies of movement. cells were exposed to ef in galvanotaxis apparatus and their reaction was recorded. in some experiments cells were pre-incubated with erk1/2 or btk-1 inhibitors. the phosphorylation of erk1/2 and btk-1 was determined by western blot analysis. proteomic analysis was performed by ultimate 3000rs lc nanosystem coupled with a q-exactive mass spectrometer. both blebbing (bc) and lamellipodial (lc) cells show cathodal migration in a physiological ef (1 v/cm). comparative analysis of bc and lc cells proteomes revealed about 150 differential proteins. functional analysis in ingenuity analysis pathway allowed to determine the statistically significant signalling pathways in which these proteins are engaged. among the most distinctively regulated pathways are tec kinase and erk/ mapk signalling activated in lc but not bc. it was found that btk-1 is required for directional movement of lc but not for bc cells. moreover, ef induced stronger and faster btk-1 phosphorylation in lc than bc cells. in contrast erk 1/2 activity was not necessary for electrotaxis of lc cells and ef did not induce erk 1/2 phosphorylation. our results reveal that both lamellipodia and membrane blebs can efficiently drive electrotactic migration of wc256 cells but it is mediated by different signalling pathways. this work was supported by a grant from the national science centre 2012/07/b/nz3/02909, poland. newborn screening for congenital hypothyroidism in turkey: a regional evaluation € o. demirelce 1 , n. y. saral 1 , f. b. aksungar 1,2 , a. coskun 1,2 , m. serteser 1,2 , i. unsal 1,2 1 acibadem labmed, istanbul, turkey, 2 acibadem university, istanbul, turkey congenital hypothroidism (ch) is the most common congenital endocrine disorder and the most important cause of preventable mental retardation. it is important to begin the treatment within 2 weeks before the development of brain damage. tsh based newborn screening programs are shown to be useful for implementing early treatment of ch. in this study, regional results of ch screening program in turkey between 2006 and 2015 were assessed retrospectively. we have evaluated the results of marmara, central anatolia, aegean and mediterranean regions in which our laboratories are located. screening was based on tsh determination in dried blood spot specimens. tsh limits determined to be 10 lu/ml for cut off point and 25lu/ml for clinical decision point. tsh was measured using enzyme immune assay (eia). blood spot tsh data for 65857 newborns during this time period were evaluated. permanent or transient ch was determined according to the results of thyroid function tests. confirmed ch cases were based on local endocrinologists' report and initiation of thyroxine treatment. the frequency of neonatal tsh levels were found to be under the cut off level of 10lu/ml in 63513 (96.44%), between 10 and 25lu/ml in 2287 (3.47%) and above the level of 25lu/ml in 57 (0.09%) babies, respectively. recall rate was 3.5%. ch cases of neonatal tsh levels greater than 25lu/ml were 26. the incidence of ch of this group was 1:2532. there were no significant differences in the number of congenital hypothyroidism between males and females (p > 0.05). the preliminary results of our study indicate that the incidence of ch in our region is higher than the worldwide reports as has been proved by preceding studies. iodine deficiency, dyshormonogenesis, highly consanguineous population, may contribute to the high incidence of ch in turkey. newborn screening of ch must be developed for detecting true cases and tsh cut off point must be reviewed for decreasing redundant recall rate. in silico analysis of the first complete genome sequence of lactobacillus acidipiscis species k. papadimitriou 1 , m. kazou 1 , v. alexandraki 1 , b. pot 2 , e. tsakalidou 1 1 agricultural university of athens, athens, greece, 2 institut pasteur de lille, lille, france introduction: lactic acid bacteria (lab) constitute a significant group of microorganisms for the food industry, as they play a key role in food fermentation and consequently in human health. lactobacillus acidipiscis aca-dc 1533 is a gram-positive, motile, rod-shaped lab isolated from traditional greek kopanisti cheese. here we present the in silico analysis of the first complete genome sequence of l. acidipiscis in order to explore the biology of the species. materials and methods: sequencing of l. acidipiscis genome was performed using the hiseq 2000 and pacbio rsii sequencing platform technologies and the genome assembly was validated against an nhei optical map of the l. acidipiscis genome. protein-coding sequences were predicted by glimmer, rrna genes by rnammer and trna genes by the trnascan-se server. potential genomic islands were detected using the island-viewer software tool, prophage regions by phast and the subsystem-based annotation by rast server. finally, the circular representation of l. acidipiscis genome keyed to the cog groups was constructed by cgview server. results: the sequencing analysis resulted in one continuous genomic scaffold of 2,678,726 bp with a g+c content of 39.75%. the genome contains 2,525 protein-coding genes on the chromosome covering up to 82.09% of the genome sequence, 63 trna and 18 rrna. according to the subsystem-based annotation, 1,562 protein-coding genes were assigned to 310 metabolic subsystems. the most abundant of the subsystems are related to carbohydrates (n = 287, 18.37% of total protein-coding genes) and protein metabolism (n = 173, 11.08% of total protein-coding genes). furthermore, three prophage regions were detected; one intact (43.5kb), one incomplete (14.7 kb) and one questionable (29.3 kb). discussion and conclusion: the whole genome analysis of l. acidipiscis aca-dc 1533 provided interesting information about a not well-studied species. investigation of serum irisin levels of patients with metformin taking new onset type 2 diabetes mellitus increases glucose tolerance and energy expenditure and improves carbohydrate homeostasis. metformin is a biguanidine class antidiabetic drug which inhibits liver gluconeogenesis and decreases insulin resistance and is frequently recommended in treatment of new onset type 2 diabetes mellitus (t2dm). irisin has a role in the regulation of energy metabolism pathways and its level in blood of persons with t2dm has been reported to decrease. regarding this relationship, it was aimed to reveal the effect of metformin on serum irisin levels. 20 patients with impaired oral glucose tolerance test were included to this investigation. they were recommended to take metformin and to change their life style, such as exercise and diet. their blood were taken at the beginning and after 1 month. also, a healthy control group (n = 20) was formed from persons with similar age and sexual distribution as the patient group. irisin levels of their sera were measured by enzyme-linked immunosorbent assay (elisa) method. statistical evaluation of the measurements showed no significant difference (p = 0.780) between the irisin levels of the patients at the beginning and after 1 month treatment. a similar result was found between the control and the treated groups (p = 0.170), while a significant difference (p = 0.002) was observed between the control and untreated patients groups. the results obtained from this study do not show a clear and significant change in the blood irisin levels of the patients with new onset t2dm taking metformin together with life style change. a longer period of treatment and a higher number of patients may be needed for more reliable results. thermodynamics of dna ligands binding at specific sites of telomeric g-quadruplex dna g-quadruplexes are a perspective target for anticancer therapy. for example stabilization of the telomeric g-quadruplex dna formed by single-stranded ends of the chromosomes leads to inhibition of telomerase, which is active in 90% of cancer cells. similarly, small molecules targeted to a specific g-quadruplex would inhibit various cellular processes. stoichiometry and affinity of interaction of these compounds to dna is determined by specific structural motifs within a g-quadruplex. rational design of novel chemical compounds requires an in depth knowledge of interactions between known ligands and g-quadruplex structures. experimental methods that are used for determination of thermodynamic binding parameters, such as isothermal titration calorimetry, differential scanning calorimetry, ultraviolet absorption and circular dichroism spectroscopy provide a collective characteristic for all of the ligand molecules bound to dna, while the information on ligand affinity to individual dna binding sites is lost. we propose a complimentary method for detailed analysis of thermodynamic parameters of ligand binding based on the introduction of fluorescent probes in the structure of g-quadruplex. monitoring fluorescence quenching of the fluorescent labels allows to derive binding constants of the dna-ligand interaction at a specific binding site. temperature dependence of the fluorescence quenching determines the thermodynamics of the dnaligand complex formation. since only a proximal ligand is able to quench the fluorescence, this method allows characterization of the ligand binding to a particular site the g-quadruplex structure. the study was supported by project no. 16-14-10396 of the russian science foundation. the correlation between biochemical and dynamic surface tension parameters of calves blood serum during the animal ontogenesis, as well as by various pathologies or poor diet, the imbalance of protein, mineral, lipid components is observed (the changes in all parameters of biological liquids are accompanied of these metabolism peculiarities). the dynamic surface tension (dst) of serum essentially depends on these factors and (in combination with the biochemical parameters) can provide the valuable information for evaluation of the physiological and biochemical status of the organism (can be used as an express test for animal diagnostics in future). the aim of the work was to study dst and biochemical parameters of calve serum, as well as their correlations, as the main indicators of the animals. ) of calve serum were in the range of the normal values for healthy animals and can be considered as reference data for animal science and practice. the obtained results enable us to establish correlations between the dst and biochemical parameters of calves serum. this work was supported by the russian scientific foundation (grant 14-16-00046). the middle strong correlations of dst values of calves serum with the level of total protein, albumin, billirubin, some enzymes and cholesterol, whereas only weak correlations with the other biochemical parameters (urea, calcium, magnesium, phosphorus, etc.) were found. in the veterinary science and practice such correlations are important for the estimation of the organism physiologicaland biochemical status, for general inspections of cattle before vaccination (immunization) or slaughter, for "quick separation" of healthy and ill animals in the case of infection, etc. role of protein kinase c in the regulation of astrocytic glutamine transporter sn1 in ammonia-exposed mouse cortical astrocytes (bisi; 1 lm). total pkc activity was analyzed by a direct pkc assay and phosphoserine detection by western blot (wb) analysis. protein level of sn1 and sn2, second astrocytic gln transporter belonging to system n, in a membrane fraction was also analyzed. the total uptake and system n-mediated (l-ala and l-leu-inhibitable) gln uptake was tested. treatment of astrocytes with ammonia resulted in a decrease of pkc activity, whereas pma treatment increased pkc activity in ammonia-independent way. bisi treatment reversed fully, and ammonia partially, the pma-induced pkc activity. pma treatment resulted in only a slight decrease in sn1 protein level in both control and ammonia-treated astrocytes, while a decrease of total and system n-mediated gln uptake were noted in control astrocytes, an effect not exacerbated by ammonia. in turn, cotreatment with pma and bisi reversed the decrease of total gln uptake and showed tendency towards increase in system nmediated gln transport. the results suggest that: a) ammonia changes the dominating direction of system n transport from release to uptake, which may be related to decreased phosphorylation or to alterations in relative phosphorylation by different pkc isoforms. this inference remains to be verified in further studies; b) changes in system n transporter function induced by ammonia appear to involve mechanisms other than changes in transporter expression. evidence for human ghrelin ghs-r1a and orexin ox1 heteroreceptor complex formation in a heterologous system ghrelin and orexin are two peptides implicated in the regulation of energy balance and modulation of food-related motivation at the level of the midbrain dopamine reward system. their function in the hypothalamic arcuate nucleus and the ventral tegmental area (vta) has already been described, but the modulation at the level of receptors remains unclear. the action of these peptides is mediated by g-protein-coupled receptors (gpcrs): ghrelin 1a and 1b (ghs-r1 a , ghs-r1 b ) for ghrelin, and orexin 1 and 2 (ox 1 , ox 2 ) for orexin. traditional approaches to know the mechanism of neurotransmission of dopaminergic neurons in the mesolimbic system have focused on targeting neuronal receptors as single entities. from the discovery that gpcrs for neuromodulators may form heteroreceptor complexes, our hypothesis is that ghrelin and orexin receptors may interact and form novel functional units that may specifically participate in the central regulation of food intake and energy balance. as a proof of concept we have investigated the potential of human ghs-r1 a and ox 1 receptors to form heterocomplexes. formation of ghs-r1 a -ox 1 receptor heteromers in transfected hek293t cells was detected by bioluminescence resonance energy transfer (bret) and proximity ligation (pla) assays. furthermore, a negative crosstalk was identified in cells co-expressing both receptors by assessing mitogen-activated protein kinase (mapk) and adenylyl cyclase (camp) pathways, and by a label-free dynamic mass redistribution assay. experiments in sources endogenously expressing ghs-r1 a and ox 1 receptors are needed to know the functional relevance of the heteromer. from the negative crosstalk here identified, it is tempting to speculate that ghs-r1 a -ox 1 receptor heteromers are important players in mediating the response to the combination of different orexigenic signals. lysosomal storage diseases which are related to deficiency of specific lysosomal hydrolases resulted to clinical aspects due to accumulation of substrates in different tissues. since dried blood spot (dbs) is non-invasive, low-cost, easy transportable, acceptable enzyme stability compared to leucocyte and/or fibroblast culture, it's recommended as a first screening test. however the false positive rate with dbs sample is higher compared to other samples. we aimed to investigate any possible effect of leucocyte number on enzyme activity in dried blood samples in a retrospective study. we re-evaluated the lysosomal enzyme activity results in regard to leucocyte number among data within last 1 year. enzyme activities had measured by using fluorometric and lc msms method. we determined the correlations between the lysosomal enzyme activities of alpha glycosidase, glycocerebrosidase, alpha galactosidase, sphingomyelinase, galactocerebrosidase and alpha-l-iduronidase in healthy population (n = 220). while glycocerebrosidase and galactocerebrosidase positively correlated with the number of neutrophils, alpha galactosidase, sphingomyelinase and alpha-l-iduronidase positively correlated with the number of lymphocytes. alpha glycosidase activity showed a correlation both lymphocytes and neutrophils. the patients having the glycocerebrosidase enzyme activity which was lower than 0.6 nmol/ml/hour (which is accepted as the cut off value to recall the patients) existed significantly lower number of leukocyte, lymphocyte and neutrophil compared to those of patients having higher enzyme activity than 0.6. our data indicated that the enzyme activity in dried blood samples including low leucocyte number might be found lower than reference intervals resulting in false positive diagnosis. therefore we suggest that the laboratory scientists should evaluate the number of leucocyte levels while they were interpreting data. using dna-markers for estimation of genetical variability of two kazakh sheep breeds a. mussayeva 1,2 , b. bekmanov 1,2 , a. amirgalieva 2 , k. dosybaev 1,2 , z. orazymbetova 1,2 , r. zhapbasov 2 , a. zhomartov 2 , n. zumadillaev 3 , n. zumadillaev 3 1 llp "kazcytogen", almaty, kazakhstan, 2 "institute of general genetics and cytology" sc mes, almaty, kazakhstan, 3 branch "scientific research institute of sheep" llp "kazakh research institute of animal husbandry and feed", almaty, kazakhstan to compare the frequencies of different microsatellite loci in sheep breeds subpopulations genomic structure of edilbay and kazakh archaromerinos was investigated. different methods for homogeneity testing of two breeds were elaborated. inter simple sequence repeats (issr) pcr analysis of the breeds studied displayed species and breed specific fragments with different frequencies (population frequency more than 0.4) there were found rarely met fragments (frequency lower than 0.4). the combinations of these fragments present the specific issr-spectra which arrange genofond profiles of breeds. using panels of microsatellits (recommended by isag) 2 breeds (5 populations) were characterized. informative value and resolving capacity of the sum of 32 str-loci were estimated. wide polymorphism of alleles length was demonstrated both when the breeds were compared and within the breeds. 10 informative markers were chosen for both two breeds, 7 markers being used for both breeds, while other 3 markers were informative for one of the breed only. when the animals of one breed were compared unique alleles which were met only within one of populations were of much interest. for example the allele 174 of bm1824 was met in birlik population of edilbay breed as often as in 59% of animals while in two other populations there were no this allele. in kumtekey population one can meet 70% animals having particular locus (dyms1), while in the other population (cf ablay) this locus was not met at all. basing on genetical distances obtained using fragment analysis phylogenetic relationships between populations were estimated. so for example edilbay population of cf ajar has the larger distance from two other populations (birlik and bayserke-agro) than each of them from one another. two subpopulations of kazakh arkharomerinos breed (cf kumtekei and cf ablay) also have the genetical difference. how preeclampsia affects oxidant status and antiinflammatory potential of breast milk? preeclampsia is a pregnancy syndrome associated with hypertension, proteinuria and edema, leading to maternal morbidity/mortality and preterm delivery. in this study we aimed to investigate if the breast milk of preeclamptic mothers is effected in oxidative status and anti-inflammatory activity in comparison to the breast milk of mothers with healthy pregnancies. for the aim of the study, hyaluronidase and myeloperoxidase activities (mpo), total oxidant status (tos), total antioxidant status (tas), oxidative stress index (osi) and tbars levels were measured in breast milks of 20 preeclamptic mothers and 20 mothers with healthy pregnancies as control group. when the control group and preeclamptic group were compared, hyaluronidase activity, tas, tos and osi levels showed statistically significant differences in the preeclamptic group. hyaluronidase activity was significantly higher in the preeclamptic mothers' breast milk (734 vs 594 u/ml, p = 0.046). while tos levels were significantly higher in the preeclampsia group (7.48 vs 4.51 lmol/l, p = 0.001), the tas levels were significantly higher in the control breast milks (0.557 vs 0.813 mmol/l, p = 0.011). as expected osi levels (tos/tas ratio) were significantly higher in the preeclampsia group. even though the mean levels were higher in preeclamptic group, the difference in mpo activities and tbars levels did not show statistic significance. oxidant status parameters also suggest that preeclampsia effects in both ways by increasing oxidant status and also decreasing antioxidant capacity shifting the balance to the increased oxidant stress side. as the results showed that the preeclampsia group had higher hyaluronidase activity, this can be interpreted as preeclamptic mothers' milk have higher inflammatory potential as this enzyme enhances inflammation by catalyzing the depolymerization of certain acidic glycosaminoglcans. p-mis-030 investigation of relationship between postprandial lipemia and erythrocyte membrane cholesterol level postprandial lipemia is a metabolic condition related to an increase in plasma triglycerides. remnant-like lipoprotein particles are predominant in postprandial phase and they play an important role in development of atherosclerosis. cholesterol is a prominent component of erythrocyte membranes and regulates the membrane functions such as viscosity and permeability. free cholesterol derived from erythrocytes is thought to participate in the atherosclerotic plaque formation. in the current study, it was aimed to investigate the relationship between postprandial lipemia and erythrocyte membrane cholesterol level in healthy subjects. study group included 87 subjects (39 female and 48 male with age range of 18-55 years). then these individuals were divided into three groups according to the values of area under curve (auc) calculated by using triglyceride levels at the fasting state and at 2nd, 4th and 6th hours after the high fat diet (ottt). lipid and erythrocyte membrane cholesterol (emc) values were compared between groups with low and high ottt response. while tc, tg, ldl-c and emc were significantly higher, hdl-c was significantly lower in high ottt response group than low ottt response group. it was not observed any statistically significant difference when compared emc values between women and men study groups. on the other part, it was seen positive correlation between emc and auc (r = 0.33, p = 0.002), tg (r = 0.26, p = 0.016), tc (r = 0.30, p = 0.005), ldl-c (r = 0.29, p = 0.007) in the total study group. it was concluded that, postprandial lipemia may show atherosclerotic tendency not only with atherogenic lipid profile but also with increasing emc. p-mis-031 eu-openscreen: the european infrastructure for chemical biology b. stechmann, p. gribbon eu-openscreen, fmp leibniz institute for molecular pharmacology, berlin, germany small molecules that can be applied as chemical 'tool' compounds (or 'probes') have become indispensable in basic research for the elucidation of fundamental biological mechanisms. they act directly with the protein-of-interest and often allow for the interrogation of biological processes that cannot be properly studied with traditional genetic or rna interference approaches. eu-openscreen (www.eu-openscreen.eu) is the largest emerging academic chemical biology research infrastructure initiative in europe and will provide access for molecular and cell biologists to screening infrastructure, well-characterized highquality chemical libraries, and facilities for medicinal chemistry services for compound optimization. molecular biologists who have a robust and suitable biological assay and are interested in collaboratively developing chemical tool compounds to validate their targets-of-interest are welcome to work with eu-openscreen. selected assays are screened against a collection of more than 100,000 compounds, incl. confirmatory and counter screening, ic/ec50 determination, sar (structure-activity relationships) and qc of confirmed hit compounds. eu-openscreen will start operations in 2017, but it can already look back on a growing number of transnational activities: joint screening projects, exchange of local compound libraries, development of new design principles for its compound collection; exchange of experimental data through its pilot database etc. steps towards an arthrobacter nicotinovorans based biotechnology for production of 6hidroxy-nicotine as the archetypal agonist of nachr, nicotine stands up as a powerful scaffold for developing new alzheimer disease therapeutic agents in form of nicotine derivatives. in this context, arthrobacter nicotinovorans pao1 and its wide range of nicotinederivatives produced when grown on nicotine have a huge biotechnological potential. indeed, the metabolic intermediate 6-hydroxy-nicotine (6hnic) produced by a. nicotinovorans pao1 was shown to bind to the nachrs, and by modulating their function, to sustain spatial memory formation in a rat model of ad. the current work presents the first attempts to produce and isolate 6hnic by using a genetically engineered a. nicotinovorans strain. the growth and the 6hnic accumulation were compared for two strains: 1. a. nicotinovorans pao1 wild type strain and 2. a genetically engineered a. nicotinovorans pao1 strain (part2ndh) containing the nicotine-dehydrogenase (ndh) genes cloned in the nicotine inducible part2 vector. the growth curves were followed spectrophotometrically. the consumption of nicotine and accumulation of 6hnic were monitored by hplc using a mn nucleodur 100-3 c18ec column and 0.1 m sulfuric acid at a flow rate of 1 ml/minute. the growth curve of the part2ndh strain shows that the bacteria grow slower when compared with the wt. as a result, in the wt strain, the nicotine is quickly depleted from the medium and only low amounts of 6hnic are observed. although the sds-page analysis of the total protein extracts from the part2ndh strain did not show clear signs of ndh overexpression, the enzyme is produced and is active, allowing a 5 fold accumulation of 6hnic in the growth medium. the first attempts to purify ndh from the part2ndh strain using imac were nevertheless unsuccessful. in conclusion, using the part2ndh strain for 6hnic production is feasible. further improvements of the growth condition and strain are envisioned (i.e. knocking the ndh downstream genes; adding inhibitors for the downstream enzymes). studies on the impact of butyrylcholinesterase (bche) on the symptoms and progression of cognitive impairments like alzheimer's disease (ad) or other neurodegenerative disruptions speak in favour of selective bche inhibitors as a new approach in future ad pharmacotherapy. some derivatives of quinine and quinidine, present in the cinchona species bark, have already been identified as selective bche inhibitors with respect to acetylcholinesterase (ache); therefore, further investigation of these compounds might result in promising leads for enhanced anti-ad drugs. we synthesised ten quaternary derivatives of cinchonines and their corresponding pseudo-enantiomeric cinchonidines. quaternization of quinuclidine moiety was carried out with groups diverse in size: methyl and differently meta and para substituted benzyl groups. all of the compounds were prepared in good yields, characterized by standard analytical spectroscopy methods, and were tested for their bche and ache inhibition potency. the inhibition potency of the compounds was defined by the dissociation constants of the enzymeàinhibitor complex (ki). all of the tested compounds reversibly inhibited both human bche and ache. the compounds inhibited bche with ki constants in the range of 0.04-30 lm, and ache in the range 2.5-70 lm. five cinchonidines displayed a 95-510 times higher inhibition selectivity to bche over ache, and four of them were potent bche inhibitors with ki constants up to 100 nm. bche affinity toward the studied compounds depended on the size of the substituent on the nitrogen of the quinuclidinium part of the molecule and on the resonance stabilization of the substituent at the quaternized nitrogen. based on the presented results, cinchonidine cd-(pbr) can be pointed out as a potent and selective bche inhibitor that could be considered for further research in alzheimer disease pharmacotherapy. exposure to nmda (100 lm) for 72 h increased the expression of kir4.1 mrna and decreased that aqp4-and gs mrna. the expression of kir4.1 was decreased by 72 h exposure to glu (2 mm) and tnfa (50 ng/ml). at 8 h incubation, nmda induced a decrease of kir4.1 expression in the presence but not in the absence of calcium in the medium. nmda did not alter the expression of nmda receptor subunits. tnfa increased the expression of the nr1 subunit, and decreased that of nr2b mrna. glu decreased the expression of 3 out of 7 subunits. the study demonstrates, to our knowledge for the first time, that prolonged exposure of astrocytes to nmda alters the expression of mrna coding for critical astrocytic proteins. the dependence of the decrease of kir4.1 mrna expression on extracellular calcium suggests the ionotropic nature of nmda receptor stimulation. the effects of nmda receptor stimulation occurred by a mechanism bypassing changes in subunit composition of the nmda receptor. experiments are under way to establish whether the tnfa-induced changes in the expression of nmda receptor subunits contribute to modulation of nmda receptor stimulation by inflammation. the importance of education in reducing preanalytical errors the preanalytical phase includes the request of test, the preparation of patient, the obtaining of sample from the patient, the transport of the sample to the laboratory, and the pretreatment of sample. the preparation of patient and the obtaining of sample are considered as the most common error sources. in order to reduce preanalytical errors, we aimed to provide training for phlebotomists and to also determine their knowledge level about the preanalytical phase before and after these training. it was given the training related with preanalytic phases to 130 pediatric nurses and 350 adult nurses, other phlebotomists in march. the surveys which are made before and after the training were consisted of 20 questions that are related with demographyic features and preanalytic phases. in order to determine the effects of training to the preanalytic phase, the preanalytic error rates before (in february) and after (in april) traning was calculated with the formula of: (the number of rejected samples/the number of total samples)x100. the average age of participants was 35 ae 9 years. it was not found significant difference between their correct answers rate before the training and the education degree of the participants. the correct answer rate before the training was 59% and after the training it was 92%, which showed an increase of 55%. the preanalytic error rates which were 0.61% in february were decreased to 0.40% in april. in our study, the positive results were obtained through the training aimed to reduce the preanalytical errors. by providing regular training to the phlebotomists and also providing pretraining to the beginners, the updating of their information about preanalytic phase can be achieved. in this way, the loss of labor and economic related to preanalytical errors can be avoided and the accurate results can be obtained in short time. curcumin, the active compound of turmeric (curcuma longa) has antiinflammatory, antioxidative and antitumour effects. unfortunately, curcumin has a poor absorption and low stability. both can be solved by encapsulation of curcumin using a proper technique like electrospray. it was reported that piperin, the active compound of black pepper, enhances the intestinal absorption of curcumin and thus its bioavailability. due to these facts it was aimed in this study to nanoencapsulate turmeric extract in order to enhance its absorption and stability. for that purpose, it was encapsulated with the maize protein zein, chitosane and black pepper extract by varying the voltage and flow rate of electrospray and the concentration of the compounds. the nanocapsules were characterised by measuring their particle size and with help of sem photographs. the particle size of the final nanocapsule formulation was 550 nm and had a sufficient stability over a period of 6 months, visually determined. by encapsulating turmeric extract into double layer nanocapsules with help of black pepper extract, zein and chitosane, the turmeric extract could be protected from degradation, which was observed for the pure turmeric extract in form of clearing its yellow colour. analysis of the human genome reveals that potential g-quadruplex sequences are enriched in promoters of the oncogenes. growing body of evidence suggests that g-quadruplexes (g4) may play putative roles in various biological processes, such as the regulation of gene expression. consequently, targeting the oncogenic g-quadruplexes using small molecules is an alternative strategy for the potential treatment of cancers. porphyrin derivatives are promising class of drug in this respect, being nucleic acids binders and generators of reactive oxygen species under visual light irradiation. interaction between porphyrin derivatives and g4 dna from oncogene promoter region has been studied in vitro. we applied chemical probing, circular dichroism spectroscopy and uv melting techniques in order to map the oxidized bases, monitor structural rearrangements and evaluate stability of the resulting dna structures. specifically, we observed that g4 dna is considerably more susceptible to lightinduced modification than duplex dna; 5 0 -terminal tetrads of the g4 dna are preferably oxidized; structural changes induced by oxidation result in decrease of the thermodynamic stability of the g4 dna. irreversibility of these effects on dna make porphyrin derivatives perspective lead compounds for rational design of ligands targeting human oncogenes. the study was financially supported by project no. 16-14-10396 from the russian science foundation. resistin levels in denervated obese rats n. saglam 1 , t. ahmedi rendi 1 , c. kahraman 2 , a. alver 1 1 department of medical biochemistry, faculty of medicine, karadeniz technical university, trabzon, turkey, 2 school of health, d€ uzce university, d€ uzce, turkey the sympathetic nervous system is an important factor affecting the metabolic and secretory function of the white adipose tissue. resistin is mainly expressed by mononuclear cells, also it is expressed by adipocytes, pancreatic cells, and muscle. resistin induces insulin resistance and glucose intolerance in mice. resistin plasma levels depend on fat depots size and sex. resistin levels decrease in short-term fasting in mice, then it increase refeeding. also, it increase as a response to fed with the high fat diet. in our study we aimed to determination of the effect of high-fat diet and denervation on serum resistin levels in rats. in this study 4 experimental groups were formed each consisted of 8 rats. during 10 weeks, first two groups are fed with high-fat diet and other two groups are fed with standart diet which they purchased from research diets company. at the beginning of the feeding periods, retroperitoneal fat tissiues of animals assigned to the first and the third groups were denervated. second and fourth groups were not denervated. at the end of the 10 week feeding periods, blood collected from rats and blood resistin concentration was determinated by elisa. in denervated and fed with high fat diet groups serum resistin levels higher than control groups (p < 0.05). according to our literature research, there are no studies demonstrating the relationship between resistin and the sympathetic nervous system. also, denervation may lead to increase in serum resistin levels. the amount of resistin is possibly reduced by b -adrenergic activation. in conclusion, it was concluded that there is differences on serum resistin levels depending on diet in bilateral denervation of retroperitoneal fat tissues of rats. stress activated protein kinases regulates the ribosomal frameshift rate in est3 gene, encoding subunit of telomerase s. t€ urkel, s. sarica uludag university, bursa, turkey est3 gene (ever shorter telomere) of s. cerevisiae encodes one of the essential subunits of telomerase enzyme. expression of est3 gene is regulated at the translation level by +1 programmed ribosomal frameshift (prf). it is known that the physiological stresses affect telomere length. in this study, we have investigated the effects of stress activated protein kinases snf1p (ampk) and gcn2p (eif2 kinase) on the prf rate in est3 gene. prf rate of est3 gene was quantified in plasmid based expression system. expression vectors were transformed in to the wild type and mutant yeast strains that deleted for snf1, or gcn2 genes. yeast cells were grown in normal conditions or subjected to acid stress, osmotic stress, or glucose limitations to activate protein kinases gcn2p, and snf1p, respectively. prf rate of est3 gene was measured as 13% in the normal growth conditions in the wild type cells. but, the prf rate of the wild type strain grown in glucose limited conditions decreased more than 10-fold, giving less than 1% prf rate. contrary to glucose limitation, osmotic or acid stress activated frameshift rate by 2-fold in the wild type cells and prf rate increased to 25%. when the prf rate was analyzed in gcn2 and snf1 mutants, frame shift rate of est3 was 4-5% in normal growth conditions. when these mutants were subjected to acidic or osmotic stress, prf rate activated slightly. we have also shown that gcn1p and gcn20p, positive regulator of gcn2p, is also essential for the regulation of prf in est3 in response to stress conditions. it is clear that the basal level expression of est3 is highly dependent on the gcn2p kinase complex. gcn2p is also associates with ribosomes, indicating that gcn2p might have a significant function in connecting the stress signals to biosynthesis of the full length est3 peptide. this regulation might also link the biosynthesis of functional telomerase and telomere replications to cell physiology through protein kinases such as snf1p and gcn2p. inflammation might have a role in erosive esophagitis but not in non-erosive reflux disease the relationship between inflammatory activation mechanisms and acid-peptic injured esophageal tissue is not clear. we evaluated whether there are differences between inflammation and tight junctional proteins such as e-cadherine among subtypes of gastroesophageal reflux disease. the aim of this study was to investigate any possible role of inflammation in pathologic mechanism of reflux disease by determining the inflammatory markers in injured esophageal tissue as well as serum of patients. three groups (erosive-ee, n = 18; nonerosive-nerd, n = 12; healthy controls-hc, n = 13) were evaluated with upper gastrointestinal endoscopy. the esophageal biopsies and blood samples were collected. serum e-cadherine levels, nfkb, chitotirosidase (chit), myeloperoxidase (mpo) activities in serum and homogenized tissues were determined. nkfb levels in tissue was significantly higher in subjects with ee (4.9 ae 2.53 ng/mg.prt) versus hc (2.95 ae 1.51 ng/mg.prt, p = 0.018). mpo tissue activities in ee group were significantly lower (0.07 ae 0.06 u/mg.prt) than hc (0.23 ae 0.22 u/mg.prt, p = 0.025) while mpo serum levels were higher in ee (1.15 ae 1.63 ul) versus hc (0.56 ae 0.69 ul, p = 0.045). tissue chit levels were three fold increased in ee versus hc (p = 0.071). none of these measurements showed any differences in nerd group. nfkb and mpo levels had a negative correlation (r=à0.408, p = 0.005) in tissue. nfkb and ecad levels had a positive correlation in serum (r = 0.642, p < 0.0001). inflammatory process might play a pivotal role in injured mechanism only in erosive esophagitis but not in nerd. noninflammatory mechanisms might be responsible such as hypersensitivity in patients with non-erosive reflux disease. d-dimer (a fibrin degradation product) test is used to aid in the diagnosis of intravascular coagulation. the aim of this study is to investigate the correlation between d-dimer levels and other inflammatory markers including procalcitonin. anonymized data on d-dimer, fibrinogen, hscrp, wbc, neutrophil% (neut%) and procalcitonin levels from 50,107 patients (mean age ae sd, 53.37 ae 20.6) were used for the correlation (excel analyze-it v4.60.4) and linear regression (pasw statistics 18 v18.0) analysis between the measured parameters. there was a significant (p < 0.05) age-dependent increase in d-dimer levels between different age groups. patients with the highest d-dimer levels were also found to have an increased frequency of hscrp levels. d-dimer levels showed a significant correlation with hscrp, wbc and neut%. a model describing the positive association between these parameters were built. the resulting equation is as follows: d-dimer = (hscrp*0.054) + (0.011*age) + (0.006*wbc) + (0.04*neut%)à0.929. correlation analysis between procalcitonin and d-dimer levels gave pearson's correlation coefficient of 0.159. our results suggest that the age-dependent variations should be taken into account while interpreting d-dimer test results. in addition, neut% ratio was found to be the most important parameter for estimating d-dimer levels. our equation can be used when the d-dimer test is not available or for control purposes only. in the field of cancer research great hope lies in finding more powerful and selective way for the direct elimination of cancer cells. this task can be solved by means of nanobiotechnology. recent progress in this field has arisen interest in a carbon nanostructurefullerene c 60 . fullerene exhibits not only unique physico-chemical properties and biological activity but also a significant potential to serve as a nanocarrier for selective drug delivery into cancer cells. the aim of this study is to analyze a unique tool for cancer therapy. the main idea is realized by the non-covalent conjugation of c 60 with the well-known anticancer drug -doxorubicin (dox). two types of conjugate with different c 60 -dox ratio (1:1 and 2:1) were studied. conjugates absorbance and fluorescence, size distribution as well as a mass data were recorded utilizing optical and analytical equipment (microplate reader, zetasizer, lc-ms/ms and maldi-tof). in vitro studies were performed including evaluation of c 60 -dox conjugate effects on human leukemic cells (jurkat, ccrf-cem, thp1 ad molt-16) viability. conjugates accumulation and distribution within cancer cells was monitored using fluorescent microscopy accompanied with fluorescence-activated cell sorting. it was evidently proven that both c 60 -dox conjugates were stable and could be used as reliable candidates for biological application. cellular accumulation and distribution studies showed that conjugation of dox with fullerene promoted its entry into leukemic cells. accumulation of dox in the form of conjugates within cancer cells was intensified compared to the free drug. the results show that conjugated dox is more cytotoxic and the value of its ic50 are lower compared with the free dox. obtained results confirm nanocarrier function of fullerene c 60 and the perspective of its application for optimization of doxorubicin efficiency against leukemic cells. comperative investigation of protective effects of tea and tea-related wastes on reducing potaential of h2o2-induced erythrocytes tea processing waste (tpw) formed during the tea production process in tea factories is up to 50,000 tones/year in turkey. tpw is one of the abundant available phenolic biomass among plantal wastes. in this study, black and green teas and their wastes were used. the aim of the study is to determinate the phenolic content and the radical scavenging activities of the samples, and to measure their effects on hydrogen peroxide-induced erythrocyte damage due to analyzing the reducing potential of erythrocyte involving glutathione reductase (gr), glutathione peroxidase (gpx) activities and reduced glutathione (gsh) content. total polyphenol content of samples was determined as mg catechine per dry mass by using folin-ciocalteau reactive and dpph radical scavenging activity was estimated by cuendet method as equivalent catechine standard. in erythrocyte, gsh level was measured by method of sedlak and lindsay while gr and gpx activities were assayed by the methods of bergmeyer and beutler, respectively. the highest phenolic content was observed in green tea and its wastes (p < 0.01) whereas black fiber waste had the lowest phenolic content. therefore, the highest radical scavenging activity and gsh level were detected in green tea and its wastes (p < 0.01). erythrocyte with the extracts of the teas and their wastes had the similar enzyme activities for both gpx and gr. in sum, the teas and wastes have antioxidant activity but, green tea and its leaf waste hade higher antioxidant activity than other samples. the tea wastes might be evaluated as many of protective health products, particularly in cosmetic fields thus, these by-products no application for any area is expected to become an economical value. fluorouracil (5-fu) is a chemotherapeutic drug classified as an "antimetabolite". it works through irreversible inhibition of thymidylate synthase. chemical derivatization of 5-fu with carbohydrtates is being investigated widely in order to enhance its bioavailability, therapeutic efficiency and to reduce its toxicity. however, water solubility of the newly derived compounds is usually very low. so, in order to obtain a pharmaceutically relevant formulation they need to be formulated appropriately. in this study, we prepared micellar delivery system for the new tetra-o-acetylglycose derivative of 5-fu synthesized via "click reaction", namely f1-[{1 0 -(2″,3″,4″,6″-tetra-o-acetyl-b-dglycopyronosyl)-1 0 h-1 0 ,2 0 ,3 0 -triazole-4 0 -yl}methyl]5-fluorouracil. since the water solubility of this compound is very limited, we tested its solubility in several pharmaceutically relevant solvents by visual estimation after stiring increasing amount of the compound in 1 ml of solvent for 48 h. to estimate the carcinogenic potential of this compound, salmonella/microsome mutagenicity assay (ames test) was performed in four histidine-requiring strains of s. typhimurium, tester strains ta98, ta 1537 (for the detection of frameshift mutations) ta100 and ta 1535 (for detection of base pair substitutions) according to the oecd guideline 471. the drug was solubilized (500 lg/ml) with no precipitation in lutrol ò -f68/ethanol/water (2.25:2.25:5.5 , wt/wt) micelles (7.3 ae 1.0 nm). the results of ames test were negative so the compound neither produced frame shift mutations nor base pair mutations in s. typhimurium strains. the results imply that the new compound can be dissolved in aqueous micellar delivery system in order to be used for further studies, and that it was not mutagenic in the tested s. typhimurium strains. in conclusion, the formulation of the newly synthesized compound is not carcinogenic, and can be evaluated for anticancer activity in vitro and in vivo. integral metabolism parameters of dairy goats during reproductive cycle periods d. solovyeva, e. zarudnaya, s. zaitsev moscow savmb, moscow, russia study of the goat metabolism at different periods of the reproductive cycle allows to correct feeding ration, to increase the age of the productive use of animals and to receive high-quality products. the aim of the work was to determine the metabolic parameters of blood serum of goats, expressed in terms of biochemical parameters and interfacial tensiometry and study their relationship to metabolic processes in the body goats depending on the age and the period of the reproductive cycle. the 90 healthy goats were divided into 5 groups. the dynamic surface tension (dst) parameters were obtained from dependences of a surface tension (r) vs. time (t): at t?0 (r 0 ), at t = 0.02 s (r 1 ), t = 1 s (r 2 ) and t?∞ (r 3 ). this work was supported by the russian scientific foundation (14-16-00046). all animals had 4-5% fat content. the contents of total protein (5.4%), albumin (8.4%) and urea (8.8%) are higher for the lactating animals as compared to the normal goat values. the levels of total cholesterol (18.0%) and creatinine (6.2%) are higher for the lactating animals. in lactating animals have the highest level of, which along with high phosphorus level talks about the intensification of energy processes during lactation. the correlations were found between the biochemical and dst parameters of the goat blood: lipids or cholesterol levels with r 0 (r = à0.40), r 1 (r = à0.72), r 2 (r = à0.89); total protein or albumin levels with r 1 (r = à0.42), r 2 (r = à0.56), r 3 (r = à0.90); aminotransferase activity with r 2 (r = à0.47), r 3 (r = à0.63). the correlations were found between the total protein and albumin levels with k 0 (r = 0.54), k 1 (r = 0.44); glucose levels and r 1 (r = 0.47), r 2 (r = 0.43). thus, the dst and biochemical parameters of goats have strong correlation relationships that are important for biomedical and veterinary applications. the relation of the severity of atherosclerotic disease with oxidative stress in patients with stable coronary artery disease h. sezen harran university, sanliurfa, turkey introduction: because, to the best of our knowledge, the relationship of total oxidant status (tos) and total antioxidant status (tas) with the severity of stable coronary artery disease (cad) has not been investigated in the literature so far, the present study was conducted to address this issue. materials and methods: this study consisted of 349 consecutive patients and controls who underwent coronary angiography. for each patient, the total gensiniscore (gs) was calculated andthose with a gs of >20 were classified as the high gs group (hgg), and those with a gs less than 20 were defined as the low gs group (lgg). the total oxidant status (tos) and total antioxidant status (tas) levels were measured using the erelmethod. the osi, which is an indicator of the oxidative balance, was calculated as the percentage ratio of tos to tas. results: the tas was lower in the hgg than lgg. the tos and osi were higher in the hgg than lgg. the correlation analysis showed that gs was negatively associated with the tas and positively with the tos and the osi. the multivariate analysis showed that age, tos, and hdl-c were independent variables for a high gs. the cut-off level of 10.9 lmol h 2 o 2 equiv./ l for serum tos levels predicted high gs with a sensitivity of 70% and a specificity of 56%. discussion: information on the severity of atherosclerosis is requiredtopredicttheprognosis of an individualpatientandtodetermine the proper treatment modality. the gs system has beenproventodemonstratethe severity of atherosclerotic disease. inthepresentstudy, thepatientswith a high gs had increasedlevels of oxidants. inaddition, tos was an independentindicator of theseverity of atherosclerosis. the optimal cut-offvaluefor tos topredict high-gens score was 10.9 (sensitivity 70% and specificity 56%). conclusions: the results suggest that the severity of atherosclerosis in stable cad is associated with increased oxidative status. evaluation of roemerine as a multidrug resistance pump inhibitor f. g. avci 1 , c. velioglu 1 , e. recber 1 , c. unsal 2 , g. gulsoy 2 , b. sariyar akbulut 1 1 marmara university, istanbul, turkey, 2 istanbul university, istanbul, turkey efflux by multidrug resistance (mdr) pumps is a common defense mechanism used against antimicrobials. by pumping the drugs out, these pumps significantly reduce the efficacy of drugs. one approach to overcome this limitation is offered by the combinatorial therapies where drugs are co-administered with together with pump inhibitors. by simply preventing the efflux of the drug, the presence of inhibitors enhance drug efficacy. (-)-roemerine is an aporphine type alkaloid with significant antibacterial (against bacillus cereus, escherichia coli) and antifungal (against candida strains) activities. interestingly, (-)-roemerine was also found to enhance the cytotoxic response mediated by vinblastine in multidrug-resistant kb-v1 cells. in the same study, this finding was linked to its possible interaction with p-glycoprotein, a eukaryotic mdr pump. taking this finding as the starting point, the current study investigates the potential of roemerine as an inhibitor of the p-glycoprotein homologue pump, bmra, in bacillus subtilis 168. the antimicrobial agent berberine was used as the model agent since its efficacy is reduced by efflux through mdr pumps. to this end, bacillus subtilis 168 cells were subjected to 100 lg/ml berberine, a value well below the mic. this concentration only slightly retarded growth for 2 hours but then cells resumed their regular growth. upon addition of 25 lg/ml (-)-roemerine to the bacillus subtilis 168 cells treated with berberine, growth pattern changed, indicating possible interaction with bmra. further investigation for the change in the expression of bmra was achieved with real time pcr analysis. glucose oxidase is an enzyme that catalyzes the oxidation of glucose to d-glucono-1,5-lactone and hydrogen peroxide. we hypothesized that enzyme would cause a double negative effect on cancer cells, by reducing the presence of glucose in cancer microenvironment and producing reactive oxygen species. to increase enzyme stability and enhance cellular uptake we encapsulated the enzyme with a thin acrylamide layer. the purpose of this work was to optimize the synthesis of these glucose oxidase nanoparticles and investigate their effect on cancer cells. nanoparticles containing single glucose oxidase were synthesized in two steps; first by introducing the vinyl groups onto the surface of enzyme by acyloylation followed by polymerization step with acrylamide monomers. encapsulated enzymes are approximately 150 nm in size and retain most of their activity. after the optimization of nanoparticles, the anticancer potency of these nanoparticles was in vitro tested in mcf-7 breast cancer cell line. according to results, both nanoparticles and free enzyme are capable of inhibiting viability of cancer cells in a similar manner at very low concentrations. currently we are investigating mechanisms involved in this viability inhibition. initial results demonstrated that glucose supplement does not rescue cells from death induced by the activity of glucose oxidase, suggesting an oxidative stress related cause of inhibition. further studies are required to elucidate the exact mechanism. until now there is no determined advantage of glucose oxidase encapsulation against proteolysis. however, encapsulation may induce the accumulation of enzyme in cancer microenvironment. furthermore results suggest that glucose oxidase has a high effect on the viability of mcf-7 breast cancer cells indicating that this enzyme may have a potential use in cancer treatment. studies on the interaction of human phospholipid scramblase 1 with c-terminal domain of topoisomerase iia u. sivagnanam, s. n. gummadi applied and industrial microbiology lab, bhupat and jyoti mehta school of biosciences, indian institute of technology madras, chennai, india human phospholipid scramblase 1 (hplscr1) is a multifunctional protein that plays key roles in several cellular processes including apoptosis, tumorigenesis, anti-viral defense, cell signalling and several protein-protein interactions. it has been shown that hplscr1 interacts with the c-terminal domain of topoisomerase iia (topo iia) and enhances its decatenation activity in vitro. the interacting region in topo iia was identified but till date, no reports exist on the binding region in hplscr1. this study aims to identify the region of hplscr1 that interacts with topo iia. to identify the topo iia interacting sites in hplscr1, nterminal deletion constructs of hplscr1 viz δ25-hplscr1, δ50-hplscr1, δ75-hplscr1, δ100-hplscr1 and δ160-hplscr1 were generated by pcr, cloned, overexpressed and purified to homogeneity using ni 2+ -nta purification. the cterminal domain (ctd) of topo iia was cloned in pgex6p-1 and was expressed as a gst fusion protein. gst pull down assays will be performed with the deletion constructs of hplscr1 and the gst-ctd-topo iia. the binding region in hplscr1 will be confirmed by peptide competition assays. our initial results show that the decatenation activity of topo iia was enhanced when the topo ii was pretreated with hplscr1. δ100-hplscr1 did not show any enhancement of the decatenation activity compared to full length hplscr1. hence, the binding region could be in the 1-100 region of hplscr1. further deletions were done in the 1-100 region of hplscr1 as described earlier. gst-pull down assays and decatenation assays will be performed for the deletion constructs to narrow down the region of hplscr1 that binds to topo iia. we conclude that hplscr1 interacts with and enhances the activity of topo iia and the 1-100 region of hplscr1 is critical for enhancement of decatenation activity. further work is under progress to identify the exact topo iia binding region of hplscr1 and the physiological relevance of this interaction in the cell. a. ugur kurtoglu 1 , v. aslan 2 , e. kurtoglu 2 1 department of biochemistry, antalya education and research hospital, antalya, turkey, 2 department of hematology, antalya education and research hospital, antalya, turkey beta-thalassemia is a common autosomal recessive disorder resulting from over 200 different mutations of the beta-globin genes. our aim was to creat a mutation map of beta thalassemia in province of antalya, turkey. in this study, mutation analysis of a total 150 of beta-thalassemia patients followed up at the thalassemia center of the antalya education and research hospital, antalya, turkey, were included. according to our results, the ivs 1.110 is the most frequent mutation type in our province same as other geographical regions of turkey. the most frequent mutations in heterozygous or homozygous patients are ivs 1.110, ivs 1.6, ivs 2.1 and ivs 1.1. our results indicate the importance of micromaping and epidemiology studies of thalassemia, which will assist in establishing the national prevention and control program in turkey. keywords: beta-thalassemia, beta-globin gene, mutation p-mis-052 investigation of the in vitro effects of some antibiotics on the purified beta-glucosidases from the rat liver n. t€ urkmen 1 , h. kara 2 1 karadeniz technical university medical biochemistry department, trabzon, turkey, 2 balikesir university veterinary faculty biochemistry department, balikesir, turkey beta-glucosidases catalyzes the hydrolysis of the glycosidic bonds to terminal non-reducing residues in b-d-glucosides and oligosaccharides.b-glucosidases are widely distributed in the living world.b-glucosidases which in mammals, primarily found in the liver and kidneys;lysosomal b-glucosidase (gba1),non-lysosomal b-glucosidase (gba2), cytosolic b-glucosidase (gba3),intestinal lactase-phloriz the hydrolase (lph). liver tissues of wistar-albino rats were homogenized with homogenizer in the extraction buffer and crude extract was obtained after centrifugation.ammoniumsulfate precipitation range designated crude extract was purified by sepharose 4b-ltyrosine-1-naphthylamine hydrophobic gel.commercially availabled antibiotics were prepared with substrate buffer.it was investigated inhbition effects of cefuroximesodium, ampicillin-sulbactam, amoxicillin trihydrate/potassium clavulanate, cefazolin sodium, gentamicin sulfate and ceftriaxone disodium antibiotics onto gba2.inhibition types and k i values of related antibiotics were determined with p-npg substrate.lineweaver-burk plot was used for that purpose. rat liver gba2 was purified at 30.2-fold with 43.4% yield.gba2 was illustrated 58 and 110 kda at sds-page.ic 50 value of ampicillin/sulbactam antibiotic for gba2 was found 62.97 mg/ml with competitive type inhibition and other antibiotics didn't inhibit. purfication methods are being used in the literature for the purified b-glucosidase from different sources.purified gba2 was illustrated 58 and 110 kda at sds-page.about molecular weight of bglucosidases is presented different information in the literat€ ure. this has been reported because of acid beta glucosidases are abnormal migration at the acrylamide or agarose gels.it was investigated inhbition effects of various antibiotics onto purified gba2.ampicillin/sulbactam antibiotic inhibited to purfied gba2 at the competitive type.similiar antibiotics studies have been made in the literature for different enzymes. effect of glutamine on insulin resistance and endoplasmic reticulum stress g. aydogdu 1 , p. b. sermikli 1 , a. abbasi taghidizaj 2 , e. yilmaz 2 1 ankara university, institute of science, ankara, turkey, 2 ankara university, biotechnology institute, ankara, turkey obesity and diseases are one of the most important public health problems of the world.excess fat storage in adipocytes leads to the release of increased amounts of non-esterified fatty acids, glycerol, hormones, cytokines, which are factors involved in the development of insulin resistance that cause type 2 diabetes. one of the major differences between obese and lean individuals is the amino acid concentration in the circulation. although there are many studies about the amino acid metabolism associated with insulin resistance in obese individuals, the effect of glutamine metabolism in insulin resistance mechanisms are not well understood yet. glutamine can be used as fuel and its levels in tissues and circulation can regulate cell responsiveness to insulin and cellular metabolism. therefore, glutamine is a potentially important factor that might help us better understand insulin resistance and type 2 diabetes. to determine whether glutamine effect on insulin resistance and endoplasmic reticulum stress, 3t3-l1 cell is treated with different concentration of glutamine and analyzed by western blot for er stress markers. our results indicated that glutamine reduced endoplasmic reticulum stress and related with that attenuated insulin resistance. in case of transport of amino acids, insulin resistance, how it is affected when we have the information about the important tips on energy requirements and metabolism reach insulin resistance and type-2 diabetes treatment is likely to reveal a possible new targets. how does different lead levels affect tsh, ft3, ft4, vitamin b12 and folate? e. ozkan ankara occupational disease hospital, ankara, turkey exposure to heavy metals is increasing with the industrialization of society. one of the most intense exposure to heavy metals is pb on this issue. this study was aimed to determine the relationship between different blood pb levels and serum thyroid hormones (th), vit b12, folate. the cases were 20-65 years old, male individuals who admitted to our hospital between april 2012-march 2016 for periodic inspections because of occupational exposure to pb. the parameters of the cases were retrospectively retrieved. according to their pb levels, exposed workers (n: 9400) were divided into four subgroups; group (g) 1: 0-9.99 lg/dl, g 2: 10-19.99, g 3: 20-39.99, g 4: ≥40 . from these, the number of cases whose th levels were measured (n:3894) given respectively; g 1:3062, g 2:178, g 3:334, g 4:275 cases. also the number of cases whose vit b12 and folate levels were measured (n:2807) given respectively; g 1:2139, g 2:143, g 3:276,g 4:249 cases. levels of pb were determined by icp-ms. th, vit b12, folate were determined by cmia. between the groups formed according to pb levels, there was no significant difference in terms of average t3, tsh and vitamin b12 (p > 0.05). on the other hand there was statistically significant difference between t4 and group 1,3,4 (p < 0.05) but there was no difference between group 2 (p > 0.05). the average folate belongs to the first group was about 10% higher than the other 3 groups, and found that the difference was statistically significant (p < 0.05). there are many publications which have various results between pb levels and t3,t4, tsh. but this study is important to compare the effect of different levels of pb. up to day there was no publication about the relation between different pb levels and vit b12, folate. it was seen that there was no significant clinical relation between different pb levels and thyroid parameters, vit b12. but the low levels of folate in the high pb levels groups shows us that we need further studies about this relationship. fluorescent study of in meso crystallization of membrane proteins with the introduction of membrane protein in meso crystallization 30 years ago by landau and rosenbusch, a new era of membrane protein structural research has emerged (1). since that time this method became associated with a number of major breakthroughs in the field (2) including exceptional successes in structural studies of microbial rhodopsins and g-protein coupled receptors (3) . here we used fluorescence microscopy to study in meso crystallization process of bacteriorhodopsin. several observations provide new insights into the in meso crystallization process. the crystallization starts with formation of microcrystals, followed by growth of a dominating crystal at the expense of smaller ones and formation of a depletion zone around it. these observations demonstrate an ostwald ripening mechanism of the in meso crystal growth. the depletion zone formed around the growing crystal is consistent with the previously proposed analogy relating in meso crystallization with the crystallization in a microgravity convection-free environment. this work is supported by rsf 14-14-00995. (1) landau, e. m.; rosenbusch, j. p. proc. natl. acad. sci. u. s. a. 1996, 93, 14532à14535. (2) caffrey, m. acta crystallogr., sect. f: struct. biol. commun. 2015, 71, 3-18. (3) katritch v., cherezov v., stevens r.c. (2013) . annu rev pharmacol toxicol 53, 531-556. p-mis-058 stamp1 is critical for both ar and mtor signaling in prostate cancer cells x. sheng, y. jin, f. saatcioglu university of oslo, oslo, norway androgen receptor (ar) signaling plays a central role in the initiation and progression of prostate cancer (pca), including when the disease progresses to castration-resistant pca (crpc). the second central signaling pathway in pca, similar to various other cancers, is the pi3k-akt-mtor signaling. importantly, these two oncogenic pathways cross-regulate each other in pca cells by reciprocal feedback, thereby maintaining tumor cell survival even when one is suppressed. we have previously identified that the six transmembrane protein of prostate 1 (stamp1) promotes pca cell proliferation as well as inhibits apoptosis through, at least in part, regulating the erk mapk signaling. human pca cell lines lncap and vcap were used in the study. colony formation, soft-agar growth, prostatosphere formation assays were performed. for in vivo xenograft experiment, the cells were implanted subcutaneously into the flanks of nude mice. here, we show that stamp1 knockdown caused defects in colony formation, anchorage-independent growth and prostatosphere formation in lncap and vcap cells both in vitro, as well as tumor formation and growth in vivo. this may be due to the impaired ar and mtor signaling in these cells upon stamp1 knockdown. interestingly, in the crpc cell line 22rv1, where-stamp1 knockdown did not affect mtor signaling, there was a remarkable repression of tumor take rate and growth. these results clearly indicate that stamp1 is essential for both ar and mtor signaling, and is crucial for pca growth in vitro and in vivo. however, the detailed molecular mechanism requires further investigation. taken together, these data unveil a critical role for stamp1 in coordinating the ar and mtor signaling pathways in pca cells, solidifying the basis for its pro-survival effects in pca, including in advanced disease. quantification of 2d thin layer chromatograms using 2d gel analysis software and gel documentation system o. kaynar, m. ileriturk, d. kaynar, s. kurt ataturk university, erzurum, turkey introduction: thin layer chromatography (tlc) is an important chromatographic technique that is widely used as a cost-effective method for rapid-sensitive analysis of compounds in plants, animals, and humans. however, one dimentional (1d) tlc is not sufficient for the separation of complex compounds. therefore, two-dimentional (2d) tlc was developed. the quantitative evaluation of plates are performed with tlc scanners or documentation systems. however, these systems specific for 1d plates, and cannot be adapted to quantitative evaluations of 2d plates. in this study, the applicability of the gel documentation systems and 2d analysis software for the analysis of 2d tlc plates were examined. material and method: 2d tlc of lipids: 1st dimension: methyl acetate/n-propanol/chloroform/methanol/0.25% kcl (25/ 25/28/10/7 v/v); 2nd dimension: chloroform/methanol/acetic acid/ water (90/40/12/2 v/v); detection: charring. 2d tlc of aminoacids: 1st dimension: 1.5% (v/v) formic acid; 2nd dimension: toluene/glacial acetic acid (10:1 v/v); detection: uv. phospholipid and aminoacid standards, each include 6 different classes were developed by 2d tlc. plates visualized with biorad geldoc xr, and band volumes on plates were calculated with biorad pdquest 2d gel analysis software. for the method validationa) 5 plates containing same 6 lipid classes were developed in the same day, and results were used for the calculation of intra-assay cv;cv% = average of each sample standard deviation/mean of sample 9 100 b) 6 plates containing same 6 lipid classes were developed in 6 different days, and results were used for the calculation of interassay cv; cv% = standard deviation of each sample average/mean of the plates 9 100 results: volume of each phospholipid and aminoacid had less than 10% intra and inter-assay cv. conclusion: gel documentation system with 2d gel analysis software can be used for the quantitative analysis of the 2d tl plates both at uv and visible light. the role of na + k + atpase activity in the vasodilatory effect of n-acetylcysteine introduction: spasm occurred at the stage of and after the preparation of arterial grafts used in coronary artery bypass surgery (cabg) is effective on morbidity and mortality in the first 24 hours of postoperative patients. n-acetyl cysteine (nac) that vasodilatory effect is known,may be considered as a suppressor agent for vasospasm developing during cabg. however, for the prevention of complications that may arise during or after cabg mechanism of these vasodilatory effects should be described. this study was aimed to investigate the role of atpase enzyme on the vasodilatory effect of nac. materials and methods: in this study, 28 adult male wistar albino rats were used. rats were separated into four groups as control rats (g1), 2 mm nac (g2), 5 mm nac (g3) and 10 mm nac (g4). a portion of the thoracic aorta isolated from rats was used for the relaxation response recording, and the other portion was used for measurement of nakatpase activity. isolated smooth muscle rings are suspended in the 20 ml organ bath containing krebs solution for relaxation responses. in all groups, level of smooth muscle contraction were allowed to reach a plateau by adding 60 mm kcl to the organ bath. then, in the first 10 minutes of application relaxation responses which created by adding nac to the medium were recorded and the maximum relaxation responses were measured. nak atpase activity was determined using the mazzanti method. groups means were compared by one-way analysis of variance (anova). the threshold for statistical significance was set at .05. results: the contraction force decreased in all nac dose groups compared to control group and this reduction was statistically significant (p < 0.05). similarly, nak atpase activity is also decreased in a dose dependent manner (p < 0.05). the findings obtained in this study suggest that vasodilator effect of nac formed in thoracic aortic smooth muscle was associated with the activity of the enzyme na k atpase. in the presented study, we isolated and characterized a novel feather-degrading bacterium that shows keratinolytic activity. a bacillus uk69, which was isolated from the soil samples taken from farmland on kahramanmaras sutcu imam university campus, showed high keratinolytic activity when cultured on feather meal medium. the enzyme activity was studied in the ph range of 5.0-12.0. the optimum temperature for keratinase activity was investigated by varying the incubation temperature between 20°c and 80°c. optimum keratinolytic activity was observed at 60°c and ph 10.5. the enzyme was stable at 60°c. the activity was investigated in the presence of some chemicals, including sds, tween 80, dmso, triton x-100, edta, nacl, zncl2, cacl2, glucose. the keratinolytic activity was inhibited by all chemicals tested to some degree. the molecular weight of keratinase was determined by polyacrylamide gel (10%) using standard molecular weight marker and estimated about 37 kda by sds-page. the keratinase isolated from bacillus uk69 could be used in biotechnological processes i.e. feather degradation, wastewater treatment and in industrial processes, such as detergent, food and leather industries. introduction: hemoglobinopathies, including thalassemia, abnormal hemoglobins, constitutes a major group of inherited disorders of hemoglobin synthesis. the reduced or absent of the beta (b) or alpha (a) globin chains of the adult human hemoglobin molecule results beta or alpha thalassemias, leading to imbalanced a-globin/non a-globin chains. the aim of this study was to give a quik desicion with a/b-globin mrna ratio for sequencing of a or b gene, when the anemia is not detectable. materials and methods: mrna and cdna extraction of 25 bthalassemia and 15 a-(including two of 3.7 kb del./hbs) thalassemia subjects and normal controls were accomplished using the high pure rna isolation kit and transcriptor first strand cdna synthesis kit, respectively, following the manufacturer's instructions. we used cdna as a template in the real-time pcr amplification using primers specific for a, b globin genes. amplification was performed in a lightcycler ò 480 instrument. the a/b-globin mrna ratio of each sample was calculated based on the 2 àddct method. results: a/b-globin mrna ratios calculated in a-thalassemia subjects relative to normal control as a result of numbers of defective a-globin genes. the a/b-globin mrna ratio was found higher in b-thalassemia subjects. coinheritance of a-thalassemia in hb s subjects concluded a stabile a/b-globin mrna ratio as per a-thalassemia or b-thalassemia subjects. discussion and conclusion: instability in a/b-globin chains is a significant factor of thalassemia disease severity and can be used before deciding type of gene sequencing when the anemia is not detectable. this study indicates that imbalance in globin gene expression could be demonstrated by measuring a/b-globin mrna ratio, which was conveniently and accurately determined by qrt-pcr and give an information about globin gene function which gene should be correct to investigate an individual for globin gene mutation. p-mis-067 self-assembling peptides mimic supramolecular biochirality r. garifullin 1,2 , m. o. guler 1 1 bilkent university unam, institute of materials science and nanotechnology, ankara, turkey, 2 kazan federal university, institute of fundamental medicine and biology, kazan, russia supramolecular chirality is rooted in asymmetric spatial arrangement of structural elements (e.g. molecules or units with higher hierarchy). self-assembled systems giving rise to this kind of chirality are of great importance because they closely resemble natural biological systems and potentially can lead to new advanced functional materials. in the process of self-assembly, both molecularly chiral and achiral structural units can organize into chiral nanostructures. chiral arrangement of chromophore molecules in space is known to result in emergence of chiroptical properties of a chromophore. organization of pigment-protein complexes into macrodomains in green plants gives rise to biochirality emanating from long-range chiral order of complexes. owing to this order, macrodomains start to absorb circularly polarized light intensively and thus exhibit huge circular dichroism (cd) signal. in our study, a simple approach which was aimed at mimicking the biochirality phenomenon makes use of self-assembling peptide amphiphiles and their interactions with pyrene chromophore. designed peptide amphiphiles are capable of self-assembly into nanofibers with chiral interior, which in principle gives an opportunity to achieve long-range chiral order. two modes of interactioncovalent and noncovalentwere utilized in order to induce supramolecular chirality. covalent interaction mode included direct covalent attachment of pyrene to peptide sequence. upon self-assembly of peptide amphiphile into nanofibers intense circular dichroism phenomenon was observed. noncovalent interaction mode envisioned encapsulation of pyrene molecules in the hydrophobic core of nanofibers of another peptide amphiphile. co-assembly of peptide amphiphile and pyrene molecules led to chiral order and intense cd signal. in addition, it was possible to control the sign of cd signals by using either of peptide isomers, l or d. p-mis-068 pon1 activity in hdl subgroups of obese, overweight and normal weight subjects objective: the aims of this study were isolation of hdl-c subgroups by using precipitation method, determination of pon-1 activity in both total and hdl3 subgroups, and evaluation of performance characteristics of pon-1 activity measurement method in newly diagnosed obese, overweight and normal subjects. material and methods: the study population consists of newly diagnosed 71 obese, 40 overweight and 30 normal subjects. fasting morning blood samples were taken from all study groups. hdl3 subgroup was obtained by heparin-mn-dextran sulphate precipitation method and cholesterol was measured with direct (homegenous) hdl-c method. hdl2-c concentrations were calculated with the subtraction of hdl3-c from total hdl-c. hdl3-c and total pon-1 activity were determined by using eckerson method. non-hdl3 pon-1 activitiy was calculated with subtraction of hdl3 pon-1 activity from total pon-1 activity. results: total hdl-c, hdl2-c and hdl3-c concentrations and the activity of total pon-1 and hdl3 pon-1 were found lower in obesity according to overweight and normal subjects (p < 0.001). negative correlations were found between body mass index and hdl3-c, total pon-1 and hdl3 pon-1 (r = à0.244, p < 0.005; r = à0.247, p < 0.005; r = à0.199, p < 0.05, respectively). conclusion: our findings indicated that hdl-c metabolism and lipoprotein associated antioxidant defense mechanisms were adversely affected with obesity. in conclusion we think that precipitation method using for separating hdl3 subgroup, is simple and cost effective for routine applications in clinical laboratories. besides hdl3-c measurements, pon1 activity, measurement of total and hdl3-c subgroup might be helpful to evaluate the atherosclerotic process in obese subjects. keywords: obesity, body mass index, paraoxonase, hdl subgroup, cholesterol p-mis-069 hepatitis e virus antibody prevelance among persons who work with animals in north cyprus introductions: hepatitis e infection is a major cause of viral hepatitis in many developing countries. the objectives of the present study was to determine the seroprevelance of hev infection in peoples who work with animals in northern cyprus. materials and methods: prevelance of hev infection were determined in study 4 group population: persons without occupational exposure to animals; persons who work with animals; veterinarian and butcher. a total of 400 blood samples were collected. all serum samples were tested elisa using a commercially available kit according to the manufacturer's instructions. ti-test were used for istatistical analyses. p > 0.05 was accepted as significant value. results: in a study of 400 blood donors (334 male, 66 female), the overall prevelance of anti-hev igg antibodies were 3.0%. the blood samples were collected 5 different areas. the prevelance of anti-hev igm antibodies was 0.25% and he was 44 years and acting a butcher during 20 years. the prevelance of anti-hev igg of women were approximately two fold higher than men. no significant difference in anti-hev prevelance was observed between the age of the blood donors. according to the anti-hev igg prevelance, the without occupation expose to animal animal were 1%, the animal husbandry were 7% and the veterians and the butcher were 2% were found. discussion: the prevelance of anti-hev in the north cyprus (3%) was found low such as the prevelance of the turkey (5%). the prevelance of anti-hev igg in animal husbandry were higher that the other groups because of they may be more spend of time and contact with animals. the prevelance of igm results suggested that the possibility of outbreaks may be low in north cyprus. conclusion: this study was the first seroprevelance analysis of north cyprus according to the population number.the further studies could be included the seroprevelance of anti-hev from the animals. most errors in the clinical laboratory occur in the preanalytical phase the aim of this study was to investigate the causes and rates of rejected samples, regarding to certain test groups in our laboratory. this study was designed on the rejected samples between january 2015 and january 2016. clinical chemistry, coagulation, hormone, cardiac markers, total urine evaluation and other (ethanol level, hba1c, hb electrophoresis, neonatal bilirubin, drug level, blood gas, fecal occult blood) test groups were included. the total number of specimen and rejected samples was obtained from the hospital information system retrospectively. types of inappropriateness were evaluated as follows: erroneous coding, clotted specimen, hemolysis, insufficient volume, incorrect patient, incorrect tube and inappropriate specimen. it was determined that 873343 blood samples were sent to our laboratory in one-year period. 0.37% of them were rejected because of preanalytical errors. erroneous coding was found as the most common rejection cause (33%). rejection rates of clotted specimen, hemolysis, insufficient volume, incorrect patient, incorrect tube and inappropriate specimen were found to be 11%, 15%, 19%, 2%, 4% and 16% respectively. in our study, erroneous coding was the most common cause of preanalytical errors. education of medical secretaries is relevant and important as can be seen in the decrease of sample errors and the resulting quality improvement. glycosylated hemoglobin test (hba1c) is important for screening, diagnosing, and monitoring diabetes and prediabetes. however, hba1c levels may dependent on patient ethnicity suggesting that the diagnostic cut-offs should be evaluated for specific populations. therefore, our aim in this study was to evaluate the efficiency of hba1c for predicting diabetes in comparison to oral glucose tolerance test (ogtt) results for turkish population. the study included anonymous lab results (acibadem labmed laboratories in turkey) of 6270 patients (3913 female, 2351 male) aging 40.4 ae 11.6 years (15-86) who had an initial diagnosis of diabetes. glucose and insulin levels during ogtt were measured after the initial administration of 75 g sugar (0hour), 1-hour and 2-hour. these parameters were statistically analyzed in comparison to simultaneous hba1c results. glucose measurements at 1 hour had better distinction power (p < 0.05) between these individual groups than initial and 2-hour glucose measurements. the average hba1c (%) levels for healthy, pre-diabetic and diabetic individuals were 5.4 ae 0.4, 5.7 ae 0.4 and 6.2 ae 0.7, respectively. roc curve analysis showed 23.4% sensitivity and 98.2% specificity for the clinically accepted hba1c cut-off value of 6.5%. hba1c cut-off value of 5.9% had a higher sensitivity of 67.8% and comparable specificity of 85.7%. the highest discrimination power between healthy, pre-diabetic and diabetic individuals was observed at glucose concentration at 1-hour after sugar administration in ogtt test as opposed 2-hours generally used for diagnosis. low sensitivity was observed for the clinically adapted 6.5% cut-off value of hba1c. the cut-off value of 5.9% for hba1c was found to be more sensitive with comparable specificity than the 6.5% cut-off values for diabetes screening in our population. our results suggest that 5.9% for hba1c should be considered for diabetes cutoff value for turkish population. induction of the glutathione-dependent detoxification capacity is involved in the hepatoprotective effect of silymarin against acetaminophen-induced hepatotoxicity y. kim, d. kwon, c. ahn seoul national university, seoul, south korea recent findings in this laboratory showed that silymarin was capable of promoting hepatic glutathione (gsh) synthesis via a modification of the transsulfuration reactions in the liver. to investigate its pharmacological significance, we examined the hepatoprotective effect of silymarin against liver injury induced by acetaminophen (apap). adult male mice were treated with silymarin (200 mg/kg, po) every 12 hours for a total of 3 doses prior to an apap challenge (500 mg/kg, ip). the apap-induced liver injury was assessed by histopathological examination and measurement of changes in plasma enzyme activities, lipid peroxidation and formation of nitrotyrosine protein adducts in the liver. plasma levels of apap and its major metabolites were monitored for 24 hours to estimate the metabolic transformation of apap. also protein and activity of the major cyp subtypes involved in the metabolic activation of apap into a toxic metabolite were determined in liver of the mice treated with silymarin only. silymarin pretreatment attenuated the apap-induced liver injury significantly when determined 24 hours later. plasma concentrations of apap, apap-glucuronide or apap-sulfate in plasma were not changed, but thiol conjugates of apap, such as apap-glutathione, apap-cysteine and apap-n-acetylcysteine, were elevated significantly in the mice pretreated with silymarin. however, silymarin treatment did not affect protein expression of cyp2e1, cyp1a2, or cyp3a11 in the liver. also hepatic microsomal enzyme activities measured using p-nitrophenol, ethoxyresorufin and erythromycin as substrates, were not increased by silymarin, indicating that the elevation of apap-thiol conjugates should be attributed to an augmentation of the gsh conjugation capacity. it is suggested that silymarin may protect the liver against an electrophilic substance-induced toxicity by increasing gsh availability which would enhance the detoxifying capacity of liver cells. prostate cancer (pca) is the second leading cause of death among men in western countries. we have previously found that the six transmembrane protein of prostate 1 (stamp1) promotes pca cell proliferation as well as inhibits apoptosis through, at least in part, regulating the erk/mapk signaling. we also found that stamp1 is highly mobile in pca cells and shuttles between the plasma membrane and the golgi, often found in vesiculotubular structures in the cytosol. using advanced imaging techniques, we have now characterized the trafficking of stamp1 from the plasma membrane to early endosomes in lncap cells, by analysing its dynamic targeting to the three main endocytosis pathways: clathrin-mediated endocytosis, caveolae/lipid rafts, and the arf6-dependent pathway. we found that stamp1 fused to cyan fluorescent protein (cfp-stamp1) is present at the plasma membrane where it accumulates in punctate structures. live cell confocal imaging showed that these puncta were dynamic over time indicating that stamp1 may be constitutively delivered to the plasma membrane and removed from it by endocytosis. co-expression of cfp-stamp1 with various fluorescent protein markers revealed that cfp-stamp1 puncta corresponded to lipid rafts that were labelled with caveolin-1-rfp or antibodies against flotillin. live cell imaging showed that cfp-stamp1 and caveolin-1-rfp disappeared at the same time from the same region of the plasma membrane suggesting that lipid rafts are likely to be responsible for stamp1 internalization. notably, stamp1 was absent from other endocytosis structures such as clathrin-coated pits/vesicles. further work is needed to determine whether stamp1 internalization is required for its function, such as its link to erk signaling, and whether interference with lipid rafts influences stamp1 effects on pca cell proliferation and survival. antithrombin-iii, mpv and plasma total homocysteine levels in behcet's disease introduction: behcet's disease is a multi-systemic and chronic inflammatory vasculitis of unknown etiology characterized by recurrent oral and genital ulcers, uveitis, arthritis, arterial aneurysms, venous thrombosis and skin lesions. platelet indices such as mean platelet volume (mpv) is a standart indicator of platelet function in disease pathophysiology. antithrombin, a glycoprotein synthesized in the liver, is the major plasma inhibitor of thrombin thus modulating blood coagulation. antithrombi-iii (at-iii) is a enzyme even moderate deficiency significantly increases the risk of thrombosis. homocysteine (hcy), that is formed during the metabolism of methionine. several clinical studies have clarified that elevated blood hcy levels are related to atherosclerotic disease. in our study, we investigated ovocystatin is one of the best characterized members of cystatin superfamily of protease inhibitors, and it has been frequently used for pathophysiological studies as the model protein, representative for this superfamily. its application has been supported by high structural similarity to human cystatin c as well as several common biological activities. as regard to biological activity, cystatins, including ovocystatin, are best characterized as inhibitors of cysteine proteases of papain family (c1), such as cathepsins b, h, l and s. these inhibitors participate in intra-and extracellular control of proteolytic events, both in physiological and pathological states. in the recent decade also new activities of cystatins, not assigned to inhibition of papain-like cysteine cathepsins, were found. these activities are associated with an alternative active center for legumain-type proteases in the molecule. here we report a chemical modification of ovocystatin that disables the anti-papain activity of the inhibitor but does not affect its anti-legumain activity. the chemical knockout has been obtained by reaction with 2-hydroxy-5-nitrobenzyl bromide (hnbb) that covalently modifies the trp104 residue in the molecule. the reaction has been monitored by uv-vis and fluorescence spectroscopy. the anti-papain activity of the inhibitor has been measured colorimetrically against bana as a substrate. the anti-legumain activity was assessed fluorometrically using z-ala-ala-asn-amc. the reacted inhibitor exhibited an additional, characteristic for hnbb, band at 410 nm in uv-vis scan. accordingly, an ablation of trp fluorescence was also observed. the molecule fully retained the anti-legumain activity, while only residual antipapain activity (10%) was observed. the modified ovocystatin can be a useful molecular tool for studying the physiological and pathological processes specifically associated with legumain activity. 3 departments of medicine (hematology/oncology) and biochemistry and molecular biology, university of louisville, james graham brown cancer center, louisville, ky, united states 6-phosphofructo-2-kinase/fructose-2,6-bisphospatase (pfkfb) family of enzymes are responsible for the conversion of fructose-6-phosphate (f6p) to fructose-2,6-bisphosphate (f2,6bp) and vice versa, and f2,6bp is an allosteric activator of phosphofructokinase-1 (pfk1), a rate-limiting enzyme of glycolysis. among the four identified pfkfb isozymes (pfkfb1-4), pfkfb2 is the least studied isozyme in human cancers. there exists two different splice variants of pfkfb2, variant-1 and variant-2, coding two different isoforms, isoform a and b, respectively. in this study, we first analyzed the effect of k-ras(g12d)induced oncogenic transformation on pfkfb2 expression in pancreatic duct cells. we found that oncogenic k-ras induction in immortalized pancreatic duct cells (ipde) was associated with decreases in total pfkfb2 mrna and protein expressions (mrna; ipde: 1 ae 0.15; ipde+kras: 0.78 ae 0.24 and protein; ipde: 1 ipde+kras: 0.55). we then, checked individual expressions of splice variants and observed that while pfkfb2 splice variant-1 (p2-v1) expression was reduced by k-ras induction (ipde:100; ipde+kras:81.50), pfkfb2 splice variant-2 (p2-v2) expression was increased (ipde:100; ipde+kras:125.70). then, we checked effects of p2-v1 and p2-v2 on glycolytic phenotype of ipde and ipde+kras cells. over-expression of pfkfb2 variants increased f2,6bp concentration (p2-v1: 1.96; p2-v2: 1.72 fold; compared to empty vec), glucose uptake (p2-v1: 16%; p2-v2: 30%) and glycolysis (p2-v1: 20%; p2-v2: 30%) in ipde+k-ras cells. we next analyzed the subcellular localizations of pfkfb2 isoforms and observed that both pfkfb2 isozymes localize to the nucleus, with more prominent nuclear localization of p2-v1 compared to p2-v2. also, nuclear localization ratio of p2-v2 increases after oncogenic transformation with mutant k-ras. taken together, these results suggest that pfkfb2 may have a role in the glycolytic phenotype of pancreatic cancers characterized with hyperactive k-ras signaling. effects of p38 map kinase inhibitors on mda-mb-231 cell line introduction: p38 mapk phosphorylates serine and/or threonine residues of the target proteins. the activation of p38 mapk leads to cell growth, differentiation, survival or apoptosis. in this study, we tested the effect p38 mapk sb203580 and sb202190 on mda-mb-231 cells to further elucidate the controversial role of p38 mapk on cell proliferation or cell migration. materials and methods: mda-mb-231 cancer line was cultured in rpmi-1640 supplemented with 10% fbs. the cytotoxic and cell migration effects of sb203580 and sb202190 inhibitors were tested by mtt assay and wound assay, respectively. the effects of both inhibitors on proliferation and adhesion of md-mb-231 cells were determined by icelligence system. results: it was found that sb202190 p38 map kinase inhibitor was more effective than sb203580. however, no significant effects of low doses of 1 lm and 5 lm of both inhibitors were seen on cell proliferation as compared to the dmso-treated control cells for up to 96 hours as determined by icelligence system. on the other hand, both sb203580 and sb202190 significantly prevented cell proliferation at a concentration of 50 lm. both sb203580 and sb202190 significantly reduced cell migration in a time-dependent manner at a concentration of 50 lm. then, we tested whether each p38 mapk inhibitors have any effect on cell adhesion during a treatment period of 3 hours using icelligence system. only 50 lm concentration of sb202190 reduced cell adhesion for about 1.5 hour (p < 0.001). conclusion: p38 mapk inhibitors sb203580 and sb202190 differentially affect cell proliferation, survival and migration. acknowledgements: this study is financially supported by dumlupınar university, scientific research project no 2015-85. mutagenicity of a series efficacious benzoxazine derivativesa new approach to evaluate ames test data e. foto 1 , f. zilifdar 1 , s. yilmaz 2 , t. sarac ßbasi 1 , i. yalc ßin 2 , n. diril 1 1 hacettepe university, ankara, turkey, 2 ankara university, ankara, turkey testing safety of drug candidates is as crucial as evaluating their efficacy in early drug development. we previously synthesized a series of 1,4-benzoxazine-3-one derivatives showing significant antimicrobial, in vitro anticancer, topoisomerase i inhibitory activities and studied their several mechanisms of action. in this present study, we have evaluated mutagenic activities of these compounds and their potential metabolites. moreover, we aimed to develop a new statistical algorithm available for structureactivity relationship analysis to identify the regions responsible for the activity. to evaluate mutagenicity of the compounds, ames salmonella/microsome test was used. salmonella typhimurium ta98 and ta100 strains were used to detect for frameshift and basepair substitution mutagens, respectively. additionally, mutagenicity of potential metabolites of them were evaluated by adding metabolic activation system (s9) which was prepared from a pool of male sprague dawley rats. results were evaluated with student's-t test. following regression model estimation analysis, we detected minimum mutagenic doses of all tested compounds for generating a 3d-common features pharmacophore model with hiphop method. according to the results, only bs12, bs13, bs16 and bs17 exhibited strong mutagenic effects on both strains in the presence and absence of s9. additionally bs10, bs7, bs1 and bs15 (in the absence of the s9), bs18, bs4 and bs7 (in the presence of the s9) showed weak mutagenic effects on ta98. hiphop analysis results revealed that mutagenicity was increased in the presence of aromatic desactivating groups which might form hydrogen bonds at the position of r3 and hydrophobic groups at the position of r2 of the benzene ring in the structure of benzoxazine. the new statistical approach developed in this study can be useful for assessing the ames test data available for structure activity relationship analyses. background: recently more than thirty different diseases can screen simultaneously with expanded newborn screening (nbs) programs by tandem ms.expanded nbs with tandem ms is performed routinely at akdeniz university hospital central laboratory since 2008.the aim of this study was to evaluate our nbs results with some second-tier and confirmatory tests. materials and methods: nbs results (n = 1863) were evaluated in dried blood samples which sent to our laboratory for the study between august 2014 and august 2015. electrospray ionisation (esi)triple quadrupole mass spectrometer (shimadzu lc-ms/ms 8030,japan) was used for nbs analysis,acylcarnitine and amino acid profile were screened with mrm (multiple reaction monitoring) spectrum within 2 minutes.second-tier tests were performed as urine organic acid analysis by gas chromatography-mass spectrometry (gc-ms),plasma and urine quantitative amino acid analysis by high pressure liquid chromatography (hplc).pathological nbs results were assessed in three separate groups as amino acid metabolism disorders, fatty acid oxidation defects and organic acidemias. results: metabolic diseases were found in 69 (3.70%) patients by the second-tier tests performed.there were detected amino acid metabolism disorders in 13,organic acidemia in 42,fatty acid oxidation defects in 14 patients. conclusions: the reason of high positive results in our laboratory could explain that our study includes both screening and monitoring of previously diagnosed metabolic patients.nbs is performed in only a few centers in turkey although there were the national screening programs included nbs in many foreign countries.more expanded nbs programmes in our country is required to start treatment of patients before irreversible damage is not occured. although many reports indicate the involvement of calpain in several human pathologies, it is not yet clarified how the protease can recognize the substrates to digest and how can escape to its natural inhibitor calpastatin. answers to these questions have been obtained by identifying specific intracellular localizations of calpain and its substrates and analyzing the interactions of the protease with calpastatin. these studies were carried out using human sknbe neuroblastoma cells. protein-protein interactions and intracellular localization of calpain and the related proteins were determined by immunoprecipitation and isolation of membrane microdomains. we have observed that small amounts of calpain-1 are localized in lipid rafts microdomains together with n-methyl-d-aspartate receptor (nmdar) containing nr1/nr2b subunits. immunoprecipitation experiments have demonstrated that nmdar containing nr1/nr2b subunits, calpain-1, hsp90 and neuronal nitric oxide synthase (nnos) but not calpastatin and calpain-2 are present in specific protein complexes. thus, in this localization calpain activity is regulated by hsp90 that reduces the affinity for ca 2+ of the protease. cell stimulation with nmdar agonists induces calpain activation that specifically cleaves the subunits nr2b of the receptor promoting changes in lipid rafts organization and internalization of nmdar without affecting cell viability. moreover, in these conditions, also nnos is digested and converted in the active form by calpain-1. our data suggest a physiological role of calpain-1 at specific cell sites. the protease inserted in lipid rafts microdomains is in strict contact with its targets and escapes to calpastatin which is not inserted in these structures. following an increase in ca 2+ influx, the activated protease regulated by hsp90, promotes the removal of nmdar from the plasma membranes, decreasing ca 2+ entrance through this receptor-channel and protecting cells from ca 2+ overloading. tissue transglutaminase 2 (tg2) is a multifunctional protein complex that can act as a crosslinking enzyme, gtpase/atpase, protein kinase and protein disulfide isomerase. at the cell surface, tg2 was shown to be involved in adhesion, migration, invasion, growth, epithelial mesenchymal transition and hence implied in the metastatic development of many different tumor types. renal cell cancer (rcc) is one of the most common type of cancer in adult males that generally grows as a single tumor within a kidney. our previous findings indicate that the increased expression of tg2 in rcc results in tumor metastasis with a significant decrease in disease-and cancer-specific survival outcome. herewith, the role of tg2 in cell migration of rcc was investigated in this study by transducing the model rcc mouse cell line renca with a series of tg2 mutant constructs. renca cells were transduced by lentiviral particles encoding wttg2, transaminase-defective tg2-c277s form with low gtpbinding affinity, gtp-binding deficient form tg2-r580a, and transaminase-inactive tg2-w241a. in order to investigate the role of tg2 transamidating and gtpase activity in cell migration, scattering assay was used where 7 colonies for each mutant clone was followed for a time interval of 24 hours. our results showed that non-transduced control and tg2-c277s mutant renca cells demonstrated a similar migration pattern with a 20% of scatter activity. on the other hand, 52% colonies formed by renca cells overexpressing wttg2 and tg2-w241a mutant scattered away from each other. a small insignificant increase in scattering was seen in 28% of the total number of 10 colonies for renca cells overexpressing tg-r580a construct. data from this study supports that gtp-binding activity of tg2 is the drive force in migration driven scattering of renca cells, suggesting that inhibitors targeting the gtp-binding activity of tg2 may serve as a new therapeutic approach in the treatment of rcc. background: in this study, we aimed to investigate the relationship between level of vitamin d with subclinical hypothyroidism and subclinical hyperthyroidism. material and metod: study groups planned as three groups such as euthyroid (n = 11966), subclinical hypothyroid (n = 1040), subclinical hyperthyroid (n = 795). serum tsh, free t4 (ft4) and free t3 (ft3) levels were determined by chemiluminescence immunoassay and serum 25-hydroxy (oh) vitamin d 25 (oh) d level were determined by liquid chromatography-tandem mass spectrometry. euthyroidism was defined as a normal level of tsh (range, 0.4 to 4.2 miu/l), ft4 (range, 0.8 to 2.3 ng/dl) and ft3 (range, 2.3 to 4.2 ng/dl). subclinical hypothyroidism is defined as an elevated serum tsh level associated with normal total or free t4 and t3 levels. subclinical hyperthyroidism is defined as low serum tsh levels associated with normal free t4 and free t3 levels. results: subclinical hyperthyroid group had significantly higher 25 (oh) vitamin d levels compared to the euthyroid and subclinical hypothyroid groups (p < 0.05). 25 (oh) vitamin d levels in subclinical hypothyroid group was not statistically significant when compared with the euthyroid group. food processing wastes provide carbon sources in high amounts for fermentative microorganisms to produce energy. converting carbon-rich biomass into bioethanol through fermentation by microorganisms both provides energy requirement for humankind and also decrease pollutant gases like co 2 , no x and so x (ghorbani et al., 2011) . fermentation processes for bio-ethanol production could be achieved by saccharomyces cerevisiae, zymomonas mobilis, and escherichia coli. bacterial hemoglobin (vitreoscilla hemoglobin, vhb) is the first and best characterized prokaryotic hemoglobin molecule. the function of vhb is supporting the cellular respiration through binding to oxygen at microaerobic environment, transferring it to the terminal respiration oxidases (geckil et al., 2004) and thus improving growth and productivity of the microorganisms. in this study, e.coli strains fbr5, ts3 and ts4 were used as ethanologenic microorganisms. expression of vhb in ts3 is lower than in ts4 strain. for the efficient ethanol production effect of different inoculum sizes, sugar species and sugar concentrations in the growth medium were investigated. vhb expression increased effectively the viability of ts3 strain by up to 1.8x10 9 cfu per ml of fructose (3%, w/v) supplemented lb medium starting with small inoculum for fermentation. this indicates that vgb expression should be at the certain level to maintain sufficient the cell growth for ethanol production. geckil h, gencer s (2004) . production of l-asparaginase in enterobacter aerogenes expressing vitreoscilla hemoglobin for efficient oxygen uptake. applied microbiology and biotechnology 63: 691-697. ghorbani, f., younesi, h., sari, a. e., najafpour, g. (2011) . cane molasses fermentation for continuous ethanol production in an immobilized cells reactor by saccharomyces cerevisiae. ethanol production from dairy industry by product using bacterial hemoglobin t. sar, g. seker, a. g. erman, m. yesilcimen akbas gebze technical university, depertment of molecular biology and genetics, kocaeli, turkey bioethanol production from biomass has a great potential to reduce greenhouse gases emissions. ethanol has several applications in industries (chemical, medical, pharmaceutical, food etc.) in the form of raw material, solvent and fuel. one of the most abundant liquid wastes is cheese whey generated from dairy industries. whey powder is concentrated form of whey and contains lactose and also protein, lipid, minerals and vitamins. vitreoscilla hemoglobin (vhb) is the first bacterial hemoglobin. the main function of this molecule is to improve oxygen transfer to cellular oxidases and thus supporting cellular growth and productivity at low oxygen levels (kallio et al. 1994) . in this work, e. coli strains fbr5, ts3 (low level vhb expressing) and ts4 (high level vhb expressing) were used as ethanol producing microorganisms. fermentation medium containing whey powder supplemented with lb material was inoculated with these strains and incubated for 48 hours at 37°c and 180 rpm in a 1000 ml erlenmayer flask. the ethanol production was improved over 300% by using lower vhb expressing strain. the ethanol levels (v/v, %) were determined as 1.08, 4.99 and 1.51 for fbr5, ts3 and ts4 strains respectively. it is shown that the certain levels of vhb could be useful tool to increase the growth and productivity of ethanol from dairy industry wastes. kallio p.t., kim d.j., tsai p.s. and bailey j.e. (1994) . bioethanol is usually produced from cellulose, hemicellulose and lignin. the lignocellulosic wastes should be hydrolysed into fermentable sugars by using enzymes or dilute acids before microbial fermentation. acidic hydrolysis methodology is cheaper than enzymatic hydrolysis but it can cause production of some inhibitors like aliphatic acids, which affect the growth of microorganisms. vitreoscilla hemoglobin (vhb) is the first described prokaryotic hemoglobin. the recombinant strains carrying vgb gene (e. coli, p. aureginosa) which encodes vhb showed increased bacterial growth, productivity of metabolites compared to untransformant counterparts under low oxygen concentrations [nasr et al., 2001; geckil et al., 2004] . in this study, ethanologic e. coli strains fbr5, its derivative strains ts3 (vgb+) and ts4 (vgb+) were used. ts4 was constructed in such that it could express more vhb than ts3. bioethanol production by these strains in presence of lignocellulosic hydrolysates derived inhibitors was investigated. different acetic acid concentrations (2.5-10 mm) were used as inhibitors from lignocellulose hydrolysate. 10.0 mm acetic acid was used as an inhibitor. the growth of vhb expressing ts3 and ts4 strains was inhibited about 31% after 48 hours fermentation time. strain fbr5 was inhibited as high as 76% by using the same inhibitor including growth medium. it was shown that the expression of vhb could improve growth and productivity in presence of lignocellulosic inhibitors. differentiation of preadipocyte, also called adipogenesis, leads to the phenotype of mature adipocyte. however, excessive adipogenesis is closely linked to the development of obesity. thus, any drug or chemical that can inhibit adipogenesis may have preventive and/or therapeutic potential against obesity and related diseases. azd1208, an inhibitor of the family of pim kinases, is known for anti-cancer activity. here we investigated the effect of azd1208 on adipogenesis in 3t3-l1 preadipocytes. notably, azd1208 treatment led to a concentration-dependent inhibition of both lipid accumulation and triglyceride (tg) synthesis during the differentiation of 3t3-l1 preadipocytes into adipocytes with no cytotoxicity. on mechanistic levels, azd1208 strongly reduced not only the expression levels of ccaat/enhancer-binding protein-a (c/ebp-a), peroxisome proliferator-activated receptor-c (ppar-c), fatty acid synthase (fas), and perilipin a but also the phosphorylation levels of signal transducer and activator of transcription-3 (stat-3) during adipocyte differentiation. furthermore, azd1208 largely decreased leptin, but not adiponectin, mrna expressions during adipocyte differentiation. collectively, these results demonstrate that azd1208 inhibits adipogenesis in 3t3-l1 preadipocytes and the inhibition is largely attributable to the reduced expression and/or phosphorylation levels of c/ebp-a, ppar-c, fas, perilipin a, and stat-3. effect of intrauterin exposure to artificial food colourings on dna damage in rats in many research genotoxic potential of food additives has been investigated. however there are few findings about the effect of artificial food colourings (afc) on dna. in this experimental study, we aimed to analyze whether in utero exposed artificial food colourings would have effect on dna and cause damage.thirteen female rats were included to the study which were equally divided into two groups as control (cg, n = 15) and food colouring (fcg, n = 15) groups. a mixture of nine food colours were given daily to fcg by oral gavage from preconception to birth. no adverse effect level (noael) of artificial food colourings for each colouring was administered to fcg. three months after the birth, 6 offspring from each group were selected randomly as control (cg) and experiment (eg) groups. then they were sacrified under anesthesia. for performing the alkaline comet comet assay leukocytes were seperated from whole blood samples. the alkaline comet assay was performed. the extent of dna damage was assessed from the length of dna migration derived by subtracting the diameter of the nucleus from the total length of the image and graded into 5 categories and these grades were converted into arbitrary unit (au). differences between the means of data were compared by independent samples t test. the results were given as the meanaesd, p values of less than 0.05 were considered as statistically significant. although the extent of dna damage was higher in eg, the comparison of experiment (13.50 ae 1.76) and control (11.66 ae 1.36) groups showed no statistical difference (p = 0.072). relationship between glucocorticoid receptor gene polymorphisms and recurrent depression l. aydogan, i. benli, z. c. ozmen, i. butun gaziosmanpasa university medical faculty, department of biochemistry, tokat, turkey objective: sensitivity to glucocorticoids varies between individuals and these differences have been implicated in the etiology of psychiatric diseases such as depression. recent studies have found relationship between common glucocorticoid receptor (gr) gene (nr3c1) polymorphisms and unipolar or bipolar depression. the nr3c1 gene is a candidate gene affecting depressive disorder risk and management. the aim of the present study was to evaluate the relative distribution of specific polymorphisms of nr3c1 (bcl1 and rs33388) in recurrent depressive disorder (rdd) patients. methods: our study included 100 volunteers with recurrent depressive disorder and 100 healthy individuals without any mental illness. depression was assessed by hamilton and madrs depression scale. nr3c1 gene polymorphisms were detected by real-time pcr, with hybridization probe method. allele and genotype frequencies at two loci (bcli and rs33388) were investigated in rdd patients and controls. results: genotype distribution among rdd patients and the control group for bcl-1 (g/c) were as follows: cc 3% and 5%, gc 27% and 34%, gg 70% and 61%, respectively. there was not a significant difference when the frequency of the allele (p = 0.204) and genotype frequency (p = 0.382) were compared between the patients and the control. genotype distribution in the rs33388region (a/t) of the patients and controls were tt 29% and 25%, ta 49% and 54%, aa 22% and 21%, respectively. allele frequency (p = 0.841) and the genotype frequencies (p = 0.754) were not significantly different among the groups. conclusion: numerous nr3c1 gene polymorphisms were previously reported in association with modification of depressive disorders. the results of our study showed no association between gr genotype and recurrent depressive disorder. nr3c1 polymorphism does not play a role in the development of recurrent depressive disorder. thymoquinone (tq) has been shown to supress the proliferation of various tumor cells, while it is minimally toxic to normal cells. the aim of this study is to investigate the potential therapeutic effects of tq on cell proliferation, apoptosis, invasion, migration, colony formation and wound-healing in sh-sy5y human neuroblastoma cell line. sh-sy5y cell line treated with 5-400 lm tq by solving medium for 24, 48 and 72 h considering a time-and dose-dependent manner. the cytotoxic effect of tq was determined by mtt method. total rna was isolated by trizol reagent. cdna synthesis was performed by using commercial kit. mdm2, p53, p21, akt, pten, cdk4, cyclin d1, caspase-3, -8, -9, -10, bcl-2, bax, parp, bcl-xl, bid, dr4, dr5, puma, noxa, mmp-2, -9, timp-1, -2 and gapdh gene expression profiles were analysed by real-time pcr method. effects of tq in sh-sy5y cells on invasion, colony formation and cell migration were detected by matrigel-chamber, colony formation assay and woundhealing assay, respectively. statistical analysis were performed with rt 2 profiles array data analysis by using student's t test. ic 50 value of tq in sh-sy5y cells was detected as 15 lm at 48 th hours. by rt-pcr results, it was determined that tq caused a decrease in the expression of mdm2, akt, cdk4, cyclin d1, bcl-2 and mmp-2. it is also observed that tq caused a significant increase in the expression of p53, pten, caspase-3, -10, bid, dr4, puma, noxa and timp-1. it was also found that tq in sh-sy5y cells suppressed invasion, migration and colony formation by using matrigel invasion chamber, wound healing and colony formation assay, respectively. in conclusion, we demonstrate that tq significantly effect cell cycle, apoptosis, invasion, migration and colony formation of sh-sy5y cells. tq may be a potential candidate as chemotherapeutic agent for the treatment of neuroblastoma. more studies have to be performed to profile the mechanisms and genome wide effects of tq to prove its therapeutic potential. dna aptamers can achieve a very high affinity to the target due to the potential of developing broad target-binding interface. however, classic strategy selection of aptamer binders is a challenging task requiring multiple rounds of panning and post-selection optimization. we have developed fast and convenient technique for the selection of dna aptamers based on the offrate selection and tandem affinity purification (tap). we constructed and produced in e.coli recombinant chimeric protein, comprising two affinity tags (his6 and gst) separated from each other and from the target protein (anthrax protective antigen domain iv, padiv) by sumo protease recognition polypeptide and synthetic cleavage site for the anthrax lethal factor (lf). the protein bound to aptamer library is first captured by imac resin, cleaved by sumo protease, captured by gst resin and eluted by lf following the lines of the tap method. the gst-captured aptamer-target complexes were subjected to the off-rate selection using soluble padiv as the competitor. multiple selection rounds are cumbersome and can result in carryover. high abundance of moderate affinity aptamers in the resulting pools obtained by classic selection approaches suggests that the procedure to counter-select them at the beginning of panning is needed. reduction of the contact duration between the aptamer library and the target was crucial for efficient selection of high-affinity binders. on the other hand, tap prevents contamination, and bundled with the off-rate selection, allows for clean isolation of high-affinity binders with affinity in the low nanomolar range. the developed technique is applicable for efficient selection of high affinity dna binders to soluble recombinant proteins and their fragments. dna aptamers obtained will be further used for the development of diagnostic and therapeutic tools for the detection and treatment of anthrax. the work was supported by russian science foundation research grant no. 14-15-00630. the role of macab efflux pump in protection of serratia marcescens against antibiotics and oxidative stress the emergence of bacterial multi-drug resistance is a growing problem of public health worldwide. bacterial drug efflux pumps are membrane protein complexes that function to expulse drugs from the cell. they play a crucial role in the rising rates of antibiotic therapy failures. the homolog of macrolide-specific pump macab was identified in opportunistic pathogen serratia marcescens and was used in this study to characterize its role in protection against antimicrobials and other processes beyond the active efflux of antibiotics. here we used method of serial dilutions to determine minimum inhibitory concentration (mic) for s. marcescens sm6 wild type (wt) and its isogenic δmacab mutant strains. we also used h 2 o 2 survival assay to evaluate the ability of wt and the mutant strain to withstand an oxidative stress. finally, we used b-galactosidase assay to evaluate macab promotor activation in the reporter strain and followed macab expression by western blotting analysis using macab-6xhis strain. we show that in contrary to its e. coli homolog, macab pump in s. marcescens is not involved in the protection against macrolides but instead it is required for protection against aminoglycosides. we further show that similar to its salmonella typhimurium homolog, s. marcescens macab is essential for protection of bacteria against h 2 o 2 . transcriptional analyses demonstrate that while low level of macab promotor activity can be detected after 2 hours of growth in lb-broth there is at least 5-fold increase in expression in response to the presence of h 2 o 2 . on the protein level macab can be detected starting from 3 hours of growth in lb-broth and it reaches maximum expression on 16 hour of growth. our data suggest that macab pump in s. marcescens is involved in protection of bacteria against aminoglycoside antibiotics and is crucial for protection against reactive oxygen species. we are currently working on identification of macab substrate with anti-h 2 o 2 properties. antiproliferative and apoptotic effects of noscapine on mcf-7 and mda-mb-231 human breast cancer cell lines approximately 10-17% of breast cancers are negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. these are most aggressive tumor and a clinical problem because of lack of targeted therapies. noscapine is an alkaloid from opium. noscapine is a microtubule-interfering agent. it causes mitotic arrest, induces apoptosis. in this study, we aimed to investigate the effects of noscapine in mcf-7 and mda-mb-231 human breast cancer cell lines. the cytotoxic effects of docetaxel, tamoxifen, and noscapine on the mcf-7 and mda-mb-231 cell lines were analyzed by roche xcelligence system. the cells were cultured in 10% fetal bovine serum containing dulbecco's modified eagle medium at 37°c in a humidified atmosphere containing 5% co 2 . 24 h after seeding, the cells were treated with 4 different doses of docetaxel (12.5 to 100 nm), tamoxifen (12.5 to 100 lm), and noscapine (12.5 to 100 lm). cultured cells were harvested, fixed with 10% formalin, and centrifuged. pellet was blocked, fixed, and embedded in paraffin. paraffin-embedded cells blocks were sectioned at 4 lm thickness and stained with h&e, ki-67, bcl-2, cyclin-d1, and bax. sides were assessed under a light microscope. quantification of the analyzed proteins were evaluated by the percentage of positive cells. all drugs showed cytotoxic effects on both cell lines. all drugs inhibited the proliferation of breast cancer cells, but effects were dependent on time and dose. all drugs were especially more effective on mcf-7 cells. immunohistochemical examinations revealed that tamoxifen was more effective on mcf-7 cells, hovewer docetaxel and noscapine were more effective on mda-mb-231 cells. tamoxifen has more apoptotic and antiproliferative effects on mcf-7 cells. docetaxel and noscapine showed more apoptotic and antiproliferative effects on mda-mb-231 cells. noscapine may be an effective anticancer agent due to antiproliferative and apoptotic effects on breast cancer cells. negative selection of dna aptamers to reduce non-specific binding in solid-phase-based selection procedures carryover by binders specific to the components of the selection system can be a serious issue in hampering the aptamer selection campaign. solution-or "mass"-based techniques still cannot substitute classic phase-separation strategies. one approach to prevent selection of "passenger" phase-specific (plastic, beads) or blocking agent specific aptamer species is their depletion from the initial library pool. our aim was to develop the universal technique for removal of such aptamers exemplified by bsa-and casein-specific binders, while preserving the initial library complexity. the dna aptamer library was subjected to three rounds of depletion using magnetic beads with covalently attached casein and bsa. to ensure high depletion efficiency, beads were pelleted in a 15-ml centrifuge tube by a neodymium magnet through a 10-cm cushion of 20% sucrose, thus preventing weakly bound aptamers from re-populating the library. high complexity of the input library helped to avoid pcr amplification after depletion rounds preventing the library bias introduced by dna amplification. the depletion effciency was confirmed by real-time pcr. resulting oligonucleotide sub-library was analyzed for binding to the targets using solid-phase real-time pcr assay. we have shown that three rounds of panning under the conditions employed provided full depletion of the initial dna pool from nucleic acid structures capable of binding to protein competitors and hampering the process of aptamer selection. we compared selection efficiency of aptamers specific to type a botulinum neurotoxin light chain in depleted vs undepleted library. the yield of the target-specific aptamers was 10-fold higher in the library subjected to the depletion procedure. removal of undesired binders from aptamer libraries appears an important step of solid-phase selex procedure. it can become a useful approach in optimizing solid-phase selex. the work was supported by russian science foundation research grant no. 14-15-00630. epithelial mesenchymal transition (emt) is a critical trans-differentiation program driving cancer metastasis. patients showing signs of emt or presence of distant metastasis have poor prognosis. another well-known feature of decreased cancer-associated survival is the lack of anti-cancer immune responses. thus we hypothesized that the emt and anti-tumor response should be linked via altered secretion of soluble factors by metastatic cells. all cell lines were grown in dmem. emt status of crc cell lines were assessed by investigating canonical markers of emt. cytokine/chemokine expression of crc cells was performed using r&d systems antibody arrays and validated using ccl5 sandwich elisa and rt-pcr. the mechanism of action of zeb1/2 on ccl5 promoter has been studied by luciferace assay and chip. ccl5 coding region was cloned into pcdna3.1 and stably transfected into dld-1 cells. ccl5 deficient ct26 cells were generated using lentivirual shrna transduction. cells overexpressing or knock/down ccl5 were injected orthotopically into mice. t lymphocyte (til) infiltration in respect to ccl5 and sip1 expression was studied using ihc or flow cytometry. emt status catagorised 13 crc cell lines into epithelial, intermediate epithelial, intermediate mesechymal and mesenchymal. cytokine/chemokine antibody arrays showed a significant increase in ccl5 in induced dld-sip1 cells. elisa, multiplex assays and rt-pcr confirmed a significant increase of secreted ccl5 in the induced dld-sip1 cells as well as mesenchymal crc cells as compared to epithelial ones (p = 0.027). promoter studies showed that zeb1/2 bind to ccl5 promoter and and activate ccl5 gene expression. no metastasis was observed for dld-1 cells overexpressing ccl5 but significant alterations of tumour associated lymphocytes were identified in syngeneic orthotopic crc models. our data shows that ccl5 is up-regulated by emt inducing transcription factor sip1, and mesenchymal (metastatic) crc cells secrete significantly more ccl5 compared to epithelial (non-metastatic) ones. ccl5 did not induce emt per se but abundant secretion of ccl5 by metastatic crc cells was a crucial regulator of immune infiltrate in crc. inhibiting ccl5 in metastatic crc may have a therapeutic potential. barley (hordeum vulgare l.) belongs to the grass family, poaceae (gramineae). it is the fourth most important cereal crop after wheat, maize and rice and is among the top ten crop plants in the world. talbina was used to be recommended for the sick and for one who is grieving over a dead person. talbina is made by adding one or two tablespoon of barley flour (must be 100 percent wholegrain barley flour) to one-and-a-half cups of water and placed on low heat for 10-15 minutes (optional: add milk or yoghurt and sweeten with honey). the main objectives of this investigation were determine the a-tocopherol contents and antimutagenicity activity of talbina (hordeum vulgare l.). our results showed that the total tocopherol content was in the range of 0.25 to 1.03 lmol/g fw. talbina extract was shown to have greater antimutagenic activity observed in the 2500 lg/plate concentration s. typhimurium ta98. at all the doses antimutagenic response was significant at (p < 0.01) against both the strains with a percent mutagenicity decrease from 40 to 25 for ta98 followed by ta100 with percent antimutagenicity from 30 to 11. the results of the study concluded that talbina is a better antimutagenic agent than vitamin e and combination of vitamins did not produce any synergistic activity. the compounds containing thiadiazoles have diverse applications as antifungals, anticancer agents, antibacterial, antiinflammatory drugs, antidepressants and carbonic anhydrase inhibitors according to literature. in this study some novel thiadiazole compounds [(1,4,10,13)-tetrathia[4.4] (2,5)-1,3,4-thiadiazolophane; (4,16)dioxo-1,7,13,19)-tetrathia[7.7](2,5)-1,3,4-thiadiazolophane; (4,7,19,22)-tetraoxo(1, 10, 16, 25 )-tetrathia[10.10](2,5)-1,3,4-thiadiazolophane and (4,7,10,22,25,28)-hexaoxo(1, 13, 19 ,31)-tetrathia [13.13] (2,5)-1,3,4-thiadiazolophane] were used to evaluate the cytotoxicity on healthy human lymphocytes and the antibacterial activities. cytotoxicity tests were perfomed using mts assay and the trypan blue test. cells were incubated with the compounds for 72 hours. at the end of the each 24 hour, cell vitality was assessed by measuring the absorbance (490 nm) of each well using a microplate reader for mts assay. in addition, viability percents of the cells were determined after trypan blue test. as a result, the compounds showed cytotoxicity in a dose dependent manner. for the concentrations of 1:1000 of 0.5 mg/ml, the cytotoxic effect was eliminated. also, antioxidant capacity was determined using 2,2-diphenyl-1-picrylhydrazyl (dpph) reagent. moreover, the antibacterial activities of the compounds were analyzed using a microdilution test against e. coli and s.aureus. compounds having various concentrations showed different antibacterial effects against these two bacteria. arabidopsis thaliana ecotypes vary in their ability to utilize organic p substrates insufficient quantity of inorganic phosphorus in soil is an evergrowing problem that affects many fields of agriculture. unlike inorganic phosphates, organic phosphorus compounds are very common in many soil types, but plants are often unable to efficiently utilize them. to better characterize the extent of natural variation in the ability of the model plant arabidopsis thaliana to grow on organic phosphorus compounds, we grew 19 arabidopsis ecotypes on several organic and inorganic sources of phosphorus. plants were grown in liquid or solid media containing naphosphate, phytate and atp as the sole supply of phosphorus or in absence of any phosphorus source. after several weeks of growth, plants were assayed for changes in their morphological and physiological characteristics. phytate was shown to be the least preferred source of phosphorus compared to inorganic phosphate and atp. the rate of biomass accumulation in all ecotypes decreased in the following order from inorganic phosphate to atp to phytate. lateral root formation was markedly reduced in the absence of any phosphorus source or in the presence of phytate. we also showed that phosphomonoesterase activity in intact roots increased when plants were grown on atp and phytate. overall phosphorus content in leaves and roots was similar when plants were grown on atp or inorganic phosphate, but it was markedly reduced on phytate. substantial differences between ecotypes were also observed in root length, p content in ash and phosphomonoesterase activity in intact roots. our analysis of the ability of arabidopsis ecotypes to grow on several different phosphorus sources provides a unique opportunity to investigate the degree of natural variation in this plant's ability to adapt to different nutritional environments. analysis of many important morphological and physiological changes observed in these plants can further extend our understanding of the full range of plant responses to phosphorus availability. laboratory tests are important in terms of confirmation of diagnosis given by clinics and implementation of appropriate treatment protocols for patients. laboratory tests used by the clinics have been increased in parallel with time.there are many reasons for increased use of the test such as increase of elderly population, increase in standard of care, lack of information and shortening of turn around time. unnecessary laboratory testing also constitute one of the reasons for increased use of laboratory tests. in our study we aimed to investigate the unnecessary laboratory testing for fpsa test. fpsa tests which are ordered with total psa tests that values of less than 4 ng/ml or greater than 10 ng/ml were accepted as inappropriate initial testing. 9759 fpsa tests were evaluated as unnecessary laboratory testing. the clinic which ordered the maximum unnecessary laboratory testing with 3315 was urology within all the clinics. although to the restrictions about the ordering of total psa and fpsa tests there were no decrease in the number of unnecessary laboratory testing. unnecessary usage of laboratory testing may cause increase of false positive results, increase in the use of invasive testing, unnecessary drug consumption and increase of healtcare costs. some precautions may be effective in reducing unnecessary tests such as to inform clinicians about the cost of laboratory tests, to increase the clinician education programs and to develop usage of disease specific diagnostic algorithms about test ordering. local clinical validation of blood collection tubes although the tubes with gel and clot activator are widely used due to the advantages, there are ongoing discussions about the effects of the blood collection tube on clinical outcomes in the analysis of biochemical parameters. therefore, we aimed to prove the local clinical validation of the new produced blood collection tubes with low-volume. the blood samples of 40 patients who referred to the hospital phlebotomy unit were collected using holder into the 3 different tubes. first tube was 5 ml glass tube and with no additive, second was 5 ml tube with gel separator, third was 1 ml tube with gel separator. serum was separated and immediadiately analysed for 25 biochemical parameters. the difference between the analyte amounts in the different tubes was evaluated using paired t-test. the clinical significance was evaluated using significant change limit. bias (%) between the other tubes with the reference tube was also evaluated according to the ''allowable total error". when we compared the other test tubes to a glass tube which was assumed reference tube, total protein, albumin, amylase, calcium, triglyceride, cholesterol, hdl-cholesterol, total and direct bilirubin, iron, gamma glutamyl transferase, magnesium, phosphorus results were statistically significant. but the results of all the analytes were within the significant changes limit and the allowable total error was not significant. while a biochemical parameters have analysed, it may be absorbed into the gel and this may caused from factors such as the chemical structure of the gel, analyte itself, the residence time in the gel, storage temperature and volume of the sample e.g. as well as the leaking of gel material to the sample was reported to be another factor for affecting the analysis. despite these factors, we observed that neither gel-clot activator tube with low nor high volume affect the clinical results. the research of the frequency of interference in thyroid function tests interference is defined as the effect of substance in the sample which changes the correct value of laboratory results. the frequency of interference in immune techniques is varied. the frequency of interference depends on population of the study, technique for detecting the reaction and researcher's method. unexpected or inconsistent results with clinical findings should suggest the possibility of interference. in this study it is aimed to investigate the frequency of interference in thyroid function tests (tsh, ft3, ft4) which are the most common requested laboratory tests. thyroid function tests of 47915 patients are analyzed in ankara numune education and research hospital in october 2014-may 2015. five samples which had the incompatible results with clinical findings are re-evaluated just because of the suspicion of interference. the detection of interference included; repetition of test via different immune techniques, serial dilution, polyethylene glycol (peg) precipitation and incubation with heterophilic blocking tubes (hbt). the results of two different immune techniques and before/ after incubation with hbt showed no significant difference. linear curves had observed in serial dilution. after peg precipitation; below 40% of recovery had obtained in one sample, therefore it is interpreted as macro-tsh. the frequency of interference in thyroid function tests for 8-month study period was 0.01%. no information is found about the best test for defining the cross reaction. it is also aforethought that interference should not be excluded by using any single procedure. p-mis-105 development of polyclonal and monoclonal antibodies against fatty acid binding protein 4 (fabp4/ap2) a. abbasi taghidizaj, g. aydogdu, b. p. sermikli, e. yilmaz ankara university, ankara, turkey recombinant proteins and antibodies can be use for therapeutic or diagnostic purposes which produced in many different host organisms. the technique for the production of immortal cell making single antibody, fusing target antibody-forming b lymphocyte precursor with a suitable myeloma cells. the fused hybrid cells (called hybridomas), as a cancer cell will reproduce rapidly and will produce large amounts of the desired antibodies. fatty acid binding protein (fabp4) is a well characterized intracellular lipid transport protein and plays a key role in the intracellular fatty acid transport and adipose tissue metabolism. fabp4 as a adipokine that regulates glucose homeostasis and has various features for metabolic syndrome associated with obesity. in this study, production of monoclonal antibodies against immunogenic fabp4 protein made by recombinant dna technology. recombinant his-fabp4 was expressed in e.coli and purified. balb/c mice used for immunization and serum anti-fabp4 antibodies determined by enzyme-linked immunosorbent assay (elisa). hybridoma cells created by fusion of splenocytes and myeloma partner cells. after selection of antibody producing cell clones, injecting hybridomas into the peritoneal cavity in balb/c mice ascites fluids was obtained. we have selected fifteen hybridoma clones that produced antibodies specific for fabp4, as shown by western blotting and immunocytochemistry. as a result we produced mabs that will be useful for the scientific community working on fatty acid binding proteins and lipid metabolism. in near future, therapeutic approach for this antibody maybe a possibility in metabolic syndrome. thioridazine, an anti-psychotic drug, inhibits migration, invasion and epithelial mesenchymal transition in breast cancer cell lines thioridazine (thz), an antipsychotic drug, exhibits anti-angiogenic effects on breast cancer cell lines. however the mechanistic insight in exerting antiangiogenic effect is not clearly understood. the objective was to investigate the role of thz in epithelialmesenchymal transition (emt) by using cell migration assay, scratch assay, western blot (wb) and immunocytochemistry. thz treatment reduced cell viability on mda-mb-231, mcf-7 and cd44 + /cd24-cells and ic50 values of thz were found to be 9 lm, 16.4 lm and 18 lm respectively, at 24 hours. invasion potency of mcf-7, cd44 + /cd24-and mda-mb-231 cells were determined as 29%, 24%, 16.5% when compared to relevant treatment controls. migration potency of mcf-7, cd44 + /cd24-and mda-mb-231 cells was determined as 28.5%, 63.2%, 61% respectively. among the three cell lines mda-mb-231 cells display enhanced invasive and migration ability when compared to other cell lines. western blotting results demonstrate that thz significantly increases e-cadherin, cytokeratin-18, b-catenin, while inhibiting n-cadherin, vimentin, fibronectin. immunocytochemistry studies revealed decrease in e cadherin and a concomitant increase in vimentin level for all three cell lines upon treatment with thz. moreover thz significantly inhibited the cell migration, invasion and emt in mda-mb-231, mcf-7 and cd44 + /cd24 cell lines by suppressing mesenchymal markers. in conclusion, these data suggest that thz might be a novel anti-proliferative and anti-metastatic agent for treatment of breast cancer. effect of seasonal temperature and humidity on urine density in children environmental heat and humidity are important factors affecting hydration status in childhood. hereby, we aimed to investigate the effects of seasonal climate changes on urine density of children living in mediterranean climate, cyprus. 1700 healthy 0-18 year children's (850 girls, 850 boys) age, sex and urine density results were collected retrospectively for three consecutive years. the correlation of urine density with each seasonal and 12 months' average temperature and humidity has been analysed. the urine density results had a positive correlation with temperature (r = 0.083, p = 0.001) and a negative correlation with humidity (r= à0.072, p = 0.003). mean urine density in spring was higher than that of autumn (p = 0.02) and winter (p = 0.00). mean value of summer was higher than autumn (p = 0.03) and winter (p = 0.00). 0-24 months age group had lower urine density. evaluation of urine density based on gender and puberty revealed no statistically significant difference. seasonal mediterranean climate changes have an impact on urine density in children which may affect hydration status especially in infants < 2 yrs of age. during high temperature seasons ensuring adequate water intake is essential in this age group in mediterranean climate. p-mis-108 implementation related to the use of antibiotics and data sources by community pharmacists in north cyprus as the resistance to antibiotics is gaining importance in today's world;the solution to this problem is possible through a common consciousness of the doctor who prescribes antibiotics,the pharmacist who sells and the patient who consumes antibiotics. irrational use of drugs is an economic and medical problem in many developed and developing countries around the world.the aim of this study is to determine the sales ratio of non-prescription antibiotics in pharmacies which is the biggest category of the antibiotic group sold as well as the indications that lead to its' prescription. eighty-four pharmacies out of 168 pharmacies located in north cyprus were involved in the study with 50%stratified systematic sampling, questionnaires were filled and a consent form was signed by the participating pharmacists. the pharmacists involved in the study stated that non-prescribed antibiotics were demanded from the pharmacists and all except two (97.6%),responded positively to this demand. it has also been identified in the study that 41.5% of the daily sale of antibiotics in the first half of the year 2014 was non-prescribed. the most purchased antibiotics either with or without prescription was found to be the penicillin and its derivatives with 76.2% and upper respiratory tract with 86.9%. when the level of selfawareness of the pharmacists was examined, the rate is found in north cyprus to be (41.5%),compared with the studies conducted in greece,italy,malta and spain 47% and egypt 50.4%that designated the non prescribed antibiotics purchased from the public pharmacies. the rate of sale of non-prescribed antibiotics in north cyprus has been found to be at a higher level compared to the rates in many developed and developing countries. furthermore, the upper respiratory tract infections are amongst the most common viral causes which lead to a high consumption of both prescribed and non-prescribed antibiotics. this study was supported by turkish viral hepatitis prevention society. acrylamide has cytotoxic, antiproliferative and apoptotic effects on human lung adeno carcinoma cell line a549 acrylamide (aa), a widespread substance in many fields, forms in foods during high temperature processing such as baking, roasting, frying. aa is a potent neurotoxic, genotoxic and clastogenic agent being a strong electrophile and forming adduct with biological molecules or potent nucleophiles. up to now, several studies confirmed the toxicity of acrylamide to several organs. on the other hand, aa is reported to have inhibition effects both on proliferation and differentiation of different cancer cells in a time and dose-dependent manner. in addition, natural and synthetic acrylamide derivatives are also used as potent anti-cancer agents. moreover, inhibition concentration (ic50) values of aa against these cancer cells have not been investigated in detail yet. thus, the goal of this study is to investigate the cytotoxicity of aa on a549 cells including with ultrastructural and morphological effects. ic50 value of aa on a549 cells for 24 h was detected with mtt (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h-tetrazolium bromide) colorimetric assay. we evaluated morphological changes under confocal microscopy and ultrastructural changes under transmission electron microscopy (tem). our results demonstrate that aa inhibits the proliferation of a549 cells in dose-dependent manner and ic50 on a549 cells was found to be 4.6 mm for 24 hours. confocal microscopy evaluations showed that aa caused nuclear condensations, fragmentations, cytoskeleton lacerations and membrane blebbing. tem results revealed membrane blebbing, chromatin condensations and cell shrinkage. although aa is a probable carcinogen substance, it drastically inhibited cell viability in dose-dependent manner. from microscopic assessments, aa is suggested to induce apoptosis in a549 cells. in conclusion, the present study confirms the high potential of aa for cytotoxic, antiproliferative and apoptotic activity on a549 cells. however, appropriate aa dose is critical to prevent its possible adverse effects. effect of hemolysis and lipemia on some immunochemical tests in beckman coulter unicell dxi 800 immunoassay analyzer c. yilmaz, s. yildiz, m. senes, v. fidanci, d. y€ ucel ankara training and research hospital, ankara, turkey the aim of the study was to investigate the effects of in vitro hemolysis and lipemia on 25 immunoassays studied by the beckman coulter unicell dxi 800 immunoassay analyzer. we prepared a serum pool without hemolysis, lipemia and icterus. baseline serum pool concentrations of 25 tests were measured by the beckman coulter unicell dxi 800. d _ ifferent serum pools, six for hemolysis and five for lipemia, were spiked with increasing concentrations of hemoglobin (0.6, 1.2, 2.4, 4.5, 6.6 and 8.6 g/l hemoglobin) and intralipid (0.625, 1.25, 2.5, 5 and 10 g/l intralipid). the hemolysate was prepared by osmotic shock method. intralipid (20%, baxter, deerfield, il) was used to mimic the effect of lipemia. the hemolysis (h), lipemia (l) and icterus (i) indices were measured on beckman coulter au 5800. after spiking the pools, the 25 tests were measured again in duplicate on beckman-coulter dxi 800 analyzer. a change of 10% from baseline results was taken as evidence of interference and the interfered tests were also evaluated according to total analytical error based on analytical imprecision and intraindividual biological variation. we observed a positive interference due to hemolysis for folat, vitamin b12, testosterone and by lipemia for cortisol. there was a negative interference of hemolysis for ca 19.9, ca 125, ca 15.3, insulin, pth and e2, and of lipemia for progesterone, ca 19.9, vitamin b12 and pth. we found clinically significant effect (>total analytical error) of hemolysis on folate and insulin, and lipemia on cortisol. investigation of the effect of two different p38mapk inhibitors in rats subjected to isoproterenol-induced acute myocardial injury: an experimental study objective: acute myocardial infarction is a serious acute condition. in the current study, we aimed to investigate the possible effect of two different mitogen-activated protein kinase (p38mapk) inhibitors in rats subjected to isoproterenol (iso)induced myocardial injury. materials and methods: a total of 32 male wistar-albino rats were equally and randomly seperated into four groups as follows: control, iso, iso plus sb203580 andiso plus tak-715. treatment agents were orally administered and myocardial injury was induced by subcutaneous injection of iso. serum cardiac troponin-i (ctni), ischemia modified albumin (ima), heart fatty acid binding protein (hfabp) levels and paraoxonase-1 (pon-1) activity, tissue tos (total oxidant status), tas (total antioxidant status), tt (total thiol), tumor necrosis factor-a (tnf-a) levels, superoxide dismutase (sod) and glutathione peroxidase (gsh-px) activity levels were measured. tissue mrna levels of nf-jb, p38 mapk and nuclear factor erythroid 2-related factor 2 (nrf2) were analyzed. heart tissues were also immunohistochemically and histopathologically evaluated. results: both compounds have led to a decrement in myocardial damage, apoptosis, ctni, ima, hfabp, tos, and tnf-a levels, nf-jb, p38 mapk, phosphorylated c-jun n-terminal protein kinase (pjnk 1/2) expressions. on the other hand, the applied treatment increased sod, gsh-px, tas and tt levels, as well as phosphorylated extracellular signal-regulated kinase (perk 1/2) and nrf2 expressions. conclusion: data established from the current study suggest that administered agents have protective effect against cardiac injury induced by iso, which was more prominent in rats received sb203580 treatment. p38mapk inhibitors may constitute a useful choice as cardioprotective agents due to their antiinflammatory, antioxidant and anti-apoptotic effects. keywords: _ isoproterenol, myocardial infarction, myocardial ischemia, p38 mitogen-activated protein kinases, sb203580, tak-715. silicosis composes the vast majority of occupational lung diseases. silicosis, caused by inhalation of crystalline silica, is a chronic lung disease characterized by parenchymal nodules and pulmonary fibrosis. the susceptibility of patients with silicosis to infection is thought to be due to toxic effects of silica on pulmonary macrophages. ada activity is considered as a nonspecific marker of t cell activation and cellular immunity. this study aimed to compare the serum ada activity in silicosis patients with spirometric values. in this study there were 35 males in each groups which contained patients with silicosis (group 1), individuals having similar symptoms with silicosis from same occupational area (group 2) and healthy subjects (group 3). routine hematological and biochemical parameters were also measured. the serum ada activity and spirometric values (fev1, fev1%, fev1/fvc, fev1/ fvc%, fef25-75 and fef25-75%) were compared. the average age of group1, 2 and 3 are 38.5 ae 10.6, 39.5 ae 2 and 51 ae 10.5 years, respectively. there was a significant difference between group 1 and 3 in terms of the ada level (p < 0.05). there was a negative correlation between ada activity and fev1, fev1%, fev1/fvc, fev1/fvc%, fef25-75 values. elevated serum ada activity has been shown in many diseases with induced cellular immunity. despite initially toxic effects were lead to a little immunological reaction in patients with silicosis, continuation of this immunological response is important in some chronic manifestations of silicosis. the release of chemotactic factors and inflammatory mediators cause the migration of polymorphonuclear leukocytes, t lymphocytes and macrophages. in this study, the ada activity was significantly higher in patients with silicosis than others. increased immunity in patients with silicosis is being considered, increasing ada activity might be help of earlier recognition of these patients and to take better quality of life. atlantic salmon (salmo salar l.) is an important model system in evolutionary and conservation biology that provides fundamental knowledge into population persistence, adaptive response and the effects of anthropogenic change. the role of behavioral and body size variation in environmental adaptation of atlantic salmon is well known, by contrast, the underlying biochemical mechanisms are largely unknown. intracellular proteases, such as cathepsins b and d in lysosomes and calpains and proteasome in cytosol, due to their metabolic and regulatory role may contribute to phenotyping speciation of salmon young. we examined the activity of intracellular proteolytic enzymes in skeletal muscles of atlantic salmon parr from two local habitats of the varzuga river (the main channel and small tributaries) differing in hydrological and feeding parameters. calpain and proteasome activities were determined by casein or suc-llvy-amc hydrolysis in the skeletal muscles of s. salar from varzuga river (kola peninsula, russia). it is known that salmon parr originated from a common hatch became phenotypically divergent during the settle in the biotopes. reliable difference in studied enzyme activities in the salmon parr from two local habitats was found; furthermore, calpain and cathepsin b proteolytic activities were found to negatively correlate with parr body size. muscle proteolytic activity data support an idea on protease contribution to environmentally-driven adaptation and speciation process in fish. the work was supported by the russian scientific foundation, project no. 14-24-00102. the phylogenetic analyses of anthriscus (apiacea) species from turkey based on non-coding "trn" regions of chloroplast genome p. yilmaz sancar 1 , m. tekin 2 , s. civelek 1 1 firat university, elazig, turkey, 2 cumhuriyet university, sivas, turkey anthriscus pers. (apiaceae) species belongs to apiaceae family and is represented by 16 genus on the world and by 8 genus in turkey. anhriscus species are used extensively for treatment various disease such as asthma, alzheimer and show anti-tumoral, anti-microbial, antioxidant features. for determining exact species which treat disease it is necessary sorting species correctly with molecular markers to support morphological features. anthriscus species were defined by examining insufficient quantity of samples in turkey flora. besides, no detailed study was found in our country after flora study. for this reason a revision study was made with the aim of solving some systematical problems in 2013 by tekin. the result of the study provided important contribution to the systematic of the species in turkey. however a molecular study was also required for building the obtained results on a more solid ground. in this study, the aim to reveal systematic and phylogenetic relationship among species of anthriscus in turkey, by using trnl-f region in chloroplast genome. dna was isolated by ctab method and amplified in pcr by using e-f primaries. the obtained data was evaluated by mega 7.0 program and phylogenetic tree was prepared by using maximum likelihood method. according to the phylogenetic tree that we prepared by using the sequence line up of trnl-f section, it was observed that a. cerefolium, a. caucalis and a. tenerrima species completed their speciation and an isolation with other species in terms of speciation was provided. it was also observed that a. kotchi, a. sylvestris subs. sylvestris, a. sylvestris subs. nemarosa and a. lamprocarpa'nın provided hybridization among themselves but they did not complete their speciation. it was determined that a.lamprocarpa var. chelikhii which is one of the two different varieties of a. lamprocarpa is actually a new sub-species. this fact was supported by molecular data obtained from the study we made after morphologic data. introduction: excessive production of androstenedione can becaused by defects of adrenal steroid biosynthesis, tumors of ovarian and adrenal origin, polycystic ovarian syndrome, increased peripheral sensitivity to androgens, and increased peripheral production of androgens. most epidemiologic studies use enzyme-linked immunosorbentassay (elisa) to measure sex steroid hormones because they have acceptable turnaround times and arerelatively inexpensive. mass spectrometry-based methods are currently the most specific quantitative analytical methods for steroid determination. mass spectrometry methods are independent of matrix effects or cross-reactivity. in this study, a new liquid chromatography-tandem mass spectrometry (lc-ms/ms) method was developed. materials and methods: for serum androstenedione measurement, 50 ll of internal standard (d5-11 deoxycortizol) in methanol was added to 250 ll standart or serum and centrifuged at 4.500 rpm for 10 minutes to remove the precipitated proteins. supernatant was transferred to clean tubes and this procedure was performed twice. the supernatant was collected and dried under a nitrogen gas flow at 60 • c and dissolved in mobile phase. 60 ll was injected in to the ultra performance liquid chromatography analytical column for chromatography. elisa study was conducted with drg (lot. no. 50k074) brand kit. results: method comporison between lc-ms/ms and elisa was found slope value 18,412, intercept value à22.87 and r² value 0.1033. the regressione quation was elisa= à2.861782 + 4.905103 lc-ms/ms. discussion and conclusion: method comparison study presented higher results in elisa compared to lc-ms/ms. in our opinion, this might due to the interference in elisa systems. our lc-ms/ms method allows rapid, sensitive and specific determination of androgens in plasma and serum.the specificity of liquid chromatography-tandem mass spectrometry (lc-ms/ ms) offers advantages over immunoassays. heparins play an important role in cell growth, differentiation, migration and invasion. however, the molecular mechanisms of heparin mediated cellular behaviors are not well defined. to determine the effect of heparin on gene expression, we performed a cdna microarray in a hepatocellular carcinoma cell line and found that heparin regulates transcription of genes involved in glucose metabolism. in this study, we showed a new role of heparin in the regulation of thioredoxin interacting protein, which is a major regulator of glucose metabolism, in hepatocellular carcinoma cell lines. we determined the importance of a unique carbohydrate response element located on its promoter for the heparin-induced activation of thioredoxin-interacting protein and the modulatory role of heparin on nuclear accumulation of carbohydrate response element associated proteins. we showed the importance of heparin mediated histone modifications and downregulation of enhancer of zeste 2 polycomb repressive complex 2 expression for heparin mediated overexpression of thioredoxininteracting protein. when we tested biological significance of these data; we observed that cells overexpressing thioredoxininteracting protein are less adhesive and proliferative, however they have a higher migration and invasion ability. interestingly, heparin treatment increased thioredoxin-interacting protein expression in liver of diabetic rats. in conclusion, our results show that heparin activates thioredoxin-interacting protein expression in liver and hepatocellular carcinoma cells and provide the first evidences of regulatory roles of heparin on carbohydrate response element associated factors. this study will contribute future understanding of the effect of heparin on glucose metabolism and glucose independent overexpression of thioredoxin-interacting protein during hepatocarcinogenesis. prolidase activity in chronic obstructive pulmonary disease and asthma t. g€ uc ßl€ u 1 , h. s€ urer 1 , g. bilgin 2 , d. y€ ucel 1 1 ankara training and research hospital, medical biochemistry department, ankara, turkey, 2 ankara training and research hospital, chest diseases department, ankara, turkey chronic obstructive pulmonary disease (copd) is a consequence of an underlying chronic inflammatory disorder of the airways that is usually progressive and causes dysregulation in the metabolism of collagen. and asthma is a disease where there is an accumulation of collagen in the reticular basal membrane of the airway leading to chronic inflammation. prolidase has an important role in the recycling of proline for collagen synthesis and cell growth. we measured and compared prolidase activity in healthy individuals with copd and asthma patients to find out that whether its activity might reflect disturbances of collagen metabolism in the patients. 60 patients with copd, 60 patients with asthma and 20 healthy control subjects with similar age range and sex were included in our study. the patient and control groups do not have any other chronic disease. serum prolidase activity was measured in the patient and control groups. ferritin and alpha-1 antitrypsin concentrations were also compared. there was no significant difference between serum prolidaz activities of asthma and copd patients. serum prolidase activities of both copd and asthma patients were significantly lower than those of the control subjects (p < 0.05). there was no significant difference for ferritine and alpha-1 antityripsin levels between the groups. the prolidase activity is significantly lower in asthma and copd patients comparing with control subjects. the collagen metabolism may be undergone to a change in these patients. hence, there may be an effect on the accumulation of collagen in the reticular basal membrane. the results suggest that collagen turnover are altered by the development of copd and asthma in human lungs, and prolidase activity may reflect disturbances of collagen metabolism in these pulmonary diseases. monitoring serum prolidase activity may be useful in evaluating fibrotic processes and in the chronic inflammatory lung diseases in human. acyclovir molecule in the active site of e. coli purine nucleoside phosphorylase (on the basis of x-ray study) i. kuranova 1,2 , v. timofeev 1,2 , n. zhukhlistova 1 , y. abramchik 3 , t. muravieva 3 , r. esipov 3 1 shubnikov institute of crystallography of fsrc "crystallography and photonics" ras, moscow, russia, 2 national research centre "kurchatov institute", moscow, russia, 3 shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia e. coli purine nucleoside phosphorylase (pnp), which catalyzes the reversible phosphorolysis of purine ribonucleosides, belongs to the family i of hexameric pnps. due to key role in the purine sulvage pathway pnps are attractive targets for drug design against some pathogens. they also used widely in biotechnology for the synthesis of nucleoside analogues as well as for the activation of the prodrugs in anti-cancer gene therapies. the acyclovir (acv), acyclic derivative of guanosine, is antiviral drug for the treatment of some human viral infections. the crystalline complex of e. coli pnp with acyclovir was prepared by co-crystallization using counter diffusion in capillary through the gel layer. the set of x-ray data at 100 k from single crystal grown in space (sp. group p6 1 22) was collected on the spring-8 synchrotron-radiation facility (japan) and the structure was solved at 2.32 a resolution, using the molecular replacement method (pdb id 5i3c). acv molecule was located in the nucleoside binding pocket of the enzyme in two conformations. the phosphate binding site was occupied by so4 ion. the hydrogen bonds network and hydrophobic interactions stabilising acv molecule in the active site as well as the conformational changes upon ligand binding were described. the comparison of e. coli pnp/acyclovir complex and the similar complexes of bacillus subtilis pnp (pdb id 4da7) and human pnp (pdb id 1pwy) allowed to establish the peculiarities of acv binding of in the e. coli enzyme. gonadotropins are glycoprotein hormones that regulate normal growth, sexual development, and reproductive function. these are large, up to 40 kda proteins, which are synthesized and secreted by the gonadotropic cells of the anterior pituitary gland. these hormones may vary in the level of glycosylation depending on the tissue and the metabolism cycles. follicle-stimulating hormone (fsh) and upon binding to fsh receptor, a g-protein coupled receptor (gpcr), regulates the development, growth, pubertal maturation, and reproductive processes of the body. human chorionic gonadotropin (hcg) and luteinizing hormone (lh) act via a shared gpcr (lh receptor) and regulate mechanisms essential for ovulation, early pregnancy and placental function in females as well as spermatogenesis and testosterone production in males. activation of gpcrs by these hormones can be measured by monitoring formation of cellular cyclic adenosine monophosphate (camp). the level on camp was measured using a f€ orster resonance energy transfer (fret)-based biosensor tepacvv (h74) kindly provided by dr, kees jalink. the biosensor was expressed using the developed bacmam gene delivery system (recombinant baculoviruses carrying the transgene under a strong mammalian promoter). kgn cells expressing the fsh receptor and cos7 cells expressing the lh receptor served as study objects. monitoring of specific gpcr activation in living cells, allows detection of only the biologically active agonists, which has real impact in quantification of large hormones. differences in levels of hormone glycosylation may affect their biological function. investigation of this phenomena is planned for near future. detection of biological activity of gonadotropins is of importance for pharmaceutical industry, where today the concentration of recombinant proteins is mostly estimated using immunological assays only. development of a colorimetric aptasensor for the detection of peanut allergen protein ara h 1 in food samples b. bora ege university, izmir, turkey food allergy, especially peanut allergy is a life-threatening problem, and severe reactions against these foods can be observed. since unintnded consumption of non-labeled foods is the most dangerous risk, any residual allergen protein should be tested and labeled by the manufacturers. an aptamer based colorimetric test is a powerful alternative to commercially available rt-pcr and elisa test kits. the main objective of this study is to develop an aptamer based colorimetric test fort he detection of major peanut allergen protein ara h 1. ara h 1 aptamer was used to recognize any residual peanut major allergen protein ara h 1 in food samples. recombinant ara h 1 protein was produced and puirifed to be used as a target. ara h 1 aptamer was used in combination with a blocking sequence, to prevent non-specific binding event, a biotinylated complementary strand to the blocking sequence, and finally strp-hrp interaction in order to facilitate colorimetric reaction. optimal blocking sequence length was optimized and introduced to the 3 0 site of aptamer sequence to construct an aptamer-hairpin structure. liberation of the blocking sequence allows biotinylated complementary strand to bind to the blocking sequence and consequently str-hrp conjugate to achieve color development that is proportional to the target concentration. since, the aptasensor will be used for the detection of ara h 1 in food samples, total protein extraction from chocolate samples was also optimized. in order to lower the detection limit of aptasensor, aptamer coupled magnetic bead based pre-enrichment assay was aslo optimized for the total protein extraction. as a result, a sensitive, fast and reliable aptamer based colorimetric assay was developed for the detection of peanut allergen protein from food samples. moreover, the assay has the advantages like ease of application and low cost which makes the assay a promising and a powerful alternative to commercially available rt-pcr and elisa tests. the association between lipid parameters and waist circumference in female university students in turkey s. ozen, a. cort sanko university, department of nutrition and dietetics, gaziantep, turkey a high waist circumference is associated with an increased risk for type 2 diabetes, dyslipidemia, hypertension, and cvd in patients with a bmi in a range between 25 and 34.9 kg/m 2 . monitoring changes in waist circumference may be helpful, in addition to measuring bmi, since it can provide an estimate of increased abdominal fat even in the absence of a change in bmi. objective of the study was to find an association between plasma lipid profile and anthropometric parameters (waist circumference percentage of body fat and body mass index (bmi)) in abdominal obesity in turkish university students. lipid profile and anthropometric parameters of obesity were studied in a sample of 30 women. students with high bmi (>24) had higher values of low-density lipoprotein (ldl), triglycerides (tg) and cholesterol (c) than students with low bmi (<24) but these differences were not significant. high-density lipoprotein (hdl) levels were non-significantly higher in low bmi (<24) student group. waist circumference, percentage of body fat was higher in high bmi (>24) group than low bmi (<24) group. waist circumference, percentage of body fat was positively correlated with bmi in both samples (bmi (>24) and bmi (<24)). students were grouped depend on their waist circumference. healty individuals who had lower than 80 cm waist circumference had decreased tg levels compared to cardiovascular risk group who had higher waist circumference than 80 cm. this study shows an association between waist circumference, percentage of body fat, body mass index and lipid parameters in young female university students. with regard to the relationship, the screening females for central obesity to prevention of cardiovascular disease are recommended. a new biotechnological product from propolis with low allergen: anti-inflammatory effect propolis is extensively used in food industry due to its special medical properies (antioxidant, antimicrobial, antiseptic, antibacterial, anti-inflammatory and antimutagenic effects). even these positive properties it may cause some allergic reactions in consumers with allergic predispositons. previously, we demonstrated that biotransformation of propolis by some special strains of lactobacillus plantarum (10, 8014, aatc strains) might decrease the allergenic molecules in propolis. in this study, we aimed to investigate the effect of biotransformation of popolis on it's antiinflammatory activities. before biotransformation, propolis samples were treated with different solutions (10% ethanol and polyethylene glycol -peg 40%) and different method (ultrasonic treatment 300 w/25 o c/ 30 minutes) in order to facilitate solvation of solid samples which are very dense and not suitable for fermentation. fermantations were performed at 30 o c/48 hours under constant agitation conditions. the anti-inflammatory activity was determined in-vitro conditions using hyaluronidase's analysis and the xanthine oxidase activity. the highest inhibition (%) of radicals produced by xanthine oxidase was determined in solid samples treated by peg prior to biotransformation and using of l.plantarum 8014 strain during fermentation (88.43%), followed by liquid samples treated by ultrasonic method prior to transformation (86.21%). concernig the results of hyaluronidase activity (%) inhibitions, the best value were determined in the solid sample treated by peg prior to biotransformation and using of l.plantarum 8014 strain during fermentation (93.93%). results indicated that the anti-inflammatory activities of analysed samples are quite high and depending of used extraction methods prior the biotransformation and used specific strain of l.plantarum could be optimized in terms of other required parameters. faceanti-mullerian hormone is not predictive for poor neonatal outcome aim: anti-mullerian hormone (amh) is a growth factor specific to ovaries. it is commonly used to predict ovarian reserve and outcomes of fertility treatments. recently, low levels of amh have been shown to be related to hypertensive diseases of the pregnancy and the risk of preterm labor. the aim of this study was to investigate the diagnostic performance of amh levels of mothers to predict poor neonatal outcome in term pregnancies and the relationship between amh and birthweights of the newborns. materials and methods: 187 patients, having delivery beyond 37 weeks, and who did not have any other medical problems were included in the study. the patients had normal 50 g. oral glucose tolerance test results. they were divided as 3 groups, based on their newborns' birthweight as "2500 g. and 4000 g.". level of amh was determined by elisa method. results: there was not any relation with the amh of the mothers and the poor neonatal outcome of the newborns, in all 3 groups. also no siginificant difference was observed in amh levels of the patients having delivery in early term and late term periods. when the patients of the same group were evaluated; amh levels were irrelevant to age, gravidy, delivery week, body mass index, the weight gain during pregnancy, and poor neonatal outcome. conclusion: amh is not a predictive factor for poor neonatal outcome and it is not a determinant of the weight of the newborn. objectives: the aim of the study was to investigate the effects of differing amounts of hemolysis on serum high sensitvity troponin i (hs-tni), ck-mb mass and myoglobin measurements. materials and methods: we prepared serum pools having troponin i, ck-mb and myoglobulin concentrations at low (5.33 ng/l,1.5 ng/ml and 34.3 ng/ml respectively), normal (39.25 ng/l, 3 ng/ml, 197.9 ng/l respectively) and high (7345 ng/l, 22 ng/ml, 574 g/ml respectively) values. the osmotic shock method was utilized to prepare a hemolysate. hemolysate was added into serum pools increasing concentrations of hemoglobin (0.65, 1.3, 2.5, 5, 7.26 and 9 .42 g/l hemoglobin). troponin i, ck-mb (mass) and myoglobin concentrations were measured in duplicate by beckman coulter access 2 analyzer. the hemolysis indices were measured on beckman coulter au 5800. a change of 10% from baseline results was taken as evidence of interference and the interfered tests were also evaluated according to total analytical error based on analytical imprecision and intraindividual biological variation. results: we found a positive interference due to hemolysis for ck-mb (mass) at low concentrations (1.5 ng/ml), and a negative interference for myoglobin at low concentrations (34.3 ng/ml) and high concentrations (574 ng/ml). conclusions: ck-mb increase and myoglobin decrease in hemolyzed samples with hemoglobin ≥9.42 g/l, but the bias might not be clinically significant (< total analytical error) in samples. a retrospective study to determine a reliable marker for selective screening of pompe disease lysosomal storage diseases (lsd) are rare inherited metabolic disorders caused as consequence of a deficiency in a specific enzyme required for lysosomal function. pompe disease is one of these disorders with deficiency of a-1,4 glycosidase enzyme with an incidence of 1:4,500-1:33,000. as enzyme replacement therapies are available nowadays, early diagnosis is crucial and selective screening is a rational method to reach pompe patients among people who administer to healthcare with lsd suspected symptoms. this study aims to examine the relationship between basic biochemistry parameters and a-1,4-glycosidase activities retrospectively, in order to find a key parameter for selective screening of pompe disease. for this reason a-1,4glycosidase, creatine kinase (ck), creatine kinase-mb (ck-mb) activities calcium, phosphate levels of those who had been suspected to be lsd patients and administered to our laboratory for analysis are examined retrospectively. out of 134 patients's examined,14 of them were diagnosed with pompe disease depending on clinical findings & low a-1,4glycosidase activity. enzyme activities of pompe patients were 0.337 nmol/ml/hour as lsd suspected patients'activities had a mean of 2.78 nmol/ml/hour (p = 0.00).comparison of ck activity was compared results showed significant difference between pompe patients and lsd suspected patients. even though ck activity levels of the lsd suspected patients were much higher (400vs41-171u/l) than reference interval, the levels of the pompe disease patients' were still more than twice of the lsd suspected group (956vs400u/l, p = 0.02). ck-mb, ca, p levels didn't show a significant difference. a strong (-) correlation (p = 0.005 r=à0.240) was observed between a-1,4-glycosidase and ck activities (n:134). selective screening is a rational way to diagnose rare diseases. this study's results show that ck activity can be used as a key parameter to determine patients for selective screening of pompe disease within lsd suspected population. the functional effect of stem cells on the reproductive organs infertitility is considered as a major health problem of recent century. importance of stem cell is increasing so it is searched new features and supposed to be involved in the infertitility treatment where oxidative stress and apoptosis play importany role. we aimed to investigate the beneficial effect of the stem cells related to free radicals and cell death on testis and ovary. biopcy model of wound healing was created in the rat testis and ovary with ppd syringe where stem cells were delivered by injection. rats were divided into four groups including controls, sham, wound healing and wound healing with stem cell. after the creation of the wound, bone marrow-derived mesenchymal stem cells from the tibia of the mature rats and medium were administered to ovaries and testes. following the applications, ovary and testis samples were investigated for oxidative stress and apoptosis by immunohistochemistry. in comparison with the medium and stem cell applications without a medium support, it was meaningfully determined that healing effect in testicles and ovaries were spotted specifically on the seven day. tissues were analysed for these staining by h-score and h-score results were determined using one-way anova test statistically. our results show the positive effects which clinic applications can bring by displaying the great contribution of the stem cell application in the treatment of testicle and ovary damage. these findings suggest that transplantation of the mesenchymal stem cells may help to promote better enviroment for the reproductive organs by the effect on oxidative stress and apoptosis. the further studies of these results in the molecular level can lead the way to solve the problem of infertility, to increase the percentage of success in the ivf and icsi techniques and more importantly to perform a differentiation from a somatic cell to a germ cell. the antimicrobial activity of 2 (5h)-furanone derivative on staphylococcus aureus nosocomial infections caused by methicillin-resistant staphylococcus aureus strains are known to be a reason of many infectious diseases like osteomyelitis, endocarditis, sepsis etc. being organized in biofilms these bacteria become extremely resistant to antimicrobials and host immune system leading to difficulties in treatments. here we report the effect of 2 (5h)-furanone derivative possessing sulfonyl group and l-menthol moiety (f105) on biofilms formed by s. aureus atcc29213 and mrsa cells. while exhibiting relatively high minimal inhibiting concentration -mic (16 mg/l), clear synergy with a number of antibiotics was found in the checkerboard assay. thus, in the presence of 2 mg/l of f105 the mic of kanamycin was decreased 4-fold, and the mics of both erythromycin and ampicillin were lowered 2-fold. at the concentration of 80 mg/l f105 also completely inhibited the biofilm formation by s. aureus; the cell growth was suppressed by two orders of magnitude as judged by differential fluorescent staining with syto9 and propidium iodide. the addition of f105 to preformed 24 h-old biofilms increased the fraction of red-stained (dead) cells of both s. aureus atcc29213 and mrsa strains uniformly throughout the whole profile of the biofilm. the quantitative analysis of clsm microphotographs revealed that f105 at concentration of 80 mg/l led to death of up to 98% of biofilm-embedded cells. this fact suggests that f105 efficiently penetrates into the biofilm matrix and kills the cells without visible damage of biofilm structure. in summary, furanone f105 seems to be a promising compound for drugs design to treat biofilm-embedded s. aureus. this work is supported by the russian science foundation, project №15-14-00046 and the german academic exchange service (№91531398). pneumonia is an inflammatory lung disease which can be associated with inadequacy of host defense system and the proliferation of various pathogenic microorganisms into the lower respiratory tract. community acquired pneumonia (cap) is one of the leading causes of death in elderly. the incidence of pneumonia in people aged 65 and over is 3-5 times more than young adults. creactive protein (crp) is an acute-phase protein of hepatic origin that increases following interleukin-6 secretion by t cells and macrophages. procalcitonin (pct) is a peptide precursor of the hormone calcitonin, the latter being involved with calcium homeostasis. it is composed of 116 amino acids and is produced by parafollicular cells (c cells) of the thyroid and by the neuroendocrine cells of the lung and the intestine. the level of pct rises in a response to a proinflammatory stimulus, especially of bacterial origin. the aim of this study was to compare crp and pct levels in young and elderly patients with pneumonia. recently diagnosed 43 young and 46 elderly patients with pneumonia and their respective aged matched controls (n = 41, n = 42) were enrolled this study. crp and pct levels were by immunoturbidometric and by elisa methods respectively. crp and pct levels for young control and patients and elderly control and patients respectively are 2.32 ae 2.25 mg/l, 0.26 ae 0.13 ng/ml, 20.71 ae 34.61 mg/l, 0.64 ae 1.09 ng/ml, 2.32 ae 2.14 mg/l, 0.27 ae 0.07 ng/ml and 31.41 ae 31.0 mg/l, 0.49 ae 0.83 ng/ml. young patients with pneumonia have significantly higher crp and pct levels than their controls (p < 0.001 and p < 0.028). elderly patients with pneumonia have significantly higher crp levels than their controls (p < 0.001). crp and pct are important markers in the diagnosis of pneumonia. effect of serum albumin concentration on total and ionized calcium z. adiyaman, c. yilmaz, s. a. peker, d. y€ ucel ankara training and research hospital, ankara, turkey objective: the aim of the study is to investigate in vitro effect of albumin concentration on total and ionized calcium concentrations. materials and methods: a serum pool with low albumin (3.10 g/dl) and normal calcium (9.79 mg/dl) concentrations was prepared from leftover sera. from this serum pool, two parts, each of 10 ml were aliquoted. purified albumin, 0.3 g, was added to one of these pools and albumin concentration was determined as 6.1 g/dl. the low and high albumin pools were mixed at different ratios and pools with 3.89, 4.78, and 5.37 g/dl albumin concentrations. total calcium and albumin concentrations of these 5 pools were measured at a beckman-coulter au5800 analyzer and ionized calcium was measured at a radiometer abl 800 blood gas analyzer in triplicate. total and ionized calcium concentrations were evaluated as compared to those of the original pool with an albumin concentration of 3.10 g/dl. results: total calcium concentrations are increased with the increasing albumin concentrations: 1.2%, 1.58%, 2.85%, and 3.59%, respectively. whereas, ionized calcium concentrations were decreased with increasing albumin: 3.0%, 6.1%, 7.4%, and 8.6%, respectively. conclusions: when total allowable error limits based on biological variation were considered, total calcium concentrations are significantly increased at >5 g/dl albumin concentrations. ionized calcium is significantly affected by 0.8 g/dl and over albumin concentrations. a regression equation based on albumin concentration may be useful for corrected ionized calcium concentrations. relationship between lipoprotein (a) and hba1c in patients with type ii diabetes , is a complex lipoprotein consisting of ldl and apolipoprotein(a). lp(a) is a risk factor for coronary artery disease and stroke. the relationship between lp(a) and diabetes mellitus is not clear. in this study, the relationship between lp(a) and glycemic parameters such as hba1c and fasting glucose concentration was investigated. lp(a), hba1c, fasting glucose, triglyceride, total cholesterol, ldl-and hdl-cholesterol concentrations were screened retrospectively from july 2013 to july 2016. there were 1831 patients with these test results at the same time. the patients were grouped according to hba1c values: group i < 5.7% (n = 468), group ii 5.7-6.4% (n = 739), and group iii >6.5% (n = 624). the relationship between these parameters were statistically within each group and all groups. there was not a statistically significant difference between the lp(a) concentrations of group i and group ii. lp(a) concentrations of group i and ii were significantly higher than those of group iii.. _ in total, lp (a) was negatively correlated with hba1c (r = 0.09; p < 0.01), but there was not a significant correlation with fasting blood glucose. _ in groups, there was a significant and negative correlation between lp(a) and fasting glucose in only group i. the negative correlation between lp(a) and glycemic parameters is interesting in patients with diabetes. despite lp(a) is an independent risk factor for cardiovascular diseases, on the contrary to expectations, lp(a) concentrations are decreased in diabetes. effect of blood collection through intravenous lines on hemolysis erroneous results are one of the most important causes of medical errors and may lead to unnecessary investigations or inappropriate interventions. total testing process consists of preanalytical, analytical and postanalytical phases. hemolyzed specimens that one of the most common source of preanalytical errors are frequently observed in laboratory practice and associated with incorrect laboratory results. blood collection through intravenous lines frequently results in hemolysis especially at eds and icus. in this study, we aimed to compare the effect of blood drawing by using bd luer-lock adapters and injector on the hemolysis rates at the ed. 60 patients who has been admitted to the ed were included in this study. all samples were drawn from newly inserted iv lines. the first blood sample was drawn with injector and the second one was drawn with luer-lock adapters to vacuum tubes. after the centrifugation routine chemistry tests and hemolysis indices were analysed on a beckman coulter au680 analyzer for each serum tube. the statistical significance of differences between two tubes was calculated with paired samples t test and statistical significance was accepted as p < 0.05. there were statistically significant differences between the two groups of tubes for the following parameters: ldh, ck, ast, k + , total bilirubin, protein, albumin, alp, calcium and hemolysis index (p < 0.05). the use of luer-lock adapters instead of injector could reduce the hemolysis rate. because of it reduces false results and unnecessary investigations, this approach will be more appropriate and cost-effective in ed. hemolysis and test rejection: are we following a reliable process? introduction: in laboratories, some blood samples are rejected due to hemolysis. we usually cancel only some of the tests that are affected by hemolysis. however, the frequency of the test cancellation may be relative. each test is affected in different degrees of hemolysis; some of them are not even affected at all. in this study, we aim to investigate unnecessary cancellations and explain the relationship between hemolysis and test results according to their kit inserts. materials and methods: we measured hemoglobin levels of 50 hemolyzed serum using drabkin method (abbott). interference studies are conducted using clsi protocol nccls ep7-p is written in kit inserts. target values (100%) and their change due to different degree of hemolysis have been defined. results: hb concentration ranges of hemolyzed sera were found from 44 to 849 mg/dl. according to kit inserts, aspartate aminotransferase (ast) test results deviate 5.7% from the target when the degrees of hb are 62 mg/dl. when the degree of hemoglobin is 125 mg/dl, the test strays about 11.6%. potassium levels increase (108%) at 125 mg/dl hb while this increase reaches to 17.9% at 250 mg/dl hb. sodium, calcium, ck, crea, total bil, lipase are not significantly affected even at 2000 mg/dl. in lactate dehydrogenase (ldh) tests, test reporting is not allowed at any hemolysis level. alt increases 11%, at the 1000 mg/dl hb. ast and potassium results were excluded from patients' reports even though those samples had low hb. some of them were reported despite of excess hemolysis. some tests are even blocked without ever being studied. discussion: prior to the approval of the lab specialist, technicians decide whether to cancel the tests affected by the hemolysis according to the visible hemolysis based on their personal knowledge. conclusion: we should use the hemolysis index, in which standards would be defined via guidelines. this way, all technicians and specialists could know which results are false. the dna-binding hu-proteins are present in all bacteria and belong to the family of nucleoid-associated proteins. these proteins can be considered precursors to eukaryotic histones. gene knockout of hu-proteins partially inhibits the growth of bacteria, their ability to resist various stressing factors and in some cases leads to their death. since the spatial structure of hu-proteins is highly conserved it is possible to create inhibitors that will affect them in a broad spectrum of pathogenic bacteria. in the present work the preparation of the recombinant hu protein from mycoplasma gallisepticum, crystallization of this protein, and x-ray diffraction study of this protein has been reported. the crystallization conditions for studying protein were found by the hanging-drop vapor-diffusion method. found conditions have been adapted to the counther-diffusion method in the capillary. the x-ray diffaction dataset from grown crystals have been collected using synchrotron radiation. 3d-structure of the hu protein from mycoplasma gallisepticum have been determined with 3a resolution. structural features of the investigated protein are described. this work is supported by russian scientific fund (15-14-00063). a novel sensitive disposable indium tin oxide (ito)-based electrochemical immunosensor was developed for simple, rapid and sensitive biomonitoring of sox2. sox2 is a cancer biomarker and used for detecting of small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, skin cancer, prostate cancer, and breast cancer. in this study, indium thin oxide (ito) thin film was used as working electrode. carboxyethylsilanetriol was also used for electrode modifying so as to obtain self-assembled monolayers. the formed self-assembled monolayers were activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (edc)/n-hydroxysuccinimide (nhs) chemistry. edc was used as a heterobifunctional crosslinker. nhs was used in conjunction with the crosslinker edc. anti-sox2 antibody was used as a biorecognition element and it was covalently immobilized onto the ito electrode modified with carboxyethylsilanetriol. immobiliztion steps were characterized by cyclic voltammetry (cv), electrochemical impedance spectroscopy (eis), and scanning electron microscopy (sem). the optimal immobilization conditions for the best sensitivity of the new immunosensor were investigated. under optimal conditions, this immunosensor demonstrated a wide linear range (0.0025-2 pg/ml) with a detection limit as low as 0.02 ng/ml sox2. furthermore, the developed sox2 immunosensor had good storage stability, repeatability and reproducibility. in this work, we successfully fabricated disposable ito thin film based electrodes for sensing the interaction between sox2 antigen and anti-sox2 antibody by electrochemical impedance spectroscopy and cyclic voltammetry. and our developed immunosensor has an acceptable performances for the detection of sox2 antigen, exhibits low detection limit, has selective and reproducible results in immunoreaction analysis. we are thankful for the support from t € ub _ itak (the scientific and technological research council of turkey, project number: 113 z 678). applying multiple linear regression model to determine the relationship between anti mullerian hormone with age, luteinizing hormone, follicle stimulating hormone and estradiol: a data mining study introduction: anti mullerian hormone (amh) has a widely used in our life because it is a good indicator of reproductive age to estimate the time of menopause. the purpose of this retrospective data mining study is the estimate of ovarian reserve by using amh and determines relationship between other indicators which are luteinizing hormone (lh), follicle stimulating hormone (fsh), estradiol and age. materials and methods: 25.294 women members were included this retrospective data mining study who were applying to acıbadem labmed laboratory. multiple regression analysis of age related changes of amh (18-45) and lh, fsh and estradiol were investigated. beckman gen ii elisa kit was used for amh and the technique of electrochemiluminescence and roche elecsys cobas analyzer were used for the measure of other hormones. results: amh shows meaningful correlation between lh, fsh, estradiol and age but also seen there is no correlation between progesterone. after the multiple linear regression analysiz amh= 11.018-(0.220 9 age)à(0.066 9 fsh) + (0.044 9 lh)à(0.004 9 estradiol) is detected and the model's r 2 = 0.627 is also detected. conclusion: nowadays there are lots of methodology were developed the estimate the function of ovary and biological age of ovarian. age, fsh, lh and estradiol show ovarian reserve by indirectly. this study shows the mathematical relationship between amh and the other indicators and results are thought to lead to future developments. antioxidant and anticancer effect of artemisia absinthium extract on colon and endometrium adenocarcinoma cells plants have always been among the common sources of medicines that have many phytochemicals with various bioactivities, including antioxidant and anticancer activities artemisia absinthium (ar) has been used as an antipyretic, antiseptic, anthelmintic, tonic, diuretic, and for the treatment of stomachaches in turkish folk medicine. this study aimed to investigate antioxidant, cytotoxic, genotoxic and apoptotic effect of methanol extracts of ar activities on the human colon (dld-1) and endometrium (ecc-1) adenocarcinoma cell line. total phenolic, flavonoid content, and antioxidant activities were determined using suitable methods (abts, cuprac i.e). cytotoxic effects of ar on cells were determined by mtt and neutral red uptake assays. genotoxicity was evaluated by comet assay and, apoptosis induction were detected by apoptosis elisa and acridine orange staining methods at the half maximal inhibitory concentrations (ic50) levels. it was determined that extract have shown antioxidant activity in all tests and that they could be considered as a source of natural antioxidants. cytotoxic effects were concentration-time dependent. specifically, apoptotic and genotoxic effect increased at 100 and 200 lg/ml concentrations by 48 hours. we found that ar extract had antiproliferative, genotoxic and apoptotic effects on the human cancer cell lines dld-1 and ecc-1. however, further studies at molecular level are required to support our findings and to elucidate chemopreventive and chemotherapeutic effects of ar on colon and endometrium cancers. keywords: artemisia absinthium, antioxidant, anticancer, apoptosis, genotoxicity introduction: colorectal cancer is considered as a major gastrointestinal. this cancer is the second cancer related cause of death after lung cancer in worldwide. we designed a vaccine chimeric including cea and ca19-9 against colorectal cancer (ce-ca). materials and methods: the construct were analyzed by bioinformatics softwares. in this study, the ce-ca gene was optimized using the codon bias of e.coli and synthesized by biomatik company. then construct (ce-ca) was cloned into an expression vector and recombinant constructs transferred to e.coli bl21de bacterium and desired recombinant protein was expressed. recombinant protein was purified using ni-nta affinity chromatography. the content of secondary structures was obtained by circular dichroism (cd) spectrum. then recombinant protein was confirmed using western blot analysis and indirect elisa method. results: sds-page analysis showed that the recombinant protein was highly expressed and purified. western blot analysis confirmed recombinant protein. also cd spectrum confirmed predicted structures by bioinformatics tools. the elisa results showed significantly high affinity toward recombinant ce-ca protein. discussion: based on many studies, cea as potential immunogenic candidate could be considered in vaccine studies. also ca19-9 is a cell-surface antigen that has significant increase of expression in colorectal cancer, thus as marker of colorectal cancer. based in available data, these two antigens, in combination can provide specificity for production of colorectal cancer vaccine. conclusion: these findings suggest that ce-ca as potential immunogenic candidate which could be considered in future vaccine studies and detection of colorectal cancer. flow cytometric cell cycle and apoptosis analyses of some wild animal species a. tas, e. koban bostanlar tubitak, marmara research center (mrc), genetic engineering and biotechnology institute (gebi), animal genetic and reproductive biology laboratory, kocaeli, turkey cell biobanking; more specifically cryopreservation of biological diversity, is promising as a tool to preserve wild animals as well as domestic ones via nuclear transfer. in this study, we investigated the viability and cell cycle characteristics of 5 wild animal species (fallow deer, red deer, wild sheep, wolf, wild goat). auricular tissue samples were maintained in pbs+2%psa. tissues were seeded on 35 mm petri dishes containing dmem/high glucose supplemented with 20% (v/v) fcs and incubated 5%co2 in air at 95% relative humidity and at 37°c. after seeding, the medium was unchanged for 7 days and then it was changed in every 2 days for 25 days at maximum. once the cells were obtained; flow cytometric cell cycle and apoptosis analyses were done. in terms of apoptosis, all the groups showed high viability rates (over 95%) in culture when compared with the negative control (38%). the cell cycle comparisons were made between serum-starved cells and roscovitine treated cells, both for which untreated cells were used as control, which revealed different results for different species. there was no difference found between serum-starved cells and roscovitine treated cells for red deer and wolf. the serum-starved cells resulted in higher g0/g1 phase for fallow deer and wild goat. on the contrary, roscovitine treated cells resulted in higher g0/g1 phase for wild sheep. as a result; the cells obtained from wild animals had high viability and g0/g1 phase rates. therefore, they may serve as a donor cell source for nuclear transfer studies.(grant: tubitak kamag, turkey, 109g016). the interaction of different types of antibiotics with endothelial cells in the presence of nanoparticles the interaction of nanomaterials with cells and lipid bilayers is critical in many applications such as phototherapy, imaging, and drug/gene delivery. the aim of this study was to investigate the interaction of nanoparticles (fe 3 o 4 ) or nanoparticles fused with different antibiotics with cell membranes in order to reveal changes in the membrane organization. endothelial cells were used to determine the effect of different antibiotics (gentamicin, kanamycin, amikacin, penicillin, polymyxin, neomycin, cefotaxime, bacitracin, moxicillin, erythromycin, streptomycin and vancomycin) on the membrane organization. for recording the anisotropy of cell suspensions treated with antibiotics or nanoparticles fused with antibiotics we used 1-4-trimethyl-6-phenyl 1, 3, 5 hexatrien p-toluenesulfonate (tma-dph). we decided to use nanoparticles fused with antibiotics because they contain small amounts of antibiotics which makes them less toxic than simple antibiotics,which is very important in patients with genetic diseases such as cystic fibrosis, that should be treated with antibiotics for a long time. our results showed that at temperatures between 31 and 35°c simple nanoparticles decreased the membrane fluidity. at physiological temperatures (37-39°c) nanoparticles fused with antibiotics (gentamicin, vancomicin, cefotaxim, bacitracin, amoxicillin) increase more the membrane rigidity compare with simple antibiotics or nanoparticles.erythromycin, polymyxin and penicillin increase the membrane rigidity at 37°c, and at 39°c the same effect was obtained in the presence of nanoparticles fused with these antibiotics,suggesting that the nanoparticles are dependent to temperature for penetrating the membrane. in conclusion the membrane fluidity does not depend on antibiotics types, the modification are present in many antibiotics irrespective of class type.the presence of nanoparticles fused with antibiotics is very important for long term treatment. objectives: hypertension is an important cardiovascular risk factor for the development of atrial fibrillation (af). increased atrial electromechanical coupling time interval measured by tissue doppler is accepted as an important factor for prediction of af development in hypertensive patients. monoamine oxidases (maos), are enzymes which catalyze the oxidation of monoamines. 8-isoprostane is considered as an indicator of oxidative stress. mao activity and 8-isoprostane levels were measured in some diseases. however, there are no information on 8-isoprostane levels and mao activity in newly diagnosed patients with stage 1 hypertension has not been observed in a study of literature. aim: this is the first study, we aimed to evaluate the levels of mao and 8-isoprostane in newly diagnosed patients with stage 1 hypertension. the study included 60 newly diagnosed stage 1 hypertensive patients with no other systemic disease. 30 patients were selected as randomized (17 women, 13 men; range of age 46-74 years) and 30 healthy individuals as control (15 women, 15 men; range of age 44-72 years). all the underwent tissue doppler echocardiographic examination. blood samples were taken from patients and controls and, the levels of mao and 8-isoprostane in serum samples were measured by elisa. results: baseline blood pressures, electrocardiographic and echocardiographic findings, and atrial electromechanical coupling were similar in both groups (p > 0.05). compared to the control group, the activity of mao and 8-isoprostane levels were found significantly higher in patients (p < 0.05). conclusion: increased 8-isoprostane level indicate that there is oxidative stress in newly diagnosed patients with stage 1 hypertension. also, increased mao activity may be biochemical biomarkers for the diagnosis of hypertension. keywords: hypertension, monoamine oxidase, 8-isoprostane p-mis-147 determining the indirect reference intervals for complete blood count parameters in bursa, turkey reference intervals (ris) for laboratory test results are defined as the most commonly used diagnostic tool in medicine. therefore, careful determination of ris by the laboratory for use is a very important task. although c28-a3 guideline recommends the direct ris (dris) calculated from healthy subjects, ris can be calculated from laboratory data which are called as indirect ris (iris). the study was carried out at the central laboratory for clinical chemistry, teaching and research (uludag university, bursa, turkey) . the results of the laboratory analyses from 142,591 males, 215,658 females, stored for approximately one year, were used for statistical analysis. data for hospitalized patients and for ambulatory patients from the intensive care unit were eliminated. furthermore, we used evidence based criteria to enrich the health-related values. a modified bhattacharya procedure was used to estimate the iris from hospital patient data. the nested anova was used to evaluate variations among genders and ages. cell dyn analyzer (abbott diagnostics, il, us) was used for the measurements of complete blood count. the obtained iris were also compared the dris determined in our previous ri study and the ris suggested by the manufacturer. we found that the ris of rbc, hb and hct required strong gender partition and calculated the ris of rbc, hb and hct separately. the observed iris for wbc, sub-fractions of wbc and plt in both genders are in good accordance with the dris reported in previous study. age-related changes were noted for rbc, hb, and hct. the calculated iris for rbc, mcv and rdw are different from the ris suggested by the manufacturer. we believe that, using this relatively easy technique, every laboratory can produce its own iris, divided, where possible, according to sex and age and according to local conditions. these ranges can be complementary to dris obtained for reference individuals according to the ifcc recommendations. the principal sigma 70 subunit, involved in transcription of most house-keeping genes in escherichia coli, was also shown to induce rnap pausing during transcription elongation, by interacting with promoter-like motifs in the transcribed dna. such pauses were proposed to play important roles in the regulation of phage and cellular genes. e. coli contains six alternative s subunits but little is known about their ability to induce transcriptional pausing. we expressed and purified alternative s subunits of the sigma 70 family and tested their effects on transcription elongation in vitro on natural and synthetic dna templates containing consensus promoter motifs. the structure of the paused complexes was analyzed by dna footprinting methods. in vivo analysis of transcription was performed using reporter genes placed under the control of corresponding promoters. we demonstrated that the stationary phase sigma 38 subunit induced efficient rnap pausing on both synthetic and natural dna templates containing promoter-like motifs in initially transcribed regions. in contrast, the sigma 32 and sigma 28 subunits did not affect rna elongation. we showed that the sigma 38 -induced pausing depends on sigma contacts with both nontemplate dna strand and rnap core. the pausing results in formation of backtracked transcription elongation complexes which can be reactivated by gre factors that stimulate rna cleavage by rnap. our results for the first time reveal transcriptional pausing induced by an alternative s subunit. analysis of sigma 38 -dependent promoters shows that a substantial fraction of them contains potential pause-inducing motifs suggesting that such pausing may be a widespread phenomenon. we propose that sigma 38 -dependent pauses may play important roles in genetic regulation and modulate the binding of transcription repressors or activators to promoter regions. the crosstalk between streptococcus pneumoniae rnase r, ribosomes and translation c. b arria, s. domingues, c. arraiano instituto de tecnologia qu ımica e biol ogica, lisbon, portugal ribonucleases (rnases) are enzymes that ensure maturation, degradation and quality control of rna thus, contributing to the maintenance of the optimal amount of each transcript in the cells. escherichia coli rnb family of enzymes is present in all domains of life and includes rnase r, rnase ii and the eukaryotic rrp44/dis3, dis3l1 and dis3l2 proteins. in streptococcus pneumoniae only rnase r was identified. rnase r, encoded by the rnr gene, hydrolyzes rnas starting from the 3 0 end. rnase r level is increased in several stress conditions such as heat shock, stationary phase or cold shock, conditions in which most of the proteins translation is blocked. moreover, rnase r is the only exoribonuclease able to degrade highly structured rnas without the help of a helicase which is critical at low temperatures. here, we investigated the role of this enzyme by comparing the wild type strain with an rnr mutant strain. for that purpose we performed northern blots analysis of transcripts involved in translation. also, we investigated rnase r connection to the ribosome and polysome fractions using sucrose gradient polysome separation and western blots. in this study, we highlight the importance of s. pneumoniae rnase r in translation. we show that this enzyme interacts with ribosomes mostly with the 30s subunit at 37°c. moreover, in the absence of this enzyme we have observed a decrease in the amount of the 70s ribosomal subunit, concomitantly with the increase of 30s and 50s subunits. rnase r seems also to modulate the amount of the elongation factors ef-tu and ef-g transcripts. nevertheless, preliminary results further suggest other roles of rnase r in translation. modified nucleotides are present in many rna species in all domains of life. the biosynthetic pathways of such nucleotides are well studied. however, much less is known about the degradation of rnas and the salvage of modified nucleotides, their respective nucleosides or heterocyclic bases. using an e. coli uracil auxotrophic strain, we screened the metagenomic libraries for genes, which would allow the conversion of 2-thiouracil to uracil and thereby lead to the growth on a defined synthetic medium. we show that a novel gene encoding previously uncharacterized domain of unknown function (duf) is responsible for such phenotype. we have purified this recombinant protein and demonstrated that it contains a fe-s cluster. the substitution of cysteines, which have been predicted to bind such clusters, with alanines abolished the growth phenotype. we conclude that this domain is required for conversion of 2-thiouracil into uracil in vivo. this work is supported by the research council of lithuania (lmt, mip-103/2015 modified nucleotides are present in almost all classes of rna. they have great chemical diversity and are critical for rna folding, stability, interaction with cellular proteins and thereby for various cellular processes such as translation, stress response, and signaling pathways. biosynthesis of pyrimidine nucleotides and their modified derivatives in rna is well studied. nonetheless, not much is known about the cellular degradation of these compounds and the enzymes catalyzing such processes. using an e. coli uracil auxotrophic strain, we screened metagenomic libraries for genes encoding isocytosine deaminases. three novel genes were obtained, one of which encodes a protein similar to 8oxoguanine deaminases. the other two encode proteins resembling hydroxydechloroatrazine ethylaminohydrolases. we confirmed that these proteins are functional in vivo, allowing growth of e.coli on minimal medium with isocytosine. we also demonstrated that such purified recombinant enzymes catalyze the conversion of isocytosine, but not cytosine, into uracil in vitro. natural products display special attributes in the treatment and prevention of various human diseases, including cancer. a significant number of organic compounds from plants exhibit anticancer properties as attested by in vitro and in vivo studies. emerging evidence supporting the antineoplastic activity of natural compounds has rendered them promising agents in the fight against cancer. in this study, skin from limnio grape, a red greek grape variety that is indigenous to the greek island of lemnos, was extracted using mixtures of methanol, water and acetone; the apoptosis-inducing properties of these extracts were studied in the human ovarian malignant adenocarcinoma cell lines tov-21g and tov-112d. for this purpose, tov-21g and tov-112d cells were treated with limnio grape skin extracts at a range of concentrations, at 37°c, for 24, 48 and 72 hours. untreated cells incubated for the same time intervals served as controls. cell viability was determined by measuring metabolic activity (colorimetric mtt assay) and observing cell membrane integrity (cell staining with trypan blue). after the determination of the optimal concentration of the extract, total rna was extracted from treated and untreated (control) tov-21g and tov-112d cells. after determination of rna concentration and subsequent first-strand cdna synthesis, mrna expression analysis of apoptosis-related genes was performed with rt-pcr using gene-specific primers. an increasing percentage of non-viable cells was observed by increasing cell exposure time and extract concentration. distinct modulations of the expression of apoptosis-related genes at the mrna level were also observed, mainly concerning bcl2, bclx, bax, bak1 and bcl2l12, along apoptosis induction. in conclusion, the cytotoxic properties of limnio grape skin extracts against ovarian malignant adenocarcinoma cells merit further investigation. the intrinsic apoptotic pathway seems to be the major mechanism of action induced by these plant extracts. almost all eukaryotic mrnas are polyadenylated by a complex machinery that recognizes the poly (a) signal, cleaves the mrna and adds the poly (a) tail. 70% of human genes harbor multiple poly (a) signals. alternative polyadenylation (apa) generates transcript isoforms with different 3 0 utr (untranslated region) lengths due to the use of proximal or distal poly (a) signals. hence, tightly regulated apa has been observed in normal physiological settings as well as in diseases. considering that 3 0 utr shortening cases have been linked to increased protein levels, we hypothesized deregulated apa to be one of the potential cancer related mechanisms. we investigated the 3 0 utr alterations in er(+) breast cancer patients and cell models compared to normal breast tissue, using gene expression data and a probe-based quantification tool, apadetect. based on means of proximal to distal probe sets, slr (short-long ratio) were calculated as an indication apa. significance analysis of microarrays (sam) determined significant genes. the gse numbers of the datasets are gse2034, gse7390 and gse9761. we analyzed two datasets of er(+) breast cancer patient samples (n = 209, n = 135) compared to normal breast tissue (n = 82) using apadetect and sam. a total of 184 3 0 utr shortening and 253 3 0 utr lengthening events were detected in breast cancer samples compared to normal breast tissue. ontology analysis suggested almost all the 3 0 utr shortening genes were proliferation related and were indeed reported to be upregulated in breast cancer. to further investigate the connection between apa and era status, we used data from a cell line model; wild type or era transfected mda-mb-231 cells that are otherwise of triple negative nature. our results suggested that most of the genes are 3 0 utr shortened or lengthened via direct binding of era to dna. our results suggest involvement of apa mechanisms in era action mechanisms. possible link between era regulated transcription and apa remains to be elucidated. contamination of nucleic acids (na) as a result of na extraction protocols may result an inaccurate measurement of dna copy number. agarose gel electrophoresis and spectrophotometric methods are commonly used to check dna purity. however, the resolution of these methods may not be good enough for special applications such as determination of dna copy number and separation of base pairs (bp) that are close in their bp number. in this study, we have developed a new method for separating na's ranging between 75-20000 bp also detecting the impurities in dna solution in 1%, 5% and 10% ratios to the dna of interest. the developed method was validated using the in-house dna fragments of 100, 150 and 200 bp. the dna mixture analyzed using analytical hitachi elite lachrom hplc using the guard and analytical columns tskgel dna-npr, 2.5 lm, 4.6 mm id 9 0.5 cm and tskgel dna-npr, 2.5 lm, 4.6 mm id 9 7.5 cm, respectively. the validation of the analysis was performed by running each sample five times on three different days. the linearity of the detector response was established by plotting a graph to quantity versus area of 200 bp dna. the lod and loq were then measured by calculating the minimum level at which analyte can be readily detected and quantified. the ratios calculated with hplc were compared to the ratios calculated by quant-it kit. recovery values were calculated for each measurement and the uncertainty were calculated for each ratio. the method was found linear for 200 bp in the range of 0.4 ng to 800 ng dna with the regression coefficient of r 2 =0. 9992. lod and loq for the 200 bp dna was found to be 0.39 ng and 1.56 ng, respectively. the recovery values for the 1%, 5% and 10% impurity ratios were found 101.74. 97.41 and 99.47, respectively. the purity of the synthetic dna was determined by hplc and related uncertainty was calculated. the developed method is a simple alternative to electrophoresis and spectrophotometric methods with higher resolution and separation range. physical and chemical factors can disturb the conformation of proteins maturing within the cellular secretory pathway. in response to unfolded proteins the cell activates several stress signaling and adaptive response mechanisms. the aim of our study was to investigate small non-coding rnas as the potential regulators of cellular response to unfolded proteins (upr). for this, we conduct the next generation sequencing of small rna and transcriptome analysis of mrna from jurkat cells exposed to dithiothreitol (dtt), which reduces protein disulfide bounds. analysis of mirnas reveals the differential expression of 104 mirnas. we observe a decrease in the normalized amount of reads aligned to mirna loci in stressed cells. affymetrix analysis with subsequent gsea reveals downregulation of reactome mirna biogenesis pathway (fdr = 0.096). the length distribution of small rnas revealed 32 nt-peak corresponding to trna-derived fragments, amount of which was increased by 2.6-fold under dtt treatment. the trna isotypes that gave rise to almost 76% and 86% of all 32nt rna fragments in stressed and control cells, respectively, include glycine, glutamic acid, aspartic acid and valine. the vast majority of 32nt fragments produced from these trnas are precisely phased 5 0 halves with the characteristic cleavage patterns generated by rnase a angiogenin (ang). observed upregulation of tirna in stressed cells is accompanied with upregulation of ang mrna and down-regulation of angiogenin inhibitor 1 (rnh1). we speculate that translational repression, associated with observed tirna, is an additional mechanism of reducing global protein synthesis in response to dtt-induced stress. collectively, our findings reveal the increase in tirna, the differential regulation of mirna expression together with the global mirna downregulation as the most prominent small rnome reprogramming events and possible fine-tuned levels of post-transcriptional regulation upon dtt-induced cellular stress response. global gene expression changes after spinal cord injury j. k. hyun 1,2,3 , j. kim 1,2 , j. y. hong 1 1 dankook university, cheonan, south korea, 2 institute of tissue regeneration engineering (itren), cheonan, south korea, 3 the neuronal regeneration is hardly achieved spontaneously after spinal cord injury (sci), and the restoration of somatic and autonomic functions after sci is also challenging in the clinical field. the pathophysiology of sci is extremely complex and many in vitro and in vivo studies continued to report opposite results each other in spite of the same treatments, therefore a fundamental analysis such as an extensive assay of global gene expression is required to find a way for spinal cord regeneration. in this study, we aimed to detect the changes of global gene expression after spinal cord contusion in rats according to the time sequence. the spinal cord tissues at contusion site were sequenced after spinal cord contusion in rats using rna-sequencing technology. for time sequence analysis, five time points was determined; 1 hour, 1 day, 1 week, 1 month and 3 months after spinal cord contusion, and sham operated rats at each time point were used as controls. quantitative rt-pcr analysis was also performed to validate expression changes of candidate genes in each category. we found that the pattern of changes in gene expression at acute and subacute stages was quite different from that at chronic stage, especially genes associated with with neurotrophin signaling and apoptosis pathways. most of gene expression levels of inflammatory cell markers were increased and peak during acute stage (1 hour to 1 week) and maintained until chronic stage. some of regeneration-associated genes (rags) including brain derived neurotrophic factor, glial cell derived neurotrophic factor and ciliary neurotrophic factor were increased at 1 hour or 1 day after sci. we concluded that the information of gene expression level according to the time sequence after sci might be useful to determine treatment strategies for spinal cord regeneration especially in chronic stage. p-01.02.2-011 3 0 utr length isoform generation profile in a differentiation model alternative polyadenylation (apa) is the regulated selection of a specific poly(a) signal among other proximal and/or distal signals on the 3 0 utrs (untranslated region) for the endolytic cleavage and addition of a poly(a) tail to form the mature mrna. consequently, position of the poly(a) site determines the length of the 3 0 utr which is known to harbor microrna and rna binding protein sites. such apa isoforms have already been linked to altered protein levels and even functions. therefore we hypothesized apa to be one of the mechanisms to generate isoform diversity in proliferating and differentiated cells to better understand the molecular basis of cancer. we used a combinatorial in silico and in vitro approach to analyze a well known enterocyte differentiation model; caco-2 cells. initially we analyzed gene expression datasets for the proliferative and differentiated caco-2 cells using a probe based apa detection tool. to better understand the significance and to validate these results, we used proliferating and differentiated (day10) caco-2 cells and tested sample apa events by rt-qpcr. 3 0 utr isoforms were identified by using 3 0 race pcr. we identified 43 genes (32% of all apa events) to undergo 3 0 utr shortening in differentiated cells compared to proliferating cells. on the contrary 91 genes (68% of all apa events) went through 3 0 utr lengthening events. several genes have been validated to follow the pattern that was seen in apa detection tool so far. to begin understanding the mechanism behind these observations, we are investigating potential inducers of apa during the complex events of differentiation. our next aim will be to further validate and investigate the consequence of such isoform generation events both in the context of differentiation in colon cancer cells. recognition of phosphorylated threonine-4 of rna polymerase ii c-terminal domain by 3 0end processing apparatus o. jasnovidova, m. krejcikova, k. kubicek, r. stefl central european institute of technology, masaryk university, brno, czech republic rna polymerase ii has evolved an array of heptad repeats with the consensus sequence y1-s2-p3-t4-s5-p6-s7 at the c-terminal domain (ctd) of its largest subunit, rpb1. phosphorylation of serines (s2, s5, and s7) and tyrosine-1 orchestrate the binding of rna processing and transcription factors in the site of transcription. several recent studies showed that also threonine-4 site can be phosphorylated which has a number of functional consequences. to reveal the structural basis for the recognition of threonine-4 phosphorylated ctd, we set out to investigate several proteins factors that were implicated with a high levels of threonine-4 ctd phosphomarks using integrative structural biology. one of them, a factor involved in the 3 0 -end processing and transcription termination, showed a high affinity to the phosphothreonine ctd. using nuclear magnetic resonance spectroscopy (nmr), we determined its structure bound to the ctd phosphorylated at threonine-4 that reveals a direct read-out of the phosphothreonine. altogether, our data provides the first insights into the recognition of this poorly understood ctd mark that plays important role in the ctd code of rna polymerase ii. the results of this research have been acquired within ceitec 2020 (lq1601) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. introduction: the treatment of brain tumor glioblastoma (gbm) is still one of the greatest challenge. anti-inflammatory drug indomethacin (ind) mainly acting through the inhibition of cyclooxygenase (cox) has also anti-cancer activity including brain tumors. the aim was to investigate how ind effects an immortality enzyme telomerases' activity. materials and methods: monolayer and spheroid cultures of t98g human gbm cell line were used to evaluate the effects of ind (100 lm) on cell proliferation, viability, apoptosis, cell cycle, camp levels, the levels of apoptotic and anti-apoptotic proteins, morphology (sem) and ultrastructure (tem) for 72 hours. results were analyzed using the student's t-test. results: ind decreased cell proliferation (p72 < 0.01), cell viability (p72 < 0.000001), cell rate at s phase (p72 < 0.000001) and g2 + m phase (p72 < 0.001), camp levels (p72 < 0.01), the levels of pdgfr-a (p72 < 0.001), mrp-1 (p72 < 0.05), nf-jb (p72 < 0.01) and cox-2 (p72 < 0.01) in comparison to control group. ind mildly increased apoptosis (p72 < 0.001) and caspase-3 levels (p72 < 0.01). interestingly, ind increased htert levels (142%, p24 < 0.001; 154%, p72 < 0.0001). sem evaluation showed that ind led to decreased and shortened microvilli, the lost of cell interactions and the conversion of many cell shapes from spindle to oval. many cell remnants in the intercellular area, intact cell membranes, many dense lipid droplets and few autophagic vacuols in the cytoplasm were observed under tem. discussion and conclusions: the effect of ind on telomerase activity can only be found in 8 publications at pubmed research that they only showed its' inhibitory effect in colon, gastric, head and neck cancers. in contrast to previous studies, it was shown for the first time that ind increased telomerase activity in gbm cells and this increase was independent from cox-2 and other tested factors. p-02.02.2-002 interaction between fibrinogen and insulin-like growth factor binding protein-1 under physiologic conditions and influence of diabetes mellitus type 2 on this interaction n. gligorijevic, o. nedic institute for the application of nuclear energy, university of belgrade, belgrade, serbia fibrinogen is plasma glycoprotein and principle participant in blood coagulation. it interacts with many proteins, including insulin-like growth factor binding proteins (igfbps). one of them, igfbp-1, is controlled by insulin. metabolic changes due to diabetes mellitus (dm) affect igfbp-1. besides glucose regulation, igfbp-1 stimulates wound healing. we have investigated complexes formed between fibrinogen and igfbp-1, their change in dm type 2 (dm2) patients and involvement in fibrin clot. samples from adult healthy persons and dm2 patients were studied: plasma, isolated fibrinogen and fibrin. the amount of igfbp-1/fibrinogen complexes was determined using immunoblotting. immunoprecipitation and lectin affinity chromatography were used to confirm interaction between fibrinogen and igfbp-1. in vitro incubation of fibrinogen with excess glucose or methylgyoxal (mgo) was employed to demonstrate influence of glyco-oxidation on complexes. results have shown that igfbp-1/fibrinogen complexes can be differentiated from igfbp-1 oligomers and igfbp-1/alpha-2macroglobulin complexes. the amount of igfbp-1/fibrinogen complexes was lower in patients with dm2. complexes participated in fibrin clot formation, the amount being significantly lower in patients' samples. the quantity of igfbp-1 monomer in fibrin clot was greater in patients' samples. in vitro experiments revealed that complexes undergo glyco-oxidative modifications leading to their reduced formation, cross-linking and increased acidity (faster electrophoretic movement). isolated fibrinogen from patients with dm2 was additionally able to bind exogenous igfbp-1. since igfbp-1 stimulates wound healing, directly and by delivering igfs, igfbp-1/fibrinogen complexes may be seen as igfbp-1 storage instrument, ready to participate in fibrin formation and to assist in damage repair. reduction of complexes due to glyco-oxidative stress in patients with dm may be part of the mechanism responsible for impaired coagulation process. human interferon gamma (hifnc) is a proinflammatory cytokine involved in the regulation of nearly all phases of immune and inflammatory responses. its abnormal expression is associated with the aetiology of many inflammatory and autoimmune diseases. recently we have been exploring the idea to counteract the over-expression of the endogenous hifnc by competitive inhibition with inactive hifnc mutants. they are designed to have preserved affinity to the hifnc receptor, but to be deprived in their capability to trigger the intracellular signal transduction. to this end a library of mutants was created and two potential hifnc antagonists were selected for further investigations: a single point mutant k88q (q substitution for k in position 88) and a double mutant with additional substitution in the n-terminus. both mutants and the wild type hifnc were expressed in e. coli employing the established by us methodology for large scale production of aggregation-prone proteins in soluble native form. the purified mutants were screened for interferon activity (antiproliferative assay), binding affinity (isothermal titration calorimetry) and ability to compete with the wild type for the hifnc receptor (competition assay on wish cells). the selected mutants demonstrated 100 (single mutant) and 1000 (double mutant) times lower antiproliferative activity than the wild type. measuring the binding thermodynamic parameters, we proved that the receptor binding affinity of both mutants was preserved, which is an indication for their potential to compete with the wild type hifnc for its receptor. finally, the biological assay performed on wish cells showed a distinct dose-dependent competition between the wild type hifnc and the mutants. based on the results presented in this study we conclude that the two hifnc mutants are potential candidates for autoimmune therapy based on selective suppression of the endogenous hifnc activity. mesencephalic astrocyte-derived neurotrophic factor (manf) is an er (endoplasmic reticulum) stress-inducible protein and widely expressed in mammalian tissues. it has been identified as a secretory protein that protects cells against er stress-induced damage. er-stress is one of the main mechanisms that play a role in ischemia/reperfusion (i/r)-induced renal injury. recent studies demonstrated that manf can protect cardiac myocytes and cortical neurons against i/r-induced injury. moreover, it has been suggested that it has a restorative effect in ischemic injury. nevertheless, the function of manf in i/r-induced renal injury is still not known. in the present study, we investigated the function of manf by manipulation its expression level in ischemic acute renal failure model established in proximal tubular kidney cells (hk-2 cells). for this purpose, the cells were transfected with either manf sirna or manf encoding plasmids for silencing or over-expression of manf, respectively. then, the cells were exposed to hypoxia-reperfusion (h/r) induction for indicated times. evaluations of cell viability were determined with wst-1 reagent. the changes in protein levels of h/r-induced stress markers were analyzed byimmunoblotting. the results showed that the overexpression of manf has provided a significant resistance to h/r-induced cell death, whereas silencing of manf has rendered the cells more susceptible to death. it was also determined that the pretreatment of cells with manf conditioned medium caused a decrease in cell death. additionally, oxidative/nitrosative stress (os/ns) and er stress levels were decreased with over-expression of manf and increased by silencing of manf in hk-2 cells. taken together, our study suggests that manf may have a protective role against h/r-induced renal cell injury, possibly through the reducing effects on os/ns and er stress. p-02.02.2-005 his-flag tag as a fusion partner in insect expression systemgain or loss? e. krachmarova 1 , m. tileva 1 , k. maskos 2 , i. ivanov 1 , g. nacheva 1 1 institute of molecular biology "roumen tsanev", sofia, bulgaria, 2 proteros biostructures, martinsried, germany human interferon gamma (hifnc) is a glycoprotein playing major role in the regulation of innate and adaptive immunity. glycosylation is not essential for hifnc activity but is important for its stability, half-life and protease resistance in blood. the commonly used hifnc in therapy and research is produced in e. coli and therefore is not glycosylated. bearing in mind the above mentioned shortcomings of the non-glycosylated hifnc we expressed it in mammalian cells and transgenic mice, however very low yields were achieved. to obtain glycosylated hifnc, here we employed a secretory expression of n-terminal his-flag fusion protein in baculovirus-infected insect high five ò cells. this small hydrophilic tag is designed to not affect the proper folding of the target protein and to facilitate the detection and purification procedures. in parallel the same fusion was expressed in e. coli cells. the fusion proteins were purified to high degree of purity by affinity and size-exclusion chromatography. bioassay carried out on wish cells showed that the antiproliferative activity of both fusion proteins was 500 times lower than that of the native hifnc. this result shows that, in contrast to the generally hold view, the n-terminal his-flag tag interferes with the biological activity of hifnc despite of the protein glycosylation. in order to restore the biological activity we attempted to remove the his-flag tag enzymatically. surprisingly, we found that the fusion protein obtained from insect cells was resistant to enterokinase, independently of the enzyme source and experimental conditions, whereas the protein isolated from e. coli was susceptible and the tag-free protein showed fully restored biological activity. we are prone to explain the enterokinase resistance of the fusion protein from insect cells with either the specific conformation of the glycosylated protein or with the interaction of the carbohydrate residues with the enzymatic activity of the enterokinase. p-02.02.2-006 development of fluorescence assay for highthroughput screening system based on flow cytometry for directed evolution of cellobiose dehydrogenase cellobiose dehydrogenase (cdh) is an enzyme produced by phanerochaete chrysosporium and it has been already successfully cloned in other organisms. one of the most important roles of cdh is removing products of cellulose degradation. cdh is very important for biofuel and biosensor industry. for improvements of enzyme properties we have used directed evolution. the most important step is to develop screening system that reflects properties of interest. screening in microtiter plates (mtp) is expensive, time-consuming and has low throughput with a small number of variants detected (10 3 -10 4 in months). the aim of this work was the development of screening system for mutant libraries of cdh expressed on surface of yeast cells based on fluorescent enzymatic assay and flow cytometry. the screening method should be capable of screening cellobiose dehydrogenase variants mutated for higher activity and higher thermostability by error prone pcr. the fluorescent assay was beta-galactosidase (ec 3.2.1.23) also known as lactase is the enzyme that typically catalyzes hydrolysis of beta-1,4-d-galactosidic linkages in beta-d-galactosides, including disaccharide lactose, with glucose and galactose as end reaction products. this enzyme is able to catalyze synthesis of oligosaccharides, in particular galactooligosaccharides via galactosyl transfer reaction. arthrobacter sulfonivorans beta-galactosidase of unique for prokaryotes extracellular localization may find application in food industry for manufacturing lactose-free dairy products and in pharmacology as bioactive principle of medicines prescribed for patients suffering from lactase deficiency. the study was aimed at cloning of the gene encoding a. sulfonivorans beta-galactosidase, purification and characterization of the enzyme. a novel extracellular beta-galactosidase from a. sulfonivorans was recovered with an overall 207-fold purification, a 7.7% yield and specific activity 16 300 uámg à1 protein. the subunit molecular mass of the enzyme determined by sds-page analysis equalled 125 kda. it was found that the enzyme displays pi 5.35, prefers ortho-nitrophenyl-beta-galactoside as substrate (km 27 mm) and shows maximum activity at 40°c and at ph 7.5-9.5. the beta-galactosidase gene was isolated from the genomic dna library of a. sulfonivorans, sequenced, cloned and deposited in the genbank database under accession number km277894.1. it was established that the gene carries an open reading frame consisting of 3132 bp (1043 amino acids) and encodes beta-galactosidase referred to glycosyl hydrolase family 2 (cazy database). p-02.02.2-009 different splice-forms of tdrd7 protein mutated in cataract's and glaucoma's interacts with s6k1/2 o. skorokhod, v. filonenko department of cellular signalling, institute of molecular biology and genetics nas of ukraine, kyiv, ukraine ribosomal s6 kinases (s6k) are important players in cellular pi3k/mtor signalling network, deregulation of which has been associated with methabolic disorders, inflammation and cancer. previously we had identified a novel binding partner of s6k1 -tdrd7 (trap). tdrd7 is a scaffold protein detected in complexes involved in the regulation of cytoskeleton dynamics, mrna transport, protein translation, non-coding pirnas processing, transposons silensing. it was reported recently that mutations in human tdrd7 result in cataract and glaucoma formation, defined by elevated intraocular pressure (iop) and optic nerve damage. the aim of our study was to confirm s6k-tdrd7 interaplay and study its role in cells. bioinformatical analysis of tdrd7 sequence revealed the presence of potential phosphorylation sites of s6k2. using in vitro kinase assay, we have demonstrated that recombinant s6k2 phosphorylate 3 from 5 fragments of tdrd7. formation of s6k2-tdrd7 complexes in vivo was further confirmed by coimmunoprecipitation using anti-s6k2 and anti-tdrd7 antibodies generated previously in rat brain lysates. this interaction was further confirmed by confocal microscopy, oleksandr had shown that tdrd7 co-localize with s6k2 in hepg2 cells, predominantly in perinuclear region, enreached for one of the tdrd7 isoforms identified previously. moreover, we have detected that c-terminal synthetic peptides of s6k2 with methylated arg interfere with tdrd7 from hepg2 lysates. the physiological characteristics of s6k2-tdrd7 interaction and the role of this complex formation in neuropathology's development need further investigation. many biological function of placenta are performed not just a set of individual proteins, but also different oligomeric structures and complexes. herewith, activities of complexes may considerably differ from activities of individual proteins. therefore, identification and characterization of placental multi-protein complexes is an important step to understanding the placenta function. the aim of the present work was to investigate a composition and biological functions of the very stable high molecular mass multi-protein complexes (spc) from placenta of healthy mother. we isolated spcs (~1000 kda) from the soluble fraction of three human placentas. light scattering measurements and gel filtration showed that the spc is stable in presence salts, acetonitrile and triton x-100 in high concentrations, but efficiently dissociates in the presence of 8 m urea and 50 mm edta. such a stable complex is unlikely to be a random associate of different proteins. it was shown the spc includes a number of proteins with molecular weights of 2 to 180 kda. several protein components of the spc were identified, including serum albumin, transferrin, iggs, annexin a5 and other proteins. serum albumin, transferrin and protein with molecular weight 14,1 kda are the main proteins of the complex. it was shown high the spcs from three placentas possesses dnsase and catalase activities. an addition, investigation of cytotoxic effect on human cancerous cell lines has shown that the spcs reveal high cytotoxicity. antibody-cytochrome b5 fusion protein, characterization and applications for antibody development process antibodies have recently become an essential tool being a part of immunodiagnostics, therapeutics and as a valuable instrument in life science research. an enormous number of options utilizing a various tags were used to create a universal antigen-binding domain, which can be easily detectable, highly soluble and might be produced in high yields with low costs, but no multipurpose solution exists yet. we addressed the question whether a single tag could be found for enhancing solubility of recombinant fab antibody fragment and providing its detection and accurate quantification by rather simple method. a new application for hemeproteincytochrome b 5 as the antibodies fusion partner were proposed. we have constructed of recombinant fab antibody fragment cytochrome b 5 fusion protein. we have shown that cytochrome b 5 enhance expression of fab antibodies fragments in bacterial system, and could be a versatile tool for recombinant proteins folding, redox (oxidation) state studies and for their precise concentration determination in the turbid solutions. fusion fab-b5 protein has a stable red color and characteristic absorbance spectrum with the maximum absorbance at 413 nm in oxidized environment. cytochrome b 5 change its spectrum maximum depending on environmental redox potential and its folded state, so one can track these events in real time spectrophotometrically. binding activities of fab-b 5 fusion protein and hybridoma secreted immunoglobulin were measured by biolayer interferometry and elisa. no significant difference between them was revealed. due to this feature we can distinguish the chimeric protein of interest in complex mixtures and control the process of recombinant proteins expression and purification in real-time. besides, cytochrome b 5 fusion tags multiples recombinant antibody yield (from 2 to 3 times) and doesn't affect antigen-binding properties. the bb125-135 site of fibrin molecule is the site of fibrin protofibrils lateral association l. urvant palladin institute of biochemistry nas of ukraine, kyiv, ukraine previously we showed that fibrin-specific monoclonal antibody i-3c (monab i-3c) inhibited the fibrin protofibrils lateral association. we suggested that the epitope of monab i-3c in bb118-134 of coiled-coil region of fibrin molecule coincides with the site involved in fibrin protofibrils lateral association. the aim of this study was to localize the site of protofibrils lateral association in fibrin molecule using the synthetic peptides bb121-138, bb125-135 and both their scrambled version, and bb109-126 peptide. monab i-3c was isolated from hybridoma culture medium by affinity chromatography on fibrin-sepharose. turbidity analysis was used to study the effect of synthetic peptides on fibrin polymerization. the interaction between peptides and monab i-3c was investigated by spr method using plasmon-6 device. we investigated the effect of synthetic peptides which corresponded to amino acide sequences of fibrin molecule bb109-126, bb121-138, bb125-135, and the scrambled versions of bb121-138 and bb125-135 peptides on a binding to monab i-3c and on the fibrin polymerization process. in spr analysis was showed that bb121-138 and bb125-135 peptides, but not their scrambled version, binds to monab i-3c, immobilized to a chip. turbidity data showed that only bb121-138 and bb125-135 peptides caused the 2-fold decrease of the rate of the lateral association of protofibryls at the concentration 2.2 9 10 à4 m and 2.7 9 10 à4 m, respectively. both of them decreased the final clot turbidity. our data let us to suggest that the bb125-135 site is the site that involved in protofibryls lateral association. it has been recently shown that irisin immunoreactivity was altered in gastrointestinal cancers. as known hematological malignancies was one of the most common malignancies through world, but no study was present how irisin was changed in this type of cancers. therefore, purpose of this was to investigate how immunoreactivity to irisin was altered in hematological malignancies (blood cancers). we used an antibody from phoenix to demonstrate how a 12 kda band after deglycosylation of irisin altered in hematological malignancies. here we first time showed that irisin tissue immunoreactivity from acute lymphoblastic leukemia (all) and acute myelogenous leukemia (aml) patients was increased when compared with unaffected biological tissue parts. from the immune-histochemical (ihc) investigations it is concluded that hematological tissue and blood cells may be another source of irisin and increased with cancer, thus this finding might help to enlighten pathophysiology of hematological malignancies. the value of urine neutrophil gelatinaseassociated lipocalin (ngal) in acute heart failure n. serdarevic clinical centre, sarajevo, bosnia and herzegovina introduction: renal dysfunction is very common in heart failure (hf) and neutrophil gelatinase-associated lipocalin (ngal) is used as an early marker of acute renal tubular injury. recent studies have been reporting that ngal is inhibitor of inactivation of matrix metalloproteinases (mmp-9) which results in enhanced proteolytic activity with prolonged effects on collagen degradation. due to its relation to extracellular matrix degradation in myocardium and infammation, we hypothesized possible increased ngal expression in hf besides it renal dysfunction etiology. patients and methods: in study were included 30 patients hospitalised with signs and symtoms of ahf. urine samples for ngal analysis were collected at admission and analysed by the chemiluminescent microparticle immunoassay (cmia) for the quantitative determination of neutrophil gelatinase-associated lipocalin in human urine (abbott, architect analyzer). refferent range for urine ngal is 0-135 ng/ml. on admission blood samples for bnp (brain natriuretic peptide) analysis were drawn and tested by architect bnp chemiluminescent microparticle immunoassay (cmia), abbott laboratory. results: the mean age of the patients (male= 30, female= 30) was 68.86 years (sd 10.92 years). among them 18 (30%) patients was diagnosed as a hf-pef (hf with preserved ejection fraction) while 42 (70%) as a hf-ref (hf with reduced ejection fraction). mean bnp values was 1616.71 pg/ml (sd 1511.15 pg/ml) and mean lvef was 33.66% (sd 14.19%). mean urine ngal was 60.91 ng/ml (sd 78.72 ng/ml). we found significantly positive, but weak correlation among ngal and bnp only by pearson correlation test (r = 0.139, p = 0.464, wilcoxon signed rank test z = à4.782 p < 0.5). conclusion: bnp levels are elevated in hf with reduced and preserved ejection fraction. urine ngal is not elevated in acute heart failure, but it is slightly positively correlated with serum bnp values. converging evidence implicates the intermediate and medial mesopallium (imm) of the domestic chick forebrain in memory for a visual imprinting stimulus. a number of learning-related changes have been found in plasma membrane and mitochondrial proteins of imm. for broader analysis of these changes we employed two-dimensional gel electrophoresis/mass spectrometry approach and identified differentially expressed proteins in membrane-mitochondrial fraction of the imm across chicks with different estimated levels of imprinting 24 h after training. we further inquired whether the amounts of those proteins in the imm and a control region (posterior pole of the nidopallium, ppn) are correlated with memory for the imprinting stimulus. learning-related increase in the amounts of the following proteins was demonstrated in the left imm, but not in the right imm or left and right ppn: (i) membrane cognin;(ii) a protein resembling the p32 subunit of splicing factor sf2;(iii) voltage dependent anionic channel-1;(iv) dynamin-1; (v) heterogeneous nuclear ribonucleoprotein a2/b1. obtained results indicate that the molecular processes involved in learning and memory of imprinting cover a wide range of cellular activities, including stabilization of protein structures, increased mrna trafficking, synaptic vesicle recycling and specific changes in the mitochondrial proteome. the aim of this work is to study the substrate and inhibitory properties of uridine derivatives in the reactions catalyzed by e.coli up in order to shed some light on the substrate's conformation in the productive complex with the enzyme. we studied the e.coli up-catalyzed phosphorolysis of uridine and its derivatives modified in the heterocyclic base and the sugar moiety. the kinetic constants (km, ki, kcat ) of the phosphorolysis reaction of near 30 uridine derivatives were determined. the combined kinetic (nnna, 35, 2016) and structural data (acta crystallogr., d70, 3310, 2014) provide clear evidence that up binds uridine in the most energetically unfavorable conformation, which, to the best of our knowledge, has no precedents in the enzymes of nucleic acid metabolism. this is possible due to multiple interactions between the substrate and the protein environment (active site residues) mainly through hydrogen bonds. these results are important for understanding the mechanism of action of this class of enzymes. an analysis of the conformations of nucleosides in solution and rotational barriers suggests that the energy difference between the ground state of uridine and uridine complexed with up may be high as 63-69 kj/mol. the binding in a high-energy conformation results in the weakening of the glycosidic bond. the observed conformation of uridine complexed with sulfate (mimetic of phosphate) may be very similar to its conformation in the transient state. until now, foxp1 (forkhead box p1) has been identified as a tumor suppressor in several correlation studies in breast cancer. although, foxp1 is defined as a transcriptional repressor that interacts with other transcription factors in various mechanistic studies, there is no study that explains its repressor functions in breast cancer biology. here we demonstrate the repressor function of foxp1 on nfat (nuclear factor of activated t cells) and the migratory effect of this repression in mda mb 231 breast cancer cells. we performed co-immunoprecipitation experiments for the investigation of protein-protein interaction between two transcription factors. protein-protein interaction on dna was investigated with emsa and transcriptional effects of foxp1 on nfat, lusiferase reporter assay was performed. wound healing assay was used to analyse the effects of overexpression of foxp1 on tumor cell migration. our results showed that foxp1 has protein-protein interaction with nfat on dna and enhances breast cancer cell migration by repressing nfat transcriptional activity and foxp1 shows oncogenic function by regulating breast cancer cell motility. introduction: phosphodiesterase 5 (pde5) is one of phosphodiesterase lead to hydrolyzing cgmp.the cgmp signaling pathway has an important role in proliferation of cells. previous studies showed pde5 was increased in cell lines cancers thus pde5 inhibitors can used as efficacious therapeutic option for treatment of cancers. the current study was to investigation the effect of hydroalcoholic achillea.wilhelmsii extract (hawe) on the pde5 gene expression and cgmp signaling in the mcf-7 er + and mda-mb-468 er à . methods and materials: the ed50 of the hawe on both cell lines were examined by using mtt viability test then the expression of pde5 and cgmp concentration were measured in timedependent manner (in the ed50) by real-time rt-pcr and colorimetric assay respectively. results: treatment with the hawe showed, 25 lg/ml is ed50 for both cell lines and the hawe lead to reduction in pde5 mrna expression and evaluation of intracellular cgmp showed an increase pattern in the time-dependent manner. conclusion: our results showed that the hawe has anti-proliferative property in the mcf-7 and mda-mb-468, cell lines of breast cancer through the cgmp pathway, these data suggested that the hawe can be potential source for the isolation of effective anti-proliferative molecules. keywords: achillea.wilhelmsii, breast cancer, anti-proliferative, phosphodiesterase, cgmp signaling pathway. outer membrane protein g (ompg) is a stable monomeric porin having 14-stranded beta barrel form from e.coli. its exact function is not fully understood; however, it allows the passage of molecules up to 900 da in neutral ph but the pore is closed by going through a conformational change under ph 5.5. as being monomeric and having ph-dependent gating characters, it is suitable for biosensor and targeted drug delivery applications. an attempt on ompg is to create a larger pore while its stability is undisturbed. ompg-16s is obtained by adding 38 amino acids to the primary chain in order to have a 16-stranded beta barrel porin. ompg-16sl is formed by further adding 6 amino acids to loop l6 and by replacing 7 lysines with arginines. ompg-16s and ompg-16sl mutants are investigated by fourier transform infrared spectroscopy (ftir) and compared with ompg-wild type (wt) in terms of ph-dependent conformational changes and thermal stability. each mutant is prepared in na-phosphate buffer pd 5.5/7.5 and infrared spectra are recorded. further, temperature profiling are recorded for the range between 22 to 106°c. results show that both mutants are responsive to ph changes. while turning the ph from acidic to neutral, beta sheet signals shift to lower wavenumbers showing difference in secondary structure, implying the existence of closed and open states. on the other hand, mutant proteins show structural differences compared with the wt protein. porins are known for their remarkable thermal stability. the mutans retain this character by having transition temperature of $ 75°c, although this is less than the wt transition at $ 85°c. in conclusion, two mutants show signs of open and closed states as ompg-wt and even if the mutants are less stable than ompg-wt. this study shows that the attempted alterations in ompg structure are successful in terms of ph-response but it needs improvement in terms of stability when necessary. nad is a key factor in the regulation of mitochondrial metabolism. besides its vital role as redox carrier, nad serves as substrate for protein adp-ribosylation and deacetylation, modifications which modulate enzyme activities in mitochondria. these functions depend on how nad levels are maintained in this organelle. in human cells, mitochondrial nad is segregated from the cytosolic pool and can be synthesized from nmn, which is probably imported into the matrix. here, we tested whether the nudix pyrophosphatase nudt13 participates in the regulation of the mitochondrial nad pool. this enzyme has a predicted nadh pyrophosphatase zinc ribbon domain and a mitochondrial targeting sequence at its n-terminus. however, it has not yet been functionally characterized. we overexpressed nudt13 endowed with a c-terminal flag-epitope in human cells. to evaluate changes in the mitochondrial nad concentration, we used a reporter system which includes the overexpression of the catalytic domain of poly(adpribose) polymerase 1 (parp1) within the organelles (mitoparp). thereby mitochondrial nad is converted into protein-bound poly(adp-ribose) (par). the extent of par formation correlates with the mitochondrial nad availability and is detected by western blotting. our results established that nudt13 is indeed a mitochondrial protein, as it was localized exclusively to these organelles. moreover, when nudt13 was overexpressed along with the mitoparp detector system, a dramatic decrease of par was observed. the obtained results indicate that nudt13 is enzymatically active upon overexpression in the mitochondrial matrix and that it might cleave nad, thereby modulating its organellar level. however, at this point we cannot exclude the possibility of direct par cleavage by nudt13. further characterization of nudt13 will define its substrate specificity and clarify its role in mitochondrial metabolism. the incidence of increase in colorectal cancer (crc) worldwide has become a major health problem. early diagnosis and treatment of crcs are of importance for improving survival. in the present study, it was aimed to investigate chemopreventive effect of rosmarinic acid and evaluate the angiogenesis process in azoxymethane (aom)-induced crc model. male sprague-dawley rats were randomly divided into a control group, aom-induced rat colorectal cancer group (15 mg/kg body weight aom; ip, weekly for four weeks), and rosmarinic acid (5 mg/kg body weight; oral, daily for four weeks)-treated group. in addition to the standart diet of the all groups 15.8% peanut oil was added throughout the experiment. the all rats were sacrificed at the end of 30 weeks. biochemical examinations were performed in rat plasma. histopathological adenocarcinoma rates were observed in 87.5% of aom group. the incidence of adenocarcinoma was showed a reduction in the treatment group. significant increases in plasma tos and mcp-1 levels were found in the aom group compared to controls. these increases were reduced in the treatment groups but no significant. a significant increase was detected in tas levels in the treatment group when compared to the aom group. significant decreases in plasma adiponectin levels were found in the aom and the treatment groups compared to controls. in conclusion, treatment with rosmarinic acid reduced the occurrence of inflammation and was helped to maintain the oxidant-antioxidant balance in the model of aom-induced rat colon cancer. mitochondrial genome, while being strongly reduced in course of evolution, still codes for several proteins. the vast majority of them are components of the respiratory chain complexes. to produce these proteins, the system of mitochondrial translation is presented in the organelles, which is in common close to that in bacteria. translation initiation in bacterial cells is orchestrated by three protein factors called if1, if2 and if3. the orthologs of the two latter proteins are commonly found in mitochondria. however, mitochondrial if3 could not been identified in several groups of organisms, including s.cerevisiae, for a long time. recently we have shown that baker's yeast protein aim23p possesses a function of mtif3. however, the mitochondrial translation has not been stopped in the yeast strain without aim23p which is surprising taking into account the fact that if3 is obligatory for the translation in bacterial systems. instead of blocking of mitochondrial protein synthesis in absence of aim23p, we observed the translational imbalance: the synthesis rate of the complex v subunits was increased while the synthesis rate of the complex iv subunits was repressed. thus, in addition to its general role in translation initiation, aim23p might specifically affect the biosynthesis of individual mitochondrial-encoded protein species. our genetic experiments have revealed that, indeed, aim23p is almost indispensable for cox1p synthesis, and that it affects the translation of cox1 mrna through its 5 0 -utr, like classical mitochondrial translational activators. this is in accordance with our measurements of complex iv activity which is several times less in yeast lacking aim23 gene than in the wild-type. taken together, our results point on the multiple role of aim23p in mitochondrial translation: in addition to its function as mitochondrial if3, it specifically regulates the amount of complex iv subunits and its activity. p-02.02.2-024 the circulating betatrophin and irisin levels in polycystic ovary syndrome patients with and without insulin resistance introduction: polycystic ovary syndrome (pcos) is the most common endocrine/metabolic disease in women around the world, characterized by oligo-or anovulation, polycystic ovary, and/or hyper-androgenism. insulin resistance (ir) and obesity are common findings in patients with pcos. irisin is a recently identified myokine secreted from skeletal muscle in response to physical activity. irisin has been postulated to induce the differentiation of white fat tissue into brown fat tissue. betatrophin is a currently discovered new hormone proposed to stimulate b-cell proliferation. in this study we investigated the levels of irisin and betatrophin in pcos patients. materials and methods: our study group was consisted of 40 patients with pcos and 20 healthy volunteers. patients group was divided into two subgroups according to presence of ir. (pcos+ir and pcos-ir). the oral glucose tolerance test (ogtt) and the homeostatic model assessment (homa-ir) were performed to assess glucose tolerance and insulin sensitivity. irisin and betatrophin levels were measured by elisa method. results: circulating irisin was significantly higher in the pco-s+ir subgroup than the control group (p < 0.022). circulating betatrophin was significantly lower in both patients subgroups than the control group (p < 0.008). there was no negative or positive correlation between irisin and betatrophin levels. discussion: these data suggest that irisin and betatrophin may act a role together in the ir mechanism in pcos patients. butyrylcholinesterase (bche) synthesized in liver has long been associated with hyperlipidemia, type 2 diabetes and obesity. there are also reports on bche knockout mice becoming obese. the exact involvement of how bche interacts with lipids is still not clear. previously we displayed a correlation between leptin, waist circumference, fat mass and bche levels. recently, we have also shown that bche overexpression in hepg2 cells is regulated by alpha linoleic acid. as the next approach on the analysis of lipid metabolism and bche interaction, we considered the capability of bche to hydrolyze lipids. human serum bche was purified by subsequent deae-tris-acryl m and procainamide chromatography. the purified bche was utilized in a modified acid lipase assay with the acid lipase substrate 4-methylumbelliferyl palmitate (4-mu-palmitate). as the second alternative substrate trioleic acid was utilized. the triolein hydrolysis was measured by the nefa kit. verification that bche hydrolysis of these lipid substrates was not due to another esterase was done by iso-ompa inhibition studies. also, lectin binding studies with bche and rca were carried out to rule out non-specific esterase activity. using purified human serum bche and hepatic lipase as control enzyme we found that bche is able to hydrolyze the acid lipase substrate 4-methylumbelliferyl palmitate (4-mu-palmitate). we found that bche hydrolyses this molecule at ph 8 rather better than at ph 7.4. at ph values, purified human bche has a km value that was 10 times bigger than that of human pancreatic lipase. with the bigger molecule the triolein, the difference between the km values of bche and pancreatic lipase was smaller. bche seems to hydrolyze triolein with an efficacy comparable to approximately 25% that of human pancreatic lipase. our results display that another function of bche may be its lipid hydrolyzing activity. p-02.02.2-026 determination of regional reference ranges for erythropoietin with laboratory data mining serum erythropoietin (epo) levels are the main regulator factor of erythrocyte production and increase in response to hypoxia. our region is a location dominated by hypoxic conditions due to the high attitude. in this study we aimed to investigate the mean serum epo levels in the living conditions of our region. two hundred and eighty epo results from our laboratory data whose hemoglobin levels were normal were evaluated in the study. mean serum epo levels were analyzed via chemiluminescence method in beckman coulter dxi 800 auto analyzer. the epo levels of samples was 12.26 ae 9.32 mul/ml (ranged between 3.00 and 48.57) mul/ml. when we performed ae 1 sd for the studies population we determined normal serum epo levels were as 2.94-21.58 mul/ml. the upper limit determined by our results was 17% higher than that of determined by the manufacturer as 18.5 mul/ml and the lower limit determined by our results was 17% higher than that of determined by the manufacturer as 2.59 mul/ml. normal serum epo levels were considerable for our region and the upper and lower limits were higher than those of determined by the manufacturer. more detailed studies considering the physical properties of participants including a higher number participants are necessary. subclinical hypothyroidism is the precursor to hypothyroidism because it has a tendency to transform into hypothyroidism. subclinical hypothyroidism is considered one of the risk factors causing metabolic syndrome. metabolic syndrome can be characterized by plasma level of apelin released from adipocytes. in the present study, we aimed to measure serum apelin level of patients with subclinical hypothyroidism and compare them with serum apelin level from healthy individuals. our study group included 31 patients diagnosed with subclinical hypothyroidism and 23 healthy volunteers. serum samples were obtained from each participant for the measurement of apelin. these were then stored at à20•c until the time of analysis. serum apelin concentrations were determined using an enzymelinked immunosorbent assay. the mean serum apelin levels of subclinical hypothyroidism and control groups were 79 ng/l, control group 60 ng/l respectively. there was no statistically significant difference in terms of the mean apelin levels between the groups (p > 0.05). apelin levels didn't show significant correlation with bmi (p > 0.05). in the present study, no significant difference of serum apelin level was observed between patients with subclinical hypothyroidism and healthy control subjects. however, the apelin levels were higher in the patients with subclinical hypothyroidism than in the control group. the possible relationship between thyroid hormones and apelins is critical to understanding the etiopathogenesis of metabolic disorders. the mitochondrial erv1/mia40 import system does not impact cytosolic fe-s cluster protein maturation and iron regulation erv1 is a sulfhydryl oxidase that partners with the import receptor mia40 to import small cysteine-rich proteins into the mitochondrial intermembrane space. it has also been suggested that erv1 has an additional role in maturation of cytosolic fe-s cluster proteins and regulation of iron homeostasis in s. cerevisiae. however, these studies were performed on one particular erv1 mutant strain (erv1-1) that we discovered has additional defects in glutathione (gsh) metabolism. since gsh is required for iron regulation and cytosolic fe-s cluster assembly, this complicates our understanding of erv1 0 s role in these processes. we discovered that the erv1-1 strain originally tested for fe-s cluster defects was the only strain to exhibit defects in the cytosolic fe-s enzymes. mitochondrial and cytosolic fe-s protein activities in the other erv1 and mia40 mutants tested were similar to the wt control. in addition, while all the erv1 and mia40 mutants tested exhibit temperature-dependent defects in mia40 oxidation, only the erv1-1 strain has significantly reduced gsh levels and more oxidized gsh: gssg redox state. we determined that the cause of gsh depletion in the erv1-1 strain is an additional mutation in the gene encoding the glutathione biosynthesis enzyme (gsh1) that compromises gsh1 protein folding and/or stability. to address whether gsh deficiency in the erv1-1 mutant is the underlying cause for the cytosolic fe-s cluster defects and iron misregulation for this strain, we measured fe-s protein activity, iron-regulated gene expression, and iron accumulation in erv1 and mia40 mutant strains. only the erv1-1 strain exhibited iron misregulation and accumulation of mitochondrial iron, while exogenous gsh rescued these defects. these results demonstrate that the defects in cytosolic fe-s enzymes and iron homeostasis in erv1-1 are due to gsh depletion and neither erv1 nor mia40 play significant roles in cytosolic fe-s cluster assembly and iron homeostasis. human c-peptide is a 31 amino acid polypeptide, which is secreted into blood from b-cells in the pancreas where pro-insulin undergoes a post translational modification and cleaved into insulin and c-peptide. human c-peptide concentrations in blood plasma and urine reflect the level of insulin resistance associated b-cell function and can point out insulin secretory failure. the reference intervals in blood plasma and urine are 0.5-10.0 ng/ml and 40-150 ng/ml respectively. c-peptide measurement in urine and plasma provides a guide for therapy in diabetes. this study describes a method for the development and validation of picaa (peptide impurity corrected amino acid analysis) method for the determination of the purity of the human c-peptide which could be used as a reference material to measure cpeptide concentrations in plasma. two different methods were performed for the picaa; aaa-id-ms/ms for quantification of constituent amino acids following hydrolysis of the material and rp-hplc-esi-tof ms for determination of the peptide related impurities. the result of the aaa id ms/ms method was corrected for the amino acids originating from the impurities. id ms/ms-aaa was performed with zivak ò hplc and zivak ò tandem gold triple quadrupole ms equipped with a phenomenex ez:faast 4l aaa column (250 9 2 mm i.d). the mobile phase was composed of, a: 20 mm ammonium formate (af) in water, b: 10 mm af in acetonitrile (acn). the intact peptide analysis was performed by a hitachi lachrome elite hplc and bruker microtof-q mass spectrometer equipped with a capcell pak mg-ii c18 column (150 9 2 mm i.d., 5 mm particle size). the purity of the synthetic c-peptide was determined by picaa analysis and related uncertainty was calculated. traceability to si was established using the amino acid standards of which the purity was determined by tub _ itak ume using qnmr analysis. picaa is a simpler alternative to the full mass balance approach which requires large quantities of the peptide material. p-02.02.2-034 heat shock proteins: complementary therapies in brain tumors with viscum album e. onay-ucar, s. n. biltekin 1 istanbul university, faculty of science, department of molecular biology and genetics, vezneciler, istanbul, turkey cancer is one of the lethal diseases in the world. different cancer types possess overexpressed hsps levels. viscum album extracts with their anticancer and antioxidant properties are being used in cancer therapies. biochemical composition of this plant is known to vary its features depending on the host trees and time of harvest. in our previous study, it has been found that v.album inhibited hsp27 expression and induced caspase-dependent apoptosis in c6 rat gliomas. the aim of the current study is to find out whether different v.album extracts have different effects on hsps expression level and apoptosis in c6 glioma cell line or not. in this study, three different extracts of v.album were compared for their potential inhibition effects on hsps. the cytotoxic effects of extracts have been determined via mtt test. different experiment groups were set up subjected to heat shock and/or incubated without any heat shock application. overexpression of hsps was induced by heat shock at 42°c for 1 h in c6 cells. expression levels of hsps were determined by western blot analysis. the apoptosis inducing effect was also evaluated via caspase-3 activation in c6 glioma cells. pretreatment of the cells with non-toxic dose (10 lg/ml) of v.album extracts prior to heat shock, reduced significantly the expression levels of hsps. similarly, pretreatment with the extracts prior to heat shock increased apoptosis via caspase-3 activation in c6 glioma cells. these results will be utilized in the determination of the relation between extract composition and stress protein expressions. these results suggest that different extracts of v.album are able to down regulate expression of hsps, and induce apoptosis. this warrants further exploration as a potential resource of bioactive compounds that can be used in cancer therapy. future studies targeting hsps for the development of chemosensitizers may help improve the treatment of cancer in combinational therapy. biological drugs (biologics) are the fastest-growing category of therapeutics among those approved by the agencies for drugs regulation. most biologics are proteins designed for parenteral use. however, proteins are characterized by poor pharmacokinetic and safety profiles. peg-coating (poly-ethylene glycol coating) of biologics provides several benefits, including an increased half-life related to reduced renal clearance, an increased stability to degradation, and a reduced immunogenic/antigenic response. preservation of the three-dimensional structure and activity of the pegylated form is a strict requirement for human use. the recombinant proteins used for this studies (as-sod, superoxide dismutase; mmp12, matrix metalloproteases 12; ansii, l-asparaginase ii) were cloned and then over-expressed in escherichia coli. pegylation reactions were performed using commercial reagents. all the protein samples were purified and analyzed by solution and solid-state nmr (fields from 700 mhz to 950 mhz). we developed new protocols to prepare samples of pegylated proteins, demonstrating that solid-state nmr spectra of exceptionally good quality can be obtained for pegylated proteins in the sedimented state (obtained by either ultracentrifugation or rehydration of freeze-dried samples); surprisingly, sedimentation of pegylated proteins to this end has never been attempted. the spectral quality is comparable toor better thanthat of the corresponding crystalline samples. the excellent quality of the solid-state nmr spectra would make it possible to perform extensive resonance assignment and even a conventional full structure determination of biologics. the proposed method is based on the comparison of a standard twodimensional solid-state nmr spectrum of the sedimented pegylated protein with that of the crystalline state of the native proteinfor which the x-ray structure is available. all eukaryotic creatures hereditarily have natural defense mechanisms and are protected from the infections with this defense mechanism. antimicrobial peptides (amp) contain 10-50 amino acid content, are positively charged with amphipathic feature. the antimicrobial activities of amps are thought to be depended on the microbiocidal effects by binding to the surface of microorganisms and creating pores in their membranes. defensins are both effector and mediator small antimicrobial peptides of the immune system. these peptides in cationic and amphipathic structure have broad spectrum antibacterial, antifungal and antiviral features. defensins regulate the innate and acquired immune systems by suppressing proinflammatory responses during infection. mammals have three structural subfamilies of defensins. these show differences according to the trisulfide arrays in their structure and are classified as a,b, h defensins. human beings have tissue-specific six functional a defensins. human hnp-1 and hnp-4 encoded by defa1, defa3 and defa4 genes are firstly expressed in neutrophils. human hdp5 and hdp6 encoded by defa5 and defa6 are firstly expressed in paneth cells in the intestines and play important role in the defense and homeostasis. human beings have many pseudogenes such as defap and deftp in addition to these functional genes. according to literature data, defensins play an important role in defense against microbial placements on mucosal surfaces. in addition, the antimicrobial spectra of defensins include gram negative and gram positive bacteria, fungi and viruses. in addition to their antimicrobial efficiency, they can accelerate the wound healing due to their mitogenic effects on epithelium cells and fibroblasts. bile salt hydrolase (bsh) enzyme catalyzes the hydrolysis of glycine and/or taurine-conjugated bile salts into amino acid residues and the free bile acids that reduce cholesterol. however, some intestinal bacteria have an excessive deconjugation of tauro-conjugated bile salts and production of secondary bile acid having potential harmful side effects to the host. the catalytic mechanism and substrate preference of such bsh enzyme is not clear. in this study, bsh gene from lactobacillus plantarum gd2 strain was cloned, expressed, characterized in escherichia coli blr(de3) strain, and then val-58 and phe-129 amino acids, supposed to be responsible for substrate preference, were substituted for met-58 and ile-129 amino acids respectively by site directed mutagenesis. the hydrolysis activities and stability of the mutant recombinant bsh (mrbsh) enzymes were examined along with six different bile acids by ninhydrin assay and sds-page respectively. ninhydrin test results indicated that wild-type recombinant bsh (wrbsh) hydrolyzed six major human bile salts with an apparent preference towards glycine-conjugated to tauro-conjugated bile salts. however, the activities of mrbsh/phe129ile enzyme are 29%, 23%, 13%, 14%, 0% and 0% of the activity of wrbsh against to glycocholic acid (gca), glycodeoxycholic acid (gdca), glycochenodeoxycholic acid (gcdca), taurocholic acid (tca), taurodeoxycholic acid (tdca) and taurochenodexycholic acid (tcdca) respectively. the activities of val58met mrbsh enzyme are 79%, 93%, 74%, 43%, 44% and 28% of wrbsh against to gca, gdca, gcdca, tca, tdca and tcdca respectively. our findings support the suggestion that bsh enzymes recognize their substrates predominantly at the amino acid moieties and not at the cholate moieties. however, further pcr-based site-directed mutagenesis and structure-driven computational and theoretical approaches are required for the precise determination of their substrate specificities and the selection of probiotic bacteria. we deposited bacteriorhodopsin in purple membranes under applied electrical field onto ito (indium tin oxide) support. purple membranes film, highly oriented in one direction, was placed between two ito electrodes. we studied dependence of electrical properties of these films on light illumination. we argue that this setup can be used for functional studies of microbial rhodopsins. in opposite to already published results where this system was used as a photocondensor for studying functional properties of bacteriorhodopsin, we studied electric properties of such systems and we found strong light dependence of resistivity of bacteriorhodopsin in purple membranes films. optogenetics is already used in study of neuronal cells cooperation in vitro and in vivo by means of microbial rhodopsinsion pumps and channels incorporated in membranes of neurons changing their electrical potential while receiving a light quantum by laser or led source. best perspectives optogenetics will give after successful transfer to medical applications, such as the treatment of blindness, treatment of disorders like parkinson's disease etc. but to achieve these we need a broad set of tools, optogenetics tools, highly specialized to solve specific problems of neurophysiology. to the creation of such tools our work is dedicated. new optogenetic tools can be made by mutations in existing ones altering their properties (mainly spectral characteristics, selectivity and conductivity) or some promising mutations in conserved residues can be found in existing organisms. a halophilic archaeon halosimplex carlsbadens is a host of protein of our interest. according to the theoretical data based on the alignment with br and the 3d structure model of this novel protein, we suppose this protein functions like the light-driven h+ pump: all the key residues are the same or at worst have the similar properties, except one in the position 96leucine instead of the aspartic acid. a gram-positive bacteria deinococcus-thermus phylum syntheses rhodopsin with substitution of this aspartic acid to alanine. sphingomonas paucimobilis has rhodopsin where aspardic acid in position 96 is changed to serine residue. and one yet uncharacterised guillardia theta rhodopsin even has the same as br motif (d85, t89, and d96) but according to alignment is closer to chr2 even the last one motif is e85, t89, n96. it is expected that all of them will show us new properties. though the further experimental data are essential. the work is supported by rsf 16-15-00242. evaluation of some thymus proteins in patients with crimean congo hemorrhagic fever i. b€ ut€ un 1 , s. sahin 1 , f. duygu 2 1 university of gaziosmanpasa, department of biochemistry, tokat, turkey, 2 oncology education and reasearch center, ankara, turkey crimean congo hemorrhagic fever (cchf) is a tick-borne viral zoonotic disease. it has a high fatal rate (%5-30). tokat is one of the cities having the most reported cchf cases, in turkey. clinical presentation of the disease varies widely among patients. thymic peptides are small molecules synthesized by thymic epithelial cells. they play role in the immune response, as well as anti-inflammatory process. fourty patients referring to the hospital with tick-contact history and/or presenting clinical manifestations consistent withcchf and with positive pcr results for cchf virus in blood samples were included to the study. the wbc and platelet values at application and before the patients were discharged were recorded. the healthy control group consisted of age and gender matched healthy volunteer adults free of any chronic disease. thymosin alpha1 (ta1), thymuline and thymosin beta4 (tb4) were studied by the elisa method in this study. biochemical parameters were also analysed. ast and alt values were significantly higher (p < 0.01) and plt and wbc levels were significantly lower in the cchf group (p < 0.05). levels of tf, ta1 and tb4 were found to be significantly higher in cchf (p < 0.01). there was no mortality during the study period. duration of hospitalization was 6.73 ae 2.74 days. levels of tb4 were significantly correlated with duration of hospitalization (r= à0.351, p = 0.036). alt levels were significantly correlated with tf levels (r = 0.413, p = 0.010). 17 patients received ffp and apheresis for the supportive treatment, while 5 patients received only ffp and 2 patients got only apheresis. 16 patients did not get any of these blood products. there was not a statistically significant differences in thymus peptides among these treatment groups (p > 0.05). we report 40 survived cchf patients with elevated thymic peptides. pathogenesis of cchf has many points to be highlighted. thymic peptides may play role in the clinical situation of the patients with the disease. the effect of methocarbamol on the peroxiadse activity of human erythrocyte hemoglobin hemoglobin is released to blood circulation, after red blood cells lysis. it is carried in circulation by binding to haptoglobin. in normal persons, no free hemoglobin is observed in the blood, because most of hemoglobin is in the form of haptoglobin complex. in some diseases that are accompanied by hemolysis, the amount of released hemoglobin is higher than its complementary haptoglobin. as a result, free hemoglobin appears in the blood, which is a toxic compound for these patients. free hemoglobin has been showed to have peroxidase activity and considered a pseudoenzyme. in this research, the effect of methocarbamol on the peroxidase activity of human hemoglobin was studied. our results showed that the drug inhibited the pseudoenzyme by un-competitive inhibition. both k m and v max decreased by increasing the drug concentration. k i and ic 50 values were determined as 6 and 10 mm, respectively. molecular docking results showed that methocarbamol did not attach to heme group directly. a hydrogen bond connected nh 2 of carbamate group of methocarbamol to the carboxyl group of asp126 side chain. two other hydrogen bonds could be also observed between hydroxyl group of the drug and ser102 and ser133 residues of the pseudoenzyme. p-02.02.2-042 dca reduces viability and down regulates mapk protein activations in human malignant mesothelioma cells and pericardium. microarray analyze results performed in mm patients revealed that one of the most prominent changes is upregulation of many genes involved in glycolysis and the krebs cycle. dichloroacetate (dca) is an inhibitor of pyruvate dehydrogenase kinase (pdk) that enhances the oxidative activity of cells by activating pyruvate dehydrogenase (pdh) in mitochondria. dca has shown as a promising anti-neoplastic agent that re-sensitizes cancer cells to apoptosis. the aim of this study is to elucidate the coupling between pdk inhibition and mm cell proliferation and cell cycle. human malignant mesothelioma (spc212) cell line was used as a model for dca treatments. cell viability was measured by mts assay; mapk protein activations and expressions were assessed by western blotting; cell cycle profile was analyzed by flow cytometry. statistical analysis was performed by utilizing one-way anova test. results showed that dca reduced viability of spc212 cells in a concentration and time dependent manner. protein analysis indicated that mapk pathway was down regulated at concentrations greater than 50 mm. moreover, primary cell cycle analysis has indicated arrestment at g2/m phase in 24 hours. our findings corroborate with recent reports where dca treatments resulted in reduction of viability and g0/g1 and g2/ m arrest in other cell lines. abnormalities in mapk signalling play a critical role in the progression of cancer. here, we showed for the first time that dca decreased mapk activation in 24 h. our results suggest that dca is an anti-prolifertive agents for mm cells in vitro. however, it requres extra analysis with other mesothelial cells. future study will focus on investigating relation between mapk and mitochondrial apoptosis. adrenomedullin (adm) is a vasodilator peptide consisting of 52 amino acids. adm is synthesized in many tissues. and is a biologically active peptide that has various effects including vasodilatation, the regulation of vascular endothelial function and adjusting adipogenesis. hypoxia inducible factor 1 alpha (hif 1a) is a subunit of a heterodimeric transcription factor hypoxia inducible factor 1. it is the master transcriptional regulator of cellular and developmental response to hypoxia. the dysregulation and overexpression of hif1a by either hypoxia or genetic alternations have been heavily implicated in cancer biology as well as a number of other pathophysiologies. in our research, the adm and hif 1-a levels in heart, kidney and lung tissues of rats were investigated in control, hypoxia, control+adm and hypoxia+adm groups. rats in hypoxia groups were provided hypoxic environment containing of 10-12% oxygen and 88-90% nitrogen for 1 week. rats in adm groups were injected intraperitoneally in a dose of 1.25 nmol/kg for four days before the collection of the tissues. the control group was oxygenated with normal air. the control and treatment groups were formed from 7-8 animals and adm, hif 1-a levels were measured in taken tissues with immunoassay method. the aim of this study was to investigate the reaction of the organism when exposed to hypoxic conditions and the effect of adm over hif 1-a level. adm levels and hif 1-a in heart tissue were found decreased in hypoxia group, and adm levels increased in hipoxia+adm group. hif 1-a levels decreased in hypoxi+adm group. adm levels in liver tissue were found decreased in hipoxia and control+adm groups than control group. hif 1-a levels were higher in control+adm group. adm has a role in angiogenic process, and our experiment showed that adm reacts earlier than hif 1-a, and affects its synthesis. organism increases its vascularization as a reaction to hypoxic condition, and adm treatment may provide a rapid adjustment. p-02.02.2-044 covalent conjugation and characterization of immunogenic protein of toxoplasma gondii and polyacryclic acid as vaccine candidate r. c ß akir koc ß yildiz technical university, department of bioengineering, istanbul, turkey toxoplasmosis is a major medical and veterinary disease caused by toxoplasma gondii which infect approximately half of the world's population. this infectious disease especially gains importance in pregnant women and immunodeficient individuals. also t. gondii infection has economic importance. however, there are only one attenuated-live t. gondii vaccine for veterinary uses and no vaccine against t. gondii is available for humans. therefore development of an effective vaccine would be extremely valuable for preventing disease in human and veterinary medicine. subunit vaccines are very attractive vaccine candidates but there is low antigenicity problem when they are used alone. polymers themselves don't stimulate immune response while they used with antigenic structure of various infective agents enhance immune response because when proteins are covalently conjugated with hydrophilic polymers, (1) their circulatory-lives and stability (in different ph and temperature values) enhance (2) binding to proteases and clearance by the reticuloendothelial-system decreases. in this study, immunogenic protein of t.gondii and polyacrylic acid with immune stimulant properties was covalently conjugated and conjugation was demonstrated by size-exclusion chromatography (sec) and fluorescence spectroscopy. it is significant to detect time of death in case of a sudden death for medical and legal concerns. there is no known method that can be used for post mortem time detection. based on this deficiency pmi detection in narrow time frame is a big problem. in this study, we aimed to investigate and determine timedependent expressional changes of apoptotic markers by western blot technique. 14 postmortem skeletal muscle were analyzed 12hour periods in first 36-hour after death. 2nd and 3rd 12-hour periods were statistically significant (p < 0.005). keywords: post mortem interval, time of death, apoptosis. hyaluronidases are excessively found in nature and involved in numerous biological functions. hyaluronidases primarily degrade hyaluronic acid (ha) and have significant role in fertilization during acrosomal reactions. therefore, the measurement of hyaluronidase enzyme activity may provide valuable information about acrosomal function and the fertilizing ability of the sperm. the aim of this study was to investigate the semen hyaluronidase enzyme activity changes among four different sheep breeds (akkaraman, suffolk, merino, and kıvırcık). in this research, ten ram testis tissues from each sheep breed, a total of 40, were cut and collected on ice. ovine testicular hyaluronidase of four different sheep breeds was purified from a crude ammonium sulfate-precipitated fraction of an extract of ram testis. the semen hyaluronidase enzyme activity differences between the sheep breeds were examined by spectrophotometrically monitoring the appearance of ha at 232 nm. analysis of variance test was used to examine the possible mean differences among the four different sheep breeds. the observed mean differences in enzyme units for kıvırcık, suffolk, akkaraman, and merino were as follows 891.60, 817.60, 729.40, and 681.60, respectively. the observed mean differences in absorbance values for kıvırcık, suffolk, akkaraman, and merino were as follows 0.4455, 0.4088, 0.3648, and 0.3408, respectively. the results showed that the observed mean differences in enzyme units and absorbance values among the four different sheep breeds were not statistically significant. despite that, in average kıvırcık had higher values for the activity of each sample and yet it had the smallest values for standard deviation. therefore, in order to achieve higher enzyme activity and more homogenous samples kıvırcık breed should be preferred. what is extra to learn from protein drying measurements? hydrations of soluble proteins are crucial for their functionality. therefore elucidating the details of protein hydration is still of interest in the proteins' action mechanisms. this is the motivation of the present study. in order to study protein hydration, changing concentrations of the well-studied serum albumin protein was measured with the spectroscopic techniques like uv-vis and ft-ir spectroscopy. spectral data is analysed and calculations were performed on the data to extract the relevant changes in the protein. experimental parameters' variation in association with the spectral changes implies the involvement of protein structure and hydrogen bonding in the drying process. the protein's reactions may not be merely a feature of the protein structure in the common sense but it could be related directly to the protein hydration states as well. this is understandable since it is already known that enzymatic proteins lose their functionality when they are dried while this drying may or may not involve dramatic structural changes. on the other hand, here it is claimed that the role of water in gaining the functionality that was lost in the dried state is not just about enhanced diffusion processes and the dynamicity but could be related to the functionality of water in the energy transfer processes as well. investigating the cellular effects of the aldoketo reductases akr1b1 and akr1b10 in hct-116 colon cancer cells b. taskoparan, e. g. seza, m. s. ceyhan, s. banerjee middle east technical university, ankara, turkey aldo-keto reductases (akr) are nad(p)h dependent oxidoreductases are best characterized as glucose reducing agents, and have been implicated in diabetic pathophysiology. increased expression of akr has been associated with tumors of lung, breast, prostate, cervix, ovarian and colon. two members of the akr superfamily that have been associated with different cancer types are akr1b1; aldo-keto reductase family 1, member b1, and akr1b10; aldo-keto reductase family 1, member b10. both are 36-kda cytosolic reductases that are similar in both amino acid sequence identity (71%) and tertiary structure with the (a/b) 8 barrel topology. while hct-116, a colorectal cancer cell line, cells expresses akr1b1 robustly, there is no expression of akr1b10. in this study, we have stably knocked down akr1b1 through shrna technology and overexpressed akr1b10 in hct-116 cells. comparisons were made with a known akr inhibitor sorbinil. with the knock down of akr1b1, we have observed reduced cellular proliferation, enhanced apoptosis, delay in cell cycle progression, reduced expression of mitogenic proteins and a decrease in activation of the inflammatory transcription factor nuclear factor kappa b (nf-kappab). interestingly, although akr1b10 overexpression did not affect cell proliferation, apoptosis or cell cycle, some effect was observed with nf-kb signaling. our data indicate that, although closely related, akr1b1 and akr1b10 have very different contributions towards signaling pathways in colorectal cancer. comparison of different nisin quantification methods and optimization of nisin production by lactococcus lactis z. girgin ersoy, g. demir, m. f. cesur, s. tunca gedik gebze technical university, kocaeli, turkey nisin, which is produced by certain strains of lactococcus lactis, is the only bacteriocin approved by world health organization (who) as a food additive. it prevents the growth of foodborne bacteria which cause food spoilage. nisin research and applications necessitates developing an accurate and reproducible method for its quantification. the agar diffusion bioassay is the most widely used method for quantifying nisin, although it has limitations especially diffusion-related difficulties of the active substance. in the present study, "agar diffusion bioassay", "enumeration of colony forming units", "colorimetric assay" and "flow cytometry" methods were compared with each other to determine antibacterial activity of nisin on micrococcus luteus. moreover, this study also covers the results about the effect of different cultural conditions to optimize nisin production by l. lactis. galactose, lactose and their combination in m17 medium (ph 7) boosted nisin production at 25°c, as the addition of 1.5 lg/ml hemin into the fermentation broth. to our knowledge, this is the first study showing the usage of "flow cytometry" method to determine nisin activity of fermentation broth filtrates. p-02.02.2-051 coronaviral nucleocapsid protein is an antiviral target for drug development institute of genomics and bioinformatics, national chung hsing university, taichung, taiwan between 2003 and 2004, the severe acute respiratory syndrome (sars)-cov caused a worldwide epidemic and had a significant economic impact in the countries affected by the outbreak. recently, the middle east respiratory syndrome human coronavirus (mers-cov) was found in patients with severe acute respiratory tract infections in the middle east and south korea. as is true for all coronavirus infections, there are no efficacious therapies currently available against coronaviral diseases, making the development of anti-coronavirus compounds a priority. the cov genome consists of positive-sense, single-stranded rna approximately 30 kb, and it contains several genes encoding several structural and non-structural proteins that are required for progeny virion production with a conserved order. the n proteins exist in the center of the viral particle and represent a helical structure complex. nucleocapsid protein is most abundant structural protein of covs, binds the viral rna genome to form the virion core, leading to the formation of a ribonucleoprotein (rnp) complex or to a long helical nucleocapsid structure, that is important for maintaining the rna in an ordered conformation for replication and transcription. the cov n protein is also involved in the regulation of cellular processes, such as gene transcription, interferon inhibition, actin reorganization, host cell cycle progression, and apoptosis. two strategies to inhibit oligomeric n protein function have been reported. the first strategy is to discover antiviral agents that target the rna-binding site. the second one is to impair normal n protein function by interfering with monomer-oligomer equilibrium. our recent studies suggest that n proteins in infections caused by coronaviruses will be useful antiviral drug targets because they serve many critical functions during the viral life cycle. post-translational modification of vascular endothelial growth factor (vegf) in colon cancer cells s. tunc ßer, e. solel, s. banerjee middle east technical university, ankara, turkey vascular endothelial growth factor a (vegf-a), commonly referred as vegf, is a potent secreted mitogen crucial for tumor initiation and progression. the gene for vegf is translated into a number of splice isoforms that lead to 121, 165, 189 and 206 amino acid proteins, with different receptor-binding and matrixbinding properties. in the present study, we discuss the functional significance of post-translational modification/processing of vegf 165 isoform in hct-116 colon cancer cells. we also focus on the role of calcium in the post-translational modification of vegf 165 . we show that vegf 165 undergoes n-linked glycosylation in hct-116 cells. perturbation of cellular calcium may affect vegf driven malignant phenotypes. p-02.02.2-053 novel methods for modulating the activity of bcl2 family proteins in apoptosis p. rowell, j. miles, a. wilson, t. edwards apoptosis, also known as programmed cell death, is an essential cellular process, but is implicated in several human diseases, including diabetes and cancer, when it is up-or down-regulated respectively. bcl-2 family proteins are major players in the control of intrinsic, or mitochondrial apoptosis; they respond to intracellular stress signals, function through protein-protein interactions and converge on the mitochondrial outer membrane to cause membrane permeabilisation, release of cytotoxic molecules, and initiation of an apoptotic cascade that leads to cellular demise. our work aims to identify molecules able to bind and modulate the activity of several key players in the bcl-2 family, including the pro-survival members bcl-2, bcl xl and mcl1, and the death promoting family member bax. adhirons, novel non-antibody peptide display scaffolds developed at the university of leeds, have been used to construct a phage display library containing over 10 10 clones, and form a key part of the strategy to identify such molecular modulators. adhirons able to selectively bind individual bcl-2 family members have been identified, in vitro assays carried out to test for modulatory activity, and xray crystallography used to elucidate details of how they interact with their target proteins. more recently, studies have been carried out to identify adhirons able to target multiple bcl-2 family members, with the aim of selectively inhibiting defined groups of proteins in cells. this work provides opportunities to differentiate the activities carried out by different bcl-2 family proteins in apoptosis, enabling us to better understand how their dysregulation contributes to human disease. biophysical and evolutionary study of the structural flexibility of adp-dependent sugar kinases from mesophilic and psychrophilic archaea r. zamora 1 , v. castro-fern andez 1 , c. a. ramirez-sarmiento 1 , e. a. komives 2 , v. guixe 1 1 facultad de ciencias, universidad de chile, santiago, chile, 2 department of chemistry and biochemistry, university of california, san diego, united states of america the capability of extremophiles microorganisms to live at low temperatures is mainly attributed to the high structural flexibility of its enzymes. several sequence and structure features have been associated to a high structural flexibility that enables metabolic processes to occur at low temperatures. during evolution, the general mechanism adopted by these enzymes has been to reduce the free energy of the transition state rather than the michaelis constant, k m . increased structural flexibility and decreased affinity for its substrates in psychrophilic enzymes is compensated by an increase in the catalytic rate, k cat . few psychrophilic enzymes have been reported to performance the optimization of their catalytic efficiency (k cat /k m ) by decreasing k m values. we use the adp-dependent kinase sugar family of archaea as a model, to identify particular structure and sequence features of a psychrophilic enzyme that would make this enzyme more flexible than their thermostable homologues. we characterize the bifunctional psychrophilic enzyme phosphofructokinase/glucokinase from methanococcoides burtonii (mbpfk-gk) and the bifunctional mesophilic enzyme phosphofructokinase/glucokinase from methanococcus maripaludis (mmpfk-gk) by spectroscopic, biophysical and computational techniques. the comparison showed that the absence of two ion pairs is primarily responsible for the increased structural flexibility accounted in the psychrophilic model. this increase in structural flexibility is reflected in the exponential increase in the k m values with temperature. additionally, we reconstruct the sequences of all ancestral enzymes between the current enzymes and their last common ancestor, which was used to trace the occurrence of these electrostatic interactions during evolution in the adp-dependent sugar kinase family. our results suggest that electrostatic interactions are a dominant feature in the transition from psychrophilic to thermophilic environments. fondecyt 1150460. elucidating the domain swapping mechanism of the forkhead domain of human foxp1 fox transcription factors control gene transcription during key processes, such as embryogenesis and immunity, and feature a conserved dna-binding domain known as forkhead. while most forkhead domains are monomeric, solved structures of members from the p subfamily (foxp) show that they can oligomerize by domain swapping (ds), a mechanism where protein segments are exchanged between subunits leading to an intertwined dimer. the biological relevance of ds in foxp has been stressed by disease-causing mutations that impair this process. however, for many proteins ds takes days to occur and requires global protein unfolding. thus, the current mechanism impedes a conciliation of the occurrence of ds of foxp1 in a biological context. here, we elucidate the biological feasibility of this process by biophysically characterizing the ds mechanism of the forkhead domain of foxp1 using size exclusion chromatography (sec), circular dichroism, and hydrogen-deuterium exchange mass spectrometry (hdxms). our results show that ds of foxp1 occurs at micromolar protein concentrations, within hours and is energetically favored. remarkably, dimeric foxp1 follows a threestate n 2 « 2i « 2u folding mechanism, where dimer dissociation into a monomeric intermediate (i) precedes protein unfolding, in contrast to other ds proteins. using sec and hdxms, we show that the i state of foxp1 largely resembles the native state, but has a larger hydrodynamic radius and a higher deuterium uptake in regions that maintain the compact monomer, suggesting that the i state is an 'open' conformation en route of ds. finally, we compared the local flexibility of the dimer and monomer of foxp1, showing that only the hinge region connecting ds segments exhibits different deuteration rates. our results show that ds in foxp1 follows an unusual threestate folding mechanism that proceeds through transient structural changes rather than needing protein unfolding as in most ds proteins. (fondecyt 1130510, 11140601). p-02.02.2-057 the sustained release of growth factor proteins following their implantation in tissue engineering j. jang bone tissue engineering has become a promising approach for bone regeneration. however, insufficient vascularization during bone regeneration, particularly with large bone defects, results in poor and unsustainable bone formation due to central necrosis. therefore, vascularization following implantation in vivo is essential to the successful formation of new bone tissue. we evaluated the release profile of vegf from bgs using a novel fluorescence-based retention assay, which revealed that vegf loaded on bgs can be released in a sustained manner without an initial burst (near zero-order cumulative release) with a controlled release rate of 13.6% per week for up to 7 weeks. in contrast, an elisa-based release assay showed vegf to have an early burst-release profile for the first week. however, the biological activity of vegf released from the bgs was preserved over the 7-week release period, which is consistent with the sustainedrelease profile observed in the fluorescence-based retention assay. we developed a novel fluorescence-based retention assay to evaluate the release of vegf from bgs. this fluorescence-based retention assay, which detects the vegf that remains on bgs, reveals that vegf loaded on bgs can be released in a sustained manner, with a minimal initial burst, for up to 7 weeks. these results indicate that the sustained biological activity of the vegf released from bgs over the full 7-week period promotes bone regeneration, and suggest its potential use for bone tissue engineering. irisin is a recently discovered myokine which regulates energy metabolism and is associated overweight. we aimed to evaluate serum irisin levels in the patients with morbid obesity. a total of sixty patients with morbidly obese and thirty healthy control subjects were included in this study. all participants were measured body weight and height, the lipid profile, and plasma glucose, hba1c, insulin and irisin levels. irisin levels were measured by elisa method. serum irisin levels were significantly lower in morbidly obese patients than healthy controls (p < 0.05). there was no statistically significant difference in terms of age or gender. serum irisin was negatively correlated with bmi, insulin levels, and homa-ir (p = 0.006, p = 0.046, p = 0.048, respectively). our study revealed lower irisin levels in morbidly obese patients with respect to control subjects. the lower irisin levels observed in morbidly obese patients might suggest a loss of browning of subcutaneous adipose tissues. p-02.02.2-059 pka inhibition restores adenosine uptake in renal tubular epithelial cells under high d-glucose conditions w. garrido, j. catal an, s. alarc on, r. san mart ın universidad austral de chile, valdivia, chile introduction: diabetic nephropathy (dn) is the leading a cause of end-stage renal failure whose pathogenesis must to be elucidated. the progression of dn has been associated with elevated levels of adenosine. extracellular adenosine availability is regulated by the activity of the equilibrative nucleoside transporters (ents). due to the ents have putative sites of phosphorylation our objective was to evaluate the role of pka and ck2 kinases on ents activity. methods: adenosine uptake (10 lm adenosine, 60s, 22°c) was assayed in hk2 cells preincubated with 5 mm or 25 mm d-glucose for 24 h and exposed to 10 lm 8-br camp, 10 lmh89 or 100lm tbb for 1 h. plasma membrane and intracellular proteins were fractionated by the biotinylation method and ent1 and ent2 contents were quantified by western blot. results: high d-glucose in hk2 cells inhibited the uptake of adenosine. this effect was reversed using a pka inhibitor (h89) through an increased ent2 uptake activity. we noticed this pka inhibitor did not regulate the plasma membrane localization of ent1 or ent2 under normal d-glucose (5 mm) or high d-glucose conditions (25 mm). also, we saw that tbb (ck2 inhibitor) decreased the activity of ent1 and ent2 under normal glucose conditions, decreasing the localization of ent1 at cell surface, while the membrane localization of ent2 decreases under the effect of tbb and high d-glucose conditions. conclusions: pka inhibition reversed the effect of high d-glucose, increasing the uptake of adenosine mediated by ent2. this could be a new target for the restoration of adenosine levels in dn. relation between serum lipo (a), plasma fibrinogen, red cell distribution width and mean platelet volume in healthy adult men the aim of this study was to investigate the relationship between the serum lipo (a) and plasma fibrinogen, red cell distribution width (rdw) and mean platelet volume (mpv) in healthy adult men. for this purpose, 37 healthy adult men have normal physical examination and laboratory findings and not use any drug were included to the study. serum lipo (a) levels and hematologic parameters (fibrinogen, rdw-sd and mpv) were measured by autoanalyzer and commercial kits. the mean of the age of the persons was 27.2; body mass index was 24.2; serum lipo (a) level was 0.21 and plasma fibrinogen level was 1.61. there was significant positive correlation between the serum lipo (a) and plasma fibrinogen levels (r = 0.246; p = 0.025), significant positive correlation between the serum lipo (a) and rdw-sd (r = 0.267; p = 0.004) and significant negative correlation between lipo (a) and mpv (r= à0.205; p = 0.027). the plasma fibrinogen and the serum lipo (a) levels have been known as the risk factors for cad (coronary artery disease) increase together in healthy adult men. similar findings have been reported in cad patients. it has reported that elevated rdw is associated with intracoronary thrombotic burden and may be associated with the severity and instability of acute myocardial infarction. in addition, mpv is predictor of severe atherosclerosis and may be used for the prediction and identification of cardiac risks in cad patients. our findings show that elevated rdw and decreased mpv may predict to increased risk of cad in the future, in healthy adult men. follicular fluid is rich in peptides, which significantly influence the growing oocyte. due to existence of a link between kisspeptin (metastin) cells and gonadal steroids kisspeptin might manipulate the gonadotropin axis and folliculogenesis. in this context, the study was planned to investigate for the first time that the follicular fluid (ff) and serum concentration of kisspeptin in high and poor responders undergoing in vitro fertilization (ivf)/intracytoplasmic sperm injection (icsi). biological samples were collected from twenty infertile women with polycystic ovary syndrome (pcos) and 20 poor responder participants undergoing controlled ovarian stimulation (cos) with gonadotropin-releasing hormone (gnrh) antagonist protocol for ivf/icsi treatment. kisspeptin concentrations were measured in serum and follicular fluid by using elisa, whereas fsh and lh levels were detected by routine laboratory methods. it was found that kisspeptin levels were significantly lower in serum and follicular fluid of infertile women with pcos. kisspeptin levels were correlated with fsh and lh level in infertile women with pcos. it can be concluded that low level of kisspeptin might inhibit gnrh release that might cause to the inhibition of fsh and lh release and might disrupt folliculogenesis. decline in serum and ff levels of kisspeptin might be possible cause of anovulation and subfertility in pcos subjects. cryptococcus albidus d24 is a newly identified yeast isolates from petroleum area in _ izmir as a lipase producer. the molecular weight of the enzyme is 36.31 kda as found. optimum temperature was 40°c and half-life times were 180, 27, 9 and 0.59 min at 30, 40, 50 and 60°c, respectively. optimum ph value was 8.0. however, it shows significant ph stability at ph values 4.0 and lower. the existence of acetone in the solution as a solvent enhanced lipase activity. cryptococcus albidus d24 lipase was able to catalyze the esterification reaction between fructose and palmitic acid to produce fructose palmitate using acetone as the solvent. due to its stability in organic solvents, we propose that in order to increase the yield of fructose palmitate, we could immobilize d24 lipase. therefore, the effect of immobilization on kinetic parameters of d24 lipase was investigated. different immobilization materials and methods were used to find efficient support materials for d24 lipase immobilization. additionally, fructose palmitate production processes will be optimized with immobilized lipase. introduction: the diagnosis of osteoarthritis (oa) is based on clinical symptoms and radiographic findings. it is known that the pathologic changes at the molecular level in the joint cartilage tissue start before symptoms appear in oa. c1q tumor necrosis factor-related protein 1 (ctrp-1), c1q tumor necrosis factor-related protein 3 (ctrp-3) and kallistatin are related to many different cellular processes including bone and cartilage tissue metabolism. the aim of this study was to investigate any probable association between the serum ctrp-1, ctrp-3 and kallistatin levels and diagnosis and radiologic staging of knee oa patients. materials and methods: this study included 60 patients with knee oa and 30 healthy individuals for control purposes. the patient group was divided into four stages radiologically. ctrp-1, ctrp-3 and kallistatin levels were measured in serum samples of patient and control groups with elisa method, and the differences between the groups were analyzed with statistical methods. results: the levels of serum ctrp-1 in the patient group were significantly higher than in the control group (p = 0.001), serum ctrp-3 and kallistatin levels were not statistically different (in order of p = 0.251, p = 0.160). in the patient group, there was not a significant difference between serum ctrp-1, ctrp-3 and kallistatin levels and radiologic stages (respectively p = 0.811, p = 0.715, p = 0.202). there was a significant positive correlation between the radiologic stage and patient's age, body mass index, western ontario and mcmaster universities arthritis index and visual analogue scale values (respectively p = 0.001, r = 0.510; p = 0.010, r = 0.331; p = 0.001, r = 0.683; p = 0.001, r = 775). discussion and conclusion: serum ctrp-1 levels were detected significantly increased in patients with knee oa, but there was no significant difference in ctrp-3 and kallistatin levels. there was not a significant association between the radiologic stage and levels of ctrp-1, ctrp-3 and kallistatin. enzymes in microorganisms, especially termophilic bacteria are more attractive in biotechnology and molecular biology due to the higher catalytic activity. turkey is rich in geothermal resources and it is important to determine unknown microbial content of geothermal sources. in this study, water and sludge samples were taken from ayder, kizilcahamam and gonen hotsprings. firstly, ph, temperature, salt concentration, gram reaction, mobility, endospore formation, oxidase and catalase tests were carried out as conventional characterization. molecular characterizations of isolates were achieved by fames, rep-pcr and 16s rrna sequencing. finally, test isolates were evaluated according to enzyme production capability by petri dish. as result of conventional tests, isolates were determined as gram positive, mobile-rod shaped, aerobic, oxidase, catalase and endospore positive. the growth range for ph and temperature of the isolates were determined as 5-9 and 50-65°c. in consequence of the salt test, the test isolates were grown at 2-10% nacl. 19 of thermophilic isolates were selected by rep-pcr and according to 16s rrna sequencing analysis test isolates were belonging to bacillus, geobacillus, anoxybacillus and brevibacillus genus at a range of 98-99%. enzyme tests showed that, some of the isolates were able to produce protease (f17, f31, f76, f6, f99 and f19), amylase (f41, f76, f42, f98 and f10), cellulase (f17, f41, f31, f6, f99 and f19), xylanase (f17, f31, f76, f99 and f19) and lipase (f17, f31, f6, f99 and f19). it can be concluded that, geothermal regions are rich in bacillus and related genera. fame analysis was particularly insufficient for diagnosis of thermophilic microorganisms, but rep-pcr was successful in separation of organisms at species and even subspecies levels. most of our bacterial isolates have industrially important enzyme production capacities. it is a pioneer result to use bacteria for industrial applications which need higher temperatures. p-02.02.2-065 warburg effect was investigated by studying various metabolic molecules and assays in mammalian cell lines z. i. kanbagli, b. kiratli, h. cimen yeditepe university, istanbul, turkey majority of the different cancer cells switch their metabolism from oxidative phosphorylation pathway to glycolytic pathway; in order to meet excessive energy requirement, which is also called warburg effect. acetylation is one of the most crucial post-translational modifications playing key roles in metabolic reprograming. in this study, the relationship between acetylation dependent changes in energy metabolism and apoptotic pathways were investigated in pnt1a, du145, hela, hep3b, hek293t, shsy5y. immunoblotting experiments were applied by using antibodies against acetylated-lysine to examine the changes in overall acetylome. candidate proteins displaying elevated acetylation was identified with mass spectrometry based-proteomic analysis. glucose transporter 4 (glut4) was used to detect insulin-stimulated glucose transport, total oxphos rodent antibody cocktail to identify variations in complexes which are responsible for most of the atp production. caspases (casp-3, -9) to unreveal different activation levels of apoptotic pathway among the cell lines. mitochondrial membrane potential was measured by using rho-damine123 by employing confocal microscopy. the expression level of respiratory chain complex iv subunit mtco1 and casp-3 was higher in hek293t compared to other cell lines. casp-9 was upregulated in cancer cell lines, mostly in hep3b. glut-4 levels were downregulated in cancer cell lines in contrast to healthy cell lines. findings imply that these proteins might have significant roles leading to variable metabolic and apoptotic activity of each cell line during energy production. due to the results, mtco1 might be important in adaptation of different cell lines to regulate the overall respiratory chain complex activity. reduction in glut4 level demonstrates insulin desensitization in cancer cell lines, which might lead to metabolic defects in these cells. besides, since p53 has a repressive effect on glut4, it also can lead us to study about p53 levels. the effect of inhibition of pi3k/akt/mtor signaling pathway on receptor tyrosine kinase expression in breast cancer cells g. tunali, a. l. dogan department of basic oncology, hacettepe university cancer institute, ankara, turkey the increased expression and activation of receptor tyrosine kinases (rtk) (egfr, her2, her3) play important roles in breast cancer pathogenesis. her2/her3 interaction is the most potent heterodimer and it causes oncogenic pi3k/akt activation. inhibitors of pi3k/akt pathway (akt inhibitor and pi3k/mtor dual inhibitors) lead to increase in rtk levels and activities while blocking signaling pathway. in this study, the time dependent effect of dual pi3k/akt inhibitor pi-103 on receptor tyrosine kinase expressions' in breast cancer cells was investigated. two breast cancer cell lines, mda-mb-468 cells (which has egfr overexpression and pten deficiency) and skbr-3 cells (which has her2 overexpression) were evaluated for the effect of dual inhibitor. these cells were treated with dual pi3k/akt inhibitor pi-103 for different time periods (1-24 h) . egfr, her2 her3 total rtks expression and pi3k/akt pathway inhibition (p-akt and p-p70s6k expression) were evaluated by western blot. in mda-mb-468 cells, there were significant decrease in p-akt and p-p70s6k proteins' expression during the first 3 h. this inhibition was followed by reactivation of the signaling pathway after 6 h. in skbr-3 cells, p-akt and p-p70s6k proteins' expression were significantly decreased during the first 6 h. the pi3k/ akt signaling pathway in these cells were reactivated after 12 h. basal expression of egfr and her3 in mda-mb-468 cells and basal expression of her2 and her3 in skbr-3 cells were found to be very high. transient inhibition of akt and mtor protein kinase activation in tumor cells followed by reactivation of signaling pathway did not result in a time-dependent difference on egfr, her2 and her3 expression levels. these results suggest that dual pi3k/mtor inhibiton by pi-103 may trigger receptor tyrosine kinase reactivation due to the signal distruption without affecting total protein expression level. site-specific bioorthogonal reactions are one of the significant tools for discovering different aspects of biological systems in live cells. the reactions should be highly stable and rapid in physiological conditions. various chemical tools can be used in bioorthogonal reactions to monitor biological systems, therapeutics, microscopy and diagnostic applications in live cells. synthetic covalent chemistry in the study of biological systems has been used to label biomolecules selectively in their native environment. for example, small synthetic fluorophores can be added to biomolecules without disturbing other molecular biological pathways. aldehydes or ketone-based functional handles can be attached onto protein at specific sites via chemoenzymatic reactions. labeling of carboxy terminus of a-tubulin has been successfully studied in our previous studies by replacement of wild type tyrosine with unnatural amino acid 3-formyltyrosine in the presence of tubulin tyrosine ligase enzyme (ttl)-as its role can suggest whether certain cancer cells might grow more aggressively than others. in this work, we highlight the synthesis and spectroscopic properties of azacoumarin chemical probes to study tubulin-tubulin tyrosine ligase (ttl) system in live cells. significant increase in fluorescence quantum yield or a red shift on absorption and emission maxima is observed when the conjugated product is formed. bioorthogonal fluorescent labeling is such a favorable reaction to perform rapid kinetics, localization and high site-specificity in cell environment. newly synthesized azacoumarin fluorophores should therefore not only be useful for studying ttlbased biological systems, but also would enable broad range of high-yielding and fast diagnostics for future biolabeling applications in biochemistry, cell biology and beyond. binase is an extracellular ribonuclease from bacillus pumilus which shows antiviral and antitumor activities in cell cultures. however, the action of binase on intracellular functions and processes has not yet been identified. here, for the first time we report the whole transcriptome analysis of binase-treated human lung adenocarcinoma epithelial a549 cells. a plasmid-based reverse genetics system and colorimetric cell viability assay were used to identify the binase internalization and binase cytotoxicity towards a549 cell line, respectively. for the whole cell transcriptome analysis a549 cells were treated with 100 lg/ml of binase for 24 h followed by mrna extraction and library preparation. sequencing was performed on solid 5500xl wildfire next-generation sequencer. we found that binase internalized into a549 cells after 2 h of incubation. the binase at 100 lg/ml was absolutely non-cytotoxic towards a549 cell line and was active in the cell culture medium during 48 h incubation. the analysis of rna-seq data showed that among 13 thousands of protein coding transcripts 791 transcripts were up and down regulated by binase, among them 79 transcripts were induced and repressed. binase repressed the production of s100a16 and tnxb which act cancer biomarkers, scn8a and drd4 which play a crucial role in cancer metastasis and responsible for pediatric tumors, respectively. the induction of transmembrane protein transcript abcb11 by binase can help binase to internalize into the cell as abc transporters are often account for transporting drugs across the cellular membrane. binase induced the production of nlrp3, rasgrp1and alpk2 transcripts which can activate apoptosis, cytokine or t cell activation in cancer cells. thus, binase exerts different effects in cancer cells. the rnaseq data obtained will help to understand the mechanism of binase anticancer action. .72) is a specific group of phosphatases capable of hydrolyzing myo-inositol 1,2,3,4,5,6-hexakisphosphate (phytate) with the formation of less phosphorylated inositol derivatives (from mono-to pentaphosphate). three major types of phytases are recognized on the basis of the first phosphate group hydrolyzed by the enzymes: 3-phytase, 4/6-phytase, and 5-phytase. due to the stereospecific way of phosphate release from the phytate molecule by the action of phytases, these enzymes by themselves and their composition may serve as a potential alternative for production of myo-inositol phosphate isomers with therapeutic properties. chemical synthesis of these compounds is inefficient and costly. pantoea sp. strain 3.5.1 showing high phytase activity was isolated from the forest soil sample of the republic of tatarstan, russia. in this study the main objective was the cloning and expression of pantoea sp. 3.5.1 phytase gene in e. coli. first, we amplified the phytase gene (agpp) from the genomic dna of the bacteria using specific primers phmh_dir and phmh_rev. size of phytase gene corresponded to 1729 base pairs. during the optimization of amplification conditions it was found that the optimum temperature for primer annealing was 66°c. this temperature increases the specificity and efficiency of annealing. then the pcr-product of agpp gene was cloned into the pbad myc/ his vector first. on the next step we carried out subcloning of the agpp into a pet28a expression vector. multicopy plasmid pet28a/agpp contained the sequence of the phytase gene of pantoea sp. 3.5.1 under t7 promoter. the corresponding recombinant protein was expressed in e. coli as a fusion with a 6 his-tag and was detected by western blotting. recombinant phytase was purified via affine chromotography on the ni-nta column and displayed high phosphomonoesterase and phytase activities. bag-1 (bcl-2 associated athanogene) is a multifunctional protein that interacts with diverse array of cellular targets and modulates a wide range of cellular processes, including proliferation, cell survival, transcription, apoptosis, metastasis and motility. in human cells bag-1 exists as three major isoforms (bag-1s, bag-1m and bag-1l) derived by alternative translation initiation from a single mrna, which allows interactions with various molecular targets such as hsp70/hsc70 molecular chaperones, components of the ubiquitylation/proteasome machinery, bcl-2, raf-1 kinase, nuclear hormone receptors and dna. our work aims to investigate how altered bag-1 expression levels affect cell survival in mda-mb-231 (er, pr and her2/neu negative) breast cancer cell lines. we first cloned bag-1l gene to a cloning vector, later we transfected mda-mb-231 cells for overexpression of bag-1. we also used bag-1 sirna to silence bag-1 gene. western blot analysis was applied to demonstrate relative expression levels of bag-1, its interacting partners and certain proteins which are important for apoptosis pathway. we performed xtt cell viability assay for bag-1 overexpressed cells to checkbag-1's impact on cell survival, and observed enhanced survival rates on cells compared to that of the untreated cells with bag-1 overexpression. in addition, our study revealed that once bag-1 forms a complex with c-raf/b-raf/hsp70/akt/bcl-2, modulation of cell survival was observed. we believe that once the exact localization and involved molecular mechanisms of bag-1 and its isoforms are found, the role of each bag-1 isoform in cell survival can be understood better. this can further provide routes to study tumor development. the aim of this study is testing the recombinant glp1 encapsulation into a biocompatible material. we also tested if it can be a therapeutic candidate drug for type 2 diabetes. the incretin hormones, which are also named as endogenic peptide hormones have become a more attractive therapy for type 2 diabetes because of different physiological effects. in circulation, glp1 is cleaved by ddp4 in a very short time. if glp1 can be protected from cleaving, the effective time of glp1 would be increased and by this way it can replace the therapy of insulin. chemical synthesis methods of peptides are limited because of low efficiency and high cost. the production of peptides by recombinant e. coli is an alternative way because of effective production, simplicity and low cost. however, the major disadvantage derived from the recombinant e. coli is the frequent formation of inclusion body. for that reason, extra methods are needed for obtaining soluble recombinant peptides. glutathione s-transferase (gst) tag is commonly used as affinity and solubility tag to improve the solubility of recombinant peptides. in this study, we cloned and heterologously produced glp1 using the gst fusion system in e. coli. affinity purification of recombinant protein was achieved by using glutathione immobilized columns. characterization of the gst-tagged glp1 was performed by sds-page. the purity of fusion protein was found to be 65%, as confirmed by glp1 elisa kit. then, the fusion protein was encapsulated in a chitosan coated polygalacturonic acid. the different ph stability and in vitro release tests also in different phs was studied. morganella morganii is an opportunistic pathogen capable of causing a wide range of clinical infections. it is known that microbial metalloproteases play an important role in the development of bacterial infections. thus, investigation of m. morganii metalloproteases has a particular interest. bacteria were grown in lb medium at 37°c. as a bioinformatics tool blast was used. for molecular biological experiments, thermo scientific kits and sibenzyme enzymes were used. pbad/myc-his plasmid was used as an expression vector. bacterial transformation was carried out by heat shock method. bacterial cells were disrupted by sonication. gene expression products were analyzed by western blotting. to analyze the actinolytic activity of bacterial extracts sds-page electrophoresis was used. the putative metalloprotease gene (an cp004345.1) has been found in the genome of annotated strain of m. morganii kt. its amino acid sequence has partial homology (37%) with actin specific metalloproteases grimelysin from s. grimesii and protealysin from s. proteamaculans. using specific primers the gene with 99% homology was identified in the genome of clinical isolate of m. morganii 4. rt-pcr analysis showed that this gene had the maximum expression at 48 h of growth. in addition, the cellular extract of m. morganii 4 had small actinolytic activity. cloning of the gene into e. coli dh5a cells led to the synthesis of the 35 kda protein. it is known that the highest expression of serratia proteases is observed at 48 h of growth, and the molecular weight of the mature proteins is 32 kda. it was shown that metalloprotease gene of m. morganii 4 expressed at the same time of growth. moreover, the recombinant e. coli cells synthesized protein with the similar weight (35 kda) which perhaps is a mature form of the metalloprotease from m. morganii. as a result, in the genome of m. morganii 4 the metalloprotease with similar properties to grimelysin and protealysin proteases was identified. the preliminary characterization of p-ii like protein glnk from lactobacillus brevis z. iskhakova 1 , d. zhuravleva 1 , a. laykov 1 , k. forchhammer 2 , a. kayumov 1 1 kazan federal university, kazan, russia, 2 eberhard-karls university tuebingen, tuebingen, germany the p-ii proteins in bacteria, archaea and plants regulate the activity of a variety of proteins in response to specific metabolic signals which affect their structural state and interaction ability. among various bacteria belonging to lactobacillus only some species have genes encoding pii protein in the genome. here we report the preliminary characterization of pii-like protein lbrglnk from lactobacillus brevis. while the amino acid sequence alignment revealed only 50-70% homology of lbrglnk with other well studied pii proteins, lbrglnk also has the atp binding motive gdgk. trimeric structure of lbrglnk was confirmed in vitro by size exclusion chromatography, suggesting possible similarities of lbrglnk properties with pii proteins. the isothermal titration calorimetry revealed a preferential binding of adp (kd = 50 lm) over atp (kd = 357 lm) suggesting that they compete for binding to lbrglnk. neither 2-oxoglutaric acid nor other nucleotides were interacting with lbrglnk in itc measurements. the mutation gly91ala in the atp binding motive completely abolished the interaction with both adp and atp. the pull down of lbrglnk with l. brevis cell extract allowed identification of chaperonin grol, transketolase and glnr-like transcriptional regulator from merr family as most probable partner proteins for interactions with lbrglnk. this work was supported by the russian foundation for basic research (project no. 15-04-02583a background: hemophilia is a bleeding disorder due to the deficiency in coagulation factors viii (hemophilia a) or ix (hemophilia b). hemophilia patients are essentially treated with intravenous replacement of the missing or dysfunctional factors fviii or fix by recombinant proteins. these therapies often induce the generation of acquired antibodies, and thus, novel approaches are needed. most recent hemophilia strategies target the tissue factor pathway inhibitor (tfpi), which is the major inhibitor of the coagulation cascade, particularly of the extrinsic tenase complex. anti-tfpi agents have been empirically developed such as aptamers, peptides, monoclonal antibodies. we have followed a structure-based strategy, to design a mutated fxa that would show more affinity for tfpi, and thus trap this inhibitor. tfpi exists as two isoforms are folded as multi-kunitz domains related by linkers. the second kunitz type domain of tfpi (tfpi-k2) is known to bind the catalytic site of fxa. methods: the molecular complex of tfpi-k2-fxa was modeled and submitted to molecular dynamics (md), allowing the identification of low-spots interaction. modified fxa with theoretically stronger interaction with tfpi-k2 were predicted using md. the mutants and wild type proteins were expressed in hek cells, and their processing status was checked. they were tested by western blotting, by chromogenic activity using a specific substrate of fxa, by thrombin generation assay in fviii depleted plasma. finally, their binding to a tfpi-k2 peptide array was compared. results: the mutants showed better efficiency to restore thrombin generation in plasmas from hemophiliacs and displayed stronger binding to tfpi-k2 than the wild type fxa. conclusions: the proof of concept of the synergistic approaches of md and mutagenesis was obtained and an efficient tfpi trap was designed. the mutated fxa is a candidate for a new hemophilia therapy. organophosphorus acid anhydride (op) nerve agents are potent inhibitors which disrupt the mechanisms of neural transmission. organophosphorus acid anhydrolase (opaa; e.c.3.1.8.2) is a class of enzyme that potentially acts on phosphorus anhydride bonds, reported intracellularly in diverse organisms, albeit notably the enzyme belongs to alteromonas species are more extensively studied. whereas mass-transfer problem is a major issue in native whole cell biocatalysis, new anchor system derived from the n-terminal domain of ice-nucleation protein from pseudomonas syringe inav (inav-n) was used for the first time to display opaa onto the cell surface. tracing of the recombinant protein and its activity assay showed a successful presentation of opaa and its significant ability for biodegradation of organophosphorus compounds. further studies on bacterial fractions confirmed that opaa is remarkably located on the outer membrane. the specific activity of recombinant bacteria to degrade diisopropylfluorophosphate (dfp) was measured at 260 u/mg of cell wet weight, which almost all was observed in the outer membrane fraction. recombinant cells could also degrade chlorpyrifos (cp) compound in 195.6 u/mg activity. it can be concluded that inav-n anchor is efficient for targeting opaa on the cell surface and can effectively eliminate the masstransfer problem in native whole cell bioconversion system. proper spatial and temporal organization of proteins involved in cell signal transduction is crucial for the specific and efficient information transfer. scaffold proteins coordinate the action of signaling molecules by their physical binding and organization in multiprotein complex assemblies. multiple protein binding is often mediated through intrinsically disordered regions of the scaffold, where the interaction epitopes are defined by linear peptide motifs. using a hub scaffolding protein axin as a paradigm, we have employed peptide microarray technique to identify the binding epitopes for axin interaction partners at high resolution. this enabled us to design axin-derived peptides corresponding to the respective binding epitopes that compete for the interaction in vitro. by transfection of chemically stabilized competitive peptides directly into the cells, we have shown the effect of specific interaction blocking on axin-mediated signaling in vivo. our data demonstrate a proof of concept for a rational design of inhibitors of protein-protein interactions that allow specific intervention with single function of the targeted protein (i.e. recruitment to the axin complex). contrary to the inhibitors that completely disrupt the protein function (e.g. inhibition of a kinase catalytic site), this approach provides a tool for investigating specific action within the axin complex, while the other cellular functions of the targeted protein remain preserved. the results of this research have been acquired within cei-tec 2020 (lq1601) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. de novo design of an artificial biocatalyst using immunoglobulin template became rather routine procedure due to the achievements of molecular biology and crystallography. recently the 'reactibody' approach was developed based on the chemical selection of catalytic repertoires from immunoglobulin library followed by expression of these biocatalysts in eukaryotic system. in this study we structurally characterize the a5 antibody, its kappa and lambda variants, in order to understand the difference on the active site between a5 and a17 which although there are two antibodies sharing very high homology and sequence identity their active residues are located in a different region of the antibody. the structures of the a17 antibody kappa and lambda variants have been already determined, there was no structural information though about the a5 antibody. the structural analysis revealed dramatically different angle in position of nucleophilic residue tyr33 and area of solvent accessible surface. the structural difference of active center reflects on kinetics of the a5 organophosphate modification. both variants of antibody bind with organophosphate throw induce-fit mechanism, but rate of the step of induce fitting is different (k obs are 35 s à1 and 16 s à1 for a5kappa and a5lambda respectively). this observation may hint at novel means of regulation of velocity and specificity of artificial biocatalysts. this study was supported by grant #rfmefi61614x0009. translation elongation factor 1ba (eef1ba) is a component of a heavy form of translation elongation factor 1 (eef1h). it functions as a catalyst of gdp/gtp exchange in translation elongation factor 1a (eef1a) restoring its active conformation appropriate for aminoacylated trna binding. eef1ba forms a tight complex with translation elongation factor 1bc (eef1bc) via the n-terminal domain, while its c-terminal domain executes the catalytic activity. eef1bc has been shown to enhance the attributed to the c-terminal domain catalytic activity of eef1ba. this suggests that the eef1ba n-terminal domain may influence the guanine nucleotide exchange process. to test this hypothesis we prepared a set of n-terminal truncated forms of human eef1ba and checked their activity in the guanine nucleotide exchange assay on both isoforms of eef1a, eef1a1 and eef1a2. we showed that recombinant eef1ba is a non-globular monomeric protein in solution with an elongated shape by analytical ultracentrifugation approach. the truncation of the dispensable for the catalytic activity n-terminal domain of eef1ba resulted in significant acceleration of the rate of guanine nucleotide exchange in eef1a2 comparing to full-length eef1ba. similar effect on the catalytic activity of eef1ba was observed after its interaction with eef1bc. in contrast, the effect of full-length eef1ba and its truncated forms on the rate of guanine nucleotide exchange in eef1a1 was similar but relatively modest compared to eef1a2. this can be explained by higher rate of spontaneous gdp dissociation from eef1a1 comparing to eef1a2 and lower affinity of eef1a1 to eef1ba. thus, we propose that the n-terminal domain of eef1ba via flexible linker region may interfere with eef1a binding to the cterminal catalytic domain that results in a decrease of the overall rate of guanine nucleotide exchange reaction. the formation of a tight complex between eef1bc and eef1ba n-terminal domains abolishes this inhibitory effect. p-02.02.2-079 assessment of quantitative proteomics results in large-scale data-independent with fragmentation spectra reproducibility measure reduces variation and allows to use lowintensity signals organisms with reduced genomes that lack the vast majority of transcriptional or translational regulation systems tend to adapt to changing environment with a variety of subtle changes in protein abundances. as soon as relative changes for most proteins fall below 50%, the power of traditional label-free proteomic analysis rapidly becomes insufficient for robust profiling of hundreds of samples. intoduction of frament-by-fragment and sample-by-sample signal quality assesment in mrm and dia experiments helps to increase accuracy of methods and at the same time reintroduce cases which could have been excluded during bulk quality assessment due to lower signal-to-noise ratio for several fragments. 240 samples of mycoplasma gallisepticum were acquired in data-independent manner on sciex tripletof 5600+ mass-spectrometer (swath acquisition) during the year. the samples were produced from mycoplasma gallisepticum culture cultivated at different temperatures. the signals for each fragment were extracted with vendor-supplied software with the theoretical fragment ions for each peptides instead of spectral library. the results were used for relative protein quantitation in two manners the first conventional method included direct use of peptides with top 3 most intense signals. the second included selection of peptides and ions for quantification for each pair of samples based on the reproducibility of fragmentation patterns after computing the areas of chromatographic peaks for each ion. spectral angle was used as a distance measure for fragmentation patterns for clustering. further, a base set of detected ions was selected for each peptide and a subset for comparison of each pair of runs. the first method resulted in quantitation of 354 proteins across all samples with variation across lc-ms replicates was 21% on average, and the second approach led to quantitation of 515 proteins in total, 390 of them across 75% of samples, all with the variation about 11% on average. p-02.02.2-080 interaction of plasminogen fragments k 1-3 and k 5 with fibrin fragment dd t. yatsenko, v. rybachuk, l. kapustianenko, s. kharchenko, o. yusova, t. grinenko palladin institute of biochemistry of nas of ukraine, kyiv, ukraine introduction: plasminogen interaction with specific binding sites in c-terminal d-domains of fibrin molecule initiates the activation process of proenzyme and subsequent fibrin clot lysis. the sites are exposed under fibrin polymerization. plasminogen kringle domains ensure the proenzyme interactions with fibrin clot. in this study, we investigated the binding of human plasminogen kringle fragments k 1-3 and k 5 with human fibrin fragment dd and their effect on glu-plasminogen interaction with dd. results: kringle-containing fragments k 1-3 and k 5 reduce plasminogen activation by tissue-type activator on fibrin fragment dd. the level of glu-plasminogen binding to dd is decreased by 50-60% in the presence of k 1-3 and k 5. fragments k 1-3 and k 5 have high affinity to fibrin fragment dd (dissociation constant is 0.02 lm for k 1-3 and 0.054 lm for k 5). analysis of k 1-3 and k 5 binding to fibrin fragment dd with reduced disulfide bonds showed the interaction of both plasminogen fragments with c-c-chains of fragment dd. k 1-3 interacts with complex of fragment dd-immobilized k 5 as well as k 5 with complex of fragment dd-immobilized k 1-3. the plasminogen fragments do not displace each other from binding sites located in fibrin fragment dd, but can compete for the interaction. analysis of k 1-3 and k 5 binding to fibrin fragment dd with reduced disulfide bonds showed the interaction of both plasminogen fragments with aand c-chains of fragment dd. conclusions: widely known specific plasminogen-binding site located in aa148-160 region of fibrin molecule is not a single binding sequence of fibrin peripheral domains or plasminogenbinding site is not linear and contains amino acid residues from other polypeptide chains of fibrin d-domains. fibrin fragment dd contains different binding sites for plasminogen kringle fragments k 1-3 and k 5, which can be located close to each other. possible plasminogen kringle-binding sites are located in aand c-chains. p-02.02.2-081 implementation of budded baculovirus particles for characterization of ligand binding to g protein-coupled receptors a. allikalt, a. rinken university of tartu, tartu, estonia g protein-coupled receptors (gpcrs) constitute the largest class of membrane receptors involved in regulation of signal transduction into the cell in response to various extracellular stimuli. for that reason, gpcrs have become important targets for variety of drugs. as these receptors are present in native tissues at very low concentrations, efficient recombinant expression systems are needed to produce sufficient amounts of protein. we have shown that budded baculovirus particles, which display gpcrs on their surfaces can be used as a source of receptors for the investigation of ligand-receptor interactions. this expression system can be used for radioligand binding assay as well as for fluorescence anisotropy-based assay (fa). we have validated the system with budded baculovirus particles produced in spodoptera frugiperda (sf9) cells expressing human dopamine d 1 receptors using [ 3 h]sch-23390 and bodipy-fl-skf-83566 as reporter ligands for corresponding assays. this system has many advantages, for example good signal to noise ratio, homogeneity of the receptor, high expression levels and long-term stability of the receptor preparation. fa method allowed real-time monitoring of reporter ligand binding in the absence and presence of different dopaminergic ligands, giving information about their kinetic properties. association, as well as dissociation of the bodipy-fl-skf-83566 itself were rapid with an apparent half-life of t 1/2 = 38.5 ae 0.3 s for association (2 nm) and t 1/2 = 73.4 ae 3.8 s for dissociation. we determined the pharmacological profiles of different dopaminergic ligands in displacement binding assays with membranes of sf9 cells or budded baculovirus particles. the data were in good agreement for both membrane preparations tested in radioligand binding as well as in fa assay. obtained results indicate that budded baculovirus particles can be proposed as a source of gpcrs for performing fluorescence anisotropy as well as radioligand binding assays. gastrointestinal (gi) cancer includes a variety of cancer types affecting the structures and functions of the gi system, encompassing the gi tract and the accessory organs of digestion, from the esophagus, stomach, biliary system and pancreas to the intestine, rectum and anus. despite the significant advances however, much remains to be learned in the spectrum of gi cancer. several investigators have shown that both gas6 and its receptors, axl, sky, and mer are expressed in various types of cancers. however, the expression level of gas6 in gi cancer remains unclear. the aim of the study was to determine and compare plasma gas6 levels in gi cancer patients. 15 female and 27 male patients were included in the study (n = 42): 21 colorectal, 8 gastric, 4 pancreatic, 3 liver, 2 ampullary, 2 gall bladder and 2 esophageal. from all gi cancer patients, 2 ml venous blood was collected in citrate tubes before surgery. blood samples were centrifuged at 3000 g for 10 min, and plasma samples were carefully removed and stored in à80°c prior to use. the level of plasma gas6 was measured using a commercial developmental elisa kit (r&d systems, minneapolis, mn) which is validated by our laboratory. plasma gas6 levels in cancer patients were determined as follows: 1.84 ae 1.1 ng/ml in colorectal; 1.16 ae 1.1 ng/ml in gastric; 1.63 ae 0.61 ng/ml in pancreatic; 3.91 ae 0.89 ng/ml in liver; 2.15 ae 0.26 ng/ml in ampullary; 1.34 ae 0.46 ng/ml in gall bladder and 2.32 ae 1.7 ng/ml in esophageal cancer. preliminary findings indicate that there is a relation between gi cancers and plasma gas6 levels. taken together, these results suggest that gas6 may be a candidate biomarker for diagnostic use in gi cancer. inhibition of gas6 would be an attractive therapeutic target for slow down the progression of gi cancer. monday 5 september 12:30-14:30 computational biology p-03.03.2-001 computational approaches as an assay for blactam hydrolysis in class a b-lactamases c. tooke university of bristol, bristol, united kingdom b-lactam hydrolysing enzymes, in particular carbapenem-hydrolysing enzymes, are an increasing clinical threat. herein we show that molecular dynamics (md) and combined quantum mechanics/molecular mechanics (qm/mm) approaches are a predictive tool of carbepenemase activity in class a b-lactamases. b-lactam drugs are the most prescribed class of antibiotics worldwide, especially in the treatment of gram-negative pathogens such as klebsiella pneumoniae and escherichia coli. these organisms produce b-lactamases, enzymes which hydrolyse the b-lactam ring, a key resistance mechanism. class a b-lactamases have the ability to hydrolyse carbapenems, termed 'last resort' antibiotics. in particular, the kpc (klebsiella pneumoniae carbapenemase) family are the most clinically important, and recently identified natural kpc variants show increased hydrolytic activity against ceftazidime, a third generation cephalosporin. here we use computational simulations of b-lactam hydrolysis by b-lactamases. in particular, molecular dynamics (md) combined with qm/mm approaches have been used to model the deacylation of the carbapenem meropenem across 8 class a b-lactamases. this method has been extended to model cephalosporin hydrolysis across class a b-lactamases, including kpc variants. these approaches calculated the free energy barriers and correctly distinguished carbapenemases from carbapenem-inhibited enzymes. preliminary results suggest this protocol is also a predictive tool for ceftazidime hydrolysis. further, md simulations of 5 kpc variants (single and double amino acid changes) were analysed to identify structural changes in the active site, highlighting that variants differ in the size of the active site opening, corresponding with experimentally derived kcat values. these computational assays provide a predictive tool of b-lactam hydrolysis and has potential to provide insights into important mechanistic differences both across class a b-lactamases and within the same families. p-03.03.2-002 computational design of a novel polyglutamic dendrimer-based platform as an anticancer therapeutic approach poly (glutamic acid) (pg)-dendrimer as potential nanocarriers for cancer therapies, to specifically deliver tumor associated antigens (taa)mannosamine and melanato target cells and to modulate cancer antigen intracellular trafficking within the cytoplasm to promote an efficient and selective antitumor immunotherapeutic effect. the theoretical structures were obtained using x-plor software. the molecular dynamics simulation of pg-g4-dendrimer and taas was performed using desmond. the electronic properties of the structures were determined by semi-empirical methods using mopac. docking studies of taa to pg-g4-dendrimer to mannose receptor (mr1) were performed using hex 8.0.0 software. taa lumo atoms were conjugated to homo atoms of pg-g4 dendrimer using maestro software. results showed that pg-g4-dendrimer displays 64 carboxylic end groups available for covalent interaction with taas. the homo molecular orbitals of the dendrimer was located on the a-carbon of the carboxylic acid groups from backbone chain and it preferentially interacts with lumo molecular orbitals of amine group from taas. no differences in the gap energy of homo/lumo of all pg-g4-conjugates. taas bind preferentially to a-carbon of cooh of backbone chain instead of cooh from side chain. docking results showed that majority of taa conjugated pg-g4-dendrimer binds to the core of the mr1 receptor. increasing of the number of mannosamine conjugated to pg-g4-dendrimer more close and stable is the conjugated to the receptor. this system shows promising results as a novel functionalized pg-dendrimers for cancer therapy. theileria parva is one of the the economically important protozoan of the theileria genus belong to apicomplexa phylum which include plasmodium spp. and toxoplasma gondii, causative agents of malaria and toxoplasmosis respectively. this parasite is the disease agent of tick-borne east coast fever (ecf) ranks first among the tick-borne diseases of cattle in sub-saharan africa. the disease caused by the parasite affects a large proportion of domestic and wild animals and leads serious economic losses in the world. major problems in dealing with this illness are the high cost of drugs, development of resistance, and absence of effective vaccines. thus, it is important to develop an efficient and affordable antitheilerial agent. for this aim, 1-deoxy-d-xylulose-5 phosphate reductoisomerase (dxr) which subjected to identify novel drug aganist malaria and toxoplasmosis, of theileria parva was selected as potential target for improving novel inhibitors aganist ecf. a computational molecular modeling approach was conducted to determine the 3d structure of tpdxr by phyre2. energy minimisation and root mean square deviation (rmsd) was performed by 3drefine and superpose servers. to ensure the quality of modelling, stereochemistry, energy profile and residue environment of modelled structure were checked by different servers and possible ligand binding pockets were identified by metapocket 2.0 server a reliable 3d model for dxr from t. parva was modeled by using 3au9 as a template. the ca rmsd and the backbone rmsd deviations for the model and the template crystal structure were found to be 0.85 and 0.86 a, respectively. the ramachandran plot for the predicted model by rampage reveals that model shows an acceptable stereochemistry. top three considered possible binding pockets have been identified. these results have important implications for future screens aimed at finding new and safe molecular entities active against tpdxr through docking studies. p-03.03.2-004 molecular binding profile of protoberberine alkoloids on amyloid precursor proteincleaving enzyme 1 (bace1) as a drug candidate for alzheimer's diseases g. yalcin 1 , i. yildiz 2 1 biotechnology institute, ankara university, ankara, turkey, 2 department of pharmaceutical chemistry, faculty of pharmacy, ankara university, ankara, turkey alzheimer's disease (ad) is the most prevalent neurodegenerative disorder that leads to dementia and nowadays over 46 million people live with dementia worldwide. because of the prevalence and economic burden of the disease, drug development studies have picked up speed and scientists especially focused on natural products. ad is basically characterized with tau hyperphosphorylation and accumulation of amyloid b (ab) proteins. ab proteins are generated from sequential cleavages of amyloid precursor protein (app) by b and c secretases, and b-site app cleaving enzyme 1 (bace1) is a b secretase essential for ab production. the alkaloids represent a very extensive group of secondary metabolites, with diverse structures, distribution in nature and important pharmacological activities. protoberberine alkaloids, which belongs to isoquinoline alkaloid class, are widely arranged in many species of the berberidaceae, annonaceae, fumariaceae, papaveraceae, and other plant families. recent searches showed that some of the protoberberine alkaloids such as berberine, palmatine, jatrorrhizine, columbamine, magnoflorine prevents the progress of neurodegenerative disorder. however, the mechanisms of them are not absolutely clear. therefore, we have aimed to elucidate the binding and affect mechanism of these alkaloids on the bace1 open and closed forms in here. for this purpose, molecular docking studies were applied for these natural products to the both forms of bace1 by using autodock vina and it was subjected to explicit solvent simulations by amber molecular dynamic package. our preliminary studies indicate that gly34, thr72, gln73, phe108, tyr198, lys224, thr232, arg235, thr329 residues of binding pocket have affiliations with all of the mentioned alkaloids and the binding of them generates alterations on closed form of bace1. the complexity of animal milk needs to apply numerous approaches and methods for its investigations. an understanding of the processes occurring in the milk can be used, for example, for quality control of the products. fat and protein are main components of milk which have a significant influence at its colloid properties, such as dynamic surface tension (dst). the application of regression-correlation analysis to milk data enables to develop a reliable quantitative model. the aim of our investigation was to perform the regression analysis to establish the relationship between above-mentioned parameters. for this purpose, we used a statistical software packages r version 3.1.2. dst was determined by bpa -1p tensiometer. milk fat (f) and protein (p) contents were measured by analyzer bentley 150. this work was supported by the russian scientific foundation (grant 14-16-00046). obtained formulas characterized the degree of influence of fat and protein contents of a milk sample for each of the dst parameters (r 0 , r 1 , r 2 , r 3 , k 0 , k 1 ): r 0 = 61.1538 + 0.8908 * p à 1.2953 * f r 1 = 63.3297 + 0.5658 * p à 1.4132 * f r 2 = 55.4274 + 0.4585 * p à 1.0143 * f r 3 = 45.6265 + 0.34634 * p + 0.05197 * f k 0 = 6.0986 + 0.1543 * p + 0.1351 * f k 1 = 10.7832 à 0.1470 * p à 1.0302 * f these formulas show that the maximum total effect of fat and protein contents influences at r 0 and r 1 . a significant coefficient (> 1) before the fat is observed in the formula, which describes the value of the tilt of final part of the tensiogram (k 1 ). the resulting regression equations have fundamental importance. with their help it is possible to calculate the dst parameters without their experimental determination, positioning fat and protein contents data. obtained dst parameters promote more complete characterization of the properties of the milk that may be used for dairy products. p-03.03.2-006 molecular studies of scorpion toxin and its mutants interactions with voltage-gated potassium channels the voltage-gated potassium kv1.3 channel is mostly expressed in neurons and immune cells. its blockage has a high therapeutic potential, for example, specific inhibitor shk toxin is undergoing clinical trials on psoriasis. goal of the current study was an interface analysis in complexes of hybrid channel kcsa-kv1.3 with peptide blockers agitoxin and its mutant forms. 3d structure was generated by homology modeling method using complex of mutated kcsa channel with charybdotoxin (pdb-code 2a9h) as a template and equilibrated by molecular dynamic simulation in gromacs software. analysis of hydrophobic and stacking interactions, hydrogen and ionic bonds of the toxin and potassium channels was performed for representative frames with optimal toxin orientations using program platinum and apbs software package. we performed contacts energy characteristics estimation to predict key toxin residues for binding process and possible mutation sites for changing selectivity against kv1.x channels. the results of investigation are in good agreement with the experimental values of binding constants, obtained by competitive binding assays. results of the conducted investigation may find an application in fundamental science and drug design. the research was supported by the russian science foundation grant no. 14-14-00239. simulations were performed using the supercomputing center of lomonosov moscow state university. p-03.03.2-007 homology modeling and molecular docking study of the paraoxonase-1 and its polymorphic variants q/r 192 and m/l 55 for non-statin lipid lowering drugs paraxonase-1 (pon1) enzyme is an hdl associated ester hydrolase exhibiting paraoxonase, arylesterase and lactonase activity, and reduces the formation of atherosclerosis blocking the ldl oxidation and reducing levels of oxidized lipids. in this study, molecular docking approach and molecular dynamics simulation were applied for finding the affinity of non-statin lipid-lowering drugs to pon1 and its polymorphic structures pon1 q/r 192 and m/l 55. fibrates (bezafibrate, ciprofibrate, clofibrate, fenofibrate, gemfibrozil), phytosterols (beta-sitosterol, brassicasterol, campesterol, stigmasterol) and other lipid lowering drugs (ezetimibe, niacin, orlistat, probucol, and sibutramine) was obtained from pubchem database. x-ray crystallographic structure of human pon1 and its polymorphic variants pon1 q/r 192 and m/l 55 was generated via 'modeller', homology modelling software, from human-rabbit hybrid x-ray crystal structure of pon1 (pdb code: 3sre). 10 ns molecular dynamic simulation analysis was performed using gromacs 4.5.5. affinity of lipid lowering drugs to pon1 and its polymorphic variants was predicted by molecular docking approach using autodock 4.2 suite. unlike other lipid lowering drugs that they have negative δg values for affinity, probucol, orlistat and betasterol was calculated by positive δg values (18.7, 12.3 and 1.4 kcal/mol). these values suggest that they may have no affinity to pon1 q/r 192 polymorphic structure. in all drug groups, brassicasterol and stigmasterol to pon1-m/l 55 and sibutramine to pon1 q/r 192 were calculated as the highest affinity. in generally, phytosterols predicted by high affinity to pon1 and m/l 55 polymorphic structures in comparison to other lipid lowering drugs. our study demonstrated that phytosterols predicted as high affinity compounds on pon1 structures may reduce the activity of antioxidant pon1 enzyme. this study need to be supported by in vitro and in vivo detailed studies. prolactin and its cognate receptor, prolactin receptor (prlr), are involved in over 300 distinct functions in mammalians. the mammalian prlr gene consists of 10-13 exons and several 5 0 and 3 0 regulatory sequences. in this study, gaps and annotation errors in the rat prlr gene were corrected by comparing the genomes of mammals and rodents and new putative exons were identified. the rat prlr gene sequences from two different sources (rnor_6.0, nc_005101.4 and rn_celera, ac_000070.1) were used and primary analysis showed that both sequences contain several gaps (varying from 0.35 to 4 kbp), corresponding to about 5.6% (10-11 kbp) of the gene. using the rat known prlr mrna exon sequences, it was found that the rnor_6.0 prlr gene has two exon-10 (one is about 2 kbp long and the other immediately after this). comparisons of mammalian and rodent prlr gene structures showed that the 2 kbp stretch is an assembly artifact. by comparing both gene sequences (and also other available rodent prlr genes), the gaps in the rat prlr gene were reduced from 5.6% to 3.8% (from 11 kbp to 7 kbp). functional annotation of the gene revealed that r. norvegicus prlr gene could have two more additional exons, exon-12 and -13, similar to mus musculus prlr gene. in mammals, prlr mrnas contain non-protein coding exons in the 5 0 utr (exon-1 and -2). in rats, there are 5 exon-1 variants, resulting from alternative promotor usages. studies on the rat and mouse prlr genes revealed that both rodents share 4 common non-protein coding exon-1 variants. in conclusion, it is found that the rnor_6.0 version of the prlr gene has the highest number of unidentified base pairs (corresponding to 5.6% of the gene) and the second exon-10 is the assembly artifact. the rat prlr genes in both databases have several gaps and our corrected version is the best available and characterized form of the rat prlr gene. in silico affinities of some statins to paraoxonase-1 enzyme the structure of the statins (atorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin) was obtained from pub chem database, and x-ray crystal structure of pon1 (pdb id:3sre) from protein data bank. modeller software was used for homology modeling of pon1 and its polymorphic variants that's called as pon1 q/ r 192 and m/l 55. amino acid sequence of human serum pon1 (uniprot: p27169) was used as the modeller template. all molecular dynamics simulations were carried out with gro-macs 4.5.5 software. molecular docking calculations on each of the polymorphic structure of the pon1 was performed with auto dock4.2. suite. for each substrate, y71 residue showed open conformation in pon1 and m/l 55 polymorphic structures while q/r 192 polymorphic structure showed closed conformation. in comparison between structures of pon1 variants, in most cases statins had lower affinity to q/r 192 polymorphic structure than to the other variant. in this study, among statins, atorvastatin showed lowest but simvastatin highest affinities to pon1. by considering that the high affinity drugs can have reducing effect of pon1 activities, it may be more appropriate to use the low affinity statins in hyperlipidemia treatment. however, these findings need to be supported with in vivo and in vitro studies. p-03.03.2-010 self-assembly of lipidoids for sirna uptake and release mechanisms studied by molecular dynamics simulations o. acar 1 , d. alpay 2 , a. r. atilgan 1 , c. atilgan 1 1 sabanci university, istanbul, turkey, 2 northwestern university, evanston, united states small interference rna (sirna) has the ability to bind a specific mrna which provides silencing of selected genes. nanocarriers made out of self-assembled lipidoids encapsulate sirna and deliver them into target cells effectively. in this study, a library of lipidoid structures is constructed and studied by molecular dynamics (md) simulations in different solvents, including sodium acetate, to ferret out their self-assembling mechanisms. the effect of the protonation state of the head group of lipidoids on the final shape of the self-assembled carrier is also studied. we further examine the role of the size of hydrocarbon tails in the packing. we study the final topology and the geometry of the self-assembled lipidoids both in the presence and in the absence of sirna. we find that stable clusters form with as few as 20 chains. for lipidoids having neutral head groups, clusters are in the form of dense bundles, while those with charged head groups form spherical capsids which are depleted of the salt on the inside and having a salt rich phase on the outside. in the self-assembled structure, lipidoids are arranged so as to expose the nitrogen and oxygen atoms to the solvent. while partial capsids with these properties also form at lower lipidoid numbers, 200 chains are necessary to form a fully closed capsid. in the presence of the sirna, the capsid assembles around the nucleotide. the free energy to remove the sirna from the assembly is calculated via repeated steered md calculations utilizing jarzynski's equality relating it to the irreversible work along and ensemble of trajectories. we therefore determine an optimal tail length for the most stable nanostructure, paving the way for designing nanocarriers with high efficacy. milk is one of the most valuable products for humans and attracts a lot of interest of researchers in various fields such as biochemistry, biology, food science and technology. the methods of milk study are quite varied. we chose the combination of the ultrasonic and dynamic surface tension (dst) measurements with the possible correlations among the obtained parameters. the aim of this work is to study the correlation between the parameters of milk, such as a content of fat, protein, lactose, minerals, dry milk solids and dst parameters. for this purpose we used milk analyzer 'klever-1m' and tensiometer 'bpa-1p'. three groups of animals were formed from clinically healthy holstein cows at the age of 4-5 years according to the fat content in the milk sample. group i -5 cows (milk fat content 4.01 ae 0.41%), group ii -6 cows (milk fat content 3.32 ae 0.14%), group iii -11 cows (milk fat content 2.73 ae 0.20%). this work was supported by the russian scientific foundation (grant 14-16-00046). the biochemical parameters of the milk samples of all three groups are in the range of the 'normal' values for healthy holstein cows: protein content varies from 3.0% to 4.2%, lactose and mineral content varies from 4.6% to 0.7%, respectively. the dst parameters (r1, r2 and k0) for the group i have strong positive correlations with the fat content in the studied milk samples. at the same time for the groups ii and iii the fat content in the milk indicates only medium positive and weak positive correlations with the r1, r2 and k0. obtained absolute values of the dst parameters of the milk samples showed some differences between all three groups. thus, the dst parameters are changing in direct proportion to fat content in the milk sample that can be explain by the primarily role of the milk lipids in the formation of the water/fat surfaces (such as fat globules, lipid-protein particles, etc.). p-03.03.2-012 exploration of allosteric paths in caspase molecules using energy dissipation e. n. bingol, o. sercinoglu, p. ozbek sarica marmara university, istanbul, turkey caspases are highly regulated aspartate-specific cysteine proteases that have major roles in programmed cell death; apoptosis. effector caspases are at the terminal step of the pathway, hence they are considered as death switches. with the discovery of the presence of allosteric sites, these molecules attracted the attention of the pharmaceutical studies and became drug targets. as a result of the binding of small molecules to the dimeric interface, active site loops are shifted to an unfavorable position. this is associated with a network between distal allosteric sites and the active site loop. an energy dissipation model was applied in order to analyze this matter in further detail. perturbation of specific residues enable us to determine a possible signaling network in proteins using external energy as an input, while focusing on the dispersion of this energy between residues throughout the structure. molecular dynamics simulations were performed with and without energy perturbation using namd software with charmm27 force field. energy perturbation was applied by increasing the velocity of a chosen residue at the desired time step of the initial md simulation. energy change of each residue was calculated upon the application of perturbation. as a result, residue response times, corresponding to the time of the response of a residue after the perturbation of another chosen residue, are obtained. combining reponse time data with a residue interaction network, it is possible to construct a final network that shows the communication started by perturbation within the molecule. it is shown that perturbation of allosteric sites result in the disruption of the catalytic sites given in literature. our findings support this and also gives a little detail of the possible communication between distal allosteric site and the active site loops. this finding enables the usage of this methodology for similar structures where the exact allosteric mechanism is yet not known. p-03.03.2-013 effect of complex mammalian membrane models with multiple membrane components on ras protein nanoclustering a. farcas 1,2 , l. buimaga-iarinca 2 , c. floare 2 , l. janosi 2 1 faculty of physics, babes-bolyai university, cluj-napoca, romania, 2 national institute for research and development of isotopic and molecular technologies, cluj-napoca, romania ras proteins are essential for the cellular signal transduction that regulates cell proliferation and differentiation and act as binary switches between gdp and gtp forms. a wide range of human tumors are associated with defective ras protein signaling. the production of permanently activated ras proteins is correlated with mutations in ras genes. experiments and computer simulations have shown that membrane-bound ras proteins form nonoverlapping dynamic nanosized subdomains (nanoclusters) in activation state-/isoform-dependent manner. we performed coarse-grained molecular dynamics simulations to investigate the effect of complex mammalian membrane models on formation and evolution of ras nanoclusters. a fundamental part of the plasma membrane is the phospholipids bilayer, which contains phosphatidyl-choline (pc), phosphatidylethanolamine (pe), phosphatidyl-serine (ps), sphingomyelin (sm) and cholesterol (chol). the nature of lipid-lipid and protein-lipid interactions was studied in binary (pc:chol) and quinary mixtures (pc:pe:ps:sm:chol). because the polar lipids are not uniformly distributed between the two leaflets of the membrane, the construction of the plasma membrane with five-component lipid mixtures took into account the asymmetry between the outer and inner mono-layers. the phospholipids chain saturation (combined with the presence of cholesterol) constitute the dominant factor in phase separation and was, therefore, modeled in different lipid tail combinations for various headgroups. using microsecond timescale simulations of membraneembedded ras proteins, we have shown that the nanoclusters are spontaneously forming dynamic structures whose behavioral characteristics is modulated not only by the ras isoform, but also by the complexity of the membrane model. furthermore, we showed that variations in the plasma membrane lipid composition have important implications in the localization of ras protein nanoclusters. optogenetics comprises biological methods to achieve gain or loss of function of well-defined events in specific cells of living tissue by means of targetable control tools that respond to light and deliver the effector function. microbial rhodopsins (mrs) have been established as powerful light-sensitive tools for optogenetics. acting as ion pumps or channels, mrs are used to induce cell (de)polarization to control neuronal activity in a wide range of living organisms. mrs are membrane proteins found in a large clade of organisms, including eukaryotes, bacteria, and archaea. they share a common architecture of 7 transmembrane a-helices and a covalently linked retinal, which is employed to absorb photons for energy conversion or the initiation of cellular signaling. major efforts are put into screening of natural and generating of synthetic mrs with desirable properties for optogenetics, e.g. ion selectivity. however, experimental study of mrs is difficult and resource consuming owing to, among other factors, low expression levels and protein stability. thus, there is a need in developing of computational tools for identification of mrs with desirable properties. we used non-redundant atomic structures of mrs taken from protein data bank to develop a set of numerical descriptors that reflects functional properties of mrs. then, we calculated the descriptors for non-redundant sequences of mrs with known function taken from the uniprot database, resulting in the feature matrix. we applied the support vector machine and the 5fold cross-validation procedure, using the feature matrix as the training set. as a result, we obtained the classifier that discriminates mrs in terms of the ion selectivity, e.g. na + , h + , or cl à pumps, with high precision. finally, we used the derived classifier on a test set of proteins and identified mrs for the further experiment in vivo. rational design of peptides with required stability and functional activity properties becomes a real instrument for the new generation drug development. the reca bacterial protein (and human rad51 homolog) is considered to be the central catalyst of homologous recombination, a mechanism essential for the accurate repair of double-strand dna breaks. dna repair via homologous recombination requires reca nucleoprotein filaments assembly. using seqopt (http://mml.spbstu.ru/seqopt/), a novel method for a-helix sequence optimization we present the successful design of peptide sequences capable to maintain a very stable a-helix structure and to inhibit reca activity. novel a-helical 18 amino acids peptide is constructed based on reca-dna complex structure. we observed in vitro inhibition of reca atp hydrolysis, dna strand exchange reaction and reca filament formation. also, we observed lower e. coli resistance to uv and sos-response suppression in vivo. computational identification of promiscous enzyme activity for the morita-baylis-hillman reaction k. ozturk, s. sayin, n. celebi olcum yeditepe university, istanbul, turkey enzyme promiscuity attracts considerable attention in terms of enzyme evolution, protein engineering and biocatalysis. especially, development of highly efficient novel biocatalysts starting from promiscuous enzymes that have the catalytic machinery to perform desired chemistry is an intense area of research in recent years. in this work, we computationally explored the catalytic promiscuity of natural enzymes for the synthesis of morita-baylis-hillman (mbh) adducts, which display antitumoral activity against human cervical cancer cells, by mining structural protein databases using quantum mechanically optimized theoretical active site models (theozymes). catalytic interactions in the active sites of selected hit proteins with potential mbh activity were evaluated in solvated dynamic environment using molecular dynamics simulations. computational screening of the protein data bank for the quantum mechanically determined optimal arrangement of catalytic functional groups for the target mbh reaction successfully identified an enzyme with experimentally determined promiscuous mbh activity. ras proteins mediate a wide variety of signal transduction pathways that regulate cell growth, proliferation and differentiation. these proteins are small gtpases that act as binary switches between gdp-bound 'off' and gtp-bound 'on' states. oncogenic point mutations of ras are associated with~15% of all cancers and up to 90% in specific tumors and many developmental disorders. both experimental and in silico results showed that the membrane-bound ras proteins form non-overlapping dynamic nanosized subdomains called nanoclusters in an activation state-/ isoform-dependent manner. we performed coarse-grained molecular dynamics simulations in order to investigate the formation and evolution of ras nanoclusters in mammalian model membranes. ras proteins were inserted into the cytoplasmic side of the plasma membrane model (di-c16:0-phosphatidyl-choline: di-18:2-phosphatidyl-choline: cholesterol 5:3:2) where they formed highly dynamic nanoclusters, both in size and in composition. furhermore, we found that the presence of ras protein nanoclusters has a significant impact on the model membrane behavior. properties such as phase behavior, diffusion coefficient, surface tension and lipid tails order parameter are also influenced by the temperature variation of the model membrane. we have investigated dynamics in three different crystal forms of ubiquitin, as well as ubiquitin in solution, with particular emphasis on (i) conformational exchange between b turn type i and ii in the region 51-54 and (ii) rocking dynamics where protein molecules as a whole undergo subtle reorientational motion within the confines of the crystal lattice. experimentally, both motional processes have been probed using relaxation dispersion techniques, including recently developed near-rotary-resonance dipolar relaxation dispersion experiments. thereby it has been determined that rocking motion in one of the crystal forms (pdb id 3n30) occurs on the time scale of tens of microseconds, whereas the conformational exchange has characteristic time constant of ca. 100 ls. using molecular dynamics simulations, we have shown that the similarity of motional time scales is not accidental: bi↔bii exchange and rocking motion appear to be coupled. we have investigated the mechanisms of this coupling and predicted a number of point mutations that are expected to abrogate (or enhance) rocking. the crystals of ubiquitin containing these mutations have been modeled in silico. we have also investigated the interactions (in particular, crystal contacts) that control the balance between bi and bii conformations in different crystal forms. finally, we have used md simulations as a basis for chemical shift calculations and illustrated how relaxation dispersion effects can emerge as a function of bi↔bii exchange in conjunction with the rocking motion. the md simulation study was supported by rsf grant 15-14-20038. serine/threonine kinases are attractive targets in targeted cancer therapy due to their overexpression in several forms of cancer. flavonoids are highly bioactive plant secondary metabolites that are important in human health due to their antioxidant property. quercetin, a natural flavonoid derivative, has been shown to regulate several signal transduction pathways and is in phase i clinical trial as an anticancer drug. this study explored the inhibitory potential of quercetin and its derivatives using in silico methods like molecular docking and molecular dynamics simulations. quercetin and its derivatives were observed to bind to several serine/threonine kinase family proteins with binding energy significantly better than other known inhibitors and commercially available drugs. this study thus sheds light on the atomic level interactions that define the polypharmacological nature of quercetin and its ability to interfere with a number of cancer pathways. introduction: noninvasive prenatal diagnosis (nipd) of the fetal rhd status by rhd genotyping of the maternal plasma was initially applied in alloimmunized pregnant women. fetal rhesus d status detection for management of rhd incompatibility using circulating cell-free fetal dna from maternal plasma or serum is now accepted by many obstetricians in europe as reliable and useful. the aim of the study was to detect fetal rhd specific antibodies in maternal plasma using a nanopolimer based electrochemical biosensor. materials and methods: a three-electrode system, consisting of a gold electrode, an ag/agcl reference electrode and a pt counter electrode, was accommodated in a 10-ml electrochemical cell. anilin and jelatin were used for immobilization of rhd antibody. the polimerization was occured at 319 nm uv light. antibodies of rhd antigen were detected using differential pulse method at between 0.4 and 0.6 v potentials by observing the differentiations in the current values. results: the rhd status of the fetus was predicted in 20 rhdnegative pregnant women (8-36th week of pregnancy). rhd antibody were detected in maternal bood using biosensor in 15 of the fetuses. the results were confirmed with real-time pcr. the fetuses found rhd (+) for exon 5 and 7 of rhd gene by multiplex real-time pcr. discussion and conclusion: biosensors based studies might be useful, because they allow to monitor the molecular interactions in real-time providing qualitative and quantitative information, through kinetics, affinity and concentration analyses. we found that more advantages in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, specific, economic, practical and less time-consuming. fetal rhd detection at low concentrations and in the early week of pregnancy is possible with this method. p-03.03.2-022 investigation of phylogeography of cricotopus sylvestris (diptera: chironomidae) using mitochondrial and nuclear molecular markers the family chironomidae is one of the most widely distributed insect families of diptera, and this family is distributed in all continents and all habitats from the tropics to the arctic in lakes, streams and puddles. in this study, we aimed to determine the dispersal of c. sylvestris using molecular phylogenetic markers not only in turkey but in the world and to reveal from where this species may have entered to turkey in the past. c. sylvestris larvae were collected from 8 lakes across turkey. after total genomic dna extraction from body of larvae, fragments of two mitochondrial genes, cytochrome c oxidase subunit i (coi) and cytochrome b (cytb), and one nuclear gene, carbomyl phosphate synthase domain (cps) of cad, were amplified and sequenced. in addition, several sequences of these three genes of c. sylvestris from different countries of different continents such as south corea, japan, canada, denmark, and sweden were obtained from genbank. all sequences were aligned using mega 6 and bioedit version 7.0.9.0 and were used for phylogenetic analyses. neighbour-joining (nj) tree was created in mega 6 and paup 4.0b10 with 1000 bootstrap replicates. maximum likelihood (ml) analysis was performed in raxmlgui 1.0 using gtrgamma model with 1000 bootstrap replicates. beast v1.8.0 was used for bayesian analysis. our phylogenetic analyses indicated that the japanese, south corean and american c. sylvestris were different from european and turkish members. turkish members of c. sylvestris were closely related to european ones according to our bayesian, nj and ml analyses. in turkish members, c. sylvestris collected from hazar and c ß ıldır lake was more ancient than those from marmara, sapanca, c ß ıldır, aygır, beys ßehir, e girdir and sıhke lakes. in conclusion, our results clearly suggest that several transoceanic dispersal events among the continents may have occurred and that the entrance of turkish c. sylvestris to turkey may have been from southeast and northeast of the country. metagenomics is providing great help to explore world of unculturable microorganisms in the natural samples to enhance our knowledge about microbial diversity. here, we have performed metagenomic analysis of fresh water lake bacterial community using 454 pyrosequencing techniques. we have observed a wide array of bacteria from phylum proteobacteria and family enterobacteriaceae as well as very few viruses from podoviridae, siphoviridae and unclassified phages. we have conducted a metagenomics analysis with the primary focus on the examination of the community of bacteria in a fresh water lake ecosystem. roche gs flx software gave us total 156 253 reads (with an average read length of 795.274 bp). there were 15 226 contigs having > 100 bp sequence length whereas 10 481 contigs with > 500 bp sequence length. for further analysis we have taken contigs with > 500 bp only. further, we have analyzed the microbial community composition using blastn/blastx against nt/nr databases with e-value cutoff of 10 à5 . ≥70% of total contigs were mapped to the reference with ≥60% contig match coverage. the community analysis revealed that domain bacteria is predominantly present (99.8%) in the water sample, followed by eukaryota (0.02%), viruses (0.08%) and other sequences (0.02%). most abundant phyla was proteobacteria (99.8%) and the most dominant family was enterobacteriaceae (89%) followed by xanthomonadaceae (10%), vibrionaceae (0.4%), pasteurellaceae (0.1%), shewanellaceae (0.07%). we performed functional analysis of all 5974 contigs using rapid annotation using subsystems technology (rast) 4 which detected 15 319 coding sequences and 197 rnas in 619 subsystems. among the classified cds from rast showed major cds hits for enzymes involved in the subsystems amino acids and derivatives and the carbohydrate metabolism. the great diversity of microorganisms present in the lake may reflect the human activity in the area. maldi-tof mass spectrometry is a ubiquitous and widespread tool for protein identification. once the protein sequence is unavailable, unambiguous identification cannot be performed, and predictability is limited by the presence of sequenced homologous proteins. we present a statistical approach to predict a number of structural, localization and functional properties of unknown proteins by direct analysis of mass distribution shapes of their post-cleavage fragments obtained from maldi-tof mass spectrometry data. secondary structure of proteins is best predicted by their specific cleavage at the inertial hydropathy group amino acid residues (filmv), with thermolysin (afilmv) being the closest commercially available reagent, leading to distinguishing between proteins with presumably ahelixes or b-sheets with 90% accuracy. cellular localization of proteins is best predicted by their specific cleavage at the external hydropathy group amino acid residues (dehknqr), exemplified by gluc(phosphate)+lysc(dek) cleavage. protein location in the cell membrane and its localization character (monotopic/ transmembrane, single-pass/multi-pass transmembrane) are predictable with~75% accuracy by this single cleavage, with optimal combination of 3-4 cleavages improving the accuracy tõ 80%. functional prediction of proteins is the best among membrane-associated proteins with characteristic structural conformations. we attribute the differences in the mass distribution shapes to the characteristic clustering of amino acids residues with respective hydropathy properties that are involved in the formation of 3d structural conformations of proteins. the suggested approach allows for a non-parametric statistical prediction of uncharacterized proteins from their maldi-tof mass spectrometry data without knowledge or reconstruction of their primary sequence. potential applications include proteomic studies of organisms with unavailable genomic sequences and highly variable proteins analysis. the cancer genome atlas (tcga) represents a comprehensive database of genomic, transcriptomic and epigenetic alterations across more than 20 tumor types. earlier we developed cross-hub tool aimed at multi-way analysis of tcga data in the context of gene expression regulation. in the present work, the software was updated with new features that are described below. crosshub is a python-based application. one of the features of crosshub is the combining tcga rna-seq co-expression analysis to encode chip-seq data in order to reveal most possible transcription factor (tf) targets and coupling mirna-mrna co-expression to several algorithms of mirna target prediction in order to enhance its efficacy. the key feature of the updated crosshub version is the analysis of the associations between expression ratio of tf to its targets and tf mutation status. this allows identification of tfs that are functionally (in)activated with driver mutations in a particular cancer type. the second novel feature of crosshub is the analysis of associations between 'tf-to-targets' expression ratio and tumor characteristics (tnm classification, pathological stage), patient follow-up, etc. in turn, this analysis may result in the identification of 'tf-targets' functional relations that are important for disease progression, tumor invasion, response to chemotherapy. thus, crosshub was supplemented with new features that can be useful for comprehensive tcga data analysis. the updated version of crosshub is freely available at http://sourceforge.net/ projects/crosshub/. this work was financially supported by the russian foundation for basic research (grants 15-04-08731, 16-16-00114 and 15-34-70055) and ras presidium program 'molecular and cellular biology'. p-03.03.2-026 mutations leading to increased rnase production and streptomycin resistance in bacillus pumilus bacillus pumilus strain 3-19 which was derived from soil-isolated b. pumilus 7p using chemical mutagenesis is characterized by resistance to streptomycin (str, up to 500 lg/ml) and ability to produce extracellular enzymes in quantities almost 10-fold higher than the parent strain. these features make the 3-19 strain suitable for industrial production of rnase (binase) which is known for its antitumour and antiviral properties and can be used as an rna-degrading tool in molecular biology. the whole genomes of both mutant and wild-type b. pumilus strains were sequenced recently. to reveal the exact genetic features responsible for rnase overproduction and str resistance we have fulfilled comparative genome analysis of b. pumilus 7p and 3-19 strains. facilities of rast server, edgar platform and additional bioinformatics tools (plasmid finder, prophinder, bl2seq) were used. it is found that both b. pumilus genomes under study contain an intact prophage, while only wild-type strain bears a 6 kb cryptic plasmid. none of the systems is inactivated in mutant strain according to the results of metabolic reconstruction. 3.4% of total cdss differ in 3-19 strain in comparison to 7p one, 36% of them are hypothetical. the altered genes are involved in membrane transport, cell wall composition, chemotaxis, spore formation, carbohydrate metabolism, dna metabolism, translation and transcription regulation. mutation (k56n) leading to str resistance is identified in 30s ribosomal protein s12p. regulatory and coding regions of binase gene have no modifications. candidate genes which can account for binase overproduction are selected. mutation k56n is classical in str resistance and leads to enhancement of decoding accuracy while decreasing elongation speed. rnase overproduction is brought about by non-specialized mechanism since other hydrolases are also overproduced in mutant strain. genes encoding extracellular serine protease, sporulation initiation phosphotransferase f, gnat-family acetyltransferase and cell wall modifying enzymes are reported previously to increase production of degradative enzymes. the action of encoded by them proteins lead to increase of stability and release of secreted proteins to environment and to derepression of their transcription from negative regulators bacillus pumilus strain 7p has been identified on its ability to produce ribonuclease and different extracellular proteases. in order to increase inherent biosynthesis of proteases the 7p strain was screened on culture medium supplemented by streptomycin. derivative b. pumilus strain 3-19 gains the resistance to streptomycin and also shows the increased ribonuclease activity. we used genomes of both these strains to explore streptomycin susceptibility and increased activity of hydrolytic enzymes. whole-genome shotgun sequencing was performed using a combination of pyrosequencing and ion semiconductor sequencing, which provided 23x (7p) and 30x (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) overall genome coverage. assembled genome sequences of 7p and 3-19 strains included 9 and 8 scaffolds > 500 bp with a calculated genome size of 3 577 758 bp and 3 572 739 bp, respectively. the gc content was 42%. both draft genomes have been deposited at genbank (jojx00000000.2 for 7p and jhud00000000.2 for [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] . detailed comparative genomic analyses of strains have been performed. we calculated average nucleotide identity (ani) values between the genomes of our strains and 9 completed b. pumilus genomes deposited in ncbi database. two b. pumilus strains (sh-b9 and safr-032) revealed the max. ani value (95.17% and 94.65%, respectively). b. pumilus sh-b9 strain has been used as a reference for snp calling in strains 7p and 3-19. 3828 snps for the 7p strain and 862 for the 3-19 strain were classified as nonsynonymous variants. 20 radical nucleotide substitutions from the 3-19 genome were not found in 7p genome. among them, the mutation in the 56 codon of rpsl gene (coding 30s ribosomal protein s12) is probably associated with resistance to streptomycin. also, two mutations in rpob and nusa genes (coding rna polymerase and transcription termination factor rho, respectively) may be related to increased enzymes activity. both our strains contain 143 protease-coding genes. twelve of them are encoding extracellular proteases. here we propose an algorithm that can predict an antibodies mutant forms with desired specificity. this algorithm allows to determine the position and type of amino acid residue for mutagenesis. approach is based on a hybrid method of quantum and molecular mechanics (qm/mm) that allows to understand the reaction mechanism and the role of active center amino acids. catalytic antibody a17, that is able to hydrolyze pesticide paraoxon, was selected as a model. however, the hydrolysis efficiency of paraoxon by a17 antibody is only 9 m à1 min à1 , that is insufficient for using this antibody as antidote. the main fundamental goal of our study is to determine the necessary conditions for improving the binding reaction of paraoxon by catalytic antibody a17. the hybrid qm/mm method allows to study the reaction mechanism of interaction of a17 with paraoxon. it was shown that the reaction proceeds via the classical s n 2 mechanism. the key step of the reaction is the proton transfer from the catalytic residue tyr-37 to paraoxon. qm/mm approach determines position for mutagenesis -leu-47 in light chain. for one of the mutant in this position -leu47argwere predicted (i) increased probability of formation of a hydrogen bond between the catalytic moiety and paraoxon compared to the wild type antibody and (ii) smaller value of the diffusion coefficient, which reflects the best positioning of paraoxon in the active center. steady-state kinetic analysis shows that leu47arg exhibits a 70-fold increase in k 2 /k d compared to a17 (90 m à1 min à1 vs. 1.35 m à1 min à1 ). double mutant leu47arg/ser35ala also has improved constants of interaction with paraoxon in comparison with the wild type antibody, however, a single mutant leu47arg still binds paraoxon three times better, that may be due to the fact that the serine in 35 position increase the nucleophilicity of tyr37. thus, our results are in line with our computed predictions. this work was supported by rfmefi60414x0069. due to high prices of meat and meat products, low quality raw materials like offal tissues are commonly used in turkey. in the retrospect of the studies for evaluating and detection of unwanted tissues in the sample is basic histological examination. the light microscopy techniques are very strong method if a researcher qualification is enough. a new researcher-independent method must be developed. therefore, different tissues and organs constitute of unique mrna and protein. our method is based on this event, so the antigenic sites of the tissues can be detected by selected antibodies. the first set of the antibodies are for detecting muscle and adipose, consist on muscle actin and adipose triglyceride lipase. this set is used for calibration on standard meat sample. the second set of the antibodies are detecting of offal tissues, consist on trrap and casein. anti-casein antibody is selected because the mammary gland usage in grinded-meat is very common. immuno-staining started with hier (heat mediated epitope retrieval), then classical ihc method applied to slides with dab-chromogen. after all process completed the slides were photographed by las (leica application suite) on microscope. the capture settings were remained same on both sets. image capture size is 1392x1040 pixels and field of view (fov) is 449 9 335 lm. all the image files were converted to binary for threshold operation. the threshold values of first set and second set were calculated and their ratios were compared. the formula is based on the distribution (dst) of pixel intensity (int) over threshold (thrs) values on all fov (axis: the results are good enough to detect the unwanted micro-structures on 80% raw meat and 20% offal tissue. calculations proofed with imagejò. future application of this method and opencv-based software algorithm is to port the source code to a single board computer (sbc) with a digital microscope connected. monday 5 september 12:30-14:30 mechanisms of pro-inflammatory diseases p-04.02.2-001 the effects of raas inhibition in rate limiting step by aliskiren on testicular torsion injury in rats testis torsion is a urological emergency condition that results in necrosis of the testis if the condition is not treated. unfortunately treatment of testis torsion is not fully understood, therefore clinical and experimental studies are performed continuously. reninangiotensin-aldosterone system (raas) contributed to pathophysiology of several diseases. aliskiren (als) inhibits the renin on the first step of this system. our aim is to investigate the protective effect of aliskiren on unilateral testis damage caused by experimental testis torsion and detorsion. the forty-eight rats were separated into eight groups of six animals: sham, sham+als 200 mg/kg (oral) group, torsion group (tor), torsion/detorsion group (tor/det), tor+als 100 mg/kg (oral) group, tor+als 200 mg/kg (oral) group, tor/det+als 100 mg/kg (oral) group, tor/det+als 200 mg/kg (oral) group. in the tor and tor/det groups, the left testes were rotated 720°clockwise together with the spermatic cord and tunica vaginalis in the scrotal space. the left testes of the rats were subjected to torsion and detorsion during 2 h. after experimental procedures, testicular tissues were examined by histopathologic and molecular analyses. the il-1b and inos mrna expressions were increased in tor and tor/det groups when compared with sham group. both doses of aliskiren administration decreased these expressions in tor/det groups. the stereological results revealed that aliskiren administration promote the numerical density of mature spermatids in tor and tor/det groups. the numerical densities of tor/det+als100 and tor/det+als200 groups were similar and these two groups have significant difference when compared to the tor and tor/det groups. the administration of als may be useful for preventing ischemic damage on unilateral testes injury in rats. this study supported by a tubitak 3001 project, coded 114s048. 3 ) has recently been recognized as a potent immunomodulator which acts through regulation of gene expression involved in immunity response thus affecting various inflammatory and autoimmune diseases. the study was aimed at investigating hepatoprotective role of d 3 in vdr-mediated regulation of pro-inflammatory factors in diabetic liver. materials and methods: type 1 diabetes was induced in male c57bl/j6 mice by i.p. injection of high-dose stz (150 mg/kg b.w.). after 2 weeks of stz-induced diabetes animals were treated with/without d 3 (15 iu/mouse per os) for 8 weeks. blood serum 25ohd 3 was assessed by elisa. rel-a, vpf, inos and vdr expression was measured by qrt-pcr and/or western-blot. results and discussion: diabetes caused two-fold reduction of serum 25ohd 3 level, indicative of d 3 deficiency. significant alterations in d 3 -endocrine system were found as is evident from reduced expression of cyp27a1, cyp2r1, vdbp and vdr at transcriptional and translational levels. these changes were accompanied by a marked increase in mrna and protein levels of inflammation markers rel-a, vpf and inos in hepatic tissue of diabetic mice. diabetes also led to structural lesions in liver tissue. complete restoration of 25ohd 3 content and partial normalization of liver tissue structure were achieved after d 3 treatment. d 3 administration partially normalized expression of cytochromes involved in d 3 metabolism and hepatic pro-inflammatory factors. d 3 treatment prevented overexpression of rel-a and phosphorylated p65/rel-a translocation to hepatocellular nuclei that is most likely mediated through 1,25(oh) 2 d 3 and vdr. conclusion: study confirmed that diabetes-induced liver abnormalities are associated with chronic inflammation that can be linked to impaired d 3 metabolism and deficiency. our findings demonstrate protective vdr-mediated effect of vitamin d 3 against diabetes-induced liver injury. p-04.02.2-003 lavandula stoechas extract increased glucose uptake and protein levels of key signaling molecules in insulin resistant c2c12 muscle cells s. savranoglu 1 , h. ipek 2 , s. arslan 3 , h. delig€ oz 4 , a. r. t€ ufekc ßi 5 , i. demirtas 5 , t. boyunegmez t€ umer 6 1 graduate program of biology, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, 2 graduate program of bioengineering, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, 3 deparment of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, 4 department of chemical engineering, faculty of engineering, pamukkale university, denizli, turkey, 5 department of chemistry, faculty of sciences, c ß ankiri karatekin university, c ß ankiri, turkey, 6 department of molecular biology and genetics, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey introduction: the aim of this is to identify remedial effects of lavandula stoechas, anatolian traditional medicine, against metabolic disorders developed on the ground of insulin resistance. ethyl acetate extract (eae) of l. stoechas was investigated in c2c12 myotubes which were made insulin resistant by free fatty acid (ffa) treatment, for its effects on glucose uptake and as well as on the activation of akt-1 (by pakt/ akt ratio) molecule which plays a central role in insulin signaling through serine (473) phosphorylation. in addition, the protein level of lipoprotein lipase (lpl) enzyme was also evaluated. material and methods: c2c12 cells were made insulin resistant by palmitic acid (ffa) and effects of eae on p-akt (ser473)/akt ratio and lpl level were determined by sds-page/western blot. the effect of eae on glucose uptake in insulin resistant cells were determined by the 2-deoxyglucose uptake assay. results: eae at 25 and 50 lg/ml significantly increased the glucose uptake 120 and 182% compared to insulin resistant control cells. metformin at 2 mm increased this parameter up to 132%. eae increased pakt ser473/akt level 43-37% and lpl expression 50-92% for 25 and 50 lg/ml, in insulin resistant myotubes, respectively (p < 0.05). discussion: eae of l. stoechas improved impaired insulin sensitivity through both enhancing glucose uptake and activation of akt1 molecule through ser473 phosphorylation. in addition, it also considerably increased lpl level which has very important function in lipid metabolism. conclusion: overall, results demonstrated that l. stoechas contain phytochemicals which can be effective for the prevention and also treatment of insulin resistance and associated conditions. our research group is on the way for the identification of these 'bioactive' molecules with bioassay guided fractionation studies. tubitak (projectid: 112t442) supports this study. achievement of complete pain control is very difficult task, which requires a search for new molecular targets during the analgesic substances development. considering the importance of glial cells and their signaling molecules, development of new gliotropic therapeutic methods is a promising direction in pain treatment. polyunsaturated fatty acids, including docosahexaenoic acid demonstrating anti-inflammatory and antioxidant activity are of considerable interest. docosahexaenoic acid (dha, 22:6 n à 3) analgesic activity was studied using a chronic constriction injury (cci) rat model. animals were subcutaneously injected with dha emulsion at a dose of 4.5 mg/kg (125 mm/kg) daily during 2 weeks after surgery. collection of material for subsequent immunohistochemistry investigation was performed on day 28. we clearly demonstrated that the activation of neurokinin neurotransmission and nnos synthesis are coincided with the astroglial activation in the spinal cord dorsal horn (scdh) superficial lamina during neuropathic pain development. however, the detailed mechanisms of interaction between substance p (sp)-, no-ergic systems and astrocytes in the spinal cord remain to be elucidated. systemic administration of dha to cci animals reduced neurogenic pain intensity and duration, leading to an earlier stabilization of paw weight distribution and preventing the development of degenerative changes in denervated limb. this drug treatment reduced the level of the sp-and no-ergic neurotransmission and decreased astrocytosis in the scdh superficial lamina. thus, the ability of dha to affect nociception is a promising and safe alternative to current pharmaceutical therapeutics. immunohistochemistry studies carried out with the russian science foundation financial support (agreement no. 14-50-00034), obtaining dha and all manipulations with animals of the material was funded by rfbr according to the research project no. 16-34-00023 mol_a. p-04.02.2-005 circulating endothelial-derived apoptotic microparticles and aopps are related to highsensitive troponin t in patients with chronic hepatitis c infection the aim of this study was to evaluate non-standard risk factors for cardiovascular events, such as endothelial dysfunction assessed by endothelial-derived microparticles (emps) (cd144 +/ cd31 + ), advanced oxidation protein products (aopps), and low-grade inflammation, that are potentially associated with elevated levels of high-sensitivity troponin t (hs-tnt) and n-terminal pro-brain natriuretic peptide (nt-probnp) in patients with chronic hepatitis c (chc). methods and results: eighty-six chc patients and 60 healthy control subjects were enrolled in the study. circulating levels of hs-tnt, nt-probnp, aopps-albumin (the ratio of aopps to albumin content), emps (cd144 +/ cd31 + ), hs-crp, and tnf-a were assessed. compared with chc patients, the chc patients with diabetes mellitus (dm) had higher levels of emps (cd144 +/ cd31 + ) and aopps-alb, which were associated with elevated hs-tnt levels (≥13.3 pg/ml). nt-probnp positively correlated with tnf-a level in all chc patients and this correlation was stronger in diabetic patients. in multivariate logistic regression analysis, the independent factors associated with the presence of elevated hs-tnt levels were the presence of dm (p < 0.001) as well as high levels of aopps-alb, apoptotic emps (cd144 + /cd31 + /an-v + ), and nt-probnp (p = 0.04, p = 0.03, p = 0.04 respectively). conclusion: the prevalence of elevated hs-tnt were increased significantly in the diabetic patients with chronic hepatitis c. hs-tnt was related to non-standard risk factors for cardiovascular events, and circulating endothelial-derived apoptotic microparticles (cd144 + /cd31 + /an-v + ) level was an independent predictor for elevated hs-tnt levels, potentially indicating some abnormalities in the myocardium. apnea; and healthy individuals; and assessing the connection between the pain and the dimension of the sleep disorder. material and methods: 60 patients who were diagnosed with obstructive sleep apnea and 40 healthy individuals who were similar in terms of age and gender were included in this study. the patients, who were diagnosed with obstructive sleep apnea with the examination and sleep tests, were assessed according to the 1990 american college of rheumatology (acr) criteria in terms of fms. serum d vitamin level was measured by using the ultra performance liquid chromatography method. findings: when the fibromyalgia syndrome and obstructive sleep apnea and pure obstructive sleep apnea patient groups are compared with the control group, the vitamin d level was found to be low at a significant level (p = 0.038, p = 0.001, respectively). no significant difference was found between the vitamin d levels in fibromyalgia syndrome, obstructive sleep apnea and pure obstructive sleep apnea patient groups. a negative correlation was found between the number of the sensitive points and vitamin d levels in fibromyalgia syndrome patients (p = 0.013). results: it has been concluded that the obstructive sleep apnea and fibromyalgia syndrome patients have low vitamin d levels, and this situation must be considered in treatment modalities. on the other hand, the results obtained in the study make us consider that vitamin d metabolism is not influential in the pathogenesis of the fibromyalgia syndrome and obstructive sleep apnea togetherness. p-04.02.2-007 decreased chitotriosidase activity and levels in familial mediterranean fever discussion: familial mediterranean fever is an inflammatory disease. several cytokines and inflammatory mediators are playing role on pathogenesis of the disease. although ıt has been demonstrated that the increased concentrations of cht in patients with fmf. we found lower cht activity and concentrations in patients with fmf. conclusion: serum cht enzyme activity and concentrations may not be considered as a biomarker in fmf patients taking colchicine. new studies are needed to evaluate the changes of the enzyme activity, concentration and the role of cht in patients with colchicines negative patients. chronic hyperglycemic state leads to an increase in subclinical systemic inflammatory response in diabetes mellitus type 2 (dmt2) patients. inflammation-based scores, neutrophil to lymphocyte ratio (nlr), platelet to lymphocyte ratio (plr) and red blood cell distribution width to platelet ratio (rpr) are biomarkers able to quantify systemic inflammation. the aim of the study was to investigate association of the inflammation-based scores with short-and long-term glycemic control markers, and whether they could be used as indicators of glucoregulation in dmt2 patients. the cross-sectional study included 92 dmt2 patients, treated at the primary health care centre zenica from december 2015 to april 2016, distributed into groups according to glycated hemoglobin (hba1c) values: a (n = 59, hba1c ≤7.0%) and b (n = 33, hba1c > 7.0%). complete blood cell count, fasting blood glucose (fbg) and hba1c measurements were determined at the primary health care centre zenica and at the department of laboratory diagnostics, cantonal hospital zenica by standard laboratory methods. all statistical tests were performed using spss 19.0. p values fasting blood glucose and hba1c were significantly higher in the group b compared to the group a (p < 0.0005). there was no significant difference of nlr, plr and rpr between the groups (p = 0.50; p = 0.220; p = 0.525, respectively). significant correlation of inflammation-based scores with fbg and hba1c was found only between plr and hba1c in the group a of dmt2 patients (r = 0.328, p = 0.011). inflammation-based scores could gather meaningful clinical information, either diagnostic or prognostic, on a variety of hyperglycemic, inflammatory, cardiovascular and thrombotic disorders. since there was no statistically significant difference of nlr, plr and rpr between dmt2 patients with good and poor glycemic control, we conclude that these scores could not be used as indicators of glucoregulation in dmt2 patients. chronic inflamation plays a central role in the development and progression of diabetes and in the pathogenesis of its comlications. the neutrophil-lymphocyte ratio (nlr) and platelet-lymphocyte ratio (plr) are indicators of subclinical inflamation. mean platelet volume (mpv) is one of the platelet function indices that reflects the platelet production rate and stimulation. we investigated the association of nlr, plr and mpv with prediabetes and type 2 diabetes mellitus (t2dm) and determine whether or not these are reliable markers for diagnosis. we evalueted 76 people's results who were carried out oral glucose tolerance test (ogtt). acording to 2-h values of plasma glucose in the ogtt; 1. group (normal glucose tolerance: ngt): under 140 mg/dl (n = 42), 2. group (prediabetic: impared glucose tolerance (igt)): ranging from 140 mg/dl to 199 mg/dl (n = 25), 3. group (firstly diagnosed diabetic by ogtt): above 200 mg/dl (n = 9). 4. group is clear diabetic without complication (taking treatment) group (n = 34). we compered nlr, plr, mpv and some biochemical markers between four groups. there are significantly differences between all groups in nlr (p = 0.004) and plr (p = 0.021) values. nlr values are significantly higher in prediabetic (1. it is recognized that a chronic low-grade inflammation and an activation of the immune system are involved in the pathogenesis of insulin resistance and type 2 diabetes mellitus (t2d). this study aimed to analyze the long-term impact of altered metabolism in female t2d patients at the level of mediators of inflammatory response. this study included 65 femalet2d patients and 107 control subjects, which were recruited at the clinical center university of sarajevo and the general hospital tesanj. in this study the effects of glycemic control on markers of the inflammatory response crp, fibrinogen, leukocytes, sedimentation, and cytokine il-6, were analyzed. all subjects included in this study were free of evidence infections, surgery, thyroid disease, polycystic ovarian syndrome, active liver and kidney damage. all biochemical analyses were performed by employing standard ifcc protocols. results from this study demonstrated significant increase of fibrinogen (p = 0.0001), crp (p = 0.001), il-6 (p = 0.013), leukocytes (p = 0.0001) and sedimentation rate (p = 0.008) in female t2d population compared to control subjects. interestingly, a significant correlation was shown between crp and hba1c (p = 0.035), il-6 and glucose (0.032), il-6 and bmi (0.007). in our study, female t2d compared to the healthy population had significantly higher levels of fibrinogen, leukocytes, il6, crp and sedimentation. other studies conducted in female population associated elevated levels of il-6 and crp with t2d independent of other risk factors for diabetes. crp being most robust predictor of diabetes. studies have shown that crp is an important predictor of t2d for the female but not the male population. thus, our data suggest that inflammation play an important role in the pathogenesis in female diabetic population. a more detailed study on a far larger number of subjects should point out fact if they can effectively be used as biomarkers in the primary prevention of t2d in this population. objectives: bone and mineral metabolism disorders hold an important place among the complications after renal transplantation. the purpose of this study was to demonstrate the relationship between vitamin d, calcium, phosphorus metabolism with graft function and to measure 1,25(oh) 2 d 3 levels with lc-ms/ ms in renal transplant recipients. design and methods: this study included 30 renal transplant recipients (10 female, 20 male; mean age: 40.30 ae 12.86) from living related donors were transplanted. blood samples were collected immediately before and after transplantation at month 6. serum creatinine, bun, calcium, phosphorus, alkaline phosphatase, glucose, albumin, pth, 25(oh)d and 1,25(oh) 2 d 3 levels were measured. gfr values were estimated by ckd-epi. plasma 1,25(oh) 2 d 3 levels were determined in a lcms-8040 triple quadrupole tandem mass spectrometer (shimadzu corporation, japan) by mrm. spss 20.0 software was used for statistical analysis. results: although plasma 1,25(oh) 2 d 3 levels significantly increased (p = 0.0001), we did not find any significant differences for serum 25(oh)d levels after transplantation. when posttransplant levels of serum phosphorus, pth, creatinin, bun and alp levels were found to be significantly decreased (p = 0.0001, p = 0.011 for alp), we observed significantly higher calcium and gfr values (p = 0.0001). vitamin d insufficiency was present 13.3%, deficiency 36.7%, severe deficiency 50% before transplantation, insufficiency was also seen 26.7%, deficiency 50%, severe deficiency 23.3% after transplantation at month 6. conclusions: in our study, all patients were found to vitamin d deficiency and insufficiency. determination of vitamin d deficiency and consequently treatment with vitamin d supplements could lead to better graft surveys. free fatty acids (ffa) represent important link between obesity, insulin resistance, type 2 diabetes (t2d), and dyslipidemia. increased adiposity, as approximated by body mass index (bmi), correlates well with increased serum levels of leptin-adipocyte derived hormone implicated in the regulation of adipose mass and alterations in insulin action and secretion. the main objective of the present study was to investigate the potential association of serum ffas with leptin levels in healthy and newly diagnosed type 2 diabetic subjects. this study involved 13 newly diagnosed type 2 diabetics and 13 healthy subjects. all participants in the study were free of evidence of hepatitis, viral infection or active liver and kidney injury. for biochemical analyses of glucose, glycosylated hemoglobin (hba1c), and lipid profile, standard ifcc protocols were used. analysis of free fatty acids (ffas) was done by gas chromatography, while serum leptin levels were determined by the elisa kit. in addition to the expected differences in glucose, hba1c, and bmi, our results also showed significant differences in leptin, myristoleic, palmitic, linolenic, arachidic, and arachidonic acids between t2d and control subjects. in healthy subjects, a significant correlation was demonstrated between glucose and lauric, arachidic, arachidonic acid levels, body weight, and bmi. newly diagnosed diabetics showed significant association between glucose and lauric, myristoleic and linolenic acid levels; with leptin being associated with myristic and palmitoleic acid levels. interestingly, in all participants, significant association was found between glucose and hba1c, glucose and leptin, myristoleic, arachidic, and bmi as well as between leptin, arachidic acid, and bmi. thus, our data point out association of different types of ffas with leptin levels in newly diagnosed type 2 diabetics. however, further studies should be done in larger number of patients to confirm our results. rheumatoid arthritis (ra) and ankylosing spondylitis (as) are chronic inflammatory diseases with distinct clinical manifestations in many ways. the aim of this study is to evaluate the serum levels of molecules which may be used as markers for angiogenesis and vascular leakage in the processes of two clinically different pictures, ra and as. 30 ra patients, 30 as patients and 30 healthy volunteers with mean age of 30-50 were included in the study. serum levels of vegf, angiopoetin-2 and tie-2 were measured by enzymelinked immuno-sorbentassay (elisa) using a commercially available kit. serum nitricoxide levels were evaluated by the griessreaction. serum vegf, ang-2 and no levels were significantly higher in the as group ml, p < 0.001; p < 0.001; p < 0.05). no differences were found between as and ra for tie-2 (p > 0.05). vegf, ang-2, tie-2 and no levels were positively correlated in both as and ra patients (p < 0.001), but no correlation was detected between clinically activation index das-28, basda _ i scores and laboratory measurements such as sedimentation, crp and anti-ccp (p > 0.05). when the diagnostic performance of the parameters were evaluated with the roc analysisonly the performance of the ang-2 in as patients was sufficient (auc (95% cl): 0.718, p < 0.05). elevation of angiogenic factors in the serums of as and ra patients supports the role of angiogenesis in the etiopathogenesis of these diseases. however, lack of relationship between disease activity leads to not to use these factors as a marker for clinical follow-up. only ang-2 measurements may be useful for the differantial diagnosis. the evaluation of ischemia modified albumin as an early biomarker of acute myocardial infarction introduction: acute myocardial infarction (ami), remains a leading cause of morbidity and mortality worldwide. early diagnosis of ami is very important because early treatment may reduce the extent of injury to the myocardium. currently, biomarkers of myocardial necrosis such as myoglobin, ck-mb and troponins are highly sensitive and exhibit good specificity. however, these biomarkers increase after tissue injury, approximately 4-6 h after the cardiac event and detect only the consequences of prolonged ischemia. recently, ischemia modified albumin (ima) has been assessed and found to be very useful for the diagnosis of myocardial ischemia and it is considered as a serum biomarker. the aim of the present study was to evaluate the serum level of ima and determine the relation between patients with ami and control group, in order to verify its potential as a novel marker for early detection of mi. materials and methods: the study was performed with 25 patients and 37 healthy controls. blood samples from all subjects were collected by venipuncture in plain tubes, and immediately centrifuged at 4000 g for 10 min at 4°c. the serum samples were stored at à20°c until analysis. the serum levels of ima were determined using the cusabio biotech human ischemia modified albumin, elisa kit according to manufacturer's instructions. the results are given as international units/milliliter (iu/ml). results: our findings revealed that ima showed no significant difference between the groups. conclusion: our results suggest that ima assay is not a sensitive marker for early detection of ischemic hearth disease and cannot be used alone for the diagnosis of ami. prospective studies are needed to identify ima's potential as a biomarker for ami. p-04.02.2-015 neutrophil-to-lymphocyte ratio and platelet-tolymphocyte ratio in polycystic ovary syndrome polycystic ovary syndrome is a complex and multifactorial disease with metabolic dysfunction and the etiopathogenesis is not well established. emerging data suggest that adiposity and chronic low-grade inflammation are involved in the development of the metabolic dysfunction. neutrophil-to-lymphocyte ratio (nlr) and platelet-to-lymphocyte ratio (plr) have recently been investigated as two new inflammatory markers used in the assessment of systemic inflammation in many diseases. the purpose of the study was to investigate their relation with pcos patients. the study population consisted of 44 patients with polycystic ovary syndrome and 23 healthy women controls. nlr and plr obtained by dividing absolute neutrophil to absolute lymphocyte count and absolute platelet count to absolute lymphocyte count, respectively. the neutrophil count (4.41 ae 1.6 vs. 3.77 ae 1.23, p < 0.05) and platelet count (320.34 ae 55.11 vs. 283.96 ae 52.17, p < 0.05) were higher in patients with pcos compared to the control group. lymphocyte count was 2.08 ae 0.43 in pcos patient and 2.30 ae 0.57 in control group. the nlr and plr of pcos patients were significantly higher compared to those of the controls (2.18 ae 0.92, 1.69 ae 0.57 p < 0.05, 158.31 ae 32.85, 128.59 ae 31.94 p < 0.05, respectively). in this study we found nlr and plr were significantly increased in patients with pcos compared to healty control. nlr and plr were two useful inflammatory markers for assessment of patients with pcos. imbalance in neurotransmission in conjunction with neuroinflammation contribute to neurological dysfunction observed during acute liver failure (alf). own observations indicate that alf in a mouse model is associated with altered expression and/or intracellular distribution of synaptic proteins. since neutralization of tgf-b1 appears to improve the neurological score in alf mice, we examined the possibility that increased levels of tgf-b1, caused by liver damage, may affect the expression of selected synaptic proteins. expression and/or cytoplasmic vs. membrane distribution of a number of functionally critical synaptic proteins in cerebral cortex and blood tgf-b1 were measured in c57bl6 mice with alf induced by single i.p. injection of aom (100 mg/kg of b.w.) and after neutralization of tgf-b1 induced by single i.p. injection of ab-tgf-b1 (1 mg/kg) 2 h before aom injection. in alf mice, blood tgf-b1 was increased, and the expression of presynaptic proteins: synaptophysin and synaptotagmin was increased in the cytosolic (s2) fraction by~45% and 30%, respectively, but was slightly depressed in the membrane (p2) fraction by~20% and~15%. aom induced an increase of postsynaptic proteins: psd-95 and nnos by~40% in p2 fraction. tgf-b1 neutralization resulted in a reduction in the expression of presynaptic proteins by~30% in s2 fraction and~20% in p2 fraction, in control animals and normalized their amount in the cytosolic fraction after aom injection, but was ineffective with regard to psd-95 and nnos. the results indicate that in alf mouse, neutralization of cytokine tgf-b1 normalizes synaptophysin and synaptotagmin expression in the synaptoplasm, without affecting their synaptic membrane content. effect of tgf-b1neutralization appear to be confined to the presynapse. 25% of acute pancreatitis (ap) patients develop severe acute pancreatitis (sap), which is is resulted in multiple organ dysfunction syndrome. an extensive inflammatory response occurs due to inflammatory mediators synthesized and secreted during sap. since preventing the inflammation in sap is important in the prognosis of the disease, new drug candidates having strong antiinflammatory effects will provide a new concept for therapeutic strategies against acute pancreatitis. non-steroidal anti-inflammatory drugs (nsaids) show their effects by inhibiting cyclooxygenases (cox-1 and cox-2) and they play an important role in the pathogenesis of acute pancreatitis. since conventional nsaids inhibit both cox-1 and cox-2, they have serious side effects on gastrointestinal system. therefore, new highly selective cox-2 inhibitors having fewer side effects are needed. in the present study, selective cox-2 inhibitory activities and cytotoxic effects of new series of 2-benzoxazolinone and thiazolo [3,2-b ]-1,2,4-triazole derivatives previously synthesized as specific cox-2 inhibitors with no side effects on gastrointestinal system were investigated. permeability of the compounds was tested by pampa using caco-2 cells. compounds were found highly selective, non-toxic and permeable. ap was induced in rats via retrograde injection of stc into the pancreatic duct system. rats were pre-treated with saline or celecoxib or the new compounds before stc injection and sacrificed 24 h later. the severity of ap was evaluated using biochemical and histopathological analyses. edema, inflammation, hemorrhage and acinar cell necrosis were detected in the pancreatic tissue of sap group. sap was remarkably increased serum lactate dehydrogenase, ast, alt, lipase and amylase activities and serum tnf-a, il-1b, il-2, il-6 and il-8 levels. tissue myeloperoxidse activity was also increased. pretreatment with the novel compounds reserved all these biochemical and histopathological parameters. alopecia areata (aa) is an inflammatory disease which is affects hair follicles, and sometimes nails. it is suggested that cytokinemediated immunity plays an important role in etiopathogenesis of aa. this study was planned to evaluate the serum ykl-40 and tgf-b1 levels of patients with aa. 40 patients with aa and 40 healthy volunteers were recruited into the study. fasting venous blood samples were collected from the participants and serum was obtained by centrifugation. serum ykl-40 and tgf-b1 levels were measured by enzyme linked immunosorbent assay (elisa). serum tgf-b1 levels in the patient group were significantly lower compared to the control group whereas serum ykl-40 levels were significantly higher in patient group. tgf-b1 levels of men and women with aa were found to be significantly lower than that of controls. while serum ykl-40 level of male control group is higher than the male patients, there were no significant differences between women groups. the increased serum ykl-40 levels in aa patients suggests that ykl-40 plays a crucial role in the pathogenesis of aa. arterial immune mediated inflammation participates centrally in all stages of the development of atherosclerosis, from the initial lesion to the end-stage thrombotic complications. although emerging evidence supports augmented cardiovascular morbidity and mortality in cutaneous psoriasis (psc) and psoriatic arthritis (psa) as compared to the general population its underlying mechanism is poorly understood. here we analyzed the inflammatory burden in recent onset of psa patients without traditional cardiovascular risk factors (cvrf) in a transversal study measuring carotid intima media thickness (cimt) (measured with ecodoppler), and proatherogenic inflammatory molecular markers like c-reactive protein (crp), interleukin 6 (il-6), and soluble intercellular adhesion molecule-1 (sicam-1) in comparison with control patients. cimt values are similar in psa (0, 59 ae 0.045*) and psc (0, 62 ae 0.10) patients. however, both of them were significant increase compared with control (0.436 ae 0, 05). regarding inflammatory markers il-6 serum levels in patients with aps was higher than pcs (18 ae 2.9) and healthy controls (16, 3 ae 2.5) but the difference did not achieve statistical significance (*p > 0.05). on other hand mean of sicam-1, value from patients with recent onset of psa is significant higher than controls. psc remain without significant changes compared to control (*p > 0.05). in addition mean value from patients with recent onset of psa is significantly higher than in controls (*p < 0.05) and psc group. overall, preliminary findings suggest for the first time that patients with early psa, without evident traditional cvrf have significant increased values of cimt, sicam-1 crp against the general population control group. this data strongly supports that early cv molecular markers are increased after the first symptoms and signs of this disease even in the absence of traditional cardiovascular risk factors. furthermore, this give new windows for a proper treatment. p-04.02.2-020 protective effect of trail against proinflammatory cytokines on pancreatic beta cells correlated with decrease in dr5 and increase in dcr1 expressions universitesi, antalya, turkey introduction: proinflammatory cytokines are known to have destructive effects on beta cells, which contribute to type 1 diabetes (t1d) development. the combinatory effects of three of these cytokines in particular, namely tnf-alpha (tnf-a), ifngamma (ifn-c), and il-1beta (il-1b), are claimed to render beta cells prone to t cell-mediated destruction. the recently identified anti-inflammatory feature of tnf-related apoptosis-inducing ligand (trail), its possible protective role in this process. in this study, the effects of applications of trail with tnf-a, ifn-ᵞ, and il-1b on beta cell viability and correlation of these effects with trail receptor expression patterns were investigated. methods: glucose-responsive insulin-secreting nit-1 mouse beta cell lines were treated with tnf-a, ifn-ᵞ, il-1b, and soluble trail (strail) individually and in various combinations. cell viabilities were determined at 24 and 48 h by mtt assay. trail ligand and receptor expression profiles on nit-1 cells, and alterations in receptor expression levels following cytokine applications were determined by western blotting analysis. results: trail treatment did not have any cytotoxic effects on nit-1 beta cells at 48 h, while increasing cell viability following il-1/ifn/trail and il-1/tnf/trail combined applications. substantial levels of death receptor 5 (dr5) expression were detected on nit-1 cells before applications, yet it displayed decreased levels at 48 h of trail treatment. lower levels of decoy receptor 1 (dcr1) expression detected prior to treatments increased significantly in contrast. discussion: the fact that trail co-treatment with tnf-a, ifn-ᵞ and il-1b increased cell viability in nit-1 beta cell lines along with reduction in dr5 death receptor expression and an increase in the decoy receptor dcr1 expression, points out to a possible protective effect of trail in insulitis, and strengthens its potential as a putative therapeutic molecule in prevention of beta cell loss. behc ßet's syndrome (bs) is a multisystemic inflammatory disorder with a strong and complex genetic background. being a prevalent disorder both in turkey and also in the ancient trade road 'silk road' countries, bs is an important cause of impairment and disability owing to its chronic and relapsing nature. besides, bs is reported to be an important cause of mortality among the young male patients. while the epidemiology of bs is substantially well documented, currently, the etiology, the molecular mechanisms underlying its pathogenesis, and the classification of the disorder remain to be elucidated. our aim was to disclose the disease mechanisms at molecular level in turkish bs patients by obtaining, comparing, and analysing the transcriptome data of bs patients with age and gender matched healthy controls. for this purpose, by using the affymetrix hg u133 plus 2.0 microarrays, peripheral blood cell mrna profiles of 30 bs patients (b) and 15 matched healthy controls (c) were obtained. following bioinformatics, gene ontology, and pathway analysis, validation experiments of the identified prominent mrnas were performed by qrt-pcr methodology. the comparison of b vs. c yielded differentially expressed gene numbers of 30 and 625 for the chosen fold changes of 2.0 and 1.5 respectively (p ≤ 0.05 for both). during gene ontology and pathway analysis, immune system process, immune system diseases, systemic lupus erythematosus, arthritis, and intestinal immune network for iga production categories/pathways were significantly enriched. clustering analysis revealed a molecular signature which accurately distinguished b and c samples, while the qrt-pcr analysis successfully validated the chosen mrna transcripts. this study documented differential expression of a large number of immune system and immune disease related genes in bs patients. the uncovering of the molecular disease mechanisms of bs will point to novel candidate molecules to be targeted for the treatment of the disorder. obesity is a public health problem in developed countries and worldwide with increasing prevalence through a relationship primarily with atherosclerotic cardiovascular diseases as well as several metabolic disturbances such as increased insulin resistance and diabetes. although several studies identified obesity as an independent risk factor for atherosclerotic cardiovascular diseases, the mechanism underlying the increased cardiovascular risk in obese patients has not been clearly delineated. adma, no, endothelin-1 and homocysteine are an indicator of endothel disfunction that plays an important role in the pathophysiology of atherosclerosis. in our study, obese children and the control group were compared in terms of adma, no, endothelin-1 and homocysteine, we also investigated whether there is a correlation between these parameters. 58 obese and 30 healthy children, participated in the study. when the obese group was compared to the healthy controls, the adma level of the obese group were significantly higher than those of the control group but there was no statistically significant difference in no, endothelin-1 and homocysteine. increased adma level might trigger the pathogenesis of atherosclerosis starting from the childhood years onward. that is why controlling obesity in this age group with diet and other treatment modalities will prevent the mortality and morbidities that will be seen in adult years. inh deficiency leads to the formation of bradykinin causing to dilation of blood vessels. furthermore, the study conducted by shagdarsuren, on the damage done by c1-esterase, demonsrates that the complement system and triglyceride levels are affected. we investigated lipid oxidation and fetuin a levels in patients with c1 _ inh deficiency. materials and methods: 44 people with c1 _ inh and 44 people without any illnesses were taken into the study. fetuin a was studied using an el _ isa kit from raybio (usa). ferrous ion oxidation-xylenol orange test was used to find looh serum levels. sh (free thiol groups) test was studied with regards to ellmans method modified by hu. _ ibm spss 20.0 was used for statistical results. results: in assessments made between the healthy and the illness groups, there was significant differences in the levels of fetuin (p = 0.035), looh (p = 0.000) and sh (p = 0.047). when pearson correlation analysis was performed, we detected a significant positive correlation between fetuin a and looh levels (r: +0.326) discussion and conclusion: in these patients, lipids is secreted from the adipose tissue. in response, anti-atherosclerotic fetuin a levels were risen. patients also possessed increased lipid peroxidation, this increase shows positive correlation with fetuin a levels. in conclusion, we identified that sh with antioxidant properties have increased levels. aim: high fructose corn syrups are found in soft drinks, juice beverages, breakfast cereals, most of the processed foods. it has been shown that high dose of fructose intake may lead to a reduction in the number of hepatocytes, deterioration of liver function, increasing reactive oxygen species and liver steatosis. the aim of this study was to explore whether caffeic acid phenethyl ester (cape) has any potential protective effect on high fructose diet-induced fatty liver model. materials and method: totally fifty rats were divided into five groups. control group, %10 fructose administered group, cape group, %10 fructose + cape administered group and ethanol group. after 6 weeks, liver oxidant and antioxidant status, and blood tnf alpha, il-6, and il-8, tissue nfkb levels were quantified. protein levels were investigated against, nfkb and p-nfkb antibodies and normalized and analyzed against b-actin antibody by western blotting. results: serum tnf-alpha, il-6, il-8 levels were found to be increased in fructose group compared with the control group (p < 0.05). in liver tissue of 10% fructose administered group, mda, protein carbonyls and no levels were higher than control group. however sod activity did not show any difference among the groups. in the fructose administered group, caspase 3 showed liver apoptosis and was considered as positive. acquired data revealed that nfkb protein level was decreased in the presence of cape while increment in nfkb protein level was observed in the fructose administered group compared with control group. in case of pnfkb antibody, increment observed in fructose only and both cape and fructose administered groups, respectively. in cape only administered group, there was a decrement in the level of pnfkb protein. conclusion: depending on further analysis, experimental findings are expected to implicate the role of cape as a protective agent on high fructose diet-induced fatty liver model in relation of inos, nfkb and p-nfkb pathways. the investigating association of hepcidin levels with iron homeostasis and inflammation variables in pregnant women with intrauterine growth restriction a. g. agg€ ul 1 , n. uzun 1 , e. c ß inar tanriverdi 2 , h. € un 1 1 agri ibrahim cecen university, agri, turkey, 2 nenehatun maternity hospital, erzurum, turkey this study was designed to investigate hepcidin levels and their associations with iron homeostasis and inflammation variables in pregnant women with intrauterine growth restriction (iugr). a total of 88 pregnant women were included in this study. pregnant volunteers were divided into two groups (30 healthy pregnant women and 58 pregnant women with iugr). serum hepcidin, total free iron, ferritin, transferrin, transferrin receptor and interleukin-6 (il-6) levels were measured by elisa. also, hemoglobin (hb) and c-reactive protein (crp) levels were determined in serum samples from the healthy pregnant women and the pregnant women with iugr. there were significant differences in hepcidin, ferritin, transferrin receptor, crp and il-6 levels between healthy pregnant women and pregnant women with iugr. hepcidin, ferritin, crp and il-6 levels in pregnant women with iugr were significantly higher than healthy pregnant women (p). the mediators of systemic inflammation in lipopolysaccharide-induced neonatal sepsis rat model sepsis is an excessive inflammatory response that causes shock, multi-organ failure and high mortality. foreign bacterias and lipopolysaccharides lead to stimulation of endothelial cells to produce biologically active mediators such as proinflammatory cytokines and chemokines, cell adhesion molecules, and growth factors. then these mediators could be act on targets, which were involved in the initiation of systemic inflammation in neonatal sepsis. our aim was to indicate a protective role of thalidomide and etanercept, which have anti-tnf-a activity on systemic inflammatory response in lipopolysaccharide (lps)-induced neonatal sepsis rat samples. thirty 7-day-old wistar rats were randomly divided into five groups: a control group that received normal saline, a sepsis group that received lps, thalidomide, etanercept and both thalidomide and etanercept treatment group that were administered with therapeutic agents 6 hrs after lps injection. the rats were sacrificed at 24 hrs after lps or normal saline injection (n = 6). hepatic tissue tnf-a, il-6, icam-1 and pdgf levels were determined by enzyme-linked immuno sorbent assay (elisa) method in all groups. in sepsis group, tissue tnf-a, il-6, icam-1 and pdgf levels were statistically significantly higher than in controls (p < 0.001). at same time, pretreatment with both thalidomide and etanercept were found statistically dramatically decreases the levels of tnf-a, il-6, and pdgf when compared to sepsis group (p < 0.001). there were no significant differences in the icam-1 levels between the all treatment groups and the sepsis group. higher liver tissue tnf-a, il-6, icam-1 and pdgf levels are associated with severe bacterial infection. these proinflammatory cytokines and angiogenic factors may be important in the endothelial dysregulation seen in sepsis. therapeutic agents used in the present study can be help to avoid devastating effects of neonatal sepsis. n-stearoylethanolamine (nse)is saturated minor compound of natural origin that represents the large family of signaling lipids n-acylethanolamines, which belong to endocannabinoid system. considering the crosstalk between obesity-induced inflammatory response and its key role in synaptic dysfunction and neurodegeneration, our current study aimed to investigate the biological effect of nse on brain tissue under high fat diet-induced insulin resistance. previously we found that nse administration to insulin resistant rats caused normalization of liver and pancreas lipid composition followed by the improvement of glucose tolerance and insulin sensitivity (decline in serum insulin level and homa-ir value). moreover, this effect of nse correlated with inhibition of nf-kb translocation into the nucleus of peritoneal macrophages and decreased pool of serum tnfa level in obesity-induced insulin resistant rats. further experiments showed that fat overload triggered significant reduction in the level of main phospholipids (phosphatidylethanolamine, phosphatidylcholine and sphingomyelin), while there were no changes in cholesterol content. nse at a dose of 50 mg/kg during 2 weeks of administration to insulin resistant rats showed a tendency to restore the phospholipid level that was accompanied by increased neural cell survival (91%) compared to rats without treatment (84%). neuroinflammation accompanied by intensive reactive oxygen species (ros) production impairs neurotransmission in a wide range of neurodegenerative pathologies. flow cytometry is used for quantitative analysis of global dna methylation, but fluorescence microscopy is mostly preferred to qualitatively reveal intranuclear localisation of dna methylation and its copattern with other markers. both methods use a similar immunostaining protocol. in this study, we aimed to compare these methods concerning the detection of the global amount of dna methylation. for this, mouse embryonic fibroblasts were cultured either with or without phenol red and then stained for dna methylation or positive controls (histone, betaactin, phosphoakt) by specific antibodies, or nonspecific control antibodies. some cells were incubated with trypan blue before or after the addition of antibodies. fluorescence intensities were measured by the green fluorescence channel (530/30 nm). autofluorescence spectrum of cells was analysed, and fluorescence channel used for dna methylation detection was changed to red (650 nm lp). a poor discrimination between signal and noise was detected due to cellular autofluorescence interfering with specific detection of dna methylation by flow cytometry but not by microscopy. it was also the case for the other markers examined. conventional advances to reduce autofluorescence such using phenol red free culture media or trypan blue quenching were not effective, but using the red channel regarding autofluorescence spectra allows detecting specific staining of dna methylation by flow cytometry. but, green channel did work well for microscopy analysis. findings show that flow cytometry detection of dna methylation requires much attention to quench cellular autofluorescence compared to detection by fluorescence microscopy. one reason could be that flow cytometry detects all cellular content, but manual image-based analysis can exclude cytosolic components. these results suggest the usability of flow cytometry and microscopy as complimentary methods for dna methylation detection, but optimisation to reduce autofluorescence is crucial for flow cytometry. objectives: lung cancers are divided in two main groups as small cell lung cancer (sclc) and non-small cell lung cancer (nsclc) . docetaxel (dtx) and cisplatine are chemotherapeutic that has an anti-tumor activity against various solid tumors. the growing resistance against dtx and cisplatine (cis) still continues to be the biggest obstacle for the treatment success of nsclc patients. deguelin (deg.) is a natural plant derivative and has an encouraging activity against a lot of human cancers. the comparison of the treatment activity of the separate and combined usage of deg., which is a potential chemotherapeutic agent, and dtx, cis which are used in standard treatment, is aimed in this study. material-method: the ic50 doses of dtx, cis and deg. on the a549 and h1299 nsclccell lines were determined via the cell vitality tests in our study. the active concentrations determined were applied to nsclc cell lines as deg., dtx, cis and their combinations. the impacts of the medicine are studied by applying flow cytometric analyzes (apoptosis, cell cycle), glutathione and reducted glutathione, colony formation, migration and angiogenesis analyzes on the treated cells and measuring the oxidative stress index (osi). statistical analyse program, rstudio (v.0.98.501) and the r-script language were used to examine the differences between the agents. the states in which the pvalue was lower than 0.05 were accepted as statistically meaningful. results: we found that deg. has pro-apoptotic, anti-migratory and cytotoxic potential on lung cancer cells. deg. amplified cis and dtx-related anti-cancer efficacy (increased apoptotic cell content and cytotoxicity, reduced migration). also, deguelin pretreatment sensitized the cells dtx-treatment (reduced ic50 values). these effects were remarkable in p53-mutant cells. conclusion: deguelin, solely, has anti-cancer potential on nsclccells. both deguelin pre-treatment and combinantion with standart chemotherapeutics result in enhanced anticancer efficacy. the 83% of the lung cancers are non-small cell lung cancers (nsclc). despite docetaxel (dtx) and cisplatine (cddp) are agents used in the standard treatments of these patients and the recent improvements in the treatments, the response and remission rates observed on the patients are relatively nominal. selenium (se) is an essential diet component and is introduced to have a preventive impact on different levels of cancer. the aim of our study is to investigate the impacts of selenium addition on anticancer feature and tumor prevention before or/and during nsclc standard treatment. the ic50 doses of dtx, cddp and selenium on the a549 and h1299 (p53 mutant) nsclc cell lines were determined via the cell vitality tests in our study. the active concentrations determined and the stipulated available concentrations were applied to cell lines as dtx, cddp, se combinations. the impacts were compared by applying flow cytometric analyzes (apoptosis, cell cycle), glutathione and reducted glutathione, western blot analyzes on the treated cells and measuring the oxidative stress index (osi) and thioredoksin reductase activity. selenium pre-treatments reduced dtx-related ic50 concentrations at lower doses in both nsclc cells. however, cddprelated ic50 concentrations reduced dose-dependent manner. selenium supplementation also altered cell-cycle charactheristics at several concentrations and combination regimens. the remarkably higher osi values were observed after dtx treatment and osi levels were found to be lower in selenium pre-treated nsclc cells. selenium sensitizes nsclc cells to dtx treatment at lower concentrations. however, this effect is obtained dose-dependent fashion for cddp regimen. breast cancer is the most common female malignancy worldwide. human epidermal growth factor receptor 2 (her2) is overexpressed in 30% of breast cancers in association with aggressive phenotypes. the prognosis of metastatic breast cancer remains poor in spite of advances in therapy. as such, her2 has long been studied as a potential target for anticancer drugs. the modulation of intracellular signaling pathways leads to altered cell metabolism that triggers tumorigenesis and adapts cells to cancer cell metabolism. this characteristic hallmark of cancer metabolism is known as warburg effect meaning energy production via enhanced glycolysis. despite of several studies in breast cancer metabolism, little detail exists on the link between her2 overexpression and warburg effect. we have committed examining the nature of aerobic glycolysis in her2 overexpression. in breast cancer cell line mcf7, her2 overexpression (mcf-her2) results in mitochondrial dysfunction with low mitochondrial membrane potential (δᴪm) and ros accumulation. intracellular iron levels are also higher in mcf7-her2 cells than vector control (mcf7-vec). additionally, mcf7-her2 cells show enhanced levels of atp and lactate in association with increase in glucose levels. we have found that complex i activity increases in mcf7-her2 and decreases in knockdown of her2 in hcc1954 cells that is her2 positive breast tumor cell line. based on these results, we conclude that there is a link between her2 overexpression and metabolic indicators of warburg effect. expression and methylation analysis revealed 11 microrna genes deregulated by methylation and new potential target genes of mir-375 and mir-127-5p in breast cancer micrornas (mirnas) and methylation of mirna genes play a great role in epigenetic deregulation in malignant tumors. the aim of our study was to assess the contribution of methylation to expression level alterations of 20 mirna genes and to search for novel potential targets of these mirnas. to analyze alterations in expression we used qpcr technique with 2 references (rnu6, rnu48) and 30 paired (tumor/normal) breast cancer (bc) samples. for methylation analysis a methylation specific pcr and the same set of bc samples were used. significant downregulation was shown for mir-125b-5p, -129-5p, -132-3p, -193a-5p, -34b-3p, -212-3p, -127-5p, and -17-5p (p ≤ 0.05, fisher's exact test) in bc. we observed 10 mirna genes to be hypermethylated and mir-191hypomethylated. hypermethylation for 3 of these mirna genes was shown for the first time: mir-132, -137, and -1258 (41-34% of bc cases). a significant correlation between methylation and expression alterations was revealed for 5 mirnas with downregulation: mir-125b-5p, -129-5p, -132-3p, -193a-5p, and -34b-3p (spearman's correlation coefficient (rs) was in the range à0.71 to à0.81, p ≤ 0.01), and for 3 mirnas with both scene (down-and upregulation) as well: mir-148a-3p, -203a, and -375 (rs = à0.72 to à0.93, p ≤ 0.01). comparative analysis of the data on expression alterations of 20 mirna genes and 6 protein-coding genes, which were predicted as targets by mirwalk 2.0, revealed the negative correlation between expression levels for some potential mirna-mrna interaction pairs. for example, for pairs mir-375/rhoa, mir-375/rassf1(a), mir-127-5p/dapk1 (rs = à038 to à0.46, p ≤ 0.05). thus, both mirnas and methylation affect regulatory networks in bc. novel potential mirna-mrna interaction pairs could be useful in the development of bc therapy approach. this work was financially supported by grant 14-15-00654 from the russian science foundation. the authors thank the n.n. blokhin cancer research center for tissue samples. clear cell renal cell cancer (ccrcc) with metastases has pour prognosis: 5-year survival is about 9%. micrornas (mirnas) and methylation of mirna genes play a great role in epigenetic deregulation in malignant tumors. the aim of our study was to find out mirnas which methylation contributed to ccrcc progression, including metastasis, and to reveal potential target genes of these mirnas. to analyze methylation status, we used a methylation specific pcr as a method and a representative set of 70 paired (tumor/ normal) ccrcc samples. we also used 19 post-mortal renal tissues from individuals without cancer history as additional control. for expression analysis we used qpcr method and 45 paired ccrcc samples. we observed 9 mirna genes (mir-124a-1/-2/-3, -9-1, -9-3, -34b/c, -129-2, -193a, -107) to be hypermethylated, (p ≤ 0.05, fisher's exact test), 3 mirna genes to be hypomethylated and mir-203a with both scene (hyper-and hypomethylation was detected). methylation of 7 mirna genes (mir-124a-2/-3, -34b/c, -129-2, -107, -148a, -203a) correlated with advanced stage and/or tumor size and/or dedifferentiation. hypermethylation of mir-129-2, mir-203a, and mir-107 significantly correlated with metastasis presence (p < 0.05, fisher's exact test). besides, preliminary data revealed the positive correlation between hypermethylation of mir-129-2 and up-regulation of 3p protein-coding genes: rarb(2), rhoa, nkiras1, and chl1, which were predicted as targets by mirwalk 2.0 (spearman's correlation coefficients (r s ) was in the range 0.35-0.53, p ≤ 0.05). in conclusion, novel supposed interactions of mir-129-2 with target genes could be useful as missing chains in signaling pathways. tests for hypermethylation of mir-129-2, mir-203a, and mir-107 could be suggested as markers of metastasis and pour prognosis of ccrcc. because of difficulty in diagnosis and treatment hc is a clinical problem: early symptoms of hc are often non-specific and surgical resection is the only curative treatment for hc. it is well known that epigenetic alterations are linked to cancer development. the purpose of this study was to determine potential mechanisms of epigenetic regulation of genes related to energy metabolism in hc. we have performed bioinformatics analysis of the cancer genome atlas (tcga) project rna-seq data with crosshub software and found a number of genes involved in glycolysis and differentially expressed in cholangiocarcinoma. qpcr analysis revealed significantly decreased expression of pgm1 and eno3 genes in a majority of hc samples which were known as up-regulated in other human cancers according to the literature date. on the basis of tcga methylation dataset (450k illumina microarrays) we supposed that cpg methylation of pgm1 and eno3 promoters may play a role in their inactivation. using bisulfite sequencing study we identified several regions within the gene promoters (pgm1:~750 bp and 330 bp upstream tss; eno3:~750 bp downstream tss) that are frequently methylated in hc samples (up to 60%, 12/20) with down-regulated pgm1 and eno3 expression. thus, we demonstrated frequent and significant pgm1 and eno3 down-regulation associated with hypermethylation of the specific regions within the gene promoters in hc. the pattern of pgm1 and eno3 gene promoter methylation suggests a possibility of ones to be used for the hc diagnosis and development new strategies for therapy. this work was financially supported by grant mк-8047.2016 .4 from the president of the russian federation. the work was performed using the equipment of eimb ras 'genome' center. introduction: the development of stomach cancer is a multifactorial and complex process and includes multiple epigenetic, genetic alterations and dietary/non-dietary factors. iodine as an antioxidant may play a protective role against gastric cancer. the aim of this study was to investigate the changes in iodine level in rat with stomach cancer induced by n-methyl-n 0 -nitro-n-nitrosoguanidine (mnng). materials and methods: a total of 62 sprague dawley rats were randomly divided into six groups. 55 rats were administered with mnng (200 lg/ml) by oral gavage on days 0, 14 and 21 to initiate stomach cancer. during the experiment, 28 rats died and those surviving were sacrificed in the 3rd, 5th, 7th, 10th and 12th months of the experimental period (group i, ii, iii, iv, v, respectively). the control group (group vi) contains 7 rat which are given only food and water for 12 months. the stomach tissue was examined histopathologically. and also, iodine levels in stomach tissue was determined using the foss method. results: a decrease in iodine level was determined in stomach cancer tissue of rats in group i-v compared with normal healthy stomach tissue in group vi. when the control (group vi) iodine level was taken as % baseline, the % iodine levels of all groups were determined as follows 71.78, 52.79, 40.76, 25.33 and 11.96 for groups i-v, respectively. the pathological diagnosis of gastric cancer was adenocarcinoma. discussion and conclusion: the iodine levels of group i were higher than those of group ii (p < 0.01) and of groups iii, iv and v (p < 0.001) and also were lower than in the control group (p < 0.001). iodine deficiency as one of the risk factors of stomach cancer strongly supports the necessity for the application of effective iodine prophylaxis in the areas with iodine deficiency. iodine supplementation might be useful in stomach cancer therapy and therefore, further research is warranted. this study was supported by ataturk university (project number: 2005/147). effect of water extract of turkish propolis on mitochondrial membrane potential in human laryngeal epidermoid carcinoma cell lines propolis is the generic name for the resinous substance collected by honeybees from the buds of various plant sources and it is used by bees to seal holes in their honeycombs, smooth out the internal walls, and protect the entrance of bee hive against intruders. the aim of this study is to investigate what kind of changes the turkish propolis cause on mitochondrial membrane potential (mmp) of human laryngeal epidermoid cell lines (hep-2), by considering its anticancer features. water extract of turkish propolis (wep, 1-3 mg/ml) and ethanolic extract of turkish propolis (eep, 0.075-3 mg/ml) were prepared and incubated with hep-2 cell lines (24, 48, and 72 h). mmp was investigated with a flourometric method by using dioc 6 (3,3 0 -dihexyloxacarbocyanine iodide). the most significant mmp decrease was seen on 72rd hour. both wep and eep extracts at all concentrations decrease mmp according to that of control. the recent studies have shown that propolis extracts have induced apoptotic cell death by decreasing mitochondrial membrane potential in various cancer cells. it was concluded that both wep and eep decreased mitochondrial membrane potentials on hep-2 cell series according to control (0 concentration) depending concentration and time. there are numerous transcription factors involved in the regulation of the inducible gene expression. thus, transcription of proinflammatory genes, steroid hormone receptors, etc. is controlled by the group of factors triggering gene expression which includes nf-kb. another group of factors is involved in the formation of the structure of the chromatin of the inducible genes regulatory regions, providing competence for gene expression. it is expected that this group of factors includes the proteins of nf1 (nuclear factor 1) family. there are few data suggesting that the nf1 factors maintain potentially active state of the chromatin of the hormone-dependent gene promoter regions. these findings initiated studies of the correlation between presence of the nf1 transcription factors on the chromatin of a gene regulatory region and the functional state of the gene in vivo. as a model we chose the rat tryptophan dioxygenase (tdo) gene which is expressed tissue-specifically in the liver under control of glucocorticoid hormones. three constitutive dnase i-hypersensitive regions are identified in the regulatory region of this gene. to conduct the study we used rat liver and kidney. the basic methods were electrophoretic mobility shift assay (emsa), immunoblotting assay and chromatin immunoprecipitation combined with real-time pcr (chip-qpcr). using emsa we found that the proteins of nf1 family interact with the constitutive dnase i-hypersensitive regions in vitro. immunoblotting assay of the protein fraction from rat liver used in emsa experiments showed the presence of the nf1-b1 isoform. chip-qpcr revealed statistically significant differences in the level of the factor nf1 enrichment of the tdo gene regulatory region between the rat liver and kidneys at p < 0.05. these data suggest the involvement of the nf1 proteins in the formation of the chromatin structure of the rat tdo gene promoter region. reciprocal (9;22) translocation and bcr-abl fusion protein that is responsible for developing leukemia are observed in more than 95% of chronic myeloid leukemia (cml) cases. epigallocathecin-3-gallate (egcg) is a green-tea flavonoid and egcg is proposed as a natural anti-cancer agent. histone modifications which contain histone deacetylases (hdac) and histone acetyltransferases (hat) are parts of epigenetic regulations. hdacs play important roles in different human malignancies including leukemia via activation of abnormal signaling pathways. hdac inhibitors have become remarkable therapeutic molecules for malignancies. the aim of this study is to determine the expression changes of leukemia-related hdacs with the treatment of egcg in k-562 cells. the cytotoxic effect of egcg on k-562 cells was determined in time and dose dependent manner by wst-1 analysis. total rna was isolated from k-562 cells. reverse transcription procedure was performed for cdna synthesis and gene expressions were detected by rt-qpcr. the expression level of hdac4, hdac6, hdac11 gene that supports cell proliferation was down-regulated 2.29, 2.56, 3.81 folds in k-562 cells treated with ic50 dose of egcg, according to control, respectively. our current findings suggest that is a polyphenol egcg may be a hopeful agent in treatment of cml by hdac inhibitory effect. chronic lymphocytic leukemia (cll) is a disorder of morphologically mature but immunologically less mature lymphocytes and is manifested by progressive accumulation of these cells in the blood, bone marrow, and lymphatic tissues. carbonic anhydrase (ca) is a metalloenzyme which is widely distributed in the living world, and it is essential for the regulation of acid-base balance. anti-ca antibodies have been reported in many disorders, such as systemic lupus erythematosus, sj€ ogren's syndrome, rheumatoid arthritis, endometriosis, idiopathic chronic pancreatitis, type 1 diabetes and graves' disease. the goal of this study was to investigate carbonic anhydrase i and ii (ca i and ii) autoantibodies in cll. 38 patients with cll and 39 healthy controls were included in the study and ca i and ii autoantibody levels were investigated by elisa. the ca i autoantibody levels of cll group were significantly higher than the healthy group (p = 0.006) while there was no statistical difference between serum ca ii autoantibody levels of the groups (p = 0.301). we found a significant positive correlation between hemoglobin and hemotocrit levels in patients with cll (r = 0.931, p = 0.0001). cut-off value of 0.072 absu for anti-ca i was associated with 92% sensitivity and 41% specificity and a cut-off value of 0.047 absu for anti-ca ii was associated with 48% sensitivity and 85% specificity for predicting cll. the ca i autoantibody levels in patients with cll were found higher compared to control group and the results suggest that ca i autoantibody may be involved in the pathogenesis of cll. genetic and epigenetic aberrations can lead to the activation of oncogenes and inactivation of tumor-suppressor genes (tsgs) followed by the development of malignant tumors. in the present work we evaluated the frequency of alterations of cpg island methylation and dna copy number in 47 paired (tumor/normal) breast cancer (bc) samples using comparative dna hybridization on noti-microarrays and original niman software. the microarrays contained 180 noti-clones associated with 188 chromosome 3 genes. expression alterations were assessed with the use of qpcr technique, ddct method and original atg software. in total, 35 noti-sites with high (15-38% of cases) hypermethylation/deletion (hm/d) frequency were revealed in bc. among genes associated with these sites, there are both known tsgs and tsg-candidates (aldh1l1, vhl, ctdspl, etc.) as well as genes, which involvement in breast oncogenesis was shown for the first time (lrrn1, foxp1, prickle2, etc.). noti-microarray data were verified selectively using bisulfite sequencing for vhl, nkiras1, itga9, lrrc3b, and ctdspl genes. several genes with high hm/d frequency (aldh1l1, ephb1, itga9, and ropn1) were tested for expression alterations using qpcr. frequent (57-90% of cases) and significant (> 2-fold) down-regulation was shown for all of them in bc. the most significant expression loss was observed for aldh1l1 geneon the average 50-fold mrna level decrease in 90% of samples. the involvement of the majority of genes with high hm/d frequency in breast oncogenesis was shown for the first time. these genes are novel tsg-candidates in bc. functional hypermethylation associated with expression loss was shown for aldh1l1, ephb1, itga9, and ropn1 genes thereby strengthening the speculation on tumor suppressor abilities of these genes. methylation and expression analyses of genes, that were revealed by noti-microarrays, were financially supported by grant 14-15-00654 from the russian science foundation. functional hypermethylation of a number of chromosome 3 genes was revealed in colon cancer using noti-microarrays cancer is a disease of genome caused by genetic and epigenetic aberrations. noti-microarrays, that were developed by prof. e.r. zabarovsky, is a unique tool that allows us to simultaneously detect hypermethylation of cpg islands and dna deletionstwo major reasons of inactivation of tumor suppressor genes (tsgs). in the present work, the frequency of chromosome 3 genetic and epigenetic alterations in colon cancer (cc) was evaluated. noti-microarrays, that contained 180 noti-clones associated with 188 chromosome 3 genes, were used for comparative (tumor/normal) hybridization of dna from 24 paired cc samples. data analysis was performed using original niman software. expression alterations were evaluated using qpcr technique and original atg software. in total, 24 noti-sites with 20% and above hypermethylation/ deletion (hm/d) frequency were revealed in cc. among genes associated with these sites, there are several known tsgs and tsg-candidates (for example, vhl, ctdspl, and itga9), but for the majority of genes, involvement in colon oncogenesis was shown for the first time (for example, lrrn1, nbeal2, and ube2e2). the highest hm/d frequency was observed for ankrd28, nkiras1/rpl15, itga9, cmtm6, and gor-asp1/ttc21a genes -38-42%. expression alterations were evaluated for 3 genes with high hm/d frequency (plcl2, prickle2, and ppp2r3a) and significant mrna level decrease (> 2-fold) associated with hypermethylation was shown for all of them in the majority of samples. a number of novel potential tsg-candidates was revealed in cc. functional hypermethylation associated with expression decrease was shown for plcl2, prickle2, and ppp2r3a genes thereby enhancing the suggestion on tumor suppressor function of these genes. this work was financially supported by in many countries, radon is the second leading cause of lung cancer, which accounts from 3% to 14% of cases. it is obvious that the population of all the developed and industrial countries in the world spend most f their time, almost 80%. therefore it is necessary to explore the obtained radiation dose, because of the presence of radon in a room due to the radon emanation from the soil and exhalation from a variety of building materials. the developed countries solve this problem of radon pollution as well as create a special monitoring services. the paper presents some data of 8 genes molecular-genetic analysis from patients with lung cancer who live in almaty located in a foothill area of tectonic faults. the object of research were blood samples obtained from patients diagnosed with lung cancer who are receiving a treatment at the almaty oncology center and living in the city of almaty, where the level of radon activity exceeds the norm approved by the international commission on radiation safety. as a control group relatively people living in the plains, characterized by a lower radon emanation have been considered. to determine mutations in the genes polymerase chain reaction with a subsequent analysis of restriction fragment length polymorphism has been conducted. the pcr products were subjected to hydrolysis by bstni restriction endonucleases haeiii, ras i. disturbances in the genes under consideration to variour types of cancer development. the analysis showed that examinees do not have mutations in the kras gene codons 12-13, which corresponds to a control group consisting of 90 people living in the city of balkhash. on the whole, molecular genetic studies have shown that 44 examined patients do not have mutations in the kras gene. one mutation was been found in the egfr gene. aim: polyps are abnormal growths of tissue that can be found in gastro intestinal system. they are most often found in the colon and rectum. most polyps are noncancerous (benign) however, because of abnormal cell growth, they can eventually become cancerous. the aim of this study is to determine the concentrations of trace element contents in colon and rectum polyp tissues and whether there is any relationship between polyp tissue element levels and the disease. material and method: the present study was conducted on total of 82 individuals including 57 patients and 25 healthy subjects. while receiving normal intestinal tissue from healthy control group; from the patient group both normal tissue and polyp samples were taken during colonoscopy procedure. the concentrations of the elements (al, cr, mn, fe, co, ni, cu, zn, as, se, ag, cd, hg and pb) were determined with induced coupled plasma-mass spectrometer. results: the mean concentrations of cr, mn, ni, se and ag in colorectal polyp tissues of patients were significantly higher than in colorectal tissues of control subjects (p is less than 0.05). on the other hand the mean concentration of cd and pb in colorectal polyp tissues of patients were significantly lower than in control colorectal tissues of control subjects (p is less than 0.05). there was no any significant difference between the groups in terms of concentrations of al, fe, co, zn, as and hg (p is more than 0.05). conclusion: the differences found in some elements between polyps and a control tissues may provide an indication about the role of trace elements in the early stage (polyps) in the colon carcinogenic process and encourages further studies to confirm the involvement of such elements in neoplastic processes. the use of herbal medicines is steadily growing, with approximately 40% of the population use herbs to treat various illnesses in the western world. vitex agnus-castus has been used since ancient times as a remedy. the aim of this study was to investigate the in vitro anticancer activities of vitexagnus-castus oil. for this purpose, the cytotoxicity of vitexagnus-castus oil in sh-sy5y cells was investigated by crystal violet staining. ec50 was found to be 0.5%(w/w) vitexagnus-castus oil for this cell line. this dose was applied to the cell for 24 h, and the cells were harvested for further studies. vitex agnus-castus oil treatment increased bax and p38 mrna levels. on the other hand, bcl-2, bcl2 l1, erk-2, jnk, caspase 3 and 8mrna expression levels were reduced significantly withvitexagnus-castus oil treatment while p53 and pten remained unchanged. these results indicate that another effector caspase such as caspase 6 or 7 may be involved apoptosis process which remains to be elucidated. moreover, mapk pathways, p38 and erk, may be involved in vitexagnus-castus oil induced apoptosis in sh-sy5y cells. these initial observations suggest that this agent might not be useful in treating cancers. further detailed studies should be carried out to elucidate the exact mechanism of vitexagnus-castus oil in neuroblastoma cell lines. melanoma is a skin cancer with a melanocyte origin that can occur in any part of the body that contain melanocytes. while melanoma is less common than other skin cancers, it causes the majority of deaths related to skin cancer. several gene expression databases have shown that interferon regulatory factor 4 (irf4) is upregulated in melanomas, and genome wide association studies linked variation at irf4 locus with skin cancers. irf4 was first identified to have roles in lymphocyte development and function. studies have identified a 'non-oncogene addiction' of malignant cells to irf4 in various hematopoietic cancer types. the aim of this study is to investigate the role of irf4 in melanoma cell lines. lentiviral vectors were used to reduce irf4 levels in melanoma cell lines. a gfp competition assay was performed to study the competitive fitness of melanoma cells with irf4 knockdown (gfp positive cells) over melanoma cells with normal irf4 levels (gfp negative cells). cell cycle profiles were investigated in melanoma cells with irf4 knockdown by propidium iodide staining. migration potential was assessed as well by wound healing assay. our preliminary data showed a decreased competitive fitness for cells with decreased irf4 levels. cell cycle profiling showed increased g0/g1 and decreased g2/m levels in irf4 knockdown cells compared to controls. wound healing assay results showed no difference between controls for cells with reduced irf4 levels. taken together, these results indicate that irf4 knockdown affects the melanoma cell lines' survival and cell cycle profile, suggesting a non-oncogene addiction of melanomas to irf4. these observations are largely similar to previous observations in hematopoietic cancers. unravelling the role of irf4 in melanoma will increase our knowledge about melanoma development and progression and thereby may lead to targeted therapy in melanoma treatment. humans are exposed to various chemicals having beneficial or toxic effects at a time in their daily lives. 7,12-dimethylbenz[a] anthracene (dmba) is a carcinogenic compound produced during the incomplete combustion of carbon-containing compounds. endosulfan is an organochlorine pesticide used against insects on food. morin is an antioxidant, antiinflammatory and chemoprotective flavonoid. this study is aimed to determine the effect of morin in the presence of dmba and endosulfan. for this purpose, 56 adult wistar male rats weighing 170-255 g were randomly selected and divided into eight groups. 25 mg/kg body weight (b.wt.) morin and 5.0 mg/kg b.wt. endosulfan were given to morin and endosulfan treated groups three times in a week. the rats in dmba treated groups were gavaged with 30.0 mg/kg b.wt. dmba three times during the administration period (54 days). cytochrome p4501a (cyp1a) associated 7-ethoxyresorufin o-deethylase (erod) and glutathione s-transferase (gst) activities were measured in rat liver cytosols and microsomes. in addition, liver tissues were evaluated by histopathological analysis. erod activities of control, morin, endosulfan, dmba, morin+endosulfan, morin+dmba, dmba+endosulfan and morin+dmba+endosulfan groups were 71 ae 7, 112 ae 6, 114 ae 8, 126 ae 6, 121 ae 9, 156 ae 12, 151 ae 15 and 185 ae 13 pmol/min/mg protein, respectively. all treatments increased erod activities. gst activities of these groups were 365 ae 14, 430 ae 27, 365 ae 24, 651 ae 57, 460 ae 10, 577 ae 25, 585 ae 23 and 649 ae 33 nmol/min/mg protein, respectively. histopathological studies showed that endosulfan and dmba induced inflammation in the liver tissues and morin reduced their effects. in conclusion, morin treatment increased the metabolism of dmba and endosulfan by inducing cyp1a activity. gst activities of morin+dmba+endosulfan group were not significantly different from those of dmba group. histopathological studies indicated that morin administration reduced the toxic effect of endosulfan and dmba in the liver cells. hepatocellular carcinoma (hcc) is the sixth most common cancer and third most frequent cause of cancer-related death worldwide. molecular mechanisms of hepatocarcinogenesis is still unclear. the impairment of epigenetic mechanisms is implicated in the development of multiple cancers, including hcc. transforming growth factor-beta has been shown to play both tumorsuppressive and tumor promoting roles. transforming growth factor-beta signaling pathway involves activation of smad2 and smad3 by the type i receptor and formation of smad2/3/4 heteromeric complexes that enter the nucleus to regulate transcription. 3-deazaneplanocin a is an inhibitor of the histone methyltransferase ezh2. we aimed to reveal the effect of 3-deazaneplanocin a on transforming growth factor-beta /smad pathway in hepg2 cell line. hepg2, a human liver cancer cell line cultured in dulbecco's minimal essential medium supplemented with 10% fbs. the cells were seeded the day before 3-deazaneplanocin a administration and then the cells were treated with 5 lm 3-deazaneplanocin a for 3 days. expression levels of genes were analyzed by roche lightcyclerò 480. gapdh was used as housekeeping gene. apoptosis assay was performed by the muse annexin v and dead cell assay kit. the unpaired t-test was used to compare variables and p < 0.05 was accepted as statistically significant. 3-deazaneplanocin treatment was significantly reduced transforming growth factor-beta, smads 2-7 in hepg2 cells (p < 0.05). we also found that 3-deazaneplanocin induces apoptosis in treated cell line (p < 0.05). as a result, 3-deazaneplanocin a may take place in treatment of hepatocellular cancer by its inhibitory effect on transforming growth factor-beta /smad pathway and inducing apoptosis in liver cancer cells. brefeldin a (bfa) is a lactone antibiotic first isolated from the fungus eupenicillium brefeldianium. bfa inhibits the transport of secreted proteins from endoplasmic reticulum (er) to golgi apparatus, leading to disruption of golgi function, accumulation of unfolded and not fully incompletely processed proteins in er. bfa also inhibits cell proliferation, phosphorylation and migration of cancer cells. therefore in this study, we investigated the effects of bfa on breast cancer cell proliferation of various phenotypes. in we observed that bfa inhibited the proliferation of all three phenotypes of breast cancer cells, but the effects of bfa were seen at different times and doses. according to time and dose, bfa was observed more effective to mcf-7 compared to other cell lines. physiological, pathological and physical factors. moreover, nlr may represent the two opposing inflammatory and immune pathways that exist together in cancer patients. we aimed to investigate nlr in breast cancer in our population. methods: using data retrieved from the medical records, 66 women diagnosed primary breast cancer met our study inclusion criteria as they had a complete blood count with leukocyte differential performed before any anti-cancer therapy. and 44 women with benign mammary neoplasm/disease, followed up in the outpatient clinics of mammary disease and confirmed with sonographical/histopathological examination, made up our controls. exclusion criteria included laboratory evidence of white blood cells count (wbc) > 10.5 9 10 9 /l. differential leukocyte counts were obtained by bc 6800 (mindray medical international ltd., china), we examined wbc, neutrophil, lymphocyte, platelet counts, and hematocrite, nlr, mean platelet volume values. results: although there is lack of evaluation of tumor-associated neutrophils and lymphocytes, higher nlr median values and lower lymphocyte mean counts (lymphopenia) were shown in women with breast cancer (p < 0.0001). there was a weak negative correlation in breast cancer between nlr values and platelet counts (r s = à0.274; p = 0.026). holmboe] is distributed throughout southern mediterranean europe from spain to the eastern mediterranean on anatolian peninsula of turkey. present study was designed to investigate the in vitro anti-cancer activities of turkish black pine essential oil. the essential oil was extracted by steam-hydrodistillation and its chemical composition analyzed by gc-ms. the major components of the essential oil were a-pinene, b-pinene and trans-b-caryophyllene, respectively. the crystal violet staining method was used to investigate the cytotoxicity of essential oil in sh-sy5y cells. ec50 was found to be 0.75% (w/w) essential oil for sh-sy5y cells. neuroblastoma cells were incubated at 37°c for 24 h. after 24 h, cells were harvested for further studies. bax and p38 mrna levels were significantly elevated in essential oiltreated cells. on the other hand, bcl-2, bcl2 l1, casp-3, casp-8, erk-2 and jnk expression were significantly downregulated. unlike these proteins, p53 and pten mrnas were not changed. in this study, apoptosis was enhanced by turkish black pine essential oil treatment which was activated by the involvement of another effector caspase subfamily, like casp-6 and casp-7. additionally, erk and p38 mapks may be associated with upregulation of the level of bax. based on these results, we suggest that p. nigra subsp. pallasiana essential oil might not be well-suited in cancer treatment. however, further detailed research is necessary to establish the exact role of p. nigra subsp. pallasiana essential oil in sh-sy5y cells. p-05.02.2-026 the protective effect of newly derivatized compound naringenin-oxime and relative to naringenin against cisplatin-induced nephrotoxicity and genotoxicity in rat background: the aim of this study was to evaluate the possible protective effect potentials of newly derivatized compound naringenin-oxime (ng-ox) relative to efficacy of free naringenin (ng) on cisplatin (cis) induced nephrotoxicity and genotoxiticity in rat. methods: totally, fifty six male wistar albino rats were equally divided into eight groups as follows: control; cis treatment (7 mg/kg b.w., i.p.), ng and ng-ox (20 mg/kg b.w., i.p daily for 10 days) alone treatment; cis + ng (20 or 40 mg/kg b.w., i.p daily for 10 days) and cis+ng-ox (20 or 40 mg/kg b.w., i.p daily for 10 days) combination treatment. at the end of the study total antioxidant capacity (tac) levels, total oxidant status (tos), lipid peroxidation (lpo), total thiol, catalase (cat) were studied in homogenate kidney. peripheral lymphocyte cell dna damage was investigated with comet assay results: the results suggest that cis induces oxidative stress resulting in increased tos and lpo reduction thiol, tac and cat in kidney and increased peripheral lymphocyte cell dna damage. the treatment with naringenin and naringenin oxime alone or with cis treatment showed a protective effect against the toxic influence of cp on peroxidation of the membrane lipids and an altering of the total thiol status in the kidney of rats. from our results we conclude that naringenin and naringenin oxime functions as a potent antioxidant and suggest that it can control cp-induced nephrotoxicity and genototoxicity and ng-ox was found more protective than that of ng on cisplatin induced toxicity in rats. keywords: naringenin, naringenin-oxime, antinephrotoxic, antigenotoxic, comet assay. introduction: oxidative damage is considered to play a pivotal role in ageing, several degenerative diseases, and carcinogenesis. lung cancer is the most common type of cancer, resulting in over 1.3 million deaths each year worldwide. accurate and reliable determination of superoxide radicals has been widely investigated using spectrophotometric, electrochemical, amperometric, polarimetric, piezoelectric technologies. among these methods, electrochemical detection is a most promising approach to achieve accurate, separate and rapid superoxide radicals monitoring with using biosensor system. materials and methods: we used a new technic for detecting superoxide radicals in samples. superoxide dismutase (sod) enzyme immobilized on the surface of gold electrode with the help of gelatin, bovin serum albumin (bsa) and glutaraldehyde (ga) crosslinker. for the biosensor preparing benzoquinone selected as a mediator in working buffer and measurements were carried out at à0.7 v. result: for the optimization studies, effect of the bsa, gelatin, glutaraldehyde, ph, buffer concentration on biosensor response. characterization of the biosensor commitment to the work process and answer reproducibility were evaluated. the analytical characteristic of the biosensor were evaluated by measuring the steady state current response to superoxide radical concentrations. the electrochemical response of the enzyme electrode was linearity gradually leveled of at higher concentration. we found that crosslinking of the sod (e.c.1.15.1.1) with glutaraldehyde could be achieved over a wide range of relative mole ratios in 50 mm phosphate buffer at ph 7.5, glutaraldehyde concentration of %2.5. discuss and conclusion: in this study, a new technique for developed sod biosensors has been developed, which features effective combination of sod/gelatin/bsa/ga modified electrode, trapping of sod and glutaraldehyde cross-linking. this technique is reliable and cost effective. the effect of astaxanthin on apoptosis and cell arrest in u87 brain cancer cell line f. s€ og€ utl€ u, b. € ozmen yelken, c ß . kayabasi, a. asik, s. gonca, r. gasimli, s. yilmaz s€ usl€ uer, c ß . biray avci, c. g€ und€ uz department of medical biology, izmir, turkey a brain tumor is a collection, or mass, of abnormal cells in your brain. brain tumors can be cancerous (malignant) or non-cancerous (benign). the brain is one of the least accessible organ that active pharmacological compounds cannot be delivered. the two physiological barriers control and block the entry and exit of endogenous, exogenous compounds. one of these is the bloodbrain barrier and the other is the blood-cerebrospinal fluid barrier. this structures maintain protection of the brain. when there is a cancer case, it can lead to problem. astaxanthin with potent antioxidant properties can cross blood-brain barrier. in our study, we aimed to evaluate the effects of astaxanthin on apoptosis, cell cycle and also migration in brain cancer cell line. in present study, xcelligence real-time cell analyzer was used so as to determine cytotoxic effect of astaxanthin in u87 cell line. changes of apoptosis and cell cycle in u87 cell line exposured to ic 50 dose of astaxanthin (19.5 nm-80 lm) are detected with annexin v-egfp apoptosis detection kit and cycle test plus dna reagent kit with facs, respectively. the result of apoptosis and cell cycle test was analysed in flow cytometry. the group to which active substance was not treated was used as controlled. the wound healing assay performed in order to measure migration ability of u87 cell line to which astaxanthin was treated or not. ic 50 dose of astaxanthin was calculated as 9.20 lm at 48 h by xcelligence rtca sp based on time and dose. astaxanthin decreased the migration ability at rate of 25% in u87 cells treated by ic 50 dose of astaxanthin. astaxanthin had no apoptotic effect on viability in u87 cell line and astaxanthin caused an increase of g2/m phase arrest (1.15 fold) and s phase arrest (1.41 fold). astaxanthin has cytotoxic effects in brain cancer. it determined that astaxantin decreases cell cycle potential at g2/m even a little. the effect of anticancer of astaxanthin should be researched further. interferon regulatory factor 4 (irf4) is a critical transcription factor in development and survival of different cell types including immune cells and melanocytes. furthermore, it has been demonstrated that irf4 expression levels are elevated in several lymphoid cancers, and irf4 is one of the key transcription factors for the survival of these cancers. several genome-wide association studies identified irf4-linked genetic variants to increased melanoma incidence. in addition results from our lab and elsewhere have shown high levels of irf4 expression in melanoma cell lines. furthermore our preliminary results suggest melanoma cells are sensitive to irf4 expression levels. however, there are no published studies about irf4 target genes in melanoma cells. in this study, we are investigating the genome-wide target genes of irf4 in melanoma cell lines via high-throughput sequencing of immunoprecipitated chromatin (chip-seq). we have identified possible irf4 binding regions in loci with known key roles in development of melanocytes from neural-crest cells. one such key factor is mitf, which is the master regulator in melanocyte development and also plays critical roles in melanoma. integrating chip-seq and rna-seq data suggests irf4 as a transcriptional regulator of genes related to progression of melanoma. objectives: aim of this study was to evaluate prognostic importance of selected laboratory parameters (c-reactive protein (crp), gama glutamiltransferaz, ferritin (fer), potassium, chloride, calcium, phosphorus, magnesium, total protein, aspartat aminotransferaz, alanin aminotransferaz (alt), ifn-c, il-6, tnf-a) in non-small cell lung cancer (nsclc). material and methods: 20 patients with nsclc who were treated with chemoradiotherapy (crt) prospectively evaluated. all patients were newly diagnosed tumour. heparinized blood samples were taken from the patients before and after the completion of crt. fer analyzed by chemiluminescence method on beckman coulter dxi 800; ifn-c, il-6, tnf-a were analyzed with elisa kits (boster biological technology) and other biochemical parameters analyzed on abbott architect c16000. post-crt and pre-crt levels compared with survival. results: the lr cox regression analysis revealed that pre-crt ferritin was significantly associated with survival of patients with nsclc (hazard ratio (hr) = 1.007, p = 0.023, 95%ci; 1.001-1.013). it was also demonstrated by lr cox regression analysis, high levels of pre-crt crp was associated with worse outcome of patients (hr = 1.017, 95%ci;1.001-1.032, p = 0.035). after crt, mean alt level was determined as 16.71. there was survival difference in nsclc patients with high post-crt alt (hr = 0.912, 95%ci; 0.841-0.990, p = 0.027). conclusions: there exists a clinically relevant relationship between pre-crt fer concentration and the prognosis of survival in patients with nsclc. elevated fer is the result of inflammation rather than body iron overload. ferritin showed negative correlation with survival so it could be a useful biomarker to indicate bad prognosis of the patients with nsclc. additionally, crp which is easy to detect and feasible for the use in the routine clinical practice should be considered in the prognosis of nsclc patients. keywords: ferritin, nonsmall cell lung cancer, survival, c-reactive protein. epigenetic therapy tries to reverse the aberrations followed to the disruption of the balance of the epigenetic signaling ways through the use of natural and synthetic compounds, active on specific targets, such as dna methyltransferases (dnmts). we previously synthesized some benzoxazole and benzamide derivatives which might have anticancer activities on account of their heterocyclic structure. our studies showed that not only these compounds caused selective cytotoxicity towards cancer cells (hela) with little or no toxicity on normal cells (l929) but also were not genotoxic. in this study, we aimed to test whether these compounds changed global demethylation profile of normal and cancer cells. we used methylation specific comet assay (msc assay) to determine global methylation levels of cells. cells were treated with the tested compounds at ic 50 concentrations for 48 h. slides were prepared as did in alkaline comet assay, then they were incubated with methylation specific restriction enzymes (mspi, hpaii) before electrophoresis. differences in global methylation levels between nontreated control cells and cells treated with compounds were compared by using tail moment data. 5-aza-c, a demethylating agent, was used as reference drug. msc assay results revealed that none of the tested 9 compounds caused hypermethylation on both cell lines. however, global methylation levels decreased statistically (p < 0.05) through both cells treated with c-2 and c-8. only c-3 decreased methylation level on l929 but not on hela. consequently, c-2 and c-8 caused demethylation on hela cells similarly with 5-aza-c at low concentrations. for the reason that dna methylation is regulated mainly dnmt enzymes in the cell, c-2 and c-8 might cause global demethylation in the cell by inhibiting dnmt activity. further studies will be done to support this prediction. overall, macrophages and some subtypes of lymphoid cells are found in tumour stroma. these cells secrete a variety of growth factors, proinflammatory cytokines and chemokines, esp. tnf-a, il-1b and il-6, causing the formation of inflammatory microenvironment around tumour cells. tnf-a and il-1b signaling increases activity of nf-kb pathway. at the same time, il-6, triggers jak-stat signaling pathway, which effector is stat3. nf-kb and stat3 activity facilitates hyperexpression of mir-nas mir-155, mir-181 and mir-21 as well as down-regulates expression of mirnas mir-15/16, mir-199 and let-7. this investigation aims to identify in what way these shifts in mirnaome can lead to epigenome reorganization supporting the cell transformation. mirna targets within gene transcripts were predicted in silico using targetscan software. transcripts of hdac2/4/8/9 and sirt1/5 genes encoding histone deacetylases carry targets for at least one of up-regulated mirnas mir-155, mir-181 or mir-21. also, these mirnas can silence ezh1, mll, mll3, nsd1, setd6/7/8, smyd1, suv39h2 genes encoding histone methyltransferases. mirna mir-21 suppresses gene encoding de novo dna methyltransferase dnmt3b. at the same time, down-regulation of mirna mir-15/16 can allow hyperexpression of gene encoding acetyltransferase elp3. these shifts impair dna and histone methylation, cause the increase of overall level of chromatin acetylation and expression and, therefore, create epigenetic background for reactivation of silent transposons, oncogenes as well as other genes important for cell transformation. immune system can paradoxically facilitate the tumour growth instead of healing. cancer-related inflammation leads to the mir-naome and epigenome shifts contributing to the tumour promotion and progression. lysine acetylation is one of the key mechanisms to regulate chromatin structure and transcriptional activation. acetyl-lysine modifications are recognized by bromodomains, which are small interaction modules found on diverse proteins including histones. among these acetyl-lysine reader proteins is the family of the bet (bromodomain and extra-terminal) proteins which contain tandem bromodomains (bd1 and bd2). the recent discovery of potent and specific inhibitors for the bet family proteins has stimulated intensive research activity in diverse therapeutic areas, especially in oncology, where bet proteins regulate the expression of key oncogenes and anti-apoptootic proteins. several bet inhibitors are currently in clinical trials and reported to exhibit promising clinical activities. however, pleiotropic nature of bet proteins regulating tissue-specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. here, we report the recent progress in the development of bet inhibitors in korea research institute of chemical technology (krict). we have identified the bet inhibitors with a novel scaffold different from the previously reported diazepine and azepine scaffolds and specific for first bromodomains (bd1s). a medicinal chemistry effort is currently made to optimize the pharmacokinetic properties of these lead compounds for further drug development. the experimental data from the biochemical and cell-based assays for these bd1-selective bet inhibitors will be presented. family of small c-terminal domain serine phosphatases (scp), which includes ctdspl, ctdsp1, and ctdsp2, plays a regulatory role in a number of vital processes. in particular, it is shown that ctdspl is capable to activate the retinoblastoma protein (rb) which is well-known tumor suppressor and one of the key cell cycle regulators. although the question on whether ctdsp1 and ctdsp2 dephosphorylate rb is open, high similarity of sequences and three-dimensional structures of phosphatases may indicate the similar function of these enzymes. in the current study expression of scp genes was evaluated by quantitative pcr in 28 non-small cell lung cancer (nsclc) samples. using original crosshub software, that combines an analysis of high-throughput sequencing data of the cancer genome atlas project (tcga) and databases of mrna-mirna interactions (targetscan, mirtarbase, etc), the involvement of mir-183-96-182 microrna cluster in co-regulation of ctdspl/1/2 genes in nsclc was predicted. the significant (5-fold on the average) and simultaneous decrease of mrna levels of ctdspl/1/2 genes was revealed in the majority of nsclc samples (82%, 23/28). such unidirectional expression change and strong positive correlation between phosphatase expression levels (r s = 0.53-0.62, p ≤ 0.01) allowed us to suggest a common mechanism of their inactivation. we evaluated the expression of predicted co-regulators of scp gene expression, mir-183-96-182 family, in examined nsclc samples. as a result, the simultaneously increased levels of all three mir-nas in most nsclc samples (82%, 23/28) and negative correlation with phosphatase gene expression was shown. the results suggest the ability of investigated phosphatases to exhibit tumor-suppressive activity and the involvement of mir-183-96-182 micrornas in the regulation of rb protein activity via inactivation of ctdspl/1/2 in nsclc. cancer is one of the leading causes of death in all around the world. cancer is defined as a disease involving abnormal cell growth with the potential to invade or spread to other parts of the body. tumor markers are substances that are produced either directly by the tumor or as an effect of the tumor on healthy tissue. tumor markers can be used for screening, determining prognosis and monitoring effectiveness of therapy and disease recurrence. the aim of this study is to investigate the frequency of tumor markers orders and the appropriateness of these requests. laboratory information systems data for 2015 were reviewed. for 2015, a total of 35874 patients and 55539 tumor marker requests were included. carbohydrate antigen 19-9, cancer antigen 125, cancer antigen 15-3, prostate specific antigen, alphafetoprotein and carcinoembryonic antigen were measured by chemiluminescence method. according to the data from the year of 2015, both positive tumor markertest resultsratio and the positive patient ratio were 14%. in the patients group with increased marker levels, 48% of the patients had no history of cancer. in the patients group with tumor marker levels in referenceranges, 40% patients with diagnosed cancer history in remission. the ratios of positive tumor markers were 13% forca 19-9, 19% for ca 125, 7.9% for psa, 25%for ca 15-3, 8.4%for afp, and 9.56% for cea. in conclusion; unnecessary test requests increase laboratory work load and health expenses. laboratory and clinical staff collaboration is crucial to increase the appropriate use of tumor markers. dna methylation is an epigenetic modification that is involved in both normal biological and disease states. hypermethylation of promoter regions of tumor suppressor genes have a role in tumor development. therefore, the measurement of promoter methylation of genes can be used for diagnosis and prognosis purposes of cancer. to detect dna methylation alterations in a sample (biopsy, blood, saliva, etc.), sensitive detection systems and optimization of the methods are needed. as a part of a collaboration project between national metrology institute of korea (kriss) and national metrology institute of turkiye (tubitak ume). dna methylation status of apc and gstp1 genes were studied. dna methylation measurements were performed using stepone real-time pcr system and results were analyzed using hrm (high resolution melting) software. the parameters effecting the quantification of dna methylation were found as primers, annealing temperature, pcr cycle number, fluorescence dye and the commercial dna methylation standards used for quantification of dna methylation. since, the accurate measurement of dna methylation is very critical in early diagnosis of cancer and choosing the right therapy, optimization of the method is required. cancer is a disease that includes heterogenic and complex molecular changes. anti-carcinogenic effects of resveratrol, a natural polyphenol, have been proved in a variety of cancer cells. considering the effects of resveratrol, the influence of the signal transduction pathways in the presence or absence of p53 of colon cancer cells is gaining importance. our aim was to investigate the effects of resveratrol in the presence or absence of p53 on cell viability, apoptotic cell death ratio and fold changes of proliferative or anti-proliferative gene expressions, which may have important effects on colon cancer, in hct116 colon carcinoma cells. ic50 doses of resveratrol were determined by wst-1 assay. the apoptotic cell death ratios in treatments of resveratrol were determined by annexin-v-fitc/pi assay for flow cytomety . the changes of ccnd1, fra1, ppard, egfr, birc5, pcna, mcl1, stat3, fos, jun, p27, atf4, trail, puma, gadd45a, rb1, faslg, tnf, socs3 , stat1 gene expressions were evaluated by real time pcr. all data were statistically analyzed by student's t test. our research has revealed that resveratrol (60 lm) causes decrease in cell viability and increase in apoptotic cell death in hct116 p53(+/+) and hct116 p53(à/à) cells significantly (p < 0.05). the fold changes of the gene expressions have shown that resveratrol has significant (p < 0.05) and different effects on the expressions of the genes related with the existence of p53 in hct116 cell lines. therefore we proposed that resveratrol might show proliferative or apoptotic effects related with p53 mutation of colon cancer cells and we predicted that unconscious consumption of resveratrol in colon cancer patient might cause adverse effects. introduction: colorectal cancer (crc) is the third most common cancer worldwide. alterations in methylation profiles of tumor suppressor genes (tsgs) have been recognized as a key mechanism in colorectal cancers. in the current study, we investigated the hypermethylation status of 25 tsgs in colorectal cancer tissues. materials and methods: formalin-fixed paraffin-embedded (ffpe) tissue samples obtained from patients with crc. methylation specific-multiplex ligation dependent probe amplification (ms-mlpa) technique was used to assess the methylation status of 25 tsgs. the findings were evaluated in terms of age, mortality, survival, positive lymph node status, lymphovascular invasion, and perineural invasion. results: hypermethylation-detected patients and hypermethylation-undetected patients were called as group 1 and group 2, respectively. hypermethylation was detected in atm, cdkn2a, and gata5 genes. mortality rate was (12.5%) in group 1 and group 2 (p > 0.05). mean 5-years survival rate in group 1 was 45 ae 3 months and mean 5-years survival in group 2 was 44 ae 3 months (p > 0.05). positive median lymph node count was 10 ae 2 for group 1 and 5 ae 1 for group 2 and the difference was statistically significant (p < 0.05). frequencies of perineural invasion and lymphovascular invasion rate in two groups were 25% (p > 0.05). discussion and conclusion: our findings suggest that tsg hypermethylation found in crc patients may increase the lymph node metastasis. further investigations with larger sample size are required to support our results. boron (b) is known to be important for cell replication and development, but the underlying mechanism remains obscure. recently b has also become important in some specific anticancer processes. some recent reports advise using of some boron compounds for the treatment of specific forms of cancer. for instance, boron-based drugs (bortezomib) are now being developed for use as therapeutic agents with anticancer activities and several other boron-based compounds are in various phases of clinical trials. it has been shown that bortezomib disrupts the regulation of cell cycle and induces apoptosis in both hematologic and solid tumor malignancies except for colon carcinoma. colorectal cancer (crc) is the third most common cancer in men and the second in women, accounting for 10% of all tumour types worldwide. cytotoxic effects of boron compounds on crc cells and changing of its effects related with p53 mutation, which is mutated 50% of cancer cases, have not take part in literature yet. for this purpose; the aim of the study was designed to investigate the effects of borax pentahydrate and disodium pentaborate decahydrate compounds on cell viability, apoptotic cell death ratio and parp protein expressions in p53 (+/+) and p53 (à/à) hct116 colon carcinoma cells lines. the effects of the boron compounds on cell viability were assessed by xtt assay and apoptotic effects and parp protein expression of the compounds were evaluated by flow cytometry and western blot analysis respectively. our results showed that borax pentahydrate (6 mm) and disodium pentaborate decahydrate (12 mm) significantly causes nearly 50% reduction of cell viability at 48 h (p < 0.05). apoptotic cell death ratios and parp expressions revealed that both of the compounds might have a potential for a candidate of anticancer agent. epithelial-mesenchymal transition (emt) is a significant event for metastasis, and could be mediated by several pathways such as pi3k/akt, map kinases and many epigenetic regulators. satb2 is an epigenetic regulator involved in emt and osteoblastic differentiation. since preliminary results indicate that there is a crosstalk between p38 and akt pathways in nsclc cells, we aimed to determine whether this crosstalk has a regulatory effects on emt and satb2 expression in nsclc cells. we used a549 and h1650 cells as a model to evaluate the effects of the crosstalk between p38 and akt on emt of nsclc cells. therefore, cell culture, inhibition of p38 activation via sb203580, transient expression assay for (ca-akt), western blot analysis, sirna transfection for satb2, wound healing and invasion assay were performed in this study. firstly, the expression statues of e-cadherin, satb2, p-p38, p38, p-akt and akt was examined in a549 and h1650 cells by western blot analysis. we observed that e-cadherin and satb2 are downregulated in a549 cells (highly active p38, lowly active akt) compared to h1650 cells (lowly active p38, highly active akt), suggesting that e-cadherin and satb2 are associated with the crosstalk between p38 and akt pathways. our results demonstrated that p38 inhibition in a549 cells leads to decreased pten expression and subsequently increased akt activation. then, we found that p38 inhibition upregulated satb2 expression, and reversed emt in a549 cells. furthermore, alone satb2 knockdown is sufficient to induce emt, and prevented the effects of p38 inhibition on emt. all these results strongly indicate that the crosstalk between p38 and akt pathways might determine satb2 expression and epithelial characters of nsclc cells, and satb2 is a critical epigenetic regulator for emt in nsclc cells. therefore, it is also need to explore how p38 and akt signalling pathways could regulate satb2 expression. this work was supported by tubitak (114s007, 215z283). introduction: lung cancer is a disease characterized by uncontrolled cell growth in the lung tissues. the most common causes of lung cancer are tobacco smoke, radon gas, asbestos, air pollution, and genetic factors. nitric oxide (no) has potential mutagenic and carcinogenic activity and may play important roles in lung cancer. endothelial no, synthesized from l-arginine by endothelial no synthase (enos), inhibits apoptosis and promotes angiogenesis and tumor cell proliferation. the aim of the present study was to examine the possible relationship between enos gene intron 4 vntr and exon 7-g894t (glu298asp) the stressful ecosystems exert strong adaptive pressure and proteins that facilitate these adaptation processes are candidate drug targets. nucleotides are the core of biochemical pathway required for cancer cell growth and replication and genetic changes will lead in oscillation in their pools. although it is questionable whether the warburg effect actually causes cancer, impairing dglucose uptake and metabolism induces oxidative metabolism. lproline (lproline) homeostasis is critical in a constellation of human diseases, in parametabolic linkage between cancer, epigenetics (phang et al. 2013 ) and bioenergetics (pallotta 2013) where degradation and biosynthesis are robustly affected by oncogenes or suppressor genes that can modulate intermediates involved in epigenetic regulation. lproline-fueled mitochondrial metabolism involves the oxidative conversion to l-glutamate by a flavin dependent lproline dehydrogenase/oxidase and a nad +dependent l-d 1 -pyrroline-5-carboxylate dehydrogenase. in saccharomyces cerevisiae an important test tube, put1p and put2p respectively help cells to respond to changes in the nutritional microenvironment by initiating lproline breakdown after mitochondrial uptake (pallotta 2014) . in this preclinical study, low molecular weight compounds were tested for inhibiting lproline mitochondrial transport and put1p/put2p catalytic activities. thus, in seeking for natural bioactive compounds targeting lproline pathway and its substrate channeling (becker's group 2016), we report data using in silico screening and in vitro researches in saccharomyces cerevisiae with genetic background atcc18790 but different phenotypic landscape induced by nutritional stress/ ph changes. cells vitality, dψ measurements, nad(p) + /nad(p) h pool and flavine turnover were determined in spectrofluorimeter microplater reader and via hplc (pallotta et al. 1998 (pallotta et al. , 1999 (pallotta et al. , 2004 pallotta 2011; di martino pallotta 2011) thus in supporting of future cancer therapies with decreasing side effects. evaluation of lymphocyte to monocyte ratio (lmr) in patients with colorectal cancer introduction: inflammation may play an important role in cancer progression and a high neutrophil to lymphocyte ratio (nlr) has been reported to be a poor prognostic indicator in several malignancies. the aim of this retrospective study was to evaluate the prognostic value of nlr, lymphocyte to monocyte ratio (lmr) and platelet to lymphocyte ratio (plr) in patients with colorectal cancer (crc). : 107 patients who were diagnosed with colorectal cancer between january 2010 and january 2016; were evaluated retrospectively. the cutoff value was determined using receiver operating characteristics curve analysis. survival analysis was performed using the kaplan-meier method and log-rank test. the cox proportional hazard model was used to identify the influence of factors related to survival. (tnm stage, tumor differentiation, age, tumour size and lmr) results: receiver operating characteristic curves showed that lmr was superior to plr and nlr as a predictive factor in patients with colorectal cancer. the cutoff value for lmr was 1.82. cancer-specific survival was not significantly different between the high-and low-lmr groups (p = 0.786). age was identified as independent prognostic factor in colorectal cancer (hazard ratio: 3.50; 95% confidence interval: 3.14-351.82; p = 0.004). discussion and conclusion: our preliminary study showed that the lmr was not an independent prognostic factor in crc patients, but additional large sample sized prospective studies will be needed to confirm these findings. the aim of this study is to investigate the effects of luteolin treatment on enzymatic activity of arginase, and ornithine and polyamine levels (putrescine, spermidine spermine) in serum and cancer tissues of ehrlich ascites breast cancer model. balb/c female mice were divided randomly into following groups: healthy control, healthy treatment, cancer control, treatment 1 and treatment 2. 0.2 ml ehrlich ascites tumor cells was inoculated subcutaneously to medial part of left hind leg. healthy treatment and treatment 1 groups, and the treatment group 2 were given 5 mg/kg and 10 mg/kg dose of luteolin, intraperitoneally, for a 12 days period, respectively. luteolin has a hydroxylated flavonoid structure and shows potent antioxidant, anti-inflammatory, and anticarcinogenic properties. luteolin not only leads to cell death in various tumors by suppressing cell survival pathways and stimulating apoptotic pathways, but also sensitize them to cytotoxic therapy. supporting various previous studies, tumor implantation to healthy mice resulted in statistically significant elevation of serum arginase and polyamine levels (p < 0.05) indicating the tumor cells as the main source of this production. furthermore, luteolin treatment abolished this increase in serum arginase and polyamine levels (p < 0.05). tissue measurements of arginase and polyamine levels indicated that luteolin treatment resulted with an increase in these parameters of tumor tissue while the serum levels of them showed a significant decrease. our results revealed that increased tissue arginase and polyamine levels might be related with estrogenic agonistic effect of luteolin on utilized tumor model in this experiment; and decreased serum levels of these parameters while there is a significant increase of them in tissue levels might be a result of a suppression of polyamine efflux from the tumor tissue by inhibitory effect of luteolin on plasma membrane polyamine transporters. hepatocellular carcinoma (hcc) is the third most common cause of cancer-related deaths. around 30-40% of hcc patients are diagnosed at an early stage of the disease. hepatic resection, liver transplantation are common strategies in hcc treatment. even if, most of the patients present advanced-stage tumors and have a restricted survival rate. for the reason, resistance against existing tumor stress conditions have been demonstrated in hcc. hypoxia, hyperglycemia are general stress sources in hcc and result in aggressive cell phenotype, resistance to apoptosis and therapeutic drugs. thioredoxin interacting protein (txnip) regulates cellular responses under stress conditions. over-expression of txnip results activation of oxidative stress and apoptosis. in cancer models txnip is considered as a tumor suppressor gene. however, its role in the development, progression of hcc and mechanisms behind it warrant further investigation. in this study expression levels of txnip were examined in 12 hcc cell lines by rt-pcr and western blotting. txnip expression was significantly high in poorly-differentiated snu-182, snu-387 and snu-423 than the well-differentiated hcc cell lines such as huh-7, hepg2 and plc/prf/5. besides, expression of txnip was examined in 16 non-hcc and 79 hcc tissue samples by immunohistochemical staining. txnip positivity was observed in 40% of well and 80% of poor differantiated hcc tissues. however, no txnip positivity was observed in non-hcc tissues. to investigate whether txnip might be involved in biological responses such as cell proliferation, motility and invasion, we used overexpression and silencing strategies. overexpression of txnip minimally inhibited adhesion and proliferation, whereas boyden-chamber motility and invasion assay showed that invasiveness of cells were increased. our findings suggest that txnip expression is increased in hcc and txnip over-expression is important for invasive phenotype during hepatocarcinogenesis. cardiovascular diseases are the leading cause of morbidity and mortality in the western world. it was shown that ischemic tolerance of the heart can be enhanced not only by ischemic or pharmacological conditioning (pre-and postconditioning), but also by adaptation to chronic hypoxia. different studies have indicated that these cardioprotective phenomena may at least partly share the same signaling pathways. the jak/stat signaling pathway has been demonstrated to participate in the development of cardioprotection by conditiong apparently through the inhibition of gsk-3b. the aim of our present study was to determine whether this pathway also takes part in cardioprotection induced by adaptation to chronic hypoxia. we investigated the effect of inhibitor of jak2 kinase (ag-490) on myocardial infarct size and the jak2/stat3 signalling pathway and other effector molecules that may participate in cardioprotection conferred by adaptation to hypoxia. adult male rats were adapted to intermittent normobaric hypoxia (10% o 2 , 3 weeks, 8 h/day) and part of them recieved ag-490 (3 mg/kg) 15 min before ischemia. control rats were kept under normoxia. infarct size was assessed in isolated perfused hearts. relative expression of the key components of the jak2/stat3 signalling system and other proteins was detected using western blotting. preliminary data indicate that administration of the jak2 inhibitor ag-490 caused a significant increase in infarct size in hypoxic rats. western blot analysis revealed changes in phosphorylation of jak2, stat3 and some other proteins involved in cardioprotection (akt, erk1/2, gsk3b). these results suggest that the jak/stat signaling pathway could participate in the development of a cardioprotective phenotype in rats exposed to chronic hypoxia. however, further research will be needed to clarify in more detail the role of this signalling pathway in the cardioprotective mechanism. p-06.01.2-002 detrimental effect of hypertension on myocardium was reversed by liver x receptor agonist gw3965 hypertension is a cardiovascular disease that causes functional and structural changes in the heart. nuclear liver x receptors (lxrs) are involved in the control of cholesterol and lipid metabolism. however, effect of lxr activation on the hypertensive heart is not well characterized. in this study, the effects of lxr agonist gw3965 on hypertension-induced damage of myocardium were investigated. hypertension was induced by deoxycorticosterone acetate (doca) injection (20 mg/kg, twice a week) following the unilateral nephrectomy in male 8-week-old wistar albino rats for 6 weeks. blood pressure was measured by using tail-cuff method. gw3965 (10 mg/kg/day, i.p.) was administered last 1 week. expression of various markers (grp78, perk, p-perk, ikb-a, nf-kb p65, tnf-a, bax, bcl-2, mmp-2) in the ventricular tissue were examined by western blotting. inflammation and fibrosis were evaluated in histopathological examination. gw3965 treatment reduced systolic blood pressure of hypertensive animals. expressions of endoplasmic reticulum stress markers grp78 and p-perk were increased by hypertension and gw3965 treatment reversed them. hypertension-induced increase in nuclear nf-kb p65 expression and decrease in ikb-a expression were reversed by gw3965 treatment. while bcl-2 expression was lower, bax level was higher than control in the hypertensive animals. in hypertensive group, fibrosis marker mmp-2 expression was augmented and gw3965 treatment reversed this elevation. hypertension-induced increase in interstitial and perivascular collagen deposition and inflammatory cell infiltration in left ventricle were prevented by gw3965 treatment. these results suggest that lxr activation by gw3965 restored the hypertension-induced structural changes of heart in the doca-salt hypertension. methylphenidate (mph) is a psychostimulant prescribed for the treatment of attention deficit hyperactivity disorder (adhd), one of the most common neurobehavioral disorders of childhood and adolescence. in fact, despite the widespread use of mph the full comprehension of its cellular/molecular mechanisms is still elusive, including its effect on blood-brain barrier (bbb). this barrier is a key structure in the central nervous system since it protects the brain and its dysfunction has been described as a critical event in several brain diseases. thus, the aim of the present study was to clarify the effects of mph on the bbb function in both physiological and adhd conditions. for that, we used a rat model of adhd, the spontaneously hypertensive (shr) rats, and wistar kyoto (wky) animals as inbred comparator strain. also, to mimic a clinical dosing schedule for adhd treatment, rats were administered for monday-friday with vehicle or mph (1.5 mg/kg/day or 5 mg/ kg/day, per os) from p28-p55. chronic mph treatment (5 mg/kg/day) promoted cortical bbb permeability in both wky and shr animals; however, more prominent in wky rats. this effect can be explained by the downregulation of claudin-5 and collagen-iv, tight junction and basal lamina protein, respectively. noteworthy, wky animals also showed an increase in the expression of caveolin-1 and in both vascular cell and intercelular adhesion molecules. these bbb alterations led to subsequent infiltration of peripheral immune cells, including cd169 + macrophages. furthermore, hippocampal bbb disruption was only observed in wky rats with 5 mg/kg of mph. here, mph decreased collagen iv expression and upregulated caveolin-1, with no alterations in claudin-5. overall, our results show that chronic exposure to mph can led to brain vascular alterations particularly under physiological conditions. this highlights the importance of an appropriate mph dose regimen for adhd, and also that mph misuse can have a negative effect. regulators of g proteins signaling (rgs) serve several cellular functions varying from tolerance, dependence, neuroprotection, transcription and tumorgenesis. despite their initial role as gtpase activating proteins, evidence suggests that rgs proteins are localized in the nucleus, interact with transcription factors thus regulating transcriptional responses. it was shown that rgs4 directly interacts with and interferes in opioid receptor (or) signaling. rgs4 is mostly expressed in brain and is implicated with brain structural alterations; however, the molecular mechanisms of how rgs4 could be involved in cellular differential functions remains unclear. based on these observations we examined whether rgs4 can regulate transcriptional responses mediated by the stat5b transcription factor. isolated neural stem cells from rgs4 à/à mice were immunostained for the mitosis marker ph3 and the mrna levels of antiapoptotic genes were determined. proliferation assays were performed with brdu staining in neuro2a cells stably expressing rgs4. the functional assays of stat5b transcriptional activation were performed in hek293 expressing either the erythropoietin receptor (epo-r) or the delta opioid receptor (d-or). the present data demonstrate that rgs4 blocks stat5b phosphorylation and transcriptional activation by interfering in stat5b heterodimerization upon epo-r or d-or activation triggered by cytokines or opioids administration. lack of functional rgs4 results in increased mrna levels of stat5b target genes such as the members of the bcl anti-apoptotic family bcl-2, bcl-6 and bcl-xl. this upregulation of stat5b inducible gene transcription results in an increased proliferation rate of neural stem cells. this study demonstrates for the first time a non-canonical function of rgs4 in stat5b mediated transcriptional responses and a novel selective role of rgs4 in transcription. role of the pre-molten globule structure in amyloid fibril formation a. eshaghi department of biology, faculty of science, islamic azad university, mashhad branch, mashhad, iran the major factor that caused extensive research on the protein fibrillation is their crucial roles in important diseases known as the amyloidosis diseases. neurodegenerative diseases, including alzheimer's, parkinson's, diabetes and huntington are the most important types of this disease. understanding the mechanisms of fibril formation and ways of treatment can be useful in reducing this type of disorder. in this project, the fibrillation of carbonic anhydrase protein was investigated as a model. carbonic anhydrase creates two stable intermediated known as pre-molten and molten globule, in different ph solution. this protein at ph between ph 3-4 molten globule structures was formed while the pre-molten form took place under ph 3. in our tests at ph 3.5 when the protein in molten globule structures only the amorphous aggregates were formed. instead, at ph 2.4 in pre-molten globule structure amyloid fibrils formed in the protein. there some reports, indicated the protein from pre-molten globule structure go toward amyloid assembly. even intrinsically unstructured proteins such as alpha-synuclein first took a structure similar to pre-molten globule and then made amyloid fibrils. it seems pre-molten globule structure have the major role in promoting to amyloid fibrils. perhaps drugs that prevent the formation of premolten globule structure have an important role in inhibiting amyloid fibrils. identification of compounds preventing the biochemical changes that underlie the epileptogenesis process and understanding the mechanism of their action is of great importance. we have previously shown that myo-inositol (mi) daily treatment for 28 days prevents certain biochemical changes that are triggered by kainic acid (ka)-induced status epilepticus (se), [1, 2] . however in these studies we have not detected any effects of mi on the first day after se. in the presented study we broadened our research and focused on ka induced other early molecular and morphological changes and influence of mi treatment on these changes. the increase in the amount of voltage-dependent anionic channel-1 (vdac-1), mitochondrial-plasma membrane cofilin and caspase-3 activity was observed in the hippocampus of ka treated rats. administration of mi 4 h later after ka treatment abolishes these changes, whereas under the same time schedule diazepam treatment has no significant influence. the number of neuronal cells in ca1 and ca3 subfields of hippocampus is decreased after ka induced se and mi post-treatment significantly lessens this reduction. no significant changes are observed in the neocortex. obtained results indicate that mi post-treatment after ka induced se could successfully target the biochemical processes involved in apoptosis, reduces cell loss and can be successfully used in the future for translational research. references 1. r. 2010. neuroscience letters, vol. 468, no. 3, pp. 277-281. 2. r. solomonia, et al; 2013. cell. mol. neurobiol. vol. 33, no extracellular deposits of amyloid-b peptide (ab) in brain parenchyma via proteolytic processing of amyloid precursor protein (app) are one of the typical characteristics of alzheimer's disease (ad). these aggregates mainly occur as a result of an increase in ab production or a decrease in its degradation. it was found that the neurotoxicity of ab aggregates is accelerated by acetylcholinesterase (ache). besides, ab-ache complex has a prominent neurodegenerative effect in brain. thus, cholinesterase inhibition and preventing ab production are current treatment strategies for ad. recent studies have shown that methylene blue (metb), a cholinesterase inhibitor with phenothiazine structure, inhibits the formation of amyloid plaques and neurofibrillary tangles. azure b, the major metabolite of metb, has been shown to inhibit ache and bche with ic50 values of 0.486 lm and 1.99 lm, respectively. in the present study, we tested whether azure b, may effectively lower the levels of ab 40/42 . we treated chinese hamster ovary cells, which stably express human wild type app and presenilin-1 (ps70) with 0-15 mm azure b or vehicle for 24 h. to determine the effect of azure b treatment on ab 40/42 levels, we used separate sandwich-based elisas and normalized to total protein levels, determined by bca protein assay. azure b treated ps70 cells were also assessed by propidium iodide in flow cytometry for cellular toxicity. we observed a significant decrease in both extracellular ab 40 and ab 42 levels with a dose range treatment of azure b in ps70 cells. ab 40 levels were reduced by 89.2% in 10 lm and 94.1% in 15 lm azure b-treated cells when compared to control. additionally, ab 42 levels were decreased by 83% in 10 lm and 93.5% in 15 lm azure b-treated cells when compared to control. overall, these preliminary results suggest that azure b may have beneficial effects for the treatment of ad. the effect of green silver nanoparticles (agnps) on the amyloid formation in alphalactalbumin and chaperone action of alphacasein a. ghahghaei, m. dehvari, j. valizadeh formation and deposition of protein fibrillar aggregates in the tissues is associated with several neurodegenerative diseases such as alzheimer's and parkinson's disorders. molecular chaperones are a family of proteins that are believed to have ability for inhibiting protein aggregation. in the present study the effect of different concentrations of green synthesis silver nanoparticles (agnps) from pulicaria undulate l. on the amyloid formation of a-lactalbumin (a-la) and chaperone action of a-casein have been investigated. the effects of the agnps were determined using light scattering absorption, tht binding assay, intrinsic fluorescence assay, ans binding assay, cd spectroscopy and sds-page. light scattering and tht assay results showed that agnps have the ability in preventing aggregation of a-la in a concentration-dependent manner. consistent with these results, sds-page results represented that by increasing the concentration of agnps the adsorption and interaction between agnps and protein have increased. light scattering and tht assay results, also, revealed that the amyloid fibrilation decreased in the presence of both agnps and a s -casein compared to presence of a s -casein alone. fluorescence results, however, show that agnps have no effect on the chaperone ability of a-casein and in fact they perform their protection of protein aggregation action independently. consistent with the above experiments, cd spectroscopy also revealed that agnps have decreased structural changes in reduced a-la in absence and presence of a-casein, both the tertiary and the secondary structure of the proteins. our finding represented that agnps have preventing effects on protein aggregation and have no effect on the chaperone ability of a s -casein. in the main, results of this study show that biosynthesized agnps mediated by >pulicaria undulate l. maybe could be affective as a therapeutic agent for inhibiting aggregation in treatment of amyloidosis disorders. pink1 is a mitochondrial kinase with multiple cellular functions. while loss-of-function mutations of pink1 gene lead to early onset parkinson's disease, its over-expresion is associated with cancer development. parkinson is a multifactorial neurogenerative disease, with a complex aethiology including various cellular stressors. it is now known that genotoxic stress also triggers the release of soluble factors able to induce changes in neighboring cells enhancing the initial lesions, process known as bystander phenomena. althrough the mechanisms are still unclear, recent studies point towards a role for mitochondria in this process. our work investigates pink1 role in intracellular and intercellular stress response, comparatively in various models: fibroblasts (mefs) and neuroblastoma (sh-sy5y) used as a tumoral model or differentiated to a neuronal phenotype. pink1 role in this process was analyzed using genetically engineered pink1 deficient cells exposed to a genotoxic agent, bleomycin. the modified cell lines showed a reduced level of basal atp production. pink1 proved to be involved in cellular vulnerability to stress. despite differences in cellular sensibility between our models, genotoxic treatment of pink1 deficient cells induced consistently higher lesions compared to corresponding wild type variant. pink1 deficient cells showed altered intercellular signaling of stress, impairing bystander phenomena induction, by suppression of signal formation in treated cells, but also by altering the capacity to respond to the signals in neighboring cells. our hypothesis is that pink1 contributes to the management of cellular stress being involved in bystander transmission of detrimental effects through intercellular communication. this is determined mainly by its role in maintaining mitochondrial homeostasy and atp levels, pink1 deficient cells lacking the amount of energy required for rapid dna repair and stress signaling transmission. p-09.02.2-008 intranasal administration of synthetic fragments from receptor for advanced glycation end products prevents memory loss in olfactory bulbectomized mice the receptor for advanced glycation end products (rage) is a member of the immunoglobulin protein superfamily. activation of rage causes brain inflammation, oxidative stress and secretion of beta-amyloid that has been recognized as an essential phase in the development of alzheimer's disease. it is known that the receptor soluble isoform (srage) which lacks the transmembrane and cytosolic domains binds to ligands and prevents negative effects of the receptor activation in in vivo and in vitro experiments. we proposed that potential ligand-binding peptide fragments from srage would demonstrate the same biological activity. we have selected and synthesized 10 peptide fragments from unstructured surface-exposed regions of rage. synthetic peptides were intranasally administrated into olfactory bulbectomized (obe) mice with neurodegeneration of alzheimer's type. we have found that only administration of rage fragment (60-76) effectively prevents the obe murine memory from impairment, leads to decrease of beta-amyloid level and blocks the development of neuronal pathology in the brain of experimental mice. six overlapping fragments of rage (60-76) peptide were synthesized in order to find a site, responding for the therapeutic effect. tests in obe mice carried out with these fragments showed that only the n-terminal part of the molecule is responsible for preventing obe mice memory from impairment. all fragments which do not include n-terminal 60-61 dipeptide have been fully inactive in these experiments. we have proposed that active peptides can interact with beta-amyloid or s100b protein preventing these ligands from binding with rage. this interaction can inhibit the development of neurodegeneration. the aim of this study was to examine effects of social isolation, enriched environment and exercise on learning in rats. the study included 36 female 25 day old wistar rats. the rats were randomly divided into four different groups; control, exercise, social isolation and the enriched environment groups. the social isolation group and the enriched environment group were housed under their specific conditions and the exercise group and the control group were housed in standard conditions during 6 weeks. the rats in the exercise group swam for 6 weeks. after 6 weeks, the rats were evaluated in the morris water maze. brain and blood samples were taken and the hippocampus tissue was dissected. bdnf and ngf levels were measured in these samples. in conlusion, while enriched environment was a positive effect on spatial learning, social isolation was a negative effect on spatial learning and increase thigmotactic behaviors. according to the analysis results ngf and bdnf levels in the hippocampus and plasma did not change with environmental conditions and exercise. time of exposure to social isolation, procedures of the enriched environment, time of exposure to the environment, type and duration of exercise and gender may affect the results. alzheimer's disease (ad) was characterized by dementia that typically begins with subtle recognition failure and poor memory. it slowly becomes more severe and, eventually, incapacitating. the cholinergic system seemed particularly susceptible to synapse loss, especially in cortical regions associated with memory and executive function (1) . recent studies showed that the main cause of the loss of cognitive functions in ad patients was a continuous decline of the cholinergic neurotransmission in cortical and other regions of the human brain (2) . acetylcholinesterase (ache) and butyrylcholinesterase (bche) are hydrolytic enzymes that act on acetylcholine (ach) to terminate its actions in the synaptic cleft by cleaving the neurotransmitter to choline and acetate. both enzymes are present in the brain and detected in neurofibrillary tangles and neuritic plaques. it was suggested that ache predominates in the healthy brain, with bche considered to play aminor role in regulating brain ach levels. however, bche activity progressively increases in patients with ad, while ache activity remains unchanged or declines. both enzymes therefore represent legitimate therapeutic targets for ameliorating the cholinergic deficit considered to be responsible for the declines in cognitive, behavioral, and global functioning characteristics of ad (3). we initiated a study to screen their acetylcholinesterase (ache, ec 3.1.1.7) inhibitory activities, which are the key enzymes taking place in pathogenesis of ad. newly synthesized chiral benzimidazole derivatives with thioure structure showed ic 50 values in the range of 11.55-36.00 nm for ache. this study was financed by turkish research council-tubi-tak (kbag 115z837). p-09.02.2-012 f3-a13 h, novel fingolimod derivative, activates camp-dependent signalling pathway in sk-n-sh cell line g. celik turgut 1 , a. sen 1 , d. doyduk 2 , y. yildirir 2 1 department of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, 2 department of chemistry, faculty of sciences, gazi university, ankara, turkey fty720, a sphingosine 1-phosphate (s1p) receptor modulator, is the first oral disease-modifying therapy to be approved for the treatment of relapsing-remitting multiple sclerosis. in this study, we have synthesized and characterized novel derivative of fty720, namely f3-a13 h, and have determined its underlying camp regulation in sk-n-sh cell lines. for this purpose, we first determined the regulation of the camp response element (cre) activity and camp concentration by f3-a13 h along with fty720 using pgl4.29 luciferase reporter assay and camp immunoassay, respectively. then, we have determined their effect on camp/pka-related gene expression profiles using custom arrays along with fty720 treatment at non-toxic doses. it was found that f3-a13 h significantly activate cre and increase camp concentration in the sk-n-sh cell line, indicating that it activates camp pathway through cell surface receptors as fty720 does. furthermore, f3-a13 h modulates the expression of the pathway related genes that are important in camp signaling pathway. in summary, our data demonstrate that the novel fty720 derivative act as a modulator of camp ultimately by influencing the gene expression via the camp and downstream transcription factor cre pathway. in conclusion, f3-a13 h might contribute future therapies for multiple sclerosis. alzheimer disease (ad) results in memory impairment and accompanied by neuroinflammation, cholinergic deficit and amyloid-beta (ab 1-42 ) accumulation in brain. we found that bacterial lipopolysaccharide (lps) injections or mice immunizations with extracellular a7 nicotinic acetylcholine receptor (a7 1-208 nachr) domain resulted in astrogliosis, decrease of a7 nachr density, accumulation of ab 1-42 in brain and episodic memory impairment. the aim was to reveal main event triggering ad-like symptoms development. c57bl/6 mice were injected with lps, immunized with recombinant a7 1-208 or a7 1-208 endoglycosidase treated to remove carbohydrates. two immunizations with 3 week interval were performed. control mice obtained complete freund's adjuvant injections. mice were tested for memory performance, blood sera were examined for presence and fine specificity of a7 1-208 -specific antibodies and brain preparations were studied for a7 nachr, ab 1-42 and il-6 levels. the original a7 1-208 ('glyc') was more immunogenic than 'deglyc', and their epitopes were recognized with different efficiency. in contrast to lps and 'glyc' a7 1-208 immunization with 'deglyc' a7 1-208 did not stimulate il-6 elevation in brain and had no proinflammatory effect. immunizations with 'glyc' or 'deglyc' a7 resulted in similar a7 nachrs decrease and ab 1-42 accumulation in brain and significant episodic memory decline comparable to those after lps injections. a7 nachr interacts directly with amyloid-beta precursor protein and facilitates its proper processing and metabolism. our data indicate that decrease of a7 nachr density caused by a7 1-208 -specific antibody is critical for ab 1-42 accumulation and episodic memory impairment while pro-inflammatory capacity of a7 1-208 -specific antibody plays secondary role in ad-like symptoms development. in vitro antioxidant and antiacetylcholinesterase activity of achillea millefolium alzheimer diseases (ad) is a neurodegenerative condition without a current effective treatment. increase in reactive oxygen species and lipid peroxidation or decrease in total antioxidant capacity causes oxidative stress-induced tissue damage. it has been suggested that decrease in oxidative stress and inhibition of acetylcholinesterase (ache) activity play a major role in the prevention and slowing of cognitive symptoms of ad. recently, studies have been directed for the discovery of medicinal plants and natural substances that are known to have natural antioxidants. achillea millefolium (a. millefolium) is a traditional herbal medicine that contains natural compounds with antioxidant activity and has been used as a carminative, diuretic, menstrual regulator and wound healer, however the mechanism of its actions are unclear. the aim of our study was to investigate the effects of a. millefolium extracts on free radical production, acetylcholinesterase (ache) activity and lipid peroxidation in vitro. methanol (me) and ethanol (ee) extracts of a. millefolium were prepared to determine (a) in vitro antioxidant capacity (by using 2,2-diphenyl-1-picrylhydrazyl assay, radical scavenging activity, phosphomolibdenum-reducing antioxidant power, ferricreducing antioxidant power, and total phenolic-total flavonoid contents), (b) effects on ache kinetics (by using a colorimetric assay) and (c) effects on sodium nitroprusside-induced lipid peroxidation in mice brain homogenates. me showed a higher antioxidant activity compared with ee in the biochemical assays tested. similarly, me demonstrated significant inhibition of ache activity that was potent than ee. both extracts dose-dependently decreased malondialdehyde content in mice brain homogenates suggesting a strong inhibition of lipid peroxidation. these results showed that a. millefolium has a high antioxidant capacity and antiache activity, indicating a potential use as an adjuvant therapy in ad. b-cells are known to play a key role in multiple sclerosis (ms) progression and autoimmune response. cxcr5 is the main b-cell chemokine receptor that under normal conditions directs their migration to specific areas of secondary lymphoid organs. in ms, areas of demyelinating lesions have been reported to attract bcells due to overexpression of cxcl13, the cxcr5 ligand. we aimed to determine whether snp rs630923 located in the promoter of cxcr5 gene and associated with high risk of multiple sclerosis could have a direct effect on of cxcr5 promoter activity. mef2c binding to dna was assessed using pull-down assay. b-cell stimulation was performed using lps, pma and ionomycin. activities of variants of cxcr5 promoters containing different rs630923 alleles were estimated using luciferase reporter assay. we determined that minor rs630923 allele creates functional mef2c-binding site within one of the regions required for the basal activity of the cxcr5 promoter. cxcr5 promoter containing minor rs630923 variant that is statistically associated with low risk of ms showed significantly decreased activity in stimulated human b-lymphoblastoid cell lines. mef2c has been reported to play an essential role in b-cell survival and b-cell responses. we determined mef2c as the main regulator of rs630923-dependent modulation of cxcr5 promoter activity in b-lymphoblastoid cell lines. this link may be directly related to pathogenic b-cell activities in multiple sclerosis. introduction: parkinson's disease (pd) is the second most common neurodegenerative progressive brain disease with increasing prevalence in aging population. the etiopathogenesis involves many cellular procesess, but is not fully elucidated yet. treatment of pd is based on levodopa and dopamine agonists, but mao-b inhibitors, comt inhibitors, amantadine or anticholinergics may be used as initial monotherapy or as adjuvant therapy. treatment related adverse drug reactions (adrs) are frequent, but cannot be predicted and/or prevented. non-motor adrs, such as nausea, somnolence, hallucinations and hypotension are frequent in dopamine agonist therapy, while dyskinesias along with motor fluctuations are the most common late adrs with levodopa. the aim of our study is to combine clinical data with genetic and epigenetic biomarkers in the algorithm for personalized approach to pd management. materials and methods: we are planning a clinical study to assess the combined impact of selected clinical, genetic and epigenetic factors on the progression of pd, adrs and treatment response. our study will have a retrospective and prospective arm. we will collect peripheral blood samples of pd patients and clinical data. single nucleotide polymorphisms (snps) in the genes involved in dopamine, neurotransmitter and drug metabolism and transport, receptors and signalling pathways will be genotyped. snps within inflammatory, neurodevelopmental, antioxidative defense, synaptic transmission and immune response pathways will also be analysed. in the prospective arm we will isolate the exosomes and check their mirna content at the time of diagnosis and after the treatment initiation. the combined effects of clinical, genetic and epigenetic factors will be analyzed using lasso penalized regression analysis. conclusions: we hope to identify genetic and/or epigenetic biomarkers that may predict progression of pd, adrs and treatment response and may support personalized tratment of pd. most evidence indicates that g protein-coupled receptors form heteromers between them and with other receptors. by allosteric mechanisms, them acquire a multiplicity of unique pharmacological and functional properties. recently, we discovered that dopamine d1 receptors (d1r) and histamine h3 receptors (h3r) form heteromers through which h3r ligands can inhibit d1r function. d1rs also physically interact and modulate ionotropic glutamate nmda receptors (nmdar). in the present work, we investigated if nmdar, d1r and h3r form a heterotrimeric complex in brain. the heteromer expression was studied in slices from both rat and mouse brain cortex by co-immunoprecipitation (co-ip) and proximity ligation assays (pla). the ability of d1r and h3r to interact with nmdar in transfected hek cells was analyzed by bioluminescence resonance energy transfer (bret) with bimolecular fluorescence complementation (bifc) experiments. heteromer properties were studied by analyzing erk1/2 phosphorylation and cell death in cortical slices. endogenous d1r-h3r heteromers were detected in rat and mouse cortical slices, where h3r ligands decreased d1r signaling (erk1/2 pathway) and were also able to block the cell death induced by overstimulation of either d1r or nmdar. by bret experiments in transfected hek cells, we demonstrated that both d1r and h3r form heteromers with nmdar subunit 1a in the presence of subunit 2b. d1r-h3r-nmdar heteromers were detected by bret with bifc. endogenous d1r-h3r-nmdar heteromers were observed in rat and mouse cortex by pla. many systems, including the glutamatergic and dopaminergic, are involved in neurodegeneration. our innovative finding is that d1r, h3r and nmdar form heteromers that may be a point of intervention for cognitive disorders in neurodegeneration. d1r-h3r-nmdar heteromers are expressed in brain cortex and a complex interaction exists between protomers in the heteromer, where h3r ligands act as a 'molecular brake' for d1r and nmdar signaling. studies conducted on obesity and hfd (high fat diet) revealed hypothalamus have crucial roles on development of metabolic diseases. after chronic over nutrition or high fat diet, as a neurodegenerative condition, premise inflammation, neural stress and development of functional impairments are observed. these studies generally focused on changes in neurons, but it's effects on brain vessels are still unknown. in this study, as a neuronal damage infrastructure, changes in hypothalamic vascularity investigated. experiment initiated with 5 weeks old total 40 male wistar rats. in order to acquire obese phenotype, the rats were fed either cafeteria diet as hfd, or normal/chow (standard diet, sd) for 9 months. intravenous glucose tolerance tests performed before sacrification. animals were exposed for 10 s to co2 and then decapitation was performed with guillotine. isolated brains were directly immersed into liquid nitrogen and stored at à80°c. the hypothalamic sections were acquired with the cryostat instrument at different. immunofluorescence was performed on serial sections through the hypothalamus using the antibody smi-71 and cd31. changes in tight junction (tj) proteins (occluding and zone occludin-1) are evaluated via western blot (wb) analysis. the hfd-treated consumed significantly more food than did control animals, when examining average food consumption per day and rats that received the hfd diet weighted significantly more at the end of 9 month diet treatment. there were no differences acquired for glucose tolerance tests. however, after hfd treatment, wb analysis have shown that tj proteins decreased even if hypothalamic micro vessel number increased and smi-71 staining have shown that increased. our primary results have shown that hfd diet can affect hypothalamic vascularity and such changes might initiate neurodegenerations and functional impairments as observed in neuroretinal degeneration in relation to vasculopathy in diabetic patients. defects of mitochondrial trafficking are common problem in many neurodegenerative diseases. its dysregulation can contribute to changes in bioenergetics profile of the cell and can lead to cell death. in our study we investigate distribution of mitochondria and their transport in primary fibroblasts derived from patients with sporadic form of alzheimer's (ad) and parkinson's (pd) diseases. our data revealed that in the most cases the velocity of mitochondrial movement is lower in ad and pd cells in comparison to the control. the most intense differences between ad, pd patients and control group are observed in the case of movement of large mitochondria. owing to the fact that mitochondrial trafficking depends on mitochondrial state, we investigated the 'age' of mitochondria. we observed a diminished mitochondrial turnover in ad and pd fibroblast. evaluation of the mitochondrial distribution within the cell in all 3 groups (ad, pd and control) showed that in the perinuclear area are accumulated 'old' and 'worn out' mitochondria, probably dedicated to remove from the cell. because mitochondrial biogenesis, shape and size depends on fusion/fission proteins we assessed their level within the cell. to summarise, our results revealed alterations in mitochondrial trafficking in fibroblasts derived from patients with alzheimer's and parkinson's diseases in comparison to the healthy control cells. carbonic anhydrases (cas, ec 4.2.1.1) is a zinc metalloenzyme that catalyzes the reversible reactions of co 2 and water. carbonic anhydrases (cas, ec 4.2.1.1) form a family of metalloenzymes that play an important function in various physiological and pathological processes. therefore, many researchers work in this field in order to design and synthesize new drugs. carbonic anhydrase activators are important as much as inhibitors. caas have polar groups to make hydrogen bond in the main body and the activation property of enzyme increaase in this way. caas are have polar groups to make hydrogen bond in the main body and the activation property increaase in this way. furthermore, recent studies suggest that ca activation may provide a novel therapy for alzheimer's disease. in this study ca activators are determined. human carbonic anhydrase isozymes ca i and ca ii are isolated from human blood erythrocyte. hca-i and hca-ii isoenzymes were purified using sepharose-4b-l tyrosine-sulfanilamide affinity colum. finally, hca-i and hca-ii isoenzymes were eluted with appropriate elution buffers. enzyme purity was checked by sds-page. the enzyme activity system contained 0.05 m tris-so 4 ph 7.4, r-nitro phenol in 1 ml total volume. effects of some macrocyclic thiacrown ethers derivates were investigated. enzyme activities were measured at constant substrate and different activator concentrations to find ac 50 value. these compounds are thought to be useful for treating alzheimer's disease. introduction: gender differences in stress models are not studied in detail. we compared different stress conditions on brain bdnf levels, in social isolation (sit) and predator scent tests (pst) in rats. bdnf levels in cortex, hippocampus and amygdala were compared, effects of chronic fluoxetine (flu) treatment were evaluated. methods: 128 rats were used. for sit, animals were kept individually for 1 month and for pst, rats were exposed to dirty cat litter for 10 min at the first day of 1 month stress. flu was given (5 mg/kg, ip) through stress. controls, stress and treated groups were evaluated in elevated plus maze (epm), anxiety scores were calculated. brain bdnf levels were determined in cortex, hippocampus and amygdala by western blot. p < 0.05 were considered significant. results: sit and pst induced anxiety in both male and female rats, females having greater anxiety scores than males (p < 0.05). flu restored anxiety scores in both sexes (p < 0.01) in two settings. male and female rats exhibited reduced cortical bdnf levels in sit (p < 0.001). pst reduced cortical bdnf in females, but increased in males. hippocampal bdnf expression was lowered in sit (p < 0.01) and pst (p < 0.001) in both sexes. female rats had 40% lower bdnf expression than males in amygdala in sit. flu did not restore cortical bdnf in females in both tests, but reduced incresed bdnf levels in males (p < 0.001). flu did not restore reduced brain bdnf in males in hippocampus and amygdala, but restored in hippocampus, in females. discussion: our findings indicate that sex differences must be considered in studies related to mood disorders of animal models, and suggest that bdnf expression in different brain regions are altered differentially in a gender-dependent manner in rats. antianxiety effect of flu is not mediated through increasing bdnf activity in cortex in both genders. increased bdnf in hippocampus and amygdala may reflect its antidepressant effect in female rats, but not in males. perineuronal nets (pnns) are special forms of neural extracellular matrix found around neuron bodies and neurites. hyaluronan and proteoglycan link protein1 (hapln1) is one of the major elements of pnns. hapln1 interacts with tenascins and aggrecan which are other essential pnns components. in most of neurodegenerative disorders caused by neuritogenesis defects, disrupted pnns structure and decreased expression of hapln1 were observed. however, the role of hapln1 in neural differentiation is unknown. the aim of this study is to determine mrna and protein levels of hapln1 during differentiation using pc12 cell line as a neural differentation model, derived from rat pheochromocytoma. after pc12 cells were stimulated to differentiate into neurons by nerve growth factor on days 3, 5 and 7; cells were collected, qrt-pcr and western blot were performed. also, in order to find out whether there is a physical interaction between hapln1 and proteins related to neuritogenesis defects, spinal muscular atrophy (sma) was used as a neurodegenerative disease model. therefore, a detailed hapln1 and survival motor protein 1 (smn1) network analysis were performed in-silico. as a result, we analyzed 3 fold increase in hapln1 mrna level compared to undifferentiated state. on the other hand, a decrease in protein level was detected. this decrease in cellular hapln1 level suggests that, hapln1 is required for formation of pnns structure, thus secreted to extracellular environment at early stage of differentiation. in addition, according to in-silico analysis, an indirect path between hapln1 and smn1 through fibulin2 (fbln2) was detected. fbln2 was also found to be an interaction partner between different matrix molecules such as aggrecan and hapln1 which form a macromolecular meshwork. the results of this study will pave the way for investigating the role of hapln1 and fbln2 in neurodegenerative disease models. also it will help us to understand the mechanism of neuritogenesis defects. determination of properties of bone marrow and tissue-derived mesenchymal stromal/stem cell population in neurofibromatosis type 1 patients neurofibromas, complex tumors deriving from schwann cells and containing fibroblasts, vascular structures and mast cells, are part of the clinical picture in nf1. the risk of malignancy is increased in nf1, wound healing is delayed and keloid formation is frequent. because multiple tissues are involved in malignant and non-malignant manifestations of nf1, we considered the mesenchymal stem/stromal cells (msc) carrying the nf1 mutation might play a role in the microenvironment. mscs affect the biological behaviour of other cells: they alter their proliferation, apoptosis and migration through various secreted growth factors, cytokines, chemokines, or by direct contact. we examined the msc of nf1 patients. methods: the adipogenic and osteogenic differentiation potential of mscs from nf1 and healthy subjects was examined in vitro and by rt-pcr. msc's migration potential was measured in the scracth assay. mscs' interaction with schwann cells and their effect on tumorigenesis was examined in co-culture by apoptosis markers on flow cytometry. results: nf1-mscs' adipogenic and osteogenic differentiation potential was lower than healthy controls as assessed by staining aizerin red s and oil red o and rt-pcr for osteopontin and collagen1. mscs cultured from dermal neurofibroma showed faster closing of the scratch compared to the same patient's normal and caf e au lait skin. on the other hand, mscs obtained from plexiform neurofibroma healed late, while mscs derived from the same patient's caf e au lait skin showed the fastest healing. hepatic encephalopathy with ammonium ions accumulation is accompanied by some disorder in the brain due to toxic material concentration being usually detoxified in the liver. one of the reasons for hyperammoniemia could be some imbalance in brain glutamine metabolisation induced by the key enzymes glutamine transferases (gts), which catalyze the reaction of glutamine transamination resulting in neurotoxic product of a-ketoglutaramate (akgm). akgm is hydrolyzed to a-ketoglutarate and ammonia by x-amidases. in the study, the dynamics of the enzymes activity in the tissues and biological liquids of experimental animals with hepatic dysfunction induced by thioacetamide (taa) was under investigation. white laboratory rats of wistar line (female, weight of 140 g) chronically intoxicated with hepatotrophic toxine of taa. every 2 weeks, some biological samples were collected to assess gt-k and x-amidase activities. x-amidase activity was the highest in the kidney tissue in the control and decreased by 70% in the experimental group. in the experimental hepatic x-amidase activity decreased by 240% compared to those in the control. the average x-amidase activity in the blood serum (0.015 nmols/ mg/min) and in the brain (0.005 nmols/mg/min) differed faintly. maximal gt-k activities were revealed in the kidneys; in the controls, it was about 250% higher than those in the experimental animals. the difference between average enzyme activities in the liver of the control and experimental animals reached 350%. the average gt-k activities in the blood serum and brain of the control and experimental animals were rather similar. the decrease in x-amidase and gt-k activities obtained in the study during hepatic pathology development could testify to imbalance of glutamine metabolism, possibly aimed at declining the level of akgm neurotoxicant under the hepatic dysfunction. acknowledgments: supported by the russian federation ministry of science and education (grant no rfme-fi60414x0116). wilson disease is an autosomal recessive disorder of copper metabolism characterized as neurodegeneration and liver abnormalities. it is caused by defects in the atp7b gene. atp7b is responsible for the sequestration of cu into secretory vesicles, and this function is exhibited by the orthologous ccc2p in the yeast. we aimed to characterize clinically-relevant novel mutations of p.t788i, p.v1036i and p.r1038g-fsx8 in yeast lacking the ccc2 gene. the patients with these mutations have copper storage abnormalities in different parts of their bodies; p.t788i mutation mainly affects the liver and the nervous system, p.v1036i mutation affects the nervous system, and p.r1038g-fsx83 mutation causes damages to the liver. to better understand the effects of these mutations on normal functions of atp7b, we cloned human atp7b gene onto a yeast expression vector and created the same mutations by site directed mutagenesis. then, wild type and mutated forms of atp7b genes were transformed into yeast cells lacking the homologous ccc2 gene for functional comparison. first, we analyzed the expression of atp7b and its variants in yeast cells by a real time pcr approach and western blot to make sure that transformed cells express the plasmids. expression of human wild type atp7b gene in ccc2d mutant yeast restored the growth deficiency and copper transport activity, however, expression of the mutant forms did not restore the copper transport functions and only partially supported the cell growth. our data support that p.t788i, p.v1036i and p.r1038g-fsx83 mutations cause functional deficiency in atp7b functions and suggest that these residues are important for normal atp7b function. in recent years, attempts were made to develop miniaturized potentiometric biosensors which is particularly important to reduce the amount of enzyme and reagents needed. the miniaturization of a biosensor is possible by using an all solid-state polymer membrane ion selective electrode which is cheap, easy to prepare and allow micro-sized construction. the use of all solidstate polymer membrane ion selective electrode as the basic sensing element also has the advantage of providing biosensors that are easy to fabricate, exhibit rapid response and have long life-times. they are also mechanically stable and allow flow-through configuration. genetical and chemical modifications for the alteration of enzyme molecule characteristics are gained considerable importance. enzymes can be modified chemically by using water-soluble polymers or some chemicals. conjugates of natural and synthetic macromolecules with enzymes provides wide usage in medicine and in many fields of biotechnology. in this study, enzyme-polymer conjugates with different molar ratios were synthesized using urease enzyme. in this study micro sized potentiometric urea sensitive biosensor has been developed in which urease-polymer conjugates were immobilized on polymeric membrane ammonium ion selective electrodes whether pvc or derivatized-pvc via glutaraldehyde cross linking reaction. biosensor is not include inner reference electrode and inner reference solution. potentiometric performance of biosensor will be examined with a computer-controlled measurement system designated. the most important features of the obtained micro sized urea biosensor by using enzyme-polymer conjugations were being highly sensitive, having long life-time, easily built at a low cost, and having short response time when compared with conventional potentiometric urea biosensor. also, these biosensors were easily built at micro-construction. this study was supported by grant from the tub _ itak research fund (project number: 114z138). creative drama technique as a new tool to increase enthusiasm and to achieve learning objective for medical students e. y. sozmen 1,2 , e. erem 3 1 faculty of medicine, ege university, izmir, turkey, 2 center for continuing education, ege university, izmir, turkey, 3 recently drama in education techniques have been implemented successfully in education program of primary and secondary high school and positive effects of these techniques on learning ability and attitude of students have been shown. the aim of this study was to organize an education program based on drama in education techniques in a special module of ege university medical faculty and to test any effect of this technique on achievement of learning objectives and student's perspectives on drama. the special module program was on the oxidative stress and antioxidants. the program covered the drama in education sessions (improvisation, role play, game) linked with learning objectives (understanding of free radical generation and free radical reactions in body, evaluation of the effect of free radical reactions in diseases as well as increase the ability usage of scientific information), laboratory work (antioxidant activity determination) and searching a special scientific topic on literature. 12 students (in 3rd year of faculty) who had taken theoretical lecture on this subject a year ago, participated in this special module. the opinions of the students on the program were obtained through a questionnaire form and the increase in knowledge was evaluated via pre/posttest. the mean of pretest point was 2.7/10, that increased to 7.4/10 in the posttest evaluation. 50% of students pointed that they enjoyed participating in drama activities in the pre-questionnaire, this rate was 100% in the final questionnaire. they all remarked that implementation of drama in education was beneficial for their communication skill, helping them to learn more about science and increased their enthusiasm to learn and discuss the scientific information. although the preparation process might take more time and need to hard work for teachers, we concluded that the drama methods as a new tool to increase of participant's interest might be proposed for students in higher education. laboratory-based performance assessment in medical education: an opportunity for connection between scientists and medical students h. tuncel cerrahpasa medical faculty, istanbul university, istanbul, turkey number of medical students who interested in basic medical sciences is declining and medical sciences literacy is falling, it is crucial to develop ways for students and scientists to connect. students need to know that science is an intensely human endeavor, and scientists need mechanisms to bring that truth to the community at large. based on continued interest and experience on the part of faculty, and on student feedback, the development of a more effective and stimulating interactive learning tool was undertaken. an in-depth knowledge of laboratory medicine principles is vital to all practicing physicians. great variation exists in the ways that medical students learn the principles of laboratory medicine. there are a number of programs for electronic media that emphasize laboratory-related skills. some of these are appropriate for medical students in the clinical years. programs that teach skills in common laboratory procedures, such as interpretation of peripheral blood smears and microscopic examination of urine sediment, have been shown to improve student performance. to ensure that important principles are addressed, medical schools should establish goals and objectives specifically related to laboratory medicine and experiment with optimal teaching and assessment methods. we also hope that this study will inspire dialogue among primary care and specialist physicians as to the proper degree of education in this area. ideally, it will encourage scientific studies that address evidence-based possibilities for improving critical laboratory medicine educational outcomes, that is, the training of physicians who optimally use laboratory diagnostics and therapeutics. engaging medical students in scholarly scientific activities and producing clinically competent and research-oriented medical workforces are essential demands, particularly in developing countries. an experimental special study module for medical undergraduate students: learning western blot analysis and detection of b-actin protein expression in tissue and cell culture samples learning, introduce basic principles of laboratory research and to present the results.b-actin is one of six actin isoforms which is mainly expressed in all eukaryotic cells. western blotting is a widely used laboratory technique to determine specific proteins and to evaluate protein expression in tissues and cells. in our study, different concentrations of rat spleen homogenates (25, 50, 75 lg/well) and 50 lg protein/well of human lung and ovary cancer cell lysates were used. the proteins were seperated by 10% sds-page, transferred to pvdf membrane, incubated with specific b-actin antibody and then with hrp-conjugated antibody. protein bands were detected with ecl and densitometric analysis of proteins was quantified by imagej software. differences in protein band intensities were compared using one way anova.a value of p < 0.05 was considered statistically significant. we detected b-actin expression in rat spleen homogenates, human lung and ovary cancer cell lysates, as a 43 kd protein. the protein band intensities were in correlation with protein concentrations. the highest concentration resulted in the highest signal intensity in rat spleen homogenates.b-actin level was higher in ovary cancer cells than in lung cancer cells, although both proteins were loaded equally. the feedback showed that students were very satisfied with this laboratory ssm. they developed their independent study skills, planned a research, worked in a laboratory, learned and performed a technique, western blotting. the hands-on experience is very important for medical undergraduate students for their future scientific career. three-dimensional structure of truncated human kv10. voltage-gated potassium channel kv10.2 belongs to the ether-ago-go family. it has been proved that its mutants are involved in development of neurological diseases and some types of tumor. directed drug design needs knowledge of details of the threedimensional channel structure. the members of the kv10-12 subfamilies are characterized by extremely long n-and c-terminal intracellular tails, which possess a number of structural domains. the n-terminal pas domain in kv10 plays an important role in activation, and is thought to alter the rate of deactivation, possibly by binding at or near the s4-s5 linker at the inner mouth of the pore. here we present an improved 3d structure of the truncated human kv10.2 channel, obtained by single particle em. this channel lacks its cytoplasmic pas domain but it still forms tetrameric particles. earlier we showed that the full length kv10.2 channel is build according to the 'hanging gondola' type, and its cytoplasmic and transmembrane parts are connected by linkers. the cytoplasmic part includes the interconnecting pas and cnbd domains. deletion of the pas domain leads to the conformational change in the cytoplasmic part of the channel. for interpretation of the 3d structures we used homology modeling and molecular dynamics simulation. there are several templates available to the moment including eag domain-cnbhd complex of the mouse eag1 channel, full-length shaker potassium channel kv1.2, c-linker/cnbhd of zelk channels and others. but there are still no templates for many fragments that led to necessity of partial de novo modeling. analysis of molecular trajectory allowed estimating dynamical characteristics of channel, supposing interdomain interactions. results of the conducted investigation have a great interest at both the academic and the industrial levels. the objective of this task program is to enable students to gain scientific attitude and skills for them to be able to deal with the problems they'll confront in future research careers. it's been emphasized in various studies that medical students are keen on conducting scientific research, and it's been stated that they need to be supported in this respect, as they lack the adequate fund of knowledge. this study aims to share information throughout a 5-year performance of the research training program (rtp) conducted at ege university, faculty of medicine. rtp is an educational program applied in addition/parallel to the bachelor's degrees for establishing scientific thought, attitude, proceeding and knowledge of the willing medical students, and enabling them to adopt study skills. the dynamic program produced by the rtp committee in 2011 has been developing each and every year via feedback received from the students. an operating principles, a manual for advisor and an introductory booklet have been laid out. applications are being accepted at the end of first academic year, making announcement to the freshmen in advance. students are being admitted to the program, taking the assessment criterias into consideration. second and third year medical students attend the didactic part of the program during the terms devoted to special study modules. thereafter, they go through the project management phase, and accomplish their projects under supervision of a faculty member until their graduation. 12 first graduates of the program, accomplishing their projects, received their certificates at the graduation ceremony of 2015 graduation. currently, there are 61 students in total from all classes who perpetuate their studies within the program. an inventive training pattern of ege university faculty of medicine, rtp experience is being maintained as a dynamic process and successfully keeps the students advised of conducting scientific researches, cultivating scientific awareness. objectives: objectives selection and verification of blood collection tubes is an important preanalytical issue in clinical laboratories. in this study comparison with the reference glass tube of ayset plastic tubes containing separator gel and assessment for routine clinical chemistry laboratory testing in samples were aimed. materials and methods: thirty-four volunteers were included in the study. samples were taken into two different tubes by two experienced technologists according to the clsi protocol [tube1: z (becton dickinson and company, franklin lakes, nj, usa); tube2: ayset (lot10069, turkey)]. glucose, urea, creatinine, ast, alt, total-cholesterol, triglycerides and high density lipoprotein-cholesterol were analyzed subsequently (olympus au2700) and randomised samples stored at 2-8°c for 24 and 48 h. 0th hour sample was analyzed immediately after collection and accepted as the reference for the comparison of the other samples. a paired t-test and wilcoxon signed rank sum test were used to test the significance of differences between the reference tube and test tubes. results: the difference between the results of reference tube and test tubes for glukose, urea, creatinine, ast, alt, total cholesterol, tg and hdl-cholesterol at 0, 24 and 48 h were statistically no significant (p = 0.09, p = 0.07, p = 0.20, p = 0.16, p = 0.13, p = 0.09, p = 0.05, p = 0.54, p = 0.58, p = 0.46, p = 0.19, p = 0.13, p = 0.66, p = 0.72, p = 0.34, p = 0.11, p = 0.16, p = 0.06, p = 0.08, p = 0.39, p = 0.23, p = 0.63, p = 0.58, p = 0.54, respectively). conclusions: no significant difference was observed between ayset tubes' results and the reference tube's results. e. akin c ß akir d€ uzen laboratuvarlar grubu, istanbul, turkey insulin resistance underlies the development of obesity which is a global health problem. obesity is a main concern of scientists because it's associated with type 2 diabetes, hypertension and some cancers. recently, inflammation centered mechanisms are deeply investigated as well as the effects of anti-inflammatory diets which are highly rich in vitamins, minerals, fibers and healthy oils. these diets are proposed to inhibit or supress the secretion of the inflammatory mediators and also improve the intestinal microflora. the aim in this study is to highlight the increasing trend of publications in regard to insulin resistance and inflammation based obesity along with the effects of antiinflammatory diets used for its treatment in the last decade. we performed a pubmed search with key words of 'obesity and insulin resistance and inflammation' (01/01/2005-15/05/ 2016) (search #1). besides, we performed another search with key words of '(anti-inflammatory diet or dietary supplement) and (obesity or insulin resistance)' (search #2) to highlight the value of anti-inflammatory diets and dietary supplements in combating inflammation based obesity and insulin resistance. search #1 revealed 6763 articles; of these 341 were clinical trials, 18 were observational studies. human studies were 4 552 while animal studies were 2 580. overall, there were 2 229 review articles and 5 meta-analysis in the field. search #2 revealed 4 255 articles of which 796 were clinical trials, 958 were review articles, 46 were meta-analysis. human studies were 2 610 and other animal studies were 1 931. the relationship of metabolic diseases with a low grade inflammatory state has opened a new area of research to understand the consequent causes of inflammation in the human body. the increasing scientific data in the field indicates that antiinflammatory diets may serve as powerful tools to solve the inflammation and the consequent insulin resistance and obesity. medical and biological illustrations for life sciences education: is 'a picture worth a thousand words' in visualizing medicine and science? medical and biological illustrations (mbi) convey complex ideas with just an image and they are powerful intersections of science and art. the clarification of complex pathways via illustrations can be effective means in education as they help the student to visualize the biomolecular world and understand the mechanisms. our aim is to illustrate how a mbi is developed over the example of mechanism of insulin action, via the phosphoinositide (pi) 3 kinase-protein kinase b (akt) pathway. organising one's thoughts and clarifying relationships and then using the optimal complexity level to illustrate the pathway most clearly are the basics of mbi. thus, we made a thorough investigation of insulin mechanism on glucose uptake in skeletal muscle and adipose tissue; a biochemical process that includes insulin receptor (ir), ir substrate, pi3 kinase, pi-dependent kinase 1 and akt. then, we found the 3d structures of molecules via protein databanks and accordingly created drawings and diagrams of each component in both molecular and macrolevels by adobe photoshopò software. graphics tablets and a compatible pc were also used in the production phase. the use of computer hardware/software enables unlimited detail in images and provides the flexibility that classical drawing techniques can not. thus, the final diagram clarifies the underlying molecular mechanisms of a biochemical pathway along with the physiologic actions. recent improvements of computer technology have resulted in the creation, and reproduction of high-quality lower cost medical art. mbi's can be used in education to explain concepts/pathways to students to enhance learning. similarly, mbi's are great tools to show mechanism/procedures to enhance patients' understanding of their medical condition. considering their unquestionable contribution to education, research and patient care, the creation of mbi's should be promoted as a graduate level course and the discipline should be represented at academic level. biochemistry is a compelling field with broad applications in many disciplines like medicine, dentistry, pharmacy and bioengineering. biochemical research increasingly combines ideas from genetics, molecular biology, etc. and collaborates with many disciplines. our objective is to conduct a scientometric analysis of the last decade's postgraduate theses in the field of biochemistry (ptb) in regard to number, collaborations, subject area distribution, etc. to discover the characteristics and trends in turkey. we searched the turkish higher education council's theses database (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) which includes master of science (msc.), doctorate (ph.d.) and specialization (s) theses in all disciplines. an electronic search with the keyword of 'biochemistry' (in the thesis subject area) was conducted, thus it brought all theses either in the biochemistry discipline or theses in other disciplines but have a biochemistry component. we performed data cleaning and further quantitative analyses in excel. we also executed word count analysis on the titles of theses to derive the main subject areas in ptb. of the total of 6374 ptb (2215 s, 2781 msc, 1339 phd theses) 37.6% was in natural sciences while 62.4% was in health sciences. the theses output-growth measured by the compund annual growth rate was 82% over the 10-years. the top 3 clinical disciplines in collaboration with biochemistry were pediatrics, surgery and cardiology, and the top 3 science disciplines were biotechnology, bioengineering and biology. oxidants-stress and antioxidants (1008), endocrine-metabolism (655) and enzymology (608) were the top research areas in ptb, followed by genetics (305) and cancer (252). scientometrics is a powerful tool to understand the direction of science and research. our ptb analysis indicated that prominent areas like stem cell, biosensors, geriatrics are somewhat lagging in turkish biochemistry research while postgraduate education and research in total is growing fast with sound collaborations. the 1st turkish in vitro diagnostic symposium evaluation objective: in vitro diagnostic (ivd) medical laboratory tests and the equipment are closely related the public health, patient safety and the safety of all who utilize these tests. it depends on auditing of the process from the production to the consumption of these materials, that they do not pose a risk to individuals and society. upon these basic requirements; 'turkish in vitro diagnostic symposium: medical laboratory tests' was held in february 2016, organized by the cooperation of turkish biochemical society branch of izmir, and dokuz eylul university health sciences institute. it was intended to shed light on some questions such as, what is the place and importance of ivd in turkey? what are the responsibilities of educational institutions?, what is the role of ministry of health? to put across the conditions of preparing a substructure that may provide achieving success in producing ivd medical devices and in this sector, in our country. material-method: 39 invited speakers attended the symposium, along with the participation of both as lecturers and attendees; ministry of health, turkish standards institution; representatives of manufacturer enterprises; representatives of enterprises manufacturing in turkey; scientists conducting considerable researches on health technology; students and representatives from some of the non-governmental organizations. in addition to the presentations, gathering up the written opinions of the participants, a report was prepared. results: the symposium that lasted for 3 days was realized with 120 participants in total,55 of which from universities;38 representatives of their companies;17 from chamber-institute-public establishments and 10 of which from public hospitals. 94 of the participants were from izmir,26 of them were coming from out of izmir. conclusion: at the end of the symposium,40% of the participants gave feedback. among the feedback selected; 86.2% of the participants evaluated the symposium overall, as successful. 75.6% of them found the symposium successful with regard to its scientific content. their feedback were 55.2% positive in terms of the symposium's contribution to the situation assessment on ivd in turkey, and 44.8% of them stated they would consider participating in the second of the ivd symposium if it is to be organized. perceptions of molecular life science master's students on their scientific and academic competencies and prospective plans for professional development the master's education (me) in molecular life sciences (mls) is aimed at strengthening the knowledge and skills base of the young scientist, preparing him for the competitive academia/industry positions. the rapidly developing pace of science and research forces the master's student (ms) to play a central role in monitoring and guiding his scientific education and professional development (pd). thus, the aim of our study was to examine the perceptions of ms of mls, regarding their scientific and academic competencies. with this data, we planned to analyse if this awareness channels ms to take action and/or matches with their prospective plans. we developed a 15 item online survey with 3 sections (demographic data, current data-contributions of me, competencies and prospective data-action for pd, future plans) and distributed it via e-mail to various postgraduate institutes. at the end of the 10-day period, 138 ms students (in the thesis phase) answered the questionnaire (female: 66.7%, male: 33.3%). the most highly rated activities that contributed to their scientific knowledge and skills gained in the me were laboratory work (73%), visits with their mentors (70%) and theoretical lessons (65%). ms expressed low levels of sufficiency both in theoretical scientific knowledge and laboratory skills (only 43% and 33% sufficiency, respectively). communication skills (80%) and team work (78%) were rated as the highest professional competencies followed by literature search and research planning (both 73%). it was striking that ms perceived themselves as quite insufficient in scientific writing (50%), data analysis (60%) and project writing (61%) while proficiency in english (55%) was the first area they wanted to take action. despite their perceptions of insufficiencies in many areas, a majority (69%) wanted to continue to phd education. these and similar surveys may lead to an increase in selfawareness in ms and the data may contribute to the revision of me. the report of the 1st turkey in vitro diagnostic symposium results the following aspects and suggestions took place in the results report prepared after the symposium: about the national ivd-tc r&d, production, forming quality assurance and innovation strategy and policy the cooperation of the university and the industry is not sufficient most of the industrialists cannot take enough advantage of the support provided by the institutions like tubitak, the ministry of science, technology and industry and the ministry of development the statistics on the ivd-tc in turkey should be carried out as soon as possible national standards should be determined in parallel with the international standards the vat rate of the exported raw materials that would be used in ivd production should be decreased. about the education and training, the job titles and positions the related graduate programs, which would focus on all steps of the whole life cycle related to the ivd-tcs one by one, are not widespread there is no 'postdoc' application in turkey. 'postdoc' staff is needed for insufficient component human resources the lecturers should not be restricted to one discipline only graduate programs on laboratory medicine are needed to be established and spread, in order to train component labor specified on the ivd-tc about research and development there are almost no researches related to product development. this should be associated with the education and training institutions about the research centers currently, there is a real infrastructure on health technology in turkey, but there are difficulties in its instituonalism the insufficient cooperation of the university and the industry does not allow the inventions to turn into products the cooperation supports of r&d, being restricted to tech-nopark and r&d centers, are open fields for improvement p-edu-014 phd training in medical education: career profiles and satisfaction levels of graduates from dokuz eylul university graduate school of health sciences this survey was carried out within the scope of special study modules that is entitled 'phd training in medical sciences' by a group of medical students in deu. the purpose of the survey to investigate the members of health sciences that have successfully completed their phd training in terms of the levels of satisfaction and the status of their career. from this scope we generated two hypothesis: we expected that graduate phd graduates are mostly involved in academy and find their satisfaction levels at moderate level as to phd education. the study was designed as cross-sectional. we reached 166 phd graduates who had graduated from deu graduate school of health sciences between 1991 and 2002 from 12 different departments via e-mail. the survey was included 27 questions, which were prepared in the light of the existing literature. among the 166 phd graduates, 55 (30%) participated in the study. through this survey, perception of phd students on supervisors' scientific and educational abilities, opinions on phd training, productivity of phd training, number of articles published, their position and related satisfaction levels after graduation were investigated. according to the results, more than half of the graduates (%52.7) are well satisficed from the education they had taken. beside this, interestingly we found that %94.5 of graduates prefers staying in the academic positions and %64.8 of them sustains their communication with their supervisors. in conclusion, most of phd graduates were contented with phd training and their career profiles. as a result of this survey, we produced a novel and precise contribution to the literature. in a further study, this survey may extend to other parts of turkey and compile the results in order to get more accurate and inclusive data. phd is an international degree that is reached by conducting an original research after finishing bachelor or master's degree. doctoral degree (phd) can open the door of academic career; on the other hand, a person with a doctoral degree is equipped to carry out important work in research, industry, or public sector. today, gradually increasing number of phd students have brought some problems in phd training. the purpose of this study is to investigate and review activities that have been done by the following international organizations: orpheus: (organization for phd education in biomedicine and health sciences) eua-cde: european university association-council on doctoral education. febs education committee: federation of european biochemical societies these three organizations have done workshops on phd training to pay attention to the following points: *a phd student must take some courses and trainings outside her/his institute, should not be limited to the institute. *the phd training programme should include transferable skills courses. *clinicians, if involved in phd training, should be given free time from their clinic duties. *with regards to potential problems with the supervisor, the institute should provide the student an advisory system. *students should be encouraged to participate in the management of doctoral programmes in the institute. *the students should be given be opportunity to select their own supervisor (thus their thesis area). the phd training has gained quality thanks to these organizations. it is advised that graduate school of health sciences should follow the recommendations and report from these organizations. itsn1 and itsn2 are genes encoded adaptor proteins with multiple isoforms participating in clathrin-mediated endocytosis (cme), mapk signaling and reorganization of actin cytoskeleton. changes in itsns expression can lead to different neurodegenerative disorders and cancers. to date little is known about regulation of itsn genes on posttranscriptional level. the aim of our work was to predict and experimentally confirm target sites for micrornas that could potentially regulate itsns expression. using 8 web servers we analyzed 3 0 utrs of short and long isoforms of human itsn mrnas and found conservative target sites for mir-34, mir-19, mir-129, mir-103/107, mir-194, mir-181 and mir-30 in 3 0 utr of itsn1-s, predicted by 5 servers, mir-203 predicted by 5 servers for itsn1-l, and mir-153, mir-148/152, mir-27, mir-144 and mir-128 predicted by 5-6 servers for itsn2-l. to elucidate potential impact on cme, mapk signaling and actin cytoskeleton regulation by these mirnas we performed enrichment analysis by diana-mirpath server and found that mir-34, mir-19, mir-103/107, mir-181, mir-30 and mir-148 were highly enriched for all analyzed pathways. using regrna 2.0 and scan for motifs services we predicted 12 types of different regulatory elements in 3 0 utr of itsn1 and itsn2: k-box and brd-box, musashi binding element for rbps musashi1 and musashi2, gu-rich element (gre) and au-rich elements (are) that regulate mrna decay. to confirm itsn1-s regulation by micrornas we cloned 3 0 utr of itsn1-s into luciferase reporter vector and transfect hek293 cells by this construction and mir-181a, mir-30a and mir-19a. for mir-181 transfected cells, we found 25-40% decrease of expression of itsn1-s 3 0 utr-bearing construction. for other mirs we did not obtain strong reproducible effect in luciferase assay. these data may confirm mir-181 target site in 3 0 utr of itsn1-s mrna but needed additional research. objective: stroke is an acute neurological disorder that is mostly caused by ischemia in central neural system. 30% of stroked patients lose their life in 1 year, and remaining 1/3 of the living patients continue to their lives as dependent to others. nihss and mrs are two scales which are used in prognosis studies because they can show stroke intensity and after stroke functional recovery. microrna's which have effects on transcription and posttranscription gene regulations are small rna molecules. their roles have been investigated on pathophysiology and treatment of diseases. in this study, it was aimed to detect changes in blood serum levels of mir-146a, -155, -210, 181b, -31, 126, -92a and let-7f of ischemic stroke patients and to investigate role in predicting prognosis methods: 21 patients diagnosed by acute ischemic stroke admitted to neurology service of g€ oztepe hospital and 16 healthy individual were included in the study. after stroke patients' blood samples were taken periodically in 1st day, 1st week, and 3rd month, and at the same time nihss and mrs scores were determined. set 8 mirna blood serum levels were measured by rt pcr results: when compared to the control group, we found that after stroke 1st day peripheric blood levels of mir-146a,-31,-92a and let-7f were significantly low; when 1st week and 1st day serum records were compared there was a significant increase in mir-126 level; and when 1st week and 3rd month records were compared we noted that there was a significant increase in mir-146a,-126 and let-7f levels. from prognosis point of view; after ischemic stroke measurements showed that mir-181b in the 1st day, mir-146a and mir-210 in the 1st week showed positive significant correlation with 3rd month mrs scores (p = 0.002, p = 0.05, p = 0.002, respectively) conclusion: according to the outcomes of this study, after stroke 1st day mir-181b, 1st week mir-146a and 1st week mir-210 levels can be stipulated to use in predicting patients' 3rd month prognosis p-01.03.3-004 inhibitory rna aptamer against lambda ci repressor showed the transcriptional activator activity s. ohuchi, b. suess tu darmstadt, darmstadt, germany because of the variety of functionalities on gene regulation and the easiness of molecular engineering, functional rnas are promising parts for the construction of genetic circuits. artificial affinity rna, or rna aptamer, is one of such genetic parts. in the previous study, an inhibitory rna aptamer against a repressor protein, tetr, was developed as a transcriptional activator [1] . the expression of this aptamer abolishes the repressor activity of tetr, resulting in the elevated gene expression under the control of tetr. because of simplicity of the mechanism, similar transcriptional activators can be generated by using rna aptamers against other repressor proteins. here, we examined the generation of an activator based on an rna aptamer against one of the most frequently applied repressor proteins, lambda phage ci. in vitro selection (selex) was performed targeting a recombinant ci protein employing an rna pool containing 40-nucleotides of a random region. after 6 rounds of selex, the pool rna showed the affinity, as well as the inhibitory activity, against ci in vitro. then, rna aptamers with the transcriptional activator activity were screened from the enriched pool in vivo employing a reporter plasmid on which the expression of a reporter gene, lacz, is repressed by ci. when the variants of the rna pool were transformed to e. coli cells harboring the reporter plasmid, about half of the transformants showed the elevated reporter expression. interestingly, all of these desired rna clones shared the same sequence. quantitative analysis indicated that 35-fold induction of the reporter expression was achieved upon the aptamer expression. our results suggested that diversity of artificial transcriptional activators can be extended by employing rna aptamers against repressor proteins to broaden parts for the construction of genetic circuits. [1] hunsicker, et al. (2009) an rna aptamer that induces transcription. chem. biol., 16: 173-180. p-01.03.3-006 microrna expression signatures between non-atherosclerotic plaque and atherosclerotic plaque in cad with humans, and parallels whole blood rnas and represent a new important class of gene regulators. the present study was designed (i) to investigate the mirna expression profile in human atherosclerotic plaques from peripheral arteries aorta as compared to non-atherosclerotic left internal mamary artery (lima); (ii) to examine the expression levels of mirna in whole with correlation mirnas of aorta tissue. material and methods: thirty-one patients with cad were enrolled in study. lima and aorta tissue samples were obtained during coronary artery bypass surgery. whole blood samples were collected before cabg surgery. each patient with cad was obtained from whole blood, aorta and limas tissues. these tissue samples were immediately soaked in rnalater solution and homogenized using a magnalyser. the rna was extracted using the trizol reagent and the mirneasyò mini-kit. the expression profiles of 738 mirnas were evaluated using highthroughput qrt-pcr. results: we found that mir-497-3p was expressed only in aorta. mir-431-5p and 433 were expressed both aorta and whole blood. 6 mirnas were significantly up-regulated in aorta when compared to lima tissue (fc > 2). 59 mirnas were significantly down-regulated in aorta compared to lima. conclusion: in conclusion, our study suggests that mir-497-3p, mir-431-5p and 433 might be a potential for cardiovascular disease development. also mir-431-5p and 433 might serve as novel non-invasive biomarkers for cad p-01.03.3-007 mir193b regulates cell proliferation and colony formation in pancreatic ductal adenocarcinoma n. gurbuz, e. isildar due to the strong metastatic potential, pancreatic cancer has the highest mortality rate of all major cancers. therefore, the investigators are in urgent need of developing the new alternative therapeutic approaches for the prevention of pancreatic cancer. mirnas, small noncoding rnas, regulates as an inducer or inhibitor in expression of key mediators related molecular mechanisms in cancer promotion. to investigate the effect of mir193b on pancreatic ductal adenocarcinoma cells, we performed the cell viability and clonogenic assays by mts and crystal violet dye, respectively, in panc-1 and miapaca-2 cells transfected with mir193b mimic. our data revealed that mir193b led to decrease the cell viability depending on enhanced mir193b doses, which are 25, 50, 100 and 150 nm, as the ratio of % 23, 60, 82 and 87, respectively, in miapaca-2 cells and as the ratio of % 40, 58, 77 and 89, respectively, in panc-1 cells compared with control condition of each cell. this inhibition mediated by mir193b was also obtained in colony formation both of pancreatic cancer cells. when the induced effect of mir193b on the death of pancreatic cancer cells was compared with gemcitabine, which is currently used as a clinical drug for pancreatic cancer patients, we determined that mir193b was more effective than gemcitabine. based on our findings, it is clearly shown that mir193b serves as a tumor suppressor in pancreatic ductal adenocarcinoma cells. we strongly believed that mir193b gene therapy might be more effective and targeted approach than classical gemcitabine therapy for pancreatic cancer patients. *this work was supported by tubitak (the scientific and technological research council of turkey) grant 114s501. breast cancer is the most common cause of cancer death in women. trastuzumab is a therapeutic agent frequently used against her2+ breast cancers, which has role in approximately 20% of invasive breast cancers. with the discovery of their activity in cancerogenesis, micrornas (mirnas) have become potential candidates to mediate therapeutic actions by targeting genes that are effective in drug response. recent studies have showed that mirnas are induced by targeted therapies. in this study, we aim to find out mirna-mediated mechanism, which is driven by common trastuzumab responsive micro-rnas in her2+ breast cancer. for this purpose, the common trastuzumab responsive mirnas were determined in treated bt474 and skbr3 cells by microarray profiling. two datasets were intersected to find out common mirnas for both cell lines. the overlapping predicted targets of common mirnas were provided by two different mirna-target prediction databases and then a mirna-gene network was built in cytoscape by using networkanalyzer plugin. the most shared target genes were chosen to be analyzed in the ebi-embl gene expression atlas for their expression patterns in breast cancer. 25 common mirnas were found to have overlapped targets in two target prediction algorithms that were used to build the mirna-gene regulatory network. 14 overlapped targets were determined as the most shared genes in the mirna-gene network. expression pattern of each shared gene in the gene expression atlas showed that 12 out of 14 the most shared target genes were strongly dysregulated in several breast cancer types. our results suggest that mirnas might show a common response to the targeted therapies and network analysis can be profitable to have a better explanation of the regulatory mechanisms between drug responsive mirnas and their target genes. revealing the mirna-potential target interactions might provide novel key players that mediate the mechanisms of action in drug treatment. chronic myeloid leukemia (cml) is a clonal disease of primitive pluripotent stem cells that identified with a specific t(9;22) reciprocal translocation that encoding bcr-abl oncoprotein. resveratrol (res) is a natural phytoalexin found in grapes and induces apoptosis, erythroid differentiation and autophagy in leukemic cells. micrornas are small, single strand, non-coding rna molecules that regulate post-transcriptional gene expression via disrupting the stabilization of target transcripts or inhibiting protein translation. in our study we aimed to determine cytotoxic effect of res in k562 human cml cell line and to evaluate the expressions of mirnas that are associated with genetics of leukemia after treatment with res; to investigate target genes of mirnas which show significant expression alterations and molecular mechanisms of res treatment. k562 cells were treated with 100 lm (ic50 dose) res during 72 h and cytotoxicity was evaluated by using wst-1 assay. the rt-qpcr is used for mirna and gene expression analysis. results showed that; res up-regulated tumor suppressor mir-214-3p level 2.87 fold and significantly downregulated hdac1 gene expression (p = 0.003) and upregulated p62/ sqsmt1 gene expression (p = 0.001), according to the control cells.p62/sqstm1 interacts with lc3 and plays role as a critical player in the autophagic degradation of the bcr-abl fusion protein. our findings showed that resveratrol acts as a hdac inhibitor targeting hdac1 and p62/sqsmt1 gene expression level. treatment with hdac inhibitors results apoptosis, cellcycle arrest, cell differentiation, anti-angiogenesis and autophagy. downregulation of hdac1 provides post-translational modification for expression of tumor supressor genes and leads to cell cycle arrest and increases apoptosis. these results could be linked to hdac1 dependent induction of gene associated with autophagy like p62/sqsmt1 and resveratrol could be a therapeutic candidate as a hdac inhibitor for cml treatment. the mirna used in this study are hsa-mir-145, hsa-let-7a, hsa-mir-29b and hsa-mir-155. thereafter, we bought pre-mirnas and their mirnas commercially. we apply them to the a549 cell line in different combination and different concentrations. these mirnas applied solely onto cells or in combination as; four of them, let7 + 145, let7 + 29b, let7 + 155, 145 + 29b, 145 + 155, 145 + let7 + 29b, 155 + let7 + 29b. the cell viability was detected by wst-1 kit in a 96 well plate elisa reader. cells were seeded as 10 000 per well, mirnas incubated with cells for 24 h in an appropriate atmosphere. according to our results some combinations and mirnas didn't alter viability, however 145 + 29b and 145 + 155 combination increased the cell viability dramatically. on the other hand let7 + 29b and 155 only applications decreased the cell viability. the other applications' viability results are not different from the control cells significantly. in this study, we used a549 cell line also called non-small lung cancer (nsclc) cell line and possibly effective mirnas on lung cancer. it is important to exhibit the mirna combinations should be effective on cancer cells' viability. the prospect combinations were determined which is crucial to develop new strategies for cancer treatment. competing endogenous rnas (cernas) act as molecular sponges for the same pool of micrornas through their mirna response elements (mre), titrate mirna levels and thereby regulate gene expression post-transcriptionally. smad interacting protein 1 (sip1), a member of the zeb family is a regulator of epithelial-to-mesenchymal transition (emt) program, which is active during embryogenesis and tumor invasion and metastasis. hence, we investigated the regulation of sip1 by cernas in hepatocellular carcinoma (hcc) cells. among hundreds of sip1 cernas listed at competive endogenous mrna database (cerdb), 14 transcripts (pten, zeb1, ptch1, creb5, acvr2b, enah, robo2, erbb4, e2f3, foxo1, rictor, klf3, ets1, cdk6) sharing at least 9 common mre sites with sip1 were selected and their expression in 9 hcc cell lines were determined by qrt-pcr. ets1 was found to be highly correlated with sip1 in hcc. furthermore, repressing sip1 expression by shrna in hcc cells resulted in decreased expression of ets1, pten and zeb1. our results suggest a possible bidirectional and post-transcriptional regulation of sip1 and its cer-nas in hcc. a meta-analysis for the identification of common microrna signatures in colorectal cancer n. sahar 1,2 , n. belder, h. ozdag 1 biotechnology institute, ankara university, ankara, turkey, 2 comsats institute of information technology, islamabad, pakistan colorectal carcinogenesis (crc) has quite frequent incidence and mortality rates worldwide, despite being studied for decades now. new biomarkers are needed to be identified in addition to the existing ones, due to heterogeneous nature of this disease. the regulatory molecular machinery of a cell, including micrornas (mirnas), contributes to this heterogeneity upon aberrant expression. herein, for a mechanistic understanding of differential gene expression in crc tissue we analyzed mirna expression profiles of 78 crc tumors against 62 normal colorectal mucosa samples, using raw data from e-mtab-752 and e-geod-35834 (affymetrix microarrays), and gse35982 and e-mtab-813 (agilent microarrays) datasets obtained from gene expression omnibus and arrayexpress. raw samples were normalized (different platforms separately) using quartile normalization in brb-array-tools. differential expression of mirnas was identified using cut-off values of p ≤ 0.05, fold change ≥ 1.5 and stringent false discovery rates. mirtarbase and mirwalk2.0 databases were explored to identify validated targets. we found thirty (including mir-21 and mir-183) and thirteen (mir-1, mir-139, mir-375, etc.) mirnas commonly upregulated and downregulated respectively, in both affymetrix and agilent microarray results. predicted targets of these mirnas frequently belong to pathways related to cancer like b-catenin, phosphoinositol-3kinase, and transforming growth factor-b, to name few. moreover, the target genes were significantly enriched in clusters related to cell cycle, cell differentiation and regulation of apoptosis. these promising results will further be compared with differentially expressed gene profiles from a cohort of turkish crc patients. hence the integrated study will refine the panel of diagnostic and prognostic crc markers. hsa-mir-x modulates motility and invasion in triple breast cancer cell line s. noyan 1 , h. g€ urdal 2 , b. g€ ur dedeoglu 1 1 biotechnology institute, ankara university, ankara, turkey, 2 department of pharmacology, ankara university, ankara, turkey breast cancer is a heterogeneous disease and expression levels of certain receptors have demonstrated subtypes which characterize clinically distinct breast tumors. a triple-negative phenotype lacks expression of er, her2 and pr and is known as basallike carcinoma. micrornas are a class of small non-coding rnas that participate in the gene expression in many biological processes. e-cadherin is an important mediator of adhesion in epithelial tissues, and loss of e-cadherin can play a critical role in tumor invasive behavior. another key player of cell integrity pip (3, 4, 5) triphosphate is generated at the leading edge of the cell and leads to cell polarization. pip3 is generated by hydrolysis of pip (4,5) bisphosphate, which is synthesized by pip5k1. any dysregulation in these molecules may support the invasive behavior of the cells. the aim of this study is to find out the role of mirna precursor (hsa-mir-x) in invasion and motility in triple negative breast cancer cells. in this study a triple-negative breast cancer cell line bt-20 was transfected with hsa-mir-x or scrambled control sirna. to check its role in motility and invasion, wound healing and invasion assays were performed respectively. cell invasion was monitored over a period of 24 h by xcelligence real-time cell analyzer using a double-plate and measuring impedance-based signals. additionally emt markers were analyzed by qrt-pcr to explain the molecular mechanisms beneath motility and invasion. we observed that cell motility and cell invasion diminished after transfection of bt-20 cells with mimic for hsa-mir-x. furthermore, qrt-pcr experiments indicated that transfection of hsa-mir-x decreased the expression level of pip5k1c while increasing the e-cadherin expression level. wound healing and invasion assays together with qrt-pcr results support the role of hsa-mir-x in cell motility and invasion. this process might be explained via e-cadherin mediated met or gsk-3-beta related inhibition of invasion. expression level of five micrornas as diagnostic markers in glioblastoma situated in the main brain lobes, but can also be found in other brain regions. while microrna (mirna) as non-coding rnas, play a crucial function in the post-transcriptional regulation of gene expression by mrna degradation or translational repression. in the present study, we aimed to investigate the contribution of gene expression of the five mirnas and to unravel their role in brain tumor cell lines, the mirnas to the risk of gbm tumor. the five gbm cell lines (crl-2365, crl-2366, crl-2948, crl-1690 and htb-15) were evaluated with non-malignant (normal) brain cell line (hcn-2) . determinations of expression level of five mirnas (mir-21, mir-101, mir-138, mir-196, and mir-222) were evaluated by monitoring quantitative rt-pcr (qrt-pcr) technique. the expression levels of four mirnas (mir-101, mir-138, mir-196, and mir-222) were significantly decreased while the expression level of mir-21 was increased in gbm cell lines according to hcn-2 cell line. consequently, these five mirnas can potentially be used as biomarkers for gbm tumor; further studies are mandatory to a better understand and confirm our preliminary findings. background: noncoding rna are known to be crucial molecules with diverse regulatory roles in neural oncology and neurodegenerative disease. the recent study suggested that lncrna anril play role in the development of neuroblastoma and alzheimer disease via binding disease-specific micrornas. material and methods: we used lncrnadisease, hmdd v2.0, mir2disease to predict lncrna-and mir-associated disease in our study. in addition, we utilize tardetscan to search lncrna-mirna interaction. results: disruptions of lncrna anril expression (also named as cdkn2b-as, locus cdkn2a/b (ink4/arf), chromosome 9p21) have been associated with the development of neuroblastoma and alzheimer disease. here, we predicted interactions between noncoding transcripts encoded by locus cdkn2a/b and their molecular partners -microrna. anril can act as decoy while containing sequences that mimic mirna target sites to titer these mirs away from their primary targets thereby act as molecular sponge. using targetscan 7.0, we predicted target sites for hsa-mir-15-p/16-p/195-p/424-p/497-p/6838-p and hsa-mir-125-5p/4319 in anril 3 0 utr. then, we used hmdd v2.0 and mir2disease databases to define if any of these mirs participate in alzheimer disease and neuroblastoma. according to both databases, mir-125 is implicated to alzheimer disease and mir-15 to neuroblastoma. as soon as anril participate in the development of both abovementioned disorders and can have microrna sponge activity, it could potentially positively regulate mir-125 and mir-15 targets by competing with them for micro-rna binding sites thus restoring the expression of target genes. in our further research we plan to experimentally validate predicted microrna sites in anril 3 0 utr. conclusions: we predicted sites for mir-125 and mir-15 in 3 0 utr of anril lncrna that could uncover its possible sponge activity in the development of neuroblastoma and alzheimer disease. aim: matrine excracted from saphora flavescens root and demonsrated that indicates pro-apoptotic and anti-proliferative effect in many types of cancer. acute lymphoblastic leukemia (all) is an acute form of leukemia, or cancer of the white blood cells which characterized by the overproduction and accumulation of lymphoblasts. mirnas play important roles in deregulated cell death mechanisms. we aimed to investigate the effects of critical mirnas during the development of matrine resistance on all cell line ccrf-cem. material-method: ccrf-cem cells were treated with different (0.5-3 mg/ml) concentrations for 72 h and cell viability measurements were carried out with xcelligence device to determine the cytotoxic effects of matrine. mirnas were extracted from treated and untreated ccrf-cem cells using the mirna isolation kit. cdna was synthesised using allin-one first strand cdna synthesis kit. expressions of 44 selected mirnas were analysed with miprofiletm custom mirna qpcr array using the applied biosystem 7500 fast real-time pcr system. results: according to the cytotoxicity assay, it was determined that 'treatment with increasing concentrations of matrine, decreased the viability of the ccrf-cem cell line. expression levels of 44 different mirnas were studied for indicated passages in two replicates. our results showed that hsa-mir-376b-3p (-37,099 fold, p = 0.008), hsamir-106-3p (-16,6795 fold, p = 0.028), hsa-mir-20a-3p (-15,926 fold p = 0.0148), hsa-mir-519a-3p (-11,7398 fold, p = 0.00534), hsa-mir-204-5p (-10,9536 fold, p = 0.0012), hsamir-30b-5p (-9,0631 fold, p = 0.0221), hsa-mir-15b-5p (-8,8971 fold, p = 0.0339), hsamir-106b-5p (-8,8561 fold, p = 0.021), hsa-mir-885-3p (-8,6139 fold, p = 0.00006), hsamir-30a-5p (-8,594 fold, p = 0.009) expression were decreased during the development of matrine resistance. conclusion: these data suggested that especially hsa-mir-376b-3p plays a critical role in the matrine response. ericd (e2f1-regulated inhibitor of cell death) is a newly found lncrna. it is located at 8q24.3. it has two exons and its transcript size is 1745 bp. ericd is regulated by e2f (transcription factor 2) and modulates the cellular response to dna damage. arid3a is a family member of the at rich interaction domain (arid) dna-binding proteins that participate in diverse biological processes like development, cell cycle control, chromatin remodeling and epigenetic regulation. both, ericd and arid3a have just opposite roles in apoptosis in case of dna damage indicating a probability of reciprocal interaction between each other. till now, there is no work related to the interaction between lncrna and arid3a in cancers. herein we try to find a probable interactive role between these in cancers. in this study, 12 different cancer cell lines, 1 osteoblast cell line and 19 different types of normal human tissues rnas were selected for expression analysis of ericd and arid3a. after rna isolation, cdna was converted from their rnas. expression profile analysis of ericd and arid3a in different cancer cell lines and normal tissues was done using imagej program for semiquantitative and 2 (-ddct) method for quantitative rt-pcr. among used cancer cell lines, ericd was highly expressed and arid3a had lower expression in u-2os (osteosarcoma), a-172 (glioblastoma) and a549 (lung cancer). at the same time, ericd expression was lower and arid3a had high expression in hfob 1.19 (osteoblast cell line) and normal tissues like brain and lung . both ericd and arid3a are cell cycle regulated and are commonly regulated by e2f. they have just opposite roles in apoptosis during dna damage. these two genes have a probability of reciprocal interaction between each other in cancer. our results indicate that both ericd and arid3a might have opposite roles in lung cancer, glioblastoma and osteosarcoma. ericd and arid3a might act as cancer promoting and tumor suppressor genes respectively in these cancers. the importance of mirna expressions in infertilty implantation process is controlled with endometrium, factors secreted by the embryos and in accordance with these factors embryo and/or endometrium via receptors on. more than 700 human microrna (mirnas) that are small noncoding rnas were shown to play an important role in intracelluler cycle regulation in both normal and pathological conditions. in this study we aim to identify mirnas and controlling molecules expressions in different time period of endometrium in fertile and infertile cases. the endometrial samples were taken from fertile and infertile patients in proliferation and early secretion periods. the samples are fixed and stained either with hematoxylen-eosin for morphological analysis or with immunohistochemistry for distributions of anti-dicer, anti-drosha, anti-eif2a and anti-eif2c. mir-17-5p, mir-23a, mir-23b, mir-542-3p, mir-21, mir-199a*, mir-705, mir-20a, mir-26a, mir-125b, mir-200a/b/c were analyzed with qrt-pcr. while dicer immunoreactivity was detected weakly only proliferation phase of fertile group, this immunoreactivity were detected strongly in both proliferation and early secretory phases of infertile group. drosha immunoreactivitiy was also weakly detected in the proliferation phase of fertile group, it was moderately detected in both proliferation and early secretory phases of infertile group. eif2a immunoreactivitiy was similar in each groups but there were a few differences between fertile and infertile group. eif2c immunoreactivitiy was negative in all groups. mir-21, mir-199a* and mir-23a were highly expressed in proliferation phase of fertile group, mir-23a and mir-125b were highly expressed in early secretion phase of infertile group. in conclusion, dicer and drosha immunoreactivities and different expression of mirna's were detected in all groups. implantation problems may be reason for different mirna expression which controlling with dicer and drosha in the infertile endometrium in both proliferation and early secretory phases. wheat is an important agricultural crop with an over 615.8 million metric tons harvesting capacity annually. drought and salinity are environmental stress factors that affect yield and quality of wheat, dramatically. there are different defense mechanisms against these stress conditions in plants. altering gene expression profiles by micrornas at post-transcriptional level is one of the most conserved mechanisms among plants. micrornas are an extensive class of noncoding rnas, approximately 22 nucleotide length which regulates the expression of genes by binding to the 3 0 -untranslated regions of specific mrnas. micrornas implicated under salt and drought stress have widely been reported in numerous plant species and wheat genomes in the last years; however, studies on einkorn wheat (triticum monococcum spp. monococcum) are not yet available. the goal of this study is identification of conserved micrornas from einkorn wheat using next generation sequencing technology and bioinformatic analysis. in this study, small rna molecules were extracted from pooled plant samples grown under normal, drought and salinity conditions. sequencing analysis revealed 75 164 unique small rna sequences obtained from 15 139 448 raw reads. after bioinformatic analysis based on comparative genomics approaches, we identified 168 putative mature microrna sequences belonging to 142 distinct microrna families. since chromosomal sequence data is not available for triticum monococcum spp. monococcum, we used available sequences from triticum urartu, a close relative, as template to extract precursor microrna sequences. 111 of precursor sequences showing 100% homology to triticum urartu genome were analyzed for secondary structure prediction using mfold software. data provided in this study is critical to investigate transcriptional regulation of genes involved in stress metabolism in einkorn wheat. the role of vim-as1, a natural antisense transcript, in cancer coding or non-coding transcript. in this regard, vim-as1 is nats located antisense to vimentin gene. in the present study, we aimed to determine tissue and cell line distribution of vim-as1. materials and method: for the tissue expressions analysis, human total rna master panel ii (containing 20 different human tissue samples) was used. total number 14 cancer cell lines and 5 normal cell lines included in the study. for the expression analysis rt-pcr and qpcr methods were used. results: as a result, expression levels of vim-as1 were found to be tissue specific. particularly, while vim-as1 was highly expressed in lung and thyroid gland tissues, its expression was not observed brain, stomach and adrenal gland tissues. also, vim-as1 was also found to be differentially expressed in cancer cell lines. vim-as1 expression was found to be lost in cal29, pc3, and hct116 and highly diminished a549 cancer cells. also, it is found to be highly expressed in bcpap, panc1 and beas2b cells. discussion: results of the current study suggest that vim-as1 may have significant role in the regulation of vim gene in thyroid gland tissues, as it is highly expressed in both thyroid gland tissues and bcpap thyroid cancer cells. moreover, vim-as1 and vim axis can be involved in the formation of lung tumors because vim-as1 was highly expressed in normal lung tissues and beas2b cells and expressed very low levels in a549 lung cancer cells. lung cancer is the leading cause of cancer related deaths in the world and approximately 90% patients with lung cancer ultimately die from metastatic disease. metastasis is the most dangerous step of cancer. in our recently published work showed that akt/nf-kb pathway is continuously active and induces cellular invasion and pten suppresses cellular invasion via inhibition of akt/nf-kb pathway. in this study we aimed to show nf-kb mediated induction of mirna expression can responsible for inducing nsclc invasion. we used chromatin immunoprecipitation (chip) assay kit for detection of tnf-a induced nf-kb mediated mirnas. therefore, h1299 and pc14 cells treated by tnf-a (30 ng/ml) for chip assay. chromatin regions, reading with chip-seq, were analyzed using bioinformatics tools. we also performed additional bioinformatics search to find nf-kb related mirnas which potentially take a role in nsclc invasion. we investigated the effects of mirna which determined at the bioinformatics analysis results on invasion using invasion chamber method. we found 16 mirnas which potentially induced by nf-kb and related with nsclc invasion. our invasion results indicate that mir-548a-3p, mir-548as-3p, mir-8078, mir-1915, mir-6814-3p, mir-548q mimics can induce cellular invasion on h1299, mir-548v, mir-548 h-5p, mir-138-5p, mir-548a-3p, mir-548as-3p, mir-8078 mimics can induce cellular invasion on pc14. we also verified our results by qrt-pcr, because we want to sure that mirnas which can induce invasion, can also transcriptionally regulated by nf-kb or not. we found that mir-548q, mir-548a-3p, mir-584as-3p, mir-1915, mir-8078 in h1299, mir-138-5p, mir-548a-3p, mir-548as-3p, mir-8078 in pc14 can induce cellular invasion by nf-kb. as a conclusion, our investigation indicate that nf-kb can induce nsclc invasion via mir-548a-3p, mir-548as-3p and mir-8078. (this study is supported by tub _ itak grand number 112s636). to understand of the molecular mechanisms of cellular invasion is very important for fight against cancer mechanisms and first step of invasion is emt. cells change phenotypical and genotypical in emt progress. in this study, we focussed on the explore molecular mechanism of tnf-alpha induced cellular invasion of nsclc. we use western blot, qrt pcr and mirna transfect methods for this purpose. we find that tnf-alfa treatment reduce the expression of pten and induce e cadherin expression in a549 cells. when we inhibit nf-kb activity by p65 targeted sirna tnf-alpha can not reduce pten expression means that tnf-alpha inhibits pten expression through on nf-kb. because tnf/nf-kb pathway change the transcriptional level of mir-548as and pten 3 0 untranslated region has recognition site for mir-548as. therefore; we transfected a549 cells by mir-548as. mir-548as transfection leads to inhibition of pten expression. our results indicate that tnf-alpha mediated activation of nf-kb can inhibit pten expression on induction of emt through on mir-548as. (this study is supported by tubitak grand nummber 112s636). introduction: the corpus luteum (cl) is an ephemeral tissue whose regulated secretion of progesterone is essential for maintenance and/or timely termination of pregnancy in rodents. our previous finding that cl of pregnant rats does not possess fas/ fasl system suggests that this tissue may undergo autophagic, but not apoptotic, cell death during regression. we here investigated the presence of autophagic system in cl and its any implications in rodent pregnancy and parturition. materials and methods: lc3 (-i and -ii) expression in cl was estimated by western blot analysis in comparison with progesterone secretion and luteal mass throughout pregnancy. lc3 was also tested by immunocytochemistry. functional implication of autophagy in this tissue was examined by local treatment with bafilomycin a1 (autophagy and v-atpase inhibitor, 6.23 pg/ 0.1 ml/ovary) on day 19 of pregnancy. reproductive, biochemical, and morphological outcomes were evaluated. results: while the expression of cytosol-associated lc3-i was not significantly altered throughout pregnancy, that of autophagosome-associated lc3-ii increased significantly from day 15, showed a peak on day 21, and decreased on day 23 (day of normal parturition). lc3-ii / i ratio had positive correlations with steroidogenic activity and cell size. immunoreactive lc3 was found to be present in the cytosol of steroidogenic cells and showed marked aggregation in cells on day 21. in vivo treatment with bafilomycin a1 resulted in unaltered delivery, but in significant reductions in steroidogenic cell size and neutrophil infiltration compared to vehicle-treated control groups. discussion and conclusion: the ratio of lc3-ii / i in cl was markedly up-regulated in the late phase of pregnancy. manifestation of this autophagy parameter was temporally matched with further structural and functional activation of cl. autophagy may contribute to activation, but not regression, of rodent cl and thus their female reproduction. apoptotic and necrotic effects of low dose bisphenol a in shsy5y neuroblastoma cells b. ayazg€ ok, t. t€ uyl€ u k€ uc ߀ ukkilinc ß faculty of pharmacy, hacettepe university, ankara, turkey bisphenol a (bpa) is a commonly used chemical in industry to make plastics. 'low-dose' term has been expressed for the first time in studies with bpa in 2001. the value of low dose bpa was received as <1lm. in this study, matrix metalloproteinases (mmps) together with their tissue inhibitors (timps), involved in tissue remodeling after i-131 therapy, have been examined in 51 patients (8m/46f) with ptc and 38 (3m/38f) with ptc+ht. peripheral blood samples were collected just before and, subsequently, at 4 days after i-131 administration (3.7 gbq). ptc+ht patients had positive titers of anti-thyroglobulin autoantibodies (tgab). the serum levels of tgab, mmp-2, mmp-9, timp-1 and timp-2 were measured by elisa. there were no significant changes in serum concentrations of mmp-2, timp-2 and mmp-2/timp-2 ratio after i-131 in the two groups. in ptc patients, i-131 administration resulted in an increase with 26% in timp-1 level (p = 0.005) and a reduction with 44% in mmp-9/timp-1 ratio (p = 0.003). in ptc+ht patients it has been observed an increase with 18% in tgab level (p = 0.001), 5% in mmp-9/timp-1 ratio (p = 0.003) and unchanged timp-1 serum concentration. tgab titers were positively correlated with mmp-9/timp-1 ratio (r = 0.51, p < 0.001). our data suggest that radioiodine therapy for ptc patients, but not for ptc+ht, modulates the balance of mmp-9/timp-1 for anti-invasion and anti-migration by augmenting timp-1. in criminal cases, the determination of the time of death of the bodies step in the analysis of events is making a big contribution. today, forensic and molecular methods utilized time of death rather than provide clear information offers potential death time interval. principally, 'time of death' is a term that should be avoided. in forensic science, the postmortem 'interval' is the term to be considered. nowadays, there is no precise molecular method that can be used alone in the determination of pmi. aim of this study is to analyze the usability of ecm components, adamts1,5 and 9 and mmp1,2 and 9 to determine pmi. for this purpose, with iliopsoas muscle tissues provided by ethical board of the ankara institute of forensic medicine, 21 cases have been included in this study, divided into 3 groups according to their pmis: '0-12 h', '12-18 h' and '18-27 h'. from these tissues, western blotting technique is used to analyse the expression of adamts1,5 and 9 and mmp1,2 and 9. it is determined that when pmi increases; adamts-1 and 9 amounts decrease. on the other hand adamts-5 amounts are examined to increased related to the interval., especially on the '12-18 h' dataset. additionally, considering metalloprotease levels, mmp-2 and 9 amounts decrease as pmi increases. unlike mmp-2 and 9; mmp-1 levels increase proportional to the interval, especially on the '18-27 h' dataset. these results are the first data on pmi determination from iliopsoas muscle tissue. in this study, it is suggested that adamts-5 and mmp-1, proteases responsible for ecm degradation, can be used to determine pmi as markers. here we present the first observations of postmortem variation of mmp and adamts activities in skeletal muscle. in recently, popular mmps and adamts s pathways of the relationship between time-dependent changes to investigate the post mortem time interval determination to shed light on the future. the role of functional polymorphisms of matrix metalloproteinases 2 and 9 in spontaneous abortion samples capillaries, which are responsible for maintenance of implantation and placental nutrition, have major effects on mechanisms underlying abortion. matrix metalloproteinases (mmps) function in various cellular pathways. they play a role in patholohical conditions, metastasis; as well as normal physiological functions like tissue and bone regeneration, physiologic function of uterus, ovulation, embryogenesis and embryo implantation. mmp2 and mmp9 (gelatinases) have key roles at organisation of extracellular matrix and affect endometrial implantation. previous studies have shown that mmp2 -1306c>t and -735c>t polymorphisms cause loss of gene function, and mmp9 -1562c>t polymorphism causes a decrease in gene expression, and also gene expression levels are lower in abortion specimens, compared to control specimens. the goal of this study was to investigate the potential effects of functional mmp2 -735c>t and -1306c>t polymorphisms, and mmp9 -1562c>t polymorphism on etiology of abortion. restriction fragment length polymorphism (rflp) method was used to analyze the polymorphisms those evaluated in the study. study group consisted of samples collected from 80 spontaneous abortion specimens, and control group consisted of peripheral blood blood samples collected from 100 healthy subjects. the risk of abortion was 2.2-fold higher in patients with heterozygous -1306c>t polymorphism (p = 0.043). combined genotype analysis showed that the risk of abortion was 3.7-fold higher for patients with normal -735c>t polymorphism, and heterozygous -1306c>t polymorphism (p = 0.021). functional polymorphisms of mmp2 and mmp9 may have a role in etiology of abortion. p-02.07.5-011 changes in the specific extracellular matrix proteins and expression of adamts proteinases and effects of hypoxia in the adriamycin-induced renal fibrosis model renal fibrosis is a common cause of renal dysfunction with chronic kidney diseases. this process is characterized by excessive production of extracellular matrix (ecm) or inhibition of ecm degradation. adamts proteinases, which are widely presented in mammals, have very critical roles in ecm remodeling. we aimed to study the role of adamts proteinases in the pathogenesis of renal fibrosis and the effets of hypoxia by studying adamts expressions in rat kidney after adriamycin (adr) administration. adr was administered intravenously in consequtive two doses (2.5 and 4 mg/kg) to the rats. in addition to control and adr groups, another rats were assigned into three groups as sham, 15 min and 45 min ischemia-reperfusion. after 2 months following the first dose, the expression of adamtss were determined in the renal tissues using western blot analysis. additionally, renal nephropathy and fibrosis were assessed pathological and immuno-histochemical staining methods. in the adr group rats developed renal dysfuntion and tissue damage in favor of renal fibrosis pathologically. it is observed that occurred remarkable changes in the expression of adamtss. it is showed that hypoxia and hypoxia time enhanced tubulointerstitial fibrosis in the rat kidney tissues. expression differences were absorved also in the hypoxia groups, and especially the expression of adamts-1 and -15 genes were showed an increase in various rates. the restricted and different expression pattern of adamts protein profiles in the adr-induced renal fibrosis suggest that adamts family members are related with development and progression of fibrosis. moreover, our findings raise the possibility that the hypoxia promotes renal fibrogenesis. the monitoring of adamts proteinases might contribute to the early diagnosis of renal fibrosis and chronic kidney disease. adams (a disintegrin and metalloproteas) are transmembrane proteins that contain a pro-domain, a metalloprotease, a disintegrin, a cysteine-rich, an epidermal-growth factor like and a transmembrane domain, as well as a c-terminal cytoplasmic tail. they are involved in cell adhesion and they have protease activities. previous studies showed that some adam proteins act in a highly diverse set of biological processes, including fertilization, neurogenesis, myogenesis, embryonic tgf-a release and the inflammatory response. although there are more researches about adam proteins, still the function of all adam proteins remain unclear. we aimed to investigate the potential link of infertilty with adam9, -10, and -17. in this study twenty four patients were included. the patients were classified as normozoospermia (ns; n = 8), oligozoospermia (os; n = 8), azoospermia (as; n = 8) groups. adam9, -10 and -17 protein levels in infertility indviduals were analysed by western blot. adam9 protein level was 1.7 fold lower in the os and as groups than in the ns group. adam10 protein level was 1.36 fold higher in the as group than in the ns group. adam17 protein level was 2 fold lower in the as group accourding to ns group. we observed no change between protein level of adam10 and adam17 of os and ns groups . in conclusion, adam proteins may have a potential role in male infertility. our study is a preliminary and first study on this issue. keywords: adam, infertility. the role of tissue metalloproteinase inhibitors thymus chemokine and thrombospondin-1 on prognosis of crimean-congo hemorrhagic fever s. bakir, m. bakir, s. ersan, a. engin cumhuriyet university, sivas, turkey crimean-congo hemorrhagic fever (cchf) is a disease which is caused by an arbovirus carried by ticks and characterized by the sudden onset of high fever, severe headache, dizziness, back and abdominal pain. cchf pathogenesis is still not resolved today to fully open. therefore, in this study, to determine the level of tck-1, timp-1 and tsp serum samples obtained from cchf patients and the control group is intended to be examined for the pathogenesis and prognosis of the disease. the study sample was created 45 patients with diagnosis of cchf. 45 healthy volunteers were chosen control group matched for gender and similar to in terms of age. tsp, tpc-1 and timp-1 levels of patients and a control group were analyzed using the human elisa kits. serum timp-1 tck-1 and tsp levels in cchf patients were measured significantly higher than the in the control group. cchf pathogenesis of today still have not provided fully open. therefore, it reveals the importance of this work. in our study, presence of high timp-1, tck-1 and tsp levels indicate important direction for pathogenesis and prognosis of cchf disease. p-02.07.5-015 activation of calpain 1 and protein kinase ca promotes a constitutive expression and release of matrix metalloproteinase 9 in peripheral blood mononuclear cells from cystic fibrosis patients matrix metalloproteinase 9 (mmp9) is physiologically involved in remodeling the extracellular matrix components but its abnormal release has been observed in several human pathologies, including cystic fibrosis. we have studied if the alteration in intracellular ca 2+ homeostasis, observed in peripheral blood mononuclear cells (pbmcs), isolated from cystic fibrosis (cf) patients homozygous for deletion of phenylalanine 508 in gene of cystic fibrosis transmembrane conductance regulator (f508del-cftr), could be involved in the abnormal presence of mmp9 in the extracellular fluids of cf patients. pbmcs were isolated from 23 healthy donors and 26 cf patients homozygous for f508del-cftr. mmp9 levels were evaluated following 2 h of cell incubation. our investigations show that all cf pbmcs analyzed constitutively express and release high levels of mmp9; conversely, in pbmcs from healthy donors, expression and secretion of mmp9 are undetectable but both events can be evoked, after 12 h of cell culture, by a possible paracrine stimulation. we have demonstrated that in cf and 12 h-cultured control pbmcs mmp9 secretion is prevented by chelation of intracellular ca 2+ and mediated by the concomitant activation of calpain and protein kinase ca (pkca) and also that mmp9 expression is mediated by the sequential activation of pkc and extracellular signal-regulated protein kinases 1 and 2 (erk1/2). moreover, the rescue of active f508del-cftr reduces the extent of mmp9 secretion, correlating the channel defect to the [ca 2+ ] i dysregulation of cf pbmcs. our results indicate that the high level of intracellular ca 2+ concentration in cf pbmcs, promoting the aberrant activation of both calpain and pkca, induces a constitutive release of mmp9. these data characterize new alterations in mononuclear leukocytes of cf patients that may be of primary importance in the progression of the disease and indicate that pbmcs may contribute to the accumulation of mmp9 in fluids of cf patients. p-02.07.5-016 aebp1/aclp is upregulated in differentiation, injury repair and fibrotic degeneration of skeletal muscle c. € ozdemir 1,2 , u. akpulat 1 , i. onbasilar 3 , c ß . kocaefe 1 1 department of medical biology, faculty of medicine, hacettepe university, ankara, turkey, 2 department of stem cell, institute of health, hacettepe university, ankara, turkey, 3 laboratory animal breeding and research unit, faculty of medicine, hacettepe university, ankara, turkey aebp1/aclp is an ambiguous gene with several attributed functions and cellular events, adipogenic differentiation, cell adhesion, pattern development and fibrosis are the well-understood. aebp1 is the short isoform that acts as a transcriptional repressor by targeting the ap2 promoter and aclp, which is the long isoform that harbors a leader sequence that directs the peptide to the extracellular compartment. the latter is known to be associated with development of the connective tissue, injury repair and fibrosis in certain pathological conditions. aebp1/aclp displays a moderate expression in skeletal muscle where the role is not known. we have investigated the spatial and temporal expression of aebp1/aclp in defined models of skeletal muscle differentiation, injury repair and fibrosis. aebp1/aclp expression is present in steady state dividing myoblasts. this basal expression is upregulated 4 folds upon the induction of differentiation in c2c12 cells. considering that differentiation and post-natal injury repair share several common aspects, we also investigated the expression of aebp1/aclp in acute injury-repair model. in the course of cardiotoxin induced injury, aebp1/aclp expression peaks up to 5 folds in the 6th day of regeneration. this time point concomitantly corresponds to tissue remodelling. since aebp1/aclp is also known to be associated with fibrotic events in chronic pathological conditions, we also have investigated its expression in tenotomy induced skeletal muscle degeneration which mimics endomysial and perimysial fibrosis. aebp1/aclp expression is upregulated up to 10 folds in early time-point samples. these results depict a novel role for aebp1/aclp in extracellular remodeling of the skeletal muscle during injury repair as well as fibrotic degeneration. the source of this expression might come from fibroadipogenic precursers which reside in endomisial area of muscle. our current efforts are focused on presenting of this endomysial expression. the mprbp gene from b. pumilus 3-19 encoding a novel secreted metalloproteinase was identified. based on the primary structure the enzyme has been classified as metzincin metalloproteinase that combines the features of two families: astacin and adamalysin. representatives of the adamalysin family previously have not been described for bacilli. the aim of the work was to elucidate the mechanisms of the gene expression regulation of a new bacillus pumilus 3-19 extracellular metalloendopeptidase. promoter region analysis revealed the presence of potential binding sites for transcription factors spo0a (sporulation) and degu (biodegradation). study of mprbp expression in strain defective in regulatory proteins degs and degu shows that the productivity of metalloproteinase biosynthesis decline in average 60% compared with the strain with a complete degs-degu system. we also studied mprbp expression in strains with a mutation in the gene degu, leading to stabilization of degu~p protein. it is known, that this mutation leads to a multiple increase in the gene expression level, positively regulated by degs-degu system. our data shows a 10-fold increase in metalloproteinase productivity in the recombinant strain. thus, deg-system participates in control of the proteinase synthesis but not only in the regulation of mprbp gene expression. the mprbp expression in the strain deficient in regulatory protein spo0a remained at the level with expression in the strain with the complete spo0a. a similar pattern we observed in the study of mprbp gene expression in strains defective in other spore-specific regulatory proteins (spo0b, spo0f, spo0k, spo0j, sigf, sigh, sigk). these data indicate that mprbp gene expression is free of spo-regulatory proteins. on this basis, we concluded that the expression of metalloproteinase gene is not correlated with the sporulation. p-02.07.5-019 paricalcitol attenuate activity and expression of matrix metalloproteinases in a rat model of renal ischemia-reperfusion injury matrix metalloproteinases (mmps) are endopeptidases involved in the degradation of extracellular matrix. they have been postulated to have a role in the pathogenesis of ischemia-reperfusion injury (iri). in the present study, we investigated the effect of paricalcitol, a synthetic vitamin d analog, on mmps in renal iri. 21 wistar albino rats were divided into three groups: sham operated, ischemia-reperfusion, and paricalcitol-pretreated. iri model was induced by bilateral clamping of renal arteries for 45 min followed by 24 h of reperfusion. the analysis of serum creatinine levels and activities/expressions of mmp-2 and -9 were performed after 24 h of iri. the effects of paricalcitol on activities and expressions of mmp-2 and mmp-9 levels were investigated by gelatin zymography and immunohistochemistry, respectively. the pathological examinations were performed to score tubular damage by light microscopy. creatinine levels increased significantly in the iri group. rats in the paricalcitolpretreated group showed significant decrease in expressions and activities of mmp-2 and mmp-9 during iri. moreover, pathological examinations displayed significantly lower score of tubular damage in paricalcitol-pretreated group. in conclusion, paricalcitol attenuated iri by downregulating the expressions and activities of mmp-2 and -9. p-02.07.5-020 the changes of matrix metalloproteinase 2, 9 activity and hyaluronic acid level in rat's heart and serum under cadmium influence o. fomenko 1 , o. shaulska 1 , y. kot 2 , g. ushakova 3 , a. shevtsova 1 1 dnipropetrovsk medical academy, dnipropetrovsk, ukraine, 2 kharkiv national university, kharkiv, ukraine, 3 dnipropetrovsk national university, dnipropetrovsk, ukraine the changes in the molecular mechanisms of the extracellular matrix degradation under toxic factors are not well known. the main goal of work was the investigation of the mmp2 and mmp9 activity and hyaluronic acid level in the heart and blood serum under cadmium influence at different doses. the 18 wister rats divided to 3 groups were used for the experiment. cdcl 2 x2.5h 2 o in doses 0.1 lg/kg and 1 lg/kg was given to rats intragastrically in drinking water during 36 days. the rats were decapitated under isoflurane anesthesia according to ethical rules; the heart was quickly removed. the relative activity [in arbitrary units (au)] of pro-and active forms of mmp9 and mmp2, total protein (tp) and hyaluronic acid levels were calculated. it was shown that low doses of exogenous cadmium (0.1 lg/ kg) lead to reduced activity of pro-and active forms of mmp9 in myocardium (7.3 ae 0.6 au/mgtp and 7.1 ae 0.6 au/mgtp compare to the 9.67 ae 0.4 au/mgtp and 9.7 ae 0.5 au/mgtp in the control rats accordingly) and in serum (0.95 ae 0.2 au/mgtp and 0.35 ae 0.05 au/mgtp compare to the 1.54 ae 0.05 au/mgtp and 1.49 ae 0.05 au/mgtp in the control rats accordingly), but pro-mmp2 activity in heart was increased (14.5 ae 1.6 au/mgtp compare to the 9.8 ae 0.6 au/mgtp in the control rats); level of ha was decreased in both tissues (0.69 ae 0.16 lg/ml and 3.63 ae 0.3 lg/ml compare to the 1.0 ae 0.13 lg/ml and 3.91 ae 0.3 lg/ml in the control rats accordingly). high doses of cadmium (1 lg/kg) caused a reliable increase of both gelatinase activity in the myocardium: mmp2 increased from 9.65 ae 0.4 au/mgtp to 14.1 ae 0.8 au/mgtp, prommp9to 12.6 ae 1.5 au/mgtp, mmp9to 15.4 ae 1.6 au/mgtp. ha level was increased in serum (4.28 ae 0.1 lg/ml) and decreased in heart (0.49 ae 0.09 lg/ml). the results indicate the dose-dependent and tissue-specific effect of cadmium on mmp-depended protein degradation and level of hyaluronic acid. a disintegrin-like and metalloproteinase domain with thrompospondin-1 repeats (adamts) are a large family of proteoglycanase that show proteolytic activity towards proteoglycans like aggrecan, brevican, neurocan, and versican. interleukin-33 (il-33) is an il-1 cytokine family member that uniquely plays a role as a cytokine and nuclear factor. it is released by necrotic epithelial cells and activated innate immune cells as an alarming danger signal. adamts and il-33 implicated in brain cancer pathogenesis. we aimed to seek the amount of adamts15 in u118 proteolytic enzymes are able to speed up wound healing by removal of the necrotic tissues and fibrin. several investigations have reported that proteases damage also the microbial biofilms formed by opportunistic bacteria including staphylococci on surfaces of chronic and acute dermal wounds. therefore, proteases are seemingly perspective enzymes for biofilm eradication by hydrolysis of both matrix proteins and adhesins, proteins providing cells attachment onto solid surface and other bacteria, as well as by the cleavage of signalling peptides of intercellular communication of gram-positive bacteria. here we report that ficin, a plant protease, efficiently degrades the structural components of biofilm matrix formed by s. aureus and s. epidermidis at concentrations of 10 lg/ml while trypsin and chimotrypsin are used as 1-2 mg/ml solution. the spatial structure of the biofilm was analyzed by atomic force microscopy. after ficin treatment, the biofilm structure became porous, with reduced viscosity. the congo red staining of the treated biofilms confirmed the hydrolysis of the protein component of the matrix. moreover, the biofilm treatment with ficin increased the antimicrobial efficiency of ciprofloxacin against biofilm-embedded cells of s. aureus and s. epidermidis. while 24 h antibiotic treatment did not lead to the increase of dead cells of neither s. aureus nor s. epidermidis embedded into the biofilm matrix, in the presence of ficin the fraction of viable cells decreased significantly. accordingly, soluble ficin appears beneficial for outer wound treatment biofilm eradication and reduces the reinfection risk. the wound-healing activity of ficin requires further investigations. this work was supported by the russian science foundation (project no 15-14-00046). resveratrol (resv) is an antioxidant that belongs to the group of plant compounds, called polyphenols. resv is defined as an antimicrobial substance that is produced by several plants (red grape skin, peanuts and berries) to protect them from rough environments like excessive ultraviolet light, infections and climate changes. as an antioxidant, this polyphenol protects the body against cardio-vascular and cancer diseases. besides, resv has anti-inflammatory, neuro-protective, anti-diabetic and other pharmacological effects. although the positive pleiotropic effects of this polyphenol are well documented, there is a huge need to understand its influence on the biophysical properties of lipid bilayer. in the present work, the interaction of resv with membranes composed of palmitoyl-docosahexaenoyl phosphatidylcholine (pdpc) or palmitoyl-oleoyl phosphatidylcholine (popc), sphingomyelin (sm) and cholesterol (ch) was investigated by means of fluorescence spectroscopy. generalized polarization of the fluorescent probe laurdan (gp) as a function of temperature was used to probe the changes in the fluidity of lipid bilayer induced by different resv concentration. the obtained results showed decreased lipid ordering from 50 to 100 lmol resv and opposite effect from 200 to 500 lmol in pdpc/sm/ch mixtures as compared to the control without resv. the interaction of resv with popc/sm/ch mixtures caused only an increase in the lipid ordering as a function of resv amount. popc and pdpc have the same head group but different fatty acid chains at sn-2. since resv changes the physicochemical properties of lipid bilayer by different ways one might suggest that the interaction of polyphenol with the membrane depends on the level of fatty acid unsaturation. this specific effect of resv on lipid organization could be related to its unique properties to prevent the cell from oxidative stress. neurodegeneration is the umbrella term for the deseases including progressive loss of structure or function up to death of neurons. beta-amyliod peptide is proteolytic fragment of the amyloid protein. the spontaneously formation of selective, voltage-dependent, ion-permeable channels in the lipid bilayers was reported as one of amyliod peptide toxicity mechanisms. the aim of our study was the investigation of the influence of the flavonoids (phloretin, phlorizin, quercetin and genistein) on the membrane activity of amyliod peptides. virtually solvent-free bilayer lipid membranes were prepared from mixtures of phospholipids in 0.1 m kcl (ph 7.4) using monolayer-opposition technique. using spectrofluorimetry we estimated prepared from phospholipids by extrusion the liposomal membrane permeability for calcein. we found that the addition of phloretin into membrane bathing solution led to an signicant increase in the channel forming activity of fragments 25-35 of amyloid peptide, fragment 25-35 of [gly35]-amyloid peptide and 106-126 of human prion protein. addition of other flavonoids did not cause any changes in the steady-state amyloid-induced current. it was found that the effect was caused by electrostatic interaction with the peptide. we found that time course of amyloid induced leakage calcein from liposome's is characterized by two components: the fast one is related to sorption of b-amyloid peptide on the membrane and the slow one is related to the oligomerization of the peptides on the surface of the lipid bilayer. addition of the phloretin simultaneously with b-amyloid peptide to the suspension of liposomes caused significant reduction in time parameters characterizing fast and slow components. from this results we can proposed that phloretin compensates the positive charge of the b-amyloid peptides and leads to the changes in their oligomerization status. the study was supported in part by rfs (14-14-00565) and sp-69.2015.4. ferritin nanocarriers: a focus on a metal-based drug encapsulated inside the protein cavity s. ciambellotti 1 , c. bernacchioni 2 , f. scaletti 3 , p. turano 1 1 cerm (magnetic resonance centre), florence, italy, 2 department of biochemical sciences, university of florence, florence, italy, 3 chemistry department 'ugo schiff', florence, italy ferritin is one of the main player involved in the iron metabolism. the biological role of ferritin is the storage of iron atoms inside the cavity preventing the uncontrolled accumulation of toxic species inside cells. ferritins are polymers constituted by 24 subunits that self-assemble giving rise to an almost spherical nanocage. in vertebrates, ferritins are formed by two distinct subunits, the heavy chain (h), with oxidoreductase activity, and the light chain (l) without catalytic activity. ferritins are promising nanocarriers for the delivery of contrast agents for diagnosis and of drugs for therapeutic purpose. their endogenous origin and the possibility to stabilize and protect the enclosed cargo inside the cavity, make ferritin a biocompatible vehicle. moreover, there are specific receptors on cells that recognize and incorporate ferritin by endocytosis, prospecting a kind of targeted-delivery. following the increasing interest in nanotechnology, we studied the interaction between different isoforms of ferritin with an antimetastatic drug, called nami-a, which is the first ruthenium derived anti-cancer drug to have entered clinical evaluation. this molecule is a metal-based prodrug that can release the metal ion ligands. here, we describe nami-a hydrolysis in the presence of recombinant homopolymers of ferritin followed spectrophotometrically. thanks to characteristic time dependent changes of spectral profile in the uv-visible region, we could detect differences in the hydrolysis process. the formation of a ru-adduct with hferritin was established by uv-visible and circular dichroism spectroscopies, as well as by kinetics measurements that showed inhibition of the ferroxidase activity of h-ferritin. crystallization trials are in progress to identify the binding site of ru by solving the x-ray structure of the complex. finally, we planned to test the cytotoxicity of ferritins pre-incubated with nami-a, using different cancer cell lines. a. cort 1 , t. ozben 2 , a. sansone 3 , s. barata-vallejo 4 , c. chatgilialoglu 5 , c. ferreri 3 1 sanko university, gaziantep, turkey, 2 akdeniz university, antalya, turkey, 3 cnr, bologna, italy, 4 universidad de buenos aires, buenos aires, argentina, 5 national center for scientific research 'demokritos', athens, greece liposomes as biomimetic models of cell membranes were used for examining some novel aspects of drug-metal induced reactivity with unsaturated lipids under oxidative conditions. the chemical basis of cis to trans transformation was ascertained by liposome experiments, using bleomycin-iron complexes in the presence of thiol as a reducing agent that by incubation at 37°c gave rise to the thiyl radical-catalysed double bond isomerisation of membrane phospholipids. the effect of oxygen and reagent concentrations on the reaction outcome was studied. as a chemical biology model for antitumoral strategies, liposomes highlight the role of cell membranes, which are not spectators but important targets of the drug effect, with synergic roles for chemotherapeutic effects. indeed, fatty acid recruitment and membrane formation are attracting a lot of interest in cancer, and in this context the loss of the natural cis geometry and the oxidationinduced lipid remodelling are worthy of deeper studies in antitumoral strategies. furthermore, the interaction between drugs and lipids can be suggestive of novel aspects of chemical reactivity for liposome carriers when circulating in vivo. gpr17 is a g-protein coupled receptor (gpcr), expressed in cells of brain, heart and kidney. it is related to the leukotriene and purinergic subfamilies of the rhodopsin-like class a gpcrs. gpr17 plays controversial role in the brain and spinal cord cells recovery after injuries. it is assumed that gpr17 is one of the cell death regulators immediately following an injury but at later stages it also takes part in tissue regeneration. there are also data implying some role of gpr17 in glucose homeostasis. drugs targeting gpr17 may help with treatments of multiple sclerosis and ischemia. the damage of rat's brain in artificially induced ischemia disease decreased after gpr17 inhibition. in addition, gpr17 takes part in myelin sheath formation, the lack of which is known to be the reason of multiple sclerosis. to better understand functional role of gpr17 and enable design of more efficient ligands we initiated structural studies of this receptor. to improve receptor stability and facilitate crystallization we created a series of chimeric constructs using different fusion partnerssmall soluble proteins inserted into the native amino acid sequence. preliminary experiments were carried out to evaluate the influence of different ligands on the receptor stability and cell surface expression in insect sf9 cells. this work was supported by russian federal target sterols are significant for the structural and dynamical features of cell membranes. among them, cholesterol is the major sterol in mammalian cell membranes whereas stigmasterol is the predominant sterol in plant membranes. stigmasterol varies structurally from cholesterol in having both an ethyl group at carbon 24 and an additional trans double bond between carbons 22 and 23. dimyristoylphosphatidylcholine (dmpc) is a widely studied synthetic lipid, which has a neutral (zwitterionic) pc headgroup and two symmetrical 14-carbon fatty acyl chains. the studies on the interactions of cholesterol and stigmasterol with dmpc membranes at molecular level are very limited. in the present study, a calorimetric comparison of the effects of the animal sterol cholesterol and the plant sterol stigmasterol on zwitterionic dimyristoylphosphatidylcholine (dmpc) multilamellar vesicles (mlvs) was investigated for the first time by using differential scanning calorimetry (dsc). our dsc studies indicate that with the inclusion of increasing cholesterol and stigmasterol concentrations from 5 to 40 mol% into pure dmpc mlvs, the pretransition disappears, the main phase transition shifts to lower temperatures and then disappears at cholesterol and stigmasterol concentration above 25 mol%. the main phase transition enthalpy (dh) is progressively reduced whereas full width at half maximum (dt ½ ) gradually increases with increasing cholesterol and stigmasterol concentrations. moreover, the main phase transition peak consists of overlapping sharp and broad components, indicating the hydrocarbon chain melting of sterol-poor and sterol-rich dmpc domains, respectively. in conclusion, this study shows clearly that both cholesterol and stigmasterol interact effectively with dmpc membranes and cause changes in their structural and functional properties. p-03.03.3-007 trh receptor mobility in the plasma membrane is affected by its interaction with its cognate signaling molecules and by cholesterol depletion r. moravcova, b. melkes, j. novotny department of physiology, faculty of science, charles university in prague, prague, czech republic g protein-coupled receptors (gpcrs) play a fundamental role in transferring information from extracellular environment to the cell interior. some gpcrs are supposed to form signaling complexes with their cognate g proteins and possibly other accessory proteins, which may facilitate the activation of g proteins and thus accelerate signal transmission. here we investigated the role of some components of thyrotropin-releasing hormone (trh) receptor signaling in hek293 cells stably expressing yfp-tagged trh receptor using fluorescence recovery after photobleaching (frap). we observed significant changes in values of the diffusion coefficients if expression of b 2 -arrestin or gb 2 subunit were suppressed by sirna. results of our frap experiments indicated significant difference between control and trh-treated cells as the diffusion coefficient markedly decreased after agonist stimulation. on the other hand, the same decline of the diffusion coefficient value was found after silencing with sirna and subsequent treatment with trh for most of the screened proteins. treatment of cells with 10 à5 m trh led to fast internalization of trh receptor, which was revealed by real-time confocal microscopy. it is known that cholesterol is an essential component of the cell membranes and it exerts modulatory effects on the functioning of various membrane proteins. disruption of plasma membrane integrity by cholesterol depletion using b-cyclodextrin significantly reduced the apparent diffusion coefficient values. interestingly, addition of trh to cells treated with b-cyclodextrin did not further reduced trh receptor mobility. it can be concluded that stimulation with agonist and/or sirna silencing of some components of the trh receptor signaling cascade significantly affects the mobility of trh receptor in the plasma membrane. p-03.03.3-008 l-opioid receptor mobility in the plasma membrane is diversely affected by biased ligands b. melkes, l. hejnova, j. novotny opioid receptors belong to the large family of g protein-coupled receptors (gpcrs), which are currently considered among the most important targets for pharmacological manipulations. during the past few years, a great deal of attention has been devoted to biased agonism. this phenomenon reflects the ability of different ligands to selectively affect the functioning of some gpcrs so they can display marked differences in their efficacies for g protein-or b-arrestin-mediated signaling. here we decided to investigate the effect of different agonists of the l-opioid receptor (l-or) on the lateral mobility of this receptor in the plasma membrane of hek293 cells which were stably transfected with l-or tagged with yellow fluorescent protein (l-or-yfp). it has been found previously that damgo stimulates g-protein-dependent signaling, endomorphine 2 stimulates arrestin-dependent signaling and morphine does not show any significant bias towards these two signaling pathways. in our experiments, we used the fluorescence recovery after photobleaching (frap) method to estimate the diffusion coefficients of l-or-yfp in the resting state and after addition of the above mentioned specific agonists. we observed that addition of damgo increased the value of the diffusion coefficient and addition of endomorphin 2 decreased the value of diffusion coefficient of l-or-yfp. addition of morphine or the l-or antagonist naloxone did not change the value of the diffusion coefficient compared to the resting state. these results indicate that different biased agonists of l-or may differently affect the mobility of this receptor in the plasma membrane. these findings provide new insights into the dynamics of l-or in the plasma membrane and contribute to better understanding of the mechanism of biased agonism at gpcrs, which is of central importance for receptor homeostasis and fine regulation of receptor activity. color tuning and adding potassium selectivity to a light-driven sodium pump k. kovalev 1,2 , v. polovinkin 1,2,3 , v. shevchenko 1,2 , v. gordeliy 1,2,3 1 moscow institute of physics and technology, dolgoprudniy, russia, 2 research centre j€ ulich, j€ ulich, germany, 3 institut de biologie structurale, universit e grenoble alpes, cnrs, and cea, grenoble, france recently a light-driven sodium pump has been discovered, characterized and tested as an inhibitory optogenetic tool. sodium pumping rhodopsin from dokdonia eikasta kr2 has an absorption maximum at 523 nm at ph 7.5 and to create more redshifted variants we analyzed available structures of the kr2 (pdb codes: 4xtl, 4xtn) and did the rational mutagenesis of residues in the retinal proximity region (i.e. m149, g153 and s254). the mutants of kr2 under investigation were: m149a, g153v, m149a/g153v, s254a, s254f, s254g, s254l, s254m, s254n, s254t, s254v, s254y. the protein mutants were expressed in escherichia coli c41 strain, expression was induced by the addition of 1 mm isopropyl b-d-1-thiogalactopyranoside. the cells were then washed trice with unbuffered 100 mm nacl or kcl solution. finally, the ph changes in cell suspensions (od600 = 8.0) were monitored. ph changes upon the addition of 30 lm of protonophore carbonylcyanide m-chlorophenylhydrazone were also measured. the following mutants completely abolished the protein function and were not used for further characterization: s254f, s254l, s254m, s254n, s254v. the remaining mutants have shown sodium pumping activity and s254a mutant has gained an additional potassium pumping activity. all functionally active mutants were purified using ni-affinity chromatography and the absorption spectra were collected for them at ph 7.5 (50 mm na/na-pi, 100 mm nacl). m149a mutant absorption maximum is blue-shifted to 519 nm. g153v and m149a/g153vblue-shift to 470 nm. s254a, a potassium pumping variant,red-shift to 545 nm. s254g, s254yred-shift to 545 nm. s254tno change in absorption maximum position. based on structural analysis of kr2 we discovered another potassium pumping variant and provided the variants with absorption maximum blue-shift up to 53 nm and red-shift up to 22 nm. human endothelin receptors belong to the g-protein coupled receptors (gpcrs) superfamily. this class is pharmacologically important, with more than 40% drugs targeting it. the human endothelin system, which includes endothelin receptors types a and b (etb and eta), plays a highly important role in the blood pressure regulation. endothelium cells produce peptides, known as endothelins 1-4, which activate endothelin receptors and launch cascades of reactions that lead to vasoconstriction or vasodilatation depending on the receptor subtype and the tissue. additionally, endothelin receptors take part in such processes as transmission of nerve impulses, development of neural crest, and regulation of acid-base-salt balance in kidneys. in order to stabilize etb receptor for crystallization trials we introduced a compact soluble protein, apocytochrome b564ril (bril), is the third extracellular loop of the receptor. bril is known to be an effective crystallization driver for gpcrs. the engineered protein was expressed using baculovirus system in sf9 insect cells, purified and subject to variety of pre-crystallization assays. localization of the overexpressed protein in insect cells was visualized via the confocal microscopy. thermal stability of the protein in the presence and absence of ligands was measured by the thermal shift assay. finally, the mobility of the receptor in lipidic cubic phase (lcp) at many different conditions was probed by the lcp-frap (fluorescence recovery after photobleaching) assay. these tests showed that the obtained protein is thermally stable, functionally active and diffuses well in lcp at certain conditions, making it a suitable candidate for proceeding to in meso crystallization trials. this work was supported by the russian science foundation (project no. 16-14-10273). mitochondrial oxidative phosphorylation is the key metabolic pathway for the production of atp. mitochondrial respiratory chain (rc) defects are some of the most commonly diagnosed inborn errors of metabolism with a diverse spectrum of clinical phenotypes. the aim of the study is to evaluate the rc enzyme activities and histopathological findings in muscle biopsies of patients with suspected mitochondrial disease. muscle biopsy samples were collected from 48 pediatric patients. the samples were homogenized in seth buffer using a glass/glass homogenizer. the activities of citrate synthase (cs) and rc enzymes (complex i, ii-iii, and iv) were measured in tissue homogenates by kinetic spectrophotometric assays by schimadzu uv spectrophotometer (uv-1800). non-collagen protein was determined by the modified lowry assay. activities of complex (c) i, ii-iii and iv (cox) were normalized by cs. histopathological investigations included h&e, modified gomori trichrome, periodic acid schiff, oil-red-o, nadh, sdh, cox and atpase stains. deficiency of rc complexes was detected in 39 biopsies (81%). c iv deficiency was most common (60%), followed by c i (40%) and c ii-iii (27%). multiple complex deficiency was present in 40% and isolated deficiency was present in 42% of the biopsies (20% c i, 20% c ii-iii, 60% c iv). cs activity was elevated in 44% of the biopsies. decreased c i/cs, c ii-iii/cs and c iv/cs ratio was observed in 44%, 42% and 52%, respectively. comparing with histological data, biochemical analysis revealed additional findings in 50% of biopsies. complex iv deficiency, either isolated or accompanied by other complex deficiencies, is most common in our cohort. rc analysis may reveal additional findings to histopathological results and careful clinical investigation with correlation of clinical, biochemical and histopathological data is mandatory for the challenging diagnosis of mitochondrial disorders in childhood. investigation of adipocytokines, activity of glut and na + /k + -atpase in rats fed glucose, fructose, starch-based sugars objective: all over the world, shows a significant increase in obesity and diabetes. intake of foods that contain fructose, glucose and starch-based sugar is a potential risk for metabolic syndrome. obesity and diabetes are important effects of high fructose corn syrup (hfcs). we aimed to research, activity of na + /k + -atpase in addition to glucose transporter (glut) 2, resistin, adiponectin and other biochemical markers in rats fed glucose, fructose and starch-based sugars. materials and methods: study was performed on rats and 3 groups were included in the study. rats were fed with chows that were given either normal diet for control group (70% carbohydrate, 20% protein and 10% fat), high fructose (70% carbohydrate (87% fructose and 13% starch), 20% protein and 10% fat), or high sucrose (70% carbohydrate (87% sucrose and 13% starch), 20% protein and 10% fat). rats were fed with chows for 8 weeks. in this process, the weight of the rats were followed. at the end of the experiment, blood is taken in all groups. level of hba1c, glucose, resistin and adiponectin were studied. glut2 and na + /k + -atpase activity were studied in the liver tissue. results: a significant increase in adiponectin levels were determined in rats fed both hfcs and sucrose (p < 0.001). a significant decrease in level of na + /k + -atpase activity were determined in rats fed both hfcs and sucrose (p < 0.001). there was no significant differance level of hba1c, glucose, resistin and glut2 in rats fed sucrose or hfcs (p > 0.05). conclusions: fructose-rich diet has an effect on changes in the atpase activity and is a major risk factor for obesity. keywords: adiponectin, fructose, glucose, high-fructose corn syrup, na + /k + -atpase, resistin. p-03.03.3-014 nucleic acid-biomembrane lipid selfassemblies: from primordial soup to novel genome organization model and cellular nonviral nanotherapies f. k. demirsoy 1 , n. eruygur 2 , e. s€ uleymanoglu 3 1 biotechnology central laboratory, biotechnology institute, ankara university, ankara, turkey, 2 department of pharmacognosy, faculty of pharmacy, cumhuriyet university, sivas, turkey, 3 department of pharmaceutical chemistry, faculty of pharmacy, gazi university, ankara, turkey turkey nucleic acid-cell membrane complexes has attracted research interest as models in gene regulation, cell cycle and division, as biosensors designs, as well as in molecular evolution. zwitterionic phospholipids, complexed with polyribonucleotides by divalent metal cations (mg2+) are considered as genosome vehicles. their formations are studied by spectroscopic, thermodynamic, interfacial and microscopic approaches to build thermodynamic and kinetic models of their structural transitions. dna forms a mg2+-driven ternary complexes with neutral liposomes both at the airwater interfaces and at vesicle surfaces. the described self-assemblies form relevant models for nuclear pore complexes and their further implications in gene expression and functions. such membrane contacts could be considered also in prokaryotic nucleoids important in bacterial segregation, whereas in eukaryotes these complexes can be regarded as focal points for transcription-translationtranslocation processes. the effects of ozone/oxygen mixture on citrate synthase and mitochondrial complex activities of striated muscle tissue of healthy mice we investigated the effects of ozone/oxygen mixture and oxygen on citrate synthase (cs) and succinate dehydrogenase (sdh) activities of striated muscle tissue of healthy mice. thirty-six mice were included the study. firstly muscle samples were taken from all mice's left thigh muscle in under anesthesia (group 0). secondly mice were randomly divided in four groups as: group 1: oxygen was given once at 3 days for 7 days, group 2: ozone/oxygen was given once at 3 days for 7 days, group 3: oxygen was given once at 3 days for 30 days, group 4: ozone/oxygen was given once at 3 days for 30 days. ozone/oxygen mixture and oxygen were given at a dose of 1 mg/ kg groups (1) (2) (3) (4) . after treatment animals were sacrificed, and muscle samples were taken and stored in à80°c for until the analyses. muscle tissues were homogenized in 0.05 m tris-hci and 0.15 m kci. cs and sdh activities were measured with spectrophotometer. cs and sdh activities were expressed as lmol/min/g tissue. cs and sdh activity results were given as mean ae sd. cs activity has been found in group 0 (37.8 ae 11.7), group 1 (46.9 ae 10.8), group 2 (34.7 ae 6.8), group 3 (32.9 ae 7.5) and group 4 (33.9 ae 8.5). sdh activity has been found in group 0 (2.50 ae 1.03), group 1 (2.37 ae 0.77), group 2 (2.52 ae 1.45), group 3 (2.24 ae 1.06) and group 4 (2.59 ae 1.25). there was no statistically significant difference among all groups in terms of cs (p > 0.130) and sdh activities (p > 0.997). there was no difference between all groups in terms of inflammation, muscle fiber size, regeneration or necrosis. vascular structures, connective tissues, lipid and glycogen content of fibers were normal. cytochrome oxidase activity was also normal. ratio of ragged blue fibers of all groups were less than 0.3%, so they were scored as 1. there was no difference among groups for ragged red fiber content. we have not found a significant effect of ozone/oxygen mixture and oxygen on cs and sdh activities of striated muscle tissue of healthy mice. lipidic cubic phases (lcps) consist of bicontinuous lipid bilayers and water channels. lpcs are widely used for membrane proteins crystallization and further determination of their spatial structures by means of x-ray crystallography. this approach was successfully used for studying g-protein coupled receptors structures. usually crystallization initiates by adding the precipitants (buffers with high salt concentrations). here we propose to use photo-switchable analogs of 1-monoolein to change lattice parameter of lpc. we synthetized a number of novel diazo-analogs of 1-monoolein. their structures were confirmed by nmr-spectroscopy and mass-spectrometry. being incorporated in phospholipid vesicles or detergent micelles they subjected to trans-to cis-isomerization under the light exposure at 365 nm. also we characterized the lcp's structures and properties by small-angle x-ray scattering on the rigaku instrument. one of the synthetized compounds, 3-(4-{-[4-(octyloxy) phenyl] diazenyl} phenoxy) propane-1,2-diol (1% mol), was incorporated into the 1-monoolein cubic phase. according to small-angle x-ray scattering data the structure of the monoolein cubic-pn3 m phase with lattice parameter 106.3 angstrem was not affected by insertion of this photo-switchable monoolein's analog. after the light exposure at 365 nm we observed trans-cis-isomerization. in the same time the cubic-pn3 m phase was not changed but the lattice parameter reduced to 98.8 angstrem. this effect on monoolein lpc is similar to the addition of a precipitant to initiate protein crystallization process. the spontaneous return to the initial lattice parameter was completed after 3 days in dark. thus, we demonstrated the possible controlling of the monoolein cubic phase lattice parameters by adding the photo-switchable diazo-derivatives of monoglyceride analogs, which can be used for crystallization of membrane proteins. evaluation of certain protein and phosphoprotein expression levels by using western blot technique in head and neck squamous cell carcinoma a. kalayci yigin 1 , t. cora 2 1 cerrahpasa faculty of medicine, istanbul university, istanbul, turkey, 2 faculty of medicine, selcuk university, konya, turkey introduction: head and neck squamous cell carcinoma (hnscc) is a primer tumor type in head and neck cancers, characterized by aggressiveness, early recurrence and metastasis. while alcohol and smoking play an important role at pathogenesis of disease, deregulation of some signaling pathways, genes and protein levels related to these pathways are considered as important at contribution of development of hnscc. materials and methods: in this study, protein and phosphoprotein expression levels of the frequently phosphorylated sites (egfr, pegfr, igf-ir, pigf-ir, pdgfrb, ppgdfrb, pten, ppten, akt and pakt) were investigated by using a western blot to confirm the expression of mrna in the context of protein levels at normal-tumor tissues of hnscc and sccl-mt1 that is a hnscc tumor cell line and hek-293 that is a normal cell line. results: as a result of western blot analysis egfr, pdgfrb and igf-1r were detected as highly overexpressed cell surface receptors in tumor tissues of hnscc. discussion and conclusion: the findings of this study revealed the overexpression of other cell surface receptors as well as egfr in hnscc. in conclution, potential pathways were identified by determining the cell surface receptors overexpressed in hnscc, these data support each other and may be important in pathogenesis of hnscc. introduction: the investigation of final products of lipid peroxidation is considered as the main mechanism involved in development of pathogenesis, diagnostics and prognosis of various parasitic illnesses. materials and methods: the concentration of lp-ap in the blood was determined in the study group considered of 129 women (65%), and 69 men (35%). results: before antiparasitic treatment, women infected with g. intestinalis showed a statistically significant 1.5 times increase of gpx activity levels; and 1.7 times ada level increase compared to the control group. after the treatment, the cat activity showed a sharp increase, whereas the ada activity decreased by 1.4 times, compared to the average level before the treatment. the results of the blood samples of the infected men with giardiasis, show the statistically significant increase in the level of all the studied parameters of lipid peroxidation, except the total primary production (tpp). the exception was the mda level, remaining significantly increased, in contrast to the control group and to the condition after antiparasitic treatment. in infected men, the level of cat activity was more than 5.8 times higher than that noted in control group. after treatment the levels of ada activity and gpx returned to the values of the control group, while the level of cat activity remained elevated. conclusion: an accumulation of primary and secondary metabolites in the course of giardiasis seems to confirm its involvement in the induction of oxidative-antioxidative homeostasis. antiparasitic treatment in giardiasis leads to normalization of the ap parameters studied in women and men, except the mda content in the blood of men. therefore, additional antioxidant treatment is advised for the antiparasitic therapy of men. in vitro effects of ethanol on rat brain synaptosome and dose-dependent antioxidative role of boric acid ethanol is a psychoactive drug that is very large and used frequently today. it has suppressive effects brain's comminication pathway. depending on its acute or chronic use and the dose, ethanol increase membrane fluidity and it causes oxidative stress. this study deals with the in vitro effects of ethanol toxicity (200 mm) and potential protective effect of different doses of boric acid (ba) (5, 10 and 25 mm) on rat brain synaptosomes. with this aim, five male spraque dawley rats are killed by decapitation under anesthesia. after the frontal cortexes of the rats are taken out, each of them is divided into four pieces. these pieces were used as a sample in five groups (control, ethanol, ethanol+5 mm ba, ethanol+10 mm ba, ethanol+25 mm ba) which include six samples. the synaptosomal fractions are prepared by the homogenization of the frontal cortex pieces and centrifugation for each samples. as markers of ethanol-induced oxidative stress in the synaptosome of the rats, malondialdehyde (mda), nitric oxide (no) and catalase (cat) levels were measured. mda levels in the ethahol group were a quantity increased compared with those in the control group but it unchanged significantly as statistically (p < 0.05). however the mda level in the ethanol+ boric acid (25 mm) group was shown to be significantly decreased compared with that in the ethanol group (p < 0.05). the cat activity of the ethanol group was significantly higher than that in the control group and cat activity of the ba (5 mm,25 mm) groups were close compared with control groups (p < 0.01). no levels in ethanol groups were decreased but unchanged compared with control groups as statistically. neverthless, no levels in ethanol+ boric acid (25 mm) groups were increased (p < 0.05). these results demonstrate that ethanol (200 mm) is capable of triggering damage to rat brain synaptosome and ba could be influential in antioxidant mechanisms against oxidative stress resulting from ethanol exposure. acute myeloid leukemia (aml) is the most common form of acute leukemia in adults and its incidence increases with age. carbonic anhydrases (cas) are zinc-containing enzymes. these enzymes catalyze a very simple physiological reaction, the inter conversion between carbon dioxide and the bicarbonate ion, and are thus involved in crucial physiological processes connected with respiration and transport of co 2 /bicarbonate between metabolizing tissues and lungs, ph and co 2 homeostasis, electrolyte secretion in a variety of tissues/organs, and biosynthetic reactions and many other physiologic or pathologic processes including reproductive tract. investigation of autoantibodies in aml patients have been popular research area in recent years. the aim of the current study was to investigate carbonic anhydrase i and ii (ca i and ii) autoantibodies in the serum of subjects with aml based on the information and considerations of autoimmune relation of acute myeloid leukemia. anti-ca i and ii antibody levels were investigated by enzyme-linked immunosorbent assay method (elisa) in serum samples of thirty patients with aml and thirty healthy peers. anti-ca i and ii antibody titers of aml group were significantly higher compared with the control group (p = 0.000), (p = 0.034), respectively. we found significant positive correlation between anti-ca i antibody and anti-ca ii antibody titers in patients with aml (r = 0.613, p = 0.000). we found significant positive correlation between anti-ca i antibody and anti-ca ii antibody titers in women and the men (r = 0.851, p = 0.000), (r = 0.503, p = 0.080), respectively. at an anti-ca i cut-off point of 0.123 absu, sensitivity was 80% and specificity 100%. at an anti-ca ii cut-off point of 0.097 absu, sensitivity was 60% and specificity 77%. the ca i and ca ii autoantibody levels in patients with aml were found higher compared to control group and the results suggest that ca i and ca ii autoantibodies may be involved in the pathogenesis of aml. aim: behc ßet's disease (bd) is a systemic autoimmune disease. recurrent oral and genital mucosal ulcers, uveitis, and skin lesions are characteristic findings for bd. platelet-lymphocyte ratio reflects a novel marker for romatological diseases. the aim of this study was to investigate the platelet/lymphocyte ratio in behc ßet's disease. methods: whole blood samples were collected from 50 healthy control and 21 patients with behc ßet's disease. the mean age for controls and patients were 40 ae 1.0 and 37 ae 13, respectively (p = 0.639). patients with chronic disease and inflammatory disorders were excluded. thrombocyte and lymphocyte counts were analyzed with abbott cell dyne heamotolgy analyzer. statistical analysis was performed with ibm spss v20. results: platelet counts were higher but not statistically significant in patients with behc ßet's disease compared to control group (289 ae 100 vs. 257 ae 57) (p = 0.088). lymphocyte counts were lower in patients with behc ßet's disease compared to control group (2.56 ae 0.58 vs. 2.69 ae 0.85) (p = 0.513). platelet/lymphocyte ratio was higher but not statistically elevated in patients with behc ßet's disease compared to control group (118 ae 41 vs. 106 ae 49) (p = 0.344) conclusions: platelet/lymphocyte ratio (nlr) and mean platelet volume (mpv) as inflammatory markers recently became popular because of their simplicity, cost effectivity and their advantages to predict clinical prognosis of specific diseases. according to this study's results, platelet/lymphocyte ratio must be analyzed in vast scale patient populations to identify the disease. objectives: systemic lupus erythematosus (sle) is a chronic relapsing autoimmune disease characterized by production of autoantibodies against a series of nuclear antigens and by chronic inflammation. in recent years, neutrophil-lymphocyte ratio (nlr) was determined to be a good indicator of inflammatory status. nlr can be easily calculated from a whole blood count. introduction: neuroblastoma, an embryonal malignancy, is the most common extracranial solid tumor of childhood. untreated neuroblastoma tumors and cell lines are reported to have reduced hla class i expression, rendering them potentially susceptible to natural killer cell killing due to lack of engagement of hla class i-specific inhibitory killer cell immunoglobulin-like receptors (kirs). the aim of this study was to investigate whether kir genes could influence the risk of neuroblastoma and prognosis of the patients. materials and methods: study group consisted of 50 neuroblastoma patients (15 male, 21 female, median age: 36 months) followed at the pediatric oncology clinic of c ß ukurova university medical faculty. control group consisted of 100 healthy children. 14 patients had stage 1, 2, 3 or 4s disease, 36 patients had stage 4 disease. 16 different kir genes were analysed by sequence specific oligonucleotide probe (ssop) analyses. statistical analysis were done using fisher's exact test. results: compared to the control group, neuroblastoma patients had lower expression of activating kir gene, kir2ds3 (p = 0.005), and higher expression of inhibitory kir gene 2dl3 (p = 0.038). additionally kir2ds3 genes were more common in patients with early stages (stage 1, 2, 3 or 4s) (p = 0.023) and kir2dl3 genes were more common in patients with stage 4 (p = 0.044). furthermore, there were no statistically significant differences between the rate of aa and bx genotypes and their centromeric/ telomeric regions of patients and controls. discussion: kir2dl3 gene can have a triggering effect in neuroblastoma pathogenesis; whereas kir2ds3 can have a protective role. investigating nk cell infiltration and kir receptors in neuroblastoma tissue samples will give more insight to the pathogenesis p-04.03.3-008 neuroprotective and immunomodulatory effects of urtica urens s. arslan 1 , g. terzioglu 1 , b. kabalay 1 , a. r. tufekci 2 , a. sen 1 , i. demirtas 2 1 department of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, 2 deparment of chemistry, faculty of arts and sciences, c ß ankiri karatekin university, c ß ankiri, turkey urtica urens (small stinging nettle) has been used for medical purposes in turkey as an alternative therapy. it has been used in the treatment of inflammation that is early, non-specific immune reaction to tissue damage or pathogen invasion, plays an important role in the initiation of neurodegenerative disorders such as multiple sclerosis. however, there are limited studies that investigate anti-inflammatory activity of urtica. therefore, aim of this study is to find out theanti-inflammatory effect of chloroform extract in caco-2 cell line. for this purpose, firstly, chloroform extract of urtica leaves was prepared. chemical composition of extract was determined by lc-ms. the effect of chloroform extract on selected pro-inflammatory and inflammatory proteins such as tumor necrosis factor-a (tnfa), nuclear factor kappa b (nf-jb), c-x-c motif chemokine 10 (cxcl10), and 11 (cxcl11) were determined. whole genome transcriptome analysis was performed by using human ht-12 v4 beadchip. extract treatment caused 35% and 40% increases in protein and mrna levels of nf-jb, respectively. on the other hand, tnf-a protein and mrna levels decreased significantly (24% and 12%, respectively). similarly, cxcl10 and cxcl11 mrna levels decreased 45% and 38%. transcriptome analysis showed that 233 probes were significantly changed (p < 0.05). pathway analysis revealed that the extract altered a group of genes involved in immune response, calcium ion homeostasis and transport, potassium channel complex, g-protein coupled receptor protein signalling pathway, etc. it is well established that calcium is very critical for brain cell death and formation of many brain disease including multiple sclerosis. these observations suggests that urtica maybe used in neurodegenerative diseases. in order to further test this hypothesis experimental autoimmune encephalomyelitis experimentsand activity guided fractionations have been still continuing. this work is supported by tubitak 111t515. p-04.03.3-009 linear low molecular weight a-1,6-glucan from bifidobacterium bifidum bim b-733d is implicated in pathogenesis of celiac disease the bifidobacteria are recognized as human commensals and widely used as probiotics. earlier, we have found (kiseleva et al., benef. microbes, 2013, 4(4) : 375-391) that bifidobacterium bifidum bim b-733d contains low molecular mass (5-7 kda) a-1,6glucans (g anti-tpo and g anti-tg ) that interact selectively with human autoantibodies to thyroid peroxidase (anti-tpo) and thyroglobulin (anti-tg), recognized markers of autoimmune thyroid disease (atd). the aim of the study was isolation and identification of b. bifidum bim b-733d biopolymers (bps) interacting selectively with autoantibodies to tissue transglutaminase (anti-ttg) and antibodies to gliadins (anti-gl), recognized markers of celiac disease (cd). we used affinity chromatography with anti-gl, size exclusion chromatography, 1 h and 13 c nmr spectroscopy, elisa with immobilized bps, tissue transglutaminase (ttg) and gliadins (gl) as positive controls. the bp isolated by affinity chromatography with anti-gl (as more available marker of cd) and size exclusion chromatography was identified by two-dimensional nmr spectroscopy as 5-7 kda linear a-1,6-glucan identical to g anti-tpo and g anti-tg . the functional activity of the bp named g anti-gl , viz., ability to interact selectively with anti-ttg and/or anti-gl was proven by elisa with (i) serum samples of cd patients containing either both anti-ttg and anti-gl without anti-tpo and anti-tg or anti-gl without anti-ttg, anti-tpo and anti-tg vs. serum samples of healthy donors without four types of antibodies and (ii) pure anti-gl vs. pure total igg (without anti-ttg, anti-gl, anti-tpo, anti-tg). since (i) serum samples of cd patients do not contain anti-ttg without anti-gl and (ii) pure anti-gl isolated by affinity chromatography with gliadins (gl) cross reacts with tissue transglutaminase (ttg), additional studies with pure anti-ttg are necessary to find out which of the two antibodies, anti-ttg and anti-gl, bind g anti-gl . in conclusion, we proved that b. bifidum bim b-733d cells contain linear low molecular mass a-1,6-glucan, g anti-gl , that interacts selectively with anti-ttg and/or anti-gl. since g anti-gl is identical to earlier found g anti-tpo and g anti-tg , we hypothesize that the a-1,6-glucan is implicated in pathogenesis of both autoimmune diseases, cd and atd. influences of elevated serum ferritin levels on insulin resistance and non-insulin-dependent diabetes mellitus (niddm) have predicted either because of increased body iron stores or influenced by several inflammatory diseases. low serum 25 hydroxyvitamin d is known to perturb cellular function in many tissues, including the endocrine pancreas, which are involved in obesity and niddm. we planned to investigate the association between 25hydroxyvitamin d with hematologic parameteres and iron status in obesity vs. diabetic patients. study groups consist of control, non-diabetic obese, obese-diabetic and lean-diabetic groups. serum triglycerides, total cholesterol, ldl-c, hdl-c, fasting glucose, hba1c, uric acid, creatinine, ggt,25-hydroxyvitamin d, insulin, crp, esr, total blood count and iron status. apart from the three parameters, there were no significant difference (p > 0.05) between groups. serum ferritin and mchc levels were significantly higher in lean-diabetic patients (p < 0.05). on the other hand, rdw are determined to be significantly lower (p < 0.001) in the non-diabetic obese group. no difference was detected in 25-hydroxyvitamin d levels between the control and the study groups (p > 0.05). non-diabetic obese patients had significantly (p < 0.05) higher levels of tg and lower levels of hdl compared to obese-diabetics. insulin levels were higher in nondiabetic obese and obese-diabetics than lean-diabetics (p < 0.05). this study provides evidence that lean diabetic patients show higher ferritin and mchc levels than obese patients. the increase in serum ferritin and mchc levels is related with altered iron metabolism at cellular level. at late mitosis, the mother cell divides, leaving two daughter cells connected by a thin intercellular bridge (icb). during abscission of the icb, the ingression of the cleavage furrow is formed, and the central spindle microtubules are compacted into the structure known as midbody (mb). the mb is situated within the icb, with the abscission usually occurring at one side of the mb. as a result, only one daughter cell inherits the post-mitotic mb. these mbs can then either accumulate in the cytoplasm or be degraded. recent studies have identified mbs as novel signaling platforms regulating stem cell fate and proliferation. indeed, stem cells as well as cancer cells were shown to accumulate post-mitotic mbs, resulting in reprogramming of the cell fate and conversion to highly-proliferative, stem cell-like phenotypes. it has been proposed that regulated macroautophagy may be playing a key role in mediating pots-mitotic mb degradation. therefore, the experimental approach involved studying the dynamics and function of a protein known as fyco1, which associates with postmitotic mbs and may regulate their degradation. in this study we identified fyco1 as a protein, which associates with post-mitotic mbs and may regulate their degradation. interestingly, fyco1 is also known to be present on autophagosomes, and overexpression of fyco1 can induce the formation of enlarged lc3-containing autophagocytic structures. here we demonstrate that fyco1 knock-down leads to defects in autophagic mb degradation, and that fyco1 functions by targeting endocytic membranes to the autophagic phagophore during early stages of mb degradation. additionally, we showed that fyco1 depletion leads to increased proliferation and cell growth in soft agar. based on all these data, we hypothesize that fyco1 mediates selective mbs degradation via endosome-dependent extension of the phagophore around the post-mitotic mbs, and that mbs may be the regulators of cancer proliferation and progression. p-05.03.3-002 proliferative effect of hypericine on human skin fibroblast cells and identification of the mechanism of action in molecular level to drawbacks associated with efficiency and viral genome integration. in order to improve reprogramming efficiency and compensate for viral transduction, new chemicals have been explored through ipsc research. the aim of this study was to investigate the proliferative effect of hypericine on human skin fibroblast cells (sf) in-vitro, and to identify the mechanism of action in molecular level. the proliferation was measured using the clonogenic and dimethylthiazol diphenyltetrazolium bromide (mtt) assays. real-time quantitative polymerase chain reaction (qrt-pcr) was performed to detect the mrna levels of cyclins (d1 and b1) and cell cycle controller genes (p53 and p21). sf cells were treated with different doses (1 nm-100 lm) of hypericine for 24 h and 48 h. a significant cell proliferation was observed in moderate concentrations (0.1-15 lm; %110-%134), but at high concentrations (25-50 lm) cytotoxic effects emerged in sf cells (ic50 = 23.62 m, r 2 = 0.915). qrt-pcr results revealed that the most proliferative dose of hypericine (15 lm) stimulates cyclin d1. the anti-proliferative activity of hypericine was accompanied by inhibition of cyclin b1 mrna, whereas it induced expression of p53 and p21 genes, and thus apoptosis was observed by dna laddering at the same dose (50 lm). overall results suggested that hypericine can compensate for viral transduction and improve reprogramming efficiency of ipscs by enforcing them in g1 phase. hence we report that hypericine can be a good candidate component for cocktails produced to trigger ipsc proliferation. glioblastoma (gbm) is the deadliest brain tumor. the mean survival time of gbm patients is approximately 12 months, increasing to 14 months after treatment with temozolomide, which is the gold standard chemotherapy. the resistance of gbm to chemotherapy seems to be associated with the blood-brain barrier (bbb) that limits the delivery of chemotherapeutics, and the presence of a population of cells that expresses stem cell-like properties, which are known to be chemo-and radioresistant,5 the glioblastoma stem cells (gscs). the difficulties imposed by these two factors could be reduced by the use of a targeted drugdelivery liposome-based strategy that allows bbb passage and reduces the side effects of chemotherapeutics. the present study evaluated the ability of the f3 peptide-targeted ph-sensitive lipid-based nanoparticle containing doxorubicin (dxr) to target gscs and non-gscs. we evaluated the expression of cell-surface nucleolin by flow cytometry, as well as of stem cell-like markers, in two gbm cell lines. we also determined the ability of gbm cell lines to specifically uptake the f3 peptide-targeted ph-sensitive lipid-based nanoparticles, by flow cytometry, and correlated it with the expression of stem cell-like markers. moreover, to ascertain the impact of intracellular delivery of chemotherapeutic drugs into gbm cell lines, cytotoxicity was further assessed by the mtt assay. our results showed that the f3 peptide-targeted ph-sensitive lipid-based nanoparticles successfully targeted glioblastoma cells and particularly gscs. in addition, the results also provided evidence of the nucleolin overexpression-dependency of this strategy, emphasizing the need to adapt the therapeutic strategy to the individual patient. this study showed that f3-targeted ph-sensitive liposomes may constitute an appropriate strategy to overcome the chemoresistance associated with glioblastoma cells. p-05.03.3-004 leukemic cell plasticiy as a resistance mechanism towards tyrosine kinase inhibitors chronic myelogenous leukemia (cml) is a hematopoietic stem cell disease characterized by the t(9;22)(q34;q11) translocation, which encodes the chimeric tyrosine kinase onco-protein, bcr-abl. the tyrosine kinase inhibitor (tki) imatinib is the first-line treatment for patients with cml. unfortunately drug resistance is one of the main problems observed. while secondary resistance is associated with bcr-abl kinase domain mutations, oncogene amplification and mechanisms interfering with intra-cellular drug concentrations; primary resistance mechanisms haven't been elucidated. we generated high dose imatinib-resistant k562 subclones (k562-ir) by clonal selection to study primary resistance mechanisms in vitro. drug resistance was shown by caspase 3 and annexin v/pi assays. we also showed cellular uptake and function of imatinib with western blot technics. k562-ir cells are not only resistant to imatinib but also to 2nd, 3rd generation tyrosine kinase inhibitors. we demonstrated that k562-ir cells have a highly adherent character, proliferate slowly and are resistant to drug-induced senescence. microarray analysis revealed that k562-ir cells differentially express tissue/organ developement and differentiation genes at high levels. we showed that k562-ir cells forms intact tumor spheroids in 3d cell culture conditions which is a marker of tumor initiating potential. cell surface maker analyses and protein analyses of k562-ir cell population, points towards an epithelial-mesenchymal plastic cell capable of adopting different morphologies. we hypotizied that imatinib and other tyrosine kinase inhibitors may cause the gain of phenotypic plasticity potential in leukemic cells, by interfering with signalling pathways; which in itself may lead to therapy resistance. hypoxia has multiple effects on cancer cells, which are critically involved in tumor progression. hypoxia leads to changes in tumor cell metabolism and can promote cancer cell survival, invasion and metastasis by its critically important role on maintenance of cancer stem cell (csc) phenotype. in this research, human cd133 + cscs isolated from human osteosarcoma cell line saos-2 using macs magnetic separation technique were characterized, and their stemness properties under hypoxic (1% o 2 ) and normoxic (21% o 2 ) conditions were compared in two and three dimensional culture conditions. two different 3d culture techniques (nanofibrous bacterial cellulose scaffolds and scaffold free microtissues) were used to evaluate effects of hypoxia on csc behavior, and the results were compared with the cell behavior in classical 2d culture systems. the morphologies of cells were examined by scanning electron microscopy (sem); rt-pcr and immunocytochemistry staining were used to examine the cancer stem cell phenotype maintenance under hypoxic and normoxic conditions. it is shown that hypoxia supports the expression of stemness markers such as oct3/4, nanog and sox2 compared to normoxic conditions in 3d cultures. although similar effects of hypoxia were observed in 2d cultured cscs, the expression levels of stem cell phenotypeindicative markers were significantly lower on 2d compared to 3d culture systems. this study is seen as an introduction to develop a more relevant 3d hypoxic cancer stem cell based tumor model to study csc behavior and tumor genesis in vitro for testing of novel cancer stem cell therapeutics and to understand signal transduction in cancer stem cells. prostate cancer (pca) is the second most frequent cause of cancer-specific mortality in the world. cancer stem cells (cscs) are a subpopulation of cells that involved in drug resistance, metastasis and recurrence of cancers. the efficacy of natural flavanone apigenin on cell survival, apoptosis and migration of cscs were evaluated. cd44 + cscs were isolated from human pca pc3 cells using a magnetic-activated cell sorting system. pc3 and cscs were treated with different concentrations of apigenin, docetaxel and combinations of the two agents for 48 h. apigenin dose dependently inhibited cscs and pc3 cell viability, and this was accompanied with a significant increase of the cell cycle inhibitors p21 and p27 (kip1). the flavonoid significantly induced apoptosis via an extrinsic caspase-dependent pathway by upregulating the mrna expressions of caspases-8, -3 and tnfa, but failed to regulate the intrinsic pathway as determined by the bax, cytochrome c and apaf-1 in cscs. in contrast to cscs, apigenin induced intrinsic apoptosis pathway as evidenced by the induction of bax, cytochrome c and caspase-3 while caspase-8, tnf-a and bcl-2 levels remained unchanged in pc3 cells. the ability of apigenin to inhibit the proliferation of cscs through apoptosis was confirmed by tali image-based cytometer. the flavanone strongly suppressed the migration rate of cscs compared to untreated cells. significant downregulation of mmp-2 and -9 exhibits the ability of apigenin treatment to suppress invasion. the expressions of pi3k/akt and nf-kb p105/ p50 were significantly decreased after 48 h apigenin treatment. taken together, these data demonstrated that flavonoid apigenin is an invaluable chemopreventive compound that inhibits proliferation, invasion and the stemness properties of cscs. this study was funded by the scientific and technological research council of turkey (tubitak, grant no. 115s356). (pi3k), are frequently found in patients with severe early-onset segmental overgrowth. whilst differences in timing and location of the founder mutation are likely to explain part of the observed disease heterogeneity, it is less clear whether and how quantitative differences in the strength and timing of pi3k activity contribute to phenotypic variability. our aim is to characterise pik3ca mutant-specific signalling as well as to explore the effects of varying the strength and/or temporal pattern of pi3k activation on downstream output specificity in the cell. we are currently employing crispr/cas9mediated gene editing in human induced pluripotent stem cells to generate isogenic disease models of three such activating pik3ca mutations. these cells will be used for signalome profiling by reverse-phase protein arrays (rppa) to compare and contrast mutant-dependent alterations to candidate signalling networks. in parallel, ongoing efforts focus on developing an endogenously expressed optogenetic p110a, allowing precise spatiotemporal control over pi3k signaling to unravel the extent to which pi3kdependent phenotypes are determined by strength of activation and/or dynamic encoding. ultimately, the outcome of this research will yield novel insight into fundamental aspects of pi3k signalling and potentially aid the development of targeted therapies for human diseases of pi3k hyperactivation. e. gov, n. kaya, k. y. arga cancer stem cells (csc) have been proposed to be the cancer initiating cells. because of their highly tumorigenic and drug resistant properties, cscs offer significant potential for developing novel anticancer drugs and therapeutic strategies. in the present study we analysed eight gene expression datasets for breast, ovarian, lung cancer and glioblastoma by comparing gene expression levels between stem cells and tumor cells and integrating them with genome scale biological networks. consequently, mutual molecular signatures (i.e: differential expressed genes, transcription factor, mirna) and biological characteristics were determined via integrative analyses, which might be feasible to uncover the mutual biological mechanism insights behind the cscs. it was identified twenty mutual differential expressed genes in four cancer types; jun and klf6 as transcription factors, egfr and cdk14 as receptors come into prominence as mutual signatures. molecules and pathways that were related to mapk, wnt, p53 signaling and pathways in cancer were the common indicators in csc types. our results provided similarities in gene expression profiles of various cscs and gave clues about the seed of tumorigenesis. this study proposed signatures and pathways that could be considered as effective therapeutic approaches in further experimental and clinical applications to eliminate subpopulation of csc. colorectal cancer (crc) is one of the leading causes of mortality worldwide. metastasis is associated with the presence of circulating tumor cells (ctcs) in the peripheral blood of cancer patients. ctc cut-off values have been shown to predict for poorer overall survival in metastatic breast (≥5), prostate (≥5), and colorectal (≥3) cancer based on assessment of 7.5 ml of blood. in our study, ctcs were detected in blood samples of colorectal cancer patients, using with our modified convenient method for the strategies of ctc enrichment and detection. 7.5 ml peripheral blood samples were firstly collected and peripheral blood mononuclear cells (pbmcs) were isolated from the fresh blood samples by ficoll gradient separation. next, the leukocytes in pbmcs were removed by magnetic microbeads conjugated with cd45 for a negative selection. finally, the retained cells were labeled with anti-epithelial cell adhesion molecule (anti-epcam), cytokeratins (ck8, ck19) and the leukocyte-specific marker as anti-cd45. all samples were analyzed by bd facs aria iii flow cytometry. in total, 10 patients and 7 healthy people were included in this study. the results showed that ctcs were not detected in the blood samples of healhty volunteers, but 3-13 ctcs were detected with ck14, 15, 16, 19-based gating strategy in the blood samples of colorectal cancer patients. it is accepted that the cut off value is 3 ctcs for colorectal cancer and ctc is negative if it is below this value or ctc is considered as a positive, if it is equal to or above this value, which might be an indication for poor prognosis. thus ctc's detection may serve a representative surrogate tumor biomarker for real-time monitoring of disease status and tailoring personalized therapy. cells were grown in culture flasks in a humidified incubator at 37°c with 5% co 2 and were used at the proliferation and confluent stages. cultured cells were exposed to the pemf and prfe. the proliferations of the cells are measured by mtt assay for the effect of emf on the cancer cells. on the other hand the wound healing was investigated by closure of the wound by the cell proliferation with cell morphology using inverted microscope images. the proliferation decreased significantly by the effect of pemf on the semi confluent mcf-7 and mda-mb-231 cells. this effect was observed more prominent on mcf-7. considering prfe therapy this effect is much more pronounced especially for mda-mb-231 comparing with pemf. the phase contrast observations of these results were consistent with mtt analyses. similarly, this effect was seen less for pemf but the proliferation was more suppressed with prfe on the wound models. it was considered that the emf applications could be effective in cancer cells, but this effect has not been studied how it occurs in invasive cancers. in our cell culture study, the appropriate emf applications were found to be effective though the inhibition of proliferation of cancer cells even in invasive cancer but with lower effect. this means that emf applications may support the existing treatment methods of cancer patients and even people who suffer from invasive cancer. metastasis is the one of the most known causes of death in patients diagnosed with cancer. circulating tumor cells (ctcs) are shed from primary tumors and circulating in the bloodstream, and thought to play a key role in metastasis. a hypothesis that ctcs may contribute to metastasis was first introduced in the mid 19th century by thomas ashworth, an austrilian pathologist. in today's research, identification and molecular characterization of ctcs are thought to be a novel target for treatment of cancer and a key factor to understand the metastatic process. existing methods of ctc capture based on the cell search system, flow cytometers, laser scanning cytometers instruments, fiber-optic array scanning technology (fast), isolation by size of epithelial tumor cells (iset), and definition fluorescence scanning microscopy. ctcs are increasingly considered as a 'liquid biopsy' and when liquid biopsy is compared to tumor tissue biopsy, liquid biopsy for ctcs detection can be carried out routinely in patients due to accessibility and ease of blood collection. also, primary tumor sampling may not reflect the actual metastatic conditions, ctcs are thought to be a novel tumor biomarker for real-time monitoring of disease status and tailoring personalized therapy. with futher works, ctcs may be used as liquid biopsies and it might provide better understanding metastatic process, new approaches in cancer diagnostics and treatment. mesenchymal stem cells (mscs) are distributed all over the organism as a source of tissue formation and regeneration. glucose is vital for the proliferation and differentiation of mscs. glucose uptake is mediated by specific glucose transporters of two families, the na-coupled glucose transporters (sglt) and glucose transporter facilitators (glut). the presence and function of glut proteins in human placental amnion derived mscs (hamscs) is unknown. we aimed to investigate the presence of glut1, glut3, glut4 proteins and genes in hamscs isolated from term placentas. mscs were isolated from human term placenta amniotic membrane, the characterization of cells were provided by flow cytometry. mscs were used to assess their chondrogenic, osteogenic and adipogenic differentiation potential. the expression of glut1, glut3 and glut4 proteins was detected in hamscs by immunofluorescence. glut1, glut3, glut4 protein and gene expression in these cells were investigated by western blot and real-time pcr, respectively. flow cytometry analysis results of isolated cells showed that they were positive for cd44, cd90, cd73, cd105 (mesenchymal stem cell markers) and hematopoietic markers cd34, cd11b, cd19, cd44 and hla-dr were negative. the presence of glut1, glut3, glut4 proteins and genes were identified in hamscs. in this study, for the first time in literature, glut1, glut3 and glut4 gene and protein presence was determined in hamscs. therefore, gluts could mediate glucose transport in human amniotic membrane mscs. proliferation and differentiation of mscs in vitro are still not optimized. further studies are required to clarify the complex mechanisms regulating the relationship between glucose and mesenchymal stem cells. disclosure of this relationship may provide a better understanding of glucose-related pathologies such as diabetes. tumors have hierarchically organized heterogeneous cell populations and a small subpopulation of cells, termed cancer stem cells (cscs), is responsible for tumor initiation, maintenance as well as drug resistance. therefore, killing the cscs along with the other cancer cells is gaining an importance. in the present study, it was aimed to evaluate the cytotoxic and apoptotic activity of a novel platin (pt) (ii) complex [pt(hepy) 2 cl 2 ] on mammospheres obtained from mcf-7 human breast cancer line. elevated expression of stemness markers were determined by western blotting. cytotoxicity was assessed using the atp viability assay. effect of the pt (ii) complex on the formation and development of mammospheres was analyzed with sphere formation (sfa) assay. apoptosis was determined via cytofluorimetric analysis (caspase 3/7 activity, annexin-v-fitc and bcl-2 activity) as well as gene expression analysis. cytotoxicity was confirmed with the atp viability assay after the treatment with zvad-fmk (an apoptosis inhibitor) and necrostatin (a necroptosis inhibitor). in addition, alterations in mitochondrial membrane potential were evaluated by jc-1 staining. mammospheres exhibited increased oct-4 and sox2 (stemness markers) expressions compared to parental mcf-7 cells. cytotoxicity by pt (ii) complex was evident in a dose-dependent fashion (1.56-100 lm) . pt (ii) complex significantly prevented mammosphere formation and disrupted mammosphere structure in a dose-dependent manner. pt (ii)-induced apoptosis was determined based on the presence of caspase 3/7 activity, annexin-v-fitc positivity and bcl-2 inactivation. apoptosis was also confirmed with increased tnfrsf10a and hrk gene expressions. in addition to apoptosis, necroptosis was also present as evidenced with increased mlkl expression. mitochondrial membrane was depolarized. in conclusion, the pt (ii) complex seems to be a powerful apoptosis-inducing compound on cancer stem cells, thereby warrants further in vivo experiments. cancer is a disease which arises from destruction of growth and proliferation mechanisms in cells and is the second leading cause of death worldwide [1] . in the development of primary cancers, the head and neck cancer is accounting for approximately 550.000 new cases annually around the world [2] . laryngeal cancer is a type of head and neck cancer in which malignant cells arise from the mucosal tissues of the larynx [3] . cancer might spread from primary tumor by getting into the lymph and blood vessel system and forms secondary tumor. greater than 90% of deaths in cancer patients are attributed to metastasis [4] . circulating tumor cells (ctc's) provide an opportunity to understand the metastatic process of cancer patients. identification and molecular characterization of ctc's in the peripheral blood of cancer patients is a promising research area in the field of biomarker development and novel treatment targeting in today's cancer research [5] . the detection of ctc methods include cell search system, flow cytometry, high-definition fluorescence scanning microscopy, fiber-optic array scanning technology, isolation by size of epithelial tumor cells, and laser scanning cytometers [6] . in our study, 7.5 ml of peripheral blood samples were collected from larynx cancer patients and healthy volunteers and the samples were analyzed by bd facs aria iii flow cytometry via biomarkers epcam, ck19, ck8 for positive selection and cd45 for negative selection [7] . according to the results of our study; ctcs were detected in larynx cancer patients by our newly modified method whereas there was no ctc's detection in the samples of controls. thus, this study may provide us monitoring of the treatment process of larynx cancer and this method might be used as diagnostic, prognostic, and predictive biomarkers in cancer therapy as a liquid biopsy. prostate cancer is the second most common cancer and the fifth leading cause of death from cancer in men 1 . circulating tumor cells (ctcs) present in the peripheral circulation of cancer patients with different solid malignancies including prostate cancer and have a potential as a liquid biopsy to monitor disease progression and response to therapies at cell and molecular level 2 . one of the general methods in ctc detection is flow cytometry 3 . radical prostatectomy is the most frequently applied procedure in the surgical management of localized prostate cancer. in this surgical operation, the surgeon removes the entire prostate gland with the seminal vesicles. a radical prostatectomy procedure can be done using the da vinci robotic system (intuitive surgical, sunnyvale, ca, usa) 4 . robotic surgery has been suggested to have fewer complications, lower risk of infections and shorter recovery period following robotic radical prostatectomy 5,6 . in this study, our aim was to detect ctcs before and after robotic radical prostatectomy in clinical localized prostate cancer patients. the ctc detection study was performed with our modified method in which 7.5 ml of peripheral blood samples were collected from each prostate cancer patient and healthy individual; the samples, using with biomarkers epcam, ck19, ck8 for positive selection and cd45 for negative selection, were analyzed by bd facs aria iii flow cytometry 7 . according to our results, we detected ctcs in the peripheral blood samples of prostate cancer patients before robotic radical prostatectomy. however, following this surgical procedure no ctc or decreased number of ctss was detected. our study might contribute to understand disease progression after robotic radical prostatectomy in clinically localized prostate cancer patients that warrants further research. keywords: circulating tumor cells, prostate cancer, flow cytometry, robotic radical prostatectomy. p-05.03.3-017 determination of effect cytotoxic, apoptotic, caspace-3 activity and mrna expression levels of apoptototic related genes of vulpinic acid on breast cancer cell lines n. kilic ß, s. aras, d. cansaran-duman ankara university, ankara, turkey breast cancer is the most common cancer types in women. several drugs used to treat breast cancer patients are developing resistance to the treatment for this reason success rate falls. therefore the discovery of alternative therapeutic agent and molecular detection of anticarcinogen effect because of treatment for cancer patients may be a source of hope for the contributions. in this study, different concentrations (1.562, 3.125, 6.25, 12.5, 25, 50 , 100 lm) vulpinic acid (va) lichen seconder metabolite was determined to cytotoxic, apoptotic effect and caspase-3 activity in breast cancer cells (mda-mb-231, mcf7, bt-474, sk-br3) and normal cell (mcf12a). in addition to the quantitative real-time pcr (qrt-pcr) using apoptose specific primers (tp-53, bcl-2, bax, birc-5, gapdh, caspase-3, caspase-7, caspase-8, caspase-9) and sybr green dye were performed to determine expression patterns of transcript level in cancer cell lines, using gapdh as a reference gene. the antiproliferative characterization of va effects identification of the gene set at molecular level and we determination role of va on apoptotic pathway. according to our study, va is demonstrated significantly (p < 0.05) effect cytotoxic, apoptotic, caspase-3 activity. beside this, dose dependent expression patterns decreased apoptose spesific genes (except of bcl-2) mrna levels from six to eleven fold change more than untreated va cell lines. va will be used as candidate molecule for effective treatment on breast cancer in the future. glycosylation largely determines the variety and functions of proteins. paucimannose, a mannosidic n-glycoepitope has long been thought to be specific for plants and invertebrates. recently, it has also been detected in mammalsin physiological conditions (stem cells) and in pathophysiological conditions (inflammation and cancer). in glioblastoma cells, paucimannose also seems to play a role in cell proliferation. glioblastoma is the most frequent brain tumor in adults with poor prognosis due to a lack of suitable treatments. we hypothesize that paucimannose could be a promising new biomarker as it is otherwise rarely found in mammals. therefore, paucimannose levels were investigated in different glioblastoma cell lines differing in their proliferation rate and tumorigenicity. the highest paucimannose levels were detected in low proliferating, nontumorigenic cells. furthermore, we found that modulation of paucimannose function by application of a specific antibody regulated cell proliferation and the capability of cells to form colonies in soft agar. these data support a functional role of paucimannosidic epitopes in tumorigenic processes. glioblastoma multiforme (gbm) is the most lethal type of malignant brain tumors. recently, gbm stem cells (gscs) have been studied in great deal and accepted that they have a legitimate role in tumor formation, development, chemo-resistance and recurrence. in this study, it is aimed to investigate new therapeutic targets within apoptosis related molecules to select and eliminate cd133+ gscs effectively. ten primary gbm cells were isolated from gbm tissue samples and they were cultured among with the 4 gbm cell lines (u87, u138, u118 and t98). cd133+ and cd133à cells were seperated by macs method via anti-cd133 (ac133) antibody from cultured cells and cell lines. rna isolation from cd133+ and (à) cells, cdna synthesis was performed. finally, by performing pcr array, mrna expression levels of 88 genes were detected. proper results were collected and analysed statistically. according to the results of pcr array; it has been found that cd133+ cells express approximately 223 fold tnfrsf21 and 20 fold tnfsf7 when they are compared with control cells. tnfsf7 binds to cd27 that is expressed on the surface of tcells. cd27 does not have a death domain, instead it has a cytoplasmic tail which binds to trafs. trafs act as adaptor molecules that are related with jnk and nf-jb signalling pathways. tnfrsf21 (dr6) is a death receptor which are known for transmitting the pro-apoptotic signals from outside to the inside of the cell. it negatively regulates t-cell activation and the release of few cytokines. as a conclusion, tnfsf7 and tnfrsf21 both are found on immune system cells, mostly on t-cells, which may mean that gbm stem cells act as a immune system cells to avoid the elimination by the immune system. to conclude, acting as an immune system cell and promoting survival via tnfsf7 and tnfrsf21, these molecules may be essential markers to target cd133+ gbm stem cells. the effect of docetaxel on p53, sin3a and mdm2 gene expression in mcf-7 breast cell line docetaxel is a cytotoxin effective in treating breast cancer. it stabilizes microtubules and causes catastrophic cell cycle arrest in g2/m. it also initiate signaling through cell death pathways that result in programmed cell death. in this study, it was aimed to investigate apoptotic and cytotoxic effects of docetaxel has on the mcf-7 breast cancer cells line. in this study, mcf-7 breast cell line was applied different doses docetaxel (10 nm, 100 nm, 1 lm, 10 lm, 100 lm) as 24 h and 48 h. mtt analysis was performed to the mcf-7 breast cancer line in control group and groups of docetaxel. afterwards, evaluation of apoptosis by tunel and levels of p53, sin3a and mdm2 gene expression by real-time pcr were determined in an order. it was observed cell variable was significant lower in docetaxel groups compared to control group (p < 0.05) in 24 h as mtt analysis. the lowest cell viabilty was determinated in group applied 100 lm docetaxel. while the lowest positive cell density was determinated in control group, it was observed apoptotic cell density gradually increased with increasing docetaxel concentration in groups treated docetaxel (p < 0.05). the highest p53, si3a and mdm2 expressions were apperared in 100 nm docetaxel group compared to control group. human alpha-fetoprotein (afp) and afp receptor binding domain (afprbd) are able to bind and internalize effectively by wide range of human tumor cells and tissues. as other vector molecules afprbd has insufficient quantity of chemical groups which can be conjugated with drugs or diagnostic agents. conjugation of vector molecules with macromolecular polymer carriers like dendrimers aims to solve this problem. our study describes influence of afprbd-dendrimer-doxorubicin conjugate surface charge on intracellular trafficking routes and toxicity. the amineterminated (g2) and acetyl-terminated (g2 12 ) 2nd generation pamam dendrimers carrying doxorubicin (dox) were used to synthesize conjugates with afprbd. unmodified by afprbd g2 and g2 12 dendrimer derivates labeled with dox were absorbed by the cells at 37°c with different efficiency. g2 12 -dox derivate characterized much slower internalization rate than nonacetylated g2-dox. only g2 12 -dox shown partial colocalization with lysosomal marker lamp2 after 4 h of incubation. internalization of afprbd-g2-dox and afprbd-g2 12 -dox did not show significant difference. at the same time, both conjugates contained afprbd wyкy almost fully associated with lamp2 already after 30 min of incubation. cytotoxicity results revealed that ic50 levels of g2 12 -dox and afprbd-g2 12 -dox coincided and demonstrated a bit higher activity against sensitive to dox skov3 and resistant skvlb cells than afprbd-g2-dox conjugate after 72 h of incubation. at the same time, after 1 h of incubation afprbd-g2-dox and afprbd-g2 12 -dox were much more than g2 12 -dox and g2-dox. we may conclude that there is significant difference in ways of dendrimers internalization by tumor cells depending on nature of surface chemical groups. on the other hand, chemical modification of dendrimer conjugated with does not afprbd influence dramatically on the protein trafficking and resulting cytotoxic effect. russian scientific foundation supported this study (no. 15-15-10013 1.40) , a key enzyme in glycolysis, catalyzes conversion of phosphoenolpyruvate (pep) into pyruvate with regeneration of adenosine triphosphate (atp). the key regulator of the metabolic alterations found in tumor cells is the glycolytic isoenzyme pyruvate kinase type m2 that is generally expressed in all proliferating cells and overexpressed in all tumor cells investigated to date. during carcinogenesis a shift in the pyruvate kinase isoenzyme equipment always takes place, such that the tissue-specific isoenzymes disappear, and m2-pk is expressed. breast carcinoma, the third most common cancer worldwide, accounts for the highest morbidity and mortality. breast cancer tissue analysis confirmed the upregulation of m2-pk in breast cancer, and high m2-pk levels were associated with poor prognosis of breast cancer patients. materials and methods: poly hema (mac) nanoploymers were immobilized by binding covalently with sulphur atoms on the gold electrod's surface. pyruvate immobilization was actualysed with cross linking reagent glutaraldehyde. biosensor was developed by preparing pottasiumferrociyanide, selected as a mediator. results: cyclic voltammograms have been carried out at between~0.4 and 0.6 v potentials vs. ag/agci. m2-pk activty was detected by using differential pulse method at between 0.3 and à0.25 v potentials by observing the differentiations in the current values. in the optimization studies, some parameters such as optimum ph, temperature, concentration of glutaraldehyde and p-hema-mac, were investigated. discussion and conclusion: the method developed for the measurement of the tumor m2-pk activity by using biosensor. we found that more advantageous in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, economic, practical and less time-consuming. piruvat kinase tumor m2-pk activity determination at low concentrations is possible with this method. p-05.03.3-023 tie2/tek: a potential biomarker for targeting glioblastoma stem cells role in angiogenesis, endothelial cell survival, proliferation, migration and adhesion. therefore, tie2/tek could be a potential target for therapeutic strategies directed against glioblastoma stem cells and their microenvironment. in this study, we investigated the gene expression levels of tie2/tek in both cd133+ gscs and cd133à gbm cells. gbm primary cells were freshly isolated from glioblastoma tissue samples and cultured in dmem supplemented with 10% fetal calf serum and 1% penicillin-streptomycin at 37°c in 5% co 2 -humidified incubator. we isolated cd133+ and cd133à cells from 10 gbm primary cells using macs system. following rna isolation from healthy brain tissues, cd133à and cd133+ cells, cdna synthesis was performed. finally, according to microarray protocol, cell surface marker panel array was applied. expression levels were analyzed using the delta delta ct method. statistical analysis was performed using spss software for windows version 13.0. tie2/tek gene expression was determined as 50.07 fold higher in cd133+ gscs than normal brain tissue (p < 0.05). morever it was determined 7.52 fold higher compared to normal brain tissue in cd133à (p < 0.05). according to our results tie2/tek expression was higher in gscs, indicating that tie2/ tek may be a potential marker for targeting cancer stem cells in gbm. this research has been supported by the scientific and technological research council of turkey (no:114s189). adenosine inhibited the breast cancer stemlike cell population through erk1/2 pathway s. m. jafari, m. aghaie cancer stem cells (cscs) are immortal tumor-initiating cells that can self-renew and drive tumorigenesis in various cancers, including breast cancer and others solid cancers. in a study indicated that extracellular atp reduces tumor sphere growth and cancer stem cell population. but at present, there are no reports available in literature on the effect of adenosine on breast cancer stem cells. in this study we evaluated the effect of adenosine inhibition and its mechanism of action in breast cancer stem cells isolated from breast cancer cell lines. our result showed that adenosine significant reduces breast cancer stem cell population. reduction of erk1/2 protein levels was also observed after treatment cancer cells with adenosine. in conclusion, our results indicate that adenosine decreases the breast cancer stem-like cell population through erk1/2 pathway. taxanes are commonly used for the treatment of many cancers as chemotherapeutic drugs that resistance to these agents has become a major clinical obstacle. taxane based chemotherapy drugs such as paclitaxel, docetaxel and cabazitaxel bind microtubules and inhibit to microtubule polymerization appear to stimulate programmed cell death. taxane-resistance to cancer has not been clearly in progression and development of drug resistance. multiple mechanisms are involved in the drug efflux proteins as multidrug resistance protein, differences in amino acid sequences among the b-tubulin isotypes. we investigated taxane resistance with different doses of paclitaxel, docetaxel and cabazitaxel in prostate cancer stem cells. we compared the expression level of apoptotic proteins, and its functional role in resistance mechanisms in cd44 + /cd133 + prostate cancer cell lines. taxane drugs were categorized as concentration-dependent or time-dependent. cabazitaxel caused a time-dependent and dose-dependent reduction in cell viability in all tested cell lines. resistance activity was consistently higher in docetaxel in prostate cancer cells compared with the other drugs. there are many different response of clonogenic formation cd44 + /cd133 + cells with resistance to docetaxel, paclitaxel and cabazitaxel in prostate cancer stem cells. the innate of prostate cancer resistances are important characterization steps and critically limits treatment outcomes therefore novel drugs must be focus on antiresistance and molecular based combinations. mesenchymal stem cells (mscs) are self-renewing cells with ability to differentiate into organized, functional network of cells. mscs isolated from various tissues including adipose tissues, bone marrow, umbilical cord, placenta and pancreas have different differentiation and proliferation potential. good knowledge of the metabolism and proliferation mechanisms of stem cells is required for stem cell therapies. glucose is an important molecule in the culture of stem cells. glucose concentration affects the differentiation and proliferation potential of stem cells. the aim of the study was to investigate the proliferation status by identifying the proliferating cell nuclear antigen (pcna) expression under normoglycemic and hyperglycemic conditions in mscs. mscs were isolated from human term placenta amniotic membrane. characterization of the isolated cells was performed using flow cytometry. chondrogenic, osteogenic and adipogenic differentiation potential of these cells were investigated. characterized cells were cultured in normoglycemic and hyperglycemic conditions for 24 and 48 h and the expression of pcna protein expression in these cells were investigated by western blot. flow cytometry analysis showed that isolated cells were positive with mesenchymal stem cell markers cd44, cd90, cd73, cd105 and negative with hematopoietic markers cd34, cd11b, cd19, cd44 and hla-dr. western blot result of pcna protein expression statistically significantly increased in human amniotic membrane mscs under hyperglycemic conditions for 24 and 48 h culture. the glucose content of stem cell medium is important because glucose is an effective molecule of the proliferation of stem cells. proliferation of mscs in vitro are still not optimized. when the relationship between glucose and stem cells be understood, it will provide a better understanding for the glucose-related pathologies such as diabetes during pregnancy. prostate cancer (pca) is the second most common type of cancer among men in the world. it is revealed that some gene, protein and metabolite sets control the pca, however the whole metabolomics changes are not completely understood yet. pca is common among older men, and this is an important health problem in developed countries. sarcosine is the n-methyl derivative of the glycine amino acid. glycine n-methyl transferase produces sarcosine from glycine. besides, it is metabolized to glycine by sarcosine dehydrogenase. in 2009, high level of sarcosine in urine was associated with pca by sreekumar et al. they identified sarcosine as a pca biomarker that was significantly increased in urine during prostate cancer progression to metastasis. following this study, several studies have been published indicating sarcosine as a pca biomarker. in our study, a preliminary biosensor system was fabricated for determination of sarcosine in urine by using sarcosine oxidase. sarcosine oxidase was immobilized on au electrode surface using gelatin as an immobilization matrix. glutaraldehyde was used as a cross-linking agent to avoid the loss of the enzymegelatin mixture. optimization and characterization studies were carried out. sarcosine concentrations were detected carefully with the developed biosensor system. the fabricated preliminary biosensor is a promising system that can allow lower detection limits after surface modifications. activation of the epithelial-mesenchymal transition (emt) program in tumor cells is associated with invasiveness and stemness. recent studies implicate emt-inducing molecules in reprogramming energy metabolism. the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (pfkfb3) regulates glycolysis by producing fructose 2,6-bisphosphate (f2,6bp). given that pfkfb3 is induced by several established emt-inducers in tumor cells, e.g. hif-1a and ras, we hypothesized that pfkfb3 may be involved in regulation of the emt in tumor cells. silencing of pfkfb3 in pancreatic adenocarcinoma cell lines panc1 and s2vp10 was achieved using specific sirna molecules. mrna and protein expressions of the cdh1 gene (encoding e-cadherin, an established epithelial marker), as well as zeb1 and snai1 genes, by real-time quantitative (q)-pcr and western blot, respectively. immunfluorescence analysis was performed to visualize e-cadherin protein expression on plasma membrane. in order to test the effect of pfkfb3 on the invasive ability of the cells, a matrigel invasion assay was performed. ectopic expression of zeb1 was achived by transfecting cells with a plasmid carrying zeb1 cdna. cells that were depleted of pfkfb3 exhibited markedly increased cdh1 mrna and e-cadherin protein expressions and reduced snai1 and zeb1 mrna expressions. immunfluorescence analysis confirmed the upregulation of the e-cadherin protein on plasma membrane. silencing of pfkfb3 caused approximately 40% reduction in matrigel invasion, compared to non-targeting sirna. inhibition of the matrigel invasion caused by pfkfb3 depletion does not appear to be associated with reduced zeb1 expression, as ectopic expression of zeb1 did not reverse the effect of pfkfb3 silencing on invasion. taken together, these data suggest that pfkfb3 may be required for the maintenance of the mesenchymal phenotype and associated traits in pancreatic adenocarcinoma cell lines. introduction: leukemias are neoplasms that arise from hematopoietic cells initially proliferate in the bone marrow, and then disseminated in the peripheral blood, spleen, lymph nodes and eventually to other tissues. lymphomas occur primarily in the lymph nodes, but can be extended in peripheral blood and bone marrow infiltrate. aim: to determine the values of haematological parameters the control and test groups. to determine the prevalence of types of chronic leukemia in relation to the experimental group. compare haematological parameters in relation to the type of chronic leukemia. materials and methods: a prospective-retrospective study included subjects who were made laboratory hematology in oj clinical chemistry and biochemistry ukcs. blood tests conducted on the hematology analyzer siemens advia 2120 hematology system and abbott cell dyn 3700 and microscopic analysis of the peripheral blood smear. results and discussion: according to the age of respondents test group was established mild form of anemia, a red blood cell count is totaled 3.69 ae 1.13x10 12 , which is signifycantly lower compared to the control group. the average number of leukocytes was significantly higher in subjects studied groups and amounted to 136.91x10 9 , with a maximum value of 580 x10 9 . in the peripheral blood of patients with chronic leucosis has established a significantly higher number of cells compared to the control group (p = 0.001), while the number of monocytes was a significantly smaller. in the group of patients with chronic leukosis largest number had chronic lymphocytic leukemia (70%), and chronic myeloid leukemia had 30% of respondents. conclusion: subjects with cll were statistically older than patients with cml, and as regards the gender structure, men have dominated in cll and cml in women. white bloodline was found that the number of leukocytes in both forms of chronic leukemia high above the reference value. p-05.03.3-032 effect of enzymatic and non-enzymatic isolation methods of endometrial stem cells on their cell proliferative potential and mesenchymal stem cell characteristics human endometrial stem cells (hescs) are responsible for the monthly renewal of the basal layer of the human endometrium by facilitating stromal and vascular regeneration. in this study, hescs were isolated with three different isolation methods including non-enzymatic and enzymatic digestion using trypsin and collagenase type 1. the effect of these three isolation methods on the acquisition of mesenchymal stem cells (msc) and on hesc proliferative potential was evaluated through flow cytometric analysis of cd surface markers and wst-1 tetrazolium salt assay. our findings indicate that hescs isolated with these three methods have statistically similar cell proliferation rate at 24 h time point. however, at 48 h time point, hescs isolated with the non-enzymatic and collagenase type 1 method displayed a higher expansion in cell number when compared to the hescs isolated with trypsin. the late passage of hescs isolated with non-enzymatic and trypsin methods showed the highest proliferation rate in comparison to the hescs obtained via collagenase type 1 isolation method at 24 h, 48 h and 72 h. the three isolation methods for the early passages of hescs had a resemblance in their msc profile with no significant difference. on the other hand, late passage hescs isolated using trypsin non-enzymatic method showed a higher cd31and lower cd44 profile. moreover, late passage of hescs isolated with non-enzymatic method displayed a significant reduction in their cell surface cd90, cd73, and cd105 surface expression levels. only hescs isolated with collagenase type 1 did not present a significant shift in their mesenchymal cd marker profile from early to late passages, taken together results from this study suggest that the longterm maintenance of mesenchymal markers can only be achieved in cell isolation with collagenase type 1, while non-enzymatic method is more suitable to obtain higher msc cell yield for immediate use. hepatocellular carcinoma (hcc) abundantly arises on the viral and/or chemical-induced cirrhosis in liver. cirrhosis is defined as one of the premalignant stage hcc in which microenvironmental changes occurred such as uncontrolled production of collagen type i and activation of hepatocyte growth factor (hgf)/c-met signaling. it has been shown that epcam+/cd133+ subpopulation of cells isolated from hcc tissue can initiate tumor at very low concentration in xenograft model and behaves as hepatic cancer stem cells. however, the molecular mechanisms supporting hepatic stem cell activation are not well understood and knowledge about the role of hgf/c-met pathway in this process is not clear. in this study, we aimed to define effect of collagen type i and hgf induction on the cell behaviours of epcam+/ cd133. epcam+/cd133+ cells were sorted by magnetic separation from huh-7 cells. then proliferation and invasion of cells were analyzed under the hgf induction as well as branching morphogenesis in vitro. after hgf stimulation, phosphorylation level of c-met increased in epcam+/cd133+ subpopulation. moreover, presence of collagen type i enhanced significantly effect of hgf stimulation in the invasion of epcam+/cd133+ cells. we also have showed that hgf stimulation increased branching tubulogenesis capacity of epcam+/cd133+ subpopulation while it did not effect proliferation of cells. these effects of hgf reverted by c-met inhibitor, su11274, in vitro. all these findings showed that hgf and collagen type i regulates aggressive phenotype as microenvironmental changes via induction of invasiveness of epcam+/cd133+ subpopulation of huh-7. in conclusion, we showed that hgf/c-met signaling causes to get more metastatic phenotype based on invasion and tubulogenesis in epcam+/cd133+ hepatic cancer stem cells in hcc and it might be possible to use c-met inhibitors to target hepatic cancer stem cells during hepatocarcinogenesis. endometriosis is defined by the migration of endometrial mesenchymal stem cells (emscs) into the peritoneal cavity or other site of body rather than uterus in a retrograde fashion. its previously known intracellular crosslinking enzyme called tissue transglutaminase (tg2) was shown to play important roles in the extracellular matrix (ecm) modelling, fibrosis, cell adhesion and migration. we have hypothesized that tg2 might be expressed in emscs and take part in the formation of endometriosis. the difference in the proliferation capacity of emsc isolated from endometrial tissue with/without endometriosis was determined using wst-1 assay and tg2 activity and expression levels were analysed by btc assay and rt-pcr. the biosynthesis and activity for mmp-2 and -9 were investigated with zymography and rt-pcr, respectively. although tg2 activity was found to be 50% less in emscs isolated from endometriotic tissue, these cells showed 9 times higher tg2 protein expression than those isolated from the control tissue without endometriosis. emscs from endometriotic tissue have 2.3 times higher tg2 and 10.3 fold higher itgb1 mrna levels when compared to the cells of healthy group. similar results were observed in sdc-4 gene expression with a 2.5fold increase. endometriotic emscs demonstrated an average of 11.5-fold increase in the mmp-2 activity while a onefold increase was evident in mmp-9 activity when compared to the healthy emscs. emscs from patient group possessed a higher proliferative ability in comparison to that of healthy subjects within 96 h. the fact that emscs from the control tissue showed lower tg2 protein levels with a high enzyme activity suggested that tg2 might be important in the development of endometriosis not only by destabilizing ecm but also enhancing the cell migration. in this context, the upregulation of tg2 along with itgb1 and sdc4 was evident in emscs of endometriosis which was possibly associated with the increase in the activity of mmp-2 and -9. recent studies have indicated that pluripotent stem cells and some stromal stem cells such as mesenchymal stem cells (msc) are metabolically different from their differentiated counterparts. in this study, the cellular mechanisms controlling metabolic changes in stem cells was investigated using wharton jelly mesenchymal/stromal stem cells (wj-mssc). wj-msscs were isolated by the explant method and cultured in dmem-f12 with 10% fbs. endothelial differentiation was induced by the addition of vegf, egf, insulin and hydrocortisone for 6 days. neuronal differentiation was achieved by using commercial neuronal differentiation medium (millipore) for 3 days. in parallel experiments, cellular metabolic activity such as lactate production was measured. the msc characterization was performed by flow cytometry using antibodies against cd90, cd105, cd73 and cd44 (bd human msc analysis kit). the differentiation process was followed by measuring the expression of cd31, cd34 for endothelial and gfap, neu and tyrozine hydroxylase proteins for neuronal cells by immunofluorescence. for gene expression, nanog, cd34 and gapdh genes were analyzed by rt-pcr. differentiation stimuli to endothelial or neuronal cells resulted in a significant decrease in msc marker proteins. expression of stem cell markers other than cd73 were decreased to 2-20%. differentiation induced the expression of cd31, cd34 for endothelial and gfap and neu proteins for neuronal cells. in vitro lactate production was decreased following differentiation in both lineages. neuronal differentiation increased glucose consumption by~48% and the extracellular calcium concentration of these cells was significantly lower than synchronous undifferentiated cells. glycolytic activity is decreased during in vitro differentiation of wj-msscs. metabolic reprogramming and glucose uptake of cells may be an early indicator of the differentiation process in wj-msscs, supporting the view on their metabolic plasticity. store-operated ca 2+ entry (soce) activated by depletion of intracellular ca +2 stores has been shown to control intracellular ca 2+ homeostasis in many physiological and pathological events. stromal interactive protein, stim1, as endoplasmic reticulum (er) ca +2 sensor and orai protein as pore-forming subunit of soc channels play crucial roles in the activation of soce channels. stim1 and orai were reported to have pathophysiological roles especially in hepatocellular carcinoma (hcc). anticancer chemotherapy frequently falls back because of these tumor-initiating subpopulations, tentatively called 'cancer stem cells'. the purpose of this study was to investigate the roles of stim1 and orai on soce in differentiation of huh-7 hccs expressing epcam and cd133 surface adhesion molecules (epcam + cd133 + ). epcam + cd133 + subpopulations in huh-7 cells were separated via flow cytometry and transfected with stim1 and orai-1 over-expressing (oe) plasmids. expression levels were confirmed by rt-pcr. changes in intracellular ca 2+ concentration were monitored via dual wavelength spectrofluorimeter in fura 2-loaded cells. in epcam + cd133 + cells, er ca 2+ release increased without any change in soce compared to that of epcam à cd133 à cells. similar results were observed in stim1-oe epcam + cd133 + cells. on the other hand, increase in orai1 has no effect on either parameter. cancer is globally one of the most death causes. recently, huge improvements occurred in the cancer diagnosis and treatment due to advanced technology, however recurrence occurs almost 30-40% of patients and their survival times decreases. in this study, we aimed to investigate of relationship between the cancer stem cells which are strongly associated with chemotherapy and radiotherapy resistance and recurrence with the non-classical mhc i antigens which have immunosuppressive properties. for this purpose, we immunohistochemically evaluated the expression patterns of cd133, cd44, nanog, oct3/4, hla-g and hla-e in the advanced stage colorectal, gastric and breast cancer and also non malign biopsy samples. we detected that the cancer stem cell markers cd133, cd44, nanog and oct3/4 significantly increased in the advanced stage cancer tissues. however, the immunosuppressive hla-g and hla-e expressions increased only in the colorectal and gastric tumor tissue. in addition to the presence of cancer stem cell like cells in the tumor tissues, increased expressions of hla-g and hla-e may indicate an immune evasive adaptation of tumor cells. according to our findings, the hla-g and hla-e may be potential therapy targets to elimination of cancer stem cells of colorectal and gastric cancers. however, more detailed studies are needed to support our findings and also to determinate of clinical values of these markers. endocannabinoids increase sdf-1 release from human mesenchymal stem cells s. k€ ose 1 , f. aerts kaya 1 , d. uc ßkan c ß etinkaya 1 , p. korkusuz 2 1 stem cell research and application center, hacettepe university, ankara, turkey, 2 department of histology and embryology, hacettepe university, ankara, turkey lipid-structured endocannabinoids are endogenous morphine ligands and present widespread receptor-mediated effects at physiological and pathological levels on the nervous system as well as many other systems. these effects are partially realized through mechanisms affecting cell growth, differentiation, apoptosis and migration at the molecular level. the hematopoietic progenitor cells (hpcs) and mesenchymal stem cells (mscs) form a distinct niche in bone marrow where they interact with each other in harmony. the stromal cell-derived factor 1 (sdf-1/cxcl12) is a chemotactic factor in bone marrow and is released from mscs and their receptor cxcr4 is found in hpcs. with these rationale in mind, we asked if hpcs and msc interaction mediates sdf-1 release via endocannabinoidal system. bone marrow mscs obtained from healthy donors and passage 3 mscs were induced with 200 ng/ml lipopolysaccaride (lps) for 4 h. antagonists for cb1 (am281) and cb2 (am630) receptors were added to cultures for 4 days. after incubation with antagonists msc culture supernatants collected and processed with human sdf-1 beta in elisa medium. analyses demonstrated direct decreasing effect of endocannabinoid receptor antagonists on sdf-1 beta release from bone marrow mscs. in conclusion, endocannabinoidal system regulates sdf-1 release on mscs and directly act on hpcs mobilization in bone marrow microenvironment (niche). this may have a clinical implication on therapeutic mobilization strategies for hscs in hematology clinical applications. implantation is invasion of the embryo into the endometrium and occurs in three stages apposition, adhesion and invasion, via the complex cellular and molecular mechanisms. during these stages, both of maternal endometrium and embryo should be appropriate for the implantation which is the beginning of pregnancy. receptivity of uterine consists in the existence of growth factors such as tgfbeta-1, igfr1, vegf. it is indicated that damages of factors relesead from endometrium and blastocyst prevent implantation. recently, stem cells can be obtained from many sources to use for therapeutic purposes and mesenchymal stem cells derived from bone marrow are the most studied. in our study, it was aimed to investigate molecules play a role in blastocyst implantation after bone marrow derived mesenchymal stem cell application into the rat endometriyum. female rats were divided into three groups which were saline (sf, n:7), media (m, n:7), stem cell in media (m+bmsc, n:7). after vaginal smear technique, female rats in estrous cylcle were injected into the uterine and periton 200 ll saline, 200 ll culture media and 1 9 10 6 cell/200 ll culture media. the pregnant female rats on the 7 day were sacrified and uterine samples removed and were stained with heamatoxylin-eosin histochemically and anti-tgfbeta-1, anti-igfr1, anti-vegf and anti-pcna immunohistochemically and obseved under light microscope. h-score results were determined using one-way anova test statistically. it was found that intraperitoneal administration of stem cells with media, was increased tgfbeta-1, igfr1, vegf and pcna parameters when compared with the intrauterine administration of stem cells. in this study, it was revealed that distribution of molecules play role in implantation were changed due to stem cell application. it is supposed that stem cell treatments can be cured the molecules caused infertility. many unconventional biochemical factors remain to be investigated for their potential effects on stem cells. among others, endogenous gasotransmitter h 2 s, generated from l-cysteine and organosulfur-compounds (oscs) metabolisms, plays very important roles in the central nervous, respiratory and cardiovascular system. slow-releasing h 2 s donors are viewed as powerful tools for basic studies and innovative pharmaco-therapeutic agents for cardiovascular and neurodegenerative diseases. exogenous h 2 s administration is able to promptly scavenge ros, activate myocardial k atp channels and increase pro-cell survival signaling, very likely activating erk and phosphatidylinositol 3-kinase (pi3k)/akt pathways. the effects of h 2 s-releasing agents on the growth of stem cells are not yet widely investigated. therefore, stem cell therapy combined with h 2 s may have great clinical relevance in cell-based therapy for regenerative medicine. the effects of slow-releasing h 2 s agents on the in vitro cell growth and differentiation of human lin à sca1 + cardiac progenitor cells (hcpc) were here studied. in particular, the effects of h 2 s-releasing agents, such as na 2 s, gyy4137 and water-soluble gsh-garlic conjugates (gsgaws), on the cell viability and differentiation of hcpc were here investigated by colorimetric assay, immune-fluorescence microscopy and western-blotting analysis. the treatment with slow-releasing h 2 s donors increased the cell proliferation in a concentration dependent manner respect to the control. moreover, the treatment with gsgaws led to an up-regulation of the expression of proteins involved in the cell adhesion and differentiation processes. these preliminary results highlight on the effects of this gasotransmitter on the stem/progenitor cells and on the possibility to develop functional 3d-systems for cardiac tissue repair, that take into account the relevant biological role of h 2 s in the cardiovascular system. p-06.02.5-002 investigation of the protective effect of boric acid and omega-3 fatty acid in model of acute myocardial infarction changes in myocardial rats ischemic heart disease being the most common cause of the mortality and morbidity in worldwide commonly results from the occlusion or narrowing of the coronary arteries by atheromatous plaque and thus is named as coronary artery disease. male sprague dawley rats were used in the present study. rats were divided into 5 groups with 10 rats in each: control, mi, mi+boric acid, mi+omega-3 and mi+boric acid+omega-3 groups. control rats were treated with 2 ml/day saline, boric acid-treated rats received 100 mg/kg/day boric acid and omega-3-treated rats received 800 mg/kg/day for 28 days by oral gavage. for the experimental mi model, 200 mg/kg izoproterenol-hcl (iso) was administered subcutaneously two times with a 24-h interval in the last 2 days of the boric acid and/or omega-3 treatments. twelve hours after the second dose of iso, general anesthesia was induced. under general anesthesia and spontaneous respiration, ecg recordings were obtained by using a computerized data recording and analysis system (mp100, biopar) and d-ii recordings were used in the analysis. compared to the control group, serum ck-mb, bnp and tnf-a levels were higher in mi group (p < 0.001, p < 0.001 and p < 0.01 respectively). in the heart tissue homogenate, biochemically measured calpain activation and mda were increased (p < 0.01 and p < 0.001, respectively) and pon1 levels were decreased (p < 0.05). according to the ecg recordings, st wave and heart rate were found to be decreased (p < 0.001 and p < 0.001, respectively). on the other hand, all above mentioned parameters were found to be improved in rats treated with boric acid and/or omega-3 after induction of mi. moreover, histological analysis including light microscopy and tem revealed a significant histological improvement in rats treated with boric acid and/or omega-3 after induction of mi. results of the present study suggest that omega-3 and/or boric acid treatment significantly decreases the cellular damage in mi. this is study is aimed at measuring the level of serum heart-type fatty acid binding protein (h-fabp) in patients presenting with diabetic ketoacidosis (dka) and diabetic ketosis (dk) and to determine its role in identifying early period cardiac ischemia by comparing this level with the level of a control group at a comparable age this study was planned to be a prospective study and it included 35 patients diagnosed with dka, 20 patients diagnosed with dk and 20 voluntary pediatric and adolescent healthy control subjects. the h-fabp, creatine kinase-mb (ck-mb) and troponin-i levels were studied in patients with dka and dk as well as in the control group at the time of presentation. for dka patients, their h-fabp values were measured once again after acidosis correction and compared with the values they had at the time of presentation there were no differences among groups in terms of sex, age, height and weight. no statistically significant differences were found among groups with respect to troponin-i values (0.06 ae 0.08, 0.07 ae 0.04 0.04 ae 0.04; p = 0.457). no statistically significant differences were found among groups with respect to ck-mb values (1.48 ae 0.91, 1.55 ae 0.9, 2.09 ae 1.37; p = 0.229). the h-fabp values of dka patients at the time of presentation were found to be statistically significantly higher than those of dk patients and control group (1.17 ae 0.79; 0.79 ae 0.5 0.69 ae 0.36; p = 0.006). the h-fabp value of the dka group at the time of presentation was found to be statistically significantly higher than the value at hour 36 after acidosis correction (1.17 ae 0.79; 0.55 ae 0.28; p = 0.0001) the fact that h-fabp levels were found to be high in pediatric patients diagnosed with dka at the time of presentation suggested that myocardial ischemia had been triggered. in diabetic patients, every ketoacidosis attack may lead to cardiac ischemia, thereby accelerating progress to necrosis. in conclusion, we would like to propose h-fabp as a potential marker for indicating myocardial ischemia. p-06.02.5-004 genome-wide analysis of hypoxic stress response in human cardiomyocytes stress in human cardiomyocytes on a genome-wide scale remains poorly understood. this study aimed to identify the gene expression patterns of adaptive response of the human cardiomyocytes (hcm) to hypoxic stress. in vitro experimental models of hypoxia mimicking in-vivo coronary ischemia, are useful tools to identify molecular pathways involved in myocardial ischemia. in the current study, we cultured ac16-hcms in dmem/f12 with 10%fbs. to simulate hypoxia model, cardiomyocytes were plated in hypoxia chamber (1%o 2 , 5%co 2 , 94%n 2 ) for 3, 6, 12, 24 h and the control group was incubated in normal conditions (5%co 2 , 95%o 2 ). cell viability was determined using mttassay. annexin-v assay was used to monitor apoptosis. gene expression profiling was analysed with affymetrix-hg-u133-plus-2 arrays. following bioinformatic and statistical analyses differentially expressed genes (deg) were classified according to gene ontology using david and kegg pathway analysis tools. according to mtt, annexin-v and hif gene expression results, hypoxia time was determined as 3 h. we identified 649 genes (279 down-regulated and 370 up-regulated) (p < 0.001, fold change ≥1.5) were differentially expressed in hypoxic-ac16 vs. ac16. degs were mainly clustered in cell proliferation, regulation of cell death, cell adhesion and response to stress. furthermore, transcriptome analyses revealed that 'metabolic, cytokinecytokine receptor interaction, hif-1 signaling, tgf-beta, cell cycle and apoptosis' pathways were involved in the hypoxic stress response of human cardiomyocytes. this study provides molecular information regarding gene expression reprogramming of human myocardial hypoxia. the pathways identified in this study may pave the road for translational medicine. this study was supported by tub _ itak project number 111s189. autologous ips cells after reprogrammed into endothelial progenitor cells (epcs) may offer several advantages in the treatment of cardiovascular disorders because of their cardiogenic and vasculogenic differentiation potential. to reach that purpose, we differentiated and characterized mouse ips cells into flk1 + , a well-recognized epc marker. further maturation of epc was characterized by the expression of cd31 and cd133 markers. purified ips cells were differentiated into flk1 + cells with the use of differentiation medium on type iv collagen-coated dishes in the absence of lif. we then analyzed flk1 gene expression and protein levels with qrt-pcr, western blot and immunocytochemical methods on days 2.5 to 7.5. flk1 + cells isolated with macs system and then recultured these cells in differentiation medium with vegf to induce epc cells. following induction, cd31 and cd133 gene expression and protein levels were analyzed with genomic and proteomic methods. after isolating these cells and aggregate overnight, we cultured cells in three-dimensional condition in collagen type i and used differentiated medium including vegf and egf. we found that flk1 expressing cell number reached to a peak level (24%) on day 5.5 followed by a progressive decline subsequently. in the second step, cd31 and cd133 positive cells were generated and enriched during day 4 of induction. we showed optimal time for harvesting flk1 + cells is day 5.5 of initial differentiation. following isolation of flk1 + progenitor cells they were further matured into functional epcs by vegf within 4 days of induction. additionally for evaluation of angiogenic potantial differentiated cells, we monitored epcs behavior along vascular formation in 3d culture. our work demonstrates that epcs could be successfully derived from ips cells and these cells have vascular formation and angiogenic potential in 3d culture. epc drived ips cells play important role in the treatment of cardiovascular disease. p-06.02.5-006 electrophysiological, biochemical and genotoxic effects of luna experience on heart tissue in rat model pesticides are widely used for the control of agricultural, industrial and domestic pests. however, the uncontrolled use of pesticides has diverse effects on ecological system and public health. fungicides are one of the pesticide type used to kill fungi or fungal spores. in this study, the effect of different doses of luna experience, a fungicide, on the cardiac electrophysiology and genotoxicity in rats were investigated. among five groups (5 mg, 10 mg, 20 mg, control and positive control for comet assay) treatment groups received by gavage doses of luna experience for 30 days. electrical activity of heart were recorded using electrophysiological recording techniques. tissue activities of paraoxanase (pon) and arylesterase (are) and level of malondialdehyde (mda) were measured using biochemical methods. comet assay was performed on heart tissue. we calculated genetic damage index (gdi) and damaged cell percent (dcp) from comet assay. it was observed that there is a significant decrease in heart rate in all treated groups as compared with control group (p < 0.05). amplitude of p wave and qrs complex did not change (p > 0.05). in all treated groups, statistically significant differences were found for values of pon, are, mda, gdi and dcp when compared to control group (p < 0.05). according to our results, exposure to different doses luna experience have a probable hazard potential for the cardiac system. the macrocyclic cage complexes iron (ii) clathrochelates are of the interest due to their bioactivity; they are able to inhibit t-7 rna polymerase, possess toxicity to leukemia cells hl-60 and suppress amyloid fibril formation. their binding to serum albumins was reported; the extreme binding affinity to albumins is observed for the compounds bearing carboxy groups. upon this interaction, clathrochelates quench protein intrinsic fluorescence and gain optical activity inducing circular dichroism (cd) signal in 350-600 nm region. here we examine the effect of spatial arrangement (isomery of substituents) of clathrochelates on their binding to globular proteins. we study 6 bis-substituted clathrochelates bearing two same or different isomers (ortho-/meta-/para-) of carboxyphenylsulfid groups. their interaction with bovine (bsa) and human serum albumins, b-lactoglobulin and lysozyme are explored by cd and protein fluorescence quenching method. the binding of compounds to albumins evoked the cd bands of the same shape, but their intensities vary up to 45 times depending on substituents isomery. in the presence of b-lactoglobulin, the intensities, shape, and positions of the induced cdbands differ for the compounds with different isomer groups. the cd bands induced by the lysozyme in the case of di-para substituted clathrochelate are shifted relatively to the bands of other isomeric compounds. the pronounced quenching of protein fluorescence by clathrochelates was observed only in the case of bsa, its intensity depends on the geometry of substituents (9-17 times). the different spatial arrangement (isomery) of carboxyphenylsulfid substituents in clathrochelates causes the distinctions in both their cd-signal induced by interaction with proteins and their effect on the protein fluorescence. the geometry of ribbed substituents is important for their binding to biomolecules (particularly proteins) and is suggested to determine the structure of the formed guest-host complex. 3d bioprinting is a new technology that revolutionized the field of tissue engineering and regenerative medicine, allowing reconstruction of living tissue and organs preferably using the patient's own cells. using a 3d printer we can design biological structures by controlling exact deposition of cells, growth factors and extracellular molecules in a spatially-controlled manner. the aim of this study was to evaluate the differentiation of human amniotic fluid stem cells (afsc) into endothelial progenitor cells using a bioinkò hydrogel photopolymerized in a 3d network resembling vascular tissue. characterization of afsc was performed by flow cytometry, followed by sorting of the cd177 + stem cell subpopulation. cd117 + stem cells were stained with cell tracker red cmtpx and then mixed with bioinkò hydrogel. printing was done using a 150 lm diameter needle, under 1 bar pressure, and 150 mm/min speed. the network models with define distance apart were printed and analyzed by fluorescent microscopy. mtt test was used to evaluate the viability of the cd117 + stem cells. our results showed that afsc remained viable as shown by mtt assay. the fluorescent microscopy images confirm the viability biochemical test showing that the cd117 + cells viability is maintained after 7 days of cultured in bioinkò hydrogel. furthermore, histological section of hydrogel showed that cells have a relatively uniform distribution forming network interactions between cells. flow cytometry assay showed that cd117+ cells expressed endothelial markers such as cd31, cd105, cd133, cd144 and vegfr2. in conclusion 3d printers are useful tools for creating three-dimensional scaffolds that mimics the cell microenvironment where different types of cells could proliferate, differentiate and crosslink with each other forming tissue-like structures. this study aims to reveal the biocompatibility, biodistribution and immunomodulatory impact on the production of inflammatory citokines of magnetite (fe3o4) nanoparticles functionalized with natural compounds with proved antimicrobial and immunomodulatory effects. co-precipitation synthesized fe3o4 were functionalized with plant-derived compounds: eucalyptol, carvone, limonene and b-pinene. characterization was done by ir, sem and hr tem, while in vitro biocompatibility was tested using endothelial human cells (fluorescence microscopy and proliferation assay). in vivo biodistribution was tested in a balbc mouse model at 2 and 7 days post-intraperitoneal injection, followed by experimental organ removal. tissue sections obtained from vital organs were stained with hematoxylin-eosin. production of inflammatory cytokines was assessed by elisa. results demonstrated that, at concentrations of 500 lg/ml, all prepared nanosystems have a good biocompatibility in vitro and in vivo, allowing the development of cultured cells and also not affecting any visible behavior and organ morphology of the mice. microscopy evaluation of the organs sections revealed that nanoparticles are not present in vital organs such as brain, heart, kidney and liver, but aggregates were visible in the lungs and spleen. at 7 days post-injection no visible aggregated were found in the lungs, few dark-brown nanoparticles clusters being visible in the red pulp of spleen. elisa results revealed that fe3o4 functionalized with carvone and limonene significantly stimulated the production of il-2, il-10 and il-6, while reducing the production of tnfa. other nanosystems din not impact significantly on the cytokine production. functional fe3o4 nanoparticles are efficient drug delivery shuttles, able to stabilize pharmacological compounds, such as plant-derived bioactives, and their biocompatibility, specific biodistribution and limited immunomodulatory effects recommend their use in pharmacological formulations. p-07.01.3-004 new approach for cell imaging with fluorescent carbon nanoparticles m. dekaliuk, k. pyrshev, o. demchenko palladin institute of biochemistry, kiev, ukraine in the nanotechnology field, much interest was focused on the new carbon nanomaterials for cell imaging. recently discovered inorganic carbon nanoparticles ('c-dots') due to their excellent fluorescence characteristics and biocompatibility have ample opportunities for their use in imaging and functional transformations in living cells. their distinctive features, such as high brightness, small sizes, high biocompatibility, small negative charge on the surface and very easy methods of their preparation present a good alternative to other nanoscale materials. the focus of our research was to determine the possibility of using c-dots as the easily available probes for apoptotic cells detection. the carbon nanoparticles were prepared from alanine, citric acid, urea, etc by hydrothermal treatment at 180 c. the studies were performed with adherent epithelial vero and hela cell lines (atcc). with these tools we demonstrate that both native and apoptotic cells can be easily visualized. the cdots uptake occurs probably by endocytosis, which allows for much larger their number to accumulate in apoptotic cells. using the different methods of sample preparation, they show the ability for labeling various structural compartments of the cell. for living cells there are the intracellular vesicles and lysosomes. in contrast, in fixed cells the nucleus is labeled preferentially. the fact that apoptotic cells accumulate strongly increased amount of cdots can be efficiently used in flow cytometry for characterizing the cell populations regarding the relative amount of apoptotic cells in different experimental conditions. the application of such cheap and easily accessible nanoparticles provides more opportunities to simplify the popular methods of cell labeling and detection. previously, our studies showed the possibility of using these nanoscale fluorophores for super resolution method sofi. a new electrochemical microbial biosensor for the fast detecting of dopamine and epinephrine based on candida tropicalis immobilized in a carbon paste electrode (cpe) modified with single wall carbon nanotube (swcnt) was described in this paper. the immobilized cells were used as a source of polyphenol oxidase (ppo) to develop voltammetric epinephrine and dopamine biosensor. voltammetric determination of phenolic compounds like epinephrine and dopamine is a simple technique available. direct oxidation of phenols can be used, but the oxidation potentials of this compounds are similar and they can not be detected distinctively. another possibility is the use of biosensors based on the polyphenol oxidase (tyrosinase) enzyme that oxidises the phenolic compounds into their corresponding quinones. by this way phenolic compounds that epinephrine and dopamine that used in this study were detected at the different potential. the effect of varying the amounts of swcnt and microorganism on the response to epinephrine was investigated to find the optimum composition of the sensor. the effects of ph and temperature were also examined. increases in biosensor responses were linearly related to dopamine concentrations between 0.1 and 1.0 mm and epinephrine concentrations between 0.01 and 1.0 mm. limits of detection of the biosensor for dopamine and epinephrine were calculated to be 0.021 and 0.0029 mm, respectively. finally, proposed systems were applied to epinephrine and dopamine analysis in pharmaceutical drugs. objective: it has started a long time ago to search for a material that can replace blood. this material does not require special storage conditions, independently of the recipient's blood group and can be applied to all individual. milk, casein derivatives, starch, saline and ringer were used for this aim in the past. the determination of toxic effect of natural hemoglobin (hb) on human, researchers have focused on development modified blood. in this work, the development of an artificial biomaterial alternative of blood for using as preoperative and operative aims was aimed. material and methods: in our study, ultrapure hb molecules are immobilized on triethanolamine coated magnetic nanoparticles using various techniques. prepared nanoparticles were characterized by ft-ir, ctem, xrd and cyclic voltammetry (cv). the cytotoxic effects of artificial blood were tried on mtt cell proliferation. results: the characteristic peaks of hemoglobin were obtained from ft-ir spectra differently from support. particles size is concluded by using debye-scherrer equation as > 80 nm from xrd spectra. sem and ctem images supported xrd result. cv results showed that hb molecule has à0.418 v cathodic potential against ag / agcl standard electrode. significant differences were not observed in the mtt results (p < 0.01). conclusion: the nanoparticles were obtained in accordance with the intended desired method. it is determined that the hemoglobin molecules give the same potential with natural blood even after 3 weeks of immobilization and carrying oxygen as natural blood. there are statistical differences between results of mtt tests due to used concentration. but, it is considered that decantation advantage of the artificial blood minimized cytotoxic effects. proteoglycans are among the most abundant molecules of the inter-cellular structure and they are present in extracellular matrices of connective tissues. these glycosylated proteins contain one or more (gag) chains that are covalently attached to the core protein and their hydrodynamic function is mainly due to the physicochemical characteristics of this gag component which provides hydration and swelling pressure to the tissue. gag levels excreted via urine are used as a marker to monitor different diseases (chronic renal disease, renal fibrosis, glomerular filtration abnormalities, bladder stones, breast and lung cancers, hypertension and diabetes, etc.) besides the well known mucopolysaccharidoses. however, their detection by using chromatographic methods is hard, because of the high polarity of negative charges and different functional groups such as acetyl sulfates that generate microheterogenity. in this study, we developed molecularly imprinted chromatographic hplc columns for specific heparan sulfate (hsa), chondroitin sulfate (cs) and dermatan sulfate (ds) detection in urine. positively charged acrylamide monomers were first polymerized by precipitation polymerization, to produce polymers which will show specific recognition for gag's via electrostatic interactions and hydrogen bond formation. these gag selective polymers were then filled in the steel hplc columns and columns eluents were chemically degraded. degradation products of gag's were examined offline column coupled with tandem mass spectrometry. the results showed that our imprinted columns separated gag's specifically and sensitively. thus, urine gag's can be specifically determined by using a gag specific molecularly imprinted column. in this study internal standart weren't used because the matrix effect was lower than 5% for each urine samples. %cv of ds, cs and hsa was calculated as; supported lipid bilayers (slb) were started to be used for cell culture studies to focus on cell adhesion, cell signaling etc. testing the stability of slbs is essential to utilize them as cell culture platforms. in this study, the stability of phosphatidylcholine (pc) lipid bilayers on glass was investigated under milli-q water, phosphate buffer saline (pbs) and dulbecco's modified eagle medium culture (dme) medium supplemented with/without serum. the stability was also checked by enriching slb with different lipids. pc-liposomes were prepared by hydrating the dried thin lipid film with pbs and then by extruding the suspension through a polycarbonate membrane. a negatively charged phospholipid, phosphatidylserine (ps, 25%); a positively charged phospholipid, dotap (50%) and cholesterol (25%) were also used for liposome preparation. liposomes were fluorescently labelled and series of slb imaging were taken for a week. in all experiments in milli-q water and pbs, the stability was conserved for 7 days. pc bilayers in medium supplemented with serum showed hole formations on the second day and their number and size increased rapidly in time. when the bilayers were prepared in medium without serum, disruption was lowered but not completely removed as a result of other factors in medium. cholesterol providing an increased rigidity to the membrane caused higher stability. positively charged bilayer structures also showed increased stability. this can be explained by decreased mobility of bilayer as a result of electrostatic interaction between positively charged molecules and negatively charged glass surfaces. decreased mobility decreases the interactions within the medium. lastly, negatively charged bilayers did not show high stability. strong repulsive forces between the negatively charged surface and bilayer probably prevented the integrity of the bilayer and increased the deformation. in recent years the use of biopolymers has gained priority in tissue engineering and biotechnology, both as dressing material and for enhancing treatment efficiency. there is a demand for new biopolymers designed with protease inhibitors and antimicrobials. ll-37 is an important antimicrobial peptide in human skin and exhibits a broad spectrum of antimicrobial activity against bacteria, fungi, and viral pathogens. using lignin which is an abundant carbohydrate polymer in nature and a polyacrylic acid, we prepared a polymer film by plastifying caprolactone and polyacyrlic acid. films were actified to immobilize ll-37. the structure was elucidated in terms of its functional groups by fourier transform infrared spectroscopy (ftir), and the morphology of the film was characterized by scanning electron microscopy (sem) before and after the immobilization process. the amount of ll-37 immobilized was determined by elisa method. 99.9% of ll-37 peptide was successfully immobilized onto the films. antimicrobial activity was determined in the film samples by quantitative antimicrobial activity method. according to the results, ll-37 immobilized film samples were effective on test organisms; gram-positive staphylococcus aureus and gram-negative escherichia coli. in bio-compatibility assays, the ability to support tissue cell integration was detected by using 3t3 mouse fibroblasts. samples were examined under transverse microscope, non-immobilized sample showed a huge cellular death, whereas ll-37 immobilized film had identical cellular growth with the control group. this dual functional film with enhanced antibacterial properties and increased tissue cell compatibility may be used to design new materials for various types of biological applications. p-07.01. [3] [4] [5] [6] [7] [8] [9] [10] in vitro modulation of the cross-talk between macrophages and osteoblasts by titania nanotube-modified ti surfaces p. neacsu, a. mazare, p. schmuki, a. cimpean bone remodeling is a dynamic process that maintains a fine balance between bone formation and resorption, and is highly influenced by the inflammatory state of the local microenvironment. therefore, a proper modulation in the cellular interactions and cytokine expression is a promising approach to achieve enhanced bone healing. as the biomaterials surface has a major impact on cellular behavior, the goal of the current study was to investigate the influence of tio 2 nanotube-modified ti surfaces (ti/tio 2 ) on the cross-talk between raw 264.7 macrophages (mf) and mc3t3-e1 preosteoblasts (ob) in mono-and co-culture systems in comparison with flat ti (cpti). raw 264.7 and mc3t3-e1 cells were seeded on the test surfaces and grown in standard culture conditions for various periods of time. for co-culture studies, the cells were cultivated using a transwell system. inflammatory mediators released by raw 264.7 cells were measured using elisa technique, while the ob capacity to produce calcified bone matrix was evaluated by alizarin red staining. in co-cultures, lps-stimulated tnf-a, il-6 and mcp-1 release was significantly increased at 24 h, while after 7 days only il-6 exhibited higher amounts when compared with mf cultures alone. moreover, the secretion of these mediators by cells exposed to ti/tio 2 was diminished, especially in lps evoked conditions. also, alizarin red staining demonstrated the presence of calcium deposits when ob were co-cultured with mf for 24 h and 7 days, whereas the presence of the mf for 4 weeks significantly inhibited mineralization. on ti/tio 2 surface elevated calcified matrix was observed, as compared with cpti. this study reveals that the overall effect of inflammation suppression induced by ti/tio 2 may contribute to the enhanced mineralization. also, chronic inflammation may inhibit or delay the regeneration process. therefore, an adequate modulation of mf and ob interactions is vital for the biomaterials success in stimulating bone regeneration. p-07.01.3-011 synthesis and characterization of the branched magnetic polymer for drug delivery systems t. tarhan 1,2 , b. tural 2 , s. tural 2 1 mardin artuklu university, mardin, turkey, 2 dicle university, diyarbakir, turkey magnetic nanoparticles (mnp) have gained a lot of attention in biomedical and industrial applications due to their biocompatibility, easy of surface modification and magnetic properties. magnetic nanoparticles can be utilized in versatile ways, very similar to those of nanoparticles in general. however, the magnetic properties of these particles add a new dimension where they can be manipulated upon application of an external magnetic field. this property opens up new applications where drugs that are attached to a magnetic particle to be targeted in the body using a magnetic field. often, targeting is achieved by attaching a molecule that recognizes another molecule that is specific to the desired target area. in recent years, the development of the systems in which drug is delivered magnetically to the target is drawing considerable attention since it is a current issue. it is possible to eliminate the most of the problems caused by high doses of chemotherapy by using the magnetic drug delivery systems. therefore, it is important to design delivery systems with high drug loading capacity. it is necessary to increase the number of reactive groups on the surface of nanoparticles in order to increase drug loading capacity. in this study, we synthesized a novel magnetic surface for drug delivery systems. magnetic dextran-nta (md-nta) was synthesized by using magnetic o-carboxymethyl dextran (ocmd) and nana-bis (carboxymethyl) -l-lysine hydrate (nta) in order to increase the number of reactive carboxyl groups on the surface of biocompatible and biodegradable magnetic dextran. magnetic material (md-nta) which was prepared and characterized by the analysis of transmission electron microscopy (tem), scanning electron microscope (sem), vibrating sample magnetometer (vsm), fourier transform infrared spectroscopy (ftir) and x-ray photoelectron spectroscopy (xps). there are three subtypes of the tgf-b protein that has been reported to be involved in tissue repair process; scar tissue formation has been reported on tissues that has been affected by tgf-b1 and 2 due to high collagen synthesis. on the other hand the other isoform tgf-b3, suppresses the dense collagen production caused by tgf-b1 and prevents the scar formation. to be able to use these growth factors local or iv route, new drug transport systems are needed to protect the bioactivity during the treatment and controlled release. for this purpose poly(lactic-co-glycolic) acid polymer which is widely used in controlled release systems was chosen as the matrix material. aim of the project was to design, formulate, prepare and optimize tgf-b3 loaded plga nanoparticular and/or plga polymeric film drug delivery systems and to test their effect on cell proliferation. tgf-b3 loaded nanoparticles was prepared with emulsion-solvent evaporation method; whereas polymeric film systems was prepared with film castingsolvent evaporation method. following the preparation tgf-b3 loaded drug delivery systems was characterized. quantification and in vitro release of the growth factor tgf-b3 was studied with elisa. hepg2 cell line was used on mtt cell proliferation assay for both tgf-b3 loaded nanoparticles and films on a time course study. nanoparticles and films were prepared and loading efficiency of the nanoparticles were found to be 42.42%. particle size, zeta index and polydispersity index for this formulation were determined as 204.9 ae 10.3 nm, 0.0381 mw and 0.380, respectively. thickness of the prepared films were 286 ae 20.16 nm. additionaly prepared nanoparticles and films were found non-toxic. tgf-b3 nanoparticles and films which were prepared in this study are planned to be used as an effective treatment strategy for wound healing after injury. this project was supported by grand 112s541 from the scientific and technological research council of turkey (tubitak). polyvinylpyrrolidone (pvp) is a biodegradable material and natural polymeric biomaterial in such studies. ganoderma lucidum is a natural material containing triterpenes, polysaccharides, adenosine, polypeptides, and amino acids. these constituents have been shown to exhibit anti-cancer properties, enhance and regulate immunity, resist oxidation and ageing, and promote metabolism and cell proliferation. composites of polyvinylpyrrolidone (pvp) have been prepared by solution intercalation method using ganoderma lucidum at different loading amounts. the characterization of pvp/ ganoderma lucidum composites was made by x-ray diffraction (xrd) and scanning electron microscopy (sem); the interactions between ganoderma lucidum and pvp was determined by ftir-atr; the thermal stability was determined by simultaneous tg/dta. hemocompatibility of the prepared composite samples were investigated by a 96-well plate spectrophotometer. in addition, contact angles and antimicrobial activity of biomaterials were also determined. ftir-atr confirms interactions formed between ganoderma lucidum and pvp. xrd and sem results give evidence that ganoderma lucidum was well dispersed and homogenously in the pvp matrix. thermogravimetric analysis indicated that introduction of clay to the polymer network resulted in an increase in thermal stability. the results of in vitro hemocompatibility test were showed that pvp/ ganoderma lucidum composites are used as biomaterial. the development of synthetic materials, textured polymers and metals and their increasing use in medicine make research of biomaterials' hemocompatibility very relevant. composite material is a multi-phase system consisted of matrix material and reinforcing material. matrix material is a continuous phase and reinforcing material is a dispersed phase. the main two bioactive components of ganoderma lucidum can be broadly grouped into triterpenes and polysaccharides. despite triterpenes and polysaccharides being widely known as the major active ingredients at anti-cancer effect. this study describes the synthesis and characterisation of biocomposites of different molecular weight of peg (polyethylene glycol) as matrix with ganoderma lucidum as a filling material at different loading (%1, %2.5, %5 wt). the composites have been prepared by solution intercalation method using ground and sieved ganoderma lucidum at 25 micron scale. the characterization of composites was made by x-ray diffraction (xrd), scanning electron microscopy (sem) and fourier transform infrared attenuated total reflectance (ftir-atr) also in this study the hemocompatibility and antibactarial properties of composite investigated. when xrd and ftir-atr results discussed, all of the composites using the different loading amunt of ganoderma lucidum (%1, %2.5 and %5 wt) were shown a homogen distribution in the matrix (peg). and an interaction have occured between matrix and filling material. the sem photos have confirmed these results. peg and composites have been detected as hemocompatible. these results showed that they can be used as biomaterials. p-07.01.3-016 evaluation of the genotoxic potential of some nanocomposites by comet assay b. yilmaz 1 , s. dogan 1 , s. celikler kasimogullari 2 1 department of molecular biology and genetics, balikesir university, balikesir, turkey, 2 department of biology, uludag university, bursa, turkey due to its similar nature to the bone, nanohydroxyapatite is a biocompatible particle and poly(methyl methacrylate) (pmma) is a polymer that has been used in dentistry and orthopedic applications for years. in this study, genotoxic potential of pmma/nanohydroxyapatite nanocomposite films composed of polymers having different molecular weights and nanohydroxyapatite fillers in different concentrations (1, 2.5 and 5%) were investigated by comet assay which is a kind of gel electrophoresis that can be used to measure dna damage in individual cells. if the dna is damaged we expect broken ends to migrate apart from the head. at the end of the assay performed after incubation with lymphocytes of healthy humans, we measured the dna damage index (ddi) and percentage of damaged cells (pdc). in addition, to prove the morphological properties of the nanocomposites scanning electron microscope was used and an interaction between the matrix and nanoparticles with a homogeneous dispersion was observed. protein adsorption on stimuli-responsive mixed pdmaema/peo polymer brushes a. bratek-skicki 1,2 , c. dupont-gillain 1 1 universit e catholique de louvain, louvain-la-neuve, belgium, 2 j. haber institute of catalysis and surface chemistry, polish academy of sciences, krakow, poland smart polymer brushes are made of macromolecules that are sensitive to stimuli from the external environment, including ph, ionic strength, temperature, etc. when stimuli-responsive polymer brushes are introduced onto material surfaces, their properties can be adjusted by tuning the environmental stimuli. these brushes can find promising applications across many areas of research, including surface science, nanotechnology, and biotechnology. in our work, the adsorption of human serum albumin (hsa, molecular weight of 66.5 kda, isoelectric point ip at ph 4.7) and lysozyme (lys, molecular weight of 14.3 kda, ip~11) was studied on polymer brushes composed of poly(ethylene oxide) (peo) and poly (2-(dimethylamine) ethyl methacrylate) (pdmaema). peo is a protein-repellent polymer and pdmaema is a polyelectrolyte bearing a variable density of positive charges depending on ph. a gold substrate was modified by these thiolated polymers according to the 'grafting to' method. the obtained polymer brushes were characterized by xray photoelectron spectroscopy, static contact angle measurements and atomic force microscopy. polymer brush formation and protein adsorption were monitored by quartz crystal microbalance. surface characterization of the mixed brushes revealed the presence of both polymers at the surface. conformational changes of pdmaema/peo brushes were experimentally evidenced, and the results indicated that the brushes collapse at ph 9.00 (pdmaema is neutral in such conditions) and were swollen at ph 3.5 (pdmaema is positively charged). protein adsorption was performed at different ph values (3.5-9 .0) and salt concentrations (0.001-0.15m). it was shown that pdmaema has a high affinity to hsa at ph above its isoelectric point. however, the adsorption of positively charged lysozyme in a wide range of ph was not observed. these results indicate that pdmaema/peo brushes are promising candidates for selective adsorption from a mixture of proteins. clay-polymer nanocomposites (cpn) developed in recent years as a new type of inorganic-organic hybrid materials that were conceived for medical uses such as tissue engineering or drug delivery [1] , [2] . the understanding of the structure and physico-chemical properties of cpn is a first step in the investigation of biomaterials, but their potential in this respect is determined by their interaction with living tissue components. in this study, pure kaolinite was intercalated with dimethyl sulfoxide (dmso) and then intercalated kaolinite was modified pyridine, 2-amino pyridine and 2,6-diamino pyridine to expand the interlayer basal spacing. modified kaolinite samples as filler and poly(vinyl chloride) (pvc) polymer as matrix were used in the nanocomposite synthesis. nanocomposites of pvc have been prepared by solvent blending method using thf as a solvent. the material characterizations were carried out by xrd, afm, ftir-atr, dta/tg and dsc. the xrd results reveal the formation of intercalation/exfoliation of modified kaolinite in the pvc matrix. ftir and afm results confirm the presence of nanomaterial in kaolinite/pvc nanocomposites. tga data show that the modified kaolinit/pvc nanocomposites have significant enhanced thermal stability. the glass transition temperature (tg) of pvc nanocomposites is higher than that of pure pvc. in addition, the antimicrobial activity of clay-polymer composites were also determined. introduction: polyhydroxyalkanoates (phas) are biocompatible and biodegradable materials obtained from microorganisms. they are produced in the cytoplasm of several bacteria as energy reserve. the physical properties of poly(3-hydroxybutyrate) (phb), which is from the group of phas, make it a competitive source to petrochemical plastics. phb has potential in order to be used in a variety of application fields such as packaging industry, printing materials, agriculture and food industry. furthermore, phb meets expectations for tissue engineering applications, since it is biocompatible, biodegradable, non-toxic and has good mechanical properties. although its many advantages, blending approach could be needed in order to fulfill all expectations of a material. due to its flexibility, polycaprolactone (pcl) is a promising candidate to be blended with phb. the aim of this study is to construct a scaffold by using phb produced by extreme alkaliphilic b. marmarensis gmbe 72 t (dsm 21297) and commercial pcl as components and investigate its properties. materials and methods: electrospinning method was used in order to construct scaffolds from blend polymer solution containing phb from b. marmarensis and commercial pcl. results: nanofiber structures were observed on scanning electron microscope (sem) images and fourier transform infrared resonance (ftir) analyses have shown characteristic peaks for both phb and pcl. discussion and conclusion: phb could be blended with other polymers in order to enrich its properties. in addition, nanofiber structure of electrospun phb-pcl blend makes it a rewarding material as scaffold for several tissue engineering applications. q fever is a zoonotic disease that is encountered widely around the world, the most common acute form of q fever shows the following symptoms; a sudden fever, shivering, lassitude, headache, anorexia. because this disease does not show specific symptoms its diagnosis is possible with laboratory tests. current diagnostic kits lack effectiveness; this is why the main goal of our studies is to come up with a new diagnostic kit that does not have disadvantages that current diagnostic kits show. with this goal, nine mile i strain (rsa 493), s serologic virulent phase i, were obtained from slovak science academy, virology institute for rickettsia reference and research from who co-operation centre. these cells were purified and lipopolysaccharide (lps) isolation from coxiella burnetii was performed. the polymeric carrier, poly (n-vinyl-2-pyrrolidone-co-acrylic acid) [p(vp-co-aa)] was synthesized and characterized. physical complexes of obtained lps and p(vp-co-aa) with varying ratios. ternary complexes of lps-cu 2+ -p(vp-co-aa) were also synthesized with copper metal mediation. structure and interaction of lipopolysaccharide-p(vp-co-aa) complexes were investigated with zeta-sizer device using zeta potential analysis and ftir spectrophotometry according to the ratios of components, reaction environment conditions and chemical structure of the polymer. the best complex ratio according to analysis results will be used in the future studies for obtaining monoclonal antibodies which will be an important step for obtaining more effective and stable diagnostic kits that can be used for q fever. this 2513 in this study; new types of water soluble polymer-biomolecule conjugates were synthesized using covalent bonding techniques between polymers and co-polymers (varying monomers of polyacrylic acid and acrylic acid) with peptides. different compositions of polymers, varying ratios of biomolecule/polymer and different molecular weight of polymer has yielded new types of bioconjugates. conjugation mechanism, composition and structure were investigated with various physicochemical methods (uv, ftir, hplc, gpc, etc.). the peptide used in this study was the antigenic peptide epitope of sheep-goat pox disease (eakssiakhfslwksyadadiknsenk). whether this peptide series was bound to polymers or whether it was bound to polymer-protein carrier; peptide-specific immunogens that were capable of producing antibodies were synthesised. it is thought that using polymer-peptide bioconjugates that contain just peptide will yield a higher peptide-specific immunogenicity compared to traditional adjuvants. in vitro and in vivo investigations of bioconjugates effectivity is planned to be done in the future studies. p-07.01.3-026 bioinert fluorinated ethylene-propylene copolymer modified for keratinocyte adhesion surface properties are crucial when adhesion of a cell to a polymeric material is required for a biomedical application. one of the methods for polymer surface tailoring is argon plasma treatment. this simple and reproducible method alters the surface properties such as roughness and wettability without affecting the bulk properties of the material. for the modification of the bioinert fluorinated ethylene-propylene (fep), related to teflonò, we employed argon plasma treatment with the powers of 3 and 8 w for 20-240 s. the human keratinocytes of the hacat cell line served as a model cell line for biocompatibility testing. we studied adhesion, proliferation and morphology of the cells on modified fep matrices as well as controls (pristine fep and standard polystyrene tissue culture dish) by means of fluorescence microscopy. further morphological details were acquired by scanning electron microscopy. furthermore, fluorescence microscopy with immunochemical labelling was used to determine the size and distribution of focal adhesions in cells grown on the modified matrices. the overall effect of the matrices on metabolic activity of cultured hacat cells was also evaluated using the wst-1 reagent. the ar plasma treatment of fep matrices improved significantly cell adhesion and proliferation and promoted spreading of the hacat cells compared to the pristine fep, on which cells were not able to spread properly. also, increased metabolic activity rates for cells cultured on modified matrices were found in comparison to the pristine fep. altogether, we found that ar plasma treatment improved the surface properties of fep to such extent, that it allows cultivation of adherent cells on its surface. we therefore propose that ar plasma treatment is a useful method for fep surface modification. p-07.01.3-027 graphene oxide enriched biomaterials display potential for tissue engineering applications tissue engineering (te) requires more efficient systems that favor local tissue regeneration with minimum cytotoxicity. materials based on natural compounds ensure biocompatibility and have better effects for regeneration. graphene oxide (go) has been shown to enhance cell adhesion and to improve the rate of cell differentiation. in this context, the aim of this study was to evaluate if the addition of different concentrations (0.25-1 wt.%) of graphene oxide (go) improves the properties of cellulose acetate (ca) materials for biocompatibility and cell differentiation, in the prospective of using these films for te applications. go-containing ca films were tested for cytocompatibility by quantitative and fluorescence microscopy assays, and compared to the ca control. cell cytoskeleton architecture in contact with biomaterials was revealed by confocal microscopy. furthermore, bioconstructs were exposed to in vitro osteogenic and adipogenic induction and monitored for 21 days. histological stainings were performed to validate differentiation. osteogenic and adipogenic markers gene expressions were assessed via qpcr. ca/go 1 wt.% revealed best biocompatibility among all tested scaffolds. adhesion proved to be dependent on the percentage of go in material's composition. cells cultivated on ca/ go 1 wt.% expressed adipogenic and osteogenic markers earlier than cells cultivated on materials with lower go content. differentiation markers displayed an increasing profile of gene expression from 7 to 21 days post induction, with higher levels registered for materials with high go content as compared to films with low go content and to the control. go added to ca materials positively influenced cell survival, proliferation and cell differentiation. ca/go films represent potential candidates for te applications. the design of appropriate scaffolds remains one of the most important challenges for te. current idea is that the cell-scaffold interaction could drive cell differentiation and be linked to gene expression and protein organization. therefore, their quality is essential and should favour cell attachment, growth, migration, in situ vascularization and release of biochemical and physical factors able to address the cell fate. moreover, for an ideal scaffolding material an adequate and interconnected porosity is relevant for facilitating cell spreading and colonization of the inner layers. a combination of optimal mechanical and biochemical properties were here utilized to design a 3d composite hydrogel scaffold (3d-chs) in order to favour cell-scaffold interactions and promote a functional differentiation of human lin à sca1 + cardiac progenitor cells (hcpc). the biocompatible peg-diacrylate (pegda) was used to prepare hybrid protein-pegda hydrogels with embedded albumin-microspheres (ms) as protein component. ms were able to modulate the mechanical and biological behavior of the scaffold acting as air-reservoir, porogen agents and potential carriers of biomolecules. an increase of the hcpc viability in the ms-concentration dependent manner was observed. moreover, the microarchitecture of the 3d scaffold also plays a key role in the stability and functionality of cellularcomposite systems. therefore, pegda-honeycomb structures were fabricated using microstereolithography process and the hcpc viability and adhesion to the microstructures were assessed. 3d-chss were synthesized embedding honeycombstructures into ms-pegda hydrogel and the effects on cell proliferation, cell-cell interactions and cellular alignment were investigated. these results could be of relevant interest for expanding the knowledge on cell-scaffold interaction processes and to promote the development and the application of 3d-chs for tissue regeneration using the emerging bioprinting technologies p-07.01.3-029 gene expressions of mesenchymal stem cells after osteogenic induction on ceraform bone substitute a. kilic s€ uloglu, e. karacaoglu, h. akel, c ß . karaaslan hacettepe university, ankara, turkey ceraformò, is a synthetic calcium phosphate ceramic with the chemical composition of hydroxyapatite 65% and tricalcium phosphate 35%, with 60-85% pore volume and 100-400 lm pore diameter. in this study adipose tissue derived mesenchymal stem cells were differentiated into osteoblast cells and loaded on cer-aformò. in order to improve cell adherence, ceraformò was covered with fibronectin. the cells were cultivated for 28-day period by osteogenic induction medium. days 1, 7, 14, 21 and 28 were selected as specific intervals for incubations. total rna was isolated and cdna was synthesized. differences in the expression of runt-related transcription factor 2 (runx2), bone morphogenetic protein-2 (bmp-2), and osteocalcin (ocn) were determined by qpcr. the peptidylprolyl isomerase a (ppia) gene was used as an internal control. according to the qpcr results, ocn gene expression was observed on the day 14th, continued to increase in day 28. bmp-2 gene expression was increased in 14, 21, 28 day compared to 7 day. on the other hand, runx2 gene expression was increased only on days 21 and 28. these findings pointed out that the osteogenic induction was successfully activated on fn coated bone material. therefore, these results can be used in bone injury treatment and related disorders. p-07.01.3-030 on the in vitro cytotoxicity of graphene oxide nanomaterials v. grumezescu 1 , i. negut 1 , f. sima 1 , e. axente 1 , l. e. sima 2 1 national institute for lasers, plasma and radiation physics, magurele, romania, 2 institute of biochemistry, romanian academy, bucharest, romania during the last decade, graphene and its derivates have proven unique physicochemical properties, several applications being continuously developed. among them, electronic, catalytic, mechanical, optical, and magnetic properties have attracted huge interests. however, the merging of graphene and graphene oxide (go) with biotechnology is still in its infancy, many challenges remaining unexplored. potential applications are related to biosensors, drug delivery or gene therapy and cells imaging. in order to use gos as drug release matrices for cancer cells targeting, it is necessary to ensure that these molecules do not affect normal cells within tissues. it was shown that the cytotoxicity of graphene nanomaterials is highly dependent on surface functionalization. studies suggest that pristine and reduced go with fewer surface functional groups tend to be more toxic than go. in striking contrast, it has been reported that functionalized graphenes, can significantly reduce the cytotoxicity even at relatively high concentrations. in this study, we report on the comparison between go and protein functionalized go when submitted to in vitro cytotoxicity tests. bovine serum albumin (bsa) was used for the noncovalent go surface conjugation. three case-studies were investigated: aqueous nano-colloids consisting of serial dilutions of both go and go-bsa conjugates, dropcasted thin films and laser-assembled thin films on glass substrates. safe concentration windows were identified by live/dead staining and mts assays for different human melanoma cell lines, while melanocytes and human dermal fibroblasts were used as normality controls. the predominant melanoma subtype is represented by cells bearing braf (v600e) activating mutation. with a view to target this specific melanoma subpopulation, we embedded braf inhibitors into go laser-deposited scaffolds and tested their anti-tumoral effect. our results evidence the high potential of these nanomaterials for biomedical applications. osteoporosis is a skeletal disorder associated with low bone mass and increase in bone fragility due to increased osteoclastic activity. binding of rank ligand to its receptor on osteoclast precursor cells results in the osteoclast differentiation. sirna is a dsrna, used to inhibit the translation of the target gene. the aim of the study is to develop an injectable sirna-delivery system targeted to the bone for osteoporosis treatment. pei (polyethyleneimine) and rank complex was loaded into poly(lactic acid-co-glycolic acid) (plga) nanocapsules which are bound to hydroxyapatite (hap)-specific elastin-like protein (elp) for targeting to bone tissue. plga nanocapsules were prepared by w/o/w double emulsion technique. affinity of elp to hap was determined by ftir. elp was coated on the nanocapsules by using the transition temperature of elp. elp on plga nanocapsules were crosslinked by genipin and binding of elp on plga nanocapsules were studied by xps and tem. the optimum ratio of n (pei) to p (sirna) in the complexes to be loaded into plga nanocapsules were studied by etbr staining and zeta potential measurements with varying n/p ratios and finally pei-dnaoligo encapsulation efficiency of the capsules was determined by picogreen reagent. xps results of elp treated plga (elp-plga) nanoparticles indicated the presence of nitrogen atom (11.7%) in the sample which appeared as a fuzzy halo in tem micrographs. n/p ratios up to 5 show negatively charged particles. when n/p was 5, the zeta potential of complex was neutralized which also resulted in larger particles compared to the others. zeta potential moved to positive values when n/p was higher than 5. the migration of polyplexes with different n/p ratios (1-10) was analyzed by gel electrophoresis. dnaoligo complexes show the same patterns of complexation wih that of sirna and thus were used in the encapsulation efficiency studies instead of sirna. the encapsulation efficiency of pei-dnaoligo in plga nanocapsules was 29%. tuesday 6 september 12:30-14:30 aging p-09.03.3-001 novel benzenesulfonamides exhibit low toxicity on zebrafish embryonic development and selectively inhibit carbonic anhydrase ix with nanomolar affinity in xenopus oocytes introduction: the toxic effects of two recently discovered inhibitors (vd12-09 and vd11-4-2) that selectively and with extraordinary strong, picomolar affinity bind to human carbonic anhydrase (ca) ix, an anticancer target, were investigated on zebrafish embryonic development and in xenopus laevis oocytes. zebrafish has emerged as a promising animal model to evaluate the toxicity of the drug candidates. xenopus oocytes do not natively possess any ca activity and thus became a convenient in vivo model system to study the ph effects and the selectivity of synthetic ca inhibitors. materials and methods: morphological changes in zebrafish treated with the compounds were studied by light-field microscopy and histological analysis. ca activity in xenopus oocytes was monitored by measuring ph in the cytosol and at the outer membrane surface and confirmed by mass spectrometry of lysed oocytes. the toxicity studies showed lc50 values to be 13 lm for vd12-09, 120 lm for vd11-4-2 and 9 lm for ethoxzolamide (eza), a non-selective ca inhibitor commonly used in clinic. the zebrafish exposed to lc50 doses of vd12-09 and vd11-4-2 showed fewer phenotypic abnormalities and less morphological changes compared to the zebrafish treated with the corresponding dose of eza. vd11-4-2 exhibited 10-25 nm ic50 for both intracellularly and extracellularly expressed ca ix in xenopus oocytes while exhibiting strong selectivity over ca ii, ca iv and ca xii. discussion: interestingly, the compounds exhibited 10-fold lower toxicity, induced fewer side effects in zebrafish than eza and the amount of vd11-4-2 needed to cause complete inhibition of ca ix enzymatic activity in xenopus oocytes was 30-fold lower than eza. conclusions: vd compounds did not lead to deleterious effects on the zebrafish embryonic development and reached the ic50 of 10 nm for ca ix in xenopus oocytes. the compounds could be further developed as anticancer drugs. cacybp/sip is present in various cells and tissues, both normal and pathological. in normal tissues, e.g. stomach or colon, cacybp/sip is weakly or barely detected whereas in gastric or colon cancer this protein is expressed at a high level. there are also data indicating that the level of cacybp/sip expression correlates with tumor metastatic potency and multidrug resistance. taking into consideration data that suggest association of cacybp/sip with many vital cellular processes, in this work we decided to investigate the possible mechanism involved in regulation of cacybp/sip gene expression, mainly by transcription factors and, on the other hand, the influence of cacybp/sip on the expression of other genes. we have shown that nfat (nuclear factor of activated t cells) influences the cacybp/sip gene expression and that overexpression of cacybp/sip has an effect on the level of ap-1 and on the activity of nfat and ap-1 transcription factors. by analyzing the cacybp/sip gene promoter sequence we also found potential binding sites for transcription factors from the stat family, which are involved in interferon signaling. microarray data indicate that indeed overexpression of cacybp/sip affects levels of the stat proteins as well as of some interferons and interleukins. based on functional analysis we have found many genes the products of which are involved in immune response. to analyze in more detail the influence of an altered level of cacybp/sip on interferon signaling pathways as well as on factors involved in expression of interleukins, including nfkappab, we plan to apply methods such as luciferase assay, real-time pcr or immunocytochemistry. one of the pathological hallmarks of alzheimer's disease (ad) is the neuritic plaques occurred as a result of the extracellular accumulation of aß peptides formed from amyloid precursor protein (app) via the ß-amyloidogenic pathway. aß42 is more prone to aggregation to form plaques and more toxic to neurons than aß40. in addition to change in app metabolism, the decline in levels of neurotransmitter acetylcholine and cholinergic dysfunction are also observed in ad. thus, current strategies for ad treatment focus on compounds with inhibitory effect on cholinesterases as well as preventive effect on aß aggregation. in our earlier studies, toluidine blue o (tbo), a phenothiazine dye, was shown to be a highly effective inhibitor of cholinesterases with k i values in nm range. we also found that intracellular app and aß42 levels are reduced in human neuroblastoma cells after treatment with tbo. additionally, an earlier study revealed that tbo has a selective inhibitory effect on tau aggregation, the other pathological characteristic of ad. the aim of this study was to investigate whether tbo may effectively lower the level of extracellular aß40/42 in an ad-like cellular model. chinese hamster ovary cells that express human wild type app and presenilin 1, namely ps70, were treated with a dose range of tbo (0-15 lm) or vehicle control for 24 h. after treatment, aß40/42 levels in cell culture media were assayed by separate sandwich-based elisas and normalized to total protein levels, determined by bca protein assay. besides, biocompatibility of tbo was evaluated in the ps70 cells using cell viability assay for flow cytometry. strikingly, all dose ranges of tbo inhibited both aß40 and aß42 secreted into the cell culture media. significant reduction for both aß species was evident at 5 lm (p < 0.05), 10 lm (p < 0.001), and 15 lm (p < 0.001) of tbo vs. vehicle control. in conclusion, these results support the idea that tbo may be used as a therapeutic in ad. monitoring the changes of key molecules participating in the osmo-regulatory response of nucleus pulposus intervertebral disc cells during stress-induced senescence e. mavrogonatou, d. kletsas ncsr 'demokritos', athens, greece introduction: intervertebral disc cells are faced with a harsh extracellular milieu characterized by hyperosmotic conditions, nutrient and oxygen deficiency because of the absence of vascularization and oxidative stress due to the accumulation of their metabolism's by-products. we have previously shown that high osmolality is anti-proliferative for disc cells through the activation of the g1 and g2 cell cycle checkpoints by p53 and p38 mapk, respectively. in addition, we have shown the participation of nine solute transporters, with the a1 subunit of na + /k + -atpase being central in this response. here we assessed the changes in the expression of these key osmo-regulatory molecules during in vitro stress-induced senescence. materials and methods: changes in cell cycle progression were assessed using flow cytometry; overall transcriptional alterations were assessed by whole-genome arrays; differences in expression at the mrna and protein level were revealed by quantitative rt-pcr and western blotting, respectively; knocking-down of selected proteins was performed by sirna. results: high osmolality led to the differential expression of > 200 genes, including nine genes encoding transporters. p38 mapk and p53 were demonstrated to differently participate in the regulation of the aforementioned transporters, while knocking-down of three selected transporters had a distinct outcome on the overall cellular response towards hyperosmotic stress. these molecules were found to show differences in their expression in senescent cells. discussion: given that the presence of senescent cells has been demonstrated in the intervertebral disc in vivo and could most probably attributed to the prevailing stressful conditions, here we showed differences in the expression profile of known key molecules for osmo-adaptation during senescence. conclusion: understanding disc cells' physiology is of outmost importance when designing cell-based therapies for disc degenerative disorders. p-09.03.3-005 smad specific e3 ubiquitin protein ligase 2 (smurf2) and its potential effects on inhibitory transmission in aging adams 1, 2, 4, 5 1 interdisciplinary program in neuroscience, bilkent university, ankara, turkey, 2 national nanotechnology research center (unam), bilkent university, ankara, turkey, 3 department of molecular biology and genetics, bilkent university, ankara, turkey, 4 molecular biology and genetics zebrafish facility, bilkent university, ankara, turkey, 5 department of psychology, bilkent university, ankara, turkey smad specific e3 ubiquitin protein ligase 2 (smurf2) is part of the tgf-b signaling pathway associated with cellular proliferation, differentiation, genomic stability and senescence. moreover, smurf2, via its downstream partners, may regulate inhibitory synaptic transmission. our research group previously found that the smurf2 transcript is significantly higher in old zebrafish brains. thus, smurf2 may alter inhibitory synaptic transmission in aged animals. the focus of this study was to examine age-related changes in smurf2 protein levels and related key inhibitory synaptic proteins; gephyrin (gep), a scaffolding protein for gaba receptors, and gaba a , an ionotropic gaba receptor subtype. additionally, the levels of those proteins were studied in a mutant zebrafish line, which lacks acetylcholinesterase (ache) and is suggested to be a delayed aging model. whole brain tissues were isolated from young, middle-aged and old male and female zebrafish brains (ab/wildtype strain), as well as from old male and female ache mutant zebrafish (ache sb55/+ ). animals were maintained and raised in standard conditions. the extracted brain tissue was homogenized in ripa buffer and subjected to western blot analysis to determine differences in the relative protein expression levels. our preliminary data indicated that smurf2 and gep levels remain stable in the aging brain (p = 0.301, p = 0.335), and in the ache mutants gep levels are increased compared to the wildtype controls (p = 0.001). further analysis of the relationships between smurf2 and gaba a levels and brain aging is ongoing. we predicted that alterations in smurf2 levels would parallel changes in key synaptic inhibitory proteins during the aging process, which was the case for the gep levels. while smurf2 may regulate inhibitory synaptic transmission, the exact roles of those synaptic proteins in the context of normal and delayed brain aging are not known well-understood and the subject of continuing study. in recent years, express the hypothesis that aged individuals are vulnerable to infectious and other inflammatory agents and they become more prone to develop majority of severe age pathologies, including cardiovascular and oncology diseases, neurodegenerative diseases, type 2 diabetes mellitus and inflammatory diseases, etc. one of the central components of immune response is the family of toll like receptors (tlr). there are several opinions that single nucleotide polymorphisms (snp) leading to a loss of function of the respective tlrs can be associated with age and increase the risk of age related diseases, especially cardiovascular diseases (cvd). however, many available studies focusing on tlr snps and cvd are with conflicting results. the aim of this study was to assess the potential interaction between genetic variants of tlr2 and tlr4 and ischemic heart disease (ihd) in kazakhstan population over 45 years old. we evaluated 148 patients with ihd and 144 healthy subjects aged 45 years and over (ethnical kazakhs and russians living in republic of kazakhstan). polymorphic loci of the genes tlr2 rs5743708 and tlr4 rs4986790 were genotyped by pcr with subsequent restriction analysis. our results indicated that the genotype and allele frequencies of tlr2 (arg753gln) and tlr4 (asp299gly) were not significantly different between the 2 groups (p ≥ 0.05). statistical analysis didn't elicit any association between studied gene polymorphisms and predisposition to ihd in individuals over 45 years old (p ≥ 0.05). for these polymorphisms, age, fasting blood sugar and serum lipid levels were not also significantly different among different genotypes in the ihd and control groups. in conclusion, the data shows that there is no interaction between tlr2 and tlr4 and ischemic heart disease (ihd) in kazakhstan population over 45 years old. we plan to include other types of polymorphisms in tlr 2 and tlr 4 genes and increase the volume of patient cohort in our future studies. p-09.03.3-007 evaluation of prognosis with total oxidant/ antioxidant status and some oxidative stress parameters in patients with acute ischemic stroke stroke is the third most common cause of death after coronary heart disease and cancer. strokes are classified into two groups according to their pathology: ischemic stroke and hemorrhagic stroke. ischemic strokes make up 87% and hemorrhagic strokes 13% of all strokes. during ischemic stroke, oxidative stress has been shown to play a major role in the occurrence and progression, formed oxidants also affect cell membranes and genetic material such asdna, rna, and various enzymatic events, and they lead to cell damage. some studies have shown oxidant-antioxidant status but have not shown the relationship with prognosis. this study has investigated the relationship between prognosis and total oxidant/antioxidant status and biochemical parameters in patients with acute ischemic stroke 58 patients, with acute ischemic stroke and 37 healthy controls we reenrolled in the study. blood samples were taken within 1st and 7th days, and after 3rd months in the patient group for analysing serum total oxidant status (tos), antioxidant status (tas), catalase, arylesterase, and thiol. prognosis was evaluated with national institutes of health stroke scale (nihss) and-modifiedrankinscale (mrs) scores. there was no significantly difference between groups by means of serum tas, tos and catalase levels. but arylesterase (p: 0.07) and thiol (p: 0.031) levels were significantly higher in first 24 h blood samplingthancontrolgroup. statistically significant negative correlation was observed between the 3rd month values of tos and nihss score (r = 0.410, p = 0.037). but there was no correlation between mrs scores and serum tas, tos, catalase, thiol and arylesterase. similarly, our findings suggested some serum oxidant levels were increased in acute ischemic stroke patients and total oxidant status might be used in evaluation of prognosis but larger studies are needed. p-09.03.3-008 amylin and preptin regulate glucose homeostasis in infertile women with polycystic ovary syndrome and poor responders undergoing ivf/icsi disrupted glucose homeostasis leads not only metabolic disturbance such as polycystic ovary syndrome (pcos), but also influences oocyte growing. this study was designed to evaluate follicular fluid (ff) and serum levels of glucoregulatory hormones, amylin and preptin, in infertile women with pcos and poor responders undergoing ivf/icsi. human follicular and serum were obtained from 20 infertile women with pcos and 20 poor responder participants undergoing controlled ovarian stimulation (cos) with gonadotropin-releasing hormone antagonist protocol for ivf/icsi treatment. ff and serum amylin and preptin levels were measured by elisa. it was found that ff and serum amylin and preptin were lower in infertile women with pcos when compared with poor responder participants. ff amylin and preptin concentrations were lower than that of the serum amylin and preptin concentrations. decreased follicular fluid amylin and preptin levels suggest that amylin and preptin may have a physiological role in follicular maturation via controlling local glucose homeostasis. despite high serum levels of amylin and preptin in pcos their low concentration within the follicle may be main culprit of defective folliculogenesis seen in pcos subjects. similar to insulin resistance in pcos subjects existence of amylin and preptin resistance support the critical role of both peptides in follicular maturation in pcos. keywords: follicular fluid; amylin; preptin; polycystic ovary syndrome; infertility. the transcription initiation on p3 promoter of xp10 bacteriophage in presence of p7 protein, a modulator of rna-polymerase activity a. shadrin g.k. skryabin institute of biochemistry and physiology of microorganisms, ras pushchino, russia many bacteriophages are able to manage the transcription system of their bacterial host for their own needs. for example, bacteriophage xp10, in the early stages of infection of xanthomonas oryzae inhibits transcriptional activity of bacterial rna-polymerase on majority of promoters via p7 protein, except of bacteriophage p3 promoter responsible for expression of the bacteriophage 'middle' class genes. the focus of this work is to study the mechanism of action of p7 protein in the transcription initiation and identification of the role of the individual elements of p3 promoter of xp10 bacteriophage, enabling x. oryzae rna-polymerase escapes inhibition by p7 protein. we have designed a set of promoter probes representing the combination of sequences of p7-resistant p3 promoter and p7sensitive t5n25 promoter. using fret-based assay it was shown that the truncated probes corresponding to promoter dna downstream à26 position, relative to the transcription initiation start site, did not lead to dissociation of the sigma-factor. longer probes, containing à35 promoter element, induce dissociation sigma-factor. the in vitro transcription experiments show that the deletion of region 4, a sigma-factor domain responsible for interaction with à35 promoter element during the transcription initiation, is not critical for inhibition of rna-polymerase by p7 protein. promoter probe with up-element of p3 promoter had affinity to x. oryzae rna-polymerase a several times higher than a probe containing the consensus up-element for e. coli rna-polymerase . summing up the results, it seems like the transcription initiation on p3 promoter of bacteriophage xp10 can escape inhibition by p7 protein through a high affinity interaction between the up-element and c-terminal domains of the alpha subunit of rna-polymerase x. oryzae. p-09.03.3-010 distribution of soluble form of glial fibrillar acidic protein in the different areas of gerbils brain during development and aging y. kovalchuk, g. ushakova oles honchar dniepropetrovsk national university, dniepropetrovsk, ukraine astrocytes are the most abundant cell type within the cns and play an important role in cns homeostasis and function. glial fibrillary acidic protein (gfap) forms the main astrocytic intermediate filament (if). the overall level gfap in different parts of the brain uneven and depends on the number of astrocyte cells. gfap is very sensitive to any kind of neurodegenerative diseases and aging. during aging, a glial reaction is observed in the human brain, as well as in rat and mouse brains. the aim of our study was to investigate the quantitative astrocytes-specific protein gfap in different areas of the gerbils brain at the first stages of postnatal development and aging. for the study 30 gerbils brains were used and divided into 5 groups (n = 6): 1: newborn animals (1 day), 2-4: 30, 90 and 180 days respectively, 5: animals aged 2 years. the animals were decapitated under mild anesthesia (thiopental), with isolated brain three divisions: the cerebellum, thalamus and hippocampus, which are then used to produce cytosolic protein fractions. the level of gfap in the obtained fractions were determined according to the method of competitive elisa. newborn gerbils found no significant content of soluble form of glial fibrillar acidic protein in all investigated parts of the brain, and a sharp increase of amount within 30 days (in cerebellumamounted to 1.12 ae 0.03 lg/100 mg tissue; to 90-180 days increased to 1.67 ae 0.11 lg/100 mg tissue, and began to grow again in older individuals aged 2 years). unlike the cerebellum, the level of sgfap in hippocampus and thalamus reached the maximum at 30 days p.d. (1.5-1.7 lg/ 100 mg tissue), and unchanged for 180 days. these results revealed that the most intensive development of astrocytes in the cerebellum to 90 p.d. of gerbils, and in the thalamus and hippocampus are formed within the first month of life. the plastid-nucleus located protein whiry1 acts as an upstream regulator of leaf senescence binding to the promoter of senescence associated genes (sags) like senescence marker gene hvs40. in order to investigate the impact of whirly1 on drought stress-induced senescence, transgenic barley plants with a knockdown of whirly1 (hvwhy1kd) were grown under untreated and drought stress conditions. the leaf senescence evolution was monitored by physiological parameters and gene expression studies of senescence and drought stress related genes. to reveal the epigenetic indexing at hvs40 at onset of drought-induced senescence in wild type (wt) and hvwhy1kd lines, stress-responsive loading with histone modifications at 6 gene regions of hvs40 (2 regions in the promoter, one region around translation start site and 3 regions located in the gene body) was analysed by chip and quantified by rtq-pcr. in barley, drought treatment caused acceleration of leaf senescence in wildtype (wt) plants, whereas why1kd lines showed a staygreen phenotype. expression of senescence-associated and drought stress responsive genes expression was delayed in hvwhy1kd indicating that whirly1 protein acts as an upstream regulator of drought stress-induced senescence. the chip results showed that drought treatment is causing in wt a significant increase in the levels of h3k9ac all over the analyzed gene regions, correlating with a massive induction of hvs40 expression, while drought stress caused no substantial increase of h3k9ac in why1kd plants. the results suggest that drought induced expression of hvs40 is under epigenetic control, and furthermore that why1 is involved in this epigenetic control level. oncolytic viral therapy is based on the capabilities of selective lysis of tumor cells and is a prospective trend in cancer disease treatment. in vitro experiments showed that plant rhabdoviruses does not have any direct cytotoxic effect upon sarcoma 37 cells, causes induction of apoptosis in these cells and does not pose any threat to somatic cells of warm-blooded animals, which makes it possible to use this virus for therapy of malignant neoplasms. buckwheat burn virus (bbv), the prototypic member of the family rhabdoviridae, contains surface glycoprotein and which is lectin-active. its carbohydrate branch can aid adhesion of lymphocytes to tumor cells. the present study has addressed the effect of bbv on cancer cell viability. all studies were carried out after 1 week of inoculated with erlich cancernome (2 9 10 6 cells/animal, i. p.) in 2 months male balb/c mice treated at once with or without plant extract with bbv (15 mg/kg, i. p.). by fluorescent microscopy and using two due staining by acredine orange and propidium iodide it was found that in the 3rd day of administration of bbv lead to increasing of necrotic and apoptotic cells on 45% and 4% respectively versus to untreated group. at the same time the viability of investigated cells was impaired too and according to flow cytometry analysis using propidium iodide the amount of dead cells was elevated by fivefold (17.7% versus 3.5% in untreated group). also as was shown in previously reports bbv decreased activity of macrophages in the early stages after injection and it may have a positive effect when using this drug in tumor therapy. when using this drug appears to slow down the possibility of a sharp activation of macrophages, and as a consequence of the development of cytotoxic effect will be prolonged. key words: rhabdoviruses, buckwheat burn virus, cancer, cell viability. plants are considered as one of most promising sources for new antimicrobials, based on the evidence of their use in folk medicine to treat various infectious diseases since ancient times. despite relatively small area size, armenia has large diversity of flora with many endemic species. the main goal of this study was the screening of various parts of 28 herbs (widely being used in armenian folk medicine) for their antimicrobial activities in order to select most prospective plants for further comprehensive studies. plant crude extracts were obtained with maceration technique using five solvents: water, methanol, chloroform, acetone and hexane. agar well diffusion assay was used to evaluate antimicrobial properties of plant crude extracts at 500 lg/ml concentration against escherichia coli vkpm-m17, pseudomonas aeruginosa grp3, bacillus subtilis wt-a1, salmonella typhimurium mdc 1754 and staphylococcus aureus mdc 5233, candida albicans wt-174 and candida guilliermondii hp-17. statistical analysis was done using graphpad prism 5.03. crude extracts of all tested plant materials expressed antimicrobial activity against at least one test strain. most of the tested extracts inhibited growth of both gram-negative and gram-positive bacteria. in contrast, only some plant materials exhibited inhibitory activity against yeast strains. according to obtained data sanguisorba officinalis, rumex confertus, hypericum alpestre, lilium armenum and agrimonia eupatoria possessed the highest and broadest antimicrobial activity. moreover, the results showed that acetone was the most effective solvent for solubilizing antimicrobial compounds from plant materials followed by methanol, chloroform, hexane and water. the results demonstrated high antimicrobial activity of medicinal plants used in armenian traditional medicine. five plant species were selected for further comprehensive studies. besides, acetone was proposed as efficient solvent in antimicrobial screening protocols. p-02.08.5-004 effects of aluminum stress on photosystem-i apoprotein a2 gene (psab) transcription level in lichen xanthoria parietina (l.) th. fr. unal € ozakc ßa ege university, izmir, turkey in this study the effects of shortrerm aluminium (al) toxicity on the lichen xanthoria parietina (l.) th.fr. were investigated at physiological and transcriptional level. lichen thalli were treated with alcl 2 in different doses (0.25, 0.5, 1 and 5 mm). lipid peroxidation and chlorophyll integrity were determined by spectrophotometer. expression level of psab gene was also investigated. chlorophyll a content was significantly (p ˂ 0.05) decreased after 48 hours treatment with 1 mm and 5 mm of al, while chlorophyll b content was increased significantly due to treatment with increased concentration of aluminum. also treatment with 0.25 and 0.5 mm al for 24 hours increased the gene expression level of psab by 35.6% and 21.3% respectively. our results indicated that aluminum treatment has decreased the chlorophyll biosynthesis and increased the lipid peroxidation depending on time and concentration. this study also demonstrates that the psi can be readily photo-inhibited by aluminum stress. in conclusion, 5 mm al exposure for 48 hours could damage the electron transport in photosystem i. p-02.08.5-005 nigella sativa reduces paracetamol-induced nephrotoxicity and oxidative stress in rats: biochemical evaluation background: nigella sativa l. (ranunculaceae) (ns) is traditionally used to treat many conditions such as inflammation. this study evaluates the effects of ns seeds ethanol extract in paracetamol-induced acute nephrotoxicity in rats. material method: forty-eight female wistar albino rats were divided into eight groups: i = sham; ii = sham + 1000 mg/kg ns; iii = sham + 140 mg/kg (n-acetyl cysteine) nac; iv = 2 g/ kg paracetamol; v = 2 g/kg paracetamol + 140 mg/kg nac; vi, vii and viii = 2 g/kg paracetamol + 250, 500 and 1000 mg/kg ns, respectively. paracetamol administration (oral) was carried out 1 h after ns and nac administrations (oral), and all animals were sacrificed 24 h later. result: urea and creatinine levels were determined in serum, while glutathione, malondialdehyde levels and superoxide dismutase activity were determined in the kidney tissues. there were significant increases in the serum levels of urea and creatinine in the paracetamol-administered group. serum levels of urea and creatinine were decreased in all groups administered ns with paracetamol. ns administration dramatically restored sod, gsh, and mda levels in the kidneys. conclusion: the results suggest ns has a significant nephroprotective activity on paracetamol-induced nephrotoxicity. it may be suggested that the antiinflammatory and antioxidant effects of ns ethanolic extract originated from different compounds of its black seeds. p-02.08.5-006 the study of problems of preservation of the birches e. shadenova 1,2 , e. zhumabekov 2 , m. sembekov 2 , m. burchaeva 2 1 institute of general genetic and cytology, almaty, 2 laboratory of genetics and reproduction of forest culture, institute of general genetics and cytology, almaty, kazakhstan nature of deciduous trees have a whole range of various medicinal properties. instead of synthetic hormone substitutes, you can use medicinal infusions and decoctions of natural phytohormones are widely used in both folk and professional medicine. one of these plants is birch, its young leaves and buds. however, they also must be used with caution because overdose of these compounds is very dangerous, not only can you not get the desired effect, but also face the opposite of his action. in our research to mass replication of plants (different types of birches (betula ajanensis, yarmolenko, jacguemontii, maximowiczii, ulmifolia, middendorffii, kelleriana, tianschanica)) we use nutritional medium excluding the application of phyto promoters in order to prevent mutation. the object of research serve as the old, the sick, being on the verge of extinction, mature trees as explant meristema. since from the moment of calling experience and most cultivation occurs at nutritional medium without hormones. as a result of molecular analysis we get without virus, genetically identical plants. molecular certification of different types of birches of interest, both in terms of organizing, and in terms of selection and genetic improvement of valuable forms, identification of lines selected from natural populations and clones obtained in vitro. relationship between clones and installed parent form by comparing profiles amplific pcr products using issr-marking. according to the results of carried out works really recovered clones obtained from one source tree, indicating the potential for certification of clones studied forms of birches pcr. a study performed in the framework of the state grant project "conservation of breeding valuable species of birches". p-02.08.5-007 fractionated triterpenoid glycosides from sea cucumber inhibit invasion and metastasis in human cancer cells sea cucumbers are slow-motioned invertebrates. holothuria polii delle chiaje, 1824 is widely distributed sea cucumber in _ izmir coastline (turkey). it secretes saponins i.e. triterpenoid glycosides (ttg) as secondary metabolites. the aim of this study is to evaluate anti-invasive and anti-migrative effects of fractionated ttgs obtained from h. polii on ht-29, t84 and upci-scc-131 cancer cell lines. the semi-purified ttgs was extracted from h. polii collected from coast of _ izmir-dikili. the four different fractions (fraction a-d) were collected by using hplc (high-performance liquid chromatography) and characterizated with maldi-ms/ ms. the fractions obtained from h. polii extract include holothurin a (1243.50 m/z) and 24-dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer (1227.50 m/z). anti-invasive and anti-migrative effects of the fractions on the cancer cell lines were detected with xcelligence rtca dp system. the results showed that fraction a-d inhibited migration and invasion of human cancer cell lines at 6th and 12th hours compared to control group. this study shows that holothurin a, 24dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer could be evaluated as promising anti-cancer agents for human cancers. acknowledgement: the authors acknowledged the scientific and technological research council of turkey (t € ub _ itak) for financial support (114z211). p-02.08.5-008 alternative splicing regulation of sr proteins in response to environmental stress in chinese cabbage serine/arginine-rich protein (sr protein) family, which acts as rna-binding protein, plays a major role in post-transcriptional regulation of pre-mrna, such as alternative splicing (as). these proteins cause pleiotropic effect by regulating as of pre-mrna in a tissue and developmental stage-specific and stress-responsive manner in arabidopsis. here, we identified 31 genes encoding sr proteins in chinese cabbage (brassica rapa chiifu-401) from brassica database and analyzed their phylogenetic relationship. b. rapa has 31 types of sr protein that are classified into common (sr, rsz and sc) and plant specific (scl, rs2z, rs and sr-like) subfamily similar with arabidopsis. interestingly, the as pattern of most sr genes changed at the late stage (14 and 21 days after germination). to verify the correlation between sr genes and environmental stress, we screened the as pattern of sr genes to various abiotic stress using rt-pcr and a microarray analysis. in particular, the expression level and the as pattern of bra015576 and bra018581 were affected significantly by heat stress. these results suggest that the as regulation by sr protein correlates with adaptation mechanism to the environmental stress in chinese cabbage. p-02.08.5-010 characterization of recombinant prolyl oligopeptidase from myxococcus xanthus and potential use in gluten hydrolysis e. k. kocaazorbaz, f. zihnioglu faculty of science, biochemistry department, ege university, izmir, turkey a recombinant prolyl oligopeptidase from myxococcus xanthus was purified with a specific activity of 224 u mg(-1) by using nickel-metal-chelate affinity chromatography and gel permeation chromatography. the recombinant enzyme had a monomeric molecular weight of 70 kda. its isoelectric point, determined by two dimension polyacryl-amide gel electrophoresis, was close to 6.3. the optimum ph and temperature was estimated as 7.5 and 37°c, respectively. the purified enzyme was stable from ph 6.0-8.5 and able to thermal stability up to 37°c. the k(m) and v(max) values were 0.2 mm and 3.42 micromol/ min per mg. the enzyme exhibited hydrolytic activity for suc-gly-ala-pna, suc-gly-pro-pna, z-gly-pro-pna, igf-1, substance p, whereas no activity for h-gly-pro-pna, h-val-ala-pna, h-arg-pro-pna, h-ala-pro-pna, glu-ala-pna, pro-pna, leu-pna. its proteolytic activity was inhibited by activesite inhibitors of serine protease, z-pro-prolinal pmsf, and metal ions, cd 2+ and hg 2+. the potential use of the enzyme was tested by the hydrolysis of the wheat gluten. the resulting gluten hydrolysate were characterized by means of their antioxidant, antibacterial, trypsin inhibition and prolyl oligopeptidase inhibition activities. keywords: serine protease, prolyl oligopeptidase, bioactive peptides, 2,4,6-trinitrobenzene sulfonic acid. proteomic analysis is probably the best approach to analyze seed germination. however, it is difficult to analyze complex samples and there are many obstacles that must be faced in order to achieve a reasonable proteome coverage. for example, the barley (hordeum vulgare) genome was fully sequenced in 2012, but the uniprot database contains less than 1000 reviewed sequences, which is approximately 16-fold less than for arabidopsis thaliana. here, to improve the barley proteome coverage, we employed several fractionation methods including polyethylene glycol precipitation, strong cation exchange chromatography, off-gel separation, sds-page and acetonitrile elution gradient. proteomic analyses were performed using an lc-ms-based analyses and an uhr q-tof mass spectrometer. the candidate peptides were targeted via selected reaction monitoring (srm) and triplestage quadrupole (tsq) mass spectrometer. in total, 4092 proteins were identified, which represents a three-to four-fold increase compared with the standard shotgun analysis of the same sample. out of these, 2240 were only accessible by one of the techniques and, besides, the detection limits were not similar. we hypothesized that an srm-based targeted analysis will allow detection and quantitation of most of these proteins, even without the application of proteome fractionation. we can conclude that all peptides from the library with ms/ms spectra of the total intensity above 10,000 are easily detectable in the total protein extracts. p-02.08.5-012 transcriptome sequencing based identification of alternative oxidase genes in white waterlily, nymphaea alba alternative oxidases (aoxs) are the terminal oxidases in the respiratory electron transport chain of plants. they reduce molecular oxygen to water with low proton translocation across the inner mitochondrial membrane. in plants, aoxs increase local tissue temperature to release volatile compounds thereby attracting pollinator insects and regulation of mitochondrial retrograde signaling pathway. regulation of retrograde signaling pathway is currently under investigation to improve cultivation studies in many plants. water lilies are aquatic ornamental and economically valuable plants classified under nymphaea family. nymphaea alba, white water-lily, has a special focus since its applications in landscaping of parks and gardens, farming as vegetable and medical applications. however, cultivation of n. alba is a challenging process. we hypothesized that by controlling alternative oxidases, success rate can be increased for n. alba cultivation. to identify alternative oxidase encoding genes in n. alba, we performed transcriptome analysis. by using transcriptome analysis data, aox gene sequences, subcellular localization of aox proteins and structural modelling of aox proteins were predicted. in 272934 transcripts, database search with trinotate tool revealed 77 transcripts with aox domains characterized in known alternative oxidases. blast analysis of these 77 sequences with known aox proteins revealed three distinct aox genes (nalba-aox1, nalba-aox2 and nalba-aox4). after subcellular localization analysis of three identified aox proteins by using targetp server tool, nalba-aox1, nalba-aox2 are predicted as mitochondrial while nalba-aox4 is localized in chloroplasts. template based structural modelling results showed that all identifed proteins are statistically similar to known structure models of corresponding aoxs. most environmental contaminants have toxic and mutagenic effects on living organisms as a result of the activation of free radical formation and inhibition of reparation activity. it is becoming relevant to search for protectors of natural origin from the effects of xenobiotics. many biologically active substances (bas) of inartificial origin are found to be antioxidants and can increase the body's resistance to the toxic and mutagenic effects of a wide range of pollutants. the aim of the study was to investigate the antioxidant and antimutagenic properties of bas from medicinal plants limonium gmelinii (plumbaginaceae) and inula britannica (compositae). the antioxidant potential of plant extracts was determined by the activity of superoxide dismutase (sod), catalase, and the content of malonic dialdehyde. mutagenic and anti-mutagenic properties of the extracts were determined in the test by counting chromosomal aberrations in root meristem of barley seeds. barley seeds were treated with an aqueous solution of unsymmetrical dimethyl hydrazine (udmh), which is highly toxic i class hazardous material, well known pro-oxidant. the results showed that udmh enhanced the process of lipid peroxidation and decreased the mitotic activity. treatment of barley seeds with extracts from i. britannica and l. gmelinii and their germination in the presence of stress factors stimulated antioxidant defenses in the primary roots of barley seeds. increase of the activity of sod and catalase, and reduction of peroxidation level of lipids were observed. cytogenetic study showed no mutagenic activity in plant extracts. when effects of plant extracts and udmh were combined there was a significant reduction in the frequency of structural mutations, induced by the toxicant. conclusion about the presence of the antioxidant and antimutagenic activity in the studied plant extracts is made. the work done within the framework of the mes project (no. gr 0115rk00378). p-02.08.5-014 comparative analysis of cytokinin dehydrogenase inhibition and trans-zeatin treatment in arabidopsis seedlings j. nov ak 1 , v. koukalov a 1 , z. medvedov a 1 , c. martin 1 , j. hradilov a 1 , l. sp ıchal 2 , b. brzobohat y 1 1 mendel university in brno, brno, 2 palack y university in olomouc and centre of the region han a, olomouc, czech republic cytokinins are plant hormones regulating many processes during plant life ranging from germination to senescence. manipulation of cytokinin levels and their impact on plant vitality, production and ability to defend against stresses is in great interest of agriculture. in this work we focused on comparison of inhibitor of the cytokinin degradation incyde (2-chloro-6-(3-methoxyphenyl)aminopurine) and exogenous application of trans-zeatin on arabidopsis thaliana seedlings. transcripts of genes regulating cytokinin metabolism were analysed by rt-qpcr analysis. classical cytokinin root essay revealed that incyde effect is comparable to that of trans-zeatin in a similar concentration-dependent manner. besides a negative effect on the primary root length, both substances induce flavonoid accumulation and an increase in the root hairs formation. histochemical staining of transgenic plants expressing glucuronidase (gus) under cytokinin-responsive promoter of arr5 gene revealed increased gus activity in cotyledons following incyde treatment suggesting diverse localization of cytokinin modulation upon trans-zeatin and incyde treatment, respectively. possible molecular differences originating in different cytokinin population and distribution following trans-zeatin or incyde treatments were monitored on the level of gene expression and via an lc-ms proteome analysis in roots and shoots of 14-day-old plantlets. rt-qpcr analysis revealed an alteration in cytokinin metabolism that could explain observed differences on the proteome level between incyde and trans-zeatin treated seedlings. pharmacologically inhibited cytokinin degradation could be very efficient tool for modulation of cytokinin levels. interestingly, the application of incyde and trans-zeatin shows a contrasting spatial and temporal pattern on molecular levels. incyde represents potent growth regulator with interesting properties useful for agriculture. p-02.08.5-015 the expression yield of prokaryotic alphaamylase is significantly magnified by molecular cloning techniques randomly hydrolyzing glycosidic bond alpha-amylase has been traditionally employed in bread and similar industries. in that regard, increasing the overall expression level of the enzyme is a crucial concern in biotechnology. to reach the goal, appropriate alpha-amylase producing species and expression vector were carefully selected. therefore, genome of bacillus subtilis was extracted and amplified by polymerase chain reaction (pcr) using specifically designed primers. subsequently, the extracted gene was inserted in expression vector pht43 and transferred to e. coli as intermediate host followed by bacillus subtilis host replacement. the recombinant vector was expressed in bacillus subtilis and the expression was evaluated by agarose gel electrophoresis. relative purification of the recombinant enzyme was performed by 50 kda filtration to remove impurities. to identify the biochemical characteristics, starch was used as specific substrate to measure enzyme activity and the enzyme was exposed to various ph and temperatures. the extra-cellular expression of alpha-amylase enzyme was successfully elevated by 5 folds in comparison to the native enzyme. the optimum temperature and ph for the enzyme was carefully determined as 70°c and 6, respectively. the enzyme was stable at 50°c, but thermal stability was dramatically decreased at higher temperatures up to 70°c. kinetic parameters were also measured; vmax was 1.998 u/ml min and km was 3.998 mg/ml. it is concluded that the elevated expression extent of recombinant alpha-amylase together with appropriate qualifications could make the clone a good choice for various industrial applications. flax seedlings of cultivars tmp1919, lira and lines g-1071/ 4_o, g-1071/4_k were treated for 4 and 24 hours with 500 lm alcl 3 solution or distilled water (control). twelve small rna libraries were constructed and sequenced using illumina gaiix. to identify known mirnas, obtained sequences were aligned with mirnas from mirbase (http://www.mirbase.org/). fold change value was calculated to identify up-and down-regulated mirnas under al stress. in total, about 40 million raw reads were obtained and 109 conserved mirnas from 26 families were identified. significant expression alterations in flax plants under al treatment were shown for mir319 and mir390. expression level of mir319 was varied in similar way in resistant and susceptible to al genotypes: mir319 was up-regulated after 4 hours of alcl 3 exposure and down-regulated after 24 hours. mir390 expression was increased after 4 hours of alcl 3 exposure and decreased after 24 hours in susceptible to al flax genotypes (lira, g-1071/4_o), while in resistant genotypes (tmp1919, g-1071/4_k) mir390 level was decreased after both 4 and 24 hours of al treatment. in other plant species, mir319 and mir390 were identified as al-responsive. mir319 targets mrna of tcp (teosinte branched/cycloidea/pcf) transcription factors, which control plant growth. mir390 targets mrna of tas3 protein, which regulates lateral root growth via degradation of arfs (auxin response factors). in flax, the involvement of mir319 and mir390 in response to al stress was shown for the first time. moreover, we revealed diverse expression alterations of mir390 in susceptible and resistant to al genotypes. this work was financially supported by grant 16-16-00114 from the russian science foundation. p-02.08.5-017 association genetics of phenylalanine ammonia lyase (pal) and cinnamyl alcohol dehydrogenase (cad) enzymes involved in lignin biosynthesis of european black poplar (populus nigra) b. taskiran, z. kaya middle east technical university, ankara, turkey populus nigra l. are considered as one of the most economically significant forest trees with respect to production of wood, biomass, and other wood-based products. while wood quality and biomass are directly associated with high cellulose content, lignin emerges as an undesirable polymer for both pulp and biofuel manufacturing industries. the aim of the study is by choosing the superior and eliminating the inferior clones to make a contribution to woody feedstock development and to improve wood quality of populus nigra. to estimate association genetics of pal and cad enzymes which have important functions in lignin biosynthesis, the important germplasms of populus nigra has been sampled from 3 year old poplar trees (285 clones x 2 replicates x 2 ramets) which were grown in behic ßbey plantation clone bank in ankara. additionally, five commercially registered clones and six foreign clones were included to the study to make comparison. the average mean values of cellulose, lignin and glucose content were calculated as 21.8 ae 16.29 lg/ml, 23 ae 4.64 lg/ml, and 35 ae 9.71 lg/ml, respectively. even though for pal and cad enzymes, data gathering process have been still resuming, particular clones have been separated from all in terms of pal and cad activities as expected. key words: populus, poplar, lignin, pal, cad, genetic variation, feedstock p-02.08.5-018 proteomic analysis of the molecular mechanisms of the response of plant seeds to pre-sowing treatment by stressors seed treatment with non-ionizing low-level radiation (nr), such as cold plasma (cp) or electromagnetic field (ef), is a modern eco-agricultural technology for stimulation of plant germination and performance. the molecular determinants of seed response to these treatments are not established and no genomic studies of plant seed response to nr have been reported. we studied the effects of pre-sowing seed treatment, using vacuum (7 min), radio-frequency ef (5-15 min) and cp (2-7 min), on germination and growth of non-oilseed helianthus annuus. to gain an insight into the molecular mechanisms underlying effect of nr on sunflower seed germination and dormancy, we estimated changes induced in the balance of plant hormones and differential protein expression. the results of the germination tests and estimation of seedling morphology showed that response develops in time and is stronger when sowing is performed in 7 days in comparison to 3 days after seed treatment. the 2d dige analysis revealed 38 differentially expressed proteoforms in kernels of seeds treated with cp or ef. proteins involved in biological processes of seed maturation, response to stress, response to abscisic acid stimulus, processes of organonitrogen compound metabolism and glucose catabolism were identified. while expression patterns for majority of the proteins were highly specific to cp and ef treated seed kernels, accumulation of several proteoforms of seed storage proteins (ssp), including vincilin-like, miraculin-like protein and albumin-8 were common for both experimental groups. this suggested that response to nr treatment could be at least partially associated to function of ssps in response to oxidative stress that protects proteins required for seed germination and seedling formation. variation of abundance of distinct proteoforms of helianthinin, vicilin-like and 11s globulin-like ssps suggested that post-translational modifications are involved in regulation of the function of ssps. p-02.08.5-019 suppression of lipopolysaccharide-induced inflammatory responses in raw 264.7 macrophages by tuber extract of cyclamen l. turkey is a prominent centre of plant diversity, being the meeting point of three main floristic zones. geophytes which have underground storage organs such as, tubers, bulbs and rhizomes. cyclamen l. is a tuberous geophyte traditionally used by some people for treating whooping cough, headaches or sinusitis, and confirmed to have antioxidant, analgesic and anti-inflammatory properties by several reports. a prolonged inflammatory response is often associated with chronic diseases such as cancer, arthritis and autoimmune disorders. recently, plant based products are used as an alternative and complementary treatment of these diseases. in this respect, the present study was aimed to determine the effects of three cyclamen tuber extracts on lps-induced inflammatory responses of murine raw 264.7 macrophages. firstly, c. cilicium (endemic), c. pseudibericum (endemic) and c. graecum subsp. anatolicum were collected from different localities of turkey. the tubers of plants were air-dried and grounded to fine powder and then extracted with ethanol. cell viability assay was performed to evaluate the nontoxic concentration in cell line by mtt assay. several measurements were performed including tnf-a, no and il-8 concentration assay by elisa after treatment compared to non treated cells to determine the anti-inflammatory activity. also, tnf-a and inos mrna levels were evaluated by quantitative rt-pcr. the cytotoxic activity which is considered safe on raw.264.7 cell were found as 0.5-5 lg/ml. studied cyclamen taxa inhibited tnf-a and il-8 release on lps stimulated-raw.264.7 in a concentration-dependent manner. among the three cyclamen tuber extracts evaluated, the highest nitrite-associated no inhibitory activity was obtained from c. pseudibericum compared to other two cyclamen l. taxa. collectively, these results suggest that cyclamen tuber extracts possess anti-inflammatory properties. p-02.08.5-020 in vitro hypoglycemic activity of ziziphus jujuba recent reports have indicated that continuous treatment with nutritional jujuba (ziziphus jujuba miller) fruit extracts in diabetic rats improved glucose utilization and produced a significant decrease in the blood glucose. in the present study, hypoglycemic activity of z. jujuba was investigated using various in vitro techniques. the hypoglycemic effect of z. jujuba in phosphate buffered saline which grown in balıkesir was studied by measuring glucose adsorption, glucose diffusion and glucose uptake by yeast cells. the glucose content in the solution measured by spectrophotometrically with commercially kits. the adsorption capacity of the z. jujuba was found to be directly proportional to the molar concentration of glucose. the glucose binding capacity of extract increased in higher glucose concentratrations. there was significant differences were observed between the adsorption capacities of z. jujuba and control samples (p < 0.05). the rate of glucose diffusion was directly proportional to the time. diffusion rate was significantly lower in the solution containing z. jujuba compared to control (p < 0.05). the extract demonstrated significant inhibitory effects on movement of glucose into external solution across dialysis membrane compared to control. the rate of glucose transport across cell membrane in yeast cells was observed to be inversely proportional to the molar glucose concentration. z. jujuba inhibited glucose transport across the yeast cells. the results showed that z. jujuba reduced glucose levels at least by three mechanisms. first by increasing glucose adsorption capacity during postprandial hyperglycemia; second by retarding glucose diffusion rate and third, at the cellular level by inhibiting glucose transport across the cell membrane. all of these decreased the absorption of glucose in the intestinal cells and the concentration of postprandial serum glucose. p-02.08.5-021 cucurbitacin b increased the anticancer effect of imatinib mesylate through inhibiton of matrix metalloproteinase-2 expression in colorectal cancer cells f. bakar ankara university, ankara, turkey several natural products have been investigated for their anticancer effects. among these, cucurbitacin b (cub) has been reported as its inhibitory effects on cancer cell proliferation. matrix metalloproteinases (mmps) belong to endopeptidase family and they are received as potential biomarkers for several types of cancer. the aim of this study is to investigate the effect of cub in combination with imatinib mesylate (im) on mmp-2 mrna expression of human sw480 colorectal carcinoma cells. the cytotoxicity analysis was performed via mtt assay. muse cytofluorimetric analysis system was performed to evaluate apoptotic cell population. the mmp-2 mrna expression was determined by quantitative real-time pcr. this study was supported by scientific and technological research council of turkey grant, sbag-114s871. data obtained from the cell culture experiments were expressed as mean ae sd and one-way anova test was applied for multiple comparisons. cub alone significantly inhibited cell growth at 10 lm and higher concentrations. the most potent effect was observed in cub-im combination treatment with 3.51 lm ic 50 value. in cub-im treated group, the apoptotic effect was higher than cub and im treated groups. cub-im induced apoptosis significantly at 10 lm concentration when compared to control and 1 lm (p < 0.05). cub alone showed inhibitory effects on mmp-2 mrna expression at 1 lm and higher doses significantly (p < 0.05). the results showed that the combination treatment of cub with imatinib synergistically inhibited human sw480 cell growth and induced apoptosis by increasing the anti-histone antibodybound nucleosom levels and annexin v binding. although cub could inhibit mmp-2 expression alone at higher treatment doses, it enhanced the inhibitory effect of im on mmp-2 synergistically in a dose dependent manner. in conclusion, this study suggests that cub combined with imatinib mesylate may enhance the effects of chemotherapy in patients with colorectal cancer. plants are most important parts of natural resources that alternatively referred to synthetic drugs for reasons such as being less side effects and lower costs. ziziphus jujuba miller (z. jujuba), a plant used in traditional medicine, is one of the most important ziziphus species belonging to rhamnacea family. the fruit and seeds of this plant are used different purposes such as antiinflammatory, antioxidant, immune-stimulant and wound healer. in this study, we investigated the antibacterial effects of z. jujuba. the aims of this study were to screen the antibacterial activity of z. jujuba. the extract was obtained from z. jujuba fruits pulverized with the aid of ball mill using 50% aqueous-ethanol solution. extracts were screened for antimicrobial activity against six different standard strains of bacteria by determining minimum inhibitory concentration (mic) according to clsi criteria. serial dilutions are made between 64 mg/ml and 0.031 mg/ml concentration range. the lowest concentration of wells that no visible growth has been accepted as mic value. materials in the mic and lower concentrated wells were transferred to 5% sheep blood agar petri dishes for calculation of minimal bactericidal concentration (mbc). the lowest concentration that no colony formation has been accepted as mbc value. jujuba showed the most potent effect on strain of s. aureus atcc 29213 is gram-positive cocci (mic: 2 mg/ml). the mic values of other gram-positive bacteria s. aureus atcc 43300, e. faecalis atcc 51219, l. monocytogenes f 1483 and m. smegmatis cmm 2067 were detected as 8, 16, 8 and 8 mg/ml respectively. mic values of gram-negative bacilli were detected as >64 mg/ml. consequently, z. jujuba was found to be effective on grampositive cocci bacteria (s. aureus atcc 29213, s. aureus atcc 43300 and e. faecalis atcc 51219). the strongest effect was observed on s. aureus atcc 29213 strain. in contrast, extract showed less effect on gram-negative bacilli. p-02.08.5-023 selective cytotoxic effect of morus rubra extract in human lung cancer cells through enhancing apoptosis and cell cycle arrest cancer is a disease that develops as a result of unlimited proliferation of abnormal cells that occurs due to loss of control over the mechanisms of normal growth and differentiation of cells. morus rubra, known as "red mulberry" belongs to family of moraceae. for many years, the fruits of morus species have been used to treat many diseases in traditional medicine. biological effects of morus species is predominantly attributed to its content of polyphenolic compounds. many studies have evaluated the cytotoxic effects of different morus species, but there is no study about cytotoxic effect of m. rubra. in this study, we aimed to evaluate the cytotoxic effect of m. rubra extract in human lung cancer cells (a549) with regard to apoptosis, cell cycle and mitochondrial membrane potential. cytotoxic effect of m. rubra extract on human lung cancer cells was determined using mtt assay. then, mechanisms of cytotoxic activity of m. rubra extract on a549 cells were examined in terms of cell cycle, apoptosis and mitochondrial membrane potential using flow cytometric methods. m. rubra extract exhibited selective toxicity against a549 cells compared to normal foreskin fibroblast cells. we determined that m. rubra extract increased cell cycle arrest at g 1 phase, the level of apoptotic cells and decreased mitochondrial membrane potential in a549 cells. our results showed that m. rubra extract has pro-apoptotic and antiproliferative effect in a549 cells. further studies are also needed to fully mechanisms underlying this effect of m. rubra extract. p-02.08.5-024 dipeptidyl peptidase iv inhibitory activity of arctium tomentosum l. a. zeytunluoglu 1 , f. zihnioglu 2 1 denizli vocational school of technical sciences, pamukkale university, denizli, 2 department of biochemistry, faculty of science, aegean university, izmir, turkey type 2 diabetes mellitus (t2dm) is rapidly growing metabolic syndrome of multiple aetiologies causing hyperglycaemia with insulin resistance at cellular level. a novel approach in the treatment of t2dm is based on preventing of rapid inactivation of the incretin hormone glucagon-like peptide-1 (glp-1) and glucose-dependent insulinotropic polypeptide (gip) by dipeptidyl peptidase-iv enzyme. in this study; dipeptidyl peptidase iv (dpp-iv; ec 3.4.14.5) inhibitory activity of the aqueous and methanolic extracts of arctium tomentosum leaves were successfully tested in vitro conditions. our study revealed that both aqueous and methanolic extracts obtained from test material had a significant dppiv enzyme inhibitory activity in changing ratio. the ic 50 values were also determined by nonlinear regression curve fit using graph pad prism 5.0 with appropriately diluted of lyophilized arctium tomentosum. diprotein-a (ile-pro-ile) was used as reference inhibitor. a. tomentosum aqueous extracts showed ic 50 4.6 lg/ml while the standard (positive control) diprotin a displayed the ic 50 value of 10.1 lg/ml. this study demonstrates that a. tomentosum aqueous extracts could be a good lead for further development as a new antidiabetic agent. p-02.08.5-026 dna recognition determinants of arabidopsis thaliana b3 transcription factors g. sasnauskas, k. kauneckaite, k. lapenas, v. siksnys institute of biotechnology, vilnius university, vilnius, lithuania transcription, one of the most important cellular processes, is regulated by transcription factors (tf), proteins that often directly interact with gene promoter sequences. tf binding to dna is mediated by various dna binding domains. the b3 tfs constitute a large, plant-specific protein family (approx. 10% of all tf proteins in the flowering plants), which is characterized by the presence of one or several small (approx. 110 amino acids) b3 dna binding domains. currently the b3 tfs are divided into four groups (lec2-abi3/val, arf, rav and rem). the preferred recognition sites were identified for representatives of all groups except the rem family. currently, only a single structure of a dna-bound b3 domain (arf1, arf family) is available, thus the mechanism of site-specific dna recognition by the lec2-abi3/val and rav b3 domains remains poorly understood. based on the arf1-dna structure (pdb 4ldx) we have built homology models of dna-bound b3 domains from a. thaliana abi3 (lec2-abi3/val family) and nga1 (rav family) transcription factors, mutated putative dna-interacting amino acid residues and characterized the dna binding ability of the purified mutants using electrophoretic mobility shift assay and a set of radiolabelled dna substrates carrying various variants of the optimal recognition site. we confirm the importance of several positively charged amino acid residues, which are conserved between the abi3/ nga1 b3 domains and structurally related dna-binding domains of bacterial restriction endonucleases ecorii, bfii and ngoavii; furthermore, we identify residues in the 'n-arm' and 'c-arm' loops that may be involved in specific interactions with the dna bases. our results therefore help us refine the homology models of the dna-bound b3 domains and in the future may help us predict the dna binding properties of currently uncharacterized b3 domains. p-02.08.5-027 immunohistochemical analysis of inhibitory effects of origanum hypericifolium oil on dipeptidyl peptidase iv in streptozotocininduced diabetic rats p. ili 1 , a. zeytunluoglu 2 1 denizli health services vocational high school, pamukkale university, denizli, 2 denizli vocational school of technical sciences, pamukkale university, denizli, turkey diabetes mellitus (dm) is a serious metabolic disorder with micro-and macro-vascular complications that result in a significant morbidity and mortality. glp-1 and gip have significant role in pancreatic beta cells and prevention of inactivation of them by dipeptidyl peptidase iv (dpp iv) inhibition is a novel approach to treatment of dm. origanum hypericifolium (lamiaceae) is an endemic turkish plant and its essential oil is mainly composed of monoterpenes including carvacrol and thymol. streptozotocin (stz) is used to induce diabetes in rats. the aim of this study is to investigate the inhibitory effects of o. hypericifolium essential oil on the dpp iv in stz-induced diabetic rats. the animals (female spraque-dawley rats) were assigned to four groups (group 1: control, group 2: stzinduced diabetic, group 3: o. hypericifolium injected, group 4: stz-induced diabetic and o. hypericifolium injected). dm was experimentally induced in groups 2 and 4 by a single intraperitoneal injection of stz at a dose of 45 mg/kg body weight. in groups 3 and 4, rats were intraperitoneally injected with o. hypericifolium oil at a daily dose of 1 ml/kg body weight for 42 consecutive days. at the end of the experimental period, all animals were sacrificed by cervical dislocation under ether anesthesia and liver and kidney tissues of each animal were rapidly excised. tissues were fixed in sainte-marie fixative. after routine histological processes, samples were embedded in paraffin, immunohistochemical staining for dpp iv was performed on sections and then they were photographed. the immunohistochemical reaction intensity differences were observed between the groups. in conclusion, the immunohistochemical distribution of dpp iv in the tissues that the test oil was applied in the diabetic rats may be important for the investigation of the inhibitory effects of oil on the enzyme. moreover, our findings suggest that o. hypericifolium oil may be used for prevention of diabetic diseases. introduction: all eukaryotic cells need microtubules for purposes of nuclear and cell division, organization of intracellular structure, and intracellular transportation, as well as ciliary and flagellar motility. microtubules are made of polymerized a/btubulin subunits. mec17 is important for microtubules, because it encodes the enzyme that adds acetyl groups to lysine 40 (k40) of tubulin. k40 is largely conserved in a-tubulins of many eukaryotes, and acetylation is thought to stabilize microtubule structure. in algae, the effect of acetylation by mec17 on flagellar motility and phototaxis has not been tested previously. materials and methods: in this study, mec17 mutant chlamydomonas reinhardtii cells were compared to wild-type cells to see the effect on flagellar motility and phototaxis. we tested phototaxis, eyespot size and quantity under the microscopy. in addition to this, we fixed cells and examined them by immunofluorescence microscopy using antibodies to tubulin, acetylated tubulin, and photoreceptor. results: we observed that some mec17 mutant cells contain more than one eyespot. we detected no acetylated-tubulin (ac-tub) by immunofluorescence. the cells still phototax and have normal motility discussion and conclusion: interestingly, mec17 cells still have the ability to phototax and they have normal flagellar motility, even though they contain occasional additional eyespots and no ac-tub. chlorella vulgaris as a model system for screening of plant growth modulators p. volynchuk 1 , e. marusich 1 , r. chuprov-netochin 1 , j. neskorodov 2 , y. mishutkina 2 , s. leonov 1 1 life sciences center, moscow institute of physics and technology, dolgoprudny, 2 center "bioengineering", russian academy of sciences, moscow, russia the discovery of new plant growth modulators became extremely important task as an alternative approach to overcome plants resistance to herbicides and pesticides, which leads to harmful action on plants and land rising, environmental and ecological problems. small molecules provide agricultural biotechnology with valuable tools, which help to circumvent the need for genetic engineering and offer unique benefits to modulate plant growth and development. we developed a system to explore molecular modes of action of plant growth modulators using chlorella vulgaris model. our model allows applying high content screening approach in 96well plate format for fast and robust effect assessment of large number of tested modulators. chlorella v. was grown in climate chamber under optimized constant temperature (20°c) and light conditions (16:8 hours/light:dark). modulating effect of tested compounds was estimated by spectrophotometric measurement of microalgae density at the beginning of the experiment (start point-0.1od) and 48 hours later. to validate our system we used known cytokinins and auxins (10 mm) as positive controls of growth stimulation. we showed that in presence of each compound the density of chlorella v. was increased in 7-11 times range, compared with only 2 times increase in control group. eight new chemicals (10 lm), which demonstrated modulation effect on nicotianatabacum l. pollen and arabidopsis thaliana models, were tested on developed chlorella v. model. positive controls showed no stimulating effect at this concentration, while tested compounds were confirmed as hits and increased the density up to 400%. we demonstrated that developed model system, based on chlorella v., is an effective system for primary screening of plant growth modulators. the main advantages of this system are short time of assay, simplicity of performance, possibility of automation and low cost. selected hits can be recommended as perspective candidates for future test on crop field. sunflower is under a big threat of downy mildew which is a fungal disease caused by plasmopara halstedii. the disease can cause up to an 80% yield loss in sunflower production. downy mildew resistance genes (r) denoted as pl has been discovered to date in sunflower. in recent years, single nucleotide polymorphism (snp) markers have become widely used in plant breeding programs. in this study snp markers have been currently developing for pl 6 , pl 8 , pl 13 , pl arg genes by competitive allele specific pcr (kasp) assay which enables bi-allelic scoring of snps and insertions/deletions (indels) at specific loci. in total 66 sequence tagged site (sts) sequences from ncbi were aligned for pl 6 , pl 8 , pl 13 , pl arg genes to identify conserved regions for each gene. based on the conserved regions, specific pcr primers were designed in order to make sequencing of these genes in five crosses and their f 2 progenies. sequence data will be used to design an allele specific primer maches the target snp and amplifies the target region with the common reverse primer provided by kasp genotyping assay. snp markers linked to pl genes which are being developed in this study, have the potential to be used in marker assisted selection (mas) for sunflower breeding programs. p-02.08.5-031 investigation of the antidiabetic effects of hibiscus sabdariffa, teucrium polium and myrtus communis in hepg2 cells line s. altundag 1,2 1 pamukkale university, denizli, 2 istanbul medeniyet university, istanbul, turkey some antidiabetic plants currently are used in alternative treatment of type ii diabetes. hibiscus sabdariffa, myrtus communis and teucirum polium plants are also known for their antidiabetic properties. hibiscus sabdariffa, myrtus communis and teucrium polium; depending on the impact on hepg -2 cells to investigate the possible mechanisms of type ii diabetes with researches on glucolysis and glucogenesis pathways gene expressions (pk m , glut-2,pepck). plants were obtained in dried state from reliable herbalists in denizli and mersin. plants treated with the extractor device. plants obtained aqueous extract was subjected to lyophilization process. human cancer cells have been used throughout the study. the cytotoxicity of the cells was measured by elisa plate reader . total rna was isolated using trızol ò solution was carried out according to the instructions of the manufacturer's (thermo scientific) recommended procedures were performed, but we have to optimize our own laboratory conditions. pk m , glut-2,pepck genes were synthesized by b _ ioneer. during our study the activation of certain genes (pk m , glut-2,pepck) were examined by real time pcr. in our study hibiscus sabdariffa, mrytus communis and teucrium polium plant to extract applied hepg -2 cell line in the gluconeogenesis and glycolysis pathways in involved in some important genes (pepck, pk m , glut-2) analyzed the expression levels . teucrum polium plant extract is applied in hepg -2 cells the glycolysis pathway genes (pk m glut-2) an increased expression also genes of gluconeogenesis pathway (pepck) were not decreased. however hibiscus sabdariffa and the expression of genes involved in glycolysis and gluconeogenesis pathway mrytus communis plants were observed to have a full effect as diabetic or hypoglycemic . in this context, it is considered that the plant teucrum polium on the line hepg -2 cells showed antidiabetic effect. p-02.08.5-032 purification of b-glucosidase from malatya apricot (prunus armeniaca l.) seeds and some of its biochemical properties h. kara 1 , s. sinan 2 , z. ekmekci 2 , y. turan 2 1 university of balikesir faculty of veterinary, balikesir, 2 university of balikesir faculty of arts and sciences, balikesir, turkey introduction: b-glucosidases are one of the key enzymes in carbohydrate metabolism and located in glycosyl hydrolases (ec 3.2.1) family. plant b-glucosidases have biotechnological significance as they are effective on glycosidic bonds of flavor and aroma precursors in plants. b-glucosidases that located in fruit seeds are important because they affect the amygdalin. aim of this study is purification and partially characterization of b-glucosidase from malatya apricot seeds. materials and methods: apricot seeds were homogenized with extraction buffer to prepare of crude extract. the enzyme protein was precipitated with 60% ammonium sulfate then purified by hydrophobic interaction chromatography using sepharose 4b-ltyrosine-1-naptylamine gel. para-nitrophenyl b-d-glucopyranoside (p-npglc) was used as substrate to determine biochemical properties of the enzyme. the optimum ph was determined using buffers between ph 2-12 and thermal optima was determined using 25-85°c temperature range. inhibitory effects was determined with 1 mm substances. results: the enzyme was 11.1-fold purified with yield of 14%. purified b-glucosidase from apricot seed was visualized about 60 kda molecular weight on sds-page. the kinetic parameters were determined against p-npglc substrate as km and vmax values of 2.5 mm and 58.1 eu, respectively. the optimum ph and temperature were determined 5.0 and 55°c respectively. effects of cacl 2 , kcl, nacl, mgcl 2 , k 2 so 4 , na 2 so 4 , cuso 4 , fecl 3 , pb(ii) acetate, agno 3 , zncl 2 and glucose on purified enzyme activity were investigated. kcl, na 2 so 4, pb(ii) acetat and cuso 4 reduced the enzyme activity. discussion and conclusion: in this study, b-glucosidase was purified from malatya apricot seed and some of its biochemical properties were determined. because this enzyme has pharmaceutical importance hydrolyzing amygdalin. the results showed that immobilized almond b-glucosidase was used to break amygdalin and release -cn compound that effective to shrink cancer mass. p-02.08.5-033 a new affinity gel for purifing polyphenol oxidase enzyme a. erg€ un 1,2 , o. arslan 2 1 balikesir university, science and technology application and research center, balikesir, 2 department of medical chemistry, faculty of science, balikesir university, balikesir, turkey polyphenol oxidase (ppo) enzyme, sometimes called as phenol oxidase, catecholase, phenolase, catechol oxidase or tyrosinase, is considered to be an o-dipenol. ppo (ec 1.14.18.1), a multifunctional copper containing metalloenzyme, is widely distributed in nature. ppo exists in many kinds of plants and fungi, such as banana, mushroom, butter lettuce, napoleon grape, potato, coffee, marula fruit, artichoke heads, longan fruit, tobacco, wheat flour. in this study, a novel affinity chromatography gel was synthesized for purifing ppo enzyme. the affinity chromatography gel was synthesized by coupling aniline as a specer arm to cnbr activeted sepharose-4b. then, p-amino benzoic acid was coupled to aniline as a ligand. ppo was purified from musa sapientum var. cavendishii (banana) by using sepharose-4b-aniline p-amino benzoic acid affinity chromatography gel. % 4.49 yield and 33.4 fold purification were achieved. sds-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent mw of 35 kda. the v max and k m of the purified enzyme were determined 42,628 u/ml min and 9.27 mm, respectively. p-02.08.5-036 phenolic content and antioxidant capacity of gamma irradiated olive leaves m. e. diken 1 , b. kocat€ urk 2 , s. dogan 1 , h. tuner 1 1 balikesir university, balikesir, 2 kavram vocational school, istanbul, turkey in this study, dried in diffirent ways (such as microwave, infrared, convection heaters and under normal atmospheric conditions) olive leaves has been used as experimental material. radiation has been applied to dried olive leaves in three diffirent dosages in room temparature. the amount of radiation to be implemented to the samples have 3, 5, 10 kgy/min. in this study, how gamma rays (radiation) effects phenolic components, total phenolic content and antioxidant capacity of dried olive leaves has been determined. the phenolic components, total phenolic content and antioxidant capacity were analysed with hplc, folin ciocalteu and dpph radical scavenging method, respectivelty. gamma rays is well known as a decontamination method for many foodstuffs and plant materials, being an environment friendly and effective technology to resolve technical problems in trade and commercialization. for this reason, nowadays it is utilized as an alternative method of sterilization. gamma rays are of ionizing radiation. when ionizing radiation interacts with matter, generally it causes a break in the molecular bonds and/or breaks the bonds between the molecules. these intermediates have unpaired electron and called free radical. gamma radiation or the radiation-induced free radicals would break or cause damage to the dna molecules of living organisms. gamma irradiation is predict to change phenolic content and antioxidant capacity in living tissues. phenolic compounds are secondary plant metabolites naturally present in fruits and vegetables. in recent years there has been a growing interest in food phenolics because of their potential health benefits mainly due to their antioxidant and free radical scavenging activity. despite the controversy about potential risks posed by genetically modified plants on human health, environment and microorganisms, cultivation area of these crops increases day by day. this increment has revealed concerns especially related to hgt. hgt studies indicated that antibiotic resistance genes in gm plants have a potential to transfer to soil microorganisms. in this study, hgt of widely used genetic elements such as regulatory sequences, from transgenic plants to bacteria was investigated. three soybean feed and four seed examples from turkish feed manufacturers' association were used for genetic analysis based on foreign gene determination. gmo analysis were conducted by camv 35s promoter-specific pcr in genomic dnas. in gm positive samples, genomic dnas sheared into appropriate fragment size by ultrasonication for the purpose of bacterial transformation. presence of 35s promotor region in fragmented dnas was proved by pcr. escherichia coli dh5a strain was transformed by fragmented dna samples according to cacl 2 method. 35s promotor sequence screened by using pcr in bacterial genomic dnas. as a result of gm screening, all feed and three of the seed samples were found to be transgenic. ultrasonication conditions were optimized for shearing dna's to 450-500 bp for bacterial transformation. fragmented dnas confirmed for carrying intact 35s promotor sequence. none of bacterial genomic dnas were found to be 35s-positive. according to the transformation results, absence of 35s promotor sequence in all tested bacterial genomic dnas, proved the dna samples belonging to gm plants can not transfer into compotent e. coli dh5 under laboratory conditions. for verification of this finding, transformation studies will continue with acinetobacter baylyi bd413 strain which is naturally competent soil bacterium for natural transformation. we believe that all of our findings will contribute to constitute transformation system which can be used as model in hgt studies. p-02.08.5-038 preliminary studies on differential methylation in a and d sub-genomes of upland cotton (gossypium hirsutum l.) four species of cotton (gossypium l.) provide raw materials for the textile industry. among the four species, two have diploid genome and another two have tetraploid genome. tetraploid genome consists of a and d sub-genomes. a sub-genome belongs to asian cotton while d-sub genome belongs to american cotton. previous studies revealed that d sub-genome of gossypium species contributes to the superior yield and quality of tetraploid gossypium l. species (atdt). dna cytosine methylation of four regions of dna sequences located on a and d sub-genomes of gossypium hirsutum l. texas marker 1 (tm-1) was investigated using bisulfite sequencing technique. among the regions studied two could not be located on subgenomes due to sequence identity match between a and d subgenomes. on the other hand two dna regions could be located on a and d sub-genomes using the blast searches. some of the dna sequences located on different sub-genomes showed polymorphic nucleotides including c/t and g/a polymorphisms. in silico analysis indicated that some alleles located on different sub-genomes of cotton have c/t and g/a polymorphisms. c/t polymorphisms between the sub-genomes could be thought as unmethylated cytosine using the bisulfite sequencing technique. this indicated that an extra attention needs to be paid in dna total cytosine methylation studies in polyploid species such as cotton using bisulfite sequencing, methylation sensitive amplification polymorphism (msap), whole-genome bisulfite sequencing (wgbs). otherwise, t in c/t polymorphism between the subgenomes could be thought as unmethylated cytosine. based on the two genomic regions we could conclude that a sub-genome may be more methylated than d sub-genome. differential methylation of genes located on different sub-genomes may provide another mechanism responsible for differential gene expression of genes located on different sub-genomes. p-02.08.5-039 cleaved minisatellite locus (cml) markers for fingerprinting of cotton cultivars grown in turkey e. u. gocer, m. karaca akdeniz university, antalya, turkey after their discovery by alec jeffreys in 1984, minisatellites have been used in genetic studies of many organisms. minisatellites, also called variable number tandem repeats (vntrs), are composed of arrays of longer repeats mostly dispersed throughout heterochromatin (centromeres and telomeres). direct amplification of minisatellite regions of dna using a single core primer is a powerful method to amplify minisatellites (damd-pcr). although the damd-pcr technique has been applied to many plant species, the level of polymorphisms in cotton (gossypium l.) is very low due to very narrow turkish cotton genetic base. the objective of this study was to improve the level of polymorphisms by cleaving minisatellite loci by restriction enzyme digestion. genomic dna samples of twenty-one turkish cultivars, pima 3-79, tm-1 and ps-7 were extracted. twenty-one minisatellite primers were screened using the damd-pcr technique. monomorphic amplicons were digested using several restriction enzymes. three to five micro liters of amplified products were digested with various restriction enzymes. digested products of minisatellite loci were separated in 3% high resolution agarose gels. comparison studies of digested and undigested markers revealed that cleaved minisatellite markers showed polymorphisms in those cotton lines that could not be differentiated by microsatellite and minisatellite markers. this approach was called cleaved minisatellite locus markers (cml). the amplification reactions of minisatellites used touch-down (td) cycling conditions. the use of td offered a simple and rapid means of optimizing polymerase chain reaction (pcr), increased specificity, sensitivity, and efficiency without the need for lengthy optimizations of minisatellite primers. the cml markers were obtained at a 55°c annealing temperature, which is a temperature higher than those used in random amplified polymorphic dna (rapd) inter-simple sequence repeat (issr) markers. p-02.08.5-040 association between cytosine methylation and tissue specific expression of microsatellites a. g. ince, m. karaca akdeniz university, antalya, turkey heritable covalent modification of dna, rna or protein without altering their primary sequences is defined epigenetics. because all biological events are influenced by epigenetics, it is one of the most important fields in science. dna methylation is one of the most important epigenetic mechanisms. dna cytosine methylation is a process by which methyl groups are added to cytosine bases of dna. microsatellites, also knows simple sequence repeats are dna sequences consisting of 1-6 nucleotide repeats. there is a large body of information regarding the relationship between microsatellite instability and abnormal gene expression, and between dna methylation and altered gene expression. however, there is limited information on cytosine methylation of microsatellites. in the present study, we investigated whether there is any association between cytosine methylation and tissue specific expression of microsatellites. genomic dna samples of various tissues and developmental stages of pepper line demre sivrisi (capsicum annuum l.) and cotton line tm-1 (gossypium hirsutum l.) were extracted. cdna samples were synthesized using mrna expressed in pepper tissues. cytosine methylation levels were investigated using bisulfite sequencing methods. screening studies of microsatellites revealed that some genes containing microsatellites were differentially expressed in tissues and developmental stages of pepper. microsatellite containing genes that expressed differently among tissues had also showed different methylation levels in cg, chg and chh (where h refers to a, c or t) contexts. methylation level differences between microsatellites were also observed. as far as our knowledge, it is the first report on differential expression of genes containing methylated microsatellites. p-02.08.5-041 pcr-lg: an alternative way to assign the chromosome location of genes/markers in cotton a. aydin, m. karaca akdeniz university, antalya, turkey assignment of genes and dna markers on chromosomes is very important in life sciences, especially for plant breeding and medicine. there are several methods for the assignment of a gene or dna sequence to a specific location on a chromosome. for example, the most widely used technique is the assignment of fluorescently-labeled gene or dna sequences (markers) on chromosomes using the fluorescently-labeled gene (for instance, fish technique). another example is the construction of a genetic map (linkage map) which orders the targeted genes along the dna strand based on recombination frequency. sequencing is the most precise technique in which coding (gene-containing) and noncoding dna region of genes could be located on a chromosome molecule. aneuploid lines could also be used to locate genes in a specific chromosome, but maintenance of these lines is difficult. here we report the use of chromosome substitution lines to indirectly locate genes/markers on chromosomes. we used chromosome substitution lines (csls) that carry a chromosome pair or chromosome arms from gossypium barbadense l. while the rest of chromosomes belong to g. hirsutum l. a total of 10 chls, a homozygous cotton line tm-1 and a double haploid line pima 3-79 were used as plant materials. twenty microsatellite primer pairs were utilized in touch-down polymerase chain reactions. we developed a method, called polymerase chain reaction to locate gene (pcr-lg), to assign genes/markers on chromosome or chromosome arm. with the use of pcr-lg approach any polymorphic genes/markers between tm-1 and pima 3-79 (g. hirsutum and g. barbadense) could be assigned to a chromosome or chromosome arm. results indicated that if 26 csls were used any polymorphic markers (genes) with known primer pairs could be assigned to cotton chromosomes. although we used cotton chromosome substitution lines to validate the proposed technique, it could be applicable all the species that have chromosome substitution lines. p-02.08.5-042 pecularities of genome variability of antarctic hairgrass deschampsia antarctica desv. from the maritime antarctic deschampsia antarctica desv. (poaceae) is the only grass species native to the antarctic region, adapted to harsh environmental conditions. however, reasons for its unique success remain unexplored. stressful environmental factors can influence a plant genome and cause changes in the chromosome number and morphology and increase genetic variation. therefore, the purpose of our research was to explore alterations in the d. antarctica genome both at the chromosomal and molecular levels and to investigate species genome stability using in vitro tissue cultures. plants used for the study were grown in vitro from seeds collected in the argentine islands region during 2008-2014. chromosome number was determined in plant root apical meristems and specimens prepared from tissue cultures. rrna genes localization were determined using the fish technique. moleculargenetic analysis was performed using pcr with polymorphic issr-primers. new forms of chromosome polymorphism, associated with aneuploidy (2n = 13-27), polyploidy (2n = 13-39) and the occurrence of additional b-chromosomes (2n = 26 + 1-3b), were observed. fish analysis also confirmed that genotypes with a different chromosome numbers varied in the number of 5s rdna and 25s rdna sites. assessment of genetic variation demonstrated a low level of diversity: differences between the plants with different chromosome numbers do not exceed the level of within-population variation. cytology analysis of d. antarctica cultured tissues revealed aneuploidy (up to 60.6 %) with predominance cells with diploid and near-diploid chromosome number irrespective of plant's initial karyotype (diploid, mixoploid or polyploid). we assume that discovered intraspecies chromosomal polymorphism is a manifestation of quick genome reaction to harsh antarctic conditions. whereas the results of molecular-genetic analysis and study of cell cultures of this species suggests on the relative stability of d. antarctica genome. p-02.08.5-043 angiotensin converting enzyme inhibitory activity of morchella esculenta (l.) pers a. zeytunluoglu 1 , i. arslan 2 1 biomedical equipment technology, pamukkale university, denizli, turkey, denizli, 2 biomedical engineering, pamukkale university, denizli, denizli, turkey hypertension is a multi-aetiological, chronic pathophysiology that leads to multi-organ dysfunctions like cardiovascular diseases, strokes, and renal complications. natural extracts play an important role in traditional medicines for the treatment of hypertension and are also an essential resource for new drug discovery. mushrooms are use as therapeutics in alternative and complementary medicine as functional food because of contain a large number of biologically active components that offer health benefits and protection against many degenerative diseases. morchella esculenta is one of the most highly priced edible mushrooms worldwide. it contains a wide range of active constituents which include tocopherols, carotenoids, organic acids, polysaccarides and phenolic compounds which exhibit a wide range of medicinal and pharmacological properties including anti-microbial, anti-inflammatory, immunustimulatory, antitumor and antioxidant. in this study; the in vitro angiotensin converting enzyme-i (ace-i) inhibitory activity of m. esculenta peptides were generated by alcalase hydrolysis were studied. the 5 kda < peptides < 10 kda in the ultrafiltration fractions displayed highest ace inhibition (85.9 ae 5.09% at 140 lg/ml). the results indicate that m. esculenta derived peptides may have potential as functional food ingredients in the prevention and management of hypertension. p-02.08.5-046 modulations of antioxidant enzymes, gsts and catalase, by salvia absconditiflora in hepg2 cell line d. irtem kartal 1 , a. altay 2 1 yuzuncuyil university, van, 2 erzincan € university, erzincan, turkey oxidative stress is considered to play a important role in the pathogenesis of aging and several degenerative diseases, such as cancer. in order to cope with an excess of free radicals produced upon oxidative stress, humans have developed several mechanisms for maintain redox homeostasis. these protective mechanisms either scavenge or detoxify ros, block their production, and include enzymatic and nonenzymatic antioxidant defenses. in enzymatic defenses include glutathione s-transferases and catalase enzymes. many epidemiological studies have revealed that there is a strong correlation between consumption of polyphenol-rich foods and the prevention of certain diseases like cancer, cardiovascular diseases and aging. phenolic compounds are abundant in all plants. so, they form an integral part of the human diet. salvia species, commonly known as sage, have been used since ancient times for more than 60 different ailments ranging from aches to epilepsy. there are around 900 species of salvia, 95 of which are represented in turkey including salvia absconditiflora. in this study, s. absconditiflora collected from metu campus (ankara, turkey) is extracted with methanol and water. effects of the water and methanol extracts on the mrna expressions of antioxidant enzymes gstm1 and catalase in hepg2 cells were investigated by q-rt-pcr technique. it was also monitored the effects of the extracts on the enzyme activities of gsts and catalase by spectrophotometrically. water and methanol extracts decreased gsts mrna expression in hepg2 cells for 48 hours and 72 hours incubation and methanol extract decrease catalase mrna expression for only 72 hours incubation. on the other hand, extracts highly increased the gsts and catalase activities in both hour incubation. overall, these results indicate that s. absconditiflora and/or its components have regulatory activities on antioxidant enzymes and they may have a potential as a therapeutic agent in the treatment of cancer. p-02.08.5-047 transcriptomics and proteomics approach to drought stress mechanism in wheat b. cevher keskin tubitak marmara research center genetic engineering & biotech. institute, kocaeli, turkey birsen cevher keskin, yasemin yıldızhan, oktay kulen, bayram y€ uksel, selma onarıcı, _ ismail t€ urkan, as ßkım hediye sekmen, u gur sezerman, bu gra € ozer identification of novel stress-responsive genes and their role in drought response is an important area for the improvement of the crops. drought-related genes were investigated in leaves and roots of three wheat genotypes after different drought stress treatments by rna sequencing (rna seq) technology and de novo assembly was performed before comparative transcriptome analysis. analyzing 311 gigabases of 100 bp paired end illumina reads from a hexaploid wheat poly(a) rna library, we identified common and new differentially expressed transcripts. selected differentially expressed genes were confirmed by qrt-pcr. we also performed root proteome analysis with nano electrospray ionization source coupled to a high-performance liquid chromatography system (nanouplcàesiàqtofàms) to identify drought-related proteins. totally 191 proteins were differentially expressed in root tissues of tolerant and non-tolerant wheat genotypes. responses of antioxidative defense system to drought stress were comparatively studied in the same wheat cultivars. similarities between protein and rna levels help increase our confidence in novel biomarkers, differences may also reveal other post-transcriptional regulatory junctures. all these analyses will allow us to get a better idea about the possible role of these genes in the drought-response mechanism. the drought-related genes that are functionally characterized could be introduced into agronomically important wheat cultivars. this work offers a resource for accelerating drought-related gene discovery and improving this important crop. p-02.08.5-048 isolation and characterization of a hexose converter from olive s. altunok 1 , e. d€ undar 2 1 department of biology, university of balikesir, balikesir, 2 department of molecular biology and genetics, college of arts and sciences, balikesir university, balikesir, turkey introduction: hexose sugars are key components of glycolysis and photosynthesis. the genes regulating their conversion into one another, is therefore, of great importance for the control of carbon metabolism. in this study, we report isolation and characterization of a cdna associated with conversion of hexoses in olive. the cdna putatively named aldolase based on bioinformatic and experimental analyses. material-methods: characterization based on nucleotide and amino acids were conducted using bioinformatic tools such as nucleotide and protein blast, bioedit, primer3, finchtv, clc genomic workbench, expasy, targetp, sosui and web promoter scan. comparison of the genomic and cdna sequence of the gene and detailed bioinformatic analyses including cellular location, hydropathy analysis, amino acid-nucleotide composition and predicted 3d structure were also conducted using the bioinformatics tools mentioned above. temporal expression pattern of the putative aldolase were conducted using real-time pcr experiments. sds and western blot analyses were completed while biochemical analyses are ongoing. oleocanthal is an important secondary metabolite that has been reported to be useful against important human diseases including cancer. the aim of this study was to identify and characterize the key biochemical and genetic components of oleocanthal biosynthesis. to determine the biochemical components of the pathway, multiple olive cultivars along with their spatial and temporal points were determined. the expression levels of multiple candidate genes were also aimed via real-time pcr. nucleotide blast and protein blast (for comparison of the similarity of the candidate genes with that of other organisms) were conducted on ncbi web page. phylogenetic tree construction, amino acid composition analysis, nucleotide composition analysis, hydropathy analysis and translations through expasy were conducted. primer3 was used to design forward and reverse primers to amplify the target genes from different olive tissues at different times. analysis of the first candiate gene with bioedit program revealed that a+t ratio was more than g+c according to the nucleotide composition analysis. according to amino acid composition analysis isoleucine, lysine and leucine were more than other amino acids while kyte&doolittle hyddropathy analysis revealed that the protein was hydrophilic. abundance of hydrophobic amino acids (leucine and isolecine) along with an abundant hydrophilic amino acid (lysine) suggest the existance of hydrophobic pockets in the protein which may mean a membrane bound protein or a sitoplasmic protein with a strong hydrophobic core. the molecular weight of the protein was 56 kda with a pi of 9.21. the protein was found to have a signal peptide. according to the sosui gramn , intracellular localization was found to be in the inner membrane. analysis of other candidate genes contiunes. acknowledgements: this study was supported by tubitak with grant number 110o108. key words: olive, olea europaea l., secologanin, polymorphism, allele diversity p-02.08.5-050 antioxidant potentials of propolis and its bioactive components, and their effects on cyp2e1 gene expression in ht-29 adenocarcinoma cell line a. altay 1 , d. irtem kartal 2 1 erzincan € university, erzincan, 2 y€ uz€ unc€ u yil university, van, turkey propolis is a resinous mixture that is collected by honeybees from plants, and is combined with beeswax and secretions from the bee's salivary glands plus some pollen. it is a rich mixture of polyphenols, flavonoid aglycones, phenolic acids and their esters. it has been used in traditional medicine for thousands of years because of these ingredients. propolis, just like honey, has been the subject of many studies due to its antimicrobial, antifungal, antiviral and hepatoprotective activities. the cytochrome p450 enzymes are involved in phase i xenobiotic and drug metabolism. cyp2e1 is present in high levels in human tumors demonstrated by qrt-pcr and immunohistochemical studies. in this study, propolis collected from datc ßa (mugla, turkey) is extracted with 70% ethanol. phenolic contents of the propolis were measured by lc-ms/ms. cytotoxic effects of the propolis on ht-29 human colon adenocarcinoma cell lines were examined via xtt colorimetric cell proliferation assay. effects of propolis extract and its main bioactive component caffeic acid on the expression of phase i detoxification enzyme cyp2e1 in ht-29 cells were investigated bu using q-rt-pcr technique. ic 50 values for dpph radicals scavenging activities of the extract was calculated as 0.042 mg/ml. tpc and tfc were determined as 168.6 mg gae/g extract and 141.8 mg qe/g extract, respectively. caffeic acid content of the extract was measured as 10.6 lg/g extract. ic 50 values for xtt assay in 48 hours incubation with the extract and caffeic acid were evaluated as 1.16 mg/ ml and 0.149 mg/ml, respectively. propolis extract and its main phenolic content caffeic acid significantly dicreased cyp2e1 mrna expression with 2.4 and 5.3 fold, respectively in ht-29 human colorectal adenocarcinoma cells for 48 hours incubation. overall, these results indicate that propolis and/or its components have regulatory activities on cyp2e1 expression and they may have a potential as a therapeutic agent in the treatment of cancer. p-02.08.5-051 effects of histone h3 lysine 9 inhibition on gene expression profile in tobacco (nicotiana tabacum l.) differentiation is the characteristic of multicellular organism. cellular dedifferentiation underlies topical issues in biology such as reprogramming in stem cell research, regeneration and nuclear cloning, and has common features in plants and animals. the state of dedifferentiation is evidenced by changes in cell morphology, genome organization and the pattern of gene expression as well as by the capability of plant tissues to differentiate into multiple types of cells depending on the type of stimulus applied. chromatin reorganization appears to be a fundamental theme in cellular dedifferentiation and reentry into the cell cycle both in plants and animals. the chemical modifications of histone proteins which are structural units of the nucleosome, generate 'codes' for the recruitment of proteins or protein complexes that affect chromatin structure and gene expression according to 'histone code' hypothesis. methylation of histone h3 at lysine 9 residue by specific methyltransferases suv39h1 in humans and kyp/suvh4 in arabidopsis generates a'code' for the recruitment of hp1 proteins. in this study, to enhance the dedifferentiation efficiency, chaetocin which inhibits suv39h1 has been used at the germination stage of tobacco seeds in in vitro conditions. our results showed that chaetocin induced callus formation from leaf discs of tobacco in the early stage of the inhibitor application. chaetocin can enhance reprogramming of plant cells in seed development treatment as callus induction. it is known that the formation of callus which is the non-differentiated cell community in plants, is a consequence of the changes in the gene expression profile. it has been found that epigenetic modifications play a crucial role in some of these changes. the definition of the genes related to callus formation by the inhibitation of an epigenetic modification -h3k9 methylation-in tobacco might be used to bear on various dedifferentiation-driven cellular processes. induction of tumor cell death by the use of some phytochemicals that consumed through diet, and derived from medicinal plants opens up new horizons for cancer treatment researches. rheum ribes species, which is studied in this research, is one of the commonly used herbs in pharmacological researches. the high content of phenolic compounds in r. ribes extracts were known to be responsible for the high antioxidant and antibacterial activities. our aim in this study is to assess cytotoxic and apoptotic changes by way of implementing methanol extract of the rheum ribes (root) to the mcf-7 breast cancer cell line. cytotoxic effect of rheum ribes extract was evaluated by using the xtt (2,3-bis(2-metoksi-4-nitro-5-sulfofenil)-2h-tetrazolyum) test. in order to determine the dose of ic50, plant extracts were applied as time and dose dependent in the range of 10-500ug. in 72nd hour, the ic50 value is determined as 400ug. to examine the apoptotic effects of the extract, total rnas were isolated from dose group and the control cells firstly, then cdnas were synthesized. expression profile of the target genes(caspase-3, caspase-7, caspase-8, caspase-9, bax, bcl-2, fas) are determined by qpcr. according to the results, when the control group compared with the cells, it was determined that, while 17.33 and 3.05-fold respectively increase in the gene expressions of caspase-3 and caspase-7 of dose group cells, 15.45-fold decrease in the gene expressions of bcl-2. no significant difference was observed in the other genes examined. based on the obtained data, we believe that methanol extract of the rheum ribes induces apoptosis by activating intrinsic pathway. as a result, this plant species can be a new and effective therapeutic candidate for the medical world in search of alternative anti-cancer approaches, and could shed light on the work to be done in this area. p-02.08.5-053 the effect of ferulic acid and 5-fluorouracil combination on apoptosis in pc-3 human prostate cancer cells prostate cancer is quite often seen in industrialized countries and has the second most common death rate due to cancer after lung cancer in men. 5-fluorouracil (5-fu) is a pyrimidine analog and cell cycle-targeting drug by inhibiting dna synthesis. it has been widely used for treatment of several cancers such as gastric, colorectal, and breast cancers. phenolic compounds found in foods are potential antioxidants of harmful oxidative processes related to cancer and also important due to induction of different mechanism such as apoptosis. ferulic acid (fa; 4-hydroxy-3-methoxycinnamic acid), a phenolic compound, is abundant in fruits and vegetables. the purpose of this study was to investigate combination effect of fa and 5-fu on apoptosis in pc-3 human prostate cancer cell line. the effects of 5-fu, fa, and combination of both of them on cell viability were determined by xtt method. total rna isolation was conducted using trizol reagent. expressions of important genes in apoptosis including casp3, casp7, casp8, casp9, bcl2, bax, fas and cycs were investigated in four groups by qpcr. the ic 50 doses of fa and 5-fu were found to be 300 lm and 60 lm for 48 hours in pc-3 cells, respectively. in order to determine combination effect, pc-3 cells were treated with <ic 50 doses (200 lm fa and 40 lm 5-fu). according to qpcr results, a significant increase in the expressions of bax and cycs genes and a decrease in the expression of bcl2 were observed in the fa group, compared with the control group cells. after the treatment with 5-fu, the expression of casp8 was significantly increased compared with the control group. furthermore, combination of fa and 5-fu significantly increased expression of casp7, casp8, fas and cycs genes. in conclusion, comparing with the single treatments, it was observed that combination of fa and 5-fu affected expression of different genes in apoptosis in pc-3 cell line. p-02.08.5-054 combination of ferulic acid and gemcitabine affects expression of some apoptotic genes in pc-3 human prostate cancer cells prostate cancer, the second causing of cancer-related death in men, is an important cancer type due to the late age of onset, slow the progression, high incidence. gemcitabine is an effective agent in several cancer types including non-small cell lung cancer, pancreatic, bladder and breast cancer. it is known that gemcitabine is used single agent or as a part of combination treatment in prostate cancer therapy. nowadays, new treatment methods have been tested for cancer therapy including natural compounds with low toxicity. ferulic acid (fa) one of these agents, an abundant phenolic compound found in various fruit and vegetables. in this study, objective was to investigate combination effect of ferulic acid and gemcitabine on apoptosis in pc-3 human prostate cancer cell line. cell viability was determined by using xtt method after the treatment with gemcitabine, fa and combination of both of them. total rna was isolated with trizol reagent. expressions of casp3, casp7, casp8, casp9, bcl2, bax, fas and cycs genes are important in apoptosis, were evaluated in four groups by qpcr. the ic 50 doses of fa and gemcitabine were found to be 300 lm and 50 lm for 48 hours in pc-3 cells, respectively. for determination of combination effect, pc-3 cells were treated with <ic 50 doses (200 lm fa and 35 lm gemcitabine). when compared with the control group, qpcr results illustrated, a significant increase in the expressions of bax and cycs genes whereas a decrease in the expression of bcl2 gene in the fa treatment group. after the treatment with gemcitabine, the expression of casp3 and fas genes were significantly increased compared with the control group. furthermore, combination of fa and gemcitabine significantly increased expression of casp3, casp7, casp8, fas and cycs genes. in conclusion, it was observed that combination of fa and gemcitabine affected different genes in apoptosis compared with the single treatments in pc-3 human prostate cancer cell line. pistacia lentiscus linn. is widely distributed in mediterranean europe, morocco and turkey. the resin part of its known as mastic gum and plant called as mastic tree. it has been referred to over centuries as having medicinal properties to treat a variety of diseases. the aim of the present study are to evaluate antioxidant, cytotoxic and antimicrobial activities of heptane, chloroform and methanol extracts from p. lentiscus leaves and mastic gum. the antioxidant activities of chloroform and methanol extracts were assayed with various methods, including tpc, dpph free radical and abts radical cation scavenging activity. also, cytotoxicity of extracts was evaluated and screened against human mcf-7, hela, a549, and ht-29 cancer cell lines and nih-3t3 cells. the viability of cells in 100 lg/ml concentration of extracts was evaluated using mtt assay. antimicrobial activity of extracts were investigated against s aureus, s epidermidis, e coli, p aeruginosa, p vulgaris, k pneumoniae, c albicans, c glabrata, c guilliermondii, c tropicalis, c parapsilosis test organims by the agar well diffusion and broth microdilution methods . according to our results, the methanol extract of p. lentiscus leaves showed the highest dpph free radical scavenging activity (ic50 = 0.16 ae 0.02 mg/ml) and abts radical cation scavenging activity (1.33 ae 0.06 mg/ml). this study also exhibited that methanol extract of p. lentiscus leaves had the highest tpc (126.7 ae 5.7 mg gallic acid equivalents/g extract). the hexane extract of mastic gum showed antifungal activity against all of the tested candida species (6-16 mm / <0.25-8 mg/ml). the methanol extracts of p. lentiscus leaves showed the highest antimicrobial activity against all of the tested bacteria except k pneumoniae strain (8-14 mm/>256 mg/ml). on the other hand, p. lentiscus showed no cytotoxicity against cancer and normal cells. the results suggested that p. lentiscus may be natural source of antioxidant and antimicrobial activities. p-02.08.5-056 antibacterial activity of and chemical composition of alcoholic extract of marjoram against some human pathogens m. ahanjan, m. rahbar mazandaran university of medical sciences, sari, iran herbs enjoy a unique value and importance in sustaining healthy communities in terms of disease prevention (1) . in this regard, marjoram is a plant of the mint family which has antibacterial properties on microorganisms (2) . the current study aims to investigate the anti-microbial activity of the alcohol extracts (i.e., methanol or ethanol) of marjoram plants on the bacteria of staphylococcus (atcc: 25923), aureus e. coli (atcc: 25922), and salmonella enterica (atcc: 13076) and p. aeroginosa through utilizing disk diffusion method. also, the minimum inhibitory concentration and the minimum bactericidal concentrationwere measured through tube. the measurement of minimum inhibitory concentration and minimum bactericidal concentration of ethanol and methanol extracts on e.coli were equal with 100 and 120 milligrams per milliliter, subsequently. moreover, the measurement of the minimum inhibitory concentration and of the minimum inhibitory concentration of marjoram ethanol extraction on staphylococcus aurous was reported to be 90 milligrams and 100 milligrams per milliliter, subsequently. in addition, the amount of ethanol and methanol extracts on salmonella enteric and p. aeroginosa was equal with 80 and 90 milligrams per milliliter, subsequently. the results showed that marjoram alcoholic extract enjoy antibacterial properties. also, among the alcoholic extracts, the ethanol extract has demonstrated to be the most effective extract on salmonella enterica and e. coli and p. aeroginosa. p-02.08.5-057 molecular analysis of serine/arginine rich sc35 splicing factor from olive b. bas 1 , e. d€ undar 2 1 depertmant of biology, university of balikesir, balikesir, 2 department of molecular biology and genetics, college of arts and sciences, balikesir university, balikesir, turkey olive (olea europaea l.) is an evergreen fruit tree adapted to mediterranean climate and rich in tannins, essential oils and organic acids. serine/arginine rich splicing factors are essential in seqeunce specific splicing of pre-mrnas. in this study we report molecular characterization of an serine/arginine rich sc35 splicing factor (oesarsc35sf) that was isolated from a cdna library constructed from olive pedicels. nucleotide blast and protein blast (for comparison of the similarity of the candidate genes from other organisms) were conducted on ncbi web page. amino acid composition analysis, nucleotide composition analysis, hydropathy analysis, open reading frame determiantion, through, molecular weight and the isoelectric points calculations were conducted using online expasy software. primer3 was used to design forward and reverse primers to amplify the target gene from different olive tissues at different times. analysis with bioedit program revealed that a+t ratio was more than that of g+c. oesarsc35sf was a protein consisting of 267 amino acids. as expected, amino acid composition analysis revealed that serines and arginines were more than other amino acids. kyte&doolittle hyddropathy analysis revealed that the protein was hydrophilic. the molecular weight of the protein was 30 kda with an isoelectric point (pi) of 11.5. the protein was found to have a signal peptide. according to the predotar analysis results, intracellular localization was found to be in the mitochondrial. the combined results suggest oesarsc35sf might function as splicing factor as its homologs from the other plants. confirming this hypothesis with futher experimental characterization including biochemical function analysis continues. acknowledgements: this study was supported by tub _ itak with grant number 110o108. keywords: olea europaea l., oesarsc35sf, alternative splcing, pre-mrna splicing, bioedit, pedicel specific cdnas. p-02.08.5-058 application of three-phase partitioning for the purification of peroxidase from kiwano (cucumis metuliferus) z. denli 1 , g. arabaci 2 1 kto karatay university faculty of medicine, konya, 2 faculty of arts and sciences, sakarya university, sakarya, turkey peroxidases are enzymes able to catalyze reduction of h 2 o 2 and oxidize various substrates. the kiwano is an oval shaped fruit which has an orange skin with lots of tiny horns. in this study, peroxidase isolated from kiwano is purified with three-phase partitioning (tpp). kiwano fruit was homogenized and obtained crude enzyme extract. the extract was saturated with 50% (w/v) ammonium sulfate ((nh 4 ) 2 so 4 ) and added t-butanol with the ratio of 1:1.5 (v/v). the lower and interfacial layer was collected. the influence of percent saturations of (nh 4 ) 2 so 4 (50, 65, 75, 85, 95%) and tbutanol ratios (1:0.5, 1:1, 1:1.5, 1:2) to the partitioning behaviour of peroxidase were analyzed. after dialyzed, the interfacial and lower phases were measured for peroxidase activity and protein content. the protein pattern of the peroxidases was evaluated by using gel electrophoresis. peroxidase activity recovery and the purification fold of interfacial and lower phases were 117.4, 1.7% and 0.84, 0.03. therefore, other experiments were continued with interfacial phase. at constant t-butanol with the ratio of 1:1.5, the enzyme activity recovery and purification fold of interfacial phase for saturations of (nh 4 ) 2 so 4 (50, 65, 75, 85%) were 40.9, 43.2, 39.2, 135% and 0.37, 0.31, 0.4, 0.91 . the interfacial phase was not dissolved in 95% (nh 4 ) 2 so 4 . at constant 85% (nh 4 ) 2 so 4 , the enzyme activity recovery and purification fold of interfacial phase for t-butanol ratio (1:0.5, 1:1, 1:1.5, 1:2) were 61.6, 48. 1, 127.6, 126.1% and 1.79, 0.6, 1.84, 1.41 . finally, at optimum conditions (% 85 (nh 4 ) 2 so 4 , t-butanol 1:1.5) after dialyzed interfacial phase, the enzyme activity recovery and the purification fold were 138.8% and 4.46. the results of gel electrophoresis showed that the molecular weight of enzyme was between 30-60 kda. the applications of tpp gave the maximum recovery of 138.8% and 4.46-fold purification. as a result, for purfication of peroxidases, tpp is a rapid, simple and economical technique. accumulating the most robust genes and proteins in elite genotypes without any harmful effect on the potential plant yield is an urgent need to enhance productivity under various stressors. among the stressors, drought is a major challenge for agricultural productivity. brachypodium distachyon, with its close relationship to agriculturally and economically important crops, is an important model plant species. although ongoing transcriptomic analyses in brachypodium distachyon available, proteomic analyses are required to obtain an integrated picture of drought response. in the current study, a comprehensive proteome analysis was conducted on brachypodium leaves under increasing levels of drought stress. to screen gradual changes upon drought stress, brachypodium leaves subjected to drought treatment for 4, 8 and 12 days were collected for each treatment day. the cellular responses were investigated through a proteomic approach involving two dimensional difference gel electrophoresis and subsequent combined tandem mass spectrometry. for the validation of transcriptional expression, the genes encoding selected proteins were examined through quantitative real-time pcr. spot detection on cye-dyed gels revealed a total of 497 distinct spots in brachypodium protein repertoire. a total of 13 differentially expressed proteins (deps), with at least 2-fold changes in abundance, were identified by mass spectrometry and classified according to their functions. the biological functions of deps included roles in photosynthesis, protein folding, antioxidant mechanism and metabolic processes, highlighting the significant degree of overlapping between metabolic alterations induced by drought stress. identified proteins in this study and understanding the molecular mechanisms of drought response and defense mechanisms in plants will contribute to the researches on development of drought tolerant crop species. p-02.08.5-060 immunohistochemical and electron microscopy investigation of tmv-based chimeric virus particles carrying conserved influenza antigen in nicotiana benthamiana recently we obtained and partially characterized set of viral vectors based on tobacco mosaic virus (tmv) genome displaying conserved influenza a m2e epitope from matrix m2 protein. purified chimeric virus particles (cvp) conferred protection of mice against lethal homologous and heterologous influenza virus challenge. we revealed significant difference in symptoms of infections caused by tmv-m2e recombinant viruses containing cysteine (cys) to serine (ser) or alanine (ala) substitutions in the human consensus m2e sequence. accumulation level of m2e-ala recombinant coat protein was significantly higher than m2e-cys/ ser (ratio 5:1). tmv-m2e-ser infection, in contrast to ala mutant, suspended growth and development of nicotiana benthamiana. non-inoculated leaves (14 d.p.i.) were fixed with ethanol and histological sections were incubated with mouse serum to m2e and secondary antibodies conjugated with either hrpo or fitc. cvps of all three mutants were detected in epidermal and stomata cells as well as in sieve elements and minor veins. electron microscopy analysis of mesophyll cells revealed typical rigid helical particles. cys/ser mutants mostly accumulated within ground cytoplasm as aggregates of discrete tubules in parallel arrangement, which were not delimited by lipid membranes. we discovered huge amount of cvps in the cytoplasm and lesser amount diffused in the central vacuole. essential part of ala particles was located in the cytoplasm, but mentioned aggregates were not found and only insignificant number of virions was revealed in vacuole. unlike wild-type tmv, none of the mutants was revealed in chloroplasts. diameters of cvps were as follows: sersingle particles in cytoplasm 13 ae 1. cyanidin is a natural anthocyanidin found in a variety of fruits (grapes, blackberry, blueberry, cherry and cranberry etc.) and vegetables (red cabbage, red onion). this polyphenolic compound is a flavonoid with significant antioxidant activity. cyanidin and its glycosides have vasoprotective effects and can interfere with inflammation, carcinogenesis, obesity, and diabetes. one important role of the macrophages is the release of pro-inflammatory mediators, such as nitric oxide, various cytokines, in response to activation signals, including chemical mediators, cytokines, and bacterial lipopolysaccharide (lps). in this study, we investigated the role of cyanidin chloride in inflammation. anti-inflammatory effects of cyanidin chloride were examined in lps-stimulated murine raw 264.7 macrophages. we observed the level of various inflammation markers such as nitric oxide (no), inducible no synthase (inos), cyclooxygenase-2 (cox-2), tumor necrosis factor-a (tnf-a) and interleukin-8 (il-8) under lps treatment with or without cyanidin chloride. cyanidin chloride inhibited not only no production but also the expression of cox-2 and inos, without any cytotoxicity. cyanidin chloride also attenuated pro-inflammatory cytokines and other inflammation-related markers such as il-8 in a dosedependent manner. in conclusion, cyanidin chloride may be beneficial for the prevention and treatment of anti-inflammatory diseases. p-02.08.5-062 the investigation of centranthus longiflorus plant extacts effects on cell proliferation and apoptosis activity in the cell lines of mcf-7 breast cancer h. askin ataturk university, erzurum, turkey introduction: in the u.s., breast cancer is the second most common cancer in women after skin cancer. current treatment of cancer can be done by surgery, chemotherapy, and radiation therapy. in addition, there is widespread use of complementary and alternative medicine in developed countries. plants and plant extracts play a critical role in the research into new anticancerogenic agents. centranthus longiflorus (cl) is used in alternative medicine for sedative and antispasmodic purposes. a plant of turkish origin, centranthus longiflorus used as traditional turkish medicine have remained uninvestigated for familial hypercholesterolemia, diabetes, coronary artery disease and cancer for their in vitro biological activity despite their use for sleep disorders. in this study, growth-inhibiting and pro-apoptotic effects of hexane, ethyl acetate and ethanol extracts of cl in mcf-7 breast cancer cell line were investigated. material and method: aerial parts of cl were collected in erzurum province. hexane, ethyl acetate and ethanol extraction were done by soxhlet extractor. the plant extracts obtained from cl was analyzed using a gc-ms system. dose-and time-dependent cytotoxic and apoptotic effects of cl were evaluated by mtt cell proliferation kit and cell death detection elisa kit, respectively. manufecturer's protocol was followed for analyses. then, apoptotic genes; caspase3, bax and p53 and antiapoptotic genes; bcl-2 and pi3 expression levels were determined by rt pcr. results: according to our results, cytotoxic effect on mcf-7 cell was only observed in 20 and 50 lg/ml doses of cl. however, any of the application doses showed an apoptotic effect on mcf-7 cells. they exhibited a necrotic effect rather than the apoptotic effect. although alterations in expression levels of these genes were determined, this alterations was statistically insignificant. discussion and conclusion: consequently, we can say that cl have a cytotoxic effect on mcf-7 breast cancer cell lines. p-02.08.5-063 reduction of the chloroplast genome and the loss of photosynthetic pathways in the mycoheterotrophic plant monotropa hypopitys, as revealed by genome and transcriptome sequencing e. gruzdev, a. mardanov, a. beletsky, v. kadnikov, e. kochieva, n. ravin, k. skryabin institute of bioengineering, research center of biotechnology of the russian academy of sciences, moscow, russia genomes of parasitic plants represent interesting model systems to study effects of relaxed selective pressure on photosynthetic function. previous genomic studies of nonphotosynthetic plants revealed reduction of their chloroplast genomes, but the corresponding changes in their nuclear genomes are less known. here we present the data on the transcriptome and the chloroplast genome of the non-photosynthetic mycoheterotrophic plant monotropa hypopitys. the chloroplast genomes were sequenced for two specimens of m. hypopitys, collected in different regions of russia. the cpdnas are 34,800 bp (mon-2kalr) and 35,336 bp (mon-1volr) long and rearranged with respect to each other. both genomes contains genes encoding ribosomal proteins, infa, matk, and 4 ribosomal rna genes. 17 and 19 trna genes were predicted in two cpdna. genes encoding nadh dehydrogenase, plastid rna polymerase, all genes related to photosynthetic apparatus, clpp, ycf1, ycf2, accd, and some genes for ribosomal proteins are missing or became pseudogenes. the reduction of gene content is associated with extensive gene order rearrangement and the lack of inverted repeats. overall, the size and gene content of m. hypopitys cpdna indicates that it is close to the end of plastid genome degradation process. in order to get insights into the changes in the nuclear genome associated with the transition to nonphotosynthetic lifestyle, we sequenced and assembled the transcriptome of m. hypopitys. as expected for holoparasites, we did not found transcripts for the nuclear genes encoding the components of photosynthetic machinery, including photosystem i and ii, cytochrome b6f complex, and ribulose bisphosphate carboxylase. contrary to the holoparasitic plant phelipanche aegyptiaca, almost all genes of chlorophyll biosynthesis pathway from protoporphyrin ix were not found in the m. hypopitys transcriptome. this work was supported by the rsf grant 14-24-00175 and rfbr grant 14-14-00749 (mon-1volr cpdna sequencing). introduction: beta-sitosterol is a substance found in plants. chemists call it a plant sterol ester. it is found in fruits, vegetables, nuts, and seeds. it is used to make medicine. beta-sitosterol is used for heart disease and high cholesterol. the federal food and drug administration (fda) allows manufacturers to claim that foods containing plant sterol esters such as beta-sitosterol are for reducing the risk of coronary heart disease (chd). centranthus longiflorus (cl) is used in alternative medicine for sleep disorders. a plant of turkish origin, cl used as folk medicine have remained uninvestigated for familial hypercholesterolemia, coronary artery disease and preventing colon cancer for their in vitro biological activity despite their use for sleep disorders. we investigated of the chemical constituents from dried aerial parts of centranthus longiflorus. material and method: aerial parts of cl were collected in erzurum province. hexane, ethyl acetate and ethanol extraction were done by soxhlet extractor. the plant extracts obtained from the aerial parts of cl was analyzed using a perkin-elmer gc-ms system. results: ten compounds were obtained and identified as butanoic acid, hexadecanoic acid (palmitic acid), 7-methyl-z-tetradecen-1-ol acetate, octadecanoic acid (stearic acid), diisobutyl phthalate, 9-octadecenamide, octacosane, nonacosane, alfa amyrin and beta sitosterol. the latter two were obtained in all extraction (hexane, ethyl acetate and ethanol). discussion and conclusion: all of these compounds are isolated from centranthus longiflorus for the first time. these findings may shed light on the design of new drugs, the cholesterollowering effect. p-02.08.5-065 role of lutein for the high light-induced inhibition of photosystem ii related reactions in thylakoid membranes of arabidopsis thaliana, wt and lut2 k. dobrev, d. stanoeva, m. velichkova, a. v. popova institute of biophysics and biomedical engineering, bulgarian academy of sciences, sofia, bulgaria photosynthetic reactions taking place in thylakoid membranes of higher plants are extremely sensitive towards different environmental stress conditions such as high and low temperature, high light intensity, uv radiation etc. carotenoids are intrinsic component of photosynthetic pigment-protein complexes and are involved in performing multiple important functions. their role of accessory pigments in absorbing sun light, participation in photoprotection via dissipation of excess absorbed light, deactivating of stress-induced reactive oxygen species and structural role are well documented and recognized. the role of lack of lutein in high light-induced alterations in structural organization and functional activity of the main pigment-protein complexes was evaluated using isolated thylakoid membranes of arabidopsis thaliana, wt and mutant lut2, deficient in lutein, subjected to photoinhibitory treatment for different periods of time. alterations in photochemical activity of photosystem i and photosystem ii were determined by a clark-type electrode in the presence of exogenous electron donors and acceptors. activity of oxygenevolving complex and of the grana and stroma situated photosystem ii reaction centers was evaluated by determination of flash oxygen yields and initial oxygen burst under constant light without donors and acceptors. low-temperature (77k) fluorescence was applied for unraveling of light-induced alterations in energy transfer and interaction between the main pigment-protein complexes. maximal quantum efficiency of psii was registered by pulse amplitude modulated fluorescence method. results obtained are discussed in respect to the importance of lutein for the organization and sensitivity of photosynthetic apparatus towards high light intensity treatment. modern agriculture relies heavily on phosphate rock fertilizer to improve phosphorus availability in many soils, but this approach is not sustainable long-term. phytate (myo-inositol hexakisphosphate) is an organic phosphorus compound often present in many soils. however, phytate can not be utilized by most plants, and its accumulation in soil leads to substantial ecological problems. phytases are enzymes that hydrolyze phytate and release inorganic phosphate. many microorganisms such as bacteria and fungi synthesize highly diverse phytases which are suitable for plant biotechnology. generation of transgenic plants expressing phytases of bacterial origin has been proposed as one option to improve plant phosphorus nutrition. in this study, we generated and characterized transgenic arabidopsis thaliana plants expressing a modified phytase gene paphyc from pantoea sp. under strong camv35s promoter. three individual transgenic a. thaliana lines expressing the bacterial phytase gene, as well as negative control plants harboring the camv35s promoter alone were identified. expression of phytase in plants was verified at both transcription and translation levels. phytase-expressing plants grown on media with phytate as the sole source of phosphorus demonstrated better than wild-type growth rates, shoot dry mass, shoot phosphorus content, as well as higher phytase activity in cell-wall extracts. overall, we show that plants expressing bacterial phytase are capable of better growth on phytate as the only source of phosphorus in laboratory conditions. further research investigating the applicability of using bacterial phytase expression to improve plant growth in soil is necessary to evaluate the different routes of solving the phosphorus deficiency problem in agriculture. p-02.08.5-067 the role of elevated temperature in photoinhibition and recovery of photosystem ii in thylakoid membranes from arabidopsis thaliana a. faik, m. gerganova, m. velitchkova institute of biophysics and biomedical engineering, bulgarian academy of sciences, sofia, bulgaria photosynthesis of higher plants is the principle process to transform light energy into biochemical usable energy. in nature, plants are exposed to the environment where light, temperature, uv-b radiation varied and very often their extreme values that are unfavorable for effective performance of photosynthetic reactions. plant are developed various strategies to cope with stress including radical scavenging enzyme system, accumulation of protective compounds, etc. pigment-protein complexes of photosystem i and photosystem ii and their light harvesting antenna, situated within thylakoid membranes, are involved in the primary reactions of photosynthesis -absorption of light, charge separation and electron transport. photosynthetic process is sensitive towards higher than optimal temperatures, the photosystem ii and oxygen evolving complexes being extremely sensitive to elevation of temperature. in present work pam fluorescence was applied to evaluate the effect of long term action of elevated temperature (38/30°c) on the quantum yield of photosystem ii, non-photochemical quenching and rdf, the latter quantifying the photosynthetic process. in addition, the activity of oxygen evolving complex was determined polarographically in the presence of exogenous electron acceptor 1,4 -benzoquinone. sds-page electrophoresis and western blot were applied to determine the damage of d1 -reaction center protein of photosystem ii. alterations of mutual organization within photosystem ii complex and its antenna and of energy interaction between them were followed by analysis of 77k steady state chlorophyll fluorescence spectra. the simultaneous application of high temperature and high light intensity resulted in a well pronounced reduction of non-photochemical quenching that restore to the initial values after recovery for 5 days at optimal conditions. d1 was also restored while quantum efficiency of photosystem ii did not recuperate to initial values. p-02.08.5-068 reorganization of the main pigment-protein complexes in thylakoid membranes from tomato (solanum lycopersicum) during long term exposure to elevated temperature the changes of earth climate resulted in unfavorable environment for plants. depending on the duration of influence of stress factors, the response of plants includes short and long term acclimation. the population, structure and organization of pigmentprotein complexes within thylakoid membranes are dynamic and flexible, thus providing for the acclimation of the photosynthetic apparatus to the changed environment. the main pigment-protein complexes, involved in energy transduction, are photosystem i, photosystem ii and light harvesting complexes. they are separated in grana and stroma regions of thylakoid membranes but it is well established that they can rearrange as a result of alterations of light intensity, temperature increase and decrease in order to balance the perception and utilization of excitation energy. in present work the effect of long term action of elevated temperature on organization and stoichiometry of main pigmentprotein complexes in the thylakoid membranes from tomato plants (solanum lycopersicum cv. m82) was investigated. three weeks old tomato grown at optimal conditions (22/20°c day/ night temperature and light intensity 250 lmol/m 2 /s) plants were exposed for 2 and 6 days to elevated temperature at 38/30°c. by means of blue-native electrophoresis the effect elevated temperature on the populations of psii (dimmer and monomers) and lhcii (monomers and trimers) was estimated and compared with the same parameters for control plants. the ability of plants to recover from this treatment was checked after 5 days under optimal conditions. the changes of content of chlorophylls and carotenoids were determined at every stage of treatment. based on the results obtained it can be concluded that one of the mechanisms for regulating the energy balance and maintenance of efficient photosynthetic process involves a change in the organization and stoichiometry of the photosystem ii and oligomer state of light harvesting complex ii. aim of the study, was to evaluate the anti-tumor effects of silymarin, curcumin and propolis on leptin-induced mcf-7 cells. mcf-7 cells were incubated various concentrations leptin (physiological, obesity and pharmacologically doses; respectively 10, 100 and 1000 ng/ml) . then different doses of silymarin (10, 20, 40, 80 lm), curcumin (10, 20, 40, 60 lm) and propolis (0.063, 0.125, 0.25, 0.5 mg/ml) were added. after 24, 48, 72 and 96 hours incubation periods 3 different area images were taken digital camera. then using dye release reagent we determined the intensity of apoptosis via colorimetric determination by elisa reader. absorbance was directly proportional the number of apoptotic cells (biocolor cell-apo percentageapoptosisassay). also, we examined the effect of these natural products on proliferation rate of leptin-induced mcf-7 cells for 1, 2, 3 and 4 hours (biovision cell proliferation kit) all experiments were carried out 3 different days, at least 3 times. all of three compounds were stimulate the apoptosis at all time points and all different doses of leptin. the differences was statistically significant at the level of p < 0.05 between 24 and 48 hours. it was found that there were not seen much cells at 72 and 96 hours time points. we thought that most of the cells were gone necrosis instead of apoptosis. the best effective doses on apoptosis of propolis was 0.063 mg/ml, silymarin and curcumin were 20 lm. also, we evaluated the effects of on proliferation rate the mcf-7 cells, we found that only propolis was effective of inhibiting proliferation at all doses of leptin induced mcf-7 cells in 1 hour. we hope this study will be a guide for the further studies in anti-cancer agent development field and show that the natural origin substances cause cancer cells apoptosis and provide targeted treatment for cancer therapy. p-02.08.5-070 investigation of some lichen-derived substances' cosmetic potential for skin protection against ultraviolet b due to the depletion of the stratospheric ozone layer and chronic exposure, the occurrence of various skin diseases have been increased in recent decades. thence, people and cosmetic companies have progressively given more importance natural sunscreen products for the protection from harmful sun rays, especially ultraviolet b rays. we, therefore, isolated some lichen-derived substances; 3-hydroxyphysodic acid and protolichesterinic acid from hypogymnia tubulosa and cetraria aculeate, respectively. chemical characterization and identification of the isolated lichen substances were accomplished by using ftir, 1 h-nmr and melting point analyses. the theoretical uv-vis spectra and 3d conformations of the isolated compounds were determined by using the gaussian 03 software with hf theory at the b3lyp/3-21g level. the dark toxicities and ultraviolet b protection capacities of the substances were lighted up as previously described [1, 2] on hacat human keratinocyte cell line by using mtt cell viability and ldh cellular membrane degradation assays. the obtained results from the assays showed that protolichesterinic acid has a more dark toxic activity on keratinocyte cells than 3hydroxyphysodic acid, and the toxic activities were found sufficient as much as 80% at the highest doses of the substances; 400 lm. however, it was observed that the cytotoxicity of the substances were reduced at the rate of approximately 15% by the irradiation. consequently, we think that the substances block the ultraviolet b rays but their cytotoxic feature is an important limitation to their usage in cosmetic industry. references [1] varol, m., t€ urk, a., candan, m., tay, t., koparal, a. t. (2016) photoprotective activity of vulpinic and gyrophoric acids towards ultraviolet b-induced damage in human keratinocytes, phytotheraphy research 30:9-15. [2] varol, m., tay, t., candan, m., t€ urk, a., koparal, a. t. (2015) a great effort and financial supports have been consumed to explore and design novel sun protection factors due to the unhindered increase of malignant and non-malignant skin diseases caused by the chronic exposure and depletion of the stratospheric ozone layer. the testing of naturally produced compounds seems to be the best and inexpensive way to search for potentially photoprotective substances. on the other hand, as photo-resistant species, lichens are still poorly exploited. norstictic acid was, therefore, isolated from the acetone extract of pleurosticta acetabulum. ftir, 1 h-nmr and melting point analyses were performed to identify the chemical features of norstictic acid. gaussian 03 software with hf theory at the b3lyp/3-21g level was also performed to determine the theoretical uv-vis spectrum and 3d conformation of the isolated compound. the dark cytotoxicity and ultraviolet b-protection capacity of norstictic acid were comparatively tested as previously described [1, 2] by using mtt cell viability and ldh cellular membrane degradation assays. as a result of the experiments, it is observed that norstictic acid has a dark-cytotoxicity as less as 25% at the highest dose of the substance; 400 lm. however, ultraviolet b-induced damage on human keratinocytes was blocked by the lower concentrations of norstictic acid as 25, 50 and 100-lm, and 20% of cells were protected according to the control experiments of irradiated cells. consequently, we think that norstictic acid might be employed as a sun protection factor at the low concentrations, and further studies should be performed. years, researchers have focused on the lichen acids because of their biological activities. it is also suggested that lichens can be used as anticancer agents. vulpinic acid, an important lichen seconder metabolite, has antimicrobial activity and strong antimutagenic, anticancer and antioxidant capacity. nanotechnology has the potential to offer solutions to current obstacles in cancer therapies, because of its unique size (1-100 nm) and large surfaceto-volume ratios. so, in this study we aimed to determine the cytotoxic and proliferative effects of vulpinic acid and magnetic nanoparticles loaded with vulpinic acid (fe 3 there are four species of wild rice known around the world. zizania aquatica l., zizania palustris l. and zizania texana hitche are found in north america, whereas zizania latifolia (griseb) turcz) is native to east asia. cwr mainly grows in the areas along the yangzi river and the huai river in china without any cultivation and domestication. cwr was an ancient grain that has been used in chinese herbal medicine to treat a variety of ailments associated with nutrition, including gastrointestinal disorders and diabetes. our previous studies have demonstrated that consuming chinese wild rice can significantly improve blood lipid profiles and ameliorate high-fat/cholesterol diet-induced insulin resistance. however, compared to the well studied common dietary white rice, active composition and the associated proteomic information of chinese wild rice have yet to be investigated. in this study, we compared and analysed the different proteins between chinese wild rice and white rice by proteomics method. our study provides insights and experimental evidence for further exploration of this ancient medical food in disease prevention and therapy. the homology between cwr and n22 is 64%, but significant differences also exist between the two. we gained new insight by analyzing the biological function of the high reliability (credibility score 67 or higher, p < 0.05) peptide mass fingerprint of cwr2-de electrophoresis revealed differences in protein composition between cwr and n22. information obtained from the pmf indicates that glutelin precursor, caffeoyl coenzyme a (coa) omethyltransferase and putative bithoraxoid-like protein can provide gene evidences for its biological function. p-02.08.5-075 mir396 and growth-regulating factors interaction during maize leaf growth under low-temperature stress g. aktug, f. aydinoglu gebze technical university, kocaeli, turkey microrna (mirna) genes are a class of non-coding small rnas about 21 nucleotide-long which are revealed as regulators of plant growth and stress responses. the mirna mir396 targets and regulates growth-regulating factors (grfs) which are plant specific transcription factors family and this regulation machinery is conserved among plant species. plant growth is a result of cell division and expansion which took place as spatial gradient zones throughout maize leaf which are meristem, elongation and mature zones. cells proliferate in meristem, migrate to elongation zone and finally reach to mature zone to get its final size. it has been shown that mir396 affects cell division by regulating grfs and changes leaf size which are determined by cell number and cell size of leaf. this study aims to investigate the role of mir396 and grfs interaction during maize leaf growth under low-temperature stress. maize seedlings were grown under low-night temperature for stress treatment to generate growth retardation and control conditions as well to make comparative analysis. length of the third and fourth leaves of seedlings was measured every day and leaf elongation rate was calculated to observe stress effects on the leaves. growth zones of fourth leaves were harvested during steady-state growth phase for determining expression level of mir396s and their target by q-rt-pcr. we mined 8 mir396 genes sharing sequence similarity and 8 grf targets. the expression analyses of mir396s and grf5 are proceeding for different growth zones. in conclusion, this is the first study investigating the regulation network between mir396s and grf5 in different developmental stages of maize leaf under low-temperature stress. oeigpd cdna was isolated from a cdna library we constructed from olive pedicels. homology searches for nucleotide, amino acids and alternative open reading frames were conducted utilizing blastn, blastp, and blastx, respectively. nucleotide sequences of homologous genes from other plants were aligned using bioedit and the number of snps were detected. the alignment was then used to generate a phylogenetic tree using mega7 program. another alignment with amino acid sequences of the homologues proteins was also generated to construct a phylogenetic tree displaying oeigpd's position among other plants. various aspects of oeigpd including amino acid composition, hydropathy analysis, isoelectric point (pi) and three dimentional structure of the protein were determined using online software at expasy. multiple primer pairs to amplify the full length open reading frame of the gene, to clone the gene into the expressing vector plate51, and to detect expression through real-time pcr were designed using primer3. amino acid composition analysis revealed that oeigpd contained serine, arginine and isoleucine predominantly while hydropathy analysis suggested it was an hydrophilic protein. isoelectric point (pi) of the protein was calculated as 4.97. the molecular weight of the protein was calculated as 11 kda. analyses continue to determine the polymorphism of oeigpd among olive cultivars, and biochemical function of the gene in olive. p-02.08.5-077 cytotoxic effect of fractionated triterpenoid glycosides from holothuria polii in human cancer cells sea cucumbers are the members of class holothuroidea and they have more than 1100 described living species all around the world. sea cucumbers secrete special secondary metabolites from their body walls and they are called triterpene glycosides (tggs). in this study, cytotoxic activity of fractionated ttgs from h. polii on different cancer cell lines were carried out. h. polii delle chiaje, 1824 samples were collected from dikili-_ izmir. the semipurified extracts were fractioned by using hplc. four different fractions (fraction a-d) were obtained. in order to characterize the fractions, maldi-tof/ms was used. the cytotoxic activity of the fraction a-d were tested on ht-29, t84 and upci-scc-131 cell lines by using xcelligence rtca sp system. the cells were treated with three different concentrations of the fractions for 48 hours. the cell index data were compared with the control group. ic50 values of the fractions for three cell lines were calculated. according to the results, the fractions have holothurin a (1243.50 m/z), 24-dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer (1227.50 m/z). the fraction d was the most effective on all cell lines with ic50 value of 10.46 ae 0.18 mg/l, 11.24 ae 0.57 mg/l and 11.13 ae 0.29 mg/l for ht-29, upci-scc-131 and t84, respectively. in conclusion, sea cucumber ttgs are promising agents for colon adenocarcinoma, oral squamous cell carcinoma and colorectal carcinoma (metastatic) treatment. p-02.08.5-078 effect of horse-chestnut (aesculus hippocastanum) seed extract on matrix metalloproteinases during diabetic wound healing impaired wound-healing in diabetics is a major public health problem. the expression and activation of matrix metalloproteinases (mmps) are also impaired in diabetic wounds according to previous studies. their main function is to degrade the various components of the extracellular matrix. also, they participate physiological processes such as inflammation, angiogenesis, tissue remodeling. horse-chestnut seeds (hc) are rich in saponins and flavonoids. it has been shown that hc has antiinflammatory, antioedema, vessel protective, and free radical scavenging properties. the aim of this study is to determine with molecular signs on cutaneous wound healing effects of the ethanol (50%) extract of hc (hce) seed in rats by excision wound model. this study was conducted on diabetic wistar albino rats, which were injected by a single dose (50 mg/kg i.p.) streptozotocin. diabetic treatment rats were applied topically 15% (w/w) ointment with hce and control rats were applied topically simple ointment, once a day during the experimental period. the gene expression levels of mmp-1, mmp-9 by qpcr and levels of nitric oxide (no), hydroxyproline and malondialdehyde in wound tissue investigated at the end of 3rd, 7th, and 14th days. wound closure was also measured. the hydroxyproline and no levels were significantly increased in the hce treated group versus control after the 3rd and 7th days. the malondialdehyde levels were significantly lower in the treatment group. mmp1 gene (associated with collagen processing and reepithelialization) expression levels in hce treated rats were increased in the 7th day while it was reduced in 14th day. mmp 9 gene (associated with inflammation and gelatinase) expression levels in hce treated rats were decreased in 7th, and 14th days compared to the control. these findings indicate that hce accelerated the cutaneous wound healing process in diabetic rats via mmp1 and mmp9 regulation. p-02.08.5-079 isolation and molecular molecular characterization of vps39/vam6 from olive b. celikkaya, e. dundar molecular biology and genetic at balikesir university, balikesir, turkey vps39/vam6 promotes aggregating and fusing of endosomes and lysosomes. it is a component of a protein complex that is found in vacuole membranes. this gene has been studied from various organisms including humans, drosophila, arabidopsis and rice. no studies on olive vps39/vam6, however, have been reported. the aim of this study is to report information of olive vps39/vam6 including expression pattern and biochemical characterization. vps39/vam6 was isolated from a cdna library we constructed from fruited olive leaves in july. to determine the putative name of the cdna, blast analyses were conducted for nucleotide, open reading frame and amino acid sequence comparisons. bioedit program was used to determine the nucleotide and amino acid composition along with its molecular weight and isoelectric point (pi). hydropathy analysis was conducted using kyte and doolittle program. phylogenetic analysis was done using mega7. cellular localization of the product was predicted using sosui gramn. the three dimentional structure of the protein was calculated using i-tasser and compared to previously known structures using cn3d. the blast and bioedit analyses revealed vps39/vam6 had 836 base pairs coding 120 amino acids with a molecular weight of 13.38 kda, and pi of 6.39. the at/gc ratio was very high (1.5) comparing to its homologs from other plants suggesting to expect significant differences of this gene's function from the others. amino acid composition analysis revealed high rates of serine, leusine and isoleucine indicating a hydrophobic property of the protein. the hydrophobic feature was confirmed by kyte and doolittle analysis while the cellular location was revealed to be extracellular. the hydrophobic nature despite extracellular location suggests it is a membrane associated protein which was confirmed by transmembrane domain analysis. as expected no signal peptide was detected. the 3d structure of the protein was similar to its previously reported homologs. p-02.08.5-080 isolation and characterization of the ribosomal l1 protein from olive s. cinarli, e. dundar department of molecular biology and genetics, balikesir university, balikesir, turkey despite as a ribosomal protein, l1 is known as the inhibitor of the cellular aging gene and it has been reported to have roles in apoptosis. the ribosomal l1 protein is larger than the lsu of ribosome and contains 2 domains as 2-layered alpha/beta domain and 3-layered alpha/beta domain. in ribosomes, it functions in translocation and orientation of trnas. although the ribosomal l1 (rl1) gene has been studied in many plants, reports on olive rl1 (oerl1) are very rare. this study presents molecular characterization of rl1 gene from olive. oerl1 was isolated from a cdna library we constructed from unfruited olive leaves in july. homology analyses were conducted using blast programs. nucleotide and amino acid compositions, molecular weight, isoelectric point (pi) and at/gc ratio were determined using bioedit and expasy programs. cellular location of the l1 protein was determined using sosui-gramn program. signal peptide detection, transmembrane domain detection, three dimensional (3d) structure analysis, and phylogenetic analysis were conducted using signalp 4.1, thhmm, i-tasser/cn3d and mega7, respectively. oel1 was found to have an open reading frame of 909 base pairs coding 220 amino acids that constitutes a molecular weight of 24.9 kda and a high pi of 9.87. lysine, leucine and valine had higher rates. the hydrophilic nature suggested by kyte and doolittle analysis despite high rates of leucine and valine suggests an amphipathic nature of the protein that can bind to both hydrophilic and hydrophobic proteins and / or function in both media. a 1.59 at/gc rate is significant comparing to that of its homologs from other plants. sitoplasmic location predicted by sosui-grann is in agreement with the hypothesis suggesting an amphyphatic nature for oerl1. likewise, no signal peptide was detected and it was predicted to have at least one transmembrane domain. further characterization of oerl1 with respect to expression pattern and biochemical function continues. p-02.08.5-081 isolation and characterization of an olive splicing factor 3b subunit 1 m. nurcin, e. dundar department of molecular biology and genetics, balikesir university, balikesir, turkey splicing factor 3b subunit 1 (sf3b1) functions in the regulation of translation and gene expression. sf3b1 forms u2 small nuclear ribonucleoprotein complex (u2 snrnp). splicing factors 3a and 3b binds pre-mrna at the 5 0 site of the intron branching point. this binding joins u2 snrnp to pre-mrna. although sf3b1 from various plants have been widely studied, no studies on olive sf3b1 (oesf3b1) have been reported. this study reports information on various aspects of oesf3b1. oesf3b1 was obtained from a cdna library we constructed from fruited olive leaves in december. it was putatively identified as a splicing factor using blastn, blastp and blastx. to determine wheter oesfb1 was a sitoplasmic protein, sosui gramn was used. tmhmm was used to detect any transmembrane domains while signal peptide analysis was conducted by signalp. i-tasser and cn3d were used to generate the calculated 3d structure and to compare it with experimentally generated models, respectively. nucleotide and amino acid compositions along with the calculated molecular weight and isoelectric point (pi) were analyzed using bioedit and online expasy software. the phylogenetic trees revealed genetic relationship of olive among other plants based on oesfb1. the orf contained 1950 nucleotides coding 254 amino acids that produce a 28.2 kda peptide with a pi of 5.94. alanine, valine and leucine were found at high ratios suggesting a hydrophobicity which was also predicted by kyte and doolittle analysis. the at rich property of oesf3b1 is not unusual comparing to most plant genes. cellular localization of the gene was suggested to be in mitochondria with no signal peptide indicating oesf3b1 could be synthesizing in mitochondria. the predicted 3d structure of oesf3b1 was similar to experimentally produced structures while some hydrophobic pockets were predicted. further characterization of the gene with respect to temporal and spatial expression pattern and biochemical function continues. p-02.08.5-082 kafirin profile of turkish originated sorghum populations r. temizg€ ul, s. yilmaz, m. kaplan, t. akar department of biology, faculty of science, erciyes university, kayseri, turkey sorghum bicolor l. is the fifth important crop in the world with its high photosynthetic activity and resistance to unfavourable conditions as high temperature, drought, salt, and ph changes. sorghum has attracted great interest due to its intensive usage both as human and animal nutrition, and contribution to resistance against many diseases. some proteins of sorghum exerts reducing effect on nutrient digestion through making connetions with other proteins and/or carbohydrates. kafirin proteins have the highest proportion in grain with a range of 77-82%. they are grouped into a (23-25 kda), b (18 kda), c (28 kda) and d (13 kda) subunits depending on molecular weight, solubility and structure. in the current study, kafirin proteins from turkey originated 282 sorghum populations were acquired through sequential extraction; first, non-prolamines were removed through application of 5% naci concentration, and second, kafirins were obtained using tertiary butanol (60%) and reducing agents. sds-page was conducted for seperating and visualising the subunits of kafirins. the a, b, c, and d subunits of populations were respectively estimated as 97, 85, 95, and 60%. of the total proteins, 74% was identified as a, 12% b, 12% c, 1% d, and %1 non-prolamines. non-prolamin group of proteins were visualised as 18 different bands ranging from 15.6 to 230 kda. c and b group of proteins were only viewed when treated with reducing agents as 2-me and dtt suggesting that they are connected with complex cross-links. however, a group of proteins visualized without using these agents due to not having intra molecular disulphide bridges and inter molecular cross-links. non prolamins, except for 47.8, 45.7, 21.7, 20.3 and 15.6 kda, were able visualised in the presence of reducing agents. transcriptomic analysis of the genes encoding analysed proteins needs to be elucidated for better understanding of the genetic diversity and biochemical characteristics of sorghum. p-02.08.5-083 untargeted metabolomic profiling of romanian and uk tomatoes varieties by high performance liquid chromatography coupled with mass spectrometry c. socaciu 1,2 1 university of agricultural sciences and veterinary medicine, cluj-napoca, 2 center for applied biotechnology ccd-biodiatech at proplanta ltd, cluj-napoca, romania tomato flesh is a rich source of many phytochemicals of high nutritional value, including a large variety of carotenoid derivatives with health promoting properties. metabolomics became the most adequate technology for an accurate chemotaxonomic classification and discriminations between different varieties, based on untargeted profiling or targeted, quantitative analysis. different varieties of tomatoes (b-carotene-rich, lycopene-rich, ketocarotenoid-rich) cultivated in romania and uk were comparatively studied using enriched fractions obtained by a preliminary fractionation of the whole pulp homogenate. two methods were applied for carotenoid extraction: a mixture of hexane/ethanol (1) and chloroform/methanol (2) . the dried extracts were dissolved in ethyl acetate and analyzed by uv-vis spectrometry and hplc-esi(+)qtof-ms (bruker gmbh). the base-peak chromatograms were processed by specific biostatistics software (data analysis and profile analysis) and the molecular identification were determined by comparison with the data base lipidomics gateway (www.lipidmaps.org). the content of carotenoids were significantly higher using extraction (2) , ranging from 4.5 to 7.6 mg/100 g. the major carotenoid derivatives, were represented by lycopene, hidroxy-lycopene, all-trans or cis-beta-carotene, echinenone, all-trans retinyl palmitate, but also sterols, phospholipids, di/tri glycerides and ceramides. the romanian varieties were more rich in polar carotenoids and lipids, in general, while the uk tomatoes proved to be enriched in non-polar derivatives, especially esterified carotenoids, keto-carotenoids and glycerides. new molecules were identified, as good discriminatory markers of each tomato variety. acknowledgements. this work was supported by a grant of the romanian national authority for scientific research and innovation, cccdi -uefiscdi, project nr. 16/2015, pncdi3. glycogen is a multi-branched polysaccharide that serves as the main form of glucose storage in the body, where the main reserves are in the liver and muscle. it has been observed that glycogen metabolism is altered in many tumor types, and that glycogen content is inversely correlated with proliferation rate. in addition, it has recently been described that when glycogen accumulation is forced in glioblastoma u87 cells in hypoxia, senescence is induced and tumor growth is inhibited in vivo. our laboratory has various animal models with different parts of the glycogen metabolism pathway affected. most notably, we have two animal models lacking glycogen: muscle glycogen synthase (gys1 ko) and liver glycogen synthase (gys2 ko) knockout animals. we isolated mouse embryonic fibroblasts (mefs) from gys1 ko to perform replicative senescence assays. in addition, we induced hepatocellular carcinomas in gys2 ko animals via n-nitrosodiethylamine (den) injections in order to track tumorigenesis in animals lacking hepatic glycogen. lastly, we performed partial hepatectomies (phx), which involves the resection of two thirds of the liver, on gys2 kos to evaluate the effect of the lack of glycogen on hepatocyte proliferation. interestingly, we have observed that glycogen levels are increased in human and mouse fibroblasts under replicative senescence, and that mefs depleted of glycogen bypass senescence and immortalize faster than wts. we have also demonstrated that senescence pathways are down regulated in mefs lacking glycogen. furthermore, gys2 kos treated with den show higher tumor burden and mortality than controls. we also evaluated the effect of glycogen on hepatocyte proliferation after phx. gys2 ko mice present faster proliferation and liver regeneration rates, when compared to wt counterparts. collectively, our preliminary data suggest that glycogen metabolism plays a crucial role in the regulation of cell cycle in both physiological and pathological states. it is established that pineal is involved in circadian regulation of testosterone secretion from leydig cells. however, the precise routes of this regulatory involvement are still unknown. as cgmp has been also regarded as modulator of steroidogenesis we sought to study the effects of pineal removal on the circadian pattern of cgmp variations and expression of the genes that encode elements of no-cgmp signaling pathway in adult rat leydig cells. the analysis was performed on testicular leydig cells obtained from pinealectomised and shame pinealectomized rats, in six time point during 24 hours. the pinealectomy was confirmed by serum melatonin eia measurement. the androgen levels were measured by ria; cgmp by eia and gene expression was quantified by rq-pcr. all results were analyzed by cosinor method. data revealed circadian transcriptional pattern of nos2, nos3 (genes encoded no producers) and pde5a (gene for cgmp remover) in leydig cells from adult rats. pinealectomy significantly increased expression of nos2 which lost rhythm and increased and delayed amplitude of nos3 expression. further, pinealectomy initiated cyclic transcription of gucy1b3 and noncyclic transcription of gucy1a3 (genes encoded cgmp producers) and increased mesor and amplitude of pde5 transcription. the transcription of prkg1, the main effector in this signaling pathway was not affected with pineal abolition. additionally, pinealectomy did not influence the circadian transcription profile of coxi2 or other investigated genes (coxi1, nrf1, nrf2a, pgc1a) related to mitochondrial function and biogenesis. finally pinealectomy reversed phase of circadian cgmp oscillation in leydig cells, increased amplitude and slightly advanced peak of serum testosterone oscillation. results suggested pineal influence on circadian rhythm of no-cgmp signaling in leydig cells. further studies based on these data are needed to better understand the relationship between pineal and circadian rhythm of testosterone production. influenza is a contagious respiratory infection caused by a variety of influenza viruses. neuraminidase inhibitors is a new class of antiviral drugs that inhibit influenza viruses. the most popular antiviral agents is oseltamivir, having a commercial name of tamiflu, within anti-influenza antivirals. as well as tamiflu is a member of neuraminidase inhibitor group drug. therefore, this study was performed to determine the effect of tamiflu on cultured human peripheral blood lymphocytes. material and methods: for examining the presence of the indirect mutagenic effect of oseltamivir in iver s9 fraction mix was used. cells were treated with 0.5, 1 and 2 lg/ml oseltamivir, the tamiflu capsule ingradient, for 24 or 48 hours in the absence or presence of an exogenous metabolic activation system. the test chemical did not demonstrate any genotoxic effect dose-dependently but it showed a weak cytotoxicity on cells in this study. on the other hand, some concentrations of tamiflu induced sce and also decreased significantly the proliferation index (p ˂ 0.05) in the absence of s9 mix. result: tamiflu did not induce significant increases of ca or micronucleated cells in vitro in cultured peripheral blood lymphocytes under the treatment conditions used but week sce induction was observed. on the other hand, the weak cytotoxic effects observed disappeared in the cultures treated in presence of the s9 mix. discuss and conclusion: tamiflu weakly induced sce at the highest concentration with/without added s9 mix in cultured human peripheral lympocytes. it could be assumed to be a sce inducer. sces can be increased by several agents that attack dna. tamiflu decreased the proliferation index and nuclear division index at some concentrations thus interferring it as being weakly cytotoxic, though this effect disappeared in the presence of s9 mix applications. this finding is important for showing the inefficiency of tamiflu metabolites on the cell cycle. introduction: chronic renal failure as a result of the progression of diabetic nephropathy is the main cause of mortality in patients with type 1 diabetes. chronic hemodialysis is a life-saving therapy for patients with strong renal disorders. the main goal of hemodialysis is toxins removal from the patient. the monitoring of hemodialysis is the best way for biomedical evaluation of correctness and efficiency of this clinical treatment. according to the published data, the markers of development of diabetes complicated with renal failure are increased levels of glucose, urea and creatinine in the patient blood. today colorimetric and spectrometric methods are most commonly used for determination of the above metabolites in biological samples. however, these methods are complex in application, have low selectivity, and require pretreatment of samples. materials and methods: we propose for levels of glucose, urea and creatinine detection the potentiometric multibiosensor based on ph-sensitive field-effect transistors and immobilized enzymes developed in our laboratory. results: we developed a potentiometric multibiosensor and studied its main analytical characteristics. linear dynamic ranges of determination of substrates were following: 0.08 -1 mm of glucose, 0.1-10 mm of urea, and 0.02 -2 mm of creatinine. it was shown that the potentiometric multibiosensor had good reproducibility, and its bioselective elements were working independently from each other, because test of substrates cross-selectivity was negative. discussion and conclusion: very sensitive, fast and selective multibiosensor for simultaneous measurement of three metabolites in a single cycle based on ph-sensitive field-effect transistors and immobilized enzymes is developed. the developed potentiometric multibiosensor was verified by quantitative analysis of glucose, urea and creatinine in blood serum of patients with diabetic nephropathy. p-03.04.4-002 ph-dependent interaction of asymmetrically charged peptides with a protein nanopore over the past two decades, the ability to use natural or artificial nanopores to probe at uni-molecular level the structural and kinetic features of various bio-molecules (peptides, dna, rna) was successfully achieved. the operating principles of the nanopore-based single-molecule technique are simple: the single macromolecule capture, entry and subsequent translocations through a free-standing, voltage-biased nanopore, depend upon the physico-chemical and topological features of the analyte. the concentration, identity volume and charge of the analyte are then deduced from the analysis of the stochastic current blockade events caused by the trafficked analyte across the nanopore. herein, we used the a-hemolysin (a-hl) nanopore and set up an experimental model providing efficient control of a-hl-peptide interactions, in the presence of a ph gradient across the nanopore. for this, we engineered a 36 amino acids long peptide containing a neutral asparagines-containing sequence, flanked by oppositely charged aminoacid patches at the n-(glutamic acids) and c-termini (arginines), whose length was set as to span a single a-hl protein. when the ph of the solution in contact to the a-hl's b-barrel opening is changed from neutral to acidic values, the electrostatic interactions between the protein's mouth and either the n-or c-terminus end of the peptide occurs, and this influences strongly the dynamics of a peptide translocating the nanopore. we further proved that during the same experiment, peptide entry into the nanopore can be set to occur with either n-or c-terminus end head on, by simply changing the sign of the transmembrane potential across the nanopore. nanopores are emerging as a powerful and broadly applicable tool in biophysics, which allows one to study the features of charged macromolecules under confinement. a few noteworthy examples are: determining the electrophoretic mobility, effective charge and diffusion coefficients of charged molecules; exploring the folding and unfolding of peptides and proteins; analyzing biopolymers trafficking, protein transport, dna translocation, rna and dna sensing and sequencing. herein, we employ single molecule analysis techniques using a wild-type ahemolysin (a-hl) protein nanopore to study the capture and translocation behavior of a short cationic peptide (20 amino acids in length) at an extremely low ph value. our experiments revealed that an effective absorbing field is created by the electroosmotic flow, against the electrophoretic force, which enables the peptide capture inside the nanopore. furthermore, our findings show that the trajectory of a single peptide can be experimentally visualized and the main steps determined: the peptide capture, reversible translocation across the pore's vestibule and lumen regions, and the peptide release from the nanopore. also, the kinetic analysis of the main steps observed allowed us to describe the free energy profile of the peptide interactions with the protein nanopore. the presented work provides evidence for the ability of controlling the dynamics of a single-peptide, its capture and passage inside a a-hl nanopore, that underlie the processes naturally occurring in cells, thus proving a powerful approach for probing single molecule biophysics phenomena, in general. changes in the physical conditions of the cancer microenvironment driven by elevated tissue growth and angiogenesis, may introduce exposure of laminar fluid flow, which effect the key factors of cancer, such as progression, immune-escaping and metastasis. conventional experimental models fail to mimic the physical cues on tumor microenvironment. microfluidic culture techniques allow precise control of fluids, simultaneous manipulation and analysis of cultured cancer cells. here, we present a platform that can be used for the investigation of the role of flow mediated mechanical stimuli on cancer cells. microfluidic cell culture platform was fabricated using polymethyl methacrylate and double-sided adhesive films with 25 9 41 mm dimensions. ovary adenocarcinoma cells (efo-27 and onco-dg-1) were used for the optimization of the platform. to understand the fluid and gas distribution patterns, specific modeling was performed. dynamic microfluidic cell culture and static conventional cell culture conditions were compared for the differences of cancer cell phenotype, such as proliferation, viability, epithelial-mesenchymal transition. we confirmed that, the proliferation and viability of cancer cells are increasing under dynamic fluid flow. the proliferation rate of ovary adenocarcinoma cells was correlated with the increase of fluid flow rate. immunocytochemical analysis showed that fluid flow causes decrease in e-cadherin expression, and increase in n-cadherin and vimentin expressions, which indicate mesenchymal phenotype of cancer cells. our results showed that, cancer cells present different characteristics due to fluid flow of tumor microenvironment. to understand the role of physical dynamics by using microfluidic culture techniques, is a key to elucidate the mechanisms underlying disease progression, and may lead to new diagnostics and therapeutic approaches. (this study was funded by turkish scientific and technical research council (tubitak-214s334). high-sensitive detection of low-affinity antibodies by immuno-pcr with supramolecular olygonucleotide-streptavidin complex detection of low affinity antibodies in blood sera and cell surface outwashes is important both in the study of molecules that bind to cellular receptors (circulating tumor cell masking antibody, for example) and medicine (diagnosis of allergy). low affinity igm and ige antibodies can not sometimes be determined by conventional methods. we using supramolecular oligonucleotide-streptavidin complex formed from single-stranded synthetic oligonucleotide (60 n) contains biotin on 5'-and 3'-ends, and sterptavidin in molar ratio 1:1. this complex represents a structure with equivalent electrophoretic mobility of 600 bp dna and preferred "valency" of streptavidin is 2. this universal immuno-pcr approach make it possible to increase a signal by using several oligonucleotides per one antibody. after the method optimization we achieved 100-1000 times highter sensitivity than elisa. to reduce the matrix effect we used 100-500 fold dilutions of sera samples. this approach achieved a significant advantage, because it allows working with small-volume samples (need only 2 mkl of serum sample). antibodies to the disaccharide galb1-3glcnac (le c ) are typical of the natural antibodies. the igm anti-le c antibodies are found in almost healthy people without the epitope specificity variation. we have shown that the concentration of igm anti-le c antibodies was higher (p ≤ 0.0005) for health donor sera (n = 45; 31.3 ae 4.3 pg/ml) compared with sera from patients with breast cancer (n = 38; 17.0 ae 4.1 pg/ml). sensitivity of igm anti-le c antibodies detection was 100 pg/sample (50 mkl) ie 6.7 9 10 7 molecules. thus for the immuno-pcr detection of antibodies the 10 3 -10 4 tumor cells are sufficient. such amount of cells seems to be a realistic one for detection of antibodies masking circulating tumor cells. this study was supported by a grant from russian science foundation (#16-14-00136) and by russian federation president scholarships donated to d. yu. riazantsev (# sp4413.2015.4 bacterial pathogen detection and identification is of crucial importance for disease diagnosis, bacterial contamination surveys and water quality assessment. we propose herein a novel method for bacterial detection based on the interaction of single gram-negative bacterial cells (i.e.: escherichia coli and pseudomonas aeruginosa) with an ahemolysin (a-hl) protein nanopore embedded in a reconstituted lipid bilayer, at neutral ph. as a consequence of an applied voltage, the negatively charged bacteria suspended in saline buffer solution are electrophoretically driven towards the pore opening, inducing reversible blockages in the ionic current through a-hl. experiments were also performed in the presence of an antimicrobial peptide, cma3, as well as in acidic environment. statistical analysis of the frequency and duration of blockage events allowed us to discriminate between the two types of bacteria. the frequency of interactions was higher for escherichia coli with respect to pseudomonas aeruginosa. adsorption of cma3 peptides on the membrane of bacteria increased the frequency of interactions with the pore, contrary to the expected effect induced by lowering the net surface charge of the cells. in experiments performed at ph = 4, the frequency of blockage events was found to be two orders of magnitude higher, with longer interaction life-times. the net negative charge (7 uncompensated aspartate residues) localized at the entrance of the pore contributes an additional electrostatic repulsion interaction between negatively charged bacterial cells and a-hl. thus, adsorption of cationic peptides at the interface will reduce this repulsive interaction. the same effect was recorded at ph = 4, when the aspartate residues are partially protonated, confirming our understanding of the previously observed results. this method could be further developed and integrated with other techniques, making nanopore-based systems a fast and reliable bacterial detection and identification tool. this study was performed to analyze the effects of tunicamycin (tm) and taurohyodeoxycholic acid (tudca) on thle-3 cells. cells were treated with tm to induce endoplasmic reticulum (er) stress and tudca was administered as an er stress inhibitor. cytotoxicity was evaluated at different times of exposure by incubating cells with increasing concentrations of either tudca, tm or both. thle3 cells were cultured in fibronectin, bovine collagen i and bovine serum albumin coated plates. cell lines were grown in begm media supplemented with epidermal growth factor, phosphoethanolamine, fetal bovine serum, 100 u of penicillinstreptomycin and maintained in a humidified incubator at 37°c and a 5% co 2 atmosphere. cell viability was measured using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay kit. cells were grown to confluence in 96well plates and incubated with 1 ll/ml dmso, 1-10 lg/ml tm, 0.1-5 mm tudca, or 10 lg/ml tm + 0.1-1 mm tudca for 18-48 hours. control cells were prepared in plates containing only medium. at the end of the incubation period, mtt was added to each well and incubation was carried out for 4 hours at 37°c. formazan production was expressed as a percentage of the values obtained from control cells. at all hours of incubation neither dmso or 1 mm tudca was cytotoxic. at 24 and 48 hours incubations 5 mm tudca and 10 lg/ml tm + 1 mm tudca were significantly cytotoxic compared to control, dmso and 1 mm tudca groups. treatment of cells with 0.5 mm tudca 8 hours before administrating 10 ug/ml tm significantly decreased the cytotoxic effect of tm. we conclude that tudca may show cytotoxic effects at 1 mm concentration when treated with tm. therefore 0.5 mm of tudca, administered 8 hours before tm treatment should be applied to protect against er stress. acknowledgement: this study was supported by a grant from the scientific and technological research council of turkey (tubitak; 214s223). recent studies reveals that history of preeclampsia is an independent risk factor for cardiac events and stroke. lipoprotein-associated phospholipase a2 (lp-pla2) is a vascular inflammatory marker associated with cardiovascular diseases (cvd). we hypothesize that vascular inflammation (lp-pla2 mass, activity, index) related genetic variations (pla2g7) increase the risk for developing future cardiovascular disease in women with pe. a group of 150 preeclamptic patients and 50 normal pregnant women were recruited from university of istanbul, cerrahpasa medical school, department of gynecology and obstetrics included into the study. the control group was matched for maternal and gestational age at time point of sampling. preeclamptic patients were starified into two groups; early-onset and late-onset according to the 32 gestational weeks. enzyme-linked immunosorbent assay procedure was used to determine the serum lp-pla2 mass level. lp-pla2 activity were determined by kinetic method. plag7 snp genotyping performed by using the sequenom massarray iplex. the rs1805017 tt genotype had a higher lp-pla2 index (p = 0.015) for early onset preeclampsia, cc genotype had a higher lp-pla2 mass and lp-pla2 index for late onset preeclampsia. no difference were found for control. the rs1809381475 gg genotype had higher lp-pla2 mass and index for late onset preeclampsia (p = 0.008, p = 0.012 respectively). stepwise logistic regression analysis performed to identify cardiovascular disease related variables that independently and significantly contributed to the presence of alleles of rs1805017 and rs1809381475 snps in early, late onset preeclampsia and control group. only lp-pla2 mass was independently and significantly associated with both snps in early onset preeclampsia. the association between lp-pla2 mass, index and rs1809381475, rs1805017 snps might be useful genetic markers to adress future cvd risk in patients with preeclampsia. introduction: b-thalassemia is one of the most monogenic autosomal recessive disorder characterized by defective production of the b-chain of hemoglobin. definition of the b-globin genotype is necessary for genetic counselling in the carriers, and for predicting prognosis and management options in the patients with thalassemia. dna-based diagnosis of b-thalassemias routinely relies on polymerase chain reaction (pcr) and gel electrophoresis. the aim of this study is to develop a new procedure, a dna-based piezoelectric biosensor, for the detection of b-thalassemia ivsi-110 mutation, the most common b-thalassemia mutation in turkey. materials and methods: b-globin gene of genomic dna isolated from whole blood, was amplified by pcr. bioactive layer was constituted by binding 2-hidroxymetacrilate metacriloamidoscystein (hema-mac) nanoploymers on the gold electrode's surface. single oligonucleotide probes specific for ivsi-110 mutation of b-thalassemia were attached to the nanopolymer via reactive cross-linker glutaraldehyde. the measurements were executed by piezoelectric resonance frequency which is caused by binding of pcr products in media with single oligonucleotide probe on the electrode surface. the results were confirmed by the conventional molecular method as arms. results: the piezoelectric resonance frequencies obtained by hybridization of the pcr products on bioactive layer were found 216 ae 12, 273 ae 6, and 321 ae 9 hz for the samples of normal b-globin, heterozygote, and homozygote of ivsi-110 mutation, respectively. discuss and conclusion: the developed biosensor serves as a specific result to ivsi-110 mutation. it could accurately discriminate between normal and ivsi-110 mutation samples. because of low costs, fast results, specificity and high detection/information effectiveness as compared with conventional methods, we can be offered this techique as an alternative to conventional molecular methods. the increasing use of nano-sized materials in the last several years has compelled the scientific community to investigate the potential hazards of these unique and useful materials. one of the most widely used nanoparticles is titanium dioxide. the objective of the research is to investigate the alterations in molecular and cellular responses in culture of primary lymphocytes to tio2 nps. human lymphocytes isolated from heparinized blood of healthy individuals were exposed to tio2 nanoparticles. viability, ros generation, the changes in the expression of genes encoding proinflammatory mediators tnf-a, il-1b and il-8 and dna damage were assessed. human lymphocytes were incubated with nanoparticles of different concentrations and viability was determined in 24 and 48 hours after treatment, respectively. cell viability was decreased by a treatment with nanoparticles in both a time-and concentration-dependent manners. the ability of tio2 to induce ros formation in lymphocytes was evaluated using dcf fluorescence as a reporter of oxidant production. the fluorescence intensity of oxidized dcf was increased in cells treated with nps. this means that ros generation occurred in response to the treatment with tio2. to investigate the expression level of mrna related to the inflammation responses in human lymphocytes real-time pcr was performed. the expression of il-1b, il-8 and tnf-a genes were increased by the exposure to nanoparticles of 10, 20 and 40 lg/ml for 24-48 hours. tio2 nanoparticles were shown to induce the dose-dependent fragmentation of dna strands. much evidence of hazardous health effects of nps has been reported. in this study, viability was reduced under the exposure to tio2. oxidative stress was elevated by the treatment with tio2 nps. oxidative stress can also trigger inflammation signals. induced by exposure to nanoparticles they may cause the translocation to the nucleus of transcription factors, which regulate proinflammatory genes, such as tnf-a, il-1b, il-8. background: endothelial cells (ec) represent one of the primary targets of the major pro-inflammatory cytokinetumor necrosis factor (tnf). development of the new approaches for the treatment of acute and chronic inflammatory conditions, including the strategies aimed to tnf neutralization, requires the usage of the adequate cellular models closely resembling the properties of the endothelium. the endothelium-derived ea.hy926 cell line expresses several inflammation and neoangiogenesis markers in response to activation factors however their expression can differ from the patterns demonstrated by primary ec. the aim of the current study was to compare the expression of the known endothelial cellular markers including receptor of vascular endothelial growth factor-2 (vegfr2) and a v b 3 -integrin on 2d and 3d cultures (spheroids) of ea.hy926. methods: the ea.hy926 cell line was used with permission from dr. edgell. the cells were cultivated in the presence of tnf (25 ng/ml) or vegf a (25 ng/ml) for 5 hours. mrna was isolated using rneasy kit from qiagen and reverse-transcribed with revertaid kit (fermentas). rt-pcr was performed with specific primers. expression of vegfr2 and a v b 3 -integrin was visualized by confocal microscopy using specific monoclonal antibodies and previously developed fluorescent hybrid proteins. results: the expression of a v b 3 -integrin and vegfr-2 increased on the 3d culture compared to 2d according to confocal microscopy and rt-pcr. the aforecited methods revealed elevated expression of a v b 3 -integrin in the 3d culture of the ea.hy926 cell line activated with tnf. also increased expression of vegfr2 in the 3d culture activated with vegf a. then by confocal microscopy, we analyzed our fluorescent hybrid proteins that bind a v b 3 -integrin and vegfr2 on the surface of 2d and 3d cultures as well as antibodies with fluorescent label. conclusions: 3d cultures of the ea.hy926 cell line represent a promising model for the inflammation studies. tumor necrosis factor (tnf) is a trimeric cytokine associated with the inflammatory response to tissue injury and found to possess a key role in rheumatoid arthritis pathogenesis. spd 304 is a highly toxic recently discovered tnf inhibitor that promotes trimer dissociation and lead to the inactivation of the protein. according to the traditional anti-tnf treatment of ra, we aim at extracellular inhibition of this pro inflammatory cytokine as an effective therapy. the project plan comprises design, synthesis and validation of candidate inhibitors (measurement of dissociation constant and aqueous solubility). because of the elevated percentage of insoluble compounds a solubility enhancement protocol has been developed. the experimental procedure was the following:: a. drug design. identification of novel drug compounds are based on two approaches: i) structure based drug design using the 3d structure of tnf and ii) design of more potent and less toxic spd304 analogues. b. drug synthesis. a series of spd304 analogues were in house synthesized while novel candidates discovered by in silico approaches were commercially available. the purity of the majority of the compounds exceeded 90%. c. solubility measurement and enhancement. samples were incubated under specific conditions that can enhance aqueous solubility and solubility measurement with a direct uv method pursued. d. measurement of the dissociation constant. a fluorescence binding assay was used in order to evaluate the inhibitory activity of the compounds. from our results it can be concluded that dmso, peg3350 and b-cyclodextrin can be used for solubility enhancement without interfering with fluorescence assay. however peg3350 -in contrast to dmso-is not suitable for isothermic titration calorimetry measurements. dissolution procedure also plays a crucial role in the levels of solubility reached. finally, it has been shown that some of the studied spd304 derivatives have better dissociation constants than spd304. the effect of exercises on serum bmp-6 levels of knee osteoarthritis cytokines. more complicated approaches are expected to focus on molecular proteins as bone morphogenetic proteins (bmps) of the transforming growth factor (tgf)-beta superfamily. bmps associated with many cellular functions, such as proliferation, differentiation, and apoptosis. bmp-6 is significantly important for the endochondral bone formation. inflamation can induced serum bmp levels in oa patients. the aim of this study is to evaluate the clinical findings of oa patients after the isokinetic exercise together with the serum levels of bmp-6 to sustain the molecular approaches for treatments. a total of 36 patients were included in this study. the groups are formed as follows: group 1, oa patients before the exercise; group 2, oa patients after the exercise; group 3, oa patients before the isokinetic exercise; group 4, oa patients after the isokinetic exercise clinical and biochemical findings were evaluated before and after 3 weeks of the exercise programme. self reported severity of pain was measured using the 100 mm visual analog scale (vas), womac scores were calculated and isokinetic knee muscle strength testing was measured using cybex dynamometer that a standardized protocol previously described was applied in a subject-specific range of motion. serum bmp-6 levels of all patients were studied by elisa method. results represented a better vas and womac scores for all exercise groups after treatment. the serum bmp-6 levels were significantly decreased in group 2 compared to group 1 (222.94 ae 44.66; 246.49 ae 38 .15 respectively, p < 0.01) and in group 4 compared to group 3 (246.74 ae 32.26; 256.23 ae 33.11 respectively, p < 0.05). there is not any statistically differences between group 2 and group 4 (p > 0.05). as a conclusion, the decreased serum levels of bmp-6 may be suggested as a biochemical marker for oa patients during exercise programmes. p-04.04.4-006 tnf-a blokade efficiently reduced severe intestinal damage in necrotizing enterocolitis c. tayman, s. aydemir, i. yakut, u. serkant, a. c ß iftc ßi g€ olbasi devlet hospital, ankara, turkey objectives: to ascertain the beneficial effects of infliximab an inhibitor of tumor necrosis factor alpha (tnf-a) on the development of nec in an experimental nec rat model. material and methods: thirty newborn sprague-dawley rats were randomly divided into three groups as nec, nec+ infliximab, and control. nec was induced by enteral formula feeding, exposure to hypoxia-hyperoxia and cold stress. pups in the nec+ infliximab group were administered infliximab at a dose of 10 mg/kg daily by intraperitoneal route from the first day until the end of the study. all pups were sacrificed on the 5th day. proximal colon and ileum were excised for histopathologic, immunohistochemical (tunel and caspase-3), and biochemical evaluation, including, total antioxidant status (tas), total oxidant status (tos), malonaldehyde (mda), and myeloperoxdase (mpo) and tnf-a activities. results: we observed better clinical sickness scores, weight gain, and survival rate in the nec+ infliximab group compared to the nec group (p < .05). histopathological and apoptosis examination (tunel and immunohistochemical evaluation for caspase-3) revealed lower damage in the nec+ infliximab group compared to the damage in the nec group (p < .01). tissue mda, mpo, tnf-a levels, and tos were significantly decreased in the nec+infliximab group, whereas tas was significantly increased in the nec + infliximab group (p < .01). conclusion: tnf-a blockade with infliximab efficiently reduced the intestinal injury and preserve the intestinal tissues from severe intestinal damage by its complex mechanisms on nec. therefore, it may be an alternative option for the treatment of nec.keywords: tnf-a; infliximab; necrotizing enterocolitis; newborn; protection; rat; treatment p-04.04.4-007 short-term diabetes causes cardiovascular inflammation: anti-inflammatory effect of resveratrol introduction: diabetes is a metabolic dysfunction and has been associated with various disorders including inflammation, cardiomyopathy and coronary artery disease. inflammation is a protective mechanism elicited by the host in response to infection, injury, and tissue damage. the aim of this study was to investigate the effect of intraperitoneally resveratrol administration on cardiac and vascular function in diabetic rats. materials and methods: diabetes was induced in sprague-dawley rats by using injection of streptozotocin (55 mg/kg, i.p.). rats were divided into group i: control, ii: control/20 mg/kg resveratrol; iii: diabetic/vehicle; and iv: diabetic/20 mg/kg resveratrol. histopathological examinations with masson's trichrome and verhoeff-van gieson staining were carried out to reveal cardiac and vascular tissue damage and inflammation. in addition to plasma glucose and cardiac & vascular mda levels were measured by standard enzymatic kits while tnf-a, il-1b, il-18 (mbl) were analyzed by elisa kit. results: final body weight decreased in all groups compared to control. in the diabetic rats, plasma glucose and vascular mda levels were enhanced while cardiac mda was unchanged compared to control. vascular tnf-a, il-1b and mbl and cardiac mbl were increased in the diabetic groups compared to control. discussion and conclusion: it has been found that resveratrol has greatly normalized altered parameters. taken together, resveratrol partly improved cardiac and vascular inflammation induced by diabetes. this may be due to the healing activity of resveratrol on pro-inflammatory markers. p-04.04.4-008 cytokine network is critical in growth hormone-induced resistance mechanism against curcumin which modulates jak/stat/ socs pathway in mda-mb-231 and mcf-7 breast cancer cells m. c ß elik, a. c ß oker g€ urkan, e. damla arisan, p. obakan yerlikaya, n. palavan unsal t.c. istanbul k€ ult€ ur university, istanbul, turkey curcumin (diferuloylmethane), a polyphenolic compound that triggers apoptotic cell death in various cancer cells such as prostate, colon, melanoma and breast cancer. a pituitary-derived hormone, growth hormone (gh) play role in elongation and differentiation of ductal epithelia into the breast terminal and buds. in this study, our aim is to determine the role of inflammation in curcumin induced apoptotic cell death via acting on jak/stat/socs pathway in wt and gh+ mda-mb-231 and mcf-7 breast cancer cell lines. according to mtt cell viability assay curcumin triggers cell viability loss in time and dose dependent manner in mda-mb-231 wt and mda-mb-231 gh+ breast cancer cell lines, respectively. selected concentrations of curcumin as 20 lm (for mcf-7) and 25 lm (for mda-mb-231) decreased cell proliferation and induced apoptosis through causing jak2 dephoshorylation, stat1, 3, 5 dimerization and acting on socs proteins expression in each cell lines. in addition, activated jak/stat/socs pathway, via forced gh expression has been suppressed following curcumin treatment for 24 hours. 20 lm curcumin-induced apoptotic cell death via dephosphorylating jak2 at tyr1007/1008 residues and decreased phospho-stat1, 3 level in both breast cancer cell lines. although curcumin dephosphorylated stat5 in both mda-mb-231 and mcf-7 wt cells, no significant effect has been observed in mda-mb-213 gh+ and mcf-7 gh+ cell lines. in consequence, although forced gh expression induced cell proliferation in mcf-7 and mda-mb-231 breast cancer cells, curcumin overcame gh-mediated resistance mechanism via acting on jak/ stat/socs signaling, which is related to pparg-induced inflammation. acknowledgment breast cancer is one of the highest cancer type among women worldwide. various enviromental and genetic factors such as age, gender, family history, metabolic diseases and gene mutations are involved in the breast cancer pathogenesis. growth hormone (gh), a pituitary derived hormone, has essential role on postnatal growth and development. it is also established that signalling route of gh and its receptor (ghr) activity is increased in different cancer types. curcumin, a nutraceutical deriatives from rhizomes of turmeric (curcuma longa), has potential therapeutic activity against cancer cells, including breast cancer. curcumin inhibits proliferation of cancer cells such as prostate, colon, melanoma, cervical and breast cancer via induction of apoptosis and inflammation. stat5, a major downstream target of gh/ghr signalling, is related to survival, proliferation and differentiation. in this study, our aim was to investigate curcumin-induced apoptotic cell death in gh overexpressed mda-mb-453 breast cancer cells via jak-stat/socs signalling and inflammatory response profile. according to mtt cell viability assay, curcumin decreased cell viability in time and dose dependent manner in wt and gh+ mda-mb-453 breast cancer cell lines. we found that 20 lm curcumin-decreased in apoptotic cell death through inactivity at jak2 which led to dimerization of stat1, stat3, stat5. concomitantly, curcumin affected stat regulating socs proteins in mda-mb-453 breast cancer cell line. in addition, we demonstrated that 20 lm curcumin induced pparg expression and altered inflammatory cytokine signalling cascade. consequently, although gh overexpression led to agressive profile in mda-mb-453 breast cancer cells, curcumin overcame this resistance. inflammation is involved in many systemic disturbances, including osteoarthicular or skin diseases, coordinating the signaling network that contributes to tissue injuries. the aim of our study is to reveal pro-inflammatory messengers at the cutaneous barrier (keratinocytes, fibroblasts, endothelial cells), simulating the dermal impact of active principles, especially polyphenols and flavones from vegetal sources: salvia officinalis, asculum hippocastanum and calendula officinalis. we focused on il6 and il8 cytokines as main mediators of inflammation progression, correlated in keratinocytes with il1a as skin irritation indicator and vegf as pro-angiogenic factor, as well as in endothelial cells with icam-1 and vcam-1 adhesion molecules expression. in order to in vitro mimic the inflammatory conditions, we used targeted stimuli for each type of cells: for fibroblasts and endothelial cells -tnfa, a systemic stimulus, single or combined with pma that activates protein kinase c and up regulates nadph oxidase, which lead to superoxide anion production; for keratinocytescontrolled uv-a and uv-b radiation, simulating the solar damages or potential uv interactions with active principles in light exposed skin. the main analysis technique was flow cytometry: beads bases assay for soluble factors and fluorescent antibodies staining. our results prove the different involvement of polyphenols and flavones in the anti-inflammatory mechanisms, depending of the vegetal source: active principles from salvia officinalis induce a strong inhibition of il6 and il8 in tnfa stimulated keratinocytes, fibroblasts and endothelial cells, reduce the icam-1 over-expression but have no effects on irradiated keratinocytes; biocomplexes from asculum hippocastanum inhibit only il8 release in stimulated fibroblasts, but protect keratinocytes from uv-a and uv-b radiation; compounds from calendula officinalis are active on il8 signaling in fibroblasts and counteracts only uv-b inflammation. ischemia and/or reperfusion injury is one of the most common causes of acute renal failure. ischemia-reperfusion associated with thrombolytic therapy, organ transplantation, coronary angioplasty, aortic cross-clamping, or cardiopulmonary bypass results in local and systemic inflammation. within the endothelium, ischemia produces expression of proinflammatory gene products (e.g. cytokines) and bioactive agents (e.g., endothelin), while preventing other "protective" gene products (e.g., thrombomodulin) and bioactive agents (e.g. nitric oxide). therefore, ischemia induces a proinflammatory state that increases tissue vulnerability to further injury on reperfusion. this experimental study was designed to investigate the protective effect of salvia l. extracts on kidneys from i/r injury. salvia lamiaceae have been used for treatment of some illnesses in turkish folk medicine. forty spraque dawley rats were divided into 5 groups (n = 8). right nephrectomy was performed to all groups. group i: control group; group ii: i/r group; group iii: i/r + 50 mg/kg salvia l.group; group iv: i/r + 100 mg/kg salvia l. group; group v: i/r + 50 mg/kg rosmarinic acid. group. salvia l. and rosmarinic acid for 7 days was given single dose as a gavage. 60 minutes ischemia, 60 minutes reperfusion were applied to groups except control. intracardiac blood samples were taken, high sensitive crp (hscrp), tumor necrosis factor-a (tnf-a), interleukin (il)-6 and interleukin ib (il-1b) levels were detected. serum hscrp levels were also determined in our clinical laboratory using routine standard methods. serum tnf-a, il-6 and il-ib levels were evaluated using an enzyme-linked immunosorbent assay technique. mean values were evaluated by statistical analysis. serum hscrp, tnf-a, il-6, and il-1b concentrations were significantly increased after renal i/r as compared to the control group. our treatment group 50 mg/kg salvia l. and 50 mg/kg rosmarinic acid especially 100 mg/kg of salvia l. were found to show a protective effect against renal structure and function. we concluded that salvia l. extracts could be beneficial in the treatment of renal ischemic injury. but 100 mg/kg salvia l. extract were more effective than 50 mg/kg salvia l. extract and used as synthetic 50 mg/kg rosmarinic acid. acne vulgaris is a common chronic inflammatory skin disease of unknown etiology. excess levels of secretory phospholipase a2 (spla2) contributes to inflammatory diseases and studies indicate that lipoprotein lipase (lpl) has differential effects on several inflammatory pathways. the aim of the present study was to assess serum activity of spla2, lpl and evaluate changes in circulating protein levels of angiopoietin-like protein 3 (angptl3), angptl4, cyclooxygenase (cox) and prostaglandin e2 (pge2). serum from 21 control subjects and 31 acne vulgaris patients with moderate and severe disease was evaluated for levels of spla2, cox, pge2, lpl, angptl3 and angptl4. disease activity was determined according to the national health service (nhs) lambeth and southwark clinical commissioning group guidelines for the management of acne. lipid profile, routine biochemical and hormone parameters were assayed by standard kit methods using autoanalyzers (beckman coulter au5800 clinical chemistry and unicel dxi 800 immunoassay systems). serum levels of spla2 and lpl were significantly increased in acne vulgaris patients compared to age and gender matched controls. no significant differences were found for cox, pge2, angptl3 and angptl4 levels between acne vulgaris patients and controls. the results of this study reveal the presence of a proinflammatory state in acne vulgaris as shown by significantly increased serum spla2 activity. increased lpl activity in serum of acne vulgaris can be protective in patients through its anti-dyslipidemic actions. to our best knowledge, this is the first study investigating spla2, lpl, angptl3 and angptl4 levels in acne vulgaris. future studies are aimed to understand the regulation of spla2 and lpl expression in acne vulgaris patients. acknowledgement: this study was supported by a grant from the scientific and technological research council of turkey (tubitak; #115s940). p-04.04.4-013 8-ohdg and hogg1 levels are as an oxidative dna damage markers in acne vulgaris treated with isotretinoin h. ecevit, m. izmirli, b. gogebakan, e. rifaioglu, d. sonmez, b. bulbul sen, t. sen, h. m. okuyan mustafa kemal university, hatay, turkey acne vulgaris is a skin disease that characterized by comedones, papules, pustules, nodules and cysts at face, back and body skin. isotretinoin is one of the treatment agents in acne vulgaris. about 4 weeks after drug treatment, the amount of sebum which is produced by sebaceous gland reduces keratinization disorder and the number of propionibacterium acnes normalizes. however, isotretinoin is known that has a wide range of side effects. in recent studies, isotretinoin treatment has been shown to increase the oxidative stress. 8-hydroxy-2 0 -deoxyguanosine (8-ohdg), an important indicator of oxidative dna damage, hydroxyl ion is bound at the 8th carbon of guanine. this structure is repaired through a base excision repair mechanism and the human 8-oxoguanine dna glycosylase 1 (hogg1) plays a key role in this processes. in this study we aimed to evaluate the dna damage and it's repair in acne vulgaris before and after 6 months of isotretinoin treatment by measuring 8-ohdg and hogg1 levels. the current study includes 43 acne vulgaris patients who are diagnosed in mustafa kemal university, department of dermatology. 8-ohdg and hogg1 levels were measured by enzymelinked immunosorbent assay (elisa) method for before and after 6 months of isotretinoin treatment. the commercial elisa kits (cloud-clone corp; usa and cell biolabs; usa) were used for the assessment of hogg1 and 8-ohdg, respectively.both 8-ohdg (p as a conclusion, isotretinoin increases dna damage and high serum 8-ohdg and hogg1 levels as a result of isotretinoin treatment may effect on the amount of reactive oxygen species. the pineal gland is a circumventricular organ which serves as a major neuroendocrine gland in the brain. its primary function is the production of melatonin which is controlled by signals from the suprachiasmatic nucleus. melatonin codes the length of the night and it is well recognized for its anti-inflammatory effects. lipopolysaccharide (lps) is the essential component in the outer surface membrane of gram-negative bacteria and act as a strong stimulator of natural and innate immunity in all eukaryotic species. furthermore, lps reduces melatonin synthesis and induces the expression of the serine protease inhibitor 3 (spi-3) in the stat3-mediated manner in pinealocytes. however, the precise function of stat3 in the cell signaling in the pineal gland is not yet known. here we investigated the effect of inhibition of stat3 on lps-induced changes in melatonin levels, expression of arylalkylamine n-acetyltransferase (aa-nat) and spi-3 in the pineal gland. experiments were performed in vitro using organotypic and primary cultures prepared from the rat pineal glands. levels of melatonin and spi-3 were determined from tissue homogenate by enzyme-linked immunosorbent assay (elisa). the pinealocytes were used to carry out sirna stat3 transfection. the successful transfection and subsequent decline in stat3 expression levels were proved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page). the changes in synthesis of aa-nat and spi-3 were studied by rt-pcr. in conclusion, lipopolysaccharide can affect the immunomodulators secreted by the pineal gland. the clarification of the effect of inhibition of stat3 on those immunomodulators is important from the clinical point of view because inhibitors of stat3 are nowadays used as tumour suppressors. silica nanoparticles have a great potential for a variety of industrial, diagnostic and therapeutic applications. in this study, we have evaluated the in vitro effects of amorphous silica nanoparticles (7 nm) using human lung mrc-5 fibroblast as model. cells were exposed to 62.5 lg/ml silica nanoparticles for 24, 48 and 72 hours. the cytotoxic and inflammatory response, and matrix metalloproteinase expression were examined. the pro-inflammatory cytokine il-1b, il-6, il-8, tumor necrosis factor (tnf-a), matrix metalloproteinases (mmp-2, mmp-9, mmp-1) and tissue inhibitor of metalloproteinase-1 (timp-1) were analyzed by western blot method. cytotoxicity was evaluated by lactate dehydrogenase (ldh) released into the culture medium by damaged cells. the level of ldh activity was increased after exposure to silica nanoparticles, in a time-dependent manner compared to control. the protein expression of il-1, il-6, il-8 and tnf-a as well as of mmp-1 and timp-1, was up-regulated whereas those of mmp-2, mmp-9 was down-regulated after 48 and 72 hours respectively. in conclusion, our data indicate that amorphous silica nanoparticles generate a cytotoxic and inflammatory response, as well as an imbalance in extracellular matrix due to the differential regulation of mmps and tissue inhibitor of metalloproteinase-1 in mrc-cells after 48 and 72 hours. p-04.04.4-017 association of fto gene variant (rs8050136) with markers of t2dm and obesity in population from bosnia and herzegovina and kosovo fto (fat mass and obesity-associated gene), recently discovered in a genome-wide association study for type 2 diabetes (t2d) encodes a 2-oxoglutarate-dependent nucleic acid demethylase and is mainly expressed in the hypothalamus. this gene may play important role in the management of energy homeostasis, nucleic acid demethylation, and regulation of body fat masse by lipolysis. the aim of this study was to analyze the association of this single nucleotide polymorphisms (snps) with clinical and biochemical parameters of obesity, t2d, prediabetes and at the level of healthy population from bosnia and herzegovina (bh). the study included 638 patients with t2d and prediabetes and 360 healthy controls both sexes, aged from 40 up to 65 years. patients were recruited at the clinical centre university of sarajevo, university hospital of clinical centre in banja luka, general hospital in te sanj and health centre in prizren. genotyping of analyzed polymorphism was performed by rt-pcr method in cooperation with the department of clinical chemistry, faculty of pharmacy, university of ljubljana (ljubljana, slovenia) and university hospital of charles university (hradec kralove, czech republic). our results did not show significant differences in genotype frequencies of the analyzed polymorphisms between patients with t2d, pre-diabetes and healthy population also, results of logistic regression analyses did not show significant association of risk a allele of fto gene polymorphism -rs8050136 with increased risk of t2d (or = 1.084, 95% ci 0.758-1.551, p = 0.659). a allele was significantly associated with higher values of hba1c, insulin, homa ir index, diastolic blood pressure and higher levels of inflammatory markers (fibrinogen and leukocytes). interestingly, a tendency of association of a allele with higher values of obesity markers (bmi, waist and hip circumference) was noted. further studies are needed on a larger population in order to confirm these results. the water extract of capparis ovata (cowe) has been shown to be used as an alternative medicine for the treatment of multiple sclerosis (ms). cowe was further fractionated and studied for additional anti-neuroinflammatory effects in sh-sy5y cells. for this purpose, the dichloromethane sub-fraction of the cowe extract was tested for its anti-inflammatory effects on selected anti-inflammatory genes believed to be important in ms pathophysiology using sh-sy5y cells. cell viability was assessed using lactate dehydrogenase (ldh) activity in the media conditioned by the crystal violet cell staining. in these cells, levels of the tumor necrosis factor-a (tnfa), nuclear factor kappa-lightchain-enhancer of activated b cells (nf-jb1), glial fibrillary acidic protein (gfap), c-x-c motif chemokine 9 and 10 (cxcl9, cxcl10), matrix metalloproteinase 9 (mmp9), chemokine (c-c) motif 5 (ccl5) and tyrosine-protein phosphatase non-receptor type 11 (ptpn11) were determined by quantitative reverse transcriptase-pcr assay (qrt-pcr). we have found out that the dichloromethane sub-fraction of cowe effectively inhibited the expression of all of the genes given above in sh-sy5y cells. thus, phytochemicals present in the dichloromethane sub-fraction of the cowe extract could be beneficial in preventing/treating neurodegenerative diseases in which neuroinflammation is part of the pathophysiology. studies are underway to identify the individual compound(s) in this subextract of the cowe extract contributing to these effects. this work is supported by tubitak 112s187 and pamukkale university paubap 2014fbe051. p-04.04.4-019 apigenin and luteoline were identified as active anti-inflammatory constitutents of lavandula stoeachas by bioassay guided fractionation h. ipek 1 , s. savranoglu 2 , a. r. t€ ufekc ßi 3 , f. g€ ul 3 , i. demirtas 3 , t. boyunegmez t€ umer 4 1 graduate program of bioengineering, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, 2 graduate program of biology, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, 3 department of chemistry, faculty of sciences, c ß ankiri karatekin university, c ß ankiri, 4 department of molecular biology and genetics, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey introduction: lavandula stoechas, in the genus of lavender, has distinct therapeutic uses among anatolian people. rather than worldwide use of its essential oil in aromatherapy, specifically the aqeous portion as decoction has been traditionally used in anatolia against the components of metabolic syndrome, all of which share a state of chronic inflammation as an underlying cause. the anti-inflammatory constiutents of l. stoechas were isolated using a bioassay guided fractionation in lipopolysaccharide (lps) inflammed raw 264.7 macrophages. materials and methods: an aqeous extract was partitioned into ethyl acetate (eae) and n-butanol fractions. the eae, determined as bioactive extract was seperated into 12 subfractions by column chromatography. e6 was identified as active subfraction subjected to sephadex column to get pure compounds which were then applied to nmr, ir, and uv analyses for structure determination. in raw 264.7 cells, the effects of extracts/fractions/subfractions/compounds on lps induced no production was determined by using griess method. the potential inhibitory effects of each compound on lps induced inos expression were determined by qpcr and western blot. results: p-coumaric acid, apigenin and luteoline were found in the e6, and the first two compounds appeared to be primarily responsible for the anti-inflammatory activity. apigenin and luteoline at 50 lm decreased no production 66 and 80%-ic 50: 56 and 26 lm-by inhibiting inos gene expression 84 and 88% as well as protein expression 94 and 99%, respectively (p < 0.05). conclusion: this is the first time that luteoline and apigenin have been found in eae of l. stoechas, and the anti-inflammatory properties of the eae can be attributed, at least in part, to the presence of these two compounds. we are on the way to gain further insight for the action mechanism of these two active principles as anti-inflammatory agent. tubitak (project id:112t442) support this work. the role of tip60 in the inflammation process immun response generates the first line of host defense during inflammation and plays an important role inducing pro-inflammatory response by generating early response against pathogens. il-6 (interleukin 6) is one of the pro-inflamatory cytokines and its expression increases during the infection to activate the jak/ stat pathway. jak/stat pathway is regulated by hamp (hepcidin antimicrobial peptide). our previous study, we reported that hamp gene expression was decreased in liver-specific tip60 conditional knockout mice, so we thought that tip60 may have a direct or indirect role on inflammation mechanism. tip60 (tat interacting protein, 60 kda) is a member of the myst enzyme family of histone acetyltransferases (hats) and plays an important role in multiple function including cellular signaling, dna repair, cell cycle and apoptosis. in this study, the quantitative gene and protein expression of il-6 were investigated by using taqman real time pcr, western blot and immunohistochemistry analysis in control group, lpsinduced inflammation group and liver-specific tip60 conditional knockout group mouse liver. according to our preliminary results, the gene and protein expression of il-6 was increased in lps-induced inflammation group (p < 0.001, p < 0.001) and liver-specific tip60 conditional knockout group mouse liver (p < 0.05, p < 0.01). our initial data suggest that tip60 may be essential for the inflammation process. this work was funded by grants from the scientific and technological research council of turkey (tubi-tak) (grant number: 114z277). although intracellular reactive oxygen species (ros) level is necessary to maintain cellular homeostasis, elevated intracellular ros level with the impact of unfavorable environmental conditions leads to oxidative stress that may cause damage to dna, proteins and lipids. in case of inflammation, organism seeks to provide cellular homeostatis by increasing ros levels via antioxidant molecules and enzymes. therefore, it was thought that there can be a direct or indirect relation between inflammation and oxidative stress. in this study, inflammation was performed by intraperitoneal injection of lipopolysaccharide (lps). the gene expression and activity of antioxidant enzyme including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glutathione s-transferase (gst), glutathione reductase (gr) and glucose 6-phosphate dehydrogenase (g6pd). additionally, any change of reduced glutathione (gsh), oxidized glutathione (gssg), malondialdehid (mda), and hydrogen peroxide (h 2 o 2 ) level are accepted as an indication for the accumulation of ros, the relative levels of them were also studied. to show our inflammation model was performed in mouse kidney with lps treatment or not, the expression of interleukin (il6), which is accepted as a inflammation marker, was investigated by real time pcr. the expression of il6 was significantly increased in lps treated group. while the level of mda and h 2 o 2 was elevated in lps treated group, gssg was decreased. no changes was seen for gsh level. the correlation was observed between enzymatic and molecular levels. while the gen expression and the enzyme activity of sod, cat, gst, gr, and g6pd were decreased, gpx was increased with inflammation. in conclusion, increasing ros level was observed in the inflammation process and, the antioxidant system was affected at the molecular and protein level. this work was funded by grants from the scientific and technological research council of turkey (tubitak) (grant number: 114z277). the aim of our study is to evaluate effect of vitamin d levels on hemogram parameters including neutrophil %, lymphocyte %, neutrophil % / lymphocyte % ratio (nlr) and mean platelet volume (mpv) in behcet's patients. fifty eight patients with diagnosis of behcet that applied to selcuk university faculty of medicine department of dermatology are recruited to the study. clinical and laboratory characteristics of the patients were obtained from hospital automation. t test was used to examine the differences between the parameters. p < 0.05 was taken to be statistically significant. there was a statistically significant difference between vitamin d values and age (p = 0.036) whereas difference was not significant between vitamin d and neutrophil %, lymphocyte %, nlr, mpv values. according to the literature, there are a lot of studies that show the relationship between vitamin d and hemogram parameters. however, contrary to the previous studies, we were unable to find any significant relationship between vitamin d and these hemogram parameters. these results serve the idea that the effects of vitamin d on the hematopoietic system should be further investigated experimentally and clinically. crimean-congo hemorrhagic fever is a tick-borne disease caused by the arbovirus and characterized by a sudden onset of high fever, severe headache, dizziness, back and abdominal pains. the exact pathogenesis of cchf has not been clarified yet. the aim of this study, clinical cases of cchf in cu, se and zn is to examine the relationship between the concentration of trace elements. the study sample consisted of 30 patients which have been diagnosed with cchf. matched for gender, 30 healthy volunteers were similar to the control group according to age. the patients and control groups, serum cu, zn and se levels were analyzed using atomic absorption spectrophotometer. cchf patients in the group, cu zn and se serum levels were significantly lower compared with the control group. in our study, the cofactor of the antioxidant enzyme cu, zn and se elements were lower. this shows us in cchf disease, a decrease in antioxidant enzyme activity, and suggest that they contribute to the immune system's degradation. p-04.04.4-024 inhibitors of mdm2 ubiquitin ligase as prospective modulators of autoimmunity e. bulatov, a. valiullina, r. sayarova, a. rizvanov kazan federal university, kazan, russia ubiquitin-proteasome system is seen as a pool of promising protein targets for therapeutic impact in many human diseases. mdm2 is an e3 ubiquitin ligase widely studied due to its wellknown role in cancerit negatively regulates p53 oncosuppressor that mediates apoptosis in tumour cells. inhibitors of p53/ mdm2 interaction have long been known as potential anticancer therapeutics. however, recent advances in the field suggest that both mdm2 and p53 might be playing a substantial role in autoimmune processes. we used a small molecule p53/mdm2 inhibitor nutlin-3a to test the effect of p53 activation on peripheral blood mononuclear cells (pbmcs) from both healthy volunteers and patients diagnosed with multiple sclerosis. in our study we employed a variety of molecular biology methods, such as immunoblotting, real-time pcr, mts cell proliferation assay, fluorescence flow cytometry and confocal microscopy. we demonstrated that disruption of p53/mdm2 interaction by nutlin-3a alters the p53 levels and also affects the lymphocyte subpopulations within pbmcs. our findings suggest that p53/mdm2 interaction inhibitors can potentially be used as prospective modulators of immune response in autoimmune diseases such as multiple sclerosis, systemic lupus erythematosus and other. the study was funded by rfbr research grant 16-34-60213 mol_a_dk. can ykl-40 be an inflammatory biomarker in vitamin d deficiency? object: the tumor necrosis factor (tnf) was found to be cytotoxic to tumor cells and to induce tumor regression in mice. except for one member, all receptors to the tnf superfamily bind tnf-related ligands and act mostly on inflammatory system. there are currently 19 tnf superfamily ligands. tnf superfamily ligands share several common features. tnf ligands are generally type ii transmembrane proteins whose extracellular domains are be divided by enzyme to create cytokines. the tnf superfamily currently consists of 29 receptors. tnf family receptors are type i or type iii transmembrane proteins that contain multiple extracellular domains. in this study, we investigated to presence, differences and effects of tnf superfamily and receptors genes in human and mice by using bioinformatics techniques. methods: the nucleotide and amino acid sequence of each protein in human and mice was determined using t-blast-n for homologous sequences. homologous sequences of human tnf family genes found an automated procedure by using psi-blast. the secondary structure of and three-dimensional of the protein were analyzed by psipred and ffas server. netcglyc 1.0 and netphos 2.0 program were used for post-translocation modifications. the web apoptosis database was also used for the lists of domains, proteins containing these domains and their associated homologs. results and conclusion: humans tnf ligands have 17 genes encoding proteins that contain a conserved carboxy-terminal domain. this family of proteins is highly conserved between humans and mice. humans contain 29 genes encoding tnffamily receptors. sequence data from the ncbi databases demonstrated the presence of 24 mouse tnf-family receptors with orthologs in humans and one additional receptor found only in mice. the differences and similarities in the tnfs genes in humans and mice will provide information for understanding the utility and limitations of the mouse models of disease and comparing of immunology outcomes. the development of left ventricular remodeling after acute myocardial infarction is a predictor of shock. the genetic influence on cardiac remodeling, and shock in the early period after acute myocardial infarction are unclear. the aim of the present study was to investigate the relationship between angiotensin converting enzyme (ace) gene polymorphism and modified shock index (msi) in the early period in patients with acute anterior myocardial infarction. overall 140 patients with a first acute ami were included in this study. dna was isolated from peripheral leukocytes. the id status was determined by pcr. based on the polymorphisms of the ace gene, they were classified into 2 groups: deletion/deletion (dd) genotype (group 1, n = 57), insertion/deletion (id), insertion/insertion (ii) genotypes (group 2, n = 83). blood pressure and pulse measurements were performed in all patients within 10 minutes admitted to coronary care unit. msi was defined as heart rate (hr) divided by mean arterial pressure (map). echocardiographic examinations were performed in accordance with the recommendations of the american echocardiography committee. one-way analysis of variance (anova) and chi-square analyses were used to compare differences among subjects with different genotypes. the study was approved by the local ethics committee, and each patient gave a written consent. there were no significant differences among clinical parameters of patients. msi was significantly higher in patients who have ace dd genotype than in patients who have ace id / ii genotypes (1.13 ae 0.52 and, 0.85 ae 0.37, p < 0.05). presentation time hypotension or developing hypotension during admission was reported to be an important predictor of intensive care unit admission besides other vital sign measurements. our results suggested that, ace gene i/d polymorphisms d allele may affect modified shock index in patients with a first acute anterior mi. glucocorticoids (gcs) are widely used in medicine, despite their side effects, e.g. osteoporosis. however, precise molecular mechanisms of gc action, especially on bone marrow (bm) cells, remain controversial. given the osteoprotective role of vitamin d 3 , the aim of our study was to examine prednisolone-induced changes in the rank (receptor activator of nuclear factor kappa-b)/rankl (rank ligand)/opg (osteoprotegerin) pathway of rat bm depending on the state of vitamin d endocrine system. female wistar rats received prednisolone (5 mg/kg b.w.) with and without 100 iu of d 3 (for 30 days). the levels of rank, rankl, opg, 1a-hydroxylase (cyp27b1) in bm were determined by western blotting. vitamin d 3 receptor (vdr) and rankl mrnas were measured by quantitative rt-pcr. 25ohd 3 content in the serum was assayed by elisa. rankand vdr-positive bm cells were quantified using flow cytometry and visualized by confocal microscopy. prednisolone induced a marked increase in rankl and rank levels, while opg level was shown to decrease. this reflects disturbances in cytokine-mediated regulation of bm progenitor cell function. data from flow cytometry indicated a significant growth in the number of rank-positive cells (hematopoietic osteoclast precursors) compared to control. these changes were accompanied by a decrease in the levels of vdr and cyp27b1, which is responsible for 1,25(oh) 2 d 3 synthesis, in bm and 25ohd 3 content in serum. co-localization of vdr and rank in mono-and multinuclear bm cells was observed, indicating a close relation between vitamin d 3 and rank/ rankl/opg pathway. vitamin d 3 co-administration prevented prednisolone-induced changes in bm cells through restoration of vitamin d 3 bioavailability and vdr signaling that resulted in a reduction of the osteoclast progenitor pool in bm. thus, prednisolone-induced imbalance in rank/rankl/ opg system components is associated with impairments of vitamin d endocrine system in bm and can be ameliorated by vitamin d 3 treatment. p-08.01.4-002 heat shock pathway in response to different stress factors the heat shock response is an emergency pathway of the cell, which mediates repair and protection from cellular stress and therefore guarantees the survival of the cell. this stress can range from heat or hypoxia to chemicals and heavy metals. it is highly conserved in all eukaryotic cells and plays an important role during atypical conditions. due to its high complexity, the pathway is not yet completely understood. most important, after activation of the pathway, is the refolding of proteins or, in case of severe misfolding, the depletion of proteins to maintain proteostasis. heat shock factor 1 encoded by the hsf1 gene is known as the main switch point in heat shock regulation. after activation it trimerizes and binds to heat shock elements in target gene promoters. one of these promoters is the hspa1a promoter (hsp72 promoter). the promoter was analyzed by dismantling it to its functional parts. especially three elements, the heat shock elements, were in the focus of this work. in first place parts of the promoter were multimerized and combined with different reporters, like luciferase, by cloning. also mutations in the natural promoter were designed by cloning. the focus now is on the heat shock elements, where hsf1 can bind as a trimer. the idea is that these different elements have various effects on different stressors like heat, chemicals (geldanamycine as hsp90 inhibitor, mg132 as proteasome inhibitor) or heavy metals (cadmium, arsenic, zinc) . this was tested on cells transiently transfected with those promoter variants. for promising variants stable cell lines were created. in these stable cell lines further experiments on mrna level can be conducted. in the last months experiments with the crispr/cas9 system were started. furthermore, experiments on transcriptional (qpcr) and translational (dual-luciferase assay) levels were done as well. in the end we hope to get a clear picture on the regulation of the hspa1a promoter by different stress factors. invasive cancer cells form membrane protrusions, invadopodia, that facilitate cell invasion and metastasis. key players invadopodia include the adaptor proteins tks4 and tks5, the actin regulators cortactin, wip and n-wasp, the kinase src and others. in spite that in the last two decades significant advances in our knowledge of the structure and development of invadopodia have been made, detailed mechanisms they are functioning is not yet available. we have identified a series of new tks4 binding partners including adaptor proteins itsn1, itsn2, crk and grb2, kinase src, amph1, bin1, plcg1 and also another member of the tks family -tks5. it may indicate the possible role of tks4 in transport and sorting of cell vesicles. current data are supported by interaction with the proteins of amph1 and bin1, as their main functions are membrane trafficking and remodeling. adaptor proteins crk, grb2 and itsns are important for the actin cytoskeleton rearrangements, endocytosis and signal transduction. moreover, we have identified and characterized new tks4 isoform -tks4-beta. we suggested that an active state of tks4 is regulated via intramolecular interactions between its proline-rich motifs and own sh3-domains. we have shown the interaction between itsns and other prominent component of invadopodia wip. data from immunofluorescent analysis revealed co-localization of itsn1 and wip at the sites of invadopodia formation and in clathrin-coated pits. we have also demonstrated that the key protein itsn1 and wip and n-wasp can form a complex in cells. together, these findings provide insights into the molecular mechanisms of invadopodia formation and identify itsns as scaffold proteins involved in this process. we have shown the interaction between itsns and other verprolin family members cr16 and wire which play an important role in the reorganization of the actin cytoskeleton. we have demonstrated that cr16 and wire interact with sh3domains of itsns in complex with actin. p-08.01.4-004 correlation between proteomic and phenazine profile of pseudomonas sp. phenazines are widely known compounds with huge variativity of biological activities which are produced by pseudomonas sp. and some other bacteria species. the results of our work shows the correlation between the changes of proteomic profile of pseudomonas aeruginosa caused by a mutagenesis and the secondary metabolism of antibiotics (phenazine) profile. different strains of pseudomonas aeruginosa were obtained using mutagenesis, after that bacterial cells were destroyed by ultrasound. protein-containing fractions were isolated using methanol-chloroform method as well as phenazines compounds were extracted from culture media using liquid phase extraction. obtained proteome was analysed by shotgun-proteomics technique. as the result of the liquid phase extraction phenazine compounds were mainly extracted to the organic phase. this phase was evaporated and re-dissolved in 100% methanol. after sample preparation obtained solutions were analyzed by hplc-agilent 1290 with quadrupole tof mass-detector. results of the analysis were compared with the library of known phenazine compounds mass-spectras generated by cfm-id online resource. obtained phenazine profiles were compared with each other and correlation with the changes in proteome was analyzed. received results promote better understanding of mechanisms of phenazine production. this data opens possibilities for targeted changes in the methabolic pathway in order to obtain phenazine compound with required biological activity. insulators are genomic elements which block enhancer-promoter interaction and prevent spreading of heterochromatin. cp190 protein is an integral component of most known drosophila insulators, it interacts directly with ctcf and pita dna-binding insulator proteins using dimeric btb-domain, but function of cp190 within insulators still remains to be elucidated. recently we described an interaction between cp190 btb-domain and cterminal domain of ctcf insulator dna-binding protein, subsequent deletion analysis allowed us to isolate 50aa fragment within ctcf c-terminal domain sufficient for interaction with cp190 btb, but deletions of flanking regions also lead to the loss of interaction with cp190 in vivo. at the same time crosslinking experiments suggest that a dimer of btb interacts with one molecule of ctcf, presuming that it could recognize two peptide fragments within ctcf c-terminal domain. we solved crystal structure of btb-domain from cp190 insulator protein at 1. 3 a resolution. overall structure is similar to other btb-domains. cp190 btb-domain has peptide-binding groove similar to that previously found in bcl6 btb domain. inspection of btb-domain surface revealed several possible binding sites for polypeptide fragments from ctcf protein. based on these observations a set of point mutations within peptide-binding groove of btb-domain has been designed and we tested ctcf-interaction abilities of these mutants using gst pull-down assay and yeast two-hybrid assay. the most significant impact was found with alanine-substitutions of hydrophobic residues whereas substitutions of hydrophilic amino acids were less effective. therefore our results support that cp190 btb-domain recognizes ctcf protein using peptide-binding groove. this study was supported by the russian science foundation (project №14-24-00166). p-08.01.4-006 comparative study of the fatty acid composition of lipids in the raw meat samples obtained from hybrid sheep one of the most important tasks in the animal biology and husbandry is to clarify the role of animal genetic diversity in providing nutrients to the diversity of animal products. the objective of our work was to study the chemical compositions of raw meat samples obtained from domestic (group i -purebred romanov sheep) and hybrid sheep (group ii -f 3 hybrids of romanov sheep with 12.5% of argali blood). the significant changes in fatty acid composition of the lipid fraction from the fat and muscle tissue of the hybrid sheep as compared to the control were found. the content of saturated fatty acids (sfas) in the fat samples of the hybrid animals was by 12.16% lower (55.12 ae 2.30%, p < 0.01), but polyunsaturated (pufas) or monounsaturated fatty acids (mufas) contents were by 0.64% and 9.43% higher (10.10 ae 0.43 and 28.78 ae 1.77 (p < 0.01), respectively) as compared to purebred romanov sheep. the most pronounced changes were found for palmitic acid (decreased from 27.70% to 14.24%) and for oleic, linoleic, arachidonic acids (increased from 20.37%, 5.58%, 0.35% to 29.98%, 6.10%, 0.70%, respectively). the last two acids together with the linolenic acids belong to the so-called essential acids and very important for the animal metabolism. a similar trend was observed on the composition of the lipid fraction of muscle tissue. sfas, pufas and mufas content in muscle tissue of hybrid sheep was 53.38 ae 0.34, 9.39 ae 1.00 and 34.16 ae 0.39%, that was 11.17% lower (p < 0.001), and 4.13% and 4.67% higher (p < 0.01) compared to purebred romanov sheep. these results emphasized the difference of the pufas/sfas ratios in fat and muscle tissues, respectively) and characterized the biological value of the lipid fraction of fat and muscle tissue. the obtained data gave evidence of the positive changes in the fatty acid compositions of the lipid fractions for the hybrid animals as compared to the purebred sheep. supported by the russian scientific foundation, no. 14-36-00039. foodborne illnesses resulting from the consumption of agricultural commodities contaminated with enteric pathogens are an increasing problem around the world. while various possibilities of produce contamination with pathogens exist, the global warming combined with a widespread use of animal manure in agriculture will likely contribute to an increased number of such outbreaks. thus, phages isolated from different agroecosystems may prove to be useful in detection/biocontrol of enterobacteria in produce. during the investigation of the impact of global warming on the diversity and co-evolutionary dynamics between microorganisms and viruses in lithuanian agroecosystems, a novel enterobacteria phage vb_ecos_nbd2 (nbd2) was isolated from agricultural soil using e. coli novablue for phage propagation. nbd2 genomic dna was isolated from cscl-purified phage particles, and was subjected to illumina dna sequencing. nbd2 is a virulent siphovirus that has a low-temperature plating profile (fails to form plaques at a temperature >30°c). the genome of nbd2 is $ 52 kb long, and has a total of 87 probable protein-encoding genes as well as 1 gene for trna ser . the genome analysis revealed that 20 nbd2 orfs encode unique proteins that have no reliable identity to database entries. among the orfs that encode proteins with matches to those in other sequenced genomes, 64 are similar to proteins from phages that infect different members of enterobacteriaceae, while 3 nbd2 orfs are most similar to those from bacteria. based on the similarity to biologically defined proteins, 32 nbd2 orfs were given a putative functional annotation, including 15 genes coding for morphogenesis-related proteins, as well as 14 associated with dna replication, recombination, and repair. phylogenetic analysis revealed that enterobacteria phage nbd2 is distantly related to phages belonging to the subfamily tunavirinae. this research was funded by a grant (no. sit-7/ 2015) from the research council of lithuania. p-08.01.4-009 the antibiotic novobiocin affects the composition of the escherichia coli proteome n. e. arenas 1 , j. williamson 2 , v. schw€ ammle 2 , s. douthwaite 2 1 universidad de cundinamarca, cundinamarca, colombia, 2 university of southern denmark, odense, denmark novobiocin (nov) is an aminocoumarin which competitively inhibits the atp binding site in the gyrase-b subunit of prokaryotic topoisomerase ii. nov remains a therapeutic choice for treating infections with bacterial pathogens that are resistant to more commonly used drugs. the aim of this study is assess the proteomic response of e. coli strain upon nov treatment. minimum inhibitory concentrations of nov were measured by standard assays. three different e. coli strains (as19, as19-rlma::aph and b) were grown aerobically in nutrient rich lb media at 37°c during one hour. the whole cell proteome (five biological replicates in each sample,) was assessed by lc-ms by using tmt labelling protocol. raw files were imported to proteome discoverer (thermo fisher scientific) and searched together with mascot against the uniprot e. coli reference proteome. mics for nov were determined to be >1000-fold higher the wild-type b-strain of e. coli than for the hypersusceptible as19 strains (1 lg/ml). whole genome comparison of the b and as19 strains were characterized by an increase in proteasome components (6 proteins), chaperones (3), error-prone dna polymerase components (4), ribosomal hibernation factors (3), heat shock response (2), electron transport coupled proton transport (8), pentose phosphate pathway (9), flagellar assembly (4), oxidative phosphorylation (10) and tca cycle (8). whereas ribosomal proteins (45), aminoacyl-trna synthetases (7), rnases (6), abc transporters (17), mismatch repair (3) and sec secretion pathway (4) were significantly down-regulated upon nov treatment. the three e. coli strains respond similarly upon nov treatment and their proteomes showed upregulation of heat shock response with changes in the components of translation and transcription, the proteasome and atp biosynthesis. the changes observed can be used to define the processes that are required for antibiotic tolerance and survival of e. coli against aminocoumarin antibiotics. postnatal growth is under control of pituitary derived hormone, growth hormone (gh) that triggers bone, fat tissue growth and development via acting on protein, carbohydrate and fat metabolism. gh functions on postnatal development by jak2/stat5 signaling following gh:gh receptor (ghr) dimerization. isolated growth hormone deficiency (ighd) is a medical condition of insufficient production of growth hormone (gh) that is caused by mutations on gh-n gene in different ethnic origin children. various mutations within gh has been determined in different populations so far, and glutamic acid to glycine (e33g), asparagine to aspartic acid (n47d), threonine to alanin (t-24a) missense mutations, alanine to serine (a13s) substitution, tryptophan to stop codon (w-7x), gaaa insertion in intron 1 of gh-n gene and both intron 1 (+83c) and deletion of 166. amino acid of gh protein phenylalanine (f166del) mutations were detected in turkish ighd children. the potential role of these mutations on cell growth, proliferation, emt via acting on gh signaling pathway has not been observed yet. all these mutations were performed on wild type gh-n gene inserted pc3.1 vector by site-direct mutagenesis and stable cell line of each gh gene mutations were generated by neomycin selection. although w-7x, e33g, f166del, a13s and n47d mutations suppresses gh signaling via acting on either jak2 dephosphorylation or stat5 downregulation, t-24a, gaaa insertion and deletion of +83c mutations have no significant effect on gh signaling. in addition, each mutation lead different growth suppression effect and colony formation potential and intracellular polyamine levels and odc expression profiles were essential role in emt potential of hek293 cell lines. as a result, w-7x, e33g, f166del, a13s and n47d mutations prevented gh signaling and cell growth and differentiation via polyamine metabolism. pulmonary embolism (pe) is a common cardiovascular emergency and affects a large number of patients. acute pe-induced oxidative stress can lead to the accumulation of specific nitroproteins that may play a role in disease progression. the impact of nitration of a single tyrosine residue often has broad implications on the activity of biologically critical proteins, which has become increasingly related to pathological conditions. in this study, we used a proteomic approach to analyze nitrated serum proteins in patients diagnosed with acute pe and healthy controls. nitrotyrosine (no2tyr)-containing proteins were immunoprecipitated from serum with a no2tyr affinity sorbent. precipitated proteins were separated by sds-page and visualized by coomassie blue staining and western blotting with mouse monoclonal anti-no2tyr antibody. among the numerous immunoreactive bands observed in disease patients, the 138 kda protein band was in-gel digested and analyzed by maldi-tof mass spectrometry (ms). mass fingerprint data sets obtained from the peptide fragment ions matched human collagen alpha-1 (iii) chain (co3a1_hu-man) with mascot algorithm analysis giving a score of 65 (p < 0.05). collagen alpha-1(iii) chain is a fibrillar collagen that is found in extensible connective tissues such as skin, lung, and the vascular system. altered metabolism of collagen and its excessive deposition in the matrix of the connective tissue is a hallmark of chronic interstitial lung diseases. collagen can be measured in serum and bronchoalveolar lavage fluid from patients with numerous chronic interstitial lung diseases. given these considerations, future studies are aimed understand the relevance of no2tyr modifications in co3a1 relating to changes in protein structure and function. recent studies have shown that the genes involved in dislipidemia represent potential loci to be associated with diabetes as a disease. recent genome wide association (gwa) studies have associated rs1260326 in gckr gene and rs4846914 in galnt2 gene with parameters of t2d and diabetic dyslipidemia. in this study, the association of these single nucleotide polymorphisms (snps) with t2d and dyslipidemia was tested in the population from bosnia and herzegovina (bh). our study involved 352 patients with t2d and 156 healthy subjects. biochemical and anthropometric parameters were measured in all participants. after dna extraction, sequenom iplex platform was used for the analysis of galnt2 polymorphism (rs4846914), while polymorphism in gckr (rs1260326) gene was analyzed by using real time pcr. our results demonstrated significant association of gckr rs 1260326 variant with waist circumference (p = 0.003) and fasting glucose levels (p = 0.003) in the control group. no such association was demonstrated for rs4846914 galnt2 gene. in the group of diabetic patients, significant association of gckr rs1260326 variant with levels of bilirubin (p = 0.004) and rs4846914 galnt2 variant with hba1c (p = 0.013) and triglyceride levels (p = 0.043) was also demonstrated. our results suggest an association of variations of gckr and galnt2 genes with specific markers of t2d and dyslipidemia. further studies would be needed in order to confirm these genetic effects in other ethnic groups as well. osteoporosis is the most common metabolic bone disorder affecting the normal bone turnover with low bone mineral density (bmd) and risk of fragility fractures. polymorphisms at the sp1 binding site of the collagen type 1 a1 (col1a1) gene is associated with low bmd. we examined the distribution of col1a1 gene polymorphism in 50 young osteoporotic women and in control group in turkish population. patients had low bmd with t score ≤2.5 sd and controls was 25 healthy women (35-57 years). mean age (51.28 ae 5.8) and (46.56 ae 6.15) respectively. the bmd, as g/cm 2 , was measured in the hip and the lumbar spine (l2-l4) with (dexa). dna was isolated from blood. col1a1 gene was analysed with genomica clinical array system. the x 2 test was used to compare allele and genotype frequences between patients and controls. mean of t score in patients was à3.06 ae 0.42. mean bmd (as g/cm 2 ) was 0.708 ae 0.073, and (1.009 ae 0.823) genotype distribution were18(36%) ss, 31 (%62)ss, 1(%2)ss for patients, and 12(48)ss, 9(36)ss, 4(16)ss for control . patients had 33(%66)s allele, 17(34%) s allele, controls had 67(67%)s allele, 33 (33%)s allele. when genotypes and bmd were compared in patients, there was no significant correlation between osteoporosis and genotypes. the allelic distribution was not significant between patients and controls p > 0.05. genotypic distribution in patients were significantly different. patients had a higher frequency of the ss(%62) than controls (ss %36) p < 0.05. this study shows that high prevalences of the ss genotype at the col1a1 locus, in osteoporosis . _ it is possible that the presence of the s allele causes variation col1a1 and col1a2 mrna's producing abnormal collagene protein. since collagen protein is major protein of bone, it is to be expected that a defect in this protein will produce bone fragility. col1a1 gene should be detected early to initiate preventative therapy for bone health. the biological activity of nigella sativa seeds is mainly attributed to its essential oil component which is pre-dominantly (30-48%) thymoquinone (tq). therapeutic effect of tq was exhibited in many diseases including inflammation, cancer, sepsis, atherosclerosis and diabetes. tq has been reported to exhibit antiproliferative effects on cell lines derived from breast, colon, ovary, larynx, lung, myeloblastic leukemia, and osteosarcoma and inhibited hormone refractory prostate cancer. tq induces apoptosis in tumor cells by suppressing nf-jb, akt activation, and extracellular signal-regulated kinase signaling pathways and also inhibits tumor angiogenesis. the aim of this study was to evaluate the anti tumor effects of tq on hepatoma cells. these antitumor assays include cell viability assay, clonogenic assay, scratch assay and molecular expression studies of death related genes. cells were treated with different concentration of tq in hep3b for cell proliferation by mtt and clonogenic assay. in addition, the metastatic character of tq was investigated by scratch assay in hep3b at 3-6 and 24 hours. the effect of tq was also evaluated at mrna level by real-time-pcr. tq was treated on the hep3b cells in three different concentration, namely 75-50 and 37.5 lm. tq showed the cell cytotoxicity in concentration and time dependent manner. the scratch assay revealed no healing in the scratched area due to the decreased cell viability. maximum permissible dose was 50 lm. proapoptotic genes, bax and bad, and autophagy genes, beclin-1 and lc3, were upregulated in hep3b cells after 24 hours treatment in contrast, antiapoptotic gene, bcl-2, expression level was decreased for hep3b cells after 24 hours. p-08.01.4-019 association of irs1 genetic variation with type 2 diabetes and insulin resistance in patients from bosnia and herzegovina insulin receptor substrate-1 (irs1) encodes the irs1 protein, a substrate for the insulin receptor tyrosine kinase and has a critical role in insulin-stimulated signaling pathways. previous studies showed that irs1 single nucleotide polymorphisms (snps) were associated with type 2 diabetes mellitus (t2d). this is the first study performed in a population from bosnia and herzegovina (bh) in which we examined the association of rs7578326 (g>a), rs2943641 (t>c) and rs4675095 (a>t) with t2d risk and related traits. our study involved 437 t2d patients and 252 healthy subjects. biochemical parameters, including but not limited to insulin, homa-ir, hba1c, glucose, and lipoprotein levels, were measured in all participants. genotyping analysis was performed by mass array sequenom iplex platform in cooperation with lund university diabetes centre, malmo, sweden. statistical analysis was done by spss 23. our results demonstrated a significant difference in frequency of rs4675095 (p < 0.001) and rs7578326 (p = 0.021) snps between t2d patients and control subjects. interestingly, here we showed a significant association of irs1 rs4675095 risk t allele with increased insulin levels (p < 0.001) and homa-ir (p < 0.001) in t2d patients. similarly, rs7578326 variant was also associated with the same markers of insulin resistance in diabetic patients, i.e. insulin levels (p = 0.029) and homa-ir (p = 0.025). no such association was demonstrated for rs2943641. however, this irs1 variant was associated with changes in lipoprotein levels, where risk c allele increased vldl (p = 0.006) and decreased hdl levels. our results suggest that irs1 variants are associated with t2d susceptibility in bh population, thus confirming similar findings in other population cohorts. furthermore, the associations of these variants with markers of insulin resistance and dyslipidemic metabolic changes point to their role as potential t2d biomarkers. the adra2a gene encodes alpha-2a adrenergic receptor which mediates adrenergic suppression of insulin. a genetic variant in adra2a was recently associated with defective b-cell function. the objective of this study was to analyze association of two adra2a polymorphisms (rs553668 a>g and rs10885122 g>t) with type 2 diabetes (t2d) and its related traits. in this study we have included 437 t2d patients and 252 healthy subjects from bosnia and herzegovina (bh). biochemical parameters, including but not limited to insulin, homa-ir, hba1c, glucose, and lipoprotein levels, were measured in all participants. genotyping analysis was performed by mass array sequenom iplex platform in cooperation with lund university diabetes centre, malmo, sweden. statistical analysis was performed by ibm spss statistics 23 software. our data showed that frequencies of both, rs10885122 and rs553668, variants were not significantly different between t2d and control subjects. however, rs553668 risk a allele appear to increase insulin levels (p = 0.0002) and homa-ir index (p = 0.00001). furthermore, this variant also seems to affect vldl levels (p = 0.004) and waist circumference (p = 0.02) in diabetic patients. the genotype analysis of rs10885122 variant demonstrated that risk g allele decreased hdl (p = 0.0004) and increased ldl levels (p = 0.014), as well as affected the waist circumference (p = 0.02) in diabetic patients. interestingly, haplotype analysis demonstrated the association of rs553668a / rs10885122g with higher homa-ir index. here we demonstrated that although both, rs10885122 and rs553668, adra2a polymorphisms were not associated with t2d risk in our cohort, they were associated with markers of dyslipidemic perturbations and insulin resistance in diabetic patients. further studies in larger cohorts are needed in order to explore these possible interactions and confirm our findings. smad-interacting protein 1 (sip1), also known as zeb2 is a member of zeb family transcription factors and was shown to regulate epithelial-to-mesenchymal transition, cell cycle, cellular senescence and cancer stemness. bipartite zing finger motifs at amino and carboxyl termini of the sip1 mediate its binding to ebox sequences in the genome. however, there are only limited data about sip1 target genes. by using a home-made anti-sip1 monoclonal antibody, clone 6e5, we conducted chip-seq in three hepatocellular carcinoma cell lines, namely snu398, plc/prf/5 and sk-hep-1 and found receptor tyrosine kinase-like orphan receptor 1 (ror1) as one of the targets. sip1 dnabinding consensus motif cacctg was found at +1 kb, +30 kb and +50 kb from ror1 transcription start site. chip experiments validated sip1 binding to all consensus motifs in the ror1 gene region. interestingly, the strongest enrichment was at +50 kb suggesting that long-range interactions play an important role in the regulation of ror1 by sip1. sip1 knockdown by shrna in high-sip1 expressing snu398 cells resulted in the repression of ror1 expression. ror1 is expressed in embryogenesis and fetal life, and is absent within most of adult normal tissues. however, overexpression of ror1 was observed in many human cancers, from hematological malignancies to solid epithelial tumors. ror1-positive cancer cells have enhanced proliferation, invasion and metastasis capacities, show resistance to apoptotic stimuli and display cancer stem cell characteristics. therefore, sip1 and ror1 act in similar pathophysiological processes. our finding that ror1 is regulated by sip1 at least in hepatocellular carcinoma cells adds another level of complexity to the molecular mechanisms of proliferation, invasion and stemness of cancer cells. hepatocellular carcinoma (hcc) is the most prevalent primary liver cancer and is one of the leading causes of cancer related deaths. smad interacting protein 1 (sip1), a member of the zeb family of emt inducers, is involved in cellular proliferation, senescence, invasion and metastasis in human tumors. however, genes regulated by sip1 in hcc are yet to be identified. we conducted a chip-seq study in high-sip1 expressing hcc cell line snu398 by using a home-made anti-sip1 antibody, clone 6e5. among 509 annotated genes, we selected six1 for further studies because of its increased expression in multiple cancers and its association with poor prognosis. sip1 dna-binding motif cacctg was found at -3 kb from transcription start site of six1 gene. chip qpcr experiment validated sip1 binding to this region with 19,7 fold enrichment. compared to healthy liver, six1 transcripts were upregulated in 8 of 9 hcc cell lines included in this study. knockdown of sip1 by shrna in snu398 cells caused upregulation of six1. immunohistochemistry studies in hcc tissue arrays showed increased expression of six1 in tumors and inverse association with sip1 expression in a tumor grade dependent manner. therefore, our results strongly suggest an inverse correlation of sip1 and six1 in hcc bone mineral density (bmd) and bone turnover are under genetic control and variations in the vitamin d receptor (vdr) are related to bmd. bmd is known to be affected by 25 -hydroxy vitamin d (25 (oh)d) and intact parathyroid hormon (ipth) levels. we aimed to determine correlation blood levels of vitamin d (vitd), ipth, and vdr gene effect in healthy turkish women. the subjects were 25 healthy women in age 35-57 years. the bmd was measured as a t score in the lumbar spine (l2-l4) with dexa. all subjects had normal t score between (-1.0 to 1.4) sd. vitd was measured by lc-20-at shimadzu. ipth was measured by chemiluminescence method, dna was isolated from blood. the fok i (vdrf-foki) and bsmi (vdrb-bsmi) polymorphisms of vdr gene was analysed with genomica clinical array system. the mean vitd level was (21.06 ae 14.54) lg/l, mean plasma ipth level was (57.96 ae 30.49) pg/ml. pearson correlation test showed no relation of vit d with bmd. there was moderately negative correlation between ipth and bmd (r = à0.3062). genotype distribution and allele frequency of subjects were as follows: 21(84%) ff), 1(4%) ff, 3 (12%) ff genotype in vdrf -fok1 gene, 7 (28%) bb, 14 (56%) bb, 4 (16%) bb in vdrb-bsmi gene. allele frequencies were f: 86%, f:14%; b:56%, b:44%. when fok1 and bsmi were combined, 52%(ff-bb) and 20% (ff-bb) were found as the most frequent genotypes. bsmi frequency was in hardy weinberg equilibrium (p > 0.5). but foki was not (p = 0). it was found that vit d, ipth levels and bmd were in normal levels in all carriers of ff genotype and in combined (ff-bb) type carrying healthy women (52%). the association vdr genotype and bmd may be different in various ethnic and geographical groups. therefore it is worthwhile to assess vdr polymorphism among turkish population. these type of distribution studies of vdr in healthy and in osteoporotic women may enlighten to earlier diagnosis and treatment planning. p-08.01.4-026 determination of hb a1c values in beta thalassemia f. g€ uzelg€ ul 1 , g. s. seydel 2 , a. e. yalin 3 , e. s€ onmez 1 , k. aksoy 1 1 c ß ukurova university, adana, 2 nigde university, nigde, 3 mersin university, mersin, turkey introduction: hemoglobinopathies are most commonly seen hereditary blood diseases worldwide. our aim was to compare the hba1c values measured on cation-exchange high performance liquid chromatography (hplc) in beta thalassemia cases. materials and methods: we collected 3 ml of whole blood k 3 edta containing tubes from forty-nine cases. arms, rflp and dna sequence analysis methodologies were carried out for determination of beta thalassemia mutations. hb a1c values were measured using the agilent 1100 hplc system. results: forty-nine diabetic and non-diabetic patients were diagnosed with beta thalassemias: twenty-one ivs1-110/ivs1-110, one ivs1-1/ivs1-1, one ivs1-5/ivs1-5, two ivs1-6/ivs1-6, two ivs2-1/ivs2-1, one fsc5/fsc5, one fsc44/fsc44, two -30/-30, two cd8/cd8, one cd36-37/cd36-37, one cd8-9/cd8-9, two cd82/cd83-g/ cd82/cd83-g, two ivs1-110/ ivs1-6, one ivs1-110/ ivs2-1, one ivs1-110/fsc44, one ivs1-110/cd39, one ivs1-110/cd8, one ivs1-110/cd8-9, one ivs1-110/ivs1-5, one ivs1-6/ ivs2-1, one ivs1-6/ ivs1-25, one ivs2-745/cd8 and one fsc5/cd37. cases were classified as diabetic (6), prediabetic (11) and non-diabetic (32) introduction: members of aurora kinase family aurora a, b and c are conservative kinases of cell cycle which are encoded by genes aura, aurb and aurc respectively. overexpression of aura and aurb was found in human cancers, especially in prostate cancer. moreover, there is the evidence that aurb interacts with one of the major oncogenic kinases -braf. little is known about implication of aurc in cancer, but it was demonstrated, that it can overlap aurb function and shares its location. we studied expression of genes of these kinases in urine of prostate cancer patients aiming to evaluate their involvement in this disease and their potential as tumor markers. materials and methods: 22 urine samples from patients with prostate cancer were gathered after prostate massage before surgical invasion. we used urine samples from 6 healthy men as control. we obtained cells from each urine sample by centrifugation and isolated rna using standard approach with phenol and guanidine thiocyanate. cdna was synthesized and taken to qpcr reactions. data was statistically analysed. results: expression of all studied genes was detected in urine of patients with prostate cancer and of healthy men. expression of aurb and aurc in cancer samples each was higher than expression of aura. the cumulative expression aurb and aurc was higher than expression of aura in 13 samples from 22. we observed positive correlation between expression of aurc and braf (rs = 0.548, p = 0.01). discussion and conclusion: previous investigation showed, that for normal prostate tissue 90% of aurora family expression was presented by aura. we suppose that presence of aurb and aurc cumulative overexpression means presence of cell cycle deviations in prostate tissue of these patients and might be further studied as prognostic marker. in this study we first showed the correlation between aurc and other carcinogenic kinase braf expression, which opens the perspective for investigation of role of aurc in carcinogenesis. bacillus marmarensis sp. nov. is an extreme obligate alkaliphile isolated from mushroom compost near marmara region of turkey. it can survive at extreme ph values up to 12.5. based on its genome sequence, metabolic pathways for 7 proteases, 6 amylases, 2 cellulases, 1 lipase, n-butanol and a biodegradable plastic poly-b-hydroxybutyrate were annotated. in addition to being a potential extracellular hydrolase producer, its ability to survive in the high ph range of 8.0 to12.5 makes it an attractive microorganism for different industrial applications. in the current study, the adaptation strategy of b. marmarensis sp. nov. to alkaline conditions was investigated using proteomic tools. the organism was grown at two different ph values, 10.0 and ph 12.0. for extraction of whole cell proteins, cells were disrupted with mp bio fast prep device. protein extracts were treated with protease inhibitors and a nuclease mix. salts were removed using a cleanup kit. obtained proteins were separated based of their isoelectric points in the first dimension and then based on their molecular weights in the second dimension. proteins maps of cells grown at these two extreme ph values showed significant differences in protein expression for alkaline adaptation. p-08.01.4-029 biochemical and proteomic analyses of normal human astrocytes and glioblastoma exposed to dichloroacetate treatment f. c. atilgan, h. cimen yeditepe university, istanbul, turkey glioblastoma (gbm) is an aggressive malignant tumor composed of astrocytes in brain tissue. gbm cells utilize glycolysis rather than oxidative phosphorylation to support rapid growth rate which is called warburg effect. dichloroacetate (dca) is an antiglycolytic agent that inhibits pyruvate dehydrogenase kinase (pdk) activity and induces apoptosis via normalizing the mitochondrial activity. this study aimed to demonstrate the metabolic alterations between the normal human astrocytes (nha) and gbm cell lines which are exposed to dca, and to identify the differentially expressed proteins by ms-based proteomic analyses. nha cell line, u87mg and u373 as gbm human cell lines were examined through analyzing the alterations in the glycolysis metabolism upon dca treatment by measuring the variations in the pyruvate levels, lactate dehydrogenase a, pdk3. mts was performed to investigate the effect of dca treatment on cell viability. immunoblotting of pgc1-a, oxphos complexes, and mitotracker green staining was employed to reveal the mitochondrial differences between normal and the cancer cells, and upon dca treatment of these cells. proteomic analyses were utilized for the identification of candidate proteins depending on the acetylation status. in this study, compared to nha, the pyruvate and ldha levels were elevated and pdk3 levels in u87mg were reduced by 14%. due to mts results, ≤10 mm dca treatment showed significant decrease in gbm cells compared to nha cells. immunoblotting and mitotracker green staining results showed increase in mitochondrial mass. elevation in the pyruvate and ldha levels and reduction in pdk3 level in u87mg and u373 cells indicates glycolysis dependent metabolic switch in energy metabolism. proteomic analyses demonstrate that most of the differentially expressed proteins comprised of metabolic enzymes. this study provides novel information about metabolic alterations existing between nha and gbm, which can inspire further studies for therapeutic applications. kidney stone is a complex disease resulting from environmental as well as hereditary factors and principally composes of approximately 75% calcium oxalate (caox) crystals, which are formed through a multi-step process. vitamin d receptor (vdr) gene encodes the nuclear hormone receptor for vitamin d3; downstream targets of this gene are chiefly contributed in mineral metabolism though the receptor regulates a variety of other metabolic pathways. calcium sensing receptor casr plays an important role in sustaining mineral ion homeostasis. the aim of this study is to profile the expression level of vdr and calcium sensing receptor (casr) genes and to unravel their role in rat kidney stone induced by ethylene glycol, in order to explain the underlying molecular mechanisms. total rna were extracted from paired sample before and after ethylene glycol treated of 50 rats. the mrna expression level of vdr and casr gene were measured employing quantitative rt-pcr (qrt-pcr). the mrna expression levels of both genes were significantly down-regulated according to before treated. in conclusion, our data suggest reduced mrna expression in vdr and casr genes might be a risk factor for kidney stone formation. further studies are necessary to verify these findings in different ethnic groups. p-08.01.4-031 apj receptor a445c gene polymorphism in turkish patients with coronary artery disease against different models of expected frequencies/counts to understand the evolutionary dynamics of saars in proteins. we obtained from ensembl the assemblies of genomes/proteomes of human and nonhuman primates (chimpanzee, gorilla, and rhesus monkey), rodents (mouse and rat), and birds (chicken and zebrafinch). the expected probabilities for the occurrence of saars based on their nucleotide frequencies in coding regions and amino acid frequencies in individual protein sequences or across the whole proteome were compared with the observed repeat occurrences. we found that with all three methods and in all eight species the correlation between observed and expected repeat counts decreased above a saar length threshold. the percentage of saar proteins for each amino acid also exhibited variability among species when both the repeat length and counts were taken into account. however, clustering based on saar characteristics generally reflected the known phylogenetic relationships between species. our comprehensive bioinformatics analyses reveal that saars show amino acid-specific occurrence patterns with respect to species as well as saar length. tissue proteins play important roles in biological metabolic processes. the qualitative and quantitative analysis of tissue proteins facilitates the understanding of molecular mechanisms that differentiate between physiologic and pathologic states. health and research institutions routinely prepare formalin-fixed paraffinembedded (ffpe) tissue blocks for histopathology. proteomics on ffpe tissue still requires standardization of tissue solubilization processes to overcome variability in protein extraction results. our aim is to compare the proteomic studies of fresh frozen and ffpe rat renal tissues. fresh frozen and ffpe preparations from renal tissues were included in this study. an adult rat was sacrificed and the dissected kidneys were divided two equal section. one immediately frozen in phosphate buffer, and the other tissue specimen not thicker than 5 mm to allow rapid penetration of the fixative put in 10% buffered formalin for 48 hours. the fresh frozen tissue was dissolved and homogenised in the cold phosphate buffer solution containing protease inhibitors. paraffin blocks were performed from formalin fixed tissue specimens. we have extracted the protein from the ffpe tissues using our previously verified method. we have utilized electrophoresis three times to compare protein yield, number, intracellular and intercellular of homogenised samples obtained from ffpe and fresh frozen kidney samples. the number of proteins identified from fresh frozen kidney tissue has generally been shown to be increased compared with ffpe tissue. decrease of the qualitative results in electrophoretic bands was found similar in all replicative studies. ffpe tissues undergo extensive cross linking between protein/ dna/rna molecules during formalin fixation, which creates inter-molecular crosslinks. on the other hand, ffpe tissues represent a valuable resource to carry out retrospective studies aimed to biomarker discovery in kidney cancer as well as other kidney diseases. background: development of atrial fibrillation (af) during the course of chronic primary mitral regurgitation (mr) is common and represents complex molecular mechanisms. however, the gene expression profile of human atrial fibrillation (af) in the setting of chronic primary mr remains uncharacterized. in the current study, we aimed to compare the gene expression profiles of patients with severe degenerative mr in sinus rhythm (sr) and af. methods: left and right atrial tissue samples were obtained from patients with chronic primary severe mr in permanent af (n = 30) and sinus rhythm (n = 30). we performed a novel micro-dissection technique for thin sections of atrial tissue samples and immediately fresh froze intra-operatively. transcriptomes of left and right atrial appendages of degenerative mitral regurgitation patients with sr versus af were compared by microarray analysis on affymetrix hgu-133 plus 2 platform. bioinformatics, data mining and pathway analyses were conducted on partek gs and webgestalt. genome-wide gene expression profiles were compared between af and sr groups among 54.675 transcripts representing 38.500 well-characterized human genes. differentially regulated genes were evaluated according to fold change (fc ≥ 1.5) with a p-value ≤0.05. results: most remarkable pathways altered in af atrial tissues compared to sr group, were extracellular matrix-receptor interaction; mapk, adipocytokine, and calcium signaling; apoptosis and cardiac muscle contraction pathways. conclusions: this is the first human study of comparative transcriptomics in left and right atrial tissues of patients with af versus sr associated with severe degenerative mr. the main findings of this multidisciplinary translational research provide novel candidate targets for the treatment and prevention of af. in order to acquire iron under iron-limiting growth conditions, bacteria employ specific mechanisms such as production and secretion of siderophores. siderophores are low molecular metalchelating compounds that contribute not only to iron scavenging, but also participate in other important processes including oxidative stress response and cell signaling. serratia marcescens, gramnegative bacterium, could be found in various environments, including wastewater, plant rhizosphere and hospital setting where s. marcescens can cause serious life-threatening infections. in this study, we performed a detailed characterization of the siderophores of the clinically important pigment-free s. marcescens strain sr41-8000 and environmental pigment-producing s. marcescens strain sm6. bioinformatic analysis of these genomes by antismash software revealed the presence of several clusters involved in non-ribosomal peptides synthesis (nrps). we found four nrps clusters in genome of s. marcescens sm6. cluster 1 has a low level of identity to enterobactin gene cluster typical for bacteria producing catechol-like siderophores. second cluster has only 4% of identity to xantholipin biosynthetic gene cluster. clusters 3 and 4 of nrps genes of s. marcescens sm6 did not show any homology to known nrps clusters. in contrast, the genome of s. marcescens sr41-8000 contains only one genetic cluster of nrps genes. this cluster does not have similarity to any of the known bacterial nrps genes. thus, genetic analysis of two isolates of s. marcescens allowed us to identify nrps genetic clusters and showed that the repertoire of these genes is different between strains. we hypothesized that the strain isolated from environment has competitive advantage over clinical isolate due to genetic diversity of siderophores. on the other side, clinical isolate has specific genetic cluster of siderophores which may promote s. marcescens growth and adaptation to the extreme niches present in medical facilities. p-08.01.4-037 the first glance on the genome's structure and activity in hibernator edible dormouse hibernation is a unique adaptive way of survival in extreme environmental conditions where mammals decrease their metabolic rate and demonstrate physical inactivity for prolonged periods of time (up to 6-8 months). remarkably, some hibernating animals have a long average lifespan and the ability to avoid muscle atrophy caused by disuse or immobilization. to identify main molecular pathways behind the protective musculoskeletal adaptation and genome structure in hibernator edible dormouse (glis glis), whole-genome analysis of mrna expression in muscles (m. soleus and m. edl) and lumbar spinal cord samples was conducted. three groups of the dormice: 1) active animals 2) hibernated animals and 3) animals immobilized for 2 weeks in laboratory, were examined. rna libraries have been sequenced using hiseq 2500 illumina platform. coupled with genome dna sequencing provided x10 coverage of the estimated genome, we have assembled de novo transcriptome of the dormice. differentially expressed genes in response to immobilization and hibernation were determined. transcriptional program of these phenotypes was similar. pathways enriched by differentially expressed genes were identified. gene expression of the key muscle proteins and muscle atrophy markers was analyzed. muscle-specific e3-ubiquitin ligases murf1 and mafbx revealed no changes in mrna expression. our study represents the first attempt to elucidate changes in transcription profiles of skeletal muscles and spinal cord during hibernation and hypokinesia in edible dormice. in corroboration to the gene expression data, they demonstrated minimal morphological evidence for muscle disuse atrophy during physical inactivity. edible dormice, thus, can be considered as a novel model organisms in investigation of the genetic mechanisms of hibernation and prevention of muscle atrophy. the work is performed according to the russian government program of competitive growth of kfu and supported by rfbr jsps_a no. 14-04-92116. in response to diverse environmental cues bacteria form complex structured communities called biofilms. the metabolic pathways activated by these cues are remarkably different depending on the species studied. however, they all lead to the formation of an extracellular matrix that holds the cells together. non-pathogenic gram-positive spore-forming soil b. subtilis strain is recognized as a model system for the study of biofilms. to discover the pathways regulating biofilm formation in b. subtilis, we studied the natural isolate of b. subtilis strain 168, and constructed the recombinant strains with knocked out genes of following regulatory proteins: abrb (global transcriptional regulator), degu (two-component response regulator of signal transduction system degs-degu), ccpa (regulator of carbon catabolism) and spooa (regulator of sporulation). in the minimal medium broth b. subtilis 168 wild-type strain forms biofilm with its maximum on 48th hour of culture growth. ph-optimum for biofilms formation by the wild-type strain is in the range of 7.4-8.0. the temperature optimum is in the range from 22°c to 45°c. this corresponds to the natural conditions of the b. subtilis habitat in rhizosphere. the level of biofilm formation by regulatory mutant strains with deleted abrb, degu, ccpa, spooa genes is on average 40% lower than by the wild-type strain. this indicates that global regulatory system controlls biofilm formation process. statistically significant differences in the levels of biofilm formation between regulatory mutants haven't been identified. ph and temperature optima of mutant strains are the same as for the wildtype strain -7,4-8 and 22°c -45°c respectively. the crataegus genus which is a member of rosaceae family, has approximately 200 species worldwide and 24 species in turkey. all plant species in this genus have the common name "hawthorn". crataegus microphylla (c. microphylla) c. koch which is characterised by having erect sepals in fruit and smaller leaves in comparison with the other species, is one of the wild edible fruits in turkey. crataegus species have been used as food and also in folk medicine for the treatment of various diseases. for this purpose, the potential biological properties of crataegus microphylla were aimed to reveal by the preliminary work. in this study, prevention of oxidative dna damage using supercoiled pbr322 plasmid dna, acetylcholinesterase, tyrosinase, a-glucosidase inhibition and antioxidant effects: 2,2diphenyl-1-picrylhydrazyl radical scavenging effect, phosphomolibdenum-reducing antioxidant power, ferric-reducing antioxidant power with total phenolic and total flavonoid contents of the c. microphylla leaves, stem barks and fruits that extracted with ethanol, methanol and water were investigated. the experiments of oxidative dna damage studies and antioxidant activities of c. microphylla extracts showed that methanol and ethanol extracts possessed a strong ability to prevent dna damage and significantly antioxidant activities. methanol extracts of stem barks from c. microphylla exhibited the highest acetylcholinesterase and tyrosinase activities (48.86 ae 4.06% and 85.57 ae 2.01%, respectively), at 200 lg/ml. in addition, ethanol extract of leaves from c. microphylla inhibited the a-glucosidase activity significantly when compared to acarbose. this study explained significant antioxidant, enzyme inhibitory, hypoglycemic, and neuroprotective activities of methanolic or ethanolic extracts prepared with stem bark and leaf from c. microphylla and also strong ability to prevent dna damage that corresponded to antioxidant potential of methanol extracts of leaf and stem bark. the yarrowia lipolytica species (yl) is nonconventional yeast widely used for recombinant protein expression due to its system of post-translation protein modification, which is the most similar to that of higher eukaryotes. yl appears the promising producer of recombinant proteins with much more complicated molecules compared to those of prokaryotic producers. however, an important feature for a producer strain of recombinant proteins is the genes, the expression of which undertakes under controlled conditions, and consequently, search of new effective inducible promoters in the yl genome is of great interest. proteome analysis of the yl cells grown at different ph values (4.0, 5.5, 9.0) showed that under alkaline conditions the amount of mitochondrial porine vdac (voltage dependent anion channel), one of the most abundant protein of the mitochondrial outer membrane, increased significantly. vdac is supposed to let reactive oxygen species out of mitochondria protecting the cell against oxidative stress. therefore, the por1 gene expression, encoding vdac should increase in the stress conditions. the promoter of the por1 gene was used to construct some new expression systems based on yl w29. a new genetic construct bearing a reporter bgalactosidase gene under control of the por1 promoter. b-galactosidase activity was assayed in the cells grown in various ph conditions and exposed to exogenous oxidants such as hydrogen peroxide, menadione, and methyl viologen. it was shown, that in h 2 o 2 and methyl viologen treated cells b-galactosidase activity increased 1.5-2.0 -fold reaching its maximum in the cells, grown at ph of 9.0. thus, we demonstrated high inducibility of the por1 promoter, which is essential for effective action of the expression system based on it and potency of application for transformed lines of producers. acknowledgments: supported by the russian foundation for basic research (grant no 16-34-00634 mol_a). aspergillus nidulans is able to detoxify and catabolize the toxic proline analogue, lazetidine-2-carboxylic acid in nature, azc serves as a plant protectant against infections and consumption. we have obtained evidence that azc is not only non-toxic for the model ascomycete aspergillus nidulans, but it can be utilized as a poor nitrogen source. in order to elucidate the molecular mechanism underlying azc detoxification, we have constructed and studied a. nidulans strains deleted in the cognate genes involved in azc detoxification in pseudomonas and saccharomyces cerevisiae. these genes, found by in silico analysis, encode a putative hydrolase, acha, and an azc acetyltransferase, ngn2, respectively. gene deletion was accomplished through double crossover. a cassette containing the~1500 bp 5' and 3' flanking sequences of each gene, with the afpyrg gene as a selection marker, was contructed. crossing the achad and the ngn2d strains isolated the achad ngn2d double mutant strain. rt-pcr was used for gene expression analysis in the wild type strain, area-loss of function and crea-derepressed mutant strains. our results clearly show that azc can be used as a poor nitrogen source by a. nidulans. this utilization requires a) acha, a putative azc hydrolase, and b) a fully active gaba catabolic pathway, as lack of either amdr or gata abolishes azc utilization. most importantly, the double mutant, achad ngn2d, shows azc toxicity, suggesting that ngn2 is a true orthologue of mpr1, able to detoxify azc, a phenotype that can be observed only in the absence of acha. as a final point, ngn2 was shown to be induced by the presence of azc and and to be under nitro the spatial genome organization plays a great role in the maintenance of the nuclear architecture and regulation of all processes occurring in the nucleus. this system is controlled by a set of special proteins having an architectural function. however, the mechanisms of their action remain unknown. among these proteins are, in particular, zad-domain-containing proteins. zinc finger-associated domain (zad) is a ubiquitous motif of c2h2 zinc finger proteins of drosophila. genes that encode zad proteins are specific for and expanded in the genomes of insects. only a few zad-encoding genes have known functions, and the role of zad is being discussed. up to date there was only one known crystal structure of zad-domain from drosophila transcription factor grauzone (grauzad). here, we present for the first time the crystal structure of the zad-domain of serendipity-d transcriptional activator of the egg-polarity gene bicoid. zad-domain was cloned, overexpressed in e. coli, purified and the structure was solved at 3.5 a by mad technique. detailed analysis of the structure proved that the protein exists in dimeric form and revealed unique spatial organization of the protein, different from those for grauzad. this work is supported in part by russian ministry of education and science grant (14.616.21.0066). mycoplasma gallisepticum is a convenient model object for studying the regulation of transcription because it has a reduced genome, lack of cell wall and many metabolic pathways and also easy to culture and non-pathogenic to humans. due to the nature of the genomic organization and the loss of many of the known regulators, the effect of disrupting the function of some proteins may be a useful tool for studying the regulation of transcription. the gene expression study was performed on agilent onecolor microarray with custom design and random-t7 polymerase primer for cdna synthesis. microarray represents 3366 probes for 678 orf including genes and ncrna. in this work, we have investigated the effect of changes in the level of gene expression of m. gallisepticum for two different types of conditions: a genetic knock-out mutants and the cell response to treatment with sub-lethal concentrations of antibiotics. we characterized transcription of m. gallisepticum when the cell responses to dysfunction of proteins with metabolic potential, possible regulators of expression, in violation of permeability of membrane by cccp, inhibition of ribosomal synthesis by tetracycline, dna gyrase by novobiocin and atp synthase by oligomycin. the data obtained allow to characterize the transcriptional response under different conditions and to identify groups of genes that change expression together. major transcriptional changes were observed in the response of cells under cccp treatment due to uncoupling of the proton gradient and further reducing the membrane potential, as well as under novobiocin treatment due to changing the topology of dna. global problem of oil pollution forces scientists to search for a new safe remediation technologies constantly. careful attention is paid to bacteria, some of which possess additional biotechnologically valuable properties, such as utilization of hydrocarbons and production of biofurfactants. in this regard, we carried out proteogenomic characterization of tsukamurella tyrosinosolvens strain ps2, which was isolated from chemical sludge and capable for alkane degradation and biosurfactant production. whole genome of the strain was sequenced on the miseq (illumina) platform, assembled and annotated. proteome on mineral medium with glucose, sucrose and hexadecane as a sole carbon and energy source was studied. shotgun proteomics approach was performed on hybrid chromatography-mass spectrometry machine (maxis impact). alkane oxidation genes (alkane-1-monooxygenase, rubredoxin and rubredoxin-reductase) under genome sequence, as well as two pathways of trehalose synthesis and genes for mycolic acids production were found. emulsification activity of cell-free culture liquid was about four times higher on hexadecane in comparison with sugars. proteomic profile was different at various culture conditions. all glycolysis genes, beginning with glucose-6-phosphate isomerase to pyruvate kinase, were found on the media with sugar. the medium with hexadecane helped to reveal enzymes involved in the beta-oxidation of fatty acids, for example 2,4-dienoyl-coa reductase, 3-ketoacyl-coa thiolase and enzymes of the initial mycolic acid synthesis pathways. thus we have established that the strain t. tyrosinosolvens ps2 utilizes sugar by glycolysis. also, the bacterium is capable for alkane oxidation followed by beta-oxidation of fatty acids. based on the proteogenomic data, we assume that the bacterium is able to synthesize trehalose lipids, namely, trehalose mycolates. obtained results could be useful to create conditions for increased biosurfactants production. gestational diabetes mellitus (gdm) is a glucose intolerance firstly diagnosed during pregnancy. in this study, we aimed to investigate the association between serum adiponectin, resistin levels and insulin resistance in gestational diabetic patients. a total of 80 patients; 40 healthy pregnant women (control group) and 40 pregnant women diagnosed with gdm (gdm group) were included in this study. serum adiponectin, resistin, glucose, insulin, hba1c levels and lipid parameters were measured. insulin resistance index homa-ir values were calculated. in this study, serum glucose, insulin, hba1c levels and homa-ir were significantly higher in gdm group compared to the control group (p = 0.038, p = 0.011, p = 0.001, p = 0.008, respectively). serum adiponectin levels were significantly lower (p < 0.001); whereas serum resistin levels were significantly higher (p = 0.004) in gdm group than in the control group. it can be concluded that resistin contributes to the formation of insulin resistance, adiponectin plays an important role in the regulation of this resistance and they also have effects on gdm pathophysiology. hematological cancers including acute myeloblastic leukemia (aml) and acute lymphoblastic leukemia (all) in terms of incidence and mortality, are the second most important cancer type in turkey. numerous studies show that cancer patients respond differently to treatment thus supporting the idea of personalized therapy need for individuals. renin angiotensin system (ras) have key roles in aml and all progression and it has been shown by many studies suggests that these system's genes might be good biomarkers for aml and all personalized therapy. we aimed to identify ras gene based homogeneous subgroups of acute leukemia and determine the most effective chemotherapoetic agent for each subgroup. after validation and verification of the results, more effective drugs can be recommended for the use in clinics for chemotherapy of aml and all. results of our preliminary studies showed that we are able to identify subgroups of aml and all as well as correlating each existing subgroup with fda approved drugs. considering the long and highly cost process of developing new drugs for cancer treatment makes the present study all the more valuable. in addition, there is a serious need for change in aml and all therapy since there is no highly effective chemotherapy protocol available for their treatment. welcome trust sanger (wts) and cancer cell line encyclopedia (ccle) databases will be used to determine subgroups of aml and all based on ras genes or whole genome expression using standard deviation and hierarchical clustering analysis. the most effective drugs for each subgroup will be identified using pearson's r correlation analysis with drug sensitivity data (ic50, ic50, amax, aare, etc.) available in same databases. further validation tests will be performed by in vitro validation using aml and all cell lines: drug sensitivity profiles will be determined and gene expression will be shown by q-rt-pcr. p-08.02.5-003 functional polymorphisms of ephx2 in a turkish population h. pinarbasi, i. sari cumhuriyet university, sivas, turkey soluble epoxide hydrolase (seh; ec 3.3.3.2) is encoded by ephx2 and catalyses the degradation of endogenous fatty acid epoxides generated by cyp450 epoxygenases. these fatty acid epoxides such as epoxyeicosatrienoic acids (eets) have been shown to posses vasodilator, anti-inflammatory, anti-platelet, anti-hypertensive, anti-apoptotic, anti-thrombotic and natriuretic effects. it has been reported that eet levels are associated with hypertension, stroke and cardiovascular diseases. individual differences in the ephx2 gene that affect the seh activity may alter the circulating levels of eets. k55r and r287q polymorphisms have been known to cause increased and decreased seh activity, respectively. therefore we aimed to determine the genotype frequencies of these two polymorphisms in a turkish population. k55r and r287q polymorphisms were determined by the real time pcr using double-dye hydrolysis probes or pcr-rflp method. the observed genotype frequencies for k55r polymorphism were 80.8% wild type (aa) and 19.2% polymorphic genotype (ag+gg) and for r287q polymorphism 81.4% wild type and 18.6% polymorphic genotype (ga+aa). the genotype distributions for both polymorphisms were in hardy-weinberg equilibrium. pregnancy is one of manifestations for thrombophilia factors, which in its turn leads to various complications of its course. one of the markers of hereditary thrombophilia is mutations in the folate cycle mtr, mtrr and mthfr genes. insufficient intake of folate during pregnancy disrupts the functioning of the genome, leading to miscarriage, violation of embryogenesis and various fetal malformations. however, results of studies on the role of hereditary thrombophilia in the occurrence of complications during pregnancy are rather contradictory. aim of this study was to determine the frequency of alleles and polymorphic variants of folate cycle genes mtr a2756g, mtrr a66g and mthfr c677t in women of kazakh ethnic group with pregnancy complications. we used real-time pcr. blood samples for dna isolation were obtained from 129 pregnant women. the main group consisted of women (n = 90) which had a history of two or more pregnancy complications in the form of pre-eclampsia, eclampsia, missed abortion, miscarriage, and etc. control group consisted of women (n = 39) with two or more normal pregnancy outcomes, and had no complications during pregnancy in history. average age of women in experimental group was 32.0 ae 0.50 years compared with control of the age 33.6 ae 0.33. the analysis of the frequency distribution of alleles of genes in experimental group of women with complications of pregnancy revealed no significant differences relative to the control group. analysis of the distribution of polymorphic variants of folate cycle genes showed significant difference between the study and control groups in the occurrence frequency of heterozygotes for the mutant allele g in the gene mtrr a66g (or = 2.89, ci 95% = 1.25-6.71; v 2 = 6.376, p < 0, 05). no significant differences in alleles between homozygous wild-type and homozygous mutant alleles were observed. this work was funded by the mes kazakhstan (gr 0115rk00287 project number). p-08.02.5-005 a study on the association between rs6918698 polymorphism in connective tissue growth factor gene and pseudoexfoliation syndrome pseudoexfoliation syndrome (pes) is a disorder of the extracellular matrix characterized by the production and progressive accumulation of an abnormal fibrillary material in many ocular tissues. pes prevalence is 11.3% above the age 40 in turkey. since pes is characterized by excessive synthesis of elastic microfibrillar components throughout the body, growth factors can have important roles in the pathophysiology of pes. human connective tissue growth factor (ctgf) is a protein expressed in a variety of tissues, including the anterior chamber of the eye. ctgf coding gene has several genetic polymorphisms. rs6918698 g/c single nucleotide polymorphism (snp) is found at position à945, in promoter region. the presence of a c allele for rs6918698 is critical for transcriptional suppression of the ctgf gene which would reduce ctgf production. aim of this study was to investigate if there is any association between pes and rs6918698 polymorphism of the ctgf gene. study population consisted of 60 patients with pes and 60 controls. blood samples were collected by g€ ulhane military medical academy, department of ophthalmology, ankara, turkey. genotypes were assigned by pcr followed by restriction fragment length polymorphism analysis. genomic dnas were isolated from whole blood samples using manual dna isolation. the frequency of ctgf rs6918698 polymorphic allele g was 0.442 in patients, and 0.450 in controls (0.967, p = 1.000). distribution of genotypes was gg: 31.7%, gc: 48.3% and cc: 20.0% among patients, while gg: 35%, gc: 40.0% and cc: 25.0% (or = 1.162, p = 0.689) in controls. statistical analysis showed that there is no significant relationship between ctgf rs6918698 snp and pes. these are the preliminary findings of a research project which is the first study analyzing the relationship between ctgf rs6918698 snp and pes. this work did not point out a role for ctgf rs6918698 in the risk for pes. a significant relationship might be found when the study population is enlarged. p-08.02.5-006 evaluation of rs11136000 single nucleotide polymorphism of clusterin gene in pseuodoexfoliation syndrome risk pseuodoexfoliation syndrome (pes), an age-related systemic disorder, is characterized by production and accumulation of abnormal fibrillar extracellular material in anterior structures of the eye. clusterin (clu) is a multifunctional glycoprotein produced and secreted by almost all cell types and is found in all body fluids and in accumulated pes material. under cellular stress conditions, clu provides inhibition of stress-induced precipitation and aggregation of misfolded proteins. clu expression level in pes patients is unexpectedly low and this could be due to single nucleotide polymorphisms (snp) on the gene coding for clu. rs11136000 c/t polymorphism has been found to be associated with alzheimer's disease and pathophysiology of alzheimer and pes are similar. this study aimed to determine whether rs11136000 snp of clu gene have a role in the development of pes. study population consisted of 60 patients with pes and 60 controls. blood samples were obtained from g€ ulhane military medical academy, department of ophthalmology, ankara, turkey. genomic dnas were isolated from whole blood of subjects using manual dna isolation. genotypes were assigned by pcr followed by restriction fragment length polymorphism analysis. t allele frequency of pes patients was 0.425 and that of controls was 0.442 (0.934, p = 0.794). the distribution of genotypes was cc: 30.0%, tc: 55.0% and tt: 15.0% among patients while cc: 28.3%, tc: 55.0% and tt: 16.7% (0.922, p = 0.841) in controls. there was no statistically significant difference between pes patients and controls in terms of tt genotype and t allele frequency. these are the preliminary findings of a research project which is the first study analyzing the relationship between clu rs11136000 snp and pes in turkish population. this work did not point out a relation for polymorphic genotype in the risk for pes. however, a relationship between clu rs11136000 polymorphism and pes can be found when we enlarge the study population. the tumor suppressor tp53 is the most frequently mutated gene in head neck squamous cell carcinoma cancer and represents a known transcription factor and tumor suppressor gene that regulates different microrna and target genes. the aim of our work is to construct the transcriptional and post-transcriptional network regulated by tp53 and to evaluate the difference at mrna and protein expression levels of the tp53 target genes in hpv negative head and neck squamous cell carcinoma (hnscc) patients with distinct tp53 mutation states and to elucidate the molecular mechanism that underlie the poor prognosis of tp53 mutation. to show the tp53 mutation landscape and its prognostic relevance for survival, we used cbioportal for cancer genomic analysis. we downloaded mutational profiles of 243 hpv negative hnscc patients. employing different databases we constructed the tp53 regulatory network. and then, to evaluate the effect on mrna, protein and microrna regulated by tp53 we used the mrna and protein expression profiles of patients from tcga. our results show that hotspot, truncating and missense mutations have statistical significance in the univariate analysis. the tp53 regulatory network show the involvement of important target involved in the progression of hnscc and the deregulation of protein expression of an important key epigenetic modifier ezh2 was significantly associated with tp53 mutational state. ezh2 is a member of the polycomb group protein enhancer zeste homolog 2 which is known to be directly repressed by tp53 and indirectly by the activation of mir-200a and mir-200b. we found a significant up-regulation of ezh2 that depend from tp53 mutation. it is important to understand the difference in mrna and protein expression of tp53 regulatory network that could depend from its mutational state. this finding suggest that ezh2 might be a potential therapeutic target for hnscc. p-08.02.5-008 next generation sequencing based approach for monitoring of minimal residual disease in acute lymphoblastic leukemia minimal residual disease (mrd) monitoring is widely used to evaluate efficiency of chemotherapy and to choose a strategy for further treatment in acute lymphoblastic leukemia (all). the most commonly used approaches for mrd detection are based on flow cytometry and qpcr. these methods have several important limitations including insufficient sensitivity, complicated experimental setup and false positivity. the newly developed next generation sequencing (ngs) approaches could overcome the existing limitations in mrd monitoring. here we describe a new mrd monitoring approach based on targeted deep sequencing of malignant rearrangements. first, we identified bcr/tcr rearrangements specific for the leukemic clones in initial bone marrow samples of 10 all patients. for this, we used sanger sequencing of the products of 19 multiplex pcr, performed with bcr/tcr specific primers combined according to the optimal frequency distribution of v/j-genes in healthy donors. second, we analyzed concentration of malignant clone rearrangements, identified at the first step, in dna samples obtained from bone marrow after 36 days of treatment. for this purpose, we performed ngs of 10 libraries for each identified leukemic rearrangement. four libraries were amplicons of bcr or tcr gene rearrangements generated using characteristic v and j segment specific primer combination. six additional libraries were amplicons of the same primer combination from the same dna sample which contained initial leukemic dna spike-in (in concentrations corresponding to 1 per 100, 1000 and 10,000 cells) for a calibration curve generation. using this approach, we analyzed 7 all clone specific rearrangements for three patients and calculated concentration of the leukemic clones by using the calibration curve. for one patient we didn't find any leukemic cells and for two patients we found 1 leukemic cell per 100,000 analyzed cells. znf365 is considered as a transcriptional target for p53 and plays an important role in the homologous recombination, mitosis, centrosome dynamics. as was shown by gwas some snps in znf365 have strong association with the risk of breast cancer (bc). however, it was unclear whether the same snps are associated with risk of bc in kazakhstan. therefore two polymorphisms (rs10995190, rs10761659) of znf365 were investigated in kazakh population in this study. the present case-control study was carried out with participation of 444 kazakh females with bc and 390 cancer-free donors. additionally, subtypes of bc, stratified by estrogen receptor (er+/à), progesterone receptor (pr+/à) and human epidermal growth factor receptor 2 (her2+/à) status were estimated. pearson p-value, odds ratio, 95% confidence interval tests were applied to data analysis. significant differences were found in allele frequency and genotype distribution at rs10995190 locus in znf365 between the patients and control groups (p = 0.013 for allele; p = 0.007 for genotype). moreover, significant association with bc was revealed for rs10995190 after dividing patients according to er+/ à, pr+/à and her2+/à status of the tumor. the g allele was associated with er+ (p = 0.01, or = 1.69, 95%ci:1.11-2.56), pr+ (p = 0.01, or = 1.78, 95%ci:1.13-2.79) and gg genotype with her2-bc carriers (p = 0.02, or = 1.66, 95%ci:1.04-2.63). also, g allele can be considered as a risk factors in er+/ pr+/her2-luminal type of tumor (p = 0.028, or = 1.79, 95% ci:1.06-3.05). our findings correlate with the data of several gwas where the association of the rs10995190 polymorphism with higher mammographic density and the risk of breast cancer have been shown. the obtained results allow us to consider g allele and gg genotype of rs10995190 as a marker of bc risk with predictive value, restricted to er, pr and her2 status of the tumor in the kazakh population. breast cancer (bc) is the most common cancer among women in most of countries. alternative variants of low-penetrance genes such as fgfr2 (rs2981582), tox3 (rs3803662), map3k1 (rs889312), lsp1 (rs381798) are shown to have high frequency in north america, south-east asia, australia, europe populations and a multiplicative effect on the development of bc. in this study was investigated assosiasion between alleles/genotypes combinations of these genes with increase/decrease of bc risk. the case-control study included 625 bc patients and 672 healthy women from kazakh and russian populations. genotyping was performed by pcr-rflp methods. combined effect of allele and genotype variations in four different genes on bc risk was assessed by apsampler algorithm. the fisher exact test, odds ratio (or) with 95% confidence intervals (95% ci) were applied to data analysis. according to obtained results combinations of allele c of tox3 rs3803662 and a of map3k1 rs889312 (p = 0.006, or = 2.2), also allele c of tox3 rs3803662 and c of lsp1 rs381798 (p = 0.02, or = 1.47) associated with increased bc risk in the russian population. consequently, combinations with c allele of tox3 rs3803662 contribute significantly to bc risk with p-value = 0.04, or = 1.86. on the contrary, tt genotype of tox3 rs3803662 with p = 0.04, or = 0.53 and its combination with allele t of lsp1 rs381798 with p = 0.03, or = 0.47 determine a bc risk reduction in russian population. in addition, a risk combination of allele c of lsp1 rs381798 and a of map3k1 rs889312 was found in kazakh population (p = 0.02, or = 1.35). studies have shown that a genetic predisposition to bc can be determined by the cumulative effect of individual alleles and genotypes and possible epistatic interactions of studied genes. obtained combinations of alleles and genotypes can be considered as complex genetic markers of bc and may be used as predictive. cancer that is caused by excessive proliferation of cells and reduced apoptosis is a pathological condition. currently, studies that are comitted with breast cancer are great important early detection and diagnosis of breast cancer. after the discovery of cisplatin as chemoterapy drug, new transition metal based complexes have been developed for treatment of cancer. in this study, anti-cancer activity of azo-azomethide ligand and its mononuclear metal complexes is studied on human cancer cell lines (mcf-7) and mouse fibroblast (l929) cell lines. cells were studied four different concentrations (12,5; 50; 75; 100 lm). xtt (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2h-tetrazolium-5-carboxanilide) protocol was applied after 24 and 48 hours. in our study 10% fetal bovine serum (fbs), 1% l-glutamine, 100 iu/ml penicillin and 10 mg/ml streptomycin in dmem (low glucose) were used. cancer cell lines in dmem medium is produced in 95% humidity and 5% co 2 incubator at 37°c. anti-cancer activity of synthesized complexes were determined on mcf-7 and l929. in the biological activity studies, synthesized compounds showed higher anticancer activity than positive control (5-fu). finally, our new synthesized complexes can be suggested that potent ajan for anti-tumuour for breast cancer drugs. large interindividual differences in response to chemotherapy present an important issue in cancer treatment. malignant mesothelioma (mm) is an aggressive tumor with poor prognosis, treated mostly with gemcitabine/cisplatin (gem/cis) or pemetrexed/cisplatin (pmx/cis) chemotherapy. as both clinical characteristics and genetic variability may affect treatment outcome, our aim was to construct and validate clinical-pharmacogenetic prediction models of treatment outcome in mm for both chemotherapy regimens and to develop an algorithm for genotype-based treatment recommendations. clinical-pharmacogenetic models were built on 71 gem/cistreated and 57 pmx/cis-treated mm patients. pharmacogenetic scores were assigned by rounding the regression coefficients. gem/cis model was validated on 66 independent mm patients. model predicting outcome of gem/cis chemotherapy included crp level, histological type, performance status, rrm1 rs1042927, ercc2 rs13181, ercc1 rs3212986, and xrcc1 rs25487. values ranged between 0 and 3.4; cutoff value of 0.75 had sensitivity of 0.62 and specificity of 0.81. patients with higher score had shorter progression-free and overall survival (p < 0.001). in the validation group, positive predictive value was 0.74 and negative predictive value was 0.56. model predicting outcome of pmx/cis chemotherapy included crp level, mthfd1 rs2236225, and abcc2 rs2273697 with scores ranging between 0 and 3.9. cutoff value of 2.7 had sensitivity of 0.75 and specificity of 0.61. patients with higher score had lower probability of good response and shorter progression-free survival (p < 0.001). clinical-pharmacogenetic models could enable stratification of mm patients based on their probability of response to gem/cis or pmx/cis and improve treatment outcome. this approach could be used for translation of pharmacogenetic testing to clinical practice as it would facilitate the selection of the best treatment option for each patient. p-08.02.5-014 evaluation of anti-diabetic potential of circiliol and circilineol using caco2 cell line diabetes mellitus is a metabolic disorder, and many people are suffering from this disease in the worldwide. oral hypoglycemic agents such as sulfonylureas and biguanides are currently used for the treatment. however, studies searching for a more effective anti-diabetic agents are being carried out continıously. based on that we aim to investigate the potential anti-diabetic effects of circilineol and circiliol isolated from teucrium alyssifolium extract, using in vitro cell culture models. for this purpose, the anti-diabetic actions were investigated by applying model caco2 (colorectal adenocarcinoma) cell line. we determined the level of ag (alpha-glucosidase), sglt1 (sodium-glucose transporter-1) and glut1-5 and glucose transport. neither ag activity nor sglt1 activity was increased with either circiliol or circilineol treatment in caco2 cells compared to positive control. similarly, neither the activity nor the expression level of glut1*-5 was increased in caco2 cell line with either circiliol or circilineol treatment relative to control. in conclusion, these results strongly suggest that circiliol and circilineol do not possess any anti-diabetic potentials.supported by tubitak 114z640 and paubap 2014fbe029 the purpose of this study was to characterize and assess the impact of a novel magnetite (fe3o4) nanosystem functionalized with the natural origin compound eugenol (e) on the pseudomonas aeruginosa virulence, biofilm formation and qs signaling in order to advance research aimed to find alternative and personalized therapeutic approaches for severe infections produced by this opportunistic pathogen. fe3o4 nanoparticles were obtained by a co-precipitation method and functionalized with analytical purity e. functionalized nanoparticles (fe3o4@e) were characterized by ir, sem, tga and hr tem. one laboratory and 9 p. aeruginosa clinical isolates were utilized in the study. growth and biofilm formation were assessed by an adapted microdilution method followed by absorbance reads and viable count analysis in dynamics (6, 12, 24 and 48 hours of treatment). soluble virulence factors production was assessed by enzyme activity evaluation of bacteria grown on specific media. the expression of qs core genes was analyzed by qrt-pcr and a luminescence assay. results demonstrated that the average size of the obtained nanosystem ranges 5-9 nm, particles are relatively homogenous and have a low tendency to form aggregates. subinhibitory concentrations of fe3o4@e limited biofilms formation in a time and strain dependent manner, and significantly inhibited the production of toxin pore forming enzymes (haemolysins and lipases) in most strains. the expression of lasi and lasr genes was three fold downregulated, while the expression of pqsr was upregulated in planktonic cultures suggesting that pqs signaling may be involved in virulence modulation after nanoparticle stimulation. the modulation of bacterial virulence and molecular signaling by functional nanoparticles utilized in subinhibitory amounts offer valuable perspectives to develop personalized antimicrobial approaches based on molecular communication control that clearly modulate pathogenicity and progression of the infectious process. p-08.02.5-016 specimen processing and handling for plasma ammonia measurement b. sarac ßligil 1,2 , s. abus ßo glu 2 , f. aky€ urek 2 , b. € ozt€ urk 2 1 selc ßuk medical school, konya, 2 biochemistry department, selcuk university faculty of medicine, konya, turkey objectives: ammonia requires special processing and handling conditions due to its' unstability. in this study, our aim to investigate a preanalytical factor (delayed analyze) affecting plasma ammonia measurement. design and methods: blood samples were obtained from 20 healthy volunteers. for determining different handling and storage conditions, the following protocols were applied: first protocol (a) transportation on ice and separation (centrifugation at 0°c) within 15 minutes of collection and analyze immediately. second protocol (b) transportation on ice and separation (centrifugation at 4°c) within 30 minutes and analyse refrigerated 2-8°c 2 hours (a1, b1) and 4 hours (a2, b2). all plasma ammonia levels was analyzed enzimatic glutamate dehiydrogenase methods by abbott architect c 8000 clinical chemistry analyzer. results: there were statistically alterations in all protocols compared to first protocol. prolonged centrifugation time for plasma ammonia lead to have higher results (38.6 versus 29.75 lg/dl, p < 0.001). in all protocols including a1, a2, b1, b2 also cause an elevation in plasma ammonia results (35.7, 37.4, 39.5 and 41.2; p = 0.032, p < 0.001, p < 0.001, p < 0.001, respectively). conclusions: ammonia concentration in the blood sample increases over time due to high concentrations in cells as erythrocyte or platelets (three fold). blood samples collected for ammonia determination should be stored on ice, and measured immediately. the wnt/b-catenin signaling pathway has been considered to be a factor in the development and progression of colorectal cancer. many studies have demonstrated that the presence of mutations or polymorphisms in ctnnb1(gene encoding b-catenin; mostly mutations in exon 3) can lead to aberrant activation of wnt/bcatenin signaling at the onset of various types of malignancies, including colorectal cancer. the aim of our study was to assess ctnnb1alteration in the patients with colorectal cancer and compared their tumor and normal tissues. a total of 30 paraffin-embedded colorectal tumor specimens were obtained from department of pathology in cerrahpasa medical faculty. also a total 30 paraffin-embedded normal tissue was used from same cases as a control group. ten-micrometer-thick tissue sections were placed on a glass slide and stained with he. then the tissue sections were dehydrated in graded ethanol solutions and dried without a cover glass. dna was extracted from the tissues with 100 ll of extraction buffer at 55°c over night. the tubes were boiled for 10 min to inactivate the proteinase k. ctnnb1 exon 3 was amplified by pcr. sscp is used to observe any difference between the 2 groups. genomic dna was isolated as described above. 10 ll of each pcr products mixed with 10 ll denaturating buffer and were denatured by heating at 95°c for 10 minutes in, and then were rapidly cooled on ice. the denatured pcr samples are run on 12% acrylamide/ bis gel in 0.59 tbe buffer for 3.5 hours at 200 v at room temperature with water cooling system. the gel was stained with silver staining method. migration and adhesion involve continuous modulation of cell motility, beta-catenin play major roles. beta-catenin gene alterations frequency range between 0 and 16% in colorectal cancer according to the different published studies. in our study no significant differences were found in the ctnnb1 exon 3 between the tumor group and normal groups. p-08.02.5-018 theranostic approach in agressive recurrent meningiomafirst experience in turkey m. o. demirkol 1 , b. uc ßar 2 1 koc ß university, istanbul, 2 american hospital, istanbul, turkey meningiomas arise from the meningothelial cells of the arachnoid membranes of the leptomeninx, which are attached to the inner layer of the dura mater. meningiomas can be classified into three grades (i-iii): grade i meningiomas which are benign, exhibit slow growth; grade ii (atypical) and grade iii (anaplastic) meningiomas, which have a much more aggressive clinical behaviour. meningiomas express non-steroid hormones, including somatostatin. in the brain, somatostatin-a cyclic tetradecapeptide neuropeptide-is believed to act as a neurotransmitter and neuromodulator. somatostatin performs its physiological functions by binding to specific receptors (sstri-sstrv). sstr2 exhibit high affinity for octreotate (tate). tate is a polar, watersoluble peptide that does not penetrate the intact bbb (brain-blood barrier). pet and scintigraphic imagings can only demonstrate somatostatin receptor positive intracranial lesions if the bbb is disrupted. in this aim, dotatate (dota-dphe1, tyr3-octreotate, tate) has been labelled with the positron emitter 68 ga and the beta and gama emitter 177 lu. in this case, we conducted a study to evaluate peptide receptor radionuclide therapy (prrt) planning based on pet/ct imaging of meningioma in the department of nuclear medicine and molecular imaging at the amerikan hospital. [ 68 ga] dota 0 -tyr 3 -oc-pet/ct has been established as the imaging modality of choice for the diagnosis and management of patient with skull-base malign meningioma (rapid progress -to 41 9 48 9 52 mm. from 31 9 37 9 35 mm. in 20 d.-after fifth operation). due to its high sstr2 selectivity, [ 177 lu]-tate showed significantly lower uptake/dose delivered to normal tissues, gatate-pet represents the imaging strategy of choice for an accurate assessment of sstr2 expression levels. although some studies have not shown a clear advantage over pet/ct, there is some evidence that it will have an advantage in selected body sites such as the head and neck, liver, and the pelvis. p-08.02.5-019 cardiovascular diseases can be treated by using 'tetr-odd-vp16' and 'hre' hypoxia inducible systems a. celik 1 , t. kaya 2 , s. cigdem 1 , m. g€ und€ uz 1 1 department of medical genetic, turgut ozal university, ankara, 2 faculty of medicine, turgut ozal university, ankara, turkey ischemia is an insufficient supply of blood to a tissue or organ, usually due to a blocked artery by a blood clot. up to now, the number one cause of death worldwide is caused by ischemia and related conditions such as heart attack or stroke. hif-1a is a transcription activator that functions as a master regulator of oxygen homeostasis. hif-1a protein levels increase under hypoxic conditions as a result of decreased o2-dependent prolyl-hydroxylation, ubiqutination and degradation. we aimed to break up clots in blood vessels and to prevent damage caused by ischemia by using hypoxia inducible systems. we added oxygen dependent degredation domain (odd) of hif1a between and in front of tetr dna binding domain and vp16 transactivation domain, so that tetr-odd-vp16 or odd-tetr-vp16 could activate transcription of tissue plasminogen activator (tpa) controlled by tetracycline response element (tre), in a hif1a independent manner. in addition, we also designed tissue plasminogen activator (tpa) under control of hypoxia response element (hre) of hif1a target genes. western blotting and immunofluorescence assay results showed the expression and nuclear localization of tetr-odd-vp16 and odd-tetr-vp16 constructs under hypoxic conditions, but not normoxic. in addition, using fluorometric reporter systems and tpa enzymatic assay we proved functionality of these constructs under hypoxic conditions. final apporoach to our project is predicting kinetic enzymatic activity of tpa during break up blood clots by using matlab. the results of the present investigation showed, the developed hypoxia responsible systems that can be engineered into endothelial cells to prevent ischemia related cardiovascular diseases. p-08.02.5-020 osteogenic potential assessment of some original scaffolds with magnetic properties new advances in bone tissue engineering demand the development of materials that can not only replace bone, but also regenerate the damaged tissue based on external or even internal stimulus. magnetic materials inside bone scaffolds are known to be a promoting factor for bone healing especially when the therapy is accompanied by application of external magnetic stimulation. based on a recent report, the presence of iron oxide in hydroxyapatite can improve the radio opacity and osteoblast proliferation. in this view, this study focuses on the development of silk fibroin-magnetite biocomposites for potential uses in bone tissue bioenegineering. such novel composites possess good mechanical properties, biocompatibility and biomineralization potential by in vitro tests and could become smart arhiectures, able to stimulate bone regeneration. a new culture model was developed by exposing a 3d cell/ scaffold bioconstruct to a continuous magnetic field during 3 weeks of osteogenic induction. in this view, mc3t3-e1 murine osteoblastes progenitor cells were seeded inside the novel silk fibroin-magnetite biocomposites and subjected to osteogenesys in a magnetic field during 21 days. osteogenic specific markers were evaluated every week in the presence and absence of the field. our results showed that the osteogenic marker's expression started earlier when mc3t3-e1 cells were exposed to the magnetic field. consequently, in our experimental model, the magnetic field had a benefic effect on the osteogenic differentiation process as mc3t3-e1 cells differentiated more efficiently in its presence. these results suggest that the bone healing process could be improved in the presence of a magnetic field. nevertheless, further in vivo studies on animal model should be employed for validation. p-08.02.5-021 impact of physical activity performed on different times of day on cardiac and skeletal muscle damage in trained and untrained male subjects s. algul, m. kara, o. ozcelik y€ uz€ unc€ u yil university, van, turkey introduction: physical activity elevates creatine kinase (ck) and creatine kinase myocardial band (ck-mb) levels which have been considered to be an indirect marker of skeletal and cardiac muscles damage. purpose: impact of physical activity performed on different times of day on serum levels of ck and ck-mb were investigated in trained and untrained male subjects. materials and methods: trained (n = 10, 18.3 ae 0.1 yr, 61.4 ae 2.3 kg) and untrained (n = 10, 18.6 ae 0.1 yr, 63.2 ae 2.4 kg) subjects performed three soccer matches (60 min) in field (30 m versus 50 m) in morning (m), afternoon (a) and at night (n) on separate days. the study protocol was approved by the local ethics committee. venous blood samples were taken at onset and at end of match. serum ck and ck-mb levels are measured using autoanalyser. data are expressed as mean ae s.e., compared by wilcoxon-signed rank and mann-whitney u-tests. p < 0.05 was accepted as statistically significant. results: ck and ck-mb levels increased in three matches in both groups (p < 0.05). importantly, there were significant increases in ck-mb levels in a and n exercises compared to m exercise (p < 0.05) in trained (10.6 ae 1.6 u/l versus 14.2 ae 1.9 u/l, 35% (m) 9.7 ae 1.3 u/l versus 18 ae 2.6 u/l, 66% (a) 9.5 ae 1.1 u/l versus 16.3 ae 2.0 u/l, 103% (n) and also untrained groups (8.6 ae 0.8 u/l versus 11.5 ae 0.8 u/l, 42% (m), 7.3 ae 0.5 u/l versus 11.9 ae 0.9 u/l, 85% (a) 7.9 ae 0.9 u/l versus 13.8 ae 0.9 u/l, 76% (n). discussion: increased metabolic stress or muscle damage during physical exercise elevate serum ck and ck-mb levels. however, higher percentage of increase in ck-mb levels in a and n exercise may reflects additional increases in cardiac muscle stress despite the similar skeletal muscle stress as indicated by ck levels. conclusion: considering the observation of higher percentage increase in ck-mb levels in untrained and also trained subjects, the caution should be taken while performing an exercise in a and n time especially in subjects who has cardiac weakness. p-08.02.5-022 regional assessment of hematological and discrimination indices of complete blood count for beta-thalassemia screening beta-thalassemia is one of the most common genetic abnormality causing health problems worldwide. blood count and film of beta thalassemia trait and iron deficiency anemia have similar features. therefore, several simple screening indices have been developed for differentiating between these diseases. it was to asaimed to assess the hematological parameters and discrimination indices in patients with betathalassemia trait who admitted to our hospital. the parameters of complete blood count (cbc) in 141 subjects (51 males and 90 females) diagnosed by mutational analysis (pcr, gene amplification, dna sequencing) between 2013 and 2015, were retrospectively screened and the thalassemia status of patients was assessed in terms of discrimination indices (eng-land&fraser (ef), green&king (gk), mentzer (m), ricerca (r), shine&lal (s-l), srivastava (s), ehsani and sirdah). the percentages of being above the cut off value were detected by ef 29.72%, gk 30.4%, m 26.35%, r 6.75%, s-l 8.78%, s 35.13%, ehsani 25.67% and sirdah 29.72%. the percentages of falsely negatives for the indices of ricerca and shine&lal were lower than others. morever, when the first three common mutations of our study were considered, 5 out of 23, 7 out of 21 and 4 out of 21 patients were up to the cut off values in terms of e&f, g&k, m, s, ehsani and sirdah indices for ivs-i-110 (g>a), ivsii-1(g>a) and heterozygous codon 8 deletion (-aa), respectively. the molecular diagnosis and prenatal detection for families at risk is important because of the difficulties of treatment in this disease. however, the use of discrimination indices may be valuable for distinguishing of thalassemia trait from iron defiency anemia when the equipment of molecular diagnosis are limited. in our study, ricerca and shine&lal had lower falsely negative results than others. nevertheless, further studies to detect diagnostic perfomance of discriminant indices should be conducted. p-08.02.5-023 novel therapeutic agents in the development of effective drug combinations to treat glioma s. avdieiev 1 , l. gera 2 , r. hodges 2 1 institute of molecular biology and genetics, kyiv, ukraine, 2 university of colorado, aurora, co, united states glial tumors are driven by multiple molecular aberrations that cannot be controlled by a single targeted agent. so, it is possible to expect that the combined multitarget anti-cancer therapy aimed simultaneously at different elements of tumor formation mechanisms will be more effective and will promote the extension of patients' life. to find out which drug combinations will enable the development of therapeutic regimens with improved effectiveness and decreased toxicity, the cytotoxic effects of several bradykinin antagonists (ba) were analyzed for different glioblastoma (gb) cell lines. among all the ba under investigation, bkm-570 appeared to be the most effective, with ic50 values of 4 lm and 3.3 lm in rat glioma c6 and human glioblastoma u251 cell lines, respectively. bkm-570 suppressed erk1/2 and akt1 phosphorylation in u251 cells. temozolomide (tmz), the firstline anti-gliomic drug used in clinics, has only a temporary positive effect and severe side effects in gb patients. we showed that the combination of bkm-570 and tmz led to significant potentiation of tmz cytotoxicity at sub-therapeutic concentrations. recombinant proteins with cytotoxic properties are promising agents for complex therapeutic applications. we revealed that the glioma-associated protein chi3l2 inhibited the viability of u251 cells more effectively than tmz. furthermore, the combination of chi3l2 and bkm-570 resulted in an additive cytotoxic effect. chi3l2-mediated decrease of cell viability was associated with a g1/s transition arrest. chi3l2 provoked the dramatic reduction of prb phosphorylation and a significant decrease of cyclin d1 expression, as well as a substantial increase in p53 level. in addition to the accumulation of p53, we observed the upregulation of cdk inhibitor p21. therefore, g1/s arrest in chi3l2-treated cells could be realized via activation of prb, downregulation of cyclin d, and activation of p53. harmful hereditary mutations in brca1 and brca2 are one of the most important risk factors of breast cancer. the aim of this study is to determine the mutations which are associated with breast cancer in people which is diagnosed breast cancer and/or have breast cancer-diagnosed family members by sanger sequencing and thus provide predictive and prognostic utility. our ongoing study is present the genetic variations in brca1 and brca2 genes in breast cancer-diagnosed 12 patients, that one of them is male, and 1 person yet healthy whose brca2 was sequenced by sanger sequencing. the data were analyzed by using seqscape software 2.6 and detected variations were compared with literature. in brca1, we determined 16 different benign genetic variations and 1 variation with unknown significance and 1 variation which has not in literature. in brca2 gene of 12 patient and 1 healthy person,14 benign variations,2 variations with unknown significance,7 variations which has not in literature and 1 mutation were determined. this mutation is c.3189-3192delgtca and is located in occr. c.9257-74a>c variation in brca2 gene, was determined in only male patient.c.32t>a variation in exon2 of brca2, was observed in only the youngest patient who has no family member with breast cancer and healthy person. while this variation takes place in literature as variation with 'uncertain clinical significance', an in silico program mutation taster speculated as 'disease causing' for this variation. also, almost all of variations with 'unknown significance' literature knowledge were determined in only one and different cases. this situation increases the possibility of being pathogenic of this variations. the our findings until now can contribute to variations with uncertain clinical significance in the literature. also the variations that have not in the literature but we suggest the possible relation with breast cancer as an estimate may be added to literature by expanding the study. p-08.02.5-025 inhibition of the recombinant human butyrylcholinesterase with paraoxon and coumarin analog of soman organophosphorus compounds (op) represent a class of extremely toxic chemicals that are used as warfare agents. uncontrolled utilization of op is highly dangerous due to their high potential to be efficient poisons in terrorist attacks. current therapy of op poisoning include intravenous administration of atropine and acetylcholine reactivators however, it does not completely eliminate brain damage effects. alternative experimental therapy against op poisoning is utilization of bioscavengers that irreversibly react with op and rapidly inactivate them. recombinant human butyrylcholinesterase (rhbche) is one of the most promising candidates as bioscavenger due to its pharmacokinetic characteristics and broad spectrum of op neutralizing activity. here we investigated in vitro inhibition of rhbche with two model oppesticide paraoxon (pox) and coumarin analog of soman (gd c ). both op lead to rapid and irreversible inactivation of rhbche that was monitored using ellman assay and fluorescence measurements. bimolecular inhibition rate constants dramatically differ between pox and gd c that could be explained by steric hindrance in soman analog. the next steps forward creation of catalytic bioscavengers based on rhbche should be done based on mechanisms of op-rhbche interactions. this work was performed in frame of grant rfme-fi60414x0069. non-hodgkin lymphomas (nhls) represent a heterogeneous group of malignancies that arise from the lymphoid system. at the present time exist a lot of drugs for the nhls therapy, but mostly all of them are unsafe and there is no consensus regarding the best treatment protocol. to increase the efficacy and safety of therapeutic b-lymphocyte depletion in lymphomas and leukemia's it would be preferable to induce the death of pathological b cells without affecting normal b cells to prevent side effects. similar to other types of cancer, nhls arise by a multistep accumulation of genetic aberrations that induce a selective growth advantage of the malignant clone. all b-cells of organism have a unique cell surface markerantigen b-cell receptor (bcr). we generate novel approach for personalized non-hodgkin lymphomas therapy based on peptide specific to malignant cells surface receptor. for this purpose we designed new lentiviral peptide library screening technique based on fluorescent reporter cells system. herein 7aa peptide library was used for screening of nhl's malignant receptor agonist. patients' lymph nodes biopsy samples mrna was used as a source of malignant bcr nucleotide sequence. variable domain of the lymphoma bcr was used for chimeric receptor generation, where bcr vh/vl part responsible for agonist recognition and bottom part of receptor was retranslate signal to the reporter gene. in this embodiment of the method, very large numbers of candidate 7aa peptides expressing lentivirus and eukaryotic reporter cells are packaged together in a format where each is capable of replication, thereby forging a direct link between genotype and phenotype. after four rounds of screening we discover peptides specifically interacted with malignant bcr's. selected peptide ligands were fused with chimeric antigen receptor for expression on t-cells. modified tcells selectively eliminate nhl's malignant cells ex vivo. this work was supported by grant rfmefi60714x0061. introduction: pesticides are used to prevent damage of unwanted insects, rodents, plant, moss and other pests. excessive use of pesticides may cause adverse effects in animals and humans. chlorpyrifosethylene (cpe) is an organophosphate pesticide, used in many agricultural products such as figs, cherries, olives worldwide; caused acute poisoning and chronically oxidative stress. rosadamascena mill (rosaceae) is a rose, used for production of rosewater (rw) and rose oil worldwide. rosaceae products are consumed in food and cosmetic industries. materials and methods: in this study, we investigated that cpe and rw effects on kidney tissues of rats. this study was included 32 adult male rats, divided into 4 groups. each group included 8 rats. i.group: control (regular feed), ii.group: cpe (0.3 mg/kg/day), iii.group: rw (100 mg/kg/day) and iv.group: cpe (0.3 mg/kg/day) + rw (100 mg/kg/day). following 15 days, kidneys were taken after sacrificed. analyzes were performed that malondialdehyde (mda), nitric oxide (no) as oxidant; superoxide dismutase (sod), glutathione reductase (gr) as antioxidant parameters. results: as compared with control, mda and no levels in cpe were a significant increase was determined (p conclusion: cpe is shown that significant increase on oxidant parameters, but significant decrease on antioxidant parameters. rw occurs opposite situation. similarly results of cpe, rw + cpe increased oxidant parameters, but decreased antioxidant parameters. these changes are lower than only cpe. these results showed that positive effects both rw and rw + cpe increasing on antioxidant parameters, also decreasing on oxidant parameters. we provide the comparative analysis of the reduced glutathione (gsh), reactive oxygen species (ros), a-tocopherol levels, an intracellular labile zn 2+ pool and esterase activity of red blood cells of patients with diagnosed components of the metabolic syndrome (ms)arterial hypertension and diabetes mellitus type ii (ah + dm + ). as the comparison groups were selected patients with one diagnosed component of msarterial hypertension (ah + dm à ) or without any diagnosed component of ms (ah -dm -). patients of all investigated groups were at the hospital treatment with a diagnosis coronary heart disease (chd) ii degree. human blood was obtained from normal donors and patients with chd ii stage. cytosolic esterase activity was assessed using calcein-am test. ros level was evaluated using cm-h 2 dcf-da. gsh level was estimated using lowry method. an intracellular labile zn 2+ pool was assessed using fluozin-3-am. investigations were performed on the specord m40, hplc system lc-20 prominence (shimadzu) and facscantoii (bd). a significant decrease of the intracellular level of labile zn 2+ in erythrocytes of patients with ah + dm + compare with ah + dm à and ah à dm à was shown. this fact confirms our assumption concerning the important role of zinc homeostasis in the etiopathogenesis of diabetes mellitus type ii. a direct relationship between the intracellular zn 2+ level modification and erythrocytes esterase activity of patients with chd ii degree was observed. moreover, in ah + dm + group of patients this relation was more marked. the unidirectional alteration in the erythrocytes redox state (gsh and a-tocopherol levels reduction, ros formation activation) was revealed at the whole of investigated chd patients groups (ah + dm + , ah + dm à , ah à dm à ). however, the pathological erythrocytes response on in vitro action of the different antioxidants (n-acetylcysteine, ascorbic acid, a-tocopherol, quercetin) had a diverse character that can be a significant test under antioxidant therapy prescription. it is known that long-term social isolation and the disorder of natural circadian rhythm is considered an important stress factors, which cause a variety of metabolic and mental disorders. it should be noted that the impact of the stresses takes up a larger area, according to, review of the action of their mechanism is one of the topical issues of modern science. it is estimated that as a result of stress the metabolic processes change in the organizm. because of this, we've studied the functionality of the antioxidant system in laboratory rat heart muscle cells and blood under psycho-emotional stress. it was found that quantitative changes of nitric oxide (no) was initiated the process of lpo, which caused oxidative stress in the cells and decreased antioxidant enzymes activity, such as catalase,sod, gpx and gr. the results suggested that psycho-emotional stress was accompanied by oxidative stress, causing a reduction in the intensity of energy metabolism in cardiac muscle cells, which was further strengthened by the fact that the activity of the enzymes involved in atp synthesis in mitochondria was reduced. also, we've studied exogenous creatine positive and negative affects on energy metabolism and blood lipid spectrum. based on this, we proposed that psychological stress is one of the factors contributing to the development of various cardiac diseases. the importance of free radical lipid transformations, which differ from the peroxidation processes, was pointed out for the first time in our laboratory studies. we have found that ros can induce free radical destruction processes of sphingolipids with c-c-bond cleavage [lipids, 2011, 46:271-276; lipid insights, 8, 1-9] . in case of acylated sphingolipids, they can undergo decomposition with c-c-bond cleavage upon direct uv irradiation [photochem. photobiol., 2012, 88:899-903] . it was of interest to establish the possibility of photosensitized decomposition reactions of not acylated sphingolipids, which do not absorb an ultraviolet. in this work we studied photosensitized reactions of sphingosines containing a free amino group, and low molecular compounds, which simulate their structure, such as aminoalcohols (serinol). as photosensitizers, the salts of transition metals, hydrogen peroxide, and acetone were used. oxygen was removed by bubbling with argon to reduce the probability of side reactions during photolysis of sphingolipids, such as oxidation processes (including oxygen reactions with alkyl radicals). we have shown that the action of uv-radiation on aminoalcohols and sphingosines in aqueous solutions in the presence of photosensitizers induces their destruction with c-c bond rupture. the main carbonyl product of sphingosines free radical destruction was an unsaturated aldehyde -2-hexadecanal. it was found, that 2-hexadecenal possesses a wide spectrum of biological activity: it promotes reorganization of the cell cytoskeleton and modifies the redox state of the cells [febs journal 282 (suppl. 1), abstracts: mem. biol. s5, lipid signaling & dynamics, p. 235.] . the results of this study can expand the frontier of research regarding free radical lipid damage, which could contribute to a better understanding of the origins of diseases associated with the activation of free radical processes in living organisms. structural basis for the 14-3-3 proteindependent inhibition of apoptosis signalregulating kinase 1 protein kinase ask1 (apoptosis signal-regulating kinase 1) is a member of the mitogen-activated protein kinase kinase kinase (map3k) family that plays a crucial role in immune and stress responses. the activity of ask1 is regulated through homo-oligomerization and interaction with other proteins including the 14-3-3 protein which binds to the phosphorylated motif located at the c-terminus of the kinase domain of ask1 and suppresses its catalytic activity through unknown mechanism. under stress conditions, ask1 is dephosphorylated at ser966 and the 14-3-3 protein dissociates. this dissociation is then one of the factors that lead to the activation of ask1. we performed low-resolution structural analysis of the kinase domain of ask1 (ask1-cd) bound to 14-3-3 using chemical cross-linking, analytical ultracentrifugation and small angle x-ray scattering. the low-resolution structural analysis shows that ask1-cd binds to the 14-3-3 protein in two to two stoichiometry through a small binding interface involving surface of 14-3-3 outside its central channel and several regions from the c-lobe of ask1-cd. the complex is dynamic and conformationally heterogeneous. phosphorus nmr and time-resolved fluorescence measurements, together with low-resolution structural analysis, indicate that binding of ask1-cd to 14-3-3 modulates conformation of ask1's activation segment. these results suggest that the 14-3-3 binding suppresses the catalytic activity of ask1 through direct structural modulation of its activation segment. our study provides new insight into the interaction between the kinase domain of ask1 and 14-3-3 and offers a plausible structural explanation for the 14-3-3 protein-dependent inhibition of ask1 kinase activity. introduction: thymoquinone (2-methyl-5-isopropyl-1,4-benzoquinone, tq) exerts a great antitumor activity against different types of cancer cells. a growing body of evidence indicates that reactive oxygen species (ros) generation followed by modulation of the akt and mapk pathways is a general mechanisms underlying the tq antitumor action. however, the data of tq effects on the mapk pathway are conflicting. to date, the activation or inhibition of the mapk protein family seems to depend on the cell type and on the tq concentration used. in order to elucidate the antitumor potential of tq against gliomas and the underlying molecular mechanism, tq influence on c6 rat glioma cells functioning was studied. results: it has been shown that the cultivation of c6 cells with tq in concentrations of 10 -100 lm during 24 hours strongly inhibits cell proliferation and induces cell death with id 50 of 60 lm. at the same time, tq induces ros generation and intracellular gsh depletion in a dose-dependent manner, that is followed by the mitochondrial potential decrease. interestingly, ros production has only cytoplasmic, but not mitochondrial origin in cells challenged with tq at the concentrations up to 100 lm. two-electron reduction of tq by dt-diaphorase attenuates tq anticancer efficiency whereby inhibition of dt-diaphorase by dicumarol increases tq-induced c6 cell death by 25 %. we analyzed mapk pathways involvement in c6 cells growth inhibition at tq treatment. it has been shown that inhibition of the erk pathway by pd98059 and jnk pathway by sp600125 does not influence on tq-induced effects. on the contrary, the specific phosphoinositide-3-kinase (pi3k) inhibitor (ly294002) abrogates tq-induced growth arrest. conclusion: antitumor effects of thymoquinone on c6 glioma cells is a result of ros generation and intracellular gsh depletion, that is followed by mitochondria disfunction, and growth arrest via pi3k pathway. assessment of oxidative stress and antioxidant defense activity parameters in patients with hiv-infection is of great importance, especially for hiv-positive women of reproductive age planning to have children. data of 53 women of reproductive age with hiv-infection analyzed: patients with hiv-monoinfection (n = 26) and patients with co-infection (hiv and hepatitis b and / or c) (n = 27). as a control we used the data of healthy women (n = 28). serum and hemolysate used as material for the study. lpo-aod products were determined by spectrophotometric and fluorometric methods. average value of initial lpo products -diene conjugates was significantly increased in the group with hiv-co-infected compared to control (1.57 times; p = 0.001) and group with hivmonoinfection (1.4-fold; p = 0.027). the level of secondary products -ketodienes and conjugated trienes increased in patients of both groups compared to control (2.16 times (p = 0.0002) and 2.43-fold (p < 0.0001), respectively). at the same time isolated double bonds and tba-active products content showed no significant changes (p > 0, 05). total antioxidant activity parameter decreased 1.28 fold (p = 0.0273) in the group with hiv-monoinfection compared to control. decrease in activity of the primary antioxidant enzyme -superoxide dismutase (p = 0.0077, compared to the control and p = 0.0035, compared with the group with hiv-monoinfection) and the level of a-tocopherol (1.22-fold to control and 1.25 fold to hiv-monoinfection) was detected in the group with hiv-coinfection. 1.29-fold higher content of retinol in hiv-coinfection group (compared to the control) revealed. in women with hiv-coinfection the oxidative stress was significantly higher than in women with hiv-monoinfection. suggested to include antioxidant supplements in the complex pathogenetic therapy in women with hiv-coinfection (hiv and hepatitis b and / or c), which will contribute to women's ability to bear children. p-09.04.4-008 acute different doses of malathion induce cholinesterase inhibition, glucogenic enzymes and histopathological change in rat liver malathion, which is an organophosphorus compound, is a widely used pesticide all over the world. despite its benefits, malathion has many toxic effects on many tissues including liver. we designed to evaluate the acute different doses of malathion on cholinesterase (che) inhibition, gluconeogenic enzymes and histopathological change of rat liver. for this purpose 4 groups were formed. rats in group 1 served as control group animals which were only given corn oil. group 2, group 3 and group 4 were administered 100, 200, 400 mg/kg of malathion, respectively, dissolved in corn oil by oral gavage. the rats were sacrificed after 24 hours following administration and the livers of rats were removed. the liver che, alanine aminotransferase (alt), aspartate aminotransferase (ast) and lactate dehydrogenase (ldh) were studied using autoanalyzer. histopathological investigation was performed using microscope. the liver che activities of group 2, group 3 and group 4 were inhibition percentage of 53%, 43%, and 51% respectively, comparison of group 1's che activity. the liver alt, ast and ldh increased in group 3 and group 4 compare to group 1 and group 2 (p < 0.013). we also observed that there was vacuolar and hydropic degeneration in liver of group 4. according to our result, acute administrations of malathion result in hepatotoxic effects with increasing doses. background and aims: an imbalance between free radicals and antioxidants is closely linked to the onset of an acute myocardial infarction (ami). the aim of this study was to investigate the antioxidant status and the lipid peroxidation in patients admitted to hospital immediately after ami. methods: the study population comprised 19 patients with ami and 14 healthy subjects. patients that had an acute myocardial infarction (ami) less than 72 hours since onset were selected for this study. antioxidant status was assessed by lactonase activity of paraoxonase (pon1 dhc), trolox equivalent antioxidant capacity (teac) and plasma uric acid level. malondialdehyde was used as marker of lipid peroxidation. results: compare with the control group, the levels of mda and pon1 dhc were significantly higher in group with ami (p < 0.005, respectively p < 0.001). elevated levels of mda (p < 0.005) were found in patients with ami compare with the control subjects. ami patients had also statistically significant reduced levels of teac (p < 0.005) comparative with healthy subjects. no statistically significance was found for plasma uric acid level in subjects from our study. conclusion: a high lipid peroxidation correlated with a decreased teac activity suggest an exacerbated oxidative stress in subjects admitted to hospital immediately after an ami. p-09.04.4-010 dealing with copper toxicity: new insights into copper detoxification in yeast copper (cu), an essential metal, is a double-edged sword, as its essential nature is counterbalanced by the toxic effect that it can exert on cells when not properly controlled. as such, organisms have evolved defence mechanisms against cu toxicity, and in the yeast saccharomyces cerevisiae, the transcription factor ace1 orchestrates several of those mechanisms, by activating cu-detoxification genes. in s. cerevisiae iron (fe) uptake is partially dependent on cu, as the membrane multicopper-oxidase fet3, which is part of the high-affinity iron uptake system, requires cu as a cofactor. aft1, the low iron-sensing transcription factor in yeast, is known to regulate the expression of fet3 gene. however, we found that a strain lacking fet3 is more sensitive to cu surplus conditions than a strain carrying the aft1 gene disrupted. this finding suggests that under such conditions another regulator came into play and controls fet3 gene expression. we next evaluated whether ace1 could be the alternative regulator of fet3 under cu excess. to test this hypothesis we first constructed the double mutant aft1ace1 and assayed its fitness under cu surplus. as expected, the double mutant is much more sensitive to cu than the single aft1 or ace1 mutants. we next monitored the expression of fet3 gene in cells lacking ace1, using yeast-one hybrid and qrt-pcr approaches. our data clearly indicates that fet3 expression is dependent on ace1 when cu is overly abundant. altogether our data unveil a novel mechanism of cu detoxification relying on the activation of fet3 by ace1 in an aft1independent way. experiments to understand the consequences of this regulation in terms of cu detoxification are currently being undertaken. in joint degeneracy, reactive oxygen species manifest their toxicity both through intrinsic reactivity and the inflammatory process activation, leading to cartilage dysfunctions, injuries of matrix proteins and cytokines stimulation. the study is focused on the identification of oxidative balance modulation (enzymes and oxygen free radicals) by a bioactive extract obtained from small sea fish. the cellular support was represented by the chondrocyte line chon-001 derived from human long bones (atcc ò crl-2846 tm ), that assure the reproducibility of a standardised biological system. the oxidative stress was induced through stimulation with il1b, a cytokine-factor that promotes the protein catabolism and also with tnfa, the initiator of pro-inflammatory activation, combined with pma, the activator of protein-kinase c, triggering of oxygen reactive species generating cascades. the antioxidant effect was compared with known antioxidants: vitamin c, x3 fatty acid, n-acetyl -cysteine. the acellular antioxidant/antiradical screening was done using two complementary techniques for total antioxidant status evaluation and completed by measuring cellular catalase (cat) and superoxidedismutase (sod) activity, correlated with intracellular hydrogen-peroxide and superoxide anion monitoring through flow cytometry. the antioxidant properties of the bioactive extract proved in acellular systems are confirmed at cellular level by the involvement of the product in the enzymatic cascade cat -sod, potentiating the catalytic action of the enzymes, and by the decline of both intracellular reactive oxygen species (the hydrogen peroxide decrease with 50%, the superoxide anion is reduced with 31% compared with the stimulated control). the demonstrated antioxidant synergy assures a complete cellular protection induced by the small sea fish extract in human condrocytes. introduction: alzheimer's disease (ad) is a progressive neurodegenerative disorder characterized by memory loss, cognitive impairment. oxidative stress is a contributory factor in ad pathogenesis. glutathione (gsh) is the main antioxidant cellular defence. the ratio of gsh/gssg shows the redox status of gsh, and plays important role in maintaining intracellular redox homeostasis. the current study was carried out to determine oxidative dna damage and ratio of gsh/gssg which plays an important role in protection of target molecules from oxidation in the patients with ad. methods: the study subjects were consisted of patients with ad (n = 67) and age matched control group (n = 42) who were treated and followed in the cerrahpasa medical faculty hospital, department of neurology and department of internal medicine/ geriatrics. dna strand breaks and h 2 o 2 -induced dna damage were determined in lymphocyte dna with comet assay. the gsh and gssg levels in the erythrocyte lysates were measured by using a commercial spectrophotometric kit. the ratio of gsh/gssg were calculated. statistical analysis was performed with spss 22 software package. results: dna strand breaks and h 2 o 2 -induced dna damage were found to be higher (p = 0.000 for all), the ratio of gsh/gssg was found to be lower (p = 0.024) in the ad group than control group. there was no significant difference between male and female for dna strand breaks and h 2 o 2 -induced dna damage in the ad group, but ratio of gsh/gssg were higher in male when compared with female (p = 0.045). no significant difference was found between the men of ad group and men of the control group for gsh/gssg ratio whereas women of the ad have a lower gsh/gssg ration than those in the women of the control group (p = 0.009). conclusion: increased systemic oxidative dna damage and dna susceptibility to oxidation may be resulted from diminished gsh/gssg ratio in ad patients. this finding shows the importance of antioxidant support in ad management. p-09.04.4-013 validation of a liquid chromatography-tandem mass spectrometry method for the measurement of lipid peroxidation product 8iso-prostaglandin f2a in urine m. kant 1 , m. akıs ß 1 , h. _ is ßlekel 1,2 1 department of medical biochemistry, school of medicine, dokuz eylul university, izmir, 2 department of molecular medicine, school of medicine, dokuz eylul university, izmir turkey 8-iso-prostaglandin f 2a (8-iso-pgf 2a ) is a reliable indicator of lipid peroxidation resulting from oxidative lipid damage and is postulated as a gold standard biomarker for the evaluation of oxidative stress. the aim of this study was to validate non-invasive and highly accurate stable isotope dilution-multiple reaction monitoring liquid chromatography-tandem mass spectrometry (sid-mrm lc-ms/ms) method for identification and quantification of 8-iso-pgf 2a . twenty four hour urine samples were collected from healthy volunteers at medical school of dokuz eylul university for analytical performance studies. lc-ms/ms analyses were performed on conventional hplc coupled to a triple quadrupole ion trap mass spectrometer equipped with a turboionspray tm source. analyst software version 1.5 were used for data analyses. mrm transitions used were m/z? 250/164 for 8-iso-pgf 2a and m/z? 255/169 for 8-iso-pgf 2a-d 4 . analytical performance of the method was evaluated by linearity, selectivity, sensitivity, precision and accuracy studies using pure standards and samples extracted from urine of healthy volunteers. the linear calibration range for 8-iso-pgf 2a was determined as 20 ng/ml by using standards ranging from 0.25-50 ng/ml. analytical sensitivity of the method was determined by lod with s/n of 3 and loq with s/n of 10. lod and loq for 8iso-pgf 2a were 2.4 9 10 à2 and 38 9 10 à2 ng/ml, respectively. the assay stability and reproducibility were assessed by the precision and accuracy of intra-and interday measurements (n = 10). the intra-and interday cvs for 8-iso-pgf 2a were 4.5% and 1.2%, respectively. sid-mrm lc-ms/ms method for absolute quantification of 8-iso-pgf 2a was optimized and validated in our laboratory and therefore this highly accurate method can successfully be applied to clinical patient samples. p-09.04.4-015 synergistic anticancer effects of sulforaphane and cisplatin through the induction of apoptosis and autophagy following oxidative stress in malignant mesothelioma cells malignant mesothelioma is characterized by poor responsiveness to current chemotherapeutic drugs, usually owing to high resistance to apoptosis. here, we investigated chemosensitizing effects of phytochemical resveratrol, in combination with cisplatin, on malignant meothelioma cells. cell viability was evaluated using mtt assay. cell apoptosis was detected with dapi staining, caspase 3/7 activity assay and flow cytometry. cell cycle distributions, ros levels and mitochondrial membrane potential were determined using flow cytometry. the expression of cell cycle-, apoptosis-, and autophage-related proteins was measured with western blotting. the combination treatment of cisplatin and resveratrol (cis/res) synergistically induced apoptosis, as evidenced by typical cell morphological changes, the appearance of a sub-g 0 /g 1 peak, an increase in the annexin v(+) cells and the cleavage of caspase-3 and parp. cis/res increased ros production and depolarization of mitochondrial membrane potential with an increase in the bax/bcl-2 ratio. these changes were attenuated by pre-treatment with n-acetylcysteine, suggesting that cis/res induced apoptosis through oxidative mitochondrial damage. compared with msto-211h cells, cis/res was less efficient in killing h-2452 cells. h-2452 cells exhibited an enhanced autophagy to cis/res, as observed by an increase in viable cells exhibiting intense lysotracker red staining and up-regulation of beclin-1 and lc3a. inhibition of autophagy by bafilomycin a1 rendered cells more sensitive to cis/res-induced cytotoxicity and this was associated with induction of apoptosis. these data indicate that the increased resistance of h-2452 cells to cis/res is closely related to the activation of self-defensive autophagy, and provide the rationale for targeting the autophagy regulation in the treatment of malignant meothelioma. stress oxidative induced by chemotherapy with cyclophosphamide (cp) causes vulnerability in sperm and decline of fertility. this study was aimed to evaluate the role of ethyl pyruvate (ep) in the amelioration of fertility and growth of primitive embryo in animals that received cp. 24 adult male mice (6-8 weeks) were randomly divided into 3 groups: control group received normal saline (0. 2 ml/day, ip), cp group received cp (15 mg/kg/week,¬ ip), the cp+ep group received ep (40 mg/kg/day, ip) along with cp, were treated for 35 days. 8 mice from each group were arranged for evaluation of sperm quality and in vitro fertilization (ivf) too. afterward, the separated oocytes from 72 ovulation stimulated mice were conducted to evaluation of ivf and embryo development. the results revealed that cp caused notable decrease in percentage of fertilization in cp group, but administration of ep along with cp caused an increase in the percentage of fertilization in comparison to cp group. the percentage of the two cell zygotes in cp+ep group, and the percentage of blastocysts in control and cp+ep groups were higher than that in cp group (p < 0.05). results showed that the total number of arrested embryos in control and cp+ep groups was decreased in comparison to cp group (p < 0.05). the percentage of arrested embryos type i, ii, and iii in cp+ep group was decreased in comparison to cp group, but that decrease was significant only in types i and ii (p < 0.05). the average motility, viability, nucleus maturity and sperm morphology were decreased significantly in cp group in comparison with control and ep groups, whereas ep caused significant increase of these parameters (p < 0.05). also, the percentage of dna damage was increased significantly in cp group in comparison with control and ep groups (p < 0.05). the results of this study indicated that the ethyl pyruvate was able to suppress free radicals and enhance the ivf and quality of sperm in cp treated animals. malathion, which is a member of organophosphate chemical family, is used to control pests and is a widely used pesticide all over the world. however it is also known to be highly toxic on many tissues including pancreas. to test this we set 4 groups to administer a single dose of malathion dissolved in corn oil via oral gavage at the doses of 100 mg/kg (group 2), 200 mg/kg (group 3) and 400 mg/kg (group 4). only plain corn oil was given to control group (group 1). the rats were sacrificed 24 hours after administration of the chemical and the pancreases of rats were removed. in an attempt to evaluate the dose dependent response, we measured amylase and lipase activities, insulin, malondialdehyde (mda), total oxidant status (tos) levels in rat pancreases. all of the parameters were measured spectrophotometrically. we found that pancreas insulin levels significantly increased in group 4 compare to group 1, besides the insulin levels of group 3 and group 4 were significantly higher than group 2 (p < 0.013). pancreas amylase and lipase activities significantly decreased in group 3 and group 4 compare to group 1 and group 2 (p < 0.013). however, there was no significant change in pancreas mda and tos levels (p > 0.05). according to correlation analysis, when pancreas amylase levels declined, lipase levels were decreased simultaneously and there was a strong positive correlation between them (p < 0.01). in addition, when the comparison was evaluated as a binary, while pancreas amylase and lipase levels diminished, pancreas insulin levels increased and a strong negative correlation between them was found (p < 0.01). according to our result, acute administrations of malathion leads to alterations of insulin, amylase, and lipase levels with a dose dependent manner, while it does not to change oxidant status. the aim of this study is to evaluate the potential toxic effects of mancozeb on the stress biomarkers such as catalase (cat) activity, malondialdehyde (mda) level and protein levels in the brain tissue of zebrafish (danio rerio). mancozeb, is a synthetic fungicide contaminating aquatic environments as a potential toxic pollutants, was investigated in the present study for acute toxicity. zebrafish groups (m-low and m-high) were exposed to different doses of mancozeb (5 mg l-1 and 7.5 mg l-1) for 120 hours except the control group. catalase (cat) activity, malondialdehyde (mda) level and total protein levels were determined by spectrophotometer. the results showed that cat activity and mda levels were decreased in all experiment groups. protein levels were increased in experiment groups when compared to the control group. in conclusion, the changes in the cat activity and mda levels were time and as well as mancozeb dose-dependent. furthermore, the biochemical parameters of mancozeb exposure on zebrafish, showed that mancozeb has significant effect on the zebrafish and/or aquatic organisims. paracetamol (para), which is antipyretic and analgesic, is widely used around the world. paracetamol can be recommended for moderate or mild pains especially in pregnancy as an analgesic. it is known that, paracetamol can cause hepatotoxicity or nephrotoxicity. we were aimed that in this study to show potential hepatoprotective and nephroprotective effect of betaine against long term paracetamol using at therapeutic doses. it has been prepared 3 groups, control, para and para+-betain groups. paracetamol and betaine were administered by gavage to pregnant rats, from first day to the last day of pregnancy (aprox. 21 day). 2 ml physiological saline (%0.9 nacl solutions), 30 mg/kg/day paracetamol, 30 mg/kg/day paracetamol and 800 mg/kg/day betain was given by orally to control, para and para+betain groups respectively. the day of the birth, newborn rats anesthetized by ether and after decapitated. 8 newborn rat's liver and kidney tissues used for biochemical analysis [malondialdehyde (mda), reduced glutathione (gsh), nitric oxide (no) and paraoxonase-arylesterase (pon-are)] and 5 rat's liver and kidney tissues used for histological studies. we showed that, mda and no levels was significantly increased, while pon activities decreased. on the other hand gsh levels and are activities was decreased but these decline wasn't significant in the liver and kidney para group. these biochemical results showed hepatotoxicity and nephrotoxicity in neonates which can be formed in long term maternal paracetamol using at therapeutic doses. also our histological findings was support these biochemical results. we used betaine against potential hepatotoxic and nephrotoxic effect of long term maternal paracetamol using at therapeutic doses for neonates. betaine has antioxidant properties and also used as a methyl donor for transsulfuration reactions in the cell. biochemical and histological examinations showed that betaine protected the tissue injury relatively. p-09.04.4-020 lipid rafts are involved in modulation of ca 2+ responses induced by glutoxim and molixan in macrophages pharmacological analogues of oxidized glutathione (gssg) disulfide-containing drugs glutoxim ò (gssg disodium salt with dmetal nanoaddition, «pharma-vam», st. petersburg) and molixan ò (complex of glutoxim with inosine nucleoside) have found clinical application as a wide range immunomodulators and hemostimulators. however, the cellular and molecular mechanisms of these drugs action are still unclear. previously we showed for the first time that glutoxim and molixan cause biphasic intracellular ca 2+ concentration ([ca 2+ ] i ) increase due to ca 2+ mobilization from thapsigargin-sensitive ca 2+ stores and subsequent store-dependent ca 2+ entry in rat peritoneal macrophages. it is known that key proteins involved in ca 2+ signaling are localized in discrete plasma membrane lipid rafts domains. lipid rafts are cholesterol and sphingolipids enriched microdomains that function as unique signal transduction platforms. thus, the aim of the present work was to elucidate the possible involvement of lipid rafts in glutoxim and molixan effects on [ca 2+ ] i in macrophages. [ca 2+ ] i measurements were performed with fura-2am microfluorimetry. to examine the involvement of lipid rafts in the effect of gssg-based drugs on [ca 2+ ] i we used methyl-bcyclodextrin (mbcd), widely used to remove cholesterol from membranes, thus disrupting the lipid raft domains. we have shown for the first time that macrophage preincubation with 10 mm mbcd for 1 hours before molixan addition causes significant inhibition of both ca 2+ mobilization (on average, by 77.6 ae 9.2%) and subsequent ca 2+ entry (on average, by 82.3 ae 10.5%), induced by molixan. similar results were obtained in experiments with glutoxim. thus, we have demonstrated for the first time that mbcd significantly decreases both phases of glutoxim-or molixan-induced ca 2+ responses in macrophages. the results suggest that intact rafts are required to initiate complex signaling cascade activated by glutoxim or molixan and leading to [ca 2+ ] i increase in macrophages. plant-derived natural substances (phytochemicals) with potent pro-apoptotic activity towards cancer cells in vitro are considered as promising nutraceuticals in anticancer therapy. nevertheless, due to their relatively low bioavailability, administration of high doses of nutraceuticals that are not achievable in vivo seems to exert potentially negligible physiological effects in clinical trials. thus, there is a need for revealing novel cytophysiological effects of low doses of phytochemicals towards cancer cells. in the present study, we have considered thirty one nutraceuticals and selected four phytochemicals acting at low micromolar range (5 to 20 lm) against phenotypically different mcf-7, mda-mb-231 and sk-br-3 breast cancer cells, namely diosmin, sulforaphane, ursolic acid and betulinic acid. nutraceuticals inhibited cell proliferation and caused changes in the cell cycle that was accompanied by elevated levels of p53, p21, p16 and/or p27. apoptosis (annexin v staining, multicaspase and mitopotential assays) was observed exclusively when nutraceuticals were used at the concentration of 20 lm, whereas at the concentration of 5 lm, stress-induced premature senescence was noticed (sa-b-gal activity). nutraceuticals diminished the levels of glut-1 and selected glycolytic enzymes. nutraceuticals promoted oxidative and nitrosative stress as judged by increased production of total reactive oxygen species, total and mitochondrial superoxide, nitric oxide and protein carbonylation. nutraceuticals also stimulated dna single and double strand breaks that was accompanied by atm phosphorylation and to a lesser extent by histone h2ax phosphorylation and 53bp1 foci formation. taken together, several responses to nutraceutical treatment were observed in breast cancer cells that may reflect the heterogeneous nature of cancer cell populations. this study was supported by grant from the national science center, 2013/11/d/nz7/00939. nucleolus is thought to be a stress sensor and oxidative and ribotoxic stimuli may cause the inhibition of rrna synthesis by the inactivation of transcription factor tif-ia/rrn3 that is accompanied by the relocation of nucleolar proteins and p53-based cell cycle arrest and/or apoptosis. as nutraceutical-mediated modulation of nucleolar activity may be considered an attractive anticancer strategy, in the present study, we have investigated the effects of three selected nutraceuticals, namely sulforaphane, ursolic acid and betulinic acid on nucleolus state in three breast cancer cell lines (mcf-7, mda-mb-231 and sk-br-3). we found that low dose nutraceutical treatment resulted in p21-mediated inhibition of cell proliferation, a decrease in rrna synthesis, shifts in lamin a/c and b1 pools, changes in the nucleolar protein levels and their carbonylation, and changes in nucleolus size and number. breast cancer cells differed in erk activity that resulted in different patterns of erk activation/inhibition, phosphorylation status of s6 and autophagy induction upon nutraceutical stimulation. nutraceuticals also affected dna methylation parameters, namely the levels of dnmt1, dnmt3a and dnmt3b that resulted in both global dna hypo-and hypermethylation. taken together, after nutraceutical treatment, nucleolus-centered cellular response was revealed in breast cancer cells of different phenotypic characteristic that may be considered a potential target of anticancer therapy. this study was supported by grant from the national science center, 2013/11/d/nz7/00939. p-09.04.4-023 rate of apoptosis in human macrophages infected with leishmania tropica promastigotes infection of the cells with parasites or exposing cells to heat stress induces a cellular stress. in the present study human macrophages are infected with leishmania tropica promastigotes and exposed to heat stress. the measurement of cytoplasmic histone-associated dna-fragments was carried out using elisa technique. visualization of apoptotic cells was performed by the terminal deoxynucleotidyl transferase dutp nick end labeling staining method (tunel). degree of oxidative stress on cell is evaluated by measuring nitric oxide (no), malondialdehyde (mda), reducte glutathion (gsh) levels and superoxide dismutase (sod) activities. results of the elisa technique showed that infection of macrophages with promastigotes induced apoptosis rate significantly (p < 0.001), heat stress however decreased the rate of apoptosis in infected macrophages remarkably (p < 0.001). high levels of apoptosis rate in infected macrophages and drastic decdrease in apoptosis in heat subjected macrophages infected with promastigotes are confirmed by visualisation of apoptotic cells using tunel method. levels of glutathion (gsh) in infected macrophages decreased significantly (p < 0.05), while malondialdehyde (mda) levels increased notably (p < 0.05). however, no statistical significant alterations were detected in the nitric oxide (no) values and superoxide dismutase (sod) activities. results of the present study indicates that infection of human macrophages with leishmania tropica induces a cellular stress response, characterized by decreased values of gsh and increased levels of mda. increased rate of apoptosis in infected macrophages may be due to the increased cellular stress caused by leishmania tropica amastigotes. decreased rate of apoptosis measured in heat exposed macrophages infected with promastigotes indicates an extention in life span of macrophages. measurements of the parameters characterizing the redox and inflammatory processes in blood are essential for diagnosis and prognosis of type 2 diabetes mellitus (t2dm), but also for monitoring the effectiveness of medical treatments. along with other biochemical effects, hormonal imbalance leads to modified transport function of erythrocytes due to changes in enzyme systems involved in upholding of cellular homeostasis through a rapid degradation of altered proteins, being the second line of defense against the free radicals, which degrade and eliminate the damaged molecules. some of these enzymes are hemoglobin peroxidase (pa) and esterase (ea). the aim of this research study was to identify new parameters with a potential role of biochemical markers in t2dm like hemoglobin peroxidase and esterase activity from erythrocyte. our data showed that pathological processes involved in t2dm imply an increased enzymatic activity of pa (73.85%), which correlates with increased levels of ea (47.9%) and glycated hemoglobin (hba1c) (13.51%), compared with control group. the variables hba1c, pa and ea are correlated: the identified pearson correlation coefficients r = 0.637 and r = 0.573 respectively, have an associated probability of p < 0.001 and p < 0.006 a value that indicates a strong positive correlation between the dependent variables pa and ea and independent variable hba1c. based on these results we concluded that together with glycosylated hemoglobin assay, all the studied parameters can be successfully used as extra test for diabetes associated with oxidative stress and disorders in erythrocyte functions or blood rheology. the radioprotective effects of propolis and caffeic acid phenethyl ester on radiationinduced oxidative/nitrosative stress in brain tissue s. taysi 1 , e. demir 2 , k. cinar 3 1 gaziantep university, school of medicine, gaziantep, 2 haran university, sanliurfa, 3 department of neurosurgery, medical school, gaziantep, turkey head and neck cancer patients treated with radiotherapy suffer severe side effects during and following their treatment. efforts to decrease toxicity of irradiation to normal tissue, organs and cells have led to searching for cytoprotective agent. investigations for effective and non-toxic compounds with radioprotective capability led to increasing interest in antioxidant such as propolis and caffeic acid phenethyl ester (cape). the aim of this study was to evaluate the antioxidant and radioprotective effects of propolis and cape on radiation-induced oxidative/nitrosative stress in the brain tissue. fourty sprague-dawley rats were randomly divided into five groups. group 1 (irradiation (ir) + propolis) received total cranium irradiation and propolis was given orally through an orogastric tube daily. group 2 (ir+cape) received total cranium irradiation plus cape, was dissolved in dimethyl sulfoxide (dmso) just before giving to the rats, intraperitoneally (ip) every day. group 3 (ir) received 5 gy of gamma irradiation as a single dose to total cranium plus 1 ml saline daily. group 4 received daily plain dmso. group 5 received daily plain saline. at the end of the 10 day time period, xanthine oxidase (xo), nitric oxide synthase (nos) activities, nitric oxide (no•) and peroxynitrite (onoo -) levels were significantly higher in ir group compared to all other groups. in conclusion, the results suggest the radioprotective ability of propolis and cape involving prevention of radiation-induced oxidative/ nitrosative damage. p-09.04.4-027 role of leptin and adiponectin in obesityassociated oxidative stress e. becer 1 , a. c ß irakoglu 2 1 near east university, nicosia, cyprus, 2 istanbul university, istanbul, turkey objective: increased oxidative stress is one of the major characteristics of obesity and obesity-related complications. adipokines also induce the production of reactive oxygen species and generating oxidative stress in physiological and pathological conditions of obesity. the aim of this study was to determine the association of leptin and adiponectin levels with body mass index, lipid parameters and oxidative stress biomarkers in obesity. methods: the study included 150 obese and 120 non-obese subjects. plasma leptin and adiponectin levels (ng/ml) were measured using commercially available enzyme-linked immunosorbent assay kits. serum lipid, superoxide dismutase, malondialdehyde and antropometric parameters were measured. results: obese and non-obese subjects did not differ in age, while plasma glucose, total cholesterol, triglycerides, ldl cholesterol and leptin levels were significantly higher and mean hdl cholesterol and adiponectin levels were significantly lower in obese than non-obese subjects. the plasma leptin (p < 0.001) and adiponectin (p = 0.003) levels were significantly correlated with bmi in both obese and non-obese subjects. in obese subjects, leptin levels were significantly correlated with superoxide dismutase (p < 0.01) and malondialdehyde (p < 0.01). strikingly, adiponectin was significantly correlated with superoxide dismutase (p = 0.02) and malondialdehyde (p = 0.0012) levels in obese group. conclusion: our results suggest that leptin and adiponectin levels are associated with defective antioxidant status and increased lipid peroxidation which may have implications in the development of obesity related health problems. clinical trials of biologically active plant substances show a significant preventive effect in cancer, cardiovascular diseases and peptic ulcer disease in the form of both nutritional supplements and therapeutic intervention. anthocyanins contained in dark berries show great antioxidant potential, with the most important including a reduction in oxidative degradation of lipids or tyrosine oxidation by peroxynitrite. our previous studies of the antioxidant properties of extracts from vaccinium corymbosum, aronia melanocarpa and sambucus nigra, however, indicated that their activities largely depend on the method of extraction. while quantitative determination of anthocyanins pointed to a disproportionately larger content of anthocyanins in isolates from lyophylized berries, their scavenging activities against hydroxyl radicals was surprisingly the lowest. inflammatory processes, vascular damage, atherosclerosis and others are caused by oxidativenitrosative stress, so we tested their efficiency to scavenge no degradation products. we found that only purified extracts of lyophilized berries showed the most significant effects against no degradation products, with efficacy of around 50%. an extract from aronia showed greater than 60% efficiency, and a net ethanol extract from all the berries showed a 30% effect. cleaned ethanol extracts showed the lowest effects, while aronia initiated production at a concentration of 25 mg/l. conversely, all acetone extracts consistently initiated no degradation products. these findings are in complete contrast to their determined action against reactive oxygen species. in summary, it follows that a particularly adjusted lyophilized extract of the berries could be responsible for the increased biological activity of no and the observed biological and pharmacological activities of anthocyanins in circulatory disorders. the study was supported by grant vega 1/0782/15 and 1/0018/16. p-09.04.4-032 attenuation of dysfunction, oxidative stress and apoptosis by resveratrol in benzo(a)pyrene exposed ins1-e 832/13 pancreatic beta cell line s. c ß elik, b. baysal faculty of medicine, afyon kocatepe university, afyonkarahisar, turkey diabetes is one of the most important problems in the world. this disease is a very important health problem due to affects many different organs and systems. it has been well established that, environmental pollutants had deleterious effects on glucose metabolism, and caused insulin resistance and type 2 diabetes. with this investigation, it was aimed to investigate the effects of benzo(a)pyrene on pancreatic beta cells and treatment affects of resveratrol. in this study, rat ins-1e beta cell line was used. after reaching the appropriate number of cells culture operations by cell culture, benzo(a)pyrene (20 lm, 24 hours) application have been made after resveratrol (80 lm, 48 hours) application. after incubations oxidative stress, insulin secretion (in cell and in medium), cell proliferation and apoptosis were analysed in cells by biochemical and molecular techniques. b(a)p application resulted in no increase and resveratrol also increased this level of no. resveratrol increased the tas levels decrased by b(a)p, and tos levels were also increased by resveratrol interestingly. osi levels determined with tas and tos levels, has no significant change between groups. gsh levels were decreased by b(a)p while resveratrol increased its' level to control level. mrna expression levels of beta cell functions related genes ins-1, ins-2 and sirt-1 were increased by resveratrol treatment. insulin levels in cell and in medium were increased after resveratrol treatment. mrna expression level of foxo-1 gene was increased while pdx-1 was decreased by resveratrol treatmeant. b(a)p suppressed the mrna expression of p53 gene, but resveratrol increased. the effects of b(a)p on pancreatic beta cells and the protective effects of resveratrol on this cells were investigated in vitro with this research firstly. the results obtained from this research showed that oxidative changes, functional impairment and carcinogenetic effects of b(a) p in pancreatic beta cells could be blocked by resveratrol. the protective effect of vitamin e (alphatocopherol) on ischemia-reperfusion injury in rat liver ischemia-reperfusion (i/r) process is usually used during transplantation and resection of the liver but liver dysfunction and cellular death due to lack of oxygen in tissues, changes in the balance of oxidant/antioxidant in favor of oxidants, and inflammatory response are inevitable during this process. in the present study, it was aimed to investigate whether vitamin e(alpha-tocopherol), an antioxidant agent, has a protective effect against liver ischemia reperfusion injury in rats by using morphometric methods. for this purpose, 21 adult sprague-dawley male rats were divided into 3 groups as; control, ischemia / reperfusion (i/r), and vitamin e+ischemia/reperfusion (evit +i/r). in experimental groups, the total hepatic ischemia was applied for 45 minutes followed by a 24 hour of reperfusion. in the treatment group, 7 days before ischemia 40 mg / kg dose of vitamin e was administered intraperitoneally once a day. after the termination of the reperfusion, the rats were perfused by cardiac way and liver tissues were dissected. following volume and weight calculations, the livers were subjected to the standard histological preparation methods and embedded in paraffin. serial sections at 5 lm thickness were obtained from these blocks, stained with hematoxylineosin, and analyzed with morphometric methods. in light microscopic examinations of the i/r group, irregularity in lobules, dilatation in central veins and sinusoids, extensive areas of necrosis and pycnotic nuclei were seen in hepatocytes. the volume density of sinusoids to liver parenchyma was estimated as 16% in the control group, whereas it was 36% in the i/r group. this value was decreased to 32% in the evit + i/ r group. however, no significant difference was found among the groups in the lobule area calculated by the point counting method. these results show that intraperitoneal vitamin e administration for 7 days prior to ischemia partially inhibits damage caused by ischemia-reperfusion injury in the liver. the leaves, fruit and bark of annona muricata, a member of the annonaceae family, are commonly used in the traditional medicine of tropical and subtropical regions. in recent years, many studies have shown their anti-cancer, anti-convulsant, antiarthritic, anti-parasitic, anti-malarial, and anti-diabetic activities. it should be noted that these characteristics have been described using different types of extracts from different parts of the plant. our studies have focused on the systematic characterization of activities most easily accessible from an aqueous extract of leaves, with hitherto documented antihypertensive and hepatoprotective effects. we found that the extract shows almost 54% efficiency against hydroxyl radicals. with increasing concentrations, the effectiveness weakened, reaching a second peak (40%) at a concentration of 50 mg/l. the scavenging activity against no degradation products maintained a continuously increasing trend with a maximum at a concentration of 100 mg/l. surprisingly, the extract initiated peroxynitrite production in a similar trend, except at 50 mg/l, where it scavenged peroxynitrites with relatively high efficiency, up to 34%. these findings are consistent with the elevated levels of reduced glutathione detected after incubation of liver mitochondria with extract to a maximum concentration of 50 mg/l, with subsequent sharp decline. the activity of glutathione s-transferase was decreased, although not significantly. this indicates a reduction of metabolic processes of compounds, allowing their action over a longer period of time. in a live system, even antihypertensive effects can be observed. however, a significant outflow of gsh to create the gsh adducts of active substances, and particularly s-nitrosoglutathione from increased production of peroxynitrites, can cause liver toxicity. the study was supported by grant vega 1/0782/15 and 1/0018/ 16. the role of polyamine metabolism in curcumin induced apoptosis via reactive oxygene species (ros) generation in growth hormone (gh) overexpressed t47d breast cancer cells r. genc ß, a. coker gurkan, e. d. arisan, p. obakan yerlikaya, n. palavan unsal, n. palavan unsal t.c istanbul k€ ult€ ur € universitesi, istanbul, turkey autocrine growth hormone (gh) signaling triggers cell proliferation, growth, metastasis and drug resistance in cancer cells. polyamines (pas) play an essential role in cell cycle, proliferation, growth and carcinogenesis of various cancer types such as prostate, colon and breast cancer. odc (ornithine decarboxylase) is the key enzyme in the pa biosynthetic pathway that is under control of antizyme (az) and antizyme inhibitor (azi) activity. pa catabolic enzymes polyamine oxidase (pao) and spermine/spermidine acetyle transferase (ssat) by-products triggers reactive oxygene species (ros) generation and apoptotic cell death. curcumin decreased cell viability in dose and time dependent manner in t47d wt and gh+ cells. although forced gh expression induced cell proliferation, 20 lm curcumin inhibited cell invasion. curcumin (20 lm) induced apoptosis by acting on intrinsic and extrinsic pathway in both cell lines. moreover, curcumin supressed odc, azi expression in wt and gh+ t47d cancer cells. although curcumin decreased az expression in t47d wt cancer cell, increased az expression was determined in t47d gh cancer cell. pao and ssat expressions were upregulated in t47d gh+ cells. concomitantly, putrescine levels were increased in t47d gh+ cancer cell compared to wt cells and curcumin depleted spermidine and spermine levels in wt and gh + t47d cells. curcumin induced-apoptotic cell death via ros generation and co-treatment of n-acetyl cysteine (nac) overcame curcumin effect. conclusion, forced gh expression triggers cell proliferation and growth via acting on polyamine metabolism and curcumin-triggered ros generation was prevented by nac treatment in t47d wt and gh+ breast cancer cells. acknowledgment: this study was supported by tubitak 1001 research project (project no: 113z791). the radio-protective effects of propolis and nigella sativa oil on oxidative/nitrosative stress in liver tissue of rats exposed to total head irradiation s. taysi our purpose is to investigate propolis and nigella sativa oil (nso) for their antioxidant effects on the liver tissue of rats exposed to ionizing radiation. a total of 32 sprague-dawley rats were divided into five groups to test the radioprotective effectiveness of of propolis and nso administered by orogastric tube. appropriate control group was also studied. biochemical parameters in liver tissue of rats were determined by spectrophotometer. xanthine oxidase (xo), nitric oxide synthase (nos), superoxide dismutase (sod) activities, nitric oxide (no•) and malondialdehyde (mda) levels were higher in ir group while glutathione-s-transferase (gst), glutathione peroxidase (gsh-px) level in the ir group were lower in this group when compared to the other groups. gst activity in ir plus propolis group was statistically higher than in all other groups. propolis and nso clearly protect liver tissue from radiationinduced oxidative stress. the effects of royal jelly on the antioxidant parameters in the breast tissues of the rats with breast carcinoma treated with paclitaxel or not effects of royal jelly on the breast tissue antioxidant parameters in rats with breast carcinoma treated with paclitaxel or not. 8-12 weeks old female sprague-dawley rats (n = 37) included in current study were divided into 5 groups: control group (n = 8) with healthy rats; breast cancer group (n = 8); breast cancer group (n = 7) treated with 15 mg/kg paclitaxel injection (once a week for 3 weeks); breast cancer group (n = 7) treated with 100 mg/kg royal jelly (by oral gavage for 30 days); and finally breast cancer group (n = 7) treated 100 mg/kg royal jelly in addition to 15 mg/kg paclitaxel injection. rats with breast carcinoma was obtained at 150th days after a single dose injection of 50 mg/kg n-methyl-n-nitrosourea (mnu). all cancer groups were followed by 30 days with treatment of paclitaxel and/or royal jelly. the antioxidant parameters in rat breast tissues, superoxide dismutase (sod) and catalase (cat) activities were determined by spectrophotometric colorimetric methods and glutathione (gsh) by high performance liquid chromatography (hplc). all the antioxidant parameters decreased in breast cancer group without any treatment (p < 0.05). but, statistically non significant increases were observed by paclitaxel and royal jelly treatment (p > 0.05). this study indicated that royal jelly supplementation can not be sufficient to increase the antioxidant parameters in breast cancer. we are going to continue to identify the effects of royal jelly on breast cancer detail. the effects of n-acetylcysteine on microsomal and serum paraoxonase 1 activities at high fat diet induced obese rats obesity is a chronic disease that develops from the interaction between genotype and environmental factors and increase in the accumulation of body fat. it is related with glucose and lipid metabolism disorders, cardiovascular diseases and increased oxidative stress. paraoxanase 1 (pon1) is an enzyme which plays a protective role in oxidative stress, inflammation and liver diseases. it has been suggested that pon1 has protective effects against high fat diet induced obesity and obesity related disorders. n-asetylcysteine (nac) is a potent antioxidant due to its ability to stimulate glutathione synthesis. the aim of this study was to evaluate the microsomal and serum pon1 enzyme activities (paraoxonase, arylesterase and lactonase) at high fat diet induced obese rats in the presence of nac. this study consisted of control, obese and nac-supplemented obese (2 g/l nac) groups. eighteen sprague-dawley rats were randomized into three groups (n = 6). control rats were fed by standart food and obese and nac groups were fed by high fat diet. the microsomal and serum paraoxonase, arylesterase and lactonase activities were determined by colorimetric methods. serum paraoxonase, arylesterase and lactonase activities decreased in obese and nac groups when compared to control groups. on the other hand microsomal paraoxonase, arylesterase and lactonase activities increased in nac group when compared to control and obese groups. however there was no statistically significant difference between the groups. it has been concluded that the microsomal and serum paraoxonase 1 enzyme activities did not change at high fat diet induced obese rats in the presence of n-asetylcysteine. reactive oxygen species, playing an active role in the early and late course of acute pancreatitis, lead to dysfunction of cell membrane and releasing of lysosomal enzymes, and thereby to the injury in pancreatic cells. gallic acid, found in vegetables such as green tea, is an active component which has antioxidant, antiinflammatory, antiviral, anticancer activities. the aim of this study was to investigate the effects of gallic acid in experimental acute necrotizing pancreatitis (anp) model in rats. eighteen male sprague-dawley rats were divided into three groups (6 rats in each group). group 1: sham + saline; group 2: anp induced by intraductal glycodeoxycholic acid and intravenous cerulein; and group 3: anp + gallic acid (100 mg/kg/day, intraperitoneal). at the end of 18th hours, pancreas histopathology was examined. the levels of serum amylase as a diagnostic marker of pancreatitis, interleukin-6 (il-6), total antioxidant status (tas), nitrite + nitrate, total thiols as antioxidant marker and thiobarbituric acid reactive substances (tbars) to measure malondialdehyde (lipid peroxidation product) were determined by spectrophotometric methods. serum amylase, il-6, plasma tbars levels were significantly higher but total thiols levels were lower than sham group in anp group without treatment (p < 0.05). however; tas and nitrite + nitrate levels did not show any significant difference (p > 0.05). on the other hand, while serum amylase, il-6 and tbars levels were lower, total thiols levels higher in gallic acid treatment group than in the untreated anp group, but statistically insignificant (p > 0.05). in conclusion, gallic acid treatment is beneficial but not sufficient to treat the acute necrotizing pancreatitis in rats. p-09.04.4-043 evaluation of oxidant/antioxidant system parameters, il-6 and il-10 levels in amniotic fluid of pregnancies with down sydrome b. _ imge erg€ uder 1 , s. bahsi 1 , t. bahsi 2 , v. topc ßu 2 , a. bakir 2 1 ankara universty faculty of medicinel, ankara, 2 zekai tahir burak research and training, hospital genetic center, ankara, turkey introduction and aim: down sydrome (ds) can be diagnosed at high-risk of down syndrome pregnancies by invasive prenatal testing. in this study we aimed to demonstrate antioxidant/oxidant system markers, il6 and il10 levels in amniotic fluid samples of pregnancies affected by ds. materials and methods: for this purpose we collected amniotic fluid samples from 18 pregnancies affected by down sydrome and 36 normal healthy pregnancies who applied to zekai tahir burak research and training hospital genetic center and were proceeded with amniocenthesis. in the amniotic fluid samples; malondialdehyde (mda), superoxide dismutase (sod), glutathion peroksidase (gsh-px) xhantine oksidase (xo), catalase (cat), adenozine deaminase (ada), nitric oxide (no), nitric oxide senthase (nos) enzymatic activities were evaluated by spectrophotometric methods, il6 and il10 levels are evaluated by elisa. for statistical analysis student's t-test and spearman corralation analusis are used. results: it was found that sod levels are significantly elevated in study group compared to control group (p < 0.05). besides this, in study group, cat and il-6 levels are found singnificantly lower than control group (p < 0.05). we couldn't find any significant difference between two groups in terms of mda, gsh-px, xo, no, nos, ada ve il-10 levels (p > 0.05). discussion and conclusion: there is an important decrease in inflammation compared to normal pregnancie in the amniotic fluid of pregnancies having ds. based on these results, sod enzyme may be used as a marker for prenatal diagnosis of ds. for this purpose these experiments should be tried on larger sample groups. the aim of our work was to compare prooxidant and antioxidant properties of linalool, which is the oxygenated monoterpene compound reported to be a major volatile component of the essential oil of several aromatic species, in hep g2 cells. cytotoxicity of linalool was assayed by celltiter-blue ò cell viability assay. malondialdehyde levels result in membrane damage in hep g2 cells were assayed by using fluorometric method. hep g2 cells were incubated with linalool at 24, 48 and 72 hours. the viability of hep g2 cells decreased in a manner dependent upon concentration and incubation time. the ic 50 values were calculated 81.5 lg/ml (24 hours), 72.7 lg/ml (48 hours) and 64.7 lg/ml (72 hours). but, cell viability of hep g2 cells increased when the cells preincubated with linalool at lower concentrations (˂ic 50 ) against h 2 o 2 cytotoxicity. membrane-damaging effects of linalool were increased with accelerating concentrations. on the other hand, membrane damaging effect of h 2 o 2 was decreased when the cells preincubated with linalool before h 2 o 2 incubation. oxygenated monoterpene linalool had both prooxidant and antioxidant properties showing membrane damaging and protective effects on hep g2 cells depend on concentration. postprandial lipemia is primarily characterized by increasing triglyceride levels after the lipid rich meal. postprandial lipemia may cause oxidative stress by increasing free radical production and increasing oxidative stress could be responsible for the development of many diseases. plasma oxidant-antioxidant status was evaluated in healthy individuals with postprandial hypertriglyceridemia generated by performing oral triglyceride tolerence test (ottt). the study group included 86 subjects (38 female and 48 male). ferric reducing ability of plasma (frap), total thiol and thiobarbituric acid reactive substances (tbars) levels were determined by colorimetric methods at fasting and 2nd, 4th and 6th hours following ottt. the levels of frap and thiol were significantly higher in males than females (p = 0.0001 and 0.042, respectively). thiol levels decreased significantly in both gender at postprandial 2nd, 4th and 6th hours as compared with fasting condition (p = 0.0001). while tbars levels increased at postprandial 2nd hour, that was only significant for male individuals (p = 0.0001). it has been concluded that postprandial lipemia may change oxidant-antioxidant balance in favor of oxidants and gender is an important criteria while assessing the oxidant-antioxidant status in postprandial lipemia p-09.04.4-046 ischemia modified albumin and c-reactive protein levels in prediabetes prediabetes is a state of abnormal glucose homeostasis characterized by the presence of impaired fasting glucose, impaired glucose tolerance, or both. the aim of this study was assess serum ischemia modified albumin (ima) in prediabetes and determine its correlation with other risk factors for chronic complications such as inflammation and hyperglycemia. glucose, insulin, total cholesterol, hdl cholesterol, triglycerides, albumin, c-reactive protein (crp) and ima were measured in 30 patients with prediabetes and 30 controls. prediabetes patients had higher levels of ima and crp in comparison with control subjects but there was no significant difference between groups for ima. significant positive correlation was observed between crp and fasting glucose, insulin. there was no significant correlation between ima levels and the parameters tested. we have shown higher level of crp in prediabetes. these results support the hypothesis that chronic inflammation may be involved in development of hyperglycaemia. p-09.04.4-048 the effects of s _ io 2 nanoparticles of rat uterine smooth muscle specially used in textile field sio 2 nanoparticles on uterus smooth muscle was aimed to be researched. in this study 64 wistar albino female rats were used. rats were separated in 4 groups as control, dose 1 (250 lg/ml), dose 2 (500 lg/ml) and dose 3 (1000 lg/ml). nanoparticle's size was chosen as 20 nm. preparations of four groups were evaluated for biochemical and histological examinations. all isolated uterine smooth muscle stripts except the controls were treated with sio 2 for two hours. in biochemical examinations in order to evaluate oxidative stress level of malondialdehit (mda), activity of superoxide dysmutase (sod) and glutathione peroxidase (gsh-px) were measured. in histological examinations via electron microscope ultrastructure of uterus was examined as well as apoptotic cells detected with immunofluorescent labeling method. intergroups differences were defined by statistical analysis. while mda level increased depending on the dosage, sod level was decreased depending on the dosage. gsh-px rate was decreased for each dosage with respect to control. however, no significant difference is detected between groups. in electron microscopic examination no changes were observed in uterus ultrastructure with compare to control. however, in immunofluorescent labeling it was detected that apoptosis increased in dosage groups with compare to control group. as a result, it was thought that application of sio 2 nanoparticles, in 20 nm size and in 250, 500 and 1000 lg/ml dosages caused of oxidative stress and apoptosis. this results suggested that sio 2 has toxic effects on uterine smooth muscle. uterine myomas are the most common benign pelvic tumors arising from myometrium. they are rarely associated with mortality but responsible for significant morbidity and have adverse effects on quality of life especially in reproductive age women. reactive oxygen species and superoxide dismutases, as well as sex steroids play important roles in the reproductive physiology processes. in addition, oxidative stress and impaired antioxidant defense system have been linked to pathophysiology of various diseases including malignant gynecological disorders. clinical investigations indicate that women with myoma may have increased risk of developing malignant tumors particularly sarcomas. the present study aimed to investigate the possible role of oxidant and antioxidant status in myomas. blood and urine samples of 21 myoma patients were collected. activities of erythrocyte antioxidant enzymes [copper-zinc superoxide dismutase (cuzn-sod), catalase (cat), glutathione peroxidase (gpx1)] and levels of lipid peroxidation biomarkers [plasma malondialdehyde (mda) and urine 8-epi-prostaglandin f 2a (8-epi-pgf2a)] were determined. the results were compared with those of 21 controls. the groups were matched in terms of age, body mass index, smoking habit, coexisting chronic diseases, menopausal status and sex steroid hormone levels. all antioxidant enzyme activities were higher (37% for cuzn-sod, p = 0.003; 52% for cat, p0.05) and the levels of lipid peroxidation biomarkers were lower (%59 for mda, p = 0.011 and 43% for 8-epi-pgf2a, p > 0.05) in myoma patients compared to controls. correlation analyses showed a significant negative correlation between erythrocyte cuznsod activity and plasma mda levels (r = -0.431, p = 0.005). the decreased lipid peroxidation may be the consequence of elevated antioxidant enzyme activities and the data suggests a protective role of activated antioxidants especially cuznsod and cat in patients with myoma. p-09.04.4-050 investigation of ischemia-modified albumin levels in patient with acute limb ischemia introduction: acute limb ischemia commonly occurs as a result of embolus caused by cardiac origin and which may end up with limb loss or even death if left untreated. thrombosis are usually seen where the arteries give branches and tendency to atherosclerosis is more serious at these sites. involvement of several arteries in either embolus or in situ thrombosis limits the adequacy of collateral circulation. restriction of blood flow due to arterial stenosis or occlusion often leads patients to complain of muscle pain on walking. any further reduction in blood flow causes ischemic pain at rest, which affects the foot. early recognition may prevent limb loss or death. ischemia can alter the capacity of the amino terminus of the albumin to bind free metal atoms such as cobalt, copper and nickel. this new, chemically changed albumin is called ischemia modified albumin (ima). ima is a sensitive marker of myocardial ischemia, skeletal muscle ischemia, pulmonary embolism, mesenteric ischemia and stroke. therefore, in this study it was aimed to investigate the ima level in acute limb ischemia. materials and methods: in this study, 24 patients with acute limb ischemia (li group; mean age 62 years) and 34 healthy individuals (control group; mean age 42 years) were included. ima levels were detected in control and li group by elisa (organo teknika, avusturya) using ima el _ isa kit. results: ima values were compared with nonparametric methods mann whitney u test, and significantly decreased ima level was statistically significantly different between li group and control group (p < 0.001). conclusion: there is a significant increase in serum ima in limb ischemia, so that alterations also might be clinically useful in the diagnosis of limb ischemia, but should be supported with further studies. object: polycystic ovary syndrome (pcos) is a multifaceted disorder with a pathogenetic pathway that is not fully understood yet. apart from hormonal derangements, insulin signaling defects and adipose tissue dysfunction, oxidative stress, defined as an imbalance derived from excessive formation of oxidants in the presence of limited antioxidants defenses, has been actively implicated in the etiology of the syndrome. the aim of this study was to determine of serum myeloperoxidase activity (mpo), paraoxonase 1 activity (pon1) and arylesterase activity (are) in patients with pcos. material and methods: the study was carried out on 150 women consisted of 103 patients with pcos and 47 healthy ones as control. serum pon1 activities were measured spectrophotometrically using diethyl-p-nitrophenylphosphate as substrate. phenylacetate was used as substrate for are measurement, and are activity was determined by measuring absorbance of the resulting phenol at 270 nm. molar absorptivity coefficients were used in the calculation of pon and are activities as 1 nmol phenol/ml serum/min. result: the mpo and are activities were significantly lower in the patient groups when compared with the control group (72.09 ae 60.66 -119.74 ae 90.81 u/ml p < 0.001, 47.43 ae 12.21 -83.93 ae 26.38 u/ml p < 0.001, recpectively). the pon1 activities are higher in the patient group (145.55 ae 98.23 u/ml) compared to the control group (153.33 ae 101.90 u/ml) are found, but are not statistically significant. conclusion: lower serum mpo and are activities might contribute to the increased susceptibility for the development of diseases risk in women with pcos. because free oxygen radicals are thought to contribute to the complication of many chronic diseases, the pcos may be related to oxidative stress. subclinical hypothyroidism, defined as an elevated serum thyroid stimulating hormone level associated with serum thyroid hormone concentrations within the reference range. free radicalmediated oxidative stress has been implicated in the pathogenesis of thyroid disorders. the ischemia-modified albumin (ima) has been proposed as a marker of protein oxidative damage, which has been found to reflect hypoxic stress. this study aimed to investigate the influence of subclinical hypothyroidism on serum ima levels. thirty-one subclinical hypothyroidism patients and 27 control subjects were enrolled in the study. albumin, ima were measured and ima/albumin ratio was calculated. to determine the ima levels the measurement method based on albumin cobalt binding assay was used. serum ima levels of patients with subclinical hypothyroidism were 0.58 ae 0.08 absu, ima levels of control subjects were 0.49 ae 0.08 absu. ima levels were significantly higher in patients with subclinical hypothyroidism patients than in control subjects (p < 0.0001). when ima values were normalized for albumin concentrations, the ima/albumin ratio was also significantly elevated in patient group compared to control group (p < 0.01). ima levels are increased in patients with thyroid dysfunction. elevated levels of ima can be a clinically useful marker of protein oxidative damage in subclinical hypothyroidism. p-09.04.4-054 the effects on endothelial dysfunction of quercetin in streptozocin-induced diabetic rats excessively produced in pathologic conditions. ultimately, imbalance between oxidants and antioxidants results with oxidative stress (os). in this study, we investigated some os parameters in standard (20% protein, 70% (7% sucrose) carbohydrate, 10% lipid) and sucrose (20% protein, 70% (35% sucrose) carbohydrate, 10% lipid) diet fed bdnf heterozygous mice liver tissues. the male c57bl6 strain wild type (+/+) and bdnf heterozygous (+/à) mice (5 weeks) were obtained. the animals were fed ad libitum by special standard and sucrose diets. twenty four mice were divided into four groups and each group consist six mice. all mice were fed for 16 weeks. first group involved in c57bl6 wild type mice and fed by standard diet. second group contained c57bl6 bdnf heterozygous mice and fed by standard diet. third group consisted c57bl6 wild type mice and fed by sucrose diet. fourth group involved in c57bl6 heterozygous mice and fed by sucrose diet. in first group, mda levels, sod and cat activities were higher than other groups. in second group, cat activities were lower than other groups. but, we could not find any statistically significant differences between all groups about mda, sod, cat levels in bdnf heterozygous mice liver tissues. in conclusion, standard and sucrose diet feeding may not affect mda, sod and cat levels in bdnf heterozygous mice liver tissues. brain-derived neurotrophic factor (bdnf) is member of neurotrophin family which plays critical roles in the development, differention, survival, maintenance of the central and peripheral nervous systems. bdnf also contributes to food intake and body weight control. bdnf heterozygous mice display increased body weight and mild hyperphagia. expression of bdnf is not limited to the brain, it also express some peripheral tissues like adipose tissue, liver, kidney, skeletal muscle, heart. even though roles of bdnf are well known relatively in central nervous systems, effects of this protein is not clear in peripheral tissues. as mentioned before, it is expressed in organs involved in energy, lipid and glucose homeostasis, including the liver, adipose and muscle tissues, but its role there remains unclear. in this study, we aimed to investigate role of bdnf on liver oxidative stress parameters in heterozygous mice model fed fat diet induced obese mice. in this study, we used c57bl/6 mice inbred strain wild type and bdnf heterozygous (+/à) mice. animals were divided to two groups: wild type (n = 5) and bdnf heterozygote mice (n = 5). the animals were fed ad libitum by high-fat diet during 4 month. weight gain was recorded every 15th days. in liver tissues were measured malondialdehyde (mda), superoxide dismutase (sod) and catalase (cat) by spectrophotometric methods. liver mda levels decreased in obese bdnf heterozygous mice compared to obese wild type group and statistically significant difference between groups. bdnf heterozygous mice cat activities were higher than the other group and this difference was statistically significant. there was no statistically significant difference between the groups in terms of sod activities. it has been concluded that the mda levels and sod enzymes activities changed at high-fat diet induced obese bdnf heterozygous mice compared to wild type mice liver tissues. p-09.04.4-060 disturbances of microelements profile in serum of overweight/obese adult females with acute and persistent pro-inflammatory chlamydia pneumoniae infection p-09.04.4-061 determination of reactive oxygen species induced dna damage using modified cupric reducing antioxidant capacity (cuprac) colorimetric method s. uzunboy, s. demirci c ß ekic ß, r. apak department of chemistry, istanbul university, faculty of engineering, istanbul, turkey reactive oxygen species (ros) term is a common name of a group of species. hydroxyl radical and singlet oxygen can be taken into account as ros samples. ros may be formed as a result of endogenous or exogenous reasons. although ros have some beneficial functions, they should be balanced by antioxidants (aox). excessive amounts of ros can attack biological macromolecules including dna. dna damage is usually related with mutagenic and carcinogenic changes. that's why determination of dna damage is so important and there are a great many studies in literature comprising different techniques. one of the most common of them is the 'comet assay'. but application of the method and interpretation of the results is not easy. investigation of certain dna damage products is also very common. these methods usually need expensive instrumentation such as using tandem mass spectrometry. on the other hand, depicting total dna damage on a certain product may cause misinterpretations. in the presented study, dna was decomposed by hydroxyl radicals produced by fenton method. in the study while dna is not cuprac reactive the oxidation products can react with the cuprac reagent. the effect of aox was also investigated. for this purpose, selected aox compounds were added to the reaction medium. because of their radical scavenging effect, the cuprac absorbance decreased in the presence of aox. in the presence and absence of aox, absorbance differences were calculated. the calibration graphs between final concentration and absorbance differences were drawn for each aox. gallic acid was determined as the most effective one among the tested aox samples. for statistical comparison with the presented study, tbars was used as reference method. direct use of dna as a probe material to determine oxidative damage may be an advantage to understand dna hazard in biological systems. the proposed method can be applied in all laboratories having a spectrometer as a cost-effective and simple procedure. p-09.04.4-062 effects of alpha-1 antagonists on oxidative system of rat heart tissue benign prostate hyperplasia is a progressive process occurring in the stromal and epithelial components of the prostate. alpha-1 receptor blocking agents are used for relaxation of the smooth muscles in the prostatic stroma. our aim was to investigate the effects of alpha-1 antagonists on oxidative system of rat heart tissue. 33 male wistar albino rats were divided into 5 groups randomly. 1) tamsulosin (1 mg/kd/day), 2) terazosin (5 mg/kg/day), 3) doksazosin (25 mg/kg/day), 4) alfuzosin (10 mg/kg/day), 5) control. all drugs were administered every other day single doze via oral. rats were sacrificed after 30 days. heart tissue was taken for biochemical analysis. malondialdehyde (mda), nitric oxide (no), protein carbonyl (pc) levels and superoxide dismutase (sod), glutathione peroxidase (gsh-px) enzyme activities were determined in supernatant samples. there was not an significant difference between terazosin, doxazosin, alfuzosin, tamsulosin groups in means of sod, mda and gsh-px levels. no levels were significantly different between tamsulosin group and the control group (p = 0.004). in addition, tamsulosin group and terazosin group were also significantly different (p = 0.032). according to these results we can say that tamsulosin group had higher no levels than control and terazosin group. tamsulosin's enhancer effect on no levels leads to relaxation of the heart muscle and vascular relaxation, and so fewer side effects than other alpha antagonists. the effect of rat liver tissue radical metabolism and the protective role of hippophae rhamnoides l. on cold and immobilization stress model cold and immobilization stress is a widely used model for study the changes that occur on oxidant-antioxidant balance. hippophae rhamnoides l. (seabuckthorn; sbt) a unique and valuable plant has recently gained worldwide attention, mainly for its medicinal and nutritional potential. this study was aimed to investigate the protective role of sbt which is a natural herbal product with high antioxidant content on oxidative and nitrosative stress induced by cold and immobilization stress in rats. 32 wistar albino rats were divided into 4 groups randomly. control (i.p. physiological saline), sbt (i.p. 200 mg/kg/48 hours sbt), stress (i.p. physiological saline; 6 hours cold + immobilization stress) and sbt + stress (i.p. 200 mg/kg/48 hours sbt; 6 hours cold + immobilization stress) groups were formed. 3nitrotyrosine levels were determined by elisa whereas total antioxidant capacity, total thiol, total glutathione, nitrite-nitrate levels and superoxide dismutase and glutathione peroxidase activities were measured by colorimetric methods. sbt + stress group nitrite-nitrat (p = 0.0001), total glutathione (p = 0.0001) levels and glutathione peroxidase activities (p = 0.0001) were found to be significantly higher whereas superoxide dismutase activity was found to be lower (p = 0.007) when compared to stress group. there was no significant differences between stress group total thiol and total antioxidant capacity levels compared with stress + sbt group. stress + sbt group oxidative and nitrosative stress marker 3-nitrotyrosine level was found to be significantly higher when compared with control group (p = 0.002) whereas there was no significant differences between stress and stress + sbt groups. all this data show that sbt has antioxidant properties on cold and immobilization induced oxidative and nitrosative stress in rat liver tissue. obesity is a major health problem with growing incidence and accompanying complications. its relation with diminished cognitive functions was reported. this study aims to evaluate the effects of obesity induced oxidative stress and metabolic alterations on the cognitive functions of children and adults. 33 children and adolescents with obesity (age: 8-18); and 33 age and gender matched healthy subjects were enrolled. all subjects completed the battery tests of cnsvs via computer. the scores were compared by using commercial software (ibm spss statistics 19). biochemical parameters, malondialdehyde (mda) and protein carbonyl (pc) levels were estimated. mda and pc levels were significantly higher in subjects with obesity (0.78 ae 0.16 lmol/l;198.30 ae 84.45 nmol/ml) than the controls (0.5 ae 0.10 lmol/l; 125.35 ae 43.52 nmol/ml) (<0.001). there was statistically significant difference between study and control groups on all cognitive performance domains. significant correlation was detected between mda, pc levels and the cognition indexes. children with obesity should be evaluated for the cognitive functions, together with the metabolic follow-up. obesity induced oxidative stress may be the reason of the diminished cognition in children as well as the changes in the lipid profile and inflammation, but we need larger study groups to lighten these complex process. p-09.04.4-067 relative contribution of nitric oxide synthase (nos) isoforms to oxidative/nitrosative stress in the cerebral cortex of rat with acute liver failure (alf) acute liver failure (alf) is associated with deregulation of nmda/cgmp/no signaling and oxidative/nitrosative stress in the brain. however, the relative roles of the different nos isoforms and the mechanisms underlying alterations in their activities during alf are not fully clear. here we investigated gene and protein expression of nos isoforms, nos activity, enos uncoupling and total no production in cerebral cortex of rats with thioacetamide (taa)-induced alf. sprague dawley rats (250-280 g) received three i.p. injections of taa (300 mg/kg) at 24 hours intervals. the brain cortex expression nos isoforms (enos/inos/nnos) was measured by real-time pcr and western blot, nos activity was tested by monitoring the conversion of radiolabeled arginine to citrulline. reactive oxygen species (ros) were quantified in the presence of nos substrate l-arginine, using the carboxy-h 2 dcfda probe. no was measured with the griess procedure. the enos expression was decreased, whereas the enos dimmer/monomer ratio and nnos/inos expression were elevated in taa treated rats. while the total nos activity was decreased, the inos activity was elevated and no concentration tended to increase. ros production was elevated by taa. unspecific nos inhibitors l-name and l-nna attenuated ros production in both control and taa rats, but with higher efficiency in the latter case. ca 2+ chelation had almost the same effect as pharmacological nos inhibition suggesting that ca 2+ -independent inos activity is not the main source of ros. incubation with high dose of tetrahydrobiopterin (bh4) with which is critical for enos dimerization and subsequent no production also reduced ros production indicating the enos uncoupling phenomenon in taa cortex. the study points to enos downregulation due to lowered protein expression and uncoupling as a novel mechanism contributing to enhanced superoxide o 2 anion formation, and confirms the role of inos/nnos in enhancing no synthesis in alf-affected brain. introduction: diabetes mellitus (dm) is an endocrine disorder of world which is characterized by altered blood glucose levels and related complications including hepatic injury. myrtus communis l. (mc) is widely used by diabetic patients in the folk medicine of turkey as well as they are used worlwide. it is known that of leaves, oil and fruit of myrtus communis l. (mc) have therapeutic effects on diabetes mellitus (dm). this study was aimed to analyze the possible antidiabetic and hepatoprotective effects of mc berries in streptozotocin (stz) induced diabetic rats. materials and methods: a total of thirty rats composed of six groups as each included five rats were used. 40 mg/kg stz was injected once to animals to induce dm. after stz injection, rats were exposed to three different ethanol extracts of mc berries (250, 500 and 1000 mg/kg) by oral gavage during 14 days. alanine aminotransferase (alt) and aspartate aminotransferase (ast) levels were determined in serum and glutathione (gsh), malondialdehyde (mda) levels and superoxide dismutase (sod) activity were determined in liver tissue. results: mc administration provided significant reducement in the altered serum glucose, ast and alt levels in all diabetic groups. mc extract showed significant antioxidant activity by altering sod activity and gsh level and reducing mda levels in diabetic rats compared to controls (p < 0.05). serum ast and alt levels were reduced by mc administration in all diabetic groups. mc administration provided significance increment in sod activity and gsh level, and significant reduction in mda levels compared to controls (p < 0.05). the maximum hypoglycemic and antioxidant effects were observed at 1000 mg/kg dose of mc. background: human serum paraoxonase 1 (pon1) is a calcium dependent esterase that hydrolyzes organophosphates and also arylesters such as phenyl acetate. pon1 prevents ldl oxidation by hydrolyzing lipid peroxides. pon1 is inhibited by various chelating agents, heavy metal ions, and sulfhydryl reagents. in our study we investigated the effect of calcium on ldl oxidation of purified pon1 q192r isoenzymes. methods: pon1 q192r isoenzymes were partially purified from human serum. both allozymic forms were treated by preincubation with 1 mm edta for 15 minutes. ldl oxidation was induced by copper ions. formation of thiobarbituric acid-reacting substances (tbars) was used as a measure of lipid peroxidation. homocysteine thiolactonase (htlase activity) and arylesterase activities were measured spectrophotometrically by using homocysteine thiolactone and phenylacetate as the substrates. results: addition of 1 mm edta to partially purified hdl-pon1 q192r isoenzymes inhibited 100% of htlase and arylesterase activities. inactivation of pon1 for arylesterase/htlase activity by the addition of edta did not reduce the abilities of both allozymic forms in protecting ldl from oxidation. conclusion: ca +2 -dependent inhibition of pon1 q192r arylesterase/htlase by using the metal chelator edta, did not alter pon1's ability to inhibit ldl oxidation. pon1's ability to protect ldl from oxidation may not require calcium. p-09.04.4-070 evaluation of cholinesterase inhibitory effect, anti-radical and anti-lipid peroxidation activities of mentha pulegium i. hamad 1, 2 1 college of applied medical sciences, aljouf university, aljouf, saudi arabia, 2 faculty of medicine, bahri university, khartoum, sudan introduction: many studies indicated that intake of dietary and medicinal plants is effective in preventing or suppressing many diseases, therefore, there is a growing interest in plant'sbioactive compounds. mentha pulegium, is widely used in gulf countries in herbal teas or as additives in commercial spice mixtures for many foods to offer aroma and flavor. the aim of this study is to investigate the in vitro radical activity, the total phenol and flavonoid content, anti-lipid peroxidation and the cholinesterase inhibitory effects of mentha pulegium methanol extract. methods: the acetylcholinesterase and butyrylcholinesterase inhibitory potentials of extracts, were evaluated by colorimetric assay. the in vitro antioxidant activity was measured by dpph assay, the total phenols content was measured by folin-ciocalteau assay, the flavonoids content by the alcl 3 colorimetric method, and the protective effect of menthe mentha pulegium extracts against lipid peroxidation was evaluated using a liposome oxidation system. results: the methanol extract showed a scavenging activity nearly equivalent to vitamin c which is attributed to its high phenolic and flavonoid contents. the extract possessed protective effect against lipid peroxidation in a dose dependent manner. the methanol extract shows very little anticholinesterase activity as compared to the standard compound, physostigmine. conclusion: results presented here indicate that mentha pulegiumpossess strong antioxidant activity and protective effects and they can therefore be used as a natural additive in food, cosmetic and pharmaceutical industries. type 2 diabetes mellitus is a long term metabolic disorder that is characterized by hyperglycemia and insulin resistance. because of the hyperglycemia and free radicals, diabetes can cause cellular instability. micronuclei is a sensitive indicator of genetic damage and a marker of dna damage. micronuclei is also a morphological marker of chromosomal instability. in this study, we aimed to evaluate the frequencies of micronuclei in papanicolaou stained buccal cells of type 2 diabetic patients. a total of 30 type 2 diabetic patients and 30 healthy individuals were involved into our study. buccal smear samples that belong to these individuals were stained by using papanicolaou method for cytologic examination and the stained slides were evaluated by light microscopy (olympus bx-51). cells with micronuclei in each papanicolaou stained buccal smear sample were counted under light microscopy. the frequency of micronucleated epithelial cells were seen as significantly higher in type 2 diabetic patients than the control group (p < 0.05). one of the boron compounds is sodium perborate tetrahydrate (nabo 3 .4h 2 o), which the most widely used solid peroxygen compounds. it is used in safety bleach formulations, detergents and tooth powders. as known these products are commonly used in daily life. however, the actions on blood antioxidant defenses of sodium tetraborate against reactive oxygen species are not identified yet. it is reported that oxidative stress caused by ros damages. in this study, we searched enzyme activities of superoxide dismutase (sod), catalase (cat), glutathione-s-transferase (gst), glutathione reductase (gr), glutathione peroxidase (gsh-px) and glucose-6-phosphate dehydrogenase (g6pd) also the effect sodium perborate tetra hydrate on activities of these enzymes from human erythrocyte under in vitro conditions. according to our findings sodium perborate tetrahydrate caused significant (p < 0.05) increasing in the cat activity from red blood cell. the other antioxidant enzyme activities (sod, gst, gr, gsh-px and g6pd) did not show any changing by influence of sodium perborate tetrahydrate. metabolism of obese individuals could be exposed to risk of chronic low-grade pro-inflammatory effect and oxidative stress. some inflammatory and oxidative markers have been studied recently. plasma total antioxidant status (tas) and total oxidant status (tos) parameters can be non-invasive markers of diseases such as fatty liver disease, laparoscopic procedures (pneumoperitoneum), accompanying inflammatory condition like urinary tract infection, diabetic neuropathy, chronic hepatitis. the study groups have been comprised of two groups with normal to over-weight children. tas and tos levels were detected and the oxidative stress index (osi) was computed as a marker of the grade of oxidative stress. the over-weight group displayed higher levels of fasting glucose, insulin resistance, the body mass index. also, we know that insulin resistance leads to increased lipolysis and free fatty acid output. higher tos as well as crp is related to the group, also lower tas than other group is shown. crp levels in plasma were positively correlated with insulin and glucose levels. in addition, there was a significant relationship between osi and insulin resistance in the over-weight group. tas and tos are together more accurate sings of oxidative and antioxidative status of people. as well as a raise over weight-related subclinical inflammation and a fall antioxidant capacity is significant even in children. this condition may eventually develop the risk of long-term vascular damage. the effects of hydrogen peroxide pretreatment on antioxidant enzyme activities in calli tissues of two eggplant genotypes under salinity o. yasarkan 1 , e. aky€ uz 1 , g. baysal furtana 2 , s. s. ellialtioglu 3 , r. tipirdamaz 4 1 nezahat g€ okyigit botanic garden, istanbul, 2 department of biology, faculty of sciences, gazi university, ankara, 3 department of horticulture, faculty of agriculture, ankara university, ankara, 4 department of biology, faculty of sciences, hacettepe university, ankara, turkey the effects of hydrogen peroxide (h 2 o 2 ) pre-treatment on catalase (cat) and superoxide dismutase (sod) were investigated and lipid peroxidation measured as malondialdehyde (mda) content of the calli from salt-sensitive (artvin) and salt-tolerant (mardin) eggplant genotypes under salinity stress. the seeds from each genotypes were germinated on ms medium for 4 weeks and hypocotyl tissues from these plantlets were used as explants for calli induction on ms medium including 1 mg/l 2,4-d and 0.1 mg/l kinetin. as for the pre-treatment, the subcultured calli tissues were transplanted on the mediums containing 50 and 100 lm h 2 o 2 for 48 hours and then transplanted on the mediums including 150 mm nacl for 24 hours. antioxidant enzyme analysis and mda measurement was carried out for the control, nacl-only, h 2 o 2 -only and h 2 o 2 pre-treated tissues. pre-treatment with h 2 o 2 decreased the deleterious effects of salt stress on mda contents. in comparison with salt stressed groups, h 2 o 2 pre-treatment with or without nacl reduced mda content especially in artvin. comparing two genotypes, a decrease was observed on sod activity in artvin genotype and an increase in mardin genotype by comparison of salt stressed groups. higher increase on sod activity was observed in 100 lm h 2 o 2 + nacl groups on each genotypes. comparing two genotypes, a decrease was observed on sod activity in artvin genotype and an increase in mardin genotype by comparison of salt stressed groups. higher increase on sod activity was observed in 100 lm h 2 o 2 + nacl groups on each genotypes. the result showed pre-treatment of 100 lm h 2 o 2 induced acclimation of the plants to salinity. in addition, 100 lm h 2 o 2 pre-treatment, as a stress signal, could trigger the activation of antioxidant enzymes in calli and in this way alleviated the oxidative damage in calli growth under salinity. the investigation of effects of ghrelin and cannabinoid cb1 receptor agonist and antagonist on oxidant and antioxidant mechanisms on brain tissues of penicillininduced epileptic rats the aim of this study is to investigate the individual effects of ghrelin and cannabinoid type1 (cb1) receptor agonist acea, the antagonist am-251 and the interaction of these two different systems on oxidant and antioxidant systems in the brain, cerebellum and brain stem tissues of penicillin-induced epileptic rats. in this study 56 male wistar albino rats were used weighing 20-250 g. each group was consisted of 8 rats. study groups: 1: control, 2:penicilin(500 iu), 3:penicillin(500 iu) + ghrelin(1 lg), 4:penicillin(500 iu) + am-251(0.25 lg), 5:penicilin (500 iu) + acea(7.5 lg), 6:penicillin(500 iu) + am-251(0.25 lg) + ghrelin (1 lg), 7:penicillin(500 iu) + acea(7.5 lg) + ghrelin(1 lg). than the levels of mda, gpx and sod are measured in plasma and tissue samples of these rats. penicillin was found to be induced lipid peroxidation in the brain, cerebellum and brain stem tissues in our study. ghrelin and acea, which both have anticonvulsant effects, were shown to be effective in reversing the oxidative damage caused by penicillin and proconvulsant am251 was found to further increase the oxidative stress caused by penicillin in these tissues. ghrelin also was found to suppress the oxidative stress caused by am251 in the cerebellum tissue but it did not contribute to antioxidant effects produced by acea. since the role of oxidative stress in epilepsy has been established, it may be suggested that ghrelin and acea may have anticonvulsant effects via their antioxidant features. the discovery of inhibitors for enzymes that metabolize endogenous ghrelin and cannabinoids through new studies may contribute to the improvement of seizure resistance in epilepsy. accelerated atherosclerosis in patients with ankylosing spondylitis (as) give rise to increased cardiovascular morbidity and mortality. endothelial dysfunction could be the initial process in the development of atherosclerosis. human endothelial cell-specific molecule-1 (endocan) is a novel human endothelial cell-specific molecule. therefore, we assessed serum endocan levels and carotid intimamedia thickness (cimt) as a surrogate marker of atherosclerosis in patients with as. a total of 57 patients with a diagnosis of as according to newyork ctriteria and 50 control subjects were included in our study. serum endocan, interleukin-6 (il-6), tumor necrozis factor-a (tnf-a), c reactive protein (crp) and cimt were measured in all participants. serum endocan, il-6, tnf-a levels were measured with elisa. the other parameters were done by routine biochemical methods. as patients exhibited increased serum endocan levels and cimt compared to matched controls (p < 0.05). whereas, serum il-6, tnf-a were similar between grous. in patient with as, there were no significant differences between active and inactive patients by means of il-6, tnf-a, endocan and cimt. in as group, cimt correlated with disease duration and age (r = 0.597, p = 0.000; r = 0.721, p = 0.000). we could not find any significant correlation between serum endocan levels and parameters studied. our study shows increased cimt in as patients without traditional risk factors such as increased bmi, lipid profile compared to controls. although we found increased circulating endocan levels in patients with as, the other factors could affect increased atherosclerosis in this population because of lack of correlation between endocan and cimt. probable biomarkers could be related to increased cimt in patients with as should be investigated in larger study groups. keywords: ankylosing spondylitis, atherosclerosis, carotid intima media thickness, endocan p-09.04.4-079 investigation of pentose phosphate pathway and oxidative stress in erthrocyte infected babesia ovis a. bildik, t. karagenc ß, p. a. ulutas, n. aysul, h. aksit adnan menderes university, aydin, turkey introduction: babesia infections occur in cattle, sheep, goat, horse, dog, cat pig and rodents. in this study, the effects of babesia ovis living and present in the erythrocytes to glucose metabolism was researched. at the same time, biochemical parameters were also associated with parasitemia. materials and methods: babesia ovis (israel) cell culture was provided from dr. abel martin gonz alez oliva (portugal). culture passaged 48 or 72 hours according to parasitemia state (8-10%). biochemical analyses were performed in erythrocyte culture in which parasitemia between 1% and 16%. cell counts and hemoglobin concentration of erythrocytes culture suspension were measured at cell counter instrument and than it was washed 3 times with physiological saline, erythrocyte suspensions were stored at-80oc analysis. gssg (oxidize glutathione), gsh (reducte glutathione), nadph, glukoz 6 p dehydrogenaz, gshpx (glutathione peroxidase), gshrx (glutathione reductase) were determined by commercial kits. all experiments were done in duplicate, the results were calculated by the number of erythrocytes. results: parasitemia was positively correlated with gsh, nadph and gshrx (p < 0.05). a correlation between other biochemical parameters was not observed. discussion: the pentose phosphate pathway in erythrocytes has an important role such as to provide pentose sugar required for the synthesis of nucleic acid, to reduce glutathione, to produce nadph and to protect from methemoglobin accumulation. in studie sthat naturally infected erythrocytes with babesia parasites, it was seen to be caused to oxidative stress, however gsh results in these investigation were obtained differently . conclusion: according to the results of this study that performed in vitro, it can be suggest that their glutathione metabolism and pentose phosphate pathway of parasites may active.key words: babesiosis, gsh, gssg, nadph, g6pdh, gshpx, gshrx p-09.04.4-080 in vitro protective effect of betaine on peroxidative injury caused by ethanol and aspirin exposure on rat brain synaptosomes i. sogut 1 , g. kanbak 2 1 istanbul bilim university vocational school of health services, istanbul, 2 eskisehir osmangazi university medical school department of biochemistry, eskisehir, turkey aspirin intake of specific daily doses are advised by doctors to postmenapausal women and men above 40 years of age to prevent heart attack and even cancer in recent times. in this study, the aim is to investigate the in vitro cytototoxic effects of different doses of ethanol (50 mm, 100 mm ve 200 mm) alone and together with 100 lg/ml aspirin, and possible protective role of 1 mm betaine on rat brain synaptosomes. 15 male sprague dawley rat forebrains were divided into equal pieces and pooled to form 10 study groups. synaptosomal fractions extracted from pooled rat brains were incubated with different doses of ethanol, aspirin and betaine, and malondialdehyde (mda) levels, an important indicator of cellular damage, were measured. a significant increase (p < 0.05) was observed in mda level of 200 mm ethanol group compared to control group. different doses of ethanol (50 mm, 100 mm ve 200 mm) + aspirin exposure significantly increased (p < 0.001) mda levels compared to controls, whereas betaine administration significantly decreased (p < 0.001) lipid peroxidation caused by ethanol + aspirin treatment. we conclude that ethanol and ethanol + aspirin administration increases lipid peroxidation in rat brain synaptosomes while betaine helps prevent this peroxidative membrane injury.keywords: aspirin, betaine, ethanol, malondialdehyde (mda) p-09.04.4-081 analyses of mitochondrial biogenesis in hepatocellular carcinoma treated with berberine f. aygenli, h. c ß imen yeditepe proteomics and mass spectrometry group (yediprot), genetics and bioengineering, yeditepe university, istanbul, turkey objective: berberine (bbr) has been demonstrated to have anticancer activities against various cancer types, particularly hepatoma. in this project, we aimed to reveal the effect of bbr treatment on mitochondrial biogenesis through sirtuins and hif-1a in hepatocellular carcinoma cell line, hep3b under hypoxia. method: hep3b cells were subjected to normoxia (21% o 2 ) and hypoxia (1% o 2 ) in the presence or absence of bbr treatment. the amount of bbr was optimized via cell viability (mts) assay under normoxia. then, immunoblotting experiments were performed to identify the effect of bbr on hif-1a, pgc-1a, and sirtuins involved in mitochondrial stress. the variation in the oxphos complexes and the level of reactive oxygen species (ros) were also measured to investigate the effect of bbr on mitochondrial energy stress state. results: here, we present that cell viability was significantly decreased at 25 lm. bbr treatment has shown significant reduction in hif-1a and sirt6 which responsible for up-regulation of glycolysis. also, succinate dehydrogenase (cii) and cytochrome c oxidoreductase (ciii) of the oxphos complexes were downregulated without any change in nadh dehydrogenase (ci) or atp synthase (cv). bbr significantly abolished to oxidative stress under hypoxia, which was demonstrated as a reduction in the level of reactive oxygen species by decreasing on sirt3 expression. bbr induces the overexpression of sirt1 and its deacetylated-pgc-1a, which might be an indicator of being a potent protective agent against hypoxia by normalizing mitochondrial function and inducing mitophagy in impaired mitochondria caused by deficiency of glycolysis and oxphos. conclusion: detailed information about the communication between hif-1a and sirtuins and their relation to mitochondrial energy production was provided with the alteration of their activity by bbr treatment. it is highly expected that bbr and its derivatives might become important during the development of supplemental therapies. introduction: reactive oxygen species are involved in a variety of biological phenomena, such as carcinogenesis, inflammation and aging. among the targets of ros, dna appears most important in tumor biology since it is firmly established that cancer is a genetic disease. ros induce several kinds of dna damage, including strand breakage and dna-protein cross-linkage. fruit of zizyphus jujuba, a traditional chinese herb widely consumed in asian countries, has been reported to possess several vital biological activities. this study intends to evaluate their antioxidant activity on glioblastoma cells. materials methods: cell survival was quantified by colorimetric mtt assay. human gliblastoma cells were pretreated with 100 lm h 2 o 2 after 30 minutes 100 lm ziziphus jujuba essential oil was added to the cells for three hours. then, the cell homogenates were taken and glutathione, total oxidant and total antioxidant capacity and nitric oxide levels were estimated using spectrophotometric methods. results: ziziphus jujuba treated cells could prevent intracellular glutathione from being depleted following an exposure of h 2 o 2 . also our data suggest that ziziphus jujuba is effective in preventing h 2 o 2 induced oxidative stress and nitric oxide levels. discussion: some research showed that h 2 o 2 was over produced in the pathological process of acute and chronic neuronal toxicity, the toxicity effect of b-amyloid on the cultured neuron and neuronal cell line was mediated by h 2 o 2 . the traditional medicine recommend several medicinal plants for providing relief from various inflammatory diseases. many research has been reported that the essential oil from seeds of helping to prevent the oxidative stress and neuronal diseases in brain. introduction: toxicity by oxygen radicals has been recommended as a major cause of cancer, heart disease, and aging. oxygen radicals and other oxidants appear to be toxic in large part because they start the chain reaction of lipid peroxidation. most of the analytical techniques for peroxide determination are generally time consuming and not very suitable for routine or on line analysis. we aimed to design a new biosensor for rapid determining of oxidant agent hydrogen peroxide. materials and methods: all reagents were of analytical grade unless stated otherwise, and were purchased from sigma aldrich. firstly, the 2-hidroxymetacrilate metacriloamidoscystein nanoploymers were immobilized by binding covalently with sulphur atoms on the gold electrod's surface. free nh 2 groups of catalase enzyme make schiff bases between nanopolymer's carbonyl groups, then immobilization was actualysed with cross linking reagent glutaraldehyde. we developed a biosensor system preparing ferrociyanide, selected as a mediator, in the buffer solution. results: polyhemamac nanopolymer and catalase complex were immobilized by glutaraldehite to construct a hydrogen peroxide biosensor. the responses of the biosensor are therefore proportional to the oxidation peaks of the complex at +0.019 v potential. the cyclic voltammograms obtained from the experiments showed that, pottasiumferrociyanide mediator complex positively affected the biosensor responses for hydrogen peroxide determination. discussion and conclusion: as a result of this study, the method developed by the catalase enzyme electrode was found to be more advantageous in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, economic, practical and less time-consuming. since biosensor technology provides economical, practical, specific and sensitive results for the determination of hydrogen peroxide, it was improved very efficiently. p-09.04.4-085 impact of amoxicillin, gentamicin and cefazolin sodium antibiotics on antioxidant gene expression and enzymatic activities in mouse liver p. g€ uller 1 , h. budak 2 , m. sisecioglu 2 , m. c ß iftci 3 1 department of chemistry, faculty of science, atat€ urk university, erzurum, 2 department of molecular biology and genetics, faculty of science, atat€ urk university, erzurum, 3 department of chemistry, faculty of arts and sciences, bing€ ol university, bing€ ol, turkey reactive oxygen species (ros) are highly reactive molecules, which are produced by living organisms as a natural byproduct of the normal metabolism and environmental factors. living organisms have the antioxidant defence systems to block harmful effects of ros. the imbalance between oxidants and antioxidants is termed oxidative stress. the antioxidant defence mechanisms are divided into two groups as enzymatic and nonenzymatic defences. enzymatic defence mechanisms consist of enzymes like superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glucose-6-phosphate dehydrogenase (g6pd) and glutathione s-transferase (gst). the present study was designed to determine the effects of gentamicin, amoxicillin and cefazolin sodium antibiotics on the hepatic antioxidant system and to determine any possible correlation between enzymatic and molecular levels. for this reason, effects of these antibiotics on the transcription of the antioxidant system has been investigated by real time pcr, and then the enzyme activity of these enzymes have been measured in whole liver homogenate obtained from control group and the drug administered groups mice. our results demonstrate that administering antibiotics led to crucial inhibition of all antioxidant enzyme activity. while significant transcriptional activation for sod and cat was seen in the gentamicin treated group, the transcription of gst and g6pd was decreased. however transcriptional activation was seen for sod and cat in amoxicillin administered group, the transcription of gst was decreased as compared with the control group. in the cefazolin sodium treated group, while cat and gst transcription were elevated significantly, the expression of sod and g6pd were decreased. in conclusion, gentamicin, amoxicillin and cefazolin sodium affect the hepatic antioxidant system at the molecular and protein level. this work was supported by scientific research project of ataturk university of turkey (grant no: 2013/296). p-09.04.4-086 protective effects of curcumin supplementation on oxidant/antioxidant system changes created by organic phosphorus pesticide poisoning organic phosphorus pesticides (opp), widely used in agriculture or as insecticides in home, cause adverse health effects. chlorpyrifos is one of the most commonly used opp. we aimed to investigate the possible protective effects of curcumin (cur) supplementation, the principal curcuminoid of turmeric, on poisoning symptoms and oxidant/antioxidant system changes caused by chlorpyrifos. adult sprague-dawley rats were used. cur (30, 100 and 300 mg/kg) were administered orally for 5 days. on the sixth day, chlorpyrifos (279 mg/kg, s.c.) was administered. twenty four hours after chlorpyrifos administration, body weights, locomotor activities and body temperatures of rats were measured. following the measurements, rats were decapitated and the blood, brain and liver tissue samples were taken and prepared for the biochemical and histopathological measurements. chlorpyrifos administration increased the malondialdehyde (mda) levels but decreased catalase (cat), superoxide dismutase (sod), glutathione reductase (grx) concentrations and reduced/oxidized glutathione (gsh/gssg) ratio in the blood samples, brain and liver tissues compared with the control group (p < 0.05-0.001). the concentration of advanced oxidation protein products (aopp) were increased only in the brain tissue after chlorpyrifos administration (p < 0.001). cur administration reduced all of these changes (p < 0.05-0.001). similarly, cur at the doses of 300 mg/kg reduced the decreases in body weight, body temperature and locomotor activity with chlorpyrifos (p < 0.001). additionally, the histopathological damage scores induced by chlorpyrifos (p < 0.05-0.01) were decreased by the administration of cur (p < 0.05-0.01). our findings suggest that cur supplementation can ameliorate the poisoning effects of chlorpyrifos via supporting the antioxidant mechanisms and cur could be used for protective purposes against oxidative stress and tissue damage caused by chlorpyrifos. the effect of ogtt applied for screening in pregnancy on adenosine deaminase and xanthine oxidase activity in normal and prediabetic pregnant women z. c. ozmen, k. deveci, i. benli department of biochemistry, gaziosmanpasa university medical faculty, tokat, turkey objective: it was reported that the activities of adenosine deaminase (ada) were different in normal pregnant women and pregnant women with gestational diabetes mellitus (gdm). it was also stated that the activity of xanthine oxidase (xo) was increased in pregnant women with gdm. the objective of this study was to evaluate if glucose have effects on oxidative stress in prediabetic women by affecting ada and ox after 50 g ogtt which was applied in pregnant women for screening. methods: the serum specimens of 39 pregnant women who applied to the outpatient clinic of the obstetrics and gynecology department and had 50 g ogtt, were used in this study. ada and xo activities were analyzed in the serum specimens taken from the normal (n = 20) and prediabetic pregnant women (n = 19) in the 0th and 60th minutes of ogtt. ada and xo activities were measured with the spectrophotometric method and the u/l enzyme activity was calculated. findings: there was no significant difference between the 0th and 60th minutes regarding the ada activities in the normal and prediabetic pregnant woman groups. however, we observed a significant difference between 0th and 60th minutes regarding the xo enzyme activity in normal pregnant women (p = 0.001). in normal pregnant women, the median xo enzyme activity in the 0th minute was 0.29 (0.11-1.33) u/l and it was 0.8 (0.25-2.26) u/l in the 60th minute. nevertheless, there was no correlation between the xo activity and glucose level. as to the prediabetic pregnant women, there was no significant difference between the xo enzyme activities in 0th and 60th minutes. the results of our study showed that the xo activity increased as a response to ogtt in the normal pregnant women compared with the prediabetic pregnant women. this finding made us think that the oxidative stress caused by ogtt did not affect the xo response in prediabetic pregnant women and that there would be some adaptive mechanisms against the chronic exposure to high level glucose. rainbow trout (oncorhynchus mykiss) aquaculture continuously increases in turkey. the objective of the present study is to increase the productivity in fish farming of rainbow trout just via intervention in physical cases without the effects of any chemicals and investigate whether this conditions cause oxidative stress. in this experiment eight tanks were used and 44 rainbow trout larvae were placed in each tank and these tanks were illuminated with light in different wavelengths; natural sunlight, and incandescent long-wave (red light), medium-wave (green light) and shortwave (blue light) led lights. the experiment took 64 days. biochemical changes in rainbow trout exposed to light in different wavelengths (red, green, blue) were analysed via the variations in gr, gst, g6pd, gpx, sod and cat enzyme activities, which are significant for enzymatic antioxidant defence system and in ache activity, which plays an important role on central nervous system. maximum activity change in liver tissue was observed for gst and g6pd enzymes in fish grown under green light and for sod enzyme in fish grown under blue light. in gill tissue, sod and g6pd activities were affected the most, and in brain tissue, these were gst and sod activities. it was observed that the average weight of the fish increased 1.2 times under red and blue lights and 1.3 times under green light. the highest weight increase was observed under green light, however, antioxidant enzyme activities increased in the liver and gills and decreased in the brain tissue under this light condition. in conclusion, it was observed that productivity was 1.2 times under red light when compared with control group and it was determined that the fish grown under red light can tolerate oxidative stress more than other wavelength. p-09.04.4-089 effect of nigella sativa on biliary obstructioninduced oxidative stress and apoptosis in rats human safety concerns, since that these agents may not only cause acute toxicity via inhibition of acetylcholinesterase but they can also induce delayed toxicity in the nervous system. a key interest to the current work is the potential correlation between gene expression and cytoskeletal protein changes in differentiating neural cells exposed to sub-lethal neurite outgrowth inhibitory concentrations of specific ops, which was addressed by analysing the underlying changes in the levels of cytoskeletal gene expression and protein levels. to assess the molecular effects of op exposure, phenyl saligenin phosphate (psp), chlorpyrifos (cpf) and its metabolite chlorpyrifos oxon (cpfo) were applied at the point of induction of differentiation of rat c6 glioma and mouse n2a neuroblastoma cells and incubated for 24 hours. at sub-lethal concentrations (1, 3, 10 lm) all three ops used in this study were able to inhibit the development of neurites with no significant effect on cell viability, as determined by neurite outgrowth and mtt reduction assays. to understand the possible effects of ops on cytoskeletal gene expression, primers for genes encoding glial fibrillary acidic protein (gfap), biii tubulin, growth associated protein 43 (gap43) and neurofilament heavy chain (nefh) were optimized for qpcr analysis and the levels of the corresponding proteins were detected by western blot analysis. exposure to ops caused in most cases a reduction in the levels of cytoskeletal proteins, and the results from qrt-pcr analysis also indicated reductions in the gene expression of gfap in c6 cells, and of nefh and biii tubulin in n2a cells, in a dose dependent manner. thus, the observed changes in protein levels are at least partly due to altered gene expression. curcumin is extracted from a perennial herbaceous plant known as curcuma longa. in recent years, considerable interest has been focused on curcumin due to its use to treat a wide variety of disorders without any side effects. earlier studies have shown that curcumin has anti-apoptotic, anti-inflammatory, antiproliferative, antiangiogenic, anticancer and antiplatelet activities. the goal of the present study was to investigate the effects of curcumin on peroxy radical-induced oxidative changes in human platelets. 15 healthy volunteers were enrolled in the study. none of the study participants were on anticoagulation therapy. citrated venous blood samples were centrifuged at 200 g for 10 minute to obtain platelet-rich plasma (prp). the platelet pellet was washed and suspended with tris-nacl buffer. then, platelets were incubated with h 2 o 2 absence and presence of curcumin (50-500 lg/ ml) for 1 hours at 37°c. to determine the preventive effects of curcumin on the oxidative stress and apoptosis induced by peroxy radicals in human platelets were determined by measuring levels of of lipid peroxidation, total antioxidant capacity, caspase 3, 8 and 9 activities, and mitochondrial membrane potential. additionally, we also studied the effects curcumin on platelet aggregation induced by adp. pre-treatment of platelets with curcumin caused a marked reduction in oxidative stress, activation and apoptotic markers in a dose-dependent manner. on the other hand, pre-treatment of platelets with increasing doses of curcumin resulted in inhibition of platelet aggregation induced by adp. in the light of our findings, we suggest that curcumin may have a therapeutic potential to prevent platelet activation related disorders. people have been using mushrooms in the treatment of diseases as well as food, for centuries. most of the edible and inedible mushroom species were used in important medical studies and their effects were begun to be used in the treatment of diseases. this study focuses on the hepatoprotective effects of tricholoma anatolicum, which is endemic specie in turkey, against oxidative stress based on hydrogen peroxide (h 2 o 2 ) on hepg2 liver cancer cell line. t. anatolicum used in this study was extracted with the help of ultrasonication and fraction methods. then the cytostatic effects of extracts on hepg2 cells were explored and their hepatoprotective effects were determined. moreover, various concentrations of aqueous extract (ehta) of t. anatolicum were determined by hepg2 cells's 24-48-72 hours effect analysis on their cellular morphology, xtt and real-time cell analysis in of xcelligence device. ehta extract's cell pathway (apoptosis and necrosis) effects on hepg2 cells were determined with flow cytometry method with the help of annexin v-apc and 7aad fluorescent dye. finally, the phenolic compounds found in ehta extracts were determined with the help of hplc methods. according to xtt cytotoxicity analysis, the ehta extract values were determined as follows: 24 hours ic 50 > 2000 lg/ml, 72 hours ic 50 . furthermore, according to the real-time cell analysis made with xcelligence, ehta extracts were found to be; 24 hours ic 50 = 241.539 lg/ml, 48 hours ic 50 = 418.135 lg/ml, 72 hours ic 50 = 285.694 lg/ml. increasing concentrations of ehta extracts were determined to direct hepg2 cells to apoptosis. moreover, considering the hplc analysis -according to the reference point of 100 mg in 100 g sample-within ehta extracts, catechins and vanillic acid peaked. the final results revealed that t. anatolicum's effect on hepg2 was cytostatic at low doses, and cytotoxic at high doses. p-09.04.4-093 relationship between serum ceruloplasmin levels and coronary blood flow background: there is growing evidence that oxidative stres plays an important role in the development of the slow coronary flow (scf) phenomenon. ceruloplasmin (cp) is a copper containing metalloenyzme which has antioxidant functionthrough its ferroxidese 1 activity and is associated with cardiovascular diseases. we aimed to investigate the relationship between scf and serum cp level. methods: patients who underwent elective coronary angiography and had no significant epicardial coronary disease were included in the study. patients who had thrombolysis in myocardial infarction frame counts (tfcs) above the normal cutoffs were considered to have scf and those within normal limits were considered to have normal coronary flow (ncf). a total of 90 patients (55 subject as scf and 35 subjects as ncf) were analyzed. 5 ml blood samples were taken from the groups to study ceruloplasmin activity. serum ceruloplasmin levels were determined spectrophotometrically. results: the serum cp levels were statistically lower in scf group than in the ncf group (414 ae 79 versus 469 ae 99 ng/ml, p = 0.009). also there was a significant correlation between serum cp levels and tfcs (r = à0.378, p = 0.013). conclusion: the findings of this study suggests that patients with scf had lower serum cp levels correlated with tfcs. we concluded that reduced serum cp levels might represent a biochemical marker of scf. introduction: sleeve gastrectomy (sg) has been used for the surgical treatment of morbid obesity, as a first step or definitive treatment. alterations of thyroid hormones in gastrointestinal surgery were previously studied. the aim of the present study was to determine the effects of triiodothyronine (t3) supplementation on oxidative stress parameters in anastomotic tissue level. materials and methods: twenty-four male wistar albino rats were divided into control (n 12), and experimental (n 12) groups. rats were underwent a sg, with a hand-sewn suture. experimental group rats received a single dose of t3 (400 mg/100 g) in postoperative day. rats were sacrificed on postoperative day 7. serum thyroid stimulating hormone (tsh), free t3 (ft3), and free thyroxine (ft4) were analysed using elisa. each tissue was homogenized in ice-cold pbs (ph: 7.4) and centrifuged at 2000 rpm for 20 minutes (4°c) to avoid contamination with cellular debris. the supernatants were used to measure total oxidant status (tos), total antioxidant status (tas), nitric oxide (no) and malondialdehyde (mda) levels. all tissue parameters were analysed by spectrophotometric methods. oxidative stress index (osi) values were calculated. results: rats given t3 hormone had not decline in ft3 levels compared with the control groups. a significant decrease in ft4 levels was found in t3 given rats on postoperative day 7. whereas tissue tos levels did not alter by thyroid hormone treatment, tas levels significantly decreased. osi values were not statistically different in tissues. tissue no levels were also similar in both groups. mda levels increased in t3 given rats compared with the control group. discussion and conclusion: this study showed that anastomosis after sleeve gastrectomy is associated with decreased ft4 level. although tos levels and osi values were similar in both groups, t3 supplementation induced lipid peroxidation by increasing tissue mda levels that might deplete tissue antioxidant level. reactive oxygen species (ros) are reactive chemical molecules, which are produced by living organisms as a natural byproduct of the normal metabolism and environmental factors. although intracellular ros level is essential molecules for the signal transduction pathways, elevated intracellular level of ros leads to oxidative stress that causes damage to dna, proteins and lipids. therefore, excessive ros levels have to be eliminated by antioxidant defence systems. tip60 (tat interacting protein, 60 kda) is a histone acetyltransferases (hats) that catalyses multiple functions in metabolism such as dna repair, apoptosis, etc. we thought that if tip60 has a role in signal transduction and apoptosis, it might have direct or indirect relationship with the antioxidant system. the present study was designed to determine the impact of tip60 gene on the hepatic antioxidant enzymes including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), and glutathione reductase (gr) both gene and protein level. for this reason, quantitative gene expression analysis on the antioxidant system has been investigated by real time pcr, quantitative protein expression has been investigated by western blot analysis, and then the activity of these enzymes have been measured in whole liver homogenate collected from control and liver-specific tip60 conditional knockout mice. additionally, since any change of reduced glutathione (gsh), oxidized glutathione (gssg), malondialdehid (mda), and hydrogen peroxide (h 2 o 2 ) level in the cell might be an indication for the accumulation of ros, the relative levels of them were also studied. our data showed that the absence of tip60 affects the antioxidan system both gene and protein level. in conclusion, our initial data suggest that tip60 may be essential for the (ros) homeostasis and redox regulation. curcumin is a major chemical component produced from the rhizome of the plant curcuma longa. lt has been demonstrated that curcumin has an antioxidant, anti-inflammatory, and antiproliferative effects and, protects tissues against ischemia/reperfusion (i/ r) injury. i/r has detrimental effects on transplanted organs including uteri. the major consequence of l/r injury is oxidative stress leading to the generation of ros. uterine transplantation (ut) has been gaining popularity around the worid in the past few years. the aim of our study was to examine the antiapoptotic effects of curcumin on uterine l/r injury. the rats were randomized into three groups of seven rats each, group i consisted of rats that did not receive any treatment, group ll exposed to 0.5 hour of lschemia and 1 hour of reperfusion, group iii of rats that received intraperitonealy curcumin (200 mg/kg) 0.5 hour before the induction of i/r. then, the rat uterine tissue levels of mda, tac, and activities of caspase 3, 8 and 9 were measured. furthermore, the apoptotic index was determined immunohistochemically by the tunel method using light microscopy. biochemical analysis results showed that curcumin decreased the mda and caspase-3 and 9 ieveis, and increased the uterine tissue levels of tac but, caspase 8 activity did not changed by curcumin suggesting that curcumin induces apoptosis via intrinsic apoptotic pathway. on the other hand, an high apoptotic index was observed in i/r group (27.37 ae 11.56%) and decreased after treatment with curcumin (8.69 ae 7.49%). in conclusion, we demonstrated the protective effect of curcumin on apoptosis immediately after reperfusion induction in uteri and we can say that curcumin could improve ir injury and decrease apoptotic index. we propose that curcumin may be a novel approach for improvement of uteri i/r injury. glutathione and the related enzymes belong to the defence system of the tissues against chemical and oxidative stress. these enzymes especially glutathione s-transferase are often overexpressed in tumor cells and are regarded as a contributor to their drug resistance and are thought to play an important role in cancer progression. the purpose of this study is to evaluate the protective effects of chlorophylline as an antioxidant molecule which has inhibitory effects on gst p1-1 on chemically-induced breast cancer model. in our previous work, we had observed that this molecule led to proliferation in breast cancer cells. in this study, n-methyl-n-nitrosourea (mnu) used for inducing carcinogenesis in eighteen, 21-day-old female sprague-dawley rats. chlorophylline and mnu solutions were injected intraperitoneally when the rats were 21, 28, 35 and 42 days old. their weight and tumor diameters were measured throughout the 5 months study period. at the end of the study, all animals were sacrificed and determined both glutathione levels and related enzymes activities (gluathione s transferase, glutathione reductase and glutathione peroxidase) in tumor and tissues such as liver, kidney, heart and spleen were studied and analyzed. as a result, in breast cancer model, glutathione and related enzyme activities were protected by chlorophylline treatment whereas mnu made them decreased compared to the control group. in conclusion, chlorophylline with antioxidant features decreased the toxic effect of mnu by regeneration of glutathione and enhancement of its related enzyme activities. the use of antioxidant molecules, because of proliferative effects and defence-oriented behaviours, should be discussed in cancer therapy. p-09.04.4-100 effect of overexpression of bacillus catalase on lactococcus lactis nisin production z. girgin ersoy, s. tunca gedik gebze technical university, kocaeli, turkey nisin, has been used commercially (e234) in food preservation for approximately 40 years. it's the only bacteriocin which is approved by world health organization as a food additive. the fundamental problem that limits nisin usage in food preservation is low product yield by producer strains. because of high commercial potential of nisin, studies about increasing the production efficiency of nisin is kept in the forefront in recent years. since nisin biosynthesis and bacterial growth are occuring in parallel to each other, conditions that promote growth are also expected to encourage nisin production. it is known that, when supplied with exogenous heme, lactococcus lactis cells can respire under aerobic conditions and produce higher energy which in turn cause higher biomass. however, aerobic conditions also cause oxidative stress since catalase enzyme, which detoxify hydrogen peroxide, is absent in l. lactis. in this study, to complete the missing component of the defence mechanism of l. lactis, catalase (kate) gene of aerobic bacterium bacillus subtilis was overexpressed in facultative anaerobe l. lactis cells. for this, kate gene of b. subtilis was amplified by polymerase chain reaction (pcr). plasmid constructions were established in e. coli by using an e. coli-l. lactis shuttle vector and then the recombinant plasmid was transferred to l. lactis cells by electroporation. the presence of catalase activity in the recombinant strain grown on the solid medium was first detected by dropping hydrogen peroxide directly on the cells, then with enzyme assays. fermentation studies are going on to determine nisin production of the recombinant strain. to the best of our knowledge, this study presents the first preliminary results that shows the effect of overexpression of catalase gene on nisin production. cancer is among the leading causes of morbidity and mortality worldwide. chemotherapy is one of the major cancer treatment strategies. remarkably, natural products have garnered increased attention in the chemotherapy drug discovery field because they are biologically friendly and have high therapeutic effects. humic acid (ha) is a natural product which is forming during decomposition of organic matter in humus. in recent years, there are some resarches on the medical use of humic acid. the present study investigated anticancer effects of ha in several human cancer cell lines. ha was purchased from sigma-aldrich. in this study, we used several human cancer cell lines: the human breast cancer cell line, mcf-7, colon cancer cell line, ht-29, lung adenocarcinoma cell line, a549, and servical cancer cell line, hela. the cells were maintained in dmem medium supplemented with 10% heatinactivated fbs and 1% penicillin/streptomycin. cells were grown in petri dishes in a humidified atmosphere containing at 37•c. five different concentrations (100 ug/ml, 50 ug/ml, 25 ug/ ml, 10 ug/ml, 5 ug/ml) were prepared using a stock solution of ha. the cell proliferation and migration was measured. on the other hand, the apoptotic mechanisms induced by ha in cancer cells were investigated using "apoptosis antibody array kit". the effects of ha on cancer cell lines were evaluated over 72 hours. according to our results, ha induced a decrease in ht-29, a549 and hela cell numbers in a dose-dependent and time-dependent manner. contrary to this, ha induced proliferation of mcf-7 cells in dose dependent manner. ha inhibited cell migration in a dose dependent manner except mcf-7 cell line. it was also determined apoptotic pathways in cancer cells induced by ha. it was concluded that ha has an inhibitory effect on certain some cancers. since the effect of ha on tumor progression is unknown, further studies are needed to clarify the rol of ha on cancer activity. p-09.04.4-102 chronic immobilization stress in rats: fluoxetine and amisulpride protects against chronic immobilization stress-induced biochemical alterations in the present study, the effects of amisulpride and fluoxetine on serum total sialic acid (tsa) and lipid bound sialic acid (lsa) levels was investigated in the rats exposed to chronic immobilization stress. the study was administered using 40 male wistar albino rats weighing 200-250 g. rats were divided into five groups (n = 8/ group). group i comprised the control group, group ii was exposed with saline + immobilization stress (30 minutes daily immobilization stress for 15 days and 0.5 ml saline was administered perorally 30 minutes before immobilization), group iii was exposed amisulpride (10 mg/kg/day) + immobilization stress, group iv was exposed fluoxetine (10 mg/kg/day) + immobilization stress and v. group was exposed amisulpride (10 mg/kg/ day) + fluoxetine (10 mg/kg/day) + immobilization stress. statistical analysis showed that the saline + stress, amisulpride + stress and amisulpride + fluoxetine + stress groups was significantly higher than the control group with regards to tsa levels (p < 0.05). whereas, the fluoxetine group was significantly lower than the group regarding tsa levels (p < 0.05). on the other hand, saline group was significantly higher than the control group with regards to lsa level (p < 0.05). whereas, no significant differences in lsa levels were observed in the amisulpride, fluoxetine and amisulpride + fluoxetine groups, as compared to the control group (p > 0.05). the present study demonstrated beneficial effect of fluoxetine and amisulpride on the concentration levels of lsa and tsa in stress. p-09.04.4-104 protective effect of borax and boric acid on total sialic acid and lipid-bound sialic acid levels against 3-methylcholanthrene and benzo(a)pyrene induced oxidative stress in rats s. ekin 1 , g. oto, f. g€ ok, y. karakus, d. yildiz 1 y€ uz€ unc€ u yil university, van, turkey the present study was performed to investigate total sialic acid (tsa) and lipid bound sialic acid (lsa) levels as possible in vivo chemoprotective effect of borax (bx) and boric acid (ba) again-st3-methylcholanthrene (3-mc) and benzo(a)pyrene (b(a)p) induced oxidative stress in rats. the rats were divided into nine groups of six rats each. group i: control, untreated animals were given % 0.9 nacl, group ii: the b(a)p were administered 25 mg/kg via ip. four times. group iii: the 3-mc-treated animals were administered 25 mg/kg via ip. four times, group iv: ba was given 300 mg/l/day with water. group v: bx was given 300 mg/l/day with water. group vi: b(a)p 25 mg/kg via ip four times + ba 300 mg/l/day dosage with water. group vii: 3-mc 25 mg/kg via ip four times + ba 300 mg/l/day with water. group viii: b(a)p 25 mg/kg via ip four times + bx 300 mg/l/ day dosage with water. group ix: 3-mc 25 mg/kg via ip four times + bx 300 mg/l/day with water. the experimental period was continued for 150 days. statistical analysis showed that the 3-mc + ba group was significantly higher than the control group with regards to tsa and lsa levels p < 0.001, p < 0.001,p p-09.04.4-105 effects of aluminum exposure on trace elements in rat tissues b. ozturk kurt, s. ozdemir department of biophysics, cerrahpasa medical faculty, istanbul university, istanbul, turkey aluminum (al) is the most abundant metal and the third most abundant element in the earth's crust. people are constantly exposed to al which is found in most rocks, soils, waters, air and foods, due to a result of an increase in industrialization and improving technology practices. the study was designed to examine the possible effects of aluminum exposure in different durations on trace elements in rat tissues. twenty-four healthy male wistar rats weighed 180-200 g were randomly divided into three groups: control group (gc) received only drinking water, short-term group (gs) and long-term group (gl). the study groups were orally exposed to 40 mg/kg body weight alcl 3 in drinking water for 8 and 16 weeks, respectively. at the end of the treatment period, rats were sacrificed and the kidney, liver, brain and cerebellum tissues were removed to analyse the levels of al, ar, b, ni, si, cr, cu, fe, mg, mn, se, cu and zn by icp-oes. the statistically significant increase were determined in cerebellum al, cu, as, b and cr levels in gl according to the gc. while as levels were statistically increased, ni levels were decreased in gl in the kidney and liver. while cu, mg and cr levels were higher, se and b levels were lower in the gs than gc in the brain. there were no significant difference in si and mn levels. as a result of our study, it may be concluded that al accumulation may lead to changes in tissue trace element levels. tacal, o., 266 tacal, ö., 321 take, g., 412 takic, m.m., 417 taldykbayev, z., 416 talim, b., 292 tamashevski, a., 382 tamer, f., 144 tamer, l., 245, 279, 395 taneva, s., 209 taniyan, g., 275, 306 tanrisev, m., 288 tanriverdi, e.c., 245 tanriverdi, k., 279 tarhan, m., 161 tarhan, t., 315 tartar, s., 387 tas, a., 204, 293 taskin, a., 406 taskin, t., 339 taskiran, e., 271 taskiran, b., 328 taskoparan, b., 221 taspinar, r., 301, 302 tastan, ö., 271 tatli, ö., 340 tauraite, d., 206 tay, t., 344 tayman, c., 354 taysi, s., 389, 392 teker, h.t., 270 tekes, s., 361 tekin neijman, s., 410 tekin, m.h., 188, 297 tekin, g., 191, 256 tekin, m., 196 tekin, n., 155 tekin, g., 256 telci, d., 187, 307 telefoncu, a., 151 temel, h.e., 158, 159, 160 temelie, m., 267 temizgül, r., 347 temlyakova, e., 168 teplova, v., 372 terashima, r., 284 tercan avci, s., 197 terekhov, s., 381 tereshenko, o., 304 terzi gulel, g., 149 terzi, e., 300, 302 terzi, e., 301 terzioglu, g., 296 terzioglu, o., 272 testoni, g., 348 tetik vardarli, a., 282 tevdoradze, t., 148 tevzadze, l., 148 tezcan, ö., 308 tezcanli kaymaz, b., 282 thielens, n., 228 thomaidou, d., 265 thomas, a., 228 thornton, j.d., 216 ticea, a.c., 168 tikhonova, a., 287 tileva, m., 209, 210 timofeev, v., 198, 202 timofeev, v., 202 timofeeva, e., 383 tipirdamaz, r., 401 tiryaki, m., 300, 302 tok, m., 140 tokay, e., 365 toker, a., 360, 402 tokgun, o., 284 toksoy öner, e., 138 tokyol, ç., 390 toman, r., 317 tomasi, a., 410 tombul, n., 333, 337 tooke, c., 231 topaloglu, h., 292 topbas, m., 200 topcu, b., 247 topcu, c., 292 topcu, g., 144, 358, 377 topçu, v., 393 topcu, c., 417 topçuoglu, c., 193, 416 topel, h., 197, 264 toprak, b., 223 toprak, m.s., 351 torac, e., 384 tosner, z., 383 tosun, m., 308, 354 toy, h., 143 toymentseva, a., 239 tozkoparan, b., 246 trabulus, d.c., 256 trantirek, l., 229 trantirkova, s., 229 trchounian, a., 165, 324 trizna, e., 200, 289 troshagina, d., 371 tro t, m., 269 truncaite, l., 149, 363 tsakalidou, e., 170 tsarkov, d., 233 tsarkova, m., 233, 235 tsigara, e.g., 206 tsigara, m.g., 206 tsverava, l., 266 tsvetkova, e., 253 tsyba, l., 362 tsyganov, d., 158 tsymbal, d., 165 tüfekçi, a.r., 241, 358 tufekci, a.r., 296 tufenkci, h., 139 tuglu, m.i., 300, 309 tuglu, i., 200 tuli, a., 142, 182, 237, 403 tuli, a., 352 tulubas, f., 247 tunali, g., 226 tunca gedik, s., 143, 408 tunca gedik, s., 221 tunçdemir, m., 396 tuncel, h., 378 tuncel, h., 273 tunçer, s., 222 tuncer, e., 161, 261 tuncer, z.s., 395 tuncer, b., 133 tuncer, e., 273 tuncez akyurek, f., 185 tunçez akyürek, f., 359 tuner, h., 333 tüney kizilkaya, i., 325 tural, b., 315 tural, s., 315 turan, i., 330 turan, m., 161, 261 turan, v., 173 turan, y., 333 turan, c., 411 turano, p., 217, 290 türel, s., 329, 341 turhan, t., 193, 242 turhan, y., 316, 317 turk, s., 373 turkan, a., 219 türkcan kayhan, c., 135 turkekul, k., 299 türkel, s., 176 turkeri, o.n., 245 turkeri, n., 141 turkkan, b., 257 turkmen, s., 401 türkmen, n., 180 turkon, h., 223 tutar, y., 161 tüten, a., 351 tutkun, e., 196, 275 tüylü küçükkilinç, t., 285 tuz, m.u., 321 twardovska, m., 335 tyapkina, o., 371 tzartos, s., 268 photoprotective activity of vulpinic and gyrophoric acids towards ultraviolet b-induced damage in human keratinocytes evaluation of the sunscreen lichen substances usnic acid and atranorin pleiotropic anticancer activity of selected nutraceuticals against mcf-7 bucharest, 3 national institute for marine research and development "grigore-antipa uk in some adult and elderly populations the acute and/or persistent infection with the common intracellular respiratory pathogen chlamydia pneumoniae (chl) may be associated with increased risk of developing obesity or cardiovascular disorders. thus, 19 microelements modifying oxidative stress status were determined by icp-ms/ms in the hno 3 diluted serum samples collected from chl-positive adult females (n = 39) living in urbanized area in poland. chl infection was confirmed by igg+ antibody elisa and real-time pcr assay. all females were classified under their body-mass index values to the normal-weight (nw), over-weight (ow) and obese group (ob) although there are many drugs currently used in the treatment of peptic ulcer, such a drug providing radical treatment without side effects is not available. since oxidative stress is involved in peptic ulceration, this study was designed to investigate antiulcerogenic and antioxidant effects of hippophae rhamnoides l. ether extract on indomethacine-induced stomach ulcer in rats. materials and methods: thirty-five sprague dawley male rats (weights ranging 180-220 g) were randomly divided into 7 groups, as each composed of 5 rats. after hippophae rhamnoides l. leaf ether extracts of 100 mg/kg, 250 mg/kg and 500 mg/kg doses and 20 mg/kg doses of famotidin orally administered, 25 mg/kg doses of indomethacine were orally applied to rats in order to make ulcer. on the sixth hour of indomethacin administration all rats were sacrificed using thiopental (50 mg/kg). the stomachs were removed, and ulcer areas were evaluated macroscopically. superoxide dismutase activity (sod), glutathione (gsh) and malondialdehyde (mda) levels in stomach tissues of rats were determined by elisa method with respective kits conclusions: we can conclude that the ether extract of hippophae rhamnoides l. leaves reduces free radical formation and has antiulcerogenic effects on stomach tissue control group; 2 hours torsion/4 hours detorsion group (t/d); all other groups were saturated for four days egcg, cape and egcg+cape (10lml/kg). sections were taken from bouine's-fixed and paraffin-embedded testicular tissue blocks and stained with h&e. immunohistochemistry was applied for the detection of pi3k, akt and mtorc. intensities were evaluated as mild (1), moderate (2) or strong (3). serum 8ohdg, plasma mda levels were analyzed using elisa method. results were analyzed by anova statistical test. testis samples in control group exhibited normal histological morphology. disorganization and separation of seminiferous tubule cells and accompanying interstitial edema and vessel dilation were while mda level decreased significantly in cape+egcg group, 8ohdg level showed significant increase in cape group. in conclusion, cape and egcg exerted protective effects on tt. effects may be achieved through pi3k/ akt/mtorc pathway involved in cell proliferation, angiogenesis, apoptosis. prophylactic use of egcg prior to tt surgery improved testicular morphology, therefore could prevent destructive effects of tt lmol/l), c18 (0.58 ae 0.278 lmol/l)), respectively. conclusions: this study is important for konya region, mitochondrial fatty acid b-oxidation disorders studies subject areas. this study is the first study to assess acylcarnitine levels of patients living in our region. we believe that our results will be useful for future studies. key words: acylcarnitine, mass spectrometry, dried blood spot p-09.04.4-137 binding of fas and cu(ii) ions to hsa changes its cys34 thiol group antioxidant capacity and carbonylation pattern with methylglyoxal binding of cu(ii) ion (0.1 mol/mol hsa) led to increase of k' value if fish oil extract was present, but for other fas k' value decreased. the content of free hsacys34-sh decreased for 10% after cu(ii) ion binding, and during 24 hours incubation at 37°c, it was further decreased for 10% (stearic acid, mixfas) and 20% (myristic, fish oil extract, oleic acid). carbonylation of fa-hsa-cu(ii) complexes with mg (20 mol/ mol hsa), lead to decrease in cys34-sh content depending on fa present: 30%-35% for myristic and stearic acid, 40% for oleic acid and mixfas and 40% for fish oil extract. carbonylation of fa-hsa-cu complexes could contribute to further enhancement of oxidative and carbonyl stress in diabetes as well as other diseases 151 pirto ek p-05.03.3-007 anti-proliferative and inducing apoptosis of the hydro alcholic achelia. wilhelmsii extract on human breast adenocarcinoma cell lines mcf-7 and mda-mb-468 background: vitamin d deficiency is associated with several conditions and/or diseases like inflammation, atherosclerosis, cardiovascular disease and mortality. several studies showed that lower vitamin d levels were associated with high serum levels of inflammatory biomarkers. ykl-40 is a glycoprotein, secreted by macrophages, neutrophils and different cell types. it is also associated with inflammation and pathological tissue remodeling. in this study, we aimed to evaluate relationship between the vitamin d deficiency and ykl-40 levels. methods: our study group includes 45 subjects with vitamin d deficiency (group 1) and 40 age and sex-matched healthy subjects with normal serum levels of vitamin d (group 2). plasma 25 (oh) vitamin d levels were measured with liquid chromatography-tandem mass spectrometry (lc-ms/ms) method. plasma ykl-40 analysis was performed by elisa. serum hs-crp levels were measured with nephelometric method. results: plasma vitamin d levels below 20 ng/ml were accepted as vitamin d deficiency. although we could not find any significant differences by means of serum hs-crp levels between groups (p > 0.05), plasma ykl-40 levels were significantly higher in group 1 than group2 (p < 0.05). conclusions: in literature, vitamin d deficiency is associated with inflammation. in our study, we found similar hs-crp levels between groups and higher ykl-40 levels in group 1. vitamin d deficiency may be related to increased ykl-40 levels in terms of causing chronic inflammation.keywords: vitamin d deficiency, ykl-40, inflammation. evaluation and comparison of tnf-family ligands and receptors genes in mice and humans by bioinformatics techniques stance called plaque builds up inside the coronary arteries. apelin is a novel endogenous peptide with inotropic and vasodilatory properties and is the ligand for the angiotensin receptor-like 1 (apj) receptor. we aimed to determine genotype and allele frequencies of apj receptor a445c gene polymorphism in turkish patients with cad and healthy controls by rflp-pcr. this study was performed on 159 unrelated cad patients and 62 healthy controls. we obtained aa, ac and cc genotype frequencies in cad patients as 41.5%, 49.1% and 9.4%, respectively. in the control group, frequencies of genotypes were found as 35.5% for aa, 48.4% for ac and 16.1% for cc. we did not observe difference in apj receptor a445c polymorphism between cad patients and healthy controls (v 2 = 2.178; df = 2; p = 0.336). the a allele was encountered in 66% (210) of the cad and 59.7% (74) of the controls. the c allele was seen in 34% (108) of the cad and 40.3% (50) of the controls. allele frequencies were not significantly different between groups (v 2 = 1.57; df = 1; p = 0.225). the frequencies of apj receptor a445c genotype were not significantly different between control and patients. individuals with cc genotypes had significantly higher weight, systolic and diastolic blood pressure levels and systolic blood pressure than other genotypes, p ≤ 0.05. in addition, hdl-c level was found decreased, but this reduction was not statistically significant. contrarily, the low levels of weight, sbp, dbp and tc were statistically significant in the subjects with aa genotype in cad. in conclusion, cc genotype carriers may have more risk than other genotypes in the development of hypertension in cad. we suggest that this polymorphism may not be a marker of cad, but it may cause useful in function of the apelin/apj system and may be a genetic predisposing factor for diagnostic processes and can be helpfull in finding new treatment strategies. p-08.01.4-032 comparative genomics/proteomics analyses of single amino acid repeat containing proteins across different vertebrate taxa a. g. keskus, o. konu department of molecular biology and genetics, bilkent university, ankara, turkeyconsecutive runs of single amino acids lead to overrepresentation of certain physicochemical properties in protein sequences. researchers also demonstrated a link between single amino acid repeat (saar) containing proteins and neurodegenerative diseases as well as biological functions. moreover, saar frequencies were shown to vary across species based on selected orthologous proteomes and/or proteins. hence, analysis of whole proteomes across multiple vertebrate taxa may provide additional species-and sequence-specific trends for saars. in addition, there is a need for testing the observed saar occurrencesthe aim of this study is to evaluate the effect of quercetin (q) on liver injury secondary to cerulein induced-acute pancreatitis (ap). for this reason, rats were randomly divided into four groups (8 rats for each group) control group received physiological saline, four time and dimethyl sulfoxide, two time, at 1 hours intervals, intraperitoneally (i.p.). cerulein group received cerulein (50 lg/ kg-rat weight, in physiological saline), four times, and dimethyl sulfoxide (1%), two times, at 1 hour intervals, i.p. quercetin pretreatment (q+cer) group received quercetin (100 mg/kg-rat weight, in dimethyl sulfoxide) one time, one hour before cerulein treatment and physiological saline, one time, six hour after cerulein treatment. quercetin post-treatment (cer+q) group received dimethyl sulfoxide, one time, one hour before cerulein treatment and quercetin, one time, six hour after cerulein treatment. cerulein treatment increased significantly vascular congestion in hepatic cells. quercetin treatment also decreased significantly vascular congestion. the liver mda and carbonyl levels in cerulein group were significantly higher than the control group (p < 0.01, p < 0.001, respectively). the mda and carbonyl levels in q+cer group decreased significantly compared to the cer group (p < 0.001). the mda, carbonyl, mpo levels in cer+q group were significantly lower than the cer group (p < 0.001). the gssg/gsh ratio of q+cer and cer+q groups were significantly lower than the cer group (p < 0.05, p < 0.001, respectively). the sod activity in cer group was significantly lower than the control group, but the sod activity in q+cer and cer+q groups was significantly higher than the cer group (p < 0.05).this study shows that quercetin treatment was reduced the severity of liver injury secondary to cerulein induced-ap as reflected by changes in the parameters of hepatic oxidant and antioxidant. p-09.04.4-029 identification of water extract of propolis components by using different columns in gas chromatography-mass spectrometry propolis is a natural material obtained by honey bees from various plants. protective effect of propolis against damages of free radicals is due to different compounds within propolis. the aim of this study is to identify qualitatively and quantitatively the chemical composition of water extract of propolis (wep) provided by erzurum region using rtx-1 and rtx-5 ms column of gas chromatography-mass spectrometry (gc-ms) and to compare with two columns.in this study, wep of 25 mg/ml was prepared, cleared by membrane filter of 0.45 lm and freezed at à80°c. then, it was lyophilized up to dry form and derivatized to apply for gaseous form. 7 mg of dry extract was reacted with 350 ll pyridin + 700 ll bis-trimethylsilyl trifluoroacetamide (bstfa) mixture including 1% trimethylchlorosilane (tmcs) and incubated for 30 minutes at 100°c. all analyses were performed with shimadzu gcms-qp2010 ultra by using a flame ionisation detector (fid). rtx-1 and rtx-5 ms capillary columns and helium for carrier gas at a flow time of 1 ml/minute were used. injection was applied on split mode at 250°c. derivatized propolis sample was injected as 2 ll, initial column temperature was adjusted as 60°c, then increased to 240°c with increments of 3°c. total analysis time was determined as 62 minutes. relative percent amount of separated compounds was calculated from total ion chromatogram with computerised integrator. all components were defined by using nist and wiley libraries.peaks obtained from rtx-1 column were much more than those of rtx-5 ms. on the other hand, analyses performing with both two columns have similar carbohydrate, aromatic acid and other acid contents.consequently chemical constituents of wep were determined qualitatively and quantitatively with gc-ms. it was concluded that rtx-1 column among both columns differentiating for polarities may produce more compounds in the propolis analysis. introduction: aquatic environment can be mostly contaminated by mixtures of metals. biochemical parameters have gain importance to characterize the effects of metals on aquatic organisms. glutathione s-transferase (gst) and its substrate glutathione (gsh) are important parameters of antioxidant defense system of fish metabolism due to their vital role in xenobiotic conjugation. objective: the goal of the current study to evaluate the changes in gst and gsh levels in response to cd, zn and cd+zn effects after 14 and 28 exposure days. materials and methods: fish were obtained from cukurova university fish culturing pools (turkey). fish were exposed to 1.0 lg/ml of cd (cdcl 2 .h 2 o) and zn (zncl 2 ), and their mixture, for 0, 14 and 28 days. at the end of the exposure period, liver tissues were dissected and homogenized in a phosphate buffer (ph 7.4). homogenates were centrifuged at 10,000 g (30 min, +4°c). supernatants were stored at à80°c until the analysis. one-way anova was used to compare data (mean ae se) followed by duncan's test (p < 0.05). results: gst and gsh changes were recorded as decreases after all metal exposures at day 14. although 28 day exposure was found as less effective, combined effects caused significant decreases in gst and gsh levels. also longer exposure durations were appeared to be more effective in that situation. conclusions: significant decreases in gst and gsh levels could be occurred due to increased reactive oxygen species caused by metals particularly their combined effects. metal type, their single and combined effects and also exposure duration should be also taken into account when considering the antioxidant system response. gst and gsh might be considered as sensitive biomarker in toxicity assessment of metal mixtures.financial support: this study was supported by a grant from c ß ukurova university (turkey).background/aims: the activation of lectin-like oxidized low density lipoprotein receptor-1 (lox-1) on endothelial cells leads to intracellular oxidative stress and inflammation and a feed-forward cycle of injury in diabetes, since both oxidized low density lipoprotein (oxldls) and glucose increase lox-1 expression. quercetin (qr) is part of a subclass of flavonoids called flavonols. polyphenolic compounds affect the development of atherosclerosis not only through antioxidant properties but also by modulation of serum lipids, thereby influencing the immune and inflammatory processes associated with the development of atherogenic diseases. we investigated the effects of dietary qr on endothelial dysfunction mediated by oxidized low density lipoprotein (oxldl)/lectin-like oxidized low density lipoprotein receptor-1 (lox-1) in animal model of type 2 diabetes mellitus. methods: we compared 4 groups of male adult wistar albino rats: a control group, an untreated diabetic group, diabetic groups treated with qr, and qr group. diabetes was induced by a single injection of stz (50 mg/kg). animals were kept in standard condition. in 30 day after inducing diabetic, serum was collected for biochemical parameters. glucose, lipid profiles, microalbuminuria, oxldl and lox-1 levels were determined. results: serum triglyceride, ldl, vldl levels in diabetic control group (without treatment) was significantly higher than control group (normoglycemic untreated group). supplementation with quercetin decreased serum total cholesterol and increased hdl-cholesterol compared with the control group. serum oxldl and lox-1 levels in diabetic control group (without treatment) were significantly higher than control group (normoglycemic untreated group). conclusions: consumption of quercetin reduced oxldl and lox-1 levels. thus, quercetin could be effective in improving hyperglycemia, dyslipidemia, and endothelial damage in type 2 diabetes. p-09.04.4-055 investigation of some oxidative stress parameters in bdnf heterozygous mice liver tissue a. bodur 1 , i. ince 1 , i. abidin 2 , a. alver 1 objectives: the aim of this study was to evaluate the possible protective effects of nigella sativa (ns) against cholestatic oxidative stress and liver damage in the common bile duct ligated rats. methods: a total of 24 male wistar albino rats were divided into three groups:sham control, bile duct ligation (bdl) and bdl+received ns; each group contain 8 animals. the rats in ns treated groups were given ns (0.2 ml/kg) once a day orally for 4 weeks starting 3 days prior to bdl operation. results: the changes demonstrating the bile duct proliferation and fibrosis in expanded portal tracts include the extension of proliferated bile ducts into lobules, mononuclear cells, and neutrophil infiltration into the widened portal areas were observed in bdl group. treatment of bdl with ns attenuated alterations in liver histology. the alpha smooth muscle actin (a-sma), transforming growth factor beta (tgf-b1) and the activity of tunel in the bdl were observed to be reduced with the ns treatment. bdl significantly increased the tissue hydroxyproline (hp) content, malondialdehyde (mda) levels, and decreased the antioxidant enzyme (superoxide dismutase (sod) and glutathione peroxidase (gpx)) activities. ns treatment significantly decreased the elevated tissue hp content and mda levels and prevented the inhibition of sod and gpx enzymes in the tissues. conclusion: the data indicate that ns attenuates bdl-induced cholestatic liver injury, bile duct proliferation, fibrosis, apoptosis and oxidative stress. the hepatoprotective effect of ns is associated with antioxidative potential. organophosphorous compounds (ops) are used widely as pesticides for agricultural purposes, as oil additives or as flame retardants. however, due to their widespread use, there are major introduction: in this study, the antiulcerogenic effect of a water extract (cawe) obtained from a spices sample, cinnamomum aromaticum, was investigated using indomethacin-induced ulcer models in rats. materials and methods: experimental groups consisted of six rats. antiulcerogenic activities of 50, 100, 200 and 400 mg/kg body wt. doses of the cawe were determined by comparing the negative (treated only with indomethacin) and positive (famotidine) control groups. results and discussion: although all doses of the cawe showed significant antiulcerogenic activity as compared to negative control groups, the highest activity was observed with 400 mg/kg body wt. doses (45%). the cawe showed similarly antioxidant activity when compared with trolox and ascorbic acids used as positive antioxidants. in addition, the activities of catalase (cat) and myeloperoxidase (mpo) enzymes were determined in the stomach tissues of rats and compared with those of the negative and positive control groups to expose the effects of these enzymes on antiulcerogenic activity. the enzymatic activities of cat and mpo and lipid peroxidation (lpo) level in indomethacin-administrated tissues were increased significantly by indomethacin in comparison to control groups. these enzymes and lpo level were decreased, however, by the cawe. in contrast to lpo level, cat and mpo activities, glutathione (gsh) level was decreased by indomethacin and increased by all doses of cawe and famotidine. the present results indicate that the cawe has a protective effect in indomethacin-induced ulcers, which can be attributed to its antioxidant potential. introduction: thiol groups (-sh) are important anti-oxidants and essential molecules protecting organism against the harmful effects of oxidative stress. the aim of our study is to evalute thiol-disulphide homeostasis with a novel and automated method in patients with prostate cancer (pc) before and after radical prostatectomy (rp). material and methods: 18 patients with prostate cancer and 17 healty control subjects were enrolled into the study. plasma samples were collected from patients before rp and 6 months after the rp operation. thiol-disulphide homeostasis was determined with a recently developed novel method. prostate specific antigen, albumin, total thiol, native thiol, disulphide and total antioxidant status (tas) were evaulated and compared between the groups. results: native thiol levels were 419.8 ae 54.87 lmol/l in the control group, 350.7 ae 46.35 lmol/l in the patients before rp, and 364.3 ae 46.88 lmol/l in patients after rp. native thiol, total thiol and tas levels were significantly higher in the control group than the patients before rp (p values < 0.001). native thiol, total thiol and tas levels were higher 6 months after rp compared to before rp in patients, but these changes were not significant statictically (p values 0.3, 0.3 and 0.09 respectively). discussion and conclusion: our study demonstrated that antioxidant defense mechanism was weakened as indicated by the decreased thiol levels in the patients with pc. increased oxidative stress in prostate cancer patients may cause metabolic disturbance and have a role in the pathogenesis of prostate cancer. p-09.04.4-108 is there any relation between 50 g oral glucose challenge test and serum total oxidant-antioxidant status in pregnant woman? the purpose of this study was to test the hypothesis that any degree of antepartum screening for gestational diabetes mellitus with oral 50 g glucose challenge test (gct) should be associated with oxidant-antioxidant status.in this prospectif study, oral glucose challenge test was applied to 25 pregnant women aged 25-40 years and at 24-28 weeks of gestation. plasma glucose concentrations were measured initial, 1 hour and in addition to test 2 hours after ingestion of 50 g glucose. at the same time serum insulin, cortisol, total antioxidant status (tas), total oxidant status (tos) levels were measured and the oxidative stress index (osi) was calculated.ten pregnant women (forty percent) had a positive glucose challenge test (gct). a positive moderate relation with initial and 1 hour serum total antioxidant status (tas) levels (r = 0.68) and the oxidative stress index (osi) (r = 0.67) was found. there was a positive weak correlation with initial and 2 hours total oxidant status (tos) levels (r = 0.22) but statistically significance difference was not found (p > 0.05).in this study after ingestion of 50 g glucose serum total antioxidant status (tas), serum oxidant status levels (tos) and serum oxidative stress index (osi) levels were higher than the initial levels.the results of this study suggest that antepartum screening for gestational diabetes mellitus with 50 g oral glucose challenge test (gct) weakly associated with oxidant-antioxidant status and to confirm this results the longer follow-up studies with more participants are necessary.wheat (triticum ssp.), cultivated for centuries in the middle-east, central asia, europe, and north-africa, is one leading staple crops around the world, and its marginally grown ancestor einkorn (triticum monococcum ssp. monococcum), possesses rich gene resources for wheat improvement and have bioactive compounds reducing and preventing chronic diseases such as diabetes, cancer, alzheimer, and cardio vascular diseases, beside their nutritional properties. however, as more attention has been given to wheat cultivars with strong gluten, protein content, starch composition, and resistance to biotic and abiotic stresses in bread wheat and yellow-colored pasta product in durum wheat health compounds such as fibers, phytochemicals, and bioactives have been underestimated so far. the aim of this study was, then, to examine the total phenolics and flavonoids, quantify their phenolic acids, a-tocopherol by high performance liquid chromatography (hplc), and their 2,2-diphenyl-1-picrylhydrazyl (dpph) scavenging activity of bread (triticum aestivum l.), durum (triticum turgidum ssp. durum desf.) wheat cultivars and einkorn (triticum monococcum ssp. monococcum) wheat populations collected from different provinces (bolu and kastamonu) of turkey. ferulic acid (148.67-764.04-lg/g), p-coumaric (5.06-54.09-lg/g), and total phenolic content (ranged 2.06-8.11-lmol gae/g) of einkorn populations were significantly higher than bread and durum wheat cultivars. results suggested the possibility of production of einkorn wheat populations, and hopefully cultivars rich in particular health beneficial component(s) may provide benefit to the consumers. in addition, higher phenolic content of einkorn may offer novel wheat genetic resources for the improvement of new wheat cultivars and the development of wheat-based functional foods. oxidative damage due to ischemia and acute kidney injury (aki) after coronary artery bypass graft (cabg) surgery are the leading complication during this process. in the kidney, ischemia/ reperfusion injury contributes to aki that is a clinical syndrome with rapid kidney dysfunction and high mortality rates. some animal and clinical studies have demonstrated an increase in serum and urinary neutrophil gelatinase-associated lipocalin (ngal) expression after renal ischemic injury.in this study, our aim was to investigate the relationship betwwen ngal and oxidative stress parameters due to ischemia caused by total perfusion time (tpt) in patients who have undergone on-pump cabg. materials and methods: the study was conducted in 30 patients who received on-pump cabg at university of istanbul, cerrahpasa medical faculty, department of cardiovascular surgery. blood samples were collected prior to surgery and after 2 hours following the termination of cardiac pulmonary bypass (cpb) . following centrifugation, serum samples were separeted and stored at -80 c until analysis. serum ngal, ima (ischemia modified alb€ umin), pco (protein carbonyl), nt (nitrotyrosine), lpd (lipid peroxide) levels were determined by elisa procedure. results and discussion: serum ngal, pco, nt levels in after 2 hours following cpb were significantly higher than the before surgery (p < 0.001, p < 0.001, p serum ngal levels in after 2 hours following cpb was found to have positive correlation with ima, pco, nt, and lpo levels (r = 0.59, p < 0.01; r = 0.77, p < 0.001; r = 0.68, p < 0.001; r = 0.77, p < 0.001 respectively). ngal levels were positively correlated with total perfusion time after cbp (r = 0.37, p < 0.05).the results of our study show that, increased ngal levels 2 hours after cpb were positively correlated with oxidative stress parameters and total perfusion time. investigation of the effects of thymoquinone against indomethacine induced gastric damage in rats introduction: incidence and high cost of acute stomach mucosa damages make this issue a very interesting issue for study. for this reason, it is aimed to investigate the effects of different dosage of thymoquinone (tq) against indomethacine induced gastric damage in rats. material and method: in our study, six groups of 36 wistrar male rats were used. groups are named as healthy, ind control, famotidine (40 mg/kg) and three different doses of tq (0.5, 1 and 2 mg/kg). while any treatment or drug administration will done on healthy group, model was generated to other groups by giving fam or tq with tap water via oral gavage. 5 min later, 25 mg/kg ind administrated to each rat. animals were sacrified about 6 hour later and stomach samples of each groups were collected for macroscopic study and gsh levels measurements. results: lower doses of tq is more effective and all tq groups exhibit reduced ulcer region with respect to the ind control group. gsh level of ind control group is lower than healthy group. the gsh level of tq, especially in lower doses, and fam groups statistically exhibit an increase in gsh level. conclusion: it is observed that ind induced gastric damage cause ulcer and increase in free radical. it is determined that lower doses of tq (0.5, 1 and 2 mg/kg) is also exhibit a protective effect on ind induced model. it is tought that quinone in tq structure have a strong redox feature and this feature clean up the free radicals caused by ind, it reduces the oxidative stress and protect the stomach from ulcer. anzer honey is the most famous honey in turkey with many endemic species flowers. the anzer plateau is located rize province of eastern black sea region. in this study, antioxidant and anti-hyaluronidase and anti-urease activities were investigated of the plateau bee pollen. the antioxidant capacity was determined by total phenolic content (tpc), total flavonoids contents (tfc), ferric reducing antioxidant power (frap) and 2,2diphenyl-1-picrylhydrazyl (dpph) radical scavenging activity. atherosclerosis is the leading cause of mortality worldwide, and as a chronic inflammatory disease, caused by a complex interplay between inflammatory and oxidative events.quercetin, a plant derived flavonoid and a well-known antioxidant, has shown great promises with regards to its protective effects against oxidative stress.however due to its physicochemical properties, the optimum pharmacokinetic behavior is a challenging issue.herein, we aimed to fabricate quercetin loaded solid lipid nanoparticle (quer-sln) to improve the bioavailability and therapeutics efficiency. furthermore the in-vitro capacity of quer-sln for ameliorating tnf-a induced oxidative stress in human endothelial vein cell (huvec) was evaluated. quer-slns were prepared by simple hot homogenizing method and characterized by means of drug loading (dl), encapsulation efficiency (ee), cytotoxicity, size, zeta potential and morphology. antioxidant activity of plain quercetin and quer-slns were then investigated using intracellular reactive oxygen species (ros) detection method (dcfh-da assay) by facs flowcytometryin conclusion, the results here showed superior control of oxidative stress by quercetin nanosystem as compared to plain quercetin. precirol based slns as a biocompatible/biodegradable lipid, may provide a novel drug delivery system for quercetin with improved beneficiary impact in atherosclerosis. objective: zinc is known as an antioxidant essential trace element. we aimed to evaluate the dose-dependent effects of zinc on the oxidant-antioxidant system in liver, kidney and brain tissues of rats and the histological alterations in the absence of oxidative stress (os). material and methods: thirty-nine female weighing about 150 gr wistar albino rats were divided into four experimental groups as ad libitum (al) diet (control), al diet + 3 mg/kg zn sulfate (low dose; group 1), al diet + 12 mg/kg zn sulfate (middle dose; group 2) and al diet + 25 mg/kg zn sulfate (high dose; group 3). zn sulfate solutions were administered 0.2 ml/day orally for 13 days and in day 14 rats were sacrificed and tissues were excised for detecting malondialdehyde (mda), advanced oxidation protein products (aopp), superoxide dismutase (sod), glutathione peroxidase (gsh-px), glutathione reductase (gr), and glutathione-s-transferase (gst) activities. histological evaluation was also performed to confirm the effects of zinc. results: in liver tissues aopp levels decreased in all groups receiving zinc as compared to the control group. liver mda levels were increased in group 1 and 3; sod and gsh-px levels were both increased while gst levels were decreased in all groups compared to control. gr levels were increased only in group 2. in kidney; aopp level was decreased only in group 1 and sod level was only decreased in group 2 as compared to control while gr levels were increased in all doses of zinc. in brain; aopp, gsh-px and gr levels were decreased in all groups receiving zinc as compared to control group. sod activity in brain tissues was increased by the administration of middle dose of zinc (group 2). gst level was decreased in only group 1 conclusions: the biochemical and histological findings of this study suggest that zinc has various effects on liver, kidney and brain tissues in the absence of os.key words: zinc, liver, kidney, brain introduction: this study aimed to investigate the effect of diabetes mellitus (dm) on oxidative stress and antioxidant capacity in humour aqueous (ha) and venous serum using total antioxidant capacity (tac) and total oxidative stress (tos) levels in serum and ha in cataract patients. materials and methods: in this study patients were divided into two groups. group 1 was composed of patients with type 2 dm and cataract and group 2 was composed of patients with cataracts who are not accompanied by dm and cataract patients who are not accompanied by systemic diseases. each group consisted of 20 patients, totally 40 patients were included in the study. the ha which was collected from the eyes at the beginning of the cataract surgery and venous blood serum collected from the same patients were analyzed. in both groups, ha and serum tac and tos levels were measured with elisa. results: : serum tac levels in the dm group were significantly lower than in the control group (p < 0.05). tos serum levels in dm group was statistically higher than the control group (p < 0.05). differences between tac and tos levels were not statistically significant when compared the two groups' ha results (p > 0.05). group 1, divided into two subgroups according to their hba1c levels, there was no statistically significant difference between the subgroups when hba1c levels were compared with the relationship between serum and ha's tac and tos levels (p > 0.05). there was not an association between the gender, age and the levels of tac-tos in both groups (p > 0.05). discussion and conclusion: presences of dm is the only risk factor for increase of oxidative stress and decrease of anti-oxidant capacity in patients without a systemic complication of dm and diabetic retinopathy. in our study, diabetic patients without retinopathy showed similar ha tos and tac levels to healthy individuals, this finding indicates that blood-aqueous barrier is protected in these patients. the effect of ferulic acid against testicular ischemia/reperfusion injury in rats u. sac ßik 1 , g. erbil 1 , z. c ß avdar 2 , c. ural 2 1 department of histology and embriyology, school of medicine, dokuz eyl€ ul university, izmir, 2 department of molecular medicine, health science of institute, dokuz eyl€ ul university, izmir, turkeytestis torsion is one of the urologic emergencies occurring frequently in neonatal and adolescent period. testis is sensitive to ischemia/reperfusion (i/r) injury and, therefore, ischemia and consecutive reperfusion cause an enhanced formation of reactive oxygen species (ros) that result in testicular cell damage and apoptosis. ferulic acid, known as an antioxsidant, is a phenolic acid found in seeds and leaves of the plants. we aimed to investigate potential protective effect of ferulic acid against testis i/r injury.thirty five wistar rats were randomly divided into 5 groups; control, ethyl alcohol, ischemia, i/r, i/r-ferulic acid groups. animals were exposed to 2 hours of ischemia followed by 2 hours of reperfusion. ferulic acid was administered (100 mg/ kg) before reperfusion intravenously. testicular cell damage was examined by h-e staining and pas. tunnel, active caspase-3, inos and mpo were evaluated by immunostaining. malondialdehyde (mda), glutathione (gsh) levels, glutathione peroxidase (gpx) and superoxide dismutase (sod) activities were assessed by biochemical methods.histological evaluation showed that ferulic acid pretreatment reduced significantly testicular cell damage and decreased tun-nel, caspase 3 positive cells; inos and also mpo expression. in addition, ferulic acid administration decreased significantly the mda levels increased by i/r. morever, ferulic acid increased significantly the sod activity levels, which was decreased by i/r. there were no statistically significant differences in the levels of gsh and gpx activity in all groups.the present results suggest that ferulic acid is a potentially beneficial agent in protecting testicular i/r. background: in heart failure (hf), angiotensin antagonists (aa), beta-blockers (bb), spironolactone, diuretics and acetylsalicylic acid are often used. top 3 pharmaceutical groups reduce mortality. on the increased oxidative stress (os) in patients with hf, it is known to have beneficial effects of certain groups of drugs. however, the net effect of these drugs in os is unknown. the aim of this study was to investigate the effects of drugs used in hf on os. materials and methods: 133 patients were included in the study. all of the patients had systolic heart failure and all of them were under treatment. drugs used by the patients were recorded. the levels of total antioxidant status (tos), total oxidant status (tas), the enzymatic activity of ceruloplasmin, paraoxonase-1 and arylestherase were measured according to erel's method. serum total thiol levels were measured with sh modified hu method and the lipid hydroperoxide levels were measured with the ferrous ion oxidation xylenol orange assay. the percentage tos / tas was determined as osi. results: in patients treated with acetylsalicylic acid (asa), spironolactone, beta blocker and furosemide, there were increased tos, decreased tas and osi (p < 0.05). in patients treated with angiotensin blockers, increased tas and looh, and decreased sh were found (p < 0.05). in patients treated with nitrates and ccb, tos and osi were found decreased. correlation analysis showed that increased tas correlated with the use of angiotensin blockers, asa, furosemide and beta blocker positively; and with the tos and osi level correlated with the use of spironolactone, asa spironolactone and furosemide (p < 0.05). conclusion: current medical agents that are being used in hf are effective in reducing os in hf patients. one of the effective mechanisms to reduce the mortality of some of these drugs may decrease os.key words: heart failure, drug use, oxidative stress p-09.04.4-119 across adjacent ring formed titanium phthalocyanine-mediated photodynamic therapy alters and degrades filamentous actin cytoskeleton and internal membranes photodynamic therapy (pdt) is widely accepted as a promising and minimally invasive treatment strategy due to its applicability on a wide range of cancer diseases. this clinically approved treatment method relies on the dramatic production of singlet oxygen and reactive oxygen species (ros) in target tissue to evoke apoptotic cell death [1] . we, therefore, focused on the intracellular ros accumulation, internal membrane degradation, filamentous actin cytoskeleton alteration and nucleus morphology changes induced by pdt-mediated across adjacent ring formed titanium phthalocyanine which was previously synthesized bis(ethane-1,1p-phenol-2,2-p-phenoxy) phthalocyaninatotitanium (iv).characterization of the synthesized metallophthalocyanine was accomplished by using uv-vis, ir, 1 h-nmr and maldi-tofmass spectroscopies. the dark and pdt-mediated activities of bare and phosphonolipids (max. 5%) charged titanium phthalocyanine (0.625, 1.25, 2.5, 5 and 10 lm) were determined on a549 human lung carcinoma and hacat human keratinocyte cell lines by using intracellular ros assay, dioc(6), tritc-phalloidin and dapi staining protocols. waltmann pdt 1200 l was used as the non-toxic light source at 100 j/cm 2 fluence and 150 mw/ cm 2 fluence rate. the experiments showed that pdt-mediated titanium phthalocyanine leads to significant and concentration dependent reactive oxygen species accumulation. moreover, internal membrane degradation, apoptotic bodies on nucleus and filamentous actin cytoskeleton alteration were observed. consequently, the activity mechanism of pdt-mediated titanium phthalocyanine seems to be in a tight relationship with ros accumulation-mediated internal membrane degradation, filamentous actin cytoskeleton alteration and apoptotic pathways activation. introduction: retinal vein occlusion (rvo) is a common retinal vascular disorder that can affect visual acuity and cause blindness in elder population. sulphur containing aminoacids such as cysteine (cys), cysteinylglycine, glutathione, homocysteine and c-glutamylcysteine are reported to be associated with the pathogenesis of rvo. thiols are organosulfur compounds that are formed of a carbon-bonded sulfhydryl group. sulphur containing aminoacids slightly contribute the composition of plasma thiol pool. thiols can undergo oxidation reaction via oxidants and form disulphide bonds our purpose is to research the relationship between a novel oxidative stress marker serum dynamic thioldisulphide homeostasis and retinal vein occlusion. materials and methods: 22 rvo patients and 22 controls were included in the study. native thiol, total thiol, disulphide levels are measured in the serum samples of rvo and control group by using an automated method described by erel et al. also disulphide/native thiol and disulphide/total thiol ratios were calculated. results: there were no significant difference between the rvo and control group in native thiol, total thiol, disulphide disulphide/native thiol and disulphide/total thiol ratios.(p > 0.05 for all) conclusion: our study is the first report evaluating the dynamic thiol-disulphide homeostasis in rvo patients by a newly developed method by erel et al. further large sample sized studies investigating the levels of sulphur containing aminoacids may additionally be planned to verify this study. purpose: the purpose of this study was to evaluate markers of systemic oxidative stress and antioxidant capacity in subjects with severity of osas. methods: a total of 106 osa patients were included in the study (18 controls, 14 with mild, 14 with moderate, and 60 with severe osa). patients were grouped according to apnea-hypopnea index (ahi) as mild, moderate and severe osa. patients with ahi<5 served as control group. known risk factors for oxidative stress, such as age, sex, obesity, smoking, hypelipidemia, and hypertension, were investigated as possible confounding factors. plasma arylesterase, total oxidative stress (tos), total antioxidant capacity (tac), total thiol, catalase (cat) levels were measured for all patients. results: the mean age was 52.49 ae 12.9 years and 40.6% (43/ 106) of the study population was female. plasma arylesterase, tos, tac, total thiol, and cat plasma values were not different between mild, moderate, severe osa groups and controls (p > 0.05). catalase levels were significantly lower in women patients with severe osa compared to healthy women controls (p < 0.05). there was a negative correlation between ahi and serum total thiol levels (r = à0.289, p < 0.05) in severe osa groups. conclusionthe present prospective study provides evidence that osa might be associated with decreased antioxidant burden possibly via catalase way. results: the sera 8-ohdg, mda and il-6 were significantly higher in diabetic group than control group (p < 0.05). although there was a notable positive correlation between mda and 8-ohdg, there was no a relationship between 8-ohdg or mda with il-6. discussion: in agreement with previous studies our data illustrated that high levels of oxidative stress is associated with increased production of oxidized lipids and nucleobases in diabetic patients compared to control group. also enhanced proinflammatory cytokine, il-6, induced inflammmation in these patients.conclusion: oxidative stress and inflammation play pivotal roles in the development of diabetes and can cause major complications in dm. so we suggest that early detection of these measurable indicatores can help to diagnosis the severity or presence of some complication in diabet. the effect of quercetin on erythrocyte glucose-6-phosphate dehydrogenase enzyme activity in ethanol treated rats in this study, we aimed to evaluate the effects of ethanol on erythrocyte (g-6-p-d) enzyme activity and the effects of quercetin on erythrocyte g-6-p-d activity in the recovery of the effects of ethanol.rats were randomly divided into four groups. the control group (n = 6) received physiological saline. the quercetin group (n = 7) received quercetin (120 mg/kg/ day) via i.g. route the alcohol group (n = 7) received ethanol (80% v/v, 1 ml/day) via i.g. route. the alcohol + quercetin group (n = 5) received 1 ml of ethanol (80% v/v) 2 hours after quercetin treatment (120 mg/ kg/day). experimental procedures were peformed for 30 days. erythrocyte g-6-p-d activity was found to be higher in the quercetin group than those in the alcohol group (p < 0.001). in the alcohol group, the erythrocyte g-6-p-d activity was found to be significantly decreased than those in the control group (p < 0.001). statistically significant differences were observed in erythrocyte g-6-p-d activity between the alcohol group and the alcohol + quercetin group (p < 0.001).as a conclusion, our results demonstrate that ethanol decreased erythrocyte g6pd activity and quercetin was found to be beneficial in the prevention of toxic effect raised by ethanol.key words: erythrocyte, ethanol, g6pd, oxidative stress, quercetin p-09.04.4-126 effects of the sulphasalazine to the cerebral hypoxia reperfusion injury in rat background: cerebral ischemia/ reperfusion (i/r) injury is still a difficult process to treat and rehabilitate today. this study was designed to investigate beneficial effects of sulfasalazine in cerebral i/r injury in rat. methods: except control group (n = 5), 20 wistar albino rats were divided into four groups for acute and chronic stage investigation of i/r injury, and temporary aneurysm clips were attempted to both internal carotid arteries for duration of 30 minutes. four hours later, except control, sham-a, sham-c groups, 40 mg/kg once a day sulfasalazine was administered to animals, orally. animals were sacrified and then necrotic neuronal cells of hippocampal ca1, ca2, and ca3 region, and cortical necrotic neurons, perivascular edema, pyknotic neuronal cells, irregularities of intercellular organization (iio) were counted and scaled histopathologically. tissue il-1b, il-6, malonyldialdehyde (mda), myeloperoxidation (mpo), no, and tnf-a levels were measured by using elisa, too. results: sulfasalazine could reduce perivascular edema, iio, cortical and hippocampal neuronal cell death in both stages. it could decreased mda in acute stage, but not reduce il-1b, il-6, mpo, no, and tnfa levels. it could increased il-1b levels in chronic stage but not affect to il-6, mpo, mda, no, tnf-a levels.conclusion: sulfasalazine could improve histopathological architecture of hypoxic tissue in both stages of i/r injury. it could inhibit lipid peroxidation cascades in acute stage but not affect to tissue mpo, no, il-6, and tnf-a levels in any stage in rat. these results suggested that therapeutic mechanisms of sulfasalazine should be investigated by using more specific laboratory methods in future studies.key words: antiinflamatory, cerebral hypoxia reperfusion injury, sulfasalazine, stroke. camel and horse milk xanthine oxidase (xo) was found to catalyze the reduction of nitrate and nitrite to nitric oxide (no) under aerobic condition. to date, mammalian nitrate reductase (nar) and nitrite reductase (nir) have not been identified. no, a gas, is found to control a seemingly, limitless range of functions in animals.one assay was used to determine nar and nir activities of milk xo: (1) nitrite formation from nitrate by nar, and (2) nitrite utilization by nir. nitrite concentrations were determined by using sulfanylamide and n-(1-naphtyl)-ethylenediamine, which form red color measured at 485 nm.these activities of the milk xo require nadh as a physiological electron donor. high xo, nar and nir activities are detected only after heat treatment (80°c, 5 min) of the fresh milk in the presence of molybdate. in both camel and horse milk nar activity of xo was almost two times higher than its nir activity. it is well known that xo can be reversibly converted from the dehydrogenase form to the oxidase through the oxidation of sulfhydryl groups. cysteine and, to a lesser extent, glutathione increased nir activity of milk xo but not its nar activity. the mechanism of this increase of nir activity remains unclear and is currently under study. substitution of tungsten for molybdenum under above conditions gave no detectable nar and nir activity of milk xo. the molybdenum site-directed inhibitor, tungsten inhibited in a dose-dependent manner. therefore, nitrate and nitrite are clear to interact with mo center of xo.camel and horse milk are traditional drinks in central asia and kazakhstan. therefore, it is very important that xo provide a mechanism for generation of no in camel and horse milk where nitric oxide synthase, no producing enzyme, does not exist. p-09.04.4-128 the influence of phytomedicine on metabolic processes of white rats undergone to ionized radiation the study of peroxide lipids oxidation (plo) process is used as one of stability parameters of organism's changes and as a key mechanism for understanding of adaptation reactions and of pathogenesis of different diseases. it's determined by high biological activity of products which are formed in the plo reactions, in this relation lipids with high contents of fat acids play important role. to investigate the influence of phytomedicine eminium regelii on the metabolic processes (peroxide lipids oxidation) of white rats' organism in conditions of ionized radiation.the animals were exposed to ionizing radiation (gamma-radiation 60 co) on the radiotherapeutic equipment teragam in a dose of 6 gy and received phytomedicine eminium regelii in a dose of 2.5 mg/kg orally within 14 days following the ionizing radiation exposure. gamma-rays caused the increase of lipid peroxidation (lpo) primary (dc) and secondary products' (mda) concentrations in spleen, liver, thymus and adrenal glands.treatment by phytomedicine resulted in contents of dc decreased in 3 times in spleen, in 7 times in thymus, in 5 times in adrenal glands, in liver in 4 times, in lymph nodes of small intestine it in 3 times. mda decreased in liver up to 6 and 3 times, in spleen in 3.2 times, in thymus in 9 times, in liver in 6 times, in adrenal glands in 12 time, no changes in lymph nodes of small intestine.the effect of phytomedicine treatment of organisms exposed to sublethal dozes of gamma-radiation results in the lpo primary and secondary products concentrations decrease in spleen, liver, thymus and adrenal glands. p-09.04.4-131 evaluation of serum levels of ischemia modified albumin (ima) in bipolar disorder patients k. € unal 1 , c. topc ßuoglu 2 , m. cingi 3 1 clinic of biochemistry, ankara polatli duatepe public hospital, ankara, 2 clinic of biochemistry, ankara numune training and research hospital, ankara, 3 clinic of psychiatry, ankara numune training and research hospital, ankara, turkey introduction: bipolar disorder is one of the most debilitating psychiatric disorders characterized by disruptive episodes of mania/hypomania and depression. considering the complex role of biological and environmental factors in the etiology of affective disorders; recent studies have focused on oxidative stress, which may damage nerve cell components and take part in pathophysiology. aim of our study is to contribute these data about oxidative stress in bipolar disorder, by detecting ischemia modified albumin (ima) levels of bipolar disorder patients in remission and also by comparing these results with healthy controls. methods: study population consisted of 35 patients meeting the diagnostic and statistical manual of mental disorders, fifth edition (dsm-5) criteria for bipolar disorder i. 36 healthy subjects were included as control group (hc). serum ischemia modified albumin (ima) levels of all participants were determined. results: statistical analysis on serum ischemia modified albumin (ima) levels did not show any significant difference between bipolar disorder patients in remission and healthy controls.conclusion: studies on oxidative stress in bipolar disorder have reached controversial results up till now. in this study, no statistically significant difference was detected between oxidative parameters of bipolar disorder patients in remission and healthy controls. in order to evaluate oxidative stress in bipolar disorder comprehensively, further studies are needed. keywords: bipolar disorder, ischemia modified albumin (ima), oxidative stress p-09.04.4-133 xanthine oxidase and adenosine deaminase activity in patients with familial mediterranean fever (fmf) objective: fmf is an autosomal recessive dissease which is characterized by recurrent fever and inflammation of serous membranes. in this study we measured serum adenosine deaminase (ada) and xanthine oxidase (xo) levels in fmf cases. method: serum ada levels were measured with a sensitive colorimetric method described by giusti and xo levels were analysed by the method of worthington in 30 fmf patients and 30 healthy controls. results: there was a significant difference in xo and ada levels between controls and cases. ada and xo levels were higher in patents with fmf. humic acid (ha) is a natural product which is forming during decomposition of organic matter in humus. in recent years, there are some resarches on the medical use of ha. the present study was undertaken in order to evaluate the anticancer properties of the ha using a prostate cancer and osteosarcome cell lines pc-3, sjsa1 as an in vitro model system.ha was purchased from sigma-aldrich. the cells were maintained in dmem medium supplemented with 10% heat-inactivated fbs and 1% penicillin/streptomycin. cells were grown in petri dishes in a humidified atmosphere containing at 37•c. five different concentrations (100 ug/ml, 50 ug/ml, 25 ug/ml, 10 ug/ ml, 5 ug/ml) were prepared using a stock solution of ha. we measured cell proliferation and migration to understand of progression effects of ha in pc-3 and sjsa1 cell lines in vitro.according to our results, ha treatment caused cytotoxicity and induced cell death in vitro in pc-3 cells with an ic50 value of 67.9 lg/ml. contrary to this, ha induced proliferation of sjsa1 cells in dose dependent manner. ha demonstrated the highest proliferatif activity against sjsa1 cells with an ic50 value of 100 > lg/ml. on the other hand, cell migration was reduced in pc-3 cell line and interestingly, migration was accelerated in sjsa1 cell line.our study may provide new insights into the regulatory effect of ha in cancer, but further studies are needed to clarify the role of ha in cancer pathogenesis. the febs journal 283 (suppl. 1) (2016) key: cord-015394-uj7fe5y6 authors: nan title: scientific abstracts date: 2008-12-23 journal: reprod sci doi: 10.1177/19337191080150020102 sha: doc_id: 15394 cord_uid: uj7fe5y6 nan by reduced placental oxygenation, hypoxia-induced oxidative stress is a predominant mechanism. we investigated the effect of hypoxic pregnancy, with and without antioxidant treatment, on placental and maternal circulatory indices of oxidative stress in rats. methods: on pregnancy day 6 , wistar rats were randomised into: normoxia (21% o 2 7 litters), hypoxia (14% o 2 8 litters) and hypoxia + vit c (14% o 2 + 500mg.100 ml -1 vit c in water, 6 litters). on day 20, dams were anaesthetised, maternal blood taken, pups measured and weighed, placentae weighed and frozen. only placentae from two male pups from any one litter were investigated. blood was processed for ascorbate, urate, l-cysteine and glutathione (gsh) measurement. placental protein was analysed for heat shock protein 70 (hsp70; western). results: hypoxia + vit c did not affect maternal food or water intake. vit c elevated maternal ascorbate by 70 % of baseline; a similar increment to human trials (poston et al. 2006) . hypoxia elevated placental hsp 70 and maternal plasma urate and l-cysteine, but decreased gsh. vit c in hypoxic pregnancies prevented all stimulated effects, but the reduction in gsh persisted. hypoxic pups had a reduced ponderal index and elevated head diameter: body weight ratio; effects also prevented by vit c. fetal oxygen uptake in normal and gdm pregnancies. emanuela taricco, tatjana radaelli, veronica cozzi, gabriele rossi, danila puglia, giorgio pardi, irene cetin. department of obstetrics gynecology "l. mangiagalli", irccs policlinico, mangiagalli e regina elena, milan, italy. background. diabetes in pregnancy has been associated with alterations of fetal growth probably due to increased nutrient availability and placental transport. fetal hypoxia and acidemia have been reported in pregestational diabetic pregnancies with poor glicemic control but this is still uncertain in well controlled patients. the role of placental function and the relationship between maternal and fetal circulation is crucial for efficient exchanges of oxygen and nutrients. since umbilical blood flow can be obtained by us in utero, we studied normal and gdm pregnancies in order to evaluate fetal oxygen uptake. methods. 21 normal (n) and 21 gdm pregnancies were studied at term, at the time of elective caesarean section. umbilical vein volume flow (qumb) was measured by us before caesarean section and blood samples from umbilical vein (uv) and artery (ua) were obtained. blood gases and acid-base balance were evaluated. results. average fetal weights were similar in both groups (n=3274±62; gdm=3381±111 g) while placental weights were significantly different (n=474±16; gdm=571±35 g). n and gdm pregnancies showed similar values of qumb and qumb/kg of fetal weight. (qumb: 245.3±19.0 in n and 242.2±18.5 ml/min in gdm; qumb/kg: 74.6±5.0 in n and 71.4±4.6 ml/min/kg in gdm). in fetuses from gdm pregnancies a significant reduction in o 2 sat, o 2 cont and po 2 and a significant increased lactate conc was found in both uv and ua compared to n (table 1) . o 2 umb uptake (n=0.78±0.08; gdm=0.56±0.08 mmo/l/min) and o 2 umb uptake/kg (n=0.24±0.02; gdm=0.16±0.02 mmo/l/ min/kg) were significantly lower in gdm compared to n fetuses. conclusions. our data indicate that fetuses from gdm pregnancies show a significant reduction in oxygen supply despite a normal blood flow/kg of fetal weight. these data may suggest that a good maternal metabolic control is not sufficient to ensure normal placental oxygen supply and/or utilization by the gdm fetus. background: synthetic glucocorticoids (sgc) are given to mothers at risk of preterm delivery to promote fetal lung maturation. evidence is emerging indicating long-term effects of such treatment on endocrine function and behavior in offspring. however, virtually nothing is known concerning potential transgenerational influences on growth, endocrine function and behaviour. we hypothesize that repeated treatment of grandmothers (f0) with sgc will alter hypothalamic-pituitary-adrenal (hpa) function in f2 offspring with no manipulation of the f1 pregnancy. methods: pregnant guinea pigs (f0) were subcutaneously injected with betamethasone (beta; 1mg/kg) or vehicle (veh) on gestational days 40/41, 50/51 60/61. adult f1 female offspring from each group were mated with control males. hpa function was assessed in adult f2 offspring by non-invasive measurement of salivary cortisol concentrations: 1) under basal conditions, 2) during and following exposure to psychological stress (high frequency strobe light) or psychological/physical stress (forced swim) and, 3) following dexamethasone suppression. results: there was no effect of beta (f0) on bodyweight from birth to adulthood in f2 offspring. basal salivary cortisol in the betaf2 females was lower than in the vehf2 group in the morning but not the afternoon; there were no differences in male f2 offspring. in contrast, both male and female betaf2 failed to mount adrenocortical responses to psychological stress compared to vehf2 offspring that mounted robust responses (p<0.02). swim stress induced robust adrenocortical responses in all groups (p<0.0001), however, the response was consistently lower in betaf2 offspring. beta exposure also led to a significant difference (p<0.003) in the cortisol response to dexamethasone suppression in female (f2) offspring, with a similar trend in males. conclusion: prenatal exposure to sgc (f0) causes transgenerational programming of adrenocortical function in adult f2 offspring. grand maternal exposure to beta results reduced basal adrenocortical activity in betaf2 female offspring, and caused stress hypo-responsiveness in both males and females. dexamethasone suppression tests indicate altered central glucocorticoid feedback. these findings have important ramifications for the management of human preterm labor. support: canadian institutes of health research. in the uterine and umbilical vasculatures, we hypothesized that their remodeling would also be blunted in pregnant enos -/-mice, leading to an elevated vascular resistance and decreased blood flow to the placenta contributing to fetal growth restriction. methods: utero-and umbilical-placental blood velocity waveforms and umbilical arterial diameters were measured using 40 mhz ultrasound biomicroscopy in control (c57bl/6j) and enos -/-mice at 17.5 days of pregnancy (n=6 mothers). spiral artery and fetal capillary morphologies, and uterine arterial diameters were evaluated from vascular corrosion casts. tissues were collected for hydroxyprobe-1 and actin immunohistochemistry to identify hypoxic and smooth muscle regions. we calculated resistance index ((s-d)/s) from systolic (s) and diastolic (d) velocities, and blood flow from mean velocity and vessel area. results: calculated uterine blood flow normalized to the weight of the uterus and its contents was 55% lower (p<0.05) in pregnant enos -/-mothers due to large reductions in uterine artery diameter (-33%, p<0.001) and mean velocity (-17%, p<0.05). uterine arterial resistance index was 52% higher (p<0.001), and the spiral arteries were less coiled and contained more smooth muscle actin in enos -/-mice than controls. more intense hypoxic immunoreactivity was detected in the spongiotrophoblast and trophoblast giant cell layers of the junctional zone of enos -/-placentas, whereas fainter staining was only detected in the spongiotrophoblast cell layer in controls. in the umbilical circulation, flow normalized to fetal weight was not significantly changed although the resistance index was slightly elevated (4%, p<0.05) and capillary lobule length was reduced by (-28%, p<0.05). fetal organs showed increased hypoxic immunoreactivity suggesting reduced organ oxygen delivery in enos -/-fetuses. conclusions: results suggest that enos plays an important role in uterine and spiral artery remodeling and in augmenting utero-placental blood flow during pregnancy. it also appears to enhance oxygen delivery to the placenta and fetus. enos may contribute to normal fetal growth by these mechanisms. integrin 5 methods: hpmcs isolated from term placentas were assessed for their phenotype markers, mutilineage capacity, and the expression of integrin molecules. the hpmcs were induced to endothelial cell differentiation in the presence of endothelial cell growth medium 2 with 2% of fcs and 50 ng/ml vegf for 14 to 21 days. the angiogenesis ability of these cells was demonstrated by using an in vitro angiogenesis kit and in vivo chick chorioallantoic membrane assay from ten-day-old embryos. blocking antibodies specific to integrin 4 , associated with gdm. this study was adequately powered to detect association in the caucasian and asian group. the absence of association suggests that gdm and t2dm may have more divergent molecular pathophysiology than previously suspected. background: uterine endometrium has a unique cycle of physiological angiogenesis. in mice, endothelial progenitor cells (epc) contribute to endometrial angiogenesis being incorporated after oestradiol administration. objective: to determine whether circulating © epc number and function vary through the menstrual cycle in response to changes in circulating sex steroid concentrations. methods: ten healthy, nulliparous, pre-menopausal, non-smoking women (mean age 31 years) with regular menses (27-29 days) were studied. venous blood was collected during menstrual, follicular, periovulatory and luteal phases of a single cycle (days 1-3, 6-9, 13-15, 20-22) . cepcs, serum oestradiol (e) and progesterone (p) were measured at each phase. cepcs were quantified by phenotype using flow cytometry (leukocytes co-expressing cd133, kdr and cd34) and function by the epc-colony forming unit (cfu) assay. epc-cfus were stained for endothelial markers including uptake of acetylated low-density lipoprotein, binding of lectin (ulex europaeus) and endoglin. results: luteal p was >30 nmol/l in all women. cepc numbers increased in the menstrual and follicular phases being 3-fold higher in the follicular compared to periovulatory phase (p<0.05). there was no significant variation in cepc function over the menstrual cycle. there was no correlation between serum e or p levels and cepc number or function. conclusion: cepcs number but not function (epc-cfu assay) vary during the menstrual cycle with numbers increasing during the menstrual and follicular phase and falling in the periovulatory and luteal phase of the menstrual cycle. this may represent mobilisation of cepcs from the bone marrow and subsequent incorporation into the endometrium. neither function nor cepc number correlate with serum p or e. our previous studies demonstrated that tissue factor (tf), which binds factor vii to act as a potent pro-coagulant and angiogenic factor, is over-expressed in endometriotic lesions. thus, we determined whether tf could serve as a target for the elimination of pre-established ectopic human endometrial growth in a mouse model. icon is composed of a mutated factor vii (fvii) domain targeting tf and an igg1 fc (fvii/igg1 fc) effector domain that activates antibody-dependent immune responses against tf bearing endothelial cells. methods: athymic, ovariectomized and estrogen-treated mice received intraperitoneal (i.p.) injections of 1.0 mg of proliferative phase human endometrial tissue derived from normal (disease-free) women. twelve days after inoculation to establish lesions, icon protein (10 ug) was delivered i.p. once a week for 4 weeks. after sacrifice, animals were subject to gross inspection. residual endometriotic tissue was formalin fixed, paraffin embedded and immunostained for von willebrand's factor (vwf) and tf. results: compared to control mice, treatment with 10ug of icon abolished all lesions in 7 of 10 mice and reduced both size (2.33 to 1.4mm) and number of lesions (1.9 to 1 per diseased mouse) in the remaining mice. moreover, residual lesions from icon treated mice were atrophic and displayed significant reductions in vessel areas of 87% +/-26% (p=0.002, mean +/-sem, n=3) as determined by vwf immunostaining. no hemorrhagic or thrombotic sequelae were observed in icon treated mice. conclusions: unlike other treatments that target developing angiogenesis, icon can target both developing and established human endometriotic lesions in athymic mice. the gross and microscopic vessel analysis suggests that icon directly or indirectly destroys endometriotic vessels. thus, icon presents a novel, non-toxic therapy for endometriosis. compared to the miscarriage and top groups. ihc for crisp3 demonstrated increased secretion of crisp3 in the glandular epithelium and expression in the leucocytes of the tubal uterine decidua. conclusions: there are differences in decidual gene expression in tubal compared to iu pregnancies. we believe that potential biomarkers of tubal pregnancy can be discovered by focusing on secreted proteins associated with uterine decidualization. one of these proteins, crisp3, is significantly increased in decidua of tubal ectopic pregnancies and we are currently investigating its expression pattern in sera from women with tubal compared to iu pregnancies. progesterone and hoxa 10 regulate gaba-a pi receptor expression, membrane translocation and activation. homayoun sadeghi, hugh s taylor. obstetrics, gynecology and reproductive sciences, yale school of medicine, new haven, ct, usa. objective the expression of the gaba-a pi receptor has been previously described in the human endometrium in both luminal epithelium and stroma. it is upregulated during stromal decidualization in the rat and in the implantation window of human endometrium. the gaba receptor is modulated by progesterone metabolites, with the resultant opening of the receptor ion channels which allow the water flux necessary for trophoblast attachment. here we identified regulators of pi subunit receptor gene expression and activity. the well-differentiated human endometrial adenocarcinoma cell line (ishikawa) and human endometrial stromal cells (hesc) were transfected with hoxa10, treated with progesterone, or treated with vehicle or empty plasmid controls. gaba-a pi receptor mrna upregulation was evaluated by real time rt-pcr. protein expression was evaluated using immunohistochemistry. results gaba-a pi receptor mrna expression was increased with either progesterone treatment (31%, p=0.049) or hoxa10 transfection (11%, p=0.027). coadministration of progesterone along with increased hoxa10 transfection had no additive effect on the expression of gaba-a pi receptor mrna (p=0.98). gaba-a pi receptor protein expression was similarly increased by each treatment. either hoxa10 or progesterone independently caused translocation of the gaba receptor from the cytoplasm to the cell membrane in ishikawa cells. conclusion gaba-a pi receptor expression is increased in the human luminal epithelium and stroma in the window of implantation. activation of the pi subunit leads to opening of ion channels, likely allowing flux of water into the epithelial cells and out of the uterine lumen. progesterone and hoxa 10 each increase both pi subunit receptor expression and membrane translocation. the lack of additive effect suggests progesterone induced pi subunit receptor expression is likely mediated indirectly through progesterone's regulation of hoxa 10 expression. finally, after receptor expression and translocation, progesterone mediated gaba receptor ion channel activation mediates water resorption necessary for implantation. cancer. suzy davies, donghai dai, kimberly k leslie. obstetrics and gynecology, university of new mexico, albuquerque, nm, usa. objectives: endometrial cancer is the most frequent gynecologic cancer in women. it affects an estimated 40,000 women in the us every year, and long term outcomes for patients with advanced stage or recurrent disease are poor. targeted molecular therapy against the vascular endothelial growth factor (vegf) and its receptors constitute a new therapeutic option for patients that is now under study by the gynecologic oncology group in a phase 2 trial, gog 229e (now in second stage accrual). the goal of our work was to assess the potential effectiveness of vegf/vegfr blockade in preclinical endometrial cancer models. methods: these studies employed two agents, bevacizumab (avastin, a vegfa blocking antibody) and vandetanib (zactima, a tyrosine kinase inhibitor of egfr and vegfr2). ic 50 experiments were performed on endometrial cancer cells in culture using four established cell line models. xenografted athymic mice were also employed to test the ability of compounds to inhibit tumor growth in vivo. tumors were isolated from controls and treated animals, mrna was extracted, and affymetrix gene expression arrays were performed to determine the genes consistently modulated by treatment. results: compared to vandetanib with an ic 50 of 1.15 m, bevacizumab showed little activity on cell proliferation in vitro, and cell numbers were not reduced by 50% using concentrations up to 2.5 m. however, bevacizumab demonstrated robust activity in the athymic mouse model, resulting in a significant decrease in tumor formation and growth compared to vehicle treated animals when dosed bi-weekly in a concentration of 0.2 mg/mouse ip. tumors from this model demonstrated that eighteen genes were consistently up or down regulated in the presence of bevacizumab. among the regulated transcripts was microrna21, which was significantly down-regulated. microrna21 is an anti-apoptotic factor, and its inhibition by bevacizumab predicts for increased expression of the tumor suppressor pten, decreased cell proliferation, and a reduced capacity for metastasis. conclusions: these studies confirm that the vegf pathway is a good target for new therapies against endometrial cancer. blocking this pathway not only inhibits angiogenesis, but also results in changes in gene expression that enhance apoptosis and reduce cellular proliferation and tumor invasion. we collected fibroid and adjacent normal tissues following hysterectomy with patient consent and institutional irbs. to date, 840 data points for each gene from 420 arrays have been collected from 42 patients. the fluorescence readings were log-transformed and normalized with the robust quartile normalization method. quality control of the normalized data was performed to remove 13 arrays that deviated from twice the inter-quartile range calculated from the 420 array signals. results: the custom microarray profiling identified 38 genes that were differentially expressed between normal and fibroid tissues (p <0.01; fdr<0.13). among them, 12 genes encode receptor tks, 15 genes encode tk ligands, and 11 genes encode cell cycle and apoptosis proteins. clustering analysis of the mean log2 ratios of these 38 genes has led to the division of the 42 patients into two major groups. thirty four (81%) belong to a group characterized by the significant downregulation of the cyr61 (ccn1) gene in fibroid tissues. the cyr61-down group can be further divided into two sub-groups based on the expression of another tk ligand, efn4a. other receptor differentially expressed tk did not segregate with the three defined sub-groups. finally, there was no sub-group segregation based on age of the patient, menstrual phase, or weight of the fibroid. conclusion: our results have demonstrated that tyrosine kinases and their ligands are uniquely differentially expressed between normal and fibroid tissues. unique tyrosine kinase ligands in our population were cyr61 and efn4a, and these markers created three molecular classification groups based on their differential expression. these results support the hypothesis that tyrosine kinase ligands are involved in fibroid growth, and may offer targets for a strategy to avoid hysterectomy. microvascular perfusion sonographic imaging to detect early stage ovarian cancer. joanie mayer hope, 1 arthur c fleischer, 2 brian day, 1 stephanie v blank, 1 bhavana pothuri, 1 robert wallach, 1 john p curtin, 1 david a fishman. 1 1 gynecologic oncology, new york university school of medicine, new york, ny, usa; 2 radiology, vanderbilt university medical center, nashville, tn, usa. objective: epithelial ovarian cancer (eoc) is the 4th leading cause of death in us women due to the inability to detect early stage disease. recent sonographic developments involving harmonics, pulse inversion, and the use of contrast agents justify the hope that depiction of aberrant tumor microvascularity associated with early disease can occur. this study utilizes these new techniques to assess the unique microvascularity associated with early stage eoc. methods: we used pulse inversion harmonic microvascular imaging (mvi) technology to depict differences between benign and malignant ovarian lesions. contrast enhanced harmonic transvaginal (tv) sonography was performed using the philips iu22 scanner after intravenous injection of 1 g of definity (bristol-myer-squibb). split screen real-time images were acquired displaying conventional sonographic views adjacent to harmonic, low mechanical index images. morphologic features (thickened wall, papillary excrescence, calcifications) and aberrant vascularity were noted. q-lab quantification of wash-in, peak enhancement, and wash-out times as well as area-under-thecurve (corresponding to microvessel perfusion) were compared using student's t-tests. results: to date, contrast enhancement patterns of 93 ovaries have been analyzed, 78 benign and 15 malignant. of the malignancies (3 fallopian tube, 12 ovarian: 3 stage i, 12 stage iii), 12/15 women were correctly identified using conventional tv imaging and 3 others were identified using mvi 15/15. all benign lesions were correctly identified by mvi 78/78 while tv detected 81 normals (3 false negatives). the lesions detected as malignant by mvi were 2-stage i fallopian tube and 1-stage i eoc. contrast enhancement kinetics of malignant lesions demonstrated similar wash-in (26.1 ± 6.3 vs 24.9 ± 7.6, p = 0.7), greater peak enhancement (23.3 ± 2.8 vs 12.3 ± 3.9, p < 0.01 ), longer wash-out (139.9 ± 43.6 vs 46.3 ± 19.7 (p < 0.01), and greater perfusion (2012.9 ± 532.9 vs 523.8 ± 318, p < 0.01) when compared to benign lesions. conclusion: contrast enhancement patterns are significantly different in benign vs. malignant ovarian masses. this technique has clear potential in differentiating benign from malignant lesions and for detecting occult stage i disease. identification and characterization of mir-199a as regulator of ikk expression and its function in ovarian cancer cells. rui chen, ayesha b alvero, thomas rutherford, gil mor. department of obstetrics and gynecology, yale university school of medicine, new haven, ct, usa. introduction: the proinflammatory environment associated with tumor growth and chemoresistance is produced by both immune cells and cancer cells. the nf-b pathway plays a critical role mediating the capacity of cancer cells to produce pro-inflammatory cytokines. recently, we described a group of epithelial ovarian cancer (eoc) cells characterized by ikk expression as the main factor promoting nf-b activation and cytokine production. in this study we evaluated the regulation of ikk in eoc cells. we describe the identification and characterization of mir-199a as a regulator of ikk expression, thus indirectly of nf-b activity and function. materials and methods: 11 human eoc cell lines were established from malignant ovarian cancer ascites. protein expression were determined by western blotting. ikk mrna was measured by rt-pcr. cytokines were profiled by the luminex 200 system. ikk transfection was done with roche fugene6 transfection reagent. mirna microarray was done with invitrogen ncode multi-species mirna microarray kit. mir-199a qrt-pcr was performed with invitrogen ncode sybr greener mirna qrt-pcr analysis kit. mirna transfection was done with ambion siport neofx agent. the ikk 3'-utr luciferase reporter plasmid was established based on ambion pmir-report mirna expression reporter vector. results: ikk expression was associated with nf-b cyclic activity and the ability of type i eoc cells (but not type ii) to produce inflammatory cytokines. transfection of ikk into type ii eoc cells reversed their phenotype. mirna microarray identified 18 mirnas differentially expressed in type i versus type ii cells, one of which, mir-199a, had 3 putative binding sites in the 3'-utr of ikk mrna. mir-199a introduction into type i cells inhibited ikk expression, and direct inhibition through ikk 's 3'-utr was confirmed by luciferase assay. conclusion: we describe for the first time the identification of ikk as a potential key switch between chemo-resistant and chemo-sensitive phenotypes, by regulating nf-b activity in eoc cells. furthermore, we identified mir-199a as a direct regulator of ikk expression. ikk expression may represent an adaptational stage in tumor progression allowing cancer cells to create their own inflammatory environment. these findings may provide novel molecular targets and potential markers for individual therapy selection. regulation of cd55 in the rat uterus by nitric oxide and the involvement of p13 kinase pathway. uma yallampalli, rebakah elkins, pawel goluszko, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. objective. cd55 is expressed in many cell types including uterine cells and has been shown to play an important role in protecting against compliment attack. we have previously shown in endometrial cell lines that cd55 levels were down regulated by nitric oxide (no) . in this study we extend our previous observations to determine if no down regulates cd55 in rat uterine tissues and assess its mechanisms of action. methods. non pregnant rats (200g; b wt) were bilaterally ovariectomized under ketamine anesthesia. groups of ovariectomized rats were implanted with alzet mini pumps to deliver nitro-l-arginine melthylester (l-name) at 10 or 50/mg/rat/day in saline or saline alone. uteri were obtained from these rats at 48 or 72 hours after infusion. in another set of experiments uteri were obtained from ovariectomized rats and cut in to small pieces and incubated in vitro with either l-name (3mm), diethylenetriamine-no 1mm) or worthmanin (0.1 m) in mem without phenol red for 24 hours. uterine tissues were homogenized in trizol and mrna levels for cd55 were measured using rt-pcr and expressed as a ratio to 18s. results. results show that in vivo treatment with l-name to ovariectomized rats caused elevations in uterine cd55 mrna levels in a time and dose dependent manner with maximal responses seen with 50mg l-name and at 72 hours. in vitro studies show that deta-no suppressed cd55 mrna levels in the rat uterus. both l-name and pi3 kinase inhibitor, worthmanin caused increases in cd55 levels in these tissues and the effects of worthmanin are reversed by deta-no. these results suggest that cd55 levels in the rat uterus are down regulated by no and are upregulated when no synthesis is inhibited by l-name. further, pi3 kinase appears to be involved in cd55 regulation in the rat uterus and no donor appears to modulate this response. lung. lakeitha r foster, 1 daniel b hardy, 2 carole r mendelson. 2 1 dept of pediatrics; 2 depts of biochemistry and ob/gyn, ut southwestern medical center, dallas, tx, usa. during 95% of human pregnancy, the maternal uterus is maintained in a state of almost complete quiescence by elevated circulating levels of progesterone (p 4 ). we previously observed that p 4 acting through the progesterone receptor (pr) serves an anti-inflammatory role and inhibits uterine contractility by antagonizing nuclear factor b activation and cyclooxygenase-2 (cox-2) expression (hardy et al., 2006) . our previous findings also suggest that the fetus provides an important signal for the initiation of labor near term through augmented secretion of the major lung surfactant protein, sp-a, into amniotic fluid (condon et al., 2004) . secreted sp-a, in turn, activates fetal macrophages which migrate to the maternal uterus where they release cytokines and promote an inflammatory response, leading to labor. in the present study, we tested the hypothesis that maternal p 4 also maintains uterine quiescence by inhibiting sp-a production by the fetal lung. age matched icr mice were injected s.c. once daily either with sesame oil (control) or p 4 (1 mg/ml) from 15 to 19 days post-coitum (dpc). as expected, treatment with p 4 delayed parturition by 24-48 h. this also was associated with a decrease in uterine cox-2 mrna expression, as compared to the vehicle-injected controls. interestingly, maternal p 4 treatment caused a marked decrease in the levels of immunoreactive sp-a protein secreted by the fetal lungs into in amniotic fluid at 19 dpc. furthermore, sp-a protein and mrna levels were reduced in the fetal lungs of p 4 -injected mothers, as compared to controls. this was associated with an inhibitory effect of p 4 treatment on cox-2 protein and mrna levels in the fetal lungs. these findings were of interest, since cox-2 expression is markedly upregulated during differentiation of human fetal lung (hfl) explants in culture (hardy et al., 2005) and endogenous and exogenous prostaglandins increase sp-a expression in hfl (acarregui et al., 1990) . collectively, these findings suggest that maternal p 4 treatment prevents increased uterine contractility, in part, by inhibiting inflammatory response pathways within the fetal lung. this, in turn, blocks the developmental induction of sp-a expression and its secretion into amniotic fluid. in this manner, maternal p 4 inhibits an important fetal signal leading to labor. supported by nih p01 hd011149; nih r37 hl050022. recent evidence suggests that leukocytes infiltrate uterine tissues at the time of parturition implicating inflammation as a key mechanism of human labor. ccl-2 is a pro-inflammatory cytokine that may contribute to the development of inflammatory reaction in the myometrium. previously we showed upregulation of rat ccl-2 gene expression in myometrium during term and ru486-induced preterm labor. also ccl-2 was elevated specifically in the gravid horn of unilaterally pregnant rats suggesting that mechanical strain imposed by the growing fetus controls its expression in the myometrium. the objective of this study was to investigate the role of mechanical stretch as a possible regulator of myometrial leukocyte infiltration and ccl-2 as a mediator of this stretch response. we also studied the effect of progesterone (p4) on the myometrial secretion of ccl-2. methods. we used primary culture of rat myometrium smooth muscle cells (smcs) to study in vitro ccl-2 gene and protein induction by static mechanical stretch. ccl-2 gene expression analysis was performed by real-time rt-pcr and immunoreactive (ir) protein content was measured by elisa assay. we used primary rat monocytes to access whether stretch-induced ccl-2 production by myometrial smcs resulted in enhanced monocyte chemotactic activity. results. myometrial cells were stretched for 2-24 hours and the supernatants collected. analysis of media conditioned by primary myometrial smcs revealed that static mechanical stretch (25% elongation for 24 hours) caused a significant accumulation in ir ccl-2 which was repressed by pretreatment with p4 (1 um). the rise in ccl-2 protein levels was preceded by a transient increase on ccl-2 mrna. the migration of primary rat monocytes in response to conditioned medium from stretched myometrial smcs was much greater than that of conditioned medium from control non-stretched cells. co-incubation with a neutralizing antibody to ccl-2 significantly reduced the chemotaxis of monocytes in response to the stretch-conditioned medium. conclusion: uterine smcs play an active role in uterine inflammation by producing chemokines and promoting the chemotaxis of immune cells into the myometrium. the blockade of this effect by p4 offers a potential explanation for the therapeutic actions of this hormone in the prevention of preterm birth. membranes during human labor. nardhy gomez-lopez, 1,2 guadalupe estrada-gutierrez, 1 lourdes vadillo-perez, 1 felipe vadillo-ortega. 1 1 direction of research, instituto nacional de perinatologia, mexico city, df, mexico; 2 escuela nacional de ciencias biologicas, ipn, mexico city, df, mexico. introduction. leukocytes arriving to the choriodecidua (chd) during labor are capable to secrete cytokines and matrix metalloproteinases that may play a role in the fetal membranes (fm) extracellular matrix degradation. objective. the aim of this work was to identify changes in the leukocyte subpopulations in the chd and fm during human labor. methods: fm were obtained from two groups of women: 1) term without labor (n=4) and 2) term with spontaneous labor (n=4). chd cells were isolated and analyzed by flow cytometry. explants of fm were embedded in paraffin and analyzed by confocal microscopy. in both techniques, cd45, cd56, cd14, cd19 and cd3 subpopulations of leukocytes were identified. intracellular mmp-9 was also identified in these cells. results: major changes in leukocytes subpopulations during labor involved a higher amount of cd3+, cd+14 and cd56+ cells, both in the chd and inside the fm. mmp-9 was associated to cd 56+ cells. cd3+ exhibited a more widespread localization in the fm and cd56+ cells were localized in the contact with the trophoblast layer during labor. conclusions: leukocyte populations changes both in the chd and fm during labor and are characterized by arrival and infiltration of specific subpopulation of lymphocytes and monocytes. nk cells are enriched in mmp-9, which may be related to a role in extracellular matrix degradation leading to the rupture of fetal membranes. women with a history of a pregnancy complicated by preeclampsia or intrauterine growth restriction (iugr) have an increased risk of future cardiovascular disease. excessive weight, particularly abdominal fat mass, is associated with cardiovascular morbidity and mortality. objectives: the aim of this study was to investigate differences in body composition and fat distribution between women with a history of preeclampsia or iugr and uncomplicated pregnancies. methods: from a genetically isolated population in the southwest of the netherlands, non-pregnant women with a history of preeclampsia (n=45), iugr (n=53) and uncomplicated pregnancies (n=106) were recruited at a mean follow up time of 10.8 years after pregnancy. body composition and fat distribution were assessed by dual energy-x-ray absorptiometry (dxa) and anthropometric measurements. results: women with a history of preeclampsia compared to controls had higher mean total-, fat-and lean mass (p <0.05) as well as higher mean indices of body mass, fat mass and lean mass (p< 0.05). no significant differences were found for these variables between women with a history of iugr and controls. women with a history of preeclampsia had higher waist circumferences and waistto-hip ratios (p<0.001) as well as excess of android fat mass and increased android-to-fat ratios (p<0.05). women after pregnancies complicated by iugr had higher waist-to-hip ratios (p <0.001). after controlling for body mass index, both women with a history of preeclampsia or iugr had higher waist circumferences (p <0.001) and waist-to-hip ratios (p <0.001) as well as smaller hip circumferences (p < 0.001). conclusion: despite differences in body mass index, both women with a history of preeclampsia and women after pregnancies complicated by iugr have a metabolically adverse fat distribution, marked by an excess of fat deposition in the abdominal region relatively to the hip region. these findings may explain, at least partly, their increased cardiovascular risk. women with a history of preeclampsia. marc ea spaanderman, 1 timo h ekhart, 2 robert aardenburg, 2 louis lh peeters. 2 1 obstetrics and gynecology, radboud university medical center, nijmegen, netherlands; 2 obstetrics and gynecology, university medical center maastricht, maastricht, netherlands. background: a history of preeclampsia is associated with persistent short-term alterations in circulatory function and remote cardiovascular disease. in this study we tested the hypothesis at least 20 years after preeclamptic pregnancy renal and central hemodynamic function is impaired as compared to women with uncomplicated pregnancy. methods: in 20 formerly preeclamptic women (pe) and 24 healthy parous controls (control) who were normotensive at 6 weeks post-partum follow up, we assessed at least 20 years after delivery blood pressure (mmhg), cardiac output (co, doppler ultrasonography, l/min) and effective renal plasma flow (erpf, pah clearance, ml/min/1.73m 2 ) after which we calculated total peripheral vascular resistance (.100 dyne.s/cm 5 ) and renal vascular resistance (.100 dyne.s/cm 5 ). data were analyzed parametrically (p<0.05). results: age and bmi were comparable between groups. blood pressure was higher in formerly pe. moreover, 40% of formerly pe women and 13% of control were hypertensive (p<0.05). although cardiac out was comparable between groups, total peripheral and renal vascular resistance were about 20% higher and erpf 15% lower in formerly pe women as compared to control. conclusion: at least 20 years after gestational hypertensive disease, women who were normotensive at direct follow up have impaired renal and central hemodynamic function and developed more often chronic hypertension. long-term follow up may also be warranted in apparently healthy formerly pe women who are normotensive at post-partum follow up. circulatory function in formerly preeclamptic women and healthy parous controls co erpf tpvr rvr formerly pe 5.0±0.6 397±61* 18±2* 120±29* control 4.7±0.8 469±80 15±2 97±25 * = p<0.05 101 pregnancy increases blood-brain barrier permeability coefficient (l p ) to lucifer yellow: role of estrogen. marchien j wiegman, 1,2 marilyn j cipolla. 1 1 neurology, ob/gyn and pharmacology, university of vermont, burlington, vt, usa; 2 ob/gyn, university medical center groningen, groningen, netherlands. background: eclampsia is similar to posterior reversible encephalopathy syndrome in which an acute rise in blood pressure causes breakthrough of autoregulation, blood-brain barrier (bbb) disruption, and cerebral edema formation. we previously showed that late-pregnant (lp) animals developed cerebral edema during breakthrough, a response that was absent in nonpregnant (np) animals. in the current study we hypothesized that pregnancy predisposes the brain to edema during acute hypertension by enhanced bbb permeability. we further hypothesized that the underlying effect of pregnancy on the bbb permeability is due to elevated estrogen levels. methods: permeability coefficients (l p ) to lucifer yellow (ly), a polar compound that does not pass through tight junctions, were compared in posterior cerebral arteries (pca) from 4 groups of sprague dawley rats: np (n=7), lp (d20; n=7), ovariectomized and implanted with 17 -estradiol (0.5mg, 21-day release) and estriol (5.0mg, 21-day release) pellets for 14 days (ovx+e; n=6), and ovariectomized and implanted with placebo pellets for 30 days (ovx; n=6). pcas were isolated, pressurized in an arteriograph, and perfused with 0.5mg/ml ly in saline. concentration changes of ly outside the vessel wall were determined at pressures from 60-200mmhg. the slope of the pressure vs. permeability curve is the rate of flux, or l p for ly. results: l p for ly was significantly increased in pcas from lp and ovx animals vs. np (p<0.05; figure 1 ). estrogen was protective of the bbb only in ovariectomized animals, decreasing l p 150% in ovx+e vs. ovx. however, pregnancy did not afford protection and had a l p that was 256% greater than np. conclusions: pregnancy significantly increases bbb permeability to ly, an effect that may predispose the brain to edema formation during acute hypertension. these data also show that estrogen modulates l p in ovariectomized animals differentially than pregnancy, suggesting that the increased bbb permeability in pregnancy is caused by a mechanism other than elevated estrogen levels. in both pre-and early pregnancy, the sympathoinhibitory response to volume expansion is blunted in formerly preeclamptic women with low plasma volume. ineke krabbendam, 1 marc ea spaanderman, 1 dorette a courtar, 2 robert aardenburg, 2 ben j janssen, 3 fred k lotgering, 1 louis lh peeters. 2 1 obstetrics and gynecology, radboud university nijmegen medical centre, nijmegen, netherlands; 2 obstetrics and gynecology, university hospital maastricht, maastricht, netherlands; 3 pharmacology and toxicology, university of maastricht, maastricht, netherlands. background: the circulation of formerly preeclamptic women with a low plasma volume (lpv) is characterized by sympathetic dominance. these women respond to a new pregnancy with an aberrant rise in atrial natriuretic peptide (anp) and a 3 times higher chance to develop recurrent gestational hypertensive disease compared to their counterparts with normal plasma volume (npv). anp has sympathicomimetic capacity. we postulate that the sympathetic overdrive in lpv-women is associated with a reduced venous capacitance. to this end, we compared the response to volume expansion (ve) in women with lpv and npv, both before and in pregnancy. method: in 24 non-pregnant normotensive formerly preeclamptic women, we measured pv (hsa i 125 indicator dilution method) at least 6 months post partum. we intravenously infused 500 ml of iso-oncotic fluid over 30 minutes. during the infusion, we recorded changes in heart rate (hr, bpm), blood pressure (bp, mmhg), cardiac output (co, l/min), sympathetic activity (lfsys, mmhg 2 , low frequency component of spontaneous fluctuations in systolic bp, portapress) and anp (nmol/l). eight women became pregnant within 1 year and were evaluated at 12 weeks gestation. changes in circulatory and autonomic function between and within groups were analyzed non-parametrically (p<0.05). results: before pregnancy, ve leads to comparable changes in hr, bp and co in women with lpv (17/24) and npv (7/24). in npv, lfsys decreased 28%, but only 7% in lpv (p<0.05). anp remained unaltered in npv, but increased in lpv. in the pregnant group, 4 women had lpv and 4 had npv. in both groups, pregnancy did not alter the response to ve. conclusion: irrespective of pregnancy, the sympathoinhibitory response to ve is diminished in lpv. these data suggest that in these women ve leads to venous overfill, giving rise to anp-release and consequently sympathetic activation, flattening the normal baroreceptor-mediated sympathoinhibitory response. we speculate that this mechanism contribute to circulatory maladaptation to pregnancy, sympathetic dominance and subsequent gestational hypertensive disease. in both pe and ht, serum sflt-1 was increased, and plgf reduced at all gestations (p<0.0001). seng levels were also increased in pe. after 28 weeks (but not before) antihypertensive treatment was associated with a significant fall in serum sflt-1 and seng, in pe only. the concentrations of both sflt-1 and seng were significantly higher in the placentas of women with pe, but not ht, compared with controls (p=0.0002). only sflt-1 was significantly reduced in the placenta in women who received antihypertensive therapy. conclusion in pe, antihypertensive therapy after 28 weeks' gestation is associated with a significant fall in serum sflt-1 and seng, and in placental sflt-1. these findings raise the possibility that these drugs may have an effect on the pathophysiology of pe other than their known antihypertensive action. the synergistic effect of soluble vegf receptor 1 pre-treatment and small doses of tnf-on endothelial cells. tereza cindrova-davies, 1 debbie a sanders, 2 olivera spasic-boskovic, 1 graham j burton, 1 d stephen charnock-jones. 2 1 dept of pdn, university of cambridge, united kingdom; 2 dept of obstetrics and gynaecology, university of cambridge, united kingdom. introduction: preeclampsia is marked by an enhanced endothelial inflammatory response manifested by maternal endothelial activation. soluble fms-like tyrosine kinase-1 (sflt-1, svegf-r1), a naturally occurring circulating antagonist of vegf-a and plgf, is one of the secreted factors implicated in the pathogenesis of preeclampsia. in women who develop preeclampsia, sflt rises sharply, preceding the onset of the clinical disease. the aim of this study was to examine the effect of a combined treatment with recombinant sflt-1 and tnf-on the activation of human umbilical cord endothelial cells (huvec) by examining leukocyte adhesion, and the expression of icam, vcam, endothelin and vwf. methods: huvec were seeded, grown overnight and pre-treated with recombinant sflt (50-250 ng/ml) in a basic 2% fbs-dmem medium for 24 hr. on day 3, low doses of tnf-(0.5 ng/ml) were applied to pre-treated cells for 6 hr. antagonism of the vegf-a action was mimicked at the protein level by pre-incubating huvec with anti-flt antibody (5-10 g/ml), anti-kdr antibody (1 g/ml), anti-vegf antibody (5 g/ml), or vegf receptor inhibitor su5614 (5 m). at the rna level, the effect of sflt was mimicked by sirna transfection of huvec with siflt or sikdr. at the end of each experiment cells were either harvested for western blotting, fixed in 2% pfa for immunofluorescence or incubated with labelled hl60 leukocyte cells, followed by fluorescent detection of adhesion. results: pre-incubation of huvec with sflt and subsequent treatment with low doses of tnf-increased the adhesion of hl60 leukocytes and increased icam-1, vcam-1, endothelin and vwf, compared to tnf-treatment alone. similar results were obtained when cells were pre-treated with su5614, anti-flt, anti-kdr or anti-vegf. transfection knock-down of flt or kdr gene also significantly increased leukocyte adhesion when small doses of tnfwere added. conclusions: pre-incubation with recombinant sflt, anti-flt, anti-kdr, anti-vegf, su5614 or knocking-down flt or kdr transcripts all antagonised the autocrine actions of vegf and/or plgf. this predisposes huvecs to be more sensitive to the effect of tnf-. our study shows that sflt and tnfcombine to induce an enhanced synergistic effect, activating endothelial cells. maternal obesity is associated with increased production of inflammatory cytokines and risk of poor perinatal outcome. inflammatory cytokines can stimulate production of reactive oxygen (ros) and nitrogen (rns) species, which can covalently modify protein function. placental oxidative and nitrative stress are increased in pathological pregnancies and associated with altered placental function. objectives: determine the effect of increasing body mass index (bmi) on placental nitrative stress, measured by the expression and localization of nitrated (nitrotyrosine) and oxidized proteins. methods: placental tissue was collected at term (37-41 wks) from lean kg/m 2 ), overweight (25-29.9 kg/m 2 ) and obese (30-40 kg/m 2 ) patients (n=5 or 6/group). tissue was sectioned for immunostaining with nitrotyrosine antibody. protein samples were either dot blotted onto nitrocellulose membrane, probed with nitrotyrosine ab, and nitrated proteins detected using ecl, or derivatized using 2,4-dinitrophenylhydrazine (dnph) (oxyblot® kit), separated on sds-page, probed with anti-dnph ab (1:150) and oxidized proteins detected using ecl. protein band intensity was measured by densitometry. oxidized proteins were selected for maldi mass spectrometry analysis. results: nitrotyrosine residues were immunolocalized primarily in the fetal capillary endothelial cells and the villous stroma, but were almost absent in the syncytiotrophoblast. by dot blot, nitrotyrosine expression differed across the three groups (p<0.003, anova) with expression in tissue from obese women being significantly increased compared to lean (p<0.005) and overweight (p<0.05, tukey test). several oxidized proteins were detected with significantly greater expression seen in the lean versus overweight groups (p<0.02, mann-whitney u). one oxidized protein was identified by maldi-ms with four peptide matches and 19.2% coverage as 3-beta hydroxysteroid dehydrogenase (3bhsd). discussion: with increasing bmi, an increase in nitrative stress appears to occur in parallel with a decrease in oxidative stress. oxidative stress is apparently reduced as ros are consumed by the interaction with rns to give nitrative stress. 3bhsd is involved in the biosynthesis of steroid hormones and glucocorticoids. its activity and hence steroid metabolism in the placenta may be regulated by oxidation with implications for fetal development. background teenagers are susceptible to delivering small-for-gestationalage (sga) infants. previous studies implicate continued maternal growth as a causal factor 1 . growing adolescent sheep have reduced fetal birthweight due to impaired placental development and nutrient transfer 2 . we hypothesized that placental function is impaired in human teenage pregnancy if there is maternal growth. methods placentas were collected from 29 teenagers (15-18 years) and 21 adults. activity of the amino acid transporter, system a, was quantified by the sodium-dependent uptake of 14 c-methylaminoisobutyric acid into placental fragments. teenagers were defined as growing (>2mm increase in kneeheight per 90 days) or non-growing. system a activity was analysed in relation to individualised birthweight centile and maternal growth. results placental system a activity was significantly lower in teenage compared to adult pregnancies (p<0.05). this was unrelated to birthweight; teenagers who delivered infants appropriate-for-gestational age (aga) had significantly lower placental system a activity compared to aga infants delivered to adults (p<0.01). growing teenagers did not deliver lower birthweight infants than non-growing teenagers (median birthweight 3550g and 3290g respectively). furthermore, system a activity in placentas from growing teenagers was significantly elevated compared to that in non-growing teenagers (p<0.01), and was similar to placental activity in adults. conclusions system a activity was reduced in placentas from teenagers compared to adults. this suggests that inherently lower placental function predisposes teenagers to sga, but that other factors (e.g. adequate nutrition) can compensate in those teenagers delivering aga infants. in contrast to our hypothesis, placental system a was elevated in growing teenagers and mimicked that of adults. this may be related to a hormonal-milieu in growing teenagers that is conducive to fetal growth, in part through stimulating placental transport. homocysteine inhibition of system a amino acid transport activity in human placenta. eleni tsitsiou, susan l greenwood, colin p sibley, stephen w d'souza, jocelyn d glazier. maternal and fetal health research group, st. mary's hospital, university of manchester, manchester, united kingdom. background: elevated plasma levels of the amino acid homocysteine (hcy) during pregnancy are associated with vascular-related complications and adverse neonatal outcomes including a reduced birthweight. fetal and maternal plasma hcy concentrations are positively correlated suggesting placental transport of hcy may be an important determinant of fetal plasma hcy. the mechanisms involved in placental hcy transport are uncharacterised. evidence that the system a amino acid transporter, which transports neutral amino acids in a na + -dependent manner, is important in promoting fetal growth and that a reduced system a activity is associated with intrauterine growth restriction (iugr), led to our hypothesis that system a provides one mechanism for placental hcy transport. this hypothesis was tested by measuring the ability of hcy to inhibit system a activity in isolated microvillous plasma membrane (mvm) vesicles and placental fragments. materials and methods: mvm vesicles and placental fragments were isolated from placentas of normal pregnancies at term. system a activity was measured at initial rate (30s and 20 min respectively) as na + -dependent 14 cmethylaminoisobutyric acid (meaib) uptake into mvm vesicles (0.165mm) or fragments (0.0085mm) in the absence (control) or presence of l-hcy and dl-hcy or model substrates. results: 20mm l-hcy (custom-synthesised) and dl-hcy (commercial source) significantly (p<0.05) inhibited na + -dependent 14 c-meaib uptake into mvm vesicles compared to control; comparable in magnitude to other model substrates (meaib, l-ala, l-ser, l-met; n=9, kruskal-wallis with dunn's multiple comparison test). l-hcy, l-met and meaib (0.05-20mm) caused a dose-dependent inhibition of na + -dependent 14 c-meaib uptake into mvm with ec50 values (in mm; mean ± se) of 0.53 ± 0.04, 0.27 ± 0.02, and 1.13 ± 0.12 respectively, n=6). na + -dependent 14 c-meaib uptake into fragments was reduced substantially in the presence of 10mm l-hcy or dl-hcy causing a 85 ± 13 and 88 ± 12% reduction (mean ± sd, n=2-3) of control respectively. conclusion: these observations suggest that hcy is a relatively high affinity substrate for system a in the human placenta. we speculate that inhibition of placental amino acid uptake by hcy could impact on fetal growth and development. supported by the mrc and action research endowment fund. 110 glucose regulates placental mtor activity and glucose deprivation down-regulates placental system l activity in an mtor-dependent manner. sara roos, 1 theresa l powell, 2 thomas jansson. 2 1 department of physiology, institute of neuroscience and physiology, gothenburg, sweden; 2 department of obstetrics and gynecology, university of cincinnati, cincinnati, oh, usa. placental amino acid transporters are down-regulated in intrauterine growth restriction (iugr). we have previously shown that mammalian target of rapamycin (mtor) regulates placental system l transporter activity and that placental mtor activity is decreased in iugr. however, the upstream regulators of placental mtor are unknown. in iugr, fetal hypoglycemia and reduced maternal glucose levels are common and the placenta may therefore be exposed to low glucose levels. hypothesis: we hypothesized that glucose availability regulates placental amino acid transporter activity mediated by changes in mtor signaling. methods: cytotrophoblast cells were isolated and cultured until syncytialization at 66 hours. cells were cultured for an additional 24 hours in culture media containing 0.5 mm, 4.5 mm, or 16 mm glucose (control), which corresponds to standard culture media. at 90 hrs, the activity of the mtor signaling pathway was assessed by measuring the protein expression of s6k1 phosphorylated at thr-389, the primary site of mtor phosphorylation. in another set of cells, system l activity was assessed by measuring the bchinhibitable uptake of 3h-leucine. results: as compared to control, phospho-thr-389-s6k1 expression was reduced by 24% in cells incubated in 0.5 mm results: fgr pregnancies were delivered earlier (39±0.2 v 32±0.6w; p<0.01), weighed less (3.5±0.2 v 1.2±0.1kg; p<0.01) and had higher s/d ratios (2.2±0.1 v 5.3±1.1; p<0.01). eaa concentrations were similar between groups, but there were differences (non-parametric testing; p<0.05) in transport rates between groups with his crossing considerably faster and ile crossing slower in fgr . the table shows fetal vein/maternal (fv/m) ratios standardized to the leu fv/m ratio for all 9 eaa for both groups. amino acids fell into 3 groups for fgr pregnancies: 1) his (ratio 1.38), 2) leu, phe, met (ratios 1), and 3) val, thr, ile, trp, lys with intermediate ratios ( 0.6 ns conclusion: this is the 1 st study to compare the relative rates of in vivo placental transport for all eaa between normal and fgr human pregnancies showing striking differences in the transport rates of two eaa. in the absence of eaa concentration differences between groups, the higher his transport rate in fgr suggests higher utilization by the placenta. vasculature of women who received exogenous estrogen compared to those who received placebo. the purpose of this investigation was to identify the gene expression in response to the estrogen, equilin, as the major component of conjugated equine estrogen and genistein, a phytoestrogen in human coronary artery endothelial cells. human coronary artery endothelial cells from a 43-year old female were used. cells were treated with estradiol [e2], equilin [eq] (0.1 nm) or genistein [gen] (5 micromolar) for 48 hours. focused oligomicroarrays for cardiovascular disease and endothelial cell function were used to study gene expression. rt-pcr was used to confirm the transcription of genes analyzed in oligoarrays. vascular adhesion molecules and genes involved in the inflammatory response were most affected with the three estrogen sources. significant reduction of integrin alphae was seen with all three 2.63, 2.06 and 16.74 for e2, eq and gen respectively. similarly nfkb1 was also reduced 2.62, 2.07 and 6.33 fold compared to controls. significant reduction of ccl2 was demonstrated with eq (3.11 fold) and gen (5.49 fold). tnfreceptor1a and 1b were only significantly decreased by gen. vcam-1 which is regulated by proatherogenic factors and upregulated mainly at atherosclerosis -prone sites, was significantly reduced (25.95-fold) with genistein, and a slight reduction was seen with e2 and no change was observed with eq. our data support the clinical findings that estrogen has favorable effects on the genes involved in atherosclerotic disease. while e2 has been shown to be slightly more effective than equilin in the modulation of gene expression, genistein was significantly more effective in the favorable expression of genes involved in particularly the inflammatory response. olga n lekontseva, sandra t davidge. physiology and obstetrics/gynecology, university of alberta, edmonton, ab, canada. introduction: the prevalence of cardiovascular disease in women dramatically rises in the postmenopausal period. although deficiency of estrogen has been implicated in the pathophysiology of systemic vascular dysfunction, the effects of estrogen on vasculature are complex and not completely understood. we have previously shown that estrogen exerts a beneficial effect on the aging vascular system by reducing circulating levels of the inflammatory cytokine tnf . tnf is a known regulator of matrix metalloproteinases (mmps), proteolytic enzymes that may modulate vascular tone through cleavage of vasoactive peptides such as big endothelin-1 (et-1). the role of estrogen in this pathway is unknown. we tested the hypothesis that in aging/estrogen deficiency, tnf -induced mmp activity mediates greater vasoconstriction, in part, through the et-1 pathway. we further hypothesized that estrogen replacement reduces vascular sensitivity to the constriction by preventing mmp activation. methods: aged (12 month old) female sprague dawley rats were ovariectomized and treated with either placebo [ovx] , 17 -estradiol [ovx+e 2 ], or the tnf inhibitor etanercept [ovx+etan] . after four weeks, resistance mesenteric arteries were isolated and studied on the pressure arteriograph. concentrationresponse to exogenous big in the absence or presence of mmp inhibitor (gm6001, 10μm) was assessed in the vessels. results: treatment of "menopausal" [ovx] rats with either estrogen or the tnf inhibitor reduced sensitivity of arteries to big et-1 (ec 50 =0.07±0.01μm in ovx versus 0.15±0.04μm in ovx+e 2 or 0.16±0.03μm in ovx+etan groups; p<0.02). although mmp inhibition attenuated maximal constriction in all of the arteries, there was a significantly greater (p<0.05) role of mmps in big et-1-induced vasoconstriction in the ovx+e 2 group (reduction in max constriction=70.3±11.8%) compared to ovx (40.7±9.1%) and ovx+etan groups (35.4±3.9%). conclusions: both estrogen and tnf inhibition reduced big et-1 vasoconstriction. however, contrary to our hypothesis, tnf is not contributing to mmp modulation of et-1 vasoconstriction. interestingly, our study demonstrates a novel role for estrogen to increase mmp contribution to big et-1 vasoactivity with the net effect being less vasoconstrictive. understanding this unique pathway of regulation by estrogen in the aged vasculature will allow for development of new therapeutic options for women. mo, usa. introduction: the etiology of pelvic organ prolapse is multifactorial, with both inherited and acquired components. the molecular mechanisms of prolapse have not been established yet. we have previously shown that lysyl oxidase (lox) expression is suppressed in uterosacral ligaments of women with pelvic organ prolapse. it has also been shown that lox is a tumor suppressor gene inactivated by methylation in human gastric cancers. hypothesis: the aim of this study was to analyze the dna sequence of the promoter region of the lysyl oxidase gene in tissues from women with pelvic organ prolapse and identify whether methylation is present. our hypothesis is that the promoters of the lox gene in women with pelvic organ prolapse have significantly more methylation sites than women without prolapse. materials and methods: genomic dna was isolated from the uterosacral ligaments of eight women with pelvic organ prolapse and 8 women without prolapse (controls). genomic dna samples were treated with ez dna methylation kit (zemo research, orange, ca). the lox gene promoter region of -246 to +74 was amplified by pcr and then cloned into pcr2.1-topo (invitrogen, carlsbad, ca) and transformed into an e. coli dh5a strain. amplified plasmid dna samples containing the lox gene promoter region from each woman were sequenced and methylated cpg islands were identified by sequence comparison. results: a total of 66 methylated cpg sites were found in the patient group with pelvic organ prolapse while only 1 methylated cpg site was found in the non-prolapse control group. conclusion: these findings suggest that methylation in the promoter region suppresses lox gene expression in women with pelvic organ prolapse. background: reports from case-control and cohort studies have suggested an inverse association between lactation and breast cancer risk, but findings have been inconsistent. methods: we conducted a prospective observational cohort study of 57,585 parous women participating in the nurses' health study ii from 1997 to 2005. our primary outcome was incident premenopausal breast cancer. results: during the study period, 621 cases of premenopausal breast cancer were diagnosed during 355,871 person-years of follow-up. women who had ever breastfed had a 24% lower incidence of premenopausal breast cancer (95% confidence interval [ci] 5-40%) compared with women who never breastfed, adjusting for parity, age at first birth, year of first birth, height, body mass index (bmi), bmi at age 18, family history, personal history of benign breast disease, participant birth weight, preterm birth, age at menarche, oral contraceptive use, physical activity, and alcohol consumption. no trend was observed with duration of lactation (p=0.52). the association between ever-breastfeeding and premenopausal breast cancer was modified by use of medication to suppress lactation (p=0.05); in analyses restricted to women who had never used suppressive medication, ever-breastfeeding was associated with a 46% (95%ci 23-63%) reduction in incident disease. the association between lactation and premenopausal breast cancer was further modified by family history of breast cancer (p=0.03). among women who had never used suppressive medication and reported a family history, those who had breastfed had a 67% lower covariate-adjusted risk of premenopausal breast cancer (95% ci 38-82%) than women who had never breastfed. among women without a family history, ever-breastfeeding was not associated with breast cancer incidence (hazard ratio 0.68, 95% ci 0.42-1.09). among women who had ever breastfed, we observed no association between breast cancer risk and duration of lactation amenorrhea or exclusive breastfeeding. conclusion: in a large, prospective cohort study, ever-breastfeeding was inversely associated with risk for premenopausal breast cancer. at the durations observed in our cohort, we observed no trend with duration of breastfeeding or lactation amenorrhea. the inverse association with ever-breastfeeding was stronger among women with a family history of breast cancer. regulatory and nkt cells at the maternal-fetal interface. thomas f mcelrath, 1 rachael a clark. 2 1 brigham women's hospital, boston, ma, usa; 2 harvard skin disease research center, brigham women's hospital, boston, ma, usa. introduction: pregnancy represents an immunologically challenging event requiring maternal tolerance of the fetal semi-allograft. an increase in decidual cd4 + cd25 + t cells has been documented but it is unclear if these represent foxp3 + t cells (tregs) . the possibility also exists that other cd3 + lineages with potential regulatory function exist within the decidua parientalis. we examined if cd4 + cd25 + cells are true foxp3 + tregs and evaluated the frequency of other t cell subsets with possible regulatory potential. methods: we extracted t cells from term deciduas of planned cesarean deliveries. cells were stained with directly conjugated monoclonal antibodies and were analyzed on a six color flow cytometer. results: we found that only a subset (median 33%) of cd25 + t cells were true foxp3 + regulatory t cells. from 65 donors, foxp3 + tregs accounted for a median of 7.1% of all cd4 + cells. these cells were memory cd45ro + t cells lacking ccr7, and l-selectin but expressing cd25, ctla-4, gitr, and hla-dr. additionally we found that a median 21% of cd3 + t cells expressed the nkt marker cd161. these cells were a mixture of cd4 and cd4 -cd8 -t cells, with variable numbers of cd8 t cells, suggesting they do not represent merely recently activated t cells. there was enrichment for t cells with nkt markers after culture on hela cells expressing cd1d, suggesting that these cell represent true nkt cells. nkt cells were of the non-classical type, with a diverse t cell repertoire (0.4% iv24jq) and also expressed cd69, hla class ii, cd45ro but were ccr7 and l-selectin low. comments: we find that only a minority of cd25-expressing t cell in the decidua are true foxp3 + tregs. because much of the work on treg in pregnancy has used cd25 as a treg marker, this suggests additional studies are needed to confirm the role of these cells in pregnancy. we find that a novel population of non-classical nkt cells exists in the human decidua. nkt cells can either promote or antagonize tolerance, depending on the immunologic context. the large number of these cells in the decidua suggests they may play a role equal or exceeding that of regulatory t cells. prolapse. marsha k guess, 1 kathleen a connell, 1 richard bercik, 1 lloyd g cantley. 2 1 obstetrics, gynecology reproductive sciences, yale university school of medicine, new haven, ct, usa; 2 internal medicine/neprhology, yale university school of medicine, new haven, ct, usa. objectives: thy-1 is a cell surface glycoprotien expressed in human fibroblasts, neurons, hematopoetic stems cells and endothelial cells. thy-1 expression affects fibroblast proliferation and migration, cell-cell, as well as, cell-matrix interactions. moreover, thy-1 expression has been shown to play a critical role in fibroblast dedifferentiation into myofibroblasts, as well as in extracellular matrix (ecm) production and fibrosis. women with pelvic organ prolapse (pop) have alterations in vaginal ecm protein expression and metabolism, as well as decreases in smooth muscle fractional content. in the current study, we evaluated thy-1 as well as the smooth muscle markers alpha-smooth muscle actin (asma) and desmin expression in women with pelvic organ prolapse compared to women with normal pelvic support (controls). methods: anterior apical vaginal wall specimens from women with pop and controls were collected at the time of hysterectomy. messenger rna and protein expression of thy-1, asma and desmin were evaluated using semiquantitative rt-pcr, real-time pcr and western blot analysis. gapdh and beta actin were used as internal controls. results: rt-pcr demonstrated the presence of thy-1, asma and desmin in vaginal tissue from women with pop and controls. further, thy-1 mrna expression was downregulated 57% in women with pop compared to controls (p = 0.004). a parallel decrease in thy-1 protein was seen in women with pop compared to controls (p=0.02). although a 44% and a 37% decease were seen in asma and desmin mrna, these differences were not statistically significant. similarly, no differences were seen in asma and desmin protein expression. conclusion: we demonstrate that there is significantly less thy-1 expression in vaginal tissue from women with pop compared to controls. differential expression of thy-1 in prolapsed vaginal tissues suggests that thy-1 may have a functional role in mediating ecm metabolism in the female genitourinary tract. hur expression is altered in ectopic endometrium. s karipcin, t altun, ua kayisli, e seli. ob gyn, yale u., new haven, ct, usa. introduction: cytokines and growth factors contribute to cyclic turnover of the normal endomterium and to the pathogenesis of endometriosis. cytokine and growth factor messenger rnas (mrnas) undergo rapid turnover that is primarily mediated by au-rich elements (are) that consist of multiple stretches of adenylate and uridylate residues located in the 3' untranslated region (3'-utr) of their mrnas. hur is a ubiquitously expressed rna-binding protein that stabilizes are containing mrnas and prolongs their expression. we hypothesized that hur may play a role in the regulation of cytokine expression during normal menstrual cycle and in endometriosis. methods: tissue sections obtained from normal (n=14) and ectopic (n=7) endometrium were immunostained for hur. staining intensity was evaluated by hscore and grouped according to menstrual cycle phase. statistical analysis was done with one-way anova. cultured stromal cells isolated from normal endometrium were treated with vehicle, estradiol (e2; 10 -8 m), or progesterone (p, 10 -8 m) for 24 and 48h, and hur expression was determined using western analysis and normalized to ß-actin. results: hur immunostaining was nuclear in endometrial cells. hur immunoreactivity was significantly lower in the early proliferative and late secretory phases (157.5 ±11.08 and 190.0 ±15.2, respectively), compared to the mid-late proliferative (270.0±8.0) and early-mid secretory phases (256.6±20.2 )(p<0.01). moreover, hur expression was significantly lower in ectopic endometrial cells when compared to normal endometrium in midlate proliferative and early and mid-secretory phases (p<0.01). progesterone suppressed hur levels significantly in cultured endometrial stromal cells at both 24 and 48 h compared to control (p<0.05) while estrogen did not cause a significant change. discussion: decreased hur levels in the late secretory and early proliferative phases are likely to contribute to degradation of cytokines and result in lower cytokine levels observed mid-cycle. late secretory decrease in hur levels may be mediated by progesterone as suggested by in vitro findings. in ectopic endometrium, persistent low expression of hur compared to normal endometrium most probably results from elevated cytokine levels associated with endometriosis. the effect of lower hur expression in ectopic endometrium on other are-containing transcripts, and on the pathogenesis of endometriosis remains to be elucidated. tissue. hong zhao, joy innes, scott reierstad, mehmet bertan yilmaz, serdar e bulun. department of obstetrics and gynecology, northwestern university, chicago, il, usa. background: aromatase is the key enzyme for estrogen biosythesis, and is encoded by the cyp19a1 gene. thus far, only three unique untranslated first exons associated with distinct promoters in the mouse cyp19a1 gene were described (brain-, ovary and testis-specific exon i). however, it remains unknown whether aromatase is expressed in other mouse tissues via previously unknown tissue-specific promoters activating new exon is. methods: real-time pcr was used to examine the aromatase expression levels in various c57bl6 mouse tissues. 5'-rapid amplification of cdna ends (5'-race) was used to determine the transcriptional start sites of cyp19a1 transcripts. promoter activity was measured using serial deletion mutants of dna fused to the luciferase reporter gene. results: real-time pcr results showed that aromatase was expressed in male gonadal fat and the expression level is lower than that in testis. the adipose tissue-specific untranslated exon i of cyp19a1 transcript was isolated using 5'-race and this novel gonadal fat-specific exon i of cyp19a1 mrna did not show sequence similarities to previously reported ones. this new adiposespecific exon i was mapped to 75 kb upstream of the translation start site in the coding exon ii. the genomic region upstream of the adipose-specific exon i was cloned into luciferase plasmids. transfection of murine 3t3-l1 cells with these plasmids showed that promoter activity was conferred by the sequence located at -1 to -343 bp upstream of the transcriptional start site. dexamethasone significantly induced activity of the adipose-specific promoter region. conclusion: taken together, our results suggest that a novel cyp19a1 transcript is regulated by a tissue-specific promoter in male murine gonadal fat. these sacrificed; reproductive and selected other organs were removed, weighed and evaluated histologically by an observer blinded to treatment. results: the table below shows that tcc augmented effects of tp on weights of accessory sex organs. histological assessment revealed that tcc induced greater glandular distention with more secretions compared to the effects of tp alone; furthermore, mitotic figures were seen only in prostates from rats exposed to tp+tcc. conclusions: tcc is a newly identified endocrine disruptor with unique and novel actions resulting in potentiation of androgen effects on sex organs. these observations underscore possible environmental risks related to exposure to tcc. background: blastocyst implantation is dependent on the differentiation of human endometrial stromal cells (hesc) into decidual cells. transforming growth factor family members have well defined roles in cell differentiation and proliferation. activin a, a tgf superfamily member, enhances hesc decidualization and localizes to decidualized cells in human endometrium. other tgf superfamily members, including bmp2, bmp4, bmp7, gdf5, gdf8 (myostatin), gdf11 and nodal, may also be present in decidual cells and therefore may also play a role during this important process. this study aimed to determine whether activin is the major family member driving decidualization or whether other family members contribute to the process. methods: broad ranging activin inhibitors (activin-m108a and sb431542) that effect receptor-ligand interactions of other tgf superfamily members were used in hesc decidualization. protein localization was examined in secretory phase endometrium and first trimester decidua by immunohistochemistry and mrna expression was examined in an ex vivo model. the secretion of candidate proteins was measured during hesc decidualization and certain recombinant proteins added during decidualization to examine their effect. results: m108a (25nm) and sb431542 (10 m) significantly reduced decidualization (60% and 77% respectively) demonstrating that activin and possibly other tgf family members are involved in decidualization in vitro. bmp2, gdf5 and tgf 1 protein were detected in decidual cells of mid-late secretory endometrium and first trimester decidua whilst all ligands except nodal, were expressed by hesc. both bmp2 and tgf 1 secretion increased during hesc decidualization and administration of both these proteins significantly enhanced decidualization in vitro. conclusions: these data support a role for activin a, bmp2 and tgf 1 in hesc decidualization. this is important as the elucidation of factors involved during decidualization will aid in better understanding implantation and fertility. abnormal chromatin remodeling in diabetic murine oocytes. laura lawrence, ann ratchford, cybill esguerra, qiang wang, kelle moley. ob/ gyn, washington university, st. louis, mo, usa. background: diabetic women experience increased miscarriages and adverse pregnancy outcomes. previous studies suggest adverse diabetic outcomes may occur earlier than the preimplantation period, particularly during oogenesis. we hypothesize that diabetes affects chromatin remodeling and chromosomal condensation in murine oocytes. methods: mii oocytes from diabetic and control mice were fixed with 4% pfa, permeabilized with 0.5% triton x-100 for 15 min, and immunostained against a-tubulin and the nucleus for 1 hr at rt. images were obtained with laser confocal scanning microscope. protein expression levels of chromatin with dimethyl h3k9 modifications were measured in nondiabetic and diabetic denuded murine oocytes at 44 hours post pmsg (0 hour) and at six hours post hcg (6 hour) via western immunoblots. mature nondiabetic denuded oocytes were fixed in 4%pfa and permeabilized with 0.5% triton x-100. they were stained via immunohistochemistry against histone protein h3 at lysine 9 (h3k9me2) and heterochromatin. results: immunohistochemistry reveals that diabetic mii oocytes have aberrant spindle formations and metaphase chromosome alignment. approximately 10/50 (20%) diabetic oocytes examined had abnormal spindles and metaphase alignments compared with only about 1/70 normal oocytes (1.4%). western blot demonstrated 3 times higher expression of dimethylated chromatin in diabetic oocytes at time 0 compared with nondiabetic oocytes. at time 6 hours, diabetic oocytes had significantly fewer h3k9 modifications than the controls. when staining mature murine nondiabetic oocytes for dimethylation of h3k9me2 by immunohistochemistry, we demonstrated h3k9me2 expression in a condensed heterochromatin ring surrounding the nucleolus, consistent with transcriptional silencing. conclusions: diabetic mii oocytes have a significant increase in abnormal spindle formation and metaphase chromosome alignment. they also have increased dimethylation compared with normal oocytes at a time point when they should be transcriptionally active, storing maternal mrna in preparation for the silencing period. in addition, after hcg injection to trigger maturation and gene silencing, the diabetic oocytes had decreased dimethylated chromatin changes. our findings suggest that diabetic oocytes may be exiting the transcriptionally active period prematurely and may ultimately experience decreased, partial, and incomplete gene silencing. and xenopus epab genes and proteins were performed. expression of human epab and pabpc1 mrna was tested in ten different somatic tissues, testes, and ovaries by rt-pcr. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. epab and pabpc1 expression in human prophase i (pi) and metaphase ii (mii) oocytes, 8-cell embryos and blastocysts was evaluated using quantitative real time pcr. results: human epab is a 625 aa protein with 77% identity and 84% similarity to mouse epab and contains 4 rna recognition motifs and a pabp domain. human epab mrna is detected in ovaries and to a lesser extent in testes and several somatic tissues including kidney, liver, and muscle. similar to its mouse orthologue, human epab mrna is expressed in pi and mii oocytes, but not in 8-cell embryos or blastocysts. pabpc1 mrna is ubiquitously present in all tissues as well as 8-cell, and blastocyst stage embryos. however, its levels are significantly lower than that of epab in oocytes. conclusions: in this study we report the identification of human epab. similar to that observed in xenopus and mouse, human epab is the predominant poly(a) binding protein in oocytes and it is replaced by pabpc1 following zga, which occurs at 4-to 8-cell stage in human. our findings suggest that the unique translational regulatory pathways that control gene expression during oogenesis and early embryo development may be common between model organisms and humans. objective: c-jun nh2-terminal kinase (jnk), a member of mitogen-activated protein (map) kinase family, is involved in cell proliferation, differentiation, and survival. fsh is required for granulosa cell proliferation and antral follicle growth but its mechanism of action in preantral stages is not well defined. we previously showed that pharmacological inhibition of jnk pathway halts invitro growth of murine preantral follicles in serum free media supplemented with fsh (100miu/ml). sigcs (spontaneously immortalized rat granulosa cell line) have characteristics similar to preantral granulosa cells and hence they were used in this study to determine whether the jnk pathway plays a key role in preantral granulosa cell proliferation. our specific aims were to determine whether: a) fsh activates jnk pathway in granulosa cells; b) the inhibition of jnk pathway blocks cell cycle progression. material and methods: sigcs were treated with 100miu/ml recombinant fsh in serum free media two days after serum starvation. activation of jnk pathway was analyzed with if and wb using phospho-jnk and phospho-cjun expression, respectively. fsh receptor expression (fshr) was analyzed with if. the inhibition of jnk pathway on cell cycle progression was analyzed by facs using a jnk inhibitor sp600125. results: fshr protein was expressed in sigc indicating that they can respond to fsh (fig 1a) . likewise by if, phospho-jnk expression was significantly increased in sigc 1 hour post fsh exposure (fig-1a) . similarly on wb, phospho-cjun expression increased as early as 30 min after fsh exposure and peaked at 4 hrs. cjun phosphorylation was abolished 1 hr after treatment with sp600125 (50mm) (fig 1b) . facs analysis showed that the inhibition of jnk by sp600125 resulted in cell cycle arrest at g2/m transition in a dose dependent fashion (fig 1c) . conclusion: these results strongly suggest that the proliferative effect of fsh on immature granulosa cells is mediated through the activation of jnk pathway. this is the first experimental observation implicating jnk signaling in granulosa cell cycle control. (nichd 043339-01). the induction of 17{alpha}-hydroxylase (cyp17) expression in granulosa cells. satin s patel, 1 victor e beshay, 1 william e rainey, 2 bruce r carr. 1 1 reproductive endocrinology and infertility, university of texas at southwestern medical center at dallas, dallas, tx, usa; 2 physiology, medical college of georgia, augusta, ga, usa. according to the traditional two-cell two-gonadotropin hypothesis of the ovary, androgen production arises exclusively from theca cells. the granulosa cells, in turn, utilize androstenedione, which is aromatized eventually to estradiol. studies involving immunohistochemical analysis of normal ovaries have shown that granulosa cells express significantly higher levels of the activator protein-1 (ap-1) transcription factor, cfos compared to theca cells, where cfos expression is virtually absent. we hypothesize that cfos functions to inhibit the expression of cyp17 in granulosa cells, thereby suppressing androgen production. hence, the inhibition of cfos activity might result in cyp17 expression in the granulosa cell. our objective was to define the role of cfos, in the regulation of cyp17 expression in granulosa cells. transformed human luteinized granulosa (hgl5) cells were utilized for all experiments. hgl5 cells were cultured in monolayer for 72 h. cells were treated for 48 h with and without pd98059 (pd), a mapkk inhibitor, which also blocks cfos expression. rna was isolated and real time rt-pcr was performed for cyp17. cfos rna interference experiments were carried out using rnaifect, cfos smartpool sirna and scrambled sirna for 48 h. rna was isolated and rt-pcr was also performed for cyp17. immunochistochemical studies were performed on normal ovaries, staining for cfos and cyp17. treatment of hgl5 cells with the mapkk inhibitor pd for 48 h, resulted in a 10-fold increase in cyp17 mrna expression compared to basal conditions. in cfos gene silenced cells, cyp17 mrna expression also increased by 10-fold compared to control sirna conditions. immunohistochemical staining for cfos and cyp17 showed significant staining of cfos in the granulosa cell layer, but absent staining for cyp17. conversely, the theca cell layer did not stain for cfos, but staining was evident for cyp17. these results suggest that the ap-1 transcription factor, cfos, may play a role in the inhibition of cyp17 expression in granulosa cells. this may provide an explanation for the lack of cyp17 expression in granulosa cells. the g2/m stages of the granulosa cell cycle. as clip-170 has been identified as an mtor substrate, we hypothesized that its function at the mitotic spindle would be positively regulated by mtor during the late g2 and m phases of the cell cycle in granulosa cells. during periods of stress (e.g., mtor inhibition), mtor would fail to phosphorylate clip-170, leading to spindle checkpoint failure and follicle undergrowth. objectives: the expression of clip-170 and mtor were evaluated. computational analysis of potential clip-170 phosphorylation sites and comparison with residues on known mtor targets were performed. clip-170 threonine 182 and serine 186 were chosen and evaluated as bona fide mtor phosphorylation sites. a preliminary assessment of the effects of mtor inhibition upon clip-170 function was performed. methods: for protein expression analyses, western blots, immunostaining of tissues and primary granulosa cells in culture were performed. computational analysis of potential clip-170 phosphorylation sites was followed by in vitro assessment of mtor kinase activity upon clip-170 and a peptide substrate. results: clip-170 was expressed in the ovarian stroma, blood vessels (including the endothelial cells of both arteries and veins), granulosa cells, and in the oocytes of primordial and growing follicles. overlapping expression was found between clip-170, mtor, and the mtor cofactors raptor and rictor in granulosa cells. this expression was conserved between the mouse and the human. evaluation of clip-170 phosphorylation supported thr 182 as a bona fide mtor target. conclusions: clip-170 was supported as an mtor substrate protein during granulosa cell mitosis. the mechanism of mtor action during granulosa cell growth and survival is likely to include the phosphorylation of clip-170 and subsequent positive regulation of mitotic spindle function. the effect of a selective oxytocin antagonist (barusiban) in threatened preterm labour: a randomised, double-blind, placebo-controlled trial. steven thornton, 1 thomas m goodwin, 2 gorm greisen, 3 morten hedegaard, 4 joan-carles arce. 5 1 warwick medical school, university of warwick, coventry, united kingdom; 2 maternal-fetal medicine, university of southern california, los angeles, usa; 3 dept of neonatology, rigshospitalet, copenhagen, denmark; 4 dept of obstetrics, rigshospitalet, copenhagen, denmark; 5 clinical research development, ferring pharmaceuticals, copenhagen, denmark. objective: a mixed oxytocin/vasopressin v 1a antagonist, atosiban, has been shown to reduce uterine contractions in placebo-controlled clinical trials and is useful in the management of preterm labour. the objective of this study was to determine the effect of a selective oxytocin antagonist, barusiban, in delaying delivery and reducing uterine contractions in women with threatened preterm labour at a late gestational age and relatively high risk of delivery. methods: this was a randomised, double-blind, placebo-controlled multicentre study in 6 countries. a total of 163 women between 34+0 and 35+6 weeks gestation, and with 6 uterine contractions of 30sec duration during 30min, cervical length 15mm, and cervical dilatation >1 and <4cm were randomised to receive a single intravenous bolus dose of either barusiban 0.3mg, 1mg, 3mg, 10mg or placebo. rescue tocolytics were prohibited. the primary end-point was percentage of women who did not deliver within 48h. uterine contractions were monitored by cardiotocography. obstetrical and neonatal outcomes were determined. results: there were no significant differences between the placebo and any barusiban group in percentage of women who did not deliver within 48h (72% in the placebo group and 65% to 88% in the barusiban groups). there was no dose-effect relationship nor an effect at 12 or 24h. none of the barusiban groups were associated with a significant reduction in number of uterine contractions compared to placebo at any time point up to 48h post-dosing. postpartum blood loss and time to established lactation were not significantly increased with barusiban. barusiban was well tolerated and was not associated with safety concerns for the women, fetus or neonates. conclusion: a single intravenous bolus of a selective oxytocin antagonist, barusiban (dose range 0.3-10mg), did not delay delivery or reduce uterine contractions compared to placebo in women with preterm labour at late gestational age and with short cervical length. the results contrast those of the mixed oxytocin/vasopressin v 1a antagonist, atosiban. prolonged delivery intervals in triplet gestations. tracy a manuck, heather l mertz, leah passmore, david c merrill. obstetrics and gynecology, wake forest university health sciences, winston-salem, nc, usa. objective: delayed interval delivery is one management strategy for previable preterm labor affecting multiple gestations. prior reports of asynchronous deliveries have examined twins and higher-order multiples as a group. this study was conducted to analyze the unique situation of asynchronous triplet deliveries. study design: cases of asynchronous triplet deliveries resulting in an ongoing twin gestation were ascertained through medline. data were abstracted and combined with two similar previously unpublished cases. patients were grouped by management with and without rescue cerclage. variables compared included use of tocolytics, antibiotic administration, gestational age at delivery of each fetus, interdelivery interval, delivery mode, birthweights, and short and long term outcomes. chi-square or t-test analyses were used where appropriate. results: fifty-one cases of asynchronous triplet deliveries met inclusion criteria and were analyzed. twenty-three patients (45.1%) underwent placement of a rescue cerclage following delivery of the first infant. these patients delivered the first fetus at a significantly earlier gestational age as compared to those patients without a cerclage (20.3 +/-3.4 weeks vs. 23.5 +/-3.5 weeks, p=0.0019). patients with a rescue cerclage had a significantly longer prolongation of the remaining twin gestation (59.4 +/-41.3 days vs. 25.9 +/-30.1 days, p=0.0016). no significant differences in use of tocolytics or antibiotics, gestational age at delivery of triplets "b" and "c," mode of delivery, short term outcome (alive at 24 hours), or long term outcome (alive at discharge) were noted, despite delivery of triplet "a" at a significantly younger gestational age. conclusion: rescue cerclage, particularly when placed following previable delivery of a first triplet, may significantly prolong the delivery interval for the remaining twin gestation. mean pregnancy prolongation (days). objective the aim of our study was to evaluate the role of amnioinfusion in pregnancies complicated by pprom. we studied 52 singleton pregnancies with pprom at <25 weeks gestation. all patients were managed conservatively with bed-rest, prophilactic antibiotics, tocolytics and steroids. only patients without vaginal bleeding and/or contractions were included: 11 patients showed an amniotic fluid pocket (afp) persistently 2 cm and did not undergo amnioinfusion (group b) whereas 41 had a maximum afp < 2 cm and were offered amnioinfusion to restore an adequate amount of amniotic fluid (group a). in 17 patients of group a amnioinfusion was successful (afp 2 cm for 48 hours following the procedure: group a1) whilst in 24 it was unsuccessful (afp<2 for 48 hours: group a2) and repeated. results were analized with the student t test for unpaired samples and with the 2 when appropriate. p values <0.05 were considered significant. results the group where amnioinfusion was not successful (group a2) showed the worst outcome (see table) . there were 6 intrauterine deaths, all in this group. pulmonary hypoplasia was present in 11/52 (22.5%) newborns (both survived and deceased) newborns, 10/11 in group a2. no maternal complications were recorded. conclusions our data confirm that a conservative-active management with amnioinfusion can be considered a reasonable option in women with pprom. in our series it was effective in preventing both neonatal death and pulmonary hypoplasia. group a1 (infusion successful) background. synthetic progestogens are effective in reducing the risk of spontaneous preterm birth in high risk singleton, but not multiple, pregnancy. we hypothesized that myometrial stretch may inhibit the response of human myometrium to progestogens. methods. myometrial strips obtained with written consent at the time of term planned cesarean section were studied using a modification of the method of young and zhang (jsgi 2004; 11:478-82) . strips were maintained in individual tubes in tissue culture media in an incubator for a period of three days. the effect of prolonged stretch was assessed by comparing strips connected to a 0.6g weight with those connected to a 2.4g weight. the effect of prolonged exposure to progestogen was studied by adding medroxyprogesterone acetate (mpa, 100nm or 1000nm). following the 3 day incubation, myometrial strips were transferred to an organ bath containing kreb's solution. all were placed under 2g tension and responses obtained to 50mm potassium then oxytocin. contractility was expressed as the ratio of the maximum response to potassium or oxytocin to the wet weight of the tissue (units = g.tension per gram), summarized as the mean (sem) and compared using student's paired t test. prolonged stretch increased the maximum response to both potassium (0.6g weight = 24.6 [5.3]; 2.4g weight = 43.1 [6.5], n=6, p=0.01) and oxytocin (0.6g weight = 30.4 [8.0]; 2.4g weight = 50.3 [7.6], n=6, p=0.01). in strips with a 0.6g weight, incubation in mpa for three days reduced the maximum response to potassium (vehicle = 30.1 [5.5]; mpa = 23.2 [5.2], n=5, p=0.02) and there was a trend towards a reduced maximum response to oxytocin (vehicle = 37.6 [8.2]; mpa = 31.5 [7.2], n=5, p=0.1). in strips with a 2.4g weight, incubation in mpa for three days had no effect on either the maximum response to potassium 1. prolonged stretch increases human myometrial contractility in vitro. 2. prolonged exposure to a progestogen inhibits the contractility of human myometrium but this effect is blocked by prolonged stretch. these properties of human myometrium may explain the failure of 17oh progesterone caproate to reduce the incidence of spontaneous preterm birth in multiple gestations. sangeeta jain, william l maner, janet l brandon, gary dv hankins, robert e garfield. obstetrics and gynecology, the university of texas medical branch, galveston, tx, usa. objective: to determine if transabdominal uterine electromyography (emg) correlates with parturition factors such as measurement-to-delivery time (mtdt), cervical dilation (cd), cervical effacement (ce), and station (s) for preterm labor patients with and without tocolysis. materials and methods: 17 pregnant preterm labor women were included. uterine electromyography (emg) was measured for 30 minutes. cd, ce, and s were assessed at or near the time of uterine emg measurement. the power density spectrum peak frequency (pdspf) was calculated on emg. patients were grouped (g1: tocolysis, n= 4; g2: no tocolysis, n=13). pearson-product-moment test was used for correlation. significant differences were sought between groups using student-t test. p<0.05 significant. results: there was a significantly higher uterine emg activity (pdspf: 0.453 ± 0.051 vs. 0.379 ± 0.016), but no difference in cd (5.125 ± 1.727 vs. 3.714 ± 1.976), for patients delivering within 6 days of emg recording compared to those who delivered later, regardless of tocolysis. there was no apparent difference in uterine emg in tocolytic vs. non-tocolytic patients, regardless of mtdt (table 1) . conclusions: uterine emg activity is significantly greater in patients in preterm labor who delivered within 6 days of measurement, making it a viable alternative diagnostic parameter for assessing the state of parturition. tocolytics may not affect uterine emg, but this should be further verified with larger studies. supported by grant nih r01-hd037480. objective: calcium sensitizers are a novel class of drugs with unique molecular and phsiological actions. levosimendan, the best characterized of these compounds and is used in the treatment of acute and chronic heart failure. levosimendan can exert an inotropic effect via sensitization of myofilaments to calcium. it also exerts a relaxant effect on vascular smooth muscle through the opening of atp-dependent potassium channels and has been shown to be a potent inhibitor of human uterine contractions in vitro. for these reasons we investigated the effects of levosimendan on uterine contractions, both spontaneous and agonist induced, in the presence of glibenclamide, a k-atp channel blocker. method: biopsies of human myometrium were obtained at elective caesarean section (n=20). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to glibenclmide (100mmol) followed by cumulative additions of levosimendan in the concentration range of 1 nmol/l to 100 mmol/l. control experiments were performed simultaneously. results: levosimendan exerted an inhibitory effect on spontaneous and oxytocin induced contractions in human m yometrium in vitro, in comparison to control experiments. the effect of levosimendan was significantly antagonized by glibenclamide with the mean maximal inhibition seen due to levosimendan greatly reduced (n=6, p<0.05). conclusion: the calcium sensitizer levosimendan exerted a potent relaxant effect on human uterine contractility in vitro. this action was antagonized by glibanclamide and this study demonstrates that the effect of levosimendan on uterine smooth muscle is mediated at least in part through the k-atp channel. introduction: the determination of the beginning and ending points for "bursts" of electrical activity occurring during uterine contractions is sometimes difficult. if bursts cannot be discerned, the preferred burst-by-burst analysis cannot be performed. one solution to is to analyze any given electrical recording in its entirety. but this approach has often lead to meaningless results when traditional analytic methods are applied. chaos analysis, using lyapunov exponents, may provide an answer. materials and methods: 46 term patients were included in the analysis: 27 were in labor (group 1), while19 were non-labor (group 2). 30 minute recordings were analyzed using "lyapunov exponent." for each pair of subsequent trajectories in phase space, only the most positive lyapunov exponent was calculated. the mean largest exponent was found by averaging over all of the trajectories in the recording. the lyapunov exponent is given in units of bits per data sample. thus a value of +1 means that the separation of nearby orbits doubles on the average in the time interval between data samples. the mean largest exponent was found for each patient recording. these values were compared using t-test (p < 0.05 considered significant). results: the mean and sd of the lyapunov exponent for all the patients was 0.0955 ± 0.0701. moreover, the lyapunov exponent calculated for each patient was positive. comparing lyapunov exponents of the two groups showed a statistically low value (low chaos) for the laboring group, compared to the non-laboring group (figure) . conclusions: there is a chaotic component associated with uterine emg traces, since small but non-negative lyapunov exponents were found in all the traces observed. the lyapunov exponent indicated significantly lower chaotic behavior in the whole emg traces of patients who were in labor than found in those who were not in labor, implying that this measure could be a good diagnostic parameter for labor, possibly eliminating the need for tedious burst-by-burst analysis. supported by grant nih r01-hd037480. comparing uterine emg to tocodynamometer for monitoring contractions. robert e garfield, lynette b mackay, sangeeta jain, william l maner. obstetrics and gynecology, the university of texas medical branch, galveston, tx, usa. objective: to determine whether uterine electromyography (emg) plots contractions similarly to tocodynamometer (toco). study design: 20 pregnant term labor patients were recorded using both uterine emg and toco simultaneously. uterine emg signals were sampled at 100hz and band-pass filtered in the 0.34 to 1.00 hz range. root-mean-square (rms) function was calculated from the uterine emg signals in order to produce a "toco-like" trace from the original emg trace. emg-generated rms contraction plots were then compared to toco contraction plots using the following criteria: contractions were assigned a marker value of "1." in-between contraction periods were assigned a "0." from these marker values, contraction rates were established. correlation was found between the contraction rates of rms and toco. temporal overlap of contractions plotted by the two methods was used to find overall percent agreement (opa), positive percent agreement (ppa), and negative percent agreement (npa). these parameters were corrected for within-patient variation using a bootstrap method. results: uterine rms contraction plots were seen to correspond with toco contraction plots (fig. 1) . corrected opa, ppa, and npa were high at 90.68%, 84.52%, and 95.77%, respectively. there was a large, statistically significant correlation between uterine emg and toco contraction frequency (fig. 2) . conclusions: the similarity between toco and uterine emg contraction plots (specifically, using rms to convert) will allow emg to be used interchangeably with toco in the clinic. supported by grant nih r01-hd037480. introduction -this is a secondary analysis of women participating in a 14 center randomized placebo controlled trial (rct) evaluating the impact of 17-ohpc in preterm birth (ptb) prevention among women with twins. objective -to evaluate the relationship between plasma 17-ohpc concentrations and gestational age (ga) at delivery in women with twins receiving weekly injections of 17-ohpc. methods -women with twins were randomized between 16-21 weeks to receive weekly im injections of either 17-ohpc (250 mg) or placebo until 35 weeks. after a minimum of five consecutive injections had been administered to assure steady state concentrations a plasma sample was collected between 24-29 weeks. the sample drawn just prior to the next scheduled injection was analyzed for 17-ohpc by hplc-ms in a blinded manner. the lower limit of quantification of 17-ohpc was 1 ng/ml. we conducted univariate analyses to assess the association of 17-ohpc concentration and ga at delivery. we also conducted a proportional hazards model to evaluate the time from sample draw to spontaneous delivery (censoring indicated preterm deliveries), and a logistic regression to evaluate ptb<35 weeks; in both analyses we adjusted for bmi, race and ga at sample draw. results -96 women assigned to 17-ohpc were included; all received all of their scheduled injections. the concentration of 17-ohpc was significantly higher in women delivering < 35 weeks compared with those women delivering >35 weeks (p=0.002, table) . concentration of 17-ohpc was significantly correlated with ga at delivery (r = -0.38, p=0.0001). each unit increase of 17-ohpc was associated with a 30% increased odds of delivering < 35 weeks (odds ratio 1.3, 95% ci,1.14-1.50, p=0.001) and a 12% increase in hazard of spontaneous delivery (hazard ratio 1.12, 95% ci, 1.05-1.19, p=0.0004) after adjusting for confounders. gestational age at delivery mean (sd) -ng/ml < 35 weeks (n=34) 13.9 (5.9) >35 weeks (n=62) 9.4 (3.5) conclusion plasma 17-ohpc concentrations after weekly injections were inversely related to ga at delivery in women with twins. since 17-ohpc induces its own metabolism it is possible that higher concentrations during initial treatment are associated with lower plasma concentrations and reduced efficacy in later pregnancy . clearly more studies are needed. objective: in many non-human species, maternal circulating progesterone levels fall prior to delivery, leading to the theory that in humans progesterone withdrawal occurs on a local and/or functional level. our objective was to characterize maternal and fetal progesterone in human preterm and term labor. methods: women between 23.7 and 34.7 weeks' gestation (cases) or term controls (37-41 weeks) with either labor with intact membranes or premature rupture of the membranes prior to labor (prom) were enrolled in a prospective case-control study. progesterone was measured by immulite assay in maternal serum collected upon enrollment and again within 60 minutes after delivery and in umbilical cord serum obtained at delivery. maternal progesterone treatment was not used in any subjects. results: 20 cases and 20 controls were studied (see table for comparisons). controls p value ga at enrollment, weeks 29.4 ± 3.2 39.3 ± 1.1 <0.0001 interval to delivery, days (median, range) 2 (0 -25) 0 (0 -1.4) <0.01 maternal progesterone at enrollment, ng/ml 125 ± 87 185 ± 64 <0.02 maternal progesterone after delivery, ng/ml 55 ± 57 47 ± 27 0.6 cord progesterone, ng/ml 878 ± 602 762 ± 457 0.5 among cases, fetal but not maternal progesterone was significantly lower in preterm labor with intact membranes (475 ± 372 ng/ml, n = 8), as compared to prom (952 ± 417, n = 12), p<0.02. this difference increased further when cases of clinical chorioamnionitis were excluded. conclusions: serum progesterone in laboring patients prior to delivery is higher at term than in the preterm period, which may be attributable to increased placental mass in late pregnancy. this disparity disappears shortly after delivery of the fetus and placenta. fetal progesterone levels are several-fold higher than peripartum maternal levels. preterm labor with intact membranes is associated with diminished fetal progesterone, a phenomenon unrelated to clinical infection. these findings suggest the possibility of fetally regulated progesterone withdrawal as a mechanism underlying preterm labor with intact membranes. [snps] and ptb. however, many of these studies are inconclusive and non reproducible. the challenge of identifying robust associations between genetic variation and either susceptibility or protection from ptb is enormous. a systematic review of literature followed by metaanalysis was performed to understand true associations between snps and ptb. methods: for systematic review, articles were chosen based on medline and embase searches (1990 ( -april 2007 and relevant articles were chosen based on stringent inclusion criteria. primarily, studies reporting genetic associations between snps in maternal dna in singleton pregnancies and spontaneous ptb were included. other criteria included, but not limited to, provided genetic data in a complete enough format so that it could be evaluated in meta-analysis and defined the clinical outcome clearly. meta-analysis was performed wherever >3 replication data sets were available results: a total of 5422 abstracts were reviewed and 88 were selected for full text review. data were extracted from 50 articles. over 100 associations were reported between snps on various candidate genes and ptb; however only 32 had replication dataset. meta-analysis documented significant association between pon2a148g (odds ratio [or]=1.64 (95%ci 1.18-2.26), pon1(rs#662)(or=1.14; ci-1.007-1.34), tnfrsf6-670a/g (fas) (or=1.52; ci-1.11-2.08) and ptb. two snps pon2s311c (or=0.56; ci-0.36-0.85) and ifn gamma (rs2430561; or-0.62; ci-0.43-0.91) documented protective effect. conclusions: systematic review concludes significant heterogeneities leading to lack of reproducible data in genetic association studies of ptb. heterogeneities are contributed predominantly by lack of adequate power, poor phenotype selection, and population admixture. the functional relevance of the risk and protective alleles needs to be verified. jignesh parvadia, 1 mounira habli, 2 jeff livingstone, 2 william polzin, 3 foong lim, 1 timothy crombleholme. 1 1 pediatric and thoracic surgery, cincinnati children's hospital medical center, cincinnati, oh, usa; 2 obstetric and gynecology, university of cincinnati, cincinnati, oh, usa; 3 obstetric and gynecology, good samaritan hospital, cincinnati, oh, usa. objective little is known about the response of ttts to treatment either by amnioreduction or selective fetospopic laser in triplet pregnancy, particularly the survival of the bystander fetuses. in order to define the response of triplet pregnancies to treatment for ttts we reviewed our experience with higher order multifetal gestations complicated by ttts. study design retrospective chart review of patients diagnosed with in high order gestation from 2004-2007 was performed. results among 254 cases of ttts 11/254(4.3%) patients with high order gestations were identified (n=11) with a mean ga at diagnosis of 20.3±0.8 weeks. 9 pregnancies (81.8%) were dichorionic triamniotic and 2(18.1%) were monochorionic triamniotic. cincinnati modification of quinterro staging was utilized to characterize recipient cardiomyopathy as mild (stage iiia, n=3), moderate (stage iiib, n=1) and severe (stage iiic or iv, n=3) categories. 4/11 (36.3%) were treated with amnioreduction alone (ar), 2/11(18.1%) with selective fetoscopic laser photocoagulation (sflp) alone, 2/11(18.1%) with ar followed by sflp and 1/11(9.09%) with ar followed by intrafetal radio frequency ablation (rfa). 1/11(9.09%) patient had a cervical cerclage. 2/11(18.1%) patients were treated but remain undelivered. mean ga at delivery was 29.5±1.04 weeks. overall survival was 21/27(77.7%) with bystander survival was 9/9(100%), donor survival 6/9(66.6%), recipient survival was 6/9(66.6%). conclusion triplet pregnancies treated for ttts have 100% survival rate for bystander fetuses and have 66.6% survival rates for donor and recipients comparable to twins treated for ttts. ga at diagnosis 20.3±0.8weeks cincinnati modification of quinterro 1(i), 1(ii), 3(iii), 3(iiia), 1(iiib), 2(iv) ga at delivery 29.5±1.04 weeks live birth -donor 6/9(66.6%)* # -recipient 6/9(66.6%)*# -bystander 9/9(100%) # birth weight -donor to assess the effect of breast feeding (bf) on perinatal outcome in relation to maternal antenatal methadone dose. study design a retrospective chart review study of 170 methadone dependent mother and infant pairs. patients were categorized into 3 groups based on maternal dose at time of delivery: group 1: dose 50 mg, group 2: dose 51-100mg, group 3: dose >100 mg. the finnegan's scoring system was used to monitor neonatal abstinence syndrome(nas). treatment for nas was initiated if there were 2 scores of 8. neonatal outcome data included:% nicu admission, % of babies discharged(d/c) at time of maternal d/c, % nas, % treated for nas and total hospital stay. data were analyzed by t-test and fisher's exact test. maternal characteristics between the 3 groups were similar. regardless of maternal methadone dose, bf infants have shorter hospital stays and higher rates of d/c at time of maternal d/c, lower incidence of nas and fewer nicu admission (table) . in all three groups, breast feeding did not impact the severity of nas as reflected in finnegan's score(fs). (table) conclusion regardless of maternal methadone dose, breast feeding improves perinatal objective: to evaluate the effects of preventive collagen plugging of the fetoscopic access port at the time of balloon removal on pregnancy outcome in fetoscopic endoluminal tracheal occlusion pregnancies. study design: fifty-one pregnancies involving fetuses with severe congenital diaphragmatic hernia (cdh) were studied. all patients underwent feto between 26-29 weeks gestational age (ga) and fetoscopic balloon removal around 34 weeks ga. at the time of balloon removal, a purified dried collagen plug was inserted through the fetoscopic access port in 23 consecutive pregnancies but not in the first 28 pregnancies considered as controls. all patients underwent post-plugging ultrasound and magnetic resonance imaging studies to evaluate for membrane dehiscence, amniotic fluid volume and fetal well being. ga at delivery, incidence of premature rupture of the membranes (pprom), bleeding at port retrieval and adverse fetal effects were compared in both groups. results: mean (sd) ga at feto [28.2 (1.8) vs. 27.5 (1.5) weeks; p= ns] and balloon removal [33.9 (0.7) vs. 33.7 (1.1) weeks; p= ns] was similar in the treatment and in the control group. incidence of pprom following the second fetoscopy was 2/23 in the study group compared to 9/28 in the control group (p<0.05). mean (sd) ga at delivery was 37.0 (1.8) weeks in the study group, compared to 36.4 (2.0) in the control group (p=0.28). bleeding from the trocar insertion site occurred in 4 cases in both groups, but clinically significant bleeding occurred only in one of the controls. membrane dehiscence was noted in 4 patient in the treatment group compared to 6 in the control group (p=ns). conclusion: preventive collagen plugging of the fetal membrane defect created by the fetoscopic access resulted in a significant reduction in pprom rates and a trend towards increased ga at birth without adverse fetal effects in feto pregnancies. wider application of this technique should be considered, but needs evaluation in larger, randomized trials. hydrops fetalis is an uncommon fetal condition characterised by the abnormal accumulation of fluid in two or more body cavities, traditionally associated with a poor prognosis. the relative rarity of this presentation has meant that published case series have consisted of small numbers. a retrospective review of case notes of all cases managed at kemh between 1995 and 2005 was performed. in western australia, kemh is the only tertiary maternity hospital incorporating a maternal-fetal medicine unit. cases were obtained from the mfm database. in the period 1995 to 2005 there was a total of 60 pregnancies affected by hydrops (incidence 0.2 per 1000 births). the average maternal age was 29 years. in 5 cases a fetal abnormality had occurred in a previous pregnancy. the median gestational age at diagnosis was 18 weeks (range 11-36 weeks). in just over half (53%) of cases, the diagnosis was confirmed prior to delivery. a post-mortem was performed on all but 2 of the babies not born alive. edema was present in at least 3 cavities in over half of cases (n=36). chromosomal anomalies included trisomy 21, trisomy 18 and turners syndrome. in all cases of infection, parvovirus b19 was implicated. cardiac arrythmias included svt and atrial flutter. cases classified as other included alpha thalassemia and syndromic disorders. in 33 cases an interruption of pregnancy was performed at a mean gestational age of 19 weeks. of those who did not elect to terminate the pregnancy, there were 15 fetal deaths in utero, 11 live borns with 1 neonatal death. for the live borns, the median gestational at delivery was 36.3 weeks (range 33 to 40.7 weeks). the causes of hydrops in live birth cases included cardiac arrythmia (n=3), infection (n=2), chromosomal abnormality (n=1), unknown (n=4) and other (n=1 (dmv) have been noted to play a role in the development of hemorrhagic and periventricular leukomalacic lesions in premature babies. since deep vein drainage system is relatively more prominent in the developmental brain than adult brain, we investigated if dmv anomalies could be associated with clastic lesions in-utero. methods two senior neuroradiologists reviewed 900 fetal brain exam performed between 2001 and 2007, seeking for unequivocal anomalies in dmv, such as periventricular venular engorgement. all mr scanning is performed at 1.5 tesla, using surface abdominal coils and single-shot fast spin-echo t2-weighted 3-4 mm thick sections, with 1.1-1.25 mm in planar spatial resolution. we found 6 cases with dmv anomalies (tab.1). most of the dmv engorgement is located at frontal lobes level. from this limited preliminary series it appears that dmv involvement plays a role in the development of periventricular leukomalacia and periventricular hemorrhagic necrosis. the observation that these lesions are mostly located at frontal level may suggest that some of the term neonates carrying sequelae of atypically located leukomalacia (i.e. deep frontal lobe) might have developed these lesions in-utero. it is of interest to notice that most of our cases were related to heart failure. therefore, central venous hypertension affecting immature deep cerebral venous system has to be taken into account. in our center, patients with an estimated risk for chromosomal abnormalities at term greater that 1 in 220 after 1 st trimester combined screening test (ft) are offered non-directive genetic counseling. the aim of this study was to evaluate the responses of women younger than 35 attending this genetic counseling session. material and methods: data from patients referred for a positive ft from september 1, 2003 to july 1, 2007 was retrieved from our database. information concerning women younger than 35 years of age at the estimated date of delivery was extracted and tabulated. results: during the study period 591 women had genetic counseling for positive ft. thirteen patients were excluded from further analysis (8 had incomplete clinical documentation and 5 had spontaneous miscarriages prior to 16 weeks gestation). four hundred and twenty-five patients were older than 35 and 153 were younger than 35 at the estimated date of delivery. table 1 depicts summary statistics for studied variables in this younger group of women. conclusions: overall this data suggests that approximately 25% of this younger group of women opted for chorionic villous sampling (cvs), 40% for amniocentesis and more than 34% declined prenatal genetic testing. moreover, this data also suggests that: 1) these women opted for cvs when the ft risk (mean = 1 in 58) almost doubled the cvs procedure related risk quoted at 1% and, 2) when the ft risk is between 1 in 100 and 1 in 220 almost half (53 out of 115) declined not only the 1st trimester cvs but also the 2nd trimester amniocentesis. we believe that understanding our patient population is important to optimize both the efficiency and efficacy of the alternative prenatal screening programs. acceptance for prenatal genetic testing after a positive first trimester combined screening test in women of less than 35 to determine the optimal diagnostic test using prenatal ultrasonography and mri for predicting pulmonary hypoplasia in fetuses with congenital diaphragmatic hernia. methods: relevant papers were identified by searching medline (1966 medline ( -2007 , embase (1988) (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) and the cochrane library (2004 issue 2). in addition, the specialist literature on the topic and reference lists were hand searched for relevant articles. studies were selected if they examined diagnostic tests for the prenatal prediction of pulmonary hypoplasia in fetuses with congenital diaphragmatic hernia. the primary outcome measure was perinatal survival. study selection, quality assessment and data abstraction were performed independently and in duplicate by separate observers. results: of a total number of 8581 articles (published studies), there were eighteen studies that fulfilled the entry criteria. six examined entirely unique heterogeneous parameters. of the remainder, all 12 examined the ultrasound measurement of lung to head ratios (lhr) at a 'cut-off of greater than or less than the thresholds of 0.6, 1.0, 1.4, 1.6. in addition, the presence of liver in the fetal thorax was included (if present) as a co-variable (liver "up"). a lhr >1.0 compared to <1.0 provided the strongest association with perinatal survival (peto or 5.73, 95% ci 3.38-9.71). the finding of "liver up"in the fetal thorax had a negative association with survival (peto or 0.25, 95% ci 0.16-0.39). only three studies provided data for lhr in conjunction with the presence of liver in the fetal thorax (peto or survival for lhr > 1.0 compared to <1.0 was or 7.65, 95% ci 3.42-17.10). data was also available for liver up and lhr > 1.4 which had a peto or of 2.2 (95% ci 0. 83-5.82 ). discussion: the data supports the view that lhr may be a useful prognostic indicator of perinatal survival in fetuses with congenital diaphragmatic hernia. however, heterogeneity still exists regarding the timing of ultrasound measurement and the use of mri. the majority of studies have small sample sizes. objective: to estimate, in fetal anaemia, the diagnostic value of fetal ultrasonography and doppler blood flow in the evaluation of fetal anaemia methods: literature from 2000 to 2007 was identified using medline and embase, the cochrane library and relevant specialist register of the cochrane collaboration, and by checking reference lists of known primary studies and review articles. studies were selected if the accuracy of the fetal ultrasound parameters or doppler studies of blood flow in the fetal vessels was estimated compared with a reference standard. diagnostic tests evaluated were ultrasound measurement of the fetal spleen and liver length and doppler studies from the umbilical vein and middle cerebral artery. data from the selected studies were abstracted as 2x2 tables comparing the diagnostic test result with the reference standard. results were pooled where appropriate. diagnostic accuracy was expressed as sensitivity and specificity. twenty-nine primary studies were identified containing suitable data. twentyone of these gave data on middle cerebral artery doppler peak systolic velocity (mca-psv) and 15 could be pooled in the meta-analysis giving a sensitivity of 0.75 (0.63-0.86) and a specificity of 0.89 (0.84-0.95) for 1078 cases in detecting severe anaemia. four studies gave data on spleen perimeter and it was possible to pool three of these giving a sensitivity of 0.20 (0.10-0.35) and a specificity of 0.28 (0.18-0.41) for 112 cases. three studies had data for liver length measurements and two were pooled. the sensitivity was 0.16 (0.05-0.36) and the specificity was 0.77 (0.46-0.95) but only 38 cases were used in the analysis. there were three studies on umbilical vein maximum velocity doppler studies and all were suitable for meta-analysis giving a sensitivity of 0.73 (0.61-0.83) and a specificity of 0.54 (0.33-0.73) with 97 cases analysed. two studies gave data on middle cerebral artery time-averaged mean velocity score giving 52 cases. the sensitivity was 0.95 (0.83-0.99) and specificity was 0.85 (0.55-0.98). discussion: middle cerebral artery peak systolic velocity dopplers remain the gold standard for non-invasive screening of fetal anaemia. middle cerebral artery time-averaged mean velocity scores require further investigation. other tests perform poorly when diagnostic accuracy is assessed. cochrane review of treatment in twin to twin transfusion syndrome. twin-twin transfusion syndrome is a condition affecting monochorionic twin pregnancies associated with a high risk of perinatal mortality and morbidity. the objective of this review was to evaluate the impact (maternal,fetal and pediatric)of treatment modalities in twin-twin transfusion syndrome. register and cochrane controlled trials register. we also searched conference proceedings and made personal contact with experts active in the area of the review. randomised and quasi-randomised studies of amnioreduction versus laser coagulation, septostomy versus laser coagulation or septostomy versus amnioreduction. eligibility was assessed by one reviewer. study authors were contacted for additional information. two studies were included. this review shows that laser coagulation of anastomotic vessels results in less fetal (rr 0.71; 95% ci 0.57, 0.89) and neonatal deaths (rr 0.30; 95% ci 0.17, 0.56) than amnioreduction ( figure 1 ). there is no difference in perinatal outcome between amnioreduction and septostomy. more babies in the laser arm are alive without neurological abnormality at six months of age (developmental delay at <2 years rr 0.70, 95% ci 0.57, 0.86)( figure 2 ). conclusions: endoscopic laser coagulation of anastomotic vessels should be considered in the treatment of all stages of twin twin transfusion syndrome to improve perinatal outcome. further research on the effect of treatment on milder forms of twin twin transfusion syndrome (quintero stage 1 and 2) are required. (produced in collaration with the cochrane collaboration, uk). (cvs) concluded that although the risks of pregnancy loss are relatively low, lack of adequate controls tends to underestimate the true added risk of prenatal invasive procedures (obstet gynecol.2007; 110 (3):687-94). the west midlands is a large region within the uk containing approximately 12% of the total uk population. the congenital anomaly register for this region is able to monitor pregnancy outcomes with accurate deminator data. results: there were 371 first trimester cvs performed, by ten operators, in the west midlands (uk) in 2005. this equates to 5.4 procedures per 1000 births. significantly higher rates were noted in areas of high socioeconomic status. the median number of procedures performed per operator was 35 (range 9-81). all operators were performing other invasive tests such as amniocentesis or cordocentesis,etc. the most common indication for cvs was: i) fetal anomaly on dating scan (24.25%) ii) abnormal (>1 in 300) risk on combined first trimester screening (22.4%) iii) molecular genetic diagnosis (18.1%) and iv) maternal request (35.25%). using a combination of first trimester scanning and cvs, 33% had abnormal karyotype/structural anomaly.the corrected loss rate (background and procedure-related) following cvs in normally formed, singleton pregnancies was 3.6% (95thci 1.1-6.0%) up to 7 days postnatal (perinatal loss) and 2.7% (95thci 0.60-4.8%) with 28 days of procedure. the proportion of cvs in which an adequate sample was not obtained was 1.1% (95th ci 0.0-2.1%). conclusions: this epidemiological study using accurate demoninator data provide interesting statistics relating to the uptake and prenatal risks of first trimester cvs. with the increasing prevalence of obesity in the last two decades, we have seen a tremendous increase in bariatric procedures in reproductive aged women. malnourishment and vitamin deficiency are common complications after gastric bypass which may impact on fetal development. we present the case of a 27 yo who underwent a duodenal switch procedure in 2005. she was discovered to be pregnant during evaluation for persistent malnutrition in 2006. multiple prenatal ultrasounds were performed; the first at 15 weeks gestation was unremarkable. the 19 week sono revealed a male fetus with shortened femurs and humeri bilaterally, nasal bridge hypoplasia, macroglossia, poorly defined hands, and possible clubbed feet. amniocentesis revealed a normal karyotype. 3d ultrasound redemonstrated the abnormal facial findings. a fetal echocardiogram was normal. the lagging long bone measurements continued to worsen, ultimately with femurs 6 weeks behind. the fetal thoracic circumference was two standard deviations below the mean, giving rise to concern for pulmonary sequelae. growth restriction was noted at the 33 wk sonogram. delivery by cesarean section was at 33 5/7 weeks secondary to nonreasuring fetal status. the birthweight was 1468gm; apgars were 1, 4, and 6. postnatal radiographs confirmed antenatal ultrasonographic findings and demonstrated evidence of epiphyseal stippling. the infant remained intubated until 12 weeks of life, after which it died secondary to respiratory complications associated with pulmonary hypoplasia. gene mapping studies have not found any point mutations on the recessive gene as an etiology of this disorder. rhizomelic chondrodysplasia punctata refers to a heterogeneous group of bone dysplasias with a familiar radiographic phenotype involving punctate calcifications and epiphyseal stippling. the etiology of this may be secondary to chromosomal abnormalities, mendelian gene disorders, or teratogens, notably warfarin. this case may be explained by vitamin k deficiency of the embryo due to maternal malabsorption after bariatric surgery. the maternal vit k level was <.03 ng/dl at time of delivery(normal > 0.10). the teratogenic effects of vitamin k deficiency in this instance highlight the need for strict counseling and screening for vitamin deficiency in those women undergoing bariatric surgery since previous obesity-related anovulation is reversed as patients lose weight, resulting in unexpected pregnancies and potentially preventable fetal abnormalities. term preeclampsia: any risk for the neonate? sindhu k srinivas, jamie bastek, christina m andrela, emmanuelle pare, michal a elovitz. obstetrics and gynecology; crrwh, university of pennsylvania, philadelphia, pa, usa. introduction: preeclampsia continues to be a major contributor to maternal morbidity and mortality worldwide. preeclampsia at term is not associated with the same risk of neonatal morbidty and mortality as preterm preeclampsia . however, neonatal outcomes in term women with preeclampsia have not been adequately studied. we sought to compare short term neonatal outcomes in term infants born to women with and without preeclampsia. methods: this study was part of a large case control study. women with preeclampsia (n=228) and term controls (n=563) were prospectively identified. infants with congenital anomalies were excluded. hospital length of stay (los), admission or transfer to the nicu, and use of mechanical ventilation or cpap within first week of life were assessed. associations between neonatal outcomes and preeclampsia were evaluated using chi-square analysis and wilcoxon rank sum test. significant confounders were controlled for using multivariable logistic regression. results: discharge day of life was significantly different between neonates born to women with preeclampsia (median =2; mean =3.6) versus those born to women with uncomplicated term deliveries (median =2; mean =2.8, p<0.001). this difference persisted even when neonates with iugr and those born to diabetic mothers were excluded (p<0.001). term infants born to women with preeclampsia have a higher odds of being admitted or transferred to the nicu (aor=2. 16 [1.36-3.43 ], p=0.001) after controlling for iugr, delivery mode, race, and gestational age at delivery. these infants also have a higher odds of mechanical ventilation (aor=8 [1. 43-44.6 ], p=0.018) and cpap use (aor=2.6 [1. 23-5.32 ], p=0.012) after controlling for the same confounders. there was no difference in ivh or nec between the two groups. conclusion: neonates born to women with preeclampsia have differences in short-term morbidity when compared to neonates born to women without preeclampsia, despite being born at term. whether this increase in neonatal morbidity is attributable to medications used in preeclampsia, such as magnesium sulfate, is unclear. these findings may have implications for patient counseling as well as hospital resource allocation. further investigation correlating these findings with long-term morbidity is warranted. could confined placental mosaicism account for adverse perinatal outcomes in ivf pregnancies? benoit c jacod, 1 gh schuring-blom, 2 kd lichtenbelt, 2 jse laven, 3 d van opstal, 4 mjc eijkemans, 1 nick s macklon. 1 1 reproductive medicine gynaecology, university medical centre, utrecht, netherlands; 2 clinical genetics, university medical centre, utrecht, netherlands; 3 obstetrics gynaecology, erasmus medical centre, rotterdam, netherlands; 4 clinical genetics, erasmus medical centre, rotterdam, netherlands. background: ivf singletons have poorer perinatal outcomes than singletons from spontaneous conceptions. this may be due to the influence of ovarian stimulation on the chromosomal constitution of the embryos which could be translated into localized chromosomal anomalies in the placenta. aim: to compare the incidence of confined placental mosaicism (cpm) in ivf/icsi pregnancies and spontaneous conceptions. methods: multicentre retrospective analysis of karyotype results obtained by chorionic villus sampling (cvs) performed because of advanced maternal age ( 36 years at 18 weeks of gestation) in the netherlands between 1995 and 2005. results: from a total of 322246 pregnancies, 20885 cvs results were analysed: 235 in the ivf/icsi group and 20650 in the control group. the mean age of women in both groups was 38.4 years (mean difference -0.08, 95% ci -0.35 -0.18). foetal karyotype was missing in 152 cases of possible cpm, all in the control group. when taking into account missing data, the incidence of cpm was lower in the ivf-icsi group than in the control group, 1.3% vs. 2.2% (odds ratio 0.58, 95% ci 0.18 -1.81) whereas the incidence of foetal chromosomal anomalies was increased 4.3% vs. 2.4% (odds ratio 1.82, 95% ci 0.96 -3.46) although both differences are not significant. conclusions: the incidence of confined placental mosaicism is not increased in ivf/icsi pregnancies compared to spontaneous conceptions. cpm probably does not account for the adverse perinatal outcomes following ivf/icsi. fetal rh d, cc and ee genotyping using fetal dna from maternal blood is not impaired by the presence of maternal alloimmunization. chad a grotegut, 1 stacey l jeronis, 2 john p gaughan, 3 enrique hernandez, 2 ossie geifman-holtzman. 2 1 obstetrics and gynecology, duke university, durham, nc, usa; 2 obstetrics, gynecology and reproductive sciences, temple university, philadelphia, pa, usa; 3 biostatistics, temple university, philadelphia, pa, usa. this study was conducted to assess the impact of maternal alloimmunization on the accuracy of fetal rh d, cc and ee genotyping from maternal blood. we performed a literature search of english-written articles describing fetal rh d, cc and ee determination from maternal blood (am j obstet gynecol 2006; 195:1163-73) . using this database, we determined the accuracy of rh d, cc and ee genotyping in the presence of maternal alloimmunization. for each subgroup, 95% confidence intervals for a proportion were calculated and compared between groups. we found 37 english-written publications reporting non-invasive rhd genotyping and 4 reporting non-invasive rh ce genotyping from maternal blood. fourteen (37.8%) of the rh d articles and three (75%) of the rh ce articles provided accuracy results in the setting of alloimmunization. the accuracy results are reported as follows: 67 .7 % of the samples were determined correctly in the presence of alloimmunization.* this accuracy was significantly lower than the accuracy reported for all rh cc samples. when only studies utilizing free fetal dna for rh cc genotyping were used (vs fetal cells), fetal cc genotype was determined correctly in 11/11 (100%, 95% ci 70.0, 100), which was similar to the overall success rate for rh cc determination. overall, there were no differences in the success of rh d, cc or ee determination in the setting of alloimmunization compared to the overall accuracy seen when free fetal dna was used. the presence of maternal alloimmunization does not reduce the accuracy of fetal rh d, cc or ee non-invasive genotyping from maternal blood utilizing free fetal dna. further research into structure and rearrangements of the rh d, cc and ee genes may further improve diagnostic accuracy of free fetal dna from maternal blood. fetal dna, most likely of trophoblast origin, is present in both the plasma of pregnant women and provides a potential basis for non-invasive fetal diagnostic tests. however, fetal dna in maternal plasma is highly contaminated with maternal dna, and this contamination is the main technical challenge in trying to accomplish non-invasive detection of fetal chromosome abnormalities. existing methods for the selective amplification of fetal dna have generally relied on specific sequence differences between the mother and fetus. as an alternative, we have developed a method for selective amplification of fetal dna that makes use of observation that trophoblast dna is globally hypomethylated in comparison with dna from other sources. in this method, a dna mixture is first digested with a methylation sensitive restriction enzyme and then amplified by linker-mediated pcr. after an initial amplification, a second isothermal rolling-circle amplification is performed. this procedure results in the differential amplification of short, relatively hypomethylated fragments. after amplification, the resulting "representations" are comparatively hybridized to a microarray consisting of oligonucleotides that correspond to restriction fragments generated by the initial digest. copy number differences are then detected through statistical analysis of array addresses that show significant amplification. to test the feasibility of this method for detecting aneuploidy, we have prepared mixtures of peripheral blood dna and first trimester trophoblast dna from either normal or from samples with trisomy 18 and trisomy 21. we present data showing that aneuploidy can be detected even when 90% of the starting dna sample was derived from a euploid source and only 10% was from an aneuploid trophoblast sample. two different approaches to data analysis are presented. one relies on prior analyses of trophoblast methylation and the second is independent of any prior knowledge or analysis. both methods provide similar ability to detect aneuploidy. future work will focus on testing whether this approach can be used for non-invasive prenatal diagnosis. chromatin immunoprecipitation and emsa analysis of nf-b and c/ ebp synergism on the otr promoter. shirin khanjani, yun s lee, vasso terzidou, mark r johnson, phillip r bennett. institute of reproductive developmental biology, imperial college, london, united kingdom. we have shown, the transient transfections of the transcription factors nf-bp65 and c/ebp leads to a synergistic increase in otr promoter activity in human myocytes. this effect is mediated through a 20bp region of the promoter between -712 to -692 from tss. we now report that this sequence binds both nf-bp65 and c/ebp in vitro and chip studies show binding of both transcription factors to be increased by il-1 in vivo. materials and methods: emsa studies were performed using a 33 bp oligonocleotide sequence (-712 to -672) , containing the 20 bp region responsible for the synergistic activation of the otr promoter and nuclear extracts from primary human myocytes treated with il-1 1ng/ml for 6 hours. for chip analysis, dna protein complexes were crosslinked and antibodies recognizing p65, c/ebp and h4 (positive control) and igg (negative control) were used for immunoprecipitation. primers were designed to amplify the region -744 to -593, which includes the 20 bp response sequence. to further confirm the interaction between nf-bp65 with c/ebp a 6xnf-b consensus/luc reporter construct was cotransfected with an expression vector for either nf-bp65 or c/ebp . results: specific nf-bp65 and c/ebp binding was seen in the emsa study. preincubation with antibodies to nf-bp65 and c/ebp led, not to supershift, but to elimination of dna binding for both nf-kb p65 and c/ebp . chip analysis confirmed increased in vivo binding of nf-b p65 and c/ebp to this region of the otr promoter following stimulation with il-1 . transfection of the nf-b/luc reporter construct with an expression vector for c/ebp caused a significant reduction in the basal promoter activity suggesting that c/ebp is binding to nf-kb. this interaction was further confirmed using a tf-tf interaction array. conclusion: these data support the role of nf-b and c/ebp in regulation of otr. the 20 bp region contains a c/ebp but not a nf-b dna binding site suggesting that c/ebp primarily binds to this part of the otr promoter but also interacts with nf-b. the emsa data shows that the 20bp region binds both nf-b and c/ebp . the loss of the supershift observed in previous emsa studies suggests that the antibodies inhibit the interaction between c/ebp and nf-b, therefore inhibiting dna binding. chip analysis supports the concept that il-1 leads to binding of nf-b and c/ebp to the 20bp region. regulation of pro-labour genes by c/ebp, nf-b and ap-1 in human uterine myocytes. suren r sooranna, 1 shirin khanjani, 2 yun s lee, 2 phillip r bennett, 2 mark r johnson. 1 1 imperial college parturition research group, imperial college, london, united kingdom; 2 irdb, imperial college, london. introduction: the transcription factors c/ebp (lap), nf-b (p65) and ap-1(c-fos and c-jun) are implicated in inflammatory processes such as parturition. the promoter regions of the pro-labour genes il-8, pghs-2 and otr contain putative transcription factor-binding sites for these transcription factors. our aim was to determine the effect of transfecting these transcription factors either alone or in combination into uterine myocytes and to determine their effects on the expression of pro-labour genes including il-8, pghs-2, otr, connexin-43 and fp. methods: primary cultures of human uterine myocytes (n=6) were grown from myometrial samples obtained at the time of elective lscs. cells were cultured in 24 well plates to 80% confluence at which point expression constructs for c/ ebp (lap), nf-b(p65), ap-1 (c-fos and c-jun) were transfected either alone or in different combinations. the empty expression vector psg5 was used as a filler construct. cells were cultured for 24 and 72h after which culture medium was collected for elisa and cells frozen at -80°c prior to rna extraction. copy numbers of il-8, pghs-2, otr, fp, connexin-43 and gapdh were measured by qpcr using a rotor-genetm (corbett research). results: 24h post transfection with c/ebp (lap), nf-b (p65), c-fos and c-jun alone or in combination showed no significant changes in pghs -2, otr and connexin-43 expression. over expression of p65 alone or together with either c-fos or c-jun increased fp expression by 56, 76 and 73% respectively (p=0.028). nf-bp65 consistently increased il-8 expression either alone (by 1108%; p=0.018) or in combination with lap, c-fos or c-jun (by 1110, 1385 and 527% respectively; n=6; p=0,028). rel a, lap, c-fos and c-jun together also increased il-8 expression (by 502%; p=0.018) and a small but significant increase was seen with a combination c-fos and c-jun (by 137%; p=0.018). the changes observed in il-8 expression were reflected in the medium il-8 concentration at 72h post transfection. in the presence of rela and c-fos il-8 increased from 3.7 ± 0.2 to 71.0 ± 16.7pg/ml of culture medium (mean ± sem; n=4). conclusions: nf-b (p65) consistently increased fp and il-8 expression in human myometrium. the data suggest that pghs-2 activation has greater dependence upon other transcription factor(s) in addition to p65. true identity of myometrial pr-c: fact or fiction? yun s lee, suren r soorana, mark r johnson, jan brosens, phillip r bennett. imperial college parturition research group, hammersmith campus, london, united kingdom. progesterone is thought to be central to maintenance of pregnancy. multiple progesterone receptor (pr) isoforms underlie complex and diverse biological action of progestins. previously two human pr isoforms have been identified: pr-b and pr-a. pr-a is n-terminally truncated form of pr-b (initiation site methionine 165). in some cells pr-a inhibits pr-b. it has been proposed that increased expression of pr-a in myometrium underlies a 'functional progesterone withdrawal'. the breast cancer cell t47d contains a 60 kda progestin-specific binding protein that is not found in pr negative cells. it was proposed that there is a downstream methionine (met595) which serves as a translation initiation site for the generation of a pr isoform of 60 kda. based on such findings condon et al (mol endo 2006) have focused on the possible role of 60kda pr-c in human parturition. they found that expression of "pr-c" using pr antibody (sc-538 santa cruz, sc) is increased in upper segment myometrium with labour and that overexpression of this protein inhibits pr-b function. we cloned the same human pr cdna into psg5 expression vector. in vivo translation produced a protein of only 39kda. furthermore overexpression of pr-c595 did not significantly decrease pr-b activity in human myocytes. we examined other downstream initiation sites, which may produce a 60 kda protein. we constructed potential pr isoforms in psg5 vector with initiation sites at met 289 and 301. in vivo translation produced proteins of approximately 70 and 62 kda respectively. to determine the effect of pr isoforms on pr-b function, myocytes were co-transfected with pr-a, pr-c289, pr-c301 and pr-c595 with constant amounts of pr-b. the progesterone dependent pre/luc was used as reporter. unlike pr-c595 both pr-c289 and pr-c301 significantly inhibited ligand dependent pr-b mediated transcriptional activity. we found in western analysis that the antibodies pgr-312 (nc) and the sc-539 (sc) detected both endogenous and overexpressed pr-a and pr-b but none of the pr-c isoforms. the sc-538 antibody detected only pr-a and pr-b very poorly. our data suggests that the sc-538 antibody would not detect any pr-c protein and that none of the commercially available antibodies in the uk do so. great care needs to be taken when over-expressing pr isoforms to ensure that proteins are of the expected size. if pr-c does exist in vivo then the 60kda but not the 39kda isoform might inhibit pr-b function. tnf receptor 1 antibody (tnf ri ab), nf-b inhibitor (nf-b activation inhibitor) and erk inhibitor (u0126) (p<0.01), but not by tnf receptor 2 antibody (tnf rii ab), p38 mapk inhibitor (sb203580) and jnk inhibitor (sp600125). by western blot analysis, we found that the protein level of tnf receptor associate factor 2 (traf2) was higher than that of tnf receptor associate factor 1 (traf1) (traf2>traf1) in untreated ct cells. however, after tnf treatment for 4h to 24h, traf1 protein level was increased, but traf2 protein level was reduced (traf1>traf2). the increase of traf 1 and decrease of traf2 were blocked by tnf ri ab, but not by tnf rii ab. we also found that tnf rapidly (within 5-30 min) and significantly increased phosphorylation of ikk , erk1/2 and jnk1/2/3 and the phosphorylation of these protein kinases by tnf was reduced significantly by tnf ri ab, but not by tnf rii ab. moreover, we found that the changes of increased traf1 and decreased traf2 in ct cells (traf1>traf2) resulted in a dramatic deficiency in phosphorylation of the above protein kinases induced by tnf compared with the normal ct cells (traf2>traf1). nuclear localization of nf-b p65 in tnf treated cells was increased compared to untreated controls. conclusion: we have demonstrated for the first time that tnf stimulates mmp-9 production in ct cells through tnf ri-trafs-ikk /erk-nf-b signaling pathways, but not through the jnk/p38-ap-1 pathway. these studies reveal steps within this pathway as possible therapeutic targets to inhibit mmp-9 expression potentially attenuating tnf -induced degradation of extracellular matrix and pre-term rupture of the fetal membranes. objective: women with preterm birth are at elevated risk of cardiovascular disease, but mechanisms that might relate these conditions are not understood. we hypothesized that women with spontaneous preterm vs. term births may have early gestation evidence of activation of the fibrinolytic cascade, as measured by the thrombin-antithrombin iii (tat) complex. we also tested if this relation may be associated with inflammation. methods: tat was measured in plasma collected <21 weeks gestation (mean 10.2 weeks, sd 3.9) among women without chronic medical conditions, preeclampsia or growth restriction who delivered singleton liveborn infants. inflammation was assessed by c-reactive protein (crp) measured in serum from the same samples. women with spontaneous preterm birth (sptb) <34 weeks (n=32) and 34-<37 weeks (n=72) were compared to women with term births >=37 weeks (n=215). high tat was defined as >5.2 ng/ml (highest quartile among women with term births) and high crp was defined as >=8 ug/ml. multinomial logistic regression was utilized to relate elevated tat and inflammation to risk of sptb subtypes. results: women with sptb were more likely to have tat concentrations in the highest quartile compared to women with term births (<34 weeks, 53.1%; 34-<37 weeks, 30.7%; >=37 weeks 24.11%, p<0.01). women with high tat concentrations had a 3.3-fold (95% ci: 1.5-7.3) increased risk for sptb <34 weeks, after adjustment for body mass index, race, age and gestational age at sampling. there was no relation between high tat and sptb 34-<37 weeks (or 1.2, 95% ci 0.7-2.3). additional adjustment for elevated crp (>=8ug/ ml) did not effect the estimates associated with tat, and elevated crp was independently related to risk for both sptb subtypes (<34 weeks, or 2.9 [1.1-8.0]; 34-<37 weeks, or 2.7 [1.3-5.6]). thus, women with high tat and elevated crp appeared to be at particularly elevated risk of sptb <34 weeks (or 8.3, 95% ci 1.8-38.1). conclusions: the thrombin-antithrombin iii complex was elevated early in gestation among women with sptb <34 weeks, perhaps secondary to microvascular injury. this effect was independent of inflammation, suggesting that the elevated fibrinolytic cascade may function independently from inflammation among women with sptb. plasma cortico-releasing hormone and cortisol concentrations and psychological stress among pregnant women. katherine p himes, hyagriv n simhan. obstetrics and gynecology, university of pittsburgh medical center, magee womens hospital, pittsburgh, pa, usa. objective: many studies have found an association between psychological stress and preterm birth. we sought to determine if women with greater psychological stress during pregnancy had higher concentrations of plasma cortico-releasing hormone (crh) or cortisol. study design: this is a secondary analysis of a multicenter case-control study, nested within an observational cohort. of 3,073 participants, plasma crh and cortisol concentrations at 24 and 28 weeks gestation were available in 178 controls who delivered after 37 weeks and 159 cases who delivered before 37 weeks. the abbreviated scale for the assessment of psychosocial status in pregnancy (asaps) was available for all women. concentrations of crh and cortisol were compared between women above and below the lowest quartile score on the asaps among cases and controls. the same analysis was done for the portion of the scale related to psychological stress. concentrations of crh and cortisol and psychological stress were also compared between black and non-black cases and controls. univariate analysis was performed with kruskal wallis or chi-square. results: there was no difference in crh or cortisol concentrations at 24 or 28 weeks among women above or below the lowest quartile on the asaps (controls:p=0.17-0.66 cases:p=0.47-0.97). greater psychological stress was not associated with higher concentrations of crh or cortisol at 24 or 28 weeks(controls:p=0.33-0.63 cases:p=0.17-0.78). crh concentrations were not different between blacks and non-blacks. among both cases and controls, cortisol concentrations at 24 and 28 weeks were lower in black women than non-black women (controls:p<0.01 cases:p< 0.01). the median cortisol concentration among control black women was 11.0 g/ml at 24 weeks compared to 14.4 g/ml among non-black women and 14.4 g/ml compared to 18.6 g/ml at 28 weeks. black women reported less psychological stress than non-black women (p= 0.001) conclusion: we found no relationship between psychological stress and plasma crh or cortisol. furthermore, while stress is hypothesized to play a role in the racial disparity of preterm birth, black women reported less psychological stress and had lower cortisol concentrations than non-black women. improved assessments of psychological stress and additional biomarkers involved in the stress response may broaden our understanding of how stress contributes to preterm birth. expression, tissular traffic and activation of mmp-9 in human fetal membranes during labor. rodrigo vega-sanchez, arturo flores, marisol castillo, nardhy gomez, felipe vadillo-ortega. direction of research, instituto nacional de perinatologia, mexico city, df, mexico. introduction. rupture of the fetal membranes (fm) during human labor occurs as a consequence of extracellular matrix degradation. this process is controlled by increased secretion and activity of matrix metalloproteinases, particularly mmp-9.several evidences suggest that mmp-9 is mainly produced by infiltrating choriodecidual leukocytes that could arrive from placental circulation. characterization of the synthesis, transport and activation of mmp-9 within the fm is critical to understand the process of membrane rupture during human labor. objectives. expression and secretion of mmp-9 in placental leukocytes, trafficking of the enzyme through the fm and one possible mechanism for its activation were analyzed. methods. leukocytes were isolated from placental blood of women after active labor. maternal leukocytes were used as controls. cells were cultured for 96 h. relative expression of mmp-9 by rt-pcr and enzyme secretion by elisa and zymography were followed at different times. to analyze the traffic of mmp-9 through the fm, fluorescein-conjugated prommp-9 was added to the choriodecidual side of the fm in an in vitro system that allows the separation of amnion and chorion. labeled mmp-9 was localized at distinct times by confocal microscopy. the protease responsible for the activation of mmp-9 was identified using neutralizing antibodies and specific inhibitors. results. no difference in the relative expression of mmp-9 in leukocytes throughout the culture was found. however, secretion of the enzyme significantly increased since 24 h (p<0.05). experiments using labeled mmp-9, repeatedly showed that after 12 h in culture, the enzyme was mainly localized within the amniotic epithelium. specific inhibition of mmp-3 significantly decreased the activation of pro-mmp-9. conclusions. our results demonstrate that the increased secretion of mmp-9 by placental leukocytes is not associated to increased gene expression, suggesting that homing of a specific leukocyte subpopulation to the choriodecidua is occurring during labor. mmp-9 can be trafficked from the choriodecidua to the amnion, suggesting a transmembranal pathway that may regulate the tissular localization of the enzyme to the area of the fm with high content of connective tissue. once secreted by the placental leukocytes, activation of mmp-9 depends mainly on mmp-3, which seems to be derived from the same leukocytes. objective: preterm labour is a major problem in terms of perinatal morbidity and mortality. the histone-deacetylase inhibitor (hdaci), trichostatin a (tsa) has been shown to have an inhibitory effect on myometrial contractility. the aim of this study was to evaluate the effect of the hdaci's suberic bishydroxymate (sbha) and valproic acid (vpa) on human uterine contractions and hence their potential role as tocolytic agents. methods: biopsies of human myometrium were obtained at elective caesarean section (n=18). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of sbha in the concentration range of 1 nmol/l to 100 mmol/l and vpa (100nmol/l-1mmol/l). control experiments were run simultaneously. results: sbha and vpa exerted a potent and cumulative inhibitory effect on spontaneous and oxytocin-induced contractions, compared to control strips. the mean maximal inhibition (mmi) values for sbha were 65.35% for spontaneous contractions (n=6; p< 0.05), and 53.53% for oxytocin-induced contractions (n=6; p<0.05). the mmi values for vpa were 36.57% for spontaneous contractions (n=6; p< 0.05), and 35.20% for oxytocin-induced contractions (n=6; p<0.05). conclusion: these results raise the possibility that hdaci's may have tocolytic potential, in addition to their current clinical indications. the inhibitory effect observed may be linked to the ability of hdac inhibitors to induce the expression of genes involved in the maintenance of myometrial quiescence via epigenetic mechanisms but may potentially also involve non-epigenetic pathways. progestin suppresses thrombin-enhanced interleukin-6 expression in term decidual cells: implications for abruption-induced preterm delivery. edward kuczynski, lynn f buchwalder, frederick schatz, charles j lockwood. obstetrics/gynecology reprod. sciences, yale university school of medicine, new haven, ct, usa. background and objective: decidual hemorrhage (abruption) promotes binding of factor vii to decidual cell (dc)-expressed tissue factor to generate thrombin. thrombin in turn induces several biological effects leading to preterm delivery via activation of cell surface protease-activated receptors (pars). interleukin-6 (il-6) is a pleiotropic proinflammatory cytokine induced by pars. this study assessed the separate and interactive effects of thrombin and medroxyprogesterone acetate (mpa) on il-6 expression in term dcs. methods: term decidua from stripped fetal membranes were isolated and the dcs were purified on a percoll gradient, grown to confluence and passaged until leukocyte-free. confluent dcs were primed in 10 -8 m estradiol (e2) of e2 +10 -7 m mpa for 7 days, then incubated in a defined medium (dm) with corresponding steroids ± thrombin. after 24 hours, il-6 levels in conditioned dm were measured by elisa and western blotting. in parallel 6-hour incubations, il-6 mrna levels were assessed by quantitative rt-pcr and normalized to -actin mrna. results: secreted il-6 levels were similar in cultures maintained in e2 alone (0.43 ± 0.14) and e2 + mpa (0.33 ± 0.06 pg/ml/ug protein; mean ± sem; n = 9). the addition of thrombin (2.5 u/ml) enhanced secreted il-6 levels by 14.3 ± 2.4 fold (p<0.05) in incubations with e2 and by 8.2 ± 1.4-fold (p<0.05) in incubations with e2 + mpa. the inhibitory effect of mpa was statistically significant (p<0.5). in confluent dcs incubated with e2 + mpa, exogenous thrombin (0.05-2.5 u/ml) elicited a concentration-dependent increase in secreted il-6 levels. hirudin acted as a pure thrombin antagonist, exerting no agonist effects alone, but counteracting thrombin-enhanced il-6 secretion. western blotting confirmed the elisa results. quantitative rt-pcr confirmed that il-6 m-rna levels corresponded to protein changes. conclusion: thrombin enhances il-6 mrna and protein expression in term dcs and progestin blunts these effects. since thrombin-generating abruption is closely associated with preterm delivery, anti-inflammatory effects induced by progestin inhibition of dc-derived il-6 may contribute to the protection against ptd, and may explain the reported protective effects of administration of 17 -oh-progesterone in recent clinical trials. introduction: catechol-o-methyltransferase (comt) catalyzes the methylation of the phenolic hydroxyl groups in a variety of catechols. during estrogen metabolism, this enzyme converts the catechol estrogen, 2-hydroxyestrogen (2ohe2), to 2-ethoxyestrogen (2meohe2). the comt substrate, 2-ohe2, can exhibit an anti-estrogenic effect in multiple biologic assays while the 2methoxyestrogen (2-meoe2) can exhibit an estrogenic effect. the biologic activities of these estrogen metabolites (2ohe 2meoe) depend upon their concentrations and tissue type. since comt activity ultimately controls levels of these metabolites, it appears to be a key factor in regulating the cellular estrogenic milieu. we have recently reported that amnion layers of human fetal membranes from laboring women exhibited 3 folds higher comt mrna expression when compared to non-laboring women (wentz et. al. sgi 2007) . objective: to investigate the impact of comt inhibition on prostaglandin e2 (pge2) production by human amniotic membrane explants. study design: explants consisting of 2-cm circular sections of the amnion layer (obtained from term pregnant women who underwent elective repeat cesarean section) were prepared and placed in tissue culture explants media at 37ºc. after a 4-hour incubation, explants were treated with the selective comt inhibitor ro 41-0960, at 10 and 50 m concentrations. the incubation media was harvested after 6 and 24-hour intervals. the levels of prostaglandin e2 (pge2) in the media were measured by sensitive elisa and were normalized against total protein concentration. results: ro 41-0960 comt inhibitor induced major reductions in pge2 production in media collected from amnion explants of human fetal membranes. in the group treated with 10 m of ro 41-9640 for 24 hours there was 72%±8% reduction of pge2 after 24 hours compared to untreated control (p<0.001). in the amnion explants treated with 50 m of ro41-9640, there was 74%±9% after 6 hours and 84%±11% after 24 hours of treatment compared to untreated control (p<0.001). conclusions: this finding indicates that comt activity in the amnion layer of human fetal membranes affects pge 2 production. by facilitating a pro-estrogenic milieu in human fetal membranes in late gestation, increased comt activity may indirectly increases production of factors associated with labor such as pge 2 . hypothesis: an extensive remodeling of the human cervical connective tissue takes place throughout pregnancy with a decrease in the total concentration of collagen and proteoglycans due to an altered higher metabolic turnover. we hypothesize that the profound changes in proteoglycan production in the human pregnant cervix can be seen in corresponding cervical fibroblasts as well. we also hypothesize that proteoglycan production in cervical fibroblasts from preterm partal women are simmilar to the production in fibroblasts from partal women. method: cervical biopsies were obtained from 5 non-pregnant women, 5 women during elective cesarean section, 6 woman after spontaneous parturition and 4 after a preterm vaginal delivery. by explant technique fibroblasts were cultured from the biopsies. produced proteoglycans were metabolically labeled with s 35 during 24 hours and then purified by ion-exchange chromatography and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. results: the total proteoglycan production decreases with approximately 50% in partal and preterm partal cell cultures. the reduction of proteoglycans in preterm partal and partal cell cultures is significant compared to non pregnant fibroblast cultures. the distribution of biglycan and perlecan are similar in partal and preterm partal cells. biglycan are significantly reduced by around 40% and perlecan are significantly increased by around 60% compared to non pregnant cultures. preterm partal cervical fibroblasts secreates significantly more heparan sulfate proteoglycans compared to non pregnant cultures. the changes in total proteoglycan production in the human pregnant cervix can be seen in corresponding cervical fibroblasts as well. both partal and preterm partal cell cultures differ in their proteoglycan production compared to their non pregnant counterpart, suggesting a role for proteoglycans in cervical ripening. the role of the oxytocin/oxytocin receptor system is not well defined in human amnion. previous studies in rabbit amnion have demonstrated an up-regulation of oxytocin receptors in the end of pregnancy and have shown that there is a large increase in the ability of ot to stimulate pge 2 production. we and others have previously shown a role for nf-b in otr regulation. in whole genome array analysis of human amnion we found that otr was the gene with the second highest increase associated with activation of nf-b (the highest being cox-2). the present work was directed towards further understanding of otr expression and function in human amnion at term. we have shown that pge 2 release by pre-labour primary human amnion cells is significantly increased after oxytocin treatment for 6 hrs (ot: 10-7 m; n=3; triplicate samples; 40 fold increase p<0.05). the expression of otr in labour (+) and labour (-) primary amnion cells was measured with real time rt-pcr. we found a significantly higher level of expression in the labour (+) cells (n=3; duplicate samples; p<0.001). western blot analysis confirmed the upregulation of otr in labouring amnion. treatment with il-1b resulted in a significant upregulation of otr which peaked with a 17 fold induction after 6 hours (p<0.001). il-1 caused a 16 fold increase in otr mrna levels in labour (-) cells, bringing the expression level up to that found in labour (+) cells. our findings provide further evidence for a role of otr in human amnion. expression of otr in human amnion is significantly increased after the onset of labour. term non laboured amnion can be stimulated with il-1 to increase otr expression to levels of laboured amnion. one of the functions of otr in amnion cells is stimulation of prostaglandin synthesis. expression of antioxidant defence proteins in human myometrium before and after the onset of labour. vasso terzidou, mandeep s kandola, shirin khanjani, jan j brosens, phillip r bennett. parturition reserach group, irdb, imperial college, london, united kingdom. background oxidative stress is a result of an imbalance between the production of reactive oxygen species (ros) and the antioxidant defence mechanisms present in biological systems. parturition and infection-induced preterm labour resemble inflammatory processes that are linked to the production of ros including super-oxide (o 2 -), hydrogen peroxide (h 2 o 2 ) and peroxynitrite. low concentrations of ros can act as second messengers in the regulation of several cellular functions. in an attempt to maintain redox homeostasis cells are equipped with machineries to both produce and scavenge ros. enzymatic scavengers include superoxide dismutase (sod), glutathione peroxidise and catalase. sod is the first enzymatic step in the defence system against oxidative stress. nuclear factor-kappa b (nf-b), a transcription factor family classically associated with inflammation, is activated in response to infection and proinflammatory cytokines, such as those prevalent during labour. labour is associated with an increase in nf-b activity in human myometrium and in fetal membranes. nf-b is known to regulate a range of genes associated with the onset of labour. in a study using affymetrix whole genome arrays analysis nf-b overexpression was associated with increased expression of sod-2 (3.5 fold). to determine changes in ros scavenging potential upon onset of labour, lower segment myometrial biopsies were taken at term before and after the onset of labour (n=7; each group). cdna was extracted from the tissues and real time rt-pcr was performed for sod-2, serum/glucocorticoidinduced protein kinase-1 (sgk-1) and the dna repair enzyme gadd45a. we found that labour onset is associated with a two fold increase in the levels of expression of each. oxidative damage at the fetomaternal interphase has been extensively studied in the placenta as part of investigations for first trimester pregnancy losses, iugr and pre-eclampsia. the role of ros is less well defined in human decidua and the underlying myometrium. our results suggest a role for oxidative stress and redox homeostasis in the maintenance of myometrial quiescene during pregnancy and onset of labour. plasma anandamide levels increase during labour induction and appear to delay labour progression. vijianitha nallendran, anthony h taylor, patricia mw lam, stephen c bell, david j taylor, justin c konje. cancer studies and molecular medicine, university of leicester, leicester, united kingdom. background: the evidence for a role of the endocannabinoid, anandamide (aea) in labour is conflicting. we previously showed elevated aea plasma levels in labouring women 1 whilst another group showed that activation of functional receptors for anandamide actually inhibited uterotonin-induced contractions through an adenylate cyclase pathway 2 . the aim of this study was to explore further the relationship between labour and plasma aea levels. methods: plasma aea levels in 20 women undergoing induction of labour for various indications at term, were measured by a sensitive isotope dilution method using hplc-ms/ms. each volunteer had an assessment of her cervix prior to the start of the induction when the first sample for aea was collected. once active labour was established (cervix 3cm dilated; contractions every 3-4 minutes), a second sample was collected. results: seventeen (85%) of the subjects were multigravida. the inductions were for postdates(n=7); decreased fetal movements(n=3), fetal growth restriction(n=2), symphysis pubis dysfunction(n=2), diabetes mellitus(n=1), pre-eclampsia(n=1), fetal cardiac complication(n=1), spontaneous rupture of newborn offspring with persistent pulmonary hypertension, despite enhanced fetal membranes(n=1). plasma aea levels increased significantly once labour was established (figure) and demonstrated in 19 (95%) of the 20 cases. the median (interquartile range) plasma aea level increased from 1.35nm (0.99-1.61) at induction to 2.24nm (1.49-2.82); *p=0.01; mann-whitney u-test) at active labour. there was a positive correlation between plasma anandamide levels taken before induction and the time taken for the women to enter into active labour (r 2 =0.41, p=0.06 pearson correlation). plasma aea levels were higher in labouring women compared to non-labouring women after the induction of labour confirming our previous observations and suggesting a direct role for this endocannabinoid in labour. further studies are required to elucidate this role. nuclear factor-kappa b (nf-b) is a transcription factor family classically associated with inflammation, activated in response to infection and proinflammatory cytokines, such as those prevalent during labour. as a cytokineinducible transcription factor it plays a key role in the expression of a variety of genes involved in inflammatory responses and cell survival. nf-b dna binding and transcriptional activity plays an important role in labour associated gene expression in myometrium. in this study we examined the range of genes regulated by nf-b in myometrium using transient transfections and wholegenome array analysis. myometrial cells were extracted from myometrial biopsies taken at the time of elective caesarean sections at term. transient transfections of primary myocytes were performed using expression vectors for nf-b p65.the amount of dna was constant and psg5 was used as a control construct. cdna was made from myocytes transfected with either nf-b or psg5. affymetrix genechip u133 microarray was performed (n=3, each group). we found that 33 genes were significantly differentially expressed between control and overexpressed nf-b samples. twenty eight of these genes were upregulated with nf-b and 5 were down regulated. several chemokines and cytokines were identified in the upregulated group. interleukin 8 demonstrated the highest, 18 fold, induction in the upregulated group, followed by tumor necrosis factor, a-induced protein (tnfaip3), chemokine ligand 2 (ccl2), chemokine ligand 5 (ccl5), pentaxin related gene (ptx3), interleukin 6 (il6) and superoxide dismutase (sod-2). nine genes were present in the nf-b group that were absent in the control group. these included chemokine ligand 20 (ccl20), chemokine ligand 11 (ccl11), chemokine ligand 2 (cxcl2) and il1 . ingenuity pathway analysis demonstrated that immune response, inflammatory, cell growth and proliferation and cell death were the main pathways involved. standardisation experiments have been performed, and the microarray results were confirmed with real time rt-pcr in several candidate genes. our results provide further support in the role of nf-b in human labour and suggest its direct link in upregulation of inflammatory genes, cytokines and chemokines, consistent with the imflammatory nature of the biochemistry of labour. inflammation is widely accepted to be a key feature of human labor. secretory leukocyte protease inhibitor (slpi), an innate immune molecule, has been shown to be an antimicrobial and anti-inflammatory protein. the aims of this study were to verify its expression and localization in human myometrium methods: specimens were obtained at time of cesarean delivery with or without labor. expression and localization of slpi was detected using immunohistochemistry. slpi expression pattern relative to nf-b p65 subunit was compared between not in labor and in labor subjects, between different tissue sections as well as in in vitro model systems including myometrial explants, uterine smooth muscle cells (usmc) and ishikawa endometrial adenocarcinoma cells. slpi was predominantly localized to the nuclei of myocytes. the observed nuclear immunoreactivity of myocytes was increased during the labor relative to not in labor, paralleled with p65 nuclear translocation. the nuclear pattern of slpi is specific to myometrium since slpi immunostaining was present exclusively in the cytoplasm of all other tissues examined, including amnion, chorion, decidua and endometrium. slpi staining was also positive in macrophages, indicated by co-localization of slpi with cd68 positive cells. treatment with il-1 or tnf-induced nuclear translocation of p65 in myometrium explants and usmc, but not in ishikawa cells. in human myometrium, slpi is predominantly localized in the nuclei of myocytes and in macrophages. the nuclear expression pattern of slpi is myometrium-specific and increased following the onset of labor and correlated with nf-b activation. further understanding of its physiological significance may suggest new strategies aimed at preventing preterm birth. application of a new proteomic technology on amniotic fluid in prom. sara consonni, 1 niccolo bosso, 2 marianna andreani, 1 agnese pizzardi, 1 fulvio magni, 2 anna locatelli. 1 1 obstetrics and gynaecology, university of milano-bicocca, monza, italy; 2 experimental medicine, university of milano-bicocca, monza, italy. objective: mass spectrometry (ms) is the obligatory tool for proteomics studies. biological samples must be purified before ms analysis due to the matrix complexity. a recent approach combines active surface prepurification with maldi-tof (matrix assisted laser desorption) analysis. clinprot technology provides the prepurification of the sample through the use of magnetic beads (mb) with activated surface. this technique can be carried out by robot in an automated way on a large number of samples. a unique example of the use of mb before maldi-tof analysis to determine proteomic profiles of amniotic fluid (af) is reported for rapid detection of fetal aneuploidies (wang 2005) . the objective of our study was to verify the applicability of clinprot prepurification before maldi-tof analysis on amniotic fluid collected noninvasively in premature rupture of membranes (prom). methods: we sampled af from vaginal posterior fornix of women with preterm prom (group 1, n=10) and term prom (group 2, n=10). samples were prepared with mb and analyzed with ms maldi-tof in order to generate proteomic profiles. results: it was possible to generate average proteomic profiles in the two study groups and the observed profiles were different. samples of af non invasively collected in prom can be analyzed by ms maldi-tof after preparation with mb. this technique allows to retain part of the eluted sample for characterization of protein peaks of interest. due to the less laborious characteristics of this method in comparison with techniques based on bidimensional electrophoresis its application can be useful in clinical proteomics. progestins accentuate the maternal but not fetal inflammatory response of women with intra-amniotic inflammation. sonya abdel-razeq, irina a buhimschi, michael cackovic, guoyang luo, antonette dulay, victor rosenberg, mert bahtiyar, errol norwitz, edmund funai, catalin buhimschi. ob./gyn. reprod.sci., yale university, new haven, ct, usa. introduction: data from animal research suggests that progestins have a marked pro-inflammatory capacity. recent studies support the administration of 17 -hydroxyprogesterone caproate in women at risk for preterm birth. we sought to determine the impact of progestins during gestation on the extent of maternal and fetal inflammatory responses in pregnant women with intraamniotic inflammation. methods: amniotic fluid, placenta and cord blood were obtained from 52 women who delivered preterm (median[range], ga: 27 [18-33] wks). an amniocentesis was done to rule out infection. women exposed to progestins (n=17) within one week prior to amniocentesis were matched to 35 controls (crl) by age, parity, history of preterm birth ga, membrane status and interval to delivery. proteomic profiling of amniotic fluid [mass restricted (mr) score] identified the presence or absence of intra-amniotic inflammation. an mr score of 3 or 4 confirmed intra-amniotic inflammation. amniotic fluid and umbilical cord interleukin-6 (il-6) levels were measured by elisa. histological chorioamnionitis was graded based on recognized criteria. results: overall, women and their fetuses exposed to progestin did not exhibit an increased inflammatory response (table) . however, sub-analysis restricted to women with mr 3 or 4 (n=18) showed that in the context of intraamniotic inflammation, progestins were associated with significantly elevated amniotic fluid il-6 levels compared to unexposed women (progestins (n=6): 77 vs. crl (n=12): 12 [0.3-94] ng/ml, p=0.027). these relationships were maintained after correction for steroid and antibiotic exposure. such significance was not found for amniotic fluid glucose, ldh, wbc count or cord blood il-6. conclusion: our results suggest that progestins may amplify the maternal, but not the fetal inflammatory response of women with intraamniotic inflammation. objective: recently progestins have been shown to reduce the incidence of recurrent preterm labor in women. progestins have long been known to inhibit preterm labor in some species including rats and are also known to delay term labor. nitric oxide (no) donors, including ng, inhibit uterine contractility and have been used as tocolytics. the aim of this study was to examine the inhibitory effects of r5020 on preterm labor in rats induced with a progesterone antagonist (onapristone, zk) with and without ng. materials and methods: charles river s-d timed pregnant rats (n=6/group) were treated with zk (3 mg/rat, s.c.) alone or vehicle (controls) on day 17 of gestation. other groups of rats were treated with zk in combination with various doses of r5020 (0.3, 1 or 2 mg/rat s.c) with and without ng (2 mg s.c. pellet) from days 18 to 19 of gestation. all rats were sacrificed on day 20 of gestation and the number of fetuses and implantation sites were counted to determine the preterm delivery rate. one way anova was used for statistical analysis. p<0.05 was considered significantly different. results: rats treated with zk alone delivered all their fetuses prematurely compared with controls (p<0.05) treated with vehicle only (ca. 3% fetuses delivered). ng treatment alone did not affect the delivery rate (p>0.05) compared to controls. similarly zk + ng did not reduce the preterm delivery rate compared to zk alone (p>0.05). however r5020 dose dependently reduced (p<0.05 at all doses) the number of fetuses delivered prematurely in response to zk and the premature delivery rate was further reduced when treatment included the combination of r5020 plus ng (p<0.05). conclusions: zk effectively induces premature delivery. premature delivery produced by zk can be effectively reduced with a r5020, a progestin known to bind with high affinity to nuclear progesterone receptors. ng by itself, at the dosage used, does not reduce the prematurity rate caused by zk. however, r5020 and ng act synergistically to reduce the preterm delivery rate. this study indicates that combinations of a progestin with an no donor may be an effective treatment for preterm labor and delivery. monkeys. pl grigsby, 1 jp rasanen, 1,2 dw sadowsky, 1 m bertolino, 3 m carbonatto, 3 e gillio tos, 3 s canali, 3 j lacy, 4 a chollet, 4 mj novy. 1 1 reprod sci, oregon primate res ctr, beaverton, or; 2 ob/gyn, oregon health sci univ, or, usa; 3 rbm, merck serono, italy; 4 merck serono, switzerland. objective: to investigate the pharmacokinetics (pk) and pharmacodynamics (pd) of as606521, a novel orally active non-peptide oxytocin (ot) antagonist in rhesus monkeys. study design: dose finding and pk/pd studies were done in chronically instrumented non-pregnant (n=3) and pregnant (120-150 dga, term 165d; n=5) monkeys. maternal and amniotic fluid compartments were serially sampled during dosing and washout. treatment phases included: ot infusion control, ot infusion + as606521, and ot rescue therapy. ot infusions (2-64 mu/kg/hr, iv) were given to determine the lowest dose required to produce stable, sub-maximal uterine contractions in each animal. as606521 was administered p.o. at 5, 10, 20mg/kg and the effects on inhibition of uterine activity (hca, mmhg.sec/hr) were compared. to verify antagonist reversibility, objectives: infection such as chorioamnionitis is thought to be the cause of premature rupture of the membrane and induce uterine contractions leading to preterm delivery. however, the mechanism for enhancement of uterine contractility is not well understood. we have reported that non-selective cation channels (nsccs) regulate pacemaker potentials to generate rhythmical contractions and should be targets of magnesium ions used for tocolysis. the purpose of this study is to investigate the changes of the expression of nsccs during normal pregnancy and the effect of inflammation in preterm. methods; atp receptors (p2x) and transient receptor potential canonical (trpc) channels were examined as uterine nsccs. the mrna was extracted from the rat myometrium of non-pregnant and pregnant rats at days 15, 18, 20 and 22. the expression of each subtype of p2x and trpc channels was measured by real time rt-pcr (abi7700) with taqman probes (abi). as an inflammatory model lipopolysaccharide (lps; 0.2 mg/kg) was injected into intraperitoneal cavity at day 18 and the tissue was sampled after six hours. results; p2x4 and p2x7 were determined to be dominant subtypes of p2x channel. the expression of p2x4 was increased by 70% at day 22, compared with day 15 and that of p2x7 was enhanced by 90%. on the other hands, trpc3 and trpc4 were detected dominantly. the expression of trpc3 was increased three times in the late stages of gestation. however, trpc4 was suppressed by 57%. in the lps treated rat myometrium cox-2 mrna expression was measured to be 52 fold higher than that of the control rat, showing inflammatory effects in the myometrium. in this model the expressions of p2x4, p2x7 and trpc3 were enhanced by 7.4, 18.6 and 24.7 times, but trpc4 was not changed. the mrna expression of p2x4, p2x7 and trpc3 channels in rat myometrium was increased in the late stages of pregnancy. these channels are suggested to be concerned with onset of labor. in the inflammatory model the expression of these channels was accelerated dramatically and these values were much higher than those in normal pregnancy. this finding supposed inflammation may enhance some types of nsccs to accelerate uterine contractility and induce preterm delivery. anandamide ( to determine the mechanism of rho protein activation during oxytocin and carbachol induced contraction, freshly prepared myometrial strips in krebs henseleit buffer were treated with oxytocin (10 nm) and carbachol (100 μm) under isometric and tension free conditions. control strips were exposed to buffer only. treated myometrial strips were solubilised and separated into membrane and cytosolic extracts and equal aliquots were immunoblotted with rhoa and rhob antibodies. rhoa translocated to the membrane after oxytocin and carbachol stimulation under both isometric and tension free conditions (p<0.05). there were no significant changes in rhob membrane to cytosol ratios relative to control. agonist induced contraction in human myometrium is associated with rhoa but not rhob membrane translocation. during pregnancy components of the intracellular camp signalling pathway show increased gene expression resulting in the maintenance of myometrial quiescence until term where a substantial decrease in expression of these genes is observed. protein kinase a regulatory subunit riia (rii ) is upregulated at both the mrna and protein levels in the human myometrium during pregnancy. this particular subunit is membrane-bound and by directing phosphorylation to myometrial cytoskeletal proteins may affect contractile machinery thus playing a role in maintaining uterine relaxation. acetylation of histones promotes a favourable chromatin environment for transcriptional activity of many genes. this process is largely inhibited by histone deacetylases (hdacs), whereby its activity leads to transcriptional repression. hdacs can be recruited to the promoter region of a gene by other transcription factors such as sp proteins. since the rii promoter contains three sp1-4 consensus binding sequences in its proximal part, we investigated whether this gene is a target for transcriptional regulation by hdacs. using dna precipitation assays we found that sp1, 3 and 4 as well as hdac1 and 2 form complexes with biotin-labelled fragments relating to sp1-4 elements in the promoter region of rii . additionally, treatment of myometrial primary cell cultures with hdac inhibitor trichostatin a (tsa), or with the methyltransferase inhibitor 5-azac resulted in increased mrna and proteins levels. further studies with full and truncated luciferase constructs of the promoter region of the rii gene in transiently transfected myometrial cells confirmed that all three sp1-4 elements are involved in the transcriptional regulation of the gene. this process involves hdacs, as 6h treatment with tsa significantly increased the luciferase signal. changes in the binding of sp proteins and hdacs to the promoter after tsa and azac treatment were investigated employing chromatin immunoprecipitation assays. alterations in the methylation status of the promoter after treatment were examined by bisulfite modification and dna sequencing. together, this study highlights the importance of chromatin modifications in the maintenance of uterine quiescence during pregnancy as well as identifying a potential mechanistic target for drugs that may reduce the incidence of preterm labour. objective: progesterone maintains pregnancy by promoting myometrial quiescence. typically progesterone effects are thought to be mediated through the classic genomic pathway. there is evidence, however, that progesterone also acts via a non-genomic pathway by interacting with specific membrane progesterone receptors (mprs) and in particular progesterone receptor membrane component -1 (pgrmc-1). the role of non-genomic progesterone actions in human pregnancy and parturition is not clearly understood. the goal of this study was to measure the extent of mpr expression in biopsy specimens of human myometrium obtained at cesarean delivery, and to determine whether expression changes with advancing gestation or the onset of labor. study design: lower uterine segment myometrial biopsies were obtained at the time of delivery from consenting women who were at term and not in labor (n=4), preterm and not in labor (n=7) and term and in labor (n=5). protein extracts were prepared and subjected to polyacrylamide gel electrophoresis and immunoblot analyses for pgrmc-1 and gapdh. abundance of pgrmc-1 protein relative to gapdh was determined by digital densitometry. we also performed immunohistochemistry (ihc) to determine the cellular localization of pgrmc-1 in the human pregnancy myometrium. results: pgrmc-1 protein was identified in each biopsy specimen. there was a 2-fold increase in pgrmc-1 protein in term compared with preterm biopsies (relative to gapdh p<0.04). the relative level of pgrmc-1 protein was not different between biopsy specimens from laboring and non-laboring women at term (compared to gapdh, p=0.8). pgrmc-1 immunoreactivity was localized to granular cytoplasmic staining. conclusion: this is the first description of the presence of pgrmc-1 protein in the human myometrium during pregnancy. its presence suggests that progesterone may influence contractility non-genomically via these receptors. the functional significance of the gestational age associated two fold increase in pgrmc-1 is unclear. changes in expression of this receptor during pregnancy may be important for the hormonal control of parturition and can be the focus of future studies. (ruddock et. al., abstract smfm, 2008) . the aim was to examine the mechanism of p4 inhibition. methods: uterine tissues from women (n=55) at term with cesarean section, were suspended in organ chambers and exposed to various agents or solvents. contractility was registered, stored, analyzed and compared before and after addition of agents or kcl. tissues were treated with p4 alone (10 -10 to 10 -3 m) or p4 bound to bovine serum albumin (bsa/p4, 10 -6 to 10 -3 m p4), a progestin with low affinity to mpr (r5020, 10 -7 -10 -3 m), or a non-sex steroid (cholesterol, 10 -5 to 10 -2 m). other tissues were pretreated with selective inhibitors of adenylate cyclase (sq 22536, 10 -5 m), guanylate cylase (odq, 10 -5 m), phosphodiesterase (rolipram, 10 -5 m), nitric oxide (no) synthases (l-name, 10 -4 m) or a nuclear p4 receptor antagonist (mifepristone, mif, 10 -5 m), followed by p4. data were analyzed by anova for statistical differences (p<0.05). results: p4 rapidly (<1 hour), effectively (100%) and dose-dependently inhibits spontaneous and kclinduced contractility (ed 50 of <10 -5 m incubation of strips with zd-6416, at concentrations up to 1 m for one hour prior to the experiment, led to no significant reduction in the total work done. however, incubation of strips with l-798106 with a concentration of 100nm for one hour prior to the experiment, led to a statistically significant reduction in the total work done after one and a half hours. furthermore, when increasing concentrations of pge 2 (10 -10 to 10 -6 ) were added to l-798106 (100nm) pre-treated strips, total work done per contraction was comparable to that of non-treated controls. similar effects were not observed in zd-6416 (100nm) pre-treated strips. since the reduction in contractility caused by l-798106 was greater than that caused by zd-6416, it is likely that the stimulating effect of pge 2 acts predominantly via ep3 receptors. taken together with our previous data showing an increase in ep3 at labour, this data shows that targetting an ep3 receptor may be a useful strategy in managaing pre-term labour. recently a truncated 46kda isoform of the er-has been described in human endothelial and testicular cells. we describe the presence of this 46kda erisoform in pregnant and immortalized non-pregnant human myometrial cells. methods: myometrial tissue obtained from non-laboring pregnant women undergoing cesarean section at term (n=4) was dissected prior to being finely minced. a portion of the tissue was placed in pbs and sonicated, while the remainder of the tissue was dissociated with collagenase prior to filtration and placement on culture plates. cells were then cultured in mem w/10% fetal bovine serum (fbs) until confluence. cultured cells were then dissociated with 0.05% typsin/edta, subsequently re-plated and grown to confluence. immunohistochemistry directed toward smooth muscle protein was used to verify myometrial phenotype.) protein was extracted and quantified. western blot was performed with mouse monoclonal anti-er-receptor antibody (santa cruz biotechnology) to the f-10 domain of the human er-receptor. an immortalized non-pregnant human myometrial cell line provided by ann word, htert, was cultured and isolated as described above. results: htert cells expressed both the truncated (46kda) and the full length (66kda) er-isoforms. fresh and cultured myometrial cells from non-labored term pregnant patients also expressed both isoforms of er-. subsequent subcultures of myometrial tissue continued to express the 46kda erisoform, yet the expression of the 66kda isoform was lost. a representative blot is below. conclusions: we demonstrate, for the first time, the presence of a 46kda erisoform in cultured and non-cultured pregnant and cultured nonpregnant human myometrial tissue. discovery of this isoform of er-in myometrial tissue could provide insight into the molecular mechanisms involved in parturition. the action of prostaglandin f 2 (pgf 2 ), a potent uterotonic stimulant that is associated with labour at term and preterm, is mediated by its receptor, ptgfr. myometrial ptgfr mrna levels fall during pregnancy and this likely plays a role in uterine quiescence. however, the mechanisms by which this occurs are poorly understood. we previously reported that pgf 2 downregulates fp mrna expression in cultured human myometrial ultr cells in a protein kinase c (pkc) dependent manner. in addition to the downregulation of mrna levels, receptor desensitization may also represent another mechanism of decreasing ptgfr activity because ligand binding to g protein coupled receptors often results in receptor internalization. we therefore hypothesized that pgf 2 treatment of ultr cells also results in pkc dependent ptgfr internalization. methods: near confluent cultured human myometrial ultr cells were treated +/-10 -7 m or 10 -6 m pgf 2 for 1, 3, 6 or 24 hr. cells were fixed with formaldehyde and visualized for localization of ptgfr by immunofluorescence. to examine the potential involvement of pkc in the process, ultr cells were treated +/-10 -6 m pgf 2 and +/-10 m myristoylated pkc inhibitor (20-28) and examined for ptgfr cellular localization as described above. results: pgf 2 treatment resulted in a dose dependent decrease in ptgfr membrane signal at 1, 3, 6 and 24 h. this decrease was dependent on pkc as cotreatment with the myristoylated pkc inhibitor (20-28) prevented the pgf 2 induced decrease in membrane ptgfr at 1 h treatment. there was no visible effect of the pkc inhibitor on ptgfr membrane signal on its own. conclusion: we conclude that pgf 2 decreases membrane levels of ptgfr protein in human myometrial ultr cells in a pkc dependent manner. these results suggest that pkc may be required for both the pgf 2 induced internalization and desensitization of ptgfr protein and downregulation of ptgfr mrna in human myometrial ultr cells. therefore pkc may play a crucial role in downregulating ptgfr expression and activity and maintaining uterine quiescence during pregnancy. rhoa is a small gtpase that acts as a molecular switch to control a variety of signalling pathways in smooth muscle, including contractility. it is thought that increases in rhoa-gtp levels facilitates phosphorylation of target proteins such as cpi-17, promoting contractility at pre-term and term labour in humans. however, in situations of acute or chronic hypoxia in the uterus, it is important that myometrial contractility and subsequent labour is not facilitated prematurely. given the importance of rhoa to a cell's response to hypoxia in other cell types, it was hypothesised that rhoa plays a central role in the mechanism controlling smooth muscle contraction in the uterus too. following acute hypoxia (0.5% o2) for one to six hours, rhoa mrna, total protein and activation (rhoa-gtp) levels were analysed, using semi-quantitative pcrs and western blot, and compared to normoxic non-pregnant human uterine smooth muscle control cells. next, we investigated whether reduced oxygen conditions affected oxytocin induced activation of rhoa, following a two hour treatment of 10 nm oxytocin. firstly, our results demonstrate that the rhoa itself is significantly activated under low oxygen conditions, resulting in phosphorylation of myosin phosphatase, myosin light chain and cofilin, three proteins known to be central in contraction and actin filament organisation. secondly, hypoxia significantly reduced the coupling of oxytocin to rhoa activation under the conditions examined. we observed a significantly reduced level of rhoa expression and activation which correlated with an increase in the level of another rhogtpase protein, rhoe. we propose that rhoa inactivation occurs through a rhoe-mediated mechanism, suggesting a balance in the activity of these two antagonistic rhogtpases in oxytocin-induced hypoxic human uterine smooth muscle cells. these results provide a possible explanation for the reduced coupling of oxytocin as a stimulant of myometrial contractions during slowly progressing labours. oxytocin-induced release of calcium ions (ca 2+ ) from sarcoplasmic reticulum (sr) and sensitisation of contractile proteins to ca 2+ have been suggested to mediate the oxytocin-induced potentiation of myometrial contractions. objective: we investigated the effects of oxytocin in the presence of nifedipine, a known inhibitor of the l-type calcium channel (ltcc). method: samples of myometrium were obtained from women undergoing term caesarean section with the approval of the local ethics committee. a standard organ bath system (ad instruments, uk) was employed to analyse contractile activity. stable spontaneous contractions were recorded for 40-60 minutes before addition of nifedipine. results: in agreement with our previous findings, application of oxytocin to spontaneously active strips produced a two-component effect: a transient tetanus-like contraction, followed by prolonged augmentation of phasic contractions. nifedipine (1um) rapidly abolishes spontaneous contractions, subsequent addition of 100nm oxytocin produced an initial, transient rise in force, approxiamately 20% compared to oxytocin alone, followed by high frequency oscillations in >50% of strips. calcium-free solutions were used to confirm that oscillations were due to ca 2+ entry. disabling the sr store using thapsigargin (1um) had no effect on oscillations, confirming the sr not to be involved. the t-type calcium channel blocker, mibefradil (1um ) showed no inhibition of oscillations. an ip 3 receptor and store-operated calcium channel inhibitor, 2-aminoethyldiphenylborate (2-apb) 50um also had no effect on oxytocin-induced oscillations. the store-operated calcium channel inhibitor skf-96365 (10um) showed partial inhibition of oscillations. conclusions: based on these results, we propose that the most likely mechanism of ca 2+ entry producing oxytocin-induced oscillations in the presence of nifedipine is the transient receptor channel-c (trpc) channel, known to be present in the human myometrium. further work needs to be completed to clarify this further. identification of stim and orai in human myometrium. a new paradigm in store-operated calcium signaling. evonne c chin-smith, 1 mark r johnson, 2 rachel m tribe. 1 1 division of reproduction and endocrinology, king's college london, london, united kingdom; 2 department of maternal fetal medicine, imperial college school of medicine, chelsea and wesminster hospital, london, united kingdom. background: recent reports have suggested that two novel proteins, stim and orai, are involved in the regulation of store-operated calcium entry. we have previously reported that members of the trpc family, putative basal and store operated calcium entry channels, are present in human myometrium and regulated by labour associated stimuli il-1beta and mechanical stretch. although stim and orai isoforms have been reported in other smooth muscle cell types, there are no published reports of stim and orai expression in human myometrium. the aim of this study was to identify mrna expression of stim1, stim2, orai1 and orai2 in human myometrium. methods: human myometrial biopsies were obtained from women undergoing elective caesarean section at term (prior to labour) with informed written consent and institutional ethics committee approval. whole myometrial tissue was either snap frozen and stored at -80 o c or used for cell culture. rna was extracted from whole tissue (n=5), primary cultured myometrial cells (n=4) and passaged (p2) myometrial cells (n=5) and stim1, stim2, orai1 and orai2 mrna expression was assesed by quantitative real-time pcr. results: all four genes were expressed in whole myometrial tissue and cells. stim1 and stim2 mrna expression in cultured myometrial smooth muscle cells (primary and passaged) was significantly reduced compared to myometrial tissue expression (p<0.05). however, there was no significant difference in either orai1 or orai2 expression in whole tissue versus cultured myometrial smooth muscle cells. conclusion: to our knowledge this is the first report of stim1/2 and orai1/2 mrna expression in human myometrium. these genes may contribute to the regulation of calcium signalling in human myometrium, but the functional significance of their expression remains to be determined. funded by the bbsrc. obstetrics and gynecology, national university of ireland, galway, galway, ireland. objective: the ability of uterine smooth muscle cells to stimulate collagen contraction has been well established as an in vitro model of myometrial contractility. devost and zingg (2007) reported that the contractility of human myometrial cell lines layered onto collagen matrices was increased by oxytocin while another group described the stimulation of human uterine smooth muscle cell contractility, cultured within collagen lattices, with endothelin-1 (dallot et al., 2003) . our study investigated the response of human primary uterine smooth muscle cells cultured within collagen lattices, to various compounds, including the non-specific depolarizing agent potassium chloride (kcl), the inflammatory cytokine tnf , the rock-1 inhibitor y-27632, oxytocin, and oxytocin plus its clinically used antagonist, atosiban. methods: human primary uterine smooth muscle cells (utsmc) (lonza) were maintained in dmem high glucose media. cells (150,000 per well) passage 3-8, were embedded in 1.5 mg/ml collagen, in 0.5 ml aliquots, in dmem-f12 media on 24 well plates (invitrogen) (dallot et al., 2003) . effects on contraction were studied by monitoring changes in gel area (alpha innotech imager, image j software (nih)). statistical significance was determined by the student t test. results: the utsmcs displayed basal contraction of the collagen gels while the non-contractile cell line hek293 cells, did not. kcl (20nm) stimulated an 11% increase in contractility (n=3, p=0.0262) while 10 nm tnf resulted in an 8.4% increase (n=3, p=0.0205), in comparison to unstimulated cells embedded in gel. the rock 1 inhibitor y-27632 (10 m) inhibited contractility, with a 17.5% decrease in collagen gel contractility (n=3, p=0.011). a 13% increase (n=4, p= 0.0146) in utsmc embedded collagen contractility was observed with 10 nm oxytocin, which was antagonized by atosiban (1 m) (n=4). conclusion: this study highlights the importance of the development and optimisation of a reproducible human in vitro myometrial contractility model, to evaluate the effect of various known labor-associated, and also unknown compounds. this should aid in our understanding of the many complex biochemical pathways involved in myometrial contractility at labor, and ultimately contribute to the prevention of preterm labor. changes in intra-mural myometrial blood flow during spontaneous labour using 3d power doppler angiography (pda). nw jones, 1,2 nj raine-fenning, 2 h mousa, 1 mj taggart, 3 k jayaprakasan, 2 gj bugg. 1 1 nottingham university hospitals nhs trust, united kingdom; 2 nottingham university, united kingdom; 3 university of newcastle upon tyne, united kingdom. methods 3d pda was used to measure the percentage (%) change in the vascularization index (vi), the flow index (fi) and the vascularization flow index (vfi) 1 in a volume of myometrium at the uterine fundus of 20 nulliparous women at term, in the first stage of uncomplicated spontaneous labour. 3d data sets were obtained during a single cycle of uterine relaxation (r1), contraction and subsequent relaxation (r2). measurements were made independantly by two authors (nwj and gjb) using vocal® (ge kretz) and the mean value was used for analysis. the results from each woman are presented as a % change of r1. data is presented as medians [interquartile range (iqr)] and analysed using non-parametric tests. the median volume (cm 3 ) of interest for r1 was 17.9cm 3 (12.6-24.4cm 3 ), for the contraction was 17. (fig.1) . the % change for all three indices between r2 and r1 was not significantly different. however, there was a significant difference between the contraction and r2 (p< 0.01). the mean intra-class correlation coefficient and 95% confidence interval (ci) of vi, fi and vfi for the authors (nwj and gjb) were 0.99 (0.98-0.99), 0.99 (0.98-0.99) and 0.98 (0.96-0.98), indicative of good inter-observer reliability. conclusion 3d pda is a useful and reliable tool in the assessment of changes in intramural myometrial blood flow, which was found to reduce significantly during a contraction but increase again during the following uterine relaxation. 1. raine-fenning nj, et al. the inter-observer reliability of 3d pda acquisition within the female pelvis. ultrasound obstet gynecol. 2004; 23:501-8. 196 the role of pgf2 on myometrial contractility; studied with a selective fp antagonist. shankari arulkumaran, 1 andre chollet, 2 phillip r bennet. 1 1 imperial college parturition research group, institute of reproductive and developmental biology, hammersmith hospital campus, london, united kingdom; 2 merck serono, geneva, switzerland. objectives: human myometrial strips established in culture will usually begin contracting after one hour. we have previously shown that stretch upregulates prostaglandin synthesis and have therefore hypothesized that spontaneous contractions occur because of stretch-related prostaglandin synthesis. methods: experiments were performed using 5x2mm human pre-labour, lower uterine segment myometrial strips in a dmt myograph 800ms in oxygenated kreb's solution, with adi powerlab software. spontaneous contractions required stretch force and initial experiments determined that the maximum number of strips attaining spontaneous contractions was greatest at a force of 5-6g. a novel antagonist to pgf2 (fpa) was studied in this model. the fpa has a ki of 6nm for fp and is 10-100 fold selective for fp compared with other prostanoid receptors. results: addition of pge 2 and pgf2 after the commencement of spontaneous contractions caused a statistically significant increase in the total work done by the strips. the effect of pge 2 was being greater than that of pgf2 . pre-incubation of the baths with a novel and selective fp antagonist (fpa) with concentrations up to 1 m (10 -6 ) did not affect the total work done by spontaneous contractions compared to non-treated controls. however, increasing concentrations of the fpa (10 -8 to 10 -5 ) decreased the total work done by 5-fold on strips treated with pgf2 beforehand in comparison the pgf2 -treated strips alone. conclusion: these data suggest that stretch and synthesis of prostaglandins is essential for spontaneous contractility in human myometrial strips. since fpa is able to block pgf2 induced but not spontaneous contractions, it is likely that pgf2 does not play a role in spontaneous myometrial contractility in vitro, although it may do so in vivo. because other factors may combine to increase contractility, the combination of an fp antagonist with other inhibitors may therefore, be a more effective strategy in reducing pre-term deliveries. objectives: brain natriuretic peptide (bnp) is synthesized in fetal membranes and inhibits oxytocin-induced contraction of preterm human myometrium. we hypothesized that bnp may be a paracrine mediator of human myometrial quiescence. we showed bnp content is higher in membranes from preterm pregnancies absent labor and significantly decreased with idiopathic preterm labor. while bnp activates natriuretic peptides receptors a (npr-a), b (npr-b), and its clearance receptor (npr-c), we have shown bnp does not inhibit myometrial contraction via npr-a or npr-b. herein, we test in part the hypothesis that bnp inhibits myometrial contractions by activating npr-c by quantitating npr-c in human myometrium at different gestations and labor status. methods: myometrial samples were obtained at the time of cesarean section after informed consent from 4 groups of patients: preterm not in labor (pt-nl), preterm in labor (pt-l), term not in labor (t-nl) and term in labor (t-l). myometrial samples were obtained from women 30-34 weeks' gestation, and term between 38 and 41 weeks. mrna for npr-a, b and c were semiquantitated by realtime pcr (normalized by 18s mrna), and npr-c protein by western blot. results: natriuretic peptide receptors a, b and c mrnas were identified in all 4 groups. while there were no differences in mrna levels among groups, npr-c protein was increased in samples from term laboring women. conclusion: since bnp inhibits the contraction of human preterm but not term myometrium independent of npr-a and npr-b, we have previously speculated that bnp activates another "unknown" receptor. herein we find that myometrial npr-c protein but not mrna increases during human labor at term. the increase in npr-c at term could compete with the unknown quiescent receptor to functionally reduce the availability of bnp and thus permit or promote myometrial activation/contraction. (supported by a grant from chilean government fondecyt 1020675). objective: bnp is synthesized within human chorion and amnion and inhibits oxytocin-induced contraction of human myometrium. bnp activates guanylate cyclase (gc) natriuretic peptides receptors a (npr-a) and b (npr-b). bnp also stimulates the clearance receptor (npr-c) whose action is not mediated by gc. the intracellular pathway of bnp/npr-c inhibition is not known. we determined that bnp does not inhibit myometrial contraction via npr-a or -b. we hypothesized that bnp inhibits myometrium by npr-c activation. we test aspects of our hypothesis by determining whether bnp/npr-c pathway inhibits myometrial contractions via myometrial nos pathway. methods: bnp and canp (specific npr-c agonist) were added to primary human myometrial cell cultures and enos/inos activity/expression determined. cell cultures (n=6) were prepared from myometrial samples of nonlaboring, term pregnant women. after confluence (10d), the cells were incubated (6h) with 100nm bnp and/or canp. nos activity was measured ( l-citruline assay) and transcription/translation semi quantitated (realtime pcr and western blotting). results: bnp had no effect on either nos expression or activity. canp significantly reduced inos but not enos mrna level. canp did not alter the protein level of nos but significantly reduced nos activity. co-incubation with bnp and canp reduced both mrna levels (p< 0.05) and significantly decreased enos, but not inos, protein without change in overall nos activity. within the female pelvis. ultrasound obstet gynecol. 2004; 23:501-8. conclusion: the activation of npr-c by canp reduces nos activity and expression. the same effect was not observed by bnp. the difference may be explained because bnp (but not canp) activates all natriuretic peptide receptors, and they may have opposite actions on nos pathway. we conclude unlikely bnp inhibits human myometrial contraction by npr-c activation via the nos pathway. ( introduction: ultr is a retroviral immortalized human uterine smooth muscle cell line which we use as a model for uterine hypertrophy studies. however, this cell line has limited usage due to a reduced rate of cell division and eventually replicative senescence in culture. one of the mechanisms of human somatic cellular senescence is un-compensated shortening of telomeres, the specialized dna structures located at the ends of eukaryotic chromosomes. introduction of human telomere reverse transcriptase (htert) has been shown to induce telomerase activity and telomere elongation, and extend life-span of normal human cells. objective: to recover ultr cell division without altering the phenotypic characteristics of smooth muscle cells by introducing htert, therefore improving ultr cell line. methods: ultr cells were transfected with a modified htert expression vector at a relatively early stage (passage 28) in the presence of selective antibiotic. ultr cell growth rate, with (ultr-ht) or without transfection, was determined by plating cells in multiple plates at a fixed density and counting cell numbers after 7 days. single cell clones of stably transfected cells were further isolated by plating in 96 well plates. expression of htert, smooth muscle specific genes (smc-sgs), and target genes was identified by rt-pcr of total rna extracted from ultr and ultr-ht cells. results: rt-pcr demonstrated successful introduction of htert into ultr cells. the growth rate of ultr-ht cells was increased and cell morphology was improved (free of the typical aneupoid appearance). at passage 37, ultr-ht cells grew 3.1 fold (p<0.0001) and 26.9 fold (p<0.0001) faster than ultr cells at passages 28 and 34, respectively. currently 8 single cell clones have been selected. ultr-ht cells express a set of smc-sgs, including -actin, caldesmon, calponin, myosin heavy chain, sm22, and smoothelin, confirming that the smooth muscle phenotype is preserved. a panel of genes involved in angiotensin ii signalling, including angiotensin ii receptors (at1/2), nox family (nox1, 4, 5 and duox1), nox-associated genes (p22phox, p47phox, p67phox, and rac1/2), was also preserved. conclusion: this is the first report of rescuing uterine smooth muscle cell replicative senescence via activation of telomerase. we have established a human uterine smooth muscle cell line which will provide an improved in vitro model for studying human myometrium. at term, myometrium is characterized by spontaneous contractions which vary in the amplitude, frequency and duration depending on specie and hormonal status. repetitive depolarization of plasma membrane followed by transient elevation in [ca 2+ ] i is thought to underlie the contractions. however, the detailed pathways which regulate the spontaneous contractility remain unclear. objective: this study was designed to elucidate the effect of protein kinase c (pkc) on the spontaneous contractions and [ca 2+ ] i transients in the myometria from term pregnant mice and women. methods: the human samples were obtained from women undergoing cesarean section with the approval from the institutional review board committee while mice myometria were dissected from the 18 days pregnant mice sacrificed according to the animal study protocol. the isometric force and [ca 2+ ] i were measured simultaneously in fura-2 loaded myometrial strips using spectrofluorometer equipped with force transducer. results: the pkc activator phorbol 12,13-dibutyrate (pdbu) applied at 10 -7 m first stimulated the amplitude of spontaneous contractions in mice and human myometria followed by their inhibition. under the pdbu treatment the frequency of the contractions was first increased and then decreased in both species. at the same time, pdbu didn't initially increase the amplitude of [ca 2+ ] i transients but attenuated it over time. however, the frequency of the transients was first increased and later decreased upon the pdbu exposure. in addition, pkc activation with pdbu resulted in the elevation of the uterine basal tone without corresponding changes in the basal level of [ca 2+ ] i in human myometrium. in mice myometrium, on the contrary, pkc activation didn't result in an increase of the muscle tone and the basal level of [ca 2+ ] i . conclusions: we propose that pkc causes bi-phasic effect on uterine spontaneous contraction in mice and human first to potentiate the amplitudes and the frequencies and later decreases them. the amplitude of [ca 2+ ] i was not initially potentiated by pdbu suggestive of the dissociation of the contractile and [ca 2+ ] i response. the stimulatory effect of pdbu on the basal muscle tone in human myometrium and the absence of the effect in mouse suggest different mode of pkc action on uterine contraction in human and mice. introduction: mechanical stretch of uterine myocytes is detected through integrins on the cell surface which form part of the focal adhesion complex, this signals through mapk, ultimately leading to the up-regulation of various pro-labour genes including pghs-2 and il-8. on integrin activation fak is recruited to the focal adhesion complex and activated by autophosphorylation at tyr-397, creating a src binding site. this promotes further fak phosphorylation at tyr-576, 577 and 925, enhancing fak catalytic activity. however fak can also act as a scaffold protein and its exact role in the expression of pro-labour genes is uncertain. in this study we used inhibitors for the kinase activity of fak and mek 1/2 in order to test the affect of fak kinase activity on expression on pghs-2 mrna. methods: primary human myometrial cell cultures were grown from myometrial biopsies taken from women undergoing elective caesarean section. cells were plated onto 6-well flexible bottom plates coated in type i collagen. cells were subjected to 11% static stretch for up to 60 minutes. cells were also incubated with either the rho kinase inhibitor y27632 or the mek 1/2 inhibitor u0126 prior to being stretched. western blots were performed using antibodies to fak phospho-397, fak phospho-925 and erk 1/2 phospho-202/204, -actin was used as a loading control. rna was also extracted and levels of pghs-2 measured using q-pcr. results: stretch increased levels of phosphorylation at both tyr-397 and tyr-925 with the greatest increases occurring at 30 and 60 minutes. however the increase at tyr-397 appeared to be greater than at tyr-925. incubation with the rho kinase inhibitor reduced phosphorylation of fak-397, however this did not affect phosphorylation of erk 1/2 or stretch induced up regulation of pghs-2 mrna. in contrast incubation with the mek 1/2 inhibitor reduced erk 1/2 phosphorylation and expression of pghs-2 mrna, whilst also reducing fak phosphorylation (n=4; p=0.05). conclusions: fak has previously been shown to be important in activation of the stretch induced mapk cascade and pghs-2 expression. however these data suggest that while stretch causes fak phosphorylation fak kinase activity is not essential to pghs-2 expression. this suggests fak may be acting as a protein scaffold. . our aim was to examine the effects of both natural progesterone and 17 hp on spontaneous myometrial contractions. myometrial biopsies were taken with informed consent and ethics approval from non-labouring women at elective caesarean section 37 weeks gestation. strips of myometrium 15 mm long ,2 mm wide were cut and suspended under a resting tension of 20 mn in organ baths of krebs gassed with 95% o 2 / 5% co 2 within 12 hours of collection. progesterone or 17 hp were added in a cumulative manner at 20-minute intervals. changes in amplitude were recorded. results were compared using anova. following equilibration for 2 hours, myometrial strips contracted in a rhythmic manner (amplitude 91.6 ±16.1 mn, n=8 pairs). progesterone (10nm-30 m) produced a concentration dependent inhibitory effect on myometrial contractions (fig1), which was greater than that of vehicle (p<0.05). maximum inhibition measured 93.3 ± 2.0% and 67.2 ±14.2% for progesterone and vehicle, respectively. 17 hp exerted an inhibitory effect, this was not significantly different from the vehicle (p>0.05). progesterone exerts an inhibitory effect on myometrial contractility in vitro. this is apparent within minutes suggesting a nongenomic action. our data are in agreement with some reports in the literature but conflict with others[3]. this acute inhibition of myometrial contractility may contribute to the mechanism by which progesterone prevents preterm birth. in contrast, we were unable to demonstrate an inhibitory effect of 17 hp on contractility despite its demonstrated ability to reduce the incidence of preterm delivery [4] . our data suggest that natural progesterone may be a more effective tocolytic agent in the acute setting than 17 hp. obstetrics and gynecology, ramathibodi hospital, mahidol university, bangkok, thailand; 2 pathology, ramathibodi hospital, mahidol university, bangkok, thailand; 3 obstetrics and gynecology, samuel lunenfeld research institute, toronto, canada. objective: chemokines has been shown to play an important role in regulating uterine function. recent evidence demonstrates that monocyte chemotactic protein (mcp) 1 level in amniotic fluid increases during spontaneous labor. the aim of this study was to examine the expression of mcp 1 in human myometrium. methods: myometrial biopsies were taken from nonpregnant women undergoing hysterectomy and term pregnant women undergoing cesarean section followed written consent and local ethics committee approval. elective cesarean section was performed before the onset of labor while emegency section was done after the onset of labor. immunolocalization (n = 5 each) was performed on paraffin sections by avidin biotin complex (abc) technique using monoclonal antibody specific to human mcp 1. reverse transcriptionpolymerase chain reaction (n = 10 each) using gene specific primer against mcp 1 and mcp 1 receptor was performed to identify mcp 1 messenger(m) rna in human myometrium . results: immunohistochemical findings demonstrated mcp 1 in human myometrial cells from nonpregnant, term pregnant women before and after the onset of labor. mcp 1 was labelled on plasma membrane and cytoplasm of myocytes from these three groups of women. similarly, mcp 1 and mcp 1 receptor mrna were found in nonpregnant and term pregnant women before and after the onset of labor. in this prospective study a group of 4075 fetuses was consecutively enrolled. one ultrasound examination was performed to each patient within 6 days from delivery. we considered fetal macrosomia a birthweight 4000g and big babies a birthweight 4500g. cut-off points for identifying the best value of fetal abdominal circumference for fetal macrosomia prediction were chosen by receiving operator characteristics' curve (roc) analysis. using the best cut-off indicated by roc analysis, specificity and sensitivity were calculated. mean gestational age at delivery was 38 +3 weeks (3 +3 sd). 1015 neonates weighted less than 2500 g, 2818 had a weight range between 2500 and 3999 g and 242 weighted more than 3999 g. the fetal ac measurement was the selected criterium to evaluate the risk of fetal macrosomia. to identify macrosomic fetuses the roc curve analysis identified a cut off of ac > 350 mm which allowed to select 867 fetuses. analysing this population we found 3183 true negative cases, 25 false negatives, 650 false positives and 217 true positives, with a sensitivity of 89.6% and a specificity of 83%. to detect big babies the roc curve analysis identified a cut off of ac > 361 mm (n.452 fetuses), reaching a sensitivity of 100% and a specificity of 90%, without false negative cases and with 413 false positives (39 true positives, 3623 true negatives). the 141 cases that weighted between 4000 and 4499 g, were included in the 413 cases considered false positives by this cut-off. conclusions. these results clearly indicated that ultrasounds alone can not be used to manage a pregnancy suspected for fetal macrosomia or big babies. in fact, to avoid shoulder dystocia and all other complications strictly related to macrosomic fetuses, clinicians should perform a large number of useless elective cesarean sections. . pathway analysis of differentially expressed genes (pa; z-score 2.0) showed up-regulation of axon guidance, folate biosynthesis, nitrogen metabolism and down-regulation of steroid biosynthesis, insulin signaling, oxidative phosphorylation, tgf-beta signaling, and ubiquinone biosynthesis pathways in cm vs cf. comparison of mnr vs c in f showed 320 genes up-and 354 down-regulated (n=3,3; p<0.05). pa showed up-regulation steroid biosynthesis, fatty acid metabolism, glycolysis/gluconeogenesis, phosphatidylinositol signaling, ketone body metabolism, and ubiquinone biosynthesis pathways and down-regulation of bile acid biosynthesis, cell adhesion molecules, dna polymerase, notch signaling and ti diabetes mellitus pathways in mnr vs c. comparison of mnr vs c in m showed 525 genes up-and 869 down-regulated (n=3,3; p<0.05). pa showed up-regulation of jak-stat signaling, autophagy, renin-angiotensin system (ras), and ubiquinone biosynthesis pathways and down-regulation of apoptosis, basal transcription, folate biosynthesis, nitrogen metabolism, protein export, and snare interaction pathways in mnr vs c. only the ubiquinone biosynthesis pathway up-regulation of mnr vs. c was common to both m and f. conclusions: ta and pa demonstrate sex-specific transcriptome expression in fetal kidneys at 0.9g. in addition, these results show sexspecificity in response to mnr. we have seen similar effects of mnr on gene expression for autophagy, apoptosis, ras, ubiquinone and cell adhesion pathways at 0.5g, suggesting these pathways may contribute to persistent affects of mnr. finally, we postulate that stress in utero may contribute to sex differences in risk of hypertension in adult life. leptin and neuropeptied y protein expression paradoxally increased in gestationall food restricted dams. louiza belkacemi, chun-hung chen, andrea jelks, michael g ross, mina desai. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: placental insufficiency is associated with marked increase in placental leptin production. this results in a rise in maternal leptin levels that serves as an early index of placental dysfunction. further increased placental leptin and suppressed neuropeptide y (npy) are associated with preeclampsia. leptin, an anorexigenic hormone and npy, an orexigenic peptide regulate food intake. importantly, leptin also serves as a placental and fetal growth factor. we have shown that maternal food restriction (mfr) results in intrauterine growth ... sf ()to:!)) df(,..)l) ...._ bf(jtolo) ... -m bf(,..lj) =-7(30.4) <(u.)) 14(48.1) )(6.7)"" 1'0~.1) !(11.7)' d.'cbom< 7(104) 2f(81 ))""" s(17l) background teenagers are more likely to deliver small-for-gestational age (sga) infants than adults, even after adjustment for socioeconomic factors 1,2 . previous studies of mostly black and hispanic subjects in the usa have suggested that maternal growth may contribute to reduced infant birthweight, due to preferential nutrient partitioning to the mother 3 . the impact of maternal growth on birthweight and nutrient partitioning in pregnant teenagers in the uk has not been examined. methods skeletal growth (change in knee-height from 1 st to 3 rd trimester), weight gain and skinfold thicknesses were measured in pregnant teenagers (n=369, 45% non-white) in london and manchester. key mediators of nutrient partitioning and metabolism: insulin-like growth factor(igf)-1, igf binding protein(bp)-1 and leptin, were measured in maternal plasma (28 weeks gestation). results maternal growth (defined as increase in knee-height >2mm/90days) was detected in 36% of pregnant teenagers. this growth was not associated with sga birth; in fact these mothers were more likely to deliver large-forgestational age (lga) infants (p<0.05). maternal weight gain and fat accrual at peripheral and central sites were greater in growers (p<0.01). these parameters correlated positively with maternal igf-1 and leptin but negatively with the igf inhibitor, igfbp-1 (p<0.001 for all). subjects delivering sga infants gained significantly less weight (p<0.01) and had lower igf-1 levels (p<0.01) than those delivering non-sga infants. conclusion maternal growth in teenage pregnancy was not associated with reduced birthweight. indeed the increased weight gain and fat accrual observed in growing teenagers may protect against sga birth and promote fetal growth. igf-1 and leptin promote fetal growth, primarily through effects on maternal metabolism and nutrient partitioning to the fetoplacental unit. these data suggest that higher maternal igf-1 and leptin in growing teenagers may provide an anabolic drive for both maternal and fetal growth. background. umbilical oxygen uptake (o 2 umb uptake) has been estimated in human pregnancies only in acute experiments at the time of caesarean section. the recently developed possibility to measure umbilical blood flow by ultrasound in utero, prompted us to study normal and iugr pregnancies in order to evaluate fetal oxygen uptake utilizing the fick principle, i.e. uptake equals umbilical blood flow times (a-v) differences. methods. thirty-six iugr pregnancies were studied at the time of elective caesarean section and compared to twenty-one controls (c) (gestational age: c=38.9±0.2 and iugr=32.0±0.5 wks). an ultrasound examination was performed within 4 hours from the caesarean section in all the recruited patients. umbilical vein absolute volume flow (qumb) was measured as the result between umbilical vein area and the time-averaged peak velocity * 0.5. blood samples from umbilical vein (uv) and artery (ua) were obtained after the delivery and blood gases and acid-base balance were evaluated. umbilical oxygen uptake was calculated as o 2 umb uptake = qumb*(uv-ua) o 2 content. results. as expected, average fetal and placental weights were significantly different in the studied groups (1287±83 and 254±32 g in iugr vs 3274±62 and 474±16 g in n) . iugr pregnancies showed a significant reduction in qumb (98.5±6.6 vs 234.8±12.1 ml/min; p<0.01) but no differences in the qumb/kg of fetal weight. iugr fetuses showed a significant reduction in o 2 sat, o 2 cont and po 2 in both uv and ua compared to n. (uv-ua)o 2 content (1.79±0.72 vs 3.3±0.24 mmol/l; p<0.001) and o 2 umb uptake/kg (0.12±0.01 vs 0.24±0.01 mmol/min/kg; p<0.01) were significantly reduced in iugr. conclusions. we here report an evaluation of fetal oxygen uptake that proved surprisingly similar to the values reported in chronically catheterized animals. however, iugr fetuses showed a significant reduction in both blood and oxygen supply: this latter was reduced more that 50% on a per kg basis. iugr fetuses therefore utilize less oxygen than normally grown fetuses. circulating levels of vitamin d and il-6 in pregnancies with iugr. calvin j hobel, 2 chander p arora, 1 adegoke adeniji, 1 priya arora, 1 susan e jackman, 1 olga miadel, 1 baldjyan lilit. 1 1 ob-gyn, cedars-sinai medical center, burns ans allen research institute, los angeles, ca, usa; 2 university of california los angeles, los angeles, ca, usa. background: any condition resulting in under exposure to sunlight, including the use of sun block or poor nutrition may result in insufficiency (37.5-80 nmol/l) or even deficiency of vitamin d (<37.5 nmol/l).vitamin d regulates placental development and function. vitamin d deficiency has been linked to increased risk of serious chronic and inflammatory diseases. objective: to determine if the circulating levels of vitamin d and interleukin -6 (il-6) in maternal plasma correlates to pregnancies resulting in fetus with intrauterine growth restriction (iugr). hypothesis: the metabolism of vitamin d initiates the biochemical cascade of events leading to the expression of il-6 and the inflammatory response in iugr births. study design: in a behavior in pregnancy study, plasma samples at all three time points were analyzed in a cohort of women for 25(oh)d using elisa. the samples were also analyzed for il-6 at three stages of pregnancy: t1 (18-20 weeks), t2 (28-30 weeks) and t3 (34-36 weeks). iugr was defined as birth weight below the tenth percentile for gestational age. none of the iugr cases had spontaneous preterm birth. results: iugr was diagnosed in 18 of 528 women with available samples from a behavior in pregnancy study (bips) . out of these subjects, 22 were selected as matched case controls. circulating levels of vitamin d (25(oh)d) were significantly lower in iugr cases at each visit (p<.001). the levels indicated deficient (29.4±4.6nmol/l) vitamin d in iugr group at t1 but sufficient vitamin d levels (83.8±5.8 nmol/l) in controls. subsequent visits also showed lower levels in the iugr cases compared to the control group (t2: 19± 3.1 nmol/l vs 45±3.6nmol /l; t3: 22 ± 4.1nmol/l vs 50± 2.9nmol /l). at all three time intervals, significantly (p<.001) higher levels of il-6 were associated with the iugr cases (415pg/ml, 1329pg/ml and 2526 pg/ml respectively) as compared to the controls (60pg/ml, 330pg/ml and 1240pg/ml respectively). conclusions: vitamin d deficiency or even insufficiency may be an unrecognized cause of iugr. it is possible that a primary non-infectious inflammatory process is activated by vitamin d deficiency. combined assessment of vitamin d deficiency and il-6 expression during different stages of pregnancy may facilitate the resognition of the risk of developing iugr. response to global 30% maternal nutrient restriction (mnr). nathan drever, thomas j mcdonald, peter w nathanielsz, cun li. obstetrics and gynecology, university of texas health science center at san antonio, san antonio, tx, usa. background: igf-ii is a major growth factor in the developing pancreas and studies in the human fetus demonstrate that the peptide localizes to b cells of the islets (j endocrinology 165:2). rodent studies show decreased fetal pancreatic growth and igf-ii and ins abundance and increased apoptosis with mnr (j endocrinology 140:10). we previously demonstrated a fall in most components of the placental and fetal baboon liver igf systems with mnr. here we have evaluated fetal pancreatic igf-ii and ins changes in response to mnr. methods: pregnant baboons were fed ad lib (ctr, n=12) or 70% of wt adjusted ctr diet (mnr, n=11) from 0.16 gestation (g) and fetuses were recovered at c-section under general anesthesia at 0.5 (n=9; 5 ctr and 4 mnr) and 0.9g (n=14; 7 ctr and 7 mnr). igf-ii and ins expression were determined by immunohistochemistry (ihc) and quantified by image analysis for fraction (area immunostained/area of the field x 100%) and density. data are expressed as mean + sem; ctr data are expressed] first; comparison made with two tailed t-test. results: at 0.5g there was no difference in igf-ii or insulin fraction or density between groups. at 0.9g, igf-ii fraction (2.57 ± 0.42 vs 1.44±0.27,p<0.05) and density (1.02x10 7 ±1.90 x10 6 vs 5.19x10 6 ±9.50x10 5 , p<0.05) and insulin fraction (2.51 ± 0.25 vs 1.58 ± 0.05, p=0.05) were reduced. conclusion: moderate mnr decreases abundance of fetal pancreatic igf-ii and ins at 0.9g. in the fetal baboon and supports the extant evidence for impaired pancreatic development with mnr seen in rodents. maintenance of liver growth in the hypoxic growth restricted fetal sheep: a role for intrahepatic glut1? sheridan gentili, janna l morrison, i caroline mcmillen. sansom institute, unisa, adelaide, south australia, australia. objective: we have previously demonstrated that there is a differential tissue response to chronic placental and fetal growth restriction. growth of fetal tissues such as the brain and the adrenal are consistently spared in the face of chronic substrate restriction, whilst we have demonstrated that the growth of the fetal liver may be either maintained or reduced. it is unclear whether the growth response of the fetal liver to hypoxia and hypoglycemia are determined by intrahepatic metabolic adaptations. hypothesis: we hypothesize that there will be a differential profile of hepatic expression of glut1, 11 hsd1, the gluconeogenic and glycolytic enzymes pepck and g3pdh and the transcription factors pgc1 and ppar in animals in which liver growth is maintained or reduced. methods: carunclectomy was performed in 19 non-pregnant ewes to induce placental restriction (pr). vascular catheters were inserted in 19 pr and 9 control (c) fetuses at 103-117d and arterial blood samples were collected for blood gas analysis. mean gestation po 2 <17mmhg was defined as hypoxic (h; normoxia, n). post mortem was performed at 140-145d. hepatic mrna expression of glut1, 11 hsd1, pepck, g3pdh, pgc1 and ppar was determined using qrt-pcr. results: four experimental groups were defined by fetal po 2 and liver growth (c-n, pr-n, pr-h and pr-h-reduced liver growth). fetal weight correlated with mean gestational po 2 (r 2 =0.67, y=0.21x+0.5, p<0.01). liver weight was significantly lower in a cohort of pr-h fetuses (c, 24.1±1.6; pr-n 22.9±0.9; pr-h 22.0±1.6; pr-h-rlg 15.8±0.3g:kg; p<0.05). glut1 expression was highest in those pr-h fetuses in which liver growth was maintained, whilst the expression of 11 hsd1, pgc1, ppar and pepck was highest in the pr-h fetuses in which liver growth was reduced (p<0.05). conclusions: in the pr-h fetuses in which liver growth was reduced, the increase in 11 hsd1 expression may be associated with an increase in hepatic exposure to cortisol and an associated increase in pepck, pgc1 and ppar . interestingly the compensatory increase in hepatic glut1 expression did not occur in fetuses in which liver growth was reduced. predicting the trajectory of fetal growth. racine n edwards-silva, 1 jeffrey gornbein, 2 calvin j hobel. 3 1 obstetrics gynecology, los angeles, ca, usa; 2 biomathematics, david geffen school of medicine at university of california, los angeles, ca, usa; 3 obstetrics gynecology, david geffen school of medicine at university of california, los angeles, ca, usa. objective: to evaluate twelve potential predictors of fetal growth trajectory defined as the rate of fetal weight change over time. study design: a longitudinal prospective study of singleton fetal growth trajectory. the twelve potential predictors considered were: fetal gender, gestational age, parity, race, bmi, age, weight at 1 st clinical exam, cumulative weight gain at each exam, smoking, and alcohol use. estimated fetal weight was computed using the hadlock formula and 4 sonographic fetal biometric parameters at 18-20 weeks, 28-30 weeks, and 34-36 weeks. the rate of change in fetal weight was defined as 60 x (fetal wt at exam j -fetal wt at exam i )(j > i)/ (gestational age at exam j -gestational age at exam i ). bivariate statistical analysis included the non-parametric spearman rank correlation and wilcoxon rank sum test. all factors were assessed multivariately using multiple linear regression. results: there were 454 multi-ethnic women included in the study, after 112 were excluded. they underwent a total of 1,277 exams. fetal gender (p=0.0048), maternal weight at 1 st exam (p=0.0055), and cumulative maternal weight gain at exam 3 (p < 0.001) were significant predictors of fetal growth trajectory. in this model, male fetuses had an average rate of fetal weight change of 46.9 grams per 60 days higher than females. the rate of fetal weight change increased by an average of 8.8 grams per 60 days for each 10 lb increase in maternal weight at the 1 st exam. the fetal growth rate increased 2.2 grams per 60 days for each 1 lb of cumulative weight gain at the 3 rd exam. conclusions: in this study, maternal weight at the 1 st exam and cumulative maternal weight gain were the significant determinants of fetal growth trajectory. adequate initial maternal weight and cumulative gestational weight gains probably ensure sufficient nourishment for normal placental growth, uteroplacental blood flow, and fetal nutrient uptake. this supports the emphasis on periconceptual nutritional counseling for all pregnant women. the implication of this study is that similar to fetal programming of adult diseases, there is nutritional programming of fetal growth trajectory. 216 high risk patients. other predictors include the known risk factors of age <15 and >40, single, tobacco/alcohol use, and african american race. interestingly, hispanic and native american patients have a lower lbw rate compared to other groups. introduction: catecholamines released by the sympathetic nervous system and adrenal medulla act via b-ars to regulate glucose and insulin function in liver, pancreas, adipose and muscle tissue. b-ar knock out mice show increased fat mass and glucose intolerance (asenslo et al.,diabetes,2005) . mnr animal models have increased sympathetic activity. we, therefore, evaluated effects of mnr on baboon fetal liver b1-ar. methods: baboons were fed as ad lib controls (ctr) or 70% of wt adjusted ctr diet (mnr) from 0.16 gestation(g) with fetuses retrieved at c-section under general anesthesia at 0.5 or 0.9g. protein expression determined by immunohistochemistry for b1-ar in the central liver lobule was quantified by image anaysis and expressed as fraction = area immuno-stained/area of the field x 100%. all data are expressed as mean + sem with ctr data presented first. liver glycogen expression was determined by the periodic accid schiiff (pas) method. comparisions were made with student's t-test with alpha level set at 0.05. results: fetal body and liver wts were not changed by mnr at either age. pas stained liver glycogen at 0.5g = 197.5 ± 1.7 vs. 162.8 ± 11.7%, p< 0.05 . b1-ar fraction was lower following mnr at 0.5g and at 0.9g (p< 0.05; fig 1) conclusions: mnr decreased b1-ar over 50% at 0.5g and 20% at 0.9g. decreased fetal liver b1-ar in mnr alters glucose metabolisam and may result in reduced lipolysis predisposing to fatty liver. infection with noncytopathic bovine viral diarrhea virus (ncpbvdv) during early bovine pregnancy (<150d gestation) results in fetal immunotolerance, persistent infection (pi) and intrauterine growth restriction (iugr). in contrast, infection after the development of adaptive immune competence (>150d gestation or postnatal) results in a transient infection (ti). we have previously reported an iugr in pi fetuses presenting as decreased body weight and ponderal index. a growth defect can be the result of many factors, including placental insufficiency and nutrient restriction, however it was hypothesized that the iugr seen in bvdv pi fetuses may be an immunopathological effect caused by the persisting virus. our two part experimental design examined the relationship between bvdv and its pi host. in experiment (exp) 1, blood cell mrna was collected from pi steers (n=2; confirmed by virus isolation), or uninfected control steers (n=3) and used to identify differentially expressed genes using microarray (affymetrix) and qtrt-pcr approaches. in exp2, bvdv naïve pregnant heifers (n=6 per group) were not infected (control) or infected with ncpbvdv on d.75 or d.175 of pregnancy creating pi and ti fetuses, respectively. fetuses were collected by c-section on d.190; infection was confirmed by elisa and qtrt-pcr. histology of placental tissue revealed no placentitis or pathology, and glucose and lactate levels in fetal serum were normal. microarray analysis revealed 294 genes that were differentially regulated in pi vs. controls (p<0.05, >1.5 fold). qtrt-pcr of steer and fetal blood revealed a significant upregulation of activators and products of the antiviral type-i interferon (ifn-i) pathway. as ifn-i can act as a growthsuppressive cytokine, a long-term upregulation may contribute to the iugr seen in persistent bvdv infection and in other viral infections observed during pregnancy. nricg 2006-03907 from the csrees. patients with a short cervix are at an increased risk for spontaneous preterm delivery. therefore, it is possible that women with a short cervix during pregnancy are also at risk to deliver an sga neonate. this study was conducted to address this question. study design: patients >14 weeks of gestation were prospectively enrolled into an observational study (08/2002 to 06/2007). transvaginal sonographic examinations were performed every 2 weeks until delivery. the shortest cervical length between 14-32 weeks was used for analysis. sga was defined as less than the tenth percentile of birth weight. results: 303 asymptomatic patients were studied. 19.8% of patients delivered an sga neonate (60/303). the median cervical length was 21 mm (0 to 55mm); 184 patients had a cervical length <25mm. of these, 17% (32/184) delivered an sga neonate. similarly, 17% (17/98) patients with a cervical length <15mm had an sga neonate. no relationship was found between a short cervix and sga (short cervix was defined as either < 25mm and <15 mm). the frequency of sga was not significantly different between women with a short cervix and those with a long cervix [17% (32/184) vs. 24% (28/119); p=0.34]. newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced background folate is an essential micronutrient for cellular growth. recommendations on periconceptional folic acid use are mainly focussed on prevention of neural tube defects, despite growing evidence that folic acid use may have positive effects on birth weight. objective to examine associations between folic acid use, intrauterine fetal growth and birth weight. design the study was embedded in the generation r study in rotterdam, the netherlands, a population-based prospective cohort study from early pregnancy onwards. methods information on folic acid use was obtained by questionnaires and categorized into three groups: 1) preconception start of folic acid use; 2) start of folic acid use in first ten weeks of gestation; 3) no folic acid use at all. fetal growth measurements included head circumference, abdominal circumference and femur length measured in mid-and late pregnancy, i.e., gestational age 18-25 and >25 weeks, respectively, and birth weight. fetal weight in mid-and late pregnancy was estimated using the haddock method. results data from 6,719 pregnant women were available. overall, folic acid use was positively associated with fetal growth. preconceptional folic acid use resulted in an increased growth of 6 grams (95%ci 2.92-9.01, p<0.001) per week from late pregnancy to birth, compared to no folic acid use. similarly, start of folic acid use in the first ten weeks of gestation resulted in an increased growth of 5 grams (95%ci 2.19-7.88, p=0.001) per week from late pregnancy to birth. both preconceptional folic acid use and folic acid use started in the first ten weeks of gestation resulted in higher birth weights of 69 grams (95%ci 39-99) and 51 grams (95%ci 23-80), respectively, compared to no folic acid use. a tendency was found for an increased risk of birth weight less than 2500 grams when folic acid was not started preconceptionally (or 1.5, 95%ci 0.9-2.4). conclusion periconceptional folic acid use is significantly associated with increased fetal growth resulting in a higher birth weight. ductus venosus isovolumetric relaxation in severely premature growth-restricted fetuses. jason l picconi, katherine drennan, farhan hanif, michael kruger, giancarlo mari. obstetrics and gynecology, wayne state university/dmc, detroit, mi, usa. objective: ductus venosus (dv) doppler waveforms are characterized by two periods in which blood velocity decreases. the first represents the isovolumetric relaxation (ir) at the end of ventricular systole and the second represents atrial contraction at the end of ventricular diastole (a). ductus venosus reversed flow (dvrf) occurring at the time of the a-wave is considered a risk factor for intrauterine fetal demise (iufd). we have previously reported that absent or reversed a-wave flow can be present for weeks before iufd occurs or delivery is performed for non-reassuring fetal testing. the guiding hypothesis for this study is that decreased flow at the time of ir in combination with absent or reversed a-wave flow allows a more accurate prediction of fetal outcome than a-wave absent or reversed flow alone. material and methods: ductus venosus doppler was serially studied in 17 severely premature iugr fetuses (estimated fetal weight < 10 th percentile and umbilical artery pulsatility index > 95 th percentile) from diagnosis until demise or delivery. ductus venosus waveforms were assessed quantitatively for peak systolic velocity (psv), isovolumetric relaxation velocity (irv), and end diastolic velocity (edv). the psv/irv + edv were compared to fetal and neonatal outcome. a kruskal-wallis one way anova, mann whitney u post-hoc test, and a roc, were used for statistical analysis. a p < .05 was considered statistically significant. results: all fetuses were delivered at < 32 weeks. six cases resulted in iufd, five cases resulted in neonatal demise (nd), and six cases resulted in neonatal survival (ns) at the time of discharge from the hospital. the psv/irv+edv correlated better than a-wave reversal of flow with perinatal outcome. a psv/irv+edv score less than -1.8 resulted in iufd, whereas a score greater than -1.8 resulted in live birth. live births segregated based on estimated gestational age, where those fetuses at less than 28 weeks resulted in nd and those fetuses at or greater than 28 weeks resulted in ns. all results were statistically significant. conclusions: the isovolumetric relaxation velocity is a novel doppler parameter in the assessment of severely premature iugr fetuses. these data indicate that assessment of irv should be considered part of the evaluation of severely iugr fetuses. background: fetal growth restriction has been linked to an increased incidence of chronic hypertension, which may be the result of extracellular matrix changes (ecm) within the vascular tree. in previous studies, ecm changes were observed in the umbilical arteries of preterm growth restricted infants. objective: to determine if there are alterations in collagen subtypes within the umbilical cords from growth restricted fetal rat pups after a period of maternal nutrient restriction. methods: timed pregnant sprague-dawley rats were fed either a 50% food restricted diet (mfr; n=5) or were fed ad libidum (control; n=5) from d10 until d21 of gestation. litter size, fetal weights and placental weights were then noted and umbilical cords from 3 randomly selected pups in each litter were snap frozen. gene expression for collagens i, iii, xiv and decorin was evaluated by real-time rt-pcr with normalization to the gadph housekeeping gene. data were analyzed from fitting general linear regression models with estimation based on the quasi-likelihood estimation (generalized estimating equations) to account for clustering of responses within litters. data are shown as cycles to amplification (ct), which is inversely proportional to mrna levels. results: no difference in median litter size was detected. however, fetal and placental weights in the mfr group were significantly less than those from control dams. no significant differences in umbilical cord gene expression for collagens i, iii, xiv or decorin were detected between mfr and control pups. conclusions: maternal food restriction does not result in any detectable alterations in collagen or decorin expression within the intact umbilical cord, despite causing significant reductions in fetal growth. control (n=5) p-value litter size (median, range) 11 (8,15) 12 (9,15) 0.59 fetal weight (mean ± sd) g 3.29 ± 0.12 4.0 ± 0.1 <0.001 placental weight (mean ± sd) g 0.73 ± 0.05 0.90 ± 0.03 0.007 collagen i (mean ± sem) ct 17.9 ± 0.13 18. we have previously demonstrated that the restriction of placental and fetal growth results in fetal hypoxia and fetal brain sparing suggesting a redistribution of cardiac output. furthermore, placentally restricted (pr) hypoxic fetuses are more dependent on their sympathetic nervous system for the maintenance of blood pressure during late gestation. nerve growth factor (ngf) plays a significant role in sympathetic innervation. hypothesis: we hypothesize that the expression of ngf will be higher in the aorta and femoral artery of the pr hypoxic compared to the control fetus. method: carunclectomy was performed in 6 non-pregnant ewes to induce pr. vascular catheters were inserted in 6 pr and 4 control (c) fetuses at 103-117d and arterial blood samples were collected for blood gas analysis. all pr fetuses introduction elevated sflt-1 levels have been shown to be a feature of pre-eclampsia and is considered to play a significant role in the pathogenesis of the condition. the role of angiogenic factors in fetal growth restriction has not been as well established. this study evaluated the levels of circulating vascular endothelial growth factor (vegf) and its soluble receptor sflt-1, in normal pregnancies and isolated placental vascular disease without evidence of pre-eclampsia. method 42 maternal peripheral venous samples were collected antenatally from two groups of pregnant women between 25-40 weeks of gestation. group a: uncomplicated normal pregnancies (n = 20) and group b: pregnancies complicated by isolated placental vascular disease( n = 22) as defined by birth weight less than 10 th centile for gestation and umbilical artery doppler s:d ratio above the 95 th centile for gestation, with no evidence of maternal preeclampsia or pre-existing hypertension. plasma vegf and sflt-1 levels were measured using standard eliza techniques. comparison between groups were performed by using one way analysis of variance. the maternal plasma sflt-1 levels (figure1) in group a: normal pregnancies increased with gestation (p < 0.001). the sflt-1 levels in group b: isolated placental vascular disease were significantly higher throughout all gestations (p = 0.002) and did not show a significant variation with gestation ( p = 0.47). the plasma vegf levels were below the detectable levels of the assay in all samples except 3 normal pregnancies. the significantly increased maternal plasma sflt-1 levels in established isolated placental vascular disease without evidence of pre-eclampsia suggest a disease of placental origin. the dysregulation of angiogenic factors may be part of a repair and regeneration process in the placenta. objectives: production of 5 -reduced neurosteroids is critical for reducing fetal vulnerability to stressors in pregnancy and inhibition of 5 -reductases (5 r) increase acute hypoxia-induced apoptosis in the fetal brain (yawno et al 2007 neurosci 146:1726 . we have developed a model of placental insufficiency that results in fetal growth restriction (gr) in the guinea pig (palliser et al 2007 repro sci 14 #381). the aim of the present study was to determine the effects of suppression of 5 -reduced steroid synthesis on apoptosis in vulnerable regions of the fetal brain and on neurosteroid synthetic enzymes in pregnancies compromised by chronic placental insufficiency. methods: placental insufficiency was induced in guinea pig dams by surgical ablation of uterine artery branches at mid gestation (term 68d). sham operated or gr dams received finasteride (a 5 r inhibitor; 25mg/kg/day) during late gestation (55d until term). activated caspase-3, a marker of apoptotic cell death, was measured by immunohistochemistry and steroidogenic enzymes, 5 r1 and cytochrome p450 side chain cleavage (p450scc) were measured by real time pcr and western blotting in fetuses (65d) and neonates 24h after birth. results: placental insufficiency significantly reduced fetal body and organ weight by 30% whilst sparing brain weight. the number of activated caspase-3 positive cells was significantly increased in the fetal hippocampus of gr fetuses and further increased in the cortex of gr fetuses receiving finasteride. the neonatal brain also exhibited changes in caspase-3 activation following gr and finasteride treatment. the fetal adrenal and the placenta responded to the compromise with increased expression of p450scc mrna and 5 r1 protein, respectively. conclusion: the combination of placental insufficiency and suppressed neurosteroid system leads to markedly increased apoptotic cell death in the fetal brain which continues to affect the neonatal brain. the fetus responds to these conditions by increasing steroid synthetic enzyme expression in the placenta and adrenal glands suggestive of a possible neuroprotective feedback process. to study the effect of smoking by mothers on the fetal and neonatal brain using non-invasive magnetoencephalography technique (meg). materials and methods: using fetal magnetoencephalography, cortical auditory evoked responses (aer) were measured from 21 fetuses ranging from 27 to 39 weeks gestational age for a total of 69 recordings. 29 measurements were taken from 10 mothers with a history of smoking (sm) and 40 from 11 mothers with no smoking history (ns). after delivery, five sm and five ns newborns had meg aer measurements twice for a total of 20 recordings. aer was quantified by cross-correlation analysis and its significance was assessed by boot-strap technique. results: aers were detectable in 35 out of 40 fetal recordings in the ns group and 23 of 29 in the sm group. the neonatal response rate was 90% for each group. the latencies were divided into three components: c1 (100-300 ms), c2 (301-600 ms) and c3 (601-900 ms). in both fetuses and neonates as well, there was a statistically significant difference (p<0.05) between the two groups in the c2-component and the sm group showed faster aer compared to the lr group. conclusion: meg technique provides a non-invasive approach to study the effects of smoking on developing fetal and neonatal brain. the observed decrease in the latency of fetuses and neonates of the smoking mothers could indicate a hypersensitive cortical response to auditory tone. adenosine (ado) modulates metabolism in adult mammals through multiple mechanisms that involve ado a1 and a2a receptors. objective: this study was designed to test the hypothesis that ado a1 and a2a receptors participate in fetal metabolic homeostasis. methods: experiments were performed in chronically catheterized fetal sheep (>0.8 term). intravascular infusion for 1 h of dpcpx (a1 receptor antagonist) or zm241385 (zm, a2a receptor antagonist) was performed alone or in concert with ado administration. the highly selective ado receptor antagonists were also infused in fetuses in which hypoxia was induced for 1 h by having the ewe breathe a hypoxic gas mixture (fio2 = 0.10). data were analyzed by two-way repeated measures of anova. results: blockade of ado a1 receptors (n=8) increased significantly (p < 0.05) fetal concentrations of glucose [control (c): 15.6 ± 1.0 (se)]; experiment (e): 17.38 ± 1.1 mg/dl] and lactate (c: 16.5 ± 2.4; e: 22.5 ± 4 mg/dl) without significantly altering insulin levels and arterial blood gases or ph. antagonism of ado a2a receptors (n=6) did not affect plasma levels of glucose, lactate, or insulin. intravenous infusion of ado (n=11), which did not alter pao2 or paco2, increased concentrations of glucose (c: 18.0 ± 0.8; e: 22.6± 1.3 mg/dl) and lactate (c: 12.2 ± 1.0; e: 20.4 ± 1.6). zm (n= 7), but not dpcpx (n=9), abolished ado-induced rise in glucose and lactate concentrations. isocapnic hypoxia (pao2 13 torr), which increases (2-3 fold) fetal plasma ado levels to those similar to ado infusion, decreased arterial ph (c: 7.331 ± 0.016; e: 7.140 ± 0.032), and increased fetal levels of glucose (c: 16.2 ± 1.3; e: 33.7 ± 7.8 mg/dl) and lactate (c: 13.3 ± 1.4; e: 97.3 ± 7.7 mg/dl) without altering changing insulin concentrations. these effects of hypoxia were not altered by dpcpx or zm. conclusions: 1) a1 receptors modulate plasma levels of glucose and lactate in normoxic fetuses; 2) a2a receptors mediate the adoinduced rise in plasma glucose and lactate; and 3) a1 and a2a receptors are not significant modulators of hypoxia-induced changes in plasma levels of glucose and lactate. supported by usphs hd-18478. objective: to investigate the dynamic changes of the relationship between leptin, adiponectin and resistin in maternal and fetal circulation during pregnancy and in the early post-natal period. materials and methods: thirty pregnant women with uncomplicated singleton pregnancy delivered at term. maternal and fetal/neonatal venous blood samples were obtained at delivery and at 72 hours from birth. leptin, adiponectin and resistin were measured by specific elisa assays. neonatal anthropometric measurements, glucose metabolism and lipid profile, blood pressure information were obtained. statistical analysis was performed by anova followed by student t test or duncan's test whenever appropriate. correlations were calculated by using the pearson coefficient. results: adipokines concentration at birth and at 72h from birth was showed in table. multivariate regression analysis showed that fetal leptin levels were positively associated with female gender and adiponectin levels, but not with anthropometric characteristics. fetal leptin and adiponectin levels were not correlated with maternal concentration, whereas fetal and maternal resistin levels were. fetal and maternal resistin concentration was positively associated with gestational age and birth weight and fetal resistin levels correlated negatively with lipid profile. after birth leptin concentration in maternal and neonatal circulation decreased dramatically and the correlation between leptin and adiponectin levels was lost. a positive correlation between resistin and leptin concentration in neonatal circulation was found at 72h from birth. conclusions: in contrast to adult life a positive correlation between leptin and adiponectin is present in fetal life. placental secretion of leptin, but not of adiponectin and resistin, contributes significantly to maternal and fetal circulating levels. significant changes in the relationship among adipokines occurred immediately after birth and may affect growth and development in early post-natal period. adipokines concentration in maternal and fetal circulation ) antagonism has been shown to normalize placental perfusion and fetal growth in several rat models of fetal growth restriction. however, direct administration of et a antagonists to newborn rats within 3 hours of delivery has been consistently associated with neonatal demise due to failure of the ductus arteriosus to close, raising concerns about the safety of their use late in pregnancy. perinatal exposure to et a antagonists (maternal administration in late gestation) and its impact on rat pup survival and oxygen saturation has not been investigated. objective: to determine the impact of a maternally administered et a antagonist on oxygen saturation in newborn and 7-day-old rat pups. methods: timed pregnant sprague-dawley rats were treated with fr139317 (12 mg/kg/day; et a antagonist) or 4.2% nahco 3 vehicle, by subcutaneous osmotic pump connected to an intravenous catheter, from gestational day 14 (term=22 days) through parturition. all five pregnant rats in each group delivered spontaneously and nursed their pups through postpartum day 7. oxygen saturation of each rat pup was measured by pulse oximeter on postpartum days 1 and 7. results are presented as means ± se. newborn 7-day neonate vehicle 92.6 ± 0.9 94.4 ± 0.8 eta antagonist 94.7 ± 0.8 91.6 ± 1.1 there were no statistically significant differences between the treatment groups. maternal administration of an et a antagonist from gestational day 14 through parturition, at a dose sufficient to ameliorate fetal growth restriction, has no adverse impact on oxygen saturation in neonatal rat pups. helen l torrance, 1 jan b derks, 1 martijn a oudijk, 1 avnesh s thakor, 2 tereza cindrova-davies, 2 frank van bel, 1 gerard ha visser, 1 graham j burton, 2 dino a giussani. 2 1 perinatal center, university medical center utrecht, netherlands; 2 department of physiology, development neuroscience, university of cambridge, united kingdom. introduction: the management of perinatal asphyxia remains a major concerns in obstetrics. umbilical cord compressions (ucc) induce fetal asphyxia and ischaemia-reperfusion (i/r). i/r increases reactive oxygen species, for instance via activation of the xanthine oxidase (xo) pathway, which may promote oxidative stress in the fetal circulation. while treatment with allopurinol of asphyxic human neonates reduced free radicals and improved cardiovascular status, treatment started postnatally was deemed too late to prevent oxidative damage (benders et al. arch dis child 91:163, 2006) . consequently, in complicated pregnancy, recommendations to treat the fetus via the mother, rather than the neonate, with allopurinol are being entertained today. we investigated the effects of maternal allopurinol treatment on indices of oxidative stress in the fetal heart following repeated ucc in sheep. methods: at 0.8 of gestation,10 surgically instrumented sheep fetuses were submitted to i/r (5 x 10 min repeated ucc) under maternal allopurinol (n=5) or saline vehicle (n=5) infusion. fetal hearts were collected 48h after i/r and snap frozen for measurement of (anti)oxidant proteins by western blot. hearts from 5 uninstrumented fetal sheep at 0.8 gestation served as controls. statistical comparisons were made using one-way anova. results: i/r episodes led to increased expression of cox-2, enos, hsp90 and decreased expression of sod and gluthatione peroxidase (gpx) in the fetal heart, findings consistent with cardiac oxidative stress. maternal treatment with allopurinol ameliorated these effects (fig. 1 objectives: hypoxic-ischemic fetal brain injury (hie) is a major cause of neonatal death and morbidity. evidence suggests that the brain cell injury associated with chronic hpx (in contrast to an acute ischemic reperfusion injury) is a complex process reflecting a series of adaptive intracellular events that are duration and gestational age dependent. we applied advanced proteomic tools as a next step toward ascertaining a more complete understanding of the impact of hpx on the fetal brain proteome. methods: time-mated guinea pigs were housed in a chamber beginning on day 50 for 14d, breathing either room air (nmx), or 10.5% or 12% o 2 (12% o 2 hpx or 10.5% o 2 hpx). on day 65 (term), the fetal brains were removed and preserved for study. total protein was extracted, and the proteome first characterized by 2d gel electrophoresis. the density of the resulting protein spots were acquired and analyzed using the gs800 densitometer and pdquest software. identified spots of interest were trypsin digested and subject to maldi mass spectrometry using the 4700 proteomics analyzer mass spectrometer for peptide mapping or sequencing. the hpx-induced protein spots were identified based on a minimum of a 2x change from nmx. results: hpx had a clear effect on the fetal brain proteome. superoxide dismutase (sod), heat shock protein 70 (hsp70), actin (actg1), interleukin-1 (il-1), and glutamine synthetase (gs) were each up regulated, while cofilin-1, brain-type creatine kinase (bb-ck), and -1 gtp binding protein were each down regulated. all protein changes were proportional to the hpx (12% o 2 hpx vs nmx, 12% o 2 hpx vs 10.5% o 2 hpx, p<0.05). conclusions: this initial application of proteomic techniques confirms that fetal brain damage secondary to chronic hpx (in contrast to acute hpx) is a complex processes characterized by fetal adaptations mediated by multiple protein activations and inactivations. while sod, il-1, and gs have each been previously investigated, the potential roles of actg1, bb-ck, beta-1 gtp binding protein, and cofilin-1 in the fetal response to hpx and the associated brain damage are unclear and represent strong candidates for future investigation. acknowlegement: this study was supported by grants from the phs (r01 hl049041-12, cpw) and cdc grant (u7dp00187-03, cpw). intermittent umbilical cord occlusion occurs in 5% of human pregnancies.given that elastogenesis within the vascular wall is in part mediated by hemodynamic conditions during development, the blood pressure response to acute hypoxic insults such as cord occlusion may alter arterial composition. elastin content of a central and peripheral artery and blood pressure responses in fetal sheep exposed to varying degrees of cord occlusion were determined. methods: over a 4 day period, near term fetal sheep received total umbilical cord occlusion (uco) lasting 1 min/ hour (mild group; n=5), 2 min/hour (moderate group; n=4), 3 min/hour (severe group; n = 6) or no occlusion (control group; n=7). fetal arterial blood samples were drawn 5 min prior to and at the end of cord occlusions. mean arterial pressure (map) was monitored continuously. the carotid and superior mesenteric arteries were excised and a colorimetric assay (biocolor) performed for determination of elastin content. results are presented as mean ± sem. results:umbilical cord occlusions produced decreases in fetal arterial oxygen pressure and oxygen saturation that were progressively more pronounced across mild, moderate and severe uco groups (p < .01). lactate concentration rose during occlusions in the moderate and severe groups, but not the mild group (p < .01).elastin content of the superior mesenteric artery did not differ between the 3 experimental groups and the control group. elastin content of the carotid artery and map response elastin content (μg/mg tissue) max ∆ in map duration of rise in map (min) control 5.7 ± 0.4 4.0 ± 1.7 -mild 7.0 ± 0.7 21.06 ± 2.5 ** 10.39 ± 0.6 moderate 7.4 ± 0.7 32.4 ± 1.6 ** † 14.3 ± 0.5 † severe 9.5 ± 1.0 * 34.5 ± 0.6 ** † † 17.4 ± 1.2 † † * p < .05; ** p < .01: experimental groups compared to control † p < .05; † † p < .01: compared to mild group the max in map was positively correlated with elastin content (r 2 = .56, p < .05). conclusions:the transient rise in blood pressure and preferential blood flow to the brain that occur in response to acute hypoxemia during severe intermittent umbilical cord occlusion induce an increase in elastin synthesis in the carotid artery.this may give rise to adaptive programming of postnatal central arterial compliance. electrocortical activity during repetitive umbilical cord occlusions (uco) with worsening acidemia in the ovine fetus near term. martin g frasch, 1 roy mansano, 2 michael g ross, 2 robert gagnon, 1 bryan s richardson. 1 1 obgyn, chri, univ of western ontario, london, canada; 2 obgyn, harbor-ucla med ctr, torrance, ca. objective: uterine contractions during labour can restrict umbilical blood flow compromising fetal oxygenation and leading to adverse neonatal outcome including newborn encephalopathy/subsequent cerebral palsy. while electronic fetal heart rate (fhr) monitoring is widely used for assessment during labour, abnormal fhr patterns as used clinically have a poor positive predictive value for concerning/significant acidosis at birth. there is limited study of fetal electrocortical activity (ecog) in animal models with induced hypoxia/ acidemia as might be seen during labour. thus, we aimed to induce repetitive ucos in fetal sheep leading to worsening acidemia to determine the predictive value of ecog activity for fetal compromise. methods: near-term fetal sheep (n=10) underwent chronic preparation with artery catheters, ecg/ecog electrodes and placement of an inflatable umbilical cord occluder. following a baseline recording period, fetuses underwent a series of mild (1min every 5min), moderate (1min every 3min) and severe (1min every 2min) ucos with each series lasting 1h or until fetal arterial ph decreased to <7.0. fetal arterial blood samples for blood gases and ph were taken at selected time points during the baseline, ucos, and recovery periods. arterial blood pressure (abp) and fhr were continuously monitored and spectral edge frequency (sef) was calculated from ecog. correlation of sef with fhr change was calculated for time intervals using sef maxima and fhr minima during uco series. data are presented as means±sem. results: repetitive ucos led to development of a marked acidosis (ph 7.36±0.03 to 6.91±0.12, p<0.001). 52±13 min prior to fetal ph drop <7.0, the sef of ecog began to increase abruptly during each fhr deceleration from 3±1 hz up to 23±2 hz (p<0.001) and was correlated to both fhr change and a decrease in fetal abp during each fhr deceleration at this time (p<0.001). conclusion: our findings suggest that in the animal model studied (1) fetal ecog activity is impaired with progressive acidemia accompanied by fhr decelerations and pathological abp decreases; (2) there is a consistent temporal relationship between the occurrence of ecog alterations and the subsequent critical drop of ph <7.0. these findings could contribute to improvement in the clinical ability to predict fetal compromise during labour using ecog/fhr monitoring. background: the ductus arteriosus (da) plays a pivotal role in fetal development and circulation. during gestation, patency of the fetal da is maintained by nitric oxide (no) and prostaglandins (pg). no and pgs are downstream effectors of estrogen (e2) and progesterone (p4). however, the roles of e2 and p4 in da regulation have not been studied. objective: we hypothesized that: e2 and p4 have opposite effects in the da; that rising e2 and falling p4 levels help trigger da closure via specific hormone receptors; and that no and/or pgs mediate da responses to e2 and p4. methods: expression of er , er , pra, prb, and the putative membrane receptors mpr-, -, -, pgrmc-1, -2, and serbp-1 was examined by rt-pcr and qpcr on days 15, 17, 19 (term), and p1. the effects of e2 and p4 (10 -9 -10 -4 m) on the d19 fetal da were examined in a cannulated microvessel myography system. e2 and p4 effects were also studied in the presence of receptor antagonists (ici 182780, ru486) and no and pg inhibitors (l-name, indomethacin). results: er and pr receptors were expressed at low levels; pgrmc-2 expression was stronger than other membrane prs, and increased with advancing gestation. under fetal o2 conditions, e2 induced rapid, concentrationdependent dilation at 10 -6 -10 -4 m (n=11); p4 induced progressive vasodilation at all doses (n=10). e2 and p4-induced dilation occurred within 60-180 seconds of exposure. while e2-dilated das did not respond to ici, ici-pretreated das failed to dilate to the same dose of e2 (n=10) suggesting antagonism of e2 effects. the vasodilatory effects of e2 were partially inhibited by pretreatment with l-name. p4-dilated das did not respond to ru486, whereas ru486pretreated das showed a small, significant response to p4 (n=10), suggesting partial antagonism or signaling via alternative, ru486-independent pathways. pre-treatment with indocin did not block the vasodilatory effects of p4. conclusions: contrary to our expectation, both e2 and p4 have dilating effects on the fetal da, via no, pgs and non-no, non-pg pathways. expression studies and the rapid response of ex vivo das are consistent with non-genomic actions via membrane receptors. hormone shifts in parturition may have longterm effects on da preparation for postnatal closure, but strategies to maintain fetal da patency or treat newborns with pda will require better understanding of this process. background. for the fetus, arterial blood gases are critical in the regulation of cerebral blood flow (cbf) and cerebral oxygenation. however, the relation of cbf, cortical tissue po 2 (tpo 2 ), and electrocorticographic (ecog) activity to arterial o 2 tension (pao 2 ) are not well defined. in an effort to elucidate these interrelations, we tested the null hypothesis that in the near-term fetus, acute hypoxic-associated cerebral oxygenation and related variables are not closely associated with ecog state. methods. by use of a laser doppler flowmeter with a fluorescent tissue o 2 probe, and with fluorescent-labeled microspheres, and with ecog electrodes, in near-term fetal sheep (n = 8) we measured laser doppler cbf (ld-cbf), tpo 2 , ecog (root mean square (rms) voltage with high voltage low frequency, hvlf versus low voltage high frequency, lvhf) and spectral edge frequency-90% (sef 90 ) in response to 40 min moderate isocapnic hypoxia. results. ld-cbf, cerebral o 2 delivery, tpo 2 , and several other variables correlated highly with ecog state. in the normoxic control fetus, in association with a shift from hvlf to lvhf ecog activity, tpo 2 decreased briefly to 7±1 from a control value of 10±1 torr; however, as ld-cbf increased 18±5%, and sef 90 increased to14±2 from 7±1%, tpo 2 returned to near normal value. with acute hypoxia (pao 2 = 12±1 torr) when in the lvhf state ld-cbf increased only 23±14%, as opposed to a 52±10% increase when in hvlf ecog state. with this degree of hypoxia, tpo 2 decreased to 3±1 torr, sef 90 remained at 8±2%, and cerebral metabolic rate for o 2 (cmro 2 ) decreased 32±5% (p<0.05). conclusions. for the near-term fetus, normoxia with changes in ecog state was associated with brief periods of decrease in tpo 2 , which were restored quickly by increased ld-cbf. in contrast, acute hypoxia was associated with a significant depression of cortical tpo 2 , cmro 2 , and ecog state, with increased ld-cbf failing to restore cortical tpo 2 . thus, we reject the null hypothesis that in such fetuses, hypoxia demonstrates no compromise in cerebral oxygenation. (supported by usphs hd-03807). releasing hormone and arginine vasopressin in the ovine fetus. charles a ducsay, 1 kanchan m kaushal, 1 malgorzata mlynarczyk, 1 kimberly hyatt, 2 dean a myers. 2 1 ctr. for perinatal biol., loma linda univ., loma linda, ca; 2 ob/gyn, univ. oklahoma hlth. sci. ctr., oklahoma city, ok. background: long term hypoxia (lth) profoundly affects the hypothalamicpituitary-adrenal axis of the fetal sheep. we previously showed that lth causes augmented corticotrope function. the present study was designed to test the hypothesis that lth enhances sensitivity to the acth secretagogues; cortiocotrophic releasing hormone (crh) and arginine vasopressin (avp) resulting in increased anterior pituitary corticotrope secretion of acth. methods: pregnant ewes were maintained at high altitude (3,820 m) from day 40 to 130-131 of gestation (dg), when they were returned to the lab and a maternal tracheal catheter was implanted. maternal po 2 was maintained at a level comparable to that observed at altitude ( 60 mmhg) by nitrogen infusion. on 132 dg, lth (n = 4) and age-matched, normoxic control (n = 4) fetuses were implantated with vascular catheters. each fetus received a 15 min infusion of either saline vehicle, 100 ng/kg of ovine crh or 20 ng/kg of avp (estimated body weight/min) in a randomized order over 3 consecutive days (137-139dg). blood samples were collected at 0 min (baseline prior to infusion), 15, 30, 60 and 90 min following the start of the infusion and analyzed for acth, as well as the acth precursors pro-opiomelanocortin and the major processing intermediate 22 kda proacth. results: vehicle had no effect on any of the measured parameters. with crh infusion, acth (pg/ml) increased in both groups over the course of the study. however, peak concentrations (at 90 min) were significantly higher in the lth group compared to control (240±17 vs. 110±14, respectively; p<0.01). acth precursor secretion (pm) was greater in lth fetuses compared to controls during the experiment (p<0.01). in response to avp, peak acth concentrations were also higher in the lth fetuses compared to control (209±51 vs. 67±20 respectively; p<0.01), however peak levels were reached at between 15 and 30 min after start of infusion with levels in both groups returning to pre-infusion values. a similar pattern was observed with precursor levels (102.1±27.9 vs. 66.1±10.2, p<0.05, lth vs. control). conclusions: lth significantly increases pituitary sensitivity to both crh and avp. this enhanced sensitivity may be mechanism of our previously observed enhanced corticotrope function. (supported my nih grants hd33147 and hd31226). martin g frasch, 1 roy mansano, 2 michael g ross, 2 robert gagnon, 1 bryan s richardson. 1 1 ob/gyn, chri, university of western ontario, london, canada; 2 ob/gyn, harbor-ucla med. ctr., torrance, ca. objective: repetitive uco leading to worsening fetal acidosis with fetal heart rate (fhr) decelerations (fhr dec ) are accompanied by pathological decreases of fetal arterial blood pressure (bp). we hypothesized this bp change may be caused by the bjr, a vagally mediated reflex with bradycardia and hypotension to reduce cardiac workload, via stimulation of cardiac chemoreceptors during systemic acidemia. methods: ten near-term fetal sheep (125 ± 2dga) underwent chronic preparation with brachial artery catheters and placement of an inflatable umbilical cord occluder. after a control period, fetuses underwent a series of mild (1 min every 5 min), moderate (1 min every 3 min) and severe (1 min every 2 min) uco each lasting 1 h or until ph decreased to < 7.0. fetal arterial blood samples were taken at selected time points during the control and uco periods. bp and fhr were continuously monitored. individual fhr nadir during each fhr dec and accompanying bp change ( bp = [bp at the time of a fhr nadir] -[bp at baseline preceding a uco series]) were determined during all uco. data are presented as means±sem. results: control period ph (7.36 ± 0.03), fhr (163 ± 5 bpm) and bp (45 ± 4 mmhg) were within the physiological range. average depth of fhr dec for all uco was 93 ± 11 bpm and increased with higher lactate concentrations (r = 0.40, p < 0.05) and lower ph (r = 0.34, p = 0.08). bp during all uco demonstrated an initial hypertensive response to fhr dec , which decreased with lower ph (r = 0.49, p < 0.05). bp increased 11 ± 2 mmhg (p < 0.05) during mild uco (ph to 7.32 ± 0.03), 4 ± 3 mmhg during moderate uco (ph to 7.19 ± 0.04, p < 0.05) and 2 ± 4 mmhg during severe uco (ph to 6.91 ± 0.12, p < 0.05). conclusion: these results suggest that bjr, a short-acting, ph dependent depressor reflex, blunts the physiologic hypertensive response to cord occlusion insults leading to worsening acidemia as might be seen in the human fetus during labour with repetitive variable fhr dec . the failure to increase bp may prevent optimal blood flow distribution responses necessary for preservation of vital organs. role of nos and pde5 in placental dysfunction following fetal bypass. mitali basu, 1 r scott baker, 1 christopher t lam, 1 kenneth e clark, 2 pirooz eghtesady. 1 1 cardiothoracic surgery, cincinnati children's hospital, cincinnati, oh, usa; 2 ob/gyn, university of cincinnati, cincinnati, oh, usa. introduction rising placental vascular resistance following fetal cardiopulmonary bypass (bypass) remains the achilles' heel of fetal cardiac surgery. we have previously shown in real-time that nitric oxide (no) production rises during bypass and falls post-bypass, while cgmp levels rise throughout. using immunohistochemical and western analysis of placenta, we examined the involvement no pathway components in this placental vascular pathophysiology. ovine fetuses at 100-110 days gestation were placed on bypass for 30 minutes and followed post-bypass for 2 hours. placental samples were collected immediately prior to bypass and at 30 and 120 min post-bypass (n=7) and compared to a group of similarly instrumented controls, (n=6). placental enos, inos and pde5 protein expression was measured using standard methods and relative expression normalized to beta-actin. statistical analysis utilized students t-test, and anova for trend and group-wise analysis, (significance at p=0.05). pre-bypass protein expression did not differ between groups. pde5 protein levels and phosphorylated pde-5 expression were both elevated 30 min post bypass, and reduced 120 min post bypass compared to sham, (p=0.01). enos levels in the bypass group increased linearly from pre-bypass to 120 min postbypass (p<0.01), and were also elevated compared with shams (p<0.01), while shams had declined significantly by 120 min post bypass, (p<0.01). similarly, phosphorylated enos expression in the bypass group increased linearly from pre-bypass to 120 min post-bypass, (p=0.058), while shams trended towards decline by 120 min post-bypass. simultaneously, placental inos expression remained stable within groups, but was lower in the bypass group at 30 and 120 min post-bypass, (p<0.04). the preceding data correlated with observed immunohistological changes in the same placental cotyledons. fetal bypass leads to significant increases in placental protein levels of pde5, phosphorylated pde-5, enos and ostensibly phosphorylated enos. increased pde5 expression may be a response to increased no and the generated cgmp. this data suggests a compensatory upregulation of pde5 and enos that eventually fails, leading to increasing placental vascular resistance and subsequent lethal placental dysfunction. angiotensin receptors (rat-ii) in neuronal nuclei of the brainstem have been implicated in integration of baroreceptor responses. in near term fetal sheep, we have previously shown that 5 days of mild chronic hypoxemia increases heart rate (hr) and blood pressure (bp). the aim of the present study was to investigate the effects of chronic mild hypoxemia on the expression of rat-ii in the medulla oblongata and on baroreflex control of the circulation. methods: at 125 days of gestational age, pregnant sheep were submitted to 5 days of hypoxemia (75% of fetal arterial po 2 ; at day 5 fetus 14±1 vs 20±0.5; mother 67±5 vs 116±3 mmhg). hr and arterial bp were continuously recorded from mother and fetus in control (n=10) and hypoxemic (n=10) animals. baroreflex sensitivity (brs) as well as hr and diastolic bp variability were analyzed (nevrokard software). brainstem was collected and rat-ii expression in the nucleus tractus solitarius (nts) and dorsal motor nucleus of the vagus (dmnx) was measured by autoradiography using 125 i-sarthran labeling. data are shown as mean±sem and were analyzed by t-test. results: hypoxemia induced a greater total binding of 125 i-sarthran in nts and dmnx resulting from an increased expression of at1 receptors. in the time-domain analysis of brs hypoxemia induces lower baroreflex sensitivity in the fetus (brs in ms/mmhg; 8.5±0.6 vs. 11.5±1.2, p<0.05). in the frequency domain, hypoxemia changes the relative power of low frequencies (lf: 0.04-0.15 hz) and high frequencies (hf: 0.15-0.4 hz) of rri and dbp with an increased value of the lf/hf ratio (rri lf/hf 3.3±0.4 vs 2±0.5, p<0.05; dbp lf/hf 2.7±0.3 vs 1.8±0.2, p<0.05). also the alpha index for baroreflex sensitivity is decreased in hypoxemic animals (alpha lf 9.2±1 vs 15.3±2.6, p<0.05; alpha hf 8.5±1.4 vs 19.8±5.3, p<0.05). no differences were observed in maternal variables. conclusions: our results suggest a link between a prenatal insult, alterations in cns receptors and functional alterations of baroreflex responses. a higher sympathetic outflow, suggested by a greater lf/hf ratio, and impaired reflex gain, both possibly mediated by increases in nts rat1, may have potential long-term consequences for the development of hypertension. hd37885;hd047584;hl51952. objective : we evaluated velocity profiles, relative wall distension rate (rwdr), and wall shear rate (wsr) of fetal descending aorta (fda) in uncomplicated singleton gestations. study design: ninety seven uncomplicated singleton fetuses were studied throughout gestation using multigate spectral doppler analysis (msda) working with gasp software. this consists of a personal computer (pc) add-on board including a single high-speed digital signal processor. the analysis of echo-signals backscattered from 256 range cells located along the axis of the interrogating ultrasound (us) beam. post-processing was accomplished using gasp software. statistical analysis consisted of spearman correlation and chi-square test. results: velocity profiles, wall distension, wall shear rate were obtained from fetal descending aorta throughout gestation establishing gestational age specific norms. wdr[%] is highly correlated with gestational age in appropriate growth fetuses (2.5±1.6 , rs=0.51 p<0.01) with linear regression with standardized coefficient of 0.4 (p<0.01). in contrast, wsr (421±97) is unchanged during the first , second and third trimesters (p=0.8).conclusion: we speculate that the relative wdr changes observed during gestation, in normally grown fetuses, may be secondary to adaptive vascular and autonomic responses and the evolving composition of the vessel wall, particularly with respect to elastin. conversely, the mean wsr for the study group was independent and constant throughout the gestation. these findings suggest that there is an increase in the diameter of the fetal aorta, which provides adaptation to the progressive flow demands, while preserving other key hemodynamic parameters. in this study, we have established normative values of rwdr and wsr, which are new rheological parameters that may be useful in distinguishing normal and pathologic hemodynamic states. objective: placental dysfunction is a key barrier to successful fetal cardiopulmonary bypass (cpb) for repair of congenital heart defects in utero. endothelial cells regulate vascular tone during fetal cpb through interactions of vasodilation by nitric oxide (no) and endothelin-1 (et-1)-mediated vasoconstriction. the objective was to determine the time during fetal cpb when endothelial cell-mediated changes occur. methods: human umbilical vein endothelial cells (huvec) were cultured in media containing 10% serum collected from ovine fetuses (n=3) that underwent 30 min of cpb, then were maintained for 120 min. serum was collected before cpb, from pump prime before initiation of cpb, 30 min on cpb, or 30 and 120 min after fetal cpb. control cells were cultured in normal fetal serum. cells were harvested 24 and 48 hr after addition of fetal serum. no production was measured in real time with an electrochemical detection system (inno-t, harvard apparatus). et-1 was measured in the culture media by elisa. results: no production by huvec after 24 and 48 hr was stimulated above control levels by fetal serum collected during and up to 120 min after fetal cpb (p< .01). serum collected from fetuses that were surgically instrumented, but not yet subjected to cpb, decreased no levels below controls (p<.01). stimulation of et-1 after 24 and 48 hr of huvec culture peaked with serum collected at 30 min after fetal cpb (p<.01 compared with control), but was elevated above control levels at each collection time point (p<.05, table 1 ). conclusions: fetal cpb releases serum proteins that elevate endothelial cell no and et-1 production during and for at least 120 min after cpb. although the specific regulatory proteins remain to be identified, the no and et-1 pathways share circulating mediators and participate in a feedback loop to modulate vascular tone. to test the hypothesis that hypoxia-induced upregulation of no is linked to cardiac inos expression, a selective inos inhibitor, l-nil (l-n6-(1-iminoethyl)-lysine), was administered in vivo to fetal guinea pigs and no levels measured in fetal hearts. methods: pregnant guinea pigs were exposed to either normal room air (normoxia; 21%o 2 ) or 10.5%o 2 (hypoxia) in a hypoxic chamber for 14 days prior to term (term=65d). l-nil was administered to pregnant normoxic and hypoxic guinea pigs via their drinking water at a dose of 1-2mg/kg/d for 10 days. at 60d gestation, pregnant sows were anesthetized and near-term fetuses removed via hysterotomy. the fetal hearts were excised, weighed, and normalized to their respective fetal body weights. left cardiac ventricles were obtained and frozen in liquid n 2 and stored at -80 o c until ready for analysis. the effect of total no product (no 2 and no 3 -, nox) of left ventricles of fetuses exposed to normoxia (n=4), hypoxia (n=5) and hypoxia plus l-nil (n=4) was quantified by a commercial fluorometric no assay kit. results: intrauterine hypoxia significantly reduced fetal body weight by 18% and increased placenta/fetal body wt by 33% as expected for hypoxic stress. hypoxia induced a slight increase in heart/fetal body wt by 4%. fetal cardiac nox levels (pmoles/mg) were increased by hypoxia (68.4+11.2) by 1.8 fold compared to normoxic controls (37.3+12.1). l-nil significantly decreased (p<0.05) nox levels in hypoxic hearts by 58% (68.4+11.2 vs 29.0+9.3; hypoxic vs hypoxic+l-nil, respectively). conclusion: l-nil inhibits inos-derived no generation in the hypoxic fetal guinea pig heart. since previous study in our lab showed a significant increase in inos expression but a decrease in enos and no change in nnos expression, we hypothesize that hypoxia upregulates cardiac no generation via the inos pathway. further study is needed to identify the important role of cardiac inos in the adaptive response of fetal hearts to chronic intrauterine hypoxia. objective: fetal asphyxia-mediated metabolic acidosis results in a ph decrease and an increase of lactate and base deficit in blood (bd blood ) and extracellular fluid (bd ecf ). it is not clear whether bd blood and bd ecf are similarly altered with worsening fetal acidosis. in the present study we sought to study the dynamic relations of bd blood and bd ecf to lactate in the ovine fetus subjected to repetitive uco with worsening acidemia. methods: ten near-term fetal sheep (125±2dga) underwent chronic preparation with brachial artery catheters and placement of an inflatable umbilical cord occluder. following a baseline recording period, fetuses underwent a series of mild (1min every 5min), moderate (1min every 3min) and severe (1min every 2min) uco with each series lasting 1h or until fetal arterial ph decreased to <7.0. fetal arterial blood was sampled at baseline, immediately before and during the first mild, moderate and severe uco and at 20min intervals during the moderate and severe uco. bd gap (bd blood -bd ecf ) was studied in relation to lactate. presented as means±sem. results: lactate correlated to bd blood (r=0.92, p<0.001). bd ecf correlated strongly with bd blood (r=1, p<0.001). bd ecf increased by 0.55±0.03 mmol/l more than bd blood from baseline to ph nadir, with bd gap therefore decreasing with increasing lactic acidemia ( fig. 1) . lactate, bd blood , bd ecf and bd gap correlated to ph (r=0.86, r=0.95, r=0.95 and r=1, respectively, all p<0.001) and ph could therefore be predicted with any of the four acid-base parameters. conclusion: the increases of bd blood and bd ecf and the decrease of bd gap with increasing lactic acidemia suggest a relatively more rapid accumulation of [h+] in ecf during uco of increasing severity. this may be because the metabolic build-up of acidosis occurs primarily in the tissues and the endothelial permeability for [h+] is impeded during increasing acidosis with atp depletion thus decreasing [h+] movement from ecf to plasma. previous studies have shown that intraperitoneal transplantation of human umbilical cord blood (hucb)-derived mononuclear cells led to the specific 'homing' of these cells to a hypoxic-ischemic brain lesion in perinatal rats. motor deficits resulting from the lesion were alleviated upon transplantation. thus, the presence of hucb cells at the lesion site seems to be a major prerequisite for their potential beneficial effect. however, the mechanisms of cell 'homing' are still unclear. in this study, we focused on elucidating mechanisms underlying the specific migration of hucb-derived mononuclear cells to the brain lesion. one possibility to induce cell 'homing' are chemotactic signals present at the lesion site. the cxc chemokine stromal derived factor-1 (sdf-1), which was previously shown to be a potent chemoattractant for directed migration of other stem and progenitor cells, is a putative candidate in our lesion paradigm. therefore we investigated the spatial and temporal expression of sdf-1 in brain hemispheres with or without hypoxic-ischemic lesion. sdf-1 expression was substantially increased at the lesion site during the investigated period of fourteen days after the insult. furthermore, hla-positive hucb cells were mainly detected in sdf-1 expressing brain regions and we were able to show that these cells express the sdf-1 receptor cxcr4 on their surface. the functional implication of sdf-1 in directing hucb cell migration was determined by application of neutralizing sdf-1 antibodies in vivo, resulting in a reduced number of hucb-derived mononuclear cells at the lesion site. with these functional effects, together with the observed timing and location of its expression, the involvement of the chemokine sdf-1 in hucb cell 'homing' seems conceivable. novel pathways in inflammation-induced fetal brain injury. michal a elovitz, jinghua chai. obstetrics and gynecology; crrwh, university of pennsylvania, philadelphia, pa, usa. intro: while survival of extremely preterm infants continues to improve, the number of children with cognitive impairment and/or cerebral palsy is increasing. if preterm birth (ptb) cannot be prevented, then strategies to identify and treat fetal brain injury in the setting of a ptb must be investigated. these studies were performed to explore novel pathways involved in fetal brain injury in the setting of inflammation-induced ptb. methods: cd-1 mice on e15 receive intrauterine injection of lipopolysaccharide (lps) or saline. 6 hours after injection, fetal brains from the left upper horn were harvested. 2 fetal brains were removed from each dam with 6 dams per treatment group. 12 separate rna samples were prepared and used for microarray (ma) analysis. all protocols were conducted as described in the affymetrix genechip expression analysis technical manual in the ma core facility using the moe 430av2 chip. data analysis was performed using significance analysis for microarray (sam). the brain samples from the same dam were considered as dependent samples. pathway analysis and functional annotation clustering tools were used with david. validations studies using qpcr with fetal brain samples (n=10-14 per group) were performed. results: while there was significant differences in gene expression between lps and saline exposed fetal brains, variability existed even between pups from the same dam. 542 genes were significantly differentially expressed between lps and saline brains (p<0.01). with a p value of <0.0001, 40 genes were differentially expressed. pathway analysis revealed significant involvement of 1) the cadeherin, cadherin-like, cell fraction and calcium binding (enrichment score 4.03) and 2) ribosomal processing, rna metabolism, pyrimidine metabolism (enrichment score 2.2). specifically, genes involved in neurogenesis, synaptic function, and neuronal and glial metabolism were most differentially regulated. qpcr confirmed observed fold changes in 6/10 genes analyzed. inflammatory pathways were not differentially regulated. conclusions: current theories regarding fetal brain injury in ptb focus on activation of inflammatory processes as essential events. this data suggests that long-term neurological injury in a ptb may be secondary to altered neuronal function, metabolism and/or communication. disruptions in these pathways should be explored as key mechanisms to adverse neurological outcomes in preterm neonates and should be targets for future investigations. regulation of microglial activation in the developing murine brain. mariya hristova, virginia zbarsky, daniel cuthill, adam wallace, donald peebles, gennadij raivich. institute for women's health, university college london, london, united kingdom. introduction: the aim of this study was to assess the process of microglial differentiation in developing white matter, an area of the brain that is particularly vulnerable to damage pre-myelination (approx 32 weeks gestation). microglia form a distinctive non-neuronal component. although related to peripheral macrophages they undergo highly specific processes of regional maturation and differentiation inside the brain with a slimming of the cell body, development of very elaborate crenulated arborised branches and downregulation of most macrophage activation markers. this process is relatively rapid in most grey matter brain regions, but is retarded in and around the subcortical white matter (swm) giving rise to the phagocytic fountains of microglia (fom). methods:we examined the process of deactivation and morphological differentiation in the cortex and swm of mice 0-28 days after birth (p0-p28) using confocal microscopy for monoclonal antibodies against alpha and beta integrin subunits and the costimulatory factor b7.2, colocalised with standard microglial marker iba1. results: strikingly, only the fom macrophages, but not cortical microglia, strongly expressed typical activation markers alpha-5, alpha-6, alpha-m, alpha-x, beta-2 and b7.2. the data for alpha-x are shown as an example in the figure; cortical microglia are shown in the grey bars and swm in black. fom activation was maximal at p0, decreased linearly over p3 and p7 and disappeared at p10. this process followed the presence of ingested phagocytic material but correlated only moderately with ramification, demonstrated by non-ramified but inactive p0 cortical microglia and formation of stubby processes in p3 fom. conclusion: these data describe strong and selective biochemical activation of fom phagocytes in p0-p7 swm, roughly equivalent to early 3 rd trimester human foetal development. this presence of highly active phagocytes in the neighbourhood of vulnerable wm could play an important role in the genesis of axonal damage in the foetus and premature neonate. 1997, pp. 13-25) . mbp is expressed in mature oligodendrocytes (jakovcevski and zecevic, glia 2005) . although myelination in the fetal sheep brain, a model extensively used to evaluate fetal development, has been described using conventional staining techniques (barlow, j comp neurol 1967) the use of specific mbp staining allows a more precise determination of the onset of myelination (antonow-schlorke et al., reprod sci 14.1, 2007, 248a) . aim: to use mbp expression to determine the trajectory of development of different white matter tracts of fetal sheep brain. methods: the ontogenetic profile of mbp expression was estimated in 47 healthy fetuses at 0.27 (n=6), 0.40 (n=5), 0.53 (n=5), 0.63 (n=6), 0.75 (n=12), 0.87 (n=9) and 0.93 (n=4) of gestation (term 150 days). after brain fixation and embedding in wax, sections at the level of the optic chiasm were stained with a monoclonal anti-mbp antibody using the abc-technique. immunohistochemical distribution of mbp was morphometrically assessed in the dorsal internal capsule and cortical white matter tracts, i.e. the lateral centrum semiovale, superficial white matter and median corpus callosum using an image analysis system (scion image 6.21, nih, usa). results: mbp expression of various fetal white matter structures started at different time points; initially in the internal capsule at 0.53 of gestation followed by the cortical white matter structures at 0.63 of gestation. cortical myelination advanced from the cortical deep white matter via superficial white matter to the corpus callosum reflecting different rates of progression. conclusions: the onset of mbp expression estimated here explicitly antedates previous observations in sheep (barlow, 1969) the way the embryonic heart functions before cardiac morphogenesis is completed is still subject of many studies. to gain more insight into the early cardiac structure-function relationship, doppler blood flow velocity waveforms at four different locations in the embryonic chicken heart during cardiovascular development were assessed. we collected waveforms using high frequency ultrasound biomicroscopy with a 55-mhz transducer at hh stages 18, 21 and 23 which are comparable to humans at 5 to 8 weeks of gestation. waveforms were obtained at the inflow tract, the primitive left ventricle, the primitive right ventricle, and at the outflow tract in ten different embryos per stage. by exploring the time relation between the waveforms, cardiac cycle events were outlined. our results demonstrate that stage and location dependent, intracardiac blood flow velocity waveforms can be obtained in the chicken embryo which reflect stage dependent pumping mechanisms. the blood flow profiles assessed at the four locations in the embryonic heart demonstrated a developmental related increase in velocity. in the primitive ventricle the passive filling wave decreased, whereas the active filling wave increased resulting in a decreased p to a ratio in the course of time. high frequency derived cardiac blood flow velocity characteristics support previous findings that the embryonic heart functions like a suction pump at early development and transforms towards another pumping mechanism at later developmental stages. these findings are of importance for the interpretation of human first trimester cardiac velocimetry studies. figure 1a . changes in the perimeter of the thymus with gestational age by fetal gender are depicted in figure 1b . background. for the fetus, the roles of arterial blood gases are recognized to be critical in the regulation of cerebral blood flow (cbf) and cerebral metabolic rate for oxygen (cmro 2 ), cortical tissue po 2 (tpo 2 ), and electrocorticographic (ecog) activity. in addition, metabolites such as adenosine (ado) are important in this regard. nonetheless, the relation of adenosine and its metabolites to these indices of cerebral oxygenation are not well defined. in an effort to elucidate these interrelations, we tested the null hypothesis that acute hypoxic-associated cerebral oxygenation and related variables are not closely associated with adenosine. the most common cause of perinatal brain injury is chronic hypoxia/ischemia. the mechanisms are complex and poorly understood. applying proteomics tools, we identified a set of hypoxia-related proteins in the fetal guinea pig (gp) brain (companion abstract). one protein, cofilin-1, which regulates the rapid cycling of actin assembly and disassembly, was dramatically altered by chronic brain hpx. herein, we test the hypothesis that confilin-1 modifications during hpx contributes to brain damage via apoptosis and is an example of an adaptive response that becomes maladaptive. methods: time-mated gps were housed in a chamber from 50d to 65d (term) with either room air (nmx) or 10.5% or 12% o 2 . fetal brains were removed and sections prepared for double staining specific to bax and dephosporylated cofilin-1. total protein was isolated and equal amounts loaded on a sds gel, separated by electrophoresis, and transfered to pvdf membranes probed with primary antibodies to dephosphorylated and phosphorylated cofilin-1, bax and bcl-xl, and incubated with second antibody. protein signals were detected, quantified by densitometry, and normalized to -actin. total rna was isolated, reverse-transcribed, and quantified by real-time pcr using sybr green i labeling. expression was calculated by the ct method using 18s for control. melt analysis was performed to confirm specificity of the amplification, and efficiency determined by the slope of the standard curve. the mitogen activated protein kinase (mapk) signalling pathway is upregulated in perinatal hypoxic-ischaemic brain. the role of the extracellular signal-regulated kinase (erk) cascade, a pivotal component of mapk signalling, remains unclear with reported actions ranging from mitogenic and trophic effects to a neuronal death-promoting role. the aim of this study was to assess the role of neuronal erk activity using selective neuronal inhibition in a perinatal model of hypoxic-ischemic brain injury. methods: we explored the effects of selective neuronal inhibition of erk activation using transgenic mice expressing dominant-negative (dn) mek1, the upstream activator of erk1/2, which was under the control of the pan-neuronal talpha1 alpha-tubulin promoter. p-erk immunoreactivity was quantified following a hypoxic inschemic (hi) insult in p7 c576bl/6 mice, caused by unilateral occlusion of the carotid artery, followed by hypoxia with 8% oxygen for 60 mins at 36°c. the volume of surviving brain on the affected hemisphere was expressed as a % relative to the control side. results: expression of the mek1 dn was associated with a reduction in erk1/2 activation following hypoxia ischaemia, as assessed by p-erk immunoreativity. compared with the wild type, littermate controls, mek1dn animals exhibited significantly decreased volume of forebrain damage brain following unilateral, 60 min hi insult (*p<0.05, **p<0.01, anova). similar protective effects of mek1dn were observed in cerebral cortex, hippocampus, thalamus and striatum when compared to wild type littermate controls and correlated with a significant reduction in microglial activation in all brain areas. conclusion: overall, these results suggest that neuronal mek1 and its downstream signals have an important death-inducing role in this model of perinatal brain injury and could serve as potential targets for therapeutic intervention. oup, 2004) . however, whether melatonin protects placento-fetal development during hypoxic or undernourished pregnancy is unknown. we investigated in rats the effects on placental and fetal growth of maternal treatment with melatonin in hypoxic and undernourished pregnancy. methods: on pregnancy day 15, wistar rats were divided: control (21% o 2 ) + melatonin (5 g.ml -1 drinking water), hypoxia (10% o 2 ) + melatonin, and undernutrition (40% reduction in food intake) + melatonin (n=7 per group). on day 20, dams were anaesthetised, the pups, placentae and fetal brain were weighed. results: relative to controls (fetal weight: 3.73±0.05g; brain/body weight ratio: 46.2±1.2mg/g; n=76), both hypoxic (2.94±0.03g; 58.4±1.1mg/g, n=80) and undernourished pregnancy (3.05±0.04g; 53.0±1.1mg/g, n=80) promoted asymmetric iugr (p<0.05), without affecting placental weight. melatonin treatment had no effect on iugr, but it decreased placental weight in normoxic (0.43+0.01, n=95), hypoxic (0.44±0.01, n=98) and undernourished (0.41±0.01, n=81) pregnancies relative to untreated controls (0.48±0.01, n=76; p<0.05). when fetal body weight was expressed relative to placental weight, a measure of placental efficiency, melatonin prevented the fall in the ratio in undernourished, but not hypoxic pregnancies (fig.1 objective: midkine (mk) is a 13-kda heparin-binding growth factor with various functions including cell proliferation, migration, differentiation and angiogenesis. mk expression is strictly regulated in temporal sequence; it is highly expressed during midgestation. we recently studied mk expression in the midgestation human fetus, and showed that the highest mk expression were observed in the adrenal gland, brain, lung and kidney. in the present study, we investigated the profile of mk in human amniotic fluid (af) and amnion (am). methods: amniotic fluid and amniotic membranes were collected at diagnostic amniocentesis, preterm no labor, and term no labor. mk protein levels were analysed by western blot. expression of transcripts encoding mk and putative mk receptors were examined by rt-pcr and real-time quantitative rt-pcr (qpcr). results: 1) western blot analysis demonstrated abundant mk protein in the human am; mk levels were higher at midgestation than at term (3-fold: 16wks vs. 37wks). 2) tissue transglutaminase known to polymerize mk was abundant in af. 3) qpcr revealed that mk mrna was not expressed in am whereas it was highly expressed in the fetal adrenal, kidney and lung (positive controls). 4) among the receptors implicated in mk signaling, lowdensity lipoprotein receptor-related protein and syndecan-4 were expressed in am while protein-tyrosine phosphatase, anaplastic lymphoma kinase, and syndecan-3 were not. conclusions: abundant mk protein in the midgestation af is likely to be derived from the fetus. mk in af may play a role in feto-amniotic communications and/or development of fetal organs exposed to af. short term hfix expression. we hypothesized that long term hfix expression could be achieved in fetal sheep using an aav vector, without stimulating an immune response to the transgenic hfix protein. we injected aav8 hfix vector (1 -9 x 10 12 p/kg) into the peritoneal cavity of fetal sheep under ultrasound guidance in early (n = 3) or late (n = 4) gestation. fetal blood was retrieved by ultrasound guided sampling from the intra-hepatic umbilical vein. fetal and lamb blood was tested for hfix expression using elisa, antibody responses using functional assays, and for liver damage up to a year after birth. vector spread was detected in maternal and fetal tissues by quantitative pcr analysis. results: the highest level hfix was detected 18 days after late ip injection (44% and 28% normal human levels). early gestation ip injection gave 8.7% and 1.8% at 3 and 21 days after injection. hfix levels dropped rapidly correlating with the increase in size of the fetal liver and lamb. however, hfix was detectable at a low levels (0.7%) 1 year after birth in early and 4 months after birth in late gestation injected lambs. up to 1 year after birth, liver function tests and bile acid levels were normal, showing no evidence of liver pathology. no functional antibodies to the hfix protein or aav vector were detectable. high vector levels were detected in the fetal liver, and other peritoneal organs; no vector was present in fetal gonadal tissue. conclusion: hfix expression is detectable up to 1 year after delivery of aav vector to the fetal sheep using a clinically applicable method. this is the first study to show long term hfix expression after fetal gene therapy in a large animal. further work will include testing for immune tolerance to exogenous hfix protein in these animals. objective: placental estrogens play a pivotal role in the endocrine control of pregnancy and may be involved in the key changes occuring during parturition. it has been established that crh interacting with crh receptor 1 has a positive effect on estrogen production throughout pregnancy. urocortin 2 (ucn 2), a novel peptide of the crh family binding exclusively crh receptor 2, is expressed by human placenta and the aim of the present study was to evaluate the influence of ucn 2 on estrogen biosynthesis in cultured trophoblast cells. methods: cultured term placental cells were treated with various concentrations of ucn2 in the presence of the estrogens precursors dehydroepiandrosterone sulphate (dhea-s), androstenedione or testosterone. estradiol secretion was measured in the culture medium using a specific elisa, p450arom mrna and protein expression were evaluated by real time pcr and western blot analysis respectively. results: the addition of ucn2, in presence of dheas significantly increased e2 levels at 4, 8 and 12 hours treatment, while in presence of androstenedione and testosterone an increase in e2 secretion was detected only at 12, 24 and 36 hours (at different ucn2 doses). both p450arom mrna and protein were up-regulated in presence of each estrogen precursor and the addition of ucn2 caused a synergistic increase. anti-sauvagine 30 (a crh type 2 receptor antagonist) resulted in a significantly attenuated ucn2 effects on e2 secretion and on p450arom mrna and protein expression. conclusions: the present study supports a possible role of ucn2 on placental e2 biosynthesis: e2 secretion and p450arom transcript and protein expression were significantly increased after ucn2 treatment. in conclusion, the crf family may play a major role on placental steroidogenesis, stimulating dheas secretion in fetal adrenals by crh and controlling placental estrogen biosynthesis through ucn2. a possible influence on the mechanisms of parturition may be hypothesized. increased expression of kiss-1 gene in preterm placenta. letizia galleri, michela torricelli, chiara voltolini, fernando m reis, felice petraglia. department of pediatrics, obstetrics and reproductive medicine, university of siena, siena, italy. introduction placenta expresses a large number of peptides involved in delivery. neuropeptides, cytokines, are expressed and secreted by human placenta at the time of preterm delivery. human placenta is a major source of kiss-1, a 54-amino-acid peptide encoded by a putative metastasis suppressor gene. kiss-1 acts on its placental g protein-coupled receptors (kiss-1r); this peptide stimulates release of oxytocin in rats, the most potent known uterine stimulant, suggesting a possible role of kiss-1 in the mechanisms of labor. aim of study the aim of this study was to evaluate placental expression of kiss-1 at preterm labor. material and methods placental tissue and plasma samples (both maternal and fetal) were collected at term in the absence of labor (tnl), at term spontaneous vaginal delivery (tl), and at preterm labor (ptl). changes in placental mrna expression were determined by real-time quantitative rt-pcr analysis. kiss-1 protein levels were measured by specific immunoenzymatic assay (elisa). results placental kiss-1 mrna expression was significantly higher (p<0.0001) in ptl than in tnl and in tl. however, maternal and fetal plasma kiss-1 levels did not differ among tnl, tl, and ptl samples. conclusion the present study showed that placental kiss-1 mrna expression is increased in preterm delivery. further studies are needed to better understand the role of kiss-1 on cascade leading term and preterm labor. placental angiogenesis is essential for placental development, which occurs under a hypoxic environment ( 2-8 % o 2 ) during normal pregnancy. it has been reported that in transformed human dermal microvascular endothelial cells, hypoxia activates erk1/2, which stabilizes hypoxia-inducible transcription factor-1 (hif-1 ). however, signaling mechanisms governing placental angiogenesis under hypoxia is largely unknown. herein, we tested whether hypoxia affected fgf2-and vegf-stimulated cell proliferation partly via activating erk1/2 and stabilizing hif-1 protein levels in human placental artery endothelial (hpae) and transformed human placental microvascular endothelial (hpme) cells. methods: cells cultured under normoxia ( 20% o 2 ) or hypoxia (3% o 2 ) for 2 days were treated with fgf2 and vegf. after 3 days, the number of cells was determined. activation of erk1/2 and levels of hif-1 protein were determined by western analysis. results: under normoxia, fgf2 and vegf stimulated (p < 0.05) cell proliferation and induced ( 5 min, p < 0.05) erk1/2 phosphorylation in hpae and hpme cells. hypoxia promoted (p < 0.05) fgf2-and vegf-stimulated cell proliferation in hpae cells, whereas hypoxia blocked (p < 0.05) such actions in hpme cells. hypoxia enhanced fgf2-, but not vegf-induced erk1/2 phosphorylation in hpae cells. in contrast, hypoxia promoted (p < 0.05) vegf-, but not fgf2-induced erk1/2 phosphorylation in hpme cells. hypoxia increased (p < 0.05) hif-1 levels in the hpae cells: first detected at 0.5 hr and maintained up to 24 hr. hpme cells had high basal levels of hif-1 (3 folds; p < 0.05) compared with hpae cells. hypoxia did not alter hif-1 levels up to 4 hr and decreased (p < 0.05) hif-1 levels at 24 hr in hpme cells, conclusions: in hpae cells, hypoxia enhances fgf2-and vegf-stimulated cell proliferation possibly partially via promoting activation of different protein kinases. this stimulatory effect is associated with increased hif-1 protein levels. however, inhibition by hypoxia on fgf2-and vegf-stimulated hpme cell proliferation is associated with relatively high hif-1 levels in the first 4 hr and decreased hif-1 levels at 24 hr. thus, these data suggest that different signaling mechanisms are involved in hypoxia-modulated growth factor-induced placental angiogenesis in which an increase in hif-1 levels plays a critical role. objective: corticotrophin releasing factor (crf) is a well established neurohormone involved in the initiation of the stress response. we recently documented that rat placenta is analogous to human placenta in the expression of crf-mrna and protein, and that placental crf release may contribute to in utero meconium passage. time-of-day variation in crf expression is a highly recognized phenomenon at the brain cellular sites of crf synthesis. we sought to determine whether crf expression in rat placenta is subject to time-of-day variation. method: time-dated pregnant rats were obtained on day 12 of gestation (term=22 days), housed in under 12h-12h light-dark conditions (lights on 0600). lab chow and water were continuously available. on day 18, pregnant rats were quickly anesthetized by exposure to isoflurane, abdomen opened and fetuses and placenta exteriorized either at 0545-0600hr (zeitgeiber time, zt0) or 1245-0100hr (zt6). individual placentas were processed either for rna extraction or immunohistochemical investigation. the levels of crf-mrna were assessed by pcr using rat specific pcr primers. pcr bands were subsequently cloned and sequenced. bouin's solution fixed paraffin sections of placenta were subjected to crf immunohistochemistry with antibodies specific to rat species and intensity of immunostaining was analyzed using image pro-plus software and expressed in arbitrary units (au). results: one specific pcr band of 339 bp size was consistently identifiable in placenta harvested at zt6 but not at zt0. nucleotide sequence of the pcr band confirmed its identity as crf-mrna. intensity of crf-immunostaining was significantly greater at zt6 in giant trophoblast (gt) cells (gt-crf: zt0= 0.176 ± 0.122 au, zt6= 0.272 ± 0.014 au, p=0.007) but not in spongiotrophoblast cells (stc) (stc-crf: zt0 = 0.218 ± 0.025 au, zt6= 0.162 ± 0.011au, p=ns) or labyrinth cells (lbc) (lbc-crf: zt0= 0.148 ± 0.003, zt6 = 0.149 ± 0.015 au, p=ns) . conclusion: similar to adult brain, rat placenta expresses crf mrna and protein. time-of-day variation of crf expression originally seen in central nervous system neurons is also identifiable in giant trophoblasts cells at zt6. these findings suggest that stress-mediated placental crf release, and potentially fetal meconium passage, may be dependent upon time-of-day. objectve: transplacental water flow is essential for the provision and maintenance of fetal body water and amniotic fluid. water flow across membranes is regulated by aquaporin (aqp) water channels in many tissues. thus aqp1, expressed in the placenta, is a candidate to regulate maternal to fetal fluid exchange. maternal beta-mimetics have been hypothesized to augment fetal growth by increasing the availability of nutrients and perhaps water. as camp acts as a second messenger to increase expression of selected aqps in other tissues, we sought to determine if betamimetics acting through camp could modulate placental aqp1, and potentially influence placental water transfer. methods: trophoblastic cell cultures were established in first trimester-derived extravillous htr-8/svneo cells and term placenta-like trophoblast carcinoma cell line jeg-3. cultures were treated with sp-camp, a membrane-permeable and phosphodiesterase resistant camp, and forskolin, an adenylate cyclase stimulator, in doses of 0.5, 5 and 50 m for 2 hrs (aqp1 mrna expression) and 20 hrs (aqp1 protein expression). for time course experiments, cells were incubated with 5 μm of either sp-camp or forskolin for 2, 10 and 20 hrs at 37°c, 5% co 2 . after cell harvest, mrna and protein expression were assayed using real time pcr and western blotting. results: sp-camp and forskolin increased aqp1 mrna expression in both cell lines after 2 hrs (p<0.05) in a dose-dependent manner. protein expression paralleled the increase seen in the mrna. 5 m of sp-camp and forskolin stimulated aqp1 mrna expression after 2 hrs in htr-8/svneo cells and after 10 hrs in jeg-3 cells (p<0.05). 5 m of either sp-camp or forskolin stimulated aqp1 protein expression in both cell lines after 10 hrs (p<0.05) and the expression remained high at 20 hrs. conclusion: aqp1 gene expression in trophoblast cells is up-regulated by camp agonists. these results suggest that maternal beta-adrenergic agonists or antagonists may modulate maternal-fetal water flux via modulation of aqp water channels. rat placenta expresses corticotrophin releasing factor-binding protein and mrna. jayaraman lakshmanan, thomas r magee, bindu cherian, sharon k sugano, hanalise huff, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: corticotrophin releasing factor-binding protein (crf-bp), a 37 kda secreted glycoprotein, was originally isolated from human plasma. it binds crf and urocortin i with an equal or greater affinity than does the crf receptor. crf-bp expression has been reported in target tissues such as brain and placenta. based on its ability to inhibit crf actions in vitro, it is speculated to function as a "gate keeper" for crf-initiated stress responses. we recently documented expression of crf and urocortin-1 in rat placenta. in the present study we sought to establish the expression of crf-bp in rat placenta by immunohistochemical and pcr-analyses. methods: placental tissues (n=20) collected from sprague-dawley pregnant rats on day 21 of gestation (term=22) were either fixed in 4% paraformaldehyde solution (ph 7.4) and paraffin embedded or processed for total rna extraction. paraffin sections of five micron thickness were subjected to immunohistochemical analysis with goat polyclonal antibodies to human crf-bp precursor (sc-1824 santa cruz biotechnology, ca) by avidinbiotin-peroxidase complex technique. the structure of cloned human crf-bp precursor exhibit significant amino acid sequence homology among all species studied. immunoreactivities on the sections were quantified using image pro 4.01 software and staining intensity (od/area) expressed as arbitrary units (au). all values are expressed as mean±sem. for pcr, cdna was synthesized and pcr amplified with a one step rt-pcr kit. fragments were gel purified, clone into plasmid vector and dna sequenced. results: the crf-bp antibody elicited strong positive staining in decidua, giant trophoblasts, spongiotrophoblasts and labyrinth cells. the results of image analyses revealed (au): decidua: 0.191±0.010, giant trophoblast cells: 0.139±0.029, spongiotrophoblasts: 0.120±0.003, fetal membranes: 0.128±0.009. pcr analyses identified a single 434 bp band, consistent with crf-bp. conclusion: our study establishes for the first time that rat placenta is analogous to humans in that both express crf-bp mrna and protein. immunohistochemical findings reveal that crf-bp protein expression occurs at multiple sites within the placenta. expression of per2 clock protein in rat placenta: an internal timer for placental functions. jayaraman lakshmanan, reuben lakshmanan, sharon k sugano, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: corticotrophin releasing factor (crf) expression time-of-day variation occurs in giant trophoblast cells on the maternal side of the rat placenta. this temporal change in crf expression pattern is very similar to that of central nervous system neurons, suggesting that placental cells may use a similar mechanism to read time of the day. the self-sustaining rhythm generating capacity of the suprachiasmatic nuclei is believed to be derived from cell-autonomous, transcriptional feed-back loops dependent on a number of canonical clock genes. in the present study we sought to determine whether rat placenta expresses period gene 2 (per 2), one of the clock/cycle related genes. methods: time-dated pregnant sprague-dawley rats were received on day 13 of gestation and housed in a controlled environment (0600-1800h lights on) with free assess to food and water. placentas collected on days 20 and 22 of gestation (term=22days) between 0930 and 11.30 am were fixed in 4% paraformaldehyde and paraffin embedded. for each gestational ages a total of 12 placentas from 6 different pregnant rats were used. paraffin sections (3 sections per placenta) were subjected to immunohistochemical analysis with antibody to per2 (1:200, santa cruz biotechnology, ca) by abc technique and 3, 3'diaminobenzidine as a chromagen. sections were examined under microscope, imunostaining quantified by image pro 4.01 software and expressed in arbitrary units (au). data are presented as mean ± sem. statistical significance was analyzed by anova with a p value < 0.05 as significant. conclusion: the present results indicate that rat placental cells, devoid of any neural innervations, express per2 clock protein, similar to central nervous system neurons. based on the differences in the relative intensities between trophoblasts and labyrinth cells on day 22 of gestation, we conclude that fetalmaternal interactions in per2 regulation disappear at term (or at the time of birth.). our findings imply that per2 may function as internal physiological modulator in placenta. to determine the mechanism(s) underlying placental time-of-day crf variation, we examined the effect of maternal administration of betamethasone (a synthetic glucocorticoid) on nuclear gc receptor expression in placental cells. method: time-dated pregnant rats (n=6) on day 21 of gestation were given a single subcutaneous injection (at 5:45 am) of betamethasone (350μg/kg body weight) while control pregnant rats (n=6) received saline. dams were exposed to isoflurane anesthesia at 10:45 am, and placentas harvested, fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochemical analysis with gc-receptor antibody (sc-8992, santa cruz biotechnology), with immunoreactive material identified by abc technique using 3, 3' diaminobenzidine as a chromagen. percentages of gc-nuclear receptor (gc-nr) positive cells and their intensities (od/area) were quantified using image pro 4.01 plus software. all values are expressed as mean ± sem. statistical analysis was by anova with p<0.05 considered significant. results: in placentas of saline exposed pregnant rats, isolated labyrinth (lb) cells (<1%) were positive to gc-nr. no positive staining for gc-nrs was seen either in giant trophoblasts (gt) or in spongiotrophoblasts (st). betamethasone administration was associated with a significant and marked increase in gc-nr staining in all three placental cell types (p<0.05). among the cells, gt (69±10%) demonstrated a greater percentage of cells expressing gc-nr than did st (34±5%) or (lb=38±5%) (p<0.05), though there was similar immunoreactive intensities (gt: 0.216±0.017, st: 0.238±0.031, lb: 1802±0.022 au; p=ns) conclusion: our findings indicate that all placental cells respond to gc with upregulation of gc-nr with an enhanced response among gt cells. these results suggest that endogenous or exogenous maternal stress-induced gc exposure may influence signaling responses within both maternal and fetal placental compartments. ovine placenta at very-preterm gestation expresses corticotrophin releasing factor (crf), crf-binding protein (crf-bp) and glucocorticoid receptors (gr). jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: in humans, the placenta is the major source of maternal and fetal plasma crf. based on its critical functions, crf is considered as a "placental clock" of parturition. we recently reported that ovine fetal as well as maternal plasma contain measurable amounts of crf at near-term but not at very preterm gestation. we interpret the absence of crf in plasma at very preterm gestation is either due to lack of placental crf expression and/or placental crf release. here, we examined the expression status of crf, crf-bp (a known specific binding protein for crf and a known regulator of crf functions in human placenta) and gr (a known positive regulator of crf expression in human placenta) in ovine placenta collected at very-preterm gestation. method: placenta harvested from time-dated pregnant ewes on 118±2 days gestation were fixed in bouins's solution and processed for paraffin embedding. paraffin sections were subjected to immunohistochemical analysis with polyclonal antibodies specific to ovine crf(1:300-1:500, phoenix pharmaceuticals, ca,) crf binding protein (1:100-1:200, sc 20630) and gr (1:100-1:200, sc: 8992, santacruz biotechnology, ca). immunoreactive material on the sections were identified as brown staining by abc technique using diaminobenzidine as chormagen. immunoreactive material was quantified by image analysis using image pro 4.01 plus software and the immuoreactive intensity (od/area) expressed as arbitrary units (au). results: strong positive staining for crf (0.363±0.033 au), crf-bp (0.505±0.003 au) and nuclear-gr (0.383±0.015au) were noticed in syncytiotrophoblast cells in all placental sections obtained from pregnant ewes at118 days gestation. control sections exhibited no positive staining. conclusion: our findings indicated that ovine placenta at very-preterm gestation expresses crf, crf-bp and gr. these results suggest that absence of measurable crf in maternal and fetal plasma at very-preterm gestation is not due to lack of placental crf expression but rather due to the absence of regulated crf secretory mechanisms. rat placenta expresses urocortin i protein and mrna. thomas r magee, michael g ross, john d richard, sharon k sugano, jayaraman lakshmanan. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: urocortin i (ucn-1), a 40 amino acid neuropeptide belongs to corticotrophin releasing factor (crf) stress hormone family. in humans, the placenta and several gestational tissues have been reported to express ucn-1 protein and ucn-1 mrna. a number of published studies indicate that ucn-1 is similar to crf in expression pattern and biological functions. we recently reported that rat placenta is a site of crf protein and crf mrna expression. in the present study we sought to determine whether rat placenta expresses ucn-1 protein and ucn-1 mrna. methods: placenta (n=18) collected from pregnant rats at 21 day gestation were either fixed in bouin's solution and processed for paraffin embedding or placed in rna later preservative and frozen for rna extraction. for immunohistochemical localization, five micron thickness paraffin sections were cut and immunostained with rabbit polyclonal antibodies to ucn-1 (1:500 to 1:750, sigma) by standard abc technique. control sections were incubated with omission of ucn-1 antibody. immunoreactive materials on the sections were identified using 3, 3' daminobenzidine as chromagen. immunostaining intensity (od/area) was quantified by image-pro plus software and expressed in arbitrary units (au). for pcr, cdna was synthesized and pcr amplified with a one step rt-pcr kit. fragments were gel purified, cloned into a plasmid vector and dna sequenced. all values are given as mean ± sem. results: ucn-1 polyclonal antibody elicited strong immunostaining in placental trophoblast cells with variable intensity. the pattern of immunostaining is as follows: giant trophoblast cells: 0.334±0.319 au, spongiotrophoblast cells: 0.318±0.013 au and labyrinth cells: 0.233±0.139 au. the relative intensity in labyrinth cells was significantly lower than the other two cell types (p< 0.01). pcr analyses revealed the presence of a single band of 379 bp, consistent with ucn-1 mrna. conclusion: similar to human placenta, rat placenta expresses ucn-1 protein and ucn-1 mrna, with most expression localized to the maternal side trophoblast cells. these results support our hypothesis that rat placenta can be used as a model to understand the role of this peptide in feto-maternal stress. the role of the nuclear hormone receptors fxr, pxr and car in placental bile acid homeostasis in normal and pathological pregnancy. victoria geenes, peter dixon, selina raguz, jenny chambers, kishore bhakoo, catherine williamson. imperial college, london, united kingdom. background: obstetric cholestasis (oc) is a pregnancy specific liver disorder characterised by raised maternal serum bile acid levels and associated with adverse fetal outcome. the aetiology of oc is complex and not fully understood, but the fetal complications are likely to result from an accumulation of bile acids in the fetal circulation. bile acids are the toxic end products of hepatic cholesterol metabolism and are synthesised from 12 weeks gestation. in common with other waste products, accumulation in the fetal compartment is prevented by excretion across the placenta into the maternal compartment. however, studies of maternal and cord serum from normal and oc pregnancies have suggested that bidirectional transfer of bile acids is possible. hepatic bile acid transport and metabolism is regulated by members of the nuclear hormone receptor family, namely fxr, pxr and car, but the mechanisms for regulating placental transfer are unknown. objectives: this study used placenta from normal and cholestatic pregnancies to investigate the expression of genes involved in hepatic bile acid homeostasis. methods: villous trophoblast samples from 6 oc and 7 normal pregnancies (np), and 3 human livers were collected and preserved in rnalater. explant cultures were prepared from a further 4 np placentas and cultured for 6 days at 8% oxygen. on day 5 they were treated with chenodeoxycholic acid, lithocholic acid or vehicle for 24 hours prior to fixing in rnalater. total rna was extracted using trizol, and reverse transcribed to cdna. quantitative real-time pcr was performed using sybr green. target gene mrna abundance was calculated from a standard curve and normalised to l19. results: the expression of fxr, pxr and car, the nuclear hormone receptors responsible for hepatic bile acid homeostasis and several fxr target genes (shp, mdr3 and bsep) was found to be very low in np, and unaffected by the presence of maternal cholestasis. furthermore, the expression of these genes could not be induced by bile acid treatment in an in vitro model. here we have shown that the nuclear hormone receptors (fxr, pxr and car) involved in hepatic bile acid homeostasis are expressed at very low levels in normal and pathological pregnancy and are thus likely to be of limited importance in placental bile acid transfer. a placental phenotype for obstetric cholestasis. victoria geenes, 1 jenny chambers, 1 jo wyatt-ashmead, 2 kishore bhakoo, 1 catherine williamson. 1 1 imperial college, london, united kingdom; 2 hammersmith hospital, london, united kingdom. background: obstetric cholestasis (oc) is a pregnancy specific liver disorder that affects 0.7% of pregnant women in the uk. biochemically, oc is characterised by liver dysfunction with elevated maternal serum bile acids (sba), and clinically by an increased risk of fetal complications including fetal distress, meconium staining of the amniotic fluid, preterm labour and sudden intrauterine death. the aetiology of oc is complex and not fully understood, but the fetal complications are likely to result from the toxic effects of bile acids, which can cause vasoconstriction in the placenta and fetal cardiac dysrrhythmias. furthermore, in a rodent model of oc, bile acids cause oxidative stress in the placenta and a reduction in litter size. these changes are absent in animals treated with ursodeoxycholic acid (udca), a drug used to treat oc. however human data are lacking. objectives: this study used whole placenta and explant cultures to investigate the effects of bile acids on human placental architecture and to establish whether udca protects the placenta against bile acid induced damage. methods: villous trophoblast samples were collected from 15 oc and 5 normal pregnancies (np) and fixed in formalin. explant cultures were prepared from a further 4 np placentas, and cultured for 6 days at 8% oxygen. on day 5 a subset were treated with udca overnight, and on day 6 the explants were treated with taurocholic acid, taurochenodeoxycholic acid or vehicle for 6 hours prior to fixing. 5 m sections were stained with haematoxyllin and eosin. slides were reviewed by a perinatal pathologist and syncytial knots (sk) counted. results: oc tissues showed marked alterations in morphology, including congestion of the terminal villi, and loosening of the stroma and fibrotic change of the membranes of the stem villi. there were significantly more sk the oc samples (mann-whitney u test p=0.024). furthermore, sk were increased in explants treated with bile acids, but not those treated with udca prior to the addition of bile acids. conclusions: here we describe several morphological abnormalities of the placenta associated with maternal cholestasis. these changes were confirmed using a placental explant model, which also showed that udca protects the placenta against bile acid induced damage. in summary, this study indicates that udca is likely to protect the fetus in oc. the placenta has complex metabolic and endocrine activities and is essential for the growth and survival of the fetus in utero. ultrasound is the most sensitive and less invasive method to evaluate placental size, morphology and function. the three-dimensional approach allows to calculate placental volume in the i and ii trimester of pregnancy. pregnancies from assisted reproduction technologies (art) show an increased rate of pathologies potentially related to placental insufficiency such as intrauterine growth restriction and preterm delivery. aim. to determine placental volume in art pregnancies in a cross sectional study in the first trimester of gestation. design. using three-dimensional ultrasound machine (ge 730) placental volume measurement from art pregnancies (n=26; 10-13 wks) were compared with data from normal controls (n=26) matched for gestational age. data were analysed with software stata 9. results. mean placental volume was 46.2 ml (sd±23.9) in art pregnancies and 58.4 ml (sd±22.9) in normal controls. mean difference resulted in 12.12192 ml (-1-25.19835) and was statistically different (p=0.047). multiple linear regression analysis showed a statistically significant interaction (p=0.04) between gestational age and case status, i.e. differences in placental volume increase significantly with advancing gestational age between cases and normal controls. mean gestational age at birth was not essentially different between the two subsets (39.1 weeks ± 1.6) however a statistically significantly lower mean fetal birthweight was found in art pregnancies: 2945 g (sd±537) vs 3226.(sd±294.4) (p=0.044). conclusions. placental volume is slightly decreased in art pregnancies. measurements of placenta volume by 3d ultrasound may play a role in identifying the degree of placental growth early in gestation in a risk population. in vitro effects of adenovirus-mediated gene transfer of insulin like growth factor-1(ad-igf-1) in trophoblast cells. mounira habli, 1 datis alae, 2 foong yen lim, 2 jignesh parvadia, 2 timothy crombleholme. 2 1 obstetrics and gynecology; 2 pediatric surgery, university of cincinnati/children's hospital, usa. objective:recently, ad-igf-1 corrected both fetal and placental weights in rat model of fetal growth restriction (fgr) . to elucidate the mechanism of action of ad-igf-1;we examined the effect of ad-igf-1 on trophoblast proliferation, differentiation and invasion. methods: rcho-1 trophoblast cell line derived from rat choricarcinoma was used. ad-igf-1 and galactosidase transgene (ad-lacz) were given at moi of 10:1 and 100:1 in all the experiments.transduction efficiency was assessed 24 hr after infection with ad-lacz by galactosidase enzyme activity. the invasiveness of rcho-1 was measured by using bdmatrigel invasion chamber kit. rcho-1 proliferation cell density was assessed by crystal violet assay. morphologic analysis of rcho-1 differentiation in response to treatment (differentiated cells are multinucleated giant cells) was assessed byimunocytochemistry staining using placental lactogen-1 antibodies(specific hormone produced by giant cells). results: 100% efficiency of gene transfer of ad-lacz to rcho-1 cells was observed at both moi's.there was no significant difference in proliferation between treated control group regardless of the dose at 12 and 24 hrs. ad-igf-1 induced morphologic differentiation of trophoblast cells compared to control (fig1) at 24 hrs.ad-igf-1 induced higher rate invasion in a dose dependent fashion as compared to control(ad-lacz) group(16% at 100moi vs 0.8% in control p<0.008)(fig2). conclusion: ad-igf-1 gene transfer induces differentiation and invasiveness of trophoblast cells. these may be the mechansim of correction of fgr in rat model of placental insufficiency. placental gene transfer may be an effective treatment strategy for placental insufficiency. types of human placenta. martina dieber-rotheneder, ursula hiden, gernot desoye, mila cervar-zivkovic. department of obstetrics and gynaecology, medical university graz, graz, austria. background and hypothesis: endothelin-1 is a polypeptide with a wide range of functions. in the placenta it acts as a potent regulator of vasotonus on endothelial and smooth muscle cells, whereas on the trophoblast it regulates cell proliferation, invasion and apoptosis. endothelin-1 action is differently signaled through two endothelin receptor (etr) subtypes, etr-a and etr-b. several alternative splice variants of etr were identified. here we hypothesize that the etr-a splicing varies with gestational age and is different in trophoblast vs endothelial cells of the human term placenta. methods: mrna of placental tissue from first trimester (pregnancy terminations, missed abortions) and term of gestation, first trimester and term trophoblasts, arterial and venous placental endothelial cells as well as cell lines representing first trimester trophoblast (ach3p) and term placental endothelial cells (hpec) were analyzed for full length etr-a and known as well as unknown splice variants by sqrt-pcr using primers spanning the exons 2-5. the predominant dna bands in agarose gel were excised and sequenced. results: all tissues and cells expressed full length etr-a. in all tissues an additional 3 4-deletion variant was identified which was not found in the isolated cells. trophoblasts expressed another yet unidentified splice variant. the endothelial cells expressed a 3-deletion variant and a novel splice variant, which was identified as partial 2 -partial 3 deletion. this novel splice variant was found regardless of the arterial or venous origin of the cells as well as in the cell line (sv40-transformed). in tissues und trophoblast no difference in splicing was found between first trimester and term. conclusion: gestational age does not alter splicing of endothelin receptor-a. splicing is different between trophoblasts and endothelial cells. the endothelial cells contain a novel splice variant. the differential splicing may allow maternal and fetal endothelin-1 to induce different effects on the two major cell types of the placenta. (supported by grant 11258, jubilee fund, austrian national bank, vienna). adrenomedullin production and secretion by human trophoblast cells are regulated by glucocorticoids and hypoxia. francesca ciardo, 1 katia pacioni, 1 emanuela marinoni, 1 giovanna corona, 1 massimo moscarini, 1 alfredo patella, 2 romolo di iorio. 1 1 department of gynecology, perinatology and child health, university "la sapienza", rome, italy; 2 department of obstetetrics and gynecology, university of ferrara, ferrara, italy. objectives: adrenomedullin (am) is produced by intrauterine tissues and is involved in the regulation of implantation, placental hemodynamics and endocrine function. circulating am is increased in pregnancy complications such as preeclampsia and intrauterine growth retardation. we investigated whether am output by human trophoblast cells is regulated by hypoxia and/or glucocorticoids. study design: trophoblast cells obtained by human placentas at term (n=7) were cultured in presence or absence of hypoxia (3% o 2 ) and treated with or without betamethasone at the dose of 10 -5 , 10 -6 , 10 -7 m. media and cells were collected at 48h and at 2, 8 and 24h from syncytiotrophoblast cultures. am was measured in cultured media by specific ria kit. protein expression in trophoblast cells was evaluated with immunohistochemistry and western blot. results: hypoxia stimulated am output and protein expression by cytotrophoblast and syncytiotrophoblast cells. betamethasone induced an increase in am production and secretion in a time-and dose-dependent manner (figure). effects of hypoxia were partially reversed by betamethasone in a dose and time-dependent fashion. conclusions: am production in trophoblast cells is up-regulated by hypoxia and glucocorticoids, independently. increased am levels in pregnancy complications characterized by placental insufficiency might derived by an increase in am placental secretion stimulated directly by hypoxia or indirectly by an increase in fetal cortisol levels. preeclampsia complicates 5-8% of pregnancies, and while associated with significant maternal and fetal morbidity and mortality, the mechanisms responsible for preeclampsia are not completely understood. recent advances suggest there are imbalances of pro-and antiangiogenic factors, present from the time of implantation affecting vascular responsiveness of the fetal placental unit and maternal vasculature. in this study, we investigate vasomotor responsiveness of placental arteries from normal and preeclamptic (pet) pregnancies using an in vitro muscle bath contractility assay. transverse rings of placental arteries were cut and equilibrated in the muscle bath containing a bicarbonate buffer aerated with 95% o 2 /5% co 2 at 37°c. viability was demonstrated by contraction to 110mm kcl prior to all experiments. cumulative logarithmic dose dependent responses to four different contractile agents: pgf2 , serotonin, carbochol, and norepinephrine were compared. placental arteries precontracted with pgf2 at half maximal concentration were relaxed with three vasorelaxants: sodium nitroprusside (snp), forskolin (fsk) and lactate (lct). agonist studies revealed contraction to both pgf2 and serotonin but not to carbochol or norepinephrine. there were no statistically significant differences between the responses of normal and pet arteries. both normal and pet arteries demonstrated heightened responsiveness to sodium nitroprusside compared with forskolin and lactate. this study reveals that the placental vessels are more sensitive to sodium nitroprusside, that mediates relaxation through nitric oxide -cyclic gmp signal transduction pathway, than forskolin that induces relaxation through the cyclic amp pathway. cancer complicates approximately one in 1,000 pregnancies. depending on the type of cancer and trimester, patients can terminate the pregnancy or choose treatment such as chemotherapy. since there is limited knowledge on the safety of chemotherapy on fetal tissues in utero, clinicians cannot efficiently analyze the risks of particular anticancer agents when deciding on a course of therapy. since carboplatin is among the most common anticancer agents used for treatment of cancer during pregnancy the primary objective of this study was to evaluate if carboplatin will readily cross to placental barrier and determine total potential platinum fetal exposure. this project utilizes an ex vivo placenta perfusion model to determine the concentration of carboplatin that crosses the human placental barrier. placentas are obtained within 15 minutes of spontaneous delivery and re-perfused. two carboplatin concentrations were selected of 1000 ng/ml and 5000 ng/ml to represent clinically relevant maternal plasma concentrations. antipyrine was used as the internal control. serial samples were collected every 10 minutes for total 60 minutes in open-open model then experiment repeated with closed circuit model. antipyrine concentrations were determined by hplc methods. platinum concentrations in media samples were determined with a validated atomic absorption assay. a cell culture-based approach will be used to determine whether cell cycle or apoptotic proteins are altered (p53, bax, bcl-2, aif, and pro-caspase-3) using western blotting as a detection method. a total of two placentas have been completed at the low carboplatin concentration and one at the high concentration. the mean carboplatin clearance was 0.67 +/-0.01 ml/min and 1.04 +/-0.15 ml/min at the low and high carboplatin concentrations, respectively. an estimated 50% increase in the transport fraction was observed in the high concentration experiments compared to the low concentrations model. fetal carboplatin exposure ranged from 60 to 300 ng/ml. the placental perfusion experiments conducted at carboplatin 1000 and 5000 ng/ml indicate that carboplatin crosses the placenta through simple diffusion. the toxicology assays are ongoing to determine potential effect on fetal tissues. preterm delivery represents one of the predominant causes of perinatal mortality and morbidity, and is one of the most unpredictable gestational disturbances. urocortin is a peptide expressed by trophoblast and gestational tissues (amnion and chorion), whose maternal levels correlate with gestational length, since they are increased at preterm delivery and decreased in post-term pregnancy, when compared to term pregnancy. the addiction of urocortin enhances contractility of human myometrial strips, suggesting a possible role on uterine contractility. high maternal urocortin levels in threatened preterm delivery correlates with the timing of delivery, suggesting a possible role in the predictivity pf preterm delivery. in the present study urocortin concentrations in amniotic fluid collected at mid gestation for amniocentesis were correlated with gestational age at delivery. a case-control study with amniotic fluid obtained from healthy 60 women undergoing amniocentesis for genetic indications was performed; urocortin concentrations were measured by a specific elisa. amniotic fluid urocortin concentrations resulted significantly lower (p= 0.0025) in women delivering preterm (n=20; ucn= 1.23 ± 0.07 pg/ml) (m ± se) than in those delivering at term (n= 40; ucn= 0.77 ± 0.11pg/ml). in conclusion, the present preliminary data showed that amniotic fluid urocortin concentrations at mid gestation may represent predictive marker of preterm delivery. human placentas throughout pregnancy. annunziata mastrogiacomo, 1 elisabetta federico, 1 francesca caprio, 1 maria teresa schettino, 1 gabriele coppola, 2 antonio de luca, 2 luigi cobellis. 1 1 department of obstetrics and gynecology, second university of naples, naples, italy; 2 department of medicine and public health, section of anatomy, second university of naples, nales, italy. hypothesis apoptosis is intimately involved in placental homeostasis, growth and remodeling and the apoptotic rates increase progressively during normal pregnancy as part of normal placental development. moreover, apoptosis increases in pregnancies complicated by some pathologies such as preeclampsia, fetal growth restriction, diabetes. in the present study, we describe differences in the expression of pro-apoptotic protein bax, in first trimester voluntary termination of pregnancy, first trimester abortion (reserved abortion), caesarean birth, spontaneous birth, preeclampsia and diabetes. material and methods human placental samples were obtained with informed consent from patients undergoing surgery such as first trimester voluntary termination of pregnancy (n=15), first trimester abortion (miscarriage) (n=15), delivery section (n=15), spontaneous birth (n=15), preeclampsia (n=15) and diabetes (n=15). the gestation period ranged from 5 to 40 weeks. the specimens were immediately fixed in formalin for immunohistochemistry. we first observed a strong increase of bax expression in the cytotrophoblast, stroma, endothelial cells and decidua of placentas of the first trimester abortion compared to the low/moderate bax immunopositivity in all the placental compartments during the first trimester voluntary termination of pregnancy. secondly, we showed a more intense immunopositivity for bax in the third trimester spontaneous birth respect to the third trimester caesarean birth. thirdly, we observed an increase of bax expression in preeclamptic placentas compared to the normal full-term placentas. on the contrary, we observed a moderate bax expression in diabetic placentas only slightly decreased compared to the normal full-term placentas. our results seem to suggest that deregulation of apoptotic turnover may lead to placental dysfunction and pathologies. objectives: maternal endothelial activation in preeclampsia (pe) is attributed to the release of unknown factors from a hypoperfused placenta. the application of proteomic technologies such as mass spectrometry promises the reward of identifying and characterising these factors. using a placental explantconditioned culture media model system we have developed a proteomics workflow to obtain relative quantification of proteins released into placental explant culture media. methods: term villous explants were cultured in serum-free conditions and exposed to differing oxygen concentrations (6% 1%) to mimic physiological and non-physiological intervillous o 2 tensions. the d4 media was concentrated, immunodepleted to remove fibrinogen (using beckman igy-12 columns) and proteins labelled using an itraq (applied biosystems) kit following the manufacturer's protocol. labelled peptides were fractionated by strong cation exchange and then analysed using lc-maldi on a 4800 maldi-tof/tof (applied biosystems) mass spectrometer. data was analysed using protein pilot 2.0 (applied biosystems). results: when matched pooled (n=6) media samples were subjected to our proteomics workflow over 590 proteins were identified with a calculated false positive identification rate of <1%. of these proteins a total of 29 display a statistically significant difference in protein levels between the 1% 6% o 2 samples (p=<0.001). from a list of 40 proteins of interest we selected 7 proteins for validation using elisa/western blotting to measure their relative abundance in both pooled and individual explant media samples. interleukin 8 is an example of a low-abundance differentially expressed protein identified in our proteomic workflow and subsequently validated by elisa; il-8 (pg/ml) in 6% o 2 : 43.7, range 30.8 to 47.3; 1% o 2 : 103.0, range 57.6 to 141.0; values are median with interquartile range. *** p<0.0001, mann-whitney test (n=40) conclusions: we demonstrate a successful reduction to practice of a relative quantitative proteomics strategy applied to placental explant-conditioned culture media. having optimised and validated this proteomic workflow we will apply the same methods to compare proteins released by normal and preeclamptic placentas. a proteomics screen of human placental microvillous syncytiotrophoblast (stb) revealed the expression of dysferlin (dysf), a membrane repair protein associated with certain muscular dystrophies (vandré et al., 2007) . a second ferlin protein, myoferlin (myof), was also discovered which has not yet been characterized in the placenta. in human c2c12 myoblasts, dysf and myof are reciprocally expressed as a function of differentiation into multinucleate syncytial myotubes, with dysf predominating in differentiated cells. we hypothesized that dysf and myof would show a similar reciprocal expression pattern in human trophoblasts as a function of syncytialization. methods term placentas from uncomplicated pregnancies were obtained with informed consent (n=8) and either fixed for immunofluorescence (if) labeling or flash frozen for subsequent immunoblotting (ib). human trophoblastic (bewo, jar, and jeg-3) and other cell lines (mrc-5, hl-60, huvec) were cultured in the absence or presence of 20 μm forskolin or solvent control for 0-3 days. dysf and myof expression was examined by rt-pcr, ib, and if. results ib validated proteomics data showing myof expression in term placenta. by if, myof labeling was predominant in apical and basal stb plasma membranes. myof was expressed in trophoblastic cell lines (bewo, jar, and jeg-3), cultured endothelium (huvec), and a fibroblast cell line (mrc-5), but was not detected in a leukemic cell line (hl-60). dysf was constitutively expressed in jar, but minimally expressed in unstimulated bewo. following forskolin-induced syncytialization, we observed a time-dependent increase in dysf expression in bewo concomitant with increasing syncytin-1 levels. by if, dysf expression was restricted to bewo cells in syncytial structures. in contrast, myof expression was robust in mononuclear bewo cells and not down-regulated over the course of 3 days of differentiation. conclusions two ferlin-family genes are expressed in trophoblasts. fusion-competent bewo cells behave similarly to cultured myoblasts and ctbs with regard to dysf expression, which is restricted to syncytializing cells. myof, in contrast, is expressed constitutively in bewo and other models. while both proteins are likely to function in stb plasma membrane repair (e.g., following syncytial sprouting), the relative contributions of each to this process awaits clarification. features of chronic placental insufficiency are significantly more common in small for gestational age placentas from infants with intrauterine growth retardation. emily king, 1 violetta kolesnikova, 1 carri tillotson, 2 jean-marie guise, 3 terry morgan. 1 1 pathology; 2 center for biostatistics; 3 obstetrics and gynecology, ohsu, portland, or, usa. background: there is a strong association between intrauterine growth restriction (iugr) and small for gestational age (sga) placentas (heinonen et al. placenta (2001), 22:399-404) . the cause is likely multifactorial, but we hypothesize that chronic uteroplacental insufficiency may play a role. our objective was to test whether sga placentas from human neonates with iugr show significantly more pathologic features of placental insufficiency compared to controls. design: we performed a retrospective review of 242 consecutive singleton placentas submitted to ohsu pathology (2005-06), excluding elective terminations and spontaneous abortions before 22 weeks gestation. clinical records were reviewed and pregnancy outcomes recorded, including: 1) neonatal sex; 2) weight (iugr calculated by routine methods), 3) trimmed weight of the placenta (sga calculated by routine methods), 4) gross placental infarctions, 5) gestational age at delivery, and 6) maternal features (e.g. race, gravida). controls were defined as cases within the series without sga or iugr (e.g. submitted for meconium, infection, etc). histologic sections were scored by two pathologists while blinded to clinical diagnoses as positive or negative for features of placental insufficiency, including accelerated villous maturation (avm), chorangiosis, and microscopic infarctions. significant associations were tested by 2 analysis and logistic regression for multiple variables. results: similar to prior reports, we observed a strong association between iugr and sga placentas ( 2 30.5; p <0.0001). this relationship was independent of maternal race, fetal sex, and parity, although it was more common in primigravidas. avm and placental infarctions were significantly more frequent in sga placentas with superimposed iugr. controls ( introduction: messenger rna expression peripheral blood cells (pbc) has been recently used as biomarkers of environmental exposures (ionizing radiation or tobacco), physiological conditions (stress) and diseases (hypertension, neurological disorders). pbc evaluation is a useful diagnostic tool in an era of individualized medicine. since there is an urgent need for non-invasive methods for determination of fetal (f) and placental (p) function, this study was designed to evaluate the genes differently and commonly expressed in p tissue and leukocytes in maternal (m) and f circulation.material and methods. p (n=5), f (n=3) and m blood (n=3) were obtained during cesarean section in pregnant baboons at term. total rna from a buffy coat pellet was isolated using a modified procedure of the qiagen rneasy mini kit (qiagen). anti-sense rna (arna) was synthesized and purified using the illumina rna amplification kit (ambion, usa). hybridization of arna to illumina sentrix human whole genome (wg-6)beadchips and subsequent washing, blocking and detection was performed using illumina's beadchip 6´2 protocol. samples were scanned on the illumina beadarrayer 500gx reader using illumina beadscan image data acquisition software (ver. 2.3.0.13). differential gene expression data analysis was performed using illumina beadstudio software (ver. 1.5.0.34). results. the detection level of gene transcripts using illumina methodology with control human rna was 6000-7000 at p<0.0001. 4329 gene transcripts were detected in f blood and 2957 in maternal blood. 1452 transcripts were uniquely expressed in fetal blood. 130 transcripts were found in m, but not in f leukocytes. the number of gene transcripts expressed in p tissue was 4825 and of these 2953 genes were not expressed in m leukocytes. conclusion. despite white blood cells trafficking through the p barrier there is a set of unique genes expressed only in p or in the m or f circulation. the application of these genes as the biomarkers of p barrier function still need to be evaluated. objective: to evaluate if a relationship exists between duration of placental exposure to meconium in vivo and histologic evidence of severity and extent of meconium uptake by macrophages. study design: from a cohort of 353/7069 (5%) consecutive singleton liveborn infants delivered at term with thick meconium-stained fluid, 193(55%) had placental histologic examination performed, and in 50/193 the timing of meconium appearance after membrane rupture was documented. placental histologic examination quantitatively evaluated the intensity of meconium uptake by resident macrophages based on the number of macrophages/field, and the extent of uptake based on histologic location, graded in a score 0 to 3. results: mean interval between meconium appearance and delivery was 136.7± 126.7 min (range 10-510). after exclusion of 6 cases in which severe placental inflammation interfered with analysis, meconium uptake by macrophages was documented in 40/44 cases at the level of amniochorionic membranes, in 34/44 cases at the placenta, and in 18/44 cases at the umbilical cord. there was no correlation between the interval meconium appearance-to-delivery in relation to presence of meconium in the membranes (p=0.53), in the placenta (p=0.78), in the cord (p=0.71), or score of severity of meconium uptake (p=0.76). the results did not change after correcting for gestational age, oligohydramnios, presence of placental acute inflammatory or vascular lesions. conclusion: there is no relationship between duration of placental exposure to meconium and the extent and intensity of its uptake by macrophages in cases with exposure up to 8.5 hours. transfer of bisphenol a across the human placenta. biju balakrishnan, 1,2 kimiora henare, 1,2 eric thorstensen, 1,2 murray d mitchell. 1,2 1 the liggins institute, university of auckland; 2 national research centre for growth and development, auckland, new zealand. introduction: there are growing concerns over the effects of developmental exposure to the xenoestrogen bisphenol a (bpa). animal studies have shown that bpa is transferred through the placenta and can cause deleterious effects to the fetus. the presence of bpa in feto-placental tissues in humans has also been reported. however, a detailed study of the time-course of bpa transfer across the human placenta has not been performed. the aim of this research was to study the transfer of bpa in ex-vivo perfused human placental tissues. methods: a dual recirculating single cotyledon perfusion was used to monitor the placental transfer of bpa. bpa (10ng/ml), antipyrine (ap) (40μg/ml), and fitc dextran (fitc-dx) (12.5μg/ml) were added to the maternal perfusate, and perfusion was continued for 4 hours. perfusate samples were collected from both reservoirs at timed intervals and analysed. bpa, ap, and fitc-dx were determined by hplc with fluorescent detection, hplc with uv detection, and spectrofluorometry respectively. the viability and metabolic activity of the placentae were assessed by measuring -hcg (elisa), glucose utilization, and lactate production (autoanalyzer). fetal pressure, ph, flow rates and fluid shifts were monitored continuously. results: the biochemical validation parameters (glucose consumption, lactate production and -hcg secretion) indicated that the placental tissue was metabolically active and viable throughout the 4-hour perfusion period. the physical parameters observed (fetal pressure <50 mmhg, ph range 7.2-7.6) were in concordance with other published works for placental perfusion. membrane integrity was confirmed by fluid shifts from either circuit of <5ml/hr, and by <2% materno-fetal transfer of fitc-dx. an observed ap transfer of 20-30% further validated our model. bpa first appeared in the fetal compartment within 30 minutes of perfusion and reached a peak of about 25-30% of maternal concentration within 3 hours of perfusion. this figure is likely to be an underestimate since it does not include conjugated bpas. conclusion: this first study of bpa transfer in ex-vivo perfused human placental tissue shows that our model can serve as a useful tool to study the transfer kinetics and metabolism of bpa in human term placentae. we found that bpa rapidly crosses the human placenta at environmentally-relevant doses, with potentially harmful effects on the human fetus. angiogenic growth factor secretion by uterine natural killer cells in co-culture with extravillous trophoblast. gendie e lash, 1 katsu naruse, 1,2 barbara a innes, 1 stephen c robson, 1 judith n bulmer. 1 1 institue of cellular medicine, newcastle university, newcastle upon tyne, tyne and wear, united kingdom; 2 obstetrics and gynaecology, nara medical university, nara, japan. objectives. uterine natural killer (unk) and extravillous trophoblast (evt) cells have been proposed to play roles in remodeling of uterine spiral arteries through secretion of various angiogenic growth factors. we have previously demonstrated that unk cells are a significant source of angiogenic growth factors within the decidua, many of which alter with gestational age. however whether the secretion of these proteins is altered by interactions of unk cells with evt is unclear. hypothesis. unk cell angiogenic growth factor secretion is regulated by evt in early pregnant decidua. methods. placental and decidual samples were collected from women undergoing termination of pregnancy with written informed consent (8-10 and 12-14 weeks gestation, n=12 each group). 0.2×10 6 cd56 + unk cells were positively selected from decidua and co-cultured with evt (0.2×10 6) or cytotrophoblast (ctb; 0.2×10 6 ) purified from the same placenta for 24h in direct or indirect contact (n=6 each group). angiogenin, ang2, pdgf-bb, fgf-basic, timp1, icam1 and vegf-a were measured by fast quant ® angiogenesis multiplex assay system, and vegf-c, ang1 and plgf by elisa. results of unk co-culture with evt or ctb (negative control) at each gestational age were analyzed with wilcoxon rank test. in addition, the effect of direct and indirect co-culture of unk cells with evt at each gestational age was compared with mann whitney u test. results. at 8-10 weeks gestation unk secretion of ang2 (p=0.03), icam1 (p=0.05) and plgf (p=0.03) was increased in the presence of evt compared with ctb. no differences were observed at 12-14 weeks gestation. in addition, at 8-10 weeks gestation unk secretion of ang2 (p=0.004), vegf-c (p=0.007) and ang1 (p=0.004) was increased in direct co-culture with evt compared with indirect co-culture. at 12-14 weeks gestation unk secretion of timp-1 (p=0.03) was reduced in direct co-culture with evt compared with indirect co-culture. conclusions. unk cell secretion of several key angiogenic growth factors was altered by direct culture with evt. these data suggest that a membrane bound molecule (such as hla-g) mediates this modulation of unk cell activity. abnormalities in placentation and impaired placental circulation can lead to fetal growth restriction. 3-d ultrasound can be used to evaluate this through the quantification of the power doppler signal that may be expressed as a percentage of colour voxels within a user-defined volume (vi: vascularisation index). we aimed to test the hypothesis that increased fetoplacental blood flow correlates with an increased vi, using the in vitro dual perfusion model of the human placental lobule. three term lobules were dually perfused through both circulations with earles bicarbonate buffer (ebb), and supplemented on the fetal-side only with adult erythrocytes, prepared to a 5% haematocrit. following initial equilibration perfusion at normal flow values, fetal-side flow was varied between 1 and 10 ml/min, whilst maternal-side flow was held at 14 ml/min. images were obtained with a 'voluson i' ultrasound machine and a neonatal transducer (pulse repetition frequency = 0.6hz, wall motion filter = low 1, and gain = 0.0). three 3-d datasets were acquired at each flow rate from each placental lobule and these were measured in triplicate using vocal. a sphere was centred on a visibly perfused cotyledon along the chorionic-decidual axis, with a diameter corresponding to placental thickness. linear regression analysis was used to assess the relationship between the total fetal-side flow and mean vi. the mean vi showed a high degree of correlation with total fetal-side flow for each lobule ( figure 1 ) suggesting increased vascular perfusion and the inclusion of perfused vessels that cross the detection threshold with increased flow. this data provides qualifying information for translation to a clinical application, where early gestational fetoplacental blood flow will be assessed to predict the onset of fetal growth restriction. anthony n imudia, 1 brian a kilburn, 1 anelia petkova, 1 samuel s edwin, 2 roberto romero, 2 d randall armant. objective survival of first trimester cytotrophoblast cells depends on their upregulation of heparin-binding egf-like growth factor (hbegf), which is downregulated in placental tissues diagnosed with preeclampsia. we have examined the expression and cytoprotective activity of hbegf in term villous explants subjected to hypoxic stress in vitro. non-pathological placentas were collected by cesarean section at term (n=6). chorionic villous explants were prepared and cultured at either 20% or 2% o 2 and treated with the hbegf antagonist crm197 or recombinant hbegf. paraffin sections were assayed for trophoblast cell death by the tunel assay, proliferation by immunohistochemical labeling of nuclear ki67 and hbegf expression by semi-quantitative immunohistochemistry. data were compared using anova and the student-newman-keuls posthoc test. results crm197 (10 mg/ml) increased trophoblast cell death after culturing villous explants 8 h at 20% o 2 (p<0.05), but only slightly affected proliferative capacity. culture at 2% o 2 increased trophoblast cell death 100% above explants incubated at 20% o 2 (p<0.05). trophoblast cell proliferation decreased after 24 h in explants cultured at either 20% or 2% o 2 (p<0.05). exogenous hbegf (1 nm) prevented the elevation of cell death during hypoxia (p<0.05) and maintained nuclear ki67 expression at 20%, but not 2%, o 2 . contrary to first trimester trophoblast, hbegf was not upregulated by hypoxia in term trophoblast. the failure of term trophoblast to elevate hbegf expression in response to hypoxia could contribute to their decreased survival at low o 2 compared to early gestation. endogenous hbegf signaling appears to facilitate survival of term trophoblast during villous explants culture. exogenous hbegf supplementation prevented cell death due to hypoxia and maintained trophoblast proliferation rates under in vitro conditions. therefore, hbegf, which is downregulated in preeclampsia, could have significant impact on trophoblast survival during late gestation. supported by the intramural research program of nichd. conspicuous meconium-laden macrophages in the chorionic plate are an independent predictor of clinically significant fetal distress. violetta kolesnikova, 1 emily king, 1 carrie tillotson, 2 jean-marie guise, 3 terry morgan. 1 1 pathology; 2 center for biostatistics; 3 obstetrics and gynecology, ohsu, portland, or, usa. background: meconium is a common indication for placental examination, present in approximately 30% of placentas routinely submitted to pathologists (beebe et al. obstet gynecol 1996; 87:771-8) . prolonged meconium exposure leads to accumulation of meconium-laden macrophages in the chorionic plate (estimated to require at least 3 hours). our objective was to test whether prolonged meconium exposure is associated with clinically significant fetal distress. design: retrospective review of 250 consecutive singleton placentas submitted to ohsu pathology (2005-06) was performed, excluding abortions before 22 weeks gestation, and placentas without representative sections of the chorionic plate. clinical records were reviewed and pregnancy outcomes recorded, including: 1) evidence of fetal distress prompting c-section (non-reassuring fetal heart rate), 2) gestational age, 3) maternal diagnosis, 4) apgar scores, and 5) neonatal length of hospital stay. routine histologic sections were independently scored by two pathologists while blinded to clinical diagnoses and outcomes. cases were scored as positive or negative for: 1) diffusely conspicuous meconium-laden macrophages in the chorionic plate (at least 1/ hpf), 2) chorioamnionitis, and 3) features of placental insufficiency. significant associations were tested by the mann-whitney u test for paired comparisons, x 2 analysis, and logistic regression for multiple variables. results: conspicuous meconium staining of the chorionic plate was common (86/250, 34%) and was significantly more frequent in c-sections performed for fetal distress (19/35 cases, 54%) (x 2 7.1; p-value <0.01). logistic regression modeling showed that this association was independent of chorioamnionitis and features of placental insufficiency. there was no association between meconium and neonatal outcome, including no difference in apgar scores or length of hospital stay. conclusions: our data support the hypothesis that meconium-laden macrophages in the chorionic plate are associated with fetal distress prompting c-section. whether meconium is the cause or consequence of this distress is uncertain. however, given the acute time frame between clinical diagnosis and c-section, we suspect prolonged meconium exposure may be a significant cause of non-reassuring fetal heart rate changes. apoptotic index in normal and intrauterine growth-restricted rat placentas. elissa scotland, tri nguyen, s chiang, radmila runic. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: intra-uterine stress caused by maternal food-restriction may have adverse fetal and placental effects. we sought to assess the effect of maternal food restriction during pregnancy on placental apoptosis. methods: rat placentas were analyzed from control dams fed ad libitum and dams 50% food-restricted from day 10 of gestation. placentas were harvested at embryonic days 16 and 20 (n=3). placentas were fixed in 4% pfa and two methods of analysis were utilized to determine the proportion of apoptotic to non-apoptotic cells within maternal food restricted and control rat placentas: immunohistochemistry (ih) using fas-ligand and in situ tunel (terminal deoxynucleotidyl transferase biotin-dutp nick end labeling). tunel: after rehydration, slides were subjected to tdt enzyme. dab was used to visualize brown apoptotic nuclei. dna-se treated slide was used as a positive control. ih: primary rabbit polyclonal fas-ligand antibody was used at 1:100 dilution, after which secondary antibody linked to peroxidase and dab staining was performed. results: food-restricted placentas demonstrated 9.29% relative apoptotic index when compared to 2.95% in the control group. the iugr iod (integrated optical density) per unit area in the placental membrane was higher than in the control group. fasl demonstrates an iod of 0.112 + 0.025 in food restricted placentas as compared to 0.049 + 0.003 per μm2 surface area in controls. apoptosis was seen in the amnion as well as trophoblast (syncytialtrophoblasts and some cytotrophoblasts). conclusion: the increased apoptotic index in maternal food restricted placentas suggests that the accompanying iugr may be a result of both maternal/fetal nutrient restriction and increased fetal stress. this study found that maternal food restriction during pregnancy affects placental apoptosis. there is more apoptosis in the iugr placental bed at e16 than at e20, with the reverse being true for the placental membrane. more research is required to statistically validate the data. the suggested next step is to evaluate fas and fas-l and to address possible mechanisms of placental apoptosis. cord. jayaraman lakshmanan, 1 avish arora, 1 lilit baldjyan, 2 sharon k sugano, 1 olga miadel, 2 adegoke adeniji, 2 michael g ross, 1 calvin j hobel. 2 1 ob-gyn, harbor-ucla medical center, torrance, ca, usa; 2 ob-gyn, cedars-sinai medical center, los angeles, ca, usa. background: crf-bp is a 37kda protein, that specifically binds corticotrophin releasing factor (crf). the structure of cloned crf cdnas in all species examined predicts that the precursor is larger than the 37kda in size and contains one n-glycosylation site. marked reductions in plasma crf-bp levels seen in pregnant women prior to both preterm and term delivery led to the notion that crf-bp is a "gatekeeper" of crf responses. recently we identified crf-bp expression in term human umbilical cord (uc) by immunohistochemical analyses. objective: to characterized the expression of crf-bp by immunohistochemical and biochemical analyses in human uc. methods: freshly obtained human preterm (28 to <37 weeks, n=6) umbilical cord (6 pieces of 3mm thickness taken at 1-2 cm intervals close to placenta) were fixed in bouin's solution and paraffin embedded. also, pieces of ucs were dissected, arteries and vein separated, weighed and frozen at -80c. for immunohistochemical localization, uc sections were subjected to immunostaining with polyclonal antibodies to human crf-bp precursors. for western blot analyses, whole uc as well as isolated arteries and vein were homogenized in a buffer containing detergents and protease inhibitors. the homogenate supernatant proteins were subjected to western analyses by standard protocol. immunoreactive protein bands were identified by chemiluminescent reagent. results: both goat and rabbit crf-bp polyclonal antibodies elicited weak to moderate positive staining in uc epithelial layers, vascular musculature and barely endothelial cells. they identified a strong 50kda band in whole uc as well as in isolated artery and venous preparations. in addition minor immunoreactive protein bands of 37, 25, 20kda in size were noticed in all three preparations. conclusion: we conclude that preterm uc expresses crf-bp. we postulate that the 50kda major band is either glycosylated crf-bp precursor or glycosylated 37kda mature protein. the low molecular weight immunoreactive proteins likely represent proteolytically processed, glycosylated crf-bp precursor or a proetolytically processed mature 37da crf-bp. uc obtained at delivery could be useful as a tool to understand the critical functions of crf-bp in feto-placental unit. objective: adenosine, known to be released from inflammatory sites and tisse ischemia, has many important biologic roles. four specific adenosine receptors have been cloned to date, termed a1, a2a, a2b, and a3. recently our study has shown that increased a3 receptor in the trophoblast of preeclamptic pregnancy was noted and non-vascular and trophoblast-mediated a3 receptor may play an important role in the pathogenesis of preeclampsia. there are evidences of impaired trophoblast invasion related to matrix metalloproteinase (mmp) in preeclampsia and the relationship between adenosine receptor and mmp in other fields. the objective of this study is to evaluate the effect of mmp expression by adenosine a3 receptor in preeclamptic villous explants at different oxygen conditions. methods: placental villous explants from normal (n=10) and preeclamptic (n=10) pregnancies were cultured at high (20%) and low (3%) oxygen levels for 5 days. explants were analyzed for mmp-2/-9 and timp-1/-2 by rt-pcr and western blot. preeclamptic villous explants in hypoxic culture condition were treated with a3 receptor agonist, cl-ib-meca and a3 receptor antagonist, mre. mmp-2/ -9 expression was determined in a time-and dose-dependent manner by rt-pcr, western blot. also mmp-2/-9 activity was evaluated by zymogram assay. results: there were significantly increased a3 receptor intensity and reduced mmp-2/-9 and timp-1/-2 expression at low oxygen level in normal and preeclamptic villous explants. interestingly, in preeclamptic villous explants, after high oxygen culture mmp-2/-9 and timp-1/-2 expression were recovered to almost same level compared to those in normal villous explants. treatment of preeclamptic villous explants with cl-ib-meca in low oxygen level resulted in a time-and dose-dependent enhanced expression of mmp-2/-9. this cl-ib-meca-induced expression of mmp-2/-9 was inhibited by pretreatment with mre. conclusion: to our knowledge, this study is the first to evaluate modulation of mmp secretion by adenosine a3 receptor in preeclamptic villous explant. our results provide evidence for the existence of functional adenosine a3 receptors in the trophoblast and suggest that adenosine a3 receptor will be investigated as a therapeutic target in preeclampsia. murray d mitchell, 1 timothy a sato, 1 anderson wang, 1 jeffrey a keelan, 1 anna p ponnampalam, 1 michelle glass. 2 1 liggins institute; 2 department of pharmacology and clinical pharmacology, university of auckland, auckland, new zealand. introduction: it is well established that prostaglandins play critical roles in multiple aspects of pregnancy and that the fetal membranes are an important site of intrauterine prostaglandin production. endocannabinoids have been implicated in the maintenance of pregnancy and parturition in women and are a source of arachidonic acid which is a substrate for the production of prostaglandins. the aim of the present study was to determine the effects of endocannabinoids on the production of prostaglandins in extraplacental membranes -amnion, chorion and decidua. methods: explants of term amnion and choriodecidua were established and treated with endogenous endocannabinoids 2-arachidonoyl glycerol (2ag) and anandamide (aea) and with the synthetic cannabinoid cp55,940, to determine the ability of these substances to modulate prostaglandin e 2 (pge 2 ) production. pge 2 was measured by radioimmunoassay. the explants were also treated with cp55,940 in the presence of either sr141716a (a selective antagonist of the cannabinoid receptor cb1) or ns398 (a cox-2 inhibitor), to determine whether any observed stimulation of pge 2 production was mediated through cox-2 activity and/or the cb1 receptor. cox-1, cox-2, cpla2 and pgdh protein levels were measured by western blotting. results: all three cannabinoids caused a significant increase in pge 2 production in amnion but not in choriodecidua. however, separated fetal (chorion) explants responded to cannabinoid treatment in a similar manner to amnion, whereas maternal (decidual) explants did not. the enhanced pge 2 production caused by cp55,940 was abrogated by co-treatment with either sr141716a or ns398, illustrating that the cannabinoid action on prostaglandin production in fetal membranes is mediated by cb1 agonism and cox-2. preliminary data from western blotting show that cannabinoid treatment results in the up-regulation of cox-2 expression. however, there was no change in cox-1 expression and no evidence either for up-regulation of cpla2 or for down-regulation of pgdh expression. conclusion: this study demonstrates a potential role for endocannabinoids in the modulation of prostaglandin production in late human pregnancy, with potentially important implications for the timing and progression of term and preterm labour and membrane rupture. inactivation of vegf receptor-2, but not vegfr-3 or vegfr-1, during the peri-implantation period prevents normal pregnancy development in the rodent through disruption of uterine angiogenesis. nataki c douglas, 1 hongyan tang, 1 raul gomez, 1 bronislaw pytowski, 2 daniel j hicklin, 2 jan kitajewski, 1 mark v sauer, 1 ralf c zimmermann. 1 1 division of reproductive endocrinology and infertility, columbia university, new york, ny, usa; 2 imclone systems, inc., new york. objective: vegf is involved in the regulation of uterine angiogenesis and implantation in both rodents and non-human primates. vegfr-1, r-2, and r-3 are expressed in the uterine decidua and are involved in the regulation of vessel formation in many systems. to determine if these receptors have a functional role in the regulation of post-implantation angiogenesis and pregnancy development, we examined the effects of blocking vegfr-1, r-2, and r-3 function. design: prospective animal laboratory material and methods: to avoid effects of vegf receptor neutralization on ovarian function, we utilized a progesterone replaced, ovariectomized mouse model. vegf receptor blocking antibodies were administered on ed 3.75, prior to embryonic expression of these receptors. embryonic development was evaluated on ed 10.5, blood vessel density and apoptosis on ed 7.5, and cellular proliferation on ed 5.5 (n=5 per time point in each group). anova with bonferroni correction was used to compare sample means. results: see tables. conclusions: neutralization of vegfr-2 and vegfr-3, but not vegfr-1 resulted in a significant reduction in cellular proliferation and decidual angiogenesis. vegfr-3 mediates decidual angiogenesis, but is not required for normal pregnancy development. in contrast, an intact vegf/vegfr-2 pathway is required for the decidual angiogenesis that mediates early pregnancy development. effect of vegfr neutralization on ed 10.5 control anti r-1 anti r-2 anti r-3 no. of implantation sites 10.7 +/-1.2 11 +/-0.9 11.0 +/-1.1 14.0 +/-0. hoxa10 encodes a transcription factor required for endometrial receptivity and embryo implantation. our objective was to identify and to characterize those molecular markers regulated by hoxa10 in multiple cellular model-systems. using microarray technologies, we identified putative hoxa10 target genes involved in early implantation. liposome-mediated transfection delivering either empty vector or the same plasmid constitutively expressing hoxa10 was introduced into newly impregnated mice during laparotomy or layered onto cultured human endometrial stromal-cells (hescs). rna products from these in vivo and in vitro transfections were used to identify targets and to validate the microarray screen employing semi-quantitative real-time pcr (qrt-pcr). we identified 82 statistically-significant genes regulated by hoxa10 overexpression of which 57 genes were down-regulated greater than 2-fold when compared to controls. cellular ontogenies of differentially-expressed genes include: cell adhesion molecules, signal transduction factors as well as metabolic regulators. furthermore, we identified the 3-phosphoglycerate dehydrogenase gene, (pgdh) whose products are regulated by hoxa10 during implantation in both murine model systems in and cell culture. this genes codes for an enzyme critical to de novo l-serine biosynthesis via a phosphorylation-dependent pathway. microarray analysis demonstrated a 2fold expression decrease when hoxa10 is overexpressed. this diminution in pgdh expression was noted in the validation experiments using qrt-pcr and corroborated in hesc cells where the mrna levels decreased to 40% when compared to controls. the repression of pgdh during the implantation window may represent a conservation of activity as secretory-phase protein synthesis may be suppressed in order to promote cellular differentiation and resultant implantation. these regulatory relationships identified in mouse implantation likely function to enhance uterine receptivity and may have a role in human implantation. objective: hoxa10 is expressed in endometrium, where it is regulated by sex steroids and is necessary for endometrial receptivity and implantation. hoxa10 is also expressed in leukocytes. here we hypothesized that hoxa10 would be regulated by sex steroids in both cell types. we further hypothesized a correlation between expression of hoxa10 in peripheral blood cell (pbcs) and endometrium in both mice and in humans. methods: real-time pcr was used to determine differential expression of hoxa10 mrna in u937 cells, a monomyelocytic cell line, in response to increasing concentrations of estradiol. to determine if hoxa10 is expressed in leukocytes in vivo, peripheral leukocyte hoxa10 mrna expression was measured over sequential estrus cycles in mature cd1 nulliparous mice and correlated to vaginal smear-cytology. additionally, peripheral leukocytes were isolated either from pregnant or normally-cycling women to assess hoxa10 mrna expression. results: there was a direct, dose-responsive correlation between exposure to increasing estradiol and hoxa10 mrna expression levels in u937 cells. in a murine model, we demonstrated that hoxa10 mrna expression-levels varies throughout the estrus cycle with a marked increase in expression following vaginal plug detection. the nadir of hoxa10 expression is prior to proestrus and increased up to 2-fold during the receptive phase. this increased expression continues throughout gestation. the heightened expression in murine leukocytederived hoxa10 mrna also is demonstrated across species. our preliminary data suggests that the greatest fold-increase of expression occurs during the window of implantation of the secretory phase in normal, cycling women. this level was sustained in pregnancy. there appears to be a trend with the highest levels of expression associated with viable gestations. women with attenuated expression profiles had non-viable gestations. conclusions: hoxa10 expression is regulated by sex steroids in both leukocytes and endometrium. the temporal pattern of peripheral hoxa10 transcript expression demonstrated in mice and humans mimics the differential rna expression documented within the uterus. leukocyte hoxa10 expression during the reproductive cycle in mice and humans is a marker of endometrial receptivity. peripheral leukocyte expression of hoxa10 mrna may correlate with implantation success. carolien m boomsma, annemieke kavelaars, marinus jc eijkemans, gijs teklenburg, bart cjm fauser, cobi heijnen, nick s macklon. reproductive medicine and gynaecology and laboratory of psychoneuroimmunology, university medical center, utrecht, netherlands. the purpose of this study is to assess the association between the intra-uterine cytokine expression profile at the time of embryo transfer and successful embryo implantation following ivf/ icsi treatment. materials and methods 210 women undergoing ivf/ icsi underwent endometrial secretion aspiration prior to embryo transfer. 16 known soluble mediators of implantation were measured using a multiplex immunoassay, namely il1ß, il5, il6, il10, il12, il15, il17, il18, tnf , vegf, ifn , eotaxin, mcp-1, ip-10, dkk-1 and hbegf. mif was determined using an elisa. the total protein concentration was measured for normalization purposes. data were log transformed to obtain normal distribution. multivariable logistic regression analysis with a backward elimination procedure (p<0.1) was used, potential confounders (age, blood contamination, embryo quality) were included in a forward stepwise model. ten mediators of the 17 analysed were detectable in 90-100% of the samples. il15 was detectable in 76% of samples, dkk-1 68%, il-10 56%, il-17 54%, il-5 35% and hbegf was detectable in 23% of samples. ifn-was not detectable in any of the samples. multivariable logistic regression showed only logmif concentrations to have a significant correlation with achieving clinical pregnancy (p =0.036). higher mif concentrations were correlated with a higher chance of conceiving. endometrial secretion analysis represents a novel means of assessing the intra-uterine milieu encountered by the embryo and offers new perspectives in the study of endometrial receptivity. in this large prospective study assessing an array of cytokines, mif was found to be significantly correlated with pregnancy. mif, macrophage migration inhibitory factor, is a cytokine with numerous proinflammatory, immunomodulatory, angiogenic and tissue remodelling properties. mif induces the synthesis and secretion of matrix metalloproteinases by endometrial cells, which may contribute to embryo invasion. its expression is particularly increased during the secretory phase, suggesting a role in reproductive processes. analysis of aspirated endometrial secretions offers a direct clinical test of endometrial receptivity which can be applied during treatment cycles without disrupting implantation. endometrial receptivity and secretory differentiation require progesterone (p). it has been hypothesized that low p levels result in delayed endometrial differentiation and infertility due to reduced receptivity. objective: test the effects of low luteal p on histologic and molecular markers of differentiation and function. methods: normal cycling women (n=12) were treated with daily leuprolide (1.0 mg/d) beginning in the midluteal phase and continuing through the protocol. after menses, subjects received transdermal estradiol (e, 0.2 mg/d) for 20 days. after day 10 of e, subjects also received daily i.m. injections of p, randomized to 10 mg/d (sub-physiological) or 40 mg/d (physiological). endometrial biopsy was performed after 10 days of combined e and p treatment. additional untreated women had biopsies performed 10 days after spontaneous lh surge. endometrial histologic dating was performed by two individuals according to the criteria of noyes et al. mrna levels were assessed using real-time rt-pcr. results: mean(s.d.) of peak and trough p serum concentrations in the 40 mg/d p group were 18.2(5.1) and 9.4(4.8) ng/dl, respectively, while those in the 10 mg/d group were 7.0(3.0) and 3.4(1.0) ng/dl. there were no differences between treatment groups for histologic dating; the mean(s.d.) histologic date was 25.7(0.8), 24.4(0.9), and 24.8(1.3) for the 10 mg, 40 mg, and spontaneous cycle groups, respectively. there also were no differences among the three groups in mrna levels of ten functional markers (er , pr, 3 integrin subunit, osteopontin, cyr61, egr-1, fkb52, c-fos, and cd55), although variability of gene expression was greater in those who received 10 mg/d p than in those who received 40 mg/d p. there was also no correlation between serum progesterone level and gene expression or histologic date. conclusions: sub-physiological levels of progesterone, in the range seen in ovulatory women, do not induce detectable changes in expression of marker genes or histological dating, although low p levels were associated with greater variability of gene expression. these data suggest that abnormalities in endometrial histologic development and function likely result from intrinsic abnormalities rather than from low levels of p secretion. (supported by unc nova carta fund and nih u54 hd-35041). endometrial ip10 attracts trophectoderm through cxcr3 interaction. simcha yagel, 1 caryn greenfield, 1 hen sela, 1 jacob hanna, 2 irit manaster, 2 orna singer, 3 ronit haimov-kochman, 1 shira natanson-yaron, 1 diana prus, 1 benjamin reubinoff, 2 debra s goldman-wohl, 1 ofer mandelboim. 2 1 obstetrics and gynecology, hadassah-hebrew university medical centers, jerusalem, israel; 2 lautenberg center for immunology, jerusalem, israel; 3 human embryonic stem cell research center, jerusalem, israel. introduction: implantation is initiated in part by attraction of the blastocyst to the endometrium lining the uterus. we hypothesized that this process is partly accomplished by chemokines expressed by the endometrium interacting with chemokine receptors on the blastocyst, suggesting that they play an important role in implantation. materials and methods: chemokine receptors were characterized in jeg cells, placental villi, primary trophoblast cell culture, trophectoderm cells derived from human es cells, blastocyst trophectoderm, and 1 st trimester placental tissue sections. expression of chemokines was tested in decidua, endometrium, and ishikawa and hec-1 cell lines. immunohistochemistry, intracellular staining, elisa, facs analysis, and rt-pcr were employed to characterize chemokine receptor and ligand expression. functional testing was performed using transwell migration assays and in a nude mouse model using a matrigel gel plug cell attraction assay followed by facs analysis. results: trophoblasts demonstrated expression of various chemokine receptors, most prominently cxcr1 and cxcr3. immunohistochemistry of trophoblast from placental villi plated on matrigel expressed cxcr3 and cxcr1 as well as hla-g. noteworthy is that trophectoderm cells derived from hes cells treated with bmp-4 and jeg cells, and blastocyst trophectoderm expressed principally cxcr3. elisa and immunohistochemistry showed that decidua and endometrium expressed chemokines ip10 and il8. migration assays demonstrated that ip10 significantly attracted various trophoblast and trophectoderm cells in vitro, and in the mouse model in vivo. taken together these results demonstrate the interaction between trophoblasts and endometrial cells is mediated by cxcr1 and cxcr3, and il8 and ip10. these interactions are important in the attraction of trophoblasts at the feto-maternal interface. however, ip10-cxcr3 is the most relevant to early implantation as only cxcr3 is expressed consistently by trophectoderm. the endometrial proteome: changes from proliferative to secretory phase. lois a salamonsen, jenny i-c chen, xian mak, peter j stanton, david m robertson, andrew n stephens. prince henry's institute of medical research, melbourne, vic, australia. global gene analyses have demonstrated major changes across the menstrual cycle, but which of these are reflected in the proteome is not known. this study aimed to globally assess proteins differentially expressed in the endometrium between the proliferative and. secretory phases. 2d page analysis with dige minimal dye labelling was conducted across the pi range 4-7 on endometrial tissue from either the mid-proliferative or midsecretory phases (n=4/group). profiles were assessed using samespots software. differentially expressed proteins were identified using maldi-tof ms and the interrelationship of proteins examined using ingenuity software. a total of 1010 spots were detected: 196 were differentially expressed (p < 0.05) with 17 spots having an overall false discovery rate <5% (q <0.05). hierarchical clustering analysis revealed that these 17 proteins lay within three main branches in the protein dendrogram. one cluster had proteins upregulated in the proliferative phase and two contained proteins up-regulated in the secretory phase. the unique protein profiles were also revealed using principle component analysis (pca): proteins clustered into two main groups, according to cycle phase. pca thus indicated similar unique protein signatures as suggested by hierarchical clustering. thirty one of the differentially expressed proteins were identified using maldi-tof ms. these proteins could be grouped into seven categories, which included structural (7), transport (4), regulatory (2), membrane (2), enzyme (2), motor (1) and others (2). proteins involved in matrix assembly and those needed for subsequent establishment of secretory endometrium were up-regulated in proliferative endometrium. proteins important for cellular organisation and communication as well as products responding to environmental stress and the immune system were highly up-regulated during the secretory phase. biological pathways were constructed based on the proteins identified. the top network for secretory endometrium clustered around tgf-b. others related to inhibition of cell death / cell viability and leukocyte extravasation. these studies provide a global approach to the cyclic changes of the endometrium and highlight the complex dynamics of protein expression in human endometrium. hormonal regulation of prokineticins in the human fallopian tube: potential regulators of embryo transport. aw horne, 1 hn jabbour, 2 p lourenco, 1 s wright, 2 s battersby, 2 arw williams, 1 hod critchley. 1 1 reproductive and developmental sciences, university of edinburgh, edinburgh, united kingdom; 2 mrc human reproductive sciences unit, queen's medical research institute, edinburgh, united kingdom. background: understanding the factors regulating embryo transport in the fallopian tube (ft) has important clinical implications. embryo retention in the tube due to ft dysfunction is thought to lead to tubal pregnancy, a considerable cause of morbidity and occasional mortality. transport of the embryo through the ft is, for the greater part, accomplished by smooth muscle contraction. a group of multi-functional proteins and their receptors, called prokineticins, have been shown to affect smooth muscle function in other tissues, such as the intestine. the expression pattern of prokineticins, and their receptors, was examined in normal human ft obtained throughout the menstrual cycle, and the effect of the sex steroids on prokineticin expression was examined in an in-vitro model of the ft. methods: ft biopsies (n=18) and sera (for measurement of oestradiol and progesterone for endocrine staging) were collected from women undergoing gynaecological procedures for benign conditions. using a combination of quantitative taqman rt-pcr and immunohistochemistry, the mrna and protein expression pattern of prokineticins, and their receptors, were examined in the ft throughout the menstrual cycle. tubal explant culture was established using surgical tissue from the biopsies and exposed to varying concentrations, and time courses, of oestrogen and progesterone. results: prokineticin 1 (pk1) and prokineticin 1 receptor (pkr1) mrna are up-regulated in the progesterone-dominant mid-secretory phase. pk1 and pkr1 protein are expressed in the epithelium, smooth muscle and around the blood vessels of the ft. stimulation of tubal explant cultures with a physiological concentration of progesterone showed an up-regulation of pk1 and pkr1. conclusions: prokineticins show temporal variation in expression in human ft and appear to be regulated by progesterone. their role in embryo transport needs to be investigated to further understanding of pregnancy complications, such as tubal pregnancy. translating mouse to human: a dynamic model of xenografted human endometrium. alex j polotsky, liyin zhu, nanette santoro, jeffrey w pollard. albert einstein college of medicine, bronx, ny, usa. in the mouse endometrium, the hormonal environment controls cellular proliferation and cell cycle activity. estradiol (e2) inhibits glycogen synthase kinase 3 beta (gsk-3 ), resulting in nuclear accumulation of cyclin d1 and progression of the cell cycle, as well as dna replication licensing. in utero administration of the gsk-3 inhibitor, licl, results in epithelial cell proliferation in the absence of e2 (zhu, pollard. pnas. 2007; 104:15847) . in this study, we derived a functional model of xenografted human endometrium to perform mechanistic studies of human endometrial proliferation. methods: human endometrial samples were obtained from volunteers aged 18-45. immuno-compromised mice were transplanted with disaggregated/ recombined human epithelial glands and stroma under the kidney capsule. after 6 weeks of out-growth, mice were ovariectomized, and replaced with e2 or licl. xenografts were harvested and processed for immunohistochemistry (ihc) and glandular labeling index (li). t test was used to compare group means. results: 40-50% of engraftments were successful, resulting in a vascularized endometrium with characteristic architecture (a, b). hoechst 33258 staining confirmed that xenografts were made up of human cells ©. e2 (e) induced significantly greater proliferation compared to control (d) as assessed by ihc for ki67, with li of 25.9±1.3 and 8.8±3.3, respectively, p =0.02). minichromosome maintenance-2, a protein involved in dna replication licensing, was more sensitive for e2-treated cells synthesizing dna than was ki67 staining (i). estrogen (g) and progesterone receptors (h) were expressed in xenotransplant tissue, the latter being up-regulated by e2. licl (f) induced proliferation similar to e2 and greater than control (li =18.4±6.4, p= 0.2 for e2 vs. licl). conclusion: xenografted human endometrium provides a dynamic model of endometrial proliferation that is well suited for translational studies. administration of licl in the absence of e2 induced glandular proliferation, supporting the notion that similar mechanisms are operative in human proliferation as in the mouse. gnrh analogs have been extensively used in assisted reproduction. although the main effects of gnrh analogs are via gnrh receptors on the pituitary gonadotrope, gnrh and gnrh receptors have been identified in many reproductive tissues including human endometrium, suggesting their potential action at endometrial level. in the present study, we examined the potential regulatory action of gnrha, la on sex steroid mediated gene expression in human endometrium. human endometrial surface epithelial (hes) cells and isolated endometrial stromal (esc) cells were used as in vitro models. all experiments lasted for 24h. the cells were treated with estradiol (e 2 , 10 nm, 24h), progesterone (p4, 10 nm, 24h), gnrha (la 1 μm, 24 h), e 2 (12h) followed by p4 (last 12h) and gnrha plus e 2 plus p4 where, gnrha added either first, second or last in order at 8h intervals. total rna was extracted, reversed-transcribed and subjected to real-time pcr simultaneously, measuring the expressions of il-1 , il-1 , il-2, il-4, il-5, il-8, il-10, il-12p35, il-12p40, il-15, ifn-and tnf . both hes and esc expressed majority of these cytokines with the exception of low to undetectable levels of il-2, il-4, il-5 and ifn-. treatment with e 2 and p4, either alone, or in combination significantly upregulated the expression of many of these cytokines at varying extend as compared to controls. however, gnrha either alone or in combination with e 2 and p4 significantly diminished e 2 , p4 or e 2 plus p4 induced mrna expression of cytokines. the suppressive effects of gnrha on some of the cytokines varied significantly by the order which gnrha was introduced into the culture medium. we conclude that gnrha by acting directly on the endometrial cells effectively suppresses the mrna expression of several key proinflammatory cytokines upregulated by ovarian steroids. our results imply that gnrha therapy during assisted reproduction may modify endometrial receptivity via downregulation of proinflammatory cytokines induced by estradiol and progesterone. supported by nih grant hd37432. introduction: endometrial angiogenesis is characterized by a rapid increase during the early proliferative phase that peaks midcycle, followed by a gradual decrease in the secretory phase, while menses involves generalized endometrial inflammation, necrosis, and vascular thrombosis. vascular endothelial growth factor (vegf), whose expression is regulated by sex steroids, is a mediator of endometrial angiogenesis. recently, myoferlin, a 230 kd transmembrane protein, was identified in endothelial cells where it mediates vegf-dependent endothelial cell proliferation, migration, and nitric oxide synthesis by affecting vegf receptor-2 function and stability. myoferlin also appears to play a role in vesicle trafficking and membrane repair. objective: to characterize myoferlin protein expression in endometrium. methods: western analysis of cultured human endometrial stromal cells (hesc) and human endometrial endothelial cells (heec) treated with physiologic concentrations of estradiol and progesterone was performed using a polyclonal rabbit antibody against myoferlin. subsequently, immunohistochemistry (ihc) was performed on human endometrium samples obtained from various stages of the menstrual cycle. results: myoferlin protein was expressed in hescs and heecs, but expression was not affected by sex steroids. ihc staining for myoferlin was specific, intense, and localized to the apical membrane of glandular epithelial cells and endothelial cells, with less intense staining in the stroma. h-score quantification showed that in endometrial endothelial cells, myoferlin protein expression was highest during the early proliferative and early secretory phases, while glandular and stromal myoferlin expression peaked during the late proliferative/early secretory phase. conclusion: myoferlin expression in human endometrium correlates with periods of greatest endometrial angiogenesis, and expression is not limited to endothelial cells, but also includes glandular and stromal cells. given the involvement of myoferlin in vegf signaling as well as membrane repair, understanding its role in human endometrium may further elucidate an understanding of endometrial development both under physiologic and pathologic conditions. the human embryo is the primary regulator of embryo-endometrial molecular cross talk during early implantation. gijs teklenburg, 1,2 cobi heijnen, 1 esther baart, 1 karima amarouchi, 1 carolien boomsma, 1 janet carver, 2 helen mardon, 2 annemieke kavelaars, 1 nick macklon. 1 1 reproductive medicine and gynaecology, and laboratory of psychoneuroimmunology, university medical center, utrecht, netherlands; 2 nuffield department of obstetrics and gynaecology, university of oxford, women's centre, john radcliffe hospital, oxford, united kingdom. introduction: uterine receptivity and implantation are controlled by locally acting trophic factors and cytokines. in humans, the regulation of the embryo-endometrial dialogue beyond the early blastocyst stage is poorly understood. we hypothesized that the interaction between a healthy conceptus and the receptive endometrium is associated with a distinct local regulation of cytokine production favouring implantation. methods: 97 human embryos, cryopreserved at day 4 after fertilization and donated for research, were thawed and cultured under standard conditions until day five. forty-two embryos from 18 donors developed to the blastocyst stage. following removal of the zona pellucida, they were placed in individual coculture on a confluent monolayer of endometrial stromal cells. on day 8, the developmental potential of each embryo was assessed as early arrested, late arrested or developing. culture supernatants were analysed for concentrations of il-1 , il-5, il-6, il-10, il-12, il-15, il-17, il-18, tnf-, mcp-1, ip-10, eotaxin and hb-egf using a multiplex immunoassay. day 8 supernatants from 29 culture systems in which no embryo had been placed were also analysed as controls. results: 11 out of 42 (26%) embryos continued to develop and were able to attach and invade into the stromal cell compartment of the co-culture environment. twelve late arrested embryos showed signs of degradation on day 8 and the totally disintegrated embryos were assigned to the early arrested group. supernatants from both early and late arrested embryo cultures contained significantly lower levels of a number of cytokines and growth factors in comparison to developing embryo cultures. moreover, the levels of these mediators in co-cultures were significantly lower than those in non-embryo control stromal cell culture supernatants. conclusion: these data suggest a pivotal role of the embryo in embryoendometrial cross talk. whether reduced mediator expression in co-cultures reflects a selective down regulation of stromal cell cytokine and growth factor production is now being investigated. epithelial cell dynamics during human implantation. hiroshi uchida, tetsuo maruyama, toru arase, masanori ono, takashi kajitani, maki kagami, hideyuku oda, sayaka nishikawa, yasunori yoshimura. department of obstetrics and gynecology, keio university school of medicine, shinjuku, tokyo, japan. epithelio-mesenchymal transition (emt) is thought to play a role in functional differentiation of endometrial epithelial cells during human implantation. molecular mechanism of epithelial sheet remodeling caused by embryo invasion remains elusive. to address this, we investigated cellular dynamics of n-cadherin and vimentin, the two representative major markers of emt, during implantation. in in vitro implantation assay using a human endometrial epithelial cell line, ishikawa, and a human choriocarcinoma cell line, jar (uchida et al., hum reprod 2007), we pre-treated human ishikawa cells with or without ovarian steroid hormones (17 -estradiol + progesterone; ep), fa-5 (n-cadherin blocking ab), or suberoylanilide hydroxamic acid (saha), one of histone deacetylase inhibitors, which has a potential to improve in vitro implantation (ibid). implantation or treatment with or without ep or saha enhanced the expression of n-cadherin and vimentin but down-regulated ecadherin. furthermore, treatment with ep or saha accelerated ishikawa cell motility and increased the number and spreading area of jar spheroids. in vitro implantation assay, the most prominent staining intensity of n-cadherin was observed just around the adhered spheroid from which its intensity decreased away. functional blockade of n-cadherin by fa-5 resulted in the complete suppression of ishikawa cell motility, the unique distribution of n-cadherin around jar spheroids, and the spreading area of jar spheroids, while it did not affect the number of the adhered spheroids. human implantation consists of the multiple steps, including apposition, adhesion and penetration. thus, these results collectively indicate that emt may take place after the apposition and that n-cadherin may be required for the remodeling and emt of the epithelial sheet during embryo invasion. n-cadherin may enhance the recruitment of spheroid-neighboring cells, suggesting its role in the covering-up of the invading embryo through acceleration of epithelial cell motility. endocannabinoid regulation in human endometrium. jessica g scotchie, marc a fritz, steven l young. obstetrics and gynecology, university of north carolina, chapel hill, nc, usa. background: research in mouse models demonstrates two cyclically regulated endocannabinoids produced in murine endometrium, anandamide and 2-arachidonoyl glycerol (2-ag); both play critical roles in murine embryonic implantation. no studies of endocannabinoids in human endometrium have been performed. objectives: determine menstrual cycle expression and localization of synthetic and degradative enzymes for anandamide and 2ag in human endometrium. methods: human endometrium was collected from volunteers across the menstrual cycle (n=43). quantitative rt-pcr was performed analyzing the expression of: n-acylphosphatidylethanolamine (nape) and fatty acid amide hydrolase (faah), the synthetic and degradative enzymes for anandamide, respectively; sn-1-diacylglycerol lipase-a (dagla), and b (daglb), the synthesis enzymes for 2ag; and monoacylglycerol lipase (magl) and cyclooxygenase-2 (cox2), the degradative enzymes for 2ag. the constitutive gene ppia was used for comparison. immunohistochemical localization of nape protein was performed using nape-pld polyclonal antibody (cayman chemical, #10005430). anova and student's t-test analysis performed on samples grouped by proliferative (pro), early, mid-, and late secretory (es, ms, ls) phases. results: mrna expression of all enzymes responsible for synthesis and degradation of anandamide and 2ag was detected throughout the cycle. no significant cyclic change in nape, faah, or daglb gene expression was seen. a decrease in dagla gene expression in the ms and ls phases compared to the pro and es phases (p=0.004) was seen. magl gene expression was higher in the secretory phase than the pro phase (p=0.004). cox2 gene expression was detected at low levels in the pro, es and ms phases, with marked increase in the ls phase (p<0.05 for all comparisons). protein localization of nape showed a cytoplasmic epithelial location, with increased staining on pro and es samples compared to ms and ls samples. immunolocalization of remaining proteins is ongoing. conclusion: this is the first report documenting the presence of endocannabinoid synthetic and degradative enzymes in human endometrium. genes controlling anandamide expression do not fluctuate significantly across the cycle. however, 2ag's degradative enzymes increase in the secretory phase, suggesting that lower 2ag levels may be advantageous for embryo implantation. our findings suggest that human endometrial endocannabinoid regulation differs from murine regulation. interleukin-1 beta (il-1 ) regulates il-6 signaling in decidua-implication in the pathophysiology of preeclampsia (pe). sj huang, 1 cf yen, 2 cp chen, 3 f schatz, 1 cj lockwood. 1 1 obstetrics, gynecology and reproductive sciences, yale university, new haven, ct, usa; 2 ob/gyn, chang gung memorial hospital, tao-yuan, taiwan; 3 ob/gyn, mackay memorial hospital, taipei, taiwan. objective: previously, we found much higher cytoplasmic immunoreactive il-6 levels in the preeclamptic decidual cells than in adjacent interstitial trophoblasts. such decidual cell-derived il-6 contributes to the systemic endothelial cell dysfunction that elicits the proteinuria and hypertension of the maternal syndrome. il-6 promotes the transition from innate to adaptive immunity. moreover, by skewing monocyte differentiation from a dendritic to a macrophage phenotype, decidual il-6 may promote the macrophage excess observed in the preeclamptic decidua. macrophages impair trophoblast decidual invasion to foster incomplete spiral artery remodeling that elicits placental ischemia and hypoxia. the current study: 1) localized il-6 mrna levels in preeclamptic versus normal decidual sections; 2) evaluated mechanisms regulating il-6 synthesis by targeting intracellular signaling pathways with specific inhibitors; 3) identified potential il-6 targets by immunolocalizing the il-6 receptor (il-6r) to specific cell types in placental bed biopsies. methods: in situ hybridization localized il-6 mrna in normal versus preeclamptic decidua. il-6r was immunolocalized in placental bed biopsies. leukocyte-free first trimester decidual cells were incubated with e2 and mpa ± il-1 (1 ng/ml) ± an inhibitor of p38 mapk (sb203580) or protein kinase c (calphostin c) or nf b (activation inhibitor iii) for 24 hrs. an elisa measured secreted il-6 levels. results: il-6 mrna was present primarily in decidual cells with increased il-6 mrna levels observed in pe. preferential expression of the il-6r was observed on decidual cells in placental bed biopsies. compared with basal il-6 levels (0.22 ± 0.14 pg/ml/ g cell protein) by decidual cells, il-1 enhanced il-6 output by decidual cells (364.55 ± 160.29 pg/ml/ g cell protein). only the p38 mapk inhibitor significantly reduced this output to 39.50 ± 8.09 pg/ml/ g cell protein (n=4, p<0.05). our results indicate that inflammatory cytokine enhances il-6 synthesis in decidual cells of the preeclamptic decidua by a mechanism involving p38 mapk. such il-6 is likely to act as an autocrine/paracrine effector via decidual cell-expressed il-6r to contribute to the macrophage excess observed in the preeclamptic decidua. heparanase is up-regulated by estrogen and during the secretory phase of the human endometrium. ronit haimov-kochman, 1 shira natanson-yaron, 1 caryn greenfield, 1 achinoam lev-sagie, 1 lichtenstein michal, 2 haya lorberboum-galsky, 2 israel vlodavsky, 3 simcha yagel, 1 arye hurwitz. 1 1 ob/ gyn, jerusalem, israel; 2 cellular biochemistry human genetics, hadassah hebrew university medical centers, jerusalem, israel; 3 cancer and vascular biology research center, technion school of medicine, haifa, israel. introduction: heparanase is an endoglycosidase that cleaves heparan sulfate (hs) proteoglycan of the extracellular matrix. the full-length proheparanase is activated by cleavage into an active isoenzyme, resulting in the release of hsbound cell-differentiation factors, such as hb-egf. the cycling endometrium involves remarkable steroid hormone-induced tissue remodeling. in vivo, increasing exposure to unopposed estrogen may lead to endometrial malignant transformation. aim: to investigate heparanase expression and regulation in the cycling endometrium. materials and methods: heparanase mrna levels were measured by quatitative rt-pcr in naturally menstruating women and in hec1a, estrogen receptor (er)-negative and ishikawa, er-positive endometrial carcinoma cell lines exposed to increasing doses of estradiol. heparanase isoenzymes were localized by immunohistochemistry using specific anitbodies in murine endometrium and human normal, hyperplastic and malignant endometrium. results: heparanase mrna level increased 100fold in secretory phase (d21) compared to proliferative phase (d10) endometrium. heparanase transcript levels increased 14fold during 8 hr culture in er positive adenocarcinoma cell line exposed to increasing doses of estradiol, but not in hec1a, er negative cell line. both heparanase isoforms were localized to murine glandular endometrium. human glandular endometrium at both proliferative and secretory phases was immunoreactive with the active isoform of heparanase. proheparanase was detected in basal membrane of endometrial glands and endometrial stroma during secretory phase. along with malignant transformation of the endometrium the presence of proheparanase increased dramatically from none in stroma of normal and hyperplastic endometrium to abundance in malignant tumors. conclusions: heparanase gene expression is higher during the window of implantation and up-regulated with estrogen in endometrial cells via er in vitro and vivo. heparanase is differentially localized in the secretory phase of the endometrium compared to the proliferative phase, suggesting a role for this molecule during the window of implantation in man. interleukin-18 (il-18) system mrna and protein expression in the human fallopian tube with ectopic implantation. hong-yuan huang, 1,2 tien-hung huang, 2 chin-jung li, 1 chyi-long lee, 1,2 hsin-shih wang, 1,2 yung-kuei soong. 1,2 1 obstetrics and gynecology, chang gung memorial hospital, kwei-shan, tao-yuan, taiwan; 2 obstetrics and gynecology, chang gung university and school of medicine, kwei-shan, tao-yuan, taiwan. objective: ectopic pregnancy, an abnormal implantation of a fertilized ovum outside the uterine cavity, has been increasing in number at a staggering pace of all pregnancies. il-18 system is one of the major cytokines involved in human endometrium during embryo implantation and might perform a defensive role against maternal immune response. very little information is available regarding the expression and synthesis of cytokines in the pathogenesis of fallopian tube with ectopic gestation. the purpose of this study is to investigate il-18 system expression in human fallopian tubes with ectopic pregnancy. methods: paired segments of human fallopian tubes with ectopic implantation site and side portion close to ectopic gestation (n=7) were collected from women undergoing laparoscopic salpingectomy after informed consent and irb approval. segments of fallopian tubes from women undergoing tubal ligation (n=4) were used as control groups. total extracted rna was reverse transcribed and amplified by pcr using specific primers for gapdh (94 bp), il-18 (144 bp), il-18bp (188 bp) and il-18r (307 bp). quantitative il-18 and il-18bp mrna expression in human fallopian tube was determined by real-time pcr. to determine the presence of il-18 system proteins, tissues were fixed and processed for immunohistochemical study. data analysis was done with anova and pearson's correlation. results: il-18 and il-18bp as well as il-18r mrna were all expressed in tubal ectopic implantation and normal tubes. according to real-time pcr with c t value quantification and 2 -ct method, a significantly higher il-18 expression in tubal ectopic implantation and lower ratio of il-18 antagonist to agonist in portion close to ectopic implantation is demonstrated in comparison to normal tubes (p<0.05). immunoreactive il-18 system at the protein levels was also present in human fallopian tubes with ectopic implantation and normal tubes. conclusions: these results suggest that fallopian tube il-18 system expression may play a crucial role during the process of early embryonic implantation. the expression and ratio of antagonist to agonist in fallopian tubes may indicate an earlier "dialogue " in human fallopian tubal gestation prior to uterine implantation. replacement. marcia c ferreira, ines kd cavallo, fernando m reis. gynecology, ufmg, belo horizonte, mg, brazil. activin a is a growth factor expressed in the endometrium, where it modulates tissue remodelling and enhances decidualization. the effects of activin a are counteracted by two binding proteins, namely follistatin and follistatin-like 3 (fstl3). while the endometrial expression of activin a increases during the secretory phase of menstrual cycle, the effects of ovarian steroids on these proteins and their mrnas has not been assessed yet in postmenopausal women or in ovariectomized animals. we have evaluated the effects of estrogen alone or estrogen plus progestin on the endometrial expression of activin beta-a subunit, follistatin and fstl3 in ovariectomized rats. adult female wistar rats (n=21) were ovariectomized and received one week later a single dose of estradiol benzoate (1.5 mg/kg body weight, i.m. injection), either alone (n=7) or associated with depot medroxyprogesterone acetate (2.4 mg/kg body weight, i.m. injection, n=7), or oil vehicle (control group, n=7). one week after the hormone or placebo treatment, the animals were sacrificed and their uteri were removed and processed by immunohistochemistry and real-time pcr. data were normalized to the expression of ribosomal phosphoprotein p0 (rpp0) and analyzed with the delta-delta ct method, anova and newman-keuls test. activin beta-a subunit mrna levels increased significantly in the uteri of rats treated with estradiol alone (7.4 fold increase over controls, p<0.05) and to the same extent in rats receiving estradiol plus medroxyprogesterone (6.1 fold increase over controls, p<0.05). this was accompanied by increase of beta-a subunit immunostaining in estradiol and estroprogestin-treated rats, which was noted only in the surface endometrial epithelium. follistatin mrna expression, conversely, showed a significant decrease in the groups treated with estrogen alone (0.5 fold compared to controls, p<0.05) and estrogen plus progestin (0.4 fold compared to controls, p<0.05), while follistatin immunostaining in the glandular epithelium was weaker in estradiol and estroprotestin-treated rats compared to controls. fstl3 expression was similar in the 3 groups. in conclusion, the expression of activin beta-a subunit increases and that of follistatin decreases following estrogen replacement in the endometrium of ovariectomized rats, and these effects are not further altered by the addition of progestin. endometrial nk cells are a unique inert nk subset until pregnancy. simcha yagel, 1 irit manaster, 2 jacob hanna, 2 ronit haimov-kochman, 1 miri godin, 2 yuval bdolach, 1 caryn greenfield, 1 shira natanson-yaron, 1 arye hurwitz, 1 debra s goldman-wohl, 1 ofer mandelboim. 2 1 obstetrics and gynecology, hadassah-hebrew university medical center, jerusalem, israel; 2 lautenberg center for tumor immunology, hadassah-hebrew university medical center, jerusalem, israel. introduction: we recently demonstrated that nk (natural killer) cells play a critical role in trophoblast migration and angiogenesis at the fetal maternal interface. nk cells populate the endometrium at the secretory phase of the menstrual cycle, the time of anticipated blastocyst implantation. peripheral blood (pb) nk cells and decidual nk (dnk) cells express a variety of activating receptors, including nkp44, nkp30 and nkp46, collectively known as natural cytotoxicity receptors (ncrs), and nkg2d which regulate nk cell killing and growth factor production. to compare endometrial nk cell (enk) activating receptor expression and function to pbnk and dnk cells and endometrial ligand expression, with a focus on their roles in blastocyst implantation. patients and methods: subjects were ivf patients undergoing natural menstrual cycles. endometrium samples were collected on treatment days 10 and 21. a lymphocyte profile of the endometrial cells and pb was performed. facs analysis was performed on isolated endometrial nk cells, pbnk cells and dnk cells for cd56, cd16, nkp44, nkp30, nkp46 and nkg2d. ncr ligand expression was characterized on adherent endometrial cells using ncr-ig fusion proteins and nkgd2-ig and nkg2d specific ligands as well as control ccmi-ig. redirected killing assays and cytokine secretion assays of ifn , vegf, plgf, and il-8 with and without il-15 were performed. results: endometrial lymphocytes of day 10 and 21 in these women are mostly cd56 bright cd16-nk cells, with a significant amount of t cells, similar to pbnk cells and in marked contrast to dnk cells. unlike pbnk and dnk cells, enk receptors do not express nkp30, nkp44. nkp46 and nkg2d are the only activating enk receptor expressed. like decidual cells, adherent stromal endometrial cells expressed the ligands for nkp30, nkp44 and nkg2d, suggesting that these nk cells have potential for activation. finally enk cells could not kill or secrete cytokines. conclusions: these findings of a unique activating receptor profile on endometrial nk cells, unlike that of dnk and pbnk, suggest that enk cells are a special local population of nk cells that change dramatically and are activated at the onset of pregnancy. variation in platelet activation throughout the menstrual cycle. fiona c denison, 1 amy o robb, 1 imogen b smith, 1 nicholas l mills, 2 hilary od critchley, 1 david e newby. 2 1 centre for reproductive biology; 2 centre for cardiovascular sciences, the university of edinburgh, united kingdom. background: platelet-monocyte aggregation (pma) is a sensitive and novel measure of platelet activation with important proinflammatory consequences including release of cytokines and chemokines. previous studies using less sensitive techniques suggest that platelet activation alters during the menstrual cycle in response to circulating concentrations of sex steroids. the effect of sex steroids on circulating (c) pmas during a single menstrual cycle is not known. objective: to determine whether cpmas, platelet surface (ps) p-selectin and plasma (p) p-selectin vary through the menstrual cycle in response to changes in circulating sex steroid concentrations. methods: 10 healthy, nulliparous, pre-menopausal, non-smoking women (mean age 31 years), with regular menses (27-29 days) were studied. subjects gave written informed consent and the study had ethical approval. serial venous blood samples were taken at menstrual, follicular, periovulatory and luteal phases of a single cycle (days 1-3, 6-9, 13-15 and 20-22). cpmas (monocytes positive for the platelet marker cd42a) were measured by flow-cytometry. psp-selectin expression was calculated on cd42a positive cells. isotype-matched controls were used. serum oestradiol (e) and progesterone (p), plasma and pp-selectin were measured by elisas. data were analysed by one-way anova with repeated measures and bonferroni's post-tests for multiple comparisons. results: luteal phase p was >30 nmol/l in all women. numbers of cpmas and expression of psp-selectin were both significantly higher during menstrual compared with periovulatory phase of the menstrual cycle (25.59±2.15 vs. 16.65±2.88%, p=0.01 and 3.95±0.77 vs 2.10±0.37%, p<0.05, respectively). there was no significant difference in pp-selectin concentration during the menstrual cycle (p=0.8). there was no correlation between levels of serum e or p and numbers of cpmas, expression of psp-selectin or pp-selectin concentration. conclusions: numbers of cpmas and expression of pspselectin are maximal at menstruation with neither numbers of cpmas nor expression of psp-selectin correlating with serum e or p levels. this study suggests that activated platelets may potentially contribute to the inflammatory response at menstruation by releasing inflammatory mediators. by angiogenic factors and peri-cellular proteases in decidual secretory endometrium (dse), decidua parietalis (dp), and basalis (db) of miscarriage patients and matched controls. comparison of these parameters between the two groups enabled hypothesizing about their correlation with the occurrence of miscarriages. methods: decidua was obtained during 1 st trimester termination of pregnancy (control group) and vacuum aspiration of missed abortions (case group). vascularization was studied by cd34-immunohistochemistry. the expression of vascular endothelial growth factor-a, placental growth factor, flt-1, kdr, angiopoietin-1, angiopoietin-2, tie-2 and the membrane-type matrix metalloproteinases mt1-, mt2-, mt3-and mt5-mmp were determined at mrna and antigen level and cd56-positive unk cells, cd68-positive macrophages, proliferation (ki67) and apoptosis (activated caspase-3) were evaluated by immunohistochemistry in consecutive serial sections. results: the decidual vascularization pattern showed differences between cases and controls: i.e. fewer vessels with larger circumference in cases, and this correlated with the differential expression of various angiogenic factors and proteases at mrna and antigen level. moreover, the endothelial protein expression of flt1, kdr, mt2-and mt5-mmp was increased at the implantation site of cases. ki67 and active caspase-3 showed similar levels in the two groups and also the immune cells, both unk cells and macrophages, showed no differences at the implantation site between both groups. conclusion: differences between cases and controls appeared not to be based on altered proliferation, apoptosis, and/or inflammation. the differences in vascularization pattern and in the expression of angiogenic factors and proteases between both study groups suggest a correlation between decidual vascularization and the occurrence of miscarriages. respond to adrenomedullin. yaun-lin dong, 1 hong y wen, janice endsley, 2 alison hogg, 2 hui-qun wang, 3 manubai nagamani, 1 chandra yallampalli. background: natural killer (nk) cells are the predominant lymphocytes present in human implantation site. decidual nk cells express perforin, an essential molecule required for lysis. formation of the placenta involves cooperation between maternal nk cells and fetal trophoblast cells that remodels the blood supply; however, the interaction between trophoblasts and decidual nk cells is largely unknown. adrenomedullin (adm) has been implicated in regulating early placental function and fetal growth. objective: to determine the role of multifunctional peptide adm in the decidual nk cells and fetal trophoblast cells interactions. methods: decidual and placental tissues were obtained from normal firsttrimester pregnancies terminated for social reasons. ethical approval to use these tissues was obtained from the irb of university of texas medical branch. cell preparations containing all decidual mononuclear cells were isolated by collagenase enzymatic disaggregation. cd56 decidual nk cells were purified by magnetic bead isolation. results: 1) immunohistochemical analysis showed that adm is expressed primarily in decidual cells and trophoblast cells at the human implantation site; 2) confocal imaging analysis demonstrated that decidual nk cells, which were identified by anti-cd56 staining, express adm receptor components crlr/ramp 2 /ramp 3 and their mrna expressions were futher confirmed by rt-pcr; 3) k562 target cell killing assay indicates that adm inhibits cytokine il-12/il-15-induced decidual nk cell cytotoxicity; and 4) immunofluorescent labeling and flow cytometric analysis revealed that adm suppresses perforin expression by decidual nk cells. conclusion: trophoblast-derived adm inhibits decidual nk cell cytotoxicity via suppressing perforin expression, thus, our results provide evidence for a new paradigm of embryonic-maternal communication involving a adm mediated interaction between decidual nk cells and fetal trophoblasts. leandro g oliveira, gendie e lash, judith n bulmer, barbara a innes, roger f searle, stephen c robson. institute of cellular medicine, newcastle university, newcastle upon tyne, tyne and wear, united kingdom. background: we have previously demonstrated that co-culture with extravillous trophoblast cells (evt) (expressing hla-g) alters cytokine secretion by uterine natural killer (unk) cells, particularly at 12-14 weeks gestation. we have also reported that unk cells can stimulate evt invasion, but only at 12-14 weeks gestation (not at 8-10 weeks gestation). in addition, unk cell cytokine profiles alter with increasing gestational age. other reports have suggested that evt or hla-g expressing cells may alter the expression of cytokines and angiogenic growth factors by unk cells. hypothesis: hla-g expressing cells alters unk secretion of cytokines. methods: cd56 + unk cells were isolated from early pregnancy decidua (8-10 and 12-14 weeks gestation, n=10 each group) using enzyme digestion and positive immunomagnetic bead separation. the human b lymphoblastoid 721.221 transfected with either hla-g1 (221g) or a mock cdna (221cdna) were obtained as a kind gift from mr r apps (university of cambridge, uk). isolated unk cells were cultured in the presence or absence of the two cell lines in either direct or indirect contact (n=5 each group and each gestational age) for 24 hours. cell supernatants were analysed for cytokines using a fastquant® th1/th2 multiplex protein assay (il-6, il-1 , il-10, il-2, il-5, tnf-, il-13, il-4, ifn-) or by standard elisa (tgf-1). the effect of direct co-culture of unk cells with 221g compared with co-culture with 221cdna at each gestational age was tested using mann whitney u test. the effect of co-culture of unk cells with 221g in both direct and indirect contact was also tested using mann whitney u test. results: there was no difference in the level of cytokines secreted by the 221g or 221cdna cells. cytokine secretion by unk cells was not altered after direct co-culture with either 221g or 221cdna cells at either gestational age. in addition, direct or indirect co-culture of 221g or 221cdna with unk did not alter cytokine secretion at either gestional age. conclusions: hla-g does not alter the secretion of cytokines by unk cells from either 8-10 or 12-14 weeks gestation. other evt or decidua derived factors (including hla-e) may be responsible for the alteration in secretion of cytokines by unks with increasing gestational age. introduction: natural killer (nk) lymphocytes are central to innate immunity and contribute to tissue homeostasis by eliminating altered cells. their nkg2d receptor pathway plays a fundamental role in target elimination through binding nkg2d ligands on the cell surface. reduction in the nkg2d ligand, ulbp2, expression is associated with immune resistance in neoplastic processes. we have previously shown that fibroblasts from adhesion tissue (at) are characterized by increased extracellular matrix molecules and inflammatory cytokines compared with normal peritoneal (np) fibroblasts. objective: to determine if there is a difference in nk lymphocyte-mediated elimination between np and at fibroblasts and to investigate potential role of nkg2d pathway in this process. material and methods: expression of nkg2d ligands; ulbp2, ulbp3, mica, and micb was evaluated by flow cytometry and western blot in primary cell cultures of fibroblasts from np and at, established from two patients. peripheral blood nk lymphocytes (cd56+cd3-) from three healthy volunteers were isolated using macs system with purity greater than 90% and kept in interleukin 15 overnight. fibroblast elimination with and without ulbp2 blocking was investigated following 3-hour co-incubation with allogeneic nk lymphocytes using our established flow cytometric cell mediated cytotoxicity assay. paired t test was used in statistical analysis. results: the flow cytometry studies showed that nkg2d ligands (ulbp2, ulbp3, mica and micb) were lower in at compared to np fibroblasts, reaching a statistical significance in ulbp2 expression (p = 0.039). western blot analysis also revealed a lower ulbp2 protein level in at than np fibroblasts. furthermore, nk lymphocyte-mediated elimination was 20% lower in at in comparison with np fibroblasts. blocking ulbp2 expression resulted in decreased nk lymphocyte-mediated np fibroblast elimination by 27%, supporting the role of nkg2d receptor pathway in the process. conclusions: our results demonstrate that nkg2d pathway is operational in at fibroblast resistance to immune elimination, and extends our prior observations of the potential role of immunological mechanisms in the pathogenesis of adhesion development. objective: galectin-1 is an anti-inflammatory lectin that has pleiotropic regulatory functions at the crossroad of innate and adaptive immunity. human galectin-1 is expressed in the placenta and immune privileged sites and it has been implicated in establishing immune tolerance. the aim of this study was to examine the evolution and placental expression of the lgals1 gene in primates. methods: seven primate nucleotide sequences were generated, aligned to 27 vertebrate orthologs from all classes and subjected to phylogenetic analysis. deduced amino acid sequences were analyzed for functionally important substitutions. placental galectin-1 expression was studied by immunohistochemistry and western blot. results: 1) the lgals1 gene had high sequence identity among all investigated species. 2) phylogenetic analysis revealed that intense purifying selection had been acting on the lgals1 gene in placental mammals (dn/ds=0.11); 3) residues responsible for sugar binding or molecule stabilizing were highly conserved in primates. 4) immunostaining showed a uniformly abundant and ubiquitous galectin-1 expression pattern in human, old and new world monkey and prosimian placentas, regardless the type of placentation. the lgals1 gene has conserved sequence and placental expression pattern in primates that may suggest its important function in maternal-fetal immune interactions. these results support the view that immune interactions at the maternal-fetal interface evolved in concert with invasive placentation and that these interactions have been maintained regardless of the degree of placental invasion in primates and other mammals. expression of interleukin-23 in human endometrium throughout the menstrual cycle and early pregnancy. yesim h uz, 1,2 william murk, 1 umit a kayisli, 1 aydin arici. 1 1 department of obstetrics, gynecology and reproductive sciences, yale university school of medicine, new haven, ct, usa; 2 department of histology and embryology, trakya university school of medicine, edirne, turkey. background: interleukin-23 (il-23) is a recently discovered heterodimeric cytokine, comprised by a novel p19 subunit and a p40 subunit shared by il-12. it has biological activities that are similar to but distinct from il-12, and is known to be involved in th1/th2 cell class switching and the regulation of cytokines such as ifn-gamma, il-12, tnf-alpha, and il-17. early pregnancy is associated with alterations in the maternal immune response, such as changes in cytokine expression, and leukocyte recruitment and subtype switching. we hypothesized that expression of il-23 in the human endometrium is menstrual cycle-and pregnancy-dependent. materials and methods: endometrial samples from women (n=44) undergoing surgery for benign gynecologic conditions, and decidual tissues from women (n=8) with clinically normal pregnancies terminated voluntarily in the first trimester, were obtained after receiving informed consent. endometrial samples were grouped according to menstrual phase. paraffin sections were stained with il-23p19 antibodies and evaluated semi-quantitatively with hscore. statistical analysis of the data was done using anova, with p<0.05 considered significant. results: il-23 immunoreactivity was predominantly located in the cytoplasm of both endometrial stromal (esc) and glandular (egc) cells. escs showed mild il-23 immunoreactivity without significant changes in intensity throughout the menstrual cycle. on the other hand, first trimester decidual cells showed significantly stronger il-23 staining compared to escs from non-pregnant endometrium (p<0.001). il-23 immunoreactivity in egcs was high in the late proliferative phase, as compared to other cycle phases and first trimester tissues (p<0.001). moreover, egcs from the early secretory phase (p<0.05) and first trimester tissues (p<0.01) showed higher il-23 immunoreactivity compared to the early proliferative and late secretory phases. conclusions: this is the first study describing il-23 expression in the human endometrium and decidua. these results suggest that il-23 has a cycledependent expression in endometrial cells and may be involved in regulating cytokine expression and immune cell modulation during the menstrual cycle and early pregnancy. gercel-taylor, douglas d taylor. obstetrics, gynecology, and women's health, university of louisville, louisville, ky, usa. objective: estrogen appears appear to be a critical regulator of the immune system. since hypoestrogenism is present in the postmenopausal woman, our objective was to determine whether t cell activation and function, defined as il-2 production and signaling molecule expressions at the transcriptional and translational levels, were affected by a low estrogen environment. design: prospective study in a university research laboratory. materials and methods: jurkat 6.1 t cells, initially grown in estrogen free media, were incubated in 4pm (representing postmenopausal levels) or 40pm (premenopausal levels) of estradiol (e 2 ) for 48 hours. cells were either resting or activated with a phorbol ester, 4 -phorbol 12 -myristate 13 -acetate (pma), and ionomycin. enzyme-linked immunosorbent spot assay (elispot) was performed to analyze production of il-2. expression of signaling protein components, cd3 and jak, were determined by western immunoblotting. real time-polymerase chain reaction was performed to quantify cd3 , jak2, and jak3 gene expression. a p value of <0.05 was considered significant. results: jurkat cells exposed to 4pm e 2 and activated exhibited significantly diminished numbers of il-2 producing colonies compared to t cells exposed to 40pm (55.7±1.2 vs. 75.3±0.7 colonies, p<0.0001). analysis of cellular cd3 and jak2 protein demonstrated that jurkat cells incubated in 4pm e 2 expressed a 1.48-fold decrease in cd3 and 1.64-fold decrease in jak compared with cells incubated in 40pm e 2 (p<0.001). these diminished protein levels appeared to be the consequence of suppressed transcription, as the mrna levels of cd3 , jak2 and jak3 were significantly decreased in jurkat cells incubated in low levels of estrogen (11.3, 213.3, and 13.3 fold, respectively, compared to 40pm). conclusions: jurkat cells exposed to low postmenopausal estrogen levels produce significantly less il-2 following activation, which was associated with a significant decrease in signaling proteins. the diminished levels of signaling proteins appear to result from decreased cd3 , jak2 and jak3 gene expression in the presence of low estrogen. these findings support the observation of decreased cellular immune response in postmenopausal women and may provide a basis for the increased risk of infections and cancer proliferation associated with aging. support: dept. of ob/gyn research seed fund. the expression pattern of 2 novel cytokines (il-24 and 29) in human fetal membranes. judith eckardt, 1,2 stephen j fortunato, 1 holger maul, 2 ramkumar menon. 1 1 the perinatal research center, nashville, tn, usa; 2 womens' hospital, university of heidelberg, heidelberg, baden-wuerttemberg, germany. objective: interleukin (il) 24 and 29 are novel cytokines produced by various immunological cells in response to microbial antigens. the functions of these cytokines in reproductive system is unknown. this study examines the expression pattern of il-24 and il-29 in human fetal membranes from preterm and term births and in in vitro in normal term membranes in response to bacterial endotoxin (lipopolysaccharide-lps). methods: fetal membranes collected (n=12) from cesareans at term (normal, not in labor) were placed in an organ explant system for 48 hours and were stimulated with lps for an additional 24 hrs. fetal membranes were also collected (n=10) either at preterm or term after vaginal deliveries. in a case -control study (preterm birth vs. normal term deliveries) amniotic fluids (af) (n=200) were collected to document the role of il-24 and il-29 in ptb. tissue expressions of il-24 and il-29 were studied by rt-pcr using specific primers. elisa documented culture media and af cytokine concentrations. statistical analysis was performed using non-parametric mann-whitney u test. results: both il-24 and il -29 expressions were seen in fetal membranes in culture (in vitro) regardless of stimulation. in vivo in membranes from preterm and term deliveries and membranes at term not in labor also documented the expression of these cytokines. culture media analysis documented higher concentration of il-24 after lps stimulation (lps-65.5 vs. 7.7 pg/ml; p=0.006) whereas no difference was noticed in il-29 concentration (lps-22.8 control-19.5 pg/ml; p=0.5) between the two groups. af analysis, regardless of the status, did not document detectable concentrations of either of the cytokines (lower limit 7.65 pg/ml for both). conclusion: this is the first study to document the expression of two novel cytokines in laboring and non laboring human fetal membranes and also in membranes from preterm deliveries. il-24 production was stimulated by lps whereas il-29 was not affected. these cytokines are not physiological components of af and their role in fetal membranes is unclear. higher il-24 concentration in response to lps but lack of its presence in term or preterm af is suggestive of an autocrine immune response during pregnancy in response to a microbial antigen. evidence for a selective migration of fetus specific cd4 + cd25 bright regulatory t cells from the peripheral blood to the decidua in human pregnancy. tamara tilburg, 1,2 dave l roelen, 2 barbara j van der mast, 1 godelieve m de groot, 1 carin kleijburg, 1 sicco a scherjon, 1 frans hj claas. 2 1 department of obstetrics, leiden university medical centre, leiden, netherlands; 2 department of immunohematologie and bloodbank, leiden university medical centre, leiden, netherlands. during pregnancy the maternal immune system has to tolerate the persistence of fetal alloantigens. many mechanisms contribute to the prevention of a destructive immune response mediated by maternal alloreactive lymphocytes directed against the allogeneic fetus. murine studies suggest that cd4 + cd25 + t cells provide mechanisms of specific immune tolerance to fetal alloantigens during pregnancy. previous studies by our group demonstrate that a significantly higher percentage of activated t cells and cd4 + cd25 bright t cells are present in decidual tissue in comparison with maternal peripheral blood in human pregnancy (1) . in this study we examined the phenotypic and functional properties of cd4 + cd25 bright t cells derived from maternal peripheral blood and decidual tissue. depletion of cd4 + cd25 bright t cells from maternal peripheral blood demonstrates regulation to a 3rd party umbilical cord blood cells comparable to non-pregnant controls, whereas the suppression capacity to umbilical cord blood cells of her own child is absent. furthermore, maternal peripheral blood shows a reduced percentage of cd4 + cd25 bright foxp3 + and cd4 + cd25 bright hla-dr + cells compared to peripheral blood of non-pregnant controls. in contrast, decidual lymphocyte isolates contain high percentages of cd4 + cd25 bright t cells with a regulatory phenotype that are able to down regulate fetus-specific and non-specific immune responses. these data suggest a preferential recruitment of fetus-specific regulatory t cells from maternal peripheral blood to the fetal-maternal interface where they may contribute to the local regulation of fetus specific responses. (1) tilburgs t, roelen dl, van der mast bj, van schip jj, kleijburg c, de groot-swings gm, kanhai hh, claas fh, scherjon sa. differential distribution of cd4 (+) cd25 (bright) and cd8 (+) cd28 (-) t-cells in decidua and maternal blood during human pregnancy. placenta. 2006 apr;27 suppl a:s47-53. 3 alicia del toro-arreola, 2 lourdes nunez-atahualpa, 4 juan velazquez-rodriguez, 5 laura gonzalez-lopez, 6 jorge i gamez-nava, 3 adrian daneri-navarro. there is evidence that the maternal immune system is influence by changes in the hormonal levels during the menstrual cycle (mc). so far, the information related to the levels of t, treg, nk cells and receptors of activation and inhibitors is scarce. aim: to analyze the populations of t, treg, nk cells and their receptors of peripheral blood of healthy women and their correlation with hormones during mc. material and methods: we studied to 10 women not using hormonal contraceptives in the day 5th of the follicular phase and 21st of the luteal phase of the mc. pbmc subsets and their receptors were determined by flow cytometry and hormone levels by chemiluminescence method. we found that the progesterone and prolactin were positive correlated (rho= 0.802, p<0.05 and rho= 0.757, p<0.05, respectively) with cd94/nkg2 in t cells and negative correlated (rho= -1.00, p<0.01 and rho= -0.857, p<0.05, respectively) with nk cells. meanwhile cortisol was positive correlated (rho= 0.857, p<0.05) with the receptor nkg2d expressed in nk cells. the results observed in this study in the luteal phase of mc on the expression of cd94/nkg2 inhibitor receptor and nkg2d activator receptor were related to a particular hormone (progesterone, prolactin and cortisol) might contribute to understanding the physiological role of the neuroendocrine axis on the expression of some receptors of the immune system in order to keep the homeostasis milieu of the mc. objective: sp-d, a key component of the innate immune system, is detected in amniotic fluid (af) and believed to originate in the fetal lung. however, sp-d is produced by other cells and therefore extra-pulmonary sources must be considered. the objective of this study was to determine the maternal and fetal plasma and af concentrations of sp-d to gain insight into the behavior of this natural antimicrobial peptide in pregnancy. moreover, we studied sp-d expression in maternal and fetal peripheral leukocytes. methods: maternal and fetal plasma and af samples were obtained from patients in the following groups: 1) term not in labor (tnl; n=20); 2) term in labor (til; n=31); 3) preterm labor without histologic chorioamnionitis (ptl; n=30) and 4) preterm labor with histologic chorioamnionitis (ptl-hc; n=27). sp-d concentration was measured by elisa. sp-d mrna expression in maternal and fetal leukocytes was evaluated by real-time qrt-pcr. flow cytometry and confocal microscopy were used to study the localization of sp-d in leukocytes. results: 1) the af sp-d concentration increased as a function of gestational age (mean, til: 52,055.7 ng/ml vs. ptl: 31,371.0 ng/ml; p<0.05); 2) in contrast, the maternal and fetal plasma sp-d concentrations decreased with advancing gestational age (mean, til: 375.8 ng/ml vs. ptl: 672.9 ng/ml; p<0.05, and til: 366.9 ng/ml vs. ptl: 1606.8 ng/ml; p<0.001, respectively); 3) the maternal plasma sp-d concentration was lower than that of fetal plasma (mean: 506.1 ng/ml vs. 1054.4 ng/ml; p<0.001); 4) however, sp-d mrna expression in maternal leukocytes was 4.8 fold higher than that of fetal leukocytes (p<0.001); 5) neutrophils (both maternal and fetal) expressed sp-d as demonstrated by flow cytometry and confocal microscopy. conclusion: 1) the concentrations of sp-d in the maternal and fetal circulation decreased with gestational age while the af concentration increased; 2) the expression of sp-d mrna is higher in maternal leukocytes than in fetal leukocytes; 3) we report for the first time that maternal and fetal neutrophils are a source of sp-d and propose that this molecule plays a role in host defense against infection and in the modulation of the maternal and fetal immune response. introduction intrauterine insemination (iui) is a fertility technique that allows for couples to have intercourse after the procedure is performed. it has been postulated that intercourse after iui may increase the pregnancy rate by either endometrial stimulation or because it may represent a second spermatic flow in the periovulatory period. in the present study we evaluate the effect of intercourse on the pregnancy rate of patients undergoing iui. material and methods: from 2004 to 2006 675 patients were enrolled in the study. every couple undergoing iui was instructed at the moment of insemination to decide whether to have or not intercourse on the same day of the procedure. all couples were abstinent three to seven days before iui. the information regarding intercourse was recorded the day after treatment. ovulatory, insulin resistant, cervical, male, tubal and endometrial factors as well as parity and time of infertility were compared between the two groups. all these factors were analyzed based on number of follicles that ovulated in each group. our principal outcome was to determine the pregnancy rate. intercourse was practiced by 65.8% of the couples. the global pregnancy rate was 15.4%. the pregnancy rate for the couples who had intercourse was 13.3% and 19.5% for those who did not have intercourse (p<0.05). even though age, parity, time of infertility and stimulation protocols were similarly distributed in both groups, the proportion of tubal and endometrial factors were higher among those who had intercourse (p<0.01). when subjects with tubal and endometrial factors were excluded, the pregnancy rate between both groups (n=463) was similar (16.1% vs 19.6% for positive and negative intercourse, respectively). the average number of ovulatory follicles was 2.12 + 1.1 for the group that had intercourse and 2.17 + 1.3 for those who did not. according to our results, intercourse after iui does not improve pregnancy rate after this procedure is performed. furthermore our study indicates that iui does not interfere with sexual intimacy since almost 66% of the couples decided to have intercourse on the same day of the procedure. . we sought to investigate the effects of gravidity on mmc and fmc in healthy, parous women. methods: mc was assayed in dna extracted from peripheral blood mononuclear cells (pbmc). hla-genotyping was first conducted and mc quantified employing a q-pcr assay targeting a non-shared maternal-or fetalspecific hla polymorphism. gravidity was dichotomized as a history of one pregnancy compared with two or more, and the prevalence of mc was analyzed using logistic regression. possible confounders were included as appropriate, including subject age and time since last pregnancy. adjustment for possible correlation between values was also made when there were repeated measures for the same subject. results: for the mmc analysis, there were 35 subjects with 73 observations. for the fmc analysis, there were 68 subjects with 145 observations. table 1 provides a summary of mmc and fmc by gravidity. mmc was significantly decreased with higher gravidity. fmc was not affected by gravidity. gravida 1 gravida 2 or more adjusted* or (95%ci) mmc 7/14 (50%) 5/48 (10%) 0.19 (0.04-0.79) fmc 13/30 (43%) 36/115 (31%) 0.49 (0.21-1.17) *adjusted for possible correlation between values within a subject, subject age, and time from last pregnancy, as appropriate. increasing gravidity is significantly associated with a decreased prevalence of mmc. despite additional sources of fmc, there does not appear to be an increase in fmc prevalence with increasing gravidity. the biology of mc is incompletely understood, and the nature of mmc and fmc are likely to be different given that acquisition of the former, but not the latter, occurs within a nascent immune system. these data raise interesting questions when considered as interactions of acquired grafts within a host, including whether emergence and persistence of one dominant source of mc may be most advantageous for an individual. anti-igd antibody treatment as a novel immunosuppressive agent for autoimmune diseases and its effects on th1/th17 gene expression. tommie g nguyen, 1 eileen d gallery, 1,2 jonathan m morris. elevated t-helper cell type-1 (th1) and type-17 (th17) cytokine expression have a role in autoimmune diseases, allograft rejection and pregnancy-related complications. thus, molecules that can shift the immunity away from th1/th17 responses toward a th2 response represent a novel therapeutic treatment for these conditions. we have previously demonstrated that pregnancy is associated with a suppression of t-bet in peripheral t cells. in this study, we examined a novel effect of anti-igd antibody on t-bet expression, th1/th17 gene expression in human peripheral blood mononuclear cells (pbmc) and its therapeutic effects in an animal model of collagen-induced arthritis. methods: human pbmc were isolated and then cultured in the presence of anti-igd antibody at various time points followed by stimulation with pma/ionomycin (p/i) for 5 hrs. gene expression was examined by rt-pcr, western blotting and elisa. for in vivo study, arthritis-prone dba/j1 mice were induced to undergo joint inflammation by intradermal injections with bovine type-ii collagen. these mice were then given 3 daily doses of 10 mg/kg of intravenous injection with anti-igd antibodies as preventive or therapeutic treatments (n = 10 per group). results: treatment with anti-igd antibodies significantly suppressed p/iinduced expression of t-bet (a master regulator of th1 immunity), tnf-(a classical pro-inflammatory th1 cytokine), and il-17 (a critical proinflammatory th17 cytokine) in human pbmc. this suppression is highly specific to these genes because anti-igd antibodies have no effects on the expression of ifn-g and il-2 (two classical th1 cytokines). in vivo experiment showed that anti-igd antibody treatment markedly reduced clinical severity of joint inflammation when comparing the clinical score of control mice group (7.8 ± 1.1, mean ± s.e.m), preventive group (5.4 ± 1.2) and therapeutic group (3.5 ± 1.3) . conclusions: our study has demonstrated that suppression of t-bet by anti-igd antibodies, similar to the changes seen in human pregnancy is a novel in vivo anti-inflammatory effect. given the essential role of t-bet, tnf-and il-17 in the pathogenesis of human autoimmune diseases, anti-igd antibodies may represent a novel immunosuppressive treatment that needs further studies and evaluation. objective: women with circulating anti-phospholipid antibodies (apl) are at risk for recurrent miscarriage, preeclampsia and preterm labor. apl antibodies directly target the placenta by binding to phospholipids or phospholipid-binding proteins expressed on the surface of viable trophoblasts. the objective of this study was to determine the effects of apl antibodies on first trimester trophoblast cells. methods: two mouse igg1 anti-human beta 2 -glycoprotein i monoclonal antibodies (mabs), designated id2 and iic5, were used in these studies. the first trimester trophoblast cell line, htr8, was incubated with either medium, a mouse igg1 control, id2 or iic5 (5-40 g/ml), in the presence or absence of unfractionated heparin (100ng/ml). trophoblast cell death and apoptosis was determined using a viability assay, hoechst staining and a caspase activity assay. cytokine production was evaluated by multiplex analysis. results: following a 48 hour incubation, significant trophoblast cell death was induced by iic5 (34.5 ± 2.8%) and id2 (46.4 ± 3.2%) at the high dose of 40 g/ml, when compared to the medium and mouse igg controls (p<0.001). hoechst staining showed that id2-and iic5-induced trophoblast cell death was a result of apoptosis. moreover, id2 and iic5 induced a significant increase in caspase-3, -8 and -9 activity (p<0.001). treatment of trophoblasts with heparin significantly inhibited the effects of iic5 and id2 on cell death by 46.5 ± 12.8% and 52.6 ± 8.6% %, respectively (p<0.005). following a 72 hour incubation at lower concentrations (20 g/ml), treatment of trophoblast cells with id2 or iic5 resulted in a significant upregulation of il-8, mcp-1, gro production (p<0.05), and a significant reduction in il-6 secretion (p<0.05). conclusion: this study demonstrates that at low levels apl antibodies can modulate trophoblast cytokine production, while at higher levels, the same antibodies induce trophoblast apoptosis in a caspase-dependent manner. these findings shed new light on the mechanisms by which apl antibodies may impact placental survival and function. antigenic targets for the diagnosis of premature ovarian failure. hc bohler, c gercel-taylor, lt ku, st nakajima, dd taylor. obstetrics, gynecology, women's health, university of louisville, louisville, ky, usa. objective: premature ovarian failure (pof) is a premature depletion of ovarian follicles before the age of 40, affecting approximately 1% of women <40 years. the involvement of autoimmune mechanisms in pof has been suggested and similar mechanisms have been postulated for other ovarian pathologies, including idiopathic infertility, polycystic ovary syndrome (pcos), or endometriosis. while the association of autoantibodies has been demonstrated for these ovarian pathologies, variation in specificity and frequency of false positivity have limited the diagnostic use of autoantibodies. the objective of this study was to develop an antigen array to differentiate antibody recognition patterns of pof from other infertility pathologies. design: prospective study in a university research laboratory. materials and methods: patients diagnosed with infertility were included in this pilot study: endometriosis (n=6), pcos (n=4) and pof (n=5). autoantibodies were assayed by dot immunoblotting using an antigen array derived from endometrial and ovarian cells. for the cellular antigen preparations, solubilized total proteins were separated by reverse phase-hplcquid chromatography and the individual proteins were blotted onto nitrocellulose membranes and reactivity visualized by peroxidase-labeled antihuman igg. results: patients with pof, endometriosis, and pcos all exhibited autoantibodies reactive with these cellular proteins. while some antigenic reactivities were shared by all infertility patients, the pattern of antigen recognition was distinct for patients with pof. patients with pof all recognized a common 6 antigenic proteins (row 2, antigens a,b,d-g). conclusions: alterations in autoreactivity are observed in patients with the diagnosis of infertility; however, distinct patterns of autoantibody recognition can be demonstrated for patients with different pathologies. while this study needs to be expanded to reliably establish the specificity, sensitivity and positive and negative predictive values, patients with pof clearly exhibit a shared recognition pattern that may be useful a diagnostic marker. cicek gercel-taylor. ob/gyn, university of louisville, louisville, ky, usa. objective: americans' consumption of nutraceuticals is one of the most rapidly expanding health markets, growing at a rate of 25% annually. multiple nutraceuticals containing phytoestrogens have been marketed as "immune boosters" despite suboptimal evidence-based medicine to support such statements. as immunomodulatory therapies should affect downstream cytokine expression, the relative effects of estradiol and genistein in regulating expression of cd3-and jak3 were tested. these markers were chosen since they are central to t cell signaling. cd3-is a critical transducer of tcr activation and regulates t cell proliferation and cytokine production. jak3 upregulation is a specific marker for hematopoietic cell stimulation. methods: to test the immunomodulatory effects of phytoestrogens and estrogen, jurkat 6.1 (t cell leukemia) cells were grown in estrogen-free, phenol red-free media for 72 hours. these cells were then exposed for 48 hours to 0 pm, 4 pm (postmenopausal), or 40 pm (premenopausual) of estradiol in the presence of increasing concentrations of genistein (0, 0.4, 1.5, 6.25, 25, and 100 m). cells were then solubilized and cellular protein quantitated. protein concentrations were standardized and western blots for each set of culturing conditions were run in triplicate. cd3-and jak3 expression were quantitated following visualization with chemiluminescence by digital pixel quantification. results: our findings show that in the absence of estradiol and at postmenopausal levels of estradiol, genistein induced a dose dependent increase in cd3-reaching a maximum of 20 fold. although cultivation of t cells in 40 pm of estradiol significantly increased the levels of cd3-and jak3 relative to hypoestrogenic conditions, the genistein mediated dose response was not observed. conclusions: these in vitro results indicate that genistein can at least partially reverse suppression of signaling molecules observed in postmenopausal estrogen environments. clinically, this suggests that phytoestrogens may have greater immunomodulatory properties for postmenopausal females than those that are premenopausal. maternal serum il-6 as a biomarker of acute immunologic rejection of pregnancy. joaquin santolaya-forgas, 1 juan deleon-luis, 2 isabel galan. 2 1 obstetrics and gynecology, brigham and women's hospital, boston, ma, usa; 2 amarillo women's health research institute, texas tech university health science center, amarillo, tx, usa. objective: markers of acute rejection of pregnancy are very scarce. in this study we aimed at determining if rapid changes in maternal serum concetration of a variety of biomarkers could be used for this purpose. we used an established baboon model for in utero stem cell therapy to introduce at 36-44 days from conception and via ultrasound-guided celocentesis, human hematopoietic stem cells with different proportions of natural killer t-cells (nk). maternal blood was collected before and 24 hours after celocentesis for quantification of hormones and il-6 using solid phase, enzyme labeled, chemiluminescent sequential immunometric assays. pearson correlation analysis was used for determination of significant changes from baseline (p<0.05). results: all 9 animals survived their pregnancies. seven animals receiving <5% concentration of nk delivered at term ( 180 days gestation) while 2 animal receiving more than 7% concentration of nk had dead fetuses on ultrasound evaluation 24 hours after celocentesis. table 1 depicts mean maternal serum concentration of the biomarkers investigated (all n.s.). figure 1 shows mean il-6 changes from baseline in continuing (n.s.) and rejected pregnancies (p<0.05). conclusions: we have described a model in which in utero graft vs host disease can be studied. these preliminary results suggest that of all the biomarkers investigated, il-6 might be the most sensitive for detection of an acute rejection of pregnancy. biomarkers of acute immunologic rejection of pregnancy biomarker unit pre-celocentesis (9) the activity of cytotoxic cd8+ t cells during pregnancy protects the mother and fetus from infection. however, pregnancy's effect on the proliferation and apoptosis of cd8+ t cells has not been clearly defined. objective: to determine if normal pregnancy changes the number of proliferating or apoptotic splenic cd8+t cells. methods: female c57bl/6 mice were used unmated (um) or underwent timed mating. one day prior to harvest, mice were i.p. injected with bromodeoxyuridine (brdu), which is incorporated into replicating dna. harvested spleens were homogenized, enumerated, and stained for cell surface expression of cd8 and t cell receptor beta chain (tcr ). apoptotic cells were detected by treatment with terminal transferase and fitc-dutp (tunel). the numbers of cd8+tcr + cells that were brdu+ or tunel+ were calculated from the percentage of positive cells obtained by flow cytometry and the absolute number of cells counted. for each experiment, the ratio of the number of positive cells in pregnant to um mice was compared by anova with dunnett's post-test. results: at day 5 of pregnancy (n=5), the number of brdu positive cd8+ t cells was two fold higher than that found in um (n=12, p<0.01). the number of proliferating cd8+ t cells continued to be non-significantly elevated at day 8 (1.6x, n=5), day 10 (1.4x, n=7), and day 12 of pregnancy (1.5x, n=6) compared to um. by day 15 of pregnancy (n=5) the number of proliferating cd8+ t cells returned to the um level, however by this time the total number of splenic cd8+ t cells was 1.5 fold higher than um (n=12 p<0.001). on gestational day 18, the number of proliferating cd8+ t cells declined further (0.3x, n=4), and the number of splenic cd8+t cells returned to the um level (n=12, p>0.05). compared to um mice, there was no significant difference in the number of cd8+ t cells undergoing apoptosis at any gestational day examined (0.7-0.9x, p>0.05). in normal murine pregnancy, the number of cd8+ t cells is increased in late gestation, and then returns to baseline at the end of pregnancy. this is due to an early increase then gradual decline in cd8+ t cell proliferation, accompanied by a steady rate of apoptosis. this argues that the maternal immune system undergoes dynamic homeostatic changes, and is not globally suppressed. supported by nihro1hd043-185 niht32ai055402. arturo cerbulo-vazquez, 1 cun li, 1,2 gene hubbard, 2 natalia e schlabritz-loutsevitch, 1,2 peter w nathanielsz. objectives: early thymocyte (t) maturation occurs in the cortex while later stages occur in the medulla. thymic epithelial cells (tec) synthesize gc and t express gr. tec may influence t cell maturation by regulating apoptosisinduced gc-gr interactions. igf-1 (also synthesized by tec) may support thymocyte prolifetarion. human fetuses of mothers in premature labor are exposed to gc. gc administered to pregnant baboons at 0.6, 0.65, and 0.7 gestation (g) alters fetal lymphocyte populations at 0.95 (g) (j repro immunol, 2006, 69:149) . we determined if fetal gc exposure alters thymic 1) structure; 2) gr and igf-i protein. methods: pregnant baboons received saline (control ctr; n=6) or betamethasone i.m. (175 μg/kg daily for two days at 0.6, 0.65 and 0.7g; gc group; n=6), c-sectioned at at 0.9g under general anesthesia and thymic gr and igf-i evaluated by immunohistochemistry. results: gr localized to medulla and a few cortical cells. igf-i localized to cortex with little medullary expression. medullary necrosis was greater in ctr than gc fetuses. t gr was located in cytoplasm. no gross differences were observed between ctr or gc fetuses for either igf-i or gr. conclusions: a) early thymocyte maturation may be supported by igf-1, b) later differentiation involves gr, and c) after exposure to gc doses equivalent to human therapy, no gross effects were detected on gr or igf, but d) natural thymic necrosis was inhibited. lindsay s christensen, peyman bizargity, elizabeth a bonney. ob/gyn, university of vermont, burlington, vt, usa. background: the exact mechanism by which bacterial products trigger preterm delivery and the immune cellular circuits involved remain unclear. our recent data in normal c57bl/6 (b6) or recombinase deficient c57bl/6 mice (rag-ko) indicates that t and b cells are not critical for lps-induced preterm delivery and stresses the importance of related innate mechanisms. rag-ko mice are more susceptible to lps, suggesting that t or b cells may control the innate response. macrophages are vital to innate immunity and produce proinflammatory cytokines that can activate prostaglandin synthesis and myometrial contraction. we questioned whether differences in susceptibility between the strains are due to differences in the uterine macrophage response to lps and thus examined macrophages at the maternal-fetal interface early after injection. objective: to compare uterine macrophages levels at 2 and 6 hours after lps injection in pregnant b6 and rag-ko mice. methods: b6 and rag-ko mice were mated and on gestation day 15, females were injected intraperitoneally with 3μg lps in 200 l saline (pbs) or 200 l pbs alone. euthanasia and uterine harvest occurred 2 or 6 hours after injection. frozen uterine sections were stained with the macrophage marker f4/80 or an isotype-matched control followed by an alexafluor 546-conjugated secondary and a nuclear stain (dapi). sections were visualized by fluorescence microscopy. for each mouse, f4/80+ dapi+ and total dapi + cells were counted in 3 areas of 1 representative section and the percentage of f4/80+ dapi+ cells was calculated. the mean percentage for at least 6 representative areas per experimental group was analyzed by anova. results: two hours post-injection, macrophages levels were similar in b6 and rag-ko mice injected with pbs (b6, n=3, 2.9±1.6; rag-ko, n=3, 2.7 ± 1.2 % + per area). lps injection increased macrophages (p<0.05) in both strains (b6, n=3, 4.4±2.9; rag-ko, n=3, 5.5 ± 1.8,); no difference was evident between strains. the percentage of f4/80+ cells was similar 6 hours post-injection (b6, n=3, 3.4± 1.9 v. rag-ko, n=3, 3.6± 2.8), and not elevated relative to the 2 hour time point. objective. decay-accelerating factor (cd55), is expressed in the plasma membranes and protects mammalian cells against the lytic action of serum complement. phosphoinositide 3-kinases (pi3ks) are involved in the regulation of cell functions by synthesizing a second messenger molecule ptdins (3,4,5) p3. akt, a serine-threonine kinase acts downstream of pi3k and regulates cell survival, growth and proliferation. the pi3k-akt activity is controlled by tumor suppressor gene pten. in this study we assessed whether the pi3k-akt activity affects the expression of cd55 in human endometrial and cervical cells. methods. endometrial and cervical cell lines which differ in the constitutive pi3k activity were used in this study. ishikawa and rl95-2 endometrial cell lines harbor pten mutation and have high levels of phosphorylated akt (p-akt). hec-1-a and kle endometrial cell lines and hela cells express wild-type pten and have minimal or no demonstrable levels of p-akt. the expression of cd55 was evaluated by rt-pcr, immunoblotting and flow cytometry. the pi3k activity was assessed by immunoblotting with anti-p-akt antibodies. the effect of inhibition of pi3k-akt pathway on cd55 expression was evaluated in cells treated with wortmannin (400 nm), ly294002 (25 mm), or with akt inhibitor sh5 (5 mm). results. immunoblotting densitometry and measurements of mean fluorescence intensities showed that the level of cd55 expression correlates with the status of pi3k-akt pathway. the cd55 expression was lowest in hec-1-a, ishikawa and rl95-2 cells which constitutively express p-akt. higher cd55 expression was found in hela cells and kle cells which express wild-type pten product and has no detectable phospho-akt. mean fluorescence intensities were 3.4-fold higher for kle cells and 12-fold higher for hela cells compared to hec-1-a cells. treatment of cells with akt inhibitor led to 1.1-1.4-fold increase in cd55 expression. the 1.1-5.7-fold increase following treatment with pi3k inhibitor wortmannin was found in ishikawa cells, rl95-2 and kle cells. conclusions. human endometrial cell lines with elevated pi3k-akt activity express lower level of cd55 compared to cell lines with intact pten gene function. these findings may indicate that structural alteration at the dna level and resultant overexpression of pi3k-akt pathway are involved in the downregulation of cd55. endometrial and cervical cells. pawel goluszko, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. objective. cell shape is determined by the cytoskeleton, which provides the mechanical support and is involved in cellular signaling. apoptotic cells undergo major morphological changes such as rounding and contraction, a process regulated by caspases, the cysteine proteases responsible for events controlling the cell disassembly. the motifs in certain cytokeratins make them substrates for caspase degradation. anti-apoptotic serine/threonine kinase akt provides a survival signal by phosphorylating downstream effector molecules including caspase-9. while studying the akt distribution in human endometrial cell line we found that akt shows filamentous pattern of staining resembling cytoskeleton organization. in this study we evaluated whether akt staining correlates with the microfilaments (mf), microtubules (mt) or intermediate filaments. endometrial ishikawa, rl95-2, hec-1-a, kle and cervical hela cell lines were used in the study. incubation with cytochalasin d, (1ug/ml) or nocodazole (1mg/ml) and labeling with bodipy fl phallacidin, anti -tubulin or anti-akt antibody were used to assess whether cytoskeleton disruptors affect akt distribution and mf and mt organization. for colocalization, cells were stained with anti-cytokeratin 18 mouse antibody followed by anti-mouse alexa 488 conjugate, and then stained with anti-akt rabbit antibody and anti-rabbit alexa 549 conjugate. the scans collected with laser scanning confocal microscope from channels filtered for alexa 488 and alexa 459 were combined digitally and evaluated with imaris colocalization analysis software. filamentous pattern of akt staining was most pronounced in ishikawa and less obvious in hec-1-a cells. treatment with cytochalasin d or nocodazole resulted in disruption of mf and mt but had no effect on cytokeratin organization and akt distribution. double staining with anti-cytokeratin-18 and anti-akt antibody showed overlapping staining for cytokeratin and akt. analysis of digitally acquired images showed highest correlation for colocalized channels in rl95-2 cells (0.87) followed by ishikawa (0.79) and hela cells (0.72). lowest correlation was found for kle (0.65) and hec-1-a cells (0.59) conclusions. this study indicates a strong colocalization pattern of serine/ threonine kinase akt with cytokeratins, and suggests a mechanism by which cytokeratins might be protected against cleavage by caspase-9 and caspase-3 in the early apoptotic stages. background: cd4+ cd25+ t regulatory cells (t-reg), express foxp3,suppress antigen-specific immune responses and are important for allograft tolerance. during pregnancy the mother tolerates an allograft expressing paternal antigens (the fetus), requiring substantial changes in immune regulation over a programmed period of time. the presence of t-reg cells (cd4+ cd25highfoxp3+) was assessed in the peripheral venous blood of 25 non-pregnant, 63 pregnant and seven postnatal healthy women by flow cytometry. human decidua was obtained by surgical termination of pregnancy in the first (n=26), second (n=16) and third (n=5) trimester of human pregnancy. paraffin sections were immunostained for foxp3 and cd25. foxp3 +cells were quantified in 5 x400 fields and results compared between first, second and third trimester samples and according to the presence of extravillous trophoblast. results: fluorometric studies of blood samples indicate an increase % of circulating cd4+ cd25highfoxp3 t-cells in pregnant (3.06% [range 1.21-4.8%]) vs. non-pregnant controls (2.58% [range 1.1 -5.03%]; p<0.05). a progression from 1st, 2nd and 3rd trimesters indicated the % of cd4+ cd25highfoxp3 t-cells was 3.01%, 2.88% and 3.14%, respectively. low numbers of foxp3+ cells were detected in all decidua samples and their distribution mirrored that of cd25+ cells. in 1st trimester samples, foxp3+ cells were often detected in lymphoid aggregates adjacent to endometrial glands. increased numbers of foxp3+ cells were detected in 1st (21.9±4.3) compared with 2nd trimester decidua (9.9±3.2; p<0.05) but there was no difference between 1st and 3rd trimester (12.0±3.1), nor between 2nd and 3 rd trimester decidua. in 1 st trimester decidua, numbers of foxp3+ cells were increased in areas without extravillous trophoblast. conclusion: normal human pregnancy is associated with an increase in the number of circulating cd4+ cd25highfoxp3 t-cells. the presence of foxp3+ cells in early gestation human decidua may be important in the initiation of materno-fetal tolerance at an autocrine level. (supported by mrc). aims: -defensins are small cationic peptides with antibiotic and antimicotic activity. hyaluronan and its degradation products have been described as endogenous ligands for tlr2 and tlr4, whose involvement in -defensin expression has been reported in different epithelial tissues and cell lines. we aim to investigate weather low molecular weight hyaluronic acid induces -defensin 2 release by keratinocytes, via tlr2 and tlr4. methods: the induction of -defensin 2 production following in vitro treatment of human keratinocytes with a low molecular weight hyaluronic acid solution was evaluated by pcr-analysis and elisa techniques. studies on the involvement of tlr2 and tlr4 in -defensin 2 production have been performed using specific blocking antibodies. results: pcr and elisa revealed an intense -defensin 2 production following hyaluronic acid treatment in human keratinocytes. the -defensin 2 production induced by hyaluronan was abolished following block of tlr2 and tlr4 by specific antibodies, demonstrating the involvement of these receptors. the same hyaluronic acid treatment did not induce activation of inflammatory genes, such as il-8, tnf-, il-1 and il-6. conclusion: our data show that hyaluronic acid is an efficient inducer ofdefensin 2 production in keratinocytes, via tlr2 and tlr4. this observation might be important to open new perspectives in the development of possible topical products containing hyaluronic acid, to improve the release ofdefensins by keratinocytes, ameliorating the self-defence of the skin in case of skin infections. therefore, one of the possible applications for this kind of topical products might be the treatment of infective vulvitis, one of the most distressing gynaecological diseases for adult women. pregnancy outcome? kiera von besser, 1 serena wu, 1 mary d stephenson. 1,2 1 obstetrics and gynecology, university of chicago, chicago, il, usa; 2 obstetrics and gynaecology, university of british columbia, vancouver, bc, canada. objective: to investigate whether gender of an ongoing pregnancy impacts the probability of a successful outcome, and, to ascertain whether the gender of prior live birth(s) impacts subsequent pregnancy outcome, among women with rm/aps. materials and method: cohort-control study. rm subjects were evaluated by mds between 1992 -2004 (stephenson, 1996 . couples who met aps criteria (wilson et al, 1999) , restricted to rm only, were followed prospectively. cohort data was compared to live birth data from the vital statistics agency of british columbia from 1999-2004. secondary sex ratios (ssrs) among successful pregnancy outcomes were calculated by dividing the number of male live births by female. sex ratios were calculated for all pregnancies 24 weeks, regardless if they ended in success or demise. pearsons 2 test with yates continuity correction was applied. results: 388 subjects were identified. 226 subjects had 319 prior live births of known gender (174 male/145 female), giving a ssr of 1.20. there were also 25 prior fetal demises 24 wks of known gender (13/12) giving a sex ratio for all prior pregnancies at 24 wks of 1.19. 252 subjects delivered 301 subsequent live births of known gender (158/143), giving a ssr of 1.08. there were also 6 subsequent fetal demises (3/3) giving a sex ratio for all subsequent pregnancies of 1.10. 112 subjects delivered both prior and subsequent live births. the ssr was 1.36 (83/61) among their prior and 1.24 (68/55) among their subsequent live births. including fetal demises, 119 subjects had ongoing prior and subsequent pregnancies. the sex ratio was 1.32 among their prior pregnancies and 1.10 among their subsequent. as the control, a ssr of 1.06 (124,982/118,093) was calculated from vital statistics data. when the prior and subsequent ssrs of the cohort were compared to each other, as well as to the control, there were no statistically significant differences. conclusions: our findings, from the largest study of its kind to date, suggest that, in patients with rm/aps, the gender of an ongoing pregnancy does not significantly affect the probability of a successful outcome, to any greater degree than it does in the general population. also, the gender of a prior ongoing pregnancy does not significantly impact the likelihood of developing rm/aps. oocyte maturation. jk friend, fb bezirci, e seli. ob gyn, yale u., new haven, ct, usa. introduction: oocyte maturation is associated with repression of transcription. during oocyte maturation, fertilization, and early embryo development until the onset of zygotic gene expression, proteins are synthesized from maternallyderived mrnas. the regulation of protein expression from these maternal mrnas is post-transcriptional, and occurs mainly via poly(a)-tail elongation. the embryonic poly(a)-binding protein (epab) is the predominant poly(a)binding protein before the activation of the zygotic genome, and plays a critical role in the activation of certain maternal mrnas, those bound by cpeb and probably pumilio. we are characterizing additional functions of epab during the process of oocyte maturation. methods and results: our model system is the xenopus laevis oocyte where oocyte maturation is induced by the addition of progesterone. our preliminary findings indicate that epab is phosphorylated, and that levels of phosphorylated epab increase upon progesterone-induced oocyte maturation. moreover, glycerol gradient centrifugation revealed that nonphosphorylated and phosphorylated epab are contained in distinct complexes that change mobility upon oocyte maturation. furthermore, oligo-dt selection for poly(a)-containing mrnas strongly suggests that these mrnas are bound exclusively by phosphorylated epab. using affinity purification, we have determined that nonphosphorylated epab exists primarily in a large protein complex prior to oocyte maturation that is later disassembled after the addition of progesterone. conclusions: based on these preliminary findings, we conclude that prior to oocyte maturation, the bulk of epab is nonphosphorylated and is found in a protein-rich complex separate from poly(a)-containing mrnas. upon oocyte maturation (when certain maternally-derived mrnas are activated for translation), the majority of epab becomes phosphorylated, and this phosphorylated form of epab is likely bound to translationally-active mrnas. we are currently investigating what kinase phosphorylates epab and whether this phosphorylation plays a role in translational up-regulation of epab-bound mrnas. introduction: reactive oxygen species (ros) play important roles in all aspects of cellular fate. nadph oxidase isoforms, a family of genetically preserved enzyme complexes, have been shown to be the main sources of ros in various cell types. however, the role of nadph oxidase isoforms in human myometrium proliferation and differentiation has not been defined. in the myometrium, different smooth muscle phenotypes maybe associated with specific physiologic functions. we have shown that angiotensin ii (angii) stimulates hypertrophy but not cell proliferation in ultr cells, an in vitro model of human myometrium. ultr cells at greater than 30 passages display a replicative senescent phenotype. by introducing human telomerase reverse transcriptase (htert) gene, we have obtained a stable cell line (ultr-ht) which has a significantly increased division rate and distinct cellular morphology than the original ultr cells. objective: to determine the relationship of expression of nadph oxidase to ultr cellular fate. methods: early and late passages (p26-34) of ultr and ultr-ht (p31-38) cells were grown on either plastic or collagen iv (cn4)-coated surfaces. ultr-ht cells were further stimulated with angii (0.1 um) for 24 hrs. expression of nadph oxidase core (nox1-5 and duox1/2) and associated subunits (p22phox, p47phox, noxo1, p67phox, noxa1, p40phox, and rac1/2), and angii receptors at1/2 was identified by rt-pcr from cellular total rna. fluorescent immunohistochemistry (ihc) was employed to determine protein expression and localization. results: the mrna level of house keeping gene -actin was unchanged by any cellular manipulation. the senescent phenotype of ultr cells was accompanied by an apparent down-regulation of nox1, p22phox, and noxa1 genes, and an up-regulation of at1/2. overexpression of htert did not reverse nox1, p22phox and noxa1 expression while cell division rate was increased. however, there was a down-regulation of nox4, at1/2 and rac2. plating ultr-ht cells on cn4 induced nox4/5 down-regulation and up-regulation of duox1/2, with no apparent change of at1/2. however, exposure to cn4 re-directed cellular response to angii such that only nox5 was induced by angii stimulation. conclusion: expression of nadph oxidase isoforms nox1, 4, 5, and duox1/2 are correlated with ultr cell differentiation and cell fate control. data also suggests that ang ii-induced myometrial hypertrophy involves nox5 mediated ros generation. fluids from reproductive women -the influence of aging. eriko y fujii, 1 masahiro nakayama. 2 1 women's health, national center for child health and development, tokyo, japan; 2 aska reproductive clinic, nara, japan. [introduction] receptor for advanced glycation end products (rage) is a multiligand type glycoprotein, and is characterized based on its ability to bind ages, adducts formed by non-enzymatic glycation and oxidation of protein and lipids. this process occurs during normal course of aging. ages/rage interaction regulates various physiological function, such as inflammation, angiogenesis through vegf inducement. a soluble form of rage (srage) works as a decoy in the body and inhibits intracellular signaling. [objectives] the balance of these factors may contribute to reproductive dysfunction by aging. we aimed to measure the ages, srage and vegf concentrations in plasma and follicular fluids from reproductive women, and examined the differences of those factors between young group and old group. [material and methods] patients' plasma and follicular fluid were collected with consent based on regulations of the ethical committee, and we measured ages (pentosidine, cml), srage and vegf in duplicate using commercially available elisa kits (fushimi co, r d and cyelex). concentrations were calculated from each standard curve, and compared between the young group under 34 years old, and the old group over 35 y.o. data were evaluated for the difference in two groups by student's t test , and the significance was determined by p<0.05. [results] 1) srage in plasma, 1517±642 pg/ml (mean±sd), n=40 in the young group was significantly higher than 1268±530 pg/ml, n=51 in the old group. there was no significant difference in plasma vegf. 2) vegf in follicular fluid was 45±22 pg /mg protein, n=37 in the young, and 56±27 pg /mg protein, n=63 in the old was increased significantly. 3) we could not see statistical difference of pentosidine nor cml concentrations between two groups in plasma and follicular fluid samples. [conclusions] it has been reported that higher concentration of vegf in follicular fluid may relate to worse pregnancy rate in art. there was a significant decrease of plasma srage in older group in our result, and because of this decrease of 'decoy', focal ages-rage-vegf signaling might be activated in older women. our results showed the possibility that ages/rage and vegf regulation may contribute to the reproductive dysfunction by aging. and leiomyosarcoma cells. qun pan, xiaoping luo, nasser chegini. ob/ gyn, university of florida, gainesville, fl, usa. as a part of a novel endogenous rna silencing machinery, a noncoding short rna strand referred to as "microrna" (mirna) has been identified to regulate the stability of the target gene expression through mrna degradation and repression. we have identified the expression of many of these mirnas in leiomyoma, myometrium, their isolated smooth muscle cells (lsmc and msmc), transformed lsmc (t-lsmc) and sklm (leiomyosarcoma cell line), including mir-21 which is predicted to target the expression of many genes, including tgf-b and tgf-b type ii receptor (tgf-brii). however, the biological significance of these mirnas in various cellular processes remains to be established. as such in the present study we examined the expression, regulation and function of mir-21 in lsmc, msmc, t-lsmc and sklm. we found that mir-21 is expressed and regulated by 17b-estridiol and medroxyprogesterone acetate (10 -8 m) in these cells (p<0.05). we further assessed the regulatory function of gain of and loss of function of mir-21 on the expression of tgf-brii. transfection of lsmc, msmc, t-lsmc and sklm with pre-mir-and anti-mir-21 oligonueclotides resulted in a significant increase and/or inhibition of mir-21 expression in these cells, respectively as determined by realtime pcr (p<0.05). over-expression of mir-21 resulted in a significant reduction, while transfection with antimir-21 increased the expression of tgfbrii mrna and protein in these cells as compared to controls (p<0.05). we concluded that mir-21 is expressed in leiomyoma and myometrial cells, its expression is regulated by the ovarian steroids and it functions by targeting the expression of tgfbrii and possibly other genes with key regulatory action on cell growth, angiogenesis, transcription regulation, ecm turnover and apoptosis that results in leiomyomas growth and regression. (supported by nih grant hd37432). objective: placenta and a number of gestational tissues are well recognized to express corticotrophin releasing factor (crf), urocortin 1 (ucn1), ucn2, ucn3 and crf -r1 and crf-r2 receptor subtypes together with crfbinding proteins locally. ucn2 and ucn3 are implicated in the reversal of stress responses initiated by crf. in the present investigation, we evaluated functions of crf and ucns by quantifying their contents in venous smooth muscle layers using image pro 4.01 in human umbilical cords collected at preterm and term gestation methods: umbilical cord specimens (3-4mm thickness, 6 pieces per umbilical cord) collected at preterm and term (n=6 each) at delivery were fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochemical analyses with polyclonal antibodies to crf (1:300), ucn1 (1:250), ucn2 (1:250) and ucn3 (1:250) (peninsula laboratory, pa and sigma aldridge, ms) by standard abc technique. immunoreactive materials on the sections were identified using 3, 3'-diaminobenzidine as a chromagen. immunostaining intensities (od/area) on uc-sections were quantified by image pro 4.01 software and expressed as arbitrary units (au). all values were expressed as mean ± sem. differences between groups were evaluated by anova, followed by the post-hoc tukey test for multiple comparison. results: antibodies to crf and ucns elicited positive immunostaining of variable intensity in venous smooth muscle layers in uc-sections of preterm and term gestations. immunostaining intensity (au) of venous smooth muscle layers at preterm (pt) and term (t) are as follows: crf-pt= 0.0451±0.0043 vs crf-t= 0.1027± 0.0242 (p<0.5); ucn1-pt=0.1736 ± 0.0108 vs ucn1-t= 0.0959± 0.0116 (p<0.01); ucn2-pt= 0.0964± 0.0063 vs ucn2-t= 0.0724 ± 0.1632 (p=ns); ucn3-pt= 0.1214 ± 0.0034 vs 0.0732± 0.0050 (p<0.05). conclusion: crf content in venous smooth muscle layer markedly increased at term, while ucn1 and ucn3 contents significantly decreased and no significant change occurred in ucn2 content. based on the opposing changes in crf vs ucn1 and ucn3 immunostaining, we speculate that crf, but not ucn1 or ucn3, is the major key player of vasodilation and function locally at the level of venous smooth muscle cells at term. ucn1 and ucn3 may perform cytoprotective functions at preterm. physiol. 1947; 150: 511-519, am j physiol. 1952; 170:72-76) . these studies showed that when ad libitum (al) feeding was resumed in cr females, fertility was sustained well beyond the age at which al-fed females became infertile. however, much of what is known on the effects of cr on fertility derives from models in which cr was initiated at weaning. further, there is large variation in how the experiments were conducted. objective: herein we tested if adult-onset cr could delay age-related infertility in females. methods: cr (40%, nia protocol as described in j geront. 1999 54a:b492-b501) was initiated in c57bl/6 female mice at 4 months and continued until 15.5 months of age, at which time al feeding was resumed. matings were initiated at 10 months of age. for mating during cr, a male mouse was housed overnight in a cage with a female and removed the next morning, so that the female mice could be fed their dietary food ration. al-fed and cr females followed the same mating regimen. the number of offspring born and that survived to weaning (day 21) were recorded. results: fertility was lost in 3 of 11 al-fed females by 10 months of age and continued to decline through 15.5 months of age. age-matched females maintained on cr during the same period exhibited poor fertility, with a total of 9 pregnancies achieved out of 12 females. although cr females showed poor fertility while on cr, their fertility improved dramatically after the reinitiation of al feeding at 15.5 months of age. while only 1 out of 8, al-fed females achieved a pregnancy between 15.5-18.5 months of age, 6 out of 10 age-matched cr-then-al-fed females achieved a total of 10 pregnancies in this 3-month time. notably, 79% of the pups born to cr-then-al-fed females between 15.5-18.5 months of age survived and developed to weaning without complications. conclusions: adult-onset cr allows maintenance of female fertility into advanced maternal age after the reinitiation of al feeding. how long fertility can be maintained and the minimum time needed for the beneficial effects of cr to be realized remain to be investigated. nonetheless, these observations suggest that there may be ways to safely extend fertility in females at ages when reproductive function is suboptimal (nih r37-ag012279). bile acids in human ovarian follicular fluid. laura p smith, 1,2 kaila deiorio-haggar, 1 jason reindollar, 3 alan s penzias, 1,2 anny usheva-simidjiyska. 3 1 ob/gyn reproductive endocrinology infertility, bidmc, boston, ma, usa; 2 boston ivf, boston, ma, usa; 3 endocrinology, bidmc, boston, ma, usa. introduction: bile acids are known to serve important functions in the hepatobiliary and gastrointestinal systems. the presence of bile acids in the human ovary and relation with fertility potential have never been previously evaluated. methods: human follicular fluid (ff) from large follicles and small follicles was obtained at vaginal oocyte retrieval. human serum was obtained before and 36 hours after human chorionic gonadotropin (h-cg). follicular fluid and serum samples were analyzed for total bile acids by spectrophotometry. bile acid concentrations were correlated with age, number of retrieved and mature oocytes, number of fertilized oocytes, and pregnancy. results: bile acid concentrations were analyzed and compared to the normal human serum bile acid concentration which ranges from 0 to 15 micromoles/l. bile acids are present in follicular fluid with a mean concentration of 15.28 micromoles/l in large follicles and 14.22 micromoles/l in small follicles (p=0.108). pre and post h-cg serum bile acid concentrations differed significantly (13.88 micromoles/l vs. 7.85 micromoles/l, p=0.004). there was a trend toward higher bile acid concentration in large follicles of young patients < 35 years old compared to older patients 40 years old (16.22 ± 5.55 vs. 13.56 ± 2.26, p=0.051). there was also a trend toward higher pre h-cg serum bile acid concentrations in older patients (4.47 ± 1.92 vs. 2.90 ± 0.72, p=0.079). there was no correlation between serum and follicular fluid bile acid concentrations and number of oocytes retrieved or number of mature oocytes, but there did appear to be a positive correlation between pre h-cg serum bile acid concentration and number of fertilized oocytes (spearman's correlation coefficient 0.678, p=0.005). conclusions: this is the first demonstration of the presence of bile acids within human ovarian follicular fluid. there may be a relationship between bile acid concentration and fertility potential. the precise function of bile acids in human ovarian follicular fluid is under investigation. objective to investigate the impact of ovarian hyperstimulation treatment on the biomarkers of the homocysteine pathway in blood and follicular fluid, and their association with the follicle diameter as a measure of follicular maturation. methods in 201 women undergoing ivf/icsi treatment blood samples were collected on cycle day 2 and the day of hcg administration. during oocyte retrieval in each woman the diameter of two follicles was measured and the corresponding follicular fluids were collected. in blood and follicular fluid total homocysteine (thcy), folate, cobalamin and pyridoxal'5'phosphate (plp) concentrations were determined. women with a serum folate 22.5 nmol/l were classified as folic acid supplemented. results ovarian hyperstimulation significantly decreased thcy and cobalamin blood levels (both p 0.001). the blood and follicular fluid concentrations of thcy, folate, cobalamin and plp were significantly correlated (all p 0.001). in the total group, a two-fold increase of thcy in follicular fluid resulted in a 0.06 mm decrease of the follicular diameter (p 0.05). in non-supplemented women this decrease was 1.64 mm (p 0.01). in supplemented women a twofold increase of follicular fluid folate resulted in a 0.74 mm decrease of the follicular diameter (p 0.05). conclusions ovarian hyperstimulation reduces thcy blood levels independent of folic acid supplementation. however, high follicular fluid thcy and folic acid supplementation may have detrimental effects on the maturation of the follicle. 2), post-fixed for 40 rain in 2% w/v osmium tetroxide in the same buffer, quickly dehydrated in a series of ethanol solutions, and embedded in epon. thin sections were stained with uranyl acetate followed by lead citrate and were observed at electron microscope. results: ten blastocysts from each group were collected and analyzed. compared to the in vivo embryos, blastocysts generated in vitro exhibited significant differences. the surface of their trophectoderm (te) layer had a reduced number of microvilli, the number of the apoptotic cells in the inner cell mass (icm) was higher and the presence of non functional mitochondria was elevated. in this study we have, for the first time, compared the ultrastructure of the in vivo and in vitro conceived blastocysts. taken together, these results suggest that both, the higher rate of apoptosis and the morphological alterations in the mitochondrial structure in ivf embryos, are associated with stress during in vitro embryo culture. therefore, these parameters can be used, in the future, as markers for the assessment of the embryo wellbeing in the ivf settings. deteriorating oocyte quality is a critical hurdle in the management of infertility, especially one associated with advancing age.here, we explore a newly discovered role of nitric oxide (no) in the sustenance of oocyte quality. methods: sibling oocytes from superovulated mice were subjected to intracytoplasmic sperm injection (icsi) with cauda-epididymal spermatozoa following exposure to either the no donor, s-nitroso n-acetyl penicillamine (snap, 0.23 m/min); a soluble guanylyl cyclase inhibitor, 1h-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (odq, 100 m) or an no synthase inhibitor, n w -nitro-l-arginine methyl ester (l-name, 1 mm). their sibling oocytes were subjected to icsi either before (young) or after culture for the corresponding period (old). outcomes of fertilization, cleavage and development to the morula and blastocyst stages were compared. some embryos from each subgroup were also subjected to tunel assay for apoptosis. results: oocytes deteriorated in their ability to undergo normal fertilization and development to morulae/blastocysts after aging in culture, as compared to their sibling cohorts subjected to icsi immediately after ovulation (p<0.05). this deterioration was prevented after oocyte exposure to snap. while, exposure to l-name or odq resulted in a significant compromise in fertilization and development to the morulae/blastocysts (p<0.05) with detection of apoptosis, which was also noted in embryos derived from aged oocytes but not in those from young or snap exposed oocytes. conclusions: no is essential to sustain oocyte fertilizability and developmental ability, and to prevent blastomere apoptosis. objective to investigate the effects of the levels of the biomarkers of the homocysteine pathway on ivf outcome. methods from 201 women undergoing an ivf or icsi procedures, two blood samples and two mono follicular fluid samples were collected for determination of folate, cobalamin, and total homocysteine (thcy). total protein was determined in follicular fluid to adjust the biomarker concentrations for follicular maturation. primary endpoint of the study was oocyte quality measured by fertilization and embryo quality (range 1-5; with 1 being best quality). secondary endpoint was the occurrence of pregnancy. results 67% of the included women used a folic acid supplement (serum folate 22.5 nmol/l). in non-supplemented women higher cobalamin levels in follicular fluid correlated with a better embryo quality (estimate -0.87; p 0.05) and higher thcy levels (median 7.1 mol/l, range 4.0-47.0) correlated with a worse embryo quality (estimate 1.01; p 0.05). in supplemented women higher follicular fluid thcy (median 6.4 mol/l, range 3.5-73.6) correlated with better embryo quality (estimate -0.58; p 0.05). the follicular fluid folate level of oocytes that did not fertilize was 1.1-fold higher than in the fluids of a fertilized oocyte (95% ci 1.00 -1.18; p 0.05). a two-fold increase of follicular folate corresponded with a 3.3 higher chance to achieve pregnancy (95% ci 1.09-9.71). conclusions cobalamin levels in follicular fluid are correlated with embryo quality. folic acid supplementation modifies the thcy and folate levels in follicular fluid and thereby affects oocyte quality. the level of folate in follicular fluid is important in the fertilization process. we recently reported that increasing relative oocyte immaturity is associated with worsening outcome, and that cycles with many immature oocytes are more common in younger women. (moore et al, asrm annual meeting, 2007) to further investigate this trend, we conducted a case/control analysis of patients with repeated cycles of high-level oocyte immaturity (hloi). methods: oocyte maturity data was collected on all icsi cycles starting in 2002. we defined a cycle with hloi as having >50% immature oocytes (>2 sd's above the median). a case was defined as a patient with hloi on more than one cycle. control subjects were age-matched and defined as having </= 35% (1sd above median) immature oocytes on all cycles. results: from 450 subjects, we identified 8 cases of recurrent hloi (comprising 19 icsi cycles) and 240 control subjects. at baseline, cases had more oocytes retrieved per cycle than controls (22.5 vs. 16.3, p<.0001). all other baseline characteristics were similar. all case subjects were younger than 37. outcomes are shown in table 1 . discussion: clinical pregnancy and live births were reduced in the case group, but more than expected based on % immaturity. during 19 cycles, there were no live births among the case group. patients with recurrent hloi represent a previously undescribed group of young patients with a very poor prognosis during icsi treatment. this study may suggest that the problem underlying the immaturity may relate to overproduction of oocytes during stimulation, a factor which may be modifiable. however, the additional trend (though not significant) that even the mature oocytes from the case patients tend to have poor outcomes suggests the possibility of a maturation defect and warrants further consideration. data on response to gonadotropins, fert rates, and implantation rates will be presented. amongst those, 23 were performed for an initial diagnosis of premature ovarian aging (poa), 6 for the diagnosis of premature ovarian failure, and 11 for repeated pregnancy loss. in women under age 42, b-fsh levels over 12 miu/ml and up to < 50 miu/ml denoted a diagnosis of poa, while levels of 50miu/ml were considered diagnostic for pof. all patients signed consent permitting review of medical records for clinical research purposes. results: day 3 fsh levels ranged from 5.6 to 30.0 miu/ml (mean 13.6 ± 7.2 miu/ml). amh levels ranged from < 0.1 to 2.7 ng/ml (mean 0.8 ± 0.8) and allele repeats ranged from 17 to 49, (mean for allele-1, 27.2 ± 4.3; allele-2, 32.0 ± 6.4). mixed model analysis of variance for all patients, adjusted for age, race, and allele-1 revealed a statistically significant correlation between b-fsh levels and number of repeats in allele-2 ( figure 1 ; p < 0.01). we did not observe a significant linear relationship between serum amh levels and cgg repeat counts, however cgg repeat counts above 32 were significantly associated with serum amh levels less than 1 ng/ml (chi square = 0.04). conclusions: triple repeats above 30, a level currently considered well within normal, are associated with evidence of premature decline in ovarian function. the evaluation of triple repeats in frm1 gene offers a first objective assessment of ovarian function and should be predictive of autologous reproductive life span in most women. objective: the levels of mis in the serum and in the ovary have been demonstrated to decrease in response to increasing doses of cisplatin. we hypothesize that ovarian stimulation also will reveal dose related decreases in ovarian and serum mis levels after cisplatin exposure, further demonstrating the follicular damage from chemotherapy. methods: adult female sprague-dawley rats were treated with saline, 4.5 mg/kg cisplatin or 6.0 mg/kg cisplatin as two weekly injections. five days following the last cisplatin injection, the rats were injected with pregnant mare serum gonadotropin (pmsg) and euthanized 54 hours following the pmsg injection. serum was collected and both ovaries were obtained for protein analysis. serum and ovarian levels of mis were determined using an elisa kit. western blot analysis, immunohistochemical analysis, and tunel analysis were also performed on the ovarian proteins. results: stimulated mis levels declined in a linear fashion in response to increasing cisplatin doses (p<0.001). ovarian protein levels measured by elisa and western blot both indicated that stimulated ovarian mis levels also decrease in a linear fashion (p=0.001 and p<0.001 respectively.) immunohistochemical analysis revealed that while the total number of follicles remains the same, both the total number of mis positive follicles, and the percentage of mis positive follicles declines in a linear fashion (p=0.001 for both). tunel analysis reveals that there is no change in the incidence of apoptosis in the stimulated ovaries from any treatment group. conclusions: the administration of pmsg after treatment with cisplatin causes a dose dependent decrease in the levels of mis in both the serum and in the ovary. serum mis levels following ovarian stimulation may be useful as a serum biomarker in this animal model to assess ovarian damage following the administration of cisplatin. correlations of micro-rnas 23a, 23b, 17-5pp, 211 and 542-3p with inflammatory and steroidogenic factors in human granulosa/cumulus cells. tannaz toloubeydokhti, orhan bukulmez, kenneth c drury, r stan williams, nasser chegini. obstetrics and gynecology, university of florida college of medicine, gaineville, fl, usa. as part of the novel endogenous rna silencing machinery, noncoding short rna strands, referred to as micrornas (mirnas), have been identified to regulate the stability of the target gene expression through degradation and repression. the expression of many of these mirnas has been identified in human tissues however, their biological significance in various cellular processes remains to be elucidated. in this cross-sectional study of human granulosa cells, we aimed to study the potential correlations between five selected mirnas, mir-23a, mir-23b, mir-542-3p, mir-211 and mir-17-5p, and mrna expressions of some of their predicted target genes namely, cyclooxygenase (cox)-2, il-1 , steroidogenic acute regulatory protein (star), aromatase and estrogen receptor (er) . granulosa/cumulus cells were obtained from 26 women (age range 23-42; mean age 34.8 ± 0.5) undergoing oocyte retrieval for assisted reproduction. total rna was extracted from these cells and reversed-transcribed and real-time pcr was performed to measure steady-state levels of mirnas and mrna transcripts. we observed that the expression of mir-23a, mir-542-3p, mir-211 and mir-17-5p displayed a significant and positive correlation with the expression of cox-2. moreover, mir-23b significantly correlated with star mrna expression, but not with other genes tested (p<0.05). with advancing age, the expression of mir-17-5p and cox-2 was significantly decreased, with cox-2 expression displaying a positive correlation with aromatase, star and er mrna expressions (p<0.05). none of the factors tested showed any significant correlations with peak estradiol levels and number of oocytes retrieved. receiver operating characteristic curve analysis revealed that female age cut-off value of 31.5 y best predicted whether mir-17-5p was below its mean arbitrary value. in conclusion, given the importance of mirnas' regulatory function in gene expression, our results suggest that mir-23b may play an important role in gene expression related to granulosa cell luteinization, while variation in mir-17-5p expression with female age may suggest its potential role as a predictor of oocyte quality and granulosa cell function. given the importance of mirnas in gene regulation, the role of mirnas in ovarian physiology and aging requires further investigation. support: nih grant 37432. introduction: a symbiotic relationship between ovarian granulosa cells (gc) and the enclosed developing oocyte is critical to reproductive efficiency. genetic modulations in gc's can lead to reproductive insufficiency, highlighting the critical role of gc's in reproductive competence. defects in the oocyte-gc repertoire in women with dor include lower e2 levels, luteal deficiency, poor fertilization rates, higher incidence of aneuploidy, lower pregnancy and higher miscarriage rates. utilizing global gene expression analyses in cumulus gc's, we attempt to enhance our understanding of biological mechanisms that may contribute to poor reproductive capacity in young women with dor. methods: 8 infertile women (<38 years old) undergoing ivf were prospectively enrolled into two groups based on ovarian reserve (normal reserve, fsh<10 dor, fsh >10miu/ml). at egg retrieval, cumulus gc's were isolated, rna extracted transcribed into cdna. microarray targets were generated cdna hybridized to affymetrix human genome u133 plus 2.0 genechips. microarray data were analyzed (array assist) and normalized (robust multichip analysis). a difference in gene expression of > 2 fold was considered biologically relevant. results: of the 8,846 genes identified to be differentially expressed in young women with dor compared to normal reserve, 215 genes demonstrated consistency of expression across five different normalization schema; 94 were down regulated and 121 up-regulated. expression of gremlin, a member of the dan family of genes known for its highly regulated expression pattern during folliculogenesis, was noted to be down-regulated 3-fold over two probe sets (-3.08) in women with dor versus normal reserve; this down-regulation was confirmed by real-time pcr (-5.33) . conclusions: this is the first demonstration linking differential expression of gremlin with etiology of infertility in women. gremlin is a downstream effector of oocyte-derived gdf-9 which facilitates cumulus cell expansion, a critical event in reproductive physiology. our finding of a significant downregulation of gremlin expression in cumulus gc's associated with dor may partly explain the physiology of poor reproductive performance. nih k24cd41978. nih 5k12rr17672. ferring pharmaceuticals. the objective of the present study was to investigate the effects of the gnrh antagonist ca on expression of mis and aromatase (cyp19) using luteinized human granulosa cells obtained during in-vitro fertilization cycles and an immortalized human granulosa cell line (hgl5). the granulosa cells were cultured +/-dibutyryl camp (1 mm) and incubated +/-the gnrh antagonist, ca, for 24-48 h. rna was isolated, reversed-transcribed and real-time pcr was performed to measure mrna transcripts for ovary-specific cypiia and mis. we observed that camp markedly induced aromatase mrna in both the primary and immortalized human granulosa cells. interestingly, camp treatment of these cells also caused an upregulation of mis mrna. objectives: bisphenol a (bpa), a known endocrine disruptor, is a chemical used as a plasticizer in the manufacture of polycarbonate plastics and epoxy resins and is present in multitude products, including the interior coating of food cans, milk containers, and baby formula bottles. bpa can leach into foods during heat processing and is known to exert a variety of endocrine-like effects on different cell types acting as an estrogen because it contains two hydroxyl groups in its diphenyl structure. in this study we focused on the effects of bpa on aromatase expression and estradiol production in the human granulosa kgn cell line. we also evaluated its effects on several transcription factors crucial in cyp19 expression. materials and methods: kgn cells were cultured in f-12dmem and were starved for 48 h before experiments. subsequently they were treated for 48 h with vehicle (control), fsh (100ng/ml), and/or bpa (20, 40, 60, 80, 100um) . messenger rna expression was quantified by real time pcr and estradiol secrection was measured in supernatants by elisa. results: fsh induced a 10-fold increase in aromatase expression. bpa induced a dose-dependent decrease in cyp19 production, with the greatest effect at 100um (p<0.001), resulting in 91+/-3 % (mean+/-sem) inhibition, compared to aromatase expression induced by fsh alone. bpa also reduced levels of estradiol secretion in a dose-dependent manner, with the greatest inhibition at 100um (p<0.01) resulting in 90+/-5% decrease. we also evaluated expression of transcription factors known to be involved in regulating the activity of the ovary-specific aromatase proximal pii promoter. interestingly factors known for induction of aromatase such us steroidogenic factor-1 and gata-4, mimic the pattern of cyp19 expression after bpa treatment, whereas, other receptors previously reported to act as aromatase inhibitor, such as ppar gamma were up-regulated by the addition of bpa. moreover, expression of creb remained virtually intact, suggesting that most likely mechanisms governing endocrine disruption by bpa are highly selective. conclusions: bpa inhibited fsh stimulated aromatase expression and downstream estradiol secretion. we propose that constant exposure to this chemical may result in endocrine disruption which may contribute to reduced fertility and early ovarian senescence. oocyte maturation occurs during folliculogenesis as a result of complex cell-tocell communications between the granulosa cells and the oocyte. maintaining the granulosa cells' spherical structure and network of gap junctions surrounding the oocyte is critical. this project tests the ability of a novel substrate-free threedimensional culture system to form viable granulosa cell spheroids. methods: after irb approval, freshly obtained follicular fluid from in vitro fertilization was obtained and granulosa cells were purified by percol gradient. nonadhesive agarose hydrogels, containing 822 cylindrical round bottom recesses 200 m in diameter, were cast from micro-molds designed using computer-assisted rapid prototyping. granulosa cells seeded at a density of 800,000 cells per gel were incubated for up to 10 days. cellular viability was assessed with live:dead assay. results: after three days in culture, granulosa cells formed spheroids of densely packed cells that were difficult to disperse with multiple pipettings. the cells remained viable for at least 10 days. conclusions: granulosa cells can be cultured in a novel substrate-free threedimensional culture system. the cells form tightly adherent spheroids that remain viable for extended culture. the cohesiveness of the cells suggests the formation of gap junctions. this is under investigation with immunohistochemistry and electron microscopy. these experiments suggest that a substrate-free threedimensional hydrogel culture system may be ideal to reassemble follicular structure important for future in vitro evidence testing and oocyte maturation. known to function as responders to pathogens, inflammation, and tissue injury. previous studies in our laboratory demonstrated that saa was produced in mouse granulosa and production was regulated by cytokines. ovulation has long been considered an inflammatory reaction and patients with chronic inflammatory conditions often experience infertility. the present study was undertaken to explore the role of saa in human ovarian function. methods: ovarian granulosa and luteal cells were obtained from surgically removed specimens and mural and cumulus granulosa-luteal cells were obtained from ivf aspirates. rna was extracted from fresh or cultured cells. some cells were treated in vitro with tnf or other cytokines for 24h. expression of saa was assessed by quantitative rt-pcr. in addition, serum levels of saa were determined using a commercial elisa in women undergoing ovulation induction (oi) and art. saa levels were measured at baseline, during oi, on the day of hcg administration and at the time of the pregnancy test. results: saa mrna was expressed in theca, granulosa, and granulosa-luteal cells. in granulosa-luteal cells both saa1 and saa2 mrnas were expressed at higher levels in cumulus compared to mural granulosa. expression of saa1 and saa2 in theca was increased following treatment with tnf in vitro. serum levels indicated that patients with ovulatory dysfunction had increased levels of saa at the time of hcg injection while patients without ovulatory dysfunction had lower saa levels as compared to the baseline level. in addition, patients undergoing oi who achieved pregnancy exhibited increased levels of saa at the time of the pregnancy test compared to baseline levels, whereas patients who did not become pregnant had lower post-cycle levels of saa. interestingly, saa levels did not change in art patients that became pregnant without undergoing oi (donor egg or frozen embryo transfer) conclusions: human ovarian cells express saa mrnas which can be altered in vitro. serum levels of saa may correlate with the responsiveness of the ovary to gonadotropins as evidenced by altered levels in women with ovulatory dysfunction, and by an increase in pregnant patients following ovarian stimulation. yeh, beom su kim, felicia hercules, jennifer peresie, armando arroyo. gynecology-obstetrics, university at buffalo, buffalo, ny, usa. objective: cisplatin is a common chemotherapeutic agent given to women for treatment for a wide variety of cancers. we hypothesize that one mechanism by which cisplatin may cause damage to ovarian structures is by decreasing the amount of anti-oxidant activity in the ovary. we examined super-oxide dismutase 2 (sod2), a critical anti-oxidant enzyme that has been shown to be affected by cisplatin in other tissues, in the ovaries of cisplatin treated animals. methods: adult female sprague dawley rats were injected with saline, cisplatin 4.5 mg/kg or cisplatin 6.0 mg/kg as 2 weekly doses. five days following the last injection, the rats were euthanized and both ovaries were excised. one ovary was processed for immunohistochemistry and the other was processed for protein analysis using western blot techniques for sod2. the anti-sod2 antibody was purchased from santa cruz. the immunohistochemical sections were scored using a semiquantitative h scoring method. results: immunohistochemistry analysis of the expression pattern of sod2 following cisplatin administration revealed that there was a significant linear decrease in a dose response pattern in the expression of sod2 in antral follicles and in corpora lutea (p<0.01 for both). no change was found in the h score of sod2 in other ovarian structures. western blot analysis of sod2 in the ovaries following increasing doses of cisplatin revealed no changes in the overall protein levels of sod2 in the ovary. conclusions: this is the first report that administration of cisplatin causes changes in the expression pattern of sod2 in antral follicles and in copora lutea. cisplatin decreases the amount of sod2 available in these structures, possibly leading to increased oxidative stress and free radical damage, thereby leading to ovarian damage found after cisplatin administration. is there evidence for aromatase activity in the stroma of postmenopausal ovaries? mf landay, rh fogle, rb allen, s patel, fz stanczyk, rj paulson. ob/gyn, usc, keck school of medicine, los angeles, ca, usa. background: following menopause, the ovaries continue to secrete androgens and estrogens. we have recently confirmed the production of androstenedione, testosterone and estradiol (e 2 ) up to ten years after menopause by measuring gradients from ovarian venous effluent and peripheral blood. anti-müllerian hormone (amh) and inhibin b have been shown to be markers of follicular activity. peripheral levels of these hormones have previously been found to be undetectable in menopause, suggesting the absence of follicular activity in the postmenopausal ovary. objective: to investigate if the postmenopausal ovary continues to demonstrate evidence of follicular activity as the source of steroid production. design: observational study materials and methods: eight subjects aged 53 ± 2.7 yr (range 48-56) were enrolled. postmenopausal status was confirmed by preoperative fsh levels of more than 40 u/l and/or amenorrhea greater than 12 months. serum was collected from the ovarian veins during total abdominal hysterectomy and bilateral oophorectomy. peripheral blood was also collected pre-operatively, intraoperatively and postoperatively. all samples were analyzed for amh and inhibin b using elisas with sensitivities of 0.05 ng/ml and 7 pg/ml, respectively. androgen and estrogen levels in these samples have previously been reported, and documented a gradient between ovarian venous effluent and peripheral serum in all cases. results: 1) six patients had no detectable follicular activity by amh and inhibin b levels. 2) one patient demonstrated detectable inhibin b levels with an 7-fold gradient between ovarian venous effluent (226 pg/ml) and peripheral blood (31 pg/ml), however no amh was detected. 3) in one patient, aged 52 and 120 months postmenopause, both amh and inhibin b were detected. peripheral inhibin b levels were high at 912 pg/ml. amh was detectable at levels of 0.95 ng/ml. conclusions: 1) in the majority of patients, continued e 2 and androgen production in the ovary occurs in the absence of follicular activity as detected by amh and inhibin b production. 2) some patients have evidence of follicular function up to ten years after menopause. 3) since e 2 is produced in post-menopausal ovaries in the absence of follicular activity, these data provide evidence for aromatase activity in the stroma of post-menopausal ovaries. to elucidate the process by which prostaglandin f2 (pgf2 ) mediates luteal regression, this study examined the role that the transcription factor yin yang 1 (yy1) and histone deacetylase 1 (hdac1) play in altering luteal cholesterol uptake by the scavenger receptor class b type i (sr-bi), intracellular cholesterol transport by steroidogenic acute regulatory protein (star), and cholesterol processing by p450 side chain cleavage enzyme (p450scc) expression. rat luteal cells (10 days post-ovulation) were treated with pgf2 (24 hr) and trichostatin a (tsa; 24 hr), a potent hdac inhibitor. protein expression was measured post-treatment by western blot and cholesterol was localized via filipin staining. star and sr-bi promoter activation was also assessed in hek 293t cells treated with yy1, myy1, a deletion mutant lacking an essential region required for transcriptional repression, and tsa. pgf2 caused a significant 2-fold decline in star (p<0.005), and smaller declines in sr-bi and p450scc which occurred concomitantly with an increase in yy1 (4-fold, p<0.001) and intracellular lipid staining (20% increase). in contrast, 100 nm tsa treatment caused a dose dependent increase in sr-bi, star, and p450scc protein levels, 3.5-fold (p<0.005), 1.4-fold (p<0.05), 4.1-fold (p<0.005), respectively, and a 2.5-fold decline (p<0.05) in intracellular lipid levels. tsa prevented the pgf2 -induced decline in sr-bi, star, and p450scc expression. promoter analysis demonstrated that wildtype yy1, but not myy1, repressed sr-bi and star activation while the addition of tsa overcame the repressive effects. this study shows the critical role that yy1 plays in pgf2 induced luteal regression by recruiting a histone deacetylase to block three essential processes in steroid production. in luteal cells yy1-mediated global repressive action prevents cholesterol metabolism by inhibiting cholesterol uptake, intracellular transport, and processing thus leading to functional and structural luteal demise. supported by nih hd35163 and nih hl78817. regulation of intermedin expression in cycling rat ovary. madhu chauhan, rebekah elkins, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: intermedin (imd) is a ct/cgrp family peptide involved in a variety of physiological functions, including vasodilatation and fetoplacental growth. this peptide is expressed in a variety of tissues such as stomach, placenta, uterus, pituitary and ovary suggesting its different functions including in reproduction. imd gene has an estrogen response element and we have shown that the plasma concentration of immuno-reactive imd is elevated in rats with pregnancy. however, expression of imd in the ovary and its regulation during estrous cycle is not known. we hypothesize that imd is expressed in the ovary and its expression is hormonally regulated throughout the estrous cycle in rat. objective: 1) to assess expression of imd mrna and its receptors components calcitonin receptor like receptor (crlr), and receptor activity modifying proteins, ramp1 and ramp3 mrna in rat ovary; on diestrus, proestrus and estrus stages of rat estrus cycle and ; 2) to demonstrate immunohistolocalization of imd, crlr, ramp1, ramp2 and ramp3 in rat ovary. methods: groups of 4 sprague-dawley non-pregnant and pregnant rats on day 18 of gestation were used in this study. non-pregnant rats were sacrificed on diestrus, proestrus and estrus stage. ovaries were collected and total rna was extracted using trizol method. rna was treated with dnase1 before performing the reverse transcriptase polymerase chain reaction (rt-pcr). immunohistolocalization of imd, ramp1, ramp2 and ramp3 proteins were assessed in tissue sections of ovaries from pregnant rats sacrificed on day 18. results: 1) imd mrna is regulated in rat estrus cycle and its expression is significantly downregulated in estrus stage compared to the diestrus and proestrus; 2)expression of imd receptor crlr is highest in the diestrus stage, followed by a decline in proestrus which further declined during estrus; 3) expression ramp1 mrna is higher in proestrus compared to diestrus and estrus (p<0.05) but there is no significant change in the ramp 3 expression during the estrus cycle and ; 4) imd, ramp1, ramp2 and ramp3 are immunolocalized in rat ovary in granulose cells of all follicles and granulosa cells of the corpus luteum. conclusion: imd mrna and protein are expressed in rat ovary suggesting a possible role for imd in ovarian function. defining and defying deterioration in oocyte quality with advancing chronological age: role of nitric oxide. pt goud, 1 ap goud, 1 mp diamond, 1 b gonik, 2 hm abu-soud. 1 1 div reprod endocrinology, dept ob/gyn, wayne state university, detroit, mi, usa; 2 div maternal and fetal medicine, sinai grace hospital, wayne state university, detroit, mi, usa. nitric oxide (no) is a ubiquitous free radical essential for oocyte maturation, function and sustenance of oocyte quality post-ovulation. the current study investigates the role of no insufficiency in the causation of oocyte quality deterioration with advancing chronological age. methods: in set 1, oocytes were retrieved from the following superovulated b6d2f1 mice: (a) young breeders (yb, n=109); (b) retired breeders (rb, n=40), and © old animals (oa, n=16), aged 6-12, 45-52, and 70-75 weeks respectively; and studied for zona pellucida dissolution time (zpdt), spindle ( -tubulin fluorescence immunocytochemistry, sigma) and chromosome morphology (dapi, vector), ooplasmic microtubule (mt) dynamics (omd) in response to taxol [1], and cortical granule (cg) status (rhodamine conjugated lectin, vector). in set 2 (n=56), oocytes from retired breeders were studied after exposure in vitro to an no-donor, s-nitroso acetyl penicillamine (snap in m-16, 0.23 m no/min , n=23, 3 h, 37°c, 5% co 2 ), while their sibling control oocytes were cultured for corresponding period under identical conditions without snap. [1]. zpdt, spindle and chromosome morphology, omd and cg status were assessed with a confocal microscope and compared between the subgroups using anova, chi square and/or fisher's exacts test and appropriate post-hoc tests. results: in set 1, a significantly fewer oocytes were retrieved per animal (mean) in rb (10) and oa (4) compared to yb (27.3, p<0.05). advancing age was also associated with a significant increase in zpdt, omd and cg loss in rb compared to yb (p<0.01). furthermore, significantly fewer oocytes from rb than yb had normal spindle and chromosome morphology (p<0.05). oocytes from oa had significant spindle and chromosome disarray, a near total cg loss and significantly harder zp (p<0.01). these oocytes also exhibited diminished omd in response to taxol although, metaphases were exquisitely sensitive to disruption with taxol. in set 2, exposure to snap in oocytes from rb resulted in a significantly lowerzpdt, omd and cg loss, and significantly higher incidence of normal spindles ( the gamma-aminobutyric acid (gaba), its biosnthetic enzyme gad and gaba-a receptors have been found in the oviduct and ovary. objective: this study examined the expression of gaba-a receptor subunit genes and gad and whether the gaba-a receptor could alter cytosolic ca2+ in gc. methods: for qrt-pcr, both human cumulus and mural gc were obtained at the time of oocyte retrieval for ivf and cultured separately. total rna was isolated separately from each gc type and from human brain ( positive control). the total rna from 16 patients was pooled for each gc type and all rna was treated with dnase i. two-step qrt -pcr was performed using gene-specific lux™ primers for all 18 gaba-a receptor subunits, gad65 and gad67 plus gapdh. single and specific qrt-pcr products were verified by melting curve analysis, gel electrophoresis, and dna sequencing. for ca2+ study, gc were cultured on coverslips. gc were loaded with fura-2-am and changes in ca2+ concentration of gc were studied using a dynamic digital ca2+ imaging system. gaba-a agonist, muscimol, was used to study any dose-dependent effects on gc. gaba-a antagonist, 10 m bicuculline, were perfused 1 min. prior to and during application of muscimol. the responses were represented as changes in the 340/380 nm fluorescence ratio over time. 50 m atp was used as positive control. results: the qrt-pcr results indicated that all gaba-a receptor subunits were expressed to various degrees in both types of gc, with the 5 expressed highest in both cell types. gaba-a receptor subunits showing the next highest expression in both cell types were 3, 3 and 2. gad67 isoenzyme was more abundantly expressed in cumulus and mural gc than gad65. ca2+ imaging showed that muscimol, had the ability to increase ca2++ in gc, about 19% gc (n=6) cells responded to muscimol. muscimol increased intracellular ca2+ in a dose-dependent manner. the muscimol responsive cells was reduced by bicuculline, from 19% to 1 % (n=6, p< 0.05). conclusion: qrt-pcr indicates that gaba-a receptor subunit gene and gad67 expression occurs in both cumulus and mural gc. the ability of bicuculline to inhibit the ca2+ response to muscimol suggests the activation of gaba-a receptor. the current study confirms the presence of functional gaba-a in gc for the first time, and suggests that gaba may exert trophic effects in the ovary via gaba-a receptor. the present study test the hypothesis that administration of l-nitro-l-arginine methyl ester (l-name), a nos inhibitor, prior to hypoxia prevents the hypoxiainduced activation of p38 mapk, erk and jnk in the cerebral cortex of the guinea pig fetus during gestation. to test this hypothesis 34 guinea pig fetuses at 35 and 60 days gestation were assigned to normoxic (nx, n=6), hypoxic (hx, n=6) and hypoxic pretreated with nos inhibitor (hx+l-name, 30mg/kg i.p., n=5) groups. hypoxia in the fetuses was induced by exposing the pregnant guinea pigs at both gestational ages to an fio 2 of 0.07 for 60 min. cerebral tissue hypoxia was documented biochemically by determining the tissue levels of atp and phosphocreatine (pcr). neuronal nuclei were isolated, purified and proteins separated by sds-page, and then probed with specific phosporylated erk, jnk and p38 antibodies. in the 35days gestation group: expression of p-p38 was 39.5±3.4 (nx), 79.1±2.8 (hx) 47.1±4.1 (hx+l-name). p-erk expression was 59.7±3.2 (nx), 109.6±6.3 (hx), 72.4±2.7 (hx+l-name). p-jnk expression was 68.3±7.2 (nx), 94.6±3.8 (hx), 55.8±9.0 (hx+l-name). in the 60 days gestation group: expression of p-p38 was 108.7±3.6 (nx), 231.1±8.3 (hx), 116.5±9.8 (hx+l-name). p-erk expression was 94.1±12.9 (nx), 226.5±9.1 (hx), 139.1±12.6 (hx+l-name). p-jnk expression was 83.2±9.5(nx), 218.2±6.9 (hx) 105.3±10.8 (hx+l-name). the data show that administration of l-name prior to hypoxia decreased the expression of phosporylated p38, erk and jnk at both gestation ages however expression of phosporylation was higher at term as compared to preterm. since a nos inhibitor prevented the hypoxia-induced phosphorylation of p38, erk and jnk in both gestational ages, we conclude that the hypoxia-induced activation of p38, erk and jnk in the cerebral cortical nuclei of preterm and term guinea pig fetus is no-mediated. we speculate that no-mediated modification of cysteine residue leading to inhibition of map kinase phosphatases results in increased activation of p38, erk and jnk in the guinea pig fetus. (nih-hd 20337, nih-hd 38079 and st. christophers foundation for children). the the endocannabinoid, anandamide (aea), is involved in the hormone-cytokine network that regulates implantation and early pregnancy maintenance with levels at the endometrial level considered a major checkpoint 1 . high levels (28nm) in culture are associated with embryo death 2 while plasma levels (>4nm) at 6 weeks in women undergoing ivf-et are associated with failed pregnancy 1 . what is uncertain is whether systemic aea levels after spontaneous conception in women presenting with threatened miscarriage are predictive of pregnancy outcome. our aim was therefore to measure plasma aea levels in women presenting with threatened miscarriage and to relate these to outcomes. methods: plasma aea levels were measured using a sensitive and previously validated hplc-ms method 3 at 6-12 weeks gestation in 45 women (nonsmokers, bmi <30kg/m 2 ) presenting with threatened miscarriage and in whom a viable pregnancy was confirmed by ultrasound scan. results: nine of the 45 women subsequently had a miscarriage. the plasma aea levels in those women who had live births was 2.85-fold lower than that in those who subsequently miscarried (1.21 ± 0.12nm versus 3.45 ± 0.31nm, mean ± sem; respectively; p<0.0001 unpaired student's t-test). the roc analysis revealed an area under the curve of 0.972 ± 0.025 with a sensitivity of 100% (95%ci of 66.3% to 100.0%) and a specificity of 94.4% (95% ci of 81.34% to 99.3%). using an aea level of 2.0nm as the optimal cut-off point, a single plasma aea measurement provided a sensitivity of 100% and a specificity of 94% with a negative predictive value of 100% and a positive predictive value of 78% for subsequent miscarriage. conclusion: these findings suggest a possible predictive role for plasma aea in women presenting with threatened miscarriage. the data also indicate that systemic aea levels may reflect local endometrial levels and therefore the local hormonal milieu in the early stages of normal pregnancies and in those complicated by threatened miscarriage. introduction: follicle stimulating hormone (fsh) mediates cyclic follicle growth and development and is widely used for controlled ovarian stimulation. the ovarian response of different women to fsh is variable, ranging from poor response to ovarian hyperstimulation and has been partly attributed to two common variants of the fsh receptor (fshr). we have previously identified four abnormal fshr splicing products (3 exon deletions and 1 intron insertion) in women with low and high response to fsh. two of the splice variants, deletion of exon 2 and deletion of exon 6, showed a correlation with low and high response to fsh, respectively. in the current study, we evaluated the functional competence of the mutant fshrs in vitro. methods: we established stable hek293 cell lines expressing wild-type (wt) and splice-variant (del) fshr under the control of the inducible tet on/off system. the cells were transfected with a camp-responsive luciferase reporter plasmid and stimulated with fsh. results: the subcellular distribution of all splicing variants was the same as the controls and the protein localized mainly on the cell surface. all four splicing variants showed markedly decreased camp activation compared to controls when stimulated with fsh. however, all variants were able to form functional heterodimers with the wt receptor when co-expressed. interestingly, the heterodimer containing the form of fshr lacking exon 2, found in patients with decreased response to fsh, resulted in lower intracellular camp compared with the wild-type homodimer. conclusion: our findings suggest that fshr variants can contribute to abnormal response to stimulation in certain women undergoing ivf treatment. the variants require the presence of wild-type receptor in order to initiate signaling in response to fsh. further analysis of this signaling cascade in granulosa cells is underway to estimate the final production of estrogen from these heterodimeric receptors. objective: to compare the proteome of human endometrium in the prereceptive versus the receptive phases of the menstrual cycle. m m: endometrial biopsies were collected 2 and 7 days after urinary lh surge in the same menstrual cycle from three fertile women (n=3). proteins were extracted using sample grinding kit (ge healthcare) and interfering substances removed using 2-d clean-up kit (ge healthcare). labelling was performed with cydye dige fluors (ge healthcare) and proteins were separated using difference gel electrophoresis (dige). for the isoelectric focusing, 24 cm ipg-strips in the nonlinear range of ph 3-11 were used. the second dimension was carried out using sds-page. differentially expressed proteins were detected by image analysis using decyder v6.5 and the statistical module eda (ge healthcare). the spots of interest were subject to protein identification based on in-gel digestion, maldi-tof/tof mass spectrometry and database searching. western blot analysis were performed in the same biopsies in order to validate some candidate proteins. results: table 1 displays the differentially protein abundance between days lh+2 and lh+7 (> 2-fold change). of these proteins, 10 were increased at lh+7 in comparison with lh+2, whereas only 2 proteins were decreased. stathmin was found more than 2-fold decrease in lh+7 compared to lh+2 in the three patients studied in the validation studies performed by western blot. conclusions: this study shows that the human endometrium has a differential protein repertoire during the window of implantation compared to the prereceptive phase. the role of these proteins in the molecular events directed to embryo implantation is under research. differential protein abundance in human endometrium ( the extracellular signal-regulated kinases 1 and 2 (erk1/2) are a mitogen-activated protein kinase (mapk) subfamily that act as key links in eukaryotic intracellular signaling transduction. activated by phosphorylation in response to specific stimuli, erk1/2 is known to play a role in the regulation of cellular proliferation and survival. the human myometrium is a tissue known to undergo cycle-dependent proliferative and apoptotic changes in response to sex steroids. we hypothesized that erk1/2 activity is involved in mediating menstrual cycle-dependent changes in the myometrium. materials and methods: immunostaining for phospho-erk and total-erk was performed on myometrial tissues obtained from normal women (n=20) at different phases of the menstrual cycle. staining intensities were evaluated by hscore. myometrial smooth muscle cells were isolated and cultured from normal women and treated with vehicle, estrogen (10-8m), and progesterone (10-8m) for 5 and 15 minutes, and then subjected to western blot analysis for p-and t-erk. statistical analysis was performed using one-way anova. results: tissue staining revealed that p-erk was mostly nuclear in all tissues, while t-erk was cytoplasmic and nuclear. p-erk staining was significantly stronger in the secretory phase and strongest at the early secretory phase, compared to other phases (p<0.05). t-erk staining intensity was moderatehigh without variation across the menstrual cycle. in cultured myometrial cells, progesterone significantly increased p-erk levels within 5 and 15 minutes (p<0.05) when compared to control. our results suggest that erk activity in the human myometrium is regulated in a menstrual cycle-dependent manner. the increased phosphorylation in the secretory phase suggests the involvement of progesterone in erk activation, as supported by our in vitro results. this increased erk activity may play a role in regulating myometrial proliferation. measures of insulin resistance (ir) including: fasting serum insulin, gir and homa. measures of plasma levels of endothelin-1 (et-1), soluble intercellular adhesion molecule-1 (sicam-1), soluble vascular cell adhesion molecule-1 (svcam-1) and high sensitivity c-reactive proteins (hscrp). results: women with pcos exhibited significantly higher levels of et-1 (p < 0.05), sicamp-1 (p < 0.05), svcam-1 (p < 0.001) and hscrp (p < 0.001) as compared with age-matched controls, respectively. positive correlations were evident between et-1 and fai (r = 0.41; p < 0.01) but et-1 negatively correlated with shbg (r = -0.36; p < 0.05). svcam-1 positively correlated with total t (r = 0.62; 0.001), hscrp correlated with: bmi (r = 0.73; p < 0.001), and homa (r = -0.39; p < 0.05), respectively. conclusions: women with pcos exhibited abnormal levels of endothelial and inflammatory markers, which appear to be inter-related to hyperandrogenaemia. (1), and a higher expression of fatty acid amide hydrolase (faah) in peripheral lymphocytes post-ovulation (2), suggest an involvement of the endocannabinoid system in menstrual cycle control. our aims were to investigate changes in plasma aea levels and in endometrial faah expression throughout the menstrual cycle. methods: plasma aea levels were measured using a hplc ms/ms method from 47 women, median age 34yrs (range 20-45) and bmi 22kg/m 2 (range 19-27), with regular menstrual cycles, with no medical problem and not on any medication for the preceding 6 months. the menstrual cycle was divided to early follicular d2-7(n=11), late follicular d8-11(n=7), ovulatory d12-15(n=10), early luteal d16-22(n-10) and late luteal phases d23-28(n=9). uterine biopsies were taken from hysterectomy specimens taken for benign conditions and subjected to immunohistochemistry for faah with polyclonal antibodies. results: aea levels were significantly higher around ovulation in comparison to the pre-ovulatory or post-ovulatory phases as shown in the figure. faah expression in the endometrial stroma was unchanged throughout the follicular phase but increased during the mid to late luteal phase reaching a maximum in the late luteal phase. a high aea level in the early follicular phase and during ovulation suggests a role for aea in ovulation. the lower levels of aea in the luteal phase, essential for successful implantation, may be regulated by increased faah expression at the uterine level. the modulation of plasma aea levels during the menstrual cycle strongly suggests that it is regulated by gonadal steroid hormones. (1 , 5, 10, 15, 20, 25, 50 and 120, as well as at pregnancy. binding activities of igfbp and their protein levels (igfbp1,2,3,4,5,6) were assessed by western ligand blot (wlb) and western blot (wb), respectively. the glyscosylation and phosphorylation status of igfbps were examined by deglycosylation treatment. our results showed that both glycosylated and unglycosylated igfbp2 elevate in fetal and newborn rat and graduately decline to a lower level at day 15 and kept lower constant level since then. interestingly, biding activity of glycosylated igfbp2 was not detected by wlb assay. the igfbp-2 cleaved products were observed after rat day age day 15, which associated with a decrease in full length of igfbp-2, suggesting endoproteolytic processing may involved in decreasing igfbp content. igfbp-3, with heterogeneous glycosylation, were appeared after age day 25 and disappeared during pregnancy, recurrence again postpartum. glycosylation of igfbp3 has no effect on its binding activity. unglycosylated and glycosylated igfbp were constant in life time. physiologically constant igfbp5 were detected by wb in protein, but not by wlb in binging activity. conclusion: highly elevated circulation igfbp2 suggest physiological role in new born and early postnatal development. its binding activity are well regulated by its posttranslation modification, such as glycosylation of igfbp2 in inactivating binding activity and possible involvement of active endoproteolytic processing in maintaining binding active igfbp-2 at low background: cigarette smoking affects hormone biosynthesis, storage, release, binding, transport and clearance, resulting in changes in circulating hormone levels. we used metabolomics to analyze the effects of cigarette smoking and second hand smoke (shs) on changes in hormone levels in women of childbearing age. methods: this is a three arm study; women aged 18-44years who are active smokers, exposed to shs (passive smokers), or non-exposed were recruited from the washington d.c. area. all women completed a detailed staffadministered questionnaire probing their medical history, occupational, lifestyle factors and diet. blood and urine samples were collected at the follicular phase. hormone profiles were determined using metabolomics for serum thyroxine and triiodothyronine and 17 steroid hormones, as well as for cotinine using isotopedilution tandem mass spectrometry (lc/ms/ms-api 5000). in addition, all samples were analyzed for serum lh, fsh, tsh and creatinine. results: the relationships of cigarette smoking, age, relative weight, and dietary intake to serum thyroxine, triiodothyronine, estradiol (e2), estrone (e1), progesterone (p), 17-hydroxyprogesterone (17ohp),dehydroepiandrosterone (dhea), dehydroepiandrosterone sulfate (dheas), androstenedione, cortisol, 11-deoxycortisol, corticosterone, testosterone, aldosterone and vitamin d3 were analyzed using lc/ms/ms. mean tsh in non-exposed, shs-exposed and smokers: 1.32, 0.86, and 1.02miu/ml respectively. similarly, mean t4 9.8, 12.23 (23% increase) (p=0.05), 9.41μg/dl (-7%) in active smokers; (active vs. exposed p=0.03). shs increased dhea levels (33% higher, p = 0.02), dheas (23% higher, p = 0.07), cortisol (21% lower, p = 0.01), aldosterone (63% lower, p = 0.02) and androstenedione (20% higher, p = 0.01). these data suggest that active smoking and shs can have a profound effect on serum t4, adrenal steroid and sex hormone concentrations in women of childbearing age. objective: to determine the prevalence and predictors of the metabolic syndrome (mbs) among in saudi women with polycystic ovary syndrome (pcos) in comparison to women without pcos and to assess the role of androgens and insulin resistance (ir) in mbs development. design: a prospective case control study. setting: tertiary referral university hospital. subjects: six hundreds saudi women living in the jeddah area were classified as follows: 300 with pcos and 300 age-matched women without pcos. interventions: blood samples were collected from all women with or without pcos between 8:00-11:00, after an overnight fast. main outcome measures: measures of abdominal obesity, blood pressure and that of serum levels of lh, fsh, tsh, ft 4 , 17-ohp, 4-a, dheas, total t, free t, shbg, insulin, hdl-c, triglycerides and plasma levels of glucose. measures ir including: fasting serum insulin, gir and homa. results: age-adjusted prevalence of mbs was higher in women with pcos (53.5%, 95% ci: 37.6-61.2%) as compared with women without pcos (14.7%, 95% ci: 10.2-18.6%) (p<0.000). in the same age group, the risk of mbs in women with pcos was greater than that for women without pcos (p<0.001). markers of ir were significantly abnormal in women with both pcos and mbs in comparison to those without mbs (p<0.001). the most common abnormal components of mbs in women with both pcos and mbs (after adjustment for age) were: decreased hdl-c (83.1 ± 10.5%); increased triglycerides (53.4 ± 7.7%); and increased bmi (38.2 ± 4.6%), respectively. the prevalence of mbs from lowest to highest tertile of free t level was 20.1, 33.7 and 54.2%, respectively; in women with both pcos and mbs. in women with pcos, 9% exhibited all 5 components of mbs; 14.1% had 4 components, and 40.5% had 3 components. conclusions: women with pcos exhibited significantly higher prevalence of mbs (3.6-fold) as compared with age-matched control without pcos. ir is a possible common pathogenetic factor for both mbs and the pcos. it is suggested that more intensive screening and/or therapy monitoring of mbs among women with pcos should be part of the therapeutic modalities of the condition. case of sisters with complete androgen insensitivity syndrome and discordant mullerian remnants. jennifer l nichols, jennifer j gell, eric j bieber. reproductive endocrinology and infertility, geisinger medical center, danville, pa, usa. complete androgen insensitivity is an x-linked recessive disorder resulting in the abnormal expression of the androgen receptor. affected individuals are most commonly phenotypically female but genotypically male. the prevalence of this disorder is 1 in 20,000 live male births. we present a case of complete androgen insensitivity in 2 members of the same family with differing residual mullerian tissue. sister a presented at age 17 for evaluation of primary amenorrhea. a karyotype revealed 46,xy. an mri of the pelvis showed a hypoplastic uterus but no ovaries. this patient underwent laparoscopic bilateral gonadectomy and hemihysterectomy. on examination under anesthesia, she was noted to have a normal vagina with no cervix noted. at laparoscopic evaluation, she was noted to have bilateral elongated gonads and what appeared to be a remnant of uterine tissue. pathology revealed portions of immature testicles and fragments of smooth muscle in what grossly appeared to be the uterine remnant. the patient's total testosterone following surgery was noted to be elevated at 2.91 ng/ml. other laboratory evaluation showed fsh 4.5 miu/ml, lh 7.04 miu/ml, free testosterone 3.9 pg/ml, and estradiol 24.6 pg/ml. approximately two years later sister b presented at age 17 for evaluation of primary amenorrhea. no uterus or ovaries were visualized on pelvic ultrasound. again a karyotype revealed 46,xy. laboratory evaluation demonstrated fsh 1.0 miu/ml, lh 13.28 miu/ml, estradiol 37.3 pg/ml, total testosterone 9.25 ng/ml, and free testosterone 11.1 pg/ml. she underwent a laparoscopic bilateral gonadectomy. no uterus, cervix or pelvic masses were identified on exam under anesthesia. at laparoscopy, both gonads were visualized and removed without difficulty but no uterus was visualized. pathology reported two testicular and epididymal-like structures. this case demonstrates the presentation and laparoscopic results of complete androgen insensitivity syndrome discovered in two siblings. although both girls are genotypically male, they differ in the presence of vestigial mullerian tissue. the case shows with complete androgen insensitivity, as an x-linked defect, one should consider apparent sisters of affected individuals, as well as offspring of unaffected individuals with a family member diagnosed. background: anandamide (n-arachidonoylethanolamine, aea) is an endocannabinoid that binds to and activates the cannabinoid receptors, cb1 and cb2 and may have important roles in the regulation of human reproduction. the traditional lipid extraction methods used for aea 1 are cumbersome, slow and of low efficiency. the aim of this study was to determine whether the use of a solid phase (spe) method of aea extraction from human plasma would offer any advantages over the traditional liquid phase (lpe) method. methods: pooled human plasma was obtained from the local blood transfusion unit and aliquots stored at -20°c prior to extraction. an internal standard of 2.5pmol of deuterated aea (aea-d 8 ) was added to each plasma sample and aea extracted from 5 aliquots on each occasion over three days using the lpe 1 and spe methods. spe was performed with waters oasis hlb 1cc/30mg cartridges on a waters vacuum manifold. cartridges were activated with methanol and water, the samples applied and washed with 40% methanol at 1ml/min. aea was eluted with 1ml acetonitrile and the eluents dried under a stream of nitrogen before reconstitution in 80 ml acetonitrile. aea levels were measured using uplc-ms/ms against authentic standards. results: these are shown in the table. conclusion: the spe method was 3-fold more efficient at extracting aea compared to the traditional lpe method. the intra-day and inter-day assay variability were similar for both techniques, although the spe method was quicker, cheaper and required less plasma to generate data similar to that from the traditional lpe method, suggesting that the spe method may be more efficient than the lpe method, and thus making it more suitable for routine analysis of multiple plasma aea samples. background: the precise role of the endocannabinoid, anandamide (narachidonoylethanolamine, aea) in reproduction has been hampered by difficulties in its accurate measurement. aea levels have previously been measured by tlc, gc-ms and hplc-ms but these are laborious. our aim was to improve the analysis of aea using uplc and tandem ms/ms with a standard isotope-dilution method 1,2 . methods: authentic non-labelled and deuterium-labelled aea (aea-d8) diluted in acetonitrile were maintained at 4°c before analysis and separation by uplc on a c 18 (2.1 x 50mm) column maintained at 40°c using a gradient of 80% a, 0.5min: 80% a, 1.5min: 0% a, 2.5min: 80% a, 3.5min: 80% a with a flow rate of 0.7ml/min. the mobile phases were a (2mm ammonium acetate, 0.1% formic acid) and b (acetonitrile, 0.1% formic acid). the analytes were quantified using multiple-reaction monitoring in positive ion mode with a quattro premier mass spectrometer. entry, collision and exit energies were -2, 17 and -17ev, respectively. calibration curves were performed in triplicate with 2.5pmol aea-d8 as the internal standard. transitions employed were 348.3>62.3 and 356.3>63.3 for aea and aea-d8, respectively. results: calibration curves (1.66 to 133fmol aea on column; n=15) were linear, producing a mean (±sd) gradient of y = 2.48±0.14x, crossing the y-axis very close to the origin (0.004±0.04 units). variability was limited, with an r 2 =0.999. measurements were precise, 133fmol aea produced a cv of only 3.7%, and the retention time was consistent at 1.67±0.0009min (n=20). the limit of quantification (signal/noise>10) was 0.22fmol on column and the limit of detection (lod) was 0.055fmol on column (signal/noise=3). accuracy for 3.33fmol, 6.65fmol and 133fmol aea was 97.5±9.5%, 98.5±6.1% and 104.5±3.2%, respectively. conclusions: the method described is an improvement over other lc-ms/ms methods 1,2 with a lower lod [0.055fmol v. 25fmol 1 or 43fmol 2 ] and shorter run time [4min v. 15min 1 or 5.4min 2 ]. thus, an improvement in terms of speed, increased sensitivity and better reproducibility will allow for a more accurate assessment of aea concentrations in a number of biological samples. objectives: the gata family of transcription factors consists of six zinc-finger proteins with a critical role in tissue-specific and developmentally-regulated gene expression. gata factors exert their effects alone and through interactions with cofactors, such as friend of gata (fog), as well as with nuclear hormone receptors, including steroidogenic factor-1 (sf-1), a well-described activator of gonadotropin gene expression. the objective of these studies was to define the role of gata family members in the gonadotrope. methods: 1) total rna was extracted from the gonadotrope cell line, l t2, and analyzed by standard rt-pcr analysis. 2) the cv-1 fibroblast cell line was transiently transfected by the calcium phosphate precipitation method with a rat -207/+5 lh promoterreporter vector as well as cmv-driven expression vectors for gata-2, gata-3, dngata-3, fog-2 and/or sf-1. results: gonadotrope l t2 cells were found to express transcripts encoding gata-2, gata-3, and gata-4 as well as fog-1 and fog-2. gata-2 and gata-3 stimulated lh gene promoter activity by approximately 5-fold (p<0.05) and synergistically increased the sf-1 response (40-fold versus 10-fold for sf-1 alone; p<0.05). the gata-mediated increase in lh gene expression was nearly eliminated with co-transfection of fog-2. similarly, co-transfection with a gata-3 dominant negative construct blunted wild-type gata-3 effects in a dose-dependent fashion. conclusions: these data demonstrate expression of both gata and the functionally related fog proteins in a well-characterized gonadotrope cell line. furthermore, they demonstrate a functional role for these factors in regulation of gonadotrope function, specifically expression of the lh gene. low-dose dexamethasone (dex) therapy early in pregnancy is used in fetuses with suspected risk of congenital adrenal hyperplasia. several adverse neurological events in prenatally treated children have been reported and the fetal hypothalamic-pituitary-adrenal (hpa) axis may be involved. aim: to investigate the immediate and long-term effects of early maternal dex administration on fetal growth and pituitary-adrenal activity in sheep. method: pregnant ewes carrying singleton fetuses (total n=119) were randomized to control (2ml saline/ewe) or dex treatment (0.14mg/kg ewe weight) consisting of four intramuscular injections at 12-hourly intervals over 48 hours on 40-41 days of gestation (dg). at 50, 100, 125, and 140dg fetal weights were recorded. i 125 -ria, qrt-pcr and in-situ hybridisation were used to measure fetal plasma cortisol and acth levels and to analyse adrenal and pituitary mrna expression. results: dex-exposed fetuses were lighter than control animals at 100dg*, but not at other times; in general fetal organ weights were similar between treatment groups. fetal plasma acth was unaffected by dex and did not differ between genders. similarly, pomc and pc-1 mrna in pars distalis were unaltered after dex. however, fetal plasma cortisol was reduced after dex in both male and females at 50dg*, was similar at 100 and 125dg, then elevated at 140dg*. plasma cortisol in female fetuses in control and after dex was significantly higher than in males. the increases in cortisol after dex at 140dg* were associated with increased fetal adrenal expression of p450c17 and 3bhsd mrna in females, reduced expression of mc2r in males, but no difference in star mrna. conclusion: we conclude that in sheep, early dex programs the developmental trajectory of the fetal hpa with increased activation directly of the adrenal, but not pars distalis function. in females this effect may be attributed to increased fetal adrenal steroidogenic activity. the effect of dex in increasing cortisol in males, albeit at a significantly lower level than in females, appears to be independent of the enzymes that we have measured.*p<0.05. synaptophysin and gonadotropin-releasing hormone (gnrh) are colocalized in rat hypothalamus. armando arroyo, 1 beom su kim, 1 amanda biehl, 1 blenna cl bett, 1,2 john yeh. 1 1 gynecology-obstetrics, university of buffalo, buffalo, ny, usa; 2 physiology and biophysics, university at buffalo, buffalo, ny, usa. the cellular and molecular mechanisms that control gonadotropin-releasing hormone (gnrh) release are not completely understood. gnrh is stored in synaptic vesicles and released by exocytosis at gnrh nerve terminals. there are currently nine families of synaptic vesicle proteins that are involved in neurotransmitter release by exocytosis. synaptophysin is one of the most common synaptic vesicle proteins present in synaptic vesicles in neurons. the hypothesis of this study is that synaptophysin is expressed in gnrh neurons. we obtained sections from the hypothalamus of female sprague dawley rats. double-label fluorescence immunohistochemistry was performed on free-floating sections. sections were incubated with a mixture of mouse monoclonal antibody against gnrh (1:200-chemicon international) and with a rabbit polyclonal antibody against synaptophysin (1:100-santa cruz biotechnology) for 16 h. after incubation the sections were washed and incubated with a mixture of alexa 488 conjugated goat antimouse and alexa 594 conjugated goat antirabbit (1:1000; molecular probes) for 2 h. slices were visualized with confocal microscopy (zeiss lsm-510). fifteen out of a total of fifteen gnrh cell bodies in the medial preoptic area and most gnrh neuron terminals in the median eminence showed intense immunostaining for synaptophysin. this is the first study to demonstrate that synaptophysin is expressed in rat gnrh neuron terminals. this suggests that synaptophysin is present in gnrh neuron vesicles. thus, gnrh release by exocytosis may be mediated by synaptic vesicle proteins. objective: our aim was to identify the effects of early gestation gc exposure on fetal and postnatal hpa axis development and function in postnatal life. method: pregnant ewes carrying singleton fetuses were randomized to control (2 ml saline/ewe) or dex groups (0.14 mg/kg), consisting of four at 12 h intervals on days 40-41 of pregnancy. at 7 months postnatal age, catheters were implanted; a bolus injection of crh (0.5 mg/body weight) and avp (0.1 mg/body weight) were administered and arterial blood samples were taken at -30, -15, 0, 5, 10, 20, 30 60, 90, 120 and 180 min. levels of hepatic cbg mrna were determined by qpcr, expressed relative to 18s rrna. plasma cortisol and cbg levels were measured by radioimmunoassay. results: both total and free cortisol levels in the dex females (n=5) were lower than in dex males (n=7) from 30 to 90 min (p 0.05) and lower than in control females (n=6) at 30 minutes (p 0.04), also in the dex-m group were higher than in control males (n=5) at 60 min (p 0.04). plasma cbg levels and cbg mrna expression were not altered by dexamethasone exposure or sex. conclusions: these findings suggest that prenatal glucocorticoid exposure alters the development of the hpa axis differentially according to the sex of the exposed fetus. to determine maternal injections of synthetic glucocorticoids in early gestation can alter expression of hippocampal corticosteroid receptors at 7 months postnatal age. method: pregnant ewes carrying singleton fetuses were randomized to control (2 ml saline/ewe) or dexamethasone treatment (0.14 mg/kg) consisting of four injections at 12 h intervals on days 40-41. hippocampal was collected from the offspring at 7 months postnatal age. levels of mrna of gr and mr were determined using qpcr and levels relatived to 18s rrna. results: dexamethasone-treated male animals (n=7) had significantly higher levels of mr gene expression than both control males (n=5; p=0.028) and females (n=6; p=0.037). gr gene expression levels were higher in treated vs. control males (p=0.043), but in females levels in treated and control animals were similar. total body, brain and hippocampus weights were similar. conclusions: maternal dexamethasone administration in early pregnancy resulted in gender-dependent changes in hippocampal gene expression when measured in the offspring seven months after birth. objective: diminished ovarian reserve (dor) affects many younger women. we analyzed superovulation with intrauterine insemination (so) cycles in vitro fertilization (ivf) cycles of women with dor to determine if women <35 with dor differ from their older counterparts. method: irb approved retrospective review of so ivf cycles from 1/2005-6/2007 with follicle stimulating hormone (fsh) levels 10 based on age <35 or 35. results: 49 so cycles were performed in 18 women. no differences were noted in clinical pregnancy. women <35 were more likely to have inseminates with a lower mean tms/ins, median tms/ins was 71.0 among pregnancy cycles compared to 28 for unsuccessful cycles. for ivf, mean total gonadotropin dosage was significantly lower in women <35, mean number of follicles 15 mm peak e2 were significantly higher in women <35. so ivf measures are in tables 1 2, respectively. conclusions: similar clinical pregnancy outcomes were seen despite age. in ivf, women <35 required less gonadotropins and generated more follicles but with no significant difference in number of mature oocytes or clinical pregnancies. of note, pregnancy rates for so in women <35 with dor are substantially lower than expected in our clinical practice. as ivf yielded a substantially higher chance of pregnancy, consideration should be made to expedite progression to ivf. to assess the effects of intraovarian injection of ad-fshr on the reproductive system of fshr (-/-) mice. methods: about 50 l containing 3x10 9 pfu of ad-hfshr were injected directly into each ovary of treated group and same amount of ad-lacz were injected into the ovaries of control animals. vaginal smears were collected and body weight was measured daily. four weeks after the injection animals were sacrificed and all organs were weighted and evaluated by h e. fsh, e2 and p4 measured before and after treatment. results: ad-hfshrtreated mice showed obvious estrogenic changes in vaginal smear while in control animals vaginal smear remained at diestrus stage. significant increase in total body weight and estrogen dependent organs weight (uterus, ovary, vagina) was observed in treated animals compare to control group (p<0.02). no significant weight changes were observed in other organs. h e evaluation of the ovaries showed significant increases in both the total number of follicles and the collective diameter of the follicles in treated animals compare to controls. on average 18 follicles/ovary were observed in ad-hfshr-treated group of which 4 follicles were at the antral stage while only 2 follicles observed in ad-lacz control group, with zero follicles at antral stage. a 2.5 to 3 folds increase in e2 and about 50% decrease of fsh observed in treated animals compared to control mice. there was no significant change in serum progesterone level between treated and control groups. conclusion: intraovarian injection of an adenovirus expressing human fshr gene is able to restore folliculogenesis and resume estrogen hormone production in female forko mice. objective: the sentinel issue surrounding multiple gestations following ivf is the inability to precisely estimate the reproductive potential of individual embryos with the currently used embryo grading systems based on embryo cleavage rate and morphology. recently, metabolomic profiling of spent culture media using raman and near-infrared spectroscopy have been reported to predict reproductive potential of embryos. in this study we applied proton nuclear magnetic resonance (¹h nmr) spectroscopy to analyze metabolomic profile of embryo culture media and to identify components of the media that correlate with reproductive potential. methods: eighteen spent media samples from embryos that failed to implant, and 16 samples from embryos that resulted in pregnancy and delivery were individually collected after embryo transfer on day 3, and evaluated using ¹h nmr. the spectra obtained were analyzed using a selective genetic algorithm (ga) to determine regions predictive of pregnancy outcome as determined by logistic regression. to avoid random correlations, a leave-one out crossvalidation was used. sensitivity and specificity of predicting pregnancy (described as implantation and delivery) were calculated. results: using the ga, two areas in the ¹h nmr spectral region were identified as most discriminatory between the two groups. viability indices calculated by ¹h nmr using these regions were significantly higher in culture media of embryos with proven reproductive potential (0.66±0.13) compared to those that failed to implant (0.33±0.16) (p<0.001). compiled outcomes from the leave-one-out cross-validation of the logistic regression using the ¹h nmr measurements resulted in a sensitivity of 96% and a specificity of 77.8%. quantification by integration showed significantly decreased glutamate levels (p=0.002) and a trend toward an increase in pyruvate levels (p=0.1) in culture media of embryos that did not cause pregnancy. conclusion: metabolomic profile of spent embryo culture media using ¹h nmr correlates with the reproductive potential of embryos. the lower glutamate levels detected in culture media of embryos that failed to implant could potentially be due to the toxicity associated with increased embryonic glutamate uptake. additional studies using complementary approaches are needed to further delineate molecular components associated with reproductive potential. objective: beta-carotene and other carotenoids are known antioxidants previously identified in human follicular fluid (ff). in addition to their inherent antioxidant properties, carotenoids have been identified as precursors of the antioxidant retinol in bovine ff. high retinol levels in bovine ff are associated with non-atretic follicles. this would suggest a possible role for retinol and its carotenoid precursors in follicular heath and the general oxidative state of the follicle. we sought to measure carotenoids and retinol in the ff of women undergoing ivf and correlate these levels with normal fertilization as a marker of follicular/oocyte health. design: prospective cohort study materials and methods: ff from a single 18-20 mm follicle was obtained from 24 women (age 29-43) undergoing ivf and intracytoplasmic sperm injection (icsi). serum was also obtained at the time of oocyte retrieval. retinol, vitamin e ( , , and tocopherol) and carotenoids ( -carotene,cryptoxanthin, lycopene and lutein/zeaxanthin) were measured using hplc. we correlated ff carotenoid and retinol levels with oocyte fertilization status following icsi. results: as previously reported, retinol, vitamin e and carotenoids were all identified in the ff. each fat-soluble vitamin level was significantly lower in ff compared to serum (p<0.001 for all analytes) and the levels were strongly correlated (r2>0.45, p<0.003 for all analytes). mean levels of ff -carotene were significantly higher in those follicles that resulted in a fertilized oocyte (0.08 +/-0.04 g/ml vs. 0.01 +/-0.01 g/ml, p=0.015). other carotenoids, retinol, and vitamin e levels did not correlate with fertilization outcomes. conclusions: our finding of a strong association between ff -carotene concentration and subsequent normal fertilization of the oocytes suggests an important antioxidant role for -carotene in the health of the human ovarian follicle/oocyte. the lack of correlation with other carotenoids, retinol and vitamin e suggests that the antioxidant properties of -carotene act by preventing singlet oxygen and scavenging the peroxyl radical and may directly influence oocyte competence. mature oocytes obtained during egg retrieval have been shown to undergo spindle depolymerization as a result of cooling. for this reason, ivf programs strive to maintain constant temperature during follicle aspiration. we sought to elucidate if there was a difference in temperature of fluid aspirated into a hand held (hh) or heating block (hb) encased collection tube. the experiment was performed in an ambient temperature of 23°c. thermistors, pinhead sized sensors with an accuracy of 1%, were used to monitor temperature differences between control fluid (cf) (sterile water 37ºc) and aspirated fluid. data was recorded using an 8-channel data acquisition system from dataq instruments. one thermistor was placed into the cf, the other was placed into an empty collection tube set in the heating block or held in a gloved hand. a 33cm, 17 gauge, single lumen needle connected to 70 cm of tubing was used to aspirate into the empty collection tube. temperature was recorded 2x/second, each experiment was repeated 3 times. baseline empty collection tube temperature was significantly cooler in the hh vs the hb group (32.99±1.39°c vs 36.61±.16°c, p=.016). two seconds after aspiration, the lowest aspirate temperature was observed, (hh 30.34±.14°c, hb 30.48±.23°c, p>.05) no difference between groups. in both groups, temperature quickly increased as aspiration progressed (hh 34.57±.38°c, hb 34.86±.33°c, p>.05). the hb group took an average of 5.28 min to return to baseline (37°c), the hh group never returned to baseline. substantial cooling of aspirated fluid occurs during oocyte retrieval, with a mean temperature decrease of 6.59±.19°c corresponding to 30.41°c. considering this dramatic decrease, the difference between temperatures in the hh vs. the hb group is negligible. current aspiration systems poorly regulate temperature, thus the choice of aspirating into a test tube warmed by hand or by heating block is inconsequential. clinical management of twin gestations with recurrent preterm labor symptoms. lesley de la torre, 1 luis roca, 1 niki b istwan, 2 debbie j rhea, 2 gary j stanziano, 2 victor hugo gonzalez-quintero. 1 1 obstetrics and gynecology, university of miami medical center, miami, fl, usa; 2 clinical research, matria healthcare, marietta, ga, usa. objective to examine pregnancy outcomes in women with twin pregnancies receiving nifedipine tocolysis (nt) who experienced recurrent preterm labor symptoms (rptlsx). study design twin pregnancies enrolled for outpatient preterm labor (ptl) surveillance services prescribed nt following an initial episode of ptl were identified from a database (n=1421). eligible for inclusion were patients later hospitalized with acute rptlsx (n=862). included were those <35 weeks' gestational age (ga), with intact membranes, remaining undelivered for >48 hours after rptlsx . pregnancy outcomes of women resuming nt (rnt group, n=418) following hospitalization were compared to those having an alteration in treatment (alttx group, n=238) to continuous subcutaneous terbutaline. per normality assumptions, either independent student´s t or mann-whitney u test statistics were used for continuous variables; pearson´s chi-square for categoric. all p-values presented as two-sided, significant at <0.05. results overall, 862 (60.7%) of twin pregnancies prescribed nt experienced rptlsx; 206 (23.9%) were not eligible for continued tocolysis. pregnancy outcomes are presented in table. conclusion rptlsx are common. in twin pregnancies receiving nt, alteration of tocolytic treatment following rptlsx had a positive impact on pregnancy prolongation and neonatal outcomes. routine cervical dilatation during elective cesarean section -is it really necessary? arie koifman, avi harlev, eyal sheiner, fernanda press, arnon wiznitzer. obstetrics and gynecology, soroka university medical center, ben-gurion university of the negev, beer-sheva, israel. objective: the purpose of this study was to examine the necessity of routine cervical dilatation during elective cesarean section. material and methods a retrospective cohort study was performed, including all cases of elective cesarean sections performed at a tertiary medical center during 2005. stratified analysis, using the mantel-haenszel technique, was done to control for confounders. results out of a total of 666 elective cesarean deliveries (cd), 348 underwent routine cervical dilatation. the overall rate of febrile morbidity was 4.2%. no significant differences in postpartum febrile morbidity were noted between the groups (5.1 and 3.1%; p=0.071). in addition, hospitalization duration did not differ between the groups (4.1±1.4 and 4.1±2.0 days p=0.95 ). about 31 % of all elective operations were repeated cd. there was no difference in febrile morbidity between the groups even in that subgroup of the elective cd. likewise, there was no difference in anemia rate between the two groups (hemoglobin 9.50 ±0.73 mg and 9.54±0.65mg p= 0.91 ). controlling for a previous vaginal delivery, using the mantel-haenszel technique, no significant association was noted between cervical dilatation and fever (weighted or=2.2; 95% ci 0.8-6.4; p=0.161). nevertheless, in a subgroup of patients following a previous vaginal delivery, cervical dilatation was significantly associated with post-operative fever (or=5. the majority of women with an unfavorable cervix undergoing iol at term deliver vaginally, even with a prolonged first stage of labor. this is important information to discuss with women prior to iol when establishing labor expectations. providers should consider the ongoing success of labor induction when contemplating a diagnosis of "failed induction". objective: this study was performed to investigate and compare changes of the lipid peroxide levels and the protein carbonyls formation in the maternal venous plasma of preterm premature rupture of membrane (pprom) during antibiotics administration. materials and methods: thirty-six pregnant women with pprom between 25 and 32 weeks of gestation were chosen for this study. eighteen patients (group 1) were treated with amoxicillin and erythromycin for 7 day period while the other 18 patients (group 2) were treated with 3 rd generation cephalosporin and erythromycin for the same period. samples of maternal blood were obtained from the two groups at before the antibiotics administration, day 3, and day 7 after the antibiotics administration. lipid peroxide levels were measured by thiobarbituric acid reaction and protein carbonyl contents were determined by the 2,4-dinitrophenylhydrazine method. results: 1. the lipid peroxide levels and protein carbonyls formation in the maternal venous plasma of pprom was significantly higher than that of normal pregnancy (4.77±0.36 vs 7.11±0.41 nmol/mg protein, p<0.01), (3.55±0.22 vs 5.69±0.30 nmol/mg protein, p<0.01). 2. there were no significant differences in the lipid peroxide levels and protein carbonyls formation of the maternal venous plasma with pprom mixed and incubated by amoxicillin, cefodizime, and erythromycin (in vitro). 3. there were no significant differences in the lipid peroxide levels and protein carbonyls formation of the venous plasma of group 1 among before the antibiotics administration, day 3, and day 7 after the antibiotics administration. 4. the protein carbonyls formation in the venous plasma of group 2 was significantly decreased at day 3 and day 7 after the antibiotics administration than that of before the antibiotics administration (6.04 ±0.44, 5.53±0.37, 7.04±0.51 nmol/mg protein, p<0.01). conclusion: in the maternal venous plasma of pprom, the lipid peroxide levels and protein carbonyls formation were increased. the results suggest that reactive oxygen species formation by inflammatory reaction is suppressed by combined treatment of 3 rd generation cephalosporin and erythromycin. objective: the aim of the present prospective cohort study was to evaluate the correlation between blood flow pulsatility index in fetal middle cerebral artery (mca) and the onset of spontaneous labor. study design: doppler evaluation of fetal mca were performed between 24 and 41 weeks gestation in 664 consecutive pregnant women with a singleton pregnancy and known gestational age. the study population was divided according to the mca pi (< 0.74 or >/= 0.74 mom) and, using survival analysis and cox regression, the two subgroups obtained were compared. in these analyses, the event was delivery (following spontaneous labor) and the time variable was time elapsed since doppler exam. results: the median time elapsed between doppler evaluation and spontaneous labor was significantly shorter in the women with mca pi lower than 0.74 mom (5.5 days, interquartile range 2-10) in comparison with the women with mca pi higher or equal than 0.74 mom (22.5 days, interquartile 5-37.5 days (p<0.001, mann-whitney test). after correction for birth weight and umbilical artery pi, survival analysis and cox regression confirmed that mca pi was independently associated with the number of days elapsed from doppler to spontaneous labor and delivery (p< 0.001, exp(b) 2.77, ci 95% 1.95-3.90). conclusions: the present data show that, at term of pregnancy, fetal cerebral resistance reduction anticipates the onset of spontaneous labor. novel calcium sensitizers and human uterine contractility. mark p hehir, audrey t moynihan, terry j smith, john j morrison. department of obstetrics and gynaecology, national university of ireland, galway, ireland. objective: the factors regulating contractility of uterine smooth muscle are central to the occurrence of preterm labour and delivery. calcium sensitizers are a novel class of drugs with unique molecular actions. levosimendan, the best characterized of these compounds, is used in the treatment of acute and chronic heart failure and is a compound which exerts a number of effects on smooth muscle. it can exert an inotropic effect via sensitization of myofilaments to calcium and also exerts a relaxant effect by opening atp-dependent potassium channels. for these reasons we hypothesized that levosimendan may have an effect on myometrial contractility and investigated its action on both spontaneous and agonist induced contractions. method: biopsies of human myometrium were obtained at elective caesarean section (n=16). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of levosimendan in the concentration range of 1 nmol/l to 100 mmol/l. two sets of control experiments were performed simultaneously as follows: 1. strips exposed to either physiological salt solution (pss) only, for spontaneous contractions, or 0.5 nmol/l oxytocin; 2. strips exposed to pss/oxytocin and vehicle for levosimendan. results: levosimendan exerted an inhibitory effect on spontaneous and agonist induced contractions, compared to control strips. the mean maximal inhibition values were as follows: 45.34 ± 5.92% for spontaneous contractions (n=6; p< 0.05) and 41.88 ± 5.40% for oxytocin-induced contractions (n=6; p<0.05). no significant difference was found between control 1 and control 2 for both spontaneous and oxytocin induced contractions. conclusion: the calcium sensitizer levosimendan exerted a potent relaxant effect on uterine contractility in vitro. this action was seen in both spontaneous and agonist induced contractions. the results from this study raise the possibility of calcium sensitizers holding potential tocolytic properties in vivo and further studies are required to investigate the potential benefits of this novel class of drugs. to determine to what degree extensive patient movement affects uterine emg signals and toco signals. study design: 11 pregnant term labor patients were recorded using both uterine emg and toco simultaneously. baseline recordings were obtained when patients were still, and these were used as control records. test recordings for both devices were obtained from all patients by asking the patients to perform movements. area under the rectified-voltage amplitude curve of the uterine emg signals and area under the curve for the toco signals were found. mean %-increase was calculated for the uterine emg and toco devices (each device %-increase values were averaged over all 11 patients). mann-whitney rank-sum test was used to look for any statistical differences in %-increase in area for uterine emg vs. toco methods (p < 0.05 considered significant). results: there was a large increase in activity (artifact) on both devices' signals during patient movement (figure 1) . both devices' traces eventually returned to baseline after the patient movement stopped. toco movement artifact was significantly higher than emg movement artifact ( table 1) . conclusions: both uterine emg and toco signals experience artifact when patients move the uterine emg electrodes and toco pressure transducer, respectively. toco seems to be more adversely affected by such movements. uterine emg may be a preferred method for monitoring contractions of laboring patients in the clinic. supported by grant nih r01-hd037480. results: 68% of obstetricians completed the questionnaire. none would counsel patients against labour unless there were contraindications. the majority would recommend labour for all indications for previous caesarean section investigated although in all instances, personal preferences were lower (p<0.04); after a failed instrumental delivery, 74% obstetricians would recommend labour but only 44% would choose that option for themselves (p<0.01). overall, female obstetricians would contemplate and recommend labour more readily than male obstetricians. labour augmentation and induction were recommended to patients more frequently (66% and 57% respectively) than chosen for personal care (57% and 52%). reluctance for labour augmentation and induction was greatest among younger consultants. conclusion: consultants have responded to consumerism and aim to meet the requests of their patients. they more readily recommend labour than they would choose for personal care, and a majority would recommend labour induction when necessary. informed patient choice is paramount rather than attaining a target figure for women attempting to labour, and the views of those requesting delivery by caesarean section should be respected. (table) . cd increased in nullipara singleton, cephalic, term pregnancy in spontaneous labor, mostly due to dystocia; nullipara singleton, cephalic term pregnancy with induced labor, mostly due to non-reassuring fetal status and dystocia; singleton, cephalic term pregnancy with previous cd, mostly due to elective cd; singleton cephalic preterm. no changes in neonatal outcome were observed. conclusion: clinical audit was useful to keep under control cd rate in our institution in comparison with italian reality, but it was not sufficient to maintain stable cd rate suggesting the need of other, multifaceted strategies. elective delivery at 39 weeks' gestation is a common obstetric intervention. the purpose of this study is to estimate the maternal mortality rates (mmr) associated with this acog -approved intervention. there are no reliable us data describing mmr by mode of delivery. therefore, we base our estimates on british data indicating a procedure related mmr of 2.06/100,000, 5.8/100,000 and 18.2/100,000 for vaginal, elective cesarean section (c/s) and emergency-unplanned c/s deliveries, respectively. a decision tree model was constructed assuming that all eligible patients (approx. 2,500,000/annually in the u.s.) would be delivered at 39 weeks by either an elective c/s or an induction of labor. we further assumed that 50% of inductions would result in a vaginal delivery. our estimates show that the annual, delivery-related maternal mortality associated with an elective delivery of all patients at 39 weeks would be 145 and 253 for elective c/s and induction of labor, respectively. because vaginal delivery results in the lowest mmr, we performed a oneway sensitivity analysis to identify the impact of changing the success rate of induction on the estimated mmr. the results indicate that once the success rate exceeds 77%, this intervention would be associated with a lower mmr as compared to elective c/s. our estimates indicate that although the overall mmr associated with elective delivery at 39 weeks is relatively low (approximately 12.5/100,000 deliveries), it is certainly not negligible. in addition, the mmr is highly dependent on the likelihood of a successful vaginal delivery following induction of labor. when the success rate for induction of labor falls below 77%, an elective c/s appears to be the safer delivery method. background: the first obstetric visit is an opportunity to provide the pregnant patient information regarding substances that can cause potential harm to the pregnancy. little is known about how obstetric care providers handle these topics. objective: to examine patient-provider communication about substance use during the first prenatal visit. methods: we audiotaped first prenatal visits and qualitatively analyzed those tapes in which patients disclosed substance use. we invited patient participants to return for a semi structured interview during which they reviewed their audiotaped conversations and described their reactions to the providers' communication styles. results: 29 providers (21 residents, 5 midwives, 3 nurse practitioners) and 51 patients participated. providers asked about smoking, alcohol and drug use in all 51 (100%) visits. 25 patients reported being smokers, 4 reported alcohol use, and 11 reported drug use. provider responses to smoking disclosures included brief discussions of smoking effects on pregnancy, encouragement to quit/cut down, and referral to smoking cessation programs. provider responses to alcohol or drug disclosures included only general statements regarding effects on pregnancy (e.g., "we find that this is bad for babies.") and referral to ultrasound/genetics for reassurance. few alcohol or drug discussions assessed whether the patient had intentions or concerns regarding continued use during the pregnancy. few discussions addressed strategies for behavioral change. none included assessment for motivations, readiness, or barriers to change. in follow up interviews, patient participants said they expected to be asked about substance use but advised providers to ask non-judgmentally. those who used alcohol/drugs wanted more information on potential effects of these substances on the pregnancy/fetus and appreciated the reassurance from referrals to ultrasound/genetics. discussion: counseling for risky behaviors in the first obstetric visits contained only limited discussion of the effects of the risky behaviors and primarily focused on referral-which may be a proxy for avoiding a difficult and time consuming conversations. background. there are conflicting results regarding the association of maternal psychiatric disorders or psychological distress due to stressful life events with pre-term birth and low birth weight. aims. to investigate the association between maternal psychiatric disorders and/or stressful life events and intrauterine growth abnormality, low birth weight or preterm birth. method. three mutually exclusive and homogeneous groups of pregnant women (20 with actual psychiatric disorder, 20 with stressful life events, and 40 healthy comparisons) underwent serial fetal ultrasound examinations and uterine and umbilical artery doppler velocimetry at 20 (+1), 28 (+1) and 34 (+1) weeks of gestational age. subjects were recruited from all consecutive women attending two antenatal clinics. the presence of any maternal medical illness, drug treatments, fetal chromosomal and/or structural malformations, were exclusion criteria. all women recruited underwent a structured interview at 18-20 weeks for the psychiatric diagnosis (mini international neuropsychiatric interview -mini) (sheehan et al, 1998) ; moreover, the 17-item hamilton rating scale for depression and the hamilton rating scale for anxiety were included in the assessment. the person who obtained obstetrical clinical data was blind to the results of psychological evaluations. results. the three groups were comparable for: age, parity, socioeconomic status, smoking, alcohol consumption, body mass index. gestational age at birth was not different in the three groups. infants of women with psychiatric disorders had significantly lower birthweight (3084 + 414 g) and higher percentage of birth weight below the 10 th centile for gestational age (30%) than infants of healthy mothers (3408 + 453 g and 5%, respectively). there was also a trend towards lower mean birth weight (3242 + 400 g) and higher incidence of birth weight below the 10 th centile (10%) in the stressful life event group. there was no significant difference among groups in the percentage of abnormal uterine or umbilical doppler results. conclusions. maternal psychiatric disorders are associated with a lower birth-weight, but the effect is unlikely to be due to abnormal utero-placental or feto-placental vascularisation. objective: the purpose of this study was to evaluate antenatal ultrasound as a tool for the detection of intrauterine growth restriction (iugr) and small for gestational age (sga) infants among subjects with elevated human chorionic gonadotropin (hcg) levels on second trimester serum screening. although iugr has been linked to elevated hcg levels, the optimal screening regimen for antenatal sonographic surveillance has not been previously established. methods: a retrospective cohort study was performed at saddleback memorial medical center where serial ultrasounds from 26 weeks-delivery are generally recommended for patients with hcg levels > 2.0 mom. all pregnancies were dated by second trimester ultrasound ± last menstrual period. subjects with an hcg > 2.0 mom, who had at least one antenatal ultrasound evaluation for iugr, were identified from an electronic ultrasound database used for clinical report generation. ultrasound data were then linked to an obstetrical birth outcomes database for relevant demographic/delivery information using unique identifiers. iugr was defined as a sonographic estimated fetal weight (efw) <10%ile for the estimated gestational age (ega). sga was defined as an actual birthweight <10%ile for the ega at the time of delivery. results: from 1999-2007, there were 659 subjects with elevated hcg levels who underwent antenatal ultrasound surveillance for iugr and who had known delivery information. a total of 1708 ultrasound examinations were performed. the median number of examinations per subject was 3 with a range from 1-5 examinations per subject. the incidence of iugr and of sga were 5.0% (n=33/659) and 7.3% (n=48/659), respectively. no fetus with iugr demonstrated absent or reverse end diastolic umbilical artery doppler flow. antenatal ultrasound examinations only identified 31.3% (n=15/48) of sga infants. however, the sensitivity for the detection of sga was 100% when an efw cut-off of 75% was used. conclusions: although the majority of sga infants did not demonstrate growth restriction on antenatal ultrasound, a sonographic efw > 75%ile appears to be a safe cut-off to rule out fetuses at risk for sga. neonatal dry lung syndrome after pprom: reason to change intrauterine referral practice in the netherlands? ellen s hoogakker, 1 christiaan v hulzebos, 2 gerda g zeeman. 1 1 obstetrics and gynecology, university medical center groningen, groningen, netherlands; 2 pediatrics, division of neonatology, university medical center groningen, groningen, netherlands. background: neonatal dry lung syndrome (dls) is a distinct clinical entity following pprom, mimicking pulmonary hypoplasia but with dramatic respiratory improvement during the first 24-48 h . its pathogenesis implies complete collapse of small airways to a degree that capillary forces impede distension by ordinary ventilatory pressures. in the netherlands, women with pprom remain admitted at a level iii nicu perinatal center until they reach 32 wks. when they are referred back to their community hospital, unless they live in the neighborhood of the tertiary center. we question this referral pattern because we still see severe respiratory problems occur > 32 wks. we sought to determine the prevalence of such morbidity, in particular dls, in women with pprom who deliver > 32 wks. methods: retrospective descriptive study of neonatal outcome data of singleton pregnancies complicated by pprom between 24-32 wks, who deliver > 32 wks (latency > 24 h) , during the 4-yr period of 2002-2006 at a single academic center. data were extracted from medical records and electronic department databases. results:108 pprom pregnancies were identified. all but 2 received at least 1 full course of steroids. 27 (25 %) delivered > 32 wks. 3 newborns born at the community hospital needed emergency transportation to a level iii nicu for respiratory morbidity. neonatal outcome data (means ± sd) are listed in the table. there were no cases of late onset sepsis, nec or perinatal mortality. conclusions: respiratory morbidity still occurs after pprom with delivery > 32 wks. further investigation of pregnancy-related characteristics, such as the presence of anhydramnios and the latency period, with regards to dls is needed. modification of current referral practice depends upon complete data derived from all dutch level iii perinatal care centers (n =10). tertiary care center (n = 8) to determine if an association exists between macrosomia (birthweight > 4000g) and perinatal outcomes in women with and without gestational diabetes mellitus (gdm). study design: this is a retrospective cohort study of 36,241 singleton pregnancies. the study cohort was stratified by the diagnosis of gdm, with the presence or absence of macrosomia as the dependent variable. perinatal outcomes examined included neonatal hyperbilirubinemia, hypoglycemia, respiratory distress syndrome (rds), shoulder dystocia and erb's palsy. chisquare tests were performed as well as multivariable analyses controlling for confounders, using p<0.05 to indicate statistical significance. results: in women diagnosed with gdm, macrosomia is associated with a higher frequency of hypoglycemia, respiratory distress syndrome, shoulder dystocia and erb's palsy. though the prevalence of these outcomes is relatively decreased in patients without gdm, they are still more prevalent in macrosomic patients. macrosomia is associated with a higher prevalence of adverse perinatal outcomes in women with and without gdm. therefore, it is important to evaluate neonates with birthweights greater than 4000 grams for hypoglycemia and unrecognized erb's palsy. conclusion a significant proportion of our obstetrical patients are obese and many do not perceive themselves to be obese. while our finding of an inverse correlation between level of education and bmi may be confounded by socioeconomic status, our results suggest that in order to address the problems of obesity in our population an important first step will be improving the education of our reproductive age women regarding normal weight gain and nutrition in pregnancy. this may have a significant impact on improving pregnancy outcomes of today's obstetrical population. objective: the aim of the study was to evaluate the impact of a "flat" oral gct upon the outcome of pregnancy. the gct was considered flat when the difference between the basal value and the after load value was 10%. methods: we prospectively analyzed the outcomes of 410 pregnancies who delivered at our department. inclusion criteria were singleton pregnancy; bmi <30 kg/m 2, absence of major risk factors for diabetes and of pregestational diabetes. 50 g 1-hour oral gct was performed at 24 -28 weeks of gestation. women were subdivided into 3 groups according to the result of the gct: group 1= negative (load glucose >10% and <140 mg/dl) 327 women (79.8%); group 2=flat (load glucose 10% than basal and <140 mg/dl) 51 women (12.4%); group 3=positive gct/negative ogtt, 32 women (7.8%). data are mean ± sd. differences were calculated with the student t test for unpaired samples and 2 test. regression analysis were performed by the least squared method. p-values were considered significant at p<0.05. results: the characteristics and obstetric outcomes in the three groups are presented in the table 1 . in all patients there was a significant linear relationship between the load and basal glucose values (load value=66.8+0.6 basal value; r=0.2; p<0.001) and between birthweight and load values (birthweight=2978.3+2.8 load value; r=0.15; p<0.02). the relationships were significant also in group 1 and 2 separately but not in group 3. in this preliminary study we found no major outcome differences in women with flat gct compared to women with normal and gct positive/ ogtt negative results. it seems important, however, to futher investigate the meaning of a flat curve in a bigger population and/or by means of metabolic studies with the use of stable isotopes. results characteristic of the study population and obstetric outcomes are presented in table 1. the probability to be primiparous and to deliver babies <10° centile decreased significantly with bmi whereas the risk of cesarean section, of post partum hemorrhage at vaginal delivery and to deliver babies >90° centile increased significantly. also the risk to develop preeclampsia and gestational diabetes was increased, although not significantly, with bmi. compared to lean and normal, obese were more likely to be hypertensive and diabetic before pregnancy (p<0.001) and to start pregnancy without any medical and obstetrical risk but obesity (85.6% vs 90.5 and 90.2%; p<0.01). african women exhibited the highest bmi and the highest rate of obesity (table 2 ). conclusions we confirm that obese women have an increased risk of prepregnancy and pregnancy complications: less than 50% have an uncomplicated pregnancy. at delivery there is an increased risk of cesarean section and post partum haemorrhage. objective: overweight and obese women often give birth to larger babies, which is associated with traumatic birth injuries and an increased risk to develop obesity, diabetes and hypertension in childhood and later in life. the mechanisms underlying fetal overgrowth in obese pregnant women are largely unknown. the aim of this study was to establish a mouse model of obesity/high fat diet in pregnancy. we hypothesized that a moderately high fat diet prior to and during pregnancy would result in increased maternal adiposity, fetal overgrowth and a metabolic profile similar to that of obese newborn offspring with persistent pulmonary hypertension, despite enhanced pregnant women. method: c57bl/6j female mice were fed control (c, 11% of energy from fat) or isocaloric high fat (hf, 32% of energy from fat) diets ad libitum for 8 weeks prior to mating and during gestation. at gestational day 18.5 maternal blood samples were obtained to measure adiponectin, leptin and cytokine levels and maternal fat pads were isolated and weighed. in a sub set of animals measurements of transplacental transport of neutral amino acids and glucose were performed in vivo under ketamine anesthesia using 3 h-meaib, and 14 c-glucose. results: no significant differences were observed in maternal pre-pregnancy bodyweight, total caloric intake, weight of the dam at e18.5 or litter size between treatment groups. fetal weight was increased in the hf group by 45% (p<0.05). maternal adiponectin levels were significantly (p<0.01, n=18) decreased (hf 45 ± 8, c 69±15 g/ml) and leptin levels increased by 60% in animals fed a high fat diet, but this difference did not reach statistical significance (n=6). adiposity (fat pad weight) was increased by 7% (p<0.05, n=10 in the dams fed high fat diet, however no difference was observed in maternal il-6 levels and neither group had measurable levels of tnf-. maternal red blood cell lipid profiles were altered in high fat animals with an increase in stearic and linoleic acids but decreased oleic acid levels. preliminary data showed that placental uptake and transfer to the fetus of glucose and meaib were increased by at least 50% in dams fed high fat diet. conclusion: this murine high fat diet model has several features consistent with human obesity in pregnancy and the maternal metabolic environment is similar to that seen in the human. the increase in placental uptake and transfer of nutrients constitute a key mechanism underlying fetal overgrowth in overweight/obesity in pregnancy. objective: epidemiological studies have demonstrated a positive relationship between maternal overnutrition and the development of the metabolic syndrome in the offspring. we previously reported that lambs of well fed ewes have increased plasma glucose levels in early life, this may be a consequence of altered hepatic glucose production. increased 11 hsd1 expression is associated with an increase in intracellular cortisol, promotion of hepatic insulin resistance and a consequential increase in gluconeogenic activity. hypothesis: we hypothesised that maternal overnutrition in late gestation in the sheep results in increased hepatic expression of 11 hsd1 in the lamb before and after birth. methods: ewes were provided with either 100% (control,c) or 160% (well fed, wf) of maintenance energy requirements from 115d gestation until delivery. post-mortem was performed on either 139±141d gestation (c=6,wf=8) or and postnatal day 30 (c=12,wf=9). plasma glucose and leptin concentrations in the fetuses and lambs were determined. the relative hepatic mrna expression of 11 hsd1 and reference gene rpp0 were determined by qrt-pcr. results: relative liver weight was significantly higher in lambs of wf ewes compared to c at 30d (p<0.05). the expression of 11 hsd1 mrna was significantly higher in the postnatal compared to the fetal lamb (p<0.001) independent of maternal nutritional treatment. however, there was no effect of maternal overnutrition on the hepatic expression of 11 hsd1 mrna before or after birth. there was no effect of prenatal nutrition on fetal or postnatal plasma cortisol concentrations. the expression of 11 hsd1 in the liver of lambs of wf, but not c ewes, was inversely related to plasma glucose concentrations in the first 24hrs after birth (r 2 =-0.78, p=0.002). we have therefore demonstrated that exposure to prenatal overnutrition results in an inverse relationship between 11 hsd1 mrna expression in the liver at 30d and plasma glucose concentrations on the first day of life. this suggests that exposure to high glucose levels before and immediately after birth results in a reduced expression of hepatic 11 hsd1. we would expect a reduction, rather than promotion of intra-hepatic cortisol production, and therefore it is unlikely to explain the hyperglycemia present in lambs of wf ewes in early life. ninety-seven (38%) of mothers were classified as obese (bmi>30) based on their first pregnancy weight. glycemic control at 36 weeks was superior in the non-obese group (table 1) . there were no differences in glycemic control during the last week of pregnancy. obesity was significantly associated with increased maternal weight gain during pregnancy. mean birth weights, ponderal indices and rates of macrosomia were significantly higher in infants born to obese women when compared to non-obese (table 1) . primary cesarean deliveries rates were comparable. the rate of neonatal hypoglycemia, hyperbilirubinemia, phototherapy and neonatal icu admissions did not differ between obese and non-obese diabetic women (table 1) . although not statistically significant, there was a trend towards an increased rate of birth injuries in the obese group. a similar comparison between obese and non-obese women treated with medication (glyburide or insulin) demonstrated a higher mean birth weight (3675g+593 vs. 3434g+520, p=.005) and higher rate of macrosomia (11 vs. 9%, p=0.04). there were no differences in glycemic control, cesarean delivery rates and other neonatal outcomes between the obese and non-obese treated with medication. conclusion: obese women with type ii and gdm give birth to larger infants than their non-obese counterparts and have a higher incidence of fetal macrosomia. there was a trend towards increased rate of birth injuries in the obese group. despite these differences other maternal and neonatal outcomes were similar which may be a reflection of glycemic control. introduction: tlrs are key components of the innate immune system which recognize conserved sequences on the surface of pathogens and trigger effector cell functions. the placenta, and more specifically the trophoblast, may play an important role in the response to infection. previously, we described the expression of tlr-3 by human trophoblast and their ability to respond to polyinosinic-polycytidylic acid (polyi:c), a synthetic double strand rna which mimics viral rna. in the present study we evaluate the effect of polyi:c in mouse pregnancy and characterize the local and systemic response. material and methods: human first trimester trophoblast cell line, htr8, was treated with polyi:c. c57b/6 wild type and tlr-3 knock out mice were injected intraperitoneally with polyi:c at 16.5 gestation day. cytokine and chemokine level were determined in supernatant and lysates using the bio-rad multiplex assay and analysis was done using the bio-plex100 is. results: polyi:c induced cytokine (il6, il1 and il1 ) and chemokine (il8, rantes, gro , mcp1 and mip1 ) secretion and production by human cultured trophoblast in a time (24-48 h) and a dose (2-25 g/ml) dependent manner. injection of polyi:c to c57b/6 wild type mice induced preterm delivery within 24 h at a dose of 9 mg/kg body weight. no effect was observed in tlr-3 knock out mice. a robust systemic (spleen and serum) and local (placenta and amniotic fluid) inflammatory response was observed 2 and 4 h following polyi:c treatment. trophoblast cell cultures from tlr-3 ko mice confirmed that the response to polyi:c is tlr-3 dependent. conclusion: we demonstrate that viral infection may trigger an immune response leading to preterm labor. furthermore we show that the trophoblast is able to recognize and respond to viruses through the expression of tlr-3. our findings provide a novel mechanism of pathogenesis of preterm labor associated with tlr-3 mediated inflammatory response. introduction: adverse neurological outcome is a major cause of neonatal morbidity after a preterm birth (ptb). a growing body of evidence demonstrates the involvement of inflammatory pathways in ptb and implicates these pathways as a causative factor in fetal brain injury. however, activations of cytokine pathways in normal labor (whether iatrogenic preterm or at term) has been observed. what remains understudied is the effect of labor on the preterm fetal brain and whether an inflammatory stimulus is essential for fetal brain injury. methods: 2 mouse models were utilized: (1) a model of intrauterine inflammation (lps into uterine horn) (n=9); controls received intrauterine saline (n=9) and (2) autism is a neurodevelopmental disorder with a strong genetic component and several known environmental risk factors, such as infection. in addition, its onset of etiology is likely to occur during prenatal development. we propose that subjecting fetal sheep via amniocentesis to the bacterial endotoxin lipopolysaccharide (lps) injected to the amniotic fluid at gestational day (gd) 110 will result in morphological alterations in the offsprings' cerebellum resembling alterations found in the cerebellum of patients with autism. using high precision design-based stereology, we investigated mean total-and layer-specific volume and mean total granule and purkinje cell (pc) number in the cerebellum of 6 lps infected animals and 3 controls. the results of the present study showed preserved volumes of the total cerebellum as well as of the molecular layer, outer and inner granular cell layers and white matter. interestingly, compared to controls, the lps infected brains showed a statistically significant increase (+20.4 %) in the mean total number of granule cells, whereas the pcs did not show any difference between the groups. these seemingly paradoxical results might be explained by (1) the so-called time of origin of these neurons, i.e. the pcs develop prenatally whereas the granule cells develop postnatally or (2) the direct correlation between pcs and granule cell number in the cerebellum. these results might contribute, as an animal model, to our understanding of the biological basis for interindividual differences in morphological alterations found in the brains of patients with autism. the previous studies have shown that there is a higher incidence of spontaneous preterm birth and poorer neonatal outcome in pregnancies with a male fetus. in vitro studies also report a sex dependent pattern in different placental enzymatic systems. we have shown previously that lactobacillus rhamnosus gr-1 supernatant is able to antagonize the actions of lps on cytokines and ptgs2 in placental trophoblasts. we hypothesize that fetal sex will influence the production of cytokines and prostaglandin regulating enzymes in lps and lactobacilli treated placental trophoblast cells. methods: term placentae were collected from women undergoing elective caesarean section. placental trophoblasts were isolated using established primary culture protocols. cells were pretreated with lactobacilli supernatant and subsequently treated with lps. pgdh and ptgs2 expression levels were measured by western blot analysis and tnf-, and il-10 concentrations measured by elisa. results: lps stimulation caused a marked increase in production of tnf-(30.9 pg/ml to 1175.3 pg/ml, n=11, p<0.05), an effect that was greater in placentae of the male fetuses (1516.6 pg/ml, n=6) compared to female fetuses (562.5 pg/ml, n=5). lactobacilli supernatant abolished this response in both sexes. lps-activated trophoblasts from placentae of the male fetuses showed an increase in il-10 production (n=5, p<0.05) and ptgs2 expression (n=4, p<0.05). however, there was no response to lps in placentae of the female fetuses. lactobacilli supernatant up-regulated pgdh (n=8) by 63%, and this effect was greater in placentae of the female fetuses (n=4). conclusion: we conclude that human placentae from pregnancies carrying male fetuses are more responsive to lps by producing more pro-and anti-inflammatory cytokines, as well as ptgs2. conversely, placentae of the female fetuses upregulate pgdh with lactobacilli treatment. these findings may explain the underlying mechanism for the higher incidence of preterm birth and adverse pregnancy outcomes seen with male fetuses in the clinical setting. , 2007) . this study investigates whether iai is associated also with altered expression of neuropilin-1. methods: (i) immunohistochemistry (ihc) was performed on tissue sections of term decidua with or without clinical / histologic evidence of iai (n=3 for each). neuropilin-1 expression was scored by an investigator blinded to the identity of the samples. (ii) cultured term dscs were retrieved from elective cesarean (n=6), purified, and depleted of leukocytes. after treatment with 10 -8 m estradiol (e2), 10 -7 m medroxyprogesterone acetate (mpa), both, or vehicle for 7 days, dscs were stimulated with il-1b (1-10 ng/ml), tnfa (1 ng/ ml), or thrombin (2.5 iu/ml) for 24h. since no elisa exists for neuropilin-1, protein expression was determined by immunocytochemistry (icc). (iii) total rna was extracted and the effect of il-1b on neuropilin-1 mrna expression measured by real-time rt-qpcr and corrected for b-actin mrna. results: neuropilin-1 expression in term decidua was increased in tissues with iai vs controls (p<0.05), and localized primarily to dscs. using icc, an increase in neuropilin-1 was noted after stimulation with il-1b and tnfa, but not thrombin. il-1b increased neuropilin-1 mrna expression in dscs by 6.5±1.6-fold (from 28.0±17.7 to 240.9±189.4 neuropilin-1 mrna/b-actin mrna; p=0.031). conclusions: iai is associated with increased expression of the vegf receptor, neuropilin-1, in term decidual tissues. il-1b and tnfa (but not thrombin) stimulated neuropilin-1 expression in term dscs, and this effect appears to be mediated at the level of gene transcription. since aberrant vegf function alters vascular permeability, these data provide a mechanism by which iai can promote 'decidual activation' and preterm labor. inflammation, university of glasgow, glasgow, united kingdom. objective: asthma is associated with inappropriate activation of airway smooth muscle, chemokine expression and accumulation of mast cells which drive smooth muscle reactivity. labour is similarly associated with smooth muscle activation and expression of cxcr1 and cxcr2 ligands. the role of mast cells in human parturition is unknown; however, mast cell products can stimulate myometrial contractions and preterm labour in animal models. we have quantified mast cells in association with human labour and determined whether they express cxcr1 and cxcr2. methods: lower segment myometrial and cervical biopsies were taken at term caesarean section from women not in labour (nil) (myometrium n=8; cervix n=9) and in labour (il) (myometrium n=8; cervix n=9). mast cells were localised in myometrial and cervical sections by icc with a primary antibody against c-kit. the number of cell transects in 10 randomly selected high-powered fields (400x) was quantified blindly by two observers for each specimen, with median density and interquartile range (iqr) calculated. backto-back icc was performed to determine whether c-kit co-localised with the chemokine receptors cxcr1 and cxcr2. results: mast cells were in close association with myometrial smooth muscle in non-labouring lower segment myometrium. labour was associated with a significant influx and increase in mast cells numbers (nil median 11.9, iqr 6.7 -19.8; il median 26.9, iqr 16.1 -40.2, p=0.025). in contrast no significant increase in mast cells was observed in cervical tissue in association with labour (nil median 19.0, iqr 4.6 -35.5; il median 7.9 iqr 3.5 -10.4, p=0.17). analysis of chemokine receptor expression demonstrated co-localisation of cxcr1 to c-kit positive cells present within the myometrium. conclusions: human labour at term is associated with an increase in mast cells within the myometrium, with close approximation to smooth muscle bundles. these mast cells express the chemokine receptor cxcr1, the ligands of which we have previously shown to be up-regulated in labouring myometrium. mast cells are not accumulated in cervix in association with labour suggesting a less critical role in cervical ripening. further analysis of the role of mast cells in modulating myometrial smooth muscle physiology is warranted. progestins and the glucocorticoid receptor in human myometrial and amnion-derived wish cells. alison j tyson-capper, stephen c robson. reproductive sciences, newcastle university, newcastle upon tyne, united kingdom. background and objectives: progesterone (p) can reduce the risk of preterm birth in some high risk women. in this context there is accumulating evidence that this, at least in part, may be due to anti-inflammatory and immunoregulatory properties of p. target tissue responsiveness to p is considered to be determined by the progesterone receptors (pr) and nuclear co-factors that directly interact with pr. pr and glucocorticoid receptors (gr) share several structural and functional characteristics, including similarities in dna sequence recognition by binding to the same hormone response elements. pr and gr interact with similar chaperones in the absence of ligand and with a similar group of co-activators in the presence of hormones; both can display comparable anti-inflammatory activities under specific physiological conditions. in this study we aimed to investigate whether the anti-inflammatory properties of progestins may be mediated by pr and gr signalling. methods: primary cultures of non-pregnant and term pregnant human myometrial (passage 1-2) and wish cells were serum starved for 24hrs and treated with 17-hydroxyprogesterone (17-hp), progesterone (p), dexamethasone (dex) and immunofluorescent staining and immunoblotting analyses performed. in some experiments cells were pre-treated with ru486 (a pr/gr antagonist) or org 31710 (a pure pr antagonist). results: in the absence of hormone gr appeared to be predominantly cytoplasmic, whereas, upon treatment with 17-hp and p (1 and 10 m) and dex (10nm) gr was abundant within the nuclei of myometrial cells. immunoblotting analyses demonstrated that levels of gr progressively increased within the nuclear fractions of both pregnant myometrial and wish cells in response to increasing concentrations of p (100nm to 10 m), and decreased sequentially within cytoplasmic fractions. in the presence of org31710 gr protein levels remained constant within the cytoplasm. there also appeared to be a slight increase in gr expression, though not statistically significant (p>0.05) within both cells types in response to p. conclusion: in this study we show that gr is activated by 17-hp and p and translocates to the nuclei of human myometrial and amnion-derived cells. in addition, levels of gr increase in response to p. whether p and 17-hp act as agonists or antagonists for gr in the regulation of hormone response genes associated with the onset of term and preterm labour remains to be elucidated. objective: using a mouse model of inflammation-induced preterm birth (ptb), we have demonstrated dramatic cytokine elevations in the uterus and placenta with concomitant, though less dramatic, cytokine elevations in the fetal liver and brain, associated with neuronal injury. because precise mechanisms of fetal injury in ptb remain unclear, we sought to examine inflammatory cell trafficking, and target organ damage by histopathologic assessment of the placenta, fetus, and fetal brain. study design: 6 hours after intrauterine infusion of saline or lps into the right lower uterine horn of cd-1 mice, the left upper horn, with the gestational sacs(gs) in situ, was removed en bloc(n=6 per group) each with 2-4 gs with15 fetuses/treatment group. specimens were fixed, bisected and processed for histology and ihc. inflammatory and hematopoietic cells were quantified using pas, gata-1 (erythroid precursors), cd3, and bm8 (macrophage-mp) within the placenta, liver, extremity mesenchyme, brain and leptomeninges. the presence of hemorrhage, necrosis, and apoptosis (h2ax stain) was assessed. erythopoietin (epo) levels were measured in brain and liver by elisa. results: more neutrophils were present in maternal decidual vessels in lps compared to saline (p=0.02). in lps-exposed, fetal mp were increased in the placenta (p=0.007), fetal extremity mesenchyme (p=0.018), fetal liver (p=0.005) and leptomeninges (p=0.013) but not in the brain or spinal cord compared to saline. no necrosis, hemorrhage or increased apoptosis was noted in the fetal brains. 69% of lps-exposed fetuses and 0% of saline-exposed had liver hemorrhages (p< 0.001). increases in nucleated erythrocytes and erythroid precursors were found in fetal vasculature of the placenta in lps-exposed (p=0.004). epo levels were not elevated in either group. conclusion: intrauterine lps infusion induces acute inflammation predominantly in the maternal circulation of the placenta. in the fetus, there is widespread mp activation, liver hemorrhage and increased erythroid precursors seen in the fetal circulation of the placenta. although histologic evidence of cns damage was not evident, the increased mps present in the leptomeninges may play an important role in inflammatory-mediated cns damage. non-toll-like innate immune proteins: do they change during pregnancy? juan m gonzalez, hua xu, ella ofori, michal a elovitz. obgyn; crrwh, university of pennsylvania, philadelphia, pa, usa. introduction: trem-1 (trigger receptors expressed on myeloid cells) is an important regulator of innate immunity. the natural ligand for trem has not been identified. activation of trem-1 in the presence of toll-like receptors results in substantial amplification of the host inflammatory response (klesney-tait et al nature immunology 2006). since inflammatory pathways are implicated in adverse pregnancy outcomes, this novel mediator of inflammation may play a critical role in preterm birth (ptb). therefore, we sought to determine trem-1 expression in the uterus, cervix, and placenta across gestation and to determine if trem-1 levels are altered by intrauterine inflammation. methods: in cd-1 mice, trem-1 was investigated in non-pregnant (np) and throughout gestation e 15, e 18 (n=3 -6 per group). uterine, cervical, and placental tissues were harvested. using an established mouse model of inflammation-induced ptb, uterine tissue was collected 6 hours after intrauterine infusion of saline (n=3) or lipopolysaccharide (lps) (n=3). for a non-pregnant model, using cd-1 mice, lps (n=3) or saline (n=3) was injected into the uterine horn following same procedures as with pregnant mice. uteri were harvested 6 hrs later. quantikine ® mouse trem-1 immunoassay was utilized for these studies. statistical analysis was performed using oneway anova followed by pair-wise comparison if statistical significance was reached (p<0.05) results: trem levels are significantly different between np and pregnant uterine tissues (p=0.002). e15 and e18 trem expression is significantly increased 2.3 and 2.5-fold compared to np (p=0.004 and 0.001 respectively). trem-1 levels in the placenta and cervix were not significantly different between e15 and e18. trem levels increased about 4-fold in the uterus after intrauterine infusion of saline or lps compared to e15 controls. in nonpregnant, trem levels were significantly different (p=0.05) with a 17-fold increase in trem expression in uteri exposed to lps or saline compared to controls. conclusions: non-toll-like innate immune proteins are differentially regulated during pregnancy compared to the non-pregnant state. the role of trem-1 in inflammation-induced ptb requires further study. research is warranted to determine if uterine up-regulation of trem in gestation is associated with an increased likelihood of responding to pathogens or severe as a protective mechanism. reduced plasma levels of vitamin d in caucasian women at term are associated with increased rate of infection. chander p arora, 1 adegoke adeniji, 1 susan e jackman, 1 babak forooghi, 1 isaac mostadim, 1 phillip yadegari, 1 calvin j hobel. 2 1 og-gyn, cedars-siani medical center, los angeles, ca, usa; 2 university of california los angeles, los angeles, ca, usa. background: vitamin d plays an important role in human pregnancy by acting as a regulator of immunity at the fetal-maternal interface. inflammatory changes associated with pro-inflammatory cytokines were reduced by vitamin d while anti-inflammatory cytokines were increased in t lymphocytes. vitamin d status has been defined as deficiency (<37.5nmol/l), insufficiency (37.5 to 80 nmol/l) and sufficiency (>80nmol/l) . objective: to assess the involvement of vitamin d in the occurrence of maternal infection during pregnacy in women with term deliveries. hypothesis: vitamin d metabolism could affect the rate of infection during pregnancy. study design plasma levels of 25(oh)d were determined by elisa and the rate of infection was recorded in a behavior in pregnancy study. in this study, 626 ethnically diverse women were evaluated at 18-20 weeks (t1), 28-30 weeks (t2) and 34-36weeks (t3). maternal infections were documented at each stage as well as at baseline visit with history of infection in current pregnancy. of these subjects who delivered at term two groups (58 with no infection during pregnancy, 101 with infection or history of infection) were matched further for non-smoking status, non-diabetics, ethnicity and maternal age. plasma from these were assayed for 25(oh)d and analyzed for the rate of maternal infection using fisher´s exact test or chi-square test. results although the women delivered at term, the levels of 25(oh)d in caucasians were significantly lower in the subjects with infection than the ones without (p<.001). women with vitamin d insufficiecy in the first trimester were more likely to develop infection during pregnancy (46.2 nmol/l ± 6.8 at t1, 28.4 nmol/l ± 4.1 at t2 and 30.2 nmol/l ± 3.6 at t3; all p<.001) but not subjects with sufficient vitamin d at t1(87.3 nmol/l ± 5.4 at t1, 27.9 nmol/l ± 3.8 at t2 and 32.8 nmol/l ± 4.6 at t3; all p<.001). conclusion the results reveal a positive association between 25(oh) d concentrations and greater risk of infection. vitamin d deficiency or even insufficiency may, therefore be involved in the pathogenesis of maternal infection during pregnancy. it is probable that vitamin d deficiency or even insufficiency could modulate the maternal susceptibility to infection during pregnancy by a proinflammatory mechanism. in vitro and in vivo observational data suggest that infection leads to caspase activation and apoptosis in the placenta and membranes, however currently there are no data on the role of apoptosis in the pathogenesis of infection associated preterm delivery. here we used group b streptococcus (gbs) as a model pathogen and examined the role of caspase dependent and independent apoptosis in preterm delivery. methods: we injected (7.5x10 8 ) heat killed group b streptococcus organisms (hk-gbs) intraperitoneally (i.p.) in 14.5 day pregnant c57b/l6 mice. the mice were euthanized at 5 hr (n=4) and 14 hr (n=6), the placentas and membranes were removed and assessed for apoptosis by tunel staining. caspase 3 activation and expression were determined by immuno-histochemistry (ih) and western blotting. the effect of apoptosis on preterm delivery was assessed by i.p. treating the pregnant mice with pbs (n=3), dmso (n=4) or pancaspase inhibitor z-vad-fmk (n=6) prior to hk-gbs and observing the animal for delivery for 48 hrs. results: there was a time dependent, gbs-induced increase in apoptosis by tunel assay and caspase 3 activation in the placenta and membranes. in addition hk-gbs-induced the expression of caspase 3 and caspase independent m-calpain in the placenta. z-vad-fmk (10 mg/kg), at the maximum concentration that did not induce maternal illness, did not prevent hk-gbsinduced preterm delivery. conclusions: caspase dependent and independent mechanisms are activated in the placenta upon exposure to gbs. systemic adminstration of a pan-caspase inhibitor did not impact upon the occurance or timing of bacterially induced preterm delivery. further studies are needed to assess the role of apoptosis in the pathogenesis of infection associated preterm delivery. early and 25 (3.2%) had a late sptb. the mean + sd gestational age at blood draw was 12 + 2 weeks. the median level of bb was higher in women with early as compared with late sptb or term births (p= 0.047 for trend). women with bb in the top quartile were 4.7 times more likely to have an early sptb as compared with women who had lower levels of bb (95% ci 1.5 to 14, p = 0.003). there was no association between bb and late sptb (rr= 0.8, 95% ci = 0.3 to 2). the adjusted or of an elevated bb for early sptb was 4.3 (95% ci = 1.3 to 14, p= 0.02). when the analysis was restricted to the 38 women with sptb the rr of an elevated bb for early sptb was 3.1 (95% ci 1.3 to 7.5, p = 0.03). conclusions: a significant relationship was found between an elevated bb in early pregnancy and early sptb suggesting inflammatory events in early pregnancy, perhaps infection-related, are part of the pathogenic mechanisms. objective: genital tract infection and/or inflammation appears to contribute to the majority of ptds preceding 30 weeks of gestation. ptd in humans has been associated with colonization and/or infection with a variety of different organisms including gram positive and negative bacteria, mycoplasma, ureaplasma, trichomonads, malaria parasites and viruses. the innate immune response to these pathogens is produced by a family of pattern-recognition cell membrane receptors known as the toll-like receptors (tlrs). these studies sought to characterize the tlr isoforms expressed in the preterm mouse uterus, and their modulation during lipopolysaccharide (lps)-induced ptd. methods: using sterile surgical technique, day-15 pregnant cd-1 mice underwent intrauterine injection of 250 g lps. subsequently, the mice were euthanized at 0, 2, 6, 12, 18 and 24 hours after lps injection. uterine tissue was harvested and placed in rnalater; subsequently total rna was isolated using the trizol reagent and genomic dna was removed using turbo dnafree. cdna was made using iscript cdna synthesis kit. pcr primers were designed using published mouse tlr gene sequences. real-time quantitative rt-pcr was performed using the power sybr green master mix and an abi prism 7000 multicycler. to confirm tlr amplicon sizes, the rt-pcr products were visualized on a 2% agarose/tbe gel stained with gelred. results: these studies have confirmed mrna expression of all 12 of the reported mouse tlr isoforms. these tlr amplicons range in size from 83 to 200 bp as expected; amplicon sequences are pending. quantitative rt-pcr studies performed using uterine tissues from five mice at each time point demonstrated that at 6 hours after lps injection, tlr3 increased 3-fold and tlr6 increased 2-fold (both p<0.05). in contrast, the expression of tlr4 (the ligand for lps) remained stable during the 24 hours after lps; the expression of tlr1, 5, 7, 8, 9, and 13 also remained stable. tlr2 expression trended upward and tlr11 trended downward, although neither was statistically significant. conclusions: these studies have confirmed expression of all 12 tlrs within the preterm pregnant mouse uterus, along with characterization of their modulation during lps-induced ptd. these observations are important because they contribute to our understanding of the immunologic signaling events leading to ptd. (funded by nih hd044747). udp-glucose and its receptor p2y14 as a new innate immune system in the female reproductive tract. toru arase, tetsuo maruyama, hiroshi uchida, takashi kajitani, masanori ono, maki kagami, hironori asada, yasunori yoshimura. obstetrics and gynecology, keio university, shinjukuku, tokyo, japan. objective: innate immune system involving toll-like receptors has recently emerged in the female reproductive tract (frt). we hypothesize that there may exist new other mucosal immunity in frt. recently, it has been reported that p2y14, a g protein-coupled p2y receptor for udp-glucose (udp-g), is involved in the lung epithelial immune system. the aim of this study is to investigate whether udp-g and p2y14 have a potential as the defense immune system in frt, in particular endometrium. materials and methods: we obtained human endometrial tissues from consenting reproductive-aged patients. the spatiotemporal expression of p2y14 in human and mouse endometrial tissues was analyzed using rt-pcr and ihc. isolated human endometrial cells and a human endometrial epithelial cell line, ishikawa, were cultured, treated with udp-g, and subjected to rt-pcr analysis for il-8 mrna expression. we also measured the il-8 secretion using elisa. small interfering rna was used to knock down p2y14 expression. the chemotactic activity of udp-g on neutrophils was tested using transwell assay with ishikawa cells. lastly, mouse uterine tissues were incubated with udp-g and subjected to rt-pcr analysis for mrna expressions of kc and mip-2, the il-8 homologues in mice. results: p2y14 was exclusively expressed in the glandular and luminal epithelium both in human and mouse uteri. treatment with udp-g induced the secretion of il-8 in ishikawa and human endometrial glandular cells, but not stromal cells, in a dose-and a time-dependent manner. p2y14 knockdown abrogated udp-g-induced il-8 production. treatment with udp-g also significantly increased the chemotaxis of neutrophils, which was attenuated by co-addition of anti-human il-8 neutralizing antibody. in the mouse uterus stimulation of udp-g significantly up-regulated the expressions of kc and mip-2 mrna. conclusions: udp-g is an endogenous molecule and released into the extracellular environment in a lytic manner after cell damage. taken together, our results collectively substantiate a model in which udp-g released from endometrial cells damaged by infection stimulates il-8 production via p2y14 in endometrial glandular epithelium, which, in turn, recruits neutrophils thereby preventing the progression of infection. thus, udp-g and its receptor p2y14 may be involved in the trans-species mucosal immune system in frt. (jsgi 2007; 14, 2(suppl) , abst #31) but in utero effects on fetal lung remain to be established. we have examined the relationship between duration of iai and subsequent azi treatment on the severity of fetal lung histopathology. we hypothesized that early treatment would prevent the development of advanced lesions, while late treatment may reduce the severity of lung damage. study design. thirteen chronically instrumented rhesus monkeys received intraamniotic inoculation of u. parvum (serovar 1; 7-14 x 10 7 cfu) at 130 ± 1.2 days gest. age (dga, mean ± sem, term=167 d). six of these animals received maternal i.v. azi (12.5mg/kg q12h or q6h for 10 d) either alone (n=3) or in combination (n=3) with dexamethasone (dex; 4mg/kg/d i.v. for 4d) and indomethacin (indo; 100mg/d p.o for 5d). tissues were obtained at cesarean section for histopathologic assessment. leukocytic infiltration of aveolar spaces and septal walls, type ii pneumocyte hyperplasia and peribronchiolar lymphocytic aggregates were scored as absent (0), minimal (1), moderate (2) and severe (3). results. inoculation-to-delivery interval was 18-25d for combined treatment groups and was similar to long duration infection without treatment (15-21d). treatment effects were tabulated as mean scores and compared as follows: control (n=3), score 1; short duration infection (3-8d; n=4), score 8; long duration infection (15-21d; n=3), score 12; short duration infection + treatment (n=5), score 4; long duration infection + treatment (n=1), score 5. conclusions. histopathologic findings of fetal pneumonia progressively worsen with duration of u. parvum iai. early maternal azi treatment prevents development of advanced lung lesions, while later treatment may reduce the severity of fetal lung damage. our results suggest that in utero treatment of iai may lower the risk of neonatal bronchopulmonary dysplasia. support: nih hd06159, rr00163. brain associated with adverse neurodevelopmental outcome. lps triggers proinflammatory responses through toll-like receptor-4 (tlr4). mitogen activated protein kinases including c-jun-n-terminal kinase (jnk) have been reported to be implicated in tlr4 signalling pathways and play important role in both onset of labor and brain injury. in the present study, we used a mouse model of intrauterine infection-associated preterm labor to determine whether the administration of specific inhibitor of jnk signaling, d-jnk inhibitory peptide (d-jnki) can (i) inhibit jnk activity in vivo, (ii) delay lps-induced preterm delivery, and (iii) improve neonatal outcome in the presence of intrauterine inflammation. intrauterine administration of tlr-4 specific lps to cd1 pregnant mice at 16 day of pregnancy caused preterm delivery after 18 to 24 h with 70% pup mortality. in vitro kinase assay demonstrated the activation of jnk in response to lps in the maternal uterus and fetal brain. furthermore, the brain specific jnk3 was found to be the major jnk isoform activated by lps in the fetal brain. co-administration of d-jnki with lps to pregnant mice delayed significantly (p<0.007) lps-induced preterm delivery and reduced pup mortality up to 15%. this was associated with inhibition of jnk activity in both maternal uterus and fetal brain. in addition, we have found that treatment with lps significantly up-regulated cox-2, cxcl1 (il-8 equivalent) and ccl2 in myometrium and this is significantly suppressed after co-administration of d-jnki. we conclude that specific inhibition of jnk signaling may have a potential of controlling preterm labor and preventing fetal brain damage as a result of infection/inflammation. the prostaglandin 15-deoxy-12,14 -prostaglandin j 2 delays lps-induced preterm delivery and reduces mortality in the mouse. intrauterine infection is a common trigger for preterm birth, and is also a risk factor for the development of neurodevelopmental abnormalities in the neonate. bacterial lipopolysaccharide (lps) binds to toll-like receptor-4 (tlr4) to activate pro-inflammatory signaling pathways. the transcription factor nuclear factor kappa b (nf-kb) is a key player in the orchestration of the inflammatory response and has a central role in parturition. we have previously shown that exposure to the anti-inflammatory cyclopentenone prostaglandin 15-deoxy-12,14 -prostaglandin j 2 (15d-pgj 2 ) inhibits il-1b-induced nf-kb activity and cox-2 expression in human myometrial and amnion epithelial cells in vitro. in the present study, we used a mouse model of intrauterine infection-associated preterm labor to determine whether the administration of 15d-pgj 2 can (i) inhibit nf-kb in vivo, (ii) delay lps-induced preterm delivery, and (iii) improve neonatal outcome in the presence of intrauterine inflammation. intrauterine administration of tlr4 specific lps to cd1 pregnant mice at 16 day of pregnancy caused preterm delivery after 18 to 24 h with 70% pup mortality. co-administration of 15d-pgj 2 with lps to pregnant mice delayed significantly (p<0.008) lps-induced preterm delivery and conferred protection from lps-induced fetal mortality up to 5%. we have looked at the expression profile of several labor associated genes in myometrium 6 hours after lps administration. (otr, connexin 23 and 46, cox-1, cox-2, cxcl1 (il-8 equivalent) and ccl2). we have found that treatment with lps significantly up-regulated cox-2, cxcl1 and ccl2 and this is significantly suppressed after with co-administration of 15d-pgj 2. western analysis for ser536-phosphorylated p65 and ikkb in-vitro kinase assay has demonstrated that lps induced activation of nf-kb at both 1 h and 6 h. co-administration of 15d-pgj 2 was associated with inhibition of nf-kb activation in both the maternal uterus and the fetal brain. conclusion 15d-pgj 2 may have potential as a therapeutic agent in the management of preterm labor and, by targeting the player nf-kb, may have the added advantage of preventing detrimental effects to the fetus that may result from infection/inflammation. synergistic macrophage response to co-activation of tlr-2 and tlr-3: mechanisms and implications for bacterial/viral co-infection. vladimir ilievski, 1 emmet hirsch. 1,2 1 department of ob/gyn, evanston northwestern healthcare, evanston, il; 2 department of ob/gyn, feinberg school of medicine, northwestern university, chicago, il. background: toll-like receptors (tlrs) recognize structural components of pathogens and initiate host defenses. tlr-2 responds to gram-positive organisms and peptidoglycan (pgn), a gram-positive cell wall constituent. tlr-3 is activated by viral infection in response to double-stranded rna. polyinosinic-cytidylic acid (poly(i:c)) is a tlr-3 ligand. we have shown that pgn and poly(i:c) have a synergistic effect on the expression of downstream genes for both tlr-2 and tlr-3. here we identify mechanisms underlying this synergy. methods: mouse peritoneal macrophages or a mouse macrophage cell line (raw 264.7) were treated in tissue culture with either pgn (1 g/ml), poly(i: c) (10 g/ml) or both pgn and poly(i:c) either simultaneously or sequentially for 5-10 hours. total rna was extracted and duplex rt-pcr was performed for inducible nitric oxide synthase (inos), interleukin 1 (il-1 ), tumor necrosis factor (tnf), the chemokine rantes and tlr-2, normalized to the housekeeping gene gapdh. results: compared to stimulation with either pgn or poly(i:c) alone, costimulation of raw 264.7 cells with both pgn and poly(i:c) resulted in synergistic expression of inos, il-1 , tnf and rantes (p<0.05 for all) at 5 and 10 hours . sequential stimulation with either pgn or poly(i:c) for 5h followed by incubation for an additional 5h with the alternate ligand also induced synergistic expression of the same rnas, albeit at lower levels than were elicited by simultaneous stimulation. in contrast, incubation with either pgn or poly(i:c) for 5h followed by medium for 5h induced minimal to no gene expression. both pgn and poly(i:c) induced tlr-2 mrna after 5h but not 10h. tlr-3 mrna was not detectable by rt-pcr. in primary peritoneal macrophages, similar synergy due to pgn and poly(i:c) was seen. conclusions: simultaneous or sequential exposure to pgn and poly(i:c) exerts a synergistic effect on the expression of inflammatory mediators in macrophages. interestingly, either one of these two tlr pathways can prime cells for activation of the other pathway. a possible mechanism for this effect may be induction of tlr-2 by either tlr-2 or tlr-3 activation. these observations have implications for bacterial/viral co-infection. this is a secondary analysis of a prospective cohort study. after irb approval, daily blood samples were obtained from pprom subjects and analyzed for il-6 by elisa. paired maternal serum il-6 levels from 34 subjects were divided into 2 groups: il-6 levels obtained 12-120 hours prior to completion of antibiotics and those obtained 12-120 hours after completion of antibiotics. the wilcoxon signed rank test was used to perform the data comparison on the analyze-it statistical software program. statistical significance was defined as p<0.05. of the 34 pprom subjects, the maternal age was 26.8 yrs; gestational age at admission was 26.7 weeks; latency was 13.1 days; gestational age at delivery was 30 weeks; and infant birth weight was 1409 grams. median maternal serum il-6 levels obtained off antibiotics were significantly higher when compared to those on antibiotics (4.9 vs. 2.7 pg/ml, p<0.02). the results of this investigation suggest that maternal serum il-6 levels rise after discontinuation of antibiotics. the optimal duration of antibiotics administration in the setting of pprom is unknown. this data suggests a role for continuation of antibiotics in women with pprom in order to prolong the latency period and potentially decrease neonatal morbidity. to identify clinical and pathologic factors associated with fetal inflammatory responses in the placenta from term parturients. methods: retrospective cohort study of consecutive term parturients with submitted placentas in 2005. placentas with histologic chorioamnionitis were divided into two cohorts: group 1-maternal inflammatory responses only, and group 2-maternal and fetal inflammatory responses. maternal and fetal inflammatory responses in the placenta were classified according to guidelines established by the amniotic fluid infection nosology committee of the perinatal section of the society of pediatric pathologists. selected demographic, intrapartum, newborn and placental characteristics were analyzed with t-tests and chi-square tests as appropriate. multiple logistic regression was used to identify independent predictors of fetal inflammatory responses in the placenta. results: of 351 consecutively submitted placentas, 210 had histologic chorioamnionitis: 155 with maternal inflammatory responses only (group 1) and 55 with both maternal and fetal inflammatory responses (group 2). fetal inflammatory responses observed in group 2 were associated with higher stages of maternal inflammatory responses (p<.001). 80% of fetal inflammatory responses were stage i (acute chorionic vasculitis or phlebitis).group 2 patients were more likely to have had amnioinfusion (29% v 13%, p=.006) and less likely induction of labor (22% v 39%, p=.02). group 2 was more likely to have had intrapartum fever (56% v 40%, p=.04) and maternal tachycardia (58% v 39%, p=.02). newborns from group 2 were more likely to have been observed for sepsis (69% v 37%, p <.001) and have an apgar score 6 at 5 minutes (7% v 1%, p=.02). a logistic regression model showed that stage ii or greater maternal inflammatory responses (or 6.3) and amnioinfusion (or 2.9) were independent predictors of fetal inflammatory responses. conclusion: higher stages of maternal inflammatory responses in the placenta and amnioinfusion were independent predictors of fetal inflammatory responses in term parturients with histologic chorioamnionitis. objective: previous studies have shown that sulfasalazaine (sasp) inhibits lipopolysaccharide (lps)-induced nf-b activation and decreases lpsstimulated interleukin-8 (il-8) production by cultured explants of placenta, amnion and choriodecidua with no effect on cell viability. bacteria-induced il-8 production in the cervix is a potential mechanism for premature cervical ripening that may lead to preterm birth. it is unclear, however, whether sasp can inhibit il-8 production by endocervical cells. therefore, we performed a series of in vitro studies to determine if sasp inhibits il-8 production by endocervical epithelial cells stimulated with bacterial pathogens associated with preterm birth. methods: human endocervical epithelial cells were incubated with 0-1.6 mm sasp overnight and then stimulated with 9 ccu/ml ureaplasma parvum, 10 8 cfu/ml escherichia coli, or 2 x 10 6 cfu/ml gardnerella vaginalis for another overnight incubation in 96-well cultures. conditioned medium was then harvested and production of il-8 was quantified by elisa. viability of the cells was ascertained at the end of the experiment with the mtt-assay. each treatment was applied in quadruplicate wells and experiments were repeated 3 times using different batches of cells for each pathogen. results were evaluated using the general linear models procedure of sas for a randomized block design. results: sasp had no detectible effect on il-8 production by endocervical cells not treated with bacteria. at the highest concentration tested (1.6 mm), sasp significantly inhibited il-8 production by cultures stimulated with e. coli (p<0.001), u. parvum (p<0.001), and g. vaginalis (p<0.001). viability of the cells, however, was significantly reduced by sasp at 0.8 and 1.6 mm in the both the presence and absence of bacteria for all experiments. conclusion: although high concentrations of sasp inhibit il-8 production by cultures of endocervical cells stimulated with pathogens associated with preterm birth, this effect may be due to toxicity of the antibiotic on the cells. the effect of 17 -hydroxyprogesterone caproate on lipopolysaccharide stimulated peripheral blood mononuclear cells from pregnant women. richard h lee, aimin li, frank z stanczyk, jinwen i lin, t murphy goodwin. obstetrics and gynecology, university of southern california, los angeles, ca, usa. introduction: 17 -hydroxyprogesterone caproate (17-ohpc) reduces the rate of recurrent preterm birth in high-risk women. however, there are lines of evidence to suggest that 17-ohpc alters inflammatory response in the setting of gram-negative infection. in a mouse model, 17-ohpc decreased the rate of preterm birth but was associated with significantly increased maternal morbidity when mice were exposed to lipopolysaccharide (lps). in non-pregnant women, 17-ohpc pre-treatment of whole blood exposed to lps significantly increased the production of tnf-. objective: to determine if 17-ohpc has an effect on the production of proinflammatory cytokines from peripheral blood mononuclear cells (pbmc) in pregnant women. methods: pbmc were isolated from fresh whole blood samples of 10 pregnant women between 16 and 24 weeks. pregnant women had no prior history of preterm birth. cells were treated with 17-ohpc (1 m) and escherichia coli lipopolysaccharide (lps, 1 g/ml) alone or in combination. after 6 and 24 hours of culture, supernatants were collected and tested for tnf-and il-6 levels by enzyme-linked immunosorbent assay (elisa). statistical analysis was performed using non-parametric tests. p < 0.05 was considered significant. results: pbmc exposed to lps significantly increased tnf-and il-6 secretion compared to untreated controls (p=0.008 and p=0.008). tnf-concentrations were not significantly different between lps and lps+17-ohpc treated cells at both 6 and 24 hours (p=0.86 and p=0.17). il-6 production was significantly increased after 6-hour treatment with lps+17-ohpc compared to lps alone (2,730 [2,150-5,090] background. recent clinical trials indicate that progestins reduce the incidence of preterm birth in some high risk pregnancies. it has been proposed that progesterone promotes uterine quiescence, in part, via its antiinflammatory properties with inhibition of pro-inflammatory gene expression. it is intriguing that progestins are clinically effective given the considerably increased background circulating levels of the hormone during pregnancy. we hypothesised that non-classical progesterone signalling pathways contribute towards mediation of the anti inflammatory effects of progestins. to the endotoxin lipopolysaccharide (lps) by activation of inflammatory pathways. myometrial cell cultures were treated with lps (1 g/ml)+/progestin (50nm). two progestin compounds were investigated. natural progesterone (p) is known to have a strong affinity with progesterone receptor (pr) analogues in contrast to 17-hydroxyprogesterone (17-hp) which, in the absence of esterification with caproate or acetate, has been reported to have no progestogenic activity at pr. the effect of p and 17-hp on the activation of two inflammatory genes known to be associated with labour (cycloxygenase 2 [cox-2], toll-like receptor 4 [tlr-4]) was evaluated. cox-2 and tlr-4 were detected at the protein and mrna levels using sds-page and rt-pcr. results. lps-induced expression of cox-2 and tlr-4 proteins were significantly inhibited by both p (p<0.01 and p<0.05, respectively) and 17-hp (p<0.01 and p<0.05, respectively). furthermore, preliminary results indicate that co-incubation with the anti-progesterone mifepristone, fail to abrogate the anti inflammatory effect associated with progestin treatment. conclusion. non-classical progesterone signalling pathways have a role in mediating the anti-inflammatory properties of progestins. further elucidation of the molecular actions of progestins may allow novel approaches to ameliorate the inflammatory response associated with preterm labour. detection and transcriptional expression of tlr-2, tlr-4 and tlr-9 at the maternal-fetal interface. jacqueline p tilak, karen zakharian, alexandra tungol, gabriel o schubiner, david m svinarich. patient care research, providence hospital, southfield, mi, usa. preterm labor and delivery remains a leading cause of neonatal morbidity and mortality and bacterial infection is considered to be the most common etiology. toll-like receptors (tlr's) and the associated components of the innate immune system may represent a first line of defense against these pathogens. tlr's belong to a family of pattern-recognition receptors that bind to highly conserved pathogen-associated molecular patterns (pamps) including lipopolysaccharide (lps), lipotechoic acid (lta) and cpg dna, and are a key component of the innate immune system. this study was undertaken to characterize the transcriptional expression and regulation of tlr-2, tlr-4 and tlr-9 within gynecologic and gestational tissues. human first trimester trophoblasts, endometrial mesoderm, ectocervix, choriocarcinoma and neonatal epithelium, were cultured in media alone or in the presence of either lps (1mg/ml) or lta (10 mg/ml) for 0, 2, 4, 6, 8 and 24 hours. total rna was isolated and semi-quantitative rt-pcr was performed using intron-spanning amplimers to tlr-2, tlr-4, tlr-9 and gapdh. following gel electrophoresis, the integrated optical densities were determined for the corresponding pcr products and normalized with respect to gapdh levels. inducible transcription of tlr-2 with lta was observed in choriocarcinoma cells (6-fold, 2h), and endometrial mesoderm cells (19-fold, 4h). tlr-4 induction with lps was observed in ectocervical cells (4-fold, 4h), choriocarcinoma cells (4-fold, 4h) and endometrial mesoderm cells (7-fold, 24h). tlr-9 induction with lps was observed in choriocarcinoma cells (32fold, 4h) and neonatal epithelial cells (11-fold, 4h). all cell lines showed at least constitutive levels of transcription for tlr-2, tlr-4 and tlr-9. these data demonstrate that tlr-2, tlr-4 and tlr-9 are transcriptionally expressed either constitutively or inducibly in both gynecologic (endometrial mesoderm, ectocervix) and gestational (chorion, trophoblast), tissues. translation of these receptors suggests that the innate immune system may function at the maternal-fetal interface to help protect the fetus against infection and prevent or diminish the likelihood of prematurity. objective: further to our development of a sheep model of intrauterine ureaplasma spp infection, we aimed to examine the capability of ureaplasma colonization in the amniotic fluid to infect the fetus and alter lung and brain development. methods: at 50 days of gestation (d, term=150 d) ewes bearing single fetuses were given a single ultrasound-guided intra-amniotic injection of (a) 2x10 7 colony forming units (cfu) of u parvum (serovar 3, n=7; serovar 6, n=8), (b) 2x10 4 cfu of u parvum (serovar 3, n=6; serovar 6, n=8) or (c) media control (n=6). at 125 d, fetuses were delivered by cesarean section. amniotic fluid and umbilical arterial blood samples were collected. fetal body weight was recorded, fetal cerebrospinal fluid (csf) collected and a descending pressurevolume curve constructed after inflation of the lungs to 40 cmh 2 o. fetal membranes were immersion fixed and the fetal brain was perfusion fixed with 4% paraformaldehyde. the fetal brain and membranes were blocked, paraffin embedded, stained and viewed under the light microscope. results: chronic intra-amniotic colonisation with u parvum serovar 3 or 6 (ureaplasmas) did not result in fetal abortion or death. amniotic fluid ureaplasma titers at delivery were not dose-dependent. chronic ureaplasma exposure did not affect fetal body or brain weights, or result in a fetal systemic inflammatory response. umbilical arterial blood gases at delivery were similar between ureaplasma-and media-exposed fetuses. chronic intra-amniotic exposure to ureaplasmas resulted in higher inflammatory cell scores in the fetal membranes compared to media controls (p<0.05). lung compliance was increased in ureaplasma-exposed fetuses compared to controls (p<0.05). there were no gross anatomical changes observed in the white or grey matter in the cerebral hemispheres of fetuses that had been exposed to ureaplasmas; even in animals (n=3) that had csf ureaplasma colonisation. conclusion: chronic ureaplasma colonisation enhances fetal lung compliance without gross anatomical changes in the fetal brain. background: an important source of vitamin d is its synthesis by skin from uv solar radiations. the skin pigment melanin absorbs uv photons thus reducing the vitamin d synthesis by more than 90% making african americans at high risk for vitamin d deficiency. low maternal vitamin d status during pregnancy has been linked to molecular pathways for adverse outcomes. objective: the nf b transcription factor regulates genes involved in inflammation and immune processes, and is proposed to play a major role in the successful outcome of pregnancy and labor. prior immunohistochemical (ihc) and biochemical studies have been conflicting regarding nf b activation in human intrauterine tissues. the aim of this study was to quantify nuclear localization of p65, the major tranactivating nf b subunit, in full-thickness fetal membranes (fm) and myometrium using ihc. methods: a retrospective analysis of paired fm, decidua, and myometrial samples was conducted using tissues collected from women in 4 cohorts: preterm no labor (pnl, n=20), preterm labor (ptl, n=20), spontaneous term labor (stl, n=19), and term no labor (tnl, n=22). subjects were delivered between gestational ages 26-36 weeks (preterm) and 37-44 weeks (term) by cesarean. paraffin sections were stained with polyclonal (rabbit) anti-human p65 and microscopically examined. myometrial samples were categorized in a blinded fashion to 3 groups of staining: no nuclear p65 (-), rare nuclear p65 (+), and >50% nuclear p65 (++). a p65 nuclear labeling index (nli; % nuclear p65/total cells) was calculated for each histological layer in full-thickness fm specimens using a blinded targeted randomization scheme consisting of 5 non-overlapping low-magnification fields. results: nuclear p65 labeling was rare in amnion and inconsistently present in chorion. in decidua, p65 nuclear labeling was observed in all cases; however, decidual nli did not vary significantly across cohorts [pnl-40% (24-55%); ptl-45% (39-57%); tnl-37% (25-43%); stl-41% (30-59%); all values are median (iqr)]. in myometrium, ++ p65 nuclear labeling was significantly associated with labor, but present only in a portion of cases (ptl-26%; stl-50%). despite a trend, decidual nli was not significantly correlated with myometrial nuclear p65 labeling: myometrial specimens classified as -, +, and ++ corresponded with decidual nli of 40% (32-55%) [median (iqr)], 37% (28-47%), and 56% (40-58%), respectively. conclusions: in a comprehensive ihc analysis, significant nuclear p65 immunolabeling was observed in myometrial cells following labor. nuclear p65 labeling in decidua was present in all cases, and did not vary significantly among the cohorts. the inflammatory cytokines interleukin-1 and tnf-increase g-csf expression in term decidual cells. objectives: chorioamnionitis (cam) elicits premature rupture of the membranes and pre-term delivery. previously, we found that the decidua from cam patients contains much higher neutrophil numbers than control decidua, but macrophage numbers are similar in both groups. granulocyte colony-stimulating factor (g-csf) enhances granulocyte colony formation chemoattraction and activation. the amniotic fluid during cam contains elevated tnf-and il-1 levels. to account for the marked influx of neutrophils infiltration in cam-complicated decidua, tnf-and il-effects were assessed on g-csf expression in term decidual cell (dc) monolayers. methods: confluent leukocyte-free term dcs from normal term deliveries were primed with 10 -8 m estradiol (e2) + 10 -7 m medroxyprogesterone acetate (mpa) for 7 days, then switched to serum-free defined medium (dm) with steroids ± tnf-or il-1 . after 24h, aliquots of conditioned dm supernatants, cell lysates and extracted rna from 6h parallel incubations were frozen. secreted g-csf was measured by elisa in conditioned dm and normalized to cell protein and mrna was assessed by quantitative real time rt-pcr and normalized to -actin mrna. results: in cultured first trimester dcs, basal g-csf levels in conditioned dm were 0.13± 0.06 pg/ml/ug cell protein [mean ± sem, n=8] and did not differ from e2+mpa basal levels. the addition of 1ng/ml of tnf-or il-1 significantly elevated g-csf output to 2.22 ± 0.97 and 454 ± 122 respectively (p<0.05), representing more than a 10-fold and 4,000-fold change; respectively. western blotting confirmed the elisa results. quantitative rt-pcr demonstrated corresponding changes in g-csf mrna levels as found for the elisa measurements. concentration-dependent effects for both tnfand il-1 from 0.01 to 10.0 ng/ml were observed. conclusions: when extrapolated to the placental milieu of cam, the marked increase in g-csf elicited in term dcs by tnf-and il-1 may provide a mechanism to account for the selective increase in decidual neutrophils versus macrophages. background. the immunological mechanisms of the female reproductive tract are unclear. toll-like receptors (tlrs), innate immune receptors that combat microbial infections and establish adaptive immunity, may play a role. infection in pregnancy has been associated with preterm birth and tlrs may modulate the host response to such infections. we hypothesised that the distribution of tlr2 and tlr4 in cervical epithelial cells may differ with pregnancy, and that oestrogen may contribute to the modulation of these receptors. aims and objectives. 3. to compare tlr2 and tlr4 protein expression, using immunocytochemistry, in the cervical epithelium of pre-menopausal non-pregnant women with pregnant women at term. methods. fresh non-pregnant (n=18) and pregnant (n=11) human cervical cells were obtained by smear and tlr2 and tlr4 expression investigated by immunocytochemistry. cervical epithelial cells from nonpregnant women obtained by smears were then coincubated with varying concentrations of estradiol, and tlr2 and tlr4 protein expression quantified by immunocytochemistry. results. using pixel-based image analysis software, we demonstrated a reduction in tlr2 expression in late pregnant compared with non-pregnant cervical epithelium (p=0.0001), whilst tlr4 did not appear to differ. there was an apparent up-regulation of tlr2 protein expression in response to 17beta-oestradiol (n=5) objective: to evaluate in vitro antimicrobial activity of cranberry-exposed urine against common urinary pathogens. subjects were pregnant women enrolled in a clinical trial evaluating the effect of daily cranberry juice cocktail or matching placebo on asymptomatic bacteriuria and other urinary tract infections. methods: 4-hour urine samples from 28 pregnant women who were randomized to cranberry or placebo in three treatment arms: a. cranberry two times daily with meals (c, c; n=10), b: cranberry in the am, then placebo at dinner (c, p; n=10), c: placebo two times daily with meals (p, p; n=8). we identified 15 non-pregnant, reproductive-aged controls, randomized them to the same treatment groups and collected 4-hour urine specimens from them. the ph of all urine specimens was adjusted to ph=7 and filtered. aliquots of each were independently inoculated with overnight culture of 10 2-3 cell/ml each of single strains of e. coli with fimbriae type i and type ii, k. pneumoniae, and c. albicans. after 24 hours of incubation for e. coli and k. pneumoniae, and 48 hours for c. albicans cfu/ml of each specimen were enumerated by subculture with quantitative plate counts in duplicate. results: there were no differences for any of the antimicrobial activities between pregnant and non-pregnant groups, or based on treatment allocation. we were able to perform antimicrobial assays on the urine of women exposed to cranberry juice or placebo, but unable to demonstrate differences based on treatment allocation or pregnancy. this may be due to beta-error. further studies are planned. supported by r21dk65827-01; clinical trials registration nct00506025. ...,.. e. coli 1.2x 10 8 .:!: 8.5x 10 8 1.2 x 10 8 .:!: 3.0 x 10 8 group a (c, c) group b ( c, p) 2.3x 10 8 + 2.7x 10 8 9.4 x 1 0 7 + 4.8 x 10 8 .33 .81 .83 group c (p, p) 1.1 x 1o"::!>s x 1o" 1.1 x 10 8~ 1.ox:1o• k. ~neumoniae group a ( c, c) 5.3 x 10 7 + 3.4 x 10 7 9.2 x 10 7 + 6.7 x 10 7 .88 .87 .91 group b (c, p) 6.0 x 10 7 + 4.8 x 10 7 6.3 x 10 7 + 3.0 x 10 7 group c (p, pl 4.7 x 10 7~2 .3x 10 7 6.9x 10 7~4 .6x 10 7 c. al bicans group a (c, c) 1.1 x 10 '+1.8x 10 7 1.9 x 10 8 + 1.0 x 10 8 group b (c, p) 1.5x 1o•+ 1.0x 10 7 2.4 x 10 8 + 1.1 x 10 6 .75 . 87 .60 group c (p, p) 4.7x 10 7~2 .3x 10 7 2.1 x 1 0 7~5 .9 x 10• objective: to measure compliance, we sought to evaluate the use of a bioassay for cranberry in the urine of women enrolled in a clinical trial designed to determine the effect of daily dosing of cranberry juice cocktail or matching placebo on the incidence of asymptomatic bacteriuria (asb) and other urinary tract infections (uti) during pregnancy. methods: we collected 4-hour urine specimens from 34 pregnant women who were randomized to ingest cranberry or placebo in three treatment arms: a: cranberry two times daily with meals (n=11), b: cranberry in the am, then placebo at dinner (n=12), c. placebo two times daily with meals (n=11). we identified 14 non-pregnant, reproductive-aged controls, randomized them to the same treatment regimens (group a: n=6, group b: n=3, group c: n=5), and collected 4-hour urine specimens from them. bacterial anti-adhesion effects of the cranberry-exposed urine were evaluated utilizing a human red blood cell hemagglutination assay specific for uropathogenic p-fimbriated e. coli. activity was quantified as 0, 50, and 100%. results: when combining all subjects and dosing regimens, the sensitivity of the assay was 25%, range 17% in the pregnant group assigned single daily dose of cranberry to 100% in the non-pregnant group assigned to multiple daily doses. the specificity ranged from 63% to 100%. conclusions: these data indicate that the bioassay can be applied to the pregnant patient population, although the sensitivity of the assay is variable. higher daily dosing appears to confer greater sensitivity. further studies are needed to evaluate the utility of this bioassay for compliance. supported by r21dk65827-01. clinical trials registration nct00093938. objective: increasing evidence suggests that inflammation plays a crucial role in initiation of labour both at term and preterm. we have previously shown upregulation of pro-inflammatory cytokines in myometrium, cervix and membranes at term labour. we have also shown that myometrium and cervix is invaded by leukocytes at the time of labour, and these leukocytes express pro-inflammatory cytokines. in this study, we aimed to test the hypothesis that pro-inflammatory cytokines and toll-like receptor 2 and 4 (tlr2 and 4) mrna expression are up-regulated in circulating leukocytes during term and preterm labour. methods: peripheral blood samples were taken from 37 pregnant women: 28-36 weeks gestation (w) preterm not in labour (ptnl) n=10; 28-36 w preterm in labour (ptl), n=7; 37-42 w term not in labour (tnl) n=10; and 37-42 w term in labour (tl) n=10. leukocytes were isolated using dextran sedimentation. total rna was isolated and reverse transcribed using high capacity cdna archive kit, and mrna expression determined by real-time pcr (abi, taqman). student's t-test was used to compare outcomes between groups. results: messenger rna expression of il-8, icam-1, mcp-1, tlr2 and tlr4 was significantly increased in tl compared to gestation matched tnl. the expression levels of il-1b, il-8 and tlr-2 were significantly greater in ptl compared with gestation matched ptnl. (table i) . conclusions: we show up-regulation of pro-inflammatory cytokine production in circulating leukocytes in both term and preterm labour. thus, the pathophysiology of labour seems to involve the upregulation of proinflammatory cytokine production in peripheral blood leukocytes, followed by their invasion into the myometrium and cervix. this work further contributes to our understanding of the pathophysiology of parturition, and suggests that peripheral blood leukocytes may be potential targets for therapeutic agents aimed at modifying the time course of parturition. introduction. the mechanisms of innate immunity and tolerance in the female reproductive tract (frt) are ill-understood but involve the pattern recognition toll-like receptors (tlrs). we have previously demonstrated high expression levels of tlr2 gene and protein in fresh human cervical epithelium. aims. in order to gain insight into the immunological role of cervical epithelial cells (cecs), we sought to determine cec responses to the following tlr-2 agonists: macrophage-activating lipopeptide (malp-2), pam 3 csk 4 , and peptidoglycan. methods. cecs were isolated by smears and compared between 7 pregnant (3 rd trimester) and 7 nonpregnant women. following cell preparation, flow cytometry was performed using a direct staining procedure with mouse anti-human tlr2 primary antibody and its igg 1, isotype control, to determine tlr-2 protein expression. a further 7 nonpregnant smear samples were each prepared, and seeded at a concentration of 100,000 cervical epithelial cells/ml into 24-well cell plates. cells were incubated at 37°c in 5% co 2 overnight with the tlr2 agonists malp-2 and pam 3 csk 4 (at concentrations of 10 and 1000ng/ml), peptidoglycan(50ng/ml), il-1 (10ng/ml, positive control) and rpmi 1640 + 1-glutamine media only (vehicle). following centrifugation, all resulting supernatants were stored at -80°c until the concentration of il-8 was determined by elisa and an array of cytokines by bead assays. results. both pregnant and nonpregnant cecs expressed tlr2 (specific mean fluorescence 4.7 vs 3.3 respectively) but did not differ (p = 0.2). unstimulated cells incubated with buffer alone demonstrated high il-8 levels (6602 pg/ml), which did not differ following stimulation with malp-2 (6217 pg/ml), pam 3 csk 4 (6449 pg/ml) or peptidoglycan (4867 pg/ml). results of an array of pro-and anti-inflammatory cytokines following incubation of cells stimulated with tlr2 agonists are pending. conclusion. high basal il-8 levels suggest constitutive expression by cecs but the physiological relevance of this intriguing observation is unclear. that cec stimulation with tlr-2 agonists did not lead to further release of il-8 may epitomise the resistance of these cells to antigenic challenge by the vaginal commensal flora. cecs may play a pivotal role in modulating the immunological tolerance necessary to minimise inappropriate inflammation in the cervix. there are several epidemiological and clinical studies that omega-3 fatty acids and related fish oils can decrease the premature labor and birth. however, the scientific mechanisms are not well elucidated. this study was carried out to investigate the effects of omega-3 fatty acids on producing pge2 and il-6, in human umbilical vascular endothelial cell(huvec) media with artificial inflammation as an infection-induced premature labor tissue model. also, if there are some significant effects with omega-3 fatty acids, we want to investigate the possible mechanisms. materials and methods; human umbilical vascular vascular cell primary culture was done. in the adequate cell confluence in each cell dish, pretreatment with various concentrations of epa, dha and troglitazone (another ppar-ligand) and incubation were done for 24 hours in 37°c, co 2 incubator. after that, 10 g/ml conc. of lipopolysaccharide(lps) was treated to the each cell dishes and incubated for 8 hours. the cell media were collected, and pge 2 and il-6 concentration were checked by elisa. the each cell lysates were collected and western blot anaysis for cyclooxygenase(cox)-2 were done. on the other hand, nuclear factor kappa b (nf b) luciferase vector was transfected and then did the same pretreatment with epa, dha and troglitazone and lps treatment to each cell dishes. after that, nf b luciferase activity was checked with luminometer. statistical analysis was done by student t-test. results: epa, dha and troglitazone decreased the pge2 (p<0.05) and il-6(p<0.01) significantly in elisa. cox-2 expression revealed the significant reduction with pretreatment of epa, dha and troglitazone in higher concentration (50, 100 m) in the western blotting (p<0.01). nf b luciferase activity were also significantly decresed with pretreatment of epa, dha and troglitazone in higher concentration (p<0.01). conclusion; this study offers some scientific mechanisms, by which omega-3 fatty acids (epa, dha) and troglitazone may be one kind of the preventive medicine for infection-induced preterm labor and delivery. also, these effects may come from the common mechanism of epa, dha and troglitazone, ppar-ligands. the next study would be how the cox-2, nf b and pparare related. (2mg/ml). cytokine expression was measured in medium using the bio-plex suspension array system (bio-rad). mean expression was compared between the two groups using t test. results: 2-aminopurine significantly inhibited lps stimulated cytokine production by human myometrium. in contrast, there were no significant differences in expression after mpa treatment, although a trend towards inhibition of proinflammatory cytokine and an increase in il-10 production was noted. introduction: intrauterine infection is now recognised as a major antecedent of white matter injury (wmi) in the preterm infant brain, which can manifest later as cerebral palsy or as varying degrees of cognitive dysfunction. wmi in these infants is characterized by damage to immature oligodendrocytes, and has been linked both to microglial activation and to elevated levels of tnfa, il-1b and il-6. we have developed a fetal sheep model for diffuse and focal wmi, based on repeated administration of e. coli lipopolysaccharide (lps) as the stimulus for an inflammatory response. methods: surgery to implant fetal vascular catheters was performed on pregnant ewes carrying single fetuses at d89-90 of gestation. fetuses received repeated iv injections of lps (100ng/kg estimated fetal weight/day) between d95 and d99. plasma levels of pro-inflammatory cytokines were determined in fetal arterial blood samples taken between d94 and d104. at d105 ewes and fetuses were euthanized and fetal brain tissue samples collected for histological analysis. results: five days after final administration of lps to fetuses we observed a pattern of wmi similar to that seen clinically, ranging from focal to diffuse injury within the periventricular region, impairment of white matter (cnpase; marker for immature/mature oligodendrocytes) tracts, and thinning of the corpus callosum, characterised by oligodendrocyte disruption and microglial proliferation in the surrounding and sub-cortical white matter. we also found a progressive rise in fetal plasma tnf levels between days 97 and 103 (day two of treatment to third day following final dose of lps). background: plasma fatty acid (fa) levels are associated with altered host inflammatory responses, blood pressure regulation, and insulin resistance, characteristic features of pregnancy-induced hypertension (pih). most studies compare the n-3 and n-6 polyunsaturated fatty acids (pufas). in addition, recent data demonstrate that saturated and trans-fas promote inflammation. based on the immunomodulatory activity of various fas, we explored their effects on placenta inflammatory responses ex vivo. methods: placenta cells were isolated from fresh human (anonymous), term placentas and treated with/without lipopolysaccharide (lps) with various fas, including saturated fas, trans-fas, and n-3 and n-6 pufas (at physiological concentrations). after an overnight treatment, tnf-alpha (tnf) production was determined. data were analyzed by analysis of variance (anova) followed by the dunnett's test. results: oleic acid (18:1n-9), a cis-monosaturated fa common in the mediterranean diet, did not induce significant placenta tnf production (fig. a) . by contrast, elaidic acid (18:1n-9), a trans-monosaturated fa, induced tnf production by 300-fold compared to vehicle (fig. a) . likewise, palmitic acid (18:0), a saturated fa, induced placenta tnf production by 800-fold (fig. a) . to mimic inflammation, placenta cells were treated with lps ex vivo in the presence/absence of fas. docosahexanoic acid (22:6n-3, dha) reduced placenta tnf production by up to 90% following stimulation (fig. b) . similarly, treatment of placenta cells with linoleic acid (18:2n-6, la) or arachidonic acid (n20:4n-6, aa) suppressed tnf production by up to 87% and 98%, respectively (fig. b) . conclusions: both saturated fas and trans-fas, which positively correlate with hypertension, induce placental tnf production. the n-6 fa precursors to prostaglandins, aa and linoleic acid, reduce placental tnf production following stimulation. likewise, dha, a product of n-3 fa metabolism commonly consumed in fish oil and associated with improved blood pressure, suppresses tnf production by stimulated placenta cells. vegf and flk1 mediated glomerulogenesis in offspring exposed to maternal hypernatremia. roy z mansano, mina desai, ahmed abdel-hakeem, thomas r magee, tazmia q henry, cynthia nast, john s torday, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: growth restricted human and animal offspring, resulting from maternal nutrient restriction or uteroplacental insufficiency, consistently demonstrate reduced glomerular number and a predisposition to adult systemic hypertension and renal compromise. in contrast, maternal water restriction (wr) produces newborns with a unique programmed phenotype of increased glomerular number. glomerulogenesis is dependent, in part, on appropriately timed and quantified vascular development. as vegf and its receptor flk1 have crucial stimulatory effects on renal vasculogenesis, we hypothesized that maternal wr-induced morphologic renal changes are secondary to vegfmediated vasculogenic effects. methods: from day 10 to term gestation, pregnant rats received either ad libitum food and water (control, n=7), or wr to produce an increment of 6 meq/l in plasma sodium (n=7). following delivery, all dams received ad libitum food and water. at d1 after birth, offspring kidneys were extracted for mrna. vegf and its receptor, flk1, mrna levels were determined using real-time rt-pcr (presented as fold difference normalized to 18s). at d21 after birth, offspring glomerular number were determined in formalin fixed 5 m sections using histomorphometric analysis. values are means±se. results: wr offspring (day 21) had higher glomerular number than control (wr 25±1, control 22±1 per mm2, p<0.01). flk1 mrna expression was increased in wr offspring kidneys (wr 6.4±2.3, control 1.0±0.3, p<0.05) as compared to controls. in contrast, vegf mrna expression in wr offspring kidney was comparable to control (wr 0.9±0.1, control 1.0±0.2, p=0.8). conclusion:prenatally wr offspring demonstrate significantly increased glomerular number. as vegf recruits angioblasts to the developing glomerulus via its receptor flk1, increased receptors (flk1) with the same level of the ligand (vegf) suggest that enhanced vasculogenesis may represent a putative mechanism for increased nephrogenesis in wr offspring. modulation of newborn vasculogenesis via vegf and flk1 expression may represent a therapeutic option for growth restricted offspring with decreased glomerular number. objective: maternal food restriction (mfr) during gestation (embryonic day 10 to birth) reduces rat kidney glomerular number by 19% at 3 weeks of age. undernutrition during human gestation leads to similar impaired nephrogenesis and increased hypertension in adults. renal development may be delineated into stages including ureteric bud branching, mesenchymal to epithelial transformation and glomerulogenesis. previously, we detected dysregulation of genes controlling nephrogenesis at gestational ages, e16-e20, suggesting that mfr has significant effects on ureteric bud branching. in the present study we evaluated the effect of this dysregulation on early nephron development in e16 fetal kidney explants from dams with mfr. methods: e16 fetal kidneys were collected from 50% mfr pregnant rats and ad libitum control rats (n=3 per group and time point), incubated in dmem/f12k medium for 0, 1, 2, 3, and 4 days, and fixed with 4% paraformaldehyde. kidneys were stained with fluorescein-labeled dolichos biflorus agglutinin (dba), images captured and terminal ureteric buds quantitated. all values are presented as mean ± sem. differences were considered significant at p<0.05. results: mfr ex-vivo kidneys at day 0 demonstrated an initial moderate reduction in terminal branches versus control (c) samples (mfr: 78±9 vs c: 86±3 terminal branch ends per kidney). both mfr and control (c) kidneys demonstrated significant (p<0.05) increases in branching in explant culture to maximum values at 4 days (mfr: 147±16 vs c: 217±16). with increasing culture days, the percent reduction in mfr branching increased with terminal branch point decreases of 9% at day 0, 14% at day 1, 24% at day 2, 20% at day 3, and 32% at day 4 (p<0.05, mfr vs c at day 4). conclusion: kidney explant cultures from mfr treated fetuses display basal and culture-based decreased branching compared to controls. this decrease in terminal ureteric buds, in combination with our previous findings of dysregulation of branching-associated kidney transcription and growth factors, suggests mfr induces early (e16) dysregulation of branching as a major mechanism of the associated nephropenia. these results indicate that early programming events in kidney development induce nephropenia and renal disease in adults. intrauterine growth restriction (iugr) has important implications for the neonate not only at the time of birth, but also as an adult. in humans and animals with iugr, the elevated risk of developing hypertension is thought to involve an upregulation of the renin-angiotensin system (ras). however, the link between the intrauterine insult and enhanced ras is not known. a novel mechanism leading to hypertension has emerged recently involving two orphan g-protein coupled receptors (gcr91 and gcr99) and their endogenous ligands, succinate and -ketoglutarate. infusion of succinate into adult mice induces hypertension, an effect which was eliminated in gcr91 knockout mice or by pretreatment with ace inhibitors. interestingly, succinate levels have been shown to increase in the circulation under conditions of oxidative stress, one of the hypothesized mediators of developmental programming of hypertension. thus, we hypothesized that gcr91 and gcr99 are upregulated in a rat model of iugr and may contribute to in utero programming of hypertension. timedpregnant rats were fed either control (c, 20% casein) or low protein (lp, 8% casein) diet throughout gestation. kidneys were collected on embryonic day 19 (e19), post-natal day 30 (d30) or 130 (d130), n 3. real-time pcr was used to compare gcr91, gcr99 and renin expression. data were standardized to a housekeeping gene (s15) and are expressed as fold increase/ decrease compared to control. a student's t-test was used to determine significance between groups (p<0.05). offspring from lp dams were significantly smaller at birth and did not display catch-up growth. in kidneys from lp fetuses, both gcr91 and gcr99 were significantly elevated compared with controls (3.6 fold and 4.7 fold respectively; p=0.02), whereas renin was 0.78 fold lower. on post-natal d30, there was a trend towards increased expression of both gcr91 (1.7 fold; p=0.07) and renin (1.6 fold; p=0.09) in offspring from lp dams. at d130, there were no significant differences between groups. in conclusion, these preliminary data suggest that in iugr offspring from lp rats, the gcr91/ 99 pathway may be enhanced, indicating a novel mechanism for the programming of hypertension. objective: in addition to excess adipose tissue, obesity is accompanied by increased fat storage in organs such as the liver. peroxisome proliferatoractivated receptors (ppars) are ligand-activated transcription factors involved in the regulation of lipid metabolism, and lipid-associated inflammatory response. obesity represents a state of chronic low-level inflammation, with ppar and ppar implicated in this process. we have previously shown that nutrient restriction in pregnancy results in intrauterine growth restricted (iugr) newborns which develop adult obesity with elevated c-reactive protein (crp) levels. as crp is derived from the liver, we hypothesized that iugr-induced obesity inhibition of hepatic ppar and ppar is associated with an increased inflammatory response. methods: control dams received ad libitum food, whereas study dams were 50% food-restricted from pregnancy day 10 to 21 to produce iugr newborns. all pups were nursed by control dams and weaned at 3 weeks to ad libitum feed. at 1 day and 9 months of age, male offspring were analyzed for hepatic ppar , ppar and crp mrna (real time rt-pcr) and protein (western blot) expression. data was normalized to -actin and presented as fold change for protein levels. at 9 months, hepatic triglyceride content was determined enzymatically. results: at 1d of age, iugr pups showed significant downregulation of ppar (0.2-fold) and ppar (0.4-fold) expression, though crp expression was significantly upregulated (4-fold). findings persisted at 9 months of age, with continued downregulation of ppar and ppar (0.5-fold) and upregulation of crp expression (5-fold). furthermore, iugr adults had significantly increased hepatic triglyceride content (600±60 vs. 373±28 mg/g liver, p<0.05). conclusions: reduced expression of hepatic ppar and ppar in iugr offspring may contribute to elevated hepatic crp levels and triglyceride content. thus, developmental hepatic dysregulation leads to programmed obesity-induced inflammation in iugr offspring. offspring. mina desai, 1 ederlen casillas, 1 guang han, 1 darran n tosh, 1,2 michael g ross. 1 1 dept. of ob/gyn, labiomed at harbor-ucla med. ctr., torrance, ca, usa; 2 dept. of physiology, univ. of adelaide, australia. objective: leptin and insulin mediate central anorexigenic signaling responses via different receptor molecules: leptin binds to the obrb receptor activating jak-stat3 and pi3k pathways, whereas insulin activates pi3k pathway by binding to its receptor (ir) and substrate (irs2). maternal food restriction in pregnancy results in iugr newborns that develop hyperphagia and adult obesity. the iugr newborns have significantly decreased plasma leptin levels with increased hypothalamic expression of basal obrb, stat3 and decreased expression of ir and irs2, suggesting altered anorexigenic pathways. we studied the response of hypothalamic leptin/insulin signal molecules to peripheral leptin in iugr newborns. methods: control dams received ad libitum food, whereas study dams were 50% food-restricted from pregnancy day 10 to 21. 1 day old male offspring were given saline or leptin (1 g/g, i.p). hypothalamus was dissected at 15, 30 and 45 minutes and protein expression of total stat3, phosphorylated stat3 (p-stat3), inhibitor of leptin signal (socs3), ir, irs2, total akt and phosphorylated akt was determined by western blot (normalized to -actin). data is compared between leptin and saline treatments in iugr and controls. results: in response to peripheral leptin, iugr newborns showed marked dysfunction in stimulated hypothalamic leptin and insulin signaling responses. (1) jak-stat3: leptin-treated controls show progressively increased pstat3 (4-fold) with initial suppression of socs3 (0.5-fold) as compared to salinetreated controls. conversely in leptin-treated iugr, the pattern is reversed such that there is sustained decline in pstat3 expression (0.3-fold) with failure to downregulate socs3. (2) pi3k pathway: leptin-treated controls showed a significant reduction in irs2 (0.3-fold) and pakt (0.5-fold) as compared to saline-treated controls. however, leptin-treated iugr newborns exhibited a paradoxical increase expression of irs2 (3-fold) and pakt (2-fold). conclusion:the iugr offspring demonstrate persistent upregulation of leptin receptor, a reduced phosphorylated stat3 (p-stat3) response in conjunction with an enhanced socs-3 response. the persistent increase in insulin responses indicates a dysfunction in dynamic signaling, leading to altered anorexigenic response and development of programmed obesity. we have previously demonstrated that maternal food restriction (mfr) in rats induces a marked increase in the expression of vegf protein in the aorta and mesenteric arterioles, accompanied by an increase in tgf-and collagen in both vessel types in adult rat offspring (am j physiol, 2007) . the aim of this study was to determine if this in vivo finding could be reproduced in an in vitro preparation. methods: two types of preparations were used in this study. we isolated endothelial cells from 3 week old male control rat aortas. these cells were used after the third passage. staining with von willerbrand factor demonstrated that these cells were pure endothelial cells. the second type of preparation was aortic explants from 3 week old male control rats. endothelial cells and aortic explants were transfected with a vegf adenovirus (106-109 viral infective particles) or a -galactosidase-adenovirus as a control. after 24 hours of culture protein was isolated from cells and explants for western blot analysis using rat specific antibodies. culture media was assayed for vegf by elisa. results: transfection of vegf adenovirus induced a dose-dependant increase in the expression of vegf protein in primary endothelial cells and aortic explants. the transfection of vegf into the endothelial cells showed a bell shaped curve, and was accompanied by an increase in media levels of vegf protein. maximal secretion of vegf was found with 107 viral infective particles. vegf adenovirus transfection induced a dose-dependant increase in c-reactive protein (crp) (inflammatory marker), and tgf-protein in both aortic explants and primary endothelial cells. these results indicate that upregulation of vegf in blood vessels induces inflammation and tgf-expression which in turn can induce collagen synthesis. thus the increased collagen expression and reduced compliance previously reported by us in vessels of mfr offspring can be explained by the over-expression of vegf which we reported. therapeutic intervention aimed at prevention of the increase in vascular vegf expression in mfr offspring could potentially prevent programmed hypertension. maternal regulation of high fat nourishment during lactation period reduce a hypertension of male offspring. hidenori takahashi, toshiaki okawa, keiya fujimori, akira sato. obgyn, fukushima med.univ., fukushima, fukushima, japan. objective: exposure to undernutrition or high fat nourishment during fetal life has been proposed as an underlying cause of adult hypertension, but the effect of maternal feeding regulation during lactation period on blood pressure of offspring is unclear. our objective was to investigate the effects of either high-fat diet (hfd) during gestation to lactation period or restrictive fed a hfd during lactation period on blood pressure in male rat offspring. we use 3 types pregnant wistar rats as fed with normal nutrition (group a), with a high fat diet (hfd) during gestation to lactation period (group b) and with hfd nutritionally restricted by feeding with 30% of the normal lactationmatched dietary intake from the day of delivery to the end of lactation period (group c). the male offspring was measured blood pressure at 12, 24 and 60 weeks by using indirect tail-cuff method. statically analysis was performed using oneway annova. results: body weight was significantly reduced in c offspring compared to a and b in male offspring at day 28 after delivery (p<0.01). at 12 weeks old, the body weight of c offspring was no difference to catch up compared to a and b offspring. systolic and diastolic blood pressures were significantly elevated at all 12, 24 and 60 weeks in offspring of b > c >a. (p<0.01, vs. a) conclusions: under high-fat nutrition during gestation to lactation period induced hypertension in male rat offspring. maternal high fat environment make a hypertensive offspring, but regulation of fat feeding during lactation period may reduce adulthood hypertension. background: uterine artery (uta) doppler velocimetry has been validated in populations of heterogeneous parity in the second trimester for prediction of obstetric outcomes requiring preterm delivery to include: fetal growth restriction, fetal demise, hypertensive disorders of pregnancy, abruption, and indicated preterm delivery. understanding that parity may affect uta doppler indices in subsequent pregnancies, we sought to validate these predictive values in the first trimester in a homogeneous population of multiparous women. study design: multiparous women undergoing first trimester screening of singleton pregnancies were enrolled and followed prospectively until delivery (n=91). these women were divided into controls, ri < 0.78 (n=74) and cases, ri 0.78 (n=17), based on prior studies. demographic, clinical, and sonographic data (including uta indices and assessment of notching) were obtained. statistical analysis included student's t test and chi square. results: cases were not significantly different from controls in terms of maternal age, ethnicity, bmi, or medical history. uta doppler indices were significantly different between the two cohorts in terms of the presence of unilateral or bilateral notching (48% vs, 55% p<0.001 and 22% vs. 68%, p<0.001, respectively). in contrast to that observed in patients of heterogenous parity previously, ri 0.78 was not associated with adverse pregnancy outcomes despite an average ri of 0.86, significantly above this threshold. conclusions: in this multiparous cohort ri was not predictive of adverse obstetrical outcome, in contrast to that observed in cohorts including nulliparous patient. parity may affect uta vasculature and obscure the ability of doppler velocimetry to predict adverse obstetric outcome in multiparous women. presence of uta doppler notching in the first trimester remained a robust predictor of adverse obstetric outcomes in multiparous patients. objective: epidemiological studies have shown that offspring exposed to preeclampsia during fetal development are more susceptible to airway disease later in life. we have shown previously that gender, but not sflt-1 over-expression during pregnancy determines higher reactivity in the offspring airways at 6 months of age. the objective of this study was to examine the effect of preeclampsia on the trachea from female and male offspring in our model of sflt-1 induced preeclampsia at 1 year of age and compare responses between the two age groups. methods: cd-1 mice at day 8 of gestation were injected via the tail vein with adenovirus carrying sflt1 (adsflt1, 10 9 pfu/100 l) or mfc (admfc, 10 9 pfu/100 l). mice were allowed to deliver. tracheas were isolated from female and male offspring at 6 months and 1 year of age, and rings were mounted in organ chambers for isometric tension recording. responses to potassium chloride (kcl, 60 mm), the mast cell degranulating agent compound 48/80 (48/80, 100 g/ml), and concentration-responses curve to acetylcholine (10 -9 -10 -5 m) were obtained. results: there was no significant difference in responses to acetylcholine, kcl, or compound 48/80 between 1 year old offspring born to the sflt1 and mfc groups. when comparing offspring within the same pregnancy exposure groups, responses to acetylcholine in adsflt1-treated group were significantly higher in 1 year old females than males. comparison between age groups by pregnancy exposure revealed that in the mfc group, 1 year old male offspring had higher responses to compound 48/80 and acetylcholine than 6 months old males. responses to kcl were significantly higher in 6 months old males than 1 year olds independent of maternal treatment during pregnancy. in females, the only difference between age groups was observed in the mfc group, where 6 months old offspring demonstrated significantly higher responses to acetylcholine compared to 1 year old offspring. conclusions: our findings did not show that airways of 1 year old offspring born to mice with a preeclampsia-like syndrome induced by sflt-1 over-expression have airway hyperreactivity. however, sex and age differences in airway responses dependent on maternal exposure during pregnancy were observed, and needs to be explored further to elucidate underlying mechanisms. objective: maternal food restriction (fr) results in iugr newborns that when normally nursed exhibit rapid catch-up growth and adult obesity. continued fr during nursing delays catch-up growth and prevents adult obesity. igf-1, which modulates growth and is secreted by the liver, may contribute to these morbidities. igf-1 is epigenetically regulated involving two promoters, alternative exon splicing and multiple transcription termination sites. we determined if hepatic igf-1 mrna levels correlate with obesity, and whether these changes are due to programmed epigenetic modification. methods: control pregnant rats received ad libitum food from gestation day 10 to 21 and lactation, whereas study dams were 50% fr. fr pups were nursed by either control (fr/adlib) or fr dams (fr/fr) and weaned to ad libitum feed. at 1 day and 9 months, male livers were analyzed for igf-1 mrna variant levels (real time rt-pcr). chromatin immunoprecipitation (chip) was performed using the antibody for h3k4 trimethyl, and associated levels of each igf-1 species were measured by pcr. results: at 9 months, obese fr/adlib males showed increased mrna levels of igf-1a, igf-1b, igf-1 exon 1, and igf-1 exon 2 as compared to controls (134±5, 165±6, 149±6, and 146±7 %). comparing fr/adlib 9 month to newborn offspring, h3/k4 was increased at igf1-promoter 1, promoter 2, exon 5, utr#3 and utr#4 (610±157, 731±167, 345±66, 1597±412, and 794±208 %), though there was no differences between control 9 month and newborns. in contrast, 9 month fr/fr males had comparable mrna levels to the controls except for igf-1b (% of control: 147±19). further, fr/fr 9 month h3/k4 was only different from newborns at utr#4 (% of newborn: 289±69). conclusion: iugr newborns with rapid catch-up growth and adult obesity have increased postnatal hepatic igf-1 mrna levels, likely a result of igf-1 histone and chromatin structure modifications to h3k4 trimethylation. conversely, iugr with delayed catch-up growth and absence of adult obesity have levels similar to that of controls. thus, modulation of the rate of iugr newborn catch-up growth may protect against igf-1 epigenetic modifications. introduction: igf-ii is synthesized as a pro-hormone (proigf-ii; 156-amino acid peptide) which is then processed into its active forms: "big" igf-ii (1-87) and mature igf-ii (1-67). these active forms are essential for placental and fetal development and have also been shown to persist into postnatal life. since maternal smoking is known to adversely affect feto-placental growth and postnatal development, we postulated that these effects might be mediated through nicotine-induced alterations in igf-ii processing. methods: in the present study, nulliparous female wistar rats (200-250g) were given nicotine (1mg/kg/day) or saline for 14 days prior to mating, during pregnancy, and throughout lactation. at gestational day 15, 18 and 21, dams were euthanized and we collected serum (fetal and maternal), amniotic fluid and recorded fetal body weight. a subset of dams were allowed to deliver at term. following parturition, serum samples from the offspring were collected at birth (pnd1) and weaning (pnd21). body weight was recorded weekly from birth to weaning. pro, "big" and mature igf-ii levels were determined by western blot analysis. results: maternal nicotine exposure during pregnancy resulted in a significant reduction in fetal body weight by gestational day 21. however, there was no effect of nicotine on fetal serum or amniotic fluid igf-ii levels at any gestational age examined. in maternal serum, mature igf-ii in control animals decreased with advancing gestational age such that igf-ii levels were lowest at gestational day 21. nicotine administration prevented this decline, which resulted in significantly higher mature igf-ii levels in nicotine-exposed mothers at gestational day 21. in postnatal life, nicotine exposed offspring had significantly lower levels of "big" igf-ii expression at weaning (pnd21). conclusions: these data demonstrate that nicotine can alter the amount of the active forms of igf-ii in the mother and the newborn. dysregulation of maternal igf-ii occurs concomitantly with suboptimal fetal growth. results from this study suggest a mechanism by which maternal smoking causes impaired fetal growth and adverse postnatal health outcomes. objective: maternal food restriction in pregnancy results in iugr newborns which develop adult metabolic syndrome. programming of both increased appetite-mediated hyperphagia and enhanced adipogenesis contribute to the development of obesity. transcription factors, peroxisome-proliferatoractivated-receptor (ppar 2), ccaat/enhancer binding-protein (c/ebp ), and sterol regulatory element binding-protein (srebp1c) regulate adipogenesis and lipogenesis. although iugr offspring exhibit acute upregulation of the adipogenesis signaling cascade prior to the development of obesity, we determined if this increased adipogenic potential was an intrinsic cellular response, and thus maintained in cell culture. we further examined the responses to adipocyte stimulators (ppar activator-ligand rosiglitazone) and inhibitors (ppar repressor-ligand badge). methods: control dams received ad libitum food, whereas study dams were 50% food-restricted from pregnancy day 10 to term. adipocytes from 1 day old iugr and controls were isolated and cell proliferation rate was determined (mtt). primary adipocyte cell cultures were established and following 100% confluence, iugr and control adipocytes were treated to two doses (1 and 10 m) of either rosiglitazone or badge for 24h. mrna and protein was extracted for expression of ppar , c/ebp , srebp1c. data was normalized to -actin and compared to the respective untreated cells. results: iugr adipocytes had significantly increased protein expression of ppar (3.5-fold) and c/ebp (2-fold) as compared to control adipocytes, though srebp1c levels were unchanged. mrna levels showed similar changes in iugr newborns. importantly, iugr adipocytes exhibited increased cell proliferation (125% of control, p<0.05) and showed greater response to rosiglitazone (2.5-fold), though similar response to badge, as the control adipocytes conclusion: iugr primary adipocytes cell culture exhibit basal phenotypic characteristic of programmed upregulation of adipogenic transcription factors which promote adipose cell proliferation. the enhanced response to the adipogenic stimulant is further evidence of the predisposition to obesity. in contrast, the normal suppressive response to the inhibitor suggests that iugr adipocytes may respond to pharmacologic approaches to prevent obesity during this period. objective: maternal nutrient restriction results in intrauterine growth restricted (iugr) newborns which develop programmed obesity despite a normal post-weaning diet. the epidemic of obesity has been attributed in part to programmed "thrifty phenotype" and exposure to "western" diets. hepatic igf-1 is epigenetically regulated involving two promoters, alternative exon splicing, and multiple transcription termination sites. iugr offspring with normal post-weaning diet have increased postnatal hepatic igf-1 mrna levels, likely a result of igf-1 histone and chromatin structure modifications to h3k4 trimethylation. we hypothesized that iugr newborns that develop programmed obesity would demonstrate discernable hepatic igf-1 changes which are distinct from diet-induced obesity. we determined igf-1 hepatic mrna levels and epigenetic characteristics in programmed (iugr) and dietinduced (dio) offspring. methods:: control pregnant rats received ad libitum food whereas study dams were 50% maternal food restricted from day 10 to 21. all pups were nursed on ad libitum fed dams. controls were weaned to high-fat (fat, 16%) diet whereas iugr were weaned to normal ad libitum diet (fat, 9%) to produce diet-induced (dio) and programmed obese groups, respectively. at 9 months, male hepatic igf-1 were analyzed for igf-1 mrna variant levels (real time rt-pcr). chromatin immunoprecipitation (chip) was performed using the antibody for h3k4 dimethyl and h3k4 trimethyl, and associated levels of each igf-1 species were measured by pcr. result: relative to dio control males, iugr had increased mrna of igf-1a, exon 1 and exon 2 (336±4, 267±4, 143±5%). chip with h3k4 dimethyl showed increased igf-1 exon 6 (215±20%) and with h3k4 trimethyl, increased igf-1 promoter 1 and promoter 2 (282±26, 1183±23%) as compared to dio controls. conclusion: adult obese iugr males exposed to normal postweaning diet have increased hepatic igf-1a mrna and h3k4 dimethylation and trimethylation of igf-1 than dio controls. changes in igf-1 in adulthood from a prenatal insult thus suggest that igf-1 is programmed during the fetal period and may be associated with programmed adult obesity. rebekah elkins, 1 pandu gangula, 2 chandra yallampalli. 1 1 obstetrics gynecology, university of texas medical branch, galveston, tx, usa; 2 internal medicine, university of texas medical branch, galveston, tx, usa. objectives: we previously reported that the offspring of rats fed 6% protein during gestation develop hypertension at two to four months and that the hypertension is exacerbated in males. this study is to evaluate: 1) changes in estrogen receptor (er) angiotensin ii subtype receptor 1 (at 1 -r) and endothelial nitric oxide synthase (enos) in the mesenteric artery and aorta of offspring and assess if these changes, if any, are gender specific. methods: pregnant sprague dawley rats were fed either 20% protein (ctrl) or 6% protein (lpd) from day 1 of gestation. the offspring were evaluated for hypertension by means of systolic blood pressure measurements. at four months for the males and nine months for the females, mesenteric artery and aorta were collected in rnalater. expression of estrogen receptor a (er-a) and b (er-b), at 1 -r, and enos were analyzed by western immunoblotting and rt-pcr and expressed relative to b-actin or 18s. results: mesenteric artery shows no differences between ctrl and lpd female offspring in at 1 -r, enos, er-a or er-b. similarly mesenteric artery shows no diet exposure related changes in at 1 -r, er-a or er-b in male offspring. however, enos expression was lower in mesenteric artery of lpd male offspring. on the other hand, in the aorta both er-a and er-b levels are lower in lpd female offspring while there were no changes in at 1 -r or enos. no changes in at 1 -r, er-a or er-b were observed in male offspring aorta of ctrl and lpd rats. conclusion: the in utero exposure to lpd results in adult hypertension in both male and female offspring. some mechanisms for hypertension include the decrease in er-a and er-b but not at1-r or enos in females, and the decrease in enos but not at 1 -r or er in males indicating gender related differences. offspring. hidenori takahashi, toshiaki okawa, keiya fujimori, akira sato. obgyn, fukushima med. univ., fukushima, fukushima, japan. our objective was to investigate the effects of either severe undernutrition during late gestation or lactation period on blood pressure and the development of vascular function in male rat offspring. we use normal pregnant wistar rats (group a), nutritionally restricted by feeding with 30% of the normal gestation-matched dietary intake from day 17 of gestation to delivery (group b) and 30% restricted after delivery to the end of lactation period (group c). the offspring was measured blood pressure at 12 and 24 weeks by using indirect tail-cuff method. rings of thoracic aorta with intact endothelium from the male offspring of a and b at 8 weeks, were equilibrated at 2 g passive tension in organ chambers filled with krebs-henseleit solution continuously bubbled with 5%co2 in air (37°, ph 7.4) for isometric tension recording. concentration-response relationships to norepinephrine (ne) and angiotensinii(atii) were obtained in the absence or presence of n(omega)-nitro-l-arginine methyl ester (l-name) or a selective atii type-1 receptor blocker (valsartan). responses to cumulative concentrations of sodium nitroprusside (snp) and to 10-5m oxyhemoglobin (hb, nitric oxide scavenger) were also determined. contractions were expressed as a percent of the reference contraction induced by potassium chloride (60 mm). statically analysis was performed using one-way annova. results: body weight was significantly reduced in b offspring compared to a and c in male offspring at day 1 (p<0.01). at 12 weeks the body weight of offspring of b was no difference to catch up compared to a and c offspring. systolic and diastolic blood pressures were significantly elevated at both 12 and 24 weeks in offspring of b > c >a. ne concentration-dependently stimulated tension of aortic rings from in a and b offspring, which was not significantly (n=6). maximal contractions to ne were significantly stimulated by l-name in a (p<0.05), but not b offspring. valsartan significantly inhibited aortic contractions by ne in r (p<0.05), but not a offspring. there was no significant difference on responses of aortic rings by atii, snp and hb in a and b offspring. conclusions: severe under nutrition during not only late gestation but also lactation period induced hypertension in male rat offspring in adulthood. fetal origin of adult hypertension might be vascular endothelial dysfunction. fujimori, akira sato. obgyn, fukushima med. univ., fukushima, fukushima, japan. objective: exposure to undernutrition during fetal life has been proposed as an underlying cause of adult hypertension, but the effect of either high fat nourishment or undernutrition during lactation period on blood pressure is unclear. our objective was to investigate the most effective maternal nourishment and feeding period for offspring induced adulthood hypertension in using high-fat diet (hfd). study design: we use 5 types pregnant wistar rats as fed with normal nutrition (group a), nutritionally restricted by feeding with 30% of the normal gestation-matched dietary intake from day 17 of gestation to delivery (group b), 30% restricted after delivery to the end of lactation period (group c), with a high fat diet (hfd) during gestation to lactation period (group d) and with hfd nutritionally 30% restricted from the day of delivery to the end of lactation period (group e). the offspring was measured body weight (bw) and measured blood pressure at 12, 24 and 60 weeks by using indirect tail-cuff method. statically analysis was performed using one-way annova. results: bw was significantly reduced in b offspring compared to another (a, c, d, e) male offspring at day 1 (p<0.01). at day 28 after delivery, bw was significantly reduced in c, e offspring compared to a, d in male offspring (p<0.01). at 12 weeks old, bw of all type offspring was no difference. systolic and diastolic blood pressures were significantly elevated at 12 weeks in offspring of d> b > e > c >a. (p<0.01, vs. a). at 24 weeks, hypertensive offspring as b>d>e>c>a (p<0.01, vs. a). at 60 weeks, d> e > b > c >a (p<0.01, vs. a). conclusions: maternal high fat environment make a hypertensive offspring, but regulation of fat feeding during lactation period may reduce adulthood hypertension. in case with normal food, restrictive feeding during late gestation is more effective than lactation period for inducing hypertensive male offspring. regulation of maternal feeding not only during late gestation but also lactation period may control adulthood hypertension. the strongest epigenetical factor of maternal nutrition is high fat feeding during pregnancy to lactation period for f1 blood pressure, respectively. or residence at high altitude, impacts fetal growth and development. in a preliminary study, we observed a significant decrease in birth weight, subsequent compensatory postnatal growth, and an increase in relative right ventricular (rv) weight at postnatal (pn) day 30 in female offspring of rats exposed to hypoxia (14,000 ft; 11.6% po 2 ) from days 12 thru 15 of gestation (dga). thus, our objective was to further elucidate the impact of prenatal hypoxia on fetal growth and postnatal development. methods: pregnant dams (hx, n=15) were hypoxic from 12-15 dga with additional control dams either fed ad libitum (al, n=10), pair-fed with the hx dams throughout gestation (pg, n=12), or only pair-fed during the window of hypoxia (ph, n=12). female offspring from hx, pg, and ph dams were cross-fostered onto additional al dams (n=8/litter) by 48 h after birth. results: at birth, there was no difference in litter size; however, body weight (bw) of the hx, pg, and ph pups was lower (p<0.05) than that of al pups, and hx pups were lighter (p<0.05) than ph pups. weight of hx offspring remained lower (p<0.05) than al pups until the termination of the study at pn120, while the pg and ph pups reached weights comparable to the al offspring by pn90. relative to bw, heart weight and left ventricular/septal (lvs) weight was not different among groups; however, right ventricular weight (rv/bw) was greater (p<0.05) in the hx offspring at pn120 as was rv/lvs (p<0.05). cardiac function was evaluated by echocardiography at pn174. rv wall thickness was 47% greater (p<0.05) in hx pups as compared to al pups, confirming the significantly higher relative rv weight observed at necropsy. pep, pep/at, and pep/et were 36%, 14%, and 31% higher respectively in the hx offspring relative to the al offspring. lv end diastolic and end systolic diameters were smaller (p<0.05) in hx and ph offspring relative to the al group. myosin heavy chain (mhc) and mrna concentrations in the rv were evaluated by qrt-pcr, and the mhc / mrna ratio was greater (p<0.05) in the hx pups. conclusion: prenatal hypoxia from 12-15 dga impacted both fetal and postnatal growth, altered postnatal heart development and function, with the primary impact being on the rv. supported by nih hd48902. reproductive sciences; 2 pharmacology experimental therapeutics, university of maryland, baltimore, md, usa. background: exposure to nicotine (nic) is a significant risk to normal fetal development. fetal nic, which readily crosses the placenta, can be acquired from pregnant mothers by smoking or nicotine replacement therapy. the impact of nic on fetal organs may be mediated directly and/or via intrauterine hypoxia (hpx) via constriction of the uterine circulation. in adult hearts, both nic and hypoxia stimulate gene expression of matrix metalloproteinases (mmp), although the study of nic and hypoxia on gene expression in fetal organs remains incomplete. because mmps are involved in the regulation of extracellular matrix turnover and cardiac remodeling, we tested the hypothesis that prenatal nic and intrauterine hpx upregulate protein expression and activity of mmp2 in the fetal guinea pig heart. methods: pregnant guinea pigs were placed in either normoxia (nmx) or hpx (10.5%o2 in chamber) for 14d prior to term (65d). in two separate groups, nic was also added to the drinking water ( 18 mg/kg/d) for 10d at a dose that generates fetal nic levels (88ng/ml cotinine) equivalent to a moderate smoker. anesthetized near-term fetuses ( 63d) were excised and weighed. left ventricles of hearts were obtained and frozen at -80c for storage. mmp2 protein levels and enzymatic activity were measured by western analysis and gel zymography, respectively. results: nic alone (nmx+nic) decreased (p<0.05) fetal body wt by 12%, increased (p<0.05) the relative fetal brain wt (brain wt/fetal body wt ratio) by 14.2% and had no effect on relative placental or fetal heart wts. hpx alone decreased (p<0.05) fetal body wt by 20.3%, increased the relative fetal brain wt by 12% but, in contrast to nic alone, increased relative placental wt by 33.8%. both mmp2 protein levels (mmp2/a-actin density values) and activity (clear band density) were increased (p<0.05) by nic alone (by 1.6 and 1.8 fold, respectively) and hpx alone (by 1.5 and 1.3 fold, respectively). in addition, both protein and activity levels of hpx hearts were further increased by nic (by 1.6 and 1.3 fold) compared to hpx alone. conclusion: prenatal nic upregulates mmp2 expression in nmx fetal hearts and is potentiated by hpx. this suggests that under conditions of intrauterine stress cardiac remodeling by mmp activation may be an important mechanism by which nic and hpx affect fetal heart function. objective: in addition to peripheral hypoglycemic effects, insulin induces central anorexigenic responses via stimulation of the phosphoinositide-3 kinase (pi3k) pathway and cellular growth by mitogen activated protein kinase (mapk) pathway. the pi3k signaling cascade is activated by insulin binding to its receptor (ir), recruiting ir substrate 2 (irs-2), and phosphorylating pi3k. activated pi3k in turn causes phosphorylation of protein kinase b/akt which subsequently modulates hypothalamic anorexigenic responses. in contrast, the mapk (erk1/erk2) signaling pathway likely involves irs-1. further, insulin signaling is inhibited by the lipid phosphatase pten. we have previously shown that maternal food restriction (mfr) during pregnancy results in iugr newborns that develop hyperphagia, obesity and insulin resistance as adults. we sought to determine if altered hypothalamic basal insulin signaling expression of pi3k and mapk pathways contribute to reduced satiety responses and thus enhanced growth in iugr newborns methods: pregnant control dams received ad libitum (n=5) food, whereas study dams were 50% mfr (n=5) from pregnancy day 10 to 21 to produce iugr newborns. at day 1, hypothalamic region was dissected and analyzed for mrna levels (real time rt-pcr) of insulin signaling components via pi3k (ir, irs2, pi3k and akt) and mapk (irs1, erk1, erk2) pathways, and pten. data is presented as fold difference normalized to beta-2-microglobulin. results: at 1d of age, iugr pups exhibited downregulation of the entire pi3k pathway with significantly decreased (p<0.05) mrna levels of ir (0.6-fold), irs-2 (0.5-fold), pi3k (0.6-fold) and akt (0.6-fold). further, iugr pups showed similar decreased mrna expression of erk1 (0.6-fold) and erk2 (0.5-fold). however pten expression was similar to the controls. conclusion: reduced insulin-mediated pi3k signaling likely contributes to the suppressed anorexigenic responses and development of obesity in iugr offspring. reduction of central mapk signaling suggests a potential maldevelopment of additional neuronal pathways in iugr offspring. objectives: in the rat, uteroplacental insufficiency restricts fetal growth and impairs mammary development further compromising postnatal growth. both male and female growth-restricted offspring have a reduced nephron endowment but only males develop hypertension with glomerular hypertrophy, which can be reversed by improving the lactational environment. this study used cross-fostering to assess the influence of the prenatal and postnatal environments on renal development and nephrogenesis. methods: bilateral uterine vessel ligation (restricted, r) or sham surgery (control, c) was performed on day 18 of gestation in wky rats. control and restricted pups were cross-fostered onto c or r mothers on postnatal day 1 (pn1). post mortem was carried out on pn1 (c and r) and pn7 (c-on-c, c-on-r, r-on-c, r-on-r). results: body and kidney weights were decreased in r and r-on-r pups on pn1 and pn7 (p<0.05). there was some evidence of accelerated pup growth for r-on-c relative to r-on-r on pn7. male, but not female, relative bmp4 mrna expression on pn1 was higher in r than c (p<0.05) while gdnf, tgf 1 and at1 receptor were not different. on pn7, wnt4 (but not at1r, vegf-a) mrna expression (males only) was relatively higher in r-on-r (p<0.05) when compared to c-on-c (p<0.05). this and the histological analyses suggests an up-regulation of nephrogenic activity with more immature nephrons (males and females) in r-on-r (p<0.05) when compared to c-on-c, while r-on-c remained intermediate. intrauterine growth-restricted pups were born lighter and with smaller kidneys. this was partially rescued by improving lactational nutrition (r-on-c) at pn7. higher bmp4 mrna expression indicates impaired branching morphogenesis in pn1 r male, but not female kidneys, suggesting the timing and/or molecular mechanisms underlying the nephron deficit may be sex specific. at pn7 there was evidence of extended and increased nephrogenic activity in r-on-r, however, this was unable to restore the later nephron deficit. improved lactation for r-on-c, which prevented the adult nephron deficit and hypertension, increased and extended nephrogenesis to a lesser degree than r-on-r suggesting that the restoration of nephron endowment was likely to have occurred prior to pn7. amy m tetrault, sarah b lieber, marya shanabrough, tamas l horvath, hugh s taylor. obstetrics, gynecology and reproductive sciences, yale university school of medicine, new haven, ct, usa. objective: classically recognized for its role in energy balance, body weight and appetite, ghrelin has also been implicated in reproduction. ghrelin (-/-) mice are infertile while administration of ghrelin to wt mice results in decreased litter size and constrained embryonic growth. here we investigate the effect of maternal ghrelin deficiency on in utero developmental programming of the female reproductive tract. hox genes determine developmental identity of the paramesonephric duct. we determined that hoxa10 is regulated by ghrelin in vitro and that in utero ghrelin deficency alters f2 hoxa10 gene expression and reproductive success. methods: wild-type females mice parented by ghrelin +/-b6d2f1 (ghrellin deficient) mice were analyzed for litter size, oocyte, and corpus luteum number. rna was extracted from the uterus of mice exposed to ghrelin deficiency in utero. ishikawa cells were treated with ghrelin with/without receptor (ghsr) antagonist, or saline and rna extracted. in both hoxa10 expression was analysed by real time rt-pcr normalized to -actin and also determined by ihc. experiments were repeated in triplicate and mrna expression compared by student's t-test. results: wild-type female offspring of ghrelin deficient dams had smaller litter sizes than controls (n=5, 4.7±3.3 pups; n= 4, 8.7±2.3 pups, respectively; p<0.01). no differences were seen in oocyte or corpus luteum number suggesting a uterine defect. hoxa10 mrna and protein expression were decreased in the uterus of the f2 females. ghsr was expressed in uterine endometrium. treatment of ishikawa cells with 1nm to 100nm ghrelin resulted in a 30 to 80% increase (p<0.01) in hoxa10 expression. treatment with ghrelin and ghsr antagonist resulted in similar increases in hoxa10 expression indicating a non-receptor mediated mechanism. conclusion: ghrelin contributes to reproductive tract developmental programming; in utero ghrelin deficiency compromises reproduction in female offspring. the developmental effects of ghrelin were mediated by alteration in hox gene expression and not through the classic ghsr receptor. obesity and decreased ghrelin may lead to defects in developmental programming of the reproductive tract. these findings demonstrate the importance of nutrition, energy utilization and appropriate ghrelin levels on normal uterine development. we have previously studied the deleterious effects of lack of the endothelial nitric oxid synthase (nos3) in mouse dams and their offspring. our laboratory demonstrated that adaptive responses in subsequent pregnancies may offset the harmful effects of the genetic deficiency of nos3. in this study we aimed to determine hepatic and renal histopathologic damage in nos3 deficient pregnant mice comparing animals carrying their first versus their second pregnancy. study design: gravid nos3 +/+wt and nos3 -/-ko mice during their first (p0) or second (p1) pregnancy were sacrificed at day 18 of gestation. livers and kidneys were stained and analyzed for the presence and extent of histopathologic lesions. results: nos3 +/+wt dams displayed a low incidence of significant renal or hepatic lesions in either the first or the second pregnancy. in nos3 -/-ko mice the incidence of liver necrosis and inflammation during the first pregnancy was 44% and 49%, respectively. in nos3 -/-ko dams sacrificed during the second gestation the incidence rates for the same lesions were 6% and 19%, respectively (p<0.05). this correlation persisted when we analyzed the relative severity of hepatic lesions between p0 and p1 animals. although a similar trend was observed, the difference between p0 and p1 animals with regards to kidney lesions did not reach the level of statistical significance in our study. conclusions: a second pregnancy in this animal model of hypertension was associated with a significantly improved hepatic histopathology compared with the first pregnancy. this observation is consistent with our previous studies showing a decrease in systemic vascular resistance in p1 versus p0 nos3 -/-ko mice. the beneficial effects of a prior pregnancy may partially underlie the phenomenon of a decreased risk of preeclampsia in multiparous versus nulliparous women. further studies are required to delineate the counterregulatory mechanisms leading to improved cardiovascular function in subsequent pregnancies in these genetically modified animals. maternal hypomethylation is associated with congenital heart defects in down syndrome. lmjw van driel, 1,2 r de jonge, 3 wa helbing, 2 bd van zelst, 3 j lindemans, 3 eap steegers, 1 rpm steegers-theunissen. 1,2,4,5 1 obstetrics gynecology; 2 pediatrics; 3 clinical chemistry; 4 epidemiolog y biostatistics; 5 clinical genetics; 6 erasmus university mc, rotterdam, netherlands. background: maternal age and hyperhomocysteinemia are risk factors for having a child with down syndrome (ds) and congenital heart defects (chds), respectively. evidence is rising that ageing is associated with a state of hypomethylation. objectives: to investigate whether the risk of a child with ds and chd is associated with maternal hypomethylation. methods: we conducted a case-control triad study at 16 months after the index-pregnancy. case-children (n=24) were included if they had ds and chd. children (n=251) without a major congenital malformation served as controls. the concentrations of s-adenosyl methionine (sam), s-adenosyl homocysteine (sah), sam/sah ratio, and homocysteine in maternal blood were measured as biomarkers for methylation. the data were analyzed using the mann-whitney u test and a logistic regression model. results: maternal age was included in the model as potential confounder. the levels and the crude and adjusted or(95%ci) of the biomarkers are shown in table 1. an increase of the sam/sah ratio with 1 unit decreases the risk of a child with ds and chd with 30 percent. moreover, every increase of 1 mol/l of homocysteine 1.1 fold increases this risk. conclusions: maternal hypomethylation is significantly associated with an increased risk of having a child with ds and chd. since, the effects are confounded by maternal age, hypomethylation can be considered as feature of ageing. the developmental origins hypothesis postulates that during critical ontogenetic periods, transient environmental stimuli perturb developmental pathways and induce permanent changes in gene expression, metabolism, and chronic disease susceptibility. one likely mechanism is via early nutritional influences on epigenetic gene modification consisting of the presence of a methyl group on the carbon 5 of a cytosine residue. this modification is responsible for an important form of gene regulation in eukaryotes. in the present study, we have tested the hypothesis that maternal low-protein diet altered epigenetic regulation of specific gene of the offspring. c57bl/6 female mice were mated and on the day the plug was detected, these females were then randomly allocated to be fed isocaloric diets consisting 18% protein or 9% protein. at delivery, offspring were killed and the livers were removed immediately, frozen in liquid nitrogen and stored at -80c. genomic sequencing after bisulfite modification is used to study site-specific dna methylation. dna methylation status of oct-4 and sphk-1 gene upstream regions in the mouse liver was analyzed. hepatic oct-4 or sphk-1 promoter methylation was not significantly different between both groups. however, dna methylation pattern of the genomic dna is specific in low-protein diet group. aberrant oct-4 and sphk-1 gene expression may cause perturbations in cell differentiation. we suggest that the epigenetic mechanism consisting of dna methylation underlies the fetal programming theory. primary human cytomegalovirus (hcmv) infection during pregnancy can have devastating consequences for both the mother and fetus. hcmv infection has been implicated in the development of pre-eclampsia and intrauterine growth retardation (iugr), as well as congenital cmv syndrome in newborns exposed in utero. previously, we have shown that hcmv infection of placental cytotrophoblasts inhibits their normal invasion, proliferation, and migration. however, the mechanisms occurring during early establishment of placental infection are largely unknown. we assessed the impact of hcmv infection on cytotrophoblasts by performing immuno-based assays for various cytokines and cellular growth factors. we detected significant cytokine dysregulation at both 24 and 48 hours after in vitro hcmv infection of cytotrophoblast cells. soluble cytokines involved in recruitment of monocytes and macrophages (gro-a, mcp-3) were downregulated at both 24 and 48 hours after infection. sdf-1, which is chemotactic for lymphocytes during early inflammation, was also decreased. these results suggest that recruitment of cells involved in the anti-viral immune response is being interrupted early in the course of infection by hcmv. additionally, a large decrease in the amount of soluble hgf was seen. hgf normally induces migration of cytotrophoblasts along the invasive pathway, and downregulation of this factor could severely affect these processes. finally, we saw increased amounts of soluble icam-1, contrasted by decreased amounts of vcam-1, indicating dysregulation of adhesion molecules that are necessary for successful placental invasion to occur. all together, these data indicate significant alterations in cytokine profiles as early as 24 hours after hcmv infection, which could provide important clues to the pathogenesis of hcmv in placental invasion and inflammation. additional studies will further elucidate this dysregulation, and determine whether these effects are due to alterations in pre-existing cellular factors or if transcriptional alterations are involved. method: studies were performed in pregnant ewes (n=6 in each group) with twin fetuses at 118-120 days (very preterm) and 140-142 days (near-term). neither mother nor fetuses were instrumented prior to the time of blood collection. maternal blood was collected from the jugular vein before sedation. anesthesia was then induced with isofluorane, the abdomen was opened, fetuses exteriorized and blood collected from umbilical cords. blood samples were transferred to plastic tubes containing ethylene-diamino tetraacetic acid and reduced glutathione, plasma separated and stored at -80c. commercial radioimmunoassay kits for ovine-crf (phoenix pharmaceuticals, b90: 7.2 ± 1.5 pg/ml) and cortisol (diagnostic laboratory, b90: 1.2 ± 0.4 ng/ml) were used according to the manufacturer's instructions. results: in very-preterm gestations, maternal and fetal plasma crf levels were undetectable. maternal, but not fetal, plasma cortisol levels were measurable (33±4 ng/ml). in near-term gestations, both cortisol and crf were measurable in maternal (crf: 86±6 pg/ml; cortisol: 55±1 ng/m) and fetal plasma (crf: 730±68 pg/ml; cortisol: 5±2 ng/ml). plasma crf levels were higher in nearterm fetuses than in their maternal ewes (p <0.001). conclusion: ovine plasma crf levels are measurable in maternal and fetal plasma in near-term but not very-preterm gestations. the absence of crf in preterm plasma, perhaps due to reduced placental expression and/or placental crf release, may contribute to the rarity of in utero meconium passage in preterm gestations. interleukin objectives: interleukin-6 (il-6) is a pro-inflammatory cytokine produced in adipose cells. recent studies suggest il-6 may be a marker of maternal obesity and in utero fetal programming. our hypotheses were 1) il-6 correlates with maternal obesity and 2) il-6 mediates the effect of maternal obesity on infant birth weight. methods: the parity, inflammation, and diabetes (pid) study is a longitudinal study of adipokine levels in a diverse sample of pregnant women. we present a cross-sectional analysis of first trimester il-6 levels from 173 non-diabetic women who underwent a live birth. the independent variable was il-6 (pg/ml), measured with monoclonal antibody elisa assays. the dependent variable was infant birth weight (gms). maternal bmi categories were: normal/underweight (< 25 kg/m 2 ); overweight (25-30 kg/m 2 ); and obese (> 30 kg/m 2 ). data on demographic and clinical factors, nutrition and physical activity were collected at baseline. average il-6 levels were compared across bmi categories using anova. the association of il-6 levels with infant birth weight was estimated using multiple linear regression, adjusting for covariates. results: average il-6 levels were significantly higher in obese women (2.6± 1.6) compared to overweight (1.2± 0.4) and normal/underweight women (1.1± 0.7) [p< 0.001]. after adjustment, il-6 levels was positively correlated with pre-pregnancy bmi [regression coefficient (rc)0.12; 95% ci: (0.05, 0.2)]. as shown in the table below, elevated levels of il-6 were statistically significantly associated with a 0.5 gm higher infant birth weight after full adjustment. each 1 unit increase in pre-pregnancy bmi was associated with a 8 gm higher infant birth weight. table 1 . association of il-6 with infant birth weight characteristic regression coefficient 95% confidence interval interleukin-6 0.5 0.02, 0.7 pre-pregnancy bmi 8 0.7, 38 gestational weight gain -3 -15, 10 bmi = body mass index; coefficients adjusted for demographics, clinical factors, pre-pregnancy bmi, gestational weight gain, non-fasting first trimester glucose levels, gestational age, nutritional intake and physical activity conclusion: il-6 and pre-pregnancy bmi were associated with infant birth weight after adjustment for covariates. our findings suggest that the effect of maternal obesity on infant birth weight may be mediated through il-6 or an alternative independent pathway. we have demonstrated that 9 -tetrahydrocannabinol (thc), in physiologically relevant concentrations, inhibits the growth and tight transcriptional control of the bewo trophoblast cell line 1 . the mechanism involved the decreased expression of the transcriptional regulator histone deacetylase 3 (hdac3). in these experiments we sought to answer the question, 'does anandamide (aea) work in the same manner as thc?' methods: the first trimester human trophoblast cell, bewo were plated at 1 or 4 x 10 5 cells /well to 96-well and 6-well plates for growth and rna experiments, respectively. after growing to 70-80% confluence, cultures were treated with varying concentrations of aea up to a maximum of 30 m for 48hr. cell numbers were determined using the xtt apoptosis/proliferation assay. total cellular rna was prepared and the relative levels of hdac3 and gapdh determined by end-point rt-pcr. aea exhibited an inhibitory effect on the bewo cell cultures, but only at concentrations in excess of 3 m where confluency was significantly reduced from 75% at 3 m to 50-60% at 15 m and 30 m (*p<0.05; one-way anova with tukey's hsd test; n= 4). cultures treated with 15 m and 30 m aea did not exhibit increased cell death or failure to attach to the substratum, as evidenced by the lack of increase in the shedding of cells into the spent medium. bewo cells treated with aea showed a dose-dependent decrease in hdac3 mrna expression with a significant effect at 0.3 m (*p<0.05; oneway anova with tukey's hsd test; n=9). at this dose, the effect of aea had reached an effective maximum decrease in hdac3 mrna levels (48%) because hdac3 mrna levels were not decreased further by either 3 m aea (45%) or 30 m aea (49%). the alteration of hdac3 gene expression by aea in bewo cells with its associated decrease in cell number suggest that the trophoblast cell may be an important target for circulating endocannabinoids during the 1 st trimester of pregnancy. the data indicates that although exocannabinoids and endocannabinoids both inhibit bewo cell growth, they do so using different transcriptional mechanisms. further understanding of the mechanism(s) by which aea alters placental physiology may lead to new strategies for the prevention of pregnancy complications such as 1 st trimester miscarriages. (1) taylor background: anandamide (aea) exerts its effects by acting on two cannabinoid receptors, cb1 and cb2 with the main regulator for aea levels being the metabolising enzyme, fatty acid amide hydrolase (faah). aea, cb1, cb2 and faah constitute the endocannabinoid system and previous studies have shown that faah and cb1 are expressed in term human placenta(1) suggesting that the endocannabinoid system might be present earlier in gestation. this study aimed to document changes in faah, cb1 and cb2 expression in the placenta during the first trimester of pregnancy. methods: first trimester samples (7 to 12 weeks gestation) were fixed in 10% neutral-buffered formalin for 4 days before embedding into paraffin wax or frozen in liquid nitrogen for rna analysis. for immunohistochemistry, faah polyclonal antibodies were used at an optimal dilution of 1:2000 in pbs, cb1 at 1:4000 and cb2 at 1:500. transcripts were measured using q-pcr with gene-specific primers. results: immunoreactive faah, cb1 and cb2 were detected in all samples. faah immunoreactivity in the syncytiotrophoblast increased between the 7th and 10th gestational week and by week 11 faah was barely detectable within large parts of the placenta. simultaneously, faah immunoreactivity increased in the mesenchymal core of the developing villi. immunoreactive cb1 and cb2 localised to the syncytiotrophoblast, cytotrophoblast and mesenchymal core with cb1 immunoreactivity showing diminished intensity after week 10, although this did not reach significance at the transcript level. cb1 immunoreactivity was absent from fetal blood cells and infiltrating maternal plasma cells, whereas cb1 and cb2 immunoreactivity was detected in endothelial cells but not in the vascular smooth muscle cells of blood vessels. the intensity of cb2 immunoreactivity in the syncytiotrophoblast differed from that of cb1 and faah in that it remained constant throughout. conclusion: the data suggest that placental faah and cb2 levels do not alter significantly during the first trimester, but alter their cellular distribution from the syncytiotrophoblast to the mesenchymal core. the significant loss of cb1 expression from the syncytiotrophoblast after the 10th week of gestation, a point of critical alteration in the developing placenta, suggests that its retention may be detrimental to normal placental development. reference: park, b. et al., (2003) hankins, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: numerous angiogenic proteins synthesized in the placenta are thought to be involved in placental vascularization and development; however, the molecular mechanism modeling the angiogenic process in early pregnancy remains elusive. calatonin gene-related peptide (cgrp) is a multifunctional peptide expressed at the human implantation site; but its influence on in vitro angiogenesis by human micro vascular endothelial cells is not known. objective: the present study was designed to determine the influence of cgrp on angiogenesis by human dermal microvascular endothelial cell (hdmvec) in vitro. methods: hdmvecs (vec technologies) were cultured in mcdb-131 complete solution containing cgrp (10 -8 m), cgrp plus its antagonist cgrp 8-37 (10 -7 m), in 6 well plates with 2x10 5 cells per well. the existence of cgrp receptor components calcitonin receptor-like receptor (crlr) and receptor activity modifying protein 1 (ramp1) was determined using immunofluorescent staining. cell proliferation was examined using methylthiazoltetrazolium (mtt) assay. the pro-angiogenic bioactivity of cgrp was evaluated using cell migration and capillary like tube formation on the matrigel. results: 1) immunofluorescent staining showed that cgrp receptor components crlr and ramp1 are abundantly expressed by hdmvecs. replacement of the primary antibodies with preimmune serum resulted in a negative staining; 2) cgrp dose-dependently (10 -10 to 10 -7 m) stimulated hdmvec proliferation, and this effect was totally blocked by cgrp antagonist, cgrp 8-37; 3) quantitative analysis for cell migration revealed that cgrp increases hdmvec migration in a dose and time-dependant manner; and 4) cgrp promotes hdmvec capillary like tube formation, and the length of capillary tube induced by cgrp (10 -8 m) was significantly increased over that of the untreated controls. this increase was observed at 12 hours of treatment and further increase was noted at 24 hours of culture. conclusion: cgrp induces in vitro angiogenesis by promoting microvascular endothelial cell proliferation, migration and capillary like tube formation. therefore, trophoblast derived cgrp at the implantation site may play a role in placental angiogenesis and fetal growth. over-expression of socs-3 gene promotes il-10 production by jeg-3 trophoblast cells. qin dong, 1,2 ruping fan, 2 yang gu, 2 david f lewis, 2 yuping wang. 2 1 biochemistry, harbin medical university, harbin, heilongjiang, china; 2 obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: suppressor of cytokine signaling-3 (socs-3) plays an important role in negative regulation of inflammatory response in biological cells. evidence has shown anti-inflammatory cytokine il-10 expression was significantly reduced in trophoblasts of preeclamptic (pe) placentas. we sought to determine if over-expression of socs-3 in placental trophoblasts could promote il-10 production. methods: full-length socs-3 open reading frame (socs-3 cdna) was generated by rt-pcr from total rna samples isolated from human leukocytes and cloned into a pzsgreen1-n1 vector, which encodes a green fluorescent protein zsgreen1. successful socs-3 cloning was confirmed by sequencing. for transfection, jeg-3 cells were placed into 6 well/cluster plates at a concentration of 1.2 x105/well. the tranfection was carried out with 1.0 g of socs-3/zsgreen1 plasmid (psocs-3/zsgreen1) for 6 hours when cell reached 45-50% confluence. siport lipid were used. jeg-3 cells transfected with zsgreen1 plasmid (pzsgreen1) only was used as control. after approximately 60 hours of transfection, cells were treated with il-6 at 1 and 10ng/ml. medium was then collected and measured for il-10 by elisa. il-10 production was calculated as the percentage of increase by psocs-3/zsgreen1 transfected cells compared to the cells transfected with pzsgreen1 only. result: il-10 production was increased by psocs-3/zsgreen1 transfected jeg-3 cells compared to the cells transfected with pzsgreen1 only when stimulated by il-6, control: 9.9% increase; 1ng/ml il-6: 28.5% increase and 10ng/ml il-6: 140% increase, p< 0.05, respectively. data are means from three independent experiments. conclusion: over-expression of socs-3 gene could promote il-10 production by placental trophoblast cells. reduced sil-6r release and increased ratio of sgp130/sil-6r production by placental tissues from women with preeclampsia. shuang zhao, 1 ruping fan, 1 jingxia sun, 2 yang gu, 1 david f lewis, 1 yuping wang. 1 1 obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa; 2 obstetrics and gynecology, first hospital, harbin medical university, harbin, heilongjiang, china. objective: placental tissue/trophoblasts release more inflammatory cytokines (il-6, il-8 and tnf ) in preeclampsia (pe) than in normal pregnancies. however, the reason for increased inflammatory cytokines released by pe placentas is not clear. soluble il-6 receptor (sil-6r) and membrane receptor il-6/gp130 complex play an important role in the negative regulation of cytokine signaling in suppressor of cytokine signaling (socs) pathway. in contrast, soluble gp130 (sgp130) is an antagonist for il-6/il-6r trans-signaling. this study was undertaken to determine sil-6r and sgp130 production by villous tissues from normal and pe placentas. methods: placentas delivered by 8 normal and 9 pe pregnant women were used in this study. placental explants were incubated with dmem for 48h. the culture medium was collected. placental villous tissue productions of sil-6r and sgp130 were measured by elisa. all samples were assayed in duplicate. data are expressed as mean ± se and analyzed by mann whitney test. a p level <0.05 was considered statistically different. results: placental tissues from pe produced significantly less sil-6r than tissues from normal pregnancies, 3.56±0.61 vs. 5.84±0.34 pg/mg of wet tissue, p<0.01. soluble gp130 production was relatively compatible between pe and normal placental tissues: 162.29±21.71 vs. 162.83± 5.56 pg/mg of wet tissue. the ratio of sgp130/sil-6r release was significantly higher in pe than in normal placentas, 49.84±6.93 vs. 28.62±0.02, p<0.01. conclusion: reduced sil-6r production and/or increased ratio of sgp130/sil-6r production by pe placentas suggest less cytokine inhibitory activity in pe placentas, which may contribute to the increased toxic cytokine production in placentas from pe. using chemiluminescence immunoassays. peter s uzelac, 1 jing dai, 2 frank z stanczyk, 2 daniel r mishell, jr. 2 1 obstetrics and gynecology, university of louisville, louisville, ky, usa; 2 obstetrics and gynecology, university of southern california, los angeles, ca, usa. objective: characterizing human chorionic gonadotropin (hcg) levels throughout normal pregnancy is critical to its use as a bio-marker for abnormal gestations. there is a paucity of data describing hcg trends during pregnancy and most of the relevant studies use older, less specific assays. our first objective was to characterize hcg levels throughout normal gestation using two different contemporary chemiluminescence immunoassays. our second objective was to compare hcg patterns in healthy gestations with pregnancies affected by diabetes, a common obstetrical complication. methods: a single blood sample was collected from 137 healthy pregnant women and 170 diabetic pregnancies. gestational age was confirmed by ultrasound. serum hcg levels were quantified by chemiluminescence immunoassays using the acs-180 and immulite systems. data was grouped in 4-week intervals until 16 weeks of gestation and 8-week intervals thereafter, with 21 to 27 samples in each interval for healthy women and 6 to 56 samples in each interval for diabetic women. paired t-test and wilcoxon rank sum test were used for statistical analysis. results: using the acs-180 system, mean hcg levels (miu/ml) for healthy pregnant women were 64,489 at 5-8 weeks, 100,421 at 9-12 weeks, 52,724 at 13-16 weeks, 16,741 at 17-24 weeks, 16,580 at 25-32 weeks, and 22,535 at 33-41 weeks. mean hcg levels obtained from the immulite system were similar. for diabetic pregnancies, mean hcg levels (miu/ml) were 45,165, 59,899, 45,448, 19,920, 20,372 and 19,525 , respectively. between 9-12 weeks of gestation, the hcg levels were significantly lower in diabetic pregnancies (p=0.02) compared to healthy controls. 3) compared to healthy pregnancies, diabetic gestations have significantly lower peak hcg levels. the cause of this difference is an area deserving further investigation. background: mouse and human placentae share several cellular and molecular features, including a haemochorial interface, allowing the murine labyrinth to be compared with the human fetal placenta. there is an autonomous embryonic ras from at least the time of implantation. ace converts angiotensin i to the active angiotensin ii (angii). angii's actions as a pro-inflammatory agent, in promoting cell migration, angiogenesis and cellular growth and apoptosis, strongly suggest a rôle in placentation. hypothesis: there would be counterregulation of the placental ras if the effect of ace were removed. this study investigated for the first time whether the knockout of somatic ace affected the expression and localisation of various components of the ras in the placentae of wild-type (wt/wt), heterozygous (wt/4) and ace knockout (4/4) mice. methods: immunohistochemistry (dako envision plus) was used to localise and semiquantify ang type 1 receptor (at1r), ang type 2 receptor (at2r), ace, and ace-2 in the three genotypes (n=3/group). placental sections were blinded to genotype; a score range of 0-4 was used. the 2 test was used (spss version 15.0) to analyse the difference in staining score by genotype. results: immunoreactivity of all antigens increased in the placental labyrinth of 4/4 mice compared to wt/wt and wt/4 mice (at1r p<0.05; at2r p<<0.001; ace p<<0.001; ace-2 p<<0.001). at2r and ace-2 displayed increased staining in the fetal vascular endothelium of 4/4 mice (at2r p<<0.001; ace-2 p=0.05), and in the cells lining the maternal central artery (at2r p<<0.001; ace-2 p<<0.001). ace-2 expression was very high in cytotrophoblast lining the maternal blood space in all genotypes. no gross structural differences were seen. comment: the antibody used did not differentiate between ace and the membrane-bound "testicular"-ace (tace). immunohistochemically-identified ace expression was upregulated in 4/4 placentae despite the loss of somatic ace, suggesting that t-ace can be expressed placentally. we believe this is the first demonstration of such expression. ace and t-ace are catalyticallysimilar in converting angi to angii. furthermore, angii acting via the at2rs is vasodilatory and ace-2 catalyses production of the vasodilatory ang (1-7); placental blood flow was presumably well-maintained and pregnancy outcome is normal in 4/4 mice. it is generally accepted that prostaglandin production plays a crucial role in both term and preterm parturition, and recently the administration of 17 alpha hydroxyprogesterone acetate has been shown useful in preventing preterm labor. what is not clear is the biochemical pathway of prostaglandin production during labor and what, if any, effect 17 alpha hydroxyprogesterone acetate has on this pathway. in order to address this question, we used immunohistochemical staining techniques to evaluate the effect of treating human placental amnion and chorion decidua with 17 alpha hydroxyprogesterone acetate. membranes from unlabored patients were obtained at cesarean section and immediately seperated into control and drug treated specimens. controls were from the same placenta and were subjected to all experimental procedures except for the addition of 17 alpha hydroxyprogesterone acetate to the culture media. specimens were compared at zero, six, and twenty four hour intervals. at the appropriate time, each specimen was formalin fixed and then paraffin blocked. tissue sections were then mounted on slides which were immunohistochemically stained using appropriate primary and secondary antibodies and standard techniques. the slides were then analyzed via light microscopy for changes in staining of three enzymes involved in prostaglandin production--cyclooxygenase 1 (cox-1), cyclooxygenase 2 (cox-2), and 15-hydroxy prostaglandin dehydrogenase (pgdh). compared to control, the slides treated with 17 alpha hydroxyprogesterone acetate had differing amounts of enzyme expression. cox-1 was relatively unchanged and pgdh was only slightly increased, but cox-2 was noticeably decreased in the treated slides. these results were time dependent. this data suggests that 17 alpha hydroxyprogesterone acetate decreases prostaglandin production in fetal membranes primarily by downregulation of the cyclooxygenase 2 enzyme. objectives: in the human placenta, proliferation, differentiation and fusion of cytotrophoblasts (ct) are essential events in the formation of the multinucleated syncytiotrophoblast, however the regulation of these processes is poorly understood. using an explant model of human first trimester placenta we have established that both igf-i and -ii enhance ct proliferation, differentiation and survival mediated via igf1r signalling. we have also shown that non-specific inhibition of protein tyrosine phosphatases inhibits igf-mediated signalling in trophoblast; therefore, we have now used sirna-mediated knockdown to investigate the role of the tyrosine phosphatase shp-2 in this pathway. methods: amaxa nucleofector technology was used to deliver shp-2 or scrambled sirna (100nm) to bewo cells or first trimester villous tissue fragments. knockdown was confirmed by q-pcr and western blotting. transfected cells and tissue were maintained in culture for 72 hours, then treated with igf-i or igf-ii (10nm) for a further 24 hours before immunohistochemical (ihc) analysis for cell proliferation (ki67, brdu) or apoptosis (m30). results: sirna-mediated knockdown of shp-2 in bewo cells (95% reduction on western blot) demonstrated that igf-induced proliferation was reduced from 67.3±3.5% to 41.9±2.4% (p<0.001, n=5). ihc analysis of tissue demonstrated that shp-2 is localised to ct. following knockdown (93% decrease by q-pcr), igf-i-and igf-ii-induced ct proliferation was decreased by 60.02±7.4% and 55.9±9.6 % respectively (p<0.001, n=4). furthermore, the ability of igf-i-and igf-ii to prevent ct apoptosis (m30 staining) was reduced by 48.3±10.1% and 39.1±7.9% respectively (p<0.05, n=4) after shp-2 knockdown. conclusions: igf stimulation of cytotrophoblast proliferation is mediated by shp-2. exogenous igf rescues cytotrophoblast from apoptosis, and this pathway is also shp-2-dependent. villous endothelial cells. emily j su, zhi-hong lin, ping yin, scott reierstad, joy innes, serdar e bulun. obstetrics and gynecology, northwestern university feinberg school of medicine, chicago, il, usa. background: within the human vascular system, estrogens have been shown to enhance vasodilatation in both normal and abnormal endothelium. estrogenic function occurs by activation of one or both of two estrogen receptors, estrogen receptor-alpha (esr1) and estrogen receptor-beta (esr2). these estrogen receptors are expressed in a wide variety of tissues. within the vasculature, estrogen receptors regulate the expression of multiple vasodilator and vasoconstrictor proteins. specifically, esr2 has been shown to be critical in maintaining normal vascular physiology in a murine model, where esr2 knock-out mice demonstrate significant systolic and diastolic hypertension. we hypothesize that within placental endothelium, estrogen plays an important role in maintaining normal vascular function that is critical for normal fetal growth and development. methods: term placentas from uncomplicated pregnancies were obtained, and the decidua was removed. an iv cannula was inserted into the umbilical vein, which was perfused with a collagenase/dispase solution. the perfusate was collected and subjected to further purification. these cells were cultured in complete medium, and after the initial passage of these cells, purity was confirmed via immunofluorescence and flow cytometric studies. estrogen receptor expression was determined in these cells via western blotting. additionally, these endothelial cells underwent treatment with varying doses of estradiol (10 -9 m to 10 -6 m), and quantitative real-time pcr was performed thereafter for mrna levels of various genes important in prostanoid production. results: western blotting demonstrated that esr2 is the only estrogen receptor expressed within villous placental endothelial cells. estradiol induced cyclooxygenase-2 (cox-2) mrna levels 3-to 6-fold, as quantified by real-time pcr (p<0.001). conversely, there was no effect of estradiol on cyclooxgenase-1 (cox-1). conclusion: these results suggest that estradiol and esr2 are important in mediating the balance of prostanoid production that is essential in maintaining placental vascular health. future studies will further delineate estrogenic effects on prostanoid production within placental endothelial cells in health and disease. supported by the smfm/aaogf scholarship award and the nih grant u54-hd40093. previously we established that proteins secreted by the decidua promote the differentiation of extravillous trophoblasts (evt) from a proliferative phenotype (characterized by cx40, her-1 and alpha5 integrin protein expression) to an invasive phenotype (characterized by her-2 and alpha1 protein expression). the ability of decidua-conditioned media (dcm) to induce trophoblast differentiation was inhibited in the presence of the her-1 receptor antagonist, ag1478. furthermore, dcm-induced jar cell migration was also attenuated in the presence of ag1478. thus, the purpose of this study is to define the role of her signaling in evt differentiation and invasion. methods: evt differentiation was assessed in placental villous explant outgrowths and jar cells using antibodies against markers of the proliferative and invasive phenotypes. trophoblast migration was assessed using jar cells in transwell migration assays. results: treatment of placental villous explants with egf, a her-1 ligand, resulted in the downregulation of her-1 and an upregulation of her-2 expression, as well as an induction of alpha1 integrin expression. pre-treatment of placental villous explants with ag1478 blocked this effect. in the jar cell line, egf treatment mimicked the differentiation-promoting effects of dcm by downregulating her-1 and upregulating her-2 expression, effects that were both blocked when jar cells were pre-incubated with ag1478. in contrast to dcm however, egf stimulation did not induce jar migration. stimulation of jar cells with hb-egf, a her-1/her-4 heterodimer ligand, induced jar migration in a dose-dependent manner. analysis of dcm using antibody arrays confirmed the presence of many members of the egf family including hb-egf. immunohistochemical assessments of placental villous explants verified the expression of her-4 in evt outgrowths and in jar cells; her-4 expression was not affected by stimulation with either egf or hb-egf. conclusions: her signaling is an important and necessary component of the invasive evt differentiation cascade. our data supports a role for her-4 signaling in the induction of the invasive evt phenotype. chandrasekhar thota, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: parathyroid hormone related peptide (pthrp) is expressed in trophoblast cells and may play a role in placental growth and function. studies conducted in pregnant rats using pthrp antagonist showed decreases in fetoplacental growth during mid gestation. objective: to assess the effects of pthrp silencing on the expression of growth factors in immortalized first trimester trophoblast cells (htr-8/svneo cells). methods: htr-8/svneo cells cultured at 37º c and 5%co2 in rpmi-1640 medium supplemented with 5% fbs were transiently transfected with 100nm of three different sirna sequences of pthrp, si1, auaccuaacucaggaaacuuu; s i 2 , g a g c u g u g u c u g a a c a u c a u u ; a n d s i 4 , caagauuuacggcgacgauuu. for control, a scrambled sirna sequence was used. at 90% confluency, cells from each well were split and transfected again in triplicates with respective sirna sequences. total rna was isolated using trizol reagent 24 h after transfection, and protein was extracted using lysis buffer 72 h after transfection. the isolated rna and protein were subjected to reverse transcription and polymerase chain reaction (rt-pcr) and western analysis, respectively, using primers and antibodies specific for pthrp, plgf, vegf and lif. the results are expressed relative to either 18s for changes in mrna expression or -actin for changes in protein expression. results: rt-pcr of total rna obtained from htr cells subjected to double transfection with sirnas for pthrp showed a significant decrease in pthrp expression. expression of growth factors plgf, vegf and lif showed decreases with all the three sirna sequences used compared to the scrambled sequence. western analysis of cell lysates obtained from htr cells subjected to transfection with sirnas for pthrp showed a significant decrease in protein expression of pthrp and vegf. however the protein expression for plgf, and lif decreased in cells transfected with only si4 sequence of pthrp. conclusions: our studies showed that transient transfection of htr cells with sirna for pthrp caused decreases in both mrna and protein expression of pthrp. our results further suggests that decrease in pthrp peptide in transfected cells resulted in a decrease in vegf, plgf and lif suggesting that pthrp may play role in regulating trophoblast cell functions. objective. maternal obesity poses an increased risk to the fetus during pregnancy, and has long term consequences for the progeny. we tested the hypothesis that maternal caloric excess effects growth-related gene expression changes in the murine placenta. methods. female c57bl/65 mice were fed a hypercaloric diet (20% fat, 38% sugar) or standard chow for six weeks prior to mating and throughout pregnancy. near-term (day 18 gestation) the dams (4 controls, 4 overfed) were sacrificed. following placental rna extraction, we used the affymetrix mouse 430a_2.0 array to measure gene expression changes. we performed pathway analysis on regulated genes. results. maternal overfeeding was associated with a two-fold increase in body fat mass. 41 probe sets, corresponding to 37 genes showed differential expression (p < 0.01); twenty-seven of which were up-regulated, and ten down-regulated, as compared to the placenta of control fed dams. of note, several genes related to obesity, diabetes, dna methylation, and the tgf beta pathway were differentially expressed. conclusions. diet-induced obesity in mice was associated with altered placental gene expression, including genes involved in tgf beta signaling and dna methylation pathways. these findings may have important implications for placental growth and epigenetic regulation. (supported by usphs hd-03807, earnest eu framework 6, tommy's the baby charity, uk). objective. maternal dietary protein restriction has been shown to have deleterious effects on placental development, and has long-term consequences for the progeny. to comprehend more completely stress responses to maternal protein restriction, we measured gene expression changes in the mouse placenta. methods. pregnant fvb/nj mice were fed an isocaloric diet containing 50% less protein than normal chow (10% vs. 20% protein content) from embryonic day 10.5 (e10.5) to e17.5. following placental rna extraction, we used the affymetrix mouse 430a_2.0 array to measure gene expression changes. we performed pathway analysis on the regulated genes, and used both qrt-pcr and immunohistochemistry to verify the results. results. the weights of the e17.5 pups were decreased 15% (p<0.05). 244 probe sets, corresponding to 235 genes, were regulated by protein restriction (p<0.001); ninety-one being up-regulated and 153 down-regulated. of particular note, several genes related to the p53 pathway were up-regulated. along with p53 itself, positive regulators of p53 (zmiz1, jmy, hipk2) and genes activated by p53 (inpp5d, cebpa) were induced. for selected genes we confirmed these results using qrt-pcr and immunohistochemistry. conclusions. by microarray analysis, we have described the genetic response to maternal protein deprivation in the mouse placenta. we observed that pups were growth restricted, and genes related to the p53 pathway were regulated. we propose a model through which intrauterine growth restriction is triggered, in part, by activation of the p53 pathway. (supported by usphs hd-03807 and the sgi medical student grant to cpg). purpose: human placental villous tissue cultures have been underused in the study of placental drug disposition. thus we assessed the utility of this model by studying the effect of time in culture on the viability and integrity of the tissue and the, expression and function of proteins involved in the formation and efflux of 1-chloro-2,4-dinitrobenzene (cdnb) conjugate 2,4-dinitrophenyl-s-glutathione (dnp-sg) as a model system for phase ii metabolism and cellular efflux. methods: placental tissue samples were obtained within 30 minutes of cesarean deliveries following normal pregnancies in three patients. villous tissue was cultured in m199 medium to 48hr. at 2, 4, 6, 10, 24, and 48hr post culture, villous tissue was preincubated without or with atpase inhibitor sodium orthovanadate, exposed to 100 μm cdnb, rinsed and incubated in buffer at 37°c to determine formation and efflux of dnp-sg, which was assayed by hplc. changes in expression of gstp1-1, abc transporter isoforms b1, c2 and g2 (abcb1, abcc2, and abcg2, resp.) were assessed by immunoblotting. lactate dehydrogenase (ldh) release, methyl tetrazolium thiazolyl blue (mtt) incorporation, and total tissue glutathione content were monitored up to 48hr. villous tissue morphology was assessed by immunohistochemistry. results: villous tissue structure and protein expression of glutathione-stransferase isoform p1-1 (gstp1-1) and abcg2 remained unchanged over 48hr in culture. expression of abcb1 and abcc2, and total tissue glutathione decreased with culture time. ldh release was unchanged up to 24hr and increased at 48hr, while mtt incorporation remained constant to 10hr and decreased at 24 and 48hr suggesting a decline in tissue integrity and viability at 48hr. however, dnp-sg formation, dnp-sg buffer/tissue ratio, and the extent of inhibition of dnp-sg efflux by sodium orthovanadate remained unchanged through 48hr. sodium orthovanadate decreased the dnp-sg buffer/tissue ratio by 70.5 ±6.90% (p<0.05), consistent with inhibition of apical abc transporters. conclusions: these results support the use of this model to study the coordinated function of metabolizing enzyme gstp1-1 and apical abc transporters in the formation and efflux of the model substrate dnp-sg. the model may be useful to study metabolism and transport of other compounds. syncytiotrophoblast. shauna f williams, ewa fik-rymarkiewicz, stacy zamudio, nicholas p illsley. obstetrics, gynecology and women's health, umd-new jersey medical school, newark, nj, usa. introduction: multiple inputs influence placental protein synthesis. nutritional, endocrine and metabolic factors have been implicated but its regulation has not been investigated. one of the factors shown to be associated with the inhibition of protein synthesis is hypoxia. the goal of this study was to determine the effects of hypoxia on a marker of placental protein synthesis. eukaryotic initiation factor 2 (eif2 ) is a subunit of eif2 which is required for initiation of translation however when phosphorylated, eif2 is unable to participate in the assembly of the initiation complex. hypoxia has been shown previously to cause increased phosphorylation of eif2 . we hypothesized that hypoxia would increase the levels of phosphorylated eif2 in term syncytiotrophoblast, thus inhibiting protein synthesis. methods: primary syncytiotrophoblast cultured from term cytotrophoblast were incubated for 18 hr in atmospheres of 1, 3, 5 or 10% o 2 or in the presence of the hypoxia-mimetic, dimethyloxalylglycine (dmog, 0.02-2.0 mm) in 20% o 2 . cell extracts were analyzed by western blotting to determine the degree of eif2 phosphorylation. results: incubation in 1, 3, or 5% o 2 did not increase eif2 phosphorylation relative to the 10% o 2 control (n=4, separate placental preparations). incubation in dmog concentrations up to 0.5 mm did not affect eif2 phosphorylation however incubation in 2.0 mm dmog increased eif2 phosphorylation by 58 ± 15% (p < 0.05, n=3). conclusions: contrary to our expectations, inhibition of protein synthesis via the eif2 regulatory pathway was not apparent except when induced by the highest concentration of dmog, consistent with severe hypoxia. thus while eif2 phosphorylation does occur, we did not observe changes at dissolved oxygen levels of 1%. these data suggest that a reduction in syncytial protein synthesis via the eif2 pathway takes place only under severe hypoxic stress. (supported by nih hd46982). the increasing prevalence of overweight and obese women of childbearing age is a growing public health concern. the impact of maternal obesity on placental aa transport, which is essential for normal fetal development, remains poorly defined. there are three sub-types of the placental na-dependent system a transporter, snat1, 2 and 4 which mediate neutral aa transport. snat2 is ubiquitously expressed in mammalian tissues and is likely responsible for the majority of placental system a activity. objective: to examine the impact of maternal obesity and over-nutrition on the fetal: maternal (f:m) aa ratio and placental protein abundance for snat2. methods: nonpregnant ewes were randomly assigned to a control (c, 100% of nrc recommendations) or obesogenic (ob, 150% of nrc) diet from -60 to 75 days of gestation (dg). under isofluorane anesthesia, maternal and fetal blood samples were collected for aa analysis by hplc from five twin bearing ewes in each dietary group. after euthanasia, placental cotyledonary (cot) tissue was separated from caruncular tissue, frozen in liquid nitrogen and stored at -80 0 c for western blot analysis. results: fetuses from ob ewes were 26% heavier (p<0.05) than those from c ewes at 75 dg (234± 7 vs. 186±7g). blood concentrations of asn, thr, cit, arg, tau, tyr, trp, val, phe, leu and orn were higher (p<0.05), or tended to be higher (met and lys, p<0.10) in ob than c ewes. in contrast, f:m ratios, for asn, ser, gln, his, gly, thr, cit, arg, b-ala, tau, ala, tyr, trp, met, val, phe, leu, orn and lys were reduced (p<0.05) in ob compared to c ewes. snat2 content in cot tissue was reduced in ob when compared to c ewes (0.5±0.1 vs. 1.0±0.2 arbitrary units; p<0.05). conclusions: maternal obesity in pregnancy reduced expression of placental snat2 protein and efficiency of placental aa transport in ewes, providing a mechanism whereby fetuses may mitigate excessive delivery of aa under conditions of maternal obesity and over-nutrition. decreased aa transport to the fetus may play a role in altered cellular structure and function. nih inbre 1p20rr16474. early gestation utero-placental hemodynamics in an ovine model of fetal growth restriction. lucia dohnal, james s barry, henry l galan, randall b wilkening, russell v anthony. perinatal research center, university of colorado health sciences center, aurora, co. objective: fetal growth restricted (fgr) pregnancies, during late gestation, exhibit altered placental hemodynamics, and reduced capacity for o 2 and nutrient transfer. it was our objective to examine utero-placental hemodynamics and o 2 uptake during early gestation in an ovine model of fgr. methods: singleton-bearing ewes were instrumented with uterine artery flow probes, uterine venous and femoral artery catheters before being placed into a highambient temperature (fgr; n=9) or normothermic (con; n=6) environment at 40 days of gestation (dga). maternal arterial and venous blood, uterine artery flow, heart rate, arterial pressure and respiration rate was collected until 55 dga, at which time umbilical venous blood, fetal weight, placental weight and tissue were harvested. data reported here were analyzed by students t-test. results: maternal respiration rate (153.3±5.4 vs 91.6±9.2 breaths/min) and arterial po 2 (91.0±1.2 vs 85.8±1.0 mmhg) were increased (p 0.01), whereas maternal heart rate (74.3±1.83 vs 88.8±0.03 beats/min), blood pressure (83.7±1.3 vs 94.0±3.2 mmhg) and arterial pco 2 (30.2±1.1 vs 35.6±0.9 mmhg) were reduced (p 0.01) in fgr pregnancies. at 55 dga, fetal weight was not different (p 0.10), but placental (total placentome) weight (85.5±15.1 vs 146.7±27.0 g) was reduced (p 0.05) in fgr pregnancies. while uterine artery (pregnant horn) flow (115.5±13.4 vs 178.4±54.3 ml/min) tended (p=0.064) to be reduced in fgr pregnancies, relative uterine artery flow (4.6±0.5 vs 5.8±0.5 ml/min/g fetus; 157.6±25.8 vs 128.1±17.3 ml/min/100g placenta) was not different (p 0.10). uterine o 2 uptake (mmol/min), relative uterine o 2 uptake (ml/min/g fetus or ml/min/100g placenta) and uterine o 2 extraction (%) were not different (p 0.10) between fgr and con pregnancies. at 55 dga, umbilical vein po 2 (mmhg), o 2 content (mm) and o 2 capacity (mm) were also not different between fgr and con pregnancies. conclusions: reduction in absolute uterine artery flow (ml/min) did not impact utero-placental o 2 uptake or transfer to the umbilical vein, and may have resulted from reductions in maternal cardiac output. relative uterine artery flow was not reduced, suggesting that uterine blood flow and delivery of o 2 to the conceptus does not mediate the ongoing placental growth restriction initiated during early gestation. supported by nih hd43089. barton c staat, 1 anna maria marconi, 2 cinzia paolini, 2 alex cheung, 1 henry l galan, 1 frederick c battaglia. 1 1 obstetrics gynecology and pediatrics, univ of colorado at denver health sciences center, aurora, co, usa; 2 dept of obstetrics and gynecology, san paolo institute of biomedical sciences, university of milano, milano, italy. objective: to determine relative contributions of transplacental flux vs fetal production for myo-inositol and mannose in normal term pregnancies using stable isotopic methodolgy. background: myo-inositol and mannose are important in biologic functions. an external supply of mannose may be required for glycoprotein synthesis. low maternal myo-inositol is associated with spina bifida. mannose concentrations are known to be higher in the mother than the fetus. in contrast, myo-inositol concentrations are higher in the fetus than the mother. what remains unknown is whether fetal levels of these polyols are a result of direct maternal transport or from conversion of glucose. design: four term uncomplicated pregnancies undergoing an elective ceasaran section were infused with 13 c labeled isotopes of glucose, myo-inositol and mannose over 2 hours prior to delivery. maternal samples were obtained prior to infusate being administered, and at 1 hour (h), 1.5h and 2h. fetal concentrations were measured from umbilical artery and vein plasma. the concentrations of labeled and unlabeled glucose, mannose and myo-inositol were measured using high pressure anion exchange chromatogrpahy permitting detection of 10 polyols and sugars at concentrations in the μm range. the feto-maternal molar percent enrichment (mpe) ratio was calculated for each glucose, mannose, and myo-inositol as the ratio between fetal plasma enrichment and the maternal plasma enrichment at steady state. steady state was calculated as the mean of the three maternal samples taken during infusion. results: the feto-maternal mpe ratios of mannose (0.985 ± 0.05, p=0.03) and glucose (0.917± 0.06, p=0.002) were not significantly different from 1.0, consistent with transplacental supply. the feto-maternal ratio for myo-inositol (0.1 ± 0.05, p=0.02) indicates little transplacental flux (10% of fetal inositol derived from maternal plasma). conclusion: in normal term pregnancies, fetal mannose and glucose concentrations are dependent upon maternal transplacental supply. in contrast, fetal myo-inositol concentration is not dependent upon transplacental supply, but fetal demands are met by placental conversion, likely from glucose. christian wadsack, 1 manuela augsten, 1 christian guelly, 2 ursula hiden, 1 ingrid lang, 3 manfred moertl, 1 uwe lang, 1 gernot desoye. 1 1 clinic ob/gyn; 2 center of med res; 3 inst cell biol, histol embryol, med univ graz, austria. background placenta and fetus need lipids for growth and synthesis functions. part of the lipids is supplied from maternal sources by transplacental transfer. recently, we identified in human placenta the high density lipoprotein (hdl) receptor scavenger receptor class b type i (sr-bi). among other functions it mediates hdl-induced ser1177-phosphorylation of endothelial nitric oxide synthase (enos) resulting in enos activation. this mechanism allows hdl to contribute to regulation of vasotonus in arteries. we hypothesized that term placental endothelial cells (ec) express sr-bi at levels different between arteries and veins. methods sr-bi was localized by ihc and quantified by qrt-pcr in rna isolated from arterial and venous vessels. arterial (eca) and venous (ecv) placental ec were rigorously characterized. sr-bi levels were measured by qrt-pcr and immunoblotting. hdl binding and uptake was measured in eca and ecv with 125 i-labelled hdl. hdl from human donors was used to stimulate ser1177 enos phosphorylation. epigenetic regulation was studied by methylationspecific pcrs for 4 cpg-rich promoter regions of sr-bi. pdzk1 a key adaptor for sr-bi mediated enos activation was measured by sqrt-pcr. in situ analyses (ihc, qrt-pcr) showed more sr-bi in arteries than in the vein. the differential expression persisted in vitro in isolated eca and ecv even after 7 passages and culture under same conditions suggesting epigenetic mechanisms regulating sr-bi. however, no methylation was found in eca or ecv. sr-bi was functional since hdl cell association was 2-fold higher in eca than in ecv (89 ± 0.4 vs 49 ± 4.1 ng hdl/mg prot). hdl did not induce ser1177 enos phosporylation in eca or ecv, which was stimulated by ionomycin about 3-fold in both cell types. pdzk1 was undetectable in eca and ecv, whereas it was expressed in placental tissue. conclusion more sr-bi is expressed in ec from arteries than from veins in situ and in vitro. this is not the result of different methylation of sr-bi promoter and, hence, unlikely an epigenetic phenomenon. mechanism of differential expression and its functional consequences for vasotonus regulation is yet unknown. the lack of pdzk1 may account for the failure of hdl to activate enos. (grants 10053, 10896, 11165 oenb). cells. juan a arroyo, brad ziebell, mi-hye park, henry l galan. obstetrics and gynecology, university of colorado andgealth sciences center, aurora, co, usa. introduction: mtor is a protein that regulates cell growth in response to nutrients and growth factors. downstream effectors of the mtor pathway include the p70 and the 4ebp1proteins. activation by phosphorylation of these proteins increases protein synthesis. given that various signaling pathways are regulated by hypoxia in human trophoblast and that mtor is expressed in human trophoblast, our objective was to determine the effects of hypoxia in the activation of mtor, p70 and 4ebp1 in cultured human trophoblast. study designs: trophoblast cell were isolated from term uncomplicated placentas using a trypsin, dnase and disapase solution. cytokeratin immunocytochemistry confirmed trophoblast cells culture purity. trophoblast cells were treated with hypoxia (2% o 2 ) or normoxia (21% o 2 ) for 24 and 48 hours. western blot for p-mtor, mtor, p-p70, p70, p-4ebp1, and 4 ebp1 were done for each time studied. results: trophoblast cells demonstrated: 1) positive staining for cytokeratin, 2) non significant differences for mtor at either 24 (1.5-fold; p= 0.128971) or 48 hours (1.4-fold, p= 0.13181), 3) no differences in p70 protein at 24 (1.1fold; p= 0.128971) or 48 hours (1.3-fold, p= 0. 0.153458), 4) no differences for 4ebp1 at either 24 or 48 hour. conclusion: we conclude that the mtor pathway is not regulated under hypoxic conditions in cultured trophoblast, which suggests that hypoxia does not affect protein synthesis in cultured human trophoblast. however, this may not reflect what happens in vivo in iugr. (supported by nih grant r01 hl071990-01a1). increased expression of phospho-mtor, phospho-p70, phospho-akt and phospho-erk in an ovine model of fetal growth restriction. juan a arroyo, brad ziebell, henry l galan. obstetrics and gynecology, university of colorado and health sciences center, aurora, co, usa. objective: both phosphorylated (p) mtor and p70 are known to be involved in protein synthesis and are regulated by physiological conditions such as fetal growth restriction (fgr). in a hyperthermic (ht) ovine model of fgr we hypothesize that mtor, p70, 4ebp1, erk and akt will be phosphorylated (activated) in the placentae of 130 age (dga) animals. study design: 4 ewes were exposed to ht conditions for 80 days to induce iugr and 4 were placed in ambient conditions. at necropsy (130 dga), placentomes were separated into the maternal (caruncle) and fetal (cotyledon) components and frozen for western blot analysis with antibodies against (p) mtor, mtor, (p) p70, p70, (p) 4ebp1, 4ebp1, (p) erk, erk, (p)akt and akt. results: compared to control animals, fgr animals had smaller fetuses (2914±201g v. 1718±433g; p=0.03) and smaller placentae (349±21g v. 169±22g; p=0.03) at 130 dga. fgr cotyledon showed an increase in p-mtor (1.8-fold; p=0.01), p-p70 (1.8-fold; p<0.008), p-erk (1.4-fold; p<0.008) and p-akt (2.6-fold; p<0.02). in contrast, caruncle (maternal) did not show any changes for the mtor pathway. conclusion: in fgr ovine pregnancies, the fetal placental tissues (cotyledons) showed upregulation of the mtor pathway for protein synthesis via phosphorylation of the p70 but not 4ebp1 while this was not seen in the maternal (caruncle) tissues. in addition neither the cotyledon or caruncle tissues at mid-gestation (95 dga) showed changes in these endpoints, which is prior to the exponential fetal growth that starts at mid-gestation. (supported by nih grant r01 hl071990-01a1). hyperuricemia has long been recognized as a common clinical finding in preeclamptic (pe) women. to date, elevated uric acid concentrations in these women have been considered a marker of disease severity. however preeclamptic pregnancies with hyperuricemia, are associated with an increased frequency of preterm birth and fetal growth restriction. over the past decade several pathogenic roles for uric acid have become evident, raising the possibility of a role(s) for uric acid in the altered vascular and placental functions associated with pe. objective: examine the effects of syncytial uric acid uptake on system a amino acid transport across the human placenta using a primary placental villous explant model. methods: placental villous explants from placentae of healthy, term pregnancies were incubated for 2 hours with uric acid (8.3 mg/dl), corresponding to concentrations of uric acid observed in pe women. these experiments were conducted in the presence or absence of probenecid (10 m), a uric acid cellular uptake inhibitor. system a amino acid transport was subsequently assayed using a radiochemical assay in which na+-dependant uptake of radio-labeled system a substrate, [14c] methyl-amino-isobutyric acid, was measured over 20 minutes. data were analyzed using a paired student's t-test and presented as mean ± sem. results: uric acid attenuated system a amino acid placental transport by 33% (±0.06%, p<0.05). this inhibitory effect of uric acid on system a activity was prevented by probenecid. conclusions: uric acid reduces placental amino acid transport at concentrations observed in pe women. this inhibitory effect of uric acid is dependant upon syncytial uptake of uric acid, being inhibited by the uric acid transporter inhibitor probenecid. these results may be relevant to the increased frequency of fetal growth restriction observed in hyperuricemic pe. additionally the results of this study, indicating a detrimental effect of hyperuricemia on placental function, also suggest a role for uric acid in the pathophysiology of pe. hyperuricemia, a well-documented clinical finding in preeclamptic women, is associated with pre-term birth and intrauterine growth restriction. uric acid is higher in women destined to develop preeclampsia as early as 10 weeks of gestation at a time when cytotrophoblast are invading decidua and myometrium and remodeling uterine spiral arterioles. we propose that elevated concentrations of uric acid may have detrimental effects on placental development in part through inhibition of trophoblast invasion through the decidua. objective: examine the effects of increasing concentrations of uric acid on trophoblast invasion through a reconstituted extracellular matrix. methods: using the in-vitro matrigel invasion assay, the effects of increasing concentrations of dissolved uric acid (4.5 mg/dl, 6.4 mg/dl and 8.3 mg/dl) on the ability of immortalized first trimester extravillous trophoblast cells (htr8-svneo) to invade through a reconstituted extracellular membrane were assessed. the concentrations of uric acid used were comparable to those measured in healthy pregnant women and preeclamptic women with an increase in uric acid of two or four standard deviations above normal. cells that successfully invaded through the matrigel membrane within 48 hours were fixed with methanol, stained with hematoxylin and counted. data were analyzed using a one-way analysis of variance with fisher's post-hoc analysis. results: uric acid attenuated trophoblast invasion in a dose-dependent fashion (p<0.05), with decreases of 29% (± 5.6%), 58% (± 3.6%) and 71% (± 2.2%) respectively compared to untreated controls. conclusions: exogenous uric acid, at physiological and pathological concentrations, is capable of attenuating trophoblast invasion through a reconstituted extracellular membrane in a dose dependent fashion. these results suggest uric acid is a potential contributor to the pathophysiology of altered placental perfusion in preeclamptic pregnancies. previous studies have shown that transforming growth factor (tgf)-is a key inhibitory factors in the invasion of early trophoblast cells, suggesting therefore that overcoming tgf-beta signaling may be necessary for successful implantation. smad ubiquitin regulatory factor 2 (smurf2), a hect type e3 ubiquitin ligase, is a key regulator of tgf-signaling pathway, targeting tgf-receptors and various smads for proteasome-mediated degradation. in this context, smurf2 has been shown to play important roles in embryonic development, cell senescence and tumor formation. as a key regulator of tgfbeta signaling, we wished to determine whether smurf2 has a physiological role during embryo implantation, especially in trophoblast invasion. we have examined the spatio-temporal expression of smurf2 in human placental villi during pregnancy. we have also investigated the possible function of smurf2 in trophoblast cell migration and invasion in a model system involving a human extravillous trophoblast cell line, htr8/svneo. our results showed that expression of smurf2 in placental villi was the highest during the first trimester and the expression decreased in the 2nd trimester. expression of smurf2 was lowest in placental villi at parturition. overexpression of smurf2 in htr8/svneo cells reduced tgf beta type i receptor levels and attenuated the inhibitory effect of tgf-on cell migration and invasion. conversely, rnai-mediated down-regulation of smurf2 resulted in significant increase of tgf-type i receptor protein levels. in contrast, the levels of smad2, another potential target of smurf2, was unchanged. in conclusion, the present study suggests that smurf2 participates in trophoblast cell migration and invasion by down-regulating the expression of tgf-type i receptor. our previous data demonstrated the extensive expression of mmp-26 in various kinds of trophoblast cells in human placenta at the early pregnancy. however, the modulation of the enzyme in trophoblasts is largely unclear. in the present study, the effects of the two types of gonadotropin releasing hormone (gnrh) on mmp-26 expression were examined in an immortalized human cytotrophoblast cell line, b6tert-1 that has been established in this lab. real-time quantitative pcr and western blot analysis revealed that both types of gnrh (gnrh i and gnrh ii) could increase mmp-26 mrna and protein levels in b6tert-1 cells in time-dependent manners. in particular, regulatory effect of gnrh i on mmp-26 expression was concentration-independent, whereas that of gnrh ii was dose-dependent. moreover, both gnrh i and gnrh ii could evidently activate jnk kinase, and sp600125, an inhibitor of a jnk kinase, reversed the up-regulation of mmp-26 induced by either gnrh i or gnrh ii. on the other hand, it is not likely that erk1/2 pathway participates in the signaling of gnrh i or gnrh ii. collectively, our observations suggest that gnrh i and gnrh ii elicit their modulation effects in human trophoblastic cells through jnk pathway leading to up-regulation of mmp-26. during the first trimester of pregnancy, the oxygen tension of the developing trophoblast cells is less than 5%. however, the majority of studies on primary trophoblast cell development have been performed at 20% oxygen. primary third-trimester trophoblast cells are believed to be nonproliferative syncytiotrophoblast cells. we have previously demonstrated that low oxygen tension dramatically affects the differentiation pathway of these cells. we now hypothesize that cell culture in low oxygen tension will improve cell growth and restore proliferation. methods: primary trophoblast cells were purified from third-trimester placenta by enzymatic dispersion and cd-9 negative selection and cultured at 20%, 5% or 2.5% oxygen tension for up to 5 days. the number of cells in culture was assessed by cell counting and by measuring genomic dna. live:dead and mtt assays were used to determine viability. proliferation was assayed with brdu and immunohistochemistry for proliferating cell nuclear antigen. to assess cellular activity, radioactivity of protein precipitated from cells cultured in the presence of tritiated leucine was measured. results: there were no obvious morphologic changes in the cells cultured in different oxygen tensions. the amount of cell loss was directly proportional to oxygen tension: at 20% oxygen 60% of the cells remain in culture; at 2.5% oxygen tension 80% of the cells remained. the cells at 2.5% oxygen tension were proliferating and had a five-fold increased metabolic activity. conclusions: it was previously believed that third-trimester trophoblast cells are non-proliferative. we have demonstrated that low oxygen tension increases the survival of primary third-trimester trophoblast cells. this may reflect the change in the differentiation pathway of these cells. however, the cells also begin to proliferate and increase their metabolic activity. trophoblast cells in vivo form a three dimensional structure which promotes critical complex cell-to-cell interactions that cannot be studied with traditional monolayer cell culture. we developed a substrate-free three-dimensional trophoblast culture system capable of studying cellular interactions without a confounding artificial matrix. methods: nonadhesive agarose hydrogels containing 822 cylindrical recesses 200 m in diameter were cast from molds designed using computer-assisted prototyping. tcl trophoblast cells were seeded into the gels (800,000 cells per) for up to 10 days. viability and cellular stress were assessed and the threedimensional structures of the spheroids were analyzed. results: tcl trophoblast cells formed uniform spheroids within three days of seeding. the spheroids remained intact after being removed from the mold. when placed in traditional cell culture dishes the cells adhered to the plate within one hour and rapidly proliferated into a monolayer. repetitive reseeding allowed easy transition between monolayer and spheroid without affecting cellular morphology. serial sectioning on days 3, 7 and 10 revealed central vacuolization forming a trophoblast vesicle with an outer rim 12.3 m (+/-1 m) thick. this rim size remained constant for at least 20 days. live:dead assay demonstrated that the outer cells remained viable and staining against proliferating cell nuclear antigen demonstrated that the cells were proliferating. the inner cells undergo apoptosis as demonstrated by caspase-3 staining. there is an abundance of vegf staining in the cells remaining in the on the inside of the sphere suggesting a gradient of nutrient or oxidative stress. the formation of a vesicle has been confirmed with confocal imaging. em imaging revealed the structure of the rim. conclusions: trophoblast cells cultured in a novel substrate-free three dimensional system form trophoblast vesicles within 7 days of seeding. these vesicles remain viable after long-term culture and can be repeatedly reformed with repetitive seeding. this new cell culture technique allows us to better study placental cell-cell interactions with the potential of forming microtissues. the transcription factor glial cell missing-1 (gcm1) mediates cell cycle arrest and differentiation of human trophoblast progenitors into villous syncytiotrophoblast and invasive extravillous cytotrophoblast (evt). micro-array analysis of total rna extracted from cultured bewo cells, in which gcm1 mrna and protein were repressed using sirna, identified tissue inhibitor of metalloproteinase-4 (timp-4) in the highest (4-fold) upregulated group of genes. confirmatory rtpcr demonstrated a 7-fold mrna induction. in placental villi, gcm1 acts as a transcription factor promoting expression of the fusogenic protein syncytin1 that mediates syncytial fusion into the overlying syncytiotrophoblast. by contrast, syncytial fusion is uncommon in evt. rather these cells invade several millimeters into the distal myometrium where they transform spiral arterioles. to investigate the role of timp4 and gcm1 in the trophoblast we assessed its mrna by qrt-pcr and protein by western blot in cellular extracts from both bewo cells grown under standard cultivation conditions (synchronized by prior thymidine exposure) and floating cultured first trimester villous explants cultured in 8% oxygen with prior exposure to either gcm1 sirna or anti-sense oligo-nucleotides to gcm1. gcm1 inhibition in the bewo system was associated with a 50-70% increase in timp-4 protein expression and alteration of cell proliferation and differentiation in both models. we are presently utilizing the explant model of evt invasion (explant tips cultured on matrigel in 3% oxygen) to test the hypothesis that gcm1 mediates metallo-proteinase expression and evt invasion via timp-4. presently we conclude that gcm1-mediated evt differentiation involves more than an arrest of mitosis and may include promotion of invasion via repression of timp-4. funding: cihr. scott h purcell, 1 jeremy d cantlon, 1 virginia d winn, 2 russell v anthony. 1,2 1 colorado state university, fort collins, co; 2 university of colorado health sciences center, aurora, co. background: periattachment factor (pf) is a nuclear protein first described in the bovine conceptus. our research in sheep has shown pf mrna concentration peaks when the conceptus is undergoing elongation and initial apposition to the endometrium, and that pf is a nuclear protein localized to the trophoblast. in silico analysis identified a human homolog, hprr15. objective: the objective of this experiment was to determine if pf was expressed in the human placenta, and to develop short-hairpin (sh) rnas for hpf to begin investigating its function. materials and methods: immunohistochemistry was performed on paraffin embedded first and second trimester human placental samples. placental sections were immuno-stained using rabbit polyclonal antiovine pf or anti-human cytokeratin-7. cytotrophoblasts from first trimester pregnancies (n=5) were subjected to an in vitro invasion assay and rna was harvested following 0, 3, and 12 h. quantitative rt-pcr was performed on these samples with intron-spanning primers for hpf, and normalized on hs15 mrna concentrations. based on the human pf sequence, four putative shrna constructs were generated and cloned into a lentiviral expression vector. bewo human choriocarcinoma cells were treated with one of four shrna contructs or an empty vector for 72 h and then rna was harvested from cells for analysis by quantitative rt-pcr. results: periattachment factor was present in the nuclei of both first and second trimester cytotrophoblasts. hpf mrna concentration increased as invasion occurred from 0, 3, to 12 h in all samples; while hypoxia decreased expression at 18 h of invasion compared to 18 h under normoxic conditions. the four lentiviral vectors expressing shrna against hpf resulted in hpf mrna concentrations at 2, 18, 83 and 97% of hpf mrna concentration with the control vector. conclusion: the presence of pf in the human placenta and the increase in pf mrna during cytotrophoblast invasion may indicate this gene plays a role during implantation. we have developed shrnas against pf that result in greater than 98% mrna knockdown and will be using these to begin to elucidate the function of pf in the human placenta, specifically during the invasion process. recently we demonstrated that infusion of imd antagonist (imd 17-47 ) in rat caused distorted labyrinth indicative of a deficient vasculature in placenta. we hypothesize that imd has a role in migration of first trimester trophoblast cell (htr-8/svneo) via regulating human leukocyte antigen (hla-g) and stimulating mek1/2/ erk1/2 phosphorylation. objectives: 1) to asses the effect of imd on migrating capacity of htr-8sv/neo cells using scratch assay in presence or absence of mek and ras/raf inhibitor, u0126 and manumycin a, respectively ; 2) to assess the effect of imd peptide on phosphorylation of mek1/2 and erk1/2 in first trimester htr cells 3) to analyze the effects of imd on the expression of human leukocyte antigen, hla-g, a critical factor involved in the invasion and vascular remodeling of spiral uterine arteries and subsequent pregnancy in human. methods: htr-8sv/neo cells were used to assess the effect of imd (10 -8 m) on the expression of hla-g mrna and phosphorylation of erk 1/2and mek1/2 protein by reverse transcriptase polymeration chain reaction (rt-pcr) and western blot analysis respectively. scratch wound assay was used to determine the migration capacity of htr cells. total rna was isolated from cells using trizol reagent and processed for rt-pcr and results are expressed relative to 18s mrna. trichloroacetic acid was used for the extraction of total protein for western blot analyses. results: our data demonstrates that, 1) imd enhances the migrating capacity of htr cells (compared to the untreated cells) and these effects are inhibited by mek and ras/raf inhibitors, u0126 and manumycin a, respectively; 2) imd (10-8 m ) stimulates phosphorylation of erk1/2 and mek1/2 proteins in htr cells, 3) imd increases the expression of hla-g mrna in htr cells. conclusion: imd promotes migration of first trimester htr cells through mek/ erk signaling pathway and modulates the expression of immunoregulatory molecule, hla-g in these cells. chymotrypsin-like protease promotes the placenta tissue release of sflt-1. yang gu, shuang zhao, david f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: the placenta is a major source of soluble vegf receptor-1 (sflt-1) in the maternal circulation during pregnancy. increased placental release of sflt-1 is believed to play an important role in the pathophysiology and pathogenesis in pe. however, the mechanism of increased placental sflt-1 release in pe is unknown. we recently reported increased chymotrypsin-like protease (clp) activity and expression in placental trophoblasts from pe. in this study, we tested if proteolytic effects of chymotrypsin may play a role in promoting sflt-1 release by placental trophoblasts. methods: placentas delivered by normal pregnant women (n = 5) were used. we tested if chymotrypsin could promote sflt-1 release by placental tissue, in which villous explants were cultured with dmem containing chymotrypsin at 1.0, 2.5, and 5.0 g/ml for 6 hours. the culture medium was then collected for measuring sflt-1. we then determined the specificity of chymotrypsin induced sflt-1 release. villous tissues were cultured with or without chymotrypsin inhibitor (ci) in culture and then the medium was collected and measured for sflt-1. soluble flt-1 was measured by elisa. all samples were assayed in duplicate. data are presented as mean ± se and analyzed by anova. a p level < 0.5 is considered as statistically different. results: 1) sflt-1 concentrations in the medium were increased when chymotrypsin was present in culture and the increased sflt-1 release induced by chymotrypsin was in a concentration-dependent manner: control: 8.25±1.39; 1.0 g/ml: 10.51±1.89; 2.5 g/ml: 13.03±2.06; and 5.0 g/ml: 16.12±2.23 (p<0.01) pg/mg tissue/hour. 2) ci could attenuate sflt-1 release. this inhibitory effect was also revealed in a concentration-dependent manner: control: 5.67±0.92; ci 0.5 g/ml: 4.96±1.30; and ci 5.0 g/ml: 3.08±0.47 (p<0.05) pg/mg tissue/hour. conclusions: increased placental sflt-1 release stimulated by chymotrypsin and decreased placental sflt-1 release inhibited by chymotrypsin inhibitor suggest that the proteolytic effect of clp may play a role in sflt-1 generation. therefore, increased clp activity in placental trophoblasts may contribute to the increased placental sflt-1 production in pe. (supported nih grants hl65997 and hd36822). the change of autophagy-related proteins, lc3 and beclin-1, by tnfa stimulation in cultured primary trophoblasts. soo-young oh, kyung hee kim, suk-joo choi, jong-hwa kim, cheong-rae roh. department of obstetrics and gynecology, samsung medical center, sungkyunkwan university school of medicine, seoul, korea. objective: our previous work have demonstrated that the expression of lc3, but not beclin-1, was increased in placentas from pregnancies complicated by severe preeclampsia (sgi 2007abstract #209) . to understand the regulatory mechanism of these autophagy-related proteins in trophoblast cells, we investigated the changes in these proteins in response to cytokine or hypoxic stimulation in cultured primary trophoblast. material and methods: primary human cytotrophoblasts obtained from normal term placenta were cultured with stimulation of tnf-a or cocl 2 for a given time and the changes of beclin-1 and lc3 were assessed using immunoblot analysis. paired t test was used for statistic analysis. results: tnf-a stimulation induced a significant increase of the expression of lc3-ii in cultured primary trophoblasts while decreasing the expression of beclin-1 (p<0.05 for each). however, cocl 2 stimulation did not induce a significant change of both lc3-ii and beclin-1. conclusions: our data suggests that tnf-a stimulation in cultured primary trophoblasts is associated with increased autophagic activity. background: thyroid hormones play vital roles in the development of the fetal brain. mutations in mct8, recently recognised as a specific thyroid hormone transporter, define a novel syndrome of severe x-linked psychomotor retardation accompanied by elevated serum t3. we previously reported that mct8 expression in n-tera-2 (nt2) cells (a human embryonal cell line with characteristics of cns precursors), as well as mct8-null jeg-3 choriocarcinoma cells, resulted in markedly reduced cell proliferation. further, the s448x mct8 mutation, as reported in males affected by severe psychomotor impairment, resulted in a similar repression of proliferation to wild type, whereas the l471p mutant failed to influence cell turnover compared with control. methods: we now examine the effect of "knocking down" mct8 via sirna and evaluate the effects of cell proliferation (mtt and [ 3 h]-thymidine assays) and tri-iodothyronine (t 3 ) uptake. results: repression of endogenous mct8 expression in nt2 cells by 90 % caused a significant increase in proliferation compared to matched-dose nonspecific sirna treatment, independent of t 3 concentration (8.6 %, 7.2 % and 8.3 % induction at 0, 10 and 50 nm t3, n=3, p<0.02). we also sought to examine the role of mct8 in t3 uptake. in jeg-3 cells, wild type mct8 induced a 1.7-fold increase in the uptake of 125 i-labelled t 3 . by contrast, mutants s448x and l471p failed to significantly augment t 3 uptake, though r271h caused a mild but significant 1.17 fold induction in uptake, hence retaining approximately 25% of wt activity. in parallel experiments, co-transfection of mu-crystallin, a t 3 binding protein, resulted in a similar increase in t3 uptake compared with control (1.9-fold; n=6; p<0.001), implying that mct8 plays only a minor role in thyroid hormone efflux in jeg-3 cells. mutants s448x, l471p and r271h showed analogous responses to those in the absence of mu-crystallin. conclusion: these results further extend the evidence of a potential role for mct8 in the modulation of cell proliferation, independent of t 3 transport. to determine predictors of failure for labor induction in women with preeclampsia. we conducted a retrospective cohort study to examine cesarean delivery rates in all the preeclamptic women at a single institution undergoing labor induction between 1987-2001 with a singleton pregnancy >=24 weeks gestational age (ga). bivariate analyses informed the creation of multivariable logistic models to predict the risk of cesarean delivery using multiple predictors (maternal age, race/ethnicity, unfavorable cervix, gestational diabetes, diabetes, and gestational age). analyses were stratified by parity. our study population included 1,123 preeclamptic women undergoing labor induction. in the bivariate analyses, the risk of cesarean delivery ranged from as low as 10.5% (p=0.01) among multiparous women 24-28 weeks ga to as high as 70.8% (p<0.01) among nulliparous women with diabetes. a total of 1,019 women had adequate data to be included in the multivariable analyses. odds ratios of the predictors are presented in the objective: preeclampsia and cardiovascular disease share many risk factors, and women with preeclampsia are at increased risk of cardiovascular mortality later in life. we investigated whether risk factors associated with cardiovascular disease and preeclampsia remain elevated months postpartum. methods: we measured plasma sflt1, endoglin, plgf, cellular fibronectin (cfn), uric acid, homocysteine, and asymmetric dimethylarginine (adma) in 30 women with uncomplicated normotensive pregnancies compared to 20 women with preeclampsia in samples collected at pre-delivery and again several months postpartum (average 10.6±6.2 months). data are mean±sd or median (interquartile range). statistical analysis was by wilcoxin rank-sum or students unpaired t-tests with statistical significance accepted at p<0.05. results: the mean concentration of sflt1, endoglin, plgf, homocysteine, adma, cfn, and uric acid were all significantly different in samples collected pre-delivery in subjects with preeclampsia compared to controls (table). adma, cfn and uric acid remained significantly higher postpartum in subjects with previous preeclampsia compared to postpartum controls (table) . conclusions: biological markers associated with altered vascular function or cardiovascular risk are elevated in women with preeclampsia, and some remain significantly higher in postpartum preeclamptic women. these data suggest that vascular dysfunction persists in women with previous preeclampsia, and may contribute to the increased risk of future cardiovascular disease. funded in part by national institutes of health nih-5mo1-rr00056 and nih-2po1-hd30367. (ajp, 2007) . as leptin is a known potent angiogenic factor we hypothesized that leptin deficiency and/or resistance to leptin-induced vegf expression might be a mechanism for reduced angiogenesis in mfr offspring. methods: pregnant sprague-dawley rats had 50% mfr from day 10 of gestation until delivery. mfr and control offspring were sacrificed on day 1 of life (p1). some tissues were used to determine the expression of leptin by western blot analysis. for culture experiments, thoracic aortas were dissected, cut into 1-2 mm explants and incubated with leptin (25-100ng/ml) in dmem (10% fbs). after 24 hours of culture, rna was extracted from the tissues and subjected to real time rt-pcr using specific rat primers for vegf, vegfr1 and r2, and ob-ra, stat3 and socs3 (18s mrna as control). culture media was analyzed for vegf protein by elisa. results: expression of leptin mrna and protein in p1 mfr aortas was significantly reduced. in culture, leptin significantly increased expression of vegf, vegfr1 and vegfr2 mrna in explants of aortas obtained from the control but not mfr tissues. as expected, control but not mfr aortic explants secreted significantly more vegf in vitro. to determine the mechanism for resistance to leptin-induced vegf in mfr offspring, we assessed expression of leptin receptor (ob-ra) in explants treated with leptin. leptin was found to induce the expression of ob-ra in aortas from both dietary groups. this upregulation of leptin receptor was accompanied by significant upregulation of stat3 and socs3 mrna in the control tissues. in contrast, in mfr explants only the 50ng/ml concentration of leptin induced an increase in stat3 mrna, and the magnitude of socs3 mrna increase by both concentrations of leptin was significantly less in the mfr explants. conclusion: these results indicate that reduced angiogenesis in mfr vessels is in part due to reduced leptin expression and ability of leptin to stimulate vegf expression. although in vitro leptin induced the expression of its receptor in both groups, it was only in the mfr group in which leptin up-regulated vegf and its receptors. our results suggest that this defect in leptin receptor function in mfr vessels is due, in part, to defects in jak/stat signaling. objective: women with a history of early-onset preeclampsia are at increased risk of developing major cardiovascular disease (cvd) related events, that have a detrimental effect on their long-term health and life expectancy. in this follow-up study, we measured established risk factors predictive of first cvd events after early-onset preeclampsia. study design: over a 10-year interval, 243 primiparous women with a history of early-onset preeclampsia (delivery <34 weeks gestation) were included and tested for major cardiovascular risk factors at least six months after delivery, in addition to a population-based control group of 374 healthy non-pregnant women. women with chronic hypertension were excluded. results: mean age was 30.5 years for cases compared to 28.3 years for controls (p<.001). after adjustment for age, we observed significantly increased mean values for weight (p=.002), body-mass index (p<.001), systolic blood pressure (p<.001), diastolic blood pressure (p<.001), total cholesterol (p=.006), ldl cholesterol (p<.001), triglycerides (p=.027), fasting blood glucose (p<.001), and lower hdl cholesterol (p<.001) in women with previous early-onset preeclampsia. no difference was found for height, smoking, diabetes, and ethnicity. estimated 10-year risk of first cvd events by framingham risk scores remained <10% for all women (low-risk). nonetheless, at mean (sd) 0.7 (1.0) years after early-onset preeclampsia, 15% of women met the criteria for metabolic syndrome, 89% of women exhibited >=1, 51% of women >=2 and 19% of women >=3 major cvd risk factors. conclusion: the majority of women with a history of early-onset preeclampsia exhibit at least one modifiable risk factor for future cvd. although most of these women are classified as low-risk according to the current aha guidelines, this is mainly due to their young age masking other, mostly modifiable, major risk factors. our data thus support life-style intervention programs aimed at primary prevention of cvd in women with a history of early-onset preeclampsia. it has recently been shown that antihypertensive drugs can stimulate cytokine release in normal and hypertensive pregnancy. there is evidence that these cytokines alter the secretion of inhibin a. inhibin a and activin a levels are increased in pre-eclampsia (pe), but it is not known if antihypertensive therapy can affect their secretion. patients and methods we recruited 129 women with hypertensive disorders in pregnancy (63 pe and 66 non-proteinuric hypertension [ht]) and 129 matched normotensive controls. inhibin a and activin a levels, before and 24-48 hours after initiating antihypertensives, were measured in serum and urine, using an elisa. the same markers were measured using validated assays in 84 placentas delivered at cesarean section at similar gestational age (29 pe, 24 ht and 31 controls). analysis the three study groups were compared using anova multiple comparisons with bonferroni post hoc testing. the data were normally distributed after logarithmic transformation. marker levels before and after antihypertensive therapy were compared using paired t-test. we compared placental concentrations between the group which received antihypertensive therapy and the group which did not, using an independent t-test. data were analysed using spss®. in pe, both serum and urine levels of inibin a and activin a were increased at all gestations (p<0.0001). activin a (but not inhibin a) level was also increased at all gestations in ht (p<0.0001). after 28 weeks' gestation (but not before), antihypertensive treatment was associated with a significant fall in both inhibin a and activin a serum levels, and urinary inhibin a, in both pe and ht. the placental concentration of inhibin a, but not activin a, was significantly higher in women with pe compared with controls (p<0.0001). there was no significant difference in either marker between controls and women with ht. the fall in serum levels of inhibin a and activin a following antihypertensive treatment after 28 weeks' gestation may indicate that these drugs have an effect on the pathophysiology of pe other than their known antihypertensive action. pre-eclampsia (pe) is a placental disease of unknown etiology. anti-angiogenic factors, such as soluble fms-like tyrosine kinase 1 (sflt-1) and soluble endoglin (seng), and pro-angiogenic factors, such as vascular endothelial growth factor (vegf) and placental growth factor (plgf), are believed to play an important role in its pathophysiology. maternal plasma concentrations of these markers are altered in pe, even weeks before the clinical manifestations. the aim of this study was to compare the concentration of these markers in placental extracts of normotensive pregnant women, and women with pe and non-proteinuric hypertension (ht). patients and methods placental samples were collected at cesarean section from women with pe (n = 29), ht (n = 24) and normotensive pregnancies of similar gestational age (n = 32). these samples were stored at -80ºc. the frozen tissue samples were homogenised and these four markers measured by specific, validated enzymelinked immunosorbent assays. analysis the three study groups were compared using anova multiple comparisons with bonferroni post hoc testing. the data were normally distributed after logarithmic transformation. data were analysed using spss®. the concentrations of both sflt-1 and seng were significantly higher in the placentas of women with pe, but not ht, compared with controls (p=0.0002). there was no significant difference in plgf concentration between controls and women with pe or ht. placental vegf concentrations in both pe and ht were higher than in controls (p<0.0001); there was no significant difference between the levels in pe and ht (p=0.3). the fact that placental concentrations of sflt-1 and seng mirrored the known rise in serum levels in pe suggests that the placenta is the main source of these circulating factors. although sflt-1 was significantly raised in pe, plgf was not reduced. this suggests that the lower levels of free plgf found in the serum of women with pe are not the result of impaired placental production or secretion, but are due to increased binding by (the increased levels of) sflt-1 in the serum. objective. the purpose of this study was to evaluate whether systematic screening with uterine artery doppler (utad) and serum biochemical markers of oxidative stress, endothelial dysfunction and vasculogenesis performed during the first trimester predict efficiently pre-eclampsia (pet), specifically early-onset pet, in an unselected chilean population. methods. this nested case-control study involved 2831 asymptomatic pregnancies scanned at 11 +0 -13 +6 week of gestation. the subjects for biochemical testing were women who were delivered due to pet (n=43) and normotensive controls (n=129) that were enrolled during the first trimester scan. mean pulsatility index (pi) of the utad was calculated. blood samples were obtained and stored at -84 o c until biochemical analysis of oxidative stress, endothelial dysfunction and vasculogenesis were performed. normally distributed data were analysed by the unpaired t test, and non-normally distributed data by the mann-whitney rank sum test. chi-square tests were used for the comparison of categorical variables. a probability level of p<0.05 was considered significant. multiple logistic regressions were used to develop a combined predictive index. results. there was 13% and 22% significantly increased of the mean pi utad in women who later developed pet or early-onset pet compared to control pregnancies during the first trimester scan. although oxidative stress and endothelial dysfunction biochemical markers were not different between all pet pregnancies and control groups, plasma levels of sflt1 (1762.1 ± 397.5 vs. 1169.4 ± 71.1 pg/ml, p<0.05) and placenta growth factor (22.0 ± 3.5 vs. 49.8 ± 5.4 pg/ml, p<0.016) were significantly higher in women who subsequently developed early-onset pet compared to controls. multivariate logistic regression showed that a combination between abnormal utad and both biochemical markers of abnormal vasculogenesis were the best predictor test for early-onset pet, being its detection rate 56% with 10% false positive rate. conclusion. this study has shown early and selective changes in markers of impaired placentation and angiogenic state in women who later developed earlyonset pet, without alteration in oxidative stress and endothelial dysfunction. supported by fondecyt 1050482. currently it is unknown whether maternal inflammatory changes are specific to pregnancy or reflect an innate susceptibility to inflammation. c-reactive protein (crp) and interleukin-6 (il-6) are markers of the acute-phase inflammatory response and predictive of future cardiovascular events. we compared crp and il-6 levels after influenza vaccination, as an in vivo model for lowgrade inflammation, in non-pregnant women with a history of early-onset preeclampsia and controls with only uneventful pregnancies. methods: forty-four women with a history of early-onset preeclampsia (delivery <34 weeks' gestation) and twenty-nine controls with at least one uneventful pregnancy received an influenza vaccination. we then compared plasma levels of crp and il-6 at baseline, 1.3 days and 3.3 days after vaccination. results: median baseline crp and il-6 levels of women with a history of early-onset preeclampsia were comparable to controls (1.6 versus 0.8 mg/l; p=0.44 and 5.0 versus 3.4 pg/l; p=0.34, respectively). however, high crp and il-6 responses to vaccination were more common in cases (ors for response >75th, >80th, >85th, >90th and >95th percentile based on the distribution of control values of 2.3, 2.7, 3.1, 4.3 and for crp [p for trend 0.11] and of 0.9, 1.4, 1.9, 2.6 and 4.5 for il-6 [p for trend 0.043], respectively). the relationship between high il-6 responses and early-onset preeclampsia persisted after adjustment for body-mass index (p for trend 0.048). conclusion: women with a history of early-onset preeclampsia more frequently exhibit an innate pro-inflammatory phenotype not specific to pregnancy. background altered maternal inflammatory responses play a role in the development of preeclampsia and the hemolysis, elevated liver enzymes and low platelets (hellp) syndrome. we examined whether allelic variants of the innate immune receptors toll-like receptor 4 (tlr4) and nucleotide-binding oligomerization domain 2 (nod2), that impair the inflammatory response to endotoxin, are related to preeclampsia and hellp syndrome. we determined five common mutations in tlr4 (d299g and t399i) and nod2 (r702w, g908r and l1007fs) in 340 primiparous women with a history of early-onset preeclampsia, of whom 177 women developed hellp syndrome and in 113 women with a history of only uneventful pregnancies as controls. in addition, we assessed plasma levels of pro-inflammatory biomarkers c-rp, il-6, sicam-1, fibrinogen and von willebrand factor in a subset of 214 women included at least six months after delivery. after adjustment for maternal age and chronic hypertension, attenuating allelic variants of tlr4 were more common in women with a history of early-onset preeclampsia than in controls (or 2.9 [95% ci 1.2-6.7]). highest frequencies for tlr4 variants were observed in women who developed hellp syndrome (adjusted or 4.1 [95% ci 1.7-9.8]). in addition, high levels of il-6 and fibrinogen were associated with a history of early-onset preeclampsia. combined positivity for any of the tlr4 and nod2 allelic variants and high levels of il-6 was 6.9-fold more common in women with a history of early-onset preeclampsia (95% ci 2.1-23.2) compared to controls. we observed an association of common tlr4 and nod2 gene variants, and pro-inflammatory phenotype with a history of early-onset preeclampsia and hellp syndrome, that suggests involvement of the maternal innate immune system in severe hypertensive disorders of pregnancy. thus, we sought changes in pbef in the serum of patients with mild and severe pe, compared with matched controls. immunodistribution of pbef in fetal membranes and placentas from similar patients was also studied. methods: serum samples (68) were collected with clinical data including; gestational age, medications, ethnicity and recognized complications. patients in labor or infection were excluded. the standard bp and proteinuria criteria was utilized to classify cases for pe grouping; no pe (n=45), mild pe (n=8 bp; 140/90-160/110, proteinuria; trace to 1+ or 5-300mg/24hr urine) and severe pe (n=15 bp; >160/110, proteinuria; >2+ or >5g/24hr, or other associated symptoms). pbef concentration was determined by eia (phoenix pharmaceuticals) in accordance with the manufacturers instructions. fetal membranes and placentas of additional patients, no pe (n=4) and with pe (n=5) were fixed and embedded in paraffin. sections (7um) were immunostained with pbef antibody 1/250 (pheonix) and treated with abc reagent (vector labs) followed by dab (0.5% mg/ml), washed, counterstained, mounted and viewed under brightfield microscopy. results: the concentrations of pbef in serum were between 5-150 ng/ml and were significantly higher in those patients with mild pe (p=0.027) and further significantly elevated in those with severe pe (p=0.003) compared with the matched controls. pbef was detected by immunocytochemistry in the placental syncytiotrophoblast and in the amnion and choriodecidua of the fetal membranes. conclusions: pbef was elevated in the serum of patients according to the degree of pe severity and may be derived from the placenta and/or fetal membranes. (supported by nih #u54rr14607-08 to the pacific research center for early human development, university of hawaii). background: platelet-monocyte aggregation (pma) is a novel sensitive measure of platelet activation and indicates a proinflammatory state (cytokine release). less sensitive techniques demonstrate platelet activation during pregnancy and pre-eclampsia (pe) but platelet activation has not been assessed by pma.objective: longitudinal study of pma in normal pregnancy and pe. methods: 25 healthy, non-smoking primigravida with an uncomplicated pregnancy and 16 primigravida women with pe were studied. pe was defined by standard definitions. informed consent was obtained and the study had ethical approval. serial venous blood collected at 16, 24, 32,37 wks in controls, at time of diagnosis in pe cases and 6 wks post-natal (pn) in all. pma, platelet surface p-selectin (psp-sel) and monocyte cd40 expression (mcd40) were analysed by flow-cytometry and plasma (p) p-sel by elisa. results: groups were matched for mean age and bmi. in controls, pmas, psp-sel and mcd40 expression and pp-sel increased with gestation and decreased post-natally (table 1) . for pe analysis, data was divided into pre-term (sampled at mean 30 wks), and term (mean 38 wks). pp-sel was lower in pre-term pe than control (normal pregnancy 32 wks; p=0.03) there was no significant difference in other measures between pe and control ( objective: much effort has been put into the evaluation of novel markers to identify pregnant women at risk for the development of pre-eclampsia. soluble endoglin (seng) and soluble fms-like tyrosine kinase 1 (sflt1), two antiangiogenic agents, appear to be involved in the pathogenesis of pre-eclampsia. despite several studies describing higher midtrimester serum concentrations of these markers in women with subsequent pre-eclampsia, information on first trimester serum levels is scarce. the aim of this study was to assess seng and sflt1 as first trimester serum markers for the prediction of pre-eclampsia. methods: sera were obtained between 11+2 and 13+6 weeks of gestation from 46 women who later developed late-onset pre-eclampsia and from controls matched for gestational age, maternal age, maternal pre-pregnancy weight, and storage. using commercially available microplate enzyme immunoanalytical methods, seng and sflt1 were determined and the results analyzed using non-parametric statistical tests. results: the serum concentration of seng was found to be increased in women with subsequent pre-eclampsia when compared to controls (mean ± sd, 5.57 ± 1.18 ng/ml versus 5.02 ± 1.01 ng/ml, p = 0.009, unpaired mann-whitney test). similarly, the serum levels of sflt1 were higher in women later developing late-onset pre-eclampsia (1764 ± 757 ng/ml) when compared to controls (1537 ± 812 ng/ml, p = 0.036). sensitivities and specificities for predicting pre-eclampsia were 63% and 57% for seng and 64% and 56% for sflt-1, respectively. the combination of the two markers by multiplication yielded a sensitivity of 64% and a specificity of 55%. conclusion: seng and sflt1, showing increased first trimester serum levels in women with subsequent pre-eclampsia, might both fulfill the characteristics of first trimester markers to predict pre-eclampsia. the combination of the two, however, did not improve the sensitivity nor the specificity compared to their individual determinations. moderate sensitivities and specifities, however, limit the clinical use of these molecules as single markers. pregnancy is associated with increased monocyte/platelet aggregate formation in whole blood. beth a bouchard, 1 adrienne schonberg, 2 gary j badger, 3 ira m bernstein. 2 1 biochemistry; 2 obstetrics and gynecology; 3 medical biostatistics, univ of vt, burlington, vt, usa. background preeclampsia is associated with increased rates of platelet clearance, changes in platelet function and platelet activation. the goal of the current study was to examine basal levels of platelet activation through pregnancy beginning prior to conception, and to examine the association of platelet activation with the development of hypertensive complications during pregnancy. methods two indices of platelet activation, platelet cd63 expression (%cd63) and monocyte/platelet aggregate (%mp) formation, were measured in whole blood by flow cytometry using specific, fluorescentlylabeled monoclonal antibodies in 16 healthy, nonsmoking women during the follicular phase of their menstrual cycle (pp, cycle day 7.5+0.9). all women subsequently conceived singleton pregnancies and were re-examined in early (ep, 11-16 wks) and late pregnancy (lp, 31-34 wks). five of these women were diagnosed with hypertensive complications (4 hypertension, 1 preeclampsia) at term although hypertension was not observed at any study time point. data are expressed as mean±sem. p<0.05 was accepted for significance. results subjects were 29.1+0.9 years old with a bmi of 23.5+0.9 kg/m 2 at the time of prepregnancy studies. a significant increase in the %mp formed over time of pregnancy was observed (p=0.015). there was little change in the %mp formed between pp and ep (pp, 6.0±3.8 % and ep, 10.4±3.8 %, p=0.384). however, the %mp increased significantly in lp (20.9±3.8 %) as compared to pp (p=0.005) and ep (p=0.041). this increase occurred independent of the development of hypertensive complications (p=0.657) and independent of pp platelet activation status. although statistically significant increases in cd63 expression were not observed, the change in cd63 expression over pregnancy correlated with the change in %mp over pregnancy (r=0.56, p=0.024) and cd63 expression correlated with %mp in lp (r=0.57, p=0.020). conclusion these combined observations suggest that pregnancy is associated with increases in levels of unstimulated platelet activation and that these increases occur in the presence or absence of subsequent hypertensive complications. furthermore, we observed a correlation between the changes in two distinct platelet activation events, %mp and cd63 expression, during pregnancy. supported by nih hl 71944. backgound sildenafil citrate (sc) has been proposed as a therapy to improve uterine perfusion in pregnancies complicated by iugr or preeclampsia. we sought to determine the effects of sc on uterine vascular resistance, uterine blood flow and cardiac output in young healthy nulliparous women. methods eleven young healthy nulligravid women were studied during the luteal phase (cycle day 22 + 5) of the menstrual cycle. women were randomized in a double-blind fashion to receive placebo (pl), or sc at a dose of 25 or 100 mg. uterine artery vascular resistance, uterine artery volumetric flow, brachial artery volumetric flow and cardiac output were measured at baseline and at 1 and 3 hours post dosing employing color doppler ultrasound. comparisons were made by anova between those randomized to pl (n=5) versus sc (n=6). p<0.05 was accepted for significance. data are expressed as mean + s.e.m. results there were no significant differences in subject age, cycle day, body mass index, uterine blood flow, brachial blood flow or cardiac output at baseline comparing the two groups. there was a tendency towards increased uterine blood flow in subjects randomized to receive sc (68% increase) compared to pl (no change), changes in uterine blood flow, brachial blood flow and cardiac output are outlined in the objective reduced maternal plasma volume in the third trimester has been associated with both fetal growth restriction and preeclampisa. we sought to determine the degree to which third trimester plasma volume is dependent on plasma volume prior to pregnancy. methods sixteen young (29.1 + 2.9 years) healthy nulligravid women had their plasma volume measured during the follicular phase (cycle day 7.5 + 3.6) of the menstrual cycle and subsequently conceived. subjects were predominantly caucasian (15/16) with a mean prepregnancy bmi of 23.5 + 3.5 kg/m 2 . plasma volume was re-estimated at 32-34 weeks gestation. all patients were placed on sodium and total calorie balanced diets for 3 days prior to each plasma volume determination. plasma volume was determined employing evans blue dilution with multiple post injection sampling time points. data are expressed as mean + s.d. results baseline prepregnant plasma volume was 2,868 + 446 ml or 123 + 18 ml/unit bmi. third trimester plasma volume was 4,306 + 663 ml representing a 51 % increase (p<0.001). the range of plasma volume expansion was 20-84% dependent upon prepregnant plasma volume. plasma volume in the third trimester of pregnancy was strongly correlated to prepregnant plasma volume r= 0.65 (p=0.006). plasma volume expansion was consistent across the range of prepregnancy plasma volume. conclusions pre-pregnancy plasma volume contributes approximately 42% of the variance in third trimester plasma volume. the observed increase is plasma volume is independent of prepregnancy volume resulting in a greater percentage increase for those starting at the lower end of the plasma volume range. as third trimester plasma volume is strongly associated with pregnancy outcome the correlation of prepregnancy plasma volume to third trimester plasma volume suggests that prepregnancy status contributes to these adverse reproductive events.this work was supported by nih hl 71944. background: hydatidiform mole is a rare disorder of pregnancy and may predispose the mother to severe morbidity. molar pregnancies are known to be associated with high risk for the development of early onset preeclampsia. in recent years, the expression of sflt-1 (soluble vegfr-1) was found to be increased in preeclampsia, and contributes to the pathogenesis of the maternal systemic disease. the objective of the present study was to examine the expression of sflt-1 in placentae from molar pregnancies. methods: placental samples from unique cases of twin pregnancies with complete molar pregnancy in one sac and developing fetus in the other sac were prospectively collected (n=2). the first set delivered at 23 weeks due to excessive bleeding. the second set delivered at 26 weeks due to severe iugr and elevated blood pressure. mrna level of sflt-1 was measured by quantitative real-time pcr using specific taqman primers and probe. protein expression of sflt-1 in placental tissue lysates were measured by western blot analysis using a polyclonal antibody against flt-1. immunohistochemistry of paraffin embedded samples was performed using specific antibody for sftl-1. results: mrna level of sflt-1 was increased by 2.3 fold in the molar placentae compared to matched controls. the placentae of the developing fetuses which were growth restricted exhibited 2.7 fold increase compared to controls. sflt-1 protein expression in the molar placentae was increased by 7.7 fold compared to controls, while the co-twin placentae exhibited a 1.4 fold increase compared to controls. immunohistochemistry revealed strong positive immunoreactivity for sflt-1 in the trophoblast layer of both molar pregnancies and iugr co-twin relative to controls. conclusion: our data suggest that sflt-1 expression is increased in placentae from molar pregnancies and thus may explain the increased risk for developing early onset preeclampsia. the expression of sflt-1 in the growth restricted twin placenta is also increased compared to controls and support our previous observation (supported by cihr and owh/igh). (n=145); 2) neonates of patients with pe (n=104); and 3) sga neonates (n=48). cord blood was collected immediately after delivery and pz plasma concentrations were measured by elisa. pz deficiency was defined as a cord plasma concentration <5 th percentile of the normal pregnancy group. non-parametric statistics were used for analysis. results: 1) cord plasma pz concentration differed significantly among the 3 study groups (kruskal wallis, p<0.001); 2) neonates of patients with pe and sga neonates had a significantly lower median cord plasma pz concentration than those delivered after normal pregnancy (pe: median 0.45 g/ml, range 0.05-1.68, p<0.001; sga: median 0.46 g/ml, range 0.06-3.56, p=0.011; normal pregnancy: median 0.6 g/ml, range 0.04-1.17); 3) there were no differences in the rate of pz deficiency among the groups; and 4) there was no relationship between placental histologic findings and median cord plasma pz concentrations between and among the sga and pe groups. conclusions: 1) at the time of delivery, the median cord plasma pz concentration was lower in sga neonates and those born to women with pe than in neonates born to normal pregnancies; 2) there was no difference in the rate of pz deficiency among the study groups, suggesting that the lower median pz cord blood concentrations in pe and sga groups may result from activation of the coagulation cascade rather than an inherited pz deficiency. objective: periconceptional multivitamin (mv) use may be related preeclampsia risk. we examined the relation between timing and frequency of periconceptional multivitamin use and the risk of preeclampsia. methods: women in the danish national birth cohort who delivered singleton liveborn infants (n=26,133) reported upon enrollment at 10.8 weeks (sd 3.6) the number of weeks of regular multivitamin use during a 12 week periconceptional period (lmp-4 to lmp+8). preeclampsia cases were identified using icd-10 codes (n=605, 2.3%). logistic regression was used to estimate the effect of frequency (number of weeks of use) and timing of use to lmp+2] and post-conception [lmp+3 to lmp+8]). results were stratified a priori by overweight status. results: overall, 18,551 women (71%) reported mv use in the periconceptional period. after adjustment for bmi, smoking, parity and chronic hypertension, infrequent mv use (<4 weeks of use) had no relation to risk (or 1.15; 95% ci 0.92,1.44) but regular use (>=8 weeks) was associated with modestly reduced risk (0.84 (0.67,1.06). similarly, when mv use was modeled as a continuous variable, each additional week of use was related to reduced risk for preeclampsia (or 0.83, 95% ci 0.67-1.02). this potential dose effect of periconceptional mv use appeared to be limited to normal weight women (bmi <25 kg/m², or 0.74; 95% ci 0.55-1.01), with no apparent effect among overweight women (bmi 25 kg/m², or 0.93; 95% ci 0.68-1.26). a total of 7,332 women reported regular mv use in both the preconception and post-conception periods, and 5,630 women reported regular use only in the post-conception period. among normal weight women, regular use in the preconception period had no effect on preeclampsia risk (or 1.29, 95% ci 0.89-1.85). in contrast, use in the post conception period was associated with reduced risk for preeclampsia (or 0.56, 95% ci 0.39-0.82). conclusions: regular periconceptional mv use was associated with a modestly reduced risk for preeclampsia among normal weight women. if causal, mv use immediately after conception appeared to be the critical exposure window. background: pre-eclampsia (pe), a disorder of pregnancy characterized by maternal inflammation, results in immune, cardiovascular and metabolic dysfunction. in non-pregnant persons, inflammatory disorders are treated with and prevented by pharmaceuticals and lifestyle methods such as physical activity (pa). while most pharmaceuticals are contraindicated for pregnant women, pa during pregnancy has been found safe, healthy and beneficial for both mother and baby. clinical evidence has found pa can beneficially affect pregnancy outcome, decrease excessive inflammation and decrease the risk of pe. epidemiological studies indicate that pa may be useful in preventing pe. unfortunately, previous studies have quantified pa based on recall of postpartum women and have not controlled for differences in women's interpretations of amount, type or intensity of pa. however, investigating pa utilizing a laboratory-based exercise intervention to control these variables inflicts difficulties translating the intervention into a community-based program that attracts and retains pregnant women in order to enhance public health. method: a retrospective study was performed to determine the rates of pe among the 9,160 women who gave birth at yale-new haven hospital (ynhh) during 2004 and 2005, and a 90 person subset of this group who performed prenatal pa in a community-based program that is evidence-based and standardized, thereby controlling for type and intensity of pa. additionally, the program is established in the community, has been offered to the public for 30 years and is internationally known. results: during 2004 during -2005 , the pe rate for the general population at ynhh was 7.8%. for the pa group, the rate was 2.2%. two women in the pa group were diagnosed with pe in the last month of pregnancy and delivered normal infants at term. no pe was observed in this group (pa group) during the second or early third trimesters nor was there any prematurity in this group. significance: these findings support the hypothesis that adequate physical activity provided in a standardized community-based group setting may provide a non-pharmacological approach for preventing pe. growth restriction. margreet plaisier, 1 esther streefland, 2 pieter koolwijk, 3 frans m helmerhorst, 1 jan jaap hm erwich. 2 1 department of gynecology and reproductive medicine, leiden university medical center, leiden, netherlands; 2 department of obstetrics and gynecology, university medical center groningen, groningen, netherlands; 3 department of physiology, vu univerity medical center, amsterdam, netherlands. objective: disturbances in decidual and placental vascular development may play a role in the pathogenesis of pregnancy complications, like pre-eclampsia (pe) or fetal growth restriction (fgr). whether the regulation of decidual vascular adaptation to implantation is altered in these illnesses, is not elucidated yet. the present study focused on the role of first-trimester angiogenic factors in the pathogenesis of pe and or fgr. methods: first-trimester decidua samples were obtained during routine chorionic villous sampling. the expression of vascular endothelial growth factor (vegf-a), placental growth factor (plgf), flt-1, kdr, angiopoietin-1 (ang-1), angiopoietin-2 (ang-2) and tie-2 mrna was determined by rt-pcr. the expression of the angiogenic factors was related to the pregnancy outcome, i.e. uncomplicated, pe or fgr. results: the first-trimester decidual tissues expressed all angiogenic factors. mrna levels of vegf-a, plgf, kdr, ang-1, ang-2 and tie-2 appeared increased in fgr cases compared to matched controls. in addition, plgf, ang-1 and tie-2 mrna appeared increased in pe cases compared to matched controls. the differential expression of angiogenic factors was more pronounced in cases with fgr than pe. the large inter-individual variation disallowed a significant outcome. conclusions: various angiogenic factors showed differential mrna expression in 1 st trimester decidua of patients developing pe or fgr in later pregnancy compared to their matched controls. the first-trimester decidual samples provided a unique opportunity to obtain information regarding the onset of pe and fgr. early 1 st trimester changes in angiogenic factor expression may well occur as a compensatory mechanism. in turn, this may set the stage for increased non-branching angiogenesis and altered decidual and placental vascular adaptation, which may be part of the pathogenesis of pe and/or fgr. complications. beth a bouchard, 1 adrienne schonberg, 2 gary j badger, 3 ira m bernstein. 2 1 biochemistry; 2 obstetrics and gynecology; 3 medical biostatistics, univ of vt, burlington, vt, usa. background preeclampsia is characterized by endothelial dysfunction. the goal of the current study was to prospectively measure plasma levels of the soluble endothelial cell adhesion molecules, sicam-1 and svcam-1, beginning prior to pregnancy and determine if subjects destined to develop hypertension complicating pregnancy had differences in the concentrations of these molecules. methods serum levels of sicam-1 and svcam-1 were measured in 16 healthy, nonsmoking women (cycle day 7.5 + 0.9, prepregnancy) by elisa. all women subsequently conceived singleton pregnancies and were re-examined in early (ep, 11-16 weeks) and late pregnancy (lp, 31-34 weeks). five of these women developed hypertensive complications (4 gestational hypertension, 1 pre-eclampsia) near term. all subjects were normotensive at all study time points. data are expressed as mean ± sem. p<0.05 was accepted as significant. results subjects were 29.1+0.9 years old with a mean bmi of 23.5+0.9 kg/m 2 at the time of prepregnancy studies. significant differences in sicam-1 levels as a function of pregnancy were observed (p=0.046) and are outlined in table 1 p=0.662 differences were dependent upon the stage of pregnancy in those women who were not diagnosed with hypertensive complications with a decrease in sicam-1 levels in ep (p=0.024) followed by an increase in sicam-1 levels in lp (p=0.044). in women with hypertension in pregnancy, these differences in sicam-1 levels were not evident (p=0.66). there were no differences in sicam-1 levels comparing women with or women without hypertensive complications prior to pregnancy (p=0.971). in contrast to sicam-1, we observed no significant differences in svcam-1 levels over pregnancy or between those with and without hypertension. conclusions these combined observations suggest that levels of the soluble adhesion molecule sicam-1 change significantly over time in normal pregnancies. subjects destined to develop hypertension did not demonstrate the early pregnancy reduction in sicam-1. supported by nih hl 71944. yuval bdolah, 1 uriel elchalal, 1 shira natanson-yaron, 1 hadas caspi, 1 tali bdolah-abram, 1 angelika bord, 1 caryn greenfield, 1 debra goldman-wohl, 1 ariel milwidsky, 1 franklin h epstein, 2 s ananth karumanchi, 2 simcha yagel, 1 drorith hochner-celnikier. 1 1 ob/ gyn, jerusalem, israel; 2 ob/gyn medicine, beth israel deaconess medical center, harvard medical school, boston, ma, usa. objectives: nulliparity is a risk factor for preeclampsia (pe) with a reported incidence of up to 4-5 times higher than multiparous pregnancies. soluble fms-like tyrosine kinase-1 (sflt1), a circulating anti-angiogenic molecule of placental origin plays a pivotal role in pe by antagonizing placental growth factor (plgf). increased sflt1 and sflt1/plgf have been shown to antedate clinical signs in pe. we therefore hypothesized, that the higher risk of pe in nulliparous pregnancies is associated with high sflt1 (or sflt1/plgf). methods: maternal serum samples from nulliparous (n=68) and multiparous (n=159) term singleton pregnancies without pe, at the time of admission to delivery room, were used. serum samples were analyzed for levels of sflt1 and plgf by elisa. statistical analysis was performed applying t-test and the kruskall-wallis test and using spss software. results: for nulliparous and multiparous pregnancies, the mean serum sflt1 levels were 13,385 ± 960 and 10,584 ± 722, (p=0.021), the mean serum plgf levels were 215 ± 15 and 249 ± 14 (p=0.117), and the mean ratios of sflt-1/plgf were 98 ± 13 and 65 ± 6 (p=0.025), accordingly. in a subgroup of 10 multigravidous nulliparous pregnancies, sflt1 levels were 14,063 ± 2295. correcting for maternal age did not alter the results. moreover, results did not differ between multiparous pregnancies with a 5-10 years interpregnancy interval compared with a 1-5 years interval. conclusions: in nulliparous pregnancies, circulating sflt1 levels and sflt1/ plgf ratios are significantly higher than in multiparous pregnancies. these findings suggest that the increased risk of pe in nulliparous pregnancies may involve anti-angiogenic imbalance. nulliparity may be more substantial than primigravity, as a risk factor for pe, suggesting that first semester abortions in primigravidas may not protect from pe in a subsequent term pregnancy. nevertheless, even 5-10 years intervals from the previous gestation do not increase the risk for pe. different normograms of angiogenesis should be used, when assessing the risk for pe in multiparous versus nulliparous pregnancies. objective: we determined whether maternal serum levels of angiogenic proteins namely soluble fms like tyrosine kinase (sflt-1), soluble endoglin (seng), and placental growth factor (plgf) -measured during the first trimester are associated with the subsequent development of placental abruption. methods: we performed a prospective, nested case-control study of women enrolled in the massachusetts general hospital obstetric maternal study (moms). first trimester serum samples from 34 placental abruption cases and 250 normal pregnancies were measured for angiogenic factors. cases and controls were matched by body mass index and age. placenta abruption was diagnosed by standard clinical findings and pathological examination of the placenta. women with confirmed preeclampsia or chronic hypertension were excluded. results: compared to controls, cases had more pregnancies, delivered infants at an earlier gestational age and with lower birth weight. first trimester levels of seng were significantly increased in cases compared to controls: 7.95 ± 0.48 ng/ml vs. 6.48± 0.15 ng/ml, p <0.05. there were no significant difference in serum levels of plgf, 40.31± 6.22 ng/ml versus 40.04± 1.79 ng/ml, p=ns, although sflt1 levels were lower in cases: 1.86 ± 1.7 ng/ml vs. 2.7±0.98 ng/ml, p=ns. in logistic regression analysis adjusted for age, race, smoking, number of pregnancies, gestational age at delivery, gestational age of blood sampling, and blood pressure at first prenatal, seng levels remained independently associated with subsequent risk (odds ratio 1.3, 95% ci 1.1-1.6) of placental abruption. examining this relationship by tertiles of seng, in the unadjusted model, women in the second (or 7.4, 95% ci 1.6-33.5) and third (or 8.8, 95% ci 1.9-39.1) tertiles were at increased risk of developing placental abruption compared with women in the lowest tertile. after adjusting for known risk factors of placental abruption, women in the second (or 13.1, 95% ci 1.3-129.09) and third (or 13.3, 95% ci 1.9 95.3) tertiles remained at increased risk for placental abruption. conclusion: increased first trimester maternal serum levels of seng are associated with increased risk of subsequent placental abruption. 4 ob/gyn, univ of vermont. shear stress is the most potent physiologic stimulus for elevating endothelial no production for flow-mediated vasodilatation. we measured in vivo shear stress during the proliferative and secretory phases and at 12 and 32 weeks of gestation hypothesizing that uterine blood flow (ubf) elevations in turn increase shear stress in early and late gestation. methods: during proliferative, secretory phases, and at 12 and 32-34 weeks of pregnancy ua blood velocity and internal radius were measured bilaterally using color doppler ultrasound. blood viscosity was measured at shear rates in excess of 60/sec. results: compared to the proliferative phase viscosity was decreased at 12 weeks and more so at 32 weeks gestation (p<0.05). 0.096 ± 0.002 0.096 ± 0.002 nsd 0.13 ± 0.004*** 0.17 ± 0.008*** ua velocity (cm/sec) 9.3 ± 0.57 15.3 ± 1.0*** 31.4 ± 3.0*** 55.7 ± 7.7*** ubf (ml/min) 15.0 ± 0.9 27 ± 2*** 101.8± 12.9*** 294.2 ± 40.3*** shear stress (dynes/cm 2 ) 20.7 ± 1.5 37.8 ± 2.62*** 37.1 ± 3.8*** 47.9 ± 6.8*** internal ua radius was not altered by the menstrual cycle, and was greater at 12 weeks (+35%) and 32 weeks (+77%). compared to proliferative, secretory phase showed significant rises in unilateral ubf and velocity that rose progressively during gestation. in contrast, shear stress increased in secretory (80%) and did not rise further in early pregnancy but by 32 weeks shear stresses was further elevated 131%. conclusions: equivalent rises in shear stress during the secretory phase and 12 weeks gestation demonstrate increases in radius and profound remodeling of uas that reflect the physiologic process of "normalization of shear". by late gestation, continued but modest rises in radius illustrate that further increases in shear stress occur almost solely due to rises in ubf via falls in down stream impedance. continued rises in shear stress into late gestation provide progressive stimuli for no production by ua endothelium. nih hl49210, hd38843, hl63101 background: hypertension during pregnancy is associated with altered uterine vascular reactivity and blood flow, although its effects on arterial myogenic behavior have not been explored. the purpose of this study was to evaluate the effects of hypertension and no inhibition on myogenic tone in pregnancy, as the ability of a vessel to constrict and dilate in response to pressure plays a key role in regulating blood flow to the uterus. methods: three groups of sprague dawley late pregnant (day 20) rats were used: control (n=5), hypertensive (0.5g/l l-name in the drinking water, n=9), and treated with l-name and hydralazine (also in the drinking water, 0.272 g/l, n=5) to prevent the blood pressure increase, yet maintain no inhibition. resistance-sized radial arteries (<150 m) were mounted in a pressure arteriograph and equilibrated at 60 mmhg (in pss containing l-nna and indomethacin) to induce a myogenic response. vessels were then subjected to pressure steps from 20 to 200 mmhg. tone (%) was calculated by comparing the vessel diameter at each pressure with the passive diameter at the same pressure (determined by incubation with 0.1mm papaverine and 10 m diltiazem). results: myogenic tone developed in controls (46±5% maximal), and was maintained over a broad range of transmural pressures . arteries from the l-name group did not develop tone at any pressure. co-treatment with hydralazine reinstated tone (33±4% maximal) over the same range of pressures as in the control group. the reduction in average placental weights in the l-name group (401 vs. 425mg, p<0.05) was restored by hydralazine (433mg, p<0.05 vs. l-name). average fetal weights were also reduced in the l-name group (1.96 vs. 2.22g, p<0.001), but only partially restored by hydralazine co-treatment (2.11g, p<0.05 vs. control and l-name groups). conclusions: surprisingly, radial uterine arteries from the l-name group did not develop tone over any range of pressures, despite the fact that matched arteries from late-pregnant controls developed significant myogenic tone. this abolishment of tone was reversed by hydralazine, which also had beneficial effects on fetal and placental growth. these results implicate hypertension rather than no inhibition as the key factor in the suppression of myogenicitiy and dysregulation of uterine blood flow. charles r rosenfeld, timothy roy. division of neonatal-perinatal medicine, ut southwestern medical center at dallas, dallas, tx, usa. background: upbf b rises 30-fold in ovine pregnancy; but the mechanisms responsible for the rise and maintenance are unclear. we (jsgi 2005) reported that uterine vascular smooth muscle bk ca k + channels contribute to uterine vasodilation and upbf b maintenance; but up-stream activators are unclear. uterine vascular prostacyclin synthesis increases in pregnancy, but cyclooxygenase inhibition does not alter upbf b (ajp 1992) . vascular nitric oxide synthase (nos) also increases; but acute inhibition with l-name decreases upbf b only 25% (jci 1996) . it is unclear if l-name doses were insufficient or if prolonged nos inhibition has a greater effect. objective: to determine if local nos inhibition with l-name dose-dependently decreases upbf b and if prolonged inhibition exerts a greater effect on upbf b . methods: 11 pregnant ewes were studied at 100-148d gestation age (ga). 4 had doseresponse studies with uterine artery l-name infusions to achieve 0.025-1.0 mg/ml over 30min. 7 had 72h arterial l-name infusions to achieve and maintain levels of 0.01 mg/ml at 107 (n=6), 122 (n=7) and 135d (n=7) ga, while measuring arterial pressure (map), heart rate (hr) and upbf b before, during (2,4,6,24,30,48,54 and 72h) and after (72, 96h) infusions. uterine arterial and venous cgmp were measured. data were analyzed by anova. results: acute nos inhibition decreased upbf b 0-20%, but was not doserelated. 72h arterial l-name infusions decreased upbf b by 2-6h at each ga (p 0.025, anova) and values returned to baseline by 96h postinfusion. sensitivity did not differ between ga (p=0.1, anova), upbf b falling 20-30% at each ga. contralateral upbf b was unaffected at all ga. map rose and hr fell during infusions at 107 and 122d ga, p 0.03; but were unaffected at 135d ga. venous-arterial cgmp concentration differences were seen at 107d and absent at 72h of l-name infusion, p=0.04. conclusions: uterine vascular nos increases in ovine pregnancy, but its inhibition decreases upbf b 30%, suggesting study doses were insufficient to fully inhibit vascular nos activity. alternatively, nos contributes to the maintenance of upbf b , but other mediators, not yet identified, are more important in activating bk ca and regulating upbf b . notably, l-name reached the systemic circulation, and although further diluted, map rose 5-10%, suggesting the systemic vasculature may be more sensitive to nos inhibition than upbf b . charles r rosenfeld, xiao-tie liu. division of neonatal-perinatal medicine, university of texas southwestern medical school, dallas, tx, usa. background: uteroplacental blood flow (upbf) rises 40-fold in ovine pregnancy, reflecting increases during pre-implantation, placentation and finally, in the last third of gestation. we reported that bk ca density in uterine vascular smooth muscle (uvsm) increases in ovine pregnancy (sgi 2007) and inhibition with tetraethylammonium ( 0.3 mm) dose-dependently decreases basal upbf 80% in the last third of pregnancy (jsgi 2005) . it is unclear how bk ca subunit expression changes and activity is regulated in pregnancy; since uterine vascular nitric oxide is increased, signaling via cgmp-dependent protein kinase g (pkg) is one potential pathway. objective: to determine simultaneous changes in uvsm bk ca density and regulatory 1-subunit expression and the cgmp signaling pathway for activation in ovine pregnancy. methods: endothelium-denuded segments of 2 nd generation uterine arteries obtained from nonpregnant (n=3), pregnant (n=14, 56-145d gestation; term 150d) and postpartum (n=8, 2-56d) sheep were used to measure expression of bk ca -and regulatory 1-subunits and signaling proteins in uvsm by immunoblot analysis and immunohistochemistry. results: uvsm bk ca density, reflected as a change in the 100 kda -subunit, rose 70% with placentation (p<0.05) and was unchanged thereafter. expression of the 39 kda regulatory 1-subunit paralleled the rise in bk ca density during placentation, increasing 50% (p<0.001), but increased another 2-fold and exponentially in the last third of pregnancy (p<0.001, r 2 =0.99, n=13). changes in subunit immunostaining in uvsm paralleled increases in protein. although uvsm soluble guanylyl cyclase was unchanged in pregnancy (p=0.8, r=0.08, n=14), pkg expression rose 2-fold (p=0.002, r=0.86, n=11) and gradually returned to nonpregnant levels by 30d postpartum (p=0.008, r=0.83, n=9). uvsm cgmp is being measured. conclusions: these are the 1 st data suggesting that increases in ovine upbf during placentation involve vascular growth and bk camediated vasodilation. the rise in upbf in the last third of pregnancy reflects bk ca -mediated vasodilation due to enhanced channel activation via increases in uvsm pkg, bk ca phosphorylation and changes in subunit stoichiometry due to an exponential rise in the regulatory 1-subunit. it is unclear what initiates and directs these changes in bk ca expression and sensitivity. relaxin (rlx) is a hormone traditionally associated with changes in the female reproductive tract during pregnacy. recent evidence suggests that rlx may play a pivotal role in regulating cardiovascular function during gestation. analogous to pregnancy, administration of recombinant human relaxin (rhrlx) to nonpregnant female rats reduces systemic vascular resistance, as well as increases global arterial compliance. additionally, we demonstrated that, concurrent with rlx´s influence on overall cardiovascular function, small renal arteries (sra) from rhrlx-treated mice and rats are characterized by reduced passive stiffness and increased arterial wall area whereas external iliac arteries (eia) are not. we hypothesized that rlx regulates arterial passive mechanical properties by altering the cellular and biochemical composition of the arterial wall. nonpregnant female mice were administered rhrlx for 5 days after which sra and eia were isolated. we measured arterial collagen, elastin, and total protein using the sircol collagen assay, the fastin elastin assay, and the pierce bca protein assay, respectively. additionally, we quantified arterial smooth muscle cell (smc) density using immunofluorescent techniques. sra isolated from rhrlx-treated mice were characterized by a significant reduction in collagen to total protein ratio (0.13±0.01 vs 0.19±0.01 μg collagen/μg protein; mean±sem; p<0.02) as well as a significant increase in smc density (6.1±0.1 vs 5.0±0.1 cells/1000 μm 2 ; p<0.001) compared to control mice with no significant change in elastin content (104±4 vs 97±14 μg elastin/mg dry weight). in contrast, there were no significant changes in collagen to total protein ratio (0.25±0.02 vs 0.28±0.04 μg collagen/μg protein), smc density (3.9±0.2 vs 4.0±0.2 cells/1000 μm 2 ) or elastin content (89±12 vs 83±5 μg elastin/mg dry weight) in eia from the rhrlx-treated mice compared to control mice. of note, comparable results were observed for rlx knock-out (rlx -/-) and wild-type mice with rlx -/mice exhibiting increased arterial collagen and decreased smc density. we conclude that the rlx-induced decrease in passive stiffness of sra that we previously reported is, at least in part, due to rlx-induced alterations in arterial wall cellular and biochemical composition. further, our findings suggest that these vessel wall remodeling effects of rlx are artery-type specific. relaxin is a peptide hormone that emanates from the corpus luteum of the ovary and circulates during pregnancy. this hormone plays an important role in renal vasodilation and hyperfiltration, two fundamental maternal adaptations in pregnancy. chronic administration of recombinant human relaxin (rhrlx) to both virgin rats and mice inhibits myogenic reactivity and increases compliance of small renal arteries, thus further mimicking pregnancy. we hypothesize that these arterial responses to rhrlx are mediated by the lgr7, and not the lgr8 receptor. both lgr7 and lgr8 receptor-deficient, and wild-type virgin mice were investigated. the mice were chronically infused with rhrlx or vehicle (veh) for 5 days. small renal arteries were isolated and mounted in a pressure arteriograph and myogenic reactivity was assessed (% change in diameter over baseline in response to a 20 mmhg step increase in intraluminal pressure). small renal arteries from rhrlx-infused lgr7 wild-type mice showed inhibited myogenic reactivity with a 6.1 ± 0.5% increase in diameter whereas the arteries from rhrlx-infused lgr7 knock-out mice exhibited robust myogenic reactivity with only a 1.2 ± 0.4% change in diameter (p=0.001). in contrast, myogenic reactivity of small renal arteries was inhibited in both the rhrlx-infused lgr8 knock-out and wild-type mice. the veh-treated lgr7 and lgr8 mice, regardless of genotype, exhibited robust myogenic reactivity. arterial compliance was also assessed for each genotype/treatment group. rhrlx infusion increased arterial compliance of small renal arteries from lgr7wild-type, but not from lgr7 knock-out mice (p=0.01). in contrast, the arteries from rhrlx-infused lgr8 wild-type and knock-out mice showed increased compliance relative to veh-infused animals. conclusion: relaxin-induced inhibition of myogenic reacitivity and increase in compliance of small renal arteries is mediated by the lgr7, and not the lgr8 receptor. smoking is associated with adverse pregnancy outcomes including fetal growth restriction. pathologic effects of smoking on maternal vasculature is a potential mechanism leading to fetal growth restriction. the objective of this study was to determine whether cigarette smoke exposure during pregnancy affects the functional properties of uterine and peripheral arteries using a gravid murine model. study design: pregnant and virgin c57bl/cj mice were exposed to whole body side stream smoke using an inhalational chamber for 4 hours/day. smoke exposure was increased from day 4 of gestation until late pregnancy (day 16-19) with mean total suspended particle levels of 63 mg/m3, representative of moderate to heavy smoking in humans. control animals were exposed to ambient room air. late pregnant and virgin mice were sacrificed and uterine, mesenteric, and renal arteries were isolated and studied in a pressure arteriograph system (n=3-6 in each group). plasma cotinine was measured by elisa. means were compared using t-test or analysis of variance. results: fetal weights were significantly reduced in mice exposed to smoke compared to control fetuses (0.88 ± 0.1g vs 1.0 ± 0.08g, p=0.02), while litter sizes were not different. cotinine levels in smoking mice were significantly elevated compared to control mice (51.9 ± 9.2 vs 4.2 ± 0.8ng/ml for virgin mice and 33.2 ± 21 vs 2.8 ± 0.4ng/ml for pregnant mice). there was no significant difference in phenylephrine responses between groups. endothelial mediated relaxation responses to methacholine were significantly impaired in both the uterine and mesenteric vasculature of pregnant mice exposed to cigarette smoke during gestation. no difference in endothelial-mediated relaxation was seen in isolated renal arteries in pregnant mice exposed to cigarette smoke, however relaxation was significantly reduced in renal arteries from smokeexposed virgin mice. conclusions: passive cigarette smoke exposure is associated with impaired vascular relaxation of uterine and mesenteric arteries in a gravid murine model. functional vascular perturbations during pregnancy, specifically reduced uterine blood flow and impaired peripheral vasodilation, may be a mechanism by which smoking results in fetal growth restriction. human chorionic gonadotropin (hcg) is essential during early human gestation for "rescue" of the corpus luteum. however, its potential contribution to the widespread maternal vasodilation of pregnancy that occurs at this stage of pregnancy remains uncertain. our objective was to use the renal circulation of conscious rats as an experimental model in which to test the vasodilatory potential of hcg. in addition, we investigated both myogenic reactivity and relaxation responses in small renal and mesenteric arteries isolated from rats, as well as in small human subcutaneous arteries using a pressure arteriograph. we chronically instrumented 10 rats for measurement of renal function. five were ovariectomized, and 5 sham-ovariectomized. after 7 days of surgical recovery, baseline glomerular filtration (gfr) and effective renal plasma flow (erpf) were measured on two separate days and the values averaged. then, an osmotic minipump containing hcg (25 iu/min) was implanted s.c. and renal function was again assessed 2 and 5 days thereafter. gfr and erpf significantly increased and calculated effective renal vascular resistance decreased from baseline in the intact (p<0.001 vs. baseline), but not ovariectomized (p=ns) rats on both days 2 and 5 of hcg administration. in the intact, but not ovariectomized rats, plasma osmolality declined and progesterone increased (both p<0.001 vs baseline). plasma hcg concentrations were 2,500 miu/ml and comparable in both groups of rats. incubation of small renal arteries from rats with recombinant human relaxin (rhrlx, 1-100 ng/ml), but not hcg (2,500-200,000 miu/ml) in vitro inhibited myogenic reactivity and relaxed phenylephrine (pe)-constricted arteries. in contrast, both rhrlx and hcg inhibited myogenic reactivity and relaxed pe-constricted small mesenteric arteries from rats and small human subcutaneously arteries. in conclusion, consistent with our earlier work showing a virtually exclusive role for relaxin in mediating the renal circulatory changes of pregnancy, hcg is likely to play little or no role. in contrast, hcg and relaxin are both likely to contribute to the vasodilation of other organ circulations during pregnancy. pierre-andre scott, 1,2 michele brochu, 2 jean st-louis. 1,2 1 research centre, chu ste-justine, montréal, qc, canada; 2 pharmacology, université de montréal, montréal, qc, canada. uterine vasculature undergoes major structural and functional changes during pregnancy. estrogens have been shown to induce increased uterine blood flow in this circulation. we have reported that estradiol (17 -e2) induced direct vasorelaxant response on smooth muscle of the uterine arteries that, for it major part, is not mediated through tissue nitric oxide. to investigate the cellular effectors mediating this vasorelaxant effect of 17 -e2, we set-up uterine arteries of non-pregnant rats in wire myograph systems for microvessels. 17 -e2 and 17 -e2 were equipotent in relaxing phe (1 μmol/l)-preconstricted uterine arteries, although the later produced significant smaller relaxations. genistein, a phytoestrogen presumed to inhibit phosphatases, also produced uterine artery relaxation with significant lower potency. to try interfere with the vasorelaxant effect of 17 -e2, tissues were preincubated with different substances. cycloheximide (protein biosysthesis inhibitor), ici 182,780 (estrogen receptor modulator), and kt5720 (pka inhibitor) did not significantly influenced the response to 17 -e2. rp-8-pcpt-cgmps (pkg inhibitor) slightly displaced the concentration-relaxation curve to 17 -e2 to the right. inhibitors of potassium channels, penitrem a (bkca) and glybenclamide (katp), showed opposite effects for 17 -e2 concentration-response curve; the former producing a right shift and the latter a small not significant left shift. these data indicated that the direct acute effect of 17 -e2 in uterine artery is the result of complex interactions within smooth muscle cells, involving potassium channels, and protein kinases and phosphatases. the renin-angiotensin-aldosterone system is paradoxically activated during pregnancy, since blood pressure decreased. earlier data showed that the high levels of aldosterone present during pregnancy may be involved in cardiovascular adaptation to pregnancy. in order to delineate this effect of aldosterone on vascular tone, potassium canrenoate, an antagonist of mineralocorticoid receptors (mr), was administered (20 mg/kg/day) to pregnant rats from the day 15 to 22 of gestation. rats were sacrificed at day 17, 19 and 22 of gestation together with untreated and day 14 pregnant rats. the thoracic aorta was quickly removed and set up in tissue baths as ring (2-3mm) preparation. as observed previously, canrenoate enhanced responsiveness to phe for all time of treatment tested, but only at day 17 (2 days treatment) for kcl responsiveness. aortic contractile responses to tea (tetraethylammonium) gradually decreased during pregnancy to almost disappear at the end of gestation. in the present results, canrenoate treatment made the response statistically different by day 17 of pregnancy. finally, the activity of na/k-atpase was measured by the relaxant effect of kcl added to physiological solution without potassium. the activity of the pump was decreased when approaching parturition compare to day 14 of pregnancy. canrenoate treatment abolished this effect. the present data show that vascular changes that occurred during pregnancy are markedly modified by treatment of rats with an antagonist of mineralocorticoid receptors, suggesting that aldosterone may be involved in vascular adaptation to pregnancy. background impaired endothelium-dependent vasodilatation has previously been demonstrated in small myometrial arteries from women with gestational diabetes. this impairment may play a role in mediating the complications observed in diabetic pregnancies. it is not known which mechanisms of endothelium-dependent vasodilatation are affected in myometrial arteries by gestational diabetes. aim to investigate mechanisms of endothelium-dependent vasodilatation in uterine arteries using a mouse model of pregnancy complicated by diabetes. methods diabetes was induced in female c57bl6/j mice (streptozotocin; 200 mg/kg) prior to mating. mice were culled at day 19 of pregnancy (term) and uterine arteries dissected, mounted on a wire myograph, normalised to 55mmhg and equilibrated (37°c; 5%co 2 /air). arteries were constricted with phenylephrine (10 m) and a concentration-response curve to the endotheliumdependent vasodilator acetylcholine (ach; 0.1nm-1 m) constructed in the presence and absence of a nitric oxide synthase inhibitor (l-nna; 10 m), a cyclooxygenase inhibitor (indomethacin; 10 m) or a combination of the two to determine the contribution of nitric oxide (no), prostacyclin and endothelium derived hyperpolarising factor (edhf) to vasodilatation. results sensitivity to ach was comparable between diabetic and vehicle treated mice (ec 50 31.4 ± 8.0nm vs 41.9 ± 15.9nm). the contribution of individual endothelium-dependent vasodilators was significantly altered in arteries from diabetic mice. at 1 m ach, edhf-mediated relaxation was significantly reduced (p=0.03, one-way anova) compared with controls (13.17 ± 4.87 vs 67.23 ± 13). in comparison, no-mediated relaxation was significantly increased (p=0.04, one-way anova) compared with controls (64.88 ± 10.78 vs 17.54 ± 5.77%). conclusions endothelium-dependent relaxation was not reduced in uterine arteries of diabetic mice compared with controls. however, there was a profound change in the contribution of endothelium-dependent vasodilators in arteries of diabetic mice. this may alter compensatory capacity as disease progresses. supported by mfn training grant (cihr). raf-1 serine/threonine protein kinase has been extensively studied as the upstream kinase linking ras activation to the mek/erk module. mek/erk has been shown to play a role in the modulation of vascular contraction. however, the role of raf kinase in vascular contraction and its possible involvement in alteration of maternal vascular function during pregnancy is not known. objectives: to determine (1) if raf kinase contributes to phenylephrine (pe)induced contractile response, (2) the role of raf kinase inhibitor (gw5074) in regulating vascular tone during pregnancy, and (3) mechanism by which gw5074 produces vasodilatation in rat mesenteric arteries. methods and results: conscious non pregnant (np) and pregnant (p) sprague dawley rats received increasing doses of pe in the absence or presence of gw5074. pe induced a dose-dependent increase in map in both np and p rats but responses were substantially depressed in pregnancy. gw5074 shifted the pe-induced dose response to the right in both np and p rats. gw5074 itself did not affect basal blood pressure. isometric tension studies in mesenteric arteries showed that gw5074 did not change the kcl-evoked contraction but significantly inhibited the contraction to pe in both np and p arteries. interestingly, at a given concentration, gw5074 produced greater inhibition of pe-induced contractile response in p than in np arteries. also, in p arteries the inhibitory effects of gw5074 were greater as the pregnancy progressed from day 18 to day 20. raf kinase expression and activity was significantly decreased in arteries of p compared to np rats. in mesenteric vascular smooth muscle cells (vsmcs), pe stimulated activation of raf kinase as indicated by phosphorylation of its immediate target, mek1/2 in a time-and dose-dependent manner. measurement of [ca 2+ ] i with fura-2 showed that gw5074-mediated inhibition of pe-induced contraction was not associated with decrease in [ca 2+ ] i . vsmcs treated with pe exhibited higher levels of the contractile proteins, p-mypt1 and p-mlc20 which was inhibited by gw5074. conclusion: to our knowledge, for the first time we show that raf kinase plays an important role in the regulation of vascular contractility. decreased expression and/or activity of raf kinase during pregnancy may explain in part, for decreased systemic vascular reactivity during late gestation. the mechanism(s) that contribute to reduced vascular sensitivity to phenyleperine (pe) during pregnancy is not well understood. pe, in addition to activating the classical contractile pathways, also stimulates growth factor pathways that results in activation of ras/mitogen-activated protein kinases. it is not clear whether these pathways play a role in the modulation of vascular contraction. hence, the present study was designed to determine 1) if ras is involved in mediating the pressor response to pe in non pregnant (np) and pregnant (p) rats, 2) if so, the mechanism by which ras protein contributes to pe-induced vascular contraction, and 3) any differential expression and/or activity of ras during pregnancy that might contribute to altered vascular response. methods: 1) mean arterial pressure (map) was measured in conscious np and p rats. 2) isometric tension was measured in mesenteric arteries of np and p rats. 3) expression of contractile proteins, p-mlc20 and p-mypt1 were studied in cultured vascular smooth muscle cells (vsmcs). 4) expression and activity of ras in mesenteric arteries of np and p rats were measured by western blot analysis. results: (1) intravenous administration of pe resulted in a dose-dependent increase in map but responses were substantially depressed in pregnancy. inhibiting ras activation with manumycin, decreased pe-induced increase in map in both np and p rats. manumycin by itself had no effects on basal map. (2) isometric contraction studies in myograph with mesenteric arteries showed that manumycin shifted pe-induced dose response curve to the right with a decrease in e max in np and p rats. higher concentration of manumycin was required to inhibit pe-induced contraction in np ( 10 μm) compared to p (1 -3 μm) rats. (3) pretreatment with manumycin inhibited pe-induced increase in the extent of phosphorylation of both mlc20 and mypt1 in mvsmcs. (4) compared to p rats, mesenteric arteries of np rats revealed increased basal and pe-induced expression and activity of ras. reduced expression of this contractile ras pathway might contribute to decreased vascular reactivity during pregnancy. conclusion: ras protein plays a role in regulation of vascular contraction. the decreased amount and activity of vascular ras may be a novel mechanism that explains the reduced vascular resistance during pregnancy. overweight affects the relaxin-induced response of mesenteric arteries. joris van drongelen, 1 arianne van koppen, 2 marc ea spaanderman, 1 paul abm smits, 2 frederik k lotgering. 1 1 obstetrics and gynecology, radboud university nijmegen medical centre, nijmegen, netherlands; 2 pharmacology and toxicology, radboud university nijmegen medical centre, nijmegen, netherlands. objective: overweight affects pregnancy-induced vascular adaptation and predisposes to gestational hypertensive disease. relaxin plays a key role in normal gestational vascular adaptation by increasing endotheliumdependent vasodilation and compliance, and reducing myogenic reactivity. we hypothesized that overweight blunts the vascular response to relaxin. the vascular responses to flow and pressure of mesenteric arteries after pre-treatment to human recombinant relaxin (rlx) or placebo (plac) were examined in overweight (ow) and normal weight (nw) rats in a pressure-perfusion myograph. overweight was established by a high-fat diet. the endothelium-dependent vasodilatation was measured in response to flow: e 50 (flow inducing 50% dilatation) and e max (maximal dilative effect). active contractile (myogenic reactivity) and passive dilative (compliance) vascular responses to pressure were determined. all vascular responses were calculated as the proportional change in diameter to 40% precontraction with u46619. results: in nw rats rlx decreased e 50 and increased e max to flow and attenuated myogenic reactivity without affecting vascular compliance. in ow plac-treated rats, compared to nw rats, an upregulated vasodilative state was present (decreased e 50 and increased e max , and lower myogenic reactivity). in ow rats rlx increased e 50 to flow and unaffected e max . rlx increased myogenic reactivity mildly in absence of changes in vascular compliance. values are presented as mean±sem; * p<0.05 between nw/ow, # p<0.05 within nw/ow. whereas rlx stimulates endothelium-dependent vasodilation to flow and myogenic reactivity in nw rats, ow overturns the flow-induced response and decreases myogenic reactivity to a lesser extent than present in nw rats. we speculate that these overweight-induced adverse effects of rlx prelude to vascular maladaptation and gestational hypertensive disease. compared to the luteal (lut) phase, uterine blood flow is increased in vivo during the follicular (fol) phase and more so during pregnancy (preg). both of these are physiologic states of high estrogen and shear stress. endothelial cells express enos and produce greater amounts of nitric oxide (no) in response to elevations of shear stress. phosphorylation of enos is a signaling marker of activation. we have recently validated lut, fol and preg uaec culture models for evaluating enos phosphorylation responses to shear stress and vascular mediators. we hypothesized that uaecs derived from fol and preg sheep will show greater enos phosphorylation than lut phase uaecs, and with more robust responses in the presence of estrogen (e 2 ). methods: uaecs were cultured until 80% confluence, and then subjected to 0 (static control), 3 or 15 dynes/cm 2 for 48hrs in the absence or presence of e 2 (10nm). western analysis was used to compare optical densities of ser635-penos (penos) normalized to total-enos (mean + sem). results: in lut uaec penos was equally increased two fold by 3 and 15 dynes/cm 2 (from 1.26 + 0.5 to 2.4 + 0.008 and 2.56 + 0.06, respectively). compared to lut uaecs, the static control fol uaecs appeared to have higher penos levels (2.2 + 1.5) and this was further increased 1.5-1.8 fold with 3 dynes/cm 2 (3.2 + 0.8) and 15 dynes/cm 2 (3.9 + 0.38). as seen with fol uaecs, preg uaec static penos (1.8 + 0.4) appeared higher than lut uaec, but unexpectedly, neither 3 nor 15 dynes/cm 2 significantly raised these levels of penos (1.76 + 0.05 and 1.6 + 0.04). regardless of shear stress level, e 2 replacement in the culture media only increased the penos levels in the nonpregnant, but not the pregnant derived uaecs. conclusions: increasing amounts of shear stress have a corresponding increase in the ratio of enos that is phosphorylated at serine635 in lut and fol uaecs. pregnant uaecs do not appear to increase the constitutive ratio of serine635 phosphorylation of enos at either 3 or 15 dynes/cm 2 . in contrast to our hypothesis, chronic treatment with e 2 for 48hrs did not augment the ratio of enos phosphorylation in pregnancy. nih hl49210, hd38843, hl87144. cells. honghai zhang, wu xiang liao, dong-bao chen. reproductive medicine, university of california san diego, la jolla, ca, usa. s-nitrosylation (sno) is a rapid, reversible, and nitric oxide (no) dependent post-translational protein modification critical for signal transduction. estrogen stimulates endothelial cell (ec) no production but yet to be determined is if this leads to increased formation of sno-proteins in ec. hypothesis: we hypothesize that estrogen stimulates the formation of sno via a receptor and endogenous no dependent pathway in huvec or ovine uterine artery ec. methods: cells on glass coverslips were treated with 1 mm of no donor gsno or dea nonoate, estradiol-17 (10 nm) in the presence of l-name (1 mm) for 30 min. the cells were fixed with methanol. in situ sno-proteins were detected by a rapid biotin derivatization method by blocking thiols by methylmethanethiosulfonate (mmts), followed by ascorbate reduction and labeling with texas red-labeled ethylmethanethiosulfonate (mtsea) and examined by fluorescence microscopy. cells ( 3x10 6 /group) were lysed in hen buffer, and then similarly prepared but labeled with biotin-mtsea. a portion of the samples were separated on sds-page and blotted with anti-biotin antibody for total sno-protein patterns. the biotin-labeled sno-proteins were captured by avidin, followed by immunoblotting with specific antibodies. results: basal sno-protein labeling was apparent in both ec types. exogenous no donated by gsno or dea nonoate significantly increased red fluorescence labeling of sno-proteins in the cytosol of both ec types. treatment with estradiol-17 also increased total sno-protein labeling in both ec types. pretreatment with l-name significantly reduced sno-protein labeling. sds-page and immunoblotting with anti-biotin antibody analyses identified multiple bands of sno-proteins. in avidin captured biotinylated samples, multiple proteins were indentified. conclusion: exogenous no donated by gsno or dea nonoate and endogenous no upon estrogen stimulation increased s-nitrosylated proteins in ec (supported by nih ro1 hl70562 and 74947). background and objective: the renin-angiotensin system (ras) functions both systemically and locally as a primary regulator of blood pressure and fluid volume and is therefore involved in the etiopathogenesis of essential hypertension as well as preeclampsia, a pregnancy-induced hypertensive disorder in pregnant women. while much progress has been made in the development of strategies to block the systemic ras and thus treat essential hypertension, relatively less advance has been achieved in the development of effective local ras inhibitors to treat preeclampsia, primarily due to the fact the conventional systemic ras inhibitors generate potential teratogenic risks to pregnant women during critical stages of their pregnancies. ginger has been widely and effectively used in pregnant women to treat pregnancyinduced nausea and vomiting with minimal adverse effects. the present study was designed to investigate whether ginger could down-regulate renin secretion by human decidual cells (the major source of renin in the tissue-based uteroplacental ras), as an initial step toward a long-term goal to develop potential safe and effective ras inhibitors for use in obstetric patients. methods: full-term normal human placentas were obtained within one hour of vaginal deliveries or cesarean sections. decidual cells were isolated from the decidua parietalis. after an initial culture for 2 3 days in a serum-containing medium, the decidual cells were treated with ginger juice at various doses in a serum-free medium for 24 hours. the culture supernatants were then harvested and subject to western blot analyses of renin protein contents. the ginger juice used was freshly prepared from the ginger roots purchased from a local grocery store and different batches of juice preparations were standardized based on their optical density values at a wavelength of 400 μm on a spectrophotometer. results: a dominant band of renin at approximately 47 kd was detected in all samples. when compared with the control (i.e. 0%), ginger juice decreased the renin protein contents in the culture supernatants in a dose-dependent manner. no significant difference in the cell morphology was observed between the control and treatment groups. conclusion: ginger root juice down-regulates renin secretion in human decidua, showing a great potential of a novel ras inhibitor, particularly, in obstetric patients. noninvasive quantification of the autonomic nervous system throughout pregnancy. dietmar schlembach, 1 karoline pickel, 1 daniela ulrich, 1 philipp klaritsch, 1 isa alkan, 2 uwe lang, 1 manfred g moertl. 1 1 obstetrics and gynecology, medical university of graz, graz, styria, austria; 2 cnsystems medizintechnik ag, graz, styria, austria. introduction: the analysis of heart rate variability (hrv), blood pressure variability (bpv), and baroreflex sensitivity (brs) has become a powerful tool for the assessment of autonomic control. although hrv analysis was initially developed for risk stratification in cardiology, the field of clinical application has broadened in recent years. the aim of our study was to investigate the adaptation of autonomic control during pregnancy based on analysis of heart rate variability and baroreceptor sensitivity. methods: 20 patients with uncomplicated pregnancy were measured with the taskforce® monitor 2040i made by cnsystems. all measurements were performed in supine position under standardized resting conditions. autonomic parameters were recorded at rest and within the "deep breathing method" as a "cardiovascular challenge test". results: throughout pregnancy a shift to higher sympathetic activity respectively a lower parasympathetic activity was observed. , and 16.15±5.54 [>30 wks]) (figure 1). during the deep breathing maneuver we could show an increase of the sympathetic / parasympathetic ratio (figure 2). the noninvasive determination of autonomic parameters throughout pregnancy is possible. these results can be used as basic parameters for classifying and assessing autonomic changes in pathological conditions in pregnancy such as hypertensive disorders. how far the characterization and challenge of these autonomic functions can be utilized for diagnosis and prediction of preeclampsia has to be shown in future studies. hemodynamic parameters measured noninvasively throughout pregnancy. daniela ulrich, 1 manfred g moertl, 1 karoline pickel, 1 philipp klaritsch, 1 isa alkan, 2 uwe lang, 1 dietmar schlembach. 1 1 obstetrics and gynecology, medical university of graz, graz, styria, austria; 2 cnsystems medizintechnik ag, graz, styria, austria. background: hemodynamic changes throughout pregnancy have been measured predominantly by invasive techniques. the discussion on the valence und the low acceptance of these invasive procedures by pregnant women demands a noninvasive method for evaluating and distinguishing cardiovascular adaptative mechanisms throughout normal and especially complicated pregnancy. method: 20 healthy patients with uncomplicated pregnancy were measured with the taskforce® monitor 2040i (cnsystems, austria) at different time points throughout pregnancy. cardiovascular parameters were recorded in supine and left lateral position under standardized conditions. results: throughout pregnancy an increase of global parameters such as heart rate (hr), blood pressure (bp), total peripheral resistance (tpr) and total peripheral resistance index (tpri) were observed. stroke volume (sv), stroke index (si), cardiac output (co), contractility index (ci), acceleration index (aci), and left ventricular ejection time (lvet) decreased throughout pregnancy (table). discussion: the noninvasive determination of cardiovascular parameters throughout pregnancy is possible and the results of this pilot study can serve as basic parameters for classifying and assessing cardiovascular changes in pathological conditions in pregnancy such as hypertensive disorders. assigning pregnant hypertensive women into hyper-and hypodynamic groups may aid in planning individual therapeutic strategies. objective: both gnrh i and gnrh ii are expressed in humans. gnrh i is produced by the hypothalamus under the regulation of gonadal steroids and stimulates pituitary gonadotropins. during pregnancy gnrh i is also produced by the placenta and affects hcg. gnrh ii, the ancient isoform, is produced by numerous human tissues including immune and reproductive tissues and circulates in blood in quantifiable levels. we have demonstrated that gnrh ii analogs directly affect fertilization and uterine function, and propose that it acts via immune regulation. during pregnancy it is known to regulate numerous hormone productions, yet the levels of gnrh ii has not been reported. in these studies we have determined the circulating levels of gnrh ii throughout pregnancy and in early pregnancy loss. study design: thirty-three women having normal pregnancies were followed prospectively. plasma samples were drawn at 8, 10, 12, 14, 16, 28, 36 weeks gestation and during labor. plasma was also collected from patients have spontaneous abortions (n=8). circulating gnrh ii and crh and gnrh i were measured by specific radioimmunoassays. results: circulating gnrh ii increased from non-pregnant levels (65+/-2 pg/ ml, mean+/-sem) to 83+/-2 pg/ml by 10-week lmp. gnrh ii concentrations continued to increase through 16 weeks gestation over the 10-week levels, although a significant increase was only attained by 28 weeks. gnrh ii continued to increase in late term, attaining levels of 99+/-1 pg/ml which was significantly higher than that of early gestation. during labor and delivery gnrh ii in maternal plasma was further increased to 106+/-2 pg/ml, i.e., 1.5 times that of 8-week gestation (73+/-4 pg/ml). circulating gnrh ii did not parallel gnrh i in early pregnancy but did in late gestation. gnrh ii did correlate with crh in early pregnancy but not in late gestation. patients having spontaneous abortion had increased circulating gnrh ii at 8-weeks lmp (85+/-3 pg/ml) as compared to normal pregnancies (73+/-4 pg/ml). conclusion: gnrh ii increased throughout pregnancy attaining highest concentrations during labor and delivery. patient with early pregnancy loss had increased gnrh ii expression. the function of gnrh ii or factors affecting this increased gnrh ii in normal or abnormal human pregnancy should be investigated. introduction: plasma levels of the endocannabinoid, anandamide (aea) decrease during the luteal phase of the menstrual cycle and early pregnancy and increase during parturition. 1 high plasma aea levels at 6 weeks in women undergoing ivf-et was associated with a failure to achieve an ongoing pregnancy 2 . what is uncertain is what happens to aea levels when the pregnancy has already failed. we aimed to quantify aea levels in women presenting in early pregnancy with a diagnosed non-viable pregnancy and to compare them to those of a viable pregnancy. methods: plasma aea was measured by a sensitive isotope dilution hplc-ms/ms method from 45 women in early pregnancy (5-12 weeks) of whom 25 had a viable pregnancy and 20 had a non-viable pregnancy. serum hcg and progesterone were measured in blood samples by standard elisa methods. results: the ages and bmi of both groups were similar (29 ± 4.9 versus 28 ± 4.7 years; mean ± sd; p=0.45; student's unpaired t-test and 24 ± 2.7 versus 25 ± 2.9 kg/m 2 ; p=0.91) respectively. plasma aea levels in women with non-viable pregnancies were significantly higher than those in women with viable pregnancies (1.68 ± 0.77 versus 1.15 ± 0.30nm; p=0.007). there was no correlation found between aea levels and hcg or progesterone levels p=0.272 and r=0.274; p=0.075, respectively) , despite progesterone being significantly lower in the non-viable group (40.0 ± 35.1 versus 61.0 ± 26.3ng/ml; p=0.027). conclusion: higher plasma aea levels were associated with early pregnancy failure, and this association appeared to be independent of serum hcg or progesterone concentrations. these data suggest that plasma aea levels are linked with pregnancy failure through a mechanism that does not involve hcg or progesterone production. the precise involvement of anandamide in early pregnancy needs to be investigated further. 6n-3) and arachidonic (aa, 20:4n-6) acids, the major brain long-chain polyunsaturated fatty acids (lc-pufa) are generated by elongation-desaturation of dietary essential fatty acids (efa). the maternal liver is principally responsible for efa elongation-desaturation. objetive. we determined effects of protein restriction in pregnancy on expression of 5d, 6d and elongases 2 and 5 (elov 2 and 5) in the maternal liver rat. methods. pregnant rats were fed control (20 % casein; c) or restricted (10 % casein; r) isocaloric diets. at day 19 gestation maternal blood and livers were collected. serum triglycerides, cholesterol and glucose were determinate with the synchron cx autoanalyser and leptin and insulin by ria. liver fat determination was performed by the soxhlet method. liver ara and dha were calculated by gas chromatography based upon retention times from methyl ester standards. liver 5d, 6d, elov 2 and 5 mrna were measured by rt-pcr and northern blot. data are m ± sem; analysis by t-test. results. liver weights did not differ in c and r. total liver fat (760 ± 39 vs 543 ± 22 mg) serum leptin (5 ± 0.1 vs 7 ± 0.7ng/ml) and insulin (0.2 ± 0.04 vs 0.9 ± 0.09 ng/ml) differed in c and r respectively (p<0.03). serum triglycerides, cholesterol, and glucose did not differ. desaturase and elongase mrna and % of maternal liver aa and dha were lower in r (fig 1) . conclusion. low liver fat content and desaturase and elongase mrna in r indicate impaired lc-pufa synthesis which may adversely impact fetal development, especially the brain. objective: to test the hypothesis that obstetrical dic results from an excessive leak of fetal material into the maternal circulation. a secondary objective was to assess maternal morbidity. methods: all cesarean hysterectomy cases for hemorrhage at our hospital from 1993 to 2002 were included. intravascular presence of fetal material was determined by two pathologists, blinded to each other and any clinical information. the percentage of those with any fetal material in the maternal circulation was calculated for each diagnosis for hemorrhage. for a given diagnosis, the percentage of intravascular fetal material in those with the given diagnosis was compared to those without that diagnosis using fisher's exact test. most patients had multiple hemorrhage diagnoses. a two sample t-test was used to evaluate the difference in mean blood loss between those with and without intravascular fetal material. results: primary outcome* 89% of patients received blood products and 40% received clotting agents. the average blood loss was 2650 cc. there were no maternal deaths and 4 injuries to adjacent organs, all to the bladder. conclusions: there was no association between the presence of intravascular fetal material and any specific hemorrhage diagnosis or amount of blood loss. although the power to detect a relationship was low, the excessive leakage of fetal material as a specific and exclusive mechanism of obstetrical dic, as postulated, seems unlikely. while 90% of the population was transfused, there were few intraoperative complications and no maternal deaths. hypertension, tel-aviv university, sheba medical center, ramat-gan, israel. objective: the joint national committee on high blood pressure (jnc7) determined blood pressure of 120-139/80-89 in adults to be prehypertension that requires health promoting life style modifications to prevent cardiovascular disease. similarly, we hypothesize that gestational prehypertension in pregnancy is associated with increased risk of pregnancy complications and its management would improve pregnancy outcome. methods: prospective and retrospective recruitment resulted in a study group consisting of 40 patients who were diagnosed in the first and early second trimester (<15 weeks) with blood pressure of 120-130/80, and a control group consisting of 87 patients with blood pressure < 120/80 at similar gestational age. comparisons between the two groups were accomplished for demographic characteristics and outcome measures using the chi-square test statistic and two-sample t-tests for categorical and continuous variables, respectively. results: all outcome measures analyzed resulted in poorer results being associated with the study group compared with control as follows: 10% versus 2% of the study and control group pregnancies, respectively, experienced preeclampsia (p=0.07); and 14% versus 0% of the study and control group, respectively, were associated with gestational hypertension (p=0.0005). 10% of the control group deliveries were associated with admission to the nicu, compared to only 3% in the control group (p=0.16); 17% versus 10% of the study and control group deliveries, respectively, were preterm (p=0.36); twenty-nine percent of the study group were cesarean deliveries, compared to 38% in the control group (p=0.05). on admission mean sbp and dbp was 132 and 76 mmhg, respectively, in the study group, compared to 122 and 70 mmhg in the control group (p=0.0003 for sbp and p=0.03 for dbp). conclusions: "gestational pre-hypertension" may be a real pregnancy condition that when is recognized, physician attention, patient close monitoring during prenatal care and delivery and early intervention in patients at risk, may all promote improved pregnancy outcome. larger scale study is in progress to evaluate and confirm these findings in the larger pregnant population. are contractions at 24-32 weeks gestation less painful than those at full term? kristy a ruis, karin blakemore, abimbola aina-mumuney, valerie jones. gynecology and obstetrics, johns hopkins hospital, baltimore, md, usa. objective to determine if gestational age plays a role in severity of pain associated with uterine contractions. study design self-reported pain from women in labor, on a scale of 1-10, is assessed and recorded in an obstetrical database by nurses at regular intervals at two hospitals within our medical system and was retrospectively reviewed from 2002-2007. pain at various dilatations (3-10cm) of women who were laboring and then delivered at 24-32 weeks' gestation was compared to women in labor at term. maternal demographics of age, race, education level, bmi, presence of support person, request for analgesia at any point in labor, and epidural placement were abstracted from maternal records. categorical data were analyzed using chi square or fisher exact test; continuous variables using student t-test. results 99 laboring patients between 24-32 weeks gestation and 409 at term were identified. term and preterm patients did not differ with respect to maternal demographics. pain reported by preterm patients was significantly less at 3-4 cm dilatation (4.8 vs. 6.3‚ p=0.01) and significantly greater at 9-10 cm (4.2 vs. 2.3‚ p=0.03). pain reported at 5-6 cm and 7-8 cm was not statistically different between the groups. of note, fewer of the preterm patients received an epidural despite the request for analgesia. conclusion preterm laboring patients between 24-32 weeks gestation may perceive less pain at 3-4 cm dilatation compared to term laboring patients; however, their pain perception at advanced dilatation was comparable to those at term despite the fact that they were less likely to have an epidural in place at the time of pain evaluation. in light of these findings, the widely accepted etiology of labor pain as the result of cervical dilatation may need to be re-examined. also, factoring in the patient's discomfort level into the evaluation for onset of early active labor may not be valid in the preterm patient. background and objective: asthma is a risk factor for adverse pregnancy outcome. this has been attributed to the effect of allergy in pregnancy. mast cell mediators can induce uterine contractility. the objective of the study was to test the hypothesis that pregnant women with asthma have different uterine electrical signals from those generated by normal pregnant women. materials and methods: uterine electromyography (emg) recordings from gestational age matched pregnant women at term were analyzed. the cases consisted of patients with asthma and the controls, those women without asthma. emg was recorded for approximately 30 minutes from surface electrodes placed upon the maternal abdomen, with the electrical signals filtered in the uterine-specific range of 0.34 to 1.00 hz to remove noise components. the recordings were analyzed in their entirety first by power spectrum analysis and then by lyapunov analysis. the power-spectrum-largest-peak frequency and the largest lyapunov exponent were calculated for each patient, and then the mean and standard deviation of these parameters was compared using student's t-test. results: patients with asthma had significant lower power spectrum frequency and higher lyapunov exponent than women without asthma. see 1) the power spectrum frequency and the lyapunov exponent used to analyze uterine emg signals were different in women with and without asthma; 2) the results suggest that uterine electrical activity is altered in women affected by asthma; 3) these findings may explain the observed increased frequency of preterm birth in women with asthma. further studies are required to dissect the mechanisms. fetal microchimeric cells trafficked into the maternal circulation persist in blood and tissues for years after pregnancy. increasing data suggest that microchimerism occurs after every pregnancy, but the biological role of fetal microchimerism or the cell types involved is unclear. while persistent fetal cells were initially implicated in autoimmune disease, animal studies suggest these fetal cells play a broader role in response to tissue injury. methods: appendix specimens were acquired from 9 women undergoing appendicectomy during pregnancy. detailed reproductive histories were obtained. fluorescence in situ hybridisation (fish) with two different probes allowed investigation of the presence of male presumed-fetal cells and nested pcr amplification of sry gene confirmed male dna in the appendix. immunostaining was used to determine the fetal cell phenotype. results: male cells were identified in appendix tissues from women with known male pregnancies (n=7) and also from a woman with no sons and a previous miscarriage of undetermined gender (n=1). no male cells were observed in the control (n=1), a woman with 3 daughters. male cells of presumed fetal origin were evenly distributed in the muscle, mucosa and submucosa layers of the appendix. morphology and co-localisation analysis suggested the identified male cells had differentiated primarily into muscle cells or lymphocytes. combined immunostaining and y-fish demonstrated male desmin+ muscle cells and cd3+ and cd19+ lymphocytes. finally, the presence of male dna in the appendix specimens was confirmed by nested pcr. male cells of presumed fetal origin were identified in the appendix of pregnant women. microchimerism frequency varied according to the reproductive history and the degree of inflammation. microchimerism rates were higher in the appendix from women with current male pregnancies than in those with previous male pregnancies and were higher in those with histological acute inflammation, when compared to milder cases. male microchimeric cells identified were of haematopoietic and mesenchymal origin. this study suggests that fetal cells are present in sites of tissue injury and may participate in tissue repair during pregnancy. collagen i 2 and collagen-binding integrins mrna expression in rat cervix during gestation. huiling ji, tanya l dailey, vit long, edward ks chien. obstetrics and gynecology, maternal fetal medicine, women and infants' hospital of rhode island, providence, ri, usa. objective cervical remodeling is associated with cell proliferation and extracellular matrix (ecm) remodeling. integrins are a family of multifunctional cell adhesion receptors which mediate ecm-cell interactions including binding collagen. of the 24 integrin (i) heterodimers, 1 1, 2 1, 10 1 and 11 1 are primary receptors for collagen. the predominant cervical collagen is collagen type 1. the purpose of this study is to investigate collagen and integrin expression in the pregnant rat cervix. the cervix was harvested from timed pregnant sprague-dawley rats. non pregnant (np)and timed pregnant day 12, 16, 18, 20, 21 and 22 animals were euthanized using a protocol approved by the iacuc. four animals were sacrificed on each day of gestation. quantitative rt-pcr was used to evaluate mrna expression and normalized to -actin. a standard curve was generated from a single sample. data was analyzed using anova and multiple comparisons testing. collagen i 2 mrna expression increased through mid-gestation but decreased to np levels on day 22. a similar pattern was seen with the integrin beta 1 subunit. the pattern of integrin alpha subunit expression was different for each subunit during pregnancy. the changes in collagen, integrin alpha 1, 10, 11 and integrin beta 1 were found to be statistically significant. (figure) . the pattern of collagen binding integrin alpha subunit expression during the rat gestation appears to vary independently of each other. the expression of the 1 subunit paralleled col 1 2 expression. collagen binding integrins may play independent roles in signaling and biomechanics during gestation. funding: women infants' hospital research fund; phs nih-ncrr p20 rr018728 p20 rr017695. background. the effects of chemotherapy on human fetal development are poorly studied, mainly due to the lack of adequate animal models in which placentation and embryological development resembles humans and pharmacokinetic studies, prenatal sonography as well as histological studies can be performed. aim of the study. to test the baboon as model for studying pharmacokinetics and fetal effects of chemotherapy administered during pregnancy. methods. experiments were performed in 11 pregnant baboons at a mean gestational age of 134 (+/-10) days. detailed ultrasound examination (biometry, doppler, screening) was performed 3 days before, at and 1 day after the drug administration. the administration of different schemes of chemotherapy and the fetal samplings occurred under endotracheal anaesthesia. maternal blood samplings, as well as percutaneous ultrasound guided fetal blood (2ml) and amniotic fluid (4ml) samplings were performed at least once immediately after drug administration. in case of fetal demise, the fetus underwent detailed macro-and microscopic examination. in the other animals mother and fetus were euthanized 24h after the experiments, with collection of blood, amniotic fluid and tissues for further analysis. results. all 11 mothers survived the experiments, one mother developed paralitic ileus. none of the fetuses died during the acute phase of the experiment but 3/11 (27%) fetuses died within 24h following the experiment. none of the animals showed significant clinical or histological signs of infection or anaemia. a total of 50 ultrasound examinations were performed in 11 fetuses, allowing the creation of sonographic growth charts in this model. at the time of drug administration the mean maternal weight was 16.8kg (range 14.0-20.2) and the estimated fetal weight was 405gr (range 317-484). sixteen out of 18 cordocenteses (89%) and all 18 amniocenteses (100%) were successful in obtaining the required samples for analysis. conclusion. the pregnant baboon can be used as a model for studies on pharmacokinetics and fetal effects of chemotherapy administered during gestation. prenatal ultrasound is comparable to the human situation, and amniocentesis and cordocentesis can be performed with a high success rate and short term survival. therapy to prevent preterm birth is limited". moreover, 17-hydroxyprogesterone (17ohp) caproate is a category d drug according to the fda (evidence of fetal harm). p is produced in the adrenal glands, gonads and brain. after 8 weeks gestation p is secreted from the placenta independently to the mother and the fetus. p is the precursor of aldosterone and after conversion to 17ohp, of cortisol and androstenedione. baboons have similar reproductive system and development to humans and are used to study mechanisms of labor and prevention of preterm labor. currently, there are no normative data on the concentration of p, 17ohp, cortisol ©, estradiol (e2) or inhibin (i) throughout their pregnancy. therefore, a doseresponse or the teratogenic effects of p or 17ohp caproate cannot be studied in a controlled manner in this animal model. the aim of this study was to generate reference values for these hormones during baboon gestation and early postpartum life. material and methods: 43 hormonal quantitative measurements were performed (immulite analyzer) results: the mean concentrations of these 5 hormones are depicted in figure 1 (maternal serum, from conception to term 180 days) and figure 2 (newborn serum) conclusion: this study provides reference values for 5 hormones quantified during the baboon gestation and early postpartum life. this may be useful for future research concerning the effects of p and 17ohp administration during pregnancy. (1)non-pregnant women eligible for ivf treatment and (2)pregnant women receiving prenatal care. the pre-9/11 samples were drawn in the year prior to 9/11/01; the post-9/11 samples were drawn within 6 months after. the pre-9/11 and post-9/11 normal pregnant samples were drawn at 20+2 weeks' gestation. the non-pregnant samples were from ivf candidates with serum e2 levels<75 newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced pg/ml and fsh serum levels<12.5 miu/ml. the samples were assayed for acth and cortisol by a commercially available immulite™ system. hsp70 and hsp90 were measured by commercially available elisa. these results were compared in the patient populations before and after 9/11. t-tests were used for analysis. statistical significance was set at p<0.05. results: in the pregnant subjects, there were no statistical differences in the mean levels of acth, cortisol, and hsp70 pre-and post-9/11 (table 1) . only serum hsp90 levels were significantly increased in the pregnant women post-9/11.* mean serum acth, cortisol, hsp70, and hsp90 levels were all significantly different in non-pregnant subjects pre-and post-9/11. conclusions: except for hsp90, serum markers for stress were unchanged in pregnant subjects after the stress of 9/11/01 in comparison to non-pregnant women whose serum hsp90, hsp70, cortisol, and acth levels were significantly different. the lack of change in serum markers of stress in pregnant women suggests that the hormonal milieu of pregnancy may buffer against acute environmental stressors. objective: in human pregnancy, maternal body composition provides an indicator of maternal nutritional status and metabolic capacity. previously, we reported that 11ß hsd-2 activity was significantly reduced in term placentas from thin women and women with a smaller mid-upper arm circumference before conception. this suggests that these fetuses may have been exposed to inappropriate levels of maternal cortisol, which is a mediator of gestation length. in this study, we hypothesize that the duration of gestation will be shorter in women who tend to be thin and have a lower mid-upper arm circumference prior to conception. methods: within the longitudinal, population-based southampton women's survey (sws), analyses were performed on 1301 women whose estimated date of conception was set using an algorithm that combined menstrual and early ultrasound scan data and who had a spontaneous onset of labour and delivered after 37 weeks of gestation. linear regression was used to examine the relationships between maternal body composition and the duration of gestation. results: within the 1301 women surveyed, lower maternal body mass index, mid-upper arm circumference and arm muscle area before conception were all associated with shorter duration of gestation at delivery (r=0.12, p=0.00001; r=0.10, p=0.0002; and r=0.09, p=0.0009, respectively). a lower subscapular skinfold thickness, sum of skinfolds and height were also associated with shorter duration of gestation (r=0.08, p=0.006; r=0.07, p=0.009 and r=0.06, p=0.03, respectively). mother's age, own birthweight and ratio of subscapular/triceps skinfold thickness were not related to the duration of gestation. conclusions: in this study, we found that thinner women with a lower midupper arm circumference and arm muscle area tended to have a shorter duration of gestation. our findings of shorter gestation length in thinner women are in keeping with our previous observation of lower 11ß hsd-2 activity in term placentas from thinner women. we conclude that metabolic capacity prior to conception could influence duration of gestation through mechanisms that include alteration in placental metabolism and fetal cortisol exposure. fecal incontinence (fi) is a debilitating condition affecting 2-10% of the us. our prior studies found that 29% of women report new onset fi after childbirth. the goal of our study was to examine the impact of fi on postpartum quality of life (qol). women reporting fi on a statewide survey who agreed to participate in a 2-year study of qol were included in the analysis. women were considered to have fi based upon the nih definition of fi. the quality of life survey was based upon the uebersax incontinence impact questionnaire and was administered every 6 months for 2 years. qol in women with fi was examined using chi square, and impact of severe fi (stool incontinence) was determined by multivariate logistic regression. results 2907 women with fi were surveyed and of those, 1247 (43%) returned at least 1 survey during the study period, completed all survey questions, and were included in the final analysis. among women with fi, 51% felt frustrated due to fi, 26% reported fi impacted their emotional health, 18.5% reported fi impacted child-caring abilities, and 16.7% reported a negative impact on social activities. qol was similar across survey periods. one out of three women with fi reported severe symptoms (incontinence of stool). women with severe symptoms were 4-7 times more likely to report negative impacts on qol compared to milder (e.g. flatus) fi after adjusting for age, parity and urinary incontinence. 33.9% felt their stool was stored elsewhere before bowel movements, 13.9% reported using digital defecation and more than half (52%) reported symptoms of urinary leakage. despite the substantial impact on postpartum quality of life, few women sought medical help with only 10% of women at 6 months, 13.5% at 1 year, and 16.7% at 2 years ever reporting their symptoms to a medical provider. postpartum women report that fecal incontinence has a substantial negative impact on their qol after delivery including their emotional health and ability to care for their newborn. despite this profound impact, few women will discuss fi with medical providers. these data suggests there may be a benefit for providers to inquire about fi at postpartum visits. objective: the objective of this study was to determine what characteristics contribute to racial/ethnic differences in breastfeeding rates. study design: a retrospective cohort study of all women who delivered a viable infant (n=26,781) was conducted. the primary outcome was breastfeeding upon discharge of the hospital. we first examined the association between race/ ethnicity and breastfeeding. next, we conducted stratified analyses examining a variety of predictors of breastfeeding within the racial/ethnic groups including maternal demographics, obstetric interventions, and perinatal complications. results: we found that both asians (or 0.53, 95% ci 0.47-0.61) and blacks (0.32, 95% ci 0.27-0.37) had statistically significant lower rates of breastfeeding, while latinas (or 1.18, 95% ci 0.99-1.41) showed a trend towards higher rates of breastfeeding. in latinas, we found that 3 rd and 4 th degree lacerations were significantly associated with lower rates of breastfeeding, but were not predictive of breastfeeding in the other racial/ethnic groups. epidural use was predictive of lower rates of breastfeeding in caucasians and blacks, but was not predictive in latinos and asians. some of the other predictors which differed between the racial/ethnic groups were obesity and induction of labor (table 1) . conclusion: race/ethnicity is significantly associated with breastfeeding, with blacks and asians having the lowest rates of breastfeeding at discharge. a variety of other factors are associated with breastfeeding and interestingly, their effect appears to differ between the racial/ethnic groups. future studies might elucidate the sociocultural and biomedical reasons that explain the differences. these results help focus our efforts during the peripartum period to advocate for mothers who have factors that might impede breast feeding. epidemiology, erasmus medical centre, rotterdam, netherlands; 4 pediatrics, erasmus medical centre, rotterdam, netherlands; 5 public health, erasmus medical centre, rotterdam, netherlands; 6 clinical genetics, erasmus medical centre, rotterdam, netherlands. background recommendations on folic acid use to prevent neural tube defects are launched in several countries. however, the adequate use of folic acid supplements during the periconception period seems to be low. objective to assess the prevalence of adequate folic acid use, defined as the preconception start of supplements, and to identify its determinants in a multi-ethnic population. design the study was embedded in the generation r study in rotterdam, the netherlands, a population-based prospective cohort study from early pregnancy onwards. methods from all women in the cohort who delivered between april 2002 and january 2006 information on folic acid use and potential determinants was obtained by questionnaires and physical examination. logistic regression models were used to identify determinants of periconception folic acid use. results. data from 6,940 pregnant women were available. of all women 37% adequately used folic acid supplements. the most important risk factors for inadequate use were unplanned pregnancy (or 9.5, p<0 .001), nonwestern ethnicity (or 3.5, ci 2.9-4.3, p <0.001) and a low educational level (or 2.5, ci 1.8-3.6, p<0.001). other risk factors were single marital status, smoking, multiparity (all p<0.001) and alcohol use (p<0.05). prior spontaneous abortion was associated with increased adequate folic acid use (p<0.001). conclusion adequate periconceptional folic acid use is low. improved preconception care and public health education programs are necessary to improve the uptake of folic acid. although methamphetamine (ma) use has been front and center of the united states drug control policy since 2005, little attention has been given to ma use in pregnancy, despite the fact that pregnant admissions to drug treatment for ma have been rising sharply. to determine trends in the prevalence of ma treatment admissions during pregnancy, we undertook a secondary analysis of the treatment episode data set (teds), an administrative data set that captures at least 70% of all known treatment admissions in the us. in particular, we investigated risk factors for ma use and how these characteristics have changed over time. demographic, geographic and substance use data were collected for the 219,335 pregnant admissions captured between 1994 and 2005. logistic regression models were constructed by year. confounding was assessed via backwards elimination with a change-in-estimate criteria of 0.1 considered substantial. trend results were reported as adjusted proportions and represented graphically. overall ma prevalence, reported as the primary drug of use upon admission, rose from 8.1% in 1994 to 24.5% in 2005. although white women had 3 times the odds of using ma in 1994 (adjusted or (95%ci) 3.2 [2.8, 3.8]), this had dropped to 1.9 [1.7, 2.1] by 2005, mostly due to an increase in latina ma use in pregnancy. pregnant women admitted for ma had fewer prior treatment admissions, were more likely to be unemployed, and less likely to use alcohol or marijuana. we found great geographic variability in use. ma was more common in the pacific region and least common in new england. there was little change in regional variation over the study time period. less is known about the perinatal effects of ma compared with other substances, yet it is the primary drug of choice for pregnant women admitted into treatment. as pregnant women occupy a unique place in drug treatment, analysis of national trend data is essential to guide both policy and research. background: epidemiologists have grouped the multiple disorders that lead to extremely preterm delivery in a variety of ways. we sought to identify characteristics that would support the combining or dividing of the disorders that lead to preterm delivery. methods: we enrolled 1,006 women who delivered a live born singleton infant between 23 and 27 completed weeks gestation at 14 tertiary centers in the united states. each delivery was classified according to the complication that prompted presentation: preterm labor, preterm premature rupture of newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced fetal membranes (pprom), preeclampsia, placental abruption, cervical incompetence, and fetal indication/intrauterine growth restriction (iugr). we compared these entities on the frequency of characteristics identified by standardized interview, chart review, histological examination of the placenta, and culture of placenta parenchyma. results: the percents of women who presented with each antenatal complication were: preterm labor (40), pprom (23), preeclampsia (18), placental abruption (11), cervical insufficiency (5), and fetal indication/iugr (3). after considering antecedents and correlates of the processes leading to preterm delivery, we observed two overarching epidemiologic patterns. the first pattern, characterized by recovery of organisms from the placenta and by histologic chorioamnionitis, tended to be associated with preterm labor, pprom, placental abruption, and cervical insufficiency. the second pattern, characterized by a paucity of organisms and inflammation and the presence of histologic features of dysfunctional placentation, tended to be associated with preeclampsia and fetal indications/iugr. conclusions: disorders leading to preterm delivery can be categorized broadly into two groups: those associated with intrauterine inflammation and those with aberrations of placentation. predictors of compliance with tuberculosis screening in pregnancy. nadav schwartz, 1 sarah wagner, 1 sean keeler, 1 may tam, 1 julian mierlak, 1,3 aaron caughey. 2 1 obstetrics and gynecology, nyu school of medicine, new york, ny; 2 obstetrics and gynecology, ucsf, san francisco, ca; 3 obstetrics and gynecology, gouverneur health care services, new york, ny. objective: poor compliance with tuberculosis screening and treatment is a major obstacle to the containment of this disease. we sought to identify predictors of ppd and cxr compliance during pregnancy. methods: a retrospective cohort study at a single institution which serves a largely immigrant population in nyc, from november 1, 2001 through june 30, 2006. data on maternal age, ethnicity, country of origin, level of education, ppd and cxr status were collected. results: of the 4,049 pregnancies, asian and hispanic race/ethnicities accounted for 50.4% and 44.0% of the population, respectively, with 11.2% being us-born. the mean age was 27.02 years and the overall ppd+ rate was 50.3%. there was 5% non-compliance with ppd testing, and 5% of ppd+ patients were non-compliant with their chest x-rays. asian women were more likely to be ppd-compliant than hispanic or caucasian women. ppd+ asian women were also more likely to be compliant with cxr. us-born women were significantly less likely to be compliant with their ppd or with their cxr. women >40 years old were less likely to be compliant with their cxr, while women with an elementary school education or less were more likely to be compliant. (see table for odds ratios and 95%ci) conclusions: age, education, immigrant status and other cultural and ethnic factors appear to play a role in compliance with tuberculosis screening. further elucidation of these effects may help clinicians target at-risk subpopulations and improve overall compliance, working towards better control of this disease. background: in the us, differential outcomes in cervical cancer among medically underserved women are linked to multiple barriers impacting loss to follow-up and failure or delay in diagnostic resolution. prior studies have found risk factors for acquisition of hpv and for inability to obtain a pap smear. no study has explored health literacy and physician communication as a key factor. methods: this research represents the formative phase of a randomized controlled trial of african american (aa) and hispanic women aimed to improve communication and abnormal pap smear follow-up in chicago. semi-structured interviews and focus groups were conducted face to face in spanish or english. each interview lasted 45 minutes, and each focus group lasted 60-90 minutes. we recruited 20 patients (10 hispanic, 10 aa) and 20 providers from a purposive sample representative of two large clinics that serve low-income women. results: all interviews were transcribed by two investigators. each interview was coded by two investigators separately and then a third investigator reviewed the transcripts and coding to achieve triangulation. codes for these themes were developed and the responses were tabulated using the coding scheme. atlas ti was utilized to analyze all qualitative data. from these data, we uncovered provider-patient challenges in cancer communication including: the providers' trade off between medical accuracy and/or literacy when communicating with their patients. for the patients, the word cancer was important to hear since they wanted the truth and needed to hear this word in order to encourage them to respond more quickly. however, many providers believed that the word cancer was too "scary" and to "extreme" of a word that may communicate too much exaggerated information. both the hispanic and aa patients did not seem to differ in their responses. conclusion: results exemplify not only the importance of health literacy and patient provider communication, but demonstrate the wealth of information gained from qualitative research-a method of data collection seldom utilized in ob/gyn investigations. funding: women's reproductive health research award: hd050121-02; r21 ca126450 (spring). population. kristine beckers, 1 isabelle guelinckx, 2 greet vansant, 2 roland devlieger. 1 1 obstetrics and gynaecology, university hospital gasthuisberg, leuven, belgium; 2 clinical nutrition, katholic university leuven, leuven, belgium. objective: to generate reference charts for weight gain during pregnancy for the different bmi-categories (underweight, normal weight, overweight, obesity), based on recent data in a homogeneous caucasian population. methods: in a retrospective study at the department of obstetrics of the leuven university hospital (belgium), weight gain and pre-pregnancy bmi were determined in 605 belgian pregnant women with accurately dateable, uncomplicated singleton pregnancies. centile curves for the different bmicategories were constructed using of the linear mixed model, one set of charts based on the absolute weight gain, another set based on the relative (expressed as percentage) weight gain. the effect of parity on weight gain was examined. results: overall mean weight gain was 14.8 ± 4.7 kg (32.63 ± 10.36 lbs). mean weight gain was 15.4 ± 4.1 kg (33.95 ± 9.04 lbs) in the underweight population, 15.1 ± 4.5 kg (33.29 ± 9.92 lbs) in the population with normal weight, 13.7 ± 5.3 kg (30.20 ± 11.68 lbs) in the overweight population and 12.0 ± 5.9 kg (26.46 ± 13.01 lbs) in the obese population. weight gain (pattern and amount) of the underweight and normal weight patients differed significantly of the overweight and obese patients. parity had a statistical, but no clinical significant influence on amount and evolution of weight gain. conclusion: by using strict inclusion criteria, bmi-category-specific reference charts were generated representing the optimal gestational weight gain, rather than the mean weight gain. this enables the weight charts to be used as a clinical tool during the counselling of pregnant women. further studies are required to assess the effectiveness of this clinical tool. female fshr(-/-) mice are sterile, because of a block in folliculogenesis at the primary follicle stage, show decrease in e2 and elevated fsh. this animal model is an appropriate model for studying hypergonadotropic ovarian dysgenesis and infertility, caused by c566t mutation in fshr gene. objective: to investigate the effects of bmt on serum hormonal levels, follicular maturation and fertility of fshr(-/-) mice. methods: fshr (-/-) mice at 6-10 weeks of age were randomized into treated versus control groups.bm from 1 syngenic female donor was injected into the tails of 2 recipients. control group received vehicle alone. vaginal smears were collected, body weight was measured daily. sample animals were sacrificed at 0, 1, 2, 3, and 4 weeks post bmt. all organs were weighted and examined by h e. fsh, e2, and p4 were measured before and after treatment. for donor cell tracking, dna was extracted from various organs. specific primer sets were designed for normal and mutant hfshr gene. pcr amplification was done and pcr products were analyzed in 1% agarose gel. results: total body weight significantly increased in treated animals(p< 0.02). significant increase in both the total number of follicles, and the collective diameter of the follicles in treated animals observed(p<0.03 and p< 0.002). six out of 8 treated animals showed estrogenic changes in daily vaginal smear. e2 level increased 2.5-7.5 times and fsh level dropped to 50% in treated animals. normal (donor allele) fshr gene was amplifiable in 6 out of 8 recipient mice, and was detected only in the ovaries and uterus but not in any other tested organs.control group did not show any changes in vaginal smear, hormonal level, and normal fshr allele. conclusion: intravenously injected syngeneic bone marrow cells were able to home to the ovaries of female fshr (-/-) mice and restore folliculogenesis and resume steroid hormone production. potential mechanisms for these observations will be discussed. conclusion: neural stem cells (nscs) give rise to progenitors, which expand by rapid proliferation until cell cycle arrest followed by differentiation along one of 3 cns cell lineages: neurons, astrocytes and oligodendrocytes. increased differentiation of neuronal cells with ins and even more with leptin suggests that iugr-associated reduction of these growth factors may contribute to impaired hypothalamic pathway development. objective: to develop a technology of modulating hucb in order to achieve neuronal cells for future potential replacement of damaged neuronal tissue. design: we developed a two-dimensional (2d) tissue culture technology for isolation and differentiation of collagen-adherent hucb neural progenitors (hucbnps). we further used the extracellular matrix protein collagen, organized in a three-dimensional (3d) gel, supplemented with neuronal conditioning medium and nerve growth factor (ngf), to facilitate ex-vivo long term neuronal differentiation of hucbnps. we developed a stable green fluorescence protein (gfp)-pc12 cell model, to be used as a positive control for monitoring neuronal outgrowth for proper evaluation of the hucbnps growth in the 3d scaffold, mimicking a neural tissue organization. results: our experimental data indicate that 3d collagen environment is neuronal biocompatible, supporting attachment, long-term survival, proliferation and differentiation of both hucbnps and gfp-pc12 cells. conclusions: improvement of the 3d technology with cultures of hucbnps that is in progress in our laboratory might be the first step in validating the concept for the feasibility of generating a neuronal 3d tissue construct for future potential treatment of neurodegenerative disorders. piane, justin tsai, gnanaratnam giritharan, emin maltepe, paolo rinaudo. reproductive sciences, university of california san francisco, san francisco, ca, usa. objective: ivf embryos are often used to generate stem cells. however, it is unknown if stem cell lines originated from in vitro generated embryos are different from stem cell originated from in vivo embryos. trophoblastic cells, due to their external position within the embryo, are most susceptible to environmental factors encountered in vitro. furthermore, our previous data showed that trophoblast transport functions may be impaired after culture in vitro and that the number of trophoblastic cells is reduced in the embryo after ivf. in this study, we assess the differentiation characteristics of ts cell lines obtained from in vivo and in vitro fertilized embryos. methods: oocytes were isolated from superovulated c57bl/6j female mice and in vitro fertilized with sperm from male c57bl/6j mice. the resulting late-cavitating blastocysts were harvested. in vivo controls were obtained by flushing the blastocysts from the uteri of superovulated pregnant mice 4 days post hcg. ts cells from the two groups were allowed to develop and maintained in vitro in the presence of fgf4 and heparin, using a feeder layer of human placental fibroblasts. ts cells were then allowed to differentiate without fgf4 and heparin, fixed on day 8, stained with -tubulin and zo-1 antibodies and then observed under fluorescence microscopy. three ts cell lines per group were analyzed and their differentiative capacity was evaluated using morphological criteria. results: in vivo and ivf derived ts exhibit a similar differentiation pattern. in particular, the number and timing of trophoblast giant cells and spongiotrophoblasts derivation is similar in the two groups. there are no obvious abnormalities in the immunologic staining morphology of the different cell lines at different time points. conclusion: there are no apparent morphological alterations in ts cells lines derived from ivf embryos as compared to in vivo embryos. this finding in an animal model increases our confidence in the reliability of human stem cells derived from ivf. as a further investigation, markers of trophoblast giant cells, spongiotrophoblast, syncytiotrophoblast and chorionic trophoblast cells will be examined and compared in the two different groups by northern blot hybridization. genomewide high density snp-cgh reveals several new deletion copy number variants on the x chromosome in pof patients. erik ah knauff, 1 cisca wijmenga, 2 ruben van 't slot, 2 lude franke, 2 bart cjm fauser. 1 1 reproduction gynecology, university medical centre, utrecht, netherlands; 2 complex genetics group, university medical centre, utrecht, netherlands. introduction: around 1% of women have a post-menopausal hormonal profile before age 40, referred to as premature ovarian failure (pof). pof could act as a genetic model for accelerated follicle loss and may render useful information about the polygenetic background of major individual differences in menopausal age. macrodeletions on the x chromosome are associated with pof but karyotyping has a maximal resolution of 10mb. when using genotyping whole genome arrays it is now possible to detect submicroscopic deletions and duplications (copy number variants (cnv)) up to 50 kb effective resolution. we have screened for microdeletions on the x chromosome in a well-phenotyped cohort of pof patients. methods: our study included 108 caucasian, 46,xx patients with spontaneous secondary amenorrhea before age 40, fsh > 40 iu/l and absence of (low) 45x/46 xx mosaicism and fmr1 premutations. dna analysis was performed using illumina 370k cnv arrays containing 370,404 probes. 12,556 probes were located on the x chromosome ( 1 probe for every 12 kb), of which 1,348 probes were specifically designed to detect known cnvs. ten samples with call rates <99% were removed from the analysis and one sample gave inconsistent x probe intensities. we screened for deletions ( 60 kb) using an algorithm detecting at least five consecutive probes with intensities (logr > -0.20) below the mean probe intensity. we identified a total of 183 x chromosomal microdeletions, divided in 84 loci and varying between 60-200 kb in size. 46 of the 97 samples showed at least one deletion on the x chromosome. 27% of the identified deletions had already been recorded in the database of genomic variants. in the newly identified newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced loci, we found 26 coding regions containing 38 genes. eight of these are established or potential pof candidate genes, including five that are clustered on the terminal xq critical pof region. conclusions: we observed abundant variation in the cnv regions, a proof-of-principle for this specially designed cnv-chip. snp comparative genomic hybridization revealed possible new deletions specific to pof on the x chromosome. data on duplications, validation and whole genome cnv analysis, compared to a control cohort, will become available soon and will be presented at the sgi annual scientific meeting. unexplained intrauterine fetal death (iufd) is associated with long qt syndrome. irene cetin, 1 patrizio antonazzo, 1 sabrina cozzolino, 1 stefania calabrese, 1 lia crotti, 2 francesca ferrari, 3 roberto insolia, 2 fabio facchinetti, 3 peter j schwartz. 2 1 dept. of obst/gynecol, univ. of milano, milano, italy; 2 molec cardiol lab, policlinico san matteo, pavia, italy; 3 mother-infant dept., univ. of modena/reggio emilia, modena, italy. introduction: in developed countries, nearly 1 in every 200 pregnancies ends in late fetal loss. many iufds can be attributed to maternal disorders, fetal pathology, placental pathology and fewer to complications of labor and delivery. however, 25-50% of cases remain unexplained. we recently demonstrated that 10% of sudden infant death syndrome (sids) cases carry functionally relevant genetic variants in long qt syndrome (lqts) genes. aim of the study was to analyze whether lqts genes are associated with iufd. materials and methods: 60 patients with iufd were enrolled in two years, as part of an italian multicentre study. iufd was defined as fetal death at 22 weeks or more of gestation according to the definition of late fetal death of the who. 32 out of 60 cases were classified as "unexplained stillbirths" according to the wigglesworth and aberdeen classifications. at birth placental and/cord samples were collected and dna extracted. the main lqts genes kcnq1, kcnh2, scn5a, kcne1 and kcne2 were screened through dhplc and sequence analysis. any amino-acid substitution identified in the samples was checked for in a control population of 122 caucasian women with uneventful pregnancies. preliminary data on the first 17 cases (gestational age of death: 23-38 weeks) are reported. results: a total of 3 missense mutations were identified in 3 of 17 stillbirths (18%), two on scn5a and one on kcnh2. the two mutations on scn5a (v1951l; p2006a) were observed in two iufd occurred at term; they had been previously associated to sids and shown to increase the late sodium current. the mutation on kcnh2 is a novel genetic variant absent in 244 reference alleles, never described in any control populations; this mutations was present in a case of iufd diagnosed at 33 weeks of gestation. we are currently performing the electrophysiological cellular studies to define its functional effect. conclusions: these preliminary data indicate that a potentially significant number of currently unexplained iufd might be caused by ion channel diseases such as lqts. the potential prevention of sids or iufd recurrence and the identification of other affected family members could have important implications for the affected families. alternative splicing of epab is regulated by exonic splicing enhancers. e seli, a yaba, o guzeloglu-kayisli, md lalioti. ob gyn, yale u., new haven, ct, usa. introduction: alternative splicing is an important mechanism by which the genome gives rise to the observed diversity of proteins. embryonic poly(a) binding protein (epab), expressed exclusively in oocytes and early embryos, mediates translation of maternal mrnas. we identified an alternatively spliced form of epab lacking exon 10 (cex10del), and investigated its regulation as a model for alternative splicing in early development. specifically, we evaluated: imprinting (expression from maternal or paternal allele only); rna editing (post-transcriptional single nucleotide substitution); and exonic splicing enhancers (eses, exonic sites that bind splicing proteins). methods: a single nucleotide polymorphism (snp) detected in exon 9 (c1290a/g) served as a marker for the parental origin of the spliced form. snp genotyping was performed by pcr amplification of exon 9 followed by restriction enzyme digestion. to evaluate imprinting, we characterized heterozygous mice (a/g) that inherited the snp from either the mother or the father. to test for rna-editing and exonic enhancer contribution we tested mice homozygous for the exon 9 snp (a/a or g/g). efficiency of alternative splicing in different genetic backgrounds was tested using real-time pcr normalized to actin expression. results: in mice heterozygous (a/g) for the exon 9 snp, cex10del was only expressed from the (a) allele. however, this was independent of the parental origin of the allele, ruling out imprinting. in mice homozygous (g/g) for the exon 9 snp, the cex10del variant also contained (g). therefore, rna editing did not occur. further sequence analysis led to the identification of an additional snp in exon 10 (c1383g/c) that co-segregated with the exon 9 snp. presence of c1383g led to the formation of an ese that binds splicing regulatory protein srp40, leading to efficient exclusion of exon 10. real time pcr revealed a five-fold increase in the expression of the cex10del alternative splicing variant in animals carrying the enhancer (homozygous g/g) for the exon 10 snp compared to those that did not (homozygous c/c) at the same locus (p < 0.001). conclusions: in this study, we found that eses mediate the alternative splicing of oocyte-specific transcripts. our findings suggest that single nucleotide polymorphisms may alter the ratio between alternative splicing variants of oocyte-specific proteins. the role that these subtle differences play in determining individual reproductive outcome remains to be identified. epigenetics and chromosomal abnormalities in human oocytes. ilse van den berg, 1,2 joop se laven, 1 robert jan galjaard, 2 j hikke van doorninck. 1,2 1 obstetrics gynaecology; 2 clinical genetics, erasmus medical center, rotterdam, netherlands. humans have a low fertility rate compared to other mammalian species. moreover their fertility declines with increasing age. both phenomena are largely due to an increasing number of chromosomal abnormalities in human oocytes during life. identifying factors that cause aneuploidy in oocytes may offer possibilities to diminish these abnormalities in vitro or in vivo. chromosomal segregation errors can result from aberrant recombination but little is known about epigenetic factors that may cause aneuploidy. epigenetic modifications such as histone acetylation and subsequent de-acetylation in oocytes are necessary for a correct progress through meiosis. if disturbed it may lead to aberrant segregation of chromosomes or chromatids due to decreased kinetochore function and imperfect spindle figures resulting in aneuploidy. mice oocyte data have shown a correlation of abnormal histone de-acetylation and aneuploidy and a correlation between age, remaining histone acetylation and aneuploidy (akiyama et al., 2006) . our research focused on human oocytes and investigated whether a similar relationship between histone acetylation, age and aneuploidy is present. human oocytes were surplus from standard ivf/icsi treatments (ic+). human oocytes showed immunostaining for histone 4, lysine 12 acetylation (h4k12) at the germinal vesicle stage and complete deacetylation at the mii stage in 45% of the oocytes, while 55% keep high levels of h4k12 acetylation. treatment of germinal vesicle oocytes with a histon deacetylase inhibitor (tsa) during in vitro maturation until mii stage resulted in high levels of acetylation. remaining acetylation in tsa treated oocytes was correlated significantly with abnormal spindle figures (tubulin staining), a hallmark of developing aneuploidy. similarly, 80% of oocytes with naturally remaining h4k12 acetylation showed abnormal spindle figures. in contrast 80% of the normal de-acetylated oocytes showed normal spindles (p=0.002). this suggests that defective de-acetylation of h4k12 in human oocytes leads to abnormal spindle figures and subsequent aneuploidy. advanced maternal age is associated with a reduction in de-acetylation during in vitro maturation and an increase in spindle abnormalities. these results may stimulate the development of assays for histone modifications as biomarkers to follow oocyte quality in in vitro maturation studies or in optimizing general ivf treatments. objective: racial disparity in preterm birth (ptb) is unexplained and genetic risk factors are suspected as a major cause. this large scale candidate gene study examines differences association of single nucleotide polymorphisms [snps] in caucasians (c) and african americans (aa) to help elucidate racial disparity in preterm birth (ptb) methods: in this case (preterm birth <36 weeks) control study (term birth > 37 weeks) maternal and fetal dna from 370 (172 cases and 198 controls) c and 279 (82 ptb and 197 term) aa were collected. a high-throughput candidate gene association study was performed examining 1442 snps in 130 genes of selected from hypothesized ptb pathways. single locus association analyses were performed separately on maternal and fetal samples. results: snps in 23 genes associated with ptb (p<0.01) were common between races with both maternal and fetal dna analyses. however, snps in 24 genes in c and 32 in aa in both maternal and fetal dna differed in its association with ptb. in c maternal dna, the single strongest association between ptb and snp was in plasminogen activator tissue (plat) gene (c-4443t; rs879293) at both allelic (p = 2.00x10 -3 ) and genotypic (p=2.0x10 -6 ) level with an odds ratio (or) of 2.80 ]. the single strongest effect in c fetal dna was observed in a snp in the interleukin-10 receptor antagonist gene (a18792g; rs17121510) for both allele (p=0.01) and genotype (p=3.34x10 -4 ), or 1.92 ). in aa, the strongest associations were in interluekin-15 (c13929t-rs10833, allele p=2.91x10 -4 , genotype p=2.0x10 -3 ) in maternal dna with an or=0.54 [ci 0.37-0.78] and in fetal dna interleukin-2 receptor b (a14481g; rs84460, allele p=1.4x10 -4 , genotype p=6.3x10 -4 ) with an or 2.36 ]. conclusion: large scale high throughput analyses of snps in candidate genes of ptb pathway reveals significant differences between races at both maternal and fetal levels. we found that the strongest single locus associations differed in the two races in both maternal and fetal dna samples. these findings support the hypothesis that underlying genetic predispositions may differ between these populations, perhaps partly explaining racial disparity in preterm birth. candidate gene association study indicates differential etiologies in gdm and in t2dm. johann urschitz, tarik sultan, kenneth ward. obgyn, jabsom, university of hawaii, honolulu, hi, usa. objective: recently several genome-wide association studies have associated multiple single nucleotide polymorphisms (snps) in multiple genes with increased risk of type 2 diabetes. as risk factors are similar between t2dm and gestational diabetes (gdm), we investigated a possible association of those 18 snps with gdm. study design: blood was collected from caucasian (100 cases, 364 controls) women who met coustan-carpenter criteria for gdm and non diabetic controls. dna was extracted and a candidate gene association study was performed (taqman, abi). results: chi 2 contingency tests were used to analyze genotype and allele frequencies in controls and gdm affected pregnancies (see table below). none of the snps showed a significant association after bonferroni correction. conclusion: several polymorphisms which displayed highly significant associations with type 2 diabetes are not associated with gestational diabetes. these results suggest that gdm is not a simple unmasking of a t2dm predisposition by the metabolic demands of pregnancy; rather it appears that different biological mechanisms are responsible for the respective diseases. introduction: histone acetylation/deacetylation plays an important role in the regulation of gene expression. histone deacetylase inhibitors (hdaci), in general, maintain gene expression, although in some cases they cause repression of specific genes. in this context we have previously shown that the hdaci tsa suppresses activation of the pro-contractile gene cox-2 in human myometrial and amnion-derived cells [reproductive sciences, vol 14, no 1 (supplement, #9)]. we have also shown that expression profiles for hdacs (1-6, 8) differ significantly within upper and lower regions of the myometrium during pregnancy and in labour. objectives: since changes in both acetylation and phosphoryation status can influence the activity of individual hdacs we aimed to define whether there are any changes in the levels of acetylation and phosphoryation of myometrial hdac 1, 2 and 8 during pregnancy and labour. methods: protein homogenates prepared from non-pregnant, term and labouring myometrium were treated +/-shrimp alkaline phosphatase and subjected to sds-page and western immunoblotting (wb) using antibodies to hdac1, 2 and 8. the acetylation status of the hdacs was assessed by a co-immunoprecipitation assay using anti-hdac and anti-acetylated lysine antibodies. results: distinct patterns of acetylation were observed for the individual hdacs; hdac 1 and 2 appeared to be more acetylated in non-pregnant and labouring lower tissues when compared to pregnant myometrium. in contrast, hdac8 appeared to be slightly more acetylated in lower uterine samples from pregnant and labouring myometrium. in experiments to evaluate changes in phosphorylation status we observed that 1) myometrial hdac1 appeared to less phosphorylated in both upper and lower labouring samples when compared to non-pregnant and pregnant samples; 2) hdac2 appeared to be more phosphorylated in labouring samples than non-pregnant and pregnant myometrium; 3) no changes in phosphorylation levels were observed for hdac8 using this test system. conclusions: the difference in hdac acetylation and phosphorylation levels in the human myometrium may indicate differential regulation of the activity of the hdacs within the distinct myometrial regions, perhaps leading to the alteration of their epigenetic effect on genes related to myometrial quiescence and contraction and the subsequent onset of both term and preterm labour. objective: native hawaiians and pacific islanders experience a higher perinatal morbidity secondary to the increased incidence and earlier onset of heart failure. this increased incidence is likely multi-factorial and includes co-morbidities and genetic factors. mutations in the human adrenergic receptor gene may play a role in the determination of heart function. mutations in the b1-adrenergic receptor (adrb1) located on chromosome 10 have been identified as a cause progressive cardiomyocyte loss leading to a dilated cardiomyopathy. the minor allele frequencies for most of these polymorphisms have been determined for caucasian, japanese, african-american and chinese as part of the hapmap project. the frequencies have not been determined in the pacific islander groups. this study helped determine the hawaiian allele frequency and determine the allele frequency in the presence of cardiac or other vascular co-morbidities such as hypertension, preeclampsia, and gestational diabetes. study design: real time pcr technology (taqman genotyping assays, applied biosystems) was used to screen maternal dna (n= 1315) from the phenotyping sample core of the pacific center for early human development (prcehd) for the rs1801253 single nucleotide polymorphisms (snps). genotype frequencies were also determined in 100% complete ethnic controls as determined by a four grandparent descent. results: chi square was used to analyze allele and genotype frequencies differences between controls and affected pregnancies. the results are summarized in the following tables. conclusion: the rs1801253 snp was not significantly associated with hypertension, preeclampsia or gdm in our overall hawaiian population. the genotype frequency in the 100% pacific islander group was significantly different from the caucasians (p=0.04) and asians (p=0.008) in our overall hawaiian population. angiotensin-converting enzyme and -adducin polymorphisms in preeclamptic mothers and fetuses. c mando, 1 p antonazzo, 1 s tabano, 2 f colleoni, 1 a martinelli, 1 s calabrese, 1 c benedetto, 3 l marozio, 3 f facchinetti, 4 m miozzo, 2 i cetin. 1 1 inst. obstetrics and gynecology, fondaz. irccs policlinico mangiagalli regina elena, univ. milan, milan, italy; 2 dept. biology and genetics, univ. milan, italy; 3 dept.obstetrics and gynecology, univ. torino, italy; univ. modena and reggio emilia, modena, italy. background the genes encoding angiotensin-converting enzyme (ace) and -adducin (add1) share the potential of influencing blood pressure. previous studies demonstrated in humans the association of hypertension with the combined effect of both ace insertion/deletion (i/d) polymorphism, which leads to a different activity of the enzyme, and add1 g460w non-sense single nucleotide polymorphism (snp). ace i-i genotype has been associated with low serum ace activity. controversial studies concerning the association of ace polymorphism with preeclampsia (pe) were reported and the possible combined effect of both ace i/d and add1 g460w polymorphisms yet remains to be investigated. population association study we genotyped 2 polymorphisms (ace i/d and add1 g460w) in 497 women: 197 with pe (119/197 with severe pe) and 300 controls. moreover, we investigated a subset of their fetuses: 24 from severe pe, 17 from mild pe and 300 controls, in order to identify specific maternal and/or fetal genotypes conferring a higher risk to develop pe. we both evaluated the single and the combined effects of ace and add1 genotypes on mother-fetus couples and singularly on mothers and fetuses. ace i/d genotype was analyzed using a pcr method; an allelic discrimination approach was performed to detect the add1 snp. in mother-fetus genotype couples, neither ace nor add1 polymorphisms are associated with pe, nor are the combination of their genotypes separately in mothers and in fetuses. nevertheless in mothers with mild pe ace i-i genotype is significantly less frequent (8% vs 13%; p<0.05) and ace i-d is significantly more frequent (59% vs 42%; p<0.02) compared to controls. ace i allele frequency is not significantly different in mild pe compared to controls (37% vs 38%). in women with mild pe the i allele seems to move from i-i to i-d genotype. it leads to the increase in mild pe women of those genotypes with a higher ace activity conferring a higher susceptibility to develop pe. this association with mild pe and not with severe pe could be due to the contribution in the latter of many other genetic and environmental factors. introduction: maternal mrnas stored in the oocyte are critical for early development as transcription ceases upon oocyte maturation, and gene expression until zygotic genome activation (zga) is mediated by translation of maternal transcripts. cytoplasmic polyadenylation element binding protein (cpeb) plays a central role in this process by stabilizing maternal mrnas and regulating their timely activation. this function has been well characterized in xenpous, and a similar role in mammals is suspected as the cpeb knockout mouse displays infertility as a result of arrested oogenesis. in order to investigate if translational regulation of maternal transcripts by cpeb is maintained in mammals, we characterized the spatial and temporal expression of cpeb-1 in the mouse and human. methods: ten different somatic tissues, testes, and ovaries were tested by rt-pcr for the expression of cpeb mrna in mouse and human. cpeb mrna expression in mouse was also tested in prophase i (pi) and metaphase ii (mii) oocytes, 1-cell, 2-cell, 4-cell, 8-cell embryos and blastocysts. in human, pi and mii oocytes, 8-cell embryos and blastocysts were evaluated. sequencing of the pcr products was performed to confirm specific amplification of cpeb. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. results and discussion: highest level of cpeb mrna expression was detected in ovaries and testis of both mouse and human. in addition, differential expression of cpeb in somatic tissues was also observed. among these, prominent expression was present in brain, where a role for cpeb in facilitating gene expression through cytoplasmic polyadenylation has been proposed. these data would suggest that translational control of mrna by cpeb may be a mechanism utilized by multiple somatic tissues to regulate gene expression. in the mouse, cpeb was expressed in pi and mii oocytes and 1-cell and 2-cell embryos, and became undetectable in 4-cell or more advanced embryos. human cpeb was expressed in pi and mii oocytes, but not in 8-cell embryos or blastocysts. zga occurs at late 2-cell stage and 4-to-8-cell stage, in mouse and human, respectively. the tightly controlled temporal expression of cpeb prior to zga in both mouse and human is consistent with a conserved role for cpeb in the regulation of maternal mrna expression during early development in mammals. background embryonic stem cells (esc) express specific transcription factors that are indicative of their ability to maintain pluripotency. these factors are activated during preimplantation embryo development. the interplay between the transcription factors pou5f1 (oct3/4) and caudal-homeobox domain 2 (cdx2) is thought to regulate inner cell mass (icm) and trophectoderm (te) differentiation; however, the mechanism of cell fate determination in mammalian embryos is poorly understood. genetic ablation of oct3/4 in mice prevents icm development, while cdx2 knockout embryos fail to implant. esc deficient in nanog, a second key regulator of pluripotency, fail to maintain pluripotency and undergo differentiation. expression of nanog in primate embryos has not been investigated, therefore the localization of oct3/4, nanog and cdx2 was determined in rhesus macaque blastocysts. methods oocytes were collected from rhesus macaque females, fertilized and cultured in vitro to the blastocyst stage. embryos were collected 144 hours post insemination, then fixed prior to incubation with primary antibodies directed against oct3/4, nanog and cdx2. following incubation with cy3 conjugated secondary antibodies, nuclei were counterstained with dapi and embryos visualized using an olympus bx41 fluorescence microscope. results nanog protein was restricted to the icm of monkey blastocysts. unlike the mouse embryo, oct3/4 protein was detected in both the icm and te, based on two different antibodies. the expression of cdx2 was localized specifically to the te. conclusions the ubiquitous pattern of oct3/4 expression is consistent with observations in human, cow and pig embryos. significantly, lack of restricted oct3/4 protein, and icm localization of nanog in primate blastocysts, suggests that nanog more specifically determines cell fate in primate embryos. these results contrast markedly with current mechanistic hypotheses, although other factors may lie upstream of nanog. importantly, this difference may underlie observations that regulatory mechanisms in esc differ between mice and primates. further investigations will focus on determining the onset of marker expression, and the upstream regulators of nanog activation. supported by nih grants 1r01hd045966 1r21rr021881, and the intramural research program of nichd. in vitro-conceived mice tend to be smaller at birth and throughout their life. luisa delle piane, annemarie donjacour, francesca di sebastiano, gnanaratnam giritharan, paolo rinaudo. obstetrics, gynecology and reproductive sciences, university of california san francisco, san francisco, ca, usa. objective: epidemiological evidence indicates that ivf is associated with an increased incidence of low birth weight. this phenomenon has not been studied in a mouse model; in addition, it is unknown if the resulting offspring show different growth pattern later in life. we therefore created an ivf mouse model to follow growth patterns till adulthood. methods: we generated 3 experimental groups of b6 mice: one cohort of in vivo generated mice (in vivo group), one cohort of in vitro generated animals (ivf group) transferred to cd1 foster mothers and one cohort of animals fertilized in vivo and transferred to cd1 foster mothers (flushed blastocysts group, fb). the fb group was generated because our preliminary results showed that embryo transfer to b6 foster mothers was not successful. all pups were delivered at term, measured and weighed at birth and then weekly up to 18 weeks. parametric tests (anova with bonferroni correction) were used as appropriate. a mixed model was used to compare growth curves. results: average litter size was 6.3 (in vivo), 3.7 (fb) and 3.5 (ivf). birth weight of male mice both ivf (1.57 mg) and fb (1.73 mg) was larger than male in vivo mice (1.30 mg) (p<0.05). ivf mice tended to be smaller than fb mice in both sexes (p= 0.0533). the bmi (body mass index) was not different among all the groups. male fb growth curves were different from in vivo mice (p<0.0001) and more importantly from ivf growth curves (p=0.0516) (fig. 1) . conclusion: the method of conception and the maternal environment play a significant role in determining birth weight in this mouse model, emphasizing that the fb group is the best control for the effects of ivf in this model. these preliminary results confirmed for the first time an ivf effect on growth and interestingly the ivf males did not show a catch-up growth. the finding of smaller ivf mice when compared to fb is both noteworthy, because it confirms human data, and worrisome, because lower birth weight is associated with long term sequelae according to the barker hypothesis. the effect of betamethasone exposure in mid-gestation on renal sodium excretion in male sheep. lijun tang, 1 luke carrey, 1 nancy valego, 1 philip deibel, 1 james perrott, 1 jorge figueroa, 1 mark chappell, 2 james c rose. 1 1 obestetrics and gynecology, wake forest university, medical school, nc, usa; 2 hypertension center, wake forest university, medical school, nc, usa. objective: whereas prenatal exposure of ovine fetuses to clinically relevant doses of glucocorticoids during the time of peak nephrogenesis results in a reduction in nephron number in adulthood, there is little information about its effect on sodium excretion. in the present study, we evaluated the effect of exposure to betamethasone on renal sodium excretion in adult male sheep. methods: we studied nineteen conscious adult rams at 1.5 years of age which were exposed to either vehicle or betamethasone at 80-81 days gestation. we implanted vascular and bladder catheters and then allowed the animals a 5-7 days recovery period prior to study. inulin and para-aminohippuric acid (pah) clearances were performed for estimating glomerular filtration rate (gfr) and renal plasma flow (rpf) respectively. following determination under basal conditions, an acute hypertonic sodium load was administrated intravenously by a continuous infusion of nacl (0.0275 meq/kg/min at 0.55 ml/min) for 60 minutes. urine was continuously collected for determination of na + excretion. results: basal gfr was decreased in steroid exposed adults (2.267 ± 0.10 ml/min/kg) compared with the vehicle animals (1.935 ± 0.08 ml/min/kg) (p=0.028). rpf was similar in the vehicle and steroid exposed group (15.483 ± 0.40 ml/min/kg vs 13.561 ± 0.80 ml/min/kg). at basal conditions, na + excretion (u na v) was similar in vehicle and steroid exposed group (16.60 ± 5.37 μmol/h vs 14.37 ± 2.46 μmol/h). this similarity was also present after normalization by body weight (0.278 ± 0.09 μmol/h/kg vs 0.234 ± 0.04 μmol/h/kg). the vehicle group excreted 53.65 ± 9.71% of the dose of na during the experiment while the betamethasone exposed group excreted only 37.07 ± 4.42% (p<0.05). gfr and prpf did not change during the experiments. these results suggest that prenatal exposure to glucocorticoids affects renal function in adult male sheep which results in a decreased basal gfr and an attenuated ability to excrete on acute sodium load. the elevated blood pressure previously observed associated with this prenatal steroid treatment may be related to the alteration in renal function. the study is supported by nih grants hd 47584, hl 68728 and hd17644. characterisation of human fetal cardiac stem cells. marah alfakir, nicholas dawe, annette meeson, stephen c robson. school of surgical reproductive sciences, newcastle university, newcastle upon tyne, united kingdom. objectives: for many patients with severe cardiac disease treatment is limited to surgical intervention and/or heart transplantation. the heart has a limited regenerative capacity but it is insufficient to repair extensive damage. cellular therapies might provide an alternative treatment. we have identified stem cells (sp cells) in the adult mouse and human heart and have shown that the mouse cardiac sp cells can differentiate along a cardiac lineage. sp cells can be identified using facs combined with hoechst 33342 dye efflux. this ability to efflux hoechst dye is due to expression of abcg2 (an abc transporter). we have hypothesized that the fetal heart would contain more cardiac stem cells, relative to the adult, and the aim of this project was to determine the spatial and temporal expression of known stem cells markers in the embryonic/fetal heart. methods: 15 human fetal hearts were collected aged between 6-16 wk. we used rt-pcr, using primers for several stem cell (abcg2, cd34, cd45) and cardiac specific (mlc-2v, mhc) markers, and ihc on frozen and paraffin sections, using a panel of antibodies (abcg2, cd34, cd45, islet1). cdna was generated from the following regions of the heart at different developmental stages: right and left atrium, right and left ventricle, and outflow tract. results: abcg2 mrna was highly expressed in all regions of the fetal heart between 7-10 wk. expression was down regulated at 11-15 wk especially in the la and lv. a similar pattern of expression was observed for cd34. cd45 showed low/moderate expression in all heart regions examined; however, this expression was absent in the lv at 13-15 wk. mrna and protein analysis showed similar results. whilst protein expression of abcg2 was robust at 7 wk (approximately 3-6%), it declined with increasing gestational age. cd34 was also strongly expressed at 7 weeks of age. there was no co-localisation between abcg2 and islet1. conclusion: abcg2 and cd34 are both expressed between 7-10 wk of age. based on this analysis, further studies are underway to isolate and characterise both the abcg2 and cd34 expressing fetal cardiac cells which might be a potential source of cardiac stem/progenitor cells. xanthine oxidase plays a significant role in the fetal cardiovascular defence to hypoxia. ja hansell, a kane, e herrera, da giussani. physiology, cambridge university, united kingdom. prenatal hypoxia remains a major concern in obstetrics. the fetal defence to hypoxia includes redistribution of the cardiac output, away from peripheral and towards essential ciculations, such as those perfusing the brain (cohn et al . ajog 120:817,1974) . the physiology underlying this response is well characterised and involves chemoreflex and endocrine responses (giussani et al. fet mat med rev 6:17, 1994) . more recently, local factors such as nitric oxide (no) and reactive oxygen species (ros), and the interaction between them, have been shown to play a role. antioxidants, such as vitamin c, scavenge hypoxia-induced ros, maintain no high and thereby depress peripheral constriction (thakor et al., sgi 2006) . in this study, we address the source of hypoxia-induced ros production and investigated the effects on the in vivo fetal femoral constrictor response to hypoxia of maternal treatment with the xanthine oxidase inhibitor allopurinol in sheep. methods: under anaesthesia, 8 sheep at 0.8 gestation, were instrumented with maternal and fetal catheters and a fetal femoral transonic probe. five days later, all animals were subjected to 2 h normoxia, 0.5 h hypoxia (mat fio 2 to reduce fetal pao 2 to ca. 10 mmhg) and 1 h recovery, either following maternal i.v. treatment with vehicle or allopurinol in low (30 mg.kg -1 over 20 min) or high (150 mg.kg -1 over 30 min ) doses. treatments finished 100 min prior to hypoxia. the low allopurinol dose was adopted from human studies (benders et al. arch dis child 91:163, 2006) . the timing of the hypoxic challenge coincided with peak concentrations in fetal sheep plasma of oxypurinol (active metabolite). we evaluated the effect of bpa exposure on uterine gene expression in a nonhuman primate model. method: a total of nine vervet monkeys (cercopithecus aethiops sabaeus) were ovariectomized. three monkeys were treated with bpa via alzet pumps (50 microgram/kg/day), two with estradiol benzoate and three received combined treatment with bpa and estradiol. the remaining monkey was untreated and served as a control. following 28 days of treatment the monkeys were hysterectomized and immunohistochemistry performed to examine endometrial gene expression. we evaluated hoxa10, proliferating cell nuclear antigen (pcna), and caspase iii gene expression as markers of differentiation, proliferation, and apoptosis respectively. differential expression was evaluated by h-score. results: exposure to a combination of estradiol and bpa resulted in increased expression of hoxa10 and pcna compared to control (p<0.01). lower, but increased, levels of hoxa10 and pcna gene expression were seen in the specimens exposed solely to estradiol (p<0.05). those specimens exposed to bpa alone demonstrated a small induction in hoxa10 expression (p<0.05) with no change in caspase iii or pcna expression when compared to the control. conclusion: bpa induced the expression of hoxa10 in the uteri of a non-human primate. exposure in the presence of endogenous estrogen further augmented estrogenic stimulation confirming that bpa is a xenoestrogen. bpa's effect of developmental gene expression may alter uterine differentiation. bpa acts as an endocrine disruptor by altering estrogen regulation on a gene required for fertility. contractility and meconium passage. jayaraman lakshmanan, 1 john d richard, 1 guo l liu, 1 sharon k sugano, 1 ahmet karadag, 2 michael g ross. 1 1 dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa; 2 dept. of pediatrics, harbor-ucla med. ctr., torrance, ca, usa. objective: endogenous glucocorticoids (gcs) increase with advancing fetal age suggesting that gcs function as a hormonal modulator of organ maturation. although fetal colonic motility and meconium passage primarily occur near term, the role of gc in colonic maturation is poorly understood. we sought to examine gc-nuclear receptor (gc-nr) expression in ovine fetal distal colon smooth muscle from very-preterm to term gestation to define the cascade of gc initiated biochemical changes as a prerequisite for maturation-associated meconium passage. methods: ovine fetal distal colonic segments removed at very-preterm (vpt: 118-120 d gestation), preterm (pt: 130-132 d), near term (nt: 140-142 d) and term (t: 146-147 d) (n=6 fetuses in each group) were fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochmical analysis with gc-receptor antibody (1:200, sc-8992, santacruz biotechnology, ca) using standard abc regimen. immunoreactive material identified by 3,3'diaminobenzidine as chromogen. the percentage of gc-nr staining in smooth muscle-enteric unit was analyzed by counting dark brown immunostained nuclei at 10 different fields at 400x magnification. all values are expressed as mean ± sem. results: the gc-receptor antibody immunostained nuclei both in smooth muscle and enteric units and the observed pattern (expressed as percentage of total nuclei) are as follows: nuclear gc expression varies with gestational age with maximal expression occurring near-term in smooth muscle and enteric neurons, in a synchronized manner. a near total disappearance of gc-nr expression occurs at term. these results suggest that peak gastrointestinal gc-nr mediated effects may occur prior to term with secondary signaling effects resulting in distal colonic motility maturation and meconium passage. the rna-binding protein hud co-localizes with choline acetyl mechanisms of in utero meconium passage. jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, octavio balbuena, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: we have previously speculated that crf, acting through its receptor crf-r1, increases gastrointestinal (gi) motility and potentiates in utero meconium passage. patients with paraneoplastic syndrome with auto-antibodies to hud, a neuronal rna binding protein, develop severe gi dysmotility, indicating a role for hud in gi motility. hud binds mrna for actylcholinesterase implying a role in the cholinergic neurotransmitter system. based on these known functions, we hypothesized that hud may regulate post-transcriptional activity in fetal colonic cholinergic neurons expressing crf-r1. method: bouin's solution-fixed paraffin-embedded sections of distal colonic segments were prepared from very preterm (vpt: 118-120 days), preterm (pt: 130-132 days), near term (nt: 140-142 days) and term (146-147 days) ovine fetuses (n=6 at each age). sections were immunostained with anti-human neuronal protein huc/hud antibodies (1:800 to 1:1000) with abc reagents and examined at 400x. neurons positive for hud staining were quantified. double immunofluorescence and laser confocal analyses evaluated co-expression of hud in neurons expressing peripheral choline acetyl transferase (pchat) (a marker for peripheral cholinergic neurons) and crf-r1 receptor. results: hud immunostaining was seen in either entire cytoplasm (c) or cytoplasm and nuclear regions (c+n) in both submucosal and myenteric neurons. a greater percentage of submucosal (vpt: 55±3, pt: 53±3, nt: 54±3, and t: 57±3 %) than myenteric neurons (vpt: 35±3, pt: 42±3, nt: 38±4, t: 31±2 %) exhibited positive staining at all ages. the percentages of neurons with c+n staining in submucosal neurons were significantly lower at very-preterm gestation. confocal studies co-localized the hud staining with pchat and crf-r1 receptor immunoreactivity both in submucosal and myenteric neurons. conclusion: in the fetal enteric nervous system hud may function both as a rna-binding protein and as a nuclear cytoplasmic shuttling protein. colocalizaion studies suggest that both pchat-mrna and crf-r1 mrna species are target transcripts for hud in myenteric neurons. we speculate upregulation of hud contributes to in utero meconium passage. meconium passage during mild stressors. jayaraman lakshmanan, 1 guo l liu, 1 john d richard, 1 sharon k sugano, 1 raina khan, 1 octavio balbuena, 1 kimberly chap, 1 virender rehan, 2 michael g ross. 1 1 dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa; 2 dept. of pediatrics, harbor-ucla med. ctr., torrance, ca, usa. objective: mr exhibit a greater affinity to glucocorticoids (gc) than do glucocorticoid receptors (gr). thus low circulating gc levels may preferentially bind mr during mild-stress periods, while the high gc levels may bind gc-receptors. mr antagonism increases gc-mediated responses, suggesting that mrs have a regulatory impact on gc-mediated stress responses. we recently documented that fetal in utero meconium passage is a neurovisceral stress response. to test our hypothesis that mrs play a primary role in mediating suppressive gc effects, we examined the expression patterns of mr in ovine fetal distal colon. methods: bouin's solution fixed paraffin sections of distal colonic segments collected from ovine fetuses (n=6 for each gestational ages) at very preterm (vpt: 118-120 days gestation), preterm (pt: 130-132 days), near term (nt: 140-142 days) and term (t: 146-147days) were subjected to immunohistochemistry with polyclonal antibodies to mr. digital photos (10 field per colonic ring, 6 rings each gestational age) taken at 400x were used to count the number of intensely stained neurons, referred to as "mr-capped neurons". differences over time were determined with anova. results: mr antibody elicited a punctate staining pattern in all layers of ovine fetal distal colon. significant immunoreactive intensity was observed in submucosal and myenteric ganglia at all ages: submucosal ganglia: vpt=0.130 ±0.009, pt=0.154±0.002, nt=0.151±0.005, t=0.169±0.016; a subpopulation of enteric neurons exhibited dense staining ("mr-capped"). advancing gestation was associated with a significant decreased in the percentage of mrcapped myenteric (vpt = 49±7, pt = 27±5, nt = 19±3, t = 22±1%; p<0.05), though not submucosal ganglia neurons. results indicate a significant decrease in the percentage of mr capped myentric neurons with advancing gestation. in view of the potential inhibitory effect of mr on gc-mediated stress responses, these results suggest that the decrease in mr expression may contribute to near term and term in utero meconium passage. the ambiguity of myometrial progesterone receptor expression in pregnancy and labour. alison j tyson-capper, elizabeth a shiells, stephen c robson. surgical reproductive sciences, institute of cellular medicine, newcastle university, newcastle upon tyne, united kingdom. background and aims: in contrast to many other species, human parturition is not due to a reduction in circulating levels of progesterone (p) but appears to be related to changes in expression, ratios or signalling events of p receptors (pr-a and pr-b). the literature on pr expression in human myometrium in pregnancy and labour remains conflicting. we hypothesise this is due, at least in part, to differing specificities of the various pr antibodies employed. we carried out a comprehensive analysis of pr expression using ten 'pr-specific' antibodies that recognise different amino acid epitopes within the pr proteins. two phosphorylation-specific antibodies for pr were also included to define whether phosphorylation status of myometrial pr changes in pregnancy and in labour. methods: western immunoblotting and semi quantitative rt-pcr were undertaken using protein lysates and rna prepared from myometrial tissue from: -first (n=4) and second (n=5) trimesters, paired upper and lower segment myometrium from preterm (22-28 wks, n = 4), term not in labour (n = 10), term spontaneous labour (n = 8) and non pregnant (n = 10). results: using the same set of myometrial lysates for each antibody, we found that the specificity of individual pr antibodies, in particular those raised against internal and c-terminal epitopes of the pr proteins, varied considerably resulting in different patterns of expression. there also appeared to be temporal and spatial differences in levels of myometrial pr proteins (90-120kda/60-70kda) in pregnancy and in labour with use of the phosphorylation-specific pr antibodies, however, it remains to be resolved which pr isoforms these may be. in contrast, data using four antibodies all of which react with the amino terminal domain of pr consistently indicated that expression of pr, in particular pr-b, decreased significantly at term (p < 0.001) and in labour (p < 0.001) when compared to non-pregnant levels. a decrease in levels of pr-b protein was also observed when comparing preterm levels to non-pregnant levels. rt-pcr using primers specific to pr-b consistently indicated that levels of pr-b mrna decreased at term and in labour. conclusion: interpretation of gestation-related changes in myometrial pr expression and phosphorylation status must take into account the pr antibodies used. further studies are now underway to validate the specificity of the pr antibodies. objective to test the hypothesis that expression of fshr in mural gl cells from ivf follicles varies with the infertility diagnosis and correlates with the outcome of ovulation induction (oi). materials and methods 86 women undergoing ivf were classified as: 1. "no ovarian factor", (tubal or male factor and egg donors, nof;n= 37); 2. endometriosis (endo; n=17); 3. poor responders (pr; n=19); 4. polycystic ovary syndrome (pcos; n=13). ovulation induction was carried out using a long or microflare or antagonist protocol based on clinical parameters and gonadotrophin doses were selected based on ovarian reserve (day 3 fsh and e2 and basal antral follicle count) and adjusted to the individual response. after ultrasound guided egg retrieval, mural gl cells were isolated from pooled follicular fluids from each patient using a percoll gradient and anti-cd45 immunobeads to eliminate wbcs, viability was assessed by trypan blue. fshr was measured by rt-pcr as relative expression compared to beta actin. statistical analysis was performed with the spss using pearson´s correlation, one way anova and student´s t-test. results fshr expression in nof (137±33.6) was statistically significantly higher than in pr (57±15.2) and lower than in pcos (290±77.2). both endo (85±30.2) and pr levels of fshr were statistically significantly lower than in pcos. analysis of all cycles showed that fshr expression correlates positively with the number of total (r=0,26; p< 0,05) and mii (r=0,35; p< 0,01) oocytes and negatively with the units of fsh (r=-0,31; p< 0,01) and lh (r=-0,24; p< 0,05) administered for oi. separate analysis of nof showed a positive correlation with the number of total and mii oocytes retrieved and with estradiol levels on day of hcg but not with the total dose of gonadotropins received during oi. fshr expression correlates negatively with day 3 fsh levels in all patients except in pcos (r=-0,25; p<0,05). conclusions these results suggest that expression of fshr in the hyperstimulated ovarian follicle is "average" in normoresponders, high in high responders (pcos) and low in poor responders (pr and endo). knowledge of the level of expression of fshr might be useful to individualize gonadotrophin doses and oi protocols thus improving pregnancy rates. comparison of intracellular atp levels between non-vitrified and surviving post-vitrification/thawed human oocytes. somjate manipalviratn, 1 zhi-bin tong, 1 eric widra, 2,3 alan h decherney. 1 1 reproductive biology and medicine branch, nichd/nih, bethesda, md, usa; 2 department of obstetrics and gynecology, georgetown university hospital, washington, dc, usa; 3 shady grove reproductive science center, washington, dc, usa. objective: to compare intracellular atp level in non-vitrified and surviving post-vitrification/thawed human oocytes using an atp bioanalysis assay. design: prospective match-controlled laboratory study material and methods: all oocytes were obtained from women undergoing controlled ovarian stimulation for ivf/icsi. oocytes were discarded due to nuclear immaturity at the time of planned icsi. all immature oocytes (gv and mi) were incubated overnight. oocytes from patients with 2 or more discarded eggs which matured to mii stage were used in this study. oocytes from each women were randomly divided into 2 groups. in group 1, the oocytes were placed in 50 l of ultrapure water and kept at -80 o c for further atp analysis. in group 2, the oocytes were vitrified using 15% ethylene glycol, 15% dimethyl sulphoxide and 0.5 m sucrose as cryoprotectant. these oocytes were kept in liquid nitrogen for 5-7 days before thawing with a rapid thawing method. oocyte survival was determined by morphological assessment after 90 minutes of incubation then oocytes were placed in 50 l of ultrapure water and kept at -80 o c for further atp analysis. intracellular atp level was determined using luciferin-luciferase bioluminescent assay. result: ninety-one discarded human oocytes were obtained from 23 women. 47 oocytes were vitrified/thawed before measuring for atp content. the other 44 oocytes were measured for their atp content without undergoing vitrification/thawing process. oocyte survival rate after vitrification/thawing is 72.34% (34/47). mean oocyte atp levels in non-vitrified oocytes is significantly higher than surviving post-vitrification/thawed oocytes (table 1). coefficient of variation of luciferin-luciferase bioluminescent assay is less than 5%. conclusion: oocyte atp level in surviving post-vitrification/thawed human oocytes is significantly lower than non-vitrified oocytes. objective to study the expression of genes involved in cell proliferation and steroidogenesis in the human follicle (kl1, kl2, fshr, papp, p450) and its relationship with ivf outcome. materials and methods 89 patients with different infertility diagnosis underwent ovulation induction with either a long or microflare or antagonist protocol based on clinical parameters; the dose of gonadotrophin used for ovulation induction was selected based on the ovarian reserve (day 3 fsh and e2 and basal antral follicle count) and adjusted to the individual patient response. ultrasound guided egg retrieval was performed 36 hours after administration of 10.000 iu of hcg. mural granulosa-lutein cells (gl cells) were isolated from pooled follicular fluids (ff) from each patient using a percoll gradient and anti-cd45 immunbeads to eliminate wbcs, viability was assessed by trypan blue. the genes under study were measured by rt-pcr as relative expression compared to actin. statistical analysis was performed with the spss statistical software using pearson´s correlation and mann-whitney u. results kl2 expression correlates positively with kl1 levels (r=0,86; p< 0,01) and with genes implicated in granulosa cell function such as fshr (r=0,36; p< 0,01), papp (r=0,54; p< 0,01) and p450 (r=0,39; p< 0,01). the number of mii oocytes obtained is positively correlated with both fshr (r=0,35; p< 0,01) and papp (r=0,28; p< 0,05) expression, but not with the other genes. kl2 expression in women who became pregnant during the ivf cycle studied was higher (25,8± 10) than in non pregnant women (16,6 ± 3,1).(n=80, mann-whitney u p<0,05). conclusions patients who become pregnant have increased expression of kl2, which is associated with optimal granulosa cell proliferation and maturation. this is in accordance with the presence of lower apoptosis in granulosa cells in the same patients. syndrome during assisted reproduction treatment. k jayaprakasan, r jayaprakasan, h al-hasie, js clewes, bk campbell, ir johnson, nj raine-fenning. school of human development, university of nottingham, nottingham, united kingdom. objective: to test the hypothesis that an increased pre-treatment ovarian blood flow is associated with the development of ovarian hyperstimulation syndrome (ohss) and to evaluate ovarian vascularity as a predictor of ohss during invitro fertilization (ivf). methods: 150 subjects undergoing first cycle of ivf had 3d transvaginal ultrasound in the early follicular phase of the menstrual cycle preceding ivf. 50 of them developed ovarian hyper-response, defined as retrieval of 15 oocytes and ohss. 100 subjects had normal ovarian response with retrieval of 4 to 15 oocytes in the absence of ohss. antral follicle count (afc), ovarian volume (ov), and ovarian vascularity (vascularisation index, vi; flow index, fi and vascularisation flow index, vfi) were measured and an unpaired t-test was used to compare these parameters between the ohss and the control groups. multiple logistic regression analysis was used to assess the predictive value of these variables against age, bmi and basal fsh for the development of ohss. results: the ovarian vi (9.64 ±7.94 vs. 8.64 ± 7.31), fi (37.95 ± 5.5 vs. 37.95 ± 5.5) and vfi (3.77 ± 2.68 vs. 3.51 ± 3.1) were similar in both the groups. afc and ov were significantly higher (p<0.01) in the ohss group (28.90 ± 14.55 and 10.40 ± 3.77 cm 3 respectively) than in the control group (19.23 ± 9.91 and 8.94 ± 4.7 cm 3 respectively). afc was the only significant (p=0.001) predictor of ohss on multiple regression analysis (table 1) . conclusion: women developing ohss during ivf do not demonstrate an increased pre-treatment ovarian blood flow as measured by 3d ultrasound but do have a significantly higher afc, which is the only significant predictor of ohss. compared to other species, little is known about the steroid biosynthetic capacity and gene expression profiles of human cumulus cells. objective: to examine the ability of human cumulus cells in primary and long-term culture to synthesize steroids and respond to gonadotropin or camp-dependent stimulation. methods: human cumulus cells were isolated from cumulusoocyte complexes during assisted reproductive technology (art) and placed in primary culture or propagated to third passage. at subconfluence, cells were transferred into serum-free conditions in the presence of vehicle, forskolin, fsh, or hcg. at 48h, media was collected and progesterone (p4) and estradiol (e2) levels were determined by eia. aromatase activity was measured by the tritiated water assay. aromatase (cyp19) and cholesterol side-chain cleavage (cyp11a1) mrna abundance was determined by quantitative real-time pcr (qrt-pcr). transcriptional regulation of the cyp19 and cyp11a1 promoters was investigated by transient transfection of cumulus cells with promoter luciferase constructs. results: substantial amounts of p4 and e2 were synthesized by cumulus cells in culture, which were further elevated by forskolin, hcg, or fsh treatment. the presence of cyp19 and cyp11a1 mrna in cumulus cells was confirmed by qrt-pcr, and the relative mrna abundance of both transcripts was induced by forskolin, hcg, or fsh treatment. changes in cyp19 and cyp11a1 gene expression in response to camp and gonadotropin stimulation were associated with increased transcriptional regulation of both the cyp19 and cyp11a1 gene promoters. our studies demonstrate that human cumulus cells are sites of significant p4 and e2 biosynthesis and respond to camp-and gonadotropin-stimulation in vitro. the ability to examine human cumulus cells in primary and long-term culture provides a unique model system to investigate cumulus cell function, and the paracrine role of cumulus cells in oocyte development, maturation, and subsequent fertilization. background: there is increasing evidence suggesting that events occurring during fetal development may result in increased predisposition to adult conditions, such as diabetes. in vitro fertilization (ivf) is a new environmental stressor and the long-term effects of these manipulations are currently unknown. there is some evidence that lower number of insulin-secreting cells at birth signals predisposition to diabetes later in life. in this study, we compare areas of pancreatic insulin-secreting cells in newborn mice conceived in vivo versus in vitro. methods: oocytes were collected from super ovulated cf-1mice and fertilized in vitro with cauda epididymal sperm from b6d2f1/j mice. fertilized eggs were cultured in whitten medium under 5% co2 in humidified air at 37 °c for 96 h. blastocysts were transferred to the uteri of pseudo-pregnant recipients. control mice were allowed to conceive in vivo. the newborn animals were sacrificed within 24 hours of birth. pancreases were sectioned, immunostained with anti-insulin, and total areas and stained areas were compared using t-test with two-tailed distribution. p value of <0.05 was considered significant. results: there were total of 12 newborn animals: 5 females (2 ivf, 3 controls) and 7 males (3 ivf, 4 controls). percentage of total pancreatic area occupied by insulin-secreting cells was lower in female ivf (0.145 mm 2 , 0.69%) animals compared to female controls (0.079 mm 2 , 0.97%) and higher in male ivf (0.019 mm 2 , 0.40%) animals compared to controls (0.138 mm 2 , 0.25%). the average of males and females was slightly lower for ivf (0.082 mm 2 , 0.54%) than controls (0.109 mm 2 , 0.61%). none of these differences were statistically significant. there was a trend in newborn female mice towards lower amount of insulin-secreting cells in the ivf offspring, suggesting possible predisposition to diabetes later in life. this effect was not observed in newborn males. while our study failed to demonstrate consistent and significant differences in the areas of insulin-secreting cells between newborn mice conceived in vitro and in vivo, the number of animals in the study may have been too small to show significant results. larger studies are needed to further investigate this question. peter s uzelac, phyllis risch, kassi shelton, steven t nakajima. obstetrics and gynecology, university of louisville, louisville, ky, usa. objective: several fertility preservation methods currently available may not be viable options to certain patients due to their high cost and limited number of centers offering these services. for women undergoing bilateral oophorectomy, in vitro maturation (ivm) of oocytes retrieved from unstimulated whole ovary specimens may represent a more practical solution. here we describe out initial experience in offering ivm as a fertility-preserving measure to two patients undergoing total abdominal hysterectomy and bilateral salpingo-oophoectomy (tah-bso). case one was a 29 year old nulligravid with chronic pelvic inflammatory disease unresponsive to medical management. case two was a 39 year old nulligravid with stage ia endometrial carcinoma. surgery was planned for the mid-follicular phase in order to collect prophase i oocytes before the onset of late-follicular phase atresia. upon surgical removal, suction aspiration of all identifiable follicles was performed. ovaries were then serially sectioned and all tissue was examined for the presence of prophase i oocytes. results: total number of prophase i oocytes retrieved was 9 and 6, maturation rate after 24 hours was 33% (2/6) and 44% (4/9), fertilization rate after 24 hours was 100% (2/2) and 25% (1/4), maturation rate after 48 hours was 50% (2/4) and 40% (2/5) and fertilization rates after 48 hours 50% (1/2) and 50% (1/2), for case 1 and case 2 respectively. ivm of oocytes retrieved from unstimulated whole ovary specimens may be a simple and inexpensive approach to fertility preservation in women undergoing bilateral oophorectomy. by requiring minimal adjustments to in vitro fertilization protocols, this treatment could easily be implemented in centers which currently have no fertility preservation program. patient age at time of retrieval may be predictive of developmental potential. continued patient enrollment and follow-up studies on the developmental potential of embryos derived from this technique are necessary to fully evaluate its potential for a role in fertility preservation. the routine use of progesterone as luteal phase support in women with a diagnosis of polycystic ovary syndrome (pcos) undergoing ovulation induction cycles with oral agents has not been fully elucidated. we hypothesize that women with pcos utilizing either clomiphene citrate or letrozole, an aromatase inhibitor, should administer intravaginal progesterone in the luteal phase to increase pregnancy rates. design: retrospective chart review. materials and methods: cycle data from women with pcos undergoing ovulation induction with clomiphene citrate or letrozole at the university of cincinnati medical center from 2002 to 2007 were evaluated. diagnosis of pcos was based on rotterdam criteria and all other infertility diagnoses were excluded. clinical pregnancy rates (presence of fetal cardiac activity on ultrasound at 6-7 weeks gestation) in women who received intravaginal micronized progesterone (200 mg bid) following ovulation induction (with and without intrauterine insemination) were compared to those who did not receive progesterone. results: no significant differences were noted in demographic parameters, including patient age or bmi. a total of 182 cycles were evaluated in 72 women treated with clomiphene citrate. clinical pregnancies were documented in 19.5% (18/93) of cycles in the progesterone group compared to 9.9% (9/91) of the non-progesterone group (p < 0.08). forty-three cycles were evaluated in 24 patients treated with letrozole. clinical pregnancies were documented in 17.2% (5/29) of cycles in the progesterone group compared to none (0/14) in the non-progesterone group (p < 0.02). conclusions: patients with pcos who used letrozole for ovulation induction had superior clinical pregnancy rates when using intravaginal micronized progesterone compared to women who did not receive luteal phase support. there was a trend toward increased clinical pregnancy rates in pcos patients utilizing luteal support following clomiphene citrate for ovulation induction. therefore, in women with pcos undergoing ovulation induction with oral agents, luteal supplementation with progesterone should be strongly considered, especially in those using letrozole. were measured and an unpaired t-test was used to compare these parameters between poor and normal responders. multiple logistic regression analysis was used to assess the predictive value of these variables against age and basal fsh for poor ovarian response. results: the ovarian vi (7.5 ± 5.3 vs. 8.6 ± 7.9), fi (38.9 ± 6.9 vs. 37.9 ± 6.5) and vfi (3.2 ± 2.6 vs. 3.5 ± 3.4) were similar in both poor and normal responders. afc and ov were significantly lower (p<0.01) in poor responders (9 ± 3.3 and 6.3 ± 3.5 cm 3 respectively) than in normal responders (19.2 ± 9.9 and 8.9 ± 4.7 cm 3 respectively). afc was the best (p<0.001) predictor of poor ovarian response on multiple regression analysis ( objective: gay male couples increasingly seek parenthood through in vitro fertilization using an oocyte donor and a gestational carrier, but no studies describe this unique experience. the purpose of this study was to determine medical and psychosocial issues unique to gay men using art. design: qualitative analysis of semi-structured interviews with gay male couples seeking parenthood through art. materials and methods: sixteen gay males (eight couples) entering an art program were assessed through the use of a semi-structured interview. characteristics evaluated included age, relationship status, duration of their relationship, psychological health and stability, how the decision was made concerning who would donate the sperm, the decision to use an anonymous or known oocyte donor, and whether their gestational carrier was someone previously known to them or not. results: the average age of the men in this study was 40 years. all eight couples were in a committed relationship and had been together for an average of 7.3 years. five of the couples (69%) had been joined in a civil union which is legal in the state of connecticut. all subjects were psychologically stable and in good health. six couples (75%) were very clear about which partner would inseminate the oocytes. of those couples, two felt that the older partner should donate; two felt that the partner who cared more about a genetic connection to the child should donate; and two felt that the partner with "better genes" should donate. the remaining two couples chose to inseminate equal numbers of oocytes in order to transfer an embryo from each partner. all couples chose an anonymous oocyte donor, two couples chose relatives as their gestational carriers, while the others chose carriers recruited through an agency. conclusions: participants in this study were determined to become parents through assisted reproduction. they had given thoughtful consideration to the medical and psychosocial issues unique to this process including which partner would be the genetic parent, and why. clomiphene superovulation. rb allen, 1 az steiner, 2 rh fogle, 1 mj kalan, 1 rj paulson. 1 1 ob/gyn, usc keck school of medicine, la, ca, usa; 2 ob/gyn, unc, chapel hill, nc, usa. intro: micro-dose hcg has been demonstrated to increase ovulation and pregnancy rates following clomiphene administration in women resistant to clomiphene. since micro-dose hcg stimulates growth in follicles expressing the lh receptor, we hypothesized that its use following clomiphene in women with unexplained infertility would increase the number of follicles that ovulate and subsequently, pregnancy rates. m m: irb approval was obtained for this prospective pilot study of women with unexplained infertility. on day #3 a baseline ultrasound was performed and serum fsh and e2 levels were measured. subjects were given 100mg of clomiphene daily from days 3-7 and then returned for serial ultrasounds on day #8. when at least 2 follicles 12mm were present, 200 iu of hcg im daily was initiated. cycles were monitored by ultrasound every 2 days until a +lh surge occurred. all subjects had previously undergone a cycle using clomiphene alone for superovulation followed by iui and these cycles were used for comparison. results: 5 subjects, aged 33.2±2.5 yrs (mean±sem) and bmi 25.8±1.6 kg/m 2 with 7.6±3.2 yrs of infertility were enrolled. the mean fsh and e2 levels were 8.9±1.4 miu/ml and 37.4±5.9 pg/ml, respectively. there was an average of 6.2±0.2 days from starting clomiphene to the attainment of 2 follicles 12mm. 4 out of the 5 subjects had 2 follicles 12mm by day #8. a mean of 10.4±0.7 days of treatment were required until ovulation occurred. there were twice as many follicles in the micro-dose hcg cycles, although due to the small sample size this did not reach statistical significance (2.8±0.4 follicles, measuring 20.6±0.9 mm in the micro-dose hcg cycles, compared to 1.4±0.3 (p=0.05), measuring 18.6±2.4 mm (p=0.47) in the control cycles). there was no difference in the endometrial thickness at the time of ovulation between the micro-dose hcg cycles and the control cycles: 8.4±2.0 mm vs. 10.0±2.3 mm (p=0.30), respectively. all subjects had at least 2 ovulatory sized follicles in the study cycles. 40% of subjects had an iui performed, however there were no pregnancies resulting from this study. conclusions: 1) the addition of micro-dose hcg to clomiphene may allow more follicles to reach an ovulatory size, which should theoretically increase pregnancy rates 2) this pilot study demonstrates that adding micro-dose hcg may increase the effectiveness of clomiphene when given in a superovulatory protocol. jessica salas, donald maier, john nulsen, claudio benadiva, lawrence engmann. obstetrics gynecology, university of connecticut, farmington, ct, usa. objective: to evaluate the antepartum, intrapartum, and neonatal complications in patients undergoing ivf using a gnrh-antagonist protocol where a gnrhagonist was used to induce final oocyte maturation. materials and methods: a retrospective review of data from high responders undergoing their 1st or 2nd ivf cycle using a gnrh-antagonist protocol who were triggered with leuprolide acetate (study group) or hcg (control) and achieved at least a singleton pregnancy reaching the third trimester. patients younger than 40 years old were included. both groups received luteal phase support with intramuscular progesterone. the study group received estrogen patch supplementation. outcomes measured were antenatal and intrapartum complications, order of gestation, gestational age at delivery, birth weight, neonatal adverse outcomes, and congenital anomalies. pearson chi square, fisher's exact test, or independent t-test were used as appropriate. results: the baseline characteristics were different (table 1) . maternal antenatal and intrapartum complications were similar in both groups (23.4% vs 39.4%, p=0.23). there were more singletons in the study group (84.9% vs 63.8%, p<0.05). a subgroup analysis of gestational age at delivery, birth weight, neonatal complications and congenital anomalies in singletons showed no difference (table 2) . conclusions: this is the first study reporting the maternal and perinatal outcomes after gnrh-agonist trigger. differences in response to ovarian stimulation may dictate use of gnrh-agonist triggering for prevention of ohss, which may explain the differences in baseline characteristics. maternal and neonatal complications remain unaffected. table 1 lupron (n=33) objective: moderate to severe ovarian hyperstimulation syndrome (ohss) occurs in 1-5% of assisted technology cycles and carries the potential for severe complications. traditional management consists of hospitalization with intravenous fluids (ivf), bedrest, and close monitoring. early and aggressive paracentesis is an alternative method of treatment for ohss in an outpatient setting, but the economic consequences of the two treatment regimens have not been compared. design: cost-effectiveness analysis materials and methods: two scenarios, outpatient management with transvaginal paracentesis and conservative therapy with hospitalization, were compared. potential initial outcomes were analyzed for the conservative group to include hospitalization either in a ward hospital bed or the intensive care unit (icu) for an average of seven days. costs included ivf administration, ultrasound, and daily bloodwork. initial outcomes for the outpatient management group included no further therapy beyond the initial transvaginal paracentesis, bloodwork, ivfs, and ultrasound versus admission for an average of three days to a ward bed with similar management. the probability of the negative outcome for the conservative group was set at 5% (icu admission) and 10% for the outpatient group (regular hospitalization). results: the cost of conservative therapy including first tier complications ranged from a low of $8,399 to a high of $14,615. the cost of outpatient management with aggressive paracentesis and its first tier complications ranged from a low of $940 to a high of $2,060. this resulted in an estimated cost burden of $7,459 to $12,555 for conservative management with hospitalization. the main improvement factor was that patients in the outpatient management group had a much lower likelihood for prolonged hospitalization than the conservatively managed group. conclusions: aggressive outpatient treatment of moderate to severe ohss with early paracentesis appears to be a cost-effective strategy. induction. zeynep alpay, michael p diamond, michael l kruger, elizabeth e puscheck. obstetrics and gynecology, division of reproductive endocrinology and infertility, hutzel hospital, wayne state university, detroit, mi, usa. introduction: aromatase inhibitors (ai) are a new method of ovulation induction and is proposed to replace clomiphene citrate (cc) due to their reported advantages. their superior effect is thought to be due to up-regulation of the estrogen receptors and increase in the sensitivity of the endometrium, resulting in better proliferation despite low estrogen levels in the circulation. objective: to compare the effect of different ovulation induction methods, including ai, cc and gonadotropins (gt) on the endometrium in infertility patients. methods: we reviewed 321 ovulation induction cycles performed in our institution in the last one-year period retrospectively. there were 149 gt, 145 cc and 27 ai cycles. age, gravida, day 3 (d3et) and midcycle (mcet) endometrial thickness, endometrial pattern (ep) on day of hcg injection, endometrial growth during the induction cycle (d-et), which was calculated by the difference between mcet and d3et, and the pregnancy rates (pr) were compared. the ep was categorized as type a (homogenous and hyperechogenic), type b (intermediate isoechogenic pattern and a poorly defined central echogenic line), and type c (multilayered triple-line). chi-square, anova, t test and pearson correlation were used for statistical analysis. results: in all cycles, ep was closely related to the d-et (p < 0.001). this correlation appeared to be more pronounced when observed ep was compared with d-et (r = 0.311; p < 0.001) rather than with mcet (r = 0.168; p = 0.002). there was a reverse relationship between the age of the patients and ep (r = -0.215; p < 0.001). a negative correlation was also found between the gravida and ep (r = -0.28; p < 0.001). pregnancy rate was significantly correlated with ep (r = 0.193; p < 0.001). pregnancy rate was 2% in type a ep, 5.2% in b, 16% in c when all cycles were analyzed. aromatase inhibitors and gt treatments each resulted in 52% type c pattern in contrast to 33% with cc (p = 0.014 for ai vs. cc; p = 0.05 for gt vs. cc). the d-et was greater in gt cycles compared with ai or cc (p < 0.001). conclusion: these results show that ep has a strong correlation with pr in ovulation induction cycles. additionally, ai and gt treatments have similar effects on ep. both ep and the growth of the endometrium during the induction (d-et) are good prognostic variables for the successful pregnancy initiation. ovarian response in patients undergoing ovarian stimulation after myomectomy. hyacinth browne, 1,2 desiree mccarthy-keith, 1,2 barbara stegmann, 1,2 alicia armstrong. 1,2 1 reproductive biology and medicine branch, nichd, nih, bethesda, md, usa; 2 reproductive endocrinology and infertility, walter reed army medical center, washington, dc, usa. objective: the impact of myomectomy on ovarian function has not been wellstudied. other surgical treatments of fibroids, such as hysterectomy and uterine artery embolization, have shown an increase of fsh into the peri-menopausal range. the objective of this study is to examine ovarian response in infertile women undergoing ovarian stimulation after abdominal myomectomy. design: retrospective analysis. materials and methods: a retrospective analysis of all infertile women with known fibroids who had a failed art cycle, from january 2000 to 2007, followed by an abdominal myomectomy and a subsequent art cycle was performed. women served as their own controls. ovarian function pre and post-myomectomy was assessed by age, day 3 and 10 fsh levels, days of stimulation, total gonadotropins used, peak estradiol level, number of oocytes retrieved, embryos obtained, and high-grade embryos, and pregnancy outcome. quantitative results are presented as mean + sd. results: four women had a failed art cycle and underwent an abdominal myomectomy prior to a subsequent art cycle. the mean age was 35 and 36 pre-and post-myomectomy, respectively. all subjects had uterine factor infertility. two of these women also had tubal factor infertility, and one had endometriosis and male factor infertility. we found no difference in ovarian response pre and post-myomectomy. pre-myomectomy post-myomectomy age (years) 35 +3.6 36 +3.6 day 3 fsh (u/l) 6.9 + 1.7 6.1 + 0.96 day 10 fsh (u/l) 6.1 +1.9 6.2+ 2. objective: anti-mullerian hormone ( amh) is produced by developing primordial follicles. amh levels are stable through the menstrual cycle and therefore can be drawn randomly. the objective of this study was to assess the association of anti-mullerian hormone ( amh) and oocytes obtained at ivf retrieval. design: cross-sectional cohort study. materials and methods: the study cohort is comprised of thirty women in 30 cycles of in vitro fertiization (ivf). serum levels antimullerian hormone (amh) were drawn before starting an ovulation induction cycle for ivf. standard ovulation induction was performed with leuprolide flare and 450 miu/ml per day of follicle stimulating hormone. we performed linear regression of log convert4d oocyte number against the log converted amh level. serum amh was measured using an enzymatically amplified two-site immunoassay dsl-10-14400 active mis/amh elisa. statistical analysis was performed using spss version 15.0. continuous values are presented as mean std error. results: the log number of oocytes retrieved was directly related to the log value of amh (p = 0.036). conclusions: our data demonstrate an association between serum amh and oocytes produced in response to ovulation induction. using amh levels in addition to fsh levels in evaluating for ovarian reserve and pregnancy outcome may help improve predictions of reproductive performance. support: foundation for reproductive medicine. context: prediction of outcome after in vitro fertilization (ivf) can be difficult due multiple factors. human chorionic gonadotropin (hcg) levels correlate with pregnancy outcome, but data that can be used to easily counsel patients on their possible outcome is lacking. objective: to investigate the use of hcg levels along with other significant factors to predict the likelihood of an ivf pregnancy progressing to the point of detection of cardiac activity by ultrasound design: retrospective data analysis of 1377 ivf cycles performed from january 1997 to july 2007 resulting in 665 pregnancies. multiple logistic regression analysis modeling was performed to determine the factors most predictive of an ongoing early pregnancy and to assess possible confounding variables. setting: an academic fertility center. patients: patients undergoing in vitro fertilization using autologous fresh embryos. intervention: none main outcome measure: pregnancy continuation to the documentation of cardiac activity by ultrasound. results: maternal age, day 14 (post-oocyte aspiration) hcg level, and day 16 hcg levels were significant in predicting pregnancy outcome. day 14 and day 16 hcg levels were highly correlated and can be considered proxies for each other. the most accurate predictive model used only a single day 14 hcg level and maternal age. the type of fertilization method used, the cycle number for that patient, and the number of embryos transferred were not found to be significantly different. ongoing pregnancy rates were directly proportional to day 14 hcg level, and inversely proportional to maternal age. the incidence of multiple pregnancies also increased proportionally to the initial hcg level. 99% of ongoing pregnancies had hcg level > 23 miu/ml. conclusions: a single day 14 post-oocyte aspiration hcg level and maternal age are the most predictive of an ongoing ivf pregnancy. there was no difference in outcome between fertilization methods or number of embryos transferred. there is no benefit to obtaining serial hcg levels after the initial one. erk activation with u0126 (10 m)) reduced fsh stimulated cyclin d2 mrna expression by 50%. fsh has also been shown to stimulate mtor, a regulator of growth and proliferation of many cell types. inhibiting mtor activation with 10nm rapamycin for 15 min significantly reduced fsh-mediated cyclin d2 mrna expression. dht exposure for 24 hours also inhibited fsh stimulated mtor signaling, as shown by a 40% reduction in the phosphorylation of its down stream target p70s6kinase. furthermore, pretreatment with dht resulted in significantly reduced fsh-mediated tsc-2 phosphorylation, which is an upstream regulator of mtor pathway. further studies revealed that fsh mediated tsc-2 phosphorylation and mtor signaling is regulated by erk, but independent of akt. based on these results we conclude that elevated 5 reduced metabolites of androgens inhibit fsh-stimulated pka-erk pathway resulting in the inhibition of multiple mitogenic signaling pathways leading to defective follicle maturation culminating in anovulation. thus, rescuing the erk activation might serve as a potential therapeutic target to restore normal granulosa cell proliferation in hyperandrogenic states. (supported by nih grant hd-38424). searching pcos-genes: results of a genome screen for pcos in a dutch founder population. olivier valkenburg, 1 annemarie g mulders, 1 aida bertoli-avella, 2 ben a oostra, 2 joop se laven. 1 department of gynecology and obstetrics, erasmusmc, rotterdam, netherlands; 2 department of internal medicine, erasmusmc, rotterdam, netherlands. context: although the etiology of pcos is not yet fully understood, evidence has accumulated for a complex genetic background i.e. a combination of multiple genetic and environmental factors. to reduce genetic heterogeneity, this study was conducted in a genetically isolated population in the netherlands . objective: in order to identify genomic loci that are associated with pcos, a whole genome screen using highly polymorphic microsatellite markers was performed in a founder population. subjects and methods: 72 pcos patients (2003 rotterdam criteria) were identified in the founder population (rucphen, the netherlands) of ± 20.000 inhabitants. patients underwent a standardized screening procedure that included clinical, ultrasound and endocrine evaluation. all patients plus 141 unaffected first-degree family members were genotyped using 366 polymorphic markers on (microsatellites) from the abi prism ® linkage mapping set md-10 (average spacing 10 cm). association-analysis was performed with the transmission disequilibrium test (tdt, genehunter software). linkage analysis was performed in 11 clusters of 2 or more closely related patients (simwalk2). results: there was an average number of 10.1 alleles per polymorphic marker. the tdt identified two non-adjacent markers on chrome 6 (d6s262 and d6s308) that showed significant association with pcos (p 0,01). other markers that showed significant association were positioned on chromosomes 1 (d1s450), 3 (d3s1565), 4 (d4s406) , 7 (d7s530), 8 (d8s264) and seventeen (d17s784) (all p values 0.01). linkage analysis in 11 sub-pedigrees revealed no significant linkage for these, or other, loci with pcos. moreover the results of a prior report of linkage of a marker on chromosome 19 (d19s884) could not be confirmed. conclusions: the lack of consistent results suggests the absence of a consistent genetic background in this select group of pcos patients. this supports the hypothesis of a complex genetic background for pcos that allows relatively small contributions of multiple risk-genes to be involved in the pathogenesis of this syndrome. in this founder-population the genetic heterogeneity was not sufficiently reduced to find risk-loci in a genome wide screen using highly polymorphic markers with an average spacing of 10 cm. objectives: to objectively quantify uterine and endometrial blood flow in women with polycystic ovarian syndrome (pcos) and to examine if this was different in women with different phenotypic expressions of the disease. methods: transvaginal 2d and 3d ultrasound was performed in 36 women with pcos, as defined by the rotterdam criteria, and sub-group analysis conducted based on the subjects' body mass index (bmi), ovulation status, and hirsutism score. anova was used to compare the mean values between the groups. results: pcos women with clinical hyperandrogenaemia had significantly lower endometrial and subendometrial blood flow than their anovulatory normoandrogenic counterparts (table 1 ). there were no differences between lean and obese women or between anovulatory and ovulatory women with pcos. the pulsed wave doppler parameters were similar in all three phenotypic groups. conclusions: hirsute women with pcos have impaired endometrial perfusion compared to their normoandrogenic counterparts which is only evident with 3d ultrasound and not conventional pulsed wave doppler. nusayba a bagegni, 1 jill blaine, 1 anuja dokras. 2 1 obstetrics gynecology, university of iowa hospitals clinics, iowa city, ia, usa; 2 obstetrics gynecology, university of pennsylvania, philadelphia, pa, usa. background: there is conflicting evidence on the association between pcos and early and late obstetric complications. it is unclear if the reported risks are independent of bmi, preexisting hypertension and diabetes. we examined the risk of early and late obstetrical complications in a large group of women with pcos compared to controls. methods: we reviewed pregnancy records of women with pcos (rotterdam criteria, n=130) and controls (tubal infertility, n=130) after in vitro fertilization at university of iowa from 1994-2006. the wilcoxon rank sum test and fisher's exact test were used to evaluate differences between variables and logistic regression analysis was used to determine the independent risk of pcos. results: subject demographics and medical history are shown in table. the first trimester miscarriage rate was 18% in women with pcos and 15% in controls. after logistic regression analysis pcos was not associated with miscarriage (p=0.495). the prevalence of gestational dm (gdm) was similar in both groups 12% pcos vs 11% controls. pcos was not associated with gdm after adjusting for age and bmi (p=0.678). however, bmi was significantly associated with gdm after adjusting for age and pcos (p=0.012). risk of both pre-eclampsia and pih was 10% in pcos and 5% in controls, but not statistically significant after adjusting for age, bmi and twin gestation. preexisting htn showed a significant association with preeclampsia (p<0.01). there was no significant difference in preterm delivery, cesarean section, twin gestation, intrauterine fetal death and intrauterine growth restriction in the 2 groups. conclusion: despite adequate power, our study did not detect an increased risk of miscarriage in women with pcos. obesity was a significant contributor to late obstetric complications, namely gdm. these findings may warrant aggressive counseling of women with pcos on the potential benefits of weight loss prior to pregnancy. objective: to assess the prevalence of polycystic appearing ovaries (pcao) as defined by the rotterdam criteria; and to determine whether metabolic parameters differ between regularly cycling women with pcao and those with normal ovaries. studies have demonstrated a population frequency of pcao of 21-33%, with 7-24% among women with regular cycles. most were performed in a young reproductive age population; none examined the impact of age. all were performed prior to adoption of the rotterdam criteria. recent studies have shown an increased prevalence of metabolic syndrome among women diagnosed with polycystic ovarian syndrome (pcos). however studies focused on women in their 20s found no increase in bmi or fasting insulin with pcao but regular menses. participants: 222 women aged 25-45 with regular cycles (every 25-35 days) enrolled in the ova study, a population-based study of ovarian aging. each subject underwent a transvaginal ultrasound for assessment of ovarian volume and antral follicle count (afc). outcomes collected included waist measurements and hdl, ldl, total cholesterol, triglycerides, fasting glucose and insulin. pcao were determined by the rotterdam criteria (afc of 12 on one ovary or ovarian volume > 10cc). student's t-test and chi-square tests were used to assess differences between those with and without pcao for continuous and categorical variables, respectively. the prevalence of pcao decreased with increasing age (table 1) . of the 69 women with pcao, 62% met the afc criterion only, while 19% met the volume criterion only, and 19% met both. women with and without pcao did not differ in waist measurements, or in fasting lipids, insulin, or glucose. the rotterdam criteria, while less subjective than those described by adams in 1986, have led to an increased prevalence of pcao among women with regular menses, over 1/2 of women in their 20s. women with pcao do not differ from those with normal ovaries in metabolic parameters associated with pcos. consideration should be given to adopting an age-adjusted criterion for afc, or a combination of afc and ovarian volume, for diagnosing pcos. background: pcos, especially accompanied by obesity, has been reported to be associated with a characteristic dyslipidemia comprising elevated triglycerides (tgs) and depressed hdl, especially the hdl-2 fraction. this is an atherogenic profile; in fact, studies suggest low hdl-2 may correlate most strongly with cardiovascular disease risk. weight loss is a mainstay of treatment and improves all manifestations of the disease, but the optimal diet to recommend remains undetermined. preliminary studies show a high protein diet may improve total hdl levels and insulin responses. however, the effect of weight loss and dietary composition on hdl-2 levels in pcos has not been investigated. objective: to evaluate the fasting lipid profile in newly diagnosed obese pcos patients and to determine the effects of a high-protein diet with or without metformin on weight loss, hdl-2 and other lipoproteins, and menstrual cyclicity. methods: in this pilot retrospective observational study, the fasting lipid profile of 42 obese women newly diagnosed with pcos was determined. they were then placed on a high protein (80-100 g/day), low carbohydrate (30-50 g/day) diet with or without metformin (76 and 24%, respectively) and followed monthly for an average of 13 months (range 5-31). results: at diagnosis, 66% had low hdl and 89% had low hdl-2; only 29% had elevated tgs. on the diet, the patients demonstrated an average weight loss of 24.7 lbs (212.01 to 187.4, p<0.001) and decreased bmi of 4.2 kg/m2 (34.2 to 30.0, p<0.001). hdl levels increased significantly (18% increase from 46.6 to 55.0, p<0.001), especially the hdl-2 fraction (34% increase from 10.9 to 14.6, p<0.001). triglycerides decreased as well (135.0 to 107.2, p<0.027). ldl decreased but did not reach statistical significance. 29 resumed menstrual cycles, 11 were started on oral contraceptives, and 1 had a hysterectomy. 2 pregnancies occurred. no difference was seen with metformin use. conclusion: a majority demonstrated decreased hdl and hdl-2 at diagnosis. the high protein diet resulted in significant weight loss and improvement in hdl-2 levels, as well as improvements in total hdl, tgs and menstrual cyclicity. metformin produced no added benefit. prospective trials based on these data will help determine the optimal diet to reduce the significant short-and long-term morbidity in the large population of women with pcos. resistin is an adipokine that has been associated with obesity and insulin resistance in animal models. studies on the role of resistin on insulin resistance in humans have been controversial. recently resistin has been shown to exert atherosclerotic effects and elevated resistin levels have been observed in women with coronary heart disease (chd). women with polycystic ovary syndrome (pcos) are at high risk for chd. our present study investigates potential association of resistin and markers of inflammation, c-reactive protein (crp) and insulin resistance in women with pcos. methods: thirty two women with pcos participated in the study. all were hirsute and had irregular cycles. nineteen women with normal ovulatory cycles who matched the pcos patients in bmi served as controls. fasting glucose, insulin, resistin and crp levels were measured in all women. after a high carbohydrate diet for 3 days, a standard oral glucose tolerance test (ogtt) was performed. blood samples were collected for glucose and insulin before and 1, 2 and 3 hrs after 75g oral glucose. the area under the curve (auc) for insulin and glucose was calculated. women with overt diabetes were excluded from the study. results: fasting insulin levels (24.9 + 2.2 μu [±se] /ml) and insulin response to oral glucose (auc) (527.9 + 52.2μu/ml) were higher (p < 0.001) in women with pcos compared to controls. all were insulin resistant with homa-ir value > 3.9 mol x μu / l 2 . resistin levels in women with pcos (15.6 ±1.3 ng/ml) was significantly (p < 0.02) higher compared to control women (10.7 ± 1.4 ng/ml). crp levels in women with pcos (9954.2 ± 918 ng/ml) was also significantly (p < 0.001) higher than the controls (2396.2 ± 579 ng/ml). there was a significant positive correlation between resistin and crp levels (r = 0.362, p < 0.04). there was no correlation between resistin levels and fasting insulin levels or insulin auc. there was also no correlation between resistin levels and fasting glucose or glucose auc. conclusions: our results indicate that (1)women with pcos and insulin resistance have increased resistin levels, (2) there is a strong association between resistin and crp (3) elevated resistin and crp levels may predict women who are at increased risk for chd (3) ) it is unlikely that resistin plays a major role on insulin resistance in women with pcos. vuk p jovanovic, 1 enrico carmina, 2 prati vardhana, 1 michel ferin, 1 rogerio a lobo. 1 1 department of obstetrics and gynecology, columbia university, new york, ny, usa; 2 department of internal medicine, university of palermo, palermo, italy. current diagnostic criteria for pcos includes both ovulatory (ov) and anovulatory or "classic" (c) phenotypes. in an effort to further characterize differences and /or similarities between these 2 phenotypes, we studied 48 hyperandrogenic women with pcos (age 27.3±6.4, bmi 27.1±5.6) and 20 age matched controls (age 27.7±4.5, bmi 26.8±3.9). women with pcos were divided into weight matched (ov) n=14 and (c) n=34 groups. fat and weight distribution were assessed by dexa (total fat, r1 fat, trunk fat) as well as fasting levels of lh, e2, mis/amh, kisspeptin and testosterone, the adipocytokines (leptin, adiponectin, visfatin and retinol-binding-protein-4 rbp4) and serum glucose, insulin and crp. the hyperandrogenic pcos groups had characteristically altered hormone profiles compared to matched controls. although total fat mass was comparable, women with c-pcos had a significantly larger waist circumference (93.2 14.9 vs. 86 85 cm, p<0.05) trunk fat, r1 fat and %trunk and %r1 fat compared to ov-pcos (p<0.05). leptin, rbp4 and visfatin did not significantly differ among the pcos subgroups although adiponectin was lower in the c-pcos group (p<0.05). quicki was significantly lower in c-pcos (0.33±0.02 vs. 0.35±0.02, p<0.05) and insulin was higher (14.9±5.76 vs. 9.7±3.5, p<0.01). serum lh was also higher (11.3±7.8 vs. 6.5±1.7, p<0.05) but kisspeptin, testosterone and estradiol were similar. mis/amh (8.3±5.2 vs. 6.8±4.9 ng/ml, not significant) and crp (2.78±1.5 vs. 1.3±0.5, p<0.05) were higher in c-pcos. significant correlations (p<0.05) were noted among kisspeptin/rbp4 (r 0.426), mis/testosterone (r 0.451), insulin/ %trunk fat (r 0.616, p<0,01), total fat/leptin (r 0.772, p<0.01), %trunk fat/adiponectin (r -0.522). in conclusion, women with c-pcos when compared to similarly hyperandrogenic women with ov-pcos with similar bmi, have increased abdominal fat, and appear to have differences in serum lh, crp and increased insulin resistance. the latter, as well as subtle differences in the adipocytokines may explain these differences in anthropometric findings. problems of normal oogenesis and folliculogenesis but also disturbed oogenesis and folliculogenesis in polycystic ovaries are not fully understood. oocyte specific genes play an essential role in oogenesis and folliculogenesis. there are suggestions about possible role of some oocyte specific genes in etiopathophysiology of pcos. zona pellucida 4 gene (zp4) is recently identified gene, which belongs to zona pellucida genes such as zp1, zp2 and zp3. the role of zp4, contrary to above-mentioned other zp genes is not well described. the aim of this study was to analyze zp4 coding sequence and expression in patients with polycystic ovary syndrome. material included blood received from 29 patients (mean age 24,2 +/-3,23 years; mean bmi 31,4 +/-4,54 kg/m 2 ) with polycystic ovary syndrome. all patients with pcos were diagnosed with the use of eshre/asrm criteria from 2003. dna was isolated from blood cells( after separation of blood cells from serum) using a dna isolation kit (qiagen ). genomic dna was used for in vitro amplification by pcr with a specific set of primers complementary to the coding sequence of the zp4 gene. products from each pcr reaction were examined by sscp method. samples with changes detected by sscp in comparison to control probes were cloned into plasmid vector and then automatically sequenced from a total of 29 patient samples with pcos, we identified 5 nucleotide changes in the zp4 coding seguence : 4 silent nucleotide changes in exons 1,2,5 ,7, and 1 nucleotide change in the exon 5 ( position 114, t>g). the mutation in exon 5 ( t>g ) results in substitution of cystein for glycin of amino acid in position 223 of zp4 protein. in summary, our data demonstrate that zp4 nucleotide changes account for 15% of patients with pcos. relationship between serum mullerian inhibiting substance levels and insulin in women with polycystic ovary syndrome. rebecca a chilvers, shilla chakrabarthy, summer james, xin ma, manubai nagamani. ob/gyn, university of texas medical branch, galveston, tx, usa. müllerian-inhibiting substance (mis) is a member of the transforming growth factor-superfamily of growth factors. it is expressed exclusively in granulosa cells and is believed to play a role in the regulation of follicle selection and maturation. women with pcos have anovulation and most of them have hyperinsulinemia. the purpose of our study is to investigate possible association between insulin and mis in the dysregulation of folliculogenesis in pcos. methods: twenty one women with pcos who had anovulatory cycles and hyperandrogenism were recruited for the study. sixteen women with ovulatory cycles and matched the pcos patients in age and bmi served as controls. an oral glucose tolerance test (ogtt) was performed in all women. blood samples were obtained at fasting and 1, 2 and 3 hours after glucose ingestion for measurement of glucose and insulin. to investigate the effect of hyperinsulinemia on mis secretion, mis levels were measured in ten patients during the ogtt. fasting mis, testosterone, dheas, fsh, and lh levels were measured in all patients. results: fasting insulin levels (16.4 + 2.4μu/ml) vs 9.9 + 1μ u/ml [+ se]) (p < 0.03) and area under the curve (auc) of insulin (267.9 + 33.0 vs 134.0 + 12.2μu/ml) (p <0.001) were significantly increased in women with pcos compared to control women, while the glucose levels were normal indicating insulin resistance. mis levels were significantly (p < 0.001) increased in women with pcos (6.5 + 0.9 ng/ml) compared to controls (1.9 + 0.2 ng/ml). there was no correlation between age and mis levels in pcos patients while there was a highly significant negative correlation between the age and mis levels in the control women ( r = -0.822, p< 0.0001). there was significant negative correlation between mis and fasting insulin levels (r = -0.462, p < 0.03) and insulin auc during the ogtt (r = -0.485, p <0.02). there were no changes in mis levels during ogtt. conclusions: results of our study indicate that in women with pcos, (1) there is an increase in secretion of mis, (2) higher insulin levels are associated with lower mis levels, (3) acute increase in insulin levels has no effect on mis levels, (4) lower mis levels in women with severe hyperinsulinemia could be due to associated increased follicular atresia and a decrease in the ovarian reserve. further studies are needed to investigate the role of insulin on mis secretion. hiroyuki asakura, noriko tanaka, kyoko nishio. ohgimachi ladies' clinic, osaka, japan. objective: elevation of serum anti-müllerian hormone (amh) levels among polycystic ovary syndrome (pcos) patients has been reported. however, the regulatory factors of amh in pcos remain unknown. we examined correlations between amh values and various indices of insulin resistance in pcos women. methods: 40 infertile women compatible with rotterdam criteria for pcos were recruited under informed consent. the subjects underwent 75g oral glucose tolerance test (75ggtt, sampling at 0, 60, 120 minutes), and 2-step elisa for amh (sensitivity 0.7 pmol/l, inter intra-assay c.v. :12.3%, 14.2%, respectively). p<0.05 was considered as statistical significance. results: average amh level of the subjects (age:31.9+3.9 years, bmi: 21.1+3.0 kg/m 2 , mean+s.d.) was 50.4+31.1pmol/l, which was higher than normo-ovulatory women (16.7+10.5 pmol/l, n=16). amh levels had significant positive correlation with total ovarian volume by ultrasound (18.2+11.4 cc), but not with bmi, waist-hip ratio (0.85+0.06), and levels of serum lh (5.5+3.6 iu/l), total testosterone (67.3+32.8 ng/dl). although fasting glucose levels (83.1+4.9 mg/dl) were positively correlated with total ovarian volume (r=0.41), amh levels had no significant correlation with fasting insulin (4.3+2.3 iu/l), fasting glucose/insulin ration (22.8+8.4), homa-ir (0.90+0.51), and the sum of insulin levels (98.6+42.1 iu/l)during 75ggtt. conclusions: increased serum amh level of pcos and its positive correlation with total ovarian volume implies that determination of serum amh level would aid in confirming diagnosis of the ovulatory disorder. absence of relationship between amh and various clinical indices of insulin resistance suggests alternative regulatory factors of amh gene expression in the follicular compartment. reproductive tissues. amisra a nikrodhanond, keeley l mui, helen h kim. ob/gyn, university of chicago, chicago, il, usa. background: although primarily a neuroendocrine hormone, gonadotropinreleasing hormone (gnrh) is also produced in reproductive tissues, where it acts locally. the study of gnrh regulation in these tissues is limited by low levels of expression. objective: to elucidate mechanisms that regulate gnrh expression in male tissues, our objective was to identify regions of the gnrh gene that target expression in vivo. methods: transgenic mice were generated with 2 different fragments of the mouse gnrh gene promoter (-3446/+28 and -249/+28 bps), fused to the luciferase reporter gene. in these mice, luciferase activity (detected as light) reflects gnrh promoter activity. using bioluminescent imaging, gnrh promoter activity was assayed in live gnrh-luc mice and in their reproductive tissues ex vivo. to confirm imaging results, luciferase activity was also measured as relative light units (rlu) in tissue homogenates. for each dna construct, 5 male mice were examined. with whole-body imaging, bioluminescence was detected in the genital region of the gnrh-luc transgenic mice (fig.a) . in mice that incorporated the -3446luc transgene, examination of reproductive tissues ex vivo revealed bioluminescence in the prostate and penis, but not in the seminal vesicles, epididymis, or testes (fig.b) . in contrast, in the -249luc mice, bioluminescence was detected in the testes, but not in other tissues (fig.c) . examination of luciferase activity in tissue homogenates from -3446luc mice confirmed gnrh promoter activity in the prostate (3876 ± 624 rlu) and penis (576 ± 151 rlu) and absence in the testes (39 ± 11 rlu). in the -249luc mice, however, luciferase activity was detected only in the testes (1283 ± 97 rlu) and not in the prostate (65 ± 25 rlu) or penis (48 ± 10 rlu). conclusions: our studies demonstrate that gnrh is present in male reproductive tissues, but may be differentially regulated in the testes vs. prostate/penis. promoter elements, contained within the proximal -249 bp of the mouse gnrh gene, are sufficient to mediate testicular gnrh expression while -3446 bp of the gene promoter are necessary to direct expression to the prostate and penis. characterization of mouse ringo/speedy homologues. z walton, s uckac, o guzeloglu-kayisli, md lalioti, d sakkas, e seli. ob gyn, yale u., new haven, ct, usa. introduction: ringo/speedy (ringo/spy) is a recently discovered cyclindependent kinase (cdk) activator that functions similar to cyclins in controlling the cell cycle. ringo/spy plays a crucial role during meiotic maturation in xenopus by inducing meiotic g2/m progression and germinal vesicle breakdown (gvbd). more recently, padmanabhan and richter demonstrated that ringo/spy mrna is repressed in the xenopus oocyte cytoplasm by pumilio 2. upon meiotic reactivation, pumilio 2 loses its interactions with ringo/spy permitting its translation. ringo/spy is then expressed, leading to activation of cpeb by phosphorylation, which in turn elicits polyadenylation and translation activation of the mrna for a critical oocyte maturation factor, the mos kinase. in this study, we investigated the expression of ringo/spy in mouse. methods: ten different somatic tissues, testes, and ovaries were tested by reverse transcription-polymerase chain reaction (rt-pcr) for the expression of ringo/spy homologues and their alternative splicing variants. ringo/spy mrna expression was also tested in prophase i (pi) and metaphase ii (mii) oocytes, 1-cell, 2-cell, 4-cell, 8-cell embryos and blastocysts. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. results: we analyzed the two previously identified homologues of ringo/spy (a and b). the two alternative splicing variants of ringo/spy a (a1 and a2) were separately studied for their expression profile. we also identified an additional alternative splicing variant of ringo/spy b and evaluated similarly. ringo/spy a (1 and 2) were expressed in testes, ovaries, and certain somatic tissues including brain, and spleen. ringo/spy b (both variants) were only expressed in testis. ringo/spy a or b were not present in mouse oocytes or early embryos. conclusions: our findings indicate that the previopusly described ringo/spy homologues a and b are not expressed in mouse oocytes or early embryos suggesting that either an alternative cdk-activator or a yet to be identified homologue of ringo/spy may be mediating meiotic g2/m progression in mouse. testis specific expression of ringo/spy b, and previously described role of ringo/spy in meiosis suggests a role for this protein in male gametogenesis in mouse. treated with glyburide. jennifer aguayo, gladys a ramos, alethea hanley, carri r warshak, thomas r moore. reproductive medicine, university of california, san diego, san diego, ca, usa. objective: to determine the risk factors that may predict inadequate response to first-line glyburide monotherapy in pregnant women with type 2 diabetes mellitus (dm) or gdm and to assess if non-responders are at increased risk of adverse pregnancy and neonatal outcomes. study design: this was a retrospective cohort of 146 women diagnosed with type ii or gdm initially treated with glyburide at a single institution from 2001-2004. maternal characteristics, adequate glycemic control defined as more than 66% of fasting glucose <95 mg/dl and one-hour post-prandials <135 mg/dl, and neonatal outcomes were assessed. non-responders were defined as failure to achieve adequate glycemic control on maximum daily doses of glyburide or intolerance due to side effects, necessitating switch to insulin therapy. statistical methods included bivariate analyses. results: of the 146 women initially treated with glyburide, 25 (17%) failed to achieve adequate glycemic control or did not tolerate glyburide. reasons for glyburide non-response were maximum dose (20 mg/d) reached (56%), maternal hypoglycemia (28%) and other side effects (16%). there were no statistically significant differences between non-responders and responders with respect to family history of diabetes (75% vs. 60%, p=0.25), prior history of gdm (24% vs. 32%, p=0.63) and macrosomia (>4000 g) (28% vs. 24%, p=0.80) or 1 hour glucose challenge test (204+51 vs. 180+36mg/dl, p=0.07). non-responders had a higher rate of obesity (bmi>30) (71% vs. 44%, p=0.02) and earlier gestational age at initiation of therapy (24+7.7 vs. 29+7.7 wks, p=0.01). there were no differences between the groups in the mean 36-week fasting (96+17 vs. 90+14 mg/dl, p=0.15) and post-prandial glucose values (133+26 vs. 127+19 mg/dl, p=0.87). no significant differences were observed in incidence of pre-eclampsia, primary cesarean delivery or birth weight. neonatal outcomes including ponderal index, neonatal hypoglycemia, nicu admission, and birth injuries also did not differ between the groups. conclusion: women who are obese and who require earlier initiation of glyburide therapy are at increased risk of non-response to glyburide monotherapy. however, despite non-response, these women can be managed with subsequent insulin therapy to achieve similar glycemic control and pregnancy and neonatal outcomes. a different diagnostic strategy using the 100gram, 3-hour glucose tolerance test for the diagnosis of gestational diabetes mellitus. yvonne w cheng, ingrid block-kurbisch, jennifer lydell, aaron b caughey. obstetrics, gynecology and reproductive sciences, university of california, san francisco, san francisco, ca, usa. objective: to examine whether a simplified strategy using the 100gm, 3-hour glucose tolerance test (gtt) may be useful for the diagnosis of gestational diabetes mellitus (gdm). methods: this is a retrospective cohort study of women with singleton pregnancy who received the 50-gm, 1-hour glucose challenging test (gct) for initial screening and the 100gm, 3-hour gtt as confirmatory tests for the diagnosis of gdm between 1988 and 2001. various combinations of the 100gm, 3-hour gtt results were examined and compared to the diagnostic criteria for gdm established by the carpenter and coustan criteria using the receiver-operator characteristic (roc) curves. perinatal outcomes of women who would have had a false-positive or false-negative test results were compared to those who did not have gdm using the carpenter and coustan criteria. potential confounding factors were controlled for using multivariable regression models. results: 2,535 women had 100gm, 3-hour gtt results available for analysis during the study period. using gdm diagnosed by the carpenter and coustan criteria as reference, various diagnostic strategies was compared using roc curves (figure 1) . summation of the 1-hour and 2-hour gtt results with a diagnostic threshold of 334mg/dl yielded the most optimal balance between sensitivity (82.1%) and specificity (93.2%). when compared to women without gdm, women who were diagnosed with gdm by c c criteria but not by summation of 1-hour and 2-hour gtts had higher odds of operative vaginal delivery (aor=1.81, 95% confidence interval [ci] 1.03-3.27) and neonatal birthweight >4000gm (aor=1.91, 95% ci 1.03-3.57). conclusion: using only the summation of only the 1-hour and 2-hour gtt results of the 100gm 3-hour gtt offers an alternative test strategy which may be more convenient and less costly for the diagnosis of gdm. however, women who would have gdm by the carpenter and coustan criteria but not by the summation method have higher odds of having an operative vaginal delivery and neonatal birthweight >4000gm. outcome of induction of labor indicated for term prom among women with or without a uterine scar. yifat ochshorn, avital skornick rapaport, adi reches, joseph b lessing, ariel many. obstetrics gynecology, lis maternity hospital, tel aviv sourasky medical center, tel aviv, israel. objective: we aimed at comparing the outcome of induced term deliveries presenting with prom with or without a previous uterine scar. methods: the computerized files of 973 women delivered following induction of labor due to term prom, were reviewed. perinatal outcome parameters such as the mode of delivery, indication for cesarean section, rate of low 5' apgar scores and nicu admissions were compared between women with and without a previous cesarean. results: during the study period 973 women delivered in our institution following prom and induction of labor , 49 of them had a previous uterine scar. parturients of both groups (with and without a scar) were similar with regard to age, gestational age at delivery and parity. cesarean section rate was higher for the previous scar group (28% vs 17%, p<0.05). the most common indication for cesarean was arrest of dilatation and/or descent among women with previous scar accounting for 43% and 27% (p<0.05) for women with and with no uterine scar, respectively. no differences were noted in neonatal outcome parameters such as rate of low 5' apgar scores and nicu admission rate between the two groups. conclusion: induction of labor due to prom culminates in higher cesarean rate in women with one previous scar compared with parturients with no scar. perinatal outcome is similar between the two groups. acid base balance and immediate perinatal outcome of vertex compared with breech presentation in elective cesarean section. avital skornick rapaport, yifat ochshorn, joseph b lessing, yuval yaron, michael kupferminc, ariel many. obstetrics gynecology, lis maternity hospital, tel aviv sourasky medical center, tel aviv, israel. backround: apgar scores, umbilical blood ph and bicarbonate are generally lower and pco 2 levels are higher in vaginally delivered breech neonates compared to cephalic deliveries. although cesarean delivery improved apgar scores there is a debate wether it improved the acid base status. this retroprospective study compared umbilical cord blood acid-base values and perinatal outcome of elective cesarean breech-delivery with those of elective cephalic cesarean delivery and to determine whether a different metabolic status and perinatal outcome should be expected in neonates in breech presentation. study design: the study group included singleton pregnancies delivered by elective cesarean section at term between january 2003 and march 2006. computerized files of singleton breech presentation elective cesarean sections were compared to those of singleton vertex neonates delivered by elective cesarean section. demographic data included: maternal age, pregnancy week at delivery and parity. perinatal outcome measures checked were: birth weight, apgar scores at 1'and 5', umbilical cord venous and arterial ph and base excess. results: during the period between january 2003 and march 2006 there were 1411 singleton elective cesarean sections, 404 of them were breech and 1007 vertex. the mean age, gravida and parity were significantly different between groups (33.5 vs.31.8, 2.95 vs. 2.19. and 1.26 vs.0.64 respectively, p<0.001). the birth weight was significantly different -3414.8 gram for the vertex and 3189.9 gram in the breech deliveries (p<0.001) there were no differences in either apgar scores or umbilical ph between the breech and vertex neonates delivered by cesarean section at term (38-40+6w). venous and arterial po 2 and pco 2 levels were significantly different, though the differences were very small and we doubt if these differences have any clinical importance. conclusion: although vaginal breech deliveries are associated with increased risk of asphyxia during delivery, elective cesarean breech deliveries are not at increased risk for lower ph levels or apgar scores. the clinical significance of bleeding during the second trimester of pregnancy. arie koifman, 1 amalia levi, 2 yaron zaulan, 1 avi harlev, 1 eyal sheiner. 1 1 obstetrics gynecology, soroka university medical center, ben gurion university of the negev, beer-sheva, israel; 2 epidemiology and health services evaluation, faculty of health sciences, ben gurion university of the negev, beer-sheva, israel. objective: this study aimed at investigating clinical importance and pregnancy outcome in women suffering from bleeding during the second half of their pregnancies. methods: a population based study including all deliveries which took place in the soroka university medical center between the years 1988-2005 were examined. comparison was performed between patients with and without second trimester bleeding pregnancies terminated before 22 weeks, multiple gestations and women lacking prenatal care were excluded . stratified analysis, using the mantel-haenszel technique, and a multiple logistic regression model were performed . results: during the study period, 175,093 singleton deliveries occurred in our institute. of these, 2010 (1.1%) were complicated with bleeding upon admission during the second half of pregnancy. the cases were attributed to placental abruption (63.5%; n=1276) and placenta previa (36.5%; n=734). independent risk factors associated with bleeding, were oligohydramnios, polyhydramnions, (odds ratio [or]= 1.6; 95% confidence interval [ci] 1.2-2.0; p=001 and 1.5;1.2-1.8;p<0.01 respectively ), suspected intra uterine growth restriction (iugr, 3.2; 2.6-4.0; p<.001), gestational age, previos abortions and maternal age. these patients subsequently were more likely to deliver by cesarean section (cs, 72.9% vs. 12.1%, or=19.5; 95%ci 17.6-19.5; p<0.001). perinatal mortality among patients admitted due to second half bleeding was significantly higher as compared to patients without bleeding (p<.001). conclusion: bleeding upon admission during the second half of pregnancy is an independent risk factor for perinatal mortality. careful surveillance, including fetal monitoring, is suggested in these cases in order to reduce the adverse perinatal outcome. crude and adjusted odds ratios for perinatal mortality among patients with vaginal bleeding. characteristics or 95% ci p crude or for perinatal mortality 7.9 6.6-9.4 <0.001 or adjusted for: <0.001 iugr 7.5 6.3-9.0 <0.001 oligohydramnios 7.6 6.3-9.1 <0.001 premature rupture of membranes 7.9 6.6-9.7 <0.001 the predictors included neonatal pi, bmi, or birthweight while the outcomes included hypoglycemia, shoulder dystocia, acidemia and hyperbilirubenemia. roc curve analyses were utilized to evaluate the relationship between sensitivity and specificity for each of the predictors and outcomes, with an area under the curve (auc) significantly greater than 0.5 indicating a screening test that is better than chance. results: neonatal pi, bmi and birthweight are poor predictors of nicu admission, acidemia and hypoglycemia with all auc values not statistically significantly different than chance alone. they are a reasonable predictor of shoulder dystocia, with birthweight functioning the best (p<0.001). in general, neonatal anthropometric measurements do not appear to be good predictors of short term neonatal outcomes. although they are a reasonable predictor of shoulder dystocia, they are not useful clinically for this outcome since they cannot be calculated until after birth. in future studies, new models of neonatal anthropometrics should be created to better predict adverse neonatal outcomes. neonatal anthropometric measurements and their relationship to perinatal outcomes expressed as auc and 95% confidence intervals history of at least one spontaneous preterm delivery were offered protocolbased prenatal care including first-or second trimester bacterial vaginosis screening, repeated ultrasound cervical length assessment, and supportive care by specialized nurses. women with a positive test for bacterial vaginosis were treated with oral metronidazol therapy, and women with a cervical length below 2.5 cm (before 25 weeks of gestation) underwent a vaginal cerclage. progesterone administration was not provided. data on obstetrical and medical history, pregnancy, delivery and neonatal outcome were prospectively collected and evaluated. results: in total, 104 pregnant women were prospectively followedup. eighty-seven women had experienced a preterm delivery in their last pregnancy, 52 of whom had delivered before 32 weeks. three women were diagnosed previously with a bicornual or septal uterus. twelve women received metronidazol therapy and 18 women underwent a vaginal cerclage. eighty-four women (80.8%) delivered at 37 weeks or beyond. one-hundred and three women (99.0%) delivered no earlier than 30 weeks, and only one woman delivered at 23 weeks. two women gave birth to a twin (at 30 and 35 weeks, respectively). two neonatal deaths due to pulmonary hypoplasia were recorded. conclusion: women at high risk of preterm delivery who were offered protocol-based prenatal care not including progesterone therapy had a better pregnancy outcome than would be expected from previous studies. our findings may question the reported beneficial effects of prenatal progesterone administration. premature rupture of membranes. tracy a manuck, alexandra g eller, m sean esplin, robert m silver. obstetrics gynecology, university of utah health sciences, salt lake city, ut, usa. objective: historically, outcomes following expectant management of midtrimester preterm premature rupture of membranes (pprom) have been uniformly poor. thus, many patients elected pregnancy termination. however, outcomes may be improved with recent advances in neonatal medicine. our purpose was to assess outcomes in expectantly managed early pprom in an era of improved maternal and neonatal care. study design: this is a retrospective cohort of patients from 2 tertiary healthcare systems from 2002-2007 experiencing pprom 24.0 weeks gestation. patients electing immediate termination of pregnancy, carrying fetus(es) with lethal anomalies, or delivering within 12 hours of pprom were excluded. survival without major morbidity was the primary outcome. data were analyzed using student t-test and chi-square as appropriate. results: a total of 92 women carrying 117 fetuses (73 singleton, 15 twin, 2 triplet, 2 quadruplet) met inclusion criteria. only the fetus in the "ruptured sac" was studied. pprom occurred at a mean of 20.7 (+/-2.6, range 13.6-24) weeks. the average latency period was 31.8 (+/-29.4, range 0.5-105) days, with a mean delivery gestational age of 25.2 (+/-4.3, range 15.4-34) weeks. this communication provides the first report of fundal uterine necrosis following placement of multiple uterine compression sutures. only two previous published cases report occurrence of uterine necrosis following application of a uterine compression suture --both identified as the b-lynch technique --and neither of these cases report necrosis confined to the fundus. in our case, uterine atony was refractory to pharmacologic therapy and manual compression of the uterus appeared to decrease the amount of blood loss. therefore, we applied a traditional b-lynch which effectively contracted the majority of the uterus, while the fundus remained atonic. a second horizontal, square suture was then placed between the cornua and carried over the fundus (see figure one: red reflects b-lynch suture; blue represents second fundal, square stitch). the patient subsequently experienced significant, persistent fevers despite antibiotic therapy. a repeat laparotomy was performed, at which time we found uterine necrosis confined to the uterine fundus (see figure 2 : photograph taken at the time of hysterectomy). the placenta showed no histologic evidence of chorioamniotis --hence, the patient's main risk factor for fundal necrosis was the compression sutures. physicians should be aware of the risk of uterine necrosis, especially after placement of multiple compression sutures and, more specifically, after placement of a compression suture confined to the uterine fundus. background: migraines are far more common in women than in men. the incidence of migraines increases in girls after puberty, reaching an incidence of 25% in women who experience migraine at least once a year around middle age. migraine has been postulated as one of the major risk factors for stroke during pregnancy and the puerperium.triptans are a class of serotonin receptor agonists used in the treatment of migraine headaches. triptans administered in combination with other drugs have been known to precipitate serotonin syndrome, a rare but potentially life-threatening condition clinically manifested by a triad consisting of mental-status changes, autonomic hyperactivity, and neuromuscular abnormalities. objective: to determine whether triptan monotherapy is associated with serotonin syndrome methods: using data mining techniques we analyzed the entire food and drug administration's (fda) adverse event reporting system (aers) database. results: after excluding reports of concomitant serotonergic medication or other potentially confounding medication, eleven reports remained of serotonin syndrome associated with triptan use without concomitant serotonergic medication. the mean age for these eleven cases was 39.9 years; nine cases occurred in females and two occurred in males. there were no apparent instances of overdose among these eleven cases. conclusions: serotonin syndrome is a rare but serious occurrence with the use of triptan monotherapy. such cases were seen with eletriptan, rizatriptan, sumatriptan, and zolmitriptan. because of the spontaneous nature of voluntary reporting to aers, the actual number of occurrences of serotonin syndrome in patients using triptans is probably higher and cannot be assessed from aers data. users of triptan in combination with an ssri or snri should be warned of this rare but serious adverse effect. during periods of drug initiation, dose escalation, or the addition of another serotonergic agent, patients should be particularly vigilant for symptoms of concern and seek urgent medical attention if any occur. the offending agents should be withdrawn and the patients closely monitored and treated with supportive measures as required. rising maternal mortality in the midst of modern technology. oormila p kovilam, 1 jane khoury, 2 padmini c sekar, 3 ralph c buncher. 3 1 obstetrics gynecology, university of cincinnati, cincinnati, oh, usa; 2 center for epidemiology and biostatistics, cincinnati children's hospital medical center, cincinnati, oh, usa; 3 environmental health, university of cincinnatic, cincinnati, oh, usa. introduction: maternal mortality rate (mmr) is an index of overall wellbeing of the community and safe motherhood should be given utmost priority in obstetric care. as per 1990, who estimates 1% of maternal mortality occurs in developed countries 1 . this rate has established without much decline in recent years. the mmr for ohio for 1987 to 1996 was reported by cdc to be 6.3 per 100,000 live births with 95% confidence interval 5.1 to 7.6. objective: our objective was to audit the trend in maternal mortality in the state of ohio for the last 20 years. methods: information from the death certificates completed by attending physicians, medical examiners, coroners, funeral directors filed with ohio state registration offices were analyzed. we only used women who were residents of the state of ohio at the time of death, and cause of death was coded as a "complication of pregnancy, childbirth and the puerperium", icd9 codes 630 to 677 and icd10 codes o0 to o9. five year maternal mortality pattern and long term trend was assessed. results: the number of maternal deaths recorded as due to pregnancy complications varied over the years from a high of 20 in 1981 to a low of 4 in 1996 and 1997. in general the five-year mortality rate was decreasing over time until the last four years 2000 to 2003, when we may be seeing an upward trend compared to 1995 to 1999 (p=0.07). the rates and associated 95% confidence intervals (ci) are shown in the table below. years mmr 95% ci 1980 ci -1984 ci 7.9 4.6-11.1 1985 ci -1989 6.0 3. 2-8.8 1990-1994 5.8 3.0-8.6 1995-1999 4.5 1.6-7.4 2000-2003 8.4 3.3-13.5 the rate of maternal mortality is on the rise after a period of downward trend. we should develop a multidisciplinary approach to analyze and target the root causes of maternal death. introduction. women with thrombophilia are at higher risk of vte during pregnancy due to the acquired hypercoagulable state. it is likely that not only maternal veins but also placental vessels are more prone to the development of thrombosis. our objective was to compare maternal and fetal placental circulation disorders in women with intra-uterine fetal death (iufd) and thrombophilia and women without thrombophilia. methods. in a dutch multi-centre study on iufd, during the period 2002-2007 we studied 750 singleton deaths > 20 weeks of gestation for which the diagnosis of iufd was determined before labour. factor v leiden and prothrombin g20210a were tested at induction of labour. panel classification of cause according to the tulip classification 1 was performed by assessors after individual investigation of structured patient information. we studied the cause of death group "maternal and fetal placental circulation disorders": placenta bed pathology with abruption or infarction as origin of mechanism and placental parenchyma pathology with fetal thrombotic vasculopathy and massive perivillous fibrin deposition as origin of mechanism. results. of the 689 women tested for factor v leiden, 46 (6.7%) were carriers. of the 261 deaths caused by "maternal and fetal placental circulation disorders" 18 (6.9%) mothers were carriers of factor v leiden. of the 428 deaths with another cause of death 28 (6.5%) mothers were carriers of factor v leiden (p=0.88). of the 691 women tested for prothrombin g20210a, 21 (3.0%) were carriers. of the 264 deaths caused by "maternal and fetal placental circulation disorders" 9 (3.5%) mothers were carriers of prothrombin g20210a mutation. of the 427 deaths with another cause of death 12 (2.8%) mothers were carrier of prothrombin g20210a mutation (p=0.65). in women with iufd and factor v leiden or prothrombin g20210a mutation "maternal and fetal placental circulation disorders" did not seem to cause iufd more often than in non-carriers. , university medical center groningen, groningen, netherlands; 2 pathology, university medical center groningen, groningen, netherlands; 3 trial coordinating center, dept of epidemiology, university medical center groningen, groningen, netherlands; 4 hematology, university medical center groningen, groningen, netherlands. introduction. growing evidence suggests that women with thrombophilic defects may be at higher risk of fetal loss. although some paternal components to the predisposition of preeclampsia have been demonstrated it is not known whether paternal components contribute to intra-uterine fetal death (iufd). our objective was to investigate the relation between paternal thrombophilic defects and iufd. methods. in a dutch multi-centre study on iufd, from 2002-2007 we studied 750 singleton deaths > 20 weeks of gestation for which the diagnosis of iufd was determined before labour. we tested male partners of women with iufd for antithrombin (at), protein c, protein s type i and iii, factor v leiden, prothrombin g20210a (factor ii) and factor viii: ag. standard tests were performed in one laboratory. normal ranges were determined in healthy male blood donors. we compared prevalence of thrombophilic defects to reference values from the literature. . of the 642 men tested for factor v leiden, 26 (4.0%) were carrier versus 618 (96.0%) non-carriers. prevalence of factor v leiden in the normal population is 5% (p=0.27). 642 men were tested for prothrombin g20210a, 6 (0.9%) were carriers and 636 (99.1%) non-carriers. all were heterozygous. prevalence of prothrombin g20210a mutation in the normal population is 3% (p=0.002). decreased levels of antithrombin, protein c, protein s type i and iii and increased levels of factor viii: ag were observed significantly more often in male partners of women with iufd compared to the normal population. conclusion. in our iufd group the prevalence of male factor v leiden carriers was comparable to the normal population, prevalence of prothrombin g20210a mutation was lower. the difference in other factors imply an as yet unexplained association of male thrombophilia with fetal death in their partner. objective fetal macrosomia, the most important complication of gdm, increases the risk of shoulder dystocia, erb's palsy and intrauterine hypoxia. we compared frequencies of fetal macrosomia and erb's palsy in two gdm cohorts with different severity of glycemic disturbance and in healthy controls. methods we studied 898 consecutive gdm women with singleton childbirth and 2 or 3 abnormal values in 2-h ogtt with 75 g of glucose. abnormal plasma glucose values of the ogtt were: fasting 5.1, 1-h 10.0 and 2-h 8.7 mmol/l. insulin treatment was started when 2 values were 5.5 preprandially or 7.8 mmol/l postprandially in the 24-h glucose profile done within 7 days of diagnosis. if the same woman had more than 1 childbirth during the study period, only the last pregnancy was included. the control group consisted of 798 women from a nearby town with singleton childbirth in the same hospital. women with diabetes were excluded from controls. macrosomia was defined as birth weight (bw) >2.0 sd above the mean of a standard population. erb's palsy was diagnosed by pediatric surgeons. results gdm women were older, more obese, had more childbirths and more often a previous child with bw >4000g than controls. insulin was started in 367 gdm women (41.9%). c/s rate was 18.7% in controls, 27.0% in diettreated and 42.0% in insulin-treated gdm women. macrosomia rate was 2.3% in controls, 4.6% in diet-treated and 18.4% in insulin-treated gdm women (p<0.001 compared with controls or diet-treated gdm women). the frequency of macrosomia did not differ between the diet-treated gdm and control women. erb's palsy occurred in 0.3% of controls, in 1.7% of diet-treated and in 1.6% of insulin-treated gdm women. the frequency of erb's palsy was significantly higher in both gdm groups than in controls (p=0.013). by regression analysis, previous child with bw >4000g (p<0.0001), previous c/s (p=0.0006), mother's age (p=0.012), bmi (p=0.015), insulin treatment (p=0.02) and fasting plasma glucose of the 24-h profile (p=0.027) were significant independent predictors of fetal macrosomia. conclusions the 24-h glucose profile done shortly after the diagnosis of gdm clearly distinguishes between low-risk (diet-treated) and high-risk (insulintreated) gdm pregnancies for fetal macrosomia. unfavourable fetal body composition of diet-treated gdm women is the likely explanation for the high rate of erb's palsy in this group. 0.264 1'-:·:tpartq,-, 1'-:·:tpartq,-, 1'-:·:tpartq,-, 25% p=0.001). first trimester uta doppler indices were similar in the two cohorts in terms of the resistance and pulsatility indices (ri 0.63 vs. 0.67, p=0.39; pi 1.29 vs. 1.44, p=0.42) . however, bilateral notching was much more common in the cohort of prior adverse outcomes (22% vs. 45% p=0.05) as well as in patients destined to have a subsequent poor outcome (p=0.001). conclusions: not surprisingly, prior poor obstetric outcome was strongly associated with recurrent adverse obstetric outcome. uta notching was robustly associated with prior poor obstetric history as well as a recurrent poor outcome. this clinical history had no discernable influence on ri or pi. reassurance may not be offered based on first trimester uta ri and pi. anti-retroviral therapy is associated with increased blood loss and uterine atony for patients undergoing primary cesarean section. carey eppes, alice cootauco, melissa russo, jessica bienstock. gynecology and obstetrics, johns hopkins university school of medicine, baltimore, md, usa. objective: anti-retroviral therapy has been associated with gastrointestinal smooth muscle dysfunction. it has also been hypothesized that myopathies can occur secondary to anti-retroviral therapy due to mitchondrial alterations. in our experience, we have noticed an increased incidence of uterine atony in our hiv patients on anti-retroviral therapy. we sought to examine the incidence of postpartum hemorrhage and uterine atony in patients currently on anti-retroviral therapy. study design: a retrospective case-controlled study was conducted on all hiv positive pregnant women on anti-retroviral therapy undergoing a primary low segment transverse cesarean section from 1996 through 2007. these patients were obtained from an irb approved database containing hiv positive patients within our institution. patients' medical records were abstracted for demographic data, use of uterotonics, preoperative and postoperative hematocrits, and incidence of blood transfusions. controls were matched for age, parity, gestational age and surgical indication. data was analyzed using the t-test, fischer's exact test and chi square. results: there were no differences in demographics, incidence of chorioamnionitis or magnesium sulfate use between groups. patients on anti-retroviral therapy had a statistically greater decrease in hematocrit and estimated blood loss compared to controls. they also had an increased need for uterotonics and blood transfusions. conclusion: anti-retroviral therapy may impact uterine smooth muscle contractility in pregnancy, as evident by the increased incidence of uterine atony and change in hematocrit. additional research is needed to elucidate the mechanism. clinicians should be aware of the potential for uterine atony and excessive blood loss in patients on anti-retroviral therapy. background: adolescent pregnancy is frequently associated with adverse outcomes, especially small-for-gestational age (sga) deliveries. some studies have implicated maternal nutritional status in these poor outcomes. methods: in a prospective longitudinal study, 500 ethnically-diverse, pregnant adolescents were studied from booking to parturition, with collection of anthropometric and nutritional variables. blood samples (28-32 weeks' gestation; n=305 subjects) were assayed for a spectrum of nutritional biomarkers. logistic regression was used to determine significant associations between studied variables and pregnancy outcomes. results: median age at recruitment was 17.8 years (iqr:17.1-18.4). outcome data was available for 478 subjects. 214 (44.8%) had uncomplicated pregnancies. median birthweight was 3,200g (iqr: 2,831-3,530g) and median birthweight centile was 37.8% (iqr: 14.3-64.2%). 84 (17.6%) infants were born sga and 43 (9.0%) were preterm. spontaneous vaginal deliveries occurred in 71.0% of cases. there were 14 (2.9%) cases of pre-eclampsia and 34 (7.1%) admissions to neonatal care. 31.9% of subjects reported smoking at booking. iron deficiency anaemia was prevalent in 49.4% of subjects by 28-32 weeks and was strongly associated with higher infant birthweight centile (p<0.0001). 48.4% had serum 25-hydroxy vitamin d concentrations <15ng/ml, although this was not associated with any outcomes. low folate status, as indicated by low red cell folate (p=0.001), low serum folate (p=0.032) and high serum homocysteine (p=0.027) concentrations, was associated with higher rates of sga birth. conclusion: adolescent pregnancy in inner-city populations is associated with a high risk of sga and preterm birth, increasing the likelihood of health problems in later life and perpetuating social disparities in health. the association between anaemia and higher birthweight is unexplained. impaired maternal folate status may contribute to impaired fetal growth. we suggest that sga birth in this population could be reduced by the use of antenatal supplements or the mandatory fortification of flour with folic acid. amount of cigarette use pre-pregnancy and during pregnancy were self-reported and subgrouped into nonsmokers, quitting during pregnancy, and continued smoking throughout pregnancy. categorical outcomes were compared using chi-square test. multivariable logistic regression analyses were used to control for potential confounders (continued smoking compared to quitting during pregnancy). results: women who quit smoking during pregnancy had higher rates of pregnancy-associated hypertension and cesarean delivery but lower rates of preterm delivery and neonatal birthweight <2500gm compared to non-smokers. compared to women who quit smoking during pregnancy, those who continue to smoke have lower odds of pregnancy-associated hypertension and cesarean delivery, but higher odds of preterm delivery, neonatal birthweight <2500gm, and apgar score <7 at 5 minutes (see table) conclusion: quitting cigarette smoking during pregnancy appears to reduce undesirable neonatal outcomes, though an increase in pregnancy associated hypertension. these findings should be emphasized to women who are smoking during pregnancy. to achieve effective tamponade the balloon was inflated initially up to 150-200ml with incremental increases of volume by 50 ml until bleeding stops. to measure intrauterine balloon pressures we used a standard pressure transducer (edwards lifesciences) connected to the balloon port and to a pressure monitor (ge dash 3000). the pressure transducer was mounted on the bedrail at uterine level, primed with sterile saline and then connected to the balloon port of the catheter. the system was zeroed to atmospheric pressure, the stopcock of the intrauterine balloon port was opened and the pressure was recorded (p1: the total pressure on the fluid). to verify the accuracy of measurements we used another balloon catheter (reference), inflated with the same amount of saline, connected to the pressure transducer and zeroed to atmospheric pressure. the reference balloon measured p2 (the intrinsic balloon pressure caused by distension at that volume). the system was then zeroed to the reference balloon. the pressure transducer was then reconnected with the intrauterine balloon and the actual intrauterine pressure was recorded (p3). we calculated the correlation coefficient between p3 and systolic, diastolic and mean blood pressure using a linear regression (statsdirect statistical software there is a growing body of evidence to suggest that peripartum assessment of fetal or neonatal lactate levels are as good as or better than standard blood gas analysis in the prediction of neonatal outcome. in this study we have evaluated the ability of umbilical cord blood gases and lactate levels in the prediction of neonatal hypoxic-ischaemic encephalopathy (hie). department of neonatal paediatrics, king edward memorial hospital, perth, western australia, australia; 3 women and infants research foundation, king edward memorial hospital, perth, western australia, australia. objective: current evidence suggests that umbilical cord ph at delivery provides the most sensitive reflection of birth asphyxia. paired umbilical artery/vein blood gases have been routinely collected at king edward memorial hospital over the last 5 years. the objective of this study was to determine: local reference ranges; accuracy of sampling; and the rates of metabolic acidosis. of the 19,426 births (2003) (2004) (2005) (2006) , accurate paired results were available on 64% of births. over the study period there was a progressive improvements in accuracy rates of paired sampling (p<0.05). the median (2.5 th , 97.5 th centile) values for cord arterial blood gases were: ph 7.27 (7.08,7.37); po2 16.3mmhg (4.6,31.0); pco2 55.1mmhg (39.0,79.0); base excess -3.0 (-10.8, 2.7); and lactate 3.7mmol/l (1.8,7.8). there was a progressive improvement in all blood gas measures over the 4 years of this study (all p<0.05). moreover, there were significant reductions in all measures of metabolic acidosis (see table) . the progressive improvement in the measures of metabolic acidosis remained significant after multivariate analysis including obstetric, fetal, and demographic factors associated with metabolic acidosis. the introduction of universal umbilical cord blood gas analysis to all births is associated with significant improvements in all markers of metabolic acidosis. together with other compelling evidence in the literature, these data support the routine use of cord arterial and venous gases at all births; however, improved accuracy rates on paired sampling requires an ongoing education program umbilical artery indicators of acidosis, percentage of total ph < 7.0 ph < 5th centile* objective: intrapartum pcn prophylaxis aims to prevent early-onset gbs sepsis by interrupting vertical transmission from colonized mothers to their newborns. however, despite its wide clinical use, systematic pharmacokinetic evidence in support of the current pcn dosage regimen is lacking. current cdc guidelines recommend intensified surveillance and testing of infants exposed to <4h of prophylaxis. our goal was to examine the relationship between maternal time of exposure to pcn and fetal serum pcn levels among maternal-fetal dyads exposed to short durations of pcn prophylaxis (<4h) compared to those exposed to longer durations. study design: ninety-eight laboring gbs positive women were administered 5 million units (mu) of intravenous pcn to be followed by 2.5 mu every 4 hours until delivery (cdc 2002) . subjects with renal disease, multiple gestation, and preterm delivery (<37wks) were excluded. umbilical cord blood samples were collected at delivery and pcn levels measured by high-performance liquid chromatography. intra and inter-assay coefficients of variation were <3%. results: the pcn concentrations (mean±sd) by duration of prophylaxis were: <1 h, 11.6±4.5 g/ml (n=10); 1-2h, 9.7±3.4 g/ml (n=15); 2-3h, 6.6±3.8 g/ ml (n=15);3-4h, 3.6±1.8 g/ml (n=17);4-8h, 6.9±3.7 g/ml (n=11);>8h 4.1±2.6 g/ml (n=24); and for those without a second dose after 4h, 2.3±0.9 g/ml (n=6). fetuses exposed to short duration (<4h) had higher levels of pcn than those exposed to >4 h (p=0.003). in multivariable linear regression analysis, fetal pcn levels were determined by total duration of exposure, time since last dose, dosage, and number of doses, but not maternal bmi. pcn levels in cord serum increased linearly until 1 hour; thereafter, they decreased rapidly, but all groups were significantly above the minimum inhibitory concentration (mic) for gbs (0.1 g/ml)(p<0.002). furthermore, every sample individually remained 10-179 fold above the mic. conclusion: in this study, even short durations of prophylaxis achieved levels above the mic, suggesting a benefit to prophylaxis even in precipitous labors. the data also suggests that the current cdc designation of infants exposed to <4h of pcn prophylaxis as particularly at risk for gbs sepsis may be inaccurate from a pharmacokinetic standpoint. objective: 3-oh aa is a metabolite of tryptophan with pro-oxidant and proapoptotic properties and has been shown to increase in umbilical cord blood in pregnancies with intra-uterine infection. since labour-related events may also activate inflammatory pathways, we sought to determine the placental release of 3-oh aa into the umbilical circulation in labouring vs non-labouring patients at term. methods: twenty-six patients were studied (term labour n=18, and term elective cesarean section n=8) with blood sampling from a clamped segment of umbilical cord after delivery of the fetus and from the cord at its insertion into the placenta after delivery of the placenta, with subsequent measurement of blood gases/ph and 3-oh aa (isocratic hplc using fluorometric detection with assay sensitivity at 1 pmol). results: 3-oh aa measurements from respective umbilical and placental cord vessels were all variably higher in the labouring group vs the elective cesarean group patients (table 1) . for labouring group patients, the 3-oh aa levels from the umbilical vein were significantly higher than those from the umbilical artery, indicating net release from the placenta into the fetal circulation. placental vein levels were also significantly higher than those from the umbilical vein, indicating continued placental release of 3-oh aa into the cord blood after delivery of the fetus. conclusion: labour at term is associated with changes in the placental metabolism of tryptophan resulting in the increased release of 3-oh aa into the fetal circulation with the potential for pro-oxidative and apoptotic effects in many tissues, including the brain. obstetrics, gynecology, and reproductive sciences, new brunswick, nj, usa; 2 department of obstetrics and gynecology, mineola, ny, usa. objective: ischemic placental disease (preeclampsia, small for gestational age, sga and placental abruption) is a major contributor to pregnancy-related morbidity. although the placenta is considered a fetal organ, it is accepted that ischemic placental disease (ipd) can present clinically with either fetal or maternal manifestations. we hypothesized that the pattern of diagnosis (maternal versus fetal) varies by gestational age and would provide insights in to origins of indicated and spontaneous preterm birth. methods: this was a retrospective cohort study utilizing the maternallylinked reproductive history data for missouri residents , restricted to singleton live births. women who experienced spontaneous onset of labor and subsequently delivered preterm were classified as spontaneous preterm birth. medically indicated preterm birth included women who delivered preterm through a labor induction or (prelabor) cesarean delivery. ipd was classified as maternal (preeclampsia only), fetal (sga only) or both (preeclampsia with sga or abruption, and all 3 conditions). results: among term births with ipd, 22.6% presented as maternal disease only, 69.5% as fetal disease, and the remainder (7.4%) as both. among spontaneous preterm births with ipd, a greater proportion were of fetal presentation ( bethesda, md, usa; 2 dept ob/gyn, wayne state univ, detroit, mi, usa; 3 dept pathology, wayne state univ, detroit, mi, usa; 4 dept ob/gyn, soroka univ medical center, israel. objective: hemoglobin (hg) and its catabolic products have been observed in cases of amniotic fluid (af) discoloration, which is a risk factor for intraamniotic infection/inflammation (iai). the study aimed to determine the association between af fetal hg concentration and gestational age, term and preterm labor and iai. study design: this cross-sectional study included: 1) mid-trimester (n=60); 2) term not in labor (tnl) (n=21); 3) term in labor (tin) (n=47); 4) preterm labor (ptl) who delivered at term (n=89); 5) ptl without iai (n=74); 6) ptl with iai (n=78); 7) preterm prelabor rupture of membranes (pprom) with (n=48) and without iai (n=48). af fetal hg concentrations were determined by elisa. results: 1) fetal hg was detected in 80.4% of all af and 31.7% of mid-trimester samples; 2) women at tnl had a higher median af fetal hg concentration than patients at mid-trimester (19.5 ng/ml, iqr 0-49 vs 0.0 ng/ml, iqr 0.0-19.9, p=0.008); 3) no differences were found in median af fetal hg concentration among patients with and without labor at term (til: 19.1 ng/ml, iqr 0-36.2; p=0.4); 4) median af fetal hg concentration was not significantly different among the 3 ptl subgroups [ptl with iai: 114.6 ng/ml, ptl who delivered preterm: 109.6 ng/ml, ptl without iai who delivered at term: 134.21 ng/ml, p=0.71(kruskal wallis) ]; 5) in pprom, there were no differences among patients with and without iai (148.4 ng/ml, respectively; p=0.074) ; 6) median af fetal hg concentrations were significantly higher in ptl or pprom, with or without iai than in pregnant women at term, with and without labor (p<0.001 for all comparisons). conclusions: 1) immunoreactive af fetal hg increases with gestational age; 2) among women with ptl or pprom, the median af fetal hg concentration is not associated with iai; 3) the median af fetal hg concentration is higher in pregnancies complicated with ptl or pprom than in term pregnancies. the impact of latency time to delivery after preterm premature rupture of membranes (pprom) on neonatal outcome. dan nayot, 1 deborah penava, 1 barbra de vrijer, 1 orlando da silva, 2 bryan s richardson. 1 1 obstetrics and gynaecology; 2 pediatrics, university of western ontario, london, on, canada. objective: there continues to be controversy as to the management of pprom with conservative management to advance gestational age (ga) versus aggressive management with early induction to avoid chorioamnionitis. we have therefore used the perinatal and neonatal databases of a large regional patient population to determine the association of pregnancy variables with latency time to delivery after pprom and the impact of latency duration on adverse neonatal outcomes. methods: the perinatal/neonatal database of st. joseph's health care, london, ontario was used to obtain demographic and neonatal outcome information for all patients with pprom >25 and <37 weeks gestation, singleton and no major anomalies, delivering between january 1, 1996 and december 31, 2005. patients were grouped according to ga at pprom stratified for latency time <72 hrs vs >72 hrs with incidences for those pregnancy related variables and neonatal outcomes available from the database then compared with the use of logistic regression analysis. results: there were 1535 patients who met the inclusion criteria of whom 10%, 19%, and 71% had pprom at 25-28 wks , at 29-32 wks, or at 33-36 wks, respectively, and with these pprom groupings showing a stepwise decrease in the percentage of patients with latency to delivery >72 hrs, at 67%, 42%, and 10%, respectively. pregnancy related variables and neonatal outcomes for these patient groupings are as shown in table 1 . conclusion: despite a 2 to 3 fold increase in the incidence of chorioamnionitis with latency to delivery >72 hrs, a policy of conservative management to advance ga after pprom will result in decreased severe infant morbidity until 32 weeks, and moderate infant morbidity until 36 weeks. hospital. outcomes studied were prolongation of latency period with intact membranes and prom using magnesium sulfate (mgso 4 ), nifedipine, terbutaline and 2 tocolytic agents. secondary outcomes examined were maternal complications. statistical analysis performed were t-test and 2 . comparisons were expressed as odds ratios (95% ci). results: in a 9-year period, 183 twin gestations were admitted in ptl and administered 1 tocolytic agent(s) with mean gestational age of 28.8 weeks. when 2 tocolytic agents were used, maternal complications were: 7 (9.5%) with intact membranes and 1 (2.8%) with prom had pulmonary edema (or=2.67, ci 0.58-12.29); 11 (14.1%) with intact membranes and 2 (5.6%) with prom had chorioamnionitis (or=1.3, ci 0.41-4.15); 2 (2.6%) with intact membranes and 2 (5.6%) with prom had postpartum hemorrhage (or=0.43, ci 0.05-3.62). conclusion: there is no difference in prolongation of latency period achieved in twin gestations in ptl with intact membranes or prom using 2 tocolytic agents. similarly, there is no difference between the 2 groups with latency period 48 hrs. there is an increased likelihood of pulmonary edema and chorioamnionitis with use of 2 tocolytic agents. however, results are not significant due to type ii error. mean latency period and latency ≥48 hrs using single or multiple tocolytic agent ( introduction: high sensitivity crp (hscrp) is a serum marker of inflammation and has proven clinical utlility in predicting cardiovascular disease (cvd). considering the hypothesized association between preeclampsia (pre) and inflammation and cvd, it is plausible that hscrp may have utility in predicting pre. prior to widespread utilization of this marker, the affect of labor on levels of crp needs to be clarified. we assessed the association between labor and hscrp levels in term deliveries and between elevated hscrp and adverse perinatal outcomes. a secondary analysis comparing hscrp in women with preeclampsia (pre) to those without was performed. methods: women presenting for term delivery or pre were prospectively identified as part of a case-control study. clinical data and serum were collected for all subjects. a standard immunoturbidimetric assay was used to measure hscrp levels. women presenting for induction of labor or planned cesarean delivery (non-labor) were compared to women presenting in labor. a secondary analysis comparing non labor women with and without pre was performed. serum was collected prior to labor induction in the non-labor group. nonparametric comparisons were made using wilcoxon rank sum tests. mvlr was used to evaluate dichotomous outcomes and control for confounders. results: 344 women were included (non-labor group (n=146), labor group (n=120), non-labor pre (n=78)). the median and mean hscrp levels were 6 and 9 and 15 and 21 in the non-labor and labor groups respectively (p=0.005). elevated levels of hscrp in these term deliveries were not associated with chorioamnionitis (p=0.3), maternal postpartum complications (endometritis, hemorrhage, transfusion) (p=0.67), mode of delivery (p=0.3), or admission to the nicu (0.96). levels of hscrp were significantly greater in the non-labor pre group compared to non labor without pre (mean 34.5 vs.20.1, median 27 vs.6, p<0.001). conclusion: use of hscrp as a biomarker may improve clinical prediction of obstetrical complications such as pre. levels of hscrp are affected by labor and this should be taken into account when studying the utility of this biomarker. further, crp levels are elevated in women with pre even after excluding patients in labor. further investigations to determine if crp elevation in term labor is associated with adverse outcomes may be warranted. the role of hscrp as a valid and discriminating biomarker in pre should be assessed. review of the electronic labor record facilitated collection of maternal demographic, intrapartum, and newborn data. data analyzed with t-tests, chi-square tests, and calculated odds ratios with 95% cis as appropriate. a forward stepwise logistic regression analysis was used to identify predictors of histologic chorioamnionitis. results: of 351 submitted placentas, 210 had histologic chorioamnionitis (cases) and 141 did not (controls). the groups were similar with respect to age, race, gbs status, and mode of delivery. gestational age, birthweight, duration of labor and ruptured membranes, and number of vaginal exams were greater in the cases (p .01). the cases were more likely to have had epidural anesthesia (or 2.9), internal monitoring (or 2.2), fever (or 4.8), maternal tachycardia (or 1.9) and fetal tachycardia (or 3.8) and less likely to have had induction of labor (or 0.49). newborns in the histologic chorioamnionitis group were more likely to have been observed for sepsis (or 3.8 severe sepsis defined as sepsis associated with acute respiratory distress syndrome (ards) or cardiovascular dysfunction(cvd) or with 2 or more other organ dysfunction.all patients were resuscitated with fluids and treated with broad spectrum antibiotics and supportive care as needed. outcome data were: etiology, management, maternal complications, duration of icu stay and perinatal survival. results patients were young (mean age=23.6± 7.6 years) with a mean gestational age at delivery 29.5±8.9 weeks. etiologies were pyelonephritis(n=8), septic abortion(n=4), endomyometritis (n=3), chorioamnionitis (n=3), ruptured appendix(n=3), pneumonia( n=1) and one unknown. eighteen (78%) were diagnosed during antepartum and 5 (22%) postpartum period.there were 2(9%) maternal deaths and high rate of major morbidities (table) . among the 18 antepartum patients,there were,4 abortions,4 iufd,2 neonatal death for a perinatal survival rate of only 39%. conclusion pregnancies complicated with severe sepsis/septic shock are associated with substantial maternal and perinatal morbidities. the low maternal mortality rate in our study as compared to previous reports is attributed to early diagnosis and aggressive management of maternal complications. fetal loss rate, however continues to be high when septic shock develops antepartum. and tnfa levels are elevated. we developed a dynamic computer (in silico) model of pregnancy (uterine myometrial environment -infection/inflammation and endocrine crosstalk). mathematical differential equations were used to describe the interactions between molecules. infection was represented as increased levels of activated, nuclear transcription factor nf-kb. in the model ru486 inhibited both the glucocorticoid receptor and progesterone receptors. simulations were run adding different concentrations of ru486 (1.5 um= dose used in patients, 0.5 um, 0.05um) at different time points during infection (before, at the time of or after nf-kb activation). ru486 degradation kinetics was also included. the effect of ru486 on nf-kb induced il-6 and tnfa levels was assessed. results: infection induced nf-kb activation led to increased il-6 and tnfa levels. there was a subsequent increase in cortisol that led to dampening of nf-kb activation, il-6 and tnfa levels. in the presence of ru486, il-6 and tnfa levels continued to rise. the effect of ru486 on nf-kb induced il-6 and tnfa was dose dependent and was more prominent in slow metabolizers who had ru486 in the system for a longer time. addition of ru486 after the onset of nf-kb activation led to increased il-6 and tnfa levels above those observed without ru486. the addition of misoprostol (prostaglandin e1 analogue), at the concentrations used together with ru486 for medical abortion, did not add to the effect of ru486 on nf-kb induced il-6 or tnf. conclusions: ru486 has dose dependent effects on infection induced immune activation and may contribute to the pathogenesis of c sordelii induced sepsis syndrome. the increased susceptibility of neonates to infection remains a major clinical problem. we previously found that after intraperitoneal (ip) listeria monocytogenes infection, neonatal mice have an ld 50 that is 5 orders of magnitude lower than adults. we also found that the inflammatory response of neonatal mouse macrophages, but not neutrophils, in the peritoneal fluid was deficient, and that this correlated with low levels of macrophage chemokines mcp-1 and rantes. given that the liver and spleen are important organs in listeria infection, we sought to characterize the innate immune response in these organs. a sublethal dose of listeria was injected intraperitoneally into balb/c 4-6 week old adult mice and 3-5 day old neonatal mice. liver and spleen was collected at 0, 24, 48 and 72 hours, sectioned serially for staining with hematoxylin-eosin and primary antibodies (rabbit anti-listeria monocytogenes igg; mhc class ii rat anti-mouse igg, a marker for activated macrophages; f4/80 rat antimouse igg, a marker for macrophages) and secondary antibodies (cy-3 goat anti-rabbit igg; af488 goat anti-rat igg) were applied. negative controls used only secondary antibody. real time pcr was used to compare the levels of the chemokines mcp-1 and rantes in adult and neonatal liver. after ip infection, both adults and neonates showed similar influx of neutrophils to the sites of infection within the liver and spleen. at 24 hours post-infection with listeria, adult liver and spleen showed increased staining for f4/80 and class ii, which increased further and became confluent surrounding microabscesses at 48 and 72 hours. in contrast, the listeria infected neonatal mouse showed some increase in f4/80 around microabscesses but no apparent increase in staining for mhc class ii. the neonates showed greater staining for listeria at each time point. mcp-1 and rantes levels were higher in infected neonatal liver compared to adults. in the neonatal mouse, the innate immune response in the liver and spleen was characterized by a deficiency of activated macrophages. this deficiency was not correlated with hepatic expression of 2 macrophage chemokines. is there a seasonal pattern in the incidence of post-cesarean endometritis? tamula m patterson, alan tn tita, william w andrews. obstetrics and gynecology, the university of alabama at birmingham, birmingham, al, usa. objective: several theories, including one suggesting a peak in july coincident with resident turnover, postulate seasonality in post-cesarean infections. we assessed whether there is seasonal variation in endometritis. a retrospective cohort study of post-cesarean endometritis at our university-based institution using our obstetric computerized database to compare annual variation in monthly incidence patterns from 1992 to 2006. prior to establishing an average aggregate seasonal pattern for all years, years were assessed separately for a recurrent pattern of peaks and nadirs in incidence. peak incidence (or nadir) was defined as any monthly incidence that differed from the mean incidence for the year by over 25%. results: a total of 22.4% (10,966) of 48,913 deliveries from 1992 to 2006 were by cesarean. annual cesarean rates increased by an absolute rate of over 10%; while, post-cesarean endometritis rates decreased from 23% to 2%. monthly incidence of post-cesarean endometritis did not reveal a consistent recurrent pattern of peaks (figure1) or nadirs. the month of july accounted for only 4 out of a total of 40 peaks for all years. the adjacent months of june and august accounted for only 3 each. the month with the highest number of peaks was april with only 7. these findings contraindicated the establishment of an average aggregate monthly pattern for all years. the incidence of post-cesarean endometritis did not follow a seasonal pattern. chi-square analyses were used to compare associations between race (black (bl) vs non-black (nbl)) and dichotomous characteristics. student´s t-test was used to compare continuous variables. results 1,030 patients were evaluated (439 cases and 591 controls). 85% and 15% of cases and 74% and 26% of the controls were bl and nbl respectively. the baseline prevalence of chtn in bl and nbl controls was 6.15% and 2.65% (p=0.09). when comparing bl and nbl cases, bl women had a higher mean systolic blood pressure and screening bmi. bl women also had a trend toward being discharged on post partum blood pressure medicine when compared to nbl women. there was no difference in chtn, diabetes, severity of disease, iugr, or delivery <34 wks between the two groups (table) . to determine the leading causes of death in a case series of stillborn infants examined in a large hospital autopsy service, and to describe the most common post-mortem observations. study design: one hundred and sixty one stillborn infants were examined. gross pathology observations were recorded at autopsy and during the placental exam, and tissue sections were collected and examined microscopically. immediate and underlying cause of death (cod) were recorded, along with contributory cod, concomitant/significant cod, and incidental findings. statistical analysis was conducted using the software spss v.11.5. results: the immediate anatomic cod could be determined in 33.5% of all infants examined. in over 50% of these cases, cod was attributable to placental or umbilical cord findings affecting the maternal-fetal blood supply. the most prevalent among these findings were placental lesions (maternal floor infarction, placental abruption, fetal thrombotic vasculopathy), umbilical cord lesions (entanglement, true knot, compression, excessive length/twisting), and infectious/inflammatory processes (chorioamnionitis, chronic villitis objective: advanced maternal age (ama) is associated with increased risk of intrauterine fetal demise (iufd). antenatal testing (at) is widely used in clinical practice to prevent iufd due to uteroplacental insufficiency and has been suggested to reduce the risk of iufd in ama women. we sought to assess the impact of at on obstetrical interventions and compliance with new practice recommendations. methods: retrospective cohort of ama women (40 and older at their due date) who delivered at or after 32 weeks. non-exposed women delivered from july 2003 to december 2004 (when at for ama was not routinely recommended); exposed women delivered from july 2005 to december 2006 (after at for ama was introduced at our institution). subjects were identified through the perinatal database; records were abstracted for demographics, medical history, and labor/delivery variables. outcomes included rates of at and induction of labor (iol) and mode of delivery. associations between at for ama and outcomes were tested using t test and chi square. assuming a baseline rate of iol of 20%, we had 80% power to detect an increase to 35% after the introduction of at. results: 276 women met the inclusion criteria: 147 delivered before the introduction of at (non-exposed=before at) and 129 delivered after the introduction of at (exposed=after at). baseline clinical characteristics were similar in both groups. as anticipated, at was more common in the after at group than in the before at group (92% vs 27%; p<0.0001). 86 women were not eligible for or declined trial of labor, thus not "at risk" for iol and not included in all analyses. ob intervention rates were increased after at compared to before at (table 1) . the corrected iufd rates were similar in both groups (8/1000 vs 7/1000). conclusion: at an academic center, compliance with new practice recommendations was excellent. introducing at testing for a new indication seemed to increase iol and cs rates. these findings should be considered when assessing the risks:benefits ratio of antenatal testing. . we stratified indications for cesarean into the following: failed induction (cervix <4 cm dilated), arrest of dilation, arrest of descent, failed operative delivery, fetal intolerance of labor (fil), and "other" reasons (e.g., pre-eclampsia, abruption, chorioamnionitis, malpresentation). both fil and "other" reasons were more likely to occur among the medically indicated group (rr 1.7, . physicians in solo practice had higher rates of elective inductions (p<0.0001), but there was no association between cesarean and practice type. conclusions: inductions accounted for 13.1% of cesareans. these results suggest an increased risk for cs for patients undergoing medically indicated inductions at our institution. there was no association between cesarean and type of practice, whether solo or group, suggesting institutional clinical policies may be more important than practice type in determining delivery outcome after induction. further research is needed to understand how age, race/ethnicity, or other unmeasured patient factors may impact these findings. given rising rates of both cesarean delivery and inductions, this information may be pertinent to women considering elective induction prior to 41 weeks. objective: fetal demise can lead to a consumptive coagulophathy ("fetal death syndrome") traditionally attributed to the release of "tissue thromboplastin", now known as "tissue factor" (tf). tf is the most potent activator of coagulation. despite the appeal and acceptance of this proposed pathophysiology, there is no evidence supporting this view. this study was undertaken to determine if fetal death prior to development of fetal death syndrome is associated with changes in maternal plasma concentration of cd40l (a marker of platelet activation), tf and its soluble inhibitor (tfpi). methods: a cross-sectional study included the following groups: 1) women with normal pregnancy (n=71) and 2) patients with fetal demise without disseminated intravascular coagulation (n=50). plasma concentrations of scd40l, tf and tfpi were measured by elisa. standard coagulation tests were performed. non-parametric statistics were used for analysis. results: 1) patients with fetal demise had a higher median maternal plasma scd40l concentration than women with normal pregnancy (median 1213.3 pg/ml, range 118-3818 vs. median 369.5 pg/ml, range 63.5-1848.7, p<0.001); 2) there was no significant difference between the groups in the median maternal plasma tf concentration and 3) in contrast, the median maternal plasma tfpi concentration was significantly lower in patients with fetal demise than in women with normal pregnancy (median 45.6 ng/ml, range 13.6-115.2 vs. median 66.7 ng/ml, range 37.4-86.5, p<0.001). conclusions: 1) a change in the plasma concentration of tf was not demonstrated; 2) a change in the ratio of tf/tf inhibitor pathway may predispose to thrombin generation and activation of the coagulation cascade; 3) however, maternal platelet activation is present in patients with a fetal demise without fetal death syndrome and 4) the role of tf in fetal death syndrome remains to be proven. lipoic acid inhibits matrix metalloproteinase 9 production, activity and prostaglandin e 2 secretion by cultured amnion epithelial and mesenchymal cells. r moore, j novak, d kumar, j moore. case western reserve university, cleveland, oh, usa. introduction: cytokines, free radicals, matrix metalloproteinases (mmp) and prostaglandins (pg) have been implicated in processes of fetal membrane rupture and labor. dietary anti-oxidant supplementation has been suggested as a possible therapy for high risk patients, however, clincal evidence supporting the efficacy of agents such as vitamin c or n-acetylcysteine remains controversial. in fact, we have previously shown that vitamin c increases matrix metalloproteinase (mmp) 9 activity in isolated fetal membrane fragments and fails to inhibit tumor necrosis factor (tnf) induced fetal membrane weakening in vitro. in this study, we examine the effect of the naturally occurring anti-oxidant, -lipoic acid, on tnf induced mmp9 activity/protein and pge 2 secretion in isolated amnion epithelial and mesenchymal cells. methods: amnion epithelial and mesenchymal cells were pre-treated with increasing doses of -lipoic acid (0-1mm/6h), then with increasing doses of tnf (0-50ng/ml/24h). medium and cells were analyzed by gelatin zymography/western blotting for mmp9/mmp2 activities/protein. pge 2 output was determined by immunoassay. results: tnf induced a dose dependent increase in mmp9 production, secretion and activity in amnion epithelial cells. tnf (50ng/ml) induced an 18 fold increase in cellular active mmp9 production and 3 fold increase in secreted mmp9 enzyme activity by amnion epithelial cells. these increases were reduced 62-100% following 6h pre-treatment with 0.5-1.0mm -lipoic acid. mmp2 protein/activity and pge 2 secretion by amnion epithelial cells were barely detectable and unaffected by tnf and/or -lipoic acid treatment. in striking contrast, mesenchymal cells exhibited little basal or tnf induced mmp9 protein/activity. mmp2 protein/activity in mesenchymal cells were unaffected by either tnf and/or -lipoic acid. however, tnf treated mesemchymal cells exhibited a dose dependent increase in pge 2 production (13 fold increase/50ng/ml tnf/24h) that was inhibited by 70%-100% following 0. objective: nearly fifty years after the discovery of microphthalmia-associated transcription factor (mitf), its gene was identified as a specialized transcription factor that dictates cell-specific differentiation. unique mitf isoforms are generated from alternative promoter usage. an isoform of mitf (mitf-cx) is down-regulated in cervical stromal cells of the ripened cervix. further, mitf-cx inhibits il-8 gene expression and thereby suppresses signaling of the final pathway in cervical ripening. since mitf binds to canonical eboxes (canntg) in promoter regions of target genes, we sought to determine if mitf regulated its own promoter through ebox motifs. methods: the 2 kb genomic dna sequence upstream of the mitf-cx transcription start site was cloned into pgl3 luciferase reporter vectors which were co-transfected with wild type or mutmitf-cx (impaired dna binding) into cervical stromal cells or hek293 cells. at 48 h, promoter activity was determined and normalized for transfection efficiency. results: gel-shift assays conducted with oligonucleotides corresponding to 11 eboxes in the mitf-cx promoter revealed two strong binding sites (eboxes 3 -983 to -977 and 9 -329 to -324 ). specific binding was established using oligonucleotides with or without mutated ebox, antibody supershift experiments, competition with cold probe, and absence of binding to mutmitf-cx. binding specificities were confirmed in nuclear extracts from cells that overexpressed mitf-cx, but not control or mutmitf-cx. reporter gene studies indicated that mitf-cx, but not mitf-m, increased mitf-cx promoter activity 4-to 5-fold. whereas mutations in ebox 3 or 9 resulted in significant decreases in mitf-stimulated promoter activity, mitfinduced increases in promoter activity were abolished by mutations in both eboxes. conclusions: collectively these experiments indicate that mitf-cx is a vital regulator of its own promoter activity and acts in a positive feedforward loop through two specific binding sites in its promoter. moreover, isoform-specific amino acids are important to mediate mitf-induced mitf-cx promoter activity. decreasing mitf protein or mutating its promoter would interrupt this loop resulting in rapid reduction of mitf synthesis. since mitf-cx suppresses il-8 gene expression in cervical stromal cells, we suggest that preservation of mitf-cx-induced mitf gene expression is an important mechanism to maintain the "brake" on cervical ripening and ensure cervical competency during pregnancy. the role of cd44 in cervical remodeling. denisse sanchez, brenda timmons, mala mahendroo. obstetrics and gynecology, ut southwestern medical center, dallas, tx, usa. objective: prior to the onset of parturition, the uterine cervix undergoes a remodeling process from a closed, rigid structure, to one that is soft and dilatable. many changes occur, including increases in hyaluronan (ha), a glycosaminoglycan that facilitates loosening of the collagen matrix. in the postpartum period, the concentration of ha is reduced to that of the nonpregnant state. cd44, a transmembrane glycoprotein expressed in hematopoietic and epithelial cells, is a receptor for ha. cd44 expression by immune cells is important in extravasation of leukocytes into tissue. cd44 may also be required for ha catabolism through the action of hyaluronidases 1 and 2. in the cervix, cd44 is expressed in the endo-cervical epithelia as well as in immune cells localized in the stromal matrix. to study the importance of cd44 in cervical remodeling, mice with a null mutation for cd44 (cd44 -/-) were evaluated during pregnancy, parturition and postpartum. methods: changes in ha amount and size distribution were assessed using ha molecular weight gels and fluorophore assisted carbohydrate electrophoresis. to identify defects in cervical remodeling in the cd44 -/mice, differences in expression for genes regulated in the cervix were studied by quantitative real time pcr . to determine whether cd44 plays a role in the recruitment of immune cells during cervical ripening and postpartum repair, immunohistochemistry with antibodies against leukocytes was done. in the postpartum period, there is a higher ratio of high molecular weight ha relative to low molecular weight ha in the cd44 -/mice. this suggests that postpartum breakdown of ha in cd44 -/cervices is delayed. as compared to wt cervix, there was a significant increase in hyaluronidase 2 (hyal-2) mrna in the postpartum period. the distribution and relative numbers of immune cells in the cd44 -/cervix was similar to wt. conclusion: these studies provide evidence that cd44 may play a role in remodeling of the postpartum cervix back to the nonpregnant state. our current data suggests that the catabolism of ha after birth is delayed in the mutant mice and upregulation of hyal2 may compensate to allow ha removal. furthermore, the activity of hyal-2 may be dependent on cd44. little difference in the recruitment of immune cells between cd44 and wt animals suggest that cd44 expression is not required for this process. these experiments provide a greater understanding for the role of cd44 and ha in cervical remodeling. in background: during in vitro experiments we have shown that separation of amnion from choriodecidua occurs as an integral part of the process of fetal membrane (fm) rupture. although spontaneous amnion and choriodecidual separation is seen in fm after both svd and elective c/s deliveries, its etiology is uncertain. biochemical degradation at the amnion-choriodecidua interface may be a key contributing factor. our previous biomechanical studies have demonstrated that separated fm require less physical work to rupture than intact membranes. the purpose of this study was to determine whether fm separation was associated with clinical differences in the birth process. hypothesis: during term, normal labor, spontaneous separation of fm is associated with differences in the clinical parameters of labor and delivery. study design: fm from consecutive term deliveries were cut off the placental disk. separated areas of fm were cut from the intact areas. both were weighed and their weight ratios determined. maternal medical, pregnancy, and delivery data were collected and analyzed. results: 221 term fm had the following characteristics: maternal age 26±6.4 yr, gravida 3.1±2.2, gestation 39±1.1 wks, elec. cs 10.0%, duration of rom 403±417 min, duration of contractions 642±440 min, african american 45%. 40% of the fm had <10 % separation; 11% had more than 95 % separation. srom fm with >10% separation (vs. <10%) had significantly shorter duration of rom (p=0.03) and admission to birth (p=0.05) times. srom fm with >95% separation (vs. <10%) had even shorter rom (p=0.006), duration of contractions (p=0.002) and admission to birth (p=0.0003). the >95% group (vs. <10%) was further along, gestationally (p=0.05). srom fm (vs. arom) had shorter admission to birth (p=0.008), but longer rom to birth (p<0.0001) times. absence of epidural (p=0.001), srom mode of rupture (p=0.04), svd (vs. elec. c/s) (p=0.06), and the presence of meconium (p=0.002), were all associated with increased fm separation. conclusion: spontaneous separation of fetal membranes is nearly universal and is associated with increased gestation, spontaneous rupture of membranes, shorter duration of contractions, and svd. speculation: we speculate that programmed biochemical changes initiate fm separation which then facilitates rom and childbirth. whole genome array and si-rna investigation of the function of nfkb in human amnion epithelial cells. sheri e lim, 1 shirin khanjani, 1 yun s lee, 1 tg teoh, 1,2 philip r bennett. 1 1 institute of reproductive and developmental biology, imperial college, london, united kingdom; 2 maternal fetal medicine, st. mary's hospital, london, united kingdom. introduction: labour is associated with activation of nfkappab in the amnion. nfkappab increases prostaglandin synthesis through the upregulation of cyclooxygenase-2 (cox-2), which is essential to the labour process. cox-2 mrna expression increases with gestation in the amnion. primary amnion epithelial cells cultivated from tissue collected prior to the onset of labour display a spectrum of nfkappab activation, similar to the spectrum of cox-2 expression presumably relating to the nearness of labour. our aim was to investigate the full range of genes under nfkappab control in amnion epithelial cells by using whole genome arrays. methods: amnion from 20 women undergoing elective caesarean section was collected and primary cell cultures established. total rna and protein were extracted from each culture. nuclear localization of nfkappab is required for its activation. western analysis of nuclear p65 was therefore performed to identify the samples displaying the lowest and highest nfkappab activity. the corresponding rna samples displaying the three lowest and three highest nuclear p65 protein concentrations were used for whole genome analysis using affymetrix u133 arrays. results and conclusions: we identified 919 significantly regulated genes. the gene with the highest fold change was cox-2 (x44.4) followed by oxytocin receptor (x24.1), ch10orf (x19.6), integrina2 (x17.7), and connective tissue growth factor (x14.9). other significant genes included interleukin-8 (il-8) (x6.3). pathway analysis revealed the majority of other nfkappab associated genes were involved in cell signaling, turnover and proliferation. we used real-time pcr (rtq-pcr) to validate cox-2 and il-8 expression. to prove that cox-2 is directly regulated by nfkappab, primary amnion epithelial cells were then transiently transfected with nfkappab p65 sigenome smart pool and sicontrol non-targeting sirna pool. western blot analysis confirmed knockdown of nfkappab p65 associated with inhibition of cox-2 demonstrating that nfkappab is essential for cox-2 expression. objective: premature birth is a major public problem accounting for over 13,000 deaths and 30,000 surviving infants with life-long morbidity yearly. in order to develop a rational basis for treatment and prevention of premature fetal membrane (fm) failure, we first need to understand the sub-failure fm structural and mechanical behavior at near full term. methods: we utilized planar biaxial mechanical testing, which approximates the physiologic loading state, for mechanical evaluation of the fm, and a structural constitutive model approach was used to offer insight into the structure-strength of the fm by integrating information on tissue composition and structure. small angle light scattering (sals) was used to nondestructively quantify the collagen fiber architecture of both intact and separated fm layers. results: in the stress free state, the gross collagen fiber architecture of the fm and separated layers were not homogenously align but exhibited small regions of fiber alignment. the amnion layer displayed the greatest alignment. the model fit the equi-biaxial strain data well (r 2 = 0.99) and indicated that fm collagen fibers were rapidly recruited and straightened well below failure stress levels. collagen fibers were gradually recruited followed by a drastic increase in fiber recruitment. conclusion: this study provided the first data on the effective collagen fiber stiffness in the intact fm under physiologic biaxial loading, which was related to quantitative collagen fiber architectural measures. modeling results indicated that the collagen fibers became fully loaded and straighten well below physiological loading levels. failure did not occur during physiological loading, indicating that fibers do not begin to fail until all collagen fibers are fully straightened and bearing load. this result suggested modest structural reserve in the fm collagen architecture, and may be an important aspect of its failure properties. previously, we demonstrated that a physically "weak zone" exists overlying the cervix in the fm, evident of collagen remodeling and cellular apoptosis. we are currently extending the present study to include the "weak zone" tissues, allowing us to elucidate the micro-mechanical mechanisms that facilitate failure in this newly identified fm zone. supported by nih 48476. the extracellular matrix of the cervix undergoes extensive remodeling during parturition. hyaluronan (ha) is a major constituent of the extracellular matrix of the term pregnant cervix. the onset of labor is preceded by an increase in ha and after delivery, the concentration of cervical ha gradually decreases to that of the non-pregnant state. these dramatic changes suggest that ha plays an important role during parturition. hyaluronan synthase 2 (has2) is one of three known ha synthases and the most abundant isoform in the pregnant cervix. transcripts for has2 are regulated by two alternative promoters, one upstream of the first coding exon (proximal promoter) and another upstream on an untranslated exon 1 (distal promoter). the focus of the current study is to further our understanding of the transcriptional regulation of has2 during cervical ripening. methods: rna blotting was carried out using transcript specific probes corresponding to the distal and proximal promoter of the mouse has2 gene. the regulation of has2 was evaluated in a cervical epithelial cancer cell line (caski cells) which we have previously shown to express endogenous has2. regulation of has2 expression by epidermal growth factor was assessed in the caski cells by western blotting and quantitative real time pcr assessment of transcripts. results: has2 mrnas in the nonpregnant (np) and pregnant cervix are transcribed from the distal promoter upstream of exon 1. two transcripts of approximately 4.1kb and 2.8kb were detected that arise from use of 2 polyadenylation sequences. as compared to np, the expression of has2 is increased 4, 23, and 9 fold on gestation days 15, 18 and shortly postpartum respectively. has2 mrna expression is increased upon treatment of caski cells with epidermal growth factor (30ng/ml) and is suppressed in cells treated with ag1478 (20 m), an inhibitor of egf receptor phosphorylation. maximal stimulation was observed at 4 and 8 hours of treatment. conclusion: has2 is the major ha synthase expressed during cervical ripening and the majority of transcripts are driven by the distal promoter in the has2 gene. in vitro studies using caski cells suggest has2 is regulated in part by the egf signaling pathway resulting in a several fold increase in has2 expression. these results provide an understanding of has2 gene regulation at the time of cervical ripening which will ultimately enhance our understanding of the molecular mechanisms important to cervical ripening. chorion and decidua cells. chad a grotegut, bernard j canzoneri, liping feng, phil heine, amy p murtha. obstetrics and gynecology, duke university, durham, nc, usa. objective: preterm premature rupture of the fetal membranes accounts for approximately 30% of all preterm deliveries. cigarette smoking independently carries a fourfold increase risk for pprom. our laboratory has previously demonstrated that the chorion layer undergoes apoptosis in women with pprom. this study was conducted to determine if extract of cigarette smoke causes cell death in specific cells of the fetal membrane. fetal membranes were collected at the time of elective cesarean section from women without labor and at term. the chorion and decidua layers were separated and purified on a gradient spin column and then plated near confluence. cigarette smoke extract (cse) was collected in cell media and used to treat chorion and decidua cells in culture at concentrations ranging from 1% to 100% in 96-well plates. cell viability was determined at 24, 48 and 72 hours following treatment with a non-radioactive cell viability assay. data were analyzed using paired t test (analyse-it, leeds, uk). chorion and decidua cells underwent cell death when exposed to cse in a dose dependent fashion. increasing concentrations of cse resulted in increased cell death at 48 hours in both cell types (figure 1 ). at 48 hours, chorion exhibited greater percent cell death compared to decidua at concentrations of 20, 40 and 60% cse (p=0.003, 0.019, and 0.069, respectively). for any given concentration of cse, the degree of cell death increased with increasing length of exposure (36, 48 and 72 hours) for each cell type. chorion cells routinely exhibited greater percentage of cell death following treatment with cse at concentrations ranging from 20-60% compared to decidua cells. human chorion and decidua cells in primary cell culture exhibit a dose-response and time dependent cell death in the presence of cse. human chorion cells show greater sensitivity to cell death when compared to decidua cells. further studies are needed to determine the mechanisms through which these cell types undergo cell death and the implications for the differential sensitivity to cigarette smoke. yoon ha kim, 1 tae-bok song, 1 cheol hong kim, 1 jong woon kim, 1 moon kyoung cho, 1 sung yeul yang, 2 bong whan ahn. 2 1 obstetrics gynecology, chonnam national university medical school, gwangju, korea; 2 biochemistry, chonnam national university medical school, gwangju, korea. objective: to investigate the lipid peroxide levels and protein carbonyls levels in the amniotic fluid of pregnant women with preterm premature rupture of membranes (pprom). the lipid peroxide levels in the amniotic fluid of normal pregnancy (n=20) and pregnant women with pprom (n=20) were newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced measured by thiobarbituric acid reaction. the protein carbonyl contents in the amniotic fluid of normal pregnancy (n=20) and pregnant women with pprom (n=20) were determined by the 2,4-dinitrophenylhydrazine method. after amniotic fluid of them were mixed and incubated up to 5 hours with 0.2ml of 1mm moxalactam, cefodizime, amoxacillin, erythromycin, the lipid peroxide levels and protein carbonyl contents in them were measured. results: 1. the lipid peroxide levels in the amniotic fluid of pregnant women with pprom was significantly higher than that of normal pregnancy (9.74±0.48 vs. 7.20±0.38 nmol/mg protein, p<0.01). 2. the protein carbonyl levels in the amniotic fluid of pregnant women with pprom was significantly higher than that of normal pregnancy (13.0±0.33 vs. 11.27±0.17 nmol/mg protein p<0.01). 3. the lipid peroxide levels and protein carbonyls formation by moxalactam in the amniotic fluid of pregnant women with pprom was significantly higher than basal level (12.08 ± 0.81 vs. 9.74±0.48 nmol/mg protein, 20.08 ± 0.66 vs. 13.0±0.33 nmol/mg protein, p<0.01). 4. the lipid peroxide levels and protein carbonyls formation by cefodizime in the amniotic fluid of pregnant women with pprom was significantly lower than basal level (5.04 ± 0.33 vs. 9.74±0.48 nmol/mg protein, 9.76 ± 0.35 vs. 13.0±0.33 nmol/mg protein, p<0.01). 5. there were no significant differences in the levels of lipid peroxide and protein carbonyls by amoxacillin and erythromycin in the amniotic fluid of pregnant women with pprom between antibiotics-induced and basal levels. background: chorioamnionitis (cam) is a major antecedent of preterm delivery (ptd) associated with elevated amniotic fluid tnf and il1 . we hypothesized that these cytokines enhance the term decidual cell (dc) expression of the matrix metalloproteinases (mmp) 2 and 9, which can then promote ptd by degrading the extracellular matrix of the decidua, fetal membranes, and cervix. methods: immunostaining for mmp-2, mmp-9, and vimentin (a dc marker) was performed on cam-complicated (n=5) and gestational age-matched control decidua (n=5), and staining intensities were evaluated by hscore. confluent, leukocyte-free term dcs were primed with 10-8 m estradiol (e2) or e2 + 10-7 m medroxyprogesterone acetate (mpa), and then switched to a defined medium with e2 +/-mpa with or without 1 ng/ml of il1 or tnf . secreted mmp-2 and mmp-9 levels were measured by elisa (n=8), and quantitative rt-pcr assessed mmp-2 and mmp-9 mrna levels (n=4). results: tissue staining revealed that mmp-2 and mmp-9 levels in cam-complicated decidua (hscore mean±sem: 203±14 and 198±12, respectively) were significantly higher than in control decidua (136±6, and 114±6 respectively; p<0.05). in cultured term dcs incubated with e2, tnf and il1 significantly increased secreted levels of mmp-9 compared to e2 alone (pg/ml/ g protein: 162.3±110.1 and 20.4±6.5, respectively, vs. 0.5±0.2; p<0.05). in parallel incubations with e2+mpa, basal mmp-9 output was lowered by 50%, and tnf -and il1 -elicited mmp9 levels were blunted by 70% and 40%, respectively. rt-pcr confirmed that tnf and il1 increased mmp9 mrna levels (p<0.05), although mrna levels in e2+mpa incubations were not different from those of e2 alone. mmp2 levels in all treatments were similar. conclusions: mmp-2 and mmp-9 are elevated in cam decidua compared to controls. our in vitro results suggest that mmp-9 expression is enhanced by the high levels of il1 and tnf associated with cam, and that mpa may be able to blunt this effect. we have previously found a similar regulatory mechanism of mmp-1 and mmp-3 and their over-expression in cam-complicated tissues. synergy among these mmps may represent a potent pathogenic mechanism of cam that can be targeted through the therapeutic use of progestins in preventing cam-induced ptd. domerudee preechapornprasert, 1 patama promsonthi, 1 wasun chantratita, 2 mana rochanawutanon, 2 patcharee karnsombut, 2 chutatip srichunrusami, 2 stephen j lye, 3 boonsri chanrachakul. 1 1 obstetrics and gynecology, ramathibodi hospital, mahidol university, bangkok, thailand; 2 pathology, ramathibodi hospital, mahidol university, bangkok, thailand; 3 obstetrics and gynecology, samuel lunenfeld research institute, toronto, canada. objective: cervical ripening is an inflammatory process involving chemokines, cytokines and various mediators. recent study has shown that the level of monocyte chemotactic protein (mcp) 1, a chemokine, increases in amniotic fluid during spontaneous labor. the aim of this study was to examine the localization and expression of mcp 1 in human cervix before pregnancy, during pregnancy and after the onset of labor. methods: this study was approved by the local ethics committee and written informed consent was obtained from each participant. cervical biopsies were taken from 4 groups of women; nonpregnant women, first trimester pregnant women, term pregnant women with and without labor. tissue samples were fixed in 10% formal saline for paraffin section. immunohistochemistry (n = 5 each) was performed by avidin biotin complex (abc) technique using monoclonal antibody specific to human mcp 1. the mcp 1 messenger(m) rna was identified by reverse transcription-polymerase chain reaction using gene specific primer against mcp 1 and mcp 1 receptor (n = 10 each). results: immunohistochemistry demonstrated mcp 1 in cervical tissues from all four groups of women. mcp 1 was localized on plasma membrane and cytoplasm of both squamous epithelial and columnar cell lining of endocervical gland. mcp 1 and mcp 1 receptor mrna were identified in nonpregnant, first trimester and term with and without labor human cervix. conclusion: mcp 1 and mcp 1 receptor were located in cervical tissues of nonpregnant and pregnant women at different gestation both before and after the onset of labor.ongoing studies are investigating the role of this chemokine during pregnancy and labor. whether preterm cervical ripening is just an aberrant regulation in timing or whether divergent mechanisms and pathways are involved in preterm versus term cervical ripening remains to be elucidated. methods: cervical tissue was collected from 3 groups of cd-1 mice. group 1: mouse model of ptb that utilizes intrauterine infusion of lipopolysaccharide (lps). group 2: e15 dams representing preterm controls. group 3: e18.5-19 dams selected from a timed pregnant batch where half of the dams had delivered representing term cervical ripening. n=6 dams/treatment group. 18 separate rna samples were used for microarray analysis (ma). significance analysis for ma and partek software was used for biostatistical analysis. pathway analysis was performed using david. quantitative pcr was performed to confirm the most differentially regulated genes. results: using a cut-off of 64-fold change with p value of <0.0001, 368 genes in the cervix were differently regulated between the 3 groups. principal component analysis revealed three distinct groups (see graph). functional annotation clustering demonstrated the following pathways: 1) in preterm cervical ripening (e15 lps vs e15): immune and inflammation response, defense response 2) in term cervical ripening compared to preterm controls: negative regulation of cellular process and biological process, ecm, cell-cell communication. qprc confirmed the highly significant differences found in ma (see table) . conclusions: the molecular mechanisms and pathways governing preterm and term cervical ripening are distinctly different. elucidating these unique pathways can lead to improved therapeutics for prevention of ptb as well as for postdate pregnancies. cervical ripening at term involves activation of apoptotic enzymes. maria kb sennstrom, 1 valentina ciani, 2 gunvor e ekman. 1 1 obstetrics and gynecology, women and child health, karolinska institute, stockholm, sweden; 2 obstetric and gynecology, university of siena, siena, italy. aim: to investigate if the human cervical ripening at term involves programmed cell death. during the final cervical ripening the extracellular matrix dominated cervix undergoes an extensive remodelling of the tissue. inflammatory mediators such as the cytokines increase in ripening cervical tissue at term. programmed cell death, apoptosis, has been suggested as important in this process. apoptosis can be induced by inflammatory mediators such as cytokines. we also looked upon the distribution of inflammatory cells in cervical tissue. materials and methods: cervical biopsies from pregnant women at term and post partum women with fully ripened cervix were studied. biopsies from non-pregnant women served as controls. immunohistochemical analysis of the apoptotic enzyme caspase-3 and the inflammatory cell marker cd 45 was performed on paraffin embedded sections of cervical tissue. double staining was performed. mann-whitney u-test was used for statistical analysis. results: there was a significantly higher frequency of caspase-3 staining in the post partal sections from ripened cervical tissue compared to tissue from term pregnant (p=0.002) with unripe cervix and from non-pregnant patients (p=0.00067). the inflammatory cells staining for cd 45 increased in post partal and term pregnant sections compared to non-pregnant (p=0.016). there was a higher frequency of caspase-3 positive cells in the post partal tissue than of cd45 positive cells (p=0.00011). the localization of cd-45 positive staining was highest in the epithelia and basal lamina while caspase-3 staining was most pronounced in stromal tissue and around vessels. conclusion: our data show apoptotic activity in stromal tissue in fully ripened human cervix at term of pregnancy, suggesting that apoptotic mechanisms are involved in the extracellular matrix remodelling at term. the apoptotic activity is not co-localized with inflammatory cells suggesting non-infectous inflammation with apoptosis as important for cervical ripening at term. objective: cervical biomechanical responses are important for accommodating the increased stress induced by an enlarging uterus. a mechanical testing system was modified to evaluate the stress relaxation response in the pregnant cervix. methods: tissue harvested from the non pregnant and timed pregnant (days 12, 16, 20, 21, and 22) sprague-dawley rats underwent tensile testing using an instron 5800 material testing system. the testing regimen consisted of tissue extension to near maximal strain over 60 seconds followed by a period of constant strain for 60 minutes, then return to rest over 60 seconds. this cycle was repeated 2 additional times with a 60 minute rest period between cycles. strain and force measurements were recorded at 1 second intervals. 4-6 animals were used for each time point. in addition, stress and strain at the yield point was also determined for each gestational time point. results: the pregnant and non pregnant samples exhibit marked differences in response in both stress and strain. the timed pregnant tissue demonstrated progressively increased compliance and lower nominal stress compared to non pregnant tissue. peak nominal stress declined with each successive cycle. this was demonstrated in the peak nominal stress and strain values at the yield point. conclusion: the cervix becomes more distensible (compliant) but less resistant to force with increasing gestational age. ...,.. e. coli 1.2x 10 8 .:!: 8.5x 10 8 1.2 x 10 8 .:!: 3.0 x 10 8 group a (c, c) group b ( c, p) 2.3x 10 8 + 2.7x 10 8 9.4 x 1 0 7 + 4.8 x 10 8 .33 .81 .83 group c (p, p) 1.1 x 1o"::!>s x 1o" 1.1 x 10 8~ 1.ox:1o• k. ~neumoniae group a ( c, c) 5.3 x 10 7 + 3.4 x 10 7 9.2 x 10 7 + 6.7 x 10 7 .88 .87 .91 group b (c, p) 6.0 x 10 7 + 4.8 x 10 7 6.3 x 10 7 + 3.0 x 10 7 group c (p, pl 4.7 x 10 7~2 .3x 10 7 6.9x 10 7~4 .6x 10 7 c. al bicans group a (c, c) 1.1 x 10 '+1.8x 10 7 1.9 x 10 8 + 1.0 x 10 8 group b (c, p) 1.5x 1o•+ 1.0x 10 7 2.4 x 10 8 + 1.1 x 10 6 .75 . 87 .60 group c (p, p) 4.7x 10 7~2 .3x 10 7 2.1 x 1 0 7~5 .9 x 10• introduction: a growing body of evidence supports that inflammatory processes are implicated in spontaneous preterm birth (ptb). using an inflammatory and non-inflammatory mouse model of ptb, we sought to determine if activation of these inflammatory pathways are essential for ptb and/or cervical ripening to occur. methods: timed pregnant cd-1 mice were used in these two models of ptb: 1) a model of intrauterine inflammation where lipopolysaccharide (lps) is injected into the uterine horn (n=6); controls for this model received intrauterine saline (n=6) and 2) a non-infectious model of ptb using ru486 sq (150ugrams/dam) (n=5); controls for this model received no intervention (n=6) were used for these studies. for both models, 6 hours later uterine and cervical tissues were harvested. the tissues were processed for protein and rna studies. elisas were performed to assess il-6 and tnf-alpha in the uterine tissue. mrna expression of ifn , il-6, il-1 , il-10, tnf-alpha were assessed in cervical tissue from both models by quantitative pcr. results: in uterine tissues, both il-6 and tnf were significantly elevated in the lps-induced ptb when compared to control, saline, and non-infectiousinduced preterm birth (p<0.001) (figure 1). in cervical tissue, an increase in il-6, il-1beta and tnf mrnawas observed in both models, while ifn-gamma and il-10 were only increased in the lps model. (figure 2 ). conclusions: up-regulation of pro-inflammatory cytokines in the uterus do not appear to be essential for ptb. cytokine expression in the cervix is greater in an inflammatory model of ptb but is also present in a non-infectious model. these studies suggest that targeting a cytokine response in the cervix may hold the most promise in prevention of ptb. the initiation of labor at term and preterm is associated with an inflammatory response, with increased interleukins in amniotic fluid (af) and infiltration of the myometrium by neutrophils and macrophages (m ). whereas, in preterm labor, intra-amniotic infection may provide the stimulus for increased af interleukins and inflammatory cell migration, the stimulus for these events at term has remained uncertain. in studies using pregnant mice, we observed that the m that invade the maternal uterus near term arise from the fetus. furthermore, we obtained compelling evidence that surfactant protein-a (sp-a), a developmentally regulated c-type lectin secreted by the fetal lung into af near term, activates af m , which migrate to the uterus where they promote an inflammatory response culminating in labor. we propose that interactions of m surface receptors with sp-a, at term, or bacterial lipopolysaccharide at preterm, initiate changes in m phenotypic properties, resulting in the enhanced expression of genes that promote their migration to the uterus. the objectives of the present study were to analyze the numbers and phenotypic properties of mouse af m during late gestation and to identify their putative tissue source(s) of origin. to assess changes in the number of m in af during late gestation, af cells were isolated and stained for the m marker f4/80. the density of adherent f4/80 + cells greatly increased in equivalent volumes of af between 15.5 and 18.5 days postcoitum (dpc) (19.5 dpc = term). interestingly, the f4/80 + cells at 18.5 dpc were highly similar in morphology to those present in 18.5 dpc fetal lung, but distinctly different from those in fetal liver, suggesting their pulmonary origin. the af m were foam cell-like, suggesting the presence of lipid inclusions, a property shared by adult alveolar m . to further analyze gestational changes in the m population(s) in mouse af, we used flow cytometric analysis. in our initial studies, cells isolated from af were stained for f4/80 and for cd45, a pan-leukocyte marker. we observed that af from 15.5 and 18.5 dpc mice contained two sub-populations of cd45 + f4/80 + cells. studies are in progress to analyze these af m populations for expression of cell surface antigens indicative of their maturity, activation state and chemotactic properties in association with the developmental induction of sp-a synthesis and secretion by the fetal lung. april bleich, patrick keller, r ann word. ob-gyn, ut southwestern, dallas, tx, usa. the role of prs, proinflammatory cytokines, cell adhesion molecules, cox-2, and toll-like receptors in mediating cervical ripening prior to labor is not clear. the objective of this study was to quantify pr isoforms and determine the relative expression of certain inflammation-related genes in cervical stroma from nonpregnant and pregnant women in early gestation (eg), term before and after cervical ripening, and during labor. methods: standard curves of pr-b, -a, pra+b and qpcr were used to quantify total and pr-b in cervical stroma from 9 nonpregnant (proliferative, n = 4; progestin treatment, n = 5) and 35 pregnant women undergoing hysterectomy (eg, n = 7; term before ripening, n = 10; after ripening, n = 7; in labor, n = 11). cervical status was determined by modified bishop scoring. results: total pr expression was maximal in nonpregnant women in the proliferative phase (47.6 ± 8.7 pg/ug cdna) and decreased 80% by progestins (8.6 ± 2.7 pg/ug cdna). this level was maintained in stroma from pregnant women before labor (6.6 ± 1.3, eg.; 9.9 ± 1.8 before ripening; 12.5 ± 2.9 after ripening pg/ug cdna). in contrast, total pr was decreased significantly in the dilated cervix (2.4 ± 0.6 pg/ug, p < 0.05). interestingly, whereas pr-b was 30 ± 2% that of total pr in the nonpregnant cervix, pr-b mrna levels were 52 ± 4% to 70 ± 6% in cervical tissues from all pregnant women and did not vary with labor status. using immunoblot analysis and pr-specific antibodies (pgr1294), pr-b immunoreactivity was 50% that of total pr in all samples from pregnant women, and both pr-a and -b were downregulated significantly in the dilated cervix (from 44 ± 9 to 19 ± 8 units/atub). decreased expression of pr in the dilated cervix was accompanied by significant increases in il-8, mcp-1, tlr-2, cd62l, cox-2, and s100a9 mrna (all p < 0.05, anova) but not cd11b or tlr-4. pgdh mrna was decreased significantly in the dilated cervix. with the exception of cox-2, expression of these genes was similar before labor regardless of cervical ripening. conclusions: both pr and pr-b are decreased proportionately in the dilated cervix, but not during cervical ripening. further, a number of inflammatory gene products are increased dramatically in the cervix during labor, but not before. taken together, the results suggest that cervical ripening is distinct from cervical dilation and involves upregulation of cox-2 but not il-8, mcp-1, or toll-like receptors. ifn-y il-6 il-1,6 il-10 tnf-01 "p value <0.05 lps/saline ru486/controls 10.3"" 0.63 68" 6.6" 15.7* 24" 12" 5.9* 1.8 3.1" 722 association between interleukin-6 (il-6) and ilto examine association of amniotic fluid interleukin 6 (il-6) concentration with il-6 and its receptor il6-r haplotypes in term and preterm caucasians (c) and african americans (aa) samples. methods: in this study case (preterm birth -ptb [<36 weeks]) and control (term [>37weeks]) amniotic fluid (af) il-6 concentrations were analyzed for association with haplotypes of the il-6 and il6r genes in aa and c separately. in il-6, eight, and in il-6r, 22 single nucleotide polymorphisms (snps) were examined. aa and c maternal and fetal genotypes were assessed (aa:35 maternal:cases-57 controls-34 fetal: cases-54 fetal controls-; c: maternal cases-88, controls-40, fetal:cases-86; controls=36). haplotype associations were performed by using a sliding window with outcome il-6 concentration. analyses were performed separately on maternal and fetal dna. results: the strongest haplotype associations were observed in il-6r rather than in il-6. in c fetal dna il-6r haplotypes defined by markers 16311-21909 bp from the transcription start site associated most strongly with af il-6 concentrations (global p=1.6x10 -3 ) and in aa maternal il-6r haplotype markers at 16311-21909-22214-26274 (global p=2.30x10 -3 associated with il-6 concentrations. in the c fetal cases the 16311-21909 haplotype with the highest concentration was a-g (log(cytokine) = 3.67 pg/ml). in aa maternal samples the highest concentration was observed for haplotype t-t-g-c at 16311-21909-22214-26274.this was seen in both cases and controls at (case log (cytokine) = 3.84; control log(cytokine) = 3.49 pg/ml). significant associations from haplotype analyses converged on three regions of the il-6r in both races. no strong differences were observed between the haplotypes of cases with and without microbial invasion of the amniotic cavity (miac), with the exception of aa fetal samples that showed two overlapping haplotypes in il-6 that associated in cases with miac but not in cases without miac (-661 and -636; -636 and -237)(both with p < 1x10 -3 ) conclusion: differences in the af il-6 concentration in ptb do not result from single snp effect on il-6 but are a result of complex relationships between il-6 and il-6r haplotypes. these associations exhibit racial disparity. the role of tnf-in parturition. helen alexander, 1 amanda tattersall, 1 mark tattersall, 1 suren sooranna, 1 peta grigsby, 2 leslie myatt, 2 mark johnson. 1 1 obstetrics gynaecology, imperial college, london, united kingdom; 2 obstetrics gynaecology, university of cincinnati college of medcine, cincinnati, oh, usa. introduction: tumour necrosis factor-alpha (tnf-) is thought to play a role in inflammation-induced preterm labour since the decidua produces tnfin response to bacterial products and amniotic fluid tnf-concentrations are increased in the presence of intra-amniotic infection. the aims of this study were to (i) investigate the expression of myometrial tnf-and its receptors in relation to the onset of preterm and term labour; (ii) to identify which intracellular pathways are activated by tnf-; and to investigate the effect of tnf-alone and in combination with il-1 or il-8 on gene expression in uterine myocytes. methods: biopsies of human myometrium were taken at caesarean section from women before and after the onset of preterm and term labour and analysed for tnf-and its receptor mrna expression. a further 6 samples were obtained before the onset of labour (n=6) from which myocytes were isolated and cultured in 6-well plates. when cells were 85-95% confluent either tnf-at a concentration of 1ng/ml was added to the cells for 7, 15, 30, 60 and 120min and the cells analysed by western blotting or tnf-at concentrations of 0, 0.1 and 1ng/ml was added either alone or in combination with similar concentrations of il-1 or il-8 to cells for 6 hours and mrna was extracted and converted to cdna to determine il-8 and gapdh gene expression by qpcr. results: there was no change in tnf-mrna expression in relation to the onset of labour, but the expression of tnfr1 and tnfr2 mrna levels were significantly increased with gestation and further increased with the onset of labour. incubation of uterine myocytes with tnf-(1ng/ml) activated all three mapk substypes: erk, jnk, and p38, activation peaked between 7-15 minutes. preliminary data suggest that tnf-induces il-8 mrna expression but exposure to il-1 or il-8 itself did not enhance this response. conclusions: although there is no significant increase in tnf-concentration from baseline during labour we have shown tnf receptor mrna levels do increase with labour at term. exposure of isolated uterine myocytes to tnfcauses activation of all mapk subtypes and an increase in il-8 mrna expression. this enhanced mapk-dependent il-8 expression at term may be mediated via increased myometrial sensitivity to tnf-through increased tnf receptor expression. remodeling. brenda c timmons, 1 anna-marie fairhurst, 2 mala s mahendroo. 1 1 obstetrics and gynecology, ut southwestern medical center, dallas, tx, usa; 2 immunology, ut southwestern medical center, dallas, tx, usa. objective: the molecular mechanisms involved in cervical ripening are not well understood. immunohistochemical studies from our lab report a recruitment of inflammatory cells to the cervical stroma one day before birth in the mouse using a neutrophil/monocyte marker (neutrophil 7/4). in this study, we sought to identify and quantitate inflammatory cells migrating into the mouse cervix and to determine if this recruitment was affected by changes in progesterone levels. peripheral blood was also evaluated to see if changes in the cervix was paralleled in blood. methods: flow cytometric analysis was performed using cervical cells and peripheral blood obtained before and during cervical ripening along with 2-4h postpartum. dispersion of cervical cells was optimized. these cells were stained with a panel of fluorescent conjugated antibodies directed against leukocyte antigens and analyzed on an lsrii flow cytometer. cells were also sorted and stained to visualize cell morphologies. to determine the effect of progesterone on the migration of leukocytes, gestation d15 mice were treated for 13h with a progesterone receptor (pr) antagonist prior to tissue collection. results: neutrophils do not appear to increase in the cervix until after birth. monocyte (mo) numbers do increase during cervical ripening (late day 18, d18.75) and remain high through postpartum (pp). macrophages (mø) are present prior to cervical ripening and steady state levels are maintained during labor and pp. pr antagonist treatment on d15 resulted in a premature increase in mo but not neutrophils or mø. in contrast to the cervix, mo and neutrophil numbers do not significantly increase in the peripheral blood until pp. results from lymphocyte studies suggest a low level of b and t cells in the cervix. in the peripheral blood, b cells remain consistent through parturition and the t cells decrease by d18.75 and continue to decrease pp. conclusion: tissue mo are increased in the cervix during ripening. this recruitment is dependant on loss of pr function. in contrast, neutrophils are increased in the pp cervix while mø numbers appear constant. timing of changes in mo and neutrophil numbers in the peripheral blood differed from that observed in the cervix suggesting quantitation of these cell types in blood is not reflective of what is occurring in the cervix during ripening, dilation and pp repair. novel interactions between nf-b and other labour-associated transcription factors identified by a tf-tf array. shirin khanjani, yun s lee, suren r sooranna, mark r johnson, phillip r bennett. irdb, imperial college, london, united kingdom. introduction: external stimuli lead to changes in cellular gene expression through activation of inducible transcription factors. nf-b is a ubiquitous transcription factor classically associated with inflammation, which is activated in response to infection and proinflammatory cytokines such as those prevalent during the labour. the tf-tf interaction array uses a novel technology for detecting interactions between transcription factors based on binding of tfs to their own consensus dna binding sequence. materials and methods: primary myometrial cells were grown until 90-95% confluent and stimulated with 1ng/ml il-1 prior to nuclear protein extraction. the nuclear extracts were incubated with the provided set of biotin-labeled, double-stranded oligonucleotide probes, which represent a known library of cis-elements. during the incubation step, these tf probes bind to their specific tfs in the nuclear extract. next, immunoprecipitation was performed using an antibody against nf-bp65, which pulled out nf-bp65 and any tfs bound to it, bound to corresponding cis-elements. normal igg was used in a parallel experiment to represent a negative control. free cis-elements and nonspecific binding proteins were washed away and the cis-elements were finally eluted and hybridized to the array membrane, which is spotted with different tf consensus sequences. results: table 1 shows the different tfs interacting with nf-bp65 based on the degree of binding stimulated by il-1 compared to no-il-1 control. conclusion: these data show that il-1 stimulation causes nf-b to bind to a wide variety of other treansciption factors. of particular interest in the area of parturition is binding to the other pro-inlammatory tfs such as c/ebp and ap-1, which are known to regulate labour-associated genes in synergy with nf-b. the lack of association between nf-b and pr without il-1 stimulation supports the concept that with the onset of labour inflammation leads to functional progesterone withdrawal, rather than pr acting to inhibit inflammation. table 1 high ap-1, c/ebp, cbf, creb , c-myb, e2f-1, ets, ets-1/pea3,fast-1, gas/isre, hse, mef-1, mef-2, myc-max, nf-1, nfatc, nf-e1, nf-e2, pax-5, objective: pre-partum cervical ripening involves remodeling of collagen structure and inflammatory immune cell activity (jsgi 10:323,2003; reprod biol endo 3:39,2005) . although parturition is associated with systemic or local progesterone withdrawal (ajog 196:289,2007) , effects of a decline in progesterone on cervical ripening is not known. the present study tested the hypothesis that progesterone withdrawal promotes collagen degradation, innervation, and immune cell trafficking in the cervix of nonpregnant mice. methods: adult virgin female c57bl6 mice received capsules (sc) with oil vehicle (v) or estradiol (e) and progesterone (p) to simulate concentrations in pregnancy (hum reprod 12:602,1997) . after 15 days, mice in the v and e+p groups were euthanized. the p capsule was removed from some mice on day 17 (e-p) and groups killed on days 18 and 19. cervix sections were stained for collagen, nerve fibers, macrophages, or neutrophils (n=6/group/day; 3 sections/ cervix; biol reprod 61: 879,1999; jsgi 12:578,2005) . stained macrophages and neutrophils were counted (image pro-plus 6, media cybernetics). results: e+p treatment for 15 days promoted hypertrophy of the cervix compared to v controls, i.e., collagen content and structure diminished, cell nuclei density declined, and nerve fibers increased. removal of p did not affect these endpoints. for immune cells, e+p for 15, 18, or 19 days decreased immune cell numbers. by contrast, p removal increased macrophages and neutrophils in the cervix on days 18 and 19 (p<0.05, e-p vs e+p groups, respectively). the census of resident immune cells in e-p groups at 24 and 48 h after p removal equaled that in the v group. conclusions: mimicking gonadal steroid concentrations in circulation during pregnancy promotes hypertrophy and suppresses immigration of immune cells in the cervix. in this non-pregnant murine model for parturition, progesterone withdrawal recruits immune cells, but fails to promote further remodeling or hyperplasia of nerve fibers in the cervix. the findings raise the possibility that ripening of the cervix requires not only recruitment, but also activation of immune cells. whether proinflammatory activities by specific immune cells affect nerve fiber hypertrophy or neural activity, as part of the mechanism for ripening of the cervix, remains to be determined. we have previously identified and characterized a truncated pr (pr-m), that localizes to the mitochondrion by multiple experimental techniques, including confocal imaging of a recombinant gfp fusion protein, western blot analysis after cellular fractionation of nuclear pr negative t47d-y breast cancer cells and western blot analysis of purified human heart mitochondrial proteins. initial studies with nuclear pr negative mcf-10a breast epithelial cells shown to express pr-m demonstrated an increase in mitochondrial membrane potential (mmp) with progesterone/progestin treatment. these studies led to the hypothesis that progesterone modulates cellular respiration via the mitochondrial receptor, pr-m. objectives: the present studies sought to further localize pr-m to the outer, inner or matrix portion of the mitochondrion and to correlate the increase in mmp with total cellular atp production. additionally, the potency of progesterone and synthetic progestins on the change in mmp was evaluated. methods: the location of pr-m was determined by western blot analysis after fractionation of human heart mitochondria with digitonin treatment and differential centrifugation. mmp was determined in mcf-10a breast epithelial cells and a673 rhabdomyosarcoma cells by the change in fluorescent emission of jc-1. total atp was determined by a bioluminescent assay. results: western blot analysis after mitochondrial fractionation showed pr-m localization exclusively in the outer membrane. a dose-dependent increase in mmp was seen in cell lines with 30-60 min treatment with progesterone and r5020 which were inhibited by a specific pr antagonist, rti-6413-049b, and not affected by the translational inhibitor, cycloheximide. similar changes in mmp were seen with the same concentration of progesterone, mpa and r5020. progesterone/progestin treatment for 120 min led to an increase in total cellular atp without a change in cell number. conclusions: progesterone/progestin treatment results in an increase in cellular respiration in cells expressing an outer mitochondrial membrane pr and known to lack nuclear pr expression. this may represent a mechanism whereby progesterone enhances cellular energy production to meet the demands of pregnancy. introduction pge 2 is a major product of the fetal membranes, decidua and myometrium and plays an important role in cervical ripening and myometrial contractions. there are four pge 2 receptors, ep-1 and ep-3 mediate contractions whilst ep-2 and ep-4 mediate quiescence. ep-4 contains multiple consensus sequences for transcription factors known to be of importance in labour, in particular nfkappab. we therefore performed experiments to determine the effect of activation and inhibition of nfkappab upon ep-4 expression. myocytes plated in 6 well plates were treated with il-1 (1ng/ml), to activate nfkappab. myometrial cells were also transiently transfected with nfkappab p65 sigenome smart pool and sicontrol non-targeting sirna to knock down nfkappab p65. rna was extracted for amplifying ep-4 using quantitative rt-pcr with amplification of l19 as a control to normalise data. il-1 caused an increase in expression of ep-4. knock down of nfkappab using si-rna resulted in a further increase in ep-4. this data suggests that although il-1 stimulates expression of ep-4 it does so through an nfkappab independent mechanism. the upregulation of ep-4 with sirna knock down of p65 suggests that activation of nfkappab would inhibit ep-4 expression consistent with the concept that activation of nfkappab at term causes the myometrium to adopt a more contractile phenotype. introduction: a role for the pro-inflammatory cytokine il-6 is suggested in preterm and term birth, independent of the presence of infection. we previously showed that mice with a null mutation in the il-6 gene (ko) delivered one day later than wild type (wt) mice due at least in part to altered timing of uterine expression of prostaglandin (pg) f 2 receptor, ptgfr, mrna. we also observed differences in mrna expression of other uterine activation proteins (uaps), pg h synthase (pghs)-2, oxytocin receptor (otr) and connexin-43 (cx-43), suggesting multiple physiological effects for il-6 in term delivery. objective: to examine the effect of il-6 deficiency on the peri-partal uterine mrna expression of the pge 2 receptors (ep) 2, 3 and 4; the post-partum changes of all uaps; and the relationship of these to serum progesterone (p 4 ) concentrations. methods: gestational length was observed in pregnant c57bl/6 wt (n=61) and ko (n=39) mice. uap mrna levels were measured by real time rt-pcr in wt and ko dams sacrificed from d15 through delivery and up to 24h postdelivery. serum p 4 was determined by ria. data were analyzed by one-way and two-way anova using the holm-sidak test to differentiate treatment effects at p<0.05. results: birth was delayed in the il-6 ko mice (20.7±0.2d vs. 19.7±0.1d), and this affected the timing of peri-partal changes for all uaps similarly. both ep2 and ep4 (relaxatory receptors) were elevated at d18 in ko dams, but returned to low levels before delivery and were elevated at delivery (ep2) or afterwards (ep4). ep3 levels did not change. otr increased several hours before delivery in all dams. cx-43 increased at delivery, then fell, while pghs-2 increased at delivery and remained elevated afterwards. ptgfr mrna increased 3-4-fold at delivery and a further 2-3-fold after delivery, suggesting loss of pgf 2 permitted enhanced ptgfr expression. p 4 serum concentrations fell pre-partum in both groups. conclusions: il-6 ko alters the expression pattern of several pregnancy and parturition-related genes and may delay the pre-partum p 4 fall, suggesting a potential ovarian effect. a uterine role for il-6 in regulating the timing of normal term parturition cannot be ruled out. objective: recent studies have highlighted the prevalence of vitamin d deficiency in pregnant women, particularly in those from ethnic groups with darker skin who require higher levels of uv light to make parental vitamin d. as the active form of vitamin d, 1,25-dihydroxyvitamin d 3 (1,25(oh) 2 d 3 ) is a potent immunomodulator, we postulated that vitamin d deficiency may lead to dysregulated placental immunity. both trophoblast and decidua express the enzyme 1 -hydroxylase (cyp27b1) which catalyzes synthesis of 1,25(oh) 2 d 3 from the inactive pro-hormone 25-hydroxyvitamin d 3 (25ohd 3 ). in view of the role of trophoblast as a barrier site protecting the fetus against infection, we investigated the impact of cyp27b1, 25ohd 3 and 1,25(oh) 2 d 3 on innate immune responses in trophoblastic cells. methods: 3a trophoblast cell line was used to assess the effect of vitamin d on innate immune responses. the cells were treated for 24 hrs with various concentrations of 1,25(oh) 2 d 3 (1-100 nm) and antimicrobial cathelicidin expression was assessed by real time pcr. to assess whether expression of cyp27b1 was affected by pathogenic stimuli, 3a cells were treated with ligands for toll-like receptor (tlr) 1-9, cyp27b1 expression (real time pcr) and 25ohd 3 utilization were assessed. results: 3a trophoblast cell line; which shows temperature-sensitive differentiation, revealed expression of cyp27b1 with higher levels of enzyme activity under conditions of syncytiotrophoblast development. cells treated for 24 hrs with 1,25(oh) 2 d 3 showed dose-dependent induction of the antimicrobial defensin cathelicidin expression (3.5-17 fold induction), whilst cells treated pro-hormone 25ohd 3 (100 nm) showed 2.5-fold induction of cathelicidin expression. activation of tlr3 (poly i:c) and tlr4 (lipopolysaccharide) enhanced the expression of cyp27b1 (2-and 2.5-fold) and increased the sensitivity to 25ohd 3 as a consequence. conclusion: these data show that autocrine synthesis of 1,25(oh) 2 d 3 from 25ohd 3 can stimulate trophoblast immune responses in a similar fashion to macrophages. as 25ohd 3 is the major circulating form of vitamin d, we hypothesize that trophoblast innate immunity may be significantly compromised under conditions of vitamin d deficiency. introduction: secretory leukocyte protease inhibitor (slpi) is a potent 12-kda protein inhibitor of neutrophil elastase and it is a mediator of mucosal immunity and an inhibitor of nfkb regulated inflammatory responses. however, its source, function and regulation within the uterus during pregnancy and at parturition are not well defined. it has previously been shown to be present in fetal membranes and cervical mucus and in amniotic fluid, where its levels is increased from second trimester to term and with a further increase at parturition. slpi has also been shown to be responsive to progesterone in human epithelial cells. our aim was to determine the effects of il-1 on slpi gene expression in human myometrium. methods: primary human uterine myocytes were isolated from non labouring myometrium and cultured in 6 well plates and when cells were 70-80% confluent they were serum starved overnight and incubated with 1 m 6methyl-17 -hydroxy-progesterone acetate for 48h and with or without 1ng/ml il-1 for a further 6 hours. at the end of incubations rna was extracted and converted to cdna. paired upper and lower segment myometrial tissue was collected at caesarean section either before or after the onset of term or pre-term labour and frozen for extraction of rna (n=6 for ptnl, ptl, tnl and l). copy numbers of slpi, gapdh and beta-actin were measured by qpcr. results: 6 h incubation of uterine myocytes with 1ng/ml il-1 caused a marked increase in slpi by 201% (n=12; p<0.015). incubation of uterine myocytes with 1 m progesterone for 48h also increased slpi by 139% (n=12; p<0.05). incubation with il-1 for 6h in the presence of 48h with progesterone increased slpi: gapdh mrna ratio from 10.50 ± 3.37 to 33.33 ± 10.72 (mean ± sem; p<0.004). slpi expression was similar in the upper and lower segment myometrium in preterm patients. in term myometrium the slpi: beta-actin mrna ratio was increased by 50-and 200-fold in term labour versus term non labour samples in the lower and upper segment respectively (mean ± sem; p<0.05). these data show slpi is present in human myometrium and that it is increased by il-1 . progesterone also increases its expression. its expression is increased in term labour where its anti-bacterial, anti-fungal and anti-viral properties could allow it to act as an endogenous block to infections. objective: pre-b cell colony-enhancing factor (pbef) downstream mapk signaling and transcription factor activation. introduction: pbef is expressed in all layers of the human fetal membranes and the myometrium and is upregulated by labor, infection, nf-kb, ap-1 and stretching. pbef has the ability to protect a variety of cells from apoptosis, however it also appears to act as an insulin mimetic via the insulin receptor (fukuhara et al. science 2005: 307; 426-430) . its levels are elevated in obese patients, those with type 2 diabetes and in gestational diabetes. because pbef has poorly understood biological activities an investigation of mapk signaling and transcription factor activation induced by pbef has been undertaken in order to gain insights into its mechanisms of action. methods: primary aec were isolated from fetal membranes and treated with rhpbef (100ng/ml) for 1h, lysed and the resultant proteins labeled with cy3 or cy5 (amersham). there were used on a protein microarray containing 64 constituents of the mapk signaling pathways (sigma). isolated aec were transfected with luciferase constructs for nf-kb, ap-1, cre, hse and gre response elements using the exgen500 reagent. the cells were treated with rhpbef (0.1, 1.0, 10, 100 ng/ml) 4 hrs, lysed and 50ul of sample was analyzed for luciferase activity with dual-luciferase reporter assay system (promega). co-transfection with gfp and sv40 luciferase constructs to control for transfection efficiency and cell number (respectively) was also performed for each experiment. results: pbef significantly upregulated 20 mapk signaling components including 9 functioning as part of the erk pathways and 5 belonging to p38 signaling. it also activated nf-kb and camp response elements. however, it significantly down regulated 3 signaling molecules belonging to the jnk pathway that resulted in decreased c-jun phosphorylation. conclusions: pbef signaling in aec is consistent with its anti-apoptotic ability and its upregulation of the pro-inflammatory cytokines. although some mapk components responsive to pbef could be associated with insulin receptor signaling, some may not. therefore, it appears likely that pbef interacts with another potentially unique, but currently unidentified receptor. was the first effector to be characterised, but camp is now known to have other effectors, including camp receptor protein, cyclic nucleotide-gated channels and camp-guanine nucleotide exchange factor/exchange protein directly activated by camp (camp-gef/epac). two epac's have been identified and they consist of 4 functional domains: camp-binding domains, a dep domain, a ras exchange motif and a gef domain. our aim was to determine the presence of epac's in human myometrium and to study the effect of camp responses on prolabour genes such as il-8, pghs-2, otr and fp. methods: primary human uterine myocytes were isolated from non labouring myometrium and cultured in 6 well plates until 70-80% confluent. cells were serum starved overnight and incubated with 1μm methyl progesterone, 0.2mm sodium 8-bromo-camp, 0.1mm forskolin, 1μm kt5720, 75ng/ml brefeldin a and 0.1mm 8-pmeopt-2'o-me-camp either alone or in combination for 48h. rna was extracted and converted to cdna. paired upper and lower segment myometrial tissue was collected at caesarean section either before or after the onset of term or pre-term labour and frozen for extraction of rna (n=6 for ptnl, ptl, tnl and l). copy numbers of epac1, epac2, il-8, pghs-2, otr, fp, gapdh and -actin were measured by qpcr. results: epac1 and epac2 were present in the upper and lower segment of myometrium with epac1 levels being some 10-fold higher than those of epac2. there was no change with the onset of labour at or before term. treatment with il-1 for 6h significantly increased epac2 (n=12; p<0.012) but had no effect on epac1. when conditions mimicked pregnancy, (the presence of forskolin and progesterone), brefeldin a, an epac antagonist, increased basal pghs-2 and fp mrna expression (n=6; p<0.05), but had no effect on il-8 and tended to reduce otr. the il-1 -induced increase in pghs-2 and il-8, but not fp, was greater with the epac antagonist. conclusions: these data show epac's are present in human myometrium and that camp acts via epacs to reduce pghs-2 and fp expression during pregnancy. background: inflammatory events have been implicated in the process of labour. glucocorticoids mediate strong anti-inflammatory effects through binding to the glucocorticoid receptor (gr), which on activation translocates to the nucleus and either increases or decreases the expression of responsive genes thereby suppressing inflammation. objective: to characterise the expression profile for gr protein and mrna in human myometrium during fetal maturation and parturition. methods: western immunoblotting (wb) was employed to characterise gr protein expression in first (n=4) and second trimester myometrium (n=5), and in paired upper and lower segment pregnant (non-labouring, p, n=8) and labouring (l, n=8) myometrium; as compared to non-pregnant (np, n=8) control samples. immunofluorescence staining with confocal microscopy and rt-pcr were also undertaken. results: detection of gr protein by wb revealed two bands at 90-95 kda, representing the alternatively spliced isoforms, gr-and gr-. densitometric analysis showed that gr levels decreased significantly during pregnancy and remained at very low levels at term and in labour when compared with np samples (p<0.001); this decrease was seen for gr-and gr-. no significant temporal variations were observed in gr protein levels at term or during labour. gr protein was localised by immunofluorescence staining to the nuclei and cytoplasm of cells. less intense staining was apparent in p compared to np tissues; consistent with wb data. rt-pcr showed a consistent predominance of gr-to gr-mrna in all the tissues used. the observed decrease in protein expression was also mirrored at the mrna level: gr-mrna levels appeared to gradually decrease throughout gestation (p<0.05). differences between the upper and lower myometrial regions were only observed in the labouring samples, where gr-mrna levels were significantly decreased in the lower segment (p<0.05). nested pcr was additionally used to amplify gr-, but no consistent pattern of mrna expression was obtained. conclusions: these data are the first to characterise gr expression in human myometrium. spatial and temporal variations have been found with expression evolving through the three trimesters of pregnancy and in labour. further studies are now underway to evaluate whether gr contributes to the sequence of inflammatory events implicated in triggering term and preterm labour. these studies sought to determine the mechanism by which pas modulate the immune response. methods: 1) an in vitro co-culture model mixed with human cervical epithelial hela cells and pma-induced human macrophage u937 cells at epithelial/macrophage cell ratio of 5:1 was employed. 2) the co-culture was pre-treated with medroxyprogesterone acetate (mpa), progesterone (prog) and dexamethasone (dex) at concentrations of 1, 10, 100 and 1000 nm for 2 hrs followed by 2 day stimulation of 10 g/ml lipopolysaccharide (lps). these experiments were repeated using hela cells transfected with sirna for glucocorticoid receptor (gr). the production of cytokines il-1 , il-6 and il-8, il-10 and tnf were determined using elisas. 3) the co-culture was pre-treated with 100nm of each pas for 2 hours prior to lps stimulation at 10 g/ml for 0.5, 1.5, 6, 12, 24 and 48 hours. the phosphorylation of p38 mapk and gr and the expression of mapk phosphatase 1 (mkp1) were determined by western-blotting. results: il-1 , il-6 and il-8, il-10 and tnf were elevated by 3.8 fold, 1.7 fold, 1 fold, 4.9 fold and 4.6 fold in the co-culture model in response to lps(p<0.001 for all). pretreatment of mpa and dex, but not prog, inhibited the lps-induced cytokine production. the anti-inflammatory effect of mpa occurs in a dose-dependent manner. in the absence of gr, the inhibitory effect of mpa and dex on il-6, but not on il-1 , was lost. mpa and dex, but not prog, induced the phosphorylation of gr and expression of mkp1. p38 mapk was activated by lps for up to 24 hrs and pretreatment of mpa and dex attenuated this response. the ability of mpa and dex to suppress the inflammatory response in cervical tissues appears to be mediated through a gr-dependent pathway, specifically though p38 mapk. our studies suggest that prog is not a significant immunomodulator in these tissues. background: progesterone (p4) has been shown to play a critical role in maintaining pregnancy and preventing preterm birth. these effects are likely related to its immunomodulatory properties. we investigated whether camp plays a role in the immunosuppressive action of progesterone on fetal mononuclear cells. methods: umbilical cord blood mononuclear cells were isolated using density gradient centrifugation. to establish a p4 effect and optimize concentrations, cells were pretreated with p4 (10 -4 m-10 -6 m), dexamethasone (3um) or vehicle for 1 hour prior to overnight lps (10ng/ml) stimulation. supernatants were assayed for tnf-using elisa. ldh assays confirmed the absence of a cytotoxic effect. next, cells were pre-incubated with forskolin (adenylate cyclase activator) or db-camp (camp agonist) prior to lps to determine the effects of camp on tnf production. finally, cells were pretreated with rp-camp (camp antagonist) prior to p4 incubation and lps stimulation to determine whether the p4 effect was reversed by a camp antagonist. results: p4 significantly inhibited lps-induced tnf production in a dose dependent manner, with maximum suppression observed at 10 -4 m. both forskolin and db-camp suppressed lps-induced tnf production in a doserelated manner (fig 1) . finally, a dose-dependent partial reversal of tnf production was observed when cells were pretreated with rp-camp. (fig 2) conclusions: progesterone suppresses the inflammatory response in fetal mononuclear cells as measured by tnf expression. this effect is mimicked by adenylate cyclase activators and camp agonists and partially reversed by camp antagonists. this implicates the camp pathway in mediating the immunomodulatory actions of p4. objectives estrogen improves endothelial function after vascular injury via largely unknown mechanisms. endothelial progenitor cells (epcs) are known to be implicated in various vascular events requiring endothelialization. we hypothesized that estrogen and progesterone could influence the function and the change of epcs in menstrual cycle. material and methods peripheral blood mononuclear cells (pbmcs) were isolated from peripheral blood of healthy young women in each menstrual period by density gradient centrifugation with ficoll separating solution. pbmcs were seeded in endothelial basal medium with or without estrogen or progesterone. on the 4th day in culture, nonadherent cells were removed. on the 7th day in culture, adherent cells were incubated with di-ldl, fixed with paraformaldehyde, and stained with fluorescein isothiocyanate-labeled lectin. the number of ldl-and lectin-positive cells was measured as epcs using flowcytometry. the expression of estrogen and progesterone receptor mrna in epcs were measured by real time pcr in menstrual and luteal period. the number of epcs was significantly increased in the menstrual and luteal period compared with the follicular phase. estrogen and progesterone significantly increased the number of adherent epcs dose dependently in menstrual period, but not in luteal period. the expression of estrogen-alpha receptor in menstrual period was higher than luteal period. also, estrogen-beta receptor in luteal period was strongly expressed compared with menstrual period. these results suggest the expression of estrogen receptor and progesterone receptor play important roles to regulate epcs' proliferation during menstrual cycle. objective: elevated levels of fetal plasma avp are associated with the development of oligohydramnios, in part a result of avp-mediated reduction in fetal urine production. as avp urinary concentration effects are mediated via upregulation of renal tubular aqp1 water channels, we propose that avp modulates placental aqp channels, influencing bidirectional maternal-fetal water flow. we sought to study the effect of avp on trophoblast aqp1 gene expression using the first trimester-derived extravillous htr-8/svneo cells and the term placenta-like trophoblast carcinoma cells jeg-3. methods: cultures of both cell lines were treated with a physiological concentration of avp (0.1 nm) to determine aqp1 mrna and protein expression. negative controls consisted of cells incubated in medium supplemented with 1% fbs without avp. to determine whether avp regulation of aqp1 occurs by a camp signaling pathway, jeg-3 cells were preincubated with 100 μm 9-(tetrahydro-2'-furyl) adenine (sq22536), a cell-permeable camp inhibitor, before being treated with avp (0.1 nm). cells were incubated for 10 hrs for aqp1 mrna expression and for 20 hrs for protein extraction at 37°c. after harvest, real time pcr and western blotting analysis were used to detect the aqp1 mrna and protein expression levels, respectively. results: avp increased aqp1 mrna expression in both cell lines by 1.8 and 2.8 fold after 10 hrs. aqp1 protein expression paralleled the increase seen in the mrna (p<0.05). pretreatment of jeg-3 cells with sq22536 inhibitor completely blocked the stimulatory effect of avp. conclusion: aqp1 gene expression is up-regulated by avp in first trimester and term trophoblast cells, with a higher induction in the later. avp activation of aqp1 gene expression occurs via a camp mediated pathway, as the adenyl cyclase inhibitor blocked avp effects on aqp1 gene expression. these results suggest that increased fetal plasma avp may contribute to oligohydramnios by an increase in aqp-mediated fetal to maternal water flow. objective: changes in expression, ratio and activity of progesterone receptors within human myometrium have been proposed as potential contributory mechanisms for the functional progesterone withdrawal effect which precedes labour. progesterone receptor c (pr-c) is an n terminally truncated isoform of the full length progesterone receptor; pr-c has been shown to be abundant in fetal membranes, placenta and upper segment of laboring myometrium (1,2). the function and regulation of pr-c is at present unclear. in this study we aimed to investigate the regulation of pr-c in cultured human myometrial and amnion-derived wish cells. methods: myometrial primary cell cultures were prepared from non pregnant and term pregnant uteri. both myometrial and wish cell cultures were treated with natural progesterone (0, 100nm, 1mm, 10mm, 100mm) for 2, 6 and 24hrs. western immunoblotting was employed using nuclear and cytoplasmic extracts prepared from treated cells to observe the effect of progesterone on a) expression and b) subcellular localisation of pr-c within both cell types. immunofluorescent staining and confocal microscopy were also employed. experiments were also undertaken whereby nuclear and cytoplasmic extracts were subjected to shrimp alkaline phosphatase treatment to evaluate the phosphorylation status of pr-c in response to hormone. results: data indicates that a 60kda cytoplasmic protein, representing pr-c is abundant in myometrial and amnion cell cultures. we show that expression of pr-c increases in both cytoplasmic and nuclear fractions within both cell types in response to progesterone. evidence also suggests that pr-c is phosphorylated in a progesterone-independent manner. concurrent treatment with progesterone and the pr-antagonists ru486 and organon 31710 in amnion-derived cells still led to an increased abundance of pr-c but seemed to alter the phosphorylation status of pr-c. conclusion: pr-c expression and subcellular localisation within myometrial and wish cells alters in response to progesterone. speculation is generated about potential role of pr-c in reproductive tissues and its contribution to functional progesterone withdrawal. 1. taylor smooth muscle responses have been studied, data on regional blood flow (bf)distribution are scarce. we have studied the effect of a single dex course on relative bf distribution within the brain, skeletal muscle, heart, omental fat, placenta and adrenal in fetal sheep at 0.75 g, the equivalent of 30 weeks. methods: fetal sheep received amniotic, jugular, carotid and femoral artery and vein catheters at 104 days g (dg). experiments started at 110 dg in 8 dex (2 mg) and 8 saline treated controls (ctr), each receiving 4 injections 12 h apart. fetal blood pressure was recorded continuously (windaq pro+). microspheres (1.86x10 6 total; sterispheres, biopal) were injected at 11:00am into fetal jugular and femoral veins before maternal i.m. injection (t0) and 2 h after the 3rd injection (t3) in both groups. tissues were collected 12 hours after the 4th injection for assessment of bf (biopal). results: fetal bp increased above baseline after dex (6.8+1.5 mmhg; p=0.007) with no change in ctr (-0.1+0.4 mmhg). regional bf distribution is presented as a percent of the total counts per 100 g tissue (table 1) and after normalization within each animal to placentomal flow, shown previously to be unchanged by dex, in table 2 . no significant differences in flow were found. discussion: although dex altered bp, regional distribution of bf among key organs was unaffected. whether gc does not affect regional bf, or the rise in bp is the result of a non-specific, system-wide vasoconstriction, or the impact of gc on bf is similar in all organs, will be subject of further investigation that will include determination of regional vascular resistance and absolute flow. stimulator while crf-r2 is an inhibitor of gi motility at times of stress. we recently hypothesized that stress-induced in utero meconium passage in fetuses is analogous to stress-induced defecation in adult rats and likely mediated by crf pathway. in support, we demonstrated hypoxia-induced meconium passage in conjunction with marked increases in fetal plasma crf levels. as the mouse is a common experimental model, with known genome, we sought to determine the presence of crf-r1 and crf-r2 receptors in mouse fetuses. methods: time-dated cd-1 pregnant mouse were anaesthetized and fetuses (n=10) were collected at e17 (term = e19). whole gi tracts were dissected, fixed in bouin's solution, paraffin embedded and sectioned at five micron thickness. sections were immunostained with rabbit polyclonal antibodies to crf-r1 and crf-r2 by abc technique with vector abc reagents. immunoreactivity was identified as brown staining using 3',3 diaminobenzamidine as chromagen. slides were counter stained with mayer's hematoxylin and examined under the microscope. immunoreactive signals in the smooth muscle layers of gi tracts were quantified by image analysis using image pro 4.01 plus software. results: immunostaining for both crf-r1 and crf-r2 was seen in the smooth muscle layers of all lumens examined (7 lumens per section). the immunostaining intensity expressed as intensity over density (iod) in arbitrary units for crf-r1 (maximal iod: 0.369±0.024 au and minimal iod: 0.177±0.13au) and for crf-r2 (maximal iod: 0.318±0.015 au and minimal iod: 0.240±0.020 au) greatly varied between lumens. the mouse fetal gi tract expressed both crf-r1 and crf-r2 receptors, suggesting that the mouse is an appropriate model for fetal-stress induced neurovisceral motor responses. the non-uniform distribution of crf receptors in the fetal gi tract suggests the existence of regional differences in the expression patterns of crf-receptors of both types. we speculate that mouse devoid of crf and crf-r1 or r2 receptors will be useful to confirm the participation of crf pathway in stress-induced in utero meconium passage. antenatal exposure to glucocorticoids (gc) is associated with a reduction in nephron number and hypertension in adult life. one of the mechanisms for the development of hypertension is thought to be the long term effect of single nephron hyperfiltration. however, in most studies there is only a 20-30% reduction in nephron number with no change in gfr. thus, although a reduction in nephron number may be a contributing factor it is unlikely the only variable. the aim of the present study was to evaluate nephron mass, renal function and blood pressure in a cohort of adult sheep exposed antenatally to betamethasone. methods: pregnant sheep were treated with two im doses of betamethasone (bm, 0.17 mg/kg) or vehicle (ctr) 24-hs apart at 80 days gestational age and allowed to deliver at term. at 1.5 yr of age in female (f) and male (m) offspring, glomerular filtration rate (gfr) was measured as inulin clearance and effective renal plasma flow (erpf) as pah clearance. blood pressure was measured though an indwelling catheter in the femoral artery over a 48-hour period. nephron number was measured by the acid maceration technique. data mean±sem were analyzed by anova and/or two sample t test. results: antenatal bm was associated with an elevation in arterial blood pressure and a reduction in nephron number in both f and m offspring. in contrast, a reduction in gfr and erpf was present only in males. plasma and urinary electrolytes as well as urinary protein excretion were not different from ctr. conclusion: our data show that prenatal exposure to a single course of gc at 0.55 gestation has long-term effects on blood pressure regulation. interestingly, while the decrease in nephron number was of similar magnitude in m and f, evidence of alterations in renal function was present only in males. these suggest that the decrease in renal function observed in males is not solely a consequence of the decrease in nephron number. furthermore, it is possible that different mechanisms are responsible for the elevation in arterial blood pressure observed in m and f. hl 68728; hd p01 hd04784. clr l>t-x to evaluate the effects on blood pressure, urine output, sodium excretion and gfr of the intrarenal infusion of angiotensin 1-7 in the male sheep with or without prenatal exposure to b. we studied male sheep which had received either b (n=4) or vehicle (n=3) at 80-81 days gestation and were born at term. catheters were placed in the femoral artery, femoral vein, left renal artery and the bladder. the right kidney was removed. after 5 days recovery the sheep received an infusion of ang 1-7(1ng/kg/min) with or without its antagonist, [d-ala 7 ]-ang-(1-7)(d-ala, 10ng/kg/min), into the renal artery for 24 hours. blood pressure, gfr, urine output and sodium excretion were measured before and after the infusion. two-way analysis of variance (anova) was used to test mean values between the groups. with or without d-ala, map and urine output didn't change significantly during ang 1-7 infusion. however, the infusion of ang 1-7 resulted in a decrease (change from baseline) (p=0.029) in na + excretion of 0.41±0.15 meq/kg/24h in the vehicle animals and 0.12±0.05 meq/kg/24h in the b animals. there was no significant change in gfr during ang 1-7 infusion. gfr was lower in the b group during the combined ang 1-7 and d-ala infusion (91.5±3.5ml/min vs 117.0±15 in vehicle group, p=0.03). intrarenal infusion of ang 1-7 at 1ng/kg/min is followed by a significant decrease in sodium excretion but no significant change in urine output and gfr. the decrease tended to be greater in the vehicle animals and was not markedly attenuated by the antagonist d-ala. this suggests the effect of ang 1-7 may be mediated by a receptor other than the mas receptor for ang 1-7 and may be of lesser magnitude in animals exposed to b before birth. and norepinephrine (0.1-0.6 g/kg/min) and to an l-name (2 mg/kg) bolus were studied. vascular reactivity was analyzed by curve fitting of either the blood pressure or hr responses to obtain the maximal response. data are expressed as mean±sem of change from baseline. statistical significance was established using t test. results: bm-exposed animals displayed a higher blood pressure response to at-ii (atii max 142±4 vs 101±5 % of baseline, p<0.05, figure left panel). no differences in blood pressure or heart rate responses to norepinephrine infusion were detected in bm when compared to control animals (figure 1 right panel) . maximal response to l-name was also elevated in bm-exposed animals but did not reach statistical significance with the current sample size. conclusion: we have shown that antenatal gc treatment significantly increases blood pressure in sheep. here we show an enhanced in vivo response to at-ii. this response may contribute to the increased blood pressure observed. the greater response to l-name most likely represent an increased no production as a compensatory mechanism to the higher blood pressure observed. hl 68728. women at risk of delivering preterm are frequently treated with glucocorticoids to facilitate fetal lung maturation. animal studies have revealed that prenatal exposure to glucocorticoids may alter aspects of hypothalamic-pituitary adrenal functioning in later life. in this study we examined the effects of clinically relevant prenatal betamethasone treatment on the responsiveness of pituitary cells (in terms of adrenocorticotropin, acth, secretion) isolated from adult sheep. time-dated pregnant sheep were injected with betamethasone (0.17 mg/kg) or vehicle on days 80 and 81 of gestation. thereafter pregnancy was allowed to continue unimpeded and offspring were born. pituitaries were isolated from adult sheep aged between 6 and 9 months and cells were dispersed and plated at a density 2×10 5 cells per well in 48 well plates. after 48h, cells were stimulated with normal medium, 10nm arginine vasopressin (avp), 100nm avp, 1nm corticotropin releasing factor (crf) or 10nm crf for 2h. medium was collected and analyzed for acth using a commercially available kit. acth secretion (given as % increase from untreated cells) was similar between the control and betamethasone groups following 10nm avp (21.9 ± 4.3 vs. 20.4 ± 6.6) and 1nm crf (10.0 ± 2.8 vs. 10.7 ± 5.0). at higher stimulatory concentrations of both avp and crf, acth secretion remained similar in control cells (24.1 ± 4.9 and 11.8 ± 5.9 respectively), but was significantly decreased in betamethasone cells (9.9 ± 7.5 and 2.4 ± 4.3 respectively). these findings suggest that prenatal exposure to clinically relevant doses of betamethasone can alter pituitary responsiveness to both avp and crf in adulthood. this research was supported by nih grants hd47584 and hd11210. lcc is supported by an rjr-leon golberg fellowship in pharmacology and toxicology. objective: newborn meconium (mec) passage normally occurs within the first 24-48 h after birth. in contrast, more than half a million infants born annually in the us pass mec in utero. neither the physiological mechanism(s) nor causes for in utero mec passage are well understood, though both fetal maturation and stress are associated. we recently reported in utero mec passage and marked increases in plasma crf levels in term fetal rats exposed to maternal hypoxic stress. we hypothesize that stress-induced in utero mec passage is mediated by the crf pathway in a manner analogous to stress-induced defecation in adult rats. in the present investigation we examined placental crf content prior to and following maternal hypoxia, to determine the source of increased plasma crf. methods: time-dated pregnant rats (n=6) were exposed to a paradigm of graded, stepwise hypoxia on day 22 (term=22) (pediatr. res. 61: 176-179, 2007) . control pregnant rats were exposed to 21% oxygen for a similar duration as experimental animals. at the end of the study, placentas were harvested, fixed in 4% paraformaldehyde and paraffin embedded. sections (n=6 per placenta) were subjected to immunohistochemical analyses with rat/human crf antibody (peninsula laboratory) by abc technique. immunoreactivities on placental sections were quantified using the image pro 4.01 software and intensity expressed as arbitrary unit (au). all values are mean± sem. results: in control maternal rats exposed to normoxia, positive staining for crf was seen in decidua (0.206±0.015 au) and in all three major placental cells types (giant trophoblast cells: 0.143±0.019 au, spongiotrophoblast cells: 0.133±0.003 au and labyrinth cells: 0.083±0.008 au.) in contrast, in animals exposed to hypoxia, there was a total absence of crf staining in the placental cells, with only positive crf staining seen only in the decidua (0.197±0.006 au). conclusion: the total absence of crf staining in both the basal and labyrinth zones in placentas of pregnant rats subjected to hypoxic stress suggests that placental cells rapidly release crf into the maternal and fetal circulation in response to hypoxic-stress. our findings support our hypothesis that the peripheral crf pathway mediates in utero mec passage. offspring. angela g massmann, jie zhang, jorge p figueroa. department of obstetrics and gynecology, wake forest university school of medicine, winston-salem, nc, usa. objective: in rats and sheep, exposure to glucocorticoids (gc) in the perinatal period induces hypertension in adult life. recent evidence suggests that endothelin b (etb) receptor plays an important role in the regulation of sodium balance and blood pressure. renal medulla is an important site of expression and action of etb receptor. furthermore, in kidney it is the medulla (km) where the highest concentrations of immunoreactive et-1 and etb receptor are found. the aim of this study was to measure eta and etb expression in adult sheep kidney exposed antenatally to gc. methods: pregnant sheep were treated with two im doses of betamethasone (bm, 0.17 mg/kg) or vehicle (v) 24 hours apart at 80 days of gestational age and allowed to deliver at term. at 1.5 yr of age, male and female offspring exposed to either vehicle or bm were euthanized and the kidneys harvested. kidney medulla (km) was obtained by sharp dissection. eta and etb protein and mrna expression were evaluated by western blot and rnaase protection assay. data are expressed as mean±sem and were analyzed by two way anova. results: a significant decrease in etb (f=5.166, p=0.030) but not eta protein was observed in km of bm exposed male and female adult sheep. at the mrna level, bm exposure also affected etb, but not, eta expression. however, bm treated animals had higher mrna levels than control sheep (f=7.05; p=0.01). conclusion: our data show that prenatal exposure to a single course of gc at 0.55 gestation has long-term effects. the activation of etb receptor by et-1 inhibits sodium transport function in the collecting duct and the selective gene deletion of the etb in medulla results in hypertension.therefore, our finding of a significant reduction in etb protein suggests that there may be an impairment in the kidney's ability to regulate sodium reabsorption. the functional relevance of the alterations in etb protein expression as well as the discrepancies in mrna regulation need to be established. hl 68728; hd p01 hd04784. in the placenta, 11 -hydroxysteroid dehydrogenase type 2 (11 hsd2) regulates the transfer of cortisol. maternal glucocorticoid (gc) administration has been shown to affect the ovine fetal growth trajectory and regulate 11 hsd2 expression in mid and late gestation. however, little is know about the effects of gc administration in early gestation. we hypothesized that maternally administered low-dose dexamethasone (dex) in early gestation would affect 11 hsd2 expression, which will subsequently alter fetal birth weight. pregnant ewes were randomized and received 4 intramuscular injections consisting of either saline (2ml saline/ewe) or dex (0.14 mg/kg) given 12 hours apart starting on 40 days of gestation (dg). the animals were sacrificed at 50, 100, 125, and 140dg and fetal weights were recorded and placental tissue was collected. qrt-pcr was used to measure 11 hsd2 placental mrna expression. statistical analysis was done by anova with student's t-test posthoc. overall, there was no significant effect of dex treatment on placental 11 hsd2 mrna expression across gestation. however at 50dg, there was a significant increase in 11 hsd2 expression after dex (p=0.026). there was an increase in placental 11 hsd2 mrna progressively from 50dg (mean = 0.45) to 140dg (mean =1.48), independent of treatment. overall, a positive correlation between 11 hsd2 and fetal weight (r 2 =0.43) was apparent at 50dg and at 140dg (p=0.065) in both control (p=0.01) and dex (p=0.13). significant positive correlations between placental 11 hsd2 and fetal weight (r 2 =0.45) were found in females (p=0.01) and a similar trend was found in male fetuses (p=0.07). we conclude that in the sheep placenta, 11 hsd2 expression increases throughout gestation and may respond, at least transiently in early gestation, to elevations in maternal gc. the positive correlation between 11 hsd2 and fetal weight is consistent with the thesis that the fetal growth trajectory may be influenced by maternal cortisol levels and that placental 11 hsd2 is an important mediator of these effects. antenatal gc exposure induces hypertension in the young adult rat (neuroendocrinol; 1996; 64:412) and increases femoral vascular resistance in the lamb (jphysiol; 2003; 547:61) . unpublished own examinations in preparation of this study have shown age dependency of the vascular reactivity. vascular contractility of the middle cerebral artery (mca) decreased in response to k + and noradrenaline (na) between 3mo and 2,5y of age (p<0.01). vascular relaxation to acetylcholine (ach) but not to pge2 decreased during the same time (p<0.01). vascular contractility of the renal artery (ra) increased in response to k + (p<0.05). aims: to examine if antenatal gc alter vascular reactivity in the aged individual when cardiovascular diseases are predominant. we studied the ra because its vascular tone is involved in modulation of arterial pressure control (amjphysiol;2002;283:r441) and the mca because depressive disorders that are associated with dysregulation of the hpa axis (jcem;1997;82:234) increase stroke mortality for unknown reasons (amjpsychiatry; 2003; 160:1823) . to be clinically relevant, we administered dexamethasone at the dose used to enhance lung maturation in babies threaten premature labor. methods: pregnant dams received saline (n=6) or 170μg/kg dexamethasone (n=6) i.p. at day e19/20 equivalent to 2x12mg dexamethasone administered to a 70kg pregnant woman. at 2.5 years of age, vascular response of the ra and mca to endothelium-dependent and independent mediators was measured using wire myography. vessels were inspected histologically for intact endothelium. results: basal vasoconstrictory and dilatory responses to all mediators were less pronounced in the mca than in the ra probably reflecting autoregulatory properties of cerebral vessels (p<0.05). after prenatal dexamethasone exposure, contractility but not sensitivity to k + and na was enhanced in the mca and even more pronounced in the ra (p<0.05). relaxation to ach and pge2 was similarly diminished in both vessels after precontraction with na (p<0.05). conclusions: prenatal dexamethasone exposure at the dose used clinically increases renal and cerebral vascular tone in the aged rat by endotheliumdependent and independent mechanisms. this effect may be a potential mechanism of fetal programming of cardiovascular diseases in later life. betamethasone (bmz) therapy during pregnancy to improve fetal lung maturity may result in long term side effects, including hypertension, during adulthood. the kidney is an important organ which regulates systemic blood pressure via the local renin angiotensin system (ras). the major enzymes of the intrarenal ras include angiotensin converting enzyme (ace), angiotensin converting enzyme 2 (ace2), and neprilysin. the hypothesis of this study is that prenatal bmz exposure will result in alterations of the enzymes regulating the intrarenal ras. specifically, sheep that are treated with bmz in utero will have higher amounts of ace activity in kidney cortex compared to control animals. pregnant sheep of known mating date were either treated with vehicle (n=7) or with two doses of bmz (n=6), 0.17mg/kg, 24 hours apart at 80 days of gestation. renal cortex from the male offspring was harvested at 19-24 months of age. cortical membranes were then solubilized and incubated at 37°c with either 125 i-ang i or 125 i-ang ii in the presence or absence of lisinopril to inhibit ace activity, mln4760 to inhibit ace2 activity, or sch39370 to inhibit neprilysin activity. the metabolic products were separated by reversephase high performance liquid chromatography (rp-hplc). the rate of enzyme activity was quantified by calculating the area under the curve for each product and converted to fmol of product per mg protein per minute of incubation. statistical analysis was performed using student's t-test. p<0.05 was considered significant. results: bmz treatment resulted in a significant increase in ace activity compared to control animals (p=0.0073). ace2 and neprilysin levels were not significantly different between treatment groups. when expressed as a ratio of ace/ace2 activity, bmz treated animals exhibited a 2.5 fold greater proportion of ace activity versus control animals (p=0.04). this study demonstrates that bmz exposure during fetal life results in a significant increase in renal ace activity during adult life. this increase in enzyme activity would be expected to be associated with increased levels of ang ii in the kidney and na + retention, possibly underlying the development of hypertension. hd47584, hd17644. we recently hypothesized that stress-induced in utero meconium passage in term fetuses utilizes peripheral and/or central crf pathways in a manner analogous to stress-induced defecation in adult rats. as participation of central crf pathway requires the crf circuitry system in brain-gut axis, we exploited immunohistochemical techniques to address whether term fetal colon is wired by crf-nerve fibers. methods: fetal distal colon segments (n=6) were dissected from term ovine fetuses (146-147 d gestation). in addition, lumbosacral spinal cord with spinal roots and dorsal root ganglia (drg) (n=3) were dissected from ovine fetuses, and vagal nerve trunk with nodose ganglia (n=3) were dissected from mouse fetuses. paraffin sections fixed either in bouin's solution or zamboni's were subjected to immunohistochemistry with rabbit polyclonal antibodies specific to ovine-crf (ocrf). immunoreactivity on the sections was identified by standard abc technique, immunostaining quantified by image-pro plus software and expressed (intensity over area) as arbitrary units (au). results: ocrf antibody elicited strong positive staining on the serosal surface (port of entry for extrinsic nerve fibers) in distal colonic segments objective: we hypothesize that stress-induced in utero meconium passage is mediated by the crf pathway. ucn-i, similar to crf, is a potent contractility inhibitor of preterm ovine fetal distal colon, which has abundant crf-r2 receptors. both ucn-1 and crf thus may inhibit colonic motility and prevent preterm in utero meconium passage via crf-r2 receptors. however, ucn-i is reported to stimulate contractility of adult rat colonic smooth muscle strips more strongly than crf. we sought to study the pattern of ucn-i innervation in distal colon of ovine fetuses to delineate whether ucn-1 functions as a facilitator or inhibitor of colonic propulsive motility. methods: bouin's solution fixed, paraffin sections of very preterm (vpt: 118-120 days gestation), preterm (pt: 130-132 days), near term (nt: 140-142) and term (t: 146-147days) ovine fetal distal colon rings were subjected to immunohistochemistry with polyclonal antibodies to human ucn-1 (1:500 sigma) by abc system. intensity of ucn-1 immunostaining in smooth muscle layers and myenteric neurons were quantified by image-pro plus software and expressed (intensity/area) in arbitrary units (au). differences over time were assessed with anova. in all groups very preterm was significantly greater than term (p<0.05). conclusion: ucn-1 immunoreactivity in all three muscle layers, as well as myenteric and submucosal neurons, is highest at very preterm as compared to more mature gestations. these results suggest that the reduction of ucn-1 inhibitory function may facilitate stress-induced meconium passage at term. to evaluate the effect on blood pressure (bp), urinary output (uop), sodium excretion (nae) and gfr of the intrarenal infusion of ang ii in the male sheep after prenatal exposure to b. methods: we studied male sheep which had received either b (n=3) or vehicle (n=3) at 80-81 days gestation. catheters were placed in the femoral artery and vein, left renal artery and in the bladder. a right side nephrectomy was performed. after 5 days of recovery, the sheep were infused with ang ii (1 ng/kg/min) with or without candesartan (10 ng/kg/day) or pd123319 (pd 500 g loading dose and 10 ng/kg/min infusion) into the renal artery over 24 hours. bp, gfr, uop and nae were measured before and after the infusion. two-way analysis of variance was used to test mean values between the groups. results: bp and uop did not change with ang ii infusion with or without candesartan. the infusion of pd did not affect uop but did increase bp in the b sheep (p=0.03). ang ii decreased the nae in both groups(p=0.0007). candesartan increased nae among controls(controls 0.16 and b 0.56 meq/kg/h, p=0.048). pd infusion decreased nae among controls but this was not significant. baseline gfr was higher among vehicle compared to b animals (p=0.0172). during ang ii infusion there was a decrease in gfr among control compared to b sheep (-71.17 vs. -18.46 ml/min, p=0.0081). this difference was not seen during candesartan or pd infusion. conclusion: ang ii infusion led to a decrease in nae in both groups and gfr among controls but not b animals. infusion of ang ii with candesartan increased nae while pd decreased nae among controls but not b animals. this suggests that prenatal steroid exposure can alter at2 receptor mediated response in renal function by decreasing the effect of ang ii on renal sodium excretion. the differences in gfr suggest that this may be due to an effect on tubular sodium reabsorption rather than glomerular perfusion. antalarmin antagonism of acth-induced cortisol secretion by ovine adrenal cells probably not at acth receptor. nancy k valego, james c rose. center of research for ob/gyn, wake forest u. school of medicine, winston-salem, nc, usa. in addition to regulating the hpa axis, crh and its type 1 receptor (crhr-1) occur peripherally and in the female reproductive system. late in human pregnancy, a surge in placental crh probably stimulates adrenal secretion of cortisol and dheas requisite to parturition. we previously reported that, in dispersed fetal or adult ovine adrenal cortical cells, 24 hour incubation with the specific crh-r1 antagonist, antalarmin (ant), significantly reduced both cyclicamp and cortisol responses to acth, suggesting an interaction with acth-r (like crh-r1, a g-protein-coupled membrane receptor). however, forskolin (fsk; direct stimulant of adenylyl cyclase)-stimulated cortisol secretion was also attenuated by 24 hour co-incubation with ant. our objective was to clarify the effect of ant on binding of acth to its receptor after a short (2.5 hours) treatment. method: acth binding: the adrenals from adult sheep were obtained at necropsy and the cortex cells dispersed and plated @ 200000 cells/well. after 48 hours in culture, wells were rinsed 2x and refilled with serum-free dmem/ f12 containing vehicle (dmso) with or without ant. after 2.5 hours, wells were washed very gently and the binding assay (adapted from rainey et al; j biol chem,264:21474,1989) completed. triplicate vehicle or ant wells were treated with i 125 -tyrosine 23 human acth (1-39) with or without 10 -6 m acth (for non-specific binding). after 1 hour, wells were chilled, washed, and the cells lysed and counted on a gamma counter. fsk treatment: cells were treated as above except that fsk (10 -6 m) was added to the wells. after 2.5 hours, medium was removed and stored @ -80ºc for camp eia and cortisol ria. results (mean±se) of 2.5 hour incubation on acth binding and fskinduced secretion. vehicle antalarmin acth binding (net cpm) n=4 3024±415 2966±463 cortisol (ng/ml/2.5hr) n=7 144±20 126±19* (p=.012) camp (pmol/ml/2.5hr) n=12 9.1±2.0 4.8±1.1* (p=.003) * indicates significant difference from vehicle alone. conclusion: the crh-r1 antagonist, antalarmin, attenuates acthstimulated cortisol secretion but not by preventing acth receptor binding. however, its effect is early in the secretory process and is associated with a reduced camp response. objective glucocorticoids are often administered to pregnant women to prevent neonatal respiratory distress syndrome. prenatal steroid treatment increases blood pressure in adult sheep. exposure to excess corticosteroids before birth is hypothesized to be a key mechanism underlying the fetal origins of adult disease hypothesis and effects on the renin-angiotension system (ras) may modulate the steroid-induced increases in blood pressure. we therefore sought to determine if renin processing and secretion were altered in adult female sheep exposed antenatally to betamethasone ( ) and to compare them with data from males studied previously. methods pregnant sheep were randomized to receive 2 doses of 0.17 mg/kg of or vehicle, at 80 and 81 days of gestation; the offspring were studied at 6 and 18 months of age. in 17 female offspring, active renin concentration (arc) and total renin concentration in plasma were measured by ria of angiotensin i generated by incubation with excess substrate. prorenin concentration (prc) is the difference between total and active renin. nine or control exposed female animals born at term (142-148 days of gestation) were brought from the farm at 12-32 months of age, and had vascular and bladder catheters placed. five days after surgery, a sodium load of hypertonic nacl (0.0275 meq/kg/min at 0.55 ml/min) was given for 60 min. blood samples were obtained. data are expressed as mean sem and were analyzed by t test. the prc was significantly higher in the females than in the males (17.74± 1.15 vs 8.90± 1.46 p<0.0001) but there was no effect of prenatal steroid treatment. arc was similar in both genders. however, arc was a significantly greater percent of the total plasma renin concentration in the males (33.9± 12.7 vs 13.88± 3.06 p<0.0001) during the na infusion experiment, the exposed females had lower arc than did the control females (0.40± 0.08 vs 1.28± 0.20 p<0.05). conclusion the data suggest that prenatal exposure to didn't alter the processing and secretion of renin in adult female sheep. prenatal steroid treatment does not appear to alter the effect of gender on plasma renin levels in adult sheep.it seems unlikely that the elevated blood pressure seen in adult ewes after prenatal exposure is the result of increased secretion or processing of renin. supported by hd 47584 and hd17644. background: mrap is a recently discovered protein with alpha and beta isoforms, a common amino terminal region and divergent c-terminal sequences generated by alternative splicing. in human and mouse mrap plays an essential role in the generation of a functional, g-protein coupled acth receptor (melanocortin-2 receptor; mc2r) but has not been described for ruminants. we previously reported that lth fetal sheep exhibited elevated basal acth 1-39 yet decreased expression of key steroidogenic enzymes and the mc2r in the adrenal cortex while basal cortisol levels were not different from control. we hypothesized that mrap could play a key role in mediating the effect of lth as well as developmental regulation of fetal adrenal cortisol synthesis in fetal sheep. the goals of the present study were to determine the ontogenic pattern of mrap expression in the sheep fetus and to determine if lth alters mrap expression. methods: we searched the bovine genomic sequence database (www.ncbi.nlm. nih.gov/genome/guide/cow/) and found a sequence with >90% homology to the human mrap n-terminal 68 residues, with partial homology to the beta isoform in the carboxyl region ( 65%). using primers based on the bovine sequence, we confirmed the presence of mrap in the ovine fetal adrenal cortex. adrenal cortical tissue was collected from sheep fetuses from 105-120 (n=5) and near term (142-145; n=5) days of gestation (dg) as well as from lth (n=6) fetuses (exposed to high altitude hypoxia from 40 to 139-141 dg) and age-matched normoxic controls (n=6). cyclophilin was used as a housekeeping mrna. data are expressed in fg mrna/50 ng total rna. results: the expression of mrap, based on quantitative rt-pcr, was low between 105-120 dg (11.72 ± 4.2) and increased (p<0.05) approx. 5-fold near term (140-145 dg; 50.6 ± 7.3). levels of mrna for mrap were highly correlated to cyp17 mrna in individual samples. mrap expression in control (25.1 ± 2.5) and lth (26.5 ± 4.2) did not differ between groups. gender differences in hypertension are well described and there is growing evidence that the regulation of the renin angiotensin system (ras) is influenced by sex hormones. the major enzymes of the ras include angiotensin converting enzyme (ace), angiotensin converting enzyme 2 (ace2), and neprilysin. the peptide products of these enzymes have opposing actions. angiotensin ii (ang ii), the product of ace, is a potent vasoconstrictor. on the other hand, the peptide products of ace2 and neprilysin, ang (1-7) and ang (1-4), exhibit vasodilatory properties. thus, modifications in the relative proportions of these enzymes and their peptide products can result in alterations of systemic blood pressure. the purpose of this study was to describe the gender differences in angiotensin converting enzyme (ace), angiotensin converting enzyme 2 (ace2), and neprilysin (nep) enzyme activities in adult sheep kidney cortex. renal cortex from male (n=5) and female (n=8) sheep were harvested at 15-31 months of age. the tissue membranes were solubilized and incubated at 37°c with either 125 i-ang i or 125 i-ang ii in the presence or absence of lisinopril to inhibit ace activity, mln4760 to inhibit ace2 activity, or sch39370 to inhibit neprilysin activity. the metabolic products were then separated by reverse-phase high performance liquid chromatography (rp-hplc). the rate of enzyme activity was then quantified by calculating the area under the curve for each product and converted to fmol of product per mg protein per minute of incubation. statistical analysis was performed using student's t-test. p<0.05 was considered significant. results ace activity was over 4 times greater (1282 ± 103.5 vs. 303.3 ± 118.4 fmol/mg/min, p<0.0001), ace2 activity was 2.5 times greater (1826 ± 312.5 vs. 721.2 ± 86.4 fmol/mg/min, p=0.02) and nep activity was nearly 2 times greater (761.3 ± 73.09 vs. 401.5 ± 39.81 fmol/mg/min, p=0.0081) in female kidney cortex. in adult sheep, key enzymes of the intrarenal ras have signficantly greater activity in female kidney cortex. these findings suggest that there are fundamental, gender specific, differences in the regulation of the enzymes of the intrarenal ras. the physiologic significance of these findings remain to be elucidated. hd47587, hd17644. in rats and sheep exposure to glucocorticoids (gc) in the perinatal period is associated with a reduction in nephron number and hypertension in adult life. furthermore, antenatal exposure to gc alters glucose tolerance in animals and in people. the aim of the present study was to determine 1) if insulin resistance is a contributing factor for the development of hypertension in adult sheep exposed antenatally to gc and 2) if diet-induced obesity has a more pronounced effect in sheep exposed antenatally to gc. methods: pregnant sheep were treated with two im doses of betamethasone (bm, 0.17 mg/kg) or vehicle (ctr) 24-hours apart at 80 days gestational age and allowed to deliver at term. at 9 mo of age, female sheep were randomly allocated to be fed at either 100% of recommended nutritional allowance or ad libitum for three months. sheep were chronically instrumented under general anesthesia to place intravascular catheters. insulin sensitivity was evaluated both by iv glucose tolerance test (ivgtt) and euglycemic clamp (hec)techniques. for the ivgtt a 0.25 g/kg glucose bolus was used and for the hec 4mu/kg human insulin was used. data mean±sem were analyzed by anova and/or two sample t test. results: ad lib fed sheep gain > 50% of the original weight. antenatal bm was associated with an elevation in basal and ivgtt plasma insulin values. as shown on the figure, diet-induced obesity significantly increased insulin plasma levels during ivgtt in bm-exposed adult female sheep (f=18.982;p<0.001). insulin sensitivity derived from hec was significantly decreased by obesity in bm-exposed adult female sheep. conclusion: our data show that prenatal exposure to a single course of gc at 0.55 gestation has long-term effects on glucose metabolism regulation. bm-exposed sheep exhibit alterations in glucose tolerance, hyperinsulinemia and insulin resistance. these abnormalities are exagertated by diet-induced obesity. hl 68728. syngergistic induction of 11 -hydroxysteroid deyhdrogenase type 1 expression by cortisol and interleukin-1 in human fetal lung fibroblasts. z yang, 1 p zhu, 1 cm guo, 1 l myatt, 2 k sun. 2 1 school of life sciences, fudan university, shanghai, china; 2 obstetrics and gynecology, university of cincinnati, cincinnati, oh, usa. objectives: glucocorticoids acting via glucocorticoid receptor (gr), serve as crucial hormones in fetal lung maturation. glucocorticoids and proinflammatory cytokines to induce 11b-hydroxysteroid dehydrogenase type 1 (11b-hsd1) which converts inactive cortisone to active cortisol, but their effect on 11b-hsd1 expression has not been addressed in human fetal lung. we examined the interactions and mechanism of cortisol and interleukin-1b (il-1b) effect on 11b-hsd1 in human fetal lung fibroblasts (hfl-1 cells). methods: the expression of 11b-hsd1 in hfl-1 was examined with immunocytochemistry and pcr. 11b-hsd1, prostaglandin h synthase-2 (pghs-2) and cytosolic phospholipase a2a (cpla2) mrna levels in cultured human fetal lung fibroblasts treated with cortisol and il-1b were measured with real time pcr. the roles of gr and c/ebps in the effect of cortisol and il-1b were studied using a gr antagonist (ru486) and transfection of plasmid carrying c/ebp-specific dominant-negative gene (cmv500-a/cebp). results: hfl-1 cells expressed a high level of 11b-hsd1. both cortisol (10 -8 -10 -6 m) and il-1b (0.1-10 ng/ml) induced 11b-hsd1 mrna expression in a concentration-dependent manner, an effect blocked by the mrna transcription inhibitor 5,6-dichlorobenzimidazole riboside (75 μm). ru486 (10 -6 m) blocked the induction of 11b-hsd1 by cortisol. induction of 11b-hsd1 mrna expression by cortisol (10 -6 m) was synergistically increased by co-treatment with il-1b (0.1-10 ng/ml) in a concentration-dependent manner. in contrast, the induction of cpla2 and pghs-2 expression by il-1b was inhibited by cortisol, suggesting a different mechanism of interaction. transfection of the cells with c/ebp-specific dominant-negative plasmid attenuated induction of 11b-hsd1 mrna expression by either cortisol or il-1b. these data suggest induction of 11b-hsd1 expression by cortisol is a gr dependent process involving c/ebps, which also mediate induction of 11b-hsd1 expression by il-1b. conclusions: cortisol and il-1b synergistically induce 11b-hsd1 expression in human fetal lung fibroblasts. this would lead to greater local cortisol production, perhaps providing either a self-attenuating mechanism for control of inflammation or a mechanism for enhancing fetal lung maturation when the fetus is exposed to cytokines e.g. with infection-induced preterm labor. do alterations in placental 11 -hydroxysteroid dehydrogenase (11 hsd) activities explain differences in fetal hypothalamic pituitary adrenal function following periconceptional undernutrition or twinning in sheep? kl connor, 1 pl van zijl, 2 cw rumball, 2,3 al jaquiery, 2,3 je harding, 2,3 mh oliver, 2,3 fh bloomfield, 2,3 jrg challis. medicine, university of toronto. periconceptional undernutrition (pcun) leads to activation of fetal hypothalamic pituitary adrenal (hpa) function, whereas twinning results in delayed fetal hpa activation. we hypothesized that these differences in fetal hpa activity were the result of altered patterns of expression of placental of 11 hsd isozymes and hence of the maternal glucocorticoid (gc) effect on the fetus. we developed a mass spectrometric assay for the measurement of 11 hsd-1 and -2 activities and validated this method against a widely used thin layer chromatography method. sheep were randomly assigned to ad libitum (n control) concentrates throughout gestation or were undernourished (un) from 60 days before until 30 days after mating to reduce maternal body weight by 15%, with ad libitum feeding thereafter. placentomes were collected on days 50, 85, 120, and 131 of gestation (term, 147d) and 11 hsd-1 and -2 activities were determined by measuring the rate of interconversion between cortisone to cortisol. with un, there was a trend towards lower 11 hsd-2 activity at d50 (p=0.1), a significant reduction at d85 (p<0.05), but no difference at d120 or d131. 11 hsd-1 activity was not different between n and un animals at any time. there was no effect of twinning on 11 hsd-1 or -2 at d50. however, with twins both 11 hsd-1 (p=0.01) and -2 (p<0.05) activities increased at d85. 11 hsd-1 activity was reduced in twins (p=0.06) at d120 but was higher than any singleton pregnancies at d131 (p=0.06). 11 hsd-2 was not different between singletons and twins at either d120 or d131. overall, 11 hsd-2 was lower in placentae of male compared to female fetuses in late gestation; 11 hsd-1 was higher in male than female fetuses. there was no interaction with un in either sex. we conclude that pcun and twinning result in alterations of placental 11 hsd activities in sheep. modifications in these enzymes during critical periods of fetal development may affect transplacental transfer or placental generation of gcs reaching the fetus potentially influencing the timing of activation of the fetal hpa axis, fetal maturation and later life development. methods: pregnant sprague-dawley rats had 50% mfr from day 10 of gestation until delivery. control animals had ad libitum food. offspring were sacrificed on day 1 of life (p1) and at 6 months (n= 6-8 per group). adrenals were dissected and snap frozen in liquid nitrogen for later extraction of rna. real time rt-pcr using specific rat primers was used to quantify mrna levels (18s as control). we evaluated the expression of 11-beta hydroxylase (cyp11b1), aldosterone synthase (cyp11b2), acth receptor (mcr2), p450 side chain cleavage enzyme (cyp11a1), star protein, 11 -hydroxysteroid dehydrogenase type 1 (hsd1) and type 2 (hsd2), glucocorticoid receptor (gr), and mineralocorticoid receptor (nr3c2). fold changes in mrna expression in controls and mfr offspring was compared by student's t-test. results: there was a marked downregulation in expression of cyp11b1 (p=.01), cyp11b2 (p=.04), hsd2 (p=.03), p450 (p=.05), acth receptor (p=.05), star (p=.01) and nr3c2 (p=.02) mrna in p1 mfr offspring, with no changes in hsd1 and gr. gender specific differences were found in the adult mfr offspring. in the male mfr offspring the expression of hsd1 and gr were significantly upregulated with a trend towards an increase in acth receptor (p=.07) whereas in the female mfr the expression of acth receptor (p=.02) was increased and nr3c2 (p=.03) and cyp11b2 were decreased (p=.04). in combined data for adult male and female offspring the expression of acth receptor was significantly (p=.02) increased. conclusion: these results indicate that mfr has a suppressive effect on steroidogenic enzymes of the newborn offspring regardless of gender. this may be an adaptive mechanism in the fetus/newborn to offset the high circulating maternal glucocorticoids in response to undernutrition. in adult male mfr offspring, increased hsd1 indicates an increase in gc synthesis whereas in the females there were no changes in gc synthesizing enzymes with a suppression of mc synthesizing pathway. the common finding of an increased acth receptor expression in both genders would suggest an increased sensitivity of adult mfr offspring adrenals to the effects of stress. objective: during gestation the fetal adrenal undergoes phases of active growth (40-90d), quiescence (100-120d) followed by reactivation (>135d) before birth. insulin like growth factors play an important role in stimulating adrenal growth throughout late gestation. interestingly the prepartum activation of the adrenal is delayed in the female compared with the male fetus, but the mechanisms underlying this delay are unknown. hypothesis: we hypothesize that there are gender specific differences in the gestational profile of adrenal igf2 mrna expression between male and female fetuses. methods: a total of 37 twin fetuses were used in this study. post mortem was performed at either 110-112d, 125-134d or 137-145d gestation. adrenal mrna expression of igf1, igf2, igf1r, igf2r and cyp17 was determined by qrt-pcr. results: fetal weight was not different between males and females, and increased (p<0.0001) with increasing gestational age. the relative adrenal weight was lower however after 125d when compared to the earlier gestation age group (p<0.0001). adrenal igf1 mrna expression was lower (p<0.0001) at 137-145d when compared to the earlier gestation age groups. interestingly, igf1r expression was highest at 125-134d (p<0.0001). there was an interaction between the effects of age and gender on adrenal igf2 and igf2r mrna expression such that the expression was higher in males compared to females at 125-134d (p<0.01), but was not different to either the earlier or later gestational ages. there was no effect of either gender or gestational age on adrenal cyp17 mrna expression. conclusions: it has been speculated that a delay in prepartum activation of the adrenal in female fetuses may be due to gender specific differences in the intra-adrenal bioavailability of igf2. in this study we have demonstrated there is an increase in both igf2 and igf2r mrna expression in males compared to female fetuses. this may be evidence that adrenal igf2 expression plays a role in the earlier activation of the adrenal gland in male fetuses. we have previously shown that in response to a secondary stressor, in vivo cortisol secretion is elevated in long term hypoxic (lth) ovine fetus despite lower acth receptor mrna expression, and no differences in plasma adrenocorticotropic hormone (acth) levels when compared to normoxic controls. the present study was designed to determine the potential mechanism(s) of this enhanced cortisol secretion. specifically we tested the hypothesis that post receptor signaling events including camp production and expression of steroidogenic acute regulatory protein (star) are enhanced following lth. methods: for the lth group, pregnant sheep were maintained at high altitude (3,820 m) from day 30 to near term. on days 138-141 (term = 146 days), fetal adrenal glands were collected from lth (n=6) and age-matched, normoxic signal transduction pathways that control embryogenesis, cell differentiation, cell proliferation and cell death. the roles of extracellular signal-regulated kinase1/2 (erk1/2) and p38 map kinase in the differentiation and invasion of human trophoblasts have been studied. however, the in vivo expression and activation of erk1/2 and p38 at the placental bed has not been elucidated. the study group consisted of placental bed biopsy tissues obtained from the pregnancies without preeclampsia (n=24) and with preeclampsia (n=8) between 31 and 40 weeks of gestation. we evaluated the expressions and phosphorylations of erk1/2 and p38 map kinase in the invasive trophoblasts in the placental bed tissues using immunohistochemistry. results: p38 and phospho-p38 map kinase were not detected in invasive trophoblasts in cases or controls. erk1/2 and phospho-erk1/2 were positive in invasive trophoblasts albeit with variable staining. phosphorylation of erk1/2 was significantly less frequent in invasive trophoblasts in placental bed biopsies from women with preeclampsia compared with normotensive controls. conclusion: these findings suggest that preeclampsia is associated with decreased activation of erk1/2 in invasive trophoblasts in vivo. objective: placentae from preeclamptic pregnancies are characterized by an excess of immature hyperproliferative trophoblast cells, however the molecular mechanisms regulating cell cycle progression in this pathology are unclear. our aim was to examine the expression of g1 phase cell cycle regulators in normal and preeclamptic placentae and to establish whether the hyperproliferative state of trophoblast cells in preeclampsia may result from a developmental delay. methods: human placental samples were collected from normal pregnancies throughout gestation (n=60) and from severe early onset preeclamptic (n=20), and age-matched control placentae (n=15). protein expression of cyclin e1, cyclin d1, cyclin d3 and cell cycle inhibitors, p15, p16, p21, and p27 was assessed by western blot analysis. spatial and temporal localization of cyclins e1, d1, d3, p21 and p27 was determined by fluorescence immunohistochemistry. expression of cyclin e1 and cyclin d1 mrna was evaluated by qpcr analysis. results: immunohistochemical analysis showed cyclin e1 and cyclin d3 to be localized to cytotrophoblast (ct) cells of the chorionic villi; additionally cyclin e1 was also expressed in the extravillous trophoblast cells of the anchoring columns. in contrast, cyclin d1 expression was predominantly restricted to the villous stroma. cell cycle inhibitor p27 was expressed in both ct and syncytiotrophoblast (st) cells whereas p21 was restricted to the st. during normal placentation, levels of both cyclin e1 and cyclin d3 were high in the first trimester and decreased with advancing gestation. the expression of cyclin d1, p27 and p16 showed an inverse correlation to cyclins e1 and d3 whereby their expression increased towards term. levels of p21 and p15 remained constant throughout pregnancy. preeclamptic placentae showed a significant increase in both cyclin e1 and p27 and a decrease cyclin d1 and p15 expression, as compared to age-matched control tissues. interestingly, preeclampsia was associated with an increased number of cyclin e1 positive progenitor ct cells in the floating villi and an increased expression of p27 in the endothelial cells lining the villous vessels. conclusion: preeclampsia displays an altered expression of g1 phase cell cycle regulatory molecules portraying an expression profile that closely resembles that of first trimester placentae. (supported by cihr, owh/igh). we have also demonstrated elevated, hif-1-mediated placental and circulating sflt-1 at high altitude. we sought to correlate circulating levels of plgf, free vegf and sflt-1 with maternal and fetal oxygen tensions. we hypothesized that circulating sflt-1 and free vegf would be increased at 3600 m, and that plgf, known to be up-regulated by higher oxygen tension, would be decreased. methods: we collected both serum and plasma samples, the latter treated with inhibitors of platelet activation. maternal and umbilical samples were obtained from 77 and 65 healthy mother-infant pairs living at 400 m and 3600 m respectively. plgf, free vegf and sflt-1 were measured in both serum and plasma using commercially available elisa kits (r d). data were log-transformed and analyzed by unpaired student's t test or anova as appropriate. results: plgf did not differ between altitudes. free vegf (7±2 pg/ml vs. 12±2 pg/ml, p<.05) and sflt-1 (25.7±2.4 vs. 31.4 ±2.2 ng/ml) were increased at 3600 m. however concentrations of all the angiogenic growth factors were reduced when platelet activation was prevented (-35±3% plgf; -85±5% free vegf; -24±2% sflt-1, p<.001). cord blood free vegf was 100-fold greater than in the mothers, but inhibition of peripheral cell activation abolished detectable levels in 95% of the babies. similar results were obtained for sflt-1. thus, while free and total maternal circulating vegf and sflt-1 are elevated at high altitude, these increases are due to activation of peripheral blood cells. moreover, free vegf does not exist in detectable amounts in human pregnancy. there was no relationship between variation in the angiogenic growth factors and maternal or fetal oxygen tensions. the objective of this study is to identify candidate genes responsible for the variance between severe preeclampsia (spe) and hellp syndrome (hs). placental biopsies from spe (n=6) and hs (n=2) were collected. diagnosis of spe was confirmed by blood pressure and protein criteria. hs was diagnosed in preeclamptic patients who developed characteristic laboratory abnormalities. placental tissues were embedded in oct for sectioning, h e staining and rna isolation (invitrogen, carlsbad). gene expression data was obtained by hybridizing fluorescently labeled reverse transcription products to spotted cdna microarrays. the microarrays were produced by the laboratory of microarray technology at the van andel institute (grand rapids, mi) using a custom microarrayer. microarrays were scanned using an agilent g2505b scanner. images were analyzed using genepix 5.0 (axon). limmagui was used to generate lists of discriminating genes for these data, while david provided functional annotations. there were 310 differentially expressed genes between spe and hs (p<.05). among these candidate genes, 233 were up-regulated and 77 were downregulated. the most up-regulated genes are 5(3)-deoxyribonucleotidase (dnt-2), superoxide dismutase 3, hydroxy-delta-5-steroid dehydrogenase, caspase 9, and general transcription factor iih. further analysis of functional groups revealed the most enriched gene categories are related to cellular energy (mitochondria), cell cycle regulation, and protein metabolism. the underlying role of the placenta in the development of hs is currently unknown. this study shows that there is a relatively mdest number of genes that are differentially expressed between hs versus spe placentals. thes data provide the molecular context for placental changes seen in this variant of preeclampsia and provides an opportunity to study the role of the effected genes in disease variance. (14), severe preeclampsia (10), hellp syndrome (4) and eclampsia (4) and control group (29 patients) uncomplicated pregnancies. total rna was isolated and reversely transcribed to c-dna. the mrna level of tert was detected with a probe-specific real-time quantitative pcr assay using ß-actin as the reference gene. crossing point (cp) values were obtained during the pcr amplification and the relative expression level of htert equals to 2 (cp htert -cp ß-actin) . statistical analysis was performed using the student's t test. immunohistochemistry (ihc) staining was employed to localize htert protein on placenta tissue sections using abc method, incubation with rabbit anti-htert antibody followed by application of a goat anti-rabbit antibody with results evaluation using microscope. objective tgf s are involved in the regulation of trophoblast differentiation and invasion, and we have previously reported that tgf-3 is over expressed in pre-eclamptic placentae. tgf s signal via a receptor complex composed of type ii (t r-ii) and type i (t r-i) receptor. to date, seven type i receptors, designated as activin receptor-like kinase (alk1-7) have been identified. our aim was to investigate the expression pattern of alk1 and alk5 receptors, known to exert tgf signaling, in preeclamptic and iugr placentae. methods human placental tissue throughout gestation was used in order to determine the development profile of the receptors. in addition, placental tissue from preeclamptic and iugr pregnancies and from age matched controls was collected. all iugr pregnancies were characterized by absence of end diastolic velocity in the umbilical artery and had no evidence of preeclampsia. expression of alk1 and alk5 mrna was measured by real-time pcr analysis, and protein by western blot analysis using alk1 and alk5 antibodies. immunoblot analysis demonstrated a unique developmental profile whereby alk1 expression increased with advancing gestation. in preeclamptic placentae alk5 expression was significantly increased compared to preterm and term controls, whereas alk1 expression was significantly decreased. preeclamptic placentae also exhibited decreased phosphorylation of smad1, a tgf signaling molecule, which is activated by alk1 and increased phosphorylation of smad2, which is triggered by alk5. the expression of alk1 and alk5 in iugr placentae differed from that of preeclampsia as both alk1 and alk5 mrna levels were significantly increased in iugr compared to preterm and term controls. however, only alk5 expression was significantly increased in iugr placentae at the protein level, while no differences in alk1 protein levels were noted between iugr and controls. conclusions imbalance between alk1 and alk5 signaling pathways might play a role in the pathogenesis of preeclampsia and iugr. as tgf signaling via either alk1 or alk5 has been found to differentially regulate vasculogenesis, changes in these signaling pathways may contribute to the altered vasculogenesis found in these pregnancy-related disorders. (supported by cihr and owh/igh). (14), severe preeclampsia (10), hellp syndrome (4) and eclampsia (4) and 29 patients, the control group who had uncomplicated pregnancies. total protein was isolated from placental tissue. gadd45a and its downstream signal proteins--phospo-p38, phospho-mkk3/mkk6 were assessed by western blot. immunohistochemistry (ihc) staining was employed to localize the expression of gadd45a and sflt-1 proteins in placenta tissue sections using abc method. huvec cells were cultured to 80-90% confluence and were divided into 3 groups: control, stress induction (sorbitol, 0.3m, 4h), p38 inhibition (sb-203580, 1ug/ml, 5ug/ml and 10ug/ml, 1 hour) + sorbitol (0.3m, 4h). total protein was isolated from cells and the supernatant of huvec was collected. western blot was processed to detect the induction of gadd45a and phospho-p38. supernatant sflt-1 was measured with an eia kit and the results were read at 450nm wavelength. results: gadd45a protein was elevated in the preeclamptic placentas with its downstream proteins (mkk3 and p38) activation compared with control. overexpression of gadd45a and sflt-1 in preeclamptic placentas was observed with ihc staining. in huvec cells, gadd45a was induced by sorbitol, triggering the activation of the downstream p-38 pathway and the accumulation of sflt-1 in the supernatant. the up-regulation of sflt-1 secretion by inducing gadd45a was depleted when treated with p-38 inhibitor. conclusions: our study reveals that gadd45a and its down stream p-38 pathway were activated in preeclamptic placentas and this stress inducible signal pathway regulates the secretion of sflt-1, which is a key player in preeclampsia. it provides novel evidence that links placental stress to sflt-1 secretion via the gadd4. trophoblast adipose triglyceride lipase (atgl) expression is upregulated in preeclampsia. beth a plunkett, 1 jennifer a doll, 2 emily j su, 1 serdar e bulun, 1 mona cornwell, 2 susan e crawford. 2 1 obstetrics and gynecology, northwestern university, chicago, il, usa; 2 pathology, northwestern university, chicago, il, usa. objective: preeclampsia is characterized by placental endothelial cell dysfunction and elevated maternal triglyceridemia (tg). tg traverse the placenta in a process of uptake followed by lipolysis with subsequent release of fatty acids to the fetus. although three placental lipases (hormone sensitive lipase, endothelial lipase and lipoprotein lipase) have been identified, they do not account for all lipolytic activity. here, we introduce a new lipase, adipose triglyceride lipase (atgl), which is responsible for the hydrolysis of triglycerides to diglycerides in adipocytes and was recently identified as a receptor for endothelial cell modulator pigment epithelium-derived factor. the purpose of this study is to determine if expression of atgl, a potential modulator of both lipid metabolism and vasculature, is upregulated in preeclampsia. methods: immunohistochemical studies were performed on placental tissues from normal pregnancies (n=5) and those complicated by severe preeclampsia (n=5) with anti-atgl antibodies. the degree of positivity in the trophoblasts and endothelial cell was scored (1=none, 2=spotty, light, 3=consistent, dark). to determine if atgl is a product of placental endothelial cells (plec), microvascular cells were isolated from normal placental tissue. purity of the sample was confirmed using flowcytometry (>2/3 positivity for factor 8 antigen). atgl was detected immunohistochemically and via western blot using anti-atgl antibodies. results: mean atgl expression in preeclamptic trophoblast was significantly higher than normal placentas (3.0 + 0 standard deviation versus 2.3 + .39, p = 0.018). endothelial expression was not significantly different in preeclamptic (2.6 + .41) versus normal placentas (2.3 + .39, p>0.05). atgl stained intensely and demonstrated a beaded pattern in the endothelial cells, suggestive of a lipid droplet pattern. immunohistochemistry of plec and western blot analysis of cell lysates revealed strong immunopositivity for atgl, although at a smaller size than anticipated. conclusion: these findings demonstrate that a novel lipase, atgl, is produced by trophoblasts and is upregulated in severe preeclampsia. plec express high levels of atgl, suggesting that atgl could prove to be important in the vascular dysfunction and lipid abnormalities characteristic of preeclampsia. increased endothelial chymotrypsin-like protease (chymase) expression is responsible for endothelial activation in preeclampsia. yuping wang, yang gu, yanping zhang, david f lewis. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: endothelial (ec) activation is an important component of inflammatory phenotypic changes in preeclampsia (pe). our previous study showed enhanced chymotrypsin-like protease (clp)/chymase expression in the maternal vessel endothelium in women with pe. in this study, specific effect of placental-derived clp on ec activation was examined. methods: human uterine microvascular endothelial cells (utmvecs) were used. placental conditioned medium (cm) was prepared by culturing villous explants from normal and pe placentas. confluent utmvecs were treated with placental cm with or without depletion of chymotrypsin. ec adhesion molecule expressions for icam, vcam, p-selectin and e-selectin were determined by a colorimetric assay at od 450nm. depletion of chymotrypsin from cm was performed by immunoprecipitation. to further determine if activation of endogenous clp/chymase in ecs is responsible for up-regulation of p-selectin and e-selectin expression, chymase sirna was applied to ec culture before the cells were treated with normal or pe cm and then ec adhesion molecule expressions were examined. data was expressed as mean ± se and analyzed by anova. a p level < 0.05 was set for statistically different. results: 1) expressions of vcam, p-selectin and e-selectin, but not icam, were significantly increased in pe-cm treated utmvecs compared to those of normal-cm treated cells and untreated controls, p<0.01; 2) there was no difference for adhesion molecule expression in utmvecs between normal-cm treated with untreated controls; 3) utmvecs transfected with chymase sirna significantly reduced p-selectin and e-selectin expressions when exposed to pe-cm, p<0.05. conclusion: placental-derived clp/chymase is responsible for activating ecs and inducing ec adhesion molecule expression. activation of ec chymase may be directly related to the inflammatory phenotypic changes that occur in ecs in pe. (supported nih grants hd36822 and hl65997). corin is a transmembrane serine protease that is important in processing natriuretic peptides (nps) and maintaining normal blood pressure. genetically modified mice without corin function develop a syndrome during pregnancy similar to preeclampsia. corin is present in the pregnant uterus and a deficiency in the enzyme may lead to hypertension and preeclampsia. we tested the hypothesis that corin expression was increased in human myometrium from women with preeclampsia. methods: myometrium was obtained from 4 groups of women at the time of primary cesarean section: (i) preterm no labor with preeclampsia (n=3, ptspe, 28.9 weeks); (ii) preterm labor (n=3, ptl, 30.9 weeks); (iv) term no labor (n=3, tnl, 39.6 weeks); (v) term labor (n=3, tl, 39.7 weeks). microarray gene profiling was performed using affymetrix human genome u133 plus 2.0 arrays. women who were not in active labor at the time of delivery (tnl) served as the control group. conventional and real time pcr was performed to verify corin expression in the arrays was directionally accurate. corin protein expression was examined by western blot. results: compared to tnl control, corin levels decreased nearly 2-fold in myometrium from women in term labor (tl). there was a small increase in corin gene expression of preeclamptic women (ptspe) and little-to-no change in myometrium from women in preterm labor (ptl). these results were confirmed by pcr analysis. conclusion: blood volume surges during pregnancy, increasing the potential for hypertension and preeclampsia in the mother. the corin-np control system could contribute to this condition. however, there are no reports on corin message or protein levels in women with preeclampsia. our preliminary results suggest that preeclampsia is not a consequence of a corin deficiency (decreased corin preeclampsia). acknowlegement: supported by grants from the phs (r01 hl049041-12, cpw) and cdc grant (u7dp00187-03, cpw). the expression of gp130 at implantation site: implication for the pathogenesis of preeclampsia. objective: gp130 is a common shard signal transducing subunit of the il-6 cytokine family which is critical for implantation, and viewed as marker of endometrial blastocyst receptivity. preeclampsia (pe) is highly related to the restricted trophoblast invasion, which leads to impaired spiral artery remodeling. our hypothesis was that the poor placentation of pe is associated with the altered expression of gp130 at the implantation site. methods: human decidua from patients with pe and uncomplicated term deliveries (n = 9, respectively) were immunostained for gp130. the intensity and distribution of immunostaining on decidual cells and extravillous traphoblasts were evaluated with hscore. statistical analysis of the data was performed using student's t-test and kruskal-wallis one way anova on ranks followed by post hoc test. results: immunostaining of gp130was significantly higher in decidual cells of patients with pe compared with normal specimens, with hscore (median, [interquartile range]) value 180 [142.5-185.0] and 120 , respectively (p<0.05). in pe specimen, the hscore of decidual cells was also significantly higher than that of trophoblast (40 [30-50]; p<0.05). there were no difference in the comparison of hscore of trophoblasts between the pe and normal specimens, and in normal specimens between decidal cells and trophoblasts. conclusions: the increase of gp130 expression in the decidual cells of preeclamptic placenta may be implied to the pathogenesis of poor placentation in preeclampsia. we have previously demonstrated that decidual cells are the major source of renin at the human uteroplacental interface, but little is known regarding the human decidual renin expression in hypoxic conditions. therefore, the present study was undertaken to determine the effect of hypoxia on renin secretion by human decidual cells in vitro. methods: full-term normal human placentas were obtained within one hour of vaginal deliveries or cesarean sections. decidual cells were isolated from the decidua parietalis. after an initial culture for 2 3 days in a serum-containing medium, the decidual cells were exposed to normoxia or hypoxia (10% or 3% oxygen) in a serum-free medium for 24 hours. the culture supernatants were then harvested and subject to western blot analyses of renin protein contents. a dominant band of renin at approximately 47 kd was detected in all samples. when compared with the cells cultured in the normoxic condition, the cells cultured in both hypoxic conditions (i.e. 10% and 3% oxygen) had significantly lower renin protein contents in their culture supernatants. conclusion: our data for the first time show that hypoxia down-regulates renin secretion in human decidua, suggesting a link between the uteroplacental ras and oxygen tension during human gestation. the effect of nucleated fetal red blood cells derived from preeclamptic patients on endothelial progenitor cell proliferation. keiichi matsubara, emiko abe, yuko matsubara, shinji hyodo, masaharu ito. obstetrics and gynecology, ehime university school of medicine, toon, ehime, japan. objectives inadequate uteroplacental circulation results in placental ischemia and the development of preeclampsia (pe). endothelial progenitor cells (epcs) are thought to be a key player in the fetal angiogenesis. vascular endothelial growth factor (vegf), which is up regulated in pe, is involved in epcs proliferation. recently, it was reported that fetal nucleated red blood cells (nrbcs) have the capability to generate vegf. we hypothesized that nrbcs could influence epcs proliferation in the placenta and may be involved in the pathogenesis of pe. material and methods mononuclear cells (mncs) were isolated from the umbilical venous blood of normal pregnant women and preeclamptic patients by density gradient centrifugation. mncs were incubated with anti-cd71 antibody conjugated with microbeads. nrbcs were collected using a mini macs separator. nrbcs were incubated for 24 hours with or without angiotensin ii (ang ii) and erythropoietin (epo). vegf and placental growth factor (plgf) concentrations in the supernatant were measured using elisa. also, pbmcs without nrbcs were seeded in endothelial basal medium with or without nrbcs using a boyden chamber. these samples were incubated for 7 days with or without ang ii and epo. the adherent cells were incubated with di-ldl, fixed with paraformaldehyde, and stained with fluorescein isothiocyanate-labeled lectin. di-ldl and lectin positive cells was considered to represent epcs and the number was measured using flowcytometry. the number of nrbcs derived from umbilical venous blood was significantly increased in pe. both ang ii and epo significantly increased vegf concentration in the supernatant of nrbcs derived from normal pregnant women. however, ang ii and epo did not influence the nrbcs' vegf production in pe patients. plgf was not detectable in the supernatant. the number of epcs in the umbilical venous blood was significantly decreased in pe and the number was not changed by nrbcs. on the other hand, the number of epcs was significantly decreased in the culture with nrbcs. epo significantly increased the number of epcs in pe at the lower concentration of epo compared with normal pregnant women. conclusions it appears that fetal nrbcs may inhibit fetal epcs proliferation in pe. since epo reduced the inhibitory reaction of nrbcs without vegf production epo may affect epcs proliferation independently of nrbcs. relationship between the hypoxia-inducible factor-2 (hif-2 ) and the receptor svegf-r1/sflt-1: implication for phatophysiology of preeclampsia. julio e valdivia-silva, 1,3 juan c gonzalez-altamirano, 2 keisy lopezthe trophoblast invasion is critical for the establishment of the uteroplacental circulation. at early phases of this process local oxygen pressure in the placenta is lower, that pathologically in preeclampsia remain constant. because of this, is important to understand the response of placental cells against these stimuli. in the present work, we use primary cultures of trophoblast cells, fibroblasts of the villous, and human umbilical endothelial cells, isolated of preterm and term placentas (with and without preeclampsia), to explore the effect of the oxygen pressure in the expression and synthesis of vegf, svegfr-1/sflt-1, hif-1 and-2 . our results show that the low pressure of oxygen resulted in a significant increase of the mrna and the protein of the receptor svegf-r1 selectively in the cts. the vegf's expression and synthesis was raised in three cellular types, but the free protein (not bounded to svegf-r1) of the cts was diminished. on the other hand, the expression of the arnm of hif-1 or -2 in cells was comparable in all the types of placentas, nevertheless, the protein hif-2 was more increased in the cts of preeclamptic placentas. to evaluate the relation of hif-2 and the increase of the receptor svegfr-1, we used sirna-hif2 . in response to the inhibition, the expression of the receptor svegf-r1 diminished dramatically. the blockade of hif-2 did not alter vegf's expression. our data are the first that propose that the protein of the factor of transcription hif-2 is one of the molecules involved in the selective expression of the receptor svegf-r1 in trophoblast cells during hypoxia. figure: reduction of the expression to soluble receptor flt-1/svegfr-1 after transfection with sirna-hif2 in trophoblast cells. sirna= interference rna, lu=luciferase. luc-sirna was used as control. effects of estradiol on synthesis, secretion, and activation of von willebrand factor in endometrial endothelial cell. shumei zhao, chainarong choksuchat, michael s scholfield, todd d deutch, thomas d kimble, david f archer. obstetrics and gynecology, eastern virginia medical school, norfolk, va, usa. objectives: heavy menstrual bleeding (hmb) is a serious clinical condition affecting 30% of women. due to inefficient and ineffective medical interventions, women with hmb often elect endometrial ablation or hysterectomy to eliminate hmb symptoms. morbidity and loss of fertility linked to these surgical treatments support the search for more effective medical remedies. von willebrand factor (vwf), a principle initiator of blood clotting produced by endothelial cells. women with von willebrand disease (vwd), have a high incidence of hmb indicating poor clotting. these findings suggest vwf heavily impacts the amount of blood loss during menstruation. estrogen stops/reduces hmb and has been used to treat hmb in women with vwd, although the mechanism(s) is unknown. this proposal addresses the hypothesis that estradiol (e 2 ) increases the synthesis and activation of vwf, promoting clotting. materials and methods: immortalized human endometrial endothelia cells (heecs) were used for the studies. to determine if e 2 increases synthesis of vwf, we treated heecs with e 2 at 0.001μm, 0.01μm and 0.1μm for 24 hours. vwf protein and mrna levels were determined by western blotting and real time pcr, respectively. to establish if e 2 can convert vwf from an inactive to an active conformation, the release of activated vwf by cells into culture medium will be assessed by elisa. decreased adamts13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif) activity results in increased amounts of active vwf. rrelease of activated adamts13 will be determined by fret. to ascertain if e2 regulated vwf secretion is by genomic pathway, heecs will be exposed to the estrogen receptor antagonist ici 182,780. elisa and fret will assess the release of active vwf and adamts13, respectively. results: western blotting demonstrated e 2 increases vwf mrna and protein levels in a dose-dependent manner in heecs.conclusion: the project will determine if e 2 acts at the heecs to increase synthesis, secretion and activation of vwf. preliminary results show e 2 increases intracellular vwf protein and mrna levels in heecs, supporting our hypothesis that e 2 increases synthesis of vwf in heecs in vitro. if e 2 increases activated vwf, it could reduce/stop hmb by increasing activated vwf, providing justification for a clinical trial of e 2 to treat hmb. over-expression of vegf-d does not induce lymphangiogenesis in the mouse endometrium. peter a rogers, 1 jacqui f donoghue, 1 marc g achen, 2 steven a stacker, 2 jane e girling. 1 1 obstetrics gynaecology, monash university, melbourne, victoria, australia; 2 ludwig institute for cancer research, melbourne, victoria, australia. background: the human endometrial functionalis has reduced lymphatics compared to the basalis and myometrium 1 . this study examines the distribution of lymphatics in mouse uterus and investigates if over-expression of the lymphangiogenic growth factor vascular endothelial growth factor-d (vegf-d) stimulates growth of new endometrial lymphatic vessels. methods: the distribution of uterine lymphatics was examined in c57bl/ 6jxcba mice collected during the oestrus cycle, early pregnancy and following oestrogen and progesterone treatment. human 293ebna cells with/without stable transfection of vegf-d were injected into the uterine horn of nod/scid mice. uteri were collected after 4 weeks. serial sections were immunostained with lyve-1 and/or vegfr3 (lymphatic endothelial cell markers), cd31 (blood endothelial cell marker), mab286 (human vegf-d), mab1278 (human mitochondria) and pcna (proliferative cell nuclear antigen). results: lymphatic vessel profiles were mostly found in the connective tissue between the longitudinal and circular muscle layers of the myometrium. they were rare in the endometrium and only observed in 24% of the sections. when present in endometrium, lymphatic vessel profiles were usually situated adjacent to the endometrial/myometrial border. 293ebna tumours formed inside and outside the uterine horn of both the control (n=4 of 6) and vegf-d group (n=3 of 6). localization of 293ebna cells within the mouse uterus was confirmed by anti-human mitochondrial expression. vegf-d immunostaining confirmed that transfected 293ebna cells expressed vegf-d in vivo. over-expression of vegf-d did not stimulate endometrial lymphangiogenesis, although there was an increase in vessel diameter of lymphatics in the myometrium adjacent to tumours. initial analysis shows no significant effect of vegf-d 293ebna cells on endometrial blood vascular density or endothelial cell proliferation. conclusions: minimal lymphatics are present in the mouse endometrium, as is the case for lymphatic vessels in the human endometrial functionalis. the lack of endometrial lymphangiogenesis in response to vegf-d suggests the presence of an inhibitory factor limiting lymphatic growth in this tissue. in early pregnancy, decidual-derived vegf mediates angiogenesis and is required for implantation and placentation. the role of decidual vegf in later pregnancy is poorly understood. decidual hemorrhage (placental abruption) generates excess thrombin and is a major risk factor for pprom and preterm birth. this study compares immunohistochemical (ihc) localization of vegf in decidual tissue sections from term pregnancies complicated by abruption and gestational age-matched controls, and investigates the effect of thrombin on vegf expression by cultured human term decidual stromal cells (dscs). study design: ihc was performed on serial sections of term placental tissues (no labor) with (n=5) and without (n=5) abruption. purified term dscs were passaged until >99% free of cd45+ cells by facs. confluent dscs were primed with 10 -8 m estradiol (e2), 10 -7 m medroxyprogesterone acetate (mpa), both, or vehicle for 7 days. after 24h incubation in defined medium with corresponding steroids ± thrombin (0.5-2.5 iu/ml), conditioned supernatants were analyzed for vegf by elisa. extracted total rna was used to assess vegf mrna levels by quantitative rt-pcr using established primers. results: vegf expression was localized by ihc primarily to dscs in placental tissue sections, and was increased in tissues from placental abruption vs controls. in term dscs, thrombin increased vegf secretion in a dosedependent fashion irrespective of the hormonal milieu (eg, 2.17-fold stimulation by 2.5 iu/ml thrombin from 5.05±1.23 to 12.62±3.01 pg/ml per mcg protein for e2+mpa; p=0.034). this effect was abrogated by the thrombin inactivator, hirudin. vegf mrna were similarly increased by thrombin with or without steroid hormones (eg, 2.9-fold for e2+mpa; p<0.05). conclusions: placental abruption is associated with increased vegf expression in term decidual tissues in vivo with thrombin enhancing vegf mrna and protein expression in term dscs in vitro. excess thrombinmediated vegf expression in term decidua aberrantly increases endothelial cell permeability to further generate thrombin by continuous exposure of tissue factor-expressing decidual cells to circulating factor vii. thrombin-enhanced matrix metalloproteinase expression in term dscs would degrade decidual and fetal membrane extracellular matrix to induce pprom and preterm birth. basal directional release of angiotensin ii by endothelial cells stimulated by chymotrypsin-like protease (clp)/chymase. yuping wang, david f lewis, yang gu. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: chymotrypsin-like protease (clp)/chymase is a serine protease which plays a major role in angiotensin ii (ang ii) generation in the human heart. our previous study showed a higher clp activity in the maternal plasma in women with pe than in normal pregnancies. we also found enhanced chymase expression in the maternal vessel endothelium in women with pe. in this study, we determined if clp could promote endothelial cell (ec) generation of ang ii. methods: we specifically examined basal directional release of ang ii by cultured ecs. ecs were grown on cell culture insert (6 well/plate, 8 micron pore size). when ecs reached confluence, chymotrypsin (chy) at concentrations of 0.25, 0.5, 1.0, 2.5, and 5.0 g/ml were added to the upper chamber of the cell insert. after 24 hours of culture, medium in the lower chamber was collected. medium concentrations of ang ii were measured by enzymelinked immunoassay (eia). all samples were measured in duplicate. data are expressed as mean ± se and analyzed by anova. a p level < 0.05 was considered statistically different. results: chymotrypsin produced a concentration-dependent increase in basal directional release of ang ii by cultured ecs, control: 1.016 ± 0.065 g/ml; chy 0.25: 1.284 ± 0.281 g/ml; chy 0.5: 1.204 ± 2.06 g/ml; chy 1.0: 2.726 ± 0.829 g/ml; chy 2.5: 4.499 ± 1.467 g/ml (p<0.01); chy 5.0: 5.159 ± 1.087 g/ml (p<0.01), respectively. data are means from 6 independent experiments. conclusion: apical exposure of ecs with chymotrypsin-like protease could promote basal directional release of ang ii. our result implicates that in pe, elevated clp levels in the maternal circulation are very likely to affect ec generation of ang ii. basal directional released ang ii may bind to its receptor on underlying vascular smooth muscle cells and contribute to the increased vasoconstriction in pe. (supported nih grants hd36822 and hl65997). vegf stimulates angiogenesis and vasodilation critical for dramatic rises in materno-feto interface blood flows directly linked to fetal growth/survival. extracellular signal-regulated kinase (erk2/1) pathway mediates partially vegf-induced angiogenic and vasodilatory responses in placental endothelial cells (ec). it is, however, unknown how this vegf-induced signaling is organized in placental ec. objectives: ovine fetoplacental artery ec (ofpaec) and its transformed counterpart, sv40-of to test whether: 1) vegf-activated erk2/1 signaling is compartmentalized in the caveolae and disruption of caveolae interferes vegf-induced erk2/1 activation and; 2) caveolin-1, the structure protein of caveolae, regulates vegf-stimulated erk2/1 phosphorylation. methods: ofpaec or sv40-of cells were cultured in mcdb-131/10% fbs/antibiotics. serum-starved subconfluent ( 80%) cells were treated with rhvegf (0 to 100 ng/ml) for various times. caveolae were disrupted by -cyclodextrin ( -cd, 10 mm, 60 min) or caveolin-1 scaffolding domain (cav-sd, 5 m, 2 hr). sv40-of cells were used for fractionation of caveolae membranes by discontinuous sucrose gradient (45%/35%/5%) ultracentrifugation. activation of erk2/1 signaling pathway were analyzed by western-blotting with specific antibodies. results: in total cell extracts, vegf stimulates erk2/1 phosphorylation in a time-and dose-dependent manner. erk2/1 phosphorylation maximized by vegf (10 ng/ml) at 5-10 min, which was abrogated by -cd or cav-sd. all the molecules for compromising the erk2/1 signaling module, plc 1, pkc , src, ras, raf-1, mek2/1 and erk2/1, were detectable in purified caveolae membranes positive for various markers including caveolin-1, enos, flotillin-1, and -adaptin. in caveolae, vegf dramatically increased phosphorylated erk2/1 without altering total erk2/1 in a time-dependent manner similar to that in total cell extracts, which also maximized at 5-10 min. pretreatment with -cd or cav-sd blocked vegf stimulation of erk2/1 phosphorylation in caveolae. conclusion: vegf activates the erk2/1 signaling pathway in caveolae and caveolae integrity is essential for vegf-activated erk2/1 signaling pathway. we conclude that caveolae/caveolin-1 serves as a platform for compartmentalizing the vegf-induced erk2/1 signaling pathway in placental ec (hl74947 and hl70562). hypoxia upregulates gcm1 in human placenta. david mccaig, fiona lyall. institute of medical genetics, university of glasgow, glasgow, united kingdom. introduction: studies in transgenic mice have shown that a variety of genes regulate the differentiation of trophoblast cells. these genes include gcm1. gcm1 is also expressed in the human placenta. placental gcm-1 protein has been reported to be reduced in pre-eclampsia, in view of the close link between hypoxia, hypoxia-reoxygneation, pre-eclampsia, placental development and the reported reduction in gcm1 we hypothesised that gcm1 expression would be affected by hypoxia. aim: the aim of this study was to determine the effects of hypoxia on gcm1 expression in the human placenta. two model systems were used; (1) free floating villous explants and (2) cultured primary cytotrophoblast and syncytiotrophoblast cells as described previously*. methods: explants or cell cultures were exposed to either hypoxia or hypoxia followed by re-oxygenation. western blot analysis was used to assess gcm1 protein levels. bands on the gels were quantified using scanning densitometry. statistical differences (n=6 experiments for both models used) were calulated by anova and turkey's post-hoc test. results: gcm1 protein was detectable at a low level in villous explants maintained for 7h in 20% o 2 . a striking increase in gcm1 protein was observed when villous explants were incubated for 1h in 0% o 2 (p<0.002). incubation of villous explants for 1h in 0% o 2 followed by re-oxygenation for 6h in 20% o 2 resulted in a marked decline in gcm1 protein (p<0.002). expression of gcm1 was also analysed in primary cytotrophoblast and syncytiotrophoblast cultured in 18% o 2 or reduced oxygen (2% o 2 ) conditions. gcm1 protein was not detected in any of the experimental conditions used. discussion: the present study has shown that acute hypoxia increases gcm-1 protein in villous explants. the experiments with purified trophoblast do not support a role for hypoxia increasing gcm-1 in these cells under the experimental conditions used. the present findings are in keeping with the complex effects of oxygen depending on the conditions used. the observed hypoxic effects on gcm1 warrant further investigation. the effect of acute alcohol exposure on histone3-lys9 modification in the mid-gestation embryonic lung. xiangyuan wang, 1 debra wolgemuth, 1,2,3 laxmi baxi. 1 1 ob/gyn; 2 genetics development; 3 human nutrition, columbia university medical center, new york, ny, usa. objective maternal alcohol abuse during pregnancy produces an array of birth defects comprising fetal alcohol syndrome. lung development depends on a balance between cell proliferation and apoptosis. we have previously shown that the acute alcohol exposure in the mid-gestation embryo can delay lung development and induce apoptosis. acute exposure to ethanol of selected tissues in mouse embryos has been reported by others to initiate apoptosis within 12 hours after exposure and result in histone modifications. specifically, histone acetylation and deacetylation are involved in transcriptional activation and repression, respectively, but can also involve apoptosis. in the present study, we have investigated the effect of alcohol on acetylation of histone3 at lysine9 (ach3lys9) in the mid-gestation embryonic lung. pregnant c57bl/6j mice at day 13.5 of gestation (e13.5) were injected intraperitoneally with 2 doses of 25% ethanol (3.75g/kg ), 4 hr apart (alcoholexposed: ae) or with ringers solution (controls: c). ae and five c fetuses were retrieved 12 and 24 hr later and the lungs were fixed and processed for morphological evaluation and staining with rabbit polyclonal anti-ach3ly9 antibody. the entire lung tissue field was evaluated for the levels of ach3lys9 staining and scored as (-) to (++++). three areas were selected randomly from each sample and the total number of cells and staining positive cells were counted in the bronchial epithelium and in the mesenchyme. twelve hr after alcohol exposure at e13.5, the morphology of ae embryonic lungs was normal. however, high levels of ach3lys9 were detected in 50% of the bronchial epithelial cells and 50% of the mesenchymal cells. the expression level in both lineages decreased 24 hr after alcohol exposure. in the controls, the expression of ach3lys9 was virtually undetectable in both the bronchial epithelium and the mesenchymal cells. our previous study showed that ae e13.5 lungs significantly increased apoptotic cell in both bronchial epithelium and mesenchyme 16 hours after alcohol treatment. we now observe that elevated expression of ach3lys9 in the embryonic lung preceded the observation of apoptosis, suggesting that alteration in the acetylation of h3 could be one of the molecular mechanisms involved in the induction of apoptosis following acute alcohol exposure. objective: our purpose was to evaluate the effect of vitamin c and e supplementation on lipid peroxide levels, total antioxidant ability, and antioxidant levels in the umbilical venous plasma. materials and methods: women at risk for preeclampsia (nullipara, previous preeclampsia, chronic hypertension) were recruited at 15 to 20 weeksgestation and randomly assigned to receive either 1000 mg of vitamin c and 400 iu of vitamin e (study group, n=20) or placebo (control group, n=20) daily until delivery. umbilical venous blood were collected after full term delivery. lipid peroxide levels, oxygen-radical absorbance capacity (orac) values, antioxidant levels were measured by each method (thiobarbituric acid reaction, cao's method, and high performance liquid chromatography). results: 1. the lipid peroxide levels in the umbilical venous plasma of study group were significantly lower than that of control group (2.22±0.06 vs. 2.56±0.12 nmol/mg protein, p<0.05). 2. the orac values in the umbilical venous plasma of study group were significantly higher than that of control group (15010.1±649.4 vs. 11804.6±463.7 u/ml, p<0.05). 3. the -tocopherol levels in the umbilical venous plasma of study group were significantly higher than that of control group (130.2±10.5 vs. 78.7±4.4 nmol/ml, p<0.01). 4. there were no significant differences in the ascorbic acid, uric acid, -carotene, retinol, and -tocopherol levels in the umbilical venous plasma between control and study group. conclusion: this suggested that maternal vitamin c and e supplementation may affect the oxidant-antioxidants balance of the utero-placenta unit and fetus. placental tnf-related apoptosis-inducing ligand (trail) in normal pregnancy and pre-eclampsia. xilian bai, jenny e myers, philip n baker, john d aplin, ian p crocker. maternal and fetal health research group, the university of manchester. introduction: enhanced placental trophoblast apoptosis is well known to occur in pre-eclampsia. however, the potential role of membrane associated or soluble trail, an apoptosis-inducing ligand, and its death receptor, dr5, is not known. we tested the hypotheses that the trail/trail-receptor system is compromised in pre-eclampsia and that soluble trail is a circulating factor which triggers the vascular complications of pre-eclampsia. method: this study was conducted on placental samples and plasma (edta) from women with uncomplicated pregnancies (n=16) and with pre-eclampsia (n=16) at 35-40 weeks gestation. protein expression levels and tissue localisation of trail and dr5 were defined in villous tissue by western blotting and immunohistochemistry. soluble trail (strail) was measured in maternal plasma from both groups using a commercial elisa (diaclone). results: placental villous trail and dr5 protein was unaltered in preeclampsia compared to normal pregnancy. whilst there was differential distribution of trail and dr5 within the component cells, this also was unaffected in pre-eclampsia. within the villi, trail was mainly confined to the cytoplasm and perinuclear regions of cytotrophoblast, syncytiotrophoblast, stromal cells and the fetal capillary endothelium. conversely, dr5 was restricted to trophoblast only, distributed evenly between cytoplasm, plasma membranes and nucleus. strail was present in plasma from non-pregnant women of childbearing age [391.6 (312.4-646.8) , median (interquartile range), pg/ml, n=6]. however, there was no significant increase either in pregnancy [372.3 (319.6-442. 3) pg/ml, n=12] or pre-eclampsia [301.8 (209.0-435.5 ) pg/ ml, n=13] (kruskal-wallis test, p>0.05). conclusions: these results suggest that placental villous trail is not adversely regulated in pre-eclampsia. the absence of dr5 in stromal and endothelial cells may increase resistance to apoptotic stimuli in cells of the villous core. the co-localisation of trail and dr5 in trophoblast suggests a role in autocrine regulation of cell turnover in this cell type. introduction: preeclampsia is associated with apoptosis of the syncytial layer of placenta and the release of particulate and soluble factors which are deleterious to maternal endothelial function. the goal of the current study was to determine whether laser capture microdissection (lcmd) and western blotting could be used to assess levels of syncytial fas ligand (fasl), a key protein in the apoptotic cascade. methods: frozen sections of term placenta delivered from uncomplicated pregnancies at term were used for study (n=3). following staining with mayer's hematoxylin (n=3), lcmd (leica instruments) of an intact terminal villus was carried out using a focused laser pulse directed to the area of interest using a microscope. in the first round of microdissection the placental villus core consisting of fibroblast, macrophages, fetal vessels, and connective tissue, were removed. in the second round of lcmd, the syncytial layer of that same villus was removed and collected in lysis buffer containing detergent and protease inhibitors, and the number of nuclei per sample was recorded. this procedure was repeated until syncytial tissue from 10-15 villi were collected. electrophoretic separation of lysate proteins was then carried out, and western blotting and immunodetection using an anti-fasl antibody was performed. results: we observed that fasl was detected in microdissected syncytial specimens at a molecular weight of approximately 37 kda ( figure) , consistent with our previous reports. it is of note, that western blotting of samples containing approximately 5000 (lane 1), 2000 (lane 2), 1000 (lane 3), and 500 (lane 4) nuclei revealed that fasl could be reliably detected in specimens containing as few as 1000 nuclei. conclusions: since fasl is a cytokine of low to moderate abundance in placenta, this suggests that lcmd coupled with western blotting will be a valuable methodology to elucidate pathways of syncytial apoptosis and pathophysiology in pregnancies complicated by preeclampsia. supported in part by nih grant hd33909. the placenta of preeclamptic pregnancies shows oxidative and nitrative stress. we have shown low protein abundance but paradoxically high activity of inducible nitric oxide synthase (inos) in the placenta with severe preeclampsia compared with normal pregnancies. protein nitration and phosphorylation are post-translational modifications possibly involved in nos activity. objectives: examine inos localization and tyrosine phosphorylation in the preeclamptic placenta and the effect of a peroxynitrite generator (sin-1) on inos expression in primary human placental microvascular endothelial cell (hpmec) cultures. methods: placental lysates from normal (n=3), mild (n=2) and severe (n=3) preeclamptic pregnancies were western blotted for inos and what are the roles played by peroxynitrite in preeclamptic women? yoshikatsu suzuki, 1 tamao yamamoto, 1 yoshimasa watanabe, 2 takeo itoh, 2 hidetaka izumi. 3 1 obstetrics and gynecology, nagoya city university, nagoya, japan; 2 pharmacology, nagoya city university, nagoya, japan; 3 obstetrics and gynecology, izumi women's hospital, fukuoka, japan. aim preeclampsia is characterized by hypertension plus proteinuria. it is hypothesized that the endothelial cell function might be activated by placental faculty in early pregnancy and the activation might cause the vascular disease in preeclampsia. peroxynitrite, which is produced by combination with superoxide (o 2 -) and nitric oxide (no), is strong oxidant as well as o 2 -.we investigated whether or not the localization of peroxynitrite in both placenta and resistance artery might play important roles of developing preeclampsia. omental arteries or placentas were obtained from severe preeclamptic and term-normotensive pregnant women at cesarean section. they were stained by ant-nitrotyrosine (nt, a marker of peroxynitrite) antibody. furthermore, the concentration of nt was measured in omental arteries. in formed consent was obtained from all patients in written. preeclampsia was diagnosed according to the criteria of japan society of obstetrics and gynecology. the localization of nt was seen in placentas obtained from sever preeclamptic women (7/10), although it was not seen from normotensive pregnant women (0/8). the localization was also seen in placentas from 5 of 7 severe preeclamptic women with intrauterine fetal restriction. in omental arteries from both groups, the localization of nt was seen to same degree, and the concentration of nt was similar (0.788±0.161 for sever preeclampsia and 0.984±0.209 for normotensive pregnant women). from these results, it was suggested that an increase in o 2 production, a decrease in no as well as peroxynitrite production in the placentas might cause the vascular dysfunction and subsequently damage uteroplacental blood circulation in preeclampsia. however, the role played by peroxynitrite in resistance artery might be different and more complicated. background: preeclampsia (pe) is associated with shallow cytotrophoblast (ct) invasion of the decidua, leading to impaired vascular transformation and poor uteroplacental perfusion. ct invasion requires the selective proteolysis of the peri-decidual cell (dc) extracellular matrix. we hypothesized that il1 and tnf , cytokines that have been linked to pe, may induce aberrant expression of the matrix metalloproteinases (mmp) 1 and 3 in dcs, thereby preferentially degrading the decidual ecm and interfering with sequential ct invasion. methods: immunostaining for mmp-1, mmp-3, and vimentin (a dc marker) was performed on decidua from normal (n=5) and preeclamptic (n=4) women, and staining intensities were evaluated by hscore. confluent, leukocyte-free first trimester dcs were primed with 10-8 m estradiol (e2) or e2 + 10-7 m medroxyprogesterone acetate (mpa), and then switched to a defined medium with e2 +/-mpa with or without 1 ng/ml of il1 or tnf . secreted mmp-1 and mmp-3 levels were measured by elisa (n=8) and confirmed by western blotting. quantitative rt-pcr assessed mmp-1 and mmp-3 mrna levels (n=3). results: tissue staining revealed that mmp-1 and mmp-3 levels in preeclamptic decidua (hscore mean±sem: 217±41 and 205±11, respectively) were significantly higher than in normal decidua (140±22 and 148±25, respectively; p<0.05). in cultured first trimester dcs incubated with e2, tnf increased secreted mmp-1 and mmp-3 levels by 13±2 and 8±2-fold, respectively, while il1 increased them by 17±3 and 28±13 fold, respectively (p<0.05). in parallel incubations with e2+mpa, basal mmp-1 and mmp-3 output were lowered by approximately 70% and 60%, respectively, while tnf -and il1elicited mmp-1 and mmp-3 levels were blunted by 40-60%. western blotting confirmed the elisa results, and mrna levels corresponded to changes in mmp-1 and mmp-3 secreted protein levels. conclusions: over-expression of mmp-1 and mmp-3 in decidual cells may promote pe by disrupting decidual ecm and impairing normal ct invasion. the high levels of il1 and tnf associated with pe may be contributing to this over-expression. our in vitro observations that mpa blunts tnf -and il1 -elicited mmp expression suggests that exogenous progestin may offer a novel therapeutic approach in preventing pe. to produce 2-methoxyestradiol (2-me), a compound with diverse biological activities including inhibition of hif-1 , a transcription factor that mediates cellular response to hypoxia. circulating levels of 2-me and placental comt activity are significantly reduced in preeclampsia, raising the possibility that reduced production of 2-me contributes to the pathophysiology of preeclampsia by altering placental response to hypoxia. genetic variation in the comt gene is linked to comt activity and has been associated with intrauterine fetal growth restriction. we determined if a snp in exon 4 of the comt gene (rs4818), which does not change amino acid sequence (136leu136), but reduces comt mrna translation, was associated with preeclampsia. we analyzed comt genotypes in paired dna samples extracted from maternal and cord blood from normal pregnancies and pregnancies complicated by preeclampsia by allele discrimination. the study population was predominantly (>98%) african-american. the frequency of the minor rs4818 "g" allele, which is associated with low comt activity, was similar in maternal cases and controls (cases: 19%; controls: 23%, p=0.5), but was significantly greater in fetal dna from 35 pregnancies complicated by preeclampsia compared to 55 control pregnancies (g allele frequency cases: 34%; control: 16%; p<0.001). likewise, fetal carriage of the rs4818 "g" allele conferred a significantly greater risk of preeclampsia (odds ratio: 1.74; 95% c.i.: 1.22, 2.48, p<0.0045). there was also a significant discordance between paired maternal and fetal rs4818 genotypes with significantly greater discordance for the "g" allele in fetuses hosted in preeclamptic pregnancies (odds ratio: 3.17; 95% c.i.: 1.19, 8.49). we conclude that genetic variation in the comt gene is associated with risk of preeclampsia, possibly through a mechanism involving reduced production of 2-me. supported by nih p60md002256. smoking is associated with elevated adma in preeclampsia. michael p frank, 1 robert w powers. 1,2 1 magee-womens research institute, pittsburgh, pa, usa; 2 obstetrics gynecology, university of pittsburgh, pittsburgh, pa, usa. objective: cigarette smoke exposure paradoxically reduces the risk of preeclampsia. asymmetric dimethylarginine (adma) is an endogenous competitive inhibitor of nitric oxide synthase (nos), an independent risk factor for cardiovascular mortality, adma is elevated in women who develop preeclampsia, and adma has been reported to be both higher and lower in smokers. the objective of this study was to investigate the concentration of adma in pregnant smokers and nonsmokers with and without preeclampsia. study design: case-control study of 120 women with uncomplicated pregnancy (controls), and 57 women with preeclampsia matched for gestational age at sample collection. adma was measured by hplc. cigarette smoke exposure was determined by questionnaire and confirmed by plasma cotinine. data are mean±sd. analysis was by two factor anova with fishers post-hoc testing, significance accepted at p<0.05 results: as previously reported, maternal plasma adma concentrations were higher in women with preeclampsia compared to controls (p<0.001). in addition, the concentration of adma was significantly higher in preeclampsia smokers compared to controls and preeclampsia nonsmokers (p<0.05). in contrast, there was no difference in adma concentration between control smokers and nonsmokers. conclusion: these data may suggest a differential effect of cigarette smoke exposure on circulating adma concentrations between women who do and do not develop preeclampsia. previous data has suggested that cigarette smoking is associated with lower adma in low risk elderly patients, and higher adma in high-risk subjects with diabetes. therefore, the data in preeclamptic and non-preeclamptic subjects may be consistent with these studies, however, the underlying biological explanation for this differential effect has yet to be determined. objective: eclampsia is similar to hypertensive encephalopathy in which an acute elevation in blood pressure causes autoregulatory breakthrough, hyperperfusion and edema formation. we previously reported that the pressure of breakthrough was similar between nonpregnant (np) and late-pregnant (lp) rats, but only lp animals developed edema. this study tested the hypothesis that lp animals have decreased in cerebrovascular resistance (cvr) and hyperperfusion in response to breakthrough vs. np. we further hypothesized that acute hypertension would cause greater blood-brain barrier (bbb) permeability in lp rats due to elevated hydrostatic pressure. methods: in vivo models of bbb permeability and cerebral blood flow (cbf) were used in np (n=16) and lp (d19-20; n=16) rats that were either normotensive or hypertensive (np-htn, lp-htn) by infusion of phenylephrine to raise mean arterial pressure. permeability was determined in anterior and posterior brain regions by calculating the flux of 70kd dextran into the brain tissue, measured by a fluorescent spectrophotometer after flushing the vasculature with saline. cbf and cvr were measured by infusion of 15 m fluorescent microspheres and determined based on the flow rate and fluorescence intensity of a reference sample for each animal. animals were ventilated to maintain blood gases within normal ranges (po 2 >100mmhg, pco 2 =35-45mmhg). results: although the pressure change was similar between np and lp ( 83 and 85 mm hg), lp animals responded to acute hypertension with hyperperfusion. cbf increased from 86 ± 10 to 242 ± 22 ml/100g/min in np (181%) and 94±9 to 318±31ml/100g/min in lp (238%; p<0.05 vs. np). hyperperfusion in lp animals was associate with decreased cvr vs. np (0.7±0.07 vs. 0.5±0.05 mm hg/(ml/100g/min); p<0.05). bbb permeability was significantly increased in lp animals at breakthrough vs. np in both anterior and posterior brain regions. the flux of dextran in anterior and posterior brain regions for np vs. lp animals was: 113±50 vs. 362±87 for anterior (p<0.05) and 167±58 vs. 545±140 for posterior (p<0.05). conclusions: these data demonstrate that pregnancy decreases cvr and causes hyperperfusion of the brain during acute hypertension. because increases in cvr is a protective function in the brain, impairment of these mechanisms during pregnancy may predispose the brain to edema when blood pressure is elevated, as in eclampsia. introduction: this study used the in vitro dually perfused human placental lobule to test the hypothesese that placental release of vegf and the fetoplacental vasodilatory response to exogenous vegf-165 are altered by tissue oxygenations that mimic healthy and preeclamptic pregancies. methods: lobules were dually perfused for six hours under one of two oxygenation conditions, representing 'normoxia' and 'hypoxia' (n = 6 each): delivering maternal side inflow perfusate at oxygen concentrations of 15.6 % and 8 %, respectively, distributed via 22 cannulae; and fetal side inflow perfusate oxygen concentration of 2-3 % in both systems. venous perfusates were sampled and assayed, appropriate to side of release, for erythropoietin (epo), macrophage inflammatory protein-1 alpha (mip-1 alpha) (reference oxygen sensitive hormones), free vegf, svegfr-1 and plgf. in separate perfusions, fetoplacental vasodilation in response to 550 pm vegf was investigated, following preconstriction of the fetoplacental vasculature to steady state fetal-side inflow hydrostatic pressure (fihp) with the thromboxane mimetic, u46619, (n = 6 each). results: maternal-side mip-1 alpha release was higher in the 'hypoxic' than the 'normoxic' system (680 ± 119 and 531 ± 93 pg/ml, respectively, at 6 hours, mean ± se; 2-way anova: p<0.01). maternal-side epo and fetal-side soluble free vegf and svegfr-1 release were not different between groups. fetal-side release of plgf was higher in the 'hypoxic' group than the 'normoxic' group (0.13 ± 0.10 and 0.03 ± 0.01 pm, respectively, at 6 hours, mean ± se; 2-way anova: p < 0.05). there was no difference in the vasodilatory response to vegf-165 in the fetoplacental vasculature between the groups (64.8 ± 5.2 and 63.5 ± 6.0 % change in fihp, mean ± se). discussion: differences in mip-1 alpha and plgf release provide evidence for metabolic separation of the adapted systems, caused by a changed oxygen environment. our failure to observe differences in epo, vegf and svegfr-1 release may be explained by longer lag-times for up regulation of their gene expression. vegf associated endothelial signalling appears to be unaffected by 'hypoxia' in the placenta over the time course studied here. background: there is a placental renin-angiotensin system (ras) from very early pregnancy. ang iv mediates various effects by binding to its specific receptor, the at4r, the active site of which is an insulin-regulated aminopeptidase (irap). there is at4r expression in both endothelial and smooth muscle cells. ang iv at low concentrations is vasodilatory, increasing blood flow via the at4rs; it also stimulates nf-kappa beta and modulates glut-4. to date at4r expression has not been investigated in the placenta. we propose that at4r plays a part in the placental vascular development necessary for successful pregnancy, and that reduced at4r expression may be associated with inadequate vascular adaptation contributing to pre-eclampsia (pe). aim: to identify and locate at4r expression in both np and pe placentae. methods: the study had hospital ethical approval; written informed consent was obtained from all women. placental samples were obtained from 4 np and 4 pe at delivery (gestational ages: 39 and 38 weeks respectively). samples were taken from 3 areas, near cord, middle and outer edge of the placenta. paraffin embedded sections were immunostained for irap reactivity using a rabbit polyclonal antibody (gift from professor david james, garvan institute, australia). immunoreactivity of trophoblast and uterine cell populations was assessed using a semi-quantitative grading system. grade 0 = no positive labelling, 1 = 1 -25%, 2 = 26 -49%, 3 = 50 -74% and 4 = 75 -100% of cells positively labelled. median (max, min) are shown. results: 1) at4r immunostaining was prominent in the syncytiotrophoblast and hofbauer cells of all placental villi examined, with no differences in expression between sampling sites. 2) at4r positivity was reduced in near cord pe samples (2.2 (2.4,1.2)) compared to np (3.3 (4.0,2.5) p=0.029). conclusion: we have shown for the first time, dense at4rs in syncytiotrophoblast and hofbauer cells in np placentae, and their down-regulation in pe. reduced ang iv/at4r binding may contribute to increased placental vasoconstriction resulting in increased ischemia/reperfusion. this in turn may stimulate xanthine oxidase, which is itself stimulated by angii, leading to increased superoxide production. further work is needed to clearly define the role of this newly identified component of the renin-angiotensin system in normal pregnancy and pe. pre-eclampsia is associated with lower percentages of regulatory t cells in maternal blood. jr prins, 1 hm boelens, 1 jj hm erwich, 1 j heimweg, 2 s van der heide, 2 ajm van oosterhout, 2 aej dubois, 3 jg aarnoudse. 1 1 obstetrics, university medical center groningen, groningen, netherlands; 2 laboratory of allergology and pulmonary disease, university medical center groningen; 3 pediatric allergology, beatrix children's clinic, university medical center groningen. pre-eclampsia is a serious disease of human pregnancy and immunological mechanisms play a role in its pathophysiology. normal pregnancy is associated with an increase in regulatory t (treg) cells and with a predominant th2 immune profile. treg cells are a subpopulation of cd4+ lymphocytes and are specifically characterized by the lineage specific transcription factor foxp3. treg cells seem to induce immunological changes that have a protective role in maintaining normal pregnancy. we hypothesised that percentages of treg cells are decreased in pregnancies complicated by pre-eclampsia. methods in total, 18 women with pregnancies complicated by pre-eclampsia and 26 healthy pregnant controls were enrolled. to obtain control umbilical cord blood as well, control group i consisted of eighteen healthy pregnant women at term. in addition, since 13 women with pre-eclampsia delivered preterm, control group ii (peripheral blood only) consisted of women during normal pregnancy with a gestational age matched for the preterm pre-eclamptic group. treg cells were measured from whole blood using four-color flow-cytometry. women with a pregnancy complicated by pre-eclampsia had a significantly lower percentage of cd4+ foxp3+ treg cells (4.5 vs 8.8%; p<0.05). in the pre-term group the pregnancies complicated by pre-eclampsia showed a significantly lower percentage of cd4 + foxp3 + cells in the peripheral blood as compared to the healthy pregnant controls. at term this percentage was also lower but not significantly so. between pre-term and term pregnancies both complicated by pre-eclampsia no significant difference was found in the percentage of cd4 + foxp3 + treg cells. no difference was found in umbilical cord blood (7.2 vs 7.9%). conclusions our data suggest that pre-eclampsia is associated with a diminished percentage of treg cells in peripheral blood. we conclude that a deficiency of regulatory t cells may play a role in the pathophysiology of pre-eclampsia. background: preeclampsia and eclampsia are significant causes of maternal and fetal death. however, the pathophysiology of these conditions is unclear. we and others have reported that inhibition of endogenous nitric oxide (no) synthesis produces symptoms similar to preeclampsia in pregnant rats. several studies demonstrate that fetoplacental weights are altered in pregnancies of spontaneous hypertensive (shr) rats. in addition, impaired synthesis of tetrahydrobiopterin (bh4), a major co-factor for endothelial nitric oxide synthase (enos) activity and enhanced expression of enos has been observed in the pathogenesis of hypertension. in the current study, we examined whether supplementation of bh4 and sepiapterin (sep, a precursor for bh4 biosynthesis in the salvage pathway) reduces increased blood pressure and improves fetoplacental weights in shr pregnant rats. methods: groups (4-6) of shr pregnant rats were either treated with bh4, sep (20 mg/k.g body weight/day/rat, oral tablets) or vehicle (normal diet) beginning from day 12 of pregnancy until day 20 of gestation. animals were sacrificed on day 20 of gestation and fetoplacental weights were recorded immediately. western blot analysis was performed to determine vascular enos expression (enos/gapdh). results: significant (p<0.05) elevations in blood pressure (bp, mmhg) were observed in shr (shr, 141±1.42 vs. wky, 106 ± 3.04 mmhg) compared to wistar-kyoto (wky) group. supplementation of either bh4 or sep (128±2.19), significantly (p<0.05) reduced elevated bp beginning from day 14 of pregnancy. fetal but not placental weights were significantly (p<0.05) reduced in shr (2.39±0.03 grams) compared to wky (3.10±0.04) rats. bh4 (2.51±0.04) treatment partially (p<0.05) increased fetal weights compared to the shr group. vascular enos expression is significantly (p<0.05) elevated in shr (1.0±0.07) compared to wky rats (0.56±0.15). further, treatment with bh4 (0.58±0.1) but not sep (0.92±0.1) significantly (p<0.05) reduced elevated enos protein expression in shr rats. conclusions: bh4 may be beneficial treatment of preeclampsia to reduce blood pressure and improve fetal perfusion to increase fetal weights. genetic risk factor for severe preeclampsia: significance of endothelial nitric oxide synthase gene t-786->c and missense glu298asp variants. toshihiro yoshimura, 1 michihiro yoshimura, 2 masafumi nakayama. 3 1 obstetrics and gynecology, kumamoto university school of medicine, kumamoto, japan; 2 cardiovascular medicine, jikei university school of medicine, tokyo, japan; 3 cardiovascular medicine, kumamoto university school of medicine, kumamoto, japan. introduction: we recently identified two endothelial nitric oxide synthase (enos) gene polymorphisms, a glu298asp missense variant in exon 7 and a t-786-->c variant in the 5'-franking region, which are associated with coronary spasm and myocardial infarction in japanese population. and we also identified a missense glu298asp variant is associated with severe preeclampsia and placental abruption. our objective was to analyze the association between the t-786-->c and severe preeclampsia. materials and methods: the study participants included 62 patients with histories of severe preeclampsia. this is a preliminary study, therefore, the comparisons were made with the 345 general normal population. results: the analyses revealed that the frequency of the missense glu298asp variant (n=18/62, 29%) was significantly higher than the general population (n=44/345, 13 %), as we previously published. however, the frequency of the t-786-->c variant (n=9/62, 14%) was not different from the general population (n=61/345, 18 %). interestingly, only one patient had both t-786-->c and missense glu298asp variants, and she developed placental abruption. conclusion: although our sample size is small, it is very unlikely that the t-786-->c variant is associated with severe preeclampsia. the t-786-->c variant may not be a genetic susceptibility factor to severe preeclampsia. the t-786-->c may have some reproductive significance in combination with missense glu298asp variant, however huge number of patients would be needed to analyze such rare (2.5%) combination of the variance. the association between the development of preeclampsia and methylenetetrahydrofolate reductase, angiotensinogen, vascular endothelial growth factor single nucleotide polymorphism genotype combinations. hyun soo park, 1 jong kwan jun, 2 chan-wook park, 2 joong shin park, 2 bo hyun yoon, 2 hee chul syn. 2 1 obstetrics and gynecology, dongguk university international hospital, goyang-si, gyeonggi-do, republic of korea; 2 obstetrics and gynecology, seoul national university college of medicine, seoul, republic of korea. objective this study was conducted to investigate if there exists any genotype combination of multiple single nucleotide polymorphism (snp)s which is frequently found in preeclampsia patients. study design one hundred sixty two preeclampsia patients and 199 normotensive pregnant women were included in this study between jan 1998 and jul 2004. diagnosis of preeclampsia and assignment of severity were made according to the criteria by national high blood pressure education working group and american college of obstetricians and gynecologists. the patients were reclassified as early (30 weeks or before) and late-onset (31 weeks or beyond) disease. genotypes were measured with pcr-rflp for methylenetetrahydrofolate reductase (mthfr) c677t, angiotensinogen (agt) m235t, vascular endothelial growth factor (vegf) c936t with the dna extracted from maternal blood. case-control study for each snp was done and the frequencies of genotype combination were compared. anova, t-test, chi-square test, fisher's exact test and logistic regression analysis were used for statistical analysis. a p value of <0.05 was considered statistically significant. results genotypes of mthfr polymorphism showed significant difference between late onset preeclampsia and control (cc+ct/tt, or: 1.82, p<0.05) but agt and vegf polymorphism did not show statistical difference between any case-control combination. only 20 out of possible 27 genotype combinations were found and there was no statistical difference in the frequencies of genotype combination between case and control group. conclusion mthfr polymorphism might be associated with the development of preeclampsia, but there was no combination of mthfr, agt and vegf polymorphisms which is associated with the development of preeclampsia. diffuse staining and vascular smooth muscle (vsm) staining. resistance-sized vessels (10-200 μm) were evaluated. results: for mpo, the intensity of staining (fig) and the % vessels with neutrophil, diffuse and vsm staining was significantly greater for obese than for overweight or normal weight patients: % diffuse staining (64.6 ± 4.2 vs. 40.4 ± 3.9 vs. 21.8 ± 4.7, p<0.001); % vsm staining (30.2 ± 6.1 vs. 20.0 ± 5.3 vs. 4.0 ± 2.6, p<0.01). for mmp8, obese and overweight patients had a greater (p<0.001) % vessels with neutrophil, diffuse and vsm staining than normal weight patients: % diffuse staining (63.0 ± 5.5 vs. 55.2 ± 4.3 vs. 26.0 ± 3.0); % vsm staining (36.8 ± 2.9 vs. 27.4 ± 3.1 vs. 2.8 ± 1.2). conclusions: neutrophils infiltrate the systemic vasculature of obese women and release mpo and mmp8. speculation: obesity may put women at risk for pe because their vasculature may already be dysfunctional due to neutrophil infiltration and release of mpo and mmp8. hl069851. collagen is an important protein that maintains the structural integrity of tissues. disruption of vascular smooth muscle collagen could result in vascular dysfunction in women with preeclampsia. recently, neutrophil infiltration of the systemic vasculature was demonstrated in preeclamptic women. neutrophils produce inflammatory mediators, such as reactive oxygen species (ros) and tnf-. we hypothesized that neutrophils, ros and tnf-would alter expression of collagen regulating genes. methods: primary cultures of human vascular smooth muscle cells (vsmc) were seeded into t-25 flasks (40,000 cells/flask) and grown for 3 days to 70% confluence. the cells were treated for 24 hours with medium control, ros (hx, 0.05 mm + xo 0.003 u/ml), tnf-(1 ng/ml); and neutrophils (60,000) activated with arachidonic acid, 50 μm, (1:16 ratio of neutrophils to vsmc). rna was extracted from cell homogenates and analyzed for gene expression with an rt2 profiler pcr array system for human extracellular matrix genes (superarray). to determine the fold-change of gene expression, the results were first normalized to a housekeeping gene and then ct was calculated across two rt-pcr arrays where group 1 was the control and group 2 was the experimental treatment. results: table 1 . conclusions: neutrophils, ros and tnf increased mmp1 expression. interestingly, genes involved in collagen synthesis (col1a1) or inhibition of mmp-1 activity (timp1) were either not affected or down-regulated. these data suggest that neutrophil infiltration in preeclamptic women could cause vascular dysfunction by creating an imbalance between collagen synthesis and collagen breakdown favoring breakdown. hl069851, fogarty 5d43tw007692, p60md002256. objective: hypoxia increases membrane attack complex (mac) binding to cultured human trophoblasts, and mac enhances apoptosis in trophoblasts exposed to low compared to normal fio 2 . trophoblast microparticles and cellular fragments released into the maternal circulation in vivo may contribute to the systemic pathophysiology of preeclampsia. we tested the hypothesis that hypoxia induced mac deposition on cultured human trophoblasts yields microparticles and fragments coated with mac. study design: primary cytotrophoblasts from term human placentas (n=4) were cultured 24 h in 1% and 20% oxygen in dmem with 10% human serum with active mac or heat inactivated serum (control). media were centrifuged to obtain pellets of microparticles and cell debris which were immunostained for mac or exposed 6-12 h to confluent, phorbol myristate acetate differentiated u937 macrophages. the percentage of macrophages that ingested trophoblast debris was quantified by counting the number of macrophages with immunofluorescence for trophoblast cytokeratin 7 filament staining, as assessed by confocal microscopy. results: cultures exposed to normal human serum, but not heat inactivated control serum, showed mac immunofluorescence on microparticles and fragments in medium, with the highest level of mac in cultures exposed to extreme hypoxia. the maximal percentage of macrophages that ingested the trophoblast debris coated with mac from cultures with 1% oxygen was 56.2%, not different from the 52.6% from cultures exposed a 1% fio 2 and control serum. conclusion: trophoblasts exposed to hypoxia and active complement release microparticles and cellular fragments coated with mac into the extracellular environment. mac coating does not influence phagocytic removal of the debris by macrophages suggesting that placental derived, membrane bound mac could circulate to yield systemic affects on maternal endothelium. supported by nih hd29190 and hd045675. is there a role for fatty acids in the pathogenesis of pre-eclampsia? nicola j robinson, 1 laura j minchell, 1 jenny e myers, 1 philip n baker, 1 carl a hubel, 2 ian p crocker. 1 1 maternal and fetal health research group, the university of manchester; 2 obstetrics, gynecology and reproductive sciences, university of pittsburgh. objectives: women with pre-eclampsia (pe) display altered lipid metabolism as characterized by elevated circulating triglycerides and non-esterified fatty acids (nefa) and these changes are evident before the disease is clinically apparent. we have tested the hypothesis that the increased circulating levels of nefa contribute to endothelial dysfunction in pe. methods: human umbilical vein endothelial cells were incubated for 24h with pooled plasma (2%) from normal or pe pregnancies, or with palmitic, oleic and linoleic acid in culture media at the concentrations and molar ratios to albumin identified in normal (100, 113, 37 m, ratio 0.9) and pe pregnancies (140, 172, 53 m, ratio 1.6) 1,2 . lipid droplet accumulation was determined using an oil red o absorbance assay. endothelial metabolism was measured using the mtt test and mitochondrial membrane potential determined by jc-1 assay as a marker of early apoptosis. results: plasma from pe pregnancies increased endothelial cell lipid droplet accumulation compared to normal plasma (p<0.01, wilcoxon signed ranks, n=9). this change was replicated following exposure to nefa at the combined concentrations found in pe compared to normal pregnant controls (p<0.05, n=7). plasma from women with pe caused a significant decrease in mitochondrial dehydrogenase activity (mtt test; p<0.01, n=8) and a reduction in jc-1 fluorescence (p<0.05, n=7), compared to normal plasma, suggestive of mitochondrial membrane depolarization and increased cellular apoptosis. again these effects were replicated using nefa in culture medium at the levels found in pe compared to normal pregnancies (mtt test: p<0.05, n=9; jc-1 assay: p<0.05, n=10). conclusions: in endothelial cell cultures, plasma from women with pe caused increased lipid droplet accumulation, decreased cellular metabolism and increased apoptosis. these changes to cellular function were mirrored using nefa in culture medium at the concentrations and molar ratios to albumin previously reported in pe. these findings provide evidence that the changes in endothelial cell function induced by plasma from women with pe may be due to the increased nefa circulating levels and that increased palmitic, oleic and linoleic acid, in combination, could play a role in pathogenesis of pe. 1 lorentzen (1995) bjog; 2 endresen (1992) ajog. background: skewing of the maternal endothelial phenotype in pre-eclampsia (pe) is attributed to the release of unknown factors from a hypoperfused placenta. we hypothesise that factors secreted from pe placental tissue will impair endothelial cell function. we have tested the effect of soluble factors by menopausal status, there was a significant increase in bmi in pre-but not in postmenopausal women. in postmenopausal women there was insufficient power to note a statistically significant change in bmi. results are summarized with the follow-up for each group represented by "n" in the graph below. premenopausal patients were divided into hysterectomy with oophorectomy (pbso) versus hysterectomy alone (ph). the ph group showed an increase in bmi that plateaus at time 3. in the pbso group the bmi continued to increase over time. subgroup analysis comparing ph to pbso demonstrates initial weight loss in pbso but a significant increase in bmi from baseline at time 3 compared to ph. conclusions: hysterectomy appears to be associated with an increase in bmi over time. subgroup analysis suggests that, in premenopausal women, oophorectomy is more strongly associated with continuing weight gain than hysterectomy alone. purpose: obesity is implicated as a key risk factor in chronic disease, but no studies have associated central obesity to the presence of chronic abdominal and/or pelvic pain. we set out to identify the prevalence of chronic abdominal/ pelvic pain in an underserved, primarily latina population by a cross-sectional study in the olive view-ucla outpatient gynecology clinic. we sought to identify an association between the presence and severity of abdominal/pelvic pain and central obesity. methods: nonpregnant women presenting to the gynecology clinic were prospectively evaluated and grouped according to the presence of abdominal/ pelvic pain ('none-mild' or 'moderate-severe' pain). body mass index (bmi) and abdominal circumference (ac) were measured. patients with 'moderate-severe' pain completed standardized questionnaires for pelvic pain and global health scores. results: 207/276 (75%) of patients has 'none-mild' pain, and 69 (25%) had 'moderate-severe' pain. pain prevalence was not significantly associated with bmi (mean: 'none-mild' 30.5+7.7 kg/m2; 'moderate-severe' 30.6+7.6 kg/m2, p=0.95), nor was pain severity (p=0.58). pain prevalence was significantly associated with ac (mean: 'none-mild' 37.1+7.7 inches; 'moderate-severe' 40.2+7.0, p=0.004). a borderline positive association exists between ac and pain severity (p=0.077). conclusions: we demonstrate an association between both the presence and severity of chronic abdominal/pelvic pain and central obesity, independent of bmi. ac appears to be a more relevant factor than other traditional measures of habitus in patients with this chronic malady. to improve preventative care in women's health management, further evaluation of the role of central obesity in the pathogenesis of chronic pain is necessary. aims: vulvitis is one of the most frequently diagnosed gynaecological infections. we aim to assess the efficacy against infective vulvitis of a new topical medical device containing low molecular weight hyaluronic acid (lmw-ha). the ability of this molecule to stimulate -defensin 2 release in keratinocytes has been recently shown. methods: we report preliminary data regarding 20 women suffering from infective vulvitis, as assessed by a gyneacologist: patients were randomly selected to receive sphg800 (10 patients), a cream containing low-molecular weigh hyaluronic acid, or vehicle (10 patients). patients were asked to apply the cream to the vulva twice-daily for 7 days. at the end of treatment, evaluation of efficacy, tolerability and acceptability of the cream was assessed by a specific questionnaire. results: preliminary results show that patients receiving sphg800 report a significative improvement of vulvitis symptoms, in terms of itch, redness of the skin and burning, in comparison to vehicle. sphg800 also showed a good tolerability, cosmetic acceptability and symptomatic relief perceived by patients. conclusion: sphg800 seems to be efficacious in ameliorating the symptoms of infective vulvitis. this activity may be probably related to the lmw-ha presents in this formulation: in fact, low molecular weight hyaluronic acid has been recently shown to induce -defensin 2 production by human keratinocytes. since this peptide exerts antimicrobial and antimicotic activity, the improvement of symptoms assessed in patients receiving sphg800 might be linked to a reduced infective charge, attributable to the activity of -defensin 2. background: allogeneic hematopoietic stem cell transplant (hsct) is a treatment used for many malignant and nonmalignant diseases of the bone marrow and immune system. hsct may be complicated by chronic graft-versus-hostdisease (cgvhd) in 60 to 70% from matched unrelated donors. genital cgvhd complicates about 25% of hsct and may uncommonly result in labial fusion. case: 22 year old woman with a history of ewing's sarcoma and acute myelogenous leukemia, had received chemotherapy and total body irradiation (tbi) followed by a matched unrelated donor hsct. menarche occurred at 13 years of age after normal pubertal development. she menstruated regularly until cancer diagnosis. premature ovarian failure resulted after chemotherapy and tbi and oral contraceptive pills were used for hormone replacement. after transplant, she developed chronic gvhd involving the skin, eyes, mouth and joints, and concomitantly complained of vulvar pruritus. she was presumed to have a yeast infection which was treated with fluconazole without a pelvic exam. she was evaluated by a gynecologist when she was unable to insert a tampon. pelvic exam revealed dense labia minora adhesions from the clitoris to urethral meatus and posteriorly leaving a 1 cm opening at the urethra. pelvic mri revealed a normal uterus and ovaries. after 2 weeks of topical estrogen cream, the adhesions remained dense and were lysed under general anesthesia. vaginal examination revealed pale, minimally rugated, normal mucosa. cervical cytology was normal. post-operatively she used daily topical estrogen and hydrocortisone creams. at 3 months after surgery, her urinary stream was stronger. on pelvic exam, the labial opening was 4 cm, but a small posterior forchette adhesion elicited severe pain. after using dilators coated with topical steroids and estrogen, she was able to insert a tampon. conclusion: genital gvhd should be considered in women with genital tract complaints after hsct. labial fusion secondary to chronic gvhd may be treated successfully with surgery and medical therapy. support: rbmb/nichd/nih. dana r ambler, mazen e abdallah, rahi victory, michael p diamond, elizabeth e puscheck, jay m berman. obstetrics and gynecology, wayne state university/detroit medical center, detroit, mi, usa. objective: to determine which factors are predictive of a ruptured ectopic pregnancy, and whether endometrial stripe thickness can be used as an alternative to such criteria in the diagnosis of an ectopic pregnancy. design: retrospectively collected ectopic pregnancy database. setting: detroit medical center, detroit, michigan. patients or participants: 413 women with a diagnosis of an ectopic pregnancy were studied, with 90 surgically confirmed tubal rupture cases. interventions: abstracted data included hcg(iu), gestational age (days), presenting symptoms of pain and/or bleeding, hemoglobin (hgb), hematocrit (hct), historical risk factors, ultrasound-determined ectopic size, endometrial stripe thickness (mm), amount of cul de sac fluid (cds), tubal rupture at time of surgery, and estimated blood loss (ebl)(ml). covariates included demographics. results were significant when p<0.05. results: chi square analysis revealed that there is a relationship between endometrial stripe thickness and hcg levels. logistic regression models demonstrated that endometrial stripe thickness was not predictive of ectopic rupture, (or 0.98, p=0.96) . however, logistic regression, both forced and forward stepwise analysis, demonstrated that hcg (or=6.8, p<0.001), a large volume of cul-de-sac fluid (or=4.2, p<0.001), and increasing pain (or=3.5, p=0.004) were associated with increased risks of rupture. gestational age, and ectopic related risk factors were also not predictive of rupture. conclusions: endometrial stripe thickness is not a useful predictor for the diagnosis of a ruptured ectopic pregnancy. serum hcg measurement, cds fluid volume and the presence of pain are much stronger diagnostic indicators of ectopic pregnancies. clinical profile of migraineurs in a university hospital gynecology department in japan. [objectives] the changes in hormonal milieu associated with menarche, pregnancy, menopause, and post-menopause are frequently accompanied by changes in the patterns and frequency of migraine. little is known on the relationship of women's issues of migraine though the balance between estrogen and progesterone is critical in the elimination of migraine. our aim was to investigate the relations among the prevalence of migraine, the reproductive stage, and gynecologic diseases. [materials and methods]197 female patients (average age: 44.6 years old) who consulted a physician and agreed to answer about the questionnaires during september, 2006 -june, 2007 . migraineurs were diagnosed with the migraine screener by the japanese headache medical treatment promotion committee in 2005. and the patients answered questionnaires that screened about menopausal disorder and abnormal menstruation at once. they were conducted a survey in the form of a questionnaire and sometimes were taken blood samples. [results] 52 in 197 patients had migraine (26.4% average age: 40.6 years old). prevalence were 33.3% in one's twenties, higher than another ages. most patients with migraine was complicated by menstruation disorders (premenstrual syndrome 67%, dysmenorrhea 52.6%), sterility (42.4%), and severe menopausal disorder (80%). when trh and the lh-rh test were examined for the sterility patient, migraineurs had higher prolactin basal level and lh level after lord but lower fsh level before and after lord than nonmigraineurs. on the other hand, the prevalence of migraine for postmenopausal women and women who had gynecology cancer treatment was low, and there was no relation between migraine and pregnancy history. [conclusions] this study provides migraine headache is influenced by reproductive stage and that women with migraine are frequently complicated by menstruation disorder. it is thought that migraine account for abnormality of hormone milieu including abnormality of the hypothalamus-pituitary system. objective: this study evaluated the efficacy of 3 doses of estradiol (e 2 ) gel 0.1% (divigel ® ), a novel formulation of e 2 consisting of 1 mg e 2 per 1 g transdermal gel, to reduce frequency and severity of vasomotor symptoms and signs of vulvar and vaginal atrophy (vva) associated with menopause. design: 488 postmenopausal women were evaluated in a 12-week study comparing placebo to e 2 gel 0.1% at doses of 1.0 g/day, 0.5 g/day, and 0.25 g/day with estimated nominal daily deliveries of 0.027 mg, 0.009 mg, and 0.003 mg of e 2 respectively. endpoints included mean change from baseline in daily frequency and severity of moderate to severe hot flashes (msvs). vaginal ph and % superficial cells were collected at baseline and end of study. results: e 2 gel 0.1% showed statistically significant improvements from placebo gel as early as week 2 (table) that were maintained throughout treatment. signs of vva (vaginal ph and % of superficial cells) showed statistically significant improvements from baseline with all 3 doses of e 2 gel 0.1% compared to placebo. conclusion: e 2 gel 0.1% significantly decreased the frequency and severity of msvs at all doses evaluated in this trial. e 2 gel 0.1% offers multiple dosing options to individualize patient therapy, including the lowest effective dose that was studied (0.25 mg e 2 , delivering 0.003 mg e 2 /day), to treat vasomotor symptoms associated with menopause. estradiol (e 2 ) gel 0.1% (divigel ® ) is a novel formulation of e 2 consisting of 1 mg e 2 per 1 g transdermal gel for the treatment of vasomotor symptoms associated with menopause. safety and tolerability of e 2 gel 0.1% were evaluated in a large placebo-controlled trial. design: 488 postmenopausal women participated in a 12-week study comparing placebo to 0.25 g/day, 0.5 g/day, and 1.0 g/day of e 2 gel 0.1%. circulating e 2 and estrone (e 1 ) concentrations were measured. safety analyses included the incidence of adverse events (aes) and clinical laboratory evaluations, including plasma levels of sex hormone binding globulin (shbg). application site tolerability was assessed using the draize scale. results: all doses of e 2 gel 0.1% produced physiologic e 2 :e 1 ratios similar to those seen in premenopausal women. e 2 :e 1 increased from a baseline mean of 0.17 to 0.49, 0.69, and 0.95 with, respectively, the 0.25, 0.5, and 1.0 g/day doses of e 2 gel 0.1%. the most frequently reported aes were breast tenderness and postmenopausal bleeding that appeared to be dose-related and would be expected with increased circulating estrogen concentrations. there were no remarkable changes in hematology, blood chemistry, urinalysis, lipid, coagulation, and carbohydrate values following treatment with e 2 gel 0.1%. shbg levels remained unchanged after 12 weeks of treatment at all doses. the vast majority of patients had no evidence of skin irritation throughout the treatment period. mean draise scale scores after 4, 8, and 12 weeks of treatment were 0.0 for all treatment groups except for a mean value of 0.1 ± 0.34 for the 0.5 g dose group after 4 weeks of treatment. conclusion: e 2 gel 0.1% is a safe and well-tolerated therapy for the treatment of menopausal symptoms. design: pharmacokinetic parameters, dosing, efficacy, and safety information for divigel ® , elestrin ™ , evamist ™ , estrogel ® , and estrasorb ® were obtained from current prescribing information (obtained from manufacturer websites) and the data were compared. results: together, these new transdermal therapies offer multiple dosing/ delivery options and contain a wide range of e 2 (0.25 mg to 4.6 mg), with the lowest systemic daily delivery of e 2 attained by the divigel ® 0.25 g dose. following 12 weeks of treatment, across the dosing options, each treatment significantly reduced both the frequency and severity of moderate to severe vasomotor symptoms (msvms) compared to placebo. change in frequency of msvms ranged from -6.0 (divigel ® 0.25 g) to -11.1 (estrasorb ® ); change in severity scores for msvms ranged from -0.3 (divigel ® 0.25 g) to -1.7 (divigel ® 1.0 g). all treatments are safe and well tolerated. breast tenderness was the only adverse event reported in 5% of subjects, occurring with all therapies. conclusions: current treatment guidelines recommend using the lowest effective dose of estrogen for the treatment of menopausal symptoms. this side-by-side comparison of the recently available e 2 non-patch transdermal options to the standard transdermal therapies is meant to assist physicians in individualizing treatment for their patients. introduction: cdb-2914 (cdb) is a relatively new progesterone receptor modulator being clinically evaluated for contraception and treatment of fibromas. its use in an intrauterine device/system (ius) has not been reported. in this study we prepared cdb-filled intrauterine devices (cdb-ius) and evaluated their effects on endometrial growth and bleeding patterns in rhesus macaques. methods: short (1.0-1.5 cm) lengths of silastic tubing (od 2.4 mm), either empty (n=3),or filled with silicone rubber matrix containing 50% of micronized cdb (n=5), were inserted into the uterine lumens of 8 ovariectomized rhesus macaques. animals were induced to cycle by sequential treatment with systemic estradiol and progesterone (p) as reported (brenner et al, ann ny acad, 2002) . after 3.5 cycles, at the end of the follicular phase, the uterus was removed and processed. results: when systemic p treatment was withdrawn at the end of each cycle, animals with empty ius menstruated normally, while animals with cdb-ius bled little or not at all. over the whole 3.5 cycles, animals bearing a blank ius bled for an average of 11.66 ± 0.88 days while cdb treated animals bled for an average of only 1 ± 0.45 days. at the end of treatment, animals exposed to blank ius had mean endometrial wet weights of 409 ± 112 mg while the cdb-treated endometria weighed only 188 ± 30 mg. the proliferation markers ki-67 and phospho-h3 were substantially lower in the cdb-ius treated than in the blank treated animals. histologically, the cdb exposed endometria were atrophied with evidence of glandular degeneration while the blank controls were proliferative and normal. summary: in cycling rhesus macaques, a cdb-ius prevented progestational development, blocked menstruation after p withdrawal, and suppressed endometrial proliferation. if these effects are confirmed in women, the cdb-ius could provide estrogen-free and bleed-free contraception, and could help control heavy menstrual bleeding. supported by rr00163, hd 43209 and the population council. introduction: irregular uterine bleeding is a major side effect and cause for discontinuation of ltpoc use. while endometria of ltpoc-exposed women display abnormally enlarged, fragile blood vessels (bv), decreased blood flow and evidence of oxidative stress, the mechanisms by which structural and vasomotor endometrial dysfunction occurs remains unknown, in part by the difficulty of manipulating hormone levels in women. the aims of this study were 1) to validate the guinea pig (gp) as a model to study uterine effects of ltpoc and 2) to investigate ltpoc-effects on endometrial histology and oxidative stress markers. methods: oophorectomized gps were implanted s.c. with time release pellets containing either placebo (crl,n=6); estradiol (e2,n=3); medroxyprogesterone acetate (mpa,n=6) or e2+mpa (n=3). after 21 days, uterine horns were weighed and frozen or paraffin embedded. angiogenesis was assessed by quantitative image analysis of vonwillebrand factor staining and included bv density and size. oxidative stress was detected by 8isoprostane and 8-oh-deoxyguanosine (8oxog). apoptosis was investigated by the tunel method. statistical analysis was by 1-way and 2-way anova. results: gp uteri were enlarged by both e2 (p<0.001) and mpa (p=0.025). effects of mpa on uterine weight differed significantly depending on e2 levels (p<0.001), where mpa opposed the e2 effect in combined treatments. angiogenesis parameters were similarly impacted upon. thus, mpa alone increased bv density (p=0.036) and bv average area (p=0.002). the presence of e2 significantly decreased these parameters (bv density mean± sem: crl: 9.4±1.0%, e2: 10.3±1.6%, mpa: 13.6±1.1%, e2+mpa: 6.0±0.7%, p=0.002). these changes were associated with highly elevated 8-isoprostane content in e2+mpa-treated uteri compared to all other groups (p<0.001). abnormalities in the e2+mpa group were consistent with chromatin redistribution, nuclear pyknosis, karyolysis and increased nuclear 8oxog staining and a marked increase in tunel labeling. conclusions: ltpoc exposure alters endometrial vascular and tissue morphology consistent with oxidative stress and apoptosis in a complex interplay with endogenous estrogens. the gp is an excellent model for the study of ltpoc effects on the uterus. physiologic and psychological symptoms associated with injectable and oral contraceptive use. abbey b berenson, susan odom, carmen r breitkopf, mahbubur rahman. ob/gyn, utmb, galveston, tx, usa. objective: to compare physiologic and psychological symptoms over 24 mo among users of depomedroxyprogesterone acetate (dmpa), an oral contraceptive with 20 micrograms ethinyl estradiol (oc), and non-hormonal (nh) contraception. methods: a total of 604 women reported the presence of 16 symptoms prior to initiating contraception (217 dmpa, 216 oc, 171 nh) and every 6 mo thereafter for 24 mo. longitudinal relationships between symptoms and contraceptives (reference: nh), as well as race/ethnicity (non-hispanic black, non-hispanic white, and hispanic) were assessed by gee-analysis after adjusting for age, visits and baseline status of symptoms. persistence, resolution, and new development of symptoms were noted by method in 6 mo increments for 24 mo and compared with that of nh controls. the gee analyses showed that oc was protective against mastalgia (or= 0.7), cramping (or=0.5), hair loss (or=0.6), acne (or=0.4), nervousness (or=0.5) and mood swings (or=0.7). when race was considered, oc was protective for all women against acne, for whites against mastalgia and cramping, and for non-whites against nervousness. for whites, it was a risk factor for bleeding between menses. oc use resulted in resolution of acne within 6 mo in nearly 50% of those with it at baseline, but no significant resolution after 6 mo. also mastalgia (29% at baseline) was less likely to persist at 18 and 24 mo. bleeding between menses was reported for the first time at 18 mo by 11% of oc users. dmpa users of all races had an increased risk of missed periods (or=95.1), bleeding between menses (or=3.6), bleeding >20 d (or=13.6) and loss of libido (or=2.2) relative to nh users. it reduced cramping (after 12 mo) and bloating (all 24 mo). racial differences were observed with dmpa protective against mastalgia and mood swings in whites, cramping in whites and hispanics, and bloating in non-whites. it was a risk factor for loss of energy in whites only. neither method affected depressive symptoms. conclusion: side effects of these two methods are mostly related to abnormal bleeding. very low dose pills can be protective against symptoms (mastalgia, cramping, hair loss, acne, nervousness and mood swings) commonly associated with pills while dmpa protects against cramping and bloating. knowledge about racial differences will allow physicians to individualize therapy. counseling should include that some symptoms can develop after 18 mo while resolution often occurs within 6 mo. methods: twenty-four women were enrolled in irb approved prospective, randomized, cross-over trial comparing 2 months of oc (ortho cyclen) vs. tc ortho evra). the daily oc administers 35 micrograms of ee; the weekly tc contains .75 milligrams of ee. each treatment was followed by 2 months of washout and 2 months of the alternative contraceptive. blood was drawn at baseline and final week of treatment for each arm of the study. ee was quantified by ria, with preceding organic solvent extraction and celite column partition chromatography. data were analyzed by t test with boferroni's correction, p < .05. results: after two months of treatment the mean (+/-sem) ee levels for tc = 81.9 pg/ml (+/-12.0) and oc = 94.1 pg/ml (+/-9.8). the ee level is not significant different for the two medication (p = 0.42). conclusions: there is no difference in ee levels with the use of these oral and transdermal contraceptives. this suggests the transdermal contraceptive, despite the lack of the hepatic first pass effect, has similar levels of ethinyl estradiol compared to oc. the continuous elevation in ee seen with the tc route of administration, versus the episodic increases seen with the oc route, raises concerns of constant exposure to ee. this may explain the increased risk of estrogen-induced thrombotic events with this tc route of administration. impact of paracervical block, in combination with general anesthesia, on post-abortion pain. gweneth b lazenby, tod aeby. obstetrics and gynecology, university of hawaii, honolulu, hi, usa. objective to evaluate the impact of paracervical block with a long-acting local anesthetic, in conjunction with general anesthesia, on post-operative pain. methods a power analysis determined 35 patients per arm were needed to demonstrate a significant difference of 2 in mean pain scores. seventy-two patients were allocated to one of two arms using urn randomization. all patients received standardized anesthesia; intravenous sedation for gestational age under 12 weeks and general anesthesia for over 12 weeks. thirty-nine patients were randomized to receive a paracervical block with 0.5% bupivacaine and thirtythree were randomized to no local anesthesia prior to surgical abortion. patients completed visual analog scales for pain and anxiety prior to the procedure, upon awaking, 30 and 60 minutes post-operatively, and prior to discharge. data were analyzed using an anova and students t-test the experimental and control groups were equivelent in age, ethnicity, gravidity, parity, prior abortions, prior vaginal deliveries, prior c-sections, gestational age, number of laminaria, pre-operative and intra-operative dilation, operative time, estimated blood loss, and reported complications. pain and anxiety were not significantly affected by placement of a paracervical block. these data do not support the hypothesized benefit of local anesthesia, prior to surgical abortion under general anesthesia, to reduce post-operative pain. we do not recommend the routine use of a paracervical block to decrease post-operative pain. age, parity, history of abortion and contraceptive choices affect the risk of repeated abortion. oskari heikinheimo, 1 mika gissler, 2 satu suhonen. 1 1 ob gyn, university of helsinki, helsinki, finland; 2 national research and development centre for welfare and health, helsinki, finland. objective the rate of repeat abortion varies from 30 to 38% in northern europe. however, risk factors for repeat abortion are poorly understood. we characterized risk factors (demographic, as well as those related to abortion and postabortal contraception) of repeat abortion. design a prospective cohort study of 1269 women undergoing medical abortion between august 2000 and december 2002. the subjects were followed by means of finnish registry on induced abortions until december 2005; the follow-up time (mean ± sd) was 49.2 ± 8.0 months. results altogether 179 (14.1%) of the subjects requested repeat abortion within the follow-up time. in univariate analysis previous abortion, parity, young age, smoking and failure to attend the follow-up visit were associated with increased risk of repeat abortion. immediate -in contrast to postponed -initiation of any contraceptive method was linked to lower risk of repeat abortion. in comparison to combined oral contraceptives, use of intrauterine contraception was most efficacious in reducing the risk of another pregnancy termination. in multivariate analysis the effects of young age, parity, previous abortion and type of contraception on the risk of another abortion persisted. conclusions increased focus on young, parous and those with the history of an abortion may be efficacious in decreasing repeat abortion. contraceptive choices made at the time of abortion have an important effect on the rate of reabortion. postabortal use of intrauterine contraception, specifically that of lng-ius, might decrease the rate repeat abortion. objective. to determine the rate of failure and to analyze factors associated with failure for the essure permanent birth control device at the detroit medical center (dmc). methods. a chart review was conducted on patients who underwent essure placement at the dmc from january 2003 through june 2007. patient demographics, past medical and surgical history, anesthesia type, procedure time, intraoperative complications, and procedure failures were noted. data were analyzed for statistical significance using spss. results. there were 316 essure procedures attempted at the dmc from january 2003 through june 2007. of the 316 attempted procedures, there were 25 failures (7.9%). 13 of the 25 failures were attributed to difficulty visualizing the ostia (52%). other causes of failure included expulsion of the device (2), tubal spasm (1), uterine perforation (1), and tubal ostia too large for the device (1). there were 3 cases of failed placement for undocumented reasons, one case requiring a laparoscopic tubal ligation secondary to postprocedure tubal patency, and 3 post-procedure pregnancies. age, race, body mass index, gavidity, parity, history of sexually transmitted infections, medical history, history of cesarean section, tobacco or illicit drug use, anesthesia type, and physician experience with the procedure were not significantly associated with placement failure or difficulty visualizing the ostia. a longer procedure time was significantly associated with failure (40.6 vs 26.6 min, p = 0.004), and history of ectopic pregnancy was significantly associated with difficulty visualizing the ostia (33.3% vs 4.7%, p = 0.003). conclusion. the failure rate for placement of the essure permanent birth control device at the dmc is 7.9% with a pregnancy rate of 0.95%. the majority of failures may be attributed to difficulty visualizing the ostia. a history of ectopic gestation was significantly associated with difficulty visualizing the ostia; thus, it may be reasonable to advise these women that success in essure placement may be reduced. introduction. the essure permanent birth control device is a relatively new form of minimally invasive sterilization for women. under hysteroscopic guidance, a dynamically expanding micro-insert is introduced into the proximal portion of the fallopian tube. the micro-insert induces local fibrosis and ultimately occlusion of the tubal lumen. a hysterosalpingogram (hsg) is performed three months after the procedure to confirm bilateral tubal occlusion. objective. to determine the follow-up rate for the post-essure hsg for a clinic population. methods. a retrospective chart review was conducted on university health center (uhc) patients who underwent placement of the essure permanent birth control device at the detroit medical center from january 2003 through june 2007. follow-up for the post-essure hsg as well as the result of the hsg were noted for each patient. results. placement of the essure permanent birth control device was attempted in 83 uhc patients of which 79 were successfully completed. of the 79 patients, ten underwent a post-essure hsg (12.7%). the hsg was performed three to six months after placement of the essure permanent birth control device. bilateral tubal occlusion was documented in all ten patients. conclusion. despite counseling patients prior to their procedure that a hsg is needed and providing an information sheet, the follow-up rate for the post-essure hsg for this clinic population is only 12.7%. for those in whom a hsg was performed three to six months after essure placement, bilateral tubal occlusion was confirmed in all. steps and or approaches to improve compliance with post-procedure confirmation of tubal occlusion should be employed to increase follow-up in the future. towards fibroids gene therapy: adenovirus mediated delivery of herpes simplex virus 1 thymidine kinase gene/ganciclovir shrinks uterine leiomyoma in the eker rat model. memy hassan, 1 dong zhang, 3 salama salama, 1 cheryl walker, 2 hala el-mazar, 1 ayman al-hendy. 3 1 ob/gyn, utmb; 2 md anderson; 3 ob/gyn, meharry medical college, nashville, usa. aim: assessment of the efficacy of gene therapy of uterine leiomyoma in the immune-competent eker rat model using adenovirus mediated delivery of herpes simplex-1-thymidine kinase gene followed by ganciclivir treatment (ad-tk/gcv). method: female eker rats with mri-confirmed uterine fibroid lesions were randomized to a single treatment with direct intratumor injection of ad-tk/gcv, ad-lacz/gcv, or medium.the tumor volume was evaluated by serial mri scanning and confirmed with caliper measurement at time of euthanasia. sample rats were selected randomly and killed at the following time points; 10, 20 and 30 days post treatment. samples were collected from tumors, other body organs and blood to assess the safety and efficacy of the treatment. results: ad-tk/gcv treatment produced dramatic shrinkage of the total uterine fibroid volume by 75% ±16, 59% ±6 and 68%±28 of pretreatment volume at days 10, 20 and 30 respectively. the tumor size in negative control animals receiving ad-lacz/gcv continued to grow by +26%±10, +66%±23, +102% ±38 while receiving media continue to grow by + 20% ± 6 , +70% ± 8 and +110% ± 15 at same time points. ad-tk/gcv induced significant increase in caspase 3 activity, bax expression, decrease in bcl2 and parp proteins expression and increased tunnel apoptosis index. additionally ad-tk/gcv treatment decreased cyclin d1and pcna expressions. ad-tk/gcv did not produce any significant change in liver function tests or relative uterine horns weight to total body weight. the adenovirus transfection did not disseminated significantly to other distal organs except to liver and myometrium in limited number of animals. h e staining of non targeted organs did not revealed any sign of tissue damage. ad-transfection increased local cd4 and cd8 expressions as well as serum anti-ad antibodies. conclusion: ad-mediated delivery of hsv1tk gene by direct intra-tumor inoculation followed by sc treatment of gcv for ten days effectively shrinks uterine leiomyoma lesions in eker rats. this effect is mediated via induction of apoptosis and decreasing the proliferation. the treatment regimen is well tolerated. these studies provide essential preclinical data for the development of gene therapy as an alternative non-surgical treatment option for women with symptomatic uterine fibroids. inhibitors of catechol o-methyl transferase shrinks uterine fibroids in the eker rat model. memy hassan, 1 dong zhang, 3 hala el-mazar, 1 cheryl walker, 2 ayman al-hendy. 3 1 ob/gyn, utmb; 2 md anderson; 3 ob/gyn, meharry medical college, nashville, usa. background :the sex hormone dependent pattern of uterine leiomyomas and their high content of catechole -o-methy transferase (comt) raise the possibility for the development of novel treatment option using comt inhibitors.aim : to assess the potential therapeutic utility of a synthetic comt inhibitor (ro 41-0960) in the eker rat model of uterine leiomyoma. methods female eker rat were evaluated by mri to confirm the prescence uterine fibroid lesion, then randomized for sc treatment with ro 41-0960 150 mg /kg ,twice/ day for 28 days versus vehicle injection.fibroid tumor burden was evaluated by serial mri measurement and confirmed by direct caliper measurement at time of euthanasia. sample animals were euthanized at 2 and 4 weeks. at that time tumor tissue, blood and most of animal organs including long bones were collected and subjected for further evaluations. 24 hours urine samples were collected for evaluation of estrogen (e2) metabolites and bone resorption marker. results:animals treated with ro 41-0960 exhibited significantly lower uterine fibroid tumor burden (86% and 105%) of pretreatment volume at 2 and 4 weeks post treatment respectively. conversely, the tumor size in control animals continued to grow and reached 300 %, and 300 % of pretreatment size at the same time points. ro 41-0960 treatment resulted in an increase in urinary 2/16 hydroxy e2 metabolite ratio. in addition ro 41-0960 increased bax expression and decreased parp , pcna and cyclind1 experssions . all ro 41-0960 treated animals tolerated the treatment protocol with no signs of toxicity. h e staining of different body organs did not reveal any signs of tissue damage. furthermore, there was no significant change in both liver function tests (alt, ast, billirubin) and bone resorption marker , deoxypyridinoline croslinks, between treated and control rats. conclusion ro 41-0960, a synthetic selective comt inhibitor, caused immediate arrest of the growth of eker rat uterine leiomyoma. this effect might be in part due to modulation of various estrogen dependent genes regulating leiomyoma apoptosis (parp, bax) and proliferation (pcna, cyclin d1). this anti-estrogenic effect is due to the accumulation of the the antiestrogenic metabolite 2 hydroxy estrogen secondary to comt inhibition.comt inhibitors might present an alternative non-surgical option for the treatment of women with symptomatic uterine fibroids. background development of uterine leiomyomas (fibroids) is the most common pathological feature in the female reproductive tract. they negatively impact patients of virtually every gynecologist. despite such morbidity, leiomyoma development is poorly understood. we have recently demonstrated that leiomyomas have a genomic expression pattern that limits retinoic acid (ra) exposure. our group and others have demonstrated that tgf-beta regulation is altered as well. these two pathways likely play central roles in leiomyoma development. the central feature of uterine leiomyomas, the extracellular matrix (ecm), is regulated by both all-trans retinoic acid and tgf-3, focusing on versican as a critical ecm component. human uterine leiomyoma and patient-matched myometrium were obtained from surgical specimens under an irb-approved protocol. these tissues were immortalized and treated with either all-trans retinoic acid, tgf-3, or anti-tgf-3 antibody. rna was isolated for qrt-pcr. human immortalized leiomyoma cells demonstrated the same increased template expression of tgf-3 (2.96±0.71 fold), retinoic acid metabolizing protein (cyp26a1; 16.81±4.50), and versican variant v0 (7.75±2.2 fold) as was found in the progenitor tissue. when treated with all-trans ra, expression of versican variant v0 decreased to levels found in myometrial cells (0.96±0.04 fold). conversely, when treated with tgf-3, expression of versican variant v0 increased 2.98±0.22 fold. to confirm that tgf-3 was central to the overexpression of v0, we treated leiomyoma cells with anti-tgf-3 antibody, and found that baseline over-expression of v0 template was decreased to expression levels similar to untreated myometrial cells. finally, we elucidated a link between the ra and tgf-pathways by assessing the impact of ra treatment of tgf-3 expression, demonstrating that tgf-3 template decreased to levels comparable to myometrial cell expression (0.84±0.12 fold). the disrupted leiomyoma ecm, of which versican is a central component, defines the leiomyoma phenotype. in this study, the leiomyoma fibrotic phenotype regressed when treated with ra and increased when treated with tgf-3, providing the basis for novel therapeutic interventions directed at cell differentiation and ecm formation. objectives: uterine fibroids are the leading cause for hysterectomies in the us. the lack of an appropriate in vitro cell model for the initiation of fibroid growth has hindered advancement in understanding the cellular and molecular basis for the development and progression of uterine fibroids. fibrosis is the underlying mechanism of uterine fibroid formation and myofibroblasts cells are the principal fibrogenic cell type in the uterus. we sought to develop a myofibroblast in vitro cell model for analyzing the initiating molecular events of uterine fibrosis. methods: smooth muscle cells (smcs) were enzymatically isolated from the myometrium of non-pregnant women and cultured in the presence of 10% serum until 70% confluent. for the next 72 h cells were cultured in serum-free media followed by replacement with serum containing media. cells were fixed at 0, 10, 20, 30, 60 m later. cell fine structure and cytoskeletal organization were evaluated by transmission electron microscopy. smooth muscle specific alpha-actin ( -sma) and progesterone receptors (pr) were detected by western blot. results: we observed smc differentiation into myofibroblasts, marked by the presence of notched nuclei ( figure) and the increased expression of -sma 20 m after serum replacement. pr-a and pr-b were detectable at 10, 20 and 30 m. conclusions: the development of myofibroblasts is important in wound healing and fibrosis. we show for the first time that uterine myofibroblasts can be derived in culture from myometrial smcs. thus, these cells will be utilized as a model for developing "in vitro fibroids". this model will enable the study of myofibroblast activation, cytokine signaling, intracellular regulation of uterine fibrogenesis, production of extracellular matrix proteins and development of antifibrotic drugs. the presence of prs in our model enables us to evaluate pr mediated events in fibroid pathogenesis and treatment. this model will be more useful in determining the molecular biology of fibroid initiation than cell models derived from established fibroids that are already well past their initial stages of development. novel approach to genome-wide expression profiling analysis. liping feng, morgan walls, insuk sohn, millie behera, sin-ho jung, phyllis leppert. obgyn; biostatistics and bioinformatics, duke university medical center, durham, nc, usa. background: 10 microarray studies have examined the differential gene expression between uterine fibroid and normal myometrium. all previous studies considered the fibroid as a whole and analyzed only fold changes. we have developed a novel statistical approach to genome-wide expression analysis comparing two fibroid tissue sites to myometrium. methods: using affymetrix tm u133a genechip, we have compared the gene expression between c and e and matched adjacent m. data has been analyzed by considering the 3 specimens per subject and 5 subjects as individuals. we used a block one-way anova method to test if each gene was differentially expressed among the three sites. the p-value is calculated using a permutation method accounting for possible dependency among three lesions. the multiple testing issue was addressed by controlling the false discovery rate. expression values were calculated using the robust multichip average (rma) method. rma estimates are based upon a robust average of background corrected perfect/mismatch (pm) intensities. normalization was done using quantile normalization. expression values were then transformed by taking logarithm base 2. confirmatory rt-pcr was performed. results: we applied a hierarchical clustering analysis to all raw data sets and then displayed a dendrogram, where the height of each branch point indicates the similarity level at each generated cluster. identical gene expression among sites clusters together. due to a strong site effect, m tissues clustered separately from e and c combined. 45 genes were differentially expressed when we used a 0.1 q-value cut off. expression data revealed concordant changes in genes regulating cholesterol biosynthesis, gene transcription, estrogen and extracellular matrix formation when both e and c were examined. cyp19 was detected and we report for the first time that scc-112 (a cell cycle-regulated molecule) folliculin and l-selectin are differentially expressed suggesting that they may be involved in the regulation of cell growth and proliferation of uterine fibroids. conclusions: our novel robust analysis of gene expression provides new clues to the relevant pathways of fibroid development. this new statistical approach that can be used in clinical and/or translational studies to identify differentially expressed genes comparing treatment regimens, cells or tissues. objectives: thrombospondin-1 (tsp-1) is a large matricellular glycoprotein secreted by many cell types. matricellular proteins modulate interactions between cells and their environment, regulate cell adhesion and are expressed during tissue formative processes. they are especially important in fibrosis. tsp-1 plays an important role in angiogenesis and is an activator of tgf -3. in a previous study, we found that differential expression of tsp-1 in uterine fibroids may contribute to an altered healing process leading to fibrosis. this alteration in tissue response to injury initiates the development of abundant, nonaligned collagen fibrils and changes in other components of the ecm. methods: we measureed the pattern of mrna and protein expression of tsp-1 by rt-pcr and western blot in an in vitro serum-deprived differentiated myometrial cell (myofibroblast) model. specifically, smooth muscle cells (smcs) were enzymatically isolated from the myometrium of non-pregnant women and grown in primary culture to 70% confluence. then smc were serum deprived for 72 h and treated back with 10% serum for 10, 20, 30, and 60 m. cells were collected for rna and protein, and tsp-1 expression was evaluated. in addition, cells were stained using the combination of anti-cd45 and anti-smooth muscle -actin or combination of anti-cd42d and anti-smooth muscle -actin as well as the appropriate single and double negative controls. stained sections were analyzed using zeiss axio imager widefield fluorescence confocal microscopy. results: tsp-1 mrna and protein was present in cells in this serumstarvation model after the addition of serum and the expression level remained elevated for 60 m following the addition of serum. fluorescence staining analysis indicated that these cells were positive for human smooth muscle -actin, but negative for leukocyte antigen cd45 and platelet marker cd42d suggesting that the myofibroblasts cells themselves were the source of the tsp-1. conclusions: unlike skin wounds, where tsp-1 is derived from the blood macrophages, monocytes and platelets, differentiated myometrial cells appear to produce tsp-1. elucidating the roles of tsp-1 in myometrium physiology and pathobiology will increase our understanding of the etiology of uterine fibroids and may lead to improved therapies. further studies utilizing this cell model to determine the role of tsp-1 in the activation of tgf -3 are indicated. phospholipid s1p via gi, rac and rho pathways. yoel smicun, armando wu pimentel, jennifer gilman, david a fishman. obstetrics gynecology, new york university school of medicine, new york, ny, usa. objectives: sphingosine-1-phosphate (s1p) levels are elevated in serum and ascites of ovarian cancer patients. we have demonstrated that low concentration s1p enhances while high dose s1p inhibits invasion of epithelial ovarian carcinoma (eoc) cells in a dose and attachment mode dependent manner. we sought to further dissect the pathways by which s1p affects invasion, using specific inhibitors. methods: dov13 eoc cells were pretreated for 4-hrs with vehicle, 0.5 m or 20 m s1p and with inhibitors for gi, p38-mapk, rac and rock, thereafter cells were detached and tested for invasion towards 40 m lpa chemoattractant in matrigel-coated chambers. conditioned media from pretreated cells and invading cells were quantified for upa activity using colorimetric assays. the significance of results was calculated by student's t-test. results: inhibition of gi mildly increased invasion of both control ( p=0.019) and 20 m s1p treated cells ( p=0.0002). inhibition of both p38-mapk and rac did not affect 20 m s1p treated cells, in contrast invasion of control cells was mildly increased (p=0.025, 0.034). inhibition of rock, a protein effector downstream of rho, highly elevated invasion of both control and 20 m s1p treated cells (8 fold, p=0.0035, 23 fold, p=0.00037). both upa and gelatinase activities were higher in conditioned media of invading cells than of attached cells. gelatinase activity was enhanced by both concentrations of s1p (p=0.002, 0.001). ptx fully inhibited gelatinase activity of control and 0.5 m s1p treated cells, and partially of the 20 m s1p treated cells (p<0.00007). 0.5 m s1p significantly increased upa activity of attached (p=0.011) but not of invading cells. this increase was sensitive to ptx and rac inhibitor. 20 m s1p inhibited upa in both attached and invading cells (p=0.0004), this inhibition was rock dependent. conclusions: these findings suggest a strong inhibition of invasion and upa by the rho pathway, of both control and 20 m s1p treated cells. this inhibition is induced partially by upstream gi protein. increased invasion by 0.5 m s1p is associated with elevation of gelatinase activity through gi, rac and rock pathways. this suggests that attached cells and invading cells affect upa activity through different pathways. objectives: sphingosine-1-phosphate (s1p) levels are elevated in serum and ascites of epithelial ovarian cancer (eoc) patients. we have demonstrated that invasion of attached eoc cells differentially react to s1p as compared to invading cells. we examined the impact of the inhibitors for gi and rac on attached and invading eoc. methods: dov13 eoc cells were pretreated for 4-hrs with vehicle, 0.5 m or 20 m s1p and with inhibitors for gi (pertussis toxin (ptx)), and rac (nsc23766, (rac-i)), and cells were detached and assayed for invasion towards 40 m lpa in matrigel-coated chambers. to distinguish the response of attached from invading cells, inhibition of cells pretreated with inhibitors was either continued or not in the invasion chambers. conditioned media (cm) of invading cells were quantified for upa and gelatinase activity by fluorometric and colorimetric assays. significance of results was calculated by student's t-test. results: the significant (p=0.004) increase of invasion by 0.5 m s1p was inhibited by both ptx and rac-i, either by pretreatment alone or by continuous treatment (p=0.0024-0.015). however, the invasion was higher in cells inhibited continuously than cells inhibited only in dishes. both inhibitors did not affect cells treated with 20 m s1p. control cells invasion was increased (2.3 fold, p=0.0019) by continuous rac-i treatment. 0.5 m s1p increased gelatinolysis in cm of invading cells. this and control cells activity was inhibited by ptx pretreatment. continuous treatment with ptx or rac-i elevated 3 and 2 fold gelatinase activity of control and 20 m s1p treated cells. in contrast upa activity was inhibited by both 0.5 m and 20 m s1p. activity of control cells was inhibited by both pretreatment and continuous treatment with both ptx and raci. upa activity of cells treated with 0.5 m s1p was increased only by pretreatment with ptx and raci. in contrast, in cells treated with 20 m s1p upa was inhibited only by continuous inhibition with ptx and raci. conclusions: invasion of s1p induced eoc cells correlated with the gelatinase and upa activities in their microenvironment. ptx and rac-i affect attached and invading cells in different manner, inhibition of invasion and gelatinolysis of attached and increased invasion of invading cells. this suggests a dual effect of the gi-rac pathway, inhibiting attached and stimulating invasion via gelatinolysis of invading cells. lysophosphatidic acid (lpa) levels are elevated in the ascites and plasma of early-and late-stage ovarian cancer patients, underscoring the unique role this bioactive lipid plays in the pathobiology of epithelial ovarian cancer (eoc). lpa binding to its cognate g-protein-coupled receptor can transactivate the receptor tyrosine kinase, egfr, which is often overexpressed in ovarian tumors. in the current study, we investigated the role that lpa activation of egfr plays in the processing of the metalloproteinase, mmp-2, and eoc dissemination. lpa stimulates tyrosine phosphorylation of egfr in ovarian cancer cells, and egfr kinase activity is required for optimal lpa induction of pro-mmp-2 activation. lpa-dependent pro-mmp-2 activation does not require the egfr ligands, amphiregulin, b-cellulin, egf, hb-egf, and tgf-a. we previously reported that when cells are cultured at high density, lpa represses rhoa activity to induce loss of stress fibers, and these changes in actin microfilament organization contribute to pro-mmp-2 activation. in the current study, inhibition of rho-kinase/rock with y-27632 reversed the repression of lpa-stimulated pro-mmp-2 processing observed with treatment of the egfr kinase inhibitor, ag1478. correspondingly, lpa induced the loss of stress fibers, while inhibition of egfr kinase restored stress fiber formation in lpa-treated cells. this suggests that lpa acts through egfr to modulate microfilament organization and pro-mmp-2 processing. finally, lpa-induced cellular haptotactic migration and invasion are abrogated when egfr kinase activity is blocked. taken together, these results suggest that egfr signaling plays a critical role in lpa regulation of metastatic pathways by mediating changes in the cytoskeleton which impact protease activity. objectives: membrane-derived vesicles are active modulators of tumor dissemination; they promote and contribute to extracellular matrix degradation and tumor cell invasion. we have isolated vesicles from ascites of ovarian cancer patients and from ovarian cancer cells in vitro. here we analyzed the functional consequences of exposure of cancer cells to vesicles derived from a different type of malignancy in order to evaluate their proinvasive properties. methods: ovarian cancer (dov13), breast cancer (mda mb 231) and mesothelioma (hp-1) cells were grown in media supplemented with 10% fbs. after serum deprivation, 10% vesicle-free fbs was added for 4 hr to induce vesicle release. vesicles were isolated from media by differential centrifugation and quantified with a bradford assay. fusion to cells was followed by fluorescence of the lipophilic tracer dii. each cell line was stimulated with 1 and 10 g/ml of each type of vesicles and tested in a matrigel invasion assay. changes in proliferation were evaluated in an mts assay. gelatin zymography was used to assess matrix metalloproteinase activity of vesicles. results: zymographic analysis showed that vesicles contained mmp-2 and 7. fusion experiments showed that all vesicles fused to all cell types. all vesicles induced the invasion of their respective cell types in a dose-dependent manner. when vesicles were used at 1 g/ml they induced significantly high levels of invasion in all cell types tested. at 10 g/ml they significantly inhibited invasion in different cell types. both concentrations of vesicles stimulated cell proliferation in all cell types tested. conclusions: membrane derived vesicles are potent mediators of invasion for mesothelioma, breast and ovarian cancer cells in vitro. this effect occurs in a dose-dependent manner when vesicles induce invasion of the same cell type from which they were isolated. when vesicles are used to induce a different cell type, low concentrations induce invasion while high concentrations inhibit invasion, this effect was independent of cellular proliferation. these results suggest that a novel mechanism may be at play where activation of recognition of self and non-self specific pathways may determine the invasive potential of the tumor cell upon fusion to microvesicles. the identification of signaling pathways responsible for this heterotypic signaling is a current effort. vegfr-2 as a potential target to inhibit lpa-induced epithelial ovarian cancer (eoc) invasion. sonia dutta, fengqiang wang, elaine barfield, david a fishman. obstetrics and gynecology, new york university school of medicine, new york, ny, usa. objective: vegf and vegf receptors (vegfrs) play important roles in ovarian cancer metastasis. in this study, we examined the expression profiles of vegf and vegfrs (vegfr1, vegfr2, co-receptor nrp1 and nrp2) in established eoc cells lines (dov13, r182, ovca429, skov3) and an immortalized normal ovarian epithelium (iose-29). the effect of lysophosphatidic acid (lpa) on vegf and vegfrs expression and the effect of vegfr-2 silencing by rnai on lpa-induced invasion were also evaluated. methods: vegf and vegfrs expression in ovarian cancer cell lines and normal ovarian epithelium was quantified by real time pcr. cell invasion differentiate into either a pro-tumor or anti-tumor phenotype depending on the specific tumor microenvironment. previously, we described a subgroup of epithelial ovarian cancer (eoc) cells with a functional tlr-4-myd88-nf-b pathway (type i eoc cells). these cells have constitutive nf-b activity and constitutively secrete il-6, il-8, mcp-1, and gro . we hypothesize that type i eoc cells, but not type ii, can promote macrophage differentiation into a tumor-supporting immune cell. methods: eoc cell conditioned media (cm) was prepared by incubating eoc cells in log-phase growth in optimem for 48h. migration assay was done using an in vitro cell culture insert with 8 m-size pore and pkh26 red fluorescentlabeled thp-1. cytokine profile of thp-1 cells co-cultured with eoc cell cm was determined using xmap technology. modulation of response to apoptotic bodies was determined by "pre-educating" thp-1 cells with eoc cell cm for 24 h prior to exposure to apoptotic bodies (1:1 ratio with thp-1 cells). results: monocytes migrate toward eoc cells. however, migration is significantly higher towards type i eoc cells. type i, but not type ii, eoc cells alter monocytes' cytokine profile by inducing the secretion of high levels of progrowth and angiogenic cytokines il-6, il-8, mcp-1, and gro . furthermore, type i eoc cells modify monocytes response to apoptotic bodies by inducing a significant increase in the secretion of il-6, il-8, mip-1 , mip-1 , and gro (18-fold, 2.5-fold, 2-fold, 3-fold, and 11-fold respectively). conclusion: we demonstrate for the first time a differential interaction between two subtypes of eoc cells and monocytes. we showed that the microenvironment created by type i eoc cells is able to modify the function and differentiation of immune cells towards a tumor supporting phenotype. understanding the molecular mechanisms mediating this tumor-immune interaction will help to design appropriate immune therapies that will take into consideration the tumor microenvironment. objectives: the standard of treatment for patients with ovarian cancer (oc) is intravenous combination chemotherapy (ct) after primary cytoreductive surgery. although initial response is above 80%, most of these patients experience recurrence. the only approach for these patients is salvage ct which may prolong their lives for months. early detection of patients who are not responding to current therapy or are at risk of experiencing an early relapse of disease might improve response rates and survival if alternative therapy is possible. no single predictive marker has yet been proven sufficiently sensitive or specific to find a place in the daily clinic. in the present study we use a newly described biomarker test for the detection of oc (visintin et al 2007 clinical cancer research) and evaluated the ability of the test to monitor ct response methods: 55 patients with oc, figo stage i-iv, were included in this study. all patients recieved postsurgery first line combination ct (paclitaxel/carboplatin). samples were evaluated at 1) baseline (mean 35 days after surgery), prior to the first cycle of ct, and 2) after 3 cycles of ct. 20 μl serum samples were analyzed by multiplex assay (beadlyte® cancer biomarker panel kit) for six markers. changes during ct and differences in markers between patients with short time to progression (ttp) and patients with long ttp were determined. results: positive test for oc was observed in 96 % of the patients evaluated at baseline. all patients had residual disease after surgery. from patients with long ttp, 15/20 patients (specificity 75 %) had a negative test after 3 cycles. from the patients with short ttp, 13/14 had a positive test (sensitivity 93 %) p=0.000096 ( ²). the risk of experiencing a ttp shorter than 6 months when having a positive test after 3 cycles of ct was 72 %. conclusion: we describe for the first time the use of a panel of biomarkers for oc that might have an application for monitoring ct response and risk of relapse. the test detects the presence of residual disease following debulking surgery, and differentiates between long term and short term progression. a longitudinal study is performed to determine how early during ct the test can identify responder versus non responder. ksp inhibitor (arry-520) as an alternative for paclitaxel in myd88-positive epithelial ovarian cancer cells. ki h kim, 1 ayesha b alvero, 1 yanhua xie, 1 david trollinger, 2 gil mor. 1 1 obstetrics, gynecology, and reproductive sciences, yale university school of medicine, new haven, ct, usa; 2 array biopharma inc., boulder, co, usa. background: we previously described that epithelial ovarian cancer (eoc) cells ubiquitously express tlr-4, but not the adaptor protein myd88. when treated with paclitaxel, a known tlr-4 ligand, myd88-positive eoc cells exhibited nf-b activation, increased secretion of il-6, il-8, mcp-1, and gro , and increased p-erk levels . since majority of eoc patients are given paclitaxel in combination with a platinum drug, it is not only important to distinguish those patients that should not be given paclitaxel; it is also important to identify alternative chemotherapy agents that would benefit this sub-group of patients. the objective of this study is to determine if the ksp inhibitor, arry-520, can be an alternative for paclitaxel in myd88-positive ovarian cancer patients. methods: myd88-positive and myd88-negative eoc cell lines isolated from either ascites or tumor tissue were treated with increasing concentrations of arry-520 (3 to 3000 nm) or paclitaxel (0.2 to 20 m) for 24, 48, and 72 hours and cell viability was determined using the celltiter 96 aqueous one solution cell proliferation assay. cytokine profiling was performed from supernatants using xmap technology. nf-b activity was determined using a luciferase reporter system. p-erk levels were measured by western blot analysis. results: arry-520 and paclitaxel exhibited the same cytotoxic effect on myd88-negative and positive eoc cells. the ic 50 at 48h for myd88-negative eoc cells was 0.003 m and 0.2 um for arry-520 and paclitaxel, respectively. for myd88-positive eoc cells, the ic 50 at 48h was 3 m and 20 m for arry-520 and paclitaxel, respectively. however, unlike paclitaxel, arry-520 did not induce nf-b activation or enhance the secretion of il-6, il-8, mcp-1, and gro , and did not induce erk phosphorylation on myd88 positive cells. conclusions: administration of paclitaxel to patients with a myd88-positive tumor could have detrimental effects due to the paclitaxel-induced enhancement of cytokine production which promotes chemoresistance and tumor growth. arry-520 has similar anti-tumor activity in eoc cells as that of paclitaxel. however, unlike paclitaxel, it does not induce cytokine production in myd88positive eoc cells, and therefore, the ksp inhibitor arry-520 may represent an alternative to paclitaxel in this subgroup of eoc patients. introduction: nf-b activation has been associated with cell proliferation, angiogenesis, metastasis and suppression of apoptosis in ovarian cancer. in addition, nf-b activity induces the production of pro-inflammatory cytokines which may contribute to chemoresistance. inhibition of nf-b may represent a new approach to prevent tumor growth and reverse chemoresistance. in the present study we described the characterization of a novel nf-b inhibitor, eriocalyxin b (erib) that is able to re-establish the apoptotic cascade in chemoresistant ovarian cancer cells by suppressing pro-inflammatory cytokines and antiapoptotic proteins. materials and methods: eoc cell lines were isolated from malignant ovarian cancer ascites. caspase activity was determined by caspase-glotm assay. cytokine production and secretion were determined using multiplex assay. erib effect on cancer cells was evaluated in a time and dose dependent manner using celltiter 96 cell proliferation assay. protein expression was determined by western blot analysis. combination studies were done with paclitaxel and carboplatin in addition to erib treatment. nf-b activity was determined by monitoring the expression of a nf-b luciferase reporter. results: erib decreased cell viability of ovarian cancer cells with an ic50 of 0.5-1μm in 48 hours and was associated to increasing levels of caspases 8,9 and 3 activity. intracellular changes induced by erib included: 1) inhibition of nf-b activity; 2) decrease in cytokine production; 3) down regulation of anti-apoptotic proteins xiap and flip, and reversal of resistance to tnf-and fasl-mediated cell death; and 4) chemosensitization to carboplatin and paclitaxel. at present, evidence is accumulating regarding the existence of unique populations of specialized tumor-initiating, stem-like cells within various tumor types of distinct origins. these cancer stem cells (csc), with characteristics reminiscent of normal stem cells, are thought to be responsible for driving tumor growth. we propose that ovarian cancers arise from csc, and are using microarray-based technology to identify specific genes/cell surface markers associated with ovarian csc that will permit distinction of these rare cells from the remaining tumor bulk. to identify unique gene signatures associated with an ovarian tumor-initiating cell population, we have utilized an in vivo serial transplantation model. this model selects for primary human ovarian tumor cells with increased tumorigenic capacity, given that time to tumor formation decreases with successive serial transplant despite fewer cells injected. our initial studies used cells derived from a human ovarian clear cell carcinoma, serially transplanted for three passages in nod/scid mice. rna derived from these transplanted tumors was analyzed on human genome microarrays. from these analyses, several differentially expressed genes were identified. the differential expression noted for potential genes of interest is currently being validated by rt-pcr. of particular interest, expression of the transmembrane glycoprotein muc4 was found to increase both at the rna and protein level with successive transplant of this clear cell carcinoma. further studies are ongoing to determine the functional significance of muc4 and other identified differentially expressed genes in ovarian clear cell cancer. in addition, we are carrying out parallel microarray analyses in other ovarian tumor subtypes. background recent observations suggest a decreased incidence of neoplastic lesions in hiv infected individuals treated with highly active anti-retroviral therapy comprised of protease inhibitors, such as ritonavir. the polycomb group protein ezh2 is associated with aggressive human malignancies via transcriptional suppression of dna repair proteins. objective objective of the present study was to assess the antineoplastic impact of ritonavir on epithelial ovarian cancer (eoc) cell lines. methods eoc cell lines (mdah 2774 and skov-3) were treated with serial dilutions of ritonavir (1-10 mm) dissolved in dmso. normal diploid human fibroblasts served as controls. growth curves, apoptosis and cell cycle analysis were performed with cell counting kit-8, annexin v and flow cytometry. signal transduction was studied with western blotting. dna double strand breaks (dsb) were induced with 0.5 mm etoposide treatment for 16 hours. homologous recombination (hr) repair of dna damage was measured by assessment of rad51 foci formation in the nucleus of the cells as visualized by fluorescence microscopy according to previously published criteria. untreated eoc cells expressed higher levels of ezh2 and lower levels of rad51 and xrcc2 as compared to controls and in turn, had lower prevalence of rad51 repair foci formation in response to dsb. serial treatments of eoc cells with ritonavir resulted in a decrease in expression of ezh2 and an increase in expression of rad51 and xrcc2 when compared to untreated eoc cells. after induction of dsb, the rad51 repair foci formation was significantly more prevalent in eoc cells treated with ritonavir as compared to untreated eoc cells. in addition, ritonavir induced apoptosis in ovarian cancer cell lines by down-regulation of akt pathway and caused g1 cell cycle arrest mediated by down modulating levels of prb phosphorylation and depleting the cyclindependent kinase 4 and 6. ritonavir effectively induces apoptosis, cell cycle arrest and improves repair of dna damage by hr in ovarian cancer cell lines. as impaired hr is a key event in causation and progression of neoplastic lesions, ritonavir may have a role in chemoprophylaxis and treatment of human malignancies. a rare case of ovarian cystic lymphangioma. tomer singer, 1 tamer seckin, 1 noa feldman, 1 susan jormark, 2 michael divon. 1 1 department of obstetrics and gynecology, lenox hill hospital, n.y., ny, usa; 2 department of pathology, lenox hill hospital, n.y., ny, usa. cystic lymphangioma (cl) is a rare, benign malformation of the lymphatic system whose exact etiology remains uncertain. cl may arise in different sites: the spleen, the mediastinum, the axillary region, the retroperitoneum, and the mesentery. retroperitoneal cl is extremely rare and its true incidence is unknown. the majority of cases are symptomatic during childhood. clinical presentation of adult cl is variable and may be misleading. typically, this is a slow-growing tumor and it remains asymptomatic for a long period of time. it is most often found incidentally during abdominal or pelvic imaging studies, surgeries or autopsies. total surgical removal of the lesions with microscopically clear margins is the best approach when it is possible. we report, for the first time, a case of cystic lymphangioma arising from the ovary in a post-menopausal woman and present the feasibility and the advantages of laparoscopic surgery, allowing accurate diagnosis, optimal treatment and minimal risk for the patient. lgsc) is a chemoresistant ovarian neoplasm that has been molecularly linked to low malignant potential tumor (lmp), which often behaves in a non-invasive fashion. micropapillary features within lmp (lmp-mp) are associated with increased invasive behavior. the aim of this study was to determine the differential gene expression of lmp, lmp-mp, and lgsc to identify genes involved in malignant transformation and carcinogenesis. methods: laser capture microdissection was used to isolate epithelial cells from snap-frozen lmp (n=18), lmp-mp (n=9) and lgsc (n=11). rna was extracted, amplified, reverse transcribed to cdna and hybridized to affymetrix u133 plus 2 arrays. data was analyzed by significance analysis methods: medical records were reviewed for patients who underwent hysterectomy for all indications at wsu hospitals from 1/1/05 -4/30/06. pathology reports were reviewed to identify the incidence of adenomyosis. data obtained from medial records included: age, race, insurance, bmi, social history, medical history, and presenting symptoms. the correlation between adenomyosis and all the above factors was tested using the appropriate statistical methods. results: 850 patients were included. adenomyosis was confirmed by pathology in 375 patients, an incidence of 44.1%. incidence was not significantly different among races after controlling for parity. 46.3% in african americans, 35.3% in caucasians, and 40.9% in others. incidence was not statistically different between uninsured(28.0%), privately insured (47.7%), and medicaid (41.9%) patients. p=.065. incidence of adenomyosis was not different between smokers (38.9%) and nonsmokers (44.6%). p= .386. table i shows the correlation between adenomyosis and different risk factors. conclusion: adenomyosis was diagnosed in 44.1% of hysterectomy specimens. race, socioeconomic status or social habits didn't affect its incidence. diabetes and endometrial cancer were negative risk factors for adenomyosis, whereas htn, hypothyroid, breast cancer, fibroids, polyps and endometriosis didn't affect its incidence. menorrhagia, dysmenorrhea, dyspareunia, and chronic pelvic pain but not metrorrhagia had a positive correlation with adenomyosis. there is a protective adipocytokine profile. we hypothesize that this finding may precede some ill-defined threshold of fat mass and/or insulin resistance after which adiponectin decreases. the objective: to correlate reproductive hormone production with menstrual bleeding patterns among women in the menopause transition. methods: a sub-cohort of the swan study consisting of 848 women age 42-52 was studied. each woman collected daily first void urine samples for one complete menstrual cycle or 50 days (whichever came first) once a year for 3 years. urine was assayed for excreted levels of fsh, lh, estrogen metabolites and progesterone metabolites which were normalized for creatinine concentration. ovulation was detected by a validated algorithm. menstrual bleeding parameters were derived from daily calendars. correlations between bleeding characteristics, hormone concentrations, and other potential clinical predictors were analyzed using multivariate logistic regression models. results: the cohort was ethnically diverse with a median age of 47 and with 73% in the early perimenopause at the start of the study. 21% of all cycles were anovulatory. short cycle intervals (< 21 days) were associated with the early perimenopause (or 5.2, ci 2.1, 12.9) and with anovulation (or 20.3, ci 7.2, 56.8). long cycle intervals (36+ days) were associated with late perimenopause (or 8.7, ci 1.0, 79.1)and with anovulatory cycles (or 2.6, ci 1.9, 3.5). both short (1-3 days) and long (8+ days) duration of menstrual bleeding were significantly associated with anovulation (or 2.4 and 2.6, respectively). women with anovulatory cycles were less likely to report heavy menstrual bleeding than women with ovulatory cycles. menorrhagia was not associated with steroid hormone concentrations but was associated with obesity (or 4.7, ci 2.6, 8.5) and with the self-reported presence of fibroids (or 4.1, ci 1.8, 9.5). conclusions: among women in the menopause transition, abnormalities in timing of menstrual bleeding (cycle intervals or bleeding duration) have a hormonal basis and are frequently associated with anovulation. in contrast, abnormally heavy periods do not have a hormonal basis and are less likely following anovulatory cycles. heavy periods are associated with obesity and fibroids. to undetectable levels. the use is vaginal e is contraindicated, although these women have even higher rates of av. topical testosterone (tt) has successfully treated vulvar atrophy; testosterone receptors are also present in the vaginal epithelium. objective: assess the effect of tt on vaginal maturation index (mi) and relief of av symptoms. methods: 10 postmenopausal women on aromatase inhibitor with symptomatic atrophic vaginitis were enrolled in prospective, irb approved study. estradiol (e) and testosterone (t) levels, av questionnaires (score 0-3; none to most severe symptoms of dryness, irritation, and dyspareunia), gynecologic exam, and vaginal smears (for ph and vaginal maturation index, mi, by meisels criteria) were performed at baseline and after 1 month of daily treatment with 300 mcg of tt. data was assessed by t test and fischer's exact test; significant p < .05. results: e levels were undetectable at baseline and following treatment. t levels increased (mean +/-sem) from baseline (16 +/-0.8) to treatment ( 38 +/-10.9); the difference was not significant; p = .8, although one patient had an appreciable rise in serum t level. two av symptoms improved significantly with tt use; comparing baseline to treatment scores: vaginal dryness (2.2 vs. 0.75; p = .03) and dyspareunia (2.3 vs. 1; p = .02 saliva analysis is a convenient, non-invasive and rapid method for assessing estradiol (e2) levels. however, particularly in postmenopausal women, the low salivary e2 levels are often near or below the sensitivity of available assays, compromising both accuracy and precision. we present results using an extraction step prior to e2 assay, which concentrates the sample to increase sensitivity and removes potentially interfering substances. morning saliva samples were obtained from premenopausal (mid-luteal phase, n=4,651) and postmenopausal women (n=1,770) not taking hormones, and from postmenopausal women receiving oral conjugated equine estrogens (cenestin, n=119; premarin, n=439), oral micronized e2 (estrace, n=145; compounded e2, n=1618), transdermal e2 patches (climara, n=623; vivelle, n=1619); or topical e2 cream (compounded e2, n=107). e2 levels were determined by an automated enzyme immunoassay (eia) after solid phase extraction. the functional sensitivity of the assay was determined to be 0.8 pg/ml, compared with >2 pg/ml without extraction. 3.9 0.5 5.7 1.0 9.6 1 oral conjugated equine estrogens; 2 oral e2; 3 e2 patch; 4 topical cream salivary e2 levels corresponded with the hormone dosage, suggesting a reliable assessment of unbound e2 levels with each formulation, dosage and type of estrogen therapy. extraction prior to eia in an automated assay dramatically increased precision and accuracy at low concentrations. omitting the extraction step may have contributed to poor serum versus saliva correlations in other studies. this method may therefore allow reliable monitoring of estrogen therapy without the need for expensive and inconvenient blood tests. the role of fibulin-3 in pelvic organ prolapse. david d rahn, 1 jesus f acevedo, 1 patrick w keller, 1 lihua marmorstein, 2 r ann word. 1 1 ob-gyn, ut southwestern, dallas, tx, usa; 2 visual sciences, univ arizona, tuscon, az, usa. objectives: fibulin-5 (fib-5) is crucial for normal elastic fibers in the protein showed a statistically significant reduction in its expression (p= 0.01). lox, loxl1, 3 and 4 proteins were detected by immunohistochemistry in all three layers of vaginal skin biopsies: (1) stratified squamous epithelium; (2) the lamina propria and (3) the muscularis layer from both patients with pop and controls. significantly, in both groups we detected a numerous macrophages scattered throughout the vaginal thickness which displayed a very strong immunostaining to loxl1. patients with severe pop showed reduced expression of proteins regulating collagen and elastin biogenesis. our finding raises the possibility that failure of ecm homeostasis could underlie the etiology of pop in women. fibroblast proliferation is regulated by hoxa11: molecular implications for pelvic organ prolapse. kathleen a connell, marsha k guess, richard bercik, hugh s taylor. obstetrics, gynecology reproductive sciences, yale university school of medicine, new haven, ct, usa. objectives: the integrity of the extracellular matrix (ecm) is maintained by a delicate balance between collagen synthesis and degradation. previously, we have demonstrated that hoxa11 is essential for the development of the uterosacral ligament in mice and regulates the expression of collagen type iii and mmp2. we have also shown that hoxa11 is deficient in the uterosacral ligament of women with pelvic organ prolapse (pop) compared to women with normal support. the exact mechanisms by which hoxa11 regulates pelvic organ integrity and repair remains to be elucidated. the aim of this study was to determine the effect of hoxa11 expression on fibroblast proliferation, and its potential role in pop. methods: nih 3t3 cells, a murine fibroblast cell line, were seeded onto a six well plate (1x 10 5 cells/well) and transfected with either a vector carrying a hoxa11 cdna or empty vector alone as a control. immunohistochemistry using bromodeoxyuridine (brdu) was performed to evaluate cell proliferation. cell division in the uterosacral ligament (usl) was also compared in women with and without pop. usl specimens were obtained at the time of hysterectomy for benign disease. immunohistochemistry was performed on paraffin embedded sections of the usl to evaluate expression of two mitotic markers, cyclin b1 and phospho-histone 3. results: constitutive expression of hoxa11 in murine fibroblasts resulted in significantly higher proliferation. cells transfected with hoxa11 had a mean brdu incorporation of 40.8+ 8.8 cells/ 100 cells compared with 32.2 + 7.5 cells/ 100 cells in controls (p=0.03). in the usl obtained from women with and without pop, cell proliferation as determined by cyclin b1 and phospho-histone3 expression was not significantly different. cyclin b1 and phosphor-histone 3 were expressed in comparable numbers of cells in both groups. conclusion: hoxa11 is necessary for usl development and promotes proliferation of adult fibroblasts. hoxa11 may have a similar function in vivo in usl, and may regulate fibroblast proliferation during growth and the acute phase response following trauma when fibroblasts are activated to proliferate and remodel the ecm. it is likely that hoxa11 mediated proliferation of usl fibroblasts contributes to the tensile strength and resilience of these structures and thereby prevents pop. supported by nichd wrhr: 5k12hd047018-03. connective tissue composition, in term of collagen i, iii and proteoglycans content, in support and nonsupport structures of women with uterine prolapse. elisabetta trabucco, 1 gennaro acone, 1 sara d'avino, 1 marco torella, 1 gennaro trezza, 2 annamaria marenna, 1 nicola colacurci, 1 luigi cobellis. 1 1 department of obstetrics and gynecology, second university of naples, naples, italy; 2 loreto mare hospital, naples, italy. objective. connective tissue consists mainly of collagen and elastic fibers, glycoproteins and proteoglycans (pgs) and is considered an important factor of the support and nonsupport structures of the genitourinary region. it has been already demonstrated altered morphologic features in the pelvic support connective tissue in women with genital prolapse. however, analysis of nonsupport tissue may provide a more accurate reflection of body collagen. the objective of our study was compare the expression of collagen i, iii and four pgs (decorin, fibromodulin, lumican, biglycan), essential for synthesis and regular assembly and diameter of the collagen fibrils, in uterosacral ligaments and in a nonsupport tissue, the uterine cervix, between premenopausal women with uterine prolapse respect to age matched controls. we characterized uterosacral ligaments and uterine cervix of 25 premenopausal women with uterine prolapse and 16 controls. this immunohistochemical study was performed on paraffin-embedded sections. results. uterosacral ligaments of the prolapsed uteri are characterized by a higher expression of collagen iii, decorin, fibromodulin and byglican and a lower quantity of collagen i. no differences in the immunoreactivity of lumican between the two patients groups. the abnormalities of support connective tissue composition are not observed in the uterine cervix of patients with prolapse. conclusions. our results suggest an altered remodeling of connective tissue in the ligaments of premenopausal patients with prolapse, with a significant decrease in collagen i content and an increase in collagen iii and pgs expression . in the prolapse patients this abnormal collagen metabolism and organization, mainly related to the observed change in pgs expression, might affect significantly the tensile strength of the connective tissue and consequently the support that is provided by the suspensory apparatus to the uterus. the alterd remodeling of support tissues, not detectable in nonsupport tissues, may suggest a predominant role of local biomechanical stresses (childbirth, chronic straining) in the pathogenesis of prolapse respect to systemic connective tissue deficiency. objective: to achieve vaginal delivery with minimal maternal injury, vaginal smooth muscle (sm) contractility likely decreases. the objective of this study was to characterize changes in rat vaginal sm contractility in pregnancy that affords circumferential vaginal distension. methods: a tubular segment of the vagina of 8 virgin, 8 late pregnant , 8 immediate and 8 late post vaginal delivery rats were mounted in an organ bath between a force transducer and an adjustable support block. dose response curves to norepinephrine (ne) and potassium (k+) were used to assess contractility. relaxation capacity was determined in ne preconstricted vagina, using cumulative doses of sodium nitroprusside (snp). differences in active vs. passive length-tension curves were used to measure sm contractility relative to the vaginal wall forces. sm -actin levels were measured using quantitative confocal immunofluorescence. results: cumulative doses of ne induced a maximum constriction force of 33.1 ± 6 mn/g in virgin vagina while no measurable force was generated in response to ne in late pregnant or postpartum vaginas. virgin vagina relaxed with cumulative doses of snp; no measurable relaxation response was observed from pregnant or postpartum animals. in the presence of the high dose k+ (124mm), virgin vagina generated the greatest contractile force (43.92 ± 3 mn/g) vs. late pregnant vagina (22.6 ± 2.4 mn/g, p<0.001) or immediate post partum vagina (26.81 ± 4 mn/g, p=0.033). four week postpartum k+ induced forces were similar to virgin levels (43.89 ± 3 mn/g). sm force generation (difference in active vs. passive length tension curves at 3mm of displacement) was decreased in late pregnancy (15.3 ± 4 mn/g) compared with virgin (112 ± 29 mn/g, p < 0.023) and 4 week postpartum vagina (172 ± 34 mn/g, p < 0.004). the expression of sm -actin was lowest in late pregnancy (15 ± 1.5, p < 0.001) and immediate postpartum vagina (23 ± 1.5, p < 0.001) relative to virgin (33 ± 3) with a return to virgin levels 4 week postpartum (56 ± 2). conclusions: vaginal sm contractility diminishes in pregnancy. the altered contractility is mirrored by a decrease in sm -actin protein expression. near complete recovery to pre-pregnancy levels occurs by 4 weeks post partum. these functional and biochemical changes likely represent maternal adaptations designed to minimize trauma during passage of the fetus(es). background: diagnosis or exclusion of endometriosis usually requires laparoscopy and peritoneal biopsy. we have described a novel and consistent observation of small nerve fibers in the functional layer of eutopic endometrium in women with endometriosis (tokushige et al, 2006) . these nerve fibers are not present in women without endometriosis. this finding allows the possibility endometriosis in the nude-mouse and inhibited mmp-3 expression in human endometrial stroma. the present findings may lead to the development of novel treatments of endometriosis involving statins. supported by nih u54hd052668. k-ras actived immunocompetent retrograde menstruation model of endometriosis. ching-wen cheng, 1,2 di licence, 1,2 cristin g print, 1,3 d stephen charnock-jones. 1,2 1 pathology, university of cambridge, cambridge, united kingdom; 2 obstetric gynaecology, university of cambridge, cambridge; 3 department of molecular medicine pathology, university of auckland, auckland, new zealand. objective: to establish a new murine model of endometriosis that mimics the retrograde menstruation theory using immuno-competent mice. method: ovariectomised donor female k-ras v12+/-/cre +/+ /rosa26r-lacz +/+ transgenic mice were treated sequentially with steroid hormones. to induce decidualization and activation and k-ras transgene, 16 mg/kg b-nf dissolved in maize oil was injected into the uterine lumen. tissue degeneration mimicking menstruation was induced by hormone withdrawal. menstruating endometrial tissue was collected from donor mice 60 hours after last hormone treatment, re-suspended in matrigel, and implanted subcutaneously in female c57bl/6 recipients. immunohistochemical and morphometric methods were used to characterize the endometriosis-like lesions. microarray analysis (illumina, n=6 in each group), was used to study the molecular changes in "menstruating" uteri following k-ras activation. result: simple transplantation of decidualised endometrial tissue into immunocompetent animals does not lead to endometriotic lesion development but using tissue with the genetic modification described here overcomes this. viable endometriosis-like lesions are visible 28 days after implantation. these lesions are histologically similar to those seen in man with intact glands, functional blood vessels, leukocyte infiltration and collagen deposition. statistical analysis revealed that 220 transcripts were differentially regulated in the ras activated and control tissue. gene ontology (go) analysis indicated that transcripts associated with epithelial cell function and differentiation were over represented within this gene set (chloride transport and epidermis development) as were those associated with the acute inflammatory response and neutrophil chemotaxis. we developed a new model of endometriosis using immunocompetent mice to mimic retrograde menstruation induced endometriosis. this permits for the first time, the ready use of transgenic and knock-out tools to investigate the cellular and molecular mechanisms underlying endometriosis. since the animals have an intact immune system it also allows the testing of therapeutic agents that modify the inflammatory response. stra6 is expressed and induced by progesterone in the endometrium and is absent in endometriosis. mary ellen pavone, serdar e bulun, scott s reierstad, joy innes, magdy p milad, you-hong cheng. obstetrics and gynecology, northwestern univeristy, chicago, il, usa. objective: retinoic acid (ra) plays important roles in development, growth and differentiation by regulating the expression of target genes. in the mouse endometrium, ra deficiency leads to hyperkeratinization, while high concentrations of retinoids promote secretory characteristics. this leads many to believe that ra mediates important actions of progesterone in the endometrium and may account for progesterone resistance in endometriosis. the mechanism for regulation of ra production by progesterone is unknown. moreover, the conversion from retinol to retinoic acid is not different between normal endometrium and endometriotic tissue. we hypothesize that retinol intake into cells rather than conversion of retinol to ra is the critical step that determines ra activity in the endometrium and endometriosis. stra6, a widely expressed multitransmembrane domain protein and member of a group of "stimulated by retinoic acid" genes, has been identified as the retinol binding protein receptor. it is strongly expressed in adult mice in several tissues including the female genital tract. we hypothesize that ra activity in the endometrium may be regulated by stra6. here we investigate the differential expression of stra6 in the endometrium and endometriosis. design: we studied primary stromal cells isolated from the eutopic endometrium of disease free women and walls of ovarian endometriomas from women with endometriosis with respect to stra6 expression and regulation by progesterone. materials and methods primary culture of eutopic endometrial and endometriotic stromal cells were treated with 10 -7 m estradiol (e 2 ), 10 -7 m r5020, a progesterone agonist, or vehicle for 24 hours. real-time pcr was employed to quantify stra6 mrna levels. we treated cells for 48 hours and quantified protein levels by immunoblotting. results basal mrna of stra6 levels in endometrial cells (n=8), were >1,000 fold higher than those in vehicle-treated endometriotic cells (n=8, p<.001). in endometrial stromal cells (n=8), r5020 stimulated stra6 mrna levels by 1.7-3 fold. r5020 induced stra6 protein levels in endometrial stromal cells (n=3). conclusions stra6 is highly expressed and regulated by progesterone in endometrial stromal cells, whereas it is practically undetectable in endometriotic cells. stra6 may mediate important actions of progesterone in endometrium. upregulation of aromatase expression in endometrial cells disseminated into the peritoneal cavity (pc) may influence their survival and persistence via local estrogen synthesis. the inducing factors for the upregulation of aromatase in endometriosis are not well defined, but increased expression of sf-1 has been suggested to play an important role. given that the aromatase substrate androstenedione (a4) is the predominant steroid in peritoneal fluid, we aimed to determine whether a4 has a role in the regulation of aromatase expression in human endometrium. we found that culture of primary endometrial stromal cells and explants with a4 (5-10 nm) for 24 h significantly upregulated expression of aromatase mrna transcripts containing exon iia at their 5'-ends. moreover, in human endometrial surface epithelial cells (hes), dose-response studies with a4 (5-100 nm) revealed that 10 nm a4 maximally upregulated expression of both aromatase and sf-1. when tissue samples were evaluated from women with endometriosis and control endometrium, expression of aromatase mrna mirrored sf-1. treatment of hes cells (24 h) with tritiated a4 demonstrated its metabolism to estradiol (e 2 ), testosterone (t), dihydrotestosterone (dht) and androstanediol (a-diol). although equimolar concentrations of a4, t and e 2 upregulated aromatase and sf-1 mrna expression in hes cells, the nonaromatizable androgens, dht and a-diol, had no effect. a positive feedback role of estrogen in aromatase upregulation was suggested by the finding that the estrogen receptor antagonist, ici 182,780, markedly diminished aromatase and sf-1 mrna expression induced by a4. finally, chromatin immunoprecipitation revealed that a4 significantly enhanced recruitment of sf-1 to its response element (-136 bp) upstream of cyp19 exon iia. collectively, our findings strongly suggest that exposure of endometrial cells within the pc to c 19 -steroids may cause an acute upregulation of cyp19 gene expression through their aromatization to estrogens. thus, estrogen may play a critical positive feedback role in the pathogenesis of endometriosis. supported by nih r01-dk031206. the hpa axis and depression/anxiety scores in chronic pelvic pain patients. b stegmann, 1 b frank, 2 j gemmill, 1 g chrousos, 1 m ballweg, 3 p stratton. 1 1 rbmb, nichd/nih, bethesda, md; 2 som, wake forest university, winston-salem, nc; 3 endometriosis association, milwaukee, wi. stress, pain, anxiety and depression adversely affect the hypothalamic-pituitaryadrenal (hpa) axis. we examined the influence of depression and anxiety on the hpa axis response to corticotropin-releasing hormone (crh) testing in women with and without chronic pelvic pain (cpp). methods: healthy women, aged 18-50, with without cpp, with regular menses and off hormonal contraception were studied. none had pelvic infections, untreated depression, manic depression, fibromyalgia, or chronic fatigue syndrome. after ovine crh injection (1 mcg/kg), serial blood samples (-5, 0, +15, +30, +45 minutes) were obtained for acth and cortisol measurements. depression and anxiety were scored using the duke quality of life questionnaire. hpa response was abnormal if peak acth levels were > 55 pg/ml without a rise in cortisol levels. 4 subject groups were: no pain and normal acth response (npnr), pain and normal acth response (pnr), no pain with an abnormal acth response (npar) and pain with an abnormal acth response (par). student t-test was used for comparisons. results: 31 women (21 cpp, 10 controls) had a mean age of 33.5 ± 9.1 years (range: 21-47). 18 women responded normally (10 pnr, 8 npnr) and 13 women responded abnormally (11 par, 2 npar). peak and absolute rise in acth were significantly higher in abnormal (a) vs normal (n) responders; (peak: 85.2 a vs 33.5 pg/ml n, p<0.001; absolute: 51.2 a vs 27.5 pg/ml n, p=0.024). there was a significant difference within groups: peak acth: 88.5 par vs 37.1 pg/ml pnr, p=0.001; 67.5 npar vs 20.3 pg/ml npnr, p<0.001; absolute acth: 72.2 par vs 28.2 pg/ml pnr, p=0.001, 56.1 npar vs 20.4 pg/ml npnr, p<0.002). however, total cortisol level was not different between or within the 2 groups (23.2 a vs 20.0 mcg/dl n, p=0.08). mean depression score did not differ among cpp patients (34.7 par vs 35.2 pnr, p=0.95), but differed among controls (25 npar vs 4.3 npnr, p=0.01). anxiety scores did not differ between or within groups (29.5 npar vs 8.4 npnr p = 0.12; 40 par vs 39.7 pnr, p=0.66). conclusions: chronic pelvic pain is associated with an abnormal hpa response, regardless of anxiety or depression symptoms. abnormal hpa responses in control women, however, appear to be influenced by depression. the mechanism and clinical significance of these findings should be explored. support: rbmb/nichd/nih and endometriosis association. objective: the eutopic endometrium in women with endometriosis demonstrates altered endometrial receptivity and altered gene expression. it is unknow if the endometrium is defective giving rise to a predisposition toward endometriosis or alternativly if the endometriosis causes the altered endometrial receptivity. here we created experimental endometriosis in a mouse model through allotransplantation of the uterus to the peritoneal cavity in immunocompetent mice and examined the expression of several markers of endometrial receptivity in the eutopic endometrium. materials and methods: the uterus of 8-week-old cd1 female mice was transected at cervicovaginal junction and each horn divided and transplantated into the abdomidnal cavity of eight cd1 mice. seven controls received sham surgery only. after 14 weeks the uterus was removed, snap-frozen in trizol. total rna was extracted and cdna generated. quantitative real time rt-pcr using sybrgreen was performed and normalized to -actin. fold changes in normalized hoxa10, hoxa11, bteb1, emx2, igfbp1, integrin -3, total progesterone receptor (pr-ab) and progesterone receptor-b (pr-b) were assessed. all experiments were conducted in duplicate, repeated at least three times and compared using mann-whitney rank sum test. results: hoxa10, hoxa11, bteb1, emx2, and igfbp1 mrna expression showed 0.57-fold (p=0.046), 0.25-fold (p<0.001), 0.50-fold (p=0.043), 0.23fold (p=0.038), and 0.09-fold (p<0.001) decrease in the uterus of mice with experimental endometriosis compared with controls. pr-ab and pr-b mrna showed 2.7-fold (p=0.016) and 8.39-fold (p=0.006) increase in endometriosis group compared to controls, respectively, however the ratio of pr-b to pr-ab (b/ab) showed no significant change in both groups (20.98 vs. 7.53 , endometriosis vs. control, p>0.05). there was no significant change in integrin -3 mrna expression (1.09-fold, p>0.05). conclusion: we demonstrate significant changes in multiple markers of endometrial receptivity in eutopic endometrium after induction of endometriosis. these findings suggest that origionally normal endometrium can develop defects with the creation of endometriosis; an abnormal endometrium is not a prerequisite for the development of endometriosis or associated abnormalities. this data also suggest the existance of a signal conduction pathway from endometriosis that alteres endometrial gene expression. endometrial expression patterns of relaxin and relaxin receptor mrna suggest involvement of relaxin in endometriosis. sara s morelli, 1 felice petraglia, 2 gerson weiss, 1 stefano luisi, 2 pasquale florio, 2 jeff gardner, 1 andrea wojtczuk, 1 laura t goldsmith. 1 1 obstetrics, gynecology and women's health, new jersey medical school, newark, nj, usa; 2 pediatrics, obstetrics and reproductive medicine, university of siena, siena, italy. objective: in normal endometrium, relaxin is a potent inhibitor of matrix metalloproteinases, which have been implicated in the invasive process of endometriosis. we tested the hypothesis that relaxin plays a role in endometriosis by comparing expression of relaxin and its lgr7 receptor in normal human endometrium to levels in samples from patients with endometriosis. materials and methods: total rnas, extracted from ectopic (n=8) and eutopic (n=11) endometrium of patients with endometriosis and from endometrium of normal controls (n=12), were reverse transcribed into cdnas. real-time pcr was performed using primers previously shown to identify human lgr7 relaxin receptor mrna, h2 relaxin mrna, and beta-actin mrna, with sybr-green detection of double stranded dna products. the comparative c t method (2 -ct ) determined relative lgr7 and relaxin mrna expression (normalized to beta-actin mrna expression). relaxin mrna was detectable in normal endometrium from 9 of 12 (75%) control patients. in contrast, relaxin mrna was detectable in a lower proportion of samples [9 of 19 (47.4%)] from patients with endometriosis, among whom relaxin mrna was detectable in a lower proportion of ectopic samples [2 of 8 (25%)] than in eutopic samples [7 of 11 (63.6%)]. lgr7 relaxin receptor mrna was detectable in all samples, with lower expression in endometriosis samples than in endometrium from normal controls. relaxin receptor lgr7 mrna levels vary with cycle phase, with greater expression in the secretory phase (sp) than in proliferative phase (pp): in normal controls 5.9-fold higher levels in the sp than pp; and in endometriosis patients 3.6-fold higher levels in the sp than pp. in both phases, lgr7 mrna levels were lower in ectopic samples than in either eutopic samples (6.2-fold lower in pp and 19.0-fold lower in sp) or endometrium from normal controls (6.5-fold lower in pp and 15.6-fold lower in sp). eutopic endometrium had similar lgr7 mrna expression to controls throughout the cycle. decreased local expression of relaxin and relaxin receptor mrna in ectopic endometrium from patients with endometriosis throughout the menstrual cycle suggests that relaxin may be protective against endometriosis. ovarian reserve after laparoscopic cystectomy of endometriotic ovarian cysts. shinichi hayasaka, takashi murakami. obsterics and gynecology, tohoku university, sendai, japan. objective laparoscopic ovarian cystectomy is generally recommended for endometriotic ovarian cysts because it has been associated with a higher pregnancy rate and a lower recurrent rate. however, residual ovarian function after laparoscopic cystectomy of endometriotic ovarian cysts has been questioned. in this study, we retrospectively evaluated ovarian response to hyperstimulation in women selected for ivf and icsi cycles who previously underwent laparoscopic cystectomy of a monolateral endometriotic ovarian cyst. methods a retrospective review of the patients between january 2003 and september 2007 was performed. the operated ovary and contralateral intact ovary were compared in terms of number of oocytes retrieved, number of follicles with a mean diameter > 15mm at the time of hcg administration. the patients who had recurrent endometriotic ovarian cysts were excluded. in total, ten patients were identified. the mean(±sd)number of oocytes retrieved was 4.1±2.4 in the control ovary and 1.1±1.4 in the previously operated ovary(p=0.031). the mean(±sd)number of follicles with a mean diameter > 15mm was 5.6±2.5 in the control ovary and 1.8±1.1 in the previously operated ovary(p=0.0004). the age of patients and diameter of the operated ovary had little influence on the difference between the response of the control ovary and one of the previously operated ovary. laparoscopic cystectomy of endometriotic ovarian cysts is associated with a significant reduction in ovarian reserve. background pre-eclampsia (pe) is associated with systemic maternal endothelial dysfunction, which is thought to result from the presence of circulating factors released following placental damage. it is hypothesised that this occurs as a result of reduced oxygen (o 2 ) delivery. some features of placental pathology seen in pe, such as increased apoptosis, can be reproduced by culture of placental explants in 1% o 2 . metabolomics operates to study 'all' metabolites within a biological system. this strategy has previously identified differences in maternal plasma between normal pregnancies and those complicated by pe. in these experiments we used metabolomics to investigate differences in the metabolic profile of explants cultured in different o 2 tensions. hypothesis the metabolic profile of placental villous explants is altered by culture in different o 2 tensions. methods placental villous explants were cultured with either 10% serum (n=10) or in serum-free conditions (n=6) for 96h in 20%, 6% and 1% o 2 . after 96h, conditioned culture medium and tissue were collected and snap frozen. tissue was homogenised in 50% ice cold methanol prior to analysis. samples were analysed using gas chromatography-mass spectroscopy (gc-ms). samples cultured at 20%, 6% and 1% o 2 were compared to identify concentration differences in metabolites in conditioned cultured medium and tissue lysate. kruskal-wallis test was used for statistical analysis. due to the large number of metabolites identified, a p value of 0.001 was considered significant. results the mean intra-assay variability was 9.1%. there were no differences in the metabolic profile of conditioned culture medium from 6% and 20% o 2 . several metabolites differed in culture medium from 1% compared to 6% and 20% o 2 including: deoxyribose, glycerol and threionic acid. these changes were present in both serum and serum-free conditions. using these metabolites alone, culture in 1% o 2 could be completely discriminated from 6% and 20% o 2 . deoxyribose was also elevated in tissue lysate from 1% o 2 compared to 6% o 2 . conclusion this metabolomic strategy can identify differences in metabolic profile in placental tissue cultured in 1% o 2 . these novel compounds may provide further insight into pathophysiology of pe. objective a strategy of metabolic footprinting (the study of extracellular metabolites, which are related to intracellular metabolism) was used to detect a wide array of low molecular weight metabolites. metabolic profiles were employed to differentiate between placental explants cultured at different oxygen tensions. two separate studies were combined to validate initial observations. methods metabolic footprints of placental villous explants, obtained from uncomplicated pregnancies, (n=5) were cultured for 96h in 1%, 6% and 20% oxygen. conditioned culture medium was then collected and frozen, prior to analysis by uplc/ltq-orbitrap mass spectrometry in both negative and positive ion mode. samples cultured at 1%, 6% and 20% were compared to identify differences in the metabolic profiles. this procedure was repeated for 6 different explants and the results compared across the 2 studies in order to validate initial findings. approximately 350 unique peaks were detected in both sample sets in negative ion mode and 300 peaks in positive ion mode. a number of metabolites were identified as being significantly different using kruskal-wallis (pval<0.01) under different oxygen tensions. some differences were seen between the results obtained from both runs due to separate batch preparation of the medium but good reproducibility was obtained for many metabolites between batches. metabolites of biological interest included amongst others 2-deoxyribose, valine, tyrosine and aspartic acid. the largest differences were those seen between o 2 1% and o 2 6%. comprehensive metabolic profiles were detected and employed to identify differences in the metabolic footprints of explants cultured under differing oxygen conditions. a number of biologically interesting metabolites were characterized. objective: brain weight and dna content were reduced in leptin deficient (lep ob /lep ob ) mice postnatally. leptin is detected in the sera and its receptors are expressed in the brain of the mouse fetus. we examined the role of leptin in the proliferation and differentiation of neural lineage cells in the mouse fetus. methods: the number of total cells in the cerebral cortex was compared between (lep ob /lep ob ) and wild type fetuses from embryonic day (e) 14 to 18. the number of brdu positive cells was also compared on e14 and e16. brdu uptake, the ratio of viable colony number to plated cell number, the proportion of multipotent, neuronal or glial progenitor colonies, and the expression levels of hes mrna were compared between leptin-and non-treated neurosphere cells. moreover, we examined stat3 phosphorylation by leptin stimulation with elisa to investigate whether or not jak/stat3 pathway transduces the leptin signal in the prenatal period as in the adult. results: lep ob /lep ob fetuses had reduced total cell numbers in the ventricular zone (vz) on e16 and e18, and in the cortical plate on e18. the number of brdu-positive cells was reduced in vz of e14 and e16 lep ob /lep ob fetuses. brdu uptake and the number of viable colonies were increased by 2 days-leptin treatment in the neurosphere culture. the proportions of glial-restricted and multipotent precursor colonies were increased by leptin, whereas that of oligodendrocyte precursor colonies were decreased. hes1 mrna expression was enhanced in neurosphere cells by leptin. neither the amount of phosphorylated stat3 nor that of stat3 was increased in neurosphere cells by leptin stimulation. conclusion: our study suggests that leptin maintains the neural stem and glial-restricted precursor cells through upregulation of hes1 expression and increases the number of cerebrocortical cells in the mouse fetus, however, jak/stat3 pathway does not mediate the leptin signal in undifferentiated neural lineage cells. 311a vaginal wall, and >90% of fib-5 knockout (ko) mice develop pelvic organ prolapse by 20 weeks of age. in contrast, fib-1 and -2 deficiencies do not result in prolapse, and fib-4 kos die shortly after birth. herein, we evaluated the role of fib-3 in pelvic organ support. methods: two observers serially measured the degree of vaginal, perineal, and anal prolapse in 227 fib-3 ko (fib3 -/-) and in fib3 -/-mice severity of prolapse was significantly related to age, but not parity, and the average age at diagnosis was 37 wks. fib3 -/-animals with advanced prolapse had attenuated uterosacral ligaments and descent of the bladder and uterus caudal to the symphysis. pro-mmp2 (2-fold, p=0.03), active mmp2 (2-fold, p=0.06), and prommp9 (3-fold, p=0.05) were increased in vaginal tissues from mice with gross prolapse compared with age-, strain-, and cycle-matched controls. this increase in protease activity, however, was also accompanied by increased expression of fib-5 and tropoelastin (1.7-fold, p<0.001). regardless of prolapse maternal morbidtties tf23 ards-n(%) 12 (52) acute ren~m f11lure-n (%) 10(43) car~tovascular ~ysfunrtion-n ("/o) 15 ( 65) hepatc fa..lure-n (%) dt sst'dlm atl:d inlfavascul..-coagul tipalhy-n(%) 6 (26) durallon of !cu suy (days. mean+sd) 9 21:t 12 .8 hospital my (days.. me411+sd) 2. condon, j. c., hardy, d. b., kovaric, k., and mendelson, c. r. (2006) mol endocrinol 20(4), 764-775. objective: innervation of the uterine cervix is important for the process of parturition (physiol beh 63:929,1998; j histochem cytochem 52:1249 ,2004 jsgi 12:578,2005) . the hypogastric nerve is a major pathway that innervates the uterine cervix, yet its contribution to processes associated with cervical ripening and parturition is not known. the objective of this study was to determine the effect of hypogastric nerve transection on processes associated with cervical remodeling and parturition. methods: time-dated pregnant rats were sham-operated or the hypogastric nerve bilaterally transected just anterior to the inferior mesenteric ganglion on day 15 post-breeding. live pups were born spontaneously by all rats at term on days 22-23 post-breeding. on the day of birth, the cervix was excised, postfixed overnight, sectioned, and processed to evaluate collagen content and structure (nih image j). sections were also processed by immunohistochemistry to assess cell nuclei density, the census of resident macrophages, and area of tissue that contained nerve fibers. results: hypogastric neurectomy did not affect cell nuclei density, the number of macrophages, or density of nerve fibers in the cervix. the failure of hypogastric nerve transection to affect indices of cervical remodeling is consistent with the finding that duration of pregnancy and timing of birth were not different in sham-operated and hypogastrectomized rats. conclusions: nerve fibers in the hypogastric nerve that innervate the lower uterus and cervix, including sympathetic and neuropeptidergic projections, are thus not essential for birth. these novel findings provide support for the contention that innervation of the uterine cervix other than through the hypogastric nerve contributes to processes associated with cervical ripening and parturition. receptor system in prostaglandin synthesis in pregnancy. eugenie r lumbers, 1,2 della m yates, 2 carolyn m mitchell, 2 jonathan j hirst, 1,2 tamas zakar. 2,3 1 school of health sciences, university of newcastle, newcastle, nsw, australia; 2 mothers and babies research centre, hunter medical research institute, newcastle, nsw, australia; 3 obstetrics and gynaecology, john hunter hospital, school of medical practice and public health, university of newcastle, newcastle, nsw, australia. the fetal membranes and decidua contain prorenin, which requires proteolytic activation. ngyuen (j clin invest. 109:1417) described a renin/prorenin receptor that conformationally activates prorenin, so that it can form ang i from aogen. in addition, receptor-bound prorenin can stimulate intracellular pathways directly. prostaglandins (pgs) participate in the control of term and preterm labour, and renin can stimulate pg production by amnion and decidua when angiotensin's action is blocked mitchell placenta 17:299) . the prorenin receptor may mediate these effects. our aim was to find out if the prorenin receptor gene is expressed and colocalised with prorenin and prostaglandin h2 synthase-2 (pghs-2) in human placenta, amnion, chorion and decidua. we have also determined if there is any change in the expression of the prorenin and prorenin receptor genes with labor. placentae with attached membranes were collected after term birth either by elective caesarian section or by spontaneous labor, and prorenin, prorenin receptor and pghs-2 mrna levels were quantitated relative to beta-actin mrna by real-time rt-pcr in amnion, chorion, decidua and placenta. before labor, mean prorenin mrna levels were 13.8 times higher in decidua and 4.0 times higher in chorion and placenta than in amnion (p<0.001). there were no significant changes with labor. proenin receptor mrna was expressed in all tissues. proenin receptor mrna levels in prelabor amnion, chorion and placenta were similar, while levels in decidua were 50% of those in amnion (p=0.02). this low level of prorenin receptor mrna in decidua persisted after labor (p<0.001). pghs-2 mrna expression was highest in amnion; in decidua and placenta it was only 6 and 4% of amnion. similar differences were present after labor. we conclude that the decidua is the principal source of prorenin within the pregnant uterus, and all gestational tissues are targets for prorenin. decidual prorenin may affect pghs-2 expression in amnion through the prorenin receptor forming a maternal-fetal paracrine system that stimulates pg production at labor. the immuno-suppressive effects of progesterone on umbilical cord blood mononuclear cells and the effect of culture media estradiol content. nadav schwartz, 1 xiangying xue, 2 christine n metz. 2 1 obstetrics and gynecology, nyu school of medicine, new york, ny, usa; 2 feinstein institute for medical research, manhasset, ny, usa. objective: progesterone (p4) is known to supress the maternal immune response and similar effects have been seen with fetal cells. we sought to ascertain whether p4 action was modulated by the phenol red and/or estrogen content of the culture media. methods: umbilical cord blood mononuclear cells were isolated using a density gradient centrifugation. the cells were incubated with p4 (10 -4 m) 1 hour prior to overnight stimulation with lps. supernatants were assayed for tnf-using elisa. the following culture media were used: a)'red' media: rpmi+10% fbs, b)'yellow' media: phenol red-free rpmi+10% fbs, and c)'clear' media: phenol-free rpmi+10% dextran/charcoal treated fbs. finally, the 'clear' media experiments were repeated with estradiol add-back. results: p4 suppressed tnf production in all medias. tnf levels were suppressed to 39.4% (p<0.06) of controls using 'red' media and to 42.0% (p<0.03) in 'yellow' media. the degree of suppression was not as substantial using 'clear' media where tnf levels were 64.5% (p<0.02) of control. (fig 1) adding estradiol to the 'clear' media had little or no effect on the degree of tnf suppression. (fig 2) conclusions: progesterone exerts an immuno-suppressive effect on lpsstimulated fetal mononuclear cells which is more pronounced when using media containing untreated fetal bovine serum. the phenol-red/estradiol content of the culture media did not modulated the p4 effect. dextran/charcoal treatment of fbs appears to deplete the media of factors other than estradiol that are necessary for p4 to exert its full effects. culture conditions should be optimized when studying progesterone's effects on immune response. 743 ovine fetal regional blood flow following prenatal dexamethasone (dex) at 0.75 gestation (g). antonine d van heesewijk, 1 jan g nijhuis, 1 susan l jenkins, 2 peter w nathanielsz, 2 mark j nijland. 2 1 ob/gyn, academisch ziekenhuis maastricht, maastricht, netherlands; 2 ob/gyn, cpnr, uthscsa, san antonio, tx, usa. background: pregnant women at risk for premature labor receive antenatal glucocorticoids (gc) to reduce neonatal morbidity and mortality. while fetal blood pressure (bp), heart rate and vascular endothelial and control fetuses (n=6). dispersed adrenal cortical cells (2.5 x10 5 cells/tube; in duplicate) were challenged with 10 -8 m acth. samples were collected at time 0 (baseline), 5, 15, 30, and 60 min after acth treatment, and tissue and media samples were frozen for determination of cortisol, camp, and star. results: cortisol output (ng/2.5x10 5 cells) was higher in the lth group compared to the control, (p< 0.01) at 15min (3.3 ± 0.5 vs. 1.0 ± 0.1), 30min (7.7 ± 0.6 vs. 1.9 ± 0.1), and 60min (10.9 ± 0.7 vs. 2.5 ± 0.1). peak camp levels (fmol/2.5x10 5 cells) were observed at 30 min but did not differ between control (81.8 ± 10.4) and lth (77.9 ± 17.8) adrenal cells. western analysis demonstrated that star protein expression was higher in the lth adrenal cortex compared to control (p<0.05) at 0 min (1.02 ± 0.07 vs. 0.59 ± 0.05), and at 60 min (1.26 ± 0.04 vs. 0.65 ± 0.05), relative optical density units. conclusions: results from the present study taken together with those of previous in vivo studies suggest that the enhanced cortisol output in lth fetuses is the result of increased adrenal acth sensitivity. this enhanced sensitivity is not due to differences in camp generation. star, which is a key element involved in cholesterol transfer at the level of the mitochondrial membrane appears to play a key role in the enhanced cortisol response to acth in the lth group. (nih grants hd31226, hd33147 and llu-nih imsd 2r 25gm060501-05). expression. hiroaki itoh, 1 makoto kawamura, 2 shigeo yura, 2 haruta mogami, 2 tsuyoshhi fujii, 2 norimasa sagawa. 3 1 obstetrics and gynecology, national hospital organization osaka national hospital, osaka, japan; 2 gynecology and obstetrics, kyoto university graduate school of medicine, kyoto, japan; 3 obstetrics and gynecology, mie university school of medicine, tsu, japan. objective: epidemiological evidences suggested that undernutrition in utero develops risk factors for adult cardiovascular disorders, such as blood pressure increase. the present study was designed to prove the hypothesis that maternal iso-caloric high protein diet alleviates blood pressure increase in the adult offspring by affecting placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-hsd2) expression. methods: the 30 % calorie restriction was applied to pregnant mice by using either standard protein diet (spd; 20% casein protein; spd-un) or high protein diet (hpd;40% casein protein; spd-un). in some groups, these pregnant mice were sacrificed at 18.5 d.p.c.; then maternal and fetal plasma as well as placental tissues were corrected. while in other groups, systolic blood pressure was measured in the offspring at 16 wks. fetal and maternal corticosterone levels were measured by elisa. the placental 11beta-hsd2 gene expression was measured by quantitative taqman pcr. results: systolic blood pressure in the spd-un offspring at 16 weeks (106 mmhg) was higher than that in spd-nn offspring (99.3 mmhg). by contrast, systolic blood pressure in the hpd-un offspring (97.5 mmhg) was similar to that in spd-nn offspring. maternal calorie restriction with spd and hpd caused similar elevation of maternal plasma corticosterone concentrations. by contrast, fetal plasma corticosterone levels in hpd-un offspring (151 ng/ml) was lower than those in spd-un offspring (208 ng/ml), indicating the amelioration of fetal exposure to high corticosterone by maternal hpd. the placental 11beta-hsd2 gene expression in the hpd-un was 72% higher than that in spd-un, suggesting that maternal hpd augmented placental 11beta-hsd2 gene expression in the placenta and probably facilitated the inactivation of maternal cortocsterone in the process of placental transfer to fetal circulation. conclusion: the preset study suggests that maternal high protein ingestion may elevate placental 11beta-hsd2 gene expression and ameliorate blood pressure increase in the adult offspring via modification of fetal exposure to maternal corticosterone. elevated cortisol levels at birth exert critical maturational effects through action at glucocorticoid receptors, gr. in contrast, the low cortisol concentrations in the preterm fetus, before the time of increased fetal cortisol secretion, are near to the kd for cortisol at the higher affinity mineralocorticoid receptor, mr. we hypothesized that endogenous cortisol in the fetus exerts physiologic actions at mr in tissues without appreciable cortisol-inactivating enzyme, 11 hsd2, including the fetal lung and brain. we therefore tested the effect of infusion of a mr-antagonist, ru26752 (mra, 1.68 mg/h over 12h) into 120-130 day fetal sheep. we tested for effects on lung liquid production rate, lung liquid and plasma electrolytes, plasma acth and cortisol, and hematocrit at 10-12h after start of the infusion. although there was no significant difference in the liquid production rate in fetuses infused with ru26752, the ratio of na + to k + in lung liquid was significantly greater in mra-treated fetuses than in the control fetuses (34.7±1.6 vs 30.2±1.7). plasma acth was significantly greater in the mra-treated fetuses than in control fetuses at 12h (657±262 vs 171±20 pg/ml); in the mra-treated fetuses, acth concentrations at 12h were greater than in the same fetuses at 0h (124±16 pg/ml), wheras there was no increase in plasma acth in the control fetuses (0h: 100±19 pg/ml). similarly, plasma cortisol concentrations at 11 and 12h were greater in the mra infused fetuses than in control fetuses (12h: 18.2±5.5 vs 7.0±1.4 ng/ml), and cortisol increased significantly over time in the fetuses infused with mra, but not in control fetuses (0h, mra: 4.7±1.5, control: 2.9±0.5 ng/ml). infusion of mra did not alter fetal plasma electrolyte, fetal blood pressure or fetal heart rate. however, fetal packed cell volume was significantly increased at 9-12h of infusion of mra (12h: 37±1 vs 32±1 %) and fetal pco2 was significantly greater at 12h of infusion of mra as compared to control fetuses (61.0±2.0 vs 53.5±0.8). these results suggest that endogenous cortisol concentrations in the preterm fetus exert negative feedback control of acth via action at mr in the fetal brain, and play a role in regulation of fetal lung liquid composition and in fetal fluid balance. preparturient increases in fetal acth secretion are cyclooxygenase-2 (cox-2) dependent. charles e wood. dept. physiology and functional genomics, university of florida college of medicine, gainesville, fl, usa. maturation of the fetal hypothalamus-pituitary-adrenal axis is critical for the timely somatic development of the fetus and readiness for birth. in sheep, increased preparturient activity of the fetal hpa axis appears to be responsible for triggering parturition itself. this study was designed to test the hypothesis that prostaglandin generation, mediated by cox-2 in the fetal brain, is critically important for stimulation of the preparturient increase in fetal acth secretion. singleton fetal sheep were chronically catheterized with vascular, amniotic, and lateral cerebral ventricular catheters. nimesulide, a selective cox-2 inhibitor, was infused (1 mg/day, n=9), and vehicle (50% dimethylsulfoxide, n=6) was infused. arterial blood samples were drawn from fetuses at 48 hour intervals for measurement of plasma hormone concentrations and arterial blood gases. in the vehicle-treated fetuses, fetal plasma acth, pomc, and cortisol concentrations increased exponentially (p<0.001 by anova) before spontaneous parturition at 146±0.3 days. in the nimesulide-treated fetuses, fetal plasma acth and pomc were constant and did not increase prior to parturition (p=ns by anova). in contrast, fetal plasma cortisol increased independent of acth (p<0.001 by anova) and the fetuses were born spontaneously on 148±0.3 days gestation. the slight delay in spontaneous parturition in the nimesulide-treated fetuses was statistically significant (p<0.01 by mantel-cox test). intracerebroventricular nimesulide infusion did not decrease fetal plasma concentrations of prostaglandin e2, demonstrating that the action of the nimesulide was restricted to the fetal brain. fetal blood gases were normal in all of the fetuses, and there were no differences in blood gases between groups. we conclude that the spontaneous increase in fetal acth and pomc prior to parturition is cox-2 dependent. however, we also conclude that the increase in fetal plasma cortisol concentration can occur independent of increases in fetal acth, suggesting that the increase in cortisol can result from increases in adrenal sensitivity to acth. the results of this study demonstrate that the timing of parturition in the sheep is not dependent upon increased acth secretion, and the results suggest that parturition is regulated primarily by changes in adrenal sensitivity. expression of extracellular signal-regulated kinase1/2 and p38 mitogen-antiphosphotyrosine. immunolocalization of inos was performed in the same tissues. hpmec were used to determine effect of sin-1 (1mm) on inos protein expression (30 min, 4 and 8 h). results: inos protein was detected in microvascular endothelium, smooth muscle and syncytiotrophoblast of the placenta. inducible nos levels in placentas from severe preeclampsia were significantly decreased compared to normal (p<0.05) but were not different from mild preeclampsia. aligning western blots of anti-phosphotyrosine and inos revealed a phosphorylated band corresponding to the molecular weight of inos. the proportion of tyrosine phosphorylated inos was reduced by 35% in severe preeclampsia compared with normotensive. preliminary data with ip for phosphotyrosine and crossblotting with inos confirmed this finding. sin-1 treatment decreased inos protein abundance at 4 and 8 h in hpmec. conclusions: our results demonstrate decreased tyrosine phosphorylation of inos in preeclamptic placenta. phosphorylation of tyrosine in inos has been reported to negatively regulate its activity. we therefore postulate that decreased phosphorylation of inos may be responsible for the increased catalytic activity of the enzyme that we have previously observed. the decreased levels of inos observed on sin-1 treatment may be due to nitration, an effect analogous to preeclampsia, where the presence of peroxynitrite has been well established. it remains to be seen if protein nitration has an effect on enzyme stability. objective: 8-isoprostane (8-iso), a prostaglandin-like compound formed in vivo, is a reliable measure of oxidative stress which occurs in response to many different stimuli, including infection. periodontal disease is a chronic oral infection associated with fetal growth restriction and preeclampsia. our objective was to determine if maternal periodontal disease is associated with oxidative stress as measured by 8-iso. methods: a secondary analysis was conducted using prospective data from the oral conditions and pregnancy study. a cohort of healthy women enrolled <26 weeks underwent oral health examination and serum sampling. maternal periodontal disease was categorized as healthy, mild, or moderate-severe by clinical criteria. maternal serum was analyzed for 8-iso by ultra-sensitive elisa. elevated 8-iso was defined by a value 75 th %tile. maternal factors associated with elevated 8-iso were determined using chi-square or t-tests as appropriate. a logistic regression model was created to determine adjusted odds ratios (95 th %ci) for elevated 8-iso. results: 791 women had complete data for analysis. the median 8-iso level (iqr) was 1,806 (16 -81,870 pg/dl). using bivariate analysis, moderate-severe periodontal disease, non-white race, use of wic/food stamps, unmarried status, obesity, and lack of insurance were associated with elevated 8-iso. the logistic regression model for elevated 8-iso is shown below. adjusted or (95 th ci)* mild periodontal disease 1. maternal periodontal disease and utilization of public assistance are associated with oxidative stress in pregnancy. the relationship between periodontal disease, social hardship, oxidative stress, and adverse pregnancy outcome remains to be determined. antioxidant therapy could represent novel therapy for the prevention of adverse pregnancy outcome. severe transient immunological thrombocytopenia in a preeclampsia patient whose bone marrow production of platelets had been restricted. toshihiro yoshimura. obstetrics and gynecology, ntt-west kyushu general hospital, kumamoto-city, kumamoto, japan. introduction: idiopathic myelofibrosis is a chronic myeloproliferative disorder in which clonal haemopoetic stem cell proliferation is accompanied by reactive fibrosis. case report: a 23-year-old primiparous woman was referred to our hospital for prenatal care after 12 weeks of gestation. her medical history was significant for idiopathic myelofibrosis, and congenital agenesis of the spleen. the laboratory workup showed the patient's white blood cell count to be 5,500/ l; hemoglobin, 8.0 gm/dl; and platelet count, 38,000/ l. her bleeding time was normal (3 min). until the 34th week, the patient's pregnancy was uneventful. during the 35th week, however, the patient's platelet count declined to 16,000/ l, when proteinuria became evident. her bleeding time was prolonged to 13 min. the coagulation system was normal. by the 37th week, the patient's blood pressure was elevated to 150/100 mmhg, and her urinary protein excretion exceeded 2 g/day. our initial diagnosis was thrombocytopenia due to bone marrow suppression. the patient had no history of platelet transfusion, but we expected it would increase the platelet count, particularly since in the absence of destruction by the spleen, the lifespan of the platelets would be prolonged. this was not the case, however. elective induction of labor was carried out after 38 weeks. at the same time, the patient was transfused with 10 units of platelets, but her platelet count remained unchanged (20,000/ l). it was then that we realized that immunological thrombocytopenia may be involved, and massive doses (400 mg/kg) of gamma globulin were given intravenously for one day. thereafter, platelets (15 units) were again transfused, this time raising the platelet count to 110,000/ l. cesarean section was promptly carried out under general anesthesia. we later found that the patient's serum platelet associated immunoglobulin (paigg) level was positive, though antiplatelet and antinuclear antibodies were negative. the postoperative course was uneventful. the maternal platelet count declined to the pretransfusion level within 2 days after delivery, but gradually increased to the prepregnancy level by 2 weeks. comment: this idiopathic myelofibrosis patient in whom bone marrow production of platelets had been severely restricted demonstrates that immunological destruction may play a major role in the development of thrombocytopenia in preeclampsia. early l-arginine therapy improves notably the fetal growth in preeclamptic women. a randomized controlled trial. keisy lopez-molina, 1 juan c gonzalez-altamirano, 2 julio e valdivia-silva. 1,3 1 oncoinmunología y biología vascular, facultad de medicina, universidad nacional san agustín, arequipa, peru; 2 cardiología y cirugía de tórax, hospital nacional case essalud, arequipa, peru; 3 inmunología, instituto de investigaciones biomédicas, méxico, distrito federal, mexico. objective: to assess the benefit of early administration to l-arginine, the precursor to nitric oxide, on fetal growth. methods: one hundred women with preeclampsia were randomized to receive either l-arginine or placebo until day 1 postpartum. 96 live singleton infants were born after preeclamptic pregnancies and compared those with other 50 control infants. birth size was expressed as the ratio between observed and expected birth weights, and infants smaller than two standard deviations from expected birth weights were classified as small for gestational age (sga). all the women had neither previous precedents of the preeclampsia nor other factors that they cause sga. results: no significant differences existed between the groups with preeclampsia before randomization. preeclampsia was associated with a 8% (95% confidence interval [ci] 6%, 9%) reduction in birth weight. 4 women with hellp syndrome had to leave the study. the risk of sga was three times higher (relative risk [rr] = 3.4; 95% ci 2.8, 7.0) in infants born after preeclampsia without l-arginine therapy than in control pregnancies, and two times higher (relative risk [rr] = 1.8; 95% ci 0.7, 2.5). in infants born after preeclampsia with l-arginine therapy. conclusion: fetal growth improve markedly with l-arginine therapy in women with preeclampsia. introduction: although the pathogenesis of severe preeclampsia (pe) and intrauterine growth restriction (iugr) is poorly defined, inadequate remodeling of uterine spiral arties may promote reperfusion injury leading to focal infarction and reduced nutrient flow between mother and fetus. our goal was to determine whether an intravillous inflammatory cytokine cascade was associated with pe and iugr. methods: immunohistochemistry (ihc) examined il-1 and il-1 expression in 3 preterm placentas with severe pe+iugr, and 3 idiopathic preterm controls (ptc) with no evidence of clinical or histological chorioamnionitis. cultures of fibroblasts (fibs) and syncytiotrophoblasts (scts) from term placentas (n=5) were treated with il-1 and cytokine levels in conditioned media were determined using elisa or multiplex array. results were analyzed by student's t test or anova. results: the gestational age at delivery was not significantly different in pe+iugr and ptc groups (28.3±2.2 vs 27.6±2.8 wks). ihc of pe+iugr samples revealed intense villus core staining of il-1 adjacent to infarcts. villi distal to infarcts stained with a lower intensity. in contrast, staining was virtually undetectable in ptc. the pattern of il-1 staining was nearly identical in pe+iugr and ptc groups, was localized to the syncytium, and did not differ with respect to distance from infarct. to identify cellular targets of il-1 action, primary cultures of fibs and scts were incubated for 48 h in serum-free medium ± 1 ng/ml il-1 . by elisa we observed that il-1 treatment of fibs increased il-8 levels from 4±1 to 2161±346 pg/ g protein (p<0.001). similarly, multiplex array revealed that levels of il-4, il-6, and il-11 were induced 24-, 269-, and 12-fold, respectively in fibs. the presence of 200 ng/ml il-1 receptor antagonist reduced the il-1 -mediated increase in il-8 levels 95±3% (p<0.001), indicating that this response was mediated through il-1 receptor. in marked contrast, il-1 treatment did not significantly affect levels of il-4, il-6, il-8 or il-11 in scts, indicating that this response was cell type-specific. conclusions: these results indicate that increased expression of il-1 in the placental villus core in pe and iugr may promote an inflammatory cascade in fibs at this site leading to focal infarction and reduced flow of nutrients to the fetus. introduction: in human pregnancy, decreased responsiveness to angiotensin ii (angii) starts in week 10, promoting an expanded vascular bed. at the same time levels of the vasodilatory hemopexin increases 1 . during pre-eclampsia the vascular bed is contracted and the responsiveness upon angii persists. this may be due to the increased levels of extra cellular atp, a natural inhibitor of hemopexin 1 . in the present study we tested whether extra cellular atp is toxic in the pregnant condition. methods: pregnant rats were infused with either atp (750 g/kg bw; n=9) or saline (n=5) on day 14 of pregnancy (permanent jugular vein cannula/1 hr infusion). non-pregnant rats (n=8) were infused with atp identically. one day before and 1, 3, 6 days after infusion, 24 hr urine and blood samples (wbc count and hemopexin activity) were collected. seven days after the infusion, rats were sacrificed and kidney tissue processed for immunohistology. results: urinary albumin excretion was increased in pregnant atp infused rats only (fig 1) . wbc were also increased only in pregnant rats infused with atp (days 15 and 17 vs pre-infusion value). in pregnant atp infused rats intraglomerular influx of monocytes was increased, which correlated with urinary albumin excretion on the same day (r 2 = 0.66). staining of glomeruli for the angii receptor (at-1r) showed decreased at-1r expression in control pregnant rats as compared to non-pregnant rats, while at-1r expression in atp infused pregnant rats was increased as compared to control pregnant rats. hemopexin activity was increased on days 17 and 21 in control pregnant rats as compared to all other groups. discussion: these data support the notion that atp is toxic exclusively in the pregnant condition. it may be suggested that atp induced an inflammatory response, although the exact mechanism by which atp induced its effects needs further investigation. background: hepatocyte growth factor (hgf) is a multifunctional cytokine that is known to promote division, motility, invasion and morphogenesis of a wide range of cell types and to inhibit apoptosis. the effects of hgf are mediated through its interaction with the tyrosine kinase receptor c-met. in pregnancy hgf/met system is involved in the physiologic growth and development of the feto-placental unit. hgf/met effects are counteracted by transforming growth factor-1 (tgf-1), that is known to promote progressive fibrosis in human tissues. moreover tgf-1 plays a major role in trophoblast growth and differentiation. tgf-1 inhibits hgf expression as well as hgf inhibits tgf-1 expression. an understanding of the mechanisms regulating placental balance of these growth factors may provide insights into the processes that occur in complications of pregnancy, such as preeclampsia (pe) and fetal growth restriction (fgr). objective: to verify the hypothesis that hgf, c-met and tgf-1 are differently expressed in tissues of normal and pe placentas. methods: we studied 13 placentas from pregnancies complicated by pe (7 with normal fetal growth and 6 with fgr) and 5 placentas from normal pregnancies. from each placenta random samples were excised and rna was extracted; quantitative real-time rt-pcr analysis was used to investigate mrna expression of hgf, c-met and tgf-1 in pe (with or without fgr) and in normal placentas. results: gestational age, neonatal weight and placental weight were, as expected, lower in pe groups. the mrna expressions of the three molecules are higher in pe than in normal placentas, but the difference is statistically significant only for c-met. the higher values are mainly due to the increase in the pe group without fgr for c-met and tgf-1 and the increase in the pe-fgr group for hgf. conclusion: increased levels of expression of hgf/met system in pathological placentas could be explained as an attempt to repair placental damages; nevertheless placental regeneration could be inhibited by the increase of tgf-1, that promote fibrosis. placental expression of hgf, c-met and tgf-1 is increased in all pe placentas, but particularly in placentas of pe with normal fetal growth, where placental tissue is probably less compromised. obesity is a risk factor for preeclampsia (pe), but the reason for this risk is unknown. previously, we found that neutrophils infiltrated into the vasculature of pe women released myeloperoxidase (mpo) and matrix metalloproteinase 8 (mmp8), products that can cause oxidative stress and vascular dysfunction. if neutrophils infiltrate the vasculature of obese women and release mpo and/or mmp8, this may help explain why they are at risk of developing pe. hypothesis: systemic vascular tissue of obese women will have a significant presence of mpo and mmp8 as a result of neutrophil infiltration. methods: subcutaneous fat, which is highly vascularized, was obtained at abdominal surgery from 5 normal weight, 5 overweight and 5 obese women. formalin fixed, paraffin embedded 8 μm sections of fat biopsies were stained using immunohistochemistry with specific antibodies for mpo and mmp8. data were evaluated for intensity of vessel staining by visual score (0-4), density of staining using image analysis software, and % vessels with neutrophil staining, liberated from serum-free placental villous explant cultures from normal and pe pregnancies on human endothelial cells, and identified candidate mediators of their differential effects on metabolism and behaviour. methods: term placental villous tissue from normal (n=9) or pe (n=10) pregnancies were explanted for 4 days at 6% oxygen. conditioned 24h medium (day 3-4) was applied to human umbilical vein endothelial cells (huvecs). an angiogenesis assay was conducted in which tubule length and number were measured by morphometric imaging following seeding on 80% matrigel. the effect of explant conditioned medium on endothelial cell metabolism was determined by mtt and bioluminescent atp assay. the release of vasoactive metabolites (nitrite, endothelin-1, prostacyclin) from huvecs exposed to this medium was also measured. finally, a luminex bead array was used to screen the explant media for a panel of 15 chemokines/cytokines. results: in the angiogenesis assay, tubule length (p<0.05, mann-whitney u test) and number (p<0.05) were significantly decreased in pe compared to normal pregnancy medium. there was no change in mtt reduction, but endothelial cellular atp levels were also significantly reduced following exposure to pe explant medium (p<0.05). both normal and pe-derived medium stimulated huvecs to produce vasoactive metabolites. the following cytokines were detectable in the explant media: interleukin (il)-6, il-8, gro-, monocyte chemotactic protein-1 and macrophage inflammatory protein-1 (mip-1 ). higher levels of both il-8 and gro-were present in the normal medium compared to pe (both p<0.0001). mip-1 was present in pe conditioned media but undetectable in media generated from normal placental explants. conclusions: these results suggest that pre-eclampsia stimulates the release of soluble factors from the placenta which have adverse effects on endothelial cell angiogenic potential and metabolism. altered levels of several cytokines were detected in the explant medium, and the effects of these on endothelial cell function are currently being addressed. yang gu, davis f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: placentas from women with preeclampsia (pe) release more chymotrypsin-like protease (clp). the purpose of this study was to determine if placenta-derived clp was responsible for altering endothelial (ec) barrier function in pe. approaches: endothelial junctional protein complex ( -catenin/ve-cadherin/ p120) expression and junctional protein ve-cadherin distribution were examined in ecs treated with pe placental conditioned medium. methods: 1) confluent ecs were treated with pe placental conditioned medium (cm). the association of ec junctional protein complex ve-cadherin/catenin/p120 was examined by a combined immuno-precipitation (ip) and immuno-blotting (ib) assay, in which total cellular protein was immunoprecipitated with monoclonal antibody against -catenin and then immunoblotted with antibodies against ve-cadherin or p-120. 2) confluent ecs grown on cover slips were exposed to pe placental cm with or without depletion of chymotrypsin. ec junction protein ve-cadherin distribution was examined by fluorescent microscopy. results: 1) ve-cadherin and p120 are expressed in control ecs but not in ecs exposed to pe cm, which indicate that the junctional protein complex vecadherin/ -catenin/ p120 is lost in ecs exposed to pe cm. 2) ecs exposed to pe placental cm showed a discontinuous distribution and reduced expression for ve-cadherin at cell contact areas. the zipper like structure was lost and cleft was formed at cell-cell contacts. these observations indicate that the homotypic cell-cell adhesion and junction protein intracellular partner complex are disrupted. these disruptive phenomena in cells treated with pe conditioned medium were not present in control cells and in cells treated with cm after depletion of chymotrypsin. conclusion: clp released by the placenta could be a candidate agent that is responsible for disrupting ec integrity and inducing endothelial permeability in pe. (supported by nih grants hl65997 and hd36822). introduction: chronic pelvic pain (cpp) syndromes such as endometriosis, irritable bowel syndrome, and interstitial cystitis are associated with visceral hyperalgesia, and often coexist in the same patient. one possible explanation for this phenomenon is viscero-visceral cross-sensitization in which increased nociceptive input from an inflamed pelvic organ sensitizes neurons that receive convergent input from an unaffected organ to the same dorsal root ganglion (drg). nociception induces upregulation of perk and substance p. the purpose of this study was to determine, in a rodent model, whether uterine inflammation increased the number of perk-and sp-positive neurons in sensory ganglia innervating both uterus and colon. methods: colonic and uterine drgs were retrogradely labeled with fluorescent tracer dyes micro-injected into the colon and uterus. ganglia were harvested, cryoprotected and cut for fluorescent microscopy. results:approximately 7% neurons were colon-specific and 11% were uterus-specific. among these uterus-or colon-specific neurons, up to 5% of labeled drg neurons in the l1-s3 levels innervated both visceral organs. uterine inflammation increased the number of perk and sp-immunoreactive neurons in drg neurons innervating colon, uterus, and those innervating both organs. furthermore, this effect was specific as non-retrograde labeled drg neurons did not manifest a significant increase in perk or sp immunoreactive cells. conclusions: localized uterine inflammation leads to increased expression of sp and perk in uterine afferents as well as dichotomizing afferents innervating both uterus and colon, suggesting that viscero-visceral convergence is present at the level of drg primary afferent cell bodies. this visceral sensory integration may underlie the co-morbidity of female pelvic pain disorders and may provide basic information regarding the etiology of cpp syndromes. purpose: success rates of medical and surgical modalities for the treatment of severe fecal incontinence due to obstetric trauma are modest. we tested whether the intrasphincteric injection of autologous myoblasts is clinically safe and feasible for postobstetric fecal incontinence. methods: using ultrasound guidance a suspension of autologous myoblasts was injected in the anal sphincter muscle complex in three women with severe postobstetric fecal incontinence. main outcome measures were safety, feasibility and the wexner grading score. secondary outcome measures were incontinence episodes, rockwood fecal incontinenece quality of life scale, external anal sphincter morphology and anal pressure values. results: all procurement procedures and injections were performed without complications; there were no clinically or laboratory signs of infection or inflammation. three months post myoblast injection, wexner continence grading scores and rockwood incontinence scales were markedly improved and incontinence episodes reduced in all three patients. no change could be observed in sonographic anal sphincter morphology. mean anal squeeze pressure improved. conclusion: autologous myoblast injection for fecal incontinence is clinically feasible and safe; further studies will evaluate the efficacy of this approach. objective: to evaluate whether hysterectomy is associated with a change in postoperative bmi. methods: a retrospective cohort study was conducted of 548 hysterectomies performed for benign indications from june 2001 to june 2006. institutional review board approval was obtained. the data were collected by a medical student blinded to the hypothesis. basic demographic data were recorded. body mass index (bmi) was determined at 4 time points: time 0 (immediately postoperatively), time 1 (2 weeks to 5 months postoperatively), time 2 (5 months to 1 year postoperatively) and time 3 (1 to 2.5 years postoperatively). one-way anova was performed using ncss statistical software. a bonferroni multiple comparison test (p<0.05) was used to compare median changes in bmi from baseline. the starting bmi served as the control group. results: there was a statistically significant increase in bmi at time 3 compared to baseline in all women (31.9 to 33.6). when sorted was measured by matrigel invasion assay. small interference rna targeting vegfr-2 were predesigned by ambion inc. and cells were transfected using ambion siport neofx according to the optimized protocol. results: of the four receptors (vegfr-1, vegfr-2, nrp-1 and nrp-2), vegfr-2 was the predominant receptor that expressed in the more invasive cell lines (dov13, skov3 and r182). lpa, at 10-20 m, significantly induced vegfr-2 expression in dov13 and skov3 cells (p<0.05), without significantly affecting vegfr-1 expression. lpa (1-20 m) also significantly induced the expression of vegf 121 and vegf 165 (p<0.05) in dov13 and skov3 cells. by small interference rna (sirna) transfection, we demonstrated that inhibition of vegfr-2 expression could significantly decrease dov13 cells' invasiveness (p<0.001) and moderately decrease skov3 cells' invasiveness. moreover, silencing of vegfr-2 by sirna significantly suppressed lpa-induced dov13 and skov3 invasion. conclusion: these results suggest that knocking down vegfr-2 expression by rnai may be an effective strategy to inhibit lpa-induced ovarian cancer metastasis. cancer. fengqiang wang, david fishman. obstetrics and gynecology, new york university school of medicine, new york, ny, usa. objectives: expression of matrix metalloproteinases, such as mmp-1 and mmp-7, has implicated in epithelial ovarian cancer (eoc) invasion and metastasis. osteopontin (opn) was also expressed in various human cancers and associated with tumor progression, invasion and metastasis. in the present study, we examined the correlation of mmp-1, mmp-7 and osteopontin expression with tumor stage in 41 ovarian tumor tissues and 7 normal ovaries using a commercial tissue scan array. methods: complimentary dna (cdna) from normal ovaries (n = 7) and ovarian tumors (n = 41) of different stages (stage i, n = 16; stage ii, n = 3; stage iii, n = 19; stage iv, n = 3) was amplified by real time pcr using gene specific primer pairs for mmp-1, mmp-7, and osteopontin. gapdh was also amplified as a reference control. the expression level of mmp-1, mmp-7 and osteopontin was calculated as relative expression normalized to that of gapdh in each tissue sample, and the expression in sample that has the lowest target gene expression was arbitrarily set as 1. the average expression of mmp-7 in ovarian tumor tissues (14630 ± 30606) is 49 fold of that in normal ovaries (301 ± 641, p = 0.0047). using an arbitrarily set standard, 1 of 7 normal ovaries (14.3%) has elevated mmp-7 expression; in ovarian tumor tissues, the percentage is 82.9% (34 of 41), significantly higher than in normal tissues (p = 0.0008). in early stage tumors (stage i/ii, n = 19), 17 of them have elevated mmp-7 expression (89.4%, p = 0.0008 vs. normal ovaries), whereh the average expression of mmp-7 is 63 fold of that in normal ovaries (p = 0.069) and 1.8 fold of that in late stage tumors (stage iii/iv, n = 22). the mmp-1 expression in ovarian tumors (n = 41, 154 ± 632) is 28 fold of that in normal ovaries (n = 7), however, the difference is not significant (p > 0.05) due to the extremely high expression of mmp-1 in two tumor tissues. osteopontin expression in ovarian tumor tissues is 11.2 fold of that in normal ovarian tissues (11 ± 12.51, n=7 vs. 123.6 ± 160.0, n = 41, p<0.0001). in early stage (i/ii) ovarian tumors, osteopontin expression is 8.1 fold of that in normal ovaries, while in late stage (iii/iv) ovarian tumors, its expression is 13.9 fold of that in normal ovaries (p<0.05), suggesting an increase trend associated with disease stages. conclusions: our results suggest that mmp-1, mmp-7 and osteopontin alone or combined may have clinical value for ovarian cancer detection. objectives: -tubulin acetylation has been proposed to control the dynamic nature of microtubule stability. acetylated -tubulin plays a role in regulating microtubule functions in mitosis and cell migration. here, we sought to identify the relationship between post-translational -tubulin acetylation and the expression of epithelial cell adhesion molecules (ep-cam) after exposure to microtubule interacting agents in ovarian cancer cells. methods: epithelial ovarian cancer cell line, hey was treated with microtubule stabilizing agents (taxol, epothilone b and discodermolide), microtubule-destabilizing agent (vinblastine), hdac6 inhibitor trichostatin a (tsa), anti-metabolite fluorouracil (5fu), or alkalyting agents (cisplatin and carboplatin). cells were separately treated with either ic 50 or 10-fold ic 50 of each agent, and incubated at 37°c for 1 h, 24 h and 48 h. acetylation oftubulin and pan--tubulin were evaluated by western blot analysis followed by protein quantification. cell surface ep-cam expression was examined by flow cytometry. results: increased acetylation of -tubulin was seen with taxol, epothilone b, discodermolide, vinblastine and tsa. -tubulin acetylation was time and dose dependent. the highest level of -tubulin acetylation (2.5-fold) was observed with vinblastine at 10-fold ic 50 after 48 h. exposure to microtubule interacting agents and tsa resulted in increased cell surface expression of ep-cam in a time and dose dependent manner. the highest level of cell surface ep-cam expression (8.5 fold) was observed with 10-fold ic 50 of vinblastine at 48 h. the increase in acetylated -tubulin and ep-cam expression was clearly detectable after 1 h treatment. this data reveals a similar dose and time dependent increases between the acetylation of -tubulin and the increase of ep-cam expression. conclusions: these data demonstrate the promotion of -tubulin acetylation and cell surface ep-cam expression by a microtubule destabilizing agent and by microtubule stabilizing agents. interestingly, vinblastine induces the highest -tubulin acetylation and ep-cam expression. acetylation of alpha-tubulin may be associated with redistribution of cell surface antigens in ovarian cancer cells. objective: epothilone b (epob) is similar to taxol in its ability to enhance tubulin polymerization and to stabilize microtubules. epob is currently being evaluated as an antitumor agent and is in phase iii clinical trials. the tumor suppressor gene, p53 plays an important role in the induction of apoptosis by a variety of anticancer drugs. p38 mitogen-activated protein kinase is activated by a wide array of stress stimuli including chemotherapeutic agents and promotes apoptosis. since both p38 and p53 activation induces apoptosis, we hoped to evaluate the relationship between p38, p-p53 and parp cleavage, an early indicator of apoptosis, in ovarian cancer cells after treatment with epob. methods: hey cells were treated with epob (5 to 200nm) for 16h. parp cleavage product p85 as well as p-p38, p53, phospho-p53 (ser-15), p21 and survivin were determined by western blot analysis. a wild type ovarian cancer cell line hey, was treated with or without 5 m sb203580, a specific inhibitor of p-p38, followed by treatment with epob (20 or 50 nm) for 16h. lysates were prepared and western blot analysis was performed with polyclonal phospho-p38 and anti-phospho-p53 antibodies. results: epob induces p53 activation and apoptosis demonstrated by increased parp cleavage product. time course studies indicated that phosphorylation of p53 precedes phosphorylation of p53 in hey cells. the expression of p53 targeted gene, p21 (survivin) were differentially expressed depending on the dose of epob and the duration of drug exposure. pretreatment with specific inhibitor of p38 markedly inhibited the p53 phosphorylation at serine 15. conclusions: 50 nm epob (a concentration that triggered mitotic arrest) causes a decrease in p21 expression and an increase in survivin expression. epob induces parp cleavage. p38 inhibitor sb203580 inhibits epo-b -induced p53 phosphorylation. these results suggest that phosphorylation of p38 may lead to p53 activation and these signaling events may be related to epo-b induced cell death in ovarian cells. conclusions: nf-b has been shown to control the expression and function of anti-apoptotic proteins and pro-inflammatory cytokines. in the present study we demonstrated that specific inhibition of nf-b by erib reversed the anti-apoptotic state of chemoresistant ovarian cancer cells, therefore may provide a new potential venue for the treatment of ovarian cancer patients. expression in cervical cancer. madhu chauhan, chandra yallampalli. gynecology, university of texas medical branch, galveston, tx, usa. background: intermedin (imd)/adrenomedullin2 is a ct/cgrp family peptide. imd is expressed in a variety of tissues such as pituitary, stomach, placenta, uterus, and ovary .we have shown that imd plays a role in a variety of physiological functions, including vasodilatation and fetoplacental growth regulation. further we have data indicating an angiogenic activity of imd in first trimester trophoblast cells. we hypothesize that imd is expressed in cervix and may have a role in the pathology of cervical cancer objective: 1) to analyze expression of imd mrna in human cervix, rat cervix from non-pregnant and pregnant rats; 2) to assess the differences in the expression of imd in normal human cervix and cervical carcinoma tissues and 3) to analyze the effect of imd on the expression of tnf-alpha and il-1beta in human epithelial cervical carcinoma cells (hela). methods: groups of 4 sprague-dawley, non-pregnant and pregnant rats on day 18 of gestation were used in this study. cervical tissues were collected from the non-pregnant and pregnant rats, women undergoing hysterectomy and from women diagnosed with cervical carcinoma. total rna was extracted using trizol method. hela cells were grown to 80% confluency in rpmi medium supplemented with 10% fbs. the cells were starved in 1%fbs for 4 hrs followed by treatment with imd (10 -8 m) in presence or absence of imd antagonist, imda (10 -5 m). cells were further incubated for 24hrs and total rna was extracted using trizol reagent. rna was treated with dnase1 before performing the reverse transcriptase polymerase chain reaction (rt-pcr). the rt-pcr data was normalized to that of 18s. results: 1) imd mrna is expressed in rat and human cervix and in hela cells. 2) expression of imd is elevated in pregnant rat cervix as compared to the non-pregnant. 3) imd has no effect on tnf-alpha expression in hela cells treated with imd but caused a decline in the expression of il-1beta mrna and, 4) expression of imd mrna is significantly elevated (p< 0.05) in cervical carcinoma as compared to the normal cervix. conclusion: imd is expressed in both rat and human cervix and is elevated in pregnant rat suggesting that it may have a role in cervical function during pregnancy in rat. in addition we demonstrate that imd is involved in the pathology of cervical carcinoma and thus may have a clinical significance in the pathology of cervical cancer. objective: there is minimal information about the impact of treatment delays or dose reductions on chemotherapeutic treatment responses and overall outcomes in ovarian cancer patients. the primary objective of this study was to quantify the impact of treatment delays and dose reductions in an in vivo xenograft ovarian cancer model and to evaluate if the growth factors could improve overall survival. methods: heya-8 and skov3-ip xenograft mouse models to determine the effect of treatment delays or dose reductions on tumor response. results: the results indicated that full dose of paclitaxel (20 mg/kg) and carboplatin (50 mg/kg) delivered on timeevery 21 daysachieved better tumor response in both aggressive (heya-8) and metastatic (skov3-ip1) human ovarian tumors compared to the non-treatment and vehicle groups. in heya-8 mice, paclitaxel/carboplatin alone and paclitaxel/carboplatin followed by growth factor support agents including pegfilgrastim (1000 μg/kg) and darbepoetin (10μg/kg) improved overall survival rate. growth factors also improved the tolerability in heya-8 model. for skov3-ip1 mice, the treatment delays resulted in a significant reduce in overall survival time compared to full dose, on time treatment (p< 0.01). for the dose reduction group, there was a significant different survival comparing to full dose, on time treatment (p< 0.05). conclusion: treatment delays had a negative impact on tumor response and overall survival compare with treatment controls. in addition, use of growth factor agents also improved treatment response and tolerability of chemotherapy and ultimately overall survival. -415) . median age at recurrence was 73 (33-104), and median interval to recurrence 13.5 months (1-415). 81 (56%) patients died of recurrent disease and 10 (7%) of other causes. site of recurrence and was local in 92, groin in 30 and distant in 22. 58 of the local recurrences were managed with wide local excision (wle) 12 radical surgery (6 radical excisions with skin flaps, 4 posterior exenterations, 1 anterior exenteration, 1 total exenteration), 10 wle and radiotherapy (rt)/chemort, 6 chemo/chemort/rt, and 6 palliation. 12 groin recurrences were managed surgically (5 adjuvant rt), 3 rt alone, 1 chemotherapy alone, and 1 palliation. 2 patients with distant metastases were managed surgically, 10 with rt/chemo/chemort, and 9 palliation. overall median survival after recurrence was 17 months. median survival (months) by site of recurrence was: local 56, groin 12, distant 4, groin or distant 6. there was a significant difference in survival in local vs groin node recurrence (p<0.0001), local vs groin and distant (p<0.0001), and groin node vs distant (p 0.0075). 3 and 5 years after recurrence survival rates were: local 56% and 46%, groin 13% and 13% distant 0,0. conclusion patients with vscc remain at risk of recurrent disease therefore long term follow up these patients is essential. patients with local recurrence often have a good prognosis. outcomes for groin and distant recurrences are poor, but a proportion of those with groin recurrences can be salvaged. therefore early identification of local and groin recurrences is essential for improving outcomes in recurrent vscc particularly in cases with more conservative management of primary disease. introduction: endocrine disrupting chemicals (edcs) are environmental agents possessing hormone-like activity. exposure to edcs during differentiation can interfere with hormonal signaling, resulting in predisposition to some cancers. it has been suggested that some of these effects may be epigenetic, mediated by changes in dna and histone methylation. we hypothesized that 2 edcs, diethylstilbestrol (des), and bisphenol-a (bpa), may act by altering the expression of dna and histone methyltransferases (dnmts and hmts). methods: ishikawa (endometrial) and mcf7 (breast) cells were cultured in steroid-free, phenol-free media with bpa (25 m), des (5 x 10-8 m) or vehicle for 48 hours. pregnant cd-1 mice were treated with intraperitoneal injections of des (10 mg/kg) or vehicle on days 9-16 of gestation. 2 weeks after birth, offspring were sacrificed and tissue obtained. mrna was extracted, cdna was generated and quantitative real time rt-pcr was performed and normalized to -actin. all experiments were conducted in triplicate, repeated three times and compared using anova. results: 3 dnmts (dnmt1, 3a, and 3b) and 2 hmts (mll and ezh2) were screened after in vitro exposure to edcs (table 1) . ezh2 mrna expression was significantly increased in ishikawa cells after treatment with des or bpa. a similar response in ezh2 expression was seen in mcf7 cells treated with des or bpa. due to the consistent induction of ezh2 in all cells with either treatment, we examined the effect of des exposure on ezh2 expression in vivo. in adult mice exposed to des in utero, ezh2 expression was persistently increased (3.0±0.66 fold, p<0.05). conclusions: breast and uterine cell lines show increased expression of ezh2 in response to bpa or des exposure. this differential expression persists in the adult offspring of mice exposed to des in utero. ezh2 has been identified as a risk for neoplastic progression in the breast and for increased proliferation in uterine cancers. des induced ezh2 expression may be a mechanism for the increased incidence of breast and reproductive tract cancers seen in des exposed women. defects in cellular programs that control apoptosis lead to an imbalance of cell proliferation and cell death in ovarian cancer. recent evidence suggests that the use of some anti-inflammatory drugs decreases risk of ovarian cancer. natural curcumin-based anti-inflammatory therapies were shown to be beneficial in preclinical models of ovarian cancer (lin et al., 2007) . in this study, we examined the effects of plant derived curcumin on cell proliferation, apoptosis, and vegf expression in cultivated ovarian cancer cells. ovarian cancer igrov1 cells were cultured under standard conditions to study the effects of curcumin on cell kinetics and on vegf expression. cell proliferation was measured by srb and mtt assays. apoptosis was determined by measuring cytoplasmic histone-associated-dna-fragments (cell death detection elisa, roche, germany). vegf gene promoter-reporter activation and real-time quantitative reverse transcription polymerase chain reaction were used. conditioned media concentrations of vegf were measured with a commercially available enzyme-linked immunosorbent assay (quantikine, r d systems, minneapolis, mn). we observed that curcumin dose-dependently suppressed cell growth in igrov1 cancer cells (ic 50 =10um). treated cells showed a 2-3 fold increase in dna fragmentation compared to controls. curcumin also resulted in a significant decrease of vegfmrna expression and vegf protein secretion into the conditioned media in a dose-dependent manner. in this study, we have demonstrated that curcumin induces apoptosis, suppresses growth, and inhibits vegf gene and protein expression in an ovarian cancer cell line. experiments are underway to identify specific mechanism of curcumin action. curcumin may act as a chemosensitizing drug by potentiating the antitumor effects of standard treatments including taxols and platins in ovarian cancer. supported by the caldwell family foundation and the vesa w. and william j. hardman, jr. charitable foundation inc. daniel r christie, 1 faheem m shaikh, 2 john a lucas iii, 1 susan l bellis. 2 1 obsterics and gynecology, university of alabama at birmingham, birmingham, al, usa; 2 physiology and biophysics, university of alabama at birmingham, birmingham, al, usa. introduction: previous work has shown that an upregulation of the enzyme st6gal i, which is responsible for the 2,6 sialylation of 1 integrins, confers a more tumorigenic/metastatic phenotype to colon carcinoma cell lines. it is not known, however, if this is unique to colon cancer, or if it is more broadly applicable to other forms of cancers. quantitatively increased expression of st6gal i has been reported in ovarian cancers versus controls, but no biochemical or functional assays have been described to date. objective: because 1 integrins are involved in cell adhesion and migration, and because metastasis of epithelial ovarian cancers is largely an intraperitoneal dissemination, we hypothesized that upregulation in the expression of st6gal i in an ovarian cancer cell line would enhance binding with the extracellular matrix, increase invasiveness, and alter migration. methods: in the present study we forced a stable transduction of st6gal i into the ov4 ovarian tumor cell line, which we found to be lacking the st6gal i enzyme, establishing a parental (par), an empty lentiviral vector (ev), and an st6gal i expressing (st6) cell line. a collagen i cell adhesion assay was performed and quantified by staining adherent cells and measuring absorbance. a haptotactic collagen i cell migration assay was performed by seeding cells in boyden chambers lined with collagen i concentration gradient, and quantified with cell staining and absorbance measurement. cell invasion through a reconstituted basement membrane (matrigel) was quantified as previously described for the cell migration assay. results: we were able to demonstrate successful creation of the ov4-st6gal i cell line by western blot analysis. functional assays demonstrated increased adhesion to collagen i (p < 0.05), increased haptotactic collagen i cell migration (p < 0.01), and increased invasiveness (p < 0.05) in the st6 cell line as compared to par and ev when analyzed by one-way anova. conclusion: this initial study into st6gal i in ovarian cancer may have future therapeutic implications, and, in addition, lend greater insight into the intraperitoneal dissemination of disease.of microarrays (sam), arrayassist and binary tree structured vector quantization. differentially expressed genes were integrated with a database of known predicted protein-protein interactions (ophid) and key genes are being validated with real-time pcr and immunohistochemistry. results: unsupervised hierarchical clustering revealed collective clustering of all tumors, irrespective of their pathological classification. sam analysis has highlighted 47 probe sets as differentially expressed between lmp and lmp-mp, 134 probes between lmp and lgsc, and 180 probes between lmp and lmp-mp+lgsc. no differential gene expression was detected between lmp-mp and lgsc. ophid analysis demonstrated gene members of the egfr and mapk1/3 pathways to be differentially regulated between the non-invasive and the invasive tumors. to date, we have successfully validated 2 members of the mapk1/3 pathway-poly adp ribose polymerase1 (ppol) and traf family associated nf kappa b activator (tank)-using real-time pcr. conclusion: our data demonstrate that although the 3 tumors have related genetic profiles, lmp-mp and lgsc are similar to each other and different from lmp, in keeping with their clinical behavior. members of the mapk1/3 and egfr pathways appear to play a key role in low grade serous cancer. identification of novel genes associated with malignant transformation, may lead to development of more effective targeted therapy for lgsc. background: approximately 50% of pap smears with the ambiguous diagnosis of atypical squamous cells, cannot exclude high grade (asc-h), are negative for dysplasia in follow up colposcopic examination and biopsy. although hpv testing provides excellent negative predictive value (npv) (95%), the prevalence of high risk hpv infection is high in young women and the positive predictive value (ppv) in asc-h pap smears is no better than cytologic diagnosis alone (50%). recent studies have shown that immunostaining for p16 ink4a , or proex tm c, supports a diagnosis of dysplasia in surgical biopsies. our objective was to determine whether staining for p16 or proexc provides sufficient predictive value to reliably distinguish high grade dysplasia in asc-h pap smears. design: we retrospectively collected samples from liquid based pap smears diagnosed as asc-h at either ohsu or portland oregon kaiser permanente (2005-07). known hsil and negative pap cases were included as immunostaining controls. asc-h cases with followup cervical biopsies (n=90) were included for subsequent immunostaining according to manufacture's instructions. samples were also sequestered for hpv testing (pending). immunostained slides were scored as positive or negative by two independent cytopathologists (as and tm) while blinded to pap diagnoses and surgical biopsy outcomes. results: we observed excellent agreement between pathologists' scores (pairwise kappa statistic 0.85). the correlation between p16 and proexc scores was moderate (kappa 0.40). chi-square analysis comparing staining to biopsy outcome revealed a significant association between proex c positivity and cervical dysplasia (*** p<0.001 jing lin, zhenmin lei, ch v rao. ob/gyn women health, university of louisville health sciences center, louisville, ky, usa. introduction: matrix metalloproteinases (mmps) are a family of highly homologous zinc-dependent endopeptidases, which degrade all kinds of extracellular matrix proteins. the degradation process is required for tissue growth and remodelling. these enzymes are probably involved in uterine growth and devolopment and its differentiated functions. ovarian steroid hormones, estradiol (e 2 ) and progesterone (p 4 ), are known to regulate some of the uterine mmps. since it is now known that lh/hcg can directly regulate uterine growth and devolopment and its functions, we questioned whether these gonadotropins could also regulate mmps in the uterus. methods: sixty day old wild type (wt) and lh receptor knockout (lhrko) mice and 21-day e 2 and p 4 treated 30-day old animals were used. in addition, primary cultures of uterine epithelial cells from wt animals were treated for 24 hrs either with no hormone or with a single or a combination of 100 pg/ml of e 2 or p 4 , 100 ng/ml of hcg. then mmp-2, -7 and -10 mrna levels were quantified by the semiquantitative rt-pcr. results: while the uterine mmp-2 mrna levels were unaffected, mmp-7 mrna levels significantly decreased and mmp-10 mrna levels significantly increased in lhrko animals as compared with wt siblings. e 2 and p 4 treatment reversed mmp-7 and mmp-10 changes in lhrko animals. treatment of wt type primary endometrial epithelial cell cultures with hcg had no effect on mmp-10 mrna levels, but it did increase mmp-7 mrna levels. this increase was synergistic with both e 2 or p 4 . conclusion: while lh/hcg do not regulate uterine mmp-2 and mmp-10, they seem to co-regulate mmp-7 with ovarian steroid hormones. cancer. radhika gogoi, christine ardalan, dorota popiolek, leslie gold, john curtin, stephanie v blank, bhavana pothuri. new york university, new york, ny, usa. objective: megesterol, a synthetic progestin with strong androgenic properties, is used in the medical management of patients (pts) with atypical endometrial hyperplasia (aeh) or endometrial carcinoma (emca). our hypothesis was that androgen receptor (ar) expression in the endometrium of aeh and emca pts would correlate with degree of response to treatment. methods: pre-and post-treatment endometrial biopsy specimens were obtained from 8 pts treated with megesterol for aeh or emca. ar expression was investigated by immunohistochemical (ihc) analysis with appropriate positive and negative controls. ihc staining was scored for intensity (1-4) and percentage of positive cells (1-4) in the glandular and stromal compartments by a pathologist, blinded to clinical response data. a composite score utilizing both intensity and percentage of positive cells was calculated. we evaluated pre-and post-treatment ar expression in responders and nonresponders as well as ar expression in emca, aeh and normal endometrium. the mann whitney u and the wilxocon signed rank tests were utilized for statistical analysis. results: eight pts' pre-and post-treatment samples were obtained; 4 with emca and 4 with aeh. three pts had no response, 1, a partial response and 4, complete responses. ar expression in emca samples when compared to the normal endometrium was significantly lower in both the glands (mean 2.8 vs. 8; p<0.01) and the stroma (mean 2.3 vs. 5; p<0.05). although there was no statistically significant difference in glandular ar expression in the pretreatment biopsies of responders compared to nonresponders (mean 4.6 vs. 2.7; p=0.14), there was a significantly higher level of glandular ar expression in the post treatment biopsies of responders compared to non-responders (mean 7.4 vs. 3; p<0.05). furthermore, we noted a trend towards higher levels of glandular ar expression in the post-treatment versus the pre-treatment biopsies in the responders (mean 7.4 vs. 4.6; p=0.07). we noted a significant decrease in ar expression in emca compared to the normal endometrium. increased ar expression after treatment in responders suggests an important role of the ar in pts treated conservatively with progestational therapy, and needs further prospective validation as a means to predict treatment response in these pts. objectives: study the correlation between adenomyosis and some potential non-surgical risk factors. objective: o 6 -methylguanine-dna methyltransferase (mgmt) acts to repair dna damaged by alkylation of guanine residues. mgmt promoter methylation and gene silencing is seen in a variety of cancers and pre-cancerous changes. the loss of mgmt activity is associated with increased sensitivity to alkylating agents and is a favorable prognostic indicator in gliomas. we sought to determine if mgmt promoter methylation plays a role in endometrial cancer. methods: one hundred and twenty primary endometrial cancers were analyzed for mgmt promoter methylation by combined bisulfite restriction analysis (cobra). the cohort included 77 endometrioid endometrial cancers, 43 endometrial tumors of adverse histologic type, and 6 endometrial cancer cell lines. twenty one endometrioid and mixed endometrioid ovarian cancers were also analyzed. a subset of the primary tumors was analyzed for mgmt expression by immunohistochemistry. results: no mgmt promoter methylation was seen in the 120 endometrial cancers evaluated or the 6 endometrial cancer cell lines. one of the 21 ovarian cancers showed methylation. immunohistochemistry for mgmt expression is ongoing. conclusion: mgmt promoter methylation is an infrequent event in endometrial cancer. mgmt expression in tumor cells and repair of alkylguanine residues could explain in part the limited response of endometrial tumors to alkylating chemotherapy. objective: uterine sarcomas (1% of gynecological malignancies) originate from uterine mesenchyma. patients with high-risk disease (i.e. high grade) or at advanced stages have poor 5-years overall survival (< 10-30%). pelvic radiation and/or chemotherapy have not demonstrated to improve survival. identification of new therapies for this malignancy is a major goal. few in vitro models have been established to test therapeutic agents for uterine sarcoma. here we sought to establish a new human uterine sarcoma cell line and to test the effects of chemotherapeutic drugs: tnf-related apoptosis inducing ligand (trail) used alone or in combination. methods: tissue sample was obtained from a woman with uterine sarcoma undergoing hysterectomy. sarcoma cell lines were established using a published protocol for endometrial cancer. phenotypic characterization was made through the different passages (1 to 13) by western blot. levels of estrogen (er), progesterone (pr) and trail receptors were also studied (rt-pcr, w-b). cells were incubated with chemotherapy agents (cisplatin, paclitaxel and doxorubicin and trail) and cytotoxicity (mts assays) and apoptosis (flow cytometry, parp cleavage by w-b, and dna laddering) measured. results: human uterine sarcoma cell line was established from a highgrade uterine sarcoma. through the different passages the cell line remains expressing cytokeratin, vimentin, tissue factor, caveolin-1 and -actin. this cell line expresses low er and pr levels. trail receptors (r1 and r2) were also detectable (rt-pcr, w-b). cisplatin, paclitaxel and doxorubicin (5um for 24 h) produced low cell cytotoxicity (< 20%). trail (200 ng/ml for 18 h) induced about 30% cell cytotoxicity. apoptosis was confirmed by parp cleavage. doxorubicin significantly enhances trail mediated cytotoxicity (up to 80%), this was demonstrated by a significant increase in the sub g0/g1 region in the dna histogram. conclusions: we establish a human uterine sarcoma cell lines using protocols for endometrial cancer. more importantly, we demonstrated that doxorubicin enhances trail effect on this uterine sarcomas cell line. thus, this combination might be considered as a treatment for high-risk uterine sarcomas. (financial support fondecyt 1050744). defining the tumor initiating capacity of cd133 + human endometrial cancer cells. anne m friel, 1 petra a sergent, 1 christine l cummings, 2 rosemary foster, 3 current data suggest rare subpopulations of cells with tumor initiating capabilities are a common feature of solid tumors. several investigators have recently identified cd133, a cell surface marker expressed on primitive cells of neural, hematopoietic, endothelial and epithelial lineages, as a potential tumor initiating cell (tic) marker in solid tumors of the brain, colon and pancreas. in our efforts to investigate such a population in human endometrial cancers (enca), we developed an in vivo model that is based on serial transplants of tics from endometrial tumor explants. serial transplant experiments were initiated in nod/scid mice that were injected with primary human enca cells. a subset of cells derived from the generated tumor was subsequently transplanted into new recipients. tumors formed following serial transplantation retained the original histological phenotype of the primary enca, and the number of cells required to initiate tumor formation was reduced at each successive transplant stage suggesting an increase in the ratio of tics to non-tics. we exploited this model in our initial efforts to investigate the tumor initiating potential of cd133-expressing enca cells. to evaluate the tumorigenic potential of cd133 + cells, the tumor initiation capacity of xenograft derived cd133 + and cd133cells were compared following subcutaneous injection of each population into nod/scid mice. only the cd133 + fraction was tumorigenic consistent with the hypothesis that tumors are generated and maintained by a subpopulation of cells with phenotypically distinct profiles. we are further investigating the functional significance and characteristics of this fraction in vitro and in vivo. objectives. central issues in tumor biology are the understanding of factors that control tumor cell proliferation and the identification of extracellular matrix cues controlling the signaling transducing repertoire that make cancer cells proliferate and invade the host tissues. among these factors, small leucine-rich proteoglycans (srlps):decorin, fibromodulin, lumican, biglycan, are emerging as powerful modulators of angiogenesis and cell growth, by affecting several key elements including matrix assembly, growth factor binding, and receptor tyrosine kinase activity. recently has been demonstrated that srlps can act as a pan-erbb ligand and, in doing so, down-regulate the activity of one of the most potent oncogenic proteins, erbb2, whose overexpression is linked to poor prognosis and increased cancer mortality in breast, ovary, and prostate. since srlps have not been previously investigated in the endometrial cancer biology, we investigated their role in the benign and malignant endometrial neoplasia. method. the sampling has been obtained in women (n=60; mean age 50± 2.7) with endometrial hyperplasia (n=20), polyps(n=20), and cancer(n=20), during therapeutic and diagnostic procedures (hysterectomy, colpohysterectomy, hysteroscopy). physiological endometrial samples (n=20) were obtained from menopausal women (n=20; mean age 51± 2.5), during procedures for others gynaecologic indications. immunohistochemestry was the biology technique used for the detection of srlps.results. in the physiological endometrial samples immunoreactivity for decorin, fibromodulin and biglycan was intense (+++), while it was weak in polyps and hyperplasia (+) and absent (-) in cancer. no significant difference in the staining of lumican between the physiological and pathological samples. conclusions. these results could provide a mechanism by which naturally occurring proteins normally synthesized by fibroblasts and smooth muscle cells, the two key components of the tumor stroma, may play a protective role in the genesis and progression of endometrial neoplasia counteracting the growth of neoplastic cells and suppressing tumor cell-mediated angiogenesis. although further studies are necessary to understand mechanisms whereby srlps might influence endometrial cell growth and survival, these molecules may represent potential target for pharmacological cancer therapy. myostatin is a member of the tgf-beta superfamily of protein and a wellknown inhibitor of skeletal muscle proliferation. the muscular component of the uterus is the myometrium, a tissue that regulates its mass in response to different physiologic conditions under the influence of steroids. we determined the expression of myostatin mrna in immortalized pregnant human myometrial (phm1) cells and we verified its biological activity. functional assays showed myostatin induced phosphorylation of smad-2 and reduction of proliferation of phm1 cells in a time and dose-dependent manner. to investigate the physiological relevance of our in vitro findings, the expression of myostatin in rat uterus was examined at various phases of the estrous cycle. for the first time we report that myostatin is expressed in rat uterus and that levels of myostatin mrna change during distinct phases of the estrus cycle. uterine levels of myostatin peaked during late estrous and were the lowest at pro-estrous. to further examine the role of steroids in myostatin regulation, we examined the effects of gonadal steroid administration in ovariectomized (ovx) rats. ovaryectomy increased myostatin expression compared to normal cycling controls. estrogen treatment strong decreased myostatin levels while progesterone produced less robust effects on myostatin expression. these findings taken together suggest an important role for myostatin in the regulation of myometrial functionality. her2neu over-expression and pi3kinase/akt pathway activation in paget's disease of the vulva. amy stenson, 1 bita behjatnia, 1 jaime shamonki, 1 jianyu rao, 1 amer karam, 1 jonathan berek, 2 oliver dorigo. 1 1 ob/gyn and pathology, ucla, los angeles, ca, usa; 2 ob/gyn, stanford university, palo alto, ca, usa. background: paget's disease of the vulva is rare with high recurrence rates. treatment of recurrent disease is challenging due to its extent and location. non-surgical approaches have limited clinical efficacy, obviating the need for novel therapies. in contrast to paget's of the breast with well-described overexpression of her2neu, molecular features of vulvar paget's are poorly characterized. our objective was to study therapeutic targets in vulvar paget's, including the her2neu protein and the phosphorylated oncoprotein akt (pakt). in addition, detailed clinical characteristics were correlated with molecular expression. methods: specimens with vulvar paget's disease were retrieved from ucla's department of pathology. protein expression was evaluated by immunohistochemistry on slides from paraffin embedded tissue using the hercep test (dako) for her2neu expression and a standard protocol to assess expression of activated pakt. slides were scored by two independent pathologists based on a nominal scale of 0 (negative) to 3 (strongly positive). clinical data was retrieved via chart review. results: between 1995 and 2007, 25 patients with vulvar paget's were identified. median age was 65 yrs (36-83 yrs). a family history of cancer was found in 14/25 (54%), 7/25 (28%) were smokers and 13/18 (72%) had a history of hormone use. intraepithelial lesions accounted for the majority (15/25, 60%), while 6/25 (24%) demonstrated invasion and 4/25 (16%) were associated with underlying gi malignancy. 16/25 (64%) had at least one recurrence, with median time to recurrence 24 months. so far, 13 specimens were stained for her2neu and pakt. overexpression of her2neu was found in 10/13 (77%.) positive staining for pakt was evident in 10/13 (77%.) statistical analysis suggested a correlation between her2neu and pakt expression. conclusions: our study demonstrates that overexpression of her2neu and activation of the pi3kinase/pakt pathway are important features of vulvar paget's disease. to the best of our knowledge, this is the largest series evaluating these molecular pathways in vulvar paget's. our data suggest that her2neu and pakt may be important molecular targets for novel therapies using for example the monoclonal antibody trastuzumab directed against her2neu, or a pi3kinase pathway inhibitor like rapamycin. introduction: uterine leiomyomas are the most frequent tumor of the female reproductive tract and are the primary indication for hysterectomy in women worldwide. these tumors occur in up to 70% of adult women, and their prevalence is especially high in africa-american women. currently there is no effective and safe medical treatment for uterine fibroids and surgery is the main stay. epigallocatechin gallate (egcg) constitutes the main solid extract of green tea and is believed to contributes most of the antioxidant and antiinflammation capacity of green tea. egcg has been shown to have beneficial effects on prostate cancer and breast cancer by inducing apoptosis and inhibiting proliferation of cancer cells. in this study, we investigated the ability of egcg to inhibit proliferation of human leiomyoma cells (hlm) in vitro. methods: human immortalized leiomyoma cells were cultured at 37ºc in a humidified atmosphere of 5% co 2 -95% air in smbm medium supplied with 5%fbs, 0.1% insulin, 0.2% hfgf-b, 0.1% ga-1000 and 0.1% hegf(lonza). the cells were plated at density of 2×10 5 cells/well in 6-well plates and grown under the same conditions above. the monolayer cultures at approximately 70% confluence were treated with various concentrations (0, 0.1, 1.0, 10, 50, 100 and 200μm) of egcg (sigma) for 7 days. a fluorometric assay using hoechst 33258 dye (sigma) for dna quantitation was conducted at the following designed time points, day 1, 3, 5, and 7 post egcg treatment. the cells were lysed and dna content was determined using hoechst dye solution (1μg/ml). fluorescence was measured after excitation at 355nm and emission at 458nm. results: egcg exhibited marked anti-proliferation effect on the growth of hlm cells. compared with untreated control, the inhibitory effect of egcg on hlm cells was observed at 10 μm and peaked at 100 μm concentration. the difference was statistically significant (p = 0.002). evaluation of the mechanism of action of egcg is cuurently under investigation in the lab.conclusion: egcg significantly inhibited the proliferation of human leiomyoma cells in a dose-depended pattern. egcg may present a potential effective and safe medicinal treatment for uterine fibroids. objective: choriocarcinoma is an aggressive form of germ cell tumor that exhibits rapid growth with early metastases. choriocarcinomas autonomously secrete hcg which acts as an autocrine/paracrine growth factor in these cancers. we hypothesize that a novel hcg antagonist (hcg-ant) can limit tumor expansion by blocking hcg-induced expression of pro-invasive genes. we investigated if hcg-ant could alter rna expression of matrix metalloproteinases (mmp-1 and mmp-7), which facilitate basement membrane degradation and hence invasion, and metastin (kisspeptin), a strong suppressant of metastasis, in the choriocarcinoma cell line jeg-3. design: in vitro experiments using the jeg-3 cell line. materials and methods: after plating jeg-3 cells in a 24 well tray overnight in rpmi media containing 10% fbs, cells were washed twice with pbs and then cultured in 500 ul of serum-free rpmi media containing one of four treatments: 1) saline; 2) hcg (50 iu); 3) hcg (50 iu) plus hcg-ant (100 iu); or 4) hcg-ant (100 iu). rna was extracted from each well using trizol and reverse transcribed using sensiscript (qiagen). the relative expression of mmp-1, mmp-7, and metastin mrna was quantified using sybr-green based real time pcr. the expression of the housekeeping gene hprt was used to normalize expression data. results: treatment of jeg-3 cells with hcg-ant vs. hcg alone reduced expression of mmp-1 (51.95 ±15.51 vs. 85.99 ±10.56) and . hcg-ant reduced mmp-1 and mmp-7 expression by 40% and 43%, respectively (p<0.05). treatment with hcg-ant vs. hcg increased metastin expression (5.44 ±2.29 vs. 4.63 ±1.32). metastin expression was increased by 17% following hcg-ant treatment. conclusion: treatment of the jeg-3 choriocarcinoma cell line with hcg-ant reversed the increased expression of mmp-1 and mmp-7 following treatment with hcg. metastin expression was increased by hcg-ant. this data suggests that hcg antagonism is capable of altering gene expression thereby inhibiting invasion in a choriocarcinoma cell line. the role of hcg-ant as an adjuvant therapy in hcg sensitive tumors offers promise. retinoids, but not progesterone, directly induce differentiation and apoptosis of endometrial cancer cells. you-hong cheng, serdar e bulun. ob/gyn, northwestern university feinberg school of medicine, chicago, il, usa. objectives: the opposing actions of estrogen and progesterone during the menstrual cycle regulate endometrial proliferation, differentiation and secretion. the unopposed action of estrogen contributes to the development of type i endometrial cancer. however, the mechanisms for progesterone protection of estrogen-induced carcinogenesis in endometrium remain unclear. methods and results: in the current study, we demonstrated that retinoids (9-cis retinoic acid (ra) or all-trans ra (atra)) significantly inhibited basal and hormone-stimulated ishikawa cell proliferation by over 60% using mtt assay. the same experiment indicated that estrogen had no significant effect, whereas progesterone slightly induced, on cell proliferation. cell cycle analysis indicated that atra significantly increased the g1/g0 cell population by 20% and decreased s phase cells by 20%, suggesting that ra induces cell cycle arrest at the s phase. knock-down of rar alone or both rar and rxr significantly increased proliferating cell nuclear antigen (pcna) levels in epithelial ishikawa cells, suggesting that ra signaling via rar/rxr activation is critical for normal endometrial growth and differentiation. to determine whether retinoids are naturally secreted by the endometrial stromal cells, we cultured primary stromal cells and analyzed the culture media using hplc. we found that retinol is the predominant retinoid form secreted by stromal cells. the average concentration of retinol in the cultured media of eutopic endometrial stromal cells was approximately 4 to 6 ng/ml/10 5 cells (n=5). although there was less than 0.25 ng/ml/10 5 cells of all-trans retinal or atra in the cultured media, we did find a small peak for all-trans retinal and atra in the media using hplc analysis. furthermore, progesterone significantly increased secreted retinol levels from eutopic endometrial stromal cells, but decreased retinol levels secreted from endometriotic stromal cells. retinol is a precursor for ra that is converted to retinal and then to ra. conclusions: we demonstrated that progesterone signaling via pr induces endometrial stromal cells to secrete paracrine retionids that in turn control the phenotype of adjacent epithelial cells. conversely, this interaction is disrupted in diseased endometrial tissues, such as endometrial cancer and endometriosis. the effects of hormonal contraceptives on antimullerian hormone by obesity status. anne z steiner, 1 frank z stanczyk, 2 stan patel, 2 alison edelman. 3 1 ob/gyn, university of north carolina, chapel hill, nc, usa; 2 ob/gyn, usc keck school of medicine, los angeles, ca, usa; 3 ob/gyn, oregon health and sciences university, portland, or, usa. background: antimullerian hormone (amh) is emerging as a predictor of reproductive potential. serum levels of fsh, a commonly used measure of ovarian reserve, are suppressed with the use of oral contraceptives (ocs) thereby limiting its use. the impact of ocs and on serum amh levels in normal and obese women is unknown. objective: to examine the impact of ocs on serum amh levels by obesity status in reproductive-age women. materials and methods: ovulatory women, ages 18-35 years, of normal (< 25 kg/m 2 ; n = 10) and obese (> 30 kg/m 2 ; n = 9) body mass index (bmi) received a low dose oc (20 mcg ethinyl estradiol/100 mcg levonorgestrel) for two cycles. serum was obtained at three time points: after 21 days of active pills (cycle 1, day 21), at the end of the 7-day hormone-free interval (cycle 1, day 28), and during the first week of active pills in cycle 2 (cycle 2, day 5,6, or 7). amh levels were quantified by elisa; fsh and lh levels were determined by chemiluminescent immunoassay. amh at the three time points was compared using repeated measures anova. models were used to assess the relationship between amh and cycle day by obesity status. amh and gonadotropin levels were compared using spearman's correlation. results: amh levels did not differ by oc cycle day (p anova = 0.90) or by active versus placebo pill use (2.79 ng/ml ± 1.3 vs. 2.85 ng/ml ± 1.4, p=0.93) among normal bmi women. among obese women, amh levels differed by oc cycle day (p anova = 0.04), but not by use of active or placebo pill (1.63 ng/ml ± 1.2 vs.1.71 ng/ml ± 1.2, p = 0.88). across the cycle, cv(standard deviation/mean) averaged 20.2%± 3.1 in the obese and 8.8%±1.3 in the normal bmi women ( p=0.003). modeling to determine differences in amh throughout the cycles based on obesity status showed a significant interaction (p = 0.02) and lower amh levels in the obese group (p<0.001). among both bmi groups, serum amh and fsh levels did not correlate during active pills or after 7 days of placebo pills. conclusions: in young, normal bmi women serum amh levels do not appear to fluctuate during oc use. among obese women, amh levels are lower and fluctuate significantly. these fluctuations do not appear to mirror changes in gonadotropins and may complicate clinical interpretation of amh. background: menopausal hormone therapy (ht) is a confusing topic for many clinicians and patients. objective: to assess comprehension of basic clinical trial features among prospective participants for the keeps trial, designed to study the effects of ht initiated within 3 years of menopause on chd markers. methods: screening materials were provided giving an overview of study purpose, duration, medications, likelihood of receiving drug vs. placebo, ht related risks and side effects. at a subsequent interview, a 10-item questionnaire assessed the participant's level of comprehension. a score of 80% was adequate to complete the informed consent process. those scoring <80 % were re-counseled and retested. demographic variables determining the likelihood of an initial score >80% were evaluated by univariate and multivariable analyses. results: 32% (24/76) scored >80% on initial testing. all women scored >80% after re-counseling and retesting. likelihood of an initial response >80% correct was unrelated to age or time since menopause. ability to correctly respond was influenced by highest educational level attained. none of 8 women whose furthest educational level was high school scored >80% on initial testing, significantly less than those with a college education (p=0.04). a higher proportion of college graduates (13/32) scored >80% compared to those attaining further education (11/35) (p=0.43). comprehension was greatest for study purpose and duration (64/76 and 70/76 correct responses respectively) and least for questions related to results of the whi hormone trial breast cancer and chd. that e alone was not associated with an increased risk of chd (41%) or breast cancer (50%) was poorly understood. conclusion: comprehension of the risks and benefits of ht by potential research subjects is low despite provision of reading materials prior to the informed consent process. (supported by the montefiore medical center/albert einstein college of medicine site for the kronos longevity research institute and k24-hd41978 to ns). variation in menopausal symptoms within a sample of hispanic women: swan, the study of women's health across the nation. robin r green, 1 alex j polotsky, 1 aileen p mcginn, 1 carol a derby, 1 rachel p wildman, 1 lhasa ray, 1 kavitha t ram, 1 gerson weiss, 2 nanette f santoro. 1 1 albert einstein coll of med, bronx, ny; 2 univ of med and dent of new jersey, newark, nj. background: menopausal symptoms are experienced by over 75% of women. purpose: to describe symptom frequency in a sample of midlife hispanic women from different countries of origin. methods: the study of women's health across the nation (swan) recruited 277 women at baseline who self-identified as hispanic. their baseline responses to validated questionnaires regarding common menopausal symptomatology were examined. symptoms were reported over the previous two weeks and scored on a frequency scale ranging from 1 (not at all) to 5 (every day). for all analyses, symptoms were dichotomized into "absent" vs. "present" variables. responses were correlated with acculturation (4-item scale: marin, hisp j behav sci 2:183, 1987) and analyzed by sub-ethnicities: central/south american (c/s am), puerto rican (pr), dominican (dr), and cuban (cu). associations between symptoms and sub-ethnicity were tested by chi-square. logistic regression was used to test for associations with acculturation and being us-born. there was significant variation in several menopausal symptoms. while puerto-rican women had the highest likelihood of reporting trouble sleeping (or=2.2, 95%ci: 1.1-4.3) and headaches (or=2.7, 95%ci:1.4-5.3), dominican women were most likely to report vaginal dryness (or=2.8, 95%ci: 1.1-7.1) acculturation and being us-born did not explain the variation between subethnicities in any of the models tested conclusion: there appear to be significant differences among hispanic women with respect to menopausal symptomatology. these differences were not readily explained by measures of acculturation. (supported by the study of women's health across the nation (swan) has grant support national institutes of health, dhhs, through the national institute on aging, from the national institute of nursing research and the nih office of research on women's health (grants nr004061; ag012505, ag012535, ag012531, ag012539, ag012546, ag012553, ag012554, ag012495 and cd41978). purpose: to compare the relative effects of conjugated equine estrogen, raloxifene, and tamoxifen therapies on cognition among women aged 65 years or older. participants and methods: annual modified mini mental state (3ms) examinations were used to assess global cognitive function among the 7,212 women enrolled in the two randomized placebo-controlled clinical trials of the women s health initiative memory study (whims) and 300 women enrolled in co-star, the cognitive substudy of the nsabp's study of tamoxifen and raloxifen (star) trial who had baseline 3ms testing. associations between baseline cognitive risk factors common to both trials and baseline 3ms scores were assessed and interactions used to examine whether risk factor relationships differed between cohorts. factors for which relationships were similar were used as covariates in analyses comparing ontrial 3ms scores. factors for which relationships did not appear to be similar were used to stratify analyses. results: compared to placebo, each of the active therapies was associated with a small mean relative deficit in 3ms scores of 0.5 units or less, which was fairly consistent between women with and without prior hysterectomy. overall, relative deficits appeared to be most marked for tamoxifen (unadjusted p=0.003) but were also evident for raloxifene (p=0.06) and cee (p=0.02). conclusions: while unmeasured differences between trials may have confounded our analysis, these findings suggest that both tamoxifen and raloxifene may adversely affect cognitive function in older women. weight gain and increased abdominal fat have been found in women after menopause and is associated with higher levels of leptin, and decreased levels of the cardioprotective adipocytokine adiponectin. at the same time, bmd characteristically decreases. in an effort to determine the evolution and correlates of these changes, we studied 25 postmenopausal women (pm) within 4 years of menopause (age 58.4±1 yrs) of normal weight (bmi 26.3±1) and compared them to 20 weight matched (bmi 27.4±1) premenopausal (pre) controls (age 26.4±1yrs.) all subjects had bmd and body composition studies by dexa and measurements of leptin, adiponectin, visfatin and retinol-binding protein 4 (rbp4.) while total fat was similar in the 2 groups, pm had more trunk and abdominal fat (10516±800; 818±67g) compared to pre (8626±493g p<0.05; 577±33g p<0.01) pm also had greater %trunk fat and %central abdominal fat compared to pre, p<0.01.serum leptin (31.8±0.5 vs 28±5 pg/ml) and visfatin ( 10.4±1 vs. 9.4±0.5 ng/ml) were similar but adiponectin (17±1 vs. 13.3±1 g/ml) and rbp4 (28.7±2 vs. 22 .3±1 ng/ml) were higher, p<0.05 in pm. while in pre, abdominal fat correlated negatively with adiponectin (p<0.01) in pm only leptin correlated with various parameters of fat mass, p<0.02, and adiponectin did not correlate but correlated positively with age (r 0.47, p<0.05.) as expected, pm had reduced bmd at the lumbar spine and hip (0.84±0.025 vs. 0.99±0.025 g/cm2; 0.72±0.025 vs. 0.87±0.025 g/cm2, p<0.01) but there was a correlation between total and trunk fat in pm and lumbar bmd (r 0.69, 0.66, p<0.01) but not with hip bmd; or any correlations in pre. there was a correlation between leptin and lumbar bmd in pm (r 0.47, p<0.02) but not in pre. in summary, in normal weight early pm, abdominal fat is increased, but only adiponectin and rbp4 are altered with an increase in the former correlating with age. lumbar bmd correlated with fat mass in pm and is partially explained by increases in leptin. it is suggested that in spite of increasing abdominal fat in normal weight early pm, (which correlates with a higher lumbar bmd) david d rahn, jesus f acevedo, r ann word. ob-gyn, ut southwestern, dallas, tx, usa. objectives: matrix metalloprotease (mmp) activity is increased in the postpartum vagina of wild type (wt) animals, and this activity is accompanied by a burst in elastic fiber synthesis. the mechanisms that precipitate these changes are unclear. the goals of this study were to determine how vaginal distention (such as in parturition) affects elastic fiber homeostasis in the vaginal wall and the potential significance of these changes in the pathogenesis of pelvic organ prolapse. methods: nonpregnant (18) and 20 pregnant (d14) wt mice underwent either vaginal distention with a 750μl balloon x 40 min (to simulate parturition) or sham procedure. tissues were obtained at 24 and 48 h for immunoblot analysis, zymography, and histology. distention was also performed in 30 young fbln5 -/-, fbln5 +/-, and wts, and prolapse was quantified for 8 weeks. results: distention resulted in marked increases in mmp activity in nonpregnant animals (prommp9, 5.6-fold; active mmp9, 3.5-fold; prommp2, 2.4-fold; active mmp2, 3.7-fold; all p < 0.05 compared with sham) which was accompanied by fragmented and disrupted elastic fibers in the vaginal wall. abundant amounts of tropoelastin and fibulin-5 in the nonpregnant vagina were not increased further by distention. in pregnant animals (which normally have suppressed vaginal wall fibulin-5 and tropoelastin), however, distention resulted in 3-fold upregulation of both proteins (p<0.05). distention also induced increased mmps in pregnant animals (mmp-9, 1.7-fold; prommp-2, 1.7-fold; active mmp-2, 2.3-fold; all p < 0.05). thirteen young fbln5 -/-(4-6 wks prior to age of prolapse development), 11 het siblings, and 6 age-matched wts underwent serial exams after ballooning. distention induced rapid increases in perineal bulge and vaginal diameter (within 3 d) in fbln5 -/mice which never recovered. conclusions: in pregnant mice, vaginal distention results in increased protease activity in the vaginal wall but also increased synthesis of proteins important for elastic fiber assembly. distention may thereby contribute to the burst of elastic fiber synthesis in the postpartum vagina. the finding that distention results in accelerated pelvic organ prolapse in fbln5 -/animals, but not wt, indicates that distention results in loss of pelvic organ support if elastic fiber synthesis is compromised. further, the data suggest that elastic fiber synthesis is crucial for recovery of the vaginal wall from parturition/distention-induced increases in vaginal protease activity. the molecular etiology of pelvic organ prolapse (pop) is complex and not yet well understood. defects in the connective tissue, such as a decrease in extracellular matrix (ecm) protein content may predispose women to pop. our objective was to study the expression and the enzymatic activity of matrix metalloproteinases (mmps) and their tissue inhibitor (timps) in vaginal tissue of patients with advanced pop and controls. after informed consent, pre-menopausal caucasian patients affected by pop ( grade 3 by pop-q), and control patients (no pop) matched for age and bmi, undergoing vaginal and abdominal hysterectomy respectively were recruited. full thickness anterior vaginal epithelial tissue was obtained from the surgical cuff of patients and controls in the proliferative phase of the menstrual cycle. total protein was extracted using ripa lyses buffer. western immunoblot analysis was performed (patients: n=7, controls: n=6) to study the protein expression of mmp-1, -12, timp-1, -2, -4. gelatin zymography was used to quantify the activity of mmp-2 and -9. immunohistochemical analysis for timp-2 and -4 was performed on pfa-fixed vaginal biopsy tissue (n=3) in each group. both patient and control vaginal biopsy samples expressed latent and active forms of mmp-12, and mmp-1. the protein expression of the 45 kda active form of mmp-12 was significantly increased in patients with pop compared to controls (p=0.03). zymography detected the enzymatic activity of the proform and active form of both mmp-2 and mmp-9. we found a significant increase in gelatinolytic activity of both 68 kda pro-form and 62 kda active forms of mmp-2 (p=0.01 and p<0.001 respectively) as well as 87 kda active form of mmp-9 (p<0.001) in patients with pop compared to controls. timp-1 protein expression in vaginal tissue showed a significant reduction in pop patients compared to controls (p=0.002). timp-2 and -4 immunostaining were mainly localized in the smooth muscle cells at the muscularis layer of the vaginal biopsies. in vaginal tissue of patients with pop, we have shown a decrease in timp protein expression paralleled by an increase in mmp protein expression and activity. these findings reflect an active ecm remodelling that may compromise the structural integrity of the pelvic floor leading to pop. aberrant elastin and collagen synthesis may play a role in the pathogenesis of pelvic organ prolapse (pop). the lysyl oxidase (lox) family of enzymes direct cross linking of collagen and elastin polymers, however to-date no information is available on the expression and localization of these proteins in human vaginal tissue. our objectives were to study the expression and in situ localization of lox, loxl1, loxl3 and loxl4 proteins in the vaginal tissue of patients with advanced pop and asymptomatic controls. pre-menopausal caucasian patients affected by pop ( grade 3 by pop-q) and control patients (no pop) matched for age and bmi, undergoing vaginal and abdominal hysterectomy respectively were recruited. full thickness anterior vaginal epithelial tissue was obtained from the surgical cuff of patients and controls in the proliferative phase of the menstrual cycle. total protein was extracted using ripa lyses buffer and western immunoblot analysis was performed (patients: n=7, controls: n=6). pfa-fixed vaginal biopsy tissues (n=3 for each group) were used for immunohistochemical study. vaginal biopsy samples from both patient and control groups expressed all four members of lox family proteins: lox (47 kda pro-form and 35 kda active form), loxl1 (47 kda pro-form and 35 kda active form), loxl3 (83 kda) and loxl4 (84 kda). the expression of all lox family proteins was reduced in patients with pop compared to controls; however only the pro-form of lox of making the diagnosis of endometriosis from an endometrial biopsy. objective: to evaluate the diagnostic value of examining endometrial biopsy specimens for small nerve fibers in women with pelvic pain or infertility in a double-blinded prospective comparison with laparoscopy. methodology: we undertook to compare the detection of endometrial nerve fibers with laparoscopy and peritoneal biopsy for the diagnosis of endometriosis in 38 women (aged 34.4 years; range 22-45 years) who presented with chronic pelvic pain or infertility. small nerve fibers were detected in the functional layer of endometrium using immuno-histochemical staining with the highly specific pan-neuronal marker pgp9.5, using a carefully validated technique and blinded assessment of nerve fiber density. laparoscopic assessment of the presence of endometriosis and peritoneal biopsies was undertaken by three experienced gynecologic endoscopic surgeons. data from these assessments were recorded independently of endometrial findings and maintained under blinded coding until the codes were broken. results: small nerve fibers were detected in all 23 of the women in whom endometriosis was surgically diagnosed and in none of the 15 women in whom endometriosis was excluded at laparoscopy, giving the specificity and sensitivity of 100%. the density of nerve fibers in the endometriosis cases was 3.4 per mm²± 4.3 (mean ± sd). conclusions: endometrial biopsy provided a reliability of detection or exclusion of endometriosis which was equal to that of diagnostic laparoscopy carried out by experienced gynecologic laparoscopists. background: erk1/2 are mapk intracellular signaling proteins involved in cell survival and differentiation. endometriosis requires angiogenesis for ectopic implant growth. we hypothesized that the endometriotic peritoneal environment, known to be high in estrogen (e2), vegf, and cytokines such as il-8 and mcp-1, stimulates angiogenesis in human endometrial endothelial cells (heec) through erk signaling. methods: serial sections from normal (n=24) and ectopic (n=25) endometrium were immunostained for total-(t-) and phospho-(p-) erk1/2 and cd34 (an endothelial progenitor cell marker); results were quantified by computer densitometry and grouped by menstrual phase. cultured normal heec were treated with control, e2 (10-8m), il-8, mcp-1, and vegf (all 2 ng/ml) for 5 15 min, and western blotted for p-/t-erk (n=3). heec were treated with peritoneal fluid (pf; diluted 1:3 in basal media) from normal (n=4) and endometriotic (n=4) women, with or without pd98059 erk1/2 inhibitor (20 m) for 48 h, and cell viability was analyzed by mtt assay. statistical analysis was done with one-way anova. results: tissue staining revealed that ectopic cd34+ endothelial progenitor cells undergoing angiogenesis (vessel sprouting and/or angiogenic cell cord formation) exhibited the strongest levels of p-erk. heec of ectopic foci showed moderate-high p-erk staining, while surrounding mesothelial capillaries were weak for p-erk. in normal endometrium, p-erk was cycledependent, with low levels in the late secretory phase vs. other phases (p<0.05). p-erk of ectopic heec showed no cycle dependence, with moderate-high levels in all phases. t-erk in all tissues was high and invariable. in cultured heec, treatment with pf from endometriotic women significantly increased heec viability after 48 h compared to normal pf (p<0.05). this effect was abrogated by erk1/2 inhibitor. among factors known to be high in endometriotic pf, vegf increased p-erk in cultured heec in 5 and 15 min (p<0.05), while e2, il-8, and mcp-1 had no effect. conclusions: high p-erk found specifically in sprouting endothelial progenitor cells and focal ectopic capillaries suggests that erk1/2 is involved in endometriotic angiogenesis. the peritoneal microenvironment of endometriosis may be persistently stimulating erk-mediated endometrial angiogenesis through vegf. further investigation into erk1/2 inhibitors may offer a novel treatment of endometriosis. the events leading to the development of post operative adhesions are unknown, though recent observations emphasize the crucial role played by the superoxide ion generated by hypoxia. exposure of normal peritoneal fibroblasts to hypoxia caused the development of the adhesion phenotype, which is characterized by increased extracellular matrix molecules and inflammatory cytokines as well as high protein nitration and low nitric oxide. superoxide dismutases (sod) are a family of metalloenzymes that eliminates superoxide by converting it to hydrogen peroxide, protecting mammalian organisms against superoxide. objective: to determine whether sod is differentially expressed between normal peritoneal and adhesion fibroblasts and whether its expression is modulated by hypoxia. methods: fibroblasts from normal peritoneal and adhesion tissues were cultured under normal (20% o 2 ) and hypoxia (2% o 2 ) conditions for 24 and 48 hours. real time rt/pcr was utilized to measure the absolute mrna levels for sod in both cell lines before and after hypoxia exposure. results: normal peritoneal fibroblasts exhibited significantly higher basal mrna levels for sod (49.7 pg/ grna, p<0.05) as compared to adhesion fibroblasts (38.1 pg/ grna, p<0.05). short exposure to hypoxia resulted in a significant increase in sod mrna levels to 63.3 and 52 pg/ grna in normal and adhesion fibroblasts respectively, p<0.05. in contrast, long exposure to hypoxia resulted in a significant decrease in sod mrna levels to 41.5 and 25.7 pg/ grna in normal peritoneal and adhesion fibroblasts respectively, p<0.05. conclusion: sod mrna levels are lower in adhesion as compared to normal fibroblasts, both basally and following short and longer exposure to hypoxia. reduced sod expression creates an milieu with greater free radical levels, which leads to the development of the adhesion phenotype. enhancement of sod levels and/or function are likely to be of benefit for reduction of postoperative adhesion development. objectives: development of endometriosis requires ectopic attachment and proliferation of endometrial tissue. the invasive process required to establish endometriosis may involve matrix metalloproteinases (mmps), including mmp-3. recently, we have demonstrated that statins inhibit proliferation of endometrial stroma. this study evaluated the effects of simvastatin on a nude mouse model of endometriosis and on modulation of mmp-3. methods: proliferative phase human endometrial biopsies were established as organ cultures or utilized for stromal cell isolation. to establish endometriosis in the nude mouse, endometrial tissues were maintained in 1 nm estradiol (e) for 24 hrs and subsequently injected intraperitoneally into ovariectomized nude mice. mice were treated with e (8 μg, silastic capsule implants) and simvastatin (5-25 mg/kg/day) by gavage for 10 days beginning 1 day after tissue injection. control mice received e+vehicle. subsequently, animals were sacrificed and endometrial implants were evaluated. studies of endometrial stroma involved plating the cells in the presence of e (1 nm) or e+medroxyprogesterone acetate (mpa; 50 pm) with and without simvastatin (1-10 μm). some cultures additionally received interleukin-1 (il-1 , 200 ng/ml). conditioned media was subjected to western analysis for expression of mmp-3. results: in vivo studies demonstrated a dose-dependent inhibitory effect of simvastatin on number and volume of endometrial implants in mice. at the highest dose of simvastatin (25 mg/kg/day), the number of endometrial implants was decreased by 83% and the volume by 98% in comparison to the control group. isolated stromal cells expressed abundant levels of mmp-3 following treatment with e, but minimal levels in cultures additionally receiving mpa or simvastatin. although il-1 induced a dramatic increase in mmp-3 secretion from cells pretreated with e alone, treatment with either mpa or simvastatin prevented this induction. cultures receiving e+mpa+simvastatin were the most resistant to mmp-3 induction by il-1 . objective: to increase awareness of potential presentations of adenomyosis and the differential of a uterine mass. material and method: in a tertiary medical center, a patient was being evaluated for a uterine cystic mass and cyclic dysmenorrhea. the patient is a 26 year old nulliparous woman who has been complaining of cyclic dysmenorrhea for several years. the pain starts with the onset of menses and lasts around 2 weeks. the patient did not improve on contraceptives. the patient had prior laparoscopy and imaging studies which misdiagnosed the mass as either a leiomyoma or an adnexal hemorrhagic cyst. the patient underwent exploratory laparotomy and resection of the mass. results: on ultrasound, the mass appeared as an echodense cyst within the uterus. intraoperatively, a 3 cm thick-walled well circumscribed uterine chocolate cyst was identified. the resection of the cyst was performed in similarly to an ovarian cystectomy. tissue examination confirmed the diagnosis of cystic adenomyosis. following surgical intervention, the patient reported significant improvement of symptoms. conclusion: this case highlights an unusual presentation and treatment of adenomyosis and cyclic dysmenorrhea. cystic adenomyosis should be on the differential of uterine cystic mass, particularly in young women with cyclic dysmenorrhea and menorrhagia. the earlier misdiagnosis probably resulted from the unfamiliarity of physicians with this condition. similar clinical presentations may occur with congenital uterine anomalies (with an obstructed uterine segment) and cystic degeneration of a leiomyoma. the incidence and pathogenesis of cystic adenomyosis are unknown. oral contraceptives may be helpful as a first line therapy. however, resection of the adenomyotic cyst appears to be more effective, particularly in women seeking fertility. objective: it is hypothesized that abnormalities within the eutopic endometrium of females with endometriosis can cause the ectopic growth of the endometrial tissue at extrauterine sites. previous studies have shown that gene expression in eutopic endometrium of women with endometriosis is markedly different from disease-free females. inflammatory cytokines and receptor-dependent kinase pathways are widely recognized as therapeutic targets for immune disorders, which is believed to be the underlying pathogenesis of endometriosis. in this study we asked whether responses of primary eutopic endometrial cell cultures are dysregulated between females with or without endometriosis. methods: a total of 9 biopsies of endometriotic eutopic endometrium (eee) and disease-free endometrium (dfe) were obtained from proliferative phase females. the primary cell cultures established from these biopsies were treated with tnfa to induce expression of inflammatory mediators. parallel cultures were also treated with kinase inhibitors of p38, mek, pi3k and ikk. after a period of 24 or 48 hours, concentrations of il-6, mcp-1, and gm-csf in cell culture supernatants were analyzed by elisa. results: in eee, basal concentrations of mcp-1 and gm-csf were 2-3 times higher, while il-6 was 10 times higher compared to dfe. as expected, tnfa treatment stimulated higher levels of cytokine secretion in dfe mimicking disease state, however, the same treatment had almost no effect on eee. kinase inhibitors were very effective in lowering the cytokine levels of dfe, however, their effect on eee were less dramatic. conclusion: eee expresses higher levels of inflammatory cytokines under basal conditions, which is in concert with the current literature. our results validate that high il-6 levels in endometrium are diagnostic for females with disease. the increase of gm-csf, il-6 and mcp-1 following tnfa treatment was expected in dfe however; tnfa's effect was blunted in eee, which implies that eee are already highly activated. the effect of kinase inhibitors on cytokine production from eee was unaltered relative to dfe, which implies that tnfa stimulated kinase pathways are modified even in eee. endometrial cancer is the fourth leading cause of cancer in women, and hyperplasia and adenocarcinoma are more commonly seen in women with polycystic ovary syndrome (pcos). mig-6, a negative inhibitor of egf signaling, is expressed in normal secretory phase endometrium and associated with hyperplasia in mig-6 knockout mice. objective: we examined and compared endometrium from normal and pcos women for mig-6 expression and characterized its regulation using in vivo and in vitro models. methods: immunohistochemistry (ihc) and real-time pcr were performed in endometrium from normal (n=20) women and pcos (n=15) women. regulation of mig-6 was studied in ishikawa and ecc-1 endometrial cell lines, treated with sex steroids or egf. endometrial xenografts from normal and pcos women were implanted in ovxd rag2-(c) immunocompromised mice, treated with e 2 or e 2 + p pellets to mimic a natural cycle. results: mig-6 protein expression was low in the functionalis of the proliferative phase and high in the secretory phase; this pattern was reversed in the basalis layer. pcos secretory phase endometrium had significantly less mig-6 protein and mrna than normal endometrium (p < 0.01). xenografts using pcos samples had paradoxically elevated expression of mig-6 compared to normal, and appeared unresponsive to steroid treatments. mig-6 expression in endometrial cell lines was regulated by egf but not by ovarian steroids, e 2 or e 2 + p. conclusions: mig-6 expression is low in proliferating endometrium and regulated by egf. risk of hyperplasia or cancer in pcos women can be explained by altered expression of mig-6. reduced expression mig-6 provides insight into endometrial function and may lead to better treatment options for disorders of endometrial proliferation including endometriosis, adenomyosis, endometrial hyperplasia and cancer. supported in part by nih-u54-hd35041(sly), u54hd055764 (lcg) and u54hd77495 (fjd). could androgens influence human luteal cells function? anna tropea, 1 mariateresa orlando, 1 maria francesca gangale, 1 federica romani, 1 isamaria loiudice, 1 federica tiberi, 2 stefania catino, 3 antonio lanzone, 3 rosanna apa. 1 1 cattedrà di fisiopatologia della riproduzione, università cattolica del s. cuore (ucsc), roma, italy; 2 istituto scientifico internazionale (isi) "paolo vi", ucsc, roma, italy; 3 istituto di ricerca "associazione oasi maria ss onlus", troina (en), italy. in pcos patients reproductive dysfunctions are frequently observed even if ovulation occurs. an impaired luteal function could partially explain this subfertility, since luteal steroidogenesis deficiency and premature luteal degeneration have been described in pcos patients. based on the frequent observation of high plasmatic levels of androgens in pcos, we investigated whether hyperandrogenism could negatively affect luteal function.to this aim, we tested the in vitro effects of androgens on progesterone (p) and on vascular endothelial growth factor (vegf) production by human luteal cells. indeed, vegf is essential for normal luteal development and function being an important regulator of angiogenesis and vascular permeability. since prostaglandins (pgs) play a central role in modulating luteal function, the influence of androgens on pge2 and pgf2a secretion was also investigated. in order to investigate whether testosterone and androstenedione act directly or after local aromatisation, we tested the in vitro effects of estriol, estrone and 17--estradiol on p, vegf, pge 2 and pgf 2 secretion by human luteal cells. moreover, we tested the effects of testosterone and androstenedione in presence of exemestane -an aromatase inactivator. highly purified human luteal cells were cultured for 24 h with medium alone (control) or in presence of increasing concentrations of testosterone or dihydrotestosterone or androstenedione (from 10 -10 to 10 -6 m) or in presence of increasing concentrations of estriol, estrone and 17--estradiol (from 0.1 to 10 ng/ml). moreover, testosterone and androstenedione 10 -7 m were tested in presence of exemestane (from 4 to 400 nm). in the culture medium vegf, p, pge 2 and pgf 2 secretion was assayed by commercially available elisa kits. in order to evaluate the influence of androgens and estrogens on vegf mrna expression on luteal cells real-time rt-pcr has been performed. our results demonstrated that testosterone, androstenedione and dihydrotestosterone were all able to significantly reduce vegf secretion in human luteal cells, while no effect was seen on vegf mrna expression. androgens were also able to significantly decrease p and pgf 2 secretion, while an increase was observed on pge 2 production. moreover our preliminary results demonstrated that in human isolated luteal cells estriol, estrone and -estradiol at all tested doses are able to mimic androgens effects on p and pge 2 production. on the contrary estrogens were able to increased vegf release. estrogens seemed to have no effect on pgf 2  released. data regarding exemestane inhibition of testosterone and androstenedione are still in progress. increased levels of androgens were able to decrease luteal vegf, p and pgf 2 release and might be involved in pcos-luteal phase defect. nevertheless, the observed effects could probably be attributed -at least in part -to estrogens locally produced via the aromatase enzyme, rather than be directly mediated by testosterone and androstenedione. the in vitro effect of proghrelin-derived peptides on purified human luteal cells. anna tropea, 1 mariateresa orlando, 1 maria francesca gangale, 1 federica romani, 1 isamaria loiudice, 1 federica tiberi, 2 stefania catino, 3 antonio lanzone, 3 rosanna apa. 1 1 cattedrà di fisiopatologia della riproduzione, università cattolica del s. cuore (ucsc), roma, italy; 2 istituto scientifico internazionale (isi) "paolo vi", ucsc, roma, italy; 3 istituto di ricerca "associazione oasi maria ss onlus", troina (en), italy. ghrelin, well known mediator of neuroendocrine effects, has been demonstrated to have a role as signal for energy insufficiency. several evidences suggested that ghrelin might also operate as regulator of reproductive function. indeed, we recently demonstrated that both p and vegf released were significantly decreased by ghrelin in isolated human luteal cells. moreover ghrelin was also able to reduce prostaglandin (pg) e 2 and increase pgf 2 luteal cells release. in the present work we investigated whether two ghrelin-related peptides derived by the same ghrelin precursor (unacylated ghrelin and obestatin) might affect human isolated luteal cells function. conventionally regarded as an inert form of ghrelin, unacylated ghrelin has been recently proven as biologically active in specific cellular contexts. obestatin has been suggested to directly control porcine ovarian cell functions. in these assumptions, we investigated whether unacylated ghrelin and obestatin could directly affect luteal progesterone (p) release. moreover, since vascular endothelial growth factor (vegf) is known to play a critical role in luteal development and function, the influence of unacylated ghrelin and obestatin on luteal vegf, pgf 2 and pge 2 production was also analysed. highly purified human luteal cells were incubated for 24h with medium alone (control) or with hcg (100 ng/ml) or with cocl 2 (10 μm) or in presence of increasing concentrations of unacylated ghrelin (1-100 nm) and obestatin (1-500 nm). in the culture medium vegf, pgf 2 , pge 2 and p secretion was assayed by commercially available elisa kits. moreover real-time rt-pcr has been performed in order to evaluate whether unacylated ghrelin and obestatin could affect vegf mrna in human luteal cells. our preliminary results demonstrated that either unacylated ghrelin or obestatin are able to negatively affect luteal steroidogenesis. moreover, both peptides seemed to increase the release of two luteotropic factors -vegf and pge 2and to reduce pgf 2 release -a luteolytic prostanoid -from isolated human luteal cells. finally data regarding the expression of vegf mrna are still in progress. our results suggest that unacylated ghrelin and obestatin -two ghrelin-related peptides -could play a role in regulating luteal function affecting both luteal steroidogenesis and luteotropic/luteolytic imbalance. the mechanisms responsible for labor progression have yet to be fully elucidated. in a previous study, over-expression of small conductance calcium-activated k + channel isoform 3 (sk3) in transgenic mice compromised parturition, suggesting a role for sk3 channels in this process. based on these findings, we hypothesized that sk3 channel expression is reduced late in pregnancy to enable the uterus to produce the forceful contractions required for parturition. we investigated the effects of sk3 channel expression on gestation and parturition by comparing transgenic mice over-expressing mice sk3 (sk3 t/t ) mice to wild-type (wt). in wt mice, sk3 transcript and protein are significantly reduced during pregnancy. the force produced by uterine strips from sk3 t/t mice on the day of delivery was significantly less than wt or sk3 knockout control (sk3 dox ), and the contractile force reached wt levels by application of the sk3 channel inhibitor, apamin. moreover, lipopolysaccharide and ru486, which induce pre-term labor in wt mice, failed to result in completion of delivery in sk3 t/t mice. thus, stimuli that initiate parturition under normal circumstances are insufficient to coordinate the uterine contractions needed for the completion of delivery when sk3 channel activity is in excess. the mechanism(s) down-regulating this channel in the uterus during pregnancy is unknown. the sk3 gene promoter region contains two specificity protein (sp) binding sites; 1) sp1, is a transcription factor known to enhance the transcription of genes in response to estrogen, and 2) sp3, which competes for the same binding motif as sp1, reduces gene expression. sp1 protein expression in mice uteri dramatically decreases in late pregnancy, while sp3 protein level remains consistent. our data indicate that sk3 channels must be downregulated for the gravid uterus to generate labor contractions sufficient for delivery in both term and preterm mice. the changes in sk3 channel expression may be transcriptionally regulated by sp1 and sp3.